Changes in calcium-sensing receptor- and GRPC6A-mediated responses in mesenteric arteries from spontaneously hypertensive rats

Erika Harno1, Arthur H Weston1, Martial Ruat2, Robert H Dodd2, Gillian Edwards1

1University of Manchester, Manchester, UK, 2CNRS, Gif-sur-Yvette, France

Hypertension can lead to vascular damage. Since the calcium-sensing receptor (CaR) and GPRC6A have been shown to be present and potentially to have a role in vascular control of rat mesenteric arteries (Weston et. al, 2005; Harno et. al, 2008), the present study was to undertaken to compare changes in vascular responses and protein expression of CaR and GPRC6A in an animal model of hypertension.

Spontaneously hypertensive rats (SHR) and age-matched Wistar-Kyoto rats (WKY) of 10 -14 weeks had their systolic and diastolic blood pressure, heart rates and weights measured using tail-cuff plethysmography. Animals were then sacrificed in accordance with Schedule 1 before mesenteric arteries were dissected for sharp microelectrode recordings, pressure myography or Western blot analysis. Pressure myograph experiments were carried out in the presence of 100 nM iberiotoxin to inhibit BKCa and artery segments were pre-contracted with U46619. Where appropriate, vessels were incubated with 300 μM L-ornithine (GPRC6A agonist) prior to contraction. Western blotting was carried out on 8% resolving gels and membranes were incubated with antibodies for either CaR or GPRC6A before being stripped and re-probed for β-actin. Results were analysed by 2-way ANOVA or Student’s t-test as appropriate and are expressed mean ± s.e.mean. A P-value of 0.05 was considered significant.

SHR and WKY rat heart rates and body weights were similar. The SHRs had, as expected, higher systolic and diastolic blood pressures (systolic: 240 ± 9 vs. 180 ± 4 mmHg, P<0.001; diastolic: 160 ± 9 vs. 110 ± 9 mmHg, P<0.01, n=9-10). In sharp microelectrode experiments, the positive allosteric modulator of CaR and GPRC6A, calindol (1 μM), produced endothelium-dependent hyperpolarisations of mesenteric artery myocytes which were significantly reduced in SHR arteries (WKY, 8.0 ± 1.7 mV; SHR 3.3 ± 1.0 mVP<0.05, n=4). L-ornithine alone (300 μM) produced similar hyperpolarisations in arteries from SHR and WKY rats (4.5 ± 0.3 mV vs. 3.7 ± 1.4 mV, respectively, P>0.05, n=4). However, in the presence of L-ornithine, the increases in membrane potential to 300 nM calindol in SHR arteries (5.6 ± 1.3 mV) were significantly less than those in WKY vessels (12.5 ± 0.7 mV, P<0.01, n=4). Using pressure myography, there was no difference between the relaxation to calindol in WKY and SHR vessels. However, there was a difference between SHR and WKY in the slope of the curve to calindol in the presence of L-ornithine (P<0.05, n=8). In addition, there was a significant reduction in the magnitude of relaxations to both 1 μM and 3 μM calindol in the presence of L-ornithine (1 μM: SHR 48.7 ± 7.2%; WKY 76.6 ± 4.4%; 3 μM: SHR, 74.9 ± 3.0%; WKY 89.6 ± 3.4%, both P<0.01, both n=8). Quantitative Western blotting showed a reduction in the expression of CaR protein in SHR lysates (% β-actin: 38.9 ± 4.5 vs. 103.9 ± 26.5, P<0.05, n=6). However, there was no change in GPRC6A protein expression.

In conclusion, loss of CaR alone probably accounts for the reduced membrane hyperpolarisations to calindol in arteries from hypertensive animals. These changes in responses and protein expression could contribute to the development or maintenance of the hypertensive state.

This study was funded by the British Heart Foundation.

Harno, E et al. (2008) Cell Calcium 44: 210-9;

Weston, AH et al. (2005). Circ Res 97: 391-8.