Hypercholesterolemia in the rabbit induced by feeding graded amounts of low-level cholesterol. Methodological considerations regarding individual variability in response to dietary cholesterol and development of lesion type.

While a number of studies have presented detailed examinations of lesion development in the cholesterol-fed rabbit, individual variability in response to cholesterol feeding and type of lesion produced relative to the degree of cholesterol exposure is not well defined. This study analyzed such critical parameters in an attempt to further characterize the model and establish a baseline for future testing of treatments targeted at limiting atherosclerosis. For these experiments, male New Zealand White rabbits were fed atherogenic diets consisting of 0.05%, 0.10%, 0.15%, 0.20%, or 0.25% cholesterol dissolved in 6% peanut oil for 31 to 32 weeks. Raising dietary cholesterol from 0.05% to 0.15% resulted in a less than twofold stepwise increase in total plasma cholesterol (TPC) exposure (area under plasma cholesterol versus time curve), whereas further increases in cholesterol intake resulted in an exponential four- to fivefold increase in TPC exposure. Regression analysis of TPC exposure with aortic sudanophilia demonstrated a threshold of approximately 5000 cholesterol weeks; below this limit lesions were minimal, and above this value the degree of plaque correlated with TPC exposure. Furthermore, a wide biological variability occurred among rabbits with respect to individual responsiveness to dietary cholesterol. In the aorta, various types of plaques, from fatty streaks to atheromatous lesions, were observed, depending on the degree of cholesterol intake. Diets consisting of < 0.15% cholesterol resulted in the development of fatty streak lesions, while transitional lesions and atheromatous plaques were mostly found with higher cholesterol feeding. Coronary artery atherosclerosis was present in > 50% of animals fed diets > or = 0.15% cholesterol. Despite the level of TPC exposure, coronary lesions in epicardial vessels were generally the fibrous type, whereas intramyocardial arteries demonstrated predominantly intimal foam cells. In conclusion, by adjusting dietary cholesterol intake and selecting rabbits with a similar responsiveness to cholesterol, the overall cholesterol exposure can be more closely controlled to minimize the inherent individual variability among animals in this model. The nature of the target lesion must also be carefully considered, because the efficacy of some treatments may depend on the type of atherosclerotic plaque.

ACAT inhibitors CL 283,546 and CL 283,796 reduce LDL cholesterol without affecting cholesterol absorption in African green monkeys.

Previous studies with a number of selective acylcoenzyme A:cholesterol acyltransferase (ACAT) inhibitors in several animal models have demonstrated significant reductions in plasma cholesterol and, in some studies, triglyceride levels. This study was conducted to examine the effects of two ACAT inhibitors, CL 283,546 and CL 283,796, in cholesterol-high fat diet fed African green monkeys, a relevant primate model of hyperlipidemia and coronary artery atherosclerosis. Treatment with CL 283,546 or CL 283,796 resulted in significant reductions (ca. 25-30%) in total plasma cholesterol at both 10 and 30 mg/kg per day doses. This reduction in plasma cholesterol was due almost entirely to reduction in low density lipoprotein (LDL) cholesterol (ca. 45%) without significantly affecting high density lipoprotein (HDL) cholesterol, very low density lipoprotein + intermediate density lipoprotein (VLDL + IDL) cholesterol, or triglyceride concentrations. There were no significant effects on plasma concentrations of apolipoproteins A-I, E, or B and, thus, the reduction seen in LDL cholesterol appears to be due to a diminished cholesterol content of LDL particles. Our studies revealed that treatment with these compounds did not reduce cholesterol absorption, which was somewhat surprising as ACAT inhibitors are generally thought to exert their hypolipidemic effects, at least in part, by inhibition of intestinal cholesterol absorption. Our data are consistent with a principal activity of these drugs on the liver to reduce cholesteryl ester secretion in VLDL, leading to a diminished LDL-cholesterol content, and, presumably, enhanced biliary cholesterol-bile acid excretion.

Characterization of the purine-reactive site of the rat testis cytosolic adenylate cyclase.

Naturally soluble rat germ cell adenylate cyclase was inhibited by adenosine and the adenosine analogs, 9-beta-D-arabinofuranosyl adenine (AFA) and 2',5'-dideoxyadenosine (DDA), all of which inhibited hormone-sensitive adenylate cyclases at the "P" site. The IC50 values for adenosine and DDA were approximately 0.1 and for AFA, 4.0 mM. The onset of adenosine inhibition was very rapid whether adenosine was added to the enzyme reactant mixture at time zero concomitantly with the addition of substrate or after the enzyme had been activated by the addition of substrate. The adenosine analogs, N6-methyladenosine (MeA) and N6-phenylisopropyl adenosine (PIA), which interact with plasma membrane receptors ("R" receptors) for hormone-sensitive adenylate cyclase, had little effect on the activity of the cytosolic adenylate cyclase. Additionally, aminophylline, which has been shown to competitively antagonize adenosine interactions with the plasma membrane "R" receptors but not "P" site interactions, had no effect upon substrate activation of the soluble enzyme and did not prevent adenosine from inhibiting the activity of the enzyme. These data provide evidence for an adenosine regulatory site on the cytosolic enzyme which resembles the "P" site described for membrane bound-adenylate cyclase.

Phospholipids inhibit cytotoxic effects of Actinobacillus actinomycetemcomitans leukotoxin on human polymorphonuclear leukocytes.

Isolated human peripheral blood neutrophils were exposed to sonic extracts of Actinobacillus actinomycetemcomitans. Such bacterial preparations contain a potent leukotoxin which rapidly kills the leukocytes as reflected by cellular uptake of trypan blue, extracellular release of lactate dehydrogenase, or discharge of 51Cr from pre-labeled cells. Exogenous phospholipids with a glycerol skeleton esterified by fatty acids or positively charged liposomes inhibited cytotoxic phenomena. The data suggest that cell damage may involve the interaction of leukotoxin with phospholipids in the neutrophil cell membrane and that exogenous lipids either compete for or sterically block binding of the leukotoxin to these moieties in the membrane.

Photoaffinity label for the beta-adrenergic receptor: synthesis and effects on isoproterenol-stimulated adenylate cyclase.

An azide derivative of the beta-adrenergic antagonist acebutolol has been synthesized and its effect examined on the isoproterenol-stimulated adenylate cyclase [ATP pyrophosphate-lyase (cyclizing); EC 4.6.1.1] activity of rat reticulocytes. It behaved as an effective competitive antagonist (Kd = 2 X 10(-7) M) prior to photolysis. However, when the reticulocyte preparation pretreated with acebutolol azide was photolyzed, a noncompetitive inhibition of isoproterenol-stimulated adenylate cyclase was obtained. Photolysis of the azide derivative in buffer alone did not convert it to a product of higher affnity. Labeling of the beta-adrenergic receptor appeared to be irreversible; multiple washings could not reverse the inhibition produced during photolysis with the label whereas washing would completely reverse the antagonism produced by the same concentration of label prior to photolysis. The effect appears to be specific for the beta-adrenergic receptor because the inhibition could be blocked stereoselectively by propranolol and there was no inhibition of fluoride- or GMP-P(NH)P-stimulated adenylate cyclase. furthermore, no effect was observed on the glucagon-mediated stimulation of adenylate cyclase of liver membranes, whereas the catecholamine response in the same membranes was inhibited.