(Received for publication, October 11, 1996, and in revised form, February 10, 1997)
From the Departments of Oral Biology and
§ Pharmacology, University of Washington, Seattle,
Washington 98195 and the
Department of Molecular
Biosciences, University of California, Davis, California 95616
Immunoblot analysis and [3H]ryanodine binding were used to characterize and identify ryanodine receptors (RyRs) in nonexcitable mouse parotid acini. Western analysis revealed ryanodine receptor type III (Ry3R) to be the only detectable isoform in parotid microsomal membranes. Binding of [3H]ryanodine to microsomal fractions was dependent on Ca2+, salt, pH, and temperature. At 23 °C, and in the presence of 0.5 M KCl and 100 µM Ca2+, [3H]ryanodine bound specifically to membranes with high affinity (Kd = 6 nM); maximum binding capacity (Bmax) was 275 fmol/mg protein. Mg2+ and ruthenium red inhibited [3H]ryanodine binding (IC50 = 1.4 mM and 0.5 µM, respectively). 4-Chloro-3-ethylphenol enhanced the binding of [3H]ryanodine 2.5-fold; whereas ATP and caffeine were much less efficacious toward activating Ry3R (56% and 18% maximal enhancement, respectively). Bastadin, a novel modulator of the 12-kDa FK506 binding protein·RyR complex, increased [3H]ryanodine binding 3-4-fold by enhancing Kd. The immunosuppressant FK506 enhanced [3H]ryanodine receptor occupancy at >100 µM and antagonized the action of bastadin, suggesting that an immunophilin modulates Ry3R in parotid acini. These results suggest that Ry3R may play an important role in Ca2+ homeostasis in mouse parotid acini.
Increases in cellular Ca2+ occur in response to agonist stimulation in many cell types and are important in regulating a number of cellular functions. Two distinct classes of channels that mediate the release of Ca2+ from intracellular stores have been identified: one is sensitive to the second messenger inositol 1,4,5-trisphosphate and the second is sensitive to ryanodine (1). Ryanodine receptors (RyRs)1 were first identified in skeletal and cardiac muscle junctional sarcoplasmic reticulum (SR) using radiolabeled ryanodine binding analysis (2, 3) where they function as Ca2+ release channels during excitation-contraction coupling (4, 5). More recently, the availability of antibodies/antisera selective for the skeletal (Ry1R), cardiac (Ry2R), and brain (Ry3R) isoforms, in conjunction with molecular approaches, have clearly demonstrated expression of RyRs in a variety of nonmuscle tissues (6-8). In addition to expression in skeletal muscle, Ry1R has been shown to be expressed at low levels in cerebral cortex and hippocampus and exhibits especially high abundance in cerebellar Purkinje cells (9). Ry2R is expressed in cardiac tissue and throughout most of the brain, with relatively high levels of expression in the olfactory nerve layer, layer VI of the cerebral cortex, the dentate gyrus, cerebellar granule cells, the motor trigeminal nucleus, and the facial nucleus. The recently identified Ry3R isoform appears to be the major isoform in smooth muscle (10-12) and has also been found to be expressed in several nonexcitable cells, including HeLa and LLC-PKI cells, mink lung cells, and submandibular cells (7, 8). Interestingly, Ry3R was recently found to be generally expressed in mammalian brain at very low levels, raising questions about its precise function in Ca2+ signaling (6).
[3H]Ryanodine binding to receptors isolated from muscle SR and brain microsomes indicates that the RyRs are modulated by a number of physiologic and pharmacologic agents. Activators include micromolar Ca2+, millimolar caffeine and adenine nucleotides, and nanomolar ryanodine. Inhibitors include micromolar ryanodine, nanomolar ruthenium red, and millimolar Mg2+ or Ca2+ (5). In addition, the immunosuppressive agents FK506 and rapamycin, which bind to immunophilins such as FKBP-12 (12-kDa FK506 binding protein) and inhibit the immune response, have been implicated in the regulation of gating properties of the RyR in skeletal muscle (13-15), cardiac muscle (16), and neurons (17). Associated with the Ry1R, FKBP-12 has been shown to stabilize the closed conformation of the Ca2+ release channel. FK506 was found to promote dissociation of FKBP-12 from the Ry1R complex (14) and to alter SR Ca2+ transport and single channel gating behavior (15). The ability of FK506 to dissociate FKBP-12 from SR membranes was shown to be enhanced by macrocyclic bastadins isolated from the marine sponge Ianthella basta (18). Like FK506, bastadins also appear to target FKBP-12. Unlike FK506, bastadins themselves do not dissociate FKBP-12 from the SR calcium release channel complex but instead facilitate FK506-induced dissociation (18).
Although RyRs have been well characterized in skeletal and heart muscle, less is known about RyRs in nonexcitable cells. The inability to observe caffeine-induced Ca2+ release from intracellular stores has been misinterpreted to mean that RyRs are absent from some cell types. Results with caffeine are especially confusing since cells known to express the Ry3R isoform fail to respond to caffeine (10, 19), and various laboratories have reported differences in caffeine sensitivity in the same cell type (19, 20). Much of the evidence supporting the presence of RyRs in nonexcitable cells, including exocrine cells, has been based on functional studies (7, 21-23) with little information concerning expression of RyR protein (7, 11, 24). Furthermore, few studies have either demonstrated or characterized specific ryanodine binding in microsomal fractions from nonexcitable cells (7, 25-27).
The focus of the present study is to identify the expression of RyR protein(s) in nonexcitable mouse parotid acini and to characterize its ability to bind [3H]ryanodine. Western blot analysis of microsomes isolated from primary acini reveals the expression of Ry3R protein without detectable levels of Ry1R or Ry2R protein. The data presented show that high-affinity binding of [3H]ryanodine to Ry3R from parotid acini is modulated in a similar fashion to Ry1R and Ry2R from muscle SR with important differences with respect to modulation by caffeine and adenine nucleotides.
Reagents were obtained as follows. Benzamidine
and 4-chloro-3-ethylphenol (4-CEP) were from Aldrich; purified sucrose,
Silver Stain Plus, and dithiothreitol (DTT) were from Bio-Rad;
caffeine, EDTA, EGTA, HEPES, MOPS, hyaluronidase, pepstatin A,
phenylmethylsulfonyl fluoride (PMSF), leupeptin, adenosine
5-(
,
-methylene)triphosphate (AMP-PCP), TRIS, horseradish
peroxidase-conjugated goat anti-mouse secondary antibody, and ruthenium
red were from Sigma; collagenase was from Worthington; horseradish
peroxidase-conjugated donkey anti-rabbit secondary antibody was from
Amersham Corp.; CHAPS and chemiluminescent detection system were from
Pierce; aprotinin and ATP were from Boehringer Mannheim; radiolabeled
ryanodine [9,21-3H] (68.4-84.0 Ci/mmol) was from DuPont
NEN; purified ryanodine was from Calbiochem (La Jolla, CA) and Wako
(Richmond, VA); Universol scintillant was from ICN (Costa Mesa, CA);
FK506 was from Signal Transduction International (San Diego, CA);
polyvinylidene difluoride (Immobilon-P) membranes were from Millipore
(Bedford, MA); and bastadin (mixture) was from Calbiochem (San Diego,
CA). Aqueous solutions of calcium and magnesium chloride were
calibrated at mM concentration using atomic adsorption
spectroscopy. All other reagents were of analytical grade or higher.
Ry1R antibody (34 °C) was a generous gift of Dr. J. L. Sutko; Ry2R antibody (C3-33) was a generous gift of Dr. G. Meissner; Ry3R antibodies were generous gifts of Dr. G. Meissner and Dr. V. Sorrentino.
Acinar cells were isolated as small aggregates from the parotid glands of male Swiss Webster mice as described by Watson et al. (28). Briefly, parotid glands from male Swiss Webster mice (27-30 g) were removed quickly, trimmed, and minced in a siliconized dish in Krebs-Henseleit bicarbonate solution (KHB), pH 7.4, containing 0.9 mM Mg2+ and 1.28 mM Ca2+, 30 mM Hepes, 90 units/ml collagenase (CLS2), and 1 mg/ml hyaluronidase. Enzyme digestion was conducted in a rotary water bath at 37 °C for 60 min under continuous CO2/02 (5/95%) gassing. After the first 40 min of digestion, the suspension was pipetted up and down twelve times with a 10-ml plastic pipette. This was repeated two more times at approximately 5-min intervals. The pH during the dispersion was maintained at 7.2-7.4. Following digestion, the cells were centrifuged at 50 × g for 2 min, washed with buffer (KHB minus enzymes with 4% BSA, pH 7.4), filtered through two layers of nylon, and washed two additional times. Cells were suspended in the same KHB minus enzyme buffer containing 1% BSA to rest for 30 min at 37 °C with continuous gassing.
Skeletal muscle (tibia anterialis) and whole brain were dissected from the same mice for use as control tissue in some experiments. Both acinar cells and control tissues were processed immediately to obtain membrane fractions.
Microsomal Membrane PreparationMicrosomal membranes were
isolated at 4 °C from parotid acinar cells and brain by
fractionation of a 10% (wet w/v) homogenate of the cells or tissue
using differential centrifugation in isomolar sucrose as described
below. Cells were suspended in 250 mM sucrose, 10 mM HEPES, 1 mM EDTA, buffer (pH 7.4) containing
1 mM DTT, and the protease inhibitors of benzamidine (1 mM), leupeptin (1 µg/ml), pepstatin A (0.7 µg/ml), and
PMSF (0.1 mM), and homogenized in a glass mortar using a
motor-driven Teflon pestle and 10 complete passes. The homogenate was
centrifuged for 5 min at 250 × g. Homogenization of
the pellet was repeated, and the pooled 250 × g
supernatants were centrifuged for 20 min at 10,000 × g. The 10,000 × g supernatant was
centrifuged for 1 h at 100,000 × g, and the
resulting microsomal fraction (pellet) was separated from the soluble
fraction (supernatant), held overnight submerged in suspension buffer
(200 mM sucrose, 50 mM KCl, 20 mM
MOPS buffer (pH 6.8) containing the protease inhibitors) at 4 °C,
and resuspended the next day at a protein concentration of 5-11 mg/ml
by homogenization in the same buffer for immediate use or storage at
80 °C. A total membrane fraction of skeletal muscle was prepared
in homogenate medium without CHAPS according to Damiani and Margreth
(29). EGTA and EDTA, each at 2 mM concentration, were
substituted for 1 mM EDTA in homogenization buffer for the
preparation of microsomes in some experiments.
SR membranes used in Western blots as positive controls were isolated from rabbit fast skeletal muscle according to Saito, et. al. (30), rat cardiac muscle according to Feher and Davis (31), and avian pectoralis muscle according to Airey, et. al. (32).
[3H]Ryanodine Binding AssayEquilibrium saturation experiments for [3H]ryanodine binding to mouse parotid acinar cell, brain and skeletal muscle membranes were performed by titrating the radioligand concentration between 0.1 and 30 nM at constant specific activity in a final assay volume of 250 µl. Unless otherwise indicated, the binding media consisted of 0.5 M KCl, 100 µM CaCl2, 20 mM HEPES, pH 7.4, and inhibitors aprotinin (0.5 mg/ml), benzamidine (1 mM), leupeptin (1 µg/ml), pepstatin A (0.7 µg/ml), and PMSF (0.1 mM) with the protease substrate BSA (100 µg/ml) or 1 mM DTT. EC50 and IC50 values for modulators were obtained from concentration-response experiments of equilibrium binding of [3H]ryanodine added at a nonsaturating concentration of 6 nM. Modulators of [3H]ryanodine binding were added in equilibrium saturation experiments at a concentration that produced half-maximal response as determined above using a nonsaturating concentration of [3H]ryanodine. The tissue samples were incubated in binding media with or without modulators at 23 ± 0.5 °C for 16-22 h. The assay was terminated by rapid dilution of sample in 4 ml of wash buffer containing 0.5 M KCl, 20 mM HEPES, pH 7.4, 100 µM Ca2+ and by passage of sample through a Whatman GF/F glass fiber filter followed immediately by 3 × 4 ml washes of filter with the same buffer; all procedures were completed within 1 min. The filters were dried overnight and placed in vials containing scintillant, and the bound [3H]ryanodine was measured by liquid scintillation counting using a Packard Tri-Carb 2200CA analyzer. Specific bound [3H]ryanodine was calculated by subtracting nonspecific binding, measured for parallel assays in the presence of 10 µM unlabeled ryanodine, from the total bound. For acinar cell microsomal membranes, nonspecific binding averaged 81 and 57% at 0.3 and 20 nM, respectively, and was linear with the concentration of [3H]ryanodine. Free Ca2+ effects were obtained by titrating CaCl2 in binding media containing 1 mM EGTA using theoretical estimates derived from the computer program BAD3 (33). [3H]Ryanodine binding to proteins in the soluble fraction of acinar cells was determined by the PEG precipitation method as described by Shoshan-Barmatz and Zarka (34). Protein was determined by the Hartree (35) modified method of Lowry (36).
Association and Dissociation Kinetics for [3H]Ryanodine BindingFor association kinetics, acinar cell microsomal membrane (0.4 mg of protein/ml) was incubated with 5 nM [3H]ryanodine in binding media with and without 10 µM cold ryanodine at 23 and 37 °C. At time intervals ranging between 10 min and 22 h, the reaction was terminated by rapid filtration as described above.
For dissociation kinetics, acinar cell microsomal membrane (0.4 mg of protein/ml) was incubated at 23 °C with 5 nM [3H]ryanodine in binding media with and without 10 µM cold ryanodine for 16 h until association was completed. Dissociation was initiated by diluting duplicate aliquots of each treatment group by 50-fold using binding media without ryanodine. Dissociation was terminated by filtering aliquots (250 µl) at the indicated intervals of time up to 20 h, and the bound radioactivity was assayed as above.
Analysis of Binding DataEC50 and IC50 values for activators and inhibitors of [3H]ryanodine binding, respectively, were determined by linear analysis of log-logit transformation of concentration response curves. The equilibrium binding constants, Kd and Bmax, for [3H]ryanodine with and without effectors were derived by curvilinear analysis using the computer program RADLIG sub-routine EBDA (Elsevier-BIOSOFT) and depicted graphically using a Rosenthal (Scatchard) plot and linear analysis. Kinetic association and dissociation rate constants were calculated using sub-routine KINETIC of RADLIG. Mean treatment differences of effectors from control were tested for significance using the student t statistic for paired observations at p < 0.05. Values reported represent the mean ± S.E. of (N) number of independent experiments performed in duplicate unless otherwise noted.
Electrophoreses and Western Blot AnalysisConstituent proteins from membrane preparations were resolved on 3-10% gradient gels by the method of Laemmli (37). Proteins were electroblotted overnight at 30 V followed by a 60-min fast transfer at 100 V. Nonspecific antibody binding was blocked by incubating blots for 1 h at 37 °C in TTBS solution (20 mM Tris-HCl, 0.5 M NaCl, 0.5% Tween-20, pH 7.5) with the addition of 5% nonfat dry milk. Specific binding of primary antibody was performed by incubating blots for 1 h at 37 °C in TTBS buffer in the presence of 1% BSA and antibody. Resulting immunoblots were labeled with horseradish peroxidase-conjugated goat anti-mouse or donkey anti-rabbit secondary antibody for 1 h at 37 °C and then visualized using an enhanced chemiluminescence technique. Nonspecific binding of each secondary antibody to each membrane preparation was minimized by performing a dilution series in the absence of primary antibody.
For the results presented herein, microsomal membranes were prepared from isolated mouse parotid acini. The enzymatic digestion method of isolation, which has been widely used for the preparation of rat and mouse salivary cells, yielded a preparation containing 95% acinar and 5% ductal cells. The percent composition of ductal cells is consistent with earlier quantitative estimations of ductal cell volume observed in parotid tissue sections, i.e. 7.7% (38), and with visual estimates in isolated parotid cells (39-41).
Western blot analysis of microsomal membranes isolated from rat parotid
acinar cells revealed no detectable levels of specific immunoreactive
skeletal (Ry1R) or cardiac (Ry2R) protein even when the blots were visualized with the highly sensitive
chemiluminescent technique. Two antibodies selective for the
"brain" (Ry3R) isoform of the ryanodine receptor
detected a high molecular weight protein in several microsomal
preparations (n = 4 determinations; Fig. 1). The immunoreactive band migrated at a slightly
smaller apparent molecular weight than the Ry1R protomer
found in rabbit skeletal muscle.
Assay conditions of Ca2+ and KCl concentrations, pH, and
temperature were evaluated 1) for their effect on
[3H]ryanodine binding to acinar cell membranes and 2) to
select assay media conditions to test other known effectors of
[3H]ryanodine binding to the RyR. An optimal range of
free Ca2+ was required for the specific equilibrium binding
of 6 nM [3H]ryanodine to membranes in assay
media, pH 7.4, containing 0.5 M KCl at 23 °C. Specific
[3H]ryanodine binding was near detection limits in the
presence of 1 mM EGTA (8 ± 5 fmol/mg of protein,
n = 3) compared with controls in the presence of 100 µM CaCl2 without added EGTA (123 ± 10 fmol/mg of protein, n = 13). Titration of
CaCl2 in the presence of 1 mM EGTA revealed a
biphasic dependence of specific [3H]ryanodine equilibrium
occupancy and Ca2+ with a threshold of approximately 10 nM and an optimum between 10 and 100 µM
Ca2+ (Fig. 2A). The apparent
IC50 for Ca2+ was approximately 2 µM, and 10 mM Ca2+ inhibited
binding by 86%.
When KCl or NaCl were removed from the binding media containing optimum Ca2+ (100 µM), specific equilibrium binding of 6 nM [3H]ryanodine was not detectable (Fig. 2B). [3H]Ryanodine binding increased proportionately with increase in concentration of KCl to 0.5 M, reached its maximum at 0.75 M KCl, and showed little or no change with increase in KCl concentration to 1 M. NaCl, at molar equivalence up to 1 M concentration, partially replaced KCl in effectiveness. Sucrose in molar equivalent to KCl or NaCl did not significantly stimulate [3H]ryanodine binding at concentrations <1 M.
Equilbrium binding of 6 nM [3H]ryanodine was also biphasic with respect to pH. When assay pH was buffered with HEPES between 7 and 8, ryanodine binding was between 70 and 90%, respectively, of its maximum at pH 7.6 (Fig. 2C). Test of pH effects below 7 (MOPS) and above 8 (TRIS) showed binding to decrease steeply to 20% of maximum at pH 6.2 and to be relatively unaffected in alkaline solution, with pH 8.8 giving 87% of maximum binding (data not shown).
Assay temperature dramatically influenced the time course and magnitude
of [3H]ryanodine binding (Fig. 2D). At
37 °C in binding media (pH 7.4) containing 100 µM
Ca2+ and 0.5 M KCl, maximum binding of 5 nM [3H]ryanodine occurred within 2 h
with an association half-time of 45 min. Thereafter, total binding
declined to approximately 50% by 4 h and to negligible levels by
22 h. At 23 °C, the rate of [3H]ryanodine (5 nM) association was significantly slower (half-time = 100 min; kobs = 0.0069 min1) but
reached a 2-fold higher level of occupancy that remained stable for at
least 22 h (Fig. 2D). After equilibrating the membranes for 16 h and subsequent to a 50-fold dilution with binding media lacking ryanodine, the radioligand dissociated with a half-time = 595 min (k-1 = 0.0012 min
1). The
time courses of association (Fig. 2D) and dissociation (Fig.
3) of 5 nM [3H]ryanodine at
23 °C were both best described by a mono-exponential equation. The
association rate constant calculated according to the relationship
k+1 = kobs
k-1/[L] was 1.16 × 106
min
1 M
1. The equilibrium
dissociation constant calculated based on Kd = k-1/k+1 was 1 nM.
A standard assay condition that includes binding media (pH 7.4) with 100 µM Ca2+, 0.5 M KCl and temperature at 23 °C was used to characterize equilibrium binding of [3H]ryanodine to mouse parotid acinar cell membranes. The distribution of specific binding of 6 nM [3H]ryanodine among acinar subcellular fractions was compared for two experiments. [3H]Ryanodine binding to the 10,000-100,000 × g particulate (microsomal) fraction comprised 26% of the total acinar cell specific binding. Percent distribution of [3H]ryanodine binding to the 250 × g and 250-10,000 × g particulate (putative nuclear and mitochondrial) fractions averaged 17 and 57% of the total, respectively. The ratio of specific binding activity for these fractions relative to the microsomal fraction (relative specific binding activity = 1) averaged 0.92 and 0.86. No binding was detectable in the 100,000 × g soluble (cytosolic) fraction.
Overall, [3H]ryanodine equilibrium binding in the
standard assay media at 22 h averaged 132 ± 10 fmol/mg of
protein for 27 preparations of microsomal membrane; the concentration
of added [3H]ryanodine averaged 6.2 ± 0.06 nM. [3H]Ryanodine (6 nM) binding
was a linear function of protein concentration from 0.1 to 1.0 mg of
protein/ml (Fig. 4); assay concentrations of membrane
used in this study were within this range. Since this is the first
report of RyR in parotid acini, we chose to illustrate that the amount
of specific binding is directly proportional to the amount of acini
receptor in the assay.
As shown in Fig. 5A for three independent
preparations of microsomes assayed in standard media at 23 °C,
specific binding of [3H]ryanodine approached saturation
near 20 nM. Nonspecific binding was linear with increasing
concentration of radionuclide but approached 45% of total binding at
the higher concentrations of [3H]ryanodine. Nonlinear
regression analysis of equilibrium binding curves reveals the data is
best fit statistically by a mathematical model describing
[3H]ryanodine binding to a single site and results in a
linear Scatchard plot (Fig. 5B). The apparent
Kd was 6.2 ± 0.55 nM
(n = 3), and maximum occupancy
(Bmax) was 275 ± 22 fmol/mg of protein (n = 3).
In Table I, [3H]ryanodine saturation binding to mouse parotid acinar microsomal membranes is compared with binding parameters measured with membrane preparations from mouse fast twitch skeletal muscle and whole brain tissues. Brain and parotid preparations differed from those derived from skeletal muscle in having a lower percentage of specific bound radioligand and greater than 10-fold less binding sites/mg of membrane protein. Brain had the highest affinity for [3H]ryanodine, having approximately 10-fold higher binding affinity than either parotid or muscle membranes.
|
In addition to Ca2+, other compounds reported to modulate
the ryanodine-sensitive release of Ca2+ from muscle SR
(caffeine, adenine nucleotide, ruthenium red, and MgCl2),
and from the endoplasmic reticulum of nonexcitable cells (4-CEP) were
tested for their influence on [3H]ryanodine binding to
acinar cell microsomes. Typical concentration-response curves of
activators and inhibitors of specific [3H]ryanodine
binding determined at equilibrium with a non-saturating concentration
of 6 nM [3H]ryanodine are shown in Fig.
6, A and B, respectively.
Concentration estimates of effectors producing half-maximal activation
(EC50) or inhibition (IC50) are summarized in
Table II. In the presence of optimal Ca2+,
AMP-PCP (2 mM) maximally enhanced
[3H]ryanodine occupancy by 40% and exhibited an
EC50 of 1 mM. Caffeine (3-10 mM)
enhanced occupancy by 25% although the activation was too small to
calculate an EC50. In marked contrast, 200-600
µM 4-CEP enhanced [3H]ryanodine receptor
occupancy 2.5-fold, exhibiting an activation threshold of approximately
10 µM and an EC50 of 50 µM
(Fig. 6A). The activating action of 4-CEP on receptor
binding was sharply biphasic with 1 mM producing a 24%
reduction of control occupancy. Ruthenium red was 50-fold less potent
than unlabeled ryanodine toward competing with 6 nM
[3H]ryanodine (IC50 of 10 and 500 nM, respectively); whereas Mg2+ was inhibitory
at a concentration above physiological relevance (>1 mM).
Table II summarizes EC50 and IC50 values for
modulators of RyR in microsomes isolated from acinar cells.
|
The effects of caffeine and AMP-PCP in the absence and presence of an inhibitory concentration of MgCl2 are given in Table III. In the presence of 100 µM Ca2+, increases in [3H]ryanodine binding of 18 and 56% above controls were measured at 10 mM caffeine and 1 mM AMP-PCP, respectively. In the presence of 100 µM Ca2+, 1 mM Mg2+ reduced [3H]ryanodine binding by 40% and 10 mM caffeine could not fully restore occupancy to control levels. In contrast, 1 mM AMP-PCP in the presence of equimolar Mg2+ nearly restored occupancy to that seen with 1 mM AMP-PCP alone, revealing that the Mg·AMP-PCP complex maintains activating properties toward RyR.
|
Equilibrium saturation binding of [3H]ryanodine to acinar microsomes was examined in the presence and absence of modulators in binding media containing 100 µM Ca2+ and 0.5 M KCl, which was found to be optimal for the binding of [3H]ryanodine to acinar microsomes (Table IV, Fig. 7). At a concentration shown to enhance occupancy by 50%, 4-CEP decreased the value of Kd (i.e. enhanced the apparent affinity of [3H]ryanodine for its binding site) nearly 4-fold and produced a small but significant reduction in Bmax. Interestingly, the macrocyclic bromotyrosine bastadins (20 µM) significantly enhanced [3H]ryanodine binding site affinity (4-fold) without a significant change in Bmax. Ruthenium red and Mg2+ significantly reduced [3H]ryanodine binding affinity (Kd from 6 to 8.5 and 10 nM, respectively) and lowered Bmax by 42% and 18%, respectively. The effects of Mg2+ on Kd were fully restored to the control value by caffeine, and the Mg·AMP-PCP complex enhanced Kd nearly 3-fold (Table IV). Although caffeine did not significantly reverse the actions of Mg2+ on Bmax, the Mg·AMP-PCP complex enhanced Bmax by 41%.
|
The immunosuppressive macrolactam, FK506, and bastadins have been shown
to interact with the FKBP-12·Ry1 receptor complex found
in skeletal muscle (18). These compounds were used to probe the
possible involvement of immunophilins toward modulating [3H]ryanodine binding sites in acinar cell microsomes.
The effect of dimethyl sulfoxide, used as a vehicle in studies with
FK506 and bastadins, was examined and found to inhibit the equilibrium binding of 6 nM [3H]ryanodine by 9% and 37%
at final concentrations of 1% and 5% (v/v), respectively. Bastadin
enhanced [3H]ryanodine receptor occupancy in a
concentration-dependent manner with maximum stimulation of
>4-fold over dimethyl sulfoxide control observed between 100-200
µM when equilibrium binding was determined at the
nonsaturating concentration of 6 nM
[3H]ryanodine; whereas, FK506 was much less efficacious,
producing a 2.0-fold increase in occupancy at 300 µM
(Fig. 8). The EC50 for bastadin was 20 µM. Interestingly, FK506 negated the remarkable activity
of bastadin toward activating [3H]ryanodine occupancy,
resulting in a concentration-response relationship very similar to that
seen with FK506 alone (Fig. 9).
In the present study, RyRs were characterized and identified in mouse parotid acinar cells by analysis of [3H]ryanodine binding and immunoblot analysis using antibodies selective toward the three known isoforms. Western analysis revealed that the major, perhaps only, RyR isoform expressed in mouse parotid acini was the so-called brain isoform, Ry3R. Data show that cells expressing Ry3R possess very low levels of Ry3R protein. Data also showed saturable, high affinity [3H]ryanodine binding in subcellular parotid membrane fractions that was Ca2+-, salt-, pH-, and temperature-dependent. Both kinetic and equilibrium binding studies with the microsomal membrane fraction revealed that [3H]ryanodine interacts with a single population of high affinity binding sites with Kd calculated between 1 and 7 nM in equilibrium and kinetic measurements in the standard assay media employed. Attempts to demonstrate different allosteric binding conformations of low and high binding affinities by using higher concentrations of [3H]ryanodine (>30 nM) produced unacceptably high nonspecific binding (42). Given that parotid cells are composed of approximately 95% acinar cells, it is not likely that the specific [3H]ryanodine-binding sites or the immunoreactive protein toward Ry3R antibody originates from a contaminating cell other than acini. The contaminating cells would have to express very high levels of protein to account for the measurements made.
In general, the effects of temperature (42-45), alkaline pH (46-49), ions (species specific, Ca2+) (2, 44, 50, 51), and monovalent ions (46-48, 51, 52) on [3H]ryanodine binding reported here using parotid acinar cells were similar to those observed in excitable cells. [3H]Ryanodine binding showed a near absolute requirement for certain monovalent ions (K+ and Na+), even in the presence of optimum Ca2+. Since NaCl, at least partially, substituted for KCl, but sucrose, at a concentration close to physiologic molar equivalents, did not, the increase in [3H]ryanodine binding at 25 °C appears to be dependent on ionic strength as reported for smooth muscle (47), and not on osmolarity of the binding media, as reported for bullfrog skeletal muscle (53). There appears to be a temperature- and concentration-dependent monovalent ion effect on [3H]ryanodine binding with tissue-specific response. Concentrations of KCl and/or NaCl above 0.25 M are activating at 25 °C in rabbit skeletal muscle (51), bullfrog skeletal muscle (53), smooth muscle (47), and, as reported here, in acinar cells. At 37 °C, concentrations of KCl and/or NaCl are activating in rat heart muscle, but inhibitory in rabbit skeletal muscle sarcoplasmic reticulum (52). The higher sensitivity of the receptor for K+ over Na+ in parotid acinar cells, observed with less and varying magnitude in other cell types (47, 52), suggests that the larger size of the K+ cation may be a factor distinguishing the activation efficiency of monovalent ions.
The pharmacology of 1) inhibition by competing cold ryanodine (47, 49, 54, 55), ruthenium red (49, 51, 52), and MgCl2 (46, 49, 51, 54), and 2) activation by AMP-PCP (45, 46, 49, 52, 54) and caffeine in excitable cells (47, 49, 52, 54, 56) was qualitatively similar to that found in mouse parotid acinar cells. Marked differences in binding assay conditions used by investigators preclude direct quantitative comparisons of the magnitude and potency of each effector in the parotid acinar cell with other cell types. The very low efficacy of caffeine and AMP-PCP toward enhancing [3H]ryanodine occupancy may reflect an inherently low sensitivity of the Ry3R complex to these agents compared with Ry1R and Ry2R isoforms expressed in striated muscle and is consistent with a lack of caffeine responsiveness of Ry3R in other cell types (10, 19). Deletion of Ry1R expression using gene targeting has revealed a small response to caffeine (25 mM) and is maintained in dyspedic myotubes, which has been attributed to expression of Ry3R (57, 58). However, this interpretation is based solely on the presence of Ry3R mRNA in selected dyspedic myotubes (57). Since the presence of Ry3R protein has not been positively identified in dyspedic muscle, this does not preclude the possibility that in the absence of Ry1R, other effects of caffeine on the Ca2+ permeability of plasma membrane and mitochondria become apparent. However, the small effects of caffeine and AMP-PCP seen in acinar microsomes to partially and completely overcome MgCl2 inhibition of [3H]ryanodine binding, respectively, are consistent with the proposed roles of Ca2+, ATP, and Mg2+ to modulate Ca2+-induced Ca2+ release via the ryanodine-sensitive calcium channel (59, 60).
4-Chloro-phenolic compounds have been shown to release Ca2+ from ruthenium red-sensitive intracellular stores of both excitable (61, 62) and nonexcitable cells (63). We sought to verify that 4-CEP specifically interacts with the ryanodine-sensitive calcium release channel of nonexcitable cells (63) by testing for 4-CEP-enhanced [3H]ryanodine binding to mouse parotid acinar cell membranes. Our results show 4-CEP to increase [3H]ryanodine binding to parotid acinar cell membranes 2.5-fold. This supports the reported role of 4-CEP in elevating levels of cytosolic Ca2+ in nonexcitable cells by releasing Ca2+ from intracellular pools via a ruthenium red inhibitable pathway (63). The observed increase in the affinity of the acinar cell receptor for [3H]ryanodine in the presence of 4-CEP suggests that it either l) increases the rate of ryanodine association or 2) decreases the rate of dissociation. 4-CEP inhibition of [3H]ryanodine binding, which occurred at a concentration of 1 mM, may have resulted from a loss of high affinity binding sites since 4-CEP, at a concentration producing half-maximal activation, appeared to reduce the number of binding sites in the acinar cell membranes. Recent findings of Herrmann-Frank et al. (62) have shown, in excitable cells, that 4-Cl-3-methylphenol, an analog of 4-CEP, also activates [3H]ryanodine binding in SR membranes isolated from rabbit skeletal muscle. The EC50 for 4-C1-3-methylphenol equaled 112 µM; activation was monophasic with no inhibition of [3H]ryanodine binding at the highest concentration tested (l mM). They also reported 4-Cl-3-methylphenol to increase receptor affinity for [3H]ryanodine without altering binding site number. Differences observed between muscle and acinar cell in potency (112 versus 54 µM, respectively) and inhibitory properties of these 4-chloro-phenolic compounds may reflect differences in phenol group substitution, sensitivity of RyR isoforms (1 versus 3), or in binding assay conditions (62).
The known association of rabbit FKBP-12 with skeletal muscle Ry1R (13, 14) and its modulator role in binding to the Ry1R (14, 15), coupled with the potential for expression of FKBP-12 in mouse parotid acinar cells (64), prompted us to test for immunophilin modulation of RyR function in mouse parotid acinar cells. FKBP-12 is the primary target for the immunosuppressant FK506 (65, 66) that, in binding to FKBP-12, dissociates the immunophilin from skeletal muscle (14). We found that FK506 produced a concentration-dependent increase in [3H]ryanodine binding to acinar cell membranes, suggesting that a change in the open state of the RyR had occurred, presumably through an FK506-induced release of a FKBP-l2. Bastadin, which enhances FK506-induced release of FKBP-12 from the ryanodine membrane complex (18), was also found to increase [3H]ryanodine binding in acinar cell membranes in a concentration-dependent manner. The EC50 value determined in acinar cell membranes for the bastadin mixture was 20 µM. Mack et al. (18) found, for skeletal muscle, that EC50 values for bastadin 5 (2.2 µM) and bastadin 7 (6.3 µM), which are part of the bastadin mixture, were approximate1y 10- and 3-fold more potent, respectively, than the bastadin used in the present study. Saturating levels of bastadin mixture produced a 2-4-fold higher binding level in acinar cell membranes compared with a 40-50-fold higher binding in skeletal muscle sarcoplasmic reticulum. Differences in the amount of KCl (140 versus 500 mM) in the assay medium may underlie the apparent differences in efficacy of the two preparations. In parotid acinar cells, and as shown by Mack et al. (18) in skeletal muscle, bastadin was more effective in enhancing [3H]ryanodine binding than FK506. Equilibrium [3H]ryanodine saturation binding studies showed bastadin to increase RyR affinity for [3H]ryanodine in parotid acinar cell membranes comparable to bastadin-induced increases (approximately 3-fold) in ryanodine affinity in skeletal muscle (18). However, unlike skeletal muscle, an increase in maximum binding capacity was not detected in mouse parotid acinar cells. This difference also appears to be related to the assay conditions (high versus physiological salt levels and 23 °C versus 37 °C.2
FK506 inhibited bastadin-augmented [3H]ryanodine binding in acinar cells analogous to that observed in skeletal muscle, indicating a similar mechanism for FK506 and bastadin toward modulation of [3H]ryanodine binding sites. Increased [3H]ryanodine binding in the presence of FK506 and bastadin, as well as FK506 antagonism of bastadin-enhanced binding in mouse parotid acinar cell microsomal membranes, provide evidence that an immunophilin modulates receptor binding of exogenous ryanodine. Therefore, an immunophilin may modulate Ry3R function in mouse parotid acinar cells presumably through regulation of channel gating behavior. It is of interest to note that both the macrocyclic bromotyrosine bastadins and the chloroderivative of either methyl or ethylphenol share a chemical ring structure with a halogen substitution, providing perhaps some part of the modulator role these agents have in enhancing [3H]ryanodine binding to the RyR.
To compare the effects of modulators of [3H]ryanodine binding in the parotid acinar cell to those of other cell types, including both excitable and non-excitable types, requires critical evaluation of the effects of different assay conditions used by investigators to measure binding of [3H]ryanodine. The magnitude of modulator response and potency, ryanodine binding affinity, and maximal binding site number for RyRs are all influenced by the selection of binding media, salt, and concentration, as well as temperature. The assay conditions we used to obtain half-maximal binding capacity and to describe the effects of modulators on [3H]ryanodine binding over time and at equilibrium were performed at 5 and 6 nM [3H]ryanodine concentration, respectively (Kd approximated 6 nM derived from equilibrium [3H]ryanodine saturation binding experiments). Modulator concentrations approximated their EC50 or IC50 values in all equilibrium [3H]ryanodine saturation binding experiments. Within these limits, we used assay conditions that produced the highest level of [3H]ryanodine binding, i.e. 1) a monovalent ion concentration of 0.5 M and 2) a temperature of 23 °C. Salt concentrations of 0.5 and 1 M, although commonly used to obtain optimum binding (51, 67, 68), are nonphysiological. Lowering the salt concentration from 1 M to near physiological levels (200 mM) has been found to result in marked changes in the extent to which MgCl2, caffeine, and AMP-PCP affect binding properties of receptor for [3H]ryanodine (44, 54). The sensitivity of the RyR to activation by Ca2+ has also been reported to be influenced by salt concentration of binding media (6, 54).
The association of [3H]ryanodine with its high affinity site is highly dependent on temperature (Q10 > 3) (42, 45). By lowering the assay temperature from 37 to 23 °C, as reported here for acinar cell microsomes or by Carroll et al. (42) in skeletal muscle sarcoplasmic reticulum, the binding occupancy of [3H]ryanodine at equilibrium was increased, implying a temperature-dependent conformational change of the receptor occurred that favored [3H]ryanodine binding with a decrease in temperature. The observation that bastadin (mixture) did not increase Bmax in parotid acinar cells, but did in skeletal muscle (18) under similar binding conditions but different assay temperatures, suggests that the reported effect of bastadin to stabilize the high affinity binding site conformation at 37 °C may be mimicked by lowering the binding assay temperature.
A significant finding of the present study was the expression of the
Ry3R isoform in mouse parotid cells. Ry3R has
also been identified in other nonexcitable cells including HeLa,
LLC-PK1, and mink lung epithelial cells (7, 10). However, this is the
first biochemical characterization of Ry3R in the absence of detectable levels of isoforms Ry1R and Ry2R.
Thus far, the function of RyRs in nonexcitable cells is unclear.
Demonstration of a Ca2+-induced Ca2+ release
process (CICR) has been hampered by the fact that caffeine, commonly
employed as an activator of CICR, has been found to produce little or
no effect on [Cai] in some cells, including mouse parotid acini.3 In cell types expressing
Ry3R, the response to caffeine has been controversial.
Caffeine did not release Ca2+ from intracellular stores of
transforming growth factor -treated mink epithelial cells or Jurkat
T-cells (10, 19). The reason(s) for the lack of or small effects of
caffeine on [Cai] in cells known to express RyRs is
unclear. One explanation given was that caffeine insensitivity is due
to the expression of low levels of RyR. This may be the case for HeLa
cells where [3H]ryanodine binding was 17-fold less than
in rabbit brain (7). In mouse parotid cells, however,
[3H]ryanodine binding capacity was comparable with mouse
brain (54, 69). Another possibility is that lack of caffeine
sensitivity may be related to the species of animal used. Caffeine has
been shown to increase [Cai] in rat parotid and
pancreatic acinar cells (21, 22) but not in mouse parotid cells.
Similarly, endothelial cells from bovine and rabbit were also found to
differ in their responses to caffeine. Caffeine did not produce a
marked release of Ca2+ from intracellular stores in bovine
endothelial cells but was effective in rabbit aortic endothelial cells
(70).
In summary, the expression and characterization of Ry3R in mouse parotid acini supports the conclusion that this receptor subtype contributes to the changes in intracellular Ca2+ following cell stimulation. Although in mutant mice lacking Ry3R where results do not suggest an involvement of Ca2+ mobilization via Ry3R in lymphocyte proliferation, mutant mice did show increased locomotor activity that may indicate abnormal Ca2+ signaling of certain neurons (71). In nonexcitable mouse parotid cells, Ry3R may be involved in the cross-talk that occurs between the Ca2+ and cAMP pathways, i.e. Ca2+ regulation of cAMP synthesis3 suggests that an RyR plays a role in muscarinic augmentation of stimulated cAMP accumulation reported previously by Watson et al. (28).