Axoplasmic transport of [3H]ouabain binding sites and catecholamine secretion from an adrenergic nerve trunk.

The presence of a functional Na+/Ca2+ exchange system was explored in the ligated cat hypogastric nerve, a preparation that has been proposed as a model of giant noradrenergic nerve terminal free of effector cells. The rationale for this study was to monitor noradrenaline secretion from the ligated cat hypogastric nerve promoted by the increase in intracellular Ca2+ levels after ouabain blockade of Na+,K(+)-ATPase molecules present in the plasma membrane of the ligated cat hypogastric nerve. Such an increase in intracellular Ca2+ levels is achieved by activation, in "reverse mode," of the Na+/Ca2+ exchange system. In the present study, [3H]ouabain binding sites were identified on crude preparations of hypogastric nerve membranes. A single, high affinity (Kd around 10 nM), binding site was observed in both ligated and nonligated nerves. The number of binding sites increased with the time of ligation, reaching a peak of about 1 pmol/mg of protein 48 hr after ligation. Blockade of these binding sites by ouabain induced a dose-dependent, Ca(2+)-dependent release of noradrenaline, with an ED50 around 50 microM. The maximum release amounted to 9% of the total noradrenaline content in the cells. As would be expected for ouabain-induced noradrenaline secretion mediated by a Na+/Ca2+ exchange system working in reverse mode, the effect of ouabain was dependent upon the presence of Na+ in the incubation medium, reaching a plateau at an extracellular Na+ concentration of 100 mM. Calcium uptake after Ca2+ reintroduction in ouabain-treated nerves increased with time of ligation, suggesting the incorporation of Na+/Ca2+ exchange carrier molecules into the axolemma of hypogastric nerves. The similarity between ouabain-induced noradrenaline secretion from the ligated cat hypogastric nerve and from other adrenergic systems strongly supports the idea that the ligated cat hypogastric nerve is equipped with a functional Na+/Ca2+ exchange system that would contribute to the regulation of intracellular Ca2+ levels. Furthermore, these data, together with previously published reports, fully characterize, from a biochemical point of view, the ligated hypogastric nerve as a model of giant noradrenergic nerve terminal free of effector cells.     

3H]noradrenaline release evoked by selegiline ((-)-deprenyl) in the isolated main pulmonary artery of the rabbit

High concentrations of selegiline[-)-deprenyl) (greater than 10(-5) M) enhanced the nerve stimulation (2 Hz)-evoked release of [3H]noradrenaline from the isolated main pulmonary artery of the rabbit. This facilitation of stimulation-evoked [3H]noradrenaline release by selegiline was reduced by exogenous (-)-noradrenaline, an agonist of presynaptic alpha 2-adrenoceptors. This inhibitory action of (-)-noradrenaline was partly antagonized by yohimbine, a selective alpha 2-adrenoceptor blocker. When the stimulation-evoked [3H]noradrenaline release had already been increased by inhibition of Na+-pump (K+-free solution), selegiline further enhanced the nerve-evoked release of labelled neurotransmitter.     

IONIC MECHANISMS REGULATING SODIUM ENTRY INTO VASCULAR SMOOTH MUSCLE

1. Na+/H+ exchange, an ethylisopropylamiloride (EIPA)-sensitive Na+ and HCO3- dependent system and a diisothio-cyanatostilbenedisulphonic acid (DIDS)-sensitive Na+ and HCO3- transporter, contribute to sodium influx and intracellular pH (pHi) regulation in vascular smooth muscle. 2. In cultured cells from the human internal mammary artery, Na+/H+ exchange and the EIPA-sensitive Na+ and HCO3- dependent system contribute about 80% to basal sodium influx. The residual Na+ influx is both EIPA and DIDS-insensitive. 3. Sodium influx via these mechanisms influences the ability of vascular smooth muscle to synthesize protein late in the G1 phase of the mitotic cell cycle. This, in turn, affects DNA biosynthesis. 4. These Na+ exchanges/transporters have the capability to facilitate the development of vascular hypertrophy in hypertension.     

Differential control of Ca2+-dependent [3H]noradrenaline release from rat brain slices through presynaptic opiate receptors and alpha-adrenoceptors

The inhibitory actions of the Ca2+ antagonist Cd2+, morphine and noradrenaline (exogenously added + endogenously released) on electrically evoked release of [3H]noradrenaline from superfused rat neocortical slices were strongly reduced when release was enhanced by 4-aminopyridine. In the presence of 4-aminopyridine the release inhibiting effects of these drugs were restored by lowering the extracellular Ca2+ concentration. When release was enhanced by prolonging the pulse duration, only the release inhibiting effect of noradrenaline was reduced but the effects of Cd2+ and morphine were unchanged. Irrespective of the pulse duration, blockade of presynaptic alpha-adrenoceptors with phentolamine did not affect the release inhibiting effects of Cd2+ and morphine. The inhibitory effects of morphine and noradrenaline remained unchanged in Cl--free medium. Furthermore, these drugs strongly reduced the [3H]noradrenaline release induced by 20 mM K+ in the presence of tetrodotoxin. The results suggest that activation of presynaptic opiate-receptors inhibits Ca2+ entry through voltage-sensitive Ca2+ channels, whereas presynaptic alpha-adrenoceptors affect a step in the secretory process subsequent to Ca2+ influx. Moreover, the involvement of (direct) changes in Na+, K+ or Cl- permeability appears unlikely for both receptor systems.     

MODULATION OF RENAL NORADRENALINE RELEASE BY PREJUNCTIONAL RECEPTOR SYSTEMS IN VITRO

1. The amount of noradrenaline released per nerve impulse from renal sympathetic nerves can be modulated through specific prejunctional receptors. 2. Under in vitro conditions activation of alpha 1-, alpha 2-, prostaglandin, adenosine, dopamine and serotonin receptors inhibits noradrenaline release from the kidney. 3. Stimulation of prejunctional beta-adrenoceptors, probably of the beta 2-subtype, as well as stimulation of prejunctional angiotensin II receptors facilitates noradrenaline release. Moreover, neuronally released noradrenaline inhibits its own release through activation of both prejunctional alpha 1- and alpha 2-adrenoceptors. 4. Locally produced PGE2, which is formed and released via stimulation of postjunctional alpha 1-adrenoceptors, as well as adenosine, released from postjunctional sites by renal nerve stimulation (RNS), seems to inhibit noradrenaline release through their specific prejunctional receptor systems.     

The relationship between nerve terminal adenosine triphosphatases and neurotransmitter release: as determined by the use of antidepressant and other CNS-active drugs.

1 The role of adenosine triphosphatases (ATPases) in neurotransmitter release was studied using nerve terminals (synaptosomes) prepared from rat cerebral cortex as a model. 2 Amitriptyline, nortriptyline, protriptyline, desipramine and imipramine were found to inhibit ATPases at concentrations of 10(-5) M and above. The drugs inhibited both the basal and electrically evoked release of acetylcholine (ACh) and noradrenaline (NA) at concentrations of 10(-4) M and above. 3 At low concentrations of antidepressants (10(-8) and 10(-7) M) release of NA was enhanced but there was no effect on ACh release. 4 Other drugs which inhibit Na+, K+-ATPase increase basal NA release as did drugs which inhibited vesicular MG2+-ATPase. 5 A model is proposed suggesting that transmitter release/re-uptake depends on (1) active Na+, K+-ATPase at the presynaptic membrane and (2) an active synaptic vesicular MG2+-ATPase.     

Electrogenic Na+/Ca2+-exchange regulation by alpha2-receptor in peripheral sympathetic nerve

Electrical depolarisation-(2 Hz, 1 ms)-induced [3H]noradrenaline ([3H]NA) release was measured from the isolated main pulmonary artery of the rabbit in the presence of uptake blockers (cocaine, 3 x 10(-5)M; corticosterone, 5 x 10(-5)M) and after blocking the MAO-enzyme by pargyline (1,2 x 10(-4)M). Substitution of most of the external Na+ by Li+ (113 mM; [Na+]0: 25 mM) slightly potentiated the stimulation-induced release of [3H]NA in a tetrodotoxin (TTX, 10(-7)M) sensitive manner. The reverse Na+/Ca2+-exchange inhibitor KB-R7943 (3 x 10(-5)M) failed to inhibit the stimulation-evoked release of [3H]NA, but increased the resting outflow of neurotransmitter. The 'N-type' voltage-sensitive Ca2+-channel (VSCC) blocker omega-conotoxin (omega-CgTx) GVIA (10(-8)M) significantly and irreversibly inhibited the release of [3H]NA on stimulation (approximately 60-70%). The 'residual release' of NA was abolished either by TTX or by reducing external Ca2+ from 2,5 to 0,25 mM. The 'residual release' of NA was also blocked by the non-selective VSCC-blocker neomycin (3 x 10(-3)M). Direct correlation was obtained between the extent of VSCC-inhibition and the transmitter release enhancing-effect of presynaptic alpha2-receptor blocker yohimbine (3 x 10(-7)M). When the release of [3H]NA was blocked by omega-CgTx GVIA plus neomycin, yohimbine was ineffective. Inhibition of the Na+-pump by removal of K+ from the external medium increased both the resting and the stimulation-evoked release of [3H]NA in the absence of functioning VSCCs (i.e. in the presence of neomycin and after omega-CgTx treatment). Under these conditions the stimulation-evoked release of NA was abolished either by TTX or by external Ca2+-removal (+1 mM EGTA). Similarly, external Li+ (113 mM) or the reverse Na+/Ca2+ exchange blocker KB-R7943 (3 x 10(-5)M) significantly inhibited the nerve-evoked release of NA in 'K+-free' solution. KB-R7943 decreased the resting outflow of NA as well. Under conditions, in which the Na+-pump was inhibited in the absence of functioning VSCCs, yohimbine (3 x 10(-7)M) further enhanced the release of neurotransmitter, while l-noradrenaline (l-NA, 10(-6)M), an agonist of presynaptic alpha2-receptors, inhibited it. The yohimbine-induced enhancement of NA-release was abolished by Li+-substitution and significantly inhibited by KB-R7943 application. It is concluded that after blockade of VSCCs, brief depolarising pulses may reverse Na+/Ca2+-exchange and release neurotransmitter in Na+-loaded sympathetic nerves. Further, similar to that of VSCCs, the reverse Na+/Ca2+-exchange may also be inhibited by presynaptic alpha2-receptor activation.     

MODULATION OF NORADRENALINE RELEASE IN VIVO THROUGH PREJUNCTIONAL α-ADRENOCEPTORS

1. Extensive in vitro studies have suggested that noradrenaline release from sympathetic nerve endings is modulated by alpha 2-adrenoceptors on the terminal varicosities, activation of which by alpha-adrenoceptor agonists or neuronally released noradrenaline leads to inhibition of transmitter release. 2. Studies in intact animals support essentially the physiological operation of this mechanism, whereas human studies have reached mixed conclusions and more information is required.     

Presynaptic alpha-adrenoceptors and opiate receptors: inhibition of [3H]noradrenaline release from rat cerebral cortex slices by different mechanisms

The inhibitory effects of morphine and Cd2+ on electrically evoked [3H]noradrenaline release from superfused brain slices were unaffected when release was enhanced by increasing the pulse duration, while the inhibitory effect of noradrenaline and the enhancing effect of phentolamine were diminished. A similar enhancement of [3H]noradrenaline release by 4-aminopyridine reduced the modulatory effects of all drugs examined. Therefore there seem to be different mechanisms for the effect of presynaptic alpha-adrenoceptors and opiate receptors on the availability of Ca2+ for the stimulus-secretion coupling process in noradrenergic nerve terminals.     

Endothelin receptor blockade attenuates air embolization-induced pulmonary hypertension in sheep

CuCl2, ZnCl2 and NiCl2, but not CdCl2 or CoCl2, induced transmitter release from superfused rat hippocampal and striatal synaptosomes preloaded with, respectively, [3H]noradrenaline and [3H]dopamine. Cu2+ was the most potent and effective, acting in a concentration- (0.1-300 microM) and time-dependent (peak effect occurring at 2-3 min) manner. The amount of Cu2+-induced release over a 5 min period is similar to that induced by depolarization with high KCl or the K+ channel blocker 4-aminopyridine. However, the time course of the Cu2+-induced release is slower and the effect of Cu2+ is not reversed by washout. Cu2+-induced catecholamine release requires extracellular calcium (Ca2+) and is inhibited by the Ca2+ channel blocker Cd2+, and in the case of noradrenaline, by the voltage-gated Na+ channel blocker tetrodotoxin. The ability of Cu2+ to induce massive Ca2+-dependent transmitter release from brain catecholaminergic nerve terminals may contribute to the neuropathological processes associated with Cu2+ toxicity in Wilson's disease.     

POPULATION-BASED SURVEY OF HUMAN SODIUM AND POTASSIUM EXCRETION

1. During the 1989 National Heart Foundation Risk Factor Prevalence Survey a subsample in Hobart collected 24 h urine samples to measure electrolyte excretion. 2. The ranges were 30-344 mmol/24 h for Na+ excretion (mean 160 mmol/24 h for men, 124 mmol/24 h for women), and 25-119 mmol/24 h for K+ excretion (mean 77 mmol/24 h for men, 68 mmol/24 h for women). 3. As in other surveys, women excreted about 20-25% less Na+ and K+ than men, although there was no significant sex difference in the ratio of Na+/K+ excretion. 4. The recommended dietary intake (RDI) for Na+ and K+ was followed simultaneously by 19% of subjects, and 13% had a 24 h urinary Na+/K+ ratio less than or equal to 1.0. 5. Observance of the RDI limited the value of iodized salt for goitre prophylaxis. 6. Sodium excretion rates were outside the therapeutic range of thiazide diuretics in 22% of subjects. 7. Diet groups for long-term prospective cohort studies to test the prophylactic value of avoiding salt could apparently be recruited from existing subsamples of the population.     

Muscarinic inhibition of potassium-induced noradrenaline release and its dependence on the calcium concentration

1. Noradrenaline release from the isolated rabbit heart was evoked by perfusion with a medium containing 135 mM potassium and 17 mM sodium ions (high K+-low Na+). 2. The noradrenaline output in response to high K+-low Na+ was dose-dependently decreased by methacholine (0.625-320 muM) and this effect was reserved by atropine 1.44 mM. 3. Lowering the calcium concentration of high K+-low Na+ from 1.8-0.1125 mM decreased the noradrenaline output by 85%. The effect of methacholine, expressed as % inhibition of noradrenaline release, was potentiated by lowering of the calcium concentration. 4. Both at normal and lowered calcium concentrations the inhibitory action of methacholine was larger from 0-5 than from 5-10 min after perfusion with high K+-low Na+. 5. Perfusion of hearts with media containing high K+-low Na+ or normal K+-low Na+ caused noradrenaline outputs somewhat smaller than those after high K+-low Na+. The release from 0-5 min was both calcium-dependent and inhibited by methacholine. 6. High K+ and/or low Na+ solutions caused an increase in coronary perfusion pressure which was little affected by the noradrenaline released simultaneously. 7. It is concluded that activation of muscarine receptors at the terminal adrenergic fibre decreases the availability of calcium for transmitter release.     

EFFECTS OF SODIUM RESTRICTION ON CARDIAC 3,4-DIHYDROXYPHENYLETHYLENE GLYCOL AND CATECHOLAMINE LEVELS IN RATS

1. The effects of low or high sodium intake for 4 weeks on cardiac catecholamines and 3,4-dihydroxyphenylethylene glycol (DHPG) levels were studied in adult female Wistar rats. 2. Rats receiving the low sodium diet had significantly higher plasma renin activity than rats receiving the high sodium diet. 3. Dopamine concentrations in both the right and left ventricle were significantly higher in the low salt compared with the high salt rats, but noradrenaline, adrenaline and DHPG levels did not differ significantly between the two groups. 4. These data do not support previous reports that sodium restriction reduces cardiac noradrenaline release, but suggest that alterations in sodium intake may influence cardiac noradrenaline metabolism.     

Properties of the peripheral opiate receptors in the cat nictitating membrane.

The noradrenaline overflow and the contractile response elicited by nerve stimulation of the muscle were inhibited by 1 micron morphine in the cat nictitating membrane. This concentration of morphine did not modify the response of the muscle to exogenous noradrenaline. The inhibitory effect of morphine was increased by low Na+ (50 mM), whereas the capacity of naloxone as antagonist to morphine was higher with 150 mM than with 50 mM Na+. These results suggest that the peripheral opiate receptors which interact with noradrenergic neurotransmission could show a sodium allosteric transformation similar to that described for the brain opiate receptor. The effect of morphine was enhanced by manganese ion in the presence of normal Na+. The responses of the cat nictitating membrane to nerve stimulation were not altered in the presence of the protein modifying reagent DTNB [5,5'-dithiobis-(2-nitrobenzoic acid)] but the effect of morphine on the adrenergic neurotransmission was diminished by DTNB with 150 mM Na+. It is postulated that the affinity of the ligands for presynaptic receptors which regulate adrenergic neurotransmission might be modified during the physiological changes in ion concentration which accompany nerve depolarization.     

3,4-Diaminopyridine-evoked noradrenaline release in rat hippocampus: role of Na+ entry on Ca2+ pools and of protein kinase C.

Slices of rat hippocampus, preincubated with [3H]noradrenaline [(3H]NA), were superfused continuously and stimulated by addition of 3,4-diaminopyridine (3,4-DAP; 100 microM) for 10 min to the superfusion medium. An overflow of 3H evoked by 3,4-DAP (representing [3H]NA release) was measurable not only in the presence but also in the absence of extracellular Ca2+. Both the protein kinase C (PKC) activator 4 beta-phorbol 12,13-dibutyrate (4 beta-PDB) and the PKC inhibitor polymyxin B, affected mainly the evoked release in the absence of extracellular Ca2+ in a facilitatory or inhibitory manner, respectively. Moreover, in the absence of extracellular Ca2+, both the 3,4-DAP-evoked [3H]NA release and the facilitatory effect of 4 beta-PDB were abolished in the presence of tetrodotoxin or in the absence of Na+ in the superfusion medium. Ruthenium red, a blocker of mitochondrial Ca2+ reuptake, potently increased 3,4-DAP-evoked [3H]NA release in Ca(2+)-free EGTA-containing medium. The facilitatory effects of ruthenium red and 4 beta-PDB were additive. From these and earlier observations we conclude (1) that the mechanism of 3,4-DAP-evoked [3H]NA release involves both Ca2+ influx into the nerve terminals and mobilization of intraneuronal Ca2+ pools. Most probably Ca2+ release from cytoplasmic Ca2+ stores (e.g. endoplasmic reticular pools or mitochondria) is induced by Na+ ions entering the nerve endings during 3,4-DAP-evoked repetitive action potentials. (2) The facilitatory effect of phorbol ester on 3,4-DAP-evoked NA release appears to be mediated not by changes in Ca2+ influx, but by enhancement of intraneuronal events distal to Na+ ion entry and increased intracellular Ca2+ availability.     

Enhancement of arachidonic acid release and prostaglandin F(2alpha) formation by Na3VO4 in PC12 cells and GH3 cells

Both activation of phospholipase A2 causing arachidonic acid release and tyrosine phosphorylation have been proposed to be involved in neuronal functions. Previously, we reported that orthovanadate (Na3VO4), an inhibitor of tyrosine phosphatases, stimulated tyrosine phosphorylation in proteins and enhanced Ca2+-induced noradrenaline release in rat pheochromocytoma PC12 cells. However, the role of tyrosine phosphorylation on phospholipase A2 activity and/or arachidonic acid release in neuronal cells has not been well established. The effects of Na3VO4 on arachidonic acid release and prostaglandin F(2alpha) formation were investigated in two types of neuronal cell lines. In PC12 cells, addition of Na3VO4 stimulated [3H]arachidonic acid release and prostaglandin F(2alpha) formation in a concentration-dependent manner. Co-addition of 5 mM Na3VO4 enhanced ionomycin-stimulated [3H]arachidonic acid release. Na3VO4 also enhanced ionomycin-stimulated [3H]arachidonic acid release from GH3 cells, a clonal strain from rat anterior pituitary. These findings suggest that the tyrosine phosphorylation pathway regulates arachidonic acid release by phospholipase A2 and prostaglandin F(2alpha) formation in neuronal cells.     

In vivo demonstration of H3-histaminergic inhibition of cardiac sympathetic stimulation by R-alpha-methyl-histamine and its prodrug BP 2.94 in the dog

1. The aim of this study was to investigate whether histamine H3-receptor agonists could inhibit the effects of cardiac sympathetic nerve stimulation in the dog. 2. Catecholamine release by the heart and the associated variation of haemodynamic parameters were measured after electrical stimulation of the right cardiac sympathetic nerves (1-4 Hz, 10 V, 10 ms) in the anaesthetized dog treated with R-alpha-methyl-histamine (R-HA) and its prodrug BP 2.94 (BP). 3. Cardiac sympathetic stimulation induced a noradrenaline release into the coronary sinus along with a tachycardia and an increase in left ventricular pressure and contractility without changes in mean arterial pressure. Intravenous administration of H3-receptor agonists significantly decreased noradrenaline release by the heart (R-HA at 2 micromol kg(-1) h(-1): +77 +/- 25 vs +405 +/- 82; BP 2.94 at 1 mg kg(-1): +12 +/- 11 vs +330 +/- 100 pg ml(-1) in control conditions, P < or = 0.05), and increases in heart rate (R-HA at 2 micromol kg(-1) h(-1): +26 +/- 8 vs +65 +/- 10 and BP 2.94 at 1 mg kg(-1): +30 +/- 8 vs 75 +/- 6 beats min(-1), in control conditions P < or = 0.05), left ventricular pressure, and contractility. Treatment with SC 359 (1 mg kg(-1)) a selective H3-antagonist, reversed the effects of H3-receptor agonists. Treatment with R-HA at 2 micromol kg(-1) h(-1) and BP 2.94 at 1 mg kg(-1) tended to decrease, while that with SC 359 significantly increased basal heart rate (from 111 +/- 3 to 130 +/- 5 beats min(-1), P < or = 0.001). 4. Functional H3-receptors are present on sympathetic nerve endings in the dog heart. Their stimulation by R-alpha-methyl-histamine or BP 2.94 can inhibit noradrenaline release by the heart and its associated haemodynamic effects.     

Stimulation by vanadate of [3H]noradrenaline release from rabbit pulmonary artery and its inhibition by noradrenaline

Vanadate, the +5 oxidation state of vanadium, present in mammalian tissues, even in nerve tissue, and a competitive inhibitor of NaK-ATPase, significantly enhanced the release of [3H]noradrenaline evoked from rabbit isolated perfused pulmonary artery by electrical stimulation. Its effect proved to be concentration-dependent. Noradrenaline (10(-6) M) reduced the vanadate-potentiated release of [3H]noradrenaline. The effect of noradrenaline is mediated via alpha 2-adrenoceptors as evidenced by the finding that yohimbine 3 x 10(-7) M prevented its action. The effect of vanadate was dependent on external K ions. When the effect of vanadate on [3H]noradrenaline release was studied under conditions when the NaK-ATPase enzyme activity was inhibited by removal of external K for 45 min, vanadate was ineffective. This finding indicates that the effect is related to the inhibition of NaK-ATPase activity, a condition known to result in transmitter release.     

Negative inotropic and arrhythmic effects of high doses of ciguatoxin on guinea-pig atria and papillary muscles

Ciguatoxin, the toxin present in fish responsible for ciguatera, at doses equal or above the maximum positive inotropic dose in atria (greater than 0.15 mouse units/ml) induced arrhythmias in atria and papillary muscles stimulated at 1 Hz and dose-dependent negative inotropy in atria. Negative inotropy was enhanced by ouabain or by an increase in stimulation to 3 Hz, little affected by procaine or increasing Ringer [Ca2+] and reversed by lidocaine and tetrodotoxin (TTX). Ciguatoxin caused negative inotropy associated with cell depolarisation in 1.2 mM Ca2+-Ringer and additionally caused signs of Ca overload in 3.2 mM Ca2+-Ringer. Ciguatoxin induced transient after-contractions and contracture in atria which were common in 3.2 mM but not 1.2 mM Ca2+-Ringer and which were enhanced by ouabain. TTX and lidocaine abolished after-contractions and contracture while procaine was less effective. Extrasystoles consisting of short bursts of 1-2 extra contractions per sec were seen in atria and papillary muscles within 45 min of ciguatoxin being added. The effect was observed in 3.2 mM but seldom in 1.2 mM Ca2+-Ringer and was absent when low doses of propranolol or TTX were added prior to ciguatoxin. Flutter was observed in a few papillary muscles after ciguatoxin. These results suggest that the toxic effects of ciguatoxin stem from its direct action of opening myocardial Na+ channels. Extrasystoles appeared to result mainly from its effect on neural Na+ channels causing an increased release of noradrenaline from the nerves associated with the myocardium.     

Noradrenaline synthesis after sympathetic nerve activation in rat atria and its dependence on calcium but not CAM kinase II and protein kinases A or C.

1. The biosynthesis of noradrenaline following sympathetic nerve activation was investigated in rat atria. In particular the time course of noradrenaline synthesis changes, the relationship of changes in synthesis to transmitter release and the possible roles of second messengers and protein kinases were examined. 2. Rat atria incubated with the precursor [3H]-tyrosine synthesized [3H]-noradrenaline. Synthesis was enhanced following pulsatile electrical field stimulation (3 Hz for 5 min) with the bulk of the increase occurring in the first 45 min after the commencement of electrical stimulation. In separate experiments rat atria were pre-incubated with [3H]-noradrenaline and the radioactive outflow in response to electrical field stimulation (3 Hz for 5 min) was taken as an index of noradrenaline release. 3. Stimulation-induced (S-I) noradrenaline synthesis was significantly correlated to S-I noradrenaline release for a variety of procedures which modulate noradrenaline release by mechanisms altering Ca2+ entry into the neurone (r2 = 0.99): those which decreased release: tetrodotoxin (0.3 microM), Ca(2+)-free medium, lowering the frequency of nerve activation to 1 Hz, and those which increased release, tetraethylammonium (0.3 mM), phentolamine (1 microM) and the combination of phentolamine (1 microM) and adenosine (10 microM). On the strength of this relationship we suggest that Ca2+ entry is a determining factor in S-I synthesis changes rather than the amount of noradrenaline released. Indeed the reduction in noradrenaline release with the calmodulin-dependent protein (CAM) kinase II inhibitor KN-62 (10 microM) which acts subsequent to Ca2+ entry, did not affect S-I synthesis. 4. The cell permeable cyclic AMP analogue, 8-bromoadenosine 3'',5''-monophosphate (BrcAMP, 90 and 270 microM), dose-dependently increased basal [3H]-noradrenaline synthesis in unstimulated rat atria. This effect was antagonized by the selective protein kinase A (PKA) antagonist, Rp-8-chloroadenosine 3'',5''-cyclic monophosphorothioate (RClcAMPS, 300 microM), suggesting that PKA activation enhances basal noradrenaline biosynthesis in sympathetic nerve terminals. 5. The protein kinase inhibitors, KN-62 (CAM kinase II, 10 microM), RClcAMPS (PKA, 300 microM), polymyxin B (protein kinase C (PKC), 21 microM) and staurosporine (PKC, PKA and CAM kinase II, (0.1 microM) did not affect S-I synthesis, although KN-62, polymyxin B and staurosporine decreased S-I release. We conclude that S-I synthesis is triggered by Ca2+ entering the neurone but that the signalling pathway does not involve classical protein kinases and appears distinct from the steps involved in transmitter release.