alpha(1)-Adrenergic receptors (ARs) are not well defined in the central nervous system. The particular cell types and areas that express these receptors are uncertain because of the lack of high avidity antibodies and selective ligands. We have developed transgenic mice that either systemically overexpress the human alpha(1A)-AR subtype fused with the enhanced green fluorescent protein (EGFP) or express the EGFP protein alone under the control of the mouse alpha(1A)-AR promoter. We confirm our transgenic model against the alpha(1A)-AR knockout mouse, which expresses the LacZ gene in place of the coding region for the alpha(1A)-AR. By using these models, we have now determined cellular localization of the alpha(1A)-AR in the brain, at the protein level. The alpha(1A)-AR or the EGFP protein is expressed prominently in neuronal cells in the cerebral cortex, hippocampus, hypothalamus, midbrain, pontine olivary nuclei, trigeminal nuclei, cerebellum, and spinal cord. The types of neurons were diverse, and the alpha(1A)-AR colocalized with markers for glutamic acid decarboxylase (GAD), gamma-aminobutyric acid (GABA), and N-methyl-D-aspartate (NMDA) receptors. Recordings from alpha(1A)-AR EGFP-expressing cells in the stratum oriens of the hippocampal CA1 region confirmed that these cells were interneurons. We could not detect expression of the alpha(1A)-AR in mature astrocytes, oligodendrocytes, or cerebral blood vessels, but we could detect the alpha(1A)-AR in oligodendrocyte progenitors. We conclude that the alpha(1A)-AR is abundant in the brain, expressed in various types of neurons, and may regulate the function of oligodendrocyte progenitors, interneurons, GABA, and NMDA receptor containing neurons.

These data demonstrate that alpha1A-ARs protect the heart from ischemic injury through a staurosporine-sensitive signaling pathway that is independent of protein kinase C.

alpha1-Adrenergic receptors (ARs) are well-known mediators of the sympathetic nervous system, are highly abundant in the brain, but are the least understood in the central nervous system. The particular cell types in the brain that contain these receptors or their functions are not known because of the lack of high avidity antibodies and selective ligands. We developed transgenic mice that endogenously overexpress the alpha1B-AR subtype fused with the enhanced green fluorescent protein (EGFP). Endogenous expression was obtained by using a 3.4 kb fragment of the mouse alpha1B-AR promoter. Using this model, we determined cellular localization of the alpha1B-AR throughout the brain. The alpha1B-AR-EGFP fusion protein is expressed in neurons throughout the brain and in the Purkinje cells of the cerebellum. The alpha1B-AR is also expressed in NG2 oligodendrocyte precursor cells in both neonatal cell cultures and in the adult cerebral cortex, but is weakly expressed in mature oligodendrocytes. The alpha1B-AR was not observed in astrocytes or in cerebral vascular smooth muscle, cell types previously suggested to contain alpha1-ARs. We conclude that the alpha1B-AR is highly abundant throughout the brain, predominately in neurons, and may be involved in the development of the oligodendrocyte. In adult NG2 cells, implicated in stem cell-like functions, the alpha1B-AR may also play a role. This is the first report of a transgenic tagged-GPCR approach to determine in vivo localization of a receptor.

Our previous studies have demonstrated that activation of ␣ 1 -adrenergic receptors (ARs) increased interleukin-6 (IL-6) mRNA expression and protein secretion, which is probably an important yet unknown mechanism contributing to the regulation of cardiac function. Using Rat-1 fibroblasts stably transfected with the ␣ 1A -AR subtype and primary mouse neonatal cardiomyocytes, we elucidated the basic molecular mechanisms responsible for the ␣ 1 -AR modulation of IL-6 expression. IL-6 mRNA production mediated by ␣ 1 -AR peaked at 1 to 2 h. Studies of the mRNA decay rate indicated that ␣ 1 -AR activation enhanced IL-6 mRNA stability. Analysis of IL-6 promoter activity using a series of deletion constructs indicated that ␣ 1 -ARs enhanced IL-6 transcription through several transcriptional elements, including nuclear factor B (NF-B). Inhibition of ␣ 1 -AR mediated IL-6 production and secretion by actinomycin D and the increase of intracellular IL-6 levels by ␣ 1 -AR activation suggest that ␣ 1 -AR mediated IL-6 secretion through de novo synthesis. Both IL-6 transcription and protein secretion mediated by ␣ 1 -ARs were significantly reduced by chemical inhibitors for p38 mitogen-activated protein kinase (MAPK) and NF-B and by a dominant-negative construct of p38 MAPK. Serum level of IL-6 was elevated in transgenic mice expressing a constitutively active mutant of the ␣ 1A -AR subtype but not in a similar mouse model expressing the ␣ 1B -AR subtype. Our results indicate that ␣ 1 -ARs stimulated IL-6 expression and secretion through regulating both mRNA transcription and stability, involving p38 MAPK and NF-B pathways.

The role of ␣ 1 -adrenergic receptors (␣ 1 ARs) in cognition and mood is controversial, probably as a result of past use of nonselective agents. ␣ 1A AR activation was recently shown to increase neurogenesis, which is linked to cognition and mood. We studied the effects of long-term ␣ 1A AR stimulation using transgenic mice engineered to express a constitutively active mutant (CAM) form of the ␣ 1A AR. CAM-␣ 1A AR mice showed enhancements in several behavioral models of learning and memory. In contrast, mice that have the ␣ 1A AR gene knocked out displayed poor cognitive function. Hippocampal brain slices from CAM-␣ 1A AR mice demonstrated increased basal synaptic transmission, paired-pulse facilitation, and long-term potentiation compared with wild-type (WT) mice. WT mice treated with the ␣ 1A AR-selective agonist cirazoline also showed enhanced cognitive functions. In addition, CAM-␣ 1A AR mice exhibited antidepressant and less anxious phenotypes in several behavioral tests compared with WT mice. Furthermore, the lifespan of CAM-␣ 1A AR mice was 10% longer than that of WT mice. Our results suggest that long-term ␣ 1A AR stimulation improves synaptic plasticity, cognitive function, mood, and longevity. This may afford a potential therapeutic target for counteracting the decline in cognitive function and mood associated with aging and neurological disorders.

The understanding of the function of ␣ 1 -adrenergic receptors in the brain has been limited due to a lack of specific ligands and antibodies. We circumvented this problem by using transgenic mice engineered to overexpress either wild-type receptor tagged with enhanced green fluorescent protein or constitutively active mutant ␣ 1 -adrenergic receptor subtypes in tissues in which they are normally expressed. We identified intriguing ␣ 1A -adrenergic receptor subtype-expressing cells with a migratory morphology in the adult subventricular zone that coexpressed markers of neural stem cell and/or progenitors. Incorporation of 5-bromo-2-deoxyuridine in vivo increased in neurogenic areas in adult ␣ 1A -adrenergic receptor transgenic mice or normal mice given the ␣ 1A -adrenergic receptor-selective agonist, cirazoline. Neonatal neurospheres isolated from normal mice expressed a mixture of ␣ 1 -adrenergic receptor subtypes, and stimulation of these receptors resulted in increased expression of the ␣ 1B -adrenergic receptor subtype, proneural basic helix-loop-helix transcription factors, and the differentiation and migration of neuronal progenitors for catecholaminergic neurons and interneurons. ␣ 1 -Adrenergic receptor stimulation increased the apoptosis of astrocytes and regulated survival of neonatal neurons through phosphatidylinositol 3-kinase signaling. However, in adult normal neurospheres, ␣ 1 -adrenergic receptor stimulation increased the expression of glial markers at the expense of neuronal differentiation. In vivo, S100-positive glial and ␤III tubulin neuronal progenitors colocalized with either ␣ 1 -adrenergic receptor subtype in the olfactory bulb. Our results indicate that ␣ 1 -adrenergic receptors can regulate both neurogenesis and gliogenesis that may be developmentally dependent. Our findings may lead to new therapies to treat neurodegenerative diseases.

A B S T R A C T The mechanism of pemphigus acantholysis has been studied with an in vitro system. Freshly prepared human skin epidermal cells were incubated in F-10 medium which contained the immunoglobulin G fraction from either pemphigus serum or normal human serum. During 18-h incubation periods, the pemphigus antibody became bound to the surface ofthe epidermal cells, caused the destruction of 75% of the viable cells as compared to only 14% in the normal immunoglobulin G controls (trypan blue exclusion), prevented the accumulation of newly synthesized proteins by nearly 60% as determined by radioactive tracer studies, and caused a dramatic shift in distribution of the newly synthesized proteins from an insoluble cell-associated fraction to an extracellular soluble fraction. These effects on the accumulation and partitioning of newly synthesized proteins were antibody concentration-dependent. Kinetic studies showed that at a fixed pemphigus antibody concentration the inhibition of protein accumulation preceded solubilization by about 1 h, at which time rapid solubilization of up to 70% of the insoluble cellular material occurred. Several lines of evidence suggested that this phenomenon was caused by enzymatic activity. Epidermal extracts solubilized a prepared substrate of radioactively labeled insoluble epidermal cell material. This activity was heat labile and pH dependent, with pH optima ranging from 4.5 to 6.5. Enzymes with pH optima between 6 and 6.5 were recovered in the culture medium after a 2-day incubation of pure, intact epidermis with the pemphigus antibody.We propose the following hypothesis to account for pemphigus acantholysis. The pemphigus antibody reacts with the epidermal cell surface and produces such a severe disturbance that the integrity of the cell

␣ 1 -Adrenoceptor subtypes (␣ 1A -, ␣ 1B -, ␣ 1D -) are known to couple to similar signaling pathways, although differences among the subtypes do exist. As a more sensitive assay, we used oligonucleotide microarrays to identify gene expression changes in Rat-1 fibroblasts stably expressing each individual subtype. We report the gene expressions that change by at least a factor of 2 or more. Gene expression profiles significantly changed equally among all three subtypes, despite the unequal efficacy of the inositol phosphate response. Gene expressions were clustered into cytokines/growth factors, transcription factors, enzymes, and extracellular matrix proteins. There were also a number of individual subtype-specific changes in gene expression, suggesting a link to independent pathways. In addition, all three ␣ 1 -AR subtypes robustly stimulated the transcription of the prohypertrophic cytokine interleukin (IL)-6, but differentially altered members of the IL-6 signaling pathway (gp-130 and STAT3). This was confirmed by measurement of secreted IL-6, activated STAT3, and gp-130 levels. Activation of STAT3 Tyr705 phosphorylation by the ␣ 1 -ARs was not through IL-6 activation but was synergistic with IL-6, suggesting direct effects. Interestingly, ␣ 1B -AR stimulation caused the dimerization-dependent phosphorylation of Tyr705 on STAT3 but did not activate the transcriptional-dependent phosphorylation of Ser727. The ␣ 1B -AR also constitutively down-regulated the protein levels of gp-130. These results suggest that the ␣ 1B -AR has differential effects on the phosphorylation status of the STAT3 pathway and may not be as prohypertrophic as the other two subtypes.