Cyclic AMP-mediated inhibition of gallbladder contractility: role of K+ channel activation and Ca2+ signaling

We have examined the mechanisms of cAMP-induced gallbladder relaxation by recording isometric tension and membrane potential in the intact tissue, and global intracellular calcium concentrations ([Ca(2+)](i)) and F-actin content in isolated myocytes. Both the phosphodiesterase (PDE) inhibitor, IBMX (100 microM) and the adenylate cyclase activator, forskolin (2 microM) caused decreases in basal tone that exhibited similar kinetics. IBMX and forskolin both caused concentration dependent, right-downward shifts in the concentration-response curves of KCl and cholecystokinin (CCK). IBMX and forskolin elicited a membrane hyperpolarization that was almost completely inhibited by the ATP-sensitive K(+) channel (K(ATP)) channel blocker, glibenclamide (10 microM). IBMX also induced an increase in large-conductance Ca(2+)-dependent K(+) (BK) channel currents, although the simultaneous blockade of BK and K(ATP) channels did not block IBMX- and forskolin-induced relaxations. Ca(2+) influx activated by L-type Ca(2+) channel activation or store depletion was also impaired by IBMX and forskolin, indicating a general impairment in Ca(2+) entry mechanisms. IBMX also decreases [Ca(2+)](i) transients activated by CCK and 3,6-Di-O-Bt-IP(4)-PM, a membrane permeable analog of inositol triphosphate, indicating an impairment in Ca(2+) release through IP(3) receptors. Ionomycin-induced [Ca(2+)](i) transients were not altered by IBMX, but the contractile effects of the Ca(2+) ionophore were reduced in the presence of IBMX, suggesting that cAMP can decrease Ca(2+) sensitivity of the contractile apparatus. A depolymerization of the thin filament could be reason for this change, as forskolin induced a decrease in F-actin content. In conclusion, these findings suggest that multiple, redundant intracellular processes are affected by cAMP to induce gallbladder relaxation.     

cAMP-Dependent potentiation of the Ca(2+)-activated release of the anionic fluorescent dye, calcein, from rat parotid acinar cells

A recent study indicates that elevation of [Ca(2+)](i) enhances the release of calcein, an anionic fluorescent dye, from isolated exocrine acinar cells, so cytoplasmic calcein is useful for monitoring the secretion of organic anions. In this study, we investigated the effect of cAMP on the calcein release evoked by elevation of [Ca(2+)](i). Isoproterenol, forskolin and dibutyryl cyclic AMP (dbcAMP) did not induce the release of calcein from isolated parotid acinar cells, but they potentiated the carbachol-induced release of calcein. Although cytoplasmic calcein is released through an increase in [Ca(2+)](i), isoproterenol potentiated the carbachol-induced release of calcein without affecting the increase in [Ca(2+)](i) evoked by a high concentration of carbachol (10(-6) M). Charybdotoxin, a K(+) channel blocker, inhibited both the carbachol-induced release and the potentiation by isoproterenol. However, the calcein permeation pathways mediating the carbachol-induced release and the isoproterenol-potentiated release exhibited distinct sensitivities to anion channel blockers. Our results indicate that the calcein release induced by carbachol is potentiated through an increase in intracellular levels of cAMP. Although both the Ca(2+)-activated release and the cAMP-potentiated release may be coupled to Ca(2+)-activated K(+) efflux, increases in both [Ca(2+)](i) and [cAMP](i) may activate the calcein conduction pathway which is not activated by an increase in [Ca(2+)](i) alone.     

Bile acids increase intracellular Ca(2+) concentration and nitric oxide production in vascular endothelial cells

The effects of bile acids on intracellular Ca(2+) concentration [Ca(2+)](i) and nitric oxide production were investigated in vascular endothelial cells. Whole-cell patch clamp techniques and fluorescence measurements of [Ca(2+)](i) were applied in vascular endothelial cells obtained from human umbilical and calf aortic endothelial cells. Nitric oxide released was determined by measuring the concentration of NO(2)(-). Deoxycholic acid, chenodeoxycholic acid and the taurine conjugates increased [Ca(2+)](i) concentration-dependently, while cholic acid showed no significant effect. These effects resulted from the first mobilization of Ca(2+) from an inositol 1,4,5-triphosphate (IP(3))-sensitive store, which was released by ATP, then followed by Ca(2+) influx. Both bile acids and ATP induced the activation of Ca(2+)-dependent K(+) current. Oscillations of [Ca(2+)](i) were occasionally monitored with the Ca(2+)-dependent K(+) current in voltage-clamped cells and Ca(2+) measurements of single cells. The intracellular perfusion of heparin completely abolished the ATP effect, but failed to inhibit the bile acid effect. Deoxycholic acid and chenodeoxycholic acid enhanced NO(2)(-) production concentration-dependently, while cholic acid did not enhance it. The bile acids-induced nitric oxide production was suppressed by N(G)-nitro-L-arginine methyl ester, exclusion of extracellular Ca(2+) or N-(6-aminohexyl)-5-chloro-l-naphthalenesulphonamide hydrochloride (W-7) and calmidazolium, calmodulin inhibitors. These results provide novel evidence showing that bile acids increase [Ca(2+)](i) and subsequently nitric oxide production in vascular endothelial cells. The nitric oxide production induced by bile acids may be involved in the pathogenesis of circulatory abnormalities in liver diseases including cirrhosis.     

Biphasic effects of oxethazaine,a topical anesthetic,on the intracellular Ca(2+) concentration of PC12 cells

There have been few reports on the mechanism(s) of action of oxethazaine (OXZ) despite its potent local anesthetic action. Generally, local anesthetics (LAs) not only inhibit Na(+) channels but also affect various membrane functions. In the present study, using PC12 cells as a nerve cell model, the effects of OXZ on intracellular Ca(2+) concentration ([Ca(2+)](i)) were examined in relation to cytotoxicity and dopamine release. [Ca(2+)](i) was determined by the quin2 method. In resting cells, (6-10)x10(-5)M OXZ produced lactate dehydrogenase leakage, which was Ca(2+)-dependent, inhibited by metal Ca(2+) channel blockers, and preceded by a marked increase in [Ca(2+)](i). Some other LAs showed no cytotoxicity at these concentrations. In K(+)-depolarized cells, however, lower concentrations of OXZ (10(-6)-10(-7)M), that had no effect on resting [Ca(2+)](i), inhibited both the dopamine release and the increase of [Ca(2+)](i) in parallel. The inhibitory potency against the [Ca(2+)](i) increase was in the order of nifedipine>OXZ approximately verapamil>diltiazem, and OXZ acted additively on the Ca(2+) channel blockers. OXZ showed the least effect on K(+)-depolarization as determined by bisoxonol uptake. OXZ also inhibited the increase in [Ca(2+)](i) induced by S(-)-BAY K 8644, a Ca(2+) channel agonist. These observations suggested that low concentrations of OXZ interact with L-type Ca(2+) channels. The biphasic effects of OXZ on Ca(2+) movement may be due to a unique chemical structure, and may participate in and complicate the understanding of the potent pharmacological and toxicological actions of OXZ.     

Serotonin-stimulated increase in cytosolic Ca(2+) in cultured rat heart endothelial cells

This study was designed to investigate the effects of serotonin on changes in intracellular Ca(2+) concentration ([Ca(2+)](i)) in cultured rat heart endothelial cells. Serotonin stimulated a biphasic change in cytosolic Ca(2+) of rat heart endothelial cells: an initial transient increase, which primarily reflects the release of Ca(2+) from internal stores, followed by a slow rise in [Ca(2+)](i) during the incubation with serotonin. Our study also demonstrated that the pattern of the serotonin-induced increase in [Ca(2+)](i) was different from that induced by thrombin in rat heart endothelial cells. In this study, the role of [Ca(2+)](i) on endothelial paracellular barrier function was also investigated. Serotonin induced an increase in endothelial permeability which paralleled the rise in [Ca(2+)](i) and was blocked by the 5-HT(2) receptor antagonist cyproheptadine. Therefore, the serotonin-stimulated increase in cytosolic Ca(2+) and macromolecular permeability was receptor-mediated in rat heart endothelial cells. Further experiments demonstrated that the serotonin-induced increase in [Ca(2+)](i) was inhibited by the phospholipase C inhibitors, neomycin and [6-[[17beta-3-methoxyestra-1,3, 5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122). Experiments involving the rapid depletion of intracellular Ca(2+) stores and Ca(2+)-free medium demonstrated that the biphasic response of endothelial Ca(2+) to serotonin was related to the release of Ca(2+) from intracellular stores and to the influx of extracellular Ca(2+). We also suggest that serotonin-induced changes in [Ca(2+)](i) are related to Ca(2+) channels sensitive to voltage-operated and inorganic Ca(2+) channel blockers.     

Different profiles of Ca2+ responses to endothelin-1 and PDGF in liver myofibroblasts during the process of cell differentiation

BACKGROUND AND PURPOSE: Hepatic stellate cells play an important role in liver fibrosis but little is known about liver myofibroblasts located around the central vein and in the portal area. In this study, intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured to assess the response to endothelin-1 (ET-1), platelet derived growth factor (PDGF) and ATP in rat liver myofibroblasts. EXPERIMENTAL APPROACH: Rat liver myofibroblasts were compared in 'quiescent' (cultured on Matrigel-coated dishes) and 'activated' (cultured on non-coated plastic dishes) conditions. [Ca(2+)](i) was measured with the fluorescent dye fura-2 and mRNA for ET-1, PDGF and their receptors by RT-PCR. KEY RESULTS: ET-1 increased [Ca(2+)](i) in quiescent cells but not in activated cells, whereas PDGF-BB increased [Ca(2+)](i) in activated cells but not in quiescent cells. However, there was no difference between responses to ATP in quiescent or activated cells. ET-1 (in quiescent cells), PDGF-BB (in activated cells) and ATP (in both cells) all induced transient increases in [Ca(2+)](i) in the absence of extracellular Ca(2+) (with EGTA), indicating the involvement of Ca(2+) release from intracellular Ca(2+) stores. The sustained increase in [Ca(2+)](i) in the presence of external Ca(2+) in activated cells (ATP and PDGF) was significantly reduced by nicardipine, a L-type Ca(2+) channel blocker, but not in quiescent cells (ATP and ET-1). CONCLUSIONS AND IMPLICATIONS: The different pharmacological profiles of [Ca(2+)](i)-response in quiescent and activated myofibroblasts suggest that ET-1 and PDGF contribute differently to myofibroblast activation during the process of liver fibrosis.     

Nanomolar level of ouabain increases intracellular calcium to produce nitric oxide in rat aortic endothelial cells

Changes in [Ca(2+)](i) across the cell membrane and/or the sarcoplasmic reticulum regulate endothelial nitric oxide (NO) synthase activity. In the present study, we investigated the effect of ouabain, a specific inhibitor of Na(+)/K(+)-ATPase, on NO release and [Ca(2+)](i) movements in cultured rat aortic endothelial cells (RAEC) by monitoring NO production continuously using an NO-specific real-time sensor and by measuring the change in [Ca(2+)](i) using a fluorescence microscopic imaging technique with high-speed wavelength switching. The t((1/2)) (half-time of the decline of [Ca(2+)](i) to basal levels after stimulation with 10 micro mol/L bradykinin) was used as an index of [Ca(2+)](i) extrusion. A very low concentration of ouabain (10 nmol/L) did not increase the peak of NO production, but decreased the decay of NO release and, accordingly, increased integral NO production by the maximal dose-response concentration induced by bradykinin. The same dose of ouabain affected [Ca(2+)](i) movements across the cell membrane and/or sarcoplasmic reticulum induced by bradykinin with a time-course similar to that of NO release. Moreover, the t((1/2)) was significantly increased. Pretreatment of RAEC with Na(+)-free solution, an inhibitor of the Na(+)/Ca(2+) exchanger, and nickel chloride hexahydrate prevented the effects induced by bradykinin and ouabain. These observations using real-time recording indicate that a small amount of ouabain contributes to the bradykinin-stimulated increase of NO production through inhibition of plasma membrane Na(+)/K(+)-ATPase activity and an increase in intracellular Na(+) concentrations. The membrane was then depolarized, leading to a decline in the bradykinin-stimulated increase in [Ca(2+)](i) by forward mode Na(+)/Ca(2+) exchange to prolong the Ca(2+) signal time. From these results, we suggest that nanomolar levels of ouabain modulate [Ca(2+)](i) movements and NO production in RAEC.     

Triethyltin increases cytosolic Ca(2+) levels in human osteoblasts

In human osteosarcoma MG63 cells, effect of triethyltin, an environmental toxicant, on intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured by using fura-2. Triethyltin (1-50 μM) caused a rapid and sustained plateau rise of [Ca(2+)](i) in a concentration-dependent manner (EC(50)=10 μM). Triethyltin-induced [Ca(2+)](i) rise was prevented by 50% by removal of extracellular Ca(2+) but was not altered by voltage-gated Ca(2+) channel blockers. In Ca(2+)-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum (ER) Ca(2+)-ATPase, caused a monophasic [Ca(2+)](i) rise, after which the increasing effect of triethyltin on [Ca(2+)](i) was attenuated by 60%; also, pretreatment with triethyltin abolished thapsigargin-induced [Ca(2+)](i) increase. Depletion of mitochondrial Ca(2+) with carbonylcyanide m-chlorophenylhydrazone (CCCP; 2 μM) did not affect triethyltin-induced Ca(2+) release. U73122, an inhibitor of phoispholipase C, abolished ATP (but not triethyltin)-induced [Ca(2+)](i) rise. A low concentration (1 μM) of triethyltin failed to alter ATP and bradykinin-induced [Ca(2+)](i) rises. These findings suggest that triethyltin rapidly increases [Ca(2+)](i) in osteoblasts by stimulating both extracellular Ca(2+) influx and intracellular Ca(2+) release via as yet unidentified mechanism(s).     

Distinct Ca(2+) signalling mechanisms induced by ATP and sphingosylphosphorylcholine in porcine aortic smooth muscle cells

1. The increase in the cytosolic Ca(2+) concentration ([Ca(2+)](i)) following repetitive stimulation with ATP or sphingosylphosphorylcholine (SPC) in single porcine aortic smooth muscle cells was investigated using the Ca(2+) indicator, fura-2. 2. The ATP-induced [Ca(2+)](i) increase resulted from both Ca(2+) release and Ca(2+) influx. The former was stimulated by phospholipase C activation, while the latter occurred predominantly via the receptor-operated Ca(2+) channels (ROC), rather than the store-operated Ca(2+) channels (SOC) or the voltage-operated Ca(2+) channel (VOC). Furthermore, the P2X(5) receptor was shown to be responsible for the ATP-induced Ca(2+) influx. 3. A reproducible [Ca(2+)](i) increase was induced by repetitive ATP stimulation, but was abolished by removal of extracellular Ca(2+) or inhibition of intracellular Ca(2+) release using U-73122 or thapsigargin, and was restored by Ca(2+) readdition in the former case. 4. SPC only caused Ca(2+) release, and the amplitude of the repetitive SPC-induced [Ca(2+)](i) increases declined gradually. However, a reproducible [Ca(2+)](i) increase was seen in cells in which protein kinase C being inhibited, which increased the SPC-induced Ca(2+) influx, rather than IP(3) generation. 5. In conclusion, although the amplitude of the ATP-induced Ca(2+) release, measured when Ca(2+) influx was blocked, or of the Ca(2+) influx when Ca(2+) release was blocked, progressively decreased following repetitive stimulation, the overall [Ca(2+)](i) increase for each stimulation under physiological conditions remained the same, suggesting that the Ca(2+) stores were replenished by an influx of Ca(2+) during stimulation. The SPC-induced [Ca(2+)](i) increase resulted solely from Ca(2+) release and decreased gradually following repetitive stimulation, but the decrease could be prevented by stimulating Ca(2+) influx, further supporting involvement of the intracellular Ca(2+) stores in Ca(2+) signalling.     

Effects of bifemelane on the calcium level and ATP release of the human origin astrocyte clonal cell

The effect of bifemelane hydrochloride (bifemelane) was examined on human origin astrocyte clonal cells (Kings-1). Bifemelane (125 - 1,000 microM) induced a dose-dependent increase in the intracellular calcium concentration ([Ca(2+)](i)). In the highest concentration (1,000 microM), the drug caused the second large increase in [Ca(2+)](i) during the washing. The increase that occurred during the administration partially remained in the Ca(2+)-free medium and was blocked by 2-aminoethoxydiphenyl borate (2-APB), an IP(3)-receptor blocker, indicating that the source of Ca(2+) for the increase could be ascribed to the intracellular store. The increase in [Ca(2+)](i) was not observed during washing with Ca(2+)-free medium, but was observed when the washing was performed with Ca(2+)-containing medium. Bifemelane caused a dose-dependent ATP release, but histamine and carbachol, which induced a large increase in [Ca(2+)](i), had no effects on the ATP release. The effects on the [Ca(2+)](i) were blocked by pretreatment with pyridoxal phosphate-6-azophenyl-2',4' disulfonic acid, a P2-receptor antagonist. Although the mechanisms of ATP release induced by the drug have not been elucidated yet, the present results demonstrate that the increase in [Ca(2+)](i) induced by bifemelane is not due to its direct effect on the cells, but is dependent upon the ATP released from the cells.     

Characterization of large-conductance Ca2+-dependent and -independent K+ channels in HaCaT keratinocytes

To characterize ion channels expressed in cell membrane of human keratinocytes, patch-clamp recordings were carried out in HaCaT cells. Two types of large-conductance K(+) channels (about 250 pS) were measured. One type was activated by micromolar concentrations of intracellular Ca(2+) ions ([Ca(2+)](i)) and membrane depolarization, the other was [Ca(2+)](i) independent. The channels were neither dependent on intracellular ATP nor Mg(2+) nor on membrane stretch. We conclude that HaCaT keratinocytes express Ca(2+)-dependent maxi K(+) channels and still unknown large Ca(2+)-independent K(+) channels. These K(+) channels may affect the proliferation and differentiation of human keratinocytes by the influence on the resting potential, which may control the Ca(2+) influx across the cell membrane.     

Effects of trans- and cis-resveratrol on Ca2+ handling in A7r5 vascular myocytes

Although the natural polyphenol resveratrol posses a direct vasorelaxant effect, its effects on cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) in vascular cells remain still unclear. Here, we have investigated the effects of the isomers trans- and cis-resveratrol on agonist- and high-K(+)-induced [Ca(2+)](i) increases and on voltage-activated transmembrane Ca(2+) fluxes using imaging and patch-clamp techniques in vascular A7r5 myocytes. Arginine vasopressin (AVP) or angiotensin II caused a biphasic increase in [Ca(2+)](i) that was reduced by preincubation with trans-resveratrol and cis-resveratrol. Both isomers also reduced the agonist-induced increase in [Ca(2+)](i) in absence of extracellular Ca(2+). In high-K(+) Ca(2+)-free solution, reintroduction of Ca(2+) caused a sustained rise in [Ca(2+)](i) that was reduced by preincubation with trans-resveratrol or cis-resveratrol. When the isomers were applied during the plateau phase of the agonist- or the high-K(+)-induced response, a biphasic change in [Ca(2+)](i) was observed: a transient reduction of the plateau (<5 min) followed by an increase (>10 min). Finally, trans-resveratrol and cis-resveratrol inhibited voltage-dependent L-type Ca(2+) currents (I(Ca(L))). In conclusion, resveratrol isomers exert a dual effect on [Ca(2+)](i) handling in A7r5 myocytes: 1) a blockade of I(Ca(L)) and 2) an increase in [Ca(2+)](i) by depletion of intracellular Ca(2+) stores (which interferes with the agonist-induced release of intracellular Ca(2+)) and influx of Ca(2+), mainly due to activation of capacitative Ca(2+) entry, although other Ca(2+)-permeable channels are also involved. Taken together, these effects may explain, in part, the endothelium-independent vasorelaxant effects of resveratrol in rat aorta.     

Pulses of external ATP aid to the synchronization of pancreatic beta-cells by generating premature Ca(2+) oscillations

Pancreatic beta-cells respond to glucose stimulation with increase of the cytoplasmic Ca(2+) concentration ([Ca(2+)](i)), manifested as membrane-derived slow oscillations sometimes superimposed with transients of intracellular origin. The effect of external ATP on the oscillatory Ca(2+) signal for pulsatile insulin release was studied by digital imaging of fura-2 loaded beta-cells and small aggregates isolated from islets of ob/ob-mice. Addition of ATP (0.01-100 microM) to media containing 20mM glucose temporarily synchronized the [Ca(2+)](i) rhythmicity in the absence of cell contact by eliciting premature oscillations. External ATP triggered premature [Ca(2+)](i) oscillations also when the sarcoendoplasmic reticulum Ca(2+)-ATPase was inhibited with 50 microM cyclopiazonic acid and phospholipase C inhibited with 10 microM U-73122. The effect of ATP was mimicked by other activators of cytoplasmic phospholipase A(2) (10nM acetylcholine, 0.1-1 micro M of the C-terminal octapeptide of cholecystokinin and 2 microg/ml melittin) and suppressed by an inhibitor of the enzyme (50 microM p-amylcinnamoylanthranilic acid). Premature oscillations generated by pulses of ATP sometimes triggered subsequent oscillations. However, prolonged exposure to high concentrations of the nucleotide (10-100 microM) had a suppressive action on the beta-cell rhythmicity. The early effects of ATP included generation of transients induced by inositol (1,4,5) trisphosphate and superimposed on the premature oscillation or on an ordinary oscillation induced by glucose. The results support the idea that purinergic activation of phospholipase A(2) has a co-ordinating effect on the beta-cell rhythmicity by triggering premature [Ca(2+)](i) oscillations mediated by closure of ATP-sensitive K(+) channels.     

Effects of tolbutamide and N-benzoyl-D-phenylalanine (NBDP) on the regulation of [Ca2+]i oscillations in mouse pancreatic islets.

The sulfonylurea derivative, tolbutamide, and the phenylalanine derivative, N-benzoyl-D-phenylalanine (NBDP), both of which stimulate insulin secretion through interaction with the sulfonylurea receptor (SUR1), were studied for their ability to increase the [Ca(2+)](i) and to interact with the glucose-induced slow large amplitude [Ca(2+)](i) oscillations in isolated mouse pancreatic islets. Tolbutamide as well as NBDP induced [Ca(2+)](i) oscillations of extremely slow frequency. Both compounds also lowered the threshold for the glucose-induced slow large amplitude [Ca(2+)](i) oscillations and significantly reduced their frequency in intact islets as well as in single pancreatic beta cells. These [Ca(2+)](i) oscillations apparently require a glucokinase-mediated glycolytic flux. This conclusion is supported by the observations that KIC, a mitochondrial fuel, cannot replace glucose in this synergism and that mannoheptulose, an inhibitor of glucokinase and glucose-induced insulin secretion, abolishes these slow [Ca(2+)](i) oscillations. In conclusion, these compounds potentiate the effect of glucose. This additive effect is the likely result of a synergistic closing action upon the ATP-sensitive K(+) (K(ATP)) channel, mediated in the case of glucose through an action upon the channel protein itself of ATP generated in glucose catabolism and in the case of tolbutamide and NBDP upon the sulfonylurea receptor (SUR1) associated with this channel.     

Pharmacological activation of plasma-membrane KATP channels reduces reoxygenation-induced Ca(2+) overload in cardiac myocytes via modulation of the diastolic membrane potential

1. The opening of cardiac plasma-membrane ATP-sensitive K(+) channels (pmK(ATP)) can protect the heart against ischaemia/reperfusion injury. We recently demonstrated that the resting membrane potential (E(m)) of ventricular myocytes strongly modulates reoxygenation-induced Ca(2+) overload. This led to the hypothesis that activation of pmK(ATP) can influence the extent of chemically induced hypoxia (CIH)/reoxygenation Ca(2+) overload via hyperpolarization of the diastolic membrane potential of ventricular myocytes. 2. The membrane potential (E(m)) of isolated rat myocytes was determined using the perforated patch-clamp technique and DiBac(4)(3) imaging. Intracellular Ca(2+) ([Ca(2+)](i)) was monitored using FURA-2 imaging. 3. CIH/reoxygenation caused a significant depolarization of E(m) and a substantial increase in [Ca(2+)](i). The K(ATP) opener pinacidil (100 microm) and the pmK(ATP) opener P-1075 (100 microm) hyperpolarized the E(m) of normoxic myocytes. Pinacidil (100 microm) and P-1075 (10 and 100 microm), applied during reoxygenation, hyperpolarized E(m) and prevented reoxygenation-induced increases in [Ca(2+)](i). 4. Myocyte hypercontracture and death increased in parallel with an E(m) depolarization of 10-15 mV and increases in [Ca(2+)](i). Under these conditions, the selective pmK(ATP) channel inhibitor HMR 1098 further depolarized myocyte membrane potential and increased hypercontracture. 5. In conclusion, activation of pmK(ATP) channels can prevent CIH/reoxygenation-induced Ca(2+) overload via a mechanism that is dependent on hyperpolarization of diastolic membrane potential. Hyperpolarization toward normal resting membrane potential favours the Ca(2+) extrusion mode of Na(+)/Ca(2+) exchange.     

Mechanism of trypsin-induced endothelium-dependent vasorelaxation in the porcine coronary artery

1. To investigate the mechanism underlying the trypsin-induced endothelium-dependent relaxation, cytosolic Ca(2+) concentration ([Ca(2+)](i)) and tension development of smooth muscle were simultaneously monitored in the porcine coronary artery, and [Ca(2+)](i) of in situ endothelial cells were monitored in the porcine aortic valvular strips, using fura-2 fluorometry. 2. During the contraction induced by 30 nM U46619, a thromboxane A(2) analogue, 100 nM trypsin induced a rapid transient significant decrease in both [Ca(2+)](i) (from 67.9+/-5.1 to 15.7+/-4.4%) and tension (from 97.5+/-9.2 to 16.8+/-3.5%) of smooth muscle only in the presence of endothelium (100% level was assigned to the level obtained with the 118 mM K(+)-induced contraction). [Ca(2+)](i) and the tension thus returned to the levels prior to the application of trypsin by 5 and 10 min, respectively. 3. The initial phase of this relaxation was partly inhibited by 100 microM N(omega)-nitro-L-arginine (L-NOARG), and was completely inhibited by L-NOARG plus 40 mM K(+) or L-NOARG plus 100 nM charybdotoxin and 100 nM apamin, while the late phase of the relaxation was inhibited by L-NOARG alone. 4. Trypsin induced a transient [Ca(2+)](i) elevation in the endothelial cells mainly due to the Ca(2+) release from the intracellular stores, at the concentrations (1 - 100 nM) similar to those required to induce relaxation. 5. In conclusion, trypsin induced an elevation in [Ca(2+)](i) mainly due to Ca(2+) release in endothelial cells, and thereby caused endothelium-dependent relaxation. The early phase of relaxation was due to nitric oxide and hyperpolarizing factors, while the late phase was mainly due to nitric oxide in the porcine coronary artery.     

Estimation of increased concentration of intracellular Cd(2+) by fluo-3 in rat thymocytes exposed to CdCl(2)

Cadmium, an environmental pollutant, has been reported to induce apoptosis in murine lymphocytes. To reveal the mechanism of cadmium-induced apoptosis, one of important questions is whether cadmium increases intracellular concentration of Ca(2+) ([Ca(2+)](i)), Cd(2+) ([Cd(2+)](i)) or both. It is difficult to detect the increase in [Ca(2+)](i) using Ca(2+)-chelator-based fluorescent Ca(2+) indicators in the presence of Cd(2+) because of their sensitivity to Cd(2+). Therefore, the study on membrane response such as Ca(2+)-dependent hyperpolarization gives a clue to reveal whether the [Ca(2+)](i) or [Cd(2+)](i) is increased. Cadmium at concentrations of 3 μM or more dose-dependently augmented fluo-3 fluorescence in rat thymocytes, presumably suggesting an increased [Ca(2+)](i). However, the membranes were not hyperpolarized although the cells possess Ca(2+)-dependent K(+) channels. One may argue that cadmium inhibits Ca(2+)-dependent K(+) channels so that cadmium fails to hyperpolarize the membranes. It is unlikely because the [Ca(2+)](i) increased by A23187, a calcium ionophore, elicited the hyperpolarization in the presence of Cd(2+). Furthermore, the profile of cytotoxicity induced by cadmium, examined by ethidium bromide and annexin V-FITC, was different from that induced by A23187. Taken together, it is concluded that the application of cadmium increases the [Cd(2+)](i) rather than the [Ca(2+)](i) in rat thymocytes, resulting in the induction of cytotoxicity.     

Desensitization of insulin secretory response to imidazolines, tolbutamide, and quinine. II. Electrophysiological and fluorimetric studies.

Prolonged in vitro exposure (18 h) of pancreatic islets to insulin secretagogues that block ATP-dependent K(+) channels (K(ATP) channels), such as sulfonylureas, imidazolines, and quinine, induced a desensitization of insulin secretion (Rustenbeck et al., pages 1685-1694, this issue). To elucidate the underlying mechanisms, K(ATP) channel activity, plasma membrane potential and the cytosolic Ca(2+) concentration ([Ca(2+)](i)) were measured in mouse single B-cells. In B-cells desensitized by phentolamine or quinine (100 microM each) K(ATP) channel activity was virtually absent and could not be elicited by diazoxide. Desensitization by alinidine (100 microM) induced a marked reduction of K(ATP) channel activity, which could be reversed by diazoxide, whereas exposure to idazoxan (100 microM) or tolbutamide (500 microM) had no lasting effect on K(ATP) channel activity. Correspondingly, phentolamine-, alinidine-, and quinine-desensitized B-cells were markedly depolarized, whereas B-cells that had been exposed to tolbutamide or idazoxan had an unchanged resting membrane potential. The increase in [Ca(2+)](i) normally elicited by phentolamine and alinidine was suppressed after desensitization by these compounds, whereas the [Ca(2+)](i) increase by re-exposure to quinine was markedly reduced and that by tolbutamide only minimally affected as compared with control-cultured B-cells. The increase in [Ca(2+)](i) elicited by a K(+) depolarization was diminished in secretagogue-pretreated B-cells, the extent depending on the secretagogue. This effect was closely correlated with the degree of depolarization after pretreatment with the respective secretagogue. In conclusion, the apparently uniform desensitization of secretion by K(ATP) channel blockers is due to different effects at two stages located distally in the stimulus-secretion coupling: either at the stage of [Ca(2+)](i) regulation, where the increase is depressed as a consequence of a persistent depolarization (e.g. in the case of phentolamine or alinidine) and/or at the stage of exocytosis, which responds only weakly to substantial increases in [Ca(2+)](i) (in the case of tolbutamide).     

Extracellular ATP-dependent activation of plasma membrane Ca(2+) pump in HEK-293 cells

1. It is well known that extracellular ATP (ATP(o)) elevates the intracellular Ca(2+) concentration ([Ca(2+)](i)) by inducing Ca(2+) influx or mobilizing Ca(2+) from internal stores via activation of purinoceptors in the plasma membrane. This study shows that ATP(o) also activates the plasma membrane Ca(2+) pumps (PMCPs) to bring the elevated [Ca(2+)](i) back to the resting level in human embryonic kidney-293 (HEK-293) cells. 2. The duration of ATP(o)-induced intracellular Ca(2+) transients was significantly increased by PMCP blockers, La(3+) or orthovanadate. In contrast, replacement of extracellular Na(+) with NMDG(+), a membrane-impermeable cation, had no significant effect on duration, thus suggesting that Na(+)/Ca(2+) exchangers do not participate in the ATP(o)-induced Ca(2+) transient. 3. A rapid and significant decrease in [Ca(2+)](i), which was not dependent on extracellular Na(+), was induced by ATP(o) in cells pretreated with thapsigargin (TG). This decrease was blocked by orthovanadate, indicating that it was caused by PMCPs rather than sarco/endoplasmic reticulum Ca(2+) pumps (SERCPs). 4. UTP and ATPgammaS also caused a decrease in [Ca(2+)](i) in cells pretreated with TG, although they were less effective than ATP. The effect of UTP implies the involvement of both P2Y(1) and P2Y(2) receptors, while the effect of ATPgammaS implies no significant role of ectophosphorylation and agonist hydrolysis in the agonist-induced [Ca(2+)](i) decreases. 5. These results point to a role of PMCPs in shaping the Ca(2+) signal and in restoring the resting [Ca(2+)](i) level to maintain intracellular Ca(2+) homeostasis after agonist stimulation.     

Inhibition of the antigen-induced activation of RBL-2H3 cells by charybdotoxin and cetiedil

Quinidine and Ba(2+), non-selective K(+)-channel blockers, have previously been shown to inhibit antigen-induced mediator (beta-hexosaminidase) release from RBL-2H3 cells, a mucosal-type mast cell line. We therefore used selective blockers of Ca(2+)-activated and other K(+) channels to determine if there was a role for these channels in antigen-induced mediator release. Charybdotoxin and cetiedil dose-dependently inhibited beta-hexosaminidase release with IC(50) values of 133 nM and 84 microM, respectively. Charybdotoxin also inhibited the repolarization phase of the antigen-induced biphasic change in the membrane potential (IC(50) 84 nM), antigen-stimulated 86Rb(+)-efflux and increase in free intracellular calcium, [Ca(2+)](i). Iberiotoxin, margatoxin, apamin and tetraethylammonium had no effect on beta-hexosaminidase release. These results suggest that K(+) conductances play a significant role in mediator release from RBL-2H3, that these conductances are of the intermediate conductance Ca(2+)-activated K(+) channel (IK(Ca)) type, and that they are somewhat similar to those which have been described in red blood cells, though they are much less sensitive to clotrimazole.