Five muscarinic receptor genes (m1–m5) that encode distinct muscarinic receptor subtypes have been cloned. Because of their structural homology and pharmacological similarity, ligand binding probes currently available do not clearly distinguish among the subtypes. To obtain a clear distribution within the CNS of molecularly defined muscarinic receptor subtypes, seven brain regions were examined for the expression of the respective mRNAs. The most sensitive method for detecting mRNA is through amplification of the respective cDNAs. Brain regions were obtained from male Wistar rats, and total RNA was isolated. The isolates were extensively treated with RNase‐free DNase to remove any residual genomic DNA. Total RNA (1 µg) was reverse‐transcribed using random primers and reverse transcriptase. The resulting cDNA was amplified using a thermal cycler, and the polymerase chain reaction (PCR)‐amplified products were analyzed by gel electrophoresis containing ethidium bromide and visualized with fluorescent illumination. PCR‐amplified samples were also injected directly onto an HPLC anion exchange column and quantified by UV detection. Each of the five muscarinic subtypes was found in every brain region examined. The m1 subtype was most abundant in cortex and gradually declined in content caudally to the spinal cord. The m2 subtype was most abundant in thalamus‐hypothalamus and pons‐medulla. The m4 subtype was found in greatest amount in the striatum, whereas m3 and m5 were expressed consistently throughout the CNS. The combination of RT‐PCR and HPLC provides a rapid and sensitive method for quantifying the expression of mRNA coding for all five muscarinic receptor subtypes derived from the CNS.

Although the biochemical properties of soluble guanylate cyclase (sGC) have been extensively studied, little is known about the regulation of gene expression of sGC subunits by second messengers. cAMP analogues and elevating agents have been previously shown to alter gene expression in vascular cells. The aim of the present study was to investigate the effects of cAMP-elevating agents on sodium nitroprusside–stimulated sGC activity and to correlate activity changes with mRNA and protein levels in cultured rat aortic smooth muscle cells. Pretreatment of cells with 50 to 1000 μmol/L isobutylmethylxanthine or 0.01 to 10 μmol/L forskolin led to a time- and concentration-dependent decrease in sodium nitroprusside–induced cGMP accumulation, first evident after 3 hours of pretreatment with forskolin and 6 hours of pretreatment with isobutylmethylxanthine. Incubation of cells with a protein kinase A–selective inhibitor (H89 or KT 5720) partially or fully prevented the downregulation in sodium nitroprusside–induced cGMP accumulation caused by cAMP-elevating agents. Quantification of reverse transcriptase–polymerase chain reaction products by high-performance liquid chromatography revealed that mRNA for both α 1 - and β 1 -subunits of sGC were decreased in cells pretreated with isobutylmethylxanthine and forskolin but not with dideoxyforskolin (inactive analogue). Moreover, protein levels for the sGC α 1 -subunit of cells pretreated with isobutylmethylxanthine and forskolin but not with dideoxyforskolin were decreased as indicated by Western blot analysis. These data indicate that cAMP-elevating agents decrease sGC activity, possibly by decreasing mRNA or protein levels or both.

Endothelium-derived relaxing factor and exogenous nitrovasodilators are thought to produce smooth muscle relaxation by activation of soluble guanylate cyclase. To investigate whether diminished cyclic GMP (cGMP) accumulation underlies the differences in vascular reactivity to nitrovasodilators between Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR), we determined cGMP formation in aortic smooth muscle cells from the two strains. Both cultured cells and aortic rings from 12-to 14-week-old SHR accumulated greater amounts of cGMP on stimulation with exogenous nitrovasodilators (ie, sodium nitroprusside) than those from WKY rats, whereas there was no difference observed in cells from prehypertensive animals (5-to 6-week old) between the two strains. Responsiveness of smooth muscle cells to endothelium-derived relaxing factor was investigated in cocultures of bovine aortic endothelial cells (BAE) and smooth muscle cells from SHR and WKY rats. cGMP accumulation elicited by endothelium-N itrovasodilators are thought to produce vascular smooth muscle relaxation through activation of the soluble isoform of guanylate cyclase, increasing cellular cyclic GMP (cGMP).14 Although the mechanism of action of cGMP is not fully understood, it has been shown that cGMP lowers intracellular Ca 2+ levels and alters the phosphorylation pattern of cellular proteins involved in contraction. -6 It has been suggested that some of the actions of cGMP are mediated by activation of cGMP-dependent protein kinase. 7 Vascular endothelium has been shown to release, both under basal conditions and after agonist stimulation (ie, bradykinin, acetylcholine, calcium ionophore A23187), an endogenous nitrovasodilator, known as endothelium-derived relaxing factor (EDRF), which is nitric oxide 89 or a nitric oxide-containing molecule, synthesized from L-arginine.10 EDRF release produces vascular relaxation through elevation of cGMP levels in the smooth muscle."" 13 Basal release of EDRF in vivo regulates vascular tone because L-arginine analogues, which inhibit nitric oxide synthase, elevate blood pressure. In several models of hypertension, both endotheliumdependent and -independent responses are altered. 19Increased total peripheral resistance has been hypothesized to be partly due to decreased EDRF action and/or increased release of endothelium-derived constricting factors, which in the spontaneously hypertensive rat (SHR), the most commonly used model of genetic hypertension, are mediated by a cyclooxygenase pathway product.20 Studies on both endothelium-dependent and -independent relaxations in SHR have yielded conflicting results. Whereas many investigators report decreased relaxation to sodium nitroprusside (SNP) or glyceryl trinitrate (GTN) in the aortas and carotid arteries of SHR, 21 -22 others have shown unchanged or even enhanced relaxation in response to nitrovasodilators in the aorta and mesenteric resistance arteries from SHR or stroke-prone SHR compared with Wistar-Kyoto (WKY) rats. 2325 The use of different age...

A significant body of evidence exists that is consistent with the possibility that heightened cholinergic activity in certain brain regions, such as the hypothalamus, leads to increased sympathetic tone and subsequent hypertension. The increase in cholinergic activity is mediated at least in part through enhanced sensitivity of muscarinic receptors. In this study, we used the technique of reverse transcriptase-polymerase chain reaction to estimate the relative levels of mRNA encoding the five known subtypes of muscarinic receptors within the hypothalamus of spontaneously hypertensive rats (SHR), a genetic model of the disease, and their normotensive counterparts (Wistar-Kyoto rats). SHR exhibited a significant increase (40% to 50%) in the excitatory M1 subtype (confirmed by receptor binding) and a decrease in the inhibitory M4 subtype of muscarinic receptors before and during the establishment of hypertension. Such alterations may form part of the genotypic profile of inherited hypertension.

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