Isoproterenol increases sorting of parotid gland cargo proteins to the basolateral pathway

Exocrine cells have an essential function of sorting secreted proteins into the correct secretory pathway. A clear understanding of sorting in salivary glands would contribute to the correct targeting of therapeutic transgenes. The present work investigated whether there is a change in the relative proportions of basic proline-rich protein (PRP) and acidic PRPs in secretory granules in response to chronic isoproterenol treatment, and whether this alters the sorting of endogenous cargo proteins. Immunoblot analysis of secretory granules from rat parotids found a large increase of basic PRP over acidic PRPs in response to chronic isoproterenol treatment. Pulse chase experiments demonstrated that isoproterenol also decreased regulated secretion of newly synthesized secretory proteins, including PRPs, amylase and parotid secretory protein. This decreased efficiency of the apical regulated pathway may be mediated by alkalization of the secretory granules since it was reversed by treatment with mild acid. We also investigated changes in secretion through the basolateral (endocrine) pathways. A significant increase in parotid secretory protein and salivary amylase was detected in sera of isoproterenol-treated animals, suggesting increased routing of the regulated secretory proteins to the basolateral pathway. These studies demonstrate that shifts of endogenous proteins can modulate regulated secretion and sorting of cargo proteins. amylase; parotid secretory protein; polarized secretion     

Hormone-induced protein phosphorylation. II. Localization to the ribosomal fraction from rat exocrine pancreas and parotid of a 29,000- dalton protein phosphorylated in situ in response to secretagogues

In the preceding paper, we demonstrated that the endogenous phosphorylation of a protein with a molecular weight of 29,000 was enhanced by various secretagogues in rat pancreatic and parotid lobules, the phosphorylation of this protein correlating both temporally and in a dose-dependent fashion with secretory protein discharge. In the present study, we established a specific methodology to characterize this phosphoprotein. Once established, this 29,000-dalton phosphoprotein was then followed selectively and quantitatively throughout subcellular fractionation procedures. Analysis of two-dimensional polyacrylamide gels demonstrated that proteins with similar mobilities (Mr 29,000; pl greater than 8.4) were affected by cholecystokinin octapeptide and isoproterenol in rat pancreatic and parotid lobules, respectively, suggesting that the same 29,000-dalton phosphoprotein was covalently modified in both tissues. Cellular fractionation studies using differential velocity and sucrose density gradient centrifugation revealed that the 29,000-dalton phosphoprotein copurified with the rough microsomal fraction of pancreas and was highly enriched in ribosomal fractions of both pancreas and parotid. Electrophoresis in two dimensions confirmed that the 29,000-dalton polypeptide that was resolved directly from stimulated cells and from ribosomal fractions exhibited a common mobility, and apparent identity of the species was strongly suggested when the 29,000-dalton polypeptides from both sources were compared by peptide mapping following limited digestion with Staphylococcus aureus V8 protease. This phosphoprotein was tentatively identified as ribosomal protein S6 after analysis by pH 8.6/4.2 two-dimensional PAGE.     

ISOLATION AND PROPERTIES OF SECRETORY GRANULES FROM RAT ISLETS OF LANGERHANS : I. Isolation of a Secretory Granule Fraction

A method has been devised for the isolation of a secretory granule fraction from isolated rat islets of Langerhans. The islets were homogenized in buffered sucrose, and the homogenate was separated into nuclear, mitochondrial, secretory granule, and microsomal fractions by differential centrifugation. The secretory granule fraction was purified by differential centrifugation in discontinuous sucrose density gradients. A greater degree of purification could be achieved by the use of two successive gradients of this type, although the final yield was greatly reduced. Biochemical and morphological characterization of the fractions was obtained; the secretory granule fraction contained both insulin and glucagon. The limiting membranes of the granules remained intact and the general appearance of the granules was similar to that seen within the whole islet cells.     

Assembly and activation of the NADPH:O2 oxidoreductase in human neutrophils after stimulation with phorbol myristate acetate

Phagocytic leukocytes contain an activatable NADPH:O2 oxidoreductase. Components of this enzyme system include cytochrome b558, and three soluble oxidase components (SOC I, SOC II, and SOC III) found in the cytosol of resting cells. Previously, we found that SOC II copurifies with, and is probably identical to, a 47-kDa substrate of protein kinase C. In the present study we investigated the change in location of several of these oxidase components after activation of intact neutrophils with phorbol myristate acetate (PMA) and separation of subcellular fraction on sucrose density gradients. On Western blots with fractions of resting cells, the alpha subunit of cytochrome b558 was detected with a monoclonal antibody as a doublet of Mr 22,000 and 24,000 in the specific granules and as a single band of Mr 24,000 in the plasma membrane. PMA induced an increase of cytochrome b558 in the plasma membrane, including the Mr 22,000 band. PMA also induced translocation of the 47-kDa protein from the cytosol to the membrane fraction, as revealed by in vitro phosphorylation experiments. When NADPH oxidase activity was determined in a cell-free system in the presence of sodium dodecyl sulfate and GTP with plasma membranes from resting cells, cytosol from PMA-treated cells was deficient compared with cytosol from resting cells. This deficiency could be partially restored by the addition of SOC I. Concomitantly, SOC I activity appeared in the plasma membranes of PMA-treated cells. These studies support the hypothesis that PMA stimulation of neutrophils results in assembly of oxidase components from the cytosol and the specific granules in the plasma membrane with subsequent expression of NADPH oxidase activity.     

Isoproterenol-stimulated labelling of particulate proteins by using [adenylate-32P]NAD+ independent on a cAMP-dependent protein kinase in parotid acinar cells.

When saponin-permeabilized rat parotid acinar cells were incubated with [adenylate-32P]NAD+, labelling of proteins (33, 27 and 23 kDa) in particulate fractions of the cells was stimulated by isoproterenol. The effect of isoproterenol was completely blocked by a beta-antagonist. Both forskolin or cAMP mimicked the effect of isoproterenol on the labelling. However, an inhibitor of cAMPdPK failed to induce complete inhibition of the effects of isoproterenol, forskolin and cAMP. When the labelled proteins were treated with snake venom phosphodiesterase, neither [32P]5'-AMP nor [32P]phosphoribosyladenosine was released. These results suggest that covalent modification of proteins with NAD+, which is distinct from ADP-ribosylation and cAMPdPK-dependent phosphorylation, is coupled to beta-receptor-cAMP signalling system in rat parotid acinar cells.     

Secretory character of a group of isoproterenol-induced polypeptides in mouse parotid glands

The secretory nature of the isoproterenol-induced mouse parotid polypeptides C, D, E, F, and G (molecular weights 64,000, 61,000, 51,500, 38,000, and 37,000, respectively) is documented. Polypeptides C, D, E, F, and G, accumulated in response to successive daily stimulations with isoproterenol, were detected in a fraction enriched in hypertrophic parotid acinar cells. These cells, characterized by an increased content of cytoplasmic granules, maintain a secretory responsiveness to isoproterenol, which has been evidenced by light microscopy, enzymatic analysis, and unidimensional SDS-polyacrylamide gel electrophoresis. Thus, a parallelism in the loss and recovery of both secretory granules, alpha-amylase and polypeptides C, D, E, F, and G, was observed. Moreover, after secretion stimulation, polypeptides C, D, E, F, and G were detected in the fluid collected directly from parotid gland cannulation. Given the secretory character of polypeptides C, D, E, F, and G, mechanisms explaining both their progressive accumulation along the chronic administration of isoproterenol, as well as their progressive disappearance observed after suspending that treatment, are discussed.     

Phosphorylation of 3 particulate proteins in rat pancreatic acini in response to vasoactive intestinal peptide (VIP), secretin and cholecystokinin (CCK-8)

Rat pancreatic acini were preincubated with 0.4 mM 32Pi for 45 min at 37 degrees C, then exposed for 15 min to VIP, secretin or CCK-8. The incubation was terminated with a stop solution and a fraction rich in mitochondria and zymogen granules was separated from a microsome-rich fraction by differential centrifugation. After heating in the presence of SDS, beta-mercaptoethanol was added and the pattern of equivalent amounts of 32P-labelled proteins was examined by autoradiography of SDS-PAGE gels. VIP, secretin, and CCK-8 stimulated the phosphorylation of a Mr=33 K microsomal protein and that of two proteins of Mr=21 K and Mr=25 K mostly present in a fraction rich in mitochondria and zymogen granules. Stimulations were dose-dependent, the highest stimulant concentrations tested allowing 2- to 3-fold increases of phosphorylation over basal. When 1 nM CCK-8 was used simultaneously with 1 microM VIP, the cyclic AMP levels attained and the pattern of protein phosphorylation were similar to those obtained with VIP alone, and there was a potentiation of amylase secretion; when a supra-maximal 0.1 microM CCK-8 concentration was added, the VIP-induced elevation in cyclic AMP levels and the phosphorylation of the Mr=21 K and Mr=25 K proteins were partially antagonized, and no potentiation any more of secretion occurred. To conclude the in vitro phosphorylation of three particulate proteins (Mr=33 K, 25 K, and 21 K) was similarly increased in rat pancreatic acini in response to secretin and VIP (acting through cyclic AMP) and to CCK-8 (acting mostly through Ca2+).     

Compartmentalization of low molecular mass GTP-binding proteins among neutrophil secretory granules

Neutrophils contain several distinct classes of secretory granules that may sequentially fuse with the phagosome after the ingestion of particulates, or that may be differentially exocytosed after cellular activation with soluble stimuli. The exocytosis of neutrophil secretory granules has been shown to be GTP-dependent at a step distal to activation of the transductional G proteins. Inasmuch as ras-related low molecular mass GTP-binding proteins have been shown to play regulatory roles in vesicle sorting in the secretory pathway in yeast, the differential mobilization of neutrophil granules might be regulated by distinct GTP-binding proteins. We therefore explored the distribution and identity of low molecular mass GTP-binding proteins in neutrophil secretory granules and other subcellular fractions. After lysis by nitrogen cavitation, four highly resolved fractions were harvested from discontinuous Percoll gradients: a microsomal fraction enriched for plasma membranes, specific granules, primary granules, and cytosol. At least seven bands of distinct Mr were detected by probing protein blots with [32P]GTP. Microsomes contained a prominent GTP-binding band at 26 kDa and weaker ones at 24 and 22.5 kDa; specific granules contained bands at 26, 24, 22, and 20 kDa; primary granules showed bands at 24 and 23 kDa; cytosol showed strong bands at 23.5 and 19 kDa and a weak band at 26 kDa. Antiserum against ADP-ribosylation factor reacted strongly with the 19-kDa band in cytosol but with none of the membrane fractions. None of these proteins was recognized by antibodies against ras or against Sec4p. Botulinum exoenzyme C3 labeled bands of molecular mass 20 and 21 kDa in cytosol and microsomes that have distinct mobilities from all the blotted [32P]GTP-binding proteins. The highly compartmentalized subcellular distribution of the blotted [32P]GTP-binding proteins in neutrophils is consistent with a regulatory role in the differential mobilization of granule compartments during cellular activation.     

Effects of secretory stimulation on the Golgi apparatus and GERL of rat parotid acinar cells

The structure and cytochemistry of the Golgi apparatus and GERL of rat parotid acinar cells was studied after in vivo secretory stimulation with isoproterenol. Discharge of mature secretory granules was complete within 1 hr after isoproterenol injection, but immature granules in the Golgi region or near the lumen were not released. At early times (1-5 hr) after isoproterenol, acid phosphatase (AcPase) activity was markedly increased in GERL and immature secretory granules compared to uninjected controls. GERL appeared increased in extent and numerous continuities with immature granules were observed. Reaccumulation of mature secretory granules was first evident at 5 hr, and was almost complete by 16 hr after isoproterenol. Thiamine pyrophosphatase (TPPase) activity, normally restricted to the trans Golgi saccules, was frequently present in immature granules during this time. Narrow cisternae resembling GERL, occasionally in continuity with immature granules, also contained TPPase reaction product. By 16-24 hr after stimulation, the activity and distribution of AcPase and TPPase were similar to control cells. These results demonstrate the dynamic nature of the Golgi apparatus and GERL in parotid acinar cells, and emphasize the close structural and functional relationship between these two structures.     

Protein kinase C and an endogenous substrate associated with adenohypophyseal secretory granules.

Secretory granules isolated from anterior pituitary glands were examined for Ca2+/phospholipid-dependent protein kinase (protein kinase C) activity as well as the occurrence of granule-associated substrate proteins. Sheep adenohypophyses were fractionated by differential and sucrose-density-gradient centrifugation to yield a granule fraction enriched for luteinizing-hormone (lutropin)-containing secretory granules. Marker-enzyme analysis showed no detectable cytosolic contamination, although there were small amounts of plasma membranes (2-4%) and lysosomes (4-6%) associated with the preparation. As determined by histone-H1 phosphorylation after DEAE-cellulose DE-52 chromatography, protein kinase C activity with a marked dependence on Ca2+ and lipid (4-fold increase in their presence) was evident in the secretory-granule fraction. Phosphorylation in vitro of the secretory-granule fraction by endogenous and exogenous protein kinase C revealed a protein of Mr 36,000, which by two-dimensional SDS/polyacrylamide-gel electrophoresis showed multiple sites of phosphorylation. The Mr-36,000 protein was not found in cytosolic or plasma-membrane fractions and was not phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase. Several secretory-granule proteins served as substrates for the catalytic subunit, the most prominent of which were of Mr 63,000, 23,000 and 21,000. From these data, we suggest that phosphorylation of secretory-granule-associated proteins by protein kinase C and by cyclic AMP-dependent protein kinase may be important in secretion regulation in the anterior pituitary gland.     

Localization of cAMP-dependent protein kinase subunits along the secretory pathway in pancreatic and parotid acinar cells and accumulation of the catalytic subunit in parotid secretory granules following beta-adrenergic stimulation

Catalytic (C) and regulatory (RI and RII) subunits of cAMP-dependent protein kinases were localized by immunoelectron microscopy in cisternae of the rough endoplasmic reticulum (rER) and in the Golgi complex of rat pancreas or parotid cells. Zymogen granules of the exocrine pancreas showed C- and RI-immunoreactivity, secretory granules of parotid acinar cells only RII-immunoreactivity. Injection of rats with isoproterenol (IPR) increased in the parotid gland the number of acinar cells with RII-labeled granules. In addition, it led to the appearance of C-immunoreactivity in the condensing vacuoles and secretory granules with a maximum at 24 h after stimulation. This was confirmed by enzyme-linked immunosorbent assay (ELISA) determinations of C- and RII-subunits in secretory granules isolated from stimulated and control parotid glands. The amount of immunoreactive C-subunits in the secretory granules increased further following repeated injections of the beta-agonist. These findings suggest the existence of secretory forms of cAMP-dependent protein kinase R- and C-subunits and their separate regulation.     

Differential effects of polyamines on the phosphorylation of chromatin-associated proteins

Studies are presented on the nature of chromatin-associated phosphoproteins whose phosphorylation is influenced by polyamines. After labelling with 32P, chromatin-associated proteins were separated into four fractions. Fraction I comprised neutral and basic non-histone phosphoproteins, including high-mobility-group non-histones; fraction II consisted mostly of histones; fraction III consisted of a class of (salt-soluble) acidic non-histone phosphoproteins; and fraction IV consisted of residual (salt-insoluble) acidic non-histone phosphoproteins. The average relative distribution of protein in the four fractions (I-IV) was about 1:4:2:1 for both liver and prostate. However, tissue-dependent differences were observed in the incorporation of 32P in various protein fractions. In the presence of polyamines (e.g. 1 mM-spermine or 2 mM-spermidine) maximal stimulation of phosphorylation was observed in non-histone proteins of fraction I (160-180%), followed by that in non-histone proteins of fraction III (80-110%). The phosphorylation of residual non-histone proteins in fraction IV, and the small extent of phosphorylation of histones in fraction II, remained unaltered in the presence of polyamines. Thus polyamines do not stimulate the phosphorylation of all non-histone proteins; their stimulative effect is most prominent in the phosphorylation of neutral and basic non-histone proteins and a class of salt-soluble acidic non-histone proteins. In accord with our hypothesis, these differential effects of polyamines on phosphorylation of endogenous non-histone proteins may relate to the conformation of these substrates rather than to endogenous kinases.     

Postnatal Age and Protein Tyrosine Phosphorylation at Synapses in the Developing Rat Brain

The relationship between postnatal age and protein tyrosine kinase activity in synaptosomes prepared from the rat forebrain was studied. Synaptosomal particulate and soluble fractions, as well as total homogenates, the cell soluble fraction, and P3, were prepared from rats ranging in postnatal age from 5 to 60 days and analyzed for (a) tyrosine kinase activity using polyglutamyltyrosine (4:1) as the substrate, (b) the presence of endogenous substrates for tyrosine phosphorylation using polyclonal antibodies specific for phosphotyrosine, and (c) levels of pp60src. Enzyme activity, expressed per milligram of protein, in the total homogenate, P3, and both the cell and synaptosomal soluble fractions was highest in the brains of young animals (postnatal days 5-10) and decreased thereafter to adult levels. In contrast, tyrosine kinase activity in the synaptosomal particulate fraction exhibited a unique biphasic developmental profile, increasing to maxima at postnatal days 10 and 20 before decreasing to adult values. Endogenous substrates for tyrosine phosphorylation were identified by incubating subcellular fractions with 2 mM ATP in the presence of sodium orthovanadate and probing nitrocellulose blots of proteins separated by gel electrophoresis with antiphosphotyrosine antibodies. Several phosphotyrosine-containing proteins were detected in the synaptosomal particulate and P3 fractions, including proteins of Mr 180K, 145K, 120K, 100K, 77K, 68K, 62K, 54K, 52K, and 42K. In the cell soluble fraction a protein doublet of Mr 54/52K and a 120K protein were the major phosphotyrosine-containing proteins. The 54/52K doublet was the major protein tyrosine kinase substrate in the synaptosomal soluble fraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Secretory carrier membrane proteins 31-35 define a common protein composition among secretory carrier membranes.

A novel compositional overlap between membranes of exocrine and endocrine granules, synaptic vesicles, and a liver Golgi fraction has been identified using a monoclonal antibody (SG7C12) raised against parotid secretion granule membranes. This antibody binds secretory carrier membrane proteins with apparent Mr 31,000, 33,000 and 35,000 (designated SCAMPs 31, 33, 35). The proteins are nonglycosylated integral membrane components, and the epitope recognized by SG7C12 is on the cytoplasmic side of the granule membrane. SCAMP 33 is found in all secretory carrier membranes studied so far while SCAMP 35 is found in exocrine and certain endocrine granules and liver Golgi membranes and SCAMP31 only in exocrine granules. They are not related to other similar-sized proteins that have been studied previously in relation to vesicular transport and secretion. Immunocytochemical staining shows that these SCAMPs are highly concentrated in the apical cytoplasm of exocrine cells. Antigens are present not only on exocrine granules and synaptic vesicles but also on other smooth membrane vesicles of exocrine and neural origin as revealed by immunolocalization in subcellular fractions and immunoadsorption to antibody-coated magnetic beads. The wide tissue distribution and localization to secretory carriers and related membranes suggest that SCAMPs 31-35 may be essential components in vesicle-mediated transport/secretion.     

Characterization of protein-tyrosine kinase activity in the canine prostate.

Following the measurement of the phosphorylation of the substrate poly(Glu80Na,Tyr20) and the analysis of the alkali-resistant phosphorylation of endogenous proteins, the protein-tyrosine kinase of the canine prostate was partially characterized with regard to its subcellular localization, as well as certain kinetic and molecular properties. This kinase was mainly found in the cytosolic fraction (75%); however, its specific activity was similar to that of the residual enzyme present in the particulate fraction. Conditions for optimal activity of both fractions were determined. Under these conditions, several endogenous phosphoproteins (44-63 kilodaltons upon electrophoresis) were alkali resistant and phosphotyrosine was present in all of the major ones (pp63, pp57, pp52, and pp44). The particulate protein-tyrosine kinase activity was partially solubilized (58%) with 0.5% Triton X-100; this percentage was increased to 85% in the presence of 0.25 M KCl. Upon gel filtration, both cytosolic and particulate kinases showed an apparent molecular mass of 44 kilodaltons; these enzymes also phosphorylated similar major alkali-resistant phosphoproteins. The soluble protein-tyrosine kinase, with a sedimentation coefficient of 4.0S and an isoelectric point of 5.5, could be separated from arginine esterase and prostatic acid phosphatase.     

Isolation of a Golgi rich fraction from rat ventral prostate

Unmodified procedures for isolation of fractions rich in Golgi elements from other tissues have not proved applicable to the rat ventral prostate because of the tendency of membranous material to aggregate. We have devised a new procedure whereby: 1) a Golgi rich fraction from rat ventral prostate was released by a gentle two-step homogenization and isolated by centrifugation through discontinuous sucrose density gradients; 2) the specific activity of UDP-galactose: glycoprotein galactosyltransferase increased 69-fold in this fraction; 3) the isolated Golgi fraction was reasonably free from mitochondria, lysosomes, endoplasmic reticulum and plasma membranes as shown by the relatively low activities of marker enzymes; 4) the specific activities of acid phosphatase and 5'-nucleotidase in the Golgi rich fraction was 4 times greater than that in prostate homogenate. Both enzymes are secretory products and their presence in Golgi elements is probably associated with their packaging in secretory granules.     

Inhibitory effect of colchicines on amylase secretion by rat parotid glands: possible localization in the golgi area

Colchicine inhibited amylase secretion by isolated rat parotid glands only 6 h after administration of the drug in vivo. This delayed effect was not the result of the inability of the drug to reach its reaction site. When parotid glands were emptied of their secretory granules by isoproterenol treatment, the subsequent replenishment of cells with granules was inhibited by colchicines. Colchicine concomitantly produced alterations of the Golgi complexes, the cisternae of which were reduced in size and surrounded by clusters of microvesicles. Incubation of parotid glands with colchicines for prolonged durations failed to alter stored amylase secretion as stimulated by isoproterenol, but it inhibited the release of de novo synthesized enzyme. Another colchicines-binding activity, firmly bound to the particular fraction of homogenates, was found, of which a part may represent membrane located microtubular protein. An assembly-disassembly cycle of microtubules appears to exist in the parotid gland, as in the liver. However, only 14 percent of tubulin was found to be polymerized as microtubules in parotid glands as opposed to 40 percent in the liver. The present data suggest that colchicine primarily inhibits the transfer of secretory material towards or away from the Golgi complexes but not the hormone-stimulated secretion of stored amylase.     

Isolation of pig platelet membranes and granules distribution and validity of marker enzymes

A method for the subcellular fractionation of pig platelet homogenates by sucrose density gradient centrifugation is described. The procedure is simple, highly reproducible and yields two major particulate fractions and a soluble phase. One particulate fraction consists almost entirely of membrane fragments and is relatively free from granule contamination. The other particulate zone contains the platelet granules and mitochondria. The distribution on the gradients of the enzymes lactate dehydrogenase, succinate dehydrogenase, 5′-nucleotidase, leucyl β-naphthylamidase and cholinesterase has been studied and organelle localisation further substantiated by electron microscopy. The degree of solubilisation of certain marker enzymes during homogenisation has been investigated and the parallel release of these enzymes with the soluble phase marker enzyme lactate dehydrogenase, suggests they have a true biphasic location between the soluble and particulate components of the cell. No significant difference was found in the molar ratios of cholesterol to phospholipid in the subcellular fractions but the content of each lipid was twice as high in the membrane fraction as in the granule fraction.     

Enzyme modulation of the Golgi apparatus and GERL: a cytochemical study of parotid acinar cells

The distribution of thiamine pyrophosphatase (TPPase) and acid phosphatase (AcPase) has been examined in resting parotid acinar cells as well as during decreased and increased secretory granule production. In resting acinar cells, TPPase activity was restricted to the trans Golgi saccules and AcPase activity was localized in GERL and immature secretory granules. Although secretory granule production is diminished during ethionine intoxication, no significant alteration in the distribution of either TPPase or AcPase was noted. However, marked changes in enzyme localization, especially of TPPase, occurred during accelerated secretory granule production. The alterations were essentially the same for all of the conditions studied (recovery from ethionine treatment, recovery from a protein depletion diet, secretory stimulation with isoproterenol, and postnatal maturation of the parotid gland). During maximal secretory granule production, TPPase activity was localized not only in the trans Golgi saccules, but also in GERL-like cisternae and immature secretory granules. The immature secretory granules were often in continuity with the GERL-like cisternae. At the same time that the TPPase activity was increased, the AcPase activity was frequently diminished. These modulations in enzyme activity provide evidence that GERL is derived from the trans Golgi saccule.     

Modulation of aortic protein phosphorylation by benzo(a)pyrene: Implications in PAH-induced atherogenesis

The toxicity of polycyclic aromatic hydrocarbons such as benzo(a)pyrene, 7,12-dimethylbenz(a)anthracene, and 3-methylcholanthrene has been associated with alterations in the proliferation of vascular smooth muscle cells and the development of lesions of mesenchymal origin. Because phosphorylation of endogenous substrates plays a central role in the regulation of smooth muscle cell growth, the present studies were conducted to evaluate the phosphorylation pattern of medial aortic protein upon repeated in vivo exposure of Japanese quail to benzo(a)pyrene (BaP). Medial aortic homogenates from quail treated for 10 weeks with 10 mg/kg benzo(a)pyrene or vehicle were processed for in vitro measurements of protein phosphorylation. In vitro phosphorylation of endogenous or exogenous proteins stimulated in vitro by phorbol myristate acetate/phosphatidyl-serine or cyclic AMP, known activators of protein kinase C and cyclic AMP-dependent protein kinase, respectively, was examined in the cytosolic and particulate fractions of homogenates from control and treated animals. Benzo(a)pyrene treatment significantly enhanced the basal phosphorylation of Mr 113, 35, and 23 kDa proteins in the cytosolic fraction. Modest increases in the phosphorylation of Mr 71, 52, and 38 kDa were also observed under basal conditions. No changes in the basal phosphorylation of particulate proteins were observed. Phosphorylation of endogenous protein substrates by protein kinase C in the cytosolic fraction was not altered by benzo(a)pyrene treatment. In contrast, inhibition of C-kinase-mediated phosphorylation of endogenous Mr 272, 72, and 45 kDa proteins was observed in the particulate fraction of aortic homogenates from benzo(a)pyrene-treated quail relative to controls. Exogenous histone phosphorylation by PKC in the particulate, but not cytosolic fraction, was decreased by benzo(a)pyrene treatment. The effects of benzo(a)pyrene on the C-kinase system were specific, since cAMP-mediated phosphorylation of endogenous proteins, as well as exogenous histone, was not altered by benzo(a)pyrene. Interestingly, benzo(a)pyrene treatment was associated with a selective increase of Mr 200, 80, and 67 kDa proteins in the cytosolic fraction. Collectively, these data are consistent with the hypothesis that medial protein phosphorylation is a significant molecular target of benzo(a)pyrene within the vascular wall.