Desensitization of adenosine receptor-mediated inhibition of lipolysis. The mechanism involves the development of enhanced cyclic adenosine monophosphate accumulation in tolerant adipocytes.

Adipocytes contain adenosine receptors, termed A1 receptors, which inhibit lipolysis by decreasing adenylate cyclase activity. The inhibition of lipolysis by adenosine agonists in vivo acutely suppresses the plasma concentrations of free fatty acids (FFA) and triglycerides. We have found that infusions of the adenosine receptor agonist phenylisopropyladenosine (PIA) initially decreases plasma FFA concentrations; however, with prolonged exposure (6 d), rats become very tolerant to the effects of the drug. Adipocytes isolated from epididymal fat pads from PIA-infused rats have altered lipolytic responses. When lipolysis is stimulated with a relatively high concentration of isoproterenol (10(-7) M), PIA does not inhibit lipolysis in adipocytes from the infused animals. However, PIA inhibits isoproterenol-stimulated cyclic AMP (cAMP) accumulation in adipocytes from the infused rats although with decreased sensitivity compared with controls. The explanation for the impaired antilipolytic effect appears to be due to the fact that isoproterenol-stimulated cAMP accumulation is markedly increased in cells from infused rats. Indeed, basal lipolysis and lipolysis stimulated with lower concentrations of isoproterenol (10(-9), 10(-8) M) are effectively inhibited by PIA. cAMP accumulation is greatly increased in adipocytes from infused rats when stimulated by isoproterenol, ACTH, and forskolin. The results have some striking analogies to changes induced in nerve cells by prolonged exposure to narcotics. These data suggest that tolerance to PIA develops in adipocytes as a consequence of enhanced cAMP accumulation.     

Quantitative differences in the cyclic AMP-lipolysis relationships for isoproterenol and forskolin

The relationships between cyclic AMP (cAMP) levels and glycerol release (lipolysis) were determined for isoproterenol and forskolin under varied conditions in the isolated fat cell of the rat. Prevention of the inhibitory action of endogenous adenosine [by adenosine deaminase (100 mU/ml) or theophylline (3.3 X 10(-4) M)] resulted in increased levels of cAMP and increased rates of lipolysis with forskolin. However, the relationship between cAMP levels and rates of lipolysis remained the same under all conditions. N6-phenylisopropyladenosine (PIA; an analog of adenosine) abolished the increase in cAMP level produced by isoproterenol (10(-7) M) or forskolin (10(-6) M) and the lipolytic response to forskolin. However, PIA failed to inhibit completely the lipolytic response to isoproterenol. Dose-response curves to isoproterenol were determined in the presence and absence of adenosine deaminase. PIA (10(-6) M) inhibited the increase in cAMP levels under both conditions. PIA also inhibited the lipolytic responses that were associated with increases in cAMP levels, i.e., high concentrations of isoproterenol alone and isoproterenol with adenosine deaminase. A plot of cAMP levels against corresponding rates of lipolysis for all conditions agreed with previous observations that the relationship for isoproterenol differs from that for forskolin. At any concentration of cAMP the corresponding lipolytic response was greater for isoproterenol than for forskolin. The possibility of a cAMP-independent lipolytic response was discussed.     

Receptor reserve for D2 dopaminergic inhibition of prolactin release in vivo and in vitro.

The full dopamine agonist R-(-)-N-n-propylnorapomorphine (NPA) completely suppressed (ED50 0.12 micrograms/kg) serum prolactin (PRL) levels elevated by pretreatment with gamma-butyrolactone (750 mg/kg). Pretreatment with the receptor-inactivating agent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (1 and 2 x 6 mg/kg) progressively shifted the dose-response curve for NPA to the right, but PRL secretion was still maximally inhibited. Receptor inactivation elicited smaller (2-fold) dextral shifts in the ED50 for the partial agonists (+)- and (-)-3-(3-hydroxyphenyl)-N-n-propylpiperidine. These results are consistent with the presence of a sizable receptor reserve at the D2 receptor regulating PRL release in the anterior pituitary. Analogous results were obtained in vitro utilizing primary cultures of anterior pituitary cells. NPA potently inhibited basal PRL release in culture (ED50 0.06 nM, maximal inhibition 83%). Receptor alkylation with phenoxybenzamine (1 microM, 1 hr) did not affect basal PRL release but right-shifted the ED50 for NPA more than 6-fold and attenuated maximal inhibition (to 68%); both effects were significant (P less than .01). The extracellular accumulation of cyclic AMP (cAMP) stimulated by a combination of forskolin (1 microM) and 3-isobutyl-1-methyl xanthine (100 microM) required higher concentrations of NPA (ED50 0.36 nM), and the maximal effect was much smaller (46%). Phenoxybenzamine treatment did not alter either basal or forskolin-stimulated cAMP accumulation, but it reduced the maximal inhibitory response to NPA (to 13%) without shifting the ED50. Plots of receptor occupancy vs. response demonstrated a 60% receptor reserve for NPA inhibition of PRL release, but none for inhibition of cAMP production.(ABSTRACT TRUNCATED AT 250 WORDS)     

A1 and A2 adenosine receptors in rabbit cortical collecting tubule cells. Modulation of hormone-stimulated cAMP.

Adenosine analogs were used to investigate the cellular mechanisms by which adenosine may alter renal tubular function. Cultured rabbit cortical collecting tubule (RCCT) cells, isolated by immunodissection, were treated with 5'-N-ethylcarboxamideadenosine (NECA), N6-cyclohexyladenosine (CHA), and R-N6-phenylisopropyladenosine (PIA). All three analogs produced both dose-dependent inhibition and stimulation of RCCT cell cyclic AMP (cAMP) production. Stimulation of cAMP accumulation occurred at analog concentrations of 0.1 microM to 100 microM with the rank order of potency NECA greater than PIA greater than CHA. Inhibition occurred at concentrations of 1 nM to 1 microM with the rank order of potency CHA greater than PIA greater than NECA. These effects on cAMP production were inhibited by 1,3-diethyl-8-phenylxanthine and isobutylmethylxanthine. CHA (50 nM) blunted AVP- and isoproterenol-stimulated cAMP accumulation. This modulation of hormone-induced cAMP production was abolished by pretreatment of RCCT cells with pertussis toxin. Prostaglandin E2 production was unaffected by 0.1 mM CHA. These findings indicate the presence of both inhibitory (A1) and stimulatory (A2) receptors for adenosine in RCCT cells. Moreover, occupancy of the A1 receptor causes inhibition of both basal and hormone-stimulated cAMP formation through an action on the inhibitory guanine nucleotide-binding regulatory component, Ni, of the adenylate cyclase system.     

Amplification of the cyclic AMP response to forskolin in pheochromocytoma PC12 cells through adenosine A(2A) purinoceptors.

In this study, we present evidence on the ability of endogenous adenosine to modulate adenylyl cyclase activity in intact PC12 cells. The adenosine receptor antagonists PD 115199, xanthine amine congener, 8-cyclopentyl-1,3-dipropylxanthine, 8-(p-sulfophenyl)theophylline, and 3,7-dimethyl-1-propargylxanthine inhibited 10 microM forskolin-induced cyclic AMP (cAMP) accumulation, with IC(50) values of 2.76 +/- 1.16 nM, 17.4 +/- 1.08 nM, 443 +/- 1. 03 nM, 2.00 +/- 1.01 microM, and 2.25 +/- 1.05 microM, respectively. Inhibition by 2.5 nM PD 115199 was only partially reversed by increasing forskolin concentrations up to 100 microM. The addition of PD 115199 with or 60 min after forskolin caused a comparable inhibition of forskolin effect over the next hour. Both exogenous adenosine (0.1 microM) and its precursor, AMP (10 and 100 microM), significantly enhanced forskolin-induced cAMP accumulation, whereas inosine was ineffective. Forskolin activity was also potentiated by the hydrolysis-resistant adenosine receptor agonists 5'-N-ethylcarboxamido adenosine and CGS 21680 (8.9- and 12.2-fold increase, respectively). Adenosine deaminase (1 U/ml) and 8-SPT (25 microM), which nearly abolished the response to 1 microM adenosine, also reduced cAMP accumulation caused by AMP (-78 and -54%, respectively). These results demonstrate that in PC12 cells, activation of adenylyl cyclase by forskolin is highly dependent on the occupancy of A(2A) adenosine receptors and that AMP potentially contributes to the amplification of forskolin response.     

Contribution of the extracellular cAMP-adenosine pathway to dual coupling of β2-adrenoceptors to Gs and Gi proteins in mouse skeletal muscle

β(2)-Adrenoceptor (β(2)-AR) agonists increase skeletal muscle contractile force via activation of G(s) protein/adenylyl cyclases (AC) and increased generation of cAMP. Herein, we evaluated the possible dual coupling of β(2)-AR to G(s) and G(i) proteins and the influence of the β(2)-AR/G(s)-G(i)/cAMP signaling cascade on skeletal muscle contraction. Assuming that the increment of intracellular cAMP is followed by cAMP efflux and extracellular generation of adenosine, the contribution of the extracellular cAMP-adenosine pathway on the β(2)-AR inotropic response was also addressed. The effects of clenbuterol/fenoterol (β(2)-AR agonists), forskolin (AC activator), cAMP/8-bromo-cAMP, and adenosine were evaluated on isometric contractility of mouse diaphragm muscle induced by supramaximal direct electrical stimulation (0.1 Hz, 2 ms duration). Clenbuterol/fenoterol (10-1000 μM), 1 μM forskolin, and 20 μM rolipram induced transient positive inotropic effects that peaked 30 min after stimulation onset, declining to 10 to 20% of peak levels in 30 min. The late descending phase of the β(2)-AR agonist inotropic effect was mimicked by either cAMP or adenosine and abolished by preincubation of diaphragm with pertussis toxin (PTX) (G(i) signaling inhibitor) or the organic anion transporter inhibitor probenecid, indicating a delayed coupling of β(2)-AR to G(i) protein which depends on cAMP efflux. Remarkably, the PTX-sensitive β(2)-AR inotropic effect was inhibited by the A(1) adenosine receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine and ecto-5'-phosphodiesterase inhibitor α,β-methyleneadenosine 5'-diphosphate sodium salt, indicating that β(2)-AR coupling to G(i) is indirect and dependent on A(1) receptor activation. The involvement of the extracellular cAMP-adenosine pathway in β(2)-AR signaling would provide a negative feedback loop that may limit stimulatory G protein-coupled receptor positive inotropism and potential deleterious effects of excessive contractile response.     

Effects of the selective adenosine A2 receptor agonist CGS 21680 on in vitro electrophysiology, cAMP formation and dopamine release in rat hippocampus and striatum

Evaluation of adenosine A2 receptor function in the mammalian CNS has been impeded by the lack of highly selective A2 receptor agonists. The present investigations describe the actions of a recently introduced A2 selective adenosine agonist, CGS 21680 (2-[p-(carboxyethyl)phenylethylamino]-5'-N-ethylcarboxamidoadenosi ne), on various functional neural responses known to be affected by adenosine. In hippocampal slices, CGS 21680 appeared to be a weak agonist on pre- and postsynaptic measures of electrophysiological activity (putative A1 receptor mediated events) and was ineffective at stimulating the formation of cAMP (a putative A2b mediated response). 5'-N-ethycarboxamidoadenosine (NECA), which is known to act at both A2a and A2b receptors, increased hippocampal cAMP levels 4-fold. In striatal slices, CGS 21680 potently stimulated the formation of cAMP with an EC50 of 110 nM but was ineffective at inhibiting electrically stimulated dopamine release. In contrast, adenosine and cyclohexyladenosine both inhibited the stimulus-evoked overflow of dopamine. These results agree with previous receptor binding studies suggesting that CGS 21680 is a relatively selective agonist at the high affinity adenosine A2a receptor in striatum, with little intrinsic activity at the low affinity A2b site in hippocampus.     

Endogenous adenosine is an autacoid feedback inhibitor of chloride transport in the shark rectal gland.

The present studies define the physiologic role of endogenous adenosine in the perfused shark rectal gland, a model epithelia for hormone-stimulated chloride transport. Chloride ion secretion, and venous adenosine and inosine concentrations increased in parallel in response to hormone stimulation. From a basal rate of 157 +/- 26 mu eq/h per g, chloride secretion increased to 836 +/- 96 and 2170 +/- 358 with 1 and 10 microM forskolin, venous adenosine increased from 5.0 +/- 1 to 126 +/- 29 and 896 +/- 181 nM, and inosine increased from 30 +/- 9 to 349 +/- 77 and 1719 +/- 454 nM (all P less than 0.01). Nitrobenzylthioinosine (NBTI), a nucleoside transport inhibitor, completely blocked the release of adenosine and inosine. Inhibition of chloride transport with bumetanide, an inhibitor of the Na+/K+/2Cl- cotransporter, or ouabain, an inhibitor of Na+/K+ ATPase activity, reduced venous adenosine and inosine to basal values. When the interaction of endogenous adenosine with extracellular receptors was prevented by adenosine deaminase, NBTI, or 8-phenyltheophylline, the chloride transport response to secretagogues increased by 1.7-2.3-fold. These studies demonstrate that endogenous adenosine is released in response to hormone-stimulated cellular work and acts at A1 adenosine receptors as a feedback inhibitor of chloride transport.     

Functional evidence for adenosine A2 receptor regulation of phosphatidylcholine secretion in cultured type II pneumocytes

We examined the effects of P1 purinoceptor agonists on secretion of phosphatidylcholine in primary cultures of rat type II pneumocytes. Adenosine and its nonmetabolizable analogs, 5'-N-ethylcarboxyamidoadenosine (NECA), N6-phenylisopropyladenosine (PIA) and 2-chloroadenosine, increased secretion, and this effect was dependent on concentration. At the highest concentration, all agonists stimulated phosphatidylcholine secretion approximately 2-fold. NECA, with an EC50 of 8.9 X 10(-8) M, was the most potent agonist. The order of potency was NECA greater than 2-chloroadenosine = L-PIA greater than or equal to adenosine greater than D-PIA. The stimulatory effect of 10(-6) M NECA was diminished by the P1 antagonists theophylline and 8-phenyltheophylline. The degree of stimulation by the adenosine analogs as well as its time course was the same as that induced by terbutaline. The effects of the adenosine analogs and of terbutaline were not additive, suggesting a similar mode of action for the beta adrenergic and purinoceptor agonists. Terbutaline and the adenosine analogs increased intracellular cyclic AMP levels. Again, NECA was the most potent adenosine analog. For NECA, L-PIA and adenosine, there was a significant correlation between effects on secretion and on cyclic AMP content. These data suggest that the effect of adenosine on phosphatidylcholine secretion in type II pneumocytes is mediated by the A2 subtype of the P1 purinoceptor.     

Intracellular signaling in the regulation of renal Na-K-ATPase. I. Role of cyclic AMP and phospholipase A2.

We have reported that dopamine (DA) inhibits Na-K-ATPase activity in the cortical collecting duct (CCD) by stimulating the DA1 receptor, and the present study was designed to evaluate the mechanism of this effect. Short-term exposure (15-30 min) of microdissected rat CCD to DA, a DA1 agonist (fenoldopam), vasopressin (AVP), forskolin, or dibutyryl cAMP (dBcAMP), which increase cAMP content by different mechanisms, strongly (approximately 60%) inhibited Na-K-ATPase activity. 2',5'-dideoxyadenosine, an inhibitor of adenylate cyclase, completely blocked Na-K-ATPase inhibition by DA or fenoldopam, and IP20, an inhibitor peptide of cAMP-dependent protein kinase A (PKA), abolished the Na:K pump effect of all the cAMP agonists listed above. To verify whether the mechanism of pump inhibition by agents that increase cell cAMP involves phospholipase A2 (PLA2), we used mepacrine, a PLA2 inhibitor, which also abolished Na-K-ATPase inhibition by DA or fenoldopam, as well as by AVP, forskolin, or dBcAMP. Arachidonic acid (10(-7) - 10(-4) M) inhibited Na-K-ATPase activity in dose-dependent fashion. Corticosterone, which induces lipomodulin, a PLA2 inhibitor protein inactivated by PKA, equally abolished the pump effects of DA, fenoldopam, forskolin, and dBcAMP, suggesting that lipomodulin might act between PKA and PLA2 in cAMP-dependent pump regulation. We conclude that dopamine inhibits Na-K-ATPase activity in the CCD through a DA1 receptor-mediated cAMP-PKA pathway that involves the stimulation of PLA2 and arachidonic acid release, possibly mediated by inactivation of lipomodulin. This pathway is shared by other agonists that increase cell cAMP and thus stimulate PKA activity.     

Differential regulation of human basophil and lung mast cell function by adenosine.

Adenosine was found to modulate the activity of the human basophil and lung mast cell (HLMC) differently. In the basophil, adenosine inhibited the anti-IgE stimulated release of histamine and leukotriene C4 (LTC4) and increased total cell cyclic AMP (cAMP) levels. Substituted adenosine analogs had a rank order potency of: N-ethylcarboxamideadenosine (NECA) greater than 2-chloroadenosine greater than R-phenylisopropyladenosine for the inhibition of immunoglobulin E-triggered mediator release from the basophil and increases in cAMP levels. The adenosine receptor antagonist, 8-phenyltheophylline, antagonized both the NECA-induced inhibition of mediator release and elevations in cyclic nucleotide. The purinergic transport inhibitor, dipyridamole, reversed the inhibition by adenosine of histamine release but not LTC4 generation, suggesting that these two actions are mechanistically separable. Dipyridamole failed to modify the adenosine-induced elevation in cAMP. In contrast to the findings in the basophil, the response to adenosine in the HLMC was biphasic in nature. Thus, at low concentrations of the nucleoside, adenosine potentiated the release of histamine and LTC4 from immunologically activated HLMC, whereas at higher concentrations a counteractive inhibitory process was observed. Analogs of adenosine had the same effects on HLMC; NECA was more potent than R-phenylisopropyladenosine for both the potentiating and inhibitory components of the biphasic response. Low concentrations of adenosine analogs, which potentiated secretion, initiated modest elevations in cAMP levels, whereas higher concentrations, which inhibited secretion, significantly augmented cAMP levels. Although R-phenylisopropyladenosine was almost as potent as NECA at elevating cAMP in HLMC, it was not as efficacious. The NECA-induced modulation of HLMC mediator release and elevations in cAMP were antagonized by 8-phenyltheophylline.(ABSTRACT TRUNCATED AT 250 WORDS)     

alpha2C adrenoceptors inhibit adenylyl cyclase in mouse striatum: potential activation by dopamine.

alpha2C adrenoceptors occur in high density in the striatum, but the functional role of these receptors is uncertain. Mice with targeted inactivation of the alpha2C adrenoceptor gene (Adra2c-/-) and genetically related control mice expressing the wild-type alpha2C adrenoceptor (Adra2c+/+) were used to determine whether striatal alpha2C adrenoceptors modulate adenylyl cyclase activation. In striatal slices from Adra2c+/+ mice, the alpha2 adrenoceptor antagonist RX821002 facilitated forskolin-stimulated cyclic AMP accumulation in a concentration-dependent manner. In contrast, RX821002 had no effect on forskolin-stimulated cAMP accumulation in striatal slices from Adra2c-/- mice or in striatal slices from Adra2c+/+ mice treated with reserpine and alpha-methyl-rho-tyrosine to deplete monoamine neurotransmitters. Given the sparse innervation of the striatum by noradrenergic neurons, the possibility that dopamine can activate the mouse alpha2C adrenoceptor at physiologically relevant concentrations was investigated using normal rat kidney (NRK) cells transfected with the mouse alpha2A or alpha2C adrenoceptor cDNA (NRK-alpha2A or NRK-alpha2C cells). Inhibition of [3H]RX821002 binding by agonists in homogenates of transfected cells revealed an affinity of dopamine for alpha2C adrenoceptors that was higher than the affinity of norepinephrine for its cognate receptor, the alpha2A adrenoceptor. Both norepinephrine and dopamine inhibited forskolin-stimulated cAMP accumulation in intact NRK-alpha2C cells. In NRK-alpha2A cells, norepinephrine facilitated forskolin-stimulated cAMP accumulation, an effect not observed for dopamine. Together, these data demonstrate that the alpha2C adrenoceptor is negatively coupled to adenylyl cyclase and is tonically activated in mouse striatal slices. The endogenous activator of the striatal alpha2C adrenoceptor may be dopamine, as well as norepinephrine.     

Role of cyclic AMP-dependent protein kinase in the diminished beta adrenergic responsiveness of vascular smooth muscle with increasing age

Beta adrenergic receptor-mediated relaxation of blood vessels declines with age although the mechanism is unknown. We have utilized the mesenteric artery and aorta of young and older rats to investigate this problem. In vessels from 12-month-old rats there was a marked loss in relaxation mediated by beta adrenergic and adenosine receptors compared to younger rats whereas relaxation induced by muscarinic cholinergic receptors, [cyclic AMP (cAMP) independent], was not impaired. Maximal relaxation to forskolin and dibutyryl cAMP were intact in the vessels from older rats. Isoproterenol-stimulated cAMP accumulation and cAMP-dependent protein kinase activation were attenuated markedly in the vessels from the older rats. Maximal forskolin-stimulated cAMP accumulation and cAMP-dependent protein kinase activation were similar in older and young animals. There was an excellent correlation between cAMP-dependent protein kinase activity and relaxation and the relationship was similar in the two age groups. Continuous infusion of the beta adrenergic antagonist timolol for 1 week into older animals partially restored relaxation to beta adrenergic and adenosine receptor agonists in the aorta. These results suggest that the age-related loss of response to beta adrenergic receptor agonist-induced relaxation may be due in part to attenuated activation of cAMP dependent protein kinase and this change may be partially dependent on endogenous catecholamines.     

Novel short-acting A2A adenosine receptor agonists for coronary vasodilation: inverse relationship between affinity and duration of action of A2A agonists.

Several potent and selective A2A adenosine receptor agonists are currently available. These compounds have a high affinity for the A2A receptor and a long duration of action. However, in situations where a short duration of action is desired, currently available A2A receptor agonists are less than ideal. From a series of recently synthesized A2A receptor agonists, two agonists (CVT-3146 and CVT-3033) with low affinity were selected for further characterization as selective and short-acting coronary vasodilators. Both compounds were selective for the A2A adenosine receptor (AdoR) versus the A1, A2B, and A3AdoR in binding and functional studies. CVT-3146 and CVT-3033 appeared to be weak partial agonists to cause cAMP accumulation in PC12 cells, but were full and potent agonists to cause coronary vasodilation, a response that has a very large A2A receptor reserve. However, the durations of action of CVT-3146 and CVT-3033 were remarkably shorter than those of the high-affinity agonists CGS21680 or WRC0470, presumably due to the relative lower affinity of CVT-3146 and CVT-3033 for the A2A receptor. Indeed, an inverse relationship was found between the affinity of the various agonists for the A2A receptor and the duration of their actions. These data indicate that low-affinity agonists can produce a response that is of equivalent magnitude but more rapid in termination than that caused by a high-affinity agonist. Hence, the low-affinity A2A agonists CVT-3146 and CVT-3033 may prove to be superior to currently available high-affinity agonists as coronary vasodilators during myocardial imaging with radionuclide agents.     

Role of adenosine in the gastric blood flow response to pentagastrin in the rat

We tested the hypothesis that the increase in gastric mucosal blood flow during pentagastrin-stimulated acid secretion in the rat is mediated partly by endogenously generated adenosine. In in vivo microscopic studies, topical 10(-5) to 10(-3) M adenosine dose-dependently dilated the submucosal arterioles, the vessels that control mucosal blood flow. The adenosine receptor antagonist 8-phenyltheophylline, significantly reduced adenosine's vasodilatory response. An adenosine analog with a high A2 receptor affinity was 100 times more potent as a vasodilator than one with a high A1 receptor affinity but lower A2 receptor affinity. We then examined the effect of i.v. 8-phenyltheophylline, 10 mg/kg, on the pentagastrin-stimulated increase in gastric blood flow and gastric acid secretion. Mucosal blood flow was estimated by the hydrogen clearance technique. Pentagastrin increased mucosal blood flow from 26.6 +/- 2.6 to 42.7 +/- 4.9 ml/min/100 g and this was reduced to 31.9 +/- 3.1 ml/min/100 g upon the addition of 8-phenyltheophylline. Gastric acid secretion upon the addition of 8-phenyltheophylline. Gastric acid secretion was stimulated by pentagastrin and stimulated further by the addition of 8-phenyltheophylline from 2.06 +/- 0.34 mEq of H+ per min to 2.84 +/- 0.49 mEq/min. 8-Phenyltheophylline had no effect on basal mucosal blood flow or gastric acid secretion. In contrast, the nonmethylxanthine phosphodiesterase inhibitor RO 20-1724 stimulated acid secretion and increased gastric mucosal blood flow during pentagastrin administration. The data suggest that gastric submucosal arterioles contain adenosine receptors of the A2 subtype that vasodilate when activated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of caffeine and 8-phenyltheophylline on the actions of purines and opiates in the guinea-pig ileum

The antagonist effects of 8-phenyltheophylline (8-PT) and caffeine against the actions of adenosine, (-)-N6-(R-phenyl-isopropyl)-adenosine (PIA), morphine and nalorphine on the guinea pig ileum preparation were examined. Antagonism of both adenosine and PIA by caffeine and 8-PT was concentration dependent. The slopes of Schild plots for both alkylxanthines vs. adenosine were significantly less than -1 unless the adenosine reuptake blocker dipyridamole (0.1 microM) was include in the tissue bath. Under these conditions, the 95% confidence intervals of the Schild plot slopes embraced theoretical unity, suggesting competitive antagonism. The antagonism of PIA by the alkylxanthine was also competitive. Dipyridamole had no effect on the potency of PIA. The pA2 value for caffeine-adenosine was not different from that for caffeine-PIA, and the pA2 values of 8-PT-adenosine and 8-PT-PIA were similar, suggesting that these two agonists interacted with similar receptors. The pA2 values using 8-PT were approximately 1.5-fold higher than those employing caffeine, suggesting higher affinity for 8-PT at these receptors. Caffeine significantly antagonized morphine at all concentrations used (0.5-1.0 mM), but only antagonized nalorphine at the two highest concentrations. After treating ilea with the mu-specific irreversible antagonist beta-FNA (beta-funaltrexamine) (resulting in a preparation with relatively pure kappa receptor population), the antagonist effect of caffeine against morphine was reduced such that only a concentration of 1 mM resulted in significant antagonism, while the effects of caffeine against nalorphine were unchanged. 8-PT did not antagonize morphine or nalorphine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hormonal regulation of proton secretion in rabbit medullary collecting duct.

With the exception of aldosterone, little is known about the hormonal regulation of distal nephron acidification. These experiments investigated the effects of prostaglandin E2, indomethacin, lysyl-bradykinin, 8-bromo-cyclic AMP, and forskolin on proton secretion in the major acidifying segment of the distal nephron, the medullary collecting duct from inner stripe of outer medulla. Using in vitro microperfusion and microcalorimetry, net bicarbonate reabsorption (proton secretion) was measured in rabbit medullary collecting ducts before, during, and after exposure to each test substance. PGE2 reduced proton secretion 12.2%, while the following substances stimulated proton secretion: indomethacin 14.2%; 8-bromo-cyclic AMP 34.5%; forskolin 39%. Lysyl-bradykinin was without effect. These studies demonstrate that distal nephron acidification, in addition to being stimulated by aldosterone, is significantly inhibited by the hormone PGE2. The stimulation of proton secretion by cAMP suggests that other hormones known to activate adenylate cyclase may also influence distal nephron acidification.     

Role of platelet-activating factor in Chinese hamster ovary cell responses to cholera toxin.

Cholera toxin (CT)-induced intestinal secretion and Chinese hamster ovary cell (CHO) elongation involves cyclic adenosine monophosphate and protein synthesis-dependent prostaglandin formation. We previously reported inhibition of CT-induced intestinal secretion and CHO elongation by platelet-activating factor (PAF) receptor antagonists and secretion of PAF by human intestinal epithelial cells exposed to CT. Herein, we show that PAF is involved after cAMP and that PAF, like CT, mediates prostaglandin E2 synthesis in CHO cells. CT-induced CHO elongation was blocked by specific PAF receptor antagonists, BN52021 and SR27417. SR27417 blocked dibutyryl cAMP-induced CHO elongation, but did not alter CHO elongation caused by PGE2. Neither CT-stimulated cAMP accumulation nor PGE2 production was inhibited by SR27417. Both PGE2 and PAF caused significant CHO elongation, but the latter did not stimulate significant cAMP production. In addition, PAF, like CT and dibutyryl cAMP, stimulated significant PGE2 production. Finally, the protein synthesis inhibitor cycloheximide, which completely blocks the effect of CT on prostaglandin synthesis, also blocked that of PAF, suggesting that PAF also mediates protein synthesis-dependent prostaglandin formation. We conclude that PAF is involved in CHO cytoskeletal responses to CT after the accumulation of cAMP and, like CT, PAF stimulates protein synthesis-dependent prostaglandin accumulation.     

Induction of nitric oxide synthase by cyclic AMP in rat vascular smooth muscle cells.

By measurements of NO2-/NO3- (NOx) production and Northern blot analysis, we studied the effects of a membrane-permeable cAMP derivative, 8-bromo-cAMP, on the expression of inducible nitric oxide synthase (iNOS) gene and the synthesis of NOx in cultured rat vascular smooth muscle cells (VSMCs). 8-bromo-cAMP stimulated NOx production and increased steady-state levels of iNOS mRNA in rat VSMC in a time- and dose-dependent manner. NG-monomethyl-L-arginine, a NOS inhibitor, completely blocked the 8-bromo-cAMP-induced NOx production, whose effect was partially, but significantly reversed by an excess L-arginine, but not by D-arginine. Compounds that increase intracellular cAMP levels (cholera toxin, forskolin, and 3-isobutyl-1-methylxanthine), all stimulated NOx production. Dexamethasone inhibited the stimulated NOx production, as well as the induction of iNOS mRNA by cAMP. Both actinomycin D and cycloheximide completely blocked the stimulated NOx production by cAMP. Actinomycin D abolished the cAMP-induced iNOS mRNA, whereas cycloheximide remarkably increased iNOS mRNA levels in the presence and absence of 8-bromo-cAMP (superinduction). Actinomycin D, but not dexamethasone, completely abolished the cycloheximide-induced iNOS mRNA. The half-life of cAMP-induced iNOS mRNA was approximately 2 h, whereas no decay in the cycloheximide-induced iNOS mRNA was observed during 12 h. These results demonstrate that iNOS gene is upregulated by cAMP and the superinduction of iNOS mRNA is attributable to increased mRNA stability in rat VSMC.     

Adenosine receptors on human leukocytes. IV. Characterization of an A1/Ri receptor.

Adenosine (10(-9)-10(-6) mol/l) and R-phenylisopropyladenosine (10(-9)-10(-7) mol/l) partially inhibited the intracellular accumulation of cyclic AMP induced by isoproterenol, prostaglandin E1, histamine and 5'-N-ethylcarboxamidoadenosine in lymphocytes. In contrast, S-phenylisopropyladenosine, which is a poor agonist of the adenosine A1/Ri receptor, had essentially no inhibitory effect. 8-Phenyltheophylline, in low concentrations that do not inhibit cyclic AMP phosphodiesterase, completely blocked the inhibitory effect of R-phenylisopropyladenosine on the increase in cyclic AMP induced by prostaglandin E1. R-Phenylisopropyladenosine (10(-8)-10(-6) mol/l) also inhibited the cyclic AMP accumulation in lymphocytes induced by forskolin (10(-5) mol/l), which activates adenylate cyclase through direct interaction with the enzyme. We also investigated the presence of the adenosine A1/Ri receptor on human polymorphonuclear leukocytes. R-Phenylisopropyladenosine (3 x 10(-9)-10(-7) mol/l) abolished the stimulating effects of prostaglandin and forskolin on cyclic AMP accumulation in polymorphonuclear leukocytes. This effect was blocked by 8-phenyltheophylline and was not observed with the stereoisomer S-phenylisopropyladenosine. The results support the existence of an A1/Ri receptor that regulates cyclic AMP metabolism of human lymphocytes and polymorphonuclear leukocytes.