Noradrenaline Efflux Research Articles

Inhibition by clostridial neurotoxins of calciumindependent [ 3H]noradrenaline outflow from freeze-thawed synaptosomes: Comparison with synaptobrevin hydrolysis

Clostridial neurotoxins are known to inhibit regulated i.e. calcium-dependent exocytosis. In the present study we have investigated their potential role in also inhibiting calcium-independent exocytosis. Synaptosomes from rat forebrain were preloaded with [ 3H]noradrenaline and permeabilized reversibly by freezing in Ca 2+-free potassium glutamate containing dimethyl sulfoxide and the toxins to be assayed. Subsequently, outflow of radioactivity was measured in isotonic calcium-free potassium glutamate. The synaptic vesicle protein synaptobrevin-2/VAMP-2 and its toxin-dependent degradation were analysed by Western blotting. The light chain of tetanus toxin reduced the synaptosomal outflow of radioactivity, whereas the activity of the heavy chain was at the detection limit. The respective activities of the dichain toxins from Clostridium tetani and C. botulinum A, B and E were enhanced by pretreatment with dithiothreitol. Reduced single-chain tetanus toxin was less potent than reduced dichain tetanus toxin. Pretreatment with ethylene diamine tetraacetic acid as an inhibitor of Zn 2+-proteases abolished the actions of the tetanus toxin light chain and of the reduced dichain toxins. Hydrolysis of synaptobrevin-2/VAMP-2 was obtained with tetanus toxin light chain, reduced dichain tetanus toxin and C. botulinum B toxin. Its hydrolysis by single-chain tetanus toxin was less pronounced, and it was absent with botulinum toxins A and E. It is concluded that clostridial neurotoxins can not only inhibit calcium-dependent release but also affect calcium-independent outflow from synaptosomes. Since this is accompanied by selective intrasynaptosomal proteolysis of synaptobrevin, calcium-independent outflow may at least in part involve the vesicular release apparatus.

High muscle blood flows are not attenuated by recruitment of additional muscle mass.

Recent studies have demonstrated that single-leg knee extensor (KE) exercise elicits high mass-specific blood flow (Q) which, if incremented toward maximum, in the presence of additional muscle recruitment would soon outstrip the heart's pumping capacity and blood pressure would fall. Thus incremental KE exercise provides the opportunity to determine the intensity at which, if at all, quadriceps muscle hemodynamics are altered during incremental exercise that involves a substantially greater muscle mass. Leg Q was measured during incremental KE exercise and again with superimposed incremental two-legged knee extensor exercise with incremental arm cranking (A+L) in trained subjects (n = 5). Leg Q and vascular conductance (VC) increased with work rate (WR) to reach high levels [Q = 385.7 +/- 26 and 342.3 +/- 15 ml.min-1.100 g-1 for KE and A+L exercise, respectively; VC at 90% of maximum WR (WRmax) = 79 +/- 5 and 75 +/- 6 ml.min-1. mmHg-1 for KE and A+L exercise, respectively], but the Q/WR and VC/WR relationships in KE and A+L exercise were not different. Maximum O2 consumption (VO2max) and the VO2max/WR relationship of the quadriceps were also unaffected by the additional muscle mass recruited. Despite a significantly greater net femoral venous norepinephrine (NE) outflow at both 90 and 100% of WRmax in A+L exercise (WRmax = 4,216 +/- 1,601 and 901 +/- 99 ng/ml for A+L and KE exercise, respectively; P < 0.05), leg Q continued to rise linearly with WR.(ABSTRACT TRUNCATED AT 250 WORDS)

Neurochemical and physiological effects of cocaine oscillate with sequential drug treatment: possibly a major factor in drug variability.

Variability in response to drug treatment is a poorly understood problem with severe consequences for both the individual and the health care delivery system. Our data suggest that one source of variability may be inherent in the way physiological systems normally respond to repeated drug exposures. We report that for a wide array of endpoints-amphetamine-evoked, in vitro striatal dopamine efflux, amphetamine and K(+)-evoked efflux of heart norepinephrine and nonevoked plasma levels of corticosterone and glucose-repeated, in vivo cocaine (15 mg/kg IP) administration to male rats precipitated successive oscillations in the magnitude or direction of the organism's responsiveness to subsequent cocaine administration. This capacity of cocaine to produce oscillations in response to successive administrations appears to be due to its foreign/stressful aspect rather than its specific pharmacological properties.

α-Adrenoceptor Modulation of Norepinephrine and ATP Release in Isolated Kidneys of Spontaneously Hypertensive Rats

The present study investigates sympathetic cotransmission and its alpha-adrenoceptor-mediated modulation in kidneys of spontaneously hypertensive rats (SHR, 12 to 14 weeks) and age-matched normotensive Wistar-Kyoto rats (WKY). In the presence of cocaine and corticosterone, renal nerve stimulation at 1 Hz (30 seconds) induced a greater outflow of norepinephrine in SHR (4.2 +/- 0.2 pmol/g kidney) than in WKY (3.0 +/- 0.2 pmol/g kidney). The alpha 2-adrenoceptor antagonist rauwolscine (0.01 to 1 mumol/L) increased the stimulation-induced norepinephrine outflow to a greater extent in SHR than in WKY. In contrast, the alpha 1-adrenoceptor antagonist prazosin (0.03 to 3 mumol/L) increased the stimulation-induced norepinephrine outflow to a greater extent in WKY than in SHR. This difference was not observed in the presence of the P1-purinoceptor antagonist 8-(p-sulfophenyl)theophylline (100 mumol/L). Stimulation at 4 Hz (30 seconds) induced an outflow of ATP (SHR, 12.7 +/- 3.3 pmol/g kidney; WKY, 16.7 +/- 2.1 pmol/g kidney; perfusion solution without cocaine and corticosterone). Prazosin (0.03 mumol/L) markedly reduced pressor responses to stimulation and inhibited the induced ATP outflow by 60% to 70%. When prazosin (0.03 mumol/L) was present, rauwolscine (0.1 mumol/L) increased the induced outflow of norepinephrine and ATP and markedly enhanced prazosin-resistant pressor responses. These pressor responses were abolished by the P2-purinoceptor antagonist suramin (300 mumol/L). The results demonstrate an increased alpha 2-adrenoceptor-mediated automodulation of norepinephrine release in SHR kidneys caused by increased intrasynaptic norepinephrine levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasma metadrenalines: do they provide useful information about sympatho-adrenal function and catecholamine metabolism?

1. The clinical utility of plasma metadrenalines for examination of sympatho-adrenal function and catecholamine metabolism was assessed from plasma measurements of these metabolites in a number of clinical conditions (hypertension, cardiac failure, bilateral adrenalectomy and X-chromosomal deletions of the gene for monoamine oxidase), and before and during activation of sympathetic outflow or infusions of noradrenaline and adrenaline. 2. Plasma concentrations of normetadrenaline were less than 25% of those of noradrenaline, concentrations of metadrenaline and adrenaline were similar and those of sulphate-conjugated metadrenalines were 20- to 30-fold higher than free metadrenalines. Hypertensive patients had elevated plasma concentrations of adrenaline, noradrenaline and conjugated but not free metadrenalines. Cardiac failure patients had 2- to 4-fold increases in plasma noradrenaline and free and conjugated normetadrenaline. Adrenalectomy resulted in undetectable plasma concentrations of adrenaline, 91-97% decreases in free and conjugated metadrenaline and a 40% decrease in normetadrenaline relative to noradrenaline. Patients with X-chromosomal deletions of the gene for monoamine oxidase had 6- and 16-fold increases in plasma free and conjugated normetadrenaline and 2- and 4-fold increases in free and conjugated metadrenaline. 3. Infusion of catecholamines increased plasma concentrations of free metadrenalines by less than 6% of increases in precursor amines, indicating that most plasma normetadrenaline (84%) and metadrenaline (90%) is derived from metabolism of catecholamines before their entry into the circulation. Considerable O-methylation of catecholamines within the adrenals explains why sympatho-adrenal activation resulted in smaller proportional increases in plasma metadrenalines than catecholamines. 4. Plasma metadrenalines provide supplementary information about sympatho-adrenal activity to that provided by catecholamines, but are more useful for examination of the extraneuronal inactivation of catecholamines, particularly detection of neurochemical phenotypes in genetic disorders of catecholamine metabolism. Significant formation of metadrenalines within chromaffin tissue explains why measurements of plasma metadrenalines provide an extraordinarily sensitive method for diagnosis of phaeochromocytoma.

Contrasting effects of the imidazol(in)e alpha 2-adrenoceptor agonists, medetomidine, clonidine and UK 14,304 on extraneuronal levels of noradrenaline in the rat frontal cortex: evaluation using in vivo microdialysis and synaptosomal uptake studies.

1. In vivo microdialysis in halothane-anaesthetized rats and synaptosomal [3H]-noradrenaline uptake studies in vitro were used to evaluate the effects of imidazole (medetomidine) and imidazoline (clonidine and UK 14,304) alpha 2-adrenoceptor agonists on extraneuronal levels of noradrenaline in the frontal cortex. 2. Levels of noradrenaline in the dialysate were increased by a depolarizing concentration of K+ (60 mM for 20 min) and substantially attenuated by reducing Ca2+ supply in the perfusate. These results suggest that spontaneous efflux of noradrenaline in the cortex is regulated predominantly by cation-dependent exocytotic mechanisms. 3. At a low perfusion concentration (0.5 microM), medetomidine, clonidine and UK 14,304 all reduced the level of noradrenaline in cortical dialysates. Continuous perfusion of the selective alpha 2-adrenoceptor antagonist, atipamezole (0.5 microM) caused a sustained increase in noradrenaline efflux and reversed the inhibitory effects of medetomidine. All these changes are consistent with drug actions at presynaptic alpha 2-adrenoceptors. 4. Higher concentrations of medetomidine (5-50 microM), but not clonidine or UK 14,304, evoked a non-desensitizing increase in noradrenaline efflux. This effect was not antagonized by 0.5 microM atipamezole. 5. The tricyclic noradrenaline reuptake inhibitor, desmethylimipramine (0.5-50 microM), increased noradrenaline efflux in a concentration-dependent manner. 6. The specific uptake of [3H]-noradrenaline into cortical synaptosomes was inhibited by medetomidine and desmethylimipramine with IC50 values of approximately 7 microM and 8 microM respectively. Neither clonidine nor UK 14,304 inhibited [3H]-noradrenaline uptake. 7. These results indicate that micromolar concentrations of the selective alpha2-adrenoceptor agonist,medetomidine, can augment extraneuronal levels of noradrenaline in the rat frontal cortex; this effect seems to involve an inhibition of noradrenaline reuptake rather than an action at alpha2-adrenoceptors.

Influence of percutaneous transluminal coronary angioplasty on cardiac release of endothelin, neuropeptide Y and noradrenaline

In the present study, the cardiac outflow of endothelin, noradrenaline and neuropeptide Y was investigated in 13 patients undergoing first time coronary angioplasty (PTCA) due to stenosis of the left anterior descending coronary artery. During PTCA there was an increase in the coronary sinus levels of endothelin but no detectable changes in neuropeptide Y or noradrenaline concentrations. It is therefore concluded that endothelial damage rather than myocardial ischaemia is the cause of endothelin release during PTCA.

Actions of morphine on noradrenaline efflux in the rat locus coeruleus are mediated via both opioid and α2 adrenoceptor mechanisms

A recent report showed that morphine inhibited [3H]clonidine binding to human platelet alpha 2 receptors. As the analgesic effects of morphine and clonidine are clinically additive, we investigated the possibility that morphine might stimulate alpha 2 receptors or alpha 2 mechanisms in rat locus coeruleus (LC) slices. Stimulated LC noradrenaline efflux was measured by fast cyclic voltammetry. Cumulatively applied morphine 10(-8)-10(-4) mol litre-1 had no effect on noradrenaline efflux evoked by pseudo-single-pulse stimulations (20 pulses at 100 Hz) while the alpha 2 agonist dexmedetomidine 2 x 10(-10)-10(-7) mol litre-1 decreased efflux of noradrenaline in a concentration-dependent manner. Administration of single concentrations of morphine 10(-6)-10(-4) mol litre-1 significantly decreased efflux of noradrenaline (by 22% and 17%, respectively) and attenuated the effect of dexmedetomidine in a concentration-dependent fashion. Morphine 10(-6)-10(-4) mol litre-1 also decreased efflux of noradrenaline on long stimulus trains (50 pulses at 50 Hz). These data suggest that the analgesic potentiation of alpha 2 and opioid agonists is not mediated via LC alpha 2 receptors.

Electrically-evoked release of taurine in the rat vas deferens: evidence for a purinoceptor-mediated effect.

Release of taurine evoked by electrical stimulation (2700 pulses; 5 Hz; 10 mA unless stated otherwise) and its dependence on noradrenaline and ATP was studied in isolated, perifused rat vas deferens. Outflow of noradrenaline was also measured in some experiments. The basal outflow of taurine averaged 3.90 +/- 0.32 nmol/g tissue per min. Electrical stimulation increased the outflow to about 4 times basal values. The electrically-evoked overflow averaged 128.0 +/- 11.7 nmol/g. An increase in current strength to 40 mA increased the evoked overflow by about 50%. At either current strength, the evoked overflow of taurine (and noradrenaline) was abolished by tetrodotoxin. Ca(2+)-deprivation blocked the overflow of taurine elicited by 10 mA and increased the overflow elicited by 40 mA pulses (but abolished noradrenaline overflow under either condition). Neither prazosin nor pretreatment of the rats with reserpine reduced electrically-evoked overflow of taurine (although reserpine pretreatment abolished evoked noradrenaline overflow). Tyramine (100 mumols/l; 9 min) caused an overflow of taurine 36% of that caused by electrical stimulation (but an overflow of noradrenaline 3 times higher than that evoked by electrical stimulation). Exogenous noradrenaline (9 min) caused a concentration-dependent overflow of taurine with a maximal effect at 162 mumol/l, amounting to 33% of the electrically-evoked overflow. alpha,beta-Methylene ATP (19 mumols/l) elicited an overflow of taurine that faded despite continued exposure to the drug and amounted to 62% of the response to electrical stimulation. Thirty minutes after the start of application of alpha,beta-methylene ATP, electrically-evoked overflow of taurine was greatly reduced. Suramin (100 mumols/l) also reduced taurine overflow in response to electrical stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Further studies on the mechanism of pulmonary efflux of 5-hydroxytryptamine and noradrenaline

The aim of this study was to investigate the transporters that mediate 5-hydroxytryptamine (5HT) and noradrenaline (NA) efflux from pulmonary endothelial cells (PEC). Rat lungs were isolated and perfused with Krebs containing 1 nM 3H-5-HT or 3U_hlA at ‘47Or fnr In min Imatahnli-rinn nnmimalc inhihitaA I, I”_ u. Y, v I”, I” 11,111 \“*‘...U”“L”I~ ““Ly”“,~, ““““,rru, and were then perfused for 21 min with Krebs. Venous effluent samples (1 -min) were collected during this efflux period. When 18 PM tryptamine was added at the 15th min of efflux, there was a rapid increase in the fractional rate of loss (FRL) of 3H-5HT, with a peak effect of 0.0408 -C 0.0078 min-’ (n=6), that was abolished if 1 PM citalopram (Cit) was present throughout efflux (n = 4). but was unaffected (n= 4) by 1 PM desipramine ,n,, \A/hrsn 1 ,A” Ar\n~m;nn ,,-,A \n,ac ~A_-,n.-, a+ +hr, 1 E+h _:m (V~U,. ““llG,l 1 p,“t U”pY”““r \Yr¶, vvua cluuru ar LIB= 1.311, I,,,,, of efflux, there was a rapid increase in the 3H-NA FRL, with a peak effect of 0.0285 + 0.0031 min-’ (n=4), that was reduced by 69% by 1 PM Des (n=4) and was unaffected by 1 /IM Cit (n=4). Hence, 5-HT efflux from PEC of rat lungs is mediated by the 5-HT transporter (5-HTT) and not by uptake, (U,), whereas NA efflux is mediated by U, and not the 5-HTT. Further experiments showed that 50% of the maximal DAinduced increase in FRL of 3H-NA occurred at 0.266 ,uM DA (95% CL: 0.186-0.372 /IM; n = 18). The processes contributing to NA efflux from the PEC were investigated in experiments in which monoamine oxidase was only partially inhibited and the efflux of both 3H-NA and its deaminated metabolite were measured before and after addition of 0.35 NM (n=5) or 1.5 PM (n = 6) Des. Analysis of the data showed that, in rat lungs, U, contributes 81% and diffusion 19% to the total NA efflux aid 90% of this NA is subject to reuptake into PEC by U,.

α2 D-Adrenoceptors modulate renal noradrenaline release in normotensive and spontaneously hypertensive rats

The aim of the present study was to investigate α-adrenoceptor modulation of neurotransmission in kidneys of spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rat (WKY) and to classify the prejunctional α 2-adrenoceptor subtype involved. Kidneys of SHR (12–14 weeks) and age-matched WKY were isolated and the sympathetic nerves were stimulated electrically. The renal nerve stimulation-induced outflow of endogenous noradrenaline was measured by high-pressure liquid chromatography with electrochemical detection (HPLC-ECD). In the presence of uptake blockade by cocaine and corticosterone, the renal nerve stimulation-induced outflow of endogenous noradrenaline was significantly greater in SHR than in WKY at 1 Hz (688 ± 42 pg/g vs. 525 ± 46 pg/g) and 4 Hz (5265 ± 495 pg/g vs. 3544 ± 245 pg/g). At a stimulation frequency of 1 Hz the α 2-adrenoceptor agonist UK 14304 (0.01-1 μM) potently inhibited the renal nerve stimulation-induced outflow of noradrenaline with a pEC 50 of 7.49 (7.32–7.68) and a maximum of 91% in SHR and with a pEC 50 of 7.46 (7.30–7.74) and a maximum of 92% in WKY. α-Adrenoceptor antagonist affinity estimates (p K B values) against UK 14304 at the prejunctional α 2-autoreceptors were determined. The rank order of affinities (phentolamine > rauwolscine > 2-(2,6-dimethoxyphenoxyethyl) aminomethyl-1,4-benzodioxane HCl (WB 4101) > prazosin) was identical in SHR and WKY, and comparison with data from radioligand binding, molecular cloning and functional studies in other tissues and cell lines indicates that prejunctional α 2-autoreceptors of SHR and WKY kidneys are of the α 2 D subtype.

Previous stress increases in vivo biogenic amine response to swim stress

In vivo microdialysis was used to determine biogenic amines in medial prefrontal cortex of rats exposed to eight minutes of swim stress on two consecutive days. On the first day of stress, norepinephrine (NE) efflux increased by 183% over baseline after stress, while dopamine (DA) and serotonin (5-HT) remained stable throughout. On the second day of stress, a robust increase was observed in all 3 neurotransmitters measured, with (NE), (DA), and (5-HT) increasing by 310%, 441% and 496% respectively, and remaining elevated for an hour or more after stress. This suggests that the first exposure to swim stress, while not causing dramatic changes in biogenic amine release, may sensitize biogenic amines in medial prefrontal cortex to subsequent swim stress. Our results also serve as preliminary data concerning the neurochemical changes which might underlie the forced swimming model of "behavioral despair".

Cardiac Noradrenaline Release Accelerates Adenosine Formation in the Ischemic Rat Heart: Role of Neuronal Noradrenaline Carrier and Adrenergic Receptors

In the present study the hypothesis was tested that local noradrenaline release contributes to adenosine formation in myocardial ischemia. Therefore, in ischemic non-working rat hearts either adrenergic receptors or ischemia-evoked noradrenaline release were blocked. Noradrenaline and adenosine were determined in the effluent using HPLC-methods. Following 20 min of stop of perfusion flow both the β-adrenergic receptor antagonist bisoprolol (91.6 ± 10.5 nmol/g) and the inhibitor of ischemia/induced noradrenaline release desipramine (108.5 ± 12.5 nmol/g) caused a suppression of adenosine release (control: 140.9 ± 7.3 nmol/g). To examine the time-course of the release, further experiments were performed at constant perfusion flow with energy metabolism blocked by cyanide together with removal of glucose from the perfusion buffer. This condition resulted in a nearly simultaneous release of adenosine and noradrenaline from the hearts. The β-adrenoceptor blocking agents atenolol and bisoprolol postponed the release of adenosine, whereas the α-antagonists prazosin and yohimbine had no effect on adenosine release induced by cyanide. None of the adrenergic receptor blockers affected the release of noradrenaline. The inhibitors of the neuronal noradrenaline carrier (uptake), desipramine, oxaprotiline, and cocaine suppressed the release of noradrenaline during cyanide administration, indicating a carrier-mediated efflux of noradrenaline. Reduction of extracellular noradrenaline by these agents coincided with a delay of adenosine release (cumulative release within 20 min—control: 251.2 ± 13.9, desipramine: 172.1 ± 15.3, oxaprotiline 36.5 ± 5.8, cocaine: 111.8 ± 23.6 nmol/g). Desipramine and cocaine were also used during administration of exogenous noradrenaline in normoxic hearts, to confirm specificity of their action. Under these condition, desipramine and cocaine increased the concentration of extracellular noradrenaline, which paralleled by an augmentation of adenosine release into the effluent of the hearts (control: 32 ± 8, desipramine: 103 ± 15, cocaine: 79 ± 9 nmol/g). Comparable to the findings in energy depleted hearts, only the administration of β-antagonists suppressed adenosine formation during noradrenaline infusion in normoxic hearts. In conclusion, in ischemic hearts adenosine formation is accelerated by the release of endogenous noradrenaline acting primarily via β-adrenergic receptor stimulation.

Outflow of dopamine and noradrenaline originating from l-DOPA and [formula omitted] in rat renal tissues

1. The present study has examined the formation and outflow of newly-formed dopamine (DA) and noradrenaline (NA) in slices of the renal cortex of rats given l-α-3,4-dihydroxyphenylalanine ( l-DOPA) (10, 30 or 100 mg/kg i.p.) and l-threo-3-(3,4- dihydroxyphenyl)serine ( l-DOPS) (10, 30 or 100 mg/kg i.p.), respectively. The outflow of 3,4-dihydroxyphenylacetic acid (DOPAC) and 3,4-dihydroxyphenylglycol (DOPEG), the deaminated metabolites of DA and NA, respectively, was also measured. 2. The accumulation of both newly-formed DA and DOPAC in renal tissues after the administration of l-DOPA was found to be dose dependent; after 30 or 100 mg/kg l-DOPA, the levels of both DA and DOPAC were, respectively, 3- and 20-fold those observed after the administration of 10 mg/kg l-DOPA. The outflow of DA and DOPAC in kidney slices of rats treated with l-DOPA was found to progressively decline with time and reflected the DA and DOPAC tissue contents. The rate constant (k) for DOPAC efflux ( k = 0.0097) was higher ( P < 0.01) than that for DA efflux ( k = 0.0033) and did not depend on the dose of l-DOPA. DOPAC/DA perifusate ratios were 2-fold those occurring in the tissues. 3. The levels of NA in renal tissues of rats given 30 and 100 mg/kg l-DOPS were, respectively, 3- and 6-fold those observed after the administration of 10 mg/kg l-DOPS. The administration of l-DOPS was also found to be accompanied by the accumulation in renal tissues of DOPEG; this was, however, not dose dependent. 4. The outflow of NA progressively declined with time and also reflected the tissue contents of NA; the outflow of DOPEG in the first collection period was found not to differ from those in subsequent collection periods. The rate constant for the loss of DOPEG ( k = 0.0483) was 10-fold that for the loss of NA ( k = 0.038). The DOPEG/NA ratio in the tissue was significantly lower than that in perifusate samples. In conclusion, the results presented here show that in cortical slices of the rat kidney a ranking order was obtained for the rate constants of efflux of k DOPEG ⪢ k DOPAC > k DA = k NA suggesting that the outflow of DA, NA, DOPAC and DOPEG formed in tubular epithelial cells depends on their relative lipophilicity, as has been described in other cell types loaded with these catechol derivatives. These results also show that the amount of DA and NA which is leaving the compartment where the synthesis has occurred is a constant source for deamination into, respectively, DOPAC and DOPEG.

Norepinephrine and dihydroxyphenylglycol effluxes from sympathetic nerve endings during hypoxia and reoxygenation in the isolated rat heart.

The present experiments were carried out in isolated rat hearts perfused according to the Langendorff method at a constant pressure of 10 kPa. The aim was to measure norepinephrine (NE) overflow and its deaminated metabolite dihydroxyphenylglycol (DOPEG) by changing the composition of the buffer perfusing the heart to simulate hypoxia. When aerobic and glycolytic pathways were simultaneously reduced, NE and DOPEG overflow increased 711 and 145%, respectively, after 30 min, compared with control values of 0.45 +/- 0.06 and 0.66 +/- 0.7 ng.min-1.g-1 of heart (n = 8, p < 0.05). Whereas NE leakage decreased sharply after reoxygenation and glucose addition, DOPEG continued to increase up to 260% after 5 min of normal reperfusion. This mechanism was calcium independent and inhibited by 80% with desipramine (1 microM), confirming the role of the uptake I carrier, which reversed its normal transport direction. Neuropeptide Y, a marker of exocytotic release, did not increase in the perfusate with the progression of hypoxia, which supports the hypothesis of a nonexocytotic release. Tyramine (1 microM) significantly enhanced NE outflow by displacing the amine from its storage vesicles through a calcium-independent mechanism, indicating that a pool of NE was still available. In the presence of 1 microM clorgyline (a monoamine oxidase A inhibitor) but not deprenyl (a monoamine oxidase B inhibitor), NE outflow increased 934% and DOPEG only 40% at 30 min (n = 6, p < 0.05 versus control hearts).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of angiotensin II on noradrenaline release in the human forearm.

The aim was to test the hypothesis that in normal humans angiotensin II would stimulate local release of noradrenaline under basal conditions or during a sympathetic stimulus provided by lower body negative pressure (LBNP). Nine healthy volunteers received intra-arterial infusions of angiotensin II, 5 ng.min-1, into the non-dominant forearm. Forearm blood flow (strain gauge plethysmography) and regional noradrenaline spillover (using the tracer methodology of Esler) were measured during angiotensin II alone, LBNP alone, and LBNP plus angiotensin II. Angiotensin II and LBNP decreased forearm blood flow comparably: from 3.1(SD 1.5) to 2.4 (0.9) ml.100 g-1.min-1 during angiotensin II, p < 0.05; and from 3.3(1.5) to 2.5(1.0) ml.100 g-1.min-1 during LBNP, p < 0.05 (p = NS, A-II v LBNP). Angiotensin II had no effect on forearm venous noradrenaline or regional noradrenaline spillover. LBNP increased venous noradrenaline outflow from the forearm, from 1.6(0.40) to 2.1(0.6) nmol.min-1 (p < 0.05), while regional noradrenaline spillover tended to increase, rising from 1.5(0.8) to 2.0(1.0) nmol.100 ml-1.min-1. Angiotensin II did not enhance forearm blood flow or noradrenaline responses to LBNP. In the human forearm, mildly vasoconstrictor infusions of angiotensin II do not increase local release of noradrenaline, either alone or during mild LBNP. At least under these conditions, angiotensin II would not appear to be a potent influence on local sympathetic activity.

Functional relation between nitric oxide and noradrenaline for the modulation of vascular tone in rat mesenteric vasculature

As previously reported, N omega-nitro-L-arginine (L-NNA), an inhibitor of nitric oxide (NO) synthesis, decreased transmural field stimulation (TFS)-induced noradrenaline overflow from the isolated perfused rat mesenteric vasculature attached to the intestine. The decrease was attenuated by L-arginine. This suggests that NO may increase noradrenaline release (Yamamoto et al. 1993). The present experiments with this preparation were done in order to monitor changes in vascular perfusion pressure caused by TFS or by noradrenaline infusion in parallel with those in the noradrenaline outflow caused by TFS in the presence of atropine (0.1 mumol/l) (to block acetylcholine-induced release of endothelial NO) and of indomethacin (3 mumol/l) (to inhibit L-NNA-induced production of vasoconstrictor prostanoids). (1) TFS (2-10 Hz) caused a frequency-dependent increase in noradrenaline overflow and perfusion pressure. (2) L-NNA (10 and 30 mumol/l) caused a concentration-dependent inhibition of TFS-induced noradrenaline overflow, whereas the TFS-induced pressure increase was augmented by L-NNA in a concentration-dependent manner. At any given concentration of L-NNA, the potentiation of vasoconstriction by L-NNA became greater in magnitude as the frequency of the TFS was raised. (3) Infusion of noradrenaline (0.38-6 nmol) caused a dose-dependent increase in perfusion pressure up to a value comparable with that caused by TFS. The pressure increase in response to noradrenaline infusion was also enhanced by L-NNA, relatively, to a greater extent than the enhancement, by L-NNA, of the pressure response to TFS.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemical evidence for the regulation of central noradrenergic activity by 5-HT 1A and 5-HT 2 receptors: Microdialysis studies in the awake and anaesthetized rat

Here we have studied the effect of various 5-HT 1A and 5-HT 2 receptor-selective drugs on noradrenaline release in the hippocampus of anaesthetized and awake rats using microdialysis. In the anaesthetized rat, administration of the 5-HT 1A agonists buspirone, gepirone and ipsapirone increased noradrenaline levels in the microdialysates. However, the common metabolite of these compounds, 1-PP (an α-2 adrenoceptor antagonist with low affinity for 5-HT 1A receptors), also increased noradrenaline efflux whilst the 5-HT 1A receptor agonist 8-OH-DPAT and MDL 73005EF, which are not metabolized to 1-PP, did not. In the awake rat, buspirone but also 8-OH-DPAT increased noradrenaline efflux. A similar effect was observed in response to MDL 73005EF and the 5-HT 1A ligand NAN-190. Since the latter two drugs have weak intrinsic activity at the post-versus presynaptic 5-HT 1A receptor, a presynaptic mechanism (inhibition of 5-HT release) was implicated. The 5-HT 2 receptor may be important to this mechanism as noradrenaline increased following administration of the 5-HT 2 receptor antagonists, ritanserin and ICI 170,809. In conclusion, our data indicate that there are clear differences in the effects of 5-HT 1A and 5-HT 2 receptor-selective drugs on noradrenaline efflux in hippocampus of the anaesthetized versus awake rat. Our findings are reconcilable with the hypothesis that in the awake (but not anaesthetized) rat, release of noradrenaline in hippocampus is influenced by an inhibitory tone mediated via 5-HT 2 receptors. If this inhibitory tone is removed, either by decreasing 5-HT release through activation 5-HT 1A autoreceptors or by blocking postsynaptic 5-HT 2 receptors, noradrenaline release increases.

Antiepileptics inhibit cortical N-methyl- D-aspartate-evoked [ 3H]norepinephrine efflux

The antiepileptic drugs phenytoin, valproic acid and phenobarbital were examined for their ability to inhibit N-methyl- D-aspartate (NMDA)-stimulated [ 3H]norepinephrine efflux from rat brain cortical slices. All three drugs inhibited efflux at varying concentrations. Valproic acid was the most potent and inhibited efflux at 0.01 mg/ml. Phenytoin and phenobarbital inhibited efflux at 0.1 mg/ml. These results indicate that some antiepileptic drugs are capable of inhibiting NMDA receptor function in the therapeutic range.

The potential of intact human platelets for sulfoconjugation of norepinephrine in vitro

The aim of the present study was to investigate the potential of human platelets for sulfoconjugation of plasma norepinephrine. For this purpose the uptake and the efflux of norepinephrine and norepinephrine sulfate were studied in vitro in intact human platelets. Incubation of platelets with 0.5 μmol/1 norepinephrine led to a rapid uptake of norepinephrine within 1 min (22.76±4.14 pmol/2.5 × 10 9 platelets/min), whereby 63.7% was transformed to norepinephrine sulfate. The substrate kinetics showed a different slope of norepinephrine uptake with increasing norepinephrine concentrations in the incubation medium (0.1−0.5 μmol/1). At 0.1 μmol/1 norepinephrine the uptake was more efficient than at higher norepinephrine concentrations. Incubation with norepinephrine sulfate slightly decreased intraplatelet norepinephrine sulfate (p≤0.01). Incubation of human platelets in a catecholamine free medium led to an efflux of norepinephrine and norepinephrine sulfate (21.8%, 11.2% of total intraplatelet content, respectively). The present findings indicate a net uptake of norepinephrine into the platelets. At physiological norepinephrine concentrations the uptake seems to be achieved predominantly by an active transport mechanism. Moreover, data indicate a net efflux of norepinephrine sulfate. Therefore it is concluded that human platelets might contribute to the formation of plasma norepinephrine sulfate by norepinephrine uptake, intraplatelet sulfoconjugation and release of norepinephrine sulfate.