Resensitization of hepatocyte glucagon-stimulated adenylate cyclase can be inhibited when cyclic AMP phosphodiesterase inhibitors are used to elevate intracellular cyclic AMP concentrations to supraphysiological values

Treatment of intact hepatocytes with glucagon led to the rapid desensitization of adenylate cyclase, which reached a maximum around 5 min after application of glucagon, after which resensitization ensued. Complete resensitization occurred some 20 min after the addition of glucagon. In hepatocytes which had been preincubated with the cyclic AMP phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), glucagon elicited a stable desensitized state where resensitization failed to occur even 20 min after exposure of hepatocytes to glucagon. Treatment with IBMX alone did not elicit desensitization. The action of IBMX in stabilizing the glucagon-mediated desensitized state was mimicked by the non-methylxanthine cyclic AMP phosphodiesterase inhibitor Ro-20-1724 [4-(3-butoxy-4-methoxylbenzyl)-2-imidazolidinone]. IBMX inhibited the resensitization process in a dose-dependent fashion with an EC50 (concn. giving 50% of maximal effect) of 26 +/- 5 microM, which was similar to the EC50 value of 22 +/- 6 microM observed for the ability of IBMX to augment the glucagon-stimulated rise in intracellular cyclic AMP concentrations. Pre-treatment of hepatocytes with IBMX did not alter the ability of either angiotensin or the glucagon analogue TH-glucagon, ligands which did not increase intracellular cyclic AMP concentrations, to cause the rapid desensitization and subsequent resensitization of adenylate cyclase. It is suggested that, although desensitization of glucagon-stimulated adenylate cyclase is elicited by a cyclic AMP-independent process, the resensitization of adenylate cyclase can be inhibited by a process which is dependent on elevated cyclic AMP concentrations. This action can be detected by attenuating the degradation of cyclic AMP by using inhibitors of cyclic AMP phosphodiesterase.

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N 6-(Phenylisopropyl)adenosine prevents glucagon both blocking insulin's activation of the plasma-membrane cyclic AMP phosphodiesterase and uncoupling hormonal stimulation of adenylate cyclase activity in hepatocytes

Biochemical Journal ◽

10.1042/bj2220177 ◽

1984 ◽

Vol 222 (1) ◽

pp. 177-182 ◽

Cited By ~ 12

Author(s):

A V Wallace ◽

C M Heyworth ◽

M D Houslay

Keyword(s):

Plasma Membrane ◽

Cyclic Amp ◽

Adenylate Cyclase ◽

Phosphodiesterase Inhibitor ◽

Cyclase Activity ◽

Adenylate Cyclase Activity ◽

Maximal Effect ◽

Hormonal Stimulation ◽

Cyclic Amp Phosphodiesterase ◽

Phenylisopropyl Adenosine

Glucagon (10nM) prevented insulin (10nM) from activating the plasma-membrane cyclic AMP phosphodiesterase. This effect of glucagon was abolished by either PIA [N6-(phenylisopropyl)adenosine] (100nM) or adenosine (10 microM). Neither PIA nor adenosine exerted any effect on the plasma-membrane cyclic AMP phosphodiesterase activity either alone or in combination with glucagon. Furthermore, PIA and adenosine did not potentiate the action of insulin in activating this enzyme. 2-Deoxy-adenosine (10 microM) was ineffective in mimicking the action of adenosine. The effect of PIA in preventing the blockade by glucagon of insulin's action was inhibited by low concentrations of theophylline. Half-maximal effects of PIA were elicited at around 6nM-PIA. It is suggested that adenosine is exerting its effects on this system through an R-type receptor. This receptor does not appear to be directly coupled to adenylate cyclase, however, as PIA did not affect either the activity of adenylate cyclase or intracellular cyclic AMP concentrations. Insulin's activation of the plasma-membrane cyclic AMP phosphodiesterase, in the presence of both glucagon and PIA, was augmented by increasing intracellular cyclic AMP concentrations with either dibutyryl cyclic AMP or the cyclic AMP phosphodiesterase inhibitor Ro-20-1724. PIA also inhibited the ability of glucagon to uncouple (desensitize) adenylate cyclase activity in intact hepatocytes. This occurred at a half-maximal concentration of around 3 microM-PIA. However, if insulin (10 nM) was also present in the incubation medium, PIA exerted its action at a much lower concentration, with a half-maximal effect occurring at around 4 nM.

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Effects of suboptimal levels of extracellular calcium on the regulation of the cyclic AMP phosphodiesterase-inhibitor system and membrane differentiation in Dictyostelium discoideum

Journal of Cell Science ◽

10.1242/jcs.90.4.691 ◽

1988 ◽

Vol 90 (4) ◽

pp. 691-700 ◽

Cited By ~ 1

Author(s):

M.B. Coukell ◽

A.M. Cameron

Keyword(s):

Cyclic Amp ◽

Dictyostelium Discoideum ◽

Cyclic Gmp ◽

Phosphodiesterase Inhibitor ◽

Wild Type ◽

Cyclic Amp Phosphodiesterase ◽

Dose Dependent Fashion ◽

Inhibitor System ◽

Membrane Bound ◽

Dose Dependent

When starved wild-type amoebae of Dictyostelium discoideum were washed and incubated in 1 mM-EGTA, their ability to induce soluble cyclic AMP phosphodiesterase (PD) activity in response to either millimolar cyclic AMP or a series of nanomolar cyclic AMP pulses was reduced by 55–75%. Supplementation of EGTA-treated cells with exogenous Ca2+ stimulated PD induction in a dose-dependent fashion (EC50 = 100–200 nM free extracellular Ca2+), and enzyme production was maximal at about 1 microM free Ca2+. Ca2+ depletion also strongly impaired production of the phosphodiesterase inhibitor (PDI). In contrast, other than delaying their appearance by about 1 h, EGTA had little effect on the induction by cyclic AMP pulses of cell surface markers such as contact sites A and membrane-bound PD activity. Similar changes in both the soluble and membrane activities were observed with strain NP368, a mutant that overproduces cyclic GMP when stimulated by cyclic AMP. Thus, Ca2+ depletion does not appear to inhibit PD and PDI production by reducing intracellular cyclic GMP. To determine whether Ca2+ depletion alters signal transduction, two mutants that produce the soluble PD activities constitutively were examined. Suboptimal concentrations of free extracellular Ca2+ were found to inhibit PD production in these cells to the same degree and with the same concentration dependence as low Ca2+ inhibited PD induction by cyclic AMP in wild-type cells. These results suggest that Ca2+ depletion by EGTA probably inhibits PD and PDI production indirectly by perturbing an intracellular Ca2+ pool(s) rather than by altering a surface cyclic AMP-receptor-mediated process.

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Dexamethasone and 8-bromo-cyclic AMP depress the incorporation of [3H]thymidine into mouse condylar cartilage by different pathways

Journal of Endocrinology ◽

10.1677/joe.0.1090209 ◽

1986 ◽

Vol 109 (2) ◽

pp. 209-213 ◽

Cited By ~ 3

Author(s):

Z. Kraiem ◽

G. Maor ◽

M. Silbermann

Keyword(s):

Cyclic Amp ◽

Thymidine Incorporation ◽

Phosphodiesterase Inhibitor ◽

Inhibitory Effect ◽

Significant Inhibition ◽

Condylar Cartilage ◽

Camp Analogue ◽

Dose Dependent Fashion ◽

Cartilage Cells ◽

Dose Dependent

ABSTRACT We examined whether cyclic AMP (cAMP) affects the incorporation of [3H]thymidine into cartilage cells and, if so, whether this action could be related to the inhibitory effect of glucocorticoid hormones on the growth of ossifying cartilage. Incorporation of [3H]thymidine into trichloroacetic acid-precipitable material by mouse cartilage was measured concomitantly with the concentration of cAMP. Dexamethasone (1 μmol/l) significantly (P < 0·05) depressed the incorporation of [3H]thymidine. The cAMP analogue 8-bromo-cAMP (0·01–1 mmol/l) also depressed the incorporation of the radionucleotide in a dose-dependent fashion. When various concentrations of 8-bromo-cAMP were added with dexamethasone (1 μmol/l), no apparent changes took place compared with the effect of dexamethasone alone. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0·2-1 mmol/l) elicited an inhibitory effect on [3H]thymidine incorporation and a stimulatory influence on cartilage cAMP concentrations. Dexamethasone, at doses (0·01–1 μmol/l) causing significant inhibition of [3H]thymidine incorporation, failed to increase cartilage levels of cAMP. It seems, therefore, that the depressive effect of dexamethasone on [3H]thymidine incorporation in condylar cartilage is not mediated through an increase of cAMP in the tissue. J. Endocr. (1986) 109, 209–213

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Theophylline Inhibition of Nucleoside Transport and its Relation to Cyclic AMP in Skeletal Muscle

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y74-140 ◽

1974 ◽

Vol 52 (6) ◽

pp. 1063-1073 ◽

Cited By ~ 8

Author(s):

Yin-Tak Woo ◽

J. F. Manery ◽

E. E. Dryden

Keyword(s):

Skeletal Muscle ◽

Cyclic Amp ◽

Phosphodiesterase Inhibitor ◽

Inhibitory Effect ◽

Nucleoside Transport ◽

Intracellular Uptake ◽

Frog Skeletal Muscle ◽

Dibutyryl Cyclic Amp ◽

Cyclic Amp Phosphodiesterase ◽

Dose Dependent

Using [14C]inosine and [3H]sorbitol, the effect of theophylline on inosine uptake was studied. Theophylline inhibited the intracellular uptake of inosine by isolated, frog skeletal muscle in a dose-dependent way. An inhibitory effect was also observed for the uptake of labelled adenosine, uridine, hypoxanthine, and adenine, but not for ribose. The inhibition was not readily reversible and was noncompetitive in nature. It was not secondary to the contracture of the muscle produced by the drug, because various treatments known to cause contracture had no effect on inosine transport. Also, papaverine (0.3 mM) significantly inhibited inosine transport without affecting the contractile properties of the muscle. Although theophylline is a cyclic AMP phosphodiesterase inhibitor, no relation could be found between inhibition of inosine uptake and cyclic AMP. N8,O2′-Dibutyryl cyclic AMP (1 mM) was ineffective. Though isoproterenol (10 μg/ml) increased the cyclic AMP concentrations in the muscle by 26-fold in the presence of theophylline and 3-fold in the absence of the drug, it did not influence inosine transport. Tracing the label into various intracellular nucleotides after incubation of the muscle with [14C]inosine suggested that theophylline inhibited inosine transport rather than inosine metabolism.

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

AJP Renal Physiology ◽

10.1152/ajprenal.1986.250.1.f103 ◽

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.

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Adenosine potentiates lutropin-stimulated cyclic AMP production and inhibits lutropin-induced desensitization of adenylate cyclase in rat Leydig tumour cells

Biochemical Journal ◽

10.1042/bj2300211 ◽

1985 ◽

Vol 230 (1) ◽

pp. 211-216 ◽

Cited By ~ 7

Author(s):

C J Dix ◽

A D Habberfield ◽

B A Cooke

Keyword(s):

Cyclic Amp ◽

Adenylate Cyclase ◽

Phosphodiesterase Inhibitor ◽

Tumour Cells ◽

Nucleoside Transport ◽

Adenosine Uptake ◽

Nucleoside Transport Inhibitor ◽

Phenylisopropyl Adenosine ◽

Kinetics Of ◽

Dose Dependent

The action of adenosine on lutropin (LH)-stimulated cyclic AMP production and LH-induced desensitization of adenylate cyclase in rat Leydig tumour cells was investigated. Adenosine and N6-(phenylisopropyl)adenosine caused a dose-dependent potentiation of LH-stimulated cyclic AMP production at concentrations (0.01-10 microM) which alone did not produce an increase in cyclic AMP production. However, 2-deoxyadenosine had no effect either alone or in combination with LH on cyclic AMP production. The potentiation produced by adenosine was unaffected by concentrations of the specific nucleoside-transport inhibitor dipyridamole, which inhibited [3H]adenosine uptake by up to 90%. The phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine, but not RO-10-1724, inhibited the adenosine-induced potentiation. In the presence of adenosine, the kinetics of LH-stimulated cyclic AMP production were linear with time up to 2h, compared with those with LH alone, which showed a characteristic decrease in rate of cyclic AMP production after the first 15-20 min. Consistent with the altered kinetics, adenosine also inhibited the LH-induced desensitization of adenylate cyclase. These results suggest that adenosine has effects on rat tumour Leydig cells through receptors on the external surface of the plasma membrane. This receptor has characteristics similar to those of the R-type receptors, which have been shown either to stimulate or to inhibit adenylate cyclase. However, the effects of adenosine in the present studies does not involve a direct inhibition or activation of adenylate cyclase, but may involve an as yet undefined receptor-mediated modulation of adenylate cyclase.

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Forskolin and phosphodiesterase inhibitors release adenosine but inhibit morphine-evoked release of adenosine from spinal cord synaptosomes

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y91-133 ◽

1991 ◽

Vol 69 (6) ◽

pp. 877-885 ◽

Cited By ~ 4

Author(s):

D. Nicholson ◽

T. D. White ◽

J. Sawynok

Keyword(s):

Spinal Cord ◽

Cyclic Amp ◽

Phosphodiesterase Inhibitor ◽

Phosphodiesterase Inhibitors ◽

Dorsal Spinal Cord ◽

Adenosine Release ◽

Basal Release ◽

Phosphodiesterase Isoenzymes ◽

Dorsal Spinal ◽

Ventral Spinal Cord

The effects of forskolin, Ro 20-1724, rolipram, and 3-isobutyl-1-methylxanthine (IBMX) on morphine-evoked release of adenosine from dorsal spinal cord synaptosomes were evaluated to examine the potential involvement of cyclic AMP in this action of morphine. Ro 20-1724 (1–100 μM), rolipram (1–100 μM), and forskolin (1–10 μM) increased basal release of adenosine, and at 1 μM inhibited morphine-evoked release of adenosine. Release of adenosine by Ro 20-1724, rolipram, and forskolin was reduced 42–77% in the presence of α, β-methylene ADP and GMP, which inhibits ecto-5′-nucleotidase activity by 81%, indicating that this adenosine originated predominantly as nucleotide(s). Significant amounts of adenosine also were released from the ventral spinal cord by these agents. Ro 20-1724 and rolipram did not significantly alter the uptake of adenosine into synaptosomes. Although Ro 20-1724 and rolipram had only limited effects on the extrasynaptosomal conversion of added cyclic AMP to adenosine, IBMX, a phosphodiesterase inhibitor with a broader spectrum of inhibitory activity for phosphodiesterase isoenzymes, significantly inhibited the conversion of cyclic AMP to adenosine and resulted in recovery of a substantial amount of cyclic AMP. As with the non-xanthine phosphodiesterase inhibitors, IBMX increased basal release of adenosine and reduced morphine-evoked release of adenosine. Adenosine released by IBMX was reduced 70% in the presence of α, β-methylene ADP and GMP, and release from the ventral spinal cord was 61% of that from the dorsal spinal cord. Collectively, these results indicate that forskolin and phosphodiesterase inhibitors release nucleotide(s) which is (are) converted extrasynaptosomally to adenosine. For forskolin, Ro 20-1724, and rolipram, the nucleotide released could be cyclic AMP. Morphine releases adenosine per se, and forskolin and phosphodiesterase inhibitors reduce this release. The lack of increase in the action of morphine with phosphodiesterase inhibitors in particular does not support a role for stimulation of cyclic AMP production by morphine in the release of adenosine. The reduction in morphine-evoked release of adenosine by forskolin and phosphodiesterase inhibitors suggests either (a) that a reduction in cyclic levels by morphine promotes adenosine release, or (b) that cyclic AMP interferes with the release process.Key words: forskolin, Ro 20-1724, 3-isobutyl-1-methylxanthine, cyclic AMP, morphine, adenosine release, spinal cord.

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Heat treatment induces an increase in intracellular cyclic AMP content in human epidermoid A-431 cells

Biochemical Journal ◽

10.1042/bj2760683 ◽

1991 ◽

Vol 276 (3) ◽

pp. 683-689 ◽

Cited By ~ 19

Author(s):

J G Kiang ◽

Y Y Wu ◽

M C Lin

Keyword(s):

Heat Treatment ◽

Cyclic Amp ◽

Adenylate Cyclase ◽

G Proteins ◽

Phosphodiesterase Inhibitors ◽

Metabolic Inhibitors ◽

Thermally Induced ◽

Heat Treated ◽

Atp Breakdown ◽

In Cells

The basal level of intracellular cyclic AMP (cAMPi) in A-431 cells incubated at 37 degrees C in Na(+)-containing Hanks solution is 2086 +/- 139 fmol/10(6) cells. When cells are exposed to 45 degrees C for 10 min, cAMPi increases by 40 +/- 4%, and then returns to basal levels within 30 min. Incubating cells in Ca(2+)-free or Mg(2+)-free Hanks solution has no effect on the heat-induced increase in cAMPi, but the increase is inhibited by acid-loading cells to intracellular pH 7.0 or 6.8. In unheated cells, cAMPi increases by 16 +/- 8%, 53 +/- 7%, or 39 +/- 8%, when incubated with isobutyl-1-methylxanthine (1 mM), Ro 20-1724 (0.5 mM), or theophylline (1 mM) respectively. However, heat treatment further elevates cAMPi in cells treated with phosphodiesterase inhibitors, indicating that heat treatment and phosphodiesterase inhibitors elevate cAMPi by a different pathway(s). Heat treatment increases adenylate cyclase activity 2.5-fold. When forskolin (150 microM), an adenylate cyclase stimulator, is applied to cells, the basal cAMPi increases 28 +/- 6-fold compared with controls. Subsequent heating of these cells lowers cAMPi levels to 7.0 +/- 0.5 times that in control cells. This decrease is prevented by pretreatment with pertussis toxin (30 ng/ml, 24 h), suggesting that G-proteins are involved in the process of heat-induced cAMPi increase. 2-Deoxy-D-glucose (10 mM), NaN3 (10 mM) and 2,4-dinitrophenol (1 mM) also increase cAMPi in A-431 cells. However, application of these metabolic inhibitors to cells before heat treatment does not result in cAMPi levels greater than that observed in cells with heat alone. Similar observations are obtained in heat-treated cells previously exposed to adenosine, but not to AMP or ADP. These data are the first to suggest that thermally induced increase in cAMPi is due to a combination of activation of adenylate cyclase and G-proteins, and an increase in adenosine owing to ATP breakdown caused by hyperthermia.

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