Multiple non-specific effects of sphingosine on adenylate cyclase and cyclic AMP accumulation in S49 lymphoma cells preclude its use as a specific inhibitor of protein kinase C

Recent studies with phorbol esters have suggested that protein kinase C (PKC) may play a role in the regulation of adenylate cyclase in mammalian cells. Since D-sphingosine has been reported to specifically inhibit PKC in many cell types, we evaluated its effects on stimulation of cyclic AMP accumulation by adrenaline in S49 lymphoma cells. We found sphingosine to have multiple non-specific effects which could not be explained by an inhibition of PKC. These effects included: (i) inhibition by sphingosine (50 microM) of adrenaline-stimulated cyclic AMP accumulation and sphingosine permeation of the cells which rendered them leaky to ATP; (iii) sphingosine (20 microMs) augmentation of adrenaline-stimulated cyclic AMP accumulation; (iii) inhibition by sphingosine of adrenaline-stimulated adenylate cyclase in isolated membranes by up to 95%; and (iv) sphingosine (20 microM) inhibition of cellular mechanisms for the elimination of cyclic AMP. These results demonstrate the importance of evaluating the non-specific effects of sphingosine before concluding that its actions are the consequences of a specific inhibition of PKC.

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Stimulation by glucose of cyclic AMP accumulation in mouse pancreatic islets is mediated by protein kinase C

Biochemical Journal ◽

10.1042/bj2530229 ◽

1988 ◽

Vol 253 (1) ◽

pp. 229-234 ◽

Cited By ~ 15

Author(s):

P Thams ◽

K Capito ◽

C J Hedeskov

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Cyclic Amp ◽

Adenylate Cyclase ◽

Pancreatic Islets ◽

Time Course ◽

Ionophore A23187 ◽

Kinase C ◽

Cyclic Amp Accumulation ◽

Mouse Pancreatic Islets

The mechanism of glucose-stimulated cyclic AMP accumulation in mouse pancreatic islets was studied. In the presence of 3-isobutyl-1-methylxanthine, both glucose and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), an activator of protein kinase C, enhanced cyclic AMP formation 2.5-fold during 60 min of incubation. Both TPA-stimulated and glucose-stimulated cyclic AMP accumulations were abolished by the omission of extracellular Ca2+. The Ca2+ ionophore A23187 did not affect cyclic AMP accumulation itself, but affected the time course of TPA-induced cyclic AMP accumulation, the effect of A23187 + TPA mimicking the time course for glucose-induced cyclic AMP accumulation. A 24 h exposure to TPA, which depletes islets of protein kinase C, abolished the effects of both TPA and glucose on cyclic AMP production. Both TPA-induced and glucose-induced cyclic AMP productions were inhibited by anti-glucagon antibody, and after pretreatment with this antibody glucose stimulation was dependent on addition of glucagon. Pretreatment of islets with TPA for 10 min potentiated glucagon stimulation and impaired somatostatin inhibition of adenylate cyclase activity in a particulate fraction of islets. Carbamoylcholine, which is supposed to activate protein kinase C in islets, likewise stimulated cyclic AMP accumulation in islets. These observations suggest that glucose stimulates islet adenylate cyclase by activation of protein kinase C, and thereby potentiates the effect of endogenous glucagon on adenylate cyclase.

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Parathyroid hormone induces protein kinase C but not adenylate cyclase in adult cardiomyocytes and regulates cyclic AMP levels via protein kinase C-dependent phosphodiesterase activity

Biochemical Journal ◽

10.1042/bj3100439 ◽

1995 ◽

Vol 310 (2) ◽

pp. 439-444 ◽

Cited By ~ 55

Author(s):

K D Schlüter ◽

M Weber ◽

H M Piper

Keyword(s):

Protein Kinase C ◽

Parathyroid Hormone ◽

Protein Kinase ◽

Cyclic Amp ◽

Adenylate Cyclase ◽

Phosphodiesterase Activity ◽

Beta Adrenoceptor ◽

Kinase C ◽

Adult Cardiomyocytes ◽

Cyclic Amp Accumulation

Adult ventricular cardiomyocytes have been identified as target cells for parathyroid hormone (PTH) but little is known about its signal transduction in these cells. In the present study the influence of PTH on cyclic AMP accumulation and the activity of protein kinase C (PKC) in cardiomyocytes was evaluated. A mid-regional synthetic fragment of PTH, PTH-(28-48), which exerts a hypertrophic effect on cardiomyocytes, increased the activity of membrane-associated PKC in a dose-dependent manner (1-100 nM). Activated membranous PKC was dependent on Ca2+ and sensitive to an inhibitor of Ca(2+)-dependent isoforms of PKC. When adenylate cyclase was stimulated by the addition of isoprenaline, a beta-adrenoceptor agonist, PTH-(28-48) antagonized cyclic AMP accumulation. This antagonistic effect of PTH-(28-48) could be mimicked by activation of PKC with a phorbol ester and inhibited by isobutylmethylxanthine, a phosphodiesterase inhibitor. An N-terminal synthetic fragment, PTH-(1-34), which includes an adenylate cyclase-activating domain, did not stimulate the accumulation of cyclic AMP in cardiomyocytes. The results demonstrate that in adult cardiomyocytes PTH (1) is able to stimulate PKC, (2) is not able to cause accumulation of cyclic AMP and (3) functionally antagonizes the effect of beta-adrenoceptor stimulation to increase cellular cyclic AMP concentrations via PKC-dependent phosphodiesterase activity.

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Protein kinase C activating phorbolesters enhance the cyclic AMP response to parathyroid hormone, forskolin and choleratoxin in mouse calvarial bones and rat osteosarcoma cells

Bioscience Reports ◽

10.1007/bf01136854 ◽

1991 ◽

Vol 11 (4) ◽

pp. 203-211 ◽

Cited By ~ 1

Author(s):

Maria Ransjö

Keyword(s):

Protein Kinase C ◽

Parathyroid Hormone ◽

Protein Kinase ◽

Cyclic Amp ◽

Adenylate Cyclase ◽

Phorbol Esters ◽

Osteosarcoma Cells ◽

Kinase C ◽

Cyclic Amp Accumulation ◽

Umr 106

The protein kinase C-(PKC) activating phorbol esters 12-O-tetradecanoylphorbol-13-acetate (TPA; 100 nmol/l) and phorbol 12, 13-dibutyrate (PDBU; 100 nmol/l) enhanced basal cyclin AMP accumulation in cultured neonatal mouse calvaria. The cyclic AMP response to parathyroid hormone (PTH; 10 nmol/l) and the adenylate cyclase activators forskolin (1–3 μmol/l) and choleratoxin (0.1 μmg/ml) was potentiated in a more than additive manner by TPA and PDBU. In contrast, phorbol 13-monoacetate (phorb-13; 100 nmol/l), a related compound but inactive on PKC, had no effect on basal or stimulated cyclic AMP accumulation. In the presence of indomethacin (1μmol/l), TPA and PDBU had no effect on cyclic AMP accumulation in calvarial bones per se, but were still able to cause a significant enhancement of the response to PTH, forskolin and choleratoxin. PTH-, forskolin- and choleratoxin-stimulated cyclic AMP accumulation in rat osteosarcoma cells UMR 106-01 was synergistically potentiated by TPA and PDBU, but not by phorb.-13. These data indicate that PKC enhances cyclic AMP formation and that the level of interaction may be at, or distal to, adenylate cyclase.

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Regulation of GH3 pituitary tumour-cell adenylate cyclase activity by activators of protein kinase C

Biochemical Journal ◽

10.1042/bj2620829 ◽

1989 ◽

Vol 262 (3) ◽

pp. 829-834 ◽

Cited By ~ 12

Author(s):

L A Quilliam ◽

P R M Dobson ◽

B L Brown

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Cyclic Amp ◽

Adenylate Cyclase ◽

Cyclase Activity ◽

Adenylate Cyclase Activity ◽

Maximal Response ◽

Kinase C ◽

Cyclic Amp Accumulation ◽

Stimulation Of

The influence of protein kinase C (PKC) activation on cyclic AMP production in GH3 cells has been studied. The stimulation of cyclic AMP accumulation induced by forskolin and cholera toxin was potentiated by 4 beta-phorbol 12,13-dibutyrate (PDBu). Moreover, PDBu, which causes attenuation of the maximal response to vasoactive intestinal polypeptide (VIP), also induced a small right shift in the dose-response curve for VIP-induced cyclic AMP accumulation. PDBu-stimulated cyclic AMP accumulation was unaffected by pretreatment of cells with pertussis toxin or the inhibitory muscarinic agonist, oxotremorine. PDBu stimulation of adenylate cyclase activity required the presence of a cytosolic factor which appeared to translocate to the plasma membrane in response to the phorbol ester. The diacylglycerol-generating agents thyroliberin, bombesin and bacterial phospholipase C each stimulated cyclic AMP accumulation, but, unlike PDBu, did not attenuate the stimulation induced by VIP. These results suggest that PKC affects at least two components of the adenylate cyclase complex. Stimulation of cyclic AMP accumulation is probably due to modification of the catalytic subunit, whereas attenuation of VIP-stimulated cyclic AMP accumulation appears to be due to the phosphorylation of a different site, which may be the VIP receptor.

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Adenylate cyclase, cyclic AMP and extracellular-signal-regulated kinase-2 in airway smooth muscle: modulation by protein kinase C and growth serum

Biochemical Journal ◽

10.1042/bj3060723 ◽

1995 ◽

Vol 306 (3) ◽

pp. 723-726 ◽

Cited By ~ 4

Author(s):

N Moughal ◽

P A Stevens ◽

D Kong ◽

S Pyne ◽

N J Pyne

Keyword(s):

Smooth Muscle ◽

Protein Kinase C ◽

Protein Kinase ◽

Cyclic Amp ◽

Adenylate Cyclase ◽

Airway Smooth Muscle ◽

Phospholipase D ◽

Extracellular Signal Regulated Kinase ◽

Kinase C ◽

Extracellular Signal

Bradykinin and phorbol 12-myristate 13-acetate stimulate adenylate cyclase activity in serum-depleted cultured airway smooth muscle via a protein kinase C (PKC)-dependent pathway. The probable target is the type II adenylate cyclase, which can integrate coincident signals from both PKC and Gs. Therefore, activation of Gs (by cholera-toxin pre-treatment) amplified the bradykinin-stimulated cyclic AMP signal and concurrently attenuated the partial activation of extracellular-signal-regulated kinase-2 (ERK-2) by bradykinin. We have previously demonstrated that, in order to induce full activation of ERK-2 with bradykinin, it is necessary to obliterate PKC-stimulated cyclic AMP formation. We concluded that the cyclic AMP signal limits the magnitude of ERK-2 activation [Pyne, Moughal, Stevens, Tolan and Pyne (1994) Biochem. J. 304, 611-616]. The present study indicates that the bradykinin-stimulated ERK-2 pathway is entirely cyclic AMP-sensitive, and suggests that coincident signal detection by adenylate cyclase may be an important physiological route for the modulation of early mitogenic signalling. Furthermore, the direct inhibition of adenylate cyclase activity enables bradykinin to induce DNA synthesis, indicating that the PKC-dependent activation of adenylate cyclase limits entry of cells into the cell cycle. These studies suggest that the mitogenicity of an agonist may be governed, in part, by its ability to stimulate an inhibitory cyclic AMP signal pathway in the cell. The activation of adenylate cyclase by PKC appears to be downstream of phospholipase D. However, in cells that were maintained in growth serum (i.e. were not growth-arrested), bradykinin was unable to elicit a PKC-stimulated cyclic AMP response. The lesion in the signal-response coupling was not at the level of either the receptor or phospholipase D, which remain functionally operative and suggests modification occurs at either PKC or adenylate cyclase itself. These studies are discussed with respect to the cell signal regulation of mitogenesis in airway smooth muscle.

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