Abstract

Introduction

Materials & Methods

Results

Discussion & Conclusion

References

Discussion
Board

SERCA 3 study

A mouse genomic library (129SVJ in Lambda Fix vector, Stratagene) was screened with parts of the rat SERCA 3 cDNA. Selected genomic clones were subcloned and a restriction map constructed. Fragments corresponding to the 3' end of the gene were sequenced in both directions (Sequenase kit, USB).

Total RNA from various rat and mouse tissues were isolated using the guanidinium thiocyanate procedure (RNA instapure, Eurogentec). RNase protection assay was performed on 20 µg of total RNA from adult rat aorta and trachea. A 807 bp Sma I-Pst I fragment corresponding to nt 2471-3277 of the rat cDNA RK 8-13 [3] was used to prepare the cRNA probe. The full-lenght probe, purified on denaturing polyacrylamide gel, was hybridized to total RNA at 39°C overnight. Unreacted cRNA was digested by 50 U of RNase T1 at room temperature for 30 min, following RPA II procedure (Ambion, Clinisciences). Yeast tRNA was used as a negative control. After digestion, protected fragments were separated by electrophoresis and exposed to X-ray film (Kodak, France).

Five µg of total RNA from mouse tissues were used for reverse transcription using Ready-To-Go You-Prime first-Strand beads (Pharmacia Biotech) and 0.2 µg of random hexamers (Boehringer Mannheim). First strand DNA was amplified in presence of AmpliTaq DNA polymerase (Thermus aquaticus; Perkin Elmer) and 40 pmoles of each specific primer hybridized at 64°C. PCR products were separated on 12% polyacrylamide gel, stained with vistra green (Amersham Life Science) and then electroblotted onto nylon membrane (Parablot NY plus; Macherey-Nagel). Hybridization was done at 39°C overnight with an internal oligonucleotide (nt 2925-2941 ) The membrane was exposed to the phosphorimager screen which was scan on a Storm (Molecular Dynamics)

SERCA 2a and SERCA 2b expression study

Rat aortic smooth muscle cells (RASMC) were isolated from thoracic aorta (aortic cross excluded) of 180 to 200g male Wistar rats. The endothelial cells were removed by scraping and the adventitia removed with forceps. The medial layer was then cut into small pieces and the RASMC isolated by enzymatic digestion with collagenase (CLS 2, Worthington 50U/ml) and pancreactic elastase (0.25 mg/ml, Sigma) for 2 hours at 37°C. The cells were suspended in DMEM containing 10% fetal calf serum and plated onto glass coverslips coated with collagen I (rat tail, Sigma), they were incubated at 37°C in an atmosphere containing 5% CO₂, 95% air, and the culture medium was changed every two days.

RASMC from primary cultures, plated onto dish glass coverslips coated with collagen I, were incubated in DMEM containing 10% fetal calf serum and 3µM fura-2/AM for 30 min at 37°C in an atmosphere containing 5% CO2/95% air. For experiments in Ca²⁺-free medium, [ Ca²⁺]e = 0, CaCl₂ was omitted and 0.1mM EGTA was added. The coverslips were placed in a thermostatically controlled holder (34°C) on the stage of a Zeiss Axiovert 35 microscope set up for epifluorescence microscopy. Cells were continuously superfused with control or test solutions by six inlet tubes converging on the coverslip chamber. The perfusion rate was 1.5 to 2 ml/min and the chamber volume was about 0.2 ml. The medium was continuously removed and replaced by aspiration.

The antibodies specific for SERCA 2a and SERCA 2b (a gift from F. Wuytack, Leuven, Belgium) were raised in rabbit against the C-terminal portion of the rat sequence and have been described elsewhere [12]. The cells were fixed in paraformaldehyde (4%) for 20 min at room temperature, washed in phosphate-buffered saline (PBS) and in PBS containing 50mM NH₄Cl, and then permeabilized with 0.1% Triton X-100 (6 min). The cells were washed three times, then incubated with diluted antibodies (PBS containing 3% bovine serum albumin): a-SERCA 2a and a-SERCA 2b (1/250), for 120 min at room temperature. The cells were then incubated with a 1/200 dilution of biotinylated anti-rabbit Ig (Amersham) and with Streptavidin-Texas Red (1/200). Coverslips were mounted in glycerol/PBS medium (Citifluor, UKC, Chem. Lab. Canterbury) and fluorescence detected with a Leitz microscope equipped with epifluorescence optics.

Total RNA from aorta or from RASMC in culture (1 µg) was used for reverse transcription in a medium containing PCR buffer II (Perkin Elmer, 50 mM KCl, 10 mM Tris-HCl, pH 8.3), 5mM MgCl₂, 1mM of each dNTP (Promega), 20U RNasin (Perkin Elmer), 50U Murine Moloney Virus reverse transcriptase (Perkin Elmer), 2.5 µM random hexamers (Boehringer Mannheim). Two PCR reactions were set up from each first strand reaction and SERCA 2b was used as a standard for each reaction. A control without reverse transcriptase was included in each experiment. The reactions were performed in a final volume of 100 µl containing 2mM MgCl₂, 2.5U AmpliTaq DNA polymerase (Perkin Elmer) and 0.5 µM of each of the specific upstream and downstream primers which annealing temperature was 60°C. The PCR products were subjected to electrophoresis in an 8% polyacrylamide gel and were stained using VISTRA-green (Amersham). The gels were scanned with a phosphor-fluorimager (Storm, Molecular Dynamics).

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