Modulation of vascular tonus by the endothelium in experimental diabetes

The role of the vascular endothelium in the contractile response of aortas from streptozotocin-induced diabetic rats was investigated using selected agents. Contractile response to KCl was not affected by removal of the endothelium in both diabetic and control groups, but was diminished in the diabetic rats compared to the control rats. Contractile response to clonidine markedly increased after removal of the endothelium in the control group, with the increment being less in the diabetic group. After removal of the endothelium, contractile response to clonidine was poorer in the diabetic group than the control group. Vascular relaxation induced by acetylcholine disappeared when the endothelium was removed in both diabetic and control groups. The degree of reaction to acetylcholine did not significantly differ between the two groups. These results suggest that in diabetic rats, abnormality of the endothelium-dependent vascular relaxation is specific for α₂ receptor while that of the vascular smooth muscle reactivity is not receptor-specific.     

Role of the endothelium in the development of reactive hyperemia

The experiments on anesthetized dogs demonstrated that reaction of the femoral vessels reactive hyperemia essentially decreased after chemical inhibition of endothelium by saponin, inhibition of lipoxygenase by quercetin and guanylate cyclase by methylene blue. Reaction was increased after cyclooxygenase inhibition by indomethacin. We concluded that the endothelium plays an important role in reaction of reactive hyperemia by endothelium-derived relaxing factor release.     

Simultaneous in situ monitoring of intracellular Ca2+ and NO in endothelium of coronary arteries

We developed an in situ assay system to simultaneously monitor intracellular Ca(2+) concentration ([Ca(2+)](i), fura 2 as indicator) and nitric oxide (NO) levels [4,5-diaminofluorescein as probe] in the intact endothelium of small bovine coronary arteries by using a fluorescent microscopic imaging technique with high-speed wavelength switching. Bradykinin (BK; 1 microM) stimulated a rapid increase in [Ca(2+)](i) followed by an increase in NO production in the endothelial cells. The protein tyrosine phosphatase inhibitor phenylarsine oxide (PAO; 10 microM) induced a gradual, small increase in [Ca(2+)](i) and a slow increase in intracellular NO levels. Removal of extracellular Ca(2+) and depletion of Ca(2+) stores completely blocked BK-induced increase in NO production but had no effect on PAO-induced NO production. However, a further reduction of [Ca(2+)](i) by application of BAPTA-AM or EGTA with ionomycin abolished the PAO-induced NO increase. These results indicate that a simultaneous monitoring of [Ca(2+)](i) and intracellular NO production in the intact endothelium is a powerful tool to study Ca(2+)-dependent regulation of endothelial nitric oxide synthase, which provides the first direct evidence for a permissive role of Ca(2+) in tyrosine phosphorylation-induced NO production.     

Constriction to hypoxia-reoxygenation in isolated mouse coronary arteries: role of endothelium and superoxide.

The aim of the present study was to determine the role of endothelium and superoxide in the responses of isolated mouse coronary arteries to hypoxia-reoxygenation. Isolated mouse coronary artery was cannulated, pressurized at 60 mmHg, and constantly superfused with recirculating Krebs-Ringer bicarbonate solution for continuous measurement of intraluminal diameter (ID) by video microscopy. Under a no-flow condition, hypoxia (0% O(2), 30 min) caused vasoconstriction. Reoxygenation caused a further vasoconstriction (ID change from 111.4 +/- 11.1 to 91 +/- 16.5 microm) that was significantly reduced by removal of endothelium (ID change from 105.4 +/- 27 to 109.9 +/- 23.4 microm). Cu/Zn superoxide dismutase (150 U/ml) did not alter the hypoxic vasoconstriction but abolished the reoxygenation-caused endothelium-dependent vasoconstriction. Hypoxia-reoxygenation markedly enhanced the generation of superoxide that was significantly reduced by either removing the endothelium or treated these endothelium-intact vessels with superoxide dismutase. These results suggest that, in isolated mouse coronary arteries, hypoxia causes vasoconstriction that is independent of endothelium, whereas reoxygenation causes vasoconstriction that is mediated by enhanced generation of superoxide from endothelium.     

Cellular mechanisms of transitory smooth muscle contraction of the coronary arteries during hypoxia: role of intracellular Ca2+

In experiments on isolated porcine and canine coronary artery rings it was shown that vascular smooth muscle (VSM) during hypoxia (decreasing bath PO2 with 147 to 20-15 mm Hg) response to biphasic constriction-dilation reaction. Transient hypoxic contractions (THC) of VSM preserved completely in Ca2+-free solution and partially (up 50-60%) in the presence of Ca2+-channel blockers, but abolished by procaine. THC of VSM skinned by saponin significantly depressed at depletion of Ca2+-store sarcoplasmic reticulum (SR) by caffeine nd abolished after SR destruction. THC is not linked with Na+-K+-ATPase inhibition because it preserved (or increased) at ouabain treatment. THC significantly depressed under selective glycolysis blockade by monoiodoacetic acid and pyruvate and also after inositol-1 monophosphatase inhibition by lithium (the phase of hypoxic relaxation of VSM was augmented in this condition). Our results indicate that transient contraction of coronary arteries under hypoxia may be mediated mainly by release of Ca2+ from SR and linked obviously with production of inositol-1,4,5-trisphosphate. The participation of glycolysis in this process is unknown.     

Thrombin-induced increase in albumin permeability across the endothelium

We studied the effect of thrombin on albumin permeability across the endothelial monolayer in vitro. Bovine pulmonary artery endothelial cells were grown on micropore membranes. Morphologic analysis confirmed the presence of a confluent monolayer with interendothelial junctions. Albumin permeability was measured by the clearance of 125I-albumin across the endothelial monolayer. The control 125I-albumin clearance was 0.273 +/- 0.02 microliter/min. The native enzyme, alpha-thrombin (10(-6) to 10(-10) M), added to the luminal side of the endothelium produced concentration-dependent increases in albumin clearance (maximum clearance of 0.586 +/- 0.08 microliter/min at 10(-6) M). Gamma (gamma) thrombin (10(-6) M and 10(-8) M), which lacks the fibrinogen recognition site, also produced a concentration-dependent increase in albumin clearance similar to that observed with alpha-thrombin. Moreover, the two proteolytically inactive forms of the native enzyme, i-Pr2 P-alpha-thrombin and D-Phe-Pro-Arg-CH2-alpha-thrombin, increased the 125I-albumin clearance (0.610 +/- 0.09 microliter/min and 0.609 +/- 0.02 microliter/min for i-Pr2 P-alpha-thrombin and D-Phe-Pro-Arg-CH2-alpha-thrombin at 10(-6) M, respectively). Since the modified forms of thrombin lack the fibrinogen recognition and active serine protease sites, the results indicate that neither site is required for increased albumin permeability. The increase in albumin clearance with alpha-thrombin was not secondary to endothelial cell lysis because lactate dehydrogenase concentration in the medium following thrombin was not significantly different from baseline values. There was also no morphological evidence of cell lysis. Moreover, the increase in 125I-albumin clearance induced by alpha-thrombin was reversible by washing thrombin from the endothelium. The basis for the increased albumin permeability following the addition of alpha-thrombin appears to be a reversible change in endothelial cell shape with formation of intercellular gaps.     

Role of ceramide in TNF-alpha-induced impairment of endothelium-dependent vasorelaxation in coronary arteries

The present study tested the hypothesis that ceramide, a sphingomylinase metabolite, serves as an second messenger for tumor necrosis factor-alpha (TNF-alpha) to stimulate superoxide production, thereby decreasing endothelium-dependent vasorelaxation in coronary arteries. In isolated bovine small coronary arteries, TNF-alpha (1 ng/ml) markedly attenuated vasodilator responses to bradykinin and A-23187. In the presence of N(G)-nitro-L-arginine methyl ester, TNF-alpha produced no further inhibition on the vasorelaxation induced by these vasodilators. With the use of 4,5-diaminofluorescein diacetate fluorescence imaging analysis, bradykinin was found to increase nitric oxide (NO) concentrations in the endothelium of isolated bovine small coronary arteries, which was inhibited by TNF-alpha. Pretreatment of the arteries with desipramine (10 microM), an inhibitor of acidic sphingomyelinase, tiron (1 mM), a superoxide scavenger, and polyethylene glycol-superoxide dismutase (100 U/ml) largely restored the inhibitory effect of TNF-alpha on bradykinin- and A-23187-induced vasorelaxation. In addition, TNF-alpha activated acidic sphingomyelinase and increased ceramide levels in coronary endothelial cells. We conclude that TNF-alpha inhibits NO-mediated endothelium-dependent vasorelaxation in small coronary arteries via sphingomyelinase activation and consequent superoxide production in endothelial cells.     

Role of myocardium and endothelium in coronary vascular smooth muscle responses to hypoxia

Hypoxia triggers a mechanism that induces vasodilation in the whole heart but not necessarily in isolated coronary arteries. We therefore studied the role of cardiomyocytes (CM), smooth muscle cells (SMC), and endothelial cells (EC) in coronary responses to hypoxia (PO(2) of 5-10 mmHg). In an attempt to determine the factor(s) released in response to hypoxia, we inhibited the contribution of adenosine, ATP-sensitive K(+) channels, prostaglandins, and nitric oxide. Isolated rat septal artery segments without (-T) and with a layer of cardiac tissue (+T) were mounted in a double wire myograph, and constriction was induced. Hypoxia induced a decrease in isometric force of 21% and 61% in -T and +T segments, respectively (P < 0.05). EC removal increased the relaxation to hypoxia in -T segments to 33% but had the same effect in +T segments (61%). Only one of the inhibitors, the adenosine antagonist in +T segments, partially affected the relaxation due to hypoxia. The role of adenosine is thus limited and other mechanisms have to contribute. We conclude that hypoxia induces a relaxation of SMC that is augmented by the presence of CM and blunted by the endothelium. A single mediator does not induce those effects.     

Effects of tumor necrosis factor on the tonus of cerebral arteries]

It was determined that tumor necrosis factor (TNF) is capable of decreasing the local brain's blood flow on 45.6% (in the concentration of 6 micrograms/kg); to make a spasm of the pial arteries on 39.6%. In vitro experiments TNF increased the amplitude of the rhythmical and the tonic contractions of the brain's arteries smooth muscles (3.6 X 10(-8) M). The direct action of the TNF in the vascular wall is endothelium-dependent.     

Sympathetic and parasympathetic regulation of the heart in transitory coronary insufficiency

Experiments were made on 480 rats and 60 rabbits with transitory insufficiency (duration of myocardial ischemia 10.40 and 120 min the length of subsequent reperfusion 40-60 min). It was discovered that there was natural development of the phenomenon of limitation of drawing the heart in direct and reflectory reactions of the circulation system within the increase of duration of local ischemia and the beginning of the subsequent reperfusion. The phenomenon of "limitation", which is realized with the involvement of sympathetic and parasympathetic mechanisms, promotes a decrease of the extent of heart alteration during its local ischemia and increase of reparative process in the heart at the reperfusion period.     

Regulatory functions of the coronary endothelium

In this brief review three functions of the coronary endothelium are surveyed: (a) its barrier and exchange function, (b) the prevention of coagulation and platelet aggregation, and (c) its role in vasoregulation. Impairment of these functions can occur in ischemia, hypertension, arteriosclerosis and inflammation. (Mol Cell Biochem116: 163–169, 1992)     

Cluster microvilli in coronary endothelium

Summary Examination of cardiac vascular endothelium with scanning electron microscopy, and transmission electron microscopy of previously scanned tissue, revealed several regions of the coronary venous system that contained cluster microvilli. These consisted of 2–15 microvillous projections that emanated radially from a common base or were grouped into a fan-like arrangement. Although rare, these clusters, when present, were widely distributed over the endothelial cell surface.Dr. Smolich is the recipient of a postgraduate scholarship from the National Health and Medical Research Council of Australia