Biphasic regulation of macrophage attachment by activators of cyclic adenosine monophosphate-dependent kinase and protein kinase C

A method is described that enabled us to study the adhesiveness of J-774 murine macrophages. Cell attachment was stimulated by activators of kinase C (i.e., phorbol esters) as well as kinase A (cyclic adenosine monophosphate; cAMP). This novel effect of cAMP was observed when its levels were increased via receptor triggering (prostaglandin E1, beta-adrenergic agonists), activation of Ns (cholera toxin), or inhibition of phosphodiesterase (Ro 20-1724) or when the kinase was directly activated by Br8-cAMP. The simultaneous treatment with kinase A and kinase C activators at the time of attachment resulted in a partially additive response. On the other hand, preincubation of the cells in suspension with one of the activators rendered them refractory to subsequent stimulation at the onset of the adhesion assay, whatever agent was used. Such a refractoriness was also observed in cells preincubated with oleoyl-acetyl-glycerol (OAG). On the other hand, when added at the time of attachment, this near-physiological activator of kinase C evoked a biphasic response: the early stimulation of cell attachment was followed by an accelerated rate of "detachment." In conclusion, kinase C and kinase A play a role in the sequence of events leading to cell adhesion. The cross desensitization observed is distal and takes place at or beyond the kinase step.     

Combinative ligand-receptor interactions: effects of cAMP, epinephrine, and met-enkephalin on RAW264 macrophage morphology, spreading, adherence, and microfilaments

Cell surface ligand-receptor interactions play a central role in the regulation and expression of macrophage function. Included among these macrophage membrane receptors are the beta-adrenergic and opioid receptors. We studied the abilities of epinephrine, met-enkephalin, forskolin, and adenosine 3':5' cyclic monophosphate (cAMP) analogues to affect macrophage morphology, spreading, and adherence. Cell spreading was quantitated by measuring the perimeters of adherent cell images recorded by videomicroscopy. Epinephrine induced a dose-dependent decrease in macrophage spreading; at 10(-5) M epinephrine the mean perimeter was 10.4 +/- 0.3 microns in comparison to 15.0 +/- 1.0 microns for controls. The inhibition of spreading can be blocked by the antagonist propranolol. On the other hand, met-enkephalin induced a dose-dependent increase in macrophage spreading, with a perimeter of 18.5 +/- 1.0 microns at 10(-8) M. Since catecholamines and opioids are simultaneously released from chromaffin cells of the adrenal, we examined the combinative effects due to treatment with both ligands. When macrophages were exposed to 10(-5) M epinephrine and 10(-8) M met-enkephalin, cell morphology and spreading were indistinguishable from that due to 10(-5) M epinephrine alone. The epinephrine dose-response curve in the presence of 10(-8) M met-enkephalin was similar to that of epinephrine alone. The beta-adrenergic receptor is apparently capable of diminishing or abrogating the opioid receptor signal(s). These combinative and epinephrine-mediated effects may be at least partially accounted for by the action of cAMP. Forskolin and the cAMP analogues N6-2'-O-dibutyryladenosine 3':5' cyclic monophosphate (dbcAMP) and 8-bromoadenosine 3':5' cyclic monophosphate (Br-cAMP) affected cell morphology and spreading in the same fashion as epinephrine. These differences in morphology and spreading behavior were accompanied by changes in the distribution of F-actin, as judged by phalladicin staining and fluorescence microscopy. We suggest that cAMP and microfilaments play important roles in receptor-mediated neuroregulation of macrophage function.     

Regulation of protooncogenes c-fos and c-myc expressions by protein tyrosine kinase, protein kinase C, and cyclic AMP mitogenic pathways in dog primary thyrocytes: a positive and negative control by cyclic AMP on c-myc expression

The proliferation of dog thyrocytes in primary culture is stimulated by three distinct intracellular signaling pathways: (1) the thyrotropin or forskolin-cyclic AMP-mediated cascade which is compatible with the differentiated state of the cell; (2) the protein kinase C pathway activated by diacylglycerol and phorbol esters; and (3) a protein tyrosine kinase system activated by epidermal growth factor. The two latter pathways also induce dedifferentiation. The activation of the three cascades induced the expression of the protooncogenes c-fos and c-myc with dose-response curves similar to those for DNA synthesis. After TPA and EGF, the time courses of stimulation of c-fos and c-myc were the same as those for mitogenically stimulated fibroblasts. However, after the cyclic AMP stimulation, c-myc expression was biphasic with an enhancement at 1 h followed by a down-regulation. A similar inhibition by cyclic AMP was also observed on the increased c-myc expression induced by EGF. This down-regulation is suppressed by cycloheximide, which suggests the involvement of a neosynthesized or a labile protein intermediate. The action of cyclic AMP on c-myc mRNA levels could be related to the opposite requirements of the stimulation of both proliferation and differentiation expression by the cyclic AMP pathway in the differentiated thyrocytes.     

Regulation of fungal proteolysis on cyclic AMP-dependent protein kinase,cyclic AMP phosphodiesterase,glycogen synthase and histones

Limited proteolysis of catalytic and regulatory subunits of cyclic AMP-dependent protein kinase (A-pk), cyclic AMP phosphodiesterase, glycogen synthase, and histones by fungal protease (type XIX) was analyzed by the digested peptide bands in SDS polyacrylamide gel electrophoresis. The modulatory effects on proteolysis by nucleotides, polypeptides, and phospholipids may greatly depend on the intrinsic nature of substrates. The proteolysis of the regulatory subunit of A-pk and glycogen synthase was not regulated by nucleotides and nucleic acids. In comparison, phosphatidyl serine, cardiolipin, and pepstatin A stimulated the proteolysis of the catalytic subunit of A-pk. Whereas, DNA (Hind III digest), t-RNA, GTP, phosphatidyl serine, sphingosine inhibited the proteolysis of cyclic AMP phosphodiesterase. Moreover, MS2 RNA, DNA, t-RNA, dGTP, Phosphatidyl serine, phosphatidyl inositol, antipain, and chymostatin exerted inhibitory proteolytic effect on histone VIII-S. Some of these agents also had similar inhibitory effect on other types of histones (types III-S and VII-S). The inhibitory effect of phosphatidyl serine on proteolysis of histone may be due to their interaction which was monitored by the drastic increase of uv absorbance.     

Control of platelet protein kinase C activation by cyclic AMP

Experiments were performed to elucidate the role of adenosine 3': 5'-cyclic monophosphate (cAMP) in the control of platelet protein kinase C (PKC) activation. Platelet aggregation and secretion in response to 4 beta-phorbol 12-myristate 13-acetate (PMA) or 1-oleoyl-2-acetylglycerol (OAG) were inhibited by dibutyryl cAMP in a dose-dependent manner. Inhibition of these functional activities paralleled a decrease in the PMA-induced phosphorylation of the Mr 47,000 substrate (p47) of PKC by pre-incubation of platelets with dibutyryl cAMP. These changes were also observed when platelet cAMP was increased by prostacyclin (PGI2), forskolin, or theophylline. The ADP scavenger creatine phosphate/creatine phosphokinase (CP/CPK) and the cyclooxygenase inhibitor indomethacin also diminished the aggregation and p47 phosphorylation responses to PMA or OAG. Pre-incubation of platelets with dibutyryl cAMP significantly potentiated the inhibition of aggregation and p47 phosphorylation effected by CP/CPK and indomethacin. These results are consistent with the model that PMA- or OAG-induced activation of platelets is amplified by secreted ADP and that the response to secreted ADP is inhibited by cAMP. Furthermore, the findings that increased intracellular cAMP inhibits PMA- or OAG-induced p47 phosphorylation in excess of that due solely to CP/CPK, and that cAMP significantly potentiates the effects of ADP removal and inhibition of cyclooxygenase in blocking p47 phosphorylation suggest that cAMP also exerts non-ADP-mediated inhibitory effects on PKC in intact platelets.     

The phosphorylation of p68, a calcium-binding protein associated with the human syncytiotrophoblast submembranous cytoskeleton, is modulated by growth factors, activators of protein kinase C and cyclic AMP

The phosphorylation of the lipocortin-related protein, p68, found in Ca2+-dependent association with the submembranous cytoskeleton has been studied using isolated human placental syncytiotrophoblast plasma membrane vesicles. p68 undergoes rapid, cation-independent phosphorylation in unstimulated membrane vesicles which was inhibited, in a dose-dependent manner, by insulin, platelet-derived growth factor, macrophage colony stimulating factor, protein kinase C-activating phorbol esters and phosphatidylinositol-specific phospholipase C. Epidermal growth factor had no effect on overall p68 phosphorylation. Transferrin induced an increase in p68 phosphorylation. However, phosphotyrosine was detected in p68 after treatment with epidermal growth factor, macrophage colony stimulating factor or transferrin, whereas a reduction in p68 phosphorylation appeared to be restricted to serine. cAMP and both cholera and pertussis toxins inhibited p68 phosphorylation. Both toxins were synergistic with the effects of insulin and platelet-derived growth factor whilst being antagonistic to the effect of transferrin. Epidermal growth factor and both human and equine immunoglobulin G, all of which alone did not affect overall p68 phosphorylation, reduced cholera or pertussis toxin-induced inhibition of p68 phosphorylation. Several phosphatase inhibitors failed to prevent macrophage colony stimulating factor-induced reduction of p68 phosphorylation. These results indicate that (i) p68 is a potential substrate of receptor tyrosyl kinases, (ii) p68 is not phosphorylated by protein kinase C or cAMP-dependent kinase and (iii) p68 phosphorylation is inhibited by activation of multiple pathways including those employing diacylglycerol or cAMP as second messengers.     

Metamorphosis inHydractinia: Studies with activators and inhibitors aiming at protein kinase C and potassium channels

Summary Metamorphosis of planula larvae involves an activation of morphogenetically quiescent cells. The present work extends a previous study [Leitz T and Müller WA (1987) Dev Biol 121:82–89] on the participation of the phosphatidylinositol/diacylglycerol/protein kinase C system. Metamorphosis is stereospecifically induced by diacylglycerols, 1,2,-sn-dioctanoylglycerol (diC₈) being by far the most effective substance. K-252a and sphingosine, inhibitors of mammalian protein kinases C, profoundly inhibited metamorphosis. Phorbolester-binding studies and the corresponding Scatchard plots revealed a specific and saturable binding of [³H]phorbol 12,13-dibutyrate to a single site of particulate fractions ofHydractinia with a specific binding affinityK _d = 50 nM. K⁺ ionophores stimulated Cs⁺ — but inhibited diC₈-induced metamorphoses, K⁺-channel blockers enhanced the inducing action of Cs⁺ or diC₈. On the basis of these data and observations of others we propose that the activation ofHydractinia larvae takes place in some cells at the anterior end as a result of activation of a kinase-C-like enzyme, which directly or indirectly leads to the closure of K⁺ channels. Closure of these channels then causes depolarisation and, thus, release of an internal signal. This hypothesis unifies notions about the role of K⁺ channels and of the phosphatidylinositol system in initiation of metamorphosis inHydractinia.     

Regulation of VL30 gene expression by activators of protein kinase C

The mouse genome contains a retrovirus-like sequence, designated VL30, which is expressed at high levels in transformed cells and which can be induced by exogenously supplied epidermal growth factor (EGF). Binding of EGF to the EGF receptor produces changes in intracellular calcium levels and phospholipase activity which indirectly lead to activation of protein kinase C. We treated AKR-2B cells, Swiss 3T3 cells, and the 3T3 variants NR6 (EGF receptorless) and TNR9 (phorbol ester nonresponsive) with various phorbol ester tumor promoters and with the synthetic diacylglycerol sn-1,2-dioctanoylglycerol. Tumor-promoting phorbol esters (e.g. 12-O-tetradecanoyl phorbol acetate (TPA] increased the level of VL30 expression. Stimulation with either TPA or EGF produced a similar time course of VL30 expression. TPA induced VL30 expression in the EGF-receptorless NR6 cell line, indicating that neither EGF ligand-receptor binding nor phosphorylation of the EGF receptor was required for induction of VL30 expression. Protein synthesis was not required for the TPA-mediated increase in VL30 expression, as pretreatment with cycloheximide did not block or reduce the TPA effect. VL30 expression was also stimulated by treatment with sn-1,2-dioctanoylglycerol, an analog of a probable endogenous activator of protein kinase C. These results suggest that activation of protein kinase C plays a direct role in regulating VL30 expression.     

Effects of protein kinase C activation on human platelet cyclic AMP metabolism

Treatment of intact human platelets with the tumour-promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA), specifically inhibited PGD2-induced cyclic AMP formation without affecting the regulation of cyclic AMP metabolism by PGI2, PGE1, 6-keto-PGE1, adenosine or adrenaline. This action of PMA was: (i) concentration-dependent; (ii) not mediated by evoked formation or release of endogenous regulators of adenylate cyclase activity (thromboxane A2 or ADP); (iii) mimicked by 1,2-dioctanoylglycerol (DiC8) but not by 4 alpha-phorbol 12,13-didecanoate (which does not activate protein kinase C); (iv) attenuated by Staurosporine. These results indicate that activation of protein kinase C in platelets may provide a regulatory mechanism to abrogate the effects of the endogenous adenylate cyclase stimulant PGD2 without compromising the effects of exogenous stimulants of adenylate cyclase (PGI2, 6-keto-PGE1, adenosine).     

Differential activation and inhibition of lymphocyte proliferation by modulators of protein kinase C: diacylglycerols, "rationally designed" activators and inhibitors of protein kinase C

The tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) can enhance or inhibit lymphocyte proliferation. Enhancement correlated with increased interleukin 2 (IL-2) production and activation of protein kinase C while inhibition correlated with decreased IL-2 and downregulation of protein kinase C activity (D.S. Grove and A.M. Mastro, Cancer Res. 51, 82-88). In this study, various activators and inhibitors of protein kinase C were used in order to try to separate the effects of TPA on this enzyme from its effects on IL-2 production and determine if protein kinase C activity was directly or indirectly related to IL-2 production. 1,2-Dioctanoylglycerol, 1-oleoyl-2-acetyl-glycerol, phospholipase C, and two "rationally designed" activators, 6-(N-decylamino)-4-hydroxy-methylindole and 3-(N-acetylamino)-5-(N-decyl-N-methylamino)-benzyl alcohol, were tested. Some activators enhanced proliferation in the presence of a Ca2+ ionophore, ionomycin, but not concanavalin A. Some activators suppressed proliferation and downregulated protein kinase C. Others neither downregulated protein kinase C nor inhibited IL-2 production and proliferation. However, inhibition or downregulation of protein kinase C activity always correlated with decreased IL-2 and depressed proliferation. Thus, the evidence in this and the previous study suggests that activation of protein kinase C is directly related to IL-2 production in activated T cells.     

Regulation of mannose 6-phosphate/insulin-like growth factor II receptor distribution by activators and inhibitors of protein kinase C

The tumor-promotor phorbol dibutyrate (PDBt) increases the binding of a neoglycoprotein containing mannose 6-phosphate (Man6P) and of insulin-like growth factor II (IGF-II) to the Man6P/IGF-II receptor at the cell surface. This effect is dependent on time and concentration and is also seen with synthetic 1-oleoyl-2-acetyl-sn-glycerol, but not with 4 alpha-phorbol, an inactive tumor-promoter. The increase is due to a 3-4-fold increase in the number of cell-surface, receptors, accompanied by a 1.6-fold increase in ligand-binding affinity. The internalization rate of the Man6P/IGF-II receptor is not affected by PDBt, suggesting that the redistribution of these receptors to the cell surface is due to an accelerated externalization rate. The redistribution of Man6P/IGF-II receptors did not impair the sorting of newly synthesized Man6P-containing ligands while uptake of these ligands is 2-4-fold increased. Inactivation or down regulation of protein kinase C decreased the binding of the Man6P-containing neoglycoprotein to 65% of controls. Incubation of cells with Man6P, IGF-I, IGF-II or epidermal growth factor induces a rapid redistribution of Man6P/IGF-II receptors to the plasma membrane [Braulke, T., Tippmer, S., Neher, E. & von Figura, K. (1989) EMBO J. 8, 681-686]. Incubation with PDBt prevented the effect of growth factors but not that of Man6P on receptor redistribution. Inactivation of protein kinase C did not affect the Man6P/IGF-II receptor redistribution induced by Man6P and growth factors. These data suggest that Man6P, growth factors and activation of protein kinase C by phorbol esters and diacylglycerols modulate Man6P/IGF-II receptor cell-surface binding by at least two independent mechanisms, receptor redistribution as well as an increase of binding affinity, which might be involved in regulation of endocytosis of ligands.     

A cell-based reporter assay for the identification of protein kinase C activators and inhibitors

Transmission of extra cellular signals across biological membranes results in the generation of lipid metabolites which in turn influence specific cellular events such as cell growth or differentiation. Many of these lipid messengers can activate protein kinase C (PKC) isozymes of which one function is to perpetuate the extracellular signals to the nucleus by phosphorylating other targets proteins. We have engineered mammalian cell lines to identify and evaluate activators and inhibitors of PKC-dependent and independent signal transduction pathways. The A31 mouse fibroblast cell line, has been stably transfected with a construct containing a triplet repeat of the TPA response element (TRE) upstream of a thymidine kinase promoter fused to the human growth hormone (hGH) gene. A31 cells containing this reporter construct exhibit significant increases in hGH secretion following stimulation by phorbol esters or other mitogens. The levels of hGH secretion are modulated in this system using different pharmacological agents. We demonstrate that this assay can be used to identify specific and general inhibitors as well as activators of the signal transduction pathway mediated by PKC isozymes. (Mol Cell Biochem141: 129–134, 1994)     

Differential signaling of cyclic AMP: opposing effects of exchange protein directly activated by cyclic AMP and cAMP-dependent protein kinase on protein kinase B activation.

The recent discovery of Epac, a novel cAMP receptor protein, opens up a new dimension in studying cAMP-mediated cell signaling. It is conceivable that many of the cAMP functions previously attributed to cAMP-dependent protein kinase (PKA) are in fact also Epac-dependent. The finding of an additional intracellular cAMP receptor provides an opportunity to further dissect the divergent roles that cAMP exerts in different cell types. In this study, we probed cross-talk between cAMP signaling and the phosphatidylinositol 3-kinase/PKB pathways. Specifically, we examined the modulatory effects of cAMP on PKB activity by monitoring the specific roles that Epac and PKA play individually in regulating PKB activity. Our study suggests a complex regulatory scheme in which Epac and PKA mediate the opposing effects of cAMP on PKB regulation. Activation of Epac leads to a phosphatidylinositol 3-kinase-dependent PKB activation, while stimulation of PKA inhibits PKB activity. Furthermore, activation of PKB by Epac requires the proper subcellular targeting of Epac. The opposing effects of Epac and PKA on PKB activation provide a potential mechanism for the cell type-specific differential effects of cAMP. It is proposed that the net outcome of cAMP signaling is dependent upon the dynamic abundance and distribution of intracellular Epac and PKA.     

Regulation of the Epstein-Barr virus C promoter by AUF1 and the cyclic AMP/protein kinase A signaling pathway

EBNA2 is an Epstein-Barr virus (EBV)-encoded protein that regulates the expression of viral and cellular genes required for EBV-driven B-cell immortalization. Elucidating the mechanisms by which EBNA2 regulates viral and cellular gene expression is necessary to understand EBV-induced B-cell immortalization and viral latency in humans. EBNA2 targets to the latency C promoter (Cp) through an interaction with the cellular DNA binding protein CBF1 (RBPJk). The EBNA2 enhancer in Cp also binds another cellular factor, C promoter binding factor 2 (CBF2), whose protein product(s) has not yet been identified. Within the EBNA2 enhancer in Cp, we have previously identified the DNA sequence required for CBF2 binding and also determined that this element is required for efficient activation of Cp by EBNA2. In this study, the CBF2 activity was biochemically purified and microsequenced. The peptides sequenced were identical to the hnRNP protein AUF1. Antibodies against AUF1 but not antibodies to related hnRNP proteins reacted with CBF2 in gel mobility shift assays. In addition, stimulation of the cellular cyclic AMP (cAMP)/protein kinase A (PKA) signal transduction pathway results in an increase in detectable CBF2/AUF1 binding activity extracted from stimulated cells. Furthermore, the CBF2 binding site was able to confer EBNA2 responsiveness to a heterologous promoter when transfected cells were treated with compounds that activate PKA or by cotransfection of plasmids expressing a constitutively active catalytic subunit of PKA. EBNA2-mediated stimulation of the latency Cp is also increased in similar cotransfection assays. These results further support an important role for CBF2 in mediating EBNA2 transactivation; they identify the hnRNP protein AUF1 as a major component of CBF2 and are also the first evidence of a cis-acting sequence other than a CBF1 binding element that is able to confer responsiveness to EBNA2.     

K-252 compounds, novel and potent inhibitors of protein kinase C and cyclic nucleotide-dependent protein kinases

K-252 compounds (K-252a and b isolated from Nocardiopsis sp. (1) and their synthetic derivatives) were found to inhibit cyclic nucleotide-dependent protein kinases and protein kinase C to various extents. The inhibitions were of the competitive type with respect to ATP. K-252a was a non-selective inhibitor for these three protein kinases with Ki values 18-25 nM. K-252b showed a comparable potency for protein kinase C (Ki, 20nM), whereas inhibitory potencies for cyclic nucleotide-dependent protein kinases were reduced. KT5720 and KT5822 selectively inhibited cAMP-dependent (Ki, 60nM) and cGMP-dependent (Ki, 2.4nM) protein kinases, respectively.     

Characterization of the yeast BMH1 gene encoding a putative protein homologous to mammalian protein kinase II activators and protein kinase C inhibitors.

We describe the identification and characterization of the BMH1 gene from the yeast Saccharomyces cerevisiae. The gene encodes a putative protein of 292 amino acids which is more than 50% identical with the bovine brain 14-3-3 protein and proteins isolated from sheep brain which are strong inhibitors of protein kinase C. Disruption mutants and strains with the BMH1 gene on multicopy plasmids have impaired growth on minimal medium with glucose as carbon source, i.e. a 30-50% increase in generation time. These observations suggest a regulatory function of the bmh1 protein. In contrast to strains with an intact or a disrupted BMH1 gene, strains with the BMH1 gene on multicopy plasmids hardly grew on media with acetate or glycerol as carbon source.     

Regulation of N-acetylaspartate and N-acetylaspartylglutamate biosynthesis by protein kinase activators

The neuronal dipeptide N-acetylaspartylglutamate (NAAG) is thought to be synthesized enzymatically from N-acetylaspartate (NAA) and glutamate. We used radiolabeled precursors to examine NAA and NAAG biosynthesis in SH-SY5Y human neuroblastoma cells stimulated with activators of protein kinase A (dbcAMP; N6,2'-O-dibutyryl cAMP) and protein kinase C (PMA; phorbol-12-myristate-13-acetate). Differentiation over the course of several days with dbcAMP resulted in increased endogenous NAA levels and NAAG synthesis from l-[(3)H]glutamine, whereas PMA-induced differentiation reduced both. Exogenously applied NAA caused dose dependent increases in intracellular NAA levels, and NAAG biosynthesis from l-[(3)H]glutamine, suggesting precursor-product and mass-action relationships between NAA and NAAG. Incorporation of l-[(3)H]aspartate into NAA and NAAG occurred sequentially, appearing in NAA by 1 h, but not in NAAG until between 6 and 24 h. Synthesis of NAAG from l-[(3)H]aspartate was increased by dbcAMP and decreased by PMA at 24 h. The effects of PMA on l-[(3)H]aspartate incorporation into NAA were temporally biphasic. Using short incubation times (1 and 6 h), PMA increased l-[(3)H]aspartate incorporation into NAA, but with longer incubation (24 h), incorporation was significantly reduced. These results suggest that, while the neuronal production of NAA and NAAG are biochemically related, significant differences exist in the regulatory mechanisms controlling their biosynthesis.     

Compartments of cyclic AMP and protein kinase in mammalian cardiomyocytes

We have studied the compartmentation of cyclic AMP action in purified ventricular cardiomyocytes prepared by collagenase perfusion of adult rabbit hearts. Incubation of purified adult myocytes with 1 microM isoproterenol causes rapid accumulation of intracellular cyclic AMP in both soluble (2.3 leads to 7.7 pmol/ mg of protein) and particulate (3.0 leads to 9.2) fractions of cell homogenates (3000 X g for 5 min), increases in the total activity and activity ratio of soluble cyclic AMP-dependent protein kinase (0.21 leads to 0.66), a decrease in protein kinase activity remaining in the particulate fraction (47 leads to 30%), and an increase in the activity ratio of glycogen phosphorylase (0.15 leads to 0.47). Incubation of myocytes with 10 microM prostaglandin E1 (PGE1) leads to a comparable increase in soluble cyclic AMP (2.3 leads to 5.8 pmol/mg of protein) and activation of soluble cyclic AMP-dependent protein kinase (0.21 leads to 0.39) but does not result in any change in cAMP or protein kinase in the particulate fraction and fails to cause an activation of glycogen phosphorylase. PGE1 does not inhibit the effects of isoproterenol; when myocytes are incubated with both isoproterenol and PGE1, the accumulation of cyclic AMP, activation of cAMP-dependent protein kinase and phosphorylase b leads to a conversion are equal to that achieved with isoproterenol alone. Perturbation of cellular calcium using the ionophore A23187, verapamil, or high or low extracellular calcium did not alter the ability of isoproterenol to cause activation of particulate cAMP-dependent protein kinase or influence the inability of PGE1 to do so. Activation of adenylate cyclase by forskolin (30 microM) caused immediate activation of both soluble and particulate cAMP-dependent protein kinase leading to rapid activation of phosphorylase. We conclude that the hormonally specific compartmentation of cyclic AMP and cAMP-dependent protein kinase that occurs in intact heart (Hayes, J. S., Brunton, L. L., Brown, J. H., Reese, J. B., and Mayer, S. E. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 1570-1574) is not explained on the basis of cellular heterogeneity but has a subcellular basis within the cardiomyocyte.     

Regulation of platelet cytosolic free calcium by cyclic nucleotides and protein kinase C

PAF elicits a rapid, concentration-dependent elevation of platelet cytosolic free calcium ([Caf]), measured by quin2. Elevation of [Caf] is transient, and the rate of reversal increases with agonist concentration. Adenylate cyclase stimulants (PGI2, PGD2) and 8-bromo cAMP; a guanylate cyclase stimulant (sodium nitroprusside) and 8-bromo cGMP; and a protein kinase C stimulant (phorbol myristate acetate) block the elevation of [Caf] induced by PAF, and accelerate its reversal. These results suggest that cAMP, cGMP and 1,2-diacylglycerol (DAG) could act as second messengers to regulate [Caf] in platelets. As PAF is known to stimulate platelet phosphoinositide hydrolysis (ergo DAG formation) but fails to elevate platelet cAMP or cGMP, it is proposed that DAG, via activation of protein kinase C, may act as an endogenous modulator of platelet [Caf]: an action that contributes to the role of DAG as a bi-directional regulator of platelet reactivity.