Visceral hypersensitivity in female but not in male serotonin transporter knockout rats.

BACKGROUND: Visceral hypersensitivity occurs in irritable bowel syndrome (IBS), particularly in women. Serotonin signaling, including reduced serotonin transporter (SERT) expression, may be disrupted in IBS patients. We studied SERT gene knockout (KO) rats to determine if they exhibited sex-related alterations in visceral sensitivity. METHODS: We measured serotonin in the colonic mucosa using HPLC and amperometric microelectrode techniques. Visceral sensitivity was assessed using the electromyographic visceromotor response (VMR) in response to colorectal balloon distention (CRD). We studied the electrophysiologic properties of colon projecting sensory neurons in vitro using whole-cell recordings. KEY RESULTS: Mucosal serotonin levels were not different among male and female WT and SERT KO rats. Serotonin oxidation currents in vitro were larger (P < 0.05) in tissues from male and female SERT KO compared with WT rats. Oxidation currents in male and female WT, but not SERT KO, rats were increased (P < 0.05) by the SERT inhibitor fluoxetine (1 µmol L(-1) ). The VMR to CRD was increased in female but not in male SERT KO rats (P < 0.05); this response varied with the estrous cycle. Colon projecting sensory neurons from female SERT KO rats fired more action potentials compared with neurons from female WT rats. There were no differences in action potential firing in neurons from male WT and SERT KO rats. CONCLUSIONS & INFERENCES: Increased colonic extracellular serotonin in female SERT KO rats is associated with visceral hypersensitivity and hyperexcitability of colon projecting sensory neurons. The SERT KO rat is a model for studying interactions between serotonin, sex and visceral sensation.

Differential serotonin transport is linked to the rh5-HTTLPR in peripheral blood cells.

The human serotonin transporter (SERT) gene possesses a 43-base pair (bp) insertion-deletion promoter polymorphism, the h5-HTTLPR. Genotype at this locus correlates with variation in anxiety-related personality traits and risk for major depressive disorder in many studies. Yet, the complex effects of the h5-HTTLPR, in combination with closely associated single-nucleotide polymorphisms (SNPs), continue to be debated. Moreover, although SERT is of high clinical significance, transporter function in vivo remains difficult to assess. Rhesus express a promoter polymorphism related to the h5-HTTLPR. The rh5-HTTLPR has been linked to differences in stress-related behavior and cognitive flexibility, although allelic variations in serotonin uptake have not been investigated. We studied the serotonin system as it relates to the 5-HTTLPR in rhesus peripheral blood cells. Sequencing of the rh5-HTTLPR revealed a 23-bp insertion, which is somewhat longer than originally reported. Consistent with previous reports, no SNPs in the rh5-HTTLPR and surrounding genomic regions were detected in the individuals studied. Reductions in serotonin uptake rates, cell surface SERT binding, and 5-hydroxyindoleacetic acid/serotonin ratios, but not SERT mRNA levels, were associated with the rh5-HTTLPR short allele. Thus, serotonin uptake rates are differentiable with respect to the 5-HTTLPR in an easily accessible native peripheral tissue. In light of these findings, we foresee that primary blood cells, in combination with high sensitivity functional measurements enabled by chronoamperometry, will be important for investigating alterations in serotonin uptake associated with genetic variability and antidepressant responsiveness in humans.

Postnatal development of the serotonin signaling system in the mucosa of the guinea pig ileum.

BACKGROUND: Serotonin is an important neurohumoral molecule in the gut but its signaling system is not fully developed in the neonatal gastrointestinal (GI) tract. This study aimed to evaluate the postnatal maturation of serotonin signaling in the small intestine. METHODS: In vitro amperometry for real-time measurement of serotonin at the mucosal surface, immunoblot, immunohistochemistry and high-performance liquid chromatography (HPLC) were used to examine serotonin handling in ileal segments from guinea pigs of different ages. KEY RESULTS: Extracellular serotonin levels significantly declined over the first three postnatal weeks, after which the levels increased and reached their maximum at 9 weeks postnatally. Serotonin levels were insensitive to the inhibition of the serotonin transporter (SERT) until the animals reached 3 weeks old. Measurement of serotonin and its metabolite 5-hydroxyindole acetic acid (5-HIAA) in the mucosa revealed that the serotonin turnover was significantly lower in neonates. Immunoblot and immunohistochemistry showed that SERT expression was extremely low in the neonatal period. Serotonin staining in cross-section showed that enterochromaffin (EC) cells were preferentially localized in the crypt region in neonates and the number of EC cells was significantly higher in 9-week-old animals. CONCLUSIONS & INFERENCES: SERT expression is low in the neonatal intestine and serotonin signaling matures postnatally. Extracellular serotonin levels decrease during the first three neonatal weeks as SERT expression increases. Extracellular serotonin levels increase after 3 weeks (weaning) possibly due to an increase in EC cell numbers. Postnatal maturation of serotonin signaling coincides with dietary changes in the developing guinea pig.

Inhibitory neuromuscular transmission to ileal longitudinal muscle predominates in neonatal guinea pigs.

BACKGROUND: Inhibitory neurotransmission to the longitudinal muscle is more prominent in the neonatal than in the adult guinea pig ileum. METHODS: Inhibitory neuromuscular transmission was investigated using in vitro ileal longitudinal muscle myenteric plexus (LMMP) preparations made from neonatal (< or =48 h postnatal) and adult ( approximately 4 weeks postnatal) guinea pigs. KEY RESULTS: Amperometric measurements of nicotine-induced nitric oxide (NO) release (measured as an oxidation current) from myenteric ganglia revealed larger currents in neonatal (379 +/- 24 pA) vs adult (119 +/- 39 pA, P < 0.05) tissues. Nicotine-induced oxidation currents were blocked by the nitric oxide synthase (NOS) inhibitor, nitro-l-arginine (NLA, 100 micromol L(-1)). Nicotine-induced, NLA-sensitive oxidation currents could be detected in the tertiary plexus of neonatal but not adult tissues. Immunohistochemistry demonstrated stronger NOS immunoreactivity in neonatal compared with adult myenteric ganglia. Western blot studies revealed higher levels of NOS in neonatal compared with adult LMMP. Cell counts revealed that the total number of myenteric neurons in the small intestine was greater in adults than in neonatal guinea pigs, however, the ratio of NOS : Calbindin neurons was significantly higher in neonatal compared with adult tissues. CONCLUSIONS & INFERENCES: Nitric oxide signaling to the longitudinal muscle is stronger in neonatal compared with adult guinea pig ileum. Nitric oxide synthase-containing neurons are diluted postnatally by cholinergic and other, as yet unidentified neuronal subtypes.

Antioxidant treatment restores prejunctional regulation of purinergic transmission in mesenteric arteries of deoxycorticosterone acetate-salt hypertensive rats.

Norepinephrine (NE) and ATP are co-released by periarterial sympathetic nerves. In mesenteric arteries (MA) from deoxycorticosterone-acetate (DOCA)-salt hypertensive rats, ATP, but not norepinephrine, release is impaired suggesting that their release may be regulated differently. We tested the hypothesis that different calcium channels contribute to ATP and norepinephrine release from sympathetic nerves in vitro in MA from normotensive and DOCA-salt hypertensive rats and that oxidative stress disrupts prejunctional regulation of co-transmission. Excitatory junction potentials (EJPs) were used to measure ATP release. Norepinephrine release was measured amperometrically with carbon-fiber microelectrodes. CdCl2 (30 microM) inhibited norepinephrine release in sham and DOCA-salt arteries by 78% and 85%, respectively. The N-type calcium channel antagonist, omega-conotoxin GVIA (CTX, 0.1 microM) inhibited norepinephrine release by 50% and 67% in normotensive and DOCA-salt arteries, respectively while CTX blocked EJPs. The P/Q-type calcium channel antagonist omega-agatoxin IVA (ATX; 0.03 microM) reduced norepinephrine release in sham but not DOCA-salt arteries and increased EJPs in sham but not DOCA-salt arteries. ATX did not increase EJPs in sham arteries in the presence of the alpha(2)-adrenergic receptor antagonist, yohimbine (1 microM). alpha(2)-Autoreceptor-sensitive EJP facilitation is impaired in DOCA-salt hypertension but this response is restored in DOCA-salt rats treated chronically with the antioxidant, apocynin. Apocynin restored alpha(2)-autoreceptor regulation of norepinephrine release. We conclude that ATP released from periarterial sympathetic nerves is controlled directly by N-type calcium channels. Norepinephrine release is controlled by N and P/Q type calcium channels. Norepinephrine release controlled by P/Q channels acts at alpha(2)-adrenergic receptors to inhibit norepinephrine release suggesting that there may be multiple pools of norepinephrine in periarterial sympathetic nerves. Regulation of norepinephrine release by alpha(2)-autoreceptors and P/Q-type channels is impaired in DOCA-salt hypertension and alpha(2)-autoreceptor function is disrupted by oxidative stress.

Electrochemical monitoring of nitric oxide released by myenteric neurons of the guinea pig ileum.

Nitric oxide (NO) released by myenteric neurons in isolated segments of guinea pig ileum was monitored in vitro using continuous amperometry. NO was detected as an oxidation current recorded with a boron-doped diamond microelectrode held at 1 V vs a Ag|AgCl reference electrode. This potential was sufficient to oxidize NO. Longitudinal muscle-myenteric plexus (LMMP) and circular muscle strip preparations were used. In the LMMP preparation, NO release was evoked by superfusion of 1 mumol L(-1) nicotine, which activates nicotinic acetylcholine receptors expressed by myenteric neurons and myenteric nerve endings. The oxidation current was ascribed to NO based on the following observations: (i) no response was detected at less positive potentials (0.75 V) at which only catecholamines and biogenic amines are oxidized, (ii) the current was abolished in the presence of the nitric oxide synthase antagonist, N-nitro-l-arginine (l-NNA) and (iii) oxidation currents were attenuated by addition of the NO scavenger, myoglobin, to the superfusing solution. In the LMMP preparation, stimulated release produced a maximum current that corresponded nominally to 46 nmol L(-1) of NO. The oxidation currents decreased to 10 and 2 nmol L(-1), respectively, when the tissue was perfused with tetrodotoxin and l-NNA. Oxidation currents recorded from circular muscle strips (stimulated using nicotine) were threefold larger than those recorded from the LMMP. This study shows that NO release can be detected from various in vitro preparations of the guinea pig ileum using real-time electroanalytical techniques.

Diamond optically transparent electrodes: demonstration of concept with ferri/ferrocyanide and methyl viologen.

A new type of optically transparent electrode is reported on--an electrically conductive diamond thin film. The electrode was free-standing (0.38 mm thick and 8 mm in diameter), mechanically polished to a 7-nm rms roughness over a 10-microm linear distance, boron-doped (0.05% B/C in the reactant gas mixture), and mounted in a thinlayer transmission cell. The electrode has a short-wavelength cutoff of approximately 225 nm, which is the indirect band gap of the material, and transmits light out to at least 1,000 nm. In theory, the electrode has an optical window from 225 nm well out into the far-infrared, except for the boron acceptor band and the intrinsic multiphonon absorptions. The electrode was used to electrooxidize ferrocyanide to ferricyanide, and the absorbance change associated with the formation of the oxidized product (lambdamax = 420 nm) was spectroscopically monitored. The electrode was also used to electroreduce methyl viologen (MV2+) to the cation radical (MV+*) and the neutral (MV0). The depletion of MV2+ (lambdamax = 257 nm) and formation of MV+* (lambdama = 398 and 605 nm) were spectroscopically monitored.

Anthraquinonedisulfonate electrochemistry: a comparison of glassy carbon, hydrogenated glassy carbon, highly oriented pyrolytic graphite, and diamond electrodes.

The electrochemistry of anthraquinone-2,6-disulfonate (2,6-AQDS) at glassy carbon (GC), hydrogenated glassy carbon (HGC), the basal plane of highly oriented pyrolytic graphite (HOPG), and boron-doped diamond was investigated by cyclic voltammetry and chronocoulometry. Quantitative determination of the surface coverage and qualitative assessment of the physisorption strength of 2,6-AQDS adsorption on each of these electrodes were done. The diamond and HGC surfaces are nonpolar and relatively oxygen-free, with the surface carbon atoms terminated by hydrogen. The polar 2,6-AQDS does not adsorb on these surfaces, and the electrolysis proceeds by a diffusion-controlled reaction. Conversely, the GC and HOPG surfaces are polar, with the exposed defect sites terminated by carbon-oxygen functionalities. 2,6-AQDS strongly physisorbs on both of these surfaces at near monolayer or greater coverages, such that the electrolysis proceeds through a surface-confined state. Less than 40% of the initial surface coverage can be removed by rinsing and solution replacement, reflective of strong physisorption. The results show the important role of the surface carbon-oxygen functionalities in promoting strong dipole-dipole and ion-dipole interactions with polar and ionic molecules such as 2,6-AQDS. The results also support the theory that diamond electrodes may be less subject to fouling by polar adsorbates, as compared to GC, leading to improved response stability in electroanalytical measurements. The relationship between the 2,6-AQDS surface coverage, the double-layer capacitance, and the heterogeneous electron-transfer rate constant for Fe(CN)(6)(3)(-)(/4)(-) for these four carbon electrodes is presented.

Enhanced Signal-to-Background Ratios in Voltammetric Measurements Made at Diamond Thin-Film Electrochemical Interfaces.

Large signal-to-background (S/B) ratios for the Fe(CN)(6)(3)(-)(/4)(-) and IrCl(6)(2)(-)(/3)(-) redox couples in KCl have been observed in cyclic voltammetric measurements made at a conductive diamond thin-film electrode without any conventional surface pretreatment. The S/B ratios were a factor of ∼16 and 8 larger at diamond than at freshly polished glassy carbon (GC) for Fe(CN)(6)(3)(-)(/4)(-) and IrCl(6)(2)(-)(/3)(-), respectively. The polycrystalline diamond film, grown on a p-Si(100) substrate, possessed significant cubic {100} faceting, as evidenced by AFM images, and was of high quality, as indicated by Raman spectroscopy. The high degree of electrochemical activity without surface pretreatment, the enhanced S/B ratios, and the excellent response stability demonstrate that diamond might be an attractive new electrode material for electroanalysis.