H. Herzlinger

Identification and characterization of angiotensin II receptors in rat epididymal adipocyte membranes

To better understand the role of the mitogenic vasoactive peptide angiotensin II (AII) in growth and differentiation, we have investigated the existence of membrane receptors for this peptide in rat adipocytes. Following isolation of epididymal fat cells, membrane protein was removed and incubated with varying concentrations of 125I-AII with or without submicromolar concentrations of unlabeled AII. Binding of AII was highly specific, rapid, and reversible. Scatchard analysis indicated that adipocyte membranes contain a high-affinity AII receptor with a Kd of 0.90 nmol/L and a binding site concentration (Bmax) of 53.7 fmol/mg protein. Additional pharmacologic analyses resulted in a rank order potency for peptide agonists and antagonists similar to that reported for the vascular receptor. Determination of subtype specificity with selective organic compounds indicated that the epididymal adipocyte receptor was displaced at low concentrations of DuP753, a selective AT1 subtype antagonist. These studies have successfully identified and characterized a high-affinity membrane receptor for AII in fat cells, further establishing adipose tissue as a peripheral site containing regulatory components of the local renin-angiotensin system.

Transforming growth factor alpha and atrial natriuretic peptide in white adipose tissue depots in rats

Abstract. To detect the presence in adipose tissue of peptides known to affect tissue growth and to investigate potential regional differences, epididymal and perirenal adipose tissue depots from male Sprague‐Dawley rats were separated into adipocyte and stroma‐vascular fractions by collagenase digestion, sequential centrifugation and filtration. Identity and integrity of the fractions were demonstrated by light and electron microscopy, while dose‐response curves for angiotensin‐converting enzyme (ACE) were performed, revealing maintained functional capacity of the stroma‐vascular fraction. ACE, atrial natriuretic peptide (ANP), and transforming growth factor‐alpha (TGF‐alpha) concentrations were significantly greater in epididymal than perirenal stroma‐vascular tissue. Adipocyte fractions from both depots contained significant concentrations of ANP and TGF‐alpha. There was no detectable ACE in the adipocyte fractions, indicating that no contaminating stromal‐vascular cells were present in these fractions. These data show significant concentrations of peptides with effects on growth in subfractions of adipose tissue and demonstrate regional differences in concentrations between fat depots.

Effect of the time interval between blood sampling and assay on serum angiotensin I-converting enzyme activity from captopril-treated rats

Intra-arterial injection of captopril (1 mg/kg) effectively lowered arterial blood pressure in aorta-coarcted hypertensive rats along with an associated reduction of serum angiotensin I-converting enzyme (ACE) activity. ACE activity in serum samples from captopril-treated animals that were assayed within 60 min after collection was inhibited 93%. However, this inhibition progressively decreased as the interval between time of assay and blood collection increased. This information would appear to be of considerable value in planning experiments for the determination of serum ACE activity from captopril-treated animals.

Aortic vascular and atrial responses to (±)‐1‐O‐octadecyl‐2‐acetyl‐glyceryl‐3‐phosphorylcholine

1 The effects of (±)‐1‐O‐octadecyl‐2‐acetyl‐glyceryl‐3‐phosphorylcholine (octadecyl‐AGPC) were studied in three types of aortic vascular smooth muscle preparations, namely, strips, rubbed and unrubbed rings, and an atrial preparation in normotensive rats. 2 In the resting tension state, octadecyl‐AGPC did not elicit significant contractions in either rubbed or unrubbed ring preparations at concentrations lower than 1 × 10−4m. However, at a concentration of 3 × 10−4 m, octadecyl‐AGPC markedly contracted both types of ring preparations. This contractile response was partially antagonized by pretreatment with reserpine and completely blocked by phentolamine (1 × 10−6m). 3 In preparations contracted with noradrenaline (NA), octadecyl‐AGPC elicited biphasic responses in intact ring preparations; an initial relaxation followed by contraction. Octadecyl‐AGPC induced only a slight contraction in strips and a slight relaxation in the rubbed ring preparation. 4 Octadecyl‐AGPC did not elicit any significant effect on chronotropy or inotropy at concentrations up to 3 × 10−5 m. When the concentration was 1 × 10−4 m, octadecyl‐AGPC produced significant positive chronotropic and inotropic effects on spontaneously beating right and electrically driven left atrial preparations, respectively. Both effects were blocked by propranolol (5 × 10−8m); reserpine pretreatment antagonized only the chronotropic response. 5 In [3H]‐dihydroalprenolol ([3H]‐DHA) binding studies, octadecyl‐AGPC had a Kd of 427.85 μm and thus was much less potent than isoprenaline (Kd = 465.10 nm) or propranolol (Kd = 4.4 nm) in displacing [3H]‐DHA in rat cardiac membrane preparations. 6 In conclusion, relaxation and contraction induced by octadecyl‐AGPC in aortic preparations is an indirect rather than a direct effect. An unknown factor released from endothelial cells is responsible for aortic smooth muscle relaxation by octadecyl‐AGPC while released NA appears to be responsible for aortic vascular contraction and for the positive chronotropic and inotropic effects in the atrial preparations.

A COMPARISON OF CARDIAC REACTIVITY AND β‐ADRENOCEPTOR NUMBER AND AFFINITY BETWEEN AORTA‐COARCTED HYPERTENSIVE AND NORMOTENSIVE RATS

1 The effects of noradrenaline (NA) and isoprenaline on isolated atria from aorta‐coarcted hypertensive rats (AHR) at early (6 day) and chronic (28 day) stages of hypertension were studied and compared with time‐matched, sham‐operated, normotensive rats (SNR). The number and affinity of β‐adrenoceptors ((−)‐[3H]‐dihydroalprenolol binding sites) were also studied in cardiac membranes prepared from these animals. 2 Six and 28 days after complete ligation of the abdominal aorta between the two renal arteries, rats became hypertensive with significantly greater arterial blood pressures than time‐matched SNR. 3 At both stages of hypertension, the atrial inotropic or chronotropic effects of NA and isoprenaline from hypertensive rats were similar to time‐matched SNR. Moreover, no differences in atrial reactivity were observed between the early and chronic stages of hypertension. 4 Irrespective of the stage of hypertension, cardiac membranes from the AHR contained the same number of β‐adrenoceptors as time‐matched SNR. In addition, the receptor affinity for the radioligand within each group was equivalent. However, the chronic stage hypertensive rats and their time‐matched controls contained fewer β‐adrenoceptors and these receptors had greater affinity for the radioligand when compared with cardiac membranes from rats at the early stage of hypertension and their controls. 5 The observed equivalent chronotropic and inotropic responses to NA and isoprenaline between the hypertensive and normotensive rats in both stages of hypertension may be explained in terms of similar receptor number and receptor binding affinity. 6 The reduced number of β‐adrenoceptors with greater binding affinity in day 28 normotensive or hypertensive rats may be a compensatory mechanism for these animals to maintain normal cardiac function with increasing age.

Disposition of catecholamines in cardiovascular tissues of aorta coarcted hypertensive rats

Aorta-coarcted hypertensive rats and sham-operated normotensive rats were compared in order to assess the contribution of sympathetic nervous system activity to the elevated blood pressure in these rats at an early (6 days) and chronic (42 days) stage of hypertension. Norepinephrine (NE), epinephrine (E) and dopamine (DA) levels were quantitated in plasma, heart and vascular tissues (aorta, inferior vena cava, mesenteric artery and vein) using a radioenzymatic procedure. Body weight was significantly reduced and mean arterial blood pressure (MABP) significantly increased in the coarcted rats at both stages of hypertension. Plasma catecholamines did not differ at either stage of hypertension. The NE content of the heart and mesenteric artery was significantly decreased in the coarcted rats at both stages of hypertension but unchanged in the other vessels studied. E and DA levels in the heart and all vasculature analyzed remained unaltered at both stages of hypertension. The present results suggest that neither E nor DA makes a major contribution to the development and maintenance of hypertension in the aorta-coarcted rat. The observation of the reduced cardiac NE concentration in the coarcted rats together with literature reports of similar observations in other animal models of hypertension suggests that myocardial NE depletion is a common feature of the hypertension and not dependent on the methodology used to produce that hypertension.

Effects of continuous angiotensin I-converting enzyme blockade on blood pressure, sympathetic activity and renin-angiotensin system in stroke-prone spontaneously hypertensive rats

Abstract The purpose of this study was to examine the effects of continuous angiotensin converting enzyme (ACE) blockade in stroke-prone spontaneously hypertensive rats (sp-SHR) on the renin-angiotensin system and on sympathetic activity. The pressor response to angiotensin II (AII) and norepinephrine (NE) were also examined after chronic blockade of ACE and compared to that of saline-treated controls. Captopril treatment had no effect on body weight. Serum ACE was significantly reduced on day 1; an effect that persisted through day 6 and day 10. Plasma renin activity (PRA) was elevated significantly on day 1 and remained at this high level throughout the 10 day observation period. Plasma NE was not altered by the chronic ACE blockade except on day 1, where there was a slight elevation of plasma NE in both groups. Pressor responses to AII and NE were not changed after chronic captopril treatment. It is observed that chronic inhibition of the renin-angiotensin system with captopril in sp-SHR resulted in a reduction of blood pressure, reduced serum ACE activity and elevated PRA. The constant plasma NE levels suggest that chronic inhibition of the renin-angiotensin system does not affect sympathetic activity. This study also indicates that long term inhibition of ACE does not alter pressor responses to either AII or NE.

Effects of CL 115,347, (±)-15-deoxy-16-vinyl-PGE2 methyl ester, on cardiovascular responses and plasma catecholamines in pithed stroke-prone spontaneously hypertensive rats during sympathetic stimulation

The effect of CL 115,347, a topically active antihypertensive PGE2 analog, and PGE2 on changes in blood pressure (BP), heart rate (HR) response and plasma epinephrine (E) and norepinephrine (NE) levels induced by stimulation of the sympathetic spinal cord outflow were studied in pithed stroke-prone spontaneously hypertensive rats (SHRSP). Surgical pithing significantly reduced plasma E but not NE levels suggesting that the sympathoadrenal medullary system differentially affects E and NE release. Sympathetic stimulation of the spinal cord of pithed SHRSP increased HR, BP, plasma E and NE levels. Topically applied CL 115,347 (0.001-0.2 mg/kg) dose-dependently decreased BP, while intravenously infused PGE2 (30 micrograms/kg/min) did not alter BP except for a brief initial drop. Topical application of CL 115,347 (0.1 mg/kg) also inhibited BP responses to sympathetic stimulation without effects on HR or plasma E or NE levels. Intravenous infusion of PGE2 (30 micrograms/kg/min) inhibited both BP and HR responses to spinal cord stimulation but did not alter plasma catecholamine levels. These studies in SHRSP suggest that CL 115,347 and PGE2 modulate cardiovascular responses mainly via postjunctional effects, but act differently on the cardiovascular elements, viz. CL 115,347 acts primarily on blood vessels while PGE2 acts on blood vessels and heart.

Evidence for the lack of interaction between (±)‐ 1‐O‐octadecyl‐2‐acetylglyceryl‐3‐phosphorylcholine and α‐adrenoceptors in vivo and in vitro

1 The interactions of (±)‐1‐O‐octadecyl‐2‐acetylglyceryl‐3‐phosphorylcholine (octadecyl‐AGPC) with α‐adrenoceptors were studied in rat mesenteric artery, cat nictitating membrane and on the blood pressure of the cat and spontaneously hypertensive (SH) rat. 2 Using a direct radioligand α‐adrenoceptor binding assay in particulate fractions of rat mesenteric arteries, octadecyl‐AGPC was found to be 5 × 107 and 75 times less potent than prazosin and noradrenaline (NA), respectively, in displacing (2,6‐dimethoxyphenoxyethyl)‐aminomethyl‐1,4‐benzodioxane ([3H]‐WB 4101 — a selective probe for the identification of α‐adrenoceptors). 3 In the cat, intravenous infusions of octadecyl‐AGPC, which produce a hypotensive response, did not attenuate nictitating membrane contractions in vivo in response to intravenous injections of NA, adrenaline (Ad) or to electrical stimulation of the postganglionic fibres of the superior cervical ganglion. In these experiments, the pressor responses to NA or Ad were not affected by octadecyl‐AGPC. Phentolamine, on the other hand, attenuated nictitating membrane contractions and blood pressure responses to Ad or NA. 4 In the SH rat, octadecyl‐AGPC decreased mean arterial blood pressure (MABP). After an intravenous dose of phentolamine which lowered MABP, the depressor response to octadecyl‐AGPC was reduced. When MABP in the phentolamine‐treated SH rat was restored to its initial level with an infusion of angiotensin II (AII), the depressor response to octadecyl‐AGPC was restored to its original magnitude. The effectiveness of α‐adrenoceptor blockade under these experimental conditions was monitored with intravenous NA and Ad. 5 Thus, based on radioligand binding studies and pharmacological studies, it is concluded that octadecyl‐AGPC lacks the ability to interact with α‐adrenoceptors.