Ryanodine receptor expression is associated with intracellular Ca2+ release in rat parotid acinar cells

Xuejun Zhang1, Jiayu Wen1, Keshore R. Bidasee2, Henry R. Besch Jr.2, and Ronald P. Rubin1

1 Department of Pharmacology and Toxicology, School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, New York 14214; and 2 Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, Indiana 46202-5120

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The ryanodine receptor mediates intracellular Ca2+ mobilization in muscle and nerve, but its physiological role in nonexcitable cells is less well defined. Like adenosine 3',5'-cyclic monophosphate and inositol 1,4,5-trisphosphate, cyclic ADP-ribose (0.3-5 µM) and ADP (1-25 µM) produced a concentration-dependent rise in cytosolic Ca2+ in permeabilized rat parotid acinar cells. Adenosine and AMP were less effective. Ryanodine markedly depressed the Ca2+-mobilizing action of the adenine nucleotides and forskolin in permeabilized cells and was likewise effective in depressing the action of forskolin in intact cells. Cyclic ADP-ribose-evoked Ca2+ release was enhanced by calmodulin and depressed by W-7, a calmodulin inhibitor. A fluorescently labeled ligand, 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3,4-diaza-s-indacene-3-propionic acid-glycyl ryanodine, was synthesized to detect the expression and distribution of ryanodine receptors. In addition, ryanodine receptor expression was detected in rat parotid cells with a sequence highly homologous to a rat skeletal muscle type 1 and a novel brain type 1 ryanodine receptor. These findings demonstrate the presence of a ryanodine-sensitive intracellular Ca2+ store in rat parotid cells that shares many of the characteristics of stores in muscle and nerve and may mediate Ca2+-induced Ca2+ release or a modified form of this process.

cyclic adenosine diphosphate-ribose; adenosine 3',5'-cyclic monophosphate

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

IN NONEXCITABLE CELLS, Ca2+ signaling depends on Ca2+ influx and a regenerative release of Ca2+ from intracellular stores. The inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) and the ryanodine receptor (RyR) modulate the two channels responsible for mobilizing intracellular Ca2+ (3). IP3 is generated from phospholipase C-mediated breakdown of membrane-bound polyphosphoinositides. Ryanodine binds to a family of receptors (RyR) that are subtypes characterized for skeletal muscle (type 1, RyR1), cardiac muscle (type 2, RyR2), and a widely distributed brain isoform (type 3, RyR3) (27). Unlike the IP3R, a natural ligand(s) that regulates the RyR Ca2+-mobilizing mechanism has not been identified, although cyclic ADP-ribose (cADPR), a naturally occurring NAD+ metabolite, has been proposed to serve as an endogenous messenger (16).

A major portion of the work on the RyR has been carried out on skeletal or cardiac muscle preparations, although specific ryanodine-binding sites localized to nonexcitable cells and cell lines have also been identified (2, 5, 29). In single nonmuscle cells the RyR, along with the IP3R, is thought to be responsible for Ca2+ spiking, which is presumed to reflect Ca2+-induced Ca2+ release (CICR) (3). This process involves the influx of extracellular Ca2+, which induces Ca2+ release from the sarcoplasmic reticulum (SR) or endoplasmic reticulum (ER) to cause cellular activation (7, 8).

Our previous studies demonstrated that adenosine 3',5'-cyclic monophosphate (cAMP) produced by beta -adrenergic receptor activation potentiates the alpha -receptor-mediated rise in intracellular Ca2+ in intact rat parotid acinar cells (19) and mobilizes Ca2+ from permeabilized cells (23). The findings that the Ca2+ response to cAMP was sensitive to ryanodine and insensitive to heparin, a blocker of the IP3R, provided preliminary evidence for the presence of a Ca2+-release channel that is not regulated by IP3 (23). To probe further the possibility that the parotid cell possesses an RyR Ca2+ release channel for regulating Ca2+, we utilized a number of physiological and nonphysiological ligands, including cyclic nucleotides, adenine nucleotides, ryanodine, calmodulin (CaM), and caffeine, which regulate the RyR in excitable cells (27). In addition, the expression and distribution of the RyR were examined by reverse transcriptase polymerase chain reaction (RT-PCR) and by monitoring of 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3,4-diaza-s-indacene-3-propionic acid succinimidyl ester (BODIPY)-ryanodine fluorescence. The data support the idea of an RyR in rat parotid cells that resembles the RyR of a ryanodine-sensitive Ca2+ release channel that exists in muscle and nerve.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Materials. Adenine nucleotides, adenosine, forskolin, 3-isobutyl-1-methylxanthine (IBMX), and IP3 were purchased from Sigma Chemical (St. Louis, MO). Fura 2, fura 2-acetoxymethyl ester, and BODIPY 409/503 SE were obtained from Molecular Probes (Eugene, OR). cADPR, ryanodine, CaM, and W-7 were purchased from Calbiochem (San Diego, CA).

Preparation of permeabilized cells. Rat parotid acinar cells were prepared and isolated as previously described (23). For permeabilization, rat acinar cells (8-10 × 106 cells/ml) were washed twice with a cytosol-like medium containing 140 mM KCl, 20 mM NaCl, 20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 3 mM ATP, 10 mM phosphocreatine, and 10 U/ml creatine phosphokinase (pH 7.2). After permeabilization with saponin at room temperature (23), the cells were centrifuged and resuspended in high-K+ buffer containing 5 µM oligomycin, 10 µM antimycin, and 6 mM ATP.

Measurement of Ca2+ release. Ca2+ levels were determined in permeabilized cells preloaded with fura 2, as previously described (23). Fluorescence was measured in a spectrophotometer (model RF-5000, Shimadzu). This single-excitation-wavelength instrument yielded values comparable to those obtained with a dual-excitation-wavelength instrument. Stimulated Ca2+ release is defined as the maximal concentration of Ca2+ (Delta [Ca2+]) determined after the addition of stimulating agent minus the concentration of Ca2+ measured in the absence or presence of any pretreatment, including ryanodine. Basal Ca2+ release from untreated permeabilized cells averaged 366 ± 14 nM (n = 66). In a few experiments, Ca2+ mobilization was measured in intact parotid cells after exposure to 5 µM fura 2-acetoxymethyl ester for 30 min (19).

Fluorescence microscopic analysis. Confocal fluorescence microscopy was carried out on intact parotid cells utilizing a new fluorescent ryanodine derivative prepared by coupling an activated ester of BODIPY 493/503 SE to glycyl ryanodine using the method previously described (12). The 50% inhibitory concentration (IC50) for the resultant BODIPY-ryanodine was evaluated from competition binding assays, as described elsewhere (11). Cells were incubated in total darkness at 37°C in HEPES-buffered Krebs-Ringer solution (pH 7.4) with various concentrations of BODIPY-ryanodine. At the end of the incubation period, the cells were washed twice with Krebs-Ringer solution to remove unbound ligand and placed on a slide under a coverslip. The cells were initially viewed using epifluorescence optics of a Nikon upright Optiphot microscope, and confocal imaging was performed with a laser scanning microscopy system (model MRC-1024, Bio-Rad) configured with a Nikon microscope and a krypton-argon laser (488 nm). A ×40 oil immersion objective was first employed to give overall views, then zoom ×2 optics were used to provide a higher-magnification view. The system was operated by a Compaq Pentium 100 computer, and photographs were processed using a disublimation printer (model NP-1600, Codonic). Concentration and time course analysis indicated that fluorescence intensity reached a plateau after a 3-h exposure to 200 nM BODIPY-ryanodine (data not shown). Therefore, these parameters were chosen for our studies. Displacement experiments to determine binding specificity were carried out by incubating cells with 200 nM BODIPY-ryanodine + 2 µM ryanodine for 3 h.

mRNA analysis and RT-PCR. mRNA was isolated from rat parotid acinar cells and brain using TRIzol reagent (GIBCO Life Technologies, Grand Island, NY). First-strand cDNA was synthesized using a SuperScript II ribonuclease H+ RT kit (GIBCO). Although rat sequences are not available in the GenBank, rat RyR type-specific primers were designed on the basis of the comparative sequence of the novel mouse brain RyR1 and the partial sequence of rat skeletal muscle RyR1, which was graciously provided by Dr. Deborah Bennett (Cambridge University, Cambridge, UK). The sequences of the primers were as follows: 5'-GGTGGCCTTCAACTTCTTCL-3' (sense) and 5'-ACTTGCTCTTGTTGGTCTCCG-3' (anitsense). By use of these primers, RyR cDNAs were amplified by PCR with a thermocycler (model 2400, Perkin Elmer) for 45 cycles with 1 unit of Pfu polymerase. Reactions were carried out as follows: denaturation for 1 min at 95°C, annealing for 2 min at 60°C, and extension for 2.5 min at 74°C. An aliquot of each PCR reaction was subjected to electrophoresis through a 2% agarose gel, and DNA was visualized by ethidium bromide staining. The DNA product was purified and sequenced in both directions by the CAMBI Nucleic Acid Facility (DNA Sequencing Service, State University of New York at Buffalo).

Statistical analysis. Values are means ± SE. Comparisons between control and experimental samples were made using two-tailed unpaired Student's t-test. Differences were accepted as significant at P < 0.05.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Effects of forskolin. Forskolin, which raises parotid cell cAMP levels by directly activating adenylyl cyclase (6), produced a concentration-dependent enhancement of Ca2+ release from saponin-permeabilized parotid acinar cells (Fig. 1). The Ca2+-mobilizing action of forskolin was enhanced by preincubating cells with the phosphodiesterase inhibitor IBMX (Fig. 1). These findings demonstrate that Ca2+ release from permeable cells can be regulated by endogenously generated cAMP levels as well as by the addition of cAMP (23). However, cAMP was much less efficacious in this regard than IP3 (Fig. 1) (23). Like cAMP, IBMX enhanced the Ca2+ response to IP3 (Fig. 1). This potentiation appears to involve sensitization of the IP3R by protein kinase A-dependent phosphorylation (23).


View larger version (27K):
[in this window]
[in a new window]
 
Fig. 1.   Concentration-dependent increase in forskolin (FSK)-stimulated Ca2+ release from fura 2-loaded permeabilized parotid cells in presence (+) and absence (-) of 3-isobutyl-1-methylxanthine (IBMX, 200 µM). Response to a submaximal concentration of inositol 1,4,5-trisphosphate (IP3) is also presented for comparison. Values (means ± SE of 5 experiments) represent peak Ca2+ release measured after ~1 min with basal values subtracted (Delta [Ca2+]). * Significantly different from corresponding control group (P < 0.05).

Effects of cADPR on Ca2+ release. Another cyclic nucleotide, cADPR, which has been proposed as an endogenous regulator of CICR mediated by RyRs (16), produced a concentration-dependent rise in Ca2+ release. The time course of a representative experiment is shown in Fig. 2, and a quantitative assessment is provided in Fig. 3. The stepwise nature of the Ca2+ mobilized by cADPR, like that of cAMP, is consistent with a quantal (all-or-none) release mechanism (Fig. 2) (23). Graded effects were observed at 0.3-5 µM (Figs. 2 and 3). A higher concentration of cADPR (25 µM) produced no further elevation in Ca2+ release (data not shown).


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 2.   Concentration-dependent increase in Ca2+ release produced by cyclic ADP-ribose (cADPR). Trace is representative of 12 traces obtained from 11 independent preparations.


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 3.   Ryanodine inhibition of cADPR-stimulated Ca2+ release. Permeabilized parotid cells were pretreated with 10 µM ryanodine or vehicle for 5 min before addition of increasing concentrations of cADPR. Vertical bars represent values for peak Ca2+ mobilization obtained in presence and absence of ryanodine. Values are means ± SE of 4 independent experiments. Ryanodine (10 µM) alone increased basal Ca2+ release by 34.2 ± 8.5 nM. * Significantly different from corresponding control group (P < 0.05).

Effects of other adenine-containing compounds. The Ca2+-mobilizing action of cADPR was shared by other adenine-containing compounds. Exposure to 1-25 µM ADP produced a concentration-dependent rise in Ca2+ mobilization (Fig. 4A). At 25-500 µM, the adenine nucleoside adenosine also elicited a concentration-dependent elevation in Ca2+ release (Fig. 4B). By contrast, consistent responses to AMP were observed only at >= 500 µM (data not shown).


View larger version (11K):
[in this window]
[in a new window]
 
Fig. 4.   Effects of ADP (A) and adenosine (B) on Ca2+ release. Peak Ca2+ release was measured after 30 s. Values are means ± SE of 4 independent experiments.

A comparison of the relative potencies of adenine-containing compounds in promoting Ca2+ mobilization is provided in Table 1. The data show that cADPR was most potent in this regard and ADP was somewhat less potent, although it was greater than adenosine, cAMP, or AMP. It is also of interest that cADPR was ~200-fold more potent than cAMP and 10-fold more potent than IP3 (Table 1).

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Comparative EC50 values for various agents in enhancing intracellular Ca2+ release

Effects of ryanodine. To investigate potential mechanisms whereby intracellular Ca2+ release may be activated, the effects of ryanodine were examined. Ryanodine (10 µM) attenuated Ca2+ release elicited by the adenylate cyclase activator forskolin in permeabilized cells (Fig. 5A), thus indicating that ryanodine can block the response to endogenously generated cAMP, just as it markedly depressed the response to added cAMP (23). Experiments were also carried out to determine whether ryanodine could block the stimulatory effects of forskolin in intact parotid cells. Figure 5B shows that forskolin was able to elicit a concentration-dependent stimulation of Ca2+ mobilization that was reduced ~50% by ryanodine. These latter findings confirm earlier work demonstrating that cAMP modulates Ca2+ homeostasis in intact parotid cells (19) and provide a physiological basis for the actions of ryanodine in permeabilized cells.


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 5.   Inhibition of forskolin-stimulated Ca2+ release by ryanodine. A: permeabilized cells were pretreated with 10 µM ryanodine or vehicle for 5 min before addition of increasing concentrations of forskolin. B: intact cells were pretreated with 50 µM ryanodine for 30 min before addition of forskolin. Values are means ± SE of at least 4 determinations from 3-4 independent experiments with basal values subtracted. Ryanodine (10 µM) alone increased basal Ca2+ release in permeabilized cells by 36 ± 5 nM. In intact cells, basal Ca2+ release averaged 86 ± 9 and 205 ± 15 nM in absence and presence of 50 µM ryanodine, respectively. * Significantly different from corresponding control group (P < 0.05).

Ryanodine also depressed the Ca2+-mobilizing action of cADPR in permeabilized cells (Fig. 3). Even at the maximal stimulating concentration of cADPR (3 µM), ryanodine attenuated Ca2+ release by ~70%. Ryanodine (10 µM) also blocked Ca2+ mobilization induced by ADP and adenosine (data not shown). However, the blockade was statistically significant only at the lowest concentration of adenosine employed (25 µM), thus suggesting the competitive nature of the blockade.

In excitable and nonexcitable cells, ryanodine at relatively low concentrations (10 nM-10 µM) is reported to cause activation of the Ca2+ release channel, whereas, at >10 µM, ryanodine eventually blocks channel activation (24, 30). In permeabilized parotid cells, 2-100 µM ryanodine produced only a modest graded enhancement of Ca2+ release within the limited time frame of the experiments (data not shown). No blockade of channel activation was observed, despite the fact that ryanodine attenuated cADPR- and forskolin-stimulated Ca2+ release (cf. Figs. 3 and 5). Further studies are underway to evaluate this phenomenon.

Role of CaM in Ca2+ mobilization. Pretreatment of permeabilized parotid cells with CaM enhanced Ca2+ release induced by cADPR (Fig. 6A). With 1 µM cADPR, CaM augmented Ca2+ mobilization by twofold. The addition of 5 µM CaM alone resulted in a 25% rise in Ca2+ levels (data not shown), which is probably the result of the addition of a small amount of contaminating Ca2+ contained in the CaM buffer. In addition, W-7, a CaM inhibitor, reduced the Ca2+ response to increasing concentrations of cADPR (Fig. 6B). Thus, in the presence of 70 µM W-7, the response to 1 and 5 µM cADPR was diminished by >50%.


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 6.   Effects of calmodulin (CaM) and W-7 on cADPR-induced Ca2+ release. Permeabilized parotid cells were pretreated with 5 µM CaM (A) or 70 µM W-7 (B) for 5 min before addition of increasing concentrations of cADPR. Peak Ca2+ release was obtained in presence and absence of CaM or W-7. Values are means ± SE of 4 independent experiments. Addition of CaM and W-7 in absence of cADPR increased basal Ca2+ release by 57.6 ± 7.5 and 71.3 ± 3.4 nM, respectively. * Significantly different from corresponding control group (P < 0.05).

Effects of caffeine. Caffeine (1 mM), a pharmacological activator of the RyR (5, 26, 30), produced only a modest elevation of cytosolic Ca2+ (~20%). Caffeine also attenuated Ca2+ release induced by cADPR by ~50% (Fig. 7A). By contrast, the ability of IP3 to raise Ca2+ levels was augmented (Fig. 7A). These actions of caffeine mimicked those of forskolin in the ability to depress the Ca2+ response to cADPR and enhance the response to IP3 (Fig. 7B).


View larger version (30K):
[in this window]
[in a new window]
 
Fig. 7.   Effects of caffeine and forskolin on cADPR- and IP3-mediated Ca2+ release. Permeabilized parotid cells were pretreated with 1 mM caffeine (A) or 2 µM forskolin (B) for 5 min before addition of cADPR or IP3. Peak Ca2+ release was obtained in presence and absence of caffeine or forskolin. Values are means ± SE of 4 independent experiments. Caffeine and forskolin alone increased basal Ca2+ release by 34.0 ± 5.3 and 18.6 ± 2.3 nM, respectively. * Significantly different from corresponding control group (P < 0.05).

Detection of ryanodine-binding sites. Figure 8 shows that BODIPY-ryanodine competitively inhibited binding of [3H]ryanodine to rabbit muscle SR vesicles with an IC50 of 100 nM. The fluorescently labeled ligand was subsequently employed to study the expression and distribution of the RyR in parotid cells. Intact cells were utilized for this study, since the high-Mg2+-low-Ca2+ medium used for permeabilized cells markedly reduces the affinity of ryanodine for specific binding sites (21). Confocal microscopy revealed that cells exposed to BODIPY-ryanodine displayed a granular fluorescence throughout the cell body that was excluded from the nucleus and plasma membrane (Fig. 9A). The localization of the fluorescence was consistent with the presence of binding sites within the ER. Under our experimental conditions, 89 ± 7% of the cells exposed to BODIPY-ryanodine exhibited fluorescence (mean of 8 images from 3 different preparations). Competition experiments were also performed to determine whether BODIPY-ryanodine binding was specific. Figure 9B shows that fluorescence intensity was markedly decreased in cells incubated with BODIPY-ryanodine + 2 µM ryanodine. Less than 10% of control cells exhibited significant fluorescence. These results document the existence of specific binding sites for ryanodine in rat parotid acinar cells.


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 8.   Displacement by 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3,4-diaza-s-indacene-3-propionic acid succinimidyl ester (BODIPY)-ryanodine of [3H]ryanodine from ryanodine receptor Ca2+ release channel of rabbit skeletal muscle junctional sarcoplasmic reticulum vesicles. Values for BODIPY-ryanodine experiments are means ± SE for at least 3 separate assays, each done in duplicate, using 2 different membrane preparations. Displacement of [3H]ryanodine by unlabeled ryanodine is shown for comparison and represents 19 separate assays using 6 different preparations. [Ryanoid], ryanoid concentration.


View larger version (68K):
[in this window]
[in a new window]
 
Fig. 9.   Fluorescence localization of ryanodine-binding sites in rat parotid acinar cells. Representative confocal images of cells labeled with BODIPY-ryanodine (A) or BODIPY-ryanodine + ryanodine (B) are shown.

Expression of RyR mRNA. The pharmacological and fluorescence experiments demonstrating the presence of RyR in rat parotid cells provided the impetus for extending this study to identify the type of RyR expressed in these cells. After primers were designed according to the partial sequence of rat skeletal muscle RyR1 or novel mouse brain RyR1 (see MATERIALS AND METHODS), the amplified mRNA from rat parotid cells failed to yield detectable PCR product. However, by use of primer pairs according to a sequence common to rat skeletal muscle RyR1 and mouse brain RyR1, the amplified cDNA from parotid and brain mRNA yielded a band of the expected size of 293 base pairs in parotid and brain (Fig. 10A).


View larger version (63K):
[in this window]
[in a new window]
 
Fig. 10.   A: reverse transcriptase-polymerase chain reaction (RT-PCR) detection of ryanodine receptors (RyR) in rat parotid cells and brain. RT-PCR analysis with specific primers common to novel mouse brain type 1 RyR (RyR1) and rat skeletal muscle RyR1, showing their expression in rat parotid cells (lane 1) and brain (lane 2). cDNA template concentration for PCR amplification of rat parotid cells was 10 times higher than that of brain. Molecular weight marker (M) is phi X174/Hae III. B: comparison of nucleotide sequence for putative RyR in rat parotid (RPR) with novel mouse brain RyR1 (MBR). star  Common regions.

The PCR product from rat cells analyzed by DNA sequencing was highly homologous to rat skeletal muscle RyR1 (98%) and mouse brain RyR1 (94%; Fig. 10B). Like the novel mouse brain RyR1 (1), the PCR product from rat parotid cells also has a high degree of homology to rabbit RyR1 (88%).

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The present study utilized three complementary techniques (pharmacological, fluorescent, and molecular) to demonstrate that parotid cells contain an RyR that mediates intracellular Ca2+ release. The genesis of this work emanated from an earlier study that showed that the ability of cAMP to induce intracellular Ca2+ release involves an RyR-mediated mechanism (23). Further experimentation revealed that the ability of cAMP to activate the RyR is shared not only by another cyclic nucleotide, cADPR, but also by a number of other adenine-containing compounds. Analysis of the concentration-response data for a series of adenine-containing compounds demonstrated a wide disparity in Ca2+-releasing activity. However, most significantly, apart from cADPR, the rank order of potency of these compounds (ADP > adenosine > cAMP > AMP; Table 1) was remarkably similar to that reported in muscle preparations (33). Adenine appears to be the preferred ligand for the nucleotide-binding site on the RyR in parotid cells and muscle preparations, since GTP and guanosine 3',5'-cyclic monophosphate are ineffective in both systems (21, 23), and the putative nucleotide-binding site possesses a much higher affinity for adenine nucleotides (33).

The fact that cADPR proved to be the most potent of the adenine-containing compounds examined is also consistent with findings in other model systems where it mobilizes intracellular Ca2+ in the nanomolar concentration range (18). The cADPR concentration for half-maximal stimulation of Ca2+ release from permeabilized parotid cells (0.6 µM) compares favorably with values derived from isolated cardiac SR (0.15 µM) and brain microsomes (0.11 µM) (22). The ability of cADPR to potentiate CICR in sea urchin eggs (15) and activate the cardiac RyR (5) bears on the possibility that cADPR represents a physiologically relevant second messenger, although the physiological relevance of the cADPR effect on cardiac RyR is controversial (25). Its relative potency in parotid cells supports this supposition, as does the finding that this cyclic nucleotide elicits Ca2+ spiking in intact exocrine cells (28). However, additional experimentation is needed to determine whether parotid cells are capable of synthesizing cADPR and regulating its cellular levels during periods of Ca2+ mobilization.

The additional result that the potency of cADPR was >10-fold greater than that of IP3 but ~3-fold less efficacious may be a consequence of a less efficient coupling mechanism for cADPR or the fact that RyR are less concentrated in the ER of parotid cells. Although receptor density was not determined in this study, the latter alternative gains favor by the knowledge that the density of RyR in the submicrosomal fraction of intestinal smooth muscle is 10-fold less than that of IP3 receptors (32).

The ability of ryanodine to block the Ca2+-mobilizing action of cADPR in parotid cells, as well as the actions of other adenine nucleotides, is in agreement with previous work that demonstrated that cADPR releases Ca2+ from cardiac SR and rat brain microsomes by a mechanism that is sensitive to inhibition by ryanodine (22, 25, 31). Furthermore, the fact that ryanodine was able to block the Ca2+-releasing action of forskolin in intact cells documents the physiological relevance of the findings in permeabilized cells. On the other hand, the characteristic property of ryanodine to open the RyR channel in low concentrations and block the channel in higher concentrations (14, 24) at functionally separate sites (11) was not observed in parotid cells. The observation that CaM potentiates cADPR-induced Ca2+ mobilization from parotid cells is also in accord with the finding that CaM has a potentiating effect on CICR in skinned muscle fibers at submicromolar Ca2+ concentrations, although CaM does inhibit CICR at higher Ca2+ concentrations (13). The apparent discrepancy that CaM serves an apparent permissive role in parotid cells but is required for cADPR action in sea urchin egg homogenates (17) may be resolved by the fact that CaM is tightly associated with parotid membranes (4), thereby enabling permeabilized parotid cells to retain sufficient CaM to sustain the activation of Ca2+ release.

Further insight into the mechanism by which adenine nucleotides regulate the Ca2+ release channel of parotid cells is gleaned from the knowledge that cADPR, ADP, cAMP, and adenosine promote Ca2+ mobilization from permeabilized cells incubated in a medium that contains millimolar concentrations of ATP and an ATP-regenerating system. This medium, which also contains several mitochondrial inhibitors, is utilized for permeabilized parotid cells to alleviate any influence of intact cells. Under such experimental conditions the adenine moiety probably exerts its Ca2+-releasing action at a locus other than the ATP-binding site on the RyR. In this context, it is known that two or more nucleotide-binding sites exist in each RyR monomer with distinct affinities (33). The physiological implications of the present studies may pertain to the ability of adenine nucleotides, as well as CaM, to serve modulatory roles by sensitizing the mechanism that allows Ca2+ influx to activate Ca2+ release, i.e., CICR. Such a model has been proposed for CICR in muscle (5, 26).

Caffeine has been widely employed to activate CICR in excitable cells (26, 30), and the property of adenine nucleotide-induced Ca2+ release from muscle preparations is shared by caffeine (5). However, attempts to duplicate with caffeine the stimulatory effects of the adenine nucleotides in permeabilized parotid cells yielded inconclusive results. The effects of IBMX that we observed in parotid cells appear to be due to phosphodiesterase inhibition to raise cAMP levels, rather than a direct action on a caffeine-sensitive Ca2+ pool (26). Thus caffeine behaved like forskolin in attenuating intracellular Ca2+ release induced by cADPR, presumably because cAMP and cADPR activate and thereby deplete the same intracellular Ca2+ pool. Moreover, the ability of caffeine to enhance the sensitivity of the Ca2+-releasing activity of IP3 was shared by forskolin and exogenous cAMP (cf. Fig. 7 and Ref. 23). The fact that caffeine lacks the ability to mobilize Ca2+ in certain nonexcitable cells supports the assertion that caffeine cannot be used to demonstrate the presence of musclelike RyR in nonexcitable cells (2, 20). However, Foskett and Wong (9) reported that caffeine can induce ryanodine-sensitive Ca2+ oscillations in intact parotid cells depleted of their IP3-sensitive store by thapsigargin. Thus other experimental strategies are required to ascertain whether parotid cells manifest a caffeine-sensitive Ca2+ store with properties comparable to those found in excitable cells.

Because ryanodine binds specifically to the Ca2+-gated Ca2+ release channels in the SR (or ER) of cells (5), the validity of the pharmacological studies could be confirmed by using a specific ligand (ryanodine) derivatized with a fluorophore to identify the distribution of ryanodine receptors in parotid acinar cells. The granular distribution of the fluorescence within the cell body indicated that these receptors were clustered within a discrete cytoplasmic domain, perhaps representing the ER. The binding of BODIPY-ryanodine was time dependent and specific, in that it was displaced by 10-fold excess ryanodine. The additional fact that binding was attained at nanomolar concentrations of BODIPY-ryanodine, which were comparable to its IC50 value for skeletal muscle receptors (100 nM), is consistent with a high-affinity binding site. Thus these experiments provide a second line of evidence that demonstrates the location of RyR in parotid acinar cells.

In addition, molecular techniques were employed to identify the type of RyR present in parotid cells. Using appropriate RyR primers, we succeeded in demonstrating the expression of an RyR in rat acinar cells that is highly homologous to a rat skeletal muscle RyR1 (98%), novel mouse brain RyR1 (94%), and rabbit skeletal muscle RyR1 (88%). A brain RyR type has previously been identified in human Jurkat T lymphocytes, which, like the RyR of the rat parotid, is ryanodine sensitive and caffeine insensitive (10). In examining the DNA sequences of rat types 1 and 2 RyR; mouse types 1, 2, and 3 RyR; novel brain type 1 RyR; and rabbit types 1, 2, and 3 RyR, it is of interest to note that the most highly conserved region is in the COOH terminus, which is the Ca2+-binding domain. Thus the RyR channel of rat parotid cells exhibits properties that are similar to those previously described in excitable and nonexcitable cells.

Our earlier work utilizing intact parotid cells revealed that the dual activation of the cAMP- and Ca2+-phosphoinositide pathways by norepinephrine, the physiological neurotransmitter, is associated with an elevated Ca2+ response and augmented amylase secretion (19). The present and a previous study (23) utilized permeabilized cells to define the nature and biochemical basis of the coupling between cAMP- and IP3-mediated Ca2+ release. On the basis of these findings, we conclude that parotid cells possess a minimum of two types of intracellular Ca2+ channels. One channel type is activated by IP3; the other, which comprises the RyR, is regulated by ryanodine, adenine nucleotides, cyclic nucleotides, and CaM. Furthermore, regenerative increases in intracellular Ca2+ during agonist-induced IP3 generation may be mediated by RyR-mediated CICR or a modified form of this process. The identification of a second basic process involved in intracellular Ca2+ release in parotid acinar cells presages new insights into what appears to be a highly regulated system for Ca2+ signaling.

    ACKNOWLEDGEMENTS

We are grateful to Dr. Robert Summers (Dept. of Anatomical Sciences and Cell Biology, University of Buffalo) for expert assistance and advice in conducting the experiments employing confocal microscopy.

    FOOTNOTES

This work was funded by National Institute of Dental Research Grant DE-059654 and by the Showalter Trust.

Address for reprint requests: R. P. Rubin, Dept. of Pharmacology and Toxicology, School of Medicine and Biomedical Sciences, State University of New York at Buffalo, 102 Farber Hall, Buffalo, NY 14214.

Received 17 December 1996; accepted in final form 21 May 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Ayabe, T., G. S. Kopf, and R. M. Schultz. Regulation of mouse egg activation: presence of ryanodine receptors and effects of microinjected ryanodine and cyclic ADP ribose on uninseminated and inseminated eggs. Development 121: 2233-2244, 1995[Abstract/Free Full Text].

2.   Bennett, D. L., T. R. Cheek, M. J. Berridge, H. DeSmedt, J. B. Parys, L. Missiaen, and M. D. Bootman. Expression and function of ryanodine receptors in nonexcitable cells. J. Biol. Chem. 271: 6356-6362, 1996[Abstract/Free Full Text].

3.   Berridge, M. J. Inositol trisphosphate and calcium signalling. Nature 361: 315-325, 1993[Medline].

4.   Campos Gonzales, R., J. F. Whitfield, A. L. Boynton, J. P. MacManus, and R. H. Rixon. Prereplicative changes in soluble calmodulin of isoproterenol-activated rat parotid glands. J. Cell. Physiol. 118: 257-261, 1984[Medline].

5.   Coronado, R., J. Morrissette, M. Sukhareva, and D. M. Vaughan. Structure and function of ryanodine receptors. Am. J. Physiol. 266 (Cell Physiol. 35): C1485-C1504, 1994[Abstract/Free Full Text].

6.   Dreux, C., V. Imhoff, C. Huleux, S. Busson, and B. Rossignol. Forskolin, a tool for rat parotid secretion studies: 45Ca efflux is not related to cAMP. Am. J. Physiol. 251 (Cell Physiol. 20): C754-C762, 1986[Abstract/Free Full Text].

7.   Endo, M. Calcium release from the sarcoplasmic reticulum. Physiol. Rev. 57: 71-108, 1977[Free Full Text].

8.   Fabiato, A. Effects of ryanodine in skinned cardiac cells. Federation Proc. 44: 2970-2976, 1985[Medline].

9.   Foskett, J. K., and D. Wong. Free cytoplasmic Ca2+ concentration oscillations in thapsigargin-treated parotid acinar cells are caffeine- and ryanodine-sensitive. J. Biol. Chem. 266: 14535-14538, 1991[Abstract/Free Full Text].

10.   Hakamata, Y., S. Nishimura, J. Nakai, Y. Nakashima, T. Kita, and K. Imoto. Involvement of the brain type of ryanodine receptor in T-cell proliferation. FEBS Lett. 352: 206-210, 1994[Medline].

11.   Humerickhouse, R. A., H. R. Besch, Jr., K. Gerzon, L. Ruest, J. L. Sutko, and J. T. Emmick. Differential activating and deactivating effects of natural ryanodine congeners on the calcium release channel of sarcoplasmic reticulum: evidence for separation of effects at functionally distinct sites. Mol. Pharmacol. 44: 412-421, 1993[Abstract].

12.   Humerickhouse, R. A., K. R. Bidasee, K. Gerzon, J. T. Emmick, S. Kwon, J. L. Sutko, L. Ruest, and H. R. Besch, Jr. High affinity C10-Oeq ester derivatives of ryanodine. Activator-selective agonists of the sarcoplasmic reticulum calcium release channel. J. Biol. Chem. 269: 30243-30253, 1994[Abstract/Free Full Text].

13.   Ikemoto, T., M. Iino, and M. Endo. Effect of calmodulin antagonists on calmodulin-induced biphasic modulation of Ca2+-induced Ca2+ release. Br. J. Pharmacol. 118: 690-694, 1996[Abstract].

14.   Lai, F. A., M. Misra, L. Xy, H. A. Smith, and G. Meissner. The ryanodine receptor-Ca2+ release channel complex of skeletal muscle sarcoplasmic reticulum. J. Biol. Chem. 264: 16776-16785, 1989[Abstract/Free Full Text].

15.   Lee, H.-C. Potentiation of calcium- and caffeine-induced calcium release by cyclic ADP-ribose. J. Biol. Chem. 268: 293-299, 1993[Abstract/Free Full Text].

16.   Lee, H.-C. Modulator and messenger functions of cyclic ADP-ribose in calcium signaling. Recent Prog. Horm. Res. 51: 355-388, 1996[Medline].

17.   Lee, H.-C., R. Aarhus, and R. M. Graeff. Sensitization of calcium-induced calcium release by cyclic ADP-ribose and calmodulin. J. Biol. Chem. 270: 9060-9066, 1995[Abstract/Free Full Text].

18.   Lee, H.-C., A. Galione, and T. F. Walseth. Cyclic ADP-ribose: metabolism and calcium mobilizing function. Vitam. Horm. 48: 199-257, 1994[Medline].

19.   McKinney, J. S., M. S. Desole, and R. P. Rubin. Convergence of cyclic AMP and phosphoinositide pathways during rat parotid secretion. Am. J. Physiol. 257 (Cell Physiol. 26): C651-C657, 1989[Abstract/Free Full Text].

20.   McNulty, T. J., and C. W. Taylor. Caffeine-stimulated Ca2+ release from the intracellular stores of hepatocytes is not mediated by ryanodine receptors. Biochem. J. 291: 799-801, 1993[Medline].

21.   Meissner, G. Ryanodine receptors/Ca2+ release channels and their regulation by endogenous effectors. Annu. Rev. Physiol. 56: 485-508, 1994[Medline].

22.   Meszaros, L., J. Bak, and A. Chu. Cyclic ADP-ribose as an endogenous regulator of the non-skeletal type ryanodine receptor Ca2+ channel. Nature 364: 76-79, 1993[Medline].

23.   Rubin, R. P., and M. A. Adolf. Cyclic AMP regulation of calcium mobilization and amylase release from isolated permeabilized rat parotid cells. J. Pharmacol. Exp. Ther. 268: 600-606, 1994[Abstract].

24.   Shoshan-Barmatz, V. High affinity ryanodine binding sites in rat liver endoplasmic reticulum. FEBS Lett. 263: 317-320, 1990[Medline].

25.   Sitsapesan, R., S. J. McGarry, and A. J. Williams. Cyclic ADP-ribose, the ryanodine receptor and Ca2+ release. Trends Pharmacol. Sci. 16: 386-391, 1995[Medline].

26.   Sitsapesan, R., and A. J. Williams. Mechanisms of caffeine activation of single calcium-release channels of sheep cardiac sarcoplasmic reticulum. J. Physiol. (Lond.) 423: 425-439, 1990[Abstract].

27.   Sorrentino, V., and P. Volpe. Ryanodine receptors, how many, where, and why? Trends Pharmacol. Sci. 14: 98-103, 1993[Medline].

28.   Thorn, P., O. Gerasimenko, and O. H. Petersen. Cyclic ADP-ribose regulation of ryanodine receptors involved in agonist evoked cytosolic Ca2+ oscillations in pancreatic acinar cells. EMBO J. 13: 2038-2043, 1994[Abstract].

29.   Tunwell, R. E. A., and F. A Lai. Ryanodine receptor expression in the kidney and a non-excitable kidney epithelial cell. J. Biol. Chem. 271: 29583-29588, 1996[Abstract/Free Full Text].

30.   Verkhratsky, A., and A. Shmigol. Calcium-induced calcium release in neurones. Cell Calcium 19: 1-14, 1996[Medline].

31.   White, A. M., S. P. Watson, and A. Galione. Cyclic ADP-ribose induced Ca2+ release from rat brain microsomes. FEBS Lett. 318: 259-263, 1993[Medline].

32.   Wibo, M., and T. Godfraind. Comparative localization of inositol 1,4,5-trisphosphate and ryanodine receptors in intestinal smooth muscle: an analytical subfractionation study. Biochem. J. 297: 415-423, 1994[Medline].

33.   Zarka, A., and V. Shoshan-Barmatz. Characterization and photoaffinity labelling of the ATP binding site of the ryanodine receptor from skeletal muscle. Eur. J. Biochem. 213: 147-154, 1993[Abstract].


AJP Cell Physiol 273(4):C1306-C1314
0363-6143/97 $5.00 Copyright © 1997 the American Physiological Society