Complex estrogenic regulation of catechol-O-methyltransferase (COMT) in rats.

Catechol-O-methyltransferase (COMT) activity depends on gender, age and physiological status suggesting that estrogen may regulate COMT activity. In fact, estrogens down-regulate the function of COMT promoters in cell cultures. On the other hand, COMT may play an important role in estrogen-induced cancers due to its ability to inactivate estrogen metabolites and thereby lowering the levels of these potential carcinogens. In this study, we explored the effect of estrogen on COMT activity in vivo in rats. Male and female Wistar rats received 14-day treatments with either estradiol (100 µg/kg/day; s.c.) or tamoxifen (500 µg/kg/day; s.c.), respectively; in addition ovariectomized rats were studied. COMT activity and COMT protein expression were measured from various brain- and peripheral tissues. Although we found a regulatory function of estrogen, its effects were sex and tissue dependent. Antagonizing the effects of estrogen via tamoxifen increased COMT protein expression in several central and peripheral tissues. However, amounts of COMT protein and COMT activities did not always match. Generally, COMT activities were quite resistant to the effects of tamoxifen and estradiol. Estradiol, unexpectedly, doubled the amount of COMT protein in the prostate but exhibited down-regulatory function in the prefrontal cortex and kidneys. Ovariectomy by itself, however, had only minor effects on COMT activity and expression. It is noteworthy that the estrogen down-regulation and tamoxifen up-regulation of COMT were best substantiated in the prefrontal cortex and kidneys where COMT is physiologically important for dopamine metabolism.

Identification and characterization of the imidazoline I2b-binding sites in the hamster brown adipose tissue as a study model for imidazoline receptors.

The imidazoline-type compound, MPV-1743, has been found to activate nonshivering thermogenesis (NST) in brown adipose tissue (BAT) of the genetically obese Zucker rats. The regulation of NST in BAT is linked to the catecholamine metabolism, and the imidazoline I2-binding sites have been found on the monoamine oxidase, a catecholamine metabolising enzyme. In this study, the I2-binding sites of hamster BAT have been characterised using a receptor binding assay with 3H-idazoxan as a radioligand, and the interaction of MPV-1743 with these I2-binding sites has been studied using the enantiomers of MPV 1743, that is, MPV 2088 and MPV 2089. Cirazoline was used to determine the specific binding of 3H-idazoxan to the imidazoline I2-binding sites. Rauwolscine was added in the 3H-idazoxan binding assay in order to inhibit any binding to potential alpha2-adrenergic sites. In the presence of rauwolscine mask 3H-Idazoxan labelled a population of non-adrenergic binding sites expressing the properties of the imidazoline I2b-receptor subtype similar to that found in the rat liver (cirazoline >> guanabenz = amiloride >> clonidine). The binding of 3H-idazoxan to the I2b-binding sites could be displaced by the imidazole compounds with the following affinities: detomidine (KiHigh 9.2 nM; KiLow 3200 nM), MPV-2088 (KiHigh 19 nM; IKiLow 760 nM) and MPV-2089 (KiHigh 190 nM; KiLow 1300 nM), atipamezole (3500 nM) and dexmedetomidine (Ki 8400 nM). These results have shown that the hamster BAT contains the imidazoline I2b-binding sites with heterogeneous binding properties for some test compounds. In addition, the enantiomers of MPV 1743, that is, MPV 2088 and MPV 2089, had high affinity to these BAT imidazoline I2b-binding sites. Therefore, it is suggested that the regulation of NST in the hamster BAT may be an attractive model to study the role of imidazoline I2b-binding sites.

Increased p53 levels without caspase-3 activity and change of cell viability in 6-hydroxydopamine-treated CV1-P cells.

The effect of 6-hydroxydopamine (6-OHDA) on the level of p53 protein, the activity of caspase-3 and the nuclear morphology-based assessment of cell viability were compared in the nonneuronal CV1-P fibroblast and neuronal SH-SY5Y neuroblastoma cells. The level of p53 protein was increased in the low-dose range (<100 micromol/L) in both cell types, particularly in fibroblasts. In the neuroblastoma cells, a moderate p53 increase paralleled the elevated caspase-3 activity and apoptotic cell behavior. Interestingly, in the fibroblasts at the low 6-OHDA concentrations, p53 remained high during the whole experiment, and there was neither significant caspase-3 activity nor cell death. In the high-dose range (>100 micromol/L), the increase of p53 was reduced and the cell death was predominantly necrotic as judged from the nuclear morphology in both fibroblasts and neuroblastoma cells. Also, the caspase-3 activity was reduced in SH-SY5Y cells. In contrast to some earlier reports, we have shown that the actual 6-OHDA sensitivity of nonneuronal cells may be equal or even higher than that in neuronal cells if the enhancement of p53 levels is used as a criterion for the response. However, the 6-OHDA toxicity was clearly higher in the neuronal than in fibroblast cells.

Efficient transfection of novel bovine papillomavirus 1 expression plasmids.

A series of novel bovine papillomavirus type 1 (BPV-1)-based expression plasmids was constructed and characterized in vitro as a starting point for the development of an in vivo gene therapeutic method. The order of transfection efficiency for different pBPVlacZ plasmids was pCGalBPV > pTKBPV > pSRalphaBPV in CV1-P cells. In the absence of selection pressure, the expression of pCGalBPVlacZ and pTKBPVlacZ was associated with long-term maintenance. In a comparison of pBPVlacZ with pSVlacZ, expression was maintained up to 12-17 and 8-12 days, respectively. The transfection of pBPVlacZ plasmids was efficient in secondary and primary, dividing and nondividing, neural and nonneural, and human cells and, furthermore, independent of the cell cycle as seen in growing as well as resting cells. All these characteristics are likely to be relevant for in vivo conditions, under which the percentage of proliferating cells could be quite low. In conclusion, the pBPV plasmids were efficiently delivered and expressed in different host cells, and therefore their performance in gene therapy is worth testing.

Effects of histamine H(3)-ligands on the levodopa-induced turning behavior of hemiparkinsonian rats.

Histamine H(3)-receptors act as heteroreceptors on many neurons. The effects of H(3)-ligands (an agonist, R-alpha-methylhistamine and an antagonist, thioperamide) on levodopa-induced turning behavior in a rat model of Parkinson's disease were quite similar to those seen with alpha(2)-adrenoceptor ligands (dexmedetomidine and atipamezole). R-alpha-methylhistamine clearly reduced contralateral turning behavior but the increase of turning behavior after thioperamide was less clear. The lack of effect of H(3)-ligands, in contrast to alpha(2)-ligands, on the amphetamine-induced ipsilateral turning behavior points to different roles or neuronal distribution of these two presynaptic receptors. We propose that in this lesion model, H(3)-receptors modify those pathways participating striatal outflow.

Anti-obesity effect of MPV-1743 A III, a novel imidazoline derivative, in genetic obesity.

MPV-1743 A III ((+/-)-4-(5-fluoro-2,3-dihydro-1H-inden-2-yl)-1H-imidazole) is a novel imidazoline derivative. In this study, it was shown to bind with high affinity to alpha2-adrenoceptor subtypes alpha2A (IC50) = 0.66 +/- 0.06 nM), alpha2B (IC50) = 3.8 +/- 0.53 nM), alpha2C (IC50) = 3.1 +/- 0.61 nM) in the recombinant S115 cells and to alpha2D (IC50 = 0.94 +/- 0.10 nM) in the rat submandibular gland. MPV-1743 A III also showed remarkably high affinity to alpha1-adrenoceptors (IC50 = 150 +/- 12 nM) in the rat cerebral cortex and to imidazoline I2b-binding sites (IC50) = 150 +/- 5.0 nM) in the rat liver. The functional alpha2-adrenoceptor antagonistic effect of MPV-1743 A III was demonstrated by studying the ability of orally administered MPV-1743 A III to reverse and prevent the alpha2-adrenoceptor agonist detomidine-induced mydriasis in rat. The anti-obesity effect of MPV-1743 A III was investigated in genetically obese (fa/fa) Zucker rats in two different phases of obesity. Chronic treatment with MPV-1743 A III (0.3 3 mg/kg per day p.o. for 3 weeks) dose dependently decreased weight gain in early-phase obesity. In fully established obesity, GDP binding to mitochondria and expression of uncoupling protein mRNA were increased in brown adipose tissue by MPV-1743 A III indicating an activation of non-shivering thermogenesis. The present study shows that MPV- 1743 A III has a modest anti-obesity effect in the genetic rodent model of obesity. The relative importance of alpha2- and alpha1-adrenoceptors and imidazoline I2b-binding sites in mediating the effects of MPV-1743 A III needs further evaluation.

Decreased liver type I 5'-deiodinase and increased brown adipose tissue type II 5'-deiodinase activity in 2,3,7,8-tetrachlorobibenzo-p-dioxin (TCDD)-treated Long-Evans rats.

The effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was studied on the activity of type I 5'-deiodinase (5' DI) in liver and of type II 5'-deiodinase (5' DII) in brown adipose tissue (BAT) microsomes of Long-Evans rats. TCDD decreased the activity of liver 5' DI and increased that of BAT 5' DII. It also reduced the concentration of serum total thyroxine (T4) without affecting triiodothyronine (T3). These effects of TCDD on 5'-deiodinases and serum thyroid hormones are compatible with partial hypothyroidism. Based on our results, it is not possible to decide unequivocally whether TCDD affects thyroid hormone status and 5'-deiodination by a direct action or a feedback mechanism. However, these data are important because they demonstrate that TCDD affects thyroid status in a tissue-specific manner and that its effects on thyroid hormone metabolism are much more complex than currently assumed.

Alpha 2A-adrenergic regulation of cyclic AMP accumulation and lipolysis in human omental and subcutaneous adipocytes.

OBJECTIVE: To characterize differences in alpha 2-adrenergic regulation between subcutaneous and omental adipocytes which could offer a possibility of pharmacological intervention in the metabolic syndrome. DESIGN: Both subcutaneous and omental adipocytes were isolated from 32 patients. Adipocytes were incubated in the presence of adrenoceptor agonists, and cyclic AMP and glycerol levels were measured. alpha 2-Adrenoceptors of isolated plasma membranes were characterized. RESULTS: Adrenaline increased cyclic AMP levels about two-fold in omental adipocytes but had almost no effect in subcutaneous fat cells. The inhibition of cyclic AMP accumulation and glycerol release by UK-14304 and dexmedetomidine was less pronounced in omental adipocytes. The maximal effect of isoprenaline on cyclic AMP levels and glycerol release was similar at the two sites. The subcutaneous and omental alpha-adrenoceptors had similar affinities to 3H-RX821002 and showed characteristics of the alpha 2A subtype. The receptor densities were 220 +/- 21 and 460 +/- 84 fmol/mg of protein (means +/- s.e.m.) in omental and subcutaneous membranes, respectively. CONCLUSION: Inhibition of cyclic AMP accumulation and lipolysis by alpha 2A-adrenoceptors is less pronounced in omental than subcutaneous adipocytes which could be due to differences in receptor number. These differences in alpha 2A-adrenergic regulation could be of value in the treatment of the metabolic syndrome.

Effects of thyroid hormone on norepinephrine signaling in brown adipose tissue. I. Beta 1- and beta 2-adrenergic receptors and cyclic adenosine 3',5'-monophosphate generation.

Brown adipose tissue (BAT) thermogenesis is activated by the sympathetic nervous system. BAT responses to norepinephrine are blunted in hypothyroidism and are rapidly restored by thyroid hormone. We examined in rats the effects of thyroid hormone on BAT beta 1- and beta 2-adrenergic receptors (AR) expression and capacity to generate cAMP in response to adrenergic stimulation. Both are reduced in hypothyroidism. The reduction in cAMP generation is equal to or greater than that in beta 1,2-AR; it is the same whether cAMP production is stimulated with norepinephrine, selective beta 3-AR agonists, or forskolin; and it is not affected by the inhibition of phosphodiesterase. Both beta 1,2-AR and the capacity to generate cAMP were slowly corrected by thyroid hormone. T3 normalized beta 1,2-AR between 1 and 2 days, whereas the improvement in cAMP generation lagged 1 or 2 days behind. Within 2 days of acclimation of athyreotic rats at 30 C, the number of beta 1,2-AR reached the euthyroid level, whereas exposure to 4 C decreased these receptors. We reached the following conclusions: 1) BAT beta 1,2-AR and capacity to generate cAMP are reduced in hypothyroidism; 2) the latter, however, is not explained by the reduction in beta 1,2-AR, but, rather, reflects a fault at the postreceptor level; 3) the reduction in beta 1.2-AR number is largely caused by the cold stress derived from the low metabolic rate of the hypothyroid state; and 4) the slow restoration of both receptor number and capacity to generate cAMP after T3 are not consistent with these defects being a significant factor in the previously reported blunted uncoupling protein responses to adrenergic stimulation in hypothyroidism.

Thyroid hormone and norepinephrine signaling in brown adipose tissue. II: Differential effects of thyroid hormone on beta 3-adrenergic receptors in brown and white adipose tissue.

UNLABELLED: beta 3-adrenergic receptors (AR) are predominantly expressed in brown (BAT) and white adipose tissue (WAT), being more abundant in the former. There is growing interest in these receptors because of their potential to be pharmacologically targeted to control energy expenditure and lipid accretion. We have examined, in BAT and WAT of rats, the effect of thyroid hormone on the expression of beta 3-AR and their contribution to cAMP generation. Receptor density was assessed with the nonselective beta 3-AR ligand [125I]-cyanopindolol ([125I]-CYP) and the highly selective beta 3-AR agonist CL3216,243. beta 3-AR messenger RNA (mRNA) was analyzed by Northern blotting of total BAT and WAT RNA. Generation of cAMP by brown adipocytes in response to norepinephrine (NE) and CL316,243 was measured by RIA. In BAT, beta 3-AR can account for as much as 50% of the maximal cAMP response to NE, whereas in WAT these receptors probably account for all the effect of NE. In hypothyroidism. BAT beta 3-AR number is increased, as are beta 3-AR mRNA (4- to 6-fold) and the relative contribution of these receptors to the maximal cAMP production. In contrast, both beta 3-AR number and mRNA levels are reduced in WAT of hypothyroid rats. The injection of T3 to hypothyroid rats reverts the changes in beta 3-AR within 24 h, and T3 excess causes a greater than 90% reduction in beta 3-AR mRNA in BAT but a 5-fold increase in WAT. In both tissues, hypothyroidism is associated with a marked reduction in the capacity to generate cAMP, but this is not completely restored even after 2 days of a receptor-saturating dose of T3. CONCLUSIONS: 1) thyroid hormone differentially affects the expression of beta 3-AR in BAT and WAT; 2) these effects of T3 are both rapid and marked and seem to take place at a pretranslational level; 3) in both tissues there is a postreceptor defect in the generation of cAMP that is corrected by T3 much later after the changes in beta 3-AR are reversed; and 4) thyroid hormone is among the known factors that most vigorously affect the expression of beta 3-AR.

Increased alpha 1-adrenoceptor density in brown adipose tissue indicates recruitment drive in hypothyroid rats.

The effects of hypothyroidism on whole body thermogenesis, brown adipose tissue recruitment state, and alpha 1-adrenergic receptor density were investigated. Treatment of rats with methimazole for 4-5 wk led, as expected, to reduction of growth and resting metabolic rate. The thermogenic response to norepinephrine injection was practically abolished. Generally, only small effects of hypothyroidism on brown adipose tissue were observed: total protein content, mitochondrial GDP binding capacity, and total content of the uncoupling protein thermogenin were not altered. The density of beta-adrenergic receptors (estimated with [3H]CGP-12177 as a ligand) was also unchanged. However, the density of alpha 1-adrenergic receptors (estimated with [3H]prazosin) was markedly increased; in other physiological conditions, such an increase has been associated with an increased degree of recruitment of the tissue. These data indicate that brown adipose tissue in the subthermoneutral hypothyroid animal, probably due to homeostatic mechanisms, is exposed to an increased sympathetic stimulation, leading to an increased alpha 1-adrenoceptor density. However, other features of recruitment are only poorly induced, probably due to attenuation of the beta-adrenergic signaling mechanism. The increased alpha 1-adrenergic receptor density may be responsible for certain altered features of brown adipose tissue in hypothyroid animals, such as peroxisomal recruitment and perhaps also for maintenance of the thermogenin content. The results also indicate that the increased alpha 1-adrenergic density generally seen in recruitment would not result from chronic beta-adrenergic stimulation of the tissue but may be controlled via another regulatory pathway, e.g., via the alpha 1-adrenergic pathway itself.

Triiodothyronine causes rapid reversal of alpha 1/cyclic adenosine monophosphate synergism on brown adipocyte respiration and type II deiodinase activity.

Previous studies have shown that thyroid status affects the response of brown adipose tissue (BAT) to the sympathetic nervous system. For example, hypothyroidism is associated with the development of a marked synergism between alpha 1- and beta-adrenergic pathways to stimulate type II iodothyronine 5'-deiodinase activity. Hypothyroidism also attenuates the respiratory response (thermogenesis) of isolated brown adipocytes to norepinephrine. To explore the interactions of the sympathetic nervous system and thyroid status in these cells, we compared the thermogenic and 5'-deiodinase responses to adrenergic agonists in isolated brown adipocytes from hypothyroid rats during treatment with 3,5,3'-triiodothyronine (T3). The fivefold synergism of alpha 1- and beta-adrenergic catecholamines to increase the deiodinase activity was progressively reduced, reaching a control euthyroid value of unity after 5 days of T3 treatment. Hypothyroidism reduced both the O2max (twofold to threefold) and increased the concentration of agonist required for 50% stimulation (10-fold) for both norepinephrine and forskolin. In hypothyroid cells, there was a twofold synergism between the alpha 1-agonist cirazoline and forskolin to increase respiration, which was blocked by prazosin and reproduced by the calcium ionophore, A23187. This synergistic effect of the alpha 1-agonist was lost within 2 days of T3 administration. These studies identify a second Ca(2+)-dependent intra-adrenergic synergism, which functions to ameliorate the reduced cyclic adenosine monophosphate (cAMP) responsiveness of the hypothyroid brown adipocyte.

Alpha 1- and beta-adrenergic receptors in brown adipose tissue and the adrenergic regulation of thyroxine 5'-deiodinase.

The density of alpha 1- and beta-adrenergic receptors and the adrenergic regulation of the type II thyroxine 5'-deiodinase were investigated in brown adipose tissue. The density of the alpha 1- and beta-adrenergic receptors was determined in membranes of brown fat from animals in different physiological states, i.e. in cold-acclimated, neonatal, cafeteria-fed, genetically obese and hypothyroid animals. The density of the alpha 1-adrenergic receptors in brown fat correlated well with the recruitment state of the tissue: the receptor density was increased when the brown fat was recruited and decreased when the brown fat was atrophied. The density of the beta-adrenergic receptors did not correlate with the recruitment state. The regulation of the conversion of thyroxine (T4) to triiodothyronine (T3) by type II 5'-deiodinase in vitro was investigated in brown adipocytes from euthyroid and hypothyroid rats. alpha 1- and beta-adrenergic stimulation enhanced the activity of the 5'-deiodinase in a synergistic way. The primary signal seemed to be beta-adrenergic stimulation and cyclic AMP, whereas the role of the alpha 1-adrenergic receptors was a potentiation of the cyclic AMP effect via Ca2+ and protein kinase C. The maximal response of the 5'-deiodinase needed all these three different signals. Synergistic potentiation of the beta-adrenergic stimulation by alpha 1-adrenergic stimulation was seen especially in brown adipocytes from hypothyroid rats. Also the normal stimulation of the 5'-deiodinase activity as such was enhanced due to hypothyroidism. The role of synergism was investigated further in cell respiration (oxygen consumption) and cyclic AMP accumulation. In brown adipocytes from hypothyroid rats, the beta-adrenergic respiration was potentiated by alpha 1-adrenergic stimulation. An inhibitory effect of the alpha 1-adrenergic antagonist prazosin was found even in the cyclic AMP accumulation in brown adipocytes of hypothyroid rats, although no significant synergism could be demonstrated. It was suggested that the increased density of the alpha 1-adrenergic receptors may be of significance for nonshivering thermogenesis in certain physiological situations (e.g. in cold acclimation) via two different but synergistic mechanisms, i.e. the regulation of thyroxine 5'-deiodinase and of adenylate cyclase.

Alpha 1- and beta-adrenergic agents cause synergistic stimulation of the iodothyronine deiodinase in rat brown adipocytes.

Previous studies have shown that norepinephrine causes a marked increase in the iodothyronine 5'-deiodinase activity in dispersed brown adipocytes. This stimulation required mRNA and protein synthesis and was 3- to 4-fold greater in cells from hypothyroid than in those from euthyroid rats. To investigate the cause of this differential response, we incubated dispersed brown adipocytes with catecholamines, a specific alpha 1-agonist, forskolin, and (Bu)2cAMP alone and in combination. Our results show a synergistic effect of alpha 1- and beta-adrenergic catecholamines to increase the deiodinase, which leads to 2-fold (euthyroid) to 4-fold (hypothyroid) higher enzyme activities in the presence of both agonists than can be accounted for by additive effects of the two agents. Since alpha 1-agonists cause minimal stimulation alone, this response is due to an enhancement of the cAMP effect. The alpha 1 effect is mimicked by the calcium ionophore A23187, but not by phorbol ester alone. After 2-h exposure to beta-adrenergic agents or forskolin, hypothyroid cells had a reduced cAMP response, but alpha 1-agonists did not reverse this. These results demonstrate a complex interrelationship between alpha 1- and beta-adrenergic agonists and thyroid status in the regulation of deiodinase in the brown adipocyte. The increase in intracellular calcium due to an alpha 1-agonist markedly enhances the effects of cAMP on deiodinase activation, permitting a beta-adrenergic effect despite the impaired cAMP generation characteristic of hypothyroid adipocytes. This unexpected enhancement of the beta-adrenergic pathway in the hypothyroid state may be especially relevant for maintaining maximum T3 production, which is required for the normal thermogenic function of this tissue.

Effect of thyroid status on catecholamine stimulation of thyroxine 5'-deiodinase in brown adipocytes.

We examined type II 5'-iodothyronine deiodinase activation by adrenergic agonists in dispersed brown adipocytes from euthyroid and hypothyroid rats. In euthyroid cells, basal deiodinase activity was 30-100 fmol I-.h-1.10(6) cells-1 and increased four- to fivefold during exposure to norepinephrine, an effect that was enhanced by alprenolol. In cells from hypothyroid rats, norepinephrine caused a three- to fourfold greater deiodinase stimulation than occurred in euthyroid cells but alprenolol inhibited the response. In euthyroid cells, phenylephrine caused greater stimulation than did norepinephrine, but this was inhibited by alprenolol. Isoproterenol and 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) inhibited the phenylephrine response but were modestly stimulatory alone. Although both alpha 1- and beta-adrenergic agonists increased deiodinase activity modestly in hypothyroid cells, in combination they caused a marked synergistic stimulation. This synergism was induced by 8-BrcAMP and forskolin, as well as by isoproterenol. The stimulation of deiodinase in both cell types was due to an increase in Vmax without an alteration in the Km and required mRNA synthesis. The markedly greater deiodinase response of the hypothyroid brown adipocyte to catecholamines may serve to enhance the impaired thermogenic response of this tissue to cold exposure.

Alpha 1- and beta-adrenergic receptors in brown adipose tissue of lean (Fa/?) and obese (fa/fa) Zucker rats. Effects of cold-acclimation, sucrose feeding and adrenalectomy.

1. The populations of alpha 1- and beta-adrenergic receptors in brown adipose tissue (BAT) of genetically obese Zucker rats (fa/fa) were studied with [3H]prazosin and [3H]CGP-12177 respectively. 2. The density of alpha 1-adrenergic receptors in BAT was significantly lower in obese than in lean Zucker rats, both at 2-4 months of age and at 6 weeks of age. The density of beta-adrenergic receptors was identical in BAT of lean and obese 6-week-old Zucker rats. 3. Cold-acclimation increased the alpha 1-receptor density significantly in BAT of both lean and obese Zucker rats, and the number of beta-receptors was also somewhat increased. 4. Sucrose feeding did not affect the density of alpha 1-receptors in BAT of lean or obese Zucker rats, but it increased beta-receptor density. 5. Adrenalectomy restored the density of alpha 1-adrenergic receptors in BAT of obese Zucker rats to the value observed in lean rats. 6. It is concluded that there is a direct correlation between alpha 1-receptor density and tissue recruitment, and that alpha 1-receptor density is thus positively correlated with sympathetic activity. beta-Receptor density is apparently better correlated with feeding conditions.

Increased alpha 1-adrenergic receptor density in brown adipose tissue of cold-acclimated rats and hamsters.

Binding sites for [3H]prazosin were characterized in crude membrane fractions from rat brown adipose tissue. Based on agonist (norepinephrine approximately equal to phenylephrine much greater than isoprenaline) and antagonist (prazosin much greater than yohimbine greater than propranolol) potencies to compete with [3H]prazosin, the binding sites were identified as alpha 1-receptors, not previously described in rat brown adipose tissue. As the [3H]prazosin binding sites could be observed in isolated brown fat cell preparations, they were probably postsynaptic. The effect of cold acclimation was studied in crude membrane fractions from control and cold-acclimated (4 degrees C) rats and hamsters. Cold acclimation did not change the affinity of the receptor for agonists and antagonists, but there was a significant increase in the number of alpha 1-receptors (per mg protein), both in rat (100% increase) and hamster (40% increase) brown fat. Based on these results and on earlier results on beta-receptors from this and other laboratories, it is suggested that activation of brown adipose tissue is associated with an increase in the relative density of alpha 1-receptors (i.e. in the alpha 1/beta ratio) and an increased significance of alpha 1-adrenergic pathways for the function of the tissue.

Increased alpha 1-adrenergic receptor density in brown adipose tissue of cafeteria-fed rats.

A possible general corollary between alpha 1-receptor density in brown adipose tissue and the degree of activation of the tissue was investigated. For this purpose, the effect of cafeteria feeding on alpha 1-adrenergic receptors in brown adipose tissue of seven-week-old female rats was studied by the use of the alpha 1-antagonist (3H)-prazosin. In cafeteria-fed rats, the KD of the alpha 1-receptor for (3H)prazosin was unchanged (about 0.35 nM), but the receptor density was doubled (up to 40 fmol per mg of membrane protein). This was also observed when the results were expressed per unit of a plasma-membrane marker (5'-nucleotidase). It was concluded that an increased alpha 1-receptor density is seen not only in cold-acclimated rats, but also in other conditions where brown fat is activated, and a possible general physiological significance of alpha 1-adrenergic pathways in brown adipose tissue is discussed.

Parallel increases in amount of (3H)GDP binding and thermogenin antigen in brown-adipose-tissue mitochondria of cafeteria-fed rats.

In order to investigate the possible existence of a 'masked' (i.e. non-GDP-binding) form of thermogenin (the brown-adipose-tissue specific, 32 000 Da so-called "uncoupling" protein), rats were fed a routine pellet diet or, in addition to this, a cafeteria diet. Brown-adipose-tissue mitochondria isolated from the cafeteria-fed animals showed as expected an increased (3H)GDP binding capacity (from 0.26 to 0.41 nmol/mg protein; an increase of 57%). However, when analysed by a quantitative enzyme-linked immuno-assay system for thermogenin, the mitochondria also showed an increased content of thermogenin (from 14.9 to 20.5 micrograms per mg; an increase of 38%). The ratio between thermogenin and GDP binding was 61 000 and 53 000 g/mol in the two cases; these values were not significantly different and were in good agreement with suggestions that thermogenin binds 1 GDP per thermogenin dimer. It was concluded that under the conditions investigated, there was no reason to assume the existence of a masked form of thermogenin.