Localization of Na+–K+-ATPase α/β, Na+–K+–2Cl-cotransporter 1 and aquaporin-5 in human eccrine sweat glands

In order to evaluate the function of the repaired or regenerated eccrine sweat glands, we must first localize the proteins involved in sweat secretion and absorption in normal human eccrine sweat glands. In our studies, the cellular localization of Na⁺–K⁺-ATPase α/β, Na⁺–K⁺–2Cl-cotransporter 1 (NKCC1) and aquaporin-5 (AQP5) in eccrine sweat glands were detected by immunoperoxidase labeling. The results showed that Na⁺–K⁺-ATPase α was immunolocalized in the cell membrane of the basal layer and suprabasal layer cells of the epidermis, the basolateral membrane of the secretory coils, and the cell membrane of the outer cells and the basolateral membrane of the luminal cells of the ducts. The localization of Na⁺–K⁺-ATPase β in the secretory coils was the same as Na⁺–K⁺-ATPase α, but Na⁺–K⁺-ATPase β labeling was absent in the straight ducts and epidermis. NKCC1 labeling was seen only in the basolateral membrane of the secretory coils. AQP5 was strongly localized in the apical membrane and weakly localized in the cytoplasm of secretory epithelial cells. The different distribution of these proteins in eccrine sweat glands was related to their functions in sweat secretion and absorption.     

Bicarbonate secretion by rat bile duct brush cells indicated by immunohistochemical localization of CFTR, anion exchanger AE2, Na+/HCO3 − cotransporter, carbonic anhydrase II, Na+/H+ exchangers NHE1 and NHE3, H+/K+-ATPase, and Na+/K+-ATPase

The function of brush cells (BCs) is unknown. In a previous study, the rat common bile duct was examined by ultrastructural cytochemical methods for localizing HCO₃⁻, Cl⁻, and Na⁺ ions. All ion precipitates increased in or on BCs after secretin or meal stimulation, and it was proposed that BCs may secrete NaHCO₃. In this study, immunohistochemical localization of proteins known to be important in HCO₃⁻ secretion was investigated in the rat common bile duct. Immunoreactivity of proteins involved in Cl⁻/HCO₃⁻ exchange reaction, cystic fibrosis transmembrane conductance regulator (CFTR) and Cl⁻/HCO₃⁻ exchanger (AE2), was found on the microvilli (MV) and along the basolateral membrane (BLM) of BCs. The proteins involved in HCO₃⁻ production, Na⁺/HCO₃⁻ cotransporter (NBC), was found along the BLM but was absent on the MV, whereas carbonic anhydrase II (CA II) was observed on the MV and along the BLM. Of proteins responsible for the extrusion of H⁺, Na⁺/H⁺ exchanger 1 (NHE1) was localized along the BLM whereas Na⁺/H⁺ exchanger 3 (NHE3) was found on the MV and BLM. Activity of H⁺/K⁺-ATPase was found along the BLM and on the MV, and Na⁺/K⁺-ATPase was localized along the BLM. The immunoreactivity of most of these proteins was absent or weak in principal cells. These results strongly suggest that BCs are a significant source of HCO₃⁻ secretion.     

The cellular localization of Na+/H+ exchanger 1, cystic fibrosis transmembrane conductance regulator,potassium channel,epithelial sodium channel γ and vacuolar-type H+-ATPase in human eccrine sweat glands

The secretory portions of human eccrine sweat glands secrete isotonic fluid into the lumen and then the primary fluid is rendered hypotonic during its passage to the skin surface. During the processes of sweat secretion and absorption, many enzymes and proteins play important roles. In the study, the cellular localizations of Na⁺/H⁺ exchanger 1 (NHE1), cystic fibrosis transmembrane conductance regulator (CFTR), potassium channel (KC), epithelial sodium channel γ (γENaC) and vacuolar-type H+-ATPase (V-ATPase) in human eccrine sweat glands and epidermis were detected using immunofluorescence labeling. The results revealed that in the secretory coils, the basolateral membranes showed evidence of CFTR, NHE1 and KC activities, the apical membranes showed the activities of KC and NHE1, and the nucleus showed γEaNC and V-ATPase activities; in the duct, the peripheral and luminal ductal cells showed evidence of CFTR, NHE1 and KC, the apical membranes showed the activities of CFTR and NHE1, and the nucleus showed γEaNC, V-ATPase and KC activities. The cellular localization of these proteins in eccrine sweat glands is helpful to better understand the mechanisms of sweat secretion and absorption.     

Time resolved kinetics of the guinea pig Na–Ca exchanger (NCX1) expressed in Xenopus oocytes: voltage and Ca2+ dependence of pre-steady-state current investigated by photolytic Ca2+concentration jumps

Kinetic properties of the Na–Ca exchanger (guinea pig NCX1) expressed in Xenopus oocytes were investigated by patch clamp techniques and photolytic Ca²⁺ concentration jumps. Current measured in oocyte membranes expressing NCX1 is almost indistinguishable from current measured in patches derived from cardiac myocytes. In the Ca–Ca exchange mode, a transient inward current is observed, whereas in the Na–Ca exchange mode, current either rises to a plateau, or at higher Ca²⁺ concentration jumps, an initial transient is followed by a plateau. No comparable current was observed in membrane patches not expressing NCX1, indicating that photolytic Ca²⁺ concentrations jumps activate Na–Ca exchange current. Electrical currents generated by NCX1 expressed in Xenopus oocytes are about four times larger than those obtained from cardiac myocyte membranes enabling current recording with smaller concentration jumps and/or higher time resolution. The apparent affinity for Ca²⁺ of nonstationary exchange currents (0.1 mM) is much lower than that of stationary currents (6 μM). Measurement of the Ca²⁺ dependence of the rising phase provides direct evidence that the association rate constant for Ca²⁺ is about 5 × 10⁸ M⁻¹ s⁻¹ and voltage independent. In both transport modes, the transient current decays with a voltage independent but Ca²⁺-dependent rate constant, which is about 9,000 s⁻¹ at saturating Ca²⁺ concentrations. The voltage independence of this relaxation is maintained for Ca²⁺ concentrations far below saturation. In the Ca–Ca exchange mode, the amount of charge translocated after a concentration jump is independent of the magnitude of the jump but voltage dependent, increasing at negative voltages. The slope of the charge–voltage relation is independent of the Ca²⁺ concentration. Major conclusions are: (1) Photolytic Ca²⁺ concentration jumps generate current related to NCX1. (2) The dissociation constant for Ca²⁺ at the cytoplasmic transport binding site is about 0.1 mM. (3) The association rate constant of Ca²⁺ at the cytoplasmic transport sites is high (5 × 10⁻⁸ M⁻¹s⁻¹) and voltage independent. (4) The minimal five-state model (voltage independent binding reactions, one voltage independent conformational transition and one very fast voltage dependent conformational transition) used before to describe Ca²⁺ translocation at saturating Ca²⁺ concentrations is valid for Ca²⁺ concentrations far below saturation.