The role of calcium in follicle-stimulating hormone signal transduction in Sertoli cells.

Sertoli cells are hormonally regulated by follicle-stimulating hormone (FSH) acting upon a G-protein-linked cell surface FSH receptor. FSH increases intracellular cyclic AMP but the involvement of other signal transduction mechanisms including intracellular calcium in FSH action are not proven. Using freshly isolated rat Sertoli cells we measured cytosolic free ionized calcium levels by dual-wavelength fluorescence spectrophotometry using the calcium-sensitive fluorescent dye Fura2-AM. The cytosolic calcium concentration in unstimulated Sertoli cells was 89 +/- 2 nM (n = 151 experiments) and was markedly increased by either calcium channel ionophores (ionomycin, Bay K8644) or plasma membrane depolarization consistent with the presence of voltage-sensitive and -independent calcium channel in Sertoli cell membranes. Ovine FSH stimulated a specific, sensitive (ED50, 5.0 ng of S-16/ml), and dose-dependent (maximal at 20 ng/ml) rise in cytosolic calcium commencing within 60 s to reach levels of 192 +/- 31 nM after 180 s and lasting for at least 10 min. The effect of FSH was replicated by forskolin, cholera toxin, and dibutyryl cyclic AMP, suggesting that cyclic AMP may mediate the FSH-induced rise in cytosolic calcium. The FSH-induced rise in cytosolic calcium required extracellular calcium and was abolished by calcium channel blockers specific for dihydropyridine (verapamil, nicardipine), nonvoltage-gated (ruthenium red) or all calcium channels (cobalt). Thus FSH action on Sertoli cells involves a specific, rapid, and sustained increase in cytosolic calcium which requires extracellular calcium and involves both dihydropyridine-sensitive, voltage-gated calcium channels and voltage-independent, receptor-gated calcium channels in the plasma membranes of rat Sertoli cells. The replication by cyclic AMP of the effects of FSH suggests that calcium may be a signal-amplification or -modulating mechanism rather than an alternate primary signal transduction system for FSH in Sertoli cells.     

Effect of BHT 920 on calcium-activated K+ channels in renal epithelioid MDCK cells.

In Madin Darby canine kidney (MDCK) cells, epinephrine has been shown to increase intracellular calcium, activate calcium-dependent K+ channels and hyperpolarize the cell membrane. The present study has been performed to test for the possible involvement of alpha 2-adrenergic receptors. To this end, the effects of alpha 2-adrenoceptor agonist BHT 920 have been studied on cell membrane potential, ion channel activity and intracellular calcium: Similar to epinephrine, BHT 920 hyperpolarizes the cell membrane, increases intracellular calcium and activates inwardly rectifying K+ channels (single channel slope conductances 30-80 pS). Half-maximal hyperpolarization is achieved at concentrations between 10 and 100 nmol/l. The hyperpolarizing effect of BHT 920 is abolished in the presence of alpha 2-adrenoceptor antagonist yohimbine (100 nmol/l) but not in the presence of alpha 1-adrenoceptor antagonist prazosin (100 nmol/l). At extracellular calcium activity below 100 nmol/l BHT 920 still leads to a transient hyperpolarization of the cell membrane but, in contrast to epinephrine, is unable to significantly increase intracellular calcium or significantly activate the calcium-sensitive K+ channels. The observations indicate that stimulation of alpha 2-receptors participates in the epinephrine-induced increase of intracellular calcium, channel activation and hyperpolarization.     

Involvement of calcium in prothoracicotropic stimulation of ecdysone synthesis in Galleria mellonella

The cytosolic free calcium was measured with Fura-2 in single prothoracic gland cells of Galleria larvae. During the last two larval instars calcium concentration correlated with ecdysone secretion by the glands. Addition of prothoracicotropic hormone (PTTH) from brains of Galleria larvae to prothoracic glands in vitro induced a significant increase in calcium in the gland cells. This effect of PTTH was abolished by removal of extracellular calcium, or by the addition of lanthanum or of the calcium channel antagonists nicardipine and verapamil. The calcium channel agonist Bay K 8644 evoked an increase in intracellular calcium. TMB-8, an inhibitor of intracellular calcium mobilization, did not block the PTTH-stimulated rise in calcium concentration or ecdysone production, indicating that intracellular calcium stores are not involved in the calcium-mediated ecdysone synthesis. Moreover, PTTH seems to exert its action by influencing dihydropyridine-sensitive calcium channels in the plasma membrane. © 1996 Wiley-Liss, Inc.     

Maitotoxin-Induced Intracellular Calcium Rise in PC 12 Cells: Involvement of Dihydropyridine-Sensitive and ω-Conotoxin-Sensitive Calcium Channels and Phosphoinositide Breakdown

The biological activities of maitotoxin are strictly dependent on the extracellular calcium concentration and are always associated with an increase of the free cytosolic calcium level. We tested the effects of voltage-sensitive calcium channel blockers (nicardipine and omega-conotoxin) on maitotoxin-induced intracellular calcium increase, membrane depolarization, and inositol phosphate production in PC12 cells. Maitotoxin dose dependently increased the cytosolic calcium level, as measured by the fluorescent probe fura 2. This effect disappeared in a calcium-free medium; it was still observed in the absence of extracellular sodium and was enhanced by the dihydropyridine calcium agonist Bay K 8644. Nicardipine inhibited the effect of maitotoxin on intracellular calcium concentration in a dose-dependent manner. The maitotoxin-induced calcium rise was also reduced by pretreating cells with omega-conotoxin. Pretreatment of cells with maitotoxin did not modify 125I-omega-conotoxin and [3H]PN 200-110 binding to PC12 membranes. Nicardipine and omega-conotoxin inhibition of maitotoxin-evoked calcium increase was reduced by pertussis toxin pretreatment. Maitotoxin caused a substantial membrane depolarization of PC12 cells as assessed by the fluorescent dye bisoxonol. This effect was reduced by pretreating the cells with either nicardipine or omega-conotoxin and was almost completely abolished by the simultaneous pretreatment with both calcium antagonists. Maitotoxin stimulated inositol phosphate production in a dose-dependent manner. This effect was reduced by pretreating the cells with 1 microM nicardipine and was completely abolished in a calcium-free EGTA-containing medium. The findings on maitotoxin-induced cytosolic calcium rise and membrane depolarization suggest that maitotoxin exerts its action primarily through the activation of voltage-sensitive calcium channels, the increase of inositol phosphate production likely being an effect dependent on calcium influx. The ability of nicardipine and omega-conotoxin to inhibit the effect of maitotoxin on both calcium homeostasis and membrane potential suggests that L- and N-type calcium channel activation is responsible for the influx of calcium following exposure to maitotoxin, and not that a depolarization of unknown nature causes the opening of calcium channels.     

Intracellular calcium in prothoracic glands of Manduca sexta

Cytosolic free calcium was measured in individual prothoracic gland cells of Manduca larvae with Fura-2. During the last larval instar there was no correlation between intracellular calcium concentration and ecdysteroid secretion by the glands. The addition of prothoracicotropic hormone (PTTH) from brains of Manduca larvae to prothoracic glands in vitro resulted in a significant increase in the calcium concentration of the gland cells. The effect of PTTH was inhibited by the inorganic calcium channel antagonists, cadmium, lanthanum and nickel, and by the antagonist of T-type calcium channels, amiloride, whereas all the other antagonists tested failed to block the action of PTTH. TMB-8, an inhibitor of intracellular calcium mobilization, did not reduce the PTTH-induced rise in calcium, which suggests that IP(3)-dependent intracellular calcium stores are not involved in the calcium-mediated stimulation of ecdysteroid synthesis. Moreover, PTTH is thought to increase intracellular calcium in prothoracic glands of Manduca by influencing calcium channels in the plasma membrane.     

The relative contributions of extracellular and intracellular calcium to secretion from tumor mast cells. Multiple effects of the proton ionophore carbonyl cyanide m-chlorophenylhydrazone

The proton ionophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) inhibited antigen-stimulated secretion and calcium influx in rat basophilic leukemia cells. In a glucose-free solution the inhibitory effects of CCCP were due to a decrease in the intracellular ATP concentration; however, when glucose was present there was no decrease in ATP. Instead, we found that in a glucose-containing saline solution, CCCP inhibited antigen-stimulated calcium uptake because it depolarized the plasma membrane, which in rat basophilic leukemia cells inhibits antigen-stimulated calcium uptake. In the presence of glucose, relatively low concentrations of CCCP inhibited calcium uptake while higher concentrations were required to inhibit secretion. In contrast, the initial antigen-stimulated rise in cytoplasmic calcium, measured with the fluorescent calcium indicator quin2, was not inhibited by CCCP. This suggests that the release of calcium from intracellular stores might, in some cases, be sufficient to support antigen-stimulated secretion. In the presence of CCCP the pH gradient becomes important for regulating the membrane potential across the plasma membrane. When cells were depolarized with CCCP and the external pH was increased, the membrane potential returned to resting levels and antigen-stimulated calcium uptake was restored. Inhibition of antigen-stimulated secretion by higher concentrations of CCCP could also be reversed by increasing the external pH.     

Dephosphorylation of Synaptosomal Proteins P96 and P139 Is Regulated by Both Depolarization and Calcium, but Not by a Rise in Cytosolic Calcium Alone

Depolarization of intact synaptosomes activates calcium channels, leads to an influx of calcium, and increases the phosphorylation of several neuronal proteins. In contrast, there are two synaptosomal phosphoproteins labeled in intact synaptosomes with 32Pi, termed P96 and P139, which appear to be dephosphorylated following depolarization. Within intact synaptosomes P96 was found in the cytosol whereas P139 was present largely in membrane fractions. Depolarization-stimulated dephosphorylation was fully reversible and continued for up to five cycles of depolarization/repolarization, suggesting a physiological role for the phenomenon. The basal phosphorylation of these proteins was at least partly regulated by cyclic AMP, since dibutyryl cyclic AMP produced small but significant increases in P96 and P139 labeling, even in the presence of fluphenazine at concentrations that inhibited calcium-stimulated protein kinases. Depolarization-dependent dephosphorylation was independent of a rise in intracellular calcium, since agents such as guanidine and low concentrations of A23187, which increase intracellular calcium without activating the calcium channel, did not initiate P96 or P139 dephosphorylation. These agents did sustain increases in the phosphorylation of a number of other proteins including synapsin I and protein III. The results suggest that the phosphorylation of these two synaptosomal proteins is intimately linked to the membrane potential and that their dephosphorylation is dependent on both the mechanism of calcium entry and calcium itself, rather than simply on a rise in intracellular free calcium.     

Toluene inhibits voltage-sensitive calcium channels expressed in pheochromocytoma cells

Commercial solvents such as toluene are commonly used as drugs of abuse by children and adolescents. The cellular and molecular sites and mechanisms of actions of these compounds are not well studied but their effects on behavior resemble those of central nervous system depressants such as alcohol, barbiturates and benzodiazepines. In this study, the effects of toluene on voltage-sensitive calcium channels (VSCCs) were measured in pheochromocytoma cells. The KCl-induced rise in intracellular calcium as measured by calcium imaging was almost completely blocked by the dihydropyridine calcium channel antagonist nifedipine verifying that undifferentiated pheochromocytoma cells express mainly the L-type of calcium channel. Toluene (0.3–3000 μM) by itself did not affect intracellular calcium levels in resting cells but dose-dependently inhibited the KCl-induced rise in calcium. This inhibition was substantially reversed upon washout of the toluene-containing solution. KCl-dependent increases in intracellular calcium in cells differentiated with nerve growth factor (NGF) were largely insensitive to nifedipine. Toluene produced a greater inhibition of the KCl response in NGF treated cells as compared with undifferentiated cells. A similar finding was obtained when whole-cell patch-clamp-electrophysiology was used to directly monitor the effects of toluene on voltage-activated calcium currents in undifferentiated and differentiated cells. These results show that dihydropyridine sensitive and insensitive calcium channels are inhibited by toluene and may represent important sites of action for this compound.     

Casein binds to the cell membrane and induces intracellular calcium signals in the enteroendocrine cell: a brief communication

Dietary protein but not amino acids stimulates cholecystokinin (CCK) secretion in rat mucosal cells. However, the dietary protein sensory mechanisms and the intracellular signal pathway in the enteroendocrine cells have not yet been clarified. The relationship between dietary protein binding to cell membrane and intracellular calcium responses were examined in the CCK-producing enteroendocrine cell line STC-1. The binding of solubilized STC-1 cell membrane to proteins was analyzed using a surface plasmon resonance sensor. Intracellular calcium concentrations of STC-1 cell suspensions loaded with Fura-2 AM were measured using a spectrafluorophotometer system with continuous stirring. Intracellular calcium concentrations in STC-1 cells were increased by exposure to alpha-casein or casein sodium, but not to bovine serum albumin. Solubilized STC-1 membranes bound to alpha-casein and casein sodium but did not bind to bovine serum albumin. alpha-Casein demonstrated higher membrane binding and intracellular calcium stimulating activities than casein sodium. Thus, protein binding to the STC-1 cell membrane and intracellular calcium responses were correlated. Intracellular calcium responses to alpha-casein were suppressed by an L-type calcium channel blocker. These results suggest that casein, a dietary protein, binds to a putative receptor on the CCK-producing enteroendocrine cell membrane and elicits the subsequent intracellular calcium response via an L-type calcium channel.     

Calcium entry through receptor-operated channels in bovine pulmonary artery endothelial cells

The activation of endothelial cells by endothelium-dependent vasodilators has been investigated using bioassay, patch clamp and 45Ca flux methods. Cultured pulmonary artery endothelial cells have been demonstrated to release EDRF in response to thrombin, bradykinin, ATP and the calcium ionophore A23187. The resting membrane potential of the endothelial cells was -56 mV and the cells were depolarized by increasing extracellular K+ or by the addition of (0.1-1.0 mM)Ba2+ to the bathing solution. The electrophysiological properties of the cultured endothelial cells suggest that the membrane potential is maintained by an inward rectifying K+ channel with a mean single channel conductance of 35.6 pS. The absence of a depolarization-activated inward current and the reduction of 45Ca influx with high K+ solution suggests that there are no functional voltage-dependent calcium or sodium channels. Thrombin and bradykinin were shown to evoke not only an inward current (carried by Na+ and Ca2+) but also an increase in 45Ca influx suggesting that the increase in intracellular calcium necessary for EDRF release is mediated by an opening of a receptor operated channel. High doses of thrombin and bradykinin induced intracellular calcium release, however, at low doses of thrombin no intracellular calcium release was observed. We propose that the increased cytosolic calcium concentration in endothelial cells induced by endothelium dependent vasodilators is due to the influx of Ca2+ through a receptor operated ion channel and to a lesser degree to intracellular release of calcium from a yet undefined intracellular store.     

Role of Ca2+ and aquaporin-2 in regulation of basolateral water permeability of collecting ducts in the rat kidney

The role of AQP2,3 and intracellular calcium in vasopressin-induced increase in the water permeability of the basolateral cell membrane in microdissected rat kidney OMCD was studied. It was shown that increase in the water permeability of the basolateral membranes correlated with increase in the content of AQP2 and AQP3 in the membrane fraction isolated from outer kidney medulla. Preliminary loading of cells with BAPTA-AM which binds intracellular Ca2+ abolished the increase in the water permeability and prevented the rise of the AQP2 content in response to dDAVP. BAPTA was ineffective to block the enhancement of AQP2 content in membrane fraction in presence of dDAVP. These results suggest that the increase in intracellular calcium activity and the enhanced content of AQP2 in plasma membrane are important for the antidiuretic effect of dDAVP.     

Calcium transport in human salivary glands: a proposed model of calcium secretion into saliva

Salivary calcium plays a vital role in bio-mineralization of dental enamel and exposed dentin. In order to elucidate the yet unknown cellular and molecular mechanisms of calcium secretion in human salivary glands the presence of various relevant plasma membrane transport systems for calcium were investigated. Using an RT-PCR approach, expression of the epithelial calcium channel (CaT-Like), the calcium binding protein (calbindin-2), the endoplasmic reticulum pumps (SERCA-2 and -3), and the plasma membrane calcium ATPases (PMCA-1, -2, and -4), were found in parotid and submandibular glands. Immunohistochemistry revealed that CaT-Like is located in the basolateral plasma membrane of acinar cells; while calbindin-2, SERCA-2 and SERCA-3 were found inside the acinar cells; and PMCA-2 was found in the apical membrane and in the secretory canaliculi between the cells. Based on these findings, we propose the following model of calcium secretion in human salivary glands: (1) calcium enters the acinar cell at the basolateral side via calcium channel CaT-Like (calcium influx); (2) intracellular calcium is taken up into the endoplasmic reticulum by SERCA-2 and possibly SERCA3 or bound to calbindin-2 (intracellular calcium pool); and (3) calcium is secreted by PMCAs at the apical plasma membrane (calcium efflux).Evamaria Kinne-Saffran deceased on 6 December 2002     

Early response of cultured lepidopteran cells to exposure to delta-endotoxin from Bacillus thuringiensis: involvement of calcium and anionic channels.

The role of ion channels in the initial steps following exposure of SF-9 lepidopteran insect cells in culture to the delta-endotoxin CryIC from the insecticidal bacterium Bacillus thuringiensis was investigated using single ionic channel measurements and microspectrofluorescence of the calcium-sensitive probe fura-2. It was found that: (1) the toxin triggers an immediate rise in intracellular calcium; (2) the surge is due to calcium entering the cells via calcium channels; (3) the toxin recruits or introduces anionic channels in the cell's plasma membrane in a time-dependent manner. These channels, not seen in the absence of the toxin, are induced by toxin exposure to either side of the cell membrane. They have a conductance of 26 picosiemens (pS) and are mainly permeable to chloride. This study provides the first evidence of the primary role of calcium and chloride ions in the action of delta-endotoxin on cultured insect cells.     

Calcium waves induced by large voltage pulses in fish keratocytes.

Intracellular calcium waves in fish keratocytes are induced by the application of electric field pulses with amplitudes between 55 and 120 V/cm and full width at half-maximum of 65-100 ms. Calcium concentrations were imaged using two-photon excited fluorescence microscopy (Denk et al., 1990 Science. 248:73-76; Williams et al. 1994 FASEB J. 8:804-813) and the ratiometric calcium indicator indo-1. The applied electric field pulses induced waves with fast calcium rise times and slow decays, which nucleated in the lamellipodium at the hyperpolarized side of the cells and, less frequently, at the depolarized side. The effectiveness of wave generation was determined by the change induced in the membrane potential, which is about half the field strength times the cell width in the direction of the field. Stimulation of waves began at voltage drops across the cell above 150 mV and saturated at voltage drops above 300 mV, where almost all cells exhibited a wave. Waves were not induced in low-calcium media and were blocked by the nonselective calcium channel blockers cobalt chloride and verapamil, but not by specific organic antagonists of voltage-sensitive calcium channel conductance. Thapsigargin stopped wave propagation in the cell body, indicating that calcium release from intracellular stores is necessary. Thus a voltage pulse stimulates Ca2+ influx through calcium channels in the plasma membrane, and if the intracellular calcium concentration reaches a threshold, release from intracellular stores is induced, creating a propagating wave. These observations and the measured parameters (average velocity approximately 66 micron/s and average rise time approximately 68 ms) are consistent with a wave amplification model in which[equation, see text] determines the effective diffusivity of the propagating molecules, D approximately 300 micron2/s (Meyer, 1991. Cell. 64:675-678).     

Critical intracellular Ca2+ dependence of transient receptor potential melastatin 2 (TRPM2) cation channel activation

TRPM2 is a member of the melastatin-related TRP (transient receptor potential) subfamily. It is expressed in brain and lymphocytes and forms a cation channel that is activated by intracellular ADP-ribose and associated with cell death. In this study we investigated the calcium dependence of human TRPM2 expressed under a tetracycline-dependent promoter in HEK-293 cells. TRPM2 expression was associated with enhanced hydrogen peroxide-evoked intracellular calcium signals. In whole-cell patch clamp recordings, switching from barium- to calcium-containing extracellular solution markedly activated TRPM2 as long as ADP-ribose was in the patch pipette and exogenous intracellular calcium buffering was minimal. We suggest this effect reveals a critical dependence of TRPM2 channel activity on intracellular calcium. In the absence of extracellular calcium we observed concentration-dependent activation of TRPM2 channels by calcium delivered from the patch pipette (EC(50) 340 nM, slope 4.9); the maximum effect was at least as large as that evoked by extracellular calcium. Intracellular dialysis of cells with high concentrations of EGTA or 1,2-bis(o-Aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) strongly reduced the amplitude of the extracellular calcium response, and the residual response was abolished by a mixture of high and low affinity calcium buffers. TRPM2 channel currents in inside-out patches showed a strong requirement for Ca(2+) at the intracellular face of the membrane. We suggest that calcium entering via TRPM2 proteins acts at an intracellular calcium sensor closely associated with the channel, providing essential positive feedback for channel activation.     

Regulation of the intracellular calcium concentration in MMQ pituitary cells by dopamine and protein kinase C.

The MMQ pituitary cell line, which expresses a membranal dopamine receptor, was used to examine the individual contributions of dopamine and protein kinase C (PKC) to control of the intracellular calcium concentration. The calcium concentrations, monitored with the fluorescent dye Indo-1, increased in response to elevated K+, BAY K8644, and maitotoxin, implicating the presence of voltage-dependent calcium channels. Dopamine had no detectable independent effect, but significantly inhibited the rise in intracellular calcium mediated by activation of voltage-dependent calcium channels; this dopaminergic action was prevented by haloperidol. Acute pharmacological activation of PKC for 60 s inhibited the stimulatory effects of these calcium channel activators, and this acute inhibitory action was abolished by prior depletion of PKC. In contrast, however, PKC depletion did not alter the calcium response to BAY K8644 or maitotoxin. Thus, MMQ cells appear to have voltage-dependent calcium channels which, at rest, are either at low density or in a closed state. The rise in intracellular calcium resulting from stimulation of the channels is under inhibitory control by an apparent D-2 dopamine receptor. When pharmacologically activated, phorbol diester-sensitive PKC limits the rise in the cellular calcium level associated with calcium uptake. In the absence of pharmacological activation, however, this enzyme system does not appear to play a role in the cellular calcium response to BAY K8644 or maitotoxin.     

cDNA cloning and characterization of an osmotically sensitive TRP channel from ascidian eggs

The transient receptor potential (TRP) ion channels are thought to be involved in the entry of calcium ion into cells. In this study, we isolated a cDNA clone, HrTRPV, that shows high homology to Caenorhabditis elegans OSM-9, a TRPV subfamily member of the TRP family, from a Halocynthia roretzi fertilized egg cDNA library. We analyzed its properties using HrTRPV-transfected cells. Upon reduction of extracellular osmolarity, the intracellular calcium concentration was found to increase in HrTRPV-transfected cells. This increase in intracellular calcium concentration was dependent on the presence of extracellular calcium ion and was inhibited by treatment with gadolinium ion, a stretch-activated calcium channel blocker. Thus, these results indicate that ascidian egg HrTRPV is an osmotically sensitive TRP channel.     

Calcium buffering and protection from excitotoxic cell death by exogenous calbindin-D28k in HEK 293 cells

Calbindin-D28k (CaBP) is a calcium-binding protein found in specific neuronal populations in the mammalian brain that, as a result of its proposed calcium-buffering action, may protect neurons against potentially harmful increases in intracellular calcium. We have stably transfected HEK 293 cells with recombinant human CaBP in order to determine the influence of this protein upon transient increases in intracellular ionic calcium concentration ([Ca(2+)](i)) induced either by transient transfection of the NR1 and NR2A subunits of the N-methyl-D-aspartate (NMDA) receptor and brief exposure to glutamate, photolysis of the caged calcium compound NP-EGTA, or exposure to the Ca(2+)]-ionophore 4-Br-A23187. The presence of CaBP did not significantly reduce the peak [Ca(2+)](i)stimulated by glutamate activation of NMDA receptors but significantly prolonged the recovery to baseline values. Flash photolysis of NP-EGTA in control cells resulted in an almost instantaneous increase in [Ca(2+)](i)followed by a bi-exponential recovery to baseline values. In cells stably expressing CaBP, the peak [Ca(2+)](i)levels were not statistically different from the controls, however, there was a significant prolongation of the initial portion of the slow recovery phase. In cells exposed to 4-Br-A23187, the presence of CaBP significantly reduced the rate of rise of [Ca(2+)](i), reduced the peak response, slowed the rate of recovery, and reduced the depolarization of mitochondria. In studies of delayed, Ca(2+)]-dependent cell death, CaBP transfected cells exhibited enhanced survival 24h after a 1-h exposure to 200 microM NMDA. However, necrotic cell death observed after the first 6h was not prevented by the presence of CaBP. These results provide direct evidence for a Ca(2+)-buffering effect of CaBP which serves to limit Ca(2+)entry and the depolarization of mitochondria, thereby protecting cells from death mediated most likely by apoptosis.     

Vasoconstrictor hormones depolarize renal glomerular mesangial cells by activating chloride channels

Mesangial cells are smooth muscle-like cells of the renal glomerulus which contract and produce prostaglandins in response to vasopressin and angiotensin. These responses serve to regulate the glomerular capillary filtering surface area. We have used the membrane potential-sensitive fluorescent dye bis-oxonol and the intracellular fluorescent calcium-sensitive probe Indo-1 to study the changes in membrane potential (Em) and intracellular free calcium concentration ([Ca2+]i) in cultured rat mesangial cells in response to vasoconstrictor hormones. Basal [Ca2+]i was 227 +/- 4 nM, and stimulation by maximal concentrations of either vasopressin or angiotensin resulted in a transient 4-6-fold rise. Resting membrane potential was 45.8 +/- 0.9 mV and vasoconstrictor hormones caused a depolarization of 14-18 mV. The following extracellular ion substitutions indicated that chloride efflux was the predominant ion flux responsible for depolarization: 1) depolarization persisted when sodium in the medium was substituted with N-methylglucamine; 2) substitution of medium sodium chloride with sodium gluconate, which enhances the gradient for chloride efflux, augmented vasoconstrictor-stimulated depolarization; 3) suspension of cells in potassium chloride medium resulted in depolarization, following which, stimulation by either vasopressin or angiotensin resulted in hyperpolarization; and 4) this hyperpolarization did not occur when potassium gluconate medium was used to depolarize the cells. The calcium ionophore ionomycin also resulted in membrane depolarization. However, prevention of the rise in [Ca2+]i by prior exposure to ionomycin in calcium-free medium or by loading mesangial cells with the intracellular calcium buffer BAPTA did not abrogate the depolarization response to vasoconstrictor hormones. This indicates that a rise in intracellular calcium is not necessary for depolarization. In contrast, prior depolarization of the cells using varying concentrations of KCl in the external medium, which dissipated the electrochemical gradient for chloride efflux, resulted in a corresponding prolongation of the transient calcium response to vasopressin and angiotensin. These findings indicate that angiotensin and vasopressin depolarize mesangial cells by activating chloride channels and that this activation can occur by both calcium-dependent and -independent mechanisms. In addition, activation of chloride channels with resulting depolarization may serve to modulate the calcium signal.     

Sequence, cloning, expression and characterisation of the 81-kDa surface membrane protein (P80) of Mycoplasma agalactiae

In response to heat-stable enterotoxin of Vibrio cholerae non-O1, the initial rise of cytosolic Ca(2+) occurred with activation of IP(3). Chelation of extracellular Ca(2+) with EGTA and suspension of cells in Ca(2+) free buffer both demonstrated the involvement of internal stores in the rise of [Ca(2+)]i. Cells pretreated with dantrolene resulted in decrease of [Ca(2+)]i response which suggested that the rise of intracellular level of Ca(2+) was mostly due to the mobilization from IP(3) sensitive stores. When the cytosolic Ca(2+) was chelated by loading the cells with BAPTA, NAG-ST could not induce Ca(2+) entry to the cell as assessed by Mn(2+) quenching of fura-2 fluorescence which suggested that calcium influx across the plasma membrane depends upon initial rise of this bivalent cation that maintained the sustained phase of [Ca(2+)]i response. Addition of toxin to the fura-2-loaded cells, preincubated with lanthanum chloride, resulted in reduction of [Ca(2+)]i level with a short duration of irregular sustained phase further suggesting that the influx of Ca(2+) across the plasma membrane might be through the calcium channel.