Synthesis and prostaglandin-like activity of 2-(trans-3-hydroxy-1-octenyl)-3-indoleheptanoic acid.

The synthesis of 2-(trans-3-hydroxy-1-octenyl)-3-indoleheptanoic acid (1) is described. The title compound appeared to show a weak prostaglandin-like activity in two different systems. It contracted rat stomach fundus strips and guinea-pig ileum preparations only at concentrations about 10(3)- and 10(2)-fold higher, respectively, than PGE1. Moreover, it stimulated adenylate cyclase from rat liver plasma membrane, but the relative potency was 4--5 X 10(2)-fold lower than the natural compound. The title compound showed also a certain degree of PGE1 antagonism.

Evidence for increased release of prostaglandins of E-type in response to orthodromic stimulation in the guinea-pig superior cervical ganglion.

Prostaglandins of the E type (PGEs) stimulate cyclic adenosine 3',5'-monophosphate (cAMP) biosynthesis both in isolated preparations of rat, guinea-pig and rabbit superior cervical ganglia (SCG) and in calf SCG slices. Electrical stimulation of preganglionic nerve fibers of the guinea-pig SCG remarkably increased PGE release and cAMP biosynthesis. These effects were blocked by reducing the Ca2+ to Mg2+ ratio in the incubation medium. Atropine (1 microM) and phentolamine (10 microM) inhibited PGE biosynthesis and significantly reduced cAMP levels.

Full and partial agonistic behaviour and thermodynamic binding parameters of adenosine A1 receptor ligands.

The inhibitory effect of forskolin-stimulated 3',5'-cyclic monophosphate (c-AMP) synthesis in isolated rat adipocytes has been measured for eight typical adenosine receptor agonists. The percent inhibition was evaluated using concentrations of each compound corresponding to 100 times their Ki, inhibitory binding constants, determined in parallel by [3H]N6-cyclohexyladenosine binding to adenosine A1 receptors on adipocyte rat membranes which guaranteed full receptor occupancy. Some drugs were able to inhibit the forskolin-induced c-AMP accumulation by 100% (full agonists, intrinsic activity = 1) but others only to a lesser extent, ranging from 80% to 50% (partial agonists, intrinsic activities in the 0.8-0.5 interval). These efficacy data were correlated with the thermodynamic binding parameters (free energy, enthalpy and entropy) obtained in rat brain membranes by equilibrium constant measurements and van 't Hoff plots in the 0 degrees-30 degrees C range. The positive correlation between intrinsic activity and entropy changes (r = 0.88, n = 8, P < 0.01) points to the possibility of obtaining information about the full spectrum of activities of adenosine analogues from in vitro binding assays and suggests some considerations on the possible drug-receptor interaction mechanism.

Presynaptic muscarinic receptors in guinea pig superior cervical ganglion.

This study characterizes the presynaptic muscarinic cholinergic receptors associated with the modulation of the electrically-evoked acetylcholine output from guinea pig superior cervical ganglion preincubated with [3H]choline. The M1-selective agonist pilocarpine had no effect while carbachol and oxotremorine strongly decreased the evoked outflow of tritium. Atropine increased such evoked release of [3H]acetylcholine whereas the M1-selective antagonist pirenzepine was ineffective. Moreover, atropine but not pirenzepine antagonized the inhibitory effect of carbachol. These results suggest that the guinea-pig superior cervical ganglion is equipped with presynaptic inhibitory muscarinic receptors of the M2 subtype.

Opiates modulation of cAMP levels and PGE2 binding in mammalian sympathetic ganglia.

The effects of opiates on cyclic adenosine monophosphate (cAMP) levels have been studied in sympathetic ganglia of guinea-pig, rat and rabbit. D-[Ala2]-Met-enkephalinamide inhibits cAMP synthesis in guinea pig and rat but not in rabbit ganglia, while morphine is always ineffective. In the presence of the enkephalin plus prostaglandin E2 (PGE2) a synergistic increase of the nucleotide levels is observed in guinea pig. This effect is not induced by morphine plus PGE2 and is not shared by rat and rabbit. In guinea pig alpha-endorphin inhibits both basal and PGE2-stimulated cAMP synthesis. In the same preparation the enkephalin increases [3H]PGE2 binding. In guinea pig ganglia a cooperative effect of the enkephalin and PGE2 on the cAMP system is suggested.

Alpha-adrenoceptor-mediated inhibition of acetylcholine release in guinea-pig superior cervical ganglion.

The effect of different substances on the electrically evoked acetylcholine (ACh) output from guinea-pig superior cervical ganglion (SCG) preincubated with [3H]choline was investigated. Forskolin significantly reduced neurotransmitter release and dose-dependently enhanced cyclic AMP (cAMP) levels. Phenylephrine and clonidine, alpha 1-and alpha 2-adrenergic agonists respectively, caused a significant inhibition of ACh output from presynaptic nerve terminals. In the presence of RMI 12330A, phenylephrine but not clonidine failed to reduce neurotransmitter release. These results suggest that cAMP and different alpha-adrenergic receptors are involved in the regulation of cholinergic transmission.

Alpha-adrenoceptor-mediated glucose release from perifused catfish hepatocytes.

In fish liver catecholamines bind to beta-adrenoceptors (AR) and increase glucose release via cAMP augmentation. Alpha1-AR have recently been shown to mediate IP3 and Ca2+ elevation in catfish and eel hepatocytes, although their coupling to a physiological response has remained doubtful. We have perifused isolated catfish hepatocytes in Bio-Gel P4 columns with epinephrine in the presence of prazosin and/or propranolol, alpha- and beta-AR antagonists, respectively. Ten nM epinephrine stimulated glucose release approximately 3-fold, and this effect was completely antagonized by the simultaneous presence of both alpha- and beta-AR blockers. The two AR antagonists separately inhibited about one-third and two-third of the total stimulation, respectively. Through alpha-AR occupancy, epinephrine provoked a significant increase of glucose release whereas no stimulation was detected in Ca2+-depleted hepatocytes. Glucose release was strongly elevated by both ionomycin and dibutyryl cAMP. These results represent the first direct evidence that alpha-AR transduction pathway is involved in epinephrine-induced glucose release from fish hepatocytes.

Non-selectivity of yohimbine for adrenergic receptors in fish liver.

Most studies on adrenergic receptors (AR) have been performed on mammalian tissues, but the adrenergic ligands routinely utilized seem not always suitable for specific interaction with fish tissues. Here we report that in isolated catfish hepatocytes, yohimbine, usually thought to act as a specific antagonist for AR of the alpha2 subtype, at high concentrations, increases adenylyl cyclase activity and synergistically enhances the forskolin-induced enzyme stimulation. Such effects are counteracted by the beta-AR antagonist propranolol, but not by the alpha-AR antagonist phentolamine. Moreover, yohimbine seems to antagonize both alpha1- and alpha2-adrenergic ligand-binding in catfish liver membrane in a manner somewhat different from the mammalian systems. Together with previous evidence that yohimbine blocks the rise of intracellular calcium induced by epinephrine via alpha1-AR, the present results seem to indicate that this compound is not a suitable tool for studying alpha2-AR in fish liver.

The role of circulating catecholamines in the regulation of fish metabolism: an overview.

The physiological role of the catecholamines (CA), adrenaline and noradrenaline in fish has been frequently reviewed, but the metabolic consequences of these hormones have received less attention. The purpose of this review is to examine the recent literature dealing with CA actions on whole fish and tissue metabolism. The CA increase glucose production both in vivo and in vitro, at least in isolated hepatocytes. Although the data are less clear, lipid mobilization is also a consequence of elevated circulating CA. The difficulty with using the whole fish for such studies is that CA may alter other circulating hormone levels, CA turnover in the circulation quickly, and it is difficult to define precisely the tissue being affected. Much of our understanding is derived, therefore, from the study of isolated tissues, and especially the hepatocyte. Catecholamines stimulate both glycogenolysis and gluconeogenesis in hepatocytes isolated from a large number of fish species. This review examines the steps involved in the signal transduction system, from the binding of CA to alpha- and beta-adrenoceptors to the ultimate effects of specific enzyme phosphorylation. Recent literature demonstrates that the complexity of the adrenoceptor system noted for mammals, also is expressed in fish. Adrenoceptor subtypes are specific to species, to tissues and to function of the tissues, and these issues are discussed especially as they are related to external and to internal stressors. Future research will pursue better definitions of the adrenoceptor systems, molecular biology of the components of these receptor systems and development of alternative cell models. There still remains a poor explanation of the reason for the diversity of adrenoceptor systems, and there are a number of fish systems that may provide unique opportunities to understand this question.

Coexistence of alpha1 and beta adrenergic receptors in the liver of the frog Rana esculenta, the toad Bufo bufo, the lizard Podarcis sicula campestris, and the turtle Pseudemys picta elegans.

In mammals and birds the characteristics of alpha1 adrenergic receptors and their biological role in liver metabolism have been clearly described, although the predominance of receptor subtypes varies with species. In contrast, the actual presence of hepatic alpha1 adrenergic receptors in fish, amphibians, and reptiles has been questioned. Only recently has their existence been demonstrated in some fish species and also in the wood frog Rana sylvatica. The present study assessed the presence of alpha1 adrenergic binding sites on hepatic membranes of frogs, toads, lizards, and turtles using the specific alpha1 adrenergic receptor antagonist [3H]prazosin; for comparison, the binding of the specific beta adrenergic receptor antagonist [3H]CGP-12177A was evaluated in the same preparations. alpha1 Adrenergic receptors are indeed present in the liver of the ectotherms examined. Specific binding is saturable, reversible, and linear as a function of tissue concentration. The binding data indicated the presence of two classes of binding sites displaying high and low affinities with Kds in the nanomolar and micromolar ranges, respectively. The present study provides the first evidence for the presence of alpha1 adrenergic receptors in the liver of toad, lizard, and turtle while confirming their existence in another species of frog, Rana esculenta.

Ca2+-activated ATPase of rat liver plasma membrane.

Rat liver plasma membranes hydrolyze ATP in the presence of Ca2+. The rate of hydrolysis is different when Mg2+ions are present in the incubation system. Several parameters differentiate Ca2+-ATPase from Mg2+-ATPase: a) the Km of ATP hydrolysis for Ca2+ (2.25 x 10(-4) M) is lower than for Mg2+ (2.14 x 10(-3) M); b) the shape of the activation curve is hyperbolic in the presence of Ca2+ and sigmoid in the presence of Mg2+; c) Mg2+-ATPase shows two different values of activation energy while Ca2+-ATPase presents only a single value; d) Ca2+-ATPase is inhibited, while Mg2+-ATPase is unaffected by cyclic AMP. Ca2+-ATPase is localized on the plasma membrane and is not inhibited by cysteine. It does not hydrolyze substrates different from nucleotides triphosphate, such as glucose-1-phosphate or alpha-glycero-phosphate. The enzyme is probably related to a mechanism of calcium transport.

Identification of alpha-adrenergic receptors in catfish liver and their involvement in glucose release.

This study aimed to characterize alpha-adrenergic receptors in catfish hepatocytes in which catecholamine-induced calcium transients have been observed. alpha-Adrenergic binding sites were studied in purified liver membranes using the specific alpha 1-receptor antagonist [3H]prazosin. At 22 degrees C specific binding of [3H]prazosin is saturable, reversible, and linear as a function of tissue concentration; the association reaches a maximum at 15 min, and the half-time for dissociation is about 4 min. Analysis of binding data suggests a single class of binding sites with a Kd of 1.6 nM and a Bmax of 182.1 fmol/mg protein. The relative potency of selective adrenoceptor ligands points to the presence in catfish liver of alpha-adrenergic receptors in addition to the beta-adrenergic receptor population previously characterized. Moreover, the binding data have been correlated to the glucose release from isolated hepatocytes suspended and perifused in a BioGel column, suggesting the involvement of alpha-adrenergic receptors in the glycogenolytic response to catecholamines in catfish hepatocytes.

Elevated levels of prostaglandin E2 in Yoshida hepatoma and the inhibition of tumour growth by non-steroidal anti-inflammatory drugs.

Prostaglandin (PG) E2 biosynthesis in Yoshida hepatoma (AH 130) was evaluated by radioimmunoassay. When hepatoma cells were incubated in vitro, the levels of PGE2 in the medium were similar to those found in hepatocytes for the first 2 h; this was followed by a rapid increase in PGE2 formation, and the 6h incubation levels were 4-fold higher than in hepatocytes. Addition of sodium arachidonate markedly and dose-dependently stimulated PGE2 synthesis; the increase was largely prevented by the addition of indomethacin (1 microM) or L 8027, a prostaglandin synthetase inhibitor. Experiments in vivo indicated that indomethacin treatment of tumour-bearing rats significantly reduced the tumour mass. When rats were injected with PGE2 after receiving the drug, the number of tumour cells was very similar to that of untreated animals. This, as well as the inhibition of tumour growth by acetylsalicylic acid, strongly suggests that the inhibition of PG biosynthesis by anti-inflammatory drugs and the inhibition of tumour proliferation may be closely associated events. It was also found that injections of indomethacin very significantly prolonged survival of hepatoma-bearing rats. Since PGE2 does not appear to affect the cyclic AMP levels of hepatoma cells, it is possible that hepatoma may use PGE2 to subvert the immune system. This could help to explain the effectiveness of anti-inflammatory drugs in the control of tumour growth.

Characterization of [3H]CGP 12177 binding to beta-adrenergic receptors in intact eel hepatocytes.

The aim of this study was to characterize [3H]CGP 12177 (CGP) binding to beta-adrenergic receptors in isolated hepatocytes of the European eel (Anguilla anguilla), in which the involvement of cAMP in epinephrine-induced glucose release has been previously observed. Specific binding of CGP was saturable, reversible, and linear as a function of cell number. Analysis of binding data suggested a single class of binding sites, with a Kd of 1.31 nM and a number of approximately 7000 beta-adrenergic receptors per cell. The potency order of specific inhibition of [3H]CGP binding was CGP > propranolol > or = alprenolol >> butoxamine > or = atenolol, while phentolamine and prazosin failed to significantly displace the tracer at concentrations up to 100 microM. The binding kinetics of CGP were closely related to its biological effect. In fact, the drug dose-dependently counteracted the enhancement of intracellular cAMP levels induced by epinephrine in isolated hepatocytes with a Kd of 1.06 nM. Moreover, it antagonized the hormone-induced stimulation of adenylyl cyclase activity in hepatic membranes as well as of glucose release from cells. These data clearly show that beta-adrenergic receptors are coupled to the adenylyl cyclase/cAMP transduction pathway in eel liver.

Effects of oxotremorine and RMI 12330 A on [3H]acetylcholine release and adenylate cyclase activity in guinea pig superior cervical ganglion.

There is considerable evidence that adenosine 3',5'-cyclic monophosphate (cAMP) is involved in the modulation of synaptic transmission in the guinea pig superior cervical ganglion (SCG). Presynaptic muscarinic receptors are known to attenuate, when activated, acetylcholine (ACh) release in the periphery as well as in the brain. Thus, the possible relationship between ganglionic adenylate cyclase activity and the output of ACh from electrically stimulated ganglia, preloaded with [3H]choline, was investigated. The muscarinic agonist oxotremorine significantly reduced in a dose-dependent manner the electrically evoked neurotransmitter release. The adenylate cyclase inhibitor N-(cis-2-phenylcyclopentyl)azacyclotridecan-2-imine hydrochloride (RMI 12330 A) also decreased ACh output. The inhibitory effects of these two drugs were additive. In crude ganglion membrane fractions oxotremorine significantly inhibited adenylate cyclase activity. The results indicate that drugs capable of inhibiting adenylate cyclase, significantly decrease ACh output from preganglionic nerve terminals in guinea pig SCG.

Adenylyl cyclase activity and glucose release from the liver of the European eel, Anguilla anguilla.

The properties of adenylyl cyclase (AC) in liver membranes of the European eel (Anguilla anguilla) and the involvement of cAMP in glucose release from isolated hepatocytes in response to catecholamines were studied. Basal enzyme activity seemed essentially unaffected by GTP, while a biphasic response to increasing nucleotide concentrations was obtained in the presence of epinephrine. Eel liver AC was dose-dependently stimulated by guanosine 5'-O-(3-thiotriphosphate) and inhibited by guanosine 5'-O-(2-thiodiphosphate). AC activity, intracellular cAMP levels, and glucose release from isolated hepatocytes were significantly enhanced by NaF, forskolin, epinephrine, and phenylephrine. The rise in cAMP production stimulated by catecholamines was counteracted by propranolol, but not by phentolamine. Catecholamine-induced glucose output was instead partially antagonized by both phentolamine and propranolol. Complete inhibition was obtained only by the simultaneous presence of the two adrenergic antagonists. Glucose release from the cells was induced by dibutyryl cAMP and by the calcium ionophore ionomycin. In summary, these data provide the first characterization of eel liver AC system and suggest a direct role for cAMP in the catecholamine-dependent glucose output. Furthermore, the involvement of calcium ions in this cellular response is hypothesized.

Alpha-mediated changes in hepatocyte intracellular calcium in the catfish, Ictalurus melas.

Adult catfish, Ictalurus melas, hepatocytes respond to alpha-agonists by increasing intracellular free calcium concentrations ([Ca2+]i) in a dose-dependent manner. Basal [Ca2+]i were approximately 78 nM in catfish hepatocytes; this value was increased by 213, 243, 131, and 238 nM in the presence of epinephrine (Epi; 10(-6) M), norepinephrine (NE; 10(-5) M), phenylephrine (PE; 10(-4) M), and isoproterenol (Iso; 10(-4) M), respectively. The binding constants were 3.4 x 10(-8), 2.6 x 10(-7), 9.8 x 10(-7), and 6.1 x 10(-5) M for Epi, NE, PE, and Iso, respectively, which is the same order of potency for these agonists reported for the alpha 1-adrenoceptor system of mammalian hepatocytes. The Epi-induced changes in [Ca2+]i were antagonized equally by phentolamine (PH; 10(-8) M) and yohimbine (10(-8) M) but poorly by propranolol (Prop; 10(-6) M), as indicated by their respective inhibitory constants. Epi (10(-7) M) induced a time-dependent increase in hepatocyte adenosine 3',5'-cyclic monophosphate concentration that was antagonized by Prop but not PH. There were, however, no clear agonist-induced changes in the activities of glycogen phosphorylase (total, a, or %a) under these conditions. These studies provide evidence that Epi and other adrenergic agonists increase [Ca2+]i by an alpha- or alpha 1-like adrenoceptor pathway in catfish hepatocytes, but the biochemical effector system responding to changes in [Ca2+]i has yet to be elucidated.

Hormone responsiveness of isolated catfish hepatocytes in perifusion system is higher than in flasks incubation.

Hepatocytes were isolated from catfish (lctalurus melas) by conventional collagenase digestion. Sensitivities of liver cells isolated from the same fish to the glycogenolytic action of epinephrine, mammalian glucagon, catfish glucagon, catfish glucagon-like peptide, synthetic fragment 19-29 of anglerfish glucagon I, fragment 19-29 of anglerfish glucagon II, and anglerfish glucagon II were compared in two different systems: perifusion in a Bio-Gel P4 column and flask incubation. Both experimental procedures were continued for a total of 100-120 min, while hormones were applied simultaneously to both preparations for 10 min. Effluent fractions from the columns and incubation media from the flasks were collected for glucose determination. The hormonal effects were clearly enhanced in perifused cells compared to those in cells incubated in flasks, the effect being especially evident at physiological concentrations of hormones. The hormonal effects in both systems were dose-dependent. Epinephrine and mammalian glucagon (10 nM), applied separately to the same column, produced two different peaks, glucagon causing more glucose production than epinephrine. In the presence of 0.4 mM glucose in the perifusion system, hormonal effects were diminished, implying that glucose accumulation during incubation of liver cells in flasks might affect hormonal effects. The results obtained in this study indicate that piscine hepatocytes suspended and perifused in a Bio-Gel column are more sensitive to physiological concentrations of glycogenolytic hormones and may represent a new tool for experimental studies of fish liver metabolism and its hormonal regulation.