Endogenous Adenosine Regulates the Apparent Efficacy of 1-Aminocyclopentyl-1S,3R-Dicarboxylate Inhibition of Forskolin-Stimulated Cyclic AMP Accumulation in Rat Cerebral Cortical Slices

Abstract: In rat cerebral cortical slices, the 1-aminocyclopentyl-1 S ,3 R -dicarboxylate (1 S ,3 R -ACPD) isomer of the selective metabotropic excitatory amino acid agonist ACPD inhibited forskolin-stimulated cyclic AMP (cAMP) accumulation in a concentration-dependent manner with a maximal inhibition of 51 ± 3% and a half-maximally effective concentration of 8.8 ± 3.4 μ M . Similarly, 1 R ,3 S -ACPD inhibited the forskolin response in a concentration-dependent manner, but with an inhibition of 80 ± 5% at 3 μ M . In addition to inhibiting forskolin-stimulated cAMP levels, 1 S ,3 R -ACPD, but not 1 R ,3 S -ACPD, enhanced the cAMP response to A_2b adenosine receptor activation. In the presence of 1.2 U/ml of adenosine deaminase (included to reduce the contribution of endogenous adenosine), the efficacy of 1 S ,3 R -ACPD was increased (88 ± 3% inhibition), but the potency was unchanged. The adenosine receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine also increased the inhibitory effect of 100 μ M 1 S ,3 R -ACPD, from 57 ± 1 to 78 ± 5%. These results indicate that endogenous adenosine plays an important role in regulating the apparent efficacy of 1 S ,3 R -ACPD inhibition of forskolin-stimulated cAMP accumulation in rat cerebral cortical slices and that previous studies in rat hippocampus and hypothalamus in the absence of added adenosine deaminase may have underestimated the efficacy of this compound.     

Olfactory marker protein interacts with adenosine nucleotide derivatives

Olfactory marker protein (OMP) is a genetic signature for mature olfactory receptor neurons (ORNs). Recently, it has been proposed that OMP directly captures odour-induced cAMP to swiftly terminate the olfactory signal transduction to maintain neuronal sensitivity. In the present study, we show that OMP can also interact with other adenosine nucleotides as ATP, ADP and AMP with different affinities. We performed bioluminescent resonant energy transfer (BRET) assay to measure the binding actions of the adenosine nucleotide derivatives in competition to cAMP. Amongst all, ATP showed the bell-shape affinity to OMP in the presence of cAMP; ADP and AMP showed fewer affinities to OMP than ATP. In the absence of cAMP analogues, ATP alone bound to OMP in a dose dependent manner with a lower affinity than to cAMP. Thus, OMP possessed different affinities to ATP in the presence or absence of cAMP. OMP may interact differentially with ATP and cAMP depending on its supply and demand along the cAMP-associated signalling in the limited spaces of cilia of ORNs.     

On the presence of adenosine 3′, 5′ -cyclic monophosphate in moss (Funariahygrometrica)

Partially purified nucleotide fraction of moss containing [14C]-labelled putative adenosine 3′, 5′ -cyclic monophosphate (cAMP) and marker authentic [3H] -cAMP was characterized by chemical deamination and also by the enzymatic hydrolysis with beef heart cyclic nucleotide phosphodiesterase. A significant conversion of marker authentic [3H] -cAMP into [3H] -inosine 3′, 5′ -cyclic monophosphate (cIMP) and [3H] -5′ adenosine monophosphate was observed by respective treatments. In contrast, the [14C] -labelled putative cAMP from control and theophylline-treated moss tissue was insensitive to chemical deamination and enzymatic hydrolysis. Apparently, the [14C] -labelled product which comigrates with authentic [3H] -cAMP does not represent true cAMP. Both the methods employed for characterization of the labelled putative cAMP were sensitive enough to detect picomole quantities of authentic [3H] -cAMP. Lack of detectability of prelabelled [14C] -cAMP in our preparations implies that the tissue may contain authentic cyclic AMP below the picomole levels. Thus, the attributed physiological role to adenosine 3′, 5′ -cyclic monophosphate in moss tissue appears somewhat skeptical.     

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.     

Roles of nucleotide substructures in the regulation of cystathionine β-synthase domain-containing pyrophosphatase

BackgroundRegulatory cystathionine β-synthase (CBS) domains are ubiquitous in proteins, yet their mechanism of regulation remains largely obscure. Inorganic pyrophosphatase which contains regulatory CBS domains as internal inhibitors (CBS-PPase) is activated by ATP and inhibited by AMP and ADP; nucleotide binding to CBS domains and substrate binding to catalytic domains demonstrate positive co-operativity.Methods: Here, we explore the ability of an AMP analogue (cAMP) and four compounds that mimic the constituent parts of the AMP molecule (adenine, adenosine, phosphate, and fructose-1-phosphate) to bind and alter the activity of CBS-PPase from the bacterium Desulfitobacterium hafniense.ResultsAdenine, adenosine and cAMP activated CBS-PPase several-fold whereas fructose-1-phosphate inhibited it. Adenine and adenosine binding to dimeric CBS-PPase exhibited high positive co-operativity and markedly increased substrate binding co-operativity. Phosphate bound to CBS-PPase competitively with respect to a fluorescent AMP analogue.ConclusionsProtein interactions with the adenine moiety of AMP induce partial release of the internal inhibition and determine nucleotide-binding co-operativity, whereas interactions with the phosphate group potentiate the internal inhibition and decrease active-site co-operativity. The ribose moiety appears to enhance the activation effect of adenine and suppress its contribution to both types of co-operativity.General significanceOur findings demonstrate for the first time that regulation of a CBS-protein (inhibition or activation) is determined by a balance of its interactions with different chemical groups of the nucleotide and can be reversed by their modification. Differential regulation by nucleotides is not uncommon among CBS-proteins, and our findings may thus have a wider significance.     

Crucial Positively Charged Residues for Ligand Activation of the GPR35 Receptor

GPR35 is a G protein-coupled receptor expressed in the immune, gastrointestinal, and nervous systems in gastric carcinomas and is implicated in heart failure and pain perception. We investigated residues in GPR35 responsible for ligand activation and the receptor structure in the active state. GPR35 contains numerous positively charged amino acids that face into the binding pocket that cluster in two distinct receptor regions, TMH3-4-5-6 and TMH1-2-7. Computer modeling implicated TMH3-4-5-6 for activation by the GPR35 agonists zaprinast and pamoic acid. Mutation results for the TMH1-2-7 region of GPR35 showed no change in ligand efficacies at the K1.32A, R2.65A, R7.33A, and K7.40A mutants. However, mutation of arginine residues in the TMH3-4-5-6 region (R4.60, R6.58, R3.36, R(164), and R(167) in the EC2 loop) had effects on signaling for one or both agonists tested. R4.60A resulted in a total ablation of agonist-induced activation in both the β-arrestin trafficking and ERK1/2 activation assays. R6.58A increased the potency of zaprinast 30-fold in the pERK assay. The R(167)A mutant decreased the potency of pamoic acid in the β-arrestin trafficking assay. The R(164)A and R(164)L mutants decreased potencies of both agonists. Similar trends for R6.58A and R(167)A were observed in calcium responses. Computer modeling showed that the R6.58A mutant has additional interactions with zaprinast. R3.36A did not express on the cell surface but was trapped in the cytoplasm. The lack of surface expression of R3.36A was rescued by a GPR35 antagonist, CID2745687. These results clearly show that R4.60, R(164), R(167), and R6.58 play crucial roles in the agonist initiated activation of GPR35.     

Adenosine receptor-adenylate cyclase system in the trout testis: involvement in the regulation of germ cell proliferation

To ascertain the presence of adenosine receptors in the trout testis, cells isolated from testes at different spermatogenetic stages were cultured in the presence or absence of adenosine, adenosine receptor agonists, or antagonists and of cAMP analogs, for up to 20 min, or 20 hr, or 4.5 days. Cyclic AMP production was then assayed or 3H-thymidine incorporation was measured. Cellular content of cAMP was enhanced by adenosine, by the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA), and by 2-p(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS-21680), an adenosine A2A receptor-selective agonist. The increase in cAMP induced by the adenylate cyclase activator L-858051 was inhibited by the adenosine A1)receptor-selective agonists R-N6-(2-phenylisopropyl)adenosine (R-PIA) and N6-cyclopentyladenosine (CPA). These effects were antagonized by the two adenosine A2)receptor antagonists 3,7-dimethyl-1-propargylxanthine (DMPX) and 8-(3-chlorostyryl)caffeine (CSC), and by the adenosine A1)receptor-selective antagonist 8-cyclopentyl-1,3dipropylxanthine (CPX), respectively. Increase in the cAMP content induced by adenosine was inhibited by the cell permeable adenylate cyclase inhibitor 2',5'-dideoxyadenosine. These data suggest that A(1) and A(2) adenosine receptors which respectively inhibit and stimulate adenylate cyclase activity are present on trout testicular cells (unidentified), while the presence of A3 adenosine receptor subtype was not apparent. 3H-thymidine incorporation decreased in the presence of the adenylate cyclase activator L-858051 and of the cAMP analogs 8-CPT cAMP and Sp-5,6-DCI-cBiMPS, regardless of the presence or absence of the phosphodiesterase inhibitor RO 20-1724. This suggests that an increase in testicular cAMP may act as a negative growth regulator for the mitotic germ cells. In agreement with these data, the activation of A2 stimulatory receptors inhibited short-term (20 hr) DNA synthesis. However, the activation of A1 inhibitory receptors had the same effect. This suggests that events, cAMP-dependent or independent, induced by the activation of testicular adenosine receptors, may participate in the regulation of trout male germ cell proliferation.     

Adenosine Receptor Activation and the Regulation of Tyrosine Hydroxylase Activity in PC 12 and PC 18 Cells

We compared the response of rat PC12 cells and a derivative PC18 cell line to the effects of adenosine receptor agonists, antagonists, and adenine nucleotide metabolizing enzymes. We found that theophylline (an adenosine receptor antagonist), adenosine deaminase, and AMP deaminase all decreased basal cyclic AMP content and tyrosine hydroxylase activity in the PC12 cells, but not in PC18 cells. Both cell lines responded to the addition of 2-chloroadenosine and 5'-N-ethylcarboxamidoadenosine, adenosine receptor agonists, by exhibiting an increase in tyrosine hydroxylase activity and cyclic AMP content. The latter finding indicates that both cell lines contained an adenosine receptor linked to adenylate cyclase. We found that the addition of dipyridamole, an inhibitor of adenosine uptake, produced an elevation of cyclic AMP and tyrosine hydroxylase activity in both cell lines. Deoxycoformycin, an inhibitor of adenosine deaminase, failed to alter the levels of cyclic AMP or tyrosine hydroxylase activity. This suggests that uptake was the primary inactivating mechanism of adenosine action in these cells. We conclude that both cell types generated adenine nucleotides which activate the adenosine receptor in an autocrine or paracrine fashion. We found that PC12 cells released ATP in a calcium-dependent process in response to activation of the nicotinic receptor. We also measured the rates of degradation of exogenous ATP, ADP, and AMP by PC12 cells. We found that the rates of metabolism of the former two were at least an order of magnitude greater than that of AMP. Any released ATP would be rapidly metabolized to AMP and then more slowly degraded to adenosine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Accumulation of Cyclic AMP Elicited by Vasoactive Intestinal Peptide Is Potentiated by Noradrenaline, Histamine, Adenosine, Baclofen, Phorbol Esters, and Ouabain in Mouse Cerebral Cortical Slices: Studies on the Role of Arachidonic Acid Metabolites and Protein Kinase C

In mouse cerebral cortical slices, noradrenaline (NA) potentiates cyclic AMP (cAMP) accumulation elicited by vasoactive intestinal peptide (VIP) through alpha 1-adrenergic receptors. This synergism is inhibited by indomethacin, and the prostaglandins E2 and F2 alpha mimic the effect of NA. In the present study, we observed that the synergism between VIP and NA is not inhibited by the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) or the diacylglycerol-lipase inhibitor RHC 80267, thus further stressing the role of phospholipase A2 activation. Various neuroactive agents that potentiate the stimulatory effect of VIP on cAMP formation were also examined. As with NA, the potentiation by histamine and adenosine is inhibited by indomethacin. In contrast to NA, histamine, and adenosine, the synergistic interaction between phorbol esters and VIP on cAMP formation is abolished by H-7 but not by indomethacin. The potentiation by baclofen, a gamma-aminobutyric acidB receptor agonist, is partially inhibited by the 5-lipoxygenase inhibitor nafazatrom. The synergism between ouabain and VIP is reduced by H-7 but not by indomethacin and nafazatrom. These data indicate that the stimulation of cAMP formation elicited by VIP is under the modulation of various neuroactive agents that trigger diverse intracellular mechanisms to potentiate the effect of the peptide.     

Two-state allosteric modeling suggests protein equilibrium as an integral component for cyclic AMP (cAMP) specificity in the cAMP receptor protein of Escherichia coli

Activation of the cAMP receptor protein (CRP) from Escherichia coli is highly specific to its allosteric ligand, cAMP. Ligands such as adenosine and cGMP, which are structurally similar to cAMP, fail to activate wild-type CRP. However, several cAMP-independent CRP variants (termed CRP*) exist that can be further activated by both adenosine and cGMP, as well as by cAMP. This has remained a puzzle because the substitutions in many of these CRP* variants lie far from the cAMP-binding pocket (>10 A) and therefore should not directly affect that pocket. Here we show a surprising similarity in the altered ligand specificity of four CRP* variants with a single substitution in D53S, G141K, A144T, or L148K, and we propose a common basis for this phenomenon. The increased active protein population caused by an equilibrium shift in these variants is hypothesized to preferentially stabilize ligand binding. This explanation is completely consistent with the cAMP specificity in the activation of wild-type CRP. The model also predicts that wild-type CRP should be activated even by the lower-affinity ligand, adenosine, which we experimentally confirmed. The study demonstrates that protein equilibrium is an integral factor for ligand specificity in an allosteric protein, in addition to the direct effects of ligand pocket residues.     

Extracellular cAMP inhibits D1 dopamine receptor expression in CAD catecholaminergic cells via A2a adenosine receptors

The expression of D1 dopamine (DA) receptor gene is regulated during development, aging, and pathophysiology. The extracellular factors and signaling mechanisms that modulate the expression of D1 DA receptor have not been well characterized. Here, we present novel evidence that endogenous D1 DA receptor expression is inhibited by extracellular cAMP in the Cath.A Derived (CAD) catecholaminergic neuronal cell line. CAD cells express the multi-drug resistance protein 5 transporters and secrete cAMP. Addition of exogenous cAMP decreases D1 receptor mRNA and protein greater than fourfold in 24 h. The cAMP-induced decrease of D1 receptor mRNA levels is blocked by cGMP and by 1,3-dipropyl-8-(p-sulfo-phenyl)xanthine, an inhibitor of ecto-phosphodiestrase. Extracellular AMP, a metabolite of cAMP, also independently decreased D1 receptor mRNA levels. Inhibitors of ecto-nucleotidases, alpha,beta-methyleneadenosine 5'-di-phosphate and GMP, completely blocked the decrease of D1 receptor mRNA by extracellular cAMP, but only partially blocked the decrease induced by extracellular AMP. Levamisole, an inhibitor of tissue non-specific alkaline phosphatase, completely blocked the AMP-induced decrease of D1 receptor mRNA. The extracellular cAMP, AMP, and adenosine (ADO)-induced decrease in D1 receptor mRNA expression are mediated by A2a ADO receptor subtype. The results suggest a novel molecular mechanism linking activation of A2a ADO receptors with inhibition of D1 DA receptor expression.     

Regulation of Cyclic AMP Formation in Cultures of Human Foetal Astrocytes by β2-Adrenergic and Adenosine Receptors

Two cell cultures, NEP2 and NEM2, isolated from human foetal brain have been maintained through several passages and found to express some properties of astrocytes. Both cell cultures contain adenylate cyclase stimulated by catecholamines with a potency order of isoprenaline greater than adrenaline greater than salbutamol much greater than noradrenaline, which is consistent with the presence of beta 2-adrenergic receptors. This study reports that the beta 2-adrenergic-selective antagonist ICI 118,551 is approximately 1,000 times more potent at inhibiting isoprenaline stimulation of cyclic AMP (cAMP) formation in both NEP2 and NEM2 than the beta 1-adrenergic-selective antagonist practolol. This observation confirms the presence of beta 2-adrenergic receptors in these cell cultures. The formation of cAMP in NEP2 is also stimulated by 5'-(N-ethylcarboxamido)adenosine (NECA) more potently than by either adenosine or N6-(L-phenylisopropyl)adenosine (L-PIA), which suggests that this foetal astrocyte expresses adenosine A2 receptors. Furthermore, L-PIA and NECA inhibit isoprenaline stimulation of cAMP formation, a result suggesting the presence of adenosine A1 receptors on NEP2. The presence of A1 receptors is confirmed by the observation that the A1-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine reverses the inhibition of isoprenaline stimulation of cAMP formation by L-PIA and NECA. Additional evidence that NEP2 expresses adenosine receptors linked to the adenylate cyclase-inhibitory GTP-binding protein is provided by the finding that pretreatment of these cells with pertussis toxin reverses the adenosine inhibition of cAMP formation stimulated by either isoprenaline or forskolin.     

AMP and adenosine are both ligands for adenosine 2B receptor signaling

Adenosine is considered the canonical ligand for the adenosine 2B receptor (A_2BR). A_2BR is upregulated following kidney ischemia augmenting post ischemic blood flow and limiting tubular injury. In this context the beneficial effect of A_2BR signaling has been attributed to an increase in the pericellular concentration of adenosine. However, following renal ischemia both kidney adenosine monophosphate (AMP) and adenosine levels are substantially increased. Using computational modeling and calcium mobilization assays, we investigated whether AMP could also be a ligand for A_2BR.The computational modeling suggested that AMP interacts with more favorable energy to A_2BR compared with adenosine. Furthermore, AMPαS, a non-hydrolyzable form of AMP, increased calcium uptake by Chinese hamster ovary (CHO) cells expressing the human A_2BR, indicating preferential signaling via the G_q pathway. Therefore, a putative AMP-A_2BR interaction is supported by the computational modeling data and the biological results suggest this interaction involves preferential G_q activation. These data provide further insights into the role of purinergic signaling in the pathophysiology of renal IRI.     

Mutagenesis of the cyclic AMP receptor protein of Escherichia coli: targeting positions 72 and 82 of the cyclic nucleotide binding pocket.

The 3', 5' cyclic adenosine monophosphate (cAMP) binding pocket of the cAMP receptor protein (CRP) of Escherichia coli was mutagenized to substitute leucine, glutamine, or aspartate for glutamate 72; and lysine, histidine, leucine, isoleucine, or glutamine for arginine 82. Substitutions were made in wild-type CRP and in a CRP*, or cAMP-independent, form of the protein to assess the effects of the amino acid substitutions on CRP structure. Cells containing the binding pocket residue-substituted forms of CRP were characterized through beta-galactosidase activity and by measurement of cAMP binding activity. This study confirms a role for both glutamate 72 and arginine 82 in cAMP binding and activation of CRP. Glutamine or leucine substitution of glutamate 72 produced forms of CRP having low affinity for the cAMP and unresponsive to the nucleotide. Aspartate substituted for glutamate 72 produced a low affinity cAMP-responsive form of CRP. CRP has a stringent requirement for the positioning of the position 72 glutamate carboxyl group within the cyclic nucleotide binding pocket. Results of this study also indicate that there are differences in the binding requirements of cAMP and cGMP, a competitive inhibitor of cAMP binding to CRP.     

腺嘌呤核苷脱氨酶及核苷运转系统对精子运动的调节作用

 腺嘌呤核苷(ADO)和它的类似物2-Cl-ADO对牛附睾尾部精子的运动有刺激作用,为了探讨ADO及其类似物对精子运动调节作用的机理,我们从ADO受体,核苷运转系统(NTS)及腺苷脱氨酶(ADA)三个方面对牛附睾尾部的精子进行了研究。我们发现腺苷受体不存在于牛精子膜上,但ADA和NTS以膜蛋白的形式结合在精子膜上,并对精子体内的ADO浓度起调节作用。我们的结论是ADO及其类似物对牛精子运动的调节作用是首先通过精子膜上的ADA和NTS影响精子体内的ADO浓度,进而ADO又通过调节钙离子浓度刺激牛精子运动。     

P1 and P2 purinergic receptor signal transduction in rat type II pneumocytes

Extracellular ATP is a potent agonist of surfactant phosphatidylcholine (PC) exocytosis from type II pneumocytes in culture. We studied P1 and P2 receptor signal transduction in type II pneumocytes. The EC50 for ATP on PC exocytosis was 10(-6) M, whereas the EC50 for ADP, AMP, adenosine, and the nonmetabolizable ATP analogue alpha,beta-methylene ATP was 10(-4) M. The rank order of agonists for PC exocytosis was ATP greater than ADP greater than AMP greater than adenosine greater than alpha,beta-methylene ATP. The rank order of agonists for phosphatidylinositol (PI) hydrolysis was ATP greater than ADP, whereas AMP, adenosine, and alpha,beta-methylene ATP did not stimulate PI hydrolysis. ATP (10(-4) M) caused a 15-fold increase in adenosine 3',5'-cyclic monophosphate (cAMP) production, and the nonmetabolizable adenosine analogue 5'-N-ethylcarboxyamidoadenosine (10(-6) M) increased cAMP production threefold. The effects of both these agonists on cAMP production were completely inhibited by the adenosine antagonist 8-phenyltheophylline (10(-5) M). The effects of ATP (10(-4) M) on PC exocytosis were inhibited 38% by 10(-5) M 8-phenyltheophylline. Thus, ATP regulates PC exocytosis by activating P2 receptors, which stimulate PI hydrolysis to inositol phosphate, as well as by activating P1 receptors, which stimulate cAMP production. Interactions between the P1 and P2 pathways may explain the high potency of extracellular ATP as an agonist of PC exocytosis.     

Adenosine Receptors Activate Adenylate Cyclase and Enhance Secretion from Bovine Adrenal Chromaffin Cells in the Presence of Forskolin

Cells of the adrenal medulla release not only catecholamines but also high concentrations of neuropeptides and nucleotides. Chromaffin cells, like many neuronal cells, have a diversity of receptors: adrenergic receptors, peptide receptors, histamine receptors, and dopamine receptors. We recently reported that these cells have nucleotide receptors that can mediate inhibition of the secretory response. The present studies show that adenosine, in the presence of enabling concentrations of forskolin, can potently enhance response to nicotinic stimulation. Neither adenosine nor forskolin alone produces a significant effect. A marked rise in intracellular cyclic AMP (cAMP) concentration is associated with the enhancement of secretion caused by forskolin plus adenosine. A phosphodiesterase inhibitor, Ro 20-1724, used together with forskolin produces significant increases in both cellular cAMP content and catecholamine secretion. However, the adenosine agonist 5'-N-ethylcarboxyadenosine elevates cellular cAMP content in the presence of forskolin without having any positive effect on secretion. This finding suggests that the rise in cAMP level may not be the sole cause of the increase in secretion by adenosine.     

In rat hepatocytes, different adenosine receptor subtypes use different secondary messengers to increase the rate of ureagenesis

In rat hepatocytes, the role of cAMP and Ca(2+) as secondary messengers in the ureagenic response to stimulation of specific adenosine receptor subtypes was explored. Analyzed receptor subtypes were: A(1), A(2A), A(2B) and A(3). Each receptor subtype was stimulated with a specific agonist while blocking all other receptor subtypes with a battery of specific antagonists. For the A(1) and A(3) adenosine receptor subtypes, the secondary messenger was the cytoplasmic Ca(2+) concentration ([Ca(2+)](cyt)). Accordingly, the A(1) or A(3)-mediated increase in [Ca(2+)](cyt) and in ureagenic activity were both inhibited by chelating Ca(2+) with either EGTA or BAPTA-AM. Also, Gd(3+) blocked both the increase in [Ca(2+)](cyt) and ureagenesis, suggesting that a Ca(2+) channel may be involved in the response to both A(1) and A(3). A partial effect was observed with the sarcoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin. The concentration of cyclic AMP ([cAMP]) increased in response to stimulation of either the A(2A) or the A(2B) adenosine receptor subtypes, while it decreased slightly in response to stimulation of either A(1) or A(3). The stimulation of either the A(2A) or A(2B) adenosine receptor subtypes resulted in an increase in [cAMP] and an ureagenic response which were not sensitive to EGTA, BAPTA-AM, Gd(3+) or to thapsigargin. In addition, the adenylyl cyclase inhibitor MDL12,330A blocked the ureagenic response to A(2A) and A(2B), but not the response to either A(1) or A(3). Our results indicate that in the ureagenic liver response to adenosine, the secondary messenger for both, the A(1) and A(3) adenosine receptor subtypes is [Ca(2+)](cyt), while the message from the A(2A) and A(2B) adenosine receptor subtypes is relayed by [cAMP].