Involvement of Adenosine-Sensitive Cyclic AMP-Generating Systems in Cobalt-Induced Epileptic Activity in the Rat

An injection of cobalt chloride solution into the unilateral sensorimotor cortex of rats induced electrographic epileptic activity, which was followed by a peripheral motor disturbance. Brain slices were prepared from the cortical region including the injection site and from the other cortical regions of rats between 8 and 50 days after the injection. In the cortical slices, we examined cyclic AMP accumulations elicited by adenosine and its stable analogue 2-chloroadenosine. Adenosine and 2-chloroadenosine at their maximal dose increased cyclic AMP accumulation six- to 10-fold and 10–15-fold, respectively, and the elicitation was markedly inhibited by the adenosine antagonist 8-phenyltheophylline. The cyclic AMP accumulation was increased in the primary epileptic region of the cortex adjacent to the injection site of cobalt chloride solution, whereas it was unchanged in the other cortical regions. The increase in cyclic AMP accumulation was observed regardless of the presence or absence of the adenosine uptake inhibitor dipyridamole, the phosphodiesterase inhibitor DL-4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone, and adenosine deaminase. Such an increased accumulation of cyclic AMP in the primary epileptic cortex was detected as early as 8 days after the injection. The cyclic AMP accumulation continued to increase and reached a peak level 17–19 days after the injection, and it returned to the control levels after 40–50 days, in correspondence with the electrographic and behavioral findings. It is concluded that alterations in adenosine receptormediated generation of cyclic AMP in the primary epileptic cortex are closely associated with the central process of cobalt-induced epilepsy. In general, the adenosine-sensitive cyclic AMP-generating systems may serve as a common mechanism in experimental models of epilepsy.     

Regional Difference in Responsiveness of Norepinephrine-Sensitive Cyclic AMP-Generating Systems of Rat Cerebral Cortex with Iron-Induced Epileptic Activity

Responsiveness of norepinephrine (NE)-sensitive cyclic AMP (cAMP)-generating systems was determined in slices from different areas of the rat cerebral cortex in which FeCl2 solution was injected unilaterally into the sensorimotor cortex to induce epileptic activity. In anterior cortical areas of rats in which the appearance of electrographic isolated spikes was dominant either ipsilaterally or contralaterally to the injection site 8-10 days after the injection, the cAMP accumulations elicited by NE and an NE-phentolamine combination were greater on the side of dominant spike activity than on the other. In anterior cortical areas of rats showing dominant spike activity on either side of the cortex 31-60 days after the injection, the cAMP accumulation elicited by NE was smaller on the dominant side than on the other. In anterior cortical areas of rats showing nearly equal spike activity on the two sides 31-60 days after the injection, the cAMP accumulations elicited by NE and an NE-phentolamine combination were greater on the side ipsilateral to the injection site than on the other. In anterior and posterior cortical areas of rats in which the appearance of spike and wave complexes, as well as isolated spikes, was detected 31-60 days after the injection, the cAMP accumulations elicited by NE and combinations of NE and phentolamine or propranolol were greater on the side ipsilateral to the injection site than on the other. The elicitation by an NE-propranolol combination, but not by an NE-phentolamine combination, of cAMP accumulation was almost completely inhibited by 8-phenyltheophylline.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of adenosine receptor-mediated generation of cyclic AMP in slices of rat cerebral cortex with chronic epileptic activity

Cyclic AMP accumulations elicited by adenosine analogues 2-chloroadenosine (2-CADO),R-N ⁶-phenylisopropyladenosine (R-PIA), andN ⁶-cyclohexyladenosine (CHA) were investigated in cortical slices of chronic iron-induced epileptic rats. Cyclic AMP accumulation was elicited 9-to 18-fold by 2-CADO and it was elicited 5-to 7-fold by eitherR-PIA or CHA; 2-CADO was more potent thanR-PIA or CHA in eliciting cyclic AMP accumulation. The adenosine analogues elicited cyclic AMP accumulation in a dose-dependent manner, and the elicitation was inhibited by the adenosine antagonist 8-phenyltheophylline. The 2-CADO-elicited accumulation of cyclic AMP was greatly increased in the cortical region on the primary epileptic side, while theR-PIA-or CHA-elicited accumulation did not change in any cortical region. The deviation detected only in the 2-CADO-elicited accumulation of cyclic AMP may be due to the difference in relative potency for adenosine receptors of the adenosine analogues. The results suggest that adenosine receptormediated generation of cyclic AMP is altered in the primary region of iron-induced epileptic cortex, in which heterogeneous alterations in different adenosine receptor subtypes may occur in the epileptic process.     

Differential inhibition of cyclic AMP and cyclic GMP hydrolysis in rat renal cortex.

Adenosine 3′,5′-monophosphate (cyclic AMP) and guanosine 3′,5′-monophosphate (cyclic GMP) metabolism in rat renal cortex was examined. Athough the cyclic AMP and cyclic GMP phosphodiesterases are similarly distributed between the soluble and particulate fractions following differential centrifugation, their susceptibility to inhibition by theophylline, dl-4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro 20-1724), and 1-methyl-3-isobutylxanthine (MIX) are quite different. Ro 20-1724 selectively inhibited both renal cortical-soluble and particulate cyclic AMP degradation, but had little effect on cyclic GMP hydrolysis. Theophylline and MIX effectively inhibited degradation of both cyclic nucleotides, with MIX the more potent inhibitor. Effects of these agents on the cyclic AMP and cyclic GMP content of cortical slices corresponded to their relative potency in broken cell preparations. Thus, in cortical slices, Ro 20-1724 (2 mm) had the least effect on basal (without agonist), carbamylcholine, and NaN₃-stimulated cyclic GMP accumulation, but markedly increased basal and (parathyroid hormone) PTH-mediated cyclic AMP accumulation, MIX (2 mm) which was as effective as Ro 20-1724 in potentiating basal and PTH-stimulated increases in cyclic AMP also mediated the greatest augmentation of basal, carbamylcholine, and NaN₃-stimulated accumulation of cyclic GMP. By contrast, theophylline (10 mm) which was only 12% as effective as Ro 20-1724 in increasing the total slice cyclic AMP content in the presence of PTH was much more effective than Ro 20-1724 in potentiating carbamylcholine and NaN₃-mediated increases in cyclic GMP. These results demonstrate selective inhibition of cyclic nucleotide phosphodiesterase activities in the rat renal cortex and support the possibility of multiple cyclic nucleotide phosphodiesterases in this tissue. Furthermore, both cyclic nucleotides appear to be rapidly degraded in the renal cortex.     

Adenosine-Elicited Accumulation of Adenosine 3'', 5''-Cyclic Monophosphate in the Chick Embryo Retina

The cyclic AMP level of 17-day-old chick embryo retina increased from 20 to 331 pmol/mg protein when the tissue was incubated for 20 min in the presence of 4-(3-butoxy-4-methoxybenzyl-2-imidozolinone) (RO 20-1724). The addition of 0.5 mM-3-isobutyl-1-methylxanthine (IBMX) or 0.5 units/ml of adenosine deaminase (EC 3.5.4.4) to the medium reduced the increase of cyclic AMP content from 20 to 100 pmol/mg protein. Dipyridamole did not interfere with the rise of the retinal cyclic AMP level observed with RO 20-1724. The EC50 of 6-amino-2-chloropurine riboside (2-chloroadenosine)-elicited accumulation of cyclic AMP of retinas incubated in the presence of RO 20-1724 plus adenosine deaminase was approximately 1 microM. When retina incubation was carried out in the presence of 0.5 mM-IBMX, the 2-chloroadenosine dose-response curve was shifted to the right two orders of magnitude. Maximal stimulation of the cyclic AMP level of 17-day-old chick embryo retina incubated in the presence of 0.5 mM-IBMX was observed at 1 mM-adenosine concentration. This effect was not blocked by dopamine antagonists. Guanosine and adenine did not affect the retinal cyclic AMP level. AMP and ATP had a slight stimulatory effect. Adenosine response of embryonic retina increased sharply from the 14th to the 17th embryonic day. A similar, but not identical adenosine effect was observed in cultured retina cells.     

Inhibition of 3',5'-nucleotide phosphodiesterase in guinea pig cerebral cortical slices

Several compounds have been tested for their activity as inhibitors of 3′,5′-nucleotide phosphodiesterase in brain cortical slices from guinea pig. SQ 20,009 (1-ethyl-4-isopropylidenehydrazino)-1H-pyrazolo (3,4-b)pyridine-5-carboxylate, ethylester, hydrochloride), a very potent inhibitor of 3′,5′-nucleotide phosphodiesterase from rat and rabbit brain shows only moderate activity as 3′,5′-nucleotide phosphodiesterase inhibitor when tested in brain slices. It enhances cyclic AMP accumulation only when slices are stimulated by histamine. It does not affect cyclic AMP levels when histamine/norepinephrine are used as stimuli of cyclic AMP formation and decreases the activity of adenosine as stimulant slightly. Ro 20–1724 (4-(3-butoxy-4-methoxy)-2-imidazolidinone) a potent inhibitor of canine cerebral cortex PDE activity effectively augments the increase in cyclic AMP under all stimulating conditions mentioned, as does to a somewhat smaller extent the more water soluble Ro 20–2926 (4-(3-ethoxy-ethoxy-4-methoxy)-2-imidazolidinone). Dose-response curves for Ro 20–1724 under three stimulating conditions of increased cyclic AMP formation (0.1 mm histamine, 0.1 mm histamine/0.1 mm norepinephrine, 0.1 mm adenosine) yield an ED₅₀ of about 20 μm in all instances. A significant increase over respective controls is seen even at 1 μm Ro 20–1724 (histamine/norepinephrine). The drugs may be useful as tools for studying the regulation of cyclic AMP levels in the central nervous system.     

Expression of A1 Adenosine Receptors Modulating Dopamine-Dependent Cyclic AMP Accumulation in the Chick Embryo Retina

Dopamine and 2-chloroadenosine independently promoted the accumulation of cyclic AMP in retinas from 16-day-old chick embryos. The two compounds added together either in saturating or subsaturating concentrations were not additive for the accumulation of the cyclic nucleotide in the tissue. This fact was shown to be due to the existence of an adenosine receptor that mediates the inhibition of the dopamine-dependent cyclic AMP accumulation in the retina. Adenosine inhibited, in a dose-dependent fashion, the accumulation of cyclic AMP induced by dopamine in 12-day-old chick embryo retinas, with an IC50 of approximately 1 microM. This effect was not blocked by dipyridamole. N6-(l-Phenylisopropyl)adenosine, (l-PIA) was the most potent adenosine analog tested, showing an IC50 of 0.1 microM which was two orders of magnitude lower than its stereoisomer d-PIA (10 microM). The maximal inhibition of the dopamine-elicited cyclic AMP accumulation by adenosine and related analogs was 70%. The inhibitory effect promoted by adenosine was blocked by 3-isobutyl-1-methylxanthine (IBMX) or by adenosine deaminase. Adenine was not effective; whereas ATP and AMP promoted the inhibition of the dopamine effect only at very high concentrations. Apomorphine was only 30% as effective as dopamine in promoting the cyclic AMP accumulation in retinas from 11- to 12-day-old embryos and 2-chloroadenosine did not interfere with the apomorphine-mediated shift in cyclic AMP levels. In the retinas from 5-day-old posthatched chickens dopamine and apomorphine were equally effective in eliciting the accumulation of cyclic AMP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of adenosine 3':5'-monophosphate-dependent protein kinase in cerebral cortex

Alterations in the cyclic AMP-dependent protein kinase activity ratio in response to putative neurotransmitters and other cyclic AMP-elevating agents in intact cerebral cortical slices and Krebs-Ringer particulate preparations from cerebral cortex were examined. Both norepinephrine (30 microM) and forskolin (20 microM) produced a time-dependent increase in intracellular levels of cyclic AMP in cerebral cortical slices which was paralleled by an increase in both cyclic AMP and the protein kinase activity ratio. The increases were maximal at 5 min. and remained elevated for at least 15 min. Forskolin, norepinephrine, adenosine and isoproterenol produced a concentration-dependent increase in both cyclic AMP and the protein kinase activity ratio, however, the degree of increase observed was dissimilar. Thus, a 5-fold change in intracellular cyclic AMP resulted in only a 2-fold increase in the activity ratio. Of the agents examined, forskolin produced the most marked change in the activity ratio (from 0.23 to 0.78 at 100 microM) while isoproterenol at 100 microM produced only a 50% increase in the activity ratio. The half-time for the decline in forskolin elicited elevations of either the activity ratio or cyclic AMP was about 4-6 min. In the presence of the phosphodiesterase inhibitor, Ro 20-1724, both were significantly prolonged being 60-70% of the maximum observed immediately after forskolin stimulation, at 15 min. Potentiation of forskolin elicited increases in the activity ratio by Ro 20-1724 were also observed but the increase in the activity ratio was maximal at 7.5 min. while cyclic AMP accumulations continued to rise during the entire 15 min. incubation. Particulate preparations from cerebral cortex were found to contain a cyclic AMP-dependent protein kinase which could be activated 2 to 3-fold with either forskolin, norepinephrine, or adenosine. Unlike the intact brain slice the changes in protein kinase activity ratio and intracellular levels of cyclic AMP in cell-free particulate preparations were similar in both time and degree.     

Change of Cyclic AMP Level in Synaptosomes from Cerebral Cortex; Increase by Adenosine Derivatives

The endogenous level of cyclic AMP in incubated synaptosomes from cerebral cortex of guinea pigs was investigated after the addition of various agents to the incubation medium. It appeared that the synaptosomal suspension already contained exogenous adenosine. Preincubation with theophylline or with adenosine deaminase (ADase) decreased both the exogenous level of adenosine and the intrasynaptosomal level of cyclic AMP. The level of cyclic AMP was reincreased by the addition of adenosine agonists, especially 2-chloroadenosine. This increase was antagonized by deoxyadenosine and was not inhibited by dipyridamole. These results suggest that the adenosine derivatives in the synaptic cleft regulate the level of cyclic AMP in nerve terminals through adenosine receptor on the presynaptic membrane. ADP, ATP, dopamine, and histamine also stimulate the formation of cyclic AMP in the ADase-treated synaptosomes.     

Adenosine-elicited accumulation of cyclic AMP in brain slices: potentiation by agents which inhibit uptake of adenosine

The uptake and incorporation of low concentrations of radioactive adenosine into guinea pig cerebral cortical slices is effectively inhibited by dipyridamole, hexobendine, papaverine, 6-($\text{p}$-nitrobenzylthio) guanosine, 5′-deoxy-adenosine and N⁶-phenyladenosine and ineffectively inhibited by other adenosine analogs such as 2-chloroadenosine, 3′-deoxyadenosine and tubercidin or by phosphodiesterase inhibitors such as theophylline, isobutylmethylxanthine, and N, 0-dibutyrylcyclic AMP. When uptake of 10–20

adenosine is inhibited 50–70% by dipyridamole, hexobendine, papaverine or 6-($\text{p}$-nitrobenzylthio)-guanosine, the adenosine-elicited accumulation of cyclic AMP is potentiated 2–3 fold. Potentiation of the effects of low concentrations of adenosine by various agents parallels more closely their efficacy as inhibitors of adenosine uptake rather than their potency as phosphodiesterase inhibitors. Amine-elicited accumulations of cyclic AMP are enhanced by hexobendine, dipyridamole, papaverine and 6-($\text{p}$-nitrobenzylthio) guanosine and this enhancement is blocked by an adenosine antagonist, theophylline. The stimulatory effects of the adenosine analogs, 5′-deoxyadenosine, 2-chloroadenosine and N⁶-phenyladenosine are blocked by theophylline and potentiated by hexobendine. The results are compatible with the hypothesis that the specific inhibition of uptake of adenosine potentiates adenosine or amine-elicited accumulations of cyclic AMP by increasing the effective extracellular concentration of adenosine within the slice. The inhibition or stimulation of cyclic AMP accumulation by adenosine analogs is consonant with differential activities as agonist or antagonist at an extracellular adenosine receptor.     

Adenosine Receptors Mediating Cyclic AMP Productioin the Rat Hippocampus

In the transversely cut rat hippocampus, adenosine caused a dose-dependent increase in the accumulation of [3H]cyclic AMP from [3H]ATP. Adenosine breakdown products were inactive. AMP was somewhat less effective than adenosine, and its effect could be partially, but not completely, abolished by alpha, beta-methylene-ADP and GMP, which inhibited its metabolism by 5'-nucleotidase. The effect of adenosine was unaffected by inhibitors of adenosine deaminase, but enhanced by several inhibitors of adenosine uptake. Some analogues of adenosine, including N6-phenylisopropyladenosine (PIA), 2-chloroadenosine and adenosine 5'-ethylcarboxamide (NECA), were more active than adenosine, whereas others such as 2-deoxyadenosine and 9-(tetrahydro-2-furyl)adenine (SQ 22536) actually inhibited the response. The effect of PIA was highly stereospecific. The action of adenosine was inhibited by several alkylxanthines, the most potent of which was 8-phenyltheophylline. [3H]Cyclohexyladenosine (CHA) bound specifically to cell membranes from the rat hippocampus. The extent of binding was similar to that found in other cortical areas. The relative potency of some adenosine analogues and alkylxanthines to displace labelled CHA was essentially similar to their potency as effectors of the cyclic AMP system. Adenosine contributed to the cyclic AMP-elevating effect of alpha-adrenoceptor-stimulating drugs and several amino acids, but not to that seen with isoprenaline. The cyclic AMP increase seen following depolarization was only partially adenosine-dependent. The present results demonstrate that the rat hippocampus contains adenosine receptors mediating cyclic AMP accumulation and that these receptors have similar characteristics to those mediating pyramidal cell depression. Adenosine-induced cyclic AMP accumulation may be used as a biochemical correlate to electrophysiology and as a convenient parameter to assess the influence of drugs on adenosine mechanisms in the rat hippocampus.     

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

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 microM), rolipram (1-100 microM), and forskolin (1-10 microM) increased basal release of adenosine, and at 1 microM inhibited morphine-evoked release of adenosine. Release of adenosine by Ro 20-1724, rolipram, and forskolin was reduced 42-77% in the presence of alpha,beta-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 alpha,beta-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.(ABSTRACT TRUNCATED AT 250 WORDS)     

An Investigation of the Low Intrinsic Activity of Adenosine and Its Analogs at Low Affinity (A2) Adenosine Receptors in Rat Cerebral Cortex

The potencies and intrinsic activities of adenosine analogs for stimulating cyclic AMP accumulation in slices of rat cerebral cortex were examined. 5'-N-Ethylcarboxamidoadenosine (NECA) caused the greatest increase in cyclic AMP accumulation (19.2-fold). 2-Chloroadenosine (2-CAD) induced a similar increase, but adenosine and six other analogs caused much smaller increases. All agonists tested had similar potencies in activating this response. Inhibition of adenosine uptake with 10 microM dipyridamole did not affect the maximal response to any agonist, although the potency of adenosine was increased approximately threefold. Each analog was also able to block partially the stimulation of cyclic AMP accumulation caused by NECA. Levels of cyclic AMP accumulation in the presence of NECA plus another analog were similar to those observed when the analog alone was present, as expected for partial agonists. Furthermore, the EC50 value for R-(-)-N6(2-phenylisopropyl)adenosine in increasing cyclic AMP accumulation was similar to the KI value for inhibiting the response to NECA. The EC50 value for adenosine was substantially higher than the KI value for inhibiting the response to NECA; however, in the presence of dipyridamole, the two values were more closely correlated. The response to NECA was blocked by 8-phenyltheophylline, 1,3-diethyl-8-phenylxanthine, and 8-p-sulfophenyltheophylline, with KI values from 1 to 10 microM. The results suggest that adenosine analogs stimulate cyclic AMP accumulation in cerebral cortex through low-affinity receptors, but that some analogs only partially activate these receptors. Adenosine itself may also be a partial agonist, or its actions may be obscured by simultaneous activation of another receptor.     

Regulation of Depolarization-Dependent Release of Neurotransmitters by Adenosine: Cyclic AMP-Dependent Enhancement of Release from PC12 Cells

We have used pheochromocytoma cells, clone PC12, as a model system for studying the effects of adenosine on neurosecretion. Exposure of the cells to adenosine or 2-chloroadenosine caused immediate activation of adenylate cyclase, increases in cellular cyclic AMP content, and inhibition of SAM-dependent phospholipid N-methylation and protein carboxymethylation. However, the effects on methylation were only observed with concentrations of adenosine 100 times greater than those that elevated cyclic AMP. Exposure of the cells to adenosine and 2-chloroadenosine did not alter the release of [3H]norepinephrine [(3H]NE) in the absence of depolarization. However, depolarization-dependent release of [3H]NE was markedly elevated by short (1-20 min) pretreatments with adenosine or 2-chloroadenosine. The enhancement of release was observed irrespective of the nature of the depolarizing stimulus (elevated K+, carbamylcholine, or veratridine). Release of [3H]acetylcholine in response to elevated K+ also was increased by adenosine pretreatment. These effects of adenosine and 2-chloroadenosine on neurotransmitter release closely paralleled elevation of cellular cyclic AMP but not inhibition of methylation. Taken together, the results show that adenosine, probably acting through adenosine receptors coupled to stimulation of adenylate cyclase, is able to modulate the neurosecretory process in PC12 cells. Furthermore, the enhancement of release occurred even though the extent of depolarization (measured as 86Rb+ flux through the acetylcholine receptor channel) and the amount of 45Ca2+ which entered upon depolarization were unchanged. Therefore, the enhancement of release produced by elevated cyclic AMP appeared to reflect increased efficiency of the stimulus-secretion coupling process.     

Effects of exogenous adenosine deaminase and erythro-9-(2-hydroxy-3-nonyl) adenine on intracellular cyclic AMP levels in C1300 murine neuroblastoma in tissue culture

The cyclic AMP content of dense cultures of C1300 murine neuroblastoma cells (clone N2a) was elevated after incubation for short periods of time in minimal volumes of serum-free medium (SFM) containing Ro 20 1724, a potent nonxanthine phosphodiesterase inhibitor. This elevation was prevented by theophylline, an adenosine antagonist, and was retarded by dipyridamole or benzylthioinosine, inhibitors of nucleoside transport. Cyclic AMP was also elevated by erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), a potent adenosine deaminase inhibitor. This effect of EHNA was more pronounced in dense cultures, in small volumes of bathing medium, and was antagonized by dipyridamole. The addition of adenosine deaminase to growth medium or SFM lowered the cyclic AMP levels attained after the addition of Ro 20 1724. We conclude that N2a cells continually release adenosine into the growth or bathing medium $\text{via}$ the nucleoside transport system and that sufficient concentrations may be achieved to tonically stimulate adenylate cyclase and influence processes controlled by the cyclic AMP:cyclic AMP-dependent protein kinase system.     

Cyclic nucleotide hydrolysis in the thyroid gland. General properties and key role in the interrelations between concentrations of adenosine 3':5'-monophosphate and guanosine 3':5'-monophosphate.

Phosphodiesterase activities of horse (and dog) thyroid soluble fraction were compared with either cyclic AMP (adenosine 3':3'-monophosphate) or cyclic GMP (guanosine 3':5'-monophosphate) as substrate. Optimal activity for cyclic AMP hydrolysis was observed at pH 8, and at pH 7.6 for cyclic GMP. Increasing concentrations of ethyleneglycol bis(2-aminoethyl)-N,N'-tetraacetic acid inhibited both phosphodiesterase activities; in the presence of exogenous Ca2+, this effect was shifted to higher concentrations of the chelator. In a dialysed supernatant preparation, Ca2+ had no significant stimulatory effect, but both Mg2+ and Mn2+ increased cyclic nucleotides breakdown. Mn2+ promoted the hydrolysis of cyclic AMP more effectively than that of cyclic GMP. For both substrates, substrate velocity curves exhibited a two-slope pattern in a Hofstee plot. Cyclic GMP stimulated cyclic AMP hydrolysis, both nucleotides being at micromolar concentrations. Conversely, at no concentration had cyclic AMP any stimulatory effect on cyclic GMP hydrolysis. 1-Methyl-3-isobutylxanthine and theophylline blocked the activation by cyclic GMP of cyclic GMP of cyclic AMP hydrolysis, whereas Ro 20-1724 (4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone), a non-methylxanthine inhibitor of phosphodiesterases, did not alter this effect. In dog thyroid slices, carbamoylcholine, which promotes an accumulation of cyclic GMP, inhibits the thyrotropin-induced increase in cyclic AMP. This inhibitory effect of carbamoylcholine was blocked by theophylline and 1-methyl-3-isobutylxanthine, but not by Ro 20-1724. It is suggested that the cholinergic inhibitory effect on cyclic AMP accumulation is mediated by cyclic GMP, through a direct activation of phosphodiesterase activity.     

Cyclic AMP-Generating Systems in Cell-Free Preparations from Guinea Pig Cerebral Cortex: Loss of Adenosine and Amine Responsiveness Due to Low Levels of Endogenous Adenosine

The accumulations of radioactive cyclic AMP elicited by adenosine, norepinephrine, and histamine in adenine-labeled vesicular entities of a particulate fraction from guinea pig cerebral cortex are greatly reduced as a result of prolonged preincubation. The presence of adenosine deaminase during preincubations largely prevents the loss of adenosine, norepinephrine and histamine responses. Adenosine deaminase was inactivated by deoxycoformycin prior to stimulation of cyclic AMP accumulation by adenosine or amines. If adenosine deaminase is not inactivated, responses to norepinephrine are not significant and histamine responses are reduced by 50%. Adenosine deaminase cannot restore responsiveness of the cyclic AMP-generating systems. It is proposed that, in particulate fractions of guinea pig cerebral cortex, low levels of adenosine cause a slow loss of receptors and/or coupling of receptors to cyclic AMP-generating systems.     

Negative control of TSH action by iodide and acetylcholine: mechanism of action in intact thyroid cells.

Iodide, a substrate of thyroid metabolism, and acetylcholine depress cyclic AMP intracellular content and secretion in dog thyroid slices under TSH stimulation. A direct or indirect pseudocompetitive effect at the level of TSH receptor interaction has been rejected. Iodide and carbachol, both inhibited cyclic AMP accumulation in TSH stimulated dog thyroid slices but only the effect of carbachol was suppressed in the presence of isobutylmethylanthine. Ro 20-1724 did not relieve either inhibitory effect. Carbachol greatly enhanced cyclic AMP disposal in TSH prestimulated slices after the cut off of hormone action by a trypsin treatment. This effect was also suppressed by isobutylmethylxanthine but not by Ro 20-1724. No action of iodide could be evidenced on cyclic AMP disposal in similar slices, although a clear effect after the same time of iodide action was observed on cyclic AMP accumulation. Neither carbachol, nor iodide depresses ATP levels in these slices. The data suggest that carbachol exerts its action through an activation of cyclic AMP disappearance probably by an activation of cyclic AMP phosphodiesterase and that iodide, through an oxidized intermediate, experts its inhibitory effect at the level of cyclic AMP synthesis.     

Role of Extracellular Adenosine in Ethanol-Induced Desensitization of Cyclic AMP Production

Abstract: The decrease in receptor-stimulated cyclic AMP production after chronic ethanol exposure was suggested previously to be secondary to an ethanol-induced increase in extracellular adenosine. The present study was undertaken to ascertain whether a similar mechanism was responsible for the ethanol-induced desensitization of cyclic AMP production in PC12 pheochromocytoma cells. The acute addition of ethanol in vitro significantly increased both basal cyclic AMP content and extracellular levels of adenosine. A 4-day exposure to ethanol decreased basal as well as 2-chloroadenosine- and forskolin-stimulated cyclic AMP contents. No change in cyclic AMP content was observed after a 2-day exposure of PC12 cells to ethanol. Inclusion of adenosine deaminase during the chronic ethanol treatment significantly decreased extracellular levels of adenosine, yet the percentage decrease in 2-chloroadenosine- and forskolin-stimulated cyclic AMP levels after chronic ethanol exposure was not changed by the inclusion of the adenosine deaminase. Similar results were obtained when the chronic treatment was carried out with serum-free defined media. The ethanol-induced desensitization could not be mimicked by chronic exposure of PC12 cells to adenosine analogues. A 24-h exposure of PC12 cells to 2-chloroadenosine resulted in a decrease in the subsequent ability of this adenosine analogue to stimulate cyclic AMP content, but basal and forskolin-stimulated cyclic AMP levels were increased. Similar results were obtained after a 4-day exposure of PC12 cells to 2-chloroadenosine or 5'- N -ethylcarboxamido-adenosine. The present results indicate that the ethanol-induced decrease in receptor-stimulated cyclic AMP content in PC12 cells is not due to an increase in extracellular adenosine.     

Activation of cyclic AMP-dependent protein kinase and stimulation of protein phosphorylation in response to adenosine in C-1300 murine neuroblastoma

DEAE-cellulose chromatography of the 20,000g supernatant fraction of homogenates of C-1300 murine neuroblastoma (clone N2a) yields one major and two minor peaks of cyclic AMP-dependent protein kinase activity. Assessment of the endogenous activation state of the enzyme(s) reveals that the enzyme is fully activated by the treatment of whole cells with adenosine (10 μM) in the presence of the phosphodiesterase inhibitor Ro 20 1724 (0.7 mM). This treatment produces a large elevation in the cyclic AMP content of the cells. The treatment of whole cells with adenosine alone (1–100 μM) or Ro 20 1724 alone (0.1–0.7 mM) produces minimal elevations in cyclic AMP but nevertheless causes significant activations of cyclic AMP-dependent protein kinase. The autophosphorylation of whole homogenates of treated and untreated cells was studied using [γ-³²P] ATP, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Treatments which activate cyclic AMP-dependent protein kinase selectively stimulate the incorporation of ³²P into several proteins. This stimulation is most prominent in the 15,000-dalton protein band. The addition of cyclic AMP to phosphorylation reactions containing homogenate of untreated cells stimulates the phosphorylation of the same protein bands. These results indicate that adenosine may have regulatory functions through its effect on the cyclic AMP: cyclic AMP-dependent protein kinase system.