Morphine decreases 3H-norepinephrine release and increases endogenous norepinephrine levels in the isolated cat superior cervical ganglion

The isolated cat superior cervical ganglion (SCG) was labeled in vitro with either 3H-norepinephrine (3H-NE) or 3H-choline and stimulated through its preganglionic trunk. The release of 3H-NE and 3H-acetylcholine (3H-ACh) elicited by the stimulation was measured under control conditions and in the presence of drugs. The incubation during 30 min with 10 microM morphine lead to a 70% decrease in the amount of 3H-NE released in response to the preganglionic stimulation (10 Hz, 80 V, during 5 min). No further decrease in 3H-NE release was produced by a 10 times higher concentration of morphine. The reduction in 3H-NE release caused by morphine was coincident with a 60% increase in the endogenous content of NE. Both effects of morphine were entirely prevented by an antagonist of opioid receptors, 1.0 microM naltrexone. The opioid antagonist did not modify by itself either the stimulation-induced release of 3H-NE or the endogenous content of NE. The basal efflux of 3H-NE was not altered by morphine. In ganglia labeled with 3H-choline, morphine (10 and 100 microM) did not modify either the basal efflux of 3H-ACh or the release of 3H-ACh evoked by stimulation of the preganglionic trunk (5 Hz, 40 V, during 5 min). These observations suggest that in the cat SCG morphine has a direct action on the dendrites of the postganglionic neuron which store and release NE. The effects of morphine in vitro on 3H-NE release and on the tissue levels of NE may be mediated through the interaction with dendritic opioid receptors.     

Plasma catecholamine concentrations in normotensive rats of different strains and in spontaneously hypertensive rats under basal conditions and during cold exposure

Under basal conditions, the levels of circulating norepinephrine (NE) and epinephrine (E) were higher in normotensive Wistar rats of different origins than in Sprague-Dawley rats. Since the decline of ³H-NE concentration in the plasma after i.v. injection was similar in Wistar and in Sprague-Dawley rats, the higher levels of endogenous NE in the former strain probably reflect greater NE release from sympathetic nerve terminals. In normotensive Sprague-Dawley and Wistar rats, plasma NE rose to various extents during cold exposure (4°C), depending on the basal plasma NE levels. Compared with normotensive Wistar Kyoto rats (WKY), spontaneously hypertensive rats (SHR) had similar basal plasma E and NE concentrations, similar rates of ³H-NE disappearance, but more rapid increases to higher values of plasma NE during cold exposure. It is concluded that the basal rate of peripheral catecholamine release does not seem to be the main determining factor for arterial blood pressure in the various rat strains and that the sympathetic neuronal system of SHR is more responsive to cold exposure than that of WKY rats.     

Regulation of catecholamine release in human adrenal chromaffin cells by β-adrenoceptors

The adrenal gland plays a fundamental role in the response to a variety of stress situations. After a stress condition, adrenal medullary chromaffin cells release, by exocytosis, high quantities of catecholamine (epinephrine, EP; norepinephrine, NE), especially EP. Once in the blood stream, catecholamines reach different target organs, and induce their biological actions through the activation of different adrenoceptors. Adrenal gland cells may also be activated by catecholamines, through hormonal, paracrine and/or autocrine system. The presence of functional adrenoceptors on human adrenal medulla and their involvement on catecholamines secretion was not previously evaluated. In the present study we investigated the role of β(1)-, β(2)- and β(3)-adrenoceptors on catecholamine release from human adrenal chromaffin cells in culture. We observed that the β-adrenoceptor agonist (isoproterenol) and β(2)-adrenoceptor agonist (salbutamol) stimulated catecholamine (NE and EP) release from human adrenal chromaffin cells. Furthermore, the β(2)-adrenoceptor antagonist (ICI 118,551; 100 nM) and β(3)-adrenoceptor antagonist (SR 59230A; 100 nM) inhibited the catecholamine release stimulated by isoproterenol and nicotine in chromaffin cells. The β(1)-adrenoceptor antagonist (atenolol; 100 nM) did not change the isoproterenol- neither the nicotine-evoked catecholamine release from human adrenal chromaffin cells. Moreover, our results show that the protein kinase A (PKA), protein kinase C (PKC), mitogen-activated protein kinase (MAPK) and phospholipase C (PLC) are intracellular mechanisms involved in the catecholamine release evoked by salbutamol. In conclusion, our data suggest that the activation of β(2)- and β(3)-adrenoceptors modulate the basal and evoked catecholamine release, NE and EP, via an autocrine positive feedback loop in human adrenal chromaffin cells.     

The renin-angiotensin system and noradrenaline release in the hypothalamus and medulla oblongata

The effect of angiotensin II (AII) and 48 h bilateral nephrectomy on the 3H-norepinephrine (3H-NE) and 3H-NE metabolites release in vitro was studied in slices of male Wistar rat hypothalamus and medulla oblongata. The total 3H outflow of radioactivity was higher in AII exposed tissues than in nephrectomized ones of both organs. The 3H-NE and 3H-NE metabolites remanent radioactivity in the tissues increased in both the soluble cytoplasmatic fractions and the granular vesicle ones, in the two organs from the nephrectomized rats. The ratio between granular and cytoplasmatic NE and granular and cytoplasmatic radioactive metabolites was not noticeably altered in any of the groups. The release of 3H-NE caused by AII and the opposite effect by nephrectomy, agree with the inverse relationship demonstrated between endogenous NE content in the central nervous system and AII plasmatic levels. AII might act on presynaptic NE receptors of the cellular membrane. The relationship between the renin-AII system and the central nervous system catecholamines could be involved in the control of development and maintenance of the renal arterial hypertension.     

Modulation of catecholamine release by endogenous adenosine in the rat adrenal medulla

Adenosine was shown to inhibit norepinephrine (NE) release from sympathetic nerve endings. The purpose of this study was to examine whether endogenous adenosine restrains NE and epinephrine release from the adrenal medulla. The effects of an adenosine receptor antagonist, 1,3-dipropyl-8-(p-sulfophenyl) xanthine (DPSPX), on epinephrine and NE release induced by intravenous administration of insulin in conscious rats were examined. Plasma catecholamines were measured by HPLC with an electrochemical detector. DPSPX significantly increased plasma catecholamine in both control rats and rats treated with insulin. The effect of DPSPX on plasma catecholamine was significantly greater in rats treated with insulin. Additional experiments were performed in adrenalectomized rats to investigate the contribution of the adrenal medulla to the effect of DPSPX on plasma catecholamine. The effect of DPSPX and insulin on epinephrine in adrenalectomized rats was significantly reduced compared with that of the controls. Finally, we tested whether endogenous adenosine restrains catecholamine secretion partially through inhibiting the renin-angiotensin system. The effect of DPSPX on plasma catecholamine in rats pretreated with captopril (an angiotensin-converting enzyme inhibitor) was reduced. These results demonstrate that under basal physiological conditions, endogenous adenosine tonically inhibits catecholamine secretion from the adrenal medulla, and this effect is augmented when the sympathetic system is stimulated. The effect of endogenous adenosine on catecholamine secretion from the adrenal medulla is achieved partially through the inhibitory effect of adenosine on the renin-angiotensin system.     

Cell Cycle-Dependent Modulation of Biosynthesis and Stimulus-Evoked Release of Catecholamines in PC12 Pheochromocytoma Cells

Catecholamine biosynthesis and its stimulus-evoked release in PC12 pheochromocytoma cells were studied as a function of cell cycle by means of HPLC with electrochemical detection. We found that 3,4-dihydroxyphenylethylamine (dopamine) levels in PC12 cells remained constant throughout the period of cell cycle. In contrast, the noradrenaline content was dependent on the cell cycle: it increased during the S + G2 phase followed by a decrease in the M phase. These results were confirmed further by measuring the activities catalyzing the catecholamine biosynthesis. Thus, activities of tyrosine 3-monooxygenase and 3,4-dihydroxyphenylalanine decarboxylase were independent of the cell cycle, whereas both soluble and membrane-bound dopamine beta-monooxygenase activities were modulated during the cell cycle. On the other hand, release of the catecholamines stimulated with 50 mM KCl increased in the G1 phase, reached a maximum in the late G1, and then gradually decreased in later periods. We also found that carbamylcholine-induced release of the catecholamines occurred maximally in the early S + G2 phase followed by a decrease during the M phase. Cell cycle dependence of the catecholamine release was in good agreement with that of 45Ca2+ uptake. Thus, this study provides evidence that the catecholamine biosynthesis and its release in PC12 cells are modulated during the period of cell cycle.     

Chronic depolarization stimulates norepinephrine transporter expression via catecholamines

Chronic depolarization increases norepinephrine (NE) uptake and expression of the norepinephrine transporter (NET) in sympathetic neurons, but the mechanisms are unknown. Depolarization of sympathetic neurons stimulates catecholamine synthesis, and several studies suggest that NET can be regulated by catecholamines. It is not clear if the depolarization-induced increase in NET is because of nerve activity per se, or is secondary to elevated catecholamines. To determine if induction of NET mRNA was a result of increased catecholamines, we used pharmacological manipulations to (i) inhibit tyrosine hydroxylase activity in neurons depolarized with 30 mm KCl, thereby preventing increased catecholamines, or (ii) stimulate tyrosine hydroxylase activity in the absence of depolarization. Inhibiting the depolarization-induced increase in catecholamines prevented the up-regulation of NET mRNA, but did not block the increase in tyrosine hydroxylase (TH) mRNA. Furthermore, stimulating catecholamine production in the absence of depolarization elevated NE uptake, NET protein, and NET mRNA in sympathetic neurons. Similarly, elevating endogenous catecholamines in SK-N-BE2M17 neuroblastoma cells increased NE uptake and NET expression. These data suggest that chronic depolarization of sympathetic neurons induces NET expression through increasing catecholamines, and that M17 neuroblastoma cells provide a model system in which to investigate catechol regulation of NET expression.     

Angiotensin II Increases Catecholamine Release from Bovine Adrenal Medulla but Does Not Enhance That Evoked by K+ Depolarization or by Carbachol

The effect of angiotensin II on catecholamine release from bovine adrenal medulla has been investigated. In retrogradely perfused, isolated bovine adrenal glands, angiotensin II increased basal efflux of catecholamines, but the presence of angiotensin II did not increase the release of catecholamines evoked either by bolus injections of the secretagogue carbachol or by depolarization with a perfusing solution containing a raised concentration of K+. In chromaffin cells maintained in primary tissue culture, angiotensin II increased 3H release from cells preloaded with [3H]-noradrenaline but did not enhance the release evoked by carbachol or by depolarization with K+. The increase in 3H release evoked by angiotensin II from chromaffin cells in tissue culture was inhibited by its analogue antagonist Sar1,Ala8-angiotensin II (saralasin) and was entirely dependent on the presence of Ca2+ in the experimental medium. These findings suggest that, in the chromaffin cells of the bovine adrenal medulla, angiotensin II acts on specific receptors to cause a calcium-dependent catecholamine release but triggers no additional response that acts synergistically with depolarizing or nicotinic stimuli to augment catecholamine release.     

Coordinate regulation of catecholamine uptake by rab3 and phosphoinositide 3-kinase.

Previously we observed that rab3 GTPases modulate both the secretion of catecholamines from PC12 neuroendocrine cells and the steady-state accumulation of exogenous norepinephrine (NE) into these cells (Weber, E., Jilling, T., and Kirk, K. L. (1996) J. Biol. Chem. 271, 6963-6971). Here we addressed the mechanisms by which these monomeric GTPases stimulate NE uptake by PC12 cells including their effects on uptake kinetics, their sites of action (secretory granule membrane versus plasma membrane), and the involvement of rab3-interacting proteins in this process. We observed that rab3B stimulated the rate and maximal accumulation of radiolabeled NE into large dense core vesicles within intact PC12 cells. rab3A and rab3B also increased NE uptake into large dense core vesicles in digitonin-permeabilized PC12 cells, which indicates that these GTPases stimulate catecholamine uptake at the level of the secretory granule membrane. In an attempt to identify rab3B targets that may mediate this effect on NE uptake, we found that rab3B interacts directly with phosphoinositide 3-kinase (PI3K) in a GTP-dependent fashion and that PI3K activity was elevated in PC12 cells overexpressing rab3B. Furthermore, two structurally distinct inhibitors of PI3K (wortmannin and LY294002) inhibited NE uptake in intact as well as digitonin-permeabilized PC12 cells, but had no effect on calcium-evoked NE secretion. Our results indicate that rab3 and PI3K positively and coordinately regulate NE uptake in PC12 neuroendocrine cells at least in part by stimulating the secretory vesicle uptake step.     

Ionic mechanisms involved in the release of 3H-norepinephrine from the cat superior cervical ganglion

It has previously been reported that in the isolated cat superior cervical ganglion (SCG) labeled with tritiated norepinephrine (3H-NE), the stimulation of the preganglionic trunk at 10 Hz as well as the exposure to 100 microM exogenous acetylcholine (ACh), produced a Ca++-dependent release of 3H-NE. The present results show that a Ca++-dependent release of 3H-NE was produced also by exposure to either 50 microM veratridine or 60 mM KCl. Tetrodotoxin (0.5 microM) abolished the release of 3H-NE induced by preganglionic stimulation, ACh and veratridine but did not modify the release evoked by KCl. The metabolic distribution of the radioactivity released by the different depolarizing stimuli showed that the 3H-NE was collected mainly unmetabolized. In the cat SCG neither the release of 3H-NE evoked by KCl nor the endogenous content of NE was modified by pretreatment with 6-OH-dopamine (6-OH-DA). On the other hand, this chemical sympathectomy depleted the endogenous content of NE in the cat nictitating membrane, whose nerve terminals arise from the SCG. The data presented suggest that the depolarization-coupled release of NE from the cat SCG involves structures that are different to nerve terminals and that contain Na+ channels as well as Ca++ channels.     

Biochemical characteristics of C-6 glial cells

C-6 glial cells were studied in culture with respect to morphological and biochemical changes under several experimental conditions. Doubling time was 33 hr for cells plated at either 0.5 or 1.0×10⁶ cells per flask. Markedly reduced cell growth and astrocyte-like appearance were observed following dibutyryl cyclic AMP (DBcAMP) treatment. An inverse relationship between cell density and DNA, RNA, and protein content per cell was observed. AChE and BuChE activities were also inversely related to cell density, and treatment with DBcAMP increased enzyme activity, but did not alter the cell density relationship. Uptake of³H-norepinephrine also decreased with increasing cell density. In DBcAMP-treated cells,³H-NE uptake was markedly lower than in nontreated controls, and cortisol treatment decreased the uptake of³H-NE in DBcAMP-treated cells further still. We interpreted the foregoing changes to indicate that cellular activity is cell density-dependent.     

Effect of chronic and acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration on the function and structure of cultured chromaffin cells

Cultured bovine adrenal chromaffin cells were treated chronically with various concentrations of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in the culture medium for 2–8 days or acutely for 10–15 min. Culture of cells with MPTP for periods of 2–8 days resulted in a marked loss of total cellular catecholamines and a parallel reduction in secretory response, but not the ratio of stimulated to unstimulated secretion. By the eighth day in culture, at the highest MPTP concentration (1000 μM), cell catecholamine content and secretion were only about 10% that of untreated cells. The proportion of total cellular catecholamines secreted was not altered by MPTP, suggesting that the secretory process was unaffected by the drug. The loss of secretory output was not prevented by inhibitors of monoamine oxidase or catecholamine uptake, drugs known to prevent MPTP-induced damage to central dopaminergic neurons. The subcellular organelles of MPTP-treated cells appeared relatively normal except for extensive depletion of the vesicle contents, in agreement with the biochemical data. The severity of the depletion appeared to be lessened in cells treated with monoamine oxidase inhibitors.Short term exposure to MPTP at concentrations less than 100 μM had little effect on secretion induced by carbachol. Higher concentrations of MPTP increased unstimulated release and reduced stimulated release. Pretreatment of the cells with MPTP resulted in a lasting reduction in their subsequent secretory responsiveness. MPTP alone, at concentrations greater than 100 μM induced catecholamine release that was unaffected by pretreatment of the cells with monoamine oxidase inhibitors or the catecholamine uptake inhibitor desipramine. MPTP-induced secretion by intact cells was calcium-dependent, while the small increase by permeabilized cells was not.     

Characterization of norepinephrine binding to plasma proteins of the dog and the rabbit

The binding of 3H-norepinephrine (L-3H-NE, 1.0 X 10(-9) M) to plasma proteins of the dog and the rabbit was studied under controlled conditions. Destruction of NE occurred less rapidly at 22 degrees than at 37 degrees. Binding measured at 22 degrees was equivalent to that at 37 degrees, while binding measured at 0 degree was greater than that at 37 degrees. Therefore, losses of plasma NE were minimized by incubation of samples at 22 degrees for no longer than 30 minutes. L-3H-NE binding was examined in the absence and presence of 10(-9) to 10(-2) M non-labeled L-NE, DL-NE, DL-normetanephrine (NM), DL-epinephrine (E), dopamine (DA), and catechol (C). Specific binding of L-3H-NE varied in the range of NE concentrations (L-3H-NE + non-labeled NE) from 10(-9) M (18.7 +/- 3.1%, rabbit; 25.6 +/- 4.8%, dog) to 10(-6) M (10.8 +/- 3.1%, rabbit; 15.2 +/- 3.6%, dog). Calculated binding constants (KD) were consistent with binding to circulating proteins such as globulins or albumin (4.2 +/- 1.2 X 10(-5) M, rabbit; 5.4 +/- 1.7 X 10(-5) M, dog). In plasma from both species, non-labeled DL-NE, L-NE, E, DA, and C, but not NM (from 10(-9) to 10(-2) M) each significantly displaced L-3H-NE from its binding site in a manner similar to displacement produced by non-labeled NE. The results demonstrate that 1) NE is bound to plasma proteins, although to a lesser extent than had been reported by other investigators; and 2) the binding of catecholamines to plasma proteins may be mediated by the catechol ring.     

Regulation of noradrenergic function by inflammatory cytokines and depolarization

Although the sympathetic neurons innervating the heart are exposed to the inflammatory cytokines cardiotrophin-1 (CT-1), interleukin-6 (IL-6) and tumor necrosis factor alpha (TNFalpha) after myocardial infarction, the effects of these cytokines on noradrenergic function are not well understood. We used cultured sympathetic neurons to investigate the effects of these cytokines on catecholamine content, the tyrosine hydroxylase co-factor, tetrahydrobiopterin (BH4), and norepinephrine (NE) uptake. CT-1, but not IL-6 or TNFalpha, suppressed NE uptake and catecholamines in these neurons, whereas CT-1 and, to a lesser extent, IL-6 decreased BH4 content. CT-1 exerted these effects by decreasing tyrosine hydroxylase, GTP cyclohydrolase (GCH) and NE transporter mRNAs, while IL-6 lowered only GCH mRNA. The neurons innervating the heart are also activated by the central nervous system after myocardial infarction. We examined the combined effect of depolarization and cytokines on noradrenergic function. In CT-1-treated cells, depolarization caused a small increase in BH4 and NE uptake, and a large increase in catecholamines. These changes were accompanied by increased TH, GCH and NE transporter mRNAs. CT-1 and depolarization regulate expression of noradrenergic properties in an opposing manner, and the combined treatment results in elevated cellular catecholamines and decreased NE uptake relative to control cells.     

Effect of 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) on monoamine neurotransmitters in mouse brain & heart

The effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was studied on dopamine (DA), norepinephrine (NE), serotonin (5HT) and γ-aminobutyric acid (GABA) neurons in mouse brain and on NE neurons of mouse heart. MPTP (45 mg/kg) was administered s.c. to mice twice daily for 2 consecutive days. This dosage regimen produced a decrease in the forebrain concentrations of DA and NE at 7 and 20 days after injection. In contrast, the forebrain concentrations of 5HT and GABA were not significantly decreased at either time. MPTP administration also produced a marked decrease in the uptake of ³H-DA into striatal slices and ³H-NE into cerebral cortical slices. In contrast, the uptake of ³H-NE into hypothalamic slices and the uptake of ³H-5HT into slices from several brain regions were not altered. MPTP initially reduced the concentration of NE in the heart, but unlike the persistent decreases in the forebrain concentrations of NE and DA, the NE concentration in the heart returned to control levels at approximately 20 days after MPTP administration. These results, showing that MPTP can produce a long lasting and selective decrease in the forebrain concentrations of NE and DA and in the uptake of radioactive DA and NE into brain slices, suggest that MPTP can cause the destruction of catecholamine neurons in mouse brain. In contrast, MPTP administration does not appear to produce long term changes in either 5HT or GABA neurons.     

Reserpine-induced reduction in norepinephrine transporter function requires catecholamine storage vesicles

Treatment of rats with reserpine, an inhibitor of the vesicular monoamine transporter (VMAT), depletes norepinephrine (NE) and regulates NE transporter (NET) expression. The present study examined the molecular mechanisms involved in regulation of the NET by reserpine using cultured cells. Exposure of rat PC12 cells to reserpine for a period as short as 5 min decreased [³H]NE uptake capacity, an effect characterized by a robust decrease in the V_max of the transport of [³H]NE. As expected, reserpine did not displace the binding of [³H]nisoxetine from the NET in membrane homogenates. The potency of reserpine for reducing [³H]NE uptake was dramatically lower in SK-N-SH cells that have reduced storage capacity for catecholamines. Reserpine had no effect on [³H]NE uptake in HEK-293 cells transfected with the rat NET (293-hNET), cells that lack catecholamine storage vesicles. NET regulation by reserpine was independent of trafficking of the NET from the cell surface. Pre-exposure of cells to inhibitors of several intracellular signaling cascades known to regulate the NET, including Ca²⁺/Ca²⁺–calmodulin dependent kinase and protein kinases A, C and G, did not affect the ability of reserpine to reduce [³H]NE uptake. Treatment of PC12 cells with the catecholamine depleting agent, α-methyl-p-tyrosine, increased [³H]NE uptake and eliminated the inhibitory effects of reserpine on [³H]NE uptake. Reserpine non-competitively inhibits NET activity through a Ca²⁺-independent process that requires catecholamine storage vesicles, revealing a novel pharmacological method to modify NET function. Further characterization of the molecular nature of reserpine's action could lead to the development of alternative therapeutic strategies for treating disorders known to be benefitted by treatment with traditional competitive NET inhibitors.     

PRIMARY CULTURES OF DISSOCIATED SYMPATHETIC NEURONS : II. Initial Studies on Catecholamine Metabolism

Initial studies are reported on the catecholamine metabolism of low-density cultures of dissociated primary sympathetic neurons. Radioactive tyrosine was used to study the synthesis and breakdown of catecholamines in the cultures. The dependence of catecholamine synthesis and accumulation on external tyrosine concentration was examined and a concentration which is near saturation, 30 µM, was chosen for further studies. The free tyrosine pool in the nerve cells equilibrated with extracellular tyrosine within 1 h; the total accumulation of tyrosine (free tyrosine plus protein, catecholamines, and metabolites) was linear for more than 24 h of incubation. Addition of biopterin, the cofactor of tyrosine hydroxylase, only slightly enhanced catecholamine biosynthesis by the cultured neurons. However, addition of reduced ascorbic acid, the cosubstrate for dopamine β-hydroxylase, markedly stimulated the conversion of dopamine (DA) to norepinephrine (NE). Phenylalanine, like tyrosine, served as a precursor for some of the DA and NE produced by the cultures, but tyrosine always accounted for more than 90% of the catecholamines produced. The DA pool labeled rapidly to a saturation level characteristic of the age of the culture. The NE pool filled more slowly and was much larger than the DA pool. The disappearance of radioactive NE and DA during chase experiments followed a simple exponential curve. Older cultures showed both more rapid production and more rapid turnover of the catecholamines than did younger cultures, suggesting a process of maturation.     

Catecholamines in human saliva.

Catecholamines are readily detectable in human saliva but their origin is unclear. Norepinephrine (NE) was stable in saliva stored at 4 degrees for 2 hours but 11 +/- 3% degraded after storage at 25 degrees for 1 hour. We intravenously infused 3H-NE into humans and measured levels of 3H-NE and its metabolites in both saliva and forearm venous plasma (a site whose plasma NE levels reflect both local uptake and release of NE). 3H-NE levels in saliva continued to rise for 1 hour even though forearm plasma levels had plateaued by 5 min. By 65 min into the infusion the ratio of 3H-NE:non-radioactive NE was similar in saliva and forearm venous plasma. The ratio of NE:epinephrine (E) was similar in saliva and forearm venous plasma at all time points. Chewing induced salivation, and at least tripled the amount of NE, E and 3H-NE released into saliva per minute, but decreased their concentration in saliva by as much as one half. Saliva NE level was unaltered after 15 min of standing but was increased by 31% after 1 hour of upright posture. Our data imply that the NE present in human saliva comes from both the bloodstream and from salivary sympathetic nerves. The finding that diffusion of blood NE into saliva takes roughly 1 hour to complete suggests that NE in saliva is a poor index of acute changes in sympathetic activity.     

Alteration by methadone of catecholamine uptake and release in isolated rat adrenomedullary storage vesicles

Incubation of isolated rat adrenomedullary storage vesicles with methadone produced inhibition of ³H-epinephrine uptake and promotion of release of endogenous catecholamines. Neither effect was seen using morphine, nor could morphine antagonize methadone-induced catecholamine release, suggesting that these actions are not mediated by opiate receptors. Inhibition of uptake by methadone appeared to contain a competitive component with a lower Ki for methadone compared to the Km for ³H-epinephrine. Despite competitive inhibition by methadone, the maximal uptake capacity (analogous to Vmax) as determined by double-reciprocal plots, was increased by the drug, probably as a result of greater availability of intravesicular storage sites because of the drug-induced of release endogenous catecholamines. Agents which enhance or block catecholamine transport into vehicles had no effect on the catecholamine release by methadone, indicating that the latter is separable from the action on uptake. These alterations of catecholamine uptake and release may play a role in the effects of methadone on the adrenal medulla $\text{in vivo}$.     

Effects of angiotensin II and bilateral nephrectomy on norepinephrine catabolism in central nervous system.

Effects of angiotensin II (AII) on norepinephrine (NE) catabolism in hypothalamus and medulla oblongata of male rats were studied. 3H-NE uptake, 3H-NE/3H-NE metabolites ratio (NE/MET) and monoamineoxidase (MAO) activity were measured in vitro in both organs. Lack of circulating AII was elicited by means of 48 h bilateral nephrectomy. Pargyline and bilateral nephrectomy increased NE uptake and NE/MET ratio, while in nephrectomized plus pargyline treated groups and additive effect on these results was observed in both organs. All decreased the NE/MET ratio. Pargyline reversed the latter effects of AII. The peptide increased MAO activity in both organs, while bilateral nephrectomy decreased the activity of the enzyme. The results showed that AII modulates NE catabolism by means of MAO activity, eventually at the presynaptic noradrenergic ending sites in the central nervous system.