Haeyoung Kong

Differential expression of messenger RNAs for somatostatin receptor subtypes SSTR1, SSTR2 and SSTR3 in adult rat brain: Analysis by RNA blotting and in situ hybridization histochemistry

The messenger RNAs encoding three somatostatin receptor subtypes, SSTR1, SSTR2 and SSTR3, were detected in rat by RNA blotting and in situ hybridization histochemistry to identify the sites of synthesis and expression of these somatostatin receptor subtypes. RNA blotting revealed that SSTR1 messenger RNA of 3.8 kilobases was highly expressed in cerebral cortex, hippocampus, midbrain and hypothalamus. In situ hybridization histochemistry revealed that SSTR1 messenger RNA was localized to discrete layers of the cerebral cortex, the piriform cortex and the dentate gyrus of the hippocampus. SSTR1 messenger RNA was expressed at low levels in the cerebellum and pituitary and was not detectable in striatum or other peripheral organs. At least two SSTR2 messenger RNAs were detected by RNA blotting of 2.4 and 2.8 kilobases which correspond to the size of the spliced and unspliced forms of this receptor messenger RNA. SSTR2 messenger RNA detected by in situ hybridization is diffusely expressed in cerebral cortex and amygdala but is discretely localized to dentate gyrus in the hippocampus, medial habenula and ventromedial and dorsomedial nuclei and arcuate nucleus of the hypothalamus. The levels of SSTR2 messenger RNA are very low in the cerebellum and were not observed in the striatum or peripheral tissues other than the pituitary or adrenal gland. A single SSTR3 messenger RNA of 4.0 kilobases was seen in hippocampus, cerebral cortex, midbrain, hypothalamus and pituitary. However, the tissue with the highest levels of SSTR3 messenger RNA is the cerebellum with messenger RNA localized to the granule cell layer. The expression of the three different somatostatin receptor messenger RNAs are distinct but overlapping. Such distinct expression may contribute to the selective biological roles of the receptor subtypes.

Expression and pharmacological characterization of a stimulatory subtype of adenosine receptor in fetal chick ventricular myocytes.

Ventricular and atrial myocytes cultured from chick embryos 14 days in ovo were used as model systems to study cardiac adenosine receptors. In membranes of ventricular cultures, blocking of the A1-adenosine receptor pathway by the A1-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) or by pertussis toxin treatment of the myocyte resulted in a significant adenosine agonist-mediated stimulation of the adenylate cyclase activity. The maximal increases in adenylate cyclase activity caused by the equipotent or the A2-adenosine receptor-selective agonists (from 52.1 +/- 3% to 63 +/- 10% [mean +/- SEM]) were significantly greater than those caused by the A1-selective agonists (from 11 +/- 5% to 34.6 +/- 7%) (p less than 0.01, by t test, n = 4-8). However, in membranes of atrial myocytes, when A1-subtype had been blocked, the various adenosine agonists had no effect on the adenylate cyclase activity. Whether the stimulatory adenylate cyclase-coupled adenosine receptor is also capable of stimulating contractility in the intact ventricular myocyte was next investigated. In ventricular but not in atrial cells, the various adenosine agonists caused an increase in the contractile amplitude in the presence of DPCPX or in myocytes preexposed to pertussis toxin. The increase in contraction amplitude caused by each agonist was expressed as percent of maximum (maximum is the increase in contractility caused by 2.4 mM calcium). In the pertussis toxin-treated myocyte, the maximal increases caused by the equipotent or A2-agonists (NECA, MECA, CV-1808, and CGS21680, from 49.6 +/- 3% to 52.5 +/- 6%, n = 8-12) were significantly greater than those elicited by the A1-agonists (2-CADO, S-PIA, R-PIA, and DCCA, from 12 +/- 4% to 37 +/- 3%, n = 8) (p less than 0.05, by t test). These data demonstrated that a stimulatory adenosine receptor, likely the A2-adenosine receptor, was present on the ventricular but not the atrial myocytes and was linked directly to a stimulation of the cardiac contractility. The functional effects mediated by the A1-subtype became manifested in the presence of isoproterenol, as evidence by an inhibition of the isoproterenol-stimulated increases in adenylate cyclase activity and in cardiac contractility by adenosine agonists. Thus, both subtypes of adenosine receptors, each mediating opposing responses, were present on the ventricular myocytes, whereas only the A1-subtype was found in the atria. The presence of a stimulatory functional A2-adenosine receptor may help explain the absence of a direct negative inotropic response to adenosine in the ventricle.

Differential agonist modulation of the cloned opioid receptors reveals distinct cellular mechanisms of receptor regulation

Abstract Tolerance is a limitation to the clinical use of opioids. A cellular basis of tolerance may involve receptor downregulation/desensitization after exposure to agonists. We have investigated the effects of agonist pretreatments of cells expressing the cloned rat mu, and mouse delta and kappa receptors on both subsequent radioligand binding and coupling to adenylyl cyclase. Agonist pretreatment of cells expressing kappa and delta receptors diminished subsequent labelling of thoroughly washed membranes with agonist. Antagonist binding to the delta receptor was also diminished, but antagonist binding to the kappa receptor was unaffected. Neither agonist nor antagonist binding to the mu receptor was affected by agonist pretreatment. The loss of agonist binding to the delta and kappa receptors was paralleled by a loss of the ability of agonists to inhibit forskolin-stimulated cAMP accumulation. The mu receptor, in contrast, did not functionally desensitize. Desensitization of the kappa, but not delta, receptor could be blocked by cotransfection of the receptors with a dominant negative mutant of β-adrenergic receptor kinase. The mechanisms underlying the development of tolerance to opioid agents are likely multiple and divergent for the different receptor types.

Molecular biology of kappa and delta opioid receptors

The endogenous opioids, enkephalin and dynorphin induce their biological actions by interacting with delta and kappa receptors. To investigate the molecular mechanisms by which these peptides induce their physiological effects, we have cloned the kappa and delta opioid receptors from a mouse brain cDNA library. The kappa and delta receptors are 380 and 372 amino acids, respectively, and are 61% identical and 71% similar (1). They have relatively little similarity in amino acid sequence with any other receptors except somatostatin receptors. We have analyzed the pharmacological specificities of these receptors following their transient expression in COS cells or in PC12 and CHO cells stably expressing the individual receptors