Increased activity of L-type Ca2+ channels exposed to serum from patients with type I diabetes.

Type I diabetes [insulin-dependent diabetes mellitus (IDDM)] is an autoimmune disease associated with the destruction of pancreatic beta cells. Serum from patients with IDDM increased L-type calcium channel activity of insulin-producing cells and of GH3 cells derived from a pituitary tumor. The subsequent increase in the concentration of free cytoplasmic Ca2+ ([Ca2+]i) was associated with DNA fragmentation typical of programmed cell death or apoptosis. These effects of the serum were prevented by adding a blocker of voltage-activated L-type Ca2+ channels. When the serum was depleted of immunoglobulin M (IgM), it no longer affected [Ca2+]i. An IgM-mediated increase in Ca2+ influx may thus be part of the autoimmune reaction associated with IDDM and contribute to the destruction of beta cells in vivo.

PKC-dependent stimulation of exocytosis by sulfonylureas in pancreatic beta cells.

Hypoglycemic sulfonylureas represent a group of clinically useful antidiabetic compounds that stimulate insulin secretion from pancreatic beta cells. The molecular mechanisms involved are not fully understood but are believed to involve inhibition of potassium channels sensitive to adenosine triphosphate (KATP channels) in the beta cell membrane, causing membrane depolarization, calcium influx, and activation of the secretory machinery. In addition to these effects, sulfonylureas also promoted exocytosis by direct interaction with the secretory machinery not involving closure of the plasma membrane KATP channels. This effect was dependent on protein kinase C (PKC) and was observed at therapeutic concentrations of sulfonylureas, which suggests that it contributes to their hypoglycemic action in diabetics.

Targeted delivery of antisense oligonucleotides to pancreatic ß-cells.

Antisense oligonucleotide (ASO) silencing of the expression of disease-associated genes is an attractive novel therapeutic approach, but treatments are limited by the ability to deliver ASOs to cells and tissues. Following systemic administration, ASOs preferentially accumulate in liver and kidney. Among the cell types refractory to ASO uptake is the pancreatic insulin-secreting ß-cell. Here, we show that conjugation of ASOs to a ligand of the glucagon-like peptide-1 receptor (GLP1R) can productively deliver ASO cargo to pancreatic ß-cells both in vitro and in vivo. Ligand-conjugated ASOs silenced target genes in pancreatic islets at doses that did not affect target gene expression in liver or other tissues, indicating enhanced tissue and cell type specificity. This finding has potential to broaden the use of ASO technology, opening up novel therapeutic opportunities, and presents an innovative approach for targeted delivery of ASOs to additional cell types.

Intracellular pH, cytosolic calcium concentration and electrical activity in RINm5F insulinoma cells.

The addition of L-lactate or acetate to RINm5F cells caused a transient intracellular acidification, an increase in [Ca2+]i and induced electrical activity. The subsequent withdrawal of lactate or acetate resulted in an intracellular alkalinization with no apparent changes in [Ca2+]i nor electrical activity. Intracellular alkalinization and acidification by application by application and withdrawal of NH4Cl were both accompanied by transient increases in [Ca2+]i in the absence of electrical activity. The induction of electrical activity by lactate was associated with the appearance of inward whole cell currents. Changes in intracellular pH may affect [Ca2+]i though not necessarily by altering plasma membrane potential. The inward currents associated with lactate application may represent an organic anion conductance contributing towards the stimulation of electrical activity by organic acids.

Inhibition of ATP-regulated K(+)-channels by a photoactivatable ATP-analogue in mouse pancreatic beta-cells.

The effects of a photoactivable (DMNPE-caged) ATP-analogue on ATP-regulated K(+)-channels (KATP-channel) in mouse pancreatic beta-cells were investigated using the inside-out patch configuration of the patch-clamp technique. The caged precursor caused a concentration-dependent reduction of channel activity with a Ki of 17 microM; similar to the 11 microM obtained for standard Mg-ATP. The small difference in the blocking capacity between the precursor and ATP is probably the reason why no change in channel activity was observed upon photolysis of the caged molecule and liberation of ATP. It is suggested that the part of the ATP molecule involved in the blocking reaction of the KATP-channel is not sufficiently protected in DMNPE-caged ATP making this compound unsuitable for studying the rapid kinetics of ATP-induced KATP-channel inhibition.

A point mutation inactivating the sulfonylurea receptor causes the severe form of persistent hyperinsulinemic hypoglycemia of infancy in Finland.

Mutations in genes encoding the ATP-regulated potassium (K(ATP)) channels of the pancreatic beta-cell (SUR1 and Kir6.2) are the major known cause of persistent hyperinsulinemic hypoglycemia of infancy (PHHI). We collected all cases of PHHI diagnosed in Finland between 1983 and 1997 (n = 24). The overall incidence was 1:40,400, but in one area of Central Finland it was as high as 1:3,200. Haplotype analysis using polymorphic markers spanning the SUR1/Kir6.2 gene cluster confirmed linkage to the 11p region. Sequence analysis revealed a novel point mutation in exon 4 of SUR1, predicting a valine to aspartic acid change at amino acid 187 (V187D). Of the total cases, 15 affected individuals harbored this mutation in heterozygous or homozygous form, and all of these had severe hyperinsulinemia that responded poorly to medical treatment and required subtotal pancreatectomy. No K(ATP) channel activity was observed in beta-cells isolated from a homozygous patient or after coexpression of recombinant Kir6.2 and SUR1 carrying the V187D mutation. Thus, the mutation produces a nonfunctional channel and, thereby, continuous insulin secretion. This unique SUR1 mutation explains the majority of PHHI cases in Finland and is strongly associated with a severe form of the disease. These findings provide diagnostic and prognostic utility for suspected PHHI patients.

ATP-sensitive potassium channels and efaroxan-induced insulin release in the electrofusion-derived BRIN-BD11 beta-cell line.

The properties of ATP-sensitive K+ (K(ATP)) channels were explored in the electrofusion-derived, glucose-responsive, insulin-secreting cell line BRIN-BD11 using patch-clamp techniques. In intact cells, K(ATP) channels were inhibited by glucose, the sulfonylurea tolbutamide, and the imidazoline compounds efaroxan and phentolamine. Each of these agents initiated insulin secretion and potentiated the actions of glucose. K(ATP) channels were blocked by ATP in a concentration-dependent manner and activated by ADP in the presence of ATP. In both intact cells and excised inside-out patches, the K(ATP) channel agonists diazoxide and pinacidil activated channels, and both compounds inhibited insulin secretion evoked by glucose, tolbutamide, and imidazolines. The mechanisms of action of imidazolines were examined in more detail. Pre-exposure of BRIN-BD11 cells to either efaroxan or phentolamine selectively inhibited imidazoline-induced insulin secretion but not the secretory responses of cells to glucose, tolbutamide, or a depolarizing concentration of KCl. These conditions did not result in the loss of depolarization-dependent rises in intracellular Ca2+ ([Ca2+]i), K(ATP) channel operation, or the actions of either ATP or efaroxan on K(ATP) channels. Desensitization of the imidazoline receptor following exposure to high concentrations of efaroxan, however, was found to result in an increase in SUR1 protein expression and, as a consequence, an upregulation of K(ATP) channel density. Our data provide 1) the first characterization of K(ATP) channels in BRIN-BD11 cells, a novel insulin-secreting cell line produced by electrofusion techniques, and 2) a further analysis of the role of imidazolines in the control of insulin release.

Hyperinsulinism of infancy: the regulated release of insulin by KATP channel-independent pathways.

Hyperinsulinism of infancy (HI) is a congenital defect in the regulated release of insulin from pancreatic beta-cells. Here we describe stimulus-secretion coupling mechanisms in beta-cells and intact islets of Langerhans isolated from three patients with a novel SUR1 gene defect. 2154+3 A to G SUR1 (GenBank accession number L78207) is the first report of familial HI among nonconsanguineous Caucasians identified in the U.K. Using patch-clamp methodologies, we have shown that this mutation is associated with both a decrease in the number of operational ATP-sensitive K+ channels (KATP channels) in beta-cells and impaired ADP-dependent regulation. There were no apparent defects in the regulation of Ca2+- and voltage-gated K+ channels or delayed rectifier K+ channels. Intact HI beta-cells were spontaneously electrically active and generating Ca2+ action currents that were largely insensitive to diazoxide and somatostatin. As a consequence, when intact HI islets were challenged with glucose and tolbutamide, there was no rise in intracellular free calcium ion concentration ([Ca2+]i) over basal values. Capacitance measurements used to monitor exocytosis in control and HI beta-cells revealed that there were no defects in Ca2+-dependent exocytotic events. Finally, insulin release studies documented that whereas tolbutamide failed to cause insulin secretion as a consequence of impaired [Ca2+]i signaling, glucose readily promoted insulin release. Glucose was also found to augment the actions of protein kinase C- and protein kinase A-dependent agonists in the absence of extracellular Ca2+. These findings document the relationship between SUR1 gene defects and insulin secretion in vivo and in vitro and describe for the first time KATP channel-independent pathways of regulated insulin secretion in diseased human beta-cells.

Cloning and functional expression of the cDNA encoding a novel ATP-sensitive potassium channel subunit expressed in pancreatic beta-cells, brain, heart and skeletal muscle.

A cDNA clone encoding an inwardly-rectifying potassium channel subunit (Kir6.2) was isolated from an insulinoma cDNA library. The mRNA is strongly expressed in brain, skeletal muscle, cardiac muscle and in insulinoma cells, weakly expressed in lung and kidney and not detectable in spleen, liver or testis. Heterologous expression of Kir6.2 in HEK293 cells was only observed when the cDNA was cotransfected with that of the sulphonylurea receptor (SUR). Whole-cell Kir6.2/SUR currents were K(+)-selective, time-independent and showed weak inward rectification. They were blocked by external barium (5 mM), tolbutamide (Kd = 4.5 microM) or quinine (20 microM) and by 5 mM intracellular ATP. The single-channel conductance was 73 pS. Single-channel activity was voltage-independent and was blocked by 1 mM intracellular ATP or 0.5 mM tolbutamide. We conclude that the Kir6.2/SUR channel complex comprises the ATP-sensitive K-channel.

Cloning and functional expression of the cDNA encoding an inwardly-rectifying potassium channel expressed in pancreatic beta-cells and in the brain.

A cDNA clone encoding an inwardly-rectifying K-channel (BIR1) was isolated from insulinoma cells. The predicted amino acid sequence shares 72% identity with the cardiac ATP-sensitive K-channel rcKATP (KATP-1;[6]). The mRNA is expressed in the brain and insulinoma cells. Heterologous expression in Xenopus oocytes produced currents which were K(+)-selective, time-independent and showed inward rectification. The currents were blocked by external barium and caesium, but insensitive to tolbutamide and diazoxide. In inside-out patches, channel activity was not blocked by 1 mM internal ATP. The sequence homology with KATP-1 suggests that BIR1 is a subunit of a brain and beta-cell KATP channel. However, pharmacological differences and the lack of ATP-sensitivity, suggest that if, this is the case, heterologous subunits must exert strong modulatory influences on the native channel.

Separate processes mediate nucleotide-induced inhibition and stimulation of the ATP-regulated K(+)-channels in mouse pancreatic beta-cells.

The mechanisms by which nucleotides stimulate the activity of the ATP-regulated K(+)-channel (KATP-channel) were investigated using inside-out patches from mouse pancreatic beta-cells. ATP produces a concentration-dependent inhibition of channel activity with a Ki of 18 microns. The inhibitory action of ATP was counteracted by ADP (0.1 mM) and GDP (0.2 mM) but not GTP (1 mM). Stimulation of channel activity was also observed when ADP, GDP and GTP were applied in the absence of ATP. The ability of ADP and GDP to reactivate KATP-channels blocked by ATP declined with time following patch excision and after 30-60 min these nucleotides were without effect. During the same time period the ability of ADP and GTP to stimulate the channel in the absence of ATP was lost. In fact, ADP now blocked channel activity with 50% inhibition being observed at approximately 0.1 mM. By contrast, GDP remained a stimulator in the absence of ATP even when its ability to evoke channel activity in the presence of ATP was lost. These observations show that nucleotide-induced activation of the KATP-channel does not involve competition with ATP for a common inhibitory site but involves other processes. The data are consistent with the idea that nucleotides modulate KATP-channel activity by a number of different mechanisms that may include both regulation of cytosolic constituents and direct interaction with the channel and associated control proteins.

Hyperinsulinism of infancy: towards an understanding of unregulated insulin release. European Network for Research into Hyperinsulinism in Infancy.

Insulin is synthesised, stored, and secreted from pancreatic beta cells. These are located within the islets of Langerhans, which are distributed throughout the pancreas. Less than 2% of the total pancreas is devoted to an endocrine function. When the mechanisms that control insulin release are compromised, potentially lethal diseases such as diabetes and neonatal hypoglycaemia are manifest. This article reviews the physiology of insulin release and illustrates how defects in these processes will result in the pathophysiology of hyperinsulinism of infancy.

Alpha 2-adrenoreceptor stimulation does not inhibit L-type calcium channels in mouse pancreatic beta-cells.

The effects of alpha 2-adrenergic stimulation on the Ca(2+)-current in mouse pancreatic beta-cells were investigated using the patch-clamp technique. When using the conventional whole-cell recording configuration (dialysis of cell interior with pipette solution), addition of adrenaline (1 microM) or the alpha 2-adrenergic agonist clonidine (5 microM) failed to reduce the Ca(2+)-current, irrespective of whether intracellular GTP (or GTP gamma S) was present or not and at both physiological (1.3 mM) and elevated (10.2 mM) Ca(2+)-concentrations. In fact, in the absence of added guanine nucleotides, the agonists tended to increase the Ca(2+)-current amplitude in the presence of the higher Ca(2+)-concentration. Ca(2+)-channel activation measured at 1.3 mM Ca2+ was not affected by clonidine. Half-maximal activation was observed at approximately -20 mV. In addition, when Ca(2+)-currents were recorded from intact beta-cells, using the perforated patch whole-cell configuration, clonidine (1 microM) also failed to detectably affect the Ca(2+)-current. It is therefore suggested that the inhibition of beta-cell electrical activity and insulin-secretion produced by alpha 2-adrenoreceptor stimulation does not result from suppression of the L-type Ca(2+)-current.

Calcium-independent potentiation of insulin release by cyclic AMP in single beta-cells.

How does cyclic AMP potentiate insulin secretion from pancreatic islet beta-cells? This question is fundamental to understanding how hormones such as glucagon, which elevates cAMP, stimulate insulin secretion and so contribute to the normal secretory response of the islet. It is well established that a rise in the cytoplasmic Ca2+ concentration ([Ca2+]i) is essential for insulin secretion and therefore cAMP has been proposed to act by elevating [Ca2+]i. But studies on permeabilized beta-cells indicate that cAMP increases insulin release even when [Ca2+]i is held constant. We have used microfluorimetry and the patch-clamp technique to measure changes simultaneously in Ca2+ currents, [Ca2+]i and exocytosis in a single beta-cell in response to cAMP. We show here that cAMP, through activation of protein kinase A, increases Ca(2+)-influx through voltage-dependent L-type Ca2+ channels, thereby elevating [Ca2+]i and accelerating exocytosis. More importantly, cAMP also promotes insulin release by a direct interaction with the secretory machinery, which accounts for as much as 80% of its effect.

The sulphonylurea receptor confers diazoxide sensitivity on the inwardly rectifying K+ channel Kir6.1 expressed in human embryonic kidney cells.

1. We have examined the effects of diazoxide and intracellular ATP (ATPi) on whole-cell currents in HEK293 cells transfected transiently with the inwardly rectifying K+ channel Kir6.1 (uKATP1) or cotransfected with Kir6.1 and the sulphonylurea receptor (SUR1). 2. Kir6.1 currents were unaffected by the K+ channel opener diazoxide or by dialysis with 0.3 mM ATPi. 3. Kir6.1-SUR1 currents increased in amplitude when cells were dialysed with 0.3 mM ATP, but not with 5 mM ATP. This activation may be explained by the loss of endogenous ATP from the cell when the intracellular solution contains 0.3 mM ATP. Kir6.1-SUR1 currents were also activated by diazoxide; this activation was greater with 0.3 mM ATP1 than with 5 mM ATP1. 4. We conclude that SUR1 is required to confer both diazoxide and ATP sensitivity on Kir6.1.

Inhibition of L-type calcium channels by internal GTP [gamma S] in mouse pancreatic beta cells.

Pretreatment of pancreatic beta cells with pertussis toxin resulted in a 30% increase in peak whole-cell Ca2+ currents recorded in the absence of exogenous intracellular guanine nucleotides. Intracellular application of 90 microM GTP[gamma S], by liberation from a caged precursor, resulted in 40% reduction of the peak Ca2+ current irrespective of whether the current was carried by Ca2+ or Ba2+. Effects on the delayed outward K+ current were small and restricted to a transient Ca(2+)-dependent K+ current component. Inhibition by GTP[gamma S] of the Ca2+ current was not mimicked by standard GTP and could not be prevented either by pretreatment with pertussis toxin or by inclusion of GDP[beta S] or cyclic AMP in the intracellular medium. The inhibitory effect of GTP[gamma S] could be counteracted by a prepulse to a large depolarizing voltage. A similar effect of a depolarizing prepulse was observed in control cells with no exogenous guanine nucleotides. These observations indicate that inhibition of beta cell Ca2+ current by G protein activation results from direct interaction with the channel and does not involve second-messenger systems. Our findings also suggest that the beta cell Ca2+ current is subject to resting inhibition by G proteins.

Cytoplasmic calcium transients due to single action potentials and voltage-clamp depolarizations in mouse pancreatic B-cells.

Changes in the cytoplasmic free calcium concentration ([Ca2+]i) in pancreatic B-cells play an important role in the regulation of insulin secretion. We have recorded [Ca2+]i transients evoked by single action potentials and voltage-clamp Ca2+ currents in isolated B-cells by the combination of dual wavelength emission spectrofluorimetry and the patch-clamp technique. A 500-1000 ms depolarization of the B-cell from -70 to -10 mV evoked a transient rise in [Ca2+]i from a resting value of approximately 100 nM to a peak concentration of 550 nM. Similar [Ca2+]i changes were associated with individual action potentials. The depolarization-induced [Ca2+]i transients were abolished by application of nifedipine, a blocker of L-type Ca2+ channels, indicating their dependence on influx of extracellular Ca2+. Following the voltage-clamp step, [Ca2+]i decayed with a time constant of approximately 2.5 s and summation of [Ca2+]i occurred whenever depolarizations were applied with an interval of less than 2 s. The importance of the Na(+)-Ca2+ exchange for B-cell [Ca2+]i maintenance was evidenced by the demonstration that basal [Ca2+]i rose to 200 nM and the magnitude of the depolarization-evoked [Ca2+]i transients was markedly increased after omission of extracellular Na+. However, the rate by which [Ca2+]i returned to basal was not affected, suggesting the existence of additional [Ca2+]i buffering processes.

Stimulation of the KATP channel by ADP and diazoxide requires nucleotide hydrolysis in mouse pancreatic beta-cells.

1. The mechanisms by which ADP and the hyperglycaemic compound diazoxide stimulate the activity of the ATP-regulated K+ channel (KATP channel) were studied using inside-out patches isolated from mouse pancreatic beta-cells maintained in tissue culture. 2. The ability of diazoxide and ADP to increase KATP channel activity declined with time following patch excision and no stimulation was observed after 15-40 min. 3. Activation of KATP channels by ADP required the presence of intracellular Mg2+. The stimulatory effect of ADP was mimicked by AMP but only in the presence of ATP. Replacement of ATP with the non-hydrolysable analogue beta, gamma-methylene ATP did not interfere with the ability of ADP to stimulate KATP channel activity. By contrast, enhancement of KATP channel activity was critically dependent on hydrolysable ADP and no stimulation was observed after substitution of alpha,beta-methylene ADP for standard ADP. 4. The ability of diazoxide to enhance KATP channel activity was dependent on the presence of both internal Mg2+ and ATP. Diazoxide stimulation of KATP channel activity was not observed after substitution of beta,gamma-methylene ATP for ATP. However, in the presence of ADP, at a concentration which in itself had no stimulatory action (10 microM), diazoxide was stimulatory also in the presence of the stable ATP analogue. 5. The stimulatory action of diazoxide on KATP channel activity in the presence of ATP was markedly enhanced by intracellular ADP. This potentiating effect of ADP was not reproduced by the stable analogue alpha,beta-methylene ADP and was conditional on the presence of intracellular Mg2+. A similar enhancement of channel activity was also observed with AMP (0.1 mM). In the absence of ATP, diazoxide was still capable of stimulating channel activity provided ADP was present. This effect was not reproduced by AMP. 6. In both nucleotide-free solution and in the presence of 0.1 mM ATP, the distribution of the KATP channel open times were described by a single exponential with a time constant of approximately 20 ms. Addition of ADP or diazoxide resulted in the appearance of a second component with a time constant of > 100 ms which comprised 40-70% of the total number of events. Under the latter experimental conditions, the open probability of the channel increased more than fivefold relative to that observed in the presence of ATP alone.(ABSTRACT TRUNCATED AT 400 WORDS)

Ca(2+)- and GTP-dependent exocytosis in mouse pancreatic beta-cells involves both common and distinct steps.

1. The effects of GTP and Ca2+ on secretion from single pancreatic beta-cells were studied using capacitance measurements as an indicator of exocytosis. 2. GTP or GTP gamma S produced a concentration-dependent increase in cell capacitance in the absence of intracellular calcium. There was no effect of cyclic AMP or BAPTA an GTP-induced secretion. 3. In the absence of GTP, the relationship between intracellular calcium concentration and the maximum rate of secretion was fitted by the Hill equation with a slope factor of 2.5 and half-maximal activation at 1.6 microM intracellular Ca2+. Similar values were obtained in the presence of GTP gamma S, suggesting GTP does not alter the sensitivity of the secretory machinery to Ca2+. 4. GDP beta S alone had no effect on cell capacitance but caused a dose-dependent inhibition of exocytosis induced by infusion of either GTP gamma S or Ca2+, suggesting both stimuli involve G-protein activation. GDP beta S was without effect on exocytosis evoked by depolarization-mediated Ca2+ entry. 5. The time course of exocytosis following rapid elevation of GTP gamma S by photolysis of a caged precursor was dependent on the intracellular Ca2+ and cyclic AMP concentrations. 6. Our results are interpreted in terms of a model in which the secretory pathways stimulated by Ca2+ and GTP contain both common and separate parts.

Properties of cloned ATP-sensitive K+ currents expressed in Xenopus oocytes.

1. We have studied the electrophysiological properties of cloned ATP-sensitive K+ channels (KATP channels) heterologously expressed in Xenopus oocytes. This channel comprises a sulphonylurea receptor subunit (SUR) and an inwardly rectifying K+ channel subunit (Kir). 2. Oocytes injected with SUR1 and either Kir6.2 or Kir6.1 exhibited large inwardly rectifying K+ currents when cytosolic ATP levels were lowered by the metabolic inhibitors azide or FCCP. No currents were observed in response to azide in oocytes injected with Kir6.2, Kir6.1 or SUR1 alone, indicating that both the sulphonylurea receptor (SUR1) and an inward rectifier (Kir6.1 or Kir6.2) are needed for functional channel activity. 3. The pharmacological properties of Kir6.2-SUR1 currents resembled those of native beta-cell ATP-sensitive K+ channel currents (KATP currents): the currents were > 90% blocked by tolbutamide (500 microM), meglitinide (10 microM) or glibenclamide (100 nM), and activated 1.8-fold by diazoxide (340 microM), 1.4-fold by pinacidil (1 mM) and unaffected by cromakalim (0.5 mM). 4. Macroscopic Kir6.2-SUR1 currents in inside-out patches were inhibited by ATP with a Ki of 28 microM. Kir6.1-SUR1 currents ran down within seconds of patch excision preventing analysis of ATP sensitivity. 5. No sensitivity to tolbutamide or metabolic inhibition was observed when SUR1 was coexpressed with either Kir1.1a or Kir2.1, suggesting that these proteins do not couple in Xenopus ocytes. 6. Our data demonstrate that the Xenopus oocyte constitutes a good expression system for cloned KATP channels and that expression may be assayed by azide-induced metabolic inhibition.