The effect of the internal Na concentration on the electrogenicity of the insulin-stimulated Na,K-pump in frog skeletal muscles

Insulin induced a hyperpolarization of the membrane by stimulating the Na,K-pump in frog skeletal muscles. The Na,K-pump activity was dependent on the internal Na concentration. As the internal Na concentration was raised from 5 mmol/kg muscle water to 18 mmol/kg muscle water, the magnitude of the insulin-induced increase in the ouabain-sensitive Na efflux (an index of the Na,K-pump activity) rose by 5-fold and the magnitude of the insulin-induced hyperpolarization rose by 8.5-fold. On the other hand, the specific membrane resistance was not significantly changed by a rise in the internal Na concentration. The Na/K coupling of the Na,K-pump was calculated at low, normal or high internal Na concentration by using the values of the insulin-induced changes in the ouabain-sensitive Na efflux and the membrane potential. As a result of the calculation, it was suggested that in frog skeletal muscles the Na/K coupling would increase with a rise of the internal Na concentration.     

Effects of external K concentration on the electrogenicity of the insulin-stimulated Na,K-pump in frog skeletal muscle

Summary Insulin hyperpolarized the membrane of frog skeletal muscle by stimulating the electrogenic Na,K-pump. At external K concentrations of 1, 2, 5 and 10mm, both the insulin-induced hyperpolarization and the insulin-stimulated ouabain-sensitive Na efflux (an index of Na, K-pump activity) were observed. By increasing the external K concentration, the insulin-stimulated Na efflux increased, but the magnitude of the insulin-induced hyperpolarization decreased; i. e., although the activity of the insulin-stimulated Na,K-pump increased, on the contrary, the magnitude of the hyperpolarization decreased. To clarify the causes of this phenomenon, the specific membrane resistance was measured and found to decrease upon increasing the external K concentration.One of the reasons for the decrease in magnitude of the hyperpolarization is the decrease in the specific membrane resistance. However, the decrease in magnitude of the hyperpolarization with a rise of the external K concentration, which increased the insulin-stimulated Na,K-pump activity, cannot be explained only by the decrease in the specific membrane resistance. It is suggested that the decrease in magnitude of the hyperpolarization is mainly caused by a decrease in the electrogenicity of the insulin-stimulated Na,K-pump upon an increase in the external K concentration. The conclusion of the present study is that the electrogenicity of the insulin-stimulated Na,K-pump in muscles is variable and decreases with increasing the external K concentration.     

Diverse effects of insulin-induced hyperpolarization on 3-O-methyl-D-glucose (3-O-MG) transport in frog skeletal muscles

It has been suggested that the insulin-induced hyperpolarization might be a mediator of the stimulatory action of insulin on glucose transport. The purpose of the present study was to investigate the relationship between the insulin-induced hyperpolarization and the stimulatory action of insulin on glucose transport in skeletal muscle. Satorius muscles dissected from bullfrogs (Rana catesbeiana) were used. Insulin induced a hyperpolarization of the membrane and an increase in the 3-O-Methyl-D-glucose (3-O-MG) uptake and extrusion. In the presence of valinomycin, insulin had no significant effect on the membrane potential. Insulin still had the stimulatory action on both the 3-O-MG uptake and extrusion even in the presence of valinomycin, under whose condition insulin had no significant effect on the membrane potential. The magnitude of the stimulatory action of insulin on the 3-O-MG uptake in the presence of valinomycin was smaller than that in the absence of valinomycin. The magnitude of the stimulatory action of insulin on the 3-O-MG extrusion was, on the contrary, larger than that in the absence of valinomycin. The abolishment of the insulin-induced hyperpolarization decreased the 3-O-MG uptake and increased the 3-O-MG extrusion. The observation in the present study concludes that insulin has two different actions on glucose transport. One of them is developed through the insulin-induced hyperpolarization, which increases the 3-O-MG uptake and decreases the 3-O-MG extrusion. The other action is irrelevant of the insulin-induced hyperpolarization and stimulates both the 3-O-MG uptake and extrusion.     

Effects of acute sodium omission on insulin release, ionic flux and membrane potential in mouse pancreatic B-cells

The effects of acute omission of extracellular Na+ on pancreatic B-cell function were studied in mouse islets, using choline and lithium salts as impermeant and permeant substitutes, respectively. In the absence of glucose, choline substitution for Na+ hyperpolarized the B-cell membrane, inhibited 86Rb+ and 45Ca2+ efflux, but did not affect insulin release. In contrast, Li+ substitution for Na+ depolarized the B-cell membrane and caused a Ca2+-independent, transient acceleration of 45Ca2+ efflux and insulin release. Na+ replacement by choline in the presence of 10 mM glucose and 2.5 mM Ca2+ again rapidly hyperpolarized the B-cell membrane. This hyperpolarization was then followed by a phase of depolarization with continuous spike activity, before long slow waves of the membrane potential resumed. Under these conditions, 86Rb+ efflux first decreased before accelerating, concomitantly with marked and parallel increases in 45Ca2+ efflux and insulin release. In the absence of Ca2+, 45Ca2+ and 86Rb+ efflux were inhibited and insulin release was unaffected by choline substitution for Na+. Na+ replacement by Li+ in the presence of 10 mM glucose rapidly depolarized the B-cell membrane, caused an intense continuous spike activity, and accelerated 45Ca2+ efflux, 86Rb+ efflux and insulin release. In the absence of extracellular Ca2+, Li+ still caused a rapid but transient increase in 45Ca2+ and 86Rb+ efflux and in insulin release. Although not indispensable for insulin release, Na+ plays an important regulatory role in stimulus-secretion coupling by modulating, among others, membrane potential and ionic fluxes in B-cells.     

Characterization of the cyclic AMP-independent actions of somatostatin in GH cells. I. An increase in potassium conductance is responsible for both the hyperpolarization and the decrease in intracellular free calcium produced by somatostatin

The neuropeptide somatostatin causes membrane hyperpolarization and reduces the intracellular free calcium ion concentration ([Ca2+]i) in GH pituitary cells. In this study, we have used the fluorescent dyes bisoxonol (bis,-(1,3-diethylthiobarbiturate)-trimethineoxonol) and quin2 to elucidate the mechanisms by which these ionic effects are triggered. Addition of 100 nM somatostatin to GH4C1 cells caused a 3.4 mV hyperpolarization and a 26% decrease in [Ca2+]i within 30 s. These effects were not accompanied by changes in intracellular cAMP concentrations and occurred in cells containing either basal or maximally elevated cAMP levels. To determine which of the major permeant ions were involved in these actions of somatostatin, we examined its ability to elicit changes in the membrane potential and the [Ca2+]i when the transmembrane concentration gradients for Na+, Cl-, Ca2+, and K+ were individually altered. Substitution of impermeant organic ions for Na+ or Cl- did not block either the hyperpolarization or the decrease in [Ca2+]i induced by somatostatin. Decreasing extracellular Ca2+ from 1 mM to 250 nM abolished the reduction in [Ca2+]i but did not prevent the hyperpolarization response. These results show that hyperpolarization was not primarily due to changes in the conductances of Na+, Cl-, or Ca2+. Although the somatostatin-induced decrease in [Ca2+]i did require Ca2+ influx, it was independent of changes in Na+ or Cl- conductance. In contrast, elevating the extracellular [K+] from 4.6 to 50 mM completely blocked both the somatostatin-induced hyperpolarization and the reduction in [Ca2+]i. Furthermore, hyperpolarization of the cells with gramicidin mimicked the effect of somatostatin to decrease the [Ca2+]i and prevented any additional effect by the hormone. These results indicate that somatostatin increases a K+ conductance, which hyperpolarizes GH4C1 cells, and thereby secondarily decreases Ca2+ influx. Since the somatostatin-induced decrease in [Ca2+]i is independent of changes in intracellular cAMP levels, it may be responsible for somatostatin inhibition of hormone secretion by its cAMP-independent mechanism.     

Protein kinase Cdelta mediates insulin-induced glucose transport in primary cultures of rat skeletal muscle

Insulin activates certain protein kinase C (PKC) isoforms that are involved in insulin-induced glucose transport. In this study, we investigated the possibility that activation of PKCdelta by insulin participates in the mediation of insulin effects on glucose transport in skeletal muscle. Studies were performed on primary cultures of rat skeletal myotubes. The role of PKCdelta in insulin-induced glucose uptake was evaluated both by selective pharmacological blockade and by over-expression of wild-type and point-mutated inactive PKCdelta isoforms in skeletal myotubes. We found that insulin induces tyrosine phosphorylation and translocation of PKCdelta to the plasma membrane and increases the activity of this isoform. Insulin-induced effects on translocation and phosphorylation of PKCdelta were blocked by a low concentration of rottlerin, whereas the effects of insulin on other PKC isoforms were not. This selective blockade of PKCdelta by rottlerin also inhibited insulin-induced translocation of glucose transporter 4 (GLUT4), but not glucose transporter 3 (GLUT3), and significantly reduced the stimulation of glucose uptake by insulin. When overexpressed in skeletal muscle, PKCdelta and PKCdelta were both active. Overexpression of PKCdelta induced the translocation of GLUT4 to the plasma membrane and increased basal glucose uptake to levels attained by insulin. Moreover, insulin did not increase glucose uptake further in cells overexpressing PKCdelta. Overexpression of PKCdelta did not affect basal glucose uptake or GLUT4 location. Stimulation of glucose uptake by insulin in cells overexpressing PKCdelta was similar to that in untransfected cells. Transfection of skeletal myotubes with dominant negative mutant PKCdelta did not alter basal glucose uptake but blocked insulin-induced GLUT4 translocation and glucose transport. These results demonstrate that insulin activates PKCdelta and that activated PKCdelta is a major signaling molecule in insulin-induced glucose transport.     

Insulin-like and non-insulin-like selenium actions in 3T3-L1 adipocytes

In insulin-sensitive 3T3-L1 adipocytes, selenium stimulates glucose transport and antilipolysis and these actions of selenium, like insulin actions, are sensitive to wortmanin, an inhibitor of phosphatidylinositol-3-kinase (PI3K). Selenium stimulates PI3K activity that is sustained up to 24 h. Selenium after 5-10 min increases tyrosine phosphorylation of selective cellular proteins, but after 24 h overall tyrosine phosphorylation is increased. Tyrosine phosphorylation of insulin receptor substrate 1 is detected when enriched by immunoprecipitation with anti-PI3K antibody. Selenium, however, does not stimulate insulin receptor tyrosine kinase activity. Selenium also increases phosphorylation of other insulin signaling proteins, including Akt and extracellular signal regulated kinases. Selenium-stimulated glucose transport is accompanied by increases in glucose transporter-1 content in the plasma membrane. These data are consistent with similar selenium action in glucose transport in 3T3-L1 fibroblasts expressing mainly GLUT1. In chronic insulin-induced insulin resistant cells, selenium unlike insulin fully stimulates glucose transport. In summary, selenium stimulates glucose transport and antilipolysis in a PI3K-dependent manner, but independent of insulin receptor activation. Selenium exerts both insulin-like and non-insulin-like actions in cells.     

Regulation of Na+-K+-ATPase: effect of Mg and Ca ions

The participation of Mg2+ and Ca2+ in complicated mechanisms of Na+, K(+)-ATPase regulation is discussed in the survey. The regulatory actions of Mg2+ on Na+, K(+)-ATPase such as its participation in phosphorylation and dephosphorylation of the enzyme, ADP/ATP-exchange inhibition, cardiac glycosides and vanadate binding with the enzyme, conformational changes induction during ATPase cycle are reviewed in detail. Some current views of mechanisms of above mentioned Mg2+ regulatory effects are discussed. The experimental evidence of Ca2+ immediate influence on the functional activity of Na+, K(+)-ATPase (catalytic, transport and glycoside-binding) are given. It's noted that these effects are based on the conformational changes in the enzyme and also on the phase transition in membrane induced by Ca2+. Unimmediate action of Ca2+ on Na+, K(+)-ATPase is also discussed, especially due to its effect on other membrane systems functionally linked with Na(+)-pump (for instance, due to Na+/Ca(+)-exchanger activation). It's concluded that Mg2+ and Ca2+ as "universal regulators" of the cell effectively influence the functional activity and conformational states of Na+, K(+)-ATPase.     

Regulation of insulin release by ionic and electrical events in B cells

This review article is an attempt to schematize the major alterations in ionic fluxes and B cell membrane potential that underlie the changes in insulin release brought about by glucose and by other stimulators or inhibitors. Glucose metabolism in B cells leads to closure of K channels in the plasma membrane. The resulting decrease in K+ permeability causes depolarization with activation of voltage-dependent Ca channels. An increase in Ca2+ influx ensues, which raises the cytoplasmic concentration of free Ca2+ and ultimately triggers insulin release. Tolbutamide induces a similar sequence of events by a direct action on K channels. In contrast, diazoxide antagonizes the effects of glucose by increasing K+ permeability of the B cell membrane. Among amino acids, leucine largely mimics the effects of glucose, whereas arginine depolarizes the B cell membrane because of its transport in a positively charged form.     

Insulin signalling and insulin actions in the muscles and livers of insulin-resistant, insulin receptor substrate 1-deficient mice.

We and others recently generated mice with a targeted disruption of the insulin receptor substrate 1 (IRS-1) gene and demonstrated that they exhibited growth retardation and had resistance to the glucose-lowering effect of insulin. Insulin initiates its biological effects by activating at least two major signalling pathways, one involving phosphatidylinositol 3-kinase (PI3-kinase) and the other involving a ras/mitogen-activated protein kinase (MAP kinase) cascade. In this study, we investigated the roles of IRS-1 and IRS-2 in the biological action in the physiological target organs of insulin by comparing the effects of insulin in wild-type and IRS-1-deficient mice. In muscles from IRS-1-deficient mice, the responses to insulin-induced PI3-kinase activation, glucose transport, p70 S6 kinase and MAP kinase activation, mRNA translation, and protein synthesis were significantly impaired compared with those in wild-type mice. Insulin-induced protein synthesis was both wortmannin sensitive and insensitive in wild-type and IRS-1 deficient mice. However, in another target organ, the liver, the responses to insulin-induced PI3-kinase and MAP kinase activation were not significantly reduced. The amount of tyrosine-phosphorylated IRS-2 (in IRS-1-deficient mice) was roughly equal to that of IRS-1 (in wild-type mice) in the liver, whereas it only 20 to 30% of that of IRS-1 in the muscles. In conclusion, (i) IRS-1 plays central roles in two major biological actions of insulin in muscles, glucose transport and protein synthesis; (ii) the insulin resistance of IRS-1-deficient mice is mainly due to resistance in the muscles; and (iii) the degree of compensation for IRS-1 deficiency appears to be correlated with the amount of tyrosine-phosphorylated IRS-2 (in IRS-1-deficient mice) relative to that of IRS-1 (in wild-type mice).     

Insulin-like actions of nickel and other transition-metal ions in rat fat-cells.

NiC12 (1-6mM) decreased adrenaline and glucagon-stimulated lipolysis in rat fat-cells, and also considerably stimulated [U-14C]glucose incorporation into fat-cell lipids. 2. These insulin-like effects were also observed with CuCl, CuCl2, CoCl2 and (to a lesser extent) with MnCl2. 3. NiCl2 was less effective in mimicking insulin effects on [U-14C]fructose metabolism than on glucose utilization. 4. It is tentatively suggested that these transition-metal ions may mimic actions of insulin at the fat-cell plasma membrane which decrease lipolysis and stimulate glucose transport, but do not mimic certain other effects of the hormone on intracellular metabolic processes. 5. These results are discussed with reference to suggestions that redistributions of cellular Ca2+ are associated with insulin action in fat-cells.     

Calcium dynamics and homeostasis in a mathematical model of the principal cell of the cortical collecting tubule

Calcium (Ca) dynamics are incorporated into a mathematical model of the principal cell in the cortical collecting tubule developed earlier in Strieter et al. (1992a. Am. J Physiol. 263:F1063-1075). The Ca components are modeled after the Othmer-Tang model for IP(3)-sensitive calcium channels (1993, in Experimental and Theoretical Advances in Biological Pattern Formation, 295-319). There are IP(3)-sensitive Ca channels and ATP-driven pumps on the membrane of the endoplasmic reticulum. Calcium enters the cell passively down its electrochemical gradient. A Ca pump and Na/Ca exchange in the basolateral membrane are responsible for the extrusion of cytoplasmic calcium. Na/Ca exchange can also operate in reverse mode to transport Ca into the cell. Regulatory effects of cytoplasmic Ca on the apical Na channels are modeled after experimental data that indicate apical Na permeability varies inversely with cytoplasmic Ca concentration. Numerical results on changes in intracellular Ca caused by decreasing NaCl in the bath and the lumen are similar to those from experiments in Bourdeau and Lau (1990. Am. J Physiol. 258:F1497-1503). This match of simulation and experiment requires the synergistic action of the Na/Ca exchanger and the Ca regulated apical Na permeability. In a homogeneous medium, cytoplasmic Ca becomes oscillatory when extracellular Na is severely decreased, as observed in experiments of cultured principal cells (Koster, H., C. van Os and R. Bindels. 1993. Kidney Int.43:828-836). This essentially pathological situation arises because the hyperpolarization of membrane potential caused by Na-free medium increases Ca influx into the cell, while the Na/Ca exchanger is inactivated by the low extracellular Na and can no longer move Ca out of the cell effectively. The raising of the total amount of intracellular Ca induces oscillatory Ca movement between the cytoplasm and the endoplasmic reticulum. Ca homeostasis is investigated under the condition of severe extracellular Ca variations. As extracellular Ca is decreased, Ca regulation is greatly impaired if Ca does not regulate apical ionic transport. The simulations indicate that the Na/Ca exchanger alone has only limited regulatory capacity. The Ca regulated apical sodium or potassium permeability are essential for regulation of cytoplasmic Ca in the principal cell of the cortical collecting tubule.     

Tension development of normal and hypertrophied-failing papillary muscles following rapid stimulation

Recent studies have shown that numerous cellular alterations exist in hypertrophied-failing (HF) cardiac muscle. Of particular interest is the finding of an altered ability of the Na-K pump to regulate membrane potential in this tissue during periods of transient stimulation. The present study was designed to determine if this altered Na-K pump function is in any way related to the ability of this tissue to develop force. Along these lines the rate of stimulation (6/min) of normal and hypertrophied-failing right ventricular papillary muscles from cats was increased to 60/min for 90 sec. This procedure was repeated in solutions with low Na+, low Na+ and Ca++, and Ouabain. These solutions were utilized to vary the ionic load on the Na-K pump and the Na-Ca exchanger. The results demonstrate that the pattern of changes in tension in HF papillary muscles seen following periods of rapid stimulation are significantly different from those of normal muscles. The pattern of changes in mechanical performance were found to be similar to the membrane potential changes described in previous studies. In addition, lowering the Na+ load presented to HF muscles returned the characteristic pattern of changes in tension, following drive, toward normal. Ouabain was found to inhibit the changes in tension development following increased rates of stimulation that are thought to be produced by activation of the Na-K pump. The results suggest that the ability of the Na-K pump to maintain normal transmembrane ionic gradients may be altered in HF muscles. This alteration appears to be capable of affecting cellular Ca++ possibly through the Na-Ca exchange system.     

Correlation of the effect of Ca+2 on Na+ and K+ permeability and membrane potential of Ehrlich ascites tumor cells

The effect of Ca+2 on the transport and intracellular distribution of Na+ and K+ in Ehrlich ascites tumor cells was investigated in an effort to establish the mechanism of Ca+2-induced hyperpolarization of the cell membrane. Inclusion of Ca+2 (2 mM) in the incubation medium leads to reduced cytoplasmic concentrations of Na+, K+ and Cl- in steady cells. In cells inhibited by ouabain, Ca+2 causes a 41% decrease in the rate of net K+ loss, but is without effect on the rate of net Na+ accumulation. Net K+ flux is reduced by 50%, while net Na+ flux is unchanged in the transport-inhibited cells. The membrane potential of cells in Ca+2-free medium (-13.9 +/- 0.8 mV) is unaffected by the addition of ouabain. However, the potential of cells in Ca+2-containing medium (-23.3 +/- 1.2 mV) declines in one hour after the addition of ouabain to values comparable to those of control cells (-15.2 +/- 0.7 mV). The results of these experiments are consistent with the postulation that Ca+2 exerts two effects on Na+ and K+ transport. First, Ca+2 reduces the membrane permeability to K+ by 25%. Second, Ca+2 alters the coupling of the Na/K active transport mechanism leading to an electrogenic hyperpolarization of the membrane.     

Metabolic regulation of apical sodium permeability in toad urinary bladder in the presence and absence of aldosterone

In the present study, further evidence was adduced for energy-dependent regulation of passive apical transport of Na in toad bladder epithelium. In potassium-depolarized preparations studied by current-voltage analysis, additions of pyruvate or glucose to the media of substrate-depleted bladders evoked proportionate increases in the transepithelial Na current and in apical Na permeability. These responses were large in aldosterone pretreated hemibladders and almost absent in the aldosterone-depleted preparations or when hormonal action was blocked by spironolactone or cycloheximide. The substrate-induced increases in apical Na permeability were fully reversed by appropriate metabolic inhibitors, i.e. 2-deoxyglucose and oxythiamine. Moreover, the inhibitory effect of 2-deoxyglucose was bypassed by the addition of pyruvate to the serosal medium. Thus apical Na permeability is clearly sensitive to the supply of cellular energy. The possibility that changes in intracellular free Na activity may mediate metabolic regulation of apical Na permeability was evaluated by prolonged exposure to Na-free mucosal and serosal media, with and without inhibition of the Na/K-pump by ouabain. The stimulatory and inhibitory effects of pyruvate, 2-deoxyglucose and oxythiamine on Na currents and Na conductances were preserved under these circumstances. Furthermore, reduction of serosal Ca to a minimal level of 3 microM, was without effect on the response to metabolic inhibition. These experiments demonstrate the existence of Na-independent metabolic regulation of apical Na transport and imply that neither basal-lateral nor mitochondrial Na/Ca exchange is required for this regulatory process under the imposed conditions. The possibility that a Na-independent, Ca transport mechanism in mitochondria or endoplasmic reticulum may be involved in metabolic regulation of apical Na transport, however, remains to be evaluated.     

Insulin-induced changes in membrane potential and 3-O-methylglucose uptake at various external K concentrations in frog skeletal muscle

Insulin induced a hyperpolarization of the membrane and stimulated the 3-O-methylglucose (3-O-MG) uptake in frog skeletal muscle. In the present study, the relationship between the insulin-induced changes in the membrane potential and the 3-O-MG uptake was investigated. The stimulatory action of insulin on the 3-O-MG uptake was mediated by two different mechanisms. One of them was dependent on the change in the membrane potential and the other was independent of the change in the membrane potential. Both values of the insulin-induced changes in the membrane potential and the 3-O-MG uptake were diminished by increasing the external K concentration. One of the causes for the diminution of the 3-O-MG uptake with a rise of the external K concentration would be the decrease in the magnitude of the insulin-induced hyperpolarization.     

Effects of chloride deficiency on the pancreatic B-cell response to acetylcholine

Muscarinic stimulation of pancreatic B-cells markedly amplifies insulin secretion through complex mechanisms which involve changes in membrane potential and ionic fluxes. In this study, normal mouse islets were used to evaluate the role of Cl- ions in these effects of acetylcholine (ACh). Whatever the concentration of glucose, the rate of 36Cl- efflux from islet cells was unaffected by ACh. Replacement of Cl- by impermeant isethionate in a medium containing 15 mM glucose did not affect, or only slightly decreased, the ability of ACh to depolarize the B-cell membrane and increase electrical activity, to accelerate 45Ca2+ and 86Rb+ efflux from islet cells, and to amplify insulin release. In the absence of extracellular Ca2+, a high concentration of ACh (100 microM) mobilized intracellular Ca2+ and caused a transient release of insulin and a sustained acceleration of 86Rb+ efflux. None of these effects was affected by Cl- omission or by addition of furosemide, a blocker of the Na+, K+, 2Cl- cotransport. Isethionate substitution for Cl- in a medium containing a nonstimulatory concentration of glucose (3 mM) barely reduced the depolarization of B-cells by ACh, but inhibited the concomitant increase in 86Rb+ efflux. We have no explanation for the latter effect that was not mimicked by furosemide. In conclusion, ACh stimulation of pancreatic B-cells, unlike that of exocrine acinar cells, is largely independent of Cl- and is insensitive to furosemide. The acceleration of ionic fluxes produced by ACh does not involve the Na+, K+, 2Cl- cotransport system.     

Measuring ion transport activities in Xenopus oocytes using the ion-trap technique

The ion-trap technique is an experimental approach allowing measurement of changes in ionic concentrations within a restricted space (the trap) comprised of a large-diameter ion-selective electrode apposed to a voltage-clamped Xenopus laevis oocyte. The technique is demonstrated with oocytes expressing the Na(+)/glucose cotransporter (SGLT1) using Na(+)- and H(+)-selective electrodes and with the electroneutral H(+)/monocarboxylate transporter (MCT1). In SGLT1-expressing oocytes, bath substrate diffused into the trap within 20 s, stimulating Na(+)/glucose influx, which generated a measurable decrease in the trap Na(+) concentration ([Na(+)](T)) by 0.080 +/- 0.009 mM. Membrane hyperpolarization produced a further decrease in [Na(+)](T), which was proportional to the increased cotransport current. In a Na(+)-free, weakly buffered solution (pH 5.5), H(+) drives glucose transport through SGLT1, and this was monitored with a H(+)-selective electrode. Proton movements can also be clearly detected on adding lactate to an oocyte expressing MCT1 (pH 6.5). For SGLT1, time-dependent changes in [Na(+)](T) or [H(+)](T) were also detected during a membrane potential pulse (150 ms) in the presence of substrate. In the absence of substrate, hyperpolarization triggered rapid reorientation of SGLT1 cation binding sites, accompanied by cation capture from the trap. The resulting change in [Na(+)](T) or [H(+)](T) is proportional to the pre-steady-state charge movement. The ion-trap technique can thus be used to measure steady-state and pre-steady-state transport activities and provides new opportunities for studying electrogenic and electroneutral ion transport mechanisms.     

Hyperpolarization of mouse skeletal muscle plasma membrane induced by extracellular NADH

Extracellularly applied NADH, but not NAD or NADPH, increases the resting membrane potential from -74.1 to -76.6 mV in freshly isolated muscles in the presence of K+ in the incubation medium and from -64.6 to -72.9 mV in muscles equilibrated for 4-6 h in a K+-free solution. The NADH-induced hyperpolarization is blocked by pretreatment of muscles with ouabain, and the inhibitors of plasma membrane NADH dehydrogenase (adriamycin, azide, PCMB, atebrine, DIDS and bleomycin). The effect of NADH is accompanied by the disappearance of NADH from the incubation medium and by decreased membrane resistance. We conclude that NADH hyperpolarization is due to the enhancement of passive membrane permeability, apparently for K+, which might result from the conformational changes in the plasma membrane during the NADH dehydrogenase reaction. The possibility is discussed that NADH dehydrogenase mediates transport of K+ out from the cell using a pathway connected with the transmembrane Na+/K+ pump.     

Galanin inhibits glucose-stimulated insulin release by a mechanism involving hyperpolarization and lowering of cytoplasmic free Ca2+ concentration

The intrapancreatic neuropeptide galanin has been demonstrated to lower plasma insulin levels in vivo. The effects of this peptide on insulin secretion, cytoplasmic free Ca2+ concentration and membrane potential have now been studied in vitro. Glucose-stimulated insulin secretion was inhibited by galanin under these conditions, indicating a direct effect of the peptide on the beta-cells. The neuropeptide reversed both the increase in membrane potential and cytoplasmic free Ca2+ in response to glucose stimulation. At a non-stimulatory concentration of the sugar, galanin induced a slight hyperpolarization without any effect on cytoplasmic free Ca2+. Galanin did not affect K+-induced increase in cytoplasmic free Ca2+, excluding a direct inhibitory effect on the voltage-activated Ca2+ channels. The results indicate that galanin inhibition of glucose-stimulated insulin release involves hyperpolarization with a subsequent decrease in cytoplasmic free Ca2+.