Dominant role of smooth muscle L-type calcium channel Cav1.2 for blood pressure regulation

Abstract

Blood pressure is regulated by a number of key molecules involving G-protein-coupled receptors, ion channels and monomeric small G-proteins. The relative contribution of these different signaling pathways to blood pressure regulation remains to be determined. Tamoxifen-induced, smooth muscle-specific inactivation of the L-type Cav1.2 Ca2+ channel gene in mice (SMAKO) reduced mean arterial blood pressure (MAP) in awake, freely moving animals from 120 +/- 4.5 to 87 +/- 8 mmHg. Phenylephrine (PE)- and angiotensin 2 (AT2)-induced MAP increases were blunted in SMAKO mice, whereas the Rho-kinase inhibitor Y-27632 reduced MAP to the same extent in control and SMAKO mice. Depolarization-induced contraction was abolished in tibialis arteries of SMAKO mice, and development of myogenic tone in response to intravascular pressure (Bayliss effect) was absent. Hind limb perfusion experiments suggested that 50% of the PE-induced resistance is due to calcium influx through the Cav1.2 channel. These results show that Cav1.2 calcium channels are key players in the hormonal regulation of blood pressure and development of myogenic tone.

Fig. 1. SMC-specific inactivation of the Ca_v1.2 gene. (A) Schematic representation of the wild-type (WT), the knockout (L1) and the conditional Ca_v1.2 alleles (L2). The numbers indicate the exon number. SMC-specific activation of tamoxifen-inducible Cre recombinase [SM-Cre ER^T2(ki)-Cre] results in the deletion of Ca_v1.2 exons 14 and 15. Restriction sites are A, Acc65I; B, BamHI; C, ClaI; EI, EcoRI. (B) Detection of lacZ gene expression in tibialis arteries of a tamoxifen-treated SM-Cre ER^T2(ki); Rosa-lacZ mouse. Blue staining indicates Cre-mediated recombination. (C) RT–PCR analysis of mesenteric arteries of +/L2 mice before (–Tam) and after (+Tam) tamoxifen treatment. L2/Wt and L1 bands represent L2/wild-type and L1 transcripts, respectively. The L1 band after tamoxifen treatment (+Tam) is generated by Cre-mediated recombination; the remaining L2/WT band is due to the remaining WT allele after recombination. (D) Western analysis of proteins from tibialis arteries using an anti-Ca_v1.2 antibody demonstrates the absence of Ca_v1.2 protein (arrowhead) in vessels from SMAKO mice. β-actin (∼43 kDa) was used as loading control.

Fig. 2. Kinetics and pharmacology of smooth muscle L-type I_Ba. (A and B) Results obtained with SMCs isolated from the tibialis artery of control (Ctr) and SMAKO mice (KO), respectively. Current voltage (I–V) relationships were recorded in the absence and presence of 1 µM Bay K 8644 or nisoldipine. The HP was 80 mV. Cells were depolarized to potentials between –60 and +60 mV with 10 mV increments for 100 ms at 0.2 Hz.

Fig. 3. Reduced arterial blood pressure in SMAKO mice. Blood pressure was measured in conscious, freely moving mice by telemetry. (A) Original tracing showing the effect of tamoxifen application (1 mg/day for 5 days) leading to Cre-mediated Ca_v1.2 gene recombination on MAP in a SMAKO mouse. (B) Basal blood pressure and effect of an i.p. bolus (200µl) injection of PE (10 µg/kg), AT2 (5 µg/kg), isradipine (Isra; 0.5 mg/kg) and Y-27632 (30 mg/kg) on MAP in awake control (Ctr) and SMAKO (KO) mice analyzed 21–28 days after tamoxifen injection. (C) Original tracing of an experiment statistically analyzed in (B). Arrows indicate time of i.p. injection of compounds.

Fig. 4. Decreased isometric force generation of aortic rings from SMAKO mice. (A) Original tracings and (B) statistical analysis of contractions of isolated aortic rings from control (Ctr) and SMAKO (KO) mice. Force generation in response to 85 mM K⁺ (K85) and 1 µM PE measured with or without 1 µM isradipine (Isra) or 5 µM Y-27632 as indicated. Force in (B) is normalized to vessel length. (C) Relaxation of aortic rings pre-contracted with 1 µM PE by 1 µM isradipine and 5 µM Y-27632. Force levels are expressed as a percentage of the maximum contractile force in SMAKO mice. *P < 0.05 versus the same condition in control mice.

Fig. 5. Tibialis arteries from SMAKO mice show a blunted response to vasocontracting agonists. (A) Original tracings showing the effect of superfusion with 85 mM K⁺ (K85) and 1 µM PE on luminal diameter in vessels from control (Ctr) and SMAKO (KO) mice. (B and C) Contractile response of tibialis arteries from control mice before and after application of 1 µM isradipine (Isra) and from SMAKO mice. Vessels were superfused with (B) 1 µM PE or (C) 500 nM AT2. *P < 0.01.

Fig. 6. Pressure- and depolarization-induced myogenic tone is absent in SMAKO mice. (A) Myogenic responses of tibialis arteries of SMAKO (KO) mice and control mice (Ctr) before and after treatment with isradipine (Isra). Myogenic tone is expressed as a percentage of the difference to the passive vessel diameter in Ca²⁺-free solution at each given pressure. (B) Myogenic response at 60 mmHg of control (Ctr) and SMAKO (KO) tibialis arteries superfused with 85 mM K⁺ (85K) or 5.6 mM K⁺ (PK) with or without 1 µM isradipine (Isra). The myogenic response is shown as a percentage of the diameter in control vessels at 5.6 mM K⁺ concentration.

Fig. 7. SMAKO mice analyzed in a perfused hind limb model show reduced tone and vasoconstrictor responses. Both hind limbs were perfused via the infrarenal aorta abdominalis with modified Krebs–Henseleit solution. (A) Original tracing showing the effect of bolus (200 µl) applications of PE on perfusion pressure in the absence or presence of 1 µM isradipine (Isra) and 5 µM Y-27632 in a control mouse. Numbers above the arrows indicate the concentration of the agonist applied with each bolus (log mol). Flow rate was adjusted before application of PE to compensate perfusion pressure reduction by the vasorelaxing compounds. (B) Parameters of the perfused hind limb of control mice with and without 1 µM isradipine and of SMAKO mice. Peripheral resistance is expressed as perfusion pressure divided by flow rate. (C and D) Vasocontraction response at constant flow to bolus (200 µl) applications of PE (log mol) in the absence or presence of 1 µM isradipine (Isra) and 5 µM Y-27632 in (C) control mice (Ctr) or (D) SMAKO mice. Vasocontraction response is shown as ΔPerfusion pressure [mmHg] (change in perfusion pressure) at constant flow.