Peter Cervoni

VPA-985, a nonpeptide orally active and selective vasopressin V2 receptor antagonist.

The introduction of the thiazides as orally-active diuretics about forty years ago (1), and other more effective low-ceiling diuretics thereafter, revolutionized the treatment of edema, ascites, hypertension and related diseases. Later, the addition of potent high-ceiling (loop) and potassium-sparing diuretics provided clinicians with a wide choice of diuretics to eliminate retained sodium and water (2). However, it was soon evident that many patients became refractory to these saluretic agents and some developed hyponatremia (serum sodium < 130 mEq/L) (3, 4, 5). Hyponatremia also occurs in the syndrome of inappropriate antidiuretic hormone secretion (SIADH), in patients with congestive heart failure (CHF), liver cirrhosis with ascites, renal failure, and many other disorders where the plasma vasopressin concentrations are inappropriately high for any given plasma osmolality. Under these conditions, an aquaretic (water diuretic), not a conventional diuretic, would be the drug of choice to promote the excretion of the retained body water and to normalize plasma osmolality and sodium concentration (6, 7, 8). As vasopressin (AVP, antidiuretic hormone (ADH)) acting at V2 receptors in the collecting ducts controls water re-absorption (7, 8), considerable effort has been spent over many years to develop vasopressin Vz receptor antagonists or agents that could inhibit the release of vasopressin from the posterior pituitary (8,9). Many peptide vasopressin analogs have been developed as vasopressin V2 receptor antagonists, and two of them, SK&F 101926 and SK&F 105494, showed excellent V2 antagonistic activity in many animal models, including nonhuman primates. Unfortunately, in humans, both compounds behaved as vasopressin V2 agonists (9). Recently, three nonpeptidic and orally active vasopressin receptor antagonists have been described in the literature. The first two, OPC-31260 (10), and SR 121463A (11), are V2 selective, while the third compound, YM087 (12), is a dual V1a/V2 receptor antagonist.

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.

Hypotensive and vasodilatory activity of (±) 1-0-octadecyl-2-acetyl glyceryl-3-phosphorylcholine in the normotensive rat

Synthetic (+/-) 1-O-octadecyl-2-acetyl-glyceryl-3-phosphorylcholine (octadecyl-AGPC) in microgram/kg doses given intravenously effectively and potently lowered mean arterial blood pressure in conscious and anesthetized normotensive rats. The hypotensive activity was much more pronounced in the anesthetized rat than in the conscious rat. The hypotension was associated with a significant elevation in plasma renin activity (PRA). In the rat in which the hindquarters were perfused, octadecyl-AGPC given intraarterially effectively decreased the perfusion and systemic pressures in a dose-dependent manner. Pharmacological blockade with specific cholinergic, histaminergic or beta-adrenergic receptor antagonists, did not block or attenuate the octadecyl-AGPC-induced reduction in perfusion or systemic pressure. These results suggest that the hypotensive activity of octadecyl-AGPC in the normotensive rat is the result of direct vasodilation and not the result of cholinergic, histaminergic or beta-adrenergic receptor interaction.

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.

Endothelium-dependent basilar and aortic vascular responses in normotensive and coarctation hypertensive rats.

The responsiveness of acetylcholine (ACh), nitroglycerin (NG) and norepinephrine (NE) (aorta only) in both basilar arteries (BA) and thoracic aortic (TA) rings from coarctation hypertensive rats (CHR) were studied and compared to their sham-operated normotensive control rats (SNR). The effects of these agents were also evaluated in TA or BA with and without endothelium from naive normotensive rats (NNR). Blood pressure (BP) and plasma renin activity (PRA) of CHR were significantly higher than their time-matched SNR. Endothelium removal from TA of NNR significantly enhanced NE and NG sensitivity and reduced the maximum ACh relaxation. Removal of BA endothelium of NNR abolished ACh-induced relaxation but had no effect on NG-induced relaxation. In BA from CHR at any stage of hypertension studied, the sensitivity and maximum relaxation induced by ACh or NG were not significantly different than their respective time-matched SNR. ACh sensitivity of TA did not change in 1 Day CHR but decreased in 4 and 14 Day CHR. NG sensitivity increased, did not change and decreased in 1, 4 and 14 Day CHR, respectively. NE sensitivity increased in all stages of hypertension. These data suggest that in coarctation-induced hypertension there is a complex progression of events in TA which is modulated by different mechanisms as evidenced by the changes in the effects of NE, ACh and NG at various stages of hypertension. The results also suggest that the vascular endothelium of TA but not of BA may provide an acute protective mechanism to counteract the imbalance created by the increased sensitivity of smooth muscle cells to contractile agonists in the early stage of hypertension. However, persistent hypertension appears to override this mechanism.

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.

Effect of Experimental Obesity and Subsequent Weight Reduction upon Circulating Atrial Natriuretic Peptide

Abstract The effect of obesity and weight reduction upon circulating concentrations of atrial natriuretic peptide was assessed in an experimental model of the disease. Obese rats weighing in excess of 750 g were compared with formerly obese animals subjected to a 15-week period of caloric restriction resulting in a 40% reduction in body weight. Mean adipocyte size was significantly reduced with weight loss, as was estimated body fat. Mean arterial blood pressure remained normotensive for both groups, but a significant reduction in heart rate was associated with weight reduction. Circulating atrial natriuretic peptide was significantly elevated in the lean rats, which also exhibited decreased plasma renin activity and a negative sodium balance. Analysis of heart to body weight ratios implied that an obesity-associated, volume-induced cardiac hypertrophy remained even after the normalization of body fat. These results suggest that the diuresis and natriuresis accompanying weight reduction may be facilitated by atrial natriuretic peptide, which was elevated in part due to a persistent left ventricular hypertrophy following the transition from the obese to lean condition.

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.

Effect of Contractile Activity on Rat Skeletal Muscle β-Adrenoceptor Properties

Abstract The effect of fiber type and endurance exercise training on skeletal muscle β-adrenoceptor properties were assessed using a direct radioligand binding technique. Six separate muscles, composed of a variety of different fiber types, were examined in treadmill trained and sedentary rats. In trained animals, sarcolemmal preparations from heart and slow twitch soleus muscle exhibited a significantly greater receptor concentration than membranes from white fast twitch glycolytic fibers of the vastus lateralis. No significant changes were observed between trained and sedentary rat muscle β-adrenoceptor density (βmax, fmole/mg protein) or affinity (Kd , nM) within each muscle type, despite significantly increased myocardial/body weight ratios and skeletal muscle enzyme adaptations associated with the exercise program. These results suggest that muscle β-adrenoceptor properties may be influenced in part by the motor nerve innervation to that muscle, and are further discussed with respect to a possible relationship between exercise intensity and receptor regulation.