ABSTRACT
The alpha1-adrenergic receptors (alpha1ARs) play an important role in mediating sympathetic neurotransmission in peripheral organ systems; however, central alpha1ARs are not well characterized. Additionally, due to the lack of sufficiently subtype-selective drugs or high avidity antibodies, the contribution of each alpha1AR subtype to various central functions is currently unclear. Transcription regulation through alpha1AR subtypes in the CNS is also unknown. Of interest, transgenic mice that systemically overexpress the alpha1BAR show central symptoms that include age-progressive impaired mobility, neurodegeneration and susceptibility to epileptic seizure. To investigate the molecular basis of this phenotype, oligonucleotide microarray studies of whole brains of various ages were carried out to compare gene expression profiles between transgenic and normal brains. The results indicated changes in expression of apoptotic, calcium regulatory, neurodegenerative and genes involved in neurotransmission. Defects in regulation of intracellular calcium are known to play a role in cell death; thus, these genes may provide clues as to the molecular basis of alpha1BAR-induced neurodegeneration. Epilepsy is a disorder that can be caused by an imbalance between excitatory (e.g. glutamate) and inhibitory (e.g. GABA) signals. Microarray analysis of transgenic brains showed increased N-methyl-d-aspartate (NMDA) receptors and decreased GABAA, which were confirmed with immunohistochemistry, western blot and radioligand binding studies. The alpha1BAR also co-localized with the glutamatergic distribution, suggesting a glutamate imbalance as a molecular rationale for the epileptic seizures.
Subject(s)
Apoptosis/genetics , Neurodegenerative Diseases/genetics , Receptors, Adrenergic, alpha-1/physiology , Animals , Cerebral Cortex/metabolism , Female , Gene Expression , Gene Expression Profiling , Hippocampus/metabolism , Male , Mice , Mice, Transgenic , Neurodegenerative Diseases/metabolism , Neurodegenerative Diseases/pathology , Oligonucleotide Array Sequence Analysis , Receptors, Adrenergic, alpha-1/genetics , Receptors, GABA-A/metabolism , Receptors, N-Methyl-D-Aspartate/metabolismABSTRACT
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.
Subject(s)
Adrenergic Agonists/pharmacology , Adrenergic alpha-1 Receptor Agonists , Signal Transduction , Adrenergic Antagonists/pharmacology , Adrenergic alpha-1 Receptor Antagonists , Animals , Cells, Cultured , Humans , Immunohistochemistry , Inositol Phosphates , Mitogen-Activated Protein Kinases/metabolism , Rats , Receptors, Adrenergic, alpha-1/metabolism , Receptors, Cell Surface/metabolism , TransfectionABSTRACT
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.
