Lilia E. Ziganshina

Effects of extracellular pH on agonism and antagonism at a recombinant P2X2 receptor

Under voltage‐clamp conditions, the activity of agonists and antagonists at a recombinant P2X2 receptor expressed in Xenopus oocytes was examined at different levels of extracellular pH (pHe). In normal Ringer (Mg2+ ions absent), the amplitude of submaximal inward currents to ATP was increased by progressively lowering pHe (8.0–5.5). ATP‐responses reached a maximum at pH 6.5 with a 5 fold increase in ATP‐affinity; the apparent pKa was 7.05±0.05. Receptor affinity for ATP was lowered when extracellular Ca2+ ions were replaced with equimolar Mg2+ ions. However, the amplitude of the ATP‐responses was still enhanced under acidic conditions, reaching maximal activity at pH 6.5 with a 5 fold increase in ATP‐affinity; the apparent pKa was 7.35±0.05. ATP species present in the superfusate (for the above ionic conditions and pH levels) were calculated to determine the forms of ATP which activate P2X2 receptors: possible candidates include HATP, CaHATP and MgHATP. However, levels of these protonated species increase below pH 6.5, suggesting that receptor protonation rather than agonist protonation is more important. The potency order for agonists of P2X2 receptors was: ATP>2‐MeS‐ATPATPγS> ATPαS>>CTPBzATP, while other nucleotides were inactive. EC50 and nH values for full agonists were determined at pH 7.4 and re‐examined at pH 6.5. Extracellular acidification increased the affinity by approximately 5 fold for full agonists (ATP, 2‐MeSATP, ATPγS and ATPαS), without altering the potency order. The potency order for antagonists at P2X2 receptors was: Reactive blue‐2>trinitrophenol‐ATPPalatine fast blackCoomassie brilliant bluePPADS>suramin (at pH 7.4). IC50 values and slopes of the inhibition curves were re‐examined at different pH levels. Only blockade by suramin was affected significantly by extracellular acidification (IC50 values: 10.4±2 μM, at pH 7.4; 78±5 nM, at pH 6.5; 30±6 nM, at pH 5.5). In summary, a lowered pHe enhanced the activity of all agonists at P2X2 receptors but, with the exception of suramin, not antagonists. Since a lowered pHe is also known to enhance agonist activity at P2X receptors on sensory neurones containing P2X2 transcripts, the sensitization by metabolic acidosis of native P2X receptors containing P2X2 subunits may have a significant effect on purinergic cell‐to‐cell signalling.

Full sensitivity of P2times2 purinoceptor to ATP revealed by changing extracellular pH

A full pharmacological characterization was carried out on a recombinant ATP‐gated ion channel (P2times2 purinoceptor) expressed in Xenopus oocytes. This slowly‐desensitizing neuronal P2times2 purinoceptor, activated by ATP (EC50 = 4.6 ± 1 μm at pH 7.4; n 4), showed the agonist potency order: ATP ≥ 2‐ MeSATP = ATPγS ≥ ATPαS < < Bz‐ATP. The receptor affinity for ATP was enhanced 5–10 fold by acidifying the bathing solution (to pH 6.5) but was diminished 4–5 fold in an alkaline solution (pH 8.0). The maximum activity of P2times2 purinoceptors and the activity order of a series of nucleotides were unaltered by changing extracellular pH. Interestingly, ATP sensitivity at a recombinant P2Y1, purinoceptor remained unaltered with changing extracellular pH. These results indicate that acidotic conditions in the synaptic cleft could strengthen purinergic transmission at neuronal P2times2 purinoceptors.

Effects of P2-purinoceptor antagonists on degradation of adenine nucleotides by ecto-nucleotidases in folliculated oocytes of Xenopus laevis

The aim of the present study was to examine the effects of a number of P2-purinoceptor antagonists on degradation of adenine nucleotides by Xenopus laevis oocyte ecto-nucleotidase. Folliculated oocytes readily metabolize all three naturally-occurring nucleotides, the order of preferential substrates being ATP >ADP > AMP. The degradation of ATP and ADP was decreased significantly in the presence of several P2X- and P2Y-purinoceptor antagonists, including suramin, PPADS, Cibacron blue, Coomassie Brilliant blue, Evans blue, Trypan blue, Congo red, and PIT (each compound was used at 100 microM). All these compounds inhibited the degradation of ATP by up to 60%, whereas the hydrolysis of ADP was inhibited by Congo red and PIT by 75-80%. In addition, DIDS (100 microM) and TNP-ATP (100 microM) selectively inhibited the breakdown of ATP, and sodium azide (10 mM) selectively inhibited the breakdown of ADP. The enzymatic breakdown of either ATP or ADP was unaffected by 8-pSPT (100 microM), an antagonist of P1-purinoceptors, or by oxidized ATP (100 microM), an antagonist of P2Z-purinoceptors. The degradation of AMP was prevented completely by PIT (100 microM) and ingibited significantly by Congo red (100 microM). In conclusion, the present study shows that most of currently available antagonists of P2-purinoceptors inhibit the enzymatic breakdown of extracellular ATP and ADP. The inhibitory effect on ecto-nucleotidase activity should be taken into account when these antagonists are used in pharmacological experiments.

Effects of cyclopiazonic acid on contractility and ecto-ATPase activity in guinea-pig urinary bladder and vas deferens.

1 Cyclopiazonic acid (CPA), an inhibitor of sarcoplasmic ATPase, was tested on guinea‐pig urinary bladder and vas deferens for its ability: (1) to modify contractile responses to electrical field stimulation (EFS), exogenous ATP, α,β‐methylene ATP (α,β‐meATP), carbachol, noradrenaline (NA), histamine, and KCl; (2) to affect ecto‐ATPase activity; (3) to modify the release of ATP evoked by EFS. 2 In the urinary bladder, CPA (10 μm) potentiated contractile responses to EFS, exogenous ATP (100 μm), α,β‐meATP (1 μm), carbachol (0.5 μm), histamine (30 μm) and KCl (30 μm). In the vas deferens, CPA (10 μm) potentiated responses to EFS, ATP, α,β‐meATP, NA (100 μm) and KCl. CPA at a concentration of 1 μm had no effect on ATP‐induced relaxation of carbachol‐precontracted guinea‐pig taenia coli, and at a concentration of 10 μm it markedly increased spontaneous contractile activity of taenia. 3 Ecto‐ATPase was estimated to have Vmax and Km values of 0.98 nmol Pi 30 min−1 mg−1 wet tissue and 881 μm ATP in the urinary bladder, and 0.75 nmol Pi 30 min−1 mg−1 wet tissue and 914 μm ATP in the vas deferens, respectively. CPA at a concentration of 10 μm significantly inhibited ecto‐ATPase activity by 18% in the urinary bladder and by 24% in the vas deferens. 4 In the guinea‐pig vas deferens, CPA significantly potentiated ATP release evoked by EFS from 2.2 ± 0.8 (6) pmol ATP min−1 g−1 wet tissue to 35.2 ± 4.8 (6) pmol ATP min−1 g−1 wet tissue (P < 0.01). 5 In conclusion, the potentiation of contractile responses of the guinea‐pig urinary bladder and vas deferens by CPA has a non‐specific character. CPA inhibited ecto‐ATPase activity and increased ATP release, but these effects do not appear to contribute to the potentiation of P2x‐purinoceptor‐mediated responses since the contractile actions of all the agonists studied were potentiated to the same extent.

Acute paw oedema formation induced by ATP: Re-evaluation of the mechanisms involved

ATP-induced inflammation was investigated using subplantar injection in the mouse hind paw. The order of efficacy of purinoceptor agonists for inducing paw oedema (30 nmol per paw) was ATP=α,β-methylene ATP=2-methylthio ATP > adenosine > UTP > ADP > AMP. Diadenosine polyphosphates effectively induced paw oedema formation with an order of efficacy of: P1, P4-di(adenosine-5′)tetraphosphate =P1,P5-di(adenosine-5′)-pentaphosphate= P1,P6-di(adenosine-5′) hexaphosphate ≫ ATP=P1,P3-di(adenosine-5′)triphosphate > P1,P2-di(adenosine-5′)pyrophosphate. Systemic administration of P2-purinoceptor antagonists (30–100 μmol/kg), suramin, 4,4′-diisothiocyanatostilbene-2,2′-disulphonate, pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid and cibacron blue, reduced the intensity of ATP-induced oedema. At 30 μmol/kg 8-(p-sulfophenyl)theophylline (non-selective adenosine receptor antagonist), 3,7-dimethyl-1,1-propargyl-xanthine (adenosine A2 receptor antagonist), triprolidine (histamine H1 receptor antagonist), ranitidine (histamine H2 receptor antagonist) and ketanserin (5-hydroxytryptamine 5-HT2 receptor antagonist), but neither 8-cyclopentyl-1,3-dipropylxanthine (adenosine A1 receptor antagonist), nor indomethacin (cyclooxygenase inhibitor) inhibited the ATP-induced swelling. Topical (100 nmol per paw), but not systemic (100 μmol/kg) administration of NG-nitro-L-arginine methyl ester (nitric oxide synthase inhibitor) reduced the intensity of the ATP-induced paw oedema. These results show that ATP can induce an inflammatory oedematous reaction and contribute to our understanding of the underlying mechanisms.

Effects of divalent cations and La3+ on contractility and ecto-ATPase activity in the guinea-pig urinary bladder

1 Several cations (Ba2+, Cd2+, Co2+, Cu2+, Mn2+, Ni2+, Zn2+ and La3+, all as chloride salts, 1–1000 μm) were tested in the guinea‐pig urinary bladder for their ability to: (i) modify contractile responses to electrical field stimulation (EFS), ATP, α,β‐methylene ATP (α,β‐meATP), carbachol (CCh), and KCl; (ii) affect ecto‐ATPase activity. 2 Ba2+ (10–1000 μm) concentration‐dependently potentiated contractile responses evoked by EFS (4–16 Hz), ATP (100 μm), α,β‐meATP (1 μm), CCh (0.5 μm), and KCl (30 mm). Ni2+ at concentrations of 1–100 μm also potentiated contractility of the urinary bladder, but at concentrations tested its effect was not concentration‐dependent. Cu2+ at a concentration of 10 μm and Cd2+ at a concentration of 1 μm potentiated responses to all stimuli, except KCl. Ni2+ at a concentration of 1000 μm and Cd2+ at a concentration of 100 μm inhibited contractions evoked by all stimuli, and at a concentration of 1000 μm Cd2+ abolished any contractions. Responses to ATP and α,β‐meATP were selectively inhibited by Cu2+, Zn2+ or La3+, each at a concentration of 1 mm. 3 Cu2+, Ni2+, Zn2+ and La3+ (100–1000 μm) concentration‐dependently inhibited ecto‐ATPase activity in the urinary bladder smooth muscle preparations, while Ba2+ and Mn2+ were without effect, and Cd2+ and Co2+ caused significant inhibition only at a concentration of 1000 μm. 4 There was no correlation between the extent of ecto‐ATPase inhibition and the effect on contractile activity of any of the cations. 5 In conclusion, the ability of some divalent cations to inhibit ecto‐ATPase activity and to potentiate or inhibit contractile responses in the guinea‐pig urinary bladder appear to be independent effects.

Potentiation of uterine effects of prostaglandin F2α by adenosine 5'-triphosphate

OBJECTIVE: To investigate the interaction of exogenous adenosine 5′-triphosphate (ATP), a P2 receptor agonist, with prostaglandin F2α (PGF2α) 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 × 10−4 mol/L), PGF2α (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 F2α at an initial rate of 7.5 μg/min, whereas the women in the ATP group (16 patients) received prostaglandin F2α concomitantly with ATP (0.45 nmol/min, intravenously). RESULTS: Adenosine 5′-triphosphate at concentrations of 10−6 –3 × 10−4 mol/L and PGF2α 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 PGF2α (P < .05, 2-way analysis of variance). Patients receiving intravenous infusion of ATP as a supplement to PGF2α 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 μg and 3,394.2 ± 1,951.9 μg, 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 PGF2α on pregnant human uterus in vitro and in vivo and thus could be a useful supplemental drug to increase uterine contractility at labor. LEVEL OF EVIDENCE: I

DIFFERENTIAL DEGRADATION OF EXTRACELLULAR ADENINE NUCLEOTIDES BY FOLLICULATED OOCYTES OF XENOPUS LAEVIS

The degradation of extracellular ATP and ADP by Xenopus oocytes was studied to investigate whether one or two ecto-enzymes are responsible for breakdown of both nucleotides. At a concentration of 100 microM, the half-life of ATP and ADP was 33 and 40 min, respectively. Degradation of ATP caused an initial fast and then a sustained accumulation of ADP in the buffer, while the concentration of AMP in the buffer increased slowly, but progressively, in a relatively linear manner. The rates of degradation of ATP and ADP were similar at pH levels between 7 and 10, but the velocity of breakdown of ATP was significantly higher than that of ADP at pH of 5-6. In divalent cation-free buffer, the addition of 0.1 mM of Ca2+, but not equimolar Mg2+, significantly potentiated the degradation of ATP by oocytes while, in the case of ADP, each of these divalent cations were able to potentiate its degradation. The rate of hydrolysis of ATP and its kinetic constants were not significantly different in the presence or absence of ADP (50 microM). In conclusion, differences in pH- and cation-dependency, and absence of inhibitory effect of ADP on degradation of ATP suggest that degradation of ATP and ADP by oocytes is provided by separate enzymes, namely Ca2+/Mg(2+)-dependent ecto-ATPase and ecto-ADPase, rather than by one ecto-enzyme.

Attitudes to pharmaceutical promotion techniques among healthcare professionals in the Republic of Tatarstan, Russia

Purpose: To study attitudes of health care professionals towards pharmaceutical promotion. Methods: Intervention study. 161 questionnaires were collected and analyzed after anonymous surveys of health-care professionals (physicians and residents). Results: Nearly half of surveyed participants (53% of physicians and 44% of residents) communicated with pharmaceutical representatives 1–2 times a week. The most widespread marketing technique was pen-gifting: 93.3% of physicians and 94.7% of residents admitted receiving pens at least once a year. 63.3% of physicians and 78.5% of residents had dinners at conferences once or more often during the last year. 3.2% of physicians and 12.5% of residents believed that pharmaceutical representatives had no influence on prescribing practices, about 60% of responders admitted ‘minor influence’, while 30% reported ‘major influence’. However only 10.2% of physicians and 3.8% of residents noted significant influence of pharmaceutical promotion on their own prescribing practice. The majority of responders indicated pharmaceutical advertising materials (information from the last conference, information from pharmaceutical reps) as one of determining factors for their pharmacotherapy choice. 42.9% of physicians and 77.8% of residents considered acceptance of gifts from pharmaceutical representatives to be ethical. About half of responders considered that development of restricting policies on interactions of health care professionals with pharmaceutical representative be not needed. Post-survey of a small proportion of participants revealed two-fold increase in the number of residents who considered pharmaceutical advertising to have major influence on their colleagues’ prescribing practice. There was a trend to decrease in the number of residents who considered trade/money agreements between physicians and pharmaceutical industry to be appropriate and to an increase in the number of opponents of trade/money agreements. Conclusions: Promotion techniques are widely and deeply integrated in everyday routine of health care professionals. Physicians are inclined to underestimate influence of pharmaceutical promotion on their own prescribing practice as compared with influence on their colleagues. The majority of responders use promotional information for prescribing decision-making being unaware of ethical implications of promotional interactions and unresponsive to restriction policies. Residents seem to be more responsive to anti-promotional educational intervention.

Effects of α,β-unsaturated sulphones and phosphonium salts on ecto-ATPase activity and contractile responses mediated via P2x-purinoceptors

Abstract 1. 1. In the guinea-pig urinary bladder and vas deferens, several α,β-unsaturated sulphones and phosphonium salts that were tested inhibited ecto-ATPase activity. The sulphones were more active in the bladder but the phosphonium salts were more effective in the vas deferens. 2. 2. These compounds either potentiated or inhibited purinergic contractile responses in the guinea-pig urinary bladder and vas deferens. 3. 3. α,β-Unsaturated sulphones and phosphonium salts represent a new promising class of compounds, capable of modulating purinergic neurotransmission.