Protein kinase C-alpha(1b)-adrenoceptor coimmunoprecipitation: effect of hormones and phorbol myristate acetate

alpha(1b)-Adrenoceptors immunoprecipitated with protein kinase C alpha, delta, and epsilon isoforms under basal conditions and such coimmunoprecipitations were increased in cells treated with phorbol myristate acetate. The increased coimmunoprecipitations induced by phorbol myristate acetate were concentration-dependent and reached their maxima 1 to 2 min after the addition of the tumor promoter. No coimmunoprecipitation of protein kinase C zeta and alpha(1b)-adrenoceptors was detected. Norepinephrine, endothelin-1, lysophosphatidic acid and epidermal growth factor were also able to increase the coimmunoprecipitation of protein kinase C isoenzymes and alpha(1b)-adrenoceptors. These data support the idea that protein kinase-receptor complexes might form and could be relevant in receptor desensitization.     

Protein phosphatase-protein kinase interplay modulates alpha 1b-adrenoceptor phosphorylation: effects of okadaic acid

In the present work we studied the effect of protein phosphatase inhibitors on the phosphorylation state and function of alpha(1b)-adrenoceptors. Okadaic acid increased receptor phosphorylation in a time- and concentration-dependent fashion (maximum at 30 min, EC(50) of 30 nM). Other inhibitors of protein phosphatases (calyculin A, tautomycin and cypermethrin) mimicked this effect. Staurosporine and Ro 31-8220, inhibitors of protein kinase C, blocked the effect of okadaic acid on receptor phosphorylation. Neither genistein nor wortmannin altered the effect of okadaic acid. The intense adrenoceptor phosphorylation induced by okadaic acid altered the adrenoceptor-G protein coupling, as evidenced by a small decreased noradrenaline-stimulated [(35)S]GTPgammaS binding. Okadaic acid did not alter the noradrenaline-stimulated increases in intracellular calcium or the production of inositol trisphosphate. Our data indicate that inhibition of protein phosphatases increases the phosphorylation state of alpha(1b)-adrenoceptors; this effect seems to involve protein kinase C. In spite of inducing an intense receptor phosphorylation, okadaic acid alters alpha(1b)-adrenergic actions to a much lesser extent than the direct activation of protein kinase C by phorbol myristate acetate.     

Okadaic acid increases the phosphorylation state of alpha1A-adrenoceptors and induces receptor desensitization

Okadaic acid, a protein phosphatase inhibitor, and phorbol myristate acetate, an activator of protein kinase C, increased the phosphorylation state of alpha1A-adrenergic receptors. The effects of these agents were of similar magnitude but that of okadaic acid developed more slowly. Wortmannin (inhibitor of phosphoinositide 3-kinase), but not staurosporine (inhibitor of protein kinase C), abolished the effect of okadaic acid on the alpha1A-adrenoceptor phosphorylation state. The effect of phorbol myristate acetate on this parameter was blocked by staurosporine and only partially inhibited by wortmannin. Okadaic acid markedly increased the co-immunoprecipitation of both the catalytic and regulatory subunits of phosphatidylinositol 3-kinase and of Akt/protein kinase B with the adrenoceptor and only marginally increases receptor association with protein kinase C epsilon. Okadaic acid induced desensitization of alpha1A-adrenoceptors as evidenced by a decreased ability of noradrenaline to increase intracellular calcium. Such desensitization was fully reverted by wortmannin. Our data indicate that inhibition of serine/threonine protein phosphatases increases the phosphorylation state of alpha1A-adrenergic receptor and alters the adrenoceptor function.     

Human alpha1D-adrenoceptor phosphorylation and desensitization

Rat-1 fibroblast were transfected with a plasmid containing the cDNA of the human alpha(1D)-adrenoceptor. A cell line was isolated that stably expressed the receptor as evidenced by BMY 7378-sensitive noradrenaline-induced increases in intracellular calcium concentration. The effect of noradrenaline was blocked by active phorbol esters; such blockade was mediated by protein kinase C (PKC) as evidenced by its inhibition by staurosporine or the downregulation of this protein kinase. Radioligand binding experiments showed expression of receptors with high affinity for [3H]tamsulosin (K(D) 0.30 +/- 0.05 nM) but low density (B(max) 35 +/- 4 fmol/mg protein). The receptors had the expected orders of potency for agonists (adrenaline = noradrenaline > oxymetazoline) and antagonists (BMY 7378 > 5-methyl-urapidil = phentolamine). Photoaffinity labeling identified the receptor as a band of M(r) 70-80kDa, which could be immunoprecipitated with a selective anti-alpha(1D)-adrenoceptor antiserum. In cells metabolically labeled with radioactive phosphate the adrenoceptor was identified as a phosphoprotein whose phosphorylation state was increased by the agonist, noradrenaline, and by phorbol myristate acetate. The data indicate that the human alpha(1D)-adrenoceptor function was regulated through phosphorylation by PKC.     

Roles of c-Src in alpha1B-adrenoceptor phosphorylation and desensitization

1 The role of the protein tyrosine kinase, c-Src, on the function and phosphorylation of alpha1B-adrenoceptors (alpha1B-AR) and their association with G-protein-coupled receptor kinase (GRK) isozymes was studied. 2 Inhibitors of this kinase (PP2 and Src Inhibitor II) decreased ( approximately 50-75%) noradrenaline- (NA) and phorbol myristate acetate-mediated receptor phosphorylation. Expression of a dominant-negative mutant of c-Src similarly reduced receptor phosphorylation induced by the natural agonists, active phorbol esters and endothelin-1 (ET-1). 3 c-Src, GRK2, GRK3 and GRK5 coimmunoprecipitate with alpha1B-ARs in the basal state. In cells treated with NA or phorbol myristate acetate the amount of coimmunoprecipitated GRK2 and GRK3 increased ( approximately 2- to 3-fold), while treatment with ET-1 only augmented the amount of coimmunoprecipitated GRK2 ( approximately 2-fold). The Src inhibitor, PP2, markedly attenuated all these increases. 4 Cell pretreatment with PP2 amplified the increase in intracellular-free calcium observed with NA, in the basal state and after the stimulation (desensitization) induced by ET-1. 5 The data suggest a role of c-Src in alpha1B-AR desensitization/phosphorylation and in the interaction of these ARs with GRKs.     

Lysophosphatidic acid modulates alpha(1b)-adrenoceptor phosphorylation and function: roles of Gi and phosphoinositide 3-kinase

The effect of lysophosphatidic acid on the phosphorylation and function of alpha(1b)-adrenoceptors transfected into rat-1 fibroblasts was studied. This phospholipid mitogen increased in a concentration-dependent fashion (EC(50) approximately 50 nM) the phosphorylation of these adrenoceptors. Lysophosphatidic acid-induced alpha(1b)-adrenoceptor phosphorylation was relatively rapid (t(1/2) approximately 1 min), intense (2.5-fold), and sustained for at least 60 min. The effect of lysophosphatidic acid was blocked by pretreatment with pertussis toxin. The alpha(1b)-adrenoceptor phosphorylation induced by lysophosphatidic acid was not blocked by genistein, a tyrosine kinase inhibitor, but it was inhibited by inhibitors of protein kinase C (bisindolylmaleimide I, staurosporine, and Ro 31-8220) and phosphoinositide 3-kinase (wortmannin and LY 294002). The ability of norepinephrine to increase cytosol calcium concentration was markedly decreased in cells previously challenged with lysophosphatidic acid. Norepinephrine-induced [(35)S]GTPgammaS binding in membrane preparations was used as an index of the functional coupling of the alpha(1b)-adrenoceptors and G proteins. Norepinephrine-stimulated [(35)S]GTPgammaS binding was markedly decreased in membranes from cells pretreated with lysophosphatidic acid. This effect of lysophosphatidic acid was blocked by pretreatment with wortmannin or staurosporine. Our data indicate that: 1) activation of lysophosphatidic acid receptors induce phosphorylation of alpha(1b)-adrenoceptors; 2) this effect is mediated through pertussis toxin-sensitive G proteins, phosphatidylinositol 3-kinase, and protein kinase C; and 3) the phosphorylation of alpha(1b)-adrenoceptors induced by the lipid mitogen is associated to adrenoceptor desensitization.     

Phosphorylation, desensitization and internalization of human alpha1B-adrenoceptors induced by insulin-like growth factor-I

The effect of insulin-like growth factor-I (IGF-I) on human alpha(1B)-adrenoceptor function, phosphorylation state and cellular location was studied. Rat-1 fibroblasts were transfected with a plasmid construction containing enhanced green fluorescent protein joined to the carboxyl terminus of the human alpha(1B)-adrenoceptor. Receptors were identified by radioligand binding and photoaffinity labeling, and were immunoprecipitated with an antiserum generated against the enhanced green fluorescent protein. The receptor was functional, as evidenced by noradrenaline action on intracellular calcium and inositol phosphate production. IGF-I had no significant effect by itself on these parameters but markedly reduced the effects of noradrenaline. IGF-I induced alpha(1B)-adrenoceptor phosphorylation, which was markedly reduced by the following agents: pertussis toxin, a metalloproteinase inhibitor, diphtheria toxin mutant CRM 197, an epidermal growth factor (EGF) receptor intrinsic kinase activity inhibitor, and by phosphoinositide 3-kinase and protein kinase C inhibitors. IGF-I action appears to involve activation of a pertussis toxin-sensitive G protein, shedding of heparin-binding EGF and autocrine activation of EGF receptors. G protein subunits and phosphotyrosine residues stimulate phosphoinositide 3-kinase activity leading to activation of protein kinase C, which in turn phosphorylates alpha(1B)-adrenoceptors. Confocal fluorescent microscopy showed that alpha(1B)-adrenoceptors fussed to the green fluorescent protein were located in plasma membrane and intracellular vesicles in the basal state. IGF-I induced receptor redistribution favoring the intracellular location; this effect was blocked by hypertonic sucrose and concanavalin A. Our data show that IGF-I induces alpha(1B)-adrenoceptor desensitization associated to receptor phosphorylation and internalization.     

Peroxovanadate induces alpha 1B-adrenoceptor phosphorylation and association with protein kinase C

Peroxovanadate induced a marked increase in the phosphorylation state of alpha(1B)-adrenoceptors. The effect was dose-dependent (EC(50) approximately 2 microM) and rapid, reaching its maximum in 5 min and remaining at this level for 30 min. Hydrogen peroxide also increased alpha(1B)-adrenoceptor phosphorylation but to a lesser extent, in an ephemeral fashion, and only at high (millimolar) concentrations. The effect of peroxovanadate was blocked by inhibitors of protein kinase C such as staurosporine and rottlerin and only partially reduced by genistein and inhibitors of phosphoinositide 3-kinase. Protein kinase C alpha, delta and epsilon are associated with the alpha(1B)-adrenoceptor under basal conditions, as reflected by coimmunoprecipitation. Such association was increased by peroxovanadate for all isoforms. In contrast, hydrogen peroxide increased only the association of the epsilon isoform to the adrenoceptor. Peroxovanadate decreased the ability of noradrenaline to increase intracellular calcium, indicating that the receptor phosphorylation induced has functional consequences.     

Roles of the α1A-adrenergic receptor carboxyl tail in protein kinase C-induced phosphorylation and desensitization

Noradrenaline- and tetradecanoyl phorbol acetate (TPA)-induced phosphorylation and functional desensitization of the following receptors were studied: (1) wild-type bovine α(1A)- and hamster α(1B)-adrenergic receptors (ARs), (2) chimeric ARs in which the carboxyl terminus tails were exchanged (α(1AB)- and α(1BA)-ARs), and (3) carboxyl terminus-truncated α(1A)-ARs fussed to enhanced green fluorescent protein. Noradrenaline and TPA pronouncedly increased α(1B)-AR phosphorylation while TPA markedly desensitized these receptors. In contrast, TPA-induced desensitization and TPA- and noradrenaline-induced phosphorylation of α(1A)-ARs were clearly of lesser magnitude. Chimeric ARs with exchanged carboxyl terminus tails showed that the extent of phosphorylation reflected the carboxyl domain rather than the receptor core. Surprisingly, there was no correlation between phosphorylation and functional desensitization, i.e., activation of protein kinase C clearly desensitized both chimeric receptors to a similar extent. Interestingly, TPA and noradrenaline increased carboxyl terminus-truncated α(1A)-AR phosphorylation and TPA also induced receptor desensitization. We were unable to detect carboxyl terminus-truncated α(1A)-AR internalization after 5-min stimulations with noradrenaline or TPA. Our results suggest the following: (a) the α(1A)-AR carboxyl terminus tail was not essential for signaling or desensitization; (b) carboxyl terminus tail exchange "transplanted" the phosphorylation pattern of the receptors, but the functional consequences of such a transplant were very limited; (c) α(1A)-AR desensitization was not associated to receptor internalization.     

Angiotensin AT(1) receptor phosphorylation and desensitization in a hepatic cell line. Roles of protein kinase c and phosphoinositide 3-kinase

Desensitization and phosphorylation of the endogenous angiotensin II AT(1) receptor were studied in clone 9 liver cells. Agonist activation of AT(1) receptors blunted the response to subsequent addition of angiotensin II. Partial inhibition of the angiotensin II-induced calcium response was observed when cells were pretreated with dibutyryl cyclic AMP, tetradecanoyl phorbol acetate (TPA), vasopressin, or lysophosphatidic acid. All of these desensitization processes were associated with receptor phosphorylation. Angiotensin II-induced AT(1) receptor phosphorylation was partially blocked by the protein kinase C inhibitor bisindolylmaleimide I and by phosphoinositide 3-kinase inhibitors (wortmannin and LY294002); the actions of these inhibitors were not additive. Pertussis toxin pretreatment of cells also partially inhibited angiotensin II-induced AT(1) receptor phosphorylation. TPA-induced AT(1) receptor phosphorylation was completely blocked by bisindolylmaleimide I. AT(1) receptor phosphorylation was also induced by vasopressin and lysophosphatidic acid, and these effects were partially inhibited by bisindolylmaleimide I. Angiotensin II increased Akt/PKB (protein kinase B) phosphorylation and protein kinase C membrane association. The effect on Akt/PKB phosphorylation was blocked by phosphoinositide 3-kinase inhibitors. These findings indicate that clone 9 cells exhibit both homologous and heterologous desensitization in association with AT(1) receptor phosphorylation. In these hepatic cells, angiotensin II-induced receptor phosphorylation involves pertussis toxin-sensitive and -insensitive G proteins, and is mediated in part through protein kinase C and phosphoinositide 3-kinase.     

Protein kinase C involvement in maintenance and modulation of noradrenaline release in the mouse brain cortex.

1. The role of protein kinase C in the modulation of noradrenaline release was investigated in mouse cortical slices which were pre-incubated with [3H]-noradrenaline. The aim was to investigate the hypothesis that protein kinase C is activated during high levels of transmitter release to maintain transmitter output. 2. The protein kinase C activators, phorbol myristate acetate (0.01-0.3 microM) and to a greater extent 4 beta-phorbol 12,13-dibutyrate (0.01-0.3 microM) significantly enhanced stimulation-induced noradrenaline release whereas 4 alpha-phorbol 12,13-dibutyrate (0.1 microM) which does not activate protein kinase C was without effect. The effect of the protein kinase C activator, phorbol myristate acetate, on noradrenaline release was attenuated by the protein kinase C inhibitor, polymyxin B (21 microM) which by itself inhibited stimulation-induced noradrenaline release. 3. Protein kinase C was down-regulated by 10 h exposure of the cortical slices to 4 beta-phorbol 12,13-dibutyrate (1 microM). In this case the facilitatory effect of 4 beta-phorbol 12,13-dibutyrate (0.1 microM) on noradrenaline release was abolished as was the inhibitory effect produced by polymyxin B. This indicates that polymyxin B was acting selectively at protein kinase C. 4. The inhibitory effect of polymyxin B on noradrenaline release, when expressed as a percentage of the appropriate frequency control, was constant at 1, 5 and 10 Hz. Furthermore, the ratio of release at 5 Hz to that at 10 Hz was not altered by protein kinase C down-regulation, indicating that there is no additional effect of protein kinase C at higher stimulation frequencies. 5. When transmitter release was elevated by blocking alpha 2-adrenoceptor auto-inhibition with idazoxan (0.1 microM) or K+ channels with tetraethylammonium (300 microM), the elevation in transmitter release was significantly attenuated by protein kinase C down-regulation, suggesting an involvement of protein kinase C. 6. We conclude that protein kinase C is involved in the modulation of noradrenaline release over a wide range of stimulation frequencies, in addition to a role when noradrenaline release is elevated by presynaptic mechanisms.     

Okadaic acid enhances human T cell activation and phosphorylation of an internal substrate induced by phorbol myristate acetate.

Okadaic acid is a potent tumor promoter and an inhibitor of serine/threonine-specific protein phosphatases. We studied the effect of okadaic acid in human T cell activation and phosphorylation of internal substrates. Okadaic acid at up to 4 nM enhanced phorbol myristate acetate (PMA)-induced proliferation and CD25 (IL-2 receptor, p55) expression, although it showed no activation by itself. Okadaic acid induced hyperphosphorylation of a 60 kDa protein in T cells as well as non-T cells, as reported in fibroblasts and keratinocytes. Preincubation with 4 nM okadaic acid enhanced PMA induced phosphorylation of the 80 kDa protein, an internal substrate of protein kinase C in T cells. These results suggest that okadaic acid inhibited dephosphorylation of protein kinase C specific substrates, and as a result, enhanced T cell activation mediated by protein kinase C pathway.     

Regulation of histamine H1 receptor-mediated phosphoinositide hydrolysis by histamine and phorbol esters in DDT1 MF-2 cells

The regulation of histamine-stimulated phosphoinositide turnover by histamine and phorbol esters was examined in intact DDT1 MF-2 cells grown in suspension culture. Histamine increased the incorporation of 32P into phosphatidylinositol (PI) in these cells, and this stimulation was inhibited by the H1 antagonist diphenhydramine but not by the H2 antagonist cimetidine. Pretreatment of cells with histamine or with phorbol 12-myristate 13-acetate (PMA) or other activators of protein kinase C induced a marked decrease in the subsequent stimulation by histamine. PMA, but not histamine, also decreased the ability of epinephrine to stimulate PI labelling through alpha 1-adrenoceptors. Thus, histamine appears to induce homologous desensitization of histamine H1 receptor-mediated PI turnover, whereas direct activation of protein kinase C in the absence of receptor occupancy by agonist induces nonspecific heterologous desensitization of both histamine H1- and alpha 1-adrenoceptor-mediated responses.     

Modulation of α1B-adrenoceptor expression by agonist and protein kinase inhibitors

The agonist-induced up-regulation of α_1B-adrenoceptors in clone H99 of transfected Chinese hamster ovary cells that we reported previously (Zhu et al., 1996) was further investigated. Studies with a larger number of clones revealed that the up-regulation observed in H99 cells is atypical and that most other clones exhibit down-regulation under the same conditions. The role of protein kinases in the up-regulation of α_1B-adrenoceptors in clone H99 was further investigated. Surprisingly, the protein kinase inhibitor staurosporine induced a similar up-regulation. Neither the selective protein kinase C inhibitor GF109203X nor the activator phorbol 12-myristate, 13-acetate altered receptor expression. The tyrosine kinase inhibitors genistein and its weaker analog daidzein did not induce up-regulation but blocked the up-regulation induced by epinephrine and by staurosporine. Up-regulation was blocked by the protein synthesis inhibitor cycloheximide. These studies suggest multiple mechanisms by which different protein kinases can modulate the expression of transfected α_1B-adrenoceptors.     

Activation of protein kinase C selectively inhibits the gamma-aminobutyric acidA receptor: role of desensitization.

The effects of protein kinase C (PKC) activators on gamma-aminobutyric acidA (GABAA) receptor function were studied by two-electrode voltage-clamp in Xenopus oocytes expressing brain mRNA or subunit cDNAs and in isolated mouse brain cerebellar membrane vesicles (microsacs), using 36Cl- uptake. Both oocytes and microsacs showed transient (desensitizing) and sustained (nondesensitizing) GABAA receptor responses. In oocytes expressing brain mRNA, the PKC activator phorbol myristoyl acetate (PMA), but not the inactive analog phorbol 12-monomyristate, inhibited both transient and sustained GABA-gated chloride currents. The inhibition by PMA was concentration dependent, with an EC50 of approximately 5 nM, and resulted in a decrease in the efficacy, but not the potency, of GABA. Additionally, PMA inhibited GABA-gated chloride currents in oocytes expressing alpha 1 beta 1 gamma 2L subunit cDNAs. The effect of PMA on recombinant receptors was significantly antagonized by PKC inhibitory peptide (PKCI). In the microsac preparation, the PKC activators (-)-7-octylindolactam V and PMA inhibited the sustained phase of 36Cl- flux without altering the transient phase. The action of PMA was blocked by kinase inhibitors and by depletion of Mg-ATP and was mimicked by protein phosphatase inhibitors. These results demonstrate that activation of PKC inhibits GABAA receptor function, and the results from the microsac experiments suggest that PKC-dependent phosphorylation preferentially inactivates a nondesensitized form or state of the receptor.     

Signaling properties of human α1D-adrenoceptors lacking the carboxyl terminus: intrinsic activity, agonist-mediated activation, and desensitization

α₁-Adrenoceptors are differentially regulated by protein kinase C-mediated phosphorylation. The most sensitive member of this family is the α_1D-subtype, which is also characterized by a constitutive activity and a reduced expression at the plasma membrane controlled by the amino terminus. Information on the structural domains that determine the function and regulation of this receptor subtype is scarce. Therefore, the function and phosphorylation of amino terminus-truncated (Δ1–79, (ΔN)) α_1D-adrenoceptors were studied and compared with those of α_1D-adrenoceptors truncated both at the amino and carboxyl termini (Δ1–79 and Δ441–572, (ΔN–ΔC)). These receptors were stably expressed in rat-1 fibroblast, at relatively high density (≈2 pmol/mg of membrane protein), and showed intrinsic activity that was markedly increased by noradrenaline. Interestingly, activation of protein kinase C markedly attenuated (desensitized) the function of both ΔN and ΔN–ΔC α_1D-adrenoceptors. These receptors were photolabeled and immunoprecitated with an antibody directed against an influenza hemagglutinin epitope inserted at the amino termini. Metabolic labeling with radioactive phosphate and receptor immunoprecipitation studies indicated that these receptors are phosphoproteins whose phosphorylation state is increased by noradrenaline and by activation of protein kinase C. Our data indicate that carboxyl terminus-truncated α_1D-adrenoceptors are fully functional and subjected to regulation by phosphorylation. The roles of the carboxyl termini differ among α₁-adrenoceptor subtypes.     

Alpha1-adrenoceptors down-regulate ClC-2 chloride channels in epithelial cells from the acutely denervated jejunum

Acute sympathetic denervation of the small intestine up-regulates alpha1-adrenoceptors on villus enterocytes and activation of these alpha1-adrenoceptors inhibits chloride secretion. We tested whether alpha1-adrenoceptor-mediated inhibition of chloride secretion was the result of reduced ClC-2 chloride channel expression. Phorbol myristate acetate (PMA) (a protein kinase C (PKC) activator) had no effect on ClC-2 levels. In contrast, alpha1-adrenoceptor activation significantly decreased ClC-2 protein levels in both the villus (1.58+/-0.19 to 0.75+/-0.19 arbitrary units) and crypt (1.69+/-0.15 to 0.37+/-0.23 arbitrary units) epithelial cells from the acutely denervated jejunum but not innervated controls. These data suggest that inhibition of chloride secretion following alpha1-adrenoceptor activation in the acutely denervated small intestine may be through ClC-2 down-regulation.     

Transmembrane signalling associated with ganglioside-induced CD4 modulation

Ganglioside (GM1) treatment of CD4+ human CEM lymphoma cells stimulated transient phosphoinositide (PI) breakdown, production of inositol phosphates (IP), protein phosphorylation and rapid decrease of CD4 surface expression. A comparison between the actions of GM1 and other agents that affect these signal transduction pathways demonstrated a distinct mechanism for GM1-induced decrease of CD4. GM1 stimulated both phospholipase C activity and protein phosphorylation but had no effect on either cellular cAMP levels or tyrosine kinase activity. Phorbol myristate acetate (PMA) stimulated protein phosphorylation and caused a significant decrease in surface display of CD4. Both of these processes were blocked by pretreating cells with the protein kinase C (PKC) inhibitor H7. These results demonstrate that GM1 stimulates PI turnover and induces a rapid decrease of CD4 surface expression by processes that do not activate adenylate cyclase or tyrosine kinase. They further demonstrate that the mechanism for GM1-induced decrease of CD4 is distinct from the CD4 internalization processes mediated by PKC activity.     

Coupling to protein kinases A and C of adenosine A2B receptors involved in the facilitation of noradrenaline release in the prostatic portion of rat vas deferens

In the prostatic portion of rat vas deferens, the non-selective adenosine receptor agonist NECA (0.1-30 microM), but not the A(2A) agonist CGS 21680 (0.001-10 microM), caused a facilitation of electrically evoked noradrenaline release (up to 43 +/- 4%), when inhibitory adenosine A(1) receptors were blocked. NECA-elicited facilitation of noradrenaline release was prevented by the A(2B) receptor-antagonist MRS 1754, enhanced by preventing cyclic-AMP degradation with rolipram, abolished by the protein kinase A inhibitors H-89, KT 5720 and cyclic-AMPS-Rp and attenuated by the protein kinase C inhibitors Ro 32-0432 and calphostin C. The adenosine uptake inhibitor NBTI also elicited a facilitation of noradrenaline release; an effect that was abolished by adenosine deaminase and attenuated by MRS 1754, by inhibitors of the extracellular nucleotide metabolism and by blockade of alpha(1)-adrenoceptors and P2X receptors with prazosin and NF023, respectively. It was concluded that adenosine A(2B) receptors are involved in a facilitation of noradrenaline release in the prostatic portion of rat vas deferens that can be activated by adenosine formed by extracellular catabolism of nucleotides. The receptors seem to be coupled to the adenylyl cyclase-protein kinase A pathway but activation of the protein kinase C by protein kinase A, may also contribute to the adenosine A(2B) receptor-mediated facilitation of noradrenaline release.     

Protein kinase C inhibits TRH-stimulated phosphoinositide hydrolysis in GH3 cells

The GH3 pituitary tumor line expresses TRH receptors that stimulate phosphoinositide hydrolysis and hormone secretion. After protein kinase C was identified in GH3 cells by direct labeling with [3H]phorbol dibutyrate (PDB), the response to phorbol ester and TRH pretreatment on subsequent TRH-stimulated inositol phosphate (IP) accumulation was found to be inhibitory. Both phorbol myristate acetate (PMA) and PDB were effective in this regard at low nM concentrations within a few minutes, whereas phorbols that do not stimulate protein kinase C were without effect. Furthermore, the mono-, bis- and tris-phosphate forms of IP were all reduced by an average of 30-40% after 5 min of PMA. TRH concentration-response studies indicated a clear change in TRH efficacy induced by PMA. Finally, preincubation with TRH itself was also capable of reducing the subsequent response to TRH. Because TRH receptor action is thought to activate protein kinase C by producing diacylglycerol, these data indicate a negative feedback system via protein kinase C operative during continuous exposure to TRH in GH3 cells.