Alain H. Altamirano-Espinoza

Analysis of anandamide- and lysophosphatidylinositol-induced inhibition of the vasopressor responses produced by sympathetic stimulation or noradrenaline in pithed rats.

The endocannabinoid system exhibits multiple functions in cardiovascular regulation mainly by cannabinoid (CB1 and CB2) receptors, vanilloid TRPV1 receptors and, probably, by the orphan G protein-coupled receptor 55 (GPR55). Hence, the role of these receptors was investigated in Wistar pithed rats on anandamide- and lysophosphatidylinositol (LPI)-induced inhibition of the vasopressor responses induced by preganglionic (T7-T9) stimulation of the vasopressor sympathetic outflow or i.v. bolus injections of noradrenaline. The corresponding frequency- and dose-dependent vasopressor responses were analyzed before and during i.v. continuous infusions of anandamide (CB1, CB2, TRPV1 and GPR55), JWH-015 (CB2) and LPI (GPR55) in animals receiving (i.v.) the antagonists NIDA41020 (CB1), AM630 (CB2), capsazepine (TRPV1) and/or cannabidiol (GPR55). Anandamide (0.1-3.1 μg/kg min) inhibited the vasopressor responses by electrical stimulation, but not those by noradrenaline; while LPI (5.6-10 μg/kg min) inhibited both responses. In contrast, JWH-015 (5.6-10 μg/kg min) failed to induce sympatho-inhibition. Anandamide-induced sympatho-inhibition was: (i) dose-dependently blocked by 31 and 100 μg/kg NIDA41020; (ii) slightly blocked by 310 μg/kg AM630 or 31 μg/kg cannabidiol; and (iii) unaffected by 310 μg/kg capsazepine. Moreover, LPI-induced inhibition of both vasopressor responses was blocked and abolished by 10 and 31 μg/kg cannabidiol, respectively, and weakly blocked by 100 μg/kg NIDA41020. Thus, the sympatho-inhibition by anandamide is primarily mediated by cannabinoid CB1 and, minimally, by cannabidiol-sensitive receptors. In contrast, LPI-induced inhibition of both responses seems to be mainly mediated by postjunctional cannabidiol-sensitive (presumably endothelial GPR55) receptors.

The role of dopamine D2, but not D3 or D4, receptor subtypes, in quinpirole‐induced inhibition of the cardioaccelerator sympathetic outflow in pithed rats

Quinpirole (a dopamine D2‐like receptor agonist) inhibits the cardioaccelerator sympathetic outflow in pithed rats by sympathoinhibitory D2‐like receptors. The present study was designed to identify pharmacologically the specific D2‐like receptor subtypes (i.e. D2, D3 and D4) involved in this sympathoinhibition by quinpirole.

The role of pre-junctional D2 -like receptors mediating quinpirole-induced inhibition of the vasodepressor sensory CGRPergic out-flow in pithed rats.

Calcitonin gene‐related peptide (CGRP) released from perivascular sensory nerves plays a role in the regulation of vascular tone. Indeed, electrical stimulation of the perivascular sensory out‐flow in pithed rats produces vasodepressor responses, which are mainly mediated by CGRP release. This study investigated the potential role of dopamine D1‐like and D2‐like receptors in the inhibition of these vasodepressor responses. For this purpose, male Wistar pithed rats (pre‐treated i.v. with 25 mg/kg gallamine and 2 mg/kg min. hexamethonium) received i.v. continuous infusions of methoxamine (20 μg/kg min.) followed by physiological saline (0.02 ml/min.), the D1‐like receptor agonist SKF‐38393 (0.1–1 μg/kg min.) or the D2‐like receptor agonist quinpirole (0.03–10 μg/kg min.). Under these conditions, electrical stimulation (0.56–5.6 Hz; 50 V and 2 ms) of the thoracic spinal cord (T9–T12) resulted in frequency‐dependent vasodepressor responses which were (i) unchanged during the infusions of saline or SKF‐38393 and (ii) inhibited during the infusions of quinpirole (except at 0.03 μg/kg min.). Moreover, the inhibition induced by 0.1 μg/kg min. quinpirole (which failed to inhibit the vasodepressor responses elicited by i.v. bolus injections of exogenous α‐CGRP; 0.1–1 μg/kg) was (i) unaltered after i.v. treatment with 1 ml/kg of either saline or 5% ascorbic acid and (ii) abolished after 300 μg/kg (i.v.) of the D2‐like receptor antagonists haloperidol or raclopride. These doses of antagonists (enough to completely block D2‐like receptors) essentially failed to modify per se the electrically induced vasodepressor responses. In conclusion, our results suggest that quinpirole‐induced inhibition of the vasodepressor sensory CGRPergic out‐flow is mainly mediated by pre‐junctional D2‐like receptors.

Pharmacological evidence that histamine H3 receptors inhibit the vasodepressor responses by selective stimulation of the rat perivascular sensory CGRPergic outflow

This study has investigated whether pharmacological activation of Gi/o coupled histamine H3/H4 receptors inhibits the rat vasodepressor sensory outflow. For this purpose, 100 male Wistar rats were pithed, artificially ventilated and pretreated (i.v.) with: 25mg/kg gallamine, 2mg/kg/min hexamethonium and 20μg/kg/min methoxamine, followed by i.v. continuous infusions of physiological saline (0.02ml/min) or immepip (3.1, 10 or 31μg/kg/min; a histamine H3/H4 receptor agonist). Under these conditions, electrical stimulation (0.56-5.6Hz; 50V and 2ms) of the spinal cord (T9-T12) resulted in frequency-dependent vasodepressor responses, which were: (i) unchanged during the infusions of saline or immepip (3.1μg/kg/min); and (ii) significantly but, surprisingly, not dose-dependently inhibited by 10 and 31μg/kg/min immepip. Moreover, the sensory-inhibition by 10μg/kg/min immepip (which failed to inhibit the vasodepressor responses by i.v. bolus injections of α-CGRP; 0.1-1µg/kg) was: (i) essentially unaltered after i.v. administration of saline (1ml/kg) or blocking doses of the antagonists ketotifen (100μg/kg; H1), ranitidine (1000μg/kg; H2) or JNJ7777120 (310μg/kg; H4); and (ii) abolished after i.v. thioperamide (310µg/kg; H3). In conclusion, our results suggest that immepip-induced inhibition of the vasodepressor sensory outflow is mainly mediated by prejunctional activation of histamine H3 receptors.

Olcegepant blocks neurogenic and non‐neurogenic CGRPergic vasodepressor responses and facilitates noradrenergic vasopressor responses in pithed rats

Olcegepant (BIBN4096BS) is a selective non‐peptide CGRP receptor antagonist with acute antimigraine properties. Since systemic vascular tone is modulated by perivascular (primary sensory CGRPergic and sympathetic) nerves, this randomized study investigated in pithed rats the effect of acute i.v. treatment with olcegepant on the neurogenic and non‐neurogenic: (i) CGRPergic vasodepressor responses; and (ii) noradrenergic vasopressor responses. The pithed rat is an experimental model predictive of systemic (cardio) vascular side effects.

Role of pre-junctional CB1, but not CB2 , TRPV1 or GPR55 receptors in anandamide-induced inhibition of the vasodepressor sensory CGRPergic outflow in pithed rats.

Stimulation of the perivascular sensory outflow in pithed rats produces vasodepressor responses mediated by CGRP release. Interestingly, endocannabinoids such as anandamide (which interacts with CB1, CB2, TRPV1 and GPR55 receptors) can regulate the activity of perivascular sensory nerves in dural blood vessels by modulating CGRP release. Yet, as no publication has reported whether this mechanism is operative in the healthy systemic vasculature, this study has specifically analysed the receptors mediating the potential inhibitory effects of the cannabinoid (CB) receptor agonists anandamide (non‐selective), JWH‐015 (CB2) and lysophosphatidylinositol (GPR55) on the rat vasodepressor sensory CGRPergic outflow (an index of systemic vasodilatation). Healthy pithed rats were pre‐treated with consecutive i.v. continuous infusions of hexamethonium, methoxamine and the above agonists. Electrical spinal (T9–T12) stimulation of the vasodepressor sensory CGRPergic outflow or i.v. injections of α–CGRP produced frequency‐dependent or dose‐dependent vasodepressor responses. The infusions of anandamide in a dose‐dependent manner inhibited the vasodepressor responses by electrical stimulation (remaining unaffected by JWH‐015 or lysophosphatidylinositol), but not those by α–CGRP. After i.v. administration of antagonists, the inhibition by 3.1 μg/kg min anandamide was: (i) potently blocked by 31–100 μg/kg NIDA41020 (CB1), (ii) unaffected by 180 μg/kg AM630 (CB2), 31 μg/kg cannabidiol (GPR55) or 31–100 μg/kg capsazepine (TRPV1) and (iii) slightly blocked by 310 μg/kg AM630. The above doses of antagonists were enough to block their respective receptors. These results suggest that anandamide‐induced inhibition of the vasodepressor sensory CGRPergic outflow is mainly mediated by pre‐junctional activation of CB1 receptors, with no pharmacological evidence for the role of CB2, TRPV1 or GPR55 receptors.

Differential cardiac sympatho-inhibitory responses produced by the agonists B-HT 933, quinpirole and immepip in normoglycaemic and diabetic pithed rats

This study compared the cardiac sympatho‐inhibitory responses produced by agonists at α2‐adrenergic (B‐HT 933), dopamine D2‐like (quinpirole) and histamine H3/H4 (immepip) receptors between normoglycaemic and streptozotocin‐pretreated (diabetic) pithed rats. Intravenous (i.v.) continuous infusions of B‐HT 933, quinpirole or immepip were used in normoglycaemic and diabetic pithed rats to analyse their sympatho‐inhibitory effects on the electrically‐stimulated cardioaccelerator sympathetic outflow. Both in normoglycaemic and diabetic animals, B‐HT 933 (until 100 μg/kg per minute) and quinpirole (until 10 μg/kg per minute) inhibited the tachycardic responses to electrical sympathetic stimulation, but not those to i.v. bolus of exogenous noradrenaline. These sympatho‐inhibitory responses were more pronounced in diabetic than in normoglycaemic animals. Accordingly, the areas under the curve for 100 μg/kg per minute B‐HT 933 and 10 μg/kg per minute quinpirole in diabetic rats (1065 ± 70 and 920 ± 35, respectively) were significantly smaller (P < .05) than those in normoglycaemic rats (1220 ± 45 and 1360 ± 42, respectively). In contrast, immepip infusions produced cardiac sympatho‐inhibition in normoglycaemic (until 10 μg/kg per minute), but not in diabetic (until 100 μg/kg per minute) animals. Our results suggest that in diabetic pithed rats: (i) the more pronounced cardiac sympatho‐inhibition to B‐HT 933 and quinpirole may be probably due to up‐regulation of α2‐adrenergic and dopamine D2‐like receptors, respectively; (ii) the histamine H3/H4 receptors do not seem to play a sympatho‐inhibitory role; and (iii) there is a differential participation of α2‐adrenergic and dopamine D2‐like receptors, which may certainly represent therapeutic targets for the treatment of diabetic complications such as cardiovascular autonomic neuropathy.