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To test the hypothesis that protein kinase C (PKC)beta-induced reactive oxygen species (ROS) underlie the vascular dysfunction in diabetes, we examined the effects of (S)-13[(dimethylamino)-methyl]-10,11-14,15-tetrahydro-4,9:16,21-dimetheno-1H,13H-dibenzo[e,k]pyrrolo[3,4-h][1,4,13]oxadi-azacyclohexadecene-1,3(2H)-dione (LY333531; LY), a specific PKCbeta inhibitor, on arachidonic acid (AA)-mediated dilation in small coronary arteries from streptozotocin-induced diabetic rats. This study was designed to determine whether diabetes impairs AA-induced vasodilation of small coronary arteries and whether this defect could be blunted by dietary treatment with LY. Coronary diameter was measured using videomicroscopy in isolated pressurized vessels. In controls, AA dose dependently dilated coronary arteries, with 1 muM producing 54.7 +/- 3.1% and 30 microM producing 72.0 +/- 3.0% dilation (n = 9). In diabetic rats, 1 microM AA only produced 31.4 +/- 3.8% (n = 8; p < 0.01 versus control) and 30 microM 43.8 +/- 3.7% dilation (n = 8; p < 0.001 versus control). Nitroprusside-mediated vasodilations were similar in control and diabetic rats. In contrast, in diabetic rats receiving LY, AA-mediated coronary dilations were normal. In controls, AA-mediated vasodilation was inhibited by miconazole (an inhibitor of cytochrome P450 epoxygenase) and by iberiotoxin (IBTX, an inhibitor of the large conductance Ca(2+)-activated K(+) channel), but miconazole and IBTX had no effects in diabetic vessels. In diabetic rats receiving LY, the effects of miconazole and IBTX were similar to control. Superoxide dismutase restored responses to AA in diabetic vessels but had no effect in vessels from control or diabetic rats on LY. These results suggest that AA-mediated vasodilation in rat coronary arteries are impaired in diabetic rats due to increases in generation of ROS. LY protects against these defects in diabetes through inhibition of PKCbeta-mediated production of ROS.

Calcium ion (Ca2+) influx through voltage-gated Ca2+ channels is important for the regulation of vascular tone. Activation of L-type Ca2+ channels initiates muscle contraction; however, the role of T-type Ca2+ channels (T-channels) is not clear. We show that mice deficient in the alpha1H T-type Ca2+ channel (alpha(1)3.2-null) have constitutively constricted coronary arterioles and focal myocardial fibrosis. Coronary arteries isolated from alpha(1)3.2-null arteries showed normal contractile responses, but reduced relaxation in response to acetylcholine and nitroprusside. Furthermore, acute blockade of T-channels with Ni2+ prevented relaxation of wild-type coronary arteries. Thus, Ca2+ influx through alpha1H T-type Ca2+ channels is essential for normal relaxation of coronary arteries.

The objectives of the present study were to 1) examine mechanisms involved in endothelium-dependent responses of coronary arteries from normal mice and 2) determine whether vascular responses of coronary arteries are altered in two genetic models of hypercholesterolemia [apolipoprotein E (apoE)-deficient mice (apoE -/-) and combined apoE and low-density lipoprotein receptor (LDLR)-deficient mice (apoE + LDLR -/-)]. Plasma cholesterol levels were higher in both apoE -/- and apoE + LDLR -/- compared with normal mice on normal and high-cholesterol diets (normal chow: normal 110 +/- 5 mg/dl, apoE -/- 680 +/- 40 mg/dl, apoE + LDLR -/- 810 +/- 40 mg/dl; high-cholesterol chow: normal 280 +/- 60 mg/dl, apoE -/- 2,490 +/- 310 mg/dl, apoE + LDLR -/- 3,660 +/- 290 mg/dl). Coronary arteries from normal (C57BL/6J), apoE -/-, and apoE + LDLR -/- mice were isolated and cannulated, and diameters were measured using videomicroscopy. In normal mice, vasodilation in response to ACh and serotonin was markedly reduced by 10 microM Nomega-nitro-L-arginine (an inhibitor of nitric oxide synthase) or 20 microM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; an inhibitor of soluble guanylate cyclase). Vasodilation to nitroprusside, but not papaverine, was also inhibited by ODQ. Dilation of arteries from apoE -/- and apoE + LDLR -/- mice on normal diet in response to ACh was similar to that observed in normal mice. In contrast, dilation of arteries in response to serotonin from apoE -/- and apoE + LDLR -/- mice was impaired compared with normal. In arteries from both apoE -/- and apoE + LDLR -/- mice on high-cholesterol diet, dilation to ACh was decreased. In apoE + LDLR -/- mice on high-cholesterol diet, dilation of coronary arteries to nitroprusside was increased. These findings suggest that dilation of coronary arteries from normal mice in response to ACh and serotonin is dependent on production of nitric oxide and activation of soluble guanylate cyclase. Hypercholesterolemia selectively impairs dilator responses of mouse coronary arteries to serotonin. In the absence of both apoE and the LDL receptor, high levels of cholesterol result in a greater impairment in coronary endothelial function.

Activation of ATP-sensitive K+ channels is involved in the coronary vascular response to decreases in perfusion pressure and ischemia. Since activation of ATP-sensitive K+ channels in collateral vessels may be important in determining flow to collateral-dependent myocardium, the ability of collaterals to respond to activation of the channel was tested. In the beating heart of dogs, we compared responses of non-collaterals less than 100 microns in diameter to collaterals of similar size using computer-controlled stroboscopic epi-illumination of the left ventricle coupled to a microscope-video system. Aprikalim, a selective activator of ATP-sensitive K+ channels (0.1-10 microM) produced similar dose-dependent dilation of non-collaterals and collaterals. Relaxation was decreased by inhibition of ATP-sensitive K+ channels with glibenclamide, but not by inhibition of nitric oxide synthase with nitro-L-arginine. Bradykinin (10-100 microM) produced similar dilation of non-collaterals and collaterals which was decreased by nitro-L-arginine but not glibenclamide. Thus, in microvascular collaterals, relaxation to both nitric oxide and activation of ATP-sensitive K+ channels is similar to non-collaterals.

Coronary artery disease often occurs in patients with hypertension. The present study was designed to evaluate coronary vascular function in isolated coronary arteries of spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats and to determine the effect of antihypertensive treatment on coronary vascular responsiveness. Male SHR and WKY rats (12 to 14 weeks old) were divided into control and hydralazine-treated (120 mg/L drinking water for 10 days) groups. After 10 days, arterial pressure and heart rate were recorded while rats were conscious and unrestrained. Left ventricular coronary arteries (200 to 300 microns diameter) were isolated and intraluminal diameter was continuously recorded while vessels were maintained at a constant intraluminal pressure of 40 mm Hg. Relaxation of coronary arteries to both acetylcholine and nitroprusside was slightly, but significantly, enhanced in vessels from SHR compared to WKY rats. The enhanced relaxation was a specific effect, since isoproterenol induced similar relaxation in coronary arteries from SHR and WKY rats. Contraction to phenylephrine, but not endothelin-1, was augmented in coronary arteries from SHR compared to WKY rats. Treatment with hydralazine significantly lowered arterial pressure in SHR and WKY rats, but did not alter the enhanced contraction to phenylephrine or the enhanced relaxation to acetylcholine and nitroprusside in coronary arteris from SHR. These results indicate that coronary arteries of 12 to 14 week-old SHR do not have impaired endothelium-dependent relaxation, but to exhibit enhanced alpha-adrenoceptor-mediated contraction that is not reduced by lowering arterial pressure.

Previous studies in which an isolated heart or in situ constant pressure preparation was used suggested a minimal role for adenosine in autoregulatory control of coronary circulation. These results, however, are controversial, and the role of adenosine in autoregulation of flow in heart is uncertain. To test the hypothesis that adenosine mediates microvascular dilation in response to reduction in perfusion pressure (PP), we performed experiments in 41 open-chest chloralose-anesthetized dogs. Internal diameters (ID) of epicardial small arterioles < 100 mumol were measured with an intravital microscope and stroboscopic epiillumination synchronized to cardiac cycle. PP was reduced by graded stenoses of the left anterior descending coronary artery (LAD, mild stenosis PP = 60 mm Hg; critical stenosis PP = 40 mm Hg) and complete occlusion. 8-Phenyltheophylline (8-PT 10 microM) or adenosine deaminase (ADA 10 U/min) was topically superfused onto the heart. Arteriolar dilation induced by topically applied adenosine < or = 10 microM was completely blocked by 8-PT. Without 8-PT (vehicle group), mild critical stenosis and complete occlusion caused arteriolar dilation (percentage of change in diameter 8.6 +/- 2.6, 16.0 +/- 2.7, and 13.6 +/- 4.8%). 8-PT did not inhibit this dilation (8.5 +/- 2.8, 16.1 +/- 4.6, 15.1 +/- 5.7%, NS vs. vehicle group). Topically applied ADA significantly inhibited intravenously (i.v.) administered adenosine-induced arteriolar dilation. Without ADA, arteriolar dilation occurred (16.6 +/- 3.0, 28.2 +/- 4.3, 15.4 +/- 6.2%, at each PP). However, ADA did not inhibit dilation induced by gradual stenoses (10.6 +/- 1.4, 24.2 +/- 4.3, 17.5 +/- 6.9%, at each PP, NS vs. vehicle group).(ABSTRACT TRUNCATED AT 250 WORDS)

The purpose of this study was to examine mechanisms involved in the response of native collaterals to coronary occlusion. In anesthetized dogs native collaterals were identified as vessels coursing between the left anterior descending and left circumflex arteries using fluorescence angiography. After a left anterior descending occlusion in 12 dogs, collaterals < 100 microns in diameter progressively dilated by 21 +/- 4% (n = 12) 1 min after occlusion and by 39 +/- 6% 15 min after occlusion. Collaterals > 100 microns in diameter did not dilate after coronary occlusion. NG-nitro-L-arginine (1 mg/min intracoronary) caused constriction under basal conditions in collaterals < 100 microns but did not prevent the dilation of collaterals after occlusion. In contrast, glibenclamide (10(-5) M), an inhibitor of ATP-sensitive potassium channels, had no effect on baseline diameter of collaterals < 100 microns diameter but completely prevented dilation of collaterals after occlusion. We conclude that collaterals are not maximally dilated immediately after a coronary occlusion but rather progressively dilate for at least 15 min after an occlusion. This dilation of native collaterals after an occlusion is not mediated by release of an endothelium-derived relaxing factor derived from L-arginine but is mediated by activation of ATP-sensitive K+ channels.

The purpose of this study was to elucidate the contribution of endothelium-derived relaxing factor (EDRF) derived from arginine to acetylcholine (ACh)-induced coronary arteriolar vasodilatation in vivo. Experiments were performed in 62 open-chest anesthetized dogs. Internal diameters of small arterioles (less than 120 microns) and large arterioles (greater than 120 microns) were measured using an intravital microscope and stroboscopic epiillumination synchronized to the cardiac cycle. Topically administered NG-monomethyl-L-arginine (L-NMMA, 3 x 10(-4) M) constricted small arterioles (-10.7 +/- 3.1% from control diameter, P less than 0.05), but L-NMMA did not produce vasoconstriction in large arterioles. ACh, in the absence of L-NMMA, caused a dose-dependent vasodilatation in both small and large arterioles. In large arterioles, L-NMMA completely abolished the ACh-induced vasodilatation (10(-5) M topical ACh: from 13.3 +/- 3.0 to -2.0 +/- 1.5%, P less than 0.05; 10(-4) M ACh: from 20.9 +/- 3.9 to -3.0 +/- 1.9%, P less than 0.01). In small arterioles, L-NMMA only partially inhibited the vasodilatation (10(-5) M ACh: from 35.4 +/- 4.0 to 19.0 +/- 2.7%, P less than 0.05; 10(-4) M ACh: from 42.5 +/- 4.8 to 22.6 +/- 3.1%, P less than 0.05). L-Arginine (10(-3) M topically) reversed L-NMMA inhibition of ACh-induced vasodilatation. Persistent dilatation of small arterioles also occurred when NG-nitro-L-arginine rather than L-NMMA was administered.(ABSTRACT TRUNCATED AT 250 WORDS)

Recent studies have demonstrated that a nitroso compound derived from L-arginine (Arg) may be the endothelium-derived relaxing factor (EDRF) released from vascular endothelium. Synthesis of EDRF from L-Arg is inhibited by analogues of Arg such as NG-monomethyl-L-arginine (L-NMMA) and N omega-nitro-L-arginine (L-NNA). We tested the role of compounds derived from Arg in the constriction of the proximal left anterior descending coronary artery (LAD) to serotonin in vivo by measuring responses before and during infusion of L-NMMA or L-NNA. In open-chest anesthetized dogs the LAD was perfused at constant pressure (80 mmHg) from a reservoir. Large-artery diameter was measured with piezoelectric crystals, and coronary flow was measured with an in-line electromagnetic flow probe. Intracoronary serotonin (5 and 50 micrograms/min) caused a dose-dependent constriction of the proximal LAD and increase in coronary flow. Intracoronary L-NMMA (2 mg/min) or L-NNA (2 mg/min) augmented the constriction to serotonin, whereas the increase in coronary flow was blunted only by L-NNA. L-Arg (10 mg/min, intracoronary) alone did not alter either the large-artery constriction or the increase in flow to serotonin; however, it prevented the enhanced constriction to serotonin following L-NMMA. Constriction to the endothelium-independent agent prostaglandin F2 alpha was not affected by L-NMMA. We conclude that a metabolite of L-Arg modulates the large coronary artery response to serotonin in vivo.

In open-chest anesthetized dogs acute hypertension causes neutrophil and platelet adhesion to vascular endothelium and selectively potentiates constriction to serotonin in proximal coronary arteries. To examine underlying mechanisms, canine left anterior descending coronary arteries subjected to 15 min hypertension (LAD-HYP) and control left circumflex coronary arteries (CX) perfused at normal pressure were studied in organ chambers. In endothelium-intact LAD-HYP rings, constriction to serotonin was potentiated fourfold compared with control CX rings but was similar in denuded LAD-HYP and CX vessels. Endothelium-dependent relaxation to acetylcholine was not affected by acute hypertension. In LAD-HYP rings 10 microM LY 83583 (which depletes guanosine 3',5'-cyclic monophosphate and inhibits effects of endothelium-derived relaxing factor) augmented constriction to serotonin twofold. LY 83583 did not affect the serotonin response in hypertensive rings whose endothelium was mechanically removed. Blockade of either leukotriene D4 (LTD4) receptors (either with LY 171883 or SKF 102992) or thromboxane A2 (TxA2) receptors (with SQ 29548) partially blunted constriction to serotonin. Combined LTD4- and TxA2-receptor blockade completely normalized serotonin-induced constriction in LAD-HYP rings. In preconstricted LAD-HYP rings, relaxations to serotonin were markedly impaired but were restored by addition of ketanserin. Normalization of relaxation to serotonin in hypertensive vessels by ketanserin is likely due to inhibition of 5-hydroxytryptamine2 (5-HT2) receptors on platelet membranes. In conclusion, augmented constriction to serotonin in canine epicardial vessels exposed to acute hypertension is not due to an impairment of endothelium-dependent relaxation to the amine but to concomitant release of leukotrienes and TxA2 from leukocytes and platelets adhering to damaged endothelium. Activation of 5-HT2 serotonergic receptors on platelet membranes could be a possible trigger mechanism.

We examined the relative contribution of endothelial and vascular smooth muscle-derived prostaglandins and endothelium-derived relaxing factor in modulating both the large coronary artery and resistance vessel responses to thromboxane in vivo. Vascular responses to the thromboxane analogue U46619 were measured in four separate experimental protocols: 1) The vascular responses were measured in the presence and absence of intact endothelium to examine the role of endothelium-derived vasodilators. 2) Responses were measured in the presence of intact endothelium before and after inhibition of cyclooxygenase with indomethacin to examine the role of endothelial and vascular smooth muscle-derived prostaglandins. 3) Responses were measured after endothelial removal before and after indomethacin to examine the role of vascular smooth muscle-derived prostaglandins. 4) Responses were measured after indomethacin and before and after removal of endothelium to examine the role of endothelium-derived relaxing factor. In anesthetized dogs (n = 41) that underwent constant pressure perfusion of the left anterior descending coronary artery (LAD), LAD diameter was measured with sonomicrometer crystals, and coronary flow was measured with an electromagnetic flow probe. Intracoronary infusion of U46619 (0.01-1.0 microgram/min) produced a dose-dependent constriction of LAD. Constriction of the LAD was augmented after endothelial removal, after indomethacin treatment in both the presence and absence of endothelium, and after removal of the endothelium in the presence of indomethacin. Inhibition of prostaglandin synthesis had the greatest effect of augmenting constriction of LAD to thromboxane. Coronary flow was decreased by U46619 only in the presence of indomethacin.(ABSTRACT TRUNCATED AT 250 WORDS)

Recent studies in isolated epicardial coronary artery rings have shown that the endothelium modulates vasomotor responses to certain endogenous neurohumoral agents. It is not known whether the endothelium plays a role in large coronary vasoregulation in the intact coronary circulation. Accordingly, we examined effects of endothelial removal on vasoconstrictor responses of the proximal coronary artery in anesthetized adult mongrel dogs. The left anterior descending artery was perfused at 100 mm Hg with arterial blood from a pressurized reservoir. Coronary diameter was measured continuously with 7-MHz sonomicrometer crystals attached to the adventitia of the artery. Fifteen minutes after mechanical disruption of the endothelium with a balloon-tipped catheter, baseline diameter was unchanged from a control value of 2.60 +/- 0.13 mm (mean +/- SE, n = 17). Endothelial denudation resulted in a dose-dependent potentiation of the constrictor response to intracoronary 5-hydroxytryptamine (1-50 micrograms/min, n = 8). With the endothelium intact, a 5 micrograms/min infusion of 5-hydroxytryptamine reduced diameter by 24 +/- 14 micron, while a 50 micrograms/min dose reduced diameter by 54 +/- 25 micron. After endothelial removal, the decrease in diameter averaged 74 +/- 18 microns at 5 micrograms/min and 132 +/- 29 micron at 50 micrograms/min, indicating a 10-fold increase in the sensitivity to 5-hydroxytryptamine. The constrictor responses to angiotensin II (1 and 5 micrograms/min, n = 7) and phenylephrine (1-5 micrograms/min, n = 7) were not altered by endothelial removal. Thus, endothelial removal selectively potentiates the constrictor responses to 5-hydroxytryptamine in the intact coronary circulation of the dog.

We tested the hypothesis that acute coronary artery hypertension may damage vascular endothelium and alter vasomotor responses to humoral agents. We examined effects of intracoronary infusion of the endothelium-dependent agent serotonin and two endothelium-independent agents, angiotensin II and methoxamine, on large coronary artery diameter in the blood perfused dog heart. Responses were examined before and 30 minutes after brief periods of coronary hypertension (200 mm Hg for 10 seconds to 15 minutes). In open-chest anesthetized dogs, the left anterior descending coronary artery was perfused at constant pressure. Coronary diameter (D) was measured with piezoelectric crystals. At a control perfusion pressure of 80 mm Hg, serotonin produced dose-dependent constriction of the large coronary artery (mean +/- SEM; delta D = -22 +/- 10 microns at 5 micrograms/min; -108 +/- 50 microns at 50 micrograms/min). Increasing perfusion pressure to 200 mm Hg increased flow 515 +/- 79% and coronary diameter 509 +/- 9 microns. After 15 minutes of hypertension, when coronary diameter had returned to baseline values, the constriction of the large artery to serotonin was potentiated (delta D = -89 +/- 33 microns at 5 micrograms/min; -207 +/- 45 microns at 50 micrograms/min; p less than 0.05). Hypertension for 1-5 minutes potentiated constrictor responses of large coronary arteries for at least 2 1/2 hours. Removal of endothelium prevented effects of hypertension on constrictor responses of large arteries to serotonin. Hypertension did not alter constrictor responses to angiotension II (1 and 2.5 micrograms/min) or methoxamine (50 and 100 micrograms/min) or the dilator response to acetylcholine (40 micrograms/min). Acute hypertension altered endothelial morphology. There were small endothelial craters following 10 seconds of hypertension, and disruption of endothelial junctions with leukocyte adherence following 1-15 minutes of hypertension. We conclude that acute hypertension alters constrictor responses of large coronary arteries to serotonin by impairing endothelial function and not by directly affecting vascular smooth muscle. These effects of acute hypertension on vascular reactivity are selective in that they do not involve non-endothelium-dependent agents or the endothelium-dependent agent, acetylcholine. The effect of hypertension also persists long after pressure is restored to normotensive levels.

We tested the hypothesis that flow-mediated endothelium-dependent dilation can attenuate humorally mediated constriction of large coronary arteries in vivo. Accordingly, we measured constriction of large coronary arteries to serotonin when flow was allowed to increase and when flow was constant in the presence and absence of endothelium. The left anterior descending coronary artery of anesthetized dogs was perfused at constant pressure (100 mmHg) and diameter measured with an ultrasonic dimension gauge. Coronary flow was measured with an in-line electromagnetic flow probe. Flow-mediated dilation was demonstrated by measuring diameter (D) after increased flow in response to adenosine (1 mg/min ic). Adenosine (n = 9) increased flow (341 +/- 69%) and resulted in large artery dilation [diameter change (delta D) = 101 +/- 54 microns], which was abolished by maintaining constant flow (distal snare) or by removing endothelium. Serotonin (n = 9, 50 micrograms/min ic) increased flow (298 +/- 45%) while simultaneously decreasing large artery diameter (delta D = -58 +/- 22 microns). When flow was kept constant, serotonin produced a greater constriction (delta D = -173 +/- 29 microns). After the removal of endothelium (n = 10), constrictor responses to serotonin were similar when flow increased (delta D = -84 +/- 13 microns) and when flow was kept constant (delta D = -79 +/- 23 microns). We conclude that flow-mediated dilation of large coronary arteries can attenuate constriction to serotonin and that this effect is dependent on endothelium.