BACKGROUND: A Nerve ligation injury may produce a pain syndrome that includes tactile allodynia. Reversal effects on tactile allodynia have been demonstrated after an intrathecal administration of adenosine analogues or morphine. Adenosine receptor agonists have been known to have antinociceptive and antiallodynic effects in many animal and human studies. We examined the drug interactions between morphine and adenosine agonists in a rat model of a nerve ligation injury. METHODS: Male Sprague Dawley rats were prepared with a tight ligation of the left lumbar 5 th and 6 th spinal nerves and chronic lumbar intrathecal catheter implantation for drug administration. We measured the tactile allodynia by applying von Frey filaments ipsilateral to the lesioned hindpaw. Thresholds for paw withdrawal were assessed. Morphine (1 - 30ng), adenosine (1 - 30ng) and R-PIA (0.1 - 10ng) were administered to obtain the dose-response curves and the 50% effective dose (ED50). Fractions of ED50 values were administered to establish the ED50 of drug combinations. Drug interactions were evaluated by the fractional and isobolographic analyses. Allodynic thresholds for left lesioned hindpaw withdrawal to the von Frey hairs test were assessed and converted to % maximal possible effect (%MPE). RESULTS: The antiallodynic effect of morphine, adenosine, and R-PIA were produced in a dose dependent manner. The antiallodynic effects of combinations showed a similar pattern. Isobolographic analysis revealed a synergistic interaction for the morphine-R-PIA combination but not for the morphine-adenosine combination. However, fractional analysis produced a synergistic result for two combination groups. CONCLUSIONS: The results demonstrated that intrathecal co-administration of adenosine A1 receptors agonist and morphine showed the synergistic effect on nerve ligation injury induced allodynia.
Adenosine* ; Animals ; Catheters ; Drug Combinations ; Drug Interactions ; Hair ; Humans ; Hyperalgesia ; Injections, Spinal ; Ligation* ; Male ; Models, Animal ; Morphine* ; Purinergic P1 Receptor Agonists* ; Rats* ; Rats, Sprague-Dawley ; Receptor, Adenosine A1 ; Receptors, Purinergic P1* ; Spinal Nerves

BACKGROUND/AIMS: Adenosine is an endogenous modulator of nociception. Its role in visceral nociception, particularly in visceral hyperalgesia, has not been studied. The aim of this study was to determine the effects of adenosine receptor agonists in a model of visceral hyperalgesia. METHODS: The visceromotor response (VMR) in rats to colorectal distension (CRD; 80 mmHg, 20 seconds) was quantified by electromyographic recordings from the abdominal musculature. Three hours after the intracolonic administration of zymosan (25 mg/mL, 1 mL), VMRs to CRD were measured before and after either subcutaneous or intrathecal administration of an adenosine receptor agonist. RESULTS: Subcutaneous injection of 5'-N-ethylcarboxyamidoadenosine (NECA; an A1 and A2 receptor agonist), R(-)-N6-(2-phenylisopropyl)-adenosine (R-PIA; a selective A1 receptor agonist), or CGS-21680 hydrochloride (a selective A2a receptor agonist) dose-dependently (10-100 mg/kg) attenuated the VMR to CRD, although hindlimb weakness occurred at the higher doses tested. Intrathecal administration of NECA or R-PIA dose-dependently (0.1-1.0 microgram/kg) decreased the VMR, whereas CGS-21680 hydrochloride was ineffective over the same concentration range. Higher intrathecal doses of the A1/A2 receptor agonist NECA produced motor weakness. CONCLUSIONS: Adenosine receptor agonists are antihyperalgesic, but also produce motor weakness at high doses. However, activation of the spinal A1 receptor significantly attenuates the VMR to CRD without producing motor weakness.
Adenosine ; Adenosine-5'-(N-ethylcarboxamide) ; Animals ; Hindlimb ; Hyperalgesia ; Injections, Subcutaneous ; Nociception ; Purinergic P1 Receptor Agonists ; Rats ; Receptors, Purinergic P1 ; Zymosan

The aim of this study is to investigate the effect of isosorbide-5-mononitrate (ISMN) on the electric field stimulation induced sympathetic purinergic vasoconstriction of the rabbit saphenous arterial rings. Isometric vasoconstrictive responses to electric field stimulation and to exogenous noradrenaline and adenosine triphosphate were recorded. We found that the vasoconstrictive responses to electric field stimulation (15 V, 1 ms pulse duration, 2 - 16 Hz) were frequency-dependant in the rabbit saphenous arterial rings, and abolished by tetrodotoxin (0.1 micromol x L(-1)). The alpha1-adrenoceptor antagonist prazosin (1 micromol x L(-1)) did not affect the vascular responses to the electric field stimulation (2 -8 Hz). After a combination treatment with both alpha,beta-meATP (3 micromol x L(-1), desensitizing P2X1 receptors) and prazosin (1 micromol x L(-1)), the vasoconstrictive responses to electric field stimulation were abolished. When the arterial preparation was treated with ISMN (one preparation was exposed to only one concentration of ISMN), ISMN at 0.1 mmol x L(-1) significantly inhibited the vasoconstriction induced by electric stimulation at 8 Hz, 0.3 and 1.0 mmol x L(-1) significantly inhibited the vasoconstrictive responses to electric stimulation at 2 - 16 Hz. The highest concentration of ISMN (1.0 mmol x L(-1)) reduced the vasoconstrictive responses by 46% (2 Hz), 47% (4 Hz), 34% (8 Hz) and 22% (16 Hz), separately. ISMN (0.3 and 1.0 mmol x L(-1)) did not affect the vascular responses to exogenous noradrenaline (0.01-100 micromol x L(-1)) and adenosine triphosphate (1 mmol x L(-1)). It is reasonable to suggest that ISMN inhibits the purinergic vasoconstriction induced by sympathetic nerve stimulation via a prejunctional mechanism.
Adenosine Triphosphate ; analogs & derivatives ; pharmacology ; Adrenergic alpha-Antagonists ; pharmacology ; Animals ; Arteries ; drug effects ; Delayed-Action Preparations ; Electric Stimulation ; Isosorbide Dinitrate ; administration & dosage ; analogs & derivatives ; pharmacology ; Male ; Norepinephrine ; pharmacology ; Prazosin ; pharmacology ; Purinergic P2 Receptor Agonists ; Rabbits ; Receptors, Purinergic P2X ; Vasoconstriction ; drug effects

An important property of the intestine is the ability to secrete fluid. The intestinal secretion is regulated by a number of substances including vasoactive intestinal peptide (VIP), ATP and different inflammatory mediators. One of the most important secretagogues is adenosine during inflammation. However, the controversy concerning the underlying mechanism of adenosine-stimulated Cl- secretion in intestinal epithelial cells still continues. To investigate the effect of adenosine on Cl- secretion and its underlying mechanism in the rabbit colon mucosa, we measured short circuit current (ISC) under automatic voltage clamp with DVC-1000 in a modified Ussing chamber. Adenosine, when added to the basolateral side of the mucosa, increased ISC in a dose-dependent manner. The adenosine-stimulated ISC response was abolished when Cl- in the bath solution was replaced completely with gluconate. In addition, the ISC response was inhibited by a basolateral Na-K-Cl cotransporter blocker, bumetanide, and by apical Clchannel blockers, dephenylamine-2-carboxylate (DPC), 5-nitro-2-(3-phenyl-propylamino)-benzoate (NPPB), glibenclamide. Amiloride, an epithelial Na+ channel blocker, and 4,4-diisothiocyanato-stilbene-2,2-disulphonate (DIDS), a Ca2+-activated Cl- channel blocker, had no effect. In the mucosa pre-stimulated with forskolin, adenosine did not show any additive effect, whereas carbachol resulted in a synergistic potentiation of the ISC response. The adenosine response was inhibited by 10 micrometer H-89, an inhibitor of protein kinase A. These results suggest that the adenosine-stimulated ISC response is mediated by basolateral to apical Cl- secretion through a cAMP-dependent Cl- channel. The rank order of potencies of adenosine receptor agonists was 5'-(N-ethylcarboxamino)adenosine(NECA) > N6-(R-phenylisopropyl)adenosine(R-PIA)>2-(p-(2-carbonylethyl)-phenyl-et hylamino)-5'-N-ethylcarboxaminoadenosine(CGS21680). From the above results, it can be concluded that adenosine interacts with the A2b adenosine receptor in the rabbit colon mucosa and a cAMP-dependent signalling mechanism underlies the stimulation of Cl- secretion.
Adenosine ; Adenosine Triphosphate ; Amiloride ; Baths ; Bumetanide ; Carbachol ; Colforsin ; Colon* ; Cyclic AMP-Dependent Protein Kinases ; Epithelial Cells ; Glyburide ; Inflammation ; Intestinal Secretions ; Intestines ; Mucous Membrane* ; Purinergic P1 Receptor Agonists ; Receptors, Purinergic P1 ; Vasoactive Intestinal Peptide

PURPOSE: To evaluate changes in the tear meniscus area and tear meniscus height over time in patients with dry eye syndrome, using anterior segment spectral-domain optical coherence tomography after the instillation of 3% diquafosol ophthalmic solution. METHODS: Sixty eyes from 30 patients with mild to moderate dry eye syndrome were included. Tear meniscus images acquired by anterior segment spectral-domain optical coherence tomography were analyzed using National Institutes of Health's image-analysis software (ImageJ 1.44p). Tear meniscus area and tear meniscus height were measured at baseline, 5 minutes, 10 minutes, and 30 minutes after instillation of a drop of diquafosol in one eye and normal saline in the other eye. Changes in ocular surface disease index score, tear film break-up time, corneal staining score by Oxford schema, and meibomian expressibility were also evaluated at baseline, and after 1 week and 1 month of a diquafosol daily regimen. RESULTS: Sixty eyes from 30 subjects (mean age, 29.3 years; 8 men and 22 women) were included. In eyes receiving diquafosol, tear volume was increased at 5 and 10 minutes compared with baseline. It was also higher than saline instilled eyes at 5, 10, and 30 minutes. Changes in tear volume with respect to baseline were not statistically different after the use of diquafosol for 1 month. Ocular surface disease index score, tear film break-up time, and Oxford cornea stain score were significantly improved after 1 week and 1 month of daily diquafosol instillation, but meibomian expressibility did not change. CONCLUSIONS: Topical diquafosol ophthalmic solution effectively increased tear volume for up to 30 minutes, compared to normal saline in patients with dry eye syndrome.
Academies and Institutes ; Cornea ; Dry Eye Syndromes* ; Humans ; Male ; Ophthalmic Solutions ; Purinergic P2Y Receptor Agonists ; Tears* ; Tomography, Optical Coherence*

BACKGROUND: Nerve ligation injury in rats produces a pain syndrome that includes mechanical allodynia. Intrathecal administration of cholinesterase inhibitors or adenosine receptor agonists have anti-allodynic effects in this model. Therefore, we tested the interaction between intrathecal neostigmine and N(6)-cyclohexyladenosine (CHA) in a rat behavioral model of neuropathic pain. METHODS: Male Sprague-Dawley rats were prepared with tight ligation of the spinal nerves for producing allodynia and with a lumbar intrathecal catheter for drug administration. Allodynia thresholds for hindpaw withdrawal against mechanical stimuli were assessed and converted to percent maximal possible effect. Neostigmine (0.3-10 microgram) and CHA (0.03-3 microgram) were administered to obtain the dose-response curves and the 50% effective dose (ED(50)). Equal fractions (1/2, 1/4 and 1/8 ED(50)s) of the two drugs were administered to establish the ED(50) of neostigmine-CHA combination. Side effects were also assessed. The drug interaction was evaluated by isobolographic and fractional analyses. RESULTS: Neostigmine, CHA, and the neostigmine-CHA combination dose-dependently produced anti-allodynia effects. Side effects such as sedation and motor weakness were similar in the three groups. In the isobolographic analysis, the experimental ED(50) for the combination of neostigmine-CHA lay far below and to the left of the theoretical additive line. Fractional analysis indicated that the total combination fraction of the two drugs was 0.39. CONCLUSIONS: Intrathecal co-administration of neostigmine and CHA showed a synergistic anti-allodynia effect.
Adenosine ; Animals ; Catheters ; Cholinesterase Inhibitors ; Drug Interactions ; Humans ; Hyperalgesia ; Injections, Spinal ; Ligation ; Male ; Neostigmine ; Neuralgia ; Purinergic P1 Receptor Agonists ; Rats ; Rats, Sprague-Dawley ; Spinal Nerves

BACKGROUNDThe amiloride-sensitive epithelial sodium channel α-subunit (α-ENaC) is an important factor for alveolar fluid clearance during acute lung injury. The relationship between adenosine receptor A(2a) (A(2a)AR) expressed in alveolar epithelial cells and α-ENaC is poorly understood. We targeted the A(2a)AR in this study to investigate its role in the expression of α-ENaC and in acute lung injury.

METHODSA549 cells were incubated with different concentrations of A(2a)AR agonist CGS-21680 and with 100 µmol/L CGS-21680 for various times. Rats were treated with lipopolysaccharide (LPS) after CGS-21680 was injected. Animals were sacrificed and tissue was harvested for evaluation of lung injury by analysis of the lung wet-to-dry weight ratio, lung permeability and myeloperoxidase activity. RT-PCR and Western blotting were used to determine the mRNA and protein expression levels of α-ENaC in A549 cells and alveolar type II epithelial cells.

RESULTSBoth mRNA and protein levels of α-ENaC were markedly higher from 4 hours to 24 hours after exposure to 100 µmol/L CGS-21680. There were significant changes from 0.1 µmol/L to 100 µmol/L CGS-21680, with a positive correlation between increased concentrations of CGS-21680 and expression of α-ENaC. Treatment with CGS-21680 during LPS induced lung injury protected the lung and promoted α-ENaC expression in the alveolar epithelial cells.

CONCLUSIONActivation of A(2a)AR has a protective effect during the lung injury, which may be beneficial to the prognosis of acute lung injury.

Acute Lung Injury ; metabolism ; Adenosine ; analogs & derivatives ; pharmacology ; Animals ; Blotting, Western ; Cell Line ; Epithelial Sodium Channels ; genetics ; metabolism ; Humans ; Male ; Phenethylamines ; pharmacology ; Pulmonary Alveoli ; cytology ; metabolism ; Purinergic P1 Receptor Agonists ; pharmacology ; Rats ; Receptors, Purinergic P1 ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo study the therapeutic efficacy and mechanism of adenosine receptor agonist N(6)-cyclopentyladenosine (CPA) against acute dichlorvos poisoning.

METHODSSoon after a certain doses of dichlorvos were given to mice and rats by gastrogavage, physiological saline, CPA, atropine or pralidoxime chloride were administered to different groups. Toxic signs and survival rate were recorded and cholinesterase (ChE) activities and acetylcholine (ACh) concentrations in whole blood were determined in treatment group with CPA and non-treatment group after dichlorvos was given to rats by gastrogavage.

RESULTS(1) The alleviated and delayed appearance of toxic signs as well as obvious prolongation of survival time was observed in CPA treatment group compared with non-treatment group. (2) ChE activities in both treatment group with CPA [(0.49 +/- 0.05) U/ml] and non-treatment group [(0.52 +/- 0.04) U/ml] were significantly lower than that [(1.56 +/- 0.15) U/ml] of the control group (P < 0.01), but there was no significant difference between treatment group and non-treatment group (P > 0.05). (3) ACh concentration [(204.24 +/- 20.48) microg/ml] in whole blood of treatment group with CPA was significantly lower than that [(230.91 +/- 25.61) microg/ml] of non-treatment group (P < 0.05).

CONCLUSIONCPA has therapeutic efficacy against acute dichlorvos poisoning, which is probably through the decrease in ACh concentration.

Acetylcholine ; blood ; Acute Disease ; Adenosine ; analogs & derivatives ; therapeutic use ; Animals ; Cholinesterases ; blood ; Dichlorvos ; poisoning ; Insecticides ; poisoning ; Male ; Mice ; Mice, Inbred Strains ; Poisoning ; blood ; drug therapy ; Purinergic P1 Receptor Agonists ; Rats ; Rats, Sprague-Dawley

This study is to observe the difference in pharmacological characteristics between circular smooth muscles of rat isolated gastric body and gastric fundus, and to investigate the effects of nucleoside and nucleotide on circular smooth muscle of the rat gastric body and the involved receptors. Circular muscle strips of the rat gastric body and gastric fundus were prepared, and contractile responses to agonists were investigated with a technique of drug-receptor interaction in functional system. There was no significant difference between the circular muscle strips of the gastric body and gastric fundus in the responses to KCl, and no difference in EC50 values of contractile responses for 5-HT and His between the two kinds of preparations (P > 0.05). However, Emax values of contractile responses to 5-HT and His [(0.81 +/- 0.26) and (0.88 +/- 0.27) g] in gastric body were significantly smaller than those in gastric fundus [(2.67 +/- 0.61) and (1.90 +/- 0.68) g, P < 0.01], and EC50 value of CCh produced contractile response [(0.45 +/- 0.15) micromol x L(-1)] in gastric body was significantly higher than that in gastric fundus [(0.20 +/- 0.09) micromol x L(-1), P < 0.01]. In precontracted circular muscle strips of the gastric body, ATP (0.1-3000 micromol x L(-1)) produced only a contractile response concentration-dependently, but the same concentration of ATP induced a biphasic response (relaxation followed by a contraction) in precontracted circular muscle strips of the gastric fundus. ATP, UTP, ADP, 2-MeSATP and alpha,beta-MeATP produced contractile responses concentration-dependently in circular muscle strips of the rat gastric body. The EC50 value for 2-MeSATP [(7.2 +/- 5.2) nmol x L(-1)] was about 500 times lower than that for Ach [(3.47 +/- 1.20) micromol x L(-1)]. The rank order of potency for the contraction was 2-MeSATP>ADP>ATP=UTP>alpha,beta-MeATP>adenosine. The contractile responses to ATP and UTP were not significantly affected by phentolamine, propranolol, atropine or tetrodotoxin. In conclusion, there is a significant difference in pharmacological characteristics between the circular smooth muscles of the rat gastric body and gastric fundus and nucleotides might be important mediators responsible for the contraction via a specific P2Y receptor in circular smooth muscle of the rat gastric body.
Adenosine ; pharmacology ; Adenosine Diphosphate ; pharmacology ; Adenosine Triphosphate ; analogs & derivatives ; pharmacology ; Animals ; Gastric Fundus ; drug effects ; physiology ; Male ; Muscle Contraction ; drug effects ; Muscle, Smooth ; drug effects ; physiology ; Purinergic P2 Receptor Agonists ; Rats ; Rats, Wistar ; Receptors, Purinergic P2 ; drug effects ; Stomach ; drug effects ; physiology ; Thionucleotides ; pharmacology ; Uridine Triphosphate ; pharmacology

OBJECTIVERecent studies showed that adenosine played important roles in vasodilation. This study aimed to investigate the effects of adenosine, its A1 and A2b receptor agonists on pulmonary artery hypertension (PAH) induced by chronic hypoxia in rats by continuously subcutaneous administration with an osmotic pump for 14 days, and to see if rennin angiotensin system and inducible nitric oxygen synthase (iNOS)/nitric oxide (NO) mediate the effects.

METHODFifty-six male SD rats were randomly assigned to seven groups. Each group included eight rats. They were normoxic group, hypoxic group, adenosine-treated group [adenosine was administered at a dose of 150 µg(kg·min) under the hypoxic condition], adenosine A1 receptor agonist CPA-treated group [CPA was administered at a dose of 20 µg/(kg·min) under the hypoxic condition], CPA plus selective adenosine A1 antagonist DPCPX-treated group [CPA and DPCPX were administered simultaneously under the hypoxic condition, the dose of CPA was the same as the above, and the dose of DPCPX was 25 µg/(kg·min)], adenosine A2b receptor agonist NECA-treated group [NECA was administered at a dose of 30 µg/(kg·min) under the hypoxic condition], NECA plus selective adenosine A2b receptor antagonist MRS-treated group[ NECA and MRS1754 were administered simultaneously under the hypoxic condition, the dose of NECA was the same as the above, and the dose of MRS1754 was 50 µg/(kg·min)]. Osmotic pumps containing adenosine or selective adenosine A1 receptor agonist (CPA), or nonselective but potent adenosine A2b receptor agonist (NECA) were placed subcutaneously 7 days after hypoxia and continuously administered the agents for 14 days.Mean pulmonary artery pressure (mPAP) was detected after administration of the agents. Then blood samples were taken from heart for measurement of renin activity, angiotensin II (AngII) and endothelin-1 (ET-1) concentration by radioimmunoassay, NO by measuring nitrate. Small pulmonary arteries were prepared for immunoreactivity staining of proliferating cell nuclear antigen (PCNA) and iNOS.

RESULT(1) Chronic hypoxia induced PAH [mPAP: (31.38 ± 3.42) mm Hg]. Adenosine or CPA or NECA administered for 14 days by subcutaneous route attenuated the mPAP [(21.17 ± 3.56) mm Hg, (22.88 ± 2.95) mm Hg, (19.81 ± 2.39) mm Hg, respectively], which showed significant difference when compared with hypoxia group (P < 0.05 respectively). (2) Plasma rennin activity and AngII level in hypoxia group [(2.51 ± 0.25) ng/(ml·h), (83.01 ± 9.38) pg/ml] were significantly higher than that in normoxic group (P < 0.05, respectively).(3) Adenosine treatment decreased the rennin activity and AngII level when compared with hypoxic group(P < 0.05, respectively);CPA and NECA attenuated respectively the rennin activity and AngII level of rats induced by chronic hypoxia (P < 0.05, respectively). (4) Adenosine administration for 14 days attenuated the wall thickness induced by chronic hypoxia (P < 0.05). CPA showed no effect on wall thickness, but NECA significantly attenuated the wall thickness (P < 0.05). (5) The number of iNOS staining positive cells in small pulmonary artery was higher in hypoxia group than in that in normoxic rats (23.75 ± 7.91 vs. 8.00 ± 2.20, P < 0.05). Adenosine or CPA, or NECA administration increased respectively the iNOS expression in rats treated with chronic hypoxia. Chronic hypoxia caused significant decrease of nitric oxide level. Adenosine treatment increased the nitric oxide level in rats treated with chronic hypoxia. CPA and NECA also increased respectively the nitric oxide level in rats treated with chronic hypoxia. Chronic hypoxia caused significant increase of ET-1 level. The ET-1 level in rats treated with adenosine, CPA or NCEA respectively were lower than that in chronic hypoxia rats (P < 0.05). (6) Adenosine treatment partially attenuated the number of PCNA-positively stained cells. NECA treatment also attenuated the PCNA expression, but CPA showed no effect.

CONCLUSIONAdenosine and its agonists CPA, NECA administered continually by subcutaneous route attenuate mPAP of rats induced by chronic hypoxia. CPA attenuates mPAP through reduction of RA/AngII activity and balance of NO/ET-1 level. NECA attenuates mPAP by inhibiting PCNA expression and proliferation of mooth muscle of pulmonary artery.

Adenosine ; administration & dosage ; pharmacology ; Angiotensin II ; blood ; Animals ; Disease Models, Animal ; Endothelin-1 ; metabolism ; Hypertension, Pulmonary ; drug therapy ; etiology ; metabolism ; Hypoxia ; complications ; Male ; Nitric Oxide ; blood ; Nitric Oxide Synthase Type II ; metabolism ; Proliferating Cell Nuclear Antigen ; metabolism ; Pulmonary Artery ; drug effects ; physiopathology ; Purinergic P1 Receptor Agonists ; administration & dosage ; pharmacology ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Renin ; blood