Knockout of the alpha 1A/C-adrenergic receptor subtype: the alpha 1A/C is expressed in resistance arteries and is required to maintain arterial blood pressure

Abstract

alpha 1-adrenergic receptors (ARs) play a major role in blood pressure regulation. The three alpha 1-AR subtypes (A/C, B, and D) stimulate contraction of isolated arteries, but it is uncertain how different subtypes contribute to blood pressure regulation in the intact animal. We studied the role of the alpha 1A/C subtype by using gene knockout. alpha 1A/C knockout (KO) mice were viable and overtly normal. The LacZ reporter gene replaced alpha 1A/C coding sequence in the KO, and beta-galactosidase staining was present in resistance arteries and arterioles, but not in the thoracic aorta or its main branches. By tail cuff manometer and arterial catheter in conscious mice, alpha 1A/C KO mice were hypotensive at rest, with an 8-12% reduction of blood pressure dependent on alpha 1A/C gene copy number. A61603, an alpha 1A/C-selective agonist, caused a pressor response that was lost in the KO and reduced but significant in heterozygous mice with a single copy of the alpha 1A/C. A subtype-nonselective agonist [phenylephrine (PE)] caused a pressor response in KO mice, but the final arterial pressure was only 85% of wild type. The baroreflex was reset in the KO, and heart rate variability was decreased. After baroreflex blockade with atropine, PE increased blood pressure but did not change heart rate. Cardiac and vascular responses to the beta-AR agonist isoproterenol were unchanged, and the arterial lumen area was not altered. We conclude that the alpha 1A/C-AR subtype is a vasopressor expressed in resistance arteries and is required for normal arterial blood pressure regulation. alpha 1A/C-selective antagonists might be desirable antihypertensive agents.

Figure 1

Targeted deletion of exon 1 of the α1A/C-adrenergic receptor gene. (A) Targeting strategy. A LacZ-Neo cassette replaced exon 1 of the α1A/C and introduced an EcoRV site. (B) Southern with EcoRV in an F2 litter. Probe B reveals a 16-kb band with the WT allele and a 11.2-kb band with the KO. Probe A to exon 1 is negative in the KO. (C) RT-PCR. Primers from exons 1 and 2 (arrows) were used with 0.5 μg of total RNA from heart and brain. Ethidium bromide staining shows a 524-bp product in WT but not in KO (Upper), as confirmed by Southern with a probe to exon 1 (Lower). (D) β-Galactosidase in E12.5 embryos. Blue staining of E. coli β-galactosidase is proportional to LacZ copy number in WT (no copies), Het (one copy), and KO (two copies).

Figure 2

β-Galactosidase staining localizes arterial expression of the α1A/C. β-Galactosidase was stained in situ (blue), and the major arteries (A–D) or skin from the chest (E) were dissected and photographed. Skin in E is from the epidermal side after removing the fur. (F) A skin section was counterstained with hematoxylin and eosin to show a hair follicle.

Figure 3

Carotid artery pressure with A61603 and PE in conscious α1A/C KO mice. Drugs were infused through a right jugular vein catheter in conscious, unrestrained mice 24 h after catheterization of the left carotid artery. Mice were the same as Table 2 (Carotid artery catheter). In A, the zero dose values were with 50 μl of saline.

Figure 4

Altered baroreflex in α1A/C KO mice. HR data from PE infusions (Fig. 3) are plotted against PE dose (Upper) and MAP (Lower).

Figure 5

Blood pressure and HR with atropine and isoproterenol in α1A/C KO mice. The protocol was as in Fig. 3. Isoproterenol (3 μg/kg) was given after the response to PE had returned to baseline. Then atropine (1 mg/kg) was given to block the baroreflex. After 4 min, when parasympathetic blockade was complete and MAP and HR had stabilized, PE was again infused at a maximum dose (100 μg/kg). In these experiments, baseline HR was WT 551 ± 12 (n = 18), KO 587 ± 24 (n = 14), P = not significant. Baseline MAP was WT 128 ± 3, KO 115 ± 2, P = 0.006.