β-Adrenergic Receptor Subtypes in Melanophores of the Marine Gobies Tridentiger trigonocephalus and Chasmichthys gulosus

The subtype of β-adrenergic receptors in melanophores of the marine gobies Tridentiger trigonocephalus and Chasmichthys gulosus was studied. Pigment of denervated melanophores in isolated, split caudal fins was preliminarily aggregated by incubating the specimens in a physiological saline containing 10 μM phentolamine and 30–100 μM verapamil or 2–10 nM melatonin, and the responses of the melanophores to a β-adrenergic agonist added to the incubating medium were recorded photoelectrically. The β-adrenergic agonists noradrenaline, adrenaline, isoproterenol, salbutamol and, dobutamine were all effective in evoking a dispersion of melanophore pigment in the presence of phentolamine and verapamil or melatonin. The pigment-dispersing effect of noradrenaline (β₁-selective agonist) was inhibited by metoprolol (β₁-selective antagonist), propranolol, and butoxamine. Whereas, the effect of salbutamol (β₂-selective agonist) was hardly inhibited by metoprolol, though it was considerably inhibited by propranolol and ICI-118551. It was estimated that β₁- and β₂-adrenergic receptors coexist at ratios of 8.6:91.4, in the melanophore of Tridentiger trigonocephalus, and 25:75, in the melanophore of Chasmichthys gulosus, through the analyses of Hofstee plots of the effects of the β-adrenergic drugs. It was suggested that the relation between the pigment-dispersing effect of a β-adrenergic agonist on the melanophores and the concentration of the drug follows mass action kinetics, when the effect is mainly caused by the activation of β₂-adrenergic receptors of the melanophores. However, when it is mainly caused by the activation of β₁-adrenergic receptors of the melanophores, the relation does not follow mass action kinetics.     

长江口纹缟虾虎鱼胚胎发育及早期仔鱼生长与盐度的关系

研究了长江口纹缟虾虎鱼(Tridentiger trigonocephalus)胚胎发育及早期仔鱼生长与盐度的相关性。纹缟虾虎鱼胚胎发育时,盐度4与6试验组效果较好,受精率分别达(76.28±2.73)%与(74.63±5.49)%,孵化率分别达(66.95±2.99)%与(62.15±2.18)%,出膜时间分别为(228.8±5.9)h与(221.5±5.2)h,器官发育特征较明显,畸形率较低。纹缟虾虎鱼仔鱼发育时,初孵仔鱼全长(2.534±0.078)mm,卵黄囊长径(0.421±0.047)mm,短径(0.369±0.038)mm,5日龄时卵黄囊基本消失,初步形成消化器官、循环器官和运动器官等。盐度8与10水体孵育的仔鱼畸形率达5―20%,其他试验组略低。盐度4与6试验组仔鱼的器官发育速度快于其他3个试验组。盐度0―6试验组纹缟虾虎鱼仔鱼生存活力指数（SAI）为41.83―46.83,仔鱼活力较好,而盐度8―10试验组SAI值为23.43―29.13,活力较差。综合各项指标表明,在盐度0―10范围内,长江口纹缟虾虎鱼胚胎发育的适宜盐度为4―6,早期仔鱼生长的适宜盐度为2―4。     

Differences in the activity rhythms of juvenile gobiid fish,Chasmichthys gulosus,from different tidal localities

Activity patterns of intertidal gobiid fish,Chasmichthys gulosus, collected from different tidal localities, were recorded in a constant environment. Gobies collected from a rocky shore with a large tidal range and a regular tidal regime displayed a ca. 12 hour rhythm in their activity pattern. Gobies from a rocky shore with a small tidal range and an irregular tidal regime, however, did not clearly display any such rhythm. The ecological implications of such activity rhythms are discussed from the viewpoints of feeding and avoidance of predators.     

长江口纹缟虾虎鱼早期发育对生态因子的适应性

纹缟虾虎鱼广泛分布于长江口水域,是许多水生动物的重要饵料,在长江口水生生态系统中具有重要地位.2007年在人工繁殖基础上系统研究了长江口纹缟虾虎鱼(Tridentiger trigonocephalus)胚胎与仔鱼的发育特征及其与温度、光照、pH等生态因子的关系.结果表明,温度上升时纹缟虾虎鱼胚胎发育速度加快,受精率与孵化率下降,17～21℃是纹缟虾虎鱼胚胎发育的适宜温度,胚胎发育有效积温模型为:N=3128.3/(T-4.36),温度系数Q10值为1.33～2.84;随着温度升高,仔鱼器官发育速度与卵黄囊被吸收速度加快,但存活率下降.不同光照周期中,长光照组胚胎比自然光照组与长黑暗组发育快,但孵化率低;仔鱼亦是长光照组比自然光照组与长黑暗组发育快.pH升高时纹缟虾虎鱼胚胎孵化率升高,发育速度加快;高pH组(pH 7.5～9.5)水体中仔鱼发育较快,活力较好,发育特征亦更加明显,畸形率较低.综合各项指标表明,长江口纹缟虾虎鱼早期发育的适宜生态条件为:温度17～21℃,每昼夜光照12h以上,pH 7.5～9.5.长江口生态环境变动可能会对纹缟虾虎鱼早期发育及其资源量产生一定影响.     

5-HT receptor subtypes as key targets in mediating pigment dispersion within melanophores of teleost,Oreochromis mossambicus

The presence of distinct class of 5-HT receptors in the melanophores of tilapia (Oreochromis mossambicus) is reported. The cellular responses to 5-HT (5-hydroxytryptamine), 5-HT₁, and 5-HT₂, agonists on isolated scale melanophores were observed with regard to pigment translocation within the cells. It was found that 5-HT exerted rapid and strong concentration dependent pigment granule dispersion within the melanophores. The threshold pharmacological dose of 5-HT that could elicit a measurable response was as low as 4.7 × 10^? 12 M/L. Selective 5-HT₁ and 5-HT₂ agonists, sumatriptan and myristicin were investigated and resulted in dose-dependent pigment dispersion. The dispersing effects were effectively antagonized by receptor specific antagonists. It is suggested that 5-HT-induced physiological effects are mediated via distinct classes of receptors that possibly participate in modulation of pigmentary responses of the fish.     

Localization of cholecystokinin receptor subtypes in the endocine pancreas.

This study was undertaken to clarify the controversy in the literature about pancreatic localization of the cholecystokinin (CCK) CCK(A) and CCK(B) receptors. With antibodies used by other investigators, we first established their specificity by Western blotting, indirect immunofluorescence, and confocal microscopy with each antibody's peptide antigen. Co-localization assays between the CCK receptors and the pancreatic hormones insulin, glucagon, and somatostatin revealed that the CCK(A) RAbs 1122 and R1-2 recognized insulin and glucagon cells in rat, pig, and human pancreas but not in the somatostatin cells. Conversely, the three CCK(B) RAbs tested, 9262, 9491, and GR4, identified the somatostatin cells. Abs 9491 and GR4 occasionally co-localized with glucagon, a feature that never occurred with Ab 9262. Finally, the specificity of Ab 9262 for the pancreatic CCK(B) R was confirmed in six different species. It co-localized with somatostatin but never with glucagon in these species. Our data suggest the use of Abs 1122 and 9262 to specifically identify and localize pancreatic CCK(A) and CCK(B) receptors, respectively. Confusion in the literature may result from the lack of specificity of most antibodies used, as established in this study.     

beta-Adrenergic modulation of muscarinic cholinergic receptor expression and function in developing heart

Imbalances of beta-adrenoceptor (beta-AR) and muscarinic ACh receptor (mAChR) input are thought to underlie perinatal cardiovascular abnormalities in conditions such as sudden infant death syndrome. Administration of isoproterenol, a beta(1)/beta(2)-AR agonist, to neonatal rats on postnatal days (PN) 2-5 caused downregulation of cardiac m(2)AChRs and a corresponding decrement in their control of adenylyl cyclase activity. Terbutaline, a beta(2)-selective agonist that crosses the placenta and the blood-brain barrier, was also effective when given either on PN 2-5 or during gestational days 17-20. Terbutaline failed to downregulate brain m(2)AChRs, even though it downregulated beta-ARs; beta-ARs and m(2)AChRs are located on different cell populations in the brain, but they are on the same cells in the heart. Destruction of catecholaminergic neurons with neonatal 6-hydroxydopamine upregulated cardiac but not brain m(2)AChRs. These results suggest that perinatal beta-AR stimulation shifts cardiac receptor production away from the generation of m(2)AChRs so that the development of sympathetic innervation acts as a negative modulator of cholinergic function. Accordingly, tocolytic therapy with beta-AR agonists may compromise the perinatal balance of adrenergic and cholinergic inputs.     

Multiple muscarinic receptor subtypes in the canine pulmonary circulation.

The vascular response to the muscarinic receptor agonist acetylcholine (ACh) in the presence of selected antagonists was examined in the isolated blood-perfused canine left lower lung lobe under conditions of normal (resting) and elevated vascular tone. At normal vascular tone, ACh (1-5 mumol) produced a dose-dependent increase in pulmonary arterial pressure (Ppa), total pulmonary vascular resistance (PVR), and downstream resistance (Rds) without altering upstream resistance (Rus). Pirenzepine (50 and 100 nM), the prototype M1-selective antagonist, and gallamine, an M2-selective antagonist, as well as atropine (50 nM) and secoverine (100 nM), nonselective antagonists, attenuated (P less than 0.05) the ACh-induced increase in Ppa and Rds. With elevated vascular tone induced by serotonin infusion, ACh produced a dose-dependent increase in Ppa in 19 of 25 lobes, although Rus decreased while Rds increased in all lobes. At high vascular tone, pirenzepine or gallamine attenuated the ACh-induced increase in Rds, whereas Rus was not affected. Secoverine and atropine antagonized ACh-induced increases in both Rds and Rus. The pA2 values (i.e., the negative log antagonist concentration requiring a doubling of ACh dose for an equivalent increase in Rds) for gallamine, pirenzepine, secoverine, and atropine were 6.1 +/- 0.1, 7.4 +/- 0.1, 8.3 +/- 0.2, and 10.2 +/- 0.3, respectively. These results suggest that 1) ACh increases PVR in the dog by constricting the venous segments (downstream) of the pulmonary circulation via activation of pulmonary vascular muscarinic receptors under conditions of both normal and elevated vascular tone, 2) both M1- and non-M1-muscarinic receptor subtypes appear to participate in mediating the ACh-induced increase in Rds, and 3) ACh moderately relaxes the upstream (arterial) vessels, especially under conditions of elevated tone.     

Characterization of angiotensin II receptor subtypes in the rat spleen.

Quantitative autoradiography was used to determine the subtype of ANG receptors in the red pulp of the rat spleen. The AT1 antagonist DuP 753 competed for ANG binding with high affinity; binding was abolished by dithiothreitol. The AT2 competitor CGP 42112 A showed lower affinity, and the AT2 competitor PD 123177 did not affect binding at 10(-5) M. These data indicated the presence of only AT1 receptors. AT1 receptor number was similar in immature (2 weeks old) and adult (8 weeks old) rats. Binding was sensitive to guanine nucleotides, suggesting an association with G-proteins. Angiotensin II, at a dose of 10(-7) M, stimulated inositol phosphate formation 33% over control values in spleen from 8-week-old rats. This effect was significantly blocked by 10(-5) M DuP 753. We suggest a possible role of AT1 receptors in the regulation of splenic volume, blood flow, and lymphocyte function.     

Distinguishing bombesin receptor subtypes using the oocyte assay.

Physiological responses to mammalian bombesin-like peptides were studied in Xenopus oocytes injected with mRNA isolated from Swiss 3T3 cells and rat esophagus in order to identify and characterize bombesin receptor subtypes. Both groups respond similarly to either gastrin releasing peptide or neuromedin B, but only the response to neuromedin B in oocytes expressing the esophagus mRNA is not blocked by a specific gastrin releasing peptide receptor antagonist, des-Met-[D-Phe6]Bn(6-13) ethyl ester. Complete desensitization of gastrin releasing peptide-evoked responses in oocytes expressing esophagus mRNA does not abolish neuromedin B-evoked responses. A single application of neuromedin B abolishes responses to subsequently applied gastrin releasing peptide in oocytes expressing esophagus, but not Swiss 3T3, mRNA. RNA blot hybridization studies using a Swiss 3T3 gastrin releasing peptide receptor cDNA probe show no detectable hybridization in esophagus mRNA samples. These data suggest that a gastrin releasing peptide receptor is expressed in the esophagus and that it is distinct from that expressed in Swiss 3T3 cells and may represent a third subtype of mammalian bombesin receptor.     

beta-Adrenergic receptor induction in HeLa cells: synergistic effect of 5-azacytidine and butyrate

³H-Labeled leukotriene C₃ was efficiently taken up by the isolated, perfused rat liver and excreted into the bile. The isolated, perfused kidney eliminated leukotriene C₃ from the perfusate slower and excreted only a fraction of the radioactivity into the urine. Isolated hepatic, intestinal and renal cells also took up leukotriene C₃, the renal cells being the most effective in accumulating the label. Anthglutin, an inhibitor of γ-glutamyl transferase, decreased the uptake by kidney cells but had no effect on the uptake by the other cell types. In liver cells, the uptake rate was sensitive to temperature and to cellular ATP content. Chromatographic analyses indicated that renal cells metabolized leukotriene C₃ more rapidly than hepatic and intestinal cells. Leukotriene D₃ and E₃ were formed during the incubations with kidney cells, whereas intestinal cells produced mainly more polar metabolites.     

Down-regulation of melanocortin receptor signaling mediated by the amino terminus of Agouti protein in Xenopus melanophores.

Agouti protein and Agouti-related protein (Agrp) regulate pigmentation and body weight, respectively, by antagonizing melanocortin receptor signaling. A carboxyl-terminal fragment of Agouti protein, Ser73-Cys131, is sufficient for melanocortin receptor antagonism, but Western blot analysis of skin extracts reveals that the electrophoretic mobility of native Agouti protein corresponds to the mature full-length form, His23-Cys131. To investigate the potential role of the amino-terminal residues, we compared the function of full-length and carboxyl-terminal fragments of Agrp and Agouti protein in a sensitive bioassay based on pigment dispersion in Xenopus melanophores. We find that carboxyl-terminal Agouti protein, and all forms of Agrp tested, act solely by competitive antagonism of melanocortin action. However, full-length Agouti protein acts by an additional mechanism that is time- and temperature-dependent, depresses maximal levels of pigment dispersion, and is therefore likely to be mediated by receptor down-regulation. Apparent down-regulation is not observed for a mixture of amino-terminal and carboxyl-terminal fragments. We propose that the phenotypic effects of Agouti in vivo represent a bipartite mechanism: competitive antagonism of agonist binding by the carboxyl-terminal portion of Agouti protein and down-regulation of melanocortin receptor signaling by an unknown mechanism that requires residues in the amino terminus of the Agouti protein.     

The marine phycotoxin gymnodimine targets muscular and neuronal nicotinic acetylcholine receptor subtypes with high affinity

Gymnodimines (GYMs) are phycotoxins exhibiting unusual structural features including a spirocyclic imine ring system and a trisubstituted tetrahydrofuran embedded within a 16-membered macrocycle. The toxic potential and the mechanism of action of GYM-A, highly purified from contaminated clams, have been assessed. GYM-A in isolated mouse phrenic hemidiaphragm preparations produced a concentration- and time-dependent block of twitch responses evoked by nerve stimulation, without affecting directly elicited muscle twitches, suggesting that it may block the muscle nicotinic acetylcholine (ACh) receptor (nAChR). This was confirmed by the blockade of miniature endplate potentials and the recording of subthreshold endplate potentials in GYM-A paralyzed frog and mouse isolated neuromuscular preparations. Patch-clamp recordings in Xenopus skeletal myocytes revealed that nicotinic currents evoked by constant iontophoretical ACh pulses were blocked by GYM-A in a reversible manner. GYM-A also blocked, in a voltage-independent manner, homomeric human alpha7 nAChR expressed in Xenopus oocytes. Competition-binding assays confirmed that GYM-A is a powerful ligand interacting with muscle-type nAChR, heteropentameric alpha3beta2, alpha4beta2, and chimeric alpha7-5HT(3) neuronal nAChRs. Our data show for the first time that GYM-A broadly targets nAChRs with high affinity explaining the basis of its neurotoxicity, and also pave the way for designing specific tests for accurate GYM-A detection in shellfish samples.     

G-protein-coupled receptors: the new dopamine receptor subtypes.

The family of genes encoding G-protein-coupled dopamine receptors continues to grow with the recent cloning of a fifth member. The availability of these clones has revolutionized the dopamine receptor field. Expression of individual dopamine receptors is permitting the detailed analysis of their pharmacology and coupling to second messenger systems, while probes based on the receptors' nucleotide sequences are being used to gain new insights into their tissue distribution and genetics.     

Distribution and function of muscarinic receptor subtypes in the ovine submandibular gland.

The effects of muscarinic receptor antagonists on responses to electrical stimulation of the chorda-lingual nerve were determined in pentobarbitone-anesthetized sheep and correlated to the morphology of tissue specimens. Stimulation at 2 Hz continuously, or in bursts of 1 s at 20 Hz every 10 s, for 10 min induced similar submandibular fluid responses (19 +/- 3 vs. 21 +/- 3 microl x min(-1) x g gland(-1)), whereas vasodilatation was greater during stimulation in bursts (-52 +/- 4 vs. -43 +/- 5%; P < 0.01). Continuous stimulation at 8 Hz induced substantially greater responses (66 +/- 9 microl x min(-1) x g gland(-1) and -77 +/- 3%). While atropine (0.5 mg/kg iv) abolished the secretory response at 2 and 20 Hz (1:10 s), a small response persisted at 8 Hz (<5%). The "M1-selective" antagonist pirenzepine (40 microg/kg iv) reduced the fluid response at all frequencies tested (P < 0.05-0.01), most conspicuously at 2 Hz (reduced by 69%). Methoctramine ("M2/M4-selective"; 100 microg/kg iv; n = 5) had no effect on fluid or the vascular responses but increased the protein output at 2 (+90%, P < 0.05) and 8 Hz (+45%, P < 0.05). The immunoblotting showed distinct bands for muscarinic M1, M3, M4, and M5 receptors, and immunohistochemistry showed muscarinic M1 and M3 receptors to occur in the parenchyma. Thus muscarinic M1 receptors contribute to the secretory response to parasympathetic stimulation but have little effect on the vasodilatation in the ovine submandibular gland. Increased transmitter release caused by blockade of neuronal inhibitory receptors of the M4 subtype would explain the increase in protein output.     

Distribution of endothelin receptor subtypes ETA and ETB in the rat kidney.

The endothelin (ET) receptor system is markedly involved in the regulation of renal function under both physiological and pathophysiological conditions. The present study determined the detailed cellular localization of both ET receptor subtypes, ET(A) and ET(B), in the vascular and tubular system of the rat kidney by immunofluorescence microscopy. In the vascular system we observed both ET(A) and ET(B) receptors in the media of interlobular arteries and afferent and efferent arterioles. In interlobar and arcuate arteries, only ET(A) receptors were present on vascular smooth muscle cells. ET(B) receptor immunoreactivity was sparse on endothelial cells of renal arteries, whereas there was strong labeling of peritubular and glomerular capillaries as well as vasa recta endothelium. ET(A) receptors were evident on glomerular mesangial cells and pericytes of descending vasa recta bundles. In the renal tubular system, ET(B) receptors were located in epithelial cells of proximal tubules and inner medullary collecting ducts, whereas ET(A) receptors were found in distal tubules and cortical collecting ducts. Distribution of ET(A) and ET(B) receptors in the vascular and tubular system of the rat kidney reported in the present study supports the concept that both ET receptor subtypes cooperate in mediating renal cortical vasoconstriction but exert differential and partially antagonistic effects on renal medullary function.     

beta-Adrenergic receptor kinase. Activity of partial agonists for stimulation of adenylate cyclase correlates with ability to promote receptor phosphorylation

The beta-adrenergic receptor (beta AR) kinase is a recently discovered enzyme which specifically phosphorylates the agonist-occupied form of the beta-adrenergic receptor. We have utilized the agonist-dependent nature of this phosphorylation reaction to characterize the ability of partial agonists to interact with the receptor. Partial agonists were tested for their ability to: 1) stimulate adenylate cyclase activity in a three-component reconstituted system, and 2) promote phosphorylation of beta AR by beta AR kinase. There is an excellent correlation between the ability of partial agonists to stimulate adenylate cyclase activity and promote receptor phosphorylation by beta AR kinase (y = 1.02x-0.01, r = 0.996, p less than 0.001). Peptide maps of receptor phosphorylated by beta AR kinase in the presence of full or partial agonists are virtually identical with the partial agonist pattern reduced in intensity. Moreover, kinetic studies of beta AR phosphorylation by beta AR kinase suggest that partial agonists alter the Vmax of the reaction with little, if any, effect on the Km. These results suggest that at steady state partial agonists transform a smaller portion of the receptor pool into the conformationally altered or activated form which serves as the substrate for beta AR kinase, although they do not completely rule out the possibility that a partial conformational change is occurring.