-
[show abstract]
[hide abstract]
ABSTRACT: Electroretinography (ERG) is widely used in clinical work and research to
assess the retinal function. We evaluated an easy to build ERG setup adapted
for small animals comprising two contact lens electrodes with a built-in
light-emitting diode and a custom-made amplification system. The system’s
sensitivity was tested by monitoring ERG in albino rat eyes subjected to
mild ischemia. Flash ERG was recorded by two contact lens electrodes
positioned on the rat’s corneas and used alternately as test or reference.
The a- and b-wave amplitudes, a-wave latency, b-wave implicit time and
oscillatory potentials (OPs) were analyzed. Ischemia was achieved by elevating
the intraocular pressure in the eye’s anterior chamber. ERG was recorded on
post-ischemia (PI) days −1, 1, 3 and 7. Morphological changes were analyzed
on hematoxylin/eosin stained 5 μm sections of control 7d PI retinas. In
control eyes, ERG exhibited a pattern similar to a standard recording. Retinas
subjected to mild ischemia preserved ordered layered morphology, exhibiting
approximately 30% loss of ganglion cells and no changes in gross morphology.
By day 3 PI, ischemia caused an increase in the a-wave amplitude (from
34.9 ± 2.7 to 45.4 ± 4.3μV), a decrease in the b-wave amplitude (from 248 ±
13 to 162 ± 8 μV), an increase in a-wave latency (from 11.1 ± 0.3 to
17.3 ± 1.4 ms) and b-wave implicit time (from 81.0 ± 1.6 to 90.0 ± 2.5 ms),
and attenuation of OPs. The described setup proved sensitive and reliable for
evaluating subtle changes in the retinal function in small animals.
Physiological Measurement 06/2012; 33236. · 1.68 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Electroretinography (ERG) is widely used in clinical work and research to assess the retinal function. We evaluated an easy to build ERG setup adapted for small animals comprising two contact lens electrodes with a built-in light-emitting diode and a custom-made amplification system. The system's sensitivity was tested by monitoring ERG in albino rat eyes subjected to mild ischemia. Flash ERG was recorded by two contact lens electrodes positioned on the rat's corneas and used alternately as test or reference. The a- and b-wave amplitudes, a-wave latency, b-wave implicit time and oscillatory potentials (OPs) were analyzed. Ischemia was achieved by elevating the intraocular pressure in the eye's anterior chamber. ERG was recorded on post-ischemia (PI) days -1, 1, 3 and 7. Morphological changes were analyzed on hematoxylin/eosin stained 5 µm sections of control 7d PI retinas. In control eyes, ERG exhibited a pattern similar to a standard recording. Retinas subjected to mild ischemia preserved ordered layered morphology, exhibiting approximately 30% loss of ganglion cells and no changes in gross morphology. By day 3 PI, ischemia caused an increase in the a-wave amplitude (from 34.9 ± 2.7 to 45.4 ± 4.3 µV), a decrease in the b-wave amplitude (from 248 ± 13 to 162 ± 8 µV), an increase in a-wave latency (from 11.1 ± 0.3 to 17.3 ± 1.4 ms) and b-wave implicit time (from 81.0 ± 1.6 to 90.0 ± 2.5 ms), and attenuation of OPs. The described setup proved sensitive and reliable for evaluating subtle changes in the retinal function in small animals.
Physiological Measurement 05/2012; 33(6):1053-2. · 1.68 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Parthenogenetic agents that evoke cytosolic calcium concentration ([Ca2+]i) oscillations similar to those evoked by sperm, mimic fertilization more faithfully than agents that trigger a single [Ca2+]i transient. Strontium chloride (SrCl2) binds to and activates the Ca2+-binding site on the inositol 1,4,5-trisphosphate receptor and evokes [Ca2+]i oscillations. Although SrCl2 has been reported to activate mouse eggs, little is known regarding the pattern of the [Ca2+]i oscillations it evokes in rat eggs and their effect on the early events of egg activation: cortical granule exocytosis (CGE) and completion of meiosis (CM). In the current study we investigated the effect of various concentrations of SrCl2 (2, 4 or 6 mM) on [Ca2+]i, by monitoring [Ca2+]i oscillations in fura-2-loaded rat eggs. Treatment with 2 mM SrCl2 was optimal for inducing the first [Ca2+]i transient, which was similar in duration to that triggered by sperm. However, the frequency and duration of the subsequent [Ca2+]i oscillations were lower and longer in SrCl2-activated than in sperm-activated eggs. The degree of CGE was identical in eggs activated by either sperm or SrCl2, as assessed by semi-quantitative immunohistochemistry combined with confocal microscopy. Evoking 1, 2 or 10 [Ca2+]i oscillations (8, 15 or 60 min in SrCl2 respectively) had no effect on the intensity of fluorescent CGE reporter dyes, while 60-min exposure to SrCl2 caused a delay in CM. Our results demonstrate that SrCl2 is an effective parthenogenetic agent that mimics rat egg activation by sperm, as judged by the generation of [Ca2+]i oscillations, CGE and CM.
Reproduction (Cambridge, England) 11/2005; 130(4):467-74. · 3.09 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Recently, we demonstrated the feasibility of a chemical synthetic lethality screen in cultured human cells. We now demonstrate the principles for a genetic synthetic lethality screen. The technology employs both an immortalized human cell line deficient in the gene of interest, which is complemented by an episomal survival plasmid expressing the wild-type cDNA for the gene of interest, and the use of a novel GFP-based double-label fluorescence system. Dominant negative genetic suppressor elements (GSEs) are selected from an episomal library expressing short truncated sense and antisense cDNAs for a gene likely to be synthetic lethal with the gene of interest. Expression of these GSEs prevents spontaneous loss of the GFP-marked episomal survival plasmid, thus allowing FACS enrichment for cells retaining the survival plasmid (and the GSEs). The dominant negative nature of the GSEs was validated by the decreased resident enzymatic activity present in cells harboring the GSEs. Also, cells mutated in the gene of interest exhibit reduced survival upon GSE expression. The identification of synthetic lethal genes described here can shed light on functional genetic interactions between genes involved in normal cell metabolism and in disease.
Nucleic Acids Research 11/2001; 29(20):E100. · 8.03 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We coexpressed Kaposi's sarcoma-associated herpesvirus G protein-coupled receptors (KSHV-GPCRs) with thyrotropin-releasing hormone (TRH) receptors or m1-muscarinic-cholinergic receptors in Xenopus oocytes and in mammalian cells. In oocytes, KSHV-GPCR expression resulted in pronounced (81%) inhibition (heterologous desensitization) of Ca(2+)-activated chloride current responses to TRH and acetylcholine. Similar inhibitions of cytoplasmic free Ca(2+) responses to TRH were observed in human embryonic kidney HEK 293 EM cells and in mouse pituitary AtT20 cells. Further study of oocytes showed that this inhibition was partially reversed by interferon-gamma-inducible protein 10 (IP-10), an inverse agonist of KSHV-GPCR. The basal rate of (45)Ca(2+) efflux in oocytes expressing KSHV-GPCRs was 4.4 times greater than in control oocytes, and IP-10 rapidly inhibited increased (45)Ca(2+) efflux. In the absence of IP-10, growth-related oncogene alpha caused a further 2-fold increase in (45)Ca(2+) efflux. In KSHV-GPCR-expressing oocytes, responses to microinjected inositol 1,4,5-trisphosphate were inhibited by 74%, and this effect was partially reversed by interferon-gamma-inducible protein 10. Treatment with thapsigargin suggested that the pool of calcium available for mobilization by TRH was decreased in oocytes coexpressing KSHV-GPCRs. These results suggest that constitutive signaling by KSHV-GPCR causes heterologous desensitization of responses mediated by other receptors, which signal via the phosphoinositide/calcium pathway, which is caused by depletion of intracellular calcium pools.
Journal of Biological Chemistry 04/2001; 276(10):7122-8. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The synthetic lethality screen is a powerful genetic method for unraveling functional interactions between proteins in yeast. Here we demonstrate the feasibility of a chemical synthetic lethality screen in cultured human cells, based in part on the concept of the yeast method. The technology employs both an immortalized human cell line, deficient in a gene of interest, which is complemented by an episomal survival plasmid expressing the gene of interest, and the use of a novel double-label fluorescence system. Selective pressure imposed by any one of several synthetic lethal metabolic inhibitors prevented the spontaneous loss of the episomal survival plasmid. Retention or loss over time of this plasmid could be sensitively detected in a blind test, while cells were grown in microtiter plates. Application of this method should thus permit high throughput screening of drugs, which are synthetically lethal with any mutant human gene of interest, whose normal counterpart can be expressed. This usage is particularly attractive for the search of drugs, which kill malignant cells in a gene-specific manner, based on their predetermined cellular genotype. Moreover, by replacing the chemicals used in this example with a library of either DNA oligonucleotides or expressible dominant negative genetic elements, one should be able to identify synthetic lethal human genes.
Genome Research 03/2001; 11(2):266-73. · 13.61 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Occlusion has previously been used to treat psoriatic plaques and was shown to improve the condition. We investigated the consequences of applying a mechanical stress, in vitro, on the HaCaT keratinocyte cell line. A mechanical load applied to cells can be mimicked by a hyperosmotic stimulus. Exposure of HaCaT keratinocytes to different hyperosmotic solutions (final osmolarity in the range 350-600 mOsm, produced by sucrose addition) resulted in an inhibition of cell proliferation after 96 h of treatment. As keratinocyte maturation is regulated by calcium levels, we measured hyperosmotic-stimulus-induced changes of intracellular calcium ([Ca2+]i) by single-cell image analysis employing FURA-2/AM. The hyperosmotic stimulus produced a rapid transient 2.6-fold elevation of [Ca2+]i followed by a gradual decay to the basal level. The transients originated from extracellular as well as from intracellular calcium pools and did not respond to voltage-sensitive calcium channel blockers. The hyperosmotic stimulus was shown to increase the cellular expression of involucrin, a differentiation marker, following 72 h of incubation, as measured by flow cytometry. Treatment of cells with the [Ca2+]i chelator BAPTA/AM almost completely blocked the [Ca2+]i elevation, but did not alter cellular growth or the induction of differentiation observed after hyperosmotic stimulus. It is suggested that treatment of keratinocytes with hyperosmotic stimulus can induce short-time effects (calcium transients) as well as long-term cellular maturation.
Journal of Investigative Dermatology 11/2000; 115(4):714-8. · 6.31 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We studied rapid desensitization of the thyrotropin-releasing hormone receptor (TRH-R) or the m1-muscarinic receptor (m1-R) to a short challenge of threshold TRH concentration and persistent desensitization due to constitutive activity of a mutant TRH-R. Xenopus oocytes expressing TRH-Rs and/or m1-Rs were challenged for 15 s with threshold concentrations of TRH ([TRH]) and then immediately with supraoptimal [TRH] or acetylcholine ([ACh]). The threshold challenge caused desensitization of 50 - 57% of responses to subsequent supraoptimal stimulation with TRH or ACh. The homologous desensitization was reversible within 60 s after removal of the agonist. The protein kinase C (PKC) inhibitor, chelerythrine, inhibited the control responses by 30 - 40%, without affecting the desensitized responses. Chelerythrine or the phosphatase inhibitor, okadaic acid, had little effect on the kinetics of resensitization, indicating limited involvement of PKC. In oocytes coexpressing wild type TRH-Rs or m1-Rs with a constitutively active TRH-R mutant (C335Stop TRH-R), a persistent desensitization (33 - 57%) of the responses to TRH or ACh was observed. Additionally, there was a complete loss of the rapid desensitization induced by threshold [TRH]. Chlorodiazepoxide (CDE), a competitive binding antagonist of TRH-Rs and an inverse agonist of C335Stop TRH-Rs, abolished the persistent desensitization induced by C335Stop TRH-Rs and enabled the rapid desensitization, conferring the wild type phenotype on C335Stop TRH-Rs. Chelerythrine had qualitatively the same effect as CDE. In conclusion, unlike the rapid desensitization, the persistent desensitization caused by the constitutively active C335Stop TRH-Rs is largely mediated by PKC. It abrogates, however, the rapid desensitization, suggesting a common mechanistic step(s).
British Journal of Pharmacology 06/2000; 130(2):315-20. · 4.41 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: To investigate the role of endogenously generated nitric oxide (NO) in the relaxation of bovine iris sphincter.
Isolated bovine sphincters were mounted on an isometric tension apparatus. Contraction-relaxation response was elicited by electrical field stimulation (ES; 12 Hz, 50-msec duration, 70-80 V). Relaxation was arbitrarily defined as maximal decrease of tension below prestimulation baseline after cessation of ES. We also determined the tissue levels of cyclic guanosine monophosphate (cGMP) by radioimmunoassay.
ES produced a biphasic response: contraction followed by relaxation. After cessation of ES, the muscle relaxed to below the initial baseline tension. Tetrodotoxin (TTX) abolished most of the contraction and all the relaxation response. Atropine blocked most of the contraction component, leaving the relaxation component unchanged. Prazosin and bupranolol (alpha1-adrenergic and beta-adrenergic antagonists, respectively) also did not affect the relaxation component of the response. Neither substance P nor its antagonist (N-acetyl-L-tryptophane 3,5-bis (trifluoromethyl)-benzyl ester; ATTB) inhibited or mimicked the response. The nitric oxide synthase (NOS) inhibitors Nomega-nitro-L-arginine methyl ester (L-NAME), Nomega-nitro-L-arginine (L-NNA), and aminoguanidine dose-dependently inhibited the relaxation response by 50% to 70%. The free radical scavenger 2-(4-carboxyphenyl) 4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (carboxy-PTIO) and the guanylyl cyclase inhibitor methylene blue also abrogated 70% and 45% of the relaxation response, respectively. ES caused an increase in muscle cGMP from 2.3+/-0.3 to 3.9+/-0.5 picomoles per muscle. L-NNA or L-NAME significantly decreased the tissue cGMP content (to 1.2+/-0.1 picomoles per muscle) and prevented the increase caused by ES.
The relaxation component of the iris sphincter response to ES is a distinct nonadrenergic, noncholinergic, ES-induced event. Most of the relaxation is mediated by the endogenously generated NO-guanylyl cyclase-cGMP cascade.
Investigative Ophthalmology & Visual Science 04/2000; 41(3):880-6. · 3.60 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The divalent cation requirements of NOS activity in bovine retina homogenate supernatant were investigated. Supernatants were assayed under standard conditions (in mM: EDTA 0.45, Ca2+ 0.25, Mg2+ 4.0). In order to investigate the enzyme's dependence on divalent cations, the tissue homogenate was depleted of di- and trivalent cations by passing it over a cation-exchange column (Chelex 100). Surprisingly, NOS activity was 50-100% higher in this preparation. However, addition of either EDTA (33 microM) or EGTA (1 mM) almost fully inhibited NOS activity, suggesting a requirement for residual divalent metal cation(s). Phenanthroline or iminodiacetic acid at low concentrations had little effect on activity, suggesting no requirement for Fe2+, Zn2+ or Cu2+. Ca2+ had a moderate stimulatory effect, with an optimum activity around 0.01 mM. Mg2+ or Mn2+ had little effect at concentrations < 0.25 mM. However, in the presence of EDTA, Mn2+ or Ca2+ markedly stimulated NOS activity with the optimum at 0.1 mM. At high concentrations (> 0.1-0.2 mM), all divalent cations tested (Ba2+, Zn2+, Co2+, Mn2+, Mg2+, Ca2+), as well as La3+, dose-dependently inhibited NOS activity. We propose that retinal NOS requires low concentrations of naturally occurring divalent metal ions, most probably Ca2+, for optimal activity and is inhibited by high di- and trivalent metal concentrations, probably by competition with Ca2+.
Molecular and Cellular Biochemistry 01/2000; 204(1-2):11-6. · 2.06 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 1. C335Stop is a constitutively active mutant of the TRH receptor (TRH-R). To investigate the mechanism of the decreased responsiveness of C335Stop TRH-R, we studied cellular Ca2+ concentrations ([Ca2+]i) in AtT20 cells stably transfected with C335Stop TRH-R cDNA, or Ca2+-activated chloride currents in Xenopus laevis oocytes expressing this mutant receptor after injection of cRNA. The competitive TRH-R binding antagonist, chlorodiazepoxide (CDE), was used as an inverse agonist to study the contribution of constitutive activity to desensitization. 2. Acute treatment with CDE resulted in a rapid (within minutes) decrease in [Ca2+]i and an increase in the response amplitude to TRH with no measurable change in receptor density. Conversely, removal of chronically administered CDE caused a rapid increase in [Ca2+]i and a decrease in TRH response amplitude. 3. CDE abolished heterologous desensitization induced by C335Stop TRH-R on muscarinic m1-receptor (ml-R) co-expressed in Xenopus oocytes. 4. Chelation of extracellular calcium with EGTA caused a rapid decrease in [Ca2+]i and a concomitant increase in the response to TRH in AtT20 cells expressing C335Stop TRH-Rs. 5. Chelerythrine, a specific inhibitor of protein kinase C (PKC), reversed the heterologous desensitization of the response to acetylcholine (ACh). The phosphoserine/phosphothreonine phosphatase inhibitor, okadaic acid, abolished the effect of chelerythrine. 6. Down-regulation of PKC by chronic exposure to phorbol 12-myristate 13-acetate (PMA) or acute inhibition with chelerythrine caused a partial resensitization of the response to TRH. 7. Western analysis indicated that the alpha subtype of protein kinase C was down-regulated in cells expressing C335Stop TRH-Rs. Following a 5 min exposure to PMA, the residual alphaPKC translocated to the particular fraction. 8. We propose that cells expressing the constitutively active mutant TRH-R rapidly desensitize their response, utilizing a mechanism mediated by an increase in [Ca2+]i and PKC.
British Journal of Pharmacology 04/1999; 126(5):1097-106. · 4.41 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The mongoose is resistant to snake neurotoxins. The mongoose muscle nicotinic acetylcholine receptor (AChR) alpha-subunit contains a number of mutations in the ligand-binding domain and exhibits poor binding of alpha-bungarotoxin (alpha-BTX). We characterized the functional properties of a hybrid (alpha-mongoose/beta gamma delta-rat) AChR. Hybrid AChRs, expressed in Xenopus oocytes, respond to acetylcholine with depolarizing current, the mean maximal amplitude of which was greater than that mediated by the rat AChR. The IC50 of alpha-BTX to the hybrid AChR was 200-fold greater than that of the rat, suggesting much lower affinity for the toxin. Hybrid AChRs exhibited an apparent higher rate of desensitization and higher affinity for ACh (EC50 1.3 vs. 23.3 microM for the rat AChR). Hence, changes in the ligand-binding domain of AChR not only affect the binding properties of the receptor, but also result in marked changes in the characteristics of the current.
FEBS Letters 08/1998; 431(3):411-4. · 3.54 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Xenopus oocytes respond to trypsin with a characteristic chloride current, virtually indistinguishable from responses mediated by a large number of native and expressed G protein-coupled receptors. We studied the involvement of G proteins of the Galphaq family as possible mediators of this and other G protein-coupled receptor-mediated responses in Xenopus oocytes. We have cloned the third member of the Galphaq family, Xenopus Galpha14, in addition to the previously cloned Xenopus Galphaq and Galpha11 (Shapira, H., Way, J., Lipinsky, D., Oron, Y., and Battey, J. F. (1994) FEBS Lett. 348, 89-92). Amphibian Galpha14 is 354 amino acids long and is 93% identical to its mammalian counterpart. Based on the Galpha14 cDNA sequence, we designed a specific antisense DNA oligonucleotide (antiGalpha14) that, together with antiGalphaq and antiGalpha11, was used in antisense depletion experiments. 24 h after injection into oocytes, either antiGalphaq or antiGalpha14 reduced the response to 1 microg/ml trypsin by 70%, whereas antiGalpha11 had no effect. A mixture of antiGalphaq and antiGalpha14 virtually abolished the response. These data strongly suggest that Galphaq and Galpha14 are the exclusive mediators of the trypsin-evoked response in Xenopus oocytes. Similar experiments with the expressed gastrin-releasing peptide receptor and muscarinic m1 receptor revealed the coupling of Galphaq and Galpha11, but not Galpha14, to these receptors in oocytes. These results confirm the hypothesis that endogenous members of the Galphaq family discriminate among different native receptors in vivo.
Journal of Biological Chemistry 08/1998; 273(31):19431-6. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Bovine iris sphincter in vitro responded to beta-adrenergic stimulation with pronounced relaxation (EC50 of isoproterenol = 0.3 nM), which was potentiated by the cAMP phosphodiesterase inhibitor, isobutylmethylxanthine, and mimicked by the adenylyl cyclase activator, forskolin. The beta1/beta2 antagonist, propranolol, exhibited low potency with calculated Ki of 200 nM. The beta3-selective antagonist, bupranolol, exhibited a biphasic inhibition profile, with calculated Kis of approximately 20-50 and 200-300 nM. The beta3-selective agonist, BRL 37344, elicited 70% of maximal relaxation (EC50 = 30 nM). When relaxation was induced by BRL 37344, bupranolol exhibited much higher potency (calculated Ki = 1 nM). Our data suggest that the beta-adrenergic relaxation response in bovine iris sphincter is mediated by a mixed population of beta-adrenergic receptors, with a predominant contribution of atypical, most likely beta3 subtype, receptors.
FEBS Letters 07/1998; 429(3):356-8. · 3.54 Impact Factor
-
Annals of the New York Academy of Sciences 06/1998; 841:97-100. · 3.15 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The mongoose AChR alpha-subunit has been cloned and shown to be highly homologous to other AChR alpha-subunits, with only six differences in amino acid residues at positions that are conserved in animal species that bind alpha-bungarotoxin (alpha-BTX). Four of these six substitutions cluster in the ligand binding site, and one of them, Asn-187, forms a consensus N-glycosylation site. The mongoose glycosylated alpha-subunit has a higher apparent molecular mass than that of the rat glycosylated alpha-subunit, probably resulting from the additional glycosylation at Asn-187 of the mongoose subunit. The in vitro translated mongoose alpha-subunit, in a glycosylated or non-glycosylated form, does not bind alpha-BTX, indicating that lack of alpha-BTX binding can be achieved also in the absence of glycosylation.
FEBS Letters 05/1998; 426(2):212-6. · 3.54 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Cartilage is exposed to mechanical loads, generating at the level of single chondrocytes a hyperosmotic stimulus (HOS). The direct effect of HOS on second messenger pathways in avian chondrocytes was evaluated by fluorimetric and image analysis techniques. HOS caused an immediate intracellular acidification of 0.07 +/- 0.02 pH units (n = 7), followed by an initial pH recovery rate of 0.033 +/- 0.04 pH units/min towards the pre-stimulus baseline values. Concomitantly, the intracellular calcium ([Ca2+]i) responded with a transient rise from baseline value of 84.7 +/- 7.4 nM to peak level of 403.1 +/- 51.0 nM (n = 16, p < 0.001). The calcium response was abolished by two calmodulin inhibitors chlorpromazine and W-7. Since these inhibitors are known to be specific ligands of a S-100 protein, its intracellular staining was determined following HOS. The amount of immunodetectable S-100 protein was significantly increased following exposure to HOS (p < 0.05), and did not require an increase of [Ca2+]i. It appears that compression of cartilage is transduced into HOS of chondrocytes, and further elicits its effects through transient intracellular elevation of protons and calcium ions accompanied by increased staining of S-100 protein.
Biochemical and Biophysical Research Communications 11/1996; 227(2):368-73. · 2.48 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: GTP gamma S1 binding experiments in a particulate preparation from Xenopus oocytes revealed two binding sites at pH = 6.9: A high affinity site (Kd = 77 +/- 4 nM) and a low affinity site (Kd = 8.74 +/- 0.05 microM). Alkaline pH (8.5) caused a significant increase in the dissociation constants of both sites (160 +/- 46 nM and 30.7 +/- 1.6 microM, respectively). In purified plasma membrane preparation, alkaline pH increased the rate of dissociation of GTP gamma S. We have previously proposed that the activation of a G-protein by the agonist-occupied receptor is rate-limiting in the kinetics of hormone-induced responses (Lipinsky et al., 1993; Pflugers Arch., 425:140-149). We have, therefore, assayed the latencies of responses evoked by TRH at different pH, in oocytes expressing the TRH receptor. A change in the medium pH was reflected by an approximately tenfold smaller change in cellular pH (pHi). Alkalinization of the medium (from pH 7.4 to 8.5) caused a shortening of latency (by 45%), whereas acidification to pH = 6.0 prolonged it (by 87%). Moreover, alkalinization decreased the latency and increased the rate of responses to microinjected GTP gamma S, but did not change the latency of responses to microinjected InsP3. These results show that activation of plasma membrane receptors coupled to G-proteins, concurrent with a change in pHi, can alter the kinetic pattern of physiological responses, thus affecting the ultimate physiological output of the cell. This finding suggests that a change of pH, is a novel potential mechanism for modulation of responses mediated by G-proteins.
Journal of Cellular Physiology 11/1996; 169(1):167-74. · 3.87 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Penetration of the oocyte by a spermatozoon is the first in the series of events resulting in the transition of the egg from a quiescent to a proliferative state. A critical regulatory role for intracellular calcium ([Ca2+]i) ion activity has been demonstrated in all species studied so far. On the other hand, it has been demonstrated that the intracellular pH (pHi) changes, but only in a small number of species. This change also has been proposed as one of the most important events in egg activation. The present study was undertaken to monitor pHi in rat eggs during fertilization, using the membrane-permeable indicator BCECF-AM and fluorescence ratio imaging. Furthermore, we proposed to evaluate the relationship between pHi and [Ca2+]i changes during egg activation. We found that the ovulated rat egg has a cytoplasmic pH significantly different from that of the follicular oocyte. Insemination with capacitated sperm resulted in a microscopically visible sperm attachment, yet no change in pHi was observed. Eggs double-loaded with fura-2-AM and BCECF-AM before insemination were used to measure [Ca2+]i and pHi simultaneously. Eggs with a normal pattern of [Ca2+]i transients (i.e., fertilized eggs) did not show any change in pHi at least for 30 min following sperm binding. Data for eggs fertilized in vivo were recorded at later times after sperm binding; these served to exclude the possibility of a transient change that occurs between sperm-egg interaction and the pronuclear stage. We conclude that the pHi of rat eggs does not change during fertilization and therefore that fertilization-induced [Ca2+]i changes do not affect pHi in these eggs.
Biology of Reproduction 09/1996; 55(2):461-8. · 4.01 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Agonist-induced calcium (Ca) mobilization is accompanied by Ca efflux, presumably reflecting the rise in Ca concentration at the cytosolic surface of the cell membrane. We studied the relationship between Ca efflux and intracellular Ca mobilization in Xenopus oocytes. Elevation of cytosolic Ca by a direct injection of 1 nmol 45CaCl2 resulted in a typical Ca-activated chloride current, but not in 45Ca efflux. This demonstrated that a Ca rise at the cytoplasmic surface of the membrane is not sufficient to produce an increased efflux. Co-injection of inositol 1,4,5-trisphosphate (InsP3), to prevent rapid Ca sequestration, also failed to cause Ca efflux. Smaller amounts of labelled Ca (0.05 nmol) equilibrated with Ca stores in a time-dependent pattern with an optimum at 2 h after injection. In contrast, Ca taken up from the medium was immediately available for agonist- or InsP3-induced efflux. Emptying the agonist-sensitive stores with thapsigargin (TG) did not affect chloride currents induced by Ca injection, indicating that these currents were due to direct elevation of Ca at the plasma membrane, rather than Ca-induced Ca release from InsP3-sensitive stores. Agonist-induced depletion of Ca stores enhanced uptake from the extracellular medium and the subsequent release of the label by an agonist. Similar protocol when the label was injected into the oocytes, failed to affect agonist induced efflux. We suggest that, under physiological conditions, agonist-dependent Ca extrusion or uptake in oocytes is executed exclusively via a functionally restricted compartment, which is closely associated with both agonist-sensitive Ca stores and the plasma membrane.
Cell Calcium 04/1996; 19(3):201-10. · 3.77 Impact Factor
