Insights into the functional roles of α₁‐adrenoceptor subtypes in mouse carotid arteries using knockout mice

Abstract

1 a 1 -Adrenoceptor (AR) subtypes in mouse carotid arteries were characterised using a combination of agonist/antagonist pharmacology and knockout (KO) mice. 2 Phenylephrine (PE) was most potent in the a 1B -KO (pEC 50 ¼ 6.970.2) followed by control (pEC 50 ¼ 6.370.06) and a 1D -KO (pEC 50 ¼ 5.570.07). Both N-[5-(4,5-dihydro-1H-imidazol-2yl)-2-hydroxy-5,6,7,8-tetrahydronaphthalen-1-yl] methanesulphonamide hydrobromide (A-61603) and 5-hydroxytryptamine (5-HT) were more potent in the a 1D -KO (pEC 50 ¼ 7.470.27 and 7.470.05, respectively) than the control (pEC 50 ¼ 6.970.09 and 6.970.08, respectively) and equipotent with the control in the a 1B -KO (pEC 50 ¼ 6.770.07 and 6.870.04). Maximum responses to PE and A-61603 were reduced in the a 1D -KO compared to control; there was no difference in maximum responses to 5-HT. 3 In control arteries, prazosin and 5-methylurapidil acted competitively with pA 2 of 9.6 and 7.5, respectively. BMY7378 produced antagonism only at the highest concentration used (100 nM; pK B 8.3). 4 Prazosin, 5-methylurapidil and BMY7378 acted competitively in a 1B -KO carotid arteries with pA 2 of 10.3, 7.6 and 9.6, respectively. 5 In the a 1D -KO, against PE, 5-methylurapidil produced a pA 2 of 8.1. pK B values were calculated for prazosin (10.6) and BMY7378 (7.0). Against A-61603, 5-methylurapidil had a pA 2 of 8.5, prazosin 8.6, while BMY7378 had no effect. 6 In conclusion, the a 1B -KO mediates contraction solely through a 1D -ARs and the a 1D -KO through a 1A -ARs. Extrapolating back to the control from the knockout data suggests that all three subtypes could be involved in the responses, but we propose that the a 1D -AR causes the contractile response and that the role of the a 1B -AR is mainly regulatory.

International Union of Basic and Clinical Pharmacology. LXVII. Recommendations for the Recognition and Nomenclature of G Protein-Coupled Receptor Heteromultimers

“…Such a finding provides a good explanation for the long-standing mystery in the ␣ 1 adrenoceptor field: that ␣ 1D receptors could not be detected in native tissue using high-affinity binding of BMY 7378 (Yang et al, 1997(Yang et al, , 1998, despite a high level of mRNA expression. This proposal is also consistent with a number of data reported with KO animals, such as increased affinity of the ␣ 1D selective antagonist BMY 7378 in ␣ 1B knockout mice (Deighan et al, 2005) or the potent BMY 7378 antagonist activity in left ventricular phenylephrineinduced pressure observed in ␣ 1A -␣ 1B double KO mice. Taken together, these different findings are consistent with the existence of ␣ 1B -␣ 1D heterodimers in native tissue.…”

The α1D-Adrenergic Receptor: Cinderella or Ugly Stepsister

“…For example, knockout studies in which the gene for one or more ␣ 1 -AR subtypes has been inactivated have hinted at potential crosstalk between the various subtypes. Thus, in keeping with the present findings, it has been demonstrated that removal of the ␣ 1B -AR leads to the appearance of binding sites with high affinity for BMY-7378 (Daly et al, 2002;Deighan et al, 2005;Hosoda et al, 2005). This suggests that the ␣ 1D -AR is indeed expressed in the vasculature and contributes to maintenance of blood pressure-a contention that is also evident from the reduced blood pressure and impaired vasoconstrictor responses to norepeinephrine of ␣ 1D -knockout mice (Tanoue et al, 2002)-but that its expression is masked by the coincident expression of the ␣ 1B -AR.…”

Heterodimers of α1B- and α1D-Adrenergic Receptors Form a Single Functional Entity

“…In addition, a recent study using ␣ 1 -AR knockout mice found that ␣ 1D -AR and ␣ 1D -/␣ 1B -AR knockout mice had a significant decrease in mean arterial blood pressure, whereas ␣ 1B -AR knockout mice did not, suggesting that ␣ 1D -/␣ 1B -ARs may act cooperatively to regulate blood pressure (Hosoda et al, 2005). Additional evidence for a physiological role of ␣ 1B -AR/␣ 1D -AR heterodimers was provided from functional studies of isolated mouse carotid arteries, in which the potency of phenylephrine was significantly decreased in ␣ 1D -AR knockout mice yet unchanged in ␣ 1B -AR knockout mice Downloaded from (Deighan et al, 2005). These findings raise the possibility that specific heterodimers respond supermaximally to agonist stimulation.…”

“…However, BMY 7378 detected only a single population of low-affinity binding sites in radioligand binding experiments (Bockman et al, 2004), which is consistent with our findings suggesting that coexpression of ␣ 1B -and ␣ 1D -ARs results in the masking of high-affinity ␣ 1D -AR binding sites. In addition, the affinity of BMY 7378 in inhibiting phenylephrine-mediated contraction was found to be significantly increased in isolated carotid arteries from ␣ 1B -AR knockout mice (Deighan et al, 2005), and phenylephrinestimulated increases in left ventricular-developed pressure were only inhibited by BMY 7378 in ␣ 1A -/␣ 1B -AR double knockout mice (Turnbull et al, 2003). These unusual findings could be explained by a model wherein the knockout of ␣ 1B -ARs from native tissues results in the unmasking of ␣ 1D -AR binding sites with high affinity for BMY 7378, which would be predicted from the results of our cellular studies reported here.…”