Xinjun He

POLARIZED DISTRIBUTION OF KEY MEMBRANE TRANSPORT PROTEINS IN THE RAT SUBMANDIBULAR GLAND

Abstract Immunofluorescence labelling and confocal microscopy were employed to examine the polarized distribution of several membrane transport proteins believed to be essential for salivary secretion in the rat submandibular gland. The Na+/K+-ATPase, Na+/H+ exchanger isoform 1 (NHE1), and the secretory Na+/K+/2Cl–cotransporter isoform were all found in the basolateral membranes of acinar and intralobular duct cells. Anion exchanger isoform 2 (AE2) was found only in the basolateral membranes of acinar cells, while AE1 was absent from glandular epithelial cells. Aquaporin 5 was detected in the apical membranes of acinar cells, while the cystic fibrosis transmembrane conductance regulator was found only in apical membranes of intralobular duct cells. NHEs 2 and 3 were found in the apical membranes of both acinar and intralobular duct cells. Our results are generally consistent with the expected distribution of most transporters based on previous physiological and pharmacological experiments. However, the apical localization of NHEs 2 and 3, and the presence of the secretory isoform of the Na+/K+/2Cl–cotransporter in intralobular duct cells were not predicted.

Systemic action of human growth hormone following adenovirus-mediated gene transfer to rat submandibular glands

We have previously suggested that although salivary glands function in an exocrine manner they might none the less offer a useful way to deliver therapeutic proteins systemically. As a direct functional test of this hypothesis, we constructed a recombinant adenovirus (AdCMVhGH) encoding human growth hormone (hGH) and then studied the biological action of hGH produced following transfer of the hGH gene to rat submandibular glands. At 48 h following infusion of AdCMVhGH into these glands via cannulation of the main excretory duct, serum levels of hGH were approximately 16 ng/ml and rat insulin-like growth factor-1 was elevated approximately 25%. Moreover, serum chemistry profiles of rats subjected to in vivo gene transfer displayed alterations in the BUN:creatinine ratio and triglyceride levels presumably reflecting the anabolic actions of the hGH. These results provide the first demonstration of systemic biological action from a transgene product secreted in an endocrine fashion from the salivary glands.

Muscarinic receptor regulation of Ca2+ mobilization in a human salivary cell line

We have studied receptor-mediated Ca2+ mobilization in an established exocrine epithelial cell line (HSG-PA) derived from a human submandibular gland. These cells possess a single class of high-affinity muscarinic cholinergic receptors identified using [3H]-quinuclidinylbezilate (Kd=0.17±0.07 nmol/l;Bmax=37±2 fmol/mg protein;n=3). The muscarinic agonist carbachol elicits a concentration dependent increase of [3H]-inositol trisphosphate in HSG-PA cells (100 μmol/l; >2 fold by 30 s). Carbachol also results in a rapid, ∼5-fold increase in cytosolic [Ca2+]. This response is made up of two components, one arising from the release of intracellular Ca2+ (La3+ insensitive; independent of extracellular [Ca2+]), the other from the entry of extracellular Ca2+ (La3+ sensitive; dependent on extracellular [Ca2+]). These Ca2+ mobilizing mechanisms are completely blocked by the muscarinic antagonist atropine (10 μmol/l) but unaffected by several voltage-dependent Ca2+ channel antagonists (verapamil, nifedipine, diltiazem) and by membrane depolarization (incubation in 55 mmol/l KCl). These results demonstrate that HSG-PA cells respond to muscarinic stimulation by mobilizing Ca2+ from an intracellular store and via a receptor-operated Ca2+ entry pathway.

A polarized salivary cell monolayer useful for studying transepithelial fluid movement in vitro

Abstract There are no reported, convenient in vitro models for studying polarized functions in salivary epithelial cells. Accordingly, we examined three often-used salivary cell lines for their ability to form a polarized monolayer on permeable, collagen-coated polycarbonate filters. Only the SMIE line, derived from rat submandibular gland, had this ability. The SMIE cell monolayer exhibited junctional complexes, with a tight-junction-associated protein, ZO-1, localized to cell–cell contact areas. The Na+/K+-ATPase α1-subunit was detected predominantly in the basolateral membranes, while the Na+/H+ exchanger isoform 2 appeared primarily in the apical membranes. Using adenovirus-mediated cDNA transfer, SMIE cells were shown to be capable of routing marker proteins (β-galactosidase ± a nuclear targeting signal, α1-antitrypsin, aquaporin-1) to appropriate locations. Furthermore, this salivary cell monolayer provided a convenient tool for studying aquaporin-1-mediated, osmotically directed, transepithelial fluid movement in vitro. Thus, SMIE cells appear to be a useful experimental model with which to study some polarized functions in a salivary epithelial cell line.

Evidence for two modes of Ca2+ entry following muscarinic stimulation of a human salivary epithelial cell line.

SummaryWe have investigated muscarinic receptor-operated Ca2+ mobilization in a salivary epithelial cell line, HSG-PA, using an experimental approach which allows independent evaluation of intracellular Ca2+ release and extracellular Ca2+ entry. The carbachol (Cch) dose response of intracellular Ca2+ release indicates the involvement of a single, relatively low-affinity, muscarinic receptor site (K0.5≅10 or 30 μm, depending on the method for [Ca2+]i determination). However, similar data for Ca2+ entry indicate the involvement of two Cch sites, one consistent with that associated with Ca2+ release and a second higher affinity site withK0.5≤2.5 μm. In addition, the Ca2+ entry response observed at lower concentrations of Cch (2.5 μm) was completely inhibited by membrane depolarization induced with high K+ (>55mm) or gramicidin D (1 μm), while membrane depolarization had little or no effect on Ca2+ entry induced by 100 μm Cch. Another muscarinic agonist, oxotremorine-M (100 μm; Oxo-M), like Cch, also induced an increase in the [Ca2+]i of HSG-PA cells (from 72±2 to 104±5nm). This response was profoundly blocked (∼75%) by the inorganic Ca2+ channel blocker La3+ (25–50 μm) suggesting that Oxo-M primarily mobilizes Ca2+ in these cells by increasing Ca2+ entry. Organic Ca2+ channel blockers (verapamil or diltiazem at 10 μm, nifedipine at 1 μm), had no effect on this response. The Oxo-M induced Ca2+ mobilization response, like that observed at lower doses of Cch, was markedly inhibited (∼70–90%) by membrane depolarization (high K+ or gramicidin D). At 100 μm Cch the formation of inositol trisphosphate (IP3) was increased 55% above basal levels. A low concentration of carbachol (1 μm) elicited a smaller change in IP3 formation (∼25%), similar to that seen with 100 μm Oxo-M (∼20%). Taken together, these results suggest that there are two modes of muscarinic receptor-induced Ca2+ entry in HSG-PA cells. One is associated with IP3 formation and intracellular Ca2+ release and is independent of membrane potential; the other is less dependent on IP3 formation and intracellular Ca2+ release and is modulated by membrane potential. This latter pathway may exhibit voltage-dependent gating.

β-Adrenergic responsiveness in a human submandibular tumor cells line (A253)

SummarySalivary epithelial functions are regulated by the autonomic nervous system. In this regard, we have been studying the morphology and neuroreceptor composition of A253, an immortal cell line isolated from a human submandibular carcinoma (Giard et al., JNCI, 51:1417–1421, 1973). Phase contrast and electron microscopic observation indicate that A253 cells are of epithelial origin. Physiologically, A253 cells posses β-adrenergic, but not α-adrenergic or muscarinic-cholinergic receptors. The β-adrenergic receptors (BARs) are composed primarily of a single class of high affinity, β2-subtype receptors as judged by [3H]dihydroalprenolol antagonist binding studies. The BARs are functional inasmuch as isoproterenol stimulation increases both intracellular cAMP content and [3H]mannose incorporation into endogeneous glycoproteins. Differences in ultrastructure and neuroreceptor composition between A253 and other immortal salivary tumor cell lines are discussed.

The effect of N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) on muscarinic receptor-induced Ca2+ mobilization in a human salivary epithelial cell line

We have investigated the effect of W-7, a calmodulin (CaM) antagonist, on Ca2+ mobilization in a human salivary epithelial cell line, HSG-PA, after muscarinic receptor stimulation. In a medium containing 1.5 mmol/l Ca2+, W-7 reduced both the maximum peak increase in cytosolic Ca2+ ([Ca2+]i) which follows stimulation by carbachol (Cch, 100 μmol/l) and the sustained nature of the response. Using an experimental approach which allows separate visualization of the intracellular Ca2+ release and extracellular Ca2+ entry phases, W-7 was shown preferentially to inhibit Ca2+ release. At 100 μmol/l W-7, Cch-induced Ca2+ release was completely inhibited, but Cch-induced Ca2+ entry was partially (∼40%) maintained. This W-7 residual Ca2+ entry response was abolished when cells were depolarized with high K+ or gramicidin D. W-7 also substantially inhibited Cch-induced inositol trisphosphate (IP3) production (∼75%). W-5, a less potent CaM antagonist than W-7, had markedly smaller effects on Cch-induced Ca2+ mobilization and IP3 formation. W-7 (100 μmol/ l) completely blocked (comparable to 10 μmol/l atropine) the binding of the muscarinic antagonist [3H] quinuclidinyl benzilate (QNB) to muscarinic receptors on cell membranes, whereas Cch (at 100 μmol/l) had minimal effects on ligand binding. W-7 and W-5 were equipotent in their ability to inhibit [3H] QNB binding. These results suggest that W-7 reduces Ca2+ mobilization in HSG-PA cells by a mechanism which likely involves the antagonism of a CaM regulatory step(s) but may also involve at least a partial blockade of the muscarinic receptor. In addition, in the presence of W-7, Ca2+ entry can occur via a receptor-operated Ca2+ pathway which is modulated by membrane potential.

Characteristics of α1-adrenergic receptors in a rat salivary cell line, RSMT-A5

Abstract 1. 1. Membrane preparations obtained from RSMT-A5, an established rat submandibular cell line, possess α1-adrenergic receptors as judged by [3H]prazosin binding studies. Scatchard analysis reveals a single class of binding sites with an equilibrium dissociation constant (Kd) of 0.052 nM and a receptor density (Bmax) of 104 fmol/mg protein. 2. 2. These receptors are functional since stimulation by epinephrine results in an increased rate of phosphatidylinositol turnover which is blocked by both phentolamine and phenoxybenzamine. 3. 3. Phenoxybenzamine (10−5 M) irreversibly blocks 95% of the receptors within 2 min. A kinetic analysis shows that the receptors subsequently reappear at a rate of 6 fmol/mg protein/h. The calculated rate constant for receptor clearance (Kc) is 0.027 hr−1, and the half life ( t 1 2 ) of the receptor is 26 hr. 4. 4. These results are comparable to those reported for α1-adrenergic receptors in the BC3H-1 smooth muscle cell line and suggest that α1-adrenergic receptors in cultured cells, from diverse tissues, display similar characteristics.