2--3--o-(2-4-6-trinitrophenyl)adenosine-5--triphosphate and 5--adenylyl-(beta-gamma-methylene)diphosphonate

Experiments with selective agonists and antagonists of purinoceptors allowed us to evaluate the subtype of P2X receptors. We showed that the myocardium of 14- 100-day-old rats contains functionally active P2X1 receptors. These receptors are involved in the realization of the positive inotropic effect of the atria and ventricles. Selective P2X1 receptor agonist beta,gamma-methylene-ATP induced a dose-dependent increase in the strength of atrial and ventricular contractions. P2X1 receptor antagonist TNP-ATP abolished the effect of the agonist in rats of all age groups.

Topics: Adenosine Triphosphate; Aging; Animals; Heart; Myocardial Contraction; Myocardium; Purinergic P2 Receptor Agonists; Purinergic P2 Receptor Antagonists; Rats; Receptors, Purinergic P2; Receptors, Purinergic P2X

The pathophysiological mechanisms of orofacial deep-tissue pain is still unclear. Previously, P2X receptors (P2XR) in sensory neurons have been shown to play a role in the signal transduction of cutaneous pain. We investigated the functional significance of P2X3R in relation to orofacial deep-tissue pain caused by monoarthritis of the temporomandibular joint (TMJ). Monoarthritis was induced by the injection of complete Freund's adjuvant (CFA) into the unilateral TMJ of the rat. The pain associated with monoarthritis was assessed by the pressure pain threshold (PPT), which was defined as the amount of pressure required to induce vocalization. Fifteen days after CFA-treatment, changes in PPT were examined after injection of P2XR agonists or antagonists into the TMJ. The number of cells expressing P2X3R in trigeminal ganglia (TG) was investigated by immunohistochemistry. Inflamed TMJ showed a continuous decline in PPT during the experimental period (P<0.001). Injection of alpha,beta-meATP, an agonist of P2X1,3,2/3R, dramatically reduced the bilateral PPTs of both inflamed and non-inflamed TMJs (P<0.01) although beta,gamma-me-l-ATP, a selective agonist of P2X1R, did not. The decreased PPTs of inflamed TMJ were reversed either by PPADS, an antagonist of P2X1,2,3,5,1/5,4/5R, or by TNP-ATP, an antagonist of P2X1,3,2/3,1/5R. Immunohistochemically, the number of P2X3R-positive cells increased in the small cell group in TG (P<0.01), whereas there was no change in medium or large cell groups after the CFA-injection. Retrograde tracing confirmed that TMJ neurons in the TG exhibited P2X3R immunoreactivity. Our results suggested that P2X3R plays an important role in orofacial pressure pain caused by monoarthritis of TMJ.

Topics: Adenosine Triphosphate; Analysis of Variance; Animals; Arthritis; Cell Count; Drug Interactions; Facial Pain; Freund's Adjuvant; Functional Laterality; Immunohistochemistry; Male; Neurons; Pain Threshold; Purinergic P2 Receptor Agonists; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Rats; Rats, Inbred Lew; Receptors, Purinergic P2; Receptors, Purinergic P2X3; Stilbamidines; Temporomandibular Joint Disorders; Time Factors; Trigeminal Ganglion

Organisms lacking Gln-tRNA synthetase produce Gln-tRNA(Gln) from misacylated Glu-tRNA(Gln) through the transamidation activity of Glu-tRNA(Gln) amidotransferase (Glu-AdT). Glu-AdT hydrolyzes Gln to Glu and NH(3), using the latter product to transamidate Glu-tRNA(Gln) in concert with ATP hydrolysis. In the absence of the amido acceptor, Glu-tRNA(Gln), the enzyme has basal glutaminase activity that is unaffected by ATP. However, Glu-tRNA(Gln) activates the glutaminase activity of the enzyme about 10-fold; addition of ATP elicits a further 7-fold increase. These enhanced activities mainly result from increases in k(cat) without significant effects on the K(m) for Gln. To determine if ATP binding is sufficient to induce full activation, we tested a variety of ATP analogues for their ability to stimulate tRNA-dependent glutaminase activity. Despite their binding to Glu-AdT, none of the ATP analogues induced glutaminase activation except ATP-gammaS, which stimulates glutaminase activity to the same level as ATP, but without formation of Gln-tRNA(Gln). ATP-gammaS hydrolysis by Glu-AdT is very low in the absence or presence of Glu-tRNA(Gln) and Gln. In contrast, Glu-tRNA(Gln) stimulates basal ATP hydrolysis slightly, but full activation of ATP hydrolysis requires both Gln and Glu-tRNA(Gln). Simultaneous monitoring of ATP or ATP-gammaS hydrolysis and glutaminase and transamidase activities reveals tight coupling among these activities in the presence of ATP, with all three activities waning in concert when Glu-tRNA(Gln) levels become exhausted. ATP-gammaS stimulates the glutaminase activity to an extent similar to that with ATP, but without concomitant transamidase activity and with a very low level of ATP-gammaS hydrolysis. This uncoupling between ATP-gammaS hydrolysis and glutaminase activities suggests that the activation of glutaminase activity by ATP or ATP-gammaS, together with Glu-tRNA(Gln), results either from an allosteric effect due simply to binding of these analogues to the enzyme or from some structural changes that attend ATP or ATP-gammaS hydrolysis.

Topics: Adenosine Triphosphate; Bacillus subtilis; Binding Sites; Chromatography, High Pressure Liquid; Enzyme Activation; Hydrolysis; Kinetics; Nitrogenous Group Transferases; RNA, Transfer, Amino Acyl; Substrate Specificity