AJP - Cell Physiology, Vol 263, Issue 6 C1289-C1294, Copyright © 1992 by American Physiological Society
Muscarinic receptors in MDCK cells are coupled to multiple messenger systems
D. Mohuczy-Dominiak and L. C. Garg
Division of Nephrology, Hypertension, and Transplantation, University of Florida College of Medicine, Gainesville 32610.
Our studies on Madin-Darby canine kidney (MDCK) cells have demonstrated
that high-affinity specific muscarinic receptors coupled to the
phosphoinositide system are present in these cells. To determine whether
muscarinic receptors in MDCK cells are linked negatively to the adenylate
cyclase system, we measured the effect of muscarinic agonists and
antagonists on vasopressin-, isoproterenol-, and forskolin-stimulated
adenosine 3',5'-cyclic monophosphate (cAMP) formation. Vasopressin produced
a maximum stimulation of cAMP formation of 13 pmol.10(6) cells-1.2 min-1 at
10(-7) M. Isoproterenol and forskolin stimulated cAMP formation production
to 21 pmol.10(6) cells-1.2 min-1 and 64 pmol.10(6) cells-1.10 min-1,
respectively, at 10(-4) M. The effects of vasopressin, isoproterenol, and
forskolin were blocked by arecoline, a cholinergic agonist, in a
concentration-dependent manner. The arecoline response was blocked by
treatment of the cells with pertussis toxin. The inhibition by arecoline of
forskolin-stimulated cAMP formation was reversed by various muscarinic
antagonists in the following order of potency:
4-diphenyl-acetoxy-N-methylpiperidine > p-fluorohexahydrosiladifenidol
> pirenzepine > methoctramine. This order of potency of muscarinic
antagonists is similar to that observed in our radioligand binding studies
and is consistent with the M3 subtype of muscarinic receptors. Our results
indicate that muscarinic receptors in MDCK cells are coupled negatively to
the adenylate cyclase system via pertussis toxin-sensitive G protein. It is
concluded that this intracellular system may at least be partially
responsible for the action of cholinergic agonists in these cells and in
the kidney.
