Milrinone Relaxes Pulmonary Veins in Guinea Pigs and Humans

Introduction

The phosphodiesterase-III inhibitor milrinone improves ventricular contractility, relaxes pulmonary arteries and reduces right ventricular afterload. Thus, it is used to treat heart failure and pulmonary hypertension (PH). However, its action on pulmonary veins (PVs) is not defined, although particularly PH due to left heart disease primarily affects the pulmonary venous bed. We examined milrinone-induced relaxation in PVs from guinea pigs (GPs) and humans.

Material and Methods

Precision-cut lung slices (PCLS) were prepared from GPs or from patients undergoing lobectomy. Milrinone-induced relaxation was studied by videomicroscopy in naïve PVs and in PVs pre-constricted with the ET_A-receptor agonist BP0104. Baseline luminal area was defined as 100%. Intracellular cAMP was measured by ELISA and milrinone-induced changes of segmental vascular resistances were studied in the GP isolated perfused lung (IPL).

Results

In the IPL (GP), milrinone (10 µM) lowered the postcapillary resistance of pre-constricted vessels. In PCLS (GP), milrinone relaxed naïve and pre-constricted PVs (120%) and this relaxation was attenuated by inhibition of protein kinase G (KT 5823), adenyl cyclase (SQ 22536) and protein kinase A (KT 5720), but not by inhibition of NO-synthesis (L-NAME). In addition, milrinone-induced relaxation was dependent on the activation of K_ATP-, BK_Ca ²⁺- and K_v-channels. Human PVs also relaxed to milrinone (121%), however only if pre-constricted.

Discussion

Milrinone relaxes PVs from GPs and humans. In GPs, milrinone-induced relaxation is based on K_ATP-, BK_Ca ²⁺- and K_v-channel-activation and on cAMP/PKA/PKG. The relaxant properties of milrinone on PVs lead to reduced postcapillary resistance and hydrostatic pressures. Hence they alleviate pulmonary edema and suggest beneficial effects of milrinone in PH due to left heart disease.     

Levosimendan Inhibits Peroxidation in Hepatocytes by Modulating Apoptosis/Autophagy Interplay

Background

Levosimendan protects rat liver against peroxidative injuries through mechanisms related to nitric oxide (NO) production and mitochondrial ATP-dependent K (mitoK_ATP) channels opening. However, whether levosimendan could modulate the cross-talk between apoptosis and autophagy in the liver is still a matter of debate. Thus, the aim of this study was to examine the role of levosimendan as a modulator of the apoptosis/autophagy interplay in liver cells subjected to peroxidation and the related involvement of NO and mitoK_ATP.

Methods and Findings

In primary rat hepatocytes that have been subjected to oxidative stress, Western blot was performed to examine endothelial and inducible NO synthase isoforms (eNOS, iNOS) activation, apoptosis/autophagy and survival signalling detection in response to levosimendan. In addition, NO release, cell viability, mitochondrial membrane potential and mitochondrial permeability transition pore opening (MPTP) were examined through specific dyes. Some of those evaluations were also performed in human hepatic stellate cells (HSC). Pre-treatment of hepatocytes with levosimendan dose-dependently counteracted the injuries caused by oxidative stress and reduced NO release by modulating eNOS/iNOS activation. In hepatocytes, while the autophagic inhibition reduced the effects of levosimendan, after the pan-caspases inhibition, cell survival and autophagy in response to levosimendan were increased. Finally, all protective effects were prevented by both mitoK_ATP channels inhibition and NOS blocking. In HSC, levosimendan was able to modulate the oxidative balance and inhibit autophagy without improving cell viability and apoptosis.

Conclusions

Levosimendan protects hepatocytes against oxidative injuries by autophagic-dependent inhibition of apoptosis and the activation of survival signalling. Such effects would involve mitoK_ATP channels opening and the modulation of NO release by the different NOS isoforms. In HSC, levosimendan would also play a role in cell activation and possible evolution toward fibrosis. These findings highlight the potential of levosimendan as a therapeutic agent for the treatment or prevention of liver ischemia/reperfusion injuries.     

Actions of Hydrogen Sulfide and ATP-Sensitive Potassium Channels on Colonic Hypermotility in a Rat Model of Chronic Stress

Objective

To investigate the potential role of hydrogen sulphide (H₂S) and ATP-sensitive potassium (K_ATP) channels in chronic stress-induced colonic hypermotility.

Methods

Male Wistar rats were submitted daily to 1 h of water avoidance stress (WAS) or sham WAS (SWAS) for 10 consecutive days. Organ bath recordings, H₂S production, immunohistochemistry and western blotting were performed on rat colonic samples to investigate the role of endogenous H₂S in repeated WAS-induced hypermotility. Organ bath recordings and western blotting were used to detect the role of K_ATP channels in repeated WAS.

Results

Repeated WAS increased the number of fecal pellets per hour and the area under the curve of the spontaneous contractions of colonic strips, and decreased the endogenous production of H₂S and the expression of H₂S-producing enzymes in the colon devoid of mucosa and submucosa. Inhibitors of H₂S-producing enzymes increased the contractile activity of colonic strips in the SWAS rats. NaHS concentration-dependently inhibited the spontaneous contractions of the strips and the NaHS IC₅₀ for the WAS rats was significantly lower than that for the SWAS rats. The inhibitory effect of NaHS was significantly reduced by glybenclamide. Repeated WAS treatment resulted in up-regulation of Kir6.1 and SUR2B of K_ATP channels in the colon devoid of mucosa and submucosa.

Conclusion

The colonic hypermotility induced by repeated WAS may be associated with the decreased production of endogenous H₂S. The increased expression of the subunits of K_ATP channels in colonic smooth muscle cells may be a defensive response to repeated WAS. H₂S donor may have potential clinical utility in treating chronic stress- induced colonic hypermotility.     

Activation of cGMP-dependent protein kinase stimulates cardiac ATP-sensitive potassium channels via a ROS/calmodulin/CaMKII signaling cascade

Background

Cyclic GMP (cGMP)-dependent protein kinase (PKG) is recognized as an important signaling component in diverse cell types. PKG may influence the function of cardiac ATP-sensitive potassium (K_ATP) channels, an ion channel critical for stress adaptation in the heart; however, the underlying mechanism remains largely unknown. The present study was designed to address this issue.

Methods and Findings

Single-channel recordings of cardiac K_ATP channels were performed in both cell-attached and inside-out patch configurations using transfected human embryonic kidney (HEK)293 cells and rabbit ventricular cardiomyocytes. We found that Kir6.2/SUR2A (the cardiac-type K_ATP) channels were activated by cGMP-selective phosphodiesterase inhibitor zaprinast in a concentration-dependent manner in cell-attached patches obtained from HEK293 cells, an effect mimicked by the membrane-permeable cGMP analog 8-bromo-cGMP whereas abolished by selective PKG inhibitors. Intriguingly, direct application of PKG moderately reduced rather than augmented Kir6.2/SUR2A single-channel currents in excised, inside-out patches. Moreover, PKG stimulation of Kir6.2/SUR2A channels in intact cells was abrogated by ROS/H₂O₂ scavenging, antagonism of calmodulin, and blockade of calcium/calmodulin-dependent protein kinase II (CaMKII), respectively. Exogenous H₂O₂ also concentration-dependently stimulated Kir6.2/SUR2A channels in intact cells, and its effect was prevented by inhibition of calmodulin or CaMKII. PKG stimulation of K_ATP channels was confirmed in intact ventricular cardiomyocytes, which was ROS- and CaMKII-dependent. Kinetically, PKG appeared to stimulate these channels by destabilizing the longest closed state while stabilizing the long open state and facilitating opening transitions.

Conclusion

The present study provides novel evidence that PKG exerts dual regulation of cardiac K_ATP channels, including marked stimulation resulting from intracellular signaling mediated by ROS (H₂O₂ in particular), calmodulin and CaMKII, alongside of moderate channel suppression likely mediated by direct PKG phosphorylation of the channel or some closely associated proteins. The novel cGMP/PKG/ROS/calmodulin/CaMKII signaling pathway may regulate cardiomyocyte excitability by opening K_ATP channels and contribute to cardiac protection against ischemia-reperfusion injury.     

Nicorandil attenuates monocrotaline-induced vascular endothelial damage and pulmonary arterial hypertension

Background

An antianginal K_ATP channel opener nicorandil has various beneficial effects on cardiovascular systems; however, its effects on pulmonary vasculature under pulmonary arterial hypertension (PAH) have not yet been elucidated. Therefore, we attempted to determine whether nicorandil can attenuate monocrotaline (MCT)-induced PAH in rats.

Materials and Methods

Sprague-Dawley rats injected intraperitoneally with 60 mg/kg MCT were randomized to receive either vehicle; nicorandil (5.0 mg·kg⁻¹·day⁻¹) alone; or nicorandil as well as either a K_ATP channel blocker glibenclamide or a nitric oxide synthase (NOS) inhibitor N ^ω-nitro-l-arginine methyl ester (l-NAME), from immediately or 21 days after MCT injection. Four or five weeks later, right ventricular systolic pressure (RVSP) was measured, and lung tissue was harvested. Also, we evaluated the nicorandil-induced anti-apoptotic effects and activation status of several molecules in cell survival signaling pathway in vitro using human umbilical vein endothelial cells (HUVECs).

Results

Four weeks after MCT injection, RVSP was significantly increased in the vehicle-treated group (51.0±4.7 mm Hg), whereas it was attenuated by nicorandil treatment (33.2±3.9 mm Hg; P<0.01). Nicorandil protected pulmonary endothelium from the MCT-induced thromboemboli formation and induction of apoptosis, accompanied with both upregulation of endothelial NOS (eNOS) expression and downregulation of cleaved caspase-3 expression. Late treatment with nicorandil for the established PAH was also effective in suppressing the additional progression of PAH. These beneficial effects of nicorandil were blocked similarly by glibenclamide and l-NAME. Next, HUVECs were incubated in serum-free medium and then exhibited apoptotic morphology, while these changes were significantly attenuated by nicorandil administration. Nicorandil activated the phosphatidylinositol 3-kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK) pathways in HUVECs, accompanied with the upregulation of both eNOS and Bcl-2 expression.

Conclusions

Nicorandil attenuated MCT-induced vascular endothelial damage and PAH through production of eNOS and anti-apoptotic factors, suggesting that nicorandil might have a promising therapeutic potential for PAH.     

Nicorandil prevents right ventricular remodeling by inhibiting apoptosis and lowering pressure overload in rats with pulmonary arterial hypertension

Background

Most of the deaths among patients with severe pulmonary arterial hypertension (PAH) are caused by progressive right ventricular (RV) pathological remodeling, dysfunction, and failure. Nicorandil can inhibit the development of PAH by reducing pulmonary artery pressure and RV hypertrophy. However, whether nicorandil can inhibit apoptosis in RV cardiomyocytes and prevent RV remodeling has been unclear.

Methodology/Principal Findings

RV remodeling was induced in rats by intraperitoneal injection of monocrotaline (MCT). RV systolic pressure (RVSP) was measured at the end of each week after MCT injection. Blood samples were drawn for brain natriuretic peptide (BNP) ELISA analysis. The hearts were excised for histopathological, ultrastructural, immunohistochemical, and Western blotting analyses. The MCT-injected rats exhibited greater mortality and less weight gain and showed significantly increased RVSP and RV hypertrophy during the second week. These worsened during the third week. MCT injection for three weeks caused pathological RV remodeling, characterized by hypertrophy, fibrosis, dysfunction, and RV mitochondrial impairment, as indicated by increased levels of apoptosis. Nicorandil improved survival, weight gain, and RV function, ameliorated RV pressure overload, and prevented maladaptive RV remodeling in PAH rats. Nicorandil also reduced the number of apoptotic cardiomyocytes, with a concomitant increase in Bcl-2/Bax ratio. 5-hydroxydecanoate (5-HD) reversed these beneficial effects of nicorandil in MCT-injected rats.

Conclusions/Significance

Nicorandil inhibits PAH-induced RV remodeling in rats not only by reducing RV pressure overload but also by inhibiting apoptosis in cardiomyocytes through the activation of mitochondrial ATP-sensitive K⁺ (mitoK_ATP) channels. The use of a mitoK_ATP channel opener such as nicorandil for PAH-associated RV remodeling and dysfunction may represent a new therapeutic strategy for the amelioration of RV remodeling during the early stages of PAH.     

The functional expression of extracellular calcium-sensing receptor in rat pulmonary artery smooth muscle cells

Background

The extracellular calcium-sensing receptor (CaSR) belongs to family C of the G protein coupled receptors. Whether the CaSR is expressed in the pulmonary artery (PA) is unknown.

Methods

The expression and distribution of CaSR were detected by RT-PCR, Western blotting and immunofluorescence. PA tension was detected by the pulmonary arterial ring technique, and the intracellular calcium concentration ([Ca²⁺]_i) was detected by a laser-scanning confocal microscope.

Results

The expressions of CaSR mRNA and protein were found in both rat pulmonary artery smooth muscle cells (PASMCs) and PAs. Increased levels of [Ca²⁺]_o (extracellular calcium concentration) or Gd³⁺ (an agonist of CaSR) induced an increase of [Ca²⁺]_i and PAs constriction in a concentration-dependent manner_. In addition, the above-mentioned effects of Ca²⁺ and Gd³⁺ were inhibited by {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 (specific inhibitor of PLC), 2-APB (specific antagonist of IP₃ receptor), and thapsigargin (blocker of sarcoplasmic reticulum calcium ATPase).

Conclusions

CaSR is expressed in rat PASMCs, and is involved in regulation of PA tension by increasing [Ca²⁺]_i through G-PLC-IP₃ pathway.     

Pulmonary arterial dysfunction in insulin resistant obese Zucker rats

Background

Insulin resistance and obesity are strongly associated with systemic cardiovascular diseases. Recent reports have also suggested a link between insulin resistance with pulmonary arterial hypertension. The aim of this study was to analyze pulmonary vascular function in the insulin resistant obese Zucker rat.

Methods

Large and small pulmonary arteries from obese Zucker rat and their lean counterparts were mounted for isometric tension recording. mRNA and protein expression was measured by RT-PCR or Western blot, respectively. K_V currents were recorded in isolated pulmonary artery smooth muscle cells using the patch clamp technique.

Results

Right ventricular wall thickness was similar in obese and lean Zucker rats. Lung BMPR2, K_V1.5 and 5-HT_2A receptor mRNA and protein expression and K_V current density were also similar in the two rat strains. In conductance and resistance pulmonary arteries, the similar relaxant responses to acetylcholine and nitroprusside and unchanged lung eNOS expression revealed a preserved endothelial function. However, in resistance (but not in conductance) pulmonary arteries from obese rats a reduced response to several vasoconstrictor agents (hypoxia, phenylephrine and 5-HT) was observed. The hyporesponsiveness to vasoconstrictors was reversed by L-NAME and prevented by the iNOS inhibitor 1400W.

Conclusions

In contrast to rat models of type 1 diabetes or other mice models of insulin resistance, the obese Zucker rats did not show any of the characteristic features of pulmonary hypertension but rather a reduced vasoconstrictor response which could be prevented by inhibition of iNOS.     

Deletion of CDKAL1 affects mitochondrial ATP generation and first-phase insulin exocytosis

Background

A variant of the CDKAL1 gene was reported to be associated with type 2 diabetes and reduced insulin release in humans; however, the role of CDKAL1 in β cells is largely unknown. Therefore, to determine the role of CDKAL1 in insulin release from β cells, we studied insulin release profiles in CDKAL1 gene knockout (CDKAL1 KO) mice.

Principal Findings

Total internal reflection fluorescence imaging of CDKAL1 KO β cells showed that the number of fusion events during first-phase insulin release was reduced. However, there was no significant difference in the number of fusion events during second-phase release or high K⁺-induced release between WT and KO cells. CDKAL1 deletion resulted in a delayed and slow increase in cytosolic free Ca²⁺ concentration during high glucose stimulation. Patch-clamp experiments revealed that the responsiveness of ATP-sensitive K⁺ (K_ATP) channels to glucose was blunted in KO cells. In addition, glucose-induced ATP generation was impaired. Although CDKAL1 is homologous to cyclin-dependent kinase 5 (CDK5) regulatory subunit-associated protein 1, there was no difference in the kinase activity of CDK5 between WT and CDKAL1 KO islets.

Conclusions/Significance

We provide the first report describing the function of CDKAL1 in β cells. Our results indicate that CDKAL1 controls first-phase insulin exocytosis in β cells by facilitating ATP generation, K_ATP channel responsiveness and the subsequent activity of Ca²⁺ channels through pathways other than CDK5-mediated regulation.     

Formononetin,an isoflavone,relaxes rat isolated aorta through endothelium-dependent and endothelium-independent pathways

We evaluated the vasorelaxation effects of formononetin, an isoflavone/phytoestrogen found abundantly in Astragalus mongholicus Bunge, on rat isolated aorta and the underlying mechanisms involved. Cumulative administration of formononetin, genistein, daidzein and biochanin A relaxed phenylephrine-preconstricted aorta. Formononetin and biochanin A caused a similar magnitude of relaxation whereas daidzein was least potent. Mechanical removal of endothelium, L-NAME (100 μM) and methylene blue (10 μM) suppressed formononetin-induced relaxation. Formononetin increased endothelial nitric oxide (NO) synthase (eNOS), but not inducible NO synthase, activity with an up-regulation of eNOS mRNA and p-eNOS^Ser1177 protein expression. In endothelium-denuded preparations, formononetin-induced vasorelaxation was significantly reduced by glibenclamide (3 μM) and iberiotoxin (100 nM), and a combination of glibenclamide (3 μM) plus iberiotoxin (100 nM) abolished the relaxation. In contrast, formononetin-elicited endothelium-independent relaxation was not altered by ICI 182,780 (10 μM, an estrogen receptor (ERα/ERβ) antagonist) or mifepristone (10 μM, a progesterone receptor antagonist). In single aortic smooth muscle cells, formononetin caused opening of iberiotoxin-sensitive Ca²⁺-activated K⁺ (BK_Ca) channels and glibenclamide-sensitive adenosine triphosphate (ATP)-dependent K⁺ (K_ATP) channels. Thus, our results suggest that formononetin caused vascular relaxation via endothelium/NO-dependent mechanism and endothelium-independent mechanism which involves the activation of BK_Ca and K_ATP channels.     

Pulmonary Hypertension in Wild Type Mice and Animals with Genetic Deficit in KCa2.3 and KCa3.1 Channels

Objective

In vascular biology, endothelial K_Ca2.3 and K_Ca3.1 channels contribute to arterial blood pressure regulation by producing membrane hyperpolarization and smooth muscle relaxation. The role of K_Ca2.3 and K_Ca3.1 channels in the pulmonary circulation is not fully established. Using mice with genetically encoded deficit of K_Ca2.3 and K_Ca3.1 channels, this study investigated the effect of loss of the channels in hypoxia-induced pulmonary hypertension.

Approach and Result

Male wild type and K_Ca3.1^−/−/K_Ca2.3^T/T(+DOX) mice were exposed to chronic hypoxia for four weeks to induce pulmonary hypertension. The degree of pulmonary hypertension was evaluated by right ventricular pressure and assessment of right ventricular hypertrophy. Segments of pulmonary arteries were mounted in a wire myograph for functional studies and morphometric studies were performed on lung sections. Chronic hypoxia induced pulmonary hypertension, right ventricular hypertrophy, increased lung weight, and increased hematocrit levels in either genotype. The K_Ca3.1^−/−/K_Ca2.3^T/T(+DOX) mice developed structural alterations in the heart with increased right ventricular wall thickness as well as in pulmonary vessels with increased lumen size in partially- and fully-muscularized vessels and decreased wall area, not seen in wild type mice. Exposure to chronic hypoxia up-regulated the gene expression of the K_Ca2.3 channel by twofold in wild type mice and increased by 2.5-fold the relaxation evoked by the K_Ca2.3 and K_Ca3.1 channel activator NS309, whereas the acetylcholine-induced relaxation - sensitive to the combination of K_Ca2.3 and K_Ca3.1 channel blockers, apamin and charybdotoxin - was reduced by 2.5-fold in chronic hypoxic mice of either genotype.

Conclusion

Despite the deficits of the K_Ca2.3 and K_Ca3.1 channels failed to change hypoxia-induced pulmonary hypertension, the up-regulation of K_Ca2.3-gene expression and increased NS309-induced relaxation in wild-type mice point to a novel mechanism to counteract pulmonary hypertension and to a potential therapeutic utility of K_Ca2.3/K_Ca3.1 activators for the treatment of pulmonary hypertension.     

Osteopenia due to enhanced cathepsin K release by BK channel ablation in osteoclasts

Background

The process of bone resorption by osteoclasts is regulated by Cathepsin K, the lysosomal collagenase responsible for the degradation of the organic bone matrix during bone remodeling. Recently, Cathepsin K was regarded as a potential target for therapeutic intervention of osteoporosis. However, mechanisms leading to osteopenia, which is much more common in young female population and often appears to be the clinical pre-stage of idiopathic osteoporosis, still remain to be elucidated, and molecular targets need to be identified.

Methodology/Principal Findings

We found, that in juvenile bone the large conductance, voltage and Ca²⁺-activated (BK) K⁺ channel, which links membrane depolarization and local increases in cytosolic calcium to hyperpolarizing K⁺ outward currents, is exclusively expressed in osteoclasts. In juvenile BK-deficient (BK^−/−) female mice, plasma Cathepsin K levels were elevated two-fold when compared to wild-type littermates. This increase was linked to an osteopenic phenotype with reduced bone mineral density in long bones and enhanced porosity of trabecular meshwork in BK^−/− vertebrae as demonstrated by high-resolution flat-panel volume computed tomography and micro-CT. However, plasma levels of sRANKL, osteoprotegerin, estrogene, Ca²⁺ and triiodthyronine as well as osteoclastogenesis were not altered in BK^−/− females.

Conclusion/Significance

Our findings suggest that the BK channel controls resorptive osteoclast activity by regulating Cathepsin K release. Targeted deletion of BK channel in mice resulted in an osteoclast-autonomous osteopenia, becoming apparent in juvenile females. Thus, the BK^−/− mouse-line represents a new model for juvenile osteopenia, and revealed the BK channel as putative new target for therapeutic controlling of osteoclast activity.     

Diminished Neurogenic Femoral Artery Vasoconstrictor Response in a Zucker Obese Rat Model: Differential Regulation of NOS and COX Derivatives

Objective

Peripheral arterial disease is one of the macrovascular complications of type 2 diabetes mellitus. This study addresses femoral artery regulation in a prediabetic model of obese Zucker rats (OZR) by examining cross-talk between endothelial and neural factors.

Methods and Results

Arterial preparations from lean (LZR) and OZR were subjected to electrical field stimulation (EFS) on basal tone. Nitric oxide synthase (NOS) and cyclooxygenase (COX) isoform expression patterns were determined by immunohistochemical labelling and Western blotting. Results indicate significantly reduced noradrenergic contractions in preparations from OZR compared with those of LZR. Functional inhibition of endothelial NOS (eNOS) indicated a predominant role of this isoform in LZR and its modified activity in OZR. Neural (nNOS) and inducible NOS (iNOS) were activated and their expression was higher in femoral arteries from OZR. Neurotransmission modulated by large-conductance Ca²⁺-activated (BK_Ca) or voltage-dependent (K_V) K⁺ channels did not seem compromised in the obese animals. Endothelial COX-1 and COX-2 were expressed in LZR and an additional adventitial location of COX-2 was also observed in OZR, explaining the higher COX-2 protein levels detected in this group. Prostanoids derived from both isoforms helped maintain vasoconstriction in LZR while in OZR only COX-2 was active. Superoxide anion inhibition reduced contractions in endothelium-intact arteries from OZR.

Conclusions

Endothelial dysfunction led to reduced neurogenic vasoconstriction in femoral arteries from OZR. In a setting of obesity, NO-dependent nNOS and iNOS dilation activity could be an alternative mechanism to offset COX-2- and reactive oxygen species-mediated vasoconstriction, along with impaired endothelial NO relaxation.     

Chronic Antidiabetic Sulfonylureas In Vivo: Reversible Effects on Mouse Pancreatic β-Cells

Background

Pancreatic β-cell ATP-sensitive potassium (K_ATP) channels are critical links between nutrient metabolism and insulin secretion. In humans, reduced or absent β-cell K_ATP channel activity resulting from loss-of-function K_ATP mutations induces insulin hypersecretion. Mice with reduced K_ATP channel activity also demonstrate hyperinsulinism, but mice with complete loss of K_ATP channels (K_ATP knockout mice) show an unexpected insulin undersecretory phenotype. Therefore we have proposed an “inverse U” hypothesis to explain the response to enhanced excitability, in which excessive hyperexcitability drives β-cells to insulin secretory failure without cell death. Many patients with type 2 diabetes treated with antidiabetic sulfonylureas (which inhibit K_ATP activity and thereby enhance insulin secretion) show long-term insulin secretory failure, which we further suggest might reflect a similar progression.

Methods and Findings

To test the above hypotheses, and to mechanistically investigate the consequences of prolonged hyperexcitability in vivo, we used a novel approach of implanting mice with slow-release sulfonylurea (glibenclamide) pellets, to chronically inhibit β-cell K_ATP channels. Glibenclamide-implanted wild-type mice became progressively and consistently diabetic, with significantly (p < 0.05) reduced insulin secretion in response to glucose. After 1 wk of treatment, these mice were as glucose intolerant as adult K_ATP knockout mice, and reduction of secretory capacity in freshly isolated islets from implanted animals was as significant (p < 0.05) as those from K_ATP knockout animals. However, secretory capacity was fully restored in islets from sulfonylurea-treated mice within hours of drug washout and in vivo within 1 mo after glibenclamide treatment was terminated. Pancreatic immunostaining showed normal islet size and α-/β-cell distribution within the islet, and TUNEL staining showed no evidence of apoptosis.

Conclusions

These results demonstrate that chronic glibenclamide treatment in vivo causes loss of insulin secretory capacity due to β-cell hyperexcitability, but also reveal rapid reversibility of this secretory failure, arguing against β-cell apoptosis or other cell death induced by sulfonylureas. These in vivo studies may help to explain why patients with type 2 diabetes can show long-term secondary failure to secrete insulin in response to sulfonylureas, but experience restoration of insulin secretion after a drug resting period, without permanent damage to β-cells. This finding suggests that novel treatment regimens may succeed in prolonging pharmacological therapies in susceptible individuals.     

Acute Simvastatin Inhibits KATP Channels of Porcine Coronary Artery Myocytes

Background

Statins (3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitors) consumption provides beneficial effects on cardiovascular systems. However, effects of statins on vascular K_ATP channel gatings are unknown.

Methods

Pig left anterior descending coronary artery and human left internal mammary artery were isolated and endothelium-denuded for tension measurements and Western immunoblots. Enzymatically-dissociated/cultured arterial myocytes were used for patch-clamp electrophysiological studies and for [Ca²⁺]_i, [ATP]_i and [glucose]_o uptake measurements.

Results

The cromakalim (10 nM to 10 µM)- and pinacidil (10 nM to 10 µM)-induced concentration-dependent relaxation of porcine coronary artery was inhibited by simvastatin (3 and 10 µM). Simvastatin (1, 3 and 10 µM) suppressed (in okadaic acid (10 nM)-sensitive manner) cromakalim (10 µM)- and pinacidil (10 µM)-mediated opening of whole-cell K_ATP channels of arterial myocytes. Simvastatin (10 µM) and AICAR (1 mM) elicited a time-dependent, compound C (1 µM)-sensitive [³H]-2-deoxy-glucose uptake and an increase in [ATP]_i levels. A time (2–30 min)- and concentration (0.1–10 µM)-dependent increase by simvastatin of p-AMPKα-Thr¹⁷² and p-PP2A-Tyr³⁰⁷ expression was observed. The enhanced p-AMPKα-Thr¹⁷² expression was inhibited by compound C, ryanodine (100 µM) and KN93 (10 µM). Simvastatin-induced p-PP2A-Tyr³⁰⁷ expression was suppressed by okadaic acid, compound C, ryanodine, KN93, phloridzin (1 mM), ouabain (10 µM), and in [glucose]_o-free or [Na⁺]_o-free conditions.

Conclusions

Simvastatin causes ryanodine-sensitive Ca²⁺ release which is important for AMPKα-Thr¹⁷² phosphorylation via Ca²⁺/CaMK II. AMPKα-Thr¹⁷² phosphorylation causes [glucose]_o uptake (and an [ATP]_i increase), closure of K_ATP channels, and phosphorylation of AMPKα-Thr¹⁷² and PP2A-Tyr³⁰⁷ resulted. Phosphorylation of PP2A-Tyr³⁰⁷ occurs at a site downstream of AMPKα-Thr¹⁷² phosphorylation.     

ATP-sensitive potassium channel (KATP channel) expression in the normal canine pancreas and in canine insulinomas

Background  

Pancreatic beta cells express ATP-sensitive potassium (K_ATP) channels that are needed for normal insulin secretion and are targets for drugs that modulate insulin secretion. The K_ATP channel is composed of two subunits: a sulfonylurea receptor (SUR 1) and an inward rectifying potassium channel (Kir_6.2). K_ATP channel activity is influenced by the metabolic state of the cell and initiates the ionic events that precede insulin exocytosis. Although drugs that target the K_ATP channel have the expected effects on insulin secretion in dogs, little is known about molecular aspects of this potassium channel. To learn more about canine beta cell K_ATP channels, we studied K_ATP channel expression by the normal canine pancreas and by insulin-secreting tumors of dogs.     

Distinct Kinin-Induced Functions Are Altered in Circulating Cells of Young Type 1 Diabetic Patients

Aims/Hypothesis

We aimed to understand early alterations in kinin-mediated migration of circulating angio-supportive cells and dysfunction of kinin-sensitive cells in type-1 diabetic (T1D) patients before the onset of cardiovascular disease.

Methods

Total mononuclear cells (MNC) were isolated from peripheral blood of 28 T1D patients free from cardiovascular complications except mild background retinopathy (age: 34.8±1.6 years, HbA_1C: 7.9±0.2%) and 28 age- and sex-matched non-diabetic controls (H). We tested expression of kinin receptors by flow cytometry and migratory capacity of circulating monocytes and progenitor cells towards bradykinin (BK) in transwell migration assays. MNC migrating towards BK (BK^mig) were assessed for capacity to support endothelial cell function in a matrigel assay, as well as generation of nitric oxide (NO) and superoxide (O₂ ⁻*) by using the fluorescent probes diaminofluorescein and dihydroethidium.

Results

CD14^hiCD16^neg, CD14^hiCD16^pos and CD14^loCD16^pos monocytes and circulating CD34^pos progenitor cells did not differ between T1D and H subjects in their kinin receptor expression and migration towards BK. T1D BK^mig failed to generate NO upon BK stimulation and supported endothelial cell network formation less efficiently than H BK^mig. In contrast, O₂ ⁻* production was similar between groups. High glucose disturbed BK-induced NO generation by MNC-derived cultured angiogenic cells.

Conclusions/Interpretation

Our data point out alterations in kinin-mediated functions of circulating MNC from T1D patients, occurring before manifest macrovascular damage or progressed microvascular disease. Functional defects of MNC recruited to the vessel wall might compromise endothelial maintenance, initially without actively promoting endothelial damage, but rather by lacking supportive contribution to endothelial regeneration and healing.     

Identification and characterization of Ca2+-activated K+ channels in granulosa cells of the human ovary

Background  

Granulosa cells (GCs) represent a major endocrine compartment of the ovary producing sex steroid hormones. Recently, we identified in human GCs a Ca²⁺-activated K⁺ channel (K_Ca) of big conductance (BK_Ca), which is involved in steroidogenesis. This channel is activated by intraovarian signalling molecules (e.g. acetylcholine) via raised intracellular Ca²⁺ levels. In this study, we aimed at characterizing 1. expression and functions of K_Ca channels (including BK_Ca beta-subunits), and 2. biophysical properties of BK_Ca channels.     

ATP-sensitive potassium channel (K<Subscript>ATP</Subscript>channel) expression in the normal canine pancreas and in canine insulinomas

Background

Pancreatic beta cells express ATP-sensitive potassium (K_ATP) channels that are needed for normal insulin secretion and are targets for drugs that modulate insulin secretion. The K_ATP channel is composed of two subunits: a sulfonylurea receptor (SUR 1) and an inward rectifying potassium channel (Kir_6.2). K_ATP channel activity is influenced by the metabolic state of the cell and initiates the ionic events that precede insulin exocytosis. Although drugs that target the K_ATP channel have the expected effects on insulin secretion in dogs, little is known about molecular aspects of this potassium channel. To learn more about canine beta cell K_ATP channels, we studied K_ATP channel expression by the normal canine pancreas and by insulin-secreting tumors of dogs.

Results

Pancreatic tissue from normal dogs and tumor tissue from three dogs with histologically-confirmed insulinomas was examined for expression of K_ATP channel subunits (SUR1 and Kir_6.2) using RT-PCR. Normal canine pancreas expressed SUR1 and Kir_6.2 subunits of the K_ATP channel. The partial nucleotide sequences for SUR1 and Kir_6.2 obtained from the normal pancreas showed a high degree of homology to published sequences for other mammalian species. SUR1 and Kir_6.2 expression was observed in each of the three canine insulinomas examined. Comparison of short sequences from insulinomas with those obtained from normal pancreas did not reveal any mutations in either SUR1 or Kir_6.2 in any of the insulinomas.

Conclusion

Canine pancreatic K_ATP channels have the same subunit composition as those found in the endocrine pancreases of humans, rats, and mice, suggesting that the canine channel is regulated in a similar fashion as in other species. SUR1 and Kir_6.2 expression was found in the three insulinomas examined indicating that unregulated insulin secretion by these tumors does not result from failure to express one or both K_ATP channel subunits.

     

The Inotropic Effect of the Active Metabolite of Levosimendan,OR-1896, Is Mediated through Inhibition of PDE3 in Rat Ventricular Myocardium

Aims

We recently published that the positive inotropic response (PIR) to levosimendan can be fully accounted for by phosphodiesterase (PDE) inhibition in both failing human heart and normal rat heart. To determine if the PIR of the active metabolite OR-1896, an important mediator of the long-term clinical effects of levosimendan, also results from PDE3 inhibition, we compared the effects of OR-1896, a representative Ca²⁺ sensitizer EMD57033 (EMD), levosimendan and other PDE inhibitors.

Methods

Contractile force was measured in rat ventricular strips. PDE assay was conducted on rat ventricular homogenate. cAMP was measured using RII_epac FRET-based sensors.

Results

OR-1896 evoked a maximum PIR of 33±10% above basal at 1 μM. This response was amplified in the presence of the PDE4 inhibitor rolipram (89±14%) and absent in the presence of the PDE3 inhibitors cilostamide (0.5±5.3%) or milrinone (3.2±4.4%). The PIR was accompanied by a lusitropic response, and both were reversed by muscarinic receptor stimulation with carbachol and absent in the presence of β-AR blockade with timolol. OR-1896 inhibited PDE activity and increased cAMP levels at concentrations giving PIRs. OR-1896 did not sensitize the concentration-response relationship to extracellular Ca²⁺. Levosimendan, OR-1896 and EMD all increased the sensitivity to β-AR stimulation. The combination of either EMD and levosimendan or EMD and OR-1896 further sensitized the response, indicating at least two different mechanisms responsible for the sensitization. Only EMD sensitized the α₁-AR response.

Conclusion

The observed PIR to OR-1896 in rat ventricular strips is mediated through PDE3 inhibition, enhancing cAMP-mediated effects. These results further reinforce our previous finding that Ca²⁺ sensitization does not play a significant role in the inotropic (and lusitropic) effect of levosimendan, nor of its main metabolite OR-1896.