Growth factor expression in aorta of normotensive and hypertensive rats.

Hypertension causes biochemical and morphological changes in the vessel wall by unknown mechanisms. Locally produced substances may have a role in mediating these vascular changes. We have studied the expression of platelet-derived growth factor (PDGF) B chain and PDGF A chain, insulin-like growth factor (IGF)-I and IGF-II, endothelial cell growth factor (ECGF), basic fibroblast growth factor (bFGF), and transforming growth factor-beta (TGF-beta) in aortic tissue from normotensive rats and rats made hypertensive by deoxycorticosterone (DOC)/salt treatment. Using Northern blotting, we found that genes for each of these growth factors were transcriptionally active in the aorta of both normotensive and hypertensive rats. TGF-beta aortic mRNA levels increased up to threefold as a result of DOC/salt hypertension. In contrast, no major changes in the expression of either PDGF chain, IGF-I or II, ECGF, or bFGF were detectable. The results indicate that at least seven genes coding for growth factors that were shown previously to influence growth and function of vascular cells in vitro, are expressed in rat aorta in vivo. These findings support the hypothesis that synthesis and release of growth factors in the arterial wall are involved in autocrine and/or paracrine regulatory mechanisms. In addition, the increased expression of TGF-beta in vivo may have a role in mediating the aortic changes induced by hypertension.

Fibronectin expression in the normal and hypertrophic rat heart.

We examined changes in the expression of fibronectin during the induction of cardiac hypertrophy by L-triiodothyronine administration and by mineralocorticoid- and salt-induced experimental hypertension. By use of Northern and Western blotting procedures, fibronectin was localized mainly in the atria of normal rat hearts. Atria contained 10- and 5-fold higher relative concentrations of fibronectin mRNA and protein, respectively, compared with ventricles. During the progression of cardiac hypertrophy induced by L-triiodothyronine over a 10-d period, there was a progressive increase in fibronectin mRNA for the first 6 d followed by a return to control levels. The major change could be accounted for by changes in ventricular mRNA, which increased about four- to sixfold. In contrast, protein levels in ventricles increased progressively over the 10-d treatment period. Ribonuclease protection analysis indicated that the relative amounts of fibronectin isoforms containing exons designated EIIIA and EIIIB increased during the progression of hypertrophy. When cardiac hypertrophy was induced by mineralocorticoid and salt treatment, increases in ventricular fibronectin mRNA and protein and the induction of alternatively spliced forms of fibronectin were also observed. However, the extent and temporal pattern of fibronectin expression differed between the two experimental models.

Rapid expression of heat shock protein in the rabbit after brief cardiac ischemia.

The effect of brief myocardial ischemia on the expression of heat shock protein (HSP 70) was examined in an in vivo rabbit model of myocardial ischemia using Northern blotting. Functional studies were carried out in the open-chested anesthetized rabbit. The large marginal branch of the left circumflex was occluded four times for 5 min. Using piezoelectric crystals implanted midwall in the ischemic zone, end-diastolic length, end-systolic length, and percent segmental shortening were assessed. Expression of HSP 70 was measured by Northern blotting. A single 5-min coronary occlusion doubled the expression of HSP 70 whereas four cycles of 5 min of ischemia/5 min of reperfusion resulted in a threefold increase in HSP 70 mRNA (P less than 0.001). Measurements with the piezoelectric crystals showed mild myocardial dysfunction concomitant with the increase in HSP 70. This increase in HSP 70 mRNA after repetitive brief ischemia was transient, occurring as early as 1 h and returning to baseline by 24 h after ischemia. Western blot analysis with a monoclonal antibody to HSP 70 was used to compare sham and postischemic myocardial HSP 70 levels. Changes in the amount of HSP 70 were evident as early as 2 h and were even more striking at 24 h.

Platelet-derived growth factor receptors on macrovascular endothelial cells mediate relaxation via nitric oxide in rat aorta.

The effects of platelet-derived growth factor (PDGF) were studied in isolated rings of rat aorta contracted submaximally to phenylephrine. The BB isoform of PDGF elicited relaxation in rings with endothelium and further contraction in rings without endothelium. Both the endothelium-dependent relaxation and endothelium-independent contraction occurred at concentrations known to induce PDGF receptor-mediated responses in cultured cells. Furthermore, the relaxation was isoform specific. This conclusion is supported by the unique ability of PDGF-BB to induce endothelium-dependent relaxations, as well as by studies showing isoform specific, concentration-dependent desensitization of PDGF-BB relaxation. The relaxation induced by PDGF-BB was prevented by N omega-nitro-L-arginine. It was also observed that endothelium-independent contractions to the AB and AA isoforms of PDGF were less than those to PDGF-BB. Contrary to the widely held view that PDGF receptors are not present on the endothelium of macrovessels, these studies provide evidence for an endothelium-dependent, nitric oxide mediated relaxation of rat aorta caused by PDGF via PDGF beta beta-receptors.

Fibronectin biosynthesis in the rat aorta in vitro. Changes due to experimental hypertension.

This study was undertaken to determine if changes in fibronectin biosynthesis accompany the phenotypic changes that occur in aortic tissue following experimental hypertension. An in vitro procedure was developed to measure fibronectin synthesis in aortic rings obtained from normotensive or hypertensive rats. There was a three to sixfold increase in fibronectin biosynthesis by aortic rings taken from rats treated with deoxycorticosterone/salt for 7 and 21 d, the change being more pronounced at 21 d. In contrast, there was no major change at either time point in net incorporation into total protein. Studies comparing fibronectin biosynthesis in aortic rings from Wistar rats and spontaneously hypertensive rats at ages between 10 and 40 wk showed increased fibronectin biosynthesis in older animals of both strains, but only slight differences between strains. Studies using rats infused with angiotensin II showed a correlation between blood pressure elevation and increased aortic fibronectin biosynthesis. Western blot analysis of aortic extracts showed that the fibronectin content was increased in the hypertensive models. The in vitro procedure for measuring fibronectin biosynthesis appears to provide a reliable reflection of in vivo changes in fibronectin expression, and the methodology could prove useful for studying the factors influencing protein expression in vascular tissue.

Angiotensin II-induced cardiac fibrosis in the rat is increased by chronic inhibition of nitric oxide synthase.

These studies were performed to determine if the effects of angiotensin II infusion on the development of cardiac fibrosis could be modified by the chronic inhibition of nitric oxide synthase activity. NG-nitro-L-arginine-methyl ester (L-NAME) was administered to adult Wistar rats in drinking water (40 mg/kg per d). Although blood pressure was maintained at hypertensive levels after 2 wk, cardiac hypertrophy or fibrosis did not occur. Angiotensin II, given for 3 d at a dose which induced little or no blood pressure elevation and minimal if any fibrosis, caused significant fibrosis when given to a rat pretreated for 2 wk with L-NAME. This marked fibrosis did not occur if angiotensin II was given shortly after L-NAME treatment was begun or briefly after discontinuation of L-NAME. The fibrosis that occurred with combined treatment was characterized by increased immunodetectable fibronectin, the presence of inflammatory cells within interstitial and perivascular regions, and increased steady state mRNA levels for matrix genes and atrial natriuretic protein. The data indicated a regulatory role for nitric oxide in modulating the angiotensin II-induced cardiac fibrosis and suggest a potentially important autocrine or paracrine role for nitric oxide in fibroblast proliferation.

Relationship of an abnormal plasma lipoprotein to protection from atherosclerosis in the cholesterol-fed diabetic rabbit.

Alloxan-diabetic rabbits develop a pronounced hypercholesterolemia and hypertriglyceridemia in response to cholesterol feeding. Despite higher levels of plasma cholesterol, these animals have much less atherosclerosis than cholesterol-fed nondiabetics. To determine whether this effect is due to properties of the lipoproteins, we compared chemical, physical, and metabolic characteristics of a very low density lipoprotein (VLDL) fraction (d less than 1.019 g/ml) from the diabetic and nondiabetic cholesterol-fed rabbits. The molar ratio of triglyceride to cholesteryl ester in the particles from diabetic animals ranged from 2:1 to 6:1, and this ratio remained constant in subfractions from individual rabbits. Triglyceride from nondiabetic control animals was a minor component. Differential scanning calorimetry showed a distinct order-disorder phase transition for cholesteryl ester at approximately 42 degrees C in the fractions from control animals, whereas in fractions from most of the diabetics no such transition was observed, indicating that both triglyceride and cholesteryl ester are present in the core of the same particle. The relative amount of apoprotein E in particles from diabetic animals was much less than that of cholesterol-fed controls. The ability of the lipoproteins from both groups to stimulate cholesteryl ester formation in mouse peritoneal macrophages also was tested. Lipoproteins from cholesterol-fed controls stimulated cholesteryl ester formation in a dose-dependent manner, but particles from the diabetic group had little or no effect. The results suggest that the presence of unusual VLDL particles in diabetic cholesterol-fed rabbits is responsible, at least in part, for the reduced incidence of atherosclerosis in this animal model.

A single myocardial stretch or decreased systolic fiber shortening stimulates the expression of heat shock protein 70 in the isolated, erythrocyte-perfused rabbit heart.

The regulation of heat shock protein 70 (HSP 70) expression was examined in the isolated, red blood cell-perfused rabbit heart by Northern and Western blot analysis. In the isovolumic (balloon in left ventricle), isolated perfused heart, HSP 70 mRNA was increased threefold after 30 min and sevenfold at 2 and 4 h compared to normal, nonperfused hearts. To further elucidate the etiology of the increase in HSP 70 mRNA, the effects of decreased systolic shortening (isovolumic heart) and of a single ventricular stretch were examined. Perfusion without the application of a stretch or the presence of a balloon resulted in no increase in HSP 70 mRNA; while a single stretch resulted in a threefold increase in HSP 70 mRNA. These changes were accompanied by an increase in HSP 70 protein by Western blot analysis. To elucidate the signalling mechanism mediating the increase in HSP 70, hearts were perfused with H7, a protein kinase C inhibitor. H7 did not prevent the induction of HSP 70. These results indicate that initiation of expression of myocardial HSP 70 can be stimulated by a single myocardial stretch or by prevention of systolic shortening. These mechanisms may contribute to the rapid expression of HSP 70 after coronary occlusion when dyskinesis, reduced systolic shortening, and increased diastolic segment length all occur.

Rapid expression of fibronectin in the rabbit heart after myocardial infarction with and without reperfusion.

The expression of fibronectin in the repair process after myocardial infarction was studied using two protocols of coronary occlusion in the rabbit: a permanent occlusion or 3 h of occlusion followed by reperfusion (too late for salvage). We found a rapid and progressive increase in cardiac fibronectin expression in the infarcted region of the ventricle. Steady-state mRNA levels for fibronectin increased 13- and 16-fold, respectively, in the permanent and reperfused infarcts 1 d postinfarction. Immunological detection of the protein with a polyclonal antibody against plasma fibronectin showed significant increases of the protein fibronectin in the infarcted myocardium by day 3 in the reperfused group and by day 5 in the permanent coronary occlusion group. Ribonuclease protection assays established the induction of EIIIB containing fibronectin mRNA in both models by day 1 and use of a monoclonal antibody showed an increase in the EIIIA isoform 2 d postinfarction. Increases in steady-state mRNA levels for several collagen types were found in both groups, but these changes occurred after those noted for fibronectin. Thus fibronectin mRNA and protein expression increased rapidly postinfarction suggesting a functional role in the repair process.

Properties of acyl-CoA:cholesterol O-acyltransferase in aortic microsomes from atherosclerotic rabbits.

Acyl-CoA:cholesterol O-acyltransferase (EC 2.3.1.26) was in microsomal fractions from atherosclerotic rabbit aortic tissue. Activity was increased over 70-fold following 8-11 weeks of cholesterol feeding. Comparison of the lipid composition of aortic microsomal fractions from control and cholesterol-fed animals showed a 2-fold increase in the molar ratio of unesterified cholesterol to phospholipid in the cholesterol-fed group, although no change in phospholipid content or composition was found. Aortic microsomes were fractionated by sucrose density gradient centrifugation. Acyl-CoA:cholesterol O-acyltransferase was localized in fractions containing neutral alpha-glucoside activity but was clearly separated from 5'-nucleotidase activity. The cholesteryl ester formed during in vitro incubation of incubations of microsomes with either [1-14C]-oleoyl CoA or [7-3H]cholesterol was localized in the same region of the density gradient as acyl-CoA:cholesterol O-acyltransferase. The studies indicate that the increased acyl-CoA:cholesterol O-acyltransferase activity found in cells from aortic atherosclerotic lesions is due to both an increased amount of enzyme and to an expanded pool of microsomal cholesterol which is available for esterification.

Esterification of cholesterol and 25-hydroxycholesterol by rat liver microsomes.

The properties of an enzyme in rat liver microsomes was described that catalyzed the formation of 25-hydroxycholesteryl ester in the presence of labeled sterol and oleoyl-CoA. The reaction was similar in several respects to that of cholesteryl ester formation by acyl-CoA: cholesterol acyltransferase. Trypsin pretreatment of microsomes inhibited the esterification of both sterols and a similar dose-dependent inhibition was produced by addition of progesterone and several androgens. Microsomes with an enhanced cholesterol content resulting from in vivo treatment with ethinyl estradiol showed increased esterifying activity towards both cholesterol and 25-hydroxycholesterol. Esterification of endogenous microsomal cholesterol was increased by the addition of 25-hydroxycholesterol, concomitant with 25-hydroxycholesteryl ester formation. To assess the relationship between the association of sterols with membranes and sterol ester formation, microsomes were preincubated with either sterol, reisolated by ultracentrifugation in a density gradient and then analyzed chemically or enzymatically. Cholesterol and 25-hydroxycholesterol both associated with microsomes and the added sterol was subsequently esterified. Maximal esterification was only partially dependent on the amount bound. Progesterone, which inhibited sterol esterification, did not bind to microsomes and no inhibition was observed in reisolated microsomes, indicating that the inhibition produced by progesterone was reversible.

Binding of lysophosphatidylcholine to the rat liver fatty acid binding protein.

In this study, lysophosphatidylcholine (lysoPC) was shown to bind to a fatty acid binding protein isolated from rat liver. To demonstrate the binding, lysoPC was incorporated into multilamellar liposomes and incubated with protein. For comparison, binding of both lysoPC and fatty acid to liver fatty acid binding protein, albumin, and heart fatty acid binding protein were measured. At conditions where palmitic acid bound to liver fatty acid binding protein and albumin at ligand to protein molar ratios of 2:1 and 5:1, respectively, lysoPC binding occurred at molar ratios of 0.4:1 and 1:1. LysoPC did not bind to heart fatty acid binding protein under conditions where fatty acid bound at a molar ratio of 2:1. Competition experiments between lysoPC and fatty acid to liver fatty acid binding protein indicated separate binding sites for each ligand. An equilibrium dialysis cell was used to demonstrate that liver fatty acid binding protein was capable of transporting lysoPC from liposomes to rat liver microsomes, thereby facilitating its metabolism. These studies suggest that liver fatty acid binding protein may be involved in the intracellular metabolism of lysoPC as well as fatty acids, and that functional differences may exist between rat liver and heart fatty acid binding protein.

Cholesteryl ester and triglyceride hydrolysis by an acid lipase from rabbit aorta.

The properties of the triglyceride- and cholesteryl ester-hydrolyzing activity by an acid lipase from rabbit aortic tissue were compared under different experimental conditions. Radiolabeled cholesteryl oleate or triolein was incorporated into phospholipid vesicles by sonication and the resulting preparations were used for in vitro studies. No distinction was observed between triglyceride lipase and cholesterol esterase activity in the aortic cytosol fraction following either thermal inactivation, inhibition by a mercurial, fractionation by ammonium sulfate or acid precipitation, or DEAE-cellulose chromatography. Addition of rabbit lipoproteins to the assay system resulted in inhibition of both cholesterol esterase and triglyceride lipase activity. Parallel changes in the hydrolysis of both substrates also were observed when exogenously added lipids were added to the incubation system in various physical states. Specificities of the enzyme system towards different cholesteryl esters were examined. No differences in the rate of hydrolysis were observed between cholesteryl oleate, palmitate and linoleate. The data suggest that a single acid lipase, presumably of lysosomal origin, has broad specificity towards triglycerides and cholesteryl esters, and may play a role in the hydrolysis of these lipids during intralysosomal degradation of lipoproteins.

Binding of acyl-CoA to liver fatty acid binding protein: effect on acyl-CoA synthesis.

The ability of purified rat liver and heart fatty acid binding proteins to bind oleoyl-CoA and modulate acyl-CoA synthesis by microsomal membranes was investigated. Using binding assays employing either Lipidex 1000 or multilamellar liposomes to sequester unbound ligand, rat liver but not rat heart fatty acid binding protein was shown to bind radiolabeled acyl CoA. Binding studies suggest that liver fatty acid binding protein has a single binding site acyl-CoA which is separate from the two binding sites for fatty acids. Experiments were then performed to determine how binding may influence acyl-CoA metabolism by liver microsomes or heart sarcoplasmic reticulum. Using liposomes as fatty acid donors, liver fatty acid binding protein stimulated acyl-CoA production, whereas that from heart did not stimulate production over control values. 14C-labeled fatty acid-fatty acid binding protein complexes were prepared, incubated with membranes, and acyl-CoA synthetase activity was determined. Up to 70% of the fatty acid could be converted to acyl-CoA in the presence of liver fatty acid binding protein but in the presence of heart fatty acid binding protein, only 45% of the fatty acid was converted. Liver but not heart fatty acid binding protein bound the acyl-CoA formed and removed it from the membranes. The amount of product formed was not changed by additional membrane, enzyme cofactors, or incubation time. Additional liver fatty acid binding protein was the only factor found that stimulated product formation. Acyl-CoA hydrolase activity was also shown in the absence of ATP and CoA. These studies suggest that liver fatty acid binding protein can increase the amount of acyl-CoA by binding this ligand, thereby removing it from the membrane and possibly aiding transport within the cell.

Peptidyl dipeptidase in rabbit brain microvessels.

The activity of peptidyl dipeptidase (peptidyldipeptide hydrolase, EC 3.4.15.1), also known as angiotensin-converting enzyme, was studied in small blood vessel preparations isolated from rabbit brain. The vascular preparation contained arterioles and capillaries and was essentially free of extravascular material. Enzymatic activity was demonstrated in microvessel homogenates using both hippuryl-histidyl-leucine and tritium-labeled angiotensin I as substrates. Activity in the microvessels was dependent on the presence of chloride ion and was sensitive to inhibition by converting enzyme inhibitors previously shown to be effective in both vivo and in vitro. Specific activity in the micro-vessels was approximately 20 times that in homogenates of brain, and was almost 60% of that found in rat lung homogenates. The data were consistent with an endothelial localication for peptidyl dipeptidase in the cerebral vasculature and supports the proposal that this enzyme has a physiological role in extrapulmonary vascular beds.

Persistent activation of nuclear factor-kappaB by interleukin-1beta and subsequent inducible NO synthase expression requires extracellular signal-regulated kinase.

The role of extracellular signal-regulated kinase (ERK) was studied in the signaling pathway by which interleukin-1beta (IL-1beta) increases the expression of inducible NO synthase (iNOS) in rat vascular smooth muscle cells. IL-1beta induced a rapid and transient activation of nuclear factor-kappaB (NF-kappaB), followed by a prolonged activation of NF-kappaB that was required to induce iNOS expression. Either PD98059 or U0126, selective inhibitors of ERK activation, did not influence IL-1beta-induced early activation but effectively reduced the prolonged activation of NF-kappaB and significantly reduced IL-1beta induction of iNOS. Transfection with antisense, but not sense, phosphorothioate-modified oligodeoxynucleotides directed toward ERK also reduced IL-1beta-induced prolonged NF-kappaB activation and iNOS expression. IkappaBbeta, but not IkappaBalpha degradation, induced by IL-1beta was markedly attenuated when ERK activation was inhibited and could be partially responsible for the persistent NF-kappaB activation. These data suggest that ERK activity is required for persistent NF-kappaB activation by IL-1beta that is necessary for iNOS gene expression.

Angiotensin II and Cardiac Fibrosis.

The renin-angiotensin system has been linked to the pathogenesis of cardiac fibrosis through the use of experimental animal models and by clinical studies. This review emphasizes recent findings implicating the direct action of angiotensin II on cardiac cell types, particularly the cardiac fibroblast, as a causative factor in the development of cardiac fibrosis. The possible influence of other factors acting in concert with angiotensin II either to increase or to attenuate the fibrotic process is also discussed. Nitric oxide is considered as an example of a paracrine agent that can antagonize either the hemodynamic or cellular effects of angiotensin II during both physiological and pathological processes. (Trends Cardiovasc Med 1996;6:193-198).

N-Acetyl-L-cysteine potentiates interleukin-1beta induction of nitric oxide synthase : role of p44/42 mitogen-activated protein kinases.

We have reported previously that N-acetyl-L-cysteine facilitated interleukin-1beta-induced nitric oxide synthase (iNOS) expression in rat vascular smooth muscle cells. The present study compares the effect of N-acetyl-L-cysteine with other antioxidants and tested the possibility that N-acetyl-L-cysteine potentiates iNOS induction by a mechanism involving activation of p44/42 mitogen-activated protein kinases (MAPKs). The effect of N-acetyl-L-cysteine on potentiating interleukin-1beta-induced nitrite production and iNOS expression was mimicked either by the enantiomers, L-cysteine and D-cysteine, or by a non-thiol-containing antioxidant, L-ascorbic acid. Interleukin-1beta activated p44/42 MAPK, and this activation was enhanced in the presence of N-acetyl-L-cysteine. Inhibition of p44/42 MAPK phosphorylation by the selective inhibitor PD98059 clearly inhibited iNOS expression induced by interleukin-1beta either in the absence or in the presence of N-acetyl-L-cysteine. These observations, combined with previous results, indicate that p44/42 MAPK activation is required for interleukin-1beta induction of iNOS and that N-acetyl-L-cysteine may act as a reducing agent and facilitate interleukin-1beta-induced iNOS expression through a reduction/oxidation-related mechanism involving potentiation of cytokine activation of the p44/42 MAPK signaling pathway.

N-acetyl-L-cysteine enhances interleukin-1beta-induced nitric oxide synthase expression.

The effect of N-acetyl-L-cysteine on interleukin-1beta-induced nitric oxide synthase expression was studied in rat vascular smooth muscle cells to determine if the reduction/oxidation state would modulate cytokine-induced changes. Interleukin-1beta induced the production of nitrite, a stable metabolite of nitric oxide in a time- and dose-dependent manner. Cytokine-induced nitrite production was enhanced by the addition of N-acetyl-L-cysteine in a dose-dependent manner, with a >50% increase produced by the addition of 1 mmol/L N-acetyl-L-cysteine. There was no influence on nitrite production when the cells were treated with N-acetyl-L-cysteine alone. Northern and Western blot analyses revealed that the upregulation of interleukin-1beta-induced nitric oxide production by N-acetyl-L-cysteine resulted from an enhanced expression of inducible nitric oxide synthase. Interferon-gamma or tumor necrosis factor-alpha when used alone had no influence on nitrite production in the absence or presence of N-acetyl-L-cysteine. Nitrite accumulation was higher by the cells treated with interleukin-1beta combined with either interferon-gamma or tumor necrosis factor-alpha compared with those treated with interleukin-1beta alone. N-Acetyl-L-cysteine upregulated nitrite production and inducible nitric oxide synthase expression induced by combination treatment with interleukin-1beta and either interferon-gamma or tumor necrosis factor-alpha. However, N-acetyl-L-cysteine had no significant influence in cytokine-induced activation of nuclear factor-kappaB or signal transducer and activator of transciption-1, as assessed by electrophoretic mobility shift assays. These results demonstrate that N-acetyl-L-cysteine possibly acted as a thiol-containing reducing agent and facilitated the expression of inducible nitric oxide synthase in rat vascular smooth muscle cells by cytokines through a mechanism that is independent of nuclear factor-kappaB or signal transducer and activator of transciption-1.

Properties of angiotensin-converting enzyme in intact cerebral microvessels.

Angiotensin-converting enzyme (ACE) was studied in preparations of microvessels isolated from rabbit cerebral cortex. Activity was determined by measuring the degradation of hippuryl-histidyl-leucine (Hip-His-Leu) by the intact microvessels in a physiological salt solution at pH 7.4. ACE activity was dependent on both substrate and chloride ion concentration and was inhibited by captopril in a manner similar to that observed previously with tissue homogenates. Angiotensin I was rapidly degraded by the intact microvessels, even in the presence of 10(-6)M captopril. An advantage of the methodology employed was the ability to pretreat the microvessels and then assess the effect of pretreatment by transfer to a postincubation assay system. Pretreatment with a hyperosmolar urea solution did not change ACE activity or cause release of ACE from the microvessels, although lactic dehydrogenase and lysosomal enzymes were released. Pretreatment with captopril caused a lag in the subsequent degradation of Hip-His-Leu, presumably reflecting dissociation of inhibitor from the cell-associated enzyme. ACE activity was unaffected by hypoxic or anoxic incubation conditions. The ability to measure ACE activity of the microvessels in vitro provides a unique opportunity to study the properties of the enzyme in intact cerebrovascular endothelial cells.