Laboratoire de Physiologie Cellulaire et Pharmacologie Moléculaire, Centre National de la Recherche Scientifique Enseignement Supérieur Associé 5017, Université de Bordeaux II, 33076 Bordeaux Cedex, France
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ABSTRACT |
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In rat portal vein myocytes, Ca2+ signals can be generated by inositol 1,4,5-trisphosphate (InsP3)- and ryanodine-sensitive Ca2+ release channels, which are located on the same intracellular store. Using a laser scanning confocal microscope associated with the patch-clamp technique, we showed that propagated Ca2+ waves evoked by norepinephrine (in the continuous presence of oxodipine) were completely blocked after internal application of an anti-InsP3 receptor antibody. These propagated Ca2+ waves were also reduced by ~50% and transformed in homogenous Ca2+ responses after application of an anti-ryanodine receptor antibody or ryanodine. All-or-none Ca2+ waves obtained with increasing concentrations of norepinephrine were transformed in a dose-response relationship with a Hill coefficient close to unity after ryanodine receptor inhibition. Similar effects of the ryanodine receptor inhibition were observed on the norepinephrine- and ACh-induced Ca2+ responses in non-voltage-clamped portal vein and duodenal myocytes and on the norepinephrine-induced contraction. Taken together, these results show that ryanodine-sensitive Ca2+ release channels are responsible for the fast propagation of Ca2+ responses evoked by various neurotransmitters producing InsP3 in vascular and visceral myocytes.
inositol 1,4,5-trisphosphate receptors; cytosolic calcium; confocal microscopy
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INTRODUCTION |
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NEUROTRANSMITTERS INDUCE contraction of smooth muscle cells initially by mobilizing Ca2+ from the intracellular Ca2+ store through inositol 1,4,5-trisphosphate (InsP3)-gated Ca2+ channels (11, 31). Recent data have suggested that intracellular Ca2+ signals are organized as a hierarchy (4, 6, 8, 18, 29). The opening of individual Ca2+ release channels gives rise to fundamental events referred to as blips in the case of InsP3 receptors or quarks for the ryanodine receptors. The next level of organization is represented by small groups of InsP3- or ryanodine-sensitive channels releasing Ca2+ as localized units to give puffs and sparks, respectively. Propagated Ca2+ waves may be obtained by recruitment of a variable number of these elementary events. Typical examples of homologous hierarchical Ca2+-signaling systems have been demonstrated in nonexcitable and excitable cells (4, 6, 18).
In smooth muscle, Ca2+ signals can be generated by InsP3 and ryanodine receptors (31), and there are indications that these two Ca2+ release channel types are located on the same intracellular store in some smooth muscle cells (15). In vascular myocytes, Ca2+ sparks may appear spontaneously (2, 23, 28) or may be triggered by activation of L-type Ca2+ currents (1). Repetitive activation of Ca2+ sparks associated with the progressive recruitment of isolated Ca2+ release channels is needed to trigger propagated Ca2+ waves in rat portal vein myocytes (1). In these myocytes, elementary Ca2+ events, similar to Ca2+ puffs, have never been observed by increasing the InsP3 concentration by flash photolysis of the caged compound (5). Moreover, when ryanodine receptors are inhibited, activation of InsP3 receptors does not trigger propagated Ca2+ waves (5). Therefore, it remains to be established whether a cooperativity between InsP3- and ryanodine-sensitive Ca2+ release channels to induce propagated Ca2+ waves in response to activation of membrane receptors is a physiological mechanism in smooth muscle cells.
The present study shows that, in vascular myocytes, norepinephrine activates InsP3- and ryanodine-sensitive channels to induce propagated Ca2+ waves and maximal contraction. After an initial Ca2+ release from InsP3 receptors, Ca2+ sparks are activated, leading to an increase in amplitude and upstroke velocity of the norepinephrine-induced Ca2+ responses.
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EXPERIMENTAL PROCEDURES |
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Cell preparation, solutions, and membrane current recordings. Wistar rats (150-160 g) were stunned and killed by cervical dislocation. The portal vein and duodenum were removed quickly, cut into several pieces, and incubated for 10 min in low-Ca2+ (40 µM) physiological solution, then 0.8 mg/ml collagenase, 0.25 mg/ml pronase E, and 1 mg/ml BSA were added at 37°C for 20 min. The solution was removed, and the pieces of vein were incubated again in a fresh enzyme solution at 37°C for 20 min. Tissues were then placed in enzyme-free solution and triturated using a fire-polished Pasteur pipette to release cells. Cells were stored on glass coverslips and used on the same day or maintained in short-term primary culture in medium 199 containing 5% FCS, 2 mM glutamine, 1 mM pyruvate, 20 U/ml penicillin, and 20 µg/ml streptomycin; they were kept in an incubator gassed with 95% air-5% CO2 at 37°C and used within 30 h.
The normal physiological solution contained (in mM) 130 NaCl, 5.6 KCl, 1 MgCl2, 1.7 CaCl2, 11 glucose, and 10 HEPES, pH 7.4. Ca2+-free solution was prepared by omitting CaCl2 and adding 0.5 mM EGTA. The basic pipette solution contained (in mM) 130 CsCl and 10 HEPES with 30 µM fluo 3 and 30 µM fura red or 60 µM fluo 3 alone; pH was adjusted to 7.3 with CsOH. Substances were externally applied by pressure ejection from a glass pipette for the period indicated on the records. All the experiments were carried out at 28 ± 1°C. Voltage-clamp and membrane current recordings were made with a standard patch-clamp technique by use of a patch-clamp amplifier (model EPC-7, List, Darmstadt-Eberstadt, Germany). Resistance of patch pipettes was 4-6 MConfocal microscopy and fluorescence measurements.
A confocal scanning head (model MRC 1000, Bio-Rad, Paris, France) was
coupled to an inverted microscope (Diaphot, Nikon, Tokyo, Japan). In
all experiments a Nikon Plan Apo ×60, 1.4 NA objective lens was
used. The iris aperture was set to 40-50% of maximum, providing
axial (z) resolution of ~1.5 µm
and x-y resolution of 0.4 µm.
Illumination was provided by a 25-mW argon ion laser (Ion Laser
Technology, Salt Lake City, UT). The excitation wavelength (488 nm) was
selected using interference filters. For some experiments, two
fluorescent dyes, fluo 3 and fura red (30 µM each) were dialyzed into
the cells through the patch pipette, as previously reported (1, 2). The
emitted fluorescence was collected at wavelengths >515 nm, and fluo 3 and fura red fluorescences were separated by a dichroic mirror. Each
fluorescence beam was filtered and detected by photomultiplier tubes,
allowing simultaneous measurements of the fluorescence emitted by fluo
3 and fura red for dual-emission imaging (19). Dividing the
fluorescence records pixel by pixel resulted in ratio images. For
calibrating the fluo 3/fura red fluorescence, cells were dialyzed with
pipette solutions adjusted to six different free ionized
Ca2+ concentrations with
appropriate ratios of EGTA to
CaCl2 (final EGTA concentration
was always 10 mM). Fluorescence ratios were measured when
Ca2+ equilibration was reached
(within 5-8 min). The calibration curve was fitted using a
least-squares analysis program. The operational dissociation constant
(Kd) for the
fluo 3-fura red mixture was 260 nM on our setup. In flash photolysis
experiments with caged InsP3, fluo
3 (60 µM) was used alone. For other experiments, cells were loaded by
incubation in physiological solution containing 1 µM fluo 3-AM for 1 h at room temperature. These cells were washed and allowed to cleave
the dye to the active fluo 3 compound for 1 h. In the presence of
fluo 3 alone, intracellular Ca2+
concentration
([Ca2+]i)
was estimated from the fluorescence ratio (R, calculated as F/Frest, where F is fluorescence
and Frest is resting fluorescence) by use of the following equation:
[Ca2+]i = Kd · R/{(Kd/[Ca2+]rest + 1)
R} (8), where
Kd is the
dissociation constant of the indicator (316 nM) and
[Ca2+]rest
is resting
[Ca2+]i,
estimated at 45 nM in control conditions and 55 nM in the presence of
10 µM caged InsP3. Image
acquisition and data analysis were performed by using COMOS, TCSM, and
MPL-1000 software (Bio-Rad). Images were acquired in the line-scan mode
of the confocal microscope; this mode repeatedly scanned a single line
through the cell every 2 ms. In this line-scan image the spatial
average fluorescence can be measured in a 2-µm region on the
x-axis, illustrating temporal changes
of
[Ca2+]i
in cell volumes of ~1 µm3.
Flash photolysis. Caged InsP3 [D-myo-InsP3, P4(5)-1-(2-nitrophenyl)ethyl ester] or caged Ca2+ (DM-nitrophen) was introduced into the cell via the patch pipette, with 3-4 min allowed for equilibration. Photolysis was produced by a 1-ms pulse from a xenon flash lamp (Hi-Tech Scientific, Salisbury, UK) focused to a ~2-mm-diameter spot around the cell. Light was band-pass filtered with a UG11 glass between 300 and 350 nm. Flash intensity could be adjusted by varying the capacitor-charging voltage between 0 and 385 V, which corresponded to a change in the energy input into the flash lamp from 0 to 240 J. On flash photolysis, Ca2+ or InsP3 was released within 2 ms. A small percentage of conversion of caged compounds (~10%) was useful if repetitive pulses were applied to obtain similar responses. Flash intensities up to 37 J could be applied repetitively without altering the reserve of caged InsP3 (10 µM) or DM-nitrophen (1 mM, in the presence of 0.25 mM CaCl2) and, consequently, the amount of photoreleased compounds.
Immunocytochemistry. Myocytes were immunostained as previously described (21), except donkey serum was used instead of FCS. Myocytes were incubated in the presence of anti-ryanodine and anti-InsP3 receptor antibodies (at 1:100 and 1:200 dilution, respectively) for 20 h at 4°C, and the secondary antibodies [donkey anti-rabbit IgG conjugated to FITC or donkey anti-mouse IgG conjugated to tetramethylrhodamine isothiocyanate (TRITC) diluted 1:200] were incubated for 3 h at 20°C. Thereafter, cells were mounted in Vectashield.
Contraction. Isometric contraction of longitudinal strips from rat portal vein were recorded in an experimental chamber, as described previously (24), by means of a highly sensitive isometric force transducer (model 801 AME, Akers). The circulating physiological solution was maintained at 30 ± 1°C.
Microsomal membrane preparation.
Microsomal membranes of portal vein, heart, and cerebellum from Wistar
rats were prepared by homogenization with a Kontes potter in a solution
containing 20 mM Tris · HCl, 1 mM EGTA, and 1 mM
phenylmethylsulfonyl fluoride, pH 7.4. The homogenate was centrifuged
at 1,200 rpm for 10 min at 4°C. Microsomal membranes were obtained
as a pellet by centrifugation of the supernatant at 40,000 rpm for 90 min at 4°C. Microsomal membranes were then resuspended in the
buffer and stored at 80°C. Protein concentration was
determined according to Bradford (7).
Immunoblotting. For Western blotting analysis, microsomal proteins were separated on 5% SDS-PAGE minigels and transferred to polyvinylidene difluoride membranes for 16 h at 30 V in a transfer buffer containing 192 mM glycine and 25 mM Tris · HCl (pH 8.3). Membranes were blocked for 1 h in blocking buffer containing 20 mM Tris · HCl and 3% BSA (pH 7.4) and then incubated overnight with the primary antibody at 1:200 (rabbit anti-InsP3 receptor antibody) or 1:100 (mouse anti-ryanodine receptor antibody) dilution. After extensive washing, membranes were incubated for 2 h with the secondary antibody coupled to the peroxidase (anti-rabbit or anti-mouse, 1:5,000 dilution). Specific antigen detection was performed using H2O2 and diaminobenzidine to detect peroxidase activity on polyvinylidene difluoride membranes and the Kodak EDAS 120 (Rochester, NY).
[3H]ryanodine and [3H]InsP3 binding assays. [3H]ryanodine binding to microsomal membranes of rat portal vein was measured, as reported previously (26), in a medium containing 1 M KCl, 25 mM HEPES (pH 7.8 at 37°C), 1 mM dithiothreitol, 0.1 mM CaCl2, 1 mg/ml BSA, and 0.1 mM phenylmethylsulfonyl fluoride. [3H]ryanodine was used in the concentration range of 2-30 nM. After a 3-h incubation at 37°C, aliquots were filtered through Whatmann GF/C glass fiber filters and washed three times with 5 ml of ice-cold binding buffer. The filters were placed in scintillation vials filled with 4 ml of liquid scintillation cocktail, shaken for 1 h, and counted in a Packard 1500 Tri-Carb. Nonspecific binding was measured in the presence of 10 µM ruthenium red and subtracted before calculation. At 20 nM [3H]ryanodine, nonspecific binding was <50% of total binding.
[3H]InsP3 binding was measured in a medium containing 0.1 M KCl, 50 mM Tris · HCl (pH 8.3 at 2-4°C), and 1 mM EGTA. [3H]InsP3 was used in the concentration range of 2-200 nM. After a 10-min incubation on ice, binding reactions were terminated by centrifugation (15,000 g, 15 min, 4°C). The supernatant was aspirated, and the pellet was rinsed quickly with 0.2 ml of ice-cold binding buffer. Pellets were solubilized with 0.1 ml of Soluene-100 (55°C, 30 min). After transfer into scintillation vials with 4 ml of liquid scintillation cocktail, radioactivity was counted (Packard 1500 Tri-Carb). Nonspecific binding was measured in the presence of a 1,000-fold excess of InsP3 over [3H]InsP3 concentration. At 100 nM [3H]InsP3, nonspecific binding was <55% of total binding.Chemicals and drugs. Collagenase was obtained from Worthington (Freehold, NJ); pronase E, BSA, norepinephrine, ACh, prazosin, heparin, ruthenium red, D-myo-InsP3, Triton X-100, and sodium azide from Sigma Chemical (St. Louis, MO); medium 199 from Flow Laboratories (Puteaux, France); FCS from Flobio (Courbevoie, France); ryanodine, indo 1-AM, caged InsP3 [D-myo-InsP3, P4(5)-1-(2-nitrophenyl)ethyl ester], and DM-nitrophen from Calbiochem (Meudon, France); caffeine from Merck (Nogent sur Marne, France); fluo 3, fluo 3-AM, and fura red from Molecular Probes Europe (Leiden, The Netherlands); and [3H]ryanodine (68 Ci/mmol) and [3H]InsP3 (20 Ci/mmol) from Du Pont NEN (Boston, MA). Oxodipine was a gift from Dr. Galiano (Instituto de Investigación y Desarrollo Químico Biológico, Madrid, Spain).
Antibodies directed against InsP3 and ryanodine receptors were added to the pipette solution to allow dialysis of the cell after a breakthrough in whole cell recording mode forAnalysis of data. Values are means ± SE. Significance was tested by Student's t-test. P < 0.05 was considered significant. Concentration-response curves were analyzed by a nonlinear least-squares fitting program. Ca2+ responses were fitted by the Boltzmann equation. Binding data were analyzed with the program GraphPad Prism (version 2.0, GraphPad Software, San Diego, CA) for one- or two-site model.
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RESULTS |
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Characterization of anti-InsP3 and
anti-ryanodine receptor antibodies on InsP3-
and Ca2+-induced
Ca2+ releases.
To study the role of InsP3 and
ryanodine receptors in triggering propagated
Ca2+ waves in smooth muscle cells,
we first analyzed the properties of antibodies directed against the
COOH terminus of InsP3 and ryanodine receptors. Because these antibodies recognize all the isoforms of each receptor, we chose to use rat heart and cerebellum as
positive controls. Figure
1A
shows a Western blot analysis on samples of rat portal vein, heart, and
cerebellum. The anti-ryanodine receptor antibody recognized a
high-molecular-weight band (~500,000) in cardiac and portal vein
membranes, whereas the anti-InsP3
receptor antibody recognized a lower-molecular-weight band (~250,000)
in cerebellum and portal vein membranes. No cross-reactivity of the antibodies with the two proteins was detectable. These values are in
good agreement with those previously reported for ryanodine and
InsP3 receptors (3, 9).
Immunodetection of InsP3 and ryanodine receptors in 0.5-µm cell confocal sections was performed with these antibodies, and the binding sites were detected with FITC
and TRITC, respectively. As illustrated in Fig.
1B, the two types of receptors
appeared to be distributed in the whole confocal sections, with spots
of ryanodine receptors in areas corresponding to the cell periphery and
to infoldings of the plasma membrane in close association with the
sarcoplasmic reticulum.
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A common intracellular
Ca2+ store
activated by caffeine and neurotransmitters.
The experiments described below were carried out under
Ca2+-free conditions to eliminate
the contribution of extracellular
Ca2+, so that the release of
Ca2+ from intracellular stores
could be resolved. Repeated stimulations by 10 µM ACh or
norepinephrine, which induced
InsP3 accumulations (16) on indo
1-loaded myocytes, did not produce a second rise in
[Ca2+]i
after recovery in Ca2+-free
solution (n = 5; Fig.
4A).
Similarly, successive applications of 10 mM caffeine, which acted at
the ryanodine receptors, produced a first
Ca2+ response, whereas a second
application was ineffective (n = 5; Fig. 4A). To determine the extent of
overlap between ACh- and caffeine-sensitive
Ca2+ stores, cells were exposed to
combinations of agents to determine whether prior exposure to one agent
would diminish a subsequent response to the other agent. As illustrated
in Fig. 4B, prior exposure to ACh
completely eliminated subsequent responses to caffeine
(n = 4). Similarly, prior applications
of caffeine suppressed subsequent responses to ACh
(n = 4).
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Effects of anti-InsP3 and anti-ryanodine
receptor antibodies on norepinephrine-induced
Ca2+ release.
To verify whether InsP3 and
ryanodine receptors participate in the
Ca2+ responses evoked by
neurotransmitters, we tested the effects of these antibodies on the
Ca2+ release evoked by various
concentrations of norepinephrine in the continuous presence of 1 µM
oxodipine. Under these conditions, the norepinephrine-induced
Ca2+ release was inhibited by
prazosin in a concentration-dependent manner (data not shown),
indicating that this response was activated by
1-adrenoceptors, as previously
demonstrated (17). Figure 5 illustrates
line-scan images of voltage-clamped myocytes and the time courses of
the Ca2+ responses evoked by
norepinephrine when increases in
[Ca2+]i
were analyzed in 2-µm regions. The delay between application of
norepinephrine and the onset of the
Ca2+ response was estimated to be
1.1 ± 0.1 s (n = 10). At 1 µM,
norepinephrine induced localized and transient
Ca2+ responses
(n = 13; Fig.
5A, see Fig. 7) or propagated
Ca2+ waves
(n = 5; see Fig. 7). In the presence
of 1 µM norepinephrine, spatially localized
[Ca2+]i
transients are obtained in >70% of the cells
(n = 18). It can be postulated that
norepinephrine stimulates these
Ca2+ signals rather than increases
the number of spontaneous Ca2+
releases, which are detected in only 30% of the cells tested in
control conditions (23). When norepinephrine was applied at 10 µM,
propagated Ca2+ waves were
recorded in all the cells tested (n = 19; Fig. 5B). Intracellular
application of an anti-ryanodine receptor antibody (10 µg/ml) for
7-8 min removed the localized and transient
Ca2+ responses activated by 1 µM
norepinephrine; however, a small and slow increase in
[Ca2+]i
persisted (Fig. 5C). This antibody
also transformed the norepinephrine-induced propagated
Ca2+ waves in reduced
Ca2+ responses showing a uniform
increase in
[Ca2+]i,
whatever the region of the line-scan image (Fig.
5D). These results indicate that the
localized and transient Ca2+
responses activated by 1 µM norepinephrine and removed by the anti-ryanodine receptor antibody correspond to
Ca2+ sparks, as previously
identified in these vascular myocytes (1, 2). They also show that
propagated Ca2+ waves evoked by
norepinephrine are dependent on activation of ryanodine-sensitive
Ca2+ release channels, whereas
activation of InsP3-gated channels alone gives rise to slow and small
Ca2+ responses showing a similar
time course in all regions of the cell.
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[3H]ryanodine and
[3H]InsP3
binding on rat portal vein membranes.
InsP3 binding to rat portal vein
microsomal preparations was measured over a broad range of
InsP3 concentrations (2-200
nM), but as shown in Fig. 8, Scatchard
plots of these data were nonlinear. The
InsP3-binding data could be fitted
by assuming the presence of two
InsP3-binding sites. The
calculated Kd
values were 1.4 and 132.5 nM, with 90% of the binding sites being low
affinity. The maximal binding capacity
(Bmax) corresponding to the
total number of InsP3-binding
sites (high-affinity + low-affinity sites) was 1.59 ± 0.21 pmol/mg
protein (n = 3).
[3H]ryanodine binding
was performed on the same membranes used for [3H]InsP3
binding. The Kd
and Bmax values of
[3H]ryanodine binding
were 8.7 ± 0.1 nM and 5.70 ± 0.65 pmol/mg protein
(n = 5), respectively. These results
show that the Bmax of
InsP3 in rat portal vein myocytes
is rather low: three to four times less than that of ryanodine.
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Effects of ryanodine receptor inhibition on
Ca2+ release
evoked by various neurotransmitters in non-voltage-clamped vascular and
visceral myocytes.
To ensure that the inhibition of norepinephrine-induced
Ca2+ responses by ryanodine
receptor inhibitors was not due to a dilution of the
InsP3 concentration when the cells
were dialyzed with the pipette solution, portal vein and duodenal
myocytes were loaded with 1 µM fluo 3-AM or indo 1-AM, respectively,
and not voltage clamped. In the continuous presence of 1 µM
oxodipine, norepinephrine (10 µM) or ACh (10 µM) evoked propagated
Ca2+ waves, the amplitude and
upstroke velocity of which are listed in Table
1. In the presence of 10 µM ryanodine for
10-12 min in the extracellular medium, the amplitude and maximal
upstroke velocity of the neurotransmitter-induced
Ca2+ responses (measured in a
2-µm region of the line-scan image) were significantly reduced (Table
1). In duodenal myocytes the ACh-induced global rise in
[Ca2+]i
was also reduced (~35%) in the presence of 10 µM ryanodine. Taken
together, these results indicate that, in non-voltage-clamped vascular
and visceral myocytes, inhibition of ryanodine-sensitive Ca2+ release channels results in
neurotransmitter-induced Ca2+
responses of reduced amplitude.
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Effects of ryanodine receptor inhibition on neurotransmitter-induced
contraction.
Involvement of ryanodine-sensitive
Ca2+ release channels in
neurotransmitter-induced contraction was first investigated by
measuring the variation of the scanned line length through the myocyte
after application of 10 µM norepinephrine (Fig.
9A).
Within 1 s after norepinephrine ejection, the increase in
[Ca2+]i
was observed without any noticeable variation in the scanned line
length (L0).
Then the scanned line length decreased as the myocyte contracted
(L1) before it
returned to initial length within 5-10 s (not shown). The
variation in cell length was normalized to the resting value and
expressed as (L0 L1)/L0.
The ratio was equal to zero when
L1 = L0 and became
positive when L1 < L0, then
reflecting a contraction. After application of 10 µM ryanodine (in
non-voltage-clamped myocytes) or 10 µg/ml anti-ryanodine receptor antibody (in voltage-clamped myocytes), the ratio
(L0
L1)/L0 was significantly reduced (by ~50%) compared with control conditions (Fig. 9B), suggesting that the
norepinephrine-induced contraction was reduced. Second, we recorded
isometric contractions from thin isolated strips of portal vein smooth
muscle (24). After pretreatment with 10 µM ryanodine for 30 min, the
norepinephrine-induced contractions were reduced by ~45% compared
with control conditions, as shown in Fig.
9C. These results support the role of
a functional interaction between
InsP3- and ryanodine-sensitive
Ca2+ release channels in the
amplitude of the norepinephrine-induced contraction in vascular
myocytes.
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DISCUSSION |
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The main conclusion from these results is that Ca2+ responses evoked by neurotransmitters in rat portal vein and duodenal myocytes depend on activation of InsP3- and ryanodine-sensitive Ca2+ release channels. Evidence supporting this conclusion is as follows. 1) Propagated Ca2+ waves evoked by norepinephrine in portal vein myocytes were blocked by intracellular applications of heparin and an anti-InsP3 receptor antibody. 2) The anti-ryanodine receptor antibody and ryanodine that completely inhibited Ca2+ signals evoked by flash-photolytic Ca2+ jumps decreased by ~50% the InsP3- and norepinephrine-induced Ca2+ waves without affecting the Ca2+ content of the store. 3) When the upstroke velocity of norepinephrine-evoked Ca2+ waves was analyzed in 2-µm regions of the line-scan image, the maximal value was obtained in the region of the line-scan image corresponding to the initiation site of the Ca2+ wave. Whatever the regions analyzed, inhibition of ryanodine receptors strongly reduced the upstroke velocity to a similar value, indicating that this homogeneous remaining component corresponded to activation of InsP3-sensitive Ca2+ channels. 4) A similar inhibition of the Ca2+ responses evoked by ACh was obtained with ryanodine treatment in portal vein and duodenal myocytes. These results show, for the first time, that activation of ryanodine-sensitive Ca2+ release channels is necessary for triggering neurotransmitter-evoked propagated Ca2+ waves of high amplitude and fast velocity that are, however, initiated by activation of InsP3-sensitive Ca2+ channels.
On the basis of functional experiments and immunodetection of InsP3 and ryanodine receptors, our results are consistent with the idea that a single intracellular store is mobilized by neurotransmitters and caffeine. First, after depletion of the intracellular Ca2+ store in Ca2+-free solution by maximal concentrations of caffeine or ACh, subsequent applications of both agents were ineffective. Similarly, when Ca2+-ATPases were blocked with thapsigargin, which induced Ca2+ leak from the intracellular Ca2+ store, ACh- and caffeine-induced Ca2+ responses were abolished. Second, after pretreatment of cells in Ca2+-containing solution with ryanodine and caffeine to deplete and prevent refilling of the intracellular Ca2+ store, the responses to ACh and caffeine were abolished. In addition, the basal [Ca2+]i was increased in the presence of ryanodine and caffeine, suggesting that depletion of the Ca2+ store leads to increased Ca2+ entry into the cell (25). Third, immunodetection of InsP3 and ryanodine receptors in 0.5-µm cell confocal sections showed that the two types of receptors were distributed in the whole sections. However, spots of ryanodine receptors are detected in several areas, whereas InsP3 receptor immunostaining did not reveal any fluorescence spots. Therefore, it can be postulated that the density of ryanodine receptors may be higher than that of InsP3 receptors. This hypothesis is supported by binding experiments to rat portal vein microsomal preparations which indicate that the Bmax is three to four times higher for [3H]ryanodine than for [3H]InsP3. [3H]InsP3 and [3H]ryanodine binding sites have been identified in guinea pig ileal smooth muscle, but with InsP3 receptors more abundant than ryanodine receptors (33). However, most of the [3H]InsP3 binding was detected in circular muscle cells, whereas the binding in longitudinal muscle cells was weak (27). Therefore, it can be proposed that the functional implications of ryanodine receptors in neurotransmitter-induced Ca2+ responses depend on their relative density compared with InsP3 receptor density. In smooth muscles displaying a higher density of InsP3 receptors than of ryanodine receptors, the Ca2+ responses to neurotransmitters could mainly depend on activation of InsP3 receptors. Comparative experiments in a greater number of tissues are, however, needed to expand this conclusion to all smooth muscles.
All-or-none Ca2+ responses evoked by neurotransmitters and flash-photolytic InsP3 jumps have been previously reported in permeabilized basophilic leukemia cells (22) and guinea pig portal vein smooth muscle cells (12). These observations have been explained by a Ca2+-dependent positive-feedback control of InsP3-induced Ca2+ release (12). This possibility seems unlikely in rat portal vein myocytes, since concentration-response curves to norepinephrine with Hill coefficients close to unity are obtained after blockade of ryanodine receptors with the anti-ryanodine receptor antibody or with high concentrations of ryanodine. In addition, the amplitude and the upstroke velocity of the norepinephrine-evoked Ca2+ responses were strongly reduced when ryanodine receptors were blocked. Activation of ryanodine channels by Ca2+ (CICR component) leads to Ca2+ responses with a high upstroke velocity (~7 µM/s) but limited amplitude (~100 nM). The fast upstroke velocity may be linked to a quasi-instantaneous release of Ca2+ in the whole scanned line that synchronously activates all the ryanodine-sensitive channels, whereas the limited amplitude of the Ca2+ response may depend on a very brief release of Ca2+ by flash photolysis of caged Ca2+ and on the Ca2+ buffering power of the cytoplasm. Accordingly, the amplitude of the maximal CICR component is reduced by ~30-50% within 0.7 s after the photolytic Ca2+ jumps (Figs. 2B and 3B). Interestingly, the CICR component obtained by subtracting the InsP3-induced Ca2+ response in the presence of anti-ryanodine receptor antibody from that in the absence of the antibody shows time course and amplitude parameters similar to those of the CICR component obtained with flash photolysis of caged Ca2+. In contrast, the specific InsP3-induced Ca2+ response (obtained in the presence of the anti-ryanodine receptor antibody) is slower (~0.3 µM/s) and more maintained than the CICR component. These observations are in agreement with the fact that, when InsP3 is released by flash photolysis or by activation of phospholipase C in response to receptor stimulation, the maximal amplitude of the InsP3-induced Ca2+ response seems to correspond to the addition of InsP3 and CICR components. However, the maximal upstroke velocity of the CICR component activated by flash photolysis of caged InsP3 or norepinephrine is slower (~3.5 µM/s) than that activated by flash photolysis of caged Ca2+ (~7 µM/s). A possible explanation is that a diffuse activation of discrete InsP3-sensitive Ca2+ channels does not provide a sufficient Ca2+ trigger to activate all the ryanodine-sensitive channels across the scanned line. In addition, when a critical [Ca2+]i threshold has been reached to open the ryanodine-sensitive channels (Fig. 3B), a part of the Ca2+ store has been released as a result of the previous InsP3-sensitive channel opening. Inasmuch as the open probability of the ryanodine-sensitive channels has been shown to be modulated by the luminal Ca2+ concentration (20), a decrease in the Ca2+ content might account for the decrease in the upstroke velocity of the Ca2+ responses evoked by flash photolysis of caged InsP3 or norepinephrine. In addition, the observation that the InsP3-induced Ca2+ release reduces the upstroke velocity of the CICR component supports the existence of a single functional Ca2+ store. Our results also suggest that the InsP3 receptors are less sensitive to Ca2+ than the ryanodine receptors. This possibility is supported by the observation that the [Ca2+]i threshold for triggering Ca2+ sparks in response to activation of L-type Ca2+ current or flash-photolytic Ca2+ jumps has been estimated to be 75-95 nM in rat portal vein myocytes (1). A critical [Ca2+]i threshold for inducing Ca2+-dependent positive feedback of InsP3-induced Ca2+ has been estimated at ~150-180 nM in various cell types (6, 13). This value is larger than the [Ca2+]i level corresponding to the transition between the two upstroke velocity components of the norepinephrine-induced Ca2+ waves obtained in some cells (~95 nM). Therefore, our results support the idea of a positive cooperativity between InsP3- and ryanodine-sensitive Ca2+ channels in rat portal vein and duodenal myocytes; i.e., Ca2+ release through ryanodine-sensitive channels is critical for generation of Ca2+ waves evoked by neurotransmitters that are known to induce an increase in InsP3 concentration (16). The cooperativity between InsP3- and ryanodine-sensitive Ca2+ channels may not only depend on their relative proportion but also on their topical organization in the sarcoplasmic reticulum. Immunodetection of Ca2+ release channels in confocal sections shows that InsP3 receptors are distributed homogeneously on the sarcoplasmic reticulum, even within the specialized areas showing clusters of ryanodine receptors. Elementary Ca2+ events, such as Ca2+ sparks, have been identified in smooth muscle cells and attributed to the opening of clusters of ryanodine-sensitive channels (1, 28). In contrast, elementary Ca2+ events corresponding to activation of clusters of InsP3-gated Ca2+ channels have never been observed in portal vein myocytes (5), suggesting that these cells may lack clustered InsP3 receptor units. Therefore, it is likely that the small increase in [Ca2+]i due to opening of InsP3-gated channels is able to activate the ryanodine receptors located in the vicinity of these InsP3 receptors, then producing Ca2+ sparks and the subsequent Ca2+ waves. Such a microarchitecture of ryanodine and InsP3 receptors has been proposed in the sarcoplasmic reticulum of rat portal vein myocytes (5) but remains, however, to be established in other smooth muscle cells. The physiological role of the Ca2+-amplifying mechanism described in this study is also supported by contraction experiments, in isolated cells and intact strips, showing that the norepinephrine-induced contractions are reduced by ~40-50% after inhibition of the ryanodine-sensitive Ca2+ release channels.
In conclusion, we have shown that, in portal vein and duodenal myocytes, ryanodine-sensitive Ca2+ release channels are involved in Ca2+ responses initiated by an increase in cytosolic InsP3. This amplifying mechanism may be involved in a variety of cells expressing both types of Ca2+ release channels located on the same intracellular store in response to various constrictors that stimulate InsP3 generation.
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ACKNOWLEDGEMENTS |
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We thank N. Biendon and J.-L. Lavie for technical assistance and J.-L. Morel for the experiments on duodenal myocytes.
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FOOTNOTES |
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This work was supported by grants from Centre National de la Recherche Scientifique and Centre National des Etudes Spatiales, France.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: J. Mironneau, Laboratoire de Physiologie Cellulaire et Pharmacologie Moléculaire, CNRS ESA 5017, Université de Bordeaux II, 146 rue Léo Saignat, 33076 Bordeaux Cedex, France (E-mail: jean.mironneau{at}esa5017.u-bordeaux2.fr).
Received 11 September 1998; accepted in final form 8 March 1999.
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