Intracellular pH and the stimulus-secretion coupling in insulin-producing RINm5F cells.
ABSTRACT: The regulation of intracellular pH (pHi) and its role in the insulin-secretory process were evaluated, by using the clonal insulin-secreting cell line RINm5F. Glyceraldehyde, lactate and dihydroxyacetone decreased pHi, but only the first two released insulin. In the presence of extracellular Na+ the cells counteracted the acidification. Blocking the Na+/H+ exchange in acidic cells resulted in a drastic further lowering of pHi, an effect not obtained under basal conditions. Whereas glyceraldehyde depolarized the cells, lactate was without effect. Dihydroxyacetone hyperpolarized the cells in the presence of extracellular Na+, but this effect disappeared when Na+ was excluded from the medium. Stimulation with glyceraldehyde resulted in increased free cytoplasmic Ca2+ concentration ([Ca2+]i). Dihydroxyacetone and lactate had no effect on [Ca2+]i in the presence of Na+, but lactate induced a decrease in [Ca2+]i in Na(+)-deficient medium. In RINm5F cells the activity of the Na+/H+ antiport could not be augmented by activation of protein kinase C (PKC). Hence, in insulin-secreting cells a PKC-insensitive Na+/H+ antiport is the major mechanism restoring a decrease in pHi. Acidification itself does not affect membrane potential, but may directly interact with the mechanisms regulating exocytosis.
Project description:The effects of hepoxilin A3 (HxA3), a 12-lipoxygenase metabolite of arachidonic acid, on cytosolic calcium ([Ca2+]i), intracellular pH (pHi), transmembrane potential and right-angle light scattering in human neutrophils were investigated. A rapid, transient elevation of [Ca2+]i was observed with HxA3 which was dependent on the concentration used. The effect of HxA3 on [Ca2+]i was blocked by pertussis toxin, suggesting involvement of receptors coupled to GTP-binding proteins. Experiments in Ca2(+)-free medium and using intracellular Ca2+ chelators indicated that HxA3 mobilized Ca2+ from intracellular stores. At similar concentrations, HxA3 altered pHi, producing an initial acidification followed by an alkalinization. The initial acidification was decreased in cells loaded with a Ca2+ chelator. In the presence of N-ethyl-N-(1-methylethyl)amino amiloride, an inhibitor of the Na+/H+ antiport, HxA3 induced a greater acidification but failed to elicit the recovery phase, suggesting that the latter is due to activation of the antiport. HxA3 also depolarized the membrane potential, although this effect was small. A decrease in right-angle light scattering, qualitatively similar to that observed with chemotactic peptides, was seen with HxA3, indicating that the 12-lipoxygenase metabolite can induce shape changes in neutrophils. At the concentrations used for the above effects, HxA3 was unable to generate a respiratory burst. These findings suggest that hepoxilins, which are formed by stimulated neutrophils, may have a role as messengers in neutrophil activation.
Project description:D-Glyceraldehyde was transported into HIT-T15 cells at a linear rate for approx. 2 min and appeared to be unsaturable up to a concentration of 50 mM. Evidence was obtained for an electrogenic component of uptake of the triose. The rate of D-glyceraldehyde transport was also reduced in the absence of Na+, suggesting that a component of uptake was Na(+)-linked. Transport of D-glyceraldehyde could be prevented by N-ethylmaleimide but not significantly by p-chloromercuribenzenesulphonic acid, L-glyceraldehyde, nor by a number of inhibitors of known transport systems. However, D-glyceraldehyde transport was inhibited by alpha-cyano-4-hydroxycinnamate, an inhibitor of some anion transport systems. D-Glyceraldehyde caused a marked depolarization of HIT-T15 cells accompanied by a rise in cytosolic [Ca2+] and [Na+] and a gradual intracellular acidification. The glyceraldehyde-induced rise in cytosolic [Na+] and intracellular acidification, but not the depolarization or rise in cytosolic [Ca2+], were reduced by dithiothreitol and 5-aminoguanidine, compounds which form chemical adducts with alpha-ketoaldehydes. Incubation of HIT cells with either D- or L-glyceraldehyde resulted in the formation of large amounts of D-lactate, the end product of methylglyoxal metabolism via the glyoxalase pathway. It is suggested that the depolarizing action of glyceraldehyde is the result, at least in part, of its electrogenic transport, probably via Na(+)-coupled entry into HIT cells involving an unidentified transport system. The intracellular acidification and a component of the increase in cytosolic [Na+] may be largely due to the presence of one or more dicarbonyl contaminants in the glyceraldehyde preparation.
Project description:In this paper, we demonstrate that low concentrations (0.5-2.5 microM) of 1,2-sn-dioctanoylglycerol (DiC8), a potent diacylglycerol used in many previous studies to probe the role of protein kinase C (PKC) in cell activation, cause cytosolic alkalinization of human, mouse and pig T lymphocytes through PKC-mediated activation of the Na+/H+ antiport. However, at higher concentrations (greater than or equal to 12.5 microM), the effect on cytosolic pH (pHi) is reversed, resulting in a marked cytosolic acidification, followed by a gradual return of pHi to baseline values. DiC8 also induces marked changes in cytosolic free calcium concentrations ([Ca2+]i), initially by releasing calcium from intracellular stores, followed by a net transmembrane influx of calcium. The DiC8-induced cytosolic acidification, the resultant return to baseline pH and the increase in [Ca2+]i are independent of activation of PKC. Unlike many other agents which increase [Ca2+]i, DiC8 does not induce phosphatidylinositol hydrolysis with the resultant production of inositol phosphates. Other compounds known to activate PKC, including the closely related diacylglycerol analogues, 1,2-sn-dihexanoylglycerol and 1,2-sn-didecanoylglycerol, phorbol esters and mezerein, did not induce changes in [Ca2+]i or cytosolic acidification in T lymphocytes. Thus the action of DiC8 on intact lymphocytes is different from that of phorbol esters and other diacylglycerols, and is specific to the length of the acyl chains. Because changes in [Ca2+]i are often associated with cell proliferation and cell differentiation, some effects of DiC8 on intact cells may be a consequence of changes in [Ca2+]i.
Project description:The effects of thyrotropin-releasing hormone (TRH) and 12-O-tetradecanoylphorbol 13-acetate (TPA) on cytosolic pH (pHi) were studied on GH4C1 pituitary cells loaded with the fluorescent pH indicator bis(carboxyethyl)carboxyfluorescein (BCECF) and the fluorescent Ca2+ indicator quin2. TRH, which was minimally effective at around 10(-9) M, and TPA, 100 nM, produced very small elevations in pHi of about 0.05 pH units from the normal basal resting pHi of GH4C1 cells of around 7.05. The effects were more marked after acid-loading the cells using 1 micrograms of nigericin/ml. Preincubation with amiloride or replacing the extracellular Na+ with choline+ completely blocked the elevations stimulated by TRH or TPA, consistent with an activation of the Na+/H+ antiport mechanism. The effects were completely independent of the cytoplasmic free calcium concentration ([Ca2+]i). The calcium ionophore ionomycin produced an elevation in [Ca2+]i with no concomitant effect on pHi, and amiloride, although completely inhibiting the pH change stimulated by TRH, failed to affect the initial stimulated [Ca2+]i transient. Although the data are consistent with an elevation in pHi by TRH which is caused by stimulation of a protein kinase C and subsequent activation of the antiporter, the rapidity of the onset of the pHi response to TRH could not be mimicked by a combination of TPA and ionomycin. These results, together with previous findings which show that secretion can be mimicked by TPA and ionomycin, suggest that TRH-stimulated Na+/H+ exchange plays no part in the acute stimulation of secretion, but that TRH increases the pH-sensitivity of the antiport system during increased synthesis of prolactin and growth hormone.
Project description:1. Docosahexaenoic acid (DHA) induced rapid (t1/2=33 s) and dose-dependent decreases in pHi in BCECF-loaded human (Jurkat) T-cells. Addition of 5-(N,N-dimethyl)-amiloride, an inhibitor of Na+/H+ exchanger, prolonged DHA-induced acidification as a function of time, indicating that the exchanger is implicated in pHi recovery. 2. Other fatty acids like oleic acid, arachidonic acid, eicosapentaenoic acid, but not palmitic acid, also induced a fall in pHi in these cells. 3. To assess the role of calcium in the DHA-induced acidification, we conducted experiments in Ca2+-free (0% Ca2+) and Ca2+-containing (100% Ca2+) buffer. We observed that there was no difference in the degree of DHA-induced transient acidification in both the experimental conditions, though pHi recovery was faster in 0% Ca2+ medium than that in 100% Ca2+ medium. 4. In the presence of BAPTA, a calcium chelator, a rapid recovery of DHA-induced acidosis was observed. Furthermore, addition of CaCl2 into 0% Ca2+ medium curtailed DHA-evoked rapid pHi recovery. In 0% Ca2+ medium, containing BAPTA, DHA did not evoke increases in [Ca2+]i, though this fatty acid still induced a rapid acidification in these cells. These observations suggest that calcium is implicated in the long-lasting DHA-induced acidosis. 5. DHA-induced rapid acidification may be due to its deprotonation in the plasma membrane (flip-flop model), as suggested by the following observations: (1) DHA with a -COOH group induced intracellular acidification, but this fatty acid with a -COOCH3 group failed to do so, and (2) DHA, but not propionic acid, -induced acidification was completely reversed by addition of fatty acid-free bovine serum albumin in these cells. 6. These results suggest that DHA induces acidosis via deprotonation and Ca2+ mobilization in human T-cells.
Project description:The relationship between endothelin-1 (ET-1)-induced stimulation of Na(+)-H+ exchange and intracellular free Ca2+ ([Ca2+]i) was examined in primary cultures of porcine coronary artery smooth muscle cells. Intracellular pH (pHi) and [Ca2+]i were measured using 2,7-bis-carboxyethyl-5(6)-carboxyfluorescein and the acetoxymethyl ester of fura-2 respectively. In HCO3(-)-free buffer (pH = 7.4), ET-1 (0.1-50 nM) induced a sustained, dose-dependent increase in pHi. ET-1 (10 nM) increased pHi from 6.83 +/- 0.01 to 6.93 +/- 0.02 (P < 0.01). The alkalinization was blocked by the Na(+)-H+ exchange inhibitor, 5-(N-ethyl-N-isopropyl)amiloride (EIPA, 3 microM) or by substitution of Na+ with N-methylglucamine or choline chloride (P < 0.05). Recovery of pHi in response to acidification, induced by washout of a 20 mM NH4Cl prepulse, was > 90% inhibited by EIPA (3 microM), confirming the presence of an ET-1-responsive Na(+)-H+ exchanger. Coronary smooth muscle cells responded to ET-1 with a dose-dependent, biphasic increase in [Ca2+]i which was not inhibited by manipulations (EIPA pretreatment or Na(+)-free media) shown to block the Na(+)-H+ exchanger. The ET-1-mediated alkalinization was not inhibited by removal of extracellular Ca2+ ([Ca2+]o). However, complete blockade of the ET-1-mediated [Ca2+]i response using the intracellular Ca(2+)-chelator, [bis-(2-amino-5-methylphenoxy)ethane-NNN'N'-tetraacetic acid tetraacetoxymethyl ester] (MAPTAM), in [Ca2+]o-free media, demonstrated that an increment in [Ca2+]i is required for activation of the Na(+)-H+ exchanger by ET-1. The ET-1-induced rise in [Ca2+]i was not associated with a rise in pHi in the presence of CO2/HCO3-. We conclude that: (1) activation of Na(+)-H+ exchange by ET-1 requires an increment in [Ca2+]i; and (2) ET-1 stimulates EIPA-sensitive Na(+)-H+ exchange, but this stimulation does not modulate ET-1-induced changes in [Ca2+]i.
Project description:BACKGROUND:Alterations of bioelectrical properties of cells and tissues are known to function as wide-ranging signals during development, regeneration and wound-healing in several species. The Drosophila follicle-cell epithelium provides an appropriate model system for studying the potential role of electrochemical signals, like intracellular pH (pHi) and membrane potential (Vmem), during development. Therefore, we analysed stage-specific gradients of pHi and Vmem as well as their dependence on specific ion-transport mechanisms. RESULTS:Using fluorescent indicators, we found distinct alterations of pHi- and Vmem-patterns during stages 8 to 12 of oogenesis. To determine the roles of relevant ion-transport mechanisms in regulating pHi and Vmem and in establishing stage-specific antero-posterior and dorso-ventral gradients, we used inhibitors of Na+/H+-exchangers and Na+-channels (amiloride), V-ATPases (bafilomycin), ATP-sensitive K+-channels (glibenclamide), voltage-dependent L-type Ca2+-channels (verapamil), Cl--channels (9-anthroic acid) and Na+/K+/2Cl--cotransporters (furosemide). Either pHi or Vmem or both parameters were affected by each tested inhibitor. While the inhibition of Na+/H+-exchangers (NHE) and amiloride-sensitive Na+-channels or of V-ATPases resulted in relative acidification, inhibiting the other ion-transport mechanisms led to relative alkalisation. The most prominent effects on pHi were obtained by inhibiting Na+/K+/2Cl--cotransporters or ATP-sensitive K+-channels. Vmem was most efficiently hyperpolarised by inhibiting voltage-dependent L-type Ca2+-channels or ATP-sensitive K+-channels, whereas the impact of the other ion-transport mechanisms was smaller. In case of very prominent effects of inhibitors on pHi and/or Vmem, we also found strong influences on the antero-posterior and dorso-ventral pHi- and/or Vmem-gradients. For example, inhibiting ATP-sensitive K+-channels strongly enhanced both pHi-gradients (increasing alkalisation) and reduced both Vmem-gradients (increasing hyperpolarisation). Similarly, inhibiting Na+/K+/2Cl--cotransporters strongly enhanced both pHi-gradients and reduced the antero-posterior Vmem-gradient. To minor extents, both pHi-gradients were enhanced and both Vmem-gradients were reduced by inhibiting voltage-dependent L-type Ca2+-channels, whereas only both pHi-gradients were reduced (increasing acidification) by inhibiting V-ATPases or NHE and Na+-channels. CONCLUSIONS:Our data show that in the Drosophila follicle-cell epithelium stage-specific pHi- and Vmem-gradients develop which result from the activity of several ion-transport mechanisms. These gradients are supposed to represent important bioelectrical cues during oogenesis, e.g., by serving as electrochemical prepatterns in modifying cell polarity and cytoskeletal organisation.
Project description:1. An insulin-producing cell line, RINm5F, derived from a rat insulinoma was studied. 2. The cellular content of immunoreactive insulin was 0.19 pg/cell, which represents approx. 1% of the insulin content of native rat beta-cells, whereas that of immunoreactive glucagon and somatostatin was five to six orders of magnitude less than that of native alpha- or delta-cells respectively. 3. RINm5F cells released 7-12% of their cellular immunoreactive-insulin content at 2.8 mM-glucose during 60 min in Krebs-Ringer bicarbonate buffer. 4. Glucose utilization was increased by raising glucose from 2.8 to 16.7 mM. There was, however, no stimulation of immunoreactive-insulin release even when glucose was increased from 2.8 to 33.4 mM. A small stimulation of release was, however, found when glucose was raised from 0 to 2.8 mM. 5. Glyceraldehyde stimulated the release of immunoreactive insulin in a dose-dependent manner. 6. At 20 mM, leucine or arginine stimulated release at 2.8 mM-glucose. 7. Raising intracellular cyclic AMP by glucagon or 3-isobutyl-1-methylxanthine stimulated release at 2.8 mM-glucose with no additional stimulation at 16.7 mM-glucose. 8. Stimulation of immunoreactive-insulin release by K+ was dose-related between 2 and 30 mM. Another depolarizing agent, ouabain, also stimulated release. 9. Adrenaline (epinephrine) inhibited both basal (2.8 mM-glucose) release and that stimulated by 30 mM-K+. 10. Raising Ca2+ from 1 to 3 mM stimulated immunoreactive-insulin release, whereas a decrease from 1 to 0.3 or to 0.1 mM-Ca2+ lowered the release. 11. These findings could reflect a relatively specific impairment in glucose handling by RINm5F cells, contrasting with the preserved response to other modulators of insulin release.
Project description:Na+/Ca2+ exchange in the B cell was recently characterized by measuring intracellular-Na(+)-dependent 45Ca2+ uptake in isolated rat pancreatic islet cells. The aim of the present study was to investigate the regulation of this process. Extracellular pH (pHo) and intracellular pH (pHi) markedly affected Na+/Ca2+ exchange. A fall of 0.04 unit in pHi decreased the exchange by 45%, whereas a rise of 0.13 unit increased the uptake by 70%. Mitochondrial poisons (oligomycin, antimycin A and 2,4-dinitrophenol) inhibited reverse Na+/Ca2+ exchange by about 25-50%. The exchanger displayed a low Q10 (temperature coefficient), indicating that it is only indirectly dependent on metabolic energy. The phorbol ester phorbol 12-myristate 13-acetate did not affect Na+/Ca2+ exchange. Likewise, lowering the extracellular K+ concentration did not inhibit 45Ca2+ uptake. In conclusion, the pHi and the metabolic state of the cell may represent important modulatory signals by which insulin secretagogues such as glucose could regulate reverse Na+/Ca2+ exchange in the B cell. The process does not appear to co-transport K+ nor to be influenced by protein kinase C.
Project description:The effects of D-glyceraldehyde on the hepatocyte contents of various metabolites were examined and compared with the effects of fructose, glycerol and dihydroxyacetone, which all enter the glycolytic/gluconeogenic pathways at the triose phosphate level. D-Glyceraldehyde (10 MM) caused a substantial depletion of hepatocyte ATP, as did equimolar concentrations of fructose and glycerol. D-Glyceraldehyde and fructose each caused a 2-fold increase in fructose 1,6-bisphosphate and the accumulation of millimolar quantities of fructose 1-phosphate in the cells. D-Glyceraldehyde caused an increase in the glycerol 3-phosphate content and a decrease in the dihydroxyacetone phosphate content, whereas dihydroxyacetone increased the content of both metabolites. The increase in the [glycerol 3-phosphate]/[dihydroxyacetone phosphate] ratio caused by D-glyceraldehyde was not accompanied by a change in the cytoplasmic [NAD+]/[NADH] ratio, as indicated by the unchanged [lactate]/[pyruvate] ratio. The accumulation of fructose 1-phosphate from D-glyceraldehyde and dihydroxyacetone phosphate in the hepatocyte can account for the depletion of the intracellular content of the latter. Presumably ATP is depleted as the result of the accumulation of millimolar amounts of a phosphorylated intermediate, as is the case with fructose and glycerol. It is suggested that the accumulation of fructose 1-phosphate during hepatic fructose metabolism is the result of a temporary increase in the D-glyceraldehyde concentration because of the high rate of fructose phosphorylation compared with triokinase activity. The equilibrium constant of aldolase favours the formation and thus the accumulation of fructose 1-phosphate.