Sympathetic Transmitter Release Research Articles

α-Adrenergic inhibition of sympathetic neurotransmitter release mediated by modulation of N-type calcium-channel gating

In sympathetic neurons, catecholamines interact with prejunctional alpha-adrenergic receptors to reduce delivery of transmitter to postjunctional target organs. This autoinhibitory feedback is a general phenomenon seen in diverse neurons containing a variety of transmitters. The underlying mechanisms of alpha-adrenergic inhibition are not clear, although decreases in cyclic AMP and cAMP-mediated phosphorylation have been implicated. We have studied depolarization-induced catecholamine release and calcium-channel currents in frog sympathetic neurons. Here we show that alpha-adrenergic inhibition of transmitter release can be explained by inhibition of Ca2+-channel currents and not by modulation of intracellular proteins. Noradrenaline strongly reduces the activity of N-type Ca2+ channels, the dominant calcium entry pathway triggering sympathetic transmitter release, whereas L-type Ca2+ channels are not significantly inhibited. The down-modulation of N-type channels involves changes in rapid gating kinetics but not in unitary flux. This is the first detailed description of inhibition of a high-voltage activated neuronal Ca2+ channel at the single-channel level. The coupling between alpha-adrenergic receptors and N-type channels involves a G protein, but not a readily diffusible cytoplasmic messenger or protein kinase C, and may be well suited for rapid and spatially localized feedback-control of transmitter release.

Coffee, catecholamines and cardiac arrhythmia.

It is generally agreed that a high release of noradrenaline in the heart may elicit cardiac arrhythmia. Furthermore, administration of aminophylline to patients with ischaemic heart disease frequently elicits ventricular premature beats and ventricular tachycardia. Recent data from animal experiments demonstrate that methylxanthines (aminophylline, caffeine, theophylline) can facilitate noradrenaline release from sympathetic nerve endings. Such facilitation of transmitter release is amplified when myocardial oxygenation is impaired, stressing its clinical significance in relation to ischaemic heart disease. If a similar amplification is also operative in man, a dose of methylxanthine causing a very modest increase in myocardial sympathetic transmitter release in the heart of a healthy subject may be sufficient to cause a three- to six-fold elevation in the local myocardial concentration of noradrenaline in a patient with ischaemic heart disease. We hypothesize that such hypoxia-induced facilitation of sympathetic transmitter release lies behind the reported connection between cardiac events and methylxanthines, for instance sudden cardiac death following coffee consumption.

Role of the coronary endothelium in the regulation of sympathetic transmitter release in isolated rabbit hearts

The roles of endothelium-derived relaxing factor (EDRF) and endothelin in the regulation of vascular tone are intensely studied at present. Since factors which directly affect vascular tone also frequently modulate sympathetic transmitter release, we found it of interest to study whether EDRF or endothelin displays such modulatory activity as well. Isolated rabbit hearts were perfused according to Langendorff, and the release of transmitter induced by sympathetic nerve stimulation was estimated by analysis of the effluent content of noradrenaline (NA) with liquid chromatography. The activity of EDRF spontaneously formed in the heart was counteracted by addition of haemoglobin (Hb, 2.4-15 g l-1) or facilitated by addition of superoxide dismutase (SOD, 14-140 U ml-1), to the perfusion solution. In other experiments authentic endothelin (0.1-10 nm) was given to the heart. Nerve stimulation (5 Hz for 30 s) elicited a release of NA into the cardiac effluent amounting to 319 +/- 28 pmol (n = 53). Hb lowered the coronary flow to 68 +/- 9% (P less than 0.01) and impaired the outflow of NA to 62 +/- 9% of control (P less than 0.01). SOD facilitated coronary flow by 11 +/- 4% (P less than 0.005), and augmented the outflow of NA by 15 +/- 6% (P less than 0.05). Endothelin dose-dependently inhibited the coronary flow, with an IC50 of about 1 nM, and in parallel decreased the efflux of NA. Mechanical obstruction of the coronary flow induced an attenuation of the efflux of NA that was quantitatively similar to the flow reduction.(ABSTRACT TRUNCATED AT 250 WORDS)

Presynaptic α-adrenoceptor regulation of transmitter release in the conscious rat

1. 1. Blood pressure (BP) and heart rate (HR) were recorded in conscious, adrenal demedulated rats. The rats were subjected to a 1-min period of mild “stress”, induced by jet-air directed into the experimental cage. The plasma content of noradrenaline (NA), dopamine (DA) and 3, 4-dihydroxy-phenylacetic acid (DOPAC) was determined 1 min after termination of “stress”. 2. 2. The presynaptic α 2-adrenoceptor antagonist yohimbine (YOH) (10 −7 − 10 −6 mol/kg, given 5 min prior to “stress”) did not alter the increase in BP and HR, induced by “stress”, when compared to control rats (receiving NaCl instead of YOH at the corresponding time). 3. 3. Pre-treatment with atropine (ATR) (2.5 mg/kg) 5 min before YOH (10 −6 mol/kg) or NaCl increased HR but not BP significantly in both groups of rats. “Stress”, as above, gave a significant prolongation of the increase in HR in rats receiving YOH, when compared to control rats. The increase in BP was not significantly altered, compared to controls. 4. 4. The neuronal re-uptake inhibitor desroethylimipramine (DMI) (0.1 mg/kg) was given together with ATR (2.5 mg/kg) 5 min before YOH (10 −6 mol/kg) or NaCl in one group of rats. This treatment induced a significant increase in HR but did not affect BP. “Stress”, induced as above, extended the duration of the increase in HR in the YOH-treated rats, compared to controls. The increase in BP was not significantly different between the YOH-treated rats and the controls. 5. 5. Plasma levels of NA, DA and DOPAC were measured 1 min after termination of “stress” in rats pre-treated with ATR (2.5 mg/kg) and DMI (0.1 mg/kg) 10 min before “stress”. Five minutes prior to “stress”, the animals received YOH (10 −6 mol/kg) or NaCl. The group receiving YOH showed significantly higher plasma levels of NA, compared to controls. DA and DOPAC levels were not affected. 6. 6. Our findings support the theory that presynaptic α 2-adrenoceptor modulation of sympathetic transmitter release exists in vivo. It is also indicated that maintenance of homeostasis is more dependent on other mechanisms, such as vagal influence.

Changes in calcitonin release during sympathetic nerve degeneration after superior cervical ganglionectomy of rats.

To assess the role of peripheral sympathetic nerves in the regulation of calcitonin release, rats subjected to superior cervical ganglionectomy (SCGx) 16-28 h earlier were used. The time periods selected allowed us to examine C cell response during the supraliminal release of sympathetic transmitter that accompanies anterograde degeneration of nerve varicosities as well as during the neural paralysis that ensues thereafter. At the time intervals examined, SCGx did not result in significant changes of basal serum calcitonin or Ca levels. The intraperitoneal administration of CaCl2 brought about an impending increase of serum Ca to the same extent in SCGx and sham-operated rats. A significant depression of calcitonin release was observed in rats killed around the time of nerve terminal degeneration (16-21 h post SCGx) but not about 10 h later. Additionally a delay to achieve a maximal calcitonin response was apparent during nerve degeneration. Injection of the alpha-adrenoceptor blocker phenoxybenzamine significantly increased basal calcitonin levels and restored the depressed calcitonin response to hypercalcemia seen in SCGx rats. Treatment with the beta-adrenoceptor-blocker propranolol counteracted phenoxybenzamine activity but was unable to modify per se calcitonin release in SCGx or sham-operated rats. Basal Ca levels and their increase after intraperitoneal CaCl2 were similar in all examined groups regardless of the drug injected. In an additional experiment phenoxybenzamine injected into SCGx rats in doses one-fifth those employed earlier still reversed both the depression in maximal calcitonin response as well as the delay to attain maximal release after CaCl2, but was unable to affect basal calcitonin levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Facilitation of noradrenaline release by gallamine in the rat salivary gland.

The effect of gallamine on spontaneous and stimulation-evoked overflow of tritium was studied in the submandibular gland of the rat. The gland was perfused retrogradely and labeled with 3H-noradrenaline. The stimulation-evoked (1 Hz for 60 s) overflow of tritium was facilitated by increasing concentrations of gallamine (0.3-20 mM). None of the concentrations of gallamine increased the spontaneous overflow of the tritium. The facilitatory effect of gallamine was observed in 0.3 to 5 mM calcium medium; the maximum facilitation was observed at the normal concentration of calcium (2.5 mM). The facilitatory effect of gallamine was inversely related to the frequency of stimulation (10-fold facilitation at 1 Hz and 3-fold at 10 Hz). Stimulation of the salivary gland by a single pulse (1 ms duration) in the normal medium did not evoke an overflow of tritium; however, the same stimulus produced a marked increase in the overflow in the presence of gallamine. The facilitatory action of gallamine on the release of sympathetic transmitter is ascribed to the enhanced availability of calcium ions to the secretory process resulting from blockade of potassium conductance during nerve activity.

Influence of Felodipine and Verapamil on the Sympathetic Transmitter Release and on the Pre- and Post-Junctional Effects of Exogenous Noradrenaline in the Perfused Rat Kidney

The effects of various concentrations (0.01 to 10 mumol/L) of felodipine and verapamil on the peripheral sympathetic function were studied in perfused rat kidney preparations in vitro. Neither of these agents inhibited stimulus-induced 3H-noradrenaline (norepinephrine) release (at 0.5 and 2.0 Hz). Higher concentrations of felodipine (10 mumol/L) and verapamil (1 and 10 mumol/L) significantly potentiated the transmitter release at both frequencies of stimulation. Unlike phentolamine, felodipine 10 mumol/L enhanced the ability of exogenous noradrenaline to inhibit 3H-noradrenaline-release, indicating that this vasodilator did not antagonise prejunctional alpha 2-receptors. The effects of verapamil 1 mumol/L were similar to that of felodipine in this respect; however, verapamil in higher concentrations (10 mumol/L) produced slight but significant attenuation of exogenous noradrenaline-induced inhibition of the transmitter release. Both felodipine and verapamil produced concentration-dependent inhibition of the vasoconstrictor responses to exogenous noradrenaline. It is concluded from these studies that the effects of these agents on the transmitter release may not have any clinical significance, but their ability to attenuate vascular effects of noradrenaline could contribute to their antihypertensive properties.

Clonidine and noradrenaline fail to inhibit vagal induced bradycardia

Guinea pig isolated right atria were used to test for inhibitory prejunctional α-adrenoceptors on terminal cardiac vagal varicosities. These receptors have been considered to be involved in reciprocal inhibition of transmitter release. Electrical field pulses delivered during the atrial refractory period caused biphasic changes in atrial period. An immediate bradycardia was followed by a more gradual tachycardia. An estimation of both parasympathetic and sympathetic transmitter release following a single stimulation was made possible by the tempral separation of the peak brahycardia and tachycardia. In one set of atria two stimulus-response curves were constructed with a 30 min interval. The sympathetic (tachycardia) stimulus-response curve was stable, but the second parasympathetic (bradycardia) curve was significantly attenuated compared with the first. In a second set of atria clonidine (1 μM) was added to the bathing solution after the first curve. In these tissues the tachycardia phase of the responses was almost abolished in the second curve. The parasympathetic responses were similar to those in the time control tissues. In a third set of tissues phentolamine (1 μM) was added after the first curve. This caused a significant increase in the resting period (bradycardia). There was no significant difference between the first and the second curves in these tissues. In a fourth group of atria propranolol (1 μM) was added after the first curve. This caused a significant attenuation of the sympathetic responses, but the parasympathetic responses were not different from the first curve. A third set of stimuli was applied after half of the tissues were exposed to noradrenaline (1 μM). The third curves were not different from the second curves with or without the presence of noradrenaline. These experiments suggest that there are no inhibitory prejunctional α-adrenoceptors on the terminal vagal varicosities in the guinea pig right atrium. This finding provides evidence against the hypothesis of reciprocal inhibition of transmitter release.

Does presynaptic regulation of sympathetic transmission occur within a limited range of neuronal activity?

Recent reports indicate that the presynaptic negative feedback mechanism involved in regulating sympathetic neurotransmission is only functional within a narrow range of neuronal activity. The specific aim of this report is to examine the release of sympathetic transmitter at varying frequencies of stimulation, and the effects of alpha-adrenoceptor antagonists on the overflow. The experiments were carried out in the 3H-noradrenalin-labeled heart of the guinea pig. The overflow of tritium (per pulse) increased from a stimulation frequency of 0.125 to 10 Hz and declined at 30 Hz. Phentolamine or yohimbine produced facilitation of the overflow from 0.125 to 10 Hz; the effect was more pronounced at lower frequencies of stimulation. We conclude that the presynaptic alpha-adrenoceptor-mediated negative feedback mechanism operates over a wide range of cardiac sympathetic nerve activity.

Does Presynaptic Regulation of Sympathetic Transmission Occur Within a Limited Range of Neuronal Activity?

Recent reports indicate that the presynaptic negative feedback mechanism involved in regulating sympathetic neurotransmission is only functional within a narrow range of neuronal activity. The specific aim of this report is to examine the release of sympathetic transmitter at varying frequencies of stimulation, and the effects of α-adrenoceptor antagonists on the overflow. The experiments were carried out in the3H-noradrenaline-labeled heart of the guinea pig. The overflow of tritium (per pulse) increased from a stimulation frequency of 0.125 to 10 Hz and declined at 30 Hz. Phentolamine or yohimbine produced facilitation of the overflow from 0.125 to 10 Hz; the effect was more pronounced at lower frequencies of stimulation. We conclude that the presynaptic α-adrenoceptor-mediated negative feedback mechanism operates over a wide range of cardiac sympathetic nerve activity.

Relative location of alpha- and beta-adrenoceptors to sites of release of sympathetic transmitter in the rabbit facial vein.

We studied alpha- and beta-adrenoceptor-mediated responses of rabbit facial vein rings to adrenergic stimulation to determine the location of the two types of receptors in relation to the sympathetic nerve terminals. Transmural electrical nerve stimulation (TNS) at low frequency elicited large beta-receptor-mediated relaxation responses in rings pretreated with phentolamine (6 x 10(-7)M). These responses were significantly greater than the corresponding alpha-receptor-mediated contractions in rings pretreated with propranolol (10(-6)M). Blockade of neuronal uptake with desmethylimipramine (DMI, 10(-7)M) increased significantly the neurogenic relaxation but had little effect on neurogenic contractility. DMI pretreatment caused a shift to the left (x 5.6) in the relaxant dose-response curve to exogenous l-norepinephrine (NE). The NE contractile dose-response curve was also shifted to the left after DMI pretreatment but by a significantly smaller amount (x 3.1). Neurogenic activation of beta-receptors evoked almost maximal relaxations in facial vein rings (85% at 2 Hz), whereas the maximum neurogenic contraction was approximately half the maximum contraction with exogenous NE (40% at 6 Hz). These results imply that the beta-adrenoceptors in the rabbit facial vein are located in close proximity to sites of adrenergic transmitter release and neuronal reuptake, whereas the alpha-receptors are more distant.

Prostaglandin-mediated inhibition of noradrenaline release: V. A comparison of the neuroinhibitory effect of three prostaglandins: E 2, I 2, and 6-keto-PGF 1α

The effect of PGE 2, PGI 2, and 6-keto-PGF 1α respectively on the contractile response of the isolate , field-stimulated guinea pig vas deferens was investigated. All three PGs were capable of inhibiting the contractile responses of the vas deferens, but the concentrations required varied considerably: PGE 2 was about 700 times more active than PGI 2 and about 4600 times more active than 6-keto-PGF 1α in this respect. It is suggested that PGI 2, although formed in tissues with sympathetic innervation, does not play a physiological role as inhibitor of sympathetic transmitter release.

Prostaglandin-mediated inhibition of noradrenaline release: III. Separation of prostaglandins released from stimulated hearts and analysis of their neurosecretion inhibitory capacity

Isolated rabbit hearts were infused with 14C-arachidonic acid and subjected to sympathetic nerve stimulation. Prostaglandins in the cardiac effluent were extracted and separated using thin layer chromatography. Other hearts were infused with un-labelled arachidonic acid and the effluent was assayed for neurosecretion inhibitory capacity on the field-stimulated guinea pig vas deferens, and for anti-aggregatory activity on ADP-induced platelet aggregation. PGs in the effluent from hearts infused with un-labelled arachidonic acid were extracted and separated on TLC, and the different fractions were assayed for neurosecretion inhibitory activity. Sympathetic nerve stimulation after preincubation with 14C-AA elicited outflow of four different peaks of 14C-labelled PGs: one chromatographing close to PGF 2α (probably mainly 6-keto-PGF 1α), and three peaks corresponding to PGA 2/PGB 2, PGD 2, and PGE 2 respectively. The cardiac interstitial effluent contained anti-aggregatory material which was inactivated by heat treatment, and thus probably identical to PGI 2. The cardiac effluent also contained material with neuro-secretion inhibitory activity, which was resistant to heat treatment. Fractional assay of the TLC separated cardiac effluent demonstrated that the neurosecretion inhibitory activity chromatographed with PGE 2 only. It has earlier been observed that endogenous PGs inhibit trans-mitter release in sympathetically stimulated organs. On the basis of the current data we suggest that PGE 2 is the only physiological inhibitor of sympathetic transmitter release.

Uptake and Retention of 14C‐Bretylium in Degenerating Postganglionic Sympathetic Nerves of the Rat

Abstract The uptake and retention of 14C‐bretylium was studied in rat salivary glands and irides in vivo at different time intervals after sympathetic denervation or decentralization. The uptake of bretylium was increased on the denervated side during a time period which precedes the degeneration release of sympathetic transmitter, but was later on reduced. On the other hand, the retention of bretylium on the denervated side was already markedly reduced during the time period preceding the onset of the degeneration transmitter release. In experiments with chronically denervated salivary glands, or glands atrophied by means of excretory duct ligation, a pronounced extraneuronal accumulation was observed. The extraneuronal accumulation of bretylium may partly mask the changes in disposition of the drug induced by the sympathetic denervation. The results are in accordance with the hypothesis that bretylium must be associated with special sites at the adrenergic nerve terminals in order to exert its degeneration delaying effect.

Potentiation of the Contractile Response of Isolated Aortae to Transmural Stimulation by Angiotensin

Electrical transmural stimulation (0.3 msec duration at frequencies of 5, 20 and 100/sec) was given to spirally-cut strips of the ascending aorta from rabbits. Angiotensin (5 × 10−11 to 10−9 M) significantly potentiated the contractile response to transmural stimulation but did not influence the response to exogenously-applied noradrenaline. In response to angiotensin, percent increase in the duration of contraction induced by transmural stimulation was approx. proportional to that in the developed tension, whereas a prolongation of the duration by cocaine greatly exceeded the increase in the tension. The contractile response to transmural stimulation was suppressed by 5 × 10−6M bretylium. The inhibitory1 effect of bretylium was not antagonized by angiotensin when administered prior to bretylium and after the bretylium-induced inhibition had been established. Cocaine reversed the inhibition by bretylium to a potentiation. The Mg++-induced inhibition of the response to transmural stimulation was not reversed by angiotensin but by excess Ca++. It appears that the angiotensin-induced potentiation of the contractile response to transmural neural stimulation is due mainly to a facilitation of the release of noradrenaline and that angiotensin does not participate in Ca++-Mg++ interactions relating to the release of sympathetic transmitters.

Potentiation of the contractile response of isolated aortae to transmural stimulation by angiotensin.

Abstract Electrical transmural stimulation (0.3 msec duration at frequencies of 5, 20 and 100/sec) was given to spirally-cut strips of the ascending aorta from rabbits. Angiotensin (5 × 10−11 to 10−9 M) significantly potentiated the contractile response to transmural stimulation but did not influence the response to exogenously-applied noradrenaline. In response to angiotensin, percent increase in the duration of contraction induced by transmural stimulation was approx. proportional to that in the developed tension, whereas a prolongation of the duration by cocaine greatly exceeded the increase in the tension. The contractile response to transmural stimulation was suppressed by 5 × 10−6M bretylium. The inhibitory1 effect of bretylium was not antagonized by angiotensin when administered prior to bretylium and after the bretylium-induced inhibition had been established. Cocaine reversed the inhibition by bretylium to a potentiation. The Mg++-induced inhibition of the response to transmural stimulation was not reversed by angiotensin but by excess Ca++. It appears that the angiotensin-induced potentiation of the contractile response to transmural neural stimulation is due mainly to a facilitation of the release of noradrenaline and that angiotensin does not participate in Ca++-Mg++ interactions relating to the release of sympathetic transmitters.

Colchicine-induced delay of the degeneration release of sympathetic transmitter in the conscious rat.

Colchicine-induced delay of the degeneration release of sympathetic transmitter in the conscious rat.

Release of prostaglandin-like material from dog kidney during nerve stimulation

Canine kidneys were autoperfused in situ at constant flow in 28 experiments. Renal venous effluent was examined for (PLM) prostaglandin-like material following acidic lipid extraction and thin layer chromatography or by continuous bioassay utilizing the bloodbathed organ technique. There was a low basal efflux of PLM from the kidney which was markedly enhanced during the renal vascular constriction elicited by renal nerve stimulation at 2-10 cycles/second or by infusion of norepinephrine. The PLM was tentatively identified as an E-type prostaglandin based on chromatographic behavior and bioassay. The present study indicates that PLM is released from the dog kidney during an increase in renal vascular resistance. Release is not necessarily dependent upon changes in total renal blood flow although alterations in the distribution of intrarenal blood flow might be involved. (authors)

Interactions between inhibitors of the axonal amine pump and bretylium in the degeneration release of sympathetic transmitter.

AbstractLundberg, D. Interactions between inhibitors of the axonal amine pump and bretylium in the degeneration release of sympathetic transmitter. Acta physiol. scand. 1970. 78. 503–521.The time course of the degeneration contraction of periorbital smooth muscles has been studied in conscious rats given different inhibitors of the axonal mine pump either alone or in combination with bretylium. Desmethylimipramine (DMI) or cocaine at 20 mg/kg did not influence the time of onset of the degeneration contraction while Lu 3–010 (10 mglkg) caused a somewhat premature start. Lu 3–010, DMI or protriptyline at 10 mg/kg but not trimipramine or cocaine at 20 mglkg antagonized bretylium. They reduced and prevented (DMI) the bretylium‐induced delay. DMI reduced the delay by a non‐competitive mechanism. The antagonism to bretylium does not seem to be explainable by simple interference with the axonal amine pump. Indirect evidence suggests that the duration of the delay induced by bretylium is related to the rate of disappearance of bretylium from critical sites in the nerve endings. –DMI and Lu 3–010 seem to have somewhat different actions on the degenerating adrenergic nerve terminals.