(1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid attenuates N-methyl-D-aspartate-induced neuronal cell death in cortical cultures via a reduction in delayed Ca2+ accumulation.

The effects of (1S,3R)-ACPD, a selective metabotropic glutamate receptor agonist, on NMDA-induced 45Ca2+ accumulation and delayed neuronal cell death were determined using primary cerebrocortical cultures. Exposure to (1S,3R)-ACPD alone, although causing small increases in 45Ca2+ accumulation, was not neurotoxic. The presence of (1S,3R)-ACPD during exposure to NMDA attenuated the resulting sustained accumulation of 45Ca2+ and delayed neuronal cell death. Reductions in sustained Ca2+ accumulation were associated both with Ca2+ efflux, in the absence of cell death, and inhibition of delayed intracellular Ca2+ accumulation. The protective effects of (1S,3R)-ACPD on NMDA-induced cell death were inhibited by pretreatment of cultures with pertussis toxin. These results suggest that activation of metabotropic glutamate receptors may stimulate intracellular processes capable of limiting sustained elevations in intracellular calcium and the resulting excitotoxic neuronal damage.

Excitatory amino acid receptor-stimulated phosphoinositide turnover in primary cerebrocortical cultures.

1. Characterization of excitatory amino acid-induced accumulation of [3H]-phosphoinositides was carried out in primary cerebrocortical cultures isolated from foetal rats. 2. All of the excitatory amino acid receptor agonists examined caused concentration-dependent enhancement of phosphoinositide (PI) formation. The most potent excitatory amino acid receptor agonists were quisqualate, (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid ((1S,3R)-ACPD), ibotenate and glutamate with mean EC50 values of 0.9 +/- 0.4 microM, 15 +/- 5 microM, 15 +/- 3 microM and 41 +/- 8 microM respectively. 3. The selective ionotropic receptor antagonists kynurenic acid (1 mM), 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline (NBQX, 10 microM) and (+/-)-4-(3-phosphonopropyl)-2 piperazinecarboxylic acid (CPP, 100 microM), failed to block responses to quisqualate, (1S,3R)-ACPD or glutamate. D,L-2-Amino-3-phosphonopropionate (D,L-AP3) did not block 1S,3R-ACPD or quisqualate-induced PI turnover, but had an additive effect with quisqualate or (1S,3R)-ACPD. 4. Exposure of cultures to agonists in the absence of added extracellular calcium reduced the maximal quisqualate response by approximately 45%, revealing a two-component concentration-response curve. Concentration-response curves to ibotenate and glutamate became flattened by omission of extracellular calcium, whereas (1S,3R)-ACPD-stimulated PI turnover was unaffected. 5. Pretreatment of cultures with pertussis toxin markedly inhibited PI responses evoked by (1S,3R)-ACPD. 6. These results suggest that excitatory amino acid-stimulated PI turnover in cerebrocortical cultures is independent of ionotropic receptor activation and is mediated via specific G-protein-linked metabotropic receptors. The partial dependence of the responses to quisqualate, ibotenate and glutamate on the presence of extracellular calcium suggests that the effects of these agonists may be mediated by more than one receptor subtype.

The effects of capsaicin, bradykinin, PGE2 and cicaprost on the discharge of articular sensory receptors in vitro.

The responses of articular sensory receptors to capsaicin, bradykinin, PGE2, and the selective IP-receptor agonist cicaprost were studied in a rat isolated hindlimb in vitro preparation. Long-term maintenance of normal sensory receptor function was achieved in vitro under conditions of combined superfusion and slow perfusion. Response characteristics to mechanical or chemical stimuli on articular sensory receptors identified in this study did not differ to those reported in vivo. This preparation lacks complex effects mediated via spinal or central reflex mechanisms and allows greater control over the physiological environment of the receptors being studied. These results support the conclusion that the effects of capsaicin, bradykinin and the prostanoids are mediated by distinct pharmacological receptors associated with articular sensory nerve endings. The potent potentiating effects of cicaprost on bradykinin-induced excitation suggests that these actions are mediated via IP-receptors.

Prostanoid-induced potentiation of the excitatory and sensitizing effects of bradykinin on articular mechanonociceptors in the rat ankle joint.

Responses of articular mechanonociceptors to intra-arterial injections of either bradykinin alone or in combination with prostaglandin E2, prostaglandin I2 or the selective I-type prostaglandin receptor agonist cicaprost were investigated electrophysiologically in anaesthetized rats. Bradykinin excited 76% of the mechanonociceptors studied and increased their responsiveness to repeated mechanical stimuli in 70% of units. Tachyphylaxis of these responses was evident in all cases. Injections of minimally effective doses of prostaglandin I2 or cicaprost excited the mechanonociceptors and increased their responsiveness to mechanical stimuli. Injections of prostaglandin E2 evoked only small increases in spontaneous discharge. Potentiation of bradykinin-evoked excitation was seen for combined injections of bradykinin with minimally effective or subthreshold doses of cicaprost in 86% of units, prostaglandin I2 in 40% of units and prostaglandin E2 in 56% of units. Combined injections of bradykinin and minimally effective or subthreshold doses of prostanoid agonist caused an increase in the responsiveness of mechanonociceptors to mechanical stimuli greater than that caused by either drug alone in 80% of units for cicaprost, 80% for prostaglandin I2 and 100% for prostaglandin E2. The relative potencies of prostaglandin I2, cicaprost and prostaglandin E2, suggest that prostanoid-induced excitation and sensitization of articular mechanonociceptors is mediated primarily by receptors for the naturally occurring prostanoid prostaglandin I2 (I-type P-receptors). Prostaglandin E2 may be important in potentiation of the sensitizing effects of bradykinin on mechanonociceptor responsiveness.

Effect of varying levels of glucose on oxygen deprivation-induced delayed neuronal cell death in primary cerebrocortical cultures.

Calcium accumulation and neuronal injury of rat cortical cell cultures in vitro were examined following oxygen deprivation under conditions of normal and low glucose. 45Ca2+ uptake and lactate dehydrogenase (LDH) release, measured at 12 and 24 h after oxygen deprivation, were significantly elevated in cultures exposed to combined oxygen deprivation and low glucose (1.7 or 0.6 mM). Although those cultures deprived of oxygen combined with no glucose displayed delayed increases in 45Ca2+ influx, no significant elevation in LDH release at 24 h was observed.

Effects of paracetamol and aspirin on neural activity of joint mechanonociceptors in adjuvant arthritis.

1. The effects of paracetamol and lysine acetylsalicylate (L-AS) on high-threshold mechanonociceptors have been investigated by recording neural activity from the inflamed ankle joint in anaesthetized rats with mild adjuvant-induced monoarthritis. 2. Paracetamol (50 mg kg-1, i.v.) and L-AS (100 mg kg-1, i.v., equivalent to 50 mg kg-1 aspirin) both caused a maximal reduction of about 40% in mechanically-evoked discharge and of 30% in ongoing (spontaneous) activity by about 15 min after the injection: a second dose of either drug did not have any significant additional effect on discharge. 3. The prostanoid IP receptor agonist, cicaprost (0.1-0.5 micrograms), increased both mechanically-evoked and ongoing discharge to pre-paracetamol levels when injected close-arterially 30-50 min after paracetamol, whereas prostaglandin E2 (PGE2) was relatively ineffective at restoring activity. 4. The results suggest that prostacyclin (PGI2) contributes to the sensitization of high-threshold joint mechanonociceptors in adjuvant-induced monoarthritis, and that paracetamol and L-AS both act to reduce discharge by inhibiting the synthesis of prostacyclin in the joint capsule. 5. Paracetamol has a direct peripheral action affecting joint capsule mechanonociceptors in rat adjuvant-induced arthritis which is very similar to that of the soluble aspirin preparation, L-AS. These findings, together with the existing literature concerning the anti-arthritic effects of paracetamol, are relevant to the treatment of chronic inflammatory disorders such as rheumatoid arthritis.

PGI2-induced activation and sensitization of articular mechanonociceptors.

The effects of PGE2 PGI2 and the stable PGI2 analogue cicaprost on the afferent discharge of ankle joint mechanonociceptors were studied in the anaesthetized rat. Close-arterial injection of PGI2 (0.01-0.1 micrograms) or cicaprost (0.05-5 micrograms) caused both sensitization to mechanical stimulation and excitation of the majority of mechanonociceptors, whereas PGE2 (0.03-3 micrograms) had only weak effects on a small number of nociceptive units. These results suggest the existence of specific PGI2 sensitive receptors (IP receptors) on rat sensory afferent nerves, and support the hypothesis that in the rat endogenous PGI2 plays an important role in the lowering of nociceptive thresholds in inflamed joints.

The role of PGE2 in the sensitization of mechanoreceptors in normal and inflamed ankle joints of the rat.

The role of PGE2 in the sensitization of high-threshold tarsal joint mechanoreceptors (putative nociceptors) has been investigated in 11 arthritic and 16 normal rats. Injections of a low dose of Freund's complete adjuvant at multiple sites into the tissues surrounding the ankle joint induced a chronic unilateral monoarthritis in the injected limb. Measurements of both spontaneous activity and responses of tarsal joint mechanoreceptors to repeated graded mechanical stimuli were made. All of the mechanoreceptors examined had afferent fibres with conduction velocities in the C- or A-delta range. Using this new model of joint inflammation we have shown that lysine acetylsalicylate reduces the mechanical sensitivity of these joint mechanoreceptors and reduces the spontaneous activity in afferent nerve fibres. Prostaglandin E2 is unable to restore either the spontaneous activity in the afferent axon or the mechanical sensitivity of tarsal joint mechanoreceptors after lysine acetylsalicylate in the arthritic rat. Similarly, PGE2 does not sensitize or excite tarsal joint mechanoreceptors in the normal rat. In the normal rat, however, PGE2 potentiates the excitatory action of bradykinin and enhances the sensitizing effect of bradykinin on the responses of joint mechanoreceptors to mechanical stimulation when both substances are injected simultaneously. These results indicate that PGE2 is not important in the sensitization of these joint mechanoreceptors in this model of chronic joint inflammation but that in other circumstances PGE2 may be able to contribute to a sensitization of joint mechanoreceptors by enhancing the action of bradykinin.

The effects of 5-HT on articular sensory receptors in normal and arthritic rats.

1. The effects of intra arterial (i.a.) injections of 5-hydroxytryptamine (5-HT, 1-100 micrograms) on the discharge of (a) identified articular high threshold mechanoreceptors and (b) unidentified chemosensitive receptors in the ankle joint have been studied electrophysiologically in anaesthetized normal and arthritic rats. Recordings were made from a fine branch of the medial plantar nerve. 2. 5-HT increased the mechanical responsiveness of high threshold nociceptive mechanoreceptors with C and A delta fibre afferents in both normal and adjuvant-arthritic rats. Receptors in arthritic joints were more sensitive to 5-HT than were those from normal joints. 3. 5-HT produced a complex response from both types of articular receptors following i.a. injection. Two separate components were identified: (a) a fast transient burst of activity was obtained within 10 s of this injection in 66% of units from normal animals and 45% from arthritics, followed by (b) a delayed slow longer-lasting excitation seen in 62% of the units examined from normals and 77% of units from arthritic rats. 4. Increased mechanoreceptor responsiveness produced by 5-HT was reduced or abolished by the 5-HT3 receptor antagonists studied (MDL 72222, ICS 205-930, or GR 38032F, in single doses of 100 micrograms kg-1, i.a.). 5. Fast excitation showed marked tachyphylaxis and was antagonized by MDL 72222, ICS 205-930 or GR 38032F. It was unaffected by ketanserin (100 micrograms kg-1, i.a.). Delayed excitation was reduced or abolished by ketanserin but was unaffected by the 5-HT3-receptor antagonists. 6. Administration of MDL 72222, ICS 205-930 or GR 38032F caused short lasting (< 5 min) reductions in background activity from both types of unit recorded in arthritic rats, as well as in normal rats in which activity had increased following administration of 5-HT. Ketanserin caused similar reductions in background activity in chemosensitive units, but had no effect on mechanoreceptors. 7. At least two types of receptor are involved in the actions of 5-HT on articular sensory receptors with fine afferent fibres. Increased mechano-responsiveness involves a 5-HT3-receptor as does fast excitation. Delayed excitation probably involves a 5-HT2-receptor. Endogenous 5-HT appears not to play a crucial role in sensitization of high threshold mechanoreceptors in this model of chronic inflammation and arthritis, although its local release may potentiate the actions of other inflammatory mediators on sensory receptors in the ankle joint.

Arterial chemoreceptor involvement in salicylate-induced hyperventilation in rats.

1. The extent to which peripheral arterial chemoreceptors are involved in the respiratory stimulant action of salicylates has been investigated in rats. 2. Injection of sodium salicylate (200 mg kg-1, single dose i.v.) caused a rapid transient hyperventilation that was not obtained when the carotid chemoreceptors were denervated by section of the carotid sinus nerves. A delayed (10 min) increase in respiration occurred regardless of whether or not the carotid nerves were sectioned. 3. Intravenous infusions of sodium salicylate (0.5 or 4 mg kg-1 min-1) caused hyperventilation in barbiturate-anaesthetized rats. The threshold dose for respiratory stimulation was significantly lower when the carotid sinus nerves were intact than when they were bilaterally sectioned, and the same pattern was observed following intravenous injections of sodium salicylate (cumulative doses) in anaesthetized and conscious rats. 4. Bilateral sectioning of the vagosympathetic nerve trunks did not significantly affect hyperventilation evoked by salicylate, suggesting that this response does not involve actions of salicylate on sensory receptors innervated by these nerves. 5. Administration of salicylate close-arterial to a carotid body, by local perfusion or cross-perfusion of a carotid sinus, led to an increase in respiration when the ipsilateral carotid nerve was intact, but not when it was sectioned. 6. Neuropharmacological studies on anaesthetized rats showed that chemosensory discharge, recorded from a sectioned carotid nerve, increased in response to salicylate injections with a similar dose-response pattern to the hyperventilation. Salicylate had no effect on baroreceptor discharge. 7. We conclude from our experiments that arterial chemoreceptors do contribute to salicylate-induced hyperventilation, and are almost exclusively responsible for the initial phase of the response in rats. Later increases in breathing are independent of reflexes from arterial chemoreceptors and result from actions at other sites, including the CNS. The therapeutic implications of our results are discussed.

A study of 5-HT-receptors associated with afferent nerves located in normal and inflamed rat ankle joints.

Neural recordings were made from sensory fibres in a nerve supplying the ankle joint in normal rats and in rats with a novel monoarticular arthritis. The responses of mechanically and chemically sensitive units to intra-arterial injections of 5-HT were measured. In most cases the mechanosensitivity of sensory receptors in the ankle joint was not altered by 5-HT. However, 5-HT produced an increase in afferent activity in units which were identified as C-fibres on the basis of action potential amplitude and duration. The receptive fields of these chemosensitive units were not located. The responses of these units to 5-HT were dose dependent and were abolished by the 5-HT2-antogonist, ketanserin, but not by the 5-HT3-receptor antagonist, MDL 72222. The responses of chemosensitive units to injections of 5-HT were similar in normal and arthritic rats although the response was slightly prolonged in arthritic animals.