Bradykinin and electrical stimulation increase prostaglandin production in the rat vas deferens.

The epididymal portion of the rat vas deferens produced prostaglandins (PG) E(2), F(2alpha)and 6-keto F(1alpha). Electrical stimulation (ES, 0.1 Hz, 1 ms) increased such production by 100%, and similar results were obtained in the presence of 1.0 microM bradykinin (Bk). When both stimuli were applied simultaneously, the increases in PG production were 1100% for PGE(2), 800% for PGF(2alpha)and 400% for PG6-keto F(1alpha). Prazosin abolished the effect of ES on PG production. A selective Bk B(2)-receptor antagonist abolished the increase in PG production induced by Bk, both in non-stimulated and in ES tissues. Bk (1.0 microM) elicited contractile responses in non-stimulated as well as in ES tissues, responses that were not modified in the presence of 10 microM indomethacin. In conclusion, the effects of Bk on prostaglandin production appears to depend on the activation of B(2) receptors, while the increase in prostaglandin release induced by ES, and the effects observed with both stimuli simultaneously, should be mediated by the release of noradrenaline and the subsequent activation of alpha(1) adrenoceptors.

Involvement of GABA and glutamate receptors in the blood pressure responses to intrathecally injected sodium nitroprusside in anesthetized rats.

In pentobarbital-anesthetized rats the intrathecal (i.t.) injection of the nitric oxide (NO) donor, sodium nitroprusside (125, 250 and 500 nmol), induced a dose-dependent hypotensive response followed by a dose-dependent pressor effect. The pressor response to sodium nitroprusside (250 nmol) was reduced to 30% of the control value by the selective antagonist for AMPA/kainate receptors, 6.7-dinitroquinoxaline-2,3-dione (50 nmol, i.t.), whereas it was not modified by the selective NMDA receptor antagonist, 2-amino-5-phosphono-valeric acid (30 nmol, i.t.). The hypotensive effect of sodium nitroprusside was antagonized by the GABA(A) receptor antagonists, bicuculline (4.4 nmol, i.t.) and picrotoxin (4.4 nmol, i.t.), and also by the GABA(B) receptor antagonist, 2-hydroxy saclofen (113 nmol, i.t.). The blood pressure responses to sodium nitroprusside were not modified by blockade of muscarinic receptors with methyl atropine (164 nmol, i.t.), or of nicotinic receptors with hexamethonium (211 nmol, i.t.), of alpha1-adrenoceptors with prazosin (3.1 nmol, i.t.), of alpha2-adrenoceptors with yohimbine (2.8 micromol/kg, i.v.), of 5-HT receptors with methysergide (5.1 micromol/kg, i.v.), or of glycine receptors with strychnine (65 nmol, i.t.). It is concluded that NO generated from sodium nitroprusside in the spinal cord exerts inhibitory and excitatory effects on blood pressure probably through the release of GABA and glutamate, respectively. The inhibitory action on blood pressure involves the stimulation of spinal GABA(A) and GABA(B) receptors whereas the excitatory response to glutamate appears to be mediated through the activation of spinal AMPA/kainate receptors.

Possible participation of spinal nitric oxide in the control of the blood pressure in anesthetized rats.

In pentobarbital-anesthetized rats the intrathecal (i.t.) injection of the nitric oxide (NO) precursor, L-arginine (10 and 20 micromol), elicited a decrease in the mean blood pressure (MBP) whereas the inhibitor of the NO synthase (NOS) N(G)-nitro-L-arginine methyl ester (L-NAME; 0.1-10 micromol) produced a dose-dependent pressor effect. The pressor response to L-NAME was prevented by pretreatment with L-arginine. Neither D-arginine nor D-NAME modified the MBP. The NO donor sodium nitroprusside (SNP; 0.125 and 0.25 micromol, i.t.) induced a hypotensive response followed by a pressor effect. The dual response to SNP as well as the hypotensive effect of L-arginine were abolished by the guanylate cyclase inhibitor Methylene blue (0.3 micromol, i.t.). Nicotinic ganglionic blockade by hexamethonium (10 mg/kg, i.v.) reduced the hypotensive effects of both L-arginine and SNP and prevented almost completely the pressor effects of either L-NAME or SNP. The pressor effect of L-NAME was abolished by 2-amino-5-phosphonovaleric acid (APV; 30 nmol, i.t.), a selective antagonist of glutamate receptors of the NMDA subtype. These results suggest that in the spinal cord of pentobarbital-anesthetized rats NO exerts both inhibitory and excitatory effects on the preganglionic sympathetic nerve activity related to the control of the BP. The synthesis of NO appears to be tonically activated through the stimulation of spinal glutamate receptors of the NMDA subtype.

Hypotensive and hypertensive effects of catecholamines intrathecally injected in anesthetized rats.

The cardiovascular effects of catecholamines intrathecally (i.t.) injected at the T12-L1 level were analyzed in pentobarbital anesthetized rats. Volumes of injection were not greater than 3 microliters. Noradrenaline in doses ranging from 0.03 to 0.3 micrograms (i.t.) did not alter the mean blood pressure (MBP) while higher doses (1, 3 and 10 micrograms, i.t.) caused a dose-dependent increase in MBP. Adrenaline induced hypotensive effects at low doses (0.03-0.3 micrograms i.t.) and pressor effects at high doses (3 and 10 micrograms, i.t.). Neither adrenaline nor noradrenaline modified the heart rate. The pressor responses to both catecholamines were antagonized by the alpha 1-adrenoceptor blocker prazosin (0.05-1 microgram, i.t.) and by the selective alpha 1A-adrenoceptor antagonist 5-methyl urapidil (10 and 15 micrograms, i.t.). In contrast, these pressor effects were not modified by the alpha 1B-adrenoceptor antagonist chloroethylclonidine (90 micrograms i.t.). In animals pretreated with 1 microgram prazosin (i.t.), low doses of noradrenaline (0.03 and 0.1 microgram, i.t.) caused a hypotensive effect. Prazosin (1 microgram i.t.) failed to alter the hypotension caused by 0.1 microgram adrenaline. The hypotensive response induced by either 0.1 microgram noradrenaline (in the presence of prazosin) or 0.1 microgram adrenaline was blocked by the alpha 2-adrenoceptor antagonist yohimbine (1 mg/kg, i.v.), by the GABA-A antagonists bicuculline (3.2 micrograms, i.t.) and picrotoxin (2.7 micrograms, i.t.), and by the GABA-B antagonist 2-hydroxy saclofen (30 micrograms, i.t.). The glycine-receptor antagonist strychnine (25 micrograms, i.t.) did not modify the hypotension induced by either noradrenaline (in the presence of prazosin) or adrenaline. These findings suggest that in the low thoracolumbar spinal cord of pentobarbital-anesthetized rats, noradrenaline and adrenaline have excitatory as well as inhibitory effects on the control of the BP. The pressor responses of high doses of i.t. injected catecholamines could be mediated by the activation of spinal alpha 1A-adrenoceptors, although the participation of alpha 1B-adrenoceptors cannot be rule out entirely. The hypotensive responses induced by low doses of i.t. injected catecholamines seem to involve the activation of spinal alpha 2A-adrenoceptors and the stimulation of an inhibitory GABAergic neuron in the spinal cord.

Possible participation of histamine H3 receptors in the modulation of noradrenaline release from rat spinal cord slices.

Rat spinal cord slices prelabelled with [3H]noradrenaline were superfused with a medium containing 1 mu M desipramine plus 0.3 mu M phentolamine. Histamine (0.01-10 mu M) and the selective histamine H3 receptor agonist R-(-)-alpha-methylhistamine (0.001-10 mu M) caused a concentration-dependent decrease in the release of radioactivity evoked by electrical field stimulation (0.8 Hz, 20 mA, 2 min). The inhibitory effect of histamine was not modified by either pyrilamine (1 mu M) or ranitidine (10 mu M), but it was antagonized by burimamide (1 mu M). The inhibitory action of histamine (1 mu M) was attenuated by pertussis toxin (3 mu g/ml) and was abolished by N-ethylmaleimide (30 mu M). Neither forskolin (10 mu M) nor rolipram (100 mu M), nor the combination of both drugs, modified the inhibitory effect of histamine. Histamine (1 mu M) did not modify the overflow of tritium induced by electrical stimulation in the absence of phentolamine. The present results suggest that in the rat spinal cord the release of noradrenaline elicited by electrical stimulation is negatively modulated by histamine, probably through the activation of histamine H3 receptors. This modulatory mechanism is likely to involve the participation of regulatory Go/Gi proteins.

Activation of 5-HT2 receptors inhibits the evoked release of [3H]noradrenaline in the rat spinal cord.

1. The participation of 5-HT2 receptors in the modulation of the evoked release of [3H]noradrenaline from rat spinal cord slices has been examined. 2. In rat spinal cord slices preincubated with [3H]noradrenaline, the alpha 2-receptor agonist clonidine (10(-6) mol/l) decreased the release of tritium evoked by field stimulation (600 pulses at 5 Hz, 20 mA, 2 msec), while the alpha 2-antagonist yohimbine (10(-6) mol/l) increased it. 3. The 5-HT2/5-HT1c agonist DOI (3 x 10(-7) mol/l) decreased the evoked release of tritium, an effect which was prevented by ketanserin (10(-7) mol/l). 4. It is suggested that in addition to presynaptic alpha 2-adrenoceptors, there are 5-HT2 receptors which modulate the release of noradrenaline in the rat spinal cord.

Release of 5-hydroxytryptamine, dopamine and noradrenaline in the rat vas deferens in the presence of compound 48/80, veratridine or K+.

1. The release of 5-hydroxytryptamine (5-HT), dopamine and noradrenaline (NA) from the rat isolated vas deferens was estimated by high performance liquid chromatography with electrochemical detection. 2. Compound 48/80, which is known to release 5-HT and histamine from mast cells, induced a concentration-related, rapid overflow of 5-HT from the rat isolated vas deferens, releasing about 50% of the 5-HT present in the tissue at a concentration of 300 micrograms ml-1. The drug did not induce the release of either dopamine or NA. 3. The depolarizing agents veratridine (10 microM) or K+ (100 mM) induced the release of 5-10% of the 5-HT present in the vas deferens, but did not evoke the overflow of 5-HT from isolated mast cells. 4. Veratridine induced the release of a greater proportion of the dopamine than of the NA contained in the vas deferens (41.0 +/- 5.5 vs 17.9 +/- 2.0%, after 20 min of exposure to the drug, n = 6). In contrast, K+ evoked the release of both amines equally. 5. It is concluded that in the rat vas deferens 5-HT is stored in mast cells and also in cells responsive to depolarizing stimuli. It is suggested that 5-HT could be a neuromodulator or a neurotransmitter in this organ. Veratridine and K+ induce the release of dopamine in addition to NA.

Regional distribution of dopamine, 5-hydroxytryptamine, and noradrenaline in the rat vas deferens.

The concentrations of dopamine (DA), 5-hydroxytryptamine (5-HT) and noradrenaline (NA) in the rat vas deferens divided in eight or four sections were determined by high performance liquid chromatography with electrochemical detection. Dopamine and NA had the same regional distribution; their concentrations were maximal near the prostatic end and decreased towards the epididymis. The concentration of 5-HT also decreased from the prostatic to the epididymal end, but 5-HT did not follow the same regional distribution as DA and NA. Reserpine (0.02 or 0.2 mg/kg, i.p., 24 hr) and 6-hydroxydopamine (2 x 80 mg/kg, i.v., 6 days) decreased the contents of DA and NA; the concentrations of both amines were modified to a similar extent. Reserpine also diminished the content of 5-HT. Pargyline (200 mg/kg, i.p., 2 hr) increased the concentration of 5-HT while p-chlorophenylalanine (300 mg/kg, oral, 3 days) decreased the contents of the amine in some sections of the vas deferens. This study suggests that DA and NA co-exist in the same sympathetic neurons. Some of the 5-HT could be stored in mast cells as previously proposed, but the finding that tissue content of 5-HT changes after inhibiting the deamination or synthesis of the amine suggests that other source(s) of 5-HT distinct from mast cells exist in the rat vas deferens.

Pre- and postsynaptic effects of p-tyramine and p-octopamine in the prostatic portion of the rat vas deferens.

The effect of p-tyramine and p-octopamine on the twitch responses of the prostatic portion of the rat vas deferens to electrical stimulation (0.025 Hz) were compared with the effects of noradrenaline. In tissues with normal monoamine oxidase (MAO) activity, the three amines increased the height and duration of the twitch contractions. When MAO activity was inhibited by pargyline (10 mumol/l), p-tyramine and p-octopamine had mixed excitatory-inhibitory effects on the twitches, while noradrenaline had mostly excitatory effects along the whole range of concentrations assayed (0.158-15.8 mumol/l). Selective blockade of alpha 1- and alpha 2-adrenoceptors, by corynanthine and yohimbine, respectively, showed that the excitatory effect of the amines depended on the activation of alpha 1-adrenoceptor and that the inhibitory action was related to the activation of alpha 2-adrenoceptors. Pretreatment with reserpine (5 mg/kg, 24 h; 2.5 mg/kg, 2 h before the experiment) largely prevented the effects of p-tyramine and p-octopamine, but the amines still modified the twitch responses to field stimulation. The addition of corynanthine and yohimbine to the bathing fluid revealed a considerable activation of alpha 1-excitatory and alpha 2-inhibitory adrenoceptors. Cocaine (10 mumol/l) did not antagonize, but rather enhanced the inhibitory effects of p-tyramine and p-octopamine in tissues with normal contents of noradrenaline. Moreover, cocaine did not antagonize the inhibition caused by p-tyramine, and enhanced the inhibition induced by p-octopamine in the prostatic portion of the vasa deferentia from reserpine-pretreated animals. These results suggest that in this tissue, at least when MAO activity is inhibited, p-tyramine and p-octopamine behave similarly.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of deuterium substitution on the chronotropic responses to some sympathomimetic amines in the isolated rat atria.

In spontaneously beating rat atria the potencies for the chronotropic effects of the following deuterated phenylethylamine derivatives were higher than the potencies of the corresponding non-substituted (protio-) amines: alpha,alpha,d2-beta-phenylethylamine; alpha,alpha,beta,beta-d4-p-tyramine; alpha,alpha,beta,beta-d4-m-tyramine; alpha,alpha,beta-d3-p-octopamine. In contrast, alpha,alpha,beta-d3-noradrenaline and alpha,alpha,beta-d3-m-octopamine were equipotent with the corresponding protio-amines. Experiments performed in atria depleted of endogenous noradrenaline by pretreatment with reserpine and in atria exposed to the monoamine oxidase (MAO) inhibitor pargyline indicated: a. p-octopamine had both direct and indirect effects, but the chronotropic responses to p-octopamine in tissues with normal MAO activity depended mostly on the direct action of the amine; deuterium substitution enhanced the indirect component of action of p-octopamine; b. m-octopamine possessed considerable indirect effects while d3-m-octopamine behaved as an amine of direct action. The substitution of deuterium for hydrogens in the alpha-carbon of the alkyl-side chain of phenylethylamines decreases the rate of deamination by MAO. Therefore, the results obtained with all the amines, except for m-octopamine and alpha, alpha,p-d3-m-octopamine, could be interpreted in terms of the direct, indirect or mixed action of those compounds and/or of the influence that MAO activity has on the chronotropic responses to these amines. The results obtained with protio- and deuterio-m-octopamine suggested that deuterium substitution, either at the alpha- or the beta-carbon, can alter some other mechanisms in addition to the enzymatic deamination.

Effects of acute and short-term treatment with antihypertensive drugs in sinoaortic denervated rats.

Sinoaortic denervation (SAD) produced a marked increase of the systolic blood pressure (SBP). Clonidine (50 micrograms kg-1, i.p.) reduced SBP in SAD but not in sham rats. L-alpha-methyldopa (alpha-MD) (50 mg kg-1, i.p.) also induced a more effective hypotensive action in SAD than sham rats. The withdrawal of alpha-MD in SAD rats after the first treatment was not abrupt and the hypotension persisted for several days, but after the second treatment the withdrawal induced a rapid rebound hypertension. Our results suggest that SAD increases the response to the hypotensive agents. An alteration in the availability of alpha-MD to accumulate or synthesize the active metabolites was also observed after second treatment.

Mechanisms involved in the blocking effects of pimozide on smooth muscle contraction.

Pimozide, a dopamine-receptor antagonist, shifted to the right the concentration-response curves (CRC) induced by (-)-noradrenaline in the isolated posterior mesenteric artery of the cat. Since this antagonism could be related to a blockade of vascular alpha-adrenoceptors, we compared the effects of pimozide (0.20 microM) with those of phentolamine (0.31 microM). The onset of the blockade by pimozide on the (-)-noradrenaline-induced contractions was slower when compared with that of phentolamine. In addition, the effects of pimozide did not reverse by washing while those of phentolamine did. Pimozide but not phentolamine inhibited the contractions elicited by potassium and it decreased the maximum responses of the curves. This effect was partially reversed by CaCl2 10.2 mM. Pimozide displaced the CRC induced by CaCl2 to a greater extent than phentolamine. Even if a blocking effect on alpha-adrenoceptors cannot be discarded, our results suggest that pimozide may interfere with calcium in the arterial smooth muscle. Furthermore, vascular smooth muscle seems to be more sensitive than non-vascular tissue to the action of pimozide.

Stimulation of presynaptic beta-adrenoceptors enhances [3H]-noradrenaline release druing nerve stimulation in the perfused cat spleen.

1 The effects of isoprenaline, propranolol and phosphodiesterase inhibitors on (3)H-transmitter overflow elicited by low frequency nerve stimulation were determined in the isolated perfused spleen of the cat.2 (-)-Isoprenaline (0.14, 1.4, and 14 nM) produced a concentration-dependent increase in [(3)H]-transmitter overflow evoked by nerve stimulation at 1 Hz and was more effective at 1 Hz than at 2 hertz.3 A concentration of propranolol (0.1 muM), devoid of neurone blocking activity, blocked this effect of (-)-isoprenaline. These results are compatible with the presence of beta-adrenoceptors in the noradrenergic nerve endings of the cat spleen.4 (+)-Isoprenaline (140 nM) failed to increase the release of radioactivity induced by nerve stimulation, indicating that the beta-adrenoceptor mediating the facilitation of transmitter release was stereospecific.5 The increase in (3)H-transmitter overflow induced by nerve stimulation during exposure to the phosphodiesterase inhibitor, papaverine (27 muM) was more pronounced than that obtained in the presence of 3-isobutyl-1-methyl xanthine (IBMX) 0.5 mM. The facilitation in transmitter release induced by papaverine was not correlated with the granular effect produced by this drug.6 In the presence of papaverine, the concentration-effect curve for (-)-isoprenaline on transmitter release was shifted to the left and its maximum was increased. In addition, propranolol significantly reduced the enhancement in noradrenaline release obtained by exposure to papaverine under conditions in which the granular effect produced by the phosphodiesterase inhibitor was even greater than in the absence of the beta-blocker.7 It is concluded that activation of presynaptic beta-adrenoceptors in the perfused cat spleen leads to an enhancement in transmitter release which appears to be linked to an increase in cyclic adenosine 3',5'-monophosphate levels in noradrenergic nerve endings.