Medullary pathways of cardiovascular responses to 5-HT2 and 5-HT3 receptor stimulation in the rat nucleus tractus solitarius.

As previously found for the baroreceptor reflex, microinjection of kynurenic acid (KYN, 2 nmol), a glutamate receptor antagonist, into the caudal ventrolateral medulla (CVL) blocked the hypotension and bradycardia elicited by microinjections of a 5-HT2 receptor agonist, 2,5-dimethoxy-4-iodoamphetamine (DOI, 0.5 pmol) into the nucleus tractus solitarius (NTS). In addition, as previously observed with the sympathetic chemoreflex response, microinjections of KYN into the RVI, blocked the increase in blood pressure elicited by microinjections of 1-m-(chlorophenyl)-biguanide (1200 pmol), a 5-HT1 receptor agonist, into the NTS. These results suggest that medullary pathways involved in the responses to 5-HT2 and 5-HT3 receptor stimulation in the NTS are similar to those that mediate the baroceptor and chemoreceptor reflex responses, respectively.

Thioesterification of 2-arylpropionic acids by recombinant acyl-coenzyme A synthetases (ACS1 and ACS2).

2-Arylpropionic acids are a class of frequently used nonsteroidal anti-inflammatory drugs exhibiting a potent inhibition of cyclooxygenase isoforms supported by the (+)S-enantiomer alone. Nevertheless, some of these compounds in the (-)R configuration may undergo extensive inversion of configuration to their antipode. The key molecular basis for this mechanism invokes the stereoselective formation of the coenzyme A (CoA) thioester of the 2-arylpropionic acid by long-chain acyl-CoA synthetases (ACSs). In this report, rat recombinant ACS1 and ACS2 enzymes, constitutively highly expressed in adult rat liver and brain, respectively, have been overproduced in Escherichia coli strains and purified to homogeneity to investigate the involvement of these enzymes in the thioesterification of fenoprofen and ibuprofen. Recombinant ACS1 efficiently catalyzed both nonsteroidal anti-inflammatory drugs with Michaelis-Menten parameters of K(M) = 1686 +/- 93 microM, V(max) = 353 +/- 45 nmol/min/mg protein for (-)R-ibuprofen and K(M) = 103 +/- 12 microM, V(max) = 267 +/- 10 nmol/min/mg protein for (-)R-fenoprofen, and exhibited a marked stereoselectivity in favor of the (-)R-enantiomer. Recombinant ACS2, a closely related sequence with ACS1, exhibited a lower enzymatic efficacy from 7- to 130-fold for (-)R-ibuprofen and (-)R-fenoprofen, respectively. On the basis of these findings and considering the level of tissue expression of the different long-chain ACSs, ACS1 appears to be the major enzyme involved in the first step of the chiral inversion of 2-arylpropionic acids. Nevertheless, the participation of other ACS isoforms of minor quantitative importance could not be excluded in the thioesterification of xenobiotics.

In vitro study of fenoprofen chiral inversion in rat: comparison of brain versus liver.

The extent and the overall stereoselectivity of the combined steps involved in the chiral inversion of fenoprofen, a non-steroidal anti-inflammatory drug, was investigated in rat brain microsomes and cytosol. Results were compared with those obtained with the same liver subcellular compartments. Brain microsomes catalysed the stereoselective activation of the R(-)-enantiomer to its coenzyme A thioester with a specific activity approximately 10-fold less than that obtained with liver microsomes. Rat brain microsomes and cytosol mediated the racemization and hydrolysis of both R(-)- and S( + )-fenoprofenoyl-CoA. In brain fractions the epimerase activity was lower than in liver, whereas the hydrolysis process appeared more efficient. Thus, the data indicated that the three-step mechanism occurred in brain subcellular compartments leading to a minor chiral inversion of fenoprofen compared with that in liver.

Microinjection of a serotonin3 receptor agonist into the NTS of unanesthetized rats inhibits the bradycardia evoked by activation of the baro- and chemoreflexes.

In the present study we investigated the effects of microinjection into the commissural nucleus tractus solitarius (NTS) of unanesthetized rats of 2-methylserotonin (2-methyl-5-HT), a 5-HT3 receptor agonist, on the cardiac component of the baro- and chemoreflexes. The study was performed in conscious freely moving rats in order to avoid the possible effects of anesthetics on the cardiovascular responses to microinjection of neuroactive substances into the NTS. The baroreflex (phenylephrine, 0.5-2.0 micrograms/kg, i.v.) and the chemoreflex (potassium cyanide, 40 micrograms/rat, i.v) were activated in different groups of rats before and after bilateral microinjection of 2-methyl-5-HT into the NTS. Microinjections of 2-methyl-5-HT (5 nmol/50 nl) into the NTS produced a significant increase in basal mean arterial pressure (101 +/- 3 versus 125 +/- 8 mmHg), no changes in basal HR and a significant reduction in the reflex bradycardia triggered by baroreflex activation at 3 (-28 +/- 7 bpm), 10 (-35 +/- 4 bpm) and 20 min (-34 +/- 5 bpm) in comparison with the control value (-68 +/- 9 bpm). A similar reduction in the bradycardic response to chemoreflex activation was observed at 3 (-94 +/- 35 bpm), 10 (-98 +/- 38 bpm) and 20 min (-110 +/- 29 bpm) after 2-methyl-5-HT in comparison with the control value (-178 +/- 19 bpm). The effect of 2-methyl-5-HT on the basal mean arterial pressure and on the bradycardia evoked by stimulation of the baro- and chemoreflexes was blocked by pretreatment with granisetron bilaterally microinjected (500 pmol/50 nl) into the NTS. The data show that the stimulation of 5-HT3 receptors in the NTS of unanesthetized rats elicits a significant increase in basal mean arterial pressure and decreases the bradycardic response to baro- or chemoreflex activation.

Cardiovascular effects of microinjection of low doses of serotonin into the NTS of unanesthetized rats.

In the present study, we analyzed in conscious rats the effects of microinjections of serotonin (5-HT; pmol range) into the nucleus of the solitary tract (NTS) on basal mean arterial pressure (MAP) and heart rate (HR) and also on the reflex bradycardia induced by the activation of the baro- and chemoreflex evaluated 1 min after 5-HT microinjection into the NTS. The data show that unilateral microinjection of 5-HT in the picomolar range into the NTS of unanesthetized rats produced a dose-dependent decrease in MAP and HR, which was blocked by previous microinjection of ketanserin (250 pmol/50 nl) into the NTS. The changes in MAP and HR induced by 5-HT were of very short duration, with a return to baseline values a few seconds later. The cardiovascular responses to baro- or chemoreflex activation 1 min after 5-HT microinjection into the NTS did not differ from the control, indicating that low doses of 5-HT produced no effect on the cardiovascular reflexes tested at that time. The present data show that, as also observed in anesthetized rats, the microinjection of picomolar doses of 5-HT into the NTS elicits the typical cardiovascular responses to baroreceptor activation. These effects, hypotension and bradycardia, seem to be mediated by 5-HT2 receptors because both were blocked by a selective 5-HT2 receptor antagonist. However, since microinjection of 5-HT (1 pmol) into the NTS produced no changes in the cardiovascular responses to the baro- and chemoreflex activated 1 min later, the role of 5-HT2 receptors in the processing of the cardiovascular afferent messages in the NTS remains to be elucidated.

Role of serotonin3 receptors in the nucleus tractus solitarii on the carotid chemoreflex.

The effects of serotonin3 (5-HT3)-receptor stimulation in the nucleus tractus solitarii (NTS) on the cardiovagal, sympathetic, and respiratory responses to activation of carotid body chemoreceptors were investigated in anesthetized rats. The chemoreflex responses were triggered by an intravenous administration of KCN (40 microg/kg) in spontaneously breathing urethan-chloralose-anesthetized rats or by an intracarotid administration of saline saturated with 100% CO2 in pancuronium bromide-paralyzed and artificially ventilated urethan-anesthetized rats. Microinjections of 5-HT (2.5-5 nmol) or the 5-HT3 agonist 1-(m-chlorophenyl)-biguanide (CPBG, 300-1,200 pmol) into the commissural NTS blocked in a dose-dependent manner the atropine-sensitive chemoreflex bradycardia elicited by KCN. However, neither 5-HT nor CPBG affected the KCN-induced increase in respiratory volume and the CO2-induced increases in blood pressure and lumbar sympathetic nerve discharge. The inhibitory effect of 5-HT or CPBG on KCN-induced bradycardia was blocked by prior intra-NTS microinjection of a 5-HT3 antagonist, such as zacopride (100 pmol) or ondansetron (100 pmol), or the A-type gamma-aminobutyric acid (GABA(A)) antagonist bicuculline (10 pmol). In contrast, local microinjections of antagonists acting at 5-HT1 and 5-HT2 receptors, such as methysergide (100 pmol) and ketanserin (10 pmol), respectively, did not prevent the actions of 5-HT or CPBG. These data show that the stimulation of 5-HT3 receptors in the NTS exerted an inhibitory influence, probably through the activation of a local GABAergic system, on the cardiovagal component of the chemoreflex. Because similar effects of 5-HT3-receptor stimulation in the NTS were previously found on the baroreflex and Bezold-Jarisch reflex responses, it can be inferred that NTS 5-HT3 receptors play a key modulatory role in the reflex control of the heart rate.

Stimulation of 5-HT3 receptors in the NTS inhibits the cardiac Bezold-Jarisch reflex response.

Intra-atrial administration of phenylbiguanide has been shown to trigger, through the stimulation of vagal afferent C-fibers, reflex bradycardia, hypotension, and sympathoinhibition classically known as the Bezold-Jarisch (B-J) reflex (O. Krayer. Naunyn-Schmiedeberg's Arch. Exp. Pathol. Pharmacol. 240: 361-368, 1961). The effects of microinjections, into the nucleus tractus solitarius (NTS), of serotonin (5-HT) and 1-(m-chlorophenyl)-biguanide (CPBG), a potent 5-HT3 receptor agonist, on these reflex responses were studied in urethananesthetized rats. 5-HT (600 and 900 pmol) and CPBG (10-150 pmol) produced a dose-dependent inhibition of the atropine-sensitive bradycardiac component of the B-J reflex. The effect of both agonists was reversed by prior local microinjection of the 5-HT3 receptor antagonists zacopride (100 pmol) and ondansetron (100 pmol), but not by that of the 5-HT2 receptor antagonist ketanserin (10 pmol) or the mixed 5-HT1/5-HT2 receptor antagonist methysergide (100 pmol). In contrast, CPBG (150 pmol) did not affect the B-J reflex inhibition of lumbar sympathetic nerve discharge. These results show that stimulation of NTS 5-HT3 receptors produced an inhibition of the cardiovagal component of the B-J reflex without affecting its sympathetic component. Because the stimulation of these receptors also inhibits the cardiac component of the baroreflex, the present data suggest the participation of NTS 5-HT3 receptors in the mechanisms that modulate cardiac reflex responses elicited by messages from different vagal afferents.