Purification and characterization of a 22-kDa microsomal protein from rat parotid gland which is phosphorylated following stimulation by agonists involving cAMP as second messenger

Stimulation of secretion in exocrine glands by agonists involving cAMP as second messenger leads to the phosphorylation of the ribosomal protein S6 (protein I) and two other particulate proteins with apparent molecular masses of 24 kDa (protein II) and 22 kDa (protein III) [Jahn, R., Unger, C. & S?ling, H. D. (1980) Eur. J. Biochem. 112, 345-352]. This report describes the purification and characterization of protein III. Solubilization studies indicate that protein III is an intrinsic membrane protein. It could be extracted from the endoplasmic reticulum membrane only with Triton X-100, SDS or concentrated formic or acetic acid. The purification of this protein involved extraction of the microsomes with Triton X-100, removal of the detergent by acetone precipitation, extraction of water-soluble proteins, lipids and lipoproteins, and preparative SDS polyacrylamide gel electrophoresis. The protein has a basic pI (greater than 8.7). For determination of the amino acid composition of protein III and for sequencing of its amino-terminal portion, the protein was electroeluted out off the gel, the detergent removed and the protein finally purified by reversed-phase HPLC. Protein III could be phosphorylated in vitro by the catalytic subunit of the cAMP-dependent protein kinase to a degree of approximately 0.14 mol phosphate/mol protein. The only phosphopeptide obtained after in vitro phosphorylation and subsequent tryptic or chymotryptic digestion was identical with the phosphopeptide obtained after stimulation of intact rat parotid gland lobules with isoproterenol. The sequence of this peptide was Lys-Leu-Ser(P)-Glu-Ala-Asp-Asn-Arg. It was confirmed by an analysis of the synthetic peptide following in vitro phosphorylation with cAMP-dependent protein kinase. The first 41 N-terminal residues of protein III were sequenced. So far no sequence homology with other known peptides or proteins could be found.     

Beta-adrenergic regulation of contractility and protein phosphorylation in spontaneously beating isolated rat myocardial cells

Spontaneously beating heart myocytes were prepared from adult rat ventricular tissues to study the correlation between beta-adrenergic receptor-stimulated changes in contractile performance and protein phosphorylation in vitro. The plasma membrane of isolated myocardial cells was permeabilized by saponin in the presence of EGTA and Mg-ATP. The permeabilized myocytes, which formed a homogeneous cell population, retained the rod-cell morphology of heart cells in situ and showed spontaneous cyclic contractions. Their contractile activity in response to extracellularly added cAMP mimicked the effects caused by beta-adrenergic stimulation of the whole heart: both the frequency and longitudinal velocity of free contraction and relaxation of the cells increased. Similar increases were observed when beta-agonist, isoproterenol, and GTP were added to suspending medium. In addition, isoproterenol maximally enhanced the adenylate cyclase activity of the cells in the presence of GTP. Both of these effects of isoproterenol were completely blocked by the beta-antagonist propranolol. cAMP-mediated phosphorylation of proteins in the permeabilized myocytes was investigated under conditions in which the beating frequency increased. cAMP elevated the phosphorylation level of five proteins; three of them with apparent molecular masses of 24, 15, and 12 kDa were membrane proteins and the other two with apparent molecular masses of 150 and 28 kDa were myofibrillar proteins. The 24-kDa phosphoprotein dissociated into 12-kDa molecules when boiled in sodium dodecyl sulfate, suggesting that these proteins are oligomeric and monomeric forms of phospholamban. The phosphorylation of these five proteins was stimulated by isoproterenol. The effect of isoproterenol was enhanced by GTP but completely blocked by propranolol. The time course of their phosphorylation correlated well with that of the increase in the beating frequency of the cells; both were measured after the administration of isoproterenol and GTP. When propranolol was added after the start of the stimulation by isoproterenol, only phospholamban and the 15-kDa protein were rapidly dephosphorylated in close correlation with the decrease of the beating frequency. These results demonstrate for the first time that the permeabilized myocytes retain the functional beta-adrenergic receptor and cellular responses to beta-adrenergic stimulation. They also suggest that cAMP-mediated phosphorylation of proteins, possibly phospholamban and/or the 15-kDa protein, is involved in the increased contractile activity of permeabilized heart cells.     

Transcriptional activity in previtellogenic oocyte germinal vesicles from Xenopus laevis

Receptor interactions of parotid acinar cells with beta-agonists are mediated by cyclic 3',5'-monophosphate (cAMP) and expressed as cAMP-dependent protein kinase (cAPK) activation. In addition to its location in the cytoplasm, we have shown that cAPK is associated with the nuclear non-histone protein (NHP) fraction (0.35 M NaCl extract) of rat parotid acinar cells. Nuclei were prepared from isolated parotid acini with minimal contamination from other cell types or cytoplasmic components. The nuclear cAPK activity was inhibited by the thermostable inhibitor and was stimulated by the addition of exogenous cAMP to the assay, indicating that the enzyme is present in the holoenzyme form. Enzyme activity was not increased in the presence of detergent, suggesting that cAPK is not bound to the nuclear membrane. Photoaffinity-labeling studies with an 8-azido analog of cAMP showed that regulatory subunits of both type I and type II cAPK isozymes are present in parotid cell nuclei. Short-term in vitro stimulation of the acini with 10(-6) M isoproterenol did not alter cAPK activity in the nuclear fraction. These findings indicate that compartmentation of cAPK into nuclear and extranuclear locations in rat parotid acinar cells is similar to that of several other cell types which are responsive to hormonal stimulation.     

Adenylate cyclase activity in the parotid gland of the mouse after isoproterenol stimulation

Previous studies have described a decrease in the activity of adenylate cyclase in the parotid gland of isoproterenol-treated rats. In the present studies, a similar decrease was observed in mice treated with isoproterenol. Studies on the subcellular distribution of adenylate cyclase after isoproterenol stimulation of the parotid gland showed that enzyme activity was increased in the lysosomal fraction and decreased in the cellular membrane fractions. Cytochemical studies on the localization of adenylate cyclase in stimulated gland showed an increase in vesicles which contained enzyme activity and a decrease in activity at the luminal and plasma membranes. It is suggested, based on the present findings and results reported by other investigators, that after isoproterenol stimulation of the parotid gland, adenylate cyclase (along with excess membrane) is degraded by lysosomes. If this suggestion is true, then the observed decrease in adenylate cyclase would have a molecular explanation.     

Beta-adrenergic receptor stimulation induces inositol trisphosphate production and Ca2+ mobilization in rat parotid acinar cells

In dispersed rat parotid gland acinar cells, the beta-adrenergic agonist (-)-isoproterenol, but not its stereoisomer (+)-isoproterenol, induced a transient 1.6-fold (at maximum stimulation, 2 x 10(-4) M) increase in cytosolic free calcium ([Ca2+]i) within 9 s, which returned to resting levels (approximately 190 nM) by 60 s. This [Ca2+]i response was not altered by chelating extracellular Ca2+ with [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) and could be completely blocked by the beta-adrenergic antagonists propranolol (beta 1 + beta 2) and ICI 118,551 (beta 2) but not by atenolol (beta 1). The muscarinic-cholinergic agonist carbachol (at maximum stimulation, 10(-5) M) induced a 3-4-fold elevation in [Ca2+]i within 6 s, which slowly returned to resting levels by 8-10 min. The peak carbachol [Ca2+]i response was not substantially altered by the addition of EGTA to the extracellular medium. However, if the cells were first stimulated with isoproterenol in the EGTA-containing medium, the peak carbachol response was decreased approximately 54%. When carbachol was added to cells in the presence of high extracellular calcium, at the isoproterenol-stimulated [Ca2+]i peak, the resulting [Ca2+]i level was equal to that achieved when carbachol was either added alone or added after propranolol and isoproterenol. 8-Bromo-cyclic AMP induced a [Ca2+]i response similar to that elicited by isoproterenol, which was not additive to that by carbachol. Carbachol induced a approximately 3.5-fold increase in inositol trisphosphate (IP3) production in parotid cells within 30 s. 8-Bromo-cAMP, N6,O2'-dioctanoyl-cAMP, and isoproterenol consistently induced a significant stimulation in IP3 production. The half-maximal concentration of isoproterenol required for [Ca2+]i mobilization and IP3 production was comparable (approximately 10(-5) M). Isoproterenol-induced IP3 formation was blocked by propranolol. The data show that in rat parotid acinar cells, beta-adrenergic stimulation results in IP3 formation and mobilization of a carbachol-sensitive intracellular Ca2+ pool by a mechanism involving cAMP. This demonstrates an interaction between the cAMP and phosphoinositide second messenger systems in these cells.     

Secretion of α-amylase in human parotid gland epithelial cell culture

The secretions of the salivary gland system are essential for the maintenance of oral health. The nature of cell-specific secretions of the various glands and their regulation is not completely understood. The objective of this study was to establish epithelial cell cultures from the human parotid gland that exhibit the tissue-specific function of α-amylase secretion. A specimen of normal human parotid gland was obtained at surgery and used to obtain primary cultures by the explant/outgrowth procedure. The cultures were maintained in keratinocyte basal medium, supplemented with insulin (5 μg/ml), EGF (10 ng/ml), hydrocortisone (0.5 μg/ml), bovine pituitary extract (25 μg/ml), and antibiotics. The cultures were passaged using 0.125% trypsin to dissociate the cells. Phase contrast and ultrastructural observations showed that the cells were polygonal and exhibited desmosomes. Their cytoplasm contained tonofilament bundles and abundant rough endoplasmic reticulum and Golgi complexes. Immunofluorescence studies showed that all cells were positive for cytokeratins. Immunoblot analysis revealed keratins with molecular weights of 58, 56, 52, 50, 48, 46, and 40 KD, which are characteristic of secretory epithelia. The cells have been passaged 35 times so far, undergoing a cumulative 120–140 population doublings. The serially passaged epithelial cell cultures produced and secreted α-amylase, a major component of parotid gland acinar cell secretion. The β-adrenergic agonist, isoproterenol (ISP), stimulated α-amylase secretion, which was accompanied by increased intracellular concentrations of cAMP. ISP-induced stimulation of amylase and cAMP was blocked by the β-adrenergic antagonist, propranolol. Further, dibutryl cAMP also enhanced the secretion of amylase. Thus we have established a long-term epithelial cell culture model of human parotid gland epithelial cells that exhibits differentiated function and retains the intact β-adrenergic receptor system. © 1993 Wiley-Liss, Inc.     

Evidence for the involvement of cAMP-GEF (Epac) pathway in amylase release from the rat parotid gland

Amylase release from the rat parotid gland is mainly mediated in a cAMP-dependent protein kinase (PKA)-dependent manner. In the present study, amylase release mediated in cAMP-dependent and PKA-independent manners was investigated with a cAMP-regulated guanine nucleotide exchange factor (cAMP-GEF: Epac)-selective cAMP analogue, 8CPT-2Me-cAMP. The Epac was localized in the intracellular and the plasma membrane fractions. PKA activation by 8CPT-2Me-cAMP was 100-fold lower than that by cAMP. The amylase release (% of the total) from the intact parotid acinar cells was 16 and 3.6% by isoproterenol (1microM) and 8CPT-2Me-cAMP (200microM), respectively, and that from the saponin-permeabilized cells was 15 and 3% by cAMP (100microM) and 8CTP-2Me-cAMP (10microM), respectively. H-89 inhibited cAMP-induced amylase release, but did not inhibit 8CPT-2Me-cAMP-induced amylase release. These results indicated that amylase release by beta-adrenergic stimulation is mediated through both the cAMP/PKA and cAMP/Epac signal pathways.     

Characterization of high affinity GTPase activity correlated to beta-adrenergic receptor stimulation of adenylyl cyclase in rat parotid membranes.

beta-Adrenergic receptor stimulation of adenylyl cyclase involves the activation of a GTP-binding regulatory protein (G-protein, termed here Gs). Inactivation of this G-protein is associated with the hydrolysis of bound GTP by an intrinsic high affinity GTPase activity. In the present study, we have characterized the GTPase activity in a Gs-enriched rat parotid gland membrane fraction. Two GTPase activities were resolved; a high affinity GTPase activity displaying Michaelis-Menten kinetics with increasing concentrations of GTP, and a low affinity GTPase activity which increased linearly with GTP concentrations up to 10 mM. The beta-adrenergic agonist isoproterenol (10 microM) increased the Vmax of the high affinity GTPase component approx. 50% from 90 to 140 pmol/mg protein per min, but did not change its Km value (approximately 450 nM). Isoproterenol also stimulated adenylyl cyclase activity in parotid membranes both in the absence or presence of GTP. In the presence of a non-hydrolyzable GTP analogue, guanosine 5'-(3-O-thio)triphosphate (GTP gamma S), isoproterenol increased cAMP formation to the same extent as that observed with AlF-4. Cholera toxin treatment of parotid membranes led to the ADP-ribosylation of two proteins (approximately 45 and 51 kDa). Cholera toxin also specifically decreased the high affinity GTPase activity in membranes and increased cAMP formation induced by GTP in the absence or the presence of isoproterenol. These data demonstrate that the high affinity GTPase characterized here is the 'turn-off' step for the adenylyl cyclase activation seen following beta-adrenergic stimulation of rat parotid glands.     

Presence of a complex containing vesicle-associated membrane protein 2 in rat parotid acinar cells and its disassembly upon activation of cAMP-dependent protein kinase.

Amylase release from parotid acinar cells is mainly induced by the accumulation of intracellular cAMP, presumably through the phosphorylation of substrates by cAMP-dependent protein kinase (PKA). However, the molecular mechanisms of this process are not clear. In a previous study (Fujita-Yoshigaki, J., Dohke, Y., Hara-Yokoyama, M., Kamata, Y., Kozaki, S., Furuyama, S., and Sugiya, H. (1996) J. Biol. Chem. 271, 13130-13134), we reported that vesicle-associated membrane protein 2 (VAMP2) is localized at the secretory granule membrane and is involved in cAMP-induced amylase secretion. To study the formation of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex containing VAMP2 in parotid acinar cells, we prepared rabbit polyclonal antibody against the peptide corresponding to Arg(47)-Asp(64) of VAMP2 (anti-SER4256). The recognition site of anti-SER4256 overlaps the domain involved in binding target membrane SNAREs (t-SNARES). Then we examined the condition of VAMP2 by immunoprecipitation with anti-SER4256. VAMP2 was not included in the immunoprecipitate from solubilized granule membrane fraction under the control conditions, but incubation with cytosolic fraction and cAMP caused immunoprecipitation of VAMP2. The effect of cytosolic fraction and cAMP was reduced by addition of PKA inhibitor H89. Addition of both the catalytic subunit of PKA and the cytosolic fraction allowed immunoprecipitation of VAMP2, whereas the PKA catalytic subunit alone did not. These results suggest that () the t-SNARE binding region of VAMP2 is masked by some protein X and activation of PKA caused the dissociation of X from VAMP2; and () the effect of PKA is not direct phosphorylation of X, but works through phosphorylation of some other cytosolic protein.     

beta-Adrenergic receptors stimulated peroxidase secretion from rat lacrimal gland

Incubation of rat extraorbital lacrimal gland slices with the beta-agonist isoproterenol caused peroxidase secretion but no K+ release. The peroxidase secretion was inhibited by propranolol. Addition of dibutyryl cyclic AMP or adenosine 3'5'-cyclic phosphorothioate to lacrimal slices produced peroxidase secretion at a higher rate than that obtained with optimal concentration of isoproterenol. Methyl isobutylxanthine is also a strong stimulator of peroxidase secretion. Peroxidase activity was determined by a modified sensitive guaiacol method. Membrane fraction of lacrimal cells was shown to contain an isoproterenol-stimulated adenylate cyclase activity. It is therefore suggested that there is a beta-adrenergic receptor in the rat lacrimal gland and that its stimulation causes activation of an adenylate cyclase which leads to peroxidase secretion.     

Characterization of high affinity GTPase activity correlated to β-adrenergic receptor stimulation of adenylyl cyclase in rat parotid membranes

β-Adrenergic receptor stimulation of adenylyl cyclase involves the activation of a GTP-binding regulatory protein (G-protein, termed here Gs). Inactivation of this G-protein is associated with the hydrolysis of bound GTP by an intrinsic high affinity GTPase activity. In the present study, we have characterized the GTPase activity in a Gs-enriched rat parotid gland membrane fraction. Two GTPase activities were resolved; a high affinity GTPase activity displaying Michaelis-Menten kinetics with increasing concentrations of GTP, and a low affinity GTPase activity which increased linearly with GTP concentrations up to 10 mM. The β-adrenergic agonist isoproterenol (10 μM) increased the V_max of the high affinity GTPase component approx. 50% from 90 to 140 pmol/mg protein per min, but did not change its K_m value (≈ 450 nM). Isoproterenol also stimulated adenylyl cyclase activity in parotid membranes both in the absence or presence of GTP. In the presence of a non-hydrolyzable GTP analogue, guanosine 5′-(3-O-thio)triphosphate (GTPγS), isoproterenol increased cAMP formation to the same extent as that observed with AlF₄^?. Cholera toxin treatment of parotid membranes led to the ADP-ribosylation of two proteins (≈ 45 and 51 kDa). Cholera toxin also specifically decreased the high affinity GTPase activity in membranes and increased cAMP formation induced by GTP in the absence or the presence of isoproterenol. These data demonstrate that the high affinity GTPase characterized here is the ‘turn-off’ step for the adenylyl cyclase activation seen following β-adrenergic stimulation of rat parotid glands.     

Specific phosphorylation of a protein in calcium accumulating endoplasmic reticulum from rat parotid glands following stimulation by agonists involving cAMP as second messenger

Stimulation of secretion in exocrine cells by agonists involving cAMP as second messenger is associated with the phosphorylation of a specific membrane-associated 22.4-kDa protein (protein III) (Jahn et al.). Here it is shown by subcellular fractionation of rat parotid gland lobules that protein III is associated with the endoplasmic reticulum. The submicrosomal fractions containing protein III, also contain the ATP-dependent microsomal calcium pump activity. Protein III in microsomal subfractions can be phosphorylated in vitro with catalytic subunit from cAMP-dependent protein kinase. Phosphorylated protein III contains exclusively P-serine. Protein III can be removed from ER-membranes with acid chloroform-methanol or Triton X-114, but not by high salt wash indicating that it is tightly associated with the membranes. Protein III is smaller than phospholamban and, in contrast to phospholamban, resistant to heating in SDS. A relationship between phosphorylation of protein III and microsomal calcium sequestration is discussed.     

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.     

β-Adrenergic receptor stimulated peroxidase secretion from rat lacrimal gland

Incubation of rat extraorbital lacrimal gland slices with the β-agonist isoproterenol caused peroxidase secretion but no K⁺ release. The peroxidase secretion was inhibited by propranolol. Addition od dibutyryl cyclic AMP or adenosine 3′,5′-cyclic phosphorothioate to lacrimal slices produced peroxidase secretion at a higher rate than that obtained with optimal concentration of isoproterenol. Methyl isobutylxanthine is also a strong stimulator of peroxidase secretion. Peroxidase activity was determined by a modified sensitive guaiacol method. Membrane fraction of lacrimal cells was shown to contain an isoproterenol-stimulated adenylate cyclase activity. It is therefore suggested that there is a β-adrenergic receptor in the rat lacrimal gland and that its stimulation causes activation of an adenylate cyclase which leads to peroxidase secretion.     

Novel phosphorylation of aquaporin-5 at its threonine 259 through cAMP signaling in salivary gland cells

Aquaporin-5 (AQP5), a water channel, plays key roles in salivary secretion. The novel phosphorylation of AQP5 was investigated by using human salivary gland (HSG) cells and mouse salivary glands. In the HSG cells stably transfected with a wild-type mouse AQP5 construct, a protein band immunoreactive with antibody against phosphorylated PKA substrate was detected in the AQP5 immunoprecipitated sample, and its intensity was enhanced by short-term treatment of the cells with 8-bromo-cAMP, forskolin, or phorbol 12-myristate 13-acetate, but not by that with A23187 calcium ionophore. Such enhancement was inhibited in the presence of H-89, a PKA inhibitor. An AQP5 mutant (AQP5-T259A) expressed by transfection of HSG cells was not recognized by anti-phosphorylated PKA substrate antibody, even when the cells were stimulated with the protein kinase activators. Immunoblotting and immunofluorescence studies using a specific antibody detecting AQP5 phosphorylated at its Thr259 demonstrated that AQP5 was rapidly and transiently phosphorylated at the apical membrane of acinar cells in the submandibular and parotid glands after administration of isoproterenol, but not pilocarpine. Furthermore, both AQP5 and AQP5-T259A were constitutively localized at the plasma membrane in HSG cells under the resting and forskolin-stimulated conditions. These results suggest that AQP5 is phosphorylated at its Thr259 by PKA through cAMP, but not Ca(2+), signaling pathways, and that this phosphorylation does not contribute to AQP5 trafficking in the salivary gland cells.     

The influence of exogenous cyclic nucleotides and theophylline on the hydrokinetic and ecbolic function of the isolated perfused canine pancreas (author's transl)

The influence of exogenous cyclic nucleotides or theophylline either on basal or stimulated volume and protein secretion is studied on the isolated perfused canine pancreas in dependence on varied extracellular calcium concentrations. Bt2cAMP or theophylline do not influence basal secretory rates of pancreatic juice but potentiate secretin-stimulated volume output. They additionally increase basal protein secretion under exclusive secretin stimulation and potentiate dose-dependently CCK- or acetylcholine-induced protein output. The hydrokinetic and ecbolic effects of Bt2cAMP and theophylline persist in a calcium-free medium but fail in normalizing inhibited protein secretion during calcium deprivation. Bt2cGMP neither increases basal nor stimulated volume and protein secretion. The demonstrated influence of Bt2cAMP and theophylline on ductal volume and acinar protein secretion accomplishes two criteria, as suggested by Sutherland, for cAMP as second messenger for secretin and CCK or acetylcholine as well.     

Alterations in the subcellular distribution of p21ras-GTPase activating protein in proliferating rat acinar cells.

Rat parotid acinar cells undergo transient proliferation in response to chronic administration of the beta-adrenergic agonist isoproterenol or epidermal growth factor (EGF). Treatment with these agents caused an increase in tyrosine phosphorylation of p21ras-GTPase activating protein (GAP). This phosphorylation event was accompanied by a redistribution of the protein from the plasma membrane to internal membrane compartments. Separation of subcellular membranes revealed increased GAP associated with a low density population of vesicles concomitant with growth stimulation as well as to the nuclear membrane, but not the nucleoplasm. Upon cessation of hyperplasia induced by isoproterenol, phosphorylated GAP present in the plasma membrane returned to control cell levels.     

Hormonal activation of the cAMP-dependent protein kinases in AtT20 cells. Preferential activation of protein kinase I by corticotropin releasing factor, isoproterenol, and forskolin

The hormonal regulation of adenylate cyclase, cAMP-dependent protein kinase activation, and adrenocorticotropic hormone (ACTH) secretion was studied in AtT20 mouse pituitary tumor cells. Corticotropin releasing factor (CRF) stimulated cAMP accumulation and ACTH release in these cells. Maximal ACTH release was seen with 30 nM CRF and was accompanied by a 2-fold rise in intracellular cAMP. When cells were incubated with both 30 nM CRF and 0.5 mM 3-methylisobutylxanthine (MIX) cAMP levels were increased 20-fold, however, ACTH release was not substantially increased beyond release seen with CRF alone. The activation profiles of cAMP-dependent protein kinases I and II were studied by measuring residual cAMP-dependent phosphotransferase activity associated with immunoprecipitated regulatory subunits of the kinases. Cells incubated with CRF in the absence of MIX showed concentration-dependent activation of protein kinase I which paralleled stimulation of ACTH release. Protein kinase II was minimally activated. When cells were exposed to CRF in the presence of 0.5 mM MIX there was still a preferential activation of protein kinase I, although 50% of the cytosolic protein kinase II was activated. Complete activation of both protein kinases I and II was seen when cells were incubated with 0.5 mM MIX and 10 microM forskolin. Under these conditions cAMP levels were elevated 80-fold. CRF, isoproterenol, and forskolin stimulated adenylate cyclase activity in isolated membranes prepared from AtT20 cells. CRF and isoproterenol stimulated cyclase activity up to 5-fold while forskolin stimulated cyclase activity up to 15-fold. Our data demonstrate that ACTH secretion from AtT20 cells is mediated by small changes in intracellular levels of cAMP and activation of only a small fraction of the total cytosolic cAMP-dependent protein kinase in these cells is required for maximal ACTH secretion.