Stimulation-evoked Norepinephrine Release Research Articles

Tyrosine-free amino acid mixtures reduce physiologically-evoked release of dopamine in a selective and activity-dependent manner

Depletion of the catecholamine precursor tyrosine using tyrosine-free amino acid mixtures is an important tool in neuropsychological studies, and often considered dopamine selective on the basis of neuropharmacological studies. However, little is known of the effects of tyrosine depletion when catecholamine neurons are activated physiologically. Here we investigated the effect of tyrosine-free amino acid mixtures on catecholamine release evoked in vivo using a stimulation paradigm aimed to approximate the phasic firing pattern of these neurons that accompanies cognitive and behavioural change. Dopamine and noradrenaline release was monitored by microdialysis in rat medial prefrontal cortex (mPFC) and striatum (chloral hydrate anaesthesia, perfusion medium containing 1 µM cocaine). Electrical stimulation of the medial forebrain bundle (MFB) caused a short-lasting, frequency-dependent increase in dopamine and noradrenaline. A full tyrosine-free amino acid mixture reduced the release of dopamine in mPFC and striatum, across a range of stimulation frequencies, and the effect was greater as stimulation frequency increased. Similar results were obtained using a smaller tyrosine-free amino acid mixture. In the same experiments showing decreased dopamine, neither tyrosine-free mixture of amino acids significantly altered stimulation-evoked release of noradrenaline. These results show that tyrosine depletion using tyrosine-free amino acid mixtures causes a selective, activity-dependent decrease in dopamine release when dopamine neurons are driven physiologically.

Regarding Article “Receptor Activity-Modifying Protein-1 Augments Cerebrovascular Responses to Calcitonin Gene-Related Peptide and Inhibits Angiotensin II-Induced Vascular Dysfunction”

HomeStrokeVol. 42, No. 2Regarding Article “Receptor Activity-Modifying Protein-1 Augments Cerebrovascular Responses to Calcitonin Gene-Related Peptide and Inhibits Angiotensin II-Induced Vascular Dysfunction” Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessLetterPDF/EPUBRegarding Article “Receptor Activity-Modifying Protein-1 Augments Cerebrovascular Responses to Calcitonin Gene-Related Peptide and Inhibits Angiotensin II-Induced Vascular Dysfunction” Kazushi Tsuda, MD, FAHA Kazushi TsudaKazushi Tsuda Cardiovascular and Metabolic Research Center Kansai University of Health Sciences Osaka, Japan (Tsuda) Search for more papers by this author Originally published30 Dec 2010https://doi.org/10.1161/STROKEAHA.110.604272Stroke. 2011;42:e24Other version(s) of this articleYou are viewing the most recent version of this article. Previous versions: January 1, 2010: Previous Version 1 To the Editor:We read with great interest the recent article by Dr Chrissobolis and colleagues1 dealing with the cerebrovascular responses in the transgenic mice with overexpression of the receptor activity-modifying protein-1 (RAMP1) for calcitonin gene-related peptide (CGRP) receptors. The results of their study demonstrated that the responses to CGRP in carotid and basilar arteries in vitro as well as cerebral arterioles in vivo were selectively enhanced in human RAMP1 transgenic mice compared with controls. In addition, the authors presented that angiotensin II-induced oxidative stress and endothelial dysfunction was prevented after expression of RAMP1. The authors propose the hypothesis that RAMP1 may be an important mediator of vascular protection and a new therapeutic target in vascular disease.Several studies have reported that enhanced activity of the sympathetic nervous system might actively participate in the pathogenesis of vascular dysfunction. In a study we presented previously, the changes in norepinephrine release induced by CGRP was investigated in the rat central nervous system.2 In an in vitro study, we showed that CGRP inhibited the stimulation-evoked norepinephrine release in a dose-dependent manner. It was also demonstrated that a dihydropyridine-sensitive calcium channel agonist, Bay K 8644, significantly reversed the inhibitory effect of CGRP on norepinephrine release, indicating that CGRP might partially interact with dihydropyridine-sensitive calcium channels and modulate intracellular calcium mobilization. Furthermore, we showed that the inhibitory action on CGRP on norepinephrine release was significantly attenuated in spontaneously hypertensive rats compared with normotensive rats.3 In the peripheral tissues, Ohhashi and Jacobowitz4 observed that CGRP might have reduced the electric stimulation-induced contraction of rat vas deferens, suggesting that CGRP might inhibit norepinephrine release during adrenergic nerve stimulation. It can be speculated that the sympatholytic action of CGRP might be a defense against vascular dysfunction. Because angiotensin II may stimulate the sympathetic neurotransmission in the central nervous system,5 we would like to know whether changes in sympathetic nervous activity might be associated with the magnitude of the RAMP1 overexpression or whether CGRP might suppress the angiotensin II-induced sympathetic hyperactivity in the study of Dr Chrissobolis and colleagues. Further studies should be performed to assess more thoroughly the interactions between CGRP and the sympathetic nervous system and their role in the protective effect against the angiotensin II-induced vascular dysfunction in the RAMP1 transgenic mice.Kazushi Tsuda, MD, FAHA Cardiovascular and Metabolic Research Center Kansai University of Health Sciences Osaka, JapanDisclosuresNone.FootnotesStroke welcomes Letters to the Editor and will publish them, if suitable, as space permits. Letters must reference a Stroke published-ahead-of-print article or an article printed within the past 3 weeks. The maximum length is 750 words including no more than 5 references and 3 authors. Please submit letters typed double-spaced. Letters may be shortened or edited. Include a completed copyright transfer agreement form (available online at http://stroke.ahajournals.org and http://submit-stroke.ahajournals.org).

Effects of nicorandil on sympathetic neurotransmission in rat caudal artery

1. We examined the effects of nicorandil, an ATP-sensitive potassium (K(ATP)) channel opener and nitric oxide donor, on the release of noradrenaline from vascular sympathetic nerves. This effect was compared to the effect on vascular smooth muscle. 2. Caudal artery preparations from Wistar rats were electrically stimulated (1 Hz, 0.5-ms duration) and noradrenaline release in the artery was detected using an high-pressure liquid chromatography-electrochemical detection technique. The pharmacological properties of the prejunctional effect of nicorandil were determined using the nonselective K(ATP) channel blocker glibenclamide, the pancreatic beta-cell and brain-type K(ATP) channel blocker tolbutamide, and the smooth muscle-type K(ATP) channel blocker PNU-37883A. 3. Nicorandil inhibited the electrical stimulation-evoked noradrenaline release in a concentration-dependent manner. This inhibitory effect was abolished by 1 micromol/L glibenclamide and 10 micromol/L tolbutamide, but was not affected by 10 micromol/L PNU-37883A or 0.3 micromol/L ODQ. Nicorandil did not affect the noradrenaline transporter uptake 1 in the adrenergic nerve terminals. 4. Nicorandil produced a relaxation response in a concentration-dependent manner in the caudal artery pre-contracted with 0.3 micromol/L noradrenaline. This relaxation response was significantly diminished in the presence of 1 micromol/L glibenclamide, 10 micromol/L PNU-37883A and 0.3 micromol/L ODQ but not by 10 micromol/L tolbutamide. 5. These findings suggest that nicorandil inhibits noradrenaline release via the K(ATP) channels of sympathetic nerves. These channels may be pharmacologically different from those of vascular smooth muscle.

Effect of ω-Conotoxin GVIA on Noradrenaline Release from Postganglionic Sympathetic Neurones in Rabbit Aorta

The aim of the present work was to examine the effect of the selective N-type calcium blocking agent omega-conotoxin GVIA on stimulation-evoked release of noradrenaline from sympathetic nerves in rabbit isolated aorta with regard to stimulation frequency, extracellular Ca2+ concentration, and transmitter uptake. Rings of rabbit isolated aorta were preloaded with (-)-3H-noradrenaline and the fractional 3H-overflow evoked by electrical-field stimulation was determined by liquid scintillation spectrometry. Omega-conotoxin GVIA (3 x 10(-10)-3 x 10(-8) M) did not alter the spontaneous 3H-outflow. Omega-conotoxin GVIA (3 x 10(-10)-3 x 10(-8) M) caused a slowly developing reduction of stimulation-evoked 3H-overflow at 1 and 30 Hz. The Emax for the omega-conotoxin-induced inhibition was less (70%) at 30 Hz than that (96%) seen at 1 Hz. Short-term incubation with omega-conotoxin GVIA caused a subsequent steady-state inhibition. The inhibitory action of omega-conotoxin GVIA (3 x 10(-10)-3 x 10(-9) M) was inversely related to the extracellular Ca2+ concentration (6.5 x 10(-4)-2.7 x 10(-3) M). Cocaine (3 x 10(-5) M) plus corticosterone (4 x 10(-5) M), neuronal and extraneuronal uptake inhibitors, respectively, did not alter the inhibitory effect of omega-conotoxin GVIA (3 x 10(-9) M) on 3H-overflow evoked by stimulation at a frequency of either 1 or 30 Hz. It is concluded that omega-conotoxin GVIA acts on prejunctional N-type calcium channels to inhibit stimulation-evoked noradrenaline release from sympathetic neurone terminals in rabbit aorta. At a high frequency, another subtype calcium channel may possibly be involved. The action of omega-conotoxin GVIA is independent of neuronal and extraneuronal uptake mechanisms for noradrenaline, but dependent on the amount of Ca2+ to be transported across the neurilemma from the extracellular space into the neurone.

Involvement of Voltage-Dependent Ca2+ Channel β3 Subunit in the Autonomic Control of Heart Rate Variability

Noradrenaline release from sympathetic nerve terminals is dependent on Ca²⁺ entry through neuronal voltage-gated N-type Ca²⁺ channels. The accessory β₃ subunits of Ca²⁺ channels (Ca_Vβ₃) are preferentially associated with the α_1B subunit to form N-type Ca²⁺ channels, and are therefore expected to play a functional role in the stimulation-evoked release of noradrenaline. In this study, we employed Ca_Vβ₃-null, Ca_Vβ₃-overexpressing (Ca_Vβ₃-Tg), and wild-type (WT) mice to investigate the possible roles of Ca_Vβ₃ in the sympathetic regulation of heart rate in vivo. Telemetry was used to monitor the ECG and both time and frequency domain analyses were carried out to evaluate heart rate variability. In the frequency domain analysis, power spectral density of the RR interval series was computed using the fast Fourier transform algorithm. The resting heart rate was increased in Ca_Vβ₃-Tg mice compared with both Ca_Vβ₃-null and WT mice. Mice overexpressing Ca_Vβ₃ displayed decreased heart rate variability, which was measured by the time domain analysis of the standard deviation of RR intervals. In the frequency domain analysis, Ca_Vβ₃-Tg mice showed decreased spectral powers compared with WT and Ca_Vβ₃-null mice. Pharmacological blockade of β-adrenergic receptors with metoprolol decreased the heart rate in all genotypes, but the extent of the decrease was most obvious in Ca_Vβ₃-Tg mice. On the other hand, the spectral powers were decreased in response to parasympathetic blockade (atropine) in WT and Ca_Vβ₃-Tg mice. These results indicate the functional roles of Ca_Vβ₃ in regulating sympathetic nerve signaling.

Involvement of Different Calcium Channels in the Depolarization‐Evoked Release of Noradrenaline from Sympathetic Neurones in Rabbit Carotid Artery*

The calcium channels coupled to noradrenaline release from sympathetic neurones in the rabbit isolated carotid artery were examined. Rings of carotid artery were preloaded with (-)-[(3)H]noradrenaline and the fractional (3)H overflow evoked by electrical-field stimulation was determined by liquid scintillation spectrometry. The N-type Ca(2+) channel blocking agent omega-conotoxin GVIA (3x10(-9)-6x10(-8) M) reduced the stimulation-evoked (3)H overflow. The maximal inhibition was seen with 3x10(-8) M. The maximal reduction was more marked at a low (2 Hz) stimulation frequency than at a high one (30 Hz). Mibefradil (10(-6) M) irreversibly reduced the (3)H overflow evoked by field stimulation (2 Hz). At 30 Hz, the reduction was more marked than at 2 Hz. Mibefradil (3x10(-6)-10(-5) M) enhanced the passive (3)H outflow. The reduction of the stimulation (30 Hz)-evoked (3)H overflow seen with omega-conotoxin GVIA (3x10(-8) M) was enhanced by mibefradil (10(-6) M) and unaffected by nimodipine (10(-5) M) and omega-agatoxin IVA (10(-8) M). We conclude that the stimulation-evoked release of noradrenaline from sympathetic neurones in rabbit carotid artery at a low frequency (2 Hz) is mediated mainly by the N-type calcium channels. At a high frequency (30 Hz), T-type Ca(2+) channels are also involved.

Amitriptyline administration transforms tumor necrosis factor-alpha regulation of norepinephrine release in the brain

The present study demonstrates that the mixed action antidepressant drug amitriptyline enhances norepinephrine (NE) release by transforming the nature of the response of neurons to both tumor necrosis factor-alpha (TNF) as well as to an alpha(2)-adrenergic agonist in an area of the central nervous system (CNS) rich in adrenergic neurons. Administration of the antidepressant drug amitriptyline for 1 day or 14 days to rats significantly increases TNF bioactivity in total homogenates of the locus coeruleus (LC) and the hippocampus as assessed by the WEHI-13VAR bioassay. Superfusion and electrical field stimulation of rat hippocampal brain slices were used to study the regulation of NE release. Exposure to TNF, as well as activation of the alpha(2)-adrenergic autoreceptor inhibits stimulation-evoked norepinephrine (NE) release from adrenergic neurons of the CNS from naïve rats. Superfusion of hippocampal slices isolated from rats chronically (14 days) administered amitriptyline demonstrates that TNF inhibition of NE release is transformed, such that TNF facilitates NE release, dependent upon alpha(2)-adrenergic activation. Furthermore, chronic administration of amitriptyline increases stimulation-evoked NE release and decreases alpha(2)-adrenergic autoreceptor inhibition of NE release, an effect not observed with acute drug administration. These data support the hypothesis that chronic antidepressant drug administration, through regulation of TNF expression, transforms alpha(2)-adrenergic receptors such that they function to facilitate NE release, suggesting a mechanism of action of antidepressant drugs.

Calcium channels involved in noradrenaline release from sympathetic neurones in rabbit carotid artery.

Transmitter release from nerve terminals is dependent on the entry of Ca(2+) through neuronal voltage-gated calcium channels. In sympathetic neurones both N- and L-type calcium channels are present. Potassium channel blockade increases Ca(2+) entry into sympathetic neurones. We examined the participation of N- and L-type calcium channels in the stimulation-evoked release of noradrenaline from vascular sympathetic neurones. Rings of rabbit carotid artery were preincubated with [3H]-noradrenaline. Electrical field stimulation was used to evoke 3H overflow. The selective N-type calcium channel blocking agent omega-conotoxin GVIA (single concentrations: 3 x 10(-10)-10(-8) M) caused a slowly developing reduction of the stimulation-evoked 3H overflow. At 3 x 10(-8) M, omega-conotoxin GVIA caused an equilibrium block with a rapid (15 min.) onset. After 2 hr exposure to omega-conotoxin the inhibition was steady (pIC50 (-log M): 9.43; Emax: 91%). The selective L-type calcium blocking agents nifedipine (10(-7)-10(-5) M) and nimodipine (10(-8)-10(-5) M) had no effect on the stimulation-evoked 3H overflow. The calcium channel opener Bay K 8644 (10-6 M) likewise had no effect. The potassium channel blocking agent 4-aminopyridine (10-5-10-3 M) enhanced the stimulation-evoked 3H overflow up to 5 times. 4-Aminopyridine (10(-4) M) did not alter the inhibitory effect of omega-conotoxin GVIA (3 x 10(-8) M). In the presence of 4-aminopyridine (10(-4) M), nifedipine (10(-5) M) and nimodipine (10(-6) M) enhanced the 3H overflow. We conclude that the stimulation-evoked release of noradrenaline from sympathetic neurones in rabbit carotid artery is mediated by N-type calcium channels and that L-type channels are not involved even when potassium channels are blocked by 4-aminopyridine.

Chronic hypoxia alters the function of NOS nerves in cerebral arteries of near-term fetal and adult sheep.

In addition to adrenergic innervation, cerebral arteries also contain neuronal nitric oxide synthase (nNOS)-expressing nerves that augment adrenergic nerve function. We examined the impact of development and chronic high-altitude hypoxia (3,820 m) on nNOS nerve function in near-term fetal and adult sheep middle cerebral arteries (MCA). Electrical stimulation-evoked release of norepinephrine (NE) was measured with HPLC and electrochemical detection, whereas nitric oxide (NO) release was measured by chemiluminescence. An inhibitor of NO synthase, N(omega)-nitro-l-arginine methyl ester (l-NAME), significantly inhibited stimulation-evoked NE release in MCA from normoxic fetal and adult sheep with no effect in MCA from hypoxic animals. Addition of the NO donor S-nitroso-N-acetyl-dl-penicillamine fully reversed the effect of l-NAME in MCA from normoxic animals with no effect in MCA from hypoxic animals. Electrical stimulation caused a significant increase in NO release in MCA from normoxic animals, an effect that was blocked by the neurotoxin tetrodotoxin, whereas there was no increase in NO release in MCA from hypoxic animals. Relative abundance of nNOS as measured by Western blot analysis was similar in normoxic fetal and adult MCA. However, after hypoxic acclimitization, nNOS levels dramatically declined in both fetal and adult MCA. These data suggest that the function of nNOS nerves declines during chronic high-altitude hypoxia, a functional change that may be related to a decline in nNOS protein levels.

Chronic hypoxia alters prejunctional alpha(2)-receptor function in vascular adrenergic nerves of adult and fetal sheep.

The impact of development and chronic high-altitude hypoxia on the function of prejunctional alpha(2)-adrenoceptors was studied by measuring norepinephrine release in vitro from fetal and adult sheep middle cerebral and facial arteries. Blockade of prejunctional alpha(2)-adrenoceptors with idazoxan significantly increased stimulation-evoked norepinephrine release in normoxic arteries. This effect was eliminated after chronic hypoxia in cerebral arteries, with a tendency to decline in fetal facial arteries. After chronic hypoxia, the capacity to release norepinephrine declined in fetal middle cerebral arteries with a similar trend in facial arteries. Norepinephrine release was maintained in adult arteries. During development, stimulation-evoked norepinephrine release from middle cerebral and facial arteries was higher compared with adult arteries. In fetal arteries, adrenergic nerve function declined after chronic hypoxia. However, in adult arteries, adrenergic nerves adapted to chronic hypoxia by maintaining overall function. This differential adaptation of adrenergic nerves in fetal arteries may reflect differences in fetal distribution of blood flow in response to chronic hypoxic stress.

Effects of β-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.

Modulation of norepinephrine release by ATP-dependent K(+)-channel activators and inhibitors in guinea-pig and human isolated right atrium.

The aim of this study was to show, whether ATP sensitive K+ channels (KATP channels), are involved in the modulation of norepinephrine (NE) release from the sympathetic nerves innervating the guinea-pig and human right atrium. The resting and stimulation-evoked release of [3H]norepinephrine ([3H]NE) was measured from the isolated guinea-pig and human right atrium and the effect of activators and inhibitors of ATP sensitive K+ channels was studied. Cromakalim (30-300 microM), a KATP channel-agonist decreased concentration-dependently the stimulation-evoked release of NE from the guinea-pig atrium, an effect, antagonized by glibenclamide, a KATP channel-antagonist (30 microM). Diazoxide (30-300 microM), another activator of the KATP channels reduced the resting release of NE, and also attenuated the evoked release at a single concentration (100 microM), and this latter action was also counteracted by glibenclamide (30 microM). Pinacidil, increased dose-dependently the resting and stimulation-evoked release of NE in a glibenclamide-sensitive manner and reversed the inhibitory effect of cromakalim (100 microM), suggesting that it acts as an antagonist. Glibenclamide (30-300 microM), by itself enhanced the stimulation-evoked release of [3H]NE, and also increased the resting release of NE. On the other hand, 5-hydroxydecanoate, an ischemia-selective inhibitor of cardiac KATP channels did not change NE release. Adenosine, (30-300 microM), an A1-receptor agonist, clonidine (3 microM), an alpha 2-adrenoceptor agonist and oxotremorine, a muscarinic receptor agonist (30 microM) all reduced the evoked release of [3H]NE, but these effects were not modified by glibenclamide (300 microM), indicating that neuronal adenosine (A1), adrenergic (alpha 2) and muscarinic (M3) receptors do not act on KATP channels. In the human right atrium, cromakalim, and diazoxide did not affect significantly the release of [3H]NE. However, glibenclamide (30-300 microM) and pinacidil (30-300 microM) enhanced dose-dependently the evoked-release of NE, and pinacidil also augmented the resting release. Our results indicate that sympathetic nerve endings of the human and guinea-pig atrium are endowed with ATP-sensitive K+ channels. These channels responded to agonists and antagonists under the experimental conditions applied and they could modulate the release of NE thereby affecting the autonomic control of cardiac function under various physiological and pathophysiological conditions.

Impact of development and chronic hypoxia on NE release from adrenergic nerves in sheep arteries.

To examine effects of development and chronic high-altitude hypoxia on sympathetic nerve function in sheep, norepinephrine release was measured in vitro from middle cerebral and facial arteries. Capsaicin was used to test the role of capsaicin-sensitive sensory nerves; norepinephrine release was not altered by capsaicin treatment. Nomega-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthase, decreased stimulation-evoked norepinephrine release in middle cerebral arteries from normoxic sheep with no effect in hypoxic arteries or facial arteries. Thus NO-releasing nerves augmented norepinephrine release. Furthermore, the function of NO-releasing nerves declined after chronic hypoxia. Despite loss of the augmenting effects of NO, stimulation-evoked fractional norepinephrine release was unchanged after chronic hypoxia, suggesting that middle cerebral arteries adapt to hypoxia by increasing stimulation-evoked norepinephrine release. In fetal facial arteries, chronic hypoxia resulted in a decline in stimulation-evoked norepinephrine release, but there was an increase in the adult facial artery. In the adult, adaptation to chronic hypoxia is similar in both cerebral and facial arteries. However, differential adaptation in fetal adrenergic nerves may reflect differences in fetal redistribution of blood flow in the face of chronic hypoxia but could also possibly contribute to increased incidence of fetal morbidity.

Adrenergic Nerve Smooth Endoplasmic Reticulum Calcium Buffering Declines with Age

Calcium buffering capacity declines with age in sympathetic nerves of rat tail artery. To test whether smooth endoplasmic reticulum (SER) calcium buffering declines with age, effects of two SER calcium-ATPase inhibitors on norepinephrine release and intracellular calcium were determined. Thapsigargin or cyclopiazonic acid caused a significant increase in stimulation-evoked norepinephrine release from 6 month tail arteries with much less effect in 20 months. In isolated superior cervical ganglion cells, the rate of rise of calcium with K +-depolarization increased only in young cells with either cyclopiazonic acid or thapsigargin, with no effect in the old. In young cells, cyclopiazonic acid significantly influenced time to peak, rate of decline, and time to basal of K +-evoked calcium transients, but had no effect in old cells. Thapsigargin caused a significant increase in rate of decline in young, but not old, cells. These differential effects suggest an age-related decline in function of SER calcium buffering mechanisms in the sympathetic nervous system causing older nerves to become more reliant on mitochondria to buffer calcium.

Impact of age on modulation of norepinephrine release from sympathetic nerves in the rat superior mesentery artery

There is an age-related increase in stimulation-evoked fractional norepinephrine release in tail arteries of Fischer 344 rats from 6–20 months of age. Previous studies have ruled out changes in the function of uptake and subsequent metabolism mechanisms, or feedback by prejunctional α 2-adrenoceptors. The tail artery is important in thermoregulation, and there is the possibility that the previously observed increase in sympathetic nerve activity is due to age-related changes in thermoregulation as opposed to a fundamental age-related change in the regulation of sympathetic nerves. Thus, we measured stimulation-evoked norepinephrine release in another blood vessel model, the superior mesentery artery using HPLC with electrochemical detection. In this study fractional norepinephrine release was measured under three separate conditions, drug free Krebs'; in the presence of deoxycorticosterone and cocaine; in the presence of deoxycorticosterone and cocaine and the α 2-adrenergic receptor antagonist, idazoxan. The most significant finding was that fractional norepinephrine release in mesentery arteries from 20-month-old animals was higher as compared to 6 months regardless of treatment condition. Furthermore, the elevation in norepinephrine release cannot be accounted for by changes in norepinephrine content, uptake and subsequent metabolism mechanisms or changes in basal norepinephrine release. These data from the mesentery artery model confirm and support our previous work in the rat tail artery model. In addition, the data from this study suggest the possibility that there are common mechanisms underlying the age-related increase in peripheral sympathetic nerve activity.

Intracellular Calcium Buffering Declines in Aging Adrenergic Nerves

Stimulation-evoked norepinephrine release from rat tail artery adrenergic nerves increased with advancing age in the Fischer-344 rat when function of norepinephrine uptake mechanisms and prejunctional alpha-2 adrenoceptors were blocked. When calcium channels were bypassed with the ionophore, ionomycin (4 μM), norepinephrine release from aged nerves (20 months) was still elevated as compared to 6-month-old nerves. Norepinephrine release stimulated by high K + was also higher in 20-month nerves. The intracellular calcium chelator, 1,2 bis(2-aminophenoxy)ethane- N,N,N′, N′-tetraacetomethylester (BAPTA/AM), was used to determine whether age-related increases in norepinephrine release could be reversed with the addition of an artificial intracellular calcium buffer. Exposure to BAPTA/AM decreased stimulation-evoked norepinephrine release in both old and young tail arteries; however, the effect was significantly greater in older arteries. When mitochondrial calcium uptake was compromised using the uncoupler of mitochondrial oxidative phosphorylation, dinitrophenol, BAPTA caused a further decrease in stimulation-evoked norepinephrine release in 20-month tail arteries with much less effect in 6-month-old nerves. These results suggest that intracellular calcium buffering is less efficient in older nerves.

P2-purinoceptors on postganglionic sympathetic neurones.

1. Postganglionic sympathetic neurones possess both excitatory and inhibitory P2-purinoceptors. 2. The mechanisms of action of excitatory P2-purinoceptors have recently been studied on cultured sympathetic neurones of the rat. The receptors mediate fast increases in intracellular Ca2+ levels and a release of noradrenaline. They are likely to belong to the neuronal types of P2X-purinoceptors and to be located on the sympathetic nerve cell bodies or their dendrites. 3. Inhibitory P2-purinoceptors have been shown to operate at sympathetic axon terminals in isolated tissues. Adenine nucleotides decreased the stimulation-evoked release of noradrenaline by activation of these receptors. The receptors are likely to belong to the group of G-protein-coupled P2Y-purinoceptors. They mediate a negative feedback in which co-transmitter ATP inhibits subsequent sympathetic transmitter release.

Sex differences in the effects of 17β-estradiol on vascular adrenergic responses

The in vitro effects of 17β-estradiol on vascular responses to adrenergic nerve stimulation were studied in perfused tail arteries from age-matched male and female rats. Nerve stimulation resulted in vasoconstriction that was greater in male arteries. Addition of 17β-estradiol (3 × 10 −5 M) reduced the vasoconstrictor responses in both male and female arteries, but the reduction was significantly greater in the females. Gonadectomy of the animals for 1 month prior to the experiment did not alter the in vitro responses to 17β-estradiol in either males or females. 17β-Estradiol (10 −6−3 × 10 −5 M) also relaxed perfused tail arteries precontracted with KCl (50 mM); however the relaxation was not different between males and females, either intact or gonadectomized. Stimulation-evoked release of noradrenaline from adrenergic nerves of perfused tail arteries was measured, but no differences were found between males and females, nor was release modified by in vitro exposure to 17β-estradiol (10 −5 M). These results suggests that 17β-estradiol acts directly on postjunctional mechanisms to relax tail arteries of either sex. The effect of the hormone on arteries constricted by adrenergic nerve stimulation, however, is greater in females compared to males.

Effects of aging on prejunctional control of neurotransmission in the rat.

In this study, we have looked in detail at the effects of aging on neurotransmission in isolated atria and intact heart in anesthetized rats, examining adult and aged animals. There was a diminished ability of the norepinephrine reuptake blocker cocaine to increase stimulation-evoked release of norepinephrine in aged rats and in terms of increasing the norepinephrine-evoked tachycardia in anesthetized rats, suggesting that the function of the reuptake process is diminished by aging. The ability of the alpha 2 adrenergic receptor agonist xylazine to inhibit release of norepinephrine neurotransmitter was also diminished in aged rats both in isolated tissues and in vivo in anesthetized animals. In further studies, the ability of the beta-adrenergic receptor agonists isoproterenol (beta 1 and beta 2 agonist) and procaterol (selective beta 2 agonist) to increase stimulation-evoked release of norepinephrine was examined. Both agonists produced a concentration-dependent facilitation of norepinephrine release in rats atria. Although isoproterenol and procaterol increased release in tissues from young and old animals, the magnitude of the increase was significantly reduced in tissues from old rats, so that beta-adrenergic receptor mediated facilitation was diminished. In summary, aging is associated with a decline in prejunctional modulation of neurotransmission, in terms of diminished norepinephrine reuptake, diminished prejunctional alpha 2 adrenergic receptor mediated inhibition and diminished prejunctional beta 2 adrenergic receptor mediated facilitation. TABLE 2 summarizes the possible effects of aging on noradrenergic neurotransmission. Overall, neurotransmission at rest may be little changed by aging, given that postjunctional responsiveness may fall, but scope for modulation may be diminished.

Evidence for decline in intracellular calcium buffering in adrenergic nerves of aged rats

Age-related alterations in neuronal intracellular calcium regulation and neurotransmitter release have been widely reported. We have investigated the impact of age on neurotransmitter release and intracellular calcium buffering in adrenergic nerve endings of the isolated rat tail artery and on intracellular calcium in acutely dissociated cells from the superior cervical ganglion. Advancing age, from 6 to 27 months, resulted in significantly increased stimulation-evoked norepinephrine release from the isolated rat tail artery, an effect which persisted when neuronal and extraneuronal uptake were blocked with cocaine and deoxycorticosterone and presynaptic alpha adrenergic receptors were blocked with idazoxan. Alterations in extracellular calcium had significant effects on stimulation-evoked norepinephrine release, but these were much more marked in old, compared to young, arteries. Blockade of mitochondrial calcium accumulation with dinitrophenol had no significant effect on stimulation-evoked norepinephrine release from 6-month-old arteries, but in 20-month-old arteries, treatment with dinitrophenol resulted in a substantial increase in stimulationevoked norepinephrine release. However, when extracellular calcium was increased to 5 mM in 6 month-old-arteries, then addition of dinitrophenol resulted in an increase in stimulation-evoked norepinephrine release. Measurement of intracellular calcium in acutely dissociated superior cervical ganglion cells using fura-2 revealed substantial age-related differences. Peak calcium transients in 20-month-old ganglion cells depolarized with 68 mM K + were substantially higher than in 6-month-old cells. Together these findings support the hypothesis that in adrenergic nerves advancing age results in a disruption of intracellular calcium buffering leading to higher levels of intracellular calcium and increased transmitter release.