Dan Shi

A role for central A3-adenosine receptors. Mediation of behavioral depressant effects.

The behavioral effects of a selective A3 adenosine receptor agonist 3‐IB‐MECA (N 6‐(3‐iodobenzyl)‐5‐N‐methylcarboxamidoadenosine) in mice and the localization of radioligand binding sites in mouse brain were examined. Low levels of A3 adenosine receptors were detected in various regions of the mouse brain (hippocampus, cortex, cerebellum, striatum), using a radioiodinated, high‐affinity A3‐agonist radioligand [125I]AB‐MECA (N 6‐(3‐iodo‐4‐aminobenzyl)‐5‐N‐methylcarboxamidoadenosine). Scatchard analysis in the cerebellum showed that the K d value for binding to A3 receptors was 1.39 ± 0.04 nM with a B max of 14.8 ±2.1 protein. 3‐IB‐MECA at 0.1 i.p. was a locomotor depressant with > 50% reduction in activity. Although selective A1 or A2a antagonists reversed locomotor depression elicited by selective A1 or A2a agonists, respectively, the behavioral depressant effects of 3‐IB‐MECA were unaffected. 3‐IB‐MECA also caused scratching in mice, which was prevented by coadministration of the histamine antagonist cyproheptadine. The demonstration of a marked behavioral effect of A3 receptor activation suggests that the A3 receptor represents a potential new therapeutic target.

Chronic caffeine alters the density of adenosine, adrenergic, cholinergic, GABA, and serotonin receptors and calcium channels in mouse brain.

Summary1.Chronic ingestion of caffeine by male NIH strain mice alters the density of a variety of central receptors.2.The density of cortical A1 adenosine receptors is increased by 20%, while the density of striatal A2A adenosine receptors is unaltered.3.The densities of corticalβ1 and cerebellarβ2 adrenergic receptors are reduced byca. 25%, while the densities of corticalα1 andα2 adrenergic receptors are not significantly altered. Densities of striatal D1 and D2 dopaminergic receptors are unaltered. The densities of cortical 5 HT1 and 5 HT2 serotonergic receptors are increased by 26–30%. Densities of cortical muscarinic and nicotinic receptors are increased by 40–50%. The density of cortical benzodiazepine-binding sites associated with GABAA receptors is increased by 65%, and the affinity appears slightly decreased. The density of cortical MK-801 sites associated with NMDA-glutaminergic receptors appear unaltered.4.The density of cortical nitrendipine-binding sites associated with calcium channels is increased by 18%.5.The results indicate that chronic ingestion of caffeine equivalent to about 100 mg/kg/day in mice causes a wide range of biochemical alterations in the central nervous system.

INHIBITION OF PLATELET AGGREGATION BY ADENOSINE RECEPTOR AGONISTS

Abstract2-(Ar)alkoxyadenosines, which are agonists selective for the A2AAR in PC 12 cell and rat striatum membranes, are also agonists at the A2AR coupled to adenylate cyclase (AC) that mediates the inhibition of platelet aggregation. A panel of twelve well-characterized adenosine analogues stimulated human platelet AC and inhibited ADP-induced platelet aggregation at sub- to low-micromolar concentrations with a potency ranking CGS 21680 < adenosine < R-PIA. There were significant correlations between the ECso of anti-aggregatory activity and either the ECso of stimulation of platelet and PC 12 cell AC (r2 = 0.66 and 0.67, respectively) or the K1 of inhibition of [3H]NECA binding to the rat striatum membranes (r2 = 0.75). Likewise, platelet AC stimulation correlated well with stimulation of PC 12 cell AC and with [3H]NECA binding (r2 = 0.94 and 0.91, respectively). Ten 2-(ar)alkoxyadenosines stimulated platelet AC at EC50s ranging between 0.16 and 2.3 μM and inhibited platelet aggregation at EC50s ranging between 2 and 30 μM. There were no correlations between the EC50s of anti-aggregatory activity and either the EC50s of the stimulation of platelet or PC 12 AC (r2 = 0.08 and 0.06, respectively) or with the K1 of the inhibition of [3H]NECA binding to the A2aAR in rat striatum (r2 = 0.02). The EC50s of the stimulation of platelet AC correlated with those of the stimulation of PC 12 AC (r2 = 0.48), and also with the K1 of [3H]NECA binding (r2 = 0.71). Each of the 23 adenosines completely inhibited platelet aggregation and thus, functionally, all behaved as full agonists. As stimulants of PC 12 cell AC, Group A and B analogues were equally efficacious. As stimulants of platelet AC, however, the efficacy relative to NECA ( = 1.0) of Group B analogues was significantly less than that of Group A analogues, 0.49 ± 0.2 vs. 0.72 ± 0.05, P±0.01. The partial agonist activity of Group B analogues at the platelet A2AR but full agonist activity at the PC 12 cell A2aAR, as well as the relatively low correlations between platelet AC stimulation and other indices of A2aAR agonist actlVlty, suggest the platelet receptor is not a typical A2aAR. Further, the lack of a correlation between the platelet anti-aggregatory and AC stimulatory activity suggests that (a) the 2-(ar)alkoxyadenosines might affect platelet aggregation by mechanisms other than AC stimulation or (b) that the stimulation of the platelet membrane AC by 2-(ar)alkoxy-adenosines does not correspond to the accumulation of cyclic AMP in intact platelets.

Chronic effects of xanthines on levels of central receptors in mice.

Abstract1.Chronic ingestion of caffeine causes a significant increase in levels of A1-adenosine, nicotinic and muscarinic receptors, serotonergic receptors, GABAA receptors and L-type calcium channels in cerebral cortical membranes from mice NIH Swiss strain mice.2.Chronic theophylline and paraxanthine had effects similar to those of caffeine except that levels of L-type channels were unchanged. Chronic theobromine, a weak adenosine antagonist, and 1-isobutyl-3-methylxanthine (IBMX), a potent adenosine antagonist and phosphodiesterase inhibitor, caused only an increase in levels of A1-adenosine receptors. A combination of chronic caffeine and IBMX had the same effects on receptors as caffeine alone. Chronic 3,7-dimethyl-1-propargylxanthine (DMPX), a somewhat selective A2A-antagonist, caused only an increase in levels of A1-adenosine receptors. Pentoxyfylline, an adenosine-uptake inhibitor inactive at adenosine receptors, had no effect on receptor levels or calcium channels.3.A comparison of plasma and brain levels of xanthines indicated that caffeine penetrated more readily and attained somewhat higher brain levels than theophylline or theobromine. Penetration and levels were even lower for IBMX, paraxanthine, DMPX, and pentoxyfylline.4.The results suggest that effective blockade of both A1 and A2A-adenosine receptors is necessary for the full spectrum of biochemical changes elicited by chronic ingestion of xanthines, such as caffeine, theophylline, and paraxanthine.

Caffeine Analogs: Effects on Ryanodine-Sensitive Calcium-Release Channels and GABAA Receptors

Abstract1. Caffeine at 0.3–10 mM enhanced the binding of [3H]ryanodine to calcium-release channels of rabbit muscle sarcoplasmic reticulum. A variety of other xanthines were as efficacious as caffeine or nearly so, but none appeared markedly more potent.2. Caffeine at 1 mM markedly inhibited binding of [3H]diazepam to GABAA receptors in rat cerebral cortical membranes.3. Other xanthines also inhibited binding with certain dimethylpropargylxanthines being nearly fivefold more potent than caffeine.4. Caffeine at 1 mM stimulated binding of [35S]TBPS to GABAA receptors as did certain other xanthines.5. The dimethylpropargylxanthines had little effect. 1,3-Dipropy1-8-cyclopentylxan- thine at 100 μM had no effect on [3H]diazepam binding, but markedly inhibited [35S]TBPS binding.6. Structure–activity relationships for xanthines do differ for calcium-release channels and and for different sites on GABAA receptors, but no highly selective lead compounds were identified.

Chronic effects of ethanol on central adenosine function of mice.

Chronic ingestion of 5% ethanol had no significant effect on open field locomotor of NIH Swiss strain male mice, nor were the depressant effects of a non-selective adenosine receptor agonist, NECA, or the stimulant effects of a non-selective antagonist, caffeine significantly altered. The density of cerebral cortical A1-adenosine receptors and of nitrendipine binding sites on calcium channels were significantly increased after chronic ethanol, while the density of striatal A2a-adenosine receptors were unchanged. The locomotor stimulant effects of ethanol (2.5 g/kg) were slightly decreased after chronic ethanol, but were markedly reduced in mice after chronic caffeine ingestion. The results suggest some involvement of adenosine systems in the effects of ethanol.

Spiropyrrolizidines: A new class of blockers of nicotinic receptors

The spiropyrrolizidine oximes 236 and 222 and a related spiropyrrolizidine alkaloid, nitropolyzonamine, block nicotinic receptor channels in rat pheochromocytoma PC12 cells and in human medulloblastoma TE671 cells. In PC12 cells with an alpha 3 beta 4(5)-nicotinic receptor, both the spiropyrrolizidine oxime 236 and nitropolyzonamine had IC50 values of about 1.5 microM, while spiropyrrolizidine oxime 222 had an IC50 value of 2.6 microM versus carbamylcholine-elicited sodium-22 influx. In TE671 cells with an alpha 1 beta 1 gamma delta nicotinic receptor, the spiropyrrolizidine oximes 236, 222, and nitropolyzonamine had IC50 values of 9.5, 14, and 67 microM, respectively. The inhibitions by the spiropyrrolizidine oxime 236 and nitropolyzonamine appeared to be noncompetitive in nature in both cell lines. In rat cerebral cortical membranes, binding of [3H]nicotine to alpha 4 beta 2 nicotinic receptors was not inhibited significantly by 10 microM concentrations of the spiropyrrolizidine oxime 236, or by nitropolyzonamine, as expected for a noncompetitive blocker. Both compounds at 10 microM had marginal effects on a variety of central receptors, but did inhibit binding of [3H]1,3-di(2-tolyl) guanidine to sigma receptors in mouse brain membranes with IC50 values of about 0.5 microM. The spiropyrrolizidine oxime 236 at 10 microM had no effect on batrachotoxin-elicited sodium influx in guinea pig cerebral cortical synaptoneurosomes or on ATP-elicited calcium influx in PC12 cells. Such spiropyrrolizidines represent a new structural class of blockers of nicotinic receptor channels with selectivity for ganglionic-type receptors.

Pyrazolopyridines: Effect of structural alterations on activity at adenosine‐ and GABAA‐receptors

A series of 4‐substituted 1H‐pyrazolo[3,4‐b]pyridine‐ 5‐carboxylic acid derivatives related in structure to the putative anxiolytics cartazolate, tracazolate, and etazolate were assessed for affinity at A1‐ and A2A‐adenosine receptors and at GABA‐, benzodiazepine‐, and picrotoxinin‐ binding sites of the GABAA‐receptor‐channel. None of the derivatives had markedly greater affinity at A1‐receptors than cartazolate (Ki‐0.5 μM), but many had markedly lower affinity than cartazolate (Ki‐1.5 μM) at A2A‐receptors. At the benzodiazepine‐binding site of GABAA‐receptors some of the derivatives enhanced [3H]diazepam binding, as did cartazolate and GABA, some had no effect and some inhibited binding. Most of the derivatives inhibited binding of the benzodiazepine‐antagonist [3H]Ro 15‐1788. At the GABA‐binding site, only a few of the derivatives inhibited binding of the antagonist [3H]SR‐95531, as did GABA. At the picrotoxinin‐binding site, many inhibited binding of [35S]TBPS, but none were as potent as cartazolate or GABA. Analysis of the interactions indicates that stimulation of [3H]diazepam binding is allosteric and results from binding of the pyrazolopyridine at the GABA site or a subdomain of that site, while inhibition of [3H]Ro 15‐1788 binding is competitive and due to binding at the benzodiazepine site. Inhibition of [35S]TBPS binding at the picrotoxinin‐channel site appears to be allosteric through the GABA site and/or by direct competition at the channel site. Alterations in structure markedly alter the affinities of pyrazolopyridines at such sites on the GABAA‐receptor‐channel. Drug Dev. Res. 42:41–56, 1997.

Chronic NMDA receptor stimulation: therapeutic implications of its effect on adenosine A1 receptors

It is known that stimulation of adenosine A1 receptors has a modulatory effect on the excitability of postsynaptic NMDA receptors. Conversely, acute stimulation of NMDA receptors results in release of adenosine via calcium-independent mechanisms. These findings indicate a close functional relationship between these receptors. It is, therefore, possible that chronic, low level stimulation of the NMDA receptor may have a negative impact on these modulatory processes. To investigate this possibility, we have subjected C57BL mice either to an acute injection of a N6-cyclopentyladenosine (CPA, 0.01 mg/kg) or deoxycoformycin (1 mg/kg) followed by a convulsant dose of N-methyl-D-aspartate (NMDA) (60 mg/kg) or to chronic, low level (20 mg/kg i.p. daily) exposure to NMDA for 8 weeks. One day after the last injection of NMDA, animals were injected either with a convulsant dose of NMDA alone, or with either CPA at 0.001 or 0.01 mg/kg, or with 1 mg/kg deoxycoformycin followed 15 min later by 60 mg/kg NMDA. Neither CPA nor deoxycoformycin were protective when NMDA was given acutely at 60 mg/kg. Chronic treatment with NMDA alone or chronic administration of NMDA followed by 0.001 mg/kg CPA had no significant effect on mortality following a convulsant dose of NMDA. However, when the chronic regimen of NMDA was followed by either 0.01 mg/kg CPA or 1 mg/kg deoxycoformycin, mortality was reduced to 10% (CPA), or eliminated completely (deoxycoformycin). Moreover, combination of chronic NMDA treatment with either CPA (both doses) or deoxycoformycin produced a significant improvement in other measures, i.e., seizure onset, intensity of neurological impairment, and extension of time to death.(ABSTRACT TRUNCATED AT 250 WORDS)

Riboflavin: Inhibitory Effects on Receptors, G-Proteins, and Adenylate Cyclase

Riboflavin inhibited binding of both agonist and antagonist radioligands to rat brain A1‐adenosine receptors with Ki values of approximately 10 μM. In an adenylate cyclase assay with membrane preparations from either rat adipocytes or DDT MF‐2 cells, both of which contain A1‐adenosine receptors, riboflavin inhibited isoproterenol‐stimulated cyclase activity with an IC50 of approximately 20 μM. However, the inhibition of cyclase by riboflavin was not reversed by an A1‐selective antagonist, nor by pretreatment with pertussis toxin. Thus, neither A1‐receptors nor Gi‐proteins appear critically involved in the inhibition of cyclase by riboflavin. Riboflavin did block the stimulation by an adenosine analog of [35S]GTPγS binding in rat cerebral cortical membranes. However, riboflavin also inhibited the stimulation by fMLP of [35S]GTPγS binding in HL‐60 cell membranes. Riboflavin inhibited forskolin‐stimulated cyclase in membranes from DDT MF‐2 cells > rat adipocytes > PC12 cells, hamster CHO M2 cells, and wild‐type S49 cells. There was virtually no inhibition of forskolin‐stimulated cyclase in membranes of human platelets, rat cerebral cortex, or cyc‐S49 cells lacking Gs‐proteins. The calcium‐stimulated cyclase in rat cerebral cortical membranes was inhibited by riboflavin. A preincubation of membranes with riboflavin markedly enhanced the inhibition for DDT MF‐2 and wild‐type and cyc‐S49 membranes. The extent of inhibition in the different cell lines was dependent on the agent used to stimulate cyclase. Riboflavin, like the P‐site inhibitor 2′,5′‐dideoxyadenosine, was more potent and efficacious when manganese instead of forskolin was used as the stimulant. However, unlike the P‐site inhibitor, riboflavin did not markedly inhibit GppNHp‐ or fluoride‐stimulated cyclase. Riboflavin at low micromolar concentrations appears to have three possibly interrelated effects on second messenger systems subserved by G‐proteins. These are antagonism at A1‐adenosine receptors, inhibition of turnover of guanyl nucleotides at G‐proteins, and inhibition of adenylate cyclase. Drug Dev. Res. 42:98–108, 1997. Published 1997 Wiley‐Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.