Büssemaker et al., SHR and EDHF, Online data supplement MS# 03-1915

Expanded Material & Methods Section

Materials

Sodium sulfaphenazole was synthesized by Marck Bioscience AG (former Clinalfa, Läufelfingen, Switzerland). Connexin blocking GAP peptides were synthesized by Sigma-Genosys, Cambridge (UK) according to the sequence published previously1. All other compounds were from Sigma (Deisenhofen, Germany). Male spontaneously hypertensive rats (SHR) and age-matched Wistar-Kyoto rats (WKY) were obtained from Charles-River (Sulzfeld Germany).

Organ chamber experiments

Animals were killed by decapitation. The renal arteries were isolated, cleaned of connective tissue and cut into rings. Rings were mounted on stainless steel wires connected to force transducers and placed in individual organ chambers containing modified Tyrode’s solution of the following composition (in mmol/L): NaCl 132, KCl 4, CaCl2 1.6, MgCl2 1.2, NaH2PO4 0.36, NaHCO3 23.8, Ca2+-EDTA 0.05, glucose 10 (gassed with 20% O2 / 5% CO2 / 75 % N2, pH 7.4). Diclofenac (10 µmol/L) was included in all experiments to inhibit prostaglandin synthesis. Passive tension was gradually increased to 1g and each ring was challenged three times with KCl-rich (80 mmol/L) buffer. Precontraction was elicited using phenylephrine at a concentration adjusted to obtain a similar contraction in each ring (approximately 80% of initial KCl-induced contraction). When a stable contraction was obtained, cumulative concentration-relaxation curves to either acetylcholine (ACh,1 nmol/L to 10 µmol/L) or KCl (4 to 10 mmol/L) were obtained. EDHF-mediated responses were defined as that portion of the endothelium-dependent relaxation that remained in the presence of Nw-nitro-L-arginine (L-NA, 300 µmol/L) and diclofenac.

Membrane potential recordings

Renal artery rings were opened longitudinally, pinned to the sylgard base of a heated bath with the intimal side upward and superfused (5 mL min-1, 37°C) with modified Tyrode's solution. All experiments were performed in the presence of L-NA (300 µmol/L), diclofenac (10 µmol/L) and phenylephrine (30 nmol/L) to mimic conditions in the organ chamber experiments. The membrane potential was recorded with glass capillary microelectrodes (tip resistance of 80 to 120 MW) filled with KCl (3 mol/L) and connected to a high impedance amplifier (intra 767, WPI, Berlin) as described 2. Successful impalements were characterized by a sudden negative drop in potential from the baseline (zero potential reference), followed by a stable negative potential for at least 3 min. ACh (1 µmol/L) and KCl (5 mmol/L) were applied as bolus injections into the bath.

Real-time PCR analysis

The sequences for the human and rat a1, a2, a3 and g subunits of the Na+/K+ ATPase, available from the NCBI database, were compared and areas with high homology were chosen for primer design. Primers were analyzed for suitability in PCR with the Primer 3 software provided by the Whitehead Institute for Biomedical Research3 and subsequent structural analysis by the mfold software provided by the Rensselaer Polytechnic Institute4. The primers used were as follows: a1 subunit: upstream: AGGGCAGTGTTTCAGGCTAAC, downstream: CCGTGGAGGAGGATAGA(A/G)CTG (fragment size 287 bp); a2 subunit: upstream: TCCTGTGGCTCAGTGAGGAA, downstream: GCATCTCCTTGTCAAG(A/C)GGGAT, (fragment size 213 bp); a3 subunit: upstream CCTGTACTCAAGAGGGA(C/T)GTGG downstream: Cgcccttcatcactaacaggtatc (fragment size 158 bp); g subunit: upstream: gactatgaaaccgt(c/t)cgcaa, downstream: CGTCACAG(C/T)TCATCTTCATTGA (fragment size 139 bp).

Total RNA was isolated from powdered rat arteries using TriReagent (Sigma) and 1 µg of total RNA was used for reverse transcription (Superscript II, Invitrogen) with pdN6 (Amersham/Pharmacia) primers in a total volume of 50 µl. These reactions (2 µl) were used for the amplification with the oligonucleotides described above with TAQ polymerase (Sure start, Stratagene) and SybrGreen in accordance with the manufacturers instructions. The PCR conditions were as follows: initial denaturation: 95°C, 10 minutes; 40 cycles of annealing (1 minute), elongation (72°C, 1 minute) and denaturation (95°C, 1 minute) and a final elongation step at 72°C for 7 minutes. The annealing temperature for the a2 and a3 subunits was 58°C, and 55°C for the a1 and g subunits. Reactions which resulted in the amplification of a large amount of non-specific product were discarded. In each PCR experiment controls lacking the templates were performed in parallel.

Statistical analysis

Values presented are mean ± S.E.M and were compared by t-test or two-way ANOVA for repeated measurements followed by the Newman-Keuls test. N refers to the number of animals used. A P-value less than 0.05 was considerate to be significant.


Online supplemental Table I. Negative logarithm of half maximal effective concentration (pD2) and maximal relaxation (Rmax)

Strain Age Condition Agonist Fig. pD2 Rmax n

(nmol/L) (%)

Responses in the presence of diclofenac (10 µmol/L)

WKY young control ACh 1A 7.67±0.13‡ 100±0 6

SHR young 8.06±0.10‡ 98±1 6

WKY aged 7.07±0.18‡† 96±8 6

SHR aged 6.58±0.22‡† 95±3 6

Responses in the presence of diclofenac (10 µmol/L) and KCl (40 mmol/L)

WKY young control ACh 1B 7.01±0.14‡ 89±2 6

SHR young 7.16±0.15‡ 84±14 6

WKY aged 6.73±0.12‡† 85±5 6

SHR aged 6.01±0.24‡† 84±8 6

Responses in the presence of diclofenac (10 µmol/L) and L-NA (300 µmol/L)

WKY young control ACh 1C 7.21±0.08‡† 68±4† 24

SHR young 7.53±0.07‡† 95±2†‡ 24

WKY aged 6.65±0.13‡ 72±6† 6

SHR aged 6.47±0.20‡ 14±6†‡ 6

WKY young control ACh 3A 7.54±0.24 38±8 6

oubain 50 7.26±016 42±10 6

oubain 500 n.a. 7±4* 4

SHR young control ACh 3B 7.54±0.22 72±5 6

oubain 50 7.60±0.13 70±7 6

oubain 500 6.59±0.20* 81±12* 4

WKY young control ACh 4A 7.20±0.07 81±5 6

sulfaphenazole 7.32±0.07 73±6

SHR young control 7.46±0.14 93±2 6

sulfaphenazole 7.34±0.19 78±7*

WKY young control ACh 4B 6.92±0.35 72±5 6

ODYA 7.30±0.07 68±13

SHR young control 7.43±0.14 96±2 6

ODYA 7.57±0.15 91±1

WKY young control ACh 4C 7.17±0.08 81±3 6

miconazole 7.13±0.26 64±5*

SHR young control 7.53±0.16 95±2 6

miconazole 6.79±0.14* 84±5

WKY aged control ACh 5A 7.50±0.30 42±4 6

oubain 50 7.60±0.13 23±4*

SHR aged control ACh 6.58±0.22 14± 5 6

oubain 50 n.a. 97±4*

WKY aged control 1-EBIO 5E 4.15±0.30 95±2 4

SHR 4.39±0.23 93±2 4

WKY aged Cromakalim 5F 7.07±0.14 98±4 4

6.88±0.12 97±4 4

*= P<0.05 vs. control (age and strain identical) by paired T-test

†=P<0.05 WKY vs SHR (age identical) by two-way ANOVA

‡=P<0.05 Young vs. aged (strain identical) by two-way ANOVA


References

1. Chaytor AT, Martin PE, Edwards DH, Griffith TM. Gap junctional communication underpins EDHF-type relaxations evoked by ACh in the rat hepatic artery. Am J Physiol Heart Circ Physiol. 2001;280:H2441-H2450.

2. Bussemaker E, Popp R, Binder J, Busse R, Fleming I. Characterization of the endothelium-derived hyperpolarizing factor (EDHF) response in the human interlobar artery. Kidney Int. 2003;63:1749-1755.

3. Rozen S, Skaletsky H. Primer3 on the WWW for general users and for biologist programmers. Methods Mol.Biol. 2000;132:365-386.

4. SantaLucia J, Jr. A unified view of polymer, dumbbell, and oligonucleotide DNA nearest- neighbor thermodynamics. Proc.Natl.Acad.Sci U.S.A. 1998;95:1460-1465.

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