Richard M. Eglen

Distribution of the Tetrodotoxin-Resistant Sodium Channel PN3 in Rat Sensory Neurons in Normal and Neuropathic Conditions

The novel sodium channel PN3/α-SNS, which was cloned from a rat dorsal root ganglion (DRG) cDNA library, is expressed predominantly in small sensory neurons and may contribute to the tetrodotoxin-resistant (TTXR) sodium current that is believed to be associated with central sensitization in chronic neuropathic pain states. To assess further the role of PN3, we have used electrophysiological, in situ hybridization and immunohistochemical methods to monitor changes in TTXRsodium current and the distribution of PN3 in normal and peripheral nerve-injured rats. (1) Whole-cell patch-clamp recordings showed that there were no significant changes in the TTXR and TTX-sensitive sodium current densities of small DRG neurons after chronic constriction injury (CCI) of the sciatic nerve. (2) Additionally, in situ hybridization showed that there was no change in the expression of PN3 mRNA in the DRG up to 14 d after CCI. PN3 mRNA was not detected in sections of brain and spinal cord taken from either normal or nerve-injured rats. (3) In contrast, immunohistochemical studies showed that major changes in the subcellular distribution of PN3 protein were caused by either CCI or complete transection of the sciatic nerve. The intensity of PN3 immunolabeling decreased in small DRG neurons and increased in sciatic nerve axons at the site of injury. The alteration in immunolabeling was attributed to translocation of presynthesized, intracellularly located PN3 protein from neuronal somata to peripheral axons, with subsequent accumulation at the site of injury. The specific subcellular redistribution of PN3 after peripheral nerve injury may be an important factor in establishing peripheral nerve hyperexcitability and resultant neuropathic pain.

Functional role of M2 and M3 muscarinic receptors in the urinary bladder of rats in vitro and in vivo

Urinary bladder smooth muscle is enriched with muscarinic receptors, the majority of which are of the M2 subtype whereas the remaining minority belong to the M3 subtype. The objective of the present study was to assess the functional role of M2 and M3 receptors in the urinary bladder of rat in vitro and in vivo by use of key discriminatory antagonists. In the isolated bladder of rat, (+)‐cis‐dioxolane produced concentration‐dependent contractions (pEC50=6.3) which were unaffected by tetrodotoxin (0.1 μm). These contractions were antagonized by muscarinic antagonists with the following rank order of affinity (pA2) estimates: atropine (9.1) > 4‐diphenyl acetoxy‐methyl piperidine methiodide (4‐DAMP) (8.9) > darifenacin (8.5) > para fluoro hexahydrosiladifenidol (p‐F‐HHSiD) (7.4) > pirenzepine (6.8) > methoctramine (5.9). These pA2 estimates correlated most favourably (r=0.99, P<0.001) with the binding affinity (pKi) estimates of these compounds at human recombinant muscarinic m3 receptors expressed in Chinese hamster ovary cells, suggesting that the receptor mediating the direct contractile responses to (+)‐cis‐dioxolane equates with the pharmacologically defined M3 receptor. As M2 receptors in smooth muscle are negatively coupled to adenylyl cyclase, we sought to determine whether a functional role of M2 receptors could be unmasked under conditions of elevated adenylyl cyclase activity (i.e., isoprenaline‐induced relaxation of KCl pre‐contracted tissues). Muscarinic M3 receptors were preferentially alkylated by exposing tissues to 4‐DAMP mustard (40 nm, 1 h) in the presence of methoctramine (0.3 μm) to protect M2 receptors. Under these conditions, (+)‐cis‐dioxolane produced concentration‐dependent reversal (re‐contraction) of isoprenaline‐induced relaxation (pEC50=5.8) but had marginal effects on pinacidil‐induced, adenosine 3′:5′‐cyclic monophosphate (cyclic AMP)‐independent, relaxation. The re‐contractions were antagonized by methoctramine and darifenacin, yielding pA2 estimates of 6.8 and 7.6, respectively. These values are intermediate between those expected for these compounds at M2 and M3 receptors and were consistent with the involvement of both of these subtypes. In urethane‐anaesthetized rats, the cholinergic component (∼55%) of volume‐induced bladder contractions was inhibited by muscarinic antagonists with the following rank order of potency (ID35%inh, nmol kg−1, i.v.): 4‐DAMP (8.1) > atropine (20.7) > methoctramine (119.9) > darifenacin (283.3) > pirenzepine (369.1) > p‐F‐HHSiD (1053.8). These potency estimates correlated most favourably (r=0.89, P=0.04) with the pKi estimates of these compounds at human recombinant muscarinic m2 receptors. This is consistent with a major contribution of M2 receptors in the generation of volume‐induced bladder contractions, although the modest potency of darifenacin does not exclude a role of M3 receptors. Pretreatment with propranolol (1 mg kg−1, i.v.) increased the ID35%inh of methoctramine significantly from 95.9 to 404.5 nmol kg−1 but had no significant effects on the inhibitory responses to darifenacin. These data suggest an obligatory role of β‐adrenoceptors in M2 receptor‐mediated bladder contractions in vivo. The findings of the present study suggest that both M2 and M3 receptors can cause contraction of the rat bladder in vitro and may also mediate reflex bladder contractions in vivo. It is proposed that muscarinic M3 receptor activation primarily causes direct contraction of the detrusor whereas M2 receptor activation can contract the bladder indirectly by reversing sympathetically (i.e. β‐adrenoceptor)‐mediated relaxation. This dual mechanism may allow the parasympathetic nervous system, which is activated during voiding, to cause more efficient and complete emptying of the bladder.

The pharmacology and distribution of human 5‐hydroxytryptamine2B (5‐HT2b) receptor gene products: comparison with 5‐HT2a and 5‐HT2c receptors

1 Full length clones of the human 5‐HT2B receptor were isolated from human liver, kidney and pancreas. The cloned human 5‐HT2B receptors had a high degree of homology (∼80%) with the rat and mouse 5‐HT2B receptors. 2 PCR amplification was used to determine the tissue distribution of human 5‐HT2B receptor mRNA. mRNA encoding the 5‐HT2B receptor was expressed with greatest abundance in human liver and kidney. Lower levels of expression were detected in cerebral cortex, whole brain, pancreas and spleen. Expression was not detected in heart. 3 Northern blot analysis confirmed the presence of 5‐HT2B receptor mRNA (a 2.4 kB sized band) in pancreas, liver and kidney. An additional 3.2 kB sized band of hybridization was detected in liver and kidney. This raises the possibility of a splice variant of the receptor or the presence of an additional homologous receptor. 4 The human 5‐HT2B receptor was expressed in Cos‐7 cells and its ligand binding characteristics were compared to similarly expressed human 5‐HT2A and 5‐HT2C receptors. The ligand specificity of the human 5‐HT2B receptor (5‐HT > ritanserin > SB 204741 > spiperone) was distinct from that of the human 5‐HT2A (ritanserin > spiperone > 5‐HT > SB 204741) and 5‐HT2C (ritanserin > 5‐HT > spiperone = SB 204741) receptors. On the basis of a higher affinity for ketanserin and a lower affinity for yohimbine the human 5‐HT2B receptor also appeared to differ from the rat 5‐HT2B receptor. 5 These findings confirm the sequence of the human 5‐HT2B receptor and they demonstrate that the receptor has a widespread tissue distribution. In addition, these data suggest that there are differences in ligand affinities between different species homologues of the receptor. Finally, the finding of two distinct bands on the Northern blots of liver and kidney raises the possibility of splice variants or subtypes of 5‐HT2B receptors, within these tissues.

Assessment of the role of α2‐adrenoceptor subtypes in the antinociceptive, sedative and hypothermic action of dexmedetomidine in transgenic mice

1 The role of α2‐adrenoceptor (AR) subtypes in the modulation of acute nociception, motor behaviour and body temperature, has been investigated by determining the activity of the α2AR selective agonist dexmedetomidine (Dex) in mice devoid of individual α2AR subtypes through either a point (α2A) or null (α2B/α2C) mutation (‘knock‐out’). 2 In a rodent model of acute thermal nociception, the mouse tail immersion test, Dex, in wild type (WT) control animals, produced a dose‐dependent increase in the threshold for tail withdrawal from a 52°C water bath with mean ED50 values of 99.9±14.5 (α2A), 94.6±17.8 (α2B) and 116.0±17.1 (α2C) μg kg−1, i.p. 3 In comparison to the WT controls, Dex (100–1000 μg kg−1, i.p.), was completely ineffective as an antinociceptive agent in the tail immersion test in the α2AAR D79N mutant animals. Conversely, in the α2BAR and α2CAR knock‐outs, Dex produced a dose‐dependent antinociceptive effect that was not significantly different from that observed in WT controls, with ED50 values of 85.9±15.0 (P>0.05 vs WT control) and 226.0±62.7 (P>0.05 vs WT control) μg kg−1 i.p., respectively. 4 Dex (10–300 μg kg−1, i.p.) produced a dose‐dependent reduction in spontaneous locomotor activity in the α2A, α2B and α2CAR WT control animals with ED50 values of 30.1±9.0, 23.5±7.1 and 32.3±4.6 μg kg−1, i.p., respectively. Again, Dex (100–1000 μg kg−1, i.p.) was ineffective at modulating motor behaviour in the α2AAR D79N mutants. In the α2BAR and α2CAR knock‐out mice, Dex produced a dose‐dependent reduction in spontaneous locomotor activity with ED50 values of 29.1±6.4 (P>0.05 vs WT control) and 57.5±11.3 (P>0.05 vs WT control) μg kg−1, respectively. 5 Dex was also found to produce a dose‐dependent reduction in body temperature in the α2A, α2B and α2CAR WT control mice with ED50 values of 60.6±11.0, 16.2±2.5 and 47.2±9.1 μg kg−1, i.p., respectively. In the α2AAR D79N mutants, Dex had no effect on body temperature at a dose (100 μg kg−1, i.p.) that produced a significant reduction (−6.2±0.5°C; P<0.01 vs vehicle) in temperature in WT controls. However, higher doses of Dex (300 and 1000 μg kg−1, i.p) produced a small, but statistically significant decrease in temperature corresponding to −1.7±0.4°C and −2.4±0.3°C (both P<0.01 vs vehicle), respectively. In the α2BAR and α2CAR knock‐out mice, Dex produced a dose‐dependent reduction in body temperature with ED50 values of 28.4±4.8 (P>0.05 vs WT control) and 54.1±8.0 (P>0.05 vs WT control) μg kg−1, respectively. 6 In conclusion, the data are consistent with the α2AAR being the predominant subtype involved in the mediation of the antinociceptive, sedative and hypothermic actions of Dex. This profile would appear to indicate that an α2AAR subtype selective analgesic will have a narrow therapeutic window, particularly following systemic administration.

Muscarinic acetylcholine receptor subtypes in smooth muscle

Muscarinic acetylcholine M2 and M3 receptor subtypes are coexpressed in many types of smooth muscle including gastrointestinal smooth muscle, urinary bladder and vascular and airway tissue. Activation of M3 receptors, via the G protein Gq, results in increased polyphosphoinositide hydrolysis, release of Ca2+ ions from the sarcoplasmic reticulum and consequently causes contraction. Quantitation of the relative expression of M2 and M3 receptors has shown that the proportion of M2 receptors often predominates over the M3 receptor population by 4:1 or more. Although it is established that M2 receptors preferentially link, via a pertussis-toxin-sensitive G protein Gi, to inhibition of adenylate cyclase activity, relatively little is known concerning the physiological role of the M2 receptor population. In this review, Richard Eglen and colleagues discuss recent data concerning the possible role(s) of muscarinic receptor subtypes in smooth muscle and appraise the pharmacological methods for dissecting the function of muscarinic receptor subtypes in tissues co-expressing multiple receptors.

RS-102221: A Novel High Affinity and Selective, 5-HT2C Receptor Antagonist

The 5-HT2C receptor is one of three closely related receptor subtypes in the 5-HT2 receptor family. 5-HT2A and 5-HT2B selective antagonists have been described. However, no 5-HT2C selective antagonists have yet been disclosed. As part of an effort to further explore the function of 5-HT2C receptors, we have developed a selective 5-HT2C receptor antagonist, RS-102221 (a benzenesulfonamide of 8-[5-(5-amino-2,4-dimethoxyphenyl) 5-oxopentyl]-1,3,8-triazaspiro[4.5]decane-2,4-dione). This compound exhibited nanomolar affinity for human (pKi = 8.4) and rat (pKi = 8.5) 5-HT2C receptors. The compound also demonstrated nearly 100-fold selectivity for the 5-HT2C receptor as compared to the 5-HT2A and 5-HT2B receptors. RS-102221 acted as an antagonist in a cell-based microphysiometry functional assay (pA2 = 8.1) and had no detectable intrinsic efficacy. Consistent with its action as a 5-HT2C receptor antagonist, daily dosing with RS-102221 (2 mg/kg intraperitoneal) increased food-intake and weight-gain in rats. Surprisingly, RS-102221 failed to reverse the hypolocomotion induced by the 5-HT2 receptor agonist 1-(3-chlorophenyl)piperazine (m-CPP). It is concluded that RS-102221 is the first selective, high affinity 5-HT2C receptor antagonist to be described.

Central 5-HT4 receptors

Activation of the 5-HT4 receptor mediates widespread effects in central and peripheral nervous systems. Recent developments, such as the identification of novel, selective agonists and antagonists, as well the cloning of the receptor, have provided insights into the physiological role of the receptor. In this article, Richard Eglen and colleagues assess the emerging evidence relating to the function of the 5-HT4 receptor in the brain. The cerebral distribution of the receptor, along with neurochemical and electrophysiological data, suggests a role in cognition. The role of the receptor in modulation of dopamine transmission and anxiolysis is also addressed.

Characterization and distribution of putative 5-ht7 receptors in guinea-pig brain.

1 In the presence of (−)−cyanopindolol (1.0 μM) and sumatriptan (1.0 μM), 0.5 nM [3H]‐carboxamidotryptamine ([3H]‐5‐CT) labelled a single population of receptors in guinea‐pig cerebral cortex membranes. 2 5‐HT‐displaceable binding was rapid, saturable and reversible. A high affinity binding site was characterized both by equilibrium saturation (Kd = 0.76 ± 0.28 nM; Bmax = 68.1 ± 26.7 fmol mg−1 protein) and kinetic (Kd = 0.18 ± 0.05 nM) analysis. The pharmacological profile of this site was similar to the profile obtained in transfected CHO‐K1 cells expressing guinea‐pig 5‐ht7 receptors. 3 Autoradiographic analysis revealed a discrete localization of binding sites in guinea‐pig brain, with the highest density of sites in the medial thalamic nuclei and related limbic and cortical regions. Moderate levels of binding were detected in sensory relay nuclei, substantia nigra, hypothalamus, central grey and dorsal raphe nuclei. This distribution corresponded to that observed using in situ hybridization with [35S]‐UTP labelled riboprobes complementary to mRNA encoding the guinea‐pig 5‐ht7 receptor. 4 In conclusion, under appropriate conditions, [3H]‐5‐CT labelled a single population of saturable binding sites that corresponded to an endogenous 5‐ht7 receptor in guinea‐pig brain. The distribution of 5‐ht7 receptors in thalamocortical and limbic brain regions suggests a role for these receptors in sensory and affective behaviours.

Immunocytochemical localization of P2X3 purinoceptors in sensory neurons in naive rats and following neuropathic injury

P2X3 purinoceptor cellular distribution was studied in rat sensory neurons in naive animals and following peripheral nerve injury using immunohistochemical methods. Specific antiserum was raised in rabbits and characterized by Western blot, absorption assays and labeling of recombinant receptors. In naive animals, P2X3 immunoreactivity was present predominantly in a subpopulation of small-diameter sensory neurons in dorsal root ganglia. In the spinal cord, immunoreactivity was observed in the superficial laminae of the dorsal horn. Following a chronic constriction injury to the sciatic nerve, the number of P2X3 positive small and medium diameter neurons increased in dorsal root ganglia when compared with sham-operated animals. In addition, the spinal cord immunoreactivity increased in magnitude on the side ipsilateral to the ligated nerve, consistent with up-regulation of receptors in presynaptic terminals of the primary sensory neurons.

Muscarinic receptor subtypes modulating smooth muscle contractility in the urinary bladder.

Normal physiological voiding as well as generation of abnormal bladder contractions in diseased states is critically dependent on acetylcholine-induced stimulation of contractile muscarinic receptors on the smooth muscle (detrusor) of the urinary bladder. Muscarinic receptor antagonists are efficacious in treating the symptoms of bladder hyperactivity, such as urge incontinence, although the usefulness of available drugs is limited by undesirable side-effects. Detrusor smooth muscle is endowed principally with M2 and M3 muscarinic receptors with the former predominating in number. M3 muscarinic receptors, coupled to stimulation of phosphoinositide turnover, mediate the direct contractile effects of acetylcholine in the detrusor. Emerging evidence suggests that M2 muscarinic receptors, via inhibition of adenylyl cyclase, cause smooth muscle contraction indirectly by inhibiting sympathetically (beta-adrenoceptor)-mediated relaxation. In certain diseased states, M2 receptors may also contribute to direct smooth muscle contraction. Other contractile mechanisms involving M2 muscarinic receptors, such as activation of a non-specific cationic channel and inactivation of potassium channels, may also be operative in the bladder and requires further investigation. From a therapeutic standpoint, combined blockade of M2 and M3 muscarinic receptors would seem to be ideal since this approach would evoke complete inhibition of cholinergically-evoked smooth muscle contractions. However, if either the M2 or M3 receptor assumes a greater pathophysiological role in disease states, then selective antagonism of only one of the two receptors may be the more rational approach. The ultimate therapeutic strategy is also influenced by the extent to which pre-junctional M1 facilitatory and M2 inhibitory muscarinic receptors regulate acetylcholine release and also which subtypes mediate the undesirable effects of muscarinic receptor blockade such as dry mouth. Finally, the consequence of muscarinic receptor blockade in the central nervous system on the micturition reflex, an issue which is poorly studied and seldom taken into consideration, should not be ignored.