Mode of Action of Palytoxin on the Release of Acetylcholine from Rat Cerebrocortical Synaptosomes

Palytoxin (PTX; 10(-14)-10(-6) M) caused a dose-dependent increase in the release of [3H]acetylcholine ([3H]ACh), cytosolic free Ca2+ concentration ([Ca2+]i), and uptake of 22Na+ and decrease in membrane potential in rat cerebrocortical synaptosomes. The dose-response curves for the PTX-induced increases in [3H]ACh release and in [Ca2+]i were depressed by removing extracellular Ca2+ or by decreasing extracellular Na+ concentrations. The release of [3H]ACh induced by concentrations of PTX less than 10(-10) M was more dependent on the simultaneous presence of both Ca2+ and Na+ than the release induced by higher concentrations of PTX. The PTX-induced increase both in [3H]ACh release and in [Ca2+]i was almost completely abolished by the combination of Ca2+ deprivation and Na+ concentration reduction. All responses to PTX were highly resistant to 10(-6) M tetrodotoxin. These results suggest that low concentrations of PTX cause depolarization as a result of an increase in Na+ permeability through tetrodotoxin-insensitive channels. This, in turn, increases Ca2+ influx and leads to an increase in the release of ACh. It appears that at high concentrations PTX increases the release of [3H]ACh by directly increasing the influx of Ca2+ into synaptosomes and by releasing Ca2+ from intracellular storage sites via an Na(+)-Ca2+ exchange mechanism.     

Effects of palytoxin on contractile response and calcium movement in guinea-pig taenia coli

PTX (10(-8)M) induced a rapid increase followed by a gradual decrease in muscle tension in normal physiological salt solution (PSS), while it induced a slow increase in muscle tension in low-Na+ solution. These contractions were inhibited by Ca2+ channel blockers, verapamil and nicardipine. PTX rapidly increased tissue Na+ and decreased tissue K+ contents in normal PSS. In low-Na+ solution, PTX decreased tissue K+ content with a slower rate than that in normal PSS. PTX increased uptake of 45Ca2+ in normal as well as low-Na+ solutions with similar time course as the increase in muscle tension. However, 45Ca2+ uptake still remained high when the PTX-induced transient contraction ceased. These results suggest that PTX increases Ca2+ influx through voltage-dependent Ca2+ channels to cause contraction. After a prolonged exposure to PTX, however, muscle tension is uncoupled from Ca2+ influx.     

Sugar moiety of cardiac glycosides is essential for the inhibitory action on the palytoxin-induced K+ release from red blood cells

Palytoxin (PTX), a highly toxic and sugar-containing substance isolated from Palythoa tuberculosa, caused K+ release from rabbit red blood cells. Cardiac glycosides, such as ouabain, convallatoxin, cymarin, digoxin and digitoxin, inhibited the PTX-induced K+ release. Their corresponding aglycones did not inhibit the K+ release, but antagonized the inhibitory effect of the glycosides. All these cardiotonic steroids equally inhibited the activity of (Na+ + K+)-ATPase prepared from hog cerebral cortex. These results suggest that the sugar moiety of the cardiac glycosides is important for the inhibitory effect on the K+ release induced by PTX and that the inhibition is not related to their inhibitory potency on the (Na+ + K+)-ATPase activity.     

Modulation of Ca2+ channel-gated Ca2+ release by W-7 in cardiac myocytes.

Cardiac muscle excitation-contraction coupling is controlled by the Ca(2+)-induced Ca2+ release mechanism. The present study examines the effects of a calmodulin antagonist W-7 on Ca2+ current (ICa)-induced Ca2+ release in whole cell-clamped rat ventricular myocytes. Exposure of cells to W-7 suppressed ICa, but the intracellular Ca(2+)-transients showed a lesser degree of reduction, suggesting possible enhancement of Ca(2+)-induced Ca2+ release. The effects of W-7 on the efficacy of Ca2+ release were most prominent at negative potentials. At test potentials of -30 mV, 20 microM W-7 almost completely blocked ICa, but significant Ca(2+)-transients remained, thus causing a four to six-fold increase in the efficacy of Ca(2+)-induced Ca2+ release. The depolarization-dependent Ca(2+)-transients were eliminated in absence of extracellular Ca2+, blocked by Cd2+, and were absent when the sarcoplasmic reticulum was depleted of Ca2+, implicating dependency on Ca(2+)-signaling between the L-type channel and the ryanodine receptor. W-7 mediated increase in the efficacy of Ca(2+)-induced Ca2+ release was eliminated when myocytes were dialyzed with the internal solution containing gluathione (5 mM), suggesting the possible role of cellular redox state in the regulation of Ca2+ release by the calmodulin antagonist.     

Interaction of palytoxin and cardiac glycosides on erythrocyte membrane and (Na+ + K+) ATPase

Palytoxin (PTX), at extremely low concentrations (0.01-1 nM), caused K+ release from rabbit erythrocytes. Among the various chemical compounds tested, cardiac glycosides potently inhibited the PTX-induced K+ release. The order of inhibitory potency (IC50) was cymarin (0.42 microM) greater than convallatoxin (0.9 microM) greater than ouabain (2.3 microM) greater than digitoxin (88 microM) greater than digoxin (90 microM). Their corresponding aglycones, even at 10 microM, did not inhibit the K+ release, but competitively antagonized the inhibitory effect of the glycosides. All these cardiotonic steroids inhibited the activity of (Na+ + K+)-ATPase prepared from hog cerebral cortex in narrow concentration ranges (IC50 = 0.15-2.4 microM), suggesting that the inhibition of K+ release is not related to their inhibitory potency on the (Na+ + K+)-ATPase activity, and the sugar moiety of cardiac glycosides is involved in the inhibition. On the other hand PTX, at higher concentrations (greater than 0.1 microM), inhibited the (Na+ + K+)-ATPase activity. However, this inhibitory effect of PTX was not antagonized by ouabain. It is suggested that, compared with ouabain, PTX has additional binding site(s) on the (Na+ + K+)-ATPase.     

Store-Operated Ca2+ Influx and Voltage-Gated Ca2+ Channels Coupled to Exocytosis in Pheochromocytoma (PC12) Cells

Microamperometry was used to monitor quantal catecholamine release from individual PC12 cells in response to raised extracellular K+ and caffeine. K+-evoked exocytosis was entirely dependent on Ca2+ influx through voltage-gated Ca2+ channels, and of the subtypes of such channels present in these cells, influx through N-type was primarily responsible for triggering exocytosis. L-type channels played a minor role in mediating K+-evoked secretion, whereas P/Q-type channels did not appear to be involved in secretion at all. Caffeine also evoked catecholamine release from PC12 cells, but only in the presence of extracellular Ca2+. Application of caffeine in Ca2+-free solutions evoked large, transient rises of [Ca2+]i, but did not trigger exocytosis. When Ca2+ was restored to the extracellular solution (in the absence of caffeine), store-operated Ca2+ influx was observed, which evoked exocytosis. The amount of secretion evoked by this influx pathway was far greater than release triggered by influx through L-type Ca2+ channels, but less than that caused by Ca2+ influx through N-type channels. Our results indicate that exocytosis may be regulated even in excitable cells by Ca2+ influx through pathways other than voltage-gated Ca2+ channels.     

Revisiting the role of Ca2+ in Shaker K+ channel gating

Shaker K+ channels were expressed in outside-out macropatches excised from Xenopus oocytes, and the effects on gating of removal of extracellular Ca2+ were examined in the complete absence of intracellular divalent cations. Removal of extracellular Ca2+ by perfusion with EDTA-containing solution caused a small negative shift in the channel's voltage-activation curve and led to an increased nonselective leak, but did not otherwise alter or disrupt the channels. The results contradict the proposal that Ca2+ is an essential component required for maintenance of ion selectivity and proper gating of Kv-type K+ channels. The large nonselective leak in Ca2+-free conditions was found to be a patch-seal phenomenon related to F- ion in the recording pipette.     

Effects of valinomycin on calcium mobilization in vascular smooth muscle cells induced by angiotensin II

The effect of the specific potassium (K+) ionophore valinomycin on increase in intracellular calcium concentration [( Ca2+]i) was studied in vascular smooth muscle cells (VSMC). Valinomycin at more than 10(-9) M dose-dependently suppressed phasic increase in [Ca2+]i in VSMC induced by angiotensin II (AII) in both control and Ca2+-free solution, indicating that it suppressed the release of Ca2+ from intracellular Ca2+ stores. Nicorandil and cromakalim, which are both K+ channel openers, also suppressed the increases in [Ca2+]i induced by AII in the Ca2+ free solution. However, valinomycin did not suppress AII-induced production of inositol 1,4,5-trisphosphate (IP3), which is known to mediate the release of Ca2+. These results indicate that decrease of intracellular K+ induced by valinomycin suppressed the release of Ca2+ from intracellular Ca2+ stores induced by IP3.     

Effects of Na+/Ca2+ exchange induced by SR Ca2+ release on action potentials and afterdepolarizations in guinea pig ventricular myocytes

In cardiac cells, evoked Ca2+ releases or spontaneous Ca2+ waves activate the inward Na+/Ca2+ exchange current (INaCa), which may modulate membrane excitability and arrhythmogenesis. In this study, we examined changes in membrane potential due to INaCa elicited by sarcoplasmic reticulum (SR) Ca2+ release in guinea pig ventricular myocytes using whole cell current clamp, fluorescence, and confocal microscopy. Inhibition of INaCa by Na+-free, Li+-containing Tyrode solution reversibly abbreviated the action potential duration at 90% repolarization (APD90) by 50% and caused SR Ca2+ overload. APD90 was similarly abbreviated in myocytes exposed to the Na+/Ca2+ exchange inhibitor KB-R7943 (5 microM) or after inhibition of SR Ca2+ release with ryanodine (20 microM). In the absence of extracellular Na+, spontaneous SR Ca2+ releases caused minimal changes in resting membrane potential. After the myocytes were returned to Na+-containing solution, the potentiated intracellular Ca2+ concentration ([Ca2+]i) transients dramatically prolonged APD90 and [Ca2+]i oscillations caused delayed and early afterdepolarizations (DADs and EADs). Laser-flash photolysis of caged Ca2+ mimicked the effects of spontaneous [Ca2+]i oscillations, confirming that APD prolongation, DADs, and EADs could be ascribed to intracellular Ca2+ release. These results suggest that Na+/Ca2+ exchange is a major physiological determinant of APD and that INaCa activation by spontaneous SR Ca2+ release/oscillations, depending on the timing, can account for both DADs and EADs during SR Ca2+ overload.     

Differences in Ca2+-mediation of hypotonic and Na+-nutrient regulatory volume decrease in suspensions of jejunal enterocytes

We determined differences in the Ca2+ signalling of K+ and Cl- conductances required for Regulatory Volume Decrease (RVD) in jejunal villus enterocytes passively swollen (0.5 or 0.95.isotonic) compared with swelling because of the absorption of D-glucose (D-Glc) or L-Alanine (L-Ala). Cell volume was measured using electronic cell sizing. In nominally Ca(2+)-free medium containing EGTA (100 microM) RVD after 0.5 or 0.95.isotonic challenge was prevented. L-Ala swelling and subsequent RVD was influenced in Ca(2+)-free medium. Villus cells were incubated with 10 microM of the acetomethoxy derivative of 1,2.bis (2-aminophenoxy) ethane N,N,N1,N1 tetracetic acid (BAPTA-AM) and RVD after 0.5.isotonic swelling or L-Ala swelling was prevented. Niguldipine (0.1 microM), nifedipine (5 microM), diltiazem (100 microM), Ni2+, and Co2+ (1 mM) all prevented hypotonic RVD but had no effect on RVD after L-Ala addition. Charybdotoxin (25 nM) a potent inhibitor of Ca(2+)-activated K+ channels, had no effect on hypotonic RVD but prevented RVD of villus cells swollen by D-Glc. We used the calmodulin antagonists, naphthalene sulfonamide derivatives W-7 and W-13, to assess calmodulin activation of K+ and Cl- conductance in these two models. L-Ala swelling and subsequent RVD was not influenced by 25 microM W-7; hypotonic RVD was prevented by 25 microM W-7 or 100 microM W-13. The W-13 inhibition of RVD was by-passed with 0.5 microM gramicidin. Our data show that hypotonic RVD requires extracellular Ca2+ and that the K+ conductance activated is not charybdotoxin sensitive but requires calmodulin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phasic contractions of the rat portal vein depend on intracellular Ca2+ release stimulated by depolarization

The phasic contraction to phenylephrine of the rat isolated portal vein was investigated using functional studies. Phasic contractions to phenylephrine and caffeine could be produced after several minutes in Ca(2+)-free Krebs solution, which were inhibited by cyclopiazonic acid or ryanodine. The phenylephrine and caffeine contractions were abolished, however, within 10 min in Ca(2+)-free Krebs solution and by nifedipine. This indicated the Ca(2+) stores were depleted in the absence of Ca(2+) influx through voltage-gated channels. The phasic contraction to phenylephrine was also abolished by niflumic acid even in Ca(2+)-free Krebs solution. This showed that the response depended on intracellular Ca(2+) release stimulated directly by depolarization, resulting from opening of Ca(2+)-activated Cl(-) channels, but did not require Ca(2+) influx. In support of this, K(+)-induced phasic contractions were also produced in Ca(2+)-free Krebs solution. The phenylephrine but not K(+)-induced phasic contractions in Ca(2+)-free Krebs solution were inhibited by ryanodine or cyclopiazonic acid. This would be consistent with Ca(2+) release from more superficial intracellular stores (affected most by these agents), probably by inositol 1,4,5-trisphospate, being required to stimulate the phenylephrine depolarization.     

Calmodulin inhibits inositol trisphosphate-induced Ca2+ mobilization from the endoplasmic reticulum of islets

IP3-induced Ca2+ release from the endoplasmic reticulum (ER) of islets is believed to be a key intracellular event in glucose-induced insulin secretion. Calmodulin was shown to increase ATP-dependent Ca2+ steady-state and inhibit by 57.2% IP3-induced Ca2+ mobilization from the ER. Conversely, the calmodulin antagonist, N-(6-aminohexyl)-5-chloro-1-naphtalene sulfonamide (W-7), induced Ca2+ release from the ER. The combination of W-7 (100 microM) and IP3 (10 microM), resulted in a greater release of Ca2+ from the ER than either W-7 or IP3 alone. W-7 was shown not to affect the structural integrity of the ER. Our results suggest that IP3-induced Ca2+ release from the ER is regulated by a calmodulin-dependent process.     

Cellular mechanisms involved in iso-osmotic high K+ solutions-induced contraction of the estrogen-primed rat myometrium

The aim of the present study was to investigate the mechanisms involved in the contraction evoked by iso-osmotic high K+ solutions in the estrogen-primed rat uterus. In Ca2+-containing solution, iso-osmotic addition of KCl (30, 60 or 90 mM K+) induced a rapid, phasic contraction followed by a prolonged sustained plateau (tonic component) of smaller amplitude. The KCl (60 mM)-induced contraction was unaffected by tetrodotoxin (3 microM), omega-conotoxin MVIIC (1 microM), GF 109203X (1 microM) or calphostin C (3 microM) but was markedly reduced by tissue treatment with neomycin (1 mM), mepacrine (10 microM) or U-73122 (10 microM). Nifedipine (0.01-0.1 microM) was significantly more effective as an inhibitor of the tonic component than of the phasic component. After 60 min incubation in Ca2+-free solution containing 3 mM EGTA, iso-osmotic KCl did not cause any increase in tension but potentiated contractions evoked by oxytocin (1 microM), sodium orthovanadate (160 micrM) or okadaic acid (20 microM) in these experimental conditions. In freshly dispersed myometrial cells maintained in Ca2+-containing solution and loaded with indo 1, iso-osmotic KCl (60 mM) caused a biphasic increase in the intracellular Ca2+ concentration ([Ca2+]i). In cells superfused for 60 min in Ca2+-free solution containing EGTA (1 mM), KCl did not increase [Ca2+]i. In Ca2+-containing solution, KCl (60 mM) produced a 76.0 +/- 16.2% increase in total [3H]inositol phosphates above basal levels and increased the intracellular levels of free arachidonic acid. These results suggest that, in the estrogen-primed rat uterus, iso-osmotic high K+ solutions, in addition to their well known effect on Ca2+ influx, activate other cellular processes leading to an increase in the Ca2+ sensitivity of the contractile machinery by a mechanism independent of extracellular Ca2+.     

Increased intracellular Ca2+ induces Ca2+ influx in human T lymphocytes.

One current hypothesis for the initiation of Ca2+ entry into nonelectrically excitable cells proposes that Ca2+ entry is linked to the state of filling of intracellular Ca2+ stores. In the human T lymphocyte cell line Jurkat, stimulation of the antigen receptor leads to release of Ca2+ from internal stores and influx of extracellular Ca2+. Similarly, treatment of Jurkat cells with the tumor promoter thapsigargin induced release of Ca2+ from internal stores and also resulted in influx of extracellular Ca2+. Initiation of Ca2+ entry by thapsigargin was blocked by chelation of Ca2+ released from the internal storage pool. The Ca2+ entry pathway also could be initiated by an increase in the intracellular concentration of Ca2+ after photolysis of the Ca(2+)-cage, nitr-5. Thus, three separate treatments that caused an increase in the intracellular concentration of Ca2+ initiated Ca2+ influx in Jurkat cells. In all cases, Ca(2+)-initiated Ca2+ influx was blocked by treatment with any of three phenothiazines or W-7, suggesting that it is mediated by calmodulin. These data suggest that release of Ca2+ from internal stores is not linked capacitatively to Ca2+ entry but that initiation is linked instead by Ca2+ itself, perhaps via calmodulin.     

K+-depolarization induces a direct release of Ca2+ from intracellular storage sites in cultured vascular smooth muscle cells from rat aorta

Using an intracellularly trapped dye, quin 2, effects of K+-depolarization on cytosolic free calcium concentrations were recorded microfluorometrically in rat aorta vascular smooth muscle cells in primary culture. When the cells were exposed to high extracellular K+ in Ca+-free media containing 2mM EGTA, there was a transient and dose-dependent elevation of cytosolic Ca2+ concentrations. However, the concentration of the cytosolic Ca2+ was not elevated when the intracellularly stored Ca2+ was depleted by the repetitive treatment with caffeine prior to the application of high K+. Thus depolarization of plasma membrane, per se, directly induces a release of Ca2+ from intracellular storage sites in vascular smooth muscle cells, and the main fraction of this released Ca2+ is derived from the caffeine sensitive storage sites; perhaps from the sarcoplasmic reticulum.     

The L-type voltage-gated Ca2+ channel is the Ca2+ sensor protein of stimulus-secretion coupling in pancreatic beta cells

L-type voltage-gated Ca2+ channels (Cav1.2) mediate a major part of insulin secretion from pancreatic beta-cells. Cav1.2, like other voltage-gated Ca2+ channels, is functionally and physically coupled to synaptic proteins. The tight temporal coupling between channel activation and secretion leads to the prediction that rearrangements within the channel can be directly transmitted to the synaptic proteins, subsequently triggering release. La3+, which binds to the polyglutamate motif (EEEE) comprising the selectivity filter, is excluded from entry into the cells and has been previously shown to support depolarization-evoked catecholamine release from chromaffin and PC12 cells. Hence, voltage-dependent trigger of release relies on Ca2+ ions bound at the EEEE motif and not on cytosolic Ca2+ elevation. We show that glucose-induced insulin release in rat pancreatic islets and ATP release in INS-1E cells are supported by La3+ in nominally Ca2+-free solution. The release is inhibited by nifedipine. Fura 2 imaging of dispersed islet cells exposed to high glucose and La3+ in Ca2+-free solution detected no change in fluorescence; thus, La3+ is excluded from entry, and Ca2+ is not significantly released from intracellular stores. La3+ by interacting extracellularlly with the EEEE motif is sufficient to support glucose-induced insulin secretion. Voltage-driven conformational changes that engage the ion/EEEE interface are relayed to the exocytotic machinery prior to ion influx, allowing for a fast and tightly regulated process of release. These results confirm that the Ca2+ channel is a constituent of the exocytotic complex [Wiser et al. (1999) PNAS 96, 248-253] and the putative Ca2+-sensor protein of release.     

Calcium-independent desensitization of rises in intracellular free Ca2+ concentration and catecholamine release in cultured adrenal chromaffin cells

Exposure of adrenal chromaffin cells to carbamylcholine (CCh) in the absence of extracellular Ca2+ suppressed rises in intracellular free Ca2+ concentration ([Ca2+]i) induced by subsequent addition of Ca2+ into the incubation medium. The extent of the suppression was dependent on the concentration of CCh and the duration of exposure. A similar inhibitory effect of CCh was also observed in the case of catecholamine release. In contrast, pretreatment with 56 mM K+ did not affect these two responses induced by Ca2+. Recovery from the desensitized state was rapid, since the responses became normal within 3 min following washout of the maximum concentration of CCh. These results show that, in Ca2+-free medium, exposure of the cells to CCh induces desensitization as indicated by diminished rise in [Ca2+]i and reduced release of catecholamines. These phenomena were not due to direct inhibition of voltage-dependent Ca2+ channels by CCh, but seem to be due to an uncoupling of signal transduction between the nicotinic receptor and Ca2+ channel.     

Ca2+ clearance mechanisms in neurohypophysial terminals of the rat

The importance of intracellular calcium ([Ca2+]i) in the release of vasopressin (AVP) and oxytocin from the central nervous system neurohypopyhysial nerve terminals has been well-documented. To date, there is no clear understanding of Ca2+ clearance mechanisms and their interplay with transmembrane Ca2+ entry, intracellular [Ca2+]i transients, cytoplasmic Ca2+ stores and hence the release of AVP at the level of a single nerve terminal. Here, we studied the mechanism of Ca2+ clearance in freshly isolated nerve terminals of the rat neurohypophysis using Fura-2 Ca2+ imaging and measured the release of AVP by radioimmuno assay. An increase in the K+ concentration in the perfusion solution from 5 to 50 mM caused a rapid increase in [Ca2+]i and AVP release. Returning K+ concentration to 5 mM led to rapid restoration of both responses to basal level. The K+-evoked [Ca2+]i and AVP increase was concentration-dependent, reliable, and remained of constant amplitude and time course upon successive applications. Extracellular Ca2+ removal completely abolished the K+-evoked responses. The recovery phase was not affected upon replacement of NaCl with sucrose or drugs known to act on intracellular Ca2+ stores such as thapsigargin, cyclopiazonic acid, caffeine or a combination of caffeine and ryanodine did not affect either resting or K+-evoked [Ca2+]i or AVP release. By contrast, the plasma membrane Ca2+ pump inhibitor, La3+, markedly slowed down the recovery phase. The mitochondrial respiration uncoupler, carbonyl cyanide 3-chlorophenylhydrazone (CCCP), slightly but significantly increased the basal [Ca2+]i, and also slowed down the recovery phase of both [Ca2+]i and release responses. In conclusion, we show in nerve terminals that (i) Ca2+ extrusion through the Ca2+ pump in the plasma membrane plays a major role in the Ca2+ clearance mechanisms of (ii) Ca2+ uptake by mitochondria also contributes to the Ca2+ clearance and (iii) neither Na+/Ca2+ exchangers nor Ca2+ stores are involved in the Ca2+ clearance or in the maintenance of basal [Ca2+]i or release of AVP.     

Activation of purinergic P2X receptors inhibits P2Y-mediated Ca2+ influx in human microglia

Purinoceptor (P2X and P2Y) mediated Ca2+ signaling in cultured human microglia was studied using Ca2+ sensitive fluorescence microscopy. ATP (at 100 microM) induced a transient increase in [Ca2+]i in both normal and Ca(2+)-free solution suggesting a primary contribution by release from intracellular stores. This conclusion was further supported by the failure of ATP to cause a divalent cationic influx in Mn2+ quenching experiments. However, when fluorescence quenching was repeated after removal of extracellular Na+, ATP induced a large influx of Mn2+, indicating that inward Na+ current through a non-selective P2X-coupled channel may normally suppress divalent cation influx. Inhibition of Mn2+ entry was also found when microglia were depolarized using elevated external K+ in Na(+)-free solutions. The possibility of P2X inhibition of Ca2+ influx was then investigated by minimizing P2X contributions of purinergic responses using either the specific P2Y agonist, ADP-beta-S in the absence of ATP or using ATP combined with PPADS, a specific inhibitor of P2X receptors. In quenching studies both procedures resulted in large increases in Mn2+ influx in contrast to the lack of effect observed with ATP. In addition, perfusion of either ATP plus PPADS or ADP-beta-S alone caused a significantly enhanced duration (about 200%) of the [Ca2+]i response relative to that induced by ATP. These results show that depolarization induced by P2X-mediated Na+ influx inhibits store-operated Ca2+ entry resulting from P2Y activation, thereby modulating purinergic signaling in human microglia.     

Histamine-Induced increases in intracellular free Ca2+ levels in hepatoma cells

The effect of histamine on intracellular free Ca2+ levels ([Ca2+]i) in HA22/VGH human hepatoma cells were evaluated using fura-2 as a fluorescent Ca2+ dye. Histamine (0.2-5 microM) increased [Ca2+]i in a concentration-dependent manner with an EC50 value of about 1 microM. The [Ca2+]i response comprised an initial rise, a slow decay, and a sustained phase. Extracellular Ca2+ removal inhibited 50% of the [Ca2+]i signal. In Ca2+-free medium, after cells were treated with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), 5 microM histamine failed to increase [Ca2+]i. After pretreatment with 5 microM histamine in Ca2+-free medium for 4 min, addition of 3 mM Ca2+ induced a [Ca2+]i increase of a magnitude 7-fold greater than control. Histamine (5 microM)-induced intracellular Ca2+ release was abolished by inhibiting phospholipase C with 2 microM 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122), and by 5 microM pyrilamine but was not altered by 50 microM cimetidine. Together, this study shows that histamine induced [Ca2+]i increases in human hepatoma cells by stimulating H1, but not H2, histamine receptors. The [Ca2+]i signal was caused by Ca2+ release from thapsigargin-sensitive endoplasmic reticulum in an inositol 1,4,5-trisphosphate-dependent manner, accompanied by Ca2+ entry.