Severe Impairment of Salivation in Na+/K+/2Cl− Cotransporter (NKCC1)-deficient Mice

Abstract

The salivary fluid secretory mechanism is thought to require Na⁺/K⁺/2Cl⁻cotransporter-mediated Cl⁻ uptake. To directly test this possibility we studied the <i>in vivo</i> and <i>in vitro</i>functioning of acinar cells from the parotid glands of mice with targeted disruption of Na⁺/K⁺/2Cl⁻cotransporter isoform 1 (<i>Nkcc1</i>), the gene encoding the salivary Na⁺/K⁺/2Cl⁻cotransporter. In wild-type mice NKCC1 was localized to the basolateral membranes of parotid acinar cells, whereas expression was not detected in duct cells. The lack of functional NKCC1 resulted in a dramatic reduction (>60%) in the volume of saliva secreted in response to a muscarinic agonist, the primary <i>in situ</i> salivation signal. Consistent with defective Cl⁻ uptake, a loss of bumetanide-sensitive Cl⁻ influx was observed in parotid acinar cells from mice lacking NKCC1. Cl⁻/ HCO₃⁻exchanger activity was increased in parotid acinar cells isolated from knockout mice suggesting that the residual saliva secreted by mice lacking NKCC1 is associated with anion exchanger-dependent Cl⁻ uptake. Indeed, expression of the Cl⁻/ HCO₃⁻exchanger AE2 was enhanced suggesting that this transporter compensates for the loss of functional Na⁺/K⁺/2Cl⁻ cotransporter. Furthermore, the ability of the parotid gland to conserve NaCl was abolished in NKCC1-deficient mice. This deficit was not associated with changes in the morphology of the ducts, but transcript levels for the α-, β-, and γ-subunits of the epithelial Na⁺channel were reduced. These data directly demonstrate that NKCC1 is the major Cl⁻ uptake mechanism across the basolateral membrane of acinar cells and is critical for driving saliva secretion <i>in vivo.</i>