scholarly journals Extracellular ATP increases cytoplasmic free Ca2+ concentration in clonal insulin-producing RINm5F cells. A mechanism involving direct interaction with both release and refilling of the inositol 1,4,5-trisphosphate-sensitive Ca2+ pool

1990 ◽  
Vol 265 (1) ◽  
pp. 203-211 ◽  
Author(s):  
P Arkhammar ◽  
A Hallberg ◽  
H Kindmark ◽  
T Nilsson ◽  
P Rorsman ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Insulin Release ◽  
Direct Interaction ◽  
Extracellular Atp ◽  
Disodium Salt ◽  
Maximal Response ◽  
Rinm5f Cells ◽  
Plasma Membrane Permeability ◽  
Potential Formation ◽  
Patch Configuration

Effects of extracellularly applied ATP (added as disodium salt) on stimulus-secretion coupling were investigated in clonal insulin-producing RINm5F cells. Cytoplasmic free Ca2+ concentration [(Ca2+]i), electrical activity, membrane potential, formation of InsP3 and insulin release were measured. Addition of ATP in a Ca2(+)-containing medium promoted a rapid rise in [Ca2+]i, which was followed by a slow decline towards the basal level. In a Ca2(+)-free medium, the ATP-induced increase in [Ca2+]i was smaller, but still enough to elicit insulin secretion. Upon normalization of the extracellular Ca2+ concentration, the response to ATP recovered instantaneously. The presence of glucose in the incubation medium was a prerequisite to obtain a pronounced effect of ATP in the absence of extracellular Ca2+. However, glucose did not enhance the response to ATP in a Ca2(+)-containing medium. The effect of ATP was dose-dependent, with a clearly detectable increase in [Ca2+]i at 1 microM and a maximal response being obtained at 200 microM-ATP. The response to ATP was unaffected by activating adenylate cyclase by forskolin, but was abolished by 10 nM of the phorbol ester phorbol 12-myristate 13-acetate. The effects of ATP on [Ca2+]i could not be accounted for by a generalized increase in plasma-membrane permeability, as evident from the failure of the nucleotide to increase the fluorescence of the nuclear stain ethidium bromide. After stimulation with ATP there was an increase in membrane potential, in both the absence and the presence of extracellular Ca2+. Blockage of the voltage-activated Ca2+ channals with D-600, in a Ca2(+)-containing medium, decreased the effect of ATP on [Ca2+]i slightly. Patch-clamp measurements using the cell-attached patch configuration revealed that the RINm5F cells produce spontaneous action potentials, the frequency of which increased markedly on addition of ATP. Whole-cell recordings demonstrated that the increase in spike frequency was not associated with the development of an inward current, but was rather accountable for by a decrease in the activity of the ATP-regulated K+ channels. Addition of 200 microM-ATP stimulated phospholipase C activity, as evident from the formation of InsP3, both in the absence and in the presence of extracellular Ca2+. Thus in the absence of extracellular Ca2+ the stimulatory effect of ATP on insulin release can be explained by InsP3-induced mobilization of intracellularly bound Ca2+. Hence, in the RINm5F cells extracellular ATP acts in a manner similar to other Ca2(+)-mobilizing agents.(ABSTRACT TRUNCATED AT 400 WORDS)

Insulin release, cGMP, cAMP, and membrane potential in acetylcholine-stimulated islets.

1978 ◽  
Vol 235 (5) ◽  
pp. E493 ◽  
Author(s):  
E Gagerman ◽  
L A Idahl ◽  
H P Meissner ◽  
I B T�ljedal
Keyword(s):  
Membrane Potential ◽  
Cyclic Amp ◽  
Insulin Release ◽  
Cyclic Gmp ◽  
Spike Activity ◽  
Obvious Effect ◽  
Ionic Fluxes ◽  
Mouse Islets ◽  
Silent State ◽  
Esterase Inhibitor

Acetylcholine potentiated the glucose-induced insulin release from microdissected mouse islets of Langerhans but had no effect on basal insulin release. Significant potentiation was obtained with 0.1 micron acetylcholine in the presence of 10 micron eserine and with 1 micron or more acetylcholine in the absence of a choline esterase inhibitor. Carbamylcholine, too, potentiated insulin release. Potentiation was blocked by methylatropine, whereas methylatropine alone had no effect on insulin release. Acetylcholine or carbamylcholine (5-500 micron) had no obvious effect on cyclic GMP or cyclic AMP in the islets. In the presence of 11.1 mM D-glucose, the membrane potential of beta-cells oscillated slowly between a polarized silent state of -50 to -55 mV and a depolarized active state of -33 to -39 mV, at which a fast spike activity occurred. Acetylcholine made the potential stay at the plateau and induced a continuous spike activity pattern. Atropine inhibited the electrical effects of acetylcholine but not those of glucose alone. It is suggested that cholinergic potentiation of insulin release is mediated by changes of transmembrane ionic fluxes, probably without the intervention of cyclic GMP or cyclic AMP.

scholarly journals The stimulus–secretion coupling 4-methyl-2-oxopentanoate-induced insulin release

1979 ◽  
Vol 184 (2) ◽  
pp. 303-311 ◽  
Author(s):  
J C Hutton ◽  
A Sener ◽  
W J Malaisse
Keyword(s):  
Insulin Release ◽  
Metabolic Flux ◽  
Energy Charge ◽  
Hyperbolic Function ◽  
Maximal Response ◽  
Coupling Mechanism ◽  
Adenylate Energy Charge ◽  
Atp Content ◽  
45Ca Uptake ◽  
Stimulus Secretion Coupling

1. Pancreatic islet insulin secretion and 45Ca uptake showed similar responses to variation in the extracellular concentration of 4-methyl-2-oxopentanoate with a threshold at 4 mM and a maximal response at a 25 mM concentration. 2. Islet respiration, acetoacetate production and rates of substrate utilization, oxidation and amination all changed as a simple hyperbolic function of 4-methyl-2-oxopentanoate concentration and exhibited a maximal response at 25 mM. 3. The responses of ATP content, [ATP]/[ADP] ratio, adenylate energy charge and [NADH]/[NAD+] ratio were also hyperbolic in nature but were maximally elevated at lower concentrations of the secretagogue. The islet [NADPH]/[NADP+] ratio, however, was tightly correlated with parameters of metabolic flux, 45Ca uptake and insulin release. 4. NH4+ and menadione, agents that promote a more oxidized state in islet NADP, did not affect islet ATP content or the rates of [U-14C]4-methyl-2-oxopentanoate oxidation or amination, but markedly inhibited islet 45Ca uptake and insulin release. 5. It is proposed that changes in the redox state of NADP and Ca transport may serve as mediators in the stimulus-secretion coupling mechanism of insulin release induced by 4-methyl-2-oxopentanoate.

Effects of potassium channel modulators on myotropic responses of aortic rings of pregnant rats

2000 ◽  
Vol 278 (2) ◽  
pp. H567-H576 ◽  
Author(s):  
C. Cadorette ◽  
B. Sicotte ◽  
M. Brochu ◽  
J. St-Louis
Keyword(s):  
Membrane Potential ◽  
Potassium Channels ◽  
Basal Membrane ◽  
Maximal Response ◽  
Dependent Manner ◽  
Response Curves ◽  
Concentration Dependent Manner ◽  
Pregnant Rats ◽  
Potassium Channel Modulators ◽  
High Conductance

The contribution of potassium channels [ATP-sensitive potassium (KATP) and high-conductance calcium-activated potassium (BKCa) channels] in the resistance of aortic rings of term pregnant rats to phenylephrine (Phe), arginine vasopressin (AVP), and KCl was investigated. Concentration-response curves to tetraethylammonium (TEA), a nonselective K+ channel inhibitor, were obtained in the absence or presence of KCl. TEA induced by itself concentration-dependent responses only in aortic rings of nonpregnant rats. These responses to TEA could be modulated in both groups of rings by preincubation with different concentrations of KCl. Concentration-response curves to Phe, AVP, and KCl were obtained in the absence or presence of cromakalim or NS-1619 (KATP and BKCa openers, respectively) and glibenclamide or iberiotoxin (KATPand BKCa inhibitors, respectively). Cromakalim significantly inhibited the responses to the three agonists in a concentration-dependent manner in both groups of rats. Alternatively, in the pregnant group of rats, glibenclamide increased the sensitivity to all three agonists. NS-1619 also inhibited the response to all agonists. With AVP and KCl, its effect was greater in aortic rings of pregnant than nonpregnant rats. Finally, iberiotoxin increased the sensitivity to all three agents. This effect was more important in aortic rings of nonpregnant rats and was accompanied by an increase of the maximal response to Phe and AVP. These results suggest that potassium channels are implicated in the control of basal membrane potential and in the blunted responses to these agents during pregnancy.

Hyperkalemia alters EDHF-mediated hyperpolarization and relaxation in coronary arteries

1996 ◽  
Vol 271 (2) ◽  
pp. H760-H767 ◽  
Author(s):  
G. W. He ◽  
C. Q. Yang ◽  
W. F. Graier ◽  
J. A. Yang
Keyword(s):  
Nitric Oxide ◽  
Cardiac Surgery ◽  
Smooth Muscle ◽  
Membrane Potential ◽  
Organ Preservation ◽  
Maximal Response ◽  
K Channels ◽  
Porcine Coronary Artery ◽  
Preservation Solutions ◽  
Endothelium Dependent Relaxation

Hyperkalemic solutions are widely used to preserve organs for transplantation and for cardiac surgery. The present study was designed to test the hypothesis that hyperkalemia may alter endothelial function through a non-nitric oxide (NO) pathway, since preliminary studies have shown that the NO pathway may not be affected. Porcine coronary artery rings were studied in organ chambers. After incubation with 20 or 50 mM K+ for 1 h, the indomethacin- and NG-nitro-L-arginine+ (L-NNA)-resistant relaxation induced by A23187 or bradykinin, which could be further inhibited by tetraethylammonium but not glibenclamide, was significantly reduced. Incubation with hyperkalemia also significantly increased the concentration eliciting 50% of the maximal response to A23187 and bradykinin. A23187-induced hyperpolarization of the membrane potential was significantly reduced by hyperkalemic incubation. However, 1-h incubation with hyperkalemia does not affect the endothelial Ca2+ concentration. We conclude that exposure to hyperkalemia reduces the indomethacin- and L-NNA-resistant endothelium-dependent relaxation and endothelium-dependent hyperpolarization. This reduction in the relaxation and hyperpolarization is related to the endothelium-derived hyperpolarizing factor by affecting its effect on the smooth muscle cell, probably through partially depolarizing the membrane, and the Ca2(+)- activated K+ channels rather than by affecting its biosynthesis and/or release in the endothelial cell. Our study may suggest a new mechanism for coronary dysfunction after exposure to hyperkalemic cardioplegia and organ preservation solutions.

Extracellular ATP Causes Changes in Plasma Membrane Permeability of Mouse Lymphocytes

1990 ◽  
Vol 603 (1 Biological Ac) ◽  
pp. 427-428
Author(s):  
FRANCESCO VIRGILIO ◽  
ENZO PICELLO ◽  
VINCENZO BRONTE ◽  
PAOLA ZANOVELLO ◽  
DINO COLLAVO
Keyword(s):  
Plasma Membrane ◽  
Membrane Permeability ◽  
Extracellular Atp ◽  
Plasma Membrane Permeability ◽  
Mouse Lymphocytes

scholarly journals Abnormal glucose metabolism accompanies failure of glucose to stimulate insulin release from a rat pancreatic cell line (RINm5F)

1983 ◽  
Vol 212 (2) ◽  
pp. 439-443 ◽  
Author(s):  
P A Halban ◽  
G A Praz ◽  
C B Wollheim
Keyword(s):  
Glucose Metabolism ◽  
Cell Line ◽  
Insulin Release ◽  
Glucose Utilization ◽  
Rinm5f Cells ◽  
Pancreatic Cell ◽  
Abnormal Glucose Metabolism ◽  
Isolated Rat Islets ◽  
Stimulate Insulin Release ◽  
Stimulation Of

Glucose metabolism and insulin release were studied in isolated rat islets and in an insulin-producing rat cell-line (RINm5F). Intact islets displayed two components of glucose utilization, with glucose stimulation of insulin release being associated with the high-Km component (reflecting glucokinase-like activity). Glucose failed to stimulate insulin release from RINm5F cells, which only displayed a single low-Km component of glucose utilization. Only low-Km (hexokinase-like) glucose-phosphorylating activity was found for disrupted RINm5F cells. These changes in glucose metabolism may contribute towards the failure of glucose to stimulate insulin release from RINm5F cells.

scholarly journals Extracellular adenosine triphosphate increases cation permeability of chronic lymphocytic leukemic lymphocytes

Blood ◽  
1989 ◽  
Vol 73 (5) ◽  
pp. 1316-1323 ◽  
Author(s):  
JS Wiley ◽  
GR Dubyak
Keyword(s):  
Adenosine Triphosphate ◽  
Fold Increase ◽  
Normal Subjects ◽  
Peripheral Blood Lymphocytes ◽  
Extracellular Atp ◽  
Total Cell ◽  
Lymphocytic Leukemia ◽  
Maximal Response ◽  
Extracellular Adenosine ◽  
Cation Permeability

Abstract Extracellular adenosine triphosphate (ATP) is known to reversibly increase the cation permeability of a variety of freshly isolated and cultured cell types. In this study the effects of extracellular ATP were studied using peripheral blood lymphocytes (PBL) isolated from both normal subjects and from patients with chronic lymphocytic leukemia (CLL). Changes in the permeability to Na+, Rb+, and Li+ ions were measured using conventional isotope and flame photometry techniques. In addition, changes in cytosolic (Ca2+) were fluorimetrically monitored to assess possible changes in net Ca2+ influx. ATP produced a 12-fold increase in 22Na+ influx into CLL cells but only a 3.5-fold increase in this flux in PBL cells. A maximal response was produced by 0.1 mmol/L ATP in the absence of Mg2+, while a twofold molar excess of Mg2+ over ATP abolished the response. ATP had no effect on the passive (ouabain-insensitive) 86Rb+ influx into PBL cells but stimulated this flux by fivefold in the CLL cells. Li+ influx into CLL cells was also stimulated threefold by ATP. Under these same conditions ATP also produced a net increase in total cell Na and a decrease in total cell K in the CLL cells. Exclusion of two normally impermeable dyes, trypan blue and ethidium bromide, was not altered in the ATP-treated CLL cells. Finally, extracellular ATP (3 mmol/L) produced no significant change in the cytosolic (Ca2+) of normal, monocyte-depleted populations of PBL. Conversely, this same concentration of ATP produced a very rapid (complete within 30 seconds) and a significant (an average threefold peak change) increase in the cytosolic (Ca2+) of cell preparations derived from five out of nine CLL patients. In these latter CLL cells, the ATP-induced elevation in cytosolic (Ca2+) appeared to be due to a net increase in Ca2+ influx, since no elevations were observed when the extracellular (Ca2+) was reduced to less than 0.1 mmol/L. These actions of ATP were specific in that equimolar concentrations of other nucleotides were without effect. These data indicate that treatment of CLL lymphocytes with extracellular ATP4 produces large increases in cation permeability. In contrast, there is less or no ATP-induced permeabilization of normal PBL.

scholarly journals Involvement of intracellular Ca2+ and K+ in dissipation of the mitochondrial membrane potential and cell death induced by extracellular ATP in hepatocytes

1992 ◽  
Vol 288 (1) ◽  
pp. 207-213 ◽  
Author(s):  
J P Zoeteweij ◽  
B van de Water ◽  
H J de Bont ◽  
G J Mulder ◽  
J F Nagelkerke
Keyword(s):  
Cell Death ◽  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Rat Hepatocytes ◽  
Extracellular Atp ◽  
Complete Loss ◽  
Isolated Rat Hepatocytes ◽  
Isolated Mitochondria ◽  
Isolated Rat

Isolated rat hepatocytes were incubated with extracellular ATP to induce a prolonged increase in intracellular Ca2+ ([Ca2+]i) and a loss of viability within 2 h. By using video-intensified fluorescence microscopy, the effects of exposure to extracellular ATP on [Ca2+]i, mitochondrial membrane potential (MMP) and cell viability were determined simultaneously in individual living hepatocytes. The increase in [Ca2+]i on exposure to ATP was followed by a decreasing MMP; there were big differences between individual cells. Complete loss of the MMP occurred before cell death was observed. Omission of K+ from the incubation medium decreased the cytotoxicity of ATP; under these conditions, intracellular K+ was decreased by more than 80%. Treatment with nigericin also depleted intracellular K+ and decreased ATP-induced toxicity. Protection against loss of viability by means of a decrease in intracellular [K+] was reflected by maintenance of the MMP. These observations suggest that ATP-induced cell death may be caused by a mechanism that has been described for isolated mitochondria: after an increase in Ca2+ levels, a K+ influx into mitochondria is induced, which finally disrupts the MMP and leads to cell death.

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