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Biomedical subjects

J E Silva

Publications and source records attributed to J E Silva.

At least 19 recordsLinked to original sources
PubMed2005

Leaching behaviour of a galvanic sludge in sulphuric acid and ammoniacal media.

Leaching studies of a sludge produced by the physico-chemical treatment of wastewaters generated by a Ni/Cr plating plant were carried out in both sulphuric acid and ammoniacal media aiming to decide which of them would be the best treatment for this kind of waste material. The dissolution behaviour of some metals (Cu, Ni, Cr and Zn) was studied in order to assure the best metal recovery conditions in subsequent processes by the use of some separation methods such as solvent extraction and precipitation techniques. Therefore, the study here presented deals with the first chemical stage of an integrated treatment process. For the sulphuric acid leaching, maximal conversions obtained were 88.6% Cu, 98.0% Ni and 99.2% Zn for the following experimental conditions: a 100 g L(-1) acid concentration, a 5:1 liquid-to-solid ratio (L/S), a particle size less than 1 mm, a digestion time of 1h, a stirring speed of 700 rpm (all at room temperature and under atmospheric pressure). As expected, no selectivity was achieved for the sulphuric acid leaching, despite this option yielding much higher metal ion dissolution when compared with that reached by ammoniacal leaching. The use of this latter medium allowed the extraction of Cu and Ni without Cr species, but rates of conversion were only about 70% for Cu and 50% for Ni, much lower than those obtained for sulphuric acid leaching.

Ammonia↗
PubMed2005

Solvent extraction applied to the recovery of heavy metals from galvanic sludge.

In this study, a hydrometallurgical treatment involving the solvent extraction and recovery of some heavy metals from a sulphuric acid leach solution of galvanic sludge, using di-(2-ethylhexyl)-phosphoric acid (D2EHPA) and bis-(2,4,4-trimethylpentyl)-phosphinic acid (Cyanex 272), both diluted in kerosene, has been investigated. The preliminary tests revealed the necessity to remove other metal species than zinc and nickel, contained in the leach solution, and therefore, processes to cement copper and precipitate chromium were then applied to finally obtain a Zn and Ni pregnant solution prior to solvent extraction. For the experimental conditions studied, Cyanex 272 showed a good recovery of Zn after the stripping stage using H2SO4, but D2EHPA effectively promoted a higher Zn extraction than Cyanex 272 did. The dependence of the solvent extraction method on variables such as pH, contact time and concentration of extractant, as well as the effect of different concentrations of sulphuric acid on stripping, are discussed. The discussion also includes the previous conditions developed to separate the main interfering metallic species from the leach solution in order to improve the extraction and recovery of zinc by solvent extraction. The final objective has been to achieve a solution as pure as possible to recover nickel sulphate.

Metallurgy↗
PubMed2004

Effect of experimental variables on the inertization of galvanic sludges in clay-based ceramics.

The incorporation of several industrial wastes in ceramic matrixes had been attempted as an effective low expense technique for the fixation of metallic species in usable products or simply to reduce the residue volume for further disposal. However, the dominant mechanism of the inertization process and the relevant influent parameters are still unknown, mostly due to the complexity of the systems. This work reports the effect of several processing parameters such as the mixing time, the calcination temperature and duration, the relative amount of sludge, and the physical aspect of the sample (powdered or pressed pellets) on the fixing level of relevant species (SiO2), SO4(2)-, Zn, Ni, Ca, Cu, Cr) by leaching in different media (aqueous, acetate, and citrate). Statistical tools were used to define the relevance of each experimental variable on the inertization process of the used galvanic sludge. The relative amount of sludge in the mixture, the calcination temperature and the agglomeration state of the sample were found to be the most influent parameters of the inertization process. The incipient reaction between sludge and ceramic matrix components points out for the dominance of a macro-encapsulation mechanism.

Acetic Acid↗
PubMed2004

Role of the mixing conditions and composition of galvanic sludges on the inertization process in clay-based ceramics.

Hydroxide-metal sludges from electroplating industry are a potential source of environmental contamination due to their high content of heavy metals. The incorporation of these residues in a ceramic matrix can be a promising way to suppress the harmful effect of metals normally present in those sludges. This work reports the role of the mixing time between the waste and ceramic materials and of the calcination step on the fixing level of several metal-containing species (Al, Zn, Ni, Fe, Ca, Cu, Cr) after sequential leaching in different media (aqueous, acetate and citrate). A strong and/or long mixing process will promote the deagglomeration of the coarser agglomerates and then will increase the reactivity of remaining grains towards the ceramic material during the calcination. As a consequence, inertization is improved for fired samples. With non-calcined samples leaching increases as a result of increasing dispersability/availability of species.

Aluminum Silicates↗
PubMed2000

Glucocorticoids inhibit the transcriptional response of the uncoupling protein-1 gene to adrenergic stimulation in a brown adipose cell line.

Glucocorticoids may inhibit brown adipose tissue (BAT) thermogenesis acting at a central level as well as reducing the responses of the tissue to adrenergic stimulation in vivo. This latter effect is not well understood. We investigated whether or not glucocorticoids directly reduce the expression of the key molecule for BAT thermogenesis, uncoupling protein-1 (UCP1), and if so, to what extent and by what mechanisms. We used HIB-1B brown adipose cells obtained from a hibernoma. The response of UCP1 mRNA to adrenergic stimulation in these cells is qualitatively and quantitatively similar to that seen in vivo. Dexamethasone and other glucocorticoids, given simultaneously with NE, nearly abolish the ensuing UCP1 mRNA accumulation. This effect was negated by the glucocorticoid receptor antagonist RU-486. Significant inhibition is seen within the physiological range of concentrations, with ID(50)s for dexamethasone and corticosterone of 1 and 75 nM, respectively. Within the time span of the experiments, glucocorticoids did not reduce the strength of the NE signal nor did they necessitate ongoing protein synthesis or reduce the stability of mature UCP1 mRNA, but they significantly inhibited the stimulation of transcription by NE in a run-on in vitro transcription assay. These observations indicate that glucocorticoids are powerful inhibitors of the UCP1 gene response to adrenergic stimulation acting at transcriptional level, and provide further evidence for a global inhibitory effect of glucocorticoids on BAT thermogenesis.

Adipose Tissue, Brown↗
PubMed2000

Uncoupling protein-2 and -3 messenger ribonucleic acids in adipose tissue and skeletal muscle of healthy males: variability, factors affecting expression, and relation to measures of metabolic rate.

Mitochondrial uncoupling protein-2 and -3 (UCP2 and UCP3) may be involved in the modulation of resting metabolic rate and energy balance. To investigate their variability, the influence of this on the variability of energy expenditure, and potential regulatory factors of the expression of the corresponding genes, we measured their messenger ribonucleic acids (mRNAs) in muscle and white adipose tissue of lean, healthy men and correlated the abundance of these mRNAs (attomoles per microg total RNA) with measures of resting metabolic rate, hormone levels (thyroid hormones, insulin, glucagon, leptin, and catecholamines), and fuels potentially involved in energy balance regulation. We also investigated whether the thiazolidinedione, troglitazone, stimulates UCP2 and UCP3 mRNA levels to follow up on the observation that this antidiabetic drug increases the levels of expression in cultured cells. We found UCP2 and UCP3 mRNA levels to be highly variable and poorly correlated with measures of energy expenditure and with most factors affecting energy balance. Only nocturnal urinary norepinephrine excretion could explain a significant fraction of the variability in both UCP2 and UCP3 expression in muscle, but not adipose tissue. Thyroid hormone and norepinephrine excretion were found to contribute to the variability of resting metabolic rate, but this could not be explained by an effect on UCP mRNAs. Troglitazone affected neither the expression of UCPs nor the hormones or the measures of metabolic rate investigated. In conclusion, our results show that the expression of UCP2 and UCP3 genes is quite variable in healthy males and that this variability does not explain that in resting energy expenditure, and suggest that sympathetic activity is an important potential regulator of the expression of these proteins in skeletal muscle. However, the data do not support the concept that regulation of the expression of these genes is the most important level of control of UCP3 and UCP2 functions, and other levels of control have to be invoked.

Adipose Tissue↗
PubMed2000

Thiazolidinediones inhibit the expression of beta3-adrenergic receptors at a transcriptional level.

The effect of the thiazolidinediones (TZDs) darglitazone and troglitazone on beta3-adrenergic receptor (AR) expression was studied in cultured cell lines representing several tissues. After 24 h of exposing HIB-1B brown adipocytes to 30 micromol/l darglitazone or 20 micromol/l troglitazone, beta3-AR mRNA levels were reduced by 75%. This effect was significant within 1 h of exposure to a maximal dose of these drugs, with the full effect obtained within 10 h. The darglitazone ID50 was approximately 10 nmol/l, similar to the Kd of TZDs binding to peroxisome proliferator-activated receptor-gamma (PPAR-gamma). These drugs also decreased beta3-AR mRNA in 3T3-F442A white adipocytes, but not in SK-N-MC cells, which lack PPAR-gamma2. A luciferase reporter gene containing 1.4 kb of 5' flanking sequence of the mouse beta3-AR was transiently transfected, with or without PPAR-gamma2, in SK-N-MC cells. The vigorous expression of luciferase driven by the beta3-AR gene sequence was inhibited by TZDs in a PPAR-gamma2-dependent manner. The half-lives of gamma3-AR precursor RNA and mRNA were short, approximately 40 and approximately 100 min, respectively, and remained unaffected by TZD treatment. Exposure of HIB-1B cells to 30 micromol/l darglitazone was associated with reduced beta3-AR mRNA levels, as well as decreased response of uncoupling protein 1 to norepinephrine + propranolol (a beta1 beta2-AR antagonist) or the specific beta3-AR agonist CL 316, 243. Both the beta3-AR mRNA level and response to these stimuli fully recovered by 24 h of removing the drug, indicating that the beta3-AR protein and its coupling to adenylyl cyclase rapidly followed the changes in mRNA. Thus, TZDs can rapidly reduce beta3-AR expression at the transcriptional level, acting through PPAR-gamma2. The rapid turnover and responses of beta3-AR to perturbations, along with numerous other factors reported to regulate its expression, suggest a tight control of beta3-AR and function. Lastly, leptin being the only other known gene suppressed by TZDs, the present studies support a concerted lipogenic effect of these drugs.

3T3 Cells↗
PubMed1999

The physiological role of the novel uncoupling proteins: view from the chair.

The recently cloned uncoupling proteins -2 and -3 (UCP2, UCP3) cDNAs encode for proteins with 57-59% homology with brown adipose tissue uncoupling protein (UCP1) that can function as uncouplers of phosphorylation. The presentations of this Session of the Symposium were largely based on mRNA measurements, with one study showing that protein concentrations are well predicted by changes in mRNA in human muscles. Overall the observations suggest more complexity than anticipated. These observations are for the moment hard to reconcile with a simple role for these proteins in energy dissipation. Just the marked differences in tissue distribution of the two novel UCPs suggest they have different functions. It is possible that the activity, rather than the concentrations of these proteins, is regulated. The investigation of this possibility will hopefully enlighten us on their physiological role.

Adipose Tissue, Brown↗
PubMed1998

Calcium pool size modulates the sensitivity of the ryanodine receptor channel and calcium-dependent ATPase of heavy sarcoplasmic reticulum to extravesicular free calcium concentration.

UNLABELLED: We have examined calcium cycling and associated ATP consumption by isolated heavy sarcoplasmic reticulum (HSR) vesicles incubated in conditions believed to exist in resting muscle. Our goals were to estimate the magnitude of calcium cycling under those conditions and identify the main mechanisms involved in its regulation. The integrity of the HSR vesicles was documented by the retention of [14C]-sucrose and electron microscopy. HSR actively exchanged Ca2+ with the medium through a partially open ryanodine-binding channel (RyR), as evidenced by the rapid attainment of a steady-state gradient between HSR and medium, which was promptly increased by the closure of the channel with ruthenium red (RR) or collapsed by its opening with caffeine. The ATP dependency was evidenced by the sustained ATP consumption after the steady state was attained and by the abrogation of the gradient following inhibition of the pump with thapsigargin (Tg) or the omission of ATP. When HSR vesicles were incubated in a comparatively large pool of calcium (approximately 1 micromol/mg HSR protein), ATP consumption was 1-1.5 micromol x [min x mg protein](-1) at 0.1 microM free Ca2+. Under such conditions, the main regulator of the sarcoplasmic Ca2+-dependent ATPase (SERCA) was extravesicular-free Ca2+ concentration, with a four- to fivefold increase between 0.1 and 2 microM Ca2+, whereas RyR channel activity and the replenishment of the HSR vesicles had only a modest effect on ATP consumption. When calcium pool size was reduced to 0.1 micromol/mg HSR protein, a steady state was established at a lower level of HSR calcium. In spite of a slightly lower free extravesicular Ca2+ at equilibrium (approximately 0.07 microM following an initial concentration of 0.1 microM), both ATP consumption and the open probability of the RyR channel were increased by a factor of three to five. Compared to the large calcium pool, the sensitivity of both RyR channel and SERCA to extravesicular free Ca2+ concentration as well as to caffeine and RR was markedly enhanced. CONCLUSIONS: 1) In conditions present in resting muscle, HSR calcium is in dynamic equilibrium with the medium through a partially open RyR channel, which requires continuous ATP hydrolysis. 2) The availability of calcium is a major determinant of the sensitivity of both RyR channel and SERCA to free extravesicular Ca2+ and possibly other stimuli. 3) These observations are consistent with the concept that calcium cycling in resting muscle may account for a significant fraction of muscle energy demands and further suggest that restricting calcium availability may enhance the energetic demands of this process.

Adenosine Triphosphate↗
PubMed1998

Thiazolidinediones stimulate uncoupling protein-2 expression in cell lines representing white and brown adipose tissues and skeletal muscle.

Thiazolidinediones (TZD) are PPAR gamma ligands that sensitize tissues to insulin. A cDNA encoding a mitochondrial protein likely to act as uncoupler (uncoupling protein 2, UCP2) has been recently cloned. Since TZD have been reported to increase energy expenditure in animals, we have examined the effects of these drugs on the expression of UCP2 mRNA in cell lines representing white (3T3-L1 and 3T3-F442A) and brown (HIB-1B) adipose tissues and skeletal muscle (L6). Northern blots probed with a mouse UCP2 full-length cDNA showed a mRNA of 1.6 kb both in tissues and the aforementioned cells lines. Within 4 h of exposing these cells to 30 microM darglitazone, there was an increase in UCP2 mRNA which reached a plateau of 5-10 times the basal in about 8 h. In all cells TZDs (darglitazone, troglitazone) were more active than the predominantly PPAR alpha ligands WY-14,613 and clofibrate, or the non-selective ligand linoleic acid. These results indicate that TZDs can stimulate the expression of UCP2 gene probably via PPAR gamma and hence have the potential to increase energy expenditure in adult humans, in whom UCP2 is expressed ubiquitously.

Adipose Tissue↗
PubMed1998

[Importance of J. Brazy's neurobiological index. Prediction of the number and severity of complications in very low birth weight infants].

The objective of this study was to evaluate the neurodevelopment outcome of Very-Low-Birth-Weight Infants (VLBW), between 1987 and 1993, and correlate these findings with J.Brazy's Neurobiologic Risk Score (NBRS). The minimum corrected age at follow-up was 12 months. The neurodevelopmental assessment was performed using Mary Sheridan and Ruth Griffiths scales, Auditory and Visual Brainstem Evoked Responses and Stycar test. The NBRS was applied to 77 children. According to the score, three groups of risk were defined: Low < or = 4; Intermediate 5-7; High > or = 8. We obtained the following results: children with NBRS < 4, 20% had handicaps (5% of which major); children with NBRS 5-7, 41% had handicaps (23% of which major); in children with NBRS > 8, 95% had handicaps (80% of which major). The incidence of handicaps, (all grades included) was 71.4% for those weighing less than 1000 gr at birth, and 39.2% for those weighing 1000-1499 gr at birth. Major handicaps, mainly motor deficits, occurred in 26.8% of VLBW infants and minor to moderate handicaps were observed in 18.3% of patients in this group. These results were compared to J.Brazy's originals. We concluded that the NBRS, which is simple and objective to perform, is a good predictor of subsquent abnormal development in VLBW infants, allowing the infant's integration as soon as possible in high-risk follow-up programs, to place as soon as possible.

Developmental Disabilities↗
PubMed1997

Rainbow trout liver expresses two iodothyronine phenolic ring deiodinase pathways with the characteristics of mammalian types I and II 5'-deiodinases.

Deiodinases are major determinants of thyroid hormone tissue availability and disposal. The knowledge of the expression of these enzymes in lower species is important to understand evolutionary and ontogenetic aspects of thyroid hormone action and metabolism. Here we have studied outer ring deiodination in the trout liver using both reverse T3 (rT3) and T4 as substrates. The use of rT3 disclosed two enzymatic components with the characteristics of mammalian types I and II 5'-deiodinases. The high rT3-K(m) type I 5'-deiodinase activity (180 nM) has a low cofactor requirement (5 mM dithiothreitol) and is relatively sensitive to propylthiouracil inhibition, whereas the low rT3-K(m) activity was akin to the outer ring deiodination of T4 in these regards. The use of T4 exhibited only a single type of activity with a low K(m) (0.63 nM), a relatively high cofactor requirement (25 mM dithiothreitol), and propylthiouracil-resistance. Teleosts constitute a unique example of type II activity expression in the liver of an adult vertebrate. Furthermore, the Vmax of this enzyme is as high as that found in comparable homogenates from hypothyroid mammalian tissues, whereas the Vmax of the type I activity is lower than that of mammalian liver. These findings are in consonance with the peculiar kinetics of T3 in trout liver, kinetics remarkably similar to those of the mammalian pituitary, cerebral cortex, and brown adipose tissue, which also preferentially express type II deiodinase.

Animals↗
PubMed1997

3',5'-cyclic adenosine monophosphate-response sequences of the uncoupling protein gene are sequentially recruited during darglitazone-induced brown adipocyte differentiation.

Uncoupling protein-1 (UCP) is uniquely expressed in brown adipose tissue (BAT) and is essential to the thermogenic function of this tissue. The UCP gene is under the control of norepinephrine (NE) via cAMP. However, the precise delineation of the cAMP response sequences and mechanisms whereby cAMP stimulate the gene have remained elusive. A BAT tumor cell line, HIB-1B, can be differentiated into UCP-expressing brown adipocytes. We report here that when these cells are differentiated with a standard differentiation protocol including insulin, T3, hydrocortisone, IBMX, and indomethacin (standard differentiation, StD), cAMP stimulation of the rat UCP gene is largely mediated by an upstream 90-bp sequence -2,399/-2,490 (R90) with a lesser contribution of a downstream sequence -57/+114 (dnCRS). This latter is functional also in non-BAT cells, whereas the cAMP response sequence contained in R90 (upCRS) is BAT-specific. Thiazolidinediones (TZD) are a new group of drugs known to increase sensitivity to insulin and, more recently, to induce adipocyte differentiation (adipogenesis) via PPARgamma. A TZD, darglitazone (darg), can rapidly induce differentiation of HIB-1B cells, as judged by the expression of the adipocyte lipid binding protein (aP2), lipoprotein lipase (LPL), uncoupling protein (UCP) and beta3-adrenergic receptors. UCP messenger RNA (mRNA) responsive to NE is evidenced as early as one day after exposure to darg. While UCP-CAT vectors (+114/-3673 bp of rat UCP gene) are barely responsive to NE in HIB-1B preadipocytes, both darg and StD markedly enhance NE responsiveness of such constructs. However, by 3 days of exposure to darg, the responses were less vigorous than in StD cells (4- to 10-fold vs. 20- to 50-fold), and the deletion of R90 did not affect the response to NE in darg-differentiated cells, whereas this deletion caused a 75% reduction in StD cells. Prolongation of darg exposure to 5-7 days resulted in greater response of UCP mRNA to NE and 50-80% inhibition of the response of UCP-CAT vectors by the deletion of R90. Thus, darg-induced differentiation of HIB-1B cells suggests that the NE-dependent expression of the UCP gene takes place in a step-wise manner: first, the gene is "enabled," as no UCP mRNA is detected in HIB-1B preadipocytes; thereafter and transiently, the response of the gene to NE is sustained by dnCRS; finally, as differentiation progresses, a cell-specific and more powerful cis-acting sequence, upCRS, is recruited, accounting in the fully differentiated cell for most of the response to NE. These results also suggest that TZDs might increase energy expenditure by inducing terminal differentiation of BAT, and that these drugs may be useful in the differential cloning of the factors involved in the recruitment of the BAT specific cAMP response sequence.

Adipocytes↗
PubMed1997

Resting energy expenditure is sensitive to small dose changes in patients on chronic thyroid hormone replacement.

We have investigated the effects of modifying the dose of thyroxine on resting energy expenditure (REE) and on the thermic effect of glucose (TEG) in 9 randomly recruited patients on chronic treatment with this hormone. The initial dose was changed twice in each patient at 6-8 wk intervals, aiming to have a normal, a slightly reduced, and a slightly elevated serum TSH concentration. A total of 27 dose points for each measured variable (3 per patient) were gathered. Dose changes were monitored with serum free T4, T3, and TSH. At the end of each dose period, low density lipoprotein and high density lipoprotein cholesterol, triglycerides, angiotensin converting enzyme, and sex hormone binding globulin were also measured, along with a systematic assessment of symptoms and signs. The investigators involved in the measurements were blinded to the dose of T4. Serum free T4 and TSH significantly correlated to the dose in each patient and in the whole group, whereas serum T3 levels were minimally affected by the dose and did not correlate with it, with free T4 or with TSH. This latter was below normal on 9 occasions, normal in 12, and above normal in 6. Serum free T4 and T3 remained within the normal range on all except 2 occasions. REE and TEG were normalized to fat-free mass (FFM). In each patient there was a significant negative correlation between REE and TSH. This correlation was maintained when all data were pooled (r2 = 0.64; P < 0.001). Also, initial REE and its change between the highest and the lowest thyroxine dose were significantly correlated with, respectively, initial serum TSH (r2 = 0.85; P < 0.001) and the change in serum TSH between the highest and the lowest dose of T4 (r2 = 0.67; P < 0.0065). REE decreased approximately 15% when TSH increased between 0.1 and 10 mU/L. In 6 of the 9 patients, TEG increased with the reduction of the dose, and higher values were associated with higher TSH levels but without reaching statistical significance (F = 2.852, P = 0.077). None of the other indices were significantly affected by the changes in dose. These results indicate that, in patients on chronic treatment with thyroxine, REE is significantly influenced by the dose of this hormone in a dose range encompassing serum TSH concentrations that are considered acceptable in the management of hypothyroid patients. In the absence of physiological or behavioral compensations, these changes in REE may be clinically relevant.

Adult↗
PubMed1997

Regulation of the uncoupling protein gene expression.

Uncoupling protein (UCP) is essential to the thermogenic function of brown adipose tissue (BAT). The thermogenic role of this protein is due to its capacity to uncouple oxidative phosphorylation in a regulated manner. The thermogenic potential of BAT is determined by its content of UCP. The gene encoding this protein is under complex regulation. Catecholamines, via cAMP, thyroid hormone and retinoic acid, directly stimulate the gene acting upon an upstream (-2.28/-2.49 kb) enhancer sequence, but cAMP may act upon other sequences of the gene as well. CCAAT enhancer binding proteins and peroxisome proliferation activator receptor (PPAR) gamma 2 have also been implicated in the regulation of the gene acting on discrete sequences. While the thyroid hormone response and retinoic acid response elements (TRE and RARE) have been well defined, the cAMP response elements (CRE) remain elusive. The two TREs are 27 bp apart between -2.33 kb and -2.39 kb. The synergism between cAMP and thyroid hormone seems to reside in a 39 bp sequence downstream (-2.28/-2.32 kb). The most important CRE, the RARE, a cell-specific enhancer and a putative PPAR element are all concentrated in a 90 bp regulatory element of great complexity (-2.40/-2.49 kb). Other hormones, such as insulin and glucocorticoids, and IGF-I also modulate the expression of the gene but their effects seem to be largely indirect. Understanding the regulation of the UCP gene expression may facilitate the development of interventions in obesity and related disorders.

Animals↗
PubMed1996

Effects of hypothyroidism on brown adipose tissue adenylyl cyclase activity.

Hypothyroidism profoundly reduces the capacity of brown adipose tissue (BAT) to generate cAMP in response to adrenergic stimulation. Evidence obtained with isolated brown adipocytes suggests a postreceptor defect that offsets the hypothyroidism-induced increase in beta3-adrenergic receptors. The goal of the present studies was to identify the defect in the cAMP generation pathway for which we studied cAMP generation in isolated cells and purified BAT membranes from normal and hypothyroid rats. Studies with adenosine deaminase and the adenosine receptor-1 agonist r-phenyl isopropyl adenosine (R-PIA) show that hypothyroid cells are not more sensitive to adenosine (same EC50) but more inhibited by high concentrations of R-PIA. Pretreatment with pertussis toxin reduced the gap in cAMP generation between eu- and hypothyroid cells and the inhibition mediated by R-PIA, but did not normalize the cAMP response to forskolin in hypothyroid cells. Although purified euthyroid BAT membranes increased cAMP production with GTP concentrations up to submillimolar range, to plateau or slightly decrease at higher levels, hypothyroid membranes were weakly stimulated by low concentrations of GTP and markedly inhibited (>50%) at concentrations > or = 10(-4) M. When assayed at 0.3 mM ATP and 1 microM GTP, hypothyroid membranes actually generated more cAMP in response to forskolin, but this was reversed when GTP concentration was 1 mM. Immunoblotting studies showed no significant effects of hypothyroidism on the abundance of G(alpha)i or Gbeta subunits, and ADP ribosylation of G(alpha)i was only 45% increased in hypothyroidism in contrast to a 2.5-fold increase in hypothyroid white adipose tissue membranes from the same rats. Hypothyroid membranes also exhibited different kinetics regarding ATP, with higher cAMP generation at submillimolar concentrations but less at >1 mM ATP. Actually, at ATP concentrations >0.6 mM, cAMP generation was markedly inhibited in hypothyroid membranes. Fixing the concentration of free Mg++ in these experiments indicates that most of the inhibition seen in hypothyroid membranes is caused by ATP, whereas euthyroid membranes are more sensitive to changes in free Mg++. Ca++ +/- calmodulin did not stimulate adenylyl cyclase (AC) activity. On the contrary, AC activity was inhibited by Ca++ in a concentration-dependent manner, by as low as 100 nM free Ca++, and to greater extent in hypo- than in euthyroid membranes (maximal inhibition 60 vs. 25-30%). Our results suggest that, functionally, hypothyroidism causes a change in the AC of BAT membranes consistent with a relative or absolute increase in the type VI AC (AC-VI). The effects on this AC of nucleotides, Ca++, and Mg++ at concentrations prevailing in the hypothyroid brown adipocyte are probably the major factor in the reduced capacity of these cells to generate cAMP. These results also open the possibility of a novel, differential effect of thyroid hormone on AC expression, and support the concept that thyroid hormone affects the adrenergic signal transduction pathways in a tissue-selective manner.

Adenosine Diphosphate Ribose↗