Intracellular recordings were made from short segments of the muscular wall of the guinea-pig gastric antrum. Preparations were impaled using two independent microelectrodes, one positioned in the circular layer and the other either in the longitudinal layer, in the network of myenteric interstitial cells of Cajal (ICC MY ) or in the circular layer. Cells in each layer displayed characteristic patterns of rhythmical activity, with the largest signals being generated by ICC MY . Current pulses injected into the circular muscle layer produced electrotonic potentials in each cell layer, indicating that the layers are electrically interconnected. The amplitudes of these electrotonic potentials were largest in the circular layer and smallest in the longitudinal layer. An analysis of electrical coupling between the three layers suggests that although the cells in each layer are well coupled to neighbouring cells, the coupling between either muscle layer and the network of ICC MY is relatively poor. The electrical connections between ICC MY and the circular layer did not rectify. In parallel immunohistochemical studies, the distribution of the connexins Cx40, Cx43 and Cx45 within the antral wall was determined. Only Cx43 was detected; it was widely distributed on ICC MY and throughout the circular smooth muscle layer, being concentrated around ICC IM , but was less abundant in the circular muscle layer immediately adjacent to ICC MY . Although the electrophysiological studies indicate that smooth muscle cells in the longitudinal muscle layer are electrically coupled to each other, none of the connexins examined were detected in this layer.

1. Gap junctions, which are comprised of members of a family of membrane proteins called connexins (Cx), permit the transfer of electrical and chemical information between adjacent cells in a wide variety of tissues. The aim of the present study was to compare the expression of Cx37, 40 and 43 in the smooth muscle and endothelium of a large elastic artery and two smaller muscular arteries of the rat. Serial section electron microscopy was also used to determine the presence of pentalaminar gap junctions in the smooth muscle and the incidence of myoendothelial gap junctions between the smooth muscle and endothelial cells in muscular arteries of different size. 2. Using immunohistochemistry, Cx37, 40 and 43 were found in the endothelium of the aorta, caudal and basilar arteries, with Cx43 being the least abundant. Connexin 43 was readily observed throughout the muscle layers of the aorta, but was not detected in the media of the caudal or basilar arteries. Connexin 40 was not detected in the media of any of the arteries, while very fine punctate staining was observed with Cx37 antibodies in the media of the caudal and basilar arteries, but not in the aorta. 3. Real-time polymerase chain reaction showed that the expression of mRNA for Cx43 was 15-fold greater in the aorta than in the caudal artery of the rat. 4. At the ultrastructural level, small pentalaminar gap junctions (< 100 nm) were found between the fine processes of adjacent smooth muscle cells and also between the smooth muscle and endothelial cells. The incidence of myoendothelial gap junctions in the mesenteric vascular bed and in the caudal artery increased as vessel size decreased. 5. In summary, heterogeneity exists within the vascular system with regard to the distribution of gap junctions and their constituent Cx. Such variation will have important consequences for the coordination and propagation of vascular responses. In muscular arteries, in comparison with elastic arteries, Cx37 may be more important than Cx43 for cell coupling within the smooth muscle layers. The correlation between the incidence of myoendothelial gap junctions and the role of endothelium-derived hyperpolarizing factor, relative to nitric oxide, in vasodilatory responses suggests that myoendothelial gap junctions play an important physiological role in the regulation of vascular tone.

We previously reported that NADPH oxidase activity is greater in intracranial cerebral versus systemic arteries of the rat. Here, we first tested whether NADPH oxidase activity is also greater in intracranial cerebral than systemic arteries of three other animal species, i.e., mouse, rabbit, and pig. Second, using Nox2-deficient mice, we evaluated the involvement of Nox2-containing NADPH oxidases in any such regional differences. NADPH-stimulated superoxide (O2−) production by basilar, middle cerebral arteries (MCA), and common carotid arteries (CA) and thoracic aorta (AO) from rat, mouse, rabbit, and pig was measured using lucigenin-enhanced chemiluminescence. Basal production of O2− and hydrogen peroxide (H2O2) by cerebral arteries, AO, and CA from wild-type (WT) and Nox2−/− mice was measured using L-012-enhanced chemiluminescence and Amplex Red fluorescence, respectively. Western blotting was used to measure Nox2 and SOD1–3 protein expression, and immunofluorescence was used to localize Nox2, in mouse arteries. In rats, WT mice, rabbits, and pigs, NADPH-stimulated O2− production by cerebral arteries was up to 40-fold greater than that in AO and CA. In WT mice, basal O2− and H2O2 production by cerebral arteries was ninefold and ∼2.5-fold higher, respectively, than that in AO and CA and was associated with ∼40% greater expression of Nox2 protein. Nox2 immunofluorescence was localized to the endothelium, and to a lesser extent the adventitia, in all mouse arteries and appeared to be more intense in endothelium of MCA than AO or CA. In Nox2−/− mice, NADPH-stimulated O2− production by cerebral arteries was ∼35% lower than that in WT mice, whereas Nox2 deletion had no significant effect on O2− production by AO or CA. Thus NADPH oxidase activity is greater in intracranial cerebral versus systemic arteries of several animal species and is associated with higher cerebrovascular expression and activity of Nox2.

Objective-To determine the connexins (Cxs) involved in intercellular coupling within vascular muscle, the present study has quantified mRNA and protein expression for Cx37, Cx40, Cx43, and Cx45 in the caudal artery (CA) and thoracic aorta (ThA) of the rat. Methods and Results-Real-time polymerase chain reaction and immunohistochemistry identified Cx37 as the most abundantly expressed Cx in the CA, with fine punctate staining observed in the media. Conversely, mRNA for Cx43 was 40-fold greater in the ThA than in the CA, with punctate staining in the endothelium and media of the ThA but confined to the endothelium in the CA. Western blotting confirmed the differences in the relative amounts of Cx43 between the 2 vessels. For both arteries, Cx45 was expressed to a lesser degree in the media but not in the endothelium, whereas Cx40 was found only in the endothelium. Cx37, Cx40, and Cx43 were expressed in the endothelium of both vessels, although the density of Cx40 plaques was significantly greater in the CA. Conclusions-The demonstration of Cx37 as the dominant Cx in the media of the CA highlights the potential heterogeneity in

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