10 research outputs found
Role of smooth muscle cell hyperpolarization in modulation of endothelial cell spontaneous Ca2+ events
No full textActivation of hexosamine pathway impairs nitric oxide (NO)-dependent arteriolar dilations by increased protein O-GlcNAcylation.
No full textWe hypothesized that under high glucose conditions, activation of the hexosamine pathway leads to impaired nitric oxide (NO)-dependent arteriolar dilation. Skeletal muscle arterioles (diameter: ~160μm) isolated from male Wistar rats were exposed to normal glucose (NG, 5.5mmol/L) or high glucose concentrations (HG, 30mmol/L, for 2h) 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 N(G)-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 (5mmol/L, for 2h) 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
Increased permeability of pulmonary arterioles after cardiopulmonary bypass
No full textPulmonary injury is a common complication after cardiopulmonary bypass (CPB) in patients undergoing cardiac surgery. The major causes of tissue damage arise from systemic inflammatory responses, and/or factors released by the ischaemic collapsed lungs. To date, there is no evidence to demonstrate whether this CPB-related pulmonary injury can influence both the permeability and function of the pulmonary arterioles. We developed a method to isolate, cannulate and pressurize porcine pulmonary arterioles to study their vasomotor function, the leak of fluorescent dyes through the arterial wall, and the expression of key proteins. All arterioles were imaged using linescan confocal microscopy. The fluorescent indicators carboxyfluorescein (CF, 0.1 µM, mw 376.3) or FITC-dextran (1 µM, mw 59,000 - 77,000) were perfused into the lumen of arterioles, and increases in fluorescence immediately outside the arteriolar wall (permeability) measured. Tight junction (Zo-1) and caspase 3 staining was performed on the same arterioles, and imaged while still cannulated, to assess the continuity of cell-cell connections and the onset of apoptosis. Arterioles were studied from pigs which had undergone CPB or sham surgery under anaesthesia and categorized into 4 groups: control (lungs collapsed to air with no ventilation during CPB, equivalent to the current method in patients); IPP-ILFV (intermittent pulmonary perfusion with intermittent low frequency ventilation during CPB); IPP-CLFV (intermittent pulmonary perfusion with continuous low frequency ventilation during CPB); and sham (chest opened only). From each group two samples were obtained, one soon after median sternotomy (baseline) and one at 1 hour of recovery after surgery (reperfusion). All baseline and sham pulmonary arterioles had low permeability to CF and FITC-dextran. When studying reperfused arterioles, the arterioles studied following IPP-ILFV and IPP-CLFV were less permeable to CF when compared to control arterioles. The permeability to FITC-dextran was not increased in any group studied, although a TRPV4 agonist did increase permeability to dextran. Furthermore, tight junction staining showed continuous, uniform cell-cell connections in the endothelium of all baseline arterioles. Interestingly, and consistent with the CF leak data, reperfused lung arterioles from the control group did appear to have gaps in tight junction staining and some caspase 3 staining was evident. These data suggest that cardiopulmonary bypass and concomitant lung collapse leads to increased permeability of pulmonary arterioles due to damaged cell-cell adhesions, which can be prevented by intermittent pulmonary perfusion with low frequency ventilation of the lungs during CPB
A novel role for spontaneous endothelial cell calcium activity in the vascular myogenic response
No full textSmooth muscle Ca(2+) -activated and voltage-gated K+ channels modulate conducted dilation in rat isolated small mesenteric arteries.
No full textOBJECTIVE: 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, triple-cannulated, 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 (2000μm: ∼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 (2000μm: >50% maximum dilation). The enhanced conducted dilation was reflected in the hyperpolarization to ACh (2000μm: 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
Enhanced K +-channel-mediated endothelium-dependent local and conducted dilation of small mesenteric arteries from ApoE -/- mice
No full textAimsAgonists that evoke smooth muscle cell hyperpolarization have the potential to stimulate both local and conducted dilation. We investigated whether the endothelium-dependent vasodilators acetylcholine (ACh) and SLIGRL stimulated conducted dilation and whether this was altered by deficiency in apolipoprotein E (ApoE -/-).Methods and resultsIsolated mesenteric arteries were cannulated, pressurized, and precontracted with phenylephrine. Agonists were either added to the bath to study local dilation or were restricted to one end of arteries to study conducted dilation. An enhanced sensitivity to both ACh and SLIGRL was observed in mesenteric arteries from ApoE -/- mice compared with wild-type controls. Inhibition of nitric oxide (NO) synthase blocked ACh responses, but had no effect on maximum dilation to SLIGRL. SLIGRL increased endothelial cell Ca 2, hyperpolarized smooth muscle cells, and fully dilated arteries. The NO-independent dilation to SLIGRL was blocked with high [KCl] or Ca 2-activated K +-channel blockers. The hyperpolarization and dilation to SLIGRL passed through the artery to at least 2.5 mm upstream. The conducted dilation was not affected by a deficit in ApoE and could also be stimulated by ACh, suggesting NO itself could stimulate conducted dilation.ConclusionIn 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. © 2011 The Author
Arginase 1 contributes to diminished coronary arteriolar dilation in patients with diabetes.
No full textArginase 1, 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 μM) with a similar magnitude in patients with (18 ± 8%) and without (17 ± 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 μM), a selective inhibitor of arginase or application of L-arginine (3 mM), restored ACh-induced coronary dilations only in patients with DM (to 47 ± 6% and to 40 ± 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
Signaling and structures underpinning conducted vasodilation in human and porcine intramyocardial coronary arteries
No full textBACKGROUND: Adequate blood flow into coronary micro-arteries is essential for myocardial function. Here we assess the mechanisms responsible for amplifying blood flow into myogenically-contracting human and porcine intramyocardial micro-arteries ex vivo using endothelium-dependent and -independent vasodilators. METHODS: Human and porcine atrial and ventricular small intramyocardial coronary arteries (IMCAs) were studied with pressure myography and imaged using confocal microscopy and serial section/3-D reconstruction EM. RESULTS: 3D rendered ultrastructure images of human right atrial (RA-) IMCAs revealed extensive homo-and hetero-cellular contacts, including to longitudinally-arranged smooth muscle cells (l-SMCs) found between the endothelial cells (ECs) and radially-arranged medial SMCs (r-SMCs). Local and conducted vasodilatation followed focal application of bradykinin in both human and porcine RA-IMCAs, and relied on hyperpolarization of SMCs, but not nitric oxide. Bradykinin initiated asynchronous oscillations in endothelial cell Ca(2+) in pressurized RA-IMCAs and, as previously shown in human RA-IMCAs, hyperpolarized porcine arteries. Immunolabelling showed small- and intermediate-conductance Ca(2+)-activated K(+) channels (K(Ca)) present in the endothelium of both species, and concentration-dependent vasodilation to bradykinin followed activation of these K(Ca) channels. Extensive electrical coupling was demonstrated between r-SMCs and l-SMCs, providing an additional pathway to facilitate the well-established myoendothelial coupling. Conducted dilation was still evident in a human RA-IMCA with poor myogenic tone, and heterocellular contacts were visible in the 3D reconstructed artery. Hyperpolarization and conducted vasodilation was also observed to adenosine which, in contrast to bradykinin, was sensitive to combined block of ATP-sensitive (K(ATP)) and inwardly rectifying (K(IR)) K(+) channels. CONCLUSIONS: These data extend our understanding of the mechanisms that coordinate human coronary microvascular blood flow and the mechanistic overlap with porcine IMCAs. The unusual presence of l-SMCs provides an additional pathway for rapid intercellular signaling between cells of the coronary artery wall. Local and conducted vasodilation follow hyperpolarization of the ECs or SMCs, and contact-coupling between l-SMCs and r-SMCs likely facilitates this vasodilation
Pulmonary Oxidative Stress, Reperfusion Injury and Lung Tissue Damage During Cardiac Surgery:The Outcome of an Experimental Trial in a Porcine Model
No full textHuman coronary microvascular contractile dysfunction associates with viable synthetic smooth muscle cells
No full textAimsCoronary 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 ResultsIntramyocardial 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.ConclusionsThese 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
