Two endogenous gonadotropin-releasing hormones generate dissimilar Ca(2+) signals in identified goldfish gonadotropes

Ca(2+) signals are involved in the signal transduction of neuroendocrine regulators. In goldfish, two endogenous gonadotropin-releasing hormones, salmon (s)GnRH and chicken (c)GnRH-II, control maturational gonadotropin secretion. Although considerable evidence suggests that sGnRH and cGnRH-II exert their activity on goldfish gonadotropes through a single population of receptors, differences in signal transduction mechanisms between these peptides have been demonstrated. We used ratiometric Fura-2 Ca(2+) imaging of single morphologically identified gonadotropes to quantitatively compare the Ca(2+) signals evoked by sGnRH and cGnRH-II. The amplitude and the rate of rise of sGnRH- and cGnRH-II-evoked Ca(2+) signals increased with concentration. At maximal concentrations, Ca(2+) signals generated by cGnRH-II rose significantly faster than those elicited by sGnRH, while other parameters such as the maximum amplitude, average Ca(2+) increase, and latency did not differ between the two peptides. Ca(2+) signals evoked by sGnRH or cGnRH-II were often spatially restricted to one part of the cell over the duration of the response. We provide a comprehensive account of the spatial and temporal aspects, including calculated kinetics, of GnRH-evoked Ca(2+) signals in single identified gonadotropes. This is the first report of quantified differences in Ca(2+) signals generated by two endogenous GnRH neuropeptides, which may act through the same receptor population in this cell type.     

The permeant molecule urea stimulates prolactin secretion in GH4C1 cells by inducing Ca2+ influx through dihydropyridine-sensitive Ca2+ channels

Isotonic urea in medium with a normal 1.2 mM Ca²⁺ concentration induced a striking rise in both cytosolic Ca²⁺ concentration ([Ca²⁺]_i) and prolactin (PRL) secretion, each of whose peaks were proportional to the concentration of urea between 5 and 120 mM. There was a significant linear relationship between the peaks of induced [Ca²⁺]_i and PRL secretion (r = 0.99, P < 0.001). The increase in both [Ca²⁺]_i and PRL secretion was completely abolished by removal of medium Ca²⁺ or by 2 μM nifedipine. Hypertonie urea was ineffective in inducing either an increase in [Ca²⁺]_i or PRL secretion. These data support the hypothesis that plasma membrane expansion is a potent non-toxic inducer of hormone secretion and that in GH₄C₁ cells an increase in [Ca²⁺]_i produced by enhanced influx of extracellular Ca²⁺ through dihydropyridine-sensitive Ca²⁺ channels plays an important role in this phenomenon.     

Tranilast inhibits contraction and Ca movement of porcine coronary arteries

In a recent clinical study, tranilast, an anti-allergic agent, was shown to reduce the rate of coronary restenosis after percutaneous transluminal coronary angioplasty, although the mechanism of this effect is unclear. The present study was undertaken to investigate the effects of tranilast on contraction and Ca²⁺ movement of the coronary arteries. We characterized the effects of tranilast on isometric force and aequorin-estimated intracellular Ca²⁺ concentrations ([Ca²⁺]_i) of porcine coronary artery strips. Tranilast concentration-dependently (10–500 μM) inhibited histamine (3×10⁻⁵ M)-induced contraction of the coronary arteries. A similar tendency was observed in the response to high K⁺ (30 mM) stimulation. Histamine caused phasic and tonic increases in [Ca²⁺]_i, and high K⁺ caused a tonic increase in [Ca²⁺]_i of smooth muscle, both of which were significantly suppressed in the presence of tranilast. These results suggest that tranilast inhibits the contraction of coronary arteries by inhibiting both Ca²⁺ influx from extracellular environment and Ca²⁺ release from intracellular Ca²⁺ stores, which might be related to its preventive effect on restenosis after coronary angioplasty.     

Age-related alterations in Ca2+ signals and mitochondrial membrane potential in exocrine cells are prevented by melatonin

Abstract:  Information regarding age-induced Ca²⁺ signal alterations in nonexcitable cells is limited. In addition, little evidence exists on the ability of melatonin to palliate the effects of aging on Ca²⁺ signals and mitochondrial potential, a parameter involved in both Ca²⁺ signaling and aging. We studied the ability of melatonin to prevent the effects of aging on intracellular Ca²⁺ homeostasis and mitochondrial potential in exocrine cells. Pancreatic acinar cells were obtained from adult (3 months old) and aged (22–24 months old) mice by collagenase dispersion. Ca²⁺ signals, in situ mitochondrial potential and in vitro amylase secretion were determined. Secretion in response to increasing levels of the secretagogues, acetylcholine and cholecystokinin (CCK), were impaired in aged pancreatic acini. This decrease was accompanied by an inhibition in the amplitude of the peak response to maximal concentrations of the agonists, and by a decrease in the pattern of Ca²⁺ oscillations induced by postprandial levels of CCK. Both the size of the calcium pools, assessed by low levels of ionomycin, and capacitative calcium entry, induced by depletion of the stores with thapsigargin, were diminished in aged cells. These changes in Ca²⁺ homeostasis were associated with depolarization of intracellular mitochondria. Oral administration of melatonin for 3 months to aged mice restored the secretory response, the amplitude and frequency of Ca²⁺ responses, the size of intracellular calcium pools, the capacitative calcium entry, and the mitochondrial potential. In conclusion, melatonin restores secretory function, Ca²⁺ signals and mitochondrial potential of aged exocrine cells.     

Serotonin interferes with Ca2+ and PKC signaling to reduce gonadotropin-releasing hormone-stimulated GH secretion in goldfish pituitary cells

In goldfish, two endogenous gonadotropin-releasing hormones (GnRH), salmon GnRH (sGnRH) and chicken GnRH-II (cGnRH-II), are thought to stimulate growth hormone (GH) release via protein kinase C (PKC) and subsequent increases in intracellular Ca²⁺ levels ([Ca²⁺]_i). In contrast, the signaling mechanism for serotonin (5-HT) inhibition of GH secretion is still unknown. In this study, whether 5-HT inhibits GH release by actions at sites along the PKC and Ca²⁺ signal transduction pathways leading to hormone release were examined in primary cultures of goldfish pituitary cells. Under static incubation and column perifusion conditions, 5-HT reduced basal, as well as sGnRH- and cGnRH-II-stimulated, GH secretion. 5-HT also suppressed GH responses to two PKC activators but had no effect on the GH-releasing action of the Ca²⁺ ionophore ionomycin. Ca²⁺-imaging studies with identified somatotropes revealed that 5-HT did not alter basal [Ca²⁺]_i but attenuated the magnitude of the [Ca²⁺]_i responses to the two GnRHs. Prior treatment with 5-HT and cGnRH-II reduced the magnitude of the [Ca²⁺]_i responses induced by depolarizing levels of K⁺. Similar inhibition, however, was not observed with prior treatment of 5-HT and sGnRH. These results suggest that 5-HT, by direct actions at the somatotrope level, interferes with PKC and Ca²⁺ signaling pathways to reduce the GH-releasing effect of GnRH. 5-HT action may occur at the level of PKC activation or its downstream signaling events prior to the subsequent rise in [Ca²⁺]_i.. The differential Ca²⁺ responses by depolarizing doses of K⁺ is consistent with our previous findings that sGnRH and cGnRH-II are coupled to overlapping and yet distinct Ca²⁺-dependent mechanisms.     

Calcium signaling and secretion in pituitary cells

An important trigger of hormone secretion from pituitary cells is a rise in cytosolic Ca²⁺ ([Ca²⁺]_i). Pituitary cells may modulate [Ca²⁺]_i by an increased membrane flux from the extracellular space and/or by a release from intracellular stores. Both mechanisms can support exocytosis, although in different pituitary cell types one or the other mechanism may predominate. Molecular events transducing a rise in [Ca²⁺]_i into hormone secretion are still poorly understood. Here, the exocytotic machinery in pituitary cells is briefly reviewed in terms of the spatial organization of [Ca²⁺]_i elevation relative to the Ca²⁺ sensor(s).     

Ovariectomy Aggravated Sodium Induced Hypertension Associated With Altered Platelet Intracellular Ca in Dahl Rats

Our purpose was to determine the effect of ovariectomy on intracellular Ca²⁺ mobilization and platelet aggregation in sodium induced hypertension. At the age of 12 weeks ovariectomy or sham operation was performed in female Dahl-Iwai salt sensitive rats on a 0.3% NaCl diet. Four weeks later we assessed the effects of ovariectomy and an 8% NaCl diet on agonist induced intracellular Ca²⁺ mobilization in fura-2 loaded platelets and platelet aggregation. Ovariectomy enhanced the increase of systolic blood pressure and heart to body weight ratio on an 8% NaCl diet. However, thrombin evoked intracellular Ca²⁺ was not correlated with systolic blood pressure (r = −0.338, P = .17), and was lowered by sodium loading and ovariectomy (360 ± 23 to 285 ± 9, 296 ± 10 nmol/L, P < .05). Furthermore, the ionomycin induced intracellular calcium fraction in the absence of external Ca²⁺ that reflected internal Ca²⁺ discharge capacity was reduced in ovariectomized rats compared with sham operated rats on an 8% NaCl diet (648 ± 15 v 768 ± 35 nmol/L, P < .05). The internal Ca²⁺ discharge capacity was inversely correlated with systolic blood pressure (r = −0.506, P = .03). In addition to the decreased internal Ca²⁺ discharge capacity, intracellular Ca²⁺-independent platelet aggregation by phorbol 12-myristate 13-acetate, a protein kinase C activator, was significantly enhanced in hypertensive rats. We concluded that ovariectomy enhanced sodium induced hypertension associated with the decreased internal Ca²⁺ discharge capacity and increased platelet aggregation in Dahl-Iwai salt-sensitive rats.     

Estrogen receptor independent rapid non-genomic effects of environmental estrogens on [Ca2+]i in human breast cancer cells

The aim of this study was to identify and characterize an alternative pathway through which environmental estrogenic compounds may mediate their intracellular effects. Three human breast cancer cell lines were employed including MCF-7 cells, which express both ER and ERβ; MDA-MB-231 cells, which express ERβ but not ER; and SKBR-3 cells, which express neither ER nor ERβ. The effect of environmental estrogenic compounds on intracellular calcium ion concentration ([Ca²⁺]_i) was measured and compared to that of 17β-estradiol (E2). A rapid and maintained increase in [Ca²⁺]_i was observed following the application of nanomolar concentrations of environmental estrogens and E2 regardless of the expression of ER and ERβ. Removal of extracellular Ca²⁺ completely abolished the steroid-induced [Ca²⁺]_i increase. Pre-treatment of cells with the estrogen receptor (ER) antagonist ICI 182,780 had no effect on either basal [Ca²⁺]_i or the steroid-triggered [Ca²⁺]_i response.

In summary, we have demonstrated ER independent rapid non-genomic effects of environmental estrogenic compounds, at nanomolar concentrations, on [Ca²⁺]_i. The results of this study demonstrate an alternative pathway to explain potent intracellular effects of endocrine disrupting chemicals.     

Potentiators of insulin secretion modulate Ca sensitivity in rat pancreatic islets

Insulin secretion stimulated by 10 mM glucose was potentiated by forskolin, an activator of adenyl cyclase, by acetyl choline which may enhance turnover of inositol phospholipids, and by tetradecanoyl phorbol acetate (TPA), an activator of protein kinase C. None of these agents initiated insulin secretion in the presence of 2 mM glucose. Glucose-stimulated insulin secretion was markedly dependent on the concentration of extracellular Ca²⁺: at or below 10 μM Ca²⁺ no insulin secretion was evoked by glucose in freshly isolated islets. The threshold Ca²⁺ requirement was increased after culture of islets for 44 h. In both fresh and cultured islets the presence of a potentiator of secretion produced both a marked increase in the maximum rate of glucose-stimulated insulin secretion and a lowering of the requirement for extracellular Ca²⁺. Thus potentiation of insulin release involves an increase in the sensitivity of the B cell to Ca²⁺.     

Characterization of the calcium response to thyrotropin-releasing hormone in lactotrophs and GH cells

Thyrotropin-releasing hormone (TRH) acts via a G-protein-coupled receptor on lactotrophs to increase the intracellular free calcium ion concentration, [Ca²⁺]_i. The [Ca²⁺]_i response depends on both TRH concentration and the duration of TRH exposure. An initial, short-lived [Ca²⁺]_i spike results from release of Ca²⁺ from intracellular stores, whereas a later sustained [Ca²⁺]_i increase, often characterized by [Ca²⁺]_i oscillations, results from an influx of extracellular Ca²⁺ through both voltage-gated and non-voltage-gated, store-operated Ca²⁺ channels. The initial spike phase predominates at high doses of TRH, whereas the plateau phase predominates at low doses. The mechanisms underlying the complex [Ca²⁺]_i response to TRH are discussed.     

Calcium-mediated mechanisms of eicosapentaenoic acid-induced relaxation in hypertensive rat aorta

We have previously demonstrated the vasorelaxant properties of the omega-3 fatty acid, eicosapentaenoic acid (EPA), in normotensive and spontaneously hypertensive rat (SHR) aorta, although the mechanism(s) of action are not fully understood. Because endothelial dysfunction and increased intracellular free calcium concentration ([Ca²⁺]_i) are seen in hypertensive rat aorta, we investigated the potential role of Ca²⁺ signaling, endothelium and derived factors, and the opening of potassium (K⁺) channels in EPA-induced relaxation. In the presence of extracellular Ca²⁺, EPA induced significant relaxations at > 10 μmol/L (P < .01) in norepinephrine (NE) (10⁻⁶ mol/L)-contracted aortic rings and at 30 μmol/L (P <. 001) in high K⁺ (80 mmol/L)-contracted aortic rings. In the absence of extracellular Ca²⁺, EPA (10 to 30 μmol/L) inhibits the tonic component of NE-induced contraction (P < .0001). The relaxant properties of EPA in SHR aorta appear specific to Ca²⁺ release from an internal storage site associated with NE-induced tonic contraction. Further studies with the use of fura-2 to measure [Ca²⁺]_i in cultured vascular smooth muscle (VSM) cells from SHR aorta indicated that EPA (30 μmol/L)-pretreatment attenuated angiotensin II (50 nmol/L)-induced Ca²⁺ transient by 95%, suggesting that an inhibitory effect on the Ca²⁺ signaling may underlie EPA-induced relaxation of the vessel preparation. In addition, EPA per se induced an increase in [Ca²⁺]_i with a duration of approximately 20 min in VSM cells, and the effect was not altered by removal of extracellular Ca²⁺. There was no increase in the level of inositol-1,4,5-trisphosphate in response to EPA (30 μmol/L). The actions of EPA are independent of endothelium-derived factors, cyclooxygenase metabolites, and activation of K⁺ channels since endothelium removal, N^ω-nitro- -arginine methyl ester hydrochloride, (L-NAME, 100 μmol/L), indomethacin (10 μmol/L), tetraethylammonium (1 mmol/L), and glibenclamide (10 μmol/L) did not affect EPA-induced vasodilation in NE-precontracted aortic rings. These results suggest that EPA directly modulates intracellular Ca²⁺ signaling in VSM cells, and that this may contribute to the vasorelaxant effect and, at least in part, the blood pressure-lowering effect of fish oil.     

A role for the actin cytoskeleton in the initiation and maintenance of store-mediated calcium entry in human platelets

Store-mediated Ca²⁺ entry (SMCE) is a major pathway for Ca²⁺ influx in many cells, yet how depletion of the intracellular Ca²⁺ stores leads to the activation of Ca²⁺ entry across the plasma membrane is not well understood. Recent work in platelets favors a secretion-like conformational coupling mechanism involving proteins in the plasma membrane (PM) and in the membrane of the Ca²⁺ store, located in the endoplasmic reticulum (ER). The activation and maintenance of SMCE in platelets has been shown to depend on remodeling of the actin cytoskeleton, which may be required to allow trafficking of the ER toward the PM to permit coupling to occur and to stabilize this coupling once achieved. The coupling itself has been shown to involve one isoform of the inositol 1,4,5-trisphosphate receptor (IP₃RII) and the Ca²⁺-permeable channel protein, human Trp1 (hTrp1).     

Cyclic Ca2+ changes in intracellular stores of gonadotropes during gonadotropin-releasing hormone-stimulated Ca2+ oscillations.

Gonadotropin-releasing hormone induces oscillatory release of Ca2+ from inositol trisphosphate-sensitive stores of gonadotropes. Simultaneously with electrophysiological measures of cytoplasmic [Ca2+], corresponding changes in [Ca2+] within intracellular stores were monitored with a fluorescent dye, mag-indo-1. Each cycle of oscillation released only 10% of the detectable stored Ca2+. Some Ca2+ was recovered by the stores using a mechanism sensitive to inhibitors of intracellular Ca2+ ATPases, and much of the remainder was temporarily and rapidly pumped into other intracellular compartments or out of the cell. The dynamics of Ca2+ oscillations are thus more complex than a repeated emptying and refilling of a single compartment. The free concentrations measured show that intracellular Ca2+ store compartments contain strong Ca2+ buffers.     

Why are endocrine pituitary cells excitable?

Since 1975, endocrine pituitary cells have been known to be excitable neuronlike cells. Using powerful single-cell approaches, in particular the patch clamp electrophysiological recording technique and the monitoring of Ca²⁺ with fluorescent probes, solid evidence has been provided in the last 10 years that intracellular Ca²⁺ signals are produced by stimulators and inhibitors of secretion via the modulation of action potentials in isolated pituitary cells. As cytosolic Ca²⁺ changes are thought to control numerous cellular functions (for example, secretion, protein synthesis, gene expression, and proliferation) over a long time scale—milliseconds to hours—it is now time to address the long-standing question of what functions would be physiologically controlled by electrical excitability in intact pituitary tissue.     

Novel, thapsigargin-insensitive intracellular Ca(2+) stores control growth hormone release from goldfish pituitary cells

The relative contribution of intracellular Ca(2+) stores to basal and agonist-stimulated hormone release in pituitary cells is still not well understood, especially in non-mammalian vertebrates. Using ratiometric Ca(2+) imaging of single identified goldfish somatotropes, along with time-resolved measurements of growth hormone (GH) secretion, we investigated the Ca(2+)-dependent signal transduction of two endogenous regulators of GH release from the goldfish pituitary. Two gonadotropin-releasing hormones (sGnRH and cGnRH-II) initiated GH release in nominally Ca(2+) free conditions. GnRH-evoked GH release was additive to KCl-stimulated GH responses. Ca(2+) signals and GH release elicited by both GnRHs were abolished by pretreatment with TMB-8, which blocks the release of Ca(2+) from intracellular stores. GnRH-stimulated GH secretion is mediated by caffeine-sensitive intracellular Ca(2+) stores that are functionally independent from those sensitive to thapsigargin and other inhibitors of SERCA-type Ca(2+)/ATPases. The caffeine/TMB-8-sensitive Ca(2+) stores are also involved in spontaneous Ca(2+) signalling and the maintenance of prolonged GH release.     

Involvement of phospholipase C and intracellular calcium signaling in the gonadotropin-releasing hormone regulation of prolactin release from lactotrophs of tilapia (Oreochromis mossambicus)

Gonadotropin-releasing hormone (GnRH) is a potent stimulator of prolactin (PRL) secretion in various vertebrates including the tilapia, Oreochromis mossambicus. The mechanism by which GnRH regulates lactotroph cell function is poorly understood. Using the advantageous characteristics of the teleost pituitary gland from which a nearly pure population of PRL cells can be isolated, we examined whether GnRH might stimulate PRL release through an increase in phospholipase C (PLC), inositol triphosphate (IP3), and intracellular calcium (Ca(i)2+) signaling. Using Ca(i)2+ imaging and the calcium-sensitive dye fura-2, we found that chicken GnRH-II (cGnRH-II) induced a rapid dose-dependent increase in Ca(i)2+ in dispersed tilapia lactotrophs. The Ca(i)2+ signal was abolished by U-73122, an inhibitor of PLC-dependent phosphoinositide hydrolysis. Correspondingly, cGnRH-II-induced tPRL188 secretion was inhibited by U-73122, suggesting that activation of PLC mediates cGnRH-II's stimulatory effect on PRL secretion. Pretreatment with 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8), an inhibitor of Ca2+ release from intracellular stores, impeded the effect of cGnRH-II on Ca(i)2+. To further address the possible involvement of intracellular Ca2+ stores, IP3 concentrations in the tilapia rostral pars distalis (RPD containing 95-99% PRL cells) was determined by a radioreceptor assay. We found that GnRH-II induces a rapid (<5min) and sustained increase in IP3 concentration in the RPD. Secretion of tPRL(188) in response to cGnRH-II was suppressed by Ca2+ antagonists (TMB-8 and nifedipine). These data, along with our previous findings that show PRL release increases with a rise in Ca(i)2+, suggest that GnRH may elicit its PRL releasing effect by increasing Ca(i)2+. Furthermore, the rise in Ca(i)2+ may be derived from PLC/IP3-induced mobilization of Ca2+ from intracellular stores along with influx through L-type voltage-gated Ca2+ channels.     

Mechanisms of cholecystokinin-induced calcium mobilization in gastric antral interstitial cells of Cajal

AIM:To investigate the effect of sulfated cholecystokinin-8(CCK-8S) on calcium mobilization in cultured murine gastric antral interstitial cells of Cajal(ICC) and its possible mechanisms.METHODS:ICC were isolated from the gastric antrum of mice and cultured.Immunofluorescence staining with a monoclonal antibody for c-Kit was used to identify ICC.The responsiveness of ICC to CCK-8S was measured using Fluo-3/AM based digital microfluorimetric measurement of intracellular Ca2+ concentration([Ca2+]i).A confocal laser scanning microscope was used to monitor [Ca2+]i changes.The selective CCK1 receptor antagonist lorglumide,the intracellular Ca2+-ATPase inhibitor thapsigargin,the type Ⅲ inositol 1,4,5-triphosphate(InsP3) receptor blocker xestospongin C and the L-type voltage-operated Ca2+ channel inhibitor nifedipine were used to examine the mecha-nisms of [Ca2+]i elevation caused by CCK-8S.Immunoprecipitation and Western blotting were used to determine the regulatory effect of PKC on phosphorylation of type Ⅲ InsP3 receptor(InsP3R3) in ICC.Protein kinase C(PKC) activator phorbol 12-myristate 13-acetate(PMA) and inhibitor chelerythrine were used to assess the role of PKC in the CCK-8S-evoked [Ca2+]i increment of ICC.RESULTS:ICC were successfully isolated from the gastric antrum of mice and cultured.Cultured ICC were identified by immunofluorescence staining.When given 80 nmol/L or more than 80 nmol/L CCK-8S,the [Ca2+]i in ICC increased and 100 nmol/L CCK-8S significantly increased the mean [Ca2+]i by 59.30% ± 4.85%(P 0.01).Pretreatment of ICC with 5 μmol/L lorglumide inhibited 100 nmol/L CCK-8S-induced [Ca2+]i increment from 59.30% ± 4.85% to 14.97% ± 9.05%(P 0.01),suggesting a CCK1R-mediated event.Emptying of intracellular calcium stores by thapsigargin(5 μmol/L) prevented CCK-8S(100 nmol/L) from inducing a [Ca2+]i increase.Moreover,pretreatment with xestospongin C(1 μmol/L) could also abolish the CCK-8S-induced effect,indicating that Ca2+ release from InsP3R-operated stores appeared to be a major mechanism responsible for CCK-8S-induced calcium mobilization in ICC.On the other hand,by removing extracellular calcium or blocking the L-type voltage-operated calcium channel with nifedipine,a smaller but significant rise in the [Ca2+]i could be still elicited by CCK-8S.These data suggest that the [Ca2+]i release is not stimulated or activated by the influx of extracellular Ca2+ in ICC,but the influx of extracellular Ca2+ can facilitate the [Ca2+]i increase evoked by CCK-8S.CCK-8S increased the phosphorylation of InsP3R3,which could be prevented by chelerythrine.Pretreatment with lorglumide(5 μmol/L) could significantly reduce the CCK-8S intensified phosphorylation of InsP3R3.In the positive control group,treatment of cells with PMA also resulted in an enhanced phosphorylation of InsP3R3.Pretreatment with various concentrations of PMA(10 nmol/L-10 μmol/L) apparently inhibited the effect of CCK-8S and the effect of100 nmol/L PMA was most obvious.Likewise,the effect of CCK-8S was augmented by the pretreatment with chelerythrine(10 nmol/L-10 μmol/L) and 100 nmol/L chelerythrine exhibited the maximum effect.CONCLUSION:CCK-8S increases [Ca2+]i in ICC via the CCK1 receptor.This effect depends on the release of InsP3R-operated Ca2+ stores,which is negatively regulated by PKC-mediated phosphorylation of InsP3R3.     

Role of Ca2+ stores in dopamine- and PACAP-evoked growth hormone release in goldfish

Growth hormone (GH) secretion, evoked by either pituitary adenylate cyclase-activating polypeptide (PACAP) or dopamine (DA), is dependent on both voltage-sensitive calcium channels (VSCC) and cAMP signaling in goldfish. We further characterized the involvement of Ca2+ in evoked release by PACAP and DA, by examining the sensitivity of evoked GH release to perturbations of Ca2+ signaling. Both VSCC and calmodulin/calmodulin-dependent kinase are involved in PACAP signaling as had been shown for DA. In spite of this apparent dependence on VSCC, blockade of TMB-8 but not ryanodine-sensitive intracellular Ca2+ stores inhibited both PACAP- and DA-evoked GH release. Using sarcoplasmic/endoplasmic reticulum Ca-ATPases (SERCA) inhibitors, we found BHQ blocked, whereas thapsigargin (Tg) enhanced stimulated GH release, suggesting that Tg-sensitive SERCA may counteract these cAMP-mobilizing neuroendocrine regulators by sequestering [Ca2+]i. As GH secretion stimulated by two endogenous gonadotropin-releasing hormones is not affected by Tg, it appears that distinct multiple Ca2+ stores mediate the hormone releasing response to different neuroendocrine regulators.     

Depletion of intracellular Ca2+ stores sensitizes the flow-induced Ca2+ influx in rat endothelial cells

Hemodynamic shear stress elicits a rise in endothelial [Ca2+]i, which may serve as a key second messenger to regulate many flow-associated physiological and biochemical processes. In the present study, we used Mn2+ quenching of fluorescent dye Fluo3 as an assay to investigate the Ca2+ influx of rat aortic endothelial cells in response to flow. We found that the Ca2+ signaling in response to flow could be greatly influenced by the status of intracellular Ca2+ stores. Depletion of intracellular Ca2+ stores by thapsigargin (4 micromol/L) or cyclopiazonic acid (10 micromol/L) drastically sensitized the Ca2+ influx in response to flow. Ca2+-mobilizing agonist bradykinin (100 nmol/L) or ATP (100 micromol/L) had similar sensitizing effect. The effect of bradykinin or ATP was blocked by Xestospongin C and U73122, suggesting that the sensitization was related to the IP3-mediated store depletion. On the other hand, the Mn2+ quenching in response to flow was greatly reduced by ochratoxin A (100 nmol/L), an agent that could increase the filling state of intracellular Ca2+ stores. In addition, we found that depletion-sensitized Ca2+ influx in response to flow was mediated by a PKG-inhibitable cation channel and that the influx was affected by membrane potential and K+ channel activity. In conclusion, the present study argues for a critical role of intracellular Ca2+ status in determining the Ca2+ signaling in response to flow and it provides a general mechanistic explanation for the stimulatory role of blood-borne agonists on flow-induced Ca2+ influx.     

Corticotropin-releasing hormone and calcium signaling in corticotropes

Corticotropin-releasing hormone (CRH) stimulates ACTH secretion from anterior pituitary corticotropes, largely, but possibly not exclusively, via activation of the adenylyl cyclase cascade. CRH stimulates secretion by increasing Ca²⁺ influx and by Ca²⁺-independent mechanisms. As Ca²⁺ influx is largely regulated by membrane electrical properties, we review the effects of CRH on membrane excitability and changes in cytosolic Ca²⁺. We also speculate on possible pathways for CRH modulation of exocytosis by Ca²⁺ independent mechanisms.