scholarly journals Co-transfection with protein kinase D confers phorbol-ester-mediated inhibition on glucagon-stimulated cAMP accumulation in COS cells transfected to overexpress glucagon receptors

1997 ◽  
Vol 326 (2) ◽  
pp. 545-551 ◽  
Author(s):  
Edward S. TOBIAS ◽  
Enrique ROZENGURT ◽  
John M. C. CONNELL ◽  
Miles D. HOUSLAY
Keyword(s):  
Protein Kinase ◽  
Adenylate Cyclase ◽  
Protein Kinase D ◽  
Intracellular Camp ◽  
Camp Production ◽  
Camp Phosphodiesterase ◽  
Glucagon Receptor ◽  
Cos Cells ◽  
Protein Receptor ◽  
Dose Dependent Fashion

Glucagon elicited a profound increase in the intracellular cAMP concentration of COS-7 cells which had been transiently transfected with a cDNA encoding the rat glucagon receptor and under conditions where cAMP phosphodiesterase activity was fully inhibited. This was achieved in a dose-dependent fashion with an EC50 of 1.8±0.4 nM glucagon. In contrast with previous observations made using hepatocytes [Heyworth, Whetton, Kinsella and Houslay (1984) FEBS Lett. 170, 38–42], treatment of transfected COS-7 cells with PMA did not inhibit the ability of glucagon to increase intracellular cAMP levels. PMA-mediated inhibition was not conferred by treatment with okadaic acid, nor by co-transfecting cells with cDNAs encoding various protein kinase C isoforms (PKC-α, PKC-βII and PKC-ϵ) or with the PMA-activated G-protein-receptor kinases GRK2 and GRK3. In contrast, PMA induced the marked inhibition of glucagon-stimulated cAMP production in COS-7 cells that had been co-transfected with a cDNA encoding protein kinase D (PKD). Such inhibition was not due to an action on the catalytic unit of adenylate cyclase, as forskolin-stimulated cAMP production was unchanged by PMA treatment of COS cells that had been co-transfected with both the glucagon receptor and PKD. PKD transcripts were detected in RNA isolated from hepatocytes but not from COS-7 cells. Transcripts for GRK2 were present in hepatocytes but not in COS cells, whereas transcripts for GRK3 were not found in either cell type. It is suggested that PKD may play a role in the regulation of glucagon-stimulated adenylate cyclase.

scholarly journals Rat-2 fibroblasts express specific adrenomedullin receptors, but not calcitonin-gene-related-peptide receptors, which mediate increased intracellular cAMP and inhibit mitogen-activated protein kinase activity

1999 ◽  
Vol 338 (1) ◽  
pp. 15-22 ◽  
Author(s):  
Hedley A. COPPOCK ◽  
Ali A. OWJI ◽  
Carol AUSTIN ◽  
Paul D. UPTON ◽  
Mary L. JACKSON ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Northern Blotting ◽  
Mitogen Activated Protein Kinase ◽  
Intracellular Camp ◽  
Camp Production ◽  
Related Peptide ◽  
Mapk Activity ◽  
Calcitonin Gene ◽  
Adrenomedullin Receptor ◽  
Mitogen Activated Protein

Rat-2 fibroblasts demonstrate specific binding of 125I-labelled rat adrenomedullin (KD = 0.43nM; Bmax = 50fmol/mg of protein) in the absence of 125I-labelled calcitonin-gene-related peptide (CGRP) binding. Therefore Rat-2 cells were used to examine the pharmacology and signal transduction pathways of adrenomedullin receptors. We examined the effects of adrenomedullin, the CGRP receptor antagonist CGRP-(8–37) and the amylin antagonists AC187 and AC253 on receptor binding and cAMP production. AC253, AC187 and CGRP-(8–37) inhibited 125I-adrenomedullin binding, with respective IC50 values of 25±8, 129±39 and 214±56nM. Adrenomedullin dose-dependently increased intracellular cAMP (approximate EC50 = 1.0nM). CGRP-(8–37), AC253 and AC187 antagonized adrenomedullin-stimulated cAMP production at micromolar concentrations. Using kinase-substrate assays, Mono Q FPLC and ‘phospho-specific ’ Western blotting, we found that adrenomedullin alone abolished basal mitogen-activated protein kinase (MAPK) activity and dose-dependently inhibited platelet-derived-growth-factor-stimulated MAPK activity. Radioimmunoassay for adrenomedullin of media from Rat-2 cells showed a linear release of adrenomedullin-like immunoreactivity of 3.1fmol/h per 2×106 cells. Gel-filtration chromatography showed that this adrenomedullin-like immunoreactivity co-eluted with synthetic rat adrenomedullin. Northern blotting with a rat adrenomedullin cDNA probe was used to confirm the presence of adrenomedullin mRNA. However, neither Northern blotting nor reverse transcriptase–PCR showed the presence of the cloned adrenomedullin receptor (L1). We conclude that the Rat-2 cell line expresses a specific adrenomedullin receptor (coupled to cAMP production and regulation of MAPK) and secretes adrenomedullin, which may participate in a regulatory control loop.

scholarly journals Protein kinase C sensitizes olfactory adenylate cyclase.

1993 ◽  
Vol 101 (2) ◽  
pp. 183-205 ◽  
Author(s):  
S Frings
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Adenylate Cyclase ◽  
Electrical Response ◽  
Polymyxin B ◽  
Camp Production ◽  
Kinase C ◽  
Mucosal Side ◽  
Olfactory Tissue

Effects of neurotransmitters on cAMP-mediated signal transduction in frog olfactory receptor cells (ORCs) were studied using in situ spike recordings and radioimmunoassays. Carbachol, applied to the mucosal side of olfactory epithelium, amplified the electrical response of ORCs to cAMP-generating odorants, but did not affect unstimulated cells. A similar augmentation of odorant response was observed in the presence of phorbol dibutyrate (PDBu), an activator of protein kinase C (PKC). The electrical response to forskolin, an activator of adenylate cyclase (AC), was also enhanced by PDBu, and it was attenuated by the PKC inhibitor Goe 6983. Forskolin-induced accumulation of cAMP in olfactory tissue was potentiated by carbachol, serotonin, and PDBu to a similar extent. Potentiation was completely suppressed by the PKC inhibitors Goe 6983, staurosporine, and polymyxin B, suggesting that the sensitivity of olfactory AC to stimulation by odorants and forskolin was increased by PKC. Experiments with deciliated olfactory tissue indicated that sensitization of AC was restricted to sensory cilia of ORCs. To study the effects of cell Ca2+ on these mechanisms, the intracellular Ca2+ concentration of olfactory tissue was either increased by ionomycin or decreased by BAPTA/AM. Increasing cell Ca2+ had two effects on cAMP production: (a) the basal cAMP production was enhanced by a mechanism sensitive to inhibitors of calmodulin; and (b) similar to phorbol ester, cell Ca2+ caused sensitization of AC to stimulation by forskolin, an effect sensitive to Goe 6983. Decreasing cell Ca2+ below basal levels rendered AC unresponsive to stimulation by forskolin. These data suggest that a crosstalk mechanism is functional in frog ORCs, linking the sensitivity of AC to the activity of PKC. At increased activity of PKC, olfactory AC becomes more responsive to stimulation by odorants, forskolin, and cell Ca2+. Neurotransmitters appear to use this crosstalk mechanism to regulate olfactory sensitivity.

scholarly journals Role of glucose in chronic desensitization of isolated rat islets and mouse insulinoma (betaTC-3) cells to glucose-dependent insulinotropic polypeptide

2000 ◽  
Vol 165 (2) ◽  
pp. 281-291 ◽  
Author(s):  
SA Hinke ◽  
RP Pauly ◽  
J Ehses ◽  
P Kerridge ◽  
HU Demuth ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Insulin Release ◽  
Intracellular Camp ◽  
Camp Production ◽  
Insulin Granules ◽  
Continuous Application ◽  
Perifused Islets ◽  
Homologous Desensitization ◽  
Glucose Dependence

It is well documented that the release of insulin from isolated perifused islets attenuates over time, despite a continued glucose stimulation. In the current study we have shown that potentiation of insulin release by the intestinal hormone glucose-dependent insulinotropic polypeptide (GIP) is also attenuated after its continuous application. In less than 20 h of maintained stimulus with either hyperglycaemia (11.0 mM glucose) or GIP (10 nM) under hyperglycaemic conditions, insulin release returned to basal values. This was not due to loss of islet viability or reduction in the releasable pool of insulin granules, as 1 mM isobutylmethylxanthine was able to stimulate equivalent insulin release under both conditions. Further examination of chronic GIP desensitization was examined in cultured mouse insulinoma (betaTC-3) cells. GIP-stimulated cAMP production was not greatly affected by the prevailing glucose conditions, suggesting that the glucose dependence of GIP-stimulated insulin release occurs distally to the increase in intracellular cAMP in betaTC-3 cells. The GIP-stimulated cAMP response curve after desensitization was of similar magnitude at all glucose concentrations, but GIP pretreatment did not affect forskolin-stimulated cAMP production. Desensitization of the cAMP response in betaTC-3 cells was shown not to involve induction of dipeptidyl peptidase IV or pertussis toxin-sensitive G-proteins, activation of protein kinase C or protein kinase A, or modulation of phosphodiesterase activity. Homologous desensitization of the insulin-potentiating activity of GIP was found to affect both GIP-stimulated and forskolin-stimulated insulin release, indicating desensitization of distal steps in the stimulus-exocytosis cascade.

scholarly journals A role for protein kinase C-mediated phosphorylation in eliciting glucagon desensitization in rat hepatocytes

1995 ◽  
Vol 307 (1) ◽  
pp. 281-285 ◽  
Author(s):  
A Savage ◽  
L Zeng ◽  
M D Houslay
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Adenylate Cyclase ◽  
Protein Phosphatase ◽  
Rat Hepatocytes ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Glucagon Receptor ◽  
Kinase C ◽  
Phosphorylation Reaction

An immobilized hepatocyte preparation was used to show that both vasopressin and glucagon could desensitize the ability of glucagon to increase intracellular cyclic AMP concentrations. This process was not dependent on any influx of extracellular Ca2+ and was not mediated by any rise in the intracellular level of Ca2+. The protein kinase C-selective inhibitors chelerythrine, staurosporine and calphostin C acted as potent inhibitors of the desensitization process but with various degrees of selectivity regarding their ability to inhibit the desensitizing actions of glucagon and vasopressin. The protein phosphatase inhibitor okadaic acid was just as potent as vasopressin and glucagon in causing desensitization. Treatment of hepatocyte membranes with alkaline phosphatase restored to near control levels the ability of glucagon to stimulate adenylate cyclase activity in membranes from both glucagon- and vasopressin-treated (desensitized) hepatocytes. It is suggested that the desensitization of glucagon-stimulated adenylate cyclase activity involves a reversible phosphorylation reaction with the likely target being the glucagon receptor itself.

Pituitary adenylate cyclase-activating polypeptide activates KATP current in rat atrial myocytes

2001 ◽  
Vol 280 (3) ◽  
pp. H1058-H1065 ◽  
Author(s):  
Anne Baron ◽  
Dominique Monnier ◽  
Angela Roatti ◽  
Alex J. Baertschi
Keyword(s):  
Protein Kinase ◽  
Adenylate Cyclase ◽  
Neonatal Rat ◽  
Atrial Myocytes ◽  
Camp Production ◽  
Rt Pcr ◽  
Kinase C ◽  
Pituitary Adenylate Cyclase ◽  
Inhibitor Peptide ◽  
Electrophysiological Effects

Because the electrophysiological effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on the heart are little known, we studied the regulation of the atrial ATP-sensitive K+(KATP) current by PACAP on primary cultured neonatal rat atrial myocytes. PACAP-38 stimulates cAMP production with EC50 = 0.28 nmol/l ( r = 0.92, P < 0.02). PACAP-38 and PACAP-27 (10 nmol/l) have similar maximal effects, whereas 100 nmol/l vasoactive intestinal polypeptide (VIP) is 2.7 times less effective ( P < 0.05). RT-PCR shows the presence of cloned PACAP receptors PAC1 (≥2 isoforms), VPAC1, and VPAC2. PACAP-38 dose dependently activates the whole cell atrial KATP current with EC50 = 1–3 nmol/l ( n = 44). Maximal effects occur at 10 nmol/l (91 ± 15 pA/pF, n = 18). Diazoxide further increases the PACAP-activated current by 78% ( P < 0.05; n = 6). H89 (500 nmol/l), a protein kinase A (PKA) inhibitor, reduces the PACAP-activated KATPcurrent to 17.8 ± 9.6% ( n = 5) of the maximal diazoxide-induced current and totally inhibits the cAMP-induced KATP current. A protein kinase C (PKC) inhibitor peptide (50 μmol/l) in the pipette reduces the PACAP-38-induced KATP current to 33 ± 17 pA/pF ( P < 0.05, n = 6) without significantly affecting the currents induced by cAMP or VIP. The results suggest that: 1) PAC1, VPAC1, and VPAC2 are present in atrial myocytes; and 2) PACAP-38 activates the atrial KATP channels through both PKA and PKC pathways.

scholarly journals Structural and functional identification of the pituitary adenylate cyclase-activating polypeptide receptor VPAC2 from the frog Rana tigrina rugulosa

2001 ◽  
Vol 27 (2) ◽  
pp. 229-238 ◽  
Author(s):  
RL Hoo ◽  
D Alexandre ◽  
SM Chan ◽  
Y Anouar ◽  
RT Pang ◽  
...  
Keyword(s):  
Adenylate Cyclase ◽  
Amino Acid Sequence Identity ◽  
Intracellular Camp ◽  
Camp Production ◽  
Functional Identification ◽  
Peripheral Tissues ◽  
Functional Roles ◽  
Rana Tigrina ◽  
Pituitary Adenylate Cyclase ◽  
The Brain

Recently, a frog pituitary adenylate cyclase-activating polypeptide (PACAP)/vasoactive intestinal peptide (VIP) receptor (fPVR) has been characterized, and interestingly, this receptor exhibits characteristics of both mammalian PACAP type II receptors VPAC(1)R and VPAC(2)R. In order to investigate the receptors responsible for mediating the actions of VIP and PACAP in amphibians, in this report, a frog VPAC(2) receptor (fVPAC(2)R) cDNA was isolated. fVPAC(2)R shares 47.7, 46.9 and 62.5% amino acid sequence identity with fPVR, human VPAC(1)R and human VPAC(2)R respectively. Functionally, fVPAC(2)R, when expressed in CHO cells, was responsive to both frog peptides including VIP, PACAP38 and PACAP27 where the EC(50) values of these peptides in intracellular cAMP production were 0.15, 0.18 and 0.16 microM respectively. The pharmacological profiles of human peptides (VIP, PACAP38 and peptide histidine methionine) to stimulate frog and human VPAC(2)Rs were compared, and it was found that these peptides could only activate the frog receptor at micromolar concentrations. fVPAC(2)R was found to be widely distributed in various peripheral tissues as well as several regions of the brain. The presence of the receptor transcripts suggests the functional roles of the receptor in mediating the actions of PACAP and/or VIP in these tissues. As VIP and particularly PACAP27 are highly conserved peptides in vertebrate evolution, comparative studies of these peptides and their receptors in non-mammalian vertebrates should provide clues to better understand the physiology of these important peptides in human and other vertebrates.

Alpha 2-adrenoceptor stimulation and cellular cAMP levels in microdissected rat glomeruli

1986 ◽  
Vol 250 (1) ◽  
pp. F103-F108
Author(s):  
S. Umemura ◽  
D. D. Smyth ◽  
W. A. Pettinger
Keyword(s):  
Adenylate Cyclase ◽  
Phosphodiesterase Inhibitor ◽  
Camp Accumulation ◽  
Camp Production ◽  
Camp Concentration ◽  
Adrenoceptor Agonist ◽  
Dose Dependent Fashion ◽  
Camp Levels ◽  
Dose Dependent ◽  
Camp Formation

A functional role for the numerically predominant glomerular alpha 2-adrenoceptors is unknown. In other tissues, activation of alpha 2-adrenoceptors inhibits adenylate cyclase activity. We therefore examined the effect of alpha 2-adrenoceptor stimulation with (-)-epinephrine (E) on the cellular cAMP concentration in glomeruli isolated by microdissection. Parathyroid hormone (1-34 PTH), prostaglandin E2 (PGE2), histamine, serotonin, or adenosine, in the presence of 3-isobutyl-1-methylxanthine (phosphodiesterase inhibitor) and propranolol, was used to activate adenylate cyclase in single intact rat glomeruli. alpha 2-Adrenoceptors were activated with varying concentrations of E (37 degrees C, 2 min). In the presence of PTH-stimulated cAMP production, alpha 2-adrenoceptor activation with E (5 X 10(-7) to 5 X 10(-6) M) suppressed cellular cAMP levels in a dose-dependent fashion with the maximum at 30%. This suppression by E was inhibited by 5 X 10(-6) M yohimbine but not by 5 X 10(-6) M prazosin, confirming alpha 2-adrenoceptor mediation of this effect of E. Consistent with the above findings, the specific alpha 2-adrenoceptor agonist BHT933 inhibited PTH-stimulated cAMP accumulation. E also inhibited cAMP accumulation stimulated by serotonin. However, E did not suppress the PGE2-, histamine-, or adenosine-stimulated increase in cellular cAMP in the glomerulus. Activation of alpha 2-adrenoceptors inhibits cAMP formation stimulated by PTH or serotonin but not by PGE2, histamine, or adenosine in the rat glomerulus. Thus, the ability of alpha 2-adrenoceptors to inhibit adenylate cyclase appears to be hormone and probably function specific.

scholarly journals Cyclic AMP-Independent Regulation of Protein Kinase A Substrate Phosphorylation by Kelch Repeat Proteins

2005 ◽  
Vol 4 (11) ◽  
pp. 1794-1800 ◽  
Author(s):  
Ailan Lu ◽  
Jeanne P. Hirsch
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Protein Kinase A ◽  
Intracellular Camp ◽  
Invasive Growth ◽  
Camp Phosphodiesterase ◽  
Α Subunit ◽  
Substrate Phosphorylation ◽  
Kinase A ◽  
In Cells

ABSTRACT Pseudohyphal and invasive growth in the yeast Saccharomyces cerevisiae is regulated by the kelch repeat-containing proteins Gpb1p and Gpb2p, which act downstream of the G protein α-subunit Gpa2p. Here we show that deletion of GPB1 and GPB2 causes increased haploid invasive growth in cells containing any one of the three protein kinase A (PKA) catalytic subunits, suggesting that Gpb1p and Gpb2p are able to inhibit each of these kinases. Cells containing gpb1Δ gpb2Δ mutations also display increased phosphorylation of the PKA substrates Sfl1p and Msn2p, indicating that Gpb1p and Gpb2p are negative regulators of PKA substrate phosphorylation. Stimulation of PKA-dependent signaling by gpb1Δ gpb2Δ mutations occurs in cells that lack both adenylyl cyclase and the high-affinity cyclic AMP (cAMP) phosphodiesterase. This effect is also seen in cells that lack the low-affinity cAMP phosphodiesterase. Given that these three enzymes control the synthesis and degradation of cAMP, these results indicate that the effect of Gpb1p and Gpb2p on PKA substrate phosphorylation does not occur by regulating the intracellular cAMP concentration. These findings suggest that Gpb1p and Gpb2p mediate their effects on the cAMP/PKA signaling pathway either by inhibiting the activity of PKA in a cAMP-independent manner or by activating phosphatases that act on PKA substrates.

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