María del Carmen García

Enhancement of the hypotensive effects of intrathecally injected endocannabinoids by the entourage compound palmitoylethanolamide

The intrathecal (i.t.) injection of 50 and 100 nmol anandamide to urethane anesthetized rats induced a dose-dependent decrease in the mean blood pressure (-10.6+/-1.6 mmHg and -15.0+/-1.7 mmHg, respectively; n=6) whereas a lower dose of this endocannabinoid (25 nmol) was devoid of effect. Similar responses were obtained both with the non-metabolizable analog methanandamide and with the endocannabinoid N-arachidonoyldopamine. When the sub-effective dose (25 nmol) of each compound was co-injected with palmitoylethanolamide (100 nmol), significant decreases in the blood pressure were observed (-12.3+1.3 mmHg for anandamide; -12.1+/-0.8 mmHg for methanandamide; -12.1+/-0.8 mmHg for N-arachidonoyldopamine; n=4-6). Palmitoylethanolamide also enhanced the hypotensive responses to the 50 nmol-dose of both anandamide and methanandamide. The hypotensive response induced by co-administration of palmitoylethanolamide and 25 nmol anandamide was prevented both by the cannabinoid CB(1) receptor antagonist SR 144716A (20 nmol; i.t.) and by the vanilloid TRPV1 receptor antagonist capsazepine (20 nmol; i.t.) and enhanced by pretreatment with URB602 (3.5 nmol; i.t.), a putative inhibitor of palmitoylethanolamide degradation. These results suggest that in the spinal cord palmitoylethanolamide acts as an entourage compound for the hypotensive effects of i.t. administered endocannabinoids. The facilitative action of palmitoylethanolamide affects the vanilloid TRPV1 as well as the cannabinoid CB(1) receptor-mediated effects of endocannabinoids on the blood pressure control.

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.

Effects of clonidine on the experimental hypertension by abdominal aortic coarctation in rats

Cardiovascular baroreflex mechanisms and sympathetic tone could be involved in the arterial hypertension by coarctation of abdominal aorta artery (CoA). The present work analyzes the effect on the arterial pressure and heart rate (HR) of the clonidine, an alpha(2)-adrenergic central acting antihypertensive agent, after intravenous (i.v.), intracerebroventricular (i.c.v.) and intrathecal (i.t.) administration in rats anesthetized with pentobarbital (40 mg/kg i.p.).Wistar rats of both sexes (240-270 g) were used to the 7 days of the CoA or a sham operation (SO). Values of mean arterial pressure (MAP) and of HR were calculated from intraarterial recordings of blood pressure. The MAP of the CoA rats (161.5+/-5.3 mmHg, n=20) was significantly higher (P<0.01) than that of the SO rats (101.6+/-3.3 mmHg, n=20). The i.v. injection of clonidine (3-30 microg/kg) produced an increase of blood pressure in the rats SO and in the CoA animals, followed by a fall of arterial pressure in both groups of rats. Clonidine showed a small pressor effect but also a great depressor action in the hypertensive rats. Except for with the dose of 10 microg/kg, differences in cardiac response to clonidine were not seen in both groups of rats. Injection of clonidine by the i.c.v. via (10 microg) like by the i.t. (3 microg) also produced a greater fallen of the MAP in the hypertensive rats than in the controls SO animals. In conclusion, these hypertensive animals would be sensitive to the antihypertensive action of central acting alpha(2)-adrenoceptor agonist clonidine administered by different ways, suggesting a great sensitivity of the post-synaptic alpha(2)-adrenoceptor of central nervous system.

Participation of nitric oxide and N-methyl-D-aspartic acid receptors in the pressor response to intrathecal injected noradrenaline at the spinal cord of the rat.

In pentobarbital-anesthetized rats, intrathecal injection of noradrenaline (NA; 6, 18 and 60 nmol) induced a dose-dependent increase in the mean blood pressure. The pressor response to NA (18 nmol) was blocked by pretreatment with the selective antagonist for N-methyl-D-aspartic acid (NMDA) receptors, 2-amino-5-phosphonovaleric acid (30 nmol), but not by pretreatment with the selective antagonist for (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid/kainate receptors, 6,7-dinitroquinoxaline-2,3-dione (50 nmol). The pressor effect of NA was reduced after pretreatment with N(G)-nitro-L-arginine methyl ester (L-NAME, 1 micromol). The effect of L-NAME on the pressor response to NA was reverted by the precursor of nitric oxide (NO), L-arginine (5 micromol). The hypertension induced by NA was also reduced by the guanylate cyclase inhibitor methylene blue (0.3 micromol). These results suggest that spinal NMDA receptors and spinal NO are involved in the pressor response to NA.

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.

Impaired hypotensive responses induced by intrathecally injected drugs in fructose-fed rats.

Blood pressure responses to intrathecal (i.t.) injection of neurochemicals were examined in the fructose-fed rat, an experimental model of metabolic syndrome.Sprague-Dawley rats receiving either tap water or water containing 10% fructose during 8 weeks were anesthetized with sodium pentobarbital. The endocannabinoid anandamide (100 nmol; i.t.) decreased mean blood pressure in control rats (-21.2 ± 6.3 mm Hg), but had no effect in fructose-fed animals. Similarly, calcitonin gene-related peptide (CGRP; 0.125 nmol; i.t.) decreased mean blood pressure in control, but not in treated rats. The high fructose diet did not cause significant changes in the pressor effects of i.t. administered noradrenaline (100 nmol) and N-methyl-d-aspartate (30 nmol). The nitric oxide donor sodium nitroprusside (500 nmol, i.t.) induced a brief hypotension followed by a sustained increase in mean blood pressure in control rats; however, this drug only produced pressor effects in fructose-fed animals. The GABAA-receptor agonist muscimol (8.8 nmol, i.t.) and the GABAB-receptor agonist baclofen (100 nmol, i.t.) decreased mean blood pressure 30-35 mm Hg, both in control and in fructose-fed rats. Fructose potentiated the pressor effect of i.v. injected noradrenaline, but did not modify the hypotensive responses to i.v. administered sodium nitroprusside and acetylcholine.These results could suggest that, in pentobarbital-anesthetized rats, fructose feeding could alter spinal mechanisms of regulation of preganglionic sympathetic nerve activity. It is proposed that the spinal cord could be involved in the sympathetic dysfunction associated with the metabolic syndrome.