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Effect of muscarinic receptor stimulation on phosphatidylinositol turnover in rat heart and guinea pig smooth muscle

University of British Columbia

Stimulation of various cell surface receptors produces an enhanced turnover of phosphatidylinositol (PI). This phenomenon, referred to as the PI response, has been suggested to be an obligatory response of muscarinic receptor stimulation and to play a role in calcium mobilization mediated by muscarinic and other receptor systems. In this study, this hypothesis has been investigated by studying PI response in rat atria and longitudinal smooth muscle of the guinea pig ileum. It was observed that even though muscarinic receptor density is known to be similar in rat left and right atria, an increase (35%, p < 0.05) in phosphate incorporation into PI was observed in rat left atrium but not in right atrium. By contrast to the small effect of muscarinic receptor stimulation, alpha-adrenergic receptor stimulation by 0.1 mM methoxamine produced a two-fold increase in phosphate incorporation into PI in both rat left and right atria, despite the lower density of these receptors compared to the muscarinic receptors in atria. These results, along with studies showing a lack of effect of muscarinic receptor stimulation on inositol lipid breakdown, suggested that only a small population (approximately 15%) of muscarinic receptors in rat atria is coupled to PI turnover. These are termed m, receptors, by analogy to a, adrenergic receptors which enhance PI turnover and Ca²⁺mobilization. The remaining population of muscarinic receptors, termed m₂, most probably is coupled in an inhibitory manner to adenylate cyclase. While these findings support the postulate that PI response may accompany only the stimulation of those receptors whose response is mediated through calcium mobilization, the results also suggest the presence of a muscarinic receptor population whose stimulation is not coupled to PI turnover. The role of PI response in calcium mobilization was investigated in longitudinal smooth muscle of guinea pig ileum. Consistent with previous findings, muscarinic receptor stimulation of guinea pig ileum was accompanied by a PI response. Carbachol (0.1 mM) produced an atropine-sensitive incorporation of [³²P]phosphate and [³H]inositol into PI. The effect of carbachol on the breakdown of inositol lipids was studied in the presence of 10 mM lithium, which causes accumulation of inositol phosphate by inhibiting inositol phosphatase. Carbachol produced a 20-fold increase in the accumulation of inositol phosphates in the presence of lithium. Analysis of the inositol phosphates by ion-exchange chromatography indicated that, unlike many other tissues, in guinea pig ileum less than. 20% of the [³H]inositol label occurred in inositol monophosphate, while the remainder was found in the inositol polyphosphate fraction. This indicated that similar to findings in other tissues reported recently, the primary event of muscarinic receptor stimulation in guinea pig ileum may be the enhanced breakdown of polyphosphoinositides, rather than PI. The effect of phenylmethanesulfonylfluoride (PMSF), a putative inhibitor of Pl-specific phospholipase C, on carbachol and potassium-stimulated PI turnover and contraction in longitudinal smooth muscle of guinea pig ileum was studied. PMSF almost completely inhibited carbachol-stimulated inositol incorporation into PI, while it had no effect on potassium-stimulated inositol incorporation. This suggests that the two stimuli produce "PI responses" by different mechanisms. In contrast to its specific inhibition of carbachol-stimulated PI turnover, PMSF produced a nonspecific, transient inhibition of contraction of guinea pig ileum by both carbachol and potassium. The nonspecific PMSF effect on contraction suggests that it is not the result of its inhibitory effect on PI turnover. The suggestion of Walenga et al. (1980) that PMSF inhibition of PI turnover may be mediated through its inhibition of Pl-specific phospholipase C was based on indirect findings. PMSF (2 mM) produced only a 16% inhibition of the carbachol-stimulated inositol phosphate accumulation in the presence of lithium, indicating that the PMSF effect on Pl-specific phospholipase C cannot fully account for the observed inhibition by PMSF of carbachol-stimulated inositol incorporation. The results obtained do not contradict the possibility that PI breakdown is involved in calcium mobilization.

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2010-06-12

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http://hdl.handle.net/2429/25611

Pharmaceutical Sciences

University of British Columbia

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