Serotonin,norepinephrine, and histamine mediation of endothelial cell barrier function in vitro

The effects of serotonin (5-hydroxytryptamine, 5-HT), norepinephrine (NE), and histamine on endothelial cell barrier function were examined in vitro. Bovine aortic endothelial (BAE) cells grown to confluence on microcarriers formed a measurable barrier to the passage of a trypan blue dye-bovine serum albumin conjugate (TB-BSA) from the culture medium into the microcarrier matrix. Vascular smooth muscle (VSM) cells or Swiss 3T3 fibroblasts impeded TB-BSA diffusion only 42% and 56%, respectively, relative to BAE cells. These results suggest that barrier formation may be an endothelial cell-specific phenomenon. Treatment of BAE cells with histamine was associated with 2-to 3-fold increases in the rate of TB-BSA diffusion. In contrast, treatment with 5-HT or NE at concentrations ranging from normal to pathophysiological circulating plasma levels significantly impeded TB-BSA diffusion by up to 43% and 33%, respectively, relative to untreated controls. The barrier-modulating effects of the vasoactive amines were dose-dependent, cell-specific, and in some cases appear to be receptor-mediated. These results are consistent with previous reports that histamine increases vascular permeability in part by affecting diffusion between endothelial cells; they support the hypothesis that 5-HT and NE contribute to the maintenance of the endothelial barrier in vivo.     

Down-Regulation of [3H]5-Hydroxytryptamine Binding Sites in Chick Embryo Brain by Monoamine Oxidase Inhibitors or Fenfluramine and Potentiation by d,l-5-Hydroxytryptophan

Three monoamine oxidase (MAO) inhibitors--pargyline, clorgyline and deprenyl--as well as the serotonin (5-HT, 5-hydroxytryptamine) releasing agent fenfluramine were administered to developing chick embryos and the effects on [3H]5-HT binding parameters and endogenous 5-HT levels were assessed. Multiple, but not acute, pretreatments with any of the three MAO inhibitors significantly increased 5-HT levels (p less than 0.01) and decreased receptor number (Bmax) to a maximum of 20% (p less than 0.01) without affecting the affinity (KD). When d,l-5-hydroxytryptophan (d,l-5-HTP) was similarly administered there were large increases in 5-HT levels (p less than 0.01), but no significant effects on either Bmax or KD. However, if d,l-5-HTP was co-administered with any of the MAO inhibitors there was a significant (p less than 0.01) enhancement of the MAO inhibitor-induced down-regulation to a maximum of about 40%. Multiple pretreatments with fenfluramine resulted in dose-related decreases in 5-HT levels (p less than 0.01) and Bmax (p less than 0.01) without affecting KD. The largest decrease in [3H]5-HT binding sites inducible by fenfluramine treatment alone was also about 40%. When given in combination with d,l-5-HTP, there was a potentiation of the down-regulation capabilities of fenfluramine at several different dosage levels; however, maximal down-regulation was also limited to 40%. Evidence was presented suggesting that these effects were not due to endogenous 5-HT or drugs remaining in the tissue preparation. The overall evidence implies that merely increasing endogenous 5-HT levels, as by precursor administration, does not necessarily induce down-regulation unless the 5-HT is also made available as functional 5-HT.     

5-Hydroxytryptamine-Stimulated Inositol Phospholipid Hydrolysis in the Mouse Cortex Has Pharmacological Characteristics Compatible with Mediation via 5-HT2 Receptors but This Response Does Not Reflect Altered 5-HT2 Function After 5,7-Dihydroxytryptamine Lesioning or Repeated Antidepressant Treatments

5-Hydroxytryptamine (5-HT; 3 x 10(-8)-1 x 10(-5)M) produced a dose-dependent increase in phosphatidylinositol/polyphosphoinositide (PI) turnover in mouse cortical slices, as measured by following production of 3H-labelled inositol phosphates (IPs) in the presence of 10 mM LiCl. Analysis of individual IPs, in slices stimulated for 45 min, indicated substantial increases in inositol monophosphate (IP1; 140%) and inositol bisphosphate (IP2; 95%) contents with smaller increases in inositol trisphosphate (IP3; 51%) and inositol tetrakisphosphate (IP4; 48%) contents. The increase in IP3 level was solely in the 1,3,4-isomer. This response was inhibited by the nonselective 5-HT antagonists methysergide, metergoline, and spiperone. It was also inhibited by the selective 5-HT2 antagonists ketanserin and ritanserin but not by the 5-HT1 antagonists isapirone, (-)-propranolol, or pindolol. 5-HT-stimulated IP formation was also unaltered by atropine, prazosin, and mepyramine. Lesioning brain 5-HT neurones using 5,7-dihydroxytryptamine (5,7-DHT; 50 micrograms i.c.v.) produced a 210% (p less than 0.01) increase in the number of 5-HT2-mediated head-twitches induced by 5-methoxy-N,N-dimethyltryptamine (2 mg/kg). However, 5,7-DHT lesioning had no effect on 5-HT-stimulated PI turnover in these mice. Similarly, an electroconvulsive shock (90 V, 1 s) given five times over a 10-day period caused an 85% (p less than 0.01) increase in head-twitch responses but no change in 5-HT-stimulated PI turnover. Decreasing 5-HT2 function by twice-a-day injection of 5 mg/kg of zimeldine or desipramine (DMI) produced 50% (p less than 0.01) and 56% (p less than 0.01), respectively, reductions in head-twitch behaviour.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of monoamine neuromediators on the growth-related variables of <Emphasis Type="Italic">Escherichia coli</Emphasis> K-12

The monoamine neuromediators serotonin (5-HT), histamine, dopamine (DA), and norepinephrine (NE), added to an Escherichia coli K-12 strain MC 4100 culture upon inoculation, stimulate cell proliferation (determined from CFU formation) and biomass accumulation (monitored nephelometrically) during the late lag phase and the early exponential growth phase. These effects are less significant in the late exponential and stationary phase cultures. According to the concentration dependence of the stimulatory effects, the neuromediators can be classified into two groups: (i) the catecholamines DA and NE, whose effects increase almost linearly with increasing concentrations within the range of 0.1–100 μM, and (ii) histamine and 5-HT, which are characterized by bell-shaped concentration dependence curves with maxima at 0.1 (histamine) and 1 μM (5-HT). On an agar-containing medium, the growing E. coli population includes solitary cells and compact cell groups (microcolonies). In this system, both tested catecholamines exert a relatively weak stimulatory influence that manifests itself as an increase in the number of both solitary cells and cell groups, and occurs at concentrations of 10 μM and higher. In analogy to the culture grown on the liquid medium, 5-HT and histamine are distinguished by nonlinear concentration dependence curves: their effects peak at 0.1 μM (histamine) or 1 μM (5-HT); an increase in the neuromediator concentrations results in a decrease in effects that are enhanced by further increasing the concentrations to the submillimolar range. DA increases the percentage of solitary cells, whereas the other tested amines promote cell group formation. The results are interpreted in terms of specific (probably receptor-dependent) mechanisms of action in the neuromediators involved.     

Endothelial cell junctional integrity modulation by serotonin: an ultrastructural analysis

We have reported previously that exogenous serotonin (5-hydroxytryptamine, 5-HT) alters cultured bovine aortic endothelial cell (BAEC) structural integrity by modulating the assembly of stress fibers. In the present study a 5-HT stimulus-coupled change in BAEC junctional integrity was quantitated by determining the width and percentage of intercellular openings in a monolayer. BAEC treated with 5-HT at concentrations of 10(-9) M to 10(-3) M caused a significant dose-dependent decrease in interendothelial cell junctional openings compared to controls, with the greatest reduction induced at 10(-6) M (92% from control). Treatment of BAEC with histamine (10(-4) M) increased the junctional openings by 82% when compared to controls. This change could be prevented by either pretreatment of the monolayers with 5-HT or by adding 5-HT in conjunction with the histamine. To assess a direct interaction of 5-HT with actin filaments, cultured BAEC monolayers were extracted, treated with 5-HT, and processed for immunocytochemical localization of 5-HT using the Avidin-Biotin method. Electron microscopy revealed 5-HT antibody bound to actin filaments and dense in areas of filament intersection, which implies a role for internalized 5-HT in stimulating the assembly of an actin filament network. Collectively, these results suggest that 5-HT helps to regulate the endothelial junctional barrier by promoting actin filament formation and stability, which may in turn increase the junctional apposition between endothelial cells.     

Duloxetine inhibits effects of MDMA ("ecstasy") in vitro and in humans in a randomized placebo-controlled laboratory study

This study assessed the effects of the serotonin (5-HT) and norepinephrine (NE) transporter inhibitor duloxetine on the effects of 3,4-methylenedioxy-methamphetamine (MDMA, ecstasy) in vitro and in 16 healthy subjects. The clinical study used a double-blind, randomized, placebo-controlled, four-session, crossover design. In vitro, duloxetine blocked the release of both 5-HT and NE by MDMA or by its metabolite 3,4-methylenedioxyamphetamine from transmitter-loaded human cells expressing the 5-HT or NE transporter. In humans, duloxetine inhibited the effects of MDMA including elevations in circulating NE, increases in blood pressure and heart rate, and the subjective drug effects. Duloxetine inhibited the pharmacodynamic response to MDMA despite an increase in duloxetine-associated elevations in plasma MDMA levels. The findings confirm the important role of MDMA-induced 5-HT and NE release in the psychotropic effects of MDMA. Duloxetine may be useful in the treatment of psychostimulant dependence. TRIAL REGISTRATION: Clinicaltrials.gov NCT00990067.     

Effects of cyclooxygenase inhibition on pulmonary vascular responses to serotonin

The vasopressor response to graded bolus doses (50-500 micrograms) of serotonin (5-hydroxytryptamine; 5-HT) was examined in the isolated canine lower left lung lobe (LLL) perfused at constant flow with autogenous blood before and after cyclooxygenase inhibition (COI). Lobar vascular resistance (LVR) was partitioned into pre- (Ra) and postcapillary (Rv) segments by venous occlusion with lobar blood volume changes monitored gravimetrically. Before COI, 5-HT produced transient, dose-dependent increases in pulmonary arterial pressure (Ppa) of 43.8 +/- 4.8-123.0 +/- 8.5% (n = 22) and simultaneous decreases in lobar blood volume (5.5 +/- 0.5-8.2 +/- 0.6 g/100 g LLL) with nearly proportionate increases in Ra and Rv at each 5-HT dose. After the initial challenge to 5-HT, LLL's were treated either with saline (n = 7) or one of three chemically distinct cyclooxygenase inhibitors. COI with 40 microM indomethacin (n = 6) or 45 microM meclofenamate (n = 6) increased resting LVR by 36.0 +/- 8.3% (P less than 0.01; n = 12) and decreased the Ra/Rv from 1.9 +/- 0.3 to 1.1 +/- 0.2 (P less than 0.01), whereas 1 mM aspirin (n = 3) caused a fourfold increase in resting LVR without affecting Ra/Rv. After indomethacin or meclofenamate treatment, the vasopressor response to graded doses of 5-HT was markedly potentiated as Ppa increased by 71.6 +/- 7.6-207.0 +/- 24.6%. COI did not potentiate the lobar vasopressor response to graded doses (10-100 micrograms) of norepinephrine (NE, n = 6).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of age on morphology,protein synthesis and secretagogoue-evoked secretory responses in the rat lacrimal gland

This study investigated changes in the morphology and protein synthesis and protein and peroxidase secretion due to peptidergic and aminergic stimulation from rat lacrimal gland acinar cells of 3–5, 9, 12, 20 and 24 month old rats. There was a marked reduction in the presence of Golgi apparatus in the acinar cells of glands from the 24 month old rats coupled to dilatation and degeneration of rough endoplasmic reticulum, when compared to that in the acinar cells of glands from 3–5 and 12 month old rats. Following incorporation of tritiated leucine for 360 min (6 h), the amount of newly synthesised protein in acinar cells of the 12 month old rats was significantly (p < 0.01) higher than that in the acinar cells of 3–5 month old animals. However, at 20 months the amount of newly synthesised protein in these acinar cells was significantly (p < 0.01) reduced to less than that in acinar cells of both the 3–5 and 12 month old animals. Immunohistochemical and immunofluorescence studies identified the presence of substance P (SP), vasoactive intestinal peptide (VIP), histamine and 5-hydroxytryptamine (5-HT) in the lacrimal glands of 3–5 month old rats. Stimulation by either SP, VIP, histamine or 5-HT resulted in significant increases in total protein output and peroxidase release from acinar cells of the 3–5 month old rats. However, all responses to the secretagogues were reduced with ageing from 3–5 to 24 months of age. The results indicate that ageing is associated with alteration in the ability of acinar cells to synthesise and secrete proteins.     

Antagonism of Serotonin 5-HT1A Receptors Potentiates the Increases in Extracellular Monoamines Induced by Duloxetine in Rat Hypothalamus

Abstract: In the current study we examined the effects of coadministration of a serotonin 5-HT_1A antagonist, (±)-1-(1 H -indol-4-yloxy)-3-(cyclohexylamino)-2-propanol maleate (LY 206130), and a dual 5-HT and norepinephrine (NE) uptake inhibitor, duloxetine, on extracellular levels of NE, 5-HT, dopamine (DA), 5-hydroxyindoleacetic acid, and 3,4-dihydroxyphenylacetic acid in rat hypothalamus microdialysates. LY 206130 (3.0 mg/kg, s.c.) alone significantly increased NE and DA levels by 60 and 34%, respectively, without affecting 5-HT levels. Duloxetine administration at 4.0 mg/kg, i.p. alone produced no significant changes in levels of 5-HT, NE, or DA. In contrast, when LY 206130 and duloxetine were coadministered at 3.0 mg/kg, s.c. and 4.0 mg/kg, i.p., respectively, 5-HT, NE, and DA levels increased to 5.7-, 4.8-, and threefold over their respective basal levels. These data demonstrate that antagonism of somatodendritic 5-HT_1A autoreceptors and concomitant inhibition of 5-HT and NE uptake with duloxetine may promote synergistic increases in levels of extracellular 5-HT, NE, and DA in hypothalamus of conscious, freely moving rats.     

6-Hydroxydopamine induced impairment of Pavlovian conditioning in the rabbit

This study employed bilateral, intraventricular injections of 6-hydroxydopamine (6-HDA) to examine the effects of monoamine depletion on Pavlovian conditioning of the rabbit's nictitating membrane response. 6-HDA produced dose-dependent and highly correlated decreases in the rate of acquisition of conditioned responses and in the telencephalic content of 5-HT, DA, and NE. At the highest dose of 6-HDA (1340 g), 5-HT, DA, and NE were reduced by 42, 48, and 89%, respectively, and the number of trials required to achieve criterion acquisition was increased by 123%. Control experiments established that the highest dose of 6-HDA: 1) had no effect on the unconditioned nictitating membrane reflex; 2) had no effect on the threshold of the conditioned stimulus for eliciting conditioned responses; and 3) produced only a small, less than 5%, decrease in nonassociative responding. It was concluded that decreases in 5-HT, DA, and NE can impair associative learning without altering sensory or motor function.Special issue dedicated to Dr. Morris H. Aprison.     

Histamine inhibits atrial myocytic ANP release via H2 receptor-cAMP-protein kinase signaling

Changes in cyclic nucleotide production and atrial dynamics have been known to modulate atrial natriuretic peptide (ANP) release. Although cardiac atrium expresses histamine receptors and contains histamine, the role of histamine in the regulation of ANP release has to be defined. The purpose of the present study was to define the effect of histamine on the regulation of ANP release in perfused beating rabbit atria. Histamine decreased ANP release concomitantly with increases in cAMP efflux and atrial dynamics in a concentration-dependent manner. Histamine-induced decrease in ANP release was a function of an increase in cAMP production. Blockade of histamine H2 receptor with cimetidine but not of H1 receptor with triprolidine abolished the responses of histamine. Cell-permeable cAMP analog, 8-Br-cAMP, mimicked the effects of histamine, and the responses were dose-dependent and blocked by a protein kinase A (PKA)-selective inhibitor, KT5720. Nifedipine failed to modulate histamine-induced decrease in ANP release. Protein kinase nonselective inhibitor staurosporine blocked histamine-induced changes in a concentration-dependent manner. KT5720 and RP-adenosine 3',5'-cyclic monophosphorothioate, another PKA-selective inhibitor, attenuated histamine-induced changes. These results suggest that histamine decreases atrial ANP release by H2 receptor-cAMP signaling via PKA-dependent and -independent pathways.     

Effects of chronic hypoxia on pulmonary vascular responses to biogenic amines

The effects of chronic hypoxia on pulmonary vascular resistance changes (% delta Rpv) to histamine, 5-hydroxytryptamine (5-HT), norepinephrine (NE), and KCl were studied in isolated perfused lungs from control rats and rats exposed to 7, 14, and 28 days of hypoxia. Histamine, which produced linear increases in % delta Rpv with increasing doses in the control, was reversed to vasodilation by chronic hypoxia of 7 and 14 days and at 28 days, vasodilation to this amine still predominated (7 out of 10). Control responses to 5-HT were unaltered by 7 days of hypoxia but enhanced at 14 and 28 days. Control responses to NE showed either vasoconstriction or vasodilation; at 7 days of hypoxia, NE had no significant vasoactivity; however, at 14 days, vasoconstriction and vasodilation were both observed, with vasodilation being more effective. Lastly, the pressor responses to KCl were not affected by chronic hypoxia of any duration. These results suggest that chronic hypoxia: 1) does not alter pulmonary vascular contractility (KCl); 2) reduces H1 and alpha-receptor activity while enhancing H2- and beta-receptor activity; and 3) enhances the pressor responses to 5-HT by increasing either the efficacy of this amine or the number of 5-HT vasoconstrictor receptors.     

Comparison of the effects of serotonin selective,norepinephrine selective,and dual serotonin and norepinephrine reuptake inhibitors on lower urinary tract function in cats

Previous studies showed that the dual serotonin (5-hydroxytryptamine, 5-HT) and norepinephrine (NE) reuptake inhibitor, duloxetine, increases bladder capacity and urethral sphincter electromyographic (EMG) activity in a cat model of acetic acid-induced bladder irritation. The present study aimed to determine the relative importance of 5-HT versus NE reuptake inhibition for mediating these effects by examining drugs that are selective for either the 5-HT or NE system or both. Similar to duloxetine, venlafaxine (0.1 to 10 mg/kg), also a dual serotonin and norepinephrine reuptake inhibitor, produced marked increases in bladder capacity and EMG activity that were reversed by methiothepin (0.3 mg/kg). S-norfluoxetine (0.01 to 10 mg/kg), a serotonin selective reuptake inhibitor, produced small but significant increases in bladder capacity and EMG activity at doses of 3 and 10 mg/kg. Thionisoxetine (0.01 to 3.0 mg/kg), a NE selective reuptake inhibitor, produced no effects on bladder capacity or sphincter EMG activity. Surprisingly, co-administration of thionisoxetine and s-norfluoxetine up to doses of 1 mg/kg of each compound produced no effect on lower urinary tract function. These doses were the maximum that could be administered in combination due to drug-induced emergence of skeletal muscle activity in chloralose-anesthetized animals. These results indicate that there are unexplained pharmacological differences between the effects of single compounds that exhibit dual NE and 5-HT reuptake inhibition and a combination of compounds that exhibit selective NE and 5-HT reuptake inhibition on lower urinary tract function.     

Histamine activates tyrosine hydroxylase in bovine adrenal chromaffin cells through a pathway that involves ERK1/2 but not p38 or JNK

In bovine adrenal chromaffin cells (BACC) histamine promotes a rapid increase in the intracellular levels of Ca2+ together with the release of catecholamines and the phosphorylation of the catecholamine biosynthetic enzyme tyrosine hydroxylase (TH). In this study we investigated the role of the mitogen-activated protein kinases (MAPK) extracellular signal-regulated kinases (ERK1/2), stress activated protein kinase (p38) and Jun N-terminal kinases (JNK) on the histamine-induced activation and phosphorylation of TH. We found that in BACC histamine produced a rapid, long lasting and histamine type-1 (H1) receptor-dependent increase in the phosphorylation levels of ERK1/2, p38 and JNK which was accompanied by a H1 receptor-dependent increase in TH activity. This increase in TH activity was partially blocked by the MEK1/2 inhibitor U0126 but was unaffected by the p38 antagonist SB203580 or the JNK blocker JNKI1. To study the effect of MAPK inhibition on histamine-induced TH phosphorylation, we generated phospho-specific antibodies against the different phosphorylated forms of TH. Treatment with U0126 totally inhibited the histamine-induced phosphorylation of TH at Ser31, without affecting the phosphorylation of either Ser40 or Ser19. Neither SB203580 nor JNKI1 treatments produced any significant modification of the histamine-induced TH phosphorylation. Our data support the hypothesis that the up-regulation of the ERK1/2 pathway, but not that of p38 or JNK, promoted by histamine is involved in the phosphorylation of TH at Ser31 and that this phosphorylation event is required for the full activation of this enzyme.     

Contractile responses of canine isolated pulmonary lobar arteries and veins to norepinephrine, serotonin, and tyramine.

Isolated helical strips of canine intrapulmonary lobar arteries and veins (about 4 mm in diameter) undergo dose-related tension development when exposed to increasing concentrations (10(-8) - 10(-3) M) of norepinephrine (NE), serotonin or 5-hydroxytryptamine (5-HT) and tyramine (Tyr). Venous segments were generally more sensitive while the maximum tension development was greater in the arterial strips, probably owing to their greater thickness. Both strips were more sensitive to 5-HT than NE and only responded to Tyr at high concentrations. Norepinephrine and 5-HT were nearly equally efficacious, whereas Tyr was less so. Responses to the latter were slow to develop, exhibited tachyphylaxis, and were greatly inhibited by phentolamine (10(-8) M), an alpha-adrenergic blocker. Exposure to cocaine (10(-5) M) enhanced submaximal NE responses, inhibited Tyr contractions and had no consistent effect on 5-HT responses. Phentolamine (10(-8) M) was also found to inhibit NE responses without altering 5-HT probably acts on other receptors. Tyramine may, in part, act directly on alpha-adrenergic receptors but may also release NE from surviving adrenergic nerve terminals in the preparation. Cocaine inhibits this effect and potentiates responses to lower levels of NE, presumably by blocking NE uptake into nerve terminals although a post-junctional action cannot be excluded.     

Defective regulation of immune responses in respiratory syncytial virus infection

The relationship of suppressor cell numbers and function to virus-specific IgE response was determined in 72 infants with respiratory syncytial virus (RSV) infection. Monoclonal antibodies to membrane antigens were used to enumerate OKT4 and OKT8 antigen-positive cells, and suppressor cell function as quantitated by the degree of suppression of lymphocyte mitogenesis induced by incubation of lymphocyte cultures with histamine. Patients with bronchiolitis had fewer OKT8-positive cells during convalescence than patients with other forms of illness due to RSV (p less than 0.05). An inverse correlation of OKT8-positive cell numbers and peak IgE titers was observed (p less than 0.01). Histamine-induced suppression was also reduced in patients with bronchiolitis (p less than 0.05). In patients with repeated infection, improved histamine-induced suppression was associated with reduced virus-specific IgE titers and the absence of wheezing. Defects in immunoregulation may underlie previously recognized immunologic and pharmacologic abnormalities in patients with bronchiolitis.     

The role of dopamine and serotonin in the prolongation of post-decapitation convulsions in mice.

L-DOPA (320 mg/kg, i.p.) increased the duration of the clonic phase of post-decapitation convulsions (PDC) by 60% in mice pretreated with the peripheral decarboxylase inhibitor, Ro 4-4602 (50 mg/kg, i.p.). Assays of brains at the time of decapitation showed a 300% increase in dopamine (DM), an 80% reduction in serotonin (5-HT) and no change in norepinephrine (NE) levels. The effect of L-DOPA on PDC was not blocked by haloperidol (0.5 – 5.0 mg/kg), a blocker of DM receptors, nor by diethyldithiocarbamate (400 mg/kg) an inhibitor of NE synthesis. Parachlorophenylalanine (300 mg/kg × 3 days) produced an 80% reduction in 5-HT and a prolongation of PDC similar to that observed after L-DOPA. Prolongation of PDC was also seen after the 5-HT antagonists methysergide (5 mg/kg) and cinanserin (10 mg/kg), but not after cyproheptadine (10 mg/kg). The 5-HT precursor, 5-hydroxytryptophan (100 mg/kg), produced no change in PDC when used alone but inhibited L-DOPA's prolongation of PDC. The results suggest that L-DOPA acts by depleting 5-HT in bulbospinal pathways and thus enhancing reflex activity in the spinal cord.     

In vivo electrochemical evidence for simultaneous 5-HT and histamine release in the rat substantia nigra pars reticulata following medial forebrain bundle stimulation

Exploring the mechanisms of serotonin [5-hydroxytryptamine (5-HT)] in the brain requires an in vivo method that combines fast temporal resolution with chemical selectivity. Fast-scan cyclic voltammetry is a technique with sufficient temporal and chemical resolution for probing dynamic 5-HT neurotransmission events; however, traditionally it has not been possible to probe in vivo 5-HT mechanisms. Recently, we optimized fast-scan cyclic voltammetry for measuring 5-HT release and uptake in vivo in the substantia nigra pars reticulata (SNR) with electrical stimulation of the dorsal raphe nucleus (DRN) in the rat brain. Here, we address technical challenges associated with rat DRN surgery by electrically stimulating 5-HT projections in the medial forebrain bundle (MFB), a more accessible anatomical location. MFB stimulation elicits 5-HT in the SNR; furthermore, we find simultaneous release of an additional species. We use electrochemical and pharmacological methods and describe physiological, anatomical and independent chemical analyses to identify this species as histamine. We also show pharmacologically that increasing the lifetime of extracellular histamine significantly decreases 5-HT release, most likely because of increased activation of histamine H-3 receptors that inhibit 5-HT release. Despite this, under physiological conditions, we find by kinetic comparisons of DRN and MFB stimulations that the simultaneous release of histamine does not interfere with the quantitative 5-HT concentration profile. We therefore present a novel and robust electrical stimulation of the MFB that is technically less challenging than DRN stimulation to study 5-HT and histamine release in the SNR.     

Serotonin modifies cytoskeleton and brush-border membrane architecture in human intestinal epithelial cells

Serotonin or 5-hydroxytryptamine (5-HT) influences numerous functions in the gastrointestinal tract. We previously demonstrated that 5-HT treatment of Caco-2 cells inhibited Na(+)/H(+) exchangers (NHE) and Cl(-)/OH(-) exchange activities via distinct signaling mechanisms. Since regulation of several ion transporters such as NHE3 is influenced by intact cytoskeleton, we hypothesized that 5-HT modifies actin cytoskeleton and/or brush-border membrane architecture via involvement of signaling pathways. Ultrastructural analysis showed that 5-HT (0.1 muM, 1 h) treatment of Caco-2 cells caused the apical membrane to assume a convex dome shape that was associated with shortening of microvilli. To examine whether these cellular architecture changes are cytoskeleton driven, we analyzed actin cytoskeleton by fluorescence microscopy. 5-HT induced basal stress fibers with prominent cortical actin filaments via 5-HT3 and 5-HT4 receptor subtypes. This induction was partially attenuated by chelation of intracellular Ca(2+) and PKCalpha inhibition (Go6976). In vitro assays revealed that PKCalpha interacted with actin and this association was increased by 5-HT. Our data provide novel evidence that 5-HT-induced signaling via 5-HT3/4 receptor subtypes to cause Ca(2+) and PKCalpha-dependent regulation of actin cytoskeleton may play an important role in modulation of ion transporters that contribute to pathophysiology of diarrheal conditions associated with elevated levels of 5-HT.     

Serotonin-induced stimulation of progesterone production by cow luteal cells in vitro

The addition of acetylcholine or histamine (10(-7) to 10(-4) M), gamma-aminobutyric acid, a dopamine agonist, and melatonin (10(-7) to 10(-5) M) did not alter basal or LH-stimulated progesterone production (P greater than 0.05). The addition of the specific beta 2-adrenergic agonist terbutaline and salbutamol did not significantly elevate progesterone production. Treatment of luteal cells with serotonin (5-HT), 10(-6) to 10(-4) M, increased the production of progesterone (P less than 0.05). This stimulated production was inhibited by the addition of mianserin (10(-5) M, a 5-HT antagonist; P less than 0.05). Isoproterenol (10(-7) to 10(-4) M) also resulted in significant increases in progesterone production (P less than 0.05). The combined treatments of 5-HT + LH, isoproterenol + LH, or isoproterenol + 5-HT did not result in a further increase in progesterone above that observed in response to LH or isoproterenol alone (P greater than 0.05). The isoproterenol-induced progesterone production could not be blocked by butoxamine (10(-5) M, a beta 2-antagonist), or practolol (10(-5) M, a beta 1-antagonist), but was inhibited by propranolol (10(-5) M, a general beta-antagonist; P less than 0.05). The response to isoproterenol was unaffected by mianserin (10(-5) M). These results demonstrate a possible role for 5-HT in the regulation of steroidogenesis by the corpus luteum of the cow. Furthermore, these results suggest that serotonin-induced progesterone production is a receptor-mediated event.