Differential activation of protein kinases in the dorsal horn in vitro of normal and inflamed rats by group I metabotropic glutamate receptor subtypes.

Group I metabotropic glutamate receptors (mGluRs) contribute to spinal sensitization and synaptic plasticity but the underlying mechanisms are unknown. Here, group I mGluR modulation of evoked monosynaptic excitatory postsynaptic currents (EPSCs) in substantia gelatinosa (SG) neurones in vitro was investigated in juvenile rats. In addition, the role of group I mGluRs in dorsal horn neuronal Fos expression was determined in tetrodotoxin (TTX)-treated in vitro spinal cords of naïve rats and those with Complete Freund's Adjuvant (CFA) peripheral inflammation. In the majority of SG neurones, (S)-3,5-dihydroxyphenylglycine (DHPG) reduced EPSCs and this effect was inhibited by the mGluR(5) antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP). Data for paired-pulse and spontaneous miniature excitatory postsynaptic currents (mEPSCs) suggest mGluR(5) acts presynaptically to reduce transmitter release. DHPG-induced reduction of EPSC amplitude operated via PKC, but not ERK, signalling cascade. In the dorsal horn of naïve but not CFA rats, DHPG increased Fos expression and this was reduced by MPEP and both PKC and ERK inhibitors. In the CFA group, basal Fos expression was reduced by MPEP and the kinase inhibitors. These data infer a role for mGluR(5) in acute modulation of nociceptive synaptic efficacy within the dorsal horn and postsynaptic activation of transcription factors such as Fos that are implicated in activity-dependent neuroplastic adaptation. These actions are achieved by differential activation of PKC- and ERK-dependent transduction pathways.

Calcium-activated potassium channel SK1- and IK1-like immunoreactivity in injured human sensory neurones and its regulation by neurotrophic factors.

Calcium-activated potassium ion channels SK and IK (small and intermediate conductance, respectively) may be important in the pathophysiology of pain following nerve injury, as SK channels are known to impose a period of reduced excitability after each action potential by afterhyperpolarization. We studied the presence and changes of human SK1 (hSK1)- and hIK1-like immunoreactivity in control and injured human dorsal root ganglia (DRG) and peripheral nerves and their regulation by key neurotrophic factors in cultured rat sensory neurones. Using specific antibodies, hSK-1 and hIK-1-like immunoreactivity was detected in a majority of large and small/medium-sized cell bodies of human DRG. hSK1 immunoreactivity was decreased significantly in cell bodies of avulsed human DRG (n = 8, surgery delay 8 h to 12 months). There was a decrease in hIK1-like immunoreactivity predominantly in large cells acutely (<3 weeks after injury), but also in small/medium cells of chronic cases. Twenty-three injured peripheral nerves were studied (surgery delay 8 h to 12 months); in five of these, hIK1-like immunoreactivity was detected proximally but not distally to injury, whereas neurofilament staining confirmed the presence of nerve fibres in both regions. These five nerves, unlike the others, had all undergone Wallerian degeneration previously and the loss of hIK1-like immunoreactivity may therefore reflect reduced axonal transport of this ion channel across the injury site in regenerated fibres, as well as decreased expression in the cell body. In vitro studies of neonatal rat DRG neurones showed that nerve growth factor (NGF) significantly increased the percentage of hSK1-positive cells, whereas neurotrophin 3 (NT-3) and glial cell line-derived neurotrophic factor (GDNF) failed to show a significant effect. NT-3 stimulated hIK1 expression, while NGF and GDNF were ineffective. As expected, NGF increased expression of the voltage-gated sodium channel SNS1/PN3 in this system. Decreased retrograde transport of these neurotrophic factors in injured sensory neurones may thus reduce expression of these ion channels and increase excitability. Blockade of IK1-like and other potassium channels by aminopyridines (4-AP and 3,4-DAP) may also explain the paraesthesiae induced by these medications. Selective potassium channel openers are likely to represent novel therapies for pain following nerve injury.

Evidence that P2X purinoceptors mediate the excitatory effects of alphabetamethylene-ADP in rat locus coeruleus neurones.

Extracellular and whole-cell patch clamp recordings were used to study the excitatory responses elicited by purine nucleotides in pontine slices of the rat brain containing the locus coeruleus (LC). The P2 purinoceptor agonists, alphabeta-methyleneadenosine 5'-triphosphate (alphabetameATP) and adenosine 5'-O-(2-thiodiphosphate) (ADPalphabetaS), and a novel purinoceptor agonist, alphabeta-methyleneadenosine 5'-diphosphate (alphabetameADP), elicited concentration-dependent increases in the spontaneous firing rate over the concentration range (1-300 microM). On vagus nerve or dorsal root preparations alphabetameADP (100 microM) had no agonist activity. In the presence of both alphabetameATP (300 microM), ADPbetaS (300 microM) elicited a further and significant increase in the firing rate of the LC neurones, whilst neither alphabetameATP nor alphabetameADP (300 microM) elicited a further response. The P2 purinoceptor antagonists, suramin (100 microM) and pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS; 30 microM), markedly attenuated responses to all three agonists. Whole-cell recording of membrane current showed that, at - 60 mV, alphabetameATP and alphabetameADP (both 100 microM) elicited inward currents of a similar magnitude, whilst the inward currents elicited by a lower concentration of ADPbetaS (30 microM) were larger and faded in the presence of this agonist. In the presence of tetrodotoxin and a combination of other neurotransmission blockers, both alphabetameATP and alphabetameADP still produced inward currents. Based on the known selectivity of the agonists used in this study, there appear to be two distinct P2 purinoceptor types present on neurones in the LC, which correspond to the P2X and P2Y types. The responses elicited by alphabetameADP appear to be mediated through a putative P2X purinoceptor, although further work is required to determine which P2X receptor subtype(s) are involved.

New insights on P2X purinoceptors.

Significant advances in understanding of P2X purinoceptor pharmacology have been made in the last few years. The limitations of nucleotide agonists as drug tools have now been amply demonstrated. Fortunately, inhibitors of the degrading ecto-ATPase enzymes are becoming available and it has become apparent that the complete removal of all divalent cations can be used experimentally in some systems to prevent nucleotide breakdown. Despite these issues, convincing evidence for P2X receptor heterogeneity, from data with agonists, has recently been reported. A number of new antagonists at P2X purinoceptors have also recently been described which to some degree appear to be more specific and useful than earlier antagonists like suramin. It is now apparent that suramin is a poor antagonist of ATP in many tissues because it potently inhibits ATPase activity at similar concentrations to those at which it blocks the P2X purinoceptor. Advances in the use of radiolabelled nucleotides as radioligands for binding studies has allowed the demonstration of P2X purinoceptors in a variety of tissues throughout the body including the brain. These studies have also provided evidence for receptor heterogeneity. Excitingly, two P2X purinoceptor genes have been cloned but operational studies suggest that more than two types exist. The cloning studies have also demonstrated a unique structure for the P2X purinoceptor which differentiates it from all other ligand-gated ion channel receptors. Further studies on P2X purinoceptor operation and structure are needed to help resolve controversies alluded to regarding the characterization and classification of nucleotide receptors. Hopefully such studies will also lead to a better understanding of the physiological and pathological importance of ATP and its activation of P2X purinoceptors. This will require the identification of better drug tools, in particular antagonists which may also provide the basis for novel therapeutic agents.

The selective P2X purinoceptor agonist, beta,gamma-methylene-L-adenosine 5'-triphosphate, discriminates between smooth muscle and neuronal P2X purinoceptors.

The effects of the putative selective P2X purinoceptor agonist, beta,gamma-methylene-L-adenosine 5'-triphosphate (beta gamma me-L-ATP), were determined at rat neuronal and smooth muscle P2X purinoceptors. beta gamma Me-L-ATP had no effect on the extracellularly recorded membrane potential of the rat isolated vagus nerve preparation at concentrations up to 300 microM. In contrast, the archetypal P2X purinoceptor agonist, alpha,beta-methylene ATP (alpha beta meATP; 1-100 microM), produced concentration-related depolarisation responses with a mean EC50 value of 10.8 microM. The depolarising effects of alpha beta meATP were not attenuated by beta gamma me-L-ATP (100 microM). In voltage clamp experiments on single nodose ganglion neurones, ATP (100 microM), but not beta gamma me-L-ATP (1-300 microM), evoked rapid (< 20 ms onset) inward currents when applied using a concentration-clamp method. In receptor binding studies to rat brain membranes, beta gamma me-D-ATP and alpha beta meATP competed with high affinity for [3H]alpha beta meATP binding sites, with mean pIC50 values of 7.7 and 8.3, respectively. However, beta gamma me-L-ATP possessed low affinity for these sites and competed only at concentrations in excess of 10 microM (mean pIC50 value 4.1). In prostatic segments of the rat vas deferens, beta gamma me-L-ATP (1-100 microM) and alpha beta meATP (0.3-100 microM) each produced concentration-related contractile responses with mean EC50 values of 17.1 and 3.6 microM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of divalent cations on the potency of ATP and related agonists in the rat isolated vagus nerve: implications for P2 purinoceptor classification.

1. By use of a 'grease-gap' technique, the depolarizing effects of adenosine 5'-triphosphate (ATP) and ATP analogues on the rat isolated vagus nerve were determined in normal and in Ca2+/Mg(2+)-free (+ 1 x 10(-3) M ethylenediamine tetraacetic acid) physiological salt solution (PSS). 2. In normal PSS, ATP produced concentration-dependent depolarization responses but the concentration-effect curve to ATP was incomplete and a maximum effect was not achieved. The threshold concentration for depolarization was 1 x 10(-5) M and at the highest concentration tested (1 x 10(-3) M) the peak amplitude of the response to ATP only amounted to 71% of the depolarization produced by a near maximal response to 5-hydroxytryptamine (5-HT, 1 x 10(-5) M). 3. In Ca2+/Mg(2+)-free PSS, ATP produced depolarization responses at much lower concentrations and of markedly larger amplitude. Under these conditions, the threshold concentration for depolarization was 1-3 x 10(-7) M and the maximal response to ATP amounted to 526% of the response to 5-HT (1 x 10(-5) M) in normal PSS. The concentration-effect curve to ATP was sigmoid, with a defined maximum effect and a mean EC50 value of 1.2 x 10(-6) M. 4. In contrast to the effects on responses to ATP, the absence of divalent cations in the PSS did not modify the effective concentrations of either alpha, beta-methylene ATP or 5-HT. However, the maximum responses to both alpha, beta-methylene ATP and 5-HT were significantly increased in Ca2+/Mg(2+)-free PSS. 5. The depolarizing effects of several analogues of ATP were determined in Ca2+/Mg2+-free PSS.ATP-gamma-S and 2-methylthioATP were of similar potency to ATP (respective equi-effective molar ratios(EMRs) of 1.9 and 1.3, where ATP = 1) and similar maximum responses were obtained. Alpha, beta-MethyleneATP, beta, gamma-methylene ATP and ,beta, gamma-imido ATP were considerably less potent than ATP, analysis yielding mean EMRs of 48.9, 85.0 and 60.0, respectively. Maximum responses to these latter three agonists were not obtained at the highest concentrations tested (1 x 10-4-3 X 10- M). Benzoyl ATP, adenosine 5'-0-(2-thiodiphosphate) and adenosine diphosphate produced only small depolarizing responses at high concentrations (>1 x 10-4 M). Adenosine monophosphate, adenosine and uridine S'-triphosphate each had little or no depolarizing effect in Ca2+/Mg2+-free PSS.6. These data demonstrate that in the absence of divalent cations the excitatory actions of some, but not all, purine nucleotides in the rat vagus nerve are markedly potentiated. In Ca2+/Mg2+-free PSS, the rank order of agonist potencies was ATP = 2-methylthioATP = ATP-gamma-S>> alpha,beta-methylene ATP = beta, gamma imido ATP = P,y-methylene ATP. These findings are in stark contrast to our previous observations in normal PSS where the rank order of agonist potencies for these nucleotides was alpha,beta-methyleneATP> ATP-gamma-S > beta,gamma-imido ATP = beta,gamma-methylene ATP> 2-methylthioATP> ATP.7. We suggest that the two different rank orders of potency can be explained by differential metabolism involving Ca2+/Mg2+-dependent ectonucleotidases. If so, these data indicate that ATP and 2-methylthioATP are inherently more potent than alpha,beta-methylene ATP as agonists at neuronal P2X purinoceptors in the rat vagus nerve. The possible implications of these findings to the present system for subclassifying P2 purinoceptors are profound.

P2 purinoceptor antagonist properties of pyridoxal-5-phosphate.

The antagonist properties of pyridoxal-5-phosphate, a synthesis precursor of pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid, were investigated on P2 purinoceptor-mediated responses of the rat isolated vagus nerve and vas deferens. In addition, the effect of this agent was studied on high affinity tritiated alpha,beta-methylene adenosine triphosphate (alpha,beta-meATP) binding to rat vas deferens membranes, thought to represent binding to functional P2x purinoceptors. In the rat vagus nerve, pyridoxal-5-phosphate (10(-5)-10(-4) M) produced concentration-related antagonism of depolarisation responses induced by alpha,beta-meATP, measured using an extracellular recording technique. In contrast, depolarisation responses to 5-hydroxytryptamine (5-HT) were unaffected by pyridoxal-5-phosphate. In the rat vas deferens, pyridoxal-5-phosphate (10(-5)-10(-4) M) antagonised contractile responses produced by alpha,beta-meATP while contractions to phenylephrine were unaffected. However, responses of the vagus nerve and the vas deferens to alpha,beta-meATP were not antagonised by pyridoxal hydrochloride (10(-4) M). Pyridoxal-5-phosphate competed for high affinity binding of [3H]alpha,beta-meATP to homogenised membranes of the rat vas deferens with a pKi estimate of 4.91 +/- 0.12 and a Hill slope of 0.80 +/- 0.03. Pyridoxal hydrochloride only competed for binding at concentrations in excess of 10(-4) M, yielding a pKi estimate of 3.21 +/- 0.04 and a Hill slope of 1.82 +/- 0.12. These findings indicate that pyridoxal-5-phosphate acts as a specific antagonist of P2 purinoceptors in the vagus nerve and vas deferens of the rat and that the phosphate moiety is required for activity.(ABSTRACT TRUNCATED AT 250 WORDS)

The use of antagonists to characterize the receptors mediating depolarization of the rat isolated vagus nerve by alpha, beta-methylene adenosine 5'-triphosphate.

1. We have previously found that the P2x-purinoceptor agonist, alpha, beta-methylene adenosine 5'-triphosphate (alpha, beta-methylene ATP), depolarizes the rat cervical vagus nerve, measured with a 'grease-gap' extracellular recording technique. This effect was attenuated by the P2 purinoceptor antagonist, suramin. In the present study we have investigated in more detail the antagonism produced by suramin and have also investigated the actions of two other putative P2 purinoceptor antagonists, cibacron blue and pyridoxal-phosphate-6-azophenyl-2', 5'-disulphonic acid (iso-PPADS). Furthermore, we have studied the interactions between suramin and cibacron blue or iso-PPADS in an attempt to determine whether these antagonists act at a common receptor site. 2. Suramin (1 x 10(-5)-1 x 10(-4) M) produced reversible, concentration-related rightward displacements of the concentration-effect curve to alpha, beta-methylene ATP. Schild analysis of this antagonism yielded a pA2 value of 5.90 with a slope value of 0.47. 3. Cibacron blue (3 x 10(-5)-1 x 10(-4) M) also antagonized depolarizations induced by alpha, beta-methylene ATP. The antagonistic effects of cibacron blue were slow to reach equilibrium but could be readily reversed on washout. At low concentrations for antagonism, cibacron blue (1 x 10(-5) M and 3 x 10(-5) M) produced enhancement of the maximal response to alpha, beta-methylene ATP. At the highest concentration tested (1 x 10(-4) M) the concentration-effect curve to alpha, beta-methylene ATP was shifted to the right in a parallel manner, yielding a pKB estimate of 4.96. 4. Iso-PPADS (1 X 10-6 1 X 10-5- M) produced a concentration-related depression in the maxima ofthe concentration-effect curves to alpha,beta-methylene ATP. Analysis of these data by a double reciprocal plot yielded a pKB estimate of 6.02. This profile of insurmountable antagonism could not be attributed to irreversible binding of iso-PPADS to the receptor since the effect of iso-PPADS could be reversed on washing, albeit slowly.5. In the presence of suramin (1 x 10-4 M), cibacron blue (1 x 10-4 M) produced no further rightward displacement of the alpha,beta-methylene ATP concentration-effect curve. The mean agonist concentration ratios in the presence of suramin or cibacron blue alone (11.7 and 10.3, respectively) were not significantly different from the mean concentration-ratio in the presence of both antagonists (11.8). This finding suggests that high concentrations of alpha,beta-methylene ATP activate a receptor population which is resistant to blockade by either antagonist.6. The antagonistic effect of iso-PPADS (1 x 10-5 M) was partially attenuated by suramin (1I x 10-4 M).It is possible that this interaction reflects a slow dissociation of iso-PPADS from the receptor with which suramin and alpha,beta-methylene ATP interact.7. Suramin, cibacron blue or iso-PPADS had no marked effect on depolarization produced by 5-hydroxytryptamine (5-HT, 1 x 10-7-3 x 10-5 M), indicating their specificity in antagonizing responses to alpha, beta-methylene ATP.8. Responses to alpha,beta-methylene ATP were not antagonized by 8-para-sulphophenyltheophylline (3 x 10-5M), ondansetron (1 x 10-7 M), bicuculline (1 x I0-5 M), phentolamine (1 X 10-6 M) or hexamethonium(1 X 10-4 M), which are antagonists at P1-purinoceptors, 5-HT3 receptors, GABAA receptors, a-adrenoceptors and nicotinic cholinoceptors, respectively, thereby excluding the involvement of these receptors.Indomethacin (3 X 10-6 M) had no effect on responses to alpha,beta-methylene ATP.9. The results obtained with three purinoceptor antagonists confirm and extend our original supposition that alpha,beta-methylene ATP-induced depolarization of the rat vagus nerve is mediated predominantly via P2 purinoceptors, thought to be of the P2,X subtype. The finding that responses induced by high concentrations of agonist were resistant to blockade by suramin and cibacron blue, but could be attenuated by iso-PPADS, adds further weight to our speculation that the purinoceptor population in the rat vagus nerve is heterogeneous.

Characterization of purinoceptors mediating depolarization of rat isolated vagus nerve.

1. As part of a broader study to characterize neuronal purinoceptors, the effects of adenosine 5'-triphosphate (ATP) and a range of ATP analogues were investigated on the extracellularly recorded membrane potential of the rat isolated vagus nerve, using a 'grease-gap' technique. 2. ATP evoked depolarization of the rat vagus nerve. The concentration-effect curve to ATP was not monophasic: at the lower concentrations (1 x 10(-5)-1 x 10(-3) M) the curve was shallow (< 50% of the near maximal response to 5-hydroxytryptamine (5-HT)) whilst at higher concentrations the relationship between concentration and amplitude of depolarization was steeper (> 135% of the response to 5-HT at the highest concentration tested, 1 x 10(-2) M). On washout of the high drug concentrations large after-hyperpolarizations were often observed. 3. alpha,beta-methylene ATP (1 x 10(-6)-3 x 10(-4) M), beta,gamma-methylene ATP (1 x 10(-6)-1 x 10(-3) M), and 5'-adenylylimidodiphosphate (beta,gamma-imido ATP; 1 x 10(-6)-1 x 10(-3) M) were all more potent than ATP and produced large depolarizations of the rat vagus nerve at the highest concentrations tested (> 150% of the response to 5-HT). The overall rank order of potency was alpha,beta-methylene ATP > beta,gamma-methylene ATP = beta,gamma-imido ATP > ATP. 4. In contrast, 2-methylthio ATP (1 x 10(-6)-1 x 10(-3) M) produced relatively small depolarizations (< 100% of the response to 5-HT). As was the case with low concentrations of ATP, the concentration-effect curve to 2-methylthio ATP was very shallow. 5. Adenosine 5'-diphosphate (ADP), adenosine 5'-monophosphate (AMP), adenosine and adenosine 5'-O-(2-thiodiphosphate) (ADP-beta-s; all 1 x 10-6 1 x 10-3M) evoked only small depolarizations of the vagus nerve, amounting to 47 +/- 2.5%, 40.8 +/- 7.8%, 33.7 +/- 3.3% and 62.4 +/- 12.7% of the response to 5-HT, respectively. Uridine 5'-triphosphate (UTP; 1 X 10-6 1 X 10-3M) was inactive.6. The P2 purinoceptor antagonist, suramin (1 x 10-5-M-1 X 10-4 M), antagonized responses to alpha-beta-methylene ATP. The nature of this antagonism was not, however, consistent with simple competitive kinetics between agonist and antagonist. Depolarizations produced by beta,gamma-methylene ATP and beta,gamma-imido ATP were also attenuated by suramin (1 x 10-4 M), but in contrast, suramin had no effect on responses to ADP, 2-methylthio ATP, ADP-beta-S or 5-HT.7. In addition to its antagonist effects, suramin (10-4 M) markedly increased the maximum amplitude of the depolarization produced by ATP.8. It is concluded that a heterogeneous receptor population mediates depolarization of the rat vagus nerve by purine nucleotides. Importantly, the large amplitude depolarizations to alpha,beta-methylene ATP,beta,gamma-methylene ATP and beta,gamma-imido ATP are mediated via receptors that share many characteristics of the classical P2, receptor. In contrast, the relatively small depolarizing effects of ADP, ADP-beta-S and 2-methylthio ATP were suramin-resistant. Although it appears that other purinoceptors are present,these data suggest that the rat vagus nerve may serve as a useful preparation for studying the pharmacology of neuronal P2x receptors.