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

Lipinsky, Dafna; Gershengorn, Marvin C.; Oron, Yoram

doi:10.1007/bf00374514

Cited by 14 publications

(23 citation statements)

References 33 publications

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“…The effect of GSSG on Ca 2ϩ release in A7r5 cells was much smaller than reported in rat hepatocytes (20, 21, 36, 37). This finding reinforces previous conclusions that InsP 3 Rs of different sources can be differently affected by sulfhydryl reagents (33).…”

Section: Insp 3 -Induced Ca 2ϩ Releasesupporting

confidence: 92%

“…The time point, from where the fractional loss started to increase above base line, was determined and the corresponding [InsP 3 ] at that time was then taken as threshold. This threshold in standard efflux medium was always around 32 nM InsP 3 .…”

Section: Camentioning

confidence: 91%

“…The fractional loss at each InsP 3 concentration decreased both after depleting the stores by preincubating them in efflux medium (Fig. 8B) and in efflux medium containing InsP 3 (Fig. 10B).…”

Section: Concluding Remarks On the Kinetics Of Insp 3 -Induced Ca 2ϩmentioning

confidence: 95%

“…Ca 2ϩ -dependent inactivation of the channel due to the proposed local build-up of Ca 2ϩ near the channel pore is still controversial (14,26,47). We previously presented evidence that heterogeneity of the stores with respect to the expression of InsP 3 Rs also contributed to the quantal Ca 2ϩ release in A7r5 cells (11). Such heterogeneity allows the more sensitive stores to be depleted faster than the less sensitive stores in the presence of low InsP 3 concentrations.…”

Section: Concluding Remarks On the Kinetics Of Insp 3 -Induced Ca 2ϩmentioning

confidence: 99%

“…Once threshold concentrations of InsP 3 are reached and conditions for the regenerative release of Ca 2ϩ are created (2), InsP 3 mobilizes Ca 2ϩ from the nonmitochondrial stores through interaction with the InsP 3 R. The precise kinetics of the [InsP 3 ] rise in an intact cell are unknown, but it is possible that there is a gradual increase in the [InsP 3 ] before the first Ca 2ϩ response (3,4 , and cytosolic Ca 2ϩ and how a repeated application of InsP 3 acts on the threshold value. This novel technique of measuring thresholds for InsP 3 action allowed a refined analysis of the kinetic parameters controlling the mechanisms of action of several regulators of the InsP 3 R.…”

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confidence: 99%

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Threshold for Inositol 1,4,5-Trisphosphate Action

Missiaen

Smedt

Parys

et al. 1996

Journal of Biological Chemistry

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We developed a unidirectional 45 Ca2؉ efflux technique in which 60 cumulative doses of inositol 1,4,5-trisphosphate (InsP 3 ), each lasting 6 s, were subsequently added to permeabilized A7r5 cells. This technique allowed an accurate determination of the threshold for InsP 3 action, which was around 32 nM InsP 3 under control conditions. The InsP 3 -induced Ca 2؉ release was characterized by an initial rapid phase, after which the normalized rate progressively decreased. The slowing of the release was associated with a shift of the threshold to higher InsP 3 concentrations. Stimulatory concentrations of thimerosal (10 M) shifted the threshold to 4.5 nM InsP 3 and increased both the cooperativity and the maximal normalized rate of Ca 2؉ release. This low threshold was maintained when the thimerosal concentration was increased to inhibitory levels (100 M) but then the effects on the cooperativity and on the normalized rate of Ca 2؉ release disappeared. Oxidized glutathione (5 mM) was much less effective in stimulating the release and did not have an effect on the threshold or on the cooperativity. ATP (5 mM) stimulated the release despite a shift in threshold toward higher InsP 3 concentrations. Luminal Ca 2؉ did not affect the threshold for InsP 3 action but stimulated the normalized release at each InsP 3 concentration. The inhibitory effect of 10 M free cytosolic Ca 2؉ was associated with a shift in threshold to higher InsP 3 concentrations and a decreased cooperativity of the release process. We conclude that this novel technique of accurately measuring the threshold for InsP 3 action under various experimental conditions has allowed us to refine the analysis of the kinetic parameters involved in the regulation of the InsP 3 receptor.Many hormones, neurotransmitters, and growth factors induce the hydrolysis of phosphatidylinositol 4,5-bisphosphate and thereby produce inositol 1,4,5-trisphosphate (InsP 3 ) 1 as an intracellular messenger (1 MATERIALS AND METHODSA7r5 cells, an established cell line derived from embryonic rat aorta, were used between the 7th and the 17th passage after receipt from the American Type Culture Collection (Bethesda, MD) and subcultured weekly by trypsinization. The cells were cultured at 37°C in a 9% CO 2 incubator in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 3.8 mM L-glutamine, 0.9% (v/v) nonessential amino acids, 85 IU ml Ϫ1 penicillin, and 85 g ml Ϫ1 streptomycin. The cells were seeded in 12-well dishes (4 cm 2 , Costar, MA) at a density of approximately 10 4 cells cm Ϫ2 . 45Ca 2ϩ fluxes on permeabilized cells were done on a thermostated plate at 25°C. The culture medium was aspirated and replaced by 1 ml of permeabilization medium containing 120 mM KCl, 30 mM imidazole HCl (pH 6.8), 2 mM MgCl 2 , 1 mM ATP, 1 mM EGTA, and 20 g ml Ϫ1 saponin. The saponin-containing solution was removed after 10 min, and the cells were washed once with a similar saponin-free solution. 45 Ca 2ϩ uptake into the nonmitochondrial Ca 2ϩ stores was accomplished by incubati...

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Section: Insp 3 -Induced Ca 2ϩ Releasesupporting

confidence: 92%

Section: Camentioning

confidence: 91%

Section: Concluding Remarks On the Kinetics Of Insp 3 -Induced Ca 2ϩmentioning

confidence: 95%

Section: Concluding Remarks On the Kinetics Of Insp 3 -Induced Ca 2ϩmentioning

confidence: 99%

mentioning

confidence: 99%

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Threshold for Inositol 1,4,5-Trisphosphate Action

Missiaen

Smedt

Parys

et al. 1996

Journal of Biological Chemistry

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Alkaline pH facilitates the exchange of guanine nucleotides: A possible mechanism for modulation of the kinetics of responses mediated by guanine nucleotide-binding proteins

Lipinsky

Oron

1996

J. Cell. Physiol.

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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.

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Contribution of response kinetics to the response pattern: Studies of responses to thyrotropin‐releasing hormone in Xenopus oocytes

Lipinsky

Gershengorn

Oron

1995

Journal Cellular Physiology

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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.

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

Cited by 14 publications

References 33 publications

Threshold for Inositol 1,4,5-Trisphosphate Action

Threshold for Inositol 1,4,5-Trisphosphate Action

Alkaline pH facilitates the exchange of guanine nucleotides: A possible mechanism for modulation of the kinetics of responses mediated by guanine nucleotide-binding proteins

Contribution of response kinetics to the response pattern: Studies of responses to thyrotropin‐releasing hormone in Xenopus oocytes

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