40K glycoPEGylated, recombinant FVIIa: 3-month, double-blind, randomized trial of safety, pharmacokinetics and preliminary efficacy in hemophilia patients with inhibitors.

BACKGROUND: A 40K glycoPEGylated, recombinant activated factor VII (rFVIIa) bypassing agent (N7-GP) with a prolonged half-life (15 h) compared with rFVIIa was developed as a potential candidate for bleed-preventive regimens in patients with hemophilia and inhibitors. OBJECTIVES: To evaluate the safety, pharmacokinetics and preliminary efficacy of multiple doses of N7-GP in congenital hemophilia A and B patients with high-titer inhibitors. PATIENTS/METHODS: In this global, prospective, randomized, double-blinded, phase 2 trial, 25, 100 or 200 µg kg(-1)  N7-GP was administered intravenously once every second day during a 3-month, bleed-preventive regimen and compared with a preceding 3-month observation period with on-demand treatment of bleeds with rFVIIa. The primary endpoint was adverse events; secondary endpoints were evaluation of immunogenicity, pharmacokinetics and efficacy. RESULTS AND CONCLUSIONS: Overall, 23 patients were randomized and dosed (n = 8/7/8 for 25/100/200 µg kg(-1) ). N7-GP was well tolerated, with a low frequency of adverse events. No serious adverse events, immunogenic or thromboembolic events related to N7-GP were reported. The pharmacokinetic properties of N7-GP were similar to those reported in phase 1. The annualized bleeding rate (ABR) decreased in the treatment period vs. the observation period at all N7-GP dose levels. However, a dose-response relationship in the reduction could not be established in the N7-GP dose range evaluated. The ABR was also reduced at two dose levels during the last part of the observation period, and increased notably in the follow-up period irrespective of previous N7-GP dose. The trial was registered at ClinicalTrials.gov ( REGISTRATION NUMBER: NCT00951405).

Functional identification and monitoring of individual alpha and beta cells in cultured mouse islets of Langerhans.

The aim of the present study was to evaluate, by use of fluorescence microscopy and immunofluorescence stainings, the use of a fluorescent membrane potential sensitive probe as a means to identify and monitor changes in membrane potential of individual cell types in whole islets of Langerhans over time. Our work supports the use of the fluorescent probe bis-(1,3 dibutylbarbituric acid) trimethine oxonol (diBAC(4)(3)), in identification of single alpha and beta cells in the periphery of mouse pancreatic islets cultured on extracellular matrix. At a low extracellular glucose concentration (3 mM), heterogeneous staining of the islets was observed. Approximately 97% of the peripheral cells that stained brightly with diBAC(4)(3) were glucagon positive. Additional diBAC(4)(3) studies, demonstrated that an increase in glucose concentration from 3 to 10 mM is paralleled by repolarization of alpha cells and depolarization of beta cells. This suggests that reciprocity of glucagon and insulin release upon glucose stimulation is coupled to divergent changes in membrane potential of these cell types and supports the use of diBAC(4)(3) as a means to detect changes in secretion in both cell types.

Phenylcyanoguanidines as inhibitors of glucose-induced insulin secretion from beta cells.

3,5-Disubstituted-phenylcyanoguanidines have been identified as activators of SUR1/Kir6.2 potassium channels and as potent inhibitors of insulin release from pancreatic beta cells in vitro.

Polyamines in pancreatic islets of obese-hyperglycemic (ob/ob) mice of different ages.

To further evaluate the role of polyamines in insulin production and cell replication in diabetic pancreatic islets, we have studied hyperplastic islets of obese-hyperglycemic mice of different ages and normal islets of the same strain. The aims of the study were to investigate the impact of the diabetic state and aging on polyamine contents and requirements in these islets. Cultured islets from lean and obese animals contained significantly less polyamines than freshly isolated islets. Spermine-to-spermidine ratio was elevated in freshly isolated islets from young obese mice compared with those from lean mice. In islets from old obese animals, spermidine content was decreased, whereas the content of spermine was not different from that of young obese mice. The physiological significance of polyamines was investigated by exposing islets in tissue culture to inhibitors of polyamine synthesis. This treatment caused a partial polyamine depletion in whole islets but failed to affect polyamine content of cell nuclei. Insulin content was not affected in polyamine-deficient islets of obese mice, irrespective of age, in contrast to decreased islet insulin content in polyamine-depleted young lean animals. Polyamine depletion depressed DNA synthesis rate in obese mouse islets; in lean mice it actually stimulated DNA synthesis. We concluded that important qualitative and quantitative differences exist between islets from obese-hyperglycemic and normal mice with respect to polyamine content and requirements of polyamines for regulation of insulin content and cell proliferation. The results suggest that spermine may be involved in mediating the rapid islet cell proliferation noted early in obese-hyperglycemic syndrome, but changes in spermine concentration do not seem to account for the decline in islet cell DNA synthesis in aged normoglycemic animals.

Excitation wave propagation as a possible mechanism for signal transmission in pancreatic islets of Langerhans.

In response to glucose application, beta-cells forming pancreatic islets of Langerhans start bursting oscillations of the membrane potential and intracellular calcium concentration, inducing insulin secretion by the cells. Until recently, it has been assumed that the bursting activity of beta-cells in a single islet of Langerhans is synchronized across the whole islet due to coupling between the cells. However, time delays of several seconds in the activity of distant cells are usually observed in the islets of Langerhans, indicating that electrical/calcium wave propagation through the islets can occur. This work presents both experimental and theoretical evidence for wave propagation in the islets of Langerhans. Experiments with Fura-2 fluorescence monitoring of spatiotemporal calcium dynamics in the islets have clearly shown such wave propagation. Furthermore, numerical simulations of the model describing a cluster of electrically coupled beta-cells have supported our view that the experimentally observed calcium waves are due to electric pulses propagating through the cluster. This point of view is also supported by independent experimental results. Based on the model equations, an approximate analytical expression for the wave velocity is introduced, indicating which parameters can alter the velocity. We point to the possible role of the observed waves as signals controlling the insulin secretion inside the islets of Langerhans, in particular, in the regions that cannot be reached by any external stimuli such as high glucose concentration outside the islets.

A potent diazoxide analogue activating ATP-sensitive K+ channels and inhibiting insulin release.

AIMS/HYPOTHESIS: To characterise the effects of BPDZ 73 (7-chloro-3-isopropylamino-4H-1,2,4-benzothiadiazine 1,1-dioxide), a newly synthesised diazoxide analogue, on insulin secretory cells. METHODS: Measurements of 86Rb, 45Ca outflow, membrane potential, [Ca2+]i, insulin release in secretory cells as well as measurements of smooth muscle contractile activity and glycaemia were carried out. RESULTS: The analogue BPDZ 73 induced a dose-dependent decrease in insulin output. The IC50 value averaged 0.73 +/- 0.05 mumol/l. The drug increased the rate of 86Rb (42K substitute) outflow from perifused rat pancreatic islets. This effect was inhibited by glibenclamide, a KATP channel blocker. Measurements of DiBAC4(3) fluorescence further indicated that BPDZ 73 hyperpolarised the insulin secreting cells. It also decreased 45Ca outflow from pancreatic islets perifused throughout in the presence of 16.7 mmol/l glucose and extracellular Ca2+. By contrast, the drug did not affect the increase in 45Ca outflow mediated by K+ depolarisation. In single beta cells, BPDZ 73 inhibited the glucose-induced but not the K(+)-induced rise in [Ca2+]i. Moreover, in Wistar rats, i.p. injection of BPDZ 73 provoked a considerable increase in blood glucose concentration whereas diazoxide induced a modest rise in glycaemia. Lastly, the vasorelaxant properties of BPDZ 73 were slightly less pronounced than those of diazoxide. CONCLUSION/INTERPRETATION: The inhibitory effect of BPDZ 73 on the insulin-releasing process results from the activation of KATP channels with subsequent decrease in Ca2+ inflow and [Ca2+]i. The drug seems to be a KATP channel opener, more potent and more selective than diazoxide for insulin secreting cells.

Separation of the glucose-stimulated cytoplasmic and mitochondrial NAD(P)H responses in pancreatic islet beta cells.

Two-photon excitation microscopy was used to image and quantify NAD(P)H autofluorescence from intact pancreatic islets under glucose stimulation. At maximal glucose stimulation, the rise in whole-cell NAD(P)H levels was estimated to be approximately 30 microM. However, because glucose-stimulated insulin secretion involves both glycolytic and Kreb's cycle metabolism, islets were cultured on extracellular matrix that promotes cell spreading and allows spatial resolution of the NAD(P)H signals from the cytoplasm and mitochondria. The metabolic responses in these two compartments are shown to be differentially stimulated by various nutrient applications. The glucose-stimulated increase of NAD(P)H fluorescence within the cytoplasmic domain is estimated to be approximately 7 microM. Likewise, the NAD(P)H increase of the mitochondrial domain is approximately 60 microM and is delayed with respect to the change in cytoplasmic NAD(P)H by approximately 20 sec. The large mitochondrial change in glucose-stimulated NAD(P)H thus dominates the total signal but may depend on the smaller but more rapid cytoplasmic increase.

Glucose decreases Na+,K+-ATPase activity in pancreatic beta-cells. An effect mediated via Ca2+-independent phospholipase A2 and protein kinase C-dependent phosphorylation of the alpha-subunit.

In the pancreatic beta-cell, glucose-induced membrane depolarization promotes opening of voltage-gated L-type Ca2+ channels, an increase in cytoplasmic free Ca2+ concentration ([Ca2+]i), and exocytosis of insulin. Inhibition of Na+,K+-ATPase activity by ouabain leads to beta-cell membrane depolarization and Ca2+ influx. Because glucose-induced beta-cell membrane depolarization cannot be attributed solely to closure of ATP-regulated K+ channels, we investigated whether glucose regulates other transport proteins, such as the Na+,K+-ATPase. Glucose inhibited Na+,K+-ATPase activity in single pancreatic islets and intact beta-cells. This effect was reversible and required glucose metabolism. The inhibitory action of glucose was blocked by pretreatment of the islets with a selective inhibitor of a Ca2+-independent phospholipase A2. Arachidonic acid, the hydrolytic product of this phospholipase A2, also inhibited Na+, K+-ATPase activity. This effect, like that of glucose, was blocked by nordihydroguaiaretic acid, a selective inhibitor of the lipooxygenase metabolic pathway, but not by inhibitors of the cyclooxygenase or cytochrome P450-monooxygenase pathways. The lipooxygenase product 12(S)-HETE (12-S-hydroxyeicosatetranoic acid) inhibited Na+,K+-ATPase activity, and this effect, as well as that of glucose, was blocked by bisindolylmaleimide, a specific protein kinase C inhibitor. Moreover, glucose increased the state of alpha-subunit phosphorylation by a protein kinase C-dependent process. These results demonstrate that glucose inhibits Na+, K+-ATPase activity in beta-cells by activating a distinct intracellular signaling network. Inhibition of Na+,K+-ATPase activity may thus be part of the mechanisms whereby glucose promotes membrane depolarization, an increase in [Ca2+]i, and thereby insulin secretion in the pancreatic beta-cell.

Simultaneous visualization of the translocation of protein kinase Calpha-green fluorescent protein hybrids and intracellular calcium concentrations.

The alpha isoform of protein kinase C (PKCalpha) is a ubiquitous protein kinase, which, upon activation, translocates rapidly from the cytoplasm to the plasma membrane. To follow this translocation, PKCalpha was tagged with a highly fluorescent derivative of green fluorescent protein and stably expressed in baby hamster kidney cells overexpressing the muscarinic type 1 receptor. Addition of the agonist carbamylcholine triggered the onset of translocation within 1 s. Half-maximal and maximal translocation occurred after about 3 and 15 s respectively. Plasma membrane association of the fusion protein was transient and the protein returned to the cytoplasm within about 45 s. A high-resolution study showed an almost homogeneous cytoplasmic distribution of tagged PKCalpha in unstimulated cells and virtually complete translocation to the plasma membrane in response to the phorbol ester, PMA. Simultaneous visualization of intracellular calcium concentration ([Ca2+]i) and PKCalpha translocation in single cells showed a good correlation between these parameters at intermediate and high concentrations of agonist. At low agonist concentration, a small increase in [Ca2+]i was observed, without detectable translocation of PKCalpha. In contrast, PMA induced translocation of PKCalpha without any increase in [Ca2+]i. Neither cytochalasin D nor colcemid influenced the distribution or calcium-dependent translocation of tagged PKCalpha, indicating that PKCalpha translocation may be independent of both actin filaments and microtubules. The time course of PKCalpha translocation is compatible with diffusion of the protein from its cytoplasmic localization to the plasma membrane.

Dissociation between changes in cytoplasmic free Ca2+ concentration and insulin secretion as evidenced from measurements in mouse single pancreatic islets.

Simultaneous measurements of cytosolic free Ca2+ concentration and insulin release, in mouse single pancreatic islets, revealed a direct correlation only initially after stimulation with glucose or K+. Later, there is an apparent dissociation between these two parameters, with translocation of alpha and epsilon isoenzymes of protein kinase C to membranes and simultaneous desensitization of insulin release in response to glucose. Recovery of insulin release, without any concomitant changes in cytosolic free Ca2+ concentration, after addition of phorbol 12-myristate 13-acetate, okadaic acid, and forskolin supports the notion that the desensitization process is accounted for by dephosphorylation of key regulatory sites of the insulin exocytotic machinery.

Oscillations in cytoplasmic free calcium concentration in human pancreatic islets from subjects with normal and impaired glucose tolerance.

Plasma insulin levels in healthy subjects oscillate and non-insulin-dependent diabetic patients display an irregular pattern of such oscillations. Since an increase in cytoplasmic free Ca2+ concentration ([Ca2+]i) in the pancreatic beta cell is the major stimulus for insulin release, this study was undertaken to investigate the dynamics of electrical activity, [Ca2+]i-changes and insulin release, in stimulated islets from subjects of varying glucose tolerance. In four patients it was possible to investigate more than one of these three parameters. Stimulation of pancreatic islets with glucose and tolbutamide sometimes resulted in the appearance of oscillations in [Ca2+]i, lasting 2-3 min. Such oscillations were observed even in some islets from patients with impaired glucose tolerance. In one islet from a diabetic patient there was no response to glucose, whereas that islet displayed [Ca2+]i-oscillations in response to tolbutamide, suggesting that sulphonylurea treatment can mimic the complex pattern of glucose-induced [Ca2+]i-oscillations. We also, for the first time, made patch-clamp recordings of membrane currents in beta-cells in situ in the islet. Stimulation with glucose and tolbutamide resulted in depolarization and appearance of action potentials. The islet preparations responded to stimulation with a number of different secretagogues with release of insulin. The present study shows that human islets can respond to stimulation with glucose and sulphonylurea with oscillations in [Ca2+]i, which is the signal probably underlying the oscillations in plasma insulin levels observed in healthy subjects. Interestingly, even subjects with impaired glucose tolerance had islets that responded with oscillations in [Ca2+]i upon glucose stimulation, although it is not known to what extent the response of these islets was representative of most islets in these patients.

Protein kinase C modulates the insulin secretory process by maintaining a proper function of the beta-cell voltage-activated Ca2+ channels.

In the present study an attempt was made to further elucidate the molecular mechanisms whereby protein kinase C (PKC) modulates the beta-cell stimulus-secretion coupling. Regulation of Ca2+ channel activity, [Ca2+]i, and insulin release were investigated in both normal pancreatic mouse beta-cells and in similar beta-cells deprived of PKC activity. [Ca2+]i was measured with the intracellular fluorescent Ca2+ indicator fura-2 and the Ca2+ channel activity was estimated by the whole cell configuration of the patch-clamp technique. To reveal the various isoenzymes of PKC present in the mouse beta-cell, proteins were separated by one-dimensional gel electrophoresis and Western blotting was performed. The production of inositol phosphates was measured by ion-exchange chromatography and insulin release was measured radioimmunologically. Acute stimulation with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate resulted in suppression of both the carbamylcholine-induced increase in [Ca2+]i and production of inositol 1,4,5-trisphosphate. Under these conditions the increase in [Ca2+]i in response to glucose was similar to that found in control cells. When beta-cells were deprived of PKC, by exposure to 200 nM 12-O-tetradecanoylphorbol-13-acetate for 24-48 h, there was an enhanced response to carbamylcholine. This response constituted increases in both the [Ca2+]i signal and production of inositol 1,4,5-trisphosphate. Interestingly, cells with down-regulated PKC activity responded more slowly to glucose stimulation, when comparing the initial increase in [Ca2+]i, than control cells. On the other hand, the maximal increase in [Ca2+]i was similar whether or not PKC was present. Moreover, PKC down-regulated cells exhibited a significant reduction of maximal whole cell Ca2+ currents, a finding that may explain the altered kinetics with regard to the [Ca2+]i increase in response to the sugar. Both the alpha and beta 1 forms of the PKC isoenzymes were present in the mouse beta-cell and were also subjected to PKC down-regulation. Hence, either of these isoenzymes or both may be involved in the modulation of phospholipase C and Ca2+ channel activity. Since insulin release under physiological conditions is critically dependent on Ca(2+)-influx through the voltage-gated L-type Ca2+ channels, the kinetics of hormone release was expected to demonstrate a similar delay as that of the [Ca2+]i increase. Although not as pronounced, such a delay was indeed also observed in the onset of insulin release. There was, however, no effect on the total amounts of hormone released.(ABSTRACT TRUNCATED AT 400 WORDS)

Enhanced stimulus-secretion coupling in polyamine-depleted rat insulinoma cells. An effect involving increased cytoplasmic Ca2+, inositol phosphate generation, and phorbol ester sensitivity.

To extend previous observations on the role of polyamines in insulin production, metabolism, and replication of insulin-secreting pancreatic beta cells, we have studied the role of polyamines in the regulation of the stimulus-secretion coupling of clonal rat insulinoma cells (RINm5F). For this purpose, RINm5F cells were partially depleted in their polyamine contents by use of the specific ornithine decarboxylase inhibitor difluoromethylornithine (DFMO), which led to an increase in cellular insulin and ATP contents. Analysis of different parts of the signal transduction pathway revealed that insulin secretion and the increase in cytoplasmic free Ca2+ concentration ([Ca2+]i) after K(+)-induced depolarization were markedly enhanced in DFMO-treated cells. These effects were paralleled by increased voltage-activated Ca2+ currents, as judged by whole-cell patch-clamp analysis, probably reflecting increased channel activity rather than elevated number of channels per cell. DFMO treatment also rendered phospholipase C in these cells more sensitive to the muscarinic receptor agonist carbamylcholine, as evidenced by enhanced generation of inositol phosphates, increase in [Ca2+]i and insulin secretion, despite an unaltered ligand binding to muscarinic receptors and lack of effect on protein kinase C activity. In addition, the tumor promoter 12-O-tetradecanoylphorbol 13-acetate, at concentrations suggested to be specific for protein kinase C activation, evoked an increased insulin output in polyamine-deprived cells compared to control cells. The stimulatory effects of glucose or the cyclic AMP raising agent theophylline on insulin release were not increased by DFMO treatment. In spite of increased binding of sulfonylurea in DFMO-treated cells, there was no secretory response or altered increase in [Ca2+]i in response to the drug in these cells. It is concluded that partial polyamine depletion sensitizes the stimulus-secretion coupling at multiple levels in the insulinoma cells, including increased voltage-dependent Ca2+ influx and enhanced responsiveness to activators of phospholipase C and protein kinase C. In their entirety, our present results indicate that the behavior of the stimulus-secretion coupling of polyamine-depleted RINm5F insulinoma cells changes towards that of native beta cells, thus improving the usefulness of this cell line for studies of beta cell insulin secretion.

Glucose-induced increase in cytoplasmic pH in pancreatic beta-cells is mediated by Na+/H+ exchange, an effect not dependent on protein kinase C.

Glucose-induced changes in cytoplasmic pH (pHi) were investigated using pancreatic beta-cells isolated from obese hyperglycemic mice. Glucose, at concentrations above 3-5 mM, depolarized the beta-cell and increased pHi, cytoplasmic free Ca2+ ([Ca2+]i), and insulin release. This increase in pHi was dependent on the presence of extracellular Na+ and was inhibited by 5-(N-ethyl-N-isopropyl) amiloride, a blocker of Na+/H+ exchange. Stimulation of protein kinase C with phorbol ester also induced an alkalinization. However, when protein kinase C activity was down-regulated, glucose stimulation still induced alkalinization. At 20 mM glucose, 10 mM NH4Cl induced a marked rise in pHi, paralleled by repolarization, inhibition of electrical activity, and decreases in both [Ca2+]i and insulin release. Reduction in [Ca2+]i was prevented by 200 microM tolbutamide, but not by 10 mM tetraethylammonium. At 4 mM glucose, NH4Cl induced a transient increase in insulin release, without changing [Ca2+]i. Exposure of beta-cells to 10 mM sodium acetate caused a persistent decrease in pHi, an effect paralleled by a small transient increase in [Ca2+]i. Acidification per se did not change the beta-cell sensitivity to glucose, not excluding that the activity of the ATP-regulated K+ channels may be modulated by changes in pHi.

Measurements of cytoplasmic free Ca2+ concentration in human pancreatic islets and insulinoma cells.

In human pancreatic islets an increase in the glucose concentration from 3 to 20 mM raised the free cytoplasmic Ca2+ concentration [( Ca2+]i), an effect being reversible upon withdrawal of the sugar. Depolarization with a high concentration of K+ or the sulphonylurea tolbutamide also raised [Ca2+]i. Addition of extracellular ATP produced a transient rapid rise in [Ca2+]i. Oscillations in [Ca2+]i were observed in the presence of 10 mM glucose. Insulinoma cells responded to glucose and tolbutamide with increases in [Ca2+]i, whereas the sulphonamide diazoxide caused a decrease in [Ca2+]i. These findings confirm previous results obtained in rodent beta-cells.

Activation by adrenaline of a low-conductance G protein-dependent K+ channel in mouse pancreatic B cells.

Insulin is produced and secreted by the B cells in the endocrine pancreas. In vivo, insulin secretion is under the control of a number of metabolic, neural and hormonal substances. It is now clear that stimulation of insulin release by fuel secretagogues, such as glucose, involves the closure of K+ channels that are sensitive to the intracellular ATP concentration (KATP channels). This leads to membrane depolarization and the generation of Ca2(+)-dependent action potentials. The mechanisms whereby hormones and neurotransmitters such as adrenaline, galanin and somatostatin, which are released by intraislet nerve endings and the pancreatic D cells, produce inhibition of insulin secretion are not clear. Here we show that adrenaline suppresses B-cell electrical activity (and thus insulin secretion) by a G protein-dependent mechanism, which culminates in the activation of a sulphonylurea-insensitive low-conductance K+ channel distinct from the KATP channel.

Glucose-stimulated efflux of indo-1 from pancreatic beta-cells is reduced by probenecid.

Indo-1 loaded pancreatic beta-cells, isolated from obese hyperglycaemic mice, were studied with respect to cytoplasmic free Ca2+ concentration ([Ca2+]i), efflux of indicator and insulin release. In the absence of glucose there was a continuous efflux of indo-1 which increased upon stimulation with 20 mM of the sugar. The anion exchange inhibitor probenecid reduced both basal efflux of indo-1 and prevented that promoted by glucose. Measurements of [Ca2+]i and insulin release revealed similar results as previously reported with quin-2 and fura-2. Furthermore, probenecid did not influence the [Ca2+]i responses. It is thus possible to reduce efflux of indo-1 probenecid and thereby improve the measurements of [Ca2+]i in pancreatic beta-cells.

Coexpression of the platelet-derived growth factor (PDGF) B chain and the PDGF beta receptor in isolated pancreatic islet cells stimulates DNA synthesis.

Suspensions rich in pancreatic beta cells were transfected by means of electroporation or by using the liposome technique with DNA constructs coding for the B chain of platelet-derived growth factor (PDGF) and the PDGF alpha and beta receptors to induce a mitotic response in this slowly replicating cell type. Transfection with the B-chain construct induced synthesis of the PDGF B-chain homodimer (PDGF-BB) as assessed by the presence of 125I-labeled PDGF-BB competing activity in the conditioned medium of the transfected islet cells. Moreover, islet cells transfected with the PDGF beta-receptor construct exhibited increased immunofluorescence staining with a PDGF beta-receptor antibody. These cells also displayed increased 125I-labeled PDGF-BB binding compared with control transfected cells. Cotransfection with the B-chain construct or the addition of 10% fetal bovine serum or purified PDGF all induced DNA synthesis in islet cells transfected with the PDGF beta-receptor construct. Islet cells transfected with the PDGF alpha-receptor construct did not respond with stimulation of [3H]thymidine incorporation to any of the PDGF isoforms (PDGF-AA, -AB, or -BB). Cotransfection of the PDGF alpha- and beta-receptor constructs resulted in a loss of the DNA synthesis response to PDGF. The beta cells exhibited elevated levels of [3H]inositol trisphosphate after transfection with the B-chain and beta-receptor constructs, indicating activation of phospholipase C. Islet cells transfected with the different receptor constructs exhibited different patterns of tyrosine phosphorylation upon ligand activation. The results demonstrate that pancreatic islet cells can be stimulated to increase DNA synthesis by transfection with the PDGF beta-receptor gene, whereas cotransfection with the alpha-receptor gene may attenuate the growth response.

Delayed rectifying and calcium-activated K+ channels and their significance for action potential repolarization in mouse pancreatic beta-cells.

The contribution of Ca2(+)-activated and delayed rectifying K+ channels to the voltage-dependent outward current involved in spike repolarization in mouse pancreatic beta-cells (Rorsman, P., and G. Trube. 1986. J. Physiol. 374:531-550) was assessed using patch-clamp techniques. A Ca2(+)-dependent component could be identified by its rapid inactivation and sensitivity to the Ca2+ channel blocker Cd2+. This current showed the same voltage dependence as the voltage-activated (Cd2(+)-sensitive) Ca2+ current and contributed 10-20% to the total beta-cell delayed outward current. The single-channel events underlying the Ca2(+)-activated component were investigated in cell-attached patches. Increase of [Ca2+]i invariably induced a dramatic increase in the open state probability of a Ca2(+)-activated K+ channel. This channel had a single-channel conductance of 70 pS [( K+]o = 5.6 mM). The Ca2(+)-independent outward current (constituting greater than 80% of the total) reflected the activation of an 8 pS [( K+]o = 5.6 mM; [K+]i = 155 mM) K+ channel. This channel was the only type observed to be associated with action potentials in cell-attached patches. It is suggested that in mouse beta-cells spike repolarization results mainly from the opening of the 8-pS delayed rectifying K+ channel.