Release of angiotensin in response to ionic stimulation: a possible role for calmodulin in the secretory event.
Although the synthesis and distribution of the putative neurotransmitter angiotensin (ANG) has been well established, little is known about the mechanism for its release. In this study, we utilized dissociated cell cultures of fetal rat brain to examine the cellular and ionic properties of angiotensin release. Graded concentrations of 0-60 mM KCl in the presence of 5 mM CaCl2 were added to the cultured cells and the resulting angiotensin release was measured by radioimmunoassay and high-performance liquid chromatography. Levels of angiotensin release increased from 13.85 +/- 1.53 pg/mg protein to 172.64 +/- 17.49 pg/mg protein with increasing concentrations of K+. Cultures incubated with 60 mM KCl buffer that did not contain CaCl2 released 39.87 +/- 15.74 pg ANG/mg protein. To further show the link between ionic stimulation and angiotensin release and determine the potential role of extracellular sodium ions on angiotensin release, cultures were incubated with the Na(+)-channel blocker tetrodotoxin (300 nM TTX) prior to maximal stimulation with 60 mM KCl/5 mM CaCl2 in the presence of the channel antagonist. Release was attenuated following incubation with stimulating buffer containing TTX (300 nM) to 5.49 +/- 4.37 pg/mg protein. Finally, to determine the role of the calcium binding protein calmodulin in the release event, the cells were incubated with graded concentrations of W-7 (2.5-250 microM) and subsequently stimulated with 60 mM KCl/5 mM CaCl2 in the presence of the calmodulin antagonist. W-7, which displays specificity for inhibition of the Ca2+/calmodulin complex below 0.2 mM, decreased angiotensin release in a dose-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)
Gadbut
A P
AP
Department of Cell and Structural Biology, University of Illinois, Urbana 61801.
Cash
S M
SM
Dagiantis
W P
WP
Cooper
S
S
Weyhenmeyer
J A
JA
D000804:Angiotensin II / Q000378:metabolism*; D000818:Animals; D001923:Brain Chemistry / Q000187:drug effects; D002118:Calcium / Q000494:pharmacology*; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000502:physiology*; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D005260:Female; D011188:Potassium / Q000494:pharmacology*; D011247:Pregnancy; D011863:Radioimmunoassay; D051381:Rats; D011919:Rats, Inbred Strains; D013449:Sulfonamides / Q000494:pharmacology; D013779:Tetrodotoxin / Q000494:pharmacology
D002147:Calmodulin; D013449:Sulfonamides; D000804:Angiotensin II; D013779:Tetrodotoxin; C017967:W 7; D011188:Potassium; D002118:Calcium