Defective NaCl Reabsorption in Salivary Glands of Eda-Null X-LHED Mice.

Mutations in the ectodysplasin A gene ( EDA) cause X-LHED (X-linked hypohidrotic ectodermal dysplasia), the most common human form of ectodermal dysplasia. Defective EDA signaling is linked to hypoplastic development of epithelial tissues, resulting in hypotrichosis, hypodontia, hypohidrosis, and xerostomia. The primary objective of the present study was to better understand the salivary gland dysfunction associated with ectodermal dysplasia using the analogous murine disorder. The salivary flow rate and ion composition of the 3 major salivary glands were determined in adult Eda-deficient Tabby hemizygous male (Ta/Y) and heterozygous female (Ta/X) mice. Submandibular and sublingual glands of Eda-mutant mice were smaller than wild-type littermates, while parotid gland weight was not significantly altered. Fluid secretion by the 3 major salivary glands was essentially unchanged, but the decrease in submandibular gland size was associated with a dramatic loss of ducts in Ta/Y and Ta/X mice. Reabsorption of Na+ and Cl-, previously linked in salivary glands to Scnn1 Na+ channels and Cftr Cl- channels, respectively, was markedly reduced at high flow rates in the ex vivo submandibular glands of Ta/Y mice (~60%) and, to a lesser extent, Ta/X mice (Na+ by 14%). Consistent with decreased Na+ reabsorption in Ta/Y mice, quantitative polymerase chain reaction analysis detected decreased mRNA expression for Scnn1b and Scnn1g, genes encoding the ß and γ subunits, respectively. Moreover, the Na+ channel blocker amiloride significantly inhibited Na+ and Cl- reabsorption by wild-type male submandibular glands to levels comparable to those observed in Ta/Y mice. In summary, fluid secretion was intact in the salivary glands of Eda-deficient mice but displayed marked Na+ and Cl- reabsorption defects that correlated with the loss of duct cells and decreased Scnn1 Na+ channel expression. These results provide a likely mechanism for the elevated NaCl concentration observed in the saliva of affected male and female patients with X-LHED.

Late responses to adenoviral-mediated transfer of the aquaporin-1 gene for radiation-induced salivary hypofunction.

We evaluated late effects of AdhAQP1 administration in five subjects in a clinical trial for radiation-induced salivary hypofunction (http://www.clinicaltrials.gov/ct/show/NCT00372320?order=). All were identified as initially responding to human aquaporin-1 (hAQP1) gene transfer. They were followed for 3-4 years after AdhAQP1 delivery to one parotid gland. At intervals we examined salivary flow, xerostomic symptoms, saliva composition, vector presence and efficacy in the targeted gland, clinical laboratory data and adverse events. All displayed marked increases (71-500% above baseline) in parotid flow 3-4.7 years after treatment, with improved symptoms for ~2-3 years. There were some changes in [Na+] and [Cl-] consistent with elevated salivary flow, but no uniform changes in secretion of key parotid proteins. There were no clinically significant adverse events, nor consistent negative changes in laboratory parameters. One subject underwent a core needle biopsy of the targeted parotid gland 3.1 years post treatment and displayed evidence of hAQP1 protein in acinar, but not duct, cell membranes. All subjects responding to hAQP1 gene transfer initially had benefits for much longer times. First-generation adenoviral vectors typically yield transit effects, but these data show beneficial effects can continue years after parotid gland delivery.

Scientific frontiers: emerging technologies for salivary diagnostics.

Saliva, a biofluid historically well-studied biochemically and physiologically, has entered the post-genomic 'omics' era, where its proteomic, genomic, and microbiome constituents have been comprehensively deciphered. The translational path of these salivary constituents has begun toward a variety of personalized individual medical applications, including early detection of cancer. Salivary diagnostics is a late-comer, but it is catching up where dedicated resources, like the Salivaomics Knowledge Base (SKB), now have taken center stage in the dissemination of the diagnostic potentials of salivary biomarkers and other translational and clinical utilities.

Acute inhibition of brain-specific Na(+)/H(+) exchanger isoform 5 by protein kinases A and C and cell shrinkage.

Little is known of the functional properties of the mammalian, brain-specific Na(+)/H(+) exchanger isoform 5 (NHE5). Rat NHE5 was stably expressed in NHE-deficient PS120 cells, and its activity was characterized using the fluorescent pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. NHE5 was insensitive to ethylisopropyl amiloride. The transport kinetics displayed a simple Michaelis-Menten relationship for extracellular Na(+) (apparent K(Na) = 27 +/- 5 mM) and a Hill coefficient near 3 for the intracellular proton concentration with a half-maximal activity at an intracellular pH of 6.93 +/- 0.03. NHE5 activity was inhibited by acute exposure to 8-bromo-cAMP or forskolin (which increases intracellular cAMP by activating adenylate cyclase). The kinase inhibitor H-89 reversed this inhibition, suggesting that regulation by cAMP involves a protein kinase A (PKA)-dependent process. In contrast, 8-bromo-cGMP did not have a significant effect on activity. The protein kinase C (PKC) activator phorbol 12-myristrate 13-acetate inhibited NHE5, and the PKC antagonist chelerythrine chloride blunted this effect. Activity was also inhibited by hyperosmotic-induced cell shrinkage but was unaffected by a hyposmotic challenge. These results demonstrate that rat brain NHE5 is downregulated by activation of PKA and PKC and by cell shrinkage, important regulators of neuronal cell function.

Protein kinase A activation phosphorylates the rat ClC-2 Cl- channel but does not change activity.

Phosphorylation-dependent events have been shown to modulate the activity of several members of the mammalian CLC Cl- channel gene family, including the inward rectifier ClC-2. In the present study we investigated the regulation of rat ClC-2 expressed in the TSA-201 cell line (a transformed HEK293 cell line that stably expresses the SV40 T-antigen) by protein kinases. Protein kinase A activation phosphorylated ClC-2 in vivo, whereas stimulation of protein kinase C with phorbol 12-myristate 13-acetate did not. In vitro labeling studies confirmed that protein kinase A could directly phosphorylate ClC-2, and that protein kinase C and Ca2+/calmodulin-dependent protein kinase II did not. Nevertheless, protein kinase A-dependent phosphorylation of CLC-2 failed to regulate either the magnitude or the kinetics of the hyperpolarization-activated Cl- currents. Considered together, we demonstrate that protein kinase A activation results in the phosphorylation of rat ClC-2 in vivo, but this event is independent of Cl- channel activity.

What can transgenic and gene-targeted mouse models teach us about salivary gland physiology?

Thousands of genetically modified mice have been developed since the first reports of stable expression of recombinant DNA in this species nearly 20 years ago. This mammalian model system has revolutionized the study of whole-animal, organ, and cell physiology. Transgenic and gene-targeted mice have been widely used to characterize salivary-gland-specific expression and to identify genes associated with tumorigenesis. Moreover, several of these mouse lines have proved to be useful models of salivary gland disease related to impaired immunology, i.e., Sjögren's syndrome, and disease states associated with pathogens. Despite the availability of genetically modified mice, few investigators have taken advantage of this resource to better their understanding of salivary gland function as it relates to the production of saliva. In this article, we describe the methods used to generate transgenic and gene-targeted mice and provide an overview of the advantages of and potential difficulties with these models. Finally, using these mouse models, we discuss the advances made in our understanding of the salivary gland secretion process.

Mouse down-regulated in adenoma (DRA) is an intestinal Cl(-)/HCO(3)(-) exchanger and is up-regulated in colon of mice lacking the NHE3 Na(+)/H(+) exchanger.

Mutations in human DRA cause congenital chloride diarrhea, thereby raising the possibility that it functions as a Cl(-)/HCO(3)(-) exchanger. To test this hypothesis we cloned a cDNA encoding mouse DRA (mDRA) and analyzed its activity in cultured mammalian cells. When expressed in HEK 293 cells, mDRA conferred Na(+)-independent, electroneutral Cl(-)/CHO(3)(-) exchange activity. Removal of extracellular Cl(-) from medium containing HCO(3)(-) caused a rapid intracellular alkalinization, whereas the intracellular pH increase following Cl(-) removal from HCO(3)(-)-free medium was reduced greater than 7-fold. The intracellular alkalinization in Cl(-)-free, HCO(3)(-)-containing medium was unaffected by removal of extracellular Na(+) or by depolarization of the membrane by addition of 75 mM K(+) to the medium. Like human DRA mRNA, mDRA transcripts were expressed at high levels in cecum and colon and at lower levels in small intestine. The expression of mDRA mRNA was modestly up-regulated in the colon of mice lacking the NHE3 Na(+)/H(+) exchanger. These results show that DRA is a Cl(-)/HCO(3)(-) exchanger and suggest that it normally acts in concert with NHE3 to absorb NaCl and that in NHE3-deficient mice its activity is coupled with those of the sharply up-regulated colonic H(+),K(+)-ATPase and epithelial Na(+) channel to mediate electrolyte and fluid absorption.

Chloride channels and salivary gland function.

Fluid and electrolyte transport is driven by transepithelial Cl- movement. The opening of Cl- channels in the apical membrane of salivary gland acinar cells initiates the fluid secretion process, whereas the activation of Cl- channels in both the apical and the basolateral membranes of ductal cells is thought to be necessary for NaCl re-absorption. Saliva formation can be evoked by sympathetic and parasympathetic stimulation. The composition and flow rate vary greatly, depending on the type of stimulation. As many as five classes of Cl- channels with distinct gating mechanisms have been identified in salivary cells. One of these Cl- channels is activated by intracellular Ca2+, while another is gated by cAMP. An increase in the intracellular free Ca2+ concentration is the dominant mechanism triggering fluid secretion from acinar cells, while cAMP may be required for efficient NaCl re-absorption in many ductal cells. In addition to cAMP- and Ca(2+)-gated Cl- channels, agonist-induced changes in membrane potential and cell volume activate different Cl- channels that likely play a role in modulating fluid and electrolyte movement. In this review, the properties of the different types of Cl- currents expressed in salivary gland cells are described, and functions are proposed based on the unique properties of these channels.

Regulation of chloride channels in secretory epithelia.

Fluid and electrolyte secretion from secretory epithelia is a highly regulated process. Chloride channel activity at the apical membrane determines the rate and direction of salt and water secretion. Multiple classes of Cl- channels with distinct gating mechanisms are involved in moving ions and water. Secretory agonists that induce intracellular increases in two second messenger systems, cAMP and [Ca2+]i, are generally associated with secretion. However, changes in cell volume and the membrane potential may also play a role in regulating fluid and electrolyte secretion in some tissues. In this review we discuss the regulation of the different types of Cl- channels found in secretory epithelia.

Extracellular Mg2+ regulates the intracellular Na+ concentration in rat sublingual acini.

The intracellular free Na+ concentration ([Na+]i) increases during muscarinic stimulation in salivary acinar cells. The present study examined in rat sublingual acini the role of extracellular Mg2+ in the regulation of the stimulated [Na+]i increase using the fluorescent sodium indicator benzofuran isophthalate (SBFI). The muscarinic induced rise in [Na+]i was approximately 4-fold greater in the absence of extracellular Mg2+. When Na+ efflux was blocked by the Na+,K+-ATPase inhibitor ouabain, the stimulated [Na+]i increase was comparable to that seen in an Mg2+-free medium. Moreover, ouabain did not add further to the stimulated [Na+]i increase in an Mg2+-free medium suggesting that removal of extracellular Mg2+ may inhibit the Na+ pump. In agreement with this assumption, ouabain-sensitive Na+ efflux and rubidium uptake were reduced by extracellular Mg2+ depletion. Our results suggest that extracellular Mg2+ may regulate [Na+]i in sublingual salivary acinar cells by modulating Na+ pump activity.

Inhibition of Ca(2+)-dependent Cl- channels from secretory epithelial cells by low internal pH.

The actions of intracellular pH (pHi) on Ca(2+)-dependent Cl- channels were studied in secretory epithelial cells derived from human colon carcinoma (T84) and in isolated rat parotid acinar cells. Channel currents were measured with the whole cell voltage clamp technique with pipette solutions of different pH. Ca(2+)-dependent Cl- channels were activated by superfusing ionomycin to increase the intracellular calcium concentration ([Ca2+]i) or by using pipette solutions with buffered Ca2+ levels. Large currents were activated in T84 and parotid cells by both methods with pHi levels of 7.3 or 8.3. Little or no Cl- channel current was activated with pHi at 6.4. We used on-cell patch clamp methods to investigate the actions of low pHi on single Cl- channel current amplitude in T84 cells. Lowering the pHi had little or no effect on the current amplitude of a 8 pS Cl- channel, but did reduce channel activity. These results suggest that cytosolic acidification may be able to modulate stimulus-secretion coupling in fluid-secreting epithelia by inhibiting the activation of Ca(2+)-activated Cl- channels.

Regulation by extracellular Na+ of cytosolic Mg2+ concentration in Mg(2+)-loaded rat sublingual acini.

The regulation of cytosolic free Mg2+ concentration ([Mg2+]i) in Mg(2+)-loaded rat sublingual mucous acini was examined using the Mg(2+)-sensitive fluorescent indicator mag-fura-2. Loading sublingual acini with 5 mM Mg2+ elevated the [Mg2+]i from 0.35 +/- 0.01 mM to 0.66 +/- 0.01 mM. Removal of extracellular Mg2+ resulted in a significantly faster [Mg2+]i decrease in Mg(2+)-loaded acini than in unloaded acini. Membrane depolarization with high extracellular [K+] and inhibition of P-type ATPases by vanadate did not alter the [Mg2+]i decrease, indicating that the Mg2+ efflux mechanism is not electrogenic. Na(+)-free medium inhibited 80% of the [Mg2+]i decrease suggesting that a Na(+)-dependent Mg2+ efflux pathway mediates the [Mg2+]i decrease. Accordingly, the Na(+)-dependent antiport inhibitor quinidine reduced > 80% of the [Mg2+]i decrease, suggesting that the Na(+)-dependent Mg2+ efflux is mediated by the Na+/Mg2+ antiport system. Mg2+ efflux was also partly driven by K+. The [Mg2+]i decreased was significantly inhibited by carbachol, a muscarinic agonist, but not by cAMP. These results indicate that in sublingual acinar cells a Na(+)-dependent pathway mediates Mg2+ efflux and that muscarinic stimulation may regulate Mg2+ extrusion.

Nicotine increases [Ca2+]i in rat sublingual mucous acini by stimulating neurotransmitter release from presynaptic terminals.

The effects of nicotine on the intracellular free Ca2+ concentration ([Ca2+]i) were examined using the Ca(2+)-sensitive fluorescent dyes indo-1 and fura-2 in isolated rat sublingual mucous acini. Nicotine induced a dose-dependent increase in [Ca2+]i. In contrast to the muscarinic agonist carbachol-induced rise in [Ca2+]i, the nicotine-stimulated increase was abolished in a Ca(2+)-free medium, in the presence of L-type Ca2+ channel blockers (diltiazem and D888), by depolarization (high extracellular K+), and if the intracellular Ca2+ pool was first depleted with thapsigargin, an endoplasmic Ca(2+)-ATPase inhibitor. Furthermore, inhibitors of the nicotine acetylcholine receptor (mecamylamine, decamethonium, hexamethonium, tubocurarine, and alpha-bungarotoxin) blocked the nicotine-stimulated increase in [Ca2+]i without affecting the muscarinic-stimulated [Ca2+]i increase, whereas, muscarinic antagonists (atropine, pirenzepine and 4-diphenylacetoxy-N-methylpiperidine methiodide [4-DAMP]) inhibited both the nicotine- and carbachol-induced [Ca2+]i increases. Nicotine stimulation increased inositol 1,4,5-trisphosphate (IP3) content by 50%. Inhibition of the IP3-sensitive intracellular Ca2+ release pathway with 8-(diethylamino)-ocytl-3,4,5-trimethoxybenzoate (TMB-8) prevented the nicotine-induced increase in [Ca2+]i. Confocal imaging of [Ca2+]i indicated that the nicotine-induced and the carbachol-induced increases in [Ca2+]i occurred in the same cells within an acinus. However, in single sublingual acinar cells nicotine did not increase [Ca2+]i, whereas, carbachol did. Taken together, these results suggest that nicotine first triggers the release of acetylcholine from presynaptic nerve terminals associated with the dispersed sublingual acini which then activates muscarinic receptors.

Inhibitors of the intracellular Ca2+ release mechanism prevent muscarinic-induced Ca2+ influx in rat sublingual mucous acini.

The effects of inhibitors of the intracellular Ca2+ release mechanism on divalent cation fluxes were examined in acinar cells loaded with the Ca(2+)-sensitive, Mn(2+)-quenchable dye, fura-2. TMB-8 and dantrolene (DTL) dramatically inhibited the carbachol (CCh)-stimulated increase in [Ca2+]i and Mn2+ influx. These agents do not directly inhibit divalent cation entry since addition of TMB-8 or DTL after CCh stimulation did not block Mn2+ influx. TMB-8 did not influence the [Ca2+]i increase or the Mn2+ influx produced by thapsigargin. These results indicate that TMB-8 and DTL do not interfere with divalent cation influx by inhibiting a step distal to depletion of the intracellular Ca2+ pool. TMB-8 and DTL did not significantly influence the muscarinic-stimulated production of inositol trisphosphate (IP3) and inositol tetrakisphosphate (IP4), although TMB-8, but not DTL, did decrease the CCh-stimulated 1,4,5-IP3 levels approximately 55%. The above results directly demonstrate that the filling state of the intracellular Ca2+ store primarily regulates the Ca2+ entry mechanism in sublingual mucous acinar cells.

Na+ influx is mediated by Na(+)-K(+)-2Cl- cotransport and Na(+)-H+ exchange in sublingual mucous acini.

The intracellular free Na+ concentration ([Na+]i) was studied using dual-wavelength microfluorometry of the fluorescent Na+ indicator sodium-binding benzofuran isophthalate (SBFI) to determine the mechanism(s) by which muscarinic stimulation increases the Na+ content in rat sublingual mucous acini. [Na+]i was 15.5 +/- 0.7 mM in acini superfused with a Na(+)-containing medium (135 mM Na+). Application of ouabain, a Na(+)-K(+)-adenosinetriphosphatase inhibitor, resulted in an increase in [Na+]i (approximately 75% in 10 min), whereas replacement of extracellular Na+ with N-methyl-D-glucamine induced a gradual decrease in [Na+]i (approximately 55% decrease in 5 min). The recovery of [Na+]i in Na(+)-depleted acini was K+ and Cl- dependent and was inhibited by bumetanide (Bum), a specific Na(+)-K(+)-2Cl- cotransport inhibitor, and by 5-(N-methyl-N-isobutyl)amiloride (MIBA), an amiloride derivative that specifically blocks Na(+)-H+ exchange. Stimulation with a muscarinic agonist (10 microM carbachol) resulted in a dramatic increase in the [Na+]i [approximately 180%, half time (t1/2) approximately 1 min] and a net increase in Na+ content, as measured with 22Na+ (approximately 110%, t1/2 approximately 1 min). Both the initial rate of the increase in [Na+]i and the magnitude of the net increase in Na+ content were dramatically blunted by Bum and MIBA. Increasing the intracellular free Ca2+ concentration ([Ca2+]i) with ionomycin, a Ca2+ ionophore, resulted in an increase in [Na+]i. Preventing the [Ca2+]i increase by chelating cytosolic Ca2+ with bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid completely abolished the agonist-induced evaluation in [Na+]i.(ABSTRACT TRUNCATED AT 250 WORDS)

Hypogastric ganglion perinatal development: evidence for androgen specificity via androgen receptors.

The hypogastric ganglion (HG) has previously been shown to be sensitive to both the organizational and activational influences of testosterone. The current investigations examined whether testosterone exerts similar effects prenatally, whether these events are specifically controlled by androgen, and whether androgens might directly masculinize the HG. Prenatal treatment with an anti-androgen, flutamide, resulted in significant decreases in the adult levels of tyrosine hydroxylase (TH) activity, an index of postsynaptic noradrenergic ontogeny, and choline acetyltransferase (ChAT) activity, a marker for presynaptic terminal formation. In addition, testosterone propionate and dihydrotestosterone benzoate reversed the effects of neonatal castration on the development of TH and ChAT activities. In contrast estradiol benzoate was unable to restore enzyme activities. To determine whether the above observations might be produced by direct effects on the HG, androgen cytosol receptor characteristics were studied. Competition and saturation analyses demonstrate that the affinity and specificity of the androgen cytosol receptor in the HG are similar to that displayed in the pituitary, which has previously been shown to contain androgen receptors. These results suggest that the adult levels of TH and ChAT activities are organized during prenatal and early postnatal development. In addition, the organization of the HG appears to be androgen specific. The presence of cytosol androgen receptors suggests that the organizational effects of androgens are possibly induced by a direct mechanism.

Androgen-specific critical periods for the organization of the major pelvic ganglion.

Previous studies indicate that the major pelvic ganglion (PG) is dependent on testosterone for normal development. Tyrosine hydroxylase (T-OH), DOPA decarboxylase, and choline acetyltransferase (CAT) activities are significantly reduced by postnatal castration on day 10-11, while testosterone replacement therapy reversed all developmental enzyme activity deficits (Melvin and Hamill, 1987). In the present studies castration on the day of birth combined with various testosterone-replacement paradigms produced effects demonstrating that the PG is sensitive to testosterone dosage and time of administration during early postnatal development. Gonadal hormone replacement experiments show that the androgens testosterone and dihydrotestosterone were effective in restoring T-OH and CAT activity deficits produced by neonatal castration. Estrogen therapy reversed the deficits in CAT activity, but was ineffective in reversing the alterations in T-OH activity. Treatment of pregnant dams with the anti-androgen flutamide altered the ontogeny of T-OH and CAT activities in pups despite replacement therapy on the day of birth. Thus, androgen-critical periods exist prenatally as well as postnatally. These studies suggest that the organization of PG development is critically dependent on both the time of exposure and dose of testosterone. Prenatal and postnatal critical periods appear to exist. In addition, the lack of an effect of estradiol on tyrosine hydroxylase activity suggests that androgens are specifically responsible for organizing the noradrenergic development of the PG.