Sodium Oxybate for Narcolepsy: Explaining Untoward Effects and Recommending New Approaches in Light of Prevailing Receptor Pharmacology.

Objective: Gamma-hydroxybutyrate (GHB) has been an abused and illicit substance for decades, but the antinarcoleptic medication Xyrem (sodium oxybate), the sodium salt of GHB, was approved just in 2002 for increasing wakefulness. We present a case of coma induced by co-ingestion of prescription GHB and ethanol and describe the response to naloxone treatment, by first responders, without evidence of opiate exposure. The purpose of this report is to bridge updated knowledge on GHB and ethanol pharmacology with the clinical sequence of events in a patient co-ingesting these compounds and to theorize on a potentially better pharmacological approach to narcolepsy. Case Summary: The patient was a 25-year-old woman with a history of narcolepsy. She suddenly collapsed at home but became transiently responsive after being administered naloxone in the ambulance. She presented to the emergency department with apnea, poor responsiveness with a Glasgow Coma Score of 7, and urinary incontinence. While undergoing intubation, the patient spontaneously and abruptly awoke. Labs were unremarkable except a blood alcohol concentration of 0.123%. The dosage of, and adherence to, GHB was unknown in this case. Discussion: The case is described in light of the most recent pharmacological advancements on these co-ingestants. A conceptual dose-response curve is shown to facilitate understanding of the complex pharmacology of GHB. Conclusions: Approved and potential alternatives to GHB, for achieving wakefulness, are discussed. Potential new strategies should bear low to no risk of coma with accidental overdose or co-ingestion of ethanol. In addition, promising antidotes for future consideration are discussed.

Chemoprevention of prostate cancer by major dietary phytochemicals.

Prostate cancer continues to be one of the most commonly diagnosed diseases and the second leading cause of cancer-related deaths among men in the United States. Options exist to treat localized disease, including surgery, radiation therapy, and hormonal therapy, but clinical management of advanced prostate cancer is challenging. In the past few decades, chemoprevention involving naturally-occurring compounds has emerged as a promising and cost-effective approach to reduce incidence and morbidity of prostate cancer by inhibiting the precancerous events before the occurrence of clinical disease. The present review focuses on summarizing the recent advances in studies of major dietary phytochemicals and their role in prostate cancer development.

The alpha1D-adrenergic receptor is expressed intracellularly and coupled to increases in intracellular calcium and reactive oxygen species in human aortic smooth muscle cells.

BACKGROUND: The cellular localization of the alpha1D-adrenergic receptor (alpha1D-AR) is controversial. Studies in heterologous cell systems have shown that this receptor is expressed in intracellular compartments. Other studies show that dimerization with other ARs promotes the cell surface expression of the alpha1D-AR. To assess the cellular localization in vascular smooth muscle cells, we developed an adenoviral vector for the efficient expression of a GFP labeled alpha1D-AR. We also measured cellular localization with immunocytochemistry. Intracellular calcium levels, measurement of reactive oxygen species and contraction of the rat aorta were used as measures of functional activity. RESULTS: The adenovirally expressed alpha1D-AR was expressed in intracellular compartments in human aortic smooth muscle cells. The intracellular localization of the alpha1D-AR was also demonstrated with immunocytochemistry using an alpha1D-AR specific antibody. RT-PCR analysis detected mRNA transcripts corresponding to the alpha1A-alpha1B- and alpha1D-ARs in these aortic smooth muscle cells. Therefore, the presence of the other alpha1-ARs, and the potential for dimerization with these receptors, does not alter the intracellular expression of the alpha1D-AR. Despite the predominant intracellular localization in vascular smooth muscle cells, the alpha1D-AR remained signaling competent and mediated the phenylephrine-induced increases in intracellular calcium. The alpha1D-AR also was coupled to the generation of reactive oxygen species in smooth muscle cells. There is evidence from heterologous systems that the alpha1D-AR heterodimerizes with the beta2-AR and that desensitization of the beta2-AR results in alpha1D-AR desensitization. In the rat aorta, desensitization of the beta2-AR had no effect on contractile responses mediated by the alpha1D-AR. CONCLUSION: Our results suggest that the dimerization of the alpha1D-AR with other ARs does not alter the cellular expression or functional response characteristics of the alpha1D-AR.

Dominance of the alpha1B-adrenergic receptor and its subcellular localization in human and TRAMP prostate cancer cell lines.

The function and distribution of alpha1-adrenergic receptor (AR) subtypes in prostate cancer cells is well characterized. Previous studies have used RNA localization or low-avidity antibodies in tissue or cell lines to determine the alpha1-AR subtype and suggested that the alpha1A-AR is dominant. Two androgen-insensitive, human metastatic cancer cell lines DU145 and PC3 were used as well as the mouse TRAMP C1-C3 primary and clonal cell lines. The density of alpha1-ARs was determined by saturation binding and the distribution of the different alpha1-AR subtypes was examined by competition-binding experiments. In contrast to previous studies, the major alpha1-AR subtype in DU145, PC3 and all of the TRAMP cell lines is the alpha1B-AR. DU145 cells contained 100% of the alpha1B-AR subtype, whereas PC3 cells were composed of 21% alpha1 A-AR and 79% alpha1B-AR. TRAMP cell lines contained between 66% and 79% of the alpha1B-AR with minor fractions of the other two subtypes. Faster doubling time in the TRAMP cell lines correlated with decreasing alpha 1B-AR and increasing alpha1 A- and alpha1D-AR densities. Transfection with EGFP-tagged alpha1B-ARs revealed that localization was mainly intracellular, but the majority of the receptors translocated to the cell surface after extended preincubation (18 hr) with either agonist or antagonist. Localization was confirmed by ligand-binding studies and inositol phosphate assays where prolonged preincubation with either agonist and/or antagonist increased the density and function of alpha 1-ARs, suggesting that the native receptors were mostly intracellular and nonfunctional. Our studies indicate that alpha1B-ARs are the major alpha1-AR subtype expressed in DU145, PC3, and all TRAMP cell lines, but most of the receptor is localized in intracellular compartments in a nonfunctional state, which can be rescued upon prolonged incubation with any ligand.

Localization of the mouse alpha1A-adrenergic receptor (AR) in the brain: alpha1AAR is expressed in neurons, GABAergic interneurons, and NG2 oligodendrocyte progenitors.

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.

alpha1A- but not alpha1B-adrenergic receptors precondition the ischemic heart by a staurosporine-sensitive, chelerythrine-insensitive mechanism.

OBJECTIVE: Brief periods of ischemia stimulate an endogenous mechanism in the heart that protects the myocardium from subsequent ischemic injury. alpha1-Adrenergic receptors (ARs) have been implicated in this process. However, the lack of sufficiently selective antagonists has made it difficult to determine which alpha1-AR subtype protects the heart from ischemic injury. The goal of this study was to identify the alpha1-AR subtype that is involved in ischemic preconditioning. METHODS: We developed transgenic mice that express constitutively active mutant (CAM) forms of the alpha1A-AR or the alpha1B-AR regulated by their endogenous promoters. Hearts isolated from transgenic and non-transgenic mice were perfused by the Langendorff method using an ischemic preconditioning perfusion protocol or a non-preconditioning perfusion protocol prior to 30-min ischemia and 40-min reperfusion. Contractile function was continuously monitored through an intraventricular balloon. RESULTS: The contractile function of non-transgenic hearts perfused according to the ischemic preconditioning protocol completely recovered from 30-min ischemia. However, non-transgenic hearts perfused according to the non-preconditioning protocol recovered only 60% of their contractile function. The contractile function of CAM alpha1A-AR hearts, but not CAM alpha1B-AR hearts, completely recovered from 30-min ischemia even though they were perfused according to the non-preconditioning protocol. Thus, CAM alpha1A-AR hearts, but not CAM alpha1B-AR hearts, were inherently preconditioned against ischemic injury. Staurosporine, but not chelerythrine, completely reversed the preconditioning effect of CAM alpha1A-ARs. CONCLUSIONS: 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.

Differential regulation of the cell cycle by alpha1-adrenergic receptor subtypes.

Alpha(1)-Adrenergic receptors have been implicated in growth-promoting pathways. A microarray study of individual alpha(1)-adrenergic receptor subtypes (alpha(1A), alpha(1B), and alpha(1D)) expressed in Rat-1 fibroblasts revealed that epinephrine altered the transcription of several cell cycle regulatory genes in a direction consistent with the alpha(1A)- and alpha(1D)-adrenergic receptors mediating G(1)-S cell cycle arrest and the alpha(1B-)mediating cell-cycle progression. A time course indicated that in alpha(1A) cells, epinephrine stimulated a G(1)-S arrest, which began after 8 h of stimulation and maximized at 16 h, at which point was completely blocked with cycloheximide. The alpha(1B)-adrenergic receptor profile also showed unchecked cell cycle progression, even under low serum conditions and induced foci formation. The G(1)-S arrest induced by alpha(1A)- and alpha(1D)-adrenergic receptors was associated with decreased cyclin-dependent kinase-6 and cyclin E-associated kinase activities and increased expression of the cyclin-dependent kinase inhibitor p27(Kip1), all of which were blocked by prazosin. There were no differences in kinase activities and/or expression of p27(Kip1) in epinephrine alpha(1B)-AR fibroblasts, although the microarray did indicate differences in p27(Kip1) RNA levels. Cell counts proved the antimitotic effect of epinephrine in alpha(1A) and alpha(1D) cells and indicated that alpha(1B)-adrenergic receptor subtype expression was sufficient to cause proliferation of Rat-1 fibroblasts independent of agonist stimulation. Analysis in transfected PC12 cells also confirmed the alpha(1A)- and alpha(1B)-adrenergic receptor effect. The alpha(1B)-subtype native to DDT1-MF2 cells, a smooth muscle cell line, caused progression of the cell cycle. These results indicate that the alpha(1A)- and alpha(1D)-adrenergic receptors mediate G(1)-S cell-cycle arrest, whereas alpha(1B)-adrenergic receptor expression causes a cell cycle progression and may induce transformation in sensitive cell lines.

Mouse alpha1B-adrenergic receptor is expressed in neurons and NG2 oligodendrocytes.

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.

The alpha(1B)-adrenergic receptor decreases the inotropic response in the mouse Langendorff heart model.

OBJECTIVE: alpha(1)-Adrenergic receptors (ARs) are known mediators of a positive inotropy in the heart, which may play even more important roles in heart disease. Due to a lack of sufficiently selective ligands, the contribution of each of the three alpha(1)-AR subtypes (alpha(1A), alpha(1B) and alpha(1D)) to cardiac function is not clearly defined. In this study, we used a systemically expressing mouse model that overexpresses the alpha(1B)-AR to define the role of this subtype in cardiac function. METHODS: We used the mouse Langendorff heart model to assess changes in contractility under basal and phenylephrine-induced conditions. RESULTS: We find that a 50% increase of the alpha(1B)-AR in the heart does not change basal cardiac parameters compared to age-matched normals (heart rate, +/-dP/dT and coronary flow). However, the inotropic response to phenylephrine is blunted. The same results were obtained in isolated adult myocytes. The difference in inotropy could be blocked by the selective alpha(1A)-AR antagonist, 5-methylurapidil, which correlated with decreases in alpha(1A)-AR density, suggesting that the alpha(1B)-AR had caused a compensatory downregulation of the alpha(1A)-AR. CONCLUSIONS: These results suggest that the alpha(1B)-AR does not have a major role in the positive inotropic response in the mouse myocardium but may negatively modulate the response of the alpha(1A)-AR.

Genetic profiling of alpha 1-adrenergic receptor subtypes by oligonucleotide microarrays: coupling to interleukin-6 secretion but differences in STAT3 phosphorylation and gp-130.

Alpha(1)-adrenoceptor subtypes (alpha(1A)-, alpha(1B)-, alpha(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 alpha(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 alpha(1)-ARs was not through IL-6 activation but was synergistic with IL-6, suggesting direct effects. Interestingly, alpha(1B)-AR stimulation caused the dimerization-dependent phosphorylation of Tyr705 on STAT3 but did not activate the transcriptional-dependent phosphorylation of Ser727. The alpha(1B)-AR also constitutively down-regulated the protein levels of gp-130. These results suggest that the alpha(1B)-AR has differential effects on the phosphorylation status of the STAT3 pathway and may not be as prohypertrophic as the other two subtypes.

Differential cardiovascular regulatory activities of the alpha 1B- and alpha 1D-adrenoceptor subtypes.

The regulation of cardiac and vascular function by the alpha 1B- and alpha 1D-adrenoceptors (ARs) has been assessed in two lines of transgenic mice, one over-expressing a constitutively active alpha 1B-AR mutation (alpha 1B-ARC128F) and the other an alpha 1D-AR knockout line. The advantage of using mice expressing a constitutively active alpha 1B-AR is that the receptor is tonically active, thus avoiding the use of nonselective agonists that can activate all subtypes. In hearts from animals expressing alpha 1B-ARC128F, the activities of the mitogen-activated protein kinases, extracellular signal-regulated kinase, and c-Jun N-terminal kinase were significantly elevated compared with nontransgenic control animals. Mice over-expressing the alpha 1B-ARC128F had echocardiographic evidence of contractile dysfunction and increases in chamber dimensions. In isolated-perfused hearts or left ventricular slices from alpha 1B-ARC128F-expressing animals, the ability of isoproterenol to increase contractile force or increase cAMP levels was significantly decreased. In contrast to the prominent effects on the heart, constitutive activation of the alpha 1B-AR had little effect on the ability of phenylephrine to induce vascular smooth muscle contraction in the isolated aorta. The ability of phenylephrine to stimulate coronary vasoconstriction was diminished in alpha 1D-AR knockout mice. In alpha 1D-AR knockout animals, no negative effects on cardiac contractile function were noted. These results show that the alpha1-ARs regulate distinctly different physiologic processes. The alpha 1B-AR appears to be involved in the regulation of cardiac growth and contractile function, whereas the alpha 1D-AR is coupled to smooth muscle contraction and the regulation of systemic arterial blood pressure.

Gene expression profiling of alpha(1b)-adrenergic receptor-induced cardiac hypertrophy by oligonucleotide arrays.

OBJECTIVE: Cardiac hypertrophy is closely associated with the development of cardiomyopathies that lead to heart failure. The alpha(1B) adrenergic receptor (alpha(1)-AR) is an important regulator of the hypertrophic process. Cardiac hypertrophy induced by systemic overexpression of the alpha(1b)-AR in a mouse model does not progress to heart failure. We wanted to explore potential gene expression differences that characterize this type of hypertrophy that may identify genes that prevent progression to heart failure. METHODS: Transgenic and normal mice (B6CBA) representing two time points were compared; one at 2-3 months of age before disease manifests and the other at 12 months when the hypertrophy is significant. Age-matched hearts were removed, cRNA prepared and biotinylated. Aliquots of the cRNA was subjected to hybridization with Affymetrix chips representing 12,656 murine genes. Gene expression profiles were compared with normal age-matched controls as the baseline and confirmed by Northern and Western analysis. RESULTS: The non-EST genes could be grouped into five functional classifications: embryonic, proliferative, inflammatory, cardiac-related, and apoptotic. Growth response genes involved primarily Src-related receptors and signaling pathways. Transgenic hearts also had a 60% higher Src protein content. There was an inflammatory response that was verified by an increase in IgG and kappa-chained immunoglobulins by western analysis. Apoptosis may be regulated by cell cycle arrest through a p53-dependent mechanism. Cardiac gene expression was decreased for common hypertrophy-inducing proteins such as actin, collagen and GP130 pathways. CONCLUSIONS: Our results suggest a profile of gene expression in a case of atypical cardiac hypertrophy that does not progress to heart failure. Since many of these altered gene expressions have not been linked to heart failure models, our findings may provide a novel insight into the particular role that the alpha(1B)AR plays in its overall progression or regression.

Mice expressing the alpha(1B)-adrenergic receptor induces a synucleinopathy with excessive tyrosine nitration but decreased phosphorylation.

We had previously reported that systemic overexpression of the alpha(1B)-adrenergic receptor (AR) in a transgenic mouse induced a neurodegenerative disease that resembled the parkinsonian-like syndrome called multiple system atrophy (MSA). We now report that our mouse model has cytoplasmic inclusion bodies that colocalize with oligodendrocytes and neurons, are positive for alpha-synuclein and ubiquitin, and therefore may be classified as a synucleinopathy. Alpha-synuclein monomers as well as multimers were present in brain extracts from both normal and transgenic mice. However, similar to human MSA and other synucleinopathies, transgenic mice showed an increase in abnormal aggregated forms of alpha-synuclein, which also increased its nitrated content with age. However, the same extracts displayed decreased phosphorylation of alpha-synuclein. Other traits particular to MSA such as Purkinje cell loss in the cerebellum and degeneration of the intermediolateral cell columns of the spinal cord also exist in our mouse model but differences still exist between them. Interestingly, long-term therapy with the alpha(1)-AR antagonist, terazosin, resulted in protection against the symptomatic as well as the neurodegeneration and alpha-synuclein inclusion body formation, suggesting that signaling of the alpha(1B)-AR is the cause of the pathology. We conclude that overexpression of the alpha(1B)-AR can cause a synucleinopathy similar to other parkinsonian syndromes.

Differences in the cellular localization and agonist-mediated internalization properties of the alpha(1)-adrenoceptor subtypes.

The cellular localization, agonist-mediated internalization, and desensitization properties of the alpha(1)-adrenoceptor (alpha(1)-AR) subtypes conjugated with green fluorescent protein (alpha(1)-AR/GFP) were assessed using real-time imaging of living, transiently transfected human embryonic kidney (HEK) 293 cells. The alpha(1B)-AR/GFP fluorescence was detected predominantly on the cell surface. Stimulation of the alpha(1B)-AR with phenylephrine led to an increase in extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and promoted rapid alpha(1B)-AR/GFP internalization. Long-term exposure (15 h) to phenylephrine resulted in desensitization of the alpha(1B)-AR-mediated activation of ERK1/2 phosphorylation. Alpha(1A)-AR/GFP fluorescence was detected not only on the cell surface but also intracellularly. The rate of internalization of the cell surface population alpha(1A)-AR/GFPs was slower than that seen for the alpha(1B)-AR. Agonist exposure also resulted in desensitization of the alpha(1A)-AR-mediated increase in ERK1/2 phosphorylation. The alpha(1D)-AR/GFP fluorescence was detected mainly intracellularly, and this localization was unaffected by exposure to phenylephrine. Phenylephrine treatment of alpha(1D)-AR/GFP expressing cells increased ERK1/2 phosphorylation. However, this increase was not significant. Cotransfection with beta-arrestin 1 did not increase the rate or extent of agonist-stimulated alpha(1A)- or alpha(1B)-AR/GFP internalization. However, a dominant-negative form of the beta-arrestin 1, beta-arrestin 1 (319-418), blocked agonist-mediated internalization of both the alpha(1A)- and alpha(1B)-ARs. These data show that transfected alpha(1)-AR/GFP fusion proteins are functional, that there are differences in the cellular distribution and agonist-mediated internalization between the alpha(1)-ARs, and that agonist-mediated alpha(1)-AR internalization is dependent on arrestins and can be desensitized by long-term exposure to an agonist. These differences could contribute to the diversity in physiologic responses regulated by the alpha(1)-ARs.