Effects of S-11701 on accumulation, release and metabolism of norepinephrine in isolated canine saphenous veins.

1. The effects S-11701 ([morpholinyl-2)-methoxy]-8-tetrahydro-1,2,3,4 quinoline) on accumulation, overflow and metabolism of [3H]norepinephrine were investigated in isolated canine saphenous veins. 2. Saphenous veins were incubated with [3H]norepinephrine in the absence or the presence of S-11701; the drug caused a concentration-dependent inhibition of the tissue content of [3H]norepinephrine and its metabolites, except for 3-methoxy-4-hydroxymandelic acid (VMA). 3. In helical strips of canine saphenous veins previously incubated with [3H]norepinephrine and then suspended for isometric tension recording and measurement of the overflow of labelled transmitter and its metabolites, S-11701 (30 microM) significantly increased the spontaneous efflux of total 3H; this effect was almost exclusively due to an augmentation of the efflux of [3H]DOPEG. 4. During electrical stimulation (9 V, 1 Hz), S-11701 at 1 microM slightly increased the overflow of extraneuronal norepinephrine metabolites without affecting the contractile response. At the higher concentration (30 microM) the compound increased the contractive response and the overflow of 3H; the latter was due mainly to an increase in [3H]DOPEG and, to a lesser extent, in [3H]norepinephrine. 5. DMI (1 microM) did not interfere with the effects of S-11701 on DOPEG efflux. 6. These experiments indicate that in the canine saphenous vein, S-11701 causes a concentration-dependent inhibition of neuronal accumulation of [3H]norepinephrine. At higher concentrations, S-11701 enters the adrenergic nerve terminals independently of the neuronal amine carrier and displaces [3H]norepinephrine from its storage sites.     

The effects of soman on norepinephrine uptake, release, and metabolism

The effects of 7-day administration of 5 micrograms/kg/day of soman sc on norepinephrine (NE) content, and catechol-O-methyl-transferase (COMT) and monoamine oxidase (MAO) activities of three rabbit tissues were examined. Effects on NE uptake by, and electrically stimulated release from, rabbit aorta were also determined, both in the 7-day study and with acutely applied soman. Tissues examined were the thoracic aorta, mesenteric artery, and brain stem. Significant (p less than 0.05) increases following 7 days of soman were observed in NE content: thoracic aorta, 20%; mesenteric artery, 48%; and brain stem, 121%. MAO activity decreased by 29% in the thoracic aorta, 20% in the mesenteric artery, and 48% in the brain stem. COMT activity also significantly decreased in two tissues, the thoracic aorta by 18% and brain stem by 23%. Acutely applied soman reduced electrically released NE from the thoracic aorta, but 7-day soman administration increased it. Seven-day soman administration, but not acutely applied soman increased NE uptake by the thoracic aorta. Thus, 7-day soman administration increased sympathetic capability by increasing NE content, nerve stimulation evoked NE release, and NE uptake, and by decreasing MAO and COMT activity. Therefore, repeated low-dose exposure to soman might exaggerate the response to any given level of sympathetic nerve stimulation and to NE-releasing agents.     

Veratridine-induced phosphorylation and activation of tyrosine hydroxylase,and synthesis of catecholamines in cultured bovine adrenal medullary cells

Summary The mechanism of the synthesis of catecholamines by veratridine was studied in cultured bovine adrenal medullary cells. (1) Veratridine increased the phosphorylation and activity of tyrosine hydroxylase as well as the synthesis of [¹⁴C]catecholamines from [¹⁴C]tyrosine, all of which were inhibited by tetrodotoxin. Veratridine-induced activation of tyrosine hydroxylase and synthesis of [¹⁴C]catecholamines were reduced in 20 mmol/l extracellular Na⁺ or in Ca²⁺-free medium. (2) 12-O-Tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C, increased the synthesis of [¹⁴C]catecholamines. In the presence of TPA, veratridine did not produce any additional increase in [¹⁴C]catecholamine synthesis. In protein kinase C-deficient cells which were prepared by pretreatment with 1 ol/1 TPA for 24 h, TPA failed to increase [¹⁴C]catecholamine synthesis and veratridine-induced [¹⁴C]catecholamine synthesis was suppressed by 50%. (3) Polymyxin B, an inhibitor of protein kinase C and N-(6-aminohexyl)-5-chloro-l-naphthalenesulfonamide (W-7), an inhibitor of calmodulin, inhibited veratridine-stimulated synthesis of [¹⁴C]catecholamines as well as veratridine-induced influx of ²²Na⁺ and ⁴⁵Ca²⁺ with similar potencies. (4) In digitonin-permeabilized cells, polymyxin B attenuated the activation of tyrosine hydroxylase caused by Ca²⁺. These results suggest that veratridine-induced synthesis of catecholamines and activation of tyrosine hydroxylase were mediated by Ca²⁺-dependent phosphorylation of this enzyme, and protein kinase C may be responsible, at least in part, for this process. Send offprint requests to Y. Uezono at the above address     

Protein kinase A and nicotinic activation of bovine adrenal tyrosine hydroxylase.

1. Stimulation of nicotinic cholinoceptors on bovine chromaffin cells increases phosphorylation of three serine residues in tyrosine hydroxylase (TOH) and activates TOH. One of the serines is a target for protein kinase A phosphorylation, and phosphorylation of this serine is adequate alone to cause TOH activation. The role of protein kinase A in nicotinic activation of TOH was therefore investigated. 2. TOH activity was studied in situ in intact, cultured, bovine adrenal chromaffin cells, by measuring 14CO2 evolved following the hydroxylation and rapid decarboxylation of [14C]-tyrosine offered to the cells. 3. Nicotine (5 microM), forskolin (1 microM) and 8-bromo-cyclic AMP (8-Br-cyclic AMP, 1 mM) each increased TOH activity by up to 200% over 10 min. The effect of nicotine was completely abolished by removal of extracellular Ca2+. 4. TOH activation by all three drugs was blocked by H89 (3-20 microM), which inhibits protein kinase A by competing for the ATP binding site on the kinase. Adenosine 3':5'-cyclic monophosphorothioate Rp-diastereomer (Rp-cAMPS) (1 mM), an inhibitor of protein kinase A that competes with cyclic AMP for the regulatory subunit of the kinase, abolished the activation of TOH by nicotine, and reduced that by forskolin and 8-Br-cyclic AMP. Both H89 and Rp-cAMPS inhibited basal TOH activity by 50-80%. 5. A structural analogue of H89, H85 (3-20 microM), which lacks activity as a protein kinase A inhibitor, did not inhibit either the activation of TOH by nicotine (5 microM) or basal TOH activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of female sex hormones on histamine metabolism in guinea-pigs.

Determination of the urinary excretion of histamine and methylhistamine was performed in intact and gonadectomized male and female guinea-pigs. The excretion of methylhistamine varied more than that of histamine both in the same animal and between different animals. The urinary excretion of histamine and methylhistamine reflects fairly well the total histamine turnover in the animal, provided that aminoguanidine is administered to inhibit diamine oxidase activity. There was no sex difference in the excretion of histamine and methylhistamine and gonadectomy did not influence the urinary output. Female sex steroids had little ifany effect upon histamine turnover in the guinea-pig which is in contrast to the situation in rats and mice.     

Phospholipid-induced activation of tyrosine hydroxylase from rat brain striatal synaptosomes.

Lysolecithin and phosphatidylserine stimulate rat striatal tyrosine hydroxylase, partially purified from the crude synaptosomal fraction. The stimulatory effect is associated with a 3- to 4-fold decrease in K_m for 6-methyl-tetrahydropterin or tetrahydrobiopterin without an alteration in the K_m for l-tyrosine or in the V_max. In addition, the K_i for dopamine inhibition is increased approximately 3-fold. Centrifugation of the enzyme on linear sucrose gradients gave a sedimentation coefficient (S_{20, w}) of 8.6, either in the absence or presence of lysolecithin, indicating that no significant changes in the molecular weight are caused by the phospholipid. The enzyme obtained after high speed centrifugation of whole striatal tissue was activated by a combination of lysolecithin plus a cyclic AMP-ATP mixture to a greater extent than that obtained by either activating condition alone. The data presented suggest a potential regulatory function of phospholipids in the control of striatal neurotransmitter synthesis.     

Acute effects of morphine on dopamine synthesis and release and tyrosine metabolism in the rat striatum

The effect of the acute administration of morphine (60 mg/kg) on the metabolism of dopamine (DA) and tyrosine in the rat striatum were investigated using in vivo and in vitro methods. 30 min after morphine injection, the initial accumulation of ³H-DA in tissues seen after the i.v. injection of L-3,5-H-tyrosine was enhanced as well as the accumulation of ³HH₂O and ³H-DA in tissues and medium of striatal slices incubated with the ³H precursor. These results and the estimation of the conversion index of tyrosine into DA (³H-DA/tyrosine specific activity) indicated that morphine stimulated DA synthesis. This effect was not seen 2 hr later. The enhanced DA synthesis was associated with a parallel increase in the release of newly synthesized ³H-DA as indicated by the greater accumulation of ³H-DA in incubating medium of slices of morphine-pretreated rats. Therefore the increased endogenous levels of DA seen after morphine are related to the increased synthesis of the transmitter. Apart from its effect on dopaminergic neuron activity, morphine also induced marked changes in tyrosine metabolism, particularly at 2 or 2.5 hr after its injection. Tyrosine levels in plasma and in striatum were about 160% of the control levels at this time: these changes in the size of the precursor pool may explain the enhanced accumulation of ³H-tyrosine seen in plasma and striatum 10 min after the ³H-amino acid injection. Marked changes in endogenous levels of DA and tyrosine occurred in the striatum of control animals from 9.00 to 12.00 am. Acute treatment with morphine significantly affected these diurnal variations.     

Effects of beta-endorphin on norepinephrine release in hypertension

Recent studies have suggested an involvement of the endogenous opioid system in blood pressure control. The purpose of the present study was to determine the role of beta-endorphin in the regulation of sympathetic nervous activity in the central nervous system of hypertension. The effects of beta-endorphin on the electrically evoked release of [3H]norepinephrine (NE) were investigated in superfused slices of rat medulla oblongata. Beta-endorphin inhibited the stimulation-evoked NE release in a dose-dependent manner in rat medulla oblongata. In the medulla oblongata of spontaneously hypertensive rats (SHR), the inhibitory effect of beta-endorphin on the stimulation-evoked NE release was significantly smaller than in the medulla oblongata of Wistar-Kyoto rats. These results showed that beta-endorphin might reduce NE release in rat medulla oblongata. Furthermore, the lesser inhibitory effect of beta-endorphin on NE release in SHR might suggest that the opioid peptide could be involved in the regulation of central sympathetic nervous activity in hypertension.     

Effects of nitroglycerin ointment on plasma norepinephrine and cyclic nucleotides in congestive heart failure

To assess the effects of nitroglycerin ointment (NTG) on hemodynamics and autonomic nervous activity, 17 normal subjects and 13 patients with severe congestive heart failure (CHF) were studied. In 12 normal subjects, NTG significantly increased the plasma norepinephrine concentration in association with a slight reduction in systolic blood pressure and a slight increase in heart rate, plasma cyclic adenosine monophosphate (cyclic AMP) concentration, and renin activity at 1 hr. All normal subjects complained of headache or felt heavy-headed after NTG administration. In the 13 patients with CHF, NTG significantly decreased plasma norepinephrine and cyclic AMP concentrations in association with a significant increase in the cardiac index and a significant reduction in pulmonary arterial diastolic pressure, systemic vascular resistance, systolic blood pressure, and heart rate. The effects occurred at 30 min after NTG administration and continued for 3 hr. Relief from dyspnea or orthopnea in patients with CHF was observed. NTG did not change the plasma cyclic GMP concentration in normal subjects and patients with CHF. We conclude that in patients with CHF, NTG decreases the enhanced sympathetic nervous activity, with concomitant beneficial effects on hemodynamics and improvement of clinical symptoms. In contrast, NTG increases sympathetic nervous activity in normal subjects.     

Chronic desipramine treatment alters tyrosine hydroxylase but not norepinephrine transporter immunoreactivity in norepinephrine axons in the rat prefrontal cortex

Pharmacological blockade of norepinephrine (NE) reuptake is clinically effective in treating several mental disorders. Drugs that bind to the NE transporter (NET) alter both protein levels and activity of NET and also the catecholamine synthetic enzyme tyrosine hydroxylase (TH). We examined the rat prefrontal cortex (PFC) by electron microscopy to determine whether the density and subcellular distribution of immunolabelling for NET and co-localization of NET with TH within individual NE axons were altered by chronic treatment with the selective NE uptake inhibitor desipramine (DMI). Following DMI treatment (21 d, 15 mg/kg.d), NET-immunoreactive (ir) axons were significantly less likely to co-localize TH. This finding is consistent with reports of reduced TH levels and activity in the locus coeruleus after chronic DMI and indicates a reduction of NE synthetic capacity in the PFC. Measures of NET expression and membrane localization, including the number of NET-ir profiles per tissue area sampled, the number of gold particles per NET-ir profile area, and the proportion of gold particles associated with the plasma membrane, were similar in DMI- and vehicle-treated rats. These findings were verified using two different antibodies directed against distinct epitopes of the NET protein. The results suggest that chronic DMI treatment does not reduce NET expression within individual NE axons in vivo or induce an overall translocation of NET protein away from the plasma membrane in the PFC as measured by ultrastructural immunogold labelling. Our findings encourage consideration of possible post-translational mechanisms for regulating NET activity in antidepressant-induced modulation of NE clearance.     

GABAB receptor activation partially inhibits N-methyl-D-aspartate-mediated tyrosine hydroxylase stimulation in rat striatal slices.

The effect of the GABAB agonist, (+/-)-baclofen, on the N-methyl-D-aspartate (NMDA)-mediated stimulation of tyrosine hydroxylase activity was investigated in slices of rat striatum. Tyrosine hydroxylase activity was stimulated by NMDA in a concentration-dependent manner (EC50, 1.3 +/- 0.3 microM; maximum stimulation 194 +/- 7% of basal activity). The action of NMDA was reversed by the NMDA antagonist, 2-amino-5-phosphonovalerate (AP-5). (+/-)-Baclofen (100 microM) decreased the maximum effect of NMDA by 24 +/- 2% without significantly modifying its EC50. The IC50 for the inhibitory action of (+/-)-baclofen was 4.2 +/- 1.2 microM. These results show that GABAB receptor activation modulates NMDA-stimulated tyrosine hydroxylase activity, further supporting the possibility of a role of GABA in the regulation of striatal dopaminergic neurotransmission.     

Influence of dopamine synthesis on methamphetamine-induced changes in striatal and adrenal tyrosine hydroxylase activity

Summary Methamphetamine in large doses decreases striatal tyrosine hydroxylase activity. This effect is prevented by neuroleptic agents such as chlorpromazine and haloperidol which would suggest that released dopamine may be involved in the response. To test this hypothesis, we have altered dopamine synthesis with -methyl-p-tyrosine and l-Dopa and found that dopamine synthesis is necessary for the observed depression of striatal TH activity by methamphetamine. In the adrenal gland, however, the increase in TH activity by methamphetamine is not prevented by inhibition of catecholamine synthesis. It is possible that released dopamine may be inhibiting TH activity by activation of pre- or postsynaptic dopamine receptors in the neostriatum resulting in activation of the neuronal feedback pathway or released dopamine may act on dendrodendritic autoreceptors in the substantia nigra.