The Journal of Physiology

1. Parameters derived from frequency-domain analysis of heart period and blood pressure variability are gaining increasing importance in clinical practice. However, the underlying physiological mechanisms in human subjects are not fully understood. Here we address the question as to whether the low frequency variability (approximately 0.1 Hz) of the heart period may depend on a baroreflex-mediated response to blood pressure oscillations, induced by the alpha-sympathetic drive on the peripheral resistance. 2. Heart period (ECG), finger arterial pressure (Finapres) and respiratory airflow were recorded in eight healthy volunteers in the supine position with metronome respiration at 0.25 Hz. We inhibited the vascular response to the sympathetic vasomotor activity with a peripheral alpha-blocker (urapidil) and maintained mean blood pressure at control levels with angiotensin II. 3. We performed spectral and cross-spectral analysis of heart period (RR) and systolic pressure to quantify the power of low- and high-frequency oscillations, phase shift, coherence and transfer function gain. 4. In control conditions, spectral analysis yielded typical results. In the low-frequency range, cross-spectral analysis showed high coherence (> 0.5) and a negative phase shift (-65.1 +/- 18 deg) between RR and systolic pressure, which indicates a 1-2 s lag in heart period changes in relation to pressure. In the high-frequency region, the phase shift was close to zero, indicating simultaneous fluctuations of RR and systolic pressure. During urapidil + angiotensin II infusion the low-frequency oscillations of both blood pressure and heart period were abolished in five cases. In the remaining three cases they were substantially reduced and lost their typical cross-spectral characteristics. 5. We conclude that in supine rest conditions, the oscillation of RR at low frequency is almost entirely accounted for by a baroreflex mechanism, since it is not produced in the absence of a 0.1 Hz pressure oscillation. 6. The results provide physiological support for the use of non-invasive estimates of the closed-loop baroreflex gain from cross-spectral analysis of blood pressure and heart period variability in the 0.1 Hz range.

1. Electrophysiologically identified thalamocortical neurones have been intra- and extracellularly recorded in acutely prepared cats, under different anaesthetic conditions. 2. A slow (0.5-4 Hz) membrane potential oscillation was observed in thalamocortical cells recorded in motor, sensory, associational and intralaminar thalamic nuclei. The oscillation consisted of rhythmic low-threshold spikes alternating with after-hyperpolarizations. 3. About 80% of the neurones with intact cortical connections were set into the slow oscillatory mode by bringing their membrane potential to between -68 and -90 mV. The oscillation did not depend upon the occurrence of fast action potentials and did not outlast the imposed hyperpolarization. 4. Anatomical or functional disconnection from related cortical areas resulted in a membrane potential hyperpolarization of about 9 mV and in the occurrence of spontaneous slow oscillations in virtually all recorded neurones. The intrinsic nature of the phenomenon was supported by the lack of rhythmic postsynaptic potentials as the cells were prevented from oscillating by outward current injection. 5. In contrast with other thalamic nuclei, the slow oscillation has not been observed in anterior thalamic neurones despite their having similar basic electrophysiological properties. 6. Barbiturate administration suppressed the slow oscillatory mode, an effect accompanied by a decrease in the membrane input resistance. 7. Multiunit recordings of spontaneously oscillating cells showed epochs characterized by phase-related firing. This synchronous discharge was paralleled by a clear-cut build-up of field potentials in the frequency range of electroencephalogram slow or delta waves. 8. These results demonstrate that the majority of thalamocortical neurones are endowed with electrophysiological properties allowing them to oscillate at 0.5-4 Hz, if they have a membrane potential more negative than -65 mV and a high input resistance. Such a condition is physiologically achieved in the deepest stages of electroencephalogram-synchronized sleep, as a result of brain stem-thalamic as well as cortico-thalamic deafferentation. We postulate a thalamic contribution in the genesis of electroencephalogram delta waves during slow wave sleep, once independently oscillating thalamocortical cells become in phase.

Amino acid infusions increase renal blood flow (RBF) and glomerular filtration rate (GFR) and stimulate tubular reabsorption in adults. To characterize the effects of amino acids on fetal renal haemodynamics, tubular sodium reabsorption, acid-base homeostasis and plasma renin levels, 11 chronically catheterized fetal sheep aged 121 +/- 1 days (term ~150 days) were infused I.V. for 4 h with alanine, glycine, proline and serine (0.1, 0.1, 0.06 and 0.06 mmol min(-1), respectively) in 0.15 M saline at 0.165 ml min(-1). Eight control fetuses were given saline. During amino acid infusion, plasma amino acid levels increased up to 20-fold (P < 0.005). GFR increased by 50 +/- 8 % (P < 0.001); there was only a small transient increase in RBF. Proximal fractional sodium reabsorption fell from 74.6 +/- 2.9 to 55.5 +/- 5.4 % (P < 0.005). Distal sodium delivery increased, but a smaller percentage of this distal sodium load was reabsorbed (P < 0.005). Thus fractional sodium reabsorption fell from 95.5 +/- 0.9 to 81.4 +/- 2.0 % (P < 0.005). There was a large diuresis, natriuresis, kaliuresis and increase in osmolar excretion (P < 0.005). Plasma sodium and chloride concentrations fell (P < 0.005). Plasma osmolality did not change. Plasma renin levels fell (P < 0.05), cortisol levels increased (P < 0.05), and there was a compensated metabolic acidosis. Thus the fetal sheep kidney demonstrated a remarkable functional capacity to respond to amino acid infusion. The increase in filtration fraction and the lack of an increase in RBF suggest that efferent arteriolar vasoconstriction occurred, a very different response from the renal vasodilatation seen in adult animals.

1. The transduction mechanisms involved in the activation and modulation of the noradrenaline-activated cation current (Icat) were investigated with whole-cell patch clamp techniques in rabbit portal vein smooth muscle cells. 2. Intracellular application of guanosine 5-O-(3-thiotriphosphate) (GTP gamma S, 500 microM) evoked a 'noisy' inward current at -50 mV with a similar current-voltage relationship and reversal potential to the current evoked by bath application of noradrenaline (100 microM). Guanosine 5-O-(2-thiodiphosphate) (GDP beta S, 1 mM) markedly inhibited noradrenaline-activated Icat. 3. The phospholipase C (PLC) inhibitor U73122 inhibited the amplitude of the noradrenaline-activated Icat in a concentration- and time-dependent manner and the IC50 was about 180 nM. U73122 had similar effects on the cation current evoked by GTP gamma S. 4. Intracellular application of myo-inositol 1,4,5-trisphosphate (IP3, 100 microM) from the patch pipette did not activate any membrane current in cells where intracellular calcium concentration ([Ca2+]i) was buffered to 14 nM, but subsequent addition of noradrenaline evoked Icat. 5. Bath application of the 1,2-diacyl-sn-glycerol (DAG) analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG, 10 microM) activated Icat, whereas the phorbol ester phorbol 12,13-dibutyrate (PDBu, 0.1-5 microM) failed to activate Icat, in every cell examined. Icat activated by OAG after bath application of PDBu was not significantly different from OAG-activated Icat in the absence of PDBu. The DAG lipase inhibitor RHC80267 (10 microM) activated Icat in some cells, whereas the DAG kinase inhibitor R59949 (10 microM) never activated Icat. 6. Bath application of the protein kinase C inhibitor chelerythrine (1-10 microM) had no effect on either OAG-or noradrenaline-activated Icat. 7. It is concluded that noradrenaline activates Icat via a G-protein coupled to PLC and that the resulting DAG product plays a central role in the activation of cation channels via a protein kinase C-independent mechanism.

1. To examine the role of the phosphoinositide cascade triggered by disturbed Ca2+ homeostasis in ischaemic neurones, inositol 1,3,4,5-tetrakisphosphate (InsP4) was applied to the cytoplasmic face of membrane patches isolated from CA1 pyramidal neurones in the gerbil hippocampus. 2. In outside-out recordings, InsP4 induced an inward current which was increased by raising the extracellular [Ca2+]. In contrast, no clear channel openings could be observed in patches from neurones of sham-operated gerbils. 3. Open probabilities of InsP4-activated channels were significantly decreased upon application of omega-conotoxin but were not affected by omega-agatoxin or nifedipine. 4. In inside-out patches using high concentrations of Ca2+, Ba2+ or Sr2+ in the pipette solution, InsP4 enhanced inward currents. 5. Application of the isomers of InsP4 slightly enhanced the currents, but inositol 1,4,5-trisphosphate (InsP3) had no effect. 6. In the absence of InsP4 there was a single main Ba2+ current peak of 4.0 pA in amplitude, whereas upon its application two main peaks of 3.0 and 7.2 pA were present. 7. The open probabilities of these channels were apparently increased by InsP4. 8. These findings support the view that a disturbed phosphoinositide cascade occurs in the hippocampal pyramidal neurones after ischaemia and the InsP4 thus formed plays an important role in promoting the Ca2+ accumulation which results in neuronal death.

Various cardio-active stimuli, including endothelin-1 (ET-1), exhibit potent arrhythmogenicity, but the underlying cellular mechanisms of their actions are largely unclear. We used isolated rat atrial myocytes and related changes in their subcellular Ca(2+) signalling to the ability of various stimuli to induce diastolic, premature extra Ca(2+) transients (ECTs). For this, we recorded global and spatially resolved Ca(2+) signals in indo-1- and fluo-4-loaded atrial myocytes during electrical pacing. ET-1 exhibited a higher arrhythmogenicity (arrhythmogenic index; ratio of number of ECTs over fold-increase in Ca(2+) response, 8.60; n = 8 cells) when compared with concentrations of cardiac glycosides (arrhythmogenic index, 4.10; n = 8 cells) or the beta-adrenergic agonist isoproterenol (arrhythmogenic index, 0.11; n = 6 cells) that gave similar increases in the global Ca(2+) responses. Seventy-five percent of the ET-1-induced arrhythmogenic Ca(2+) transients were accompanied by premature action potentials, while for digoxin this proportion was 25 %. The beta-adrenergic agonist failed to elicit a significant number of ECTs. Direct activation of inositol 1,4,5-trisphosphate (InsP(3)) receptors with a membrane-permeable InsP(3) ester (InsP(3) BM) mimicked the effect of ET-1 (arrhythmogenic index, 14.70; n = 6 cells). Inhibition of InsP(3) receptors using 2 microM 2-aminoethoxydiphenyl borate, which did not display any effects on Ca(2+) signalling under control conditions, specifically suppressed the arrhythmogenic action of ET-1 and InsP(3) BM. Immunocytochemistry indicated a co-localisation of peripheral, junctional ryanodine receptors with InsP(3)Rs. Thus, the pronounced arrhythmogenic potency of ET-1 is due to the spatially specific recruitment of Ca(2+) sparks by subsarcolemmal InsP(3)Rs. Summation of such sparks efficiently generates delayed after depolarisations that trigger premature action potentials. We conclude that the particular spatial profile of cellular Ca(2+) signals is a major, previously unrecognised, determinant for arrhythmogenic potency and that the InsP(3) signalling cassette might therefore be a promising new target for understanding and managing atrial arrhythmia.

Phosphorylation of inositol 1,4,5-trisphosphate receptors (InsP(3)R) by PKA represents an important, common route for regulation of Ca(2+) release. Following phosphorylation of the S2 splice variant of InsP(3)R-1 (S2-InsP-1), Ca(2+) release is markedly potentiated. In this study we utilize the plasma membrane (PM) expression of InsP(3)R-1 and phosphorylation state mutant InsP(3)R-1 to study how this regulation occurs at the single InsP(3)R-1 channel level. DT40-3KO cells stably expressing rat S2- InsP(3)R-1 were generated and studied in the whole-cell mode of the patch clamp technique. At hyperpolarized holding potentials, small numbers of unitary currents (average approximately 1.7 per cell) were observed which were dependent on InsP(3) and the presence of functional InsP(3)R-1, and regulated by both cytoplasmic Ca(2+) and ATP. Raising cAMP markedly enhanced the open probability (P(o)) of the InsP(3)R-1 and induced bursting activity, characterized by extended periods of rapid channel openings and subsequent prolonged refractory periods. The activity, as measured by the P(o) of the channel, of a non-phosphorylatable InsP(3)R-1 construct (Ser1589Ala/Ser1755Ala InsP(3)R-1) was markedly less than wild-type (WT) InsP(3)R-1 and right shifted some approximately 15-fold when the concentration dependency was compared to a phosphomimetic construct (Ser1589Glu/Ser1755Glu InsP(3)R-1). No change in conductance of the channel was observed. This shift in apparent InsP(3) sensitivity occurred without a change in InsP(3) binding or Ca(2+) dependency of activation or inactivation. Biophysical analysis indicated that channel activity can be described by three states: an open state, a long lived closed state which manifests itself as long interburst intervals, and a short-lived closed state. Bursting activity occurs as the channel shuttles rapidly between the open and short-lived closed state. The predominant effect of InsP(3)R-1 phosphorylation is to increase the likelihood of extended bursting activity and thus markedly augment Ca(2+) release. These analyses provide insight into the mechanism responsible for augmenting InsP(3)R-1 channel activity following phosphorylation and moreover should be generally useful for further detailed investigation of the biophysical properties of InsP(3)R.

We investigated synergism between inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) and diacylglycerol (DAG) on TRPC6-like channel activity in rabbit portal vein myocytes using single channel recording and immunoprecipitation techniques. Ins(1,4,5)P(3) at 10 microm increased 3-fold TRPC6-like activity induced by 10 microm 1-oleoyl-2-acetyl-sn-glycerol (OAG), a DAG analogue. Ins(1,4,5)P(3) had no effect on OAG-induced TRPC6 activity in mesenteric artery myocytes. Anti-TRPC6 and anti-TRPC7 antibodies blocked channel activity in portal vein but only anti-TRPC6 inhibited activity in mesenteric artery. TRPC6 and TRPC7 proteins strongly associated in portal vein but only weakly associated in mesenteric artery tissue lysates. Therefore in portal vein the conductance consists of TRPC6/C7 subunits, while OAG activates a homomeric TRPC6 channel in mesenteric artery myocytes. Wortmannin at 20 microm reduced phosphatidylinositol 4,5-bisphosphate (PIP(2)) association with TRPC6 and TRPC7, and produced a 40-fold increase in OAG-induced TRPC6/C7 activity. Anti-PIP(2) antibodies evoked TRPC6/C7 activity, which was blocked by U73122, a phospholipase C inhibitor. DiC8-PIP(2), a water-soluble PIP(2) analogue, inhibited OAG-induced TRPC6/C7 activity with an IC(50) of 0.74 microm. Ins(1,4,5)P(3) rescued OAG-induced TRPC6/C7 activity from inhibition by diC8-PIP(2) in portal vein myocytes, and this was not prevented by the Ins(1,4,5)P(3) receptor antagonist heparin. In contrast, Ins(1,4,5)P(3) did not overcome diC8-PIP(2)-induced inhibition of TRPC6 activity in mesenteric artery myocytes. 2,3,6-Tri-O-butyryl-Ins(1,4,5)P(3)/AM (6-Ins(1,4,5)P(3)), a cell-permeant analogue of Ins(1,4,5)P(3), at 10 microm increased TRPC6/C7 activity in portal vein and reduced association between TRPC7 and PIP(2), but not TRPC6 and PIP(2). In contrast, 10 microm OAG reduced association between TRPC6 and PIP(2), but not between TRPC7 and PIP(2). The present work provides the first evidence that Ins(1,4,5)P(3) modulates native TRPC channel activity through removal of the inhibitory action of PIP(2) from TRPC7 subunits.

1. The effects of activation of guanine nucleotide-binding protein (G protein) by guanine nucleotides or sodium fluoride on the release of intracellular Ca2+ and on tension development were determined in chemically skinned strips of rabbit main pulmonary arteries (MPA). Ca2+ movements were monitored with Fura-2, as the change in free Ca2+ concentration in the bath medium surrounding the skinned MPA. 2. Sodium fluoride or non-hydrolysable analogues of GTP, guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) and guanosine 5'-[beta,gamma-imido]triphosphate (GMP-PNP), induced sustained and dose-dependent contraction of skinned MPA. GTP (100 microM) induced transient contraction of skinned MPA. GTP gamma S did not contract intact MPA. We also confirmed that inositol 1,4,5-trisphosphate (InsP3) released sufficient Ca2+ to induce contraction of skinned, but not intact, MPA. 3. Guanosine 5'-[beta-thio]diphosphate (GDP beta S), a non-hydrolysable analogue of GDP that competitively inhibits the binding of guanine nucleotides to G proteins, inhibited the contractions induced by GTP gamma S. Neomycin (1 mM) inhibited the GTP gamma S-induced contractions, but also, to a lesser extent, contractions induced by caffeine. 4. Depletion of Ca2+ from the sarcoplasmic reticulum (SR) or treatment with Triton X-100 inhibited the GTP gamma S-induced contractions. The effects of Ca2+ depletion was reversible, while that of Triton X-100 was irreversible. GTP gamma S (up to 100 microM) had no apparent effect on the pCa-tension curve of freeze-glycerinated MPA. 5. GTP gamma S- or InsP3-induced contractions occurred in the presence of 20 mM-procaine, while this agent completely blocked the contraction induced by caffeine. 6. Both GTP gamma S and InsP3 induced an increase in the Fura-2 fluorescence signal of the bath medium surrounding the skinned MPA, indicating that GTP gamma S releases intracellular Ca2+. The release of Ca2+ induced by GTP gamma S was inhibited by GDP beta S. 7. During the initial phasic contraction induced by GTP gamma S, added InsP3 had little or no additive effect, in contrast to its additive effect during the latter sustained contraction induced by GTP gamma S.(ABSTRACT TRUNCATED AT 400 WORDS)

1. Inositol 1,4,5-trisphosphate (InsP3) injected into unfertilized golden hamster eggs elicits a hyperpolarizing response (HR) that is due to stimulation of calcium-activated potassium channels in the egg plasma membrane. 2. A single injection of InsP3 gave a single HR above a threshold value of 0.3 nM. At 5 nM and above, InsP3 induced HRs with no detectable latency. At concentrations between these two values a latency was observed. The amplitude of the HR was independent of InsP3 concentration. 3. A second HR could be elicited by injection of InsP3, but five times more InsP3 was required to trigger a second HR, and 10-100 times more to give an HR of similar magnitude to the first, and there was no latency. 4. The increase in [Ca2+]i in response to an initial injection of 1 nM InsP3 could be resolved into two distinct components: a slow, early rise immediately after InsP3 injection (phase I) followed by a larger and more rapid increase (phase II). The initiation of an HR coincided with the second component of the [Ca2+]i increase. 5. Further injection of InsP3 resulted only in slow, smaller increases in [Ca2+]i that resembled phase I and often did not cause an HR. Phase II appeared to be absent. However, 100-fold greater InsP3 concentrations gave slow, larger Ca2+ transients (and HRs) with no detectable latency. 6. If large amounts of InsP3 were allowed to leak into the eggs constantly from a pipette, repetitive calcium transients were seen. Unlike the sustained repetitive responses seen at fertilization, they were often smaller than the initial transient and less well sustained. However, a subsequent transient could still be elicited on injection of very large concentrations of InsP3. 7. InsP3 can induce regenerative, all-or-none [Ca2+]i increase (CICR) in hamster eggs, often with a long latency, as well as non-regenerative increases. InsP3 injections desensitize CICR and cannot mimic all the features of Ca2+ signalling at fertilization in the hamster egg, in particular, the sensitization of CICR caused by the sperm.

Spontaneous, rhythmical contractions, or vasomotion, can be recorded from cerebral vessels under both normal physiological and pathophysiological conditions. Using electrophysiology to study changes in membrane potential, the ratiometric calcium indicator Fura-2 AM to study changes in [Ca(2+)](i) in both the arterial wall and in individual smooth muscle cells (SMCs), and video microscopy to study changes in vessel diameter, we have investigated the cellular mechanisms underlying vasomotion in the juvenile rat basilar artery. During vasomotion, rhythmical oscillations in both membrane potential and [Ca(2+)](i) were found to precede rhythmical contractions. Nifedipine depolarized SMCs and abolished rhythmical contractions and depolarizations. [Ca(2+)](i) oscillations in the arterial wall became reduced and irregular, while [Ca(2+)](i) oscillations in adjacent SMCs were no longer synchronized. BAPTA-AM, thapsigargin and U73122 hyperpolarized SMCs, relaxed the vessel, decreased basal calcium levels and abolished vasomotion. Chloride substitution abolished rhythmical activity, depolarized SMCs, increased basal calcium levels and constricted the vessel, while niflumic acid and DIDS abolished vasomotion. Ryanodine, charybdotoxin and TRAM-34, but not iberiotoxin, 4-aminopyridine or apamin, each depolarized SMCs and increased the frequency of rhythmical depolarizations and [Ca(2+)](i) oscillations. We conclude that vasomotion in the basilar artery depends on the release of intracellular calcium from IP(3) (inositol 1,4,5,-trisphosphate)-sensitive stores which activates calcium-dependent chloride channels to depolarize SMCs. Depolarization in turn activates voltage-dependent calcium channels, synchronizing contractions of adjacent cells through influx of extracellular calcium. Subsequent calcium-induced calcium release from ryanodine-sensitive stores activates an intermediate conductance potassium channel, hyperpolarizing the SMCs and providing a negative feedback pathway for regeneration of the contractile cycle.

In rabbit portal vein smooth muscle cells, store-operated Ca2+-permeable cation channels (SOCs) display multi-modal gating mechanisms. SOCs are activated by depletion of intracellular Ca2+ stores but also may be stimulated in a store-independent manner by noradrenaline acting on alpha-adrenoceptors and by diacylglycerol (DAG) via protein kinase C (PKC). In the present study we have investigated whether inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) modulates SOC activity in freshly dispersed rabbit portal vein myocytes with patch pipette recording techniques. Inclusion of 1 mum Ins(1,4,5)P3 in the patch pipette solution increased whole-cell currents evoked by the Ca2+-ATPase inhibitor cyclopiazonic acid (CPA) by about 3-fold at -80 mV. In the cell-attached configuration the cell-permeable Ca2+ chelator BAPTA-AM stimulated SOC activity and after excision of an isolated inside-out patch bath application of 1 mum Ins(1,4,5)P3 increased open channel probability (NP(o)) by approximately 3-fold. Ins(1,4,5)P3 also produced a similar increase in NP(o) of SOCs stimulated by the phorbol ester, phorbol 12,13-dibutyrate (PDBu) in inside-out patches and these channel currents had a unitary conductance of about 2 pS. The equilibrium constant of Ins(1,4,5)P3 on increasing PDBu-evoked SOC activity was about 0.4 mum. The facilitatory effect of Ins(1,4,5)P3 was also manifest as markedly increasing the rate of activation of SOCs. The synergistic effect of Ins(1,4,5)P3 was mimicked by the metabolically stable analogue 3-fluoro-Ins(1,4,5)P3 and Ins(1,4)P2, a metabolite of Ins(1,4,5)P3, but was not inhibited by the classical Ins(1,4,5)P3 receptor antagonist heparin. Finally Ins(1,4,5)P3 also increased NP(o) of SOCs activated by a PKC catalytic subunit. It is concluded that Ins(1,4,5)P3 facilitates SOC opening via a heparin-insensitive mechanism at, or close to, the channel protein.

1. Vascular smooth muscle cells were isolated from the coronary artery of the guinea-pig. At 2.5 mM [Ca2+]o and 36 degrees C, whole cell membrane currents were recorded under voltage-clamp and the concentration of ionized calcium in the cytoplasm ([Ca2+]i) was monitored by indo-1 fluorescence. 2. At -60 mV, [Ca2+]i was 143 +/- 36 mM (mean +/- S.D.) and was insensitive to clamp steps to +100 mV. During 1 min application of acetylcholine (ACh, 10 microM) [Ca2+]i increased within approximately 2 s to 1480 +/- 250 nM. During the subsequent slow decay, [Ca2+]i was transiently increased by depolarizing clamp steps and decreased during hyperpolarizing steps. [Ca2+]i transients in response to caffeine (10 mM) could not be modulated by voltage steps. The results suggest that modulation of [Ca2+]i by membrane potential involves inositol 1,4,5-trisphosphate (Ins(1,4,5)P3)-induced Ca2+ release (IICR). 3. Modulation of IICR by membrane potential did not depend on sarcolemmal Ca2+ fluxes; it persisted after block of sarcolemmal Ca2+ fluxes with 3 mM lanthanum or after a change to nominally Ca(2+)-free bathing solutions. 4. Modulation of [Ca2+]i by membrane potential was recorded during cell dialysis of 50 microM GTP-gamma-S in the absence of ACh. Cell dialysis of exogenous Ins(1,4,5)P3 (50 or 100 microM) did not mimic the effects. The sensitivity of [Ca2+]i to depolarizing clamp steps was also induced by cell dialysis of lithium ions which, presumably, inhibited the breakdown of Ins(1,4,5)P3. The results are compatible with the idea that the membrane potential modulates the liberation of Ins(1,4,5)P3. 5. Modulation of IICR by membrane potential is discussed as a new mechanism that contributes to the regulation of activator calcium and to the modulation of contraction in vascular smooth muscle cells.

1. Effects of membrane hyperpolarization induced by pinacidil on Ca2+ mobilization induced by noradrenaline (NA) were investigated by measuring intracellular Ca2+ concentration ([Ca2+]i), isometric tension, membrane potential and production of inositol 1,4,5-trisphosphate (IP3) in smooth muscle cells of the rabbit mesenteric artery. 2. Pinacidil (0.1-10 microM) concentration dependently hyperpolarized the smooth muscle membrane with a reduction in membrane resistance. Glibenclamide (1 microM) blocked the membrane hyperpolarization induced by 1 microM-pinacidil. NA (10 microM) depolarized the smooth muscle membrane with associated oscillations. Pinacidil (1 microM) inhibited this response and glibenclamide (1 microM) prevented the action of pinacidil on both the NA-induced events. 3. In thin smooth muscle strips, 10 microM-NA produced a large phasic and a subsequent small tonic increase in [Ca2+]i with associated oscillations. These changes in [Ca2+]i seemed to be coincident with phasic, tonic and oscillatory contractions, respectively. Pinacidil (0.1-1 microM) inhibited the increases in [Ca2+]i and in tension induced by NA, but not by 128 mM-K+. Glibenclamide inhibited these actions of pinacidil. Pinacidil (1 microM) also inhibited the contraction induced by 10 microM-NA in strips treated with A23187 (which functionally removes cellular Ca2+ storage sites), suggesting that membrane hyperpolarization inhibits Ca2+ influxes activated by NA. 4. In Ca2(+)-free solution containing 2 mM-EGTA, NA (10 microM) transiently increased [Ca2+]i, tension and synthesis of IP3. Pinacidil (over 0.1 microM) inhibited the increases in [Ca2+]i, tension and synthesis of IP3 induced by 10 microM-NA in Ca2(+)-free solution containing 5.9 mM-K+, but not in a similar solution containing 40 or 128 mM-K+. Glibenclamide (1 microM) inhibited these actions of pinacidil. These inhibitory actions of pinacidil were still observed in solutions containing low Na+ or low Cl-. These results suggest that pinacidil inhibits NA-induced Ca2+ release from storage sites through an inhibition of IP3 synthesis resulting from its membrane hyperpolarizing action. 5. In beta-escin-treated skinned strips, NA (10 microM) or IP3 (20 microM) increased Ca2+ in Ca2(+)-free solution containing 50 microM-EGTA and 3 microM-guanosine triphosphate (GTP) after brief application of 0.3 microM-Ca2+, suggesting Ca2+ is released from intracellular storage sites. Heparin (500 micrograms/ml, an inhibitor of the IP3 receptor), but not pinacidil (1 microM) or glibenclamide (1 microM), inhibited the Ca2+ release from storage sites induced by NA or IP3. These results suggest that membrane hyperpolarization is essential for the inhibitory action of pinacidil on the NA-induced Ca2(+)-releasing mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)

1. Single smooth muscle cells were isolated by enzymic digestion from the longitudinal muscle layer of rabbit jejunum, and the response of the cells to calcium (Ca2+) release by InsP3 (D-myo-inositol 1,4,5-trisphosphate) was studied. Changes in internal Ca2+ concentration were monitored by measuring Ca(2+)-activated K+ currents (outward currents) using the whole-cell voltage-clamp technique. 2. At break-through from cell-attached patch to whole-cell recording mode using a 100 microM-InsP3-filled pipette, cells exhibited a brief outward current which reached its peak in 1.1 s and terminated within 10 s. Following this the generation of spontaneous transient outward currents (STOCs) was inhibited. (STOCs are considered to represent bursts of openings of Ca(2+)-activated K+ channels in response to spontaneous discharges of Ca2+ from the stores.) When a pipette filled with 20 microM-InsP3 was used, similar current responses were also evoked, but some cells failed to respond. 3. The InsP3-induced outward current at membrane break-through was similar in size and time course to the outward current response of normal cells to bath-applied carbachol (CCh, 100 microM) or caffeine (20 mM). 4. Dialysis with InsP3-containing solution inhibited the caffeine-induced outward current, depending on the pipette InsP3 concentration. Inclusion of heparin (5 mg/ml) in the pipette completely prevented inhibition by InsP3 of the caffeine response and of STOC discharge. However, the InsP3-induced current at break-through remained unchanged, probably because of the slower rate of diffusion of heparin. 5. In cells dialysed with pipette solution containing 30 or 100 microM-caged InsP3, flash photolysis (producing up to 1.5 microM-InsP3) induced an outward current response after a latency of 31.0 +/- 1.8 ms (n = 15), which was followed by inhibition of STOCs. The reversal potential of the current to flash-release of InsP3 followed closely the Nernst potential for K+ ions (EK), suggesting negligible contributions from channels other than Ca(2+)-activated K+ channels. 6. Photolysis of caged InsP3 (30 or 100 microM) still produced a current response after 3-6 min in Ca(2+)-free (3 mM-EGTA added) bathing solution, but no response occurred if the cell was exposed to either caffeine (20 mM) or CCh (100 microM) to deplete Ca stores.(ABSTRACT TRUNCATED AT 400 WORDS)

We studied inositol-1,4,5-trisphosphate (IP(3)) receptor-dependent intracellular Ca(2+) waves in CA1 hippocampal and layer V medial prefrontal cortical pyramidal neurons using whole-cell patch-clamp recordings and Ca(2+) fluorescence imaging. We observed that Ca(2+) waves propagate in a saltatory manner through dendritic regions where increases in the intracellular concentration of Ca(2+) ([Ca(2+)](i)) were large and fast ('hot spots') separated by regions where increases in [Ca(2+)](i) were comparatively small and slow ('cold spots'). We also observed that Ca(2+) waves typically initiate in hot spots and terminate in cold spots, and that most hot spots, but few cold spots, are located at dendritic branch points. Using immunohistochemistry, we found that IP(3) receptors (IP(3)Rs) are distributed in clusters along pyramidal neuron dendrites and that the distribution of inter-cluster distances is nearly identical to the distribution of inter-hot spot distances. These findings support the hypothesis that the dendritic locations of Ca(2+) wave hot spots in general, and branch points in particular, are specially equipped for regenerative IP(3)R-dependent internal Ca(2+) release. Functionally, the observation that IP(3)R-dependent [Ca(2+)](i) rises are greater at branch points raises the possibility that this novel Ca(2+) signal may be important for the regulation of Ca(2+)-dependent processes in these locations. Futhermore, the observation that Ca(2+) waves tend to fail between hot spots raises the possibility that influences on Ca(2+) wave propagation may determine the degree of functional association between distinct Ca(2+)-sensitive dendritic domains.

1. The blocking action of 4-aminopyridine (4-AP) on a delayed rectifier Kv1.2 K+ channel expressed in oocytes was investigated at room temperature (22 degrees C) and physiological temperature (34 degrees C) using the double-electrode voltage clamp and patch clamp techniques. 2. At room temperature, 4-AP (100 microM) inhibition occurred only after activation of current. The rate of onset of block was dependent upon the length of time current was activated by a depolarizing step. Similarly, removal of block required current activation. The degree of steady-state block by 4-AP was not reduced by increasingly more depolarized step potentials. The degree of steady-state block also did not change over the duration of a 1 s step. 3. When channels were nearly fully inactivated, 4-AP produced no additional block of a subsequent depolarizing step, suggesting that 4-AP did not bind when channels were in the inactivated state. In single channel experiments, 4-AP decreased the mean open time in a dose-dependent manner but did not alter the single-channel current amplitude. 4. At 34 degrees C the I-V relationship and inactivation curve shifted to more negative potentials. Increasing the temperature to 34 degrees C did not alter the degree of block by 4-AP, although the rate of onset of block was greatly enhanced. 5. Results suggest that 4-AP binds to the open state of the Kv1.2 channel and is trapped when the channel closes. 4-AP cannot bind when the channel is closed or inactivated prior to the addition of the drug. C-type inactivation and 4-AP binding to the channel are mutually exclusive. A model for the proposed mechanism of action of 4-AP on the Kv1.2 channel is proposed based on experimental data.

We examined changes in ionic and gating currents in Ca(V)1.2 channels when extracellular Ca(2+) was reduced from 10 mm to 0.1 microm. Saturating gating currents decreased by two-thirds (K(D) approximately 40 microm) and ionic currents increased 5-fold (K(D) approximately 0.5 microm) due to increasing Na(+) conductance. A biphasic time dependence for the activation of ionic currents was observed at low [Ca(2+)], which appeared to reflect the rapid activation of channels that were not blocked by Ca(2+) and a slower reversal of Ca(2+) blockade of the remaining channels. Removal of Ca(2+) following inactivation of Ca(2+) currents showed that Na(+) currents were not affected by Ca(2+)-dependent inactivation. Ca(2+)-dependent inactivation also induced a negative shift of the reversal potential for ionic currents suggesting that inactivation alters channel selectivity. Our findings suggest that activation of Ca(2+) conductance and Ca(2+)-dependent inactivation depend on extracellular Ca(2+) and are linked to changes in selectivity.

Cav1.3 channels mediate Ca2+ influx that triggers exocytosis of glutamate at cochlear inner hair cell (IHC) synapses. Harmonin is a PDZ-domain containing protein that interacts with the C-terminus of the Cav1.3 α1 subunit (α1 1.3) and controls cell-surface Cav1.3 levels by promoting ubiquitin-dependent proteosomal degradation. However, PDZ-domain containing proteins have diverse functions and regulate other Cav1.3 properties, which could collectively influence presynaptic transmitter release. Here, we report that harmonin binding to the α1 1.3 distal C-terminus (dCT) enhances voltage-dependent facilitation (VDF) of Cav1.3 currents both in transfected HEK293T cells and in mouse inner hair cells. In HEK293T cells, this effect of harmonin was greater for Cav1.3 channels containing the auxiliary Cav β1 than with the β2 auxiliary subunit. Cav1.3 channels lacking the α11.3 dCT were insensitive to harmonin modulation. Moreover, the "deaf-circler" dfcr mutant form of harmonin, which does not interact with the α11.3 dCT, did not promote VDF. In mature IHCs from mice expressing the dfcr harmonin mutant, Cav1.3 VDF was less than in control IHCs. This difference was not observed between control and dfcr IHCs prior to hearing onset. Membrane capacitance recordings from dfcr IHCs revealed a role for harmonin in synchronous exocytosis and in increasing the efficiency of Ca2+ influx for triggering exocytosis. Collectively, our results indicate a multifaceted presynaptic role of harmonin in IHCs in regulating Cav1.3 Ca2+ channels and exocytosis.

1. Using the two-microelectrode, 'cut open' oocyte, and 'torn off' macropatch voltage clamp techniques, we studied the blocking effects of 4-aminopyridine (4-AP) on two cloned K+ channels expressed in Xenopus oocytes, an inactivating K+ channel isolated from ferret ventricle (FK1), and its NH2-terminal deletion mutant (delta NCO) which lacks fast N-type inactivation. 2. Experiments with a permanently charged, impermeant 4-AP derivative, 4-aminopyridine-methyliodide, indicated that the cationic form of 4-AP blocks at an intracellular site. 3. Block accumulated from pulse to pulse and was sensitive to the applied potential during hyperpolarizing deactivating pulses, indicating trapping of 4-AP in deactivated channels. For long trains of depolarizing pulses (-90 to +50 mV, 0.1 Hz), 4-AP block increased with decreasing pulse duration. Block of FK1 was much more sensitive to pulse duration than was block of delta NCO, consistent with competition between N-type inactivation and 4-AP binding. 4. To elucidate these mechanisms further, in the absence of fast N-type inactivation the following results were obtained on delta NCO channels: (1) application of 4-AP caused the appearance of apparent inactivation; (2) 4-AP, however, did not cause cross-over of deactivating tail currents; (3) 4-AP block developed with time for potentials positive to -40 mV; and (4) trapping of 4-AP by delta NCO was insensitive to the degree of C-type inactivation. 5. We conclude that the kinetics of 4-AP block of FK1 and delta NCO channels cannot be accounted for by either a pure open channel or closed channel blocking scheme.

Mutations in the TTX-sensitive voltage-gated sodium channel subtype Nav1.7 have been implicated in the painful inherited neuropathy, hereditary erythromelalgia. Hereditary erythromelalgia can be difficult to treat and, although sodium channels are targeted by local anaesthetics such as lidocaine (lignocaine), some patients do not respond to treatment with local anaesthetics. This study examined electrophysiological differences in Nav1.7 caused by a hereditary erythromelalgia mutation (N395K) that lies within the local anaesthetic binding site of the channel. The N395K mutation produced a hyperpolarized voltage dependence of activation, slower kinetics of deactivation, and impaired steady-state slow inactivation. Computer simulations indicate that the shift in activation is the major determinant of the hyperexcitability induced by erythromelalgia mutations in sensory neurons, but that changes in slow inactivation can modulate the overall impact on excitability. This study also investigated lidocaine inhibition of the Nav1.7-N395K channel. We show that the N395K mutation attenuates the inhibitory effects of lidocaine on both resting and inactivated Nav1.7. The IC50 for lidocaine was estimated at 500 microM for inactivated wild-type Nav1.7 and 2.8 mM for inactivated Nav1.7-N395K. The N395K mutation also significantly reduced use-dependent inhibition of lidocaine on Nav1.7 current. In contrast, a different hereditary erythromelalgia mutation (F216S), not located in the local anaesthetic binding site, had no effect on lidocaine inhibition of Nav1.7 current. Our observation of reduced lidocaine inhibition on Nav1.7-N395K shows that the residue N395 is critical for lidocaine binding to Nav1.7 and suggests that the response of individuals with hereditary erythromelalgia to lidocaine treatment may be determined, at least in part, by their specific genotype.

We have examined the distribution of the sensory neuron-specific Na+ channel Nav1.8 (SNS/PN3) in nociceptive and non-nociceptive dorsal root ganglion (DRG) neurons and whether its distribution is related to neuronal membrane properties. Nav1.8-like immunoreactivity (Nav1.8-LI) was examined with an affinity purified polyclonal antiserum (SNS11) in rat DRG neurons that were classified according to sensory receptive properties and by conduction velocity (CV) as C-, Adelta- or Aalpha/beta. A significantly higher proportion of nociceptive than low threshold mechanoreceptive (LTM) neurons showed Nav1.8-LI, and nociceptive neurons had significantly more intense immunoreactivity in their somata than LTM neurons. Results showed that 89, 93 and 60% of C-, Adelta- and Aalpha/beta-fibre nociceptive units respectively and 88% of C-unresponsive units were positive. C-unresponsive units had electrical membrane properties similar to C-nociceptors and were considered to be nociceptive-type neurons. Weak positive Nav1.8-LI was also present in some LTM units including a C LTM, all Adelta LTM units (D hair), about 10% of cutaneous LTM Aalpha/beta-units, but no muscle spindle afferent units. Nav1.8-LI intensity was negatively correlated with soma size (all neurons) and with dorsal root CVs in A- but not C-fibre neurons. Nav1.8-LI intensity was positively correlated with action potential (AP) duration (both rise and fall time) in A-fibre neurons and with AP rise time only in positive C-fibre neurons. It was also positively correlated with AP overshoot in positive neurons. Thus high levels of Nav1.8 protein may contribute to the longer AP durations (especially in A-fibre neurons) and larger AP overshoots that are typical of nociceptors.

Persistent tetrodotoxin-resistant (TTX-r) sodium currents up-regulated by intracellular GTP have been invoked as the site of action of peripheral inflammatory mediators that lower pain thresholds, and ascribed to the Na(V)1.9 sodium channel. Here we describe the properties of a global knock-out of Na(V)1.9 produced by replacing exons 4 and 5 in SCN11A with a neomycin resistance cassette, deleting the domain 1 voltage sensor and introducing a frameshift mutation. Recordings from small (< 25 microm apparent diameter) sensory neurones indicated that channel loss eliminates a TTX-r persistent current. Intracellular dialysis of GTP-gamma-S did not cause an up-regulation of persistent Na(+) current in Na(V)1.9-null neurones and the concomitant negative shift in voltage-threshold seen in wild-type and heterozygous neurones. Heterologous hNa(V)1.9 expression in Na(V)1.9 knock-out sensory neurones confirms that the human clone can restore the persistent Na(+) current. Taken together, these findings demonstrate that Na(V)1.9 underlies the G-protein pathway-regulated TTX-r persistent Na(+) current in small diameter sensory neurones that may drive spontaneous discharge in nociceptive nerve fibres during inflammation.

The article by Mironov et al. (2009) contained an error in Fig. 7B. Figure 7 should have appeared as follows. It should be noted that the error does not alter results or discussion in the text.

Degenerins have emerged from genetic studies in Caenorhabditis elegans as candidate mechanically gated amiloride-sensitive ion channels for transducing mechanical stimuli into cellular responses. In C. elegans muscle, the existence of a genetic interaction between the unc-105 degenerin gene and let-2, a gene encoding an alpha2(IV) collagen, raised the possibility that UNC-105 may function as a mechanically gated channel in a stretch receptor complex. However, to date, ion channel activity of UNC-105 has only been recorded in a gain-of-function mutant form in heterologous expression systems. In this study we investigated the in situ properties of UNC-105 using the whole cell configuration of the patch clamp technique on body wall muscle cells from acutely dissected C. elegans. Amiloride was found to be without effect on membrane potential of wild-type muscle cells, suggesting that the UNC-105 degenerin is electrically silent in resting muscle. Hypo-osmotic shocks induced a reversible depolarization of muscle cells but which was not affected by amiloride. Deformation of the cells by applying tension to the filamentous complex on which muscle cells remained attached or by ejecting external solution under pressure failed to induce any change of membrane potential. In gain-of-function unc-105(n506) mutant cells, an amiloride-sensitive inward Na(+) current was found to be constitutively active, leading to maintained muscle depolarization. An associated mutation in the alpha2(IV) collagen LET-2 led to the closure of the mutant UNC-105(n506) channel while a collagenase treatment of these double mutant cells caused it to re-open, giving evidence for a functional interaction between LET-2 collagen and mutant UNC-105 channel.

1. External Ca2+ block of Na+ channels was studied by a gigaohm-seal patch clamp technique in single cardiac ventricular cells from guinea-pig. Single-channel currents were recorded from cell-attached patches. 2. Increasing external Ca2+ concentrations in the patch pipette from 0.1 to 20 mM reduced the single-channel conductance of normal Na+ channels from 27 to 14 pS without causing flickering (obtained from linear regression, eight patches). 3. Exposed to external Ca2+ concentrations of 20 mM, the single-channel currents decreased at potentials negative to -60 mV in spite of an increased driving force for inward Na+ currents. 4. An external concentration of 35 mM-Mg2+, which is supposed to exert a screening of surface charges nearly equal to that of 20 mM-Ca2+ (Hille, Woodhull & Shapiro, 1975), reduced the single-Na+-channel conductance only from 26 (1 mM-Mg2+) to 20 pS (linear regression, eight patches). A weaker voltage-dependent block at potentials negative to -50 mV was observed in 35 mM-Mg2+ than in 20 mM-Ca2+. Therefore, surface charge effects cannot explain the obvious reduction of the conductance of single Na+ channels found when the external Ca2+ concentration was increased. 5. Single Na+-channel currents increased with an increase in the external Na+ concentration [( Na+]o) but showed saturation. The Na+o-single-channel current relationship could be described by i = imax/(1 + kd/[Na+]o) with imax = 5.4 pA and kd = 359 mM (seventeen patches). 6. The mean open time of Na+ channels varied between 0.18 and 0.59 ms (potentials between -80 and -20 mV). No significant changes in the mean open time could be obtained when Ca2+ was varied between 0.1 and 20 mM. 7. The piperazinylindole compound DPI 201-106 was used as a tool to prolong the open time of single Na+ channels. If the external Ca2+ concentration was increased from 0.1 to 20 mM the currents through the modified channels were reduced. The reduction of single-channel currents was accentuated at potentials negative to -60 mV (20 mM-Ca2+) similar to the control channels. 8. In contrast to non-modified Na+ channels, the mean open time of DPI 201-106-modified channels proved extremely voltage and Ca2+ dependent.(ABSTRACT TRUNCATED AT 400 WORDS)

One of the most important features of prolonged weightlessness is a progressive impairment of muscular function with a consequent decrease in exercise capacity. We tested the hypothesis that the impairment in musculo-skeletal function that occurs in microgravity results in a potentiation of the muscle metaboreflex mechanism and also affects baroreflex modulation of heart rate (HR) during exercise. Four astronauts participating in the 16 day Columbia shuttle mission (STS-107) were studied 72-71 days before launch and on days 12-13 in-flight. The protocol consisted of 6 min bicycle exercise at 50% of individual V(o2,max) followed by 4 min of postexercise leg circulatory occlusion (PECO). At rest, systolic (S) and diastolic (D) blood pressure (BP), R-R interval and baroreflex sensitivity (BRS) did not differ significantly between pre- and in-flight measurements. Both pre- and in-flight, SBP increased and R-R interval and BRS decreased during exercise, whereas DBP did not change. During PECO preflight, SBP and DBP were higher than at rest, whereas R-R interval and BRS recovered to resting levels. During PECO in-flight, SBP and DBP were significantly higher whereas R-R interval and BRS remained significantly lower than at rest. The part of the SBP response (delta) that was maintained by PECO was significantly greater during spaceflight than before (34.5 +/- 8.8 versus 13.8 +/- 11.9 mmHg, P = 0.03). The tachycardic response to PECO was also significantly greater during spaceflight than preflight (-141.5 +/- 25.2 versus - 90.5 +/- 33.3 ms, P = 0.02). This study suggests that the muscle metaboreflex is enhanced during dynamic exercise in space and that the potentiation of the muscle metaboreflex affects the vagally mediated arterial baroreflex contribution to HR control.

1. Observations were made upon five subjects who flew through 4(1/2)-6 time zones, four of them returning later to their starting point, and upon twenty-three subjects experiencing simulated 6 or 8 hr time zones shifts in either direction in an isolation unit.2. Measurements were made of plasma concentration of 11-hydroxycorticosteroids, of body temperature, and of urinary excretion of sodium, potassium and chloride. Their rhythm was defined, where possible, by fitting a sine curve of period 24 hr to each separate 24-hr stretch of data and computing the acrophase, or maximum predicted by the sine curve.3. The adaptation of the plasma steroid rhythm was assessed by the presence of a sharp fall in concentration after the sample collected around 08.00 hr. The time course of adaptation varied widely between individuals; it was usually largely complete by the fourth day after westward, and rather later after eastward, flights. After time shift the pattern often corresponded neither to an adapted nor to an unadapted one, and in a subject followed for many months after a real flight a normal amplitude only appeared 2-3 months after flight.4. Temperature rhythm adapted by a movement of the acrophase, without change in amplitude, although on some days no rhythm could be observed. This movement was always substantial even on the first day, and was usually nearly complete by the fifth.5. High nocturnal excretion of electrolyte was often seen in the early days after time shift, more notably after simulated westward flights. Adaptation of urinary electrolyte rhythms usually proceeded as with temperature, but the movement of the acrophase was slower, more variable between individuals, more erratic, and sometimes reversed after partial adaptation. On a few days there were two maxima corresponding to those expected on real and on experimental time.6. Sodium excretion was much less regular than that of potassium, but adapted more rapidly to time shift, so that the two often became completely dissociated. Chloride behaved much as sodium.7. The time course of adaptation of the plasma steroid and urinary potassium rhythms were sufficiently similar to suggest a causal connexion. The time course of adaptation of the temperature rhythm did not coincide with that of any other component considered here.

1. Microinjection of selective antibodies into superior cervical ganglion (SCG) neurones has identified the G-protein alpha-subunits mediating muscarinic receptor inhibition of M-type K+ current (IK(M)) and alpha-adrenoceptor inhibition of Ca2+ current (ICa). 2. Antibodies specific for G alpha q/11, but not those for G alpha o, reduced M-current inhibition by the muscarinic agonist oxotremorine-M, whereas anti-G alpha o antibodies, but not anti-G alpha q/11 or anti-G alpha i1-3 antibodies, reduced calcium current inhibition by noradrenaline. 3. Immunoblots with specific anti-G-protein antibodies demonstrated the presence of both G alpha q and G alpha 11, while G alpha o1 (but virtually no G alpha o2) was present. 4. We conclude that M1 muscarinic receptor inhibition of IK(M) is transduced by G alpha q and/or G alpha 11, and that G alpha o transduces alpha-adrenoceptor inhibition of ICa.

Dark noise, light-induced noise and responses to brief flashes of light were recorded in the membrane current of isolated rods from larval tiger salamander retina before and after bleaching most of the native visual pigment, which mainly has the 11-cis-3,4-dehydroretinal (A2) chromophore, and regenerating with the 11-cis-retinal (A1) chromophore in the same isolated rods. The purpose was to test the hypothesis that blue-shifting the pigment by switching from A2 to A1 will decrease the rate of spontaneous thermal activations and thus intrinsic light-like noise in the rod. Complete recordings were obtained in five cells (21 degrees C). Based on the wavelength of maximum absorbance, lambda max,A1 = 502 nm and lambda max,A2 = 528 nm, the average A2 : A1 ratio determined from rod spectral sensitivities and absorbances was approximately 0.74 : 0.26 in the native state and approximately 0.09 : 0.91 in the final state. In the native (A2) state, the single-quantum response (SQR) had an amplitude of 0.41 +/- 0.03 pA and an integration time of 3.16 +/- 0.15 s (mean +/- s.e.m.). The low-frequency branch of the dark noise power spectrum was consistent with discrete SQR-like events occurring at a rate of 0.238 +/- 0.026 rod(-1) s(-1). The corresponding values in the final state were 0.57 +/- 0.07 pA (SQR amplitude), 3.47 +/- 0.26 s (SQR integration time), and 0.030 +/- 0.006 rod(-1) s(-1) (rate of dark events). Thus the rate of dark events per rod and the fraction of A2 pigment both changed by ca 8-fold between the native and final states, indicating that the dark events originated mainly in A2 molecules even in the final state. By extrapolating the linear relation between event rates and A2 fraction to 0% A2 (100% A1) and 100% A2 (0% A1), we estimated that the A1 pigment is at least 36 times more stable than the A2 pigment. The noise component attributed to discrete dark events accounted for 73% of the total dark current variance in the native (A2) state and 46% in the final state. The power spectrum of the remaining 'continuous' noise component did not differ between the two states. The smaller and faster SQR in the native (A2) state is consistent with the idea that the rod behaves as if light-adapted by dark events that occur at a rate of nearly one per integration time. Both the decreased level of dark noise and the increased SQR amplitude must significantly improve the reliability of photon detection in dim light in the presence of the A1 chromophore compared to the native (A2) state in salamander rods.

1. The regulation of substance P (SP) responsiveness in acutely isolated nodose neurones from adult guinea-pigs was investigated using standard intracellular recording techniques. 2. In control neurones, SP produced no measurable electrophysiological effects. However, following incubation with serotonin (5-HT, 10 microM), 64% of neurones were depolarized by 10 +/- 0.6 mV (n = 84 of 132 neurones) by SP (100 nM). 5-HT-induced SP responses were inhibited by SR48968 (100 nM, n = 6), a neurokinin 2 (NK-2) receptor antagonist, but were unaffected by CP99,994 and SR142801, NK-1 and NK-3 receptor antagonists (n = 3 each), respectively. 3. 5-HT-induced unmasking of SP responses was maximal within 5 min. Increasing the 5-HT incubation time up to 120 min did not increase the mean response amplitude or the percentage of SP responsive neurones (P = 0.611 and 0.867, respectively). 4. 5-HT-induced unmasking of SP responses was dose dependent (EC50 = 14 nM). A 5-HT3 receptor agonist CPBG (1 microM), mimicked the unmasking effects of 5-HT (n = 10 of 19 neurones), while 5-CT (10 microM), a non-selective 5-HT agonist devoid of action at 5-HT3 receptors, did not (n = 18). ICS205-930 (1 microM), a 5-HT3 receptor antagonist, completely blocked the 5-HT-induced unmasking of SP responses (n = 10 of 10 neurones). 5. In 68% of the neurones tested, bath-applied 5-HT (10 microM) evoked a 178 +/- 29.5 nM increase in [Ca2+]i (n = 16), which was blocked by nominally zero [Ca2+]o (n = 4) or by ICS205-930 (1 microM, n = 4). Nodose neurones incubated with 5-HT in the presence of nominally zero [Ca2+]o did not respond to SP (n = 12 of 13 neurones) in Locke solution containing normal [Ca2+]o, indicating that the 5-HT-mediated elevation of [Ca2+]i is required for unmasking of SP responses. Calmidazolium (100 nM), a calmodulin inhibitor, inhibited the unmasking effects of 5-HT (n = 5 of 5 neurones). 6. Incubating neurones with the nitric oxide (NO) donors papaNONOate (1 mM, 15-30 min) or SNAP (50 microM, 30-60 min) unmasked depolarizing SP responses in 71% and 45% of the neurones studied, respectively. L-NMMA (30 microM), a NO synthase inhibitor, blocked 5-HT-induced unmasking of SP responses (n = 10 of 10 neurones). 7. In sum, these results suggest that stimulation of 5-HT3 receptors activates an intracellular signalling cascade that couples calcium-calmodulin and NO activation to NK-2 receptor unmasking in sensory neurones.

1. Whole-cell patch-clamp recordings of iontophoretically induced N-methyl-D-aspartate (NMDA) receptor-mediated currents (INMDA) in CA1 pyramidal cells in hippocampal slices from 1- to 40-day-old rats were used to characterize developmental changes in the Mg2+ sensitivity of NMDA receptors. 2. The dose-response relations for extracellular Mg2+ blockade of INMDA indicated a high affinity binding of Mg2+ to NMDA receptors at membrane potentials more negative than -60 mV, independent of postnatal age. 3. Depolarizing the cells unblocked NMDA receptors by decreasing their affinity for Mg2+. The efficacy of depolarization in unblocking NMDA receptors markedly increased after postnatal day 4 (P4), endowing the receptors with a greater voltage dependence. 4. The NR2B subunit-specific NMDA antagonist ifenprodil reduced INMDA in pyramidal cells of all ages. The sensitivity of INMDA to ifenprodil was greatest during the first postnatal week and decreased thereafter, indicating an enhanced contribution of NR2B subunit-containing NMDA receptors to INMDA in the first week after birth. 5. In the first postnatal week, the ifenprodil-insensitive INMDA component had a lower voltage dependence than the total INMDA. In older pyramidal cells, the voltage dependence of the ifenprodil-insensitive component and the total INMDA were similar. 6. In another set of CA1 pyramidal cells, single-cell reverse transcription and polymerase chain reaction (RT-PCR) were used to characterize concomitant developmental changes in NMDA subunit mRNA expression. The mRNA for the NR2D subunit was detected during the first postnatal week in 50 % of the cells and disappeared thereafter. The proportion of cells expressing the NR2A and NR2B subunits remained relatively constant throughout the first five postnatal weeks. 7. We conclude that NMDA receptors in hippocampal CA1 pyramidal cells are effectively blocked by Mg2+ at all ages. After 4 days they become much less sensitive to Mg2+ at depolarized membrane potentials. This postnatal switch in voltage control of Mg2+ binding to NMDA receptors may be due to the downregulation of NR2D subunit expression in developing CA1 pyramidal cells.

1. The mechanisms of manganese transport into erythroid cells were investigated using rabbit reticulocytes and mature erythrocytes and 54Mn-labelled MnCl2 and Mn2-transferrin. In some experiments iron uptake was also studied. 2. Three saturable manganese transport mechanisms were identified, two for Mn2+ (high and low affinity processes) and one for transferrin-bound manganese (Mn-Tf). 3. High affinity Mn2+ transport occurred in reticulocytes but not erythrocytes, was active only in low ionic strength media such as isotonic sucrose and had a Km of 0.4 microM. It was inhibited by metabolic inhibitors and several metal ions. 4. Low affinity Mn2+ transport occurred in erythrocytes as well as in reticulocytes and had Km values of approximately 20 and 50 microM for the two types of cells, respectively. The rate of Mn2+ transport was maximal in isotonic KCl, RbCl or CsCl, and was inhibited by NaCl and by amiloride, valinomycin, diethylstilboestrol and other ion transport inhibitors. The direction of Mn2+ transport was reversible, resulting in Mn2+ efflux from the cells. 5. The uptake of transferrin-bound manganese occurred only with reticulocytes and depended on receptor-mediated endocytosis of Mn-Tf. 6. The characteristics of the three saturable manganese transport mechanisms were similar to corresponding mechanisms of iron uptake by erythroid cells, suggesting that the two metals are transported by the same mechanisms. 7. It is proposed that high affinity manganese transport is a surface representation of the process responsible for the transport of manganese across the endosomal membrane after its release from transferrin. Low affinity transport probably occurs by the previously described Na(+)-Mg2+ antiport, and may function in the regulation of intracellular manganese concentration by exporting manganese from the cells.

1. The goal of this study was to examine the regulation of resistance in the large arteries and small vessels that supply the brain stem. 2. We used a new method in anaesthetized cats to measure blood flow to the medulla (microspheres) and pressure (servo-null) in branches of the basilar artery that supply the medulla. Resistance was determined during normocapnia, hypercapnia, hypocapnia and seizures (produced with intravenous bicuculline). 3. Pressure in arteries that supply the medulla (150 microns internal diameter) was 71 +/- 4% (mean +/- S.E. of mean) of aortic pressure and large artery resistance was 31 +/- 4% of the total resistance in the medulla. Hypercapnia and seizures decreased large artery resistance by 67 and 50%, respectively, and hypocapnia increased large artery resistance by 58%. Small vessel resistance decreased by 82% during hypercapnia and by 43% during seizures, and increased by 96% during hypocapnia. 4. Thus, resistance of the large arteries (greater than 150 microns diameter) accounts for about one-third of the total vascular resistance in the brain stem. Both large arteries and small vessels respond to alterations in arterial carbon dioxide tension and seizures, and contribute to the regulation of blood flow to the brain stem.

1. Whole-cell sodium currents (INa) were measured in mouse neuroblastoma cells (N1E-115) at different [Ca2+]i values using appropriate Ca-EGTA buffers in the pipettes. 2. INa was found to be larger at pCa 7 than at pCa 8 or 9 with a ratio of 1:0.65 or 0.55, respectively. The steady-state inactivation (h infinity curve) was independent of [Ca2+]i, thus excluding surface charge effects as a cause of the Ca2+ effect. 3. Recovery of INa from slow inactivation after changing from resting (-30 to -40 mV) to holding potential (-70 mV) occurred in a similar way at all pCa values. The Ca2+ effect appears to be independent of slow inactivation and to occur within the first 2 min of pipette buffer-cytoplasm equilibration. 4. The cell membrane capacitance (Cm) was independent of [Ca2+]i, thus excluding exo- or endocytosis of sodium channel-containing membrane as a cause of the Ca2+ effect. 5. Non-stationary fluctuation analysis was used to determine simultaneously the single channel current (iNa) and the size of INa. At pCa values of 7 and 9, iNa was identical, i.e. 0.59 and 0.58 pA, while INa/Cm differed, i.e. 41.1 and 22.2 pA pF-1, respectively. The peak open probability at 0 mV was about 0.5 for both pCa values indicating that [Ca2+]i controls the fraction of channels available for activation. 6. Since [Ca2+]i in other neurons varies between 30 and 100 nM in the resting and active state, respectively, the present data suggest a modulatory role for [Ca2+]i in neuronal excitability.

Two-pore (2-P) domain potassium channels are implicated in the control of the resting membrane potential, hormonal secretion, and the amplitude, frequency and duration of the action potential. These channels are strongly regulated by hormones and neurotransmitters. Little is known, however, about the mechanism underlying their regulation. Here we show that phosphatidylinositol 4,5-bisphosphate (PIP2) gating underlies several aspects of 2-P channel regulation. Our results demonstrate that all four 2-P channels tested, TASK1, TASK3, TREK1 and TRAAK are activated by PIP2. We show that mechanical stimulation may promote PIP2 activation of TRAAK channels. For TREK1, TASK1 and TASK3 channels, PIP2 hydrolysis underlies inhibition by several agonists. The kinetics of inhibition by the PIP2 scavenger polylysine, and the inhibition by the phosphatidylinositol 4-kinase inhibitor wortmannin correlated with the level of agonist-induced inhibition. This finding suggests that the strength of channel PIP2 interactions determines the extent of PLC-induced inhibition. Finally, we show that PIP2 hydrolysis modulates voltage dependence of TREK1 channels and the unrelated voltage-dependent KCNQ1 channels. Our results suggest that PIP2 is a common gating molecule for K+ channel families despite their distinct structures and physiological properties.

1. The superficial layers II and III of the entorhinal cortex, which form the main cortical input to the hippocampus, receive a large serotonergic projection from the raphe nuclei and express 5-HT receptors at high density. Here, we studied the effects of serotonin on the intrinsic properties and excitatory synaptic transmission of the superficial medial entorhinal cortex. 2. Intracellular and patch clamp recordings revealed that serotonin hyperpolarized only one-third of the cells, approximately, through a potassium conductance via a GTP-dependent process. 3. Serotonin depressed mixed as well as isolated alpha-amino-3-hydroxy-5-methyl-4-isoxazole- propionic acid receptor (AMPAR)- and N-methyl-D-aspartic acid receptor (NMDAR)-mediated excitatory postsynaptic potentials/currents (EPSPs/EPSCsapproximately 40 % reduction with 1 microM serotonin). 4. The effect of serotonin on EPSPs/EPSCs was similar in whole-cell versus intracellular recordings; it did not require intracellular GTP and was not visible in glutamate applications to excised patches. Miniature EPSCs recorded in the presence of tetrodotoxin and bicuculline were reduced in frequency, but not altered in amplitude. 5. The effects of serotonin on intrinsic properties and EPSPs were partially mimicked by 5-HT1A receptor agonists (+/-)-8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide (8-OH-DPAT) and 5-carboxamido-tryptamine maleate (5-CT), and reduced by 5-HT1A receptor antagonists S-(-)-5-fluoro-8-hydroxy-DPAT hydrochloride (S-UH-301), 1-(2-methoxyphenyl)-4-[4-(2-phthalimido)butyl]piperazine hydrobromide (NAN-190) and spiperone. 6. We conclude that serotonin potently suppresses excitatory synaptic transmission via 5-HT1A receptors in layers II and III of the medial entorhinal cortex by a presynaptic mechanism.