Stimulation-evoked Release Research Articles

Calcium channels involved in the inhibition of acetylcholine release by presynaptic muscarinic receptors in rat striatum.

1. The mechanism of the inhibitory action of presynaptic muscarinic receptors on the release of acetylcholine from striatal cholinergic neurons is not known. We investigated how the electrically stimulated release of [3H]-acetylcholine from superfused rat striatal slices and its inhibition by carbachol are affected by specific inhibitors of voltage-operated calcium channels of the L-type (nifedipine), N-type (omega-conotoxin GVIA) and P/Q-type (omega-agatoxin IVA). 2. The evoked release of [3H]-acetylcholine was not diminished by nifedipine but was lowered by omega-conotoxin GVIA and by omega-agatoxin IVA, indicating that both the N- and the P/Q-type (but not the L-type) channels are involved in the release. The N-type channels were responsible for approximately two thirds of the release. The release was >97% blocked when both omega-toxins acted together. 3. The inhibition of [3H]-acetylcholine release by carbachol was not substantially affected by the blockade of the L- or P/Q-type channels. It was diminished but not eliminated by the blockade of the N-type channels. 4. In experiments on slices in which cholinesterases had been inhibited by paraoxon, inhibition of [3H]-acetylcholine release by endogenous acetylcholine accumulating in the tissue could be demonstrated by the enhancement of the release after the addition of atropine. The inhibition was higher in slices with functional N-type than with functional P/Q-type channels. 5. We conclude that both the N- and the P/Q-type calcium channels contribute to the stimulation-evoked release of acetylcholine in rat striatum, that the quantitative contribution of the N-type channels is higher, and that the inhibitory muscarinic receptors are more closely coupled with the N-type than with the P/Q-type calcium channels.

Nitric oxide (NO) increases acetylcholine release from and inhibits smooth muscle contraction of guinea-pig gastric fundus

Experiments were carried out to investigate the interaction between nitric oxide (NO) and cholinergic neurotransmission in smooth muscle strips of guinea-pig gastric fundus. Electrical field stimulation (2 Hz, 1 ms, 360 shocks) evoked atropine-sensitive contractions. Dimethylphenylpiperazinium (DMPP) (100 μM), a nicotinic receptor agonist, reversed the stimulation-evoked contraction and resulted in relaxation. Nω-nitro-L-arginine (L-NNA) (100 μM), an NO synthase inhibitor, significantly increased the amplitude of stimulation-evoked contraction and abolished the effect of DMPP. Electrical stimulation increased the release of [ 3H]acetylcholine ([ 3H]ACh) from the tissue strips above the basal levels. Neither L-NNA (100 μM) nor DMPP (100 μM) alone influenced the basal release of [ 3H]ACh. Nω-nitro-L-arginine (100 μM) decreased the electrical stimulation-evoked release of [ 3H]ACh. Dimethylphenylpiperazinium increased the stimulation-evoked release of [ 3H]ACh but had no effect in the presence of L-NNA. It is suggested that in guinea-pig gastric fundus, endogenous NO released in response to field stimulation has an opposite effect at the pre- and postsynaptic sites: it increases the release of ACh from cholinergic nerve terminals but reduces smooth muscle responses to ACh.

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.

Effects of non-depolarizing neuromuscular blocking agents on norepinephrine release from human atrial tissue obtained during cardiac surgery

We have studied the effect of non-depolarizing neuromuscular blocking agents, at concentrations present in serum during anaesthesia, on release of [3H]-norepinephrine ([3H]NE) from superfused atrial appendage obtained during cardiac surgery from 48 patients. Three of the neuromuscular blocking agents (pancuronium, gallamine and rocuronium), which are known to cause an increase in heart rate during anaesthesia, increased stimulation-evoked release of [3H]NE. In contrast, (+)tubocurarine and pipecuronium, neuromuscular blocking agents that do not cause tachycardia, did not affect release of NE. Org 9487 significantly enhanced release while SZ1677 was ineffective, even at concentrations higher than those expected after administration of a 2 x ED95 dose. Atropine enhanced release. These data suggest that the axon terminals of sympathetic nerves in human heart have muscarinic heteroreceptors whose activation by acetylcholine (ACh) released from the vagal nerve reduces release of NE. This action contributes to lowering of heart rate. Therefore, any neuromuscular blocking agent with antimuscarinic actions and capable of increasing the release of NE may produce tachycardia.

Presynaptic modulation of transmitter release via α2-adrenoceptors: nonsynaptic interactions

It is generally accepted that neurochemical transmission occurring at the synapse is the primary way of sending messages from one neuron to another. Neurotransmitters released from axon terminal in a [Ca2+]0-dependent manner act transsynaptically on the postsynaptic site. The past 30 years have witnessed something of a revolution in the understanding of how neurons communicate with each other. It has been shown that the exocytotic release of transmitters from axon terminals is subject to presynaptic modulation via presynatic hetero- and auto-receptors. For example via stimulation of alpha2-adrenoceptors expressed on varicosities and coupled to G-protein the stimulation-evoked release of different transmitters can be inhibited. This review will focus on nonsynaptic interactions between axon terminals. The present data clearly show that transmitters released from axon terminals without synaptic contact play an important role in the fine tuning of communication between neurons within a neuronal circuit.

Local regulation of vasopressin and oxytocin secretion by extracellular ATP in the isolated posterior lobe of the rat hypophysis.

It is now widely accepted that ATP functions as a signalling substance in the nervous system. The presence of P2 receptors mediating the action of extracellular ATP in brain regions involved in hormonal regulation raises the possibility that a similar role for ATP might also exist in the neuroendocrine system. In this study, the release from the rat isolated neurohypophysis preparation of endogenous ATP, oxytocin and vasopressin (AVP) were measured simultaneously using luciferin-luciferase and RIA techniques. After 70 min preperfusion, electrical field stimulation caused a rapid increase in the amount of ATP in the effluent and the release of AVP and oxytocin also increased stimulation-dependently. Inhibition of voltage-dependent Na+ channels by tetrodotoxin (1 microM) reduced the stimulation-evoked release of AVP and oxytocin; however, the evoked release of ATP remained unaffected. The effect of endogenous ATP on the hormone secretion was tested by suramin (300 microM), the P2 receptor antagonist. Suramin significantly increased the release of AVP, and the release of oxytocin was also enhanced. ATP, when applied to the superfusing medium, decreased the release of AVP, but not that of oxytocin, and its effect was prevented by suramin. ATP (60 nmol), added to the tissues, was readily decomposed to ADP, AMP and adenosine measured by HPLC combined with ultraviolet light detection, and the kinetic parameters of the enzymes responsible for inactivation of ATP (ectoATPase and ecto5'-nucleotidase) were also determined (Km=264+/-2.7 and 334+/-165 microM and vmax=6.7+/-1.1 and 2.54+/-0.24 nmol/min per preparation (n=3) for ectoATPase and ecto5'-nucleotidase respectively). Taken together, our data demonstrate the stimulation-dependent release, P2 receptor-mediated action and extracellular metabolism of endogenous ATP in the posterior lobe of the hypophysis and indicate its role, as a paracrine regulator, in the local control of hormone secretion.

Neurochemical evidence of dopamine release by lateral olivocochlear efferents and its presynaptic modulation in guinea-pig cochlea

In this study, using an in vitro superfusion technique for the first time, we provide direct neurochemical evidence of the transmitter role of dopamine at the level of lateral olivocochlear efferent fibres of the guinea-pig cochlea. Our results revealed that nerve terminals are able to take up and release dopamine upon axonal stimulation. Since dopamine is thought to protect the afferent nerve fibres from damage due to acoustic trauma or ischaemia, enhancement of the release of dopamine, a potential therapeutic site of these injuries, was investigated. Positive modulation of dopamine release has been shown by a D 1 dopamine receptor agonist, an antagonist and piribedil. Furthermore, negative feedback on the stimulation-evoked release of dopamine via D 2 dopamine receptors has been excluded. Electrical stimulation of the cochlear tissue produced a significant and reproducible release of [ 3H]dopamine, which could be blocked by tetrodotoxin (1 μM) and cadmium (100 μM), proving that axonal activity releases dopamine and its dependence on Ca 2+ influx verifies its neuronal origin. Nomifensine, a high-affinity dopamine uptake blocker, prevented the tissue from taking up [ 3H]dopamine from the bathing solution, also indicating the neural origin of dopamine released in response to stimulation. SKF-38393 (a selective D 1 agonist) increased both the resting and electrically evoked release of dopamine. Piribedil (a D 3/D 2/D 1 agonist), a drug under investigation, known to prevent acoustic trauma or ischaemia-induced hearing loss, had a similar and concentration-dependent increasing effect on both resting and evoked release of dopamine. The effect of both drugs on stimulation-evoked release could be prevented by SKF-83566 (a selective D 1 antagonist). However, SKF-83566 alone enhanced the resting and axonal conduction-associated release of dopamine. D 2 agonists and antagonists failed to modulate the release of dopamine, indicating the lack of negative feedback modulation of dopamine release. Our results suggest that the release of dopamine was subjected to modulation by a D 1 receptor agonist and an antagonist. In addition, it is concluded that D 2 receptors are not involved in the modulation of dopamine release. This observation may have clinical relevance in the prevention or therapy of particular types of hearing loss, because enhanced dopaminergic input into the primary auditory neuron may inhibit the (over)excitation of this neuron by glutamatergic input from inner hair cells.

Release of immunoreactive substance P in the brain stem upon stimulation of the cranial dura mater with low pH - inhibition by the serotonin (5-HT1) receptor agonist CP 93,129.

1. The therapeutical benefit of serotonin (5-HT1) receptor agonists in the treatment of migraine headache has been attributed to their inhibitory effect on the release of pro-inflammatory neuropeptides from trigeminal afferents within the cranial meninges. The effect of 5-HT1 receptor agonists on the release of neuropeptides from central afferent terminals has not been examined so far. In the present study in the rat we therefore measured the effect of the 5-HT1B receptor agonist CP 93,129 on the stimulation-evoked release of immunoreactive substance P (ir-SP) in the spinal trigeminal nucleus. 2. To measure release of ir-SP, microprobes coated with antibody to substance P were inserted into the medulla oblongata at the level of the obex. The ipsilateral parietal dura mater encephali was exposed and stimulated with acid phosphate buffered Tyrode solution (pH 5.8). This chemical stimulus increased the release of ir-SP in the medullary dorsal horn. 3. Systemic (i.v.) administration of CP 93,129 (460 nmol kg(-1)) prior to stimulation suppressed the stimulation-evoked increase of release of ir-SP. Local administration of CP 93,129 (10 microM) to the dorsal surface of the medulla had no significant inhibitory effect on the release. 4. It is concluded that systemically applied 5-HT1 receptor agonists reduce the stimulation-evoked release of substance P from the central endings of meningeal afferents in the spinal trigeminal nucleus (medullary dorsal horn). This inhibitory effect may contribute to the antinociceptive effect of 5-HT1 receptor agonists in migraine.

Novel synthesis and release of GABA in cerebellar granule cell cultures after infection with defective herpes simplex virus vectors expressing glutamic acid decarboxylase

The inhibitory amino acid neurotransmitter γ-aminobutyric acid (GABA) is synthesized from glutamate in a single step by the enzyme glutamatic acid decarboxylase (GAD). We sought to determine whether viral vectors containing GAD cDNA could be used to enhance synthesis and stimulation-evoked release of GABA in cultures of CNS neurons. For this purpose, we generated double-cassette defective herpes simplex virus (HSV) vectors that expressed one of the two GAD isoforms (GAD65 or GAD67), and Escherichia coli LacZ. Infection of cerebellar granule cell (CGC) cultures with vectors containing GAD cDNA resulted in a significant increase in isoform-specific expression of GAD, synthesis of GABA, and stimulation-evoked GABA release. GAD65 and GAD67 vector-infected neurons exhibited a comparable profile of GABA levels, synthesis and release, as well as GAD protein distribution. In CGCs cultured for 6 days in vitro (DIV), GABA synthesized after vector-derived GAD expression was released by treatment with glutamate or veratridine, but only in a Ca 2+-independent fashion. In more mature (10 DIV) cultures, both Ca 2+-dependent, K + depolarization-induced, as well as Ca 2+-independent, veratridine-induced, GABA release was significantly enhanced by GAD vector infection. Treatment of CGCs with kainic acid, which destroys most of the GABAergic neurons (<1% remaining), did not prevent vector-derived expression of GAD nor synthesis of GABA. This suggests that defective HSV vector-derived GAD expression can be used to increase GABA synthesis and release in CNS tissue, even in the relative absence of GABAergic neurons. The use of such GAD vectors in the CNS has potential therapeutic value in neurologic disorders such as epilepsy, chronic pain, Parkinson's and Huntington's disease.

Co-release of endogenous ATP and [ 3H]noradrenaline from rat hypothalamic slices: origin and modulation by α2-adrenoceptors

The release of endogenous ATP, measured by the luciferin-luciferase assay, and of [ 3H]noradrenaline from the in vitro superfused rat hypothalamic slices were studied. ATP and [ 3H]noradrenaline were released simultaneously during resting conditions and in response to low and high frequency field electrical stimulation; the release of both substances were frequency dependent between 2 Hz and 16 Hz. The stimulation-induced release of ATP and [ 3H]noradrenaline was diminished by more than 80% under Ca 2+-free conditions. Tetrodotoxin inhibited the majority of the evoked release of both ATP and [ 3H]noradrenaline, however, it was less effective in reducing the release of [ 3H]noradrenaline, than that of ATP. Bilateral stereotaxic injection of 6-hydroxydopamine (4 μg/side) to the ventral part of the ventral noradrenergic bundle, originating from the A1 cell group in the brainstem, resulted in a 55% reduction of endogenous noradrenaline content of the hypothalamic slices, and the tritium uptake and the stimulation-evoked release of [ 3H]noradrenaline was also markedly reduced. While the basal release of ATP was not affected, the evoked release was diminished by 72% by this treatment. Perfusion of the slices with noradrenaline (100 μM) initiated rapid and continuous tritium release; on the other hand, it did not release any ATP. In contrast, 6 min perfusion of (−)nicotine and 1,1-dimethyl-4-phenyl-piperazinium iodide evoked parallel release of ATP and [ 3H]noradrenaline which was inhibited by the nicotinic receptor antagonist mecamylamine; 6-hydroxydopamine lesion of the ventral part of the ventral noradrenergic bundle did not affect the nicotine-evoked ATP and [ 3H]noradrenaline release. While CH 38083, a non subtype-selective α 2-antagonist and BRL44408, the subtype-selective α 2AD antagonist augmented the evoked release of [ 3H]noradrenaline, ARC239, a selective α 2BC antagonist was without effect. In contrast, neither of the α 2-antagonists significantly affected the evoked-release of ATP. In summary, we report here that endogenous ATP and [ 3H]noradrenaline are co-released stimulation-dependently from superfused rat hypothalamic slices. A significant part of the release of both compounds is derived from the nerve terminals, originating from the A1 catecholaminergic cell group of brainstem nuclei. Unlike that from the peripheral sympathetic transmission, noradrenaline and α 1-adrenoceptor agonists were unable to promote the release of ATP. Conversely, parallel ATP and noradrenaline release could be induced by nicotine receptor activation, but this release does not originate from the same nerve endings. The evoked-release of [ 3H]noradrenaline is inhibited by endogenous noradrenaline via α 2AD subtype of adrenoreceptors, while the release of ATP is not subject to this autoinhibitory modulation. In conclusion, our results support the view that ATP is involved in the neurotransmission in the hypothalamus, but the sources of the released ATP and noradrenaline seem to be not identical under different stimulatory and modulatory conditions.

Age-dependent changes of presynaptic neuromodulation via A1-adenosine receptors in rat hippocampal slices

Thepresynaptic neuromodulation of stimulation-evoked release of [3H]-acetylcholine by endogenous adenosine, via A1-adenosine receptors, was studied in superfused hippocampal slices taken from 4-, 12- and 24-month-old rats. 8-Cyclopentyl-1,3-dimethylxanthine (0.25 μM), a selective A1-receptor antagonist, increased significantly the electrical field stimulation-induced release of [3H]-acetylcholine in slices prepared from 4- and 12-month-old rats, showing a tonic inhibitory action of endogenous adenosine via stimulation of presynaptic A1-adenosine receptors. In contrast, 8-cyclopentyl-1,3-dimethylxanthine had no effect in 24-month-old rats. 2-Chloroadenosine (10 μM), an adenosine receptor agonist decreased the release of [3H]-acetylcholine in slices taken from 4- and 12-month-old rats, and no significant change was observed in slices taken from 24-month-old rats. In order to show whether the number/or affinity of the A1-receptors was affected in aged rats, [3H]-8-cyclopentyl-1,3-dimethylxanthine binding was studied in hippocampal membranes prepared from rats of different ages. Whereas the Bmax value was significantly lower in 2-year-old rats than in younger counterparts, the dissociation constant (Kd) was not affected by aging, indicating that the density rather than the affinity of adenosine receptors was altered. Endogenous adenosine levels present in the extracellular space were also measured in the superfusate by high performance liquid chromatography (HPLC) coupled with ultraviolet detection, and an age-related increase in the adenosine level was found.Insummary, our results indicate that during aging the level of adenosine in the extracellular fluid is increased in the hippocampus. There is a downregulation and reduced responsiveness of presynaptic adenosine A1-receptors, and it seems likely that these changes are due to the enhanced adenosine level in the extracellular space.

Dopamine, as well as norepinephrine, is a link between noradrenergic nerve terminals and splenocytes

The effect of supramaximal electric field stimulation on 3H released from rat spleen strips was studied after loading with either [ 3H]dopamine ([ 3H]DA) or [ 3H]norepinephrine ([ 3H]NE). In some experiments, [ 3H]DA and [ 3H]NE stored in the tissue or released in response to electrical stimulation were separated from their tritiated metabolites using HPLC followed by radiochemical detection. The stimulation-evoked release of 3H after loading with either derivative was subject to negative feedback modulation through α 2-adrenergic, D 2-dopamine and muscarinic acetylcholine receptors, and could be prevented by either calcium removal or tetrodotoxin blocking of Na + influx, indicating its neuronal and vesicular origin After the separation of radioactive metabolites by HPLC, both the tissue loaded with [ 3H]DA and the fractions collected during electrical stimulation contained a considerable amount of [ 3H]NE, providing evidence that the neurons it originated from were adrenergic in function. [ 3H]DA was also released during electrical stimulation. Since the spleen does not receive dopaminergic innervation, it was concluded that the noradrenergic axon terminals in the spleen were able to take up DA, convert it in part into NE, and release it as both DA and NE in response to neural activity. The ratio of [ 3H]DA and [ 3H]NE in the spleen loaded with [ 3H]DA was found to be dependent on both temperature and time of loading, and could be modulated by various drugs such as desmethylimipramine, a NE uptake blocker, and disulfiram or fusaric acid, dopamine β-hydroxylase inhibitors. The phenomenon may reveal a new mechanism by which immunocytes in the spleen can be regulated by the neuroendocrine system.

Studies on the release and extracellular metabolism of endogenous ATP in rat superior cervical ganglion: support for neurotransmitter role of ATP

The release of endogenous ATP, measured by the luciferin-luciferase assay, and the release of [ 3H]acetylcholine from the isolated superior cervical ganglion of the rat loaded with [ 3H]choline were studied simultaneously. Electrical field stimulation enhanced the release of endogenous ATP and acetylcholine in a [Ca 2+] o-dependent manner. The Na + channel blocker, tetrodotoxin (1 μM) inhibited the stimulation-evoked release of endogenous ATP and of [ 3H]acetylcholine, but did not change the resting release. The release of ATP was dependent on the frequency of stimulation between 2 and 10 Hz, when the number of shocks was kept constant (360 shocks), while acetylcholine was not released in a frequency-dependent fashion. Ten days after cutting of the preganglionic nerve of the superior cervical ganglion the stimulation-evoked release of acetylcholine and ATP was abolished and the uptake of [ 3H]choline was significantly reduced but not inhibited. Hexamethonium, (100 μM) a nicotinic acetylcholine receptor antagonist, significantly reduced the release of both acetylcholine and ATP, indicating a positive feedback modulation of ACh and ATP release. 8-Cyclopentyl-1,3-dipropylxanthine (10 nM), the selective A 1-adenosine receptor antagonist exhibited similar effect on the release of ATP and acetylcholine: both of them were augmented, showing that the stimulation-evoked release of ATP and acetylcholine are under the inhibitory control of A 1-adenosine receptors. When the temperature was reduced to 7°C to inhibit carrier-mediated processes, the resting and stimulated release of acetylcholine was not changed. Conversely, the release of ATP in response to stimulation was reduced by 79.9±5.6%, and the basal release was also almost completely blocked. Carbamylcholine by itself was able to release ATP, but not acetylcholine, in a hexamethonium-inhibitable manner, even from ganglia whose preganglionic nerve had been cut 10 days prior to experiments, suggesting that ATP release can occur in response to nicotinic receptor stimulation of postsynaptic cells. The breakdown of ATP or AMP by superior cervical ganglion was measured by high performance liquid chromatography combined with UV detection. ATP and AMP, added to the tissues, were readily decomposed: the K m (apparent Michaelis constant) and V max (apparent maximal velocity) were 475±24 μM and 3.50±0.18 nmol/min per mg for ectoATPase and 1550±120 μM and 14.5±0.9 nmol/min per mg tissue for 5′-nucleotidase. In addition, by using electron microscopic enzyme histochemistry, the presence of ectoATPase was also shown in the superior cervical ganglion. It is concluded that endogenous ATP and acetylcholine are released simultaneously in response to stimulation of preganglionic nerve terminals in the superior cervical ganglion in a [Ca 2+] o-dependent, tetrodotoxin-sensitive manner and is metabolized by ectoenzymes present in the tissue. The dissociation of the release of ATP and acetylcholine at different stimulation frequencies and temperatures shows that the release-ratio of acetylcholine and ATP can vary upon the condition of stimulation: this can reflect either the different composition of synaptic vesicles in the preganglionic nerve terminals or a significant contribution of non-exocytotic, carrier-mediated type of release of ATP to the bulk release.

ATP and [3H]noradrenaline release and the presence of ecto-Ca(2+)-ATPases in the capsule-glomerulosa fraction of the rat adrenal gland.

Both [3H]noradrenaline ([3H]NA) and ATP were released in response to supramaximal electric field stimulation in superfused rat adrenal capsule-glomerulosa preparations. The voltage-dependent potassium channel blocker 4-aminopyridine enhanced, while the ATP-sensitive potassium channel blocker glibenclamide failed to affect the stimulation-evoked release of [3H]NA. The selective alpha 2-adrenoceptor antagonist CH-38083 enhanced the evoked release of [3H]NA while the P2 receptor agonist ATP and alpha, beta-methylene-ATP failed to affect it. Neither the adenosine A1 receptor agonist N6-cyclopentyl-adenosine (CPA) nor the adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) influenced the stimulation-evoked [3H]NA release. The data showed that ATP was released from capsule-glomerulosa preparations in response to field stimulation together with but independently from [3H]NA, and that the local noradrenergic varicose axon terminals are not equipped with purinoceptors sensitive to ATP and/or adenosine. High concentrations of ATP also stimulated steroid hormone secretion in vitro, and thus may have a physiological role in this tissue. The presence of ecto-Ca(2+)-ATPases, enzymes able to terminate the effect of ATP, was demonstrated around the nerve profiles at the border of the capsule and zona glomerulosa tissue.

Effect of subtype-specific Ca(2+)-antagonists and Ca(2+)-free media on the field stimulation-evoked release of ATP and [3H]acetylcholine from rat habenula slices.

The involvement of different subtypes of voltage-sensitive (Ca2+ channels in the initiation of field stimulation-induced endogenous adenosine triphosphate (ATP) and [3H]acetylcholine ([3H]ACh) release was investigated in the superfused rat habenula slices. ATP, measured by the luciferin-luciferase assay, and [3H]ACH were released simultaneously from the tissue in response to low frequency electrical stimulation (2 Hz, 2.5 msec, 360 shocks). The N-type Ca(2+)-channel blocker omega-conotoxin GVIA (omega-CgTX, 0.01-1 microM) reduced the stimulation-evoked release of ATP and [3H]ACh in a dose-dependent manner. Similarly, the P-type Ca2+ channel antagonist omega-agatoxin IVA (omega-Aga IVA) (0.05 microM) and the inorganic Ca(2+)-channel blocker Ca2+ (0.2 mM) inhibited the outflow of both transmitters, while Ni2+ (0.1 mM) was without significant effect. A high correlation was observed between the percent inhibition of ATP release and percent inhibition of ACh release caused by the different Ca2+ antagonists. Long-term perfusion (i.e., 90 min) with Ca(2+)-free solution inhibited the evoked-release of ATP and [3H]ACh. In contrast, perfusion of slices with the same media for a shorter time (i.e., 20 min) did not reduce the release of [3H]ACh and ATP but even increased the evoked-release of ATP about fourfold. The breakdown of extracellular ATP was not blocked under low [Ca2+]0 condition, measured by the creatine phosphokinase assay and HPLC-UV technique. Application of extra- or intracellular Ca2+ chelators, and dipyridamole (2 microM), the nucleoside transporter inhibitor, did not reduce the excess release of ATP after short-term perfusion with Ca(2+)-free media. Tetrodotoxin (TTX, 1 microM), while inhibiting the majority of ATP release under normal conditions, was also unable to reduce release under low [Ca2+]0 conditions. In summary, we showed that both N- and P-type Ca2+ channels are involved in the initiation of electrical stimulation-evoked release of ATP and [3H]ACh in the rat habenula under normal extracellular calcium concentration. Under low [CA2+]0 conditions an additional release of ATP occurs, which is not associated with action potential propagation.

Alpha 2A subtype of presynaptic alpha 2-adrenoceptors modulates the release of [3H]-noradrenaline from rat spinal cord.

The modulation of noradrenaline (NA) release from rat spinal cord slices was investigated and the subtype of presynaptic alpha 2-adrenoceptors involved in the negative feedback modulation was characterized using in vitro perfusion experiments. Rat spinal cord slices were loaded with [3H]NA and the release of radioactivity at rest and in response to field stimulation was determined. The alpha 2-adrenoceptor agonists, clonidine and dexmedetomidine inhibited the stimulation-evoked release of NA from spinal cord slices, whereas alpha 2-adrenoceptor antagonists yohimbine and CH-38083 (7,8-(methylenedioxy)-14-alpha-hydroxyalloberbane HCI), enhanced it. ARC 239, a selective alpha 2B-antagonist, had no effect on the release. In contrast, BRL 44408 a selective alpha 2A-antagonist increased the release of NA. Our results indicate that the negative feedback modulation of NA release from noradrenergic fibres in the spinal cord is mediated via alpha 2A subtype of alpha 2-adrenoceptors.

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.

S-51-1 - Modulation of cerebral Monoamine release by Mao inhibitors

The ability of L-3,4-dihydroxyphenylalanine (L-DOPA) to release noradrenaline (NA) from peripheral and CNS neurons was studied using isolated rat vas deferens and in vivo frontal cortex microdialysis. Application of L-DOPA (30 μM) to vas deferens increased basal NA efflux but not electrical field stimulation-evoked release of NA when the tissue was pretreated with an inhibitor of MAO-B (clorgyline 1 μM) or an inhibitor of MAO-A and MOA-B (AGN-1133 1 μM). No effect on NA efflux was seen when the tissue was treated with rasagiline (0.005 μM; selective inhibitor of MAO-B), but rasagiline, AGN-1133 and clorgyline increased basal efflux of dopamine (DA) following L-DOPA. In microdialysis experiments, systemic administration of L-DOPA/carbidopa combination (50/12 mg.kg−1) increased the efflux of 3,4-dihydroxyphenylglycol and 3-methoxy,4-hydroxyphenylglycol but reduced that of NA. Microdialysate levels of NA, however were increased following L-DOPA/carbidopa when desipramine (1 μM) was infused locally via the probe, or following systemic administration of yohimbine (2 mg.kg−1). The data are consistent with the hypothesis that administration of L-DOPA is followed by increased axoplasmatic levels of DA which release NA from storage sites into the free cytoplasmatic pool.