Cholinergic stimulation of isolated rat parietal cells: role of calcium, calmodulin and protein kinase C.
We studied the cholinergic stimulation of isolated and enriched rat parietal cells. H+ production was indirectly measured by the uptake of 14C-aminopyrine into the parietal cells. Stimulation by carbachol required the presence of extracellular Ca2+ not only in the initial phase but also during the sustained phase of a 100-min incubation period. The response to carbachol was prevented by the Ca2+ entry blocker lanthanum IC50: 1.5 X 10(-7) mol/l). Furthermore, the dependence on Ca2+ influx of cholinergic stimulation was demonstrated by a 269% increase in total intracellular Ca2+ in response to carbachol, as determined by optical emission spectrometry. The naphthalene sulfonamides W7 and W5 which bind calmodulin and thus block the intracellular transduction of Ca2+ effects also inhibited a carbachol-induced H+ production. In the following experiments we studied the effect of agents which activate the protein kinase C, an enzyme which is supposed to play a key role in intracellular signal transduction of Ca2+-dependent effects. Phospholipase C is supposed to activate protein kinase C via induction of the phosphoinositol breakdown. In our preparation of isolated rat parietal cells, phospholipase C (4-100 mU/ml) exerted inhibition instead of amplification of the response to 10(-4) mol/l carbachol. Similarly, the direct activation of protein kinase C by 12-O-tetradecanoylphorbol-13-acetate or by 1-oleoyl-2-acetyl-sn-glycerol (both tested at 10(-7) to 10(-5) mol/l) reduced the submaximal and maximal response to 10(-5) or 10(-4) mol/l carbachol. We conclude that the cholinergic stimulation of rat parietal cells is dependent on the influx of extracellular Ca2+. Calmodulin seems to mediate intracellular Ca2+ effects during cholinergic stimulation. The activation of protein kinase C impairs carbachol-induced H+ production instead of augmenting the response. This might be due to an already maximal activation of protein kinase C by carbachol alone or to autoregulatory down-regulation by the protein kinase C of muscarinic parietal-cell receptors.
Schepp
W
W
Department of Internal Medicine II, Technical University of Munich, FRG.
Brosch
E
E
Tatge
C
C
Schusdziarra
V
V
Classen
M
M
D000632:Aminopyrine / Q000493:pharmacokinetics; D000818:Animals; D002118:Calcium / Q000502:physiology*; D002147:Calmodulin / Q000502:physiology*; D002217:Carbachol / Q000494:pharmacology*; D005260:Female; D006859:Hydrogen / Q000378:metabolism; D010295:Parietal Cells, Gastric / Q000187:drug effects / Q000378:metabolism*; D011493:Protein Kinase C / Q000502:physiology*; D051381:Rats; D011919:Rats, Inbred Strains; D010738:Type C Phospholipases / Q000502:physiology
D002147:Calmodulin; D000632:Aminopyrine; D006859:Hydrogen; D002217:Carbachol; D011493:Protein Kinase C; D010738:Type C Phospholipases; D002118:Calcium