Sharath S. Hegde

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.

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.

Muscarinic receptors in the bladder: from basic research to therapeutics.

Muscarinic receptor antagonists (antimuscarinics) serve as the cornerstone in the pharmacological management of overactive bladder (OAB) by relieving the symptoms of urgency, frequency and incontinence. These drugs operate primarily by antagonizing post‐junctional excitatory muscarinic receptors (M2/M3) in the detrusor. The combination of pharmacological and gene knockout studies has greatly advanced our understanding of the functional role of muscarinic receptors in the bladder. M3 receptors produce direct smooth muscle contraction by a mechanism that relies on entry of extracellular calcium through L‐type channels and activation of a rho kinase. M2 receptors, which predominate in number, appear to facilitate M3‐mediated contractions. M2 receptors can also produce bladder contractions indirectly by reversing cAMP‐dependent β‐adrenoceptor‐mediated relaxation, although the physiological role of β‐adrenoceptors in detrusor relaxation is controversial. Emerging evidence suggests that muscarinic receptors in the urothelium/suburothelium can modulate the release of certain factors, which in turn may affect bladder function at the efferent or afferent axis. Currently, oxybutynin, tolterodine, darifenacin, solifenacin and trospium are the five major antimuscarinics approved for the treatment of OAB. Comparative clinical studies have shown that oxybutynin and solifenacin may be marginally more effective than tolterodine, although the latter seems to be better tolerated. Pharmacokinetic–pharmacodynamic analyses using plasma concentrations of ‘total drug’ indicate that, at therapeutic doses, the clinical efficacy of darifenacin and solifenacin may be driven primarily by selective M3 receptor occupation, whereas the pharmacodynamic effects of pan‐selective molecules (such as tolterodine, trospium) may potentially involve multiple receptors, including M2 and M3. Furthermore, high M3 receptor occupation is the likely explanation for the greater propensity of darifenacin and oxybutynin to cause dry mouth and/or constipation. Although the recently introduced drugs represent a significant improvement over older drugs, especially with respect to the convenience of dosing schedule, their overall efficacy and tolerability profile is still less than optimal and patient persistence with therapy is low. Recent advances in basic research have not yet offered a clear discovery path for improving the therapeutic index of antimuscarinic molecules. There is still an unmet need for an antimuscarinic medicine with superior clinical effectiveness that can translate into better persistence on therapy.

Peripheral 5-HT4 receptors.

The 5‐HT4 receptor is a member of the seven transmembrane spanning G‐protein‐cou‐pled family of receptors. The receptor is positively coupled to adenylate cyclase and exists in two iso‐forms (5‐HT4S and 5‐HT4L) that differ in the length and sequence of their carboxy termini. The 5‐HT4 receptor is pharmacologically defined by selective agonists such as SC 53116 and RS 67506, and selective antagonists such as GR 113808, SB 204070, and RS 39604. The receptor is widely distributed in the central nervous system and peripheral tissues. In the periphery, the receptor plays an important role in the function of several organ re‐sponses including the alimentary tract, urinary bladder, heart and adrenal gland. In the alimentary tract, stimulation of 5‐HT4 receptors has a pronounced effect on smooth muscle tone, mucosal electrolyte secretion, and the peristaltic reflex. In the urinary bladder, activation of 5‐HT4 receptors modulates cholinergic/purinergic transmission. In the heart, stimulation of atrial 5‐HT4 receptors produces positive inotropy and tachycardia that can precipitate arrythmias. In the adrenal gland, agonism of 5‐HT4 receptors stimulates release of Cortisol, corticos‐terone, and aldosterone. Since its discovery in 1988, significant advances have been made in our understanding of the physiology and pharmacology of the 5‐HT4 receptor. These advances have led to the development of several selective 5‐HT4 receptor agonists and antagonists that may have therapeutic utility in the treatment of peripheral disorders such as irritable bowel syndrome, gastroparesis, urinary incontinence and cardiac arrhythmias.—Hegde, S. S., Eglen, R. M. Peripheral 5‐HT4 receptors. FASEB J. 10, 1398‐1407 (1996)

Muscarinic receptor ligands and their therapeutic potential

Over the past year, the introduction of novel ligands has accelerated the classification of muscarinic receptor subtypes and has led to a better understanding of their physiological role. Important in this respect is the recent recognition of the exquisite selectivity of a series of snake toxins, enabling better definition of the muscarinic subtype 4 receptor. Moreover, several compounds, both agonists and antagonists, are progressing in advanced clinical trials for the treatment of several conditions, including Alzheimers disease, pain, urinary incontinence and chronic obstructive pulmonary disease.

Catecholamine modulatory effects of nepicastat (RS‐25560‐197), a novel, potent and selective inhibitor of dopamine‐β‐hydroxylase

1 Inhibitory modulation of sympathetic nerve function may have a favourable impact on the progression of congestive heart failure. Nepicastat is a novel inhibitor of dopamine‐β‐hydroxylase, the enzyme which catalyses the conversion of dopamine to noradrenaline in sympathetic nerves. The in vitro pharmacology and in vivo catecholamine modulatory effects of nepicastat were investigated in the present study. 2 Nepicastat produced concentration‐dependent inhibition of bovine (IC50=8.5±0.8 nM) and human (IC50=9.0±0.8  nM)dopamine‐β‐hydroxylase. The corresponding R‐enantiomer (RS‐25560‐198) was approximately 2–3 fold less potent than nepicastat. Nepicastat had negligible affinity (>10 μM) for twelve other enzymes and thirteen neurotransmitter receptors. 3 Administration of nepicastat to spontaneously hypertensive rats (SHRs) (three consecutive doses of either 3, 10, 30 or 100 mg kg−1, p.o.; 12 h apart) or beagle dogs (0.05, 0.5, 1.5 or 5 mg kg−1, p.o.; b.i.d., for 5 days) produced dose‐dependent decreases in noradrenaline content, increases in dopamine content and increases in dopamine/noradrenaline ratio in the artery (mesenteric or renal), left ventricle and cerebral cortex. At the highest dose studied, the decreases in tissue noradrenaline were 47%, 35% and 42% (in SHRs) and 88%, 91% and 96% (in dogs) in the artery, left ventricle and cerebral cortex, respectively. When tested at 30 mg kg−1, p.o., in SHRs, nepicastat produced significantly greater changes in noradrenaline and dopamine content, as compared to the R‐enantiomer (RS‐25560‐198), in the mesenteric artery and left ventricle. 4 Administration of nepicastat (2 mg kg−1, b.i.d, p.o.) to beagle dogs for 15 days produced significant decreases in plasma concentrations of noradrenaline and increases in plasma concentrations of dopamine and dopamine/noradrenaline ratio. The peak reduction (52%) in plasma concentration of noradrenaline and the peak increase (646%) in plasma concentration of dopamine were observed on day‐6 and day‐7 of dosing, respectively. 5 The findings of this study suggest that nepicastat is a potent, selective and orally active inhibitor of dopamine‐β‐hydroxylase which produces gradual modulation of the sympathetic nervous system by inhibiting the biosynthesis of noradrenaline. This drug may, therefore, be of value in the treatment of cardiovascular disorders associated with over‐activation of the sympathetic nervous system, such as congestive heart failure.

Pharmacological characterization of muscarinic receptors in rabbit isolated iris sphincter muscle and urinary bladder smooth muscle

The pharmacological characteristics of muscarinic receptors in the rabbit iris sphincter muscle were studied and compared to M3 receptors in rabbit urinary bladder smooth muscle. (+)‐Cis‐dioxolane induced concentration‐dependent contractions of the iris sphincter muscle (pEC50=6.41±0.10, Emax=181±17 mg, n=38) and urinary bladder smooth muscle (pEC50=6.97±0.04, Emax=4.28±0.25 g, n=54). These contractions were competitively antagonized by a range of muscarinic receptor antagonists (pKB values are given for the iris sphincter muscle and the bladder smooth muscle, respectively): atropine (9.30±0.07 and 9.40±0.04), AQ‐RA 741 (6.35±0.04 and 6.88±0.03), darifenacin (9.56±0.05 and 9.12±0.05), methoctramine (5.75±0.07 and 5.81+0.06), oxybutynin (8.10±0.09 and 8.59±0.06), pirenzepine (6.79±0.05 and 6.89±0.04), secoverine (7.54±0.05 and 7.66±0.05), p‐F‐HHSiD (7.55±0.09 and 7.50±0.05) and zamifenacin (8.69±0.10 and 8.36±0.06). A significant correlation between the pKB values in the bladder and the pKB values in the iris was obtained. In both tissues, the pKB values correlated most favorably with pKi values for these compounds at human recombinant muscarinic m3 receptors. A reasonable correlation was also noted at human recombinant muscarinic m5 receptors given the poor discriminative ability of ligands between m3 and m5 receptors. Overall, the data from this study suggest that the muscarinic receptors mediating contraction of the rabbit iris sphincter muscle and urinary bladder smooth muscle are similar and equate most closely with the pharmacologically‐defined muscarinic M3 receptor.

Effects of Dopamine β-Hydroxylase Inhibition With Nepicastat on the Progression of Left Ventricular Dysfunction and Remodeling in Dogs With Chronic Heart Failure

Background—Inhibition of dopamine β-hydroxylase (DBH) results in a decrease in norepinephrine synthesis. The present study was a randomized, blinded, placebo-controlled investigation of the long-term effects of therapy with the DBH inhibitor nepicastat (NCT) on the progression of left ventricular (LV) dysfunction and remodeling in dogs with chronic heart failure (HF). Methods and Results—Moderate HF (LV ejection fraction [LVEF] 30% to 40%) was produced in 30 dogs by intracoronary microembolization. Dogs were randomized to low-dose NCT (0.5 mg/kg twice daily, n=7) (L-NCT), high-dose NCT (2 mg/kg twice daily, n=7) (H-NCT), L-NCT plus enalapril (10 mg twice daily, n=8) (L-NCT+ENA), or placebo (PL, n=8). Transmyocardial (coronary sinus–arterial) plasma norepinephrine (tNEPI), LVEF, end-systolic volume, and end-diastolic volume were measured before and 3 months after initiating therapy. tNEPI levels were higher in PL compared with NL (86±20 versus 13±14 pg/mL, P<0.01). L-NCT alone and L-NCT+ENA reduced tNEPI t...

Pharmacological characterization of RS 25259‐197, a novel and selective 5‐HT3 receptor antagonist, in vivo

1 The pharmacological effects in vivo, of RS 25259‐197, a selective 5‐HT3 receptor antagonist, have been investigated. 2 In anaesthetized rats, RS 25259‐197, administered by the intravenous, intraduodenal or transdermal route, dose‐dependently inhibited the von Bezold‐Jarisch reflex induced by 2‐methyl 5‐HT (ID50 = 0.04 μg kg−1, i.v., 3.2 μg kg−1, i.d. and 32.8 μg per chamber, respectively). In this regard, when administered intraduodenally, RS 25259‐197 was more potent and exhibited a longer duration of action than either ondansetron or granisetron. 3 In conscious ferrets, RS 25259‐197, administered intravenously or orally, dose‐dependently inhibited emesis induced by cisplatin. The ID50 estimates of RS 25259‐197 were 1.1 μg kg−1, i.v. and 3.2 μg kg−1, p.o. In this respect, RS 25259‐197 was more potent than ondansetron and equipotent with granisetron. 4 In conscious dogs, RS 25259‐197, administered intravenously or orally, dose‐dependently inhibited emesis induced by cisplatin (ID50 = 1.9 μg kg−1, i.v. and 8.5 μg kg−1, p.o.), dacarbazine (ID50 = 4.1 μg kg−1, i.v. and 9.7 μg kg−1, p.o.), actinomycin D (ID50 = 4.9 μg kg−1, i.v. and 2.5 μg kg−1, p.o.) and mechlorethamine (ID50 = 4.4 μg kg−1, i.v. and 3.0 μg kg−1, p.o.). Against each of the emetogenic agents, RS 25259‐197 was very much more potent than ondansetron. When tested at equi‐effective intravenous doses against cisplatin‐induced emesis in dogs, RS 25259‐197 had a longer duration of anti‐emetic activity (7 h) than ondansetron (4 h). At doses up to and including 1000 μg kg−1, p.o., neither RS 25259‐197 nor ondansetron was capable of inhibiting apomorphine‐induced emesis. 5 At doses up to 1000 μg kg−1, i.v., RS 25259‐197 produced no meaningful haemodynamic changes in anaesthetized dogs. 6 In summary, RS 25259‐197 is a novel, highly potent and orally active 5‐HT3 receptor antagonist in vivo. With respect to its anti‐emetic activity, RS 25259‐197 appears to be a significant improvement over ondansetron in terms of potency and duration of action.

RS 39604 : a potent, selective and orally active 5-HT4 receptor antagonist

1 Selective antagonism of 5‐HT4 receptors may provide therapeutic benefit in certain disorders of the myocardium, alimentary and lower urinary tract. We now report on RS 39604, a novel and selective 5‐HT4 receptor antagonist and compare its pharmacological properties with those of SB 204070. 2 In guinea‐pig striatal membranes, both RS 39604 and SB 204070 inhibited specific binding of [3H]‐GR 113808 in a concentration‐dependent manner yielding p Ki estimates of 9.1 and 10.9, respectively. RS 39604 displayed a low affinity (pKi <6.5) for 5‐HT1A, 5‐HT2C, 5‐HT3, α1c, D1? D2, M1 M2, AT1 B1 and opioid u receptors and moderate affinity for σ1 (pK1 = 6.8) and σ2 (pK1 = 7.8) sites. 3 In the rat isolated oesophagus, precontracted with carbachol, RS 39604 (30–300 nM) behaved as a competitive antagonist towards 5‐HT‐induced relaxation (pA2 = 9.3; Schild slope =1.0). We and others have shown previously that SB 204070 behaves as an unsurmountable antagonist in this preparation (pA2‐10.5). In the guinea‐pig isolated ileal mucosa, RS 39604 (30 nM) antagonized 5‐MeOT‐induced increase in short‐circuit current (pA2 = 9.1). 4 In anaesthetized vagotomized micropigs, RS 39604, administered by the i.v. or intraduodenal (i.duod.) route, produced dose‐dependent inhibition of 5‐HT‐induced tachycardia (ID50 = 4.7 μg kg−1, i.v. and 254.5 ug kg−1, i.duod). At maximal doses of 30 μg kg−1, i.v. and 6 mg kg−1, i.duod., the inhibitory effects of RS 39604 lasted for more than 6 h. In this preparation, SB 204070 was as potent as RS 39604 by the i.v. route but was inactive by the intraduodenal route at doses up to 3 mg kg−1. 5 In conscious mice, RS 39604, administered by the i.p. or p.o. route, produced dose‐dependent inhibition of 5‐hydroxytryptophan (5‐HTP)‐induced diarrhoea (ID50 = 81.3 ug kg−1, i.p. and 1.1 mg kg−1, p.o.). In this assay, SB 204070 was inactive by the oral route at doses up to 30 mg kg−1. 6 In anaesthetized guinea‐pigs, RS 39604 antagonized the contractile effect of 5‐HT in the proximal colon by producing parallel, dextral displacement of the dose‐response curve to 5‐HT. The mean dose‐ratios to 5‐HT at 0.1 mg kg−1, i.v., 1 mg kg−1, i.v. and 10 mg kg−1, i.duod. were 4.6, 30.7 and 10.8, respectively. SB 204070 behaved as an unsurmountable antagonist in this assay. 7 In a model of visceral pain in conscious rats, RS 39604 (0.01‐1 mg kg−1, i.v.) did not affect colorectal distension‐induced increases in arterial pressure whereas morphine (1 mg kg−1, i.v.) produced significant inhibition of the response, implying that 5‐HT4 receptors are not involved in nociception in this model. 8 The data suggest that RS 39604 is a high affinity and selective 5‐HT4 receptor antagonist that is orally active and long‐lasting in vivo. It is concluded that RS 39604 may be the preferable probe to use for investigating the physiological and pathophysiological role of 5‐HT4 receptors in vivo.