A comparison of the cardiovascular and sedative actions of the alpha-adrenoceptor agonists, FLA-136 and clonidine, in the rat

1 The degree of selective monoamine oxidase (MAO) inhibition produced by (-)-deprenyl, clorgyline, LY51641 and tranylcypromine was examined in relation to modification of tyramine and noradrenaline contractile responses of the rat isolated vas deferens. 2 All inhibitors possessed reversible alpha-adrenoceptor blocking activity, determined against noradrenaline on the denervated vas deferens. For LY51641 and tranylcypromine, antagonism was competitive, with pA2 values of 6.17 and 5.16. 3 Clorgyline, LY51641 and (-)-deprenyl (10(-5) M) inhibited the tyramine response while present in the organ bath: LY51641, which was the most potent as an alpha-adrenoceptor blocker, produced this effect at 10(-6) M. Responses to tyramine and noradrenaline were potentiated on wishing out the inhibitors, but noradrenaline potentiation was seen only when tyramine had been present in the system. 4 Tranylcypromine (10(-6) M) potentiated responses to noradrenaline and tyramine while present in the organ bath. 5 Potentiation of tyramine responses by clorgyline and LY51641 occurred at 91% and 64% inhibition of MAO type A respectively, although full potentiation of the tyramine response was elicited only when substantial inhibition of both enzyme types occurred. Selective inhibition of MAO type B by 67% (with deprenyl) was not associated with tyramine potentiation.     

The potentiating effects of 4-aminopyridine on adrenergic transmission in the rabbit vas deferens.

The effects of 4-aminipyridine on adrenergic transmission in rabbit vas deferens were investigated. 10(-5)-10(-3) M 4-aminopyridine markedly potentiated the sustained, secondary contractile response to transmural stimulation and significantly increased the amount of (+/-)-|3H]-metaraminol released at 5 Hz, but had little effect on responses to exogenous (-)-noradrenaline. 10(-5)-10(-4) M 4-aminopyridine did not significantly alter the accumulation or efflux of (-)-[3H]-noradrenaline in rabbit heart. It is suggested that 4-aminopyridine potentiated responses of rabbit vas deferens to transmural stimulation mainly by increasing transmitter release, possibly as a result of prolongation of the action potential. Responses to transmural stimulation were also potentiated by by 2- and 3-aminopyridine and by 3,4-diaminopyridine but not by pyridine, aniline, 4-dimethylaminopyridine, 4-aminoethylpyridine or 2-aminopyrimidine.     

Modification of blood pressure and nictitating membrane response to sympathetic amines by selective monoamine oxidase inhibitors, types A and B, in the cat.

The selective monoamine oxidase (MAO) inhibitors clorgyline, selegiline and AGN 1135 did not cause a change in responses of the cat nictitating membrane to preganglionic sympathetic nerve stimulation at 5 Hz. Both selective MAO-A and MAO-B inhibitors markedly potentiated nictitating membrane contractions in response to beta-phenylethylamine (PEA). However, the responses to tyramine were unchanged. The pressor responses to tyramine were potentiated by the selective MAO-A inhibitor clorgyline (2 mg kg-1) but not by selegiline (1.0 mg kg-1) and AGN 1135 (1.5 mg kg-1), selective MAO-B inhibitors. At the doses used selegiline and AGN 1135 caused a near total selective inhibition of liver and brain MAO-B, while clorgyline inhibited MAO-A only in the brain. AGN 1135, like selegiline, could be a useful drug in potentiating the action of L-DOPA in Parkinson's disease.     

Effects of amiloride on contractions and the release of tritium from rat vas deferens preloaded with [3H]-noradrenaline.

1 The effect of amiloride was studied on contractions and tritium release from rat vas deferens preloaded with [3H]-noradrenaline. 2 Amiloride had no effect on the resting tension and maximal contractile force of the vas deferens and did not alter the ED50 of noradrenaline. 3 Amiloride (10(-4)-10(-3)M) decreased the response of vas deferens to electrical stimulation dose-dependently without inhibiting the response to KCI (60mM). 4 The effect of amiloride was not prevented by preincubation of the tissue with phentolamine, propranolol, atropine or indomethacin. 5 Amiloride did not alter the spontaneous outflow of radioactivity from [3H]-noradrenaline labelled vasa deferentia. 6 Amiloride decreased the release of tritium induced by electrical stimulation or nicotine but did not inhibit the release of radioactivity induced by KCI or tyramine. 7 It is concluded that amiloride may inhibit the contractions of rat vas deferens by inhibiting the release of noradrenaline.     

Release of endogenous ATP from the vasa deferentia of the rat and guinea-pig by the indirect sympathomimetic tyramine

1 Adenosine 5′triphosphate (ATP) as well as [³H]-noradrenaline ([³H]-NA) is released by perfusion of the vas deferens with the indirect sympathomimetic tyramine (100 μM); this result is consistent with the concept of sympathetic cotransmission. 2 While tyramine produced a strong contraction in the vas deferens of the rat, it had little mechanical action in the guinea-pig vas deferens. This appears to be largely because tyramine induces considerably lower levels of release of both ATP and NA from the guinea-pig vas deferens compared to that of the rat. Furthermore, NA released by tyramine appears to release ATP from a secondary pool in the rat vans deferens, but not that of the guinea-pig, since prazosin reduced the tryamine-induced release of ATP in the rat vas deferens. 3 α, β -Methylene ATP (α, β -meATP) increased both the spontaneous release of ATP and the tyramine-evoked efflux of ATP and [³H]-NA. The basal and tyramine-induced efflux of [³H]-NA was also enhanced by the α₁-adrenoceptor antagonist, prazosin, suggesting that prejunctional α₁-adrenoceptors may modulate neurotransmitter release.     

The effect of ethacrynic acid on the guinea-pig and rat isolated vas deferens

1 The effect of ethacrynic acid (EA) was studied on guinea-pig and rat vas deferens in vitro.2 EA contracted the guinea-pig but not the rat vas deferens in a dose-dependent manner (50-800 mug/ml). Tyramine caused contraction in 10 out of 18 guinea-pig vas deferens; EA caused contraction in 17 of the preparations which did not respond to tyramine. Repeated doses of EA produced tachyphylaxis, but there was no cross tachyphylaxis to tyramine.3 The contractions produced by EA were prevented by phentolamine or reserpine pretreatment and potentiated by cocaine. A low concentration of desipramine (3 ng/ml) potentiated and higher concentrations (0.6 and 3.0 mug/ml) inhibited the response of vas deferens to EA.4 Hexamethonium (100 mug/ml) or atropine (0.1 mug/ml) did not inhibit the effect of EA, excluding the nicotinic and muscarinic receptors as the sites of action.5 The effect of noradrenaline (NA) on the guinea-pig and rat vas deferens was enhanced by EA pretreatment, which may be due to inhibition of NA uptake.6 It is concluded that EA releases NA from guinea-pig vas deferens. The mechanism of release seems to be different from that of tyramine.     

Effect of enkephalin and substance P on sympathetic nerve transmission in mouse vas deferens.

Effect of methionine-, leucine-enkephalin (met-, leu-enkephalin) and substance P on the transmission in mouse vas deferens was studied. Both met- and leu-enkephalin inhibited electrically induced contraction of vas deferens at 10(-8)-10(7) M, met-enkephalin being 1.4 times more active than leu-enkephalin. Nalorphine (10(-6) M) antagonized these effects. Substance P (10(-9)-10(-7) M) had no effect on the contraction. Met- and leu-enkephalin (10(-7)-10(-5) M) decreased the high potassium induced [3H]-norepinephrine release from vas deferens, while substance P (10(-6) M) significantly increased it. Nalorphine (10(-5) M) reversed the inhibitory effect of met-enkephalin. These results indicate that these peptides modify the transmission of sympathetic nerve in mouse vas deferens.     

Postjunctional inhibition of contractor responses in the mouse vas deferens by rat and human calcitonin gene-related peptides (CGRP).

The effects of rat and human alpha-calcitonin gene-related peptide (CGRP) were compared in the mouse and rabbit isolated vas deferens preparation contracted by either field stimulation or acetylcholine. The peptides were about equipotent at inhibiting twitch responses of the mouse vas deferens to field stimulation at 0.2 Hz (IC50 12 +/- 4 nM and 15 +/- 3 nM, rat and human alpha-CGRP respectively). Rat alpha-CGRP was less potent at inhibiting responses to 10 Hz than to either 0.2 Hz or 1.0 Hz stimulation. The potency of rat alpha-CGRP at 1.0 Hz was unaltered by halving the calcium concentration of the Krebs solution. The inhibitory effect of human alpha-CGRP was not antagonized by either propranolol (300 nM) or idazoxan (300 nM), although in the same tissues these latter two drugs reduced responses to isoprenaline and clonidine respectively. Rat alpha-CGRP (100 nM) and human alpha-CGRP (1.0 microM) did not alter the uptake of [3H]-noradrenaline (30 nM) into mice isolated vasa deferentia. Rat alpha-CGRP (3-100 nM) did not alter the fractional release per pulse (1.0 Hz, 100 pulses) of tritium from vasa preloaded with [3H]-noradrenaline, although at the same time the peptide inhibited responses of the smooth muscle to field stimulation. Rat and human alpha-CGRP were equipotent at inhibiting contractions of the mouse vas deferens evoked by acetylcholine although the peptides were less potent than against twitch responses. In the rabbit vas deferens neither rat nor human alpha-CGRP (3 nM-1 microM) inhibited either twitch responses or acetylcholine contractions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Potentiation of the effects of adenosine on isolated cardiac and smooth muscle by diazepam.

1. Adenosine (10(-7) to 3 x 10(-4) M) or 2-chloroadenosine (10(-8) to 10(-5) M) produced concentration-dependent inhibition of the responses of the rat isolated vas deferens to electrical field stimulation. In electrically driven (2 Hz) guinea-pig isolated left atria, adenosine (10(-6) to 10(-3) M) or 2-chloroadenosine (10(-8) to 10(-7) M) produced concentration-dependent decreases in isometric tension. 2. Diazepam (10(-6) and 10(-5) M) had no direct effect per se, but significantly potentiated the inhibitory action of adenosine on both tissues without altering the inhibitory effect of 2-chloroadenosine. 3. The adenosine uptake inhibitors, hydroxynitrobenzylthioguanosine (HNBTG, 10(-5) M) and dipyridamole (10(-5) M) also potentiated the inhibitory actions of adenosine in rat vas deferens, but not hose of 2-chloroadenosine. 4. Following adenosine uptake inhibition in rat vas deferens by HNBTG (10(-5) M), diazepam (10(-5) M) failed to produce any significant further potentiation of the inhibitory action of adenosine. 5. It is concluded that the potentiation of adenosine by diazepam is possibly due to an inhibition of adenosine uptake.     

Calcium mobilization and phosphatidylinositol turnover in vas deferens smooth muscle of diabetic rats

The study concerned Ca2+ channels that are receptor-operated by norepinephrine (NE) and mediate hyper-reactivity of vas deferens smooth muscle from rats with streptozotocin (STZ)-induced diabetes, and the mediatory responses of these channels, such as tension development, Ca2+ uptake and phosphatidylinositol (PI) turnover. The contractile responses induced by adrenoceptor agonists were significantly greater in diabetic rat vas deferens than in the controls. A greater Ca2+ uptake was induced by 10(-5) M NE in strips from diabetic rats than in the controls. The uptake of Ca2+ was completely inhibited by 10(-6) M prazosin but not by 10(-5) M verapamil. Enhancement of Ca2+ release by 10(-5) M NE was faster and greater in diabetic muscles than in the controls. The accumulation of [3H]inositol phosphates was increased 4-fold in the controls and 7-fold in diabetic muscles by 10(-5) M NE. This increase was completely inhibited by 10(-6) M prazosin but not by 10(-6) M yohimbine. The data suggest that vas deferens smooth muscle hyper-reactivity in diabetic rats is due to increased PI turnover mediated by alpha 1-adrenoceptors, to the release of intracellular bound Ca2+ and to an increase of Ca2+ uptake through receptor-operated Ca2+ channels.     

(-)-Deprenyl inhibits tyramine-induced noradrenaline release, but not tyramine-induced dopamine release or potassium-induced noradrenaline release, from rat brain synaptosomes.

The effect of (-)-deprenyl (selegiline), a therapeutic agent for Parkinson's disease, on the tyramine-induced release of catecholamine from rat brain synaptosomes was studied using a superfusion system. Tyramine (10(-7) to 10(-5)M) enhanced the release of [3H]noradrenaline (NA) and [3H]dopamine (DA) from forebrain and striatal synaptosomes in a dose-dependent manner. (-)-Deprenyl (5x10(-5)M) had no effect on spontaneous catecholamine release, suggesting that it has no tyramine-like catecholamine releasing effect. Pretreatment with (-)- or (+)-deprenyl (5x10(-5)M) significantly prevented the tyramine (10(-6)M)-induced NA release, but not DA release. The inhibitory action of (-)-deprenyl was not observed on potassium (15mM)-induced NA release. (-)-Desmethyldeprenyl (5x10(-5)M), a metabolite of (-)-deprenyl, and a monoamine oxidase-A (MAO-A) inhibitor, clorgyline (5x10(-5)M), failed to block the tyramine-induced NA and DA release. Although (+)-deprenyl, a potent DA uptake inhibitor, did not inhibit tyramine-induced DA release, a catecholamine uptake inhibitor nomifensine (5x10(-5)M) did. In summary, (-)-deprenyl at a dose inhibiting tyramine-induced NA release did not have any effect on tyramine-induced DA release or potassium-induced NA release.     

The effect of cocaine and imipramine on tyramine-induced release of noradrenaline-3H from the rat vas deferens in vitro

1. Tyramine 10(-4)M significantly increased release of noradrenaline-7-(3)H (NA-7-(3)H) from rat vas deferens in vitro.2. Neither cocaine 10(-5)M nor imipramine 10(-7)M-10(-6)M significantly reduced tyramine-induced release of NA-7-(3)H.3. Increasing the exposure time to cocaine and imipramine from 10 to 20 min or pre-incubating the tissue with a wide range of NA-7-(3)H concentrations (3.3-333.3 ng/ml.) did not affect the lack of inhibition by cocaine and imipramine.4. It is suggested that the tyramine receptor in rat vas deferens differs from that in other systems and that blockade of tyramine-released noradrenaline at alpha-adrenergic receptors may be the most important mechanism for tyramine antagonism by imipramine-like drugs in this tissue.     

Effect of the dihydropyridine Bay K 8644 on the release of [3H]-noradrenaline from the rat isolated vas deferens.

The effects of Bay K 8644 on the release of [3H]-noradrenaline evoked by potassium, electrical stimulation or tyramine from the rat isolated vas deferens labelled with [3H]-noradrenaline were investigated. Bay K 8644 (1 microM) by itself did not affect the spontaneous release of tritium from the rat isolated vas deferens. However, it increased the calcium-dependent release of tritium elicited by both high potassium (59 mM) and electrical field stimulation. The exposure of rat vas deferens to phentolamine (10 microM) increased the release of tritium induced by potassium (59 mM) and electrical field stimulation. Bay K 8644 (1 microM) failed to increase further the release of tritium elicited by both stimuli in preparations previously treated with phentolamine (10 microM). The calcium-independent release of [3H]-noradrenaline evoked by tyramine (10 microM) was not affected by Bay K 8644 (1 microM). The results of our study support the view that alpha2-adrenoceptors modulate noradrenaline release by restricting calcium influx into sympathetic nerve terminals through voltage-dependent channels.     

The effects of drugs inhibiting catecholamine uptake on tyramine and noradrenaline-induced contractions of the isolated rat vas deferens

1. Cocaine did not antagonize the tyramine-induced contractile response of the isolated rat vas deferens at the same concentrations which markedly potentiated the contractile response to noradrenaline.2. Imipramine and amitriptyline non-competitively antagonized the contractile response to tyramine but did not potentiate noradrenaline. Desmethylimipramine produced both potentiation of noradrenaline and antagonism of tyramine.3. Dexchlorpheniramine non-competitively antagonized the contractile response to tyramine. It also produced an atypical potentiation of noradrenaline in which lower concentrations of noradrenaline were potentiated to a greater extent than higher ones.4. Imipramine inhibited the in vitro uptake of noradrenaline-(3)H in rat vas deferens as did cocaine, desmethylimipramine and dexchlorpheniramine. These results suggest that the alpha-adrenergic blocking property of imipramine masks the potentiation of noradrenaline by uptake inhibition.5. Evidence is also presented which suggests that alpha-adrenergic blockade of released noradrenaline may be the major mechanism for tyramine inhibition by imipramine-like drugs. This may explain why cocaine, which has no real alpha blocking action, is ineffective against tyramine.     

Cocaine potentiates the responses to methacholine and noradrenaline in the rat vas deferens

The ability of cocaine (10 μM) to potentiate the contractile responses of the epididymal half of the rat vas deferens to methacholine was reversed by prazosin. Prazosin also partially reversed the ability of cocaine to increase the spontaneous overflow of ³ H following loading of the tissue with [³ H]noradrenaline. We suggest that cocaine potentiated the responses to methacholine by stimulating, directly or indirectly, α₁-adrenoceptors.     

The uptake and overflow of radiolabelled beta-adrenoceptor blocking agents by the isolated vas deferens of the rat.

1. A comparison of uptake into and overflow from the isolated vas deferens of the rat has been made between [3H]-noradrenaline ([3H]-NA), [14C]-D-sorbitol and three radio-labelled beta-adrenoceptor blocking agents, [14C]-practolol, [14C]-(+/-)-propranolol and [3H]-penbutolol. 2. The accumulation of [3H]-NA after 30 min incubation was reduced by desmethylimipramine (DMI) 1 X 10(-8)M and was also reduced in vasa from rats pretreated with 6-hydroxydopamine (6-OHDA). This was not so with [14C]-D-sorbitol. 3. 6-OHDA pretreatment of the rats reduced the uptake of [3H]-penbutolol after 30 min incubation but not that of [4C]-propranolol or [14C]-practolol. DMI 1 X 10(-8)M did not alter the tissue uptake of [14C]-propranolol, [14C]-practolol or [3H]-penbutolol. 4. Electrical stimulation of vasa preloaded with [3H]-NA caused a significantly greater increase in [3H]-NA overflow than during the resting, unstimulated periods. No such increase in overflow was observed with [14C]-sorbitol or any of the three beta-adrenoceptor blocking agents use. 5. The beta-adrenoceptor blocking agent penbutolol was shown to possess adrenergic neurone blocking activity in the isolated vas deferens of the rat. 6. It is concluded that any effect that practolol or (+/-)-propranolol have on noradrenergic neurones is brought about without the need for these drugs to gain access to the interior of the neurone.     

Effects of the glycine prodrug milacemide (2-N-pentylaminoacetamide) on catecholamine secretion from isolated adrenal medulla chromaffin cells.

1. Milacemide (2-n-pentylaminoacetamide) is a glycine prodrug which readily crosses the blood brain barrier and increases brain glycine and glycineamide. In vitro and in vivo studies, with numerous tissues, including adrenal chromaffin cells, have clearly shown that the formation of the latter metabolites is exclusively mediated by monoamine oxidase B for which milacemide is a substrate. 2. Milacemide, glycineamide and glycine caused a time- and dose-dependent release of catecholamines from bovine isolated chromaffin cells. 3. Milacemide (10(-4) M) induced catecholamine release was roughly 30% of that initiated by acetylcholine (10(-4) M), the natural secretagogue. 4. The combined effects of milacemide (10(-4) M) and acetycholine (10(-4) M) on catecholamine secretion from chromaffin cells is additive, suggesting that milacemide does not act through the normal nicotinic receptor release mechanism. 5. The release of catecholamines from chromaffin cells in response to milacemide (10(-4) M) was partially inhibited by the selective MAO-B inhibitors (-)-deprenyl (10(-7) M) and AGN 1135 (10(-6) M). This indicates that the MAO-B derived metabolites, glycineamide and glycine, contribute to the secretion of catecholamines as does milacemide itself. 6. It is apparent that release of catecholamines by glycine is mediated by its uptake into the cells since [3H]-glycine uptake and catecholamine release showed a highly significant correlation (r = 0.96).     

Effects of ginsenosides on release of [3H]norepinephrine from rat vas deferens and portal vein

The effects of ginsenosides (G) on the release of [3H]norepinephrine ([ 3H]NE) from the isolated rat vas deferens (RVD) and portal vein (RPV) preloaded with [3H]NE were studied, G (100 micrograms/ml) did not affect the spontaneous or high potassium (H-K+, 60 mmol/L)- and tyramine (Tyr 10 mumol/L)-evoked release of [3H]NE, but obviously blunted the phentolamine (Phe 10 mumol/L)-induced increase in [3H]NE release from RVD and enhanced the isoprenaline (Iso 0.1 mumol/L)-augmented [3H]NE release from RPV evoked by H-K+. It is still not known whether G can bind with adrenoceptors. We infer that G may act as a modulator in sensitizing both presynaptic alpha 2- and beta-receptors.     

Radioenzymatic assay measurement of yohimbine's influence on adrenergic neurotransmission of rat vas deferens: unique consequence of using normetanephrine as an uptake2-blocking agent

The effect of the alpha 2-receptor antagonist, yohimbine on norepinephrine overflow was studied in the transmural-stimulated isolated rat vas deferens. A radioenzymatic assay was used to measure the endogenous norepinephrine overflow. In initial studies conducted in the presence of uptake1 blocker desipramine (10(-6) M) and uptake 2 blocker, normetanephrine (10(-5) M) there was an apparent uncoupling of the influence of yohimbine on nerve-stimulated contractile response from norepinephrine overflow. These results were found to be due to the electrolytic O-demethylation of normetanephrine with the resultant generation of large quantities of norepinephrine obscuring the influence of yohimbine on nerve-stimulated norepinephrine overflow from the vas deferens. These findings serve as a warming to the use of normetanephrine as an uptake1 blocker when radioenzymatic assay is used to measure norepinephrine overflow from transmural-stimulated isolated tissue preparations. Yohimbine (10(-6) M), in the absence of uptake blockade, causes a 3-fold enhancement of nerve-stimulated norepinephrine overflow at 1 Hz and a 2-fold enhancement at 10 Hz. This report demonstrates utilization of a radioenzymatic assay to study endogenous norepinephrine overflow from rat vas deferens. Results for yohimbine are complementary to others using measurement of 3H-label overflow from [3H]norepinephrine prelabeled isolated tissue.