J. Ricardo Martinez

Characterization of Ca2+ Mobilization in the Human Submandibular Duct Cell Line A253

Abstract The regulation of Ca2+ mobilization in the human submandibular duct cell line A253 was investigated by monitoring cytosolic free Ca1+ concentrations ([Ca2+],) using the Ca2+-sensitive fluorescent indicator fura-2 and by measuring inositol 1,4,5-triphosphate (IP3) formation. An increase in [Ca2+], was elicited by ATP, isoproterenol (IPR), or vasoactive intestinal polypeptide (VIP), but not by acetylcholine, norepinephrine, or substance P, suggesting that Ca2+ mobilization is regulated by P2-purinergic, (β2-adrenergic, and VIP receptors. 1,4,5-IP3 formation was significantly increased by ATP but not by the other agonists. Exposure of the cells to a membrane permeable cAMP analog, dibutyryl-cAMP, or to the adenylate cyclase activator forskolin induced a smaller increase in [Ca2+]i, indicating that the IPR-induced Ca2+ release is not mediated by cyclic AMP. Inhibition of the endoplasmic Ca2+-ATPase with thapsigargin (TG) in Ca2+-free medium induced a 207% increase in [Ca2+]i, and a subsequent exposure to ATP caused a further increase in [Ca2+]i of 104%. Similarly, TG exposure after ATP induced a further Ca2+ release, suggesting that the TG-sensitive store and the IP3-sensitive store do not overlap. Similar results were observed by sequential exposure to TG and IPR or to ATP and IPR. Ca2+ influx across the plasma membrane was enhanced after ATP or TG, but not after IPR. Our findings show a unique pattern of Ca2+ mobilization in the A253 cell line: (i) Ca2+ mobilization is regulated by P2-purinergic, β2-adrenergic, and VIP receptors; (ii) Ca2+ release is mediated by 1,4,5-IP3and probably by an unknown mediator; (iii) TG, P2-, and β2-agonists discharge separate Ca2+ stores; and (iv) ATP and TG, but not IPR, regulate Ca2+ influx.

Effects of serum on calcium mobilization in the submandibular cell line A253.

The effects of serum on inositol 1,4,5‐trisphosphate (IP3) formation and Ca2+ mobilization in the human submandibular cell line A253 were studied. Exposure of A253 cells to fetal bovine serum (FBS) elicited a 3.3‐fold increase in IP3 formation and a concentration‐dependent transient increase in cytosolic free Ca2+ concentration ([Ca2+]i), which was similar in Ca2+‐containing and Ca2+‐free media. Newborn bovine serum (NBS), but not bovine serum albumin (BSA), induced a similar response. The Ca2+ release triggered by FBS was significantly (88%) reduced by the phospholipase C inhibitor U73122, indicating that Ca2+ release induced by FBS is through the PLC pathway. Pretreatment with the tyrosine kinase inhibitor genistein abolished the FBS‐ and NBS‐induced Ca2+ release, suggesting that tyrosine kinase plays an important role in mediating the Ca2+ release. Pre‐exposure to ATP or thapsigargin (TG) significantly reduced the FBS‐induced [Ca2+]i increase, indicating that Ca2+ release caused by FBS is from the TG‐ or ATP‐sensitive Ca2+ store. While FBS exposure elicited a large Ca2+ release, it reduced Ca2+ influx. Furthermore, FBS significantly inhibited the Ca2+ influx activated by the depletion of intracellular stores by ATP or TG. These results suggest that (1) serum elicits Ca2+ release from ATP‐ and TG‐sensitive stores, which is mediated by IP3; (2) the serum‐induced Ca2+ release may be modulated by a tyrosine kinase‐associated process; and (3) serum strongly inhibits Ca2+ influxes including the store depletion‐activated Ca2+ influx. J. Cell. Biochem. 73:458–468, 1999.

Comparison of calcium mobilization in response to noradrenaline and acetylcholine in submandibular cells of newborn and adult rats

The response of mature and immature rat submandibular cells to alpha-receptor stimulation was compared in terms of the generation of inositol triphosphate (IP3) and Ca2+ mobilization, and of how the calcium mobilization response affects acetycholine (ACh)-induced Ca2+ mobilization. In mature cells, noradrenaline (NA) caused much smaller IP3 and Ca2+ responses than ACh. However, the Ca2+ release induced by NA was enough to partially discharge an agonist-sensitive store and to reduce Ca2+ release by a subsequent ACh stimulus. Exposure to NA also caused an influx of Ca2+ in the mature cells, which was largely associated with Ca2+ entry induced by store depletion (i.e. capacitative entry). In the immature submandibular cells of newborn rats, NA caused essentially no IP3 response and a small Ca2+ release, which only partially affected the Ca2+ released by a subsequent exposure to ACh. In contrast to adult cells, immature cells did not show an increased Ca2+ influx after exposure to NA. However, prestimulation with this agonist potentiated the Ca2+ influx activated by ACh in the cells of newborn rats, but not in cells of adult rats. As both mature and immature submandibular cells have a well-developed phosphoinositide turnover response to ACh, the findings in mature cells suggest a less efficient coupling between alpha-receptors and phospholipase C, while those in immature cells suggest that this coupling is even less functional in the early stages of postnatal development. In permeabilized and 45Ca(2+)-loaded mature cells, cyclic ADP-ribose (cADPR) released 13.4% of loaded 45Ca2+ and this release was significantly reduced by pre-exposure to IP3. Similarly, pre-exposure to cADPR also reduced the IP3-induced 45Ca2+ release. It is concluded that: (1) stimulation with NA induces a smaller Ca2+ release in mature and immature submandibular cells than ACh; (2) the mediator for this small Ca2+ mobilization may be cADPR; and (3) NA stimulates capacitative Ca2+ entry in mature cells, but not in immature cells, and it also activates a Ca2+ entry pathway distinct from the one induced by store depletion, particularly in immature cells.

Effects of low concentrations of paraoxon on Ca2+ mobilization in a human parotid salivary cell-line HSY

The salivary gland is a target organ of organophosphate pesticides (OPs). Inhibition of acetylcholinesterase (AChE) by OPs leads to a decrease in acetylcholine (ACh) breakdown that results in overstimulation of muscarinic cholinergic receptors (mChR). However, OPs may also directly interact with downstream elements of the phosphoinositide (PI) signalling pathway coupled with mChR. The present study examined the effects of exposure to low concentrations of the OP paraoxon on inositol 1,4,5-trisphosphate (IP(3)) formation and Ca(2+) mobilization in response to ACh or ATP in the human parotid cell-line HSY. Exposure to 0.1 and 1 nM, but not 10 nM, paraoxon for 24 hr significantly elevated the basal cytosolic free Ca(2+) ([Ca(2+)](i)). This increase was abolished by atropine. Ca(2+) release from the IP(3)-sensitive store in response to ACh or ATP, a P2Y-nucleotide agonist, was significantly increased in cells pre-exposed to 0.1 nM paraoxon. However, IP(3) formation was inhibited by paraoxon but mChR expression was not altered. Although IP(3) receptor expression was not changed, Ca(2+) release elicited by IP(3) in streptolysin O toxin-permeabilized cells was significantly larger in cells pre-exposed to 0.1 nM paraoxon, suggesting that paraoxon increases the sensitivity of IP(3) receptors. Paraoxon exposure also induced a concentration-dependent reduction in the total capacity of intracellular Ca(2+) stores, whereas the capacity of the IP(3)-sensitive Ca(2+) store was not altered by paraoxon, as judged by discharging of the IP(3)-sensitive Ca(2+) store with thapsigargin (TG). Ca(2+) influx stimulated by ACh or ATP was also enhanced by 0.1 nM, but not 1 and 10 nM, paraoxon. On the other hand, Ca(2+) influx activated by TG was enhanced by exposure to all concentrations of paraoxon, indicating that paraoxon modulates the Ca(2+) entry pathway. These results suggest that low concentrations of paraoxon interact with elements of the PI pathway, enhancing Ca(2+) release and influx mechanisms.

Developmental Aspects of Fluid and Electrolyte Secretion in Salivary Glands

The salivary glands of rodents undergo considerable cytodifferentiation after birth and are useful models for the study of functional development, including the mechanisms of fluid and electrolyte secretion. In the rat submandibular gland, secretion of salivary fluid cannot be elicited until approximately 2 weeks of age. The currently accepted model of salivary fluid secretion indicates that this process depends on the activation, on stimulation of cholinergic receptors, of several ion transport systems, resulting in a net transport of osmotically active ions (primarily Cl- and Na+) across the acinar epithelium. This creates the necessary osmotic gradient for the transacinar movement of water. The process is associated with a signal transduction pathway involving the formation of phosphoinositide products (primarily inositol triphosphate or IP3) and the mobilization of Ca2+. The latter regulates monovalent ion conductances (K+, Cl-), which are critical for the secretory process. Immature submandibular glands and cells of early postnatal rats have a lower density of cholinergic receptors and release less K+ and Cl- than mature cells and gradually develop other ion transport systems (such as a Na, K, 2Cl cotransport system) involved in the secretory process. Surprisingly, they form more IP3 and show a larger increase in cytosolic Ca2+ when stimulated with maximal or supramaximal concentrations of agonist. Therefore, they show some interesting dissociations in the signal transduction mechanism that suggest differences in the coupling between receptors and membrane phosphoinositides, between IP3 and IP3-dependent Ca2+ stores, and between the Ca2+ signal and the monovalent ion transport systems which are critical for secretion.

Chloride secretion in the submandibular gland of adult and early postnatal rats studied by X-ray microanalysis.

Submandibular acinar cells of 1-day-old, 7-day-old, and adult rats were analyzed with X-ray microanalysis after stimulation with carbachol for different time periods (2–7 min). In unstimulated animals, marked differences in elemental content between compartments could be observed: secretory granules had a higher Ca and lower P and K content than other cell compartments. Comparison between different age groups showed significant differences for Ca, which increased with age in all compartments; Mg increased with age in the secretory granules and the apical cytoplasm. Only the glands from adult animals showed a significant effect of cholinergic stimulation: a transient decrease in Cl and K. The Cl concentration in the secretory granules decreased to 60% of the control value, which suggests that the granules release Cl upon stimulation. In young animals, no or little change in elemental distribution was observed after stimulation. This may indicate that Cl-secretion mechanisms are much less prominent in young animals. The ultrastructure of submandibular secretory granules depends on the preparation method: condensed and electrondense in freeze-substituted unfixed tissue, decondensed and more translucent in aldehyde-fixed tissue. This may indicate that the granules can transport water, and swell during the process of aldehyde fixation.

Effects of particulate and soluble (1–3)-β-glucans on Ca2+ influx in NR8383 alveolar macrophages

Particulate and soluble (1-3)-beta-glucans are effective in preventing infections by enhancing macrophage and neutrophil functions. However, the mechanisms triggering these enhanced cellular responses are essentially unknown. We recently demonstrated that zymosan, a particulate (1-3)-beta-glucan receptor agonist, caused an influx of Ca2+ in NR8383 rat alveolar macrophages (AMs) and a resulting increase in intracellular Ca2+ (Zhang et al., J. Leukoc. Biol. 62 (1997) 341-348). Since Ca2+ is important in mediating leukocyte responses, we investigated whether other (1-3)-beta-glucans also alter Ca2+ mobilization in AMs. Particulate and soluble (1-3)-beta-glucans derived from Saccharomyces cerevisiae were used in these studies. Like zymosan, particulate (1-3)-beta-glucan (WGPs) caused a concentration-dependent increase in [Ca2+]i, which was inhibited by removal of extracellular Ca2+ and by SKF96365, an inhibitor of receptor-operated Ca2+ channels. When three different soluble (1-3)-beta-glucans, with molecular weights of approximately 11,000, 150,000, and 1,000,000 Da, were tested alone for effects on Ca2+ responses, the low molecular weight (1-3)-beta-glucan produced no effect and the intermediate and high molecular weight (1-3)-beta-glucans caused only a small increase in [Ca2+]i. Interestingly, however, all three soluble (1-3)-beta-glucans could significantly reduce the Ca2+ responses induced by a subsequent exposure to either WGPs or zymosan. These results demonstrate that: 1) particulate (1-3)-beta-glucan activates Ca2+ influx in NR8383 macrophages through receptor-operated Ca2+ channels; 2) soluble (1-3)-beta-glucans do not strongly activate Ca2+ influx in these cells; and 3) soluble (1-3)-beta-glucans significantly inhibit Ca2+ influx induced by WGPs or zymosan. Soluble (1-3)-beta-glucans are likely to prevent Ca2+ influx by competitively binding to the (1-3)-beta-glucan receptors recognizing zymosan and WGPs. The smaller Ca2+ influx induced by soluble (1-3)-beta-glucans may represent only a partial activation of post-receptor signal transduction pathways necessary for inducing Ca2+ influx.

Inhibition of Ca2+ influx by pentoxifylline in NR8383 alveolar macrophages.

Pentoxifylline (PTF), a phosphodiesterase (PDE) inhibitor, can prevent inflammation and tissue damage in animal and in vitro human studies. However, the underlying mechanism remains unclear. Since Ca2+ is a critical signal regulating the release of inflammatory mediators in macrophages, the effects of PTF on Ca2+ influx were examined in NR8383 alveolar macrophages (AMs). PTF induced a dose-dependent inhibition on Ca2+ influx activated by zymosan and by protein kinase C (PKC) activators 1,2-dioctanoyl-sn-glycerol (DOG) or phorbol-12-myristate 13-acetate (PMA). The inhibition appeared to be specifically on the receptor-operated Ca2+ entry. The capacitative Ca2+ entry was not affected by PTF. The inhibition was not due to altered cAMP levels since the zymosan-activated Ca2+ influx was not affected by the adenylate cyclase activator forskolin, nor by dibutyryl cAMP. Pretreatment with protein tyrosine kinase (PTK) inhibitor genistein abolished zymosan-induced, but not DOG-induced Ca2+ influx, suggesting that PTK is an upstream element of the signaling cascade and not the target of PTF. The Ca2+ entry activated by zymosan and by PKC activators was inhibited by the mitogen-activated protein kinase (MAPK) inhibitor PD98059. Moreover, activation of MAPK by C6-ceramide (C6C) triggered a similar Ca2+ influx as elicited by zymosan and PKC activators, suggesting that MAPK is an element of the pathway. The C6C-induced Ca2+ influx was also inhibited by PTF. These results indicate that PTF blocks the receptor-operated Ca2+ influx in NR8383 AMs by inhibiting PDE which may acts as a downstream element of the signaling pathway or by direct interaction with Ca2+ channels.

Modulation of Ca2+ mobilization by protein kinase C in the submandibular duct cell line A253.

The expression of protein kinase C (PKC) isoforms and the modulation of Ca2+ mobilization by PKC were investigated in the human submandibular duct cell line A253. Three new PKC (nPKC) isoforms (δ, ε, and θ) and one atypical PKC (aPKC) isoform (λ) are expressed in this cell line. No classical PKC (cPKC) isoforms were present. The effects of the PKC activator phorbol 12-myristate-13-acetate (PMA) and of the PKC inhibitors calphostin C (CC) and bisindolymaleimide I (BSM) on inositol 1,4,5-trisphosphate (IP3) and Ca2+ responses to ATP and to thapsigargin (TG) were investigated. Pre-exposure to PMA inhibited IP3 formation, Ca2+ release and Ca2+ influx in response to ATP. Pre-exposure to CC or BSM slightly enhanced IP3 formation but inhibited the Ca2+ release and the Ca2+ influx induced by ATP. In contrast, pre-exposure to PMA did not modify the Ca2+ release induced by TG, but reduced the influx of Ca2+ seen in the presence of this Ca2+-ATPase inhibitor. These results suggest that PKC modulates elements of the IP3/Ca2+ signal transduction pathway in A253 cells by (1) inhibiting phosphatidylinositol turnover and altering the sensitivity of the Ca2+ channels to IP3, (2) altering the activity, the sensitivity to inhibitors, or the distribution of the TG-sensitive Ca2+ ATPase, and (3) modulating Ca2+ entry pathways.

Modulation of Ca2+ mobilization by protein kinase C in rat submandibular acinar cells

The effects of protein kinase C (PKC) activation and inhibition on the inositol 1,4,5‐trisphosphate (IP3) and cytosolic Ca2+ ([Ca2+]i) responses of rat submandibular acinar cells were investigated. IP3 formation in response to acetylcholine (ACh) was not affected by the PKC activator phorbol 12‐myristate 13‐acetate (PMA), nor by the PKC inhibitor calphostin C (CaC). The ACh‐elicited initial increase in [Ca2+]i in the absence of extracellular Ca2+ was not changed by short‐term (0.5 min) exposure to PMA, but significantly reduced by long‐term (30 min) exposure to PMA, and also by pre‐exposure to the PKC inhibitors CaC and chelerythrine chloride (ChC). After ACh stimulation, subsequent exposure to ionomycin caused a significantly (258%) larger [Ca2+]i increase in CaC‐treated cells than in control cells. However, pre‐exposure to CaC for 30 min did not alter the Ca2+ release induced by ionomycin alone. These results suggest that the reduction of the initial [Ca2+]i increase is due to an inhibition of the Ca2+ release mechanism and not to store shrinkage. The thapsigargin (TG)‐induced increase in [Ca2+]i was significantly reduced by short‐term (0.5 min), but not by long‐term (30 min) exposure to PMA, nor by pre‐exposure to ChC or CaC. Subsequent exposure to ionomycin after TG resulted in a significantly (70%) larger [Ca2+]i increase in PMA‐treated cells than in control cells, suggesting that activation of PKC slows down the Ca2+ efflux or passive leak seen in the presence of TG. Taken together, these results indicate that inhibition of PKC reduces the IP3‐induced Ca2+ release and activation of PKC reduces the Ca2+ efflux seen after inhibition of the endoplasmic Ca2+‐ATPase in submandibular acinar cells. J. Cell. Biochem. 72:47–55, 1999.