Endothelial coordination of cerebral vasomotion via myoendothelial gap junctions containing connexins 37 and 40

Abstract

Control of cerebral vasculature differs from that of systemic vessels outside the blood-brain barrier. The hypothesis that the endothelium modulates vasomotion via direct myoendothelial coupling was investigated in a small vessel of the cerebral circulation. In the primary branch of the rat basilar artery, membrane potential, diameter, and calcium dynamics associated with vasomotion were examined using selective inhibitors of endothelial function in intact and endothelium-denuded arteries. Vessel anatomy, protein, and mRNA expression were studied using conventional electron microscopy high-resolution ultrastructural and confocal immunohistochemistry and quantitative PCR. Membrane potential oscillations were present in both endothelial cells and smooth muscle cells (SMCs), and these preceded rhythmical contractions during which adjacent SMC intracellular calcium concentration ([Ca 2ϩ ]i) waves were synchronized. Endothelium removal abolished vasomotion and desynchronized adjacent smooth muscle cell [Ca 2ϩ ]i waves. N G -nitro-L-arginine methyl ester (10 M) did not mimic this effect, and dibutyryl cGMP (300 M) failed to resynchronize [Ca 2ϩ ]i waves in endothelium-denuded arteries. Combined charybdotoxin and apamin abolished vasomotion and depolarized and constricted vessels, even in absence of endothelium. Separately, 37,43 Gap27 and 40 Gap27 abolished vasomotion. Extensive myoendothelial gap junctions (3 per endothelial cell) composed of connexins 37 and 40 connected the endothelial cell and SMC layers. Synchronized vasomotion in rat basilar artery is endothelium dependent, with [Ca 2ϩ ]i waves generated within SMCs being coordinated by electrical coupling via myoendothelial gap junctions.connexin; electron microscopy; endothelial function; potassium channel; membrane potential VASOMOTION is an intrinsic feature of many arteries and arterioles under normal and pathological conditions and may contribute to regulation of blood flow and pressure (1,14). Both spontaneous and agonist-induced vasomotion occur when oscillations in the concentration of intracellular calcium ([Ca 2ϩ ] i ) become synchronized among adjacent smooth muscle cells, giving rise to global oscillations in [Ca 2ϩ ] i across the vessel wall (13,15,26,27,33,41,(47)(48)(49).There is clear heterogeneity in the mechanisms that underlie vasomotion, and specifically whether the endothelium plays a mandatory or modulatory role. In the rat iris arteriole, rabbit ear, and superior mesenteric artery, vasomotion is reported to be endothelium independent (3, 18, 39), whereas in the rat mesenteric, rabbit femoral artery, and hamster aorta, vasomotion is suggested to be endothelium dependent, as shown by its abolition with endothelium denudation (12,21,34,38,40).Endothelium-dependent effects on vasomotion have been attributed to the vasodilatory factors nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF). In some studies, endothelial cells play an essential role in synchronizing the activity of smooth muscle cells through the rel...

Obesity Up-Regulates Intermediate Conductance Calcium-Activated Potassium Channels and Myoendothelial Gap Junctions to Maintain Endothelial Vasodilator Function

“…E, MEGJs are ϳ12-fold higher in density in obese compared with control. As found previously for some MEGJ-associated projections in rat caudal cerebellar and mesenteric artery (Sandow et al, 2002Haddock et al, 2006), the endothelial "head" of such MEGJ-associated projections is multilobed (examples with asterisks, A and B); perhaps providing greater surface area for localized Ca 2ϩ and K ϩ channel activity compared with nonlobed projections. Such sites have characteristic pentalaminar MEGJ morphology (arrows and insets) and apparent localized IK Ca (IK1) densities (Fig.…”

“…In this regard, in addition to gap junction effects, carbenoxolone and Gap peptides have nongap junction vascular ion channel and putative calcium store activity (Coleman et al, 2001;Tare et al, 2002;Haddock et al, 2006;Dora et al, 2008). Indeed, volume-regulated anion channel block with carbenoxolone mimics Cx43 block in astroglial cells (Benfenati et al, 2009), in a similar manner to its action in rat mesenteric artery (compare carbenoxolone data and interpretation in Tare et al, 2002;Dora et al, 2008).…”

“…The distribution of SK Ca and IK Ca was examined in the rat saphenous artery by using conventional wholemount confocal immunohistochemistry (Mather et al, 2005;Haddock et al, 2006). Rats were perfused via the left ventricle with a clearing solution of 0.1% bovine serum albumin, 0.1% NaNO 3 , and 10 U/ml heparin to fully dilate the vessels and fixed with 2% paraformaldehyde in 0.1 M PBS.…”

Reduced EDHF responses and connexin activity in mesenteric arteries from the insulin-resistant obese Zucker rat

“…Gap peptides have been shown to inhibit the hyperpolarisation induced by endothelium-dependent vasodilators in rat mesenteric [39] and saphenous [6] arteries, and inhibit relaxations to acetylcholine in rabbit central ear arteries [40]. Additionally, gap peptides have been shown to abolish gap junctionmediated vasomotion in rat basilar arteries [41]. Studies have also shown that some tissues require a combination of gap peptides to cause significant attenuation of the relaxation response [10], with individual peptides observed to have either no or negligible effects.…”

Function and Regulation of Pannexin 1 Channels in the Vascular Endothelium

“…The arteries were mounted in DAPI and coverslip sealed to provide a flat surface for confocal imaging. Autofluorescence of the internal elastic lamina between the endothelium and smooth muscle was readily apparent with "holes" between the two layers of cells which has been extensively documented (e.g., [212][213][214]). In our study, the detection of punctate fluorescence in >25% of the "holes" was considered indicative of protein localization to MEJs.…”