Role of calreticulin in regulating intracellular Ca2+ storage and capacitative Ca2+ entry in HeLa cells

Protein and energy metabolism in boars of different breeds, 10 each of Hampshire, Duroc and Danish Landrace was measured in balance and respiration experiments by means of indirect calorimetry in an open-air circulation system. Measurements were performed in four periods (Period I-IV) covering the body weight range from 25 to 100 kg. In order to achieve maximum protein retention (RP) a daily intake of digestible protein > 12 g/kg0.75 and metabolisable energy > 1100 kJ/kg0.75 was assumed to be necessary. Protein retention of Danish Landrace boars was inferior to that of Hampshire and Duroc boars in Periods III and IV, and therefore, 55 measurements on Hampshire and Duroc boars fulfilling the chosen criteria for digested protein and ME intake were used for calculation of maximum protein retention, giving the following significant quadratic relationship: RP [g/d] = 11.43.W0.75-0.144.W1.50 (n = 55, RSD = 15.2, CV = 9.2%, R2 = 0.851) with a summit of 227 g/d at 135 kg BW. In Period I, when BW was below 30 kg, 12 measurements fulfilled the chosen criterion for digested protein but not for ME, and these data were used comparatively. Protein retention of boars with a low ME intake in Period I was significantly below that of boars with a high ME intake (93 g/d vs. 107 g/d; P = 0.02). In summary, the present data have shown that boars of high genetic potential have capacity for maximum protein retention of about 230 g/d, and that there was a significant quadratic relationship between protein retention and metabolic body weight, indicating that maximum protein retention was not reached until 135 kg BW. Differences in capacity for protein retention were recorded between boars of different breeds, with Duroc and Hampshire boars being superior to Danish Landrace boars. Additionally, the crucial importance of a sufficient ME supply early in the growth period was underscored by a lower protein accretion rate of boars given a daily ME supply below 1100 kJ ME/kg0.75 at an approximate BW of 25 kg.     

Dependence of the Ca2+-inhibitable adenylyl cyclase of C6-2B glioma cells on capacitative Ca2+ entry

The ability of adenylyl cyclases to be regulated by physiological transitions in Ca2+ provides a key point for integration of cytosolic Ca2+ concentration ([Ca2+]i) and cAMP signaling. Ca2+-sensitive adenylyl cyclases, whether endogenously or heterologously expressed, require Ca2+ entry for their regulation, rather than Ca2+ release from intracellular stores (Chiono, M., Mahey, R., Tate, G., and Cooper, D. M. F. (1995) J. Biol. Chem. 270, 1149-1155; Fagan, K., Mahey, R., and Cooper, D. M. F. (1996) J. Biol. Chem. 271, 12438-12444). The present study compared the regulation by capacitative Ca2+ entry versus ionophore-mediated Ca2+ entry of an endogenously expressed Ca2+-inhibitable adenylyl cyclase in C6-2B cells. Even in the face of a dramatic [Ca2+]i rise generated by ionophore, Ca2+ entry via capacitative Ca2+ entry channels was solely responsible for the regulation of the adenylyl cyclase. Selective efficacy of BAPTA over equal concentrations of EGTA in blunting the regulation of the cyclase by capacitative Ca2+ entry defined the intimacy between the adenylyl cyclase and the capacitative Ca2+ entry sites. This association could not be impaired by disruption of the cytoskeleton by a variety of strategies. These results not only establish an intimate spatial relationship between an endogenously expressed Ca2+-inhibitable adenylyl cyclase with capacitative Ca2+ entry sites but also provide a physiological role for capacitative Ca2+ entry other than store refilling.     

Differential effects of extracellular Mg2+ on thrombin-induced and capacitative Ca2+ entry in human coronary arterial endothelial cells

Receptor-mediated and capacitative Ca2+ entry are the primary Ca2+ entry pathways in endothelial cells (ECs). The mechanisms for Ca2+ entry via these pathways have not been fully elucidated. In this study, the effect of low and high external Mg2+ concentrations on these Ca2+ entry pathways was examined in human coronary arterial ECs. External Mg2+ concentration did not affect cytosolic free Mg2+ concentration. After exposure to thrombin in Ca(2+)-free medium, addition of Ca2+ to the medium caused a rise in cytosolic free Ca2+ concentration ([Ca2+]i), indicating thrombin-induced Ca2+ influx. Thrombin-induced Ca2+ influx was inhibited by not only low but also high external Mg2+ concentrations. After depletion of endoplasmic Ca2+ stores by thapsigargin, addition of Ca2+ to the medium induced an increase in [Ca2+]i, indicating capacitative Ca2+ entry. Capacitative entry was found to be accelerated by low external Mg2+ and inhibited by high external Mg2+ concentration. Results suggest that receptor-mediated Ca2+ influx requires external Mg2+ but is inhibited by increased external Mg2+ concentrations and that capacitative Ca2+ entry is reduced by external Mg2+ in human coronary arterial ECs.     

Region-specific activity of the plasma membrane Ca2+ pump and delayed activation of Ca2+ entry characterize the polarized, agonist-evoked Ca2+ signals in exocrine cells

The initial release of Ca2+ from the intracellular Ca2+ stores is followed by a second phase during which the agonist-dependent Ca2+ response becomes sensitive to the extracellular Ca2+, indicating the involvement of the plasma membrane (PM) Ca2+ transport systems. The time course of activation of these transport systems, which consist of both Ca2+ extrusion and Ca2+ entry pathways, is not well established. To investigate the participation of these processes during the agonist-evoked Ca2+ response, isolated pancreatic acinar cells were exposed to maximal concentrations of an inositol 1,4,5-trisphosphate-mobilizing agonist (acetylcholine, 10 microM) in different experimental conditions. Following the increase of [Ca2+]i, there was an almost immediate activation of the PM Ca2+ extrusion system, and maximal activity was reached within less than 2s. The rate of Ca2+ extrusion was dependent on the level of [Ca2+]i, with a steep activation at values just above the resting [Ca2+]i and reached a plateau value at 700 nM Ca2+. In contrast, the PM Ca2+ entry pathway was activated with a much slower time course. There was also a delay of 3-4 s between the maximal effective depletion of the intracellular Ca2+ stores and the activation of this entry pathway. By use of digital imaging data, the PM Ca2+ transport systems were also analyzed independently in two regions of the cells, the lumenal and the basal poles. With respect to the activation of the Ca2+ entry pathways, no significant difference existed between these two regions. In contrast, the PM Ca2+ pump displayed a different pattern of activity in these regions. In the basal pole, the pump activity was more sensitive to changes of [Ca2+]i and had a higher maximal activity. Also, in the lumenal pole, the pump became saturated at values of [Ca2+]i around 700 nM, whereas at the basal pole [Ca2+]i had a biphasic effect on the pump activity, and higher [Ca2+]i inhibited the pump. It is argued that these differences in sensitivity to the levels of [Ca2+]i and the different relationship between [Ca2+]i and the rate of extrusion at the two functional poles of the pancreatic acinar cells indicate that the plasma membrane Ca2+ ATPase might play an important role in the polarization of the Ca2+ response.     

Calmodulin regulation of Ca2+ entry in Jurkat T cells

BACKGROUND: The authors have previously demonstrated abnormalities in glucose and insulin metabolism in nondiabetic black American (BA) adults versus white American (WA) adults. Whether similar glucoregulatory alterations extend to BA adolescents remain unknown. In addition, obesity, a known risk factor for insulin resistance and hyperinsulinemia, occurs in a greater proportion of BA adults and children when compared to WA. The objective of the present study was to examine the differential effects of obesity on glucose homeostasis in BA and WA adolescents. METHODS: We examined glucose homeostasis in BA and WA adolescents using oral glucose tolerance test (OGTT), intravenous glucose tolerance test (IVGTT), and [6,6-2H2]-glucose infusion. The study consisted of four age-, sex-, and pubertal stage-matched groups: 15 lean BA, 29 lean WA, 7 obese BA, and 9 obese WA. RESULTS: Both obese groups had significantly increased insulin and C-peptide area under the curve (AUC) during OGTT and IVGTT when compared to their same-race lean counterparts. During OGTT, obese BA demonstrated greater insulin and C-peptide when compared to obese WA. During IVGTT, first- and second-phase insulin were significantly greater in obese BA versus obese WA. CONCLUSION: In summary, BA adolescents demonstrated insulin resistance which is markedly exaggerated in the face of obesity when compared to WA adolescents, implying a differential impact for obesity on glucose homeostasis that is unique to the obese BA adolescent group. In conclusion, there is a need for early aggressive weight management in obese BA adolescents.     

Ca2+-dependent capacitance increases in rat basophilic leukemia cells following activation of store-operated Ca2+ entry and dialysis with high-Ca2+-containing intracellular solution

Ca2+-dependent vesicular fusion was studied in single whole-cell patch-clamped rat basophilic leukemia (RBL) cells using the capacitance technique. Dialysis of the cells with 10 microM free Ca2+ and 300 microM guanosine 5'-O-(3-thiotriphosphate) (GTP[gamma-S]) resulted in prominent capacitance increases. However, dialysis with either Ca2+ (225 nM to 10 microM) or GTP[gamma-S] alone failed to induce a capacitance change. Under conditions of weak Ca2+ buffering (0.1 mM EGTA), activation of Ca2+-release-activated Ca2+ (CRAC) channels by dialysis with inositol 1,4,5-trisphosphate (InsP3) failed to induce a capacitance increase even in the presence of GTP[gamma-S]. However, when Ca2+ATPases were inhibited by thapsigargin, InsP3 and GTP[gamma-S] led to a pronounced elevation in membrane capacitance. This increase was dependent on a rise in intracellular Ca2+ because it was abolished when cells were dialysed with a high level of EGTA (10 mM) in the recording pipette. The increase was also dependent on Ca2+ influx because it was effectively suppressed when external Ca2+ was removed. Our results demonstrate that ICRAC represents an important source of Ca2+ for triggering a secretory response.     

Depletion of ryanodine-sensitive Ca2+ store activates Ca2+ entry in rat submandibular gland acinar cells

The existence of ryanodine-sensitive Ca2+ stores and their role in the Ca2+ entry mechanism were examined in the rat submandibular gland acinar cells, using the microfluorimetry of intracellular Ca2+ concentration ([Ca2+]i). In the presence of thapsigargin, a Ca(2+)-ATPase inhibitor of inositol (1, 4, 5) triphosphate (InsP3)-sensitive Ca2+ stores, caffeine caused an increase in [Ca2+]i, which was inhibited by treatment with ryanodine (a ligand to the Ca(2+)-induced Ca2+ release channels). In the cells treated with ryanodine, 1 mM Ca2+ addition to a Ca(2+)-free solution caused a marked increase in [Ca2+]i, which was eliminated by application of Ni2+ or SK & F 96365, suggesting a Ca2+ entry triggered by ryanodine. The maximal change in the net increase in [Ca2+]i caused by the ryanodine-coupled Ca2+ entry, was 104.0 +/- 16.0 nM, which intense was caused by 10 microM ryanodine. Emptying the InsP3-sensitive stores by treatment with thapsigargin also caused Ca2+ entry, which maximally changed [Ca2+]i by 349.6 +/- 15.1 nM. Ten mumol/liter ryanodine was confirmed to cause a release of 45Ca2+ from the parotidic microsomal fraction enriched in endopalsmic reticulum. We propose that ryanodine-sensitive Ca2+ stores are present in rat submandibular gland acinar cells. We further propose that release of Ca2+ from the ryanodine-sensitive stores, which means eventually depletion of the ryanodine-sensitive Ca2+ stores, can activate the Ca2+ entry. The ability for Ca2+ entry coupled with the ryanodine-sensitive Ca2+ stores seems to be about 30% of the ability for Ca2+ entry coupled with the thapsigargin-sensitive Ca2+ stores.     

Ca2+ entry in CHO cells, after Ca2+ stores depletion, is mediated by arachidonic acid

The synthesis and antihypertensive activity of a series of 2,4-dioxoimidazolidin-1-yl and perhydro-2,4-dioxopyrimidin-1-yl ergoline derivatives are reported. The oral antihypertensive activity was studied in spontaneously hypertensive rats (SHRs) by measuring systolic blood pressure by an indirect tail-cuff method at different times after treatment. The prolactin lowering activity (indirectly measured by the nidation test) in rats and the oral acute toxicity in mice were also studied. The results of this study revealed potent antihypertensive ergoline derivatives devoid of side-effects related to the dopaminergic stimulation and the importance of the delta 9,10 double bond for conferring high potency within these compounds.     

Ca2+ pool emptying stimulates Ca2+ entry activated by S-nitrosylation

The entry of Ca2+ following Ca2+ pool release is a major component of Ca2+ signals; yet despite intense study, how "store-operated" entry channels are activated is unresolved. Because S-nitrosylation has become recognized as an important regulatory modification of several key channel proteins, its role in Ca2+ entry was investigated. A novel class of lipophilic NO donors activated Ca2+ entry independent of the well defined NO target, guanylate cyclase. Strikingly similar entry of Ca2+ induced by cell permeant alkylators indicated that this Ca2+ entry process was activated through thiol modification. Significantly, Ca2+ entry activated by either NO donors or alkylators was highly stimulated by Ca2+ pool depletion, which increased both the rate of Ca2+ release and the sensitivity to thiol modifiers. The results indicate that S-nitrosylation underlies activation of an important store-operated Ca2+ entry mechanism.     

Dissociation of intracellular Ca2+ release and Ca2+ entry response to 5-hydroxytryptamine in cultured canine tracheal smooth muscle cells

The relationship between the agonist-sensitive Ca2+ pool and those discharged by the Ca2+ -ATPase inhibitor thapsigargin (TG) were investigated in canine tracheal smooth muscle cells (TSMCs). In fura-2-loaded TSMCs, 5-hydroxytryptamine (5-HT) stimulated a rapid increase in intracellular Ca2+ ([Ca2+]i), followed by a sustained plateau phase that was dependent on extracellular Ca2+. In such cells, TG produced a concentration-dependent increase in [Ca2+]i, which remained elevated over basal level for several minutes and was substantially attenuated in the absence of extracellular Ca2+. Application of 5-HT after TG demonstrated that the TG-sensitive compartment partly overlapped the 5-HT-sensitive stores. Pre-treatment of TSMCs with TG significantly inhibited the increase in [Ca2+]i induced by 5-HT in a time-dependent manner. Similar results were obtained with two other Ca2+ -ATPase inhibitors, cyclopiazonic acid and 2,5-di-t-butylhydroquinone. Although these inhibitors had no effect on phosphoinositide hydrolysis, Ca2+ -influx was stimulated by these agents. These results suggest that depletion of the agonist-sensitive Ca2+ stores is sufficient for activation of Ca2+ influx. Some characteristics of the Ca2+ -influx activated by depletion of internal Ca2+ stores were compared with those of the agonist-activated pathway. 5-HT-stimulated Ca2+ influx was inhibited by La3+, membrane depolarisation, and the novel Ca2+ -influx blocker 1-?beta-[3-(4-methoxyphenyl) propoxy]-4-methoxyphenethyl?-1H-imidazole hydrochloride (SKF96365). Likewise, activation of Ca2+ influx by TG also was blocked by La3+, membrane depolarisation, and SKF96365. These results suggest that (1) in the absence of PI hydrolysis, depletion of the agonist-sensitive internal Ca2+ stores in TSMCs is sufficient for activation of Ca2+ influx, and (2) the agonist-activated Ca2+ influx pathway and the influx pathway activated by depletion of the inositol 1,4,5-trisphosphate-sensitive Ca2+ pool are indistinguishable.     

Multiple mechanisms of activating Ca2+ entry in freshly isolated rabbit aortic endothelial cells

In Fura-2-loaded, freshly isolated rabbit aortic endothelial cells the Ca2+ entry pathway was investigated using the Mn2(+)-quenching technique. Acetylcholine (ACh) interaction with muscarinic receptors activated Mn2+ influx through the plasma membrane. Sarcoplasmic-endoplasmic reticulum Ca2+ ATPase blockers such as cyclopiazonic acid (CPA), thapsigargin and BHQ, which block the endoplasmic reticulum Ca2+ pump and do not interact with receptors, also activated Mn2+ influx. Mn2+ influx activated by either ACh or CPA was blocked by the following agents: SKF96365, a receptor-operated Ca2+ channel (ROC) blocker; NCDC, a PLC and ROC blocker, and genistein, a tyrosine kinase inhibitor. D600, the L-type Ca2+ channel blocker, had no significant effect on Mn2+ influx. Caffeine blocked the ACh-induced Ca2+ release but had no effect on the ACh-induced Mn2+ influx. Similarly dantrolene, which blocked intracellular Ca2+ release induced by ACh, did not affect the ACh-activated Mn2+ influx. These data suggest that ACh can activate Ca2+ influx without depletion of the ACh-sensitive intracellular Ca2+ store. It is concluded (1) that in freshly isolated endothelial cells depletion of the intracellular Ca2+ store is not necessary for ACh-activated Ca2+ influx, and (2) that receptor activation and intracellular Ca2+ store depletion may activate the same Ca2+ entry pathway through parallel mechanisms.     

Nitric oxide inhibits capacitative cation influx in human platelets by promoting sarcoplasmic/endoplasmic reticulum Ca2+-ATPase-dependent refilling of Ca2+ stores

Nitric oxide (NO) is a potent inhibitor of thrombin-induced increase in cytoplasmic free Ca2+ concentration and aggregation in platelets, but the precise mechanism of this inhibition is unclear. To measure Ca2+/Mn2+ influx in intact platelets and to monitor Ca2+ uptake into the stores in permeabilized platelets, fura-2 was used. In intact platelets, maximal capacitative Ca2+ and Mn2+ influx developed rapidly (within 30 s) after fast release of Ca2+ from the stores with thrombin (0.5 U/mL) or slowly (within 5 to 10 minutes) following passive Ca2+ leak caused by inhibition of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) with 30 micromol/L 2,5-di-(tert-butyl)-1,4-benzohydroquinone (BHQ). NO (1 micromol/L) inhibited capacitative Ca2+ and Mn2+ influx independently of the time after thrombin application. In contrast, the effect of NO on BHQ-induced Ca2+ and Mn2+ influx was observed only during the first few minutes after BHQ application and completely disappeared when capacitative cation influx reached its maximum. In Ca2+-free medium, NO reduced the peak Ca2+ rise caused by thrombin and significantly promoted Ca2+ back-sequestration into the stores. Both effects disappeared in the presence of BHQ. Inhibition of guanylate cyclase with H-(1,2,4) oxadiazolo(4,3-a) quinoxallin-1-one (10 micromol/L) attenuated but did not prevent the effects of NO on cytoplasmic free Ca2+ concentration. Inhibition of Ca2+ uptake by mitochondria did not change the effects of NO. In permeabilized platelets, NO accelerated back-sequestration of Ca2+ into the stores after inositol-1,4,5-trisphosphate-induced Ca2+ release or after addition of Ca2+ (1 micromol/L) in the absence of inositol-1,4,5-trisphosphate. The effect of NO depended on the initial rate of Ca2+ uptake and on the concentration of ATP and was abolished by BHQ, indicating the direct involvement of SERCA. These data strongly support the hypothesis that NO inhibits store-operated cation influx in human platelets indirectly via acceleration of SERCA-dependent refilling of Ca2+ stores.     

Functional coupling of secretion and capacitative calcium entry in PC12 cells

The caffeine-evoked effects on the intracellular Ca2+ concentration ([Ca2+]i) and on the release of dopamine by PC12 cells were investigated. Stimulation by caffeine resulted in a transient Ca2+ release which was followed by a sustained phase of Ca2+ entry through a non-voltage dependent pathway. Treatment with cyclopiazonic acid (CPA) or thapsigargin, inhibitors of the Ca2+ ATPase pump of the endoplasmic reticulum, resulted in only a sustained rise in [Ca2+]i in the presence of extracellular Ca2+. Pretreatment of cells with CPA or thapsigargin abolished the subsequent Ca2+ responses to caffeine. Caffeine also evoked the release of dopamine from the cells only in the presence of extracellular Ca2+, which was mimicked by CPA. These results suggest that store-dependent Ca2+ entry evoked by caffeine has an indispensable role in the secretory response in an excitable cell line, PC12 cells.     

Selective activation of Ca2+-activated K+ channels by co-localized Ca2+ channels in hippocampal neurons

Calcium entry through voltage-gated calcium channels can activate either large- (BK) or small- (SK) conductance calcium-activated potassium channels. In hippocampal neurons, activation of BK channels underlies the falling phase of an action potential and generation of the fast afterhyperpolarization (AHP). In contrast, SK channel activation underlies generation of the slow AHP after a burst of action potentials. The source of calcium for BK channel activation is unknown, but the slow AHP is blocked by dihydropyridine antagonists, indicating that L-type calcium channels provide the calcium for activation of SK channels. It is not understood how this specialized coupling between calcium and potassium channels is achieved. Here we study channel activity in cell-attached patches from hippocampal neurons and report a unique specificity of coupling. L-type channels activate SK channels only, without activating BK channels present in the same patch. The delay between the opening of L-type channels and SK channels indicates that these channels are 50-150 nm apart. In contrast, N-type calcium channels activate BK channels only, with opening of the two channel types being nearly coincident. This temporal association indicates that N and BK channels are very close. Finally, P/Q-type calcium channels do not couple to either SK or BK channels. These data indicate an absolute segregation of coupling between channels, and illustrate the functional importance of submembrane calcium microdomains.     

Regulation of capacitative Ca2+ influx in human neutrophil granulocytes. Alterations in chronic granulomatous disease

Ca2+ entry through the capacitative (store-regulated) pathway was shown to be inhibited in neutrophil granulocytes by the protein kinase C activator phorbol 12-myristate 13-acetate and the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP) by a hitherto unknown mechanism. Measuring both Ca2+ and Mn2+ entry into store-depleted cells we show in the present study that inhibition of the capacitative pathway is absent in various forms of chronic granulomatous disease. To establish the possible relationship between inhibition of the capacitative pathway and ability of O-2 production and consequent membrane depolarization, gradual changes of the membrane potential were evoked in neutrophils of healthy individuals. This was accomplished by pharmacological manipulation of the membrane potential and by variations of the concentration and type of the stimulant. Close relationship was observed between membrane depolarization and inhibition of Mn2+ entry through the capacitative transport route. Our results provide an explanation for the inhibitory action of fMLP and phorbol 12-myristate 13-acetate on capacitative cation influx and reveal that upon physiological stimulation, Ca2+ entry into neutrophils is restricted by the depolarization accompanying O-2 production.     

Rapid Ca2+ entry through Ca2+-permeable AMPA/Kainate channels triggers marked intracellular Ca2+ rises and consequent oxygen radical production

The widespread neuronal injury that results after brief activation of highly Ca2+-permeable NMDA channels may, in large part, reflect mitochondrial Ca2+ overload and the consequent production of injurious oxygen radicals. In contrast, AMPA/kainate receptor activation generally causes slower toxicity, and most studies have not found evidence of comparable oxygen radical production. Subsets of central neurons, composed mainly of GABAergic inhibitory interneurons, express AMPA/kainate channels that are directly permeable to Ca2+ ions. Microfluorometric techniques were performed by using the oxidation-sensitive dye hydroethidine (HEt) to determine whether the relatively rapid Ca2+ flux through AMPA/kainate channels expressed on GABAergic neurons results in oxygen radical production comparable to that triggered by NMDA. Consistent with previous studies, NMDA exposures triggered increases in fluorescence in most cultured cortical neurons, whereas high K+ (50 mM) exposures (causing depolarization-induced Ca2+ influx through voltage-sensitive Ca2+ channels) caused little fluorescence change. In contrast, kainate exposure caused fluorescence increases in a distinct subpopulation of neurons; immunostaining for glutamate decarboxylase revealed the responding neurons to constitute mainly the GABAergic population. The effect of NMDA, kainate, and high K+ exposures on oxygen radical production paralleled the effect of these exposures on intracellular Ca2+ levels when they were monitored with the low-affinity Ca2+-sensitive dye fura-2FF, but not with the high-affinity dye fura-2. Inhibition of mitochondrial electron transport with CN- or rotenone almost completely blocked kainate-triggered oxygen radical production. Furthermore, antioxidants attenuated neuronal injury resulting from brief exposures of NMDA or kainate. Thus, as with NMDA receptor activation, rapid Ca2+ influx through Ca2+-permeable AMPA/kainate channels also may result in mitochondrial Ca2+ overload and consequent injurious oxygen radical production.     

Relation of exocytotic release of gamma-aminobutyric acid to Ca2+ entry through Ca2+ channels or by reversal of the Na+/Ca2+ exchanger in synaptosomes

The specific inhibitor of the gamma-aminobutyric acid (GABA) carrier, NNC-711, (1-[(2-diphenylmethylene)amino]oxyethyl)- 1,2,5,6-tetrahydro-3-pyridine-carboxylic acid hydrochloride, blocks the Ca(2+)-independent release of [3H]GABA from rat brain synaptosomes induced by 50 mM K+ depolarization. Thus, in the presence of this inhibitor, it was possible to study the Ca(2+)-dependent release of [3H]GABA in the total absence of carrier-mediated release. Reversal of the Na+/Ca2+ exchanger was used to increase the intracellular free Ca2+ concentration ([Ca2+]i) to test whether an increase in [Ca2+]i alone is sufficient to induce exocytosis in the absence of depolarization. We found that the [Ca2+]i may rise to values above 400 nM, as a result of Na+/Ca2+ exchange, without inducing release of [3H]GABA, but subsequent K+ depolarization immediately induced [3H]GABA release. Thus, a rise of only a few nanomolar Ca2+ in the cytoplasm induced by 50 mM K+ depolarization, after loading the synaptosomes with Ca2+ by Na+/Ca2+ exchange, induced exocytotic [3H]GABA release, whereas the rise in cytoplasmic [Ca2+] caused by reversal of the Na+/Ca2+ exchanger was insufficient to induce exocytosis, although the value for [Ca2+]i attained was higher than that required for exocytosis induced by K+ depolarization. The voltage-dependent Ca2+ entry due to K+ depolarization, after maximal Ca2+ loading of the synaptosomes by Na+/Ca2+ exchange, and the consequent [3H]GABA release could be blocked by 50 microM verapamil. Although preloading the synaptosomes with Ca2+ by Na+/Ca2+ exchange did not cause [3H]GABA release under any conditions studied, the rise in cytoplasmic [Ca2+] due to Na+/Ca2+ exchange increased the sensitivity to external Ca2+ of the exocytotic release of [3H]GABA induced by subsequent K+ depolarization. Thus, our results show that the vesicular release of [3H]GABA is rather insensitive to bulk cytoplasmic [Ca2+] and are compatible with the view that GABA exocytosis is triggered very effectively by Ca2+ entry through Ca2+ channels near the active zones.     

Pathways mediating Ca2+ entry in rat cerebellar granule cells following in vitro exposure to methyl mercury

Metastatic disease is the most common cause of malignant liver lesion in the United States. This article focuses on the MR techniques utilized for evaluation of the liver for metastatic disease, the MR appearance of hepatic metastases, and several contrast agents being developed to further improve detection of focal hepatic lesions by MR imaging.     

Capacitative Ca2+ entry and the regulation of smooth muscle tone

In many non-excitable cells, activation of phospholipase C-linked receptors results in a biphasic increase in the cytosolic Ca2+ concentration; an initial transient increase, owing to the release of Ca2+ from the endoplasmic/sarcoplasmic reticulum (ER/SR), is followed by a much smaller but sustained elevation, which often involves capacitative Ca2+ entry, where depletion of Ca2+ within the ER signals the opening of store-operated Ca2+ channels in the plasma membrane. However, in excitable cells such as smooth muscle, the role of capacitative Ca2+ entry is less clear and the main Ca2+ entry mechanisms responsible for sustained cellular activation have been considered to be either voltage-operated or receptor-operated Ca2+ channels. Although store-regulated Ca2+ entry was known to occur following agonist activation of smooth muscle, it was believed to be important only for the re-filling of the depleted SR and not as a source of activator Ca2+ for the contractile mechanisms. Here, Alan Gibson, Ian McFadzean, Pat Wallace and Christopher Wayman review recent evidence that capacitative Ca2+ entry might indeed be important for the regulation of smooth muscle tone, and that it might provide an important for pharmacological intervention.     

Phosphorylation down-regulates the store-operated Ca2+ entry pathway of human neutrophils

We have reported previously that the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) inhibits transiently Ca2+ entry through the plasma membrane Ca2+ pathway activated by emptying the intracellular Ca2+ stores (Montero, M., García-Sancho, J., and Alvarez, J. (1993) J. Biol. Chem. 268, 13055-13061). We show here that calyculin A and okadaic acid, inhibitors of protein phosphatases 1 and 2A, prevent the spontaneous reversion of the fMLP-induced inhibition of the entry of Ca2+ and Mn2+ (used as a Ca2+ surrogate), leading to a permanently inhibited Ca2+ entry pathway. At high concentrations or long incubation times the phosphatase inhibitors were even able to inhibit the store-operated Ca2+ entry pathway (SOCP) in the absence of fMLP. Inhibition of SOCP by phorbol dibutyrate, which is not reversible, was not modified by phosphatase inhibitors. These results provide additional support for the view that fMLP inhibits SOCP through phosphorylation of either the SOCP protein or a regulatory protein and indicate that dephosphorylation mediated by protein phosphatases 1 and/or 2A restores the activity of SOCP after inhibition by fMLP. The time course of the inhibition of SOCP by fMLP was similar to the one reported previously for the transient fMLP-induced phosphorylation of a 47-kDa protein involved in the generation of respiratory burst, which was similarly affected by the phosphatase inhibitors.