Adenosine A1 Receptors In Rats Research Articles

Electroacupuncture Pretreatment Prevents Cognitive Impairment Induced by Cerebral Ischemia-Reperfusion via Adenosine A1 Receptors in Rats.

A previous study has demonstrated that pretreatment with electroacupuncture (EA) induces rapid tolerance to focal cerebral ischemia. In the present study, we investigated whether adenosine receptor 1 (A1 R) is involved in EA pretreatment-induced cognitive impairment after focal cerebral ischemia in rats. Two hours after EA pretreatment, focal cerebral ischemia was induced by middle cerebral artery occlusion for 120 min in male Sprague-Dawley rats. The neurobehavioral score, cognitive function [as determined by the Morris water maze (MWM) test], neuronal number, and the Bax/Bcl-2 ratio was evaluated at 24 h after reperfusion in the presence or absence of CCPA (a selective A1 receptor agonist), DPCPX (a selective A1 receptor antagonist) into left lateral ventricle, or A1 short interfering RNA into the hippocampus area. The expression of the A1 receptor in the hippocampus was also investigated. The result showed that EA pretreatment upregulated the neuronal expression of the A1 receptor in the rat hippocampus at 90 min. And EA pretreatment reversed cognitive impairment, improved neurological outcome, and inhibited apoptosis at 24 h after reperfusion. Pretreatment with CCPA could imitate the beneficial effects of EA pretreatment. But the EA pretreatment effects were abolished by DPCPX. Furthermore, A1 receptor protein was reduced by A1 short interfering RNA which attenuated EA pretreatment-induced cognitive impairment.

Long residence time adenosine A1 receptor agonists produce sustained wash-resistant antilipolytic effect in rat adipocytes

Elevated circulating free fatty acid (FFA) level is closely linked to the pathogenesis of insulin resistance and type 2 diabetes mellitus. Activation of the adenosine A1 receptor (A1R) inhibits lipolysis in adipocytes and hence reduces the concentration of FFA, which represents a potential target for the development of antilipolytic agents. We aimed to assess the binding affinity as well as target binding kinetics of A1R agonists and further delineate a possible relationship with their antilipolytic effect in adipocytes. Radioligand binding assays were performed to determine the affinity and kinetics of three representative A1R agonists, namely CPA, LUF6944 and LUF6941, on the rat A1R. Functional responses to these agonists were examined in both a recombinant cell system and physiologically relevant rat adipocytes. The three A1R agonists displayed similar affinity while divergent target binding kinetics on the rat A1R. Irrespective of equilibrium binding affinity, temporal analysis of receptor signaling demonstrated persistent functional responses for the long residence time agonist, despite removal of excess agonist, in both a recombinant cell system and in rat adipocytes. By contrast, such effect was less pronounced or even lost for agonists with medium or short receptor residence time, respectively. Our results indicate that ligand receptor binding kinetics rather than their affinity or potency play an essential role in regulating cellular responses. The long residence time A1R agonist produces a sustained wash-resistant antilipolytic effect in rat adipocytes and thus may represent a potential antilipolytic alternative for further investigation.

Hemorrhagic preconditioning improves vascular reactivity after hemorrhagic shock by activation of PKC[alpha] and PKC[epsilon] via the adenosine A1 receptor in rats

BACKGROUND: Our previous study demonstrated that protein kinase C (PKC) has an important protective role on vascular reactivity and calcium sensitivity after shock. Here, we investigated if hemorrhagic preconditioning could lessen shock-induced vascular hyporeactivity by activating PKC. METHODS: Using hemorrhagic-shocked rats, the protective effects of different extents of hemorrhagic preconditioning on vascular reactivity and calcium sensitivity; the roles of PKC[alpha], PKC[epsilon], and adenosine in this process; as well as hemorrhagic preconditioning-induced systemic effects were observed. RESULTS: Hemorrhage preconditioning (particularly hemorrhage involving 5% of the total estimated blood volume implemented 30 minutes before shock) significantly improved vascular reactivity and calcium sensitivity after shock. Hemorrhage preconditioning enhanced the translocation of PKC[alpha] and PKC[epsilon] from the cytoplasm to the membrane and increased the blood concentration of adenosine after shock. Antagonists of PKC[alpha], PKC[epsilon], and the adenosine A1 receptor abolished the hemorrhagic preconditioning-induced protective effects on vascular reactivity and calcium sensitivity. The adenosine A1 receptor antagonist eliminated hemorrhagic preconditioning-induced translocation of PKC[alpha] and PKC[epsilon]. Hemorrhagic preconditioning could significantly increase survival, improve hemodynamic parameters, and increase the blood flow and mitochondrial respiratory function of the liver and kidney in hemorrhagic-shock rats. CONCLUSION: Hemorrhagic preconditioning could induce the protection of vascular reactivity and calcium sensitivity after hemorrhagic shock through the adenosine–adenosine A1 receptor–PKC[alpha] and PKC[epsilon] signaling pathway and could bring further beneficial systemic effects in hemorrhagic-shock rats.

Hemorrhagic preconditioning improves vascular reactivity after hemorrhagic shock by activation of PKCα and PKCε via the adenosine A1 receptor in rats

Our previous study demonstrated that protein kinase C (PKC) has an important protective role on vascular reactivity and calcium sensitivity after shock. Here, we investigated if hemorrhagic preconditioning could lessen shock-induced vascular hyporeactivity by activating PKC. Using hemorrhagic-shocked rats, the protective effects of different extents of hemorrhagic preconditioning on vascular reactivity and calcium sensitivity; the roles of PKCα, PKCε, and adenosine in this process; as well as hemorrhagic preconditioning-induced systemic effects were observed. Hemorrhage preconditioning (particularly hemorrhage involving 5% of the total estimated blood volume implemented 30 minutes before shock) significantly improved vascular reactivity and calcium sensitivity after shock. Hemorrhage preconditioning enhanced the translocation of PKCα and PKCε from the cytoplasm to the membrane and increased the blood concentration of adenosine after shock. Antagonists of PKCα, PKCε, and the adenosine A1 receptor abolished the hemorrhagic preconditioning-induced protective effects on vascular reactivity and calcium sensitivity. The adenosine A1 receptor antagonist eliminated hemorrhagic preconditioning-induced translocation of PKCα and PKCε. Hemorrhagic preconditioning could significantly increase survival, improve hemodynamic parameters, and increase the blood flow and mitochondrial respiratory function of the liver and kidney in hemorrhagic-shock rats. Hemorrhagic preconditioning could induce the protection of vascular reactivity and calcium sensitivity after hemorrhagic shock through the adenosine-adenosine A1 receptor-PKCα and PKCε signaling pathway and could bring further beneficial systemic effects in hemorrhagic-shock rats.

REVERSAL OF NEUROMUSCULAR EFFECTS OF ADENOSINE BY SPECIFIC ADENOSINE A1-RECEPTOR ANTAGONIST IN LIVE RATS

Intravenous adenosine in-vivo was shown to potentiate the effects of non-depolarizing neuromuscular blocking agents. This study aimed to determine whether adenosine A1-receptors mediated this potentiation. The authors investigated the effects of intravenous adenosine, N6-cyclopentyladenosine, specific A1-receptor agonist, and 8-cyclopentyl-1,3-dipropylxanthine, specific A1-receptor antagonist, on neuromuscular block by vecuronium, in in-vivo rat sciatic nervetibialis anterior preparations. In the presence of 50% steady state block by vecuronium, adenosine, and N6-cyclopentyladenosine caused similar degree of depressions of twitch tension. Twitch tension returned to its pre-injection value more rapidly when 8-cyclopentyl-1,3-dipropylxanthine was given at the maximal block than when it was allowed to recover spontaneously. It was concluded that in in-vivo adenosine potentiated the neuromuscular effects of vecuronium through adenosine A1-receptors in rats.

Functional expression of rat adenosine A1 receptor in the dimorphic zygomycete Mucor circinelloides.

We have produced the rat adenosine A1 receptor in Mucor circinelloides using a translational fusion to the endogenous glucoamylase (GlaM) gene. The fusion protein produced from an episomal plasmid was correctly processed as judged by western blotting, since only a 33 kDa band was detected in membrane preparations from M. circinelloides expressing the receptor. This corresponds to the mass of the full-length receptor released from the fused GlaM protein. The presence of a high affinity binding site with a K(d) value of 0.5 nM for the receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) in membrane preparations suggests that the receptor was correctly folded and inserted into the membranes. A receptor expression level of 100-300 fmol/mg total membrane protein was achieved as judged by binding of the antagonist [(3)H]-DPCPX.

Allosteric modulation of the rat adenosine A1 receptor: Differential effects on agonist and antagonist binding

The interaction of 2-amino-3-benzoyl-thiophene derivative, PD81,723, as well as other G protein-coupled receptor (GPCR)-modulating agents such as suramin (SUR), N-ethylmaleimide (NEM), GTP, and NaCl with the rat adenosine A1 receptor was investigated using kinetic, saturation, as well as displacement experiments of various agonists, partial agonists or antagonists. PD81,723 enhanced agonist (CPA, R-PIA, NECA) binding ∼2-fold, while its effect on CPA binding was increased (4–11-fold) with other modulators present. In contrast, binding of antagonists (DPCPX, CPT, N-0840) was inhibited, while binding of partial agonists (8BCPA, MeSCPA) remained uninfluenced. The effect of PD81,723 is consistent with shifting A1 receptors to an “active” (R*) state with high affinity for agonists and low for antagonists. Further, all described allosteric modulators influenced both agonist and antagonist binding. The IC50 values observed for the agonist CPA, ranging from 4.7 nM in the presence of PD81,723 to a high value of 2.9 μM in the combined presence of NEM, NaCl, and GTP, represented a greater than 600-fold affinity shift. We suggest that the latter micromolar IC50 value may approximate CPA’s “true” affinity (KA) for the rat adenosine A1 receptor. Drug Dev. Res. 51:207–215, 2000. © 2001 Wiley-Liss, Inc.

Elucidation of structure–activity relationships of 2‐amino‐3‐benzoylthiophenes: Study of their allosteric enhancing vs. antagonistic activity on adenosine A1 receptors

Novel 2-amino-3-benzoylthiophene derivatives, with variable substitution on the thiophene as well as benzoyl ring, were synthesized and evaluated both as allosteric enhancers of agonist binding to the rat adenosine A1 receptor, and as antagonists on this receptor. Structural features were identified on the novel derivatives that favored allosteric enhancing activity, such as benzoyl lipophilic substitution and thiophene 4-alkyl substitution. In contrast, antagonistic properties were favored by thiophene 5-bulky substitution. Upon further analysis, a significant correlation was found between antagonistic activity and hydrophobic fragment constants (π values) of substituent R5, in contrast to a negative correlation with those of R4. Comparison of low energy conformations of some of the 2-amino-3-benzoylthiophene derivatives (PD81,723 and 4f) with known adenosine A1 antagonists (theophylline and 8-cyclohexyltheophylline) indicated that thiophene 5-substituents may interact with the same lipophilic domain of the adenosine A1 receptor accommodating 8-substituents of xanthine antagonists. Drug Dev. Res. 49:227–237, 2000. © 2000 Wiley-Liss, Inc.

Elucidation of structure-activity relationships of 2-amino-3-benzoylthiophenes: Study of their allosteric enhancing vs. antagonistic activity on adenosine A1 receptors

Novel 2-amino-3-benzoylthiophene derivatives, with variable substitution on the thiophene as well as benzoyl ring, were synthesized and evaluated both as allosteric enhancers of agonist binding to the rat adenosine A1 receptor, and as antagonists on this receptor. Structural features were identified on the novel derivatives that favored allosteric enhancing activity, such as benzoyl lipophilic substitution and thiophene 4-alkyl substitution. In contrast, antagonistic properties were favored by thiophene 5-bulky substitution. Upon further analysis, a significant correlation was found between antagonistic activity and hydrophobic fragment constants (π values) of substituent R5, in contrast to a negative correlation with those of R4. Comparison of low energy conformations of some of the 2-amino-3-benzoylthiophene derivatives (PD81,723 and 4f) with known adenosine A1 antagonists (theophylline and 8-cyclohexyltheophylline) indicated that thiophene 5-substituents may interact with the same lipophilic domain of the adenosine A1 receptor accommodating 8-substituents of xanthine antagonists. Drug Dev. Res. 49:227–237, 2000. © 2000 Wiley-Liss, Inc.

Working memory failure by stimulation of hippocampal adenosine A1 receptors in rats.

THE highly selective adenosine A1 receptor agonist N6-cyclohexyladenosine (CHA) significantly increased the number of errors (attempts to pass through two incorrect panels of the three panel-gates at four choice points) in the working memory task with a three-panel runway setup, when injected bilaterally at 0.3 and 1.0 microgram per side into the dorsal hippocampus. Pretreatment with the selective adenosine A1 receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 1 mg kg-1, i.p.) prevented the increase in working memory errors induced by intrahippocampal administration of CHA (1.0 microgram per side). CGS-21680, a highly selective agonist for adenosine A2 receptors, had no effect on the number of working memory errors, when administered intrahippocampally at a dose of 1.0 microgram per side. These results suggest that activation of hippocampal adenosine A1 receptors interferes with neural processes involved in working memory function.

Chronic intrathecal morphine treatment does not cause down-regulation of spinal adenosine A1 receptors in rats.

We have shown previously that systemic chronic morphine treatment causes down-regulation of spinal adenosine A1 receptors in rats. Recently, we have found that chronic supraspinal morphine treatment also causes this effect. In the present study, we investigated whether chronic spinal morphine treatment has the same effect of down-regulation of spinal adenosine A1 receptors. Adult male Sprague-Dawley rats were rendered tolerant to morphine either by multiple intrathecal (i.t.) injections or continuous i.t. infusion by osmotic pump administration for 2 or 4 days. Spinal A1-adenosine receptor binding activity was measured by using the selective A1 adenosine agonist [3H]cyclohexyladenosine. No significant decrease in [3H]cyclohexyladenosine binding was found in the spinal cord after 2 or 4 days of multiple i.t. injections of morphine. There was also no significant change in the amount of spinal [3H]cyclohexyladenosine bound after 4 days of continuous i.t. infusion of morphine by osmotic pump. From these and our previous results, it is concluded that only supraspinal chronic morphine treatment down regulates the spinal A1 adenosine receptor and this may play a role in the mechanism of supraspinal morphine tolerance but not spinal morphine tolerance.