Decreased blood pressure response in mice deficient of the alpha1b-adrenergic receptor

Abstract

To investigate the functional role of different alpha1-adrenergic receptor (alpha1-AR) subtypes in vivo, we have applied a gene targeting approach to create a mouse model lacking the alpha1b-AR (alpha1b-/-). Reverse transcription-PCR and ligand binding studies were combined to elucidate the expression of the alpha1-AR subtypes in various tissues of alpha1b +/+ and -/- mice. Total alpha1-AR sites were decreased by 98% in liver, 74% in heart, and 42% in cerebral cortex of the alpha1b -/- as compared with +/+ mice. Because of the large decrease of alpha1-AR in the heart and the loss of the alpha1b-AR mRNA in the aorta of the alpha1b-/- mice, the in vivo blood pressure and in vitro aorta contractile responses to alpha1-agonists were investigated in alpha1b +/+ and -/- mice. Our findings provide strong evidence that the alpha1b-AR is a mediator of the blood pressure and the aorta contractile responses induced by alpha1 agonists. This was demonstrated by the finding that the mean arterial blood pressure response to phenylephrine was decreased by 45% in alpha1b -/- as compared with +/+ mice. In addition, phenylephrine-induced contractions of aortic rings also were decreased by 25% in alpha1b-/- mice. The alpha1b-AR knockout mouse model provides a potentially useful tool to elucidate the functional specificity of different alpha1-AR subtypes, to better understand the effects of adrenergic drugs, and to investigate the multiple mechanisms involved in the control of blood pressure.

Figure 1

Targeted disruption of the mouse α_1b-AR gene. The structure of the wild-type α_1b-AR allele, the targeting vector (αNeoTk), and the recombinant allele after homologous recombination are shown. Exon I (filled box) encodes the receptor portion from its starting methionine to transmembrane domain VI, as indicated by the schematic receptor structure. The neo cassette (gray box) replacing exon I introduces an additional SacI restriction site, which was used for Southern analysis. Probe A (black bar) was used for Southern blot analysis after digestion of DNA with SacI. The solid bars indicate the expected fragment sizes of the wild-type and mutant α_1b-AR alleles. E, EcoRI; H, HindIII; N, NcoI; S, SacI; X, XmnI; Xb, XbaI.

Figure 2

RT-PCR analysis of the RNA from different tissues of α_1b +/+ (WT, wild type) and −/− (KO, knockout) mice. (Left) Ethidium bromide staining of the RT-PCR fragments. The α_1d, α_1b, and α_1a mRNA transcripts were detected as 650-, 470-, and 450-bp fragments, respectively. RT-PCR analysis was controlled by detection of the 390-bp fragment of the hypoxanthine-phosphoribosyl-transferase message. The DNA size markers (M) are shown on the left. The positive control (C +) indicates the RT-PCR analysis of RNA derived from COS-7 cells expressing each α₁-AR subtype. For the negative control (C −), RT-PCR analysis was performed on samples without RNA. (Right) Southern blots of the RT-PCR fragments shown on the left. The specificity of the amplified fragments was assessed using ³²P-labeled probes specific for each receptor subtype (see Materials and Methods).

Figure 3

Blood pressure response in α_1b +/+ and −/− mice. Due to the dysrythmic properties of high doses of phenylephrine and norepinephrine, absolute maximal responses were not attempted. The results are the mean ± SE of 10 dose-response curves for each genotype. For angiotensinII (AII, 50 ng/kg) and vasopressin (VP, 30 milliunits/kg) the results are the mean ± SE from six mice of each genotype. ∗, P < 0.05 and ∗∗, P < 0.001 as compared with α_1b+/+.

Figure 4

Aorta contractility in α_1b +/+ and −/− mice. The results are the mean ± SE of 13 concentration-response curves for each genotype and for both phenylephrine and serotonin (5-HT). Concentrations of phenylephrine higher than 10⁻⁶ M did not further increase, but rather decreased the contractions of the aortic rings derived from both α_1b +/+ and −/− mice. ∗, P < 0.05 as compared with α_1b+/+.