Effects of isoproterenol on aquaporin 5 levels in the parotid gland of mice in vivo

Abstract

In the membrane fraction of mouse parotid gland (PG), the protein level of aquaporin 5 (AQP5), a member of the water channel family, was increased by injection (ip) of isoproterenol (IPR), a ␤-adrenergic agonist, at 1 h, and stayed at high levels until 6 h; this change occurred simultaneously as amylase secretion. The AQP5 level then decreased and returned toward the original level at 12-48 h. After IPR injection, the AQP5 mRNA gradually increased and reached a maximum at 24 h. The facts suggest a rapid appearance of AQP5 at plasma membrane by IPR and subsequent degradation/metabolism by activation of proteolytic systems. Pretreatment of animals with two calpain inhibitors, N-Ac-Leu-Leu-methininal (ALLM) and calpeptin, as well as a protein synthesis inhibitor, cycloheximide (CHX), significantly suppressed the IPR-induced AQP5 degradation in the PG membrane fraction; such suppression was not observed by two proteasome inhibitors, MG132 and lactacystin, or the lysosome denaturant chloroquine, although most of these inhibitors increased AQP5 protein levels in unstimulated mice. The AQP5 protein was also degraded by -calpain in vitro. Furthermore, we demonstrated that -calpain was colocalized with AQP5 in the acinar cells by immunohistochemistry, and its activity in the PG was increased at 6 h after IPR injection. These results suggest that the calpain system was responsible for IPR-induced AQP5 degradation in the parotid gland and that such a system was coupled to the secretory-restoration cycle of amylase in the PG. aquaporin 5; exocytosis; -calpain; parotid gland; isoproterenol AQUAPORINS (AQPs) are members of the water channel family that facilitate water movement between in and out of cells; they are expressed in various tissues and cells throughout the body and play pivotal roles in water movement. So far, 13 different AQPs (AQP 0 -12) have been identified in mammals (27). Among them, AQP5 is the one identified first in the salivary gland (24), and its deficiency in mice has been shown to lead to a decrease in their survival rates at the embryonic stage (9, 13). Studies of the AQP5-mutant rat (18) and AQP5-knockout mice (13) as well as the model mouse of Sjögren's syndrome (26) have indicated that AQP5 plays a pivotal role in maintaining the normal physiological function of the salivary gland; i.e., some of these animals produce significantly hypertonic, viscous, and smaller volumes of saliva (13, 18). The parotid gland (PG) belongs to one of the major salivary glands and expresses abundant AQP5 in the luminal membrane of its serous acinar cells (20, 31). It was reported previously that isolated and disaggregated PG acinar cells prepared from AQP5-knockout mice showed a significant decrease in transmembrane water transport (9). These results suggest that a certain quantity of fluid secreted by mouse salivary gland is due to the transcellular water transport, in which AQP5 plays a major role.Acinar cells of the PG in mice are filled with a plenty of secretory granules containing amylase, which contributes 80% o...

Dynamics of Salivary Gland AQP5 under Normal and Pathologic Conditions

“…On the other hand, β-adrenergic agonists and their second messenger, that is, cAMP and its derivatives, are also known to increase expressions of the AQP5 mRNA and protein in MLE-12, a cultured mouse lung cell line, and induce translocation of AQP5 to the apical plasma membrane [36]. Thus Chen et al [37] examined the possibility that β-adrenergic agonists affect AQP5 in the salivary gland; that is, they studied the effects of IPR, a β-adrenergic agonist, on AQP5 in the mouse PG.…”

“…These authors imply that reduction of AQP5 starting 6 h after IPR injection would be due to proteolysis of AQP5. From both in vivo and in vitro experiments using various inhibitors the responsive enzyme for this proteolysis is suggested to be calpain [37]. Despite β-adrenergic agonist was shown to elevate the AQP5 mRNA level in the PG, it is still uncertain how AQP5 transcription is regulated by cAMP in this tissue.…”

Insight into Salivary Gland Aquaporins

“…The neural signal sent to the submandibular glands via the parasympathetic nerve innervating submandibular glands, i.e., the chorda tympani nerve, was suggested to maintain certain AQP5 expression [ 22 ]. Sympathetic activation leads to the release of adrenalin and subsequent activation of intracellular signaling cascade, leading to cyclic AMP (cAMP) increase and a subsequent increase in AQP5 RNA levels and translocation of AQP5 to the cell apical membrane [ 22 , 38 , 39 ]. Indeed, the injection of isoproterenol, a ß-adrenergic agonist, increased both Aqp5 mRNA and protein expression, as well as exocytotic translocation of AQP5 from secretory granules to the plasma membrane in mouse parotid glands [ 22 ].…”

β‐Actin protein expression differs in the submandibular glands of male and female mice

“…The SMGs of mice were isolated and homogenated in cold RIPA buffer (150 mM NaCl, 1.0% IGEPAL CA‐630, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 50 mM Tris, pH 8.0, and EDTA‐free protease inhibitor cocktail) by a glass homogenizer. After removing the tissue debris and nucleus by centrifugation at 10,000 rpm for 15 min at 4°C, the expression levels of β‐actin and GAPDH in the supernatant were determined by Western blotting as described previously (Chen et al, ). Briefly, proteins were extracted from the SMG, and protein concentrations were determined by a Bio‐Rad protein assay, using bovine serum albumin as a standard.…”