The pharmacology of 5-HT and the classification of 5-HT receptors have become increasingly complex. However, recent advances have produced a new nomenclature system for 5-HT receptors. 5-HT3 receptors are neuronal receptors coupled directly to cation channels. Recently, many selective 5-HT3-receptor antagonists including tropisetron, zacopride, ondansetron, granisetron, zatosetron, nazasetron, YM060 and YM114 (KAE-393) have been developed. Many actions attributable to the 5-HT3-receptor have been described in both the peripheral and central nervous systems, and clinical trials are already showing the potential use of these 5-HT3 receptor antagonists in a number of disorders of the gastrointestinal tract and central nervous system, such as nausea and vomiting induced by cancer chemotherapy, anxiety, depression, schizophrenia and migraine. In addition, endogenous 5-HT is suggested to be one of the substances that mediate stress-induced responses in gastrointestinal function, i.e., increase in fecal pellet output and diarrhea. Moreover, YM060, YM114 (KAE-393) and granisetron have been reported to inhibit restraint stress and 5-HT-induced increases in fecal pellet output and diarrhea in rats and mice, indicating that endogenous 5-HT may mediate stress-induced changes in bowel function through the 5-HT3 receptor. Therefore, 5-HT3-receptor antagonists are new therapeutic drugs for stress-induced gastrointestinal dysfunctions like irritable bowel syndrome (IBS).
Cholecystokinin (CCK) has a unique pharmacological profile as a “brain-gut peptide hormone”, and recent development of specific non-peptide antagonists prompted us to investigate the role of the receptors of this peptide in nervous control mechanisms of gastric acid secretion. CCK-8 induced a distinctive acid secretory response after intracerebroventricular (i.c.v.) administration, as well as after intravenous infusion in the perfused stomach preparation in rats. L364718, a CCKA-receptor antagonist, had an inhibitory effect against CCK-8 when applied i.c.v., while L365260, a CCKB-receptor antagonist, had no influence, suggesting the apparent dominant control of CCKA receptors in the central nervous system on gastric acid secretion. However, L364718 caused remarkable potentiation in the acid secretory response to the intravenous CCK-8 infusion, and L365260 significantly inhibited the CCK-8-stimulated acid secretion. The potentiation of the acid secretory response to CCK-8 by the CCKA antagonist was completely blocked by vagotomy or atropine, as well as hexamethonium. Thus it was suggested that a vagal control mechanism was involved in the potentiation. Denervation of capsaicin-sensitive primary afferent nerve by repeated capsaicin injection also decreased the potentiated response to CCKA antagonist. This seems to suggest the involvement of the vago-vagal reflex in the control of gastric acid secretion by endogenous CCK. Ketanserin, a serotonin 5-HT2/1C-receptor antagonist, showed a distinctive inhibition of acid secretion induced by CCK-8 (i.c.v.), suggesting the involvement of serotonergic control of acid secretion in the CCK-8 action. Current developments in the research in this field were briefly reviewed in relation to our findings.
The effects of aging on some drug receptor mechanisms were studied in isolated smooth muscles. Potencies of α- and β-adrenoceptor agonists increased from the young stage to the adult stage and decreased slowly thereafter to the old stage. The affinities of the adrenergic drugs on their receptors did not alter with aging. Excellent relationships between the potencies or efficacies of the agonists and the amounts of receptors or receptor reserve were found, suggesting that changes in the adrenoceptor mechanisms with aging were due to changes in the amount of receptors. However, it was also found that the decrease in the potency of β-adrenoceptor agonists in preparations from the older animals was due to the change in post β-adrenoceptor processes in responsiveness. However, the serotonin, acetylcholine (ACh) and tachykinin (NK2) receptor mechanisms did not alter with aging. Enzyme activities altered with aging. For example, activities of acetylcholinesterase and choline acetyltransferase decreased with aging. The potencies of nicotine, which accelerates ACh release, and exogenously applied ACh were modified by the change in enzyme activities with aging. Similar observations were obtained in the tachykinin (NK2) receptor mechanism. Effects of aging on physiological functions seem to be complementary. In iris dilator muscles, the potency of norepinephrine (NE), which altered with aging, was proportional in the receptor reserve. No age-related change was observed in the activity of sympathetic nerves, which innervate the iris dilator muscles. On the other hand, in iris sphincter muscles innervated by parasympathetic nerves, the ACh (muscarine) receptor mechanisms did not alter, but the activity in cholinergic nerves changed with aging.
Compounds that show their pharmacological actions via specific receptors are considered potential candidates for new drugs. Recently, several compounds that have specific binding sites and show certain pharmacological actions have been identified, but neither their binding sites, their endogenous substances, nor the functional role of the binding sites have been clarified. Regardless of the exact role of the binding sites of the compounds, research into the sites has opened up new areas of receptor investigation, and also new strategies for developing drugs. The σ-ligand is one such kind of compound, and the existence of a binding site for the ligand was first postulated to account for the psychotomimetic effects of N-allylnormetazocine and related racemic benzomorphans. The binding site of the σ-ligand is widely distributed in the central nervous system and peripheral systems. However, it still remain to be established whether σ-ligand binding sites are to be refered to as “receptors”. The classification of the ligands as agonists or antagonists at the sites and the heterogeneity and the functional role of the binding sites have not yet been clarified. Furthermore, the therapeutic targets have not been clearly determined. However, the σ-ligands have high potential for developing new drugs. One of the possibile targets of the σ-ligands as new forms of drugs is schizophrenia. Recently, we identified two potent and highly selective σ-ligands, FH-510 and NE-100. Together with the data on the binding properties and pharmacological actions of these compounds, the possibilities of the σ-ligand as a new therapeutic drug were discussed.
The receptor for γ-aminobutyric acid (GABA), a major inhibitory neurotransmitter in the mammalian brain, has been classified into GABAA and GABAB subtypes. The GABAA receptor forms a Cl- channel and is consisted of several subunits. These subunits in the brain are known to be multiple and heterogeneous in their molecular structure. Therefore, it is suggested that these multiple GABAA receptors generate various inhibitory functions in the brain. In contrast, the GABAB receptor is known to be one of the metabotropic type of receptors which generate slow inhibitory postsynaptic potentials and functionally couple with Gi/Go types of GTP-binding proteins. The GABAB receptor in the brain has been purified to homogeneity using immuno-affinity purification procedures and found to be an 80-kDa protein. The possible existence of multiplicity in the cerebral GABAB receptor has also been suggested.
Recent progress on the activation of G protein-coupled receptor kinases is reviewed. β-Adrenergic receptor kinase (βARK) is activated by G protein βγ-subunits, which interact with the carboxyl terminal portion of βARK. Muscarinic receptor m2-subtypes are phosphorylated by βARK1 in the central part of the third intracellular loop (I3). Phosphorylation of I3-GST fusion protein by βARK1 is synergistically stimulated by the βγ-subunits and mastoparan or a peptide corresponding to portions adjacent to the transmembrane segments of m2-receptors or by βγ-subunits and the agonist-bound I3-deleted m2 variant. These results indicate that agonist-bound receptors serve as both substrates and activators of βARK.
The renin-angiotensin system (RAS) is known to play a very important role in cardiovascular diseases. The present state of the research on orally active nonpeptide angiotensin II (A II)-receptor antagonists and their pharmacological characterization will be reviewed. In the late 1970s, the first nonpeptide A II-receptor antagonists were discovered among some derivatives of imidazoleacetic acid, and this was followed by the development of losartan in 1989. TCV-116, synthesized in our laboratories, is a prodrug that is converted in vivo to the active form CV-11974. TCV-116 and CV-11974 were demonstrated to be effective antagonists for many A II-induced cardiovascular actions and are effective antihypertensive agents in several animal models of hypertension. TCV-116 also demonstrated secondary benefits in treating congestive heart failure (CHF), preventing stroke, delaying the progression of renal disease and preventing the intimal thickening of vascular injury in animal models. Clinical studies confirmed the efficacy of TCV-116 in the treatment of essential hypertension. The utility of A II antagonist may extend beyond that of hypertension and CHF, as suggested by the potential usefulness of ACE inhibitors in the treatment or prevention of many other cardiovascular diseases. The A II antagonists will help to determine the role of the RAS in the physiologic regulation and in the pathophysiology of various cardiovascular diseases.
Complementary DNAs encoding δ, μ and κ-opioid receptors have now been cloned and characterized. These receptors, which are members of the superfamily of seven transmembrane spanning receptors, share a high degree of amino acid sequence similarity among these receptors. From the similarity of the sequence, it is speculated that both the 1st and 2nd extracellular loop and the 4th membrane spanning domain are supposed to be involved in the opioid binding and subtype specificity. Because of the high similarity of the cytoplasmic regions' amino acid sequence, however, it seems that the signal transductions of δ, μ and κ are very similar. In Xenopus oocytes expressing δ-opioid receptors and various kinds of GTP-binding protein α-subunits, the δ-agonist DSLET caused currents through Gi1α (or Gi2α)-phospholipase C mechanisms Neither Goα, Gqα, G11α nor G14α was involved in such δ-receptor-mediated responses. The higher concentration of DSLET (3-10 μM) showed a rapid desensitization upon repeated challenges. Such a rapid desensitization was purely homologous, and this was rescued by the pretreatment with protein kinase C inhibitor. Similar findings were also observed with μ and κ-opioid receptors. These results suggest that the phosphorylation by protein kinase C is involved in the acute tolerance.
To date, cDNA studies and purification of nicotinic acetylcholine receptors (nAChRs) have shown that there are many different subtypes of the receptors in the central and peripheral nervous systems. This review focuses on the animal and avian brain nAChRs, their diversity, pharmacological properties, distribution and regulation. nAChR subtypes are classified as αβ types and α-bungarotoxin binding proteins. Each subtype contains two or more subunits. Each subunit combination leads to a distinct pattern of sensitivity to nicotinic agonists in reconstituted systems of Xenopus oocytes. Nicotine has been shown to increase the levels of intracellular free Ca through both nAChR types. The mRNAs for each subunit are uniquely expressed in animal and avian brains, although β2 is expressed in most regions of the brains. This shows somewhat overlapping patterns of gene expression. Monoclonal antibodies against each subunit make it possible to investigate the distribution of the subunits in brains and neurons. nAChRs have been shown to be regulated by nicotine administration and phosphorylation of the receptors. The regulatory mechanisms have been extensively studied, but still remain obscure. The structural and functional diversity of neuronal nAChRs is probably important in nicotine addiction, tolerance and reverse tolerance.
Desensitization of the m3-muscarinic acetylcholine-receptor in the smooth muscle of the digestive tract is discussed together with the changes in intracellular signal transduction. Isolated single cells that show an all-or-none contractile response to acetylcholine were desensitized by treatment with 0.1 mM acetylcholine for 10 min, resulting in an increase in the threshold concentration of acetylcholine for contraction, but without changing any of the binding characteristics. Permeabilized cells showed that the desensitization is via uncoupling between the receptor and G-protein. Secretory cells (rat basophil leukemia-2H3 cells) transfected with human m3-receptor showed desensitization when treated with 0.1 mM carbachol for 30 min. The coupling between the receptor and G-protein was not impaired, but some unknown Ca2+-independent mechanism may be involved. Smooth muscle tissue was tested for its time-course of desensitization, and a novel transient resensitization was found at 1 min of incubation with 0.1 mM carbachol. This resensitization, and the desensitization prior to it, were accompanied with changes in binding affinity. However, the affinity was not changed, in parallel with desensitization afterwords, but the positive feedback loop of Ca2+-influx caused by alkalization via receptor-stimulation was suppressed. After a 30-min treatment, a Ca2+-independent mechanism caused the uncoupling and affinity decrease. Treatment for 3 hr increased the number of binding sites without recovery of the response. The desensitizing process is very diverse to achieve selectivity, but its purpose is in unity.