Cellular Mechanisms of Carbachol-Stimulated Cl− Secretion in Rat Epididymal Epithelium1

Abstract

Neurotransmitter-controlled Cl- secretions play an important role in maintenance of the epididymal microenvironment for sperm maturation. This study was carried out to investigate the effect of carbachol (CCH) on the cultured rat epididymal epithelium and the signal transduction mechanisms of this response. In normal K-H solution, CCH added basolaterally elicited a biphasic Isc response consisting of a transient spike followed by a second sustained response. Ca2+ activated Cl- channel blocker disulfonic acid stilbene (DIDS, 300 microM) only inhibited part of the CCH-induced Isc response, while nonselective Cl- channel blocker diphenylamine-dicarboxylic acid (DPC, 1 mM) reduced all, indicating the involvement of different conductance pathways. Both peaks of the CCH-induced Isc response could be significantly inhibited by pretreatment with an adenylate cyclase inhibitor, MDL12330A (50 microM). An increase in intracellular cAMP content upon stimulation of CCH was measured. All of the initial peak and part of the second peak could be inhibited by pretreatment with Ca2+-chelating agent BAPTA/AM (50 microM) and an endoplasmic reticulum Ca2+ pump inhibitor, Thapsigagin (Tg, 1 microM). In a whole-cell patch clamp experiment, CCH induced an inward current in the single cell. Two different profiles of currents were found; the first component current exhibited an outward rectifying I-V relationship in a time and voltage-dependent manner, and the current followed showed a linear I-V relationship. The carbachol-induced current was found to be partially blockable by DIDS and could be completely blocked by DPC. The above results indicate that the CCH-induced Cl- secretion could be mediated by Ca2+ and cAMP-dependent regulatory pathways.

Listeriolysin O affects barrier function and induces chloride secretion in HT-29/B6 colon epithelial cells

“…Experimental shortcomings can be ruled out, since BAPTA did partially inhibit the LLO-stimulated increase in intracellular Ca 2ϩ concentration in FURA-2 experiments and, likewise, partially inhibited a caffeine triggered I SC response in Ussing experiments. Similar observations of BAPTA only partially inhibiting Ca 2ϩ -mediated epithelial Cl Ϫ secretion have been reported before (19), and it has been suggested that BAPTA may be locally "overcharged" by rapidly increasing Ca 2ϩ concentrations in microdomains (7,49). Furthermore, BAPTA-inaccessible sites were reported in muscle cells (52).…”

Infection by Toxoplasma gondii , a severe parasite in neonates and AIDS patients, causes impaired anion secretion in airway epithelia

“…Mice were anesthetized using CO 2 , and the proximal tracheas were dissected out and put in K-H solution. After carefully removing the blood vessels and connective tissue, the trachea was longitudinally cut open with fine scissors under a stereo microscope and clamped between the two halves of an Ussing Chamber as previously described (49).…”

Sodium Coupled Bicarbonate Influx Regulates Intracellular and Apical pH in Cultured Rat Caput Epididymal Epithelium

“…NBC, a Na + dependent bicarbonate transporter, may mediate the acidification recovery. With low concentration, DIDS serve as the inhibitor of some anion channels, such as Ca 2+ - sensitive [31] and volume-sensitive Cl − channels [32]. While in high concentration, DIDS is always set as the inhibitor of HCO 3 − transporter, including Na + /HCO 3 − cotransporter [33], [34] and AE [35].…”