Selected contribution: cerebrovascular nos and cyclooxygenase are unaffected by estrogen in mice lacking estrogen receptor-alpha.

Estrogen alters reactivity of cerebral arteries by modifying production of endothelium-dependent vasodilators. Estrogen receptors (ER) are thought to be involved, but the responsible ER subtype is unknown. ER-alpha knockout (alphaERKO) mice were used to test whether estrogen acts via ER-alpha. Mice were ovariectomized, with or without estrogen replacement, and cerebral blood vessels were isolated 1 mo later. Estrogen increased levels of endothelial nitric oxide synthase and cyclooxygenase-1 in vessels from wild-type mice but was ineffective in alphaERKO mice. Endothelium-denuded middle cerebral artery segments from all animals constricted when pressurized. In denuded arteries from alphaERKO but not wild-type mice, estrogen treatment enhanced constriction. In endothelium-intact, pressurized arteries from wild-type estrogen-treated mice, diameters were larger compared with arteries from untreated wild-type mice. In addition, contractile responses to indomethacin were greater in arteries from wild-type estrogen-treated mice compared with arteries from untreated wild-type mice. In contrast, estrogen treatment of alphaERKO mice had no effect on diameter or indomethacin responses of endothelium-intact arteries. Thus ER-alpha regulation of endothelial nitric oxide synthase and cyclooxygenase-1 pathways appears to contribute to effects of estrogen on cerebral artery reactivity.

Estrogen reduces mouse cerebral artery tone through endothelial NOS- and cyclooxygenase-dependent mechanisms.

Gender and estrogen status are known to influence the incidence and severity of cerebrovascular disease. The vasoprotective effects of estrogen are thought to include both nitric oxide-dependent and independent mechanisms. Therefore, using small, resistance-sized arteries pressurized in vitro, the present study determined the effect of gender and estrogen status on myogenic reactivity of mouse cerebral arteries. Luminal diameter was measured in middle cerebral artery segments from males and from females that were either untreated, ovariectomized (OVX), or OVX with estrogen replacement (OVX + EST). The maximal passive diameters of arteries from all four groups were similar. In response to increases in transmural pressure, diameters of arteries from males and OVX females were smaller compared with diameters of arteries from either untreated or OVX + EST females. In the presence of N(G)-nitro-L-arginine methyl ester, artery diameters decreased in all groups, but diameters remained significantly smaller in arteries from males and OVX females compared with untreated and OVX + EST females. After endothelium removal or when inhibition of nitric oxide synthase and cyclooxygenase were combined, differences in diameters of arteries from OVX and OVX + EST were abolished. These data suggest that chronic estrogen treatment modulates myogenic reactivity of mouse cerebral arteries through both endothelium-derived cyclooxygenase- and nitric oxide synthase-dependent mechanisms.

Gonadal hormones affect diameter of male rat cerebral arteries through endothelium-dependent mechanisms.

Gender is known to influence the incidence and severity of cerebrovascular disease. In the present study, luminal diameter was measured in vitro in pressurized middle cerebral artery segments from male rats that were either untreated, orchiectomized (ORX), ORX with testosterone treatment (ORX+TEST), or ORX with estrogen treatment (ORX+EST). The maximal passive diameters (0 Ca(2+) + 3 mM EDTA) of arteries from all four groups were similar. In endothelium-intact arteries, myogenic tone was significantly greater in arteries from untreated and ORX+TEST compared with arteries from either ORX or ORX+EST. During exposure to N(G)-nitro-L-arginine-methyl ester (L-NAME), an NO synthase (NOS) inhibitor, myogenic tone significantly increased in all groups. The effect of L-NAME was significantly greater in arteries from untreated and ORX+EST compared with arteries from ORX and ORX+TEST rats. Differences in myogenic tone between ORX and ORX+TEST persisted after inhibition of NOS. After endothelium removal or inhibition of the cyclooxygenase pathway combined with K(+) channel blockers, myogenic tone differences between ORX and ORX+TEST were abolished. Wall thickness and forced dilation were not significantly different between arteries from ORX and ORX+TEST. Our data show that gonadal hormones affect myogenic tone in male rat cerebral arteries through NOS- and/or endothelium-dependent mechanisms.

Simulated microgravity increases myogenic tone in rat cerebral arteries.

Adaptation of the cerebral circulation to microgravity was investigated in rat middle cerebral arteries after 20 days of hindlimb unweighting (HU). Myogenic responses were measured in isolated, pressurized arteries from HU and control animals. Maximal passive lumen diameters, obtained in the absence of extracellular Ca2+ plus EDTA, were not significantly different between groups (249 vs. 258 micrometer). In physiological salt solution, arteries from both HU and control animals maintained a constant lumen diameter when subjected to incremental increases in transmural pressure (20-80 mmHg). However, the diameter of arteries from HU animals was significantly smaller than that of arteries from control animals at all pressures; this difference could be eliminated by exposure to the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester. After HU treatment, transient distensibility of the artery wall in response to pressure was also significantly decreased, whereas the frequency and amplitude of vasomotion were increased. The latter changes were not affected by NG-nitro-L-arginine methyl ester. Thus simulated microgravity increases cerebral artery myogenic tone through both nitric oxide synthase-dependent and -independent mechanisms.

Estrogen reduces myogenic tone through a nitric oxide-dependent mechanism in rat cerebral arteries.

Gender differences in the incidence of stroke and migraine appear to be related to circulating levels of estrogen; however, the underlying mechanisms are not yet understood. Using resistance-sized arteries pressurized in vitro, we have found that myogenic tone of rat cerebral arteries differs between males and females. This difference appears to result from estrogen enhancement of endothelial nitric oxide (NO) production. Luminal diameter was measured in middle cerebral artery segments from males and from females that were either untreated, ovariectomized (Ovx), or ovariectomized with estrogen replacement (Ovx + Est). The maximal passive diameters (0 Ca2+ + 1 mM EDTA) of arteries from all four groups were identical. In response to a series of 10-mmHg step increases in transmural pressure (20-80 mmHg), myogenic tone was greater and vascular distensibility less in arteries from males and Ovx females compared with arteries from either untreated or Ovx + Est females. In the presence of NG-nitro-L-arginine methyl ester (L-NAME; 1 microM), an NO synthase inhibitor, myogenic tone was increased in all arteries, but the differences among arteries from the various groups were abolished. Addition of L-arginine (1 mM) in the presence of L-NAME restored the differences in myogenic tone, suggesting that estrogen works through an NO-dependent mechanism in cerebral arteries. To determine the target of NO-dependent modulation of myogenic tone, we used tetraethylammonium (TEA; 1 mM) to inhibit large-conductance, calcium-activated K+ (BKCa) channels. In the presence of TEA, the myogenic tone of arteries from all groups increased significantly; however, myogenic tone in arteries from males and Ovx females remained significantly greater than in arteries from either untreated or Ovx + Est females. This suggests that activity of BKCa channels influences myogenic tone but does not directly mediate the effects of estrogen. Estrogen appears to alter myogenic tone by increasing cerebrovascular NO production and/or action.

Appearance of contractile endothelin-B receptors in rat mesenteric arterial segments following organ culture.

The aim of this study was to examine how different procedures for organ culture affect the expression of contractile endothelin(ET)-B receptors in a branch of the rat mesenteric artery. In fresh segments, ET-1 and ET-3 induced similar strong contractions, ET-1 being 20-fold more potent, whereas neither of the selective ETB receptor agonists, sarafotoxin 6c (S6c) nor IRL 1620, induced significant contractions. In segments cultured for 1 day, ET-3 was only 3-fold less potent as ET-1, and S6c and IRL 1620 induced concentration-dependent contractions which were about 60% of the ET-1 induced contraction. The maximum contractile response to S6c was not altered in segments cultured with foetal calf serum or in buffer solution, but was reduced to about 20% of the control value when cultured in glucose-free buffer solution. The contraction to S6c was abolished in segments placed in cold (4 degrees C) buffer solution. Removal of the endothelium had no effect on the S6c-induced contractions. Arteries cultured at isometric tension (at 2 mN) for 1 day achieved the same contractile response for ETB agonists as resting segments. Pressurized arteries (60 mmHg) did not constrict to S6c when mounted as a fresh segment but demonstrated a strong contraction after 1 day at this transmural pressure. This study suggests that the appearance of ETB receptor mediated contraction following organ culture is not dependent on specific nutrients, endothelial factors or absence of intrinsic tension, but is a metabolically active process.

Effect of melatonin in the rat tail artery: role of K+ channels and endothelial factors.

1. The role of endothelial factors and potassium channels in the action of the pineal hormone melatonin to potentiate vasoconstrictor responses was investigated in the isolated perfused tail artery of the rat. 2. Melatonin (100 nM) potentiated contractile responses to both adrenergic nerve stimulation and alpha1-adrenoceptor stimulation by phenylephrine. After removal of the endothelium, melatonin no longer caused potentiation. 3. The potentiating effect of melatonin was also lost when nitric oxide synthase was inhibited with L-NAME (10 nM). Thus potentiating effects depend on the presence of nitric oxide released by the endothelium. However, melatonin did not affect relaxation responses to acetylcholine in endothelium-intact arteries, nor did melatonin modulate relaxing responses to sodium nitroprusside in endothelium-denuded arteries. While melatonin does not appear to modulate agonist-induced release of nitric oxide nor its effect, melatonin may modulate nitric oxide production induced by flow and shear stress. 4. When the Ca2+-activated K+ channel opener, NS 1619 (10 microM), was present, potentiating effects of melatonin were restored in endothelium-denuded vessels. However, addition of the opener of ATP-sensitive K+ channels, cromakalim (3 microM), did not have the same restorative effect. Furthermore, addition of a blocker of Ca2+-activated K+ channels, tetraethylammonium (1 mM), significantly attenuated potentiating effects of melatonin. These findings support the hypothesis that melatonin inhibits the activity of large conductance Ca2+-activated K+ channels to produce its potentiating effects. 5. Thus in the rat perfused tail artery, potentiation of constriction by melatonin depends on the activity of both endothelial factors and Ca2+-activated K+ channels. Our findings suggest that melatonin inhibits endothelial K+ channels to decrease flow-induced release of nitric oxide as well as block smooth muscle K+ channels to enhance vascular tone.

Melatonin directly constricts rat cerebral arteries through modulation of potassium channels.

The pineal hormone melatonin was found to decrease luminal diameter of rat middle cerebral artery segments, pressurized in vitro, in a concentration-dependent manner (concentration that produced a half-maximal effect = 2.7 nM). Contractile responses to melatonin were inhibited by luzindole, a melatonin receptor antagonist, but not by the serotonin receptor antagonist ketanserin. Pertussis toxin abolished the effect of melatonin, which is consistent with involvement of Gi or G(o) protein-coupled receptors. The maximal effect of melatonin was increased by elevating transmural pressure. When compared at the same pressure, contractions elicited by melatonin were smaller than those elicited by serotonin but similar in magnitude to those produced by tetraethylammonium or charybdotoxin, blockers of Ca(2+)-dependent, large-conductance K+ (BKCa) channels. The effect of melatonin was significantly attenuated in the presence of BKCa channel blockers, but not by apamin, a blocker of Ca(2+)-dependent, small-conductance K+ channels. Melatonin, like tetraethylammonium, significantly reduced vasodilation produced by NS-1619, an opener of BKCa channels. Contractile responses to melatonin were diminished in the presence of elevated extracellular K+ (16 mM), but they were not significantly affected by NG-nitro-L-arginine methyl ester. The results suggest that activation of melatonin receptors on rat cerebral arteries increases vascular tone through Gi or G(o) protein-mediated inhibition of BKCa channels. Thus melatonin, which is secreted during the night, can directly influence the contractile state of cerebral arteries.

Endothelium-dependent vascular smooth muscle relaxation activated by electrical field stimulation.

Electrical field stimulation (EFS) produced relaxation of contracted arteries in the presence of tetrodotoxin. In the present study the contributions of vascular smooth muscle repolarization and endothelial release of nitric oxide to the relaxation response were investigated using isolated rat tail arteries and bovine aortic endothelial cells (BAEC). Intact and endothelium-denuded rings or intact, pressurized artery segments were contracted with either phenylephrine or KCl prior to EFS. Electrical field stimulation induced a small relaxation in denuded, phenylephrine contracted rings that was inhibited by the K+ channel blockers glibenclamide and BaCl2. In intact, phenylephrine-contracted rings, EFS induced significantly larger relaxations that were inhibited by BaCl2 as well as by L-NAME, an inhibitor of nitric oxide (NO) synthase, and methylene blue. EFS-induced relaxations were completely inhibited when BaCl2 and L-NAME or methylene blue were combined. Exposure to Ca(2+)-free buffer or diltiazem also inhibited the relaxation while ascorbic acid had no effect. Effluent from electrically stimulated BAEC caused denuded, phenylephrine contracted rings to relax. The ability of the effluent to cause relaxation was almost completely blocked by exposure of the BAEC to L-NAME or exposure of the recipient vascular smooth muscle to methylene blue; glibenclamide caused partial blockade. Simultaneous measurements of membrane potential and intraluminal pressure showed that EFS-induced membrane repolarization preceded changes in steady-state pressure. It is concluded that (1) the smooth muscle cells possess an endothelium-independent repolarization mechanism, (2) EFS causes endothelial cells of intact arteries to release NO and possibly a hyperpolarizing factor, (3) EFS of BAEC causes release of NO, and (4) EFS-induced relaxation depends on vascular smooth muscle cell membrane repolarization and endothelial cell release of vasoactive substances.

Propranolol and vagal nerve stimulation do not affect postsystolic shortening during acute myocardial ischaemia in the dog.

Changes in myocardial segment length (ultrasonic crystals) and myocardial blood flow (15(3) microns microspheres) were studied during 5 min occlusions of the left anterior descending coronary artery in open chest anaesthetised dogs, and the effects of occlusion without intervention were compared with those of occlusion during bilateral vagal nerve stimulation (n = 11) and occlusion after administration of 1 mg.kg-1 propranolol (n = 9) in the same dogs. Delineation of the perfusion beds of occluded and non-occluded arteries at necropsy verified placement of the crystals at the centres and immediately within the borders of the ischaemic areas. In untreated animals (n = 6) systolic shortening during occlusion decreased by 160(2)% (dyskinesis) in the centre zone and by 61(1)% (hypokinesis) in the border zone of ischaemia, myocardial blood flow decreased by 96(2)% in the centre and 81(2)% at the border, and the changes were reproducible over three successive occlusions. Postsystolic shortening (after peak decline of left ventricular pressure) was reproducible in control animals over three occlusions, was similar in magnitude to the magnitude of dyskinesis (centre zone) or to the degree of hypokinesis (border zone), and persisted after the release of occlusion. Vagal stimulation and propranolol decreased dyskinesis during occlusion but did not affect postsystolic shortening or collateral blood flow within the ischaemic zones. If postsystolic shortening of dyskinetic centre zone segments represents residual active shortening of these segments, as is suggested by other evidence, these results suggest that the oxygen sparing effects for very ischaemic myocardium of vagal stimulation and propranolol do not include a significant reduction in residual active shortening.

Effect of early treatment with propranolol on left ventricular function four weeks after myocardial infarction.

Left ventricular function and exercise capacity were assessed in 79 patients randomised to receive intravenous and oral propranolol (n = 44) or conventional therapy (n = 35) within four hours of onset of their first myocardial infarction. Cineangiocardiography and exercise testing were performed four weeks after infarction to allow for maximum recovery of myocardial function. Left ventriculography showed no improvement in ejection fraction or preservation of regional contractile function in patients treated with propranolol compared with controls. A trend towards smaller end diastolic volumes was seen in the propranolol group (mean (SD) 151(42) ml) compared with controls (167(42) ml). Exercise duration and frequency of angina were not significantly different in the two groups. It is concluded that limitation of infarct size by propranolol does not lead to a significant improvement in ventricular systolic function, although left ventricular dilatation may be reduced. These findings are consistent with the known effect of early intravenous beta blockade which limits infarct size by preservation of subepicardial myocardium.

Prevention of ventricular fibrillation during acute myocardial infarction by intravenous propranolol.

A trial of intravenous followed by oral propranolol, started within 4 h of onset of suspected myocardial infarction and continued over 27 h, was carried out in 735 patients; 364 received propranolol, 371 were controls. Ventricular fibrillation during the first 48 h after entry to the trial occurred in 2 treated patients and in 14 controls (p = 0.006). Rates of hospital mortality, complications other than ventricular fibrillation, and progression from threatened to completed infarction did not differ between treated and control patients. Ventricular fibrillation was not apparently prevented by prior beta-blocker treatment, which was not a reason for exclusion from the trial. This intravenous/oral propranolol regimen seems to prevent ventricular fibrillation due to evolving myocardial infarction.

Prognosis after recovery from first acute myocardial infarction: determinants of reinfarction and sudden death.

Factors associated with total cardiac mortality, sudden cardiac death and reinfarction were studied in 325 male survivors aged younger than 60 years of age (mean 50) of a first myocardial infarction (MI). All patients had undergone exercise testing and cineangiocardiography 4 weeks after MI, 24% underwent coronary artery surgery and 30% received beta-blocking therapy. Patients were followed 1 to 6 years (mean 3.5). Total cardiac mortality was best predicted by the left ventricular (LV) ejection fraction (EF) and by a coronary prognostic index. In contrast, neither the severity of coronary arterial lesions measured with a scoring system nor the results of the exercise test gave significant prediction of mortality. Of the 2 major late sequelae of MI, reinfarction could not be predicted by any clinical or cineangiocardiographic variable. However, sudden death not associated with reinfarction was significantly more common (p less than 0.001) when EF was less than or equal to 40% than when it was greater than 40%. Comparison of patients with an EF less than or equal to 40% who did or did not die suddenly showed that LV dilation (high volumes at ventriculography) was an added risk factor, but the extent of coronary occlusions and stenoses was not. It is concluded that, at least for groups of patients treated with standard modern methods after MI, the main determinant of medium-term survival is the extent of LV damage. The state of the coronary arteries and the presence of ischemic myocardium during exercise are only of secondary importance for survival.

An electrocardiographic model of myocardial ischemic injury.

Previous electrocardiographic models of myocardial ischemic injury have assumed that transmembrane potential changes are uniform throughout a region of ischemia such that injury currents arise exclusively at the boundary between normal and ischemic myocardium. In such models, the distribution and amplitude of ST segment deflections are considered to arise from a polarized surface interfacing normal and ischemic myocardium. This concept in modeling ischemic injury was derived from the application of principles of electric field theory which had been successfully applied previously to ventricular activation in which QRS potentials are considered to arise from polarized surfaces representing the relatively narrow interfaces between depolarized and nondepolarized myocardium. The present paper outlines the limitations of modeling ischemic injury as a polarized surface in terms of the failure of the predictions of such a model to be supported by the experimentally observed: 1) distribution and relative amplitude of epicardial ST segment elevation overlying a region of ischemia; 2) directional changes in epicardial ST segment elevation that occur with changes in the size of an ischemic region; and 3) nonuniform distribution of transmembrane potential changes which occur within a region of ischemia. A new electrocardiographic model of ischemic injury is formulated which accounts for the nonuniform distribution of transmembrane potential changes which occur throughout a region of ischemia. The model accurately describes experimental observations regarding ST segment deflections which had remained inconsistent with previous models.

Quantitative assessment in infarct size reduction by coronary venous retroperfusion in baboons.

Initial favorable reports in which coronary venous retroperfusion was begun after acute coronary artery occlusion have demonstrated a reversal of ischemic injury and improved left ventricular function. However, little information has been generated to document the extent to which retroperfusion may decrease ultimate histologically determined infarct size. The objective of the present study was to evaluate the effectiveness of retroperfusion in reducing infarct size by using an accurate quantitative method in which infarct size was related to the size of the anatomic perfusion bed of the occluded artery (region at risk for infarction). In an experimental group of 5 baboons, the left anterior descending coronary artery was occluded and coronary venous retroperfusion started 1 hour after occlusion. After a 4-hour period of occlusion, retroperfusion was discontinued and anterograde perfusion was simultaneously restored. A control group of 5 baboons underwent an identical procedure without retroperfusion. Twenty-four hours after occlusion, hearts were excised and the previously occluded left anterior descending coronary artery as well as the adjacent arteries were infected with microvascular dye to delineate the perfusion bed of the occluded artery. Planimetry of serial corss-sections of the left ventricle enabled the size of the perfusion bed of the occluded artery and size of the infarct to be determined. The mean percentage of the perfusion bed infarcted in the control group was 94.1 +/- 0.9 (mean +/- standard error) and in the retroperfused group was 57.4 +/- 3.5 (p less than 0.001). Hence, the results demonstrated that when retroperfusion was initiated after 1 hour of coronary occlusion, the mean percentage of the perfusion bed salvaged was increased by 36.7%.

Quantitative effect of early coronary artery reperfusion in baboons. Extent of salvage of the perfusion bed of an occluded artery.

We examined the extent to which ischemic myocardium was salvaged by reperfusion using a method that allowed expression of the volume of infarction as a percentage of the volume of the perfusion bed of the occluded artery (region at risk of infarction). In eight baboons, the left anterior descending coronary artery (LAD) was occluded for 2 hours, after which perfusion was restored. A control group of eight baboons underwent an identical protocol, but perfusion was not restored. Twenty-four hours after occlusion, microvascular dyes were injected into the LAD and adjacent arteries to delineate the perfusion bed of the occluded artery. The volume of infarction and volume of the perfusion bed were determined planimetrically. The mean percentage of the perfusion bed infarcted in the control baboons was 94.2 +/- 3.5% and 50.1 +/- 5.8% in the reperfused baboons. Hence, the mean percentage of the perfusion bed infarcted was reduced by 44.1% in the reperfused group compared with the control group (p less than 0.001). In reperfused baboons, hemorrhage occurred in the region of infarction but did not result in infarct extension. We conclude that reperfusion after 2 hours of coronary occlusion results in substantial salvage of ischemic myocardium in the baboon.

Failure of nifedipine therapy to reduce myocardial infarct size in the baboon.

The value of nifedipine in reducing the ultimate size of an infarct associated with a period of coronary occlusion followed by reperfusion was assessed. Eight baboons were administered a bolus dose of nifedipine, 5 micrograms/kg intravenously, and then a maintenance dose of 30 micrograms/kg per hour was begun 1 hour before occlusion. This regimen resulted in an 8.5 +/- 1.2 percent (mean +/- standard error) decrease in mean arterial pressure. The left anterior descending coronary artery was occluded for 2 hours and then perfusion restored. At 2 hours after reperfusion the nifedipine infusion was discontinued. Eight control baboons underwent an identical protocol without nifedipine therapy. At 24 hours after occlusion, microvascular dyes were injected into the left anterior descending coronary artery and adjacent arteries to delineate the perfusion bed of the previously occluded artery. The volume of infarction was determined with planimetry and compared with the volume of the perfusion bed of the occluded artery. The area of infarction was always contained within the perfusion bed of the occluded artery. The mean percent of the perfusion bed with infarction was 50.1 +/- 5.8 in the control group and 41.7 +/- 9.5 in the treated group (difference not significant; p greater than 0.05). In both control and treated groups of baboons hemorrhage occurred only within the region of infarction. In both groups electron microscopy revealed large electron-dense granules within the mitochondria. In conclusion nifedipine therapy during a 2 hour period of coronary occlusion followed by reperfusion did not result in any significant reduction in ultimate infarct size in the baboon.