Cl^-/HCO₃^-exchange is acetazolamide sensitive and activated by a muscarinic receptor-induced [Ca²⁺]_iincrease in salivary acinar cells

Abstract

Large volumes of saliva are generated by transepithelial Cl(-) movement during parasympathetic muscarinic receptor stimulation. To gain further insight into a major Cl(-) uptake mechanism involved in this process, we have characterized the anion exchanger (AE) activity in mouse serous parotid and mucous sublingual salivary gland acinar cells. The AE activity in acinar cells was Na(+) independent, electroneutral, and sensitive to the anion exchange inhibitor DIDS, properties consistent with the AE members of the SLC4A gene family. Localization studies using a specific antibody to the ubiquitously expressed AE2 isoform labeled acini in both parotid and sublingual glands. Western blot analysis detected an approximately 170-kDa protein that was more highly expressed in the plasma membranes of sublingual than in parotid glands. Correspondingly, the DIDS-sensitive Cl(-)/HCO(3)(-) exchanger activity was significantly greater in sublingual acinar cells. The carbonic anhydrase antagonist acetazolamide markedly inhibited, whereas muscarinic receptor stimulation enhanced, the Cl(-)/HCO(3)(-) exchanger activity in acinar cells from both glands. Intracellular Ca(2+) chelation prevented muscarinic receptor-induced upregulation of the AE, whereas raising the intracellular Ca(2+) concentration with the Ca(2+)-ATPase inhibitor thapsigargin mimicked the effects of muscarinic receptor stimulation. In summary, carbonic anhydrase activity was essential for regulating Cl(-)/HCO(3)(-) exchange in salivary gland acinar cells. Moreover, muscarinic receptor stimulation enhanced AE activity through a Ca(2+)-dependent mechanism. Such forms of regulation may play important roles in modulating fluid and electrolyte secretion by salivary gland acinar cells.

Governing effect of regulatory proteins for Cl−/HCO3− exchanger 2 activity

“…11,27 In addition, this muscarinic receptor stimulation involved in the activation of AE. 28 Thus, the SPL machinery induced by receptor stimulation may play a role in secretory processes. SPL also has additional binding modules such as those for PP1 and F-actin that can recruit other binding partners and can assemble the complex to the cytoskeleton.…”

“…At the cellular level, muscarinic receptor stimulation enhances AE activity in salivary acinar cells. 9 The global mechanism by which extracellular stimuli including activation of muscarinic receptors are converted to intracellular Ca 2C responses involves the activation of G protein-coupled receptors (GPCRs). The GPCRs recruit large complex proteins including the G a subunit, G ßg , PLCß, and regulators of G protein signaling (RGS) proteins.…”

“…It has been reported that Cl ¡ / HCO 3 ¡ exchange activity is modulated by the CA inhibitor acetazolamide. 9 Moreover, physical interaction between CA and AE is necessary for maximal AE activity, suggesting that AE2 activity is dependent on CA activity 17 and the physical interaction at the similar position of the BLM should be considered. Here, we evaluated that the BLM-associated CA XII may provide fuel to activate AE2.…”

Ae4 (Slc4a9) Anion Exchanger Drives Cl− Uptake-dependent Fluid Secretion by Mouse Submandibular Gland Acinar Cells

“…Targeted disruption of Nkcc1 produces a dramatic 65% reduction in saliva secretion by the mouse parotid gland, demonstrating that Na ϩ -K ϩ -2Cl Ϫ cotransporter 1 (Nkcc1) is the main mechanism for concentrating Cl Ϫ in acinar cells (11). A parallel HCO 3 Ϫ -dependent Cl Ϫ uptake mechanism appears to be responsible for much of the remaining Nkcc1-independent fluid secretion (12)(13)(14). The current fluid secretion model proposes that this secondary Nkcc1-independent Cl Ϫ uptake pathway is a basolateral Cl Ϫ / HCO 3 Ϫ exchanger.…”

Role of Calcium and PKC in Salivary Mucous Cell Exocrine Secretion

“…PLC activation generates inositol 1,4,5-trisphosphate (IP 3 ) (Zhang and Melvin, 1993) to release Ca 2+ from intracellular stores, followed by an influx of extracellular Ca 2+ through a store-operated Ca 2+ entry mechanism to sustain increased intracellular free calcium ([Ca 2+ ] i ) (Melvin et al, 1991). Although mechanisms associated with mucous cell ion fluxes (Nguyen et al, 2004) and cell volume (Krane et al, 2001) have been elucidated, roles for PLC and for [Ca 2+ ] i in mucous cell exocrine secretion are unclear. Moreover, the PLC product, diacylglycerol (DAG), may mediate secretion through activation of protein kinase C (PKC) (Dempsey et al, 2000).…”