Muscarinic acetylcholine receptors (mAChRs), M(1)-M(5), regulate the activity of numerous fundamental central and peripheral functions. The lack of small-molecule ligands that can block or activate specific mAChR subtypes with high selectivity has remained a major obstacle in defining the roles of the individual receptor subtypes and in the development of novel muscarinic drugs. Recently, phenotypic analysis of mutant mouse strains deficient in each of the five mAChR subtypes has led to a wealth of new information regarding the physiological roles of the individual receptor subtypes. Importantly, these studies have identified specific mAChR-regulated pathways as potentially novel targets for the treatment of various important disorders including Alzheimer's disease, schizophrenia, pain, obesity and diabetes.

The past decades have witnessed significant efforts toward the development of three-dimensional (3D) cell cultures as systems that better mimic in vivo physiology. Today, 3D cell cultures are emerging, not only as a new tool in early drug discovery but also as potential therapeutics to treat disease. In this review, we assess leading 3D cell culture technologies and their impact on drug discovery, including spheroids, organoids, scaffolds, hydrogels, organs-on-chips, and 3D bioprinting. We also discuss the implementation of these technologies in compound identification, screening, and development, ranging from disease modeling to assessment of efficacy and safety profiles.

No abstract

Small neurons of the dorsal root ganglia (DRG) are known to play an important role in nociceptive mechanisms. These neurons express two types of sodium current, which differ in their inactivation kinetics and sensitivity to tetrodotoxin. Here, we report the cloning of the alpha-subunit of a novel, voltage-gated sodium channel (PN3) from rat DRG. Functional expression in Xenopus oocytes showed that PN3 is a voltage-gated sodium channel with a depolarized activation potential, slow inactivation kinetics, and resistance to high concentrations of tetrodotoxin. In situ hybridization to rat DRG indicated that PN3 is expressed primarily in small sensory neurons of the peripheral nervous system.

Recent advances in cell biology, microfabrication techniques, and tissue engineering have enabled the development of a wide range of 3D cell culture technologies. These include multicellular spheroids, organoids, scaffolds, 696795J BXXXX10.

1 Urinary bladder smooth muscle is enriched with muscarinic receptors, the majority of which are of the M 2 subtype whereas the remaining minority belong to the M 3 subtype. The objective of the present study was to assess the functional role of M 2 and M 3 receptors in the urinary bladder of rat in vitro and in vivo by use of key discriminatory antagonists. 2 In the isolated bladder of rat, (+)-cis-dioxolane produced concentration-dependent contractions (pEC 50 =6.3) which were unaected by tetrodotoxin (0.1 mM). These contractions were antagonized by muscarinic antagonists with the following rank order of anity (pA 2 ) estimates: atropine (9.1) 4 4-diphenyl acetoxy-methyl piperidine methiodide (4-DAMP) (8.9) 4 darifenacin (8.5) 4 para¯uoro hexahydrosiladifenidol (p-F-HHSiD) (7.4) 4 pirenzepine (6.8) 4 methoctramine (5.9). These pA 2 estimates correlated most favourably (r=0.99, P50.001) with the binding anity (pK i ) estimates of these compounds at human recombinant muscarinic m 3 receptors expressed in Chinese hamster ovary cells, suggesting that the receptor mediating the direct contractile responses to (+)-cis-dioxolane equates with the pharmacologically de®ned M 3 receptor. 3 As M 2 receptors in smooth muscle are negatively coupled to adenylyl cyclase, we sought to determine whether a functional role of M 2 receptors could be unmasked under conditions of elevated adenylyl cyclase activity (i.e., isoprenaline-induced relaxation of KCl pre-contracted tissues). Muscarinic M 3 receptors were preferentially alkylated by exposing tissues to 4-DAMP mustard (40 nM, 1 h) in the presence of methoctramine (0.3 mM) to protect M 2 receptors. Under these conditions, (+)-cis-dioxolane produced concentration-dependent reversal (re-contraction) of isoprenaline-induced relaxation (pEC 50 =5.8) but had marginal eects on pinacidil-induced, adenosine 3':5'-cyclic monophosphate (cyclic AMP)-independent, relaxation. The re-contractions were antagonized by methoctramine and darifenacin, yielding pA 2 estimates of 6.8 and 7.6, respectively. These values are intermediate between those expected for these compounds at M 2 and M 3 receptors and were consistent with the involvement of both of these subtypes. 4 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 (ID 35%inh , nmol kg 71 , i.v.): 4-DAMP (8.1) 4 atropine (20.7) 4 methoctramine (119.9) 4 darifenacin (283.3) 4 pirenzepine (369.1) 4 p-F-HHSiD (1053.8). These potency estimates correlated most favourably (r=0.89, P=0.04) with the pK i estimates of these compounds at human recombinant muscarinic m 2 receptors. This is consistent with a major contribution of M 2 receptors in the generation of volumeinduced bladder contractions, although the modest potency of darifenacin does not exclude a role of M 3 receptors. Pretreatment with propranolol (1 mg kg 71 , i.v.) increased the ID 35%inh of methoctramine signi®cantly from 95.9 to 404.5 nmol kg 71 but had...

No abstract

Alterations in sodium channel expression and function have been suggested as a key molecular event underlying the abnormal processing of pain after peripheral nerve or tissue injury. Although the relative contribution of individual sodium channel subtypes to this process is unclear, the biophysical properties of the tetrodotoxin-resistant current, mediated, at least in part, by the sodium channel PN3 (SNS), suggests that it may play a specialized, pathophysiological role in the sustained, repetitive firing of the peripheral neuron after injury. Moreover, this hypothesis is supported by evidence demonstrating that selective ''knock-down'' of PN3 protein in the dorsal root ganglion with specific antisense oligodeoxynucleotides prevents hyperalgesia and allodynia caused by either chronic nerve or tissue injury. In contrast, knock-down of NaN͞SNS2 protein, a sodium channel that may be a second possible candidate for the tetrodotoxin-resistant current, appears to have no effect on nerve injury-induced behavioral responses. These data suggest that relief from chronic inf lammatory or neuropathic pain might be achieved by selective blockade or inhibition of PN3 expression. In light of the restricted distribution of PN3 to sensory neurons, such an approach might offer effective pain relief without a significant side-effect liability.