Luis E. Cobos-Puc

Evidence that some imidazoline derivatives inhibit peripherally the vasopressor sympathetic outflow in pithed rats

Imidazoline derivatives (e.g. clonidine and moxonidine) and alpha(2)-adrenoceptor agonists (e.g. B-HT 933) have been shown to inhibit sympathetically-induced [(3)H]noradrenaline release in several isolated blood vessels. The present study has compared the potential capability of agonists at imidazoline I(1/2) receptors and/or alpha(1/2)-adrenoceptors to inhibit the sympathetically-induced vasopressor responses in pithed rats. For this purpose, male Wistar rats were pithed and prepared for measurement of diastolic blood pressure and heart rate. Then, the vasopressor responses induced by either selective electrical stimulation (2 ms, 60 V; 0.03, 0.1, 0.3, 1 and 3 Hz) of the vascular sympathetic outflow (T(7)-T(9)) or i.v. bolus injections of exogenous noradrenaline (0.03, 0.1, 0.3, 1 and 3 microg/kg) were determined before and during i.v. continuous infusions of the agonists B-HT 933 (alpha(2)), clonidine (alpha(2), I(1)), moxonidine (alpha(2), I(1)), cirazoline (alpha(1), I(2)), agmatine (putative endogenous ligand of imidazoline receptors) and methoxamine (alpha(1)), or equivalent volumes of physiological saline. Electrical sympathetic stimulation elicited frequency-dependent vasopressor responses which were significantly inhibited during the continuous infusions of B-HT 933, clonidine, moxonidine, cirazoline and agmatine, but not of physiological saline. Interestingly, the vasopressor responses to exogenous noradrenaline, which remained unaffected during the infusions of physiological saline, B-HT 933, moxonidine, cirazoline and agmatine, were significantly blocked during the infusions of clonidine or methoxamine. These results suggest that B-HT 933, moxonidine, cirazoline and agmatine induced a prejunctional inhibition of the vasopressor sympathetic outflow in pithed rats, whilst clonidine inhibited the vasopressor sympathetic outflow by both prejunctional and postjunctional mechanisms.

Pharmacological characterization of the inhibition by moxonidine and agmatine on the cardioaccelerator sympathetic outflow in pithed rats

This study analysed the inhibition produced by the agonists moxonidine (imidazoline I(1) receptors>alpha(2)-adrenoceptors) and agmatine (endogenous ligand of imidazoline I(1)/I(2) receptors), using B-HT 933 (6-ethyl-5,6,7,8-tetrahydro-4H-oxazolo[4,5-d]azepin-2-amine dihydrochloride; alpha(2)-adrenoceptors) for comparison, on the rat cardioaccelerator sympathetic outflow. Male Wistar rats were pithed and prepared to stimulate the cardiac sympathetic outflow or to receive i.v. bolus of exogenous noradrenaline. Sympathetic stimulation or noradrenaline produced, respectively, frequency-dependent and dose-dependent tachycardic responses. I.v. continuous infusions of moxonidine (3 and 10 microg/kg min), agmatine (1000 and 3000 microg/kg min) and B-HT 933 (30 and 100 microg/kg min) inhibited the tachycardic responses to sympathetic stimulation, but not those to noradrenaline. The cardiac sympatho-inhibition by either moxonidine (3 microg/kg min) or B-HT 933 (30 microg/kg min) was not modified by i.v. injections of saline or the antagonists AGN192403 [(+/-)-2-endo-Amino-3-exo-isopropylbicyclo[2.2.1]heptane hydrochloride; 3000microg/kg; imidazoline I(1) receptors] or BU224 (2-(4,5-dihydroimidazol-2-yl)quinoline hydrochloride; 300 microg/kg; imidazoline I(2) receptors) and abolished by rauwolscine (300 microg/kg; alpha(2)-adrenoceptors). At the same doses of these compounds, the sympatho-inhibition to moxonidine (10 microg/kg min) and agmatine (1000 microg/kg min) was: (1) not modified by saline, AGN192403 or BU224; (2) partially blocked by rauwolscine or the combination of rauwolscine plus BU224; and (3) abolished by the combination of rauwolscine plus AGN192403. These results demonstrate that the cardiac sympatho-inhibition to: (1) 3 microg/kg min moxonidine or 30 microg/kg min B-HT 933 involves alpha(2)-adrenoceptors; and (2) 10 microg/kg min moxonidine or 1000 microg/kg min agmatine involves alpha(2)-adrenoceptors and imidazoline I(1) receptors.

Pharmacological characterization of ergotamine-induced inhibition of the cardioaccelerator sympathetic outflow in pithed rats

Ergotamine inhibits the sympathetically-induced tachycardia in pithed rats. The present study identified the pharmacological profile of this response. Male Wistar rats were pithed and prepared to stimulate the preganglionic (C7–T1) cardiac sympathetic outflow. Intravenous continuous infusions of ergotamine dose-dependently inhibited the tachycardic responses to sympathetic stimulation, but not those to exogenous noradrenaline. Using several antagonists, the sympatho-inhibition to ergotamine was: (1) partially blocked by rauwolscine (α2), haloperidol (D1/2-like) or rauwolscine plus GR127935 (5-HT1B/1D); (2) abolished by rauwolscine plus haloperidol; and (3) unaffected by either saline or GR127935. In animals systematically pretreated with haloperidol, this sympatho-inhibition was: (1) unaffected by BRL44408 (α2A), partially antagonized by MK912 (α2C); and (3) abolished by BRL44408 plus MK912. These antagonists failed to modify the sympathetically induced tachycardic responses per se. Thus, the cardiac sympatho-inhibition by ergotamine may be mainly mediated by α2A/α2C-adrenoceptors, D2-like receptors and, to a lesser extent, by 5-HT1B/1D receptors.

Pharmacological evidence that Ca2+ channels and, to a lesser extent, K+ channels mediate the relaxation of testosterone in the canine basilar artery

Testosterone induces vasorelaxation through non-genomic mechanisms in several isolated blood vessels, but no study has reported its effects on the canine basilar artery, an important artery implicated in cerebral vasospasm. Hence, this study has investigated the mechanisms involved in testosterone-induced relaxation of the canine basilar artery. For this purpose, the vasorelaxant effects of testosterone were evaluated in KCl- and/or PGF(₂α)-precontracted arterial rings in vitro in the absence or presence of several antagonists/inhibitors/blockers; the effect of testosterone on the contractile responses to CaCl₂ was also determined. Testosterone (10-180 μM) produced concentration-dependent relaxations of KCl- or PGF(₂α)-precontracted arterial rings which were: (i) unaffected by flutamide (10 μM), DL-aminoglutethimide (10 μM), actinomycin D (10 μM), cycloheximide (10 μM), SQ 22,536 (100 μM) or ODQ (30 μM); and (ii) significantly attenuated by the blockers 4-aminopyridine (K(V); 1 mM), BaCl₂ (K(IR); 30 μM), iberiotoxin (BK(Ca²+); 20 nM), but not by glybenclamide (K(ATP); 10 μM). In addition, testosterone (31, 56 and 180 μM) and nifedipine (0.01-1 μM) produced a concentration-dependent blockade of the contraction to CaCl₂ (10 μM to 10 mM) in arterial rings depolarized by 60mM KCl. These results, taken together, show that testosterone relaxes the canine basilar artery mainly by blockade of voltage-dependent Ca²+ channels and, to a lesser extent, by activation of K+ channels (K(IR), K(V) and BK(Ca²+)). This effect does not involve genomic mechanisms, production of cAMP/cGMP or the conversion of testosterone to 17β-estradiol.

Effect of some acute and prophylactic antimigraine drugs on the vasodepressor sensory CGRPergic outflow in pithed rats

AIMS This study analyzed in pithed rats the effect of several acute and prophylactic antimigraine drugs on the CGRPergic vasodepressor sensory outflow, in an attempt to investigate systemic cardiovascular effects in a model unrelated to migraine. MAIN METHODS Male Wistar pithed rats were pretreated with continuous i.v. infusions of hexamethonium (2 microg/kg.min; to block autonomic outflow) and methoxamine (15-20 microg/kg.min; to maintain diastolic blood pressure at around 130 mmHg). Under these conditions, the effect of both electrical stimulation (0.56-5.6 Hz; 50 V and 2 ms) of the spinal cord (T(9)-T(12)) or i.v. bolus injections of exogenous alpha-CGRP (0.1-1 microg/kg) were studied in animals pretreated with continuous i.v. infusions of sumatriptan (1-100 microg/kg.min), ergotamine (0.18-0.56 microg/kg.min), dihydroergotamine (1-10 microg/kg.min), magnesium valproate (1000-1800 microg/kg.min), propranolol (100-300 microg/kg.min) or their respective vehicles. KEY FINDINGS Electrical stimulation of the spinal cord and i.v. bolus injections of exogenous alpha-CGRP resulted in, respectively, frequency- and dose-dependent decreases in diastolic blood pressure without affecting heart rate. Moreover, the infusions of sumatriptan, ergotamine and dihydroergotamine, but not of magnesium valproate, propranolol or their respective vehicles, dose-dependently inhibited the vasodepressor responses to electrical stimulation. In contrast, sumatriptan (10 microg/kg.min), ergotamine (0.31 microg/kg.min) and dihydroergotamine (3 microg/kg.min) failed to inhibit the vasodepressor responses to exogenous alpha-CGRP. SIGNIFICANCE The above findings suggest that the acute (rather than the prophylactic) antimigraine drugs attenuate the vasodepressor sensory outflow mainly by prejunctional mechanisms. This may be of particular relevance when considering potential cardiovascular adverse effects by acute antimigraine drugs.

α2A-adrenoceptors, but not nitric oxide, mediate the peripheral cardiac sympatho-inhibition of moxonidine.

Moxonidine centrally inhibits the sympathetic activity through the I1-imidazoline receptor and nitric oxide. In addition, inhibits the peripheral cardiac sympathetic outflow by α2-adrenoceptors/I1-imidazoline receptors, although the role of α2-adrenoceptor subtypes or nitric oxide in the cardiac sympatho-inhibition induced by moxonidine are unknown. Therefore, the cardiac sympatho-inhibition induced by moxonidine (10μg/kgmin) was evaluated before and after of the treatment with the following antagonists/inhibitor: (1) BRL 44408, (300μg/kg, α2A), imiloxan, (3000μg/kg, α2B), and JP-1302, (300μg/kg, α2C), in animals pretreated with AGN 192403 (3000μg/kg, I1 antagonist); (2) N(ω)-nitro-l-arginine methyl ester (l-NAME; 34, 100, and 340μg/kgmin); and (3) the combinations of the highest dose of l-NAME plus AGN 192403 or BRL 44408. Additionally, the expression of the neuronal (nNOS) and inducible (iNOS) nitric oxide synthase in the stellate ganglion was determined after treatment with moxonidine (i.p. 0.56mg/kg daily, during one week). The cardiac sympatho-inhibition of 10μg/kgmin moxonidine was: (1) unaffected by imiloxan and JP-1302, under pretreatment with AGN 192403, or l-NAME (34, 100 and 340μg/kgmin) given alone; (2) partially antagonized by the combination of 340 μg/kgmin l-NAME plus BRL 44408; and (3) abolished by BRL 44408 under treatment with AGN 192403. Furthermore, moxonidine did not modify the nNOS or iNOS protein expression in the stellate ganglion, the main source of postganglionic sympathetic neurons innervating the heart. In conclusion, our results suggest that the peripheral cardiac sympatho-inhibition induced by moxonidine is mediated by α2A-adrenoceptor subtype but not by nitric oxide.

Pharmacological characterization of the mechanisms involved in the vasorelaxation induced by progesterone and 17β-estradiol on isolated canine basilar and internal carotid arteries

Progesterone and 17β-estradiol induce vasorelaxation through non-genomic mechanisms in several isolated blood vessels; however, no study has systematically evaluated the mechanisms involved in the relaxation induced by 17β-estradiol and progesterone in the canine basilar and internal carotid arteries that play a key role in cerebral circulation. Thus, relaxant effects of progesterone and 17β-estradiol on KCl- and/or PGF2α-pre-contracted arterial rings were investigated in absence or presence of several antagonists/inhibitors/blockers; the effect on the contractile responses to CaCl2 was also determined. In both arteries progesterone (5.6-180 μM) and 17β-estradiol (1.8-180 μM): (1) produced concentration-dependent relaxations of KCl- or PGF2α-pre-contracted arterial rings; (2) the relaxations were unaffected by actinomycin D (10 μM), cycloheximide (10 μM), SQ 22,536 (100 μM) or ODQ (30 μM), potassium channel blockers and ICI 182,780 (only for 17β-estradiol). In the basilar artery the vasorelaxation induced by 17β-estradiol was slightly blocked by tetraethylammonium (10mM) and glibenclamide (KATP; 10 μM). In both arteries, progesterone (10-100 μM), 17β-estradiol (3.1-31 μM) and nifedipine (0.01-1 μM) produced a concentration-dependent blockade of the contraction to CaCl2 (10 μM-10mM). These results suggest that progesterone and 17β-estradiol produced relaxation in the basilar and internal carotid arteries by blockade of L-type voltage dependent Ca(2+) channel but not by genomic mechanisms or production of cAMP/cGMP. Potassium channels did not play a role in the relaxation to progesterone in both arteries or in the effect of 17β-estradiol in the internal carotid artery; meanwhile KATP channels play a minor role on the effect of 17β-estradiol in the basilar artery.

Pharmacological analysis of the cardiac sympatho-inhibitory actions of moxonidine and agmatine in pithed spontaneously hypertensive rats.

This study shows that in spontaneously hypertensive rats (SHR) of 14-weeks-old, the sympathetically-induced, but not noradrenaline-induced tachycardic response are higher than age-matched Wistar normotensive rats. Furthermore, in SHR the sympathetically-induced tachycardic response was: (1) unaffected by moxonidine (3μg/kgmin); (2) partially inhibited by B-HT 933 (30μg/kgmin), both at the lowest doses; and (3) completely inhibited by the highest doses of B-HT 933 (100μg/kgmin), moxonidine (10μg/kgmin) or agmatine (1000 and 3000μg/kgmin) while the noradrenaline-induced tachycardic responses remained unaffected by the above compounds, except by 3000μg/kgmin agmatine. In SHR, 300μg/kg rauwolscine failed to block the sympatho-inhibition to 100μg/kgmin B-HT 933 or 10μg/kgmin moxonidine, but 1000μg/kg rauwolscine abolished, partially antagonized, and did not modify the sympatho-inhibition to the highest doses of B-HT 933, moxonidine, and agmatine, respectively, 3000μg/kg AGN 192403 or 300μg/kg BU224 given alone had no effect in the moxonidine- or agmatine-induced sympatho-inhibition, and the combination rauwolscine plus AGN 192403 but not plus BU224, abolished the sympatho-inhibition to the highest doses of moxonidine and agmatine. In conclusion, the sympathetically-induced tachycardic responses in SHR are inhibited by moxonidine and agmatine. The inhibition of moxonidine is mainly mediated by prejunctional α2-adrenoceptors and to a lesser extent by I1-imidazoline receptors, while the inhibition of agmatine is mediated by prejunctional α2-adrenoceptors and I1-imidazoline receptors at the same extent. Notwithstanding, the inhibitory function of α2-adrenoceptors seems to be altered in SHR compared with Wistar normotensive rats.

Further analysis of the inhibition by agmatine on the cardiac sympathetic outflow: Role of the α2-adrenoceptor subtypes

&NA; This study has investigated the role of the &agr;2‐adrenoceptor subtypes involved in the inhibition of the cardiac sympathetic outflow induced by intravenous (i.v) infusions of agmatine. Therefore, we analysed the effect of an i.v. bolus injections of the selective antagonists BRL 44408 (300 &mgr;g/kg; &agr;2A), imiloxan (3000 &mgr;g/kg; &agr;2B), and JP‐1302 (300 &mgr;g/kg; &agr;2C) given separately, and their combinations: BRL 44408 plus Imiloxan, JP 1302 plus imiloxan, BRL 44408 plus JP‐1302, BRL 44408 plus imiloxan plus JP‐1302 on the cardiac sympatho‐inhibition of agmatine. Also, the effect of the combination BRL 44408 plus JP‐1302 plus AGN 192403 (3000 &mgr;g/kg; I1 antagonist) was evaluated. In this way, i.v. infusions of 1000 &mgr;g/kg min of agmatine, but not 300, inhibited the tachycardic response induced by electrical stimulation. Furthermore, the antagonists used or their combinations had no effect on the electrically‐induced tachycardic response. On the other hand, the inhibitory response of agmatine was: (1) partially antagonized by BRL 44408 or JP‐1302 given separately, a similar response was observed when we administered their combination with imiloxan, but not by imiloxan alone, (2) antagonized in greater magnitude by the combination BRL 44408 plus JP‐1302 or the combination BRL 44408 plus imiloxan plus JP‐1302, and (3) abolished by the combination BRL 44408 plus JP‐1302 plus AGN 192403. Taken together, these results demonstrate that the &agr;2A‐ and &agr;2C‐adrenoceptor subtypes and I1‐imidazoline receptors are involved in the inhibition of the cardiac sympathetic outflow induced by agmatine.