María Luisa Martín

Peripheral 5-HT1D and 5-HT7 serotonergic receptors modulate sympathetic neurotransmission in chronic sarpogrelate treated rats

5-HT₂ receptor activation induces vasoconstriction, hypertension and platelet aggregation; therefore, its blocking may be useful in cardiovascular diseases, probably due to alterations in the modulation of serotonergic system. The aim of this study was to evaluate whether 5-HT₂ receptor blockade changes serotonergic modulation of sympathetic neurotransmission in pithed rats. Serotonergic modulation of sympathetic neurotransmission was investigated in Wistar rats treated with sarpogrelate, a 5-HT₂ receptor antagonist, during 14 days (30 mg/kg/day). After central nervous system destruction, we conducted electrical stimulation throughout the spinal cord flow to study the 5-HT-related products action on adrenergic system. 5-Hydroxytryptamine exerted inhibition of sympathetic outflow in sarpogrelate-treated pithed rats. This effect was mimicked and enhanced by 5-CT (5-HT₁/₇ receptor agonist). L-694,247 and AS-19, 5-HT₁D and 5-HT₇ receptor agonists respectively, reproduced this action. Pretreatment with LY310762+SB258719 (5-HT₁D and 5-HT₇ receptor antagonists, respectively) completely abolished 5-CT inhibitory action. The nature of this action was prejunctional since these agonists did not modify the pressor responses induced by exogenous noradrenaline. Western Blot analysis confirmed a higher expression of 5-HT₁D receptors in sarpogrelate-treated rats. Experimental 5-HT₂ receptor blockade induces changes in the 5-HT receptors involved in the serotonergic inhibition of sympathetic-induced pressor responses. Prejunctional activation of 5-HT₁D and 5-HT₇ receptors induces a significantly higher serotonergic inhibition on adrenergic neurotransmission in sarpogrelate-treated pithed rats. The antagonism of 5-HT₂ receptors produces an enhancement of serotonergic sympathoinhibitory effect, which may explain the beneficial effects of this blockade in cardiovascular disorders where 5-hydroxytryptamine plays a crucial role.

Diabetes-induced changes in the 5-hydroxytryptamine inhibitory receptors involved in the pressor effect elicited by sympathetic stimulation in the pithed rat

1 We investigated the effect of alloxan‐induced diabetes on the inhibitory mechanisms of 5‐hydroxytryptamine (5‐HT) in the pressor responses induced by stimulation of sympathetic vasopressor outflow in pithed rats, and analysed the type and/or subtype of 5‐HT receptors involved. 2 Diabetes was induced in male Wistar rats by a single s.c. injection of alloxan, then 4 weeks later, they were anaesthetized, pretreated with atropine and pithed. Electrical stimulation of the sympathetic outflow from the spinal cord (0.1, 0.5, 1 and 5 Hz) resulted in frequency‐dependent increases in blood pressure. 3 Intravenous infusions of 5‐HT (1–80 μg kg−1 min−1) reduced the pressor effects obtained by electrical stimulation. The 5‐HT1 receptor agonist 5‐carboxamidotryptamine, 5‐CT (5 μg kg−1 min−1), caused an inhibition of the pressor response, whereas the selective 5‐HT2 receptor agonist, α‐methyl‐5‐HT (5 μg kg−1 min−1) and the selective 5‐HT3 receptor agonist, 1‐phenylbiguanide (40 μg kg−1 min−1), did not modify the sympathetic pressor responses. 5‐HT had no effect on exogenous noradrenaline (NA)‐induced pressor responses. 4 The inhibition of electrically induced pressor responses by 5‐HT (10 μg kg−1 min−1) was unable to be elicited after i.v. treatment with methiothepin (100 μg kg−1) because of the marked inhibition produced by methiothepin alone. The 5‐HT‐induced inhibition was blocked after i.v. administration of WAY‐100,635 (100 μg kg−1) and not affected by ritanserin (1 mg kg−1), MDL 72222 (2 mg kg−1). 5 The selective 5‐HT1A receptor agonist, 8‐hydroxydipropylaminotretalin hydrobromide (8‐OH‐DPAT) (5–20 μg kg−1 min−1) but neither the rodent 5‐HT1B receptor agonist, CGS‐12066B (5 μg kg−1 min−1), nor the selective nonrodent 5‐HT1B and 5‐HT1D receptor agonist, L‐694,247 (5 and 40 μg kg−1 min−1), inhibited the electrically induced pressor response. The selective 5‐HT1A receptor antagonist, WAY‐100,635 (100 μg kg−1), blocked the inhibition induced by 8‐OH‐DPAT (10 μg kg−1 min−1). 8‐OH‐DPAT had no effect on exogenous NA‐induced pressor responses. 6 Experimental diabetes produces changes in the inhibitory effect induced by 5‐HT on electrically induced sympathetic pressor responses, such that the inhibitory action induced by 5‐HT in diabetic pithed rats is mediated by prejunctional 5‐HT1A receptors.

Pharmacological profile of 5-hydroxytryptamine-induced inhibition on the pressor effect elicited by sympathetic stimulation in long-term diabetic pithed rats

We analysed the type and/or subtype of 5-hydroxytryptamine (5-HT) receptors involved in the inhibitory mechanisms of 5-HT on the pressor responses induced by stimulation of sympathetic vasopressor outflow in long-term diabetic pithed rats. Diabetes was induced in male Wistar rats by a single subcutaneous injection of alloxan. Eight weeks later, rats were anaesthetized, pre-treated with atropine, and pithed. The effect of 5-HT on the pressor responses elicited by stimulation of the sympathetic outflow was analysed in eight-week alloxan-induced diabetic pithed rats. 5-HT (20 microg/kg/min) reduced the pressor action obtained by electrical stimulation of the sympathetic outflow. However, there was no effect on exogenous noradrenaline-induced pressor responses. 5-CT (5 microg/kg/min), 8-OH-DPAT (5 microg/kg/min), and alpha-methyl-5-HT (5 microg/kg/min), selective 5-HT(1), 5-HT(1A) and 5-HT(2) receptor agonists, respectively, reproduced the 5-HT inhibitory action. Nevertheless, infusion of 5 microg/kg/min of 1-phenylbiguanide, CGS-12066B, L-694,247, BW273C86 or MK212 (5-HT(3), 5-HT(1B), 5-HT(1D), 5-HT(2B) and 5-HT(2C) receptor agonists, respectively) had no effect on the pressor responses elicited by stimulation of the sympathetic outflow. Methiothepin (100 microg/kg) and a cocktail of WAY-100,635 (100 microg/kg) and spiperone (125 microg/kg) blocked the 5-HT inhibitory effect on the pressor action obtained by sympathetic stimulation. Moreover, WAY-100, 635 abolished the 8-OH-DPAT inhibitory effect and spiperone blocked alpha-methyl-5-HT action. In conclusion, this study revealed that long-term experimental diabetes induces changes in the receptor type/subtype involved in the 5-HT inhibitory action on the sympathetic pressor responses produced by electrical stimulation. This is mainly mediated by pre-junctional 5-HT(1A) and 5-HT(2A) receptors.

The role of endothelium-derived hyperpolarizing factor and cyclooxygenase pathways in the inhibitory serotonergic response to the pressor effect elicited by sympathetic stimulation in chronic sarpogrelate treated rats

We have demonstrated that the antagonism of 5-HT2 receptors produces an enhancement of serotonergic sympathoinhibitory effect by 5-HT1D and 5-HT7 activation. The aim of this work was to determine mechanisms involved in the 5-hydroxytriptaminergic inhibitory action on the pressor responses elicited by sympathostimulation in pithed rats treated with a 5-HT2 receptor blocker. The blockade of 5-HT2 receptors was induced by orally sarpogrelate treatment (30 mg/kg/day). Two weeks later, animals were anaesthetized and pithed. A bolus injection of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (10 µg/kg), a guanylyl cyclase inhibitor, or indomethacin (2mg/kg), a non-selective COX inhibitor, prior to the infusion of (2S)(+)-5-(1,3,5-trimethylpyrazol-4-yl)-2-(dimethylamino)tetralin, AS-19 (5 µg/kg/min) were not able to abolish its inhibitory action. However, i.v. administration of glibenclamide (20mg/kg), a blocker of ATP-sensitive K(+) channels, completely reversed AS-19 sympathoinhibitory action. The inhibitory effect of 2-[5-[3-(4-methylsulfonylamino)benzyl-1,2,4-oxadiazol-5-yl]-1H-indol-3-yl]ethanamine, L-694,247 (5 µg/kg/min) was abolished by indomethacin, whereas pretreatment with ODQ had no effect. Nimesulide (3mg/kg), a COX-2 inhibitor, completely reversed the inhibitory action of L-694,247, whereas 1-[[4,5-bis (4-methoxyphenyl)-2-thiazolyl]carbonyl]-4-methylpiperazine hydrochloride (FR122047) (3mg/kg), a COX-1 inhibitor, partially blocked this action. The sympathoinhibition by 5-HT (20 µg/kg/min) could not be elicited after i.v. treatment with indomethacin plus glibenclamide. In conclusion, these results suggest that in chronic sarpogrelate-treated rats, the inhibitory serotonergic effect of the pressor responses induced by electrical stimulation of the sympathetic outflow via 5-HT7 and 5-HT1D receptor activation is mediated by KATP channel-mediated smooth muscle hyperpolarization and the COX pathway, respectively.

Peripheral 5-HT1A and 5-HT7 Serotonergic Receptors Modulate Parasympathetic Neurotransmission in Long-Term Diabetic Rats

We analyzed the modulation of serotonin on the bradycardia induced in vivo by vagal electrical stimulation in alloxan-induced long-term diabetic rats. Bolus intravenous administration of serotonin had a dual effect on the bradycardia induced either by vagal stimulation or exogenous Ach, increasing it at low doses and decreasing it at high doses of 5-hydroxytryptamine (5-HT), effect reproduced by 5-carboxamidotryptamine maleate (5-CT), a 5-HT1/7 agonist. The enhancement of the bradycardia at low doses of 5-CT was reproduced by 5-HT1A agonist 8-hydroxy-2-dipropylaminotetralin hydrobromide (8-OH-DPAT) and abolished by WAY-100,635, 5-HT1A antagonist. Pretreatment with 5-HT1 antagonist methiothepin blocked the stimulatory and inhibitory effect of 5-CT, whereas pimozide, 5-HT7 antagonist, only abolished 5-CT inhibitory action. In conclusion, long-term diabetes elicits changes in the subtype of the 5-HT receptor involved in modulation of vagally induced bradycardia. Activation of the 5-HT1A receptors induces enhancement, whereas attenuation is due to 5-HT7 receptor activation. This 5-HT dual effect occurs at pre- and postjunctional levels.

Chronic Sarpogrelate Treatment Reveals 5-HT7 Receptor in the Serotonergic Inhibition of the Rat Vagal Bradycardia.

Abstract: 5-Hydroxytryptamine (5-HT) modulates the cardiac parasympathetic neurotransmission, inhibiting the bradyarrhythmia by 5-HT2 receptor activation. We aimed to determine whether the chronic selective 5-HT2 blockade (sarpogrelate) could modify the serotonergic modulation on vagal cardiac outflow in pithed rat. Bradycardic responses in rats treated with sarpogrelate (30 mg·kg−1·d−1; orally) were obtained by electrical stimulation of the vagal fibers (3, 6, and 9 Hz) or intravenous (IV) injections of acetylcholine (1, 5, and 10 &mgr;g/kg). 5-HT7 receptor expression was quantified by Western blot in vagus nerve and right atrium. The IV administration of 5-HT (10–200 &mgr;g/kg) dose dependently decreased the vagally induced bradycardia, and agonists 5-CT (5-HT1/7), 8-OH-DPAT (5-HT1A), or AS-19 (5-HT7) (50 &mgr;g/kg each) mimicked the 5-HT–induced inhibitory effect. Neither agonists CGS-12066B (5-HT1B), L-694,247 (5-HT1D), nor 1-phenylbiguanide (5-HT3) modified the electrically-induced bradycardic responses. Moreover, SB-258719 (5-HT7 antagonist) abolished the 5-HT–, 5-CT–, 8-OH-DPAT–, and AS-19–induced bradycardia inhibition; 5-HT or AS-19 did not modify the bradycardia induced by IV acetylcholine; and 5-HT7 receptor was expressed in both the vagus nerve and the right atrium. Our outcomes suggest that blocking chronically 5-HT2 receptors modifies the serotonergic influence on cardiac vagal neurotransmission exhibiting 5-HT as an exclusively inhibitory agent via prejunctional 5-HT7 receptor.