Abstract

In a recent study from our group, we showed that the activation of the TMEM16A channel is required for muscarinic, Ca2+-dependent salivation. Furthermore, we found a novel β-adrenergic, cAMP-dependent salivation mechanism that is independent of TMEM16A, CFTR and ClC-2 Cl- channels. In contrast to the modest HCO3- -dependence displayed by the Ca2+ -dependent secretion, the cAMP-dependent secretion is severely impaired in the absence of HCO3- ions. We propose that the HCO3- dependence of the cAMP dependent secretion is given by the involvement of basolateral NBCe1 Na+-HCO3- cotransporters. Ca2+-dependent secretion by salivary gland secretory acinar cells is characterized by a reduction in cell volume upon muscarinic receptor stimulation. Conversely, acinar cells markedly increase their volume in response to the β-adrenergic receptor agonist isoproterenol. The cAMP-dependent ion-transporting proteins that promote solute accumulation and cause acinar cell swelling have not been identified yet. We propose that NKCC1 Na+-K+-2Cl- cotransporters and/or AE4 Cl-/HCO3- exchangers act as basolateral solute uptake pathway(s) required for driving cAMP-dependent fluid secretion. Acinar cell swelling caused by β-adrenergic receptor stimulation suggests the involvement of the swelling-activated, volume regulated anion channel (VRAC) in cAMP-dependent secretion as apical Cl- (and perhaps HCO3-) efflux pathway. Acinar VRAC has not been identified at the molecular level yet, but based on the similar biophysical properties such as voltage dependence and sensitivity to blockers, it is likely that is encoded by member(s) of the recently discovered LRRC8 gene family, which encode for VRAC in other cell types. Taken together, we propose the following hypothesis: ”Mouse submandibular gland displays a cAMP-dependent ion-transporting machinery that promotes saliva secretion. In contrast to the acinar cell shrinkage that occurs during calcium-dependent secretion, cAMP-mediated secretion is characterized by an increase in acinar cell volume. The cAMP-induced acinar cell swelling is caused by the activation of basolateral solute uptake pathway(s) and is critical for the activation of VRAC, which mediates anion secretion into the luminal space”. The overall goal of this project is to elucidate the molecular mechanism underlying fluid secretion by the major salivary glands in response to the stimulation of -adrenergic receptors. The main focus of this proposal is to identify the Cl- - and HCO3- -transporting proteins located at the basolateral and apical domains of acinar cells that are involved in this novel secretion mechanism. By using a multidisciplinary approach consisting of electrophysiological (patch clamp), conventional and multiphoton fluorescence microscopy (cell volume, intracellular Cl- and intra- and extracellular pH measurements) and mouse gene-targeting techniques (systemic and conditional knockout mouse models and in vivo gene knockdown in mice using shRNA-containing adeno-associated virus), we will address the following specific goals: 1. Elucidate the role of HCO3- ions in cAMP-elicited fluid secretion in mouse salivary glands. 2. Explore the role of NKCC1 cotransporters and AE4 exchangers as Cl- -transporting pathways involved in fluid secretion by salivary glands in response to β-adrenergic receptor stimulation. 3. Investigate whether the volume regulated anion channel (VRAC) present in mouse salivary gland acinar cells is encoded by members of the LRRC8 gene family. Ultimately, the information gained from this proposal will facilitate the understanding of the β-adrenergic stimulated secretion mechanism and the rational design of pharmacological therapies for treating millions of patients worldwide suffering from dry mouth (xerostomia).