No abstract

The selective angiotensin (ANG II) antagonists losartan (DuP 753) and PD 123319 have been shown to bind selectively to AT1 and AT2 subtypes, respectively. To characterize ANG II receptor subtypes in mesangial cells, washed membranes were incubated with 0.1 to 0.5 nM 125I-ANG II and increasing concentrations of competitors. The inhibition of 125I-ANG II binding by losartan and PD 123319 was biphasic, and LIGAND curve-fitting analysis revealed two populations of specific binding sites. One subpopulation comprised 86% of the total and showed high affinity for ANG II and losartan, but low affinity for the AT2 antagonists PD 123319 and CGP42112A, and thus appear identical to the recently cloned AT1 subtype. The remaining 14% of the sites showed nearly 100-fold lower affinity for losartan and 10,000-fold higher affinity for PD 123319 relative to AT1 sites. However, another AT2-selective antagonist, CGP42112A, showed little affinity for these sites. Both classes of binding sites were inhibited by guanosine 5'-O-(3-thiophosphate) and pertussis toxin treatment. We propose that there are two distinct G protein-coupled ANG II receptor subtypes (AT1A and AT1B) present in renal mesangial cells.

We mimicked human weight cycling in the obese spontaneously hypertensive rat (SHROB) model of genetic obesity. A 12-day very low calorie diet (VLCD; 16.7% of baseline calories) was alternated with 4-6 wk of ad libitum chow refeeding for three cycles. Control SHROB ate chow ad libitum. VLCD induced rapid weight loss, but during refeeding all the lost weight was regained. Final body weight was higher in cycled rats than in ad libitum controls (149 +/- 5 vs. 117 +/- 7% of initial baseline). Less weight was lost as a percent of starting body weight during each successive VLCD, which could not be explained by aging. At death, retroperitoneal fat pads were heavier in cycled SHROB than in ad libitum controls (62 +/- 3 vs. 44 +/- 4 g). During the first 2 days after each VLCD, cycled rats overate significantly relative to ad libitum controls (88 +/- 2 vs. 78 +/- 3 kcal/day), but cumulative food intake throughout the duration of the experiment did not differ (11.4 +/- 0.6 vs. 11.7 +/- 0.1 Mcal). Compared with ad libitum-fed rats, food efficiency (g body wt gain/kcal) was increased during each refeeding period. Weight cycling elevated blood pressure above the initial baseline throughout refeeding. Refeeding hypertension was abolished by ganglionic blockade with chlorisondamine. Thus weight cycling in SHROB exacerbates obesity, metabolic efficiency, abdominal fat accumulation, sympathetic activity, and hypertension.

ACh regulates the gene encoding phenylethanolamine N-methyltransferase (PNMT) in bovine adrenal chromaffin cells. In addition to stimulating catecholamine release from these cells, cholinergic agents elevate transcription of the PNMT gene. Carbachol, which activates both nicotinic and muscarinic receptors, produces 12-19-fold increases in PNMT mRNA and a 22-fold increase in epinephrine release. Selective nicotinic and muscarinic antagonists (hexamethonium and atropine) each partially reduce carbachol-stimulated increases in PNMT mRNA while a combination of both eliminates > 90% of the carbachol response, thus indicating that separable nicotinic and muscarinic components contribute to the cholinergic increase in PNMT mRNA. Muscarine alone produces a dose-dependent increase (mean sixfold) in steady state PNMT mRNA levels and stimulates the rate of transcription fivefold. Only atropine and the m3-m4-selective muscarinic antagonist 4-diphenylacetoxy-4-methyl-piperidine (4-DAMP) reduce the response to muscarine, strongly suggesting that the m4 receptor is crucial for PNMT mRNA activation. In these chromaffin cells, muscarine inhibits adenylate cyclase, antagonist bind with affinities characteristic of m4 receptors, and cDNA hybridization detects only m4 mRNAs (Fernando et al., 1991). Nicotine also induces a dose-dependent increase (mean of 8.5-fold) in PNMT mRNA levels. The importance of voltage-gated Ca2+ channels in the nicotine effect is demonstrated by the stimulatory effects of calcium ionophores on PNMT mRNA levels (two-to fivefold increase) and the ability of the L- and N-type channel blockers nifedipine and omega-conotoxin to decrease the nicotine response (by 60% and 40%, respectively). Nuclear "run-on" assays further reveal that nicotine enhances transcription of the PNMT gene (approximately fourfold). Thus, this study provides the first demonstration that both nicotinic and muscarinic stimulation modify genomic responses of bovine adrenergic chromaffin cells and identifies possible mechanisms.