A PACAP-activated network for secretion requires coordination of Ca2+ influx and Ca2+ mobilization.

Chromaffin cells of the adrenal medulla transduce sympathetic nerve activity into stress hormone secretion. The two neurotransmitters principally responsible for coupling cell stimulation to secretion are acetylcholine and pituitary adenylate activating polypeptide (PACAP). In contrast to acetylcholine, PACAP evokes a persistent secretory response from chromaffin cells. However, the mechanisms by which PACAP acts are poorly understood. Here, it is shown that PACAP induces sustained increases in cytosolic Ca2+ which are disrupted when Ca2+ influx through L-type channels is blocked or internal Ca2+ stores are depleted. PACAP liberates stored Ca2+ via inositol trisphosphate receptors (IP3Rs) on the endoplasmic reticulum (ER), thereby functionally coupling Ca2+ mobilization to Ca2+ influx and supporting Ca2+-induced Ca2+-release. These Ca2+ influx and mobilization pathways are unified by an absolute dependence on phospholipase C epsilon (PLCε) activity. Thus, the persistent secretory response that is a defining feature of PACAP activity, in situ, is regulated by a signaling network that promotes sustained elevations in intracellular Ca2+ through multiple pathways.

A PACAP-activated network for secretion requires coordination of Ca 2+ influx and Ca 2+ mobilization.

Chromaffin cells of the adrenal medulla transduce sympathetic nerve activity into stress hormone secretion. The two neurotransmitters principally responsible for coupling cell stimulation to secretion are acetylcholine and pituitary adenylate activating polypeptide (PACAP). In contrast to acetylcholine, PACAP evokes a persistent secretory response from chromaffin cells. However, the mechanisms by which PACAP acts are poorly understood. Here, it is shown that PACAP induces sustained increases in cytosolic Ca 2+ which are disrupted when Ca 2+ influx through L-type channels is blocked or internal Ca 2+ stores are depleted. PACAP liberates stored Ca 2+ via inositol trisphosphate receptors (IP3Rs) on the endoplasmic reticulum (ER), thereby functionally coupling Ca 2+ mobilization to Ca 2+ influx and supporting Ca 2+ -induced Ca 2+ -release. These Ca 2+ influx and mobilization pathways are unified by an absolute dependence on phospholipase C epsilon (PLCε) activity. Thus, the persistent secretory response that is a defining feature of PACAP activity, in situ , is regulated by a signaling network that promotes sustained elevations in intracellular Ca 2+ through multiple pathways.

Hair cell morphological patterns and polarity organization in the sea lamprey vestibular cristae.

The inner ear of the sea lamprey was examined by scanning electron microscopy, antibody labeling with tubulin, Myo7a, Spectrin, and Phalloidin stain to elucidate the canal cristae organization and the morphology and polarity of the hair cells. We characterized the hair cell stereocilia bundles and their morphological polarity with respect to the kinocilia. We identified three types of hair cells. In Type 1 hair cells, the kinocilia were slightly longer than the tallest stereocilia. This type was located along the medial bank of the crista and their polarity, based on kinocilia location, was uniformly pointed ampullipetally. Type 2 hair cells that had kinocilia that were much longer than the stereocilia, were most abundant in the central region of the crista. This type of hair cell displayed variable polarity. Type 3 hair cells had extremely long kinocilia (~40-50 µm long) and with extremely short stereocilia. They were mostly located in the lateral zone crista and displayed ampullipetal polarity. Myo7a and tubulin antibodies revealed that hair cells and vestibular afferents are distributed across the canal cristae in the lamprey, covering the area of cruciate eminence; a feature that is absent in more derived vertebrates. Spectrin shows hair cells of varying polarities in the central zone. In this zone, some cells followed the main polarity vector (lateral) like those in medial and lateral zones, whereas other cells displayed polarities that carried up to 40° from the main polarity vector.

PACAP and acetylcholine cause distinct Ca2+ signals and secretory responses in chromaffin cells.

The adrenomedullary chromaffin cell transduces chemical messages into outputs that regulate end organ function throughout the periphery. At least two important neurotransmitters are released by innervating preganglionic neurons to stimulate exocytosis in the chromaffin cell-acetylcholine (ACh) and pituitary adenylate cyclase activating polypeptide (PACAP). Although PACAP is widely acknowledged as an important secretagogue in this system, the pathway coupling PACAP stimulation to chromaffin cell secretion is poorly understood. The goal of this study is to address this knowledge gap. Here, it is shown that PACAP activates a Gαs-coupled pathway that must signal through phospholipase C ε (PLCε) to drive Ca2+ entry and exocytosis. PACAP stimulation causes a complex pattern of Ca2+ signals in chromaffin cells, leading to a sustained secretory response that is kinetically distinct from the form stimulated by ACh. Exocytosis caused by PACAP is associated with slower release of peptide cargo than exocytosis stimulated by ACh. Importantly, only the secretory response to PACAP, not ACh, is eliminated in cells lacking PLCε expression. The data show that ACh and PACAP, acting through distinct signaling pathways, enable nuanced and variable secretory outputs from chromaffin cells.

Selection and characterization of structure-switching DNA aptamers for the salivary peptide histatin 3.

In this study, single-stranded DNA aptamers that switch structural conformation upon binding to the salivary peptide histatin 3 have been reported for the first time. Histatin 3 is an antimicrobial peptide that possesses the capability of being a therapeutic agent against oral candidiasis and has recently been linked as a novel biomarker for acute stress. The aptamers were identified through a library immobilization version of an iterative in vitro process known as the Systematic Evolution of Ligands by EXponential enrichment (SELEX). Through the SELEX process, four unique aptamer candidates sharing a consensus sequence were identified. These selected sequences exhibited binding affinity and specificity to histatin 3 and in order to further characterize these aptamers, a direct format enzyme-linked aptamer sorbent assay (ELASA) was developed. The best performing candidate demonstrated an equilibrium dissociation constant (Kd) value of 1.97 ± 0.48 µM. These novel aptamers have the potential to lead to the further development of refined sensing assays and platforms for the detection and quantification of histatin 3 in human saliva and other biological media.

Synaptotagmin-7 enhances calcium-sensing of chromaffin cell granules and slows discharge of granule cargos.

Synaptotagmin-7 (Syt-7) is one of two major calcium sensors for exocytosis in adrenal chromaffin cells, the other being synaptotagmin-1 (Syt-1). Despite a broad appreciation for the importance of Syt-7, questions remain as to its localization, function in mediating discharge of dense core granule cargos, and role in triggering release in response to physiological stimulation. These questions were addressed using two distinct experimental preparations-mouse chromaffin cells lacking endogenous Syt-7 (KO cells) and a reconstituted system employing cell-derived granules expressing either Syt-7 or Syt-1. First, using immunofluorescence imaging and subcellular fractionation, it is shown that Syt-7 is widely distributed in organelles, including dense core granules. Total internal reflection fluorescence (TIRF) imaging demonstrates that the kinetics and probability of granule fusion in Syt-7 KO cells stimulated by a native secretagogue, acetylcholine, are markedly lower than in WT cells. When fusion is observed, fluorescent cargo proteins are discharged more rapidly when only Syt-1 is available to facilitate release. To determine the extent to which the aforementioned results are attributable purely to Syt-7, granules expressing only Syt-7 or Syt-1 were triggered to fuse on planar supported bilayers bearing plasma membrane SNARE proteins. Here, as in cells, Syt-7 confers substantially greater calcium sensitivity to granule fusion than Syt-1 and slows the rate at which cargos are released. Overall, this study demonstrates that by virtue of its high affinity for calcium and effects on fusion pore expansion, Syt-7 plays a central role in regulating secretory output from adrenal chromaffin cells.

CACNB2 is associated with aberrant RAS-MAPK signaling in hypertensive Dahl Salt-Sensitive rats.

Several independent genome-wide association studies (GWAS) have indicated that calcium (Ca2+) voltage-gated channel auxiliary subunit beta 2 (CACNB2) an L-type Ca2+ channel (LTCC) associated protein has strong association with hypertension. However, the molecular mechanism of CACNB2 and its role in the pathophysiology of hypertension is not clear. To address this knowledge gap, we utilized in vitro and in vivo approaches using HEK293 cells and genetically hypertensive, Dahl Salt-Sensitive (SS) rats. We demonstrated that CACNB2 over-expression in HEK293 cells triggers cell proliferation via an up-regulation of the RAS-MAPK pathway compared to non-transfected cells. These effects were likely independent of LTCC activity as treatment with nifedipine, a well-known LTCC blocker, in CACNB2 overexpressing cells failed to inhibit the RAS-MAPK pathway gene expressions or show an effect on apoptosis marker gene expression. Furthermore, the expression level of CACNB2 was up-regulated in the high salt (HS) diet fed SS rat kidneys compared to low salt diet (LS) fed group. Similar to our in vitro observation the RAS-MAPK mRNA levels were increased in HS fed SS rat kidneys, compared to LS fed group. Collectively, our data suggest that CACNB2 is associated with the increase in RAS-MAPK gene expressions and lead us to speculate that in addition to its role in regulating LTCC α1-subunit trafficking, CACNB2 might lead to aberrant RAS activation, which is one of the key cascade associated with hypertension.

Role of NAADP for calcium signaling in the salivary gland.

Coordination of intracellular Ca2+ signaling in parotid acini is crucial for controlling the secretion of primary saliva. Previous work from our lab has demonstrated acidic-organelle Ca2+ release as a participant in agonist-evoked signaling dynamics of the parotid acinar cell. Furthermore, results implicated a potential role for the potent Ca2+ releasing second messenger NAADP in these events. The current study interrogated a direct role of NAADP for Ca2+ signaling in the parotid salivary gland acinar cell. Use of live-cell Ca2+ imaging, patch-clamp methods, and confocal microscopy revealed for the first time NAADP can evoke or enhance Ca2+ dynamics in parotid acini. These results were compared with pancreatic acini, a morphologically similar cell type previously shown to display NAADP-dependent Ca2+ signals. Findings presented here may be relevant in establishing new therapeutic targets for those suffering from xerostomia produced by hypofunctioning salivary glands.

Arachidonic acid-induced Ca2+ entry and migration in a neuroendocrine cancer cell line.

BACKGROUND: Store-operated Ca2+ entry (SOCE) has been implicated in the migration of some cancer cell lines. The canonical SOCE is defined as the Ca2+ entry that occurs in response to near-maximal depletion of Ca2+ within the endoplasmic reticulum. Alternatively, arachidonic acid (AA) has been shown to induce Ca2+ entry in a store-independent manner through Orai1/Orai3 hetero-multimeric channels. However, the role of this AA-induced Ca2+ entry pathway in cancer cell migration has not been adequately assessed. METHODS: The present study investigated the involvement of AA-induced Ca2+ entry in migration in BON cells, a model gastro-enteropancreatic neuroendocrine tumor (GEPNET) cell line using pharmacological and gene knockdown methods in combination with live cell fluorescence imaging and standard migration assays. RESULTS: We showed that both the store-dependent and AA-induced Ca2+ entry modes could be selectively activated and that exogenous administration of AA resulted in Ca2+ entry that was pharmacologically distinct from SOCE. Also, whereas homomeric Orai1-containing channels appeared to largely underlie SOCE, the AA-induced Ca2+ entry channel required the expression of Orai3 as well as Orai1. Moreover, we showed that AA treatment enhanced the migration of BON cells and that this migration could be abrogated by selective inhibition of the AA-induced Ca2+ entry. CONCLUSIONS: Taken together, these data revealed that an alternative Orai3-dependent Ca2+ entry pathway is an important signal for GEPNET cell migration.

Synaptotagmin isoforms confer distinct activation kinetics and dynamics to chromaffin cell granules.

Adrenomedullary chromaffin cells respond to sympathetic nervous system activation by secreting a cocktail of potent neuropeptides and hormones into the circulation. The distinct phases of the chromaffin cell secretory response have been attributed to the progressive fusion of distinct populations of dense core granules with different activation kinetics. However, it has been difficult to define what distinguishes these populations at the molecular level. Functional segregation of granule pools may depend on selective sorting of synaptotagmin-1 (Syt-1) and synaptotagmin-7 (Syt-7), which our previous work showed are rarely cosorted to the same granule. Here we assess the consequences of selective sorting of Syt isoforms in chromaffin cells, particularly with respect to granule dynamics and activation kinetics. Upon depolarization of cells expressing fluorescent Syt isoforms using elevated K+, we find that Syt-7 granules fuse with faster kinetics than Syt-1 granules, irrespective of stimulation strength. Pharmacological blockade of Ca2+ channels reveals differential dependence of Syt-1 versus Syt-7 granule exocytosis on Ca2+ channel subtypes. Syt-7 granules also show a greater tendency to fuse in clusters than Syt-1 granules, and granules harboring Syt-1 travel a greater distance before fusion than those with Syt-7, suggesting that there is spatial and fusion-site heterogeneity among the two granule populations. However, the greatest functional difference between granule populations is their responsiveness to Ca2+ Upon introduction of Ca2+ into permeabilized cells, Syt-7 granules fuse with fast kinetics and high efficacy, even at low Ca2+ levels (e.g., when cells are weakly stimulated). Conversely, Syt-1 granules require a comparatively larger increase in intracellular Ca2+ for activation. At Ca2+ concentrations above 30 µM, activation kinetics are faster for Syt-1 granules than for Syt-7 granules. Our study provides evidence for functional specialization of chromaffin cell granules via selective expression of Syt isoforms with different Ca2+ sensitivities.

Salivary protein changes in response to acute stress in medical residents performing advanced clinical simulations: a pilot proteomics study.

CONTEXT: Quantitative changes of salivary proteins due to acute stress were detected. OBJECTIVE: To explore protein markers of stress in saliva of eight medical residents who performed emergency medicine simulations. MATERIALS AND METHODS: Saliva was collected before the simulations, after the simulations, and following morning upon waking. Proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), identified by mass spectrometry (MS), and relatively quantified by densitometry. RESULTS: Salivary alpha-amylase and S-type cystatins significantly increased, while the ∼26 kDa and low-molecular weight (MW) (<10 kDa) SDS-PAGE bands exhibited changes after stress. DISCUSSION AND CONCLUSION: Alpha-amylase and cystatins are potential salivary markers of acute stress, but further validation should be performed using larger sample populations.

cAMP-dependent recruitment of acidic organelles for Ca2+ signaling in the salivary gland.

Autonomic neural activation of intracellular Ca2+ release in parotid acinar cells induces the secretion of the fluid and protein components of primary saliva critical for maintaining overall oral homeostasis. In the current study, we profiled the role of acidic organelles in shaping the Ca2+ signals of parotid acini using a variety of imaging and pharmacological approaches. Results demonstrate that zymogen granules predominate as an apically polarized population of acidic organelles that contributes to the initial Ca2+ release. Moreover, we provide evidence that indicates a role for the intracellular messenger NAADP in the release of Ca2+ from acidic organelles following elevation of cAMP. Our data are consistent with the "trigger" hypothesis where localized release of Ca2+ sensitizes canonical intracellular Ca2+ channels to enhance signals from the endoplasmic reticulum. Release from acidic stores may be important for initiating saliva secretion at low levels of stimulation and a potential therapeutic target to augment secretory activity in hypofunctioning salivary glands.

Nicotinic Acid Adenine Dinucleotide Phosphate Plays a Critical Role in Naive and Effector Murine T Cells but Not Natural Regulatory T Cells.

Nicotinic acid adenine dinucleotide phosphate (NAADP), the most potent Ca(2+) mobilizing second messenger discovered to date, has been implicated in Ca(2+) signaling in some lymphomas and T cell clones. In contrast, the role of NAADP in Ca(2+) signaling or the identity of the Ca(2+) stores targeted by NAADP in conventional naive T cells is less clear. In the current study, we demonstrate the importance of NAADP in the generation of Ca(2+) signals in murine naive T cells. Combining live-cell imaging methods and a pharmacological approach using the NAADP antagonist Ned-19, we addressed the involvement of NAADP in the generation of Ca(2+) signals evoked by TCR stimulation and the role of this signal in downstream physiological end points such as proliferation, cytokine production, and other responses to stimulation. We demonstrated that acidic compartments in addition to the endoplasmic reticulum were the Ca(2+) stores that were sensitive to NAADP in naive T cells. NAADP was shown to evoke functionally relevant Ca(2+) signals in both naive CD4 and naive CD8 T cells. Furthermore, we examined the role of this signal in the activation, proliferation, and secretion of effector cytokines by Th1, Th2, Th17, and CD8 effector T cells. Overall, NAADP exhibited a similar profile in mediating Ca(2+) release in effector T cells as in their counterpart naive T cells and seemed to be equally important for the function of these different subsets of effector T cells. This profile was not observed for natural T regulatory cells.

Increased Glucose-induced Secretion of Glucagon-like Peptide-1 in Mice Lacking the Carcinoembryonic Antigen-related Cell Adhesion Molecule 2 (CEACAM2).

Carcinoembryonic antigen-related cell adhesion molecule 2 (CEACAM2) regulates food intake as demonstrated by hyperphagia in mice with the Ceacam2 null mutation (Cc2(-/-)). This study investigated whether CEACAM2 also regulates insulin secretion. Ceacam2 deletion caused an increase in ß-cell secretory function, as assessed by hyperglycemic clamp analysis, without affecting insulin response. Although CEACAM2 is expressed in pancreatic islets predominantly in non-ß-cells, basal plasma levels of insulin, glucagon and somatostatin, islet areas, and glucose-induced insulin secretion in pooled Cc2(-/-) islets were all normal. Consistent with immunofluorescence analysis showing CEACAM2 expression in distal intestinal villi, Cc2(-/-) mice exhibited a higher release of oral glucose-mediated GLP-1, an incretin that potentiates insulin secretion in response to glucose. Compared with wild type, Cc2(-/-) mice also showed a higher insulin excursion during the oral glucose tolerance test. Pretreating with exendin(9-39), a GLP-1 receptor antagonist, suppressed the effect of Ceacam2 deletion on glucose-induced insulin secretion. Moreover, GLP-1 release into the medium of GLUTag enteroendocrine cells was increased with siRNA-mediated Ceacam2 down-regulation in parallel to an increase in Ca(2+) entry through L-type voltage-dependent Ca(2+) channels. Thus, CEACAM2 regulates insulin secretion, at least in part, by a GLP-1-mediated mechanism, independent of confounding metabolic factors.

Crosstalk between purinergic receptors and canonical signaling pathways in the mouse salivary gland.

Isolated clusters of mouse parotid acinar cells in combination with live cell imaging were used to explore the crosstalk in molecular signaling between purinergic, cholinergic and adrenergic pathways that integrate to control fluid and protein secretion. This crosstalk was manifested by (1) ß-adrenergic receptor activation and amplification of P2X4R evoked Ca(2+) signals, (2) ß-adrenergic-induced amplification of P2X7R-evoked Ca(2+) signals and (3) muscarinic receptor induced activation of P2X7Rs via exocytotic activity. The findings from our study reveal that purinoceptor-mediated Ca(2+) signaling is modulated by crosstalk with canonical signaling pathways in parotid acinar cells. Integration of these signals are likely important for dynamic control of saliva secretion to match physiological demand in the parotid gland.

Gastro-Enteropancreatic Neuroendocrine Tumor Cell Dynamics in Liver Microvasculature.

For many cancers, liver metastasis is common and usually indicates poor prognosis. Gastro-enteropancreatic neuroendocrine tumors (GEPNETs) of the midgut are a heterogeneous group of cancers that typically remain asymptomatic until they metastasize to the liver. However, the mechanisms by which these usually indolent cancers establish distal metastasis remain unclear. To begin to elucidate this process, we performed standard in vitro assays to assess cell motility, transendothelial migration, and invasion using BON cells, a widely used model GEPNET cell line. In addition, transmission electron microscopy was used in combination with a novel ex vivo organ slice xenograft model to reveal ultrastructural details of the initial events of BON cell extravasation and re-distribution within the liver. The ultrastructural resolution of the extravasation process revealed the route, sequence, and time course by which tumor cells migrated from the sinusoidal lumen into the hepatic parenchyma in this organ slice model. Both standard in vitro assays and our organ slice model indicated that tumor cells migrated through the discontinuous sinusoidal endothelium to invade the liver parenchyma.

Activity of nicotinic acid substituted nicotinic acid adenine dinucleotide phosphate (NAADP) analogs in a human cell line: difference in specificity between human and sea urchin NAADP receptors.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca2+ mobilizing second messenger that has been identified. We have previously shown that NAADP analogs substituted at the 5-position of nicotinic acid were recognized by the sea urchin receptor at low concentration, whereas the 4- substituted analogs were not as potent. However, to date the structure-activity relationship (SAR) of these analogs has not been addressed in mammalian systems. Thus, we asked whether these structurally modified analogs behave similarly in an NAADP-responsive mammalian cell line (SKBR3) using microinjection and single cell fluorescent imaging methods. Novel "caged" 4- and 5-substituted NAADP analogs that were activated inside the cell by flash photolysis resulted in Ca2+ mobilizing activity in SKBR3 cells in a concentration dependent manner, but with reduced effectiveness compared to unmodified NAADP. The SAR in mammalian SKBR3 cells was quite different from that of sea urchin and may suggest that there are differences between NAADP receptors in different species or tissues. Importantly, these data indicate that modifications at the 4- and 5-position of the nicotinic acid ring may lead to the development of functional photoaffinity labels that could be used for receptor localization and isolation in mammalian systems.

Different roles of the cardiac Na+/Ca2+-exchanger in ouabain-induced inotropy, cell signaling, and hypertrophy.

Previous studies have shown that digitalis drugs, acting as specific inhibitors of cardiac Na(+)/K(+)-ATPase, not only cause positive inotropic effects, but also activate cell signaling pathways that lead to cardiac myocyte hypertrophy. A major aim of this work was to assess the role of Na(+)/Ca(2+)-exchanger, NCX1, in the above two seemingly related drug effects. Using a mouse with ventricular-specific knockout (KO) of NCX1, ouabain-induced positive inotropy that was evident in isolated wild-type (Wt) hearts was clearly reduced in KO hearts. Ouabain also increased Ca(2+) transient amplitudes in Wt myocytes, but not in KO myocytes. Ouabain-induced activations of ERK 1/2 were noted in Wt myocytes, but not in KO myocytes; however, ouabain activated PI3K1A and Akt in both Wt and KO myocytes. Protein synthesis rate, as a measure of hypertrophy, was increased by ouabain in Wt and KO myocytes; these drug effects were prevented by a PI3K inhibitor but not by a MEK/ERK inhibitor. Hypertrophy caused by ET-1, but not that induced by ouabain, was accompanied by upregulation of BNP gene in Wt and KO myocytes. The findings indicate 1) the necessity of NCX1 for positive inotropic action of ouabain; 2) the irrelevance of NCX1 and ERK 1/2 activation to ouabain-induced hypertrophy; and 3) that hypertrophy caused by ouabain through the activation of PI3K1A/Akt pathway is likely to be beneficial to the heart.

Human heat shock protein 105/110 kDa (Hsp105/110) regulates biogenesis and quality control of misfolded cystic fibrosis transmembrane conductance regulator at multiple levels.

Heat shock protein 105/110-kDa (Hsp105/110), a member of the Hsp70 super family of molecular chaperones, serves as a nucleotide exchange factor for Hsc70, independently prevents the aggregation of misfolded proteins, and functionally relates to Hsp90. We investigated the roles of human Hsp105α, the constitutively expressed isoform, in the biogenesis and quality control of the cystic fibrosis transmembrane conductance regulator (CFTR). In the endoplasmic reticulum (ER), Hsp105 facilitates CFTR quality control at an early stage in its biosynthesis but promotes CFTR post-translational folding. Deletion of Phe-508 (ΔF508), the most prevalent mutation causing cystic fibrosis, interferes with de novo folding of CFTR, impairing its export from the ER and accelerating its clearance in the ER and post-Golgi compartments. We show that Hsp105 preferentially associates with and stabilizes ΔF508 CFTR at both levels. Introduction of the Hsp105 substrate binding domain potently increases the steady state level of ΔF508 CFTR by reducing its early-stage degradation. This in turn dramatically enhances ΔF508 CFTR cell surface functional expression in cystic fibrosis airway epithelial cells. Although other Hsc70 nucleotide exchange factors such as HspBP1 and BAG-2 inhibit CFTR post-translational degradation in the ER through cochaperone CHIP, Hsp105 has a primary role promoting CFTR quality control at an earlier stage. The Hsp105-mediated multilevel regulation of ΔF508 CFTR folding and quality control provides new opportunities to understand how chaperone machinery regulates the homeostasis and functional expression of misfolded proteins in the cell. Future studies in this direction will inform therapeutics development for cystic fibrosis and other protein misfolding diseases.

Distinct contributions by ionotropic purinoceptor subtypes to ATP-evoked calcium signals in mouse parotid acinar cells.

There is emerging consensus that P2X4 and P2X7 ionotropic purinoceptors (P2X4R and P2X7R) are critical players in regulating [Ca²âº]i dynamics and fluid secretion in the salivary gland. In contrast, details regarding their compartmentalization and selective activation, contributions to the spatiotemporal properties of intracellular signals and roles in regulating protein exocytosis and ion channel activity have remained largely undefined. To address these concerns, we profiled mouse parotid acinar cells using live-cell imaging to follow the spatial and temporal features of ATP-evoked Ca²âº dynamics and exocytotic activity. Selective activation of P2X7Rs revealed an apical-to-basal [Ca²âº]i signal that initiated at the sub-luminal border and propagated with a wave speed estimated at 17.3 ± 4.3 µm s⁻¹ (n =6). The evoked Ca²âº spike consisted of Ca²âº influx and Ca²âº-induced Ca²âº release from intracellular Ca²âº channels. In contrast, selective activation of P2X4Rs induced a Ca²âº signal that initiated basally and propagated toward the lumen with a wave speed of 4.3 ± 0.2 µm s⁻¹ (n =8) that was largely independent of intracellular Ca²âº channel blockade. Consistent with these observations, P2X7R expression was enriched in the sub-luminal regions of acinar cells while P2X4R appeared localized to basal areas. In addition, we showed that P2X4R and P2X7R activation evokes exocytosis in parotid acinar cells. Our studies also demonstrate that the P2X4R-mediated [Ca²âº]i rise and subsequent protein exocytosis was enhanced by ivermectin (IVR). Thus, in addition to furthering our understanding of salivary gland physiology, this study identifies P2X4R as a potential target for treatment of salivary hypofunction diseases.