Activators of potassium channels enhance calcium influx into endothelial cells as a consequence of potassium currents

Summary Ca²⁺ influx into stimulated endothelial cells is attenuated by depolarization. We hypothesized that Ca²⁺ influx is driven by the membrane potential and may be enhanced by hyperpolarizing drugs like activators of K⁺ channels. Therefore we studied the effects of pinacidil, cromakalim, and cicletanine on membrane currents and on the intracellular free calcium concentration ([Ca²⁺]i) in cultured endothelial cells from porcine aorta. In patch-clamped cells, pinacidil (1 mol/l) and cromakalim (1 mol/l) elicited outward currents carried by K⁺ and significantly prolonged the Ca²⁺-dependent K⁺ currents induced by bradykinin and ATP. Peak currents in response to bradykinin were not affected. In cells loaded with the fluorescent Ca²⁺ indicator indo-1 and prestimulated with thimerosal, pinacidil (0.1–1 mol/l elicited long-lasting increases in [Ca²⁺)_i from 100 ± 10 to 550 ± 110 nmol/l. These effects were completely abolished in a medium containing 90 mmol/l K⁺. Similar results were obtained with cromakalim. Likewise, in cells stimulated with bradykinin, pinacidil raised [Ca²⁺]_i when applied during the decline of [Ca²⁺]_i after the initial peak. Cicletanine elicited K⁺ currents in resting and attenuated K⁺ currents in bradykinin-stimulated cells. It elevated [Ca²⁺]_i even in the absence of extracellular Ca²⁺ and in K⁺-rich medium. Hence, the effects of cicletanine cannot be explained by direct actions on K⁺ channels. However, our studies demonstrate that pinacidil and cromakalim elevate [Ca²⁺]_i secondary to their activation of K⁺ channels by inducing hyperpolarization and augmenting the driving force for potential-dependent Ca²⁺ influx. In this way, the two drugs may promote Ca²⁺-dependent formation of endothelium-derived relaxing factor. Send offprint requests to A. Lückhoff at the above address     

Temperature-independent Inhibition of L-type calcium currents by halothane and sevoflurane in human atrial cardiomyocytes

BACKGROUND: Cardiac L-type calcium currents (ICa,L) are affected by volatile anesthetics, possibly contributing to their side effects. Actions of anesthetics on ion channels are usually studied in vitro at room temperature. However, the solubility of anesthetic gases as well as ICa,L are markedly sensitive to the study temperature. Therefore, temperature-dependent effects of halothane and sevoflurane on cardiac ICa,L were analyzed. METHODS: ICa,L were studied at 21 degrees C and 36 degrees C with the patch clamp technique in isolated human atrial cardiomyocytes. Concentrations of anesthetics brought into solution by gassing at both temperatures were determined with gas chromatography. RESULTS: The aqueous concentrations of halothane and sevoflurane were linearly related to their concentration in the gas phase (1 to 3 minimum alveolar concentration [MAC]). At 21 degrees C, the slope of this relation was 0.52 and 0.12 mm/vol % for halothane and sevoflurane, respectively, and decreased at 36 degrees C to 0.29 and 0.09 mm/vol %, respectively. ICa,L displayed significantly higher current amplitudes at 36 degrees C than at 21 degrees C and significantly accelerated time-dependent inactivation. Halothane (1-2 MAC) and sevoflurane (1-3 MAC) evoked stronger inhibitions of ICa,L at 21 degrees C than at 36 degrees C. In spite of different temperature-dependent current amplitudes, the fractional (percent) inhibition of ICa,L showed the same linear relationship to the concentrations of halothane and sevoflurane in the bath medium at both temperatures, as revealed from present and previous experiments. CONCLUSIONS: Inhibition of ICa,L by halothane and sevoflurane is determined by the aqueous concentration of the anesthetics, independently of the temperature. Increased solubility may explain the stronger effects of the anesthetics at lower temperatures.     

Evidence of endothelial dysfunction in coronary resistance vessels in patients with angina pectoris and normal coronary angiograms

This study determines whether an impaired endothelium-mediated vasodilation in coronary resistance vessels exists in patients with microvascular angina. In 23 patients with clinically suspected coronary artery disease and smooth coronary arteries in the angiogram, coronary flow in response to an endothelium-related (acetylcholine) and endothelium-unrelated (dipyridamole) vasodilation was measured. Coronary flow was determined by the gas-chromatographic argon method (1) before, (2) with intracoronary acetylcholine infusion, and (3) after dipyridamole administered intravenously. In 8 patients, acetylcholine did not significantly increase coronary flow (from 91 +/- 28 to 118 +/- 37 ml/min.100 g), whereas flow was greatly increased after administration of dipyridamole (258 +/- 97 ml/min.100 g), indicating an endothelium-related vasodilator defect. In 6 patients, neither acetylcholine nor dipyridamole caused a significant increase in coronary flow, indicating an impaired coronary vasodilation on the vascular site. In 6 patients, coronary flow increased markedly after both administration of both acetylcholine and dipyridamole (from 81 +/- 26 to 191 +/- 68 and 234 +/- 87 ml/min.100 g). In 3 patients given acetylcholine, coronary artery constriction occurred. No significant correlation was found between the response to acetylcholine and that to dipyridamole (r = 0.40, p = not significant). The results indicate that in a subgroup of patients with smooth coronary arteries angina can be caused by an abnormality of the endothelial function in the microcirculation.     

The diagnosis of acute urethral trauma

Background

During trauma resuscitation, blind catheterization of an injured urethra may aggravate the injury by disrupting a partially torn urethra. In busy trauma centers, retrograde urethrograms (RUG) prior to catheterisation for all patients with unstable pelvic fractures presents a challenge during trauma resuscitation, and the procedure is not commonly practiced despite Advanced Trauma Life Support (ATLS) and World Health Organisation recommendations. The aim of this study was to determine the presenting clinical features of patients with urethral injuries and to predict major trauma patients needing further investigation to exclude this injury.

Methods

A retrospective review of adult major trauma patients diagnosed with urethral injuries during an 8-year period at a major trauma centre, was conducted.

Results

There were 998 major trauma patients with fractures of the pelvis over the study period, of whom 223 had pubic symphysis disruption. There were 29 patients with urethral injuries. The sensitivity of any one of the traditional signs of urethral trauma was 66.7% (95% CI: 46.0-82.8). After exclusion of patients with penetrating trauma and iatrogenic injuries, pubic symphysis disruption on initial pelvis AP X-ray and/or the clinical signs of urethral injury had a sensitivity of 100% (95% CI: 84.4-100.0) for urethral trauma.

Discussion

Reliance on clinical features alone to predict urethral injury results in a substantial proportion of missed injuries in major trauma patients. RUGs did not appear to be needed in patients with no disruption of the pubic symphysis on initial pelvis X-ray or where no signs of urethral injury are present. In the absence of clinical signs and pubic symphysis disruption, blind urethral catheterisation may be attempted.     

Composite prognostic models across the non-alcoholic fatty liver disease spectrum: Clinical application in developing countries

Heterogeneity in clinical presentation, histological severity, prognosis and therapeutic outcomes characteristic of non-alcoholic fatty liver disease(NAFLD) necessitates the development of scientifically sound classification schemes to assist clinicians in stratifying patients into meaningful prognostic subgroups. The need for replacement of invasive liver biopsies as the standard method whereby NAFLD is diagnosed, graded and staged with biomarkers of histological severity injury led to the development of composite prognostic models as potentially viable surrogate alternatives. In the present article, we review existing scoring systems used to(1) confirm the presence of undiagnosed hepatosteatosis;(2) distinguish between simple steatosis and NASH; and(3) predict advanced hepatic fibrosis, with particular emphasis on the role of NAFLD as an independent cardio-metabolic risk factor. In addition, the incorporation of functional genomic markers and application of emerging imaging technologies are discussed as a means to improve the diagnostic accuracy and predictive performance of promising composite models found to be most appropriate for widespread clinical adoption.     

LY 83583 (6-anilino-5,8-quinolinedione) blocks nitrovasodilator-induced cyclic GMP increases and inhibition of platelet activation

Summary We studied the effects and the mechanism of action of the cyclic GMP-lowering substance 6-anilino-5,8-quinolinedione (LY 83583) on cyclic GMP-mediated inhibition of platelet function. The activation of washed human platelets by thrombin was counteracted by 8-bromo-cyclic GMP and the direct activators of soluble guanylate cyclase, sodium nitroprusside and endothelium-derived relaxant factor (EDRF = nitric oxide). LY 83583 significantly antagonized the inhibitory effect of sodium nitroprusside and EDRF, but not that of 8-bromo-cyclic GMP, on thrombin-induced aggregation, ATP-release, adhesion to native endothelial cells and increase in concentration of free intracellular calcium ions. In accordance, increases in intracellular cyclic GMP by sodium nitroprusside and EDRF were attenuated by LY 83583. The inhibition of cyclic GMP-mediated effects on platelets by LY 83583 could be related to inhibition of platelet soluble guanylate cyclase, as the activation of the purified enzyme from platelets by sodium nitroprusside was directly inhibited by LY 83583. This effect of LY 83583 was attenuated in the presence of superoxide dismutase. Our findings support the hypothesis that sodium nitroprusside and EDRF inhibit platelet activation by stimulation of soluble guanylate cyclase via nitric oxide. Consequently, inhibition of nitric oxide-induced cyclic GMP formation by LY 83583, which may act by intracellular generation of superoxide anions, facilitates platelet activation.Send offprint requests to A. Mülsch at the above address     

Differential role of extra- and intracellular calcium in the release of EDRF and prostacyclin from cultured endothelial cells.

1. The effects of extracellular Ca2+ on the release of endothelium-derived relaxing factor (EDRF) and prostacyclin (PGI2), and on the intracellular free calcium concentration [( Ca2+]i), were studied in cultured bovine aortic endothelial cells. 2. Receptor-mediated stimulation of endothelial cells with bradykinin (10 nM) elicited a transient release of EDRF (assayed by its stimulant effect on purified soluble guanylate cyclase) and of PGI2 (measured by radioimmunoassay for 6-keto prostaglandin F1 alpha). 3. Bradykinin (10 nM) also increased [Ca2+]i (measured with the fluorescent probe indo-1) from 125 +/- 11 nM to 631 +/- 59 nM, with the same time course as for autacoid release. 4. In Ca2+-free medium, [Ca2+]i was still increased by bradykinin but declined faster (within 1 min) to resting levels than in the presence of extracellular Ca2+. 5. PGI2 release was almost completely abolished in Ca2+-free medium. The intracellular calcium antagonist TMB-8 evoked a similar inhibition of PGI2 release. 6. In contrast, bradykinin-induced EDRF release was not significantly affected by TMB-8 but was completely abolished in Ca2+-free medium. 7. When endothelial cells were stimulated with the receptor-independent drug thimerosal (an inhibitor of the enzyme acyl-CoA-lysolecithin-acyl-transferase; 5 microM), a long-lasting release of EDRF (greater than 90 min) and PGI2 (greater than 20 min) was observed. 8. In contrast to bradykinin stimulation, thimerosal-induced autacoid release was associated with only a slight increase of [Ca2+]i to 201 +/- 13 nM after 40 min.(ABSTRACT TRUNCATED AT 250 WORDS)     

Potassium currents induced by hydrostatic pressure modulate membrane potential and transmitter release in vestibular type II hair cells

Vestibular type II hair cells respond to increases in the hydrostatic pressure with pressure-dependent K⁺ currents. We examined whether such currents may modulate transmitter release (assessed as membrane capacitance increments) by altering membrane potentials and voltage-gated Ca²⁺ currents. Capacitance increments were dependent on voltage-gated Ca²⁺ influx. Stimulating currents (0.7 nA) in current clamp induced depolarisations that were more negative by 8.7 ± 2.1 mV when the bath height was elevated from 0.2 to 0.5 cm. In voltage clamp, protocols were used that simulated the time course of the membrane potential in current clamp at either low (control) or high hydrostatic pressure (high bath). The low bath protocol induced significantly larger Ca²⁺ currents and increases in capacitance than the high bath protocol. We conclude that pressure-dependent K⁺ currents may alter the voltage response of vestibular hair cells to an extent critical for Ca²⁺ currents and transmitter release. This mechanism may contribute to vestibular dysfunction in Meniere’s disease. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Duong Dinh and Haasler contributed equally to this work.     

TRPM2

TRPM2 is a cation channel enabling influx of Na+ and Ca2+, leading to depolarization and increases in the cytosolic Ca2+ concentration ([Ca2+]i). It is widely expressed, e.g. in many neurons, blood cells and the endocrine pancreas. Channel gating is induced by ADP-ribose (ADPR) that binds to a Nudix box motif in the cytosolic C-terminus of the channel. Endogenous ADPR concentrations in leucocytes are sufficiently high to activate TRPM2 in the presence of an increased [Ca2+]i but probably not at resting [Ca2+]i. Another channel activator is oxidative stress, especially hydrogen peroxide (H2O2) that may act through ADPR after ADPR polymers have been formed by poly(ADP-ribose) polymerases (PARPs) and hydolysed by glycohydrolases. H2O2-stimulated TRPM2 channels essentially contribute to insulin secretion in pancreatic beta-cells and alloxan-induced diabetes mellitus. Inhibition of TRPM2 channels may be achieved by channel blockers such as flufenamic acid or the anti-fungal agents clotrimazole or econazole. Selective blockers of TRPM2 are not yet available; those would be valuable for a characterization of biological roles of TRPM2 in various tissues and as potential drugs directed against oxidative cell damage, reperfusion injury or leucocyte activation. Activation of TRPM2 may be prevented by anti-oxidants, PARP inhibitors and glycohydrolase inhibitors. In future, binding of ADPR to the Nudix box may be targeted. In light of the wide-spread expression and growing list of cellular functions of TRPM2, useful therapeutic applications are expected for future drugs that block TRPM2 channels or inhibit their activation.