Role of adrenergic and cholinergic mediators in salivary phospholipids secretion.

The influence of adrenergic and cholinergic mediators on phospholipid secretion in rat sublingual salivary gland cells maintained in the presence of [3H]choline was investigated. The secretion of [3H]choline-containing phospholipids over 30 min period averaged 1.93% of the total cellular labeled phospholipids in the absence of any mediator, and was enhanced by beta-adrenergic agonist, isoproterenol, to a greater extent than the cholinergic agonists, pilocarpine and carbachol. A 2.9-fold increase in phospholipid secretion occurred with isoproterenol, while pilocarpine and carbachol evoked only 1.3-fold increase. The effect of isoproterenol was inhibited by alprenolol and that of pilocarpine and carbachol by atropine. In contrast to pilocarpine and carbachol, the enhanced phospholipid secretion due to isoproterenol was accompanied by an increase in cAMP concentration. The secretion of phospholipids was also stimulated by dibutyryl-cAMP and the protein kinase C activator, phorbol myristate acetate, but not by 4 alpha-phorbol 12,13-didecanoate which does not activate protein kinase C. Furthermore, the effects of dibutyryl-cAMP and phorbol myristate acetate were additive. The phospholipids secreted in response to isoproterenol exhibited a 52% decrease in lysophosphatidylcholine, while those secreted in response to pilocarpine and carbachol showed a 21-23% lower content of phosphatidylcholine, and were enriched in lysophosphatidylcholine (2.6-2.8-fold) and sphingomyelin (1.5-1.6-fold). The results indicate that salivary phospholipid secretion remains mainly under beta-adrenergic regulation, while the phospholipid makeup of the secretion is under cholinergic control.     

Role of adrenergic and cholinergic mediators in gastric mucus phospholipid secretion.

The role of adrenergic and cholinergic mediators in phospholipid secretion by gastric mucosal cells maintained in the presence of [3H]choline was investigated. The secretion of [3H]choline phospholipids over 30 min period averaged 1.98% of the total cellular labeled phospholipids in the absence of any mediator, and was enhanced by beta-adrenergic agonist, isoproterenol, to a greater extent than the cholinergic agonist, pilocarpine. A 2-fold increase in phospholipid secretion was achieved with isoproterenol, while pilocarpine evoked 1.3-fold increase. The stimulatory effect of isoproterenol was inhibited by alprenolol and that of pilocarpine by atropine. The phospholipids secreted in response to isoproterenol exhibited a 30% decrease in lysophosphatidylcholine, while 2.1-fold enrichment in this phospholipid occurred with pilocarpine. The results, for the first time, demonstrate the involvement of neural mediators in the regulation of phospholipid secretion in gastric mucus.     

The role of protein kinase C in cholinergic stimulation of insulin secretion from rat islets of Langerhans.

The role of the Ca2+/phospholipid-dependent protein kinase C (PKC) in cholinergic potentiation of insulin release was investigated by measuring islet PKC activity and insulin secretion in response to carbachol (CCh), a cholinergic agonist. CCh caused a dose-dependent increase in insulin secretion from cultured rat islets at stimulatory glucose concentrations (greater than or equal to 7 mM), with maximal effects observed at 100 microM. Short-term exposure (5 min) of islets to 500 microM-CCh at 2 mM- or 20 mM-glucose resulted in redistribution of islet PKC activity from a predominantly cytosolic location to a membrane-associated form. Prolonged exposure (greater than 20 h) of islets to 200 nM-phorbol myristate acetate caused a virtual depletion of PKC activity associated with the islet cytosolic fraction. Under these conditions of PKC down-regulation, the potentiation of glucose-stimulated insulin secretion by CCh (500 microM) was significantly decreased, but not abolished. CCh stimulated the hydrolysis of inositol phospholipids in both normal and PKC-depleted islets, as assessed by the generation of radiolabelled inositol phosphates. These results suggest that the potentiation of glucose-induced insulin secretion by cholinergic agonists is partly mediated by activation of PKC as a consequence of phospholipid hydrolysis.     

Protein kinase C differentially inhibits muscarinic receptor operated Ca2+ release and entry in human salivary cells

We investigated the effects of phorbol myristate acetate on muscarinic receptor-induced Ca2+ release from intracellular stores and extracellular entry in a human salivary duct cell line, HSG-PA. Phorbol myristate acetate (approximately 10(-7) M) blocked both Ca2+ release and Ca2+ entry induced by the muscarinic agonist carbachol. This blockade was the result of the activation of protein kinase C since 4 alpha-phorbol 12,13-didecanoate, which lacks the ability to activate protein kinase C, did not inhibit Ca2+ mobilization responses to carbachol. Importantly, at lower phorbol myristate acetate concentrations (approximately 10(-9) M), carbachol-induced Ca2+ release was blocked, but carbachol-induced Ca2+ entry was maintained. These results show that carbachol-induced Ca2+ entry does not occur via an intracellular store and that protein kinase C plays a role in a feedback control mechanism for muscarinic-induced Ca2+ mobilization at different levels.     

Functional and biochemical interaction of dibutyryl cyclic AMP with the phosphoinositide system in isolated rat parietal cells

The interaction of the muscarinic agonist carbachol and of dibutyryl cAMP on acid secretion and phosphoinositide second messenger metabolism were studied in rat gastric parietal cells. Compared to the added effects of each agonist alone aminopyrine uptake, a measure of acid secretion, was enhanced 2-4-fold by the combination of both compounds. In addition the ED50 for carbachol was left shifted in the presence of dibutyryl-cAMP. The cholinergic stimulation of inositol phosphate production was slightly inhibited by dibutyryl-cAMP while levels of diacylglycerol were not affected. Thus the interaction of the cAMP and the phosphoinositide systems involve potentiation and positive sensitivity modulation of the cholinergic response by cAMP which is mediated by events distal to the generation of phosphoinositide second messengers.     

Process of re-establishment of beta-adrenergic induced protein secretion after cytochalasin D inhibition in rat parotid gland. Effect of cholinergic agonist, phorbol ester and calcium.

In rat parotid gland, 3H-protein secretion is stimulated by beta-adrenergic receptor activation (via cAMP) and also by cholinergic receptor activation (via IP3, calcium and diacylglycerol). The disorganization of microfilament system by cytochalasin D induced an inhibition of beta-adrenergic induced 3H-protein secretion whereas it did not modify the cholinergic muscarinic one. Cytochalasin D induced the formation of vacuoles in the parotid cell. In this work we show that the activation of muscarinic receptors (with carbachol) partially abolished the inhibitory effect of cytochalasin D on beta-adrenergic induced secretion. Since carbachol induced both intracellular calcium increase and protein kinase C activation, we decided to test separately the effect of calcium (using the calcium ionophore A23187) and protein kinase C activation (using phorbol ester) on the inhibitory effect of cytochalasin D on beta-adrenergic induced secretion. A23187, in the presence of calcium in the external medium was able to partially abolish cytochalasin D effect (ie re-establishing protein secretion) whereas activation of protein kinase C by phorbol 12-13 di-butyrate had no effect. These results suggest that protein kinase C is not involved in re-establishing a 'normal' secretion phenomenon whereas calcium does interfere. Furthermore, our fluorescence study shows that, when cytochalasin D is present in the incubation medium, the actin network is disturbed even in the presence of carbachol. This indicates that a calcium entry in the cell is not sufficient to restore a 'normal' actin network.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanisms of release of atrial natriuretic factor. II. Effect of chronic administration of alpha- and beta-adrenergic and cholinergic agonists on plasma and atrial ANF in the rat

The effect that chronic subcutaneous infusion of alpha- and beta-adrenergic and cholinergic agonists on plasma and atrial ANF was investigated. Isoproterenol, a beta-adrenergic agonist, and carbachol, a cholinergic agonist produced a 3-fold increase in plasma ANF levels which were constant until the end of the infusion period. An increased natriuresis was observed in the same groups which was positively correlated with plasma ANF. No differences were observed in atrial content of ANF between the experimental groups. A sharp post-surgery decline in plasma ANF was observed in control, phenylephrine and epinephrine-treated groups which was maintained during the observation period of five days. This suggests that the rise in plasma ANF induced by isoproterenol and carbachol may be secondary to hemodynamic changes and not to direct receptor stimulation, and may play a role in the observed natriuresis. It is also suggested that the depression of plasma ANF may contribute to the well known post-surgery sodium retention.     

Regulation of atrial natriuretic factor-(99-126) secretion from neonatal rat primary atrial cultures by activators of protein kinases A and C

Atrial natriuretic factor (ANF) is stored in atrial myocytes as a prohormone (ANF-(1-126] and is cosecretionally processed to the circulating ANF-related peptides, ANF-(1-98) and ANF-(99-126). Recently, we have shown that the cosecretional processing of ANF can be replicated in primary cultures of neonatal rat atrial myocytes maintained under serum-free conditions and that glucocorticoids are responsible for supporting this processing activity. Activators of protein kinase C (phorbol esters and alpha-adrenergic agonists) and of protein kinase A (cAMP analogs, forskolin, and beta-adrenergic agonists) were tested for their abilities to alter the rate of ANF secretion from the primary cultures. ANF secretion was stimulated approximately 4-fold after a 1-h incubation of the cultures with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA); maximal release occurred at about 100 nM TPA. Reversed-phase high performance liquid chromatography analysis of secreted material indicated that the cells efficiently cosecretionally processed ANF under both basal and TPA-stimulated conditions. However, incubating the cultures for more than 1 h with TPA resulted in a blunted secretory response to further TPA challenge and a 40-50% decrease in the quantity of ANF in the cells. The alpha-adrenergic receptor agonist phenylephrine was also capable of stimulating ANF secretion by about 4-fold at a half-maximal dose of about 1 microM. Phenylephrine-stimulated ANF secretion was inhibited by the alpha 1-adrenergic antagonist prazosin with half-maximal inhibition occurring at approximately 1 nM. Forskolin, 8-bromoadenosine 3':5'-cyclic monophosphate, and N6-2(1)-O-dibutyryladenosine 3':5'-cyclic monophosphate inhibited basal, TPA- and phenylephrine-stimulated ANF secretion. The beta-adrenergic agonist isoproterenol partially inhibited phenylephrine-stimulated ANF secretion with the maximal effect occurring at 1 nM. These results indicate that ANF secretion from the neonatal rat atrial cultures is enhanced by activators of protein kinase C, and decreased by activators of protein kinase A, and that these secretory effects may be mediated through the actions of alpha- and beta-adrenergic receptors, respectively.     

Salivary phospholipid secretion in response to beta-adrenergic stimulation is mediated by Src kinase-dependent epidermal growth factor receptor transactivation

Communication between receptor tyrosine kinase and G protein-coupled receptor (GPCR)-mediated signaling is recognized as a common integrator linking diverse aspects of intracellular signaling systems. Here, we report that G protein-coupled beta-adrenergic receptor activation leading to stimulation of salivary phospholipid release occurs with the involvement of epidermal growth factor receptor (EGFR). Using sublingual gland acinar cells, we show that prosecretory effect of isoproterenol on phospholipid release was subjected to suppression by EGFR kinase inhibitor, PD153035, and wortmannin, an inhibitor of PI3K, but not by PD98059, an inhibitor of extracellular signal regulated kinase (ERK). Furthermore, wortmannin, but not the ERK inhibitor, caused the reduction in the acinar cell secretory responses to beta-adrenergic agonist-generated cAMP as well as adenyl cyclase activator, forskolin. The acinar cell phospholipid secretory responses to isoproterenol, moreover, were inhibited by PP2, a selective inhibitor of tyrosine kinase Src responsible for ligand-independent EGFR phosphorylation. Taken together, our data are the first to demonstrate the requirement for Src kinase-dependent EGFR transactivation in regulation of salivary phospholipid secretion in response to beta-adrenergic GPCR activation.     

Protein Kinase C is a mediator of the synthesis and secretion of osteoprotegerin in osteoblast-like cells.

Osteoprotegerin (OPG) is a member of the TNF receptor superfamily and plays a critical role in the development of osteoclasts from precursor cells. OPG is produced by a variety of cells of mesenchymal origin and has been demonstrated to be present in osteoblasts and osteocytes. However, the mechanisms of regulation of OPG production and secretion are not known. Using a highly specific polyclonal antibody, we demonstrate that OPG is synthesized and secreted by osteoblast-like cells in culture. We further show that phorbol myristate acetate, an activator of protein kinase C, activated the secretion of OPG. Further, the increased secretion of OPG correlated well with a corresponding increase in OPG mRNA abundance. In addition, OPG promoter stably integrated into an osteoblast cell line was activated by phorbol myristate acetate. The increase in OPG expression was blocked by an inhibitor of protein kinase C, although the basal OPG expression was not altered. These results suggest that activation of the protein kinase C pathway may play a critical role in OPG expression.     

Regulation of cellular alpha-MSH and beta-endorphin during stimulated secretion from intermediate pituitary cells: involvement of aspartyl and cysteine proteases in the control of cellular levels of alpha-MSH and beta-endorphin

The regulation of cellular levels of alpha-melanocyte stimulating factor (alpha-MSH) and beta-endorphin in response to stimulated secretion from intermediate pituitary cells in primary culture was investigated in this study. Regulation of the cell content of alpha-MSH and beta-endorphin occurred in two phases consisting of (a) initial depletion of cellular levels of these peptide hormones during short-term secretion (3 h) induced by isoproterenol, forskolin, or phorbol myristate acetate (PMA) which was followed by (b) long-term (24 h) increases in cellular levels of alpha-MSH and beta-endorphin in response to stimulated secretion induced by isoproterenol and PMA. In short-term experiments (3 h), cellular levels of alpha-MSH and beta-endorphin were reduced by 30-50% during stimulated secretion of these peptide hormones by isoproterenol (agonist for the beta-adrenergic receptor), forskolin that activates protein kinase A (PKA), and PMA that activates protein kinase C (PKC). Moreover, dopamine inhibited isoproterenol-induced depletion of cellular alpha-MSH and beta-endorphin. During long-term incubation of cells (24 h) with isoproterenol, cellular alpha-MSH and beta-endorphin were increased to twice that of controls (unstimulated cells). Treatment with PMA for 24 h also increased cellular levels of alpha-MSH and beta-endorphin. Moreover, cellular levels of alpha-MSH and beta-endorphin were decreased during long-term treatment of cells with an aspartyl protease inhibitor, pepstatin A, and with the cysteine protease inhibitor E64c. These results implicate aspartyl and cysteine proteases in the cellular production of alpha-MSH and beta-endorphin that requires proteolytic processing of their common precursor proopiomelanocortin (POMC). These findings demonstrate the parallel regulation of cellular levels of alpha-MSH and beta-endorphin during their cosecretion, which may involve aspartyl and cysteine proteases in the metabolism of these peptide hormones.     

Phospholipase C and phospholipase A2 are involved in the antiviral activity of human interferon-alpha

Treatment of human amniotic cells (UAC) with human interferon-alpha (Hu-IFN alpha) or phorbol myristate acetate (PMA) resulted in translocation of protein kinase C (PK-C) activity from the cytosol fraction to that of the membranes. Analysis of 32P incorporation into phospholipid fractions and studies of alterations in fatty acid content for the major phospholipids of IFN-treated cells suggest that phospholipases C and A2 are activated by Hu-IFN alpha. Addition of neomycin (an inhibitor of phospholipase C), as well as mepacrine (an inhibitor of phospholipase A2) to IFN-treated cells inhibited the antiviral activity of Hu-IFN alpha in the vesicular stomatitis virus (VSV)-UAC system used. These observations indicate that (i) activation of PK-C and (ii) diacylglycerol formation, arachidonic acid and/or lysophosphatidylcholine release are important steps in the mechanism of action of IFN.     

Role of cyclic adenosine 3',5'-monophosphate-dependent protein kinase in hormone-stimulated beta-endorphin secretion in AtT20 cells.

Secretion of beta-endorphin from mouse pituitary AtT20 cells is stimulated by a variety of compounds that raise intracellular cAMP and Ca2+. To investigate the role of cAMP-dependent protein kinases in secretion, AtT20 cells were transfected with an expression vector coding for a regulatory (R) subunit of cAMP-dependent protein kinase containing mutations in both cAMP-binding sites. Expression of the mutant regulatory subunit in stable transformants (RAB cells) results in a dominant inhibition of cAMP-dependent protein kinase activity. Isoproterenol (1 microM) or analogs of cAMP stimulated beta-endorphin secretion from AtT20 cells, but failed to stimulate secretion in RAB cells expressing the mutant R subunit. Secretion in response to CRF (100 nM) was inhibited by 80% in these mutant clones, whereas the secretory response to vasoactive intestinal peptide (VIP; 100 nM) or phorbol ester (100 nM phorbol myristate acetate) was not inhibited by the R subunit mutation. Intracellular cAMP was elevated in response to CRF (11- to 15-fold), isoproterenol (5- to 10-fold), and VIP (4- to 8-fold) in RAB cells. Similar concentrations of VIP were required to evoke beta-endorphin secretion in either RAB cells or AtT20 cells. As with most secretagogues, VIP-induced secretion was inhibited in the presence of either EGTA or a voltage-sensitive Ca2+ channel antagonist, PN200-110. The secretory response to VIP was unaffected by down-regulation of protein kinase-C. These results suggest that CRF and isoproterenol work via cAMP-dependent protein kinase to activate beta-endorphin secretion, whereas VIP can act by a different mechanism that does not involve cAMP-dependent protein kinase or protein kinase-C.     

Secretion of gastric mucus phospholipids in response to beta-adrenergic G protein-coupled receptor activation is mediated by SRC kinase-dependent epidermal growth factor receptor transactivation.

Recent advances in understanding the nature of cellular responses mediated by G protein-coupled receptor (GPCR) activation indicate that integration of the converging regulatory signals into functional cellular pathways requires epidermal growth factor receptor (EGFR) transactivation. In this study, we report that G protein-coupled beta-adrenergic receptor activation leading to stimulation in gastric mucus phospholipid secretion occurs with the involvement of EGFR. Using [14C]choline-labeled gastric mucosal cells in culture, we show that stimulatory effect of beta-adrenergic agonist, isoproterenol, on phospholipid release was subject to a dose-dependent suppression by EGFR kinase inhibitor, PD153035, as well as wortmannin, a specific inhibitor of PI3K. Both inhibitors, moreover, caused the reduction in the gastric mucosal cell phospholipid secretory responses to beta-adrenergic agonist-generated second messenger, cAMP as well as adenyl cyclase activator, forskolin. The gastric mucosal phospholipid secretory responses to isoproterenol, furthermore, were inhibited by PP2, a selective inhibitor of tyrosine kinase Src responsible for ligand-independent EGFR phosphorylation, but not by ERK inhibitor, PD98059. The inhibition of ERK, moreover, did not cause attenuation in phospholipid secretory responses to cAMP and forskolin. The findings underline the central role of EGFR in mediation of gastric mucosal secretory processes, and demonstrate the requirement for Src kinase-dependent EGFR transactivation in regulation of gastric mucosal phospholipid secretion in response to beta-adrenergic GPCR activation.     

Phorbol ester stimulates amylase secretion from rat parotid cells

Phorbol myristate acetate (PMA), a potent activator of Ca2+- and phospholipid-dependent protein kinase (protein kinase C), evoked amylase release from rat parotid cells. In dose-response studies, PMA stimulated amylase release independently of db-cAMP, but potentiated the effect of carbachol. PMA and A23187, a Ca2+ ionophore, synergistically increased amylase release. The maximum effect of carbachol was further enhanced by PMA but not by A23187, suggesting that protein kinase C is not fully activated by the muscarinic-cholinergic agonist under the condition where calcium is fully utilized for amylase secretion.     

Calcium ionophore enables soluble agonists to stimulate macrophage 5-lipoxygenase

The regulation of arachidonic acid conversion by the 5-lipoxygenase and the cyclooxygenase pathways in mouse peritoneal macrophages has been studied using particulate and soluble agonists. Particulate agonists, zymosan and latex, stimulated the production of cyclooxygenase metabolites as well as the 5-lipoxygenase product, leukotriene C4. In contrast, incubation with the soluble agonist phorbol myristate acetate or exogenous arachidonic acid led to the production of cyclooxygenase metabolites but not leukotriene C4. We tested the hypothesis that the 5-lipoxygenase, unlike the cyclooxygenase, requires activation by calcium before arachidonic acid can be utilized as a substrate. Addition of phorbol myristate acetate to macrophages in the presence of calcium ionophore (A23187) at a concentration which alone did not stimulate arachidonate metabolism resulted in a synergistic increase (50-fold) in leukotriene C4 synthesis compared to phorbol ester or A23187 alone. No such effect on the cyclooxygenase pathway metabolism was observed. Exogenous arachidonic acid in the presence of A23187 produced similar results yielding a 10-fold greater synthesis of leukotriene C4 over either substance alone without any effects on the cyclooxygenase metabolites. Presumably, calcium ionophore unmasked the synthesis of leukotriene C4 from phorbol myristate acetate-released and exogenous arachidonate by elevating intracellular calcium levels enough for 5-lipoxygenase activation. These data indicate that once arachidonic acid is released from phospholipid by an agonist, it is available for conversion by both enzymatic pathways. However, leukotriene synthesis may not occur unless intracellular calcium levels are elevated either by phagocytosis of particulate agonists or with calcium ionophore.     

Induction of desensitization by phorbol ester to beta-adrenergic agonist stimulation in adenylate cyclase system of rat reticulocytes

Treatment of rat reticulocytes with a phorbol ester, tetradecanoyl phorbol acetate (TPA), resulted in the desensitization of adenylate cyclase to the beta-adrenergic agonist stimulation depending on the dose and period of the TPA treatment. Treatment of the reticulocytes with TPA caused approximately 40% reduction in the stimulation by beta-adrenergic agonists of adenylate cyclase activity, whereas the treatment had little effect on the basal activity and the activation by fluoride and guanine nucleotide of the enzyme system. No change in the number of beta-adrenergic receptors was observed after the TPA treatment. Treatment with 1-oleoyl-2-acetyl-glycerol (OAG), an activator of protein kinase C, also caused the desensitization of reticulocyte adenylate cyclase to isoproterenol. On the other hand, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), a potent inhibitor of protein kinase C, prevented the desensitization induced by TPA. These results suggest the involvement of protein kinase C in a process of desensitization of adenylate cyclase system to beta-adrenergic agonists in rat reticulocytes.     

Potentiation of arachidonic acid release by phorbol myristate acetate in platelets is not due to inhibition of arachidonic acid uptake or incorporation into phospholipids

Activators of protein kinase C, such as tumor-promoting phorbol esters (e.g., phorbol myristate acetate), mezerein, (-)-indolactam V and 1-oleoyl 2-acetoyl glycerol, potentiate arachidonic acid release caused by elevation of intracellular Ca2+ with ionophores. This action of protein kinase C-activators required protein phosphorylation, and was attributed to enhanced hydrolysis of phospholipids by phospholipase A2 (Halenda, et al. (1989) Biochemistry 28, 7356-7363). Recently Fuse et al. ((1989) J. Biol. Chem 264, 3890-3895) reported that the apparent enhanced release of arachidonate was actually due to inhibition of the processes of re-uptake and re-esterification of released arachidonic acid. They attributed this to loss of arachidonyl-CoA synthetase and arachidonyl-CoA lysophosphatide acyltransferase activities, which were measured in membranes obtained from phorbol myristate acetate-treated platelets. In this paper, we show that phorbol myristate acetate, at concentrations that strongly potentiate arachidonic acid release, does not inhibit either arachidonic acid uptake into platelets or its incorporation into specific phospholipids. Furthermore, the fatty acid 8,11,14-eicosatrienoic acid, a competitive substrate for arachidonyl-CoA synthetase, totally blocks arachidonic acid uptake into platelets, but, unlike phorbol myristate acetate, does not potentiate arachidonic acid release by Ca2+ ionophores. We conclude that the action of phorbol myristate acetate is to promote the process of arachidonic acid release by phospholipase A2.     

Phorbol diesters promote beta-adrenergic receptor phosphorylation and adenylate cyclase desensitization in duck erythrocytes

Preincubation of duck erythrocytes with tumor promoting phorbol diesters or catecholamines leads to attenuation of adenylate cyclase activity. 12-0-Tetradecanoyl phorbol-13-acetate (TPA) and phorbol 12,13-dibutyrate treatment induced a 38% and 30% desensitization of isoproterenol-stimulated adenylate cyclase activity, respectively. In contrast, the inactive phorbol diester, 4 alpha-phorbol 12,13-didecanoate, was without effect in promoting adenylate cyclase desensitization. The catecholamine isoproterenol induced a 51% desensitization. Incubation of 32Pi labeled erythrocytes with TPA promoted a 3- to 4-fold increase in phosphorylation of the beta-adrenergic receptor as did incubation with isoproterenol. Treatment of the cells with both TPA and isoproterenol together resulted in desensitization and receptor phosphorylation which were no greater than those observed with either agent alone. These data suggest a potential role for protein kinase C in regulating beta-adrenergic receptor function.