Interaction of hydrocortisone with ATP and adenosine on nerve-mediated contractions of frog skeletal muscle.

The inhibitory effects of ATP and adenosine on the nerve-mediated contractile responses of isolated sartorius muscle of the frog, Rana ridibunda, evoked by electrical field stimulation (EFS) were studied using pharmacological organ-bath technique. The effects of hydrocortisone applied in vitro and in vivo on contractility of sartorius muscle were also examined. ATP (100 microM) significantly reduced the amplitude of contraction to EFS of sartorius muscle, while pyridoxalphosphate-6-azonphenyl-2',4'-disulfonic acid (PPADS; 10 microM), a P2 receptor antagonist, abolished inhibitory effect of ATP. A similar inhibitory effect of adenosine (100 microM) was fully antagonized by 8-(p-sulfophenyl)-theophylline (8-SPT, 100 microM), a P1 receptor antagonist. Incubation of the tissue with hydrocortisone (10 microM) caused a slight, but significant, decrease of muscle contractions. After incubation of muscle preparations with both hydrocortisone and ATP, no inhibition of muscle contractility was registered. A single injection of hydrocortisone (100 mg/kg) 12 h prior to experiments to frogs did not significantly change the nerve-mediated contractility of isolated sartorius muscle; however, it abolished the inhibitory action of ATP without changing inhibitory activity of adenosine. After treatment of frogs with hydrocortisone for 14 days (100 mg/kg/day), both ATP and adenosine retained their inhibitory action on EFS-induced contractions of the muscle, and their effects were antagonized by PPADS and 8-SPT, respectively. It is concluded that hydrocortisone has antagonistic actions against the inhibitory effects of ATP at the frog neuromuscular junction, although this effect is lost following long-term treatment with hydrocortisone.

Ovalbumin-induced sensitization affects non-quantal acetylcholine release from motor nerve terminals and alters contractility of skeletal muscles in mice.

Skeletal muscles play key roles in the development of various pathologies, including bronchial asthma and several types of auto-immune disorders, e.g. polymyositis. Since most of these maladies have an immunological/allergic element, this paper is devoted to assessing the impact of immunobiological reorganization on the functional properties of isolated skeletal muscles in mice. A combination of two methods (myography and electrophysiology) was used to evaluate extensor digitorum longus (EDL) and diaphragmatic muscle (DM) in this regard. Conventional myographic technique showed that ovalbumin-induced sensitization (OS) produced different changes in the contractile properties of EDL and DM. The amplitudes of carbachol (CCh)-induced contractions increased in DM but decreased in EDL. Those changes were inversely related to OS-mediated changes of non-quantal acetylcholine (ACh) release intensity within the muscle endplate, as shown by the electrophysiologically measured H-effect. These results clearly show that OS-mediated changes of non-quantal ACh release alter the functional properties of postjunctional ACh receptors and therefore contribute to the disturbance of CCh-induced contractility of skeletal muscles. Other mechanisms of OS-mediated changes of skeletal muscle contractility are also proposed and discussed.

Contrasting effects of P2 receptor agonists on spontaneous contractility of human fallopian tubes with and without acute inflammation.

Two groups of women were used to compare effects of P2 receptor agonists on the contractile activity of isolated human fallopian tubes with and without acute purulent inflammation. A control group included women with uterine tubes without inflammation (n=15). A study group included women, operated for unsuccessful conservative anti-inflammatory treatment of acute purulent tuboovarial formations (group with acute purulent salpingitis, n=16). Division into two groups was done according to pathohistological diagnosis. Spontaneous contractions of the isolated tubes were registered isometrically with electromechanical transducer before and after incubation of the tissues with agonists of P2 receptors-ATP, UTP, 2-methylthio-ATP and alpha,beta-methylene-ATP. In the control group neither of the agonists produced any significant effect on fallopian tube contractility. In the study group, ATP, 2-methylthio-ATP and alpha,beta-methylene-ATP significantly increased the spontaneous contractility of isolated tubes. It is suggested that higher activity of P2 receptor agonists in the uterine tubes with acute purulent inflammation is due to expression of several subtypes of P2 receptors during inflammation.

Hypotensive effect of UDP on intraocular pressure in rabbits.

Nucleotides can modify intraocular pressure (IOP). We have tested the ability of uridine-5'-diphosphate, UDP, for modulating IOP in New Zealand white rabbits. Uridine 5' diphosphate, UDP, reduced IOP by 82.9+/-2.6% compared to control. Dose-response analysis demonstrated a concentration dependent pattern which presented a pD(2) value of 7.57+/-1.45, equivalent to an EC(50) of 26.91 nM. Of all the tested P2 receptor antagonists, suramin, pyridoxalphosphate-6-azophenyl-2, 4-disulfonic acid (PPADS) and Reactive Blue 2 (RB-2), only the last two were able to reverse the action triggered by UDP. Altogether, UDP acting probably on P2Y(6) receptors present on the ciliary processes, can reduce intraocular pressure, indicating that this substance may be used for the treatment of ocular hypertension and glaucoma.

Different effects of ATP on the contractile activity of mice diaphragmatic and skeletal muscles.

Apart from acetyl-choline (Ach), adenosine-5'-trisphosphate (ATP) is thought to play a role in neuromuscular function, however little information is available on its cellular physiology. As such, effects of ATP and adenosine on contractility of mice diaphragmatic and skeletal muscles (m. extensor digitorum longa-MEDL) have been investigated in in vitro experiments. Application of carbacholine (CCh) in vitro in different concentrations led to pronounced muscle contractions, varying from 9.15+/-4.76 to 513.13+/-15.4 mg and from 44.65+/-5.01 to 101.46+/-9.11 mg for diaphragm and MEDL, respectively. Two hundred micromolars of CCh in both muscles caused the contraction with the 65% (diaphragm) to 75% (MEDL) of maximal contraction force-this concentration was thus used in further experiments. It was found that application of ATP (100 microM) increased the force of diaphragmatic contraction caused by CCh (200 microM) from 335.2+/-51.4 mg (n=21) in controls to 426.5+/-47.8 mg (n=10; P<0.05), but decreased the contractions of MEDL of CCh from 76.6+/-6.5mg (n=26) in control to 40.2+/-9.0mg (n=8; P<0.05). Application of adenosine (100 microM) had no effect on CCh-induced contractions of these muscles. Resting membrane potential (MP) measurements using sharp electrodes were done at 10, 20 and 30 min after the application of ATP and adenosine. Diaphragm showed depolarization from 75+/-0.6 down to 63.2+/-1.05, 57.2+/-0.96 and 53.6+/-1.1 mV after 10, 20 and 30 min of exposition, respectively (20 fibers from 4 muscles each, P<0.05 in all three cases). Adenosine showed no effect on diaphragmatic MP. Both agents were ineffective in case of MEDL. The effects of ATP in both tissues were abolished by suramin (100 microM), a P2-receptor antagonist, and chelerythrin (50 microM), a specific protein-kinase C (PKC) inhibitor, but were not affected by 1H-[1,2,4]-oxadiazolo-[4,3-alpha]-quinoxalin-1-one (ODQ, 1 microM), a guanylyl-cyclase inhibitor, or by adenosine-3,5-monophosphothioate (Rp-cAMP, 1 microM), a protein-kinase A (PKA) inhibitor. Besides the action on contractile activity, ATP (100 microM) led to a significant (P<0.001) depolarization of diaphragm muscle fibers from 74.5+/-2.3 down to 64+/-2.1, 58.2+/-2.2 and 54.3+/-2.4 mV after 10, 20 and 30 min of incubation, respectively. Incubation of MEDL with the same ATP concentration showed no significant change of MP. Denervation of the muscles for 28 days led to a decrease of CCh-induced contractions of diaphragm down to 171.1+/-34.5mg (n=11, P<0.05), but increased the contractile force of MEDL up to 723.9+/-82.3mg (n=9, P<0.01). Application of ATP elevated the contractility of denervated diaphragm caused by CCh up to normal values (311.1+/-79.7 mg, n=6, P>0.05 versus control), but did not significantly affect of contractility of MEDL, which became 848.1+/-62.7 mg (n=6). These results show that the effects of ATP on both diaphragmatic and skeletal muscles are mediated through P2Y receptors coupled to chelerytrin-sensitive protein-kinase C.

Term-dependency of P2 receptor-mediated contractile responses of isolated human pregnant uterus.

OBJECTIVE: The aim of the study was to test the hypothesis that in the human uterus, the effectiveness of P2 receptor-mediated contractile responses is up-regulated during pregnancy. STUDY DESIGN: Experiments were performed on myometrial samples obtained from women undergoing caesarean section at 28-30 weeks of pregnancy (3 women, Group 1), 32-34 weeks of pregnancy (6 women, Group 2) and 38-41 weeks of pregnancy (16 women, Group 3). Concentration-response relationships for a non-selective P2 receptor agonist, adenosine 5'-triphosphate (ATP), a selective P2X receptor agonist, alpha,beta-methylene-ATP (alpha,beta-meATP), and a frequency-response relationship for non-adrenergic non-cholinergic (NANC) electrical field stimulation (EFS) were obtained using routine pharmacological organ bath technique. Effects of pyridoxalphosphate azophenyl-2',4'-disulphonic acid (PPADS, 10(-5) M), a P2 receptor antagonist, were also evaluated. Parametric Student's t-test, non-parametric Wilcoxon T-test, Mann-Whitney U-test, two-way analysis of variance (ANOVA) and Krushkal-Wallis tests were used for statistical analysis. RESULTS: ATP (10(-6) to 3 x 10(-4) M), alpha,beta-meATP (10(-7) to 3 x 10(-5) M) and EFS (2-32 Hz) evoked contractions of isolated pregnant uterus in all three groups. Uterus responses to ATP were not correlated with the term of pregnancy while the amplitude of uterine contractions to alpha,beta-meATP and EFS was higher in full term pregnancy than in earlier pregnancy. PPADS antagonized uterus responses to alpha,beta-meATP and EFS, but not to ATP, in all three groups. CONCLUSION: P2X receptor-mediated contractions of human pregnant uterus to alpha,beta-meATP and EFS, but not to ATP, are increased with the progression of pregnancy.

Potentiation of uterine effects of prostaglandin F2{alpha} by adenosine 5'-triphosphate.

OBJECTIVE: To investigate the interaction of exogenous adenosine 5'-triphosphate (ATP), a P2 receptor agonist, with prostaglandin F(2alpha) (PGF(2alpha)) on pregnant women in labor as well as on isolated human pregnant uterus preparations. METHODS: For an in vitro study, myometrial samples were obtained from 27 women undergoing elective cesarean delivery at term. Concentration-response relationships for ATP (10(-8) -3 x 10(-4) mol/L), PGF(2alpha) (10(-9) -10(-5) mol/L), and their combination were obtained by using routine pharmacological organ bath technique. An in vivo study was performed with 34 pregnant women with dysfunctional abnormalities of the active stage of labor who were randomly allocated into 2 study groups. The women in the control group (18 patients) received intravenous prostaglandin F(2alpha) at an initial rate of 7.5 mug/min, whereas the women in the ATP group (16 patients) received prostaglandin F(2alpha) concomitantly with ATP (0.45 nmol/min, intravenously). RESULTS: Adenosine 5'-triphosphate at concentrations of 10(-6) -3 x 10(-4) mol/L and PGF(2alpha) at concentrations of 10(-8) -10(-5) mol/L caused concentration-dependent contractions of isolated smooth muscle preparations of the human pregnant uterus. At concentrations of 10(-6) mol/L and below, ATP had no effects on mechanical activity of the isolated uterus, but at concentrations of 10(-7) mol/L and 10(-6) mol/L, it significantly potentiated the contractile responses of the uterus induced by PGF(2alpha) (P < .05, 2-way analysis of variance). Patients receiving intravenous infusion of ATP as a supplement to PGF(2alpha) treatment, compared with those without ATP, had a significantly shorter interval from the start of the treatment to full cervical dilatation (3.31 +/- 1.49 hours and 4.67 +/- 1.11 hours in ATP and control groups, respectively; P = .014, Wilcoxon Mann-Whitney test). The total dose of prostaglandin received was significantly lower in the ATP group than that of controls (1,489.8 +/- 699.9 mug and 3,394.2 +/- 1,951.9 mug, respectively; P = .003, Wilcoxon Mann-Whitney test). No side effects of ATP treatment were observed during or after infusion. CONCLUSION: Adenosine 5'-triphosphate potentiates effects of PGF(2alpha) on pregnant human uterus in vitro and in vivo and thus could be a useful supplemental drug to increase uterine contractility at labor.

The influence of hypothermia on P2 receptor-mediated responses of frog skeletal muscle.

The contractile responses of isolated Rana ridibunda frog sartorius muscle contractions evoked by electrical field stimulation (EFS) were studied at three temperature conditions of 17, 22 and 27 degrees C. Temperature-dependent increase of muscle contractility was found. ATP (10-100 microM) concentration dependently inhibited the electrical field stimulation-evoked contractions of sartorius muscle at all three temperatures; this effect was significantly more prominent at a temperature of 17 degrees C than at other two temperatures. Adenosine (100 microM) also caused inhibition of electrical field stimulation-evoked contractions which was statistically identical at all three temperature conditions tested. A P2 receptor antagonist, pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 10 microM) reduced the inhibitory effect of ATP at all three temperatures but did not affect inhibitory action of adenosine. In contrast, 8-(p-sulfophenyl)theophylline (8-SPT, 100 microM), a nonselective P1 receptor antagonist, abolished inhibitory effects of adenosine at all three temperature conditions but did not antagonize inhibition caused by ATP. In electrophysiological experiments, ATP (100 microM) and adenosine (100 microM) temperature dependently reduced end-plate currents recorded in sartorius neuromuscular junction by voltage-clamp technique. The inhibitory effects of both agonists were enhanced with the decrease of temperature. 8-SPT (100 microM) abolished the inhibitory effect of adenosine but not ATP on end-plate currents. Suramin (100 microM), a nonselective P2 receptor antagonist, inhibited the action of ATP but not adenosine, while PPADS (10 microM) had no influence on the effects of either ATP or adenosine. It is concluded from this study that the effectiveness of P2 receptor-mediated inhibition of frog skeletal muscle contraction in contrast to that of adenosine is dependent on the temperature conditions.

Academic medicine in Russia.

Academic medicine, along with professionalism of the medical community in Russia underwent a remarkable evolution from the Revolution through the decline of the Soviet Union. The Soviet period brought about an enormous expansion of numbers of admissions to medical schools and a corresponding increase in the number of new physicians. Academic medical institutions were separated from institutions of higher learning in general and medical science was separated from the mainstream of science. Many of these features have been reversed in the past 14 years and re-professionalization of medicine has resumed.

Varicose disease affects the P2 receptor-mediated responses of human greater saphenous vein.

The aim of the present study was to investigate in vitro the differences in P2 receptor mediated responses of human greater saphenous vein (GSV) taken from patients with varicose disease and obliterating atherosclerosis. Samples of the inguinal part of the GSV were taken from the patients who underwent phlebectomia operation due to varicose disease (n=9, VD group) or femoropoplitea bypass operation using auto-vein due to obliterating atherosclerosis of lower extremities (n=11, OA group). The mechanical responses of the isolated segments of GSV to P2 receptor agonists were tested using standard organ-bath technique. ATP (10(-6)-10(-4) M), ADP (10(-6)-10(-4) M) and alpha,betamethyleneATP (10(-8)-10(-5) M) caused concentration-dependent contractions of the veins of both groups, the latter agonist being approximately tenfold more active than first two. ATP at all concentrations tested, alpha,betamethyleneATP at concentrations of 10(-6) and 10(-5) M and ADP at a concentration of 10(-6) M produced significantly higher contractions of the GSV taken from OA group than from VD group. UTP (10(-6)-10(-4) M) caused concentration-dependent contractions of the veins taken from OA group, while in VD group this agonist was virtually without effect. Adenosine (10(-6)-10(-4) M) and 2-methylthio-ATP (10(-7)-10(-5) M) had no significant contractile activity in this tissue in both groups. It is concluded from this study that there are P2 receptor and adrenoceptor mediated contractions in human greater saphenous veins, which are impaired by varicose disease, in contrast to contractions produced by histamine and carbachol which are, if anything, enhanced.

Temperature dependency of P2 receptor-mediated responses.

The P2 receptor-mediated responses of isolated guinea pig urinary bladder and vas deferens (P2X receptors) and taenia caeci (P2Y receptors) were registered at the three temperature conditions of 30, 37 and 42 degrees C. The contractile responses of both urinary bladder and vas deferens to a P2X receptor agonist alpha,beta-methylene ATP (alpha,beta-meATP; 0.01-30 microM) and to electrical field stimulation (1-64 Hz, 0.1 ms, supramaximal voltage) in the presence of atropine (0.1 microM) and phentolamine (1 microM) were markedly more prominent at a temperature of 30 degrees C than at 37 or 42 degrees C. Similarly, relaxation of carbachol-precontracted taenia caeci caused by electrical field stimulation (0.5-8 Hz, 0.1 ms, supramaximal voltage) temperature-dependently increased with decrease of temperature, while relaxation of this tissue by exogenous ATP (1-100 microM) was not affected by the temperature. A P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 1-30 microM) at all three temperature conditions concentration-dependently antagonised contractile responses to alpha,beta-methylene ATP and electrical field stimulation in both urinary bladder and vas deferens. PPADS, even at the highest concentration tested (30 microM), had no effect on the relaxant responses of the taenia caeci either to electrical field stimulation or ATP and its action was not affected by the change of temperature. It is concluded from this study that the effectiveness of P2 receptor-mediated responses in guinea pig urinary bladder, vas deferens and taenia caeci increases by decrease of temperature.

Effects of chronic specific urogenital infections on contractility of the human isolated pregnant myometrium.

The contractile activity of the isolated myometrium of pregnant women with mycoplasma, chlamydia and mixed infections has been studied by pharmacological organ bath method. We found that mycoplasma infection decreased while chlamydia or mixed infection increased myometrium contraction evoked by oxytocin or prostaglandin F2alpha. The results of this study could be important for the prediction of possible complications during pregnancy and labour in women with chronic specific urogenital infections.

Implementation of a cross-cultural evidence-based medicine curriculum.

While principles of evidence-based medicine (EBM) are increasingly prevalent in medical education curricula in Europe and North America, medical educators elsewhere face formidable barriers to its implementation. We sought to determine the feasibility of implementing a learner-centered, case-based EBM curriculum among academic physicians in Kazan, Russia, facilitated by residents participating in an international health elective. This article reports that implementation of an EBM curriculum is feasible during a resident international health elective and that mutually beneficial educational exchanges represent an opportunity for teaching the tenets of EBM abroad.

Synthesis and Structure-Activity Relationships of Pyridoxal-6-arylazo-5'-phosphate and Phosphonate Derivatives as P2 Receptor Antagonists.

Novel analogs of the P2 receptor antagonist pyridoxal-5'-phosphate-6-phenylazo-2',4'-disulfonate (PPADS) were synthesized. Modifications were made through functional group substitution on the sulfophenyl ring and at the phosphate moiety through the inclusion of phosphonates, demonstrating that a phosphate linkage is not required for P2 receptor antagonism. Substituted 6-phenylazo and 6-naphthylazo derivatives were also evaluated. Among the 6-phenylazo derivatives, 5'-methyl, ethyl, propyl, vinyl, and allyl phosphonates were included. The compounds were tested as antagonists at turkey erythrocyte and guinea-pig taenia coli P2Y(1) receptors, in guinea-pig vas deferens and bladder P2X(1) receptors, and in ion flux experiments by using recombinant rat P2X(2) receptors expressed in Xenopus oocytes. Competitive binding assay at human P2X(1) receptors in differentiated HL-60 cell membranes was carried out by using [(35)S]ATP-γ-S. A 2'-chloro-5'-sulfo analog of PPADS (C(14)H(12)O(9)N(3)ClPSNa), a vinyl phosphonate derivative (C(15)H(12)O(11)N(3)PS(2)Na(3)), and a naphthylazo derivative (C(18)H(14)O(12)N(3)PS(2)Na(2)), were particularly potent in binding to human P2X(1) receptors. The potencies of phosphate derivatives at P2Y(1) receptors were generally similar to PPADS itself, except for the p-carboxyphenylazo phosphate derivative C(15)H(13)O(8)N(3)PNa and its m-chloro analog C(15)H(12)O(8)N(3)ClPNa, which were selective for P2X vs. P2Y(1) receptors. C(15)H(12)O(8)N(3)ClPNa was very potent at rat P2X(2) receptors with an IC(50) value of 0.82 µM. Among the phosphonate derivatives, [4-formyl-3-hydroxy-2-methyl-6-(2-chloro-5-sulfonylphenylazo)-pyrid-5-yl]methylphosphonic acid (C(14)H(12)-O(8)N(3)ClPSNa) showed high potency at P2Y(1) receptors with an IC(50) of 7.23 µM. The corresponding 2,5-disulfonylphenyl derivative was nearly inactive at turkey erythrocyte P2Y(1) receptors, whereas at recombinant P2X(2) receptors had an IC(50) value of 1.1 µM. An ethyl phosphonate derivative (C(15)H(15)O(11)N(3)PS(2)Na(3)), whereas inactive at turkey erythrocyte P2Y(1) receptors, was particularly potent at recombinant P2X(2) receptors.

Structure Activity Relationships for Derivatives of Adenosine-5'-Triphosphate as Agonists at P(2) Purinoceptors: Heterogeneity Within P(2X) and P(2Y) Subtypes.

The structure-activity relationships for a variety of adenine nucleotide analogues at P(2x)- and P(2Y)-purinoceptors were investigated. Compounds formed by structural modifications of the ATP molecule including substitutions of the purine ring (C2, C8, N1, and N(6)-substituents, and a uridine base instead of adenine), the ribose moiety (2' and 3'-positions), and the triphosphate group (lower phosphates, bridging oxygen substitution, and cyclization) were prepared. Pharmacological activity at P(2Y)-purinoceptors was assayed in the guinea pig taenia coli, endothelial cells of the rabbit aorta, smooth muscle of the rabbit mesenteric artery, and turkey erythrocyte membranes. Activity at P(2X)-purinoceptors was assayed in the rabbit saphenous artery and the guinea-pig vas deferens and urinary bladder. Some of the analogues displayed selectivity, or even specificity, for either the P(2X)- or the P(2Y)-purinoceptors. Certain analogues displayed selectivity or specificity within the P(2X)- or P(2Y)-purinoceptor superfamilies, giving hints about possible subclasses. For example, 8-(6-aminohexylamino)ATP and 2',3'-isopropylidene-AMP were selective for endothelial Pzypurinoceptors over P(2Y)-purinoceptors in the guinea pig taenia coli, rabbit aorta, and turkey erythrocytes. These compounds were both inactive at P(2X)-purinoceptors. The potent agonist N(6)-methyl ATP and the somewhat less potent agonist 2'-deoxy-ATP were selective for P(2Y)-purinoceptors in the guinea pig taenia coli, but were inactive at P(2X)-purinoceptors and the vascular P(2Y)-purinoceptors. 3'-Benzylamino-3'-deoxyATP was very potent at the P(2X)-purinoceptors in the guinea pig vas deferens and bladder, but not in the rabbit saphenous artery and was inactive at P(2Y) receptors. These data suggest that specific compounds can be developed that can be utilized to activate putative subtypes of the P(2X)- and P(2Y)-purinoceptor classes.