Changes In Chloride Transport Research Articles

Effects of salt‐loading on supraoptic vasopressin neurones assessed by ClopHensorN chloride imaging

Salt-loading (SL) impairs GABAA inhibition of arginine vasopressin (AVP) neurones in the supraoptic nucleus (SON) of the hypothalamus. Based on previous studies, we hypothesised that SL activates tyrosine receptor kinase B (TrkB), down-regulating the activity of K+ /Cl- co-transporter2 (KCC2) and up-regulating Na+ /K+ /Cl- co-transporter1 (NKCC1). These changes in chloride transport would result in increased [Cl- ]i in SON AVP neurones. The study combined virally-mediated chloride imaging with ClopHensorN with a single-cell western blot analysis. An adeno-associated virus with ClopHensorN and a vasopressin promoter (AAV2-0VP1-ClopHensorN) was bilaterally injected in the SON of adult male Sprague-Dawley rats that were either euhydrated (Eu) or salt-loaded (SL) for 7days. Acutely dissociated SON neurones expressing ClopHensorN were tested for decreases or increases in [Cl- ]i in response to focal application of the GABAA agonist muscimol (100μmolL-1 ). SON AVP neurones from Eu rats showed muscimol-induced chloride influx (P<0.05;23/35). SON AVP neurones from SL rats either significantly increased chloride efflux (P<0.05;27/39) or did not change chloride flux (12/39). The SON AVP neurones that responded to muscimol appeared to be viable and expressed KCC2 and β-actin. Neurones that did not respond during chloride imaging did not show KCC2 and β-actin protein expression. The KCC2 antagonist (VU0240551,10μmolL-1 ) significantly blocked the chloride influx in cells from Eu rats but did not affect cells from SL rats. A NKCC1 antagonist (bumetanide,10μmolL-1 ) significantly blocked the chloride efflux in cells from SL rats but had no effect on cells from Eu rats. Blocking NKCC1 using bumetanide had less of an effect on the muscimol-induced Cl- influx in Eu rat neurones compared to the KCC2 antagonist. The TrkB antagonist (AnA-12) (50μmolL-1 ) and protein kinase inhibitor (K252a) (100nmolL-1 ) each significantly blocked chloride efflux in SON AVP neurones from SL rats. Salt-loading increases [Cl- ]i in SON AVP neurones via a TrKB-KCC2-NKCC1-dependent mechanism in rats.

Bioelectric effects of quinine on polarized airway epithelial cells

Quinine has been increasingly utilized as a placebo in cystic fibrosis (CF) clinical trials, including those leading to FDA approval of inhaled tobramycin, recent studies of anti-inflammatory aerosols such as glutathione, and clinical testing of hypertonic saline aerosols to augment mucous clearance. The drug effectively masks taste of experimental therapeutics, but could also confer changes in processes contributing to CF pathogenesis, including chloride secretion and paracellular ion permeability. In the Ussing chamber, concentrations of quinine (1 mg/ml) anticipated in the airways of CF subjects after aerosolization led to changes in chloride transport in Calu-3 (airway serous glandular) cell monolayers. Tissue resistance was significantly disrupted by the compound in both Calu-3 and primary airway epithelial cells in vitro. Lower doses of quinine (between 10 and 100 μg/ml) strongly inhibited the chloride secretory mechanism that utilizes CFTR, and forskolin activated I SC was reduced by approximately 24% and 44% in the presence of 10 and 100 μg/ml quinine, respectively. Our findings indicate that quinine disrupts airway epithelial functional integrity and blocks transepithelial chloride transport. The use of quinine as a taste-masking agent may have bioelectric effects relevant to CF trials using aerosolized drug delivery.

Cationic lipid-mediated CFTR gene transfer to the lungs and nose of patients with cystic fibrosis: a double-blind placebo-controlled trial

We and others have previously reported significant changes in chloride transport after cationic-lipid-mediated transfer of the cystic fibrosis transmembrane conductance regulator (CFTR) gene to the nasal epithelium of patients with cystic fibrosis. We studied the safety and efficacy of this gene transfer to the lungs and nose of patients with cystic fibrosis in a double-blind placebo-controlled trial. Eight patients with cystic fibrosis were randomly assigned DNA-lipid complex (active) by nebulisation into the lungs followed 1 week later by administration to the nose. Eight control patients followed the same protocol but with the lipid alone (placebo). Safety was assessed clinically, by radiography, by pulmonary function, by induced sputum, and by histological analysis. Efficacy was assessed by analysis of vector-specific CFTR DNA and mRNA, in-vivo potential difference, epifluorescence assay of chloride efflux, and bacterial adherence. Seven of the eight patients receiving the active complex reported mild influenza-like symptoms that resolved within 36 h. Six of eight patients in both the active and placebo groups reported mild airway symptoms over a period of 12 h following pulmonary administration. No specific treatment was required for either event. Pulmonary administration resulted in a significant (p<0.05) degree of correction of the chloride abnormality in the patients receiving active treatment but not in those on placebo when assessed by in-vivo potential difference and chloride efflux. Bacterial adherence was also reduced. We detected no alterations in the sodium transport abnormality. A similar pattern occurred following nasal administration. Cationic-lipid-mediated CFTR gene transfer can significantly influence the underlying chloride defect in the lungs of patients with cystic fibrosis.

Recovery of Chloride Uptake in Sea Wateradapted Rainbow Trout (Salmo Gairdneri) after Transfer to Fresh Water

ABSTRACT The ability of rainbow trout (Salmo gairdneri Richardson) to acclimate to sea water or partially diluted sea water has been studied many times (Houston, 1959; Parry, 1960; Eddy and Bath, 1979; Prunet and Boeuf, 1985). However, the reverse transition from sea water (SW) to fresh water (FW) has been less well studied. The transition from a hyperosmotic medium to a hypo-osmotic medium reverses the burden on the osmoregulatory organs, i.e. the gills and the kidney. SW teleost fish normally show net salt (NaCl) efflux, whereas FW fish need to acquire salt from the medium. Recent work on salmon (Salmo salar) transferred from SW to FW (Talbot and Potts, 1989; Talbot et al. 1989; Potts et al. 1989) has described the changes in sodium balance, osmoregulation and renal function in adult and juvenile fish. Potts et al. (1985) showed that salmon are capable of taking up sodium immediately on transfer to FW. Chloride uptake via the gills of FW fish is of equal importance to sodium uptake, and the two ions are thought to be transported independently (Maetz and Garcia-Romeu, 1964). This paper examines the changes in chloride transport that occur during the transition from SW to FW.

Methylprednisolone stimulation of guanylate cyclase activity in rat small intestinal mucosa: Possible role in electrolyte transport

A single, intramuscular dose of the glucocorticoid methylprednisolone (3 mg/100 g) significantly increased rat jejunal and ileal mucosal guanylate cyclase (GTP pyrophosphatelyase [cyclizing]; EC 4.6.1.2) activity and cyclic GMP concentration 6 h after treatment. This increase in guanylate cyclase activity was maximal 6 h after injection, was not associated with changes in mucosal cyclic AMP concentration, and occurred before any increase in NaK-activated adenosine triphosphatase activity could be detected. In contrast to the findings in the small intestine, no alterations in guanylate cyclase activity were present in the rat colon 6 or 72 h after methylprednisolone treatment. The increase in ileal guanylate cyclase activity and cGMP concentration coincided with increases in ileal short-circuit current, potential difference, net Cl secretion, and serosal-to-mucosal unidirectional Cl flux. In contrast, in the colon, where no changes in guanylate cyclase activity were observed, methylprednisolone did not alter electrolyte transport in vitro. A similar correlation was observed between various methylprednisolone doses, the increase in guanylate cyclase activity and the alteration in ileal short-circuit current and net Cl transport. The methylprednisoloneinduced increase in ileal guanylate cyclase activity was not prevented by pretreatment with the mineralocorticoid competitive inhibitor spironolactone, nor did the mineralocorticoid deoxycorticosterone acetate (0.5 mg/100 g) affect ileal or colonic mucosal guanylate cyclase activity. In addition, isolated enterocytes from methylprednisolone-treated animals exhibited significant increases in guanylate cyclase activity with the greatest percentage increase occurring in the crypt cell population. These results suggest that the changes in chloride transport which occurred in the small intestine 6 h after methylprednisolone administration may be due to alterations in guanylate cyclase activity, and further support the role of cGMP as an intracellular mediator of ileal chloride secretion.