Muscarinic receptors in MDCK cells are coupled to multiple messenger systems

Abstract

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.

Agonist‐stimulated calcium decreases in ovine ciliated airway epithelial cells: role of mitochondria

“…It has been well established that a single G‐protein coupled receptor can simultaneously activate multiple effectors by signalling through multiple G‐proteins (Mohuczy‐Dominiak & Garg, 1992 a,b ; Felder, 1995; Ecelbarger et al 1996), by signalling through distinct actions of the α monomer and the βγ dimer G‐protein subunits (e.g. Kofuji et al 1995), and finally by signalling through direct interaction with a variety of effectors through the C‐terminal domains (e.g.…”

Muscarinic acetylcholine receptor trafficking in streptolysin O-permeabilized MDCK cells

“…However, in intact cells, the plasma membrane clearly hinders access to the intracellular milieu to explore the regulation of intracellular mAChR trafficking. We therefore investigated the validity of a permeabilized cell system of Madin-Darbin canine kidney (MDCK) cells which endogenously express m3 mAChRs coupled to pertussis toxin-sensitive G proteins (Mohuczy-Dominiak and Garg 1992). The MDCK cells were chosen as model system as they have been extensively Used for the study of membrane-traffic regulation by G proteins (Zerial and Stenmark 1993).…”

Protein Kinase C and Gi/o Proteins Are Involved in Adenosine- and Ischemic Preconditioning—Mediated Renal Protection

“…The distal portion (S 3 segment) of the proximal tubule located in the outer medulla of the kidney is the primary site of injury in renal ischemia and reperfusion (18,19) because of its marginal oxygenation under normal physiologic conditions coupled with high basal metabolic demand (19 -21). These renal tubular cells express the A 1 AR ( 22) as well as the bradykinin (23,24), muscarinic (25,26), and opioid (27,28) receptors. Therefore, the second hypothesis of the current study was that bradykinin, muscarinic, or opioid receptors may mimic the protection induced by A 1 AR activation.…”