Changes In Cytosolic Ca2 Research Articles

Influence of CA2+ on K+ efflux during regulatory volume decrease in cultured astrocytes

The calcium (Ca2+) dependence of potassium (K+) efflux activated by hyposmolarity in cultured cerebellar astrocytes was investigated, measuring in parallel experiments (86)Rb release and changes in cytosolic Ca2+ ([Ca2+]i). Hyposmotic (50%) medium increased [Ca2+]i from 117 to 386 nM, with contributions of extracellular Ca2+ and Ca2+ from the endoplasmic reticulum. Hyposmotic medium increased (86)Rb efflux rate from 0.015 min(-1) to a maximal of 0. 049 min(-1) and a net release of 30%. This osmosensitive efflux was inhibited by Ba(2+) (0.028 min(-1)), quinidine (0.024 min(-1)), and charybdotoxin (0.040 min(-1)), but was unaffected by TEA, 4-AP, or apamin. Removal of external Ca2+ from the hyposmotic medium increased (86)Rb efflux to a maximal rate constant of 0.056 min(-1) and a net release of 38% and caused a delay of inactivation. These changes were due to the overlaping of an efflux activated by Ca2+ removal in isosmotic medium. This isosmotic 86Rb efflux was unaffected by TEA or 4-AP, reduced by verapamil, and abolished by Ba2+, nitrendipine, and Mg2+. With the swelling-induced [Ca2+]i rise suppressed by ethyleneglycoltetraacetic acid-acetoxy-methyl ester (EGTA-AM), hyposmotic (86)Rb was 30% reduced. The Ca2+ entry blockers Cd2+, Ni2+, La3+, and Gd3+ did not affect (86)Rb efflux. A 40% decrease observed with verapamil and nitrendipine was found unrelated to Ca2+, because these agents did not affect the [Ca2+]i rise and the inhibition persisted in the absence of external Ca2+. The phospholipase C blocker U-73122 did not affect [Ca2+]i nor (86)Rb efflux. Blockers of Ca2+/calmodulin W7 and KN-93 decreased (86)Rb efflux to the same extent as EGTA-AM. Ionomycin markedly potentiated (86)Rb release in hyposmotic conditions only when [Ca2+]i was raised to about 1 microM, suggesting the implication of maxi-K+ channels at this [Ca2+]i threshold, which nonetheless, was not attained during hyposmotic swelling. It is concluded that (86)Rb efflux in cerebellar astrocytes is largely (70%) Ca2+-independent and the Ca2+-dependent fraction is sustained essentially by Ca2+ released from the endoplasmic reticulum and mediated by a mechanism involving Ca2+/calmodulin.

Novel mechanisms of calcium handling by the osteoclast: A review-hypothesis.

The osteoclast is a cell that is unique in its ability to resorb bone and, in doing so, becomes exposed to unusually high millimolar Ca2+ concentrations. It is generally accepted that, during resorption, osteoclasts can "sense" changes in their ambient Ca2+ concentration. This triggers a sharp cytosolic Ca2+ increase through both Ca2+ release and Ca2+ influx. The change in cytosolic Ca2+ is transduced finally into inhibition of bone resorption. It has been shown that a type 2 ryanodine receptor isoform, expressed uniquely in the plasma membrane, functions as a Ca2+ influx channel and possibly as a Ca2+ sensor. Ryanodine receptors are ordinarily Ca2+ release channels that have a microsomal membrane location in a wide variety of eukaryotic cells, including the osteoclasts. However, only recently has it become obvious that ryanodine receptors are also expressed in osteoclast nuclear membranes, at which site they probably gate nucleoplasmic Ca2+ influx. Nucleoplasmic Ca2+ in turn regulates key nuclear processes, including gene expression and apoptosis. Here, we review the potential mechanisms underlying the recognition, movement, and effects of Ca2+ in the osteoclast. We will also speculate on the general biological significance of the unique processes used by the osteoclast to handle high Ca2+ loads during bone resorption.

Troglitazone inhibits the capacitative Ca 2+ entry in endothelial cells

To investigate the effects of troglitazone on the capacitative Ca 2+ entry, we monitored changes in cytosolic Ca 2+ concentrations ([Ca 2+] i ) induced by thapsigargin in fura-2-loaded porcine endothelial cells in situ and in primary culture. In aortic valve endothelial cells in situ, thapsigargin induced sustained elevation of [Ca 2+] i . Both troglitazone and SKF 96365 inhibited the steady state increase in [Ca 2+] i in a concentration-dependent manner. At 30 μM, troglitazone and SKF 96365 inhibited the [Ca 2+] i elevation to 19.4±3.6% and 43.9±4.5%, respectively. In aortic endothelial cells in primary culture, both troglitazone (10 μM) and SKF 96365 (100 μM) completely inhibited the thapsigargin-induced [Ca 2+] i increase. The EC 50 value of troglitazone (1.4±0.1 μM) was lower than that of SKF 96365 (10.0±3.3 μM). We suggest that troglitazone would be a useful tool to investigate the capacitative Ca 2+ entry.

Potentiation of Ca2+ signaling in endothelial cells by 11,12-epoxyeicosatrienoic acid.

Incubation of endothelium with an increased epoxyeicosatrienoic acid (EET) concentration specifically augments the endothelium-dependent relaxation ascribed to endothelium-derived hyperpolarizing factor in porcine coronary arteries (Weintraub et al., Circ Res 1997;81:258-267). Experiments were designed to test whether such sustained increased levels of EETs in the environment of endothelial cells alters Ca2+ signaling. Changes in cytosolic Ca2+ were monitored in cultured porcine aortic endothelial cells (PAECs) and in the human endothelial EA.hy926 cell line after incubation (or not) with 5 microM 11,12-epoxyeicosatrienoic acid (EET). Although the mobilization of intracellular Ca2+ induced by 2 microM thapsigargin was not affected significantly, EET treatment augmented the capacitative Ca2+ entry evoked by the Ca(2+)-ATPase) inhibitor in both cell types. Similar observations were obtained by using histamine as a stimulant in EA.hy926 cells. As assessed in PAECs, 2 micrograms/ml triacsin C, a known inhibitor of the incorporation of EETs into phospholipids, did not significantly affect the potentiating action of EETs on Ca2+ signaling in response to thapsigargin. However, in solvent-control cells, triacsin C significantly reduced both the mobilization of Ca2+ from intracellular stores and the capacitative Ca2+ entry provoked by thapsigargin. Thus the EET-potentiating effect overcomes the inhibitory action of triacsin C on Ca2+ signaling in endothelial cells. Taken together, these results demonstrate that sustained increases in EETs may amplify Ca2+ signaling. However, contrary to the EET-induced augmentation of endothelium-dependent relaxation in the porcine coronary artery, resistance of this novel action of EETs to triacsin C suggests that the mechanism involved does not depend on incorporation into phospholipids.

Calcium-dependent Regulation of Cytochromec Gene Expression in Skeletal Muscle Cells: IDENTIFICATION OF A PROTEIN KINASE C-DEPENDENT PATHWAY

Mitochondrial biogenesis can occur rapidly in mammalian skeletal muscle subjected to a variety of physiological conditions. However, the intracellular signal(s) involved in regulating this process remain unknown. Using nuclearly encoded cytochrome c, we show that its expression in muscle cells is increased by changes in cytosolic Ca2+ using the ionophore A23187. Treatment of myotubes with A23187 increased cytochrome c mRNA expression up to 1.7-fold. Transfection experiments using promoter-chloramphenicol acetyltransferase constructs revealed that this increase could be transcriptionally mediated since A23187 increased chloramphenicol acetyltransferase activity by 2.5-fold. This increase was not changed by KN62, an inhibitor of Ca2+/calmodulin-dependent kinases II and IV, and it was not modified by overexpression of protein kinase A and cAMP response element-binding protein, demonstrating that the A23187 effect was not mediated through Ca2+/calmodulin-dependent kinase- or protein kinase A-dependent pathways. However, treatment of myotubes with staurosporine or 12-O-tetradecanoylphorbol-13-acetate reduced the effect of A23187 on cytochrome c transactivation by 40-50%. Coexpression of the Ca2+-sensitive protein kinase C isoforms alpha and betaII, but not the Ca2+-insensitive delta isoform, exaggerated the A23187-mediated response. The short-term effect of A23187 was mediated in part by mitogen-activated protein kinase (extracellular signal-regulated kinases 1 and 2) since its activation peaked 2 h after A23187 treatment, and cytochrome c transactivation was reduced by PD98089, a mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibitor. These results demonstrate the existence of a Ca2+-sensitive, protein kinase C-dependent pathway involved in cytochrome c expression and implicate Ca2+ as a signal in the up-regulation of nuclear genes encoding mitochondrial proteins.

Control of action potential-driven calcium influx in GT1 neurons by the activation status of sodium and calcium channels.

An analysis of the relationship between electrical membrane activity and Ca2+ influx in differentiated GnRH-secreting (GT1) neurons revealed that most cells exhibited spontaneous, extracellular Ca(2+)-dependent action potentials (APs). Spiking was initiated by a slow pacemaker depolarization from a baseline potential between -75 and -50 mV, and AP frequency increased with membrane depolarization. More hyperpolarized cells fired sharp APs with limited capacity to promote Ca2+ influx, whereas more depolarized cells fired broad APs with enhanced capacity for Ca2+ influx. Characterization of the inward currents in GT1 cells revealed the presence of tetrodotoxin-sensitive Na+, Ni(2+)-sensitive T-type Ca2+, and dihydropyridine-sensitive L-type Ca2+ components. The availability of Na+ and T-type Ca2+ channels was dependent on the baseline potential, which determined the activation/inactivation status of these channels. Whereas all three channels were involved in the generation of sharp APs, L-type channels were solely responsible for the spike depolarization in cells exhibiting broad APs. Activation of GnRH receptors led to biphasic changes in cytosolic Ca2+ concentration ([Ca2+]i), with an early, extracellular Ca(2+)-independent peak and a sustained, extracellular Ca(2+)-dependent phase. During the peak [Ca2+]i response, electrical activity was abolished due to transient hyperpolarization. This was followed by sustained depolarization of cells and resumption of firing of increased frequency with a shift from sharp to broad APs. The GnRH-induced change in firing pattern accounted for about 50% of the elevated Ca2+ influx, the remainder being independent of spiking. Basal [Ca2+]i was also dependent on Ca2+ influx through AP-driven and voltage-insensitive pathways. Thus, in both resting and agonist-stimulated GT1 cells, membrane depolarization limits the participation of Na+ and T-type channels in firing, but facilitates AP-driven Ca2+ influx.

Anandamide-induced mobilization of cytosolic Ca2+ in endothelial cells.

1. Experiments were designed to determine whether anandamide affects cytosolic Ca2+ concentrations in endothelial cells and, if so, whether CB1 cannabinoid receptors are involved. To this effect, human umbilical vein-derived EA.hy926 endothelial cells were loaded with fura-2 to monitor changes in cytosolic Ca2+ using conventional fluorescence spectrometry methods. 2. Anandamide induced an increase in Ca2+ in endothelial cells which, in contrast to histamine, developed slowly and was transient. Anandamide caused a concentration-dependent release of Ca2+ from intracellular stores without triggering capacitative Ca2+ entry, contrary to histamine or the endoplasmic reticulum Ca2+ -ATPase inhibitor thapsigargin. 3. Anandamide pretreatment slightly reduced the mobilization of Ca2+ from intracellular stores that was evoked by histamine. The mobilization of Ca2+ from intracellular stores evoked by anandamide was impaired by 10 mM caffeine. 4. Anandamide and histamine each significantly increased NO synthase activity in EA.hy926 cells, as determined by the enhanced conversion of L-[3H]-arginine to L-[3H]-citruline. 5. The CB1 cannabinoid receptor antagonist SR141716A (1 microM) only produced a marginal reduction of the mobilization of Ca2+ produced by 5 microM anandamide. However, at 5 microM SR141716A elicited the release of Ca2+ from intracellular stores. This concentration strongly impaired the mobilization of cytosolic Ca2+ evoked by either anandamide, histamine or thapsigargin. 6. Pretreatment of the cells with either 200 microM phenylmethylsulphonyl fluoride (to inhibit the conversion of anandamide into arachidonic acid) or 400 ng ml(-1) pertussis toxin (to uncouple CB1 cannabinoid receptors from Gi/o proteins) had no significant effect on the mobilization of cytosolic Ca2+ evoked by either anandamide, or histamine. 7. Taken together the results demonstrate that anandamide mobilizes Ca2+ from a caffeine-sensitive intracellular Ca2+ store that functionally overlaps in part with the internal stores mobilized by histamine. However, a classical CB1 cannabinoid receptor-mediated and pertussis toxin-sensitive mechanism does not mediate this novel effect of anandamide in endothelial cells. 8. The mobilization of cytosolic Ca2+ in endothelial cells may account for the endothelium-dependent and NO-mediated vasodilator actions of anandamide. Due to its non-specific inhibition of Ca2+ signalling in endothelial cells, SR141716A may not be used to assess the physiological involvement of endogenous cannabinoids to endothelium-dependent control of vascular smooth muscle tone.

Transgenic aequorin monitors cytosolic calcium transients in soybean cells challenged with β-glucan or chitin elicitors

Transgenic soybean (Glycine max L.) cells expressing aequorin were used to monitor changes in cytosolic Ca2+ concentrations in response to treatment with fungal elicitors. After an apparent lag phase of about 60 s, both chitin fragments and β-glucan elicitors caused a rapid increase in cytosolic Ca2+ concentration, which peaked within 2–2.5 min of treatment. The Ca2+ concentration then decreased and reached the basal level after about 5 min in the case of the treatment with chitin fragments, while a second rise in the Ca2+ concentration with a maximum occurring after about 7–8 min was observed in the case of β-glucan treatment. Calibration of the signals showed that the elicitors enhanced the cytosolic Ca2+ concentration from resting concentrations as low as 0.1 lM to highest levels of about 2 lM. Dose-response experiments showed that the concentration of elicitors giving a Ca2+ response at the 50% level was 0.4 nM for the chitin fragment and 28 lM and 72 lM, respectively, for a synthetic hepta-β-glucoside and a fungal β-glucan fraction. The β-glucan- or N,N′,N′′,N′′′-tetraacetyl chitotetratose (CH4)-induced Ca2+ signals were inhibited by both the Ca2+ chelator 1,2-bis-(2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA) and by the Ca2+-channel inhibitor La3+. Neomycin, whose target in plant cells has not yet been clearly identified, reduced predominantly the expression of the second peak of the biphasic Ca2+ curve following β-glucan treatment. Bacterial cyclic β-glucans known to suppress β-glucan-induced phytoalexin production were also found to function as a suppressor for the Ca2+ response that was elicited by the fungal β-glucans. The results clearly show that the increase in the cytosolic Ca2+ concentration is an early and rapid event in the elicitor-sensing mechanism of soybean cells, and is probably connected with the subsequent activation of defence responses.

Agonist-dependent difference in the relationship between cytosolic Ca 2+ level and release of vascular relaxing factors in the endothelium of rabbit aortic valve

The correlation between changes in cytosolic Ca 2+ levels ([Ca 2+] i) and the release of vascular relaxing factor(s) was investigated in the endothelium of rabbit aortic valve. ATP, carbachol and thapsigargin increased endothelial [Ca 2+] i in rabbit aortic valve loaded with a leakage resistant, fluorescent Ca 2+ indicator, fura-PE3. Release of relaxing factors was bioassayed using the `sandwich' preparation in which contraction was measured in the endothelium-denuded rabbit aorta attached to the endothelial surface of the valve. Addition of ATP, carbachol and thapsigargin induced sustained relaxation of the phenylephrine-induced contraction of the aorta in the `sandwich' preparation. N G-monomethyl- l-arginine ( l-NMMA) greatly attenuated the relaxation induced by carbachol, and combined treatment with tetra- n-butylammonium completely inhibited the relaxation. These results suggest that the endothelial relaxing factors released from aortic valve are nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF). When the increase in endothelial [Ca 2+] i was plotted against the relaxation, the carbachol-induced increase in [Ca 2+] i elicited greater relaxation than did ATP or thapsigargin at a given [Ca 2+] i. This suggests that various agonists differently modulate the relationship between [Ca 2+] i and release of NO.

Vasorelaxation and inhibition of the voltage-operated Ca2+ channels by FK506 in the porcine coronary artery.

Using fura-2 fluorometry, the effects of FK506, an immunosuppressant, on changes in cytosolic Ca2+ concentrations ([Ca2+]i) and tension were investigated in porcine coronary arterial strips. The effects of FK506 on the activity of voltage-operated Ca2+ channels were examined by applying a whole cell patch clamp to the isolated smooth muscle cells of porcine coronary artery. FK506 inhibited the sustained increases in both [Ca2+]i and tension induced by 118 mM K+ depolarization and 100 nM U46619 in a concentration-dependent manner (1-30 microM). The extent of inhibition of the K+-induced contraction was greater than that of the U46619-induced contraction. The increases in [Ca2+]i and tension induced by histamine and endothelin- in the presence of extracellular Ca2+ were also inhibited by 10 microM FK506. FK506 (10 microM) had no effect on Ca2+ release induced by caffeine or by histamine in the Ca2+-free solution. FK506 (10 microM) had no effect on the [Ca2+]i-tension relationships of the contractions induced by cumulative increases of extracellular Ca2+ during K+ depolarization or stimulation with U46619. In the patch clamp experiments, FK506 (30 microM) partially inhibited the inward current induced by depolarization pulse from -80 mV to 0 mV. In conclusion, FK506 induces arterial relaxation by decreasing [Ca2+]i mainly due to the inhibition of the L-type Ca2+ channels, with no effect on the Ca2+ sensitivity of the contractile apparatus.

Modulation of Pancreatic Acinar Cell to Cell Coupling during ACh-evoked Changes in Cytosolic Ca2+

The temporal changes in cytosolic free Ca2+ ([Ca2+]i), Ca2+-dependent membrane currents (Im), and gap junctional current (Ij) elicited by acetylcholine (ACh) were measured in rat pancreatic acinar cells using digital imaging and dual perforated patch-clamp recording. ACh (50 nM-5 microM) increased [Ca2+]i and evoked Im currents without altering Ij in 19 of 37 acinar cell pairs. Although [Ca2+]i rose asynchronously in cells comprising a cluster, the delay of the [Ca2+]i responses decreased with increasing ACh concentrations. Perfusion of inositol 1,4,5-trisphosphate (IP3) into one cell of a cluster resulted in [Ca2+]i responses in neighboring cells that were not necessarily in direct contact with the stimulated one. This suggests that extensive coupling between acinar cells provides a pathway for cell-to-cell diffusion of Ca2+-releasing signals. Strikingly, maximal (1-5 microM) ACh concentrations reduced Ij by 69 +/- 15% (n = 9) in 25% of the cell pairs subjected to dual patch-clamping. This decrease occurred shortly after the Im peak and was prevented by incubating acinar cells in a Ca2+-free medium, suggesting that uncoupling was subsequent to the initiation of the Ca2+-mobilizing responses. Depletion of Ca2+-sequestering stores by thapsigargin resulted in a reduction of intercellular communication similar to that observed with ACh. In addition, ACh-induced uncoupling was prevented by blocking nitric oxide production with L-nitro-arginine and restored by exposing acinar cells to dibutyryl cGMP. The results suggest that ACh-induced uncoupling and capacitative Ca2+ entry are regulated concurrently. Closure of gap junction channels may occur to functionally isolate nearby cells differing in their intrinsic sensitivity to ACh and thereby to allow for sustained activity of groups of secreting cells.

Temporal Relationship between Action Potential and Ca2+ Transient in Characean Cells

Temporal relationship between the action potential and the change in cytosolic Ca2+ concentration was investigated in cells of four species of Characeae, Chara corallina, Nitellopsis obtusa, Nitella flexilis and Nitella axilliformis. The Ca2+ transient was detected by light emission from Ca2+-sensitive photoprotein aequorin injected into the cytoplasm. Action potential was triggered by an outward or sometimes inward electric current pulse of 20–50 ms in most cases. In all species the action potential started at almost the same time as the time at which the light emission from aequorin began to increase. Also the peak of action potential almost coincided with that of light emission, which is in contrast with the slower Ca2+ transient in Chara reported by Thiel et al. [(1997) J. Exp. Bot. 48: 609]. A discussion was made on the origin of Ca2+ transient and the ionic processes during membrane excitation.

Activation of β-adrenoceptors does not cause any change in cytosolic Ca 2+ distribution in rat parotid acinar cells

The effects of the β-adrenoceptor agonist isoproterenol on the distribution of cytosolic Ca 2+ concentrations were studied with digital imaging microscopy in fura-2-loaded rat parotid acinar cells. At concentrations <10 μM, isoproterenol did not cause any measurable change in cytosolic Ca 2+ concentration ([Ca 2+] i). Monitoring of [Ca 2+] i in selected areas of the acinar cells failed to show that stimulation with isoproterenol causes a localized rise in [Ca 2+] i at the apical region close to the lumen. As the maximum response of amylase exocytosis is observed at 0.1 or 1 μM isoproterenol [Tanimura, A., Matsumoto, Y., Tojyo, Y., 1990. Evidence that isoproterenol-induced Ca 2+-mobilization in rat parotid acinar cells is not mediated by activation of β-adrenoceptors. Biochim. Biophys. Acta, 1055, pp. 273–277], the data obtained here indicate that the isoproterenol-induced amylase exocytosis is not accompanied by Ca 2+ mobilization. The high concentration (100 μM) of isoproterenol caused a small but significant increase in [Ca 2+] i, particularly in the apical region. This response was completely attenuated by the α-adrenoceptor antagonist phentolamine, but not by the β-adrenoceptor antagonist propranolol, indicating that the isoproterenol-induced increase in [Ca 2+] i resulted from an activation of α-adrenoceptors. Further, the effect of cyclic AMP on Ca 2+ release from intracellular Ca 2+ stores was studied in saponin-permeabilized acinar cells using the lipophilic Ca 2+ indicator Calcium Green C 18. Cyclic AMP had no effect on the Ca 2+ release, while the same acinar cells responded strongly to inositol 1,4,5-trisphosphate. This result does not support the hypothesis that cyclic AMP directly stimulates Ca 2+ mobilization in rat parotid acinar cells.

Distinct subcellular localization of calcium binding S100 proteins in human smooth muscle cells and their relocation in response to rises in intracellular calcium.

Changes in cytosolic Ca2+ concentration control a wide range of cellular responses, and intracellular Ca2+-binding proteins are the key molecules to transduce Ca2+ signaling via interactions with different types of target proteins. Among these, S100 Ca2+-binding proteins, characterized by a common structural motif, the EF-hand, have recently attracted major interest due to their cell- and tissue-specific expression pattern and involvement in various pathological processes. The aim of our study was to identify the subcellular localization of S100 proteins in vascular smooth muscle cell lines derived from human aorta and intestinal smooth muscles, and in primary cell cultures derived from arterial smooth muscle tissue under normal conditions and after stimulation of the intracellular Ca2+ concentration. Confocal laser scanning microscopy was used with a specially designed colocalization software. Distinct intracellular localization of S100 proteins was observed: S100A6 was present in the sarcoplasmic reticulum as well as in the cell nucleus. S100A1 and S100A4 were found predominantly in the cytosol where they were strongly associated with the sarcoplasmic reticulum and with actin stress fibers. In contrast, S100A2 was located primarily in the cell nucleus. Using a sedimentation assay and subsequent electron microscopy after negative staining, we demonstrated that S100A1 directly interacts with filamentous actin in a Ca2+-dependent manner. After thapsigargin (1 microM) induced increase of the intracellular Ca2+ concentration, specific vesicular structures in the sarcoplasmic reticulum region of the cell were formed with high S100 protein content. In conclusion, we demonstrated a distinct subcellular localization pattern of S100 proteins and their interaction with actin filaments and the sarcoplasmic reticulum in human smooth muscle cells. The specific translocation of S100 proteins after intracellular Ca2+ increase supports the hypothesis that S100 proteins exert several important functions in the regulation of Ca2+ homeostasis in smooth muscle cells.

Pulmonary microvascular and macrovascular endothelial cells: differential regulation of Ca2+ and permeability.

Cytosolic Ca2+ concentration ([Ca2+]i) plays an important role in control of pulmonary vascular endothelial cell (ECs) barrier function. In this study, we investigated whether thapsigargin- and ionomycin-induced changes in cytosolic Ca2+ induce permeability in rat pulmonary microvascular (RPMV) versus macrovascular (RPA) ECs. In Transwell cultures, RPMVECs formed a tighter, more restrictive barrier than RPAECs to 12,000-, 72,000-, and 150,000-molecular-weight FITC-labeled dextrans. Thapsigargin (1 microM) produced higher [Ca2+]i levels in RPAECs than in RPMVECs and increased permeability in RPAEC but not in RPMVEC monolayers. Due to the attenuated [Ca2+]i response in RPMVECs, we investigated whether reduced activation of store-operated Ca2+ entry was responsible for the insensitivity to thapsigargin. Addition of the drug in media containing 100 nM extracellular Ca2+ followed by readdition media with 2 mM extracellular Ca2+ increased RPMVEC [Ca2+]i to a level higher than that in RPAECs. Under these conditions, RPMVEC permeability was not increased, suggesting that [Ca2+]i in RPMVECs does not initiate barrier disruption. Also, ionomycin (1.4 microM) did not alter RPMVEC permeability, but the protein phosphatase inhibitor calyculin A (100 nM) induced permeability in RPMVECs. These data indicate that, whereas increased [Ca2+]i promotes permeability in RPAECs, it is not sufficient in RPMVECs, which show an apparent uncoupling of [Ca2+]i signaling pathways or dominant Ca(2+)-independent mechanisms from controlling cellular gap formation and permeability.

Attenuated Influx of Calcium Ions at Nerve Endings ofcspandshibireMutantDrosophila

Previous work has shown that cysteine-string proteins (csps) are synaptic vesicle proteins that are important for evoked neurotransmitter release at Drosophila neuromuscular junctions. Indirect evidence has implicated csps in a regulatory link between synaptic vesicles and presynaptic calcium (Ca) channels. In this report, we use Ca Crimson to monitor stimulus-dependent changes of cytosolic Ca at motor nerve terminals of csp mutant Drosophila. These mutants display temperature-sensitive (TS) paralysis and a presynaptic failure of evoked synaptic transmission. We show that this TS inhibition of neuromuscular transmission is correlated with a block of Ca ion entry at nerve endings of csp mutants. These data support the hypothesis that csps mediate a regulatory interaction between synaptic vesicles and presynaptic Ca channels. Moreover, these results predict that if one depletes nerve endings of synaptic vesicles, one may see a reduction of presynaptic Ca ion entry. Defects of the dynamin gene in TS shibire mutant Drosophila interfere with synaptic vesicle recycling and lead to an activity-dependent depletion of these organelles. Our results show that Ca influx is blocked at nerve terminals of shibire mutant larvae at the same time that synaptic transmission fails in these organisms. Thus, using two completely independent Drosophila mutants, we demonstrate that synaptic vesicles and csps are vital for the function of presynaptic Ca channels.

Digital imaging of intracellular Ca 2+ signaling in rat parotid acinar cells

Utilizing digital imaging microscopy, the receptor-mediated changes in cytosolic Ca 2+ concentration ([Ca 2+] i) were studied in fura-2-loaded rat parotid acinar cells. The increase in [Ca 2+] i induced by carbachol was initiated in the apical pole of the acinar cells and then spread as a Ca 2+ wave toward the basolateral region. A similar polarization of Ca 2+ signal was observed when the acinar cells were stimulated with substance P or phenylephrine. As the microsomal Ca 2+-ATPase inhibitor thapsigargin did not produce a Ca 2+ wave, activation of phosphoinositide hydrolysis is probably essential to trigger the Ca 2+ wave. Stimulation with 1 μM isoproterenol, a concentration which causes the maximum release of amylase, had no effect on [Ca 2+] i. Extracellular ATP (0.5 mM) induced a homogeneous increase in [Ca 2+] i throughout the cells in the presence of extracellular Ca 2+ but did not change [Ca 2+] i in the absence of extracellular Ca 2+, indicating that the ATP-induced rise in [Ca 2+] i is due to Ca 2+ entry.

GABAB receptor-mediated inhibition of GABAA receptor calcium elevations in developing hypothalamic neurons.

In the CNS, gamma-aminobutyric acid (GABA) affects neuronal activity through both the ligand-gated GABAA receptor channel and the G protein-coupled GABAB receptor. In the mature nervous system, both receptor subtypes decrease neural excitability, whereas in most neurons during development, the GABAA receptor increases neural excitability and raises cytosolic Ca2+ levels. We used Ca2+ digital imaging to test the hypothesis that GABAA receptor-mediated Ca2+ rises were regulated by GABAB receptor activation. In young, embryonic day 18, hypothalamic neurons cultured for 5 +/- 2 days in vitro, we found that cytosolic Ca2+ rises triggered by synaptically activated GABAA receptors were dramatically depressed (>80%) in a dose-dependent manner by application of the GABAB receptor agonist baclofen (100 nM-100 microM). Coadministration of the GABAB receptor antagonist 2-hydroxy-saclofen or CGP 35348 reduced the inhibitory action of baclofen. Administration of the GABAB antagonist alone elicited a reproducible Ca2+ rise in >25% of all synaptically active neurons, suggesting that synaptic GABA release exerts a tonic inhibitory tone on GABAA receptor-mediated Ca2+ rises via GABAB receptor activation. In the presence of tetrodotoxin the GABAA receptor agonist muscimol elicited robust postsynaptic Ca2+ rises that were depressed by baclofen coadministration. Baclofen-mediated depression of muscimol-evoked Ca2+ rises were observed in both the cell bodies and neurites of hypothalamic neurons taken at embryonic day 15 and cultured for three days, suggesting that GABAB receptors are functionally active at an early stage of neuronal development. Ca2+ rises elicited by electrically induced synaptic release of GABA were largely inhibited (>86%) by baclofen. These results indicate that GABAB receptor activation depresses GABAA receptor-mediated Ca2+ rises by both reducing the synaptic release of GABA and decreasing the postsynaptic Ca2+ responsiveness. Collectively, these data suggest that GABAB receptors play an important inhibitory role regulating Ca2+ rises elicited by GABAA receptor activation. Changes in cytosolic Ca2+ during early neural development would, in turn, profoundly affect a wide array of physiological processes, such as gene expression, neurite outgrowth, transmitter release, and synaptogenesis.

Guard cells possess a calcium-dependent protein kinase that phosphorylates the KAT1 potassium channel.

Increasing evidence suggests that changes in cytosolic Ca2+ levels and phosphorylation play important roles in the regulation of stomatal aperture and as ion transporters of guard cells. However, protein kinases responsible for Ca2+ signaling in guard cells remain to be identified. Using biochemical approaches, we have identified a Ca(2+)-dependent protein kinase with a calmodulin-like domain (CDPK) in guard cell protoplasts of Vicia faba. Both autophosphorylation and catalytic activity of CDPK are Ca2+ dependent. CDPK exhibits a Ca(2+)-induced electrophoretic mobility shift and its Ca(2+)-dependent catalytic activity can be inhibited by the calmodulin antagonists trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide. Antibodies to soybean CDPK alpha cross-react with CDPK. Micromolar Ca2+ concentrations stimulate phosphorylation of several proteins from guard cells; cyclosporin A, a specific inhibitor of the Ca(2+)-dependent protein phosphatase calcineurin enhances the Ca(2+)-dependent phosphorylation of several soluble proteins. CDPK from guard cells phosphorylates the K+ channel KAT1 protein in a Ca(2+)-dependent manner. These results suggest that CDPK may be an important component of Ca2+ signaling in guard cells.

Light-dependent signal transduction and transient changes in cytosolic Ca2+ in a unicellular green alga

The physiological function and the molecular mechanisms of Ca 2+ -mediated signal transduction processes were studied in the unicellular green alga Eremosphaera viridis by different electrophysiological and microfluorimetric techniques. A sudden blockage of photosynthetic electron transport by darkening or inhibitors causes a transient hyperpolarization of the plasma membrane. For the alga this transient hyperpolarization seems to be an important mechanism to release monovalent ions and to drive the uptake of divalent cations. The transient hyperpolarization is due to the opening of K + channels and is caused by a rapid transient elevation of the cytosolic free Ca 2+ concentration ([Ca 2+ ] cy spike). Different agonists like caffeine or InsP 3 which are known to release Ca 2+ from internal stores in animal cells, also cause a transient hyperpolarization and a [Ca 2+ ] cy spike, similar to darkening. In Eremosphaera the transient hyperpolarization can be used as an indicator for [Ca 2+ ] cy spikes. The InsP 3 gated and the ryanodine/cADPR gated Ca 2+ channels which obviously both mediate Ca 2+ release from internal stores in Eremosphaera do not seem to be involved in the dark-induced [Ca 2+ ] cy spikes. Besides single [Ca 2+ ] cy spikes, the addition of Sr 2+ (or caffeine in the absence of divalent cations) causes repetitive [Ca 2+ ] cy spikes which may last hours and resemble [Ca 2+ ] cy oscillations observed in excitable animal cells. These observations suggest that some principal molecular mechanisms causing single or repetitive [Ca 2+ ] cy spikes are conserved from animal to plant cells.