Pituitary Hormones (FSH, LH, PRL, and GH) Differentially Regulate AQP5 Expression in Porcine Ovarian Follicular Cells

This study aimed to examine the effect of follicle-stimulating hormone (FSH), luteinizing hormone (LH), prolactin (PRL), and growth hormone (GH) on Aquaporin 5 (AQP5) expression in granulosa (Gc) and theca cells (Tc) from medium (MF) and large (LF) ovarian follicles of pigs. The results showed that GH significantly decreased the expression of AQP5 in Gc from MF in relation to the control. In the Gc of large follicles, PRL stimulated the expression of AQP5. However, the increased expression of AQP5 in the Tc of LF was indicated by GH and PRL in relation to the control. A significantly higher expression of the AQP5 protein in the Gc from MF and LF was indicated by FSH and PRL. In co-cultures, an increased expression of AQP5 was observed in the Gc from LF incubated with LH, PRL, and GH. A significantly increased expression of AQP5 was also observed in co-cultures of Tc from all type of follicles incubated with LH, whereas PRL stimulated the expression of AQP5 in Tc from MF. Moreover, AQP5 protein expression increased in the co-culture isolated from MF and LF after treatment with FSH, LH, PRL, and GH. AQP5 immunoreactivity was observed in the cytoplasm, mainly in the perinuclear region and endosomes, as well as in the cell membranes of Gc and Tc from the LF and MF.

An Experimental Study on Botulinum Toxin Type A for the Treatment of Excessive Secretion after Submandibular Gland Transplantation in Rabbits

Objectives. To investigate whether botulinum toxin type A (BTXA) could control excessive secretion after submandibular gland (SMG) transplantation in rabbits and its possible mechanisms.Methods. A new SMG transplantation model was established in rabbit. 30 successfully constructed models were randomly assigned to five groups including control group and four experimental groups. Secretion outputs were used to analyze the effect of BTXA injection on excessive secretion. Hematoxylin and eosin (HE) staining, transmission electron microscopy (TEM), Western blot, and immunofluorescence were performed to analyze its possible mechanisms.Results. After BTXA injection, a significant decrease of excessive secretion after SMG transplantation was found in 2 and 4 weeks groups, but no significant effect on 12 and 24 weeks groups. HE and TEM results showed that BTXA led to morphological and ultrastructural changes of acinar cells of transplanted SMG. Western blot results suggested that BTXA decreased the aquaporin-5 (AQP5) protein expression after BTXA injection for 2 and 4 weeks. Immunofluorescence results showed that AQP5 protein was mainly expressed in the cytoplasm after BTXA injection for 2 and 4 weeks, which might indicate that BTXA promoted AQP5 expression from the cell membrane to cytoplasm.Conclusion. BTXA could effectively control excessive secretion after SMG transplantation in rabbits.

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

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 β-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 μ-calpain in vitro. Furthermore, we demonstrated that μ-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.

Role of cAMP-PKA/CREB pathway in regulation of AQP 5 production in rat nasal epithelium

OBJECTIVES: Aquaporin 5 (AQP5) is a water-specific channel protein. In this study, we investigated the possible role of the cyclic adenosine monophosphate-protein kinase A/cyclic adenosine monophosphate response element binding protein (cAMP-PKA/CREB) pathway in the regulation of AQP5 in nasal epithelial cells. METHODS: Rat nasal epithelial cells were cultured and treated with the PKA inhibitor H89 or cAMP inducing medicine forskolin for 12 or 24 hours in vitro. AQP5 and phosphorylated CREB (p-CREB) at serine133 (Ser133) were detected by immunocytochemistry, Western blotting or real-time PCR. Experiments were repeated 10 times. RESULTS: After treatment with H89 for 12 or 24 hours, the number of cells positive for AQP5 and p-CREB (Ser133) were decreased, p-CREB (Ser133) and AQP5 protein decreased, and AQP5 mRNA decreased. After treatment with forskolin for 12 or 24 hours, the number of p-CREB (Ser133) and AQP5 positive cells increased, p-CREB (Ser133) and AQP5 protein increased, and AQP5 mRNA was increased. CONCLUSION: Both H89 (PKA inhibitor) and forskolin (cAMP inducing medicine) regulate AQP5 production through the cAMP-PKA/CREB pathway, which could influence the secretory function of the submucosal glands in nasal epithelium.

Degradation of submandibular gland AQP5 by parasympathetic denervation of chorda tympani and its recovery by cevimeline, an M3 muscarinic receptor agonist

By chorda tympani denervation (CTD, parasympathectomy), the aquaporin 5 (AQP5), but not AQP1, protein level in the rat submandibular gland (SMG) was significantly decreased, dropping to 37% of that of the contralateral gland at 4 wk. The protein levels of AQP5 and AQP1 were not significantly affected by denervation of the cervical sympathetic trunk (sympathectomy). Administration of cevimeline hydrochloride, an M3 muscarinic receptor agonist (10 mg/kg for 7 days po), but not pilocarpine (0.3 mg/kg for 7 days po), recovered the AQP5 protein level reduced by CTD and increased the AQP1 protein level above the control one. The mRNA level of AQP5 was scarcely affected by CTD and cevimeline hydrochloride administration. Administration of chloroquine (50 mg/kg for 7 days po), a denaturant of lysosomes, increased the AQP5 protein level reduced by CTD. An extract obtained from the submandibular lysosomal fraction degraded the AQP5 protein in the total membrane fraction in vitro. These results suggest the possible regulation of the AQP5 protein level in the SMG by the parasympathetic nerves/M3 muscarinic receptor agonist and imply the involvement of lysosomal enzymes, but not a transcriptional mechanism, in this regulation.