Stimulation-evoked 3H-overflow Research Articles

Effect of ω-Conotoxin GVIA on Noradrenaline Release from Postganglionic Sympathetic Neurones in Rabbit Aorta

The aim of the present work was to examine the effect of the selective N-type calcium blocking agent omega-conotoxin GVIA on stimulation-evoked release of noradrenaline from sympathetic nerves in rabbit isolated aorta with regard to stimulation frequency, extracellular Ca2+ concentration, and transmitter uptake. Rings of rabbit isolated aorta were preloaded with (-)-3H-noradrenaline and the fractional 3H-overflow evoked by electrical-field stimulation was determined by liquid scintillation spectrometry. Omega-conotoxin GVIA (3 x 10(-10)-3 x 10(-8) M) did not alter the spontaneous 3H-outflow. Omega-conotoxin GVIA (3 x 10(-10)-3 x 10(-8) M) caused a slowly developing reduction of stimulation-evoked 3H-overflow at 1 and 30 Hz. The Emax for the omega-conotoxin-induced inhibition was less (70%) at 30 Hz than that (96%) seen at 1 Hz. Short-term incubation with omega-conotoxin GVIA caused a subsequent steady-state inhibition. The inhibitory action of omega-conotoxin GVIA (3 x 10(-10)-3 x 10(-9) M) was inversely related to the extracellular Ca2+ concentration (6.5 x 10(-4)-2.7 x 10(-3) M). Cocaine (3 x 10(-5) M) plus corticosterone (4 x 10(-5) M), neuronal and extraneuronal uptake inhibitors, respectively, did not alter the inhibitory effect of omega-conotoxin GVIA (3 x 10(-9) M) on 3H-overflow evoked by stimulation at a frequency of either 1 or 30 Hz. It is concluded that omega-conotoxin GVIA acts on prejunctional N-type calcium channels to inhibit stimulation-evoked noradrenaline release from sympathetic neurone terminals in rabbit aorta. At a high frequency, another subtype calcium channel may possibly be involved. The action of omega-conotoxin GVIA is independent of neuronal and extraneuronal uptake mechanisms for noradrenaline, but dependent on the amount of Ca2+ to be transported across the neurilemma from the extracellular space into the neurone.

Calcium channels involved in noradrenaline release from sympathetic neurones in rabbit carotid artery.

Transmitter release from nerve terminals is dependent on the entry of Ca(2+) through neuronal voltage-gated calcium channels. In sympathetic neurones both N- and L-type calcium channels are present. Potassium channel blockade increases Ca(2+) entry into sympathetic neurones. We examined the participation of N- and L-type calcium channels in the stimulation-evoked release of noradrenaline from vascular sympathetic neurones. Rings of rabbit carotid artery were preincubated with [3H]-noradrenaline. Electrical field stimulation was used to evoke 3H overflow. The selective N-type calcium channel blocking agent omega-conotoxin GVIA (single concentrations: 3 x 10(-10)-10(-8) M) caused a slowly developing reduction of the stimulation-evoked 3H overflow. At 3 x 10(-8) M, omega-conotoxin GVIA caused an equilibrium block with a rapid (15 min.) onset. After 2 hr exposure to omega-conotoxin the inhibition was steady (pIC50 (-log M): 9.43; Emax: 91%). The selective L-type calcium blocking agents nifedipine (10(-7)-10(-5) M) and nimodipine (10(-8)-10(-5) M) had no effect on the stimulation-evoked 3H overflow. The calcium channel opener Bay K 8644 (10-6 M) likewise had no effect. The potassium channel blocking agent 4-aminopyridine (10-5-10-3 M) enhanced the stimulation-evoked 3H overflow up to 5 times. 4-Aminopyridine (10(-4) M) did not alter the inhibitory effect of omega-conotoxin GVIA (3 x 10(-8) M). In the presence of 4-aminopyridine (10(-4) M), nifedipine (10(-5) M) and nimodipine (10(-6) M) enhanced the 3H overflow. We conclude that the stimulation-evoked release of noradrenaline from sympathetic neurones in rabbit carotid artery is mediated by N-type calcium channels and that L-type channels are not involved even when potassium channels are blocked by 4-aminopyridine.

Prejunctional muscarinic receptor modulation of noradrenaline release from sympathetic neurones in rabbit aorta.

The prejunctional muscarinic modulation of stimulation-evoked release of 3H-noradrenaline from sympathetic neurones in rabbit aorta was examined. The role of transmitter uptake, alpha-adrenoceptor blockade, stimulation frequency and endothelium on the modulation was investigated. Rings of aorta were incubated with (-)-3H-noradrenaline and subsequently subjected to electrical-field stimulation. Fractional 3H-overflow was determined by liquid scintillation counting. Acetylcholine (10(-8)-3 x 10(-6) M) added cumulatively, reduced the stimulation-evoked 3H-overflow up to 80%. The effect of acetylcholine was the same in intact and endothelium-free aorta. The inhibitory effect of acetylcholine was inversely related to the frequency of stimulation (1-10 Hz). The maximal inhibition (%) was 80 (1 Hz), 53 (3 Hz) and 14 (10 Hz). The inhibitory effect of acetylcholine (10(-6) M) and carbachol (10(-5) M) reached a maximum 15 min. after addition and then remained almost constant. Cocaine (3 x 10(-5) M) did not alter the effect of acetylcholine. Desipramine (10(-6) M) and corticosterone (4 x 10(-5) M) attenuated the inhibition seen with low concentrations (10(-8)-10(-7) M) of acetylcholine. The acetylcholine-induced inhibition was antagonized by desipramine. Cocaine plus corticosterone attenuated the inhibition seen with high concentrations (10(-6)-3 x 10(-6) M) of acetylcholine. Rauwolscine (10(-6) M) enhanced the maximal inhibitory effect of acetylcholine. We conclude that the inhibitory effect of acetylcholine on 3H-overflow from rabbit aorta preloaded with 3H-noradrenaline is (1) inversely related to stimulation frequency; (2) independent of endothelium; (3) unaffected by neuronal and extraneuronal transmitter uptake; (4) that cocaine is not a prejunctional muscarinic antagonist; (5) that cocaine, but not desipramine, is suited as a neuronal uptake inhibitor in studies of prejunctional muscarinic receptor subtypes; and (6) and that there is an inverse interaction between prejunctional alpha2-adrenoceptors and muscarinic receptors.

Effect of adenosine on adrenergic neurotransmission and modulation by endothelium in canine pulmonary artery.

To determine the effect of adenosine on adrenergic neurotransmission in pulmonary vasculature and its modulation by endothelial cells, we studied canine pulmonary arteries under isometric conditions in vitro. Adenosine decreased the contractile responses to electrical field stimulation but had no effect on those to norepinephrine. This inhibitory effect was concentration dependent, with a rank order of potency of NECA > 2-chloroadenosine > adenosine >> APNEA (an A3-adenosine-receptor agonist) > CGS-21680 (an A2a agonist) > CCPA (an A1 agonist). Adenosine reduced the electrical field stimulation-evoked 3H overflow in superfused pulmonary artery previously soaked in [3H]norepinephrine. Pretreatment with the adenosine uptake blocker dipyridamole or the adenosine deaminase inhibitor deoxycoformycin enhanced the adenosine action, and this enhancement was not observed in the endothelium-denuded tissues. Adenosine deaminase activity was found in endothelial cells. Therefore, adenosine inhibits norepinephrine release via an A2b-receptor mechanism, an effect that may be modulated by uptake and metabolism by endothelial cells.

Prejunctional modulation by prostaglandin E2 of noradrenaline release from sympathetic neurones in rabbit aorta.

The modulating effect of prostaglandin E2 (PGE2) on the electrically-evoked 3H-overflow from rabbit isolated aorta preloaded with 3H-noradrenaline was examined. PGE2 (3 x 10(-9)-3 x 10(-7) M) inhibited the stimulation-evoked 3H-overflow (maximum inhibition: 81%; pIC50: 8.1). The inhibition was reversible and inversely related to stimulation frequency (1-30 Hz). Cocaine (3 x 10(-5) M) and corticosterone (4 x 10(-5) M) did not alter the inhibitory effect of PGE2 (3 x 10(-9)-10(-7) M). Rauwolscine (10(-6) M) enhanced the reduction caused by PGE2 (3 x 10(-9)-10(-7) M). Rauwolscine (10(-6) M) alone enhanced the 3H-overflow by 360%. Indomethacin (3 x 10(-6) M) and suprofen (4 x 10(-5) M) did not alter the PGE2 (3 x 10(-9)-10(-7) M)-induced reduction of the 3-H-overflow. Indomethacin (3 x 10(-6) M) and suprofen (4 x 10(-5) M) alone had no effect. We conclude that in the rabbit aorta (1) PGE2 modulates noradrenaline release from sympathetic neurones through a prejunctional inhibitory receptor mechanism; (2) that there is an interaction between alpha 2-adrenoceptors and EP-receptors; (3) that uptake inhibition does not affect the effect of PGE2; and (4) that the influence of endogenous prostaglandins on the noradrenaline release can be excluded.

Pharmacological characterization of histamine H 3 receptors in isolated guinea pig pulmonary artery and ileum

We characterized the histamine H 3 receptors involved in the modulation of electrical field stimulated neurogenic contraction of guinea pig pulmonary artery sympathetic, and guinea pig ileum parasympathetic preparations. Simultaneous measures of electrical field stimulation-evoked 3H overflow and tension in [ 3H]norepinephrine-loaded pulmonary artery were sensitive to tetrodotoxin (300 nM) and insensitive to hexamethonium (100 μM). Only the contractile response was inhibited by prazosin (100 nM). ( R)-α-Methylhistamine's inhibition of the pulmonary artery contraction and 3H overflow were dose-dependently antagonized by thioperamide (30–100 nM). ( R)-α-Methylhistamine also inhibited the neurogenic contractions of the isolated ileum (pD 2 = 8.2). In the pulmonary artery, the relative potency of the histamine H 3 receptor antagonists vs. ( R)-α-methylhistamine innibition of neurogenic contractions (pD 2 = 7.1) was thioperamide (pA 2 = 8.6 ± 0.1) > burimamide (pA 2 = 7.6 ± 0.2) > impromidine (pA 2 = 6.9 ± 0.02). Similarly, the relative potency of histamine H 3 receptor antagonists in the isolated ileum was thioperamide > burimamide ≥ impromidine, with pA 2 estimates of 8.7 ± 0.1, 7.3 ± 0.1 and 7.1 ± 0.1, respectively. Antagonist potencies suggest a predominant histamine H 3A-like receptor population on postganglionic sympathetic neurons innervating the pulmonary artery and parasympathetic neurons innervating the ileum longitudinal muscle.

Release of 3H-5-hydroxytryptamine from isolated rabbit aorta.

The main aim of the present investigation was to study systematically the passive and stimulation-evoked release of 3H-5-hydroxytryptamine (3H-5-HT) from rabbit isolated aorta. This was accomplished by preloading rings of aorta with 3H-5-HT (10(-6)M) and then monitoring by fractional collection the basal 3H-outflow and stimulation-evoked 3H-overflow. The basal 3H-outflow from aorta preloaded with 10(-6)M of either 3H-5-HT or (-)-3H-noradrenaline (3H-NA) leveled off about 100 min. after the onset of wash-out and remained almost constant thereafter (100-240 min.). The basal 3H-outflow from tissue preloaded with 3H-5-HT was almost 3-fold higher (70-240 min.) than that seen after preloading with 3H-NA. Cocaine (3 x 10(-5)M) did not alter the basal 3H-outflow (15-240 min.) from tissue preloaded with 3H-5-HT, while pargyline (5 x 10(-4)M) decreased it by about 66% (100-240 min.). Electrical-field stimulation (S1-S7, 200 mA, 600 pulses, 0.5 msec., 3 Hz) were applied to the tissue. The initial stimulation-evoked 3H-overflow from aorta preloaded with 3H-5-HT was higher than the subsequent ones (S1-S7; 100, 35, 35, 35, 35, 37, and 40%). Similar results to these were obtained with tissues preloaded with 3H-NA. The stimulation (S1-S7; 200 mA). 600 pulses, 0.5 msec, 3 Hz)-evoked 3H-overflow increased in an apparent linear manner with the amount of current used (50-200 mA).(ABSTRACT TRUNCATED AT 250 WORDS)

Role of cyclic AMP in stimulation-evoked release of 3H-noradrenaline from rabbit isolated ear artery.

The possible involvement of cyclic AMP in the stimulation-evoked 3H-overflow from rabbit isolated ear artery preloaded with 3H-noradrenaline was studied. Cyclic AMP (10(-5)-3 x 10(-4)M), 8-bromo-cyclic AMP (3 x 10(-4)M) and adenosine (10(-5)-3 x 10(-4)M) enhanced stimulation-evoked 3H-overflow. Dibutyryl-cyclic AMP (10(-5)-3 x 10(-4)M) had no effect. Theophylline (3 x 10(-5)M) and the phosphodiesterase inhibitor AH 21-132 (3 x 10(-5)M) did not alter the enhancement of 3H-overflow caused by either cyclic AMP or adenosine. Forskolin (3 x 10(-6)M) and the phosphodiesterase inhibitors ICI 63 197 (10(-4)M) and AH 21-132 (3 x 10(-6)-3 x 10(-5)M) increased stimulation-evoked 3H-overflow. Forskolin (10(-6)M) enhanced the effect of ICI 63 197 (3 x 10(-5)M) but it did not alter the effect of AH 21-132. It is concluded that cyclic AMP is involved in the stimulation-evoked release of noradrenaline from postganglionic sympathetic nerves in the rabbit ear artery.

Endogenous noradrenaline activates alpha 2-adrenoceptors on serotonergic nerve endings in human and rat neocortex.

Slices from human neocortex preincubated with [3H]serotonin ([3H]5-HT) were superfused and stimulated electrically to investigate whether the alpha 2-adrenoceptors on serotonergic terminals can be stimulated by endogenous noradrenaline (NA) released from neighboring noradrenergic fibers. The stimulation-evoked 3H overflow, representing action potential-induced, exocytotic release of 5-HT, was depressed by the NA uptake blocker (+)-oxaprotiline. Rauwolscine (a mixed alpha 2-adrenoceptor antagonist/5-HT autoreceptor agonist) or phentolamine [a combined alpha-adrenoceptor/5-HT autoreceptor antagonist; the latter drug in the presence of (+)-oxaprotiline] enhanced the release when the 5-HT autoreceptors had previously been blocked by metitepine. Under hypothermia the release of 5-HT was found to be decreased and that of NA to be increased; under these conditions idazoxan (an alpha 2-adrenoceptor antagonist) enhanced the release of 5-HT. In neocortex slices from rats (+)-oxaprotiline similarly depressed the release of 5-HT (measured with the same methods) as in human tissue. When rats were pretreated with 6-hydroxydopamine, the inhibitory effect of exogenous NA on 5-HT release was increased, and in slices from rats pretreated with desipramine, it was decreased. In conclusion, alpha 2-heteroreceptors can be activated by endogenous NA released from neighboring noradrenergic fibers. Because regulatory processes analogous to those in rats probably occur in humans as well, an up- or down-regulation of alpha 2-heteroreceptors in depressed patients with a (pathological) decrease or a (therapeutic) enhancement of the noradrenergic neurotransmission may also be assumed to occur.

Accumulation and release of adrenaline, and the modulation by adrenaline of noradrenaline release from rabbit blood vessels in vitro

The accumulation of (-)-3H-adrenaline (3H-A) by rabbit isolated aorta was studied. In all experiments, monoamine oxidase and catechol-O-methyltransferase were inhibited by treatment with pargyline and 3',4'-dihydroxy-2-methyl-propiophenone, respectively. The relationship between the accumulation of 3H derived from 3H-A and the duration of incubation was linear. The 3H-accumulation after 3 h incubation was 22.5 ml/g. In reserpine-treated tissue, the 3H-accumulation levelled off after 30 min and was 8.5 ml/g after 3 h. The concentration of 3H-A or (-)-3H-noradrenaline (3H-NA) and the 3H-accumulation (ml/g) were inversely related. At 10(-8) M, the 1-hour accumulation of 3H derived from 3H-A and 3H-NA was 7.8 and 15.2 ml/g, respectively. With increasing concentrations the accumulation values approached each other. The accumulation of 3H derived from 3H-A by reserpine-treated tissue also showed an inverse relationship with concentration. The accumulation of 3H derived from 3H-A was dependent on the bath temperature. Storage of tissue (0-5 days in salt solution without equilibration with 95% O2/5% CO2; 4 degrees C) did not affect the accumulation of 3H derived from 3H-A. Thereafter (7-14 days), the accumulation decreased. The inhibitory potency (IC50; -log M) of desipramine, cocaine, propranolol, isoprenaline, and normetanephrine on accumulation of 3H derived from 3H-A was found to be 8.26; 6.50; 5.48; 4.88, and 4.02, respectively. The maximal degree of inhibition was almost the same for these drugs, while that of clonidine and corticosterone was 50 and 20%, respectively. In the presence of desipramine, either clonidine, corticosterone or isoprenaline reduces the accumulation of 3H derived from 3H-A. Ouabain and iodoacetic acid, but not sodium cyanide and 2,4-dinitrophenol, reduced the accumulation of 3H derived from 3H-A. Anoxia (95% N2/5% CO2; 37 degrees C; 1-24 h) did not alter the accumulation of 3H derived from 3H-A. Glucose deprivation alone or combined with anoxia markedly reduced the 3H-accumulation. The release of 3H-A from rabbit isolated aorta was studied. This release was compared with that of 3H-NA. The stimulation-evoked 3H-overflow from aorta preloaded with 3H-A decreased with repeated stimulation. In contrast, prestimulation enhanced subsequent stimulation-evoked 3H-overflows. For both 3H-amines, the 3H-overflow increased concomitantly to the same degree with the number of pulses. The time course of 3H-overflows with either 3H-A or 3H-NA was compared.(ABSTRACT TRUNCATED AT 400 WORDS)

Lack of presynaptic modulation by isoprenaline of 3H-noradrenaline release from rabbit isolated ear artery

The aim of the present investigation was to examine whether or not presynaptic facilitatory beta-adrenoceptors are detectable on the postganglionic nerves in the rabbit isolated ear artery. Strips of rabbit central ear artery were incubated with 3H-noradrenaline (10(-7) mol/l; 30 min or 10(-6) mol/l; 60 min). Subsequently, they were washed repeatedly with physiological salt solution. The strips were subjected to electrical-field stimulation (S1-S8) and the resultant 3H-overflow was determined. When the ear artery was stimulated with 150 pulses (0.5 ms; 3 Hz; 225 mA), isoprenaline (10(-9)-10(-6) mol/l) either alone or in the presence of either rauwolscine (10(-6) mol/l) or phentolamine (10(-6) mol/l) did not alter the stimulation-evoked 3H-overflow. This was also the case in the presence of rauwolscine (10(-6) mol/l) plus either the selective phosphodiesterase inhibitor ICI 63 197 (3 x 10(-5) mol/l) or forskolin (10(-6) mol/l). When the ear artery was stimulated with 300 pulses (1 ms; 5 Hz; 225 mA), isoprenaline had no effect on the stimulation-evoked 3H-overflow. This was also the case when phentolamine (10(-6) mol/l) was present. Propranolol (10(-7)-10(-5) mol/l) did not alter the stimulation-evoked 3H-overflow. In some experiments, the stimulation current was reduced to 175 mA in order to obtain similar reference release (S3) values despite the presence of rauwolscine (150 pulses; 0.5 ms; 3 Hz). Even then, isoprenaline (10(-9)-10(-6) mol/l) did not change stimulation-evoked 3H-overflow. The results suggest that postganglionic sympathetic nerves in rabbit central ear artery do not possess presynaptic facilitatory beta-adrenoceptors.

Presynaptic modulation by adrenaline of 3H-noradrenaline release in rabbit ear artery.

The aim of the present investigation was to examine the ability of adrenaline to modulate presynaptically the stimulation-evoked release of noradrenaline from postganglionic sympathetic nerves in the rabbit ear artery. Strips of rabbit central ear artery were incubated with 3H-noradrenaline. Subsequently, they were washed repeatedly with physiological salt solution containing cocaine and corticosterone. The strips were subjected to repeated electrical-field stimulation (S1-S8, 150 pulses, 0.5 ms, 225 mA, 3 Hz) and the resultant 3H overflow was determined. Adrenaline (10(-8) to 10(-6) mol/l) and clonidine (10(-9) to 10(-6) mol/l) reduced the stimulation-evoked 3H overflow. Rauwolscine (10(-7) to 10(-5) mol/l) and phentolamine (3 x 10(-7) to 3 x 10(-5) mol/l) enhanced markedly the stimulation-evoked 3H overflow. Rauwolscine (10(-6) mol/l) abolished the inhibitory effect of low concentrations of adrenaline (10(-8) to 10(-7) mol/l) and clonidine (10(-9) to 10(-7) mol/l) and attenuated the inhibitory effect of the highest concentration (10(-6) mol/l) of clonidine, but not that of adrenaline. In some experiments, the stimulation current was reduced to 175 mA in order to obtain similar reference release (S3) values despite the presence of rauwolscine. Even then, only the highest concentration (10(-6) mol/l) of adrenaline decreased the stimulation-evoked 3H overflow. Facilitation was not seen. It is concluded that adrenaline activates inhibitory presynaptic alpha 2-adrenoceptors. Furthermore, adrenaline does not reveal the presence of presynaptic facilitatory beta-adrenoceptors in the rabbit ear artery.

Release of 3H‐Noradrenaline from Rabbit Isolated Ear Artery

The stimulation-evoked 3H-overflow from rabbit isolated ear arteries preincubated with 3H-noradrenaline was studied. Three strips were derived from each central artery. The strips were incubated (30 min.) with 3H-noradrenaline (10(-7) M) and the spontaneous 3H-outflow and stimulation-evoked 3H-overflow were followed by fractional collection. After a wash-out period (75 min.), the strips were stimulated (225 mA; 150 monophasic pulses; 3 Hz; 0.5 msec.) several times. The initial stimulation-evoked 3H-overflow (S1) was about 5-fold higher than the subsequent five 3H-overflows (S2-S6) which remained almost constant. Bretylium (10(-5) M), tetrodotoxin (10(-6) M), and omission of Ca2+ from the physiological salt solution reduced the stimulation-evoked 3H-overflow by maximally 52%, 77% and 62%, respectively. An increase in stimulation current from 50 to 225 mA caused a continuous rise in stimulation-evoked 3H-overflow, which tended to be Ca2(+)-sensitive. The stimulation-evoked 3H-overflow was frequency-dependent: at 1-4 Hz, the 3H-overflows were the same; at 8 and 16 Hz, they increased. Cocaine (3 x 10(-5) M) plus corticosterone (4 x 10(-5) M) enhanced the stimulation-evoked 3H-overflow at 1-8 Hz, while it had no effect at 16 Hz. Propranolol (3 x 10(-7) M) did not antagonize this enhancement. An increase in number of pulses from 10 to 1000 in the stimulus caused a corresponding rise in the evoked 3H-overflow. This was also the case when cocaine plus corticosterone were present.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine A1‐receptor‐mediated inhibition of evoked acetylcholine release in the rat hippocampus does not depend on protein kinase C

The possible involvement of protein kinase C and/or a lipoxygenase product in the mechanism by which adenosine inhibits release of [3H]acetylcholine evoked by electrical pulses from [3H]choline-labelled hippocampal slices was examined. For comparison, the muscarinic autoreceptors were examined using carbachol. The order of potency of adenosine analogues (CHA = R-PIA greater than NECA much greater than CGS 21680, CV 1808) indicates that the adenosine receptor responsible is of the A1 subtype. Adenosine (10 microM) and R-PIA (0.1 microM) were virtually equiactive as inhibitors and were antagonized to an equal extent by 8-CPT with a potency (IC50 approximately 25 nM) which is also compatible with A1-receptor mediation. The effects of carbachol and of R-PIA were not antagonized by the lipoxygenase inhibitor NDGA (10 or 50 microM). Stimulation of protein kinase C by the phorbol ester 4 beta-phorbol 12,13-dibutyrate caused a concentration-dependent increase in stimulation-evoked 3H overflow, but did not antagonize the presynaptic inhibitory effect of R-PIA or carbachol (0.01-1 microM). Staurosporine (0.1 microM), which inhibited the stimulating effect of phorbol dibutyrate, did not alter the effects of carbachol or R-PIA. The presynaptic effects of phorbol dibutyrate, R-PIA and adenosine were reduced by pretreatment with N-ethylmaleimide (100 microM for 10 min), which inactivates G-proteins. The evoked transmitter release was unaffected by nifedipine (1 microM) in the presence and in the absence of phorbol dibutyrate. These results indicate that adenosine, by acting at presynaptic A1-receptors, reduces transmitter release by a mechanism that involves neither an NDGA-sensitive lipoxygenase nor protein kinase C. The results also indicate that the enhancement of transmitter release by phorbol esters is due to protein kinase C activation and that a G-protein may be involved in the effect but a dihydropyridine-sensitive L-type Ca2+ channel probably is not.

Intra- and extraluminally-applied acetylcholine on the vascular tone or the response to transmural stimulation in dog isolated mesenteric arteries

Acetylcholine applied extraluminally to isolated, perfused dog mesenteric artery segments produced an endothelium-dependent depressor response when the perfusion pressure was raised by continuous infusion of noradrenaline; the potency was 1/30 to 1/60 that of intraluminal acetylcholine. Contractions induced by transmural electrical stimulation were attenuated by treatment with intra- and extraluminal acetylcholine; the inhibitory effect of intraluminal acetylcholine was greater than that of extraluminal acetylcholine. Removal of endothelium did not significantly alter the inhibitory effect. In mesenteric artery strips with endothelium, treatment with oxyhaemoglobin suppressed the relaxant response to acetylcholine but did not influence the inhibitory effect of acetylcholine on stimulation-evoked contractions. Acetylcholine reduced the 3H-overflow and contraction of superfused mesenteric artery strips, preloaded with 3H-noradrenaline, response to transmural stimulation. By the use of bioassay (dog femoral artery segment with endothelium/coronary artery strip without endothelium), the release of EDRF was first determined in the perfusate, which was introduced to dog mesenteric artery strips loaded with 3H-noradrenaline. The 3H-overflow and contraction caused by the stimulation were not attenuated by EDRF and were also observed following treatment with superoxide dismutase. Inability of the perfusate to reduce the stimulation-evoked 3H-overflow was also observed when the donor and assay tissues were treated with superoxide dismutase. It may be concluded that the inhibition by acetylcholine of the release of neuronal noradrenaline is not dependent on endothelium. Extraluminally applied acetylcholine would reach the endothelium and release EDRF, and intraluminal acetylcholine is presumed to act directly on prejunctional muscarinic receptors; however, acetylcholine appears to cross the medial layer more efficiently from intima to adventitia than in the reverse direction.

Adrenaline released as a cotransmitter does not enhance stimulation-evoked 3H-noradrenaline release from rabbit isolated aorta.

1. The aim of the present investigation was to examine if adrenaline released as a cotransmitter by field stimulation of sympathetic neurons in rabbit isolated aorta facilitates noradrenaline release by activation of presynaptic beta-adrenoreceptors. 2. Rabbit aortic rings were preincubated with adrenaline (10(-8)-3 x 10(-6) M) or noradrenaline (10(-8)-3 x 10(-6) M) prior to incubation with 3H-noradrenaline (3H-NA; 10(-7) M). Subsequently, the tissues were subjected to repeated electrical-field stimulation. 3. Preincubation of aorta with either adrenaline or noradrenaline did not change the time course of repeated stimulation-evoked 3H-overflow from tissues preloaded with 3H-NA. 4. Propranolol (10(-8)-10(-6) M) did not alter the stimulation-evoked 3H-overflow. 5. Rauwolscine (10(-7)-10(-5) M) enhanced markedly the 3H-overflow evoked by stimulation. In the presence of rauwolscine, propranolol (10(-8)-10(-6) M) and metoprolol (10(-8)-10(-6) M) did not change the 3H-overflow. 6. In experiments using rings of aorta exposed to adrenaline (10(-8) M) for 30 min in the absence of cocaine, stimulation-evoked 3H-overflow was not affected after withdrawal of adrenaline. This was also the case in the presence of propranolol (10(-6) M). 7. We conclude that adrenaline released as a cotransmitter from sympathetic nerve terminals in rabbit aorta does not facilitate noradrenaline release.

Frequency-dependence of 3H-noradrenaline release from rabbit pulmonary artery: effect of alpha-adrenoceptor antagonists and inhibitors of transmitter inactivation.

The aim of this study was to examine the modulating role of presynaptic alpha 2-adrenoceptors on transmitter release from vascular sympathetic neurones. This was done by examining the influence of removal of inactivation pathways on the effect of alpha-adrenoceptor antagonists on the release of transmitter from noradrenergic neurones. The rabbit main pulmonary artery preloaded with 3H-noradrenaline (3H-NA) was used. The artery was stimulated with 300 pulses at various frequencies (1, 3, 10 and 30 Hz). Pargyline (3 x 10(-4) M) increased the stimulation-evoked 3H-overflow at 1 and 3 Hz and decreased it at 30 Hz. U-0521 (3',4'-dihydroxy-2-methylpropiophenone; 3 x 10(-6) M) enhanced the overflow at 1 Hz and had no effect at 3-30 Hz. Corticosterone (4 x 10(-5) M) did not alter the stimulation-evoked 3H-overflow at 1-30 Hz. Cocaine (3 x 10(-6) M) enhanced the 3H-overflow slightly at 1-30 Hz. At 3 x 10(-5) M, cocaine enhanced 3H-overflow at 1 Hz and reduced it at 30 Hz. Neither corticosterone (4 x 10(-5) M) nor propranolol (10(-7) M) modified this effect of cocaine. Propranolol (10(-7) M) alone decreased the 3H-overflow at 30 Hz and had no effect at 1-10 Hz. Phenoxybenzamine (10(-6) M) and chlorpromazine (3 x 10(-6) M) potentiated the stimulation-evoked 3H-overflow at 1-30 Hz.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of isoprenaline on noradrenaline release from sympathetic neurones in rabbit isolated pulmonary artery

The aim of the present work was to demonstrate the presence of presynaptic beta-adrenoceptors in the rabbit isolated pulmonary artery by investigating the effect of isoprenaline on 3H-noradrenaline (3H-NA) release evoked by electrical field stimulation. (-)-Isoprenaline (10(-7)-10(-6) mol/l) had no effect on the 3H-overflow evoked by stimulation (3 Hz) of the pulmonary artery preloaded with 3H-NA. At 10(-5) mol/l, (-)-isoprenaline reduced the 3H-overflow by maximally 39%. (-)-Isoprenaline (10(-5) mol/l) caused an inhibition that remained almost constant with time. The same results were obtained with (-)-isoprenaline (10(-7)-3 X 10(-5) mol/l) in the presence of cocaine (3 X 10(-5) mol/l), corticosterone (4 X 10(-5) mol/l), and the catechol-O-methyltransferase inhibitor U-0521 (3',4'-dihydroxy-2-methylpropiophenone) (10(-4) mol/l). In the presence of cocaine plus corticosterone, (-)-isoprenaline (3 X 10(-10)-10(-7) mol/l) had no effect on the 3H-overflow evoked by stimulation at 1 Hz. At 10(-6) mol/l, (-)-isoprenaline slightly reduced the 3H-overflow. At 10 Hz, (-)-isoprenaline (10(-6)-3 X 10(-5) mol/l) decreased the 3H-overflow and had no effect at 10(-7) mol/l. In the presence of either rauwolscine (10(-6) mol/l), phentolamine (10(-6) mol/l) or the phosphodiesterase inhibitor ICI 63,197 (3 X 10(-5) mol/l), (-)-isoprenaline (10(-7)-10(-6) mol/l) did not enhance the stimulation-evoked 3H-overflow. (+/-)-Propranolol (10(-7)-10(-5) mol/l) did not alter the stimulation-evoked 3H-overflow.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of chlorpromazine on the metabolism of 3H-noradrenaline released from rabbit aorta.

The effect of chlorpromazine on the metabolism of 3H-noradrenaline (3H-NA) released spontaneously or by electrical-field stimulation was studied on the rabbit isolated aorta preloaded with 3H-NA. Chlorpromazine (10(-6) M) neither altered the spontaneous outflow of total tritium nor had any major effect on the distribution of the 3H-outflow on 3H-NA and its 3H-metabolites. Chlorpromazine (10(-6) M) altered the distribution in stimulation-evoked 3H-overflow. Thus, 3H-NA and 3H-NMN were increased, 3H-DOPEG was markedly decreased and 3H-DOMA and 3H-OMDA were unchanged. It is concluded that chlorpromazine is an inhibitor of uptake-1.

Prejunctional α2-adrenoceptors modulate stimulation-evoked norepinephrine release in rabbit lateral saphenous vein

Segments of rabbit lateral saphenous vein prelabelled with [ 3H]noradrenaline were perfused and superfused with physiological salt solution. Tritium overflow evoked by transmural nerve stimulation (3 Hz for 2 min) was abolished by tetrodotoxin (1 μM). The selective α 2-adrenoceptor agonist UK 14,304 inhibited stimulation-evoked 3H-overflow in a concentration-dependent manner, with an IC 50 of 71 nM. In contrast, the α 2-adrenoceptor agonist B-HT 933 had no effect on 3H-overflow in concentrations up to 10 μM. The selective α 2-adrenoceptor antagonists idazoxan and SKF 86466, as well as the non-selective α-antagonist phentolamine, facilitated the nerve stimulation evoked 3H-overflow, with an order of potency of idazoxan⩾ phentolamine> SK&F 86466. Prazosin (100 nM) had little effect on 3H-overflow. These findings suggest that stimulation-evoked neurotransmitter release is modulated via prejunctional α 2-adrenoceptors.