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Crucial Role of Type 2 Inositol 1,4,5-Trisphosphate Receptors for Acetylcholine-Induced Ca2+ Oscillations in Vascular Myocytes

Originally publishedhttps://doi.org/10.1161/01.ATV.0000089013.82552.5DArteriosclerosis, Thrombosis, and Vascular Biology. 2003;23:1567–1575

Abstract

Objective— The aim of this study was to correlate the expression of InsP3R subtypes in native vascular and visceral myocytes with specific Ca2+-signaling patterns.

Methods and Results— By Western blot and immunostaining, we showed that rat portal vein expressed InsP3R1 and InsP3R2 but not InsP3R3, whereas rat ureter expressed InsP3R1 and InsP3R3 but not InsP3R2. Acetylcholine induced single Ca2+ responses in all ureteric myocytes but only in 50% of vascular myocytes. In the remaining vascular myocytes, the first transient peak was followed by Ca2+ oscillations. By correlating Ca2+ signals and immunostaining, we revealed that oscillating vascular cells expressed both InsP3R1 and InsP3R2 whereas nonoscillating vascular cells expressed only InsP3R1. Acetylcholine-induced oscillations were not affected by inhibitors of ryanodine receptors, Ca2+-ATPases, Ca2+ influx, and mitochondrial Ca2+ uniporter but were inhibited by intracellular infusion of heparin. Using specific antibodies against InsP3R subtypes, we showed that acetylcholine-induced Ca2+ oscillations were specifically blocked by the anti-InsP3R antibody. These data were supported by antisense oligonucleotides targeting InsP3R2, which selectively inhibited Ca2+ oscillations.

Conclusions— Our results suggest that in native smooth muscle cells, a differential expression of InsP3R subtypes encodes specific InsP3-mediated Ca2+ responses and that the presence of the InsP3R2 subtype is required for acetylcholine-induced Ca2+ oscillations in vascular myocytes.

Stimulation of plasma membrane receptors results generally in the generation of inositol 1,4,5-trisphosphate (InsP3) via the activation of phospholipase C. InsP3 releases Ca2+ from intracellular stores by binding to InsP3 receptors (InsP3Rs), which form tetrameric InsP3-gated Ca2+ release channels.1 Molecular cloning studies have revealed that there are at least 3 subtypes of InsP3Rs derived from distinct genes, designated as subtype 1 (InsP3R1), subtype 2 (InsP3R2), and subtype 3 (InsP3R3).2–4 Multiple subtypes of InsP3Rs may be expressed in different cell types and form both homotetramers and heterotetramers.5,6 Therefore, expression of different InsP3Rs may support the generation of specific Ca2+ responses. Recent studies using incorporation of InsP3Rs into lipid bilayers and overexpression of recombinant InsP3Rs have suggested that the 3 InsP3R subtypes may be differentially regulated by InsP3 and Ca2+, calmodulin, ATP, and phosphorylation.7–12 In genetically engineered DT40 B cells that express either a single or a combination of InsP3R subtypes, distinct patterns of Ca2+ signals have been reported.13 However, expression of InsP3R subtypes have not been analyzed in different native cells under equivalent experimental conditions, and it has not been demonstrated that the differential expression of InsP3R subtypes may be responsible for the different Ca2+ signal patterns recorded in situ.

The aim of the present study was to correlate the functional expression of InsP3R subtypes with specific Ca2+-signaling patterns in native vascular and visceral smooth muscle cells. Our results show that the functional InsP3R subtypes expressed in rat portal vein and ureteric myocytes differ and may support different acetylcholine-induced Ca2+ responses, ie, Ca2+ oscillations and single Ca2+ responses.

Methods

Cell Preparation

The investigation conforms with the European Community and French guiding principles in the care and use of animals. Authorization to perform animal experiments was obtained from the French Ministère de l’Agriculture et de la Pêche.

Wistar rats (150 to 170 g) were killed by cervical dislocation. Isolated myocytes were obtained from portal vein and ureter by enzymatic dispersion, as previously described.14 Cells were seeded on glass slides in physiological solution and maintained in short-term primary culture in medium M199 containing FCS (2% for portal vein myocytes and 5% for ureteric myocytes), 2 mmol/L glutamine, 1 mmol/L pyruvate, 20 U/mL penicillin, and 20 μg/mL streptomycin; they were kept in an incubator gassed with 95% air and 5% CO2 at 37°C and used either within 20 to 30 hours or within 2 to 4 days for cells injected with the antisense oligonucleotides.

Microinjection of Oligonucleotides

Phosphorothioate antisense oligonucleotides (denoted with the prefix “as”) used in the present study were designed on the known cloned InsP3R sequences deposited in the GenBank sequence database with Lasergene software (DNASTAR). Sequences of all 3 InsP3R cDNAs were aligned with each other, and specific antisense oligonucleotide sequences were chosen in the region of the cDNA of interest, completely different from the sequences of the 2 other InsP3R subtypes. Then antisense sequences displaying putative binding to any other mammalian sequences deposited in GenBank were discarded. Oligonucleotides were injected into the nuclei of myocytes by a manual injection system (Eppendorf). Intranuclear oligonucleotide injection with Femtotips II (Eppendorf) was performed as previously described.15 The myocytes were then cultured for 2 to 4 days in culture medium, and the glass slides were transferred into the perfusion chamber for physiological experiments. The sequence of asInsP3R1 is ATCTGTTGTACTGTTGGCC, corresponding to nucleotides 566 to 584 of cDNA; that of asInsP3R2 is TATTTCACAATTTCTCC, corresponding to nucleotides 580 to 596 of InsP3R2 cDNA; and that of InsP3R3 is TGTGCAGAAGCTGAATCA, corresponding to nucleotides 494 to 513 of InsP3R3 cDNA.

Western Blot Analysis

Western blot analyses of rat tissue extracts and COS-1 cells (50 μg protein) were performed as previously described.16

Fluorescence Measurements

Measurements of [Ca2+]i were performed with an indo-1 setup, as described elsewhere.14 Cells were either preloaded with the membrane-permeant indo-1 AM (1 μmol/L) for 30 minutes or 50 μmol/L indo-1 was added to the pipette solution and entered the cells after establishment of the whole-cell recording mode. [Ca2+]i was estimated from the 405/480-nm fluorescence ratio using a calibration determined within cells.14 All measurements were made at 25±1°C. Voltage clamp was made with a standard patch-clamp technique using a List EPC-7 patch-clamp amplifier (Darmstadt-Eberstadt). Patch pipettes had resistances of 3 to 4 MΩ. Anti-InsP3R1, anti-InsP3R2, and anti-InsP3R3 antibodies were added to the pipette solution to allow dialysis of the cell after a breakthrough in whole-cell recording mode for at least 5 minutes, a time longer than that expected for diffusion of substances in solution.17

InsP3R Immunostaining

Myocytes were washed with PBS, fixed with 4% (vol/vol) formaldehyde and 0.5% glutaraldehyde for 10 minutes at room temperature, and permeabilized in PBS containing 3% FCS and 1 mg/mL of saponin for 20 minutes. Cells were incubated with PBS, saponin (1 mg/mL), and anti-InsP3R antibody (1 μg/mL) overnight at 4°C. Then cells were washed (4×5 minutes) and incubated with the appropriate secondary antibody conjugated to fluorescein-isothiocyanate during 45 minutes at room temperature. After washing in PBS, cells were mounted in Vectashield (Valbiotech). Images of the stained cells were obtained with a confocal microscope (Bio-Rad MRC 1024, Bio-Rad), and fluorescence was estimated by gray level analysis using IDL software (RSI) in 0.5-μm confocal sections. On each cell, fluorescence was acquired from a z-series analysis (20±5 sections) using Lasersharp software (Bio-Rad) and expressed by volume unit. Cells were compared by keeping acquisition parameters (such as gray scale, exposure time, iris aperture, gain, and laser power) constant. A similar protocol was applied to portal vein strips (longitudinal muscle) except that anti-InsP3R1 (Affinity BioReagents) and anti-InsP3R2 (Santa Cruz Biotechnology) antibodies were conjugated to Alexa 488 and Alexa 568 secondary antibodies, respectively.

Solutions

The physiological solution contained (in mmol/L) NaCl 130, KCl 5.6, MgCl2 1, CaCl2 2, glucose 11, and HEPES 10, pH 7.4, with NaOH. The basic pipette solution contained (in mmol/L) CsCl 130 and HEPES 10, pH 7.3, with CsOH. Acetylcholine and active compounds were applied to the recorded cell by pressure ejection for the period indicated on the records.

Chemicals and Drugs

Collagenase was obtained from Worthington. M199 medium, streptomycin, penicillin, glutamine, and pyruvate were from Invitrogen). FCS was from BioMedia. Indo-1, indo-1 AM, and the mouse monoclonal anti-RYR antibody (559279) were from Calbiochem, Meudon, France. Anti-InsP3R1, anti-InsP3R2, and anti-InsP3R3 antibodies (SC6093, SC7278, and SC7277) were from Santa Cruz Biotechnology (Santa Cruz, Calif), from Affinity BioReagents (anti-InsP3R1 antibody, COGER, Paris, France), from AbCys (anti-InsP3R2 antibody, Valbiotech), and from Transduction Laboratories (anti-InsP3R3 antibody, BD Sciences, Le Pont de Claix, France). Alexa 488 and Alexa 568 secondary antibodies were from Molecular Probes (Eugene, Ore). Anti-PCNA (proliferating cell nuclear antigen) antibody was from Chemicon (Temecula, Calif). All other products were from Sigma.

Data Analysis

Data are expressed as mean±SEM; n represents the number of cells or experiments. Significance was tested by means of paired Student’s t test when cells were their own control; otherwise, unpaired test was used. P<0.05 was considered significant.

Results

Ca2+ Responses Evoked by Acetylcholine

In portal vein myocytes, application of 1 μmol/L acetylcholine (ACh) for 30 seconds induced 2 types of Ca2+ responses. In 51% of cells (138 of 270 cells, 18 dissociations), ACh initiated a Ca2+ peak that was followed by regenerative oscillations (Figure 1A). The amplitude of the first response reached 218±9 nmol/L from a resting level of 66±11 nmol/L (n=138). The amplitude of oscillations varied strongly, but the frequency of oscillations was reproducible independently of the duration of ACh applications (4.0±0.4 oscillations/min, n=138). Although the percentage of oscillating cells was similar in the ACh concentration range from 1 nmol/L to 10 μmol/L, the highest and more reproducible frequency of oscillations was obtained at 1 μmol/L ACh (n=138), so that the following experiments were performed with this ACh concentration. The nonoscillating cells (49%) responded by single Ca2+ responses (Figure 1A). On application of ACh, the peak response reached 198±10 nmol/L from a resting level of 67±9 nmol/L (n=132) and was followed by a sustained plateau of 50±25 nmol/L (n=132). It is noteworthy that the duration at half-maximal amplitude of the first Ca2+ peak in oscillating cells (2.62±0.22 seconds, n=138) was smaller than that of single Ca2+ transients in nonoscillating cells (6.74±0.62 seconds, n=132). Similarly, application of noradrenaline (0.1 to 10 μmol/L) induced both Ca2+ oscillations and single Ca2+ transients in vascular myocytes (n=64). In contrast, in ureteric myocytes, Ca2+ oscillations were never observed after application of 0.1 to 10 μmol/L ACh (0 of 125 cells tested, 6 dissociations). As shown in Figure 1A, the amplitude of the transient Ca2+ response evoked by 1 μmol/L ACh was 368±17 nmol/L from a resting level of 72±9 nmol/L (n=92). This Ca2+ peak was followed by a sustained plateau of 41±15 nmol/L (n=92).

Figure 1. Increase in [Ca2+]i evoked by ACh in portal vein and ureteric myocytes. A, In portal vein myocytes, ACh (1 μmol/L) induced Ca2+ oscillations (a) or single Ca2+ response (b). In ureteric myocytes, ACh induced only single Ca2+ response (c). B, Concentration response curve for the inhibitory effects of thapsigargin (tg) applied 1 minute before ejection of 1 μmol/L ACh for 30 seconds. Δ[Ca2+]i is expressed as the ratio of measurements in presence and absence of tg. C, ACh-induced Ca2+ oscillations in control conditions and after application of 50 nmol/L tg for 1 minute. Similar results were obtained in the 9 cells tested showing Ca2+ oscillations.

Effects of different pharmacological substances and external ion solutions were tested on both amplitude (first Ca2+ peak) and frequency of Ca2+ oscillations induced by 1 μmol/L ACh in vascular myocytes (Table). Ca2+ oscillation frequency was not affected after removal of external Ca2+ for 30 seconds (in Ca2+-free solution containing 0.5 mmol/L EGTA), but the responses decreased in amplitude within 3 to 4 minutes as a consequence of the rapid depletion of the intracellular Ca2+ store. Both amplitude of the first ACh-induced Ca2+ peak and frequency of oscillations were not significantly affected in the presence of 1 μmol/L oxodipine (a specific inhibitor of voltage-dependent Ca2+ channels) or 1 μmol/L RU-360 (a selective inhibitor of the mitochondrial Ca2+ uniporter18). Although 10 μmol/L ryanodine or 10 μg/mL anti-ryanodine receptor antibody (inhibitors of ryanodine-sensitive Ca2+ release channels19) had no effect on Ca2+ oscillation frequency, they decreased the amplitude of the Ca2+ peak, as previously reported.19 Depletion of the Ca2+ store can be modulated by thapsigargin (a Ca2+-ATPase inhibitor). Different concentrations of thapsigargin were applied for 1 minute before testing the effects of 1 μmol/L ACh. Thapsigargin inhibited in a concentration-dependent manner the ACh-induced Ca2+ responses (Figure 1B). Complete inhibition was obtained with 10 μmol/L thapsigargin, indicating that the store was empty. At 50 nmol/L, thapsigargin partially depleted the Ca2+ store, because the first ACh-induced Ca2+ peak was decreased by approximately 50% (Figure 1B). Under these conditions, the number of cells producing Ca2+ oscillations in response to ACh (9 of 17 cells tested) and the frequency of Ca2+ oscillations were not significantly different from those obtained in control conditions (Table, Figure 1C). However, a second application of ACh 5 minutes later was ineffective, as expected when the agonist-sensitive Ca2+ store was completely depleted. Finally, intracellular application of 1 mg/mL heparin (an inhibitor of InsP3Rs) for 5 minutes suppressed the ACh-induced Ca2+ responses in both vascular (n=14) and ureteric (n=12) myocytes. These results indicate that the ACh-mediated Ca2+ release occurs from the sarcoplasmic reticulum and that Ca2+ oscillations can be obtained from a partially depleted Ca2+ store.

Effects of External Ca2+, Inhibitors of Ca2+ Channels, and Ca2+ Pumps on Ca2+ Oscillations Evoked by 1 μmol/L ACh in Portal Vein Myocytes

Control ConditionsExperimental Conditions
Frequency, osc/minFirst Peak Amplitude, nmol/LFrequency, osc/minFirst Peak Amplitude, nmol/L
ACh (1 μmol/L) was applied 30 seconds after removal of external Ca2+, 1 minute after application of thapsigargin (50 nmol/L), 5 minutes after application of oxodipine (1 μmol/L), and 15 minutes after application of RU-360 (1 μmol/L) or ryanodine (10 μmol/L). Anti-ryanodine receptor antibody (10 μg/mL) was applied intracellularly through the patch pipette for 5 minutes and cells were held at −50 mV. Values are mean±SEM for 9 to 19 oscillating nonpatched cells from 4 dissociations or for 5 oscillating cells held at −50 mV from 2 dissociations.
osc indicates oscillations.
*Values significantly different from those obtained in control conditions.
4.1±0.3215 ±150 mmol/L [Ca2+]e4.2 ±0.4191 ±11
4.2±0.9222 ±17Oxodipine4.9 ±0.7289 ±12
4.2±0.6235 ±31RU-3605.1 ±1.0230 ±28
5.1±1.2220 ±28Ryanodine5.0 ±1.1190 ±25
5.3±2.2225 ±60Anti-RYR antibody3.1 ±1.5122 ±21*
4.8±1.7240 ±46Thapsigargin3.9 ±1.2139 ±41*

To check the possibility that the capacitative Ca2+ entry could be different in the 2 types of vascular myocytes, Ca2+ entry was stimulated either by a pathway involving a Gq-coupled receptor (ACh) or by thapsigargin in cells identified as oscillating or nonoscillating during previous applications of ACh. After complete depletion of the Ca2+ store by applications of 10 μmol/L ACh or 10 μmol/L thapsigargin in Ca2+-free 0.5 mmol/L EGTA-containing solution, readmission of 2 mmol/L external Ca2+ caused a transient and significant increase in [Ca2+]i.20 In oscillating cells, peak Ca2+ responses activated by ACh and by store depletion were 98±17 nmol/L (n=17) and 106±11 nmol/L (n=18), respectively, whereas in nonoscillating cells, they were 100±14 nmol/L (n=19) and 101±14 nmol/L (n=43), respectively. In addition, the Ca2+ loading of the store was assessed by applications of 10 mmol/L caffeine after complete refilling of the Ca2+ store in 2 mmol/L external Ca2+ for 15 minutes. Peak amplitudes of caffeine-induced Ca2+ responses were not significantly different in oscillating (183±19 nmol/L, n=18) and nonoscillating (210±21 nmol/L, n=19) cells. Taken together, these results indicate that ACh-induced Ca2+ oscillations in vascular myocytes depend only on Ca2+ release from the sarcoplasmic reticulum through activation of InsP3Rs and that both Ca2+ content of the intracellular store and capacitative Ca2+ entry were not different in oscillating and nonoscillating vascular myocytes.

Expression of InsP3R Subtypes

Expression of InsP3R subtypes was examined by using specific antibodies directed against InsP3R1, InsP3R2, and InsP3R3 in rat portal vein and ureteric myocytes and in COS-1 cells. Western blot detection of InsP3R subtypes (Figure 2A) indicated that InsP3R1 and InsP3R2 were expressed in portal vein myocytes and InsP3R1 and InsP3R3 in ureteric myocytes. As previously reported,21 COS-1 cells expressed InsP3R2 and InsP3R3 but not InsP3R1. The molecular weight of each subtype was determined in 3 different experiments and corresponded to that expected from primary sequences.4

Figure 2. Expression of InsP3R subtypes in portal vein, ureteric, and COS-1 cells. A, Microsomal proteins (50 μg) were separated by SDS/PAGE and analyzed by Western blot with anti-InsP3R1 (1:1000, Affinity BioReagents), anti-InsP3R2 (1:100, AbCys), and anti-InsP3R3 (1:1000, Transduction Laboratories) antibodies. Molecular weights are in kilodaltons. Similar results were obtained from 3 different experiments. B, Typical confocal cell sections showing a differential expression of InsP3R subtypes in portal vein (PV1 and PV2) and ureteric (Ur) myocytes. Anti-InsP3R subtype antibodies were from Santa Cruz Biotechnology. C, Confocal micrographs of rat portal vein sections (longitudinal muscle) costained with the anti-InsP3R1 and anti-InsP3R2 antibodies (Santa Cruz Biotechnology).

Immunodetection of InsP3R subtypes in confocal sections from ureteric and portal vein myocytes was performed with a second set of antibodies from Santa Cruz Biotechnology. With these antibodies, the binding sites were revealed with the same fluorescein-isothiocyanate–conjugated secondary antibody and the specificity was attested by the use of available antigenic peptides. Nonspecific fluorescence (NSF) was determined when specific anti-InsP3R antibody was preincubated with its antigenic peptide 1 hour before application of the immunostaining protocol. When the cell fluorescence obtained with the anti-InsP3R antibody was higher than NSF, the cell was considered as positive and specific fluorescence (F-NSF) could be estimated. The antigenic peptide for InsP3R1 blocked the ability of the anti-InsP3R1 antibody to bind to its target (n=41) but did not interfere with the activity of both anti-InsP3R2 and anti-InsP3R3 antibodies (n=41). Similarly, the antigenic peptides for InsP3R2 and InsP3R3 blocked the activity of the anti-InsP3R2 and anti-InsP3R3 antibodies, respectively, but did not interfere with the ability of the anti-InsP3R1 antibody to bind to its target (n=32). Figure 2B illustrates typical immunostainings obtained in portal vein myocytes from the same dissociation with each of the anti-InsP3R subtype antibodies. All of the cells were stained with the anti-InsP3R1 antibody (Figure 2B; please see Figure IA, available in the online data supplement at http://atvb.ahajournals.org). In contrast, no specific staining was obtained with the anti-InsP3R3 antibody. Staining with the anti-InsP3R2 antibody revealed that only a fraction of myocytes expressed InsP3R2 (7 of 15 cells; Figure 2B; please see online Figure IA). A similar distribution of cell fluorescence was obtained from 6 different dissociations (please see online Figure IB), supporting the idea that all of the cells expressed InsP3R1 whereas approximately 50% of the cells expressed InsP3R2. Quantitatively, specific fluorescence with the anti-InsP3R1 antibody was observed in 271 of 288 vascular myocytes tested (6 dissociations), whereas specific InsP3R2 immunostaining was detected in 231 of 452 cells tested (6 dissociations) and InsP3R3 labeling was never observed (217 cells tested, 6 dissociations) (please see online Figure IC). In ureteric myocytes, specific InsP3R1 and InsP3R3 immunostainings were detected (Figure 2B), respectively, in 74 and 71 of 78 cells (please see online Figure IC), whereas InsP3R2 labeling was never observed (96 cells tested, 5 dissociations). In COS-1 cells, specific immunostaining was obtained for InsP3R2 and InsP3R3 (in 58 and 55 of 60 cells tested, respectively) whereas InsP3R1 staining was never observed (63 cells tested, 4 dissociations; please see online Figure IC). Coimmunostainings in confocal sections of intact portal vein muscle revealed that InsP3R2 was not expressed in all of the myocytes compared with InsP3R1 (Figure 2C). These results confirm that immunostainings with the anti-InsP3R antibodies are specific for the corresponding InsP3R subtypes and reveal a differential expression of InsP3R subtypes in vascular and ureteric myocytes.

The fact that the InsP3R2 subtype was detected by immunostaining in approximately 50% of all of the vascular cells tested prompted us to verify the expression of InsP3R1 and InsP3R2 in myocytes showing either Ca2+ oscillations or single Ca2+ responses. We found that all of the oscillating vascular myocytes expressed both InsP3R1 and InsP3R2 (n=116; 5 dissociations), whereas the nonoscillating myocytes expressed only InsP3R1 (n=121; 5 dissociations).

Specific InsP3R Isoforms Required for Ca2+ Oscillations

To assess the role of InsP3R subtypes in Ca2+ oscillations, we used both subtype-specific antibodies and inhibition of InsP3R subtypes by an antisense strategy. For the antibody experiments, the following protocol was performed. On each myocyte, ACh (1 μmol/L) was first ejected in non–voltage-clamped conditions to detect the oscillating cells, and the parameters of the Ca2+ responses were measured. Then, voltage-clamp conditions were established and a second application of ACh was applied 5 minutes later on the same cells maintained at a holding potential of −50 mV. In portal vein myocytes, the peak Ca2+ response corresponding to the first ACh application (in non–voltage-clamped cells) was 270±39 nmol/L from a resting [Ca2+]i level of 66±8 nmol/L (n=19). The amplitude of the second ACh-induced Ca2+ response (in voltage-clamped cells) was 225±21 nmol/L from a resting level of 69±9 nmol/L (n=19). Similarly, the mean frequency of Ca2+ oscillations during the second application of ACh was 4.2±0.8 oscillations/min, not different from that obtained during the first application of ACh (4.0±0.8 oscillations/min, n=19). Therefore, this protocol was used to examine the role of the InsP3R subtypes on the ACh-induced Ca2+ responses by infusing the cells through the patch pipette with the specific InsP3R subtype antibodies for 5 minutes.

We tested the anti-InsP3R antibodies in nonoscillating vascular cells expressing only InsP3R1 to determine the effective concentrations of these antibodies. In nonoscillating vascular cells, the single Ca2+ responses were inhibited in a concentration-dependent manner by intracellular applications of the anti-InsP3R1 antibody with maximal inhibition obtained at 1 μg/mL (please see online Figures IIA and IIB), whereas both anti-InsP3R2 and anti-InsP3R3 antibodies had no significant effects at concentrations up to 10 μg/mL (please see online Figures IIC and IID). The inhibitory effect of the anti-InsP3R1 antibody is specific, as shown by the absence of effect of boiled (90°C for 30 minutes) anti-InsP3R1 antibody (see online Figure IIB) or antibody preincubated with its antigenic peptide (not shown). In oscillating vascular myocytes, intracellular application of the anti-InsP3R1 antibody inhibited the amplitude of Ca2+ peaks, but oscillations were detected in all the cells tested (Figure 3A and 3B). Interestingly, in the presence of anti-InsP3R1 antibody, the frequency of Ca2+ oscillations was not significantly affected (control, 4.1±0.5 oscillations/min; in the presence of the antibody, 3.9±0.4 oscillations/min; n=6). Intracellular applications of anti-InsP3R2 antibody inhibited both amplitude and generation of Ca2+ oscillations with a maximal effect at 1 μg/mL (Figure 3C). At 1 μg/mL anti-InsP3R2 antibody, ACh was able to induce a single Ca2+ peak without oscillation (Figure 3A). By contrast, the anti-InsP3R23 antibody (1 to 10 μmol/L) had no effect on the ACh-induced Ca2+ responses (not illustrated), in agreement with the absence of detection of InsP3R3 in vascular myocytes. Similar results were obtained with the anti-InsP3R antibodies from Santa Cruz Biotechnology (not shown).

Figure 3. Effects of anti-InsP3R1 and anti-InsP3R2 antibodies on Ca2+ oscillations evoked by ACh (1 μmol/L) in portal vein myocytes. A, Typical recordings showing the effect of 1 μg/mL anti-InsP3R1 or anti-InsP3R2 antibody on Ca2+ oscillations. B, Compiled data showing the effect of increasing concentrations of the anti-InsP3R1 antibody (Affinity BioReagents) on the amplitude of the first Ca2+ peak and the percentage of oscillating cells. C, Compiled data showing the effect of increasing concentrations of the anti-InsP3R2 antibody (AbCys) on the amplitude of the first Ca2+ peak and the percentage of oscillating cells. Filled bars are obtained with boiled antibody. Δ[Ca2+]i is expressed as the ratio of measurements obtained in the presence and absence of anti-InsP3R antibody. Data are mean±SEM, with the number of cells indicated in parentheses (★, P<0.05).

Reverse transcriptase–polymerase chain reaction performed on RNA extracts prepared from rat portal vein and ureteric myocytes confirmed the expression of the different InsP3R subtypes, as identified by Western blotting. Therefore, we designed antisense oligonucleotides specifically targeting each InsP3R subtype. The time course of antisense oligonucleotides efficiency was determined by checking the ability of asInsP3R to inhibit the expression of InsP3R isoform and the amplitude of ACh-induced Ca2+ responses in vascular myocytes. Immunostaining with the anti-InsP3R1 antibody was maximally decreased 2 days after nuclear injection of 10 μmol/L asInsP3R1 in vascular myocytes located in a marked area of glass slides, whereas noninjected cells outside this marked area (used as controls) were normally immunostained (please see online Figure IIIA). Recovery began the next day, and staining of injected cells returned to control 4 days after injection of asInsP3R1. Increasing asInsP3R1 concentration to 20 μmol/L had no additional inhibitory effect on InsP3R1 staining (n=17). Based on this time scale, we verified that the Ca2+ responses were maximally decreased 2 days after injection of asInsP3R1 plus asInsP3R2 (10 μmol/L each) and recovery began the next day (please see online Figure IIIB). Nonspecific effects of antisense oligonucleotides were detected only at concentrations higher than 50 μmol/L (n=10). We also verified that each InsP3R subtype was specifically decreased by the corresponding asInsP3R 2 days after injection. Vascular myocytes injected with asInsP3R2 or asInsP3R3 were normally stained with the anti-InsP3R1 antibody (please see online Figure IIIC). In cells injected with asInsP3R, staining with the anti-InsP3R2 antibody was strongly reduced in immunopositive cells, which represented approximately 50% of the cells examined (please see online Figure IIID). In contrast, in asInsP3R1- and asInsP3R3-injected cells, stainings with the anti-InsP3R2 antibody were similar to those obtained in noninjected cells (please see online Figure IIID). These results indicate that the antisense oligonucleotides were efficient and specific in inhibiting expression of InsP3R subtypes within 2 days after nuclear injection and that they could be used to study the role of each InsP3R subtype on ACh-induced Ca2+ responses. In vascular myocytes injected with 10 μmol/L asInsP3R1, the amplitude of the first Ca2+ peak evoked by 1 μmol/L ACh was reduced by approximately 50% compared with noninjected cells, but oscillations were observed in most of the cells tested (41 of 50 cells, Figure 4). In cells injected with asInsP3R2, the amplitude of the first Ca2+ peak was slightly reduced, but the percentage of oscillating cells was strongly inhibited (7 of 72 cells). No effect on the Ca2+ peak and the percentage of oscillating cells (26 of 50 cells) was observed in cells injected with asInsP3R3 (Figures 4B and 4C). The frequency of oscillations in asInsP3R1-injected cells (4.0±0.3 oscillations/min, n=41) was not different of that in noninjected cells (4.3±0.2 oscillations/min, n=82). All of the cells used induced large Ca2+ responses when 10 mmol/L caffeine was added at the end of this protocol. Cell proliferation was not detected with the anti-PCNA antibody in both freshly dissociated and cultured vascular myocytes (n=250).

Figure 4. Effects of asInsP3R1, asInsP3R2, and asInsP3R3 antisense oligonucleotides on Ca2+ responses evoked by 1 μmol/L ACh in portal vein myocytes. A, Typical ACh-induced Ca2+ responses in control cells and in cells injected with 10 μmol/L asInsP3R1 or asInsP3R2. B, Effects of 10 μmol/L asInsP3R1, asInsP3R2, or asInsP3R23 on the amplitude of the first ACh-induced Ca2+ peaks. C, Percentage of oscillating cells in response to ACh in control cells and in cells injected with the indicated antisense oligonucleotides. Control indicates noninjected cells on the same glass slides. Data are mean±SEM, with the number of cells indicated in parentheses (★, P<0.05).

Taken together, these results suggest that Ca2+ oscillations in vascular myocytes require the presence of InsP3R2 and that InsP3R1 alone or both InsP3R1 and InsP3R3 are unable to produce Ca2+ oscillations in vascular and ureteric myocytes, respectively.

Discussion

The results of the present study establish the functional role of InsP3R subtypes in native smooth muscle cells in response to acetylcholine. We obtained evidence that endogenous expression of InsP3R2 was critical for Ca2+ oscillations in rat portal vein myocytes, whereas endogenous expression of InsP3R1 alone or both InsP3R1 and InsP3R3 was responsible for single Ca2+ responses in vascular and ureteric myocytes, respectively.

For studying the role of each InsP3R subtype in various smooth muscle cells, we used 2 complementary methods, ie, specific anti-InsP3R antibodies and antisense oligonucleotides, which specifically inhibited the expression of each InsP3R subtype. Specificity of the anti-InsP3R antibodies is based on the following observations: (1) in different cell types, Western blotting reveals a differential expression of InsP3Rs, indicating that the anti-InsP3R antibodies do not cross-react with the different InsP3R subtypes; (2) similar differential immunostainings were obtained in the same cells with these anti-InsP3R antibodies; (3) immunostaining for 1 anti-InsP3R antibody is blocked by the COOH-terminal antigenic peptide of this antibody but not by any other antigenic peptides; (4) in functional experiments, these antibodies neutralized by their antigenic peptides or by heating (95°C for 30 minutes) have no effect on ACh-induced Ca2+ responses in 2 different smooth muscle cell types; and (5) the inhibitory effects of each anti-InsP3R antibody on the Ca2+ responses are concentration-dependent, with a maximal effect of approximately 1 μg/mL. For the antisense strategy, appropriate controls have been carried out to demonstrate the efficiency of antisense oligonucleotides. We evaluated the specificity of antisense oligonucleotides to reduce the expression of the InsP3R subtypes labeled with the specific antibodies by showing (1) the specific inhibition of the targeted InsP3R subtype by 1 given antisense oligonucleotide and the lack of effect of this antisense oligonucleotide on the other InsP3R subtypes; (2) the lack of effect of antisense oligonucleotides against InsP3R3 in myocytes that did not express this subtype; and (3) the reduction of the InsP3R-mediated Ca2+ response produced by each effective oligonucleotide compared with the suppression of Ca2+ response after injection of a mixture of oligonucleotides targeting the expressed InsP3R subtypes.

Ca2+ oscillations can be supported by different Ca2+ channels and Ca2+ stores.14,22–24 We showed that the pharmacological inhibition of the mitochondrial Ca2+ uniporter, RYRs, voltage-gated Ca2+ channels, and Ca2+ influx had no effect on ACh-induced Ca2+ oscillations in rat portal vein myocytes. Because inhibition of InsP3R2 by specific antibodies and antisense oligonucleotides suppressed these responses, InsP3R2 seems necessary to support the ACh-induced Ca2+ oscillations. In vascular myocytes, distribution of InsP3R2 has been found at the plasma membrane and associated with the nucleus in proliferating cells.25,26 We show that freshly dissociated and short-term cultured rat portal vein myocytes express InsP3R1 whereas only 50% of the cells express InsP3R2 and generate Ca2+ oscillations. It is unlikely that these 2 populations of myocytes correspond to cells being in different states of development, because staining with the anti-PCNA antibody was not detected in freshly dissociated and short-term cultured myocytes. Immunostaining of intact vascular muscle revealed that both InsP3R1 and InsP3R2 were expressed and that some cells expressed only InsP3R1, confirming the results obtained in dissociated vascular myocytes.

Targeted gene knockouts of InsP3R subtypes in DT40 B cells have revealed that cells expressing different InsP3R subtypes may show distinct patterns of Ca2+ signaling.13 When expressed singly, activation of InsP3R1 or InsP3R3 triggers 1 or 2 Ca2+ peaks, whereas activation of InsP3R2 supports long-lasting, regular Ca2+ oscillations.13 In native cells, such as hepatocytes, cardiac myocytes, and pancreatic islets that show Ca2+ oscillations,27–29 the presence of InsP3R has been demonstrated in separate experiments.30 With specific antibodies and antisense oligonucleotides, we demonstrate that native vascular myocytes expressing only InsP3R2 may generate Ca2+ oscillations of small amplitude, confirming the results obtained in DT40 B cells. In agreement with these data, in native cells that do not express InsP3R2, like A7r5 cells31 or ureteric myocytes (the present study), InsP3R-mediated Ca2+ oscillations have not been observed. Conversely, Ca2+ oscillations were observed in cells expressing a high proportion of InsP3R2 (RBL-2H3 mast cells32) or expressing both InsP3R2 and InsP3R3 (NIH-3T3 cells33). In HeLa cells that show Ca2+ oscillations with a low expression of InsP3R2,33 it has been shown that these oscillations are dependent on the extracellular Ca2+ concentration,34 suggesting that Ca2+ influx may be involved in triggering Ca2+ oscillations in these cells.

Our results support a model in which Ca2+ oscillations depend on Ca2+ release from the intracellular store through both InsP3R1 and InsP3R2. Because InsP3R1 alone is unable to trigger Ca2+ oscillations, it is likely that the properties of InsP3R2 are critical for these oscillations. InsP3R2 has a higher InsP3 sensitivity than InsP3R1.13 In native cells expressing predominantly InsP3R2, a sustained inhibition is obtained by increasing [Ca2+]i,4 a result that is not found in studies using planar lipid bilayers.35 In contrast, InsP3R1 inhibition by high [Ca2+]i has been reported in both bilayers and native cells.36 Because our preliminary experiments using GFP-PHPLCδ expression indicate that the InsP3 production in vascular myocytes does not oscillate in response to 1 μmol/L ACh, we speculate that the Ca2+-dependent inhibition of InsP3R2 may be important in the rapid fall of [Ca2+]i during the first peak, as revealed by the duration at half-maximal amplitude of the first Ca2+ peak, which is shorter in oscillating cells than in nonoscillating cells. Because [Ca2+]i decreases near basal value, InsP3R2 can reopen, and a second Ca2+ peak is initiated with the concomitant participation of InsP3R1. The InsP3- and Ca2+-dependences of InsP3R1 and InsP3R2 are under investigation in native vascular myocytes, because intracellular accessory proteins can strongly modulate InsP3R activity.36

The physiological role of InsP3R2 has not been previously examined in freshly dissociated and short-term cultured vascular myocytes. However, in agonist-stimulated adult cardiomyocytes, it has been reported that activation of InsP3R may trigger additional action potentials leading to arrhythmias.37 Moreover, intracellular Ca2+ oscillations have been shown to support spontaneous contractions during early stages of cardiomyogenesis, before complete expression of ion channels.38 It is tempting to speculate that vascular myocytes expressing both InsP3R1 and InsP3R2 may function as pacemaker cells, initiating and modulating rhythmic electrical and mechanical activities in portal vein. We have previously shown that in these cells, InsP3 is generated under basal conditions and that the InsP3 production is strongly increased by neurotransmitters.39 Because vascular myocytes are electrically coupled by gap junctions, a small fraction of pacemaker cells activated by the basal InsP3 concentration may trigger the spontaneous contractile activity of the vessel. When neurotransmitters are delivered, the frequency of rhythmic contractions is strongly increased,40 as expected from an increase in the InsP3 production, which can stimulate Ca2+ oscillations in all the cells expressing both InsP3R1 and InsP3R2.

In conclusion, the differential expression of InsP3R subtypes seems a determinant factor in the generation of specific Ca2+-signaling patterns in native smooth muscle cells.

*These authors contributed equally to this study.

This work was supported by grants from Centre National de la Recherche Scientifique, Centre National des Etudes Spatiales, and Association Française contre les Myopathies, France. N.F. is supported by a grant from Région Aquitaine. The authors thank N. Biendon for secretarial assistance.

Footnotes

Correspondence to Jean Mironneau, Laboratoire de Signalisation et Interactions Cellulaires, CNRS UMR 5017, Université Bordeaux 2, 146 rue Léo Saignat, 33076 Bordeaux Cedex, France. E-mail

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