Early activation of Ca(2+)-permeable AMPA receptors reduces neurite outgrowth in embryonic chick retinal neurons.

Calcium entry through Ca(2+)-permeable AMPA/kainate receptors may activate signaling cascades controlling neuronal development. Using the fluorescent Ca(2+)-indicator Calcium Green 1-AM we showed that the application of kainate or AMPA produced an increase of intracellular [Ca(2+)] in embryonic chick retina from day 6 (E6) onwards. This Ca(2+) increase is due to entry through AMPA-preferring receptors, because it was blocked by the AMPA receptor antagonist GYKI 52466 but not by the N-methyl-D-aspartic acid (NMDA) receptor antagonist AP5, the voltage-gated Ca(2+) channel blockers diltiazem or nifedipine, or by the substitution of Na+ for choline in the extracellular solution to prevent the depolarizing action of kainate and AMPA. In dissociated E8 retinal cultures, application of glutamate, kainate, or AMPA reduced the number of neurites arising from these cells. The effect of kainate was prevented by the AMPA/kainate receptor antagonist CNQX and by GYKI 52466 but not by AP5, indicating that the reduction in neurite outgrowth resulted from the activation of AMPA receptors. Blocking Ca(2+) influx through L-type voltage-gated Ca(2+) channels with diltiazem and nifedipine prevented the effect of 10-100 microM kainate but not that of 500 microM kainate. In addition, joro spider toxin-3, a blocker of Ca(2+)-conducting AMPA receptors, prevented the effect of all doses of kainate. Neither GABA, which is depolarizing at this age in the retina, nor the activation of metabotropic glutamate receptors with tACPD mimicked the effects of AMPA receptor activation. Calcium entry via AMPA receptor channels themselves may therefore be important in the regulation of neurite outgrowth in developing chick retinal cells.     

Arachidonic acid is a physiological activator of the ryanodine receptor in pancreatic beta-cells

Pancreatic beta-cells have ryanodine receptors but little is known about their physiological regulation. Previous studies have shown that arachidonic acid releases Ca(2+) from intracellular stores in beta-cells but the identity of the channels involved in the Ca(2+) release has not been elucidated. We studied the mechanism by which arachidonic acid induces Ca(2+) concentration changes in pancreatic beta-cells. Cytosolic free Ca(2+) concentration was measured in fura-2-loaded INS-1E cells and in primary beta-cells from Wistar rats. The increase of cytosolic Ca(2+) concentration induced by arachidonic acid (150microM) was due to both Ca(2+) release from intracellular stores and influx of Ca(2+) from extracellular medium. 5,8,11,14-Eicosatetraynoic acid, a non-metabolizable analogue of arachidonic acid, mimicked the effect of arachidonic acid, indicating that arachidonic acid itself mediated Ca(2+) increase. The Ca(2+) release induced by arachidonic acid was from the endoplasmic reticulum since it was blocked by thapsigargin. 2-Aminoethyl diphenylborinate (50microM), which is known to inhibit 1,4,5-inositol-triphosphate-receptors, did not block Ca(2+) release by arachidonic acid. However, ryanodine (100microM), a blocker of ryanodine receptors, abolished the effect of arachidonic acid on Ca(2+) release in both types of cells. These observations indicate that arachidonic acid is a physiological activator of ryanodine receptors in beta-cells.     

NTP,the photoproduct of nifedipine,activates caffeine-sensitive ion channels in leech neurons

Leech P neurons possess caffeine-sensitive ion channels in intracellular Ca(2+) stores and in the plasma membrane. The following results indicate that these channels are also activated by 2,6-dimethyl-4-(2-nitrosophenyl)-3,5-pyridinedicarboxylic acid dimethyl ester (NTP), the photoproduct of the L-type Ca(2+) channel-blocker nifedipine: (1) Just like caffeine, NTP evoked Ca(2+) influx and intracellular Ca(2+) release, as well as the influx of various other divalent cations and that of Na(+). (2) In the presence of high NTP or caffeine concentrations the plasma membrane channels close, suggesting desensitization of the channel-activating mechanism. (3) Depending on the concentration, NTP and caffeine induce cross-desensitization or act additively. (4) NTP was effective in the same neurons as caffeine (P, N, Leydig, 101), and it was ineffective in neurons in which caffeine was also ineffective (AP, T, L, 8, AE). (5) In Retzius neurons, NTP and caffeine evoked intracellular Ca(2+) release but no Ca(2+) influx. Despite these parallels, the effects of NTP and caffeine were not identical, which may be due to differences in the mechanisms of channel activation or desensitization and/or to substance-specific side effects. The caffeine-sensitive ion channels were activated by NTP concentrations > or =10 microM, which is almost three orders of magnitude smaller than the threshold concentration of caffeine.     

Two modes of secretion in pancreatic acinar cells: involvement of phosphatidylinositol 3-kinase and regulation by capacitative Ca(2+) entry

In pancreatic acinar cells, muscarinic agonists stimulate both the release of Ca(2+) from intracellular stores and the influx of extracellular Ca(2+). The part played by Ca(2+) released from intracellular stores in the regulation of secretion is well established; however, the role of Ca(2+) influx in exocytosis is unclear. Recently, we observed that supramaximal concentrations of acetylcholine (>or=10 microM) elicited an additional component of exocytosis despite reducing Ca(2+) influx. In the present study, we found that supramaximal exocytosis was substantially inhibited (approximately 70%) by wortmannin (100 nM), an inhibitor of phosphatidylinositol 3-kinase. In contrast, exocytosis evoked by a lower concentration of acetylcholine (1 microM) was potentiated (approximately 45%) by wortmannin. Exocytosis stimulated by 1 microM acetylcholine in the absence of extracellular Ca(2+) was, like supramaximal exocytosis, inhibited by wortmannin. The switch to a wortmannin-inhibitable form of exocytosis depended upon a reduction in Ca(2+) entry through store-operated Ca(2+) channels, as the switch in exocytotic mode could also be brought about by the selective blockade of these channels by Gd(3+) (2 microM), but not by inhibition of noncapacitative Ca(2+) entry by SB203580 (10 microM). We conclude that supramaximal doses of acetylcholine lead to a switch in the mode of zymogen granule exocytosis by inhibiting store-dependent Ca(2+) influx.     

Capsazepine elevates intracellular Ca2+ in human osteosarcoma cells, questioning its selectivity as a vanilloid receptor antagonist

Capsazepine is thought to be a selective antagonist of vanilloid type 1 receptors; however, its other in vitro effect on different cell types is unclear. In human MG63 osteosarcoma cells, the effect of capsazepine on intracellular Ca(2+) concentrations ([Ca(2+)](i)) and cytotoxicity was explored by using fura-2 and tetrazolium, respectively. Capsazepine caused a rapid rise in [Ca(2+)](i) in a concentration-dependent manner with an EC(50) value of 100 microM. Capsazepine-induced [Ca(2+)](i) rise was partly reduced by removal of extracellular Ca(2+), suggesting that the capsazepine-induced [Ca(2+)](i) rise was composed of extracellular Ca(2+) influx and intracellular Ca(2+). In Ca(2+)-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+)-ATPase, caused a monophasic [Ca(2+)](i) rise, after which the increasing effect of capsazepine on [Ca(2+)](i) was inhibited by 75%. Conversely, pretreatment with capsazepine to deplete intracellular Ca(2+) stores totally prevented thapsigargin from releasing more Ca(2+). U73122, an inhibitor of phospholipase C, abolished histamine (an inositol 1,4,5-trisphosphate-dependent Ca(2+) mobilizer)-induced, but not capsazepine-induced, [Ca(2+)](i) rise. Overnight treatment with 1-100 microM capsazepine inhibited cell proliferation in a concentration-dependent manner. These findings suggest that in human MG63 osteosarcoma cells, capsazepine increases [Ca(2+)](i) by stimulating extracellular Ca(2+) influx and also by causing intracellular Ca(2+) release from the endoplasmic reticulum via a phospholiase C-independent manner. Capsazepine may be mildly cytotoxic.     

Glutamate-mediated extrasynaptic inhibition: direct coupling of NMDA receptors to Ca(2+)-activated K+ channels

NMDA receptors (NMDARs) typically contribute to excitatory synaptic transmission in the CNS. While Ca(2+) influx through NMDARs plays a critical role in synaptic plasticity, direct actions of NMDAR-mediated Ca(2+) influx on neuronal excitability have not been well established. Here we show that Ca(2+) influx through NMDARs is directly coupled to activation of BK-type Ca(2+)-activated K+ channels in outside-out membrane patches from rat olfactory bulb granule cells. Repetitive stimulation of glutamatergic synapses in olfactory bulb slices evokes a slow inhibitory postsynaptic current (IPSC) in granule cells that requires both NMDARs and BK channels. The slow IPSC is enhanced by glutamate uptake blockers, suggesting that extrasynaptic NMDARs underlie the response. These findings reveal a novel inhibitory action of extrasynaptic NMDARs in the brain.     

Functional voltage-gated Ca2+ channels in muscle fibers of the platyhelminth Dugesia tigrina

The presence and function of voltage-gated Ca(2+) channels were examined in individual muscle fibers freshly dispersed from the triclad turbellarian Dugesia tigrina. Individual muscle fibers contracted in response to elevated extracellular K(+) in a concentration-dependent fashion. These depolarization-induced contractions were blocked by extracellular Co(2+) (2.5 mM), suggesting that they were dependent on depolarization-induced Ca(2+) influx across the sarcolemma. A voltage-gated inward current was apparent in whole cell recordings when the outward K(+) current was abolished by replacement of intracellular K(+) by Cs(+). This inward current was amplified with increasing concentration (相似文献   

Neurotransmitter release from bovine adrenal chromaffin cells is modulated by capacitative Ca(2+)entry driven by depleted internal Ca(2+)stores

Two potential mechanisms by which the intracellular Ca(2 stores might modulate catecholamine release from bovine adrenal chromaffin cells were investigated: (i) that the cytosolic Ca(2+)transient caused by Ca(2+)release from the intracellular stores recruits additional chromaffin granules to a readily releasable pool that results in augmented catecholamine release when this is subsequently evoked, and (ii) that the Ca(2+)influx that follows depletion of intracellular stores (i.e. store-operated Ca(2+)entry) triggers release per se thereby augmenting evoked catecholamine release. When histamine or caffeine were applied in Ca(2+)-free perfusion media, a transient elevation of intracellular free Ca(2+)occurred owing to mobilization of Ca(2+)from the stores. When Ca(2+)was later readmitted to the perfusing fluid there followed a prompt and maintained rise in intracellular Ca(2+)concentrations of magnitude related to the degree of store mobilization. In parallel experiments, increased catecholamine secretion was measured under the conditions when Ca(2+)influx following store-mobilization occurred. Furthermore, the size of the catecholamine release increment correlated with the degree of Ca(2+)influx. Store-operated Ca(2+)entry evoked by mobilization with histamine and/or caffeine did not augment nicotine-evoked secretion per se; that is, it augmented evoked catecholamine release only to the extent that it increased basal catecholamine release. The nicotine-evoked catecholamine release was sensitive to cytosolic BAPTA, which, at the concentration used (50 microM BAPTA-AM), reduced release by approximately 25%. However, the increment in basal catecholamine release which followed Ca(2+)influx triggered by Ca(2+)store mobilization was not reduced by intracellular BAPTA. This finding is inconsistent with the hypothesis that the elevated cytosolic Ca(2+)from store mobilization recruits additional vesicles of catecholamine to the sub-plasmalemmal release sites to augment subsequently evoked secretion. This position is supported by the observation that histamine (10 microM) in Ca(2+)-free medium caused a pronounced elevation of cytosolic free Ca(2+), but this caused no greater catecholamine release when Ca(2+)was re-introduced than did prior exposure to Ca(2+)-free medium alone, which caused no elevation of cytosolic free Ca(2+). It is concluded that intracellular Ca(2+)stores can modulate secretion of catecholamines from bovine chromaffin cells by permitting Ca(2+)influx through a store-operated entry pathway. The results do not support the notion that the Ca(2+)released from intracellular stores plays a significant role in the recruitment of vesicles into the ready-release pool under the experimental conditions reported here.     

Lysophosphatidic acid-induced Ca(2+) mobilization in the neural retina of chick embryo

Lysophosphatidic acid (LPA) plays various roles in the regulation of cell growth as a lipid mediator. We studied the effect of LPA on intracellular Ca(2+) concentration ([Ca2+]i) with Fura-2 in the neural retina of chick embryo during neurogenesis. Bath application of LPA (1-100 microM) to the embryonic day 3 (E3) chick retina caused an increase in [Ca2+](i) in a dose-dependent manner, with an EC(50) value of 9.2 microM. The Ca(2+) rise was also evoked in a Ca(2+)-free medium, suggesting that release of Ca(2+) from intracellular Ca(2+) stores (Ca(2+) mobilization) was induced by LPA. U-73122, a blocker of phospholipase C (PLC), inhibited the Ca(2+) rise to LPA. Pertussis toxin partially inhibited the Ca(2+) rise to LPA, indicating that G(i)/G(o) protein was at least partially involved in the LPA response. The developmental profile of the LPA response was studied from E3 to E13. The Ca(2+) rise to LPA declined drastically from E3 to E7, in parallel with decrease in mitotic activity of retinal progenitor cells. The signal transduction pathway and developmental profile of the Ca(2+) response to LPA were the same as those of the Ca(2+) response to adenosine triphosphate (ATP), which enhances the proliferation of retinal progenitor cells. The coapplication of LPA with ATP resulted in enhancement of Ca(2+) rise in the E3 chick retina. Our results show that LPA induces Ca(2+) mobilization in the embryonic chick retina during neurogenesis.     

Inhibition of H2 histamine receptor-mediated cation channel opening by protein kinase C in human promyelocytic cells

Histamine, through H(2) receptors, triggers a prominent rise in intracellular free Ca(2+) concentration ([Ca(2+)](i)) in addition to an elevation of cAMP level in HL-60 promyelocytes. Here we show that the histamine-induced [Ca(2+)](i) rise was due to influx of Ca(2+) from the extracellular space, probably through nonselective cation channels, as incubation of the cells with SKF 96365 abolished the histamine-induced [Ca(2+)](i) rise, Na(+) influx, and membrane depolarization. The Ca(2+) influx was specifically inhibited by pretreatment of the cells with PMA or extracellular ATP with 50% inhibitory concentrations of 0.12 +/- 0.03 nM and 185 +/- 17 microM, respectively. Western blot analysis of protein kinase C (PKC) isoforms revealed that PMA (< or =1 nM) and ATP (300 microM) caused selective translocation of PKC-delta to the particulate/membrane fraction. Costimulation of the cells with histamine and SKF 96365 partially reduced histamine-induced granulocytic differentiation, which was evaluated by looking at the extent of fMet-Leu-Phe-induced [Ca(2+)](i) rise and superoxide generation. In conclusion, nonselective cation channels are opened by stimulation of the H(2) receptor, and the channels are at least in part involved in the induction of histamine-mediated differentiation processes. Both effects of histamine were selectively inhibited probably by the delta isoform of PKC in HL-60 cells.     

Survival promotion of mesencephalic dopaminergic neurons by depolarizing concentrations of K+ requires concurrent inactivation of NMDA or AMPA/kainate receptors

The death of dopaminergic neurons that occurs spontaneously in mesencephalic cultures was prevented by depolarizing concentrations of K+ (20-50 mM). However, unlike that observed previously in other neuronal populations of the PNS or CNS, promotion of survival required concurrent blockade of either NMDA or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate receptors by the specific antagonists, MK-801 and GYKI-52466, respectively. Rescued neurons appeared to be healthy and functional because the same treatment also dramatically enhanced their capacity to accumulate dopamine. The effects on survival and uptake were rather specific to dopaminergic neurons, rapidly reversible and still observed when treatment was delayed after plating. Glutamate release increased substantially in the presence of elevated concentrations of K+, and chronic treatment with glutamate induced a loss of dopaminergic neurons that was prevented by MK-801 or GYKI-52466 suggesting that an excitotoxic process interfered with survival when only the depolarizing treatment was applied. The effects of the depolarizing stimulus in the presence of MK-801 were mimicked by BAY K-8644 and abolished by nifedipine, suggesting that neuroprotection resulted from Ca(2+) influx through L-type calcium channels. Measurement of intracellular calcium revealed that MK-801 or GYKI-52466 were required to maintain Ca(2+) levels within a trophic range, thus preventing K+-induced excitotoxic stress and Ca(2+) overload. Altogether, our results suggest that dopaminergic neurons may require a finely tuned interplay between glutamatergic receptors and calcium channels for their development and maturation.     

The anti-anginal drug fendiline elevates cytosolic Ca(2+) in rabbit corneal epithelial cells

The effect of the anti-anginal drug fendiline on intracellular free Ca(2+) levels ([Ca(2+)](i)) in a rabbit corneal epithelial cell line (SIRC) was explored using fura-2 as a fluorescent Ca(2+) indicator. At a concentration above 1 microM, fendiline increased [Ca(2+)](i) in a concentration-dependent manner with an EC(50) value of 7 microM. The [Ca(2+)](i) response consisted of an immediate rise and an elevated phase. Extracellular Ca(2+) removal decreased half of the [Ca(2+)](i )signal. Fendiline induced quench of fura-2 fluorescence by Mn(2+) (50 microM), suggesting the presence of Ca(2+) influx across the plasma membrane. This Ca(2+) influx was abolished by La(3+) (50 microM), but was insensitive to dihydropyridines, verapamil and diltiazem. Fendiline (10 microM)-induced store Ca(2+) release was largely reduced by pretreatment with thapsigargin (1 microM) (an endoplasmic reticulum Ca(2+) pump inhibitor) to deplete the endoplasmic reticulum Ca(2+). Conversely, pretreatment with 10 microM fendiline abolished thapsigargin-induced Ca(2+) release. Fendiline (10 microM)-induced Ca(2+) release was not altered by inhibiting phospholipase C with 2 microM 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122). Cumulatively, this study shows that fendiline induced concentration-dependent [Ca(2+)](i )increases in corneal epithelial cells by releasing the endoplasmic reticulum Ca(2+) in a phospholipase C-independent manner, and by causing Ca(2+) influx.     

Pathways of cadmium influx in mammalian neurons

The influx of the toxic cation Cd2+ was studied in fura 2-loaded rat cerebellar granule neurons. In cells depolarized with Ca2(+)-free, high-KCI solutions, the fluorescence emission ratio (R) increased in the presence of 100 microM Cd2(+). This increase was fully reversed by the Cd2+ chelator tetrakis(2-pyridylmethyl)ethylenediamine, indicating a cadmium influx into the cell. The rate of increase, dR/dt, was greatly reduced (67+/-5%) by 1 microM nimodipine and enhanced by 1 microM Bay K 8644. Concurrent application of nimodipine and omega-agatoxin IVA (200 nM) blocked Cd2+ permeation almost completely (88+/-5%), whereas omega-conotoxin MVIIC (2 microM) reduced dR/dt by 24+/-8%. These results indicate a primary role of voltage-dependent calcium channels in Cd2+ permeation. Stimulation with glutamate or NMDA and glycine also caused a rise of R in external Cd2+. Simultaneous application of nimodipine and omega-agatoxin IVA moderately reduced dR/dt (25+/-3%). NMDA-driven Cd2(+) entry was almost completely prevented by 1 mM Mg2+, 50 microM memantine, and 10 microM 5,7-dichlorokynurenic acid, suggesting a major contribution of NMDA-gated channels in glutamate-stimulated Cd2+ influx. Moreover, perfusion with alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate caused a slow increase of R. These results suggest that Cd2+ permeates the cell membrane mainly through the same pathways of Ca2+ influx.     

Calcium-induced calcium release in neurosecretory insect neurons: fast and slow responses

The dynamics of intracellular free Ca(2+)([Ca(2+)](i)) changes were investigated in dorsal unpaired median (DUM) neurons of the cockroach Periplaneta americana. Activation of voltage-gated Ca(2+) channels caused a steep increase in [Ca(2+)](i). Depolarizations lasting for < 100ms led to Ca(2+) release from intracellular stores as is indicated by the finding that the rise of [Ca(2+)](i) was greatly reduced by the antagonists of ryanodine receptors, ryanodine and ruthenium red. There is a resting Ca(2+)current which is potentiated on application of a neuropeptide, Neurohormone D (NHD), a member of the adipokinetic hormone family. Ca(2+) influx enhanced in this way again caused a rise of [Ca(2+)](i) sensitive to ryanodine and ruthenium red. Such rises developed and relaxed much more slowly than the depolarization-induced signals. Ca(2+)responses similar to those induced by NHD were obtained with the ryanodine receptor agonists caffeine (20mM) and cADP-ribose (cADPR, 100nM). These Ca(2+) responses, however, varied considerably in size and kinetics, and part of the cells did not respond at all to caffeine or cADPR. Such cells, however, produced Ca(2+) rises after having been treated with NHD. Thus, the variability of Ca(2+) signals might be caused by different filling states of Ca(2+) stores, and the resting Ca(2+) current seems to represent a source to fill empty Ca(2+) stores. In line with this notion, block of the endoplasmic Ca(2+) pump by thapsigargin (1 microM) produced either no or largely varying Ca(2+) responses. The Ca(2+) signals induced by caffeine and cADPR displayed different sensitivity to ryanodine receptor blockers. cADPR failed to elicit any response when ryanodine or ruthenium red were present. By contrast, the response to caffeine, in the presence of ryanodine, was only reduced by about 50% and, in the presence of ruthenium red, it was not at all reduced. Thus, there may be different types of Ca(2+) release channels. Block of mitochondrial Ca(2+) uptake with carbonyl cyanide m -chlorophenylhydrazone (CCCP, 1 microM) completely abolished cADPR-induced Ca(2+) signals, but it did not affect the caffeine-induced signals. Taken together our findings seem to indicate that there are different stores using different Ca(2+) uptake pathways and that some of these pathways involve mitochondria.     

Functional TRPV4 channels are expressed in human airway smooth muscle cells

Hypotonic stimulation induces airway constriction in normal and asthmatic airways. However, the osmolarity sensor in the airway has not been characterized. TRPV4 (also known as VR-OAC, VRL-2, TRP12, OTRPC4), an osmotic-sensitive cation channel in the transient receptor potential (TRP) channel family, was recently cloned. In the present study, we show that TRPV4 mRNA was expressed in cultured human airway smooth muscle cells as analyzed by RT-PCR. Hypotonic stimulation induced Ca(2+) influx in human airway smooth muscle cells in an osmolarity-dependent manner, consistent with the reported biological activity of TRPV4 in transfected cells. In cultured muscle cells, 4alpha-phorbol 12,13-didecanoate (4-alphaPDD), a TRPV4 ligand, increased intracellular Ca(2+) level only when Ca(2+) was present in the extracellular solution. The 4-alphaPDD-induced Ca(2+) response was inhibited by ruthenium red (1 microM), a known TRPV4 inhibitor, but not by capsazepine (1 microM), a TRPV1 antagonist, indicating that 4-alphaPDD-induced Ca(2+) response is mediated by TRPV4. Verapamil (10 microM), an L-type voltage-gated Ca(2+) channel inhibitor, had no effect on the 4-alphaPDD-induced Ca(2+) response, excluding the involvement of L-type Ca(2+) channels. Furthermore, hypotonic stimulation elicited smooth muscle contraction through a mechanism dependent on membrane Ca(2+) channels in both isolated human and guinea pig airways. Hypotonicity-induced airway contraction was not inhibited by the L-type Ca(2+) channel inhibitor nifedipine (1 microM) or by the TRPV1 inhibitor capsazepine (1 microM). We conclude that functional TRPV4 is expressed in human airway smooth muscle cells and may act as an osmolarity sensor in the airway.     

Effect of Y-24180 on Ca2+ movement and proliferation in renal tubular cells

In canine renal tubular cells, the effect of Y-24180, a presumed specific platelet activating factor (PAF) receptor antagonist, on intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured by using fura-2 as a Ca(2+)-sensitive fluorescent probe. Y-24180 (0.1-10 microM) caused a rapid and sustained [Ca(2+)](i) rise in a concentration-dependent manner. The [Ca(2+)](i) rise was prevented by 30% by removal of extracellular Ca(2+), but was not changed by dihydropyridines, verapamil and diltiazem. Y-24180-induced Ca(2+) influx was confirmed by Mn(2+)-influx induced quench of fura-2 fluorescence. In Ca(2+)-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+)-ATPase, caused a monophasic [Ca(2+)](i) rise, after which the increasing effect of 5 microM Y-24180 on [Ca(2+)](i) was abolished; conversely, depletion of Ca(2+) stores with 5 microM Y-24180 abolished thapsigargin-induced [Ca(2+)](i) rise. U73122, an inhibitor of phoispholipase C, inhibited ATP-, but not Y-24180-induced [Ca(2+)](i) rise. Overnight treatment with Y-24180 did not alter cell proliferation rate. Collectively, these results suggest that Y-24180 acts as a potent, but not cytotoxic, Ca(2+) mobilizer in renal tubular cells, by stimulating both extracellular Ca(2+) influx and intracellular Ca(2+) release. Since alterations in Ca(2+) movement may interfere many cellular signaling processes unrelated to modulation of PAF receptors, caution must be applied in using this chemical as a selective PAF receptor antagonist.     

Modulation of Ca2+ signals by phosphatidylinositol-linked novel D1 dopamine receptor in hippocampal neurons

Recent evidence indicates the existence of a putative novel phosphatidylinositol-linked D1 dopamine receptor in brain that mediates phosphatidylinositol hydrolysis via activation of phospholipase Cbeta. The present work was designed to characterize the Ca(2+) signals regulated by this phosphatidylinositol-linked D(1) dopamine receptor in primary cultures of hippocampal neurons. The results indicated that stimulation of phosphatidylinositol-linked D1 dopamine receptor by its newly identified selective agonist SKF83959 induced a long-lasting increase in basal [Ca(2+)](i) in a time- and dose-dependent manner. Stimulation was observable at 0.1 microm and reached the maximal effect at 30 microm. The [Ca(2+)](i) increase induced by 1 microm SKF83959 reached a plateau in 5 +/- 2.13 min, an average 96 +/- 5.6% increase over control. The sustained elevation of [Ca(2+)](i) was due to both intracellular calcium release and calcium influx. The initial component of Ca(2+) increase through release from intracellular stores was necessary for triggering the late component of Ca(2+) rise through influx. We further demonstrated that activation of phospholipase Cbeta/inositol triphosphate was responsible for SKF83959-induced Ca(2+) release from intracellular stores. Moreover, inhibition of voltage-operated calcium channel or NMDA receptor-gated calcium channel strongly attenuated SKF83959-induced Ca(2+) influx, indicating that both voltage-operated calcium channel and NMDA receptor contribute to phosphatidylinositol-linked D(1) receptor regulation of [Ca(2+)](i).     

Calcium dependence of the thrombin-induced increase in endothelial albumin permeability

We examined whether the increase in endothelial albumin permeability induced by alpha-thrombin is dependent on extracellular Ca2+ influx. Permeability of 125I-albumin across confluent monolayers of cultured bovine pulmonary artery endothelial cells was measured before and after the addition of 0.1 microM alpha-thrombin. In the presence of normal extracellular Ca2+ concentration ([Ca2+]o, 1000 microM), alpha-thrombin produced a 175 +/- 10% increase in 125I-albumin permeability. At lower [Ca2+]o (100, 10, 1, or less than 1 microM), alpha-thrombin caused a 140% increase in permeability (P less than 0.005). LaCl3 (1 mM), which competes for Ca2+ entry, blunted 38% of the increase in permeability. Preloading endothelial monolayers with quin2 to buffer cytosolic Ca2+ (Cai2+) produced a dose-dependent inhibition of the increase in 125I-albumin permeability. Preincubation with nifedipine or verapamil was ineffective in reducing the thrombin-induced permeability increase. A 60 mM K+ isosmotic solution did not alter base-line endothelial permeability. alpha-Thrombin increased [Ca2+]i in a dose-dependent manner and the 45Ca2+ influx rate. Extracellular medium containing 60 mM K+ did not increase 45Ca2+ influx, and nifedipine did not block the rise in 45Ca2+ influx caused by alpha-thrombin. Ca2+ flux into endothelial cells induced by alpha-thrombin does not occur through voltage-sensitive channels but may involve receptor-operated channels. In conclusion, the increase in endothelial albumin permeability caused by alpha-thrombin is dependent on Ca2+ influx and intracellular Ca2+ mobilization.     

Induction of a sodium ion influx by progesterone in human spermatozoa

In human spermatozoa, progesterone (P(4)) induces a depolarization of the plasma membrane, a rapid calcium (Ca(2+)) influx, and a chloride efflux. The sodium ion (Na(+)) was partly responsible for the P(4)-induced depolarizing effect but was not required for calcium influx. We used fluorescent probes for spectrofluorometry to investigate whether P(4) induced a Na(+) influx and whether voltage-operated channels were involved in Na(+) and/or Ca(2+) entries. We found that 10 microM P(4) significantly increased intracellular Na(+) concentration from 17.8 +/- 2.0 mM to 27.2 +/- 1. 6 mM (P < 0.001). Prior incubation of spermatozoa with 10 microM flunarizine, a Na(+) and Ca(2+) voltage-dependent channel blocker, inhibited the sodium influx induced by 10 microM P(4) by 84.6 +/- 15.4%. The Ca(2+) influx induced by 10 microM P(4) was also significantly inhibited in a Na(+)-containing medium by 10 microM flunarizine or 10 microM pimozide (P < 0.01). In contrast, flunarizine had no inhibitory effect on the Ca(2+) influx induced by 10 microM P(4) in spermatozoa incubated in Na(+)-depleted medium. The P(4)-promoted acrosome reaction (AR) was significantly higher when spermatozoa were incubated in Na(+)-containing medium as compared to Na(+)-depleted medium. These data demonstrate that P(4) stimulates a Na(+) influx that could be involved in the AR completion. They also suggest that voltage-dependent Na(+) and Ca(2+) channels are implicated in P(4)-mediated signaling pathway in human spermatozoa.     

Regulation of store-operated calcium channels by the intermediary metabolite pyruvic acid

A rise in cytosolic Ca(2+) concentration is used as a key activation signal in virtually all animal cells, where it triggers a range of responses, including neurotransmitter release, muscle contraction, and cell growth and proliferation. A major route for Ca(2+) influx is through store-operated Ca(2+) channels. One important intracellular target for Ca(2+) entry through store-operated channels is the mitochondrion, which increases aerobic metabolism and ATP production after Ca(2+) uptake. Here, we reveal a novel feedback pathway whereby pyruvic acid, a critical rate-limiting substrate for mitochondrial respiration, increases store-operated entry by reducing inactivation of the channels. Importantly, the effects of pyruvic acid are manifest at physiologically relevant concentrations and membrane potentials. The reduction in the inactivation of calcium release-activated calcium (CRAC) channels by pyruvate is highly specific in that it is not mimicked by other intermediary metabolic acids, does not require its metabolism, is independent of its Ca(2+) buffering action, and does not involve mitochondrial Ca(2+) uptake or ATP production. These results reveal a new and direct link between intermediary metabolism and ion-channel gating and identify pyruvate as a potential signaling messenger linking energy demand to calcium-channel function.