Protein kinase regulation of muscarinic receptor signalling in colonic smooth muscle

Abstract

1 We have previously demonstrated that M2 and M3 muscarinic receptors coexist in the circular smooth muscle of canine promixal colon. Activation of receptors of the M2 subtype leads to inhibition of adenylyl cyclase activity through the GTP-binding protein, Gi, while M3 receptors are coupled to a pertussis toxin-insensitive GTP-binding protein and mediate phosphoinositide hydrolysis. 2 In the present study, the interactions between these second messenger systems were examined. Activation of either protein kinase C or adenosine 3':5'-cyclic monophosphate (cyclic AMP)-dependent protein kinase attenuated carbachol-stimulated phosphoinositide hydrolysis without affecting basal activity. Activation of both protein kinases produced greater attenuation of inositol 1,4,5-trisphosphate formation than activation of either kinase alone. 3 In contrast to its inhibitory effect on phosphoinositide hydrolysis, activation of protein kinase C had no effect on adenylyl cyclase activity. 4 Activation of protein kinase C by phorbol ester treatment resulted in the sequestration of M3 muscarinic receptors from the cell surface without effecting the M2 muscarinic receptor population. Sequestered M3 muscarinic receptors were not rapidly degraded. 5In contrast, elevation of cellular cyclic AMP decreased the affinity of cell surface muscarinic receptors for an antagonist radioligland without affecting their density. 6 Muscarinic agonist binding was not affected by either activation of protein kinase C or elevation of cellular cyclic AMP. 7 These data support the notion of negative feedback by protein kinase C and cyclic AMP-dependent protein kinase on phosphoinositide hydrolysis. In canine colonic circular smooth muscle this negative feedback regulation of inositol phosphate generation by muscarinic receptor stimulation does not appear to involve the guanine nucleotide binding protein:receptor interaction.

Sensitization of the contractile system of canine colonic smooth muscle by agonists and phorbol ester.

“…M3 receptors (18%) are also present, and these are coupled to activation of phospholipase C. Activation of phospholipase C increases phosphatidylinositol turnover producing inositol 1,4,5-trisphosphate and diacylglycerol. Presumably diacylglycerol activates PKC (Zhang & Buxton, 1993) which catalyses phosphorylation of undefined substrates that modulate smooth muscle contraction. There are many possible sites of action of PKC in smooth muscle including proteins regulating the contractile element (Nishimura, Moreland, Moreland & van Breemen, 1991;Andrea & Walsh, 1992;Sato et at.…”

“…Because agonist activation of PKC might modulate gastrointestinal smooth muscle contraction we also tested the ability of PDBu to potentiate contraction. Previous studies have shown that PKC is activated by PDBu and cholinergic stimulation of canine colonic muscles (Zhang & Buxton, 1993). To eliminate potentially confounding effects of metabolism of acetylcholine we used methacholine to stimulate muscarinic receptors.…”

“…2). When compared to the control response to ACh, the amplitude of phasic contractions was increased significantly by prior exposure to NKA ( Fig (Zhang & Buxton, 1993). To eliminate potentially confounding effects of metabolism of acetylcholine we used methacholine to stimulate muscarinic receptors.…”

Potentiation of bradykinin-induced inositol phosphates production by cyclic AMP elevating agents and endothelin-1 in cultured astrocytes

“…Elevations in CAMP level have been shown to inhibit agonist-dependent PI turnover in NG108-15, NCB-20, astrocyte, rat aorta, colon, and kidney cells (Campbell et al, 1990;Manolopoulos et al, 1991;Mctee and Dawson, 1990;Neylon and Summers, 1988;Robertson et al, 1990;Zhang and Buxton, 1993), and to enhance it in BALBc/3T3 cells and hepatocytes (Hlackmore and Exton, 1986;Olashaw and Pledger, 1988). Possible targets of PKA action responsible for the regulation of receptor-coupled PIPz hydrolysis include the receptor, a G-protein, involved in coupling receptor function to PLC, and PLC itself.…”

Isoproterenol Changes the Relationship between Cytosolic Ca2+and Contraction in Guinea Pig Taenia Caecum

“…The inhibition by forskolin and dibutyryl cyclic AMP of phosphatidyl inositol hydrolysis has been demonstrated in tracheal (28) and vascular smooth muscles (29). Activation of protein kinase A by forskolin in canine colonic smooth muscle desensitizes muscarinic receptors and inhibits phos phatydil-inositol turnover (30).…”