Regulation of the cellular localization and signaling properties of the alpha(1B)- and alpha(1D)-adrenoceptors by agonists and inverse agonists.

The regulation of the cellular distribution and intracellular signaling properties of the alpha(1B)- and alpha(1D)- adrenoceptor (alpha(1)-AR) subtypes was examined in stably transfected Rat 1 fibroblasts. In unstimulated cells, alpha(1B)-AR expression was noted primarily on the cell surface. Treatment with phenylephrine induced internalization of the alpha(1B)-AR and promoted association with arrestin 2. The internalized alpha(1B)-AR colocalized with the transferrin receptor, an endosomal marker. In unstimulated fibroblasts, the alpha(1D)-AR was detected in a perinuclear orientation and was colocalized with arrestin 2 in a compartment also containing the transferrin receptor. After treatment with prazosin, which exhibits inverse agonist properties, the alpha(1D)-AR was redistributed from intracellular sites to the cellular periphery and was no longer associated with the transferrin receptor or arrestin 2. alpha(1D)-AR-expressing cells exhibited a high degree of basal activity for both inositol phosphate formation and extracellular signal regulated kinase (ERK), which was reduced by treatment with prazosin. In these cells, phenylephrine induced a dose-dependent increase in inositol phosphate formation but had no effect on ERK activity. In alpha(1B) -AR-expressing cells, phenylephrine stimulated both inositol phosphate formation and ERK activity. These data show that: 1) there are differences in the cellular localization of the alpha(1)-AR subtypes; 2) the alpha(1B)-AR exhibits expected G protein-coupled receptor activity regarding cellular localization, agonist-mediated internalization, and coupling to second messengers; and 3) the alpha(1D)-AR is constitutively active and, as a result, is localized to intracellular compartments involved in receptor recycling.

Expression of multiple alpha1-adrenoceptors on vascular smooth muscle: correlation with the regulation of contraction.

Previous work has shown that the genes encoding each alpha1-adrenoceptor subtype are coexpressed throughout the peripheral vascular system. We have evaluated subtype-selective antibodies as tools to determine the extent of protein expression in arteries. The alpha1A-, alpha1B-, and alpha1D-adrenoceptors were detected in the medial layer of the aorta, caudal, femoral, iliac, renal, superior mesenteric, and mesenteric resistance arteries. In Rat1 fibroblasts expressing each subtype, immunoreactivity was noted both on the cell surface and in a perinuclear orientation. Intense alpha1B-adrenoceptor immunostaining was similarly localized in cultured femoral and renal vascular smooth muscle cells. Although the cellular localization appeared to be the same, immunoreactivity obtained with alpha1A- and alpha1D-adrenoceptors was much less intense than that with the alpha1B-adrenoceptor. The alpha1A-adrenoceptor selective agonist A-61603 was 22-fold more potent in activating renal artery contraction when compared with the femoral artery. The expression of each alpha1-adrenoceptor was significantly decreased by in vivo application of antisense oligonucleotides targeted against each subtype. Inhibition of the expression of only one, the alpha1A in renal and the alpha1D in femoral arteries, reduced the contractile response to naphazoline. The results show: 1) subtype-selective antibodies can be used in tissues and cell culture to localize the alpha1-adrenoceptor subtypes, 2) in addition to expression on the cell surface, the alpha1-adrenoceptors are expressed intracellularly, and 3) despite expression of all adrenoceptors, a single subtype mediates the contractile response in the femoral and renal arteries.

The agonism and synergistic potentiation of weak partial agonists by triethylamine in alpha 1-adrenergic receptor activation: evidence for a salt bridge as the initiating process.

Alpha 1-adrenergic receptor (AR) activation is thought to be initiated by disruption of a constraining interhelical salt bridge (). Disruption of this salt bridge is achieved through a competition for the aspartic acid residue in transmembrane domain three by the protonated amine of the endogenous ligand norepinephrine and a lysine residue in transmembrane domain seven. To further test this hypothesis, we investigated the possibility that a simple amine could mimic an important functional group of the endogenous ligand and break this alpha 1-AR ionic constraint leading to agonism. Triethylamine (TEA) was able to generate concentration-dependent increases of soluble inositol phosphates in COS-1 cells transiently transfected with the hamster alpha 1b-AR and in Rat-1 fibroblasts stably transfected with the human alpha 1a-AR subtype. TEA was also able to synergistically potentiate the second messenger production by weak partial alpha 1-AR agonists and this effect was fully inhibited by the alpha 1-AR antagonist prazosin. However, this synergistic potentiation was not observed for full alpha 1-AR agonists. Instead, TEA caused a parallel rightward shift of the dose-response curve, consistent with the properties of competitive antagonism. TEA specifically bound to a single population of alpha 1-ARs with a Ki of 28.7 +/- 4.7 mM. In addition, the site of binding by TEA to the alpha 1-AR is at the conserved aspartic acid residue in transmembrane domain three, which is part of the constraining salt bridge. These results indicate a direct interaction of TEA in the receptor agonist binding pocket that leads to a disruption of the constraining salt bridge, thereby initiating alpha 1-AR activation.

Immunocytochemical localization of the alpha-1B adrenergic receptor and the contribution of this and the other subtypes to vascular smooth muscle contraction: analysis with selective ligands and antisense oligonucleotides.

The contribution of the alpha-1B adrenergic receptor (AR) to vascular smooth muscle contraction has been assessed using a combination of immunological, molecular biological and pharmacological approaches. A subtype-selective antibody detected alpha-1B immunoreactivity in the medial layer of the aorta, caudal, femoral, iliac, mesenteric resistance, renal and superior mesenteric arteries. Receptor protection assays and antisense oligonucleotides were used to assess the contribution of the alpha-1B AR to contraction. The alpha-1B AR was implicated in mediating the phenylephrine-induced contraction of the mesenteric resistance artery. The alpha-1D AR was implicated in mediating the contraction of the aorta, femoral, iliac and superior mesenteric arteries. Similarly, the alpha-1A AR was implicated in mediating contraction of the caudal and renal arteries. In vivo application of antisense oligonucleotides targeted to the translational start site of the alpha-1B AR had no effect on the phenylephrine-induced contraction of the femoral or renal arteries. In contrast, antisense oligonucleotides directed against the alpha-1D AR significantly inhibited the phenylephrine response in the femoral artery but had no effect on the renal artery. Application of alpha-1A AR antisense oligonucleotides inhibited the contraction of the renal artery without effect on the femoral artery. These data show that (1) alpha-1B AR immunoreactivity is widely distributed in the same peripheral arteries in which previous studies detected its mRNA, and (2) despite this distribution, receptor protection and antisense oligonucleotide studies indicate that the alpha-1B AR mediates the contraction of only the mesenteric resistance artery.