Serotonin Concentration-response Curves Research Articles

Antispasmodic, antidepressant and anxiolytic effects of extracts from Schinus lentiscifolius Marchand leaves

Schinus lentiscifolius (Anacardiaceae) is widely used in folk medicine for treating gastrointestinal and emotional complaints but there are no scientific studies that support these uses. This work aims at evaluating the antispasmodic and central effects of S. lentiscifolius as well as the flavonoids presence in the tincture (SchT) and the composition of the essential oil (SchO). SchT inhibited the concentration-response curves (CRC) of carbachol and calcium in a non-competitive way in isolated rat intestine, bladder and uterus. SchT also non-competitively inhibited the CRC of histamine in guinea-pig intestine and the CRCs of serotonin and oxytocin in rat uterus. Isoquercetin and rutin were identified in SchT. The behavioral effects of SchT, SchO and infusion of S. lentiscifolius leaves (SchW) were tested in mice. These extracts showed an anxiolytic-like effect in the novelty-suppressed feeding test, which was reversed by flumazenil except in SchO-treated mice. Only SchO reduced the spontaneous locomotor function in the open field test. Also, SchT and SchW decreased immobility time in both, the tail suspension (TST) and forced swimming tests, while SchO produced the same effect in the TST. d-limonene and α-santalol were the main components found in SchO. The results demonstrated that extracts obtained from S. lentiscifolius leaves were effective as intestinal, urinary and uterine antispasmodics. SchT and SchW exhibited anxiolytic and antidepressant properties without sedation, whereas SchO showed also sedative properties. Therefore, the present study gives preclinical support to the traditional use of this plant for gastrointestinal and depressive or emotional symptoms.

Effects of 1-(2-trifluoromethylphenyl)-imidazole (TRIM) on receptor-independent and -dependent contractile responses in rat aorta.

This study investigates whether 1-(2-trifluoromethylphenyl)-imidazole (TRIM), originally proposed as a nitric oxide synthase inhibitor and also suggested to be an inhibitor of store-operated calcium entry in mouse anococcygeal muscle, inhibits receptor-independent and -dependent responses in rat thoracic aorta. Cyclopiazonic acid- and serotonin-induced vascular responses were investigated in aortic segments isolated from male Sprague Dawley rats using isolated tissue experiments. Changes in intracellular calcium levels were also monitored via front surface fluorescence measurements in fura-2-loaded embryonic rat vascular smooth muscle cell line A7r5. TRIM inhibited serotonin-mediated vascular contractions without affecting cyclopiazonic acid-induced responses. In addition, TRIM caused a nonlinear rightward shift in the serotonin concentration-response curve, possibly via serotonin receptor modulation. TRIM may have an impact on investigation of tissue-specific receptor-independent and -dependent vascular responses. It may also be used as a lead compound in the development of selective serotonin receptor modulators.

Interaction of Bupropion with 5-HT3A Receptors

For more than two decades, bupropion has been clinically prescribed for the treatment of depression (Wellbutrin®), and more recently for smoking cessation (Zyban®). Bupropion is conventionally described as a dual norepinephrine dopamine reuptake inhibitor, therefore effective as an antidepressant. However, we and others have shown that it also interacts with receptors of the Cys-loop superfamily, namely neuronal nicotinic acetylcholine receptors. In the continuum of exploring the molecular interactions of bupropion with other prominent members of the Cys-loop superfamily, we report here that bupropion additionally inhibits 5-hydroxytryptamine (serotonin) type 3A receptors (5-HT3ARs).5-HT3ARs are cation-conducting, homo-pentameric ligand-gated ion channels of the Cys-loop superfamily which are structurally and functionally distinct from other serotonin receptors that are G-proteins. 5-HT3 receptors are current targets for anti-emetics mainly used in cancer chemotherapy, as well as potential future targets for disorders including anxiety, schizophrenia, and Alzheimer's disease.Here we characterized our newly-identified interaction of bupropion with 5-HT3ARs with electrophysiological and radioligand binding studies. In oocytes, bupropion inhibited 5-HT3AR-mediated currents with an IC50 value of 87 µM (nH = 1.2). That 300 µM bupropion inhibited [3H]-5-HT or [3H]-granisetron (a competitive antagonist) binding by ∼ 20% indicated at best weak interaction with the agonist binding site. In contrast, the effect of different concentrations of bupropion on serotonin concentration-response curves suggested a non-competitive nature of interaction. Along these lines, importantly, the inhibition of serotonin-evoked currents recorded from oocytes was proportional to the length of preincubation time with bupropion.In summary, our results are indicative of a negative allosteric interaction of bupropion with the 5-HT3AR. Currently, experiments are underway to identify the site(s) of interaction for bupropion within the 5- HT3AR.

Lipopolysaccharide increases agonist‐induced contractile responses in Sprague Dawley rat corpus cavernosum

Chronic inflammatory conditions are frequent co‐morbidities of erectile dysfunction (ED) and elevated lipopolysaccharide (LPS) levels may increase inflammation via Toll‐like receptor 4 (TLR4) activation. An imbalance between vascular dilatation and contraction underlies ED. We hypothesized that LPS increases the agonist‐induced contractile response in corpus cavernosum (CC). The CC tissues were isolated, incubated for 6 hours in tissue culture media with 1 ug/mL LPS or vehicle, before being mounted in a muscle strip myograph for studies of isometric force generation. Contraction to 120 mM KCl did not differ between LPS and vehicle treated CC. KCl‐induced contraction data were used to normalize contraction data from the serotonin concentration response curve (30 nM‐100 uM). The CC tissue exposed to LPS exhibited enhanced contraction to serotonin (as % of KCl contraction) compared to control values. At 30 uM concentration: 67.34±5.15 vs. 43.34±6.67% (p<0.05), and at 100 uM: 77.61±5.4 vs. 56.61±6.98% (p<0.05). These results show that treatment with the TLR4 ligand LPS increased the contractile response to serotonin in rat corpus cavernosum. Thus, TLR4 activation may play a role in the etiology of ED which is a condition that has been shown to predict cardiovascular disease.Support: NIH and SMSNA.

Molecular Properties Important for Inhaled Anesthetic Action on Human 5-HT3A Receptors

Although inhaled anesthetics have diverse effects on 5-hydroxytryptamine type 3 (5-HT3A) receptors, the mechanism accounting for this diversity is not understood. Studies have shown that modulation of 5-HT3A receptor currents by n-alcohols depends on molecular volume, suggesting that steric interactions between n-alcohols and their binding sites define their action on this receptor. Electrostatic interactions also play an important role in anesthetic action on other ligand-gated receptors. We aimed to determine the contribution of molecular volume and electrostatics in defining volatile anesthetic actions on 5-HT3A receptors. Human 5-HT3A receptors were expressed in, and recorded from, Xenopus oocytes using the two-electrode voltage-clamp technique. The effects of a range of volatile anesthetics, n-alcohols, and nonhalogenated alkanes on submaximal serotonin-evoked peak currents, and full serotonin concentration-response curves were defined. Volatile anesthetics and n-alcohols, but not alkanes, smaller than 0.120 nm3 enhanced submaximal serotonin-evoked peak currents whereas all larger agents reduced currents. Most compounds tested inhibited maximal serotonin-evoked peak currents to varying degrees. However, only agents smaller than 0.120 nm3 shifted the 5-HT3A receptor's serotonin concentration-response curve to the left, whereas larger anesthetics shifted them to the right. Modulation of human 5-HT3A-mediated currents by volatile anesthetics exhibits a dependence on molecular volume consistent with the n-alcohols, suggesting that both classes of agents may enhance 5-HT3A receptor function via the same mechanism. Furthermore, the enhancing but not inhibiting effects of anesthetic compounds on 5-HT3A receptor currents are modulated by electrostatic interactions.

Phenylephrine precontraction increases the sensitivity of rabbit femoral artery to serotonin by enabling 5-HT1-like receptors.

We used selective receptor antagonists to identify the receptors mediating the isometric contractile response to serotonin in control and phenylephrine (PHE)-precontracted rabbit femoral artery rings. Serotonin, in the absence of PHE, elicited monophasic concentration-response curves (CRCs) early, but biphasic CRCs late in the course of the study. In the monophasic curves, the threshold and maximal concentrations were 10 and 1,000 microM, respectively. In biphasic CRCs, the threshold and maximal concentrations of the first phase were 0.03 and 3 microM, respectively. The respective values for the second phase were 10 and 1,000 microM. Prazosin, 0.1 microM, a selective alpha1-adrenoceptor antagonist, inhibited the monophasic curves, but only the second phase of the biphasic curves. Ritanserin, 0.01 microM, a selective 5-HT2A-receptor antagonist, shifted the first phase of the biphasic serotonin CRCs to the right but had little effect on the second phase. PHE increased the sensitivity of rabbit femoral artery response to serotonin. This amplified response to serotonin was antagonized by 0.01 microM GR 127935T, a selective 5-HT1B-receptor antagonist. The selective 5-HT1 agonist, sumatriptan, had no effect in control femoral arteries, but caused a concentration-dependent contraction after PHE precontraction. These results suggest that 5-HT1-like receptors are normally inactive or "silent" in the absence of PHE. However, in the presence of PHE, these receptors become enabled and mediate the amplified response to serotonin. The evidence also suggests that, in the absence of PHE, alpha1-adrenoceptors mediated the contractile response to serotonin in the monophasic CRCs. In the biphasic curves observed late in the study, the first phase was mediated by 5-HT2A receptors, and the second, by the alpha1-adrenergic receptors.

Inhibition of small conductance K<sup>+</sup>-channels attenuated melatonin-induced relaxation of serotonin-contracted rat gastric fundus

The aim of this study was to investigate the effects of melatonin on rat gastric fundus smooth muscle. Melatonin (10(-4) to 10(-3) M) had no effect on the basal tone of gastric smooth muscle. After precontraction with carbachol (10(-6) M) or serotonin (10(-7) M), melatonin caused a concentration dependent inhibitory action. The half maximal effect on serotonin-induced contraction was found with 1.12 +/- 0.86 x 10(-5) M of melatonin. Increasing concentrations of melatonin (10(-5) to 10(-3) M) resulted in a right shift of the serotonin concentration response curve (10(-10) to 10(-5) M). This inhibitory effect of melatonin was partially blocked in the presence of apamin (10(-10) to 10(-7) M), a specific blocker of the small conductance calcium-dependent potassium channel, but not in the presence of other potassium channel blockers like charybdotoxin (10(-8) M), glibenclamide (l0(-5) M), or tetraethylammonium (ODQ, 10(-4) M). The inhibitory effect was not changed in the presence of the neuronal blocker tetrodotoxin (10(-6) M), the selective P2-receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (3 x 10(-5) M), the nitric-oxide synthase inhibitor N-nitro-L-arginine (3 x 10(-4) M), or the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]-quinoxalin-1-one (10(-4) M), suggesting that neither the purinergic, nitrergic, nor guanylate cyclase pathways were involved. We further investigated inhibitory responses to electrical field stimulation (EFS) at different frequencies under non-adrenergic, non-cholinergic (NANC) conditions on a serotonin-induced contraction in the presence of melatonin (10)-5 to 10(-4) M). Melatonin significantly reduced these inhibitory NANC responses in higher (8-32 Hz), but not lower (05-4 Hz), frequencies (16 Hz without melatonin, 103 +/- 6.3%; melatonin 10(-5) M, 80.4 +/- 7.5%; melatonin 10(-4) M, 39.1 +/- 17.1%). Melatonin had no effect on contractile responses induced by EFS under basal tone. These results demonstrate that the inhibitory effect of melatonin in rat gastric fundus smooth muscle is apamin sensitive, but is not affected by other potassium channel blockers. This suggests that melatonin may be another transmitter candidate for the apamin sensitive responses within the gastrointestinal tract.

Effects of protein tyrosine kinase inhibitors on contractility of isolated bronchioles of the rat.

The role of protein tyrosine kinases (PTK) in modulating contractility has not been investigated in airway smooth muscle (ASM). We have examined the effects of the PTK inhibitors ST638, genistein, and tyrphostin A47 on contractions induced by carbachol, serotonin, ionomycin, and 75 mM KCl in isolated bronchioles of the rat with internal diameters of 614 +/- 16 microm (small, n = 143), and 1,433 +/- 39 microm (large, n = 57). ST638 caused a dose-dependent decrease in the maximum response to carbachol, and shifted the carbachol concentration-response curve to the right. This effect was greater in small bronchioles. Tyrphostin A47 (100 microM) and genistein (74 microM) had a similar effect to ST638. ST638 caused a concentration-dependent relaxation (EC50 approximately 7.2 microM) in bronchioles precontracted with 0.5 microM carbachol, and was maximally effective at 50 microM when tone was reduced by 82.5 +/- 3.8% in small bronchioles, and 57.2 +/- 2.8% in large bronchioles. ST638 also reduced the maximal response to serotonin, and caused a large shift to the right of the serotonin concentration-response curve. Pretreatment with ST638 (50 microM) reduced the response to 75 mM KCl in both small and large bronchioles in the presence of atropine (small: by 88.9 +/- 5.6%, n = 11; large: by 90.1 +/- 4.4%, n = 11). Tyrphostin A47 (100 microM) had a similar effect (91%). ST638 (50 microM) and tyrphostin A47 (100 microM) substantially relaxed small bronchioles contracted with 1.5 microM ionomycin (ST638: by 86.7 +/- 1.8%, n = 6; tyrphostin: by 89.3 +/- 1.7%, n = 5). We have therefore demonstrated that PTK inhibitors can suppress contraction induced by a number of different mechanisms in ASM. These results suggest that PTK signaling pathways are not only important for proliferation of ASM, but also fon contractile function.

The influence of high glucose levels and/or hyperosmolarity on rat isolated aorta

We investigated the influence of high-glucose levels (30.5 mmol/L) and/or hyperosmolarity on pharmacological responses in aortic ring preparations taken from nondiabetic rats. Moderate changes were observed for the concentration-response curves of noradrenaline and phenylephrine, but not for methoxamine and cirazoline. Maximal active forces of the concentration-response curves of potassium chloride and calcium chloride were significantly reduced by the elevated glucose levels per se, but neither the slopes of the curves nor the -logEC 50 values were affected. Concentration-response curves ofserotonin and U 46619 were not affected. For angiotensin II, the −logEC 50 values and maximal active forces of the concentration-response curves were significantly lower under both hyperglycemic and hyperosmolar conditions. The present study suggests that potential changes in contractile behavior in isolated vessels from diabetic animals cannot be attributed to high glucose levels and/or hyperosmolarity as such, but indeed reflect vascular changes associated with the diabetic state. An exception has to be made for the depolarization of aortic ring preparations of nondiabetic rat; the elevated glucose level leads to an impaired calcium influx via the (slow) L-type calcium channels.

A 5-HT 2 Receptor Mediates Serotonin-Induced Electrolyte Transport in Rat Left Colon

Serotonin (5-hydroxytryptamine, 5-HT) is a potent mediator of diarrhea in the carcinoid syndrome. However, the mechanisms of serotonin-induced intestinal secretion remain unclear. The purpose of this study was to determine whether 5-HT stimulates electrolyte transport by a direct action at a mucosal receptor. Serotonin-stimulated electrolyte transport was studied in flat sheet preparations of rat left colon using flux chambers. 5-HT stimulated a concentration-dependent rise in short-circuit current in sheets stripped of muscularis propria. The half-maximal concentration required to produce this effect was 50 μ M. The preferential 5-HT 2 antagonist ketanserin produced a rightward shift of the serotonin concentration-response curve with a pA 2 value of 7.7. Serotonin stimulated electrolyte transport by inhibiting mucosa to serosa movement of both sodium and chloride. The 5-HT 1 antagonist N -acetyl-5-hydroxytryptophyl 5-hydroxytryptophan amide and the 5-HT 3 antagonist 3-tropanyl-indole-3-carboxylate methiodide had no effect on these actions of serotonin on electrolyte transport. In contrast, ketanserin inhibited the actions of serotonin on both sodium and chloride movement and inhibited the rise in short-circuit current induced by 100 μ M 5-HT. This study demonstrates that a 5-HT 2 receptor located in the vicinity of the mucosa is involved in the regulation of serotonin-stimulated intestinal electrolyte transport.

Prostaglandin-related Microvascular Dilation in Pentobarbital- and Etomidate-anesthetized Rats

Etomidate is characterized by minimal systemic cardiovascular effects, but its effect on the microvasculature has not been assessed. We compared the microvasculature of etomidate-anesthetized animals to that of animals anesthetized with pentobarbital, since its effects on the microvasculature are known. Male Sprague-Dawley rats were anesthetized with etomidate or pentobarbital. The cremaster muscle was prepared for microscopic viewing, leaving the neural and vascular supply intact. Small arterioles were near their maximal diameters in etomidate-anesthetized rats, whereas the pentobarbital group had a large dilator capacity (maximal diameter-basal diameter/basal diameter). The effect on resting arteriolar diameters of endothelium-derived relaxing factor (EDRF) and prostaglandin synthesis inhibitors was tested. Dilator capacity was not affected by the EDRF inhibitor nitro-L-arginine, but it was significantly increased by mefenamic acid and ibuprofen in etomidate-anesthetized animals. To test whether dilator and constrictor mechanisms were normal, serotonin concentration-response curves were obtained in pentobarbital and etomidate-anesthetized animals with and without mefenamate or ibuprofen present. The dilation of small arterioles to serotonin in the etomidate groups with mefenamate or ibuprofen was not significantly different from that of the pentobarbital groups. Serotonin produced a comparable constriction of large arterioles in both anesthetic groups. The topical application of etomidate to the cremaster muscle did not affect arteriolar diameters. Thus, etomidate appears to trigger the release of dilator prostaglandins in striated muscle through a central or indirect mechanism.

Halothane Attenuates Small Arteriole Vasodilation to Serotonin (5-HT) in Skeletal Muscle

Inhaled anesthetics may have an effect on the microcirculation through selective alteration of receptor-mediated control. Halothane was chosen because its actions in the microcirculation have been determined previously. Serotonin has received recent attention as a potential mediator of vascular changes in a number of disease states. We obtained concentration-response curves for serotonin-induced constriction of large arterioles and dilation of small arterioles (less than 60 microns diameter) in the cremaster muscle of halothane-anesthetized and decerebrate rats. Cremaster muscles were prepared for microscopic viewing, leaving the neural and vascular supply intact. Serotonin concentration-response curves were obtained before and after receptor antagonist application. Large arteriole constriction was not affected by halothane. Dilation of small arterioles was decreased in halothane-anesthetized animals but enhanced in the presence of methysergide, a nonspecific antagonist. These data indicate that halothane interferes with occupancy of 5-hydroxytryptamine receptors.

Effects of preload and eicosanoid synthesis inhibition on rat aortic smooth muscle sensitivity

Concentration-response curves to serotonin and phenylephrine were obtained from aortic strips subjected to low (0.75 g) and high (3.0) preloads in the presence and absence of eicosanoid synthesis inhibitors. The sensitivity of the strips to both agonists was greater in the high preload strips. The cyclooxygenase inhibitor, indomethacin (28 μM), shifted the serotonin concentration-response curves to the right. However, the preload effect still remained. The lipoxygenase inhibitor, nordihydroguaiaretic acid (10μM), not only decreased sensitivity to serotonin and phenylephrine, but eliminated the preload effect as well. These results suggest that 1) both cyclooxygenase and lipoxygenase metabolites affect the sensitivity of isolated arterial smooth muscle to vasoactive agents, and 2) lipoxygenase, but not cyclooxygenase, metabolites may play a role in the effect of preload on arterial smooth muscle sensitivity.

Epithelial modulation of tracheal smooth muscle response to antigenic stimulation.

The role of the epithelium has been studied in the contractile responses of rat trachea. The different modulations observed are discussed in respect to vagal components of the epithelial layer. Responses of rat trachea to immunologic stimulation are shown to be dependent on the presence of the epithelium, which prolongs the relaxation stage without affecting the contractions. This prolongation is abolished by neonatal capsaicin pretreatment, whereas substance P induces a significantly greater relaxation of serotonin-precontracted intact than deepithelialized trachea. Serotonin concentration-response curves are shifted to the right in intact preparations, which is partly reversed by neonatal capsaicin pretreatment, but a hyporeactivity of the tissue exists. A relaxing factor released by the epithelium is hypothesized, possibly dependent on substance P-ergic innervation. Muscarinic cholinergic innervation slightly modulates the contractions but not the relaxations in antigen-induced responses, independently on the presence of the epithelial layer. 4-Aminopyridine induces epithelium-dependent potentiations of contractions to antigen and to serotonin, which involves acetylcholine at one step of the reaction cascade. Epithelial-dependent contracting and relaxing factors are thus suggested in rat trachea.

5HT 2 receptors in the rat portal vein: Desensitization following cumulative serotonin addition

Contractile responses to serotonin were examined in vitro in the longitudinal portal vein to determine whether such responses were mediated by the interaction of serotonin with 5HT 1 receptors (those that preferentially bind [ 3H]serotonin) or 5HT 2 receptors (those that preferentially bind [ 3H]spiperone). Using eight serotonin receptor antagonists (spiperone, metergoline, LY53857, ketanserin, trazodone, benzoctamine, 1-(1-naphthyl)piperazine, and 1-meta-methoxyphenylpiperazine), we found a significant correlation between the affinity for serotonin receptors in the rat portal vein and the ability to bind to 5HT 2, but no 5HT 1 receptors in rat frontal cortical membranes. Thus, the receptors mediating vascular contraction to serotonin in the rat portal vein were similar to those receptors defined in other vascular beds from the rat (aorta, jugular vein, and caudal artery). Furthermore, contraction resulting from the cumulative addition of serotonin in the rat portal vein was associated with desensitization (higher ED 50 value) relative to contractions produced by the non-cumulative addition of serotonin. Affinities of serotonin receptor antagonists were also lower when determined by antagonism of cumulative serotonin concentration-response curves compared to affinities obtained by antagonism of non-cumulative concentration-response curves. Thus, 5HT 2 receptor affinities of antagonists in the rat portal vein are best determined by the shift of non-cumulative responses to serotonin.

Actions of serotonin antagonists on dog coronary artery

Serotonin released from platelets may initiate coronary vasopasm in patients with variant angina. If this hypothesis is correct, serotonin antagonists without constrictor activity may be useful in this form of angina. We have investigated drugs classified as serotonin antagonists on dog circumflex coronary artery ring segments in vitro. Ergotamine, dihydroergotamine, bromocriptine, lisuride, ergometrine, ketanserin, trazodone, cyproheptadine and pizotifen caused non-competitive antagonism of serotonin concentration-response curves. In addition, ketanserin, trazodone, bromocriptine and pizotifen inhibited noradrenaline responses in concentrations similar to those required for serotonin antagonism. All drugs with the exception of ketanserin, cyproheptadine and pizotifen showed some degree of intrinsic constrictor activity. Methysergide antagonized responses to serotonin competitively but also constricted the coronary artery. The lack of a silent competitive serotonin antagonist precludes a definite characterization of coronary serotonin receptors at this time. However, the profile of activity observed for the antagonist drugs in the coronary artery differs from that seen in other vascular tissues. Of the drugs tested, ketanserin may be the most useful in variant angina since it is a potent 5HT antagonist, lacks agonist activity and has α-adrenoceptor blocking activity.