Endothelial cell (EC) Ca 2+ -activated K channels (SK Ca and IK Ca channels) generate hyperpolarization that passes to the adjacent smooth muscle cells causing vasodilation. IK Ca channels focused within EC projections toward the smooth muscle cells are activated by spontaneous Ca 2+ events (Ca 2+ puffs/pulsars). We now show that transient receptor potential, vanilloid 4 channels (TRPV4 channels) also cluster within this microdomain and are selectively activated at low intravascular pressure. In arterioles pressurized to 80 mmHg, ECs generated low-frequency (∼2 min −1 ) inositol 1,4,5-trisphosphate receptor-based Ca 2+ events. Decreasing intraluminal pressure below 50 mmHg increased the frequency of EC Ca 2+ events twofold to threefold, an effect blocked with the TRPV4 antagonist RN1734. These discrete events represent both TRPV4-sparklet-and nonsparklet-evoked Ca 2+ increases, which on occasion led to intracellular Ca 2+ waves. The concurrent vasodilation associated with increases in Ca 2+ event frequency was inhibited, and basal myogenic tone was increased, by either RN1734 or TRAM-34 (IK Ca channel blocker), but not by apamin (SK Ca channel blocker). These data show that intraluminal pressure influences an endothelial microdomain inversely to alter Ca 2+ event frequency; at low pressures the consequence is activation of EC IK Ca channels and vasodilation, reducing the myogenic tone that underpins tissue blood-flow autoregulation.endothelial cell calcium | cremaster arterioles | mesenteric arteries C a 2+ -activated K + (K Ca ) channels in arteriolar endothelial cells (ECs) are activated by intrinsic spontaneous or receptormediated Ca 2+ events, each leading to hyperpolarization of smooth muscle cells (SMCs) and vasodilation independent of nitric oxide or prostacyclin-the endothelium-dependent hyperpolarization (EDH) response. This hyperpolarization spreads both radially and longitudinally through the vascular wall via patent gap junctions to evoke local and conducted dilation, and it is central to cardiovascular function (1, 2).EDH is the predominant endothelium-dependent mechanism in smaller "resistance" arteries and arterioles. The underlying hyperpolarization is generated by two subtypes of K Ca channels found in the EC, but not SMC, membrane, the small (SK Ca ,K Ca 2.3) and intermediate (IK Ca, K Ca 3.1) conductance forms that may be activated independently of each other (3). The physiological importance of independent activation is apparent from studies with K Ca 3.1-deficient mice in which the mean blood pressure is raised by ∼7 mmHg, but further elevated by disrupting both K Ca channels (4). In mesenteric resistance arteries, IK Ca channels are focused within EC projections through the internal elastic lamina (IEL) termed myoendothelial junctions (MEJs). MEJs can contain gap junctions (MEGJs) coupling ECs to SMCs, and EDH can spread by direct electrical coupling and/or a diffusible factor (5, 6). The IK Ca channels enriched within MEJs can be activated by spontaneous inositol 1,4,5-trisphosphate (IP 3 ...

, via competing with nitric oxide (NO) synthase for the substrate L-arginine, may interfere with NO-mediated vascular responses. We tested the hypothesis that arginase 1 contributes to coronary vasomotor dysfunction in patients with diabetes mellitus (DM). Coronary arterioles were dissected from the right atrial appendages of 41 consecutive patients with or without DM (the 2 groups suffered from similar comorbidities), and agonist-induced changes in diameter were measured with videomicroscopy. We found that the endothelium-dependent agonist ACh elicited a diminished vasodilation and caused constriction to the highest ACh concentration (0.1 M) with a similar magnitude in patients with (18 Ϯ 8%) and without (17 Ϯ 9%) DM. Responses to ACh were not significantly affected by the inhibition of NO synthesis with N G -nitro-L-arginine methyl ester in either group. The NO donor sodium nitroprusside-dependent dilations were not different in patients with or without DM. Interestingly, we found that the presence of N G -hydroxy-L-arginine (10 M), a selective inhibitor of arginase or application of L-arginine (3 mM), restored ACh-induced coronary dilations only in patients with DM (to 47 Ϯ 6% and to 40 Ϯ 19%, respectively) but not in subjects without DM. Correspondingly, the protein expression of arginase 1 was increased in coronary arterioles of patients with DM compared with subjects without diabetes. Moreover, using immunocytochemistry, we detected an abundant immunostaining of arginase 1 in coronary endothelial cells of patients with DM, which was colocalized with NO synthase. Collectively, we provided evidence for a distinct upregulation of arginase 1 in coronary arterioles of patients with DM, which contributes to a reduced NO production and consequently diminished vasodilation. coronary microvessel; endothelium; endothelial nitric oxide synthase PATIENTS WITH DIABETES exhibit endothelial dysfunction, which is characterized by an impaired flow-and acetylcholine (ACh)-induced, endothelium-dependent relaxation of brachial artery (8) and forearm resistance vessels (36). Kaneda et al. (17) performed a study in which 165 patients underwent intracoronary injection of ACh and found that diabetes was the strongest predictor for ACh-induced vasospasm, a response that indicates coronary endothelial dysfunction. This and other studies indicated that diabetes is also associated with an impaired dilator function of coronary arteries, and this is manifested and measured as a reduced vasodilator or even vasoconstrictor response to ACh (5, 23). Our previous studies have demonstrated that coronary arterioles isolated from animals with experimental diabetes also exhibit impaired ACh-induced dilation, which is primarily due to the reduced synthesis and/or availability of nitric oxide (NO) (1, 2, 9, 16). The exact mechanism(s) responsible for the diminished NO production in human diabetes is still incompletely understood.L-Arginine, the substrate for NO synthase, is the precursor for NO synthesis in the vascular endothelium. Experimenta...

Thus, under normal conditions, Cav-1 limits the contribution of the BK(Ca) channel to EDHF-mediated arteriolar dilation. In obesity, a reduced expression of Cav-1 leads to greater contribution of the BK(Ca) channel to EDHF-mediated response, which seems essential for maintained coronary dilation.

In small mesenteric arteries of ApoE(-/-) mice, NO-independent dilation is enhanced. Since both NO-dependent and -independent pathways can stimulate local and conducted dilation, the potential for reducing vascular resistance is improved in these vessels.

Objective-To assess the influence of blocking smooth muscle large conductance Ca 2+ -activated K + channels and voltage-gated K + channels on the conducted dilation to ACh and isoproterenol.Materials and Methods-Rat mesenteric arteries were isolated with a bifurcation, triplecannulated, pressurized and imaged using confocal microscopy. Phenylephrine was added to the superfusate to generate tone, and agonists perfused into a sidebranch to evoke local dilation and subsequent conducted dilation into the feed artery.Results-Both ACh− and isoproterenol-stimulated local and conducted dilation with similar magnitudes of decay with distance along the feed artery (2000 µm: ~15% maximum dilation). The gap junction uncoupler carbenoxolone prevented both conducted dilation and intercellular spread of dye through gap junctions. IbTx, TEA or 4-AP, blockers of large conductance Ca 2+ -activated K + channels and voltage-gated K + channels, did not affect conducted dilation to either agonist. A combination of either IbTx or TEA with 4-AP markedly improved the extent of conducted dilation to both agonists (2000 µm: >50% maximum dilation). The enhanced conducted dilation was reflected in the hyperpolarization to ACh (2000 µm: Control, 4 ± 1 mV, n = 3; TEA with 4-AP, 14 ± 3mV, n = 4), and was dependent on the endothelium.Conclusions-These data show that activated BK Ca and K V -channels serve to reduce the effectiveness of conducted dilation.

We hypothesized that under high glucose conditions, activation of the hexosamine pathway leads to impaired nitric oxide (NO)-dependent arteriolar dilation. Skeletal muscle arterioles (diameter: ~160 μm) isolated from male Wistar rats were exposed to normal glucose (NG, 5.5 mmol/L) or high glucose concentrations (HG, 30 mmol/L, for 2 h) and agonist-induced diameter changes were measured with videomicroscopy. Western blots were performed to identify the vascular levels of protein O-linked-N-acetyl-glucosamine (O-GlcNAc) and phosphorylated endothelial NO synthase (eNOS). In arterioles exposed to HG, dilations to histamine were abolished compared to those exposed to NG (max: −6±6% and 69±9%, respectively), while acetylcholine-induced responses were not affected. Inhibition of NO synthesis with NG-nitro-L-arginine methyl ester (L-NAME) reduced histamine-induced dilations in NG arterioles, but it had no effect on microvessels exposed to HG. Dilations to the NO donor, sodium nitroprusside and constrictions to norepinephrine and serotonin were similar in the two groups. In the presence of the inhibitor of hexosamine pathway, azaserine, histamine-induced dilations were significantly augmented in arterioles exposed to HG (max: 67±2%). Moreover, exposure of vessels to glucosamine (5 mmol/L, for 2 h) resulted in reduced histamine-induced arteriolar dilations (max: 26±3%). The level of protein O-GlcNAcylation was increased, whereas the P-eNOS (Ser-1177) was decreased in HG exposed vessels. These findings indicate that a high concentration of glucose may lead to glucosamine formation, which impairs histamine-induced, NO-mediated arteriolar dilations. We propose that interfering with the hexosamine pathway may prevent microvascular complications in diabetes.

Aims Coronary microvascular smooth muscle cells (SMCs) respond to luminal pressure by developing myogenic tone (MT), a process integral to the regulation of microvascular perfusion. The cellular mechanisms underlying poor myogenic reactivity in patients with heart valve disease are unknown and form the focus of this study. Methods and Results Intramyocardial coronary micro-arteries (IMCAs) isolated from human and pig right atrial appendage (RA) and left ventricular (LV) biopsies were studied using pressure myography combined with confocal microscopy. All RA- and LV-IMCAs from organ donors and pigs developed circa 25% MT. In contrast, 44% of human RA-IMCAs from 88 patients with heart valve disease had poor (<10%) MT yet retained cell viability and an ability to raise cytoplasmic Ca2+ in response to vasoconstrictor agents. Comparing across human heart chambers and species we found that based on patient medical history and six tests, the strongest predictor of poor MT in IMCAs was increased expression of the synthetic marker caldesmon relative to the contractile marker SM-myosin heavy chain. In addition, high resolution imaging revealed a distinct layer of longitudinally-aligned SMCs between ECs and radial SMCs, and we show poor MT was associated with disruptions in these cellular alignments. Conclusions These data demonstrate the first use of atrial and ventricular biopsies from patients and pigs to reveal that impaired coronary MT reflects a switch of viable SMCs towards a synthetic phenotype, rather than a loss of SMC viability. These arteries represent a model for further studies of coronary microvascular contractile dysfunction.

Abstract:Type 2 diabetes mellitus is associated with clustering of cardiovascular risk factors that may greatly increase individuals' risk of developing coronary artery disease. Type 2 diabetes is believed to impair coronary function. However, its impact on the vasomotor function of coronary resistance vessels in humans is still debated. Reduced, preserved or even augmented dilations of coronary arterioles have been reported in subjects with type 2 diabetes. Interestingly, recent studies have suggested that reactive oxygen species (ROS), particularly hydrogen peroxide, may compensate for the loss of the vasodilatory function of coronary microvessels during disease development. Recent interventional clinical trials have yielded largely negative results, and there has even been some suggestion of harm caused by attempts to reduce ROS. Thus, it is possible that interference with ROS-related signaling might paradoxically temper the function of coronary microvessels, predisposing patients to myocardial ischemia. In this review, we aim to highlight current findings supporting a potential role for ROS in preserving coronary arteriolar dilation in type 2 diabetes mellitus.