Ca signaling and regulation of fluid secretion in salivary gland acinar cells

Neurotransmitter stimulation of plasma membrane receptors stimulates salivary gland fluid secretion via a complex process that is determined by coordinated temporal and spatial regulation of several Ca²⁺ signaling processes as well as ion flux systems. Studies over the past four decades have demonstrated that Ca²⁺ is a critical factor in the control of salivary gland function. Importantly, critical components of this process have now been identified, including plasma membrane receptors, calcium channels, and regulatory proteins. The key event in activation of fluid secretion is an increase in intracellular [Ca²⁺] ([Ca²⁺]_i) triggered by IP₃-induced release of Ca²⁺ from ER via the IP₃R. This increase regulates the ion fluxes required to drive vectorial fluid secretion. IP₃Rs determine the site of initiation and the pattern of [Ca²⁺]_i signal in the cell. However, Ca²⁺ entry into the cell is required to sustain the elevation of [Ca²⁺]_i and fluid secretion. This Ca²⁺ influx pathway, store-operated calcium influx pathway (SOCE), has been studied in great detail and the regulatory mechanisms as well as key molecular components have now been identified. Orai1, TRPC1, and STIM1 are critical components of SOCE and among these, Ca²⁺ entry via TRPC1 is a major determinant of fluid secretion. The receptor-evoked Ca²⁺ signal in salivary gland acinar cells is unique in that it starts at the apical pole and then rapidly increases across the cell. The basis for the polarized Ca²⁺ signal can be ascribed to the polarized arrangement of the Ca²⁺ channels, transporters, and signaling proteins. Distinct localization of these proteins in the cell suggests compartmentalization of Ca²⁺ signals during regulation of fluid secretion. This chapter will discuss new concepts and findings regarding the polarization and control of Ca²⁺ signals in the regulation of fluid secretion.     

Dissection of Calcium Signaling Events in Exocrine Secretion

The secretion of fluid and electrolytes by salivary gland acinar cells requires the coordinated regulation of multiple ion channel and transporter proteins, signaling components, and water transport. Importantly, neurotransmitter stimulated increase in the cytosolic free [Ca²⁺] ([Ca²⁺]_i) is critical for the regulation of salivary gland secretion as it regulates several major ion fluxes that together establish the sustained osmotic gradient to drive fluid secretion. The mechanisms that act to modulate these increases in [Ca²⁺]_i are therefore central to the process of salivary fluid secretion. Such modulation involves membrane receptors for neurotransmitters, as well as mechanisms that mediate intracellular Ca²⁺ release, and Ca²⁺ entry, as well as those that maintain cellular Ca²⁺ homeostasis. Together, these mechanisms determine the spatial and temporal aspects of the [Ca²⁺]_i signals that regulate fluid secretion. Molecular cloning of these transporters and channels as well as development of mice lacking these proteins has established the physiological significance of key components that are involved in regulating [Ca²⁺]_i in salivary glands. This review will discuss these important studies and the findings which have led to resolution of the Ca²⁺ signaling mechanisms that determine salivary gland fluid secretion.     

Tuning store-operated calcium entry to modulate Ca2+-dependent physiological processes

The intracellular calcium signaling processes are tightly regulated to ensure the generation of calcium signals with the specific spatiotemporal characteristics required for regulating various cell functions. Compartmentalization of the molecular components involved in the generation of these signals at discrete intracellular sites ensures the signaling specificity and transduction fidelity of the signal for regulating downstream effector processes. Store-operated calcium entry (SOCE) is ubiquitously present in cells and is critical for essential cell functions in a variety of tissues. SOCE is mediated via plasma membrane Ca²⁺ channels that are activated when luminal [Ca²⁺] of the endoplasmic reticulum ([Ca²⁺]_ER) is decreased. The ER-resident stromal interaction molecules, STIM1 and STIM2, respond to decreases in [Ca²⁺]_ER by undergoing conformational changes that cause them to aggregate at the cell periphery in ER-plasma membrane (ER-PM) junctions. At these sites, STIM proteins recruit Orai1 channels and trigger their activation. Importantly, the two STIM proteins concertedly modulate Orai1 function as well as the sensitivity of SOCE to ER-Ca²⁺ store depletion. Another family of plasma membrane Ca²⁺ channels, known as the Transient Receptor Potential Canonical (TRPC) channels (TRPC1-7) also contribute to sustained [Ca²⁺]_i elevation. Although Ca²⁺ signals generated by these channels overlap with those of Orai1, they regulate distinct functions in the cells. Importantly, STIM1 is also required for plasma membrane localization and activation of some TRPCs. In this review, we will discuss various molecular components and factors that govern the activation, regulation and modulation of the Ca²⁺ signal generated by Ca²⁺ entry pathways in response to depletion of ER-Ca²⁺ stores. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.     

TRPC1, Orai1, and STIM1 in SOCE: Friends in tight spaces

Store-operated calcium entry (SOCE) is a ubiquitous Ca²⁺ entry pathway that is activated in response to depletion of ER-Ca²⁺ stores and critically controls the regulation of physiological functions in miscellaneous cell types. The transient receptor potential canonical 1 (TRPC1) is the first member of the TRPC channel subfamily to be identified as a molecular component of SOCE. While TRPC1 has been shown to contribute to SOCE and regulate various functions in many cells, none of the reported TRPC1-mediated currents resembled I_CRAC, the highly Ca²⁺-selective store-dependent current first identified in lymphocytes and mast cells. Almost a decade after the cloning of TRPC1 two proteins were identified as the primary components of the CRAC channel. The first, STIM1, is an ER-Ca²⁺ sensor protein involved in activating SOCE. The second, Orai1 is the pore-forming component of the CRAC channel. Co-expression of STIM1 and Orai1 generated robust I_CRAC. Importantly, STIM1 was shown to also activate TRPC1 via its C-terminal polybasic domain, which is distinct from its Orai1-activating domain, SOAR. In addition, TRPC1 function critically depends on Orai1-mediated Ca²⁺ entry which triggers recruitment of TRPC1 into the plasma membrane where it is then activated by STIM1. TRPC1 and Orai1 form discrete STIM1-gated channels that generate distinct Ca²⁺ signals and regulate specific cellular functions. Surface expression of TRPC1 can be modulated by trafficking of the channel to and from the plasma membrane, resulting in changes to the phenotype of TRPC1-mediated current and [Ca²⁺]_i signals. Thus, TRPC1 is activated downstream of Orai1 and modifies the initial [Ca²⁺]_i signal generated by Orai1 following store depletion. This review will summarize the important findings that underlie the current concepts for activation and regulation of TRPC1, as well as its impact on cell function.     

STIM1 Regulates Platelet-Derived Growth Factor-Induced Migration and Ca2+ Influx in Human Airway Smooth Muscle Cells

It is suggested that migration of airway smooth muscle (ASM) cells plays an important role in the pathogenesis of airway remodeling in asthma. Increases in intracellular Ca²⁺ concentrations ([Ca²⁺]_i) regulate most ASM cell functions related to asthma, such as contraction and proliferation. Recently, STIM1 was identified as a sarcoplasmic reticulum (SR) Ca²⁺ sensor that activates Orai1, the Ca²⁺ channel responsible for store-operated Ca²⁺ entry (SOCE). We investigated the role of STIM1 in [Ca²⁺]_i and cell migration induced by platelet-derived growth factor (PDGF)-BB in human ASM cells. Cell migration was assessed by a chemotaxis chamber assay. Human ASM cells express STIM1, STIM2, and Orai1 mRNAs. SOCE activated by thapsigargin, an inhibitor of SR Ca²⁺-ATPase, was significantly blocked by STIM1 siRNA and Orai1 siRNA but not by STIM2 siRNA. PDGF-BB induced a transient increase in [Ca²⁺]_i followed by sustained [Ca²⁺]_i elevation. Sustained increases in [Ca²⁺]_i due to PDGF-BB were significantly inhibited by a Ca²⁺ chelating agent EGTA or by siRNA for STIM1 or Orai1. The numbers of migrating cells were significantly increased by PDGF-BB treatment for 6 h. Knockdown of STIM1 and Orai1 by siRNA transfection inhibited PDGF-induced cell migration. Similarly, EGTA significantly inhibited PDGF-induced cell migration. In contrast, transfection with siRNA for STIM2 did not inhibit the sustained elevation of [Ca²⁺]_i or cell migration induced by PDGF-BB. These results demonstrate that STIM1 and Orai1 are essential for PDGF-induced cell migration and Ca²⁺ influx in human ASM cells. STIM1 could be an important molecule responsible for airway remodeling.     

Role of STIM1 in Regulation of Store-Operated Ca<Superscript>2+</Superscript> Influx in Pheochromocytoma Cells

Changes in the local environment such as pH (acidosis/alkalosis), temperature (hypothermia/hyperthermia), and agonist (glutamate) can adversely affect neuronal function, and are important factors in clinical situations such as anesthesia and intensive care. Regulation of intracellular Ca²⁺ ([Ca²⁺]_i) is key to neuronal function. Stromal interaction molecule (STIM1) has been recently recognized to trigger store-operated Ca²⁺ entry (SOCE), an important component of [Ca²⁺]_i regulation. Using differentiated, fura-2 loaded rat pheochromocytoma (PC12) cells transfected with small interference RNA for STIM1 (or vehicle), we examined the role of STIM1 in SOCE sensitivity to temperature, pH, and glutamate. SOCE was triggered following endoplasmic reticulum depletion. Cells were washed and exposed to altered pH (6.0–8.0), altered temperature (34–40°C), or to glutamate. In non-transfected cells, SOCE was inhibited by acidosis or hypothermia, but increased with alkalosis and hyperthermia. Increasing glutamate concentrations progressively stimulated SOCE. STIM1 siRNA decreased SOCE at normal temperature and pH, and substantially decreased sensitivity to acidosis and hypothermia, eliminating the concentration-dependence to glutamate. Sensitivity of SOCE to these environmental parameters was less altered by decreased extracellular Ca²⁺ alone (with STIM1 intact). We conclude that STIM1 mediates exquisite susceptibility of SOCE to pH, temperature, and glutamate: factors that can adversely affect neuronal function under pathological conditions.     

Store-operated Ca2+ Entry in Malignant Hyperthermia-susceptible Human Skeletal Muscle

In malignant hyperthermia (MH), mutations in RyR1 underlie direct activation of the channel by volatile anesthetics, leading to muscle contracture and a life-threatening increase in core body temperature. The aim of the present study was to establish whether the associated depletion of sarcoplasmic reticulum (SR) Ca²⁺ triggers sarcolemmal Ca²⁺ influx via store-operated Ca²⁺ entry (SOCE). Samples of vastus medialis muscle were obtained from patients undergoing assessment for MH susceptibility using the in vitro contracture test. Single fibers were mechanically skinned, and confocal microscopy was used to detect changes in [Ca²⁺] either within the resealed t-system ([Ca²⁺]_t-sys) or within the cytosol. In normal fibers, halothane (0.5 mm) failed to initiate SR Ca²⁺ release or Ca²⁺_t-sys depletion. However, in MH-susceptible (MHS) fibers, halothane induced both SR Ca²⁺ release and Ca²⁺_t-sys depletion, consistent with SOCE. In some MHS fibers, halothane-induced SR Ca²⁺ release took the form of a propagated wave, which was temporally coupled to a wave of Ca²⁺_t-sys depletion. SOCE was potently inhibited by “extracellular” application of a STIM1 antibody trapped within the t-system but not when the antibody was denatured by heating. In conclusion, (i) in human MHS muscle, SR Ca²⁺ depletion induced by a level of volatile anesthetic within the clinical range is sufficient to induce SOCE, which is tightly coupled to SR Ca²⁺ release; (ii) sarcolemmal STIM1 has an important role in regulating SOCE; and (iii) sustained SOCE from an effectively infinite extracellular Ca²⁺ pool may contribute to the maintained rise in cytosolic [Ca²⁺] that underlies MH.     

Distinct Mechanisms of [Ca2+]i Oscillations in HSY and HSG Cells: Role of Ca2+ Influx and Internal Ca2+ Store Recycling

This study examined [Ca²⁺]_i oscillations in the human salivary gland cell lines, HSY and HSG. Relatively low concentrations of carbachol (CCh) induced oscillatory, and higher [CCh] induced sustained, steady-state increases in [Ca²⁺]_i and K _Ca currents in both cell types. Low IP₃, but not thapsigargin (Tg), induced [Ca²⁺]_i oscillations, whereas Tg blocked CCh-stimulated [Ca²⁺]_i oscillations in both cell types. Unlike in HSG cells, removal of extracellular Ca²⁺ from HSY cells (i) did not affect CCh-stimulated [Ca²⁺]_i oscillations or internal Ca²⁺ store refill, and (ii) converted high [CCh]-induced steady-state increase in [Ca²⁺]_i into oscillations. CCh- or thapsigargin-induced Ca²⁺ influx was higher in HSY, than in HSG, cells. Importantly, HSY cells displayed relatively higher levels of sarcoendoplasmic reticulum Ca²⁺ pump (SERCA) and inositoltrisphosphate receptors (IP₃Rs) than HSG cells. These data demonstrate that [Ca²⁺]_i oscillations in both HSY and HSG cells are primarily determined by the uptake of Ca²⁺ from, and release of Ca²⁺ into, the cytosol by the SERCA and IP₃R activities, respectively. In HSY cells, Ca²⁺ influx does not acutely contribute to this process, although it determines the steady-state increase in [Ca²⁺]_i. In HSG cells, [Ca²⁺]_i oscillations directly depend on Ca²⁺ influx; Ca²⁺ coming into the cell is rapidly taken up into the store and then released into the cytosol. We suggest that the differences in the mechanism of [Ca²⁺]_i oscillations HSY and HSG cells is related to their respective abilities to recycle internal Ca²⁺ stores. Received: 30 October 2000/Revised: 26 February 2001     

Calcium elevation-dependent and attenuated resting calcium-dependent abscisic acid induction of stomatal closure and abscisic acid-induced enhancement of calcium sensitivities of S-type anion and inward-rectifying K+ channels in Arabidopsis guard cells

Stomatal closure in response to abscisic acid depends on mechanisms that are mediated by intracellular [Ca²⁺] ([Ca²⁺]_i), and also on mechanisms that are independent of [Ca²⁺]_i in guard cells. In this study, we addressed three important questions with respect to these two predicted pathways in Arabidopsis thaliana. (i) How large is the relative abscisic acid (ABA)‐induced stomatal closure response in the [Ca²⁺]_i‐elevation‐independent pathway? (ii) How do ABA‐insensitive mutants affect the [Ca²⁺]_i‐elevation‐independent pathway? (iii) Does ABA enhance (prime) the Ca²⁺ sensitivity of anion and inward‐rectifying K⁺ channel regulation? We monitored stomatal responses to ABA while experimentally inhibiting [Ca²⁺]_i elevations and clamping [Ca²⁺]_i to resting levels. The absence of [Ca²⁺]_i elevations was confirmed by ratiometric [Ca²⁺]_i imaging experiments. ABA‐induced stomatal closure in the absence of [Ca²⁺]_i elevations above the physiological resting [Ca²⁺]_i showed only approximately 30% of the normal stomatal closure response, and was greatly slowed compared to the response in the presence of [Ca²⁺]_i elevations. The ABA‐insensitive mutants ost1‐2, abi2‐1 and gca2 showed partial stomatal closure responses that correlate with [Ca²⁺]_i‐dependent ABA signaling. Interestingly, patch‐clamp experiments showed that exposure of guard cells to ABA greatly enhances the ability of cytosolic Ca²⁺ to activate S‐type anion channels and down‐regulate inward‐rectifying K⁺ channels, providing strong evidence for a Ca²⁺ sensitivity priming hypothesis. The present study demonstrates and quantifies an attenuated and slowed ABA response when [Ca²⁺]_i elevations are directly inhibited in guard cells. A minimal model is discussed, in which ABA enhances (primes) the [Ca²⁺]_i sensitivity of stomatal closure mechanisms.     

STIM1 and Orai1 mediate thrombin-induced Ca2+ influx in rat cortical astrocytes

In astrocytes, thrombin leads to cytoplasmic Ca²⁺ elevations modulating a variety of cytoprotective and cytotoxic responses. Astrocytes respond to thrombin stimulation with a biphasic Ca²⁺ increase generated by an interplay between ER-Ca²⁺ release and store-operated Ca²⁺ entry (SOCE). In many cell types, STIM1 and Orai1 have been demonstrated to be central components of SOCE. STIM1 senses the ER-Ca²⁺ depletion and binds Orai1 to activate Ca²⁺ influx. Here we used immunocytochemistry, overexpression and siRNA assays to investigate the role of STIM1 and Orai1 in the thrombin-induced Ca²⁺ response in primary cultures of rat cortical astrocytes. We found that STIM1 and Orai1 are endogenously expressed in cortical astrocytes and distribute accordingly with other mammalian cells. Importantly, native and overexpressed STIM1 reorganized in puncta under thrombin stimulation and this reorganization was reversible. In addition, the overexpression of STIM1 and Orai1 increased by twofold the Ca²⁺ influx evoked by thrombin, while knockdown of endogenous STIM1 and Orai1 significantly decreased this Ca²⁺ influx. These results indicate that STIM1 and Orai1 underlie an important fraction of the Ca²⁺ response that astrocytes exhibit in the presence of thrombin. Thrombin stimulation in astrocytes leads to ER-Ca²⁺ release which causes STIM1 reorganization allowing the activation of Orai1 and the subsequent Ca²⁺ influx.     

Oxytocin induced a biphasic increase in the intracellular Ca2+ concentration of porcine myometrial cells: Participation of a pertussis toxin—insensitive G-protein,inositol 1,4,5-trisphosphate—sensitive Ca2+ pool,and Ca2+ channels

This study investigated the underlying mechanisms of oxytocin (OT)-induced increases in intracellular Ca²⁺ concentrations ([Ca²⁺]_i) in acutely dispersed myometrial cells from prepartum sows. A dosedependent increase in [Ca²⁺]_i was induced by OT (0.1 nM to 1 μM) in the presence and absence of extracellular Ca²⁺ ([Ca²⁺]_e). [Ca²⁺]_i was elevated by OT in a biphasic pattern, with a spike followed by a sustained plateau in the presence of [Ca²⁺]_e. However, in the absence of [Ca²⁺]_e, the [Ca²⁺]_i response to OT became monophasic with a lower amplitude and no plateau, and this monophasic increase was abolished by pretreatment with ionomycin, a Ca²⁺ ionophore. Administration of OT (1 μM) for 15 sec increased inositol 1,4,5-trisphosphate (IP₃) formation by 61%. Pretreatment with pertussis toxin (PTX, 1 μg/ml) for 2 hr failed to alter the OT-induced increase in [Ca²⁺]_i and IP₃ formation. U-73122 (30 nM to 3 μM), a phospholipase C (PLC) inhibitor, depressed the rise in [Ca²⁺]_i by OT dose dependently. U-73122 (3 μM) also abolished the OT-induced IP₃ formation. Thapsigargin (2 μM), an inhibitor of Ca²⁺-ATPase in the endoplasmic reticulum, did not increase [Ca²⁺]_i. However, it did time-dependently inhibit the OT-induced increase in [Ca²⁺]_i. Nimodipine (1 μM), a Voltage-dependent Ca²⁺ channel (VDCC) blocker, inhibited the OT-induced plateau by 26%. La³⁺ (1 μM), a nonspecific Ca²⁺ channel blocker, abrogated the OT-induced plateau. In whole-cell patch-clamp studies used to evaluate VDCC activities, OT (0.1 μM) increased Ca²⁺ Current (I_ca) by 40% with no apparent changes in the current-voltage relationship. The OT-induced increase in I_ca reached the maximum in 5 min, and the increase was abolished by nimodipine (1 μM). These results suggested that (1) activation of OT receptors in porcine myometrium evokes a cascade in the PTX-insensitive G-protein–PLC-IP₃ signal transduction, resulting in an increase in [Ca²⁺]_i; (2) the OT-induced increase in [Ca²⁺]_i is characterized by a biphasic pattern, in which the spike is predominately contributed by the intracellular Ca²⁺ release from the IP₃-sensitive pool, and to a lesser extent by Ca²⁺ influx, whereas the plateau is from increased Ca²⁺ influx; and (3) the influx is via VDCC and receptor-operated Ca²⁺ channels. © 1995 Wiley-Liss, Inc.     

Mechanisms Underlying Leakage of Calcium from the Endoplasmic Reticulum of Acinar Cells of the Submandibular Salivary Gland

In the resting state, the Ca²⁺ concentration in agonist-sensitive intracellular stores reflects the balance between active uptake of Ca²⁺, which is mediated by Ca²⁺-ATPase (SERCA), and passive leakage of Ca²⁺. The mechanisms underlying such a leakage in cells of the submaxillary salivary gland were not studied. In our experiments, we examined possible pathways of passive leakage of Ca²⁺ from the endoplasmic reticulum (ER) of acinar cells obtained from the rat submaxillary salivary gland; direct measurements of the concentration of Ca²⁺ in the ER ([Ca²⁺]_ER) using a low-affinity calcium-sensitive dye, mag-fura 2/AM, were performed. The cellular membrane was permeabilized with the help of β-escin (40 μg/ml); the Ca²⁺ concentration in the cytoplasm ([Ca²⁺] _i ) was clamped at its level typical of the resting state (∼100 nM) using an EGTA/Ca²⁺ buffer. Incubation of permeabilized acinar cells in a calcium-free intracellular milieu, as well as application of thapsigargin, resulted in complete inhibition of the uptake of Ca²⁺ with the involvement of SERCA. This effect was observed 1 min after the beginning of superfusion of the cells with the corresponding solutions and was accompanied by the leakage of Ca²⁺ from the ER; this is confirmed by a gradual drop in the [Ca₂₊]_ER. Such a leakage of Ca²⁺ remained unchanged in the presence of thapsigargin, heparin, and ruthenium red; therefore, it is not mediated by SERCA, inositol 1,4,5-trisphosphate-sensitive receptors (InsP₃R), or ryanodine receptors (RyRs). At the same time, an antibiotic, puromycin (0.1 to 1.0 mM), which disconnects polypeptides from the ER-ribosome translocon complex, caused intensification of passive leakage of Ca²⁺ from the ER. This effect did not depend on the functioning of SERCA, InsP₃R, or RyR. Therefore, passive leakage of Ca²⁺ from the ER in acinar cells of the submaxillary salivary gland is realized through pores of the translocon complex of the ER membrane. Neirofiziologiya/Neurophysiology, Vol. 37, No. 4, pp. 339–346, July–August, 2005.     

ATP-induced calcium signalling in acinar cells of the submandibular salivary gland

To study changes in the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) and the total amount of calcium in cells, we used, respectively, the fluorescent dye fura 2/AM and the metallochrome dye arsenazo III. The total amount of calcium in acinar cells after their incubation in calcium-free ATP-containing extracellular solution decreased. The action of ATP induced a dose-dependent increase in the [Ca²⁺]_i; the EC₅₀ was, on average, 130 ± ± 36 μM. Calcium transients induced by ATP demonstrated no desensitization. Against the background of a blocker of ionotropic P2X receptors, pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid, we observed a decrease in the ATP-induced calcium transients by 72%. In addition, these transients were reduced by 65% in the calcium-free milieu, while after thapsigargin-induced exhaustion of the endoplasmic reticulum store they disappeared. This is indicative of the involvement of metabotropic P2Y receptors in the formation of the above calcium transients. Therefore, P2X and P2Y receptors participate in ATP-induced calcium signalling in acinar cells of the submandibular salivary gland; activation of these channels results in a rise in the [Ca²⁺]_i. The P2X receptors to a higher extent contribute to the formation of calcium signals; the P2Y-determined increase in the [Ca²⁺]_i is smaller (equal to about 35%). Therefore, the functionally active ligand-operated ionotropic P2Y receptors and metabotropic G protein-related P2Y receptors do exist in acinar cells of the submandibular salivary gland and play an important role in the control of functioning of this gland. Neirofiziologiya/Neurophysiology, Vol. 37, Nos. 5/6, pp. 395–402, September–December, 2005.     

Processes Maintaining Calcium Homeostasis in Acinar Cells of the Rat Submandibular Salivary Gland

Using a Ca²⁺-sensitive fluorescent indicator, fura-2/AM, we recorded calcium transients in secretory cells of isolated acini of the rat submandibular salivary gland; these transients were induced by hyperpotassium-induced depolarization (after an increase in [K⁺] _e up to 50 mM) of the plasma membrane of the above cells. Calcium transients were significantly suppressed by 50 M nifedipine. Addition of 10 M carbonyl cyanide m-chlorophenylhydrazone to the normal extracellular solution was accompanied by a rise in [Ca²⁺] _i , whereas when hyperpotassium solution is used the effect was less expressed. Blockers of CA²⁺-ATPase in the cellular membrane and in the endoplasmic reticulum, eosin Y (5 M) and cyclopiazonic acid (CPA, 5 M), respectively, evoked a significant increase in [Ca²⁺] _i and a decrease in the K⁺-depolarization-induced calcium transient. Extracellular application of caffeine (2, 10, or 30 mM) was accompanied by a concentration-dependent rise in [Ca²⁺] _i . Therefore, potassium depolarization of the plasma membrane of acinar cells of the rat submandibular salivary gland activates both the voltage-dependent Ca²⁺ influx and Ca²⁺-induced Ca²⁺ release from the endoplasmic reticulum; the initial level of [Ca²⁺] _i was restored at the joint involvement of Ca²⁺-ATPases in the plasma membrane and the membranes of the endoplasmic reticulum and mitochondria.     

Autoantibodies in primary Sjögren's syndrome patients induce internalization of muscarinic type 3 receptors

Objectives. Primary Sjögren's syndrome (pSS) is a systemic autoimmune disease characterized by lymphocyte infiltration into the salivary and lachrymal glands, leading to dry mouth and eyes. The presence of functional autoantibodies against muscarinic type 3 receptor (M3R) has been reported in pSS patients. However, the pathological role of anti-M3R autoantibodies in pSS salivary dysfunction remains controversial. Methods. Purified IgGs were obtained from normal (control) and primary SS patients' sera (pSS IgG). Internalization of M3R and clathrin was analyzed by biochemical assay and immunofluorescence confocal microscopy using human submandibular gland (hSMG) cells. Cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_i) was measured by microspectrofluorimetry. Results. Incubation of hSMG cells with pSS IgG (1 mg/ml) significantly decreased M3R expression levels at the membrane. Carbachol-induced [Ca²⁺]_i transients (CICTs) in these cells were also inhibited by pSS IgG. In contrast to pSS IgG, control IgG had no effect on both the M3R expression level and CICTs. We found that binding of pSS IgG to M3R induces phosphorylation of the receptor, and that the pSS IgG-induced M3R internalization is prevented by the lysosomal inhibitor, chloroquine. In addition, pSS IgG decreased membrane clathrin expression, which was inhibited by atropine. Our immunofluorescence study further confirmed that pSS IgG induces a co-localization of M3R with clathrin and subsequent internalization of M3R. Conclusion. pSS IgG induces internalization of M3R partly through a clathrin-mediated pathway. The results suggest M3R internalization as a potential mechanism to explain the exocrinopathy seen in pSS patients.     

Purinergic P2X7 Receptors Mediate ATP-induced Saliva Secretion by the Mouse Submandibular Gland

Salivary glands express multiple isoforms of P2X and P2Y nucleotide receptors, but their in vivo physiological roles are unclear. P2 receptor agonists induced salivation in an ex vivo submandibular gland preparation. The nucleotide selectivity sequence of the secretion response was BzATP ≫ ATP > ADP ≫ UTP, and removal of external Ca²⁺ dramatically suppressed the initial ATP-induced fluid secretion (∼85%). Together, these results suggested that P2X receptors are the major purinergic receptor subfamily involved in the fluid secretion process. Mice with targeted disruption of the P2X₇ gene were used to evaluate the role of the P2X₇ receptor in nucleotide-evoked fluid secretion. P2X₇ receptor protein and BzATP-activated inward cation currents were absent, and importantly, purinergic receptor agonist-stimulated salivation was suppressed by more than 70% in submandibular glands from P2X₇-null mice. Consistent with these observations, the ATP-induced increases in [Ca²⁺]_i were nearly abolished in P2X₇^–/– submandibular acinar and duct cells. ATP appeared to also act through the P2X₇ receptor to inhibit muscarinic-induced fluid secretion. These results demonstrate that the ATP-sensitive P2X₇ receptor regulates fluid secretion in the mouse submandibular gland.Salivation is a Ca²⁺-dependent process (1, 2) primarily associated with the neurotransmitters norepinephrine and acetylcholine, release of which stimulates α-adrenergic and muscarinic receptors, respectively. Both types of receptors are coupled to G proteins that activate phospholipase Cβ (PLCβ) during salivary gland stimulation. PLCβ activation cleaves phosphatidylinositol 1,4-bisphosphate resulting in diacylglycerol and inositol 1,4,5-trisphosphate (InsP₃) production. Activation of Ca²⁺-selective InsP₃ receptor channels localized to the endoplasmic reticulum of salivary acinar cells increases the intracellular free calcium concentration ([Ca²⁺]_i).⁴ Depletion of the endoplasmic reticulum Ca²⁺ pool triggers extracellular Ca²⁺ influx and a sustained elevation in [Ca²⁺]_i. This increase in [Ca²⁺]_i activates Ca²⁺-dependent K⁺ and Cl^– channels promoting Cl^– secretion across the apical membrane and a lumen negative, electrochemical gradient that supports Na⁺ efflux into the lumen. The accumulation of NaCl creates an osmotic gradient which drives water movement into the lumen, thus generating isotonic primary saliva. This primary fluid is then modified by the ductal system, which reabsorbs NaCl and secretes KHCO₃ producing a final saliva that is hypotonic (1, 2).Salivation also has a non-cholinergic, non-adrenergic component, the origin of which is unclear (3). In addition to muscarinic and α-adrenergic receptors, salivary acinar cells express other receptors that are coupled to an increase in [Ca²⁺]_i such as purinergic P2 and substance P receptors. Like muscarinic and α-adrenergic receptors, P2 receptor activation leads to a sustained increase in [Ca²⁺]_i in salivary acinar cells (4). In contrast, substance P receptor activation rapidly desensitizes and therefore generates only a relatively transient increase in [Ca²⁺]_i (5) that is unlikely to appreciably contribute to fluid secretion. The purinergic P2 receptor family is comprised of G protein-coupled P2Y and ionotropic P2X receptors activated by extracellular di- and triphosphate nucleotides. Activation of both subfamilies of P2 receptors causes an increase in [Ca²⁺]_i. P2Y receptors increase [Ca²⁺]_i via InsP₃-induced Ca²⁺ mobilization from intracellular stores (similar to α-adrenergic and muscarinic receptors) while P2X receptors act as ligand-gated, non-selective cation channels that mediate extracellular Ca²⁺ influx (6). Salivary gland tissues express at least four isoforms of P2X (P2X₄ and P2X₇) and P2Y (P2Y₁ and P2Y₂) subtypes; however, their in vivo physiological significance has yet to be characterized (7–11).Our results revealed that ATP acts in isolation to stimulate fluid secretion from the mouse submandibular gland, but secretion is inhibited when ATP is simultaneously presented with a muscarinic receptor agonist. Ablation of the P2X₇ gene had no effect on the salivary flow rate evoked by muscarinic receptor activation, but markedly reduced ATP-mediated fluid secretion and rescued the inhibitory effects of ATP on muscarinic receptor activation. Submandibular gland acinar cells from P2X₇^–/– animals had dramatically impaired ATP-activated Ca²⁺ signaling, consistent with this being the mechanism responsible for the reduction in ATP-mediated fluid secretion in these mice. Together, these results demonstrated that ATP regulates salivation, acting mainly through the P2X₇ receptor. Activation of the P2X₇ receptor may play a major role in non-adrenergic, non-cholinergic stimulated fluid secretion.     

Dynamic regulation of guard cell anion channels by cytosolic free Ca2+ concentration and protein phosphorylation

In guard cells, activation of anion channels (I_anion) is an early event leading to stomatal closure. Activation of I_anion has been associated with abscisic acid (ABA) and its elevation of the cytosolic free Ca²⁺ concentration ([Ca²⁺]_i). However, the dynamics of the action of [Ca²⁺]_i on I_anion has never been established, despite its importance for understanding the mechanics of stomatal adaptation to stress. We have quantified the [Ca²⁺]_i dynamics of I_anion in Vicia faba guard cells, measuring channel current under a voltage clamp while manipulating and recording [Ca²⁺]_i using Fura‐2 fluorescence imaging. We found that I_anion rises with [Ca²⁺]_i only at concentrations substantially above the mean resting value of 125 ± 13 nm , yielding an apparent K_d of 720 ± 65 nm and a Hill coefficient consistent with the binding of three to four Ca²⁺ ions to activate the channels. Approximately 30% of guard cells exhibited a baseline of I_anion activity, but without a dependence of the current on [Ca²⁺]_i. The protein phosphatase antagonist okadaic acid increased this current baseline over twofold. Additionally, okadaic acid altered the [Ca²⁺]_i sensitivity of I_anion, displacing the apparent K_d for [Ca²⁺]_i to 573 ± 38 nm . These findings support previous evidence for different modes of regulation for I_anion, only one of which depends on [Ca²⁺]_i, and they underscore an independence of [Ca²⁺]_i from protein (de‐)phosphorylation in controlling I_anion. Most importantly, our results demonstrate a significant displacement of I_anion sensitivity to higher [Ca²⁺]_i compared with that of the guard cell K⁺ channels, implying a capacity for variable dynamics between net osmotic solute uptake and loss.     

Mitochondria adjust Ca signaling regime to a pattern of stimulation in salivary acinar cells

The salivary acinar cells have unique Ca²⁺ signaling machinery that ensures an extensive secretion. The agonist-induced secretion is governed by Ca²⁺ signals originated from the endoplasmic reticulum (ER) followed by a store-operated Ca²⁺ entry (SOCE). During tasting and chewing food a frequency of parasympathetic stimulation increases up to ten fold, entailing cells to adapt its Ca²⁺ machinery to promote ER refilling and ensure sustained SOCE by yet unknown mechanism. By employing a combination of fluorescent Ca²⁺ imaging in the cytoplasm and inside cellular organelles (ER and mitochondria) we described the role of mitochondria in adjustment of Ca²⁺ signaling regime and ER refilling according to a pattern of agonist stimulation. Under the sustained stimulation, SOCE is increased proportionally to the degree of ER depletion. Cell adapts its Ca²⁺ handling system directing more Ca²⁺ into mitochondria via microdomains of high [Ca²⁺] providing positive feedback on SOCE while intra-mitochondrial tunneling provides adequate ER refilling. In the absence of an agonist, the bulk of ER refilling occurs through Ca²⁺-ATPase-mediated Ca²⁺ uptake within subplasmalemmal space. In conclusion, mitochondria play a key role in the maintenance of sustained SOCE and adequate ER refilling by regulating Ca²⁺ fluxes within the cell that may represent an intrinsic adaptation mechanism to ensure a long-lasting secretion.     

Modulation of Ca2+ Influx in Leech Retzius Neurons II. Effect of Extracellular Ca2+

We investigated the cytosolic free calcium concentration ([Ca²⁺]_i) of leech Retzius neurons in situ while varying the extracellular Ca²⁺ concentration via the bathing solution ([Ca²⁺]_B). Changing [Ca²⁺]_B had only an effect on [Ca²⁺]_i if the cells were depolarized by raising the extracellular K⁺ concentration. Surprisingly, raising [Ca²⁺]_B from 2 to 10 mm caused a decrease in [Ca²⁺]_i, and an increase was evoked by reducing [Ca²⁺]_B to 0.1 mm. These changes were not due to shifts in membrane potential. At low [Ca²⁺]_B moderate membrane depolarizations were sufficient to evoke a [Ca²⁺]_i increase, while progressively larger depolarizations were necessary at higher [Ca²⁺]_B. The changes in the relationship between [Ca²⁺]_i and membrane potential upon varying [Ca²⁺]_B could be reversed by changing extracellular pH. We conclude that [Ca²⁺]_B affects [Ca²⁺]_i by modulating Ca²⁺ influx through voltage-dependent Ca²⁺ channels via the electrochemical Ca²⁺ gradient and the surface potential at the extracellular side of the plasma membrane. These two parameters are affected in a counteracting way: Raising the extracellular Ca²⁺ concentration enhances the electrochemical Ca²⁺ gradient and hence Ca²⁺ influx, but it attenuates Ca²⁺ channel activity by shifting the extracellular surface potential to the positive direction, and vice versa. Received: 23 January 2001/Revised: 23 June 2001