Article

The interaction of pH and divalent cations at the neuromuscular junction

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Abstract

1. 1. The effects of acidic pH on transmitter release were studied at the frog neuromuscular junction, using intracellular recording techniques. 2. Acid pH reduced the amplitude of the end-plate potentials (e.p.p.s) and accelerated the frequency of the miniature e.p.p.s(m.e.p.p.s). 3. At pH 6-0 the m.e.p.p. frequency was on the average 2-5 times greater than at pH 7-4. This multiplication was independent of the divalent ion concentration of the medium over a large range. 4. Reduction of the e.p.p. amplitude at low pH was the result of a decrease in m, the number of quanta of transmitter liberated by the nerve impulse. 5. The effect of low pH on m was blocked by high concentrations of Mg2+ and by lower concentrations of Mn2+ ions. This occlusion was found even when the total concentration of divalents in the bathing solution was kept constant. 6. These results indicated that H+ and Mn2+ ions bind to an acidic site which regulates Ca-mediated release of acetylcholine (ACh). The acid dissociation constant (KH) was determined using both a kinetic and a surface charge model. The pKa of the site calculated from the kinetic model was 5-7, while a pKa of 3-6 was obtained from the surface charge model. 7. It is suggested that protonation of the acidic site mentioned above reduces evoked transmitter release by blocking the influx of Ca into the nerve terminal following the nerve action potential.

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... The pK, of the acidic nerve terminal site associated with the decrease in negative surface potential by H + was estimated. The hypothesis is that this pI~ represents an acidic site at the Ca ++ channel associated with Ca ++ influx (Landau and Nachshen, 1975). The relation between the dissociation constant for H + (Kv0, the negative surface potential [ K, = (3) d-ff for 0 s and ~r at pH 7.40 and 6.40, respectively. ...
... Since the constant C is unknown, KK cannot be determined from Eq. 12. For constant values ofK + and Ca ++, i.e., [K+]1 and [Ca++]1, log C VK may be a function ofpH (-log [H+]), i.e., log C VK = -log (1 + [K+],/KK + [Ca++]l/Kc~ + [H+]/Kn) + log C [K+] on the hypothesis that u, defined only for F >> jq, is equal to n in Dodge and Rahamimoff's model (1967); n is independent ofpH (Landau and Nachshen, 1975). If it is assumed that a graph of log Fvs. ...
... A parallel shift to the right was observed when [K+]o was changed from 7.5 to 10 mM. Slopes were not significantly different: mean slopes _+ SEM were 0.49 _+ 0.09 at 10 mM K +, and 0.37 _+ 0.09 at 7.5 mM K +. using surface charge theory was consistent with reported pKa values of 3.6 and 5.7 (Landau and Nachshen, 1975). ...
Article
Inhibition of transmitter release by protons (H+) was studied at the frog neuromuscular junction at various extracellular concentrations of calcium ([Ca++]o) and potassium ([K+]o) by recording miniature end-plate potential (MEPP) frequency with the intracellular microelectrode. H+ decreased K+ -stimulated MEPP frequency. A double logarithmic graph of MEPP frequency at 7.5 mM K+ vs. [H+]o yielded a straight line with negative slope. At 10 mM K+, there was a parallel shift to the right of the graph. According to the surface charge model, K+ acts solely to depolarize the prejunctional membrane in accordance with the Nernst equation. By decreasing the prejunctional negative surface charge, H+ decreases K+ -stimulated MEPP frequency by decreasing [Ca++]o at the Ca++ channel. An estimated pKa of 4.20 may represent an acidic site at the Ca++ channel associated with Ca++ influx. As [Ca++]o increased above 1 mM for pH 7.40 and 10 mM K+, MEPP frequency decreased, i.e., the inhibitory component of dual effects of Ca++ occurred. At pH 6.40, the inhibitory component was abolished, unmasking the stimulatory effect of Ca++ on MEPP frequency. Reversal of Ca++ action by H+ could not be explained by surface charge theory alone. A double logarithmic graph of MEPP frequency vs. [K+]o at 8.5-10.5 mM was linear with a slope of 4. There were parallel shifts to the right of this graph for changes in pH from 7.40 to 6.90 and in [Ca++]o from 1 to 2.5 mM. These results are explained on the hypothesis that K+ also acts at an acidic prejunctional site to increase Ca++ -dependent quantal transmitter release. This action of K+ was inhibited by H+ and raised Ca++. Based on kinetic theory, the estimated pKa of the acidic prejunctional K+ site was 6.31. Based on free energy calculations, its cation preference was H+ greater than K+ greater than Ca++.
... terminals ( Landau and Nachshen, 1975;Cohen and Van der Kloot, 1976;Drapeau and Nachshen, 1988). There is little information, however, about how pH~ is regulated in presynaptic nerve terminals. ...
... Lowering pHo diminishes depolarization-induced Ca uptake (Nachshen and Blaustein, 1979) and Ca-dependent, evoked transmitter release ( Landau and Nachshen, 1975;Drapeau and Nachshen, 1988) and increases the rate of spontaneous transmitter release ( Landau and Nachshen, 1975;Cohen and Van der Kioot, 1975;Drapeau and Nachshen, 1988). It therefore seemed important to determine whether changes in pHo were accompanied by changes in pHi. ...
... Lowering pHo diminishes depolarization-induced Ca uptake (Nachshen and Blaustein, 1979) and Ca-dependent, evoked transmitter release ( Landau and Nachshen, 1975;Drapeau and Nachshen, 1988) and increases the rate of spontaneous transmitter release ( Landau and Nachshen, 1975;Cohen and Van der Kioot, 1975;Drapeau and Nachshen, 1988). It therefore seemed important to determine whether changes in pHo were accompanied by changes in pHi. ...
Article
Cytosolic pH (pHi) was measured in presynaptic nerve terminals isolated from rat brain (synaptosomes) using a fluorescent pH indicator, 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF). The synaptosomes were loaded with BCECF by incubation with the membrane-permanent acetoxy-methyl ester derivative of BCECF, which is hydrolyzed by intracellular esterases to the parent compound. pHi was estimated by calibrating the fluorescence signal after permeabilizing the synaptosomal membrane by two different methods. Synaptosomes loaded with 15-90 microM BCECF were estimated to have a pHi of 6.94 +/- 0.02 (mean +/- standard error; n = 54) if the fluorescence signal was calibrated after permeabilizing with digitonin; a similar value was obtained using synaptosomes loaded with 10 times less BCECF (6.9 +/- 0.1; n = 5). When the fluorescence signal was calibrated by permeabilizing the synaptosomal membrane to H+ with gramicidin and nigericin, pHi was estimated to be 7.19 +/- 0.03 (n = 12). With the latter method, pHi = 6.95 +/- 0.09 (n = 14) when the synaptosomes were loaded with 10 times less BCECF. Thus, pHi in synaptosomes was approximately 7.0 and could be more precisely monitored using the digitonin calibration method at higher BCECF concentrations. When synaptosomes were incubated in medium containing 20 mM NH4Cl and then diluted into NH4Cl-free medium, pHi immediately acidified to a level of approximately 6.6. After the acidification, pHi recovered over a period of a few minutes. The buffering capacity of the synaptosomes was estimated to be approximately 50 mM/pH unit. Recovery was substantially slowed by incubation in an Na-free medium, by the addition of amiloride (KI = 3 microM), and by abolition of the Nao/Nai gradient. pHi and its recovery after acidification were not affected by incubation in an HCO3-containing medium; disulfonic stilbene anion transport inhibitors (SITS and DIDS, 1 mM) and replacement of Cl with methylsulfonate did not affect the rate of recovery of pHi. It appears that an Na+/H+ antiporter is the primary regulator of pHi in mammalian brain nerve terminals.
... Since the low pH mediated portion of the contraction can be reproduced by the addition of HC1, HN03, or H2S04, then it is apparent that this contraction results from the action of the raised [H+] in the SHR tissue. Low pH has been associated with a negative chronstropic (Hughes and Coret 1976), and a negative inotropic action (Pannier and Brutsaert 1968) in cardiac muscle and also a lowered pH results in a reduction of neurotransmitter release (Landau and Nachshen 1975) Ca" influx in rabbit aorta (van Breemen et al. 1973 ) . It was therefore anticipated that lowering the pH of the Krebs would inhibit contractile activity in rat aorta and thus the paradoxical contraction seen in this study to raising [H+] may indicate differences in the utilization, storage, or binding of @a2+ in the SHR aorta. ...
... It has been suggested that raising [H+] results in protonation of the cell membrane, screening of cation binding sites and a consequent interference, with the binding of calcium to the cell membrane and with calcium influx into the cell (D'Arrigo 1974;Landau and Nachshen 1975 ) , and lowering [H+] has been shown to enhance calcium influx in ventricular muscle (Kohlhardt and Haap 1976). It had been reported by Nakamura and Schwartz (1972) that the Ca3+ binding capacity of skeletal muscle SR increases with increasing [H+], with an apparent optimum pH for Ca2+ binding occurring between 6.1 and 6.3; this data suggests an important role for pH in regu- lating ECC. ...
Article
Full-text available
Significant tension development resulting from the administration of lanthanum salts (La3+) to isolated aorta tissues was observed in preparations from the spontaneously hypertensive rat (SHR) but not in preparation from age-matched normotensive Wistar rats (NWR). The response to La3+ was significantly greater in tissues maintained in bicarbonate-buffered Krebs than observed in bicarbonate and phosphate-free HEPES-buffered Krebs. At least part of the LA3+ response in the bicarbonate- and phosphate-buffered solutions was due to a pH shift and could be mimicked by raising the extracellular hydrogen ion [H+] concentration. However, a direct action of La3+ on excitation-contraction coupling could be observed in HEPES-buffered solutions where the addition of La3+ also resulted in tension development but no significant pH change; this action of La3+ was found to be resistant to inhibition by maintenance in a Ca2+-free medium and (or) D-600 pretreatment suggesting an intracellular action for La3+. The paradoxical response to both H+ and La3+ in the SHR aorta suggests that the muscle membrane may be more permeable to these ions and, in addition, suggests that the membrane and (or) intracellular calcium stores in this tissue may also be more labile than those in the normotensive controls.
... We conclude that cytosolic acidification regulates spontaneous and evoked ACh release differentially in Xenopus motoneurons, increasing spontaneous ACh release but inhibiting evoked ACh release. Nelson & Sanchez, 1962;Landau & Nachshen, 1975). In contrast, the spontaneous release of transmitter at frog and rat neuromuscular junctions is increased by extracellular acidification (Hubbard, Jones & Landau, 1968;Landau & Nachshen, 1975;Cohen & van der Kloot, 1976). ...
... Nelson & Sanchez, 1962;Landau & Nachshen, 1975). In contrast, the spontaneous release of transmitter at frog and rat neuromuscular junctions is increased by extracellular acidification (Hubbard, Jones & Landau, 1968;Landau & Nachshen, 1975;Cohen & van der Kloot, 1976). The nature of these opposite effects of extracellular pH on evoked and spontaneous ACh release is unknown. ...
Article
• The effects of intracellular pH changes on the acetylcholine (ACh) release and cytoplasmic Ca2+ concentration at developing neuromuscular synapses were studied in Xenopus nerve-muscle co-cultures. • Spontaneous and evoked ACh release of motoneurons was monitored by using whole-cell voltage-clamped myocytes. Intracellular alkalinization with 15 mmNH4Cl slightly reduced the frequency of spontaneous synaptic currents (SSCs). However, cytosolic acidification following withdrawal of extracellular NH4Cl caused a marked and transient increase in spontaneous ACh release. • Another method of cytosolic acidification was used in which NaCl in Ringer solution was replaced with weak organic acids. The increase in spontaneous ACh release paralleled the level of intracellular acidification resulting from addition of these organic acids. Acetate and propionate but not isethionate, methylsulphate and glucuronate, caused an increase in intracellular pH and a marked increase in spontaneous ACh release. • Impulse-evoked ACh release was slightly augmented by intracellular alkalinization and inhibited by cytosolic acidification. • Cytosolic acidification was accompanied by an elevation in the cytoplasmic Ca2+ concentration ([Ca2+]i), resulting from both external Ca2+ influx and intracellular Ca2+ mobilization. In contrast, the increase in [Ca2+]i induced by high K+ was inhibited by cytosolic acidification. • We conclude that cytosolic acidification regulates spontaneous and evoked ACh release differentially in Xenopus motoneurons, increasing spontaneous ACh release but inhibiting evoked ACh release.
... Another possibility is that 48-day exposure to CO 2 resulted in some irreversible alterations in the functions of GABA receptors. Further, low pH per se could have reduced neuromuscular transmission (Landau and Nachshen, 1975;Takahashi and Copenhagen, 1996). Clearly, further studies are needed for mechanistic understanding of the observed reduction in the contraction force of tube feet under elevated CO 2 conditions. ...
Article
This study examined how contraction force and protein profiles of the tube feet of the sea urchin (Pseudocentrotus depressus) were affected when acclimated to 400 (control), 2000 and 10,000 μatm CO2 for 48 days. Acclimation to higher CO2 conditions significantly reduced contraction force of the tube feet. Two-dimensional gel electrophoresis showed that eight spots changed in protein volume: six up-regulated and two down-regulated. Using matrix-assisted laser desorption/ionization-quadrupole ion trap-time of flight mass spectrometry, three up-regulated spots (tubulin beta chain, tropomyosin fragment, and actin N-terminal fragment) and two down-regulated spots (actin C-terminal fragment and myosin light chain) were identified. One possible interpretation of the results is that elevated CO2 weakened contraction of the tube feet muscle through an alteration of proteome composition, mainly associated with post-translational processing/proteolysis of muscle-related proteins.
... The site of fatigue has variously been claimed to be the central nervous system (Ikai & Steinhaus, 1961), the neuromuscular junction (Landau & Nachsen, 1975) or the muscle itself (Merton, 1954). Since the classic work of Fletcher & Hopkins (1907), lactic acid accumulation has been implicated in muscle fatigue both in intact organisms (Karlsson et al. 1975 a) and isolated muscle preparations (Hill & Kupalov, 1930;Mainwood & Worsley-Brown, 1975;Fitts & Holloszy, 1976). ...
Article
Full-text available
Fatigue produced a marked increase in the lactic acid content of hind-limb muscles, the blood, and the whole animal. After 15 min of rest there was little decline of lactic acid levels but the animals could be stimulated into about 3 min of intense activity. This re-fatigue produced a further increase in lactic acid levels. Gastrocnemius muscles removed from fatigued frogs and stimulated in vitro were able to generate initial tensions similar to those in control muscles; total tension was about a third of the control value. In vitro stimulation of these muscles from fatigued frogs led to additional accumulation of lactic acid. Fatigue produced little decrease in the glycogen content of muscles in X. laevis but a marked decrease in R. pipiens. Considerable glycogen stores remained even in the muscles of re-fatigued animals. These data show that accumulation of lactic acid in muscle or blood, depletion of glycogen in muscle, or change in blood pH cannot account for fatigue in these species. Possible other causes of fatigue are discussed.
... In contrast, extracellular acidification inhibited the release of dopamine by blocking voltage-gated Ca 2 ÷ channels (Drapeau and Nachshen, 1988). A similar influence of extra-and intracellular protons on synaptic transmitter relase was reported from the frog (del Castillo et al., 1962;Landau and Nachshen, 1975;Cohen and van der Kloot, 1976) and at the rat neuromuscular junction (Hubbard et al., 1968). ...
Article
The regulation of H+ in nervous systems is a function of several processes, including H+ buffering, intracellular H+ sequestering, CO2 diffusion, carbonic anhydrase activity and membrane transport of acid/base equivalents across the cell membrane. Glial cells participate in all these processes and therefore play a prominent role in shaping acid/base shifts in nervous systems. Apart from a homeostatic function of H(+)-regulating mechanisms, pH transients occur in all three compartments of nervous tissue, neurones, glial cells and extracellular spaces (ECS), in response to neuronal stimulation, to neurotransmitters and hormones as well as secondary to metabolic activity and ionic membrane transport. A pivotal role for H+ regulation and shaping these pH transients must be assigned to the electrogenic and reversible Na(+)-HCO3-membrane cotransport, which appears to be unique to glial cells in nervous systems. Activation of this cotransporter results in the release and uptake of base equivalents by glial cells, processes which are dependent on the glial membrane potential. Na+/H+ and Cl-/HCO3-exchange, and possibly other membrane carriers, accomplish the set of tools in both glial cells and neurones to regulate their intracellular pH. Due to the pH dependence of a great variety of processes, including ion channel gating and conductances, synaptic transmission, intercellular communication via gap junctions, metabolite exchange and neuronal excitability, rapid and local pH transients may have signalling character for the information processing in nervous tissue. The impact of H+ signalling under both physiological and pathophysiological conditions will be discussed for a variety of nervous system functions.
... Although this was necessary to prevent precipitation of La3+, it also proved useful as it allowed subthreshold e.p.ps to be observed in a wider range of Ca and Sr concentrations than at normal pH. This effect may be due to the action of H+ as a modest competitive inhibitor of ACh release (Landau & Nachsen, 1975;E.M.S. unpublished). As matching levels of ACh secretion were employed to calculate affinity and efficacy, the quantitative results were not dependent upon pH (unpublished). ...
Article
1 The behaviour of the divalent cations Ca and Sr as agonists for receptors that mediate the synchronous evoked secretion of acetylcholine (ACh) was studied in the hope of determining whether the relationship between Ca binding and ACh secretion is determined only by the law of mass action or by the mathematical framework of receptor theory. Experiments were designed to evaluate the assumption that maximum effect requires occupation of all receptors by testing for the presence of spare Ca receptors on presynaptic terminals. Frog cutaneous nerve-muscle preparations were employed in conjunction with conventional electrophysiological methods.2 Curves of log [Ca] or log [Sr] against the mean number of ACh quanta released (m) were constructed to saturation. The log [Sr]-m relationship was shifted to the right and had a smaller maximum than the log [Ca]-m curve. This suggests that Ca has a higher efficacy than Sr and raises the possibility that spare binding sites are present for Ca.3 As a qualitative test for spare Ca receptors, La(3+) (>/=0.5 mum) or 2-chloroadenosine (25 mum) was employed as an irreversible antagonist of the effects of extracellular Ca on evoked ACh release. Despite the irreversible blockade of a proportion of receptors, increases in the [Ca] overcame this antagonism and produced a parallel shift in the log [Ca]-m relation to the right. This suggests an apparent receptor reserve for Ca. Antagonism of Sr-mediated ACh release by either La(3+) or 2-chloroadenosine could not be overcome by increasing the [Sr].4 As a quantitative test for spare Ca binding sites, the equilibrium affinity constant for Sr(K(Sr)) as a competitive inhibitor of Ca was determined and compared with values for K(Sr) calculated by two other methods which invoke the spare receptor assumption. All three methods produced comparable results. (K(Sr) = 0.24-0.27 mm(-1)).5 The equilibrium affinity constant for Ca (K(Ca)) was calculated by comparing reciprocal plots of the concentrations of Ca that produce equal levels of ACh release in the presence and absence of La(3+) (0.5 mum-3 mum). K(Ca) was estimated to be between 0.02 and 0.06 mm(-1).6 Efficacy (e), which is thought to reflect the ability of Ca or Sr once bound to receptors to support ACh release, was determined by the modified occupation theory of Stephenson (1956). The e(Ca) was estimated to be 9-20 and e(Sr) was 0.2-0.5.7 The experimentally determined values for K(Ca), K(Sr), e(Ca), e(Sr) along with the assumptions that spare Ca binding sites exist and that the non-linearities in the log [Ca] or log [Sr]-m curves are introduced beyond the sites of binding and efficacy were used to generate theoretical log [Me]-m curves. The theoretical relationships were similar to the experimental results.8 The results suggest that spare Ca receptors are present at motor nerve endings and that receptor theory provides an accurate quantitative description of the lumped events between Ca binding and ACh secretion. The possible physical correlates of affinity and efficacy are discussed.
... After acid loading, recovery of intracellular pH to normal levels is inhibited by anion blocking agents (Roos & Boron, 1981). At vertebrate neuromuscular junctions, acidification has been shown to decrease the average number of quanta evoked by a nerve impulse (Landau & Nachshen, 1975), yet it has no affect on the time course of evoked release (Datyner & Gage, 1980). Thus, it is possible that the synaptic depression in the presence of anion blocking reagents, which we have observed, is the result of changes in axoplasmic pH. ...
Article
1. The effects of the anion-transport blocking reagents Probenecid, 4,4'diisothiocyanostilbene-2,2'-disulphonate (DIDS), and pyridoxal phosphate on the number of quanta released (m) and the time course of the probability of quantal release, alpha(t), following an action potential were studied at single synaptic sites on the opener muscle in the crayfish leg.2. Low concentrations (10-50 muM) of DIDS or Probenecid produced decreases in m to about 30-50% of the initial value; at higher concentrations (100 muM or greater), evoked release failed. Much greater concentrations (500 muM) of pyridoxal phosphate were necessary to produce decreases in m.3. Synaptic depression in 50 muM-Probenecid was reversible by removing the agent; after treatment with 50 muM-DIDS, however, m partially regained the initial value upon removal of the drug.4. The time course of alpha(t) after an action potential was unaffected by any of the reagents.5. It is concluded that the reagents decrease the number of quanta evoked by a nerve impulse by decreasing the magnitude, but not the time course, of the average rate of quantal release following an action potential; it is suggested that nerves and other secretory cells may share a common mechanism during exocytosis that is subject to modification by these agents.
... Mn2, inhibition of m cannot be attributed chiefly to surface charge screening, but can be better accounted for assuming that Mn2+ tightly binds surface charge. A similar charge binding scheme was proposed by Landau and Nachshen (1975) to account for the effects of H+ on m. The ability of the electrostatic screening hypothesis to account for several interactions between inhibitory cations and Ca2", without the addition of new parameters, makes this model a reasonable alternative to the model of cationic inhibition of quantal release which postulates competitive binding to unspecified Ca2"-dependent release activating sites. ...
Article
We have investigated an electrostatic screening hypothesis of cationic inhibition of quantal release at the neuromuscular junction of the frog (Rana pipiens). According to this hypothesis, increasing the extracellular concentration of an inhibitory cation reduces the quantal content (m) of the end-plate potential by reducing the ability of negative surface charge to attract Ca2+ to the external surface of the presynaptic membrane. The inhibitory power of various cations should depend only on their net ionic charge and should increase strongly with increasing charge. We have demonstrated, in Ringer's solutions containing modified concentrations of Na+, Ca+, and Mg2+, that at fixed concentrations of Ca2+ and Na+ (a) the dependence of m on [Mg2+]0 is satisfactorily accounted for by electrostatic theory and (b) the dependence of m on the univalent cation concentration of the modified Ringer's solution is satisfactorily predicted from the Mg2+ inhibition of m. (Glucosamine or arginine was used to replace a fraction of the Na+ content of Ringer's solution in the latter experiments.) These results are consistent with electrostatic screening actions of Mg2+ and univalent cations in the inhibition of m. We have also re-examined the inhibition of m caused by the addition to Ringer's solution of two trace concentration divalent cations, Mn2+ and Sr2+. Our data suggest that the inhibition of m by Sr2+ at high quantal contents may also be due to surface charge screening, while the potent inhibitory actions of Mn2+ may be due to its ability to bind negative surface charge.
... The Blocking Effect of Manganese Is Enhanced in the Presence of TTX To further elucidate the effect of reducing the spike amplitude on transmitter release, the inhibitory action of Mn2" was examined. According to previous work, this ion competes with calcium for the release site, X (Meiri and Rahamimoff, 1972;Landau and Nachshen, 1975). The classic equation for release which includes the effects of competing, inhibitory ions is the following (Dodge and Rahamimoff, 1967): ...
Article
Transmitter release from frog motor terminals was studied in the presence of very low concentrations of tetrodotoxin (TTX, 4.10(-10)--6.10(-9) g/ml). TTX reversibly reduced the amplitude of the end-plate potential (epp), while leaving the amplitude distribution to follow Poisson's law. The effects of a number of divalent cations were studied in the presence of TTX. It was found that after the addition of TTX there was an increase in the constant of dissociation of calcium and strontium from a hypothetical membrane "release site," while the dissociation constants of magnesium and manganese remained unaltered. It is concluded that the release site is probably intracellular and that a reduced presynaptic spike amplitude, as well as magnesium and manganese ions, decrease the access of calcium and strontium to the site.
... Thus, as with basal dopamine release, evoked release was enhanced by cytosolic acidification. DISCUSSION Our observations that cytosolic acidification stimulated basal dopamine release from rat brain synaptosomes and that lowering pHo inhibited evoked release are consistent with previous observations in the frog (del Castillo et al., 1962; Landau and Nachshen, 1975; Cohen and van der KIoot, 1976) and the rat neuromuscular junction (Hubbard et al., 1968). In particular, the requirement for cytosolic, and not just extracellular, acidification for stimulation of basal release agrees with the finding of Cohen and van der Kloot (1976). ...
Article
We examined the effects of extracellular and intracellular pH changes on the influx of radioactive 45Ca, the concentration of ionized Ca (pCai) as monitored with the Ca-sensitive fluorescent indicator fura-2, and the efflux of dopamine in presynaptic nerve endings (synaptosomes) isolated from rat brain corpora striata and preloaded with [3H]dopamine. Cytosolic pH (pHi) was monitored by loading the synaptosomes with the H+-sensitive fluorescent indicator 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) (see Nachshen, D. A., and P. Drapeau, 1988, Journal of General Physiology, 91:289-303). An abrupt decrease of the pH of the external medium, from 7.4 to 5.5, produced a slow decrease of pHi (over a 5-min period) from an initial value of 7.2 to a steady state level of approximately 5.8. When 20 mM acetate was present in acidic media, pHi dropped as fast as could be measured (within 2 s) to a level similar to that reached (more slowly) in the absence of acetate. It was therefore possible to lower pHi over short time periods to different levels depending on whether or not acetate was present upon extracellular acidification. Extracellular acidification to pH 5.5 (in the absence of acetate) had no significant effect on pCai and dopamine release over a 30-s period (pHi = 6.4). Acidification in the presence of acetate lowered pHi to 5.8 without affecting pCai, but dopamine efflux increased approximately 20-fold. This increase in basal dopamine release was also observed in the absence of extracellular Ca. Thus, intraterminal, but not extracellular, acidification could stimulate the efflux of dopamine in a Ca-independent manner. The high Q10 (3.6) of acid-stimulated dopamine efflux in the presence of nomifensine (which blocks the dopamine carrier) was consistent with an activation of vesicular dopamine release by H+. When synaptosomes were both depolarized for 2 s in high-K (77.5 mM) solutions and acidified (in the absence of acetate), there was a parallel block of 45Ca entry and evoked dopamine release (50% block at pH 6.0 with 0.2 mM external Ca). When acetate was included in the acidic media to further reduce pHi, Ca entry remained blocked, but evoked dopamine release was increased. Therefore, extracellular, but not cytosolic, acidification inhibited the release of dopamine by blocking voltage-gated Ca channels. The stimulation by cytosolic acidification of both basal and evoked dopamine release suggests that vesicular release in resting and depolarized synaptosomes was directly activated by cytoplasmic H+.
... However, if a second charged group does play a role in divalent ion selectivity, this group might be strongly acidic, thereby remaining dissociated in the pH range we examined. Neurotransmitter release, at the frog neuromuscular junction, is reduced by lowering the pH of the bathing medium (Del Castillo et al., 1962), and it has been suggested that this reduction is caused by protonation of an acidic site (with a pKa 5.7) that blocks Ca influx into the nerve terminal (Landau and Nachshen, 1975). The present study provides direct evidence for such a mechanism in synaptosomes. ...
Article
The effects of low pH, and of alkaline earth cations, were examined on calcium uptake by pinched-off nerve terminals (synaptosomes). This uptake appears to be mediated by voltage-sensitive Ca channels (J. Physiol. 247:617, 1975). Ca uptake was measured in low (5 mM) or high (77 mM) potassium media. The extra uptake promoted by depolarizing (K-rich) media was almost maximal at pH 7.5, and decreased as the pH was lowered. Data relating depolarization-induced 45Ca uptake to pH fit a titration curve with a pKa approximately 6. Experiments in which Ca concentration and pH were both varied indicated that Ca2+ and H+ compete for a common binding site. Inhibition of depolarization-induced 45Ca uptake by the alkaline earth cations was studied to determine the apparent binding sequence for these cations in the Ca channels: Ca greater than Sr greater than Ba greater than Mg. This sequence resembles that observed for block of Ca channels in other preparations. The apparent binding sequence of the alkaline earth cations and the apparent pKa (approximately 6) of the Ca-binding site indicate that the Ca channel is a "high field strength" system. Protonation of a Ca channel binding site could explain the inhibitory effect of low pH on Ca-dependent neurotransmitter release (cf. Del Castillo et al., J. Cell. Comp. Physiol. 59:35, 1962).
... These results indicate that H + and Mn 2+ ions bind to an acidic site which regulates Ca 2+ -mediated ACh release. It was suggested that protonation of the acidic site evoked transmitter release by blocking the influx of Ca 2+ cations into the nerve terminal following the nerve action potential (Landau and Nachshen, 1975). The common denominator for most of these findings is the inhibitory effect of manganese on evoked ACh release in the presynaptic neuron. ...
Article
Information on changes in the central nervous system (CNS) cholinergic systems following exposure to manganese are considerably less extensive than that associated with other neurotransmitter systems. However, experimental and clinical evidence support the notion that cholinergic activity plays a key role in the pathophysiology of manganese-induced neurotoxicity. Manganese acts as a chemical stressor in cholinergic neurons in a region-specific manner causing breakdown of the cellular homeostatic mechanisms. In fact, a number of cholinergic synaptic mechanisms are putative targets for manganese activity: presynaptic choline uptake, quantal release of acetylcholine into the synaptic cleft, postsynaptic binding of acetylcholine to receptors and its synaptic degradation by acetylcholinesterase. Moreover, manganese significantly influences astrocytic choline transport systems and astrocytic acetylcholine-binding proteins. Thus, manganese exerts its effect on the highly dynamic reciprocal relationship between astrocytes and cholinergic neurons. Cholinergic afferents are crucial in the physiology of locomotion, cognition, emotion and behavioral response, and therefore, it is not surprising that the anatomical selectivity of most manganese-induced cholinergic effects is compatible with the clinical correlates of manganism, which involves impairment of emotional response, decline in higher cortical functions and movement disorder. Manganism, also referred to as Parkinson's-like disorder, is initially manifested by a neuropsychiatric syndrome (locura manganica), the most frequent symptoms and signs of which are compulsive behavior, emotional lability, visual hallucinations and flight of ideas, cognitive decline and memory loss. These signs and symptoms are followed by an extrapyramidal syndrome, which shares numerous clinical and pathophysiological characteristics with idiopathic Parkinson's disease (PD). This natural history of disease could be a clinical reflection of the preferential involvement of the cholinergic systems, initially in the septo-hippocampus and later in the basal ganglia. These observations highlight the importance of studying the role of the CNS cholinergic systems in manganese-mediated neurotoxicity.
Article
The authors sought to compare power output at blood lactate threshold, maximal lactate steady state, and pH threshold with the average power output during a simulated 20-km time trial assessed during cycle ergometry. Participants (N = 13) were trained male and female cyclists and triathletes, all permanent residents at moderate altitude (1,525-2,225 m). Testing was performed at 1,525 or 1,860 m altitude. Power outputs were determined during a simulated 20-km time trial (PTT), at blood pH threshold (PpHT), at maximal lactate steady state (PMLSS), and at blood lactate threshold determined by 2 methods: the highest power output that did not result in consecutive and continued increases in blood lactate concentrations from exercising baseline (PLT) and the highest power output that did not result in consecutive and continued increases of > or =1 mmol/L in blood lactate concentrations from exercising baseline (PLT1). PLT, PLT1, and PMLSS were all significantly lower than PpHT (p < .05) and PTT (p < .05). No significant difference was observed between PpHT and PTT (p > .05). Significant correlations were observed between each of the metabolic variables, PLT, PLT1, PMLSS, and PpHT, compared with PTT (p < .05). The authors conclude that, of the 4 metabolic variables, only PpHT offered an accurate reflection of PTT.
Article
Bicuculline-induced status epilepticus was studied in paralyzed rabbits ventilated with an oxygen and nitrous oxide mixture. An Oxford Instruments TMR 32-200 spectrometer was used to record phosphorus 31 nuclear magnetic resonance spectra of the in vivo brain. An array of conventional physiological variables including the electroencephalogram was simultaneously recorded. Several features were consistently observed during status epilepticus: (1) Phosphocreatine levels fell to about two-thirds of their control values and remained at that level despite a gradual decline in seizure activity; (2) intracellular pH declined and then remained constant, whereas seizure discharges declined; (3) adenosine triphosphate levels remained constant at their control values. These new, lower levels of brain phosphocreatine and intracellular pH were largely unaffected by increases in seizure activity brought about by elevation of blood pressure from levels too low to support adequate cerebral perfusion, by waning of anticonvulsant drug effect, or by repeated doses of bicuculline.
Article
The amplitude of the electrophoretically evoked end-plate potential increases with changing the pH of the bathing solution from 9.4 to 5.4 at room temperature. This change is not observed at lower temperature. The underlying current (e.p.c.I ) is slightly decreasing at room temperature by lowering the pH. The relationship between the amplitude of the e.p.c.I and membrane potential is highly non-linear at pH 9.4, while it is quite linear at pH 5.4. The time course of the e.p.c.I is changed neither by different pH, nor by different membrane potential. The data suggest that during the e.p.c.I , the mediator (ACh), the receptor (R) and the mediator-receptor complex are in equilibrium: the amplitude of the e.p.c.I will thus depend on the affinity constant of the reversible reaction between ACh and R. It is concluded that by decreasing the pH, the affinity constant is decreased.
Chapter
This chapter reviews the experiments on the relation between [Ca2+]in and transmitter release at synapses, with an emphasis on the neuromuscular junction. Then, the quantitive models of calcium action, starting with the simplest and adding variables stepwise are discussed, until a model with the minimum complexity consistent with the data is reached. The models are compared to the data for transmitter release at synapses. In the end, the implications of the model for the control of spontaneous quantal release and for synaptic facilitation are considered. The simplest realistic model for Ca2+ action in triggering the quantal release of neurotransmitter takes into account the following: the Ca2+ that enters following stimulation adds to [Ca2+]in already present in the resting terminal. Then there is a cooperative interaction in release, n Ca2+ binds to a receptor to trigger the release of a quantum, and there are a finite number of release sites. There are several reasons to believe that the mechanism of Ca2+ action in eliciting secretion is substantially different at the neuromuscular junction and the adrenal medulla.
Article
A rat cerebral cortical slice preparation was used to study the response of transmitter release to the application of the food dye, Erythrosin B, a tetraiodinated derivative of fluorescein. Erythrosin B (100 microM) stimulated net release of previously taken up [3H]norepinephrine and [3H]gamma-aminobutyric acid (GABA). The Erythrosin-induced release of GABA (the only transmitter studied) occurred in the absence of added Ca2+, and in the presence of tetrodotoxin (TTX). Ultrastructural analysis of the vesicle content of frog neuromuscular junctions treated with Erythrosin B revealed a diminution in the number of synaptic vesicles present in the nerve terminal. By using fluorescein and some halogen-substituted derivatives including Erythrosin B, it was found that incubation with the unhalogenated compound caused no net release, whereas incubation with the iodine-, chlorine- or bromine-substituted compound did cause release. It was also found that somewhat greater release induced by Erythrosin B (at 100 microM) occurred in the light than in the dark. That Erythrosin B inhibits the Na+,K+,Mg2+-ATPase was confirmed in this preparation; it did so in both light and dark. The discrepancy between release and Na+,K+,Mg2+-ATPase blockade in the dark suggests that release either occurs by some other mechanism than by Na+,K+,Mg2+-ATPase blockade, or that an additional light-dependent process contributes to the release. We conclude that Erythrosin B can presumably induce net release of transmitters generally, that release does not occur via the TTX-sensitive Na+ channel, that release via vesicles does occur, and that light somewhat enhances the release.
Article
: Dopamine synthesis regulation as a function of pH has been examined in rat brain striatal synaptosomes. Synthesis stimulation produced by lowering the incubation pH from 7.2 to 6.2 is accompanied by a significant increase in apparent A'm for tyrosine and in apparent Vmax. While these kinetic alterations are similar to those produced by the depolarizing agent veratridine, it does not appear that synthesis is stimulated at pH 6.2 via synaptosomal depolarization since (1) synthesis stimulation still occurs at pH 6.2 in a calcium-free medium in contrast to the calcium-dependency of veratridine- induced stimulation and (2) tyrosine uptake is not inhibited by incubation at pH 6.2, but is markedly inhibited by veratridine. In order to study how the regulatory properties of synaptosomal preparations vary according to pH, the ability of synaptosomal dopamine synthesis to respond to various agents was tested between pH 7.2 and 6.2. The stimulatory effects of veratridine, amphetamine, phenylethylamine and dibutyryl cyclic AMP at pH 7.2 were significantly diminished at pH 6.2. In addition, incubation at pH 6.2 antagonized the veratridine-induced inhibition of tyrosine uptake, suggesting an interference with the depolarization process. The inhibitory effects of dopamine and tyramine at pH 7.2 were also antagonized at pH 6.2. In contrast to the effects of pH 6.2 buffer, incubation at pH 6.6 does not markedly alter responses to the various drugs. The results suggest that, although basal dopamine synthesis rates can be increased by lowering the pH, synaptosomal regulatory properties are significantly altered as the pH is lowered below 6.6.
Article
1. The increase in miniature end-plate potential (m.e.p.p.) frequency in response to tetanic stimulation of the motor nerve at frog neuromuscular junctions in Ca(2+)-free, Mg(2+) EGTA-containing (0 Ca(2+)-Mg(2+) EGTA) solutions of varying tonicity has been studied. The response to stimulation is markedly increased in hypertonic solutions and is decreased in hypotonic solutions. Under these conditions changes in tonicity have comparable effects on stimulated and spontaneous quantal release.2. The tonicity was raised by adding sucrose, NaCl or glycine to the extracellular solution. The effects of the addition depended primarily on the increase in osmolarity of the solution, not on the chemical species producing it.3. The tonicity was decreased by lowering [NaCl](o). The hypotonic solution decreased the response to tetanic stimulation. When the tonicity of the solution with the low [NaCl](o) was restored to normal by adding sucrose, the response was restored to its usual level. These results suggest that in 0 Ca(2+)-Mg(2+) EGTA solutions stimulation does not enhance the probability of quantal release by raising [Na(+)](i).4. Repeated bouts of tetanic stimulation produced almost identical responses. In some instances the frequency continued to rise after the end of the tetanic stimulation, as reported by Erulkar & Rahamimoff (1978). This suggests that the stimulation of the nerve leads to the elevation within the terminal of a substance that in turn liberates an activator for quantal release.5. The Q(10) for the increase in probability of quantal release is as high as 7. High Q(10) values have also been reported for spontaneous m.e.p.p. frequencies. Tonicity and temperature appear to affect spontaneous and stimulated quantal release similarly.
Article
An in vitro preparation of the medulla oblongata of the rat was used to examine the responses to reducing pH, at constant CO2, of neurons in several identified nuclei. Neurons in an area close to the ventral surface, which is thought to be the location of pH-sensitive central respiratory chemoreceptors, did not respond differently from neurons in other medullary nuclei.
Article
Exposure of the frog neuromuscular junction at 17° C or 23° C to salines with low [Ca2+], buffered with EGTA, causes mepp frequency to fall after 4–6 min to about 20% of the control rate. Results obtained in the presence of verapamil suggest that this fall is a consequence of a lower Ca2+-influx coupled with the action of the extracellular EGTA in promoting Ca2+-efflux from the terminals. These findings confirm the suggestion that [Ca2+]i has a major role in determining mepp frequency. At 13° C, the fall in mepp frequency after addition of EGTA is preceded by a transient (1–2 min) rise in mepp rate which is not present at 17° C or in the presence of verapamil. This transient acceleration in spontaneous release is believed to be because EGTA promotes the emptying of a Ca2+-reservoir on or beneath the inner face of the membrane, thus causing a rapid Ca2+-efflux via the Ca2+-sensitive sites that trigger exocytosis of transmitter. The significance of the sensitivity of the response to temperature is discussed. The suppressive effect of higher temperatures can be reversed to some extent by hyperosmotic salines, an effect that may reflect the action of hypertonicity on the plasmalemma. It is concluded that the characteristics of the release system may change markedly at about 16° C. Ca2+-EGTA buffers are widely used; it is suggested that extracellular EGTA can also modify [Ca2+]i in cellular systems.
Article
Effects of hypoxia and pH alterations on the spontaneous contractions and responses to cholinergic stimuli or KCl were investigated in isolated human intestines. The longitudinal strip of human intestine showed spontaneous contractions. The spontaneous contraction was abolished by hypoxia with nitrogen gas substitution (95% N2 and 5% CO2) but not by substitution with acidic (pH 6.53) or alkaline (pH 7.75) solution. Contractile responses to acetylcholine (ACh) were not altered by treatment with hypoxia or pH alterations. KCl (10 mM)-induced contraction was inhibited by hypoxia but not by pH alterations. Transmural electrical stimulation elicited a transient contraction that was blocked by tetrodotoxin or atropine. Contractions induced by electrical stimulation at a low frequency (5 Hz) was not altered by hypoxia or pH alterations. The metabolic pathway related to energy generation and utilization for the spontaneous contraction and KCl-induced contraction seems to be more dependent on oxygen supply than that for ACh-induced contraction. The contractile mechanism in human intestine seems to be resistant to pH alterations.
Chapter
The brain slice preparation has been used in a wide variety of investigations since its development in the 1930s by Quastel and by Elliot (Elliot and Wolfe, 1962). Prior to the 1970s, most of this work centered on slice metabolism, with a particular focus on the metabolic consequences of electrical activity (McIlwain and Bachelard, 1971). Although these studies were fundamental to the whole development of the brain slice as a useful preparation, they had a serious drawback in that normal electrophysiological responses could not be obtained from these cortical preparations. Thus, electrical activity was mimicked by profound membrane depolarizations, produced either by high-frequency electrical stimulation or by large changes in extracellular K+ concentrations. More recently, the development of the olfactory and the hippocampal slice preparations (Yamamoto and Kurokawa, 1970; Skrede and Westgaard, 1971) has opened the door to much more sophisticated studies of the relationship between neural activity and energy metabolism than was possible with the previous cortical slice preparation. Thus, it is now possible to correlate metabolic and electrophysiological changes in different conditions and thereby determine mechanisms by which neural transmission affects metabolism and vice-versa (Yamamoto and Kurokawa, 1970).
Article
The effects of a wide variety of agents on the release of vesicles filled with neurotransmitters have been studied in synaptic junctions, particularly the frog neuromuscular junction. Most of these treatments modify [Ca2+] either transiently (altering evoked release) or in the longer term (altering the spontaneous release of single vesicles). The recent elucidation of the protein-protein interactions in the active zone during priming and exocytosis enables us to see how treatments that alter the slope of the double logarithmic plots of the number of vesicles released versus [Ca2+](o), are acting at the molecular level.
Article
The effects of external pH (pH out) variations on the Na+ and on the Ca2+ dependent fractions of the evoked amino acid neurotransmitter release were separately investigated, using GABA as a model transmitter. In [3H]GABA loaded mouse brain synaptosomes, the external acidification (pH out6.0) markedly decreased the Na+ dependent fraction of [3H]GABA release evoked by veratridine (10 μM) in the absence of external Ca2+, as well as the Ca2+ dependent fraction of [3H]GABA release evoked by high (20 mM) K+ in the absence of external Na+. The depolarization-induced elevation of [Nai ] (monitored in synaptosomes loaded with the Na+ indicator dye, SBFI) and the depolarization-induced elevation of [Cai ] (monitored in synaptosomes loaded with the Ca2+ indicator dye fura-2) were also markedly decreased at pH out 6. On the contrary, the external alkalinization (pH out 8) facilitated all the above responses. A slight increase of the baseline release of the [3H]GABA was observed when pH out was changed from 7.4 to 8. This effect was only observed in the presence of Ca2+. pH out changes from 7.4 to 6 or to 7 did not modify the baseline release of the transmitter. All the effects of pH out variations on [3H]GABA release were independent on the presence of HCO-3. It is concluded that external H+ regulate amino acid neurotransmitter release by their actions on presynaptic Na+ channels, as well as on presynaptic Ca2+ channels.
Article
Mild subclinical impairment of neuromuscular transmission can be detected with single-fibre electromyography (SFEMG) in subgroups of patients suffering from migraine and could be due to dysfunctioning Ca2+-channels on motor axons controlling stimulation-induced acetylcholine release. Acetazolamide, which is thought to ameliorate ion channel function, was shown effective in familial hemiplegic migraine and episodic ataxia type 2, both of which are associated with mutations of the neuronal Ca2+-channel gene CACNA1A, as well as in aura status. We treated therefore in an open pilot study five non-hemiplegic migraineurs showing mild SFEMG abnormalities with acetazolamide for several weeks. This was followed by a normalization of SFEMG recordings in all patients and by clinical improvement in four. These results support the assumption that the subclinical impairment of neuromuscular transmission found in certain migraineurs might be due to dysfunctioning Ca2+-channels.
Article
Involvement of cellular, ionic and hormonal interactions in the control of spontaneous and induced nuclear and cytoplasmic maturation of starfish oocytes are discussed and compared. Spontaneous oocyte nuclear maturation occurs when large ovarian follicles are isolated into seawater and this process is sensitive to the calcium and hydrogen ion concentrations in seawater. In the presence of calcium ions induction of spontaneous oocyte maturation is closely associated with loss of follicle cells from the oocyte. In the absence of calcium ions, follicle cells are also lost from the oocyte, however, spontaneous maturation is inhibited and denuded oocytes do not mature following return to normal seawater. Exposure of isolated follicle cells to calcium ions results in the release of meiosis inducing substance into the media. Treatment of isolated follicles with acidic seawater inhibits spontaneous oocyte maturation in the presence of calcium ions but without loss of follicle cells. Effects of low pH on spontaneous maturation are reversible in the presence but no absence of the follicle cells whereas oocytes exposed to calcium free media or low pH remain responsive to 1-methyladenine and undergo maturation. Immature oocytes collected in CaFSW also undergo some aspects of cytoplasmic activation (fertilization membrane elevation, cortical granule discharge) in response to sperm or divalent ionophore (A-23187). This occurs without induction of nuclear maturation. Similar activation responses are obtained when immature oocytes are collected and denuded of follicle cells in acidic seawater and not exposed to CaFSW. Results suggest that oocyte cytoplasmic maturation is not necessarily entirely dependent upon nuclear maturation. Data indicate that spontaneous maturation is mediated through calcium and hydrogen ion sensitive processes in the follicle cells, and that these processes are involved in the release of meiosis inducing substance. The evidence support the hypothesis that somatic follicle cells serve as a common mediator for hormone (RNF) induced and spontaneous maturation. Calcium ions appear to play an important role in the functional maturation of both the follicular somatic cells as well as the oocyte. Similarities between spontaneous oocyte maturation in starfish and mammals are discussed.
Article
This study was undertaken to examine the effects of hypercapnia on the excitability of respiratory motoneurons. The action of CO2 on phrenic (inspiratory) and internal intercostal (expiratory) motoneurons was compared with that exerted on non-respiratory motoneurons of the musculocutaneous nerve. The experiments were performed on spinalized (C1 segment), partially deafferented cats that were exposed to different CO2/O2 mixtures (end-tidal CO2 3 +/- 0.3, 6 +/- 0.5 and 9 +/- 0.5%). Changes in neuronal excitability were assessed by: measuring the amplitudes of antidromic field potentials recorded from a population of motoneurons; analysis of the amplitude and latency of the orthodromic response recorded from a given nerve and evoked by microstimulation within the corresponding motor nucleus; monitoring the membrane potentials during intracellular recordings from phrenic motoneurons; and recording ongoing activity of the phrenic and internal intercostal nerves. Hypercapnia (end-tidal CO2 6 +/- 0.5 or 9 +/- 0.5%) decreased the excitability of phrenic and musculocutaneous motoneurons, the effect being larger at the higher CO2 level. Internal intercostal motoneurons were generally more resistant to the effects of CO2. A depression of their excitability was observed only at end-tidal CO2 9 +/- 0.5%. The decreased excitability of phrenic motoneurons was associated with membrane hyperpolarization. It is concluded that the depressant action of CO2 is present in both respiratory and non-respiratory spinal motoneurons. The action of hypercapnia on respiratory motoneurons may oppose the excitatory effects exerted through specific chemoreflexes.
Article
Starfish (Asterias forbesi) oocytes encased within their follicle cells mature spontaneously during a portion of the normal reproductive period when released from the ovary into seawater. A previous report has shown that oocytes isolated in acidic seawater do not mature spontaneously but retain the capacity to do so when returned to normal seawater. The object of this study was to determine the mechanism by which acidic pH reversibly blocks spontaneous oocyte maturation in isolated follicles. Incidence of spontaneous oocyte maturation in follicles isolated in acidic seawater decreased as pH decreased from 7 to 4. Oocytes in which spontaneous maturation was inhibited (ASW at pH 4.7 TO 5.4) underwent germinal vesicle breakdown with the addition of 1-methyladenine. Oocytes isolated in acidic seawater (pH 4 or 5) with intact follicle cells matured spontaneously when transferred immediately to normal seawater pH 8); after four hours, 60-65% of the follicles incubated in seawater at pH 5 matured spontaneously when returned to normal seawater as compared to less than 10% of the follicles maintained at pH 4. Inhibition of spontaneous maturation was not reversible in the absence of the follicle cells. Oocytes isolated in acidic seawater with their follicle cells did not spontaneously mature when transferred to calcium-free seawater at pH 8. The results obtained support the hypothesis that acidic seawater reversibly inhibits spontaneous oocyte maturation by interfering with the release of meiosis-inducing substance from the follicle cells.
Article
In motoneurons of the isolated spinal cord of frogs (Rana esculenta) short latency depolarizations (‘VR-EPSPs’), produced by stimulation of different ventral roots, were investigated.During increased extracellular magnesium ion concentration, postsynaptic potentials were reversibly suppressed. In addition, the antidromic spike invasion of the somatodendritic membrane of motoneurons was blocked because of membrane hyperpolarization and the subsequent increment in threshold depolarization. Consequently, the antidromic focal potential diminished in amplitude and the VR-EPSPs disappeared to a similar extent. At the same time, the neuron input resistance increased and the extracellular concentration of potassium ions, [K+]e, decreased below control levels. However, antidromic spikes again invaded the somatodendritic membrane of motoneurons, despite the maintained action of magnesium ions, whenever the bulk of motoneurons were depolarized by increasing [K+]e. As a result, the VR-EPSPs reappeared, whereas all chemical PSPs remained blocked.PSPs were also blocked reversibly by application of manganese ions. No effects were seen either in [K+]e, membrane potential, passive membrane properties, or in antidromic spike invasion of motoneurons, and VR-EPSPs remained unchanged. On the basis of these results, it was concluded that the VR-EPSP is electronically propagated between different motoneurons.Electron microscopic inspection of the membranes of frog motoneuron somata and dendrites, as exposed by freeze-fracturing, revealed numerous membrane specializations of gap junction character. As a typical feature of these gap junctions, regions of densely packed particles surrounded small particle free regions.As a consequence of these findings the term VR-EPSP, as employed by Kubota and Brookhart, should be used with some reservation. If the proposed interpretation of the signal should prove to be correct, the designation ‘ventral root electrotonic junction potential’ (VR-EJP) would, in our opinion, be more appropriate.
Article
As reported by Landau & Nachshen (1975), a decrease in extracellular pH at the frog neuromuscular junction leads to an increase in min.e.p.p. frequency. 2. Decreasing the extracellular pH still increases the min.e.p.p. frequency when the bathing Ringer contains 10 mM-Ca2+, in place of the usual 2-5 mM. At the mammalian neuromuscular junction, the elevated Ca2+ blocks the effect of the pH change on the min.e.p.p. frequency (Hubbard, Jones & Landau, 1968). 3. In Cl--free solution (isethionate or methylsulphate substitution) min.e.p.p. frequency is no longer a monotonic function of decreasing pH. Instead there is an optimum pH for spontaneous release between pH 6-6 and 8-6. 4. This suggests that in Cl- containing Ringer min.e.p.p. frequency increases with increasing extracellular acidity because there is a change in the PCl of the nerve terminal leading to a depolarization. In agreement with this idea,in low Ca2+ Ringer, acid pH has little effect on the min.e.p.p. frequency. 5. Decreasing the intracellular pH by raising PCO2 produces substantial increases in the min.e.p.p. frequency. The effects are much greater than the effects of equal changes of H+ in the extracellular solution. 6. Possible explanations for the effects of increased PCO2 are discussed. Although release of Ca2+ from mitochondria or other unknown effects of intracellular pH change or molecular CO2 are possible, the results do give some support to the hypothesis that an important step in transmitter release involves an electrostatic repulsion between fixed membrane surface charges on the transmitter containing vesicles and the inner face of the nerve terminal. The surface charge density would be decreased by a lower pH in the axoplasm, and this would increase the rate of spontaneous transmitter release, in agreement with the observations.
Article
We have developed a technique to measure the pHi of single neurons in brainstem slices using a fluorescence imaging system. Slices were loaded with the pH-sensitive fluorescent dye BCECF and fluorescence was visualized by exciting the slices alternately at 500 and 440 nm. The emitted fluorescence at 530 nm was directed through an MTI GenIISys image intensifier and MTI CCD72 camera. The images were processed by Image-1/FL software. The ratio of emitted fluorescence at excitation wavelengths of 500 and 440 nm was measured and converted to pH by constructing a calibration curve using high K+/nigericin solutions at pH values ranging from 5.8 to 8.6. BCECF-loaded slices showed distinct spheres of intense fluorescence and diffuse background fluorescence. Slices labeled with a neuron-specific antibody, neuron-specific enolase, showed staining that correlated with the spheres of intense fluorescence of BCECF-loaded cells. Slices labeled with a glial-specific antibody, glial fibrillary acidic protein, showed a diffuse, background staining. Neurons that were retrograde-labeled with rhodamine beads fluoresced as large spheres that exactly correlated with the fluorescence from BCECF-loaded cells. Further, large fluorescent spheres had membrane potentials of about −60 mV and generated action potentials. These findings indicate that the large fluorescent spheres are neurons. pHi was measured in these large spheres (neurons) in the dorsal and ventral medullary chemosensitive regions, and was 7.32 ± 0.02 (n = 110) and 7.38 ± 0.02 (n = 85), respectively.
Article
1. The effect of pH changes on synaptic currents has been analysed by external recording of the miniature end-plate currents (m.e.p.c.s) or by recording in voltage-clamped end-plates the current elicited by nerve stimulation (e.p.c.). 2. Changes in pH do not appreciably effect the peak amplitude of the current produced by a single quantum or by short ionophoretic pulses of acetylcholine. 3. The time constant of decay of the m.e.p.c.s is prolonged by about 50% in acid pH and shortened by about the same amount in alkaline pH. This effect is independent of the cholinesterase activity of the end-plate. 4. In curarized preparations the decay of the e.p.c. is shorter than in Mg-blocked end-plate even in the absence of cholinesterase blocking agents. 5. The action of pH on the decays can be explained by a titration of the surface charges of the membrane which effects the voltage dependent reaction that controls the rate of closing of the synaptic channels.
Article
The hydrosmotic responses to serosal hyperosmolar solutions were studied in isolated urinary bladders of toads (Bufo marinus) at different pH values to see whether the increase in watet flux obtained with serosal hypertonicity in urinary bladders of amphibia is due to non-specific mechanisms or whether it is related to specific ionic regulation.The increase in net water flux elicited by serosal hypertonicity (Ringer + 250 mM mannitol ⋍ 430 mosM) at pH 8.2 is inhibited by lowering the pH to 6.6. The same inhibition is obtained if pH is lowered when the response is fully developed. The inhibition of the development is reversible upon return to pH 8.2, provided serosal Na+ and Ca2+ are present. The reversibility is incomplete in Ca2+-free solutions and is completely abolished in Na+-free solutions.It is concluded that the hydrosmotic response to serosal hypertonicity in the amphibian bladder is not a passive phenomenon. It is suggested that during the osmotic challenge there is an increase in a H+-sensitive Na+ permeability at the basolateral membrane which may be related to the intracellular events that lead to the permeability changes. Serosal Ca2+ could be important in the maintenance of the water permeability in the developed response to hypertonicity.A hypothesis is presented concerning the possibility of similar mechanisms in the modulation of the hydrosmotic responses to antidiuretic hormone and to serosal hypertonicity.
Article
In 1959, Goodman [9] determined the minimum number of monochromatic triangles in a complete graph whose edge set is 2-coloured. Goodman (1985) [10] also raised the question of proving analogous results for complete graphs whose edge sets are coloured with more than two colours. In this paper, for n sufficiently large, we determine the minimum number of monochromatic triangles in a 3-coloured copy of K-n. Moreover, we characterise those 3-coloured copies of K-n that contain the minimum number of monochromatic triangles.
Article
1. Histamine secretion from rat peritoneal mast cells occurs spontaneously in the absence of an external stimulus. Spontaneous secretion increases as the concentration of Sr in the extracellular medium is raised from 1 to 10 m-mole/l. Ca 0.1-10 m-mole/l. does not increase spontaneous secretion.2. Spontaneous histamine secretion in the presence of Sr occurs slowly compared with evoked histamine secretion, reaching a maximum only after more than 120 min incubation with Sr 10 m-mole/l. at 37 degrees C and pH 7.6. Phosphatidyl serine, 10 mug/ml., increases the rate of spontaneous secretion in the presence of Sr.3. The spontaneous secretion occurring in the presence of Sr is highly dependent on the extracellular H ion concentration. Maximal secretion occurs at pH 8.4 and only a very limited secretion is detected at pH below 7.6. The rate of spontaneous secretion is also greater at higher pH. Inhibition of secretion caused by lowering the pH can be reversed by raising the Sr ion concentration over a limited range.4. Intact glycolytic and oxidative metabolism is required for the spontaneous secretion of histamine in the presence of Sr. Removal of extracellular glucose inhibits the secretion by about 80%, and the further addition of inhibitors of oxidative phosphorylation almost abolishes the secretion.5. Ca, Mg and Mn all inhibit the spontaneous secretion of histamine which occurs in the presence of Sr. The antagonism of the effect of Sr by Mg appears not to be competitive.6. Dibutyryl cyclic AMP, 10 mumole/l. to 3 m-mole/l. and theophylline, 30 mumole/l. inhibit spontaneous secretion in the presence of Sr. Cyclic AMP, AMP, and cyclic GMP 10 m-mole/l. are without effect on the spontaneous secretion. The inhibitory effect of dibutyryl cyclic AMP and of theophylline are dependent on pH: greater inhibition being achieved at lower pH.
Article
The effects of imidazole, guanidine, and theophylline on spontaneous (frequency of miniature end-plate potentials) and evoked (quantum composition of end-plate potentials) transmitter release were compared in isolated sartorius muscles ofRana temporaria at different temperatures and during changes in the calcium concentration in the external solution. All three substances increased the quantum composition of the end-plate potentials and the frequency of the miniature end-plate potentials at 20°C and in 0.5 mM calcium. As regards their effect on the quantum composition the substances could be arranged in the following order: imidazole ˜ guanidine » theophylline; as regards their effect on frequency: theophylline » imidazole ˜ guanidine. Theophylline increased spontaneous release, whereas imidazole and guanidine increased evoked transmitter release more than the rest. Comparison of the effect of these substances at 20 and 7°C showed that only the action of theophylline on spontaneous release depends on temperature. The effect of imidazole and theophylline on frequency was independent of the calcium concentration in the medium. Differences in the mechanism of action of these compounds on spontaneous and evoked acetylcholine release are discussed.
Chapter
Katz (1) in his Sherrington lecture summarized our understanding of the microphysiology of the presynaptic terminals of the vertebrate neuromuscular junction (nmj). The arrival of a nervous impulse causes a transient increase in the calcium permeability of the plasma membrane and the associated entry of calcium ions triggers the synchronized release of quanta of transmitter. The magnitude of the evoked response, recorded as the endplate potential (EPP) is therefore primarily dependent on the concentration of extracellular calcium (2). There is also a spontaneous release of quantal packets of acetylcholine (ACh), recorded as the miniature endplate potentials (MEPPs), and the MEPP frequency is determined, at least in part, on the other hand, by the concentration of intracellular calcium at the terminals (3, 4). However, we still lack a firm conclusion as to how the intracellular calciumions trigger both the spontaneous and the evoked release of transmitter. Since the earlier biophysical studies of Katz and Miledl a large number of pharmacological agents have been tested on the vertebrate nmj, and the purpose of this very brief review is to attempt to determine their sites of action in the hope of discovering some of the biochemical events of exocytosis.
Article
The effects of water-soluble carbodiimide were examined at the frog neuromuscular junction. Acetylcholine sensitivity was measured using a fluid electrode technique and intracellular recording of miniature end-plate potentials. The carbodiimide blocked synaptic sensitivity by a reversible, curare-like action. Irreversible blockade was also observed, probably due to covalent binding. The conditions of reaction and irreversibility suggest that several different residues may be attacked. The inability of cholinergic antagonists to protect the receptor from attack indicates that nonspecific sites, and not the acetylcholine binding site, are involved.
Article
The effects of variations in extracellular pH have been studied on rat detrusor muscle in vitro. At pH 7.4 a continuous low amplitude spontaneous contractile activity was found. At pH 6.75 the contractions became more regular with periods of relaxation between the contractions which had increased in amplitude. At pH 7.85 the reverse was found. The results are interpreted as a membrane effect of pH. No effect of pH on amplitudes of high-K(+)-induced contractures was found. Carbachol dose-response relations and maximal contraction amplitude to carbachol was similar at pH 7.4 and 6.75. A significant depression in response to nerve stimulation was, however, noted at pH 6.75. We suggest that, while the force output of the activated detrusor smooth muscle cell is unaffected by changes in extracellular pH, a prejunctional inhibition of nerve induced contraction might occur at low pH.
Article
1.Properties of the membrane currents ofDidinium nasutum have been investigated under voltage clamp in different solutions and after deciliation.2.Theearly transient Ca2+ inward current activates in a voltage-dependent manner. Inactivation is both Ca2+ -dependent and voltage-dependent.3.Alate Ca2+ current rises with time to peak > 50 ms and decays in the order of seconds.4.Activation and inactivation of the late Ca2+ current is voltage-dependent.5.The delayed outward current is activated by voltage. The kinetics of this K+ current, but not its amplitude, are enhanced in the presence of intracellular EGTA.6.The two voltage-dependent Ca2+ channels are located in the cilia, whereas all K+ channels are restricted to the somatic membrane.
Article
Smooth muscle performs many functions that are essential for the normal working of the human body. Changes in pH are thought to affect many aspects of smooth muscle. Despite this, until recently little was known about either intracellular pH (pHi) values or pHi regulation in smooth muscle. Recent work measuring pHi with either microelectrodes or nuclear magnetic resonance spectroscopy is now providing some of this much needed information for smooth muscles. From these studies, it can be concluded tentatively that pHi is the same in different smooth muscles, approximately 7.06 (37 degrees C). This value is very close to those obtained in cardiac and skeletal muscle. It is clear that H+ is not in equilibrium across the smooth muscle membrane; i.e., pHi is regulated. Preliminary results in smooth muscle suggest that certain aspects of this regulation are different from that described for other muscle types. Changes in pHi have been found to produce marked effects on contraction in smooth muscle. Of particular interest is the fact that, unlike striated muscles, some smooth muscles can product more force during an intracellular acidification.
1.1. Transmitter mobilization and fractional release were studied in Helix pomatia. The right palliai nerve was stimulated and a synaptic potential was recorded in cell F76 in the right parietal ganglion.2.2. The extra- and intra-cellular pH were changed with Tris-maleate, CO2 or (NH4)2SO4.3.3. The time constant for the monoexponential part of mobilization decreased with reduced intracellular pH. Only a fraction of this effect could be related to an increase in the intracellular Ca-activity.4.4. Fractional release was reduced in low external pH, but was increased in low intracellular pH. Fractional release is affected more by changes in internal pH than external pH.
Article
— The disposition of newly synthesized ACh subsequent to depletion of vesicular endogenous ACh by stimulation was studied in the electromotor nerve terminals of Torpedo marmorata using [3H]acetate as a precursor of ACh. Little vesicular [3H]ACh could be isolated from tissue immediately after stimulation at 1 Hz. After 3 h post-stimulation recovery the newly synthesized [3H]ACh is found predominantly in a subpopulation of vesicles distinct from the vesicles containing most of the endogenous poorly labelled ACh. Restimulation of the tissue causes release of highly labelled ACh with a specific radioactivity (SRA) comparable to that of the newly synthesized [3H]ACh in the highly labelled subpopulation of vesicles and significantly greater than the SRA of ACh in the main vesicular pool or the total tissue.
Article
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The inhibition by Mg(++) of stimulus-evoked transmitter release is attributed to a decrease in surface potential, Psi(0), on the outer surface of the presynaptic terminal and hence a lower surface calcium concentration, [Ca(++)](0). Data on the frog neuromuscular junction are quantitatively fit by assuming that there is a negative charge density, sigma, on the outer surface of the presynaptic terminal of 6.5 x 10(13) charges per cm(2) and that simple diffuse double layer theory is applicable. No specific binding of Mg(++) or Ca(++) is required. Without any additional assumptions, the inhibitory effect of univalent cations is also quantitatively predicted.
Article
1. Effects of raising extracellular hydrogen-ion-concentration [H+]e on the action potential (AP) of isolated nodes of Ranvier in calcium-containing (1.08 mMol/l) and in calciumpoor (0.05 mMol/l) solutions were studied between ph 7 and 4.8. 2. Threshold membrane potential (i.e. maximum subthreshold response) and membrane potential at the end of the slow first falling phase increase with increasing [H+]e , especially below ph 6.5. The amplitude of the AP is slightly raised. 3. Mild lowering of ph from 7 to 6.5 in Ca-containing solutions accelerates repolarization, mainly during the slow first phase. In Ca-poor solution, small drop in ph reverses the effects of Ca-lack. 4. Below ph 6.5 to 6.0, repolarization is delayed, mainly during the slow first phase and, to a smaller extent, during the negative after-potential. At low ph the AP becomes insensitive to lack of extracellular calcium. 5. Between ph 7 and 6.5 H+-ions are suggested mainly to act in an indirect way by raising Ca-activity in or at the membrane, thus accelerating the decrease of P Na or the increase in P K. From the effects at lower ph-values it is concluded that in this range H+-ions act directly on the membrane; they would delay and decrease the change of P K during the AP.
Article
1. The effect of different polyvalent metal ions in the external solution upon the threshold membrane potential for spike initiation in crayfish axons has been studied by means of intracellular micro-electrodes. The metal ions tested included six divalent cations (Ca(2+), Mg(2+), Sr(2+), Ba(2+), Co(2+), and Ni(2+)) and three trivalent cations (La(3+), Y(3+) and Eu(3+)).2. Identical extracellular concentrations of different cations with the same valence had essentially the same effect on threshold membrane potential. However, a very low concentration of trivalent cations (about 225 muM) was found to be equivalent to a much higher divalent cation concentration (13.5 mM) as measured by their effects on threshold potential.3. Upon a tenfold increase in concentration, the threshold potential for spike initiation was shifted in a positive direction by 30.6 mV with divalent cations and by 20.8 mV with trivalent cations.4. It is shown that a hypothesis involving screening of negative charges at the axonal membrane surface, based on Gouy-Chapman theory, predicts these various experimental results rather closely.5. It is concluded that a high negative charge density, sufficient to render a screening mechanism possible, exists at the surface of crayfish axons in the region of the sodium ;gates'.6. The density of surface charges is calculated to be approximately 1e(-)/43 A(2). This calculation is discussed in connexion with the possible molecular identity of the sodium ;gates' in crayfish axons and other excitable systems.
Article
1. The changes in transmitter release produced by mitochondrial inhibitors has been studied at the frog neuromuscular junction using conventional electrophysiological techniques for stimulation and intracellular recording. 2. Inhibitors of the electron transport chain and inhibitors of oxidative phosphorylation produce an increase in the frequency of appearance of the miniature end-plate potentials. This increase in frequency is observed also in calcium-free media. Mitochondrial inhibitors also augment the amount of transmitter liberated by a nerve impulse. 3. Ruthenium red, which is an inhibitor of calcium uptake by mitochondria, increases the spontaneous transmitter release but decreases the quantal content. The latter effect of Ruthenium red is antagonized by calcium. 4. The mitochondrial content of the motor nerve terminals is, on the average, 6.59%. 5. The experimental results are explained on the hypothesis that spontaneous release of transmitter reflects the resting level of intracellular free calcium and the evoked release reflects the sum of the resting calcium and the calcium brought in by the action potential. The mitochondria play a role in transmitter release by participating in the regulation of the intracellular free Ca.
Article
Evidence is given for a high density of negative surface charge near the sodium channel of myelinated nerve fibres. The voltage dependence of peak sodium permeability is measured in a voltage clamp. The object is to measure voltage shifts in sodium activation as the following external variables are varied: divalent cation concentration and type, monovalent concentration, and pH. With equimolar substitution of divalent ions the order of effectiveness for giving a positive shift is: Ba equals Sr less than Mg less than Ca less than Co approximately equal to Mn less than Ni less than Zn. A tenfold increase of concentration of any of these ions gives a shift of +20 to +25 mV. At low pH, the shift with a tenfold increase in Ca-2+ is much less than at normal pH, and conversely for high pH. Soulutions with no added divalent ions give a shift of minus 18 mV relative to 2 mM Ca-2+. Removal of 7/8 of the cations from the calcium-free solution gives a further shift of minue 35 mV. All shifts are explained quantitatively by assuming that changes in an external surface potential set up by fixed charges near the sodium channel produce the shifts. The model involves a diffuse double layer of counterions at the nerve surface and some binding of H+ions and divalent ions to the fixed charges. Three types of surface groups are postulated: (1) an acid pKa equals 2.88 charge density minus 0.9 nm- minus 2; (i) an acid pKa equals 4.58, charge density minus 0.58 nm- minus 2; (3) a base pKa equals 6.28, charge density +0.33 nm- minus 2. The two acid groups also bind Ca-2+ ions with a dissociation constant K equals 28 M. Reasonable agreement can also be obtained with a lower net surface charge density and stronger binding of divalent ions and H+ ions.
Article
1. The frequency of miniature end-plate potentials (m.e.p.p.s) was recorded from neuromuscular junctions in rat diaphragm phrenic nerve preparations in vitro after preparations had soaked in solutions containing Ca in concentrations between 10(-10) and 10(-2)M and a similar range of [Mg].2. Ethylenediamine tetra-acetate (EDTA) and ethyleneglycol bis (beta-aminoethyl ether) tetra-acetate (EGTA) buffers were added to prepare solutions with [Ca] and [Mg] below 10(-4)M. A computer program was used to estimate the free [Ca(2+)] in these solutions, and it was shown that the effects of Ca could be attributed to the free [Ca(2+)] in the bathing solution.3. M.e.p.p.s could still be detected without difficulty after soaking preparations for 6-8 hr in solutions containing EDTA or EGTA buffers and no added Ca. The basal frequency was unchanged upon exhibition of Ca in concentrations up to 10(-5)M and/or Mg in concentrations up to 10(-3)M.4. Ca in concentrations of and above 10(-4)M accelerated m.e.p.p. frequency from the basal level. This effect reached a maximum in [Ca] of 10 mM and raising the [Ca] above this level did not further change frequency. These effects were explained by the combination of Ca molecules with a nerve terminal receptor site. It was postulated that this combination allosterically activated the spontaneous release mechanism.5. Mg could accelerate m.e.p.p. frequency in the absence of added Ca. The interactions of Ca and Mg upon m.e.p.p. frequency indicated that Ca and Mg competed for the same sites.6. Raising the [H(+)] of the bathing medium accelerated m.e.p.p. frequency. This effect was thought to be exerted partly by combination with the same receptor sites as Ca and Mg and partly by variation of the ionization of the CaCl(2) of the bathing solution.
Article
1. The relationship between the quantal content of end-plate potentials (e.p.p.s) and the bathing [Ca] and [Mg] was determined at neuromuscular junctions in the rat diaphragm in vitro.2. E.p.p.s were recorded intracellularly from preparations exposed to solutions with [Ca] between 0.05 and 10 mM and [Mg] between 0.1 and 12.5 mM. The quantal content of e.p.p.s was increased by raising the [Ca] over this range and decreased by raising the [Mg]. There appeared to be competition of Mg with Ca at three sites in the nerve terminal membrane.3. A kinetic scheme based on competition of Ca and Mg at three sites could quantitatively explain the effects of Ca and Mg upon the quantal content of e.p.p.s and also the effects of these ions upon miniature end-plate potential frequency.
Article
1. The interaction between Ca and Sr ions on quantal transmitter release at the frog neuromuscular junction was studied, using conventional electrophysiological techniques.2. While Ca ions always activate transmitter release, the activating action of Sr ions depends on the Ca ion concentration in the medium; at low [Ca], strontium ions enhance the release, but at higher [Ca] they inhibit it. It is postulated that there is a [Ca] at which Sr ions do not affect transmitter liberation.3. When Sr activates release, its effect and the effect of Ca add in a more than linear fashion.4. Magnesium ions inhibit the release induced by Sr.5. The results can be explained by assuming that Ca and Sr act on the same site, at some stage of the process of quantal transmitter release. The affinity of both ions towards the sites is approximately the same, but the effectiveness of Sr is much smaller.
Article
The effect of Ruthenium Red on synaptic transmission was examined at isolated junctions of the frog, by conventional methods for stimulation and intracellular recording. Ruthenium Red (2.5-10.0 muM) reduces the synaptic potential to subthreshold levels. An analysis of this phenomenon shows that the main action of Ruthenium Red is on the presynaptic nerve terminal where it decreases the number of quanta of transmitter liberated by the nerve impulse. It has the following additional effects: a reduction in the amplitude of the spontaneous miniature end plate potentials; an increase in their frequency; and an increase in delayed release of transmitter after a nerve impulse. Some of these results are discussed in terms of the known inhibitory action of Ruthenium Red on calcium transport across mitochondrial membranes.
Article
1. The mean quantal content of the frog end-plate potential was examined under conditions that reduced evoked transmitter release to very low values.2. When the calcium concentration of the Ringer is reduced below 10(-4)M a deviation occurs from the fourth power dependence of the mean quantal content (m) on the calcium concentration such that more quanta are released than is expected from the behaviour of m at higher calcium concentrations.3. At 10(-5)M this extra quantal release is more than two orders of magnitude greater than that predicted by the fourth power relationship.4. The calcium dependence of very low values of m was studied in low calcium Ringer in which the calcium was buffered by either citrate or EDTA. It was found that in the fourth power dependence of m on the external calcium concentration changes rather suddenly to an approximately linear dependence.5. The inclusion of small concentrations of cobalt in the Ringer was found to reduce m to very low values even in the presence of millimolar concentrations of calcium.6. The fourth power dependence of m on the external calcium concentration at high values of m was unaffected by the presence of cobalt. At low quantal contents the transition to a linear dependence on external calcium was again seen, but was shifted to calcium concentrations that did not require buffering.7. The fourth power to linear transition is discussed in terms of its relevance to the relationship between m and the m.e.p.p. frequency.
1. The puffer fish poison, tetrodotoxin (T.T.) was applied to eliminate impulse propagation in nerve and muscle fibre, and the physiological properties of the neuromuscular junction were studied under this condition. 2. Spontaneous miniature end-plate potentials of normal frequency and amplitude were recorded in the T.T.-paralysed muscle. 3. Depolarization of motor nerve endings by locally applied current still produces the usual increase in the frequency of miniature end-plate potentials (e.p.ps). 4. When brief current pulses are applied to the nerve endings e.p.ps can be evoked, whose size varies with the intensity of the current. The responses are composed, like normal e.p.ps, of a statistically varying number of miniature potentials. The response fails when calcium is removed from the bath. 5. When two identical pulses are applied at varying intervals, facilitation of the second e.p.p. occurs, similar to that observed normally with pairs of nerve impulses. 6. It is concluded that tetrodotoxin while blocking electric excitation in nerve and muscle does not interfere with the release of acetylcholine from nerve endings nor with its local action on the muscle fibre.
1. The effect of brief depolarizations focally applied to a motor nerve ending was studied. Particular attention was paid to the relation between (i) strength and duration of the pulse and (ii) the size and latency of the resulting end-plate potential. 2. The release of acetylcholine lags behind the depolarization which causes it. If pulses of less than 4 ms duration are used (at 5 degrees C), the release starts after the end of the pulse. 3. Within a certain range, lengthening the pulse increases the rate of the ensuing transmitter release. 4. Unexpectedly, lengthening the depolarizing pulse also increases the latency of the transmitter release. This finding is discussed in detail. It is regarded as evidence suggesting that entry into the axon membrane of a positively charged substance (external Ca2+ ions or a calcium compound CaR+) is the first step leading to the release of acetylcholine packets from the terminal.
Article
1. The dimethyloxazolidinedione (DMO) technique was used to estimate intracellular pH (pH(i)) in bullfrog toe muscles incubated in vitro. The control value of pH(i) was 7.16 + +/- 0.01 (S.D.).2. pH(i) was affected by changes in P(CO2) and external bicarbonate ion concentration ([HCO(3) (-)](0)). At a given P(CO2), decreasing the external [HCO(3) (-)] was more effective in lowering pH(i) than increasing the external [HCO(3) (-)] was in increasing pH(i).3. On the assumption that the changes in pH(i) were due to hydrogen ion [H(+)) movements across the membrane, a H(+) flux of 10(-13) mole/cm(2). sec was calculated. The corresponding H(+) permeability coefficient was 10(-3) cm/sec.4. The variability of the tissue CO(2) buffer value was examined.
Article
Bass and Moore [Proc. Nat. Acad. Sci. 55:1214 (1966)] proposed that the vesicles containing acetylcholine undergo Brownian motion in the nerve terminals. Acetylcholine is released whenever a vesicle touches the inner face of the axolemma of the nerve terminal. The frequency at which contact is made is limited by an energy barrier that must be overcome before the vesicle can touch the axolemma. The energy barrier has two components. (1) An electrostatic repulsion between positive, fixed charges on the vesicles and a relatively positive potential at the face of the axolemma that is generated by the resting potential. (2) A layer of water molecules held to the vesicle by the surface charge. This model is inconsistent with experimental data. A modification of the model is presented. Both the vesicle and the inner face of the axolemma are assumed to have fixed, negative surface charges that are responsible for the energy barrier. By a series of simplifications, the model leads to two predictions. (1) A plot of the ln (miniature end plate potentials/sec) as a function of the concentration of ions in the axoplasm)–0.5 should give a straight line. (2) A plot of ln (end plate potential amplitudes) as a function of (extracellular Ca++ concentration)–0.5 should give a straight line. These predictions are shown to agree reasonably well with experimental data.
Article
1. The Ca-sensitive photoprotein aequorin was injected into squid axons and the light response to stimulation or depolarizing voltage clamp pulses recorded.2. The effects of Mn(2+), Co(2+), Ni(2+), La(3+) and of the organic Ca antagonists D-600 and iproveratril on the early tetrodotoxin-sensitive and late tetrodotoxin-insensitive components of the light response were studied.3. The late tetrodotoxin-insensitive component can be blocked, reversibly, by concentrations of Mn, Co and Ni that reduce but do not block the tetrodotoxin-sensitive component. The late component can also be blocked by La(3+) and the organic Ca antagonists D-600 and iproveratril.4. Mn(2+), Co(2+), Ni(2+) and the drug D-600 all reduce the Na currents, but have little effect on either outward or inward K currents. Tetraethylammonium blocks the outward K current but has no appreciable effect on the tetrodotoxin-insensitive entry of Ca.5. Concentrations of Mn between 5 and 50 mM substantially reduce the light output during a train of action potentials; they also slightly reduce the rate of rise of the action potential.6. On pharmacological grounds it is concluded that the tetrodotoxin-insensitive component of Ca entry does not represent Ca ions passing through the K permeability channels. There must exist a potential-dependent late Ca channel that is distinct from the well known Na and K channels of the action potential. A possible function for this late Ca channel in the coupling of excitation to secretion is discussed.
Article
1. The construction and properties of a new design of pH-sensitive micro-electrode are described. The electrodes are very durable, and have a recessed configuration so that only the extreme tip, which can be as small as 1 mum in diameter, needs to enter the cell.2. The average intracellular pH in thirty-two snail neurones was 7.4. This was not in accord with a passive distribution of H(+) ions across the cell membrane.3. Changing membrane potential or external pH had only slow effects on internal pH.4. Removing external K had no effect, and removing external Na had only slow and variable effects on intracellular pH.5. Anoxia, azide and DNP all caused a slow fall in internal pH.6. External CO(2) caused large and rapid decreases in internal pH, which external bicarbonate appeared to offset slowly. Injected bicarbonate increased internal pH.7. The size of the pH changes caused by CO(2) suggested a minimum intracellular buffering power of 25 m-equiv H(+)/unit pH per l., equivalent to that of 150 mM Tris maleate, pH 7.4.8. External ammonia caused a large and rapid increase in internal pH, while the injection of ammonium ions had the opposite effect.
Article
Manzganiese ions. are potent blocking argenits of synaptic transmission at the neuromuscular junction in the frog. The main site of action is the presynaptic nerve terminal, where the ions decr-ease the amount of transmitter liberated by a never impulse. The inihibition produced by manganese is reversible.
Article
1. Conflicting values of intracellular pH (pH(i)) were obtained by Caldwell in crab muscle and by Carter et al. in rat muscle. To investigate this discrepancy, double and single barrelled pH sensitive micro-electrodes as described by Carter et al. were used both in rat muscle (in vivo and in vitro) and in large single fibres of crab (in vitro).2. The pH(i) values obtained in the superficial layers of muscle cells in rat and pH(i) values obtained in the superficial area of large single fibres in crab seemed in agreement with the Donnan equilibrium. Furthermore, when the membrane potential (E(m)) value of extracellular pH (pH(e)) value was changed, each new pH(i) value was obtained instantly.3. The pH(i) values obtained in deep layers of muscle cells in rat (pH(i) 6.68) and in a deep area of large single fibres in crab (pH(i) 6.96) did not agree with Donnan equilibrium; these pH(i) values were little influenced by modifications in E(m) or pH(e) values.4. These findings could be easily explained if the length of the sensitive portion of the pH micro-electrode exceeds the diameter of rat muscle cells and is smaller than the diameter of crab large fibres. The pH(i) values obtained superficially in the two preparations and which seem in agreement with the Donnan equilibrium are artificially low because the E(m) values recorded by the pH micro-electrodes are partly shunted.
Article
Phospholipid bilayer membranes were bathed in a decimolar solution of monovalent ions, and the conductance produced by neutral carriers of these monovalent cations and anions was used to assess the electric potential at the surface of the membrane. When the bilayers were formed from a neutral lipid, phosphatidylethanolamine, the addition of alkaline earth cations produced no detectable surface potential, indicating that little or no binding occurs to the polar head group with these ions. When the bilayers were formed from a negatively charged lipid, phosphatidylserine, the addition of Sr and Ba decreased the magnitude of the surface potential as predicted by the theory of the diffuse double layer. In particular, the potential decreased 27 mv for a 10-fold increase in concentration in the millimolar-decimolar range. A 10-fold increase in the Ca or Mg concentration also produced a 27 mv decrease in potential in this region, which was again due to screening, but it was necessary to invoke some specific binding to account for the observation that these cations were effective at a lower concentration than Ba or Sr. It is suggested that the ability of the alkaline earth cations to shift the conductance-voltage curves of a nerve along the voltage axis by 20-26 mv for a 10-fold increase in concentration may be due to essentially a screening rather than a binding phenomenon.
Article
1. Changes in ionized calcium in giant axons were followed by recording the light produced by injected aequorin.2. From the effect of injecting calcium buffers the internal concentration of ionized calcium was found to be about the same as in a mixture of 45 Ca EGTA:55 free EGTA, i.e. about 0.3 muM.3. After an axon had been exposed to cyanide for 50-100 min the velocity of the aequorin reaction increased about 500 times. This effect, which could be reversed rapidly by removing cyanide, was probably brought about by release of calcium from an internal store.4. Injecting 30 mumole ATP per litre of axoplasm into a cyanide-poisoned axon caused a transient lowering of light intensity; oligomycin blocked the effect.5. Raising external calcium or replacing external sodium by choline or lithium reversibly increased the light produced by axons injected with aequorin.6. Stimulation at 50-200 impulses/sec in a solution containing 112 mM-Ca caused the light intensity to increase to a new steady level; after stimulation the light intensity returned to its original level with a time constant of 10-30 sec. Similar but smaller effects were seen in solutions containing less external calcium. The recovery after stimulation is probably due to uptake of calcium by the internal store.7. Injecting 3 m-mole EGTA per litre axoplasm lowered the resting glow and abolished the aequorin response to stimulation.8. There was no light response to stimulation immediately after an axial injection of aequorin and the effect increased to a ;steady' level with a half-time of about 5 min. The conclusion is that the rise in calcium concentration resulting from stimulation is confined to the peripheral part of the axon and that the diffusion coefficient of aequorin in axoplasm is about 4 x 10(-7) cm(2)/sec.9. The increment in light per impulse often increased markedly during the course of a long experiment and there was also considerable variation between axons.10. If the light response to stimulation was small it was proportional to the frequency of stimulation; if large to the square of the frequency.11. Voltage-clamp experiments showed that the calcium entry associated with a depolarizing pulse could be divided into an early component which was abolished by tetrodotoxin (TTX), and a late component which was unaffected by this inhibitor.12. The time relations of the early calcium entry were consistent with its being a leak of calcium ions through the sodium channel; the permeability of the sodium channel to calcium was about 1% of the permeability to sodium.13. The late entry of calcium was little changed by injecting enough tetraethylammonium (TEA) to block the outward potassium current; it was greatly reduced by external concentrations of manganese which had little effect on the maximum potassium conductance.14. The voltage-response curve for the late entry of calcium had a well defined maximum and was similar in shape to the curve relating calcium entry to depolarization at the presynaptic ending (Katz & Miledi, 1969, 1970).
Article
1. The quantitative dependence of transmitter release on external calcium concentration has been studied at the frog neuromuscular junction, using intracellular recording and taking the amplitude of the end-plate potential (e.p.p.) as an index of the number of packets released.2. The relation between [Ca] and the e.p.p. is highly non-linear. The initial part of this relation on double logarithmic co-ordinates gives a straight line with a slope of nearly four (mean 3.78 +/- 0.2 S.D. in 28 experiments). Addition of a constant amount of Mg reduces the e.p.p. without altering the slope of the log e.p.p./log Ca relation.3. The slope of this logarithmic relation diminishes as [Ca] is raised towards the normal level.4. The results are explained quantitatively on the hypothesis that Ca ions combine with a specific site X on the nerve terminal forming CaX, and that the number of packets of acetylcholine released is proportional to the fourth power of [CaX].5. The analysis suggests that a co-operative action of about four calcium ions is necessary for the release of each quantal packet of transmitter by the nerve impulse.
Article
The effects of various divalent cations in the external solution upon the Ca spike of the barnacle muscle fiber membrane were studied using intracellular recording and polarizing techniques. Analysis of the maximum rate of rise of the spike potential indicates that different species of divalent cations bind the same membrane sites competitively with different dissociation constants. The overshoot of the spike potential is determined by the density of Ca (Sr) ions in the membrane sites while the threshold membrane potential for spike initiation depends on the total density of divalent cations. The order of binding among different divalent and trivalent cations is the following: La+++, UO2++ > Zn++, Co++, Fe++ > Mn++ > Ni++ > Ca++ > Mg++, Sr++.
Article
1. The interaction between Na and Ca ions on quantal transmitter release at the frog neuromuscular junction has been studied, using intracellular recording and averaging of responses.2. At low calcium concentrations, partial withdrawal of Na ions increases end-plate potential (e.p.p.) amplitudes and quantal content (m) and decreases the amplitude of the miniature e.p.p.s (m.e.p.p.s). Under these conditions the relation between [Ca] and m is highly non-linear. When plotted on double logarithmic co-ordinates withdrawal of [Na] causes a nearly parallel shift of this relation.3. Mutual interaction occurs between Ca, Na and Mg in transmitter release. With a constant low [Ca] in the medium, withdrawal of [Na] produces a smaller increase in m when [Mg] is high, than when [Mg] is low.4. In the presence of normal [Ca] (1.8 mM), [Na] withdrawal decreases the amplitude of the e.p.p. and produces a small decrease in m.5. The results can be explained by assuming that [Na] reduction has two mutually opposing effects on transmitter release: it makes more sites available for the action of Ca, and it lowers the amplitude of the action potential in the nerve terminals. The former effect dominates at low, the latter at high, calcium concentrations.
The effects of pH changes on transmitter release at the frog neuromuscular synapse
  • E M Landau
  • D A Nyachshen Nachshen
  • D Landau
E. M. LANDAU AND D. A. NYACHSHEN NACHSHEN, D. & LANDAU, E. M. (1974). The effects of pH changes on transmitter release at the frog neuromuscular synapse. Proc. Jerusalem Satellite Symposia on Synaptic Mechanisms, p. 39. 25th Int. physiol. Congr.
  • H R Cordes
MAHLER, H. R. & CORDES, E. H. (1966). Biological Chemistry, p. 267. Tokyo: Harper & Row.
Intracellular pH, hydrogen ion flux and hydrogen ion permeability coefficient in bullfrog toe muscle
IZUTSU, K. T. (1972). Intracellular pH, hydrogen ion flux and hydrogen ion permeability coefficient in bullfrog toe muscle. J. Physiol. 221, 15-27.