Protein kinase C-dependent diacylglycerol formation is mediated via Ca2+/calmodulin in parotid cells.
The kinetics of carbachol-induced sn-1,2-diacylglycerol (DAG) formation and the underlying mechanism(s) involved in parotid acinar cells were investigated. Supramaximal concentrations of carbachol for amylase secretion (10 microM) caused a transient rise in DAG levels at 10 s. In contrast, this rapid rise was not elicited by 1 microM carbachol, which is the maximally effective concentration for amylase secretion. Carbachol (10 microM) also increased DAG levels linearly up to 20 min, which were sustained for up to a further 10 min. DAG formation stimulated by 1 microM carbachol was biphasic; the first peak was observed after 5 min and the second after 20 min. DAG formation induced by 0.01-0.1 microM carbachol was concentration-dependent and monophasic, peaking at 5 min. The second peak evoked by carbachol was partly inhibited by Ca2+ deprivation from the extracellular space, whereas the first peak was not. Similar results were obtained in experiments using Ca2+ antagonists such as verapamil and LaCl3. The protein kinase C inhibitors, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) and staurosporine, and a calmodulin antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), significantly inhibited the second DAG peak produced by 1 microM carbachol, but did not alter the first peak. The degree of inhibition of the second peak by these antagonists was comparable. Furthermore, the inhibitory effect of staurosporine and W-7 was concentration-dependent. The A23187-induced accumulation of DAG also was abolished by both staurosporine and W-7. These data indicate that a protein kinase C-dependent mechanism(s) is involved in mediating the second DAG accumulation peak induced by 1 microM carbachol and is mainly regulated by the Ca(2+)-calmodulin complex.
Komabayashi
T
T
Department of Physiology and Pharmacology, Tokyo College of Pharmacy, Japan.
Yakata
A
A
Izawa
T
T
Suda
K
K
Rubin
R P
RP
Tsuboi
M
M
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000470:Alkaloids / Q000494:pharmacology; D000681:Amylases / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology*; D004075:Diglycerides / Q000096:biosynthesis* / Q000378:metabolism; D007546:Isoquinolines / Q000494:pharmacology; D007811:Lanthanum / Q000494:pharmacology; D008297:Male; D010306:Parotid Gland / Q000166:cytology / Q000378:metabolism*; D010879:Piperazines / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D019311:Staurosporine; D013449:Sulfonamides / Q000494:pharmacology; D014700:Verapamil / Q000494:pharmacology
C011439:1,2-diacylglycerol; D000470:Alkaloids; D002147:Calmodulin; D004075:Diglycerides; D007546:Isoquinolines; D010879:Piperazines; D013449:Sulfonamides; C028521:lanthanum chloride; C017967:W 7; D007811:Lanthanum; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D002217:Carbachol; D014700:Verapamil; D011493:Protein Kinase C; D000681:Amylases; D019311:Staurosporine; D002118:Calcium