Cellular mechanisms of angiotensin release

In this study we examined angiotensin (ANG) release from primary cultured rat brain cells in response to ion stimulation and ligands that stimulate or inhibit second messenger systems in an attempt to understand how the release of ANG is regulated.Results indicated that ANG release could be stimulated in a dose dependent fashion by increasing concentrations of K+. This suggests that ANG secretion resembles a neurotransmitter-like release as opposed to a volume transmission-like secretion. Angiotensin release was inhibited by voltage gated calcium channel (VGCC) antagonists Cd\sp{++} and $\omega$-Conotoxin GIVA ($\omega$-CgTx), but only slightly by nitrendipine. This suggests that extracellular Ca\sp{++} is involved in ANG release. Furthermore, the N-type VGCC seems to be the primary VGCC type responsible for Ca\sp{++} entrance into the cell leading to ANG release.To examine the potential effect of the Ca\sp{++} binding protein calmodulin on ANG release the cells in culture were K\sp+ stimulated in the presence of the calmodulin antagonists W-7 and W-13. The calmodulin antagonists W-7 and W-13 both blocked ANG release in a dose dependent fashion with W-13 being a more potent blocker of ANG release. These data suggest extracellular Ca\sp{++} enters the cell and activates calmodulin to facilitate ANG release.We found that ANG release could be stimulated by isoproterenol, the cholera toxin, forskolin, and 8-bromo-c-AMP. This suggests a receptor linked mechanism, whereby the $\beta$-adrenergic receptor is linked via G-protein to the activation of adenylate cyclase and c-AMP, regulating the release of angiotensin from brain cells in culture.The Ca\sp{++}/calmodulin system and the adenylate cyclase/c-AMP system reportedly (Kennedy, 1989) co-modulate each other in several systems. To examine this potential interaction, cultured cells were stimulated with isoproterenol or forskolin in the presence of W-7, W-13 or Cd\sp{++}. The release of ANG was attenuated in response to isoproterenol/W-13, isoproterenol/Cd\sp{++} and forskolin/W-7 as compared to isoproterenol or forskolin alone. However, the level of attenuation was different from that observed by W-13, W-7 or Cd\sp{++} alone. This suggests that Ca\sp{++}/calmodulin and adenylate cyclase systems are both capable of stimulating ANG release independently and that some modulatory interaction does exist between the two systems.U of I OnlyETDs are only available to UIUC Users without author permissio

Induction of the cholesterol metabolic pathway regulates the farnesylation of RAS in embryonic chick heart cells: a new role for ras in regulating the expression of muscarinic receptors and G proteins.

We propose a novel mechanism for the regulation of the processing of Ras and demonstrate a new function for Ras in regulating the expression of cardiac autonomic receptors and their associated G proteins. We have demonstrated previously that induction of endogenous cholesterol synthesis in cultured cardiac myocytes resulted in a coordinated increase in expression of muscarinic receptors, the G protein alpha-subunit, G-alphai2, and the inward rectifying K+ channel, GIRK1. These changes in gene expression were associated with a marked increase in the response of heart cells to parasympathetic stimulation. In this study, we demonstrate that the induction of the cholesterol metabolic pathway regulates Ras processing and that Ras regulates expression of G-alphai2. We show that in primary cultured myocytes most of the RAS is localized to the cytoplasm in an unfarnesylated form. Induction of the cholesterol metabolic pathway results in increased farnesylation and membrane association of RAS. Studies of Ras mutants expressed in cultured heart cells demonstrate that activation of Ras by induction of the cholesterol metabolic pathway results in increased expression of G-alphai2 mRNA. Hence farnesylation of Ras is a regulatable process that plays a novel role in the control of second messenger pathways

Demonstration of functional M(3)-muscarinic receptors in ventricular cardiomyocytes of adult rats

1. Muscarinic receptors (M-receptors) in the mammalian heart are predominantly of the M(2)-subtype. The aim of this study was to find out whether there might exist an additional myocardial non-M(2)-receptor. 2. For this purpose, we assessed, in adult rat isolated ventricular cardiomyocytes, carbachol-induced [(3)H]-inositol phosphate (IP) formation, and its inhibition by M-receptor antagonists. 3. Carbachol (10(−7)–10(−3) mol l(−1)) increased IP-formation (maximal increase: 14±3% above basal, n=6). This increase was significantly enhanced by pretreatment with pertussis toxin (PTX, 250 ng ml(−1) for 20 h): maximal increase was 31±5%, pEC(50)-value was 5.08±0.33 (n=6). 4. In PTX-pretreated cardiomyocytes 100 μmol l(−1) carbachol-induced IP-formation was inhibited by atropine (pK(i)-value: 8.89±0.10) and by the M(3)-receptor antagonist darifenacin (pK(i)-value: 8.67±0.23) but was not significantly affected by the M(1)-receptor antagonist pirenzepine (1 μmol l(−1)) or the M(2)-receptor antagonists AF-DX 116 and himbacine (1 μmol l(−1)). 5. In conclusion, in adult rat cardiomyocytes there exists an additional, non-M(2)-receptor, that is coupled to activation of the phospholipase C/IP(3)-pathway; this receptor is very likely of the M(3)-subtype