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.

Activation of salivary secretion: coupling of cell volume and [Ca2+]i in single cells.

High-resolution differential interference contrast microscopy and digital imaging of the fluorescent calcium indicator dye fura-2 were performed simultaneously in single rat salivary gland acinar cells to examine the effects of muscarinic stimulation on cell volume and cytoplasmic calcium concentration ([Ca2+]i). Agonist stimulation of fluid secretion is initially associated with a rapid tenfold increase in [Ca2+]i as well as a substantial cell shrinkage. Subsequent changes of cell volume in the continued presence of agonist are tightly coupled to dynamic levels of [Ca2+]i, even during [Ca2+]i oscillations. Experiments with Ca2+ chelators and ionophores showed that physiological elevations of [Ca2+]i are necessary and sufficient to cause changes in cell volume. The relation between [Ca2+]i and cell volume suggests that the latter reflects the secretory state of the acinar cell. Agonist-induced changes in [Ca2+]i, by modulating specific ion permeabilities, result in solute movement into or out of the cell. The resultant cell volume changes may be important in modulating salivary secretion.

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.

The calcium-activated potassium channel KCa3.1 plays a central role in the chemotactic response of mammalian neutrophils.

AIM: Neutrophils are the first cells to arrive at sites of injury. Nevertheless, many inflammatory diseases are characterized by an uncontrolled infiltration and action of these cells. Cell migration depends on volume changes that are governed by ion channel activity, but potassium channels in neutrophil have not been clearly identified. We aim to test whether KCa3.1 participates in neutrophil migration and other relevant functions of the cell. METHODS: Cytometer and confocal measurements to determine changes in cell volume were used. Cells isolated from human, mouse and horse were tested for KCa3.1-dependent chemotaxis. Chemokinetics, calcium handling and release of reactive oxygen species were measured to determine the role of KCa3.1 in those processes. A mouse model was used to test for neutrophil recruitment after acute lung injury in vivo. RESULTS: We show for the first time that KCa3.1 is expressed in mammalian neutrophils. When the channel is inhibited by a pharmacological blocker or by genetic silencing, it profoundly affects cell volume regulation, and chemotactic and chemokinetic properties of the cells. We also demonstrated that pharmacological inhibition of KCa3.1 did not affect calcium entry or reactive oxygen species production in neutrophils. Using a mouse model of acute lung injury, we observed that Kca3.1(-/-) mice are significantly less effective at recruiting neutrophils into the site of inflammation. CONCLUSIONS: These results demonstrate that KCa3.1 channels are key actors in the migration capacity of neutrophils, and its inhibition did not affect other relevant cellular functions.

Activation of calcium-dependent chloride channels in rat parotid acinar cells.

The Ca2+ and voltage dependence of Ca(2+)-activated Cl- currents in rat parotid acinar cells was examined with the whole-cell patch clamp technique. Acinar cells were dialyzed with buffered free Ca2+ concentrations ([Ca2+]i) from < 1 nM to 5 microM. Increasing [Ca2+]i induced an increase in Cl- current at all membrane potentials. In cells dialyzed with [Ca2+]i > 25 nM, depolarizing test pulses activated a Cl- current that was composed of an instantaneous and a slow monoexponential component. The steady-state current-voltage relationship showed outward rectification at low [Ca2+]i but became more linear as the [Ca2+]i increased because of a shift in Cl- channel activation toward more negative voltages. The Ca2+ dependence of steady-state channel activation at various membrane voltages was fit by the Hill equation. The apparent Kd and Hill coefficient obtained from this analysis were both functions of membrane potential. The Kd decreased from 417 to 63 nM between -106 and +94 mV, whereas the Hill coefficient was always > 1 and increased to values as large as 2.5 at large positive potentials. We found that a relatively simple mechanistic model can account for the channel steady-state and kinetic behavior. In this model, channel activation involves two identical, independent, sequential Ca2+ binding steps before a final Ca(2+)-independent transition to the conducting conformation. Channel activation proceeds sequentially through three closed states before reaching the open state. The Ca2+ binding steps of this model have a voltage dependence similar to that of the Kd from the Hill analysis. The simplest interpretation of our findings is that these channels are directly activated by Ca2+ ions that bind to sites approximately 13% into the membrane electric field from the cytoplasmic surface.

Functional differences in the acinar cells of the murine major salivary glands.

In humans, approximately 90% of saliva is secreted by the 3 major salivary glands: the parotid (PG), the submandibular (SMG), and the sublingual glands (SLG). Even though it is known that all 3 major salivary glands secrete saliva by a Cl(-)-dependent mechanism, salivary secretion rates differ greatly among these glands. The goal of this study was to gain insight into the properties of the ion-transporting pathways in acinar cells that might account for the differences among the major salivary glands. Pilocarpine-induced saliva was simultaneously collected in vivo from the 3 major salivary glands of mice. When normalized by gland weight, the amount of saliva secreted by the PG was more than 2-fold larger than that obtained from the SMG and SLG. At the cellular level, carbachol induced an increase in the intracellular [Ca(2+)] that was more than 2-fold larger in PG and SMG than in SLG acinar cells. Carbachol-stimulated Cl(-) efflux and the protein levels of the Ca(2+)-activated Cl(-) channel TMEM16A, the major apical Cl(-) efflux pathway in salivary acinar cells, were significantly greater in PG compared with SMG and SLG. In addition, we evaluated the transporter activity of the Na(+)-K(+)-2Cl(-) cotransporters (NKCC1) and anion exchangers (AE), the 2 primary basolateral Cl(-) uptake mechanisms in acinar cells. The SMG NKCC1 activity was about twice that of the PG and more than 12-fold greater than that of the SLG. AE activity was similar in PG and SLG, and both PG and SLG AE activity was about 2-fold larger than that of SMG. In summary, the salivation kinetics of the 3 major glands are distinct, and these differences can be explained by the unique functional properties of each gland related to Cl(-) movement, including the transporter activities of the Cl(-) uptake and efflux pathways, and intracellular Ca(2+) mobilization.

Membrane potential regulates Ca2+ uptake and inositol phosphate generation in rat sublingual mucous acini.

In salivary acinar cells, muscarinic-induced fluid secretion is associated with a 1,4,5-IP3 induced increase in the cytosolic free Ca2+ concentration ([Ca2+]i), which in turn activates Ca(2+)-dependent K+ and Cl- channels that modulate the membrane potential. In the present study the influence of the membrane potential on [Ca2+]i and inositol phosphates was monitored in rat sublingual mucous acini. Depolarization induced by switching from 5.8 mM extracellular K+ ([K+]e) to 116 mM [K+]e resulted in a transient increase in the [Ca2+]i measured using the Ca2+ sensitive fluorescent indicator Fura-2. This initial rapid (t1/2 approximately 5 s) increase (approximately 3-fold) in [Ca2+]i was dependent on extracellular Ca2+, insensitive to nifedipine, and followed by establishment of a 'new' resting [Ca2+]i, approximately 35% higher than the level in physiological [K+]e. Depolarization also induced a significant rise in the resting cellular inositol trisphosphate (IP3) and inositol tetrakisphosphate (IP4) contents, but not 1,4,5-IP3 content. Stimulation with 10 microM carbachol (CCh, a muscarinic agonist) produced a biphasic increase in [Ca2+]i, the initial transient phase due to mobilization of Ca2+ from an intracellular pool, and a sustained phase mediated by an influx of Ca2+. Membrane depolarization had no effect on the initial phase, while, the sustained increase in [Ca2+]i was eliminated. The CCh-enhanced quench of the Fura-2 signal by Mn2+ (an index for divalent cation entry) was reversibly inhibited by depolarization. The enhanced Mn2+ uptake induced by inhibiting microsomal Ca(2+)-ATPase with thapsigargin was similarly inhibited by membrane depolarization, consistent with the effect of depolarization primarily acting on the Ca2+ entry pathway and not on receptor coupling. Depolarization did not alter the initial CCh-induced increases in IP3, IP4 or 1,4,5-IP3 content, or the sustained increase in 1,4,5-IP3, whereas, depolarization significantly blunted (> 70%) the sustained, CCh-induced generation of IP3 and IP4. The membrane potential, therefore, appears to modulate Ca2+ activated fluid secretion by controlling the driving force for Ca2+ entry via a depletion-activated Ca2+ entry pathway. Inositol phosphate metabolism is also influenced by the membrane potential, but this effect apparently plays a minor role in regulating [Ca2+]i since 1,4,5-IP3 levels were unchanged by depolarization.

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.

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.

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.

Agonist-induced Ca2+ mobilization in the rat submandibular gland during aging and subsequent to chronic propranolol treatment.

The effects of age and chronic propranolol treatment on the agonist-induced rise in intracellular free Ca2+ ([Ca2+]i), an index for the coupling of receptor-second messenger generation, was studied using a dispersed rat submandibular gland preparation. Muscarinic stimulation (10 microns carbachol) caused a rapid (T1/2 less than 2 s) and dramatic (approximately 4.5-fold) rise in [Ca2+]i followed by a lower sustained increase (approximately 3-fold) in [Ca2+]i as measured directly with the Ca(2+)-sensitive fluorescent probe, fura-2. The magnitude and the rate of increase of the initial rise in [Ca2+]i and the level of the sustained increase in [Ca2+]i were not different between 2- an 21-month-old rats. Stimulation in a Ca(2+)-free medium reduced the initial agonist-induced increase in [Ca2+]i by approximately 35-40%, while the sustained increase was abolished by the removal of extracellular Ca2+ from cells in both young and old rats. Chronic treatment for 30 days with 20 mg/kg propranolol, a beta-adrenergic antagonist, did not significantly alter the ability of dispersed submandibular cells in old rats to mobilize Ca2+ during agonist stimulation or influence the in vivo stimulated gland output. These results suggest that the agonist-induced rise in [Ca2+]i is not altered by aging or by chronic treatment of aged rats with propranolol and, therefore, receptor-second messenger coupling remains intact.

Gonadal hormone regulation of neurotransmitter synthesizing enzymes in the developing hypogastric ganglion.

The effects of postnatal castration at 10-11 days of age were examined in the sympathetic hypogastric ganglion (HG). Assays for choline acetyltransferase (ChAT) activity, a biochemical marker for presynaptic cholinergic maturation, and the activity of tyrosine hydroxylase (T-OH), the rate limiting enzyme in postsynaptic catecholamine biosynthesis and an index of noradrenergic development, were employed to monitor HG ontogeny. After 1 postoperative week ChAT activity and T-OH activity were significantly reduced in castrated animals as compared to sham operated controls. Over the 12 week postoperative observation period T-OH activity never varied significantly from the day 10 precastration value; thus, by 12 postoperative weeks T-OH activity in the castrated animals was 3% of the control value. In contrast, ChAT activity and total ganglion protein continued to mature in the castrated animals, but at diminished rates, so that by 12 postoperative weeks both indices were approximately 40% of the control values. Testosterone replacement therapy restored both ChAT and T-OH activities to control levels. Additionally, testosterone replacement restored the control level of activity for DOPA decarboxylase, a catecholamine synthetic enzyme which is differentially regulated from T-OH. The failure of enzyme activities to develop normally subsequent to castration on postnatal day 10 suggests that testosterone regulates the postorganizational maturation of postsynaptic noradrenergic enzyme activities and presynaptic ChAT activity.

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.

Central neurotoxic effects of guanethidine: altered serotonin and enkephalin neurons within the area postrema.

This study reveals that the 5-HT and enkephalin cell body population of the area postrema (AP) is dramatically reduced via exposure to the peripherally circulating neurotoxin guanethidine. Efforts to restore cell body numbers to control levels, with colchicine and monoamine oxidase inhibitors, subsequent to guanethidine treatment are ineffective. However, 5-HT and enkephalin immunostaining of surrounding bulbar nuclei appears normal, implying that the neurotoxic effect of guanethidine is restricted to the AP. In addition, gamma-aminobutyric acid and neurotensin immunostaining within the AP appears normal, subsequent to guanethidine treatment, suggesting that the neurotoxic effect is restricted to specific AP cell populations.

Organizational role of testosterone on the biochemical and morphological development of the hypogastric ganglion.

Previous reports have demonstrated that after postnatal day 10 testosterone influences hypogastric ganglion (HG) development by 'activating' morphological and biochemical indices. We now report an 'organizational' influence on the developing HG during the first 10 postnatal days. To investigate the organizational effects of testosterone, male rats were castrated within 12 h of birth. Testosterone replacement therapy initiated following castration maintained the normal number of neurons in the HG. Conversely, delayed replacement therapy starting at day 10 or vehicle treatment only, resulted in a significant decrease in neuron number. Castration also produced a significant decrease in somal and nuclear cross-sectional areas. Testosterone replacement, whether initiated immediately or if delayed until day 10, restored somal and nuclear cross-sectional areas to normal. Tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT) activities were sensitive to both testosterone dosage and the time of administration. Testosterone decanoate administered subsequent to castration was not able to completely reverse the enzyme activity deficits, while delayed replacement therapy was even less effective in restoring enzyme activities. In contrast, higher doses of testosterone completely reversed enzyme activity deficits, and in fact produced a significant increase in TH activity. Again, delayed testosterone replacement did not fully restore deficits in enzyme activity. In summary, the hormonal environment during the first 10 days of life is critical for the organization of HG cell number; in contrast, nuclear and cell size appear to be dependent on testosterone for activation. TH and ChAT activities also appear to be organized during this dose- and time-dependent developmental period.

Biochemical and morphological effects of castration on the postorganizational development of the hypogastric ganglion.

The biochemical and morphological development of the sympathetic hypogastric ganglion (HG) was examined subsequent to postnatal castration at 10-11 days of age. Previous studies suggested that tyrosine hydroxylase (T-OH) activity, an index of noradrenergic maturation, and choline acetyltransferase (ChAT) activity, a marker for preganglionic terminal formation, were dependent on gonadal hormones during normal ontogeny. In the present studies, morphometric analyses of the HG revealed that the cross-sectional area of the cell soma and nucleus were significantly reduced following postnatal castration at day 10. Conversely, castration produced no change in the number of HG neurons. In addition, postnatal castration prevents the development of postsynaptic T-OH activity to a greater extent than ganglionic protein resulting in a significant loss of T-OH specific activity. In contrast, presynaptic ChAT activity was reduced in parallel with ganglionic protein, thus ChAT specific activity was unchanged. Testosterone replacement therapy, even in groups where treatment was delayed for up to 2 weeks after castration, completely reversed deficits in both T-OH and ChAT activities. These studies suggest that altered development of ganglion protein subsequent to postnatal castration is related to decreases in the size of neurons and not to the loss of neurons. The lack of cell loss also suggest that decreased levels of postsynaptic T-OH activity results from a loss of enzyme activity per cell and the decreased levels of ChAT activity probably represent fewer presynaptic terminals per neuron. In addition, delayed testosterone replacement subsequent to castration was effective in restoring enzyme activities suggesting an 'activational' not 'organizational' role for testosterone after postnatal day 10.

Altered sympathetic-salivary gland development: delayed response to postnatal castration.

These studies defined the normal and hormonally altered development of activity for tyrosine hydroxylase (T-OH), the rate-limiting enzyme in catecholamine biosynthesis (Levitt et al., 1965), and choline acetyltransferase activity (CAT) in the male rat superior cervical ganglion (SCG). Additionally, salivary gland weight was monitored. Two distinct developmental plateaus for postsynaptic T-OH activity exist. The first plateau represents the prepubertal level, which is significantly lower than the second postpubertal plateau. In contrast, presynaptic CAT activity displayed only a single plateau, commencing at approximately 45 days of age. The effects of postnatal castration (at 10 or 11 days of age) on the submandibular gland and T-OH activity were delayed until after puberty. No change in T-OH activity was seen at two and four post-operative weeks between control and castrated animals; however, T-OH activity was significantly less in castrated animals at 12 and 16 post-operative weeks. Testosterone replacement reversed the effect of castration on T-OH activity. Conversely, CAT activity in the SCG was unchanged by postnatal castration for at least 16 post-operative weeks, the longest time point studied. The failure of castrated animals to display the normal developmental increase in T-OH activity following puberty was comparable with the effect of castration on the development of submandibular salivary gland weight. These results suggest that in postpubertal male rats, development of T-OH activity in the superior cervical ganglion is influenced by testosterone. The parallel effects of castration on submandibular gland weight imply that testosterone regulates T-OH activity via an indirect mechanism. In contrast to noradrenergic enzyme activity and target tissue size, the ontogeny of presynaptic CAT activity appears to be insensitive to testosterone levels.

Inhibition by thiocyanate of muscarinic-induced cytosolic acidification and Ca2+ entry in rat sublingual acini.

Thiocyanate (SCN-) plays a critical part in an oral antimicrobial system by acting as a substrate for peroxidases. Salivary glands concentrate SCN- from blood up to 5 mM in saliva; however, the influence of SCN- on salivary acinar-cell function is unknown. The present study examined the effects of SCN- on the regulation of cytosolic pH (pHi) and free Ca2+ concentration ([Ca2+]i) in rat sublingual mucous acini using the pH- and Ca(2+)-sensitive fluorescent indicators, 2',7'-bis-(2-carboxyethyl)-5,6-carboxyfluorescein and fura-2, respectively. SCN- induced a concentration-dependent inhibition of the carbachol-stimulated cytosolic acidification (K1/2, approx. 1.4 mM SCN-). Cytosolic pH recovery from an acid load was not changed by substitution of Cl- by SCN-, suggesting that Na+/H+ exchange activity was not affected by SCN-. SCN- did not alter the initial carbachol-stimulated increase in [Ca2+]i; however, the sustained [Ca2+]i increase was inhibited by > 65% (K1/2, approx. 1.0 mM SCN-). Furthermore, SCN- prevented the carbachol-stimulated Mn2+ influx, indicating that it inhibits the divalent-cation entry pathway. Consistent with decreased Ca2+ mobilization being involved in the blockade of the agonist-induced acidification by SCN-, only total replacement of Cl- with SCN- significantly inhibited the acidification induced by the Ca2+ ionophore ionomycin. The permeability to SCN- through the Ca(2+)-dependent Cl- channels was 5.2-fold higher than the permeability to Cl-. These results suggest that inhibition of the agonist-induced cytosolic acidification by high-concentration SCN- may be mediated by both competitive inhibition of HCO3- efflux and by blockade of Ca2+ influx.

Mediation of the depolarization-induced [Ca(2+)]i increase in rat sublingual acini by acetylcholine released from nerve terminals.

In sublingual mucous acini, membrane depolarization induces a threefold transient increase in cytosolic free Ca(2+) concentration [(Ca(2+))i]. The underlying mechanism was examined by using the Ca(2+) sensitive fluorescent indicator fura-2. Membrane depolarization with high K+ induced a transient [Ca(2+)]i increase in acini, but not in single acinar cells. Atropine, pirenzepine and 4-diphenylacetoxy-N-methylpiperidine methiodide prevented the[Ca(2)+]i increase, suggesting the involvement of muscarinic receptor activation. Inhibition of the inositol trisphosphate (IP3)-sensitive Ca(2+) release pathway with S-(diethylamino)-octyl-3,4,5-trimethoxybenzoate prevented the depolarization-induced increase in [Ca(2+)]i. Blockade of nicotinic receptors and L-, N-, and P-type voltage-dependent Ca(2+) channels (hexamethonium, nifedipine, diltiazem, (omega-conotoxin GVIA and omega-agatoxin IVA) did not inhibit the increase in [Ca(2+)]i. However, Cd(2)+ (0.2 mM) blocked >85 percent of the [Ca2+]i increase. The depolarization-induced [Ca(2+)]i increase was also extracellular Ca(2+)-dependent. These results suggest that the membrane depolarization-induced Ca(2+) increase in sublingual acini is mediated by activating Cd(2+)-sensitive, voltage-dependent Ca(2+) channels in nerve terminals associated with the dispersed acini and stimulating release of acetylcholine, which then triggers the [Ca(2+)]i increase in acinar cells.