Solution structure of crotamine, a Na+ channel affecting toxin from Crotalus durissus terrificus venom.

Crotamine is a component of the venom of the snake Crotalus durissus terrificus and it belongs to the myotoxin protein family. It is a 42 amino acid toxin cross-linked by three disulfide bridges and characterized by a mild toxicity (LD50 = 820 micro g per 25 g body weight, i.p. injection) when compared to other members of the same family. Nonetheless, it possesses a wide spectrum of biological functions. In fact, besides being able to specifically modify voltage-sensitive Na+ channel, it has been suggested to exhibit analgesic activity and to be myonecrotic. Here we report its solution structure determined by proton NMR spectroscopy. The secondary structure comprises a short N-terminal alpha-helix and a small antiparallel triple-stranded beta-sheet arranged in an alphabeta1beta2beta3 topology never found among toxins active on ion channels. Interestingly, some scorpion toxins characterized by a biological activity on Na+ channels similar to the one reported for crotamine, exhibit an alpha/beta fold, though with a beta1alphabeta2beta3 topology. In addition, as the antibacterial beta-defensins, crotamine interacts with lipid membranes. A comparison of crotamine with human beta-defensins shows a similar fold and a comparable net positive potential surface. To the best of our knowledge, this is the first report on the structure of a toxin from snake venom active on Na+ channel.     

Modifications of human cardiac sodium channel gating by UVA light

Voltage-gated Na(+) channels are membrane proteins responsible for the generation of action potentials. In this report we demonstrate that UVA light elicits gating changes of human cardiac Na+ channels. First, UVA irradiation hampers the fast inactivation of cardiac Nav1.5 Na(+) channels expressed in HEK293t cells. A maintained current becomes conspicuous during depolarization and reaches its maximal quasi steady-state level within 5-7 min. Second, the activation time course is slowed by UVA light; modification of the activation gating by UVA irradiation continues for 20 min without reaching steady state. Third, along with the slowed activation time course, the peak current is reduced progressively. Most Na(+) currents are eliminated during 20 min of UVA irradiation. Fourth, UVA light increases the holding current nonlinearly; this phenomenon is slow at first but abruptly fast after 20 min. Other skeletal muscle Nav1.4 isoforms and native neuronal Na(+) channels in rat GH(3) cells are likewise sensitive to UVA irradiation. Interestingly, a reactive oxygen metabolite (hydrogen peroxide at 1.5%) and an oxidant (chloramine-T at 0.5 mM) affect Na(+) channel gating similarly, but not identically, to UVA. These results together suggest that UVA modification of Na(+) channel gating is likely mediated via multiple reactive oxygen metabolites. The potential link between oxidative stress and the impaired Na(+) channel gating may provide valuable clues for ischemia/reperfusion injury in heart and in CNS.     

G protein-independent activation of an inward Na(+) current by muscarinic receptors in mouse pancreatic beta-cells

Depolarization of pancreatic beta-cells is critical for stimulation of insulin secretion by acetylcholine but remains unexplained. Using voltage-clamped beta-cells, we identified a small inward current produced by acetylcholine, which was suppressed by atropine or external Na(+) omission, but was not mimicked by nicotine, and was insensitive to nicotinic antagonists, tetrodotoxin, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DiDS), thapsigargin pretreatment, and external Ca(2+) and K(+) removal. This suggests that muscarinic receptor stimulation activates voltage-insensitive Na(+) channels distinct from store-operated channels. No outward Na(+) current was produced by acetylcholine when the electrochemical Na(+) gradient was reversed, indicating that the channels are inward rectifiers. No outward K(+) current occurred either, and the reversal potential of the current activated by acetylcholine in the presence of Na(+) and K(+) was close to that expected for a Na(+)-selective membrane, suggesting that the channels opened by acetylcholine are specific for Na(+). Overnight pretreatment with pertussis toxin or the addition of guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S) or guanosine-5'-O-(2-thiodiphosphate) (GDP-beta-S) instead of GTP to the pipette solution did not alter this current, excluding involvement of G proteins. Injection of a current of a similar amplitude to that induced by acetylcholine elicited electrical activity in beta-cells perifused with a subthreshold glucose concentration. These results demonstrate that muscarinic receptor activation in pancreatic beta-cells triggers, by a G protein-independent mechanism, a selective Na(+) current that explains the plasma membrane depolarization.     

Intraspecific variation of the crotamine and crotasin genes in Crotalus durissus rattlesnakes

Crotamine is a small basic myotoxin peptide of Crotalus durissus venom, with β-defensin scafold and variable concentration in individual venoms. The crotamine gene was mapped to the end of chromosome 2 and the signal intensity differed significantly between the two homologues. In contrast to crotamine, the paralogous crotasin gene is scarcely expressed in the venom glands. In this study, we analyzed the crotamine concentrations in the venoms of a total of 23 rattlesnakes from diverse Brazilian localities by ELISA as well as the copy number of both crotamine and crotasin genes by real-time PCR. Crotamine was found to constitute 5–29% of venom proteins varying greatly among individual animals. The crotamine gene exists from 1 to 32 copies per haploid genome, whereas the crotasin gene is present from 1 to 7 copies. Furthermore, we observed that the crotamine concentration and crotamine gene copy number are positively correlated (r² = 0.68), implying the variation of crotamine in venom results from the variation of the gene copy number. Sequencing of 50 independent copies of crotamine and crotasin genes from four different rattlesnakes revealed the presence of six crotasin isoforms with a single amino acid difference from the original crotasin sequence, whereas only two additional crotamine isoforms were observed. Taken together, our results suggested that after duplication from a common ancestor gene, crotamine and crotasin may have diverged in such a way that the crotamine gene underwent repetitive duplication to increase its copy number, whereas the crotasin gene diversified its sequence.     

Insulin stimulates transepithelial sodium transport by activation of a protein phosphatase that increases Na-K ATPase activity in endometrial epithelial cells.

The objective of this study was to investigate the effects of insulin and insulin-like growth factor I on transepithelial Na(+) transport across porcine glandular endometrial epithelial cells grown in primary culture. Insulin and insulin-like growth factor I acutely stimulated Na(+) transport two- to threefold by increasing Na(+)-K(+) ATPase transport activity and basolateral membrane K(+) conductance without increasing the apical membrane amiloride-sensitive Na(+) conductance. Long-term exposure to insulin for 4 d resulted in enhanced Na(+) absorption with a further increase in Na(+)-K(+) ATPase transport activity and an increase in apical membrane amiloride-sensitive Na(+) conductance. The effect of insulin on the Na(+)-K(+) ATPase was the result of an increase in V(max) for extracellular K(+) and intracellular Na(+), and an increase in affinity of the pump for Na(+). Immunohistochemical localization along with Western blot analysis of cultured porcine endometrial epithelial cells revealed the presence of alpha-1 and alpha-2 isoforms, but not the alpha-3 isoform of Na(+)-K(+) ATPase, which did not change in the presence of insulin. Insulin-stimulated Na(+) transport was inhibited by hydroxy-2-naphthalenylmethylphosphonic acid tris-acetoxymethyl ester [HNMPA-(AM)(3)], a specific inhibitor of insulin receptor tyrosine kinase activity, suggesting that the regulation of Na(+) transport by insulin involves receptor autophosphorylation. Pretreatment with wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase as well as okadaic acid and calyculin A, inhibitors of protein phosphatase activity, also blocked the insulin-stimulated increase in short circuit and pump currents, suggesting that activation of phosphatidylinositol 3-kinase and subsequent stimulation of a protein phosphatase mediates the action of insulin on Na(+)-K(+) ATPase activation.     

Unraveling the antifungal activity of a South American rattlesnake toxin crotamine

Crotamine is a highly basic peptide from the venom of Crotalus durissus terrificus rattlesnake. Its common gene ancestry and structural similarity with the β-defensins, mainly due to an identical disulfide bond pattern, stimulated us to assess the antimicrobial properties of native, recombinant, and chemically synthesized crotamine. Antimicrobial activities against standard strains and clinical isolates were analyzed by the colorimetric microdilution method showing a weak antibacterial activity against both Gram-positive and Gram-negative bacteria [MIC (Minimum Inhibitory Concentration) of 50–>200 μg/mL], with the exception of Micrococcus luteus [MIC ranging from 1 to 2 μg/mL]. No detectable activity was observed for the filamentous fungus Aspergillus fumigatus and Trichophyton rubrum at concentrations up to 125 μg/mL. However, a pronounced antifungal activity against Candida spp., Trichosporon spp., and Cryptococcus neoformans [12.5–50.0 μg/mL] was observed. Chemically produced synthetic crotamine in general displayed MIC values similar to those observed for native crotamine, whereas recombinant crotamine was overridingly more potent in most assays. On the other hand, derived short linear peptides were not very effective apart from a few exceptions. Pronounced ultrastructure alteration in Candida albicans elicited by crotamine was observed by electron microscopy analyses. The peculiar specificity for highly proliferating cells was confirmed here showing potential low cytotoxic effect of crotamine against nontumoral mammal cell lines (HEK293, PC12, and primary culture astrocyte cells) compared to tumoral B16F10 cells, and no hemolytic activity was observed. Taken together these results suggest that, at low concentration, crotamine is a potentially valuable anti-yeast or candicidal agent, with low harmful effects on normal mammal cells, justifying further studies on its mechanisms of action aiming medical and industrial applications.     

Inhibition of Na+/Ca2+ exchanger by peroxynitrite in microsomes of pulmonary smooth muscle: role of matrix metalloproteinase-2

Treatment of bovine pulmonary artery smooth muscle microsomes with peroxynitrite (ONOO-) (100 microM) markedly stimulated matrix metalloproteinase-2 (MMP-2) activity and also enhanced Ca2+ATPase activity and ATP-dependent Ca2+ uptake. Pretreatment of the microsomes with vitamin E (1 mM) and TIMP-2 (50 microg/ml) preserved the increase in MMP-2 activity, Ca2+ATPase activity and also ATP-dependent Ca2+ uptake in the microsomes. In contrast, Na(+)-dependent Ca2+ uptake in the microsomes was inhibited by ONOO- and this was found to be reversed by vitamin E (1 mM) and TIMP-2 (50 microg/ml). However, changes caused by ONOO- in MMP-2 activity, ATP-dependent Ca2+ uptake and Na(+)-dependent Ca2+ uptake were not reversed upon pretreatment of the microsomes with a low concentration of 5 microg/ml of TIMP-2 which, on the contrary, reversed MMP-2 (1 microg/ml)-mediated alteration on these parameters. The inhibition of Na(+)-dependent Ca2+ uptake by ONOO- and MMP-2 overpowered the stimulation of ATP-dependent Ca2+ uptake in the microsomes. Treatment with ONOO- abolished the inhibitory effect of TIMP-2 (5 microg/ml) on MMP-2 (1 microg/ml) causing 14C-gelatin degradation. Overall, the present study suggests that ONOO- inactivated TIMP-2, the ambient inhibitor of MMP-2, leading to activation of the ambient proteinase, MMP-2, and subsequently stimulated Ca2+ATPase activity and ATP-dependent Ca2+ uptake, but inhibited Na(+)-dependent Ca2+ uptake, resulting in a marked decrease in Ca2+ uptake in microsomes of bovine pulmonary artery smooth muscle.     

Control of ion transport in mouse proximal and distal colon by prolactin.

The lactogenic hormone prolactin (PRL) has been known to affect Ca(2+) and electrolyte transport in the intestinal epithelium. In the present study we analyzed ion transport in mouse proximal and distal colon, and acute changes induced by PRL. In the proximal colon, carbachol activated a Ca(2+) dependent Cl(-) secretion that was sensitive to DIDS and NFA. In the distal colon, both ATP and carbachol activated K(+) secretion. Ca(2+) -activated KCl transport in proximal and distal colon was inhibited by PRL (200 ng/ml), while amiloride sensitive Na(+) absorption and cAMP induced Cl(-) secretion remained unaffected. Luminal large conductance Ca(2+) -activated K(+) (BK) channels were largely responsible for Ca(2+) -activated K(+) secretion in the distal colon, and basolateral BK channels supported Ca(2+) -activated Cl(-) secretion in the proximal colon. Ca(2+) chelating by BAPTA-AM attenuated effects of carbachol and abolished effects of PRL. Both inhibition of PI3 kinase with wortmannin and blockage of MAP kinases with SB 203580 or U 0126, interfered with the acute inhibitory effect of PRL on ion transport, while blocking of Jak/Stat kinases with AG 490 was without effects. PRL attenuated the increase in intracellular Ca(2+) that was caused by stimulation of isolated colonic crypts with carbachol. Thus PRL inhibits Ca(2+) dependent Cl(-) and K(+) secretion by interfering with intracellular Ca(2+) signaling and probably by activating PI3 kinase and MAP kinase pathways.     

Electrolyte Transport in the Mouse Trachea: No Evidence for a Contribution of Luminal K+ Conductance

Recent studies on frog skin acini have challenged the question whether Cl(-) secretion or Na(+) absorption in the airways is driven by luminal K(+) channels in series to a basolateral K(+) conductance. We examined the possible role of luminal K(+) channels in electrolyte transport in mouse trachea in Ussing-chamber experiments. Tracheas of both normal and CFTR (-/-) mice showed a dominant amiloride-sensitive Na+ absorption under both, control conditions and after cAMP-dependent stimulation. The lumen-negative transepithelial voltage was enhanced after application of IBMX and forskolin and Cl(-) secretion was activated. Electrolyte secretion induced by IBMX and forskolin was inhibited by luminal glibenclamide and the blocker of basolateral Na(+2)Cl(-)K(+) cotransporter azosemide. Similarly, the compound 293B, a blocker of basolateral KCNQ1/KCNE3 K(+) channels effectively blocked Cl(-) secretion when applied to either the luminal or basolateral side of the epithelium. RT-PCR analysis suggested expression of additional K(+) channels in tracheal epithelial cells such as Slo1 and Kir6.2. However, we did not detect any functional evidence for expression of luminal K(+) channels in mouse airways, using luminal 293B, clotrimazole and Ba(2+) or different K(+) channel toxins such as charybdotoxin, apamin and a-dendrotoxin. Thus, the present study demonstrates Cl(-) secretion in mouse airways, which depends on basolateral Na(+2)Cl(-)K(+) cotransport and luminal CFTR and non-CFTR Cl(-) channels. Cl(-) secretion is maintained by the activity of basolateral K(+) channels, while no clear evidence was found for the presence of a luminal K(+) conductance.     

Na+/Mg2+ exchange is functionally coupled to the insulin receptor

Regulation of cellular Mg(2+) levels by insulin has been shown in various tissues. However, the mechanisms for hormonal regulation of cellular Mg(2+) have not been well described. We studied the effect of insulin on Na(+)/Mg(2+) exchange in normal human cells, measuring Na(+)/Mg(2+) exchange activity as net total Mg(2+) efflux driven by an inward Na(+) gradient in Mg(2+)-loaded red blood cells (RBCs). Na(+)/Mg(2+) exchange was increased significantly by the addition of 2.4 nmol/L of insulin to the flux medium (from 0.60 +/- 0.06 mmol/L cell x h to 0.75 +/- 0.08 mmol/L cell x h [P = 0.0098, n = 44]). A dose-response curve for the effects of insulin on the exchanger activity gave an estimated EC(50) for insulin of 0.95 +/- 0.31 nmol/L and a V(max) of 0.86 +/- 0.12 mmol/L cell x h (n = 7). Kinetics of the Na(+)/Mg(2+) exchange were characterized by measuring its activity as a function of Mg(2+) and Na(+) concentrations. The K(0.5) for cellular Mg(2+) was not affected by incubation with insulin. However, the K(0.5) for extracellular Na(+) decreased from 69.9 +/- 6.3 to 40.3 +/- 8.4 mol/L (n = 5, P = 0.03) in the presence of insulin. We also studied the effect of wortmannin (WT), a PI 3-kinase inhibitor, on activity of the exchanger. WT significantly blocked the insulin-stimulated Na(+)/Mg(2+) activity (n = 6, P = 0.048), with an IC(50) of 0.5 nmol/L. LY294002, another PI 3-kinase inhibitor, likewise blocked the insulin-stimulated activity of the exchanger. Therefore, insulin regulates cellular Mg(2+) metabolism in part via an increase in the affinity for Na(+) of the Na(+)/Mg(2+) exchange and PI 3-kinase activation, suggesting another role for the PI 3-kinase pathway in insulin-mediated cellular events.     

Effects of insulin on Na and K transporters in the rat CCD

We tested the effects of insulin (2 nM, 30-60 min) on principal cells of isolated split-open rat cortical collecting ducts (CCD) using whole-cell current measurements. Insulin addition to the superfusate of the tubules enhanced Na pump (ouabain-sensitive) current from 18 ± 3 to 31 ± 3 pA/cell in control and from 74 ± 9 to 126 ± 11 pA/cell in high K-fed animals. It also more than doubled ROMK (tertiapin-Q-sensitive) K(+) currents in control CCD from 320 ± 40 to 700 ± 80 pA/cell, although it did not affect this current in tubules from K-loaded rats. Insulin did not induce the appearance of amiloride-sensitive Na(+) current in control animals, while in high K-fed animals the currents were similar in the presence (140 ± 30) and the absence (180 ± 70 pA/cell) of insulin. Intraperitoneal injection of insulin plus hypertonic dextrose decreased Na excretion, as previously reported. However, injection of dextrose alone, or the nonmetabolized sugar mannose, had similar effects, suggesting that they were largely the result of vascular volume depletion rather than specific actions of the hormone. In summary, we find no evidence for acute upregulation of the epithelial Na channel (ENaC) by physiological concentrations of insulin in the mammalian CCD. However, the hormone does activate both the Na/K pump and apical K(+) channels and could, under some conditions, enhance renal K(+) secretion.     

Gating charge immobilization caused by the transition between inactivated states in the Kv1.5 channel

Sustained Na(+) or Li(+) conductance is a feature of the inactivated state in wild-type (WT) and nonconducting Shaker and Kv1.5 channels, and has been used here to investigate the cause of off-gating charge immobilization in WT and Kv1.5-W472F nonconducting mutant channels. Off-gating immobilization in response to brief pulses in cells perfused with NMG/NMG is the result of a more negative voltage dependence of charge recovery (V(1/2) is -96 mV) compared with on-gating charge movement (V(1/2) is -6.3 mV). This shift is known to be associated with slow inactivation in Shaker channels and the disparity is reduced by 40 mV, or approximately 50% in the presence of 135 mM Cs. Off-gating charge immobilization is voltage-dependent with a V(1/2) of -12 mV, and correlates well with the development of Na(+) conductance on repolarization through C-type inactivated channels (V(1/2) is -11 mV). As well, the time-dependent development of the inward Na(+) tail current and gating charge immobilization after depolarizing pulses of different durations has the same time constant (tau = 2.7 ms). These results indicate that in Kv1.5 channels the transition to a stable C-type inactivated state takes only 2-3 ms and results in strong charge immobilization in the absence of Group IA metal cations, or even in the presence of Na. Inclusion of low concentrations of Cs delays the appearance of Na(+) tail currents in WT channels, prevents transition to inactivated states in Kv1.5-W472F nonconducting mutant channels, and removes charge immobilization. Higher concentrations of Cs are able to modulate the deactivating transition in Kv1.5 channels and prevent the residual slowing of charge return.     

Simvastatin Impairs Insulin Secretion by Multiple Mechanisms in MIN6 Cells

Statins are widely used in the treatment of hypercholesterolemia and are efficient in the prevention of cardiovascular disease. Molecular mechanisms explaining statin-induced impairment in insulin secretion remain largely unknown. In the current study, we show that simvastatin decreased glucose-stimulated insulin secretion in mouse pancreatic MIN6 β-cells by 59% and 79% (p<0.01) at glucose concentration of 5.5 mmol/l and 16.7 mmol/l, respectively, compared to control, whereas pravastatin did not impair insulin secretion. Simvastatin induced decrease in insulin secretion occurred through multiple targets. In addition to its established effects on ATP-sensitive potassium channels (p = 0.004) and voltage-gated calcium channels (p = 0.004), simvastatin suppressed insulin secretion stimulated by muscarinic M3 or GPR40 receptor agonists (Tak875 by 33%, p = 0.002; GW9508 by 77%, p = 0.01) at glucose level of 5.5 mmol/l, and inhibited calcium release from the endoplasmic reticulum. Impaired insulin secretion caused by simvastatin treatment were efficiently restored by GPR119 or GLP-1 receptor stimulation and by direct activation of cAMP-dependent signaling pathways with forskolin. The effects of simvastatin treatment on insulin secretion were not affected by the presence of hyperglycemia. Our observation of the opposite effects of simvastatin and pravastatin on glucose-stimulated insulin secretion is in agreement with previous reports showing that simvastatin, but not pravastatin, was associated with increased risk of incident diabetes.     

Glucose-induced cytosolic pH changes in beta-cells and insulin secretion are not causally related: studies in islets lacking the Na+/H+ exchangeR NHE1

The contribution of Na(+)/H(+) exchange (achieved by NHE proteins) to the regulation of beta-cell cytosolic pH(c), and the role of pH(c) changes in glucose-induced insulin secretion are disputed and were examined here. Using real-time PCR, we identified plasmalemmal NHE1 and intracellular NHE7 as the two most abundant NHE isoforms in mouse islets. We, therefore, compared insulin secretion, cytosolic free Ca(2+) ([Ca(2+)](c)) and pH(c) in islets from normal mice and mice bearing an inactivating mutation of NHE1 (Slc9A1-swe/swe). The experiments were performed in HCO(-)(3)/CO(2) or HEPES/NaOH buffers. PCR and functional approaches showed that NHE1 mutant islets do not express compensatory pH-regulating mechanisms. NHE1 played a greater role than HCO(-)(3)-dependent mechanisms in the correction of an acidification imposed by a pulse of NH(4)Cl. In contrast, basal pH(c) (in low glucose) and the alkalinization produced by high glucose were independent of NHE1. Dimethylamiloride, a classic blocker of Na(+)/H(+) exchange, did not affect pH(c) but increased insulin secretion in NHE1 mutant islets, indicating unspecific effects. In control islets, glucose similarly increased [Ca(2+)](c) and insulin secretion in HCO(-)(3) and HEPES buffer, although pH(c) changed in opposite directions. The amplification of insulin secretion that glucose produces when [Ca(2+)](c) is clamped at an elevated level by KCl was also unrelated to pH(c) and pH(c) changes. All effects of glucose on [Ca(2+)](c) and insulin secretion proved independent of NHE1. In conclusion, NHE1 protects beta-cells against strong acidification, but has no role in stimulus-secretion coupling. The changes in pH(c) produced by glucose involve HCO(-)(3)-dependent mechanisms. Variations in beta-cell pH(c) are not causally related to changes in insulin secretion.     

Interchain disulfide bonds in crotamine self-association

Crotamine, a basic neurotoxic protein, was isolated from the venom of the Southern Brazilian rattlesnake (Crotalus durissus terrificus) by gel filtration. The isolated protein showed a single band on PAGE at pH 4.5 and 7% (w/v) gel concentration, but two or more bands at 14% gel concentration, even in the presence of 4 M urea. After reduction and carboxymethylation, however, a single band was again detected. SDS-PAGE as well as ultracentrifugal analysis of the native (NC) and of the reduced and carboxymethylated (RCC) crotamine revealed a molecular weight of 4,500-5,000 for RCC and 9,000-10,000 for NC. Both components of a two-band crotamine preparation were isolated by preparative PAGE and characterized. Their particular electrophoretic mobility was retained. Their amino acid composition. N-terminal residue, and apparent toxicity were the same as those of the original sample. It was concluded that crotamine is able to form a dimer of 9,760 Da with two identical polypeptide chains crosslinked by interchain disulfide bonds and a shape not very far from spherical, which covalently binds extra subunits of 4,880 Da each.     

Lessons from genetically engineered animal models VIII. Absorption and secretion of ions in the gastrointestinal tract

Absorption and secretion of ions in gastrointestinal and other epithelial tissues require the concerted activities of ion pumps, channels, symporters, and exchangers, which operate in coupled systems to mediate transepithelial transport. Our understanding of the identities, membrane locations, and biochemical activities of epithelial ion transporters has advanced significantly in recent years, but major gaps and uncertainties remain in our understanding of their physiological functions. Increasingly, this problem is being addressed by the analysis of mutant mouse models developed by gene targeting. In this review, we discuss gene knockout studies of the secretory isoform of the Na(+)-K(+)-2Cl(-) cotransporter, isoforms 1, 2, and 3 of the Na(+)/H(+) exchanger, and the colonic H(+)-K(+)-ATPase. This approach is leading to a clearer understanding of the functions of these transporters in the living animal.     

Glucose stimulates Ca2+ influx and insulin secretion in 2-week-old beta-cells lacking ATP-sensitive K+ channels

In adult beta-cells glucose-induced insulin secretion involves two mechanisms (a) a K(ATP) channel-dependent Ca(2+) influx and rise of cytosolic [Ca(2+)](c) and (b) a K(ATP) channel-independent amplification of secretion without further increase of [Ca(2+)](c). Mice lacking the high affinity sulfonylurea receptor (Sur1KO), and thus K(ATP) channels, have been developed as a model of congenital hyperinsulinism. Here, we compared [Ca(2+)](c) and insulin secretion in overnight cultured islets from 2-week-old normal and Sur1KO mice. Control islets proved functionally mature: the magnitude and biphasic kinetics of [Ca(2+)](c) and insulin secretion changes induced by glucose, and operation of the amplifying pathway, were similar to adult islets. Sur1KO islets perifused with 1 mm glucose showed elevation of both basal [Ca(2+)](c) and insulin secretion. Stimulation with 15 mm glucose produced a transient drop of [Ca(2+)](c) followed by an overshoot and a sustained elevation, accompanied by a monophasic, 6-fold increase in insulin secretion. Glucose also increased insulin secretion when [Ca(2+)](c) was clamped by KCl. When Sur1KO islets were cultured in 5 instead of 10 mm glucose, [Ca(2+)](c) and insulin secretion were unexpectedly low in 1 mm glucose and increased following a biphasic time course upon stimulation by 15 mm glucose. This K(ATP) channel-independent first phase [Ca(2+)](c) rise was attributed to a Na(+)-, Cl(-)-, and Na(+)-pump-independent depolarization of beta-cells, leading to Ca(2+) influx through voltage-dependent calcium channels. Glucose indeed depolarized Sur1KO islets under these conditions. It is suggested that unidentified potassium channels are sensitive to glucose and subserve the acute and long-term metabolic control of [Ca(2+)](c) in beta-cells without functional K(ATP) channels.     

Up-regulation of acid-gated Na(+) channels (ASICs) by cystic fibrosis transmembrane conductance regulator co-expression in Xenopus oocytes.

Cystic fibrosis transmembrane conductance regulator (CFTR) functions as both a chloride channel and an epithelial transport regulator, interacting with Na(+) (epithelial sodium channel), Cl(-), renal outer medullary potassium channel(+), and H(2)O channels and some exchangers (i.e. Na(+)/H(+)) and co-transporters (Na(+)-HCO(3)(minus sign), Na(+)-K(+)-2Cl(-)). Acid-sensitive ion channels (ASICs), members of the epithelial sodium channel/degenerin superfamily, were originally cloned from neuronal tissue, and recently localized in epithelia. Because CFTR has been immunocytochemically and functionally identified in rat, murine, and human brain, the regulation of ASICs by CFTR was tested in oocytes. Our observations show that the proton-gated Na(+) current formed by the heteromultimeric ASIC1a/2a channel was up-regulated by wild type but not by Delta F508-CFTR. In contrast, the acid-gated Na(+) current associated with either the homomultimeric ASIC1a or ASIC2a channel was not influenced by wild type CFTR. The apparent equilibrium dissociation constant for extracellular Na(+) for ASIC1a/2a was increased by CFTR, but CFTR had no effect on the gating behavior or acid sensitivity of ASIC1a/2a. CFTR had no effect on the pH activation of ASIC1a/2a. We conclude that wild type CFTR elevates the acid-gated Na(+) current of ASIC1a/2a in part by altering the kinetics of extracellular Na(+) interaction.     

Lactate stimulates insulin secretion without blocking the K+ channels in HIT-T15 insulinoma cells.

To clarify the mechanism by which lactate affects insulin secretion, we investigated the effect of lactate on insulin secretion, cytosolic free Ca2+ ([Ca2+](i), the ATP sensitive K+ channel (K(ATP)) and the Ca2+-activated K+ channel (K(Ca)) in HIT-T15 cells, and the results were compared with those of glucose and glibenclamide. All three agents caused insulin secretion and increased [Ca2+](i), but the effects on the K+ channels were different. In cell-attached patch configurations, 10 mmol/l glucose blocked both the K(ATP) and KCa channels, while 100 nmol/l glibenclamide had no effect on KCa channels, but blocked K(ATP) channels. Lactate at a concentration of 10 mmol/l activated both the K(ATP) and KCa channels, not only in cell-attached, but also in inside-out patch configurations, indicating that the increase in [Ca2+](i) and secretion of insulin by lactate cannot be explained by the blocking of the K+ channels. Lactate, at concentrations of 10 mmol/l and 50 mmol/l decreased 45Ca2+ efflux, while glibenclamide increased the efflux. These results suggest that the lactate-induced Ca2+ increase is not due to the closing of K+ channels, but at least in part, to the suppression of Ca2+ efflux from HIT cells.     

Tranilast inhibits glucose-induced insulin secretion from pancreatic beta-cells.

Tranilast, N-(3,4-demethoxycinnamoyl)-anthranilic acid, is an anti-allergic agent identified as an inhibitor of mast cell degranulation. Recently, tranilast was shown to decrease albuminuria in a rat model of diabetic nephropathy and to ameliorate vascular hypertrophy in diabetic rats, suggesting that it may be clinically useful in the treatment of diabetic complications. However, the effects of tranilast on glucose tolerance have not been elucidated. Thus, the aim of this study is to investigate the effect of tranilast on insulin secretion in pancreatic beta-cells. Treatment with tranilast significantly suppressed insulin secretion in INS-1E cells and rat islets induced by 16.7 mmol/l glucose. Furthermore, tranilast inhibited tolbutamide-induced insulin secretion. Treatment with tranilast increased (86)Rb (+) efflux from COS-1 cells in which pancreatic beta-cell-type ATP-sensitive K (+) (K (ATP)) channels were reconstructed and suppressed the cytosolic ATP/ADP ratio in INS-1E cells. Interestingly, treatment with tranilast enhanced glucose uptake in INS-1E cells. In the present study, we demonstrated that tranilast inhibited glucose- and tolbutamide-induced insulin secretion through the activation of K (ATP) channels in pancreatic beta-cells.