Stimulation of urine production by the Malpighian (renal) tubules in Rhodnius prolixus is regulated by at least two diuretic hormones, CRF-related peptide and serotonin, that have traditionally been believed to function through the activation of cAMP-mediated intracellular second messenger pathways. In this study I demonstrate that serotonin stimulation triggered, in addition to cAMP, intracellular Ca2+ waves in the Malpighian tubule cells of R. prolixus. Treatment with the intracellular Ca2+ chelator BAPTA-AM blocked the intracellular Ca2+ waves and reduced serotonin-stimulated fluid secretion by 75%. This suggests a role for intracellular Ca2+ signaling in the excretory system of R. prolixus. Serotonin stimulated Malpighian tubules (MTs) exposed to Ca2+-free saline plus BAPTA-AM secreted an abnormal fluid, showing: increased K+ concentration, reduced Na+ concentration and lower pH. These results along with measurement of transepithelial potential (TEP) suggest that the basolateral Na+:K+:2Cl- cotransporter (NKCC) activity is reduced in tubule cells treated with BAPTA-AM, suggesting that Ca2+ is required to modulate the activity of the basolateral NKCC. Treatment with the non-hydrolysable cell-permeable cAMP analog, 8Br-cAMP, produced fluid with the same K+ and Na+ concentration and at the same secretion rate as serotonin-stimulated tubules. In addition, 8Br-cAMP triggered intracellular Ca2+ oscillations similar to those obtained with serotonin. 8Br-cAMP-stimulated tubules treated with BAPTA-AM decreased their fluid secretion by about 40% and increased Na+ concentration, similar to the effect observed on serotonin-stimulated tubules. Therefore, I conclude that the intracellular Ca2+ waves triggered by serotonin are mediated by cAMP. The role of inositol-3-phospate (InsP3) in Ca2+ release was tested by treating the tubules with the InsP3 receptor blocker xestospongin. The treatment decreased fluid secretion rate as well as the amplitude of Ca2+ waves in serotonin-stimulated tubules. These results suggest that serotonin activates the production of InsP3 and, most likely, diacylglycerol (DAG). Thus, I decided to test whether the protein kinase C (PKC) may be involved in serotonin-stimulated secretion. The PKC inhibitors chelerythrine and bisindolylmaleimide (BIM) decreased secretion fluid rate in serotonin-stimulated tubules by 50% and 70%, respectively. Fluid secreted by tubules treated with BIM showed no differences in K+ and Na+ concentrations compared to controls, however both ion fluxes decreased. The evidence suggests that PKC is involved in serotonin stimulated secretion; the mechanism is still not understood. Taken together, the results suggest that cAMP, Ca2+ and PLC-PKC pathway are involved in serotonin stimulated secretion. However cAMP stimulation is enough for maximal secretion rate. Therefore PLC-PKC must act downstream of cAMP. Based on those results we hypothesize that serotonin binds a GPCR, increasing cAMP by activation of an adenylate cyclase (AC). Subsequently, cAMP is somehow able to activate PLC, which finally produces Ca2+ release, PKC activation and NKCC upregulation.

Na+/K+-ATPase has been shown to regulate the sweating and cutaneous vascular responses during exercise; however, similar studies have not been conducted to assess the roles of the Na-K-2Cl cotransporter (NKCC) and K+ channels. Additionally, it remains to be determined if these mechanisms underpinning the heat loss responses differ with exercise intensity. Eleven young (24±4 years) males performed three 30-min semi-recumbent cycling bouts at low (30% VO2peak), moderate (50% VO2peak), and high (70% VO2peak) intensity exercise, respectively, each separated by 20-min recovery periods. Using intradermal microdialysis, four forearm skin sites were continuously perfused with either: 1) lactated Ringer solution (Control), 2) 6 mᴍ ouabain (Na+/K+-ATPase inhibitor), 3) 10 mᴍ bumetanide (NKCC inhibitor), or 4) 50 mᴍ BaCl2 (non-specific K+ channel inhibitor); sites at which we assessed local sweat rate (LSR) and cutaneous vascular conductance (CVC). Inhibition of Na+/K+-ATPase attenuated LSR compared to Control during the moderate and high intensity exercise bouts (both P˂0.01), whereas attenuations with NKCC and K+ channel inhibition were only apparent during the high intensity exercise bout (both P≤0.05). Na+/K+-ATPase inhibition augmented CVC during all exercise intensities (all P˂0.01), whereas CVC was greater with NKCC inhibition during the low intensity exercise only (P˂0.01) and attenuated with K+ channel inhibition during the moderate and high intensity exercise conditions (both P˂0.01). We show that Na+/K+-ATPase, NKCC and K+ channels all contribute to the regulation of sweating and cutaneous blood flow but their influence is dependent on the intensity of exercise.

<p>Ammonia is the primary nitrogenous waste product in teleost fish, which is produced primarily through protein metabolism. Fish experience natural elevations in internal ammonia loads, including during digestion where luminal ammonia concentrations in the intestine rise substantially. Furthermore, the intestine may absorb a portion of this ammonia, despite it being toxic to the fish. Based on this, <em>in vitro </em>techniques were employed in order to develop a model for teleost intestinal ammonia handling.</p> <p>Ammonia absorption and endogenous ammonia production occur along the entire length of the intestine. However, section-specific differences exist in terms of both endogenously produced ammonia and ammonia flux rates, with the highest rates in the anterior and mid intestine. Feeding stimulated an increase in production rates in all intestinal sections. Overall, ammonia originating from the gut may account for up to 42% of post-prandial whole-fish ammonia excretion. This could partly be attributed to the increased activity of the ammonia-producing enzyme glutamate dehydrogenase, and decreased activity of the ammonia-fixing glutamine synthetase. Furthermore, gut tissue ammonia concentrations surpassed typical chyme concentrations and were well regulated independent of high luminal ammonia, suggesting active transport across the intestinal epithelium.</p> <p>Seawater (60%) acclimation caused no substantial changes in the ammonia handling properties of the intestine. Ammonia transport in the intestine of both freshwater and seawater trout appears to occur via active means, coupled to Na⁺/K⁺ ATPase activity. Specifically, this involves Na⁺ linked transport through substitution of NH₄⁺ for K⁺on the apical Na⁺/K⁺/2Cl^- co-transporter occurring predominantly in the anterior and mid intestine, and solvent drag through fluid transport (osmotically driven by active NaCl absorption) in all sections. Additionally, Rhesus glycoprotein mediated ammonia transport likely occurs through basolateral Rhbg1, supporting previous molecular evidence. Overall this thesis illuminates the quantitative importance and mechanisms of gut ammonia transport in fish, and highlights future research avenues.</p> / Master of Science (MSc)

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Objectifs: La pathophysiologie de la maladie de Ménière (MM) demeure mal comprise. Nous avons identifié dans la littérature un groupe de protéines exprimées sur le sac endolymphatique (SEL) et impliquées dans la régulation du volume endolymphatique : l’Aquaporine-2 (AQP2), le récepteur V2R de vasopressine (AVP), le Co-transporteur de Sodium Potassium et Chlorure type 2 (NKCC2) et le canal TRP type V4 (TRPV4). Notre objectif est de déterminer si leur expression sur le SEL est altérée dans la MM, pour améliorer notre compréhension de la physiologie de l’hydrops endolymphatique. Méthodes: Recrutement des cas de MM et schwannomes vestibulaires (SV) comme contrôles, le jour de leurs chirurgies respectives. Prélèvement de biopsies de SEL et sang pour AVP. L’immunohistochimie pour AQP2, V2R, NKCC2 et TRPV4 fut effectuée, et les lames scannées pour analyse digitale de densité d’expression par un logiciel spécialisé (VIS par Visiopharm®). Résultats: Total de 27 cas MM et 23 contrôles. Les scores générés par le logiciel représentent la densité d’expression totale et relative des protéines, exclusivement sur l’épithélium du SEL. Les scores d’AQP2 sont élevés de façon significative dans la MM comparée aux contrôles (p = 0.018). Nous ne rapportons aucune variation significative pour AVP, V2R, NKCC2 et TRPV4. Conclusion: Cette étude originale évalue l’expression simultanée de AQP2, V2R, NKCC2 et TRPV4 sur le SEL dans la MM, avec un groupe contrôle (SV). Nos résultats démontrent une augmentation isolée de l’AQP2 dans la MM. Nous proposons une surexpression constitutive de cette dernière, indépendante de son axe de régulation (AVP-V2R). Une mutation somatique au niveau des séquences régulatrices pourrait justifier nos observations. / Objectives: Endolymphatic sac (ELS) pathophysiology in Ménière’s Disease (MD) remains poorly understood. We identified from the literature a group of proteins expressed on the ELS and involved in endolymph volume regulation: Aquaporin-2 (AQP2), vasopressin receptor V2R, Sodium Potassium Chloride Cotransporter type 2 (NKCC2) and TRP channel type V4 (TRPV4). Our objective was to determine whether their ELS expression was altered in MD, to better understand the pathophysiology of endolymphatic hydrops. Methods: Patients with definite MD undergoing endolymphatic duct blockage surgery were recruited, as well as controls undergoing surgery for vestibular schwannomas (VS). ELS biopsies and blood samples for plasma Arginine Vasopressin (AVP) were obtained. Immunohistochemistry for AQP2, V2R, NKCC2 and TRPV4 was performed. Slides were scanned digitally for highly sensitive pixel density analysis by specialized software (VIS by Visiopharm®). Results: 27 definite MD patients and 23 VS controls were included. Global scores generated by the software represent total and relative protein expression density of 3 staining intensity levels, exclusively on ELS epithelium. AQP2 expression density was significantly elevated in MD compared to VS (p = 0.018). There was no significant difference in plasma AVP, V2R, NKCC2 and TRPV4 expression. Conclusion: This original study evaluates simultaneous in-situ expression of AQP2, V2R, NKCC2 and TRPV4 on the human ELS in MD, with a VS control group. Our results show only AQP2 up regulation on the ELS of MD patients. We suggest a constitutively increased expression of AQP2 in MD, independent of its regulatory axis (AVP-V2R). Acquired regulator sequence mutations could support this model.