Cells Of Rat Parotid Gland Research Articles

Effects of Benzodiazepines on Acinar and Myoepithelial Cells.

Background: Benzodiazepines (BZDs), the most commonly prescribed psychotropic drugs with anxiolytic action, may cause hyposalivation. It has been previously shown that BZDs can cause hypertrophy and decrease the acini cell number. In this study, we investigated the effects of BZDs and pilocarpine on rat parotid glands, specifically on acinar, ductal, and myoepithelial cells.Methods: Ninety male Wistar rats were divided into nine groups. Control groups received a saline solution for 30 days (C30) and 60 days (C60), and pilocarpine (PILO) for 60 days. Experimental groups received lorazepam (L30) and midazolam (M30) for 30 days. Another group (LS60 or MS60) received lorazepam or midazolam for 30 days, respectively, and saline for additional 30 days. Finally, other groups (LP60 or MP60) received either lorazepam or midazolam for 30 days, respectively, and pilocarpine for additional 30 days. The expression of calponin in myoepithelial cells and the proliferating cell nuclear antigen (PCNA) in acinar and ductal cells were evaluated.Results: Animals treated with lorazepam showed an increase in the number of positive staining cells for calponin as compared to control animals (p < 0.05). Midazolam administered with pilocarpine (MP60) induced an increase in the proliferation of acinar and ductal cells and a decrease in the positive staining cells for calponin as compared to midazolam administered with saline (MS60).Conclusion: We found that myoepithelial cells might be more sensitive to the effects of BZD than acinar and ductal cells in rat parotid glands.

Radioprotective Effect of Vitamin E in Parotid Glands: a Morphometric Analysis in Rats

The aim of this study was to evaluate the radioprotective effect of vitamin E on rat parotid glands by morphometric analysis. Sixty male rats were divided into 5 groups (n=6): control, in which animals received olive oil solution; olive oil/irradiated, in which animals received olive oil and were irradiated with a dose of 15 Gy of gamma radiation; irradiated, in which animals were irradiated with a dose of 15 Gy gamma radiation; vitamin E, which received α-tocopherol acetate solution; vitamin E/irradiated, which received α-tocopherol acetate solution before irradiation with a dose of 15 Gy gamma rays. Half of the animals were euthanized at 8 h, and the remaining at 30 days after irradiation. Both parotid glands were surgically removed and morphometric analysis of acinar cells was performed. Data were subjected to two-way ANOVA and Tukey's test (α=0.05). Morphometric analysis showed a significant reduction in the number of parotid acinar cells at 30 days in olive oil/irradiated and irradiated groups. In groups evaluated over time a significant reduction was shown at 30 days in olive oil/irradiated and irradiated groups, indicating that ionizing radiation caused tissue damage. The vitamin E/irradiated group presented more acinar cells than the irradiated group, but no statistically significant difference was observed (p>0.05). In conclusion, vitamin E seems to have failed as a radioprotective agent on acinar cells in rat parotid glands.

The effects of antidepressants and pilocarpine on rat parotid glands: an immunohistochemical study

OBJECTIVES:To evaluate the effects of antidepressants and pilocarpine on the quantity of myoepithelial cells and on the proliferation index of the epithelial cells of rat parotid glands.INTRODUCTION:Hyposalivation, xerostomia, and alterations in saliva composition are important clinical side effects related to the use of antidepressants.METHODS:Ninety male Wistar rats were allocated to nine groups. The control groups received saline for 30 (group C30) or 60 days (group C60) or pilocarpine for 60 days (group Pilo). The experimental groups were administered fluoxetine (group F30) or venlafaxine for 30 days (group V30); fluoxetine (group FS60) or venlafaxine (group VS60) with saline for 60 days; or fluoxetine (group FP60) or venlafaxine (group VP60) with pilocarpine for 60 days. Parotid gland specimens were processed, and the immunohistochemical expression of calponin and proliferating cell nuclear anti-antigen on the myoepithelial and parenchymal cells, respectively, was evaluated. Analysis of variance (ANOVA), Tukey HSD and Games-Howell tests were applied to detect differences among groups (p<0.05).RESULTS:Compared with the controls, chronic exposure to antidepressants was associated with an increase in the number of positively stained cells for calponin. In addition, venlafaxine administration for 30 days was associated with an increase in the number of positively stained cells for proliferating cell nuclear anti-antigen. Fluoxetine and pilocarpine (group FP60) induced a significant decrease in the number of positively stained cells for calponin compared with all other groups.CONCLUSIONS:The number of positively stained cells for calponin increased after chronic administration of antidepressants. The proliferation index of the epithelial cells of rat parotid glands was not altered by the use of antidepressants for 60 days.

Low-level (gallium-aluminum-arsenide) laser irradiation of Par-C10 cells and acinar cells of rat parotid gland

We investigated cell response, including cell proliferation and expression of heat stress protein and bcl-2, to clarify the influence of low-level [gallium-aluminum-arsenide (Ga-Al-As) diode] laser irradiation on Par-C10 cells derived from the acinar cells of rat parotid glands. Furthermore, we also investigated amylase release and cell death from irradiation in acinar cells from rat parotid glands. The number of Par-C10 cells in the laser-irradiated groups was higher than that in the non-irradiated group at days 5 and 7, and the difference was statistically significant (P < 0.01). Greater expression of heat shock protein (HSP)25 and bcl-2 was seen on days 1 and 3 in the irradiated group. Assay of the released amylase showed no significant difference statistically between the irradiated group and the non-irradiated group. Trypan blue exclusion assay revealed that there was no difference in the ratio of dead to live cells between the irradiated and the non-irradiated groups. These results suggest that low-level laser irradiation promotes cell proliferation and expression of anti-apoptosis proteins in Par-C10 cells, but it does not significantly affect amylase secretion and does not induce rapid cell death in isolated acinar cells from rat parotid glands.

Immunolocalization of E-cadherin and αE-catenin in rat parotid acinar cell under chronic stimulation of isoproterenol

E-cadherin and αE-catenin were localized in normal and chronically isoproterenol-treated acinar cells of rat parotid gland by means of immunogold labelling of Lowicryl embedded sections. Immunostaining of both experimental groups with polyclonal antibodies to E-cadherin and αE-catenin was mainly restricted to the areas of adherens junctions. Surprisingly, in isoproterenol-treated cell αE-catenin was also found on the secretory granules periphery and appeared to encircle a secretory vesicle. In isoproterenol-induced cell hyperproliferation, the maintened presence of adherens junctions components, such as E-cadherin and αE-catenin molecules, should be an essential prerequisite for tissue integrity. Our data suggest the presence of a correlation between the organization of actin and the localization of αE-catenin in the chronically isoproterenol-treated acinar cell of rat parotid gland.

Identification of AQP5 in lipid rafts and its translocation to apical membranes by activation of M3 mAChRs in interlobular ducts of rat parotid gland

Aquaporin-5 (AQP5), an apical plasma membrane (APM) water channel in salivary glands, lacrimal glands, and airway epithelium, has an important role in fluid secretion. M(3) muscarinic acetylcholine receptor (mAChR)-induced changes in AQP5 localization in rat parotid glands were investigated with immunofluorescence or immunoelectron microscopy, detergent solubility, and gradient density floatation assays. Confocal microscopy revealed AQP5 localization in intracellular vesicles of interlobular duct cells in rat parotid glands and AQP5 trafficking to the APM 10 min after injection of the mAChR agonist cevimeline. Conversely, 60 min after injection, there was a diffuse pattern of AQP5 staining in the cell cytoplasm. The calcium ionophore A-23187 mimicked the effects of cevimeline. Immunoelectron microscopic studies confirmed that cevimeline induced AQP5 trafficking from intracellular structures to APMs in the interlobular duct cells of rat parotid glands. Lipid raft markers flotillin-2 and GM1 colocalized with AQP5 and moved with AQP5 in response to cevimeline. Under control conditions, the majority of AQP5 localized in the Triton X-100-insoluble fraction and floated to the light-density fraction on discontinuous density gradients. After 10-min incubation of parotid tissue slices with cevimeline or A-23187, AQP5 levels decreased in the Triton X-100-insoluble fraction and increased in the Triton X-100-soluble fraction. Thus AQP5 localizes in the intracellular lipid rafts, and M(3) mAChR activation induces AQP5 trafficking to the APM with lipid rafts via intracellular Ca(2+) signaling and induces AQP5 dissociation from lipid rafts to nonrafts on the APM in the interlobular duct cells of rat parotid glands.

Initial activation processes of the Na+-K+-2Cl− cotransporter and the Cl− channel in carbachol-stimulated acinar cells of rat parotid glands

Initial activation processes of the Na+-K+-2Cl− cotransporter and the Cl− channel in carbachol-stimulated acinar cells of rat parotid glands

THE IMPACT OF PHORBOL ESTER ON THE REGULATION OF AMILORIDE-SENSITIVE EPITHELIAL SODIUM CHANNEL IN ALVEOLAR TYPE II EPITHELIAL CELLS

Amiloride-sensitive sodium channel (ENaC) plays an important role in recovery from pulmonary edema. Recently, it has been shown that an activation of protein kinase C (PKC) could affect the mRNA expression of ENaC in rat parotid gland cells and A6 distal nephron epithelial cells. To determine whether an activation of PKC would regulate the mRNA expression or the function of ENaC, we stimulated rat alveolar type II epithelial cells with phorbol 12-myristate 13-acetate (PMA), a potent PKC activator, at a concentration of 100 nM. The mRNA expression of α -, β -, and γ -ENaC subunits and amiloride-sensitive current were measured. PMA inhibited the mRNA expression of all 3 ENaC subunits (α -ENaC: 56.0% ± 12.1%; β -ENaC: 62.6% ± 15.9%; γ -ENaC: 68.5% ± 10.6%, respectively) and amiloride-sensitive current (control = 7.0 ± 1.5 μ A/cm 2 ; PMA = 1.7 ± 0.9 μ A/cm 2) significantly at 24 hours. On the other hand, 4 α -phorbol didecanoate 4 α -PDD, inactive form of PMA, had no inhibitory effect on α - and γ -ENaC expression or amiloride-sensitive current. However, no significant difference was seen in β -ENaC expression between PMA and 4 α -PDD. GF 109203X, a wide-range PKC inhibitor, blocked the inhibitory effect of PMA on all ENaC subunits mRNA expression. These results suggest that an activation of PKC may play an important role in the regulation of ENaC mRNA expression and function.

Gramicidin-perforated patch analysis on HCO3- secretion through a forskolin-activated anion channel in rat parotid intralobular duct cells.

Forskolin-induced anion currents and depolarization were investigated to clarify the mechanism of HCO3- secretion in the intralobular duct cells of rat parotid glands. Anion currents of the cells were measured at the equilibrium potential of K+, using a gramicidin-perforated patch technique that negligibly affects intracellular anion concentration. The forskolin-induced anion current was sustained and significantly (54%) suppressed by glibenclamide (200 microM), a blocker of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. The anion current was markedly suppressed by addition of 1 mM methazolamide, a carbonic anhydrase inhibitor, and removal of external HCO3-. Forskolin depolarized the cells in the current-clamp mode. Addition of methazolamide and removal of external HCO3- significantly decreased the depolarizing level. These results suggest that activation of anion channels (mainly the CFTR Cl- channel located in luminal membranes) and production of cytosolic HCO3- induce the inward anion current and resulting depolarization. Inhibition of the Na(+)-K(+)-2Cl- cotransporter and the Cl(-)-HCO3- exchanger had no significant effect on the current or depolarization, indicating that the uptake of Cl- via the Na(+)-K(+)-2Cl- cotransporter or the Cl(-)-HCO3- exchanger is not involved in the responses. Taken together, we conclude that forskolin activates the outward movement (probably secretion) of HCO3- produced intracellularly, but not of Cl- due to lack of active Cl- transport in parotid duct cells, and that the gramicidin-perforated patch method is very useful to analyze anion transport.

A monoclonal antibody to the ligand-binding domain of the neurokinin 1 receptor (NK1-R) for the neuropeptide substance P

Monoclonal antibodies to the binding site of the NK1 receptor for the neuropeptide substance P were produced in mice using the complementary or antisense peptide methodology. Among several anti-peptide monoclonal antibodies, we selected the mAb12 antibody which specifically crossreacted, through its paratope, with a binding site present on membranes from rat parotid gland cells, with an affinity close to 2×10 −7 M and with membranes from CHO cells expressing human brain NK1 receptors. Immunocytochemical investigations using mAb12 revealed immunostaining whose distribution in the dorsal horns of rat spinal cord fits well with the known location of NK1 receptors. In both biochemical and immunocytochemical experiments, the competition occurring between the antibody and substance P, or a substance P-protein conjugate, indicates that mAb12 recognizes a membrane epitope located at or near the substance P binding domain on the NK1 receptor. Immunization of mice with mAb12 led to the production of specific anti-substance P antibodies, again suggesting that mAb12 shares common structural features with the neuropeptide. This monoclonal antibody can now be used in further biochemical or cytochemical characterizations of NK1 receptors. Owing to its fine specificity, mAb12 could also serve as a molecular model for designing peptides, possibly displaying pharmacological properties in the various processes in which substance P is involved, e.g. immunomodulation, inflammation or chronic pain.

Role for protein kinase in Ca2+-dependent feedback modulation of divalent cation influx in internal-Ca2+-store-depleted rat parotid gland cells.

Divalent cation (Ca2+ and Mn2+) influx, stimulated by internal Ca2+ store depletion, into rat parotid acinar cells is inhibited by conditions which increase protein phosphorylation [T. Sakai and I.S. Ambudkar (1996) Am J Physiol 271:C284-C294]. The present study examines the involvement of this protein phosphorylation and Ca2+ in the store-dependent inactivation of divalent cation entry. Internal Ca2+ store depletion, achieved by incubation (30 min) of cells in nominally Ca2+-free medium containing either carbachol or thapsigargin, stimulated Ca2+, and Mn2+, influx into cells. In either case, inclusion of 1.5 mM Ca2+ for the last 5 min of incubation resulted in a decrease in Ca2+ (33-41%) and Mn2+ (50%) influx, which could not be accounted for by internal Ca2+ store refill. The inhibition was prevented when internal-store-depleted cells were treated (prior to incubation with Ca2+) with either staurosporine or K-252a, but not with H-7 or KN-93. Refilling of internal Ca2+ store(s) in carbachol-treated cells (incubation with Ca2++atropine) induced complete inhibition of divalent cation influx, which was not prevented by treatment with protein kinase inhibitors. These data suggest the staurosporine-sensitive (and K-252a-sensitive) protein phosphorylation is not involved in Ca2+-store-refilling-dependent inactivation of Ca2+ influx but mediates a Ca2+-dependent feedback modulation of divalent cation influx in rat parotid gland acinar cells.

Changes in intracellular Ca2+ concentration produced by the alteration of intracellular pH in rat parotid acinar cells.

The effects of intracellular pH (pH(i)) on intracellular Ca2+ concentration ([Ca2+]i) vary in different cells, and mechanisms underlying these effects are still not clear. In the experiments reported here, the effects of changes in pH(i) produced by ammonium chloride and butyric acid were studied in enzymatically dispersed acinar cells of rat parotid glands. The changes in pH(i) and [Ca2+]i were estimated using the fluorescent dyes biscarboxyethyl-5,6-carboxyfluorescein (BCECF) and fura-2, respectively. pH(i) was altered using NH4Cl, butyric acid, or propionic acid while keeping the external pH constant at 7.4. NH4Cl (20 mM) applied for 4-5 min increased pH(i) from 7.18 to 7.79 (a decrease of proton concentration, [H+]i, from 66 to 16 nM) and produced a transient [Ca2+]i increase followed by a small sustained decrease. On the other hand, butyric acid (20 mM) decreased pH(i) from 7.16 to 6.81 (an increase of [H+]i from 69 to 155 nM) and produced a small sustained increase in [Ca2+]i. Washing out the butyric acid 4 min after application induced the recovery of pH(i) from 6.93 to 7.43 (a decrease of [H+]i from 118 to 37 nM) and a further transient increase in [Ca2+]i. The removal of external Ca2+ had little effect on changes in pH(i) produced by NH4Cl or butyric acid, but markedly reduced both the sustained and transient components of [Ca2+]i response. Cyclopiazonic acid (0.3 microM), an inhibitor of Ca2+ pump in intracellular stores, abolished the transient [Ca2+]i increase produced by the application of NH4Cl or withdrawal of butyric acid. These results suggest that a decrease in [H+]i, not the absolute level of [H+]i may release Ca2+ from intracellular stores.

Localization of the G-protein G(o) in exocrine glands.

The GTP-binding protein G(o) was localized immunohistochemically in the rat parotid gland and in other exocrine glands with specific G(o) antibodies. Immunohistochemical studies revealed that affinity-purified G(o alpha) polyclonal antibody (GO/85) immunoreacted primarily with duct cells of the rat parotid gland; immunoreactivity was also noted in duct cells of the rat submandibular, mouse parotid, and mouse submandibular glands. Light labeling of rat parotid and submandibular gland acinar cells was also noted. G(o alpha) antiserum (9072) differing in specificity for epitopes within G(o alpha) produced similar results. This antiserum also immunoreacted with rat submandibular duct cell secretory granule membranes. In contrast, in rat and mouse pancreas G(o alpha) antibodies immunoreacted primarily with islet cells. Duct cells were negative but there was light labeling of rat pancreatic acinar cells. The apparent duct specificity of G(o alpha) staining was further verified by demonstrating that G(o alpha) antibodies immunoreacted with HSG-PA cells, a human transformed salivary duct cell line. Specificity in immunohistochemical labeling of HSG-PA cells was confirmed by Western blot analysis. The results demonstrate that G(o) appears to be selectively expressed in the duct cells of rat parotid gland and other salivary glands. The selective enrichment of G(o) in duct cells suggests that this G-protein plays an important role in duct cell physiology.

Effects of receptor agonists on polyamine concentrations and spermidine/spermine N1-acetyltransferase activity in rat parotid gland.

The polyamine putrescine might be formed via a degradation (catalyzed by spermidine/spermine N1-acetyltransferase, SSAT) of the higher polyamines spermidine and spermine to putrescine. The involvement of different intracellular signal pathways in the regulation of putrescine formation was studied in explants and in cultured cells of rat parotid glands by using receptor agonists that activate separate second messenger systems, and measuring their effects on the concentrations of putrescine, spermidine and spermine and on the SSAT activity. The beta-adrenoceptor agonist isoprenaline, which is an activator of cAMP formation, increased the putrescine concentration and stimulated the SSAT activity. Pilocarpine, a drug that activates the muscarinic receptors and thereby enhances the phosphoinositide turnover, had no effect on either the polyamine concentrations or on the SSAT activity. Epidermal growth factor (EGF), which induces activation of a protein tyrosine kinase, had no effect on the polyamine concentrations or on the SSAT activity. The adenylate cyclase activator forskolin increased the glandular levels of putrescine. Taken together, these findings suggest that increases in putrescine concentration in cultured rat parotid gland cells are accompanied by accumulation of cAMP.

Identification and localization of G proteins in exocrine glands.

GTP-binding proteins were identified in rat parotid acinar plasma-enriched membranes (PM) by immunoblot analysis and localized immunohistochemically in the parotid gland as well as in other exocrine glands by using affinity-purified antisera specific for alpha subunits of the G proteins. Isolated rat parotid acinar PM immunoreacted strongly to antisera directed against Gs alpha, Gi alpha 1/alpha 2, Gi alpha 3, and Go alpha; the signal for Go alpha, however, was weak with crude Go antisera. Immunohistochemical studies to identify and localize Go in rat parotid tissue revealed that antisera to Go alpha immunoreacted with ductal cells. In addition, strong immunoreactivity to Go alpha antisera was noted in ductal cells of other salivary glands including rat submandibular, mouse parotid, and mouse submandibular glands. Light labeling of rat parotid and submandibular gland acinar cells was also noted. In contrast, in the rat and mouse pancreas, Go antisera immunoreacted primarily with islet cells. Ductal cells were negative, but there was light labeling of rat pancreatic acinar cells. The apparent ductal specificity of Go alpha staining was further verified by demonstrating that Go alpha antisera immunoreacted strongly with HSG-PA cells, a human transformed salivary ductal cell line. The results demonstrate that rat parotid acinar plasma membranes express a number of G proteins including Go and that Go appears to be selectively expressed in the ductal cells of rat parotid gland and other salivary glands. The selective enrichment of Go in ductal cells suggests that this G protein may play an important role in ductal cell physiology.

Anti-substance P anti-idiotypic antibodies modulate the secretory process in the rat parotid gland in vitro

Anti-idiotypic antibodies (anti-Id) obtained in rabbits in response to immunization with polyclonal anti-substance P antibodies (anti-SP) were shown to bind specifically and with high affinity to membranes from rat parotid gland cells. Whereas substace P (SP) was unable to displace anti-Id from membrane binding sites, anti-Id partly inhibited the binding of radiolabelled substance P. Like substance P, anti-Id were able to trigger protein secretion by parotid cells, i.e. to behave as physiological agonists of the neuropeptide. Under our experimental conditions, the biological effects of both ligands appear to be additive. Unlike substance P, however, anti-Id dir not potentiate the secretory responses induced by a β-adrenoceptor agonist. Taken together, the present results might indicate that anti-Id interact with epitope(s) at or/and near he peptide-combining site on the substance P receptor. These data demonstrate further the possibility of raising pharmacologically active anti-receptor antibodies through the immunological anti-idiotypic approach.

Use of anti-idiotypic antibodies as probes for in vitro and in vivo identification of substance P receptor

In order to develop immunological tools for studying the receptor of the neuropeptide substance P (SP), anti-idiotypic antibodies (anti-Id abs) were produced by immunization with anti-SP antibodies whose specificity was close to that of the SP receptor. Immunological studies revealed structural similarity between some anti-Id abs and SP itself. As a consequence, these anti-Id abs were able to bind to mammalian SP receptors. These antibodies were used in immunocytochemistry to label SP receptors both in the rat spinal cord and in rat and guinea pig peripheral tissues (parotid gland and trachea, respectively). Like SP, anti-Id abs were able to trigger protein secretion by isolated rat parotid gland cells. Finally, it was shown that anti-Id abs in vivo modulated reactivity to chemical stimuli. These antibodies therefore appear to be promising tools for further biochemical, cytochemical, and pharmacological characterization of SP receptors.

Regulation of proline-rich protein and alpha-amylase genes in parotid-hepatoma hybrid cells.

Salivary proline-rich proteins are encoded by tissue-specific multigene families, and are dramatically induced in mice, rats, and hamsters by treatment with the beta agonist isoproterenol. Salivary gland cells, however, are difficult to maintain in a differentiated state in culture and can be induced to synthesize proline-rich protein mRNAs for only a few days. In an attempt to establish a cell line in which it would be possible to regulate proline-rich protein gene transcription, rat parotid gland cells were fused with the rat hepatoma cell line, FTO-2B. Fused cells were obtained that had a frequency of 7.5 x 10(-6), which is about 125-fold greater than the reversion rate of FTO-2B. The hybrid cells exhibited induced proline-rich protein mRNA synthesis when incubated with either dibutyryl cyclic AMP or forskolin. The ability to induce these genes was maintained for at least 20 passages. Most of the fused cell populations also synthesized elevated levels of alpha-amylase mRNA, another tissue-specific gene.

Effect of NaF on cAMP accumulation, cAMP-dependent protein kinase activity in, and amylase secretion from, rat parotid gland cells.

Stimulation of amylase secretion from parotid glands by beta-adrenergic agonists is mediated by the activation of adenylate cyclase and the resultant increase in cellular cAMP. Since NaF is known to increase adenylate cyclase activity and cAMP accumulation in intact cells, we investigated whether it would stimulate amylase secretion from isolated rat parotid gland cells. The results provide evidence that the addition of NaF (0.01-10 mmol/L) increased cAMP concentration (1.5-2.8-fold) in, and amylase secretion (16-93%) from, isolated parotid gland acinar cells. NaF was found to increase cAMP-dependent protein kinase activity ratios (51-84%) in a concentration- and time-dependent manner. The data suggest that the stimulation of amylase secretion from parotid gland cells by NaF may be mediated by an increase in the cellular cAMP concentration, which exerts its effect, at least in part, by increasing the activity of cAMP-dependent protein kinase.

Quantitative immunoelectron microscopic localization of (Na+,K+)ATPase in rat parotid gland.

Distribution of (Na+,K+)ATPase on the cell membranes of acinar and duct cells of rat parotid gland was investigated quantitatively by immunoelectron microscopy using the post-embedding protein A-gold technique. In acinar cells, ATPase was localized predominantly on the basolateral plasma membranes. A small but significant amount of (Na+,K+)ATPase was, however, detected on the luminal plasma membranes, especially on the microvillar region of the acinar cells; the surface density on the luminal membrane was approximately one third of that on the basolateral membranes. In duct cells, many gold particles were found on the basolateral membrane, especially along the basal infoldings of the plasma membranes, whereas no significant gold particles were found on the luminal plasma membranes, suggesting unilateral distribution of ATPase in duct cells. We suggest that in acinar cells sodium ion is not only transported paracellularly but is also actively transported intracellularly into the luminal space by the (Na+,K+)ATPase located on the luminal plasma membranes, and that water is passively transported to the luminal space to form a plasma-like isotonic primary saliva, while in the duct cells the same ion is selectively re-absorbed intracellularly by (Na+,K+)ATPase found in abundance along the many infoldings of the basal plasma membranes, thus producing the hypotonic saliva.