Altered Expression of Sodium Transporters and Water Channels in the Submandibular Gland of Rats Treated with Nitric Oxide Synthesis Inhibitors

A role of nitric oxide (NO) in the regulation of sodium transporters and water channels in the salivary gland was investigated. Male Sprague-Dawley rats were treated with NG-nitro-L-arginine methyl ester (L-NAME, 100 mg/L drinking water) for 1 week. The control group was supplied with normal tap water. The expression of Na+,K+-ATPase, type 2 Na+/K+/2Cl- cotransporter (NKCC2), type 1 Na+/H+ exchanger (NHE1), α-subunit of epithelial sodium transporter (ENaC), and aquaporin-5 (AQP5) and aquaporin-1 (AQP1) proteins were determined in the submandibular gland by Western blot analysis. Following the treatment with L-NAME, the expression of Na+,K+-ATPase α1-subunit, NKCC2, NHE1, and ENaC α-subunit increased significantly. On the contrary, the expression of AQP5 was significantly decreased, while that of AQP1 was not significantly altered. These findings indicate that the sodium transporters and water channels may be under a tonic regulatory influence of NO in the salivary gland.


Introduction
Salivary glands are composed of acinar cells, either serous or mucous, and ductal cells of several varieties. The primary saliva is formed in the lumen of the acinar cells, where the accumulation of ions generates a transepithelial osmotic gradient driving water movement. In the second stage of secretion, sodium is actively reabsorbed while the primary saliva passes through the duct. Therefore, alterations of sodium transporters and water channels may affect the formation and the ductal modification of the saliva.
Immunofluorescence labelling and confocal microscopy revealed a polarized distribution of Na + ,K + -ATPase, the secretory isoform of Na + /K + /2Cl cotransporter (NKCC2), and type 1 Na + /H + exchanger (NHE1) in the basolateral membranes of acinar and intralobular duct cells 1) . Na + ,K + -ATPase produces an inward-directed sodium chemical gradient in the acinar cell 2) . Although sodium influx is negligible in resting cells, it is rapidly increased by activation of NKCC2, NHE1, and nonselective cation channels in the acinar cell 3) . Blockade of NKCC2 causes significant reduction of acetylcholine-stimulated secretion 4) , in which the residual secretion is attributed to the operation of Na + /H + and Cl /HCO3 antiport 5,6) . On the other hand, there have been known multiple isoforms of aquaporin (AQP) water channels in the salivary gland. Among them, AQP5 may be the only isoform of AQP playing a major role in the salivary secretion process.
Strong AQP5 labelling is located in the apical membrane of serous-type acinar cells from the rat submandibular gland 9) . The transepithelial water movement occurs through the apical AQP5 channels and paracellular pathways 10) , leading to the secretion of isotonic primary saliva. Although the presence of AQP1 and AQP8 has been also generally accepted, their role in the salivary secretion remains controversial 11) .
Over the last two decades, nitric oxide (NO) has been found to play a role in the regulation of various physiological functions. It has been also implicated in mediating the nerve-evoked vasodilator and secretory responses in the salivary gland. Parasympathetic nerve activity increases the submandibular blood flow, and elicits the flow of saliva and output of protein by mechanisms that involve in situ generation of NO in the ferret 12) . A previous study revealed that NO synthase (NOS) is not present in the acinar cells, but in neural terminals within the gland and in the apical membrane of the excretory and striated ducts, the cytoplasm of granular convoluted tubules, and, to a lesser extent, in the cytoplasm of excretory and striated ducts 13) .
However, later investigators demonstrated that acinar cells also posses endogenous NOS activity 14) . Furthermore, the salivation induced by cholinergic agonists or that produced by the chorda stimulation was decreased by inhibition of NO synthesis by N G -nitro-L-arginine methyl ester (L-NAME) in the rat submandibular gland 11,15) . Nevertheless, the role of NO in the regulation of sodium transporters and AQP channels in the salivary gland has not been established. Therefore, the present study was aimed to explore the role of NO in the regulation of sodium transporters and AQP channels in the salivary gland. Rats were inhibited of endogenous generation of NO by treatment with L-NAME, and the expression of sodium transporters and water channels was determined in the submandibular gland.

Animals
Male Sprague-Dawley rats (200-250 g) were used. The experimental group was treated with L-NAME (100 mg/L drinking water) for 1 week. The control group was supplied with normal tap water. The experimental procedure conformed to the Institutional Guidelines for Experimental Animal Care and Use.

Western blot analysis
The submandibular glands were rapidly isolated under

Drugs and statistical analysis
Drugs were purchased from Sigma, unless stated otherwise. Results are expressed as mean±SEM. The statistical significance of values between groups was determined by the unpaired t-test.

Results
In the L-NAME-treated group, the systolic blood pressure measured indirectly by the tail cuff method in a conscious state was significantly higher in the experimental group than in the control (146±9 vs 121±6 mmHg, p<0.05, n=6 each). Accordingly, the expression of eNOS and nNOS in the submandibular gland was decreased significantly (Fig. 1). The amount of -actin was not signi β ficantly altered (Fig. 2). On the contrary, the expression of 1-subunit α of Na + ,K + -ATPase was significantly increased, although that of 1-subunit remained unchang β ed (Fig. 3). The expression of NKCC2, NHE1, and -subunit of ENaC was α increased significantly (Fig. 4). The expression of AQP5 was significantly decreased, while that of AQP1 was not significantly altered (Fig. 5).

Discussion
NO has been implicated in mechanisms mediating nerve-evoked vasodilator and secretory responses in salivary glands. The salivation induced by cholinergic agonists or chorda stimulation was decreased by NO synthesis inhibition in the submandibular gland 12,13,15) . The formation of primary and secondary saliva involves transepithelial electrolyte and water movement. Therefore, it is plausible to hypothesize that NO plays a role in regulating the sodium transporters and AQP channels in the salivary gland.
Previous studies have shown that L-NAME treatment decreases the gene and protein expression of eNOS 16,17) .
The present study also demonstrated a decreased expres- On the other hand, the expression of AQP5 was significantly decreased by the treatment with L-NAME. This finding suggests that AQP5 is under tonic excitatory influence of NO in the submandibular gland. A key role of AQP5 in saliva fluid secretion has been well known.
Transgenic mice lacking AQP5 display reduced pilocarpinestimulated saliva secretion (60%), in which the secreted saliva is more hypertonic and viscous 18) . In Sjøgren's syndrome, an abnormal distribution of AQP5 has been demonstrated in the acini, which is likely to contribute to the deficiency of fluid secretion 19) . A down-regulation of AQP5 may result in a decrease of water flux in the acinar cell and hence the formation of primary saliva. In this context, the L-NAME-induced reduction of secretory responses 12,13,15) may be attributed in part to the down-regulation of AQP5. The expression of AQP1 has been demonstrated to be continuously distributed to microvasculature during embryonic and postnatal development in the rat submandibular gland 20,21) . However, the expression of AQP1 proteins was not significantly altered by the L-NAME-treatment. In transgenic mice lacking AQP1, pilocarpine-induced salivary secretion showed no defects in volume or composition of saliva 18,22) . It is unlikely that AQP1 should play a major role in salivary secretion.
Finally, it is worth to note that the systolic blood pressure was significantly increased, indicating that the animals were in a state of systemic withdrawal of tonic influences of endogenous NO. Furthermore, the alteration of sodium transporters and water channels cannot be attributed to the increased blood pressure, perse, since an attenuated salivation has not been documented in other models of hypertension.
In summary, these results indicate that sodium transporters and water channels are under tonic regulatory influence of NO in the submandibular gland. An altered NO synthesis may affect the expression of these transporters and channels, and hence the formation and secretion of saliva. Further studies will be needed to directly measure the sodium and water flux in the ductal epithelia to determine the actual contribution of each transporter.