Conserved polar residues in the transmembrane domain of the human tachykinin NK2 receptor: functional roles and structural implications.

We have studied the effects of agonist and antagonist binding, agonist-induced activation and agonist-induced desensitization of the human tachykinin NK2 receptor mutated at polar residues Asn-51 [in transmembrane helix 1 (TM1)], Asp-79 (TM2) and Asn-303 (TM7), which are highly conserved in the transmembrane domain in the rhodopsin family of G-protein-coupled receptors. Wild-type and mutant receptors were expressed in both COS-1 cells and Xenopus oocytes. The results show that the N51D mutation results in a receptor which, in contrast with the wild-type receptor, is desensitized by the application of a concentration of 1 microM of the partial agonist GR64349, indicating that the mutant is more sensitive to agonist activation than is the wild-type receptor. In addition, we show that, whereas the D79E mutant displayed activation properties similar to those of the wild-type receptor, the D79N and D79A mutants displayed a severely impaired ability to activate the calcium-dependent chloride current. This suggests that it is the negative charge at Asn-79, rather than the ability of this residue to hydrogen-bond, that is critical for the activity of the receptor. Interestingly, the placement of a negative charge at position 303 could compensate for the removal of the negative charge at position 79, since the double mutant D79N/N303D displayed activation properties similar to those of the wild-type receptor. This suggests that these two residues are functionally coupled, and may even be in close proximity in the three-dimensional structure of the human tachykinin NK2 receptor. A three-dimensional model of the receptor displaying this putative interaction is presented.

The relationship between the agonist-induced activation and desensitization of the human tachykinin NK2 receptor expressed in Xenopus oocytes.

1. Repeated applications of neurokinin A (NKA) to oocytes injected with 25 ng wild-type hNK2 receptor cRNA caused complete attenuation of second and subsequent NKA-induced responses while analogous experiments using repeated applications of GR64349 and [Nle10]NKA(4-10) resulted in no such desensitization. This behaviour has been previously attributed to the ability of the different ligands to stabilize different active conformations of the receptor that have differing susceptibilities to receptor kinases (Nemeth & Chollet. 1995). 2. However, for Xenopus oocytes injected (into the nucleus) with 10 ng wild-type hNK2 receptor cDNA, a single 100 nM concentration of any of the three ligands resulted in complete desensitization to further concentrations. 3. On the other hand, none of the ligands caused any desensitization in oocytes injected with 0.25 ng wild-type hNK2 receptor cRNA. even at concentrations up to 10 microM. 4. The two N-terminally truncated analogues of neurokinin A have a lower efficacy than NKA and it is likely that it is this property which causes the observed differences in desensitization, rather than the formation of alternative active states of the receptor. 5. The peak calcium-dependent chloride current is not a reliable measure of maximal receptor stimulation and efficacy is better measured in this system by studying agonist-induced desensitization. 6. The specific adenylyl cyclase inhibitor SQ22536 can enhance NKA and GR64349-mediated desensitization which suggests that agonist-induced desensitization involves the inhibition of adenylyl cyclase and the subsequent down-regulation of the cyclic AMP-dependent protein kinase, possibly by cross-talk to a second signalling pathway.

5-HT spinal antinociception involves mu opioid receptors: cross tolerance and antagonist studies.

The antinociceptive effects of intrathecal 5-HT, fentanyl, ICI197067 and U50488H were assessed by electrical current nociceptive threshold and tail flick latency measurements. Equieffective doses of these agonists were then given intrathecally with a range of doses of naloxone or the highly selective mu opioid antagonist, beta-funaltrexamine. Antagonist dose-response curves were plotted. Other rats were made tolerant to either fentanyl or 5-HT by intrathecal injections of these drugs seven times daily and the antinociceptive effects of intrathecal fentanyl and 5-HT were assessed in each group. All intrathecal drugs caused spinally mediated antinociception in both tests. The antinociceptive effects of intrathecal 5-HT assessed by the electrical test (ECT) but not by tail flick latency (TFL) were suppressed by both opioid antagonists at doses similar to those required to suppress all of the effects of intrathecal fentanyl. The ED50 values were 0.22 (fentanyl, ECT), 0.25 (fentanyl, TFL) and 0.18 (5-HT, ECT) mumol kg-1 for naloxone and for beta-funaltrexamine 2.2 fmol (5-HT, ECT), the same order as that required to produce similar suppression of the antinociceptive effects of fentanyl (46 amol: fentanyl, ECT; 4.6 fmol: fentanyl, TFL) and very different from the ED50 for beta-FNA suppression of the antinociceptive effects of the kappa opioid, U50488H (5.88 pmol). Cross tolerance in both directions was demonstrated between intrathecal fentanyl and 5-HT in the electrical test but not in the tail flick test. We conclude that intrathecal 5-HT caused spinally mediated antinociceptive effects revealed by electrical current and tail flick latency tests. The antinociceptive effects in the electrical test involved spinal cord mu opioid receptors.

Antinociception by intrathecal midazolam involves endogenous neurotransmitters acting at spinal cord delta opioid receptors.

Intrathecal midazolam causes antinociception by combining with spinal cord benzodiazepine receptors. This effect is reversible with doses of naloxone, suggesting involvement of spinal kappa or delta but not mu opioid receptors. The antinociceptive effects of intrathecally administered drugs in the spinal cord were demonstrated by measurements of the electrical current threshold for avoidance behaviour in rats with chronically implanted lumbar intrathecal catheters. A comparison was made of suppression by two opioid selective antagonists (nor-binaltorphimine (kappa selective) and naltrindole (delta selective)) of spinal antinociception caused by equipotent doses of opioids selective for different receptor subtypes (U-50488H (kappa), DSLET and DSBULET (delta), fentanyl (mu)) and the benzodiazepine midazolam. Nor-binaltorphimine selectively suppressed the effects of U-50488H but not midazolam or fentanyl. However, the delta selective antagonist, naltrindole, caused dose-related suppression of antinociception produced by both delta opioid agonists and midazolam with the same ED50 (0.5 nmol). We conclude that intrathecal midazolam caused spinally mediated antinociception in rats by a mechanism involving delta opioid receptor activation.

Inhibition of Ca(2+)-sensitive K+ currents in NG 108-15 cells by substance P and related tachykinins.

1. The whole-cell patch-clamp technique was used to investigate the actions of substance P and other agonists at neurokinin (NK) receptors on voltage-gated K+ and Ca+ channel currents in undifferentiated mouse neuroblastoma x rat glioma NG 108-15 cells. 2. Both substance P (0.3-30 microM) and the NK1 receptor selective agonist GR73632 (10 nM-10 microM) caused concentration-dependent inhibition of K+ currents. GR64349 and senktide (agonists at NK2 and NK3 receptors respectively) also inhibited K+ currents, but only at concentrations which were several orders of magnitude greater than GR73632, suggesting that current inhibition was mediated via NK1 receptors. 3. Substance P and GR73632 were without effect on residual K+ currents recorded in the presence of extracellular Co2+ (4 mM) to abolish the Ca(2+)-sensitive component (IKca) of the K+ current. Ca2+ channel currents, recorded with either Ba2+ or Ca2+ as charge carrier, were unaffected by NK1, NK2 and NK3 receptor ligands. 4. Iontophoretic application of GR73632 produced a current-dependent reduction of K+ currents. In the presence of the non-peptide NK1 antagonists, CP-99,994 and RP67580, and the peptide antagonist, GR82334, the current-response relationship was reversibly shifted to the right. This indicates that the response is mediated by NK1 receptors. 5. Our results indicate that activation of NK1 receptors leads to the selective inhibition of IKca in undifferentiated NG 108-15 cells.

gamma-Aminobutyric acidA receptors and spinally mediated antinociception in rats.

Experiments were performed on rats with lumbar subarachnoid catheters, using four agonist drugs [gamma-aminobutyric acid (GABA), muscimol, midazolam and 5-hydroxytryptamine (5-HT)] and two GABA(A) antagonists (bicuculline and SR-95531) given intrathecally. All four agonists caused dose-related antinociception assessed by the electrical current threshold test. These effects were spinally mediated because the agonists caused increases in nociceptive thresholds in the skin of the tail and not the neck. In the same experiments, 5-HT and GABA caused simultaneous increases in tail-flick latency and electrical current thresholds in the tail. Both GABA(A) antagonists caused dose-related suppression of the antinociceptive effects of equieffective doses of all four agonists. Tail-flick latency increases caused by 5-HT were not suppressed by bicuculline in the same experiments in which bicuculline had suppressed the electrical current threshold effects of intrathecal 5-HT. The log dose-response curves for both antagonists for suppression of GABA effects were coincident, having a very shallow slope and covering the whole range of doses effective against the other agonists. The two GABA(A) antagonists were very different in relative potency for suppression of the spinally mediated antinociceptive effects of the other three agonists. The rank order of potency for bicuculline suppression of the effects of equieffective doses of the other agonists was muscimol > 5-HT > midazolam, whereas the rank order for SR-95531 was muscimol >> midazolam > 5-HT. We conclude that there exist in the spinal cord at least three different GABA(A) receptors responsible for spinally mediated antinociception caused by intrathecal injections of midazolam, muscimol and 5-HT. These are all targets for endogenous GABA.

Opioid receptor independent inhibition of Ca2+ and K+ currents in NG108-15 cells by the kappa opioid receptor agonist U50488H.

The whole-cell patch clamp technique was used to investigate the actions of the opioid agonist U50488H on Ca2+ and K+ currents in differentiated NG108-15 cells. U50488H (5-50 microM) caused a concentration-dependent, reversible inhibition of high voltage-activated Ca2+ currents which persisted in the presence of nifedipine (2 microM), indicating a blockade of N-type Ca2+ channels. The actions of U50488H were also observed in the presence of 30 microM naloxone, which fully abolished current inhibition caused by a selective delta opioid receptor agonist. U50488H also inhibited Ca(2+)-insensitive, voltage-gated K+ currents in NG108-15 cells in the presence of naloxone. Our results indicate that U50488H can inhibit neuronal ionic channels via a mechanism which does not involve activation of kappa opioid receptors.

Antinociceptive actions of intrathecal xylazine: interactions with spinal cord opioid pathways.

We have studied rats with chronically implanted subarachnoid catheters. Xylazine, an alpha 2 adrenoceptor agonist, was injected intrathecally and nociceptive thresholds measured at two skin sites: the tail and the neck. Intrathecal xylazine (dose range 24.3-389 nmol) produced increases in electrical thresholds for nociception in the tail without any change in the neck; this observation suggested that the antinociceptive action of this drug was confined to the caudal part of the spinal cord responsible for tail innervation. The magnitude of this effect was dose-dependent. Tail flick latency also increased in these rats and the antinociceptive effects were antagonized in a dose-dependent manner by the selective alpha 2 adrenoceptor antagonist idazoxan (dose range 6.7-540 nmol). Intrathecal idazoxan also suppressed the increase in tail flick latency caused by the mu opioid agonist fentanyl (0.74 nmol) given intrathecally. This effect was also dose-dependent. The idazoxan dose-response curve for this suppression of fentanyl antinociception assessed with tail flick latency was the same as that for suppression of xylazine. In contrast, the antinociceptive effects of intrathecal xylazine were not affected by concurrent administration of opioid or GABAA antagonists. We conclude that intrathecal xylazine produced spinally mediated antinociceptive effects by combination with spinal cord alpha 2 adrenoceptors and that neither opioid nor GABA-containing propriospinal neurones were involved in the mediation of this effect. However, alpha 2 adrenoceptors in the spinal cord appear to be involved with antinociception produced by intrathecal fentanyl.

Neurokinin actions on substantia gelatinosa neurones in an adult longitudinal spinal cord preparation.

We have used an adult longitudinal spinal cord preparation to study the effects of a range of selective neurokinin analogues on single neurones located exclusively within the substantia gelatinosa. Since the preparation retained attached dorsal roots it was possible synaptically to activate the substantia gelatinosa neurones by electrical stimulation of their afferent fibres, thus providing a means of studying directly the role of neurokinins in mechanisms of primary afferent transmission. The actions of three agonists selective for the three NK receptor subtypes (NK1, GR73632; NK2, GR64349; NK3, senktide), and a highly selective antagonist at NK1 receptors (GR82334) were investigated. Experiments were performed on a total of 274 substantia gelatinosa neurones, estimates of conduction velocity for evoked responses suggested that the majority of these neurones were innervated by unmyelinated afferents. A large proportion responded to iontophoretically applied neurokinin agonists. The majority responded to NK1, fewer responded to NK2; some, although not all, of the neurones tested responded to both NK1 and NK2 agonists. In most cases the responses were excitatory, although inhibitory effects were observed in some neurones. None of the neurones tested responded to NK3 agonist. Excitatory and inhibitory actions could be demonstrated following abolition of synaptic transmission by removal of calcium, suggesting direct mechanisms for both effects. The antagonist alone failed to modify either spontaneous firing or firing in response to afferent stimulation in any of the neurones studied, even though the doses used were shown to be effective in selectively antagonising responses to the NK1 agonist, suggesting that neither relied on the endogenous release of neurokinins.

Electrophysiological properties of substantia gelatinosa neurones in a novel adult spinal slice preparation.

A novel longitudinal spinal cord slice prepared from adult rats aged 3-6 weeks is described. This preparation differs from conventional slices in that it retains multiple dorsal roots, each more than 10 mm in length, and presents a sagittal section through the laminae of the dorsal and ventral horns. The substantia gelatinosa can be visually distinguished from the other laminae of the dorsal horn, thus making the preparation particularly suited for the study of neurones located in this region. Extracellular recordings were made from 142 substantia gelatinosa cells, most of which responded to electrical stimulation of a dorsal root. Using measurements of latency it was possible to estimate conduction velocities for afferent input fibres. The effects of iontophoretically applied excitatory amino acids and of tachykinin peptides on spontaneous and afferent-evoked firing are briefly described. The preparation has applications in the investigation of mechanisms of primary afferent transmission within the dorsal horn and of transmission of afferent input between successive spinal segments.

Spinal cord effects of drugs used in anaesthesia.

1. Measurement of nociceptive thresholds at two skin sites in rats with chronic intrathecal catheters has allowed study of drug actions demonstrated to be at spinal level. 2. Using this preparation we have demonstrated segmental antinociceptive effects following intrathecal administration of agonists selective for benzodiazepine receptors, mu, kappa and delta opioid receptors, 5-hydroxytryptamine (5-HT) receptors and alpha adrenoceptors. 3. Such effects were dose related and were suppressed by appropriate selective antagonists. 4. Antagonist dose-response curves have been constructed for suppression of antinociceptive effects of various agonists. This has revealed complex interactions between spinal systems; for example midazolam produces its actions by activating an opioid system involving delta receptors.

Reversal by naloxone of spinal antinociceptive effects of fentanyl, ketocyclazocine and midazolam.

Experiments using measurement of electrical-current threshold as a nociceptive test in the skin of the tail and neck in rats demonstrated that fentanyl, ketocyclazocine and midazolam caused spinally mediated antinociception when the drugs were administered intrathecally via chronically implanted lumbar subarachnoid catheters. The benzodiazepine antagonist flumazenil selectively suppressed the midazolam response, indicating that this benzodiazepine exerted its segmental antinociceptive effect via spinal-cord benzodiazepine receptors. Naloxone blocked the responses to both opioids and also midazolam. The dose of naloxone which suppressed the midazolam response was similar to that required to suppress the response to the kappa-opioid agonist. We suggest that the segmental antinociceptive effects of fentanyl and midazolam are mediated via different pathways; the benzodiazepine exerts its antinociceptive action via a spinal-cord opioid pathway which does not involve mu-receptors.

Spinally mediated antinociception following intrathecal chlordiazepoxide--further evidence for a benzodiazepine spinal analgesic effect.

This is a report of the results of 25 experiments in five rats investigating the dose-response relationship for the antinociceptive effects of chlordiazepoxide given intrathecally in the dose range 0.03-0.9 mumol and a further 40 experiments in eight rats investigating the actions of flumazenil and naloxone on this effect. Electrical-current thresholds for pain were measured in the skin of the tail and neck of rats with previously implanted lumbar subarachnoid catheters. Intrathecal chlordiazepoxide produced spinally mediated antinociception, i.e. rises in the current threshold for pain in the tail without a significant change in the neck. This antinociceptive effect was dose dependent. Flumazenil 16.5 mumol kg-1 i.p. reduced the response caused by chlordiazepoxide 0.6 mumol by 78 +/- 6% (mean +/- SEM). By contrast, the same dose of flumazenil did not significantly affect the antinociceptive effect of an equipotent dose of intrathecal fentanyl 0.74 nmol. Naloxone 0.38 mumol kg-1 i.p. abolished the spinally mediated antinociception caused by fentanyl (96 +/- 7% suppression) but did not significantly reduce the effect of chlordiazepoxide (27 +/- 13% suppression). However, a higher dose of naloxone (6.1 mumol kg-1 i.p.) caused significant partial suppression (79 +/- 10.7%) of chlordiazepoxide spinal antinociception. We conclude that chlordiazepoxide produces an antinociceptive effect by combination with benzodiazepine receptors in the spinal cord.

Analgesia mediated by spinal kappa-opioid receptors.

The results from 44 experiments performed on 13 rats with chronically implanted lumbar subarachnoid catheters are reported. ICI 197 067 produced dose-dependent segmental analgesic effects when measurement of electrical current threshold for pain was used as the nociceptive test. Ten microliters of intrathecal ICI 197 067 (0.06 mg ml-1; 1.5 nmol) caused a significant rise in the current threshold for pain in the tail of 1.56 +/- 0.04 x control (mean +/- SEM) but no significant change in pain threshold in the neck (1.03 +/- 0.03 x control). By contrast, simultaneous measurements of tail-flick latency in these animals revealed no significant rise in pain thresholds using this nociceptive test. Intraperitoneally administered naloxone produced a dose-dependent suppression of the spinally mediated analgesic effect produced by ICI 197 067; the ED50 for this effect was 0.79 mumol kg-1, a value very close to the ED50 for naloxone antagonism of ketocyclazocine spinally mediated analgesia. We conclude that ICI 197 067 produces spinally mediated analgesia by binding to spinal-cord kappa-opioid receptors.

Convulsive thresholds in mice during the recovery phase from anaesthesia induced by propofol, thiopentone, methohexitone and etomidate.

1. Convulsive thresholds were measured with intravenous pentylenetetrazol in mice during the recovery phase after intravenous anaesthetic doses of propofol (10 and 20 mg kg-1), thiopentone (30 mg kg-1), methohexitone (10 mg kg-1), and etomidate 3 mg kg-1). 2. The convulsive threshold rose after each agent, indicating an anticonvulsant action for all the drugs tested; this declined to control values with initial half times of: 1.56 min (propofol 10 mg kg-1); 1.03 min (propofol 20 mg kg-1): 1.02 min (methohexitone); 3.35 min (etomidate); 13.7 min (thiopentone). 3. At no time during the recovery phase of any agent did the convulsive threshold fall below control values, which might indicate an epileptogenic effect of the drug. 4. The threshold was depressed below control values by intravenous administration of Ro 15-4513, a partial inverse agonist at the benzodiazepine receptor, thus indicating the ability of this pentylenetetrazol test to demonstrate a proconvulsant effect. 5. We conclude that the abnormal movements or convulsions associated with recovery from anaesthesia with short-acting intravenous anaesthetics may not be the result of an intrinsic proconvulsant action of the drugs.

On the mechanism by which midazolam causes spinally mediated analgesia.

The electrical current thresholds for pain (ECTP) in the skin of the neck and tail were measured in rats with chronically implanted lumbar subarachnoid catheters. The effects of a benzodiazepine antagonist and a gamma-aminobutyric acid (GABA) antagonist on the analgesic effects of equivalent doses of midazolam, fentanyl, and ketocyclazocine were studied. These were the minimum doses producing maximal segmental analgesia when given intrathecally (i.e., they all caused a significant and maximum increase in ECTP in the tail, which was similar for all three drugs, but no significant change in the ECTP in the neck). Flumazenil (Ro 15-1788) administration caused a parallel shift to the right of the dose-response curve for midazolam spinal analgesia. Segmental analgesia following midazolam was also significantly attenuated (P less than 0.05) when the selective GABA antagonist bicuculline was given intrathecally at the same time as midazolam. The highest dose of bicuculline used (50 pmol) caused no significant attenuation of the segmental analgesic effects of either ketocyclazocine or fentanyl. The authors concluded that the segmental analgesia produced by intrathecal midazolam is mediated by the benzodiazepine-GABA receptor complex that is involved in other benzodiazepine actions.

Anticonvulsant properties of propofol and thiopentone: comparison using two tests in laboratory mice.

Experiments were carried out in mice to assess the protection provided by thiopentone (Intraval, May and Baker) and propofol (Diprivan, I.C.I.) against epileptiform seizures induced by electroshock and pentylenetetrazol. Intraperitoneal administration of propofol 50 mg kg-1 and thiopentone 25 mg kg-1 produced similar peak behavioural effects of mild sedation and incoordination. However, at these doses propofol afforded a greater degree of protection against pentylenetetrazol than thiopentone and at greater doses both propofol and thiopentone caused marked protection. Both anaesthetics were effective also against electroshock seizures at sedative doses. We conclude that propofol has strong anticonvulsant properties.

Vascular effects of propofol: smooth muscle relaxation in isolated veins and arteries.

Isolated hepatic portal veins and aorta taken from the rat were used to investigate the direct action of the intravenous anaesthetic propofol. This compound is known to produce a fall in blood pressure in man and animals and it has been suggested that the hypotension may result from a direct vasodilator action on the veins and arterioles. In our experiments propofol caused a dose related decrease of potassium-induced tone in both types of blood vessel. However, the concentrations required to produce this effect in the experiments on veins were significantly lower than those required to produce similar changes in the isolated artery preparation. We conclude that this direct action may contribute towards the hypotensive effects of propofol.

HPLC analysis of brain and plasma for octanoic and decanoic acids.

Two methods are described for determination of octanoic and decanoic acids in plasma and brain homogenate by "high-performance" liquid chromatography with ultraviolet detection. Analysis of the underivatized acids had a detection limit of only 50 mg/L, but formation of the p-bromophenacyl ester increased the sensitivity by 100-fold, to a detection limit of 0.5 mg/L. The latter procedure gave interassay coefficients of variation of 4.1% and 4.8% for octanoic and decanoic acids, respectively. The corresponding intra-assay values were 3.95% and 4.7% (n = 6). The derivative method, applied to samples of plasma from children receiving a medium-chain triglyceride (MCT) diet, gave values in agreement with results by gas-liquid chromatography. Results have also been obtained for samples from mice, either treated with the medium-chain triglyceride diet or given infusions of sodium octanoate.