Mouse Submandibular Acinar Cells Research Articles

Involvement of sodium–glucose cotransporter-1 activities in maintaining oscillatory Cl− currents from mouse submandibular acinar cells

In salivary acinar cells, cholinergic stimulation induces elevations of cytosolic [Ca2+]i to activate the apical exit of Cl− through TMEM16A Cl− channels, which acts as a driving force for fluid secretion. To sustain the Cl− secretion, [Cl−]i must be maintained to levels that are greater than the electrochemical equilibrium mainly by Na+-K+-2Cl− cotransporter-mediated Cl− entry in basolateral membrane. Glucose transporters carry glucose into the cytoplasm, enabling the cells to produce ATP to maintain Cl− and fluid secretion. Sodium–glucose cotransporter-1 is a glucose transporter highly expressed in acinar cells. The salivary flow is suppressed by the sodium–glucose cotransporter-1 inhibitor phlorizin. However, it remains elusive how sodium–glucose cotransporter-1 contributes to maintaining salivary fluid secretion. To examine if sodium–glucose cotransporter-1 activity is required for sustaining Cl− secretion to drive fluid secretion, we analyzed the Cl− currents activated by the cholinergic agonist, carbachol, in submandibular acinar cells while comparing the effect of phlorizin on the currents between the whole-cell patch and the gramicidin-perforated patch configurations. Phlorizin suppressed carbachol-induced oscillatory Cl− currents by reducing the Cl− efflux dependent on the Na+-K+-2Cl− cotransporter-mediated Cl− entry in addition to affecting TMEM16A activity. Our results suggest that the sodium–glucose cotransporter-1 activity is necessary for maintaining the oscillatory Cl− secretion supported by the Na+-K+-2Cl− cotransporter activity in real time to drive fluid secretion. The concerted effort of sodium–glucose cotransporter-1, Na+-K+-2Cl− cotransporter, and apically located Cl− channels might underlie the efficient driving of Cl− secretion in different secretory epithelia from a variety of animal species.

Muscarinic Receptors and BK Channels Are Affected by Lipid Raft Disruption of Salivary Gland Cells

Activity-dependent fluid secretion is the most important physiological function of salivary glands and is regulated via muscarinic receptor signaling. Lipid rafts are important for G-protein coupled receptor (GPCR) signaling and ion channels in plasma membranes. However, it is not well understood whether lipid raft disruption affects all membrane events or only specific functions in muscarinic receptor-mediated water secretion in salivary gland cells. We investigated the effects of lipid raft disruption on the major membrane events of muscarinic transcellular water movement in human salivary gland (HSG) cells. We found that incubation with methyl-β-cyclodextrin (MβCD), which depletes lipid rafts, inhibited muscarinic receptor-mediated Ca2+ signaling in HSG cells and isolated mouse submandibular acinar cells. However, MβCD did not inhibit a Ca2+ increase induced by thapsigargin, which activates store-operated Ca2+ entry (SOCE). Interestingly, MβCD increased the activity of the large-conductance Ca2+-activated K+ channel (BK channel). Finally, we found that MβCD did not directly affect the translocation of aquaporin-5 (AQP5) into the plasma membrane. Our results suggest that lipid rafts maintain muscarinic Ca2+ signaling at the receptor level without directly affecting the activation of SOCE induced by intracellular Ca2+ pool depletion or the translocation of AQP5 into the plasma membrane.

Asymmetry of Rb+ Conduction Emerged under Bi-Ionic Conditions in Epithelial Maxi-K+ Channels

K(+) channels permit more than one ion within their conducting pathway at any given moment and show a saturating single-file behavior. The conduction of Rb(+) shows an unusual behavior, a so-called "Rb(+) anomaly," and it has been used to probe the mechanism of the ion conduction through K(+)-selective channels. Under the bi-ionic condition of K(+) and Rb(+), we carried out patch-clamp single-channel current measurements in MaxiK(+) channels from mouse submandibular acinar cells. Keeping only K(+) on one side of the membrane while varying fractional Rb(+) concentration on the opposite, we had a series of current-voltage relationships. It showed a characteristic inflection at which the ion conductance was divided into two components, one ascribed to pure K(+) conduction and the other to K(+) and Rb(+) bi-ionic conduction. By analyzing the latter, we depicted that (1) the bi-ionic conductance showed a characteristic reduction curve as the Rb(+) fractional concentration increased; (2) Rb(+) can bind the channel more tightly when it accesses from the outside than from the inside. Thus we conclude that such asymmetry of the Rb(+) binding determines the pattern of bi-ionic conductance reduction in K-selective channels.

Na+ entry via store-operated Ca2+ channels in mouse submandibular acinar cells.

Na+ entry via store-operated Ca2+ channels in mouse submandibular acinar cells.

Hydroxychloroquine therapy in patients with primary Sjogren's syndrome may improve salivary gland hypofunction by inhibition of glandular cholinesterase

To determine whether (i) cholinesterase activity is increased in the saliva of patients with primary Sjogren's syndrome (pSS), (ii) increased levels of cholinesterase of lymphocyte origin could interfere with the secretory activity of submandibular acinar cells, and (iii) hydroxychloroquine at therapeutic doses could interfere with cholinesterase activity. The Ellman method was used to determine the levels of salivary cholinesterase activity and the K(i) of both chloroquine and hydroxychloroquine for serum cholinesterase. The ability of lymphocyte cholinesterase to inhibit the acetylcholine (ACh)-evoked rise in [Ca(2+)](i) in mouse submandibular acinar cells was determined using fura-2 microfluorimetry. Patients with pSS had significantly higher levels of cholinesterase activity in both their unstimulated (P < 0.05) and stimulated saliva (P < 0.0001) compared with control subjects. Lymphocyte cholinesterase was capable of inhibiting the ACh-evoked rise in [Ca(2+)](i). The in vitro K(i) for hydroxychloroquine inhibition of cholinesterase was 0.38 +/- 1.4 microM. These data suggest that increased levels of cholinesterase present in the salivary glands of patients with pSS may contribute to glandular hypofunction and provide evidence that the therapeutic enhancement of salivary secretion in patients with pSS by hydroxychloroquine may be mediated by inhibition of glandular cholinesterase activity, although further in vivo investigation is needed.

Local and global calcium signals and fluid and electrolyte secretion in mouse submandibular acinar cells.

Polarized Ca(2+) signals that originate at and spread from the apical pole have been shown to occur in acinar cells from lacrimal, parotid, and pancreatic glands. However, "local" Ca(2+) signals, that are restricted to the apical pole of the cell, have been previously demonstrated only in pancreatic acinar cells in which the primary function of the Ca(2+) signal is to regulate exocytosis. We show that submandibular acinar cells, in which the primary function of the Ca(2+) signal is to drive fluid and electrolyte secretion, are capable of both Ca(2+) waves and local Ca(2+) signals. The generally accepted model for fluid and electrolyte secretion requires simultaneous Ca(2+)-activation of basally located K(+) channels and apically located Cl(-) channels. Whereas a propagated cell-wide Ca(2+) signal is clearly consistent with this model, a local Ca(2+) signal is not, because there is no increase in intracellular Ca(2+) concentration at the basal pole of the cell. Our data provide the first direct demonstration, in submandibular acinar cells, of the apical and basal location of the Cl(-) and K(+) channels, respectively, and confirm that local Ca(2+) signals do not Ca(2+)-activate K(+) channels. We reevaluate the model for fluid and electrolyte secretion and demonstrate that Ca(2+)-activation of the Cl(-) channels is sufficient to voltage-activate the K(+) channels and thus demonstrate that local Ca(2+) signals are sufficient to support fluid secretion.

Putative anti-muscarinic antibodies cannot be detected in patients with primary Sjogren's syndrome using conventional immunological approaches.

To determine whether autoantibodies directed against muscarinic M3 receptors are present in the serum of patients with primary Sjogren's syndrome (pSS), and if so whether these autoantibodies inhibit secretion by intact salivary acinar cells. IgG was purified by affinity chromatography using protein G from sera collected from 15 patients with pSS. Antibody detection was by Western blotting, whole-cell enzyme-linked immunosorbent assay (ELISA) and immunoblotting. The antisecretory activity of the IgG faction was determined using fura-2 microfluorimetry to measure changes in intracellular Ca(2+) activity ([Ca(2+)](i)) in human and mouse salivary gland acinar cells and in Chinese hamster ovary (CHO) cells transfected with human M3 receptors (CHO-M3). We found no specific M3 receptor recognition by the IgG fraction obtained from pSS patients using either Western blotting or ELISA or immunoblot techniques in which epitope conformation were preserved. Chronic exposure to pSS IgG had no effect on agonist-evoked Ca(2+) signals measured in human or mouse submandibular acinar cells or in CHO-M3 cells. However, acute application of IgG from Sjogren's syndrome patients produced a rapidly reversible reduction in the agonist-stimulated elevation in [Ca(2+)](i). These data represent the first demonstration of salivary acinar cell inhibition by pSS IgG; however, this inhibition was found to be reversible. Our data also show that pSS IgG binding to M3R cannot be visualized by conventional immunological approaches.

Role of Ins(1,4,5)P3, cADP-ribose and nicotinic acid-adenine dinucleotide phosphate in Ca2+ signalling in mouse submandibular acinar cells.

cADP-ribose (cADPr) and nicotinic acid-adenine dinucleotide phosphate (NAADP) are two putative second messengers; they were first shown to stimulate Ca(2+) mobilization in sea urchin eggs. We have used the patch-clamp whole-cell technique to determine the role of cADPr and NAADP in relation to that of Ins(1,4,5)P(3) in mouse submandibular acinar cells by measuring agonist-evoked and second-messenger-evoked changes in Ca(2+)-dependent K(+) and Cl(-) currents. Both Ins(1,4,5)P(3) and cADPr were capable of reproducing the full range of responses normally seen in response to stimulation with acetylcholine (ACh). Low concentrations of agonist (10-20 nM ACh) or second messenger [1-10 microM Ins(1,4,5)P(3) or cADPr] elicited a sporadic transient activation of the Ca(2+)-dependent currents; mid-range concentrations [50-500 nM ACh, 50 microM Ins(1,4,5)P(3) or 50-100 microM cADPr] elicited high-frequency (approx. 2 Hz) trains of current spikes; and high concentrations [more than 500 nM ACh, more than 50 microM Ins(1,4,5)P(3) or more than 100 microM cADPr] gave rise to a sustained current response. The response to ACh was inhibited by antagonists of both the Ins(1,4,5)P(3) receptor [Ins(1,4,5)P(3)R] and the ryanodine receptor (RyR) but could be completely blocked only by an Ins(1,4,5)P(3)R antagonist (heparin). NAADP (50 nM to 100 microM) did not itself activate the Ca(2+)-dependent ion currents, nor did it inhibit the activation of these currents by ACh. These results show that, in these cells, both Ins(1,4,5)P(3)R and RyR are involved in the propagation of the Ca(2+) signal stimulated by ACh and that cADPr can function as an endogenous regulator of RyR. Furthermore, although NAADP might have a role in hormone-stimulated secretion in pancreatic acinar cells, it does not contribute to ACh-evoked secretion in submandibular acinar cells.

Role of Ins(1,4,5)P3, cADP-ribose and nicotinic acid–adenine dinucleotide phosphate in Ca2+ signalling in mouse submandibular acinar cells

cADP-ribose (cADPr) and nicotinic acid–adenine dinucleotide phosphate (NAADP) are two putative second messengers; they were first shown to stimulate Ca2+ mobilization in sea urchin eggs. We have used the patch-clamp whole-cell technique to determine the role of cADPr and NAADP in relation to that of Ins(1,4,5)P3 in mouse submandibular acinar cells by measuring agonist-evoked and second-messenger-evoked changes in Ca2+-dependent K+ and Cl- currents. Both Ins(1,4,5)P3 and cADPr were capable of reproducing the full range of responses normally seen in response to stimulation with acetylcholine (ACh). Low concentrations of agonist (10–20nM ACh) or second messenger [1–10µM Ins(1,4,5)P3 or cADPr] elicited a sporadic transient activation of the Ca2+-dependent currents; mid-range concentrations [50–500nM ACh, 50µM Ins(1,4,5)P3 or 50–100µM cADPr] elicited high-frequency (approx. 2Hz) trains of current spikes; and high concentrations [more than 500nM ACh, more than 50µM Ins(1,4,5)P3 or more than 100µM cADPr] gave rise to a sustained current response. The response to ACh was inhibited by antagonists of both the Ins(1,4,5)P3 receptor [Ins(1,4,5)P3R] and the ryanodine receptor (RyR) but could be completely blocked only by an Ins(1,4,5)P3R antagonist (heparin). NAADP (50nM to 100µM) did not itself activate the Ca2+-dependent ion currents, nor did it inhibit the activation of these currents by ACh. These results show that, in these cells, both Ins(1,4,5)P3R and RyR are involved in the propagation of the Ca2+ signal stimulated by ACh and that cADPr can function as an endogenous regulator of RyR. Furthermore, although NAADP might have a role in hormone-stimulated secretion in pancreatic acinar cells, it does not contribute to ACh-evoked secretion in submandibular acinar cells.

An investigation of interactions between the immune system and stimulus-secretion coupling in mouse submandibular acinar cells. A possible mechanism to account for reduced salivary flow rates associated with the onset of Sjögren's syndrome.

To determine whether chronic exposure to lymphocyte-derived cytokines could inhibit the fluid secretory mechanism in salivary gland acinar cells and so account for the loss of gland function seen in the early stages of Sjögren's syndrome. Mouse submandibular acinar cells maintained in primary culture were exposed to a profile of cytokines produced by concanavalin A-activated splenic lymphocytes in vitro for periods up to 72 h. Agonist-evoked changes in intracellular Ca(2+) were determined microfluorimetrically in both control and cytokine-treated cells. Acinar cells maintained in primary culture in the presence of cytokines for up to 72 h were able to mobilize intracellular calcium in response to stimulus by acetylcholine in an identical fashion to those maintained in primary culture in the absence of cytokines. Acute application of the conditioned medium produced by the activated lymphocytes had an antisecretory effect on acetylcholine-evoked Ca(2+) mobilization, which was found to be mediated by cholinesterase rather than by cytokines. Neither chronic nor acute exposure to the profile of cytokines released by concanavalin A-activated splenic lymphocytes interfered in any way with the second messenger cascade and fluid and electrolyte secretion in acinar cells. Our data suggest an alternative hypothesis, in which elevated levels of cholinesterase can metabolize acetylcholine released within the salivary glands and thus prevent fluid secretion.

Amplification of the thapsigargin-evoked increase in the cytosolic free Ca2+ concentration by acetylcholine in acutely isolated mouse submandibular acinar cells.

The intracellular Ca2+ concentration was measured in single, acutely isolated, mouse submandibular acinar cells loaded with fura-2 AM. All experiments were performed in the absence of extracellular Ca2+ in order to eliminate Ca2+ influx. The microsomal ATPase inhibitor, thapsigargin, was used to release Ca2+ from intracellular stores and simultaneously prevent re-uptake into the stores. Sequential application of thapsigargin (2 microM) and the Ca2+ ionophore ionomycin (500 nM) indicated that thapsigargin was able to mobilize practically all intracellular Ca2+. Furthermore, in comparison with results obtained following inhibition of the plasma membrane Ca(2+)-ATPase by La3+ (2 mM), it may be shown that slowly unloading the intracellular Ca2+ stores using thapsigargin does not normally cause a massive, cytotoxic, increase in the cytosolic Ca2+ concentration, because Ca2+ is rapidly extruded from the cell across the plasma membrane. Application of a submaximal dose of acetylcholine (500 nM) during the rising phase of the response to thapsigargin caused a 3-4-fold increase in the amplitude of the rise in the cytosolic Ca2+ concentration without any significant alteration of the time course of the response. As thapsigargin alone is capable of mobilizing all releasable Ca2+, this increase in amplitude is most likely the result of inhibition of the Ca2+ extrusion process by acetylcholine.

Acetylcholine- and caffeine-evoked repetitive transient Ca(2+)-activated K+ and C1- currents in mouse submandibular cells.

1. Resting and acetylcholine-induced membrane currents were measured in single mouse submandibular acinar cells using the patch-clamp whole-cell current recording technique. 2. Micromolar ACh activated a large, sustained outward, Ca(2+)-dependent K+ current and a single transient inward Ca(2+)-dependent C1-current. 3. Nanomolar ACh induced a series of transients in both the K+ and C1- currents; C1- current activation was now observed throughout the period of agonist application. We consider this repetitive transient current activation better able to support sustained fluid and electrolyte secretion than the response elicited by a high dose of agonist. 4. Repetitive K+ and C1- current transients were also induced by 1 mM-caffeine, consistent with caffeine-induced Ca2+ release from the Ca(2+)-sensitive Ca2+ stores which are thought to comprise part of the pathway for activation of secretion. 5. The ACh-induced current transients were inhibited by 10 mM-caffeine, 100 microM-IBMX and 10 microM membrane-permeable cyclic AMP. Therefore, it seems likely that caffeine is able to inhibit agonist-induced calcium mobilization via a cyclic AMP-dependent pathway.

The receptor‐regulated calcium influx in mouse submandibular acinar cells is sodium dependent: a patch‐clamp study.

1. Salivary acini were isolated enzymatically from submandibular glands of adult male mice. The patch-clamp technique was employed to record K+ currents in cell-attached patches of basolateral membrane. Application of acetylcholine (10(-5) M) to the medium bathing the cells results in a pronounced and sustained activation of the K+ channels in the cell-attached patches, an effect mediated by an intracellular second messenger. In the present study we investigate the mechanism by which acetylcholine achieves activation of K+ channels. 2. The effects of acetylcholine on single-channel activity were shown to be dependent on extracellular Ca2+, i.e. due to Ca2+ influx. In cells bathed in Ca2+-free medium acetylcholine activation resulted in no increase in single-channel open probability. This blockade could be reversed by reintroduction of Ca2+ to the extracellular fluid in the continued presence of the agonist. The effects of acetylcholine in control medium (1.2 mM-Ca2+) were mimicked by the Ca2+ ionophore, A23187 (10(-8) M). 3. In K+-depolarized cells (bathed in a Na+-free, 145 mM-KCl solution) there was no evidence of any voltage-activated Ca2+ influx pathway. In the K+-depolarized cells acetylcholine application was no longer associated with any increase in the open probability of the K+ channels. K+ channels could be activated by adding A23187 (10(-8) M) to the high-K+ solution. 4. Cells bathed in another Na+-free (N-methyl-D-glucamine substituted for Na+) but non-depolarizing solution were also refractory to acetylcholine. K+ currents could, however, be activated in patches attached to these cells by application of A23187 (10(-8) M) or by the introduction of 20 mM-Na+ to the extracellular fluid in the presence of acetylcholine. The increased activity associated with the reintroduction of Na+ was totally reversed by atropine, i.e. it was receptor regulated. 5. The data presented above indicate that the cholinergic regulation of K+ channels is secondary to the receptor-regulated activation of a Ca2+ influx pathway. There is no evidence of voltage-activated Ca2+ influx in these cells. The cholinergic activation of Ca2+ influx is abolished in Na+-depleted cells. We conclude that the Na+ dependency indicates either that Na+ is involved in the gating of some voltage-independent Ca2+ channel or that Ca2+ entry is via a coupled Na+-Ca2+ co- or countertransport pathway.

A patch‐clamp study of potassium currents in resting and acetylcholine‐stimulated mouse submandibular acinar cells.

Salivary acini were enzymatically isolated from submandibular glands of adult male mice. The patch-clamp technique was employed to investigate the conductive properties and activities of a large-conductance K+ channel in both cell-attached and in excised patches of basolateral acinar cell membranes. In excised, inside out, patches with identical high-K+ solutions (145 mM-KCl) on either side of the membrane the current-voltage (I-V) plot was linear. The mean single-channel conductance was 245 +/- 4.8 ps with a single-channel permeability of 4.6 X 10(-13) cm3 s-1. At Ca2+ concentrations of 10(-9)-10(-8) M bathing the intracellular membrane face the channel was exquisitely sensitive to changes in transmembrane potential, i.e. voltage sensitive. At 10(-7) M-Ca the channel was almost always open and displayed little sensitivity to voltage. Single-channel currents were recorded in cell-attached patches. When the recording pipettes contained the high-K+ solution the I-V plots were linear and the mean single-channel conductance and permeability almost identical to that in the excised patches. The mean spontaneous resting potential of the acinar cells bathed in physiological saline (140 mM-NaCl, 4.5 mM-KCl) was -43 +/- 1.8 mV. The voltage sensitivity of the in situ K+ channel was very similar to that recorded in excised patches at 10(-9)-10(-8) M-Ca. In experiments designed to reproduce the physiological ionic gradients across the patch membrane pipettes were filled with the high-Na+ solution. The I-V plot was not linear but showed pronounced rectification at negative membrane potentials. The channel is K+ selective and the extrapolated reversal potential was close to -90 mV. The single-channel conductance at the spontaneous resting membrane potential was about 35 pS. The single-channel permeability was however only slightly reduced at 4.29 X 10(-13) cm3 s-1. It was demonstrated that current flow through the open K+ channel could be accurately modelled using constant field electrodiffusion theory. Continuous in situ recordings before and after application of the agonist acetylcholine to the solution bathing the acini revealed that acetylcholine stimulation is associated with a marked increase in the frequency and duration of K+ currents in the patch membrane. The increased current activity in the patch membrane during acetylcholine application must be mediated via an intracellular second messenger and was very similar to that observed in the excised patches on increasing ionized Ca2+ concentrations from 10(-8) to 10(-7) M.(ABSTRACT TRUNCATED AT 400 WORDS)