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Sheila Collins - One of the best experts on this subject based on the ideXlab platform.
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Regulation of the uncoupling protein gene (Ucp) by beta 1, beta 2, and beta 3-adrenergic receptor subtypes in immortalized brown adipose cell lines.
The Journal of biological chemistry, 1995Co-Authors: Elizabeth M. Rohlfs, Kiefer W. Daniel, Richard T. Premont, Leslie P. Kozak, Sheila CollinsAbstract:Immortalized brown adipocyte cell lines derived from a mouse hibernoma express all three Beta-Adrenergic receptor subtypes, including beta 3-adrenergic receptor (AR). In response to norepinephrine, cAMP production by plasma membranes from four clonal cell lines was stimulated to levels comparable with brown adipocytes isolated from interscapular brown adipose tissue (72.8-89.6 versus 97.8 pmol cAMP/min/mg of protein, respectively). All cell lines responded to the highly selective beta 3-adrenergic receptor agonist CL316,243 by stimulating adenylyl cyclase activity (3-10-fold over basal). beta 1-, beta 2-, and beta 3-adrenergic receptor mRNA was detected by Northern blotting and/or reverse transcriptase-polymerase chain reaction. Competition binding assays with the antAgonists CGP20712A and 125I-cyanopindolol showed the proportions of beta 1AR and beta 2AR in immortalized cells to be similar to brown adipocytes from tissue (cells: 35% beta 1AR, 65% beta 2AR; brown adipocytes from tissue: beta 1AR 41%, 59% beta 2AR). Expression of brown fat-specific mitochondrial uncoupling protein (Ucp) was stimulated by Beta-Adrenergic Agonists in two of the four cell lines. The ability of individual beta AR subtypes to regulate Ucp expression was examined with combinations of selective Beta-Adrenergic Agonists and antAgonists. Expression of Ucp could be induced by any of the Beta-Adrenergic receptor subtypes. However, the greatest response was obtained by stimulating all three Beta-Adrenergic receptor subtypes simultaneously (100 microM isoproterenol). Incubation of membranes from cultured cells or brown adipocytes from tissue with CL316,243 at an optimal concentration (5 microM) did not prevent norepinephrine from further stimulating adenylyl cyclase activity, suggesting that the combined activation of beta 1AR/beta 2AR, plus beta 3AR, together produced an additive cAMP response. Multiple forms of adenylyl cyclase were identified in brown and white adipocyte cell lines and tissues. Northern blot analysis detected adenylyl cyclase types 5, 6, and 10. Screening of reverse transcriptase-PCR products by DNA sequencing confirmed the identities of these forms and lower levels of additional isoforms, raising the possibility that Beta-Adrenergic receptor subtypes in adipocytes couple to distinct adenylyl cyclases. Because these cell lines display functional and phenotypic similarities to interscapular brown adipocytes, they will be a useful model to study the regulation of Beta-Adrenergic receptor expression and function, and the control of Ucp expression and activity.
Daniel Ricquier - One of the best experts on this subject based on the ideXlab platform.
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Characterization of the novel brown adipocyte cell line HIB 1B. Adrenergic pathways involved in regulation of uncoupling protein gene expression
Journal of Cell Science, 1994Co-Authors: Susanne Klaus, Lisa Choy, Odette Champigny, Anne Marie Cassard-doulcier, Susan R. Ross, Bruce M. Spiegelman, Daniel RicquierAbstract:The HIB 1B cell line, derived from a brown fat tumor of a transgenic mouse, is the first established brown adipocyte cell line capable of expressing the brown fat-specific mitochondrial uncoupling protein (UCP). UCP gene expression, which was virtually undetectable under basic conditions, was stimulated by acute catecholamine or cyclic AMP treatment to levels comparable to primary cultures of brown adipocytes. Elevation of UCP mRNA levels following stimulation was very rapid but transient, decreasing after about 4 hours with a half-life between 9 and 13 hours. Immunoblotting showed the presence of UCP in HIB 1B mitochondria, but expression was much lower than observed in BAT or primary cultures of brown adipocytes. Upon transfection of HIB 1B cells with a reporter gene containing the UCP promoter, the activity of the transgene was regulatable by cAMP and norepinephrine. Investigation of the possible adrenergic receptors involved in UCP stimulation showed that specific beta 3-adrenergic Agonists were much less effective than nonspecific Beta-Adrenergic Agonists and that mRNA levels of the atypical, fat-specific beta 3-adrenoceptor were lower than those observed in brown adipocytes differentiated in primary culture. From pharmacological evidence we conclude that beta 3-adrenergic receptors account for approximately 30-40% of catecholamine induced UCP gene stimulation, whereas about 60-70% is stimulated via the classical beta 1/2 adrenergic pathway. We conclude that HIB 1B cells represent a functional system for the study of mechanisms related to brown adipose thermogenesis.
Elizabeth M. Rohlfs - One of the best experts on this subject based on the ideXlab platform.
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Regulation of the uncoupling protein gene (Ucp) by beta 1, beta 2, and beta 3-adrenergic receptor subtypes in immortalized brown adipose cell lines.
The Journal of biological chemistry, 1995Co-Authors: Elizabeth M. Rohlfs, Kiefer W. Daniel, Richard T. Premont, Leslie P. Kozak, Sheila CollinsAbstract:Immortalized brown adipocyte cell lines derived from a mouse hibernoma express all three Beta-Adrenergic receptor subtypes, including beta 3-adrenergic receptor (AR). In response to norepinephrine, cAMP production by plasma membranes from four clonal cell lines was stimulated to levels comparable with brown adipocytes isolated from interscapular brown adipose tissue (72.8-89.6 versus 97.8 pmol cAMP/min/mg of protein, respectively). All cell lines responded to the highly selective beta 3-adrenergic receptor agonist CL316,243 by stimulating adenylyl cyclase activity (3-10-fold over basal). beta 1-, beta 2-, and beta 3-adrenergic receptor mRNA was detected by Northern blotting and/or reverse transcriptase-polymerase chain reaction. Competition binding assays with the antAgonists CGP20712A and 125I-cyanopindolol showed the proportions of beta 1AR and beta 2AR in immortalized cells to be similar to brown adipocytes from tissue (cells: 35% beta 1AR, 65% beta 2AR; brown adipocytes from tissue: beta 1AR 41%, 59% beta 2AR). Expression of brown fat-specific mitochondrial uncoupling protein (Ucp) was stimulated by Beta-Adrenergic Agonists in two of the four cell lines. The ability of individual beta AR subtypes to regulate Ucp expression was examined with combinations of selective Beta-Adrenergic Agonists and antAgonists. Expression of Ucp could be induced by any of the Beta-Adrenergic receptor subtypes. However, the greatest response was obtained by stimulating all three Beta-Adrenergic receptor subtypes simultaneously (100 microM isoproterenol). Incubation of membranes from cultured cells or brown adipocytes from tissue with CL316,243 at an optimal concentration (5 microM) did not prevent norepinephrine from further stimulating adenylyl cyclase activity, suggesting that the combined activation of beta 1AR/beta 2AR, plus beta 3AR, together produced an additive cAMP response. Multiple forms of adenylyl cyclase were identified in brown and white adipocyte cell lines and tissues. Northern blot analysis detected adenylyl cyclase types 5, 6, and 10. Screening of reverse transcriptase-PCR products by DNA sequencing confirmed the identities of these forms and lower levels of additional isoforms, raising the possibility that Beta-Adrenergic receptor subtypes in adipocytes couple to distinct adenylyl cyclases. Because these cell lines display functional and phenotypic similarities to interscapular brown adipocytes, they will be a useful model to study the regulation of Beta-Adrenergic receptor expression and function, and the control of Ucp expression and activity.
P M Quinton - One of the best experts on this subject based on the ideXlab platform.
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iontophoretic beta adrenergic stimulation of human sweat glands possible assay for cystic fibrosis transmembrane conductance regulator activity in vivo
Experimental Physiology, 2008Co-Authors: A K M Shamsuddin, M M Reddy, P M QuintonAbstract:With the advent of numerous candidate drugs for therapy in cystic fibrosis (CF), there is an urgent need for easily interpretable assays for testing their therapeutic value. Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) abolished Beta-Adrenergic but not cholinergic sweating in CF. Therefore, the Beta-Adrenergic response of the sweat gland may serve both as an in vivo diagnostic tool for CF and as a quantitative assay for testing the efficacy of new drugs designed to restore CFTR function in CF. Hence, with the objective of defining optimal conditions for stimulating Beta-Adrenergic sweating, we have investigated the components and pharmacology of sweat secretion using cell cultures and intact sweat glands. We studied the electrical responses and ionic mechanisms involved in Beta-Adrenergic and cholinergic sweating. We also tested the efficacy of different Beta-Adrenergic Agonists. Our results indicated that in normal subjects the cholinergic secretory response is mediated by activation of Ca(2+)-dependent Cl(-) conductance as well as K(+) conductances. In contrast, the Beta-Adrenergic secretory response is mediated exclusively by activation of a cAMP-dependent CFTR Cl(-) conductance without a concurrent activation of a K(+) conductance. Thus, the electrochemical driving forces generated by Beta-Adrenergic Agonists are significantly smaller compared with those generated by cholinergic Agonists, which in turn reflects in smaller Beta-Adrenergic secretory responses compared with cholinergic secretory responses. Furthermore, the Beta-Adrenergic Agonists, isoproprenaline and salbutamol, induced sweat secretion only when applied in combination with an adenylyl cyclase activator (forskolin) or a phosphodiesterase inhibitor (3-isobutyl-1-methylxanthine, aminophylline or theophylline). We surmise that to obtain consistent Beta-Adrenergic sweat responses, levels of intracellular cAMP above that achievable with a Beta-Adrenergic agonist alone are essential. Beta-Adrenergic secretion can be stimulated in vivo by concurrent iontophoresis of these drugs in normal, but not in CF, subjects.
Michael A Levine - One of the best experts on this subject based on the ideXlab platform.
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impaired beta adrenergic receptor activation of adenylyl cyclase in airway smooth muscle in the basenji greyhound dog model of airway hyperresponsiveness
American Journal of Respiratory Cell and Molecular Biology, 1993Co-Authors: C W Emala, Cynthia Black, Craig Curry, Michael A LevineAbstract:Previous studies in human asthmatics have suggested a defect in the Beta-Adrenergic pathway leading to cyclic adenosine monophosphate (cAMP) generation. Although these studies have suggested normal or increased numbers of Beta-Adrenergic receptors, limitations in the quantity of tissue available have not allowed further delineation of the biochemical or molecular mechanisms of human asthma. The basenji-greyhound (BG) dog model of nonspecific airway hyperreactivity displays similarities to human asthma, and altered functional response to Beta-Adrenergic Agonists has been previously shown in airway tissue from this model. We have now correlated this functional impairment in Beta-Adrenergic response with a decreased generation of cAMP in response to isoproterenol. Organ bath studies and adenylyl cyclase assays of trachealis muscle revealed impaired responses to isoproterenol in the BG dog as compared with control dogs. Pretreatment of muscle strips from BG dogs with isoproterenol had no effect on subsequent dose-response curves to methacholine (pD2 = 7.17 +/- 0.13, 7.34 +/- 0.12, and 7.14 +/- 0.17 for control, 10(-6) M isoproterenol, and 10(-5) M isoproterenol, respectively), while muscle strips from mongrel dogs had a significant shift in methacholine responses after isoproterenol pretreatment (pD2 = 7.91 +/- 0.23, 7.48 +/- 0.29, and 6.98 +/- 0.33 for control, 10(-6) M isoproterenol, and 10(-5) M isoproterenol, respectively). Adenylyl cyclase activity in response to isoproterenol was 54% in the BG trachealis membranes as compared with mongrels. Functional and biochemical responses to forskolin, NaF, prostaglandin, and dibutyryl cAMP, and the quantity of G,alpha were similar in the BG and control dogs.(ABSTRACT TRUNCATED AT 250 WORDS)