Characterization of Na⁺-K⁺-2Cl⁻Cotransporter Activity in Rabbit Lacrimal Gland Duct Cells

Abstract

PurposeWe recently reported that isolated duct segments from rabbit lacrimal gland (LG) were able to secrete fluid in response to secretagogues, which were blocked completely by bumetanide. This suggests the functional involvement of Na+-K+-2Cl− cotransporter (NKCC1) in ductal fluid secretion. Therefore, the aim of this study was to investigate the activity profile of NKCC1 in isolated rabbit LG duct segments.MethodsInterlobular ducts were isolated from fresh rabbit LG tissue. Microfluorometry with the ammonium (NH4+)–pulse technique was used to elicit pH changes in duct cells, and the rate of bumetanide-sensitive cytosolic acidification after addition of NH4+ was used to quantify the activity of NKCC1.ResultsWhile basal activity of NKCC1 was undetectable, low cytosolic chloride (Cl−) level and hyperosmotic challenge (390 mOsm) were able to increase the activity of NKCC1. Carbachol (100 μM) had no significant effect on NKCC1 activity. Elevation of cytosolic calcium (Ca2+) level with Ca2+-ionophore (A 23187, 1 μM) did not cause any alteration in the activity of the cotransporter while direct activation of protein kinase C (phorbol myristate acetate, 100 nM) increased its activity slightly but in a significant manner. Addition of either forskolin (10 μM), cell-permeable cAMP analogue (8-bromo cAMP, 100 μM) or vasoactive intestinal peptide (200 nM) resulted in a significant increase in the activity of NKCC1.ConclusionsThese results highlight the functional involvement of NKCC1 in LG duct secretion. These findings may facilitate our understanding of LG function and may contribute to the development of targeted pharmacologic interventions in case of dry eye disease.

Patch-Clamp Recording of K+ Current from Lacrimal Gland Duct Cells

“…The Ca 2+ -dependent K + channel identified in this study probably corresponds to the IKCa1 channel that we have identified on the apical membranes of rat lacrimal gland ducts. 7 The presence of this channel, along with the apical KCC1 transporter and CFTR, which have been identified immunohistochemically and functionally, 7,9,10,15,16 are consistent with evidence that the ducts secrete K + . The location of muscarinic receptors on the basal membrane of the duct cells, 7 and the demonstration by Katona et al 17 that isolated ducts secrete fluid in response to carbachol, support a mechanism in which cholinergic stimulation causes release of intracellular calcium 18 which activates IKCa1 channels, resulting in secretion of fluid rich in K + .…”

“…7 The secretory properties of intact ducts have been described, and the data are consistent with the proposed model. [8][9][10] Electrophysiologic studies have characterized several K + and Cl-channels in lacrimal acinar cells, [11][12][13] however, due to their relative paucity and inaccessibility, the function of ion channels of duct cells has not been studied extensively. The goal of this project was to develop a method for isolation of viable, single duct cells and demonstrate their utility for patch-clamp recording by characterizing the properties of K + channels in these cells.…”

Role of the Cation-Chloride-Cotransporters in Cardiovascular Disease

“…The Delpire group reported similar results of both 86 Rb + and FluxOR methods [ 134 ]. Other techniques include the ammonium pulse technique which uses cardiac strips to measure the acidification rate [ 135 ]. A comparative analysis of measuring cation-chloride-cotransporter activity can be found in a review by Medina et al [ 131 ].…”

Role of the Cation-chloride-cotransporters in Cardiovascular Disease

“…The Delpire group reported similar results between 86 Rb + and FluxOR [115]. Other techniques include the ammonium pulse technique which uses cardiac strips to measure acidification rate [116]. A comparative analysis of measuring cation-chloridecotransporter activity can be found in a review by Medina et al [111].…”