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Alpha-2 adrenoreceptors in brown adipose tissue of infant rats

University of British Columbia

This thesis consists of five chapters. The first chapter deals with general background and introduction. Each of the subsequent chapters are divided into sections. The first section deals with pharmacological characterization of ∝₂-adrenoceptors using various ligands. The second section pertains to the study of binding characteristics of ∝₂-adrenoceptors following chemical sympathectomy by 6-hydroxydopamine and chronic blockade of ∝₂-adrenoceptors by yohimbine injections. The third section deals with the study of guanylate cyclase system in relation to ∝₂-adrenoceptors stimulation in brown fat fragments of 7-day-old rats. The fourth section is devoted to the study of the physiological response associated with the stimulation of ∝₂-adrenoceptors in isolated adipocytes from brown fat of 7-day-old rats. Finally cyclic GMP production in obese and lean mice in relation to ∝₂-adrenoceptors stimulation was discussed in the fifth section. Binding characteristics of ∝₂-antagonists ([³H]-RX-78- 1094, [³H]-yohimbine, [³H]-rauwolscine) and agonists ([³H]-clon- idine, [³H]-norepinephrine) to ∝₂-adrenoceptors on isolated plasma membrane fragments from brown adipose tissue were studied. The binding of [³H]-yohimbine was rapid,saturable and reversible. Yohimbine, (-)-epinephrine, and clonidine displaced [³H]-yohimbine from its binding sites in that order of potency as would be expected of binding to ∝₂- adrenoceptors. A Scatchard plot of yohimbine binding showed an equilibrium constant (K[sub d]) of 18 nM and total binding capacity (B[sub max] ) of 0.15 pmol/mg protein. Binding of [³H]-RX781094 and [³H]-clonidine showed a similar pattern of rapid, stable, saturable and reversible binding. Studies on the binding of (-)[³H]-norepinephrine indicated the presence of more than one binding site. Scatchard analysis of the (-)[³H]-norepinephrine binding using (-)-epinephrine or yohimbine as the displacing agent, revealed a K[sub d] of .60.4 nM and 65.8 nM respectively, and B[sub max] values of 0.22 and 0.24 pmol/mg protein. Norepinephrine, yohimbine and ∝₂-epinephrine probably shared one common binding site; the other site with of 64.5 nM was present in much lower number (71.3 fmol/mg protein) and was specific for (-)-norepin-ephrine and yohimbine only. In addition, a Hill coefficient of 1.4 further supported the presence of two positively cooperative binding sites. Binding of (-)[³H]-dihydroalpre-nolol was displaceable by practolol and norepinephrine with a K[sub d] of 50 nM and 10 nM respectively (β₁-site) and B[sub max] of 0.19 and 0.5 pmol/mg protein. However, (-)[³H]-dihydroal-prenolol binding could also be displaced by yohimbine suggesting either a relative non-specificity of the ligand or an atypical nature of the β₁-adrenoceptors in brown fat of infant rats. It is suggested that the plasma membranes from actively proliferating brown fat of infant rats possess both β₁-and ∝₂-adrenoceptors. The physiological in vivo agonist (-)-norepinephrine may exert its effects via both or either adrenoceptor sub-type. Binding studies carried out with [³H]-yohimbine on membranes isolated from brown fat of chemically sympath- ectomized infant rats showed smaller number of high affinity yohimbine binding sites when compared to those isolated from control (saline-injected) rats of the same age. The (-)[³H]-norepinephrine binding to identical membrane preparations revealed the presence of both high (K[sub d] = 36 nM) and low (K[sub d] = 200 nM) affinity binding sites; with a Hill coefficient of 1.5. The total number of norepinephrine binding sites more than doubled after sympathectomy; this increase was caused by emergence of low affinity sites. Chronic yohimbine pre- treatment resulted in more than two-fold increase in the number of binding sites for both [³H]-yohimbine and (-)[³H]- norepinephrine. The affinity of ∝₂-adrenoceptors for yohimbine binding sites decreased whereas that for norepinephrine remained unchanged. These results not only confirm the presence of ∝₂-adrenoceptors in brown fat of developing rats but also indicate that the binding characteristics of these receptors can be altered by chemical sympathectomy and by chronic exposure of infant rats to an ∝₂ -receptor blocker. Incubation of brown fat tissue pieces with clonidine (0.2-20μM) showed a dose- and;time-' dependent elevation of tissue cyclic GMP content. The peak response occurred at the concentration of 20μM for one-month-old rats. For brown fat from one-week-old rats, the peak response occurred at 0.5 - 1μMof clonidine and 3 - 5 minutes of incubation. The response could be blocked by prior incubation with yohimbine. When tissue cyclic GMP concentration, elevated in response to clonidine incubation, was separated into releaseable and receptor-protein bound fraction, a similar trend was seen. The data supported the hypothesis that ∝₂-receptor stimulation of brown fat is linked (directly or indirectly perhaps via Ca²⁺) to guanylate cyclase activation. Earlier in vivo experiments had shown a defective response of brown fat cyclic GMP production in obese mice upon acute cold exposure and catecholamine injections as compared to control litter mates which showed a dose- and time- dependent increase. Preliminary in vitro experiments where where fragments from obese and lean mice were stimulated with clonidine, showed two-fold increase in the cyclic GMP concentration compared to non-stimulated controls. This suggested that tissue capability to respond by an increase in cyclic GMP production in obese mice is the same as that in the lean mice. Forskolin and Isobutylmethylxanthine stimulated glycerol release in isolated adipocytes from brown fat of one-week-old rats. Clonidine, prostaglandin E₂ and nicotinic acid showed inhibitory effects on glycerol release. Inhibition of glycerol release by clonidine was concentration-dependent and was antagonized by yohimbine. Inactivation of inhibitory regulatory protein (Ni) by pertussis toxin abolished the inhibitory effect of clonidine. This indicated that the inhibitory effect of clonidine on glycerol release is mediated via inhibitory protein (Ni). It was suggested that, perhaps, the anti-lipolytic effect of ∝₂-adrenoceptors may have a role in controlling the state of activity of fat cells.

Text

2010-08-07

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http://hdl.handle.net/2429/27195

Interdisciplinary Studies

University of British Columbia

UBCV

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