Nifedipine reveals the existence of two discrete components of the progesterone-induced [Ca2+]i transient in human spermatozoa

The steroid progesterone, an agonist of acrosome reaction, induces a biphasic [Ca(2+)](i)-signal in human sperm comprising an initial transient [Ca(2+)](i) elevation, and a subsequent ramp or plateau. Nifedipine, an inhibitor of voltage-operated Ca(2+) channels, inhibits progesterone-induced acrosome reaction in human sperm, but fluorimetric studies have detected no effect of this compound on the progesterone-induced [Ca(2+)](i) signal. We have used single-cell imaging to study the effects of nifedipine on [Ca(2+)](i) signalling in human sperm. Analysis of mean responses from large numbers of cells showed that treatment with nifedipine reduced the duration but not the amplitude of the progesterone-induced [Ca(2+)](i) transient. In control cells, the latency of the transient peak (maximum fluorescence) fell within the range of 30-105 s. In the presence of nifedipine, very few cells peaked "late" (>60 s after application of progesterone). Analysis of transient responses in control cells revealed characteristic "early" and "late" responses, most cells showing both "early" and "late" transients, whereas "late" transients were rare and smaller in the presence of nifedipine. Sustained responses showed strong association with late transients, and occurrence and amplitude of sustained responses were significantly reduced in nifedipine pretreated cells.These findings are consistent with the occurrence of a discrete, nifedipine-sensitive component of the progesterone-induced [Ca(2+)](i) transient that peaks 1-2 min after exposure to the hormone and is crucial for the induction of the sustained [Ca(2+)](i) signal.     

Patterns of [Ca2+](i) mobilization and cell response in human spermatozoa exposed to progesterone

Human spermatozoa stimulated with progesterone (a product of the cumulus and thus encountered by sperm prior to fertilization in vivo) apparently mobilize Ca(2+) and respond very differently according to the way in which the steroid is presented. A progesterone concentration ramp (0-3 microM) induces [Ca(2+)](i) oscillations (repetitive store mobilization) which modify flagellar beating, whereas bolus application of micromolar progesterone causes a single large transient (causing acrosome reaction) which is apparently dependent upon Ca(2+) influx. We have investigated Ca(2+)-mobilization and functional responses in human sperm exposed to 3 muM progesterone. The [Ca(2+)](i) response to progesterone was abolished by 4 min incubation in 0 Ca(2+) medium (2 mM EGTA) but in nominally Ca(2+)-free medium (no added Ca(2+); 0 EGTA) a smaller, slow response occurred. Single cell imaging showed a similar effect of nominally Ca(2+)-free medium and approximately 5% of cells generated a small transient even in the presence of EGTA. When cells were exposed to EGTA-containing saline (5 min) and then returned to nominally Ca(2+)-free medium before stimulation, the [Ca(2+)](i) transient was greatly delayed (approximately 50 s) and rise time was doubled in comparison to cells not subjected to EGTA pre-treatment. We conclude that mobilization of stored Ca(2+) contributes a 'slow' component to the progesterone-induced [Ca(2+)](i) transient and that incubation in EGTA-buffered saline is able rapidly to deplete this store. Analysis of flagellar activity induced by 3 muM progesterone showed an effect (modified beating) associated with the [Ca(2+)](i) transient, in >80% of cells bathed in nominally Ca(2+)-free medium. This was reduced greatly in cells subjected to 5 min EGTA pre-treatment. The store-mediated transient showed a pharmacological sensitivity similar to that of progesterone-induced [Ca(2+)](i) oscillations (consistent with filling of the store by an SPCA) suggesting that the transient induced by micromolar progesterone is a 'single shot' activation of the same store that generates Ca(2+) oscillations.     

Progesterone-induced increase of sperm cytosolic calcium is enhanced by previous fusion of spermatozoa to prostasomes

Ejaculated spermatozoa must undergo a number of modifications before becoming able to fertilize the oocyte. The interaction of sperm with other semen components may influence these phenomena; human semen contains vesicles of prostatic origin, called prostasomes that may fuse to sperm at slightly acidic to neutral pH values.Prostasomes contain calcium and it has been demonstrated that their fusion with spermatozoa produces a transient increase (wave) of [Ca(2+)](i) in these cells. The fusion process also transfers protein and lipid to spermatozoa. These phenomena may induce long-lasting changes of sperm properties. We test the hypothesis that spermatozoa, as modified by fusion, change their ability to undergo the progesterone-induced increase of [Ca(2+)](i) and we find that the increase of [Ca(2+)](i) produced by the fusion with prostasomes and by the stimulation with progesterone are independent and additive phenomena. We also find that spermatozoa present a stronger response to the progesterone-induced increase of [Ca(2+)](i) if they are previously made to fuse with prostasomes. This effect does not depend directly on the [Ca(2+)](i) increase due to fusion, since it is still present after the [Ca(2+)](i) has returned to resting values.     

Kinetics of the progesterone-induced acrosome reaction and its relation to intracellular calcium responses in individual human spermatozoa

Progesterone at 3 microM triggers a biphasic (transient and sustained) increase in intracellular calcium ([Ca(2+)](i)) in human sperm, which is believed to be a prerequisite for progesterone-induced acrosome reaction (AR). As very little is known about how AR occurrence, latency, and completion relate to the characteristics of the progesterone-induced [Ca(2+)](i) signal, we examined these events using fluorescence microscopy of individual living human sperm. Direct assessment of acrosomal status after calcium imaging showed no differences in kinetics or amplitude of the preceding progesterone-induced calcium responses in acrosome-reacted and acrosome-intact cells, which indicates that the amplitude of the [Ca(2+)](i) signal is not the critical determinant of AR. Chelation of extracellular calcium to arrest AR at varying times after progesterone stimulation revealed that maximal AR occurred immediately following progesterone stimulation, during the initial transient calcium influx rather than during the sustained calcium response. Attempts to follow acrosomal dispersal in real-time by staining with the acidic organelle probes LysoTracker DND-99 and dapoxyl (2-aminoethyl) sulphonamide (DAES) proved inconclusive due to heterogeneous labeling of the cell population. Surprisingly, the dye was often not confined to the acrosome but stained the whole sperm head, which suggests that only a subpopulation of human sperm cells contains a sufficiently acidic acrosome.     

Characterization of intracellular Ca(2+) increase in response to progesterone and cyclic nucleotides in mouse spermatozoa

Rises in intracellular Ca(2+) concentration ([Ca(2+)](i)) caused by progesterone, an inducer of the acrosome reaction, or by cyclic nucleotides, possible second messengers, were investigated by Ca(2+) imaging of the head of individual mouse sperm. Progesterone induced a [Ca(2+)](i) rise in a dose-dependent manner (4-40 microM), primarily in the postacrosomal region. For 20-microM progesterone, Ca(2+) responses occurred in 42% of sperm, separated into two types: transient type (60% of responding cells; duration, 1-1.5 min; mean amplitude, 335 nM) and prolonged type (40%; >3 min; 730 nM). Prolonged responses required higher doses of progesterone, and their occurrence was enhanced significantly by preincubation for 2-4 h as compared with transient responses. 8-Bromo-cGMP (0.3-3 mM) induced a [Ca(2+)](i) rise more effectively than did 8-bromo-cAMP. For 1-mM 8-bromo-cGMP, 90% of cells exhibited transient Ca(2+) responses (approximately 1 min; 220 nM), independently of the preincubation time. In Ca(2+)-free medium, most sperm showed no Ca(2+) response to progesterone and 8-bromo-cGMP. Pimozide, a Ca(2+) channel blocker, completely blocked prolonged responses and partially inhibited transient responses. These results suggest that progesterone activates at least two distinct Ca(2+) influx pathways, with fast or slow inactivation kinetics, and some sperm show both types of response. A cyclic nucleotide-mediated process could participate in the progesterone-induced [Ca(2+)](i) rise.     

The non-genomic rapid acidification in peripheral T cells by progesterone depends on intracellular calcium increase and not on Na+/H+-exchange inhibition

Progesterone is an endogenous immunomodulator that is able to suppress T cell activation during pregnancy. An increased intracellular free calcium concentration ([Ca(2+)](i)), acidification, and an inhibition of Na(+)/H(+)-exchange 1 (NHE1) are associated with this progesterone rapid non-genomic response that involves plasma membrane sites. Such acidification, when induced by phytohemagglutinin, is calcium dependent in PKC down-regulated T cells. We investigated the relationship between this rapid response involving the [Ca(2+)](i) increase and various membrane progesterone receptors (mPRs). In addition, we explored whether the induction of acidification in T cells by progesterone is a direct result of the [Ca(2+)](i) increase. The results show that the intracellular calcium elevation caused by progesterone is inhibited by SKF96365, U73122, and 2-APB, but not by pertussis toxin or U73343. The elevation is enhanced by the protein tyrosine kinase inhibitor staurosporine and the protein kinase C inhibitors Ro318220 and Go6983. These findings suggest that progesterone does not stimulate the [Ca(2+)](i) increase via the Gi coupled mPR(α). Furthermore, progesterone-induced acidification was found to be dependent on Ca(2+) entry and blocked by the inorganic channel blocker, Ni(2+). However, BAPTA, an intracellular calcium chelator, was found to prevent progesterone-induced acidification but not the inhibition of NHE1. This implies that acidification by progesterone is a direct result of the [Ca(2+)](i) increase and does not directly involve NHE1. Taken together, further investigations are needed to explore whether one or more mPRs or PGRMC1 are involved in bringing about the T cell rapid response that results in the [Ca(2+)](i) increase and inhibition of NHE1.     

Stimulation of human spermatozoa with progesterone gradients to simulate approach to the oocyte. Induction of [Ca(2+)](i) oscillations and cyclical transitions in flagellar beating

Progesterone is present at micromolar concentrations in the cumulus matrix, which surrounds mammalian oocytes. Exposure of human spermatozoa to a concentration gradient of progesterone (0-3 microM) to simulate approach to the oocyte induced a slowly developing increase in [Ca(2+)](i) upon which, in many cells, slow oscillations were superimposed. [Ca(2+)](i) oscillations often started at very low progesterone (<10 nm), and their frequency did not change during the subsequent rise in concentration. Oscillations also occurred, but in a much smaller proportion of cells, in response to stepped application of progesterone (3 microM). When progesterone was removed, [Ca(2+)](i) oscillations often persisted or quickly resumed. Superfusion with low-Ca(2+) bathing medium (no added Ca(2+)) did not prevent [Ca(2+)](i) oscillations, but they could be abolished by addition of EGTA or La(3+). Inhibitors of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPases or inositol trisphosphate signaling had no effect on [Ca(2+)](i) oscillations, but pharmacological manipulation of ryanodine receptors affected both their frequency and amplitude. Staining of live spermatozoa with BODIPY FL-X ryanodine showed localization of ryanodine binding primarily to the caudal part of the head and mid-piece. [Ca(2+)](i) oscillations did not induce acrosome reaction, but in cells generating oscillations, the flagellar beat mode alternated in synchrony with the oscillation cycle. Flagellar bending and lateral movement of the sperm head during [Ca(2+)](i) peaks were markedly increased compared with during [Ca(2+)](i) troughs. This alternating pattern of activity is likely to facilitate zona penetration. These observations show that progesterone initiates unusual and complex store-mediated [Ca(2+)](i) signaling in human spermatozoa and identify a previously unrecognized effect of progesterone in regulating sperm "behavior" during fertilization.     

Stretch-elicited calcium responses in the intact mouse thoracic aorta

Stretch-elicited intracellular calcium ([Ca(2+)](i)) changes in individual smooth muscle cells in a ring of aorta were measured simultaneously with the force developed by the ring. A phasic increase in [Ca(2+)](i) was observed in 30% of the cells and a sustained one in 10%. Depletion of intracellular calcium store by thapsigargin and caffeine decreased phasic and increased sustained calcium responses. The inhibition of calcium entry either by stretching the aorta in a calcium-free medium or by the inhibition of stretch-activated, non-selective cationic channels by 5 microM GsMtx-4 toxin, decreased the proportion of sustained [Ca(2+)](i) responses but increased transient responses. In this condition, a third of the cells responded to stretch by a bursts of [Ca(2+)](i) spikes. The decrease of calcium influx triggered the generation of burst of calcium spikes after the application of stretch steps to the vascular wall. We conclude that progressive recruitment of smooth muscle cells is the mechanism underlying the force-generating part of the myogenic response. Two types of stretch-elicited calcium responses were observed during the recruitment of the smooth muscle cells. One was a phasic calcium discharge generated by the sarcoplasmic reticulum. The second was a tonic response produced by the activation of the stretch-sensitive cationic channels allowing extracellular Ca(2+) entry.     

Endothelin-1 increases intracellular Ca(2+) in rabbit pulmonary artery smooth muscle cells through phospholipase C

In freshly isolated rabbit pulmonary artery smooth muscle cells, endothelin (ET)-1 induced a transient increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) followed by a return to the initial [Ca(2+)](i). This response was not abolished by the voltage-dependent Ca(2+) channel blocker nicardipine or removal of Ca(2+) from the bath solution but was inhibited by ryanodine and thapsigargin. This finding suggested that the increase in [Ca(2+)](i) induced by ET-1 was attributable to release of Ca(2+) from ryanodine- and inositol 1,4,5-trisphosphate-sensitive intracellular Ca(2+) stores. The transient increase in [Ca(2+)](i) induced by ET-1 was also inhibited by pretreatment with antagonists of ET type A and B (ET(A) and ET(B)) receptors (BQ-123 and BQ-788, respectively). Furthermore, the ET(B) receptor agonist IRL-1620 induced an increase in [Ca(2+)](i) that was followed by a sustained increase in [Ca(2+)](i); the sustained increase in [Ca(2+)](i) was blocked by nicardipine. Using the nystatin-perforated patch-clamp technique, we found that IRL-1620 caused an increase in Ca(2+) current that was inhibited by addition of ET-1. ET-1 did not inhibit Ca(2+) current when cells were pretreated with BQ-123. These results suggested that when both receptor types are activated, the opposing responses lead to abolition of the sustained [Ca(2+)](i) increases induced by ET(B) receptor activation. Western blot analysis confirmed expression of ET(A) and ET(B) receptors. Finally, U-73122 inhibited the ET-1-induced [Ca(2+)](i) increase, indicating that phospholipase C was involved in modulation of the ET-1-induced [Ca(2+)](i) increase in rabbit pulmonary artery smooth muscle cells.     

Disturbances in purinergic [Ca2+]i signaling pathways in a transformed rat thyroid cell line

It was previously shown that in rat thyroid PC-Cl3 cell line, a purinergic P2Y receptor increases the concentration of free cytosolic Ca(2+) ([Ca(2+)](i)) via phospholipase C activation. We here studied whether in a transformed cell line (PC-E1Araf) derived from parental PC-Cl3 cells, ATP is still able to transduce the [Ca(2+)](i)-based intracellular signal.We demonstrate the expression of mRNA for P2Y2 in both PC-Cl3 and PC-E1Araf cells; mRNAs for P2Y1, P2Y4, P2Y6 and P2Y11 were absent. In both cell lines activation of P2Y2 receptor provokes a transient increase in [Ca(2+)](i) followed by a lower sustained phase persisting for over 5min in PC-Cl3 and only 1.5 min in PC-E1Araf cells. In both cell lines the [Ca(2+)](i) reached a plateau level significantly higher than the basal [Ca(2+)](i) level persisting for over 10 min. Removal of extracellular Ca(2+) reduced the initial transient response to ATP in PC-Cl3, but not in PC-E1Araf cells, and completely abolished the plateau phase in both cell lines.In the presence of extracellular Ca(2+) thapsigargin (TG) caused a rise in [Ca(2+)](i) significantly higher in PC-Cl3 than transformed PC-E1Araf cells, while in Ca(2+)-free medium the effect of TG was similar in both cell lines. The capacitative Ca(2+)-entry in PC-Cl3 resulted significantly higher than in PC-E1Araf cells.Further studies were performed in order to investigate whether the different effects of ATP on [Ca(2+)](i) was due to variation in divalent cation plasma membrane permeability. PC-E1Araf cells showed a much lower permeability to Ca(2+), Ba(2+), Sr(2+), Mn(2+), and Co(2+) that may be responsible for the differences in purinergic Ca(2+) signaling pathway with respect to parental PC-Cl3 cells.     

Intracellular calcium responses in bovine oocytes induced by spermatozoa and by reagents

The objectives of the present study were to clarify and compare the characteristics of the transient rises in intracellular calcium concentrations ([Ca2+]i) induced either by spermatozoa or by stimulation with artificial activators in bovine oocytes. These transient rises in [Ca2+]i in oocytes matured in vitro were recorded with Ca2+ imaging using the Ca2+ indicator fura-2. During fertilization, a series of transient rises in [Ca2+]i was observed. The first Ca2+ response peaked at a concentration of 521 +/- 39 nM (n = 20) and lasted for 4 min, while the subsequent Ca2+ responses were significantly smaller and shorter, with a peak of 368 +/- 13 nM (n = 23) and a duration of 2 min. Injection of inositol 1,4,5- triphosphate (InsP3) into unfertilized oocytes caused a transient rise in [Ca2+]i in a dose-dependent manner. The maximum response was induced by 20 nA x 1 sec injection of InsP3. Thimerosal, a sulfhydryl reagent, induced the repetitive transient rises in [Ca2+]i. The peak and the duration of the rises in [Ca2+]i induced by InsP3 or thimerosal were smaller and shorter, respectively, than those of the first rise induced by spermatozoa. Ethanol and Ca2+ ionophore IA23187, which are general parthenogenetic activators of unfertilized oocytes, each induced a single transient rise in [Ca2+]i. The duration of the rise in [Ca2+]i by ethanol or Ca2+ ionophore was significantly longer than that by spermatozoa at fertilization, although the peaks were smaller. These results clarified the characteristics of the rises in [Ca2+]i induced by spermatozoa and by several artificial reagents, and showed that the first rise in [Ca2+]i induced by spermatozoa had a higher peak [Ca2+]i and a longer duration compared with each the subsequent rises in [Ca2+]i and the rises in [Ca2+]i induced by artificial reagents. These indicate that a mode like as the first rise in [Ca2+]i induced by spermatozoa is an effective trigger for artificial activation of oocytes.     

Receptor-mediated increase in cytoplasmic free calcium required for activation of pathogen defense in parsley

Transient influx of Ca(2+) constitutes an early element of signaling cascades triggering pathogen defense responses in plant cells. Treatment with the Phytophthora sojae-derived oligopeptide elicitor, Pep-13, of parsley cells stably expressing apoaequorin revealed a rapid increase in cytoplasmic free calcium ([Ca(2+)](cyt)), which peaked at approximately 1 microM and subsequently declined to sustained values of 300 nM. Activation of this biphasic [Ca(2+)](cyt) signature was achieved by elicitor concentrations sufficient to stimulate Ca(2+) influx across the plasma membrane, oxidative burst, and phytoalexin production. Sustained concentrations of [Ca(2+)](cyt) but not the rapidly induced [Ca(2+)](cyt) transient peak are required for activation of defense-associated responses. Modulation by pharmacological effectors of Ca(2+) influx across the plasma membrane or of Ca(2+) release from internal stores suggests that the elicitor-induced sustained increase of [Ca(2+)](cyt) predominantly results from the influx of extracellular Ca(2+). Identical structural features of Pep-13 were found to be essential for receptor binding, increases in [Ca(2+)](cyt), and activation of defense-associated responses. Thus, a receptor-mediated increase in [Ca(2+)](cyt) is causally involved in signaling the activation of pathogen defense in parsley.     

Norepinephrine induces slow calcium signalling in murine brown preadipocytes through the beta-adrenoceptor/cAMP/protein kinase A pathway

The mechanism of adrenergically activated calcium signalling in isolated murine brown preadipocytes (stromal-vascular fraction) was studied with Fura-2. Norepinephrine (NE) generated in preadipocytes a slow Ca(2+)-response ( approximately 10 nM/min) without a burst and a maximum, whereas in mature brown adipocytes, the quick burst reached 1.5 microM [Ca(2+)](i). Thapsigargin, which is known to discharge Ca(2+) ions from the IP(3)-sensitive stores, initiated a huge capacitative calcium entry in mature brown adipocytes but failed to stimulate a response in preadipocytes. The beta-selective antagonist nadolol almost completely prevented the effect of NE on [Ca(2+)](i), while the antagonist of alpha-adrenoceptors phentolamine caused only a approximately 25% reduction of the cellular response. Forskolin or the cell-permeable Br-cAMP caused [Ca(2+)](i) rise, which were even higher than with NE. The protein kinase A (PKA) inhibitor N-[2-(p-bromocynnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89) reduced and the phosphodiesterase inhibitors 3-isobutyl-1-methylxanthine (IBMX), N-cyclohexyl-N-(2-hydroxyethyl)-4-(6-(1,2-dihydro-2-oxoquinolyloxy))butyramide (OPC-3911), 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidone (Ro 20-1724) or the protein phosphatase inhibitor okadaic acid enhanced the NE-, isoproterenol- or forskolin-initiated cellular calcium responses. It was concluded that (i) brown preadipocytes lacked a trigger mechanism of initiation of [Ca(2+)](i) rises and (ii) the cAMP- and protein kinase A-mediated phosphorylation played an important role in the beta-adrenoceptor-initiated calcium signalling in these cells. All these features distinguish brown adipocyte precursors from differentiated brown adipocytes, where calcium signalling is initiated exclusively via alpha(1)-adrenoceptors and the trigger mechanism.     

Mobilization of intracellular Ca(2+) by endothelin-1 in rat intrapulmonary arterial smooth muscle cells

Endothelin-1 (ET-1) increases intracellular Ca(2+) concentration ([Ca(2+)](i)) in pulmonary arterial smooth muscle cells (PASMCs); however, the mechanisms for Ca(2+) mobilization are not clear. We determined the contributions of extracellular influx and intracellular release to the ET-1-induced Ca(2+) response using Indo 1 fluorescence and electrophysiological techniques. Application of ET-1 (10(-10) to 10(-8) M) to transiently (24-48 h) cultured rat PASMCs caused concentration-dependent increases in [Ca(2+)](i). At 10(-8) M, ET-1 caused a large, transient increase in [Ca(2+)](i) (>1 microM) followed by a sustained elevation in [Ca(2+)](i) (<200 nM). The ET-1-induced increase in [Ca(2+)](i) was attenuated (<80%) by extracellular Ca(2+) removal; by verapamil, a voltage-gated Ca(2+)-channel antagonist; and by ryanodine, an inhibitor of Ca(2+) release from caffeine-sensitive stores. Depleting intracellular stores with thapsigargin abolished the peak in [Ca(2+)](i), but the sustained phase was unaffected. Simultaneously measuring membrane potential and [Ca(2+)](i) indicated that depolarization preceded the rise in [Ca(2+)](i). These results suggest that ET-1 initiates depolarization in PASMCs, leading to Ca(2+) influx through voltage-gated Ca(2+) channels and Ca(2+) release from ryanodine- and inositol 1,4,5-trisphosphate-sensitive stores.     

Nitric oxide and fusion with prostasomes increase cytosolic calcium in progesterone-stimulated sperm

Spermatozoa must undergo a number of reactions before they are able to fertilize the oocyte. Among these is the acrosome reaction, which is related to an increase in cytosolic Ca2+ concentration ([Ca2+]i). It has been reported in the literature that progesterone may achieve this effect through the intervention of extragenomic receptors. Nitric oxide (NO) has been reported to affect spermatozoa; the nature of the effect depends on the concentration of the radical. In a previous paper, we reported that the fusion of spermatozoa with prostasomes may also produce a transient increase in spermatozoa [Ca2+]i; in addition, this phenomenon causes a long-lasting effect that influences the action of progesterone. In this paper, we test the effects of a NO donor (CysNO) and of fusion of the prostasome to spermatozoa on progesterone-induced [Ca2+]i increase. No effect at all was noticed in the absence of progesterone stimulation. In the presence of the hormone, both CysNO and fusion increased the progesterone effect. This phenomenon was much more evident if the two treatments were used together. We conclude that both NO and fusion with prostasomes act on the progesterone-dependent pathway additively. Probably the effects are independent.     

Increase of [Ca(2+)](i) via activation of ATP receptors in PC-Cl3 rat thyroid cell line.

In PC-Cl3 rat thyroid cell line, ATP and UTP provoked a transient increase in [Ca(2+)](i), followed by a lower sustained phase. Removal of extracellular Ca(2+) reduced the initial transient response and completely abolished the plateau phase. Thapsigargin (TG) caused a rapid rise in [Ca(2+)](i) and subsequent addition of ATP was without effect. The transitory activation of [Ca(2+)](i) was dose-dependently attenuated in cells pretreated with the specific inhibitor of phospholipase C (PLC), U73122. These data suggest that the ATP-stimulated increment of [Ca(2+)](i) required InsP(3) formation and binding to its specific receptors in Ca(2+) stores. Desensitisation was demonstrated with respect to the calcium response to ATP and UTP in Fura 2-loaded cells. Further studies were performed to investigate whether the effect of ATP on Ca(2+) entry into PC-Cl3 cells was via L-type voltage-dependent Ca(2+) channels (L-VDCC) and/or by the capacitative pathway. Nifedipine decreased ATP-induced increase on [Ca(2+)](i). Addition of 2 mM Ca(2+) induced a [Ca(2+)](i) rise after pretreatment of the cells with TG or with 100 microM ATP in Ca(2+)-free medium. These data indicate that Ca(2+) entry into PC-Cl3 stimulated with ATP occurs through both an L-VDCC and through a capacitative pathway. Using buffers with differing Na(+) concentrations, we found that the effects of ATP were dependent of extracellular Na(+), suggesting that a Na(+)/Ca(2+) exchange mechanism is also operative. These data suggest the existence, in PC-Cl3 cell line, of a P2Y purinergic receptor able to increase the [Ca(2+)](i) via PLC activation, Ca(2+) store depletion, capacitative Ca(2+) entry and L-VDCC activation.     

Differential effects of progesterone and 17beta-estradiol on the Ca(2+) entry induced by thapsigargin and endothelin-1 in in situ endothelial cells

The effects of progesterone and 17beta-estradiol on Ca(2+) signaling in in situ endothelial cells were investigated using front-surface fluorometry of fura-2-loaded strips of porcine aortic valve. Progesterone inhibited the thapsigargin-induced sustained [Ca(2+)](i) elevation (IC(50)=33.9 microM, n=4), while 17beta-estradiol added a transient [Ca(2+)](i) elevation. Progesterone and 17beta-estradiol had no significant effect on the thapsigargin-induced [Ca(2+)](i) elevations in the absence of extracellular Ca(2+). A Mn(2+)-induced decline of fluorescent intensity at 360 nm excitation was accelerated by thapsigargin. This acceleration was completely reversed by progesterone, but not by 17beta-estradiol. Progesterone inhibited, and 17beta-estradiol enhanced the endothelin-1 (ET-1)-induced [Ca(2+)](i) elevation, while both had no effect on the ET-1-induced Ca(2+) release observed in the absence of extracellular Ca(2+) or in the pertussis toxin-treated strips. Progesterone and 17beta-estradiol thus had different effects on Ca(2+) signaling, especially on Ca(2+) influx, in endothelial cells.     

Glucose-insensitivity induced by Ca(2+) toxicity in islet beta-cells and its prevention by PACAP

The present study examined whether a sustained increase in cytosolic Ca(2+) concentration ([Ca(2+)](i)) causes glucose-insensitivity in beta-cells and whether it could be modulated by pituitary adenylate cyclase-activating polypeptide (PACAP), a pancreatic insulinotropin. Rat single beta-cells were cultured for 2 days with sustained increases in [Ca(2+)](i), followed by determination of the [Ca(2+)](i) response to glucose (8.3 mM) as monitored with fura-2. High K(+) (25 mM) produced sustained increases in [Ca(2+)](i) in beta-cells, which were inhibited by nifedipine, a Ca(2+) channel blocker. After culture with high K(+), the incidence and amplitude of [Ca(2+)](i) responses to glucose were markedly reduced. This glucose-insensitivity was prevented by the presence of nifedipine or PACAP-38 (10(-13) M and 10-9) M) in high K(+) culture. PACAP-38 attenuated high K(+)-induced [Ca(2+)](i) increases. In conclusion, sustained increases in [Ca(2+)](i) induce glucose-insensitivity (Ca(2+) toxicity in beta-cells) and it is prevented by PACAP possibly in part due to its Ca(2+)-reducing capacity.