Signal propagation via gap junctions, a key step in the regulation of liver metabolism by the sympathetic hepatic nerves.

Cell-to-cell communication via gap junctions has been proposed to be involved in the metabolic actions of sympathetic liver nerves in the rat. The effects of hepatic nerve stimulation and noradrenaline-, PGF2 alpha- and glucagon infusion on glucose metabolism and perfusion flow were studied in perfused rat liver in the absence and presence of the gap junctional inhibitors, heptanol, carbenoxolone and (4 beta)phorbol 12-myristate 13-acetate (4 beta PMA). (i) Stimulation of the hepatic nerve plexus increased glucose output, decreased flow and caused an overflow of noradrenaline into the hepatic vein. (ii) Heptanol completely inhibited not only the nerve stimulation-dependent metabolic and hemodynamic alterations but also the noradrenaline overflow. Thus the heptanol-dependent inhibitions were caused primarily by a strong impairment of transmitter release. (iii) Carbenoxolone inhibited the effects of neurostimulation on glucose metabolism partially by about 50%, whereas it left perfusion flow and noradrenaline overflow essentially unaltered. (iv) 4 beta PMA reduced the nerve stimulation-dependent enhancement of glucose release by about 80% but the noradrenaline-dependent increase in glucose output only by about 30%; the increase in glucose release by PGF2 alpha and by glucagon remained essentially unaltered. 4 beta PMA reduced the nerve stimulation-dependent decrease in portal flow by about 35% but did not affect the noradrenaline-and PGF2 alpha-elicited alterations, nor did it alter noradrenaline overflow. The results allow the conclusion that gap junctional communication plays a major role in the regulation of hepatic carbohydrate metabolism by sympathetic liver nerves, but not by circulating noradrenaline, PGF2 alpha or glucagon.     

DEGENERATION OF CENTRAL AND PERIPHERAL NORADRENALINE NEURONS PRODUCED BY 6-HYDROXY-DOPA

—The effects of systemically administered 2,4,5-trihydroxyphenylalanine (6-OH-DOPA) on endogenous noradrenaline, [³H]amine uptake and fluorescence morphology has been investigated in mouse brain, heart and iris. 6-OH-DOPA in a dose of 100 mg/kg intraperitoneally caused practically no changes in these parameters. Pretreatment with a potent monoamine oxidase inhibitor (nialamide) led to a pronounced long-lasting 6-OH-DOPA induced reduction in endogenous noradrenaline, [ ³ H]amine uptake and nerve density of noradrenaline nerve terminals both in the central and peripheral nervous system. Histochemically accumulations of noradrenaline were observed in non-terminal axons. These results strongly support the view that 6-OH-DOPA can produce degeneration of both central and peripheral noradrenaline neurons. The degeneration is mediated by decarboxylation of 6-OH-DOPA to 6-OH-DA, since the effects could be abolished by decarboxylase inhibition. The effect of 6-OH-DOPA was selective on noradrenaline neurons in the brain, since neither 5-hydroxytryptamine nor dopamine neurons were affected, opening up new possibilities for studies on central noradrenaline transmitter mechanisms. In the brain there were pronounced accumulations of noradrenaline in the ascending noradrenaline axons making 6-OH-DOPA a powerful tool in the mapping of central noradrenaline pathways.     

Portal and arterial free and conjugated noradrenaline in two models of portal hypertension in rats

Free and conjugated noradrenaline concentrations were measured in portal-venous and arterial plasma from sham-operated rats or rats with portal hypertension. Two types of portal hypertension in rats were evaluated: in portal vein stenosed rats, the liver was normal, whereas cirrhosis developed in bile duct ligated rats. In cirrhotic rats, arterial free noradrenaline level was higher than in both sham-operated and portal-stenosed rats, this indicating that enhanced sympathetic nervous activity depends on the development of cirrhosis. In all groups of rats, portal venous plasma free noradrenaline was higher than arterial plasma level, indicating a production of noradrenaline by splanchnic organs. Arterial noradrenaline level may be mainly dependent on this splanchnic production in case of portal hypertension. Sulfoconjugated and glucuronoconjugated noradrenaline plasma levels were similar in the three groups of rats. This shows that alteration in conjugation is not likely to be a major factor in the abnormal circulating levels of free noradrenaline observed in cirrhotic rats.     

6-AMINODOPAMINE-INDUCED DEGENERATION OF CATECHOLAMINE NEURONS

the effects of 6-aminodopamine on central and peripheral catecholamine neurons using fluorescence histochemical and isotope techniques have been investigated. Systematic administration of 6-aminodopamine (20 mg/kg intraveneously) produced a rapid (within 1 h) and long-lasting depletion of endogenous noradrenaline in adrenergic nerves of mouse atrium and iris with a concomitant loss of [³H]noradrenaline uptake. The effects were dosedependent. Accumulations of noradrenaline in non-terminal axons were observed histochemically, indicating that 6-aminodopamine induces neuronal damage. Desipramine completely blocked the 6-aminodopamine induced noradrenaline depletion and reduction in [³H]noradrenaline uptake, indicating that 6-aminodopamine has to be taken up by the axonal ‘membrane pump’ to produce its effects. Themonoamine oxidase inhibitor, nialamide, potentiated the effect of 6-aminodopamine on [³H]noradrenaline uptake. 6-Aminodopamine did not affect the cell bodies of the adrenergic neurons and there was a reappearance of adrenergic nerves and recovery of [³H]noradrenaline uptake. 6-Aminodopamine does not seem to pass the blood-brain barrier after systemic injection. Intraventricular injection of 6-aminodopamine in rats led to a considerable reduction in endogenous whole brain noradrenaline and [³H]noradrenaline uptake in slices from cerebral cortex and hypothalamus. Similar, but less pronounced effects were observed on dopamine neurons in the caudate nucleus. Histochemically, pronounced accumulations of transmitter were observed in the axons of the catecholamine neurons. The results obtained favour the view that 6-aminodopamine is able to produce an acute and selective degeneration of catecholamine neurons similar to that seen after the neurotoxicagent, 6-hydroxydopamine. Both compounds seemed to be approximately equally potent in their neurotoxicity, although 6-aminodopamine seemed to be more generally toxic.     

Inhibition of glucose production during hepatic nerve stimulation in regenerating rat liver perfused in situ. Possible involvement of gap junctions in the action of sympathetic nerves.

To explore the possible role of gap junctions in neural regulation of hepatic glucose metabolism, the effects of hepatic nerve stimulation on metabolic and hemodynamic changes were examined in normal and regenerating rat liver which was perfused in situ at constant pressure via the portal vein with a medium containing 5 mM glucose, 2 mM lactate and 0.2 mM pyruvate. 1. The content of connexin 32, a major component of gap junctions in rat liver, decreased transiently to about 25% of the control level in regenerating liver 48-72 h after partial hepatectomy and recovered to normal by the 11th day after the operation. 2. In normal liver, electrical stimulation of the hepatic nerves (10 Hz, 20 V, 2 ms) and infusion of noradrenaline (1 microM) both increased glucose and lactate output and reduced perfusion flow. 3. In early stage of regenerating liver 48 h and 72 h after partial hepatectomy, the increase in glucose output in response to nerve stimulation was almost completely inhibited, whereas the change in lactate balance was partially suppressed and the reduction of flow rate was retained. The response of glucose output to nerve stimulation recovered by the 11th day after partial hepatectomy. In contrast, exogenous application of noradrenaline increased glucose output even in the early stage of regenerating liver. 4. The increase in noradrenaline overflow during hepatic nerve stimulation in the early stage of regenerating liver was approximately the same as in normal liver. Liver glycogen was sufficiently preserved in the early stage of regenerating liver. However, noradrenaline infusion could no more increase glucose output both in normal and in regenerating livers after 24 h of fasting that depleted liver glycogen. These results suggest that the impaired effects of sympathetic nerve stimulation on glucose metabolism observed in regenerating liver are derived neither from reduced release of noradrenaline nor from depletion of liver glycogen, but rather from transient reduction of gap junctions which assist signal propagation of the nerve action through intercellular communication in rat liver.     

Angiotensin II and noradrenaline increase PDGF-BB receptors and potentiate PDGF-BB stimulated DNA synthesis in vascular smooth muscle

The effects of angiotensin II and noradrenaline were examined on PDGF-BB and PDGF-AB induced mitogenesis in primary cultures of rat aortic smooth muscle. Incubation of the smooth muscle with either angiotensin II or noradrenaline potentiated the submaximal but not maximal mitogenic effects of PDGF-BB but not PDGF-AB. These effects on PDGF-BB stimulated mitogenesis correlated with an increase in receptor number specific for this homodimer when the smooth muscle was incubated with either angiotensin II or noradrenaline. Mitogenic concentrations of PDGF-AB did not interact with this PDGF receptor subtype. These results indicate that the mitogenic effects of PDGF-AB and -BB are elicited via different PDGF receptor subtypes. Angiotensin II and noradrenaline potentiate the mitogenic effects of PDGF-BB by increasing the steady state concentrations of membrane receptors for this homodimer.     

Quantitation and immunohistochemistry of catecholamines in the posterior segment of the eye

Summary Dopamine, noradrenaline, and adrenaline were assayed with HPLC in the light adapted retinae of carp, frog, chicken, pigeon, rat, guinea-pig, rabbit, cat, pig and cow. Dopamine varied from 0.6 to 2.6 nmol/g wet weight and was not influenced by sympathectomy. The dopamine figures agree with previously published results. Noradrenaline concentrations varied from not detectable to 0.06 nmol/g wet weight in different species. Homolateral sympathectomy significantly decreased the noradrenaline figure in rabbits. There are no previous figures for noradrenaline for most of the species. Adrenaline was not detected in any species. Immunohistochemical analysis showed noradrenaline to be present in choroidal nerves, but noradrenaline immuno-reactivity was not seen in the retina (chicken, rat, guinea-pig, rabbit, cat, cow). It is concluded that dopamine is the major catecholamine in the retina. Noradrenaline was found present only in minute amounts in the assays, and much of its was likely to stem from sympathetic nerve fibres. The study did not demonstrate any noradrenergic neurons in the retina.     

A possible mechanism for cadmium-induced hypertension in rats

The mechanism of cadmium-induced hypertension was explored by measuring noradrenaline metabolism. Cadmium $\text{in vitro}$ was shown to inhibit both monoamine oxidase and catechol-O-methyltransferase, the two enzymes which inactivate the neurotransmitters noradrenaline and adrenaline. However, rats which were injected or fed (via the drinking water) with cadmium showed that, among the tissues surveyed, these two enzymes were inhibited significantly only in the aorta. $\text{In vitro}$, cadmium was found to inhibit noradrenaline binding to membranes from the heart, lung, and kidney, while stimulating binding to aortic membranes, which suggests that the effects may be specific. These results suggest that, in the aorta, cadmium may inhibit the two catabolic enzymes of noradrenaline, while at the same time stimulating noradrenaline-binding. Thus the effects of noradrenaline on vascular smooth muscle would be increased as well as prolonged.     

Quantitation and immunohistochemistry of catecholamines in the posterior segment of the eye

Dopamine, noradrenaline, and adrenaline were assayed with HPLC in the light adapted retinae of carp, frog, chicken, pigeon, rat, guinea-pig, rabbit, cat, pig and cow. Dopamine varied from 0.6 to 2.6 nmol/g wet weight and was not influenced by sympathectomy. The dopamine figures agree with previously published results. Noradrenaline concentrations varied from not detectable to 0.06 nmol/g wet weight in different species. Homolateral sympathectomy significantly decreased the noradrenaline figure in rabbits. There are no previous figures for noradrenaline for most of the species. Adrenaline was not detected in any species. Immunohistochemical analysis showed noradrenaline to be present in choroidal nerves, but noradrenaline immuno-reactivity was not seen in the retina (chicken, rat, guinea-pig, rabbit, cat, cow). It is concluded that dopamine is the major catecholamine in the retina. Noradrenaline was found present only in minute amounts in the assays, and much of its was likely to stem from sympathetic nerve fibres. The study did not demonstrate any noradrenergic neurons in the retina.     

Muscarinic receptor activation inhibits both release and synthesis of noradrenaline in rat hippocampal synaptosomes

Rat hippocampal synaptosomes were used to investigate the effects of muscarinic cholinergic drugs on the release of [³H]noradrenaline and the rate of noradrenaline synthesis. Oxotremorine and acetylcholine caused depression of K⁺-evoked release of [³H]noradrenaline; these effects were reversed by atropine and pirenzepine. Muscarinic agonists depressed 2-chloroadenosine- and isoprenaline-stimulated noradrenaline synthesis but had no effect on 8-Br-cyclic AMP-stimulated synthesis. Oxotremorine also depressed the K⁺-acceleration of noradrenaline synthesis. The action of pirenzepine suggests that the inhibition of release and synthesis are mediated by separate muscarinic receptor subtypes.     

EVIDENCE FOR REVERSIBLE INHIBITION OF BRAIN DOPAMINE-β-HYDROXYLASE ACTIVITY IN VIVO BY AMPHETAMINE ANALOGUES1

Rats treated with d -amphctamine have a decreased ability to convert intracisternally injected radioactive dopamine to noradrenaline. This effect is dose-related and reversible by increasing the dose of injected dopamine. L-Amphetamine is equipotent to the d -isomer, but para-hydroxyamphetamine reduces noradrenaline formation only slightly. Like the amphetamines, imipramine produces significant inhibition of noradrenaline from labelled dopamine; however, dopamine content after imipramine, but not after the amphetamines, was consistently decreased, suggesting that imipramine's effect on conversion of labelled dopamine to noradrenaline is mediated by interference with membranal transport systems. Conversion of labelled tyrosine to noradrenaline increases after imipramine. In contrast, noradrenaline accumulating from labelled tyrosine is decreased by d - or l -amphetamine. The divergent effects of the amphetamines and imipramine on noradrenaline accumulation from labelled tyrosine suggest that the amphetamines' effect on in vivo dopamine-β-hydroxylase activity are not solely attributable to interference with transport and, therefore, suggest a direct effect on the enzyme.     

INHIBITION OF SYNAPTOSOMAL NORADRENALINE UPTAKE BY VERATRIDINE, GRAMICIDIN D AND VALINOMYCIN

Abstract— —The effects of brief exposures of rat brain synaptosomes to veratridine, gramicidin D and valinomycin on noradrenaline uptake were investigated. All three drugs inhibited the Na⁺-dependent component of noradrenaline uptake by synaptosomes. These effects were independent of extracellular Ca^{2 +} concentrations, indicating that the reductions were not due to the release of newly accumulated noradrenaline.
Gramicidin D reduced the V_max for noradrenaline uptake, whereas veratridine and valinomycin reduced the V^max and also increased the V^m for uptake.
Most of these findings can be explained on the basis of the effects that these drugs have on the inward-directed electrochemical gradients for Na⁺ across synaptosomal membranes, although, in the cases of veratridine and valinomycin, the elevated K_m's suggest that an impairment of noradrenaline binding to its carriers might also be involved.     

Lack of Selectivity Between the Uptake of [3H] Adrenaline and [3H]Noradrenaline into Rat Hypothalamic Slices

The uptake of [3H]adrenaline and [3H]noradrenaline into rat hypothalamic slices was compared for determination of whether adrenaline uptake was independent of uptake into noradrenergic neurones. Kinetic analysis revealed a similar high-affinity uptake process for both adrenaline and noradrenaline, with Km and Vmax values within similar ranges. These uptakes were inhibited by desipramine and maprotiline in a dose-dependent manner, but the selective dopamine and 5-hydroxytryptamine uptake inhibitors benztropine and fluoxetine, respectively, were without effect. Competition for uptake sites by unlabelled adrenaline with [3H]adrenaline and [3H]-noradrenaline and by unlabelled noradrenaline with [3H]-adrenaline and [3H]noradrenaline was very similar. Lesioning of the major adrenaline-containing cell group (C1 cell group) decreased the hypothalamic adrenaline concentration but had no effect on hypothalamic [3H]adrenaline or [3H]noradrenaline uptake. The results suggest that exogenous adrenaline is largely taken up by high-affinity sites on noradrenergic nerve terminals.     

The influence of ovarian hormones on the rat oviductal and uterine concentration of noradrenaline and 5-hydroxytryptamine

This paper describes the effects of estradiol and progesterone on the concenirations of noradrenaline and 5-hydroxytryptamine in the Wistar rat oviduct and uterus. The levels of noradrenaline and 5-hydroxytryptamine are higher in the oviduct than in the uterus whereas p-tyrosine and tryptophan are similar in both tissues. Estradiol treatment reduced the oviductal concentration of noradrenaline but not 5-hydroxytryptamine in oviduct, while the concentrations of both noradrenaline and 5-hydroxytryptamine were reduced in uterine horn. The levels of noradrenaline in the oviduct and uterus in rats in estrus were lower than those of diestrous rats. Bilateral ovariectomy produced an increase in uterine noradrenaline and 5-hydroxytryptamine levels. These changes were reversed in the presence of ovarian hormones as indicated by experiments where unilateral ovariectomy was performed. Reserpine administration reduced noradrenaline concentration in both the oviduct and the uterus but did not change oviductal or uterine 5-hydroxytryptamine.These results indicate the existence of noradrenaline within postganglionic sympathetic nerve terminals and suggest that estrogens increase the utilization and the synthesis of noradrenaline in both the oviducts and the uterine horns. With respect to 5-hydroxytryptamine the data support the concept that it is mainly associated with mast cells.     

Activation of Muscarinic and of α1-Adrenergic Receptors on Astrocytes Results in the Accumulation of Inositol Phosphates

Astrocyte-enriched cultures prepared from the newborn rat cortex incorporated [3H]myo-inositol into intracellular free inositol and inositol lipid pools. Noradrenaline and carbachol stimulated the turnover of these pools resulting in an increased accumulation of intracellular [3H]inositol phosphates. The effects of noradrenaline and carbachol were dose-dependent and blocked by specific alpha 1-adrenergic and muscarinic cholinergic receptor antagonists, respectively. The increase in [3H]inositol phosphate accumulation caused by these receptor antagonists was virtually unchanged when cultures were incubated in Ca2+-free medium, but was abolished when EGTA was also present in the Ca2+-free medium. Cultures of meningeal fibroblasts, the major cell type contaminating the astrocyte cultures, also accumulated [3H]myo-inositol, but no increased accumulation of [3H]inositol phosphates was found in response to either noradrenaline or carbachol.     

A possible mechanism for cadmium-induced hypertension in rats

The mechanism of cadmium-induced hypertension was explored by measuring noradrenaline metabolism. Cadmium $\text{in vitro}$ was shown to inhibit both monoamine oxidase and catechol-$\text{O}$-methyltransferase, the two enzymes which inactivate the neurotransmitters noradrenaline and adrenaline. However, rats which were injected or fed (via the drinking water) with cadmium showed that, among the tissues surveyed, these two enzymes were inhibited significantly only in the aorta. $\text{In vitro}$, cadmium was found to inhibit noradrenaline binding to membranes from the heart, lung, and kidney, while stimulating binding to aortic membranes, which suggests that the effects may be specific. These results suggest that, in the aorta, cadmium may inhibit the two catabolic enzymes of noradrenaline, while at the same time stimulating noradrenaline-binding. Thus the effects of noradrenaline on vascular smooth muscle would be increased as well as prolonged.     

Influence of desoxycorticosterone on the reaction of isolated segments of coronary arteries to noradrenaline in the presence of pyrogallol

The effect of the desoxycorticosterone on the noradrenaline-induced relaxation of coronary arteries waw studied in vitro, after a known inhibitor of COMT, pyrogallol. Relaxation induced by noradrenaline was enhanced by desoxycorticosterone. Relaxation in response to noradrenaline was increased by desoxycorticosterone. Pyrogallol potentiated the responses of coronary strips to noradrenaline and also reduced or abolished the enhancing effects of desoxycorticosterone. It is concluded that desoxycorticosterone enhances the reponse of coronary smooth muscle to noradrenaline by inhibiting and enzymatic pathway for the inactivation of catecolamines.     

Differential control of glycogenolysis and flow by arterial and portal acetylcholine in perfused rat liver.

The effects of acetylcholine on glucose and lactate balance and on perfusion flow were studied in isolated rat livers perfused simultaneously via the hepatic artery (100 mmHg, 25-35% of flow) and the portal vein (10 mmHg, 75-65% of flow) with a Krebs-Henseleit bicarbonate buffer containing 5 mM-glucose, 2 mM-lactate and 0.2 mM-pyruvate. Arterial acetylcholine (10 microM sinusoidal concentration) caused an increase in glucose and lactate output and a slight decrease in arterial and portal flow. These effects were accompanied by an output of noradrenaline and adrenaline into the hepatic vein. Portal acetylcholine elicited only minor increases in glucose and lactate output, a slight decrease in portal flow and a small increase in arterial flow, and no noradrenaline and adrenaline release. The metabolic and haemodynamic effects of arterial acetylcholine and the output of noradrenaline and adrenaline were strongly inhibited by the muscarinic antagonist atropine (10 microM). The acetylcholine-dependent alterations of metabolism and the output of noradrenaline were not influenced by the alpha 1-blocker prazosin (5 microM), whereas the output of adrenaline was increased. The acetylcholine-dependent metabolic alterations were not inhibited by the beta 2-antagonist butoxamine (10 microM), although the overflow of noradrenaline was nearly completely blocked and the output of adrenaline was slightly decreased. These results allow the conclusion that arterial, but not portal, acetylcholine caused sympathomimetic metabolic effects, without noradrenaline or adrenaline being involved in signal transduction.     

Changes in the development of central noradrenaline neurones following neonatal administration of 6-hydroxydopamine

Abstract— The effects of the neurotoxic compound 6-hydroxydopamine on central noradrenaline (NA) neurones have been investigated in the adult rat after systemic administration of the drug at birth. This treatment produced a permanent and selective reduction in endogenous noradrenaline, [³H]noradrenaline uptake in vitro and the number of histochemically demonstrable noradrenaline nerve terminals in the forebrain, certainly related to neuroneal degeneration. The fluorescence morphology of the noradrenaline perikarya in the locus coeruleus was not notably affected. In the pons-medulla region, the 6-hydroxydopamine treatment led to an almost two-fold increase in endogenous noradrenaline with a similar increase in [³H]noradrenaline uptake and formation of ³H-catecholamines from [³H]tyrosine. Fluorescence histochemistry revealed an increased number of noradrenaline nerve terminals which in addition showed an increased fluorescence intensity. Subcellular distribution studies of endogenous noradrenaline in pons—medulla disclosed the highest relative noradrenaline increase in the microsomal fraction after 6-hydroxydopamine at birth. Sucrose gradient centrifugations disclosed that the pons-medulla synaptosomes from 6-OH-DA treated rats sedimented at a higher sucrose concentration than those from untreated controls. It is concluded that treatment of neonate rats with 6-hydroxydopamine produces a selective degeneration of noradrenaline nerve terminals in the forebrain, especially in the cerebral cortex, whereas in the pons-medulla this treatment leads to an increased intraneuronal noradrenaline concentration due to accumulation of noradrenaline in collateral systems not affected by 6-hydroxydopamine and probably also to an increased outgrowth of noradrenaline nerve terminals.