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					                                                     MS#150 On-line Materials and Methods Page 1


                                   Baron et al, MS#150

                                         ON-LINE

                              MATERIALS and METHODS



       Atrial myocyte cultures. Atrial appendage myocytes from 2-3 day-old rats were

cultured as described previously (27). Briefly, atrial appendages were minced, dispersed both

enzymatically with 0.2% trypsin (Worthington) and mechanically by repeated pipetting in

Ca2+- and Mg2+-free medium. Cells were grown in 10% fetal calf serum-containing culture

medium, a 1:1 mixture of Dulbecco’s modified Eagles medium (DMEM) and Ham’s F-12

(Gibco), for 2-4 days in the presence of 1 µmol/L dexamethasone and 1 µmol/L

triiodothyronine in a 5% CO2 incubator. Cultured atrial myocytes were shown to synthesize

ANP by immunostaining (not shown). Between 10% to 50% of the myocytes contracted

spontaneously (at a rate of 0.1 to 1 Hz), and non-contracting myocytes could be induced to

contract by mechanical stimulation, indicating that the myocytes were normally polarized.

       Patch-clamp recording of KATP current. KATP currents were recorded on culture

days 2 to 4 at room temperature from initially beating atrial myocytes using the whole-cell

and the inside-out configurations of the patch-clamp technique (28). The cells were observed

with an inverted microscope (Diaphot TMD Nikon, Tokyo, Japan). Currents were recorded by

an Axopatch 200B patch-clamp amplifier, digitized by a TL-1 DMA interface and sampled by

a PC computer with the pCLAMP software (all from Axon Instruments, CA, USA).

Borosilicate glass patch pipettes were pulled with a BB-CH puller (Mecanex SA, Nyon,

Switzerland) and their resistance was 2-4 M for whole-cell recordings and 5-10 M for

inside-out recordings. For whole-cell recordings, the standard pipette solution contained

(mmol/L): KCl 121 ; CaCl2 1.5 ; EGTA 10 ; MgCl2 1.3 ; ATP 0-5 ; Glucose 10 ; KOH 34 ;

HEPES 10 (pH 7.45 with KOH), and the bathing solution contained (mmol/L): KCl 5 ; CaCl2
                                                      MS#150 On-line Materials and Methods Page 2


1 ; MgCl2 1 ; NaCl 118 ; Glucose 10 ; HEPES 10 (pH 7.5 with NaOH). The osmolality of the

hypotonic solution (stimulus) was 260 mOsm/kg H2O, and sucrose was added to yield a

solution of 290 mOsm/kg H2O. Currents were filtered at 2 kHz and sampled at the frequency

of 0.8 kHz. For inside-out recordings, the pipette solution contained (mmol/L): KCl 140 ;

CaCl2 1 ; MgCl2 1 ; HEPES 5 (pH 7.3 with KOH), whereas the bathing solution contained

(mmol/L): KCl 5 ; KOH 15.5 ; NaCl 135 ; MgCl2 1 ; EGTA 5 ; Glucose 10 ; HEPES 5 (pH

7.3 with KOH). Currents were filtered at 1 kHz and sampled at 6 kHz. The probability of a

channel to be opened (Po) was expressed as the time spent in the open state (To) divided by

the total recording time (T): Po = To/T. Po is usually calculated over a period of 15 seconds.

When several (N) identical channels are simultaneously recorded on the same patch, the

probability of one channel to be opened is calculated as follows: Po = (To1 + 2To2 + 3To3

+...+NToN) / NT, with ToN being the time spent by a channel at the open level N. Burst

kinetics analysis, open and close time duration histograms were only performed where a

single channel was present on the membrane patch.

       Just after the breaking of the patch membrane, the spontaneously beating myocytes

showed an initial membrane potential of -20.8 ± 1.7 mV (n = 45) and -25.6 ± 1.8 mV (n = 71)

with 1 mmol/L ATP and 5 mmol/L ATP in the patch pipette, respectively. After several

minutes of recording, the membrane potential usually increased, reaching -9.5 ± 2.4 mV (n=

10) and -16.6 ± 2.9 mV (n = 15) after 3 minutes with 1 mmol/L and 5 mmol/L internal ATP,

respectively, a change probably due to the dilution of some cytosolic compounds by the

pipette solution. The fact that atrial myocytes showed spontaneous contractile activity

suggests that the membrane resting potential was normally polarized before the cells were

patched.

       Chemicals and drugs. Ethylene glycol-bis(ß-aminoethylether)-N,N,N’,N’-tetraacetic

acid (EGTA), ATP, glyburide, diazoxide and cromakalim were all purchased from Sigma
                                                            MS#150 On-line Materials and Methods Page 3


Chemical (St Louis, MO, USA). Glyburide, cromakalim and diazoxide were dissolved in

DMSO at the stock concentration of 10, 10 and 100 mmol/L respectively.

       RT-PCR analysis for KATP channel subunits. Total RNA from 4 day-old cultured

neonatal atrial appendage myocytes was extracted by Trizol Reagent (Gibco BRL) and

subjected to RQ1 DNase (Promega) digestion. 3 µg of total RNA were used for reverse

transcriptase experiments. cDNAs were synthesized with oligo (dT) primers and Superscript

II reverse transcriptase (200U, Gibco BRL) at 42 C according to the manufacturer's

recommendation. For polymerase chain reaction (PCR) analysis of K ATP channel subunits,

primers were based on the mRNA sequences for rat Kir6.1, Kir6.2, SUR1A, SUR1B, SUR2A

and SUR2B, and for mouse SUR2C (see details in Legend to Fig. 7). The primers used were:

Kir6.1 sense primer 5'-TACATGGAGAAAGGCATCACGG-3', Kir6.2 sense primer 5'-

AGGTACCGTACTCGGGAGAGG-3',                        Kir6.1/Kir6.2            antisense           primer     5'-

GCCGTCTTCATGAAGATGCAGCC-3' (PCR products: 236 bp for Kir6.1 and 443 bp for

Kir   6.2);         SUR1A/SUR1B          sense   primer      5'-GTTCCAGCAGAAGCTCCTAG-3',

SUR1A/SUR1B antisense primer 5'-CTGTCATAGCGTACACTCAGG-3' (402 bp for

SUR1A         and       288      bp       for    SUR1B);         SUR2          sense          primer     5'-

CGAAAGAGCAGCATACTCATTA-3',                       SUR2A         specific      antisense         primer    5'-

GGTCATCACCAAAGTAGAAAAG-3' (249 bp), SUR2A/SUR2B antisense primer 5'-

CCTCTCTTCATCACAATGACC-3' (349 bp for SUR2A and 173 bp for SUR2B);

SUR2A/SUR2C           sense primer 5'-TCTTGAGCGATGAGATTGGC-3', SUR2A/SUR2C

antisense primer 5'-ACTTTTCCTTCCAGGGTCTGC-3' (358 bp for SUR2A and 252 bp for

SUR2C);       rat    gene     encoding    cytoplasmic     ß-actin     (J00691)        sense     primer   5'-

AGGCATCCTGACCCTGAAGTA-3',                                 antisense               primer                 5'-

GCCATCTCTTGCTCGAAGTCT-3' (497 bp); rat gene encoding ANP (K2062) sense primer

5'-CAGCAAAGCTTAGATCGTGC-3', antisense primer 5'-GCTTGGGATCCTTTGCGATC-
                                                      MS#150 On-line Materials and Methods Page 4


3' (535 bp). PCR was carried out in a Biometra personal cycler in a final volume of 50 µl

containing 1 µl of the RT reaction, 1.5 units of Taq polymerase (Gibco BRL), 1.5 mmol/L

MgCl2, 250 µmol/L of each dNTP and 20 pmoles of each primer. In some cases, the final

volume was reduced to 20 l with an equally successful outcome. The PCR conditions were

the following: initial denaturation at 94 C for 3 min followed by 35 cycles of denaturation at

94 C for 45 sec, annealing at 60 C for 30 sec and extension at 72 C for 1 min, with a final

extension step at 72 C for 10 min. Negative (- RT) controls were included. The PCR

products were separated by electrophoresis on a 1.5% agarose gel and visualized with

ethidium bromide under UV fluorescence. After extraction and purification of the PCR

products, their nature was confirmed by a double-strand sequencing on both strands using the

Big Dye terminator kit on an automatic ABI377 sequencer (both from Perkin Elmer).

				
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posted:11/1/2011
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Lingjuan Ma Lingjuan Ma
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