scholarly journals Characterization of the homologous and heterologous desensitization of rat Leydig-tumour-cell adenylate cyclase

1984 ◽  
Vol 220 (3) ◽  
pp. 803-809 ◽  
Author(s):  
C J Dix ◽  
A D Habberfield ◽  
B A Cooke
Keyword(s):  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
G Protein ◽  
Tumour Cell ◽  
Tumour Cells ◽  
Dependent Manner ◽  
Dibutyryl Cyclic Amp ◽  
Heterologous Desensitization ◽  
Homologous Desensitization

The homologous and heterologous desensitization of rat Leydig-tumour-cell adenylate cyclase induced by lutropin (LH) was characterized with the aid of forskolin and cholera toxin. Forskolin stimulated cyclic AMP production in a dose-dependent manner, with linear kinetics up to 2h. Forskolin also potentiated the action of LH on cyclic AMP production, but was only additive with cholera toxin. Preincubation of rat Leydig tumour cells with LH (1.0 micrograms/ml) for 1 h produced a desensitization of the subsequent LH (1.0 micrograms/ml)-stimulated cyclic AMP production, whereas the responses to cholera toxin (5.0 micrograms/ml), forskolin (100 microM), LH plus forskolin or cholera toxin plus forskolin were unaltered. In contrast, preincubation with LH for 20h produced a desensitization to all the stimuli tested. When rat Leydig tumour cells were preincubated for 1h with forskolin or dibutyryl cyclic AMP, the only subsequent response that was significantly altered was that to LH plus forskolin after preincubation with forskolin. However, preincubation for 20h with forskolin or dibutyryl cyclic AMP induced a desensitization to all stimuli subsequently tested. LH produced a rapid (0-1h) homologous desensitization, which was followed by a slower (2-8h)-onset heterologous desensitization. Forskolin and dibutyryl cyclic AMP were only able to induce heterologous desensitization. The rate of desensitization induced by either forskolin or dibutyryl cyclic AMP was similar to the rate of heterologous desensitization induced by LH. These results demonstrate that in purified rat Leydig tumour cells LH produces an initial homologous desensitization of adenylate cyclase that involves a cyclic AMP-independent lesion at or proximal to the guanine nucleotide regulatory protein (G-protein). This is followed by heterologous desensitization, which can also be induced by forskolin or dibutyryl cyclic AMP, thus indicating that LH-induced heterologous desensitization of rat Leydig-tumour-cell adenylate cyclase involves a cyclic AMP-dependent lesion that is after the G-protein.

scholarly journals Effect of pertussis toxin and neomycin on G-protein-regulated polyphosphoinositide phosphodiesterase. A comparison between HL60 membranes and permeabilized HL60 cells

1988 ◽  
Vol 256 (2) ◽  
pp. 343-350 ◽  
Author(s):  
S Cockcroft ◽  
J Stutchfield
Keyword(s):  
Cyclic Amp ◽  
G Protein ◽  
Pertussis Toxin ◽  
Dimethyl Sulphoxide ◽  
Hl60 Cell ◽  
Dependent Manner ◽  
Dibutyryl Cyclic Amp ◽  
Hl60 Cells ◽  
Permeabilized Cells ◽  
Streptolysin O

The promyelocytic HL60 cell can be differentiated with dimethyl sulphoxide or dibutyryl cyclic AMP leading to the appearance of fMetLeuPhe receptors on the cell surface. G-protein-stimulated polyphosphoinositide phosphodiesterase (PPI-pde) activity was assessed in membranes prepared from both differentiated and non-differentiated HL60 cells. Both the extent of the response and the rank order of potency of the GTP analogues to stimulate PPI-pde activation (guanosine 5′-[gamma-thio]triphosphate (GTP[S]) greater than guanosine 5′-[beta gamma-imido]triphosphate (p[NH]ppG) greater than guanosine 5′-[beta gamma-methylene]triphosphate (p[CH2]ppG) remains unchanged after differentiation with dimethyl sulphoxide. In comparison, differentiation by dibutyryl cyclic AMP leads to diminution of PPI-pde activity when stimulated by GTP[S] or fluoride, but not by millimolar concentrations of Ca2+. GTP[S]-stimulated PPI-pde in membranes is sensitive to the presence of Ca2+ (pCa 8-5). Pertussis-toxin pretreatment of intact HL60 cells leads to inhibition of both the secretory response and the formation of inositol phosphates when stimulated by fMetLeuPhe. In contrast, pertussis-toxin pretreatment has no effect on either GTP[S]- or fluoride-stimulated PPI-pde. Neomycin in a concentration-dependent manner inhibits both GTP[S] plus Ca2+ (pCa 5)-stimulated secretion and PPI-pde activation in streptolysin-O-permeabilized cells. The extent of PPI-pde activation in membranes compared with streptolysin-O-permeabilized cells reveals that the membrane preparation does not possess all the components that make up the inositide signalling system.

scholarly journals Lipopeptides activate Gi-proteins in dibutyryl cyclic AMP-differentiated HL-60 cells

1993 ◽  
Vol 296 (1) ◽  
pp. 245-251 ◽  
Author(s):  
J F Klinker ◽  
A Höer ◽  
I Schwaner ◽  
S Offermanns ◽  
K Wenzel-Seifert ◽  
...  
Keyword(s):  
Cyclic Amp ◽  
Phosphatidic Acid ◽  
G Protein ◽  
Acid Formation ◽  
Human Neutrophils ◽  
Dependent Manner ◽  
Dibutyryl Cyclic Amp ◽  
Gtp Hydrolysis ◽  
Alpha Subunits ◽  
Gi Proteins

Synthetic lipopeptides activate superoxide-anion (O2-) formation in human neutrophils in a pertussis-toxin (PTX)-sensitive manner, suggesting the involvement of G-proteins of the Gi family in the signal-transduction pathway. We compared G-protein activation by lipopeptides and the chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (fMLP) in dibutyryl-cyclic-AMP-differentiated HL-60 cells. The lipopeptide (2S)-2-palmitoylamino-6-palmitoyloxymethyl-7-palmitoyloxy heptanoyl-SK4 (Pam3AhhSK4) and fMLP activated high-affinity GTPase, i.e. the enzymic activity of G-protein alpha-subunits, in HL-60 membranes in a time- and protein-dependent manner, but they had no effect on Mg(2+)-ATPase and Na+/K(+)-ATPase. Pam3AhhSK4 and fMLP increased Vmax. of GTP hydrolysis. Pam3AhhSK4 activated GTP hydrolysis with half-maximal and maximal effects at about 2 microM and 10 microM respectively. Other lipopeptides activated GTP hydrolysis as well. Lipopeptides were less effective than fMLP to activate GTPase. In membranes from PTX-treated cells, the stimulatory effects of lipopeptides and fMLP on GTPase were abolished. In N-ethylmaleimide-treated membranes, the relative stimulatory effect of Pam3AhhSK4 on GTP hydrolysis was enhanced, whereas that of fMLP was diminished. fMLP and Pam3AhhSK4 activated GTPase in an over-additive manner in N-ethylmaleimide-treated membranes. Unlike fMLP, Pam3AhhSK4 did not enhance incorporation of GTP azidoanilide into, and cholera-toxin-catalysed ADP-ribosylation of Gi-protein alpha-subunits in, HL-60 membranes and did not induce rises in cytosolic Ca2+ concentration. Pam3AhhSK4 and fMLP stimulated phosphatidic acid formation in a PTX-sensitive manner. Pam3AhhSK4 itself did not activate O2- formation, but potentiated the stimulatory effects of fMLP. Our data suggest that (i) lipopeptides activate the GTPase of Gi-proteins, (ii) lipopeptides and fMLP activate Gi-proteins differently, (iii) lipopeptides stimulate phospholipase D via Gi-proteins, and (iv) phosphatidic acid formation is not sufficient for activation of O2- formation.

scholarly journals Inhibition by somatostatin of amylase secretion induced by calcium and cyclic AMP in rat pancreatic acini

1994 ◽  
Vol 304 (2) ◽  
pp. 531-536 ◽  
Author(s):  
H Ohnishi ◽  
T Mine ◽  
I Kojima
Keyword(s):  
Cyclic Amp ◽  
G Protein ◽  
Pertussis Toxin ◽  
Dose Response ◽  
Response Curve ◽  
Dose Response Curve ◽  
Amylase Secretion ◽  
Ionophore A23187 ◽  
Dependent Manner ◽  
Pancreatic Acini

It has recently been shown that somatostatin inhibits amylase secretion from isolated pancreatic acini by reducing cyclic AMP (cAMP) production [Matsushita, Okabayashi, Hasegawa, Koide, Kido, Okutani, Sugimoto and Kasuga (1993) Gastroenterology 104, 1146-1152]. To date, however, little is known as to the other mechanism(s) by which somatostatin inhibits amylase secretion in exocrine pancreas. To investigate the action of somatostatin independent of cAMP generation, we examined the effect of somatostatin in isolated rat pancreatic acini stimulated by 1 microM calcium ionophore A23187 and 1 mM 8-bromo-cyclic AMP (8Br-cAMP). Somatostatin inhibited amylase secretion evoked by a combination of A23187 and 8Br-cAMP in a dose-dependent manner. The maximum inhibition was obtained by 10(-7) M somatostatin, and at this concentration somatostatin inhibited the effect of A23187 and 8Br-cAMP by approximately 30%. In electrically permeabilized acini, an elevation of free calcium concentration resulted in an increase in amylase secretion and cAMP enhanced the secretion evoked by calcium. cAMP shifted the dose-response curve for calcium-induced secretion leftwards and elevated the peak value of secretion. Somatostatin inhibited the effect of cAMP on calcium-induced amylase secretion by shifting the dose-response curve to the right. To determine the involvement of a G-protein(s), we examined the effect of somatostatin in acini pretreated with pertussis toxin. Pretreatment of acini with pertussis toxin completely blocked somatostatin-inhibition of amylase-secretion evoked by A23187 and 8Br-cAMP. These results indicate that somatostatin decreases amylase secretion induced by cAMP and calcium by reducing the calcium sensitivity of exocytosis. A pertussis toxin-sensitive G-protein is also involved in this step.

Mode of Action of the Hypertrehalosaemic Peptides from the American Cockroach

1985 ◽  
Vol 40 (9-10) ◽  
pp. 670-676 ◽  
Author(s):  
Gerd Gäde
Keyword(s):  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Mode Of Action ◽  
Glycogen Phosphorylase ◽  
Fat Body ◽  
American Cockroach ◽  
Dependent Manner ◽  
Low Concentrations ◽  
First Time ◽  
Dose Dependent

Abstract Although crude extracts of cockroach (Periplaneta amencana) corpora cardiaca have been shown previously to affect the activity of adenylate cyclase and phosphorylase, we demonstrate in the present study for the first time that low concentrations (0.5 to 5 pmol) of the synthetic myoactive peptides. M I and M II, also affect these systems; these myoactive peptides are identical to the hypertrehalosaemic hormones I and II, and cause an increase in the concentration of the second messenger cyclic AMP in the fat body.In addition, both octapeptides activate fat body glycogen phosphorylase and promote breakdown of fat body glycogen. Both peptides increase the levels to haemolymph carbohydrate in a dose-dependent manner.

scholarly journals Bradykinin-dependent activation of adenylate cyclase activity and cyclic AMP accumulation in tracheal smooth muscle occurs via protein kinase C-dependent and -independent pathways

1994 ◽  
Vol 297 (1) ◽  
pp. 233-239 ◽  
Author(s):  
P A Stevens ◽  
S Pyne ◽  
M Grady ◽  
N J Pyne
Keyword(s):  
Smooth Muscle ◽  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
Phospholipase D ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Tracheal Smooth Muscle ◽  
Constitutive Activation ◽  
Cyclic Amp Accumulation

Treatment of cultured tracheal smooth-muscle cells (TSM) with phorbol 12-myristate 13-acetate (PMA) (100 nM) or bradykinin (100 nM) elicited enhanced basal and guanosine 5′-[beta gamma-imido]-triphosphate-stimulated adenylate cyclase activities in subsequently isolated membranes. Combined stimulation of cells was non-additive, indicating that both agents activate adenylate cyclase via similar routes. Both PMA (100 nM) and bradykinin (100 nM) allowed the alpha subunit of Gs to act as a more favourable substrate for its cholera-toxin-catalysed ADP-ribosylation in vitro. PMA was without effect on intracellular cyclic AMP in control cells. However, constitutive activation of Gs by treatment in vivo with cholera toxin (0.5 ng/ml, 18 h) sensitized the cells to PMA stimulation, resulting in a concentration-dependent increase in intracellular cyclic AMP accumulation (EC50 = 7.3 +/- 2.5 nM, n = 5). Bradykinin also elicited a concentration-dependent increase in intracellular cyclic AMP (EC50 = 63.3 +/- 14.5 nM, n = 3). Constitutive activation of Gs resulted in an increased maximal response (10-fold) and potency (EC50 = 6.17 +/- 1.6 nM, n = 3) to bradykinin. This response was not affected by the B2-receptor antagonist, NPC567 [which selectively blocks bradykinin-stimulated phospholipase C (PLC), with minor activity against phospholipase D (PLD) activity]. Des-Arg9-bradykinin (a B1-receptor agonist) was without activity. These results suggest that the receptor sub-type capable of activating PLD may also be stimulatory for cyclic AMP accumulation. Furthermore, pre-treatment of the cells with butan-l-ol (0.3%, v/v), which traps phosphatidate derived from PLD reactions, blocked the bradykinin-stimulated increase in intracellular cyclic AMP. These studies suggest that there may be a causal link between PLD-derived phosphatidate and the positive modulation of adenylate cyclase activity. In support of this, the concentration-dependence for bradykinin-stimulated adenylate cyclase activity was identical with that of bradykinin-stimulated phospholipase D activity (EC50 = 5 nM). Bradykinin, but not PMA, was also capable of eliciting the inhibition of cyclic AMP phosphodiesterase activity in TSM cells (EC50 > 100 nM) via an unidentified mechanism. These studies indicate that cross-regulation between the cyclic AMP pathway and phospholipid-derived second messengers in TSM cells does not occur as a consequence of PLC-catalysed PtdIns(4,5)P2 hydrolysis, but may involve, in part, PLD-catalysed phosphatidylcholine hydrolysis.

Renal metabolism of N6,O2'-dibutyryl adenosine 3',5'-monophosphate

1979 ◽  
Vol 237 (1) ◽  
pp. F75-F84
Author(s):  
R. Coulson ◽  
W. W. Harrington
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Urinary Excretion ◽  
Rat Kidney ◽  
Bond Cleavage ◽  
Dibutyryl Cyclic Amp ◽  
Renal Metabolism ◽  
Phosphate Bond ◽  
Isolated Rat

Metabolism of dibutyryl cyclic AMP was studied by including the 3H- or C-labeled nucleotide (0.1 mM, 5 mumol) in the recirculating perfusate of the isolated rat kidney. Kidneys were perfused with nucleotide for 60 min. Dibutyryl cyclic AMP was almost completely cleared from the perfusate, about one-quarter as urinary excretion principally by probenecid-sensitive secretion and about one-half as metabolism beyond 3'-phosphate bond cleavage. The principal metabolite, N6-monobutyryl adenosine, accounted for one-third of added dibutyryl cyclic AMP. The remaining metabolites were ATP, ADP AMP, and N6-monobutyryl AMP. Dibutyryl cyclic AMP (0.1 or 1.0 mM) elevated renal ATP but did not alter uricogenesis. Both dibutyryl cyclic AMP and cyclic AMP at 0.2 mM produced similar activation and subcellular redistribution of renal protein kinase. N6-monobutyryl adenosine, unlike adenosine, had no effect on the renal activity of adenylate cyclase, low Km cyclic AMP phosphodiesterase, and protein kinase. Dibutyryl cyclic AMP is like exogenous cyclic AMP in that it penetrates the rat kidney, activates protein kinase, and is metabolized to ATP (R. Coulson, J. Biol. Chem. 251: 4958-4967, 1976), but is unlike cyclic AMP in its extent of secretion and metabolism to ATP and urate and in its formation of the unique metabolites N6-monobutyryl AMP and N6-monobutyryl adenosine.

G-protein Activation Decreases Isoflurane Inhibition of N-type Ba2+Currents

2003 ◽  
Vol 99 (2) ◽  
pp. 392-399 ◽  
Author(s):  
Igor M. Nikonorov ◽  
Thomas J. J. Blanck ◽  
Esperanza Recio-Pinto
Keyword(s):  
Cholera Toxin ◽  
G Protein ◽  
G Proteins ◽  
Pertussis Toxin ◽  
Volatile Anesthetic ◽  
Cell Voltage ◽  
Dependent Manner ◽  
Protein Activation ◽  
G Protein Activation ◽  
Voltage Dependent

Background G-protein activation mediates inhibition of N-type Ca2+ currents. Volatile anesthetics affect G-protein pathways at various levels, and activation of G-proteins has been shown to increase the volatile anesthetic potency for inhibiting the electrical-induced contraction in ileum. The authors investigated whether isoflurane inhibition of N-type Ba2+ currents was mediated by G-protein activation. Methods N-type Ba2+ currents were measured in the human neuronal SH-SY5Y cell line by using the whole cell voltage-clamp method. Results Isoflurane was found to have two effects on N-type Ba2+ currents. First, isoflurane reduced the magnitude of N-type Ba2+ currents to a similar extent (IC50 approximately 0.28 mm) in the absence and presence of GDPbetaS (a nonhydrolyzable GDP analog). Interestingly, GTPgammaS (a nonhydrolyzable GTP analog and G-protein activator) in a dose-dependent manner reduced the isoflurane block; 120 microm GTPgammaS completely eliminated the block of 0.3 mm isoflurane and reduced the apparent isoflurane potency by approximately 2.4 times (IC50 approximately 0.68 mm). Pretreatment with pertussis toxin or cholera toxin did not eliminate the GTPgammaS-induced protection against the isoflurane block. Furthermore, isoflurane reduced the magnitude of voltage-dependent G-protein-mediated inhibition of N-type Ba2+ currents, and this effect was eliminated by pretreatment with pertussis toxin or cholera toxin. Conclusions It was found that activation of G-proteins in a neuronal environment dramatically reduced the isoflurane potency for inhibiting N-type Ba2+ currents and, in turn, isoflurane affected the G-protein regulation of N-type Ba2+ currents.

scholarly journals Changes in adenosine receptors during differentiation of 3T3-F442A cells to adipocytes

1988 ◽  
Vol 249 (2) ◽  
pp. 377-381 ◽  
Author(s):  
K Ravid ◽  
J M Lowenstein
Keyword(s):  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Adenosine Receptors ◽  
Positive Response ◽  
Inhibitory Effect ◽  
Dependent Manner ◽  
Similar Extent ◽  
Phenylisopropyl Adenosine ◽  
Dose Dependent ◽  
Basal Concentration

Incubation of undifferentiated 3T3-F442A cells (preadipocytes) with 5′-N-ethylcarboxamidoadenosine (NECA) increases intracellular cyclic AMP in a dose-dependent manner. The effect of NECA is antagonized by 8-phenyltheophylline, but potentiated by 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidine, an inhibitor of cyclic AMP phosphodiesterase. Incubation of preadipocytes with (-)-N6-(R-phenylisopropyl)adenosine (PIA) has no inhibitory effect on the basal concentration of cyclic AMP or on the stimulation of adenylate cyclase by isoprenaline or forskolin. Micromolar concentrations of PIA increase intracellular cyclic AMP, but with a lower potency than NECA. Similar findings are obtained with the non-differentiating cell line 3T3-C2. Thus preadipocyte 3T3-F442A cells and 3T3-C2 cells appear to express only stimulatory adenosine receptors. For some time after 3T3-F442A cells have differentiated to adipocytes, micromolar concentrations of NECA and PIA continue to increase cyclic AMP to a similar extent to that in preadipocytes, whereas nanomolar concentrations of PIA decrease the stimulatory effects of isoprenaline and forskolin on adenylate cyclase by 50%. However, several days after differentiation, the adipocytes gradually lose the major part of their positive response to NECA and reach a steady response to NECA 10 days after differentiation. The inhibition of adenylate cyclase caused by PIA remains constant for at least 2 weeks after differentiation. With membranes derived from the cells, the effects of NECA and PIA depend on GTP. These results indicate that, during the differentiation of 3T3-F442A cells to adipocytes, new inhibitory adenosine receptors are expressed, whereas the stimulatory receptors become attenuated.

scholarly journals Effect of administration of the fructose on the glycogenolytic action of glucagon. An investigation of the pathogeny of hereditary fructose intolerance

1973 ◽  
Vol 134 (2) ◽  
pp. 637-645 ◽  
Author(s):  
G. Van Den Berghe ◽  
L. Hue ◽  
H. G. Hers
Keyword(s):  
Blood Glucose ◽  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Urinary Excretion ◽  
Fold Increase ◽  
Maximal Effect ◽  
Dibutyryl Cyclic Amp ◽  
Hereditary Fructose Intolerance ◽  
Fructose Intolerance ◽  
Km Value

1. The mechanism by which the administration of fructose to patients with hereditary fructose intolerance makes them unresponsive to the hyperglycaemic action of glucagon was studied. In four patients, a 10-fold increase in the urinary excretion of cyclic AMP was induced by glucagon, but this effect was drastically decreased by the previous administration of fructose (250mg/kg). Further, the intravenous injection of 6-N,2′-O-dibutyryl cyclic AMP did not cause an increase in the blood glucose during fructose-induced hypoglycaemia. 2. The administration of a large dose of fructose (5g/kg) to mice decreased markedly both the concentration of ATP and the increase in the concentration of cyclic AMP caused by glucagon in the liver. Other ATP-depleting agents had a similar effect and a linear correlation could be drawn between the concentration of ATP and the change in cyclic AMP concentration; a half-maximal effect was obtained for a concentration of ATP close to the Km value of adenylate cyclase. 3. The administration of fructose to mice caused the inactivation of phosphorylase in the liver, but this effect was easily reversed by glucagon. 4. At a concentration of 10mm-fructose 1-phosphate and 1.5mm-Pi, purified liver phosphorylase a was inhibited by 70%. This inhibition appears to be a likely explanation for the unresponsiveness to glucagon of patients with hereditary fructose intolerance.

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