Role of presynaptic purinoceptors and cyclic AMP on the noradrenaline release in cat cerebral arteries

Field electrical stimulation (ES), K+ (50 mM) or ionophore X-537A (0.01 mM) induced tritium release from cat cerebral arteries preincubated with [3H]noradrenaline (NA). Adenosine and AMP (0.5 mM) did not modify tritium release caused by ionophore X-537A, but these agents and ATP (0.5 mM) significantly reduced that elicited by ES and K+; this reduction was antagonized by 1-methyl-3-isobutylxanthine (MIX; 0.05 mM). Inosine (0.5 mM) and the agonist of purinergic A2-receptors, 5'N-ethyl-carboxamide adenosine (NECA; 0.5 mM) had no effect, but the agonist of purinergic A2-receptors L-N6-phenylisopropyl adenosine (L-PIA; 0.1 mM) diminished tritium efflux caused by ES and K+. The adenosine inhibition of ES-induced radioactivity release was not affected by indomethacin (0.05 mM). MIX (0.05 mM) increased tritium release evoked by ES and K+. Agents that increase intracellular cyclic (c)AMP levels, such as dibutyryl cAMP (0.5 mM), the phosphodiesterase inhibitor Ro 20-1724 (0.1 mM), and the activators of adenylate cyclase, forskolin (0.005 mM) and NaF (2 mM) reduced tritium secretion elicited by ES and K+. However, the intracellular increase of cyclic GMP (cGMP) caused by 8-Br-cGMP did not affect this secretion. Dipyridamole (0.05 mM) and the adenosine deaminase inhibitor erythro-9-2-hydroxy-3 nonyl adenosine (EHNA; 0.1 mM) also produced inhibition of tritium secretion elicited by ES and K+. Dipyridamole reduced both the uptake of [3H]NA and [3H]adenosine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of cyclic AMP in the prejunctional α2-adrenoceptor modulation of noradrenaline release from the rat tail artery

Summary Experiments were designed to evaluate the effect of cyclic AMP on the electrically-induced release of noradrenaline from vascular sympathetic nerve terminals. The possible implication of the inhibition of adenylate cyclase in the negative feed-back control by prejunctional α₂-adrenoceptors of neurotransmitter release was also investigated. Rat isolated tail arteries were preincubated with [³H]-noradrenaline; the preparations were subsequently perfused/superfused with [³H]-noradrenaline-free medium and their perivascular nerves were field stimulated with 24 pulses at 0.4 Hz (0.3 ms, 200 mA). 2 compounds known to enhance the intracellular concentration of cyclic AMP, namely the membrane permeant analogue 8-Br-cAMP (10–300 μmol/l) and forskolin (0.3–10 μmol/l), an activator of adenylate cyclase, concentration-dependently enhanced the stimulation-evoked tritium overflow. The 1,9-dideoxy derivative of forskolin, which does not stimulate adenylate cyclase, was ineffective. Exposure to the cyclic AMP phosphodiesterase inhibitor rolipram 30 μmol/l produced a moderate increase (about 20%) in tritium overflow. However, in the presence of rolipram the facilitatory effect of forskolin was significantly more pronounced than in its absence. Whereas 8-Br-cAMP produced a slight concentration-dependent enhancement of the stimulation-induced vasoconstriction, forskolin and rolipram depressed it. The α₂-adrenoceptor agonist B-HT 933 (3–30 μmol/l) concentration-dependently inhibited the tritium overflow. The effect of B-HT 933 30 μmol/l was slightly, but significantly reduced in the presence of 8-Br-cAMP 100 and 300 μmol/l, but was not changed in the presence of forskolin 3 μmol/l The facilitatory effect of rauwolscine 1 μmol/l was enhanced in the presence of 8-Br-cAMP 100 μmol/l. During perfusion with 8-Br-cAMP 100 μmol/l, the current strength and frequency were decreased to 150 mA and 0.2 Hz, respectively in order to obtain similar amounts of tritium overflow to those observed in the absence of the cyclic AMP analogue with the initial stimulation parameters. Under these conditions, the inhibition of the overflow by B-HT 933 30 μmol/l and the facilitation by the α₂-adrenoceptor antagonist rauwolscine 1 μmol/l were unaltered as compared to controls under initial stimulation conditions. It is concluded that, in the rat tail artery, the terminals of perivascular sympathetic nerves are endowed with an adenylate cyclase system. Cyclic AMP is able to modulate noradrenaline release, but does not appear to play a role in the initiation of the release process itself. In addition, the results do not support the hypothesis that prejunctional α₂-adrenoceptors depress noradrenaline release through the inhibition of adenylate cyclase. Send offprint requests to B. Bucher at the above address     

Angiotensin modulation of vascular tone and adrenergic neurotransmission in cat femoral arteries

• 1.1. AI and AII induced contractions in cat femoral arteries, which were inhibited by saralasin.
• 2.2. The response to AI was reduced by captopril and endothelium removal and by chymostatin in endothelium-denuded segments.
• 3.3. AII contractions were increased by indomethacin, L-NAME and endothelium removal.
• 4.4. AII and AI facilitated the adrenergic neurotransmission. This facilitation was inhibited by saralasin and/or captopril.
• 5.5. These data suggest: (1) AI is converted into AII in the endothelial and adventitial layer; (2) the contractions caused by AI and AII are mediated by AII receptors and are modulated by endothelial release of NO and PGI₂; and (3) the existence of presynaptic AII receptors mediating the facilitation of neurotransmission caused by AI and AII.

     

Role of cyclic AMP in the release of noradrenaline from isolated rat atria

Summary The possible role of cyclic AMP (cAMP) on tritium overflow evoked by stimulation of the cardioaccelerant nerves was studied in rat atria preincubated with [³H]-noradrenaline. Addition of the activator of adenylate cyclase forskolin (1 µmol/l), or of the phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine (IBMx, 100 µmol/l), did not affect both basal and evoked overflow. However, in the presence of the ₂-adrenoceptor antagonist yohimbine (0.03 µmol/l) both forskolin and IBMx increased the stimulation-induced transmitter overflow by 49% and 141%, respectively (compared to yohimbine 0.03 µmol/l). Thus, in rat atria the cAMP-dependent facilitation of noradrenaline release is only present when the autoinhibition exerted by activation of prejunctional ₂-adrenoceptors is blocked. Propranolol (0.1 µmol/l) that did not produce any effect on noradrenaline release markedly reduced the facilitatory response induced by forskolin in the presence of yohimbine. When rats were pretreated with the ₂-adrenoceptor agonist clenbuterol (0.3 mg · kg^–1, s. c., twice daily, 14 days), a treatment which desensitizes -adrenoceptor-me-diated facilitation of noradrenaline release (Kazanietz and Enero 1989), the facilitatory effect of forskolin and IBMx in the presence of yohimbine was abolished. The results indicate that in rat atria the effect of forskolin and IBMx on noradrenaline release are only to be observed after blockade of presynaptic ₂-adrenoceptor autoinhibition. -adrenoceptor blockade or clenbuterol pre-treatment decreases the facilitatory response to forskolin and hence prejunctional -adrenoceptor-mediated enhancement of noradrenaline release is linked to the stimulation of adenylate cyclase. Correspondence to M. A. Enero at the above address     

Effect of tetraethylammonium and verapamil on noradrenaline release induced by field electrical stimulation and potassium from cat cerebral and femoral arteries

Tritium release evoked by field electrical stimulation (FES) or high potassium (K+) from cat cerebral and femoral arteries prelabelled with 3H-noradrenaline was investigated. The release induced by FES and K+ was reduced by Ca2+ suppression and tetrodotoxin (TTX) but not by verapamil in both vessels. Tetraethylammonium (TEA) increased tritium release evoked by FES and K+, when TTX plus TEA were added together radioactivity secretion induced by FES was practically abolished. These results indicate that FES or K+ induce exocytotic noradrenaline release mainly by propagated action potentials and by similar mechanisms in cerebral and femoral arteries.     

Effect of pentobarbital on the noradrenaline release induced by drugs and field electrical stimulation from cerebral and femoral arteries of the cat

The present studies showed that field electrical stimulation, high potassium (K⁺), tyramine and ionophore X537A induced tritium release from cerebral and femoral arteries of cat prelabelled with [³H]noradrenaline. The secretion caused by K⁺ or field stimulation was Ca²⁺-dependent and was antagonized by high concentrations of pentobarbital (10^?4 and 10^?3M), whereas that induced by the rest of the drugs was unchanged in the same situations. The noradrenaline uptake by these arteries was reduced by pentobarbital (10^?3M and 10^?4M). These results suggest that this barbiturate interferes with Ca²⁺ entry to the adrenergic nerve endings, and therefore antagonizes the noradrenaline release by Ca²⁺-dependent processes (exocytosis).     

Analysis of the effects of noradrenaline and tyramine in isolated middle cerebral and femoral arteries of cat

1. Tyramine and noradrenaline (NA) caused dose-dependent contractions in middle cerebral and femoral arteries of cat, which were decreased by phentolamine. 2. Gangliectomy increased the contraction evoked by NA in brain arteries. 3. Reserpine pretreatment and/or gangliectomy reduced the contraction caused by tyramine, the maximal responses being unmodified in the cerebral vessels. 4. Tyramine induced Ca2+-dependent tritium release from brain and femoral arteries, which was reduced by reserpine pretreatment and/or gangliectomy. 5. These data suggest that tyramine has a direct component, apart from an indirect one, in brain arteries. The mechanisms by which NA induces slight contraction in them, including the role of Ca2+, are postulated.     

Involvement of cyclic nucleotides in prejunctional modulation of noradrenaline release in mouse atria.

1 In mouse isolated atria previously incubated with [3H]-noradrenaline, 8-bromo-cyclic AMP (3-270 microM) produced a concentration-dependent increase in the fractional stimulation-induced outflow of radioactivity. 8-Bromo-cyclic GMP induced a lesser increase in the stimulation-induced outflow. 2 The phosphodiesterase inhibitors: M&B 22948 (90 microM); ICI 63197 (30 and 90 microM) and 3-isobutyl-1-methylxanthine (90 microM) increased the fractional stimulation-induced outflow. Together these results indicate that cyclic AMP may have a modulatory effect on noradrenaline release. 3 The inhibition of the stimulation-induced outflow produced by clonidine (0.03 microM) and its facilitation produced by phentolamine (1 microM) were unaltered in the presence of 8-bromo-cyclic AMP (90 microM). However, in the presence of 8-bromo-cyclic AMP (270 microM), the facilitatory effect of phentolamine was enhanced, but the inhibitory effect of clonidine (0.03 microM) was unaltered. In the presence of ICI 63197 (30 microM) the inhibitory effect of clonidine (0.03 microM) was unaltered, but the facilitatory effect of phentolamine (1 microM) was slightly enhanced. 4 Isoprenaline (0.003-0.1 microM) enhanced the fractional stimulation-induced outflow, an effect blocked by propranolol (0.1 microM). In the presence of 8-bromo-cyclic AMP (90 microM), the facilitatory effect of isoprenaline (0.01 microM) was blocked. In the presence of ICI 63197 (30 microM) the facilitatory effect of isoprenaline (0.003 microM) was potentiated. 5 These results suggest that whereas beta-adrenoceptor-mediated enhancement of noradrenaline release is linked to the stimulation of adenylate cyclase and enhanced formation of cyclic AMP, alpha-adrenoceptor-mediated inhibition of noradrenaline release is not linked to inhibition of adenylate cyclase activity.     

Contractile responses and noradrenaline release evoked by serotonin in cat femoral artery: Influence of pentobarbital

• 1.1. 5-Hydroxytryptamine (5-HT) induced dose-dependent contractions in the isolated cat femoral artery, which were reduced by LSD, methysergide, phentolamine and reserpine pretreatment (only at low doses).
• 2.2. Pentobarbital (PB) and Mn²⁺ relaxed the arteries previously contracted with 5-HT. These drugs reduced the contraction evoked by this amine as it was Ca²⁺-suppression.
• 3.3. High concentrations of 5-HT and K⁺ induced tritium release from vessels prelabelled with ³H-noradrenaline. Ca²⁺-deprivation and PB unmodified the release caused by 5-HT, but that elicited by K⁺ was abolished.
• 4.4. These data indicate that 5-HT-induced contraction is essentially due to direct interaction of this agent with 5-HT-receptors, and that PB interferes with Ca²⁺ entry to the cell.

     

Dopaminergic modulation of hippocampal noradrenaline release

Summary ³H-Noradrenaline release in the rabbit hippocampus and its possible modulation via presynaptic dopamine receptors was studied. Hippocampal slices were preincubated with ³H-noradrenaline, continuously superfused in the presence of cocaine (30 mol/l) and subjected to electrical field stimulation. The electrically evoked tritium over-flow from the slices was reduced by 0.1 and 1 mol/l dopamine and apomorphine, but significantly enhanced by 10 mol/l apomorphine or by 0.1 and 1 mol/l bromocriptine. If the ₂-adrenoceptor antagonist yohimbine (0.1 mol/l) was present throughout superfusion, the inhibitory effects of dopamine and apomorphine were more pronounced and even 10 mol/l apomorphine and 1 mol/l bromocriptine inhibited noradrenaline release. Qualitatively similar observations were made in the presence of another ₂-antagonist, idazoxane (0.1 mol/l). In the presence of the D2-receptor antagonist domperidone (0.1 mol/l) the inhibitory effects of dopamine were almost abolished, whereas both apomorphine (>1 mol/l) and bromocriptine (>0.01 mol/l) greatly facilitated noradrenaline release. The D2-receptor agonist LY 171555 (0.1 and 1 mol/l) significantly reduced the evoked noradrenaline release whereas the D1-selective agonist SK & F 38393 was ineffective at similar concentrations. The effects of LY 171555 were abolished in the presence of domperidone (0.1 mol/l) but remained unchanged in the presence of yohimbine or idazoxane (0.1 mol/l, each).At 1 mol/l the D2-receptor antagonists domperidone and (-)sulpiride significantly increased the evoked noradrenaline release by about 10%. However, at this concentration, domperidone (but not (-)sulpiride) affected also basal tritium outflow. Bulbocapnine and the preferential D1-receptor antagonists SCH 23390 enhanced the evoked noradrenaline release already at 0.1 mol/l. Their marked facilitatory effects (50 to 60% increase at 1 mol/l) were reduced in the presence of idazoxane (0.1 mol/l) and almost abolished in the presence of 0.1 mol/l yohimbine, whereas the increase due to 1 mol/l (-)sulpiride persisted under these conditions.The evoked tritium efflux from rabbit hippocampal slices preincubated with ³H-serotonin was not affected by dopamine receptor agonists.From our results we conclude that hippocampal noradrenaline, but not serotonin release, is modulated via D2-dopamine receptors. In addition, our results provide evidence for more or less pronounced ₂-adrenoceptor agonistic properties of dopamine and ₂-adrenoceptor antagonistic properties of apomorphine, bromocriptine, SCH 23390 and bulbocapnine in this noradrenaline release model from CNS tissue.     

Neurogenic vasoconstriction of cat cerebral and femoral arteries

1. Segments of cat posterior communicating artery and femoral artery submitted to field electrical stimulation showed frequency-dependent contractile responses which were decreased by tetrodotoxin, bretylium, and reserpine pretreatment. Phentolamine could only reduce the response of femoral artery segments. Superior cervical gangliectomy also decreased the contractions of cerebral cylinders. 2. The tritium released by electrical stimulation from both kinds of vessels preloaded with [3H]noradrenaline appeared reduced in the presence of tetrodotoxin, bretylium, and after reserpine pretreatment. It was also decreased in cerebral segments after superior cervical gangliectomy. 3. These results suggest that the electrical field stimulates the sympathetic nerve endings of these vessels.     

M3-muscarinic receptor mediates prejunctional inhibition of noradrenaline release and the relaxation in cat femoral artery.

The aim of the present study was to analyse the muscarinic receptors involved in the vasodilation elicited by acetylcholine (ACh) and the carbachol inhibition of electrically-evoked [3H]noradrenaline (NA) release in cat femoral artery. For this purpose, the following receptor antagonists were used, atropine, pirenzepine (M1-antagonist), AF-DX 116 (M2-antagonist) and 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP; M3-antagonist). The order of potency (pA2 values) of these drugs at postjunctional level was: atropine (9.7) greater than or equal to 4-DAMP (9.6) greater than pirenzepine (7.2) greater than AF-DX 116 (6.0), and at prejunctional level (pIC50 values) was: 4-DAMP (9.3) greater than atropine (8.5) greater than AF-DX 116 (7.1) greater than pirenzepine (5.9). These findings indicate that the muscarinic receptors mediating the vasodilation induced by ACh and the carbachol inhibition of NA release are of the M3-subtype.     

Characterization of the subtype of presynaptic alpha 2-adrenoceptors modulating noradrenaline release in cat and bovine cerebral arteries.

The possible existence of a heterogeneous population of alpha 2-adrenoceptors (alpha 2A and alpha 2B, demonstrated by binding studies) in adrenergic nerve endings of cat and bovine cerebral arteries modulating noradrenaline release was investigated. Electrical field stimulation elicited an increase of tritium secretion from these vessels preincubated with (+/-)-[3H]noradrenaline, which was reduced by the alpha 2-agonists, clonidine (1 microM) and B-HT 920 (0.01 and 0.1 microM), in cat cerebral arteries but only by B-HT 920 in bovine cerebral arteries. This reduction was inhibited by the antagonist of the alpha 2B-subtype, prazosin, and the antagonists of alpha 2A- and alpha 2B-subtypes yohimbine and particularly rauwolscine. The effect of B-HT 920 was partially inhibited by clonidine in bovine, but not in cat cerebral arteries. In both types of arteries, prazosin, yohimbine and the alpha 1-agonist methoxamine (all at 1 microM) failed to modify the stimulated radioactivity liberation, whereas it was increased by 1 microM rauwolscine, and by yohimbine plus prazosin in cat cerebral arteries. The basal tritium release was enhanced by rauwolscine and prazosin in cat cerebral arteries but only by the latter in bovine cerebral arteries. These results suggest: (1) the existence of presynaptic alpha 2-adrenoceptors, mainly of the alpha 2B-subtype, in these vessels negatively modulating noradrenaline release, their activity being greater in cat than in bovine cerebral arteries, and (2) clonidine has no agonistic but a weak antagonistic action in the latter vessels.     

Effect of some vasodilators on cat femoral arteries

Adenosine, cyclic-AMP (cAMP), papaverine and 1-methyl-3-isobutylxanthine (MIX) evoked dose-dependent vasodilatation in cat femoral arteries precontracted with 75 mM K⁺. The vasodilator response induced at maximal concentration used was: papaverine > MIX > adenosine = cAMP. With regard to the potency of relaxant effects (IC10) the order was MIX = cAMP = papaverine > adenosine. The dilatation elicited by papaverine, adenosine and cAMP was increased by MIX. Preincubation with adenosine enhanced the relaxation induced by MIX and reduced that produced by cAMP. These results indicate that the effects of adenosine and cAMP seem not to be mediated by specific surface receptors but by a cAMP-dependent mechanism. The interference between adenosine and cAMP could be due to competition for a similar site and/or mechanism.     

Omega-conotoxin GVIA and pharmacological modulation of hippocampal noradrenaline release

The tritium overflow evoked by electrical stimulation of rabbit hippocampal slices labeled with [3H]noradrenaline was inhibited by omega-conotoxin GVIA, a peptide modulator of the N-type voltage-sensitive calcium channel (N-VSCC). The magnitude of this inhibition was unchanged in the presence of substances which interact with N- and/or L-VSCCs (cadmium, neomycin, (-)- and (+)-202-791), alpha 2-adrenoceptors (idazoxan, UK-14304), protein kinase C (4 beta-phorbol-12,13-dibutyrate) or potassium channels (4-aminopyridine). This finding suggests that the attenuation of calcium-dependent neurotransmitter release by omega-conotoxin GVIA is relatively insensitive to alterations of such release effected by other substances.     

Behavioural effects of dibutyryl cyclic 3',5' AMP, noradrenaline and cyclic 3',5' AMP in rats

It was shown that dibutyryl cyclic AMP (DAMP) injected intraventricularly in doses of 100–200 μg in rats causes the increase of locomotor and exploratory activity and convulsions, depending on the dose. DAMP increased excitatory behavioural effects of noradrenaline (NA) injected intraventricularly in doses of 10 or 50 μg. NA in a dose of 200 μg abolished convulsions evoked by DAMP. Cyclic AMP (cAMP) injected intraventricularly in doses of 100–400 μg had no effect on the rat's behaviour. Pretreatment of rats with dimethylsulphoxide and with theophylline caused in some animals behavioural phenomena after injection of cAMP, similar to the behavioural symptoms evoked by DAMP.     

Characterization of prejunctional purinoceptors inhibiting noradrenaline release in rat mesenteric arteries

The effects of purinoceptor agonists on noradrenaline NA release by electrical stimulation in rat mesenteric arteries were examined to clarify the pharmacological properties of prejunctional purinoceptors on adrenergic nerves. Adenosine and the other P1-receptor agonists, 5'-(N-ethylcarboxamido) adenosine and 2-chloroadenosine, significantly inhibited the release of NA. Also beta,gamma-methylene ATP and 2-methylthio ATP, P2-receptor agonists, significantly inhibited NA releases. The inhibitory effect of adenosine was significantly reduced by adenosine deaminase, but those of beta,gamma-methylene ATP and 2-methylthio ATP were not affected. This suggests that the inhibitory effects of P2-receptor agonists are not due to conversion into adenosine. 1,3-Dipropyl-8-cyclopentylxanthine (DPCPX), a P1 (A1)-receptor antagonist, significantly reduced the inhibitory effects of not only the P1- but also P2-receptor agonists. Therefore, DPCPX appears to act on both prejunctional P1- and P2-receptor as an antagonist. Pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), a P2-receptor antagonist, significantly reduced the inhibitory effects of the P2-receptor agonists, but not those of the P1-receptor agonists. From these findings in the rat mesenteric artery, the P1-receptor agonist-induced inhibition of NA-release appears to be mediated via a well-known prejunctional P1-receptor of the A1-subtype, but the P2-receptor agonist-induced inhibition appears to be mediated via an unidentified purinoceptor that is blocked not only by P2-receptor antagonists but also by P1-receptor antagonists.     

Asymmetric release of cyclic AMP from guinea-pig and rabbit gallbladder

Summary The release of cyclic adenosine 3:5-monophosphate (cAMP) from guinea-pig and rabbit gallbladder was investigated in vitro. Serosal addition of prostaglandin E₁ (PGE₁) to luminally perfused guinea-pig gallbladders caused a concentration-dependent efflux of cAMP to the mucosal side, the threshold concentration of PGE₁ being 10^–7 M. The efflux of cAMP to the serosal side was 7-fold lower. A mucosal sidedness of cAMP release was also observed in stripped preparations of rabbit gallbladder mucosa mounted between two half chambers. No cAMP was found in the solutions bathing the serosal layers isolated from rabbit gallbladders. Fluid secretion was observed at 10^–7 M PGE₁, an effect mimicked by serosal, but not mucosal application of cAMP (3.3×10^–3 M). This is taken to indicate that the basolateral membrane is more easily permeated by cAMP than the apical membrane, since cAMP is believed to exerts its physiological effects from inside the cell. It is concluded that preferential release of cAMP to the mucosal side is not due to a higher permeability of the brush border membrane but rather represents an as yet undefined transport process which may be of importance for the regulation of excessive intracellular cAMP levels.