Peter Krippeit-Drews

Elevated Blood Pressure Linked to Primary Hyperaldosteronism and Impaired Vasodilation in BK Channel–Deficient Mice

Background—Abnormally elevated blood pressure is the most prevalent risk factor for cardiovascular disease. The large-conductance, voltage- and Ca2+-dependent K+ (BK) channel has been proposed as an important effector in the control of vascular tone by linking membrane depolarization and local increases in cytosolic Ca2+ to hyperpolarizing K+ outward currents. However, the BK channel may also affect blood pressure by regulating salt and fluid homeostasis, particularly by adjusting the renin-angiotensin-aldosterone system. Methods and Results—Here we report that deletion of the pore-forming BK channel &agr; subunit leads to a significant blood pressure elevation resulting from hyperaldosteronism accompanied by decreased serum K+ levels as well as increased vascular tone in small arteries. In smooth muscle from small arteries, deletion of the BK channel leads to a depolarized membrane potential, a complete lack of membrane hyperpolarizing spontaneous K+ outward currents, and an attenuated cGMP vasorelaxation associated with a reduced suppression of Ca2+ transients by cGMP. The high level of BK channel expression observed in wild-type adrenal glomerulosa cells, together with unaltered serum renin activities and corticotropin levels in mutant mice, suggests that the hyperaldosteronism results from abnormal adrenal cortical function in BK−/− mice. Conclusions—These results identify previously unknown roles of BK channels in blood pressure regulation and raise the possibility that BK channel dysfunction may underlie specific forms of hyperaldosteronism.

Oxidative stress and beta-cell dysfunction

Diabetes mellitus type 1 and 2 (T1DM and T2DM) are complex multifactorial diseases. Loss of beta-cell function caused by reduced secretory capacity and enhanced apoptosis is a key event in the pathogenesis of both diabetes types. Oxidative stress induced by reactive oxygen and nitrogen species is critically involved in the impairment of beta-cell function during the development of diabetes. Because of their low antioxidant capacity, beta-cells are extremely sensitive towards oxidative stress. In beta-cells, important targets for an oxidant insult are cell metabolism and KATP channels. The oxidant-evoked alterations of KATP channel activity seem to be critical for oxidant-induced dysfunction because genetic ablation of KATP channels attenuates the effects of oxidative stress on beta-cell function. Besides the effects on metabolism, interference of oxidants with mitochondria induces key events in apoptosis. Consequently, increasing antioxidant defence is a promising strategy to delay beta cell failure in (pre)-diabetic patients or during islet transplantation. Knock-out of KATP channels has beneficial effects on oxidant-induced inhibition of insulin secretion and cell death. Interestingly, these effects can be mimicked by sulfonylureas that have been used in the treatment of T2DM for many years. Loss of functional KATP channels leads to up-regulation of antioxidant enzymes, a process that depends on cytosolic Ca2+. These observations are of great importance for clinical intervention because they show a possibility to protect beta-cells at an early stage before dramatic changes of the secretory capacity and loss of cell mass become manifest and lead to glucose intolerance or even overt diabetes.

ABCC8 and ABCC9: ABC transporters that regulate K+ channels

The sulfonylurea receptors (SURs) ABCC8/SUR1 and ABCC9/SUR2 are members of the C-branch of the transport adenosine triphosphatase superfamily. Unlike their brethren, the SURs have no identified transport function; instead, evolution has matched these molecules with K+ selective pores, either KIR6.1/KCNJ8 or KIR6.2/KCNJ11, to assemble adenosine triphosphate (ATP)-sensitive K+ channels found in endocrine cells, neurons, and both smooth and striated muscle. Adenine nucleotides, the major regulators of ATP-sensitive K+ (KATP) channel activity, exert a dual action. Nucleotide binding to the pore reduces the activity or channel open probability, whereas Mg-nucleotide binding and/or hydrolysis in the nucleotide-binding domains of SUR antagonize this inhibitory action to stimulate channel openings. Mutations in either subunit can alter this balance and, in the case of the SUR1/KIR6.2 channels found in neurons and insulin-secreting pancreatic β cells, are the cause of monogenic forms of hyperinsulinemic hypoglycemia and neonatal diabetes. Additionally, the subtle dysregulation of KATP channel activity by a KIR6.2 polymorphism has been suggested as a predisposing factor in type 2 diabetes mellitus. Studies on KATP channel null mice are clarifying the roles of these metabolically sensitive channels in a variety of tissues.

Interference of H2O2 with stimulus‐secretion coupling in mouse pancreatic β‐cells

1 We have reported previously that in mouse pancreatic β‐cells H2O2 hyperpolarizes the membrane and increases the ATP‐sensitive K+ current recorded in the perforated patch configuration of the patch‐clamp technique. The present study was undertaken to elucidate the underlying mechanisms. 2 The intracellular ATP concentration measured by chemoluminescence was reduced by H2O2. The ADP concentration increased in parallel during the first 10 min, resulting in a pronounced decrease in the ATP/ADP ratio. 3 Consistent with these results, glucose‐stimulated insulin secretion from isolated islets was inhibited by H2O2. 4 Membrane hyperpolarization measured with intracellular microelectrodes in intact islets and inhibition of insulin secretion were counteracted by tolbutamide, indicating that the channels are still responsive to inhibitors and that the ATP concentration is not too low to trigger exocytosis. However, the sensitivity of the β‐cells to tolbutamide was reduced after treatment with H2O2. 5 H2O2 increased the intracellular Ca2+ activity ([Ca2+]i) in a biphasic manner. A first transient rise in [Ca2+]i due to mobilization of Ca2+ from intracellular stores was followed by a sustained increase, which was at least partly dependent on Ca2+ influx. The first phase seems to reflect Ca2+ mobilization from mitochondria. 6 Our results demonstrate that H2O2 interferes with glucose metabolism, which influences the membrane potential and ATP‐sensitive K+ current via the intracellular concentration of ATP. These events finally lead to an inhibition of insulin secretion despite an increase in [Ca2+]i.

Electrophysiology of Islet Cells

Stimulus-Secretion Coupling (SSC) of pancreatic islet cells comprises electrical activity. Changes of the membrane potential (V(m)) are regulated by metabolism-dependent alterations in ion channel activity. This coupling is best explored in beta-cells. The effect of glucose is directly linked to mitochondrial metabolism as the ATP/ADP ratio determines the open probability of ATP-sensitive K(+) channels (K(ATP) channels). Nucleotide sensitivity and concentration in the direct vicinity of the channels are controlled by several factors including phospholipids, fatty acids, and kinases, e.g., creatine and adenylate kinase. Closure of K(ATP) channels leads to depolarization of beta-cells via a yet unknown depolarizing current. Ca(2+) influx during action potentials (APs) results in an increase of the cytosolic Ca(2+) concentration ([Ca(2+)](c)) that triggers exocytosis. APs are elicited by the opening of voltage-dependent Na(+) and/or Ca(2+) channels and repolarized by voltage- and/or Ca(2+)-dependent K(+) channels. At a constant stimulatory glucose concentration APs are clustered in bursts that are interrupted by hyperpolarized interburst phases. Bursting electrical activity induces parallel fluctuations in [Ca(2+)](c) and insulin secretion. Bursts are terminated by I(Kslow) consisting of currents through Ca(2+)-dependent K(+) channels and K(ATP) channels. This review focuses on structure, characteristics, physiological function, and regulation of ion channels in beta-cells. Information about pharmacological drugs acting on K(ATP) channels, K(ATP) channelopathies, and influence of oxidative stress on K(ATP) channel function is provided. One focus is the outstanding significance of L-type Ca(2+) channels for insulin secretion. The role of less well characterized beta-cell channels including voltage-dependent Na(+) channels, volume sensitive anion channels (VSACs), transient receptor potential (TRP)-related channels, and hyperpolarization-activated cyclic nucleotide-gated (HCN) channels is discussed. A model of beta-cell oscillations provides insight in the interplay of the different channels to induce and maintain electrical activity. Regulation of beta-cell electrical activity by hormones and the autonomous nervous system is discussed. alpha- and delta-cells are also equipped with K(ATP) channels, voltage-dependent Na(+), K(+), and Ca(2+) channels. Yet the SSC of these cells is less clear and is not necessarily dependent on K(ATP) channel closure. Different ion channels of alpha- and delta-cells are introduced and SSC in alpha-cells is described in special respect of paracrine effects of insulin and GABA secreted from beta-cells.

Bile Acids Acutely Stimulate Insulin Secretion of Mouse β-Cells via Farnesoid X Receptor Activation and KATP Channel Inhibition

Type 2 diabetes mellitus is associated with alterations in bile acid (BA) signaling. The aim of our study was to test whether pancreatic β-cells contribute to BA-dependent regulation of glucose homeostasis. Experiments were performed with islets from wild-type, farnesoid X receptor (FXR) knockout (KO), and β-cell ATP-dependent K+ (KATP) channel gene SUR1 (ABCC8) KO mice, respectively. Sodium taurochenodeoxycholate (TCDC) increased glucose-induced insulin secretion. This effect was mimicked by the FXR agonist GW4064 and suppressed by the FXR antagonist guggulsterone. TCDC and GW4064 stimulated the electrical activity of β-cells and enhanced cytosolic Ca2+ concentration ([Ca2+]c). These effects were blunted by guggulsterone. Sodium ursodeoxycholate, which has a much lower affinity to FXR than TCDC, had no effect on [Ca2+]c and insulin secretion. FXR activation by TCDC is suggested to inhibit KATP current. The decline in KATP channel activity by TCDC was only observed in β-cells with intact metabolism and was reversed by guggulsterone. TCDC did not alter insulin secretion in islets of SUR1-KO or FXR-KO mice. TCDC did not change islet cell apoptosis. This is the first study showing an acute action of BA on β-cell function. The effect is mediated by FXR by nongenomic elements, suggesting a novel link between FXR activation and KATP channel inhibition.

Expression pattern of aquaporin water channels in the inner ear of the rat: The molecular basis for a water regulation system in the endolymphatic sac

Mammalian aquaporins constitute a family of so far 10 related water channel proteins which mediate osmotically driven water fluxes across the plasma membrane. Because regulation of the ionic composition and osmolality of inner ear fluids is of great functional significance, we investigated the expression patterns of aquaporins in five defined areas of the rat inner ear by RT-PCR. The tissues used were stria vascularis, endolymphatic sac, Reissners membrane, vestibulum and organ of Corti. Aquaporin 1 transcripts were detected in all tissues and are probably constitutive. Aquaporin 5 was only expressed in the organ of Corti and in Reissners membrane. We show that aquaporin 2, so far considered to be specific to the principal cells of the renal collecting duct, is expressed in the endolymphatic sac. Aquaporin 2 expression was not detected in any other inner ear region. The postnatal appearance of aquaporin 2 transcripts in the endolymphatic sac resembled that in the kidney, i.e. it increased postnatally until day 4. The full-length DNA for aquaporin 2 was cloned from cDNA of the endolymphatic sac. It had an irrelevant Ile54Thr mutation because it could be functionally expressed in Xenopus oocytes. Also exclusively in the endolymphatic sac of the inner ear, we detected transcripts for aquaporin isoforms 3 and 4 which are known to be expressed in the renal principal cells. In the kidney, aquaporin 2 regulation involves vasopressin-stimulated, cAMP-dependent phosphorylation of Ser256 of aquaporin 2 which is stored in cytosolic vesicles. These storage vesicles also contain a serpentine calcium/polycation-sensing receptor. Vesicle shuffling to the plasma membrane involves proteins such as vesicle-associated membrane protein VAMP2, syntaxin-4 and the small GTPase Rab3a. Using RT-PCR we were able to demonstrate the expression of all of these components. By analogy the data suggest that in the endolymphatic sac of the inner ear a system for cellular water permeability is in place which may share many similarities with that characterized in the principal cells of the renal collecting duct. These findings may have a number of interesting pharmacological implications which need to be addressed in future studies.

Oscillations of membrane potential and cytosolic Ca2+ concentration in SUR1-/- beta cells

Aims/hypothesisSUR1(ABCC8)−/− mice lacking functional KATP channels are an appropriate model to test the significance of KATP channels in beta-cell function. We examined how this gene deletion interferes with stimulus-secretion coupling. We tested the influence of metabolic inhibition and galanin, whose mode of action is controversial.MethodsPlasma membrane potential (Vm) and currents were measured with microelectrodes or the patch-clamp technique; cytosolic Ca2+ concentrations ([Ca2+]c) and mitochondrial membrane potential (ΔΨ) were measured using fluorescent dyes.ResultsIn contrast to the controls, SUR1−/− beta cells showed electrical activity even at a low glucose concentration. Continuous spike activity was measured with the patch-clamp technique, but with microelectrodes slow oscillations in Vm consisting of bursts of Ca2+-dependent action potentials were detected. [Ca2+]c showed various patterns of oscillations or a sustained increase. Sodium azide did not hyperpolarize SUR1−/− beta cells. The depolarization of ΔΨ evoked by sodium azide was significantly lower in SUR1−/− than SUR1+/+ cells. Galanin transiently decreased action potential frequency and [Ca2+]c in cells from both SUR1−/− and SUR1+/+ mice.Conclusion/interpretationThe strong dependence of Vm and [Ca2+]c on glucose concentration observed in SUR1+/+ beta cells is disrupted in the knock-out cells. This demonstrates that both parameters oscillate in the absence of functional KATP channels. The lack of effect of metabolic inhibition by sodium azide shows that in SUR1−/− beta cells changes in ATP/ADP no longer link glucose metabolism and Vm. The results with galanin suggest that this peptide affects beta cells independently of KATP currents and thus could contribute to the regulation of beta-cell function in SUR1−/− animals.

Suppression of KATP channel activity protects murine pancreatic β cells against oxidative stress

The enhanced oxidative stress associated with type 2 diabetes mellitus contributes to disease pathogenesis. We previously identified plasma membrane-associated ATP-sensitive K+ (KATP) channels of pancreatic beta cells as targets for oxidants. Here, we examined the effects of genetic and pharmacologic ablation of KATP channels on loss of mouse beta cell function and viability following oxidative stress. Using mice lacking the sulfonylurea receptor type 1 (Sur1) subunit of KATP channels, we found that, compared with insulin secretion by WT islets, insulin secretion by Sur1-/- islets was less susceptible to oxidative stress induced by the oxidant H2O2. This was likely, at least in part, a result of the reduced ability of H2O2 to hyperpolarize plasma membrane potential and reduce cytosolic free Ca2+ concentration ([Ca2+]c) in the Sur1-/- beta cells. Remarkably, Sur1-/- beta cells were less prone to apoptosis induced by H2O2 or an NO donor than WT beta cells, despite an enhanced basal rate of apoptosis. This protective effect was attributed to upregulation of the antioxidant enzymes SOD, glutathione peroxidase, and catalase. Upregulation of antioxidant enzymes and reduced sensitivity of Sur1-/- cells to H2O2-induced apoptosis were mimicked by treatment with the sulfonylureas tolbutamide and gliclazide. Enzyme upregulation and protection against oxidant-induced apoptosis were abrogated by agents lowering [Ca2+]c. Sur1-/- mice were less susceptible than WT mice to streptozotocin-induced beta cell destruction and subsequent hyperglycemia and death, which suggests that loss of KATP channel activity may protect against streptozotocin-induced diabetes in vivo.

Enhanced Glucose Tolerance by SK4 Channel Inhibition in Pancreatic β-Cells

OBJECTIVE Ca2+-regulated K+ channels are involved in numerous Ca2+-dependent signaling pathways. In this study, we investigated whether the Ca2+-activated K+ channel of intermediate conductance SK4 (KCa3.1, IK1) plays a physiological role in pancreatic β-cell function. RESEARCH DESIGN AND METHODS Glucose tolerance and insulin sensitivity were determined in wild-type (WT) or SK4 knockout (SK4-KO) mice. Electrophysiological experiments were performed with the patch-clamp technique. The cytosolic Ca2+ concentration ([Ca2+]c) was determined by fura-2 fluorescence. Insulin release was assessed by radioimmunoassay, and SK4 protein was detected by Western blot analysis. RESULTS SK4-KO mice showed improved glucose tolerance, whereas insulin sensitivity was not altered. The animals were not hypoglycemic. Isolated SK4-KO β-cells stimulated with 15 mmol/l glucose had an increased Ca2+ action potential frequency, and single-action potentials were broadened. These alterations were coupled to increased [Ca2+]c. In addition, glucose responsiveness of membrane potential, [Ca2+]c, and insulin secretion were shifted to lower glucose concentrations. SK4 protein was expressed in WT islets. An increase in K+ currents and concomitant membrane hyperpolarization could be evoked in WT β-cells by the SK4 channel opener DCEBIO (100 μmol/l). Accordingly, the SK4 channel blocker TRAM-34 (1 μmol/l) partly inhibited KCa currents and induced electrical activity at a threshold glucose concentration. In stimulated WT β-cells, TRAM-34 further increased [Ca2+]c and broadened action potentials similar to those seen in SK4-KO β-cells. SK4 channels were found to substantially contribute to Kslow (slowly activating K+ current). CONCLUSIONS SK4 channels are involved in β-cell stimulus-secretion coupling. Deficiency of SK4 current induces elevated β-cell responsiveness and coincides with improved glucose tolerance in vivo. Therefore, pharmacologic modulation of these channels might provide an interesting approach for the development of novel insulinotropic drugs.