Diclofenac does not decrease renal blood flow or glomerular filtration in elderly patients undergoing orthopedic surgery

P2X receptors for adenosine 5'-triphosphate (ATP) comprise a family of ligand-gated cation channels with distinct characteristics which are dependent on the receptor subunits (P2X1-7) expressed, and the homomeric or heteromeric assembly of protein subunits in individual cells. We describe the properties of P2X receptors expressed by cultured adult rat dorsal root ganglion cells on the basis of the time course of responses to ATP, alpha, beta-methylene adenosine 5'-triphosphate (alpha, beta-meATP) and 2-methyl-thioadenosine 5'-triphosphate (2-meSATP), and using the antagonists 2',3'-O-(2,4,6-trinitrophenyl) ATP (TNP-ATP), a novel and highly selective purinoceptor antagonist, suramin and iso-pyridocalphosphate-6-azophenyl-2',5' disulphonic acid (PPADS). ATP (10 microM) evoked inward currents in approximately 95% of neurons tested and > 80% responded with a fast transient inward current that rapidly inactivated during the continued presence of ATP. Of the remaining neurons, approximately 4% showed a sustained response and approximately 10% showed a combination of transient and sustained components. Rapid application of ATP, alpha,beta-meATP and 2meSATP demonstrated these to be full agonists of the rapidly inactivating P2X receptor (pA50 values = 5.83, 5.86 and 5.55, respectively), whilst uridine triphosphate (UTP) and 1-beta,gamma-methyleneadenosine 5'-triphosphate (1-beta,gamma-meATP) were ineffective as agonists. These rapidly inactivating responses could be inhibited by TNP-ATP, suramin and PPADS (pIC50 = 9.5, 6.5, 6.4, respectively). Using inactivation protocols, we demonstrate the presence of homomeric P2X3-like receptors and non-inactivating P2X receptors, which indicates that individual subsets of adult dorsal root ganglion neurons have distinct P2X receptor phenotypes, and that individual DRG neurons may express multiple P2X receptor subtypes.     

Functional characterisation of P2 purinoceptors in PC12 cells by measurement of radiolabelled calcium influx

The aim of this study was to determine whether 45Ca2+ influx could be used as a quantitative measure of channel activation for functional characterisation of P2X purinoceptors in cell lines. In undifferentiated PC12 cells, grown in suspension, ATP (EC50 = 45 microM), ATP gamma S (EC50 = 50 microM) and 2-meSATP (EC50 = 81 microM) but not alpha beta meATP (1 mM) stimulated 45Ca2+ influx 2-5 fold. This effect did not appear to be due to activation of P2U or P2Y purinoceptors since 1 mM UTP, ADP or ADP beta S did not produce any significant effect. Similarly, the effects of ATP were not apparently mediated through activation of P2Z purinoceptors since dibenzylATP behaved as a weak (EC50 = 191 microM) partial agonist (Maximal effect 29.5% of ATP maximum) and there was no detectable ATP-stimulated ethidium bromide uptake in the PC12 cells. ATP-stimulated 45Ca2+ influx was not affected by nifedipine suggesting that it was not secondary to activation of L-type calcium channels and rather reflected influx through a P2X purinoceptor present in these cells. The ATP-stimulated 45Ca2+ influx could be reduced by monovalent cations, presumably as a result of direct competition for influx through the cation channel, with the following rank order of potency:- guanidinium (EC50 = 16 mM) > sodium > Tris > choline > N-methyl-D-glucamine = sucrose). A number of P2 purinoceptor antagonists inhibited ATP-stimulated 45Ca2+ influx. Pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (3-300 microM), pyridoxal 5-phosphate (3-300 microM) and d-tubocurarine (30-300 microM) produced an insurmountable antagonism of responses to ATP, with no marked change in agonist EC50. Suramin (100-300 microM) and cibacron blue (30-300 microM) produced a surmountable antagonism while DIDS (4,4'-diisothiocyanatostilbene-2,2'disulfonic acid) only antagonised responses to ATP at concentrations in excess of 300 microM. The general properties of the P2X purinoceptor population identified in these cells were consistent with them being P2X2 purinoceptors. These findings suggest that ATP-stimulated 45Ca2+ influx may be used as a reliable and quantitative functional assay for characterisation of P2X purinoceptor subtypes in cell lines.     

A twin study of antisocial and neurotic symptoms in childhood

1. 2,2'-Pyridylisatogen tosylate (PIT) has been reported to be an irreversible antagonist of responses to adenosine 5'-triphosphate (ATP) at metabotropic purinoceptors (of the P2Y family) in some smooth muscles. When a recombinant P2Y1 purinoceptor (derived from chick brain) is expressed in Xenopus oocytes, ATP and 2-methylthioATP (2-MeSATP) evoke calcium-activated chloride currents (ICl,Ca) in a concentration-dependent manner. The effects of PIT on these agonist responses were examined at this cloned P2Y purinoceptor. 2. PIT (0.1-100 microM) failed to stimulate P2Y1 purinoceptors directly but, over a narrow concentration range (0.1-3 microM), caused a time-dependent potentiation (2-5 fold) of responses to ATP. The potentiation of ATP-responses by PIT was not caused by inhibition of oocyte ecto-ATPase. At high concentrations (3-100 microM), PIT irreversibly inhibited responses to ATP with a IC50 value of 13 +/- 9 microM (pKB = 4.88 +/- 0.22; n = 3). PIT failed to potentiate inward currents evoked by 2-MeSATP and only inhibited the responses to this agonist in an irreversible manner. 3. Known P2 purinoceptor antagonists were tested for their ability to potentiate ATP-responses at the chick P2Y1 purinoceptor. Suramin (IC50 = 230 +/- 80 nM; n = 5) and Reactive blue-2 (IC50 = 580 +/- 130 nM; n = 6) reversibly inhibited but did not potentiate ATP-responses. Coomassie brilliant blue-G (0.1-3 microM) potentiated ATP-responses in three experiments, while higher concentrations (3-100 microM) irreversibly inhibited ATP-responses. The results indicated that potentiation and receptor antagonism were dissociable and not a feature common to all known P2 purinoceptor antagonists. 4. In radioligand binding assays, PIT showed a low affinity (pKi < 5) for a range of membrane receptors, including: alpha 1, alpha 2-adrenoceptors, 5-HT1A, 5-HT1B, 5-HT2, 5-HT3, D1, D2, muscarinic, central benzodiazepine, H1, mu-opioid, dihydropyridine and batrachotoxin receptors. PIT showed some affinity (pKi = 5.3) for an adenosine (A1) receptor. 5. In guinea-pig isolated taenia caeci, PIT (12.5-50 microM) irreversibly antagonized relaxations to ATP (3-1000 microM); PIT also directly relaxed the smooth muscle and histamine was used to restore tone. Relaxations to nicotine (10-100 microM), evoked by stimulating intrinsic NANC nerves of taenia caeci preparations in the presence of hyoscine (0.3 microM) and guanethidine (17 microM), were not affected by PIT (50 microM, for 25-60 min). 6. These experiments indicate that PIT causes an irreversible antagonism of ATP receptors but, for recombinant chick P2Y1 purinoceptors, this effect is preceded by potentiation of ATP agonism. The initial potentiation by PIT (and by Coomassie brilliant blue-G) of ATP-responses raises the possibility of designing a new class of modulatory drugs to enhance purinergic transmission at metabotropic purinoceptors.     

P2 purinoceptors modulating noradrenaline release from sympathetic neurons in culture

ATP (1 mM) inhibited, whereas 2-methylthio-ATP (30 microM), a P2Y-selective purinoceptor agonist, increased electrically evoked release of [3H]noradrenaline from chick sympathetic neurons. The P2X-selective purinoceptor agonist alpha,beta-methylene-ATP (30 microM) had no effect. The ATP-induced inhibition of release as well as the facilitation caused by 2-methylthio-ATP was not affected by the selective adenosine (P1) receptor antagonist 8-(p-sulfophenyl)-theophylline (8-PST; 100 microM), but completely prevented by the non-selective P2 antagonist suramin (300 microM). The present data reveal a dual regulation of noradrenaline release from sympathetic neurons. Facilitation seems to be mediated by a P2Y purinoceptor, whereas inhibition is caused by a P2 purinoceptor which needs further subtype characterization.     

Pharmacological profile of a novel cyclic AMP-linked P2 receptor on undifferentiated HL-60 leukemia cells

1. Extracellular ATP (EC50=146+/-57 microM) and various ATP analogues activated cyclic AMP production in undifferentiated HL-60 cells. 2. The order of agonist potency was: ATPgammaS (adenosine 5'-O-[3-thiotriphosphate]) > or = BzATP (2'&3'O-(4-benzoylbenzoyl)-adenosine-5'-triphosphate) > or = dATP > ATP. The following agonists (in order of effectiveness at 1 mM) were all less effective than ATP at concentrations up to 1 mM: beta,gamma methylene ATP > or = 2-methylthioATP > ADP > or = Ap4A (P1, P4-di(adenosine-5') tetraphosphate) > or = Adenosine > UTP. The poor response to UTP indicates that P2Y2 receptors are not responsible for ATP-dependent activation of adenylyl cyclase. 3. Several thiophosphorylated analogs of ATP were more potent activators of cyclic AMP production than ATP. Of these, ATPgammaS (EC50=30.4+/-6.9 microM) was a full agonist. However, adenosine 5'-O-[1-thiotriphosphate] (ATPalphaS; EC50=45+/-15 microM) and adenosine 5'-O-[2-thiodiphosphate] (ADPbetaS; EC50=33.3+/-5.0 microM) were partial agonists. 4. ADPbetaS (IC50=146+/-32 microM) and adenosine 5'-O-thiomonophosphate (AMPS; IC50=343+/-142 microM) inhibited cyclic AMP production by a submaximal concentration of ATP (100 microM). Consistent with its partial agonist activity, ADPbetaS was estimated to maximally suppress ATP-induced cyclic AMP production by about 65%. AMPS has not been previously reported to inhibit P2 receptors. 5. The broad spectrum P2 receptor antagonist, suramin (500 microM), abolished ATP-stimulated cyclic AMP production by HL-60 cells but the adenosine receptor antagonists xanthine amine congener (XAC; 20 microM) and 8-sulpho-phenyltheophylline (8-SPT; 100 microM) were without effect. 6. Extracellular ATP also activated protein kinase A (PK-A) consistent with previous findings that PK-A activation is involved in ATP-induced differentiation of HL-60 cells (Jiang et al., 1997). 7. Taken together, the data indicate the presence of a novel cyclic AMP-linked P2 receptor on undifferentiated HL-60 cells.     

Diazepam-insensitive GABAA receptors on postnatal spiral ganglion neurones in culture

Using dissociated spiral ganglion cell cultures obtained from 3-day-old rat cochlea, we investigated the response of auditory neurones to gamma-aminobutyric acid (GABA) using patch-clamp techniques. In our recording conditions, GABA elicited inward currents in > 95% of the neurones which reversed around 0 mV. Similar inward currents were measured using isoguvacin, a specific agonist of GABAA receptors. GABA-gated currents were reversibly inhibited by the channel blocker picrotoxin and the GABA competitive antagonist bicuculline. These functional GABAA receptors are characterized by an insensitivity to benzodiazepines and a relatively high sensitivity to beta-carbolines and barbiturates. These results show that the GABAA receptor pharmacological properties of spiral ganglion neurones are close to those of cerebellar granule cells.     

A1 adenosine receptor modulation of electrically-evoked contractions in the bisected vas deferens and cauda epididymis of the guinea-pig

1. The effects of adenosine receptor agonists upon both electrically-evoked and phenylephrine-induced contractile responses were investigated in the bisected vas deferens and the cauda epididymis of the guinea-pig. Electrical field-stimulation (10 s trains of pulses at 9 Hz, 0.1 ms duration, supramaximal voltage) elicited biphasic and monophasic contractile responses from preparations of bisected vas deferens and cauda epididymis, respectively; these responses were abolished by tetrodotoxin (300 nM). 2. In the prostatic half of the vas deferens the A1 selective adenosine receptor agonists, N6-cyclopentyladenosine (CPA) and (2S)-N6-[2-endo-norbornyl]adenosine ((S)-ENBA) and the non-selective A1/A2 adenosine receptor agonist, 5'-N-ethylcarboxamidoadenosine (NECA) inhibited electrically-evoked contractions (pIC50+/-s.e.mean values 6.15+/-0.24, 5.99+/-0.26 and 5.51+/-0.24, respectively). The responses to CPA were blocked by the A1 adenosine receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine, DPCPX (100 nM). 3. In the epididymal half of the vas deferens NECA potentiated (at < or = 100 nM) and inhibited (at > or = 1 microM) electrically-evoked contractions. In the presence of the non-selective alpha-adrenoceptor antagonist phentolamine (3 microM), the alpha1-adrenoceptor antagonist, prazosin (100 nM), or at a reduced train length (3 s) NECA inhibited electrically-evoked contractions (pIC50 values 6.05+/-0.25, 5.97+/-0.29 and 5.71 +/-0.27, respectively). CPA (at 10 microM) also inhibited electrically-evoked contractions in this half of the vas deferens. In the presence of prazosin (100 nM), CPA also inhibited electrically-evoked contractions (pIC50 6.14+/-0.67); this effect was antagonized by DPCPX (30 nM, apparent pK(B) 8.26+/-0.88). In the presence of the P2 purinoceptor antagonist, suramin (300 microM), CPA (up to 1 microM) potentiated electrically-evoked contractions. 4. NECA, CPA and APNEA potentiated electrically-evoked contractions in preparations of cauda epididymis (pEC50 values 7.49+/-0.62, 7.65+/-0.74 and 5.84+/-0.86, respectively), the response to CPA was competitively antagonized by DPCPX (100 nM) with an apparent pK(B) value of 7.64+/-0.64. 5. The alpha1-adrenoceptor agonist phenylephrine elicited concentration-dependent contractile responses from preparations of bisected vas deferens and cauda epididymis. NECA (1 microM) potentiated responses to phenylephrine (< or = 1 microM) in the epididymal, but not in the prostatic half of the vas deferens. In preparations of epididymis NECA (1 microM) shifted phenylephrine concentration response curves to the left (4.6 fold). In the presence of a fixed concentration of phenylephrine (1 microM), NECA elicited concentration-dependent contractions of preparations of the epididymal half of the vas deferens and of the epididymis (pEC50 values 7.57+/-0.54 and 8.08+/-0.18, respectively). NECA did not potentiate responses to ATP in either the epididymal half of the vas deferens or the epididymis. 6. These studies are consistent with the action of stable adenosine analogues at prejunctional A1 and postjunctional A1-like adenosine receptors. The prejunctional A1 adenosine receptors only inhibit the electrically-evoked contractions of purinergic origin (an effect predominant in the prostatic half of the vas deferens). At the epididymis, where electrically-evoked contractions are entirely adrenergic, the predominant adenosine receptor agonist effect is a potentiation of alpha1-adrenoceptor-, but not of ATP-induced contractility.     

Properties of GABA(A) receptors in cultured rat oligodendrocyte progenitor cells

We have studied the properties of GABA responses in oligodendrocyte-type 2 astrocyte (O-2A) progenitor cells derived from primary cultures of the neonatal rat brain. In whole cell voltage clamp recordings, rapid application of 1-10 mM GABA elicited current responses in > 85% of the cells examined. The dose-response relationship pooled from nine progenitor cells was best fit by a logistic function of EC50=113 microM and Hill coefficient=0.9. In contrast to the rate of current deactivation, the rate of current activation exhibited marked concentration-dependence. Pharmacologically, GABA, muscimol and ZAPA ((Z)-3[(aminiiminomethyl)thio]prop-2-enoic acid sulphate) produced responses with ligand-specific kinetics, whereas glycine and the GABA(C) receptor agonist CACA were without effect; bicuculline methochloride acted as a competitive antagonist. Neither the amplitude nor the kinetics of currents produced by 100 microM GABA were affected by the benzodiazepine flunitrazepam (1 microM). Similarly the benzodiazepine receptor inverse agonist DMCM (1 microM) was also without effect. GABA-activated currents reversed polarity within 2 mV of the calculated Cl- equilibrium potential. With brief agonist pulses deactivation was monoexponential, however, unlike neurones the rate of deactivation was voltage-independent. Desensitisation of responses to 10 mM GABA was bi-exponential and accelerated at depolarised membrane potentials. Increasing the amount of GABA(A) receptor desensitisation (by increasing the duration of the agonist exposure) consistently produced a slowing of deactivation.     

A comparison of the effects of selective metabotropic glutamate receptor agonists on synaptically evoked whole cell currents of rat spinal ventral horn neurones in vitro

1. Whole cell synaptic currents were recorded under voltage clamp from a total of 54 ventral horn neurones held near to their resting potential by the patch clamp technique in immature rat spinal cord preparations in vitro. Twenty eight neurones were identified, by antidromic invasion from ventral roots, as motoneurones. Excitatory postsynaptic currents (e.p.s.cs) of peak amplitude -480 pA +/- 66 s.e. mean and -829 +/- 124 pA were evoked respectively from the unidentified ventral horn neurones and the motoneurones in response to maximal activation of the segmental dorsal root. 2. The e.p.s.cs were depressed reversibly by the metabotropic glutamate agonists 1S3S-1-aminocyclopentane-1,3-dicarboxylate (1S3S-ACPD) (EC50 17.1 microM +/- 0.3 s.e. mean, n = 14) and L-2-amino-4-phosphonobutanoate (L-AP4) (EC50 = 2.19 +/- 0.19 microM, n = 15). Since both agonists independently produced more than 90% depression it is likely that the receptors that mediate their effects are present on the same presynaptic terminals. 3. When the Mg2+ concentration was raised from 0.75 mM to 2.75 mM together with the addition of 50 microM D-2-amino-5-phosphonopentanoate (AP5), a treatment which would increase the proportion of monosynaptic component in the e.p.s.c. the concentration-effect plots for both 1S3S-ACPD (EC50 1.95 +/- 0.4 microM, n = 8) and L-AP4 (EC50 0.55 +/- 0.20 microM, n = 7) were shifted to the left, suggesting that monosynaptic e.p.cs of primary afferents to ventral horn neurones are more susceptible to L-AP4 and 1S3S-ACPD than are other synapses in polysynaptic pathways. 4. lS3S-ACPD (20 and 50 microM) also caused mean sustained inward currents of 95 +/- 31 pA (n = 6) and248 +/- 49 pA (n = 10) respectively. In the combined presence of AP5 (50 microM) and Mg2+ (2.75 mM) themean response to 50 microM lS3S-ACPD was reduced to 106+/- 18 pA (n = 4). In the presence of tetrodotoxin(1 microM) the corresponding value was 48 +/- 6 pA (n = 4). Similar sustained inward currents produced by N-methyl-D-aspartate (NMDA) were almost abolished to < 10 pA in the presence of AP5 and 2.75 mMMg2+. In the presence of tetrodotoxin the maximum inward current produced by NMDA was undiminished. Thus a large component of the excitatory action of lS3S-ACPD was mediated at non-NMDA receptors both directly at the patch-clamped neurones and indirectly by synaptic relay.     

Cortistatin increase of a potassium conductance in rat locus coeruleus in vitro

1. In this study we examined the effects of cortistatin, a putative endogenous ligand for somatostatin (SRIF) receptors, on the membrane properties of rat locus coeruleus (LC) neurones in vitro, by use of intracellular and whole cell patch clamp recording. We have compared the actions of cortistatin with those of SRIF and the SRIF analogue D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP). 2. When LC neurones were voltage clamped to -60 mV, application of cortistatin caused an outward current in all cells examined (n = 44), with a pEC50 of 6.62. SRIF also caused an outward current in all cells examined (n = 43), with a pEC50 of 6.93. 3. The outward currents caused by cortistatin in 2.5 mM extracellular K+ reversed polarity at -106 mV, very close to the predicted K+ reversal potential of -105 mV. Increasing extracellular K+ to 10.5 mM resulted in a shift of the reversal potential of +38 mV, a shift consistent with a K+ conductance. The conductance activated by cortistatin showed mild inward rectification. 4. Continuous application of a high concentration of SRIF (1 microM) resulted in a decrease of the outward current to a steady level of 49% of the maximum response, with a t1/2 of 131 s. Application of a high concentration of cortistatin (3 microM) during the desensitized portion of the SRIF response did not result in any further outward current. Continuous application of a high concentration of cortistatin (10 microM) resulted in a decrease of the outward current to a steady level of 42% of the maximum response with a t1/2 of 114 s. Application of a high concentration of SRIF (3 microM) during the desensitized portion of the cortistatin response produced only a small outward current. 5. Continuous application of cortistatin (3 microM) also resulted in a decrease of the outward current (by 43%, t1/2 of 136 s) and application of a high concentration of CTOP (10 microM) during the desensitized portion of the cortistatin response did not produce any outward current. Continuous application of a high concentration of CTOP (10 microM) resulted in a decrease of the outward current to a steady level of 70% of the maximum response with a t1/2 of 143 s. Application of a high concentration of cortistatin (3 microM) during the desensitized portion of the CTOP response did not result in any further outward current. 6. The actions of cortistatin (300 nM-10 microM) were not affected by the opioid antagonist naloxone (10 microM). Application of met-enkephalin during the desensitized portion of the response to a high concentration of cortistatin (3 microM) produced an outward current similar to that produced by metenkephalin application alone. 7. Thus cortistatin efficaciously activates an inwardly rectifying K+ conductance in LC neurones. These actions appear to be mediated by a population of SRIF receptors, at which CTOP is also an agonist. Cortistatin does not appear to be a ligand for mu-opioid receptors in rat LC neurons.     

Radiolabeling of the rat P2X4 purinoceptor: evidence for allosteric interactions of purinoceptor antagonists and monovalent cations with P2X purinoceptors

The rat recombinant P2X4 purinoceptor was expressed in CHO-K1 cells, and binding studies were performed using the radioligand [35S]adenosine-5'-O-(3-thio)triphosphate ([35S]ATPgammaS). In 50 mM Tris/1 mM EDTA assay buffer, pH 7.4 at 4 degrees, [35S]ATPgammaS bound with high affinity to the P2X4 purinoceptor (KD = 0.13 nM, Bmax = 151 pmol/mg of protein). The purinoceptor agonists ATP and 2-methylthioadenosine triphosphate possessed nanomolar affinity for the P2X4 purinoceptor, whereas the antagonist suramin possessed much lower affinity (IC50 = 0.5 mM). Cibacron blue was more potent than suramin but produced a biphasic competition curve, whereas d-tubocurarine potentiated binding at concentrations in excess of 10 microM. The complex effects of cibacron blue and d-tubocurarine seemed to be due to an allosteric interaction with the P2X4 purinoceptor because these compounds affected radioligand dissociation, measured after isotopic dilution with unlabeled ATPgammaS. Cibacron blue (1-100 microM) and d-tubocurarine (0.1-1 mM) produced rapid (10 sec to 5 min) decreases or increases, respectively, in the level of [35S]ATPgammaS binding measured immediately after initiation of the dissociation reaction. However, the subsequent rates of radioligand dissociation were not markedly different from those measured in their absence. Monovalent cations produced similar affects on the P2X4 purinoceptor and, like d-tubocurarine, increased [35S]ATPgammaS binding. The actions of d-tubocurarine and sodium were not additive. The findings from this study indicate that [35S]ATPgammaS can be used to label the P2X4 purinoceptor and suggest that this binding can be enhanced by monovalent cations and d-tubocurarine and may be subject to negative allosteric modulation to varying degrees by different purinoceptor antagonists.     

Effects of excitatory neurotransmitters on Ca2+ channel current in smooth muscle cells isolated from guinea-pig urinary bladder

1. A whole-cell voltage clamp technique was used to examine the effects of purinoceptor and muscarinic receptor agonists on voltage-sensitive Ca2+ channels in guinea-pig isolated urinary bladder cells. 2. When the cell membrane was clamped at the holding potential, rapid application of ATP elicited a large inward current in normal solution containing 2.5 mM Ca2+, and reduced the subsequent Ca2+ channel current evoked by a depolarizing pulse (0 mV). Carbachol (CCh) elicited little membrane current, but similarly reduced the Ca2+ current. 3. When purinoceptor agonists were rapidly applied during conditioning depolarizations at +80 mV, an outward current was elicited, and the Ca2+ channel current evoked by the subsequent test potential of 0 mV was not affected. Application of CCh at +80 mV also elicited an outward current, but it reduced the subsequently evoked Ca2+ current. 4. The inhibitory effect of muscarinic agonists on the Ca2+ channel current was attenuated by caffeine (10 mM). 5. In Ca(2+)-free, low-Mg2+ solution, a Na+ current flowing through voltage-dependent Ca2+ channels was evoked by depolarization. This current was not reduced by bath application of purinoceptor agonists (ATP and alpha,beta-methylene ATP). 6. These results suggest that the main effect of purinoceptor stimulation is opening of non-selective cation channels, and that muscarinic stimulation triggers Ca2+ release from intracellular stores. Voltage-sensitive Ca2+ channels are inactivated through an increase in intracellular Ca2+ induced by either activation of purinoceptor or muscarinic receptors.     

Effects of bis(2-chloroethyl)sulfide on ATP receptor-mediated responses of the rat vas deferens: possible relationship to cytotoxicity

Extracellular ATP is a broad-spectrum cytotoxic agent that produces effects via cell surface P2 purinoceptors. The ligand-gated P2X purinoceptor subtype has very high sequence homology with the RP-2 gene, which encodes for apoptosis. The P2X RNA found in rat vas deferens is expressed preferentially by apoptotic thymocytes. P2X purinoceptor-mediated phasic (twitch) motor responses of the isolated rat vas deferens to neurogenic or exogenous ATP were rapidly, specifically and irreversibly potentiated by bis(2-chloroethyl)sulfide (HD 10-100 microM). Both untreated and HD-potentiated neurogenic responses were Ca++ dependent, blocked in the absence of Ca++ plus 0.1 mM EGTA, by the neuronal Ca++ channel blocker omega-conotoxin-MVIIC (3 microM), by the P2 purinoceptor antagonist suramin (100 microM) and by tetrodotoxin (100 nM). HD also potentiated the effects of ATP on isolated guinea pig taenia caecum, where the nucleotide acts at G protein-coupled P2Y purinoceptor subtypes to cause relaxation. HD failed to inhibit the metabolism of ATP by ecto-ATPase in vas deferens or to cause the release of endogenous ATP. Potentiation of the twitch response to electric field stimulation by HD was attenuated or eliminated in tissues excised from rats previously challenged with topically applied HD, suggesting that HD absorbed into the systemic circulation had already effected maximal potentiation of ATP responses before in vitro testing. The physiological consequences of HD-induced potentiation of the extracellular actions of ATP are discussed in relation to apoptosis and necrosis.     

2',3'-O-(2,4,6- trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP)--a nanomolar affinity antagonist at rat mesenteric artery P2X receptor ion channels

1. P2X receptor activation by alpha,beta-meATP evoked inward currents in acutely dissociated rat mesenteric artery smooth muscle cells and contractions of whole artery rings. 2. The selective P2X1 and P2X3 receptor antagonist TNP-ATP inhibited P2X receptor mediated inward currents in response to 3 microM alpha,beta-meATP (an approximately EC90 concentration) with an IC50 of approximately 2 nM. This provides further evidence that the P2X receptor underlying membrane depolarisation associated with P2X receptor activation can be accounted for by the expression of P2X1 receptors. 3. TNP-ATP inhibited alpha,beta-meATP induced contractions with an IC50 of approximately 30 microM and had non-specific effects on smooth muscle contraction. 4. The reduced potency of TNP-ATP in whole tissue experiments probably reflects the breakdown of TNP-ATP by nucleotidases. Thus, TNP-ATP is of limited use in whole tissue experiments as a P2X receptor antagonist.     

Co-existence of P2Y-and PPADS-insensitive P2U-purinoceptors in endothelial cells from adrenal medulla

1. We have studied the effects of purinoceptor stimulation on Ca2+ signals in bovine adrenomedullary endothelial cells. [Ca2+]i was determined with the fluorescent probe fura-2 both in population samples and in single, isolated, endothelial cells in primary culture and after subculturing. 2. In endothelial cells, maintained in culture for more than one passage, several purinoceptor agonists elicited clear [Ca2+]i transient peaks that remained in the absence of extracellular Ca2+. Adenosine 5'-triphosphate (ATP) and uridine 5'-triphosphate (UTP) were equipotently active, with EC50 values of 8.5 +/- 0.9 microM and 6.9 +/- 1.5 microM, respectively, whereas 2-methylthioadenosine 5'-triphosphate (2MeSATP), adenosine 5'-(alpha, beta-methylene)triphosphate (alpha, beta-MeATP) and adenosine(5')tetraphospho(5')adenosine (Ap4A) were basically inactive. Adenosine 5'-O-(2-thiodiphosphate) (ADP beta S) was a weak agonist. The apparent potency order was UTP = ATP > ADP beta S > 2MeSATP > alpha, beta-MeATP. 3. Cross-desensitization experiments revealed that UTP or ATP, added sequentially at concentrations of maximal effect, could completely abolish the [Ca2+]i response to the second agonist. ADP beta S exerted only a partial desensitization of the response to maximal ATP, in accordance with its lower potency in raising [Ca2+]i. 4. The effect on [Ca2+]i of 100 microM ATP in subcultured cells was reduced by only 25% with 100 microM suramin pretreatment and was negligibly affected by exposure to 10 microM pyridoxalphosphate-6-azophenyl-2', 4'-disulphonic acid (PPADS). The concentration-effect curve for ATP was not significantly affected by PPADS, but was displaced to the right by a factor of 6.5 by 100 microM suramin. 5. In primary cultures, clear [Ca2+]i responses were elicited by 2MeSATP. Suramin totally and selectively blocked 2MeSATP responses, whereas UTP-evoked [Ca2+]i transients were mainly unaffected by suramin or PPADS. Over 80% of cells tested showed responses to both 2MeSATP and UTP. The [Ca2+]i response to UTP was not desensitized in the presence of 2MeSATP. 6. ATP and UTP stimulated the release of preloaded [3H]-arachidonic acid ([3H]-AA), both in the presence and in the absence of extracellular Ca2+, by approximately 135% with respect to basal levels. Suramin and PPADS enhanced, rather than inhibited, the [3H]-AA releasing effect of ATP by 2.5 times. Suramin also potentiated the effect of the calcium ionophore A23187. 7. These results indicate that endothelial cells from adrenomedullary capillaries co-express both P2Y- and P2U-purinoceptors. P2Y-purinoceptors are lost in culture with the first passage of the cells. The P2U-purinoceptor subtype present in these cells is insensitive to PPADS and thus similar to that found in aortic endothelial cells.     

Selectivity and activity of adenine dinucleotides at recombinant P2X2 and P2Y1 purinoceptors

1. Adenine dinucleotides (Ap3A, x = 2-6) are naturally-occurring polyphosphated nucleotidic substances which are found in the CNS and are known to be released in a calcium-dependent manner from storage vesicles in brain synaptosomes. The selectivity and activity of adenine dinucleotides for neuronally-derived recombinant P2 purinoceptors were studied using P2X2 and P2Y1 subtypes expressed in Xenopus oocytes. 2. For the P2Y1 subtype derived from chick brain, Ap3A was equipotent and as active as ATP (EC50 values: 375 +/- 86 nM and 334 +/- 25 nM, respectively). Ap4A was a weak partial agonist and other dinucleotides were inactive as agonists. None of the inactive dinucleotides were antagonists nor modulated the activity of Ap3A and ATP. 3. For the P2X2 subtype derived from rat PC12 cells, Ap4A was as active as ATP but less potent (EC50 values: 15.2 +/- 1 microM and 3.7 +/- 0.7 microM, respectively). Other adenosine dinucleotides were inactive as either agonists or antagonists. 4. Ap5A (1-100 nM) potentiated ATP-responses at the P2X2 subtype, showing an EC50 of 2.95 +/- 0.7 nM for this modulatory effect. Ap5A (10 nM) shifted the concentration-response curves for ATP to the left by one-half log10 unit but did not alter the Hill co-efficient for ATP (nH = 2.1 +/- 0.1). Ap5A (10 nM) failed to potentiate Ap4A-responses but did enhance the efficacy of the P2 purinoceptor antagonist, suramin, by 12 fold at the P2X2 subtype. 5. In conclusion, the results show that ionotropic (P2X2) and metabotropic (P2Y1) ATP receptors which occur in the CNS are activated selectively by naturally-occurring adenine dinucleotides which are known to be released with nucleotides from storage vesicles. The observed potentiation of P2X2-responses by Ap5A, where co-released with ATP by brain synaptosomes, may have a functional bearing in purinergic signalling in the CNS.     

Fragile mitochondrial DNA: the missing link in the apoptotic neuronal cell death in Parkinson''s disease

Experiments were undertaken to compare effects of the NMDA and non-NMDA receptor antagonists, AP5 (40 microM) and NBQX (10 microM), on glutamate-induced firing in supraoptic oxytocin (OT) and vasopressin (VP) neurones in vitro. In putative OT neurones NBQX caused a significantly greater reduction in firing than AP5, whilst in putative VP neurones both antagonists reduced activity powerfully and to a similar extent. The relatively small effect of AP5 in putative OT neurones was unaffected by the removal of extracellular magnesium. These results suggest that glutamate-induced firing in putative OT neurones is predominantly controlled by non-NMDA receptors.     

Opposing actions of CSW and RasGAP modulate the strength of Torso RTK signaling in the Drosophila terminal pathway

The NMDA receptor antagonistic effects of budipine were assessed using concentration- and patch-clamp techniques on cultured striatal, hippocampal, cortical and superior colliculus neurones. Inward current responses of striatal neurones to NMDA (200 microM) at -70 mV were antagonized by budipine in a concentration-dependent manner (50% inhibitory concentration (IC50) 59.4 +/- 10.7 microM, n = 17) with 24 times lower potency than memantine but similar potency to amantadine. In striatal neurones, budipine blocked outward currents at +70 mV with an IC50 of 827 microM, suggesting that the binding site is less deep in the channel (delta = 0.45) than for memantine. However, more detailed analysis of the fractional block by budipine 300 microM in hippocampal neurones gave a delta-value of 0.90, but revealed that 28% block is mediated at a voltage-independent site. This voltage-insensitive site was accessible in the absence of agonist. Budipine exhibited concentration-dependent open channel blocking kinetics (kappa(on) = 0.71 x 10(4) M(-1) s(-1)) whereas the fast offset rate was concentration-independent (kappa(off) = 0.63 s(-1)). Calculation of the ratio kappa(off)/kappa(on) revealed an apparent Kd value of 88.7 microM. Budipine, memantine and amantadine had similar effects against NMDA-induced currents in cultured hippocampal, cortical and superior colliculus neurones, although amantadine was somewhat more potent in cultured striatal neurones. The relevance of NMDA receptor antagonism to the anti-Parkinsonian effects of budipine remains to be established.     

The role of histamine H1, H2 and H3 receptors on enteric ascending synaptic transmission in the guinea pig ileum

The role of histamine H1-, H2- and H3-receptors was studied on neural transmission in ascending excitatory pathways of the guinea pig ileum. A two-compartment (oral and anal compartments) bath was used: ascending neural pathways were activated by electrical stimulation in the anal compartment and the resulting contraction of the circular muscle in the oral compartment was recorded. Drugs were applied in the anal compartment and each agonist was evaluated in the presence of the antagonists of the other two receptors. In the presence of cimetidine (10 microM) and thioperamide (1 microM), histamine (0.03-3 microM) depressed the nerve-mediated contractions (5-70% inhibition, P <.05-.01). The inhibitory effect of histamine was antagonized by mepyramine. At the higher concentrations (10 and 30 microM), histamine elicited contractions of the circular muscle in the oral compartment, and these were abolished by mepyramine (1 microM) and tetrodotoxin (0.6 microM). The H2 agonists dimaprit (30 and 100 microM) and amphamine (0.1-300 microM) produced small contractions of the circular muscle in the oral compartment. These contractile responses were abolished by tetrodotoxin (0.6 microM) and cimetidine (10 microM). The H3 agonist R-alpha-methylhistamine (0.001-1 microM) inhibited (2-58%, P <.05) the nerve-mediated contractions. This inhibitory effect was antagonized by the H3 antagonist thioperamide. These results indicate that 1) histamine, acting at H1 receptors, at lower concentrations depresses synaptic transmission, although at higher concentrations activates the enteric excitatory ascending pathway; 2) activation of H2 receptors by H2 agonists stimulates the enteric excitatory ascending pathways and 3) activation of H3 receptors inhibits synaptic transmission.