VIP and muscarinic synergistic mucin secretion by salivary mucous cells is mediated by enhanced PKC activity via VIP-induced release of an intracellular Ca²⁺ pool

Abstract

Mucin secretion by salivary mucous glands is mediated predominantly by parasympathetic acetylcholine activation of cholinergic muscarinic receptors via increased intracellular free calcium ([Ca²⁺]_i) and activation of conventional protein kinase C isozymes (cPKC). However, the parasympathetic co-neurotransmitter, vasoactive intestinal peptide (VIP), also initiates secretion, but to a lesser extent. In the present study, cross-talk between VIP- and muscarinic-induced mucin secretion was investigated using isolated rat sublingual tubuloacini. VIP-induced secretion is mediated by cAMP-activated protein kinase A (PKA), independently of increased [Ca²⁺]_i. Synergistic secretion between VIP and the muscarinic agonist, carbachol, was demonstrated but only with submaximal carbachol. Carbachol has no effect on cAMP ± VIP. Instead, PKA activated by VIP releases Ca²⁺ from an intracellular pool maintained by the sarco/endoplasmic reticulum Ca²⁺-ATPase pump. Calcium release was independent of phospholipase C activity. The resultant sustained [Ca²⁺]_i increase is additive to submaximal, but not maximal carbachol-induced [Ca²⁺]_i. Synergistic mucin secretion was mimicked by VIP plus either phorbol 12-myristate 13-acetate or 0.01 μM thapsigargin, and blocked by the PKC inhibitor, Gö6976. VIP-induced Ca²⁺ release also promoted store-operated Ca²⁺ entry. Synergism is therefore driven by VIP-mediated [Ca²⁺]_i augmenting cPKC activity to enhance muscarinic mucin secretion. Additional data suggest ryanodine receptors control VIP/PKA-mediated Ca²⁺ release from a Ca²⁺ pool also responsive to maximal carbachol. A working model of muscarinic and VIP control of mucous cell exocrine secretion is presented. Results are discussed in relation to synergistic mechanisms in other secretory cells, and the physiological and therapeutic significance of VIP/muscarinic synergism controlling salivary mucous cell exocrine secretion.

INTRODUCTION

Human salivary glands are differentiated by their composition of exocrine cell types, pattern of innervation and contributions of salivary fluid and organic components [65]. Mucous glands comprised of major sublingual and minor mucous glands contribute about 70% of salivary gel-forming mucins, with the additional 30% from submandibular mucous cells [68]. Gel-forming mucins function in lubrication, hydration, bacterial clearance and selective adherence of bacteria to tooth surfaces [16]. To study the control of salivary mucous cell secretion rat sublingual glands may be used since they are strikingly similar histologically to human sublingual and minor mucous glands [13]. These glands are composed primarily of mucous cells arranged into tubuloacinar structures with a simple duct system and relatively few serous demilune cells, located at the distal end of mucous tubuloacinar structures [13]. Tubuloacinar mucous cells of major sublingual and minor mucous glands receive robust parasympathetic innervation, but a paucity of sympathetic innervation [15]. In parotid and submandibular glands sympathetic release of noradrenaline predominantly mediates exocrine protein secretion via β₁-adrenergic receptor activation of adenylate cyclase and protein kinase A (PKA) [75]. In contrast, sublingual mucin secretion is primarily under muscarinic cholinergic control, whereas the β-adrenergic agonist, isoproterenol, is without effect [14] but may instead be responsible for protein secretion from the minor population of serous demilune cells that cap tubuloacini and from striated ducts [49,79].

It was demonstrated previously that muscarinic-induced mucous cell exocrine secretion in rat sublingual glands is mediated by M1 and M3 muscarinic cholinergic receptor subtypes coupled to G proteins, G_q and G₁₁, to activate phosphatidylinositol 1,4,bisphosphate (PIP₂)-specific phospholipase C (PLC) [62]. PLC generates diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP₃), the latter interacting with IP₃ receptors (IP₃Rs) to release Ca²⁺ from intracellular stores followed by influx of extracellular Ca²⁺ via store-operated Ca²⁺ entry (SOCE), resulting in a sustained increase in intracellular free calcium concentration ([Ca²⁺]_i) [112]. DAG and increased [Ca²⁺]_i together mediate exocrine secretion via activation of protein kinase C (PKC-α) [17].

Parasympathetic nerves supplying salivary secretory elements release the neuropeptide, vasoactive intestinal peptide (VIP), in addition to acetylcholine [75]. In parotid and submandibular glands, VIP can induce protein secretion via cAMP activation of PKA [75]. VIP also elicits mucin secretion from isolated sublingual tubuloacini [15]. Because cross-talk between cAMP and Ca²⁺ pathways leads to synergistic protein secretion by parotid and submandibular acinar cells [75], and given the predominant parasympathetic control of mucous cells, the current study explores synergism between VIP- and muscarinic-induced mucin secretion from sublingual tubuloacini and further interrogates signaling mechanisms responsible for synergism. Results are discussed in relation to known mechanisms of synergism in other secretory cells and the physiological significance of VIP/muscarinic synergism controlling salivary mucous cell exocrine secretion.

MATERIALS AND METHODS

Materials:

All materials were purchased from Millipore Sigma (Burlington, Massachusetts) unless otherwise noted.

Animals, isolation of sublingual tubuloacini and measurement of mucin secretion:

Specific pathogen and sialodacryoadenitis virus free male Wistar rats (2 months old, 175–200 g) were obtained from Charles River Laboratories (Kingston facility, Stone Ridge, NY). For each preparation of isolated tubuloacini, 4–6 rats were killed by exsanguination after CO₂ anesthesia. The University of Rochester IACUC committee approved all animal procedures. Tubuloacini were isolated as described previously [14]. Briefly, tubuloacini were dispersed enzymatically (collagenase and hyaluronidase) from freshly extracted sublingual glands after filtration through nylon mesh and centrifugation (200g × 1 min). To pellet clusters of 2–4 mucous cells for assessing intracellular Ca²⁺, the resultant supernatant was centrifuged 1,000g × 3 min. Mucous cell mucin glycoproteins (Muc19) of tubuloacini were radiolabeled endogenously with [³H]glucosamine for 2 h, followed by the distribution culture wells and challenged with drugs or control solvents. Tubuloacini were pelleted and homogenized. Mucin glycoproteins in the supernatants and homogenates were acid-precipitated and counted. Mucin secretion was normalized as the percentage of the total (supernatant plus pellet) precipitable disintegrations per min (dpm) in the supernatant, and expressed as % total precipitable dpm released. Each condition was performed in triplicate. Mucin specificity of this assay was reported previously [105]. See Appendix, page 1, for details.

Development of Concentration-Response Curves and Calculation of EC₅₀ Values

Mucin secretion concentration-response curves and calculation of the EC₅₀ (concentration of agonist at half the maximal secretory response) was performed by fitting mean values to the logistic function: Y = [(N − M)] / [(1 + (X / EC₅₀)^B] + M; where Y is the secretory response; X, the concentration of agonist (nmol/L); N, the secretory response when X = 0; M, the secretory response when X = infinity (e.g., maximal response); B, slope factor or Hill slope.

Intracellular cAMP measurements:

Isolated tubuloacini were evenly distributed to 60 mm petri dishes and incubated 15 min at 37°C. Drugs or control solvents were added and tubuloacini incubated for an additional 15 min. Soluble ice-cold extracts (67% ethanol in 6.7 mM HCl) from cell homogenates were then prepared, dried and the residue assayed for cAMP by RIA (Biomedical Technologies, Inc., Stoughton, MA). To normalize results among samples, the remaining pellets were assayed for DNA and results expressed as pmol cAMP/μg DNA. See Appendix, page 2, for details.

Intracellular calcium:

Intracellular free Ca²⁺ concentrations were estimated using the calcium-sensitive dye, fura-2 (Molecular Probes). Isolated tubuloacini were incubated in LRL medium containing 2 μM fura-2/AM for 30 min at 37°C, then rinsed twice with PBS solution containing 1 mg/ml BSA and 1 mg/ml glucose, resuspended in LRL medium and maintained for no more than 4 h in an incubator gassed with 40% O₂-5% CO₂-55% air at 37°C. The medium (LRL) consisted of Dulbecco’s modified Eagle’s medium-Ham’s nutrient mixture F-12 (1:1) plus β-retinyl acetate (0.1 μmol/L), transferrin (5 μg/mL), selenious acid (2 ng/mL), insulin (1 μg/mL), hydrocortisone (0.1 μg/mL), soybean trypsin inhibitor (0.1 mg/mL), bovine serum albumin (BSA, 0.5 mg/mL), and gentamicin (50 μg/mL). Aliquots (1 ml) of fura-2 loaded tubuloacini were centrifuged (400g for 15 sec) as needed and each pellet gently resuspended in Modified Earle’s-Hanks’ solution (MEH) consisting of 110 mM NaCl, 24 mM NaHCO₃, 20 mM HEPES, 5.4 mM KCl, 1.2 mM CaCl₂, 0.8 mM MgSO₄, 0.4 mM KH₂PO₄, 0.33 mM NaH₂PO₄, 10 mM glucose, pH 7.4. Tubuloacini were then allowed to attach to a coverslip within a perfusion chamber mounted on the stage of a Nikon inverted microscope interfaced to a fluorometer (AR-CM, SPEX Industries, Edison, NJ, USA). A Nikon CF Fluor X40 1.3-NA oil immersion objective incorporating a pinhole turret was used to isolate 5–8 mucous cells. Perfusion solutions were gassed continuously with 95% O₂-5% CO₂ at approximately 23°C and superfused at a rate of 4 ml/min. Fluorescence readings were obtained using 340 nm and 380 nm excitation filters and a 505 nm emission filter. Fluorescence ratios were converted to estimates of [Ca²⁺]_i using the equation [Ca²⁺]_i = K_d [(F − F_min)/(F_max − F)] [37]. F_min and F_max were determined by in situ calibration. To obtain minimum fluorescence, F_min, tubuloacini were superfused with Ca²⁺-free MEH solution containing 3 mM EGTA and 2 μM ionomycin and for F_max tubuloacini were superfused with normal MEH solution containing 1.2 mM Ca²⁺ and 2 μM ionomycin. A dissociation constant, K_d, for fura-2 of 224 nM was used as determined in 115 mM KCl, 20 mM NaCl, 10 mM K-MOPS, pH 7.05, l mM free Mg²⁺ with increasing Ca²⁺ concentrations [37] and used previously by us [17] and others in sublingual cells [66,112,113] as well as in parotid cells [24,32], and in pancreatic ducts [3]. Reported sustained increases in intracellular calcium were measured at 2.5 min after stimulation, unless indicated. For Mn²⁺ quench studies, a 360-nm excitation filter was used. At this wavelength (isosbestic point), fura-2 is unsensitive to changes in [Ca²⁺]_i; therefore, a decrease in fura-2 fluorescence represents Mn²⁺ binding (entry). Quenching of the fura-2 signal by Mn²⁺ was expressed as the fluorescence divided by the original starting fluorescence (F/F₀).

Confocal Fluorescence Imaging of Mucous Cell Calcium:

Changes in intracellular Ca²⁺ of individual mucous cells were assessed using an ACAS570 (Meridian Instruments, Inc) confocal interactive laser cytometer. Tubuloacini were loaded with 10 μM fluo-3/AM (Molecular Probes, Eugene, OR, USA) for 30 min at 37°C, washed with PBS containing 1 mg/ml glucose and 1 mg/ml BSA, then resuspended in MEH solution. Tubuloacini were then allowed to attach to a coverslip within a perfusion chamber mounted on the scanning stage of an Olympus IMT-2 inverted microscope. Fluorescence pseudo-color optical sections (approximately 16 μM z-axis) were recorded from mucous cells. Digital images were uploaded into the “kinetics” software package supplied by the manufacturer. Fluo-3 was excited with the argon-ion laser tuned to 488 nm, and fluorescence emission detected using a 510 nm long pass dichroic filter. Data was normalized to correct for background.

Localization of Ryanodine Receptors:

Localization of ryanodine receptors in sublingual tubuloacinar cells. Freshly isolated tubuloacini were fixed 1 h in 4% paraformaldehyde-PBS, washed 10 min in Tris-buffered saline, pH 7.2 (TBS) followed by three 15 min washes in TBS containing 0.3% Triton X-100 and 2% goat serum, then incubated overnight in Basal Medium Eagle medium containing 1.67% bovine serum albumin and 1% soybean trypsin inhibitor. BODIPY FL-X ryanodine (Molecular Probes, Eugene, OR) was then added to 5 nM final concentration (with and without addition of 1 μM ryanodine) and tubuloacini incubated 3 h. Fixation, washes and labeling were at 4°C with gentle rocking. Tubuloacini were washed in TBS and mounted with GVA Mount (Thermo Fisher Scientific, Waltham, MA), coverslipped and examined with a Nikon Eclipse E800 microscope (Nikon Inc., Melville, NY) with a 63x objective under DIC (differential interference contrast) or fluorescence (488-nm excitation and 515-nm emission) optics. Digital images were captured with a Spot 2 digital camera and software (Diagnostic Instruments, Sterling Heights, MI).

Addition of Drugs

Drugs were applied as either 100-fold or 1,000-fold stock solution. DMSO and ethanol were used as solutions only when necessary and added to give final concentrations of 0.1% and 1%, respectively. In control experiments, these concentrations exhibited no effects on either mucin secretion or intracellular calcium measurements. Drugs dissolved in water (100-fold stock): forskolin (DMB-forskolin, 7β-Deacetyl-7β-[γ-(morpholino) butyryl]-HCl), atropine, 8-bromo-cAMP, ryanodine, BODIPY FL-X ryanodine (6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl) amino)hexanoic acid ryanodine), H89, caffeine. Drugs dissolved in DMSO (1000-fold stock): BAPTA/AM, PMA (phorbol-12-myristate-13-acetate), Gö6976, KT5720, fura-2/AM, fluo-3/AM. Drugs dissolved in 100% ethanol (100-fold stock): thapsigargin, U73122, U73343. Drugs dissolved in PBS (100-fold stock): carbachol, VIP, isobutylmethylxanthine (IBMX), VIP antagonist (VIPhyb; Bachem, Torrance, CA).

Statistics:

Statistical comparisons, unless indicated, were by one-way ANOVA with Bonferroni post-hoc test. All calculations were performed using Prism 8 for OS X (v. 8.1.2), GraphPad Software, Inc., San Diego, CA. Unless indicated, each experiment was from a separate preparation of tubuloacini.

RESULTS

Synergistic mucin secretion between VIP and carbachol

VIP stimulates exocrine mucin glycoprotein secretion from rat sublingual tubuloacini in a concentration dependent manner with an EC₅₀ of 0.24 nM (Fig. 1a). In a recent study, we reported that the a half-maximal concentration of carbachol inducing mucin secretion was 0.3 μM [17]. We currently find that challenging tubuloacini with increasing VIP in the presence of 0.3 μM carbachol produces synergism at higher VIP concentrations. As a result, maximal VIP-induced secretion is more than doubled, but the VIP EC₅₀ (0.34 nM) little changed. Tubuloacini were also challenged with increasing carbachol alone and in the presence of 5 nM VIP (Fig. 1b). Carbachol, alone induces a maximal secretion nearly 3-fold greater than VIP, and with an EC₅₀ of 0.39 μM, in agreement with previous results [17]. However, synergism is observed with 5 nM VIP combined with 0.3 and 1 μM carbachol. Collectively, there is a decrease the EC₅₀ for carbachol to 0.13 μM (Fig. 1b). VIP therefore increases the sensitivity of muscarinic-induced mucin secretion, but does not alter the maximal response to carbachol. From Fig. 1b, peak synergism is observed with 5 nM VIP + 0.3 μM carbachol. These two agonist concentrations were therefore used in further studies of synergism, and as shown in Table 1, results in mucin secretion more than 50% greater than the additive sums of each agonist alone. In Fig. 1c and Fig. 1d are controls verifying each agonist alone or together stimulate mucin secretion at a linear rate for at least 45 min.

Figure 1.

Synergistic mucin secretion between VIP and carbachol. a. Mucin secretion in response to increasing VIP (open circles) or by increasing VIP in the presence of 0.3 μM carbachol (closed circles). Values are mean ± SE of 6 experiments for each curve. Asterisks indicate mucin secretion is greater than the calculated additive sum for secretion induced by VIP and by 0.3 μM carbachol separately. b. Mucin secretion induced by increasing carbachol, either without (open circles) or with 5 nM VIP (closed circles). Values are mean ± SE from 3 experiments. Asterisks indicate mucin secretion is greater than the calculated additive sum for secretion induced by carbachol and by 5 nM VIP separately. c. Time course of basal and VIP-induced mucin secretion. Results are means ± SE of 6 experiments. d. Time course of mucin secretion in response to 0.3 μM carbachol (Carb) and 0.3 μM carbachol + 5 nM VIP. Results are means ± SE of 6 experiments.

Table 1.

Synergistic exocrine secretion in response to VIP and carbachol

Secretion	Basal	5 nM VIP	0.3 μM Carbachol	5 nM VIP + 0.3 μM Carbachol	Agonist-Induced Additive Sum
Total Secretion	7.0 ± 0.3	*11.9 ± 0.7	*14.2 ± 0.6	*†25.8 ± 1.3	–
Agonist-Induced	–	4.9 ± 0.5	7.2 ± 0.5	‡18.8 ± 1.1	12.1 ± 3.8

Values are means ± SD from 30 experiments performed in triplicate and expressed as % total precipitable dpm released.

^*p < 0.001 versus basal;

^†p < 0.0001 versus VIP or carbachol;

^‡p < 0.0001 versus the additive sum for agonist-induced secretion by VIP and carbachol alone.

Cyclic AMP mediates VIP-induced mucin secretion via protein kinase A, but is not modulated by carbachol

As shown in Fig. 2a, 0.5 mM 8-bromo-cAMP (8-Br-cAMP) induces mucin secretion equivalent to 5 nM VIP, as did the adenylate cyclase activator, forskolin. Although VIP and 10 μM forskolin each increase intracellular cAMP, forskolin induces cAMP levels higher than VIP without inducing additional mucin secretion (Fig. 1a), suggesting VIP-activated cAMP levels are maximal for secretion. Moreover, secretion induced by VIP or forskolin, but not carbachol, is blocked by the protein kinase A (PKA) inhibitors, H89 and KT5720 (Fig. 2b) indicating cAMP activates PKA to mediate VIP-induced secretion. Both 10 μM forskolin as well as 0.5 mM 8-Br-cAMP mimic the synergistic mucin secretory effects of VIP (Fig. 2c). Furthermore, carbachol at 0.3 μM or at 10 μM has no effect on basal or VIP-induced intracellular cAMP, even when potentiated by the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), indicating synergism is not due to muscarinic enhancement of cAMP (Fig. 2d).

Figure 2.

Cyclic AMP via protein kinase A mediates mucin secretion induced by VIP. a. 8-bromo-cAMP (8-Br-cAMP) and DMB-forskolin (Forsk) mimic mucin secretion induced by VIP. Open bars are mean ± SE of 3 experiments. Grey bars are mean ± SE of 5 experiments. * p ≤ 0.05 versus basal secretion. Closed bars are verification of increased intracellular cAMP levels by VIP or DMB-forskolin. * p ≤ 0.01 versus basal. † p ≤ 0.05 versus basal and versus VIP. b. Mucin secretion induced by 5 nM VIP or 10 μM Forskolin (Forsk), but not by 10 μM carbachol (Carb), is inhibited by the protein kinase A inhibitors, H89 and KT5720. Values are mean ± SE from 3 experiments. H89 and KT5720 were added 20 min prior to secretagogue. * p ≤ 0.05 versus control basal secretion. c. Secretion induced by 5 nM VIP and synergism with 0.3 μM carbachol (Carb) is mimicked by 10 μM DMB-Forskolin (Forsk) or by 0.5 mM 8-Br-cAMP. Values are mean ± SE from 5 experiments. * p ≤ 0.05 for secretion above basal versus the calculated additive sum for secretion induced by Carb and by either 5 VIP, 8-Br-cAMP or Forsk separately. d. Intracellular cAMP levels in tubuloacini unchallenged (Basal) or challenged with 5 nM VIP ± either 0.3 μM carbachol (Carb, open bars) or 10 μM carbachol (gray bars). Closed bars, tubuloacini treated as in the open bar experiment except 1 μM IBMX added 15 min before agonist. Values are mean ± SE from 3 (open bars), 4 (gray bars) or 5 (closed bars) experiments. * p ≤ 0.01, and † p ≤ 0.001 versus basal.

VIP increases [Ca²⁺]_i via the cAMP-PKA pathway

VIP in many cell systems increases [Ca²⁺]_i [21], thus prompting examination of the effects of VIP on mucous cell [Ca²⁺]_i. As shown in Fig 3a, VIP induces a sustained and concentration-dependent increase in [Ca²⁺]_i with an EC₅₀ of approximately 1.0 nM, about 4-fold greater than that for VIP induction of mucin secretion. In Fig. 3b are representative traces of the effects of the VIP antagonist, VIPhyb [55], and the muscarinic antagonist, atropine, on VIP-induced [Ca²⁺]_i. Collective results (Fig. 3c) demonstrate VIPhyb reverses the effects of VIP on sustained [Ca²⁺]_i, suggesting VIP enhances [Ca²⁺]_i directly via VIP receptors. In contrast, 10 μM atropine, a concentration that blocks mucin secretion by 10 μM carbachol [13], does not affect VIP-induced [Ca²⁺]_i, indicating increased [Ca²⁺]_i by VIP is not via indirect activation of muscarinic receptors. Furthermore, both forskolin and 8-Br-cAMP each stimulate [Ca²⁺]_i (see representative traces in Fig. 3d) that mimics 5 nM VIP (Fig. 3e). H89 blocks VIP-induced [Ca²⁺]_i, but not that induced by 0.3 μM or 10 μM carbachol (Fig. 3f), suggesting PKA mediates VIP-induced [Ca²⁺]_i. In contrast, the PLC inhibitor, U73122, blocks [Ca²⁺]_i induced by carbachol, but not by VIP (Fig. 3g), indicating VIP-increased [Ca²⁺]_i is not through a PLC-mediated mechanism (e.g., IP₃Rs).

Figure 3.

VIP increases [Ca²⁺]_i via the cAMP-PKA pathway. Unless indicated, basal values of [Ca²⁺]_i were taken immediately prior to secretagogue addition and sustained values measured 2.5 min after challenge with secretagogue. a. Sustained [Ca²⁺]_i induced by increasing VIP concentrations. Values are mean ± SE from 3 experiments. Insert: Representative tracings of responses to VIP. b. Representative traces of [Ca²⁺]_i response to 5 nM VIP and subsequent addition of either 10 μM atropine (dashed line), or 2 μM VIP antagonist, VIPhyb (solid line). c. Collective results of experiments shown in Fig. 3b. Values are mean ± SE of 4 experiments for each experimental set. * p ≤ 0.01 versus basal. Values for conditions in the presence of antagonist were taken 2 min after initiation of perfusion with VIP + antagonist. d. Representative traces of [Ca²⁺]_i response to 10 μM DMB-forskolin (Forsk) or 0.5 mM 8-Bromo-cAMP (8-Br-cAMP). e. Collective results of three sets of experiments as presented in Fig. 3d, and also include results with 5 nM VIP. Values are mean ± SE of 6 experiments for VIP and 3 experiments each for Forsk and 8-Br-cAMP. * p ≤ 0.01 versus basal. f. PKA inhibitor, H89, inhibits VIP-induced [Ca²⁺]_i, but not in response to 0.3 μM or 10 μM carbachol (Carb). Values are mean ± SE from 4 experiments (control) and 5 experiments with H89. H89 was added 20 min prior to agonist. * p ≤ 0.05 versus control. g. The phospholipase C inhibitor, U73122 (10 μM), but not its inactive analog, U73343 (10 μM), blocks sustained [Ca²⁺]_i induced by carbachol (Carb) but not VIP. Values are means ± SE of 5 experiments for each experimental set. * p < 0.05 versus basal. U73122 and U73343 were added 15 min before agonist.

VIP-induced [Ca²⁺]_i does not function in VIP-stimulated secretion, but is required for synergism via PKC

In Fig. 4a is a representative trace showing subsequent addition of VIP further increases [Ca²⁺]_i in response to 0.3 μM carbachol. Because these measurements of [Ca²⁺]_i represent average values from approximately 5–8 mucous cells within the central region of a single tubuloacinus, [Ca²⁺]_i within individual cells were assessed by confocal microscopy to exclude the possibility of VIP and carbachol activating different cells. As shown in Fig. 4b, the focal plane encompasses the cytoplasm of at least three individual cells. These cells display VIP-induced [Ca²⁺]_i, with a further increase after challenge with carbachol, therefore indicating VIP and carbachol modulate [Ca²⁺]_i in the same cell. Although 0.3 μM carbachol is half-maximal for mucin secretion, we further showed previously [17] that in the context of [Ca²⁺]_I, 0.3 μM carbachol represents about 20% of maximal whereas 1 μM and 10 μM carbachol increase [Ca²⁺]_i 40% and 100% of maximal, respectively. Sustained [Ca²⁺]_i responses to increasing carbachol ± 5 nM VIP were therefore tested. As shown in Fig. 4c, in the presence of VIP there is an additional increase in sustained [Ca²⁺]_i with 0.3 μM and 1 μM carbachol, but not with maximal carbachol (10 μM), consistent with the absence of synergistic mucin secretion with 10 μM carbachol (Fig 1b). We also previously found that increased [Ca²⁺]_i and mucin secretion by 10 μM carbachol is blocked by perfusion of tubuloacini in Ca²⁺-free medium containing 0.1 mM EGTA and preloading cells with BAPTA/AM (1,2-bis (o-aminophenoxy) ethane-N,N,N’,N’-tetraacetic acid tetra (acetoxymethyl) ester) [17]. As shown Fig. 4d, these same conditions block increased [Ca²⁺]_i induced by 5 nM VIP + 0.3 μM carbachol. However, blocking increased [Ca²⁺]_i does not hinder VIP-induced mucin secretion (Fig. 4e and Fig. 4f), but abolishes synergistic secretion (Fig. 4f). VIP-induced [Ca²⁺]_i may therefore promote synergism by interacting with the muscarinic-mediated signaling pathway. Muscarinic-stimulated mucin secretion is mediated by activation of cPKCs, as evidenced earlier [17] in part by inhibition via the selective cPKC inhibitor, Gö6976 [63]. As shown in Fig. 4g, Gö6976 has no effect on VIP-induced secretion, but blocks synergistic mucin secretion as well as inhibiting carbachol-induced secretion, consistent with a role for cPKCs in synergism. Muscarinic-induced activation of cPKCs was also shown [17] to involve potentiating interactions between DAG and increasing [Ca²⁺]_i that can be mimicked by the DAG analogue, phorbol 12-myristate 13-acetate (PMA) and by increasing [Ca²⁺]_i with the SERCA pump inhibitor, thapsigargin. And we further demonstrated [17] that 0.01 μM thapsigargin increases [Ca²⁺]_i by approximately 50 nM (equivalent to 5 nM VIP in the current study) but to have little if any effect on mucin secretion. Moreover, thapsigargin at 1 μM was shown [17] to increase [Ca²⁺]_i to a level equivalent to that induced by a maximal carbachol, and alone stimulates mucin secretion to about 40–50% of the maximal carbachol-induced rate via partial activation of cPKCs. As shown in Fig. 4h, synergistic mucin secretion is induced by 0.01 μM thapsgargin + 0.3 μM carbachol, whereas only an additive effect is observed with 1 μM thapsgargin + 0.3 μM carbachol. The DAG analogue, phorbol 12-myristate 13-acetate (PMA) was also reported [17] to stimulate mucin secretion, with a maximal concentration (1 μM PMA) inducing about a third of maximal carbachol-induced secretion. As shown in Fig. 4i, both submaximal (0.1 μM) and maximal (1 μM) PMA interact with 5 nM VIP to promote synergistic mucin secretion.

Figure 4.

VIP-induced [Ca²⁺]_i does not function in VIP-stimulated secretion, but is required for synergism via PKC. a. Representative trace of the [Ca²⁺]_i response by mucous cells to 0.3 μM carbachol (Carb) and subsequent perfusion with 0.3 μM carbachol + 5 nM VIP. b. Relative agonist-induced changes in [Ca²⁺]_i of individual mucous acinar cells. Shown are Fluo-3 emission confocal images on the same focal plane (16 μm z-axis) of at least four branching, distally truncated tubuloacini radiating out from centrally localized interconnecting ducts, all within very loose and partially digested connective tissue. Mucous cells measure about 25 μm from their basal to luminal border and are about 20 μm wide. Thus, a 16 μm confocal z-axis captures completely the cytoplasm of only a select few mucous cells within each truncated tubuloacini. Images were taken at 3 time points: (i) just before 5 nM VIP addition (51 s), (ii) at 185 s when the average VIP-induced signal stabilizes, and (iii) at 296 s when the response stabilizes to perfusion with 5 nM VIP + 0.03 μM carbachol (Carb). Trace of “Average Units” versus “Time” is average fluorescence signal in the area demarcated by the white line in each image. Vertical lines indicate times when each image was taken. c. Results of three sets of experiments of sustained [Ca²⁺]_i responses to perfusion of mucous cells with 0.3 μM, 1 μM or 10 μM carbachol, followed by perfusion with carbachol + 5 nM VIP. Values are mean ± SE of 5 experiments for each experimental set. * p ≤ 0.01 versus basal and † p ≤ 0.01 versus carbachol alone. d. Representative traces from 3 separate experiments of tubuloacini challenged with the indicated agonist in either Ca²⁺-containing medium or tubuloacini first preloaded for 30 min with 2 μM BAPTA/AM and challenged with VIP + carbachol (Carb) in Ca²⁺-free medium containing 0.1 mM EGTA (Ca²⁺-free + BAPTA). e. Time courses of mucin secretion, either unstimulated (Basal) or in response to 5 nM VIP, in either Ca²⁺-containing medium or under Ca²⁺-free + BAPTA conditions. Results are mean ± SE of 4 experiments. f. Both secretion induced by carbachol alone and synergistic mucin secretion are inhibited by Ca²⁺-free + BAPTA conditions. Values are mean ± SE of 3 experiments. Asterisks indicate mucin secretion is greater than the calculated additive sum for secretion induced by VIP and by 0.3 μM carbachol separately (13.5 ± 1.2). g. Gö6976 (5 μM) inhibits carbachol-induced mucin secretion and synergism between carbachol and VIP. Values are mean ± SE of 6 experiments. † p ≤ 0.01 versus carbachol without Gö6976; ‡ p > 0.05 versus carbachol alone without Gö6976. Asterisk indicates mucin secretion above basal is greater (p ≤ 0.05) than the calculated additive sum for secretion induced by 0.3 μM carbachol and by 5 nM VIP separately (11.6 ± 0.8). h. Synergistic mucin secretion is observed with 0.3 μM carbachol (Carb) in combination with 0.01 μM thapsigargin (Thaps), but not with 1 μM Thaps. Values are mean ± SE of four experiments. Asterisk indicates mucin secretion above basal is greater (p ≤ 0.05) than the calculated additive sum for secretion induced by 0.3 μM carbachol and by 0.01 μM Thaps separately (7.8 ± 0.5). i. Synergistic mucin secretion is induced by 5 nM VIP + 0.3 μM carbachol (Carb) or by 5 nM VIP in combination with submaximal (0.1 μM) or maximal (1 μM) of phorbol 12-myristate 13-acetate (PMA). Values are mean ± SE of 4 experiments. † p ≤ 0.01 and * p ≤ 0.05 each indicate mucin secretion above basal is greater than the calculated additive sum for secretion induced by 5 nM VIP and by either 0.3 μM carbachol (Carb) or PMA separately. Additive sums: Carb & VIP, 12.5 ± 0.6; VIP & 0.1 μM PMA, 8.2 ± 0.4; VIP & 1 μM PMA, 11.1 ± 0.3.

The intracellular Ca²⁺ pool sensitive to VIP is both separate from, and shared with the carbachol-sensitive pool

In Ca²⁺-free medium, challenge of mucous cells with 5 nM VIP or with 10 μM forskolin results in a transient increase in [Ca²⁺]_i, consistent with VIP/cAMP-mediated Ca²⁺ release from intracellular stores followed by subsequent depletion via plasma membrane Ca²⁺ channels (Fig. 5a). Subsequent conversion to Ca²⁺-containing medium induces a rapid and sustained increase in [Ca²⁺]_i (Fig. 5a), further suggesting VIP, via cAMP, promotes opening of plasma membrane Ca²⁺ channels. To confirm VIP induces the influx of extracellular Ca²⁺, quenching of intracellular fura-2 fluorescence by Mn²⁺ was exploited as an indicator of Ca²⁺ entry. Cells in Ca²⁺-containing medium display an increase in the rate of fluorescence quenching by Mn²⁺ in the presence of 5 nM VIP (trace 2 versus trace 1), suggesting VIP increases Ca²⁺ entry (Fig. 5b). Moreover, fluorescence quenching is greatly accelerated when mucous cells are perfused continuously in Ca²⁺-free medium containing 0.1 mM EGTA (trace 4) versus Ca²⁺-containing medium (trace 2), indicating enhanced Ca²⁺ entry upon depletion of the VIP-sensitive intracellular Ca²⁺ pool. Removal of extracellular Ca²⁺ upon addition of Mn²⁺ (trace 3) did not alter the initial rate of quenching as observed in Ca²⁺-containing medium (trace 2), indicating rapid quenching in trace 4 is not due to highly selective Mn²⁺ entry compared to Ca²⁺. Further efforts were directed at interrogation of the relationship between intracellular Ca²⁺ pools sensitive to VIP and to carbachol by first perfusing tubuloacini with maximal carbachol (10 μM) in Ca²⁺-free medium containing 0.1 mM EGTA to deplete the carbachol-sensitive pool. Subsequent challenge with maximal 5 nM VIP did not increase [Ca²⁺]_i (black trace, Fig. 5c). Conversely, initial depletion of the VIP-sensitive Ca²⁺ pool with maximal VIP does not prevent subsequent Ca²⁺ release in response to 10 μM carbachol (grey trace, Fig. 5c), but does reduce the peak response to carbachol compared to when mucous cells are first challenged with carbachol (compare white and black bars in Fig. 5d). The VIP-sensitive Ca²⁺ pool is thus shared with the carbachol-sensitive Ca²⁺ pool. Furthermore, the VIP-sensitive pool is maintained by SERCA pumps, as slow depletion of intracellular Ca²⁺ by thapsigargin abolishes the Ca²⁺ response to VIP (Fig. 5e).

Figure 5.

The intracellular Ca²⁺ pool sensitive to VIP is shared with, but is also distinct from the carbachol-sensitive pool. a. Intracellular Ca²⁺ response to 5 nM VIP or 10 μM DMB-forskolin (Forsk) in Ca²⁺-free medium containing 0.1 mM EGTA is transient, but recovers and is sustained upon addition of 1.2 mM CaCl₂ to the perfusion medium. Shown are traces from a single experiment with DMB-forskolin and a representative trace from 3 experiments with VIP. b. Mn²⁺ quenching of intracellular fura-2 fluorescence to assess Ca²⁺ entry in VIP-challenged cells. Fura-2 emission was monitored at 505 nm and excitation at 360 nm, its Ca²⁺-insensitive isosbestic point. Fluorescence quenching was initiated by addition of 50 μM MnCl₂ to the perfusion medium and is expressed as fluorescence divided by starting fluorescence (F/F₀) versus time. A; indicates time when 5 mM VIP was added to the perfusion medium. B; indicates time when perfusion with MnCl₂ was initiated, under 1 of 4 conditions: 1, Ca²⁺-containing medium; 2, 5 nM VIP in Ca²⁺-containing medium; 3, Ca²⁺-free medium containing 0.1 mM EGTA; 4, 5 nM VIP in Ca²⁺-free medium containing 0.1 mM EGTA. Traces are representative of 4 separate experiments. c. Representative traces of [Ca²⁺]_i in mucous cells perfused with Ca²⁺-free medium containing 0.1 mM EGTA. Black trace: Cells first challenged with maximal 10 μM carbachol (C), followed later by prefusion with 5 mM VIP (V). Gray trace: Cells challenged with 5 nM VIP (V) followed by 10 μM carbachol (C). d. Combined results of basal and peak [Ca²⁺]_i responses from experiments shown in Fig. 5c. Basal [Ca²⁺]_i recorded just before addition of the first agonist. Note, because there consistently was no response to VIP in experimental set 1 (open bars), peak values were taken 30 sec after VIP addition. Values are mean ± SE of 4 (open bars), 3 (closed bars) experiments. * p ≤ 0.001 versus peak carbachol response in experimental set 1 (open bars). e. Depletion of intracellular thapsigargin-sensitive Ca²⁺ stores in Ca²⁺-free medium containing 0.1 mM EGTA abolishes Ca²⁺ release induced by subsequent VIP. Trace is representative of 3 experiments. f. Combined results of experiment shown in Fig. 5e (means ± SE of 3 separate experiments). Basal [Ca²⁺]_i recorded just before addition of 1 μM thapsigargin (Thaps). Note, because there consistently was no response to VIP, “After 5 nM VIP” values were taken 30 sec after VIP addition.

Evidence for regulation of the VIP-sensitive Ca²⁺ pool by ryanodine receptors

Evidence presented above suggests the VIP-sensitive Ca²⁺ pool is mediated via the cAMP-PKA pathway independently of PLC. Furthermore, in mouse parotid glands, Ca²⁺ release dependent on PKA is from ryanodine receptor (RyR)-sensitive Ca²⁺ stores [84]. Whether RyR channels contribute to VIP-mediated intracellular Ca²⁺ release in mucous cells was therefore explored. Ryanodine is known to open RyR channels but slows the kinetics of channel openings and closings with increasing concentrations, eventually blocking Ca²⁺ release at concentrations ≥10 μM [12]. In rat sublingual gland tubuloacini increasing concentrations of ryanodine progressively attenuated VIP-induced [Ca²⁺]_i with complete inhibition at 10 μM (see representative traces in Fig. 6a and collective results in Fig. 6b). In addition, ryanodine (10 μM) prevents increased [Ca²⁺]_i upon challenge with VIP, but does not affect carbachol-induced [Ca²⁺]_i (see representative traces in Fig. 6c and 6d, respectively, and collective results in Fig. 6e). Inhibition of VIP-induced [Ca²⁺]_i by 10 μM ryanodine is not due to inhibition of Ca²⁺ entry, as Mn²⁺ influx after VIP challenge is unimpeded by 10 μM ryanodine (Fig. 6f). In the context of mucin secretion, 10 μM ryanodine does not affect secretion induced by VIP or by carbachol alone but abolishes synergism (Fig. 6g). Caffeine is an activator of RyRs, lowering the threshold concentration of Ca²⁺ sequestered in the endoplasmic reticulum to induce quantal Ca²⁺ release [50]. As shown in Fig. 6h, caffeine (10 μM) induced a leftward shift of more than 0.5 log units in the [Ca²⁺]_i response to increasing VIP concentrations. Because caffeine is also a methylxanthine alkaloid with phosphodiesterase activity [33], the increased sensitivity of the [Ca²⁺]_i response to VIP by caffeine could instead be explained by increased intracellular cAMP. However, 1 μM of the phosphodiesterase inhibitor, IBMX, has no effect on the [Ca²⁺]_i response to increasing concentrations of VIP (Fig. 6h) whereas in Fig. 2D, comparison of basal cAMP levels in the two data sets without IBMX (0.29 ± 0.07 and 0.17 ± 0.05 pmol/μg DNA) versus the set with 1 μM IBMX (0.60 ± 0.15 pmol/μg DNA) suggests IBMX has a small effect on intracellular cAMP (p = 0.091 and p = 0.041, respectively, two-tailed unpaired t test). This effect on cAMP levels is supported by the modest but significant increase in mucin secretion induced by 1 μM IBMX (Fig. 6i). If the leftward shift in the [Ca²⁺]_i response to VIP by caffeine is indeed due to increased cAMP, then the expected increase in cAMP would be greater than that induced by 1 μM IBMX, and further expected to also induce a leftward shift in mucin secretory response to VIP. As shown in Fig. 6j, 10 μM caffeine has no effect on the EC₅₀ of VIP stimulation of mucin secretion. Also, mucin secretion induced by VIP (EC₅₀ = 0.23 nM, Fig. 6j) is at least as sensitive, if not more sensitive than the [Ca²⁺]_i response to VIP (EC₅₀ = 1.02 nM, Fig. 6h). Because both responses are mediated by cAMP, the leftward shift by caffeine on the [Ca²⁺]_i response, but not mucin secretion, further indicates caffeine’s effect is not due to the [Ca²⁺]_i response being more sensitive to increased intracellular cAMP. It is therefore unlikely that the marked leftward shift in the calcium response results from increased cAMP, but is instead due more likely to caffeine activation of ryanodine receptors. Interestingly, low concentrations of ryanodine (1 to 10 nM) were without detectable effects on [Ca²⁺]_i (not shown), whereas ryanodine binding sites were localized primarily in the posterior basolateral region of mucous cells after light fixation in paraformaldehyde (Fig. 7).

Figure 6.

Initial evidence supporting regulation of the VIP-sensitive Ca²⁺ pool by ryanodine receptors. a. A representative trace of attenuation of [Ca²⁺]_i induced by 5 nM VIP with subsequent addition of increasing concentrations of ryanodine. b. Collective results of experiment shown in Figs 6A. Values are means ± SE of 4 separate experiments. [Ca²⁺]_i were taken 2.5 min after VIP addition, and that for ryanodine taken 6 min after addition. Comparisons of VIP versus VIP + ryanodine are * p ≤ 0.05; † p ≤ 0.01; ‡ p ≤ 0.001. c. Representative traces of ryanodine (10 μM) blocking the sustained [Ca²⁺]_i induced by subsequent addition of VIP. d. Representative traces of ryanodine (10 μM) not affecting sustained [Ca²⁺]_i induced by subsequent addition of 10 μM carbachol (Carb). e. Collective results of experiments shown in Figs 6c and 6d. Values in each experimental set are means ± SE of 4 separate experiments. Basal, [Ca²⁺]_i just before adding agonist. Agonist responses taken 2.5 min after adding agonist, either with (closed bars) or without (open bars) 10 μM ryanodine added 15 min before agonist. * p ≤ 0.05 versus control. f. Ryanodine (10 μM) does not block Mn²⁺ quenching of intracellular fura-2 fluorescence of cells challenged with 5 nM VIP. Traces are representative of 4 paired experiments conducted with two preparations of tubuloacini. g. Ryanodine (10 μM) inhibits synergistic secretion induced by 5 nM VIP + 0.3 μM carbachol (Carb). Values are means ± SE of 3 separate experiments. * p ≤ 0.01 for secretion above basal versus the calculated additive sum for secretion induced by VIP and by carbachol separately, under control conditions (13.4 ± 0.8). Ryanodine added 15 min before agonists. h. Caffeine (10 μM) but not 1 μM IBMX causes a leftward shift in sustained [Ca²⁺]_i induced by increasing concentrations of VIP. Each curve is the result of 4 separate experiments. Caffeine and IBMX added 15 min before VIP. i. Mucin secretion in response to increasing concentrations of IBMX. Values are means ± SE of 3 experiments. * p ≤ 0.05 and † p ≤ 0.01 versus basal. j. Caffeine (10 μM) does not alter mucin secretion induced by increasing concentrations of VIP. The calculated EC₅₀ for VIP is 0.23 nM and 0.26 nM for VIP + 10 μM caffeine. Each curve is the result of 3 separate experiments. k. Localization of ryanodine binding sites in mucous cells fixed previously in 4% paraformaldehyde and after incubation in 5 nM BODIPY FL-X ryanodine. 1) differential interference contrast (DIC) image of 5 mucous cells in cross-section, with the lumen indicated by an asterisk. 2) fluorescence from BODIPY FL-X ryanodine in the mucous cells in panel 1. 3) merged image of 1 and 2. 4) and 5) negative control DIC and fluorescence images of mucous cells incubated in 5 nM BODIPY FL-X ryanodine, plus 1 μM ryanodine to block specific BODIPY FL-X ryanodine binding. Scale bar in panel 1 is 10 μm.

Figure 7.

Working model of mechanisms mediating muscarinic and VIP control of mucous cell exocrine secretion. Initial steps of acetylcholine (ACh) activation of muscarinic M1 and M3 receptor subtypes to activate PKC (α and possibly βI isoforms) and apical translocation to initiate granule docking and priming required for persistent granule secretion (2) is described in the Introduction. However, the initial muscarinic-induced IP₃-mediated release of Ca²⁺ from intracellular stores can alone induce release of a small fusion-competent pool of secretory granules at the apical membrane (1), and further activates store-operated Ca²⁺ entry (SOCE) via Orai1 in the plasma membrane (PM) through release of stromal activation molecule 1 (STIM1) and possibly STIM2 (presumably from endoplasmic reticulum, ER, membranes). STIM2 may increase STIM1-Orai1 channel complexation at lower agonist concentrations. Orai1-mediated Ca²⁺ entry recruits TRPC1 Ca²⁺ channels to the PM, potentially modulating Ca²⁺ influx. Ca²⁺ influx sustains increased [Ca²⁺]_i and helps replenish Ca²⁺ stores via thapsigargin-sensitive SERCA pumps. VIP activates VIP receptors (VIPR), exchange of GTP for GDP on α_s-subunits of G_s, dissociation of GTP-bound α_s to activate adenylyl cyclase (AC) and generation of cyclic adenosine monophosphate (cAMP). Protein kinase A (PKA) activation by cAMP initiates exocrine secretion by primed apical granules (3), independent of increased [Ca²⁺]_i, and further initiates granule docking and priming from a subset of vesicles (4), possibly with a specific vesicle-associated membrane protein (e.g., VAMP-8). PKA increases the Ca²⁺ permeability of ryanodine receptors (RyRs) controlling ER Ca²⁺-stores within the posterior basolateral region, presumably in response to direct phosphorylation of RyRs by PKA linked via an A-kinase-anchoring protein. Depletion of RyR-stores activates SOCE channels (Orai1 and possibly TRPC1) ostensibly via STIM1/2. Activation of RyRs may also occur via Ca²⁺-induced Ca²⁺ release (CICR) through robust muscarinic-induced Ca²⁺ release from more apical IP₃R-mediated Ca²⁺-stores. However, VIP-induction of RyR-mediated Ca²⁺ release does not induce CICR from IP₃Rs, possibly due to spatiotemporal segregation and the smaller Ca²⁺ pool of RyR stores, as well as Ca²⁺ uptake via SERCA pumps and surrounding mitochondria and Golgi. Increased [Ca²⁺]_i in response to VIP is additive to that in response to submaximal muscarinic stimulation to further enhance cPKC activity and exocrine secretion. However, release of the smaller RyR Ca²⁺ pool in the posterior basolateral region, without an increase in DAG, is alone insufficient to activate cPKC-mediated secretion. For more details see reviews by Ambudkar, [2], Trebak and Putney [99] and Liu et al. [57].

DISCUSSION

VIP-induced exocrine mucin secretion

VIP receptors are expressed by mucous cells of human labial [98] and submandibular glands [51], and induce submandibular mucous cell exocytosis [20]. The VIP EC₅₀ (0.24 nM) for mucin secretion by rat sublingual tubuloacini is consistent with VPAC₁ receptors of seromucous cells of murine submandibular glands [40], rat parotid serous cells [19,43] and murine sublingual glands [49]. However, maximal secretion induced by VIP/cAMP is a third of maximal muscarinic-induced secretion. Interestingly, exocrine secretion by sublingual mucous cells challenged by VIP alone is unaffected by removal of extracellular Ca²⁺, similar to that reported for cAMP-mediated exocytosis by colonic goblet cells [31] and pancreatic acinar cells [85]. In contrast, removal of extracellular Ca²⁺ from parotid acinar cells reduces VIP-induced exocrine secretion by 40% [86], thus indicating differences between salivary exocrine cell types in VIP-mediated exocytosis. We previously showed that increased [Ca²⁺]_i alone can promote the transient release of a small pool of tubuloacinar mucins, presumably from primed apical vesicles [17]. However, the [Ca²⁺]_i independence of a linear rate of VIP-induced secretion suggests PKA directly activates steps associated with the translocation, fusion and release of mucous vesicles [41]. Indeed, VIP activates PKA in canine pancreatic duct cells, without an increase in [Ca²⁺]_i, and potentiates Ca²⁺-induced exocrine secretion by accelerating granule docking/priming. This enhances the pool of releasable granules by increasing the Ca²⁺ sensitivity of final granule fusion [47]. Exocytosis involves vesicle membrane proteins of the synaptotagmin (SYT) family, small GTP-binding proteins and interactions between vesicle-membrane-associated proteins (VAMPs) of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor family (SNAREs), with targeted plasma membrane proteins (tSNAREs) of the syntaxin and SNAP25 families [94,107]. Various members of these families have been identified in exocrine cells, including salivary acinar cells [23,34,36,46,67,91,102,104,107]. Of specific interest, VAMP8 and VAMP2 are expressed among exocrine acinar cells and function in exocrine secretion mediated by either the Ca²⁺ or the cAMP pathway, respectively [34,46,67,94,104]. In pancreatic acini, VAMP2 and VAMP8 associate with distinct vesicles responsible for ≈50% of the secretory response [67]. Therefore, control of a subpopulation of vesicles by VIP/cAMP/PKA in mucous cells may be the result of associated VAMPs, tSNAREs and/or synaptotagmins different from Ca²⁺/PKC controlled vesicles. However, because VIP has no additive effect on mucin secretion at maximal carbachol concentration, the pool of mucin granules responsive to PKA must also be responsive to muscarinic activation of PKC [16]. Interestingly, the small GTP-binding protein, Rap1, is localized to zymogen granules of mouse pancreatic acinar cells and its activation mediates amylase secretion in response to both cAMP and carbachol [107], but in parotid acinar cells it functions only in cAMP-mediated secretion [18]. Further determination of signaling mechanisms controlling the translocation and plasma membrane fusion of mucous vesicles by both the cAMP and PKC pathways are thus required to elucidate their shared control of a subpopulation of exocrine vesicles.

VIP-induced [Ca²⁺]_i

In sublingual mucous cells, VIP induces Ca²⁺ release from intracellular stores maintained via Ca²⁺ influx by the thapsigargin-sensitive SERCA pump. However, VIP-induced Ca²⁺ release is mediated by a cAMP/PKA pathway (i.e., increased by cAMP, inhibited by H89) and with a sustained increase in [Ca²⁺]_i less than a fifth of that induced by carbachol, similar to mucin release by rat conjunctival goblet cells [54]. Similarly, VIP increases [Ca²⁺]_i in mucous acinar cells of human salivary labial glands [74] and in rat parotid acini mobilizes Ca²⁺ via cAMP, presumably from an intracellular pool distinct from the carbachol-sensitive pool [86]. Furthermore, 1 nM VIP induces cytosolic Ca²⁺ oscillations via cAMP in murine pancreatic acinar cells [48], and cAMP mobilizes intracellular Ca²⁺ in canine submandibular cells [25].

We provide initial evidence to suggest Ca²⁺ release by VIP is independent of IP₃Rs (i.e., not blocked by U73122) and is instead mediated by ryanodine Ca²⁺ channels as shown by inhibition with increasing ryanodine, plus a caffeine-mediated leftward shift in VIP-induced Ca²⁺ release without an effect on VIP-stimulated mucin secretion. VIP-mediated Ca²⁺ release may involve PKA complexation with RyRs via an A-kinase-anchoring protein, similar to that observed for type 3 RyRs in arachidonic acid-mediated amylase secretion by mouse parotid acini [22,84]. PKA can directly phosphorylate RyRs to increase their open probability by dissociation of FK506-binding protein, FKBP12 [73]. Such a mechanism is consistent with cAMP induction of Ca²⁺ release by microsomal vesicles from rat parotid acinar cells and inhibition of increased [Ca²⁺]_i by ryanodine and H-89 [73]. Conversely, a role for cyclic ADP-ribose in activation of RyRs cannot be ruled out, as it lowers RyR sensitivity to Ca²⁺, thus inducing Ca²⁺-induced Ca²⁺ release (CICR) [106]. Correspondingly, rat parotid acinar cells express type 1 RyRs [53], while cyclic ADP-ribose, cAMP or forskolin each mediate Ca²⁺-release that is attenuated by ryanodine [114]. These results are reminiscent of cardiac ventricular myocytes in which cAMP/PKA signaling activates ADP-ribosyl cyclase (CD38), possibly via PKA-phosphorylation of CD38 [106]. Another potential mediator participating in the VIP/cAMP-induced increase in [Ca²⁺]_i is a member of the family of EPACs (exchange protein directly activated by cAMP) [83]. For example, in the pancreatic β-cell line (MIN6) EPAC2 via IP₃Rs, and PKA via RyRs, function synergistically in mediating CICR induced by glucagon-like peptide-1 [100]. Moreover, VIP-mediated increase in cAMP in pancreatic acinar cells activates p21-activated kinase 4 to increase Na⁺, K⁺-ATPase via EPAC [82], and EPAC activation accelerates muscarinic-induced Ca²⁺ waves [87]. In parotid acinar cells EPAC may function, at least in part, in potentiation of increased [Ca²⁺]_i in response to stimulation of P2X receptors [4]. In the context of exocrine secretion, evidence indicates EPAC1 in parotid acinar cells partially mediates β-adrenergic-induced amylase release, independent of PKA [88,108]. Our results also indicate VIP further induces SOCE, which in serous and seromucous cells is mediated by depletion of IP₃ stores and the plasma membrane Ca²⁺ channel, Orai1, under control of the Ca²⁺-sensor, stromal interaction molecule 1 (STIM1). Orai1-mediated Ca²⁺ entry further controls Ca²⁺ entry through the PM Ca²⁺ channel, TRPC1 [2]. Because SOCE mediated by RyRs in skeletal muscle also utilizes STIM1 and Orai1 [109] these components and TRPC1 may function in VIP-induced SOCE in mucous cells. However, activation of VIP receptors in gastric cells results in extracellular Ca²⁺ entry via TRPV4 channels [95], raising the possibility that VIP uses a different mechanism for Ca²⁺ entry.

With respect to RyRs, we localized ryanodine binding sites within the posterior basolateral region of mucous cells, similar to that reported for rat parotid acini [53]. In parotid cells RyRs may function in VIP-induced Ca²⁺ release, whereas IP₃Rs are concentrated near the apical pole, triggering muscarinic-induced Ca²⁺ signals [7,86]. Also similar to rat parotid acini [53], a [Ca²⁺]_i response to low ryanodine concentrations was not detected, due possibly to rapid channel opening and closing to limit Ca²⁺ release from this small Ca²⁺ pool combined with efficient Ca²⁺ uptake by ER SERCA pumps, mitochondria and/or Golgi[93]. Because the VIP-sensitive Ca²⁺ pool is depleted by thapsigargin and maintained by SERCA pumps, we posit this store is harbored within the ER lumen, although other sources cannot be excluded, including lysosomes, endosomes, Golgi and secretory granules [78,103]. For example, apically localized serous exocrine granules in parotid acinar cells represent acidic organelles that can release Ca²⁺ by cAMP-mediated NAADP [42], presumably upon activation of CD38 to activate lysosomal two-pore channels [56]. In contrast, NAADP does not contribute to muscarinic-induced Ca²⁺ currents in rat submandibular seromucous acinar cells [39]. Although mucous granules are acidic and with a high Ca²⁺ concentration [29], Ca²⁺ is required for tight packaging of these extremely large and negatively charged molecules [77]. Mucous granules therefore likely do not represent a releasable Ca²⁺ pool.

Evidence in pancreatic acinar cells [78], neurons [11] and vascular smooth muscle cells [81] indicates the ER is a single pool of Ca²⁺ that can readily diffuse and equilibrate within its continuous luminal network. However, we find the VIP-sensitive Ca²⁺ pool is depleted by 5 nM VIP within 150 sec, and subsequent carbachol addition initiates Ca²⁺ release after [Ca²⁺]_i returns to baseline, albeit from a smaller Ca²⁺ pool shared between VIP and carbachol which is functionally distinct from the carbachol-sensitive pool. Although the presence of a shared Ca²⁺ pool explains why VIP has no additive effect on [Ca²⁺]_i at maximal carbachol, the larger and contiguous carbachol-sensitive pool would be expected to gradually replenish the VIP-sensitive pool under Ca²⁺-free conditions, eventually depleting the pool responsive to carbachol. Evidence for functionally distinct ER Ca²⁺ pools is not unique. For example, colonic myocytes appear to contain separate Ca²⁺ stores, one expressing only RyRs and refilled from cytosolic Ca²⁺, and another expressing both IP₃ Rs and RyRs that is refilled from extracellular Ca²⁺ [30]. More recent results suggest the ER of hippocampal neurons contain a distinct RyR-sensitive Ca²⁺ pool refilled by voltage-gated Ca²⁺ channels versus a IP₃R pool refilled by Orai2 channels [9,76]. Conversely, Rainbow et al., [81] working with vascular myocytes provides evidence that apparently distinct Ca²⁺ pools are the result of reduced sensitivity of IP₃Rs and RyRs to ligand activation as the luminal [Ca²⁺] decreases. As a result, one channel type may stop responding before the other, although significant residual luminal Ca²⁺ remains. Likely contributing to this Ca²⁺ retention and control of IP₃Rs and RyRs are luminal Ca²⁺-binding proteins associated with the Ca²⁺ channels, which may further function to help prevent excessive depletion of luminal Ca²⁺ and activation of ER stress [38]. We therefore hypothesize that the ER of mucous cells is a continuous network of a single pool of Ca²⁺, but with apical expression of IP₃Rs and co-expression of IP₃Rs and RyRs in the basolateral region. The basolateral ER may be a single compartment containing both RyRs and IP₃Rs or is divided into two compartments, one containing RyRs and the other IP₃Rs. However, in both cases IP₃Rs and RyRs of the basolateral ER are expected to be interconnected functionally via CICR, with Ca²⁺ released via IP₃Rs initiating RyR-mediated Ca²⁺ release, thus accounting for Ca²⁺ release from the VIP-sensitive pool upon maximal activation of IP₃-mediated signaling by carbachol. On the other hand, the small induction of [Ca²⁺]_i by VIP activation of cAMP/PKA does not function in mucin secretion. Perhaps the basolateral localization of the smaller RyR Ca²⁺ pool, combined with robust Ca²⁺ uptake, hinders VIP/RyR-mediated CICR from the more apical IP₃ stores that control Ca²⁺-induced exocytosis. Additional studies are therefore required to elucidate PKA-dependent and putative PKA-independent signaling pathways functioning in VIP-induced mucin secretion, including RyR and IP₃R expression, their localization, spatial-temporal localization of Ca²⁺ signals and mechanisms of Ca²⁺ entry. Presented in Fig. 7 is a working model of mechanism controlling muscarinic- and VIP-induced mucous cell exocrine secretion as a guide for future studies.

VIP and muscarinic synergistic secretion

There are few reports of synergism between VIP and muscarinic agonist in salivary glands, with studies focusing on fluid or protein secretion from intact submandibular or serous parotid glands of cats [26,27,44,45,52,60,61,101]. Mature rat submandibular gland acinar cells are seromucous, histologically, and secrete both glutamine/glutamic acid-rich protein (45–50 KD) and a small ≈115 KD mucin, Muc10 [69], in contrast to cat submandibular glands with distinct cell populations of serous and mucous acini, similar to “mixed” submandibular glands of humans [79]. In addition, the abundant and long oligosaccharides of high-molecular weight gel-forming mucins largely mask mucin detection in standard protein assays [13]. It is thus likely that mucous cell exocrine secretion in the above studies of cat submandibular glands was poorly accounted for. The current study is therefore the first to elucidate VIP and muscarinic synergism in exocrine secretion in a representative salivary mucous gland in which mucous cells are devoid of significant sympathetic innervation. Synergism in the above previous studies was explained as either VIP-induced vasodilation [60] or muscarinic enhancement of VIP-induced cAMP [28]. However, our results with isolated tubuloacini preclude a vasodilatory effect, and carbachol has no effect on VIP-induced cAMP levels. VIP also reportedly increased carbachol affinity to muscarinic receptors ≈1,000-fold [59], which if functioning in mucous cells should have resulted in a leftward shift in the carbachol concentration-response curve by VIP, rather than a more restricted increased slope.

In contrast, there are many reports of synergistic interactions between the cAMP and [Ca²⁺]_i in secretory cells in the context of either fluid secretion [1,10,111] or exocrine secretion [5,8,47,64,72,80,89,97,110], including those specifically reporting interactions between VIP and muscarinic agonist [5,10,64,89]. However, much less is known of mechanisms associated with reported synergistic effects. Synergism in the volume of secretions from airway glands involve VIP activation of the apical CFTR (cystic fibrosis transmembrane conductance regulator) combined with carbachol stimulation of basolateral K⁺ channels [10]. In rat parotid and submandibular acinar cells, β-adrenergic-mediated cAMP/PKA has little, if any, effect on [Ca²⁺]_i but instead augments muscarinic-induced [Ca²⁺] resulting in synergistic fluid secretion [90]. This synergism is due to a lowering of the threshold for IP₃-mediated Ca²⁺ release via PKA phosphorylation of InsP₃R1 and InsP₃R2 and/or by cAMP sensitization of IP₃R1–3 to IP₃ [7,96] resulting in reduced affinity for IRBIT (IP₃ receptors binding protein release with IP₃), which once released is free to activate ion transporters [1]. Such a mechanism does not appear to mediate the synergistic effects of VIP-induced [Ca²⁺]_i in sublingual mucous cells, as VIP induces a relatively modest increase in [Ca²⁺]_i that is necessary for synergism, but is independent of PLC activation. Instead, our results are consistent with VIP augmentation of muscarinic-induced mucin secretion by increasing [Ca²⁺]_i via PKA-mediated release of Ca²⁺ from an intracellular Ca²⁺ pool, resulting in enhancement of muscarinic-induced cPKC activity and exocytosis, thus increasing muscarinic agonist sensitivity. Six key results support this scenario: 1) VIP is synergistic with submaximal carbachol in stimulation of mucin secretion; 2) VIP-induced [Ca²⁺]_i is additive to carbachol-induced [Ca²⁺]_i at submaximal carbachol; 3) VIP and carbachol increase [Ca²⁺]_i within the same cell; 4) VIP-induced [Ca²⁺]_i does not function in VIP-stimulated mucin secretion; 5) Increased [Ca²⁺]_i via thapsigargin that is equivalent to VIP-induced [Ca²⁺]_i results in synergistic mucin secretion with submaximal carbachol; and 6) VIP-induced [Ca²⁺]_i interacts with PMA to produces synergistic mucin secretion, presumably via enhanced cPKC activity [17].

Synergy between VIP and muscarinic agonist in mucous cell exocrine secretion is physiologically significant in the context of salivary mucous gland function in humans. The multiple minor submucosal mucous glands lining the oral cavity (e.g., labial, palatine, buccal, retromolar) in addition to sublingual glands provide secretions rich in mucins that help keep tissues hydrated and lubricated and provide innate immune functions such as binding of select bacteria to oral surfaces and/or their clearance [6]. Indeed, secreted mucins, MUC7 (≈ 150–200 kDa) and the hydroscopic gel-forming mucin MUC5B (≥ 1,000 kDa), were shown to be important constituents of the mucosal pellicle overlaying oral epithelial cells [35,70]. With lower parasympathetic nerve activity between meals, synergy between VIP and acetylcholine is predicted to help maintain this protective layer. Further elucidation of mechanisms mediating VIP/muscarinic synergism, as mentioned above, may further identify therapeutic targets for patients with hyposalivary function, including Sjögren’s syndrome patients and patients after radiation treatment for head-and-neck cancers. For example, targeting the VIP/cAMP/PKA pathway may enhance mucin secretion in response to the necessarily low doses of the pharmacotherapeutic, pilocarpine, by increasing the sensitivity of remaining functional mucous cells to this muscarinic agonist [71]. Additional effects of VIP in increasing vasodilation would further support mucin secretion [92]. Moreover, VIP in murine Sjögren’s syndrome models has trophic effects, is anti-inflammatory [40] and reduces disease-associated cytokines [58].