Two new classes of conopeptides inhibit the alpha 1-adrenoceptor and noradrenaline transporter

Cone snails use venom containing a cocktail of peptides ('conopeptides') to capture their prey. Many of these peptides also target mammalian receptors, often with exquisite selectivity. Here we report the discovery of two new classes of conopeptides. One class targets alpha (1)-adrenoceptors (rho -TIA from the fish-hunting Conus tulipa), and the second class targets the neuronal noradrenaline transporter (chi -MrIA and chi -MrIB from the mollusk-hunting C. marmoreus). rho -TIA and chi -MrIA selectively modulate these important membrane-bound proteins. Both peptides act as reversible non-competitive inhibitors and provide alternative avenues for the identification of inhibitor drugs.

Amino Acids involved in differences in the pharmacological profiles of the rat and human noradrenaline transporters

It has been suggested from a previous study in our laboratory that differences in the pharmacology of the species variants of the noradrenaline transporter (NET) are the result of four non-conservative amino acid exchanges from the total of 26 amino acids that are divergent between the rat NET (rNET) and human NET (hNET). The aim of this study was to examine the effects of changing the rNET at each of these four amino acid residues, which markedly alter local charge distribution, to the amino acid found in hNET. Site-directed mutagenesis was used to create mutant cDNAs from rNET cDNA. The mutant NETs (rK71), rE62K, rK375N and rR612Q), rNET and hNET were expressed in transiently transfected COS-7 cells to determine the effects of the mutations on the differing pharmacological properties of the species variants. The ratios of V-max for noradrenaline uptake and B-max for nisoxetine binding (which are a measure of the turnover number of the transporter, i.e. the number of transport cycles per min) were greater for rNET and rR612Q than for hNET, rK71), rE62K and rK375N. The K-m of noradrenaline was lower for hNET, rK713, rE62K and rK375N than for rNET or rR612Q. There were no differences between the K-i values for inhibition of noradrenaline uptake by nisoxetine for rNET, hNET or the mutants, but the K-i values of cocaine were lower for hNET, rE62K and rR612Q than rNET or rK375N. Hence, the study showed that: (1) the aspartate 7. lysine 62 and asparagine 375 amino acid residues are important in determining the lower substrate translocation by hNET than rNET; (2) the aspartate 7 and lysine 62 residues in the N-terminus of hNET determine the higher affinities of substrates for the hNET than the rNET; and (3) the lysine 62 and glutamine 612 residues in the N- and C-termini, respectively, of hNET Lire determinants of the higher cocaine affinity for the hNET than rNET.

Functional significance of a highly conserved glutamate residue of the human noradrenaline transportere

The aim of the study was to investigate the role of glutamate residue 113 in transmembrane domain 2 of the human noradrenaline transporter in determining cell surface expression and functional activity. This residue is absolutely conserved in all members of the Na+- and Cl--dependent transporter family. Mutations to alanine (hE113A), aspartate (hE113D) and glutamine (hE113Q) were achieved by site-directed mutagenesis and the mutants were expressed in transfected COS-7 or HEK-293 cells. Cell surface expression of IIE113A and hE113D, but not hE113Q, was markedly reduced compared with wild type, and functional noradrenaline uptake was detected only for the hE113Q mutant. The pharmacological properties of the hE113Q mutant showed very little change compared with wild type, except for a decrease in V-max values for noradrenaline and dopamine uptake of 2-3-fold. However, the hE113D mutant showed very marked changes in its properties, compared with wild type, with 82-260-fold decreases in the affinities of the substrates, noradrenaline, dopamine and MPP+, and increased Na+ affinity for stimulation of nisoxetine binding. The results of the study show that the size and not the charge of the 113 glutamate residue of the noradrenaline transporter seems to be the most critical factor for maintenance of transporter function and surface expression.

Changes in the vascular reactivity of the isolated tail arteries of spontaneous and renovascular hypertensive rats to endogenous and exogenous noradrenaline.

We have investigated the changes in the responses to noradrenaline of isolated tail arteries of spontaneously hypertensive (SHR) and renovascular hypertensive rats (Wistar-Kyoto: two-kidney, one-clip model, WKY:2K1C) compared with normotensive (Wistar-Kyoto, WKY) rats. Renovascular hypertension was induced by 4 weeks' unilateral renal artery clipping. Arteries were vasoconstricted with exogenous noradrenaline, electrical field stimulation or high potassium. The effects of the latter two stimuli were abolished by reserpine and so were presumably dependent on the presence of endogenous noradrenaline. In the SHR the maximal vasoconstriction produced by all three stimuli was greater than in WKY. Dose-response curves were steeper and there was no change in threshold. Vascular mass was greater. We interpret these results as showing an increase in vascular reactivity in the SHR caused by structural adaptation. The WKY:2K1C responses to noradrenaline could also be explained in terms of structural adaptation but there was no increase in vascular mass. Sensitivity to potassium and electrical stimulation was decreased, suggesting a defect in vascular neurotransmission. This was supported by the observations of a decreased arterial noradrenaline content and of decreased sensitivity to cocaine.

Vasoactive intestinal peptide, pituitary adenylate cyclase-activating peptide, and noradrenaline induce the transcription factors CCAAT/enhancer binding protein (C/EBP)-beta and C/EBP delta in mouse cortical astrocytes: involvement in cAMP-regulated glycogen metabolism.

We have described previously a transcription-dependent induction of glycogen resynthesis by the vasoactive intestinal peptide (VIP) or noradrenaline (NA) in astrocytes, which is mediated by cAMP. Because it has been postulated that the cAMP-mediated regulation of energy balance in hepatocytes and adipocytes is channeled at least in part through the CCAAT/enhancer binding protein (C/EBP) family of transcription factors, we tested the hypothesis that C/EBP isoforms could be expressed in mouse cortical astrocytes and that their level of expression could be regulated by VIP, by the VIP-related neuropeptide pituitary adenylate cyclase-activating peptide (PACAP), or by NA. We report in this study that in these cells, C/EBP beta and C/EBP delta are induced by VIP, PACAP, or NA via the cAMP second-messenger pathway. Induction of C/EBP beta and -delta mRNA by VIP occurs in the presence of a protein synthesis inhibitor. Thus, c/ebp beta and c/ebp delta behave as cAMP-inducible immediate-early genes in astrocytes. Moreover, transfection of astrocytes with expression vectors selectively producing the transcriptionally active form of C/EBP beta, termed liver-enriched transcriptional activator protein, or C/EBP delta enhance the glycogen resynthesis elicited by NA, whereas an expression vector producing the transcriptionally inactive form of C/EBP beta, termed liver-enriched transcriptional inhibitory protein, reduces this resynthesis. These results support the idea that C/EBP beta and -delta regulate gene expression of energy metabolism-related enzymes in astrocytes.

3-Hydroxybutyrate coinfused with noradrenaline decreases resulting plasma levels of noradrenaline in Wistar rats

Pentobarbital-anaesthetized male Wistar rats were infused with 6microgkg-1min-1 of noradrenaline. The infusion was supplemented with 8.5 mgkg-1min-1 of D-3-hydroxybutyrate (3-OHB) for 15 min in order to determine its effect on the adrenergic response of the rat. Plasma levels of noradrenaline rose to a plateau of approximately 50 nmoll-1 with infusion. In the group infused with noradrenaline alone, noradrenaline levels were maintained for 1h. Supplementation with 3-OHB induced a decrease in plasma noradrenaline level that was inversely correlated with 3-OHB level. Aortic and interscapular brown adipose tissue temperatures increased with noradrenaline infusion, but the rise was arrested by 3-OHB; replacing 3-OHB with glucose had no effect. Infusion of saline, glucose or 3-OHB in the absence of noradrenaline did not induce a rise in temperature in either tissue. Blood 3-OHB concentration increased to 1.2 mmoll-1 during 3-OHB infusion, decreasing rapidly at the end of infusion. Blood glucose levels increased with noradrenaline infusion; the presence of high 3-OHB levels decreased glucose concentration. The effects observed were transient and dependent on 3-OHB concentration; these effects may help explain most of the other effects of noradrenaline described here. The role of 3-OHB as a regulator of adrenergic responses seems to be part of a complex fail-safe mechanism which prevents wasting.

High concentrations of KCl release noradrenaline from noradrenergic neurons in the rat anococcygeus muscle

The aim of the present study was to investigate the effects of high concentrations of KCl in releasing noradrenaline from sympathetic nerves and its actions on postsynaptic alpha-adrenoceptors. We measured the isotonic contractions induced by KCl in the isolated rat anococcygeus muscle under different experimental conditions. The contractile responses induced by KCl were inhibited by alpha-adrenoceptor antagonists in 2.5 mM Ca2+ solution. Prazosin reduced the maximum effect from 100 to 53.9 ± 10.2% (P<0.05) while the pD2 values were not changed. The contractile responses induced by KCl were abolished by prazosin in Ca2+-free solution (P<0.05). Treatment of the rats with reserpine reduced the maximum effect induced by KCl as compared to the contractile responses induced by acetylcholine from 339.5 ± 157.8 to 167.3 ± 65.5% (P<0.05), and increased the pD2 from 1.57 ± 0.01 to 1.65 ± 0.006 (P<0.05), but abolished the inhibitory effect of prazosin (P<0.05). In contrast, L-NAME increased the contractile responses induced by 120 mM KCl by 6.2 ± 2.3% (P<0.05), indicating that KCl could stimulate the neurons that release nitric oxide, an inhibitory component of the contractile response induced by KCl. Our results indicate that high concentrations of KCl induce the release of noradrenaline from noradrenergic neurons, which interacts with alpha1-adrenoceptors in smooth muscle cells, producing a contractile response in 2.5 mM Ca2+ (100%) and in Ca2+-free solution, part of which is due to a direct effect of KCl on the rat anococcygeus muscle.

Loss of noradrenaline transporter sites in frontal cortex of rats with acute (ischemic) liver failure.

There is increasing evidence that central noradrenaline (NA) transport mechanisms are implicated in the central nervous system complications of acute liver failure. In order to assess this possibility, binding sites for the high affinity NA transporter ligand [3H]-nisoxetine were measured by quantitative receptor autoradiography in the brains of rats with acute liver failure resulting from hepatic devascularization and in appropriate controls. In vivo microdialysis was used to measure extracellular brain concentrations of NA. Severe encephalopathy resulted in a significant loss of [3H]-nisoxetine sites in frontal cortex and a concomitant increase in extracellular brain concentrations of NA in rats with acute liver failure. A loss of transporter sites was also observed in thalamus of rats with acute liver failure. This loss of NA transporter sites could result from depletion of central NA stores due to a reserpine-like effect of ammonia which is known to accumulate to millimolar concentrations in brain in ischemic liver failure. Impaired NA transport and the consequent increase in synaptic concentrations and increased stimulation of neuronal and astrocytic noradrenergic receptors could be implicated in the pathogenesis of the encephalopathy and brain edema characteristic of acute liver failure.

Antidepressant treatment and emotional processing: can we dissociate the roles of serotonin and noradrenaline?

The ability to match individual patients to tailored treatments has the potential to greatly improve outcomes for individuals suffering from major depression. In particular, while the vast majority of antidepressant treatments affect either serotonin or noradrenaline or a combination of these two neurotransmitters, it is not known whether there are particular patients or symptom profiles which respond preferentially to the potentiation of serotonin over noradrenaline or vice versa. Experimental medicine models suggest that the primary mode of action of these treatments may be to remediate negative biases in emotional processing. Such models may provide a useful framework for interrogating the specific actions of antidepressants. Here, we therefore review evidence from studies examining the effects of drugs which potentiate serotonin, noradrenaline or a combination of both neurotransmitters on emotional processing. These results suggest that antidepressants targeting serotonin and noradrenaline may have some specific actions on emotion and reward processing which could be used to improve tailoring of treatment or to understand the effects of dual-reuptake inhibition. Specifically, serotonin may be particularly important in alleviating distress symptoms, while noradrenaline may be especially relevant to anhedonia. The data reviewed here also suggest that noradrenergic-based treatments may have earlier effects on emotional memory that those which affect serotonin.

Cardiovascular effects of noradrenaline microinjected into the insular cortex of unanesthetized rats

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Inhibition of pilocarpine-induced salivation in rats by central noradrenaline

Peripheral treatment with cholinergic or adrenergic agonists results in salivation and the possibility of synergy between cholinergic and adrenergic efferent mechanisms in the control of salivation has been proposed. Central injections of the cholinergic agonist pilocarpine also induce salivation, while the effects of central injections of noradrenaline (norepinephrine) are not known. Here (a) the effects of intracerebroventricular (icv) injection of noradrenaline on the salivation induced by icv or intraperitoneal (i.p.) injection of pilocarpine and (b) the receptors involved in the effects of central noradrenaline on pilocarpine-induced salivation were investigated. Male Holtzman rats with a stainless-steel guide cannula implanted into the lateral ventricle were used. Rats were anaesthetized with tribromoethanol (200 mg/kg body weight) and saliva was collected on small, preweighed cotton balls inserted into the animal's mouth. Noradrenaline (40, 80 and 160 nmol/l mul) injected icv reduced the salivary secretion induced by pilocarpine (0.5 mumol/l mul) injected icv. Noradrenaline (80 and 160 nmol/l mul) injected icv also reduced the salivation induced by pilocarpine (4 mumol/kg) injected i.p. Previous treatment with the alpha(2)-adrenergic receptor antagonists RX 821002 (40, 80 and 160 nmol/l mul) or yohimbine (160 and 320 nmol/l mul) abolished the inhibitory effect produced by icv injection of noradrenaline on pilocarpine-induced salivation in rats. Prazosin (alpha(1)-adrenergic receptor antagonist) injected icv did not change the effect of noradrenaline on pilocarpine-induced salivation. Prior icv injection of only RX 821002 (80 or 160 nmol/l mul) or yohimbine (320 nmol/l mul) increased pilocarpine-induced salivation. The results show that (1) contrary to its peripheral effects, noradrenaline acting centrally inhibits cholinergic-induced salivation in rats; (2) central mechanisms involving alpha(2)-adrenergic receptors inhibit pilocarpine-induced salivation. (C) 2002 Elsevier B.V. Ltd. All rights reserved.

Noradrenaline and mixed alpha(2)-adrenoceptor/imidazoline-receptor ligands: effects on sodium intake

The effect of noradrenaline, and mixed ligands to alpha(2)-adrenoceptors (alpha(2)-AR) and imidazoline receptors (IR), injected intracerebroventricularly (i.c.v.), on sodium intake of sodium depleted rats, was tested against idazoxan, a mixed antagonist ligand to alpha(2)-AR and IR. The inhibition of sodium intake induced by noradrenaline (80 nmol) was completely reversed by idazoxan (160 and 320 nmol) injected i.c.v. The inhibition of sodium intake induced by mixed ligands to alpha(2)-AR and IR, UK14,304, guanabenz and moxonidine, was antagonized from 50 to 60% by idazoxan i.c.v. The results demonstrate that noradrenaline, a non-ligand for IR, acts on alpha(2)-AR inhibiting sodium intake. The possibility that either alpha(2)-AR or IR mediate the effect of mixed agonists on sodium intake remains an open question. (C) 1999 Elsevier B.V. B.V. All rights reserved.