Beta-adrenergic upregulation of the Na(+)-K(+)-2Cl- cotransporter in rat parotid acinar cells.

Abstract

We used the pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5(6')-carboxyfluorescein to monitor the recovery of the intracellular pH (pH,) of rat parotid acini from an NH+-induced alkaline load. This recovery was markedly inhibited by the loop diuretic bumetanide and by Cl-removal, indicating that it is largely due to NH' entry via the basolateral Na'-K+-2Cl-cotransporter. The rate of recovery of pHi was enhanced threefold by pretreatment (37.5 s) with isoproterenol (K112 = 21.5 nM) or norepinephrine (in the presence of phentolamine), and blocked by the #,-specific antagonist atenolol, indicating an upregulation of cotransport activity by ,81-adrenergic stimulation. The effect of isoproterenol was prevented by protein kinase inhibitors and mimicked by cAMP analogues, and by maneuvers known to increase cytosolic cAMP levels in these cells, consistent with the involvement ofprotein kinase A. Physiologically, such an upregulation of the acinar Na'-K+-2CI-cotransporter would lead to an increase in acinar chloride uptake across the basolateral membrane, and consequently, an increase in overall chloride and fluid secretion. Prevention of this upregulation by t-blockers and possibly by other commonly used clinical agents may account for the dry mouth and dry eyes experienced by some patients taking these medications. (J.

A fluid secretion pathway unmasked by acinar-specific Tmem16A gene ablation in the adult mouse salivary gland

“…The DCPIB and NPPB sensitivity of the IPR-induced secretion and the acinar cell swelling observed on IPR treatment suggest that VRAC might be involved in this secretory process. Although the importance of these latter observations will require further investigation, it can be speculated that IPR activates basolateral Na + /K + /2Cl − cotransporters (36,37), causing solute accumulation and cell swelling. Such swelling would be expected to activate the cell volume-sensitive Cl − channels present in acinar cells (21) and to provide an apical Cl − efflux pathway important for β-adrenergic, cAMP-stimulated fluid secretion.…”

A Highly Conserved Motif at the COOH Terminus Dictates Endoplasmic Reticulum Exit and Cell Surface Expression of NKCC2

“…Calibration of the 2Ј,7Ј-bis(carboxyethyl)-5,6-carboxyfluorescein excitation ratio was accomplished using the nigericin technique. Na-K-2Cl cotransport activity was measured as bumetanide-sensitive NH 4 influx, as previously described (23,24). Cells were first bathed at 37°C in a CO 2 -free Hepes/Tris-buffered medium containing 135 mM NaCl, 10 mM Hepes (pH 7.4), 5 mM KCl, 1 mM MgCl 2 , 0.8 mM KH 2 PO 4 , 0.2 mM K 2 HOP 4 , 1 mM CaCl 2 , and 10 mM BaCl 2 to measure base-line pH i .…”

“…Ϫ transport activity was assessed by estimating the rate of intracellular acidification caused by entry into the cells of NH 4 ϩ via this transport mechanism after abrupt application of NH 4 Cl to the cells (23,24). As illustrated in Fig.…”

NKCC2 Surface Expression in Mammalian Cells

“…To address this, we first verified whether Myc-NKCC2 fusion protein expressed at the cell plasma membrane is functional. Na-K ϩ (NH 4 ϩ )-2Cl transport activity was assessed by estimating the rate of intracellular acidification caused by entry into the cells of NH 4 ϩ via this transport mechanism after abrupt application of 20 mM NH 4 Cl to the cells (31,32,59). Importantly, previous studies conducted in MTAL (31,32) and OK cells (60) clearly demonstrated that neither Na/H (NH 4 ϩ ) exchanger nor Na-K(NH 4 ϩ )-ATPase are involved in the NH 4 ϩ -induced intracellular acidification observed under these experimental conditions.…”