Phorbol esters promote alpha 1-adrenergic receptor phosphorylation and receptor uncoupling from inositol phospholipid metabolism.

Abstract

DDT, MF-2 cells, which are derived from hamster vas deferens smooth muscle, contain a1-adrenergic receptors (54,800 ± 2700 sites per cell) that are coupled to stimulation of inositol phospholipid metabolism. Incubation of these cells with tumor-promoting phorbol esters, which stimulate calcium-and phospholipid-dependent protein kinase, leads to a marked attenuation of the ability of a,-receptor agonists such as norepinephrine to stimulate the turnover of inositol phospholipids. This turnover was measured by determining the 32p content of phosphatidylinositol and phosphatidic acid after prelabeling of the cellular ATP pool with 32p;. These phorbol ester-treated cells also displayed a decrease in binding affinity of cellular al receptors for agonists with no change in antagonist affinity. By using affinity chromatography on the affinity resin Affi-Gel-A55414, the a, receptors were purified =300-fold from control and phorbol ester-treated 32P1-prelabeled cells. As assessed by NaDodSO4/polyacrylamide gel electrophoresis, the Mr 80,000 a,-receptor ligandbinding subunit is a phosphopeptide containing 1.2 mol of phosphate per mol of a, receptor. After phorbol ester treatment this increased to 3.6 mol of phosphate per mol of a, receptor. The effect of phorbol esters on norepinephrinestimulated inositol phospholipid turnover and a,-receptor phosphorylation showed the same rapid time course with a til2 < 2 min. These results indicate that calcium-and phospholipid-dependent protein kinase may play an important role in regulating the function of receptors that are coupled to the inositol phospholipid cycle by phosphorylating and deactivating them.The mechanisms whereby hormones regulate cellular metabolism are of great current biochemical interest. In addition to the well-delineated, second messenger-generating adenylate cyclase system, another potentially quite general pathway has been demonstrated recently. This pathway involves receptor-mediated stimulation of the breakdown of membrane polyphosphoinositides (phosphatidylinositol phosphates) to diacylglycerol and inositol trisphosphate (1-5).Both products are potential second messengers serving to stimulate calcium-and phospholipid-dependent protein kinase (protein kinase C) (6, 7) and to mobilize intracellular calcium (8, 9), respectively. Factors known to stimulate this pathway include agonists such as acetylcholine (10), angiotensin (11), and catecholamines acting via a1-adrenergic receptors (1,(12)(13)(14)(15)(16).A powerful approach to the study of this signal transfer pathway has been the use of the tumor-promoting phorbol esters, which appear to mimic endogenously produced diacylglycerol in activating protein kinase C (17, 18), thus bypassing the receptor signal. By using this approach it has been demonstrated recently that receptors for epidermal growth factor (19, 20), insulin (21, 22), and somatomedin C (21) are either directly or indirectly phosphorylated by protein kinase C. Although in a few of these systems receptor phosphorylation has been shown to...

Effect of catecholamines on Na/H exchange in vascular smooth muscle cells.

“…In the latter case, the potency order, receptor involvement, and effects of angiotensin II and TPA were identical to the results reported for catecholamine-stimulated Na/H exchange. This is the first report of catecholamine stimulation of inositol phosphate release in VSMC, although others have made a similar observation using vas deferens smooth muscle cells (10). In addition, these data are the first to demonstrate that NE can enhance angiotensin II-stimulated inositol phosphate release in VSMC (although others have measured the effect of angiotensin II [5,11,21]).…”

“…To identify the mechanism by which catecholamines activate Na/H exchange, we measured catecholamine effects on the two second messenger systems known to be linked to adrenergic receptors, that is cAMP (26) and inositol phosphate release (10). cAMP was measured by radioimmunoassay (22) with the acetylation modification (7).…”

“…Finally, adrenergic receptors have been shown to be linked to two second messenger systems: adenylate cyclase (26) and inositol phosphate release (10). Thus, due to their physiological relevance, the wide variety of available receptor probes, and their linkage to second messengers, we chose to study catecholamine effects on Na/H exchange as a means of understanding the regulation of the system.…”

Permissive stimulation of Ca(2+)-induced phospholipase A2 by an adenosine receptor agonist in a pertussis toxin-sensitive manner in FRTL-5 thyroid cells: a new ‘cross-talk’ mechanism in Ca2+ signalling.

“…In a separate experiment, noradrenaline-induced arachidonic acid release was also increased by the down-regulation of protein kinase C (Table 1, Experiment 2). These results suggest that, when receptor-mediated phospholipase C activation induces arachidonic acid release, the endogenously activated protein kinase C seems to play a negative rather than a positive role in arachidonic acid release, as shown for the ac-adrenergic receptor [21].…”

Inhibition of formation of cyclic AMP and cyclic GMP by vasopressin in smooth-muscle cells is insensitive to pertussis toxin

“…It is possible that, when protein kinase C is activated either by exogenously added phorbol esters or by endogenously formed diacylglycerol (in response to AVP or NaF), it might phosphorylate some key regulatory protein(s) in the cyclic nucleotide pathway, resulting in an attenuated response. Protein kinase Cmediated phosphorylation of al-and fl-adrenergic receptors resulted in an attenuated response mediated by these receptors [34][35][36]. Further work is required to identify these phosphorylated proteins.…”