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Y Oron

Publications and source records attributed to Y Oron.

At least 37 records · Page 2Linked to original sources
PubMed1996

Intracellular pH of rat eggs is not affected by fertilization and the resulting calcium oscillations.

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.

Animals↗
PubMed1996

Evidence for contribution of effector organ cellular responses to the biphasic dynamics of heat acclimation.

The involvement of cellular processes in the biphasic dynamics of heat acclimation was studied. Key steps in the cholinergic signal transduction pathway for water secretion were measured in the submaxillary gland of acclimating [2-day short-term heat acclimation (STHA) and 30-day long-term heat acclimation (LTHA) at 34 degrees C] or acute heat-stressed (2 h at 40 degrees C) rats in vitro. Both the carbamylcholine (CCh)-induced maximal fractional rate and the total 86Rb+ efflux, reflecting K+ efflux and water transport, transiently decreased in STHA (P < 0.001). In LTHA, the total K+ efflux increased (P < 0.001), whereas the maximal fractional rate of efflux increased only slightly. During STHA, the density of the high-affinity binding site of the muscarinic receptors (MRs) increased by 50% and their affinity for the muscarinic antagonist [3H]-N-methylscopolamine decreased transiently by 87%. Basal cytosolic Ca2+ concentration ([Ca2+]i) decreased (P < 0.05), but the peak CCh-induced [Ca2+]i increase resembled the control values. In LTHA, MR density continued to increase (100%; P < 0.05), whereas affinity resumed control values. Basal and CCh-induced [Ca2+]i increases returned to control levels. We conclude that glandular cellular processes follow a biphasic pattern with major apparent changes attributable to events distal to the [Ca2+]i rise. This was further validated by employing heat stress, which produced qualitatively different effects on the MR profile with a decrease in 86Rb+ efflux comparable to STHA. Hence, although heat-induced changes in the proximal components of the signal transduction pathway may contribute to altered regulatory span, the predominant apparent cellular effect is on the distal part of the pathway.

Acclimatization↗
PubMed1995

Nitric oxide synthase inhibitors protect rat retina against ischemic injury.

Elevation of the ocular pressure in the anterior chamber of the rat eye caused major ischemic damage, manifested as changes in retinal morphology. The two most affected structures were the inner plexiform layer, which decreased in thickness by 90%, and the number of ganglion cells, which decreased by 80%. Pretreatment of the animals with N omega-nitro-L-arginine, a nitric oxide (NOS) inhibitor, almost completely abolished the ischemic damage. Administration of aminoguanidine, a NOS inhibitor selective for the inducible enzyme, partially abolished the ischemic damage. Moreover, administration of the NOS inhibitors 1 h after ischemia, also protected the retina from damage, suggesting that similarly acting drugs could be used clinically to limit ischemic injury in humans. We conclude that NOS, and therefore NO, may be involved in the mechanism of ischemic injury to the retina.

Animals↗
PubMed1995

Multiple effects of opiates on intracellular calcium level and on calcium uptake in three neuronal cell lines.

The present study examines the modulation by opiates of intracellular calcium levels and calcium entry, using fura-2 imaging and 45Ca2+ uptake, in three neuronal cell lines. We show that opiates (10(-7)-10(-5) M morphine and 10(-9)-10(-7) M etorphine) exert both inhibitory and excitatory effects on KCl-induced elevation in intracellular calcium level in SK-N-SH, NG108-15 and NMB cell lines. In addition, opiates elevate basal (non KCl-stimulated) intracellular calcium level in all three cell cultures. 45Ca2+ uptake is augmented by opiates in SK-N-SH cells and this stimulatory effect is not blocked by pertussis toxin. In NMB cells, an additional inhibitory effect of opiates on basal calcium takes place: opiates reduce intracellular calcium level as measured by fura-2, and decrease calcium influx as detected by 45Ca2+ uptake. The heterogeneity in the opioid regulation of calcium could not be attributed to the type of opioid drug, neither to its concentration nor to the experimental conditions, since neighboring cells within the same culture responded differently.

Calcium↗
PubMed1995

Hyperosmotic modulation of the cytosolic calcium concentration in a rat osteoblast-like cell line.

1. The effects of hyperosmotic stress on cytosolic calcium concentration ([Ca2+]i) were studied by ratio image analysis in single cells of an osteoblast-like bone cell line (RCJ 1.20) loaded with fura-2 AM. 2. The ratio (340 nm/380 nm) of steady-state [Ca2+]i in resting osteoblasts kept in Hepes-buffered medium was 0.82 +/- 0.04. A hyperosmotic stimulus (200 mosmol l-1 sucrose) produced a [Ca2+]i transient with a peak ratio of 1.28 +/- 0.09, which decayed with an apparent half-life (t1/2) of 42.7 +/- 2.6 s. 3. The hyperosmotically induced [Ca2+]i transients were insensitive to verapamil, diltiazem or nifedipine, which excludes the involvement of dihydropyridine-sensitive Ca2+ channels in the process. Non-specific Ca2+ channel blockers (Mn2+, Ni2+, La3+ or Gd3+) partially abolished the hyperosmotically induced [Ca2+]i elevation, indicating the contribution of extracellular Ca2+ influx. 4. A hyperosmotic stimulus applied in Ca(2+)-free medium (0.5 mM EGTA) lowered the [Ca2+]i peak to a ratio of 0.96 +/- 0.08 (P < 0.001) compared with a Ca(2+)-containing medium. This suggests that the [Ca2+]i increase is due to extracellular influx, as well as release from an intracellular Ca2+ pool. 5. Application of thapsigargin (0.5 microM), a specific inhibitor of endoplasmic reticulum Ca(2+)-ATPase, in Ca(2+)-free medium caused transient [Ca2+]i elevation to peak ratios of 1.33 +/- 0.09, and completely abolished the [Ca2+]i response to a hyperosmotic stimulus. This implies the existence of a thapsigargin-sensitive intracellular pool of Ca2+ that is mobilized by hyperosmotic stimulus.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Truncation of the thyrotropin-releasing hormone receptor carboxyl tail causes constitutive activity and leads to impaired responsiveness in Xenopus oocytes and AtT20 cells.

We studied the activity of a truncated thyrotropin-releasing hormone receptor (TRH-R), which lacks the last 59 amino acids of the carboxyl tail, where Cys-335 was mutated to a stop codon (C335Stop) (Nussenzveig, D. R., Heinflink, M., and Gershengorn, M. C. (1993) J. Biol. Chem. 268, 2389-2392). In Xenopus laevis oocytes expressing C335Stop TRH-Rs, TRH binding was higher, whereas chloride current, 45Ca2+ efflux, and [Ca2+]i responses evoked by TRH were 23, 39, and 21%, respectively, of those in oocytes expressing wild type mouse pituitary TRH-Rs (WT TRH-Rs). In oocytes expressing C335Stop TRH-Rs, basal 45Ca2+ efflux and [Ca2+]i were twice those in oocytes expressing WT TRH-Rs; chelation of Ca2+ caused a rapid increase in holding current, which is consistent with basal activation; and coexpression with other receptors caused inhibition of the responses to the other cognate agonists. In AtT20 pituitary cells stably expressing C335Stop TRH-Rs, thyrotropin-releasing hormone (TRH)-independent inositol phosphate formation was 1.32 +/- 0.11-fold higher, basal [Ca2+]i was 1.8 +/- 0.2-fold higher, and the [Ca2+]i response to TRH was much lower than in cells expressing WT TRH-Rs. We conclude that a TRH-R mutant truncated at Cys-335 exhibits constitutive activity that results in desensitization of the response to TRH.

Animals↗
PubMed1995

Contribution of response kinetics to the response pattern: studies of responses to thyrotropin-releasing hormone in Xenopus oocytes.

In Xenopus oocytes injected with total rat pituitary GH3 cell RNA, thyrotropin-releasing hormone (TRH) causes the activation of the inositol lipid transduction pathway and the induction of chloride conductance via calcium-activated channels (Oron et al., 1987, Mol. Endocrinol., 1:918-925). This response exhibits characteristic prolonged latency (Oron et al., 1988, Proc. Natl. Acad. Sci. U.S.A., 85:3820-3824; Lipinsky et al., 1993, Pflugers Arch., 425:140-149). We examined the role of agonist diffusion in the extracellular medium in the generation of latency and the determination of response amplitude. An increase in the viscosity of the medium markedly prolonged the latency and decreased the amplitude of the response. Moreover, an increase in the viscosity of the medium in the immediate vicinity of the oocyte had a major effect on both the latency and the amplitude of the response, which appeared to be a result of desensitization rather than restricted diffusion of chloride to the medium. Extrapolation to [TRH] infinity yielded a diffusion-dependent latency value of 0 and a diffusion-independent latency value of 4 seconds. In low viscosity medium, at all TRH concentrations, diffusion contributed less than 2% to the latency of the response. This implied that events distal to ligand binding are responsible for a major part of latency. Analysis of the dependence of latency and amplitude of the response on [TRH] yielded Hill coefficients markedly smaller than unity, suggesting postreceptor negative modulation of the response. Preincubation of cells with a specific inhibitor of protein kinase C, chelerythrine, increased the Hill coefficients to unity and changed the shape of the Hill plot of response amplitudes. Our results suggest that at low agonist concentrations, even in a low viscosity medium, the prolonged latency allows negative effects on both latency and amplitude by a simultaneous activation of a protein kinase C.

Alkaloids↗
PubMed1995

Low temperature and fertilization-induced Ca2+ changes in rat eggs.

In mammalian eggs, activation by sperm that leads to resumption of meiosis is characterized by an explosive transient increase in intracellular calcium ion concentration ([Ca2+]i), followed by [Ca2+]i oscillations. In addition to the spermatozoon, various treatments can induce parthenogenetic activation, accompanied by an elevation of [Ca2+]i. It has been reported that cooling can induce egg activation, yet the mechanism of this phenomenon has not been elucidated. In the present study we followed changes in egg [Ca2+]i (measured by Fura-2 fluorescence ratio imaging) during activation by cooling, using conditions that ensure a low rate of spontaneous activation. Our present findings demonstrate that cooling induces egg activation as manifested by [Ca2+]i transient(s) and second polar body extrusion. Seventy-eight of 104 eggs responded to cooling with increased [Ca2+]i. Thirty-five percent of the responding eggs displayed a single [Ca2+]i transient, while 65% exhibited at least two [Ca2+]i transients within the time window of the experiment (30-40 min). Twenty-two percent of these eggs displayed high-frequency oscillations (intervals of 3.5-5.9 min). In these eggs, the overall pattern of calcium dynamics was similar to that observed in eggs activated by sperm, as judged by the transient's intervals, duration, and a gradual increase in the amplitude of successive transients. The amplitudes of [Ca2+]i transients, however, were 2-3 times lower. We propose that cooling affects [Ca2+]i homeostasis to produce fertilization-like changes in [Ca2+]i, possibly associated with parthenogenetic activation. Moreover, great care should be exercised to prevent temperature changes during egg handling.

Animals↗
PubMed1995

Desensitization of the response to thyrotropin-releasing hormone in Xenopus oocytes is an amplified process that precedes calcium mobilization.

Consecutive challenges with thyrotropin-releasing hormone (TRH) of oocytes expressing the TRH receptor (TRH-R) resulted in a pronounced desensitization, manifested as a decrease in chloride current amplitude and an increase in response latency. Exposure to low concentrations of TRH resulted in a marked decrease in the amplitude of the subsequent response to a higher concentration of the agonist, even though the second challenge was given before the onset of the response to the first challenge (within 3 - 15 s). Cellular calcium concentration ([Ca]i) did not increase within this interval, suggesting that calcium was not involved in the desensitization process. The latency of the second response, however, was either unchanged or shortened, implying additive effects of processes initiated by the first challenge. A longer interval (30 s) between the two challenges brought about a more pronounced decrease in amplitude and a prolongation of response latency. The calcium mobilization initiated by a second challenge with a high concentration of the agonist exhibited a longer latency, a lower rate of [Ca]i increase and a lower amplitude. Stimulation of co-expressed cholinergic-muscarinic ml receptors with a low concentration of acetylcholine resulted in a pronounced desensitization of the TRH response (heterologous desensitization). Activation of protein kinase C by beta-phorbol 12-myristate, 13-acetate resulted in a dose-dependent inhibition of the response to TRH, suggesting that protein kinase C was involved in desensitization. Chelerythrine, a specific inhibitor of protein kinase C, abolished a large part of the desensitization. A mutant of the TRH-R that lacks protein kinase C consensus phosphorylation sites in the C-terminal region, exhibited desensitization.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

A constitutively active mutant thyrotropin-releasing hormone receptor is chronically down-regulated in pituitary cells: evidence using chlordiazepoxide as a negative antagonist.

A carboxyl-terminus truncated mutant of the guanine nucleotide-binding (G) protein-coupled TRH receptor (TRH-R) was previously shown to exhibit constitutive, i.e. TRH-independent, activity (C335Stop TRH-R). Chlordiazepoxide (CDE), a known competitive inhibitor of TRH binding to wild-type (WT) TRH-Rs, is shown to compete for binding to C335Stop TRH-Rs also. More importantly, CDE is shown to be a negative antagonist of C335Stop TRH-Rs. CDE rapidly caused the basal rate of inositol phosphate second messenger (IP) formation to decrease in AtT-20 pituitary cells stably expressing C335Stop TRH-Rs (AtT-C335Stop cells), but not in cells expressing WT TRH-Rs (AtT-WT cells). Similar observations were made in HeLa cells transiently expressing C335Stop or WT TRH-Rs. CDE inhibition of IP formation was shown to be specific for TRH-Rs using GH4C1 cells expressing both TRH-Rs and receptors for bombesin. In these cells, CDE inhibited TRH-stimulated IP formation, but had no effect on bombesin-stimulated IP formation. The effects of chronic administration of CDE were studied. Preincubation of AtT-C335Stop cells, but not AtT-WT cells, with CDE for several hours caused an increase in cell surface receptor number (up-regulation) that led to increased TRH stimulation of inositol phosphate formation and elevation of intracellular free Ca2+. Preincubation with CDE did not affect methyl-TRH binding affinity or TRH potency in cells expressing AtT-C335Stop or in AtT-WT cells. We conclude that CDE is a negative antagonist of C335Stop TRH-Rs and that constitutively active C335Stop TRH-Rs are down-regulated in AtT-20 pituitary cells in the absence of agonist.

Animals↗
PubMed1994

The hemispheric functional expression of the thyrotropin-releasing-hormone receptor is not determined by the receptors' physical distribution.

The thyrotropin-releasing-hormone receptor (TRH-R) is a member of a family of the G-protein-coupled receptors that share structural similarities and exert their physiological action via the inositol lipid signal-transduction pathway. The TRH-R when expressed in Xenopus oocytes exhibits marked preference of the response (increased chloride conductance) for the animal hemisphere. Whereas the rat TRH-R functional distribution was strongly asymmetric (animal/vegetal ratio = 9.5), the mouse TRH-R exhibited a significantly lower ratio (3.9). Truncation of the last 59 amino acids of the C-terminal region of the mouse TRH-R did not lead to any changes in the functional hemispheric distribution. Despite the polarization of response, receptor number was similar on both hemispheres. Moreover, the apparent half-life of the functional expression of the TRH-R was approx. 4 h on both hemispheres when the expression was inhibited by a specific antisense oligonucleotide. Inhibition of total protein synthesis with cycloheximide affected hemispheric responses mediated by each of the three TRH-Rs tested in a qualitatively different way. These results suggest that an additional, rapidly degraded, protein modulates the functional hemispheric expression of the TRH-Rs.

Animals↗
PubMed1994

Neuromedin B receptor, expressed in Xenopus laevis oocytes, selectively couples to G alpha q and not G alpha 11.

G-proteins of the q family have been implicated as mediators of bombesin receptors action. We cloned Xenopus G alpha q and G alpha 11 and specifically disrupted the synthesis of either protein with selective antisense oligonucleotides. G alpha q antisense inhibited responses mediated by neuromedin B receptor (NMB-R) by 74%, though not by gastrin-releasing peptide receptor (GRP-R). G alpha 11 antisense had little effect on either GRP-R- or NMB-R-mediated responses. This suggests that NMB-R couples to G alpha q, and that GRP-R and NMB-R show distinct G-protein coupling preferences in the Xenopus oocyte.

Amino Acid Sequence↗
PubMed1994

Independent external calcium entry and cellular calcium mobilization in Xenopus oocytes.

We studied cellular calcium (Ca) mobilization and Ca entry from the medium following injection of various inositol phosphates (IPs) or activation of thyrotropin-releasing hormone receptors (TRH-Rs) in oocytes injected with TRH-R cRNA. We determined the order of potency of various IPs for evoking the rapid depolarizing current in Ca-free medium, which reflects the mobilization of cellular Ca. The most potent compound was inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), followed by inositol 1,2,4,5-tetrakisphosphate (Ins(1,2,4,5)-P4), which displayed 91% of the activity of Ins(1,4,5)P3, while inositol 1,3,4,6-tetrakisphosphate (Ins(1,3,4,6)P4) had only 29% effect. All other IPs used in the present study exhibited responses that were 40% or less than those elicited by Ins(1,4,5)P3. Cellular Ca mobilization was confirmed by 45Ca2+ efflux for Ins(1,4,5)P3, Ins(1,2,4,5)P4 and Ins(1,3,4,6)P4, or by Fura-2 ratio imaging studies for the latter. In parallel, we assayed the ability of these compounds to promote Ca entry into the cell, as reflected by Ca-evoked depolarizing current or Fura-2 imaging. These assays revealed a different order of potency, where Ins(1,4,5)P3 > inositol 4,5-bisphosphate (Ins(4,5)P2) > Ins(1,3,4,6)P4 = Ins(1,2,4,5)P4. All other inositol phosphates were largely ineffective. Heparin inhibited the response to TRH by 67% while Ca entry was inhibited only by 22%. The latency of the response to TRH was significantly shorter in the presence of extracellular Ca, suggesting Ca entry preceded the response, i.e. major depletion of Ca stores. These results strongly suggest that the activation of Ca entry is largely independent of cellular Ca mobilization and may be mediated by a receptor for an unidentified phosphorylated compound, different from that for Ins(1,4,5)P3 on the endoplasmic reticulum.

Animals↗
PubMed1993

Prolonged, repetitive calcium transients in rat oocytes fertilized in vitro and in vivo.

Zona-free rat oocytes inseminated with capacitated sperm, under conditions that allow polyspermic fertilization, exhibited a rapid, transient elevation of cellular calcium (from 147 +/- 10 to 607 +/- 55 nM, n = 19, measured by Fura 2 fluorescence ratio imaging) immediately after sperm attachment. This peak was followed by a series of dramatic calcium transients of high amplitude (maximal 847 +/- 32 nM) and frequency (range 2.1 +/- 0.07 - 3.9 +/- 0.07 min), which continued for several hours. A similar pattern was seen also in zona-free oocytes fertilized with low sperm density (i.e. producing mainly monospermic attachment) and in zona-enclosed oocytes fertilized in vitro. Moreover, single or repetitive calcium transients were observed in rat oocytes fertilized in vivo. These findings indicate that in normal fertilization in vivo, sperm-oocyte interaction initiates a prolonged train of cyclical calcium changes in the oocyte. This activity may be necessary for the early events in the fertilization process.

Animals↗
PubMed1993

Latency in the inositol lipid transduction pathway: the role of cellular events in responses to thyrotropin-releasing hormone in Xenopus oocytes.

To dissect the cellular events responsible for the prolonged latency of the response to thyrotropin-releasing hormone (TRH) in Xenopus oocytes we interfered with different steps of the signal transduction pathway. Preincubation of oocytes with cis-vaccenic acid (a membrane-fluidizing agent) shortened the latency, suggesting a contribution of membranal processes. TRH-induced depletion of cellular calcium stores prolonged latency (up to threefold), which returned to control levels upon repletion of the stores. Injection of D-2,3-diphosphoglycerate (PGA), which inhibits inositol (1,4,5)-trisphosphate (InsP3) dephosphorylation, alone evoked a small, prolonged depolarizing current and significantly shortened the latency of the response to TRH. Injection of guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), which inactivates guanine nucleotide-binding regulatory proteins, decreased the amplitude of the response and increased latency. Injection of guanosine 5-O-(3-thiotriphosphate) (GTP gamma S) immediately before the challenge with TRH did not shorten the latency of the response. Decreasing the effective receptor density with chlordiazepoxide, an antagonist of the TRH receptor, resulted in an extension of latency, whereas the expression of a large number of TRH receptors by injection of RNA transcribed from cloned receptor DNA (10-100 ng/oocyte) shortened the latency to below 2 s. Our results suggest that the latency of the response to TRH reflects the activation of a late step in the signal transduction sequence, most likely the release of calcium by InsP3. We propose that this process is kinetically controlled by an early rate-limiting event, involving the activation of a guanine nucleotide-binding protein by the TRH receptor.

2,3-Diphosphoglycerate↗
PubMed1993

Differential effects of cytoskeletal agents on hemispheric functional expression of cell membrane receptors in Xenopus oocytes.

1. We studied the effects of three cytoskeleton-disrupting agents, colchicine (COL), vinblastine (VIN), cytochalasins, on the functional hemispheric expression of native muscarinic and acquired thyrotropin-releasing hormone receptors TRH-Rs). Responses in oocytes of common donors, which express M3-like receptors (M3Rs), were not affected by either COL or VIN on the animal hemisphere. The functional expression of M3Rs on the vegetal hemisphere was inhibited by 50%. Cytochalasin B caused a uniform inhibition (by 31-33%) of receptor functional expression on either hemisphere. 2. Oocytes of variant donors express predominantly M1-like receptors (M1Rs) on the animal and M3Rs on the vegetal hemisphere. In these oocytes, both COL and VIN caused approximately 50% inhibition of functional expression on either hemisphere. Cytochalasin B caused more extensive, though variable inhibition on both hemispheres. Both antitubulin agents had no effect on the functional expression of the TRH-Rs on either hemisphere. Cytochalasin B, however, caused an extensive inhibition of the functional expression of this receptor (by 70-75%). 3. Induction of maturation of oocytes (7-hr incubation with progesterone) resulted in a 66% decrease in the response to TRH, reflecting mainly a decrease on the animal hemisphere. Maturation in the presence of colchicine had no further effect on the activity measured on the animal hemisphere but caused a major increase in the activity on the vegetal hemisphere. This resulted in a dramatic change in animal/vegetal activity ratio (4.8 +/- 1.5 to 0.8 +/- 0.2). 4. It appears that while antitubulin drugs affect the functional expression of the three receptors at the two hemispheres differently, disruption of the microfilaments interferes uniformly with receptor functional expression. We suggest that microfilaments may be involved in a common component of the signal transduction pathway in oocytes or in the anchoring of receptors coupled to the guaninine nucleotide-binding regulatory proteins. Moreover, progesterone-induced changes in the functional organization of the signal transduction pathway appear to be controlled to a large extent by the tubulin component of the cytoskeleton.

Amino Acids↗
PubMed1993

Calcium entry in Xenopus oocytes: effects of inositol trisphosphate, thapsigargin and DMSO.

Agonist- and inositol 1,4,5-trisphosphate (InsP3)-evoked responses in Xenopus oocytes utilize calcium mobilized from cellular stores as well as from the medium. We studied the effect of the status of Ca stores on InsP3-induced Ca entry. Thapsigargin (TG) caused a net increase of 45Ca2+ efflux from oocytes in a time and dose dependent manner (31 and 54% of total label, at 30 and 60 min, respectively). Incubation with TG (60 min) resulted in a complete loss of the response to InsP3 implying that InsP3-sensitive Ca stores were depleted. Challenge with 1.8 mM Ca2+ resulted in a large depolarizing chloride current (1231 +/- 101 nA) which was not further potentiated by InsP3. This suggested that extensive depletion of cellular Ca stores is sufficient to induce maximal entry of extracellular Ca (Cao). Following the injection of InsP3, a much more limited loss of cellular Ca was sufficient to produce large Ca entry. Dimethyl sulfoxide (DMSO) alone, the vehicle used to dissolve TG, did not cause increase in either efflux of 45Ca2+, nor in the Cao-evoked Cl- current. It did, however, markedly potentiate this current following the injection of InsP3. DMSO moderately inhibited InsP3-induced 45Ca2+ efflux from oocytes. Hence, apparent potentiation of Ca entry can be observed without additional depletion of cellular Ca. We conclude that Ca entry may be induced via either stimulation with InsP3 and limited Ca depletion or depletion of a specific and, possibly small, cellular Ca store alone. The mechanism of DMSO potentiation is unknown, but may be important in view of the universal use of this solvent as vehicle.

Animals↗