F. M. Bumpus

Angiotensin II-forming pathways in normal and failing human hearts.

Reduced preload and afterload to the heart are important effects of angiotensin converting enzyme (ACE) inhibitors in the treatment of congestive heart failure. However, since angiotensin II (Ang II) directly increases the strength of myocardial contraction, suppression of Ang II formation by ACE inhibitors could potentially reduce the beneficial effects of Ang II on the failing heart. To study how ACE inhibition suppresses cardiac Ang II formation in man, we characterized ACE-dependent and ACE-independent Ang II-forming pathways in eight normal and 24 failing human hearts obtained at cardiac transplantation. Ang II-forming activity in left ventricular (LV) membrane preparations was assessed by measuring the conversion of [125I]angiotensin I (Ang I) to [125I]Ang II. LV [125I]Ang II-forming activity in normal hearts (35.5 +/- 2.7 fmol/min/mg, n = 8) was not different from that in hearts from patients with ischemic cardiomyopathy (25.5 +/- 2.9 fmol/min/mg, n = 9) and was 48% lower (p less than 0.001) in hearts from patients with idiopathic cardiomyopathy (18.5 +/- 1.9 fmol/min/mg, n = 15).(ABSTRACT TRUNCATED AT 250 WORDS)

Cellular localization and regional distribution of an angiotensin II-forming chymase in the heart.

The human heart is a target organ for the octapeptide hormone, angiotensin II (Ang II). Recent studies suggest that the human heart contains a dual pathway of Ang II formation in which the major Ang II-forming enzymes are angiotensin I-converting enzyme (ACE) and chymase. Human heart chymase has recently been purified and its cDNA and gene cloned. This cardiac serine proteinase is the most efficient and specific Ang II-forming enzyme described. To obtain insights into the cardiac sites of chymase-dependent Ang II formation, we examined the cellular localization and regional distribution of chymase in the human heart. Electron microscope immunocytochemistry using an anti-human chymase antibody showed the presence of chymase-like immunoreactivity in the cardiac interstitium and in cytosolic granules of mast cells, endothelial cells, and some mesenchymal interstitial cells. In the cardiac interstitium, chymase-like immunoreactivity is associated with the extracellular matrix. In situ hybridization studies further indicated that chymase mRNA is expressed in endothelial cells and in interstitial cells, including mast cells. Tissue chymase levels were determined by activity assays and by Western blot analyses. Chymase levels were approximately twofold higher in ventricles than in atria. There were no significant differences in chymase levels in ventricular tissues obtained from non-failing donor hearts, failing ischemic hearts, or hearts from patients with ischemic cardiomyopathy. These findings suggest that a major site of chymase-dependent Ang II formation in the heart is the interstitium and that cardiac mast cells, mesenchymal interstitial cells, and endothelial cells are the cellular sites of synthesis and storage of chymase. In the human heart, because ACE levels are highest in the atria and chymase levels are highest in ventricles, it is likely that the relative contribution of ACE and chymase to cardiac Ang II formation varies with the cardiac chamber. Such differences may lead to differential suppression of cardiac Ang II levels during chronic ACE inhibitor therapy in patients with congestive heart failure.

A New, Long-Lasting Competitive Inhibitor of Angiotensin

An analog of angiotensin II, [Sar1, Ile8]-angiotensin II, has a potent and long-lasting competitive antagonistic effect against angiotensin II when tested for its myotropic action on the isolated rabbit aorta and for its effect on blood pressure in anesthetized cats and dogs. Compared to [Ile8]-angiotensin II, the new analog has equal antagonistic potency on the isolated system but a much greater potency in vivo. It is assumed that sarcosine in position 1 protects the peptide against enzymatic degradation and enhances its half-life. This study demonstrates that the modification in both positions 1 and 8 are important for the in vivo antagonistic potencies of angiotensin analogs.

Angiotensins and the failing heart. Enhanced positive inotropic response to angiotensin I in cardiomyopathic hamster heart in the presence of captopril.

We examined the hypothesis that the positive inotropic effect of angiotensin I (Ang I) may be retained in the presence of angiotensin converting enzyme inhibitors so that it may have a direct beneficial effect on the heart. Accordingly, isolated perfused hearts (Langendorff preparation) of 300-day-old cardiomyopathic hamsters (a model of spontaneous cardiomyopathy) and age-matched normal hamsters (controls) were infused with Ang I in the presence of captopril; propranolol was added to the perfusing medium to block catecholamine-mediated effects of angiotensins on the heart. Left ventricular developed pressure and the rate of increase in left ventricular developed pressure increased significantly (p less than 0.001) in both the cardiomyopathic and the normal hamster heart despite concomitant reduction in myocardial flow rate favoring a direct inotropic effect of Ang I in both normal and myopathic hearts; these changes were significantly higher by almost threefold in the cardiomyopathic than in the normal hamsters (p less than 0.01) and were blocked by the angiotensin II (Ang II) antagonist [Sar1,Thr8]Ang II. Comparing dose-left ventricular contractility response curves for Ang I and Ang II, ED50 for responses was identical in both normal and myopathic hearts, whereas peak responses to Ang II were double those to Ang I in normal hearts but were almost identical in the myopathic hearts. Binding of [125I]Ang II in six cardiomyopathic and four normal hamster hearts was of high affinity, but there was no evidence for Ang I-saturable high-affinity binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Competitive antagonism of 8-ala-angiotensin II to angiotensins I and II on isolated rabbit aorta and rat ascending colon

Abstract 8-Alanine angiotensin II has a specific competitive antagonistic effect against angiotensins I and II when assayed on the rabbit isolated aortic strip and rat colon; but by itself it has no myotropic action on these smooth muscle preparations even et very high concentrations. In pentobarbital anesthetized cats, 8-ala-angiotensin II does not antagonize the pressor and intestinal inhibitory effect of angiotensins I and II. The possible mechanism of these effects of 8-ala-angiotensin II is discussed.

Regulation of angiotensin II in rat adrenal gland.

Levels of angiotensin II immunoreactivity In the rat adrenal gland are over one hundredfold higher than those in plasma. It is unclear, however, whether the major source of adrenal angiotensin II immunoreactivity is intracellular synthesis by a local renin-angiotensin system, uptake by angiotensin II receptors, or both. Our studies show that angiotensin II immunoreactivity in the adrenal gland is predominantly attributable to angiotensin II (>75%). Angiotensin III (16%) and other angiotensin II fragments are also present. The majority of angiotensin II immunoreactivity (73%), renin activity (73%), and angiotensin II receptor binding activity (66%) in the adrenal gland is located in the capsular glomerulosa cell layers. Dehydration produced by 2% NaCl imbibition decreased these activities in the capsular-glomerulosa. In the fasciculate-medullary regions of the adrenal gland, dehydration decreased renin activity but not angiotensin II immunoreactivity or angiotensin II receptor binding activity. Combined data from control and dehydrated rats showed a close correlation of the capsular-glomerulosa angiotensin n immunoreactivity with angiotensin II receptor binding activity (r =0.94, p <0.001) and a weaker, nonsignificant correlation with renin activity (r = 0.66, p <0.1). In the fasciculata-medullary cell layers, no significant correlations were found between angiotensin II immunoreactivity and either renin or angiotensin n receptor binding activity. These data demonstrate that functionally distinct layers of the rat adrenal gland differentially regulate angiotensin II receptors and the renin-angiotensin system. These data also suggest that the majority of angiotensin II immunoreactivity in the adrenal capsular-glomerulosa is derived from receptor-mediated sequestration of extracellular angiotensin II by its receptors and is not due to intracellular synthesis of the peptide.

Chemical Assay of Phospholipid Renin Preinhibitor in Canine and Human Blood

A phospholipid previously shown to be converted to a lysophospholipid renin inhibitor has been demonstrated in the plasma and red cells of man and the dog. This inhibitor precursor, designated preinhibitor, is chromatographically identical to the compound previously isolated from kidneys of dogs and hogs. A method for its quantitative measurement in plasma and red cells is presented. Normal dogs tested so far have an average of 119.6 μg of preinhibitor phospholipid/ml of blood; 16.3 μg of this is in the plasma. These concentrations are constant (in red cells even increased) 48 hr after bilateral nephrectomy. To assess the physiological importance of preinhibitor, it may be necessary to measure its active lyso derivative as well as the lipase(s) most directly involved in maintaining blood levels of these two phospholipids.

A potent competitive antagonist of angiotensin II

8-Isoleucine-angiotensin II has a specific competitive antagonistic effect against angiotensin II both in vitro isolated rabbit aorta and rat uterus and in vivo on the rat and cat blood pressure. It also competitively antagonizes the inhibitory effect of angiotensin II on the cat terminal ileum. Using Schilds parameters to describe competitive antagonism, 8-isoleucine-angiotensin II has a pA2 value of 9.21, a log K2 value of 8.86 at a molar concentration of 10−9.34 and a log K2 value of 9.47 at a concentration of 10−7.835. Based on these data it is concluded that 8-isoleucine-angiotensin II is the most potent competitive inhibitor of angiotensin II so far reported.

Lung Perfusion with Angiotensins I and II: Evidence of Release of Myotropic and Inhibitory Substances

Perfusion of isolated rat and guinea pig lungs with angiotensin I or II in Krebs9 solution results in significant losses of peptide activity. This was not prevented by angiotensin cofactor. In addition to being converted to the octapeptide during passage through the lung, angiotensin I released a myotropic substance which is different from serotonin, epinephrine, norepinephrine, and prostaglandin E 1 and F 2α . This substance was not one of the several fragments of angiotensin I tested. There was a decreased responsiveness of rabbit aorta following repeated exposure to lung perfusate containing angiotensin I. Since repeated direct exposure to this peptide and metabolic fragments in Krebs9 solution did not produce this effect, it was assumed to be caused by a substance released from the lungs.

Evidence for the existence of a family of biologically active angiotensin I-like peptides in the dog central nervous system.

A family of angiotensin I-like peptides has been derived from endogenous precursors present in dog cerebrospinal fluid after incubation with species homologous renin. These peptides are immunologically and pharmacologically similar to [Ile5]angiotensin I, and have molecular weights ranging between 1300 and 2200 daltons. The presence of precursors in the cerebrospinal fluid able to generate various biologically active angiotensin I-like peptides dissimilar to plasma angiotensin I supports the concept of a local angiotensin I-forming system in the brain.