Objective-Despite the role that extracellular matrix (ECM) plays in vascular signaling, little is known of the complex structural arrangement between specific ECM proteins and vascular smooth muscle cells. Our objective was to examine the hypothesis that adventitial elastin fibers are dominant in vessels subject to longitudinal stretch. Methods and Results-Cremaster muscle arterioles were isolated, allowed to develop spontaneous tone, and compared with small cerebral arteries. 3D confocal microscopy was used to visualize ECM within the vessel wall. Pressurized arterioles were fixed and stained with Alexa 633 hydrazide (as a nonselective ECM marker), anti-elastin, or anti-type 1 collagen antibody and a fluorescent nuclear stain. Exposure of cremaster muscle arterioles to elastase for 5 minutes caused an irreversible lengthening of the vessel segment that was not observed in cerebral arteries. Longitudinal elastin fibers were demonstrated on cremaster muscle arterioles using 3D imaging but were confirmed to be absent in cerebral vessels. The fibers were also distinct from type I collagen fibers and were degraded by elastase treatment. Key Words: cell physiology Ⅲ extracellular matrix Ⅲ microcirculation Ⅲ vascular biology Ⅲ elastin T he extracellular matrix (ECM) contains a number of proteins, including collagen, elastin, laminin, fibronectin, vitronectin, glycoproteins, and proteoglycans. In addition to providing a mechanically dynamic structural scaffold, the ECM is involved in physiological processes such as cell growth, differentiation, and migration. With respect to the vasculature, recent studies have demonstrated a number of these ECM proteins to actively signal through outside-in means into both vascular smooth muscle and endothelial cells, particularly conveying mechanical signals. For example, fibronectin binding through cell surface integrins modulates the activity of smooth muscle cell (SMC) ion channels (voltage-gated Ca 2ϩ channels and large conductance Ca 2ϩ -activated K ϩ channels) 1-3 and affects local cellular contractions. 4 Similarly, ECM protein-integrin activation of various intracellular signaling mechanisms underlies endothelial cell mechanotransduction to stimuli such as shear stress. 5,6 Despite these demonstrated roles of the ECM in vascular cell signaling, relatively little is known of the complexities of the in situ arrangement between specific ECM proteins and arteriolar SMCs. Given the above examples, it is likely that the structural arrangement of the vessel wall ECM proteins, particularly at the microvascular level, has an impact on how local mechanical forces are transmitted, sensed, and responded to and, ultimately, how effectively a vessel is able to alter its diameter. Conclusion-TheseAdding to difficulties in understanding the complexity of the extracellular components of the vessel wall is an apparent regional heterogeneity. Importantly, regional differences involve both variation in matrix composition and structural arrangement. Thus, in contrast to skeletal muscle arterioles...

Matchkov VV, Moeller-Nielsen N, Dam VS, Nourian Z, Boedtkjer DM, Aalkjaer C. The ␣2 isoform of the Na,K-pump is important for intercellular communication, agonist-induced contraction, and EDHF-like response in rat mesenteric arteries. Am J Physiol Heart Circ Physiol 303: H36 -H46, 2012. First published May 4, 2012 doi:10.1152/ajpheart.00673.2011The specific role of different isoforms of the Na,K-pump in the vascular wall is still under debate. We have previously suggested that the ␣2 isoform of the Na,K-pump (␣2), Na ϩ , Ca 2ϩ -exchange (NCX), and connexin43 form a regulatory microdomain in smooth muscle cells (SMCs), which controls intercellular communication and contractile properties of the vascular wall. We have tested this hypothesis by downregulating ␣2 in cultured SMCs and in small arteries with siRNA in vivo. Intercellular communication was assessed by using membrane capacitance measurements. Arteries transfected in vivo were tested for isometric and isobaric force development in vitro; [Ca 2ϩ ]i was measured simultaneously. Cultured rat SMCs were well-coupled electrically, but 10 M ouabain uncoupled them. Downregulation of ␣ 2 reduced electrical coupling between SMCs and made them insensitive to ouabain. Downregulation of ␣2 in small arteries was accompanied with significant reduction in NCX expression. Acetylcholine-induced relaxation was not different between the groups, but the endotheliumdependent hyperpolarizing factor-like component of the response was significantly diminished in ␣2-downregulated arteries. Micromolar ouabain reduced in a concentration-dependent manner the amplitude of norepinephrine (NE)-induced vasomotion. Sixty percent of the ␣ 2-downregulated arteries did not have vasomotion, and vasomotion in the remaining 40% was ouabain insensitive. Although ouabain increased the sensitivity to NE in the control arteries, it had no effect on ␣ 2-downregulated arteries. In the presence of a low NE concentration the ␣ 2-downregulated arteries had higher [Ca 2ϩ ]i and tone. However, the NE EC50 was reduced under isometric conditions, and maximal contraction was reduced under isometric and isobaric conditions. The latter was caused by a reduced Ca 2ϩ -sensitivity. The ␣2-downregulated arteries also had reduced contraction to vasopressin, whereas the contractile response to high K ϩ was not affected. Our results demonstrate the importance of ␣ 2 for intercellular coupling in the vascular wall and its involvement in the regulation of vascular tone. Na ϩ , Ca 2ϩ exchanger; smooth muscle cell synchronization; short interfering RNA; norepinephrine contractility; endothelium-dependent hyperpolarizing factor THE ELECTROGENIC NA,K-PUMP modifies numerous cellular pathways by modulating Na ϩ -coupled transport. The functional significance of the Na,K-pump is dependent upon the isoform. The catalytic ␣-subunit exists in three isoforms (5). These isoforms have different affinities for cardiac glycosides, kinetics, and regulation but show high structural homology and are difficult to distinguish at a fu...

Key points• The plasma membrane large-conductance Ca 2+ -activated, K + channel (BK Ca ) is a major ion channel contributing to the regulation of membrane potential.• Activation of large-conductance Ca 2+ -activated K + channel by both depolarization and increased intracellular Ca 2+ results in hyperpolarization that acts to limit agonist and mechanically induced vasoconstriction in small arteries.• Using patch-clamp techniques we demonstrate that regional differences exist in how BK Ca is regulated, particularly with respect to its Ca 2+ sensitivity.• Using single-channel recordings and siRNA to manipulate protein subunit expression, it is argued that the β1-subunit plays a more dominant role in cerebral blood vessels as compared with small arteries from skeletal muscle.• Subtle differences in the regulation of membrane potential in different vascular beds allow local blood flow and pressure to be closely adapted to the tissue's metabolic needs.Abstract β1-Subunits enhance the gating properties of large-conductance Ca 2+ -activated K + channels (BK Ca ) formed by α-subunits. In arterial vascular smooth muscle cells (VSMCs), β1-subunits are vital in coupling SR-generated Ca 2+ sparks to BK Ca activation, affecting contractility and blood pressure. Studies in cremaster and cerebral VSMCs show heterogeneity of BK Ca activity due to apparent differences in the functional β1-subunit:α-subunit ratio. To define these differences, studies were conducted at the single-channel level while siRNA was used to manipulate specific subunit expression. β1 modulation of the α-subunit Ca 2+ sensitivity was studied using patch-clamp techniques. BK Ca channel normalized open probability (NP o ) versus membrane potential (V m ) curves were more left-shifted in cerebral versus cremaster VSMCs as cytoplasmic Ca 2+ was raised from 0.5 to 100 μM. Calculated V 1/2 values of channel activation decreased from 72.0 ± 6.1 at 0.5 μM Ca 2+ i to −89 ± 9 mV at 100 μM Ca VSMCs. Functionally, this leads both to higher Ca 2+ sensitivity and NP o for BK Ca channels in the cerebral vasculature relative to that of skeletal muscle.

Our findings provide a new mechanism for hypoosmotic-stress-induced cardiomyocyte Ca2+ entry and cell damage in the aged heart. These finding have potential implications in treatment of elderly populations at increased risk of myocardial infarction and I/R injury.