The function of alpha- and beta-adrenoceptors of the saphenous artery in caveolin-1 knockout and wild-type mice

Abstract

Background and purpose: Adrenoceptors can associate with cardiac caveolae. To investigate the function of vascular caveolae, adrenoceptor-mediated effects were compared in the saphenous artery of caveolin-1 knockout (cav-1KO) and wild-type (WT) mice.

Experimental approach: Electronmicroscopy was used to detect caveolae. Real-Time quantitative PCR was used for adrenoceptor subtypes. Catecholamine-evoked contractions and relaxations were studied in arterial segments.

Key results: Caveolae were found in arterial smooth muscle from WT but not from cav-1KO mice. Arterial mRNA levels for the adrenoceptors alpha1A, alpha1B, alpha1D, beta1, beta2 and beta3 were similar in cav-1KO and WT. (-)-Noradrenaline contracted cav-1KO (-log EC50M=7.1) and WT (-log EC50M=7.3) arteries through prazosin-sensitive receptors. Maximum (-)-noradrenaline-evoked contractions were greater in cav-1KO than WT arteries. (-)-Isoprenaline relaxed WT arteries (-log EC50M=7.3) more potently than cav-1KO arteries (-log EC50M=6.8); the effects were antagonized partially and similarly by the beta2-selective antagonist ICI118551 (50 nM). The (-)-isoprenaline-evoked relaxation was partially antagonized by the beta1-adrenoceptor-selective antagonist CGP20712 (300 nM) in WT but not cav-1KO arteries. The beta3-adrenoceptor-selective antagonist L748337 (100 nM) partially antagonized the relaxant effects of (-)-isoprenaline in cav-1KO but not in WT arteries. BRL37344 partially relaxed arteries through beta3-adrenoceptors in cav-1KO but not WT. The relaxant effects of BRL37344 were decreased by the NO synthase inhibitor OmegaL-nitroarginine.

Conclusions and implications: The function of arterial alpha1- and beta2-adrenoceptors is similar in cav-1KO and WT mice. beta1-adrenoceptor-mediated relaxation in WT is lost in cav-1KO and replaced by the appearance of beta3-adrenoceptors.

Figure 1

Electron microscopy of vascular smooth muscle cells (VSMC). WT: cell membranes of VSMC of WT mice exhibit numerous invaginations (caveolae, indicated by arrows). Caveolin-1 knockout mice (Cav-1KO): cell membranes of VSMC of cav-1KO mice show shallow indentations (arrows) but no caveolae.

Figure 2

Expression of subunits of adrenoceptors in murine arteria saphena (Aa. saphena). Mean values from three experiments with pooled data from two mice each. (a) Agarose gel electrophoresis of the samples after OneStep RT real-time PCR: notice that there are two splice variants of β₃-adrenoceptor. (b) Amount of α-adrenoceptor-mRNA normalized to total mRNA. (c) Amount of β-adrenoceptor-mRNA normalized to total mRNA. Data from the two β₃-adrenoceptor splice variants were pooled.

Figure 3

Concentration–effect curves for (−)-noradrenaline and antagonism by prazosin on Aa. saphena of WT and cav-1KO mice. Numbers refer to arterial preparations/number of mice.

Figure 4

Effects of cocaine on Aa. Saphenae of WT and cav-1KO mice. (a) Original tracing of an experiment on a cav-1KO artery. (b) Addition of 3 μM cocaine leads to contraction of the vessels. This effect is significantly stronger in cav-1KO compared with WT mice (P=0.0002). In both groups of arteries, the effect was washed out with PSS containing 3 μM cocaine. c=control; NA=(−)-noradrenaline (10 μM). Numbers refer to arterial preparations/number of mice.

Figure 5

Relaxant effects of SNP (30 μM) on Aa. saphenae precontracted with 60 mM KCl. Numbers refer to arterial preparations/number of mice.

Figure 6

Concentration–effect curves of (−)-isoprenaline in Aa. saphenae of WT and cav-1KO mice. (a) Curves showing reduction in wall tension. (b) Curves normalized to SNP-evoked relaxation. Numbers refer to arterial preparations/number of mice.

Figure 7

Comparison of the effects of β-blockers on (−)-isoprenaline-evoked relaxation in WT and cav-1KO arteries. For −log EC₅₀M and E_max data, see Table 2.

Figure 8

Comparison of the effects of β-blocker combinations on (−)-isoprenaline-evoked relaxation in WT and cav-1KO arteries. For −log EC₅₀M and E_max data, see Table 2.

Figure 9

Concentration-effects curves for the β₃-adrenoceptor-selective agonist BRL37344. (a) Antagonism of BRL37344-evoked relaxations by L748337 in cav-1KO. (b) Partial reduction of the relaxant effects of BRL37344 by L-NAME. For −log EC₅₀M and E_max data, see Table 2.