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Salivary gland dysfunction is a feature in diabetes and hypertension. We hypothesized that sodium-glucose cotransporter 1 (SGLT1) participates in salivary dysfunctions through a sympathetic- and protein kinase A (PKA)-mediated pathway. In Wistar-Kyoto (WKY), diabetic WKY (WKY-D), spontaneously hypertensive (SHR), and diabetic SHR (SHR-D) rats, PKA/ SGLT1 proteins were analyzed in parotid and submandibular glands, and the sympathetic nerve activity (SNA) to the glands was monitored. Basal SNA was threefold higher in SHR (P < 0.001 vs. WKY), and diabetes decreased this activity (~50%, P < 0.05) in both WKY and SHR. The catalytic subunit of PKA and the plasma membrane SGLT1 content in acinar cells were regulated in parallel to the SNA. Electrical stimulation of the sympathetic branch to salivary glands increased (~30%, P < 0.05) PKA and SGLT1 expression. Immunohistochemical analysis confirmed the observed regulations of SGLT1, revealing its location in basolateral membrane of acinar cells. Taken together, our results show highly coordinated regulation of sympathetic activity upon PKA activity and plasma membrane SGLT1 content in salivary glands. Furthermore, the present findings show that diabetic- and/or hypertensive-induced changes in the sympathetic activity correlate with changes in SGLT1 expression in basolateral membrane of acinar cells, which can participate in the salivary glands dysfunctions reported by patients with these pathologies. diabetes mellitus; sodium glucose cotransporter 1; hypertension; protein kinase A; [beta]-adrenergic activity doi: 10.1152/ajpendo.00395.2010.

Perry C, Baker OJ, Reyland ME,Grichtchenko II. PKC[alpha][beta][gamma]- and PKC[delta]-dependent endocytosis of NBCel-A and NBCel-B in salivary pasrotid Acinar cells. Am J Physiol Cell Physiol 297: C1409-C1423, 2009. First Published September 30, 2009; doi: 10.1152.ajpcell.00028.2009.--We examined membrane trafficking of NBCe1-A and NBCe1-B variants of the electrogenic [Na.sup.+]-HC[O.sup.3.sub.-] cotransporter (NBCe1) encoded by the SLC4A4 gene, using confocal fluorescent microscopy in rat parotid acinar cells (ParC5 and ParC10). We showed that yellow fluorescent protein (YFP)-tagged NBCe1-A and green fluorescent protein (GFP)-tagged NBCe1-B are colocalized with E-cadherin in the basolateral membrane (BLM) but not with the apical membrane marker zona occludens 1 (ZO-1). We inhibited constitutive recycling with monensin and W13 and detected that NBCe1-A and NBCe1-B accumulated in vesicles marked with the early endosomal marker early endosome antigen-1 (EEA1), with a parallel loss from the BLM. We observed that NBCe1-A and NBCe1-B undergo massive carbachol (CCh)-stimulated redistribution from the BLM into early endosomes. We showed that internalization of NBCe1-A and NBCe1-B was prevented by the general PKC inhibitor GF-109203X, the PKC[alpha][beta][gamma]-specific inhibitor Go-6976, and the PKC[delta]-specific inhibitor rottlerin. We verified the involvement of PKC[delta] by blocking CChinduced internalization of NBCe1-A-cyan fluorescent protein (CFP) in cells transfected with dominant-negative kinase-dead (Lys376Arg) PKC[delta]-GFP. Our data suggest that NBCe1-A and NBCe1-B undergo constitutive and CCh-stimulated endocytosis regulated by conventional PKCs (PKC[alpha][beta][gamma]) and by novel PKC[delta] in rat epithelial cells. To help develop a more complete model of the role of NBCe1 in parotid acinar cells we also investigated the initial phase of the secretory response to cholinergic agonist. In an Ussing chamber study we showed that inhibition of basolateral NBCe1 with 5-chloro-2,3-dihydro-3-(hydroxy-2-thienylmethylene)-2-oxo-1H-indole-1- carboxamide (tenidap) significantly decreases an initial phase of luminal anion secretion measured as a transient short-circuit current ([I.sub.sc]) across ParC10 cell monolayers. Using trafficking and functional data we propose a model that describes a physiological role of NBC in salivary acinar cell secretion. internalization; model of saliva secretion; Ussing chamber; SLC4A4 doi: 10.1152/ajpcell.00028.2009

Paradoxically, bilateral transection of the chorda tympani nerve (CTX) raises the taste discrimination threshold for the free fatty acid, linoleic acid (LA), yet the chorda tympani nerve (CT) is unresponsive to lingual application of LA alone. LA may require a background of saliva to activate taste cells, since CTX decreases saliva production through denervation of the submaxillary and sublingual salivary glands. To assess the role of saliva, we measured LA taste discrimination thresholds for animals whose submaxillary and sublingual salivary glands were removed and also recorded CT responses to LA mixed in artificial saliva. Partial desalivation shifted LA discrimination thresholds from between 5.5 and 1l [micro]M to between 11 and 22 [micro]M. However, this effect was not as pronounced as previously seen with CTX animals. Surprisingly, the CT was unresponsive to LA mixed with artificial saliva, suggesting that artificial saliva may lack components necessary for LA taste. Additionally, fats may primarily enhance other tastes. We previously reported that LA increases CT responses to monosodium glutamate (MSG). Thus we also recorded CT whole nerve responses to taste mixtures of LA and sodium chloride (NaCl), sucrose (SUC), citric acid (CA), or quinine hydrochloride (QHCl) in anesthetized rats. We found that LA increased CT responses to NaCl but did not alter CT responses to SUC, CA, and QHCl. Thus CT recordings either lack the sensitivity to detect small changes to SUC, CA, and QHCl or LA may affect CT responses to MSG and NaCl only, perhaps by specifically modulating gustatory processing of [Na.sup.+]. fat taste; free fatty acids; electrophysiology; electrolytes doi: 10.1152/ajpregu.00217.2009

The circadian clock in the suprachiasmatic nucleus (SCN) maintains phase synchrony among circadian oscillators throughout the organism. Environmental light signals entrain the SCN, but timed, limited meal access acts as an overriding time cue for several peripheral tissues. We present data from a peripheral oscillator, the submaxillary salivary gland, in which temporal restriction of meals fails to entrain gene expression. In day-fed rats, submaxillary gland rhythms in expression of the clock gene Period1 (Per1) stay entrained to the light cycle (peaking at night) or become arrhythmic. This result suggests that feeding cues compete weakly with light cycle cues to set the phase of clock genes in this tissue. Since the submaxillary glands receive sympathetic innervation originating in the SCN, which relays light cycle cues to other oscillators, we attempted to assess the role of this neural input in phase control of submaxillary Per1 expression. We sympathetically denervated the submaxillary glands before subjecting rats to daytime-restricted feeding. After denervation, Per1 rhythms in all submaxillary glands shifted phase 180[degrees] and entrained to daytime feeding. These results support the hypothesis that peripheral oscillators may receive multiple signals contributing to their phase of entrainment. Sympathetic efferents from the SCN can relay light cycle information, while other external cues may reach tissues through other efferents or nonneural pathways. In an abnormal, disruptive regimen such as daytime-restricted feeding, these different signals compete. Arrhythmicity may result if one signal is not clearly dominant. Elimination of the dominant signal (e.g., surgical sympathectomy) may allow a secondary signal to control phase. submaxillary gland; Period1; sympathectomy; restricted feeding

A greater than twofold diversity in the expression level of aquaporin 5 (AQP5) has been observed in the membrane fraction of the submandibular gland (SMG) in Sprague-Dawley rats (Murdiastuti K, Miki O, Yao C, Parvin MN, Kosugi-Tanaka C, Akamatsu T, Kanamori N, and Hosoi K. Pflugers Arch 445: 405-412, 2002). In the present study, breeding between brother and sister rats was repeated within high AQP5 producers and low ones to obtain inbred offspring. High- and low-producer rats from 3rd to 18th generations were used for experiments. By Western blotting, levels of AQP5 proteins in the parotid and lacrimal glands, and lungs were all low in low producers, whereas they were all high in high producers, implying genetic variations of the gene for this water channel. Despite this implication, AQP5 mRNA levels were almost the same between the two groups by Northern blotting, suggesting the irrelevance of transcriptional regulation for this diversity. AQP5 cDNAs from the SMGs of the two groups were sequenced. The nucleotide sequence of AQP5 cDNA from low producers indicated the existence of a point mutation at nt 308 (G308A), leading to a replacement of [sup.103]Gly with [sup.103]Asp in the third transmembrane domain, but no alteration was detected in the Kozak area. The existence of such a mutation was confirmed by the assessment of genomic DNA also. This mutation may have resulted in an abnormal membrane insertion or ineffective trafficking of AQP5, since the rats having this mutation showed extremely low membrane expression of AQP5 in the SMG acinar cells and decreased water secretion from their salivary glands. water channel; salivary gland

The [beta]-adrenergic receptor agonist isoproterenol exerts growth-promoting effects on salivary glands. In this study, activation of ERKs, members of the mitogen-activated protein kinase family, by isoproterenol was examined in a human salivary gland cell line (HSY). Immunoblot analysis indicated that isoproterenol ([10.sup.-5] M) induced transient activation of ERK1/2 (4.4-fold relative to basal at 10 min) similar to that caused by EGF (6.7 fold). Isoproterenol, like EGF, also induced phosphorylation of the EGF receptor. However, inhibition of EGF receptor phosphorylation by the tyrphostin AG-1478 only partially attenuated isoproterenol-induced ERK phosphorylation, whereas EGF-responsive ERK activation was completely blocked. The [G.sub.i] inhibitor pertussis toxin also caused partial inhibition of isoproterenol-stimulated ERK activation. The cAMP analog 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP) and the cAMP-elevating agents IBMX and cholera toxin produced transient ERK1/2 activation, similar to the effect of isoproterenol, in HSY cells. The stimulatory effects of isoproterenol and cAMP on ERK phosphorylation were not reduced by the PKA inhibitor H-89, whereas the Src family inhibitor 4-amino5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidase (PP2) and transfection of a dominant-negative Src construct diminished isoproterenol-induced ERK activation. Isoproterenol induced marked overexpression of the cell growth-related adhesion molecule CD44, and this effect of isoproterenol was abolished by the ERK pathway inhibitor PD-98059. In summary, we show a dual mechanism of isoproterenol-induced ERK phosphorylation in HSY cells--one pathway mediated by EGF receptor transactivation and the other by an EGF receptor-independent pathway possibly mediated by cAMP. Our results also suggest that isoproterenol-induced growth of salivary tissue may involve ERK-mediated CD44 expression. mitogen-activated protein kinase; CD44

To investigate the interaction between the ion channels and transporters in the salivary fluid secretion, we measured the membrane voltage ([V.sub.m]) and intracellular concentrations of [Ca.sup.2+], [Na.sup.+] ([[Na.sup.+]].sub.c]), [Cl.sup.-], and [H.sup.+] ([pH.sub.i]) in rat submandibular gland acini (RSMGA). After a transient depolarization induced by a short application of acetylcholine (ACh; 5 [micro]M, 20 s), RSMGA showed strong delayed hyperpolarization ([V.sub.h, ACh]; -95 [+ or -] 1.8 mV) that was abolished by ouabain. In the HC[O.sup.-.sub.3]- free condition, the [V.sub.h.ACh] was also blocked by bumetanide, a blocker of [Na.sup.+]-[K.sup.+]-2[Cl.sup.-] cotransporter (NKCC). In the presence of HC[O.sup.-.sub.3] (24 meq, bubbled with 5% C[O.sub.2]), however, the [V.sub.h,ACh] was not blocked by bumetanide, but it was suppressed by ethylisopropylamiloride (EIPA), a [Na.sup.+]/[H.sup.+] exchanger (NHE) inhibitor. Similarly, the ACh-induced increase in [[Na.sup.+]].sub.c] was totally blocked by bumetanide in the absence of HC[O.sup.-.sub.3], but only by one-half in the presence of HC[O.sup.-.sub.3]. ACh induced a prominent acidification of [pH.sub.i] in the presence of HC[O.sup.- .sub.3], and the acidification was further increased by EIPA treatment. Without HC[O.sup.-.sub.3], an application of ACh strongly accelerated the NKCC activity that was measured from the decay of [pH.sub.i] during the application of N[H.sup.+.sub.4] (20 mM). Notably, the ACh-induced activation of NKCC was largely suppressed in the presence of HC[O.sup.-.sub.3]. In summary, the ACh-induced anion secretion in RSMGA is followed by the activation of NKCC and NHE, resulting an increase in [[Na.sup.+]].sub.c]. The intracellular [Na.sup.+.sub.4]-induced activation of electrogenic [Na.sup.+]/[K.sup.+]-ATPase causes [V.sub.h,ACh]. The regulation of NKCC and NHE by ACh is strongly affected by the physiological level of HC[O.sup.-.sub.3]. [Na.sup.+]-[K.sup.+]-2[Cl.sup.-] cotransporter; [Na.sup.+]/[H.sup.+] exchanger; acetylcholine; secretion; membrane voltage

Although [Ca.sup.2+]-activated [K.sup.+] ([K.sub.Ca]) channels distinct from maxi-[K.sup.+] channels have been suggested to contribute to muscarinically stimulated [K.sup.+] currents in salivary acinar cells, the molecular nature of the channels is unclear. Using electrophysiological and RT-PCR techniques, we have now investigated the involvement of SK4/IK1-like channels in native [K.sub.Ca] currents in bovine parotid acinar (BPA) cells. [Ca.sup.2+]-dependent [K.sup.+] efflux from perfused bovine parotid tissues was not inhibited by a maxi-[K.sup.+] channel blocker, tetraethylammonium (TEA). Whole cell recordings from BPA cells showed a TEA-insensitive [K.sub.Ca] conductance, which was highly permeable to [Rb.sup.+]. In inside-out macropatches, TEA-insensitive [Rb.sup.+] currents were activated by [Ca.sup.2+] with half-maximal values of 0.4 [micro]M. 1-Ethyl-2-benzimidazolinone (1-EBIO) increased the [Ca.sup.2+] sensitivity of the currents. The calmodulin antagonists trifluoperazine, calmidazolium, and W-7 inhibited the [Ca.sup.2+]-activated [Rb.sup.+] currents. In outside-out macropatches, [Ca.sup.2+]-activated [Rb.sup.+] currents were inhibited by [Ba.sup.2+], quinine, clotrimazole, and charybdotoxin but not by d-tubocrarine or apamin. RT-PCR analysis showed transcripts of SK4/IK1 in BPA cells. These results collectively suggest that SK4/IK1-like channels mediate the native [K.sub.ca] currents in BPA cells. patch clamp; salivary secretion; HC[O.sup.-.sub.3] transport

Experimental evidence shows that Na(super +)/H(super +) exchanger (NHE) genes play important functions in parotid acinar and ductal cells in rats. This observation was confirmed in an experiment that investigated the molecular and pharmacological properties of NHE in rat parotid salivary gland cells. Results showed that NHE1 is the dominant NHE isoform in acinar cells while NHE3 is a significant contributor to pH regulation in ductal cells. In addition, NHE activity was suppressed by low concentrations of the amiloride derivative ethylisopropyl amiloride in the case of acinar cells superfused with a physiological salt solution.

The ontogeny of the aquaporin proteins AQP1, AQP3, AQP4 and AQP5 in the distal lung, trachea, nasopharynx and salivary glands of rat were investigated to shed light on the possible roles of aquaporins in the physiology of these tissues. Results showed significant complexity of aquaporin protein expression in respiratory tract and glandular epithelial. Also, the aquaporins reached peak expression at different postnatal days and were involved in the removal of fluid from the interstitium of the developing organs.

The expression and biological function of the 25-kDa leucine-rich parotid secretory protein (PSP) were analyzed in non-obese diabetic (NOD) mice. Analysis of NOD mice tissues and organs indicated the presence of PSP messenger RNA in lacrimal glands including PSP gene transcription in the NOD heart and pancreas. However, abnormal regulation of PSP gene transcription were detected in NOD tissues. Furthermore, the 25-kDa leucine-rich glycoprotein exhibited potential antimicrobial functions in murine saliva and tears.

The expression and distribution P2Y G protein-coupled receptors in rat salivary gland cells were analyzed by reverse transcriptase-polymerase chain reaction. The expression of P2Y receptors in rat salivary glands was developmentally regulated via postranslational and translational modification. Furthermore, salivary gland cells from older rats exhibited lower levels of P2Y receptor messenger RNA expression compared to the salivary gland cells of postnatal rats.

The effects adenosine 3'5'-cyclic monophosphate (cAMP) on tetraethylammonium (TEA)-insensitive, Ca2+-activated K+ conductance were analyzed during the potentiation of the A-23187 Ca2+ ionophore. Analysis of perfused intact rat submandibular glands indicated the role of cAMP-related agents in enhancing basolateral K+ efflux and the initial phase of biphasic fluid secretion induced by A-23187. Furthermore, cAMP-mediated potentiation of Ca2+-activated fluid secretion in rat submandibular gland was followed by the potentiation of TEA-insensitive, Ca2+-activated K+ conductance.

Secretion experiments with mucous acini cells isolated from rat sublingual glands were conducted to determine the role of M1 and M3 muscarinic receptor subtypes in exocrine secretion. The specific interaction of M1-toxin with acinar M1 receptors and the inability of M1-toxin to affect acinar secretion in response to nonmuscarinic secretagogues exhibited the role of M1 receptors in salivary gland exocrine secretion. Furthermore, the role of M1 receptors in exocrine secretion was exhibited by the 40% reduction in maximal secretory response of acini cells incubated with M1-toxin.

The intracellular hydrogen-ion concentration (pH) in salivary glands and the nonspecific cation channels of polyphosphoinositide-specific P2z receptors were analyzed by acid load technique in rat submandibular cells. The rat submandibular cells exhibited lower pH after treatment with ammonium chloride. The pH recovery rate was also affected by ethylisopropylamiloride which inhibited Na2+/H+ exchange. Furthermore, the increased rate of pH recovery after treatment with adenosine triphosphate was mediated by P2z purigenic receptors and nonspecific cation channels.

Interferon (IFN)-gamma is a class of gamma interferons that prevents the proliferation of a number of cell types. Human salivary gland (HSG) cells treated with fetal bovine albumin serum showed rapid increases in internal calcium concentration but treatment with IFN-gamma in the presence of tumor necrosis factor (TNF)-alpha suppressed this proliferative effect. Likewise, carbachol- and thapsigargin-stimulated increases in intracellular Ca2+ stores decreased upon treatment with IFN-gamma plus TNF-alpha. These results suggest that IFN-gamma and TNF-alpha causes the depletion of intracellular Ca2+ stores in HSG cells.

Basolateral Na+-K+-Cl- transport appears as the main tool driving Cl- secretion and fluid secretion in labial acinar cells. Human labial acini stimulated with carbachol showed little or no intracellular acidification, which occurs in other salivary glands. Beta-adrenergic stimulation depressed Na+/H+ exchange activity in human labial acini while muscarinic stimulation enhanced it. Muscarinic simulation had no effect on Na+-K+-Cl- cotransport activity and beta-adrenergic stimulation only slightly increased it.

A experimental technique involving conductive transport pathway-mediated flux increase helps study purified rat submandibular intralobular duct membrane vesicles' 22Na+ transport properties. An Na+-channel sensitive to amiloride and having pharmacological homology with 'low-amiloride affinity channels' is present in these salivary intralobular ducts. This Na+ transport pathway is not affected by temperature.

Adenovirus vectors can efficiently deliver genes to various cell types in the salivary glands of rats in vivo, to cell lines derived from salivary glands in vitro and to human salivary glands ex vivo. Treatment of the recombinant adenovirus vectors, Ad alpha 1 AT comprising of the human alpha(sub 1) antitrypsin, a secreted protein, and Ad.RSV beta gal comprising of the intracellular protein beta-galactosidase to salivary gland cell lines in vitro exhibits exogenous gene expression.

Luo, W., L. R. Latchney, and D. J. Culp. G protein coupling to [M.sub.1] and [M.sub.3] muscarinic receptors in sublingual glands. Am J Physiol Cell Physiol 280: C884-C896, 2001.--Rat sublingual gland [M.sub.1] and [M.sub.3] muscarinic receptors each directly activate exocrine secretion. To investigate the functional role of coreceptor expression, we determined receptor-G protein coupling. Although membrane proteins of 40 and 41 kDa are ADP-ribosylated by pertussis toxin (PTX), and 44 kDa proteins by cholera toxin (CTX), both carbachol-stimulated high-affinity GTPase activity and the GTP-induced shift in agonist binding are insensitive to CTX or PTX. Carbachol enhances photoaffinity labeling ([[Alpha]-[sup.32]P]GTP-azidoaniline) of only 42-kDa proteins that are subsequently tractable to immunoprecipitation by antibodies specific for G[[Alpha].sub.q] or G[[Alpha].sub.11] but not G[[Alpha].sub.12] or G[[Alpha].sub.13]. Carbachol-stimulated photoaffinity labeling as well as phosphatidylinositol 4,5-bisphosphate ([PIP.sub.2]) hydrolysis is reduced 55% and 60%, respectively, by [M.sub.1] receptor blockade with m1-toxin. G[[Alpha].sub.q/11]-specific antibody blocks carbachol-stimulated [PIP.sub.2] hydrolysis. We also provide estimates of the molar ratios of receptors to G[[Alpha].sub.q] and G[[Alpha].sub.11]. Although simultaneous activation of [M.sub.1] and [M.sub.3] receptors is required for a maximal response, both receptor subtypes are coupled to G[[Alpha].sub.q] and G[[Alpha].sub.11] to stimulate exocrine secretion via redundant mechanisms. salivary glands; muscarinic cholinergic receptors; mucous cells; m1-toxin; G[[Alpha].sub.q/11]; exocrine secretion

Zhang, Bin-Xian, Chih-Ko Yeh, Tazuko K. Hymer, Meyer D. Lifschitz, and Michael S. Katz. EGF inhibits muscarinic receptor-mediated calcium signaling in a human salivary cell line. Am J Physiol Cell Physiol 279: C1024-C1033, 2000.--The effects of epidermal growth factor (EGF) on intracellular calcium ([[[Ca.sup.2+]].sub.i]) responses to the muscarinic agonist carbachol were studied in a human salivary cell line (HSY). Carbachol ([10.sup.-4] M)-stimulated [[[Ca.sup.2+]].sub.i] mobilization was inhibited by 40% after 48-h treatment with 5 x [10.sup.-10] M EGF. EGF also reduced carbachol-induced [[[Ca.sup.2+]].sup.i] in [Ca.sup.2+]-free medium and [Ca.sup.2+] influx following repletion of extracellular [Ca.sup.2+]. Under [Ca.sup.2+]-free conditions, thapsigargin, an inhibitor of [Ca.sup.2+] uptake to internal stores, induced similar [[[Ca.su.2+]].sub.i] signals in control and EGF-treated cells, indicating that internal [Ca.sup.2+] stores were unaffected by EGF; however, in cells exposed to thapsigargin, [Ca.sup.2+] influx following [Ca.sup.2+] repletion was reduced by EGF. Muscarinic receptor density, assessed by binding of the muscarinic receptor antagonist L-[benzilic-4,4'-[sup.3]HCN]quinuclidinyl benzilate ([[.sup.3]H]QNB), was decreased by 20% after EGF treatment. Inhibition of the carbachol response by EGF was not altered by phorbol ester-induced downregulation of protein kinase C (PKC) but was enhanced upon PKC activation by a diacyl-glycerol analog. Phosphorylation of mitogen-activated protein kinase (MAP kinase) and inhibition of the carbachol response by EGF were both blocked by the MAP kinase pathway inhibitor PD-98059. The results suggest that EGF decreases carbachol-induced [Ca.sup.2+] release from internal stores and also exerts a direct inhibitory action on [Ca.sup.2+] influx. A decline in muscarinic receptor density may contribute to EGF inhibition of carbachol responsiveness. The inhibitory effect of EGF is mediated by the MAP kinase pathway and is potentiated by a distinct modulatory cascade involving activation of PKC. EGF may play a physiological role in regulating muscarinic receptor-stimulated salivary secretion. carbachol; signal transduction; protein kinase C; mitogen-activated protein kinase; epidermal growth factor