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Background and purposeThe calcium-activated potassium channel KCa3.1 is expressed in the vascular endothelium where its activation causes endothelial hyperpolarization and initiates endothelium-derived hyperpolarization (EDH)-dependent dilatation. Here, we investigated whether pharmacological activation of KCa3.1 dilates skeletal muscle arterioles and whether myoendothelial gap junctions formed by connexin40 (Cx40) are required for EDH-type dilatations and pressure depressor responses in vivo.Experimental approachWe performed intravital microscopy in the cremaster muscle microcirculation and blood pressure telemetry in Cx40-deficient mice.Key resultsIn wild-type mice, the KCa3.1-activator SKA-31 induced pronounced concentration-dependent arteriolar EDH-type dilatations, amounting to ∼40% of maximal dilatation, and enhanced the effects of ACh. These responses were absent in mice devoid of KCa3.1 channels. In contrast, SKA-31-induced dilatations were not attenuated in mice with endothelial cells deficient in Cx40 (Cx40(fl/fl):Tie2-Cre). In isolated endothelial cell clusters, SKA-31 induced hyperpolarizations of similar magnitudes (by ∼38 mV) in Cx40(fl/fl):Tie2-Cre, ubiquitous Cx40-deficient mice (Cx40(-/-)) and controls (Cx40(fl/fl)), which were reversed by the specific KCa3.1-blocker TRAM-34. In normotensive wild-type and Cx40(fl/fl):Tie2-Cre as well as in hypertensive Cx40(-/-) animals, i.p. injections of SKA-31 (30 and 100 mg·kg(-1)) decreased arterial pressure by ∼32 mmHg in all genotypes. The depressor response to 100 mg·kg(-1) SKA-31 was associated with a decrease in heart rate.Conclusions and implicationsWe conclude that endothelial hyperpolarization evoked by pharmacological activation of KCa3.1 channels induces EDH-type arteriolar dilatations that are independent of endothelial Cx40 and Cx40-containing myoendothelial gap junctions. As SKA-31 reduced blood pressure in hypertensive Cx40-deficient mice, KCa3.1 activators may be useful drugs for severe treatment-resistant hypertension.

Two private, heterozygous mutations in two functionally related genes, GUCY1A3 and CCT7, are identified in an extended family with myocardial infarction; these genes encode proteins that work together to inhibit platelet activation after nitric oxide stimulation, suggesting a link between impaired nitric oxide signalling and myocardial infarction risk. Signal failure raises cardiac disease risk Next-generation sequencing in families with multiple affected individuals has revolutionized the identification of rare and private (single family or limited group) mutations. Here, the authors identify two private, heterozygous mutations in two functionally related genes, GUCY1A3 and CCT7, in an extended family with myocardial infarction. The products of these genes work together to induce vasodilation and inhibit platelet activation upon stimulation with nitric oxide. Starting with a severely affected family, this work identifies a link between impaired nitric oxide signalling and myocardial infarction risk, possibly through accelerated thrombus formation. Reversal of this signalling defect may provide a new therapeutic strategy for reducing the risk of myocardial infarction. Myocardial infarction, a leading cause of death in the Western world.sup.1, usually occurs when the fibrous cap overlying an atherosclerotic plaque in a coronary artery ruptures. The resulting exposure of blood to the atherosclerotic material then triggers thrombus formation, which occludes the artery.sup.2. The importance of genetic predisposition to coronary artery disease and myocardial infarction is best documented by the predictive value of a positive family history.sup.3. Next-generation sequencing in families with several affected individuals has revolutionized mutation identification.sup.4. Here we report the segregation of two private, heterozygous mutations in two functionally related genes, GUCY1A3 (p.Leu163Phefs*24) and CCT7 (p.Ser525Leu), in an extended myocardial infarction family. GUCY1A3 encodes the [alpha]1 subunit of soluble guanylyl cyclase ([alpha]1-sGC).sup.5, and CCT7 encodes CCT[eta], a member of the tailless complex polypeptide 1 ring complex.sup.6, which, among other functions, stabilizes soluble guanylyl cyclase. After stimulation with nitric oxide, soluble guanylyl cyclase generates cGMP, which induces vasodilation and inhibits platelet activation.sup.7. We demonstrate in vitro that mutations in both GUCY1A3 and CCT7 severely reduce [alpha]1-sGC as well as [beta]1-sGC protein content, and impair soluble guanylyl cyclase activity. Moreover, platelets from digenic mutation carriers contained less soluble guanylyl cyclase protein and consequently displayed reduced nitric-oxide-induced cGMP formation. Mice deficient in [alpha]1-sGC protein displayed accelerated thrombus formation in the microcirculation after local trauma. Starting with a severely affected family, we have identified a link between impaired soluble-guanylyl-cyclase-dependent nitric oxide signalling and myocardial infarction risk, possibly through accelerated thrombus formation. Reversing this defect may provide a new therapeutic target for reducing the risk of myocardial infarction., Author(s): Jeanette Erdmann [sup.1] [sup.2] , Klaus Stark [sup.3] [sup.4] , Ulrike B. Esslinger [sup.3] [sup.5] , Philipp Moritz Rumpf [sup.6] [sup.7] , Doris Koesling [sup.8] , Cor de Wit [...]

Hypoxic pulmonary vasoconstriction (HPV) is a physiological mechanism by which pulmonary arteries constrict in hypoxic lung areas in order to redirect blood flow to areas with greater oxygen supply. Both oxygen sensing and the contractile response are thought to be intrinsic to pulmonary arterial smooth muscle cells. Here we speculated that the ideal site for oxygen sensing might instead be at the alveolocapillary level, with subsequent retrograde propagation to upstream arterioles via connexin 40 (Cx40) endothelial gap junctions. HPV was largely attenuated by Cx40-specific and nonspecific gap junction uncouplers in the lungs of wild-type mice and in lungs from mice lacking Cx40 ([Cx40.sup.-/-]). In vivo, hypoxemia was more severe in [Cx40.sup.-/-] mice than in wild-type mice. Real-time fluorescence imaging revealed that hypoxia caused endothelial membrane depolarization in alveolar capillaries that propagated to upstream arterioles in wild-type, but not [Cx40.sup.-/-], mice. Transformation of endothelial depolarization into vasoconstriction involved endothelial voltage-dependent α1G subtype [Ca.sup.2+] channels, cytosolic phospholipase [A.sub.2], and epoxyeicosatrienoic acids. Based on these data, we propose that HPV originates at the alveolocapillary level, from which the hypoxic signal is propagated as endothelial membrane depolarization to upstream arterioles in a Cx40-dependent manner., Introduction Hypoxic pulmonary vasoconstriction (HPV) is a fundamental physiological mechanism by which the lung optimizes ventilation/perfusion (V/Q) matching, redirecting blood flow from poorly to better ventilated areas (1). Yet in [...]

We previously found that deletion of connexin 40 (Cx40) causes a misdirection of renin-expressing cells from the media layer of afferent arterioles to the perivascular tissue, extraglomerular mesangium, and periglomerular and peritubular interstitium. The mechanisms underlying this aberrant renin expression are unknown. Here, we questioned the relevance of cyclooxygenase-2 (COX-2) activity for aberrant renin expression in Cx40-deficient kidneys. We found that COX-2 mRNA levels were increased three-fold in the renal cortex of Cx40-deficient kidneys relative to wild-type (wt) kidneys. In wt kidneys, COX-2 immunoreactivity was minimally detected in the juxtaglomerular region, but renin expression was frequently associated with COX-2 immunoreactivity in Cx40-deficient kidneys. Treatment with COX-2 inhibitors for 1 wk lowered renin mRNA levels in wt kidneys by about 40%. In Cx40-deficient kidneys, basal renin mRNA levels were increased two-fold relative to wt kidneys, and these elevated mRNA levels were reduced to levels of untreated wt mice by COX-2 inhibitors. In parallel, renin immunoreactive areas were clearly reduced by COX-2 inhibitors such that renin expression vanished and decreased significantly in the periglomerular and peritubular extensions. Notably, COX-2 inhibitor treatment lowered plasma renin concentration (PRC) in wt kidneys by about 40% but did not affect the highly elevated PRC levels in Cx40-deficient mice. These findings suggest that aberrant renin-producing cells in Cx40-deficient kidneys express significant amounts of COX-2, which contribute to renin expression in these ceils, in particular, those in the periglomerular and peritubular position. Apparently, these disseminated cells do not contribute to the enhanced renin secretion rates of Cx40-deficient kidneys. gap junctions; juxtaglomerular cell; prostaglandins

Vascular coordination in the microcirculation depends on gap junctional intercellular communication (GJIC), which is reflected by the conduction of locally initiated vasomotor responses. However, little is known about the regulation of GJIC in vivo. We hypothesized that endothelial NO regulates GJIC and therefore studied whether conduction of constrictions and dilations along the vessel wall is modulated by modifying the level of microcirculatory NO. Arterioles were focally stimulated using high [K.sup.+] or acetylcholine in the cremaster muscle in situ, and diameter changes were assessed at the local and remote upstream sites by intravital microscopy. Local stimulation with [K.sup.+] initiated a constriction that conducted along the arteriole with diminishing amplitude (length constant [lambda]: 371 [+ or -] 42 [micro]m). After [N.sup.[omega]]-nitro-L-arginine (L-NNA), [lambda] increased to 507 [+ or -] 30 [micro]m, indicating that GJIC is attenuated by endogenous NO. Exogenous NO, but not adenosine, reduced [lambda] after L-NNA in a reversible, concentration-dependent, and mainly cGMP-dependent manner as assessed by inhibition of soluble guanylate cyclase. In endothelial NO synthase-deficient mice, [lambda] was 530 [+ or -] 80 [micro]m and thus similar to that in wild-type mice after L-NNA. Exogenous NO likewise reduced [lambda] in these mice. The effects of NO were comparable to those of wild-type animals in Cx40-deficient mice, which excludes Cx40 as a specific target of NO. In contrast to constrictions, the amplitude of conducted dilations on acetylcholine did not diminish up to 1,300 [micro]m and were not altered by L-NNA or exogenous NO. We conclude that endogenously released NO attenuates the conduction of vasoconstrictions most likely due to a modulation of gap junctional conductivity. We suggest that this effect is specific for smooth muscle cells, which probably transmit constricting signals, and involves connexins other than Cx40. This mechanism may support the dilatory potency of NO by preventing the conduction of remote vasoconstrictions into areas with basal or activated NO release. conducted responses; gap junctional communication; connexins; nitric oxide doi:10.1152/ajpheart.01061.2006

Vasomotor reactions upon focal stimulation of arterioles have been shown to be conducted along the vascular wall. Such a conduction, which is assumed to reflect the spread of electrical signals, may contribute to coordination of responses within a vascular segment. We aimed to identify which endothelial autacoid(s) act as mediators of the local and conducted dilator responses, respectively. To this end, arterioles in the hamster cremaster microcirculation were locally stimulated with endothelium-dependent [acetylcholine (ACh)] or endothelium-independent dilators [sodium nitroprusside (SNP)], and the resulting changes in diameter were measured using a videomicroscopy technique at the site of application and up to 1.4 mm upstream at distant sites. Experiments were also performed after blockade of nitric oxide (NO) synthase, cyclooxygenase, P-450 monooxygenase, or [K.sup.+] channels. Dilations upon ACh (71 [+ or -] 3%) were conducted rapidly ( endothelium-dependent dilation; gap junctions; signal transmission; endothelium-derived hyperpolarizing factor

The function of endothelium-derived hyperpolarizing factor (EDHF) along the vascular tree under stimulated ACh or basal conditions was investigated in hamster muscles using intravital microscopy. It was found that Ach-induced hyperpolarization of the vascular smooth muscle was reduced by pentobarbital. Moreover, a considerable part of the ACh dilation in the microcirculation seems to be regulated by EDHF. Results also suggested that EDHF contributes to the control of the basal tone and is a product of the cytochrome P-450 pathway.