The receptors responsible for heat production in brown adipose tissue in the young rabbit

It is known that adrenergic agonists stimulate thermogenesis in the brown fat of the young rabbit but the receptors responsible for mediating the response have not been identified. The infusion of either noradrenaline or isoproterenol (1-2 micrograms . kg-1 X min-1) produced an increase in subcutaneous temperature (0.93 +/- 0.15 and 1.22 +/- 0.10 degrees C, respectively over the interscapular brown fat. At low doses (0.4 microgram . kg-1 X min-1) only isoproterenol was effective. The thermogenic response to isoproterenol was blocked by atenolol, a beta 1-adrenergic antagonist. Neither salbutamol or terbutaline, both beta 2-agonists, produced a temperature increase. Collectively, these data suggest that stimulation of beta 1-adrenoceptor is primarily responsible for the thermogenic activity of brown fat in the rabbit. However, it was found that 53% of the increase in temperature could be blocked by prazosin, an alpha 1-antagonist. Phentolamine was not effective as a blocker. Although a maximal brown fat thermogenic response can be achieved by stimulating the beta-adrenoceptors, the alpha-adrenoceptors appears to play at least an auxiliary role in young rabbit.     

The noradrenaline content and innervation of brown adipose tissue in the young rabbit

This work examined the noradrenaline content of brown adipose tissue, the metabolic response to endogenous noradrenaline released during tyramine infusion, and the innervation of brown fat at the electron microscopic level in the young rabbit. The noradrenaline content (ng/g) of the interscapular and cervical fat deposits ranged from 256 +/- 51 to 343 +/- 59 and 399 +/- 18 to 694 +/- 92, respectively, in four groups of rabbits (1-2, 7-8, 12-13, and 25-27 days of age). There was considerable variation amongst animals in each age group, but no evidence of a major increase or decrease in noradrenaline content during the first 4 weeks of life. Intravenous infusion of tyramine (100 micrograms X kg-1 X min-1) increased plasma noradrenaline concentration, oxygen consumption, and blood flow to brown fat. Thus noradrenaline released from endogenous sites, as well as injected noradrenaline, will initiate the thermogenic response of brown fat. Ultrastructurally, unmyelinated axons that were not organized in a fascicle were observed adjacent to the adipocytes in the late gestation fetus. By 1 week of age of axons were surrounded by Schwann cell cytoplasm which formed a fascicle. However, no evidence of myelination was found up to 21 days of age. Collectively, the data indicate that the brown adipocyte is fully responsive at 1-2 days of age even though myelination of the nerves is incomplete, and that the incomplete development of the sympathetic nerves at birth is not a factor in the synthesis of noradrenaline in the very young rabbit. In addition, brown fat of the newborn rabbit is not as thermogenically active as the brown fat of the cold-acclimated rat.     

Phosphorylation sites and inactivation of branched-chain alpha-ketoacid dehydrogenase isolated from rat heart, bovine kidney, and rabbit liver, kidney, heart, brain, and skeletal muscle

Branched-chain alpha-ketoacid dehydrogenase complex was isolated from rat heart, bovine kidney, and rabbit liver, heart, kidney, brain, and skeletal muscle. Phosphorylation to approximately 1 mol Pi/mol alpha-subunit of the alpha-ketoacid decarboxylase component was linearly associated with 90-95% inactivation. The complex from some tissues (i.e., from rabbit kidney and heart, and rat heart) showed 30-40% more phosphate incorporation for an additional 5-10% inactivation. Reverse-phase HPLC analysis of tryptic digests of 32P-labeled complexes from all of the above tissues revealed two major (peaks 1 and 2) and one minor (peak 3) phosphopeptide which represent phosphorylation sites 1, 2, and a combination of 1 and 2, respectively. These phosphopeptides, numbered according to the order of elution from reverse-phase HPLC, had the same elution time regardless of the tissue or animal source of the complex. The amino acid sequence of site 1 from rabbit heart branched-chain alpha-ketoacid dehydrogenase was Ile-Gly-His-His-Ser(P)-Thr-Ser-Asp-Asp-Ser-Ser-Ala-Tyr-Arg. Regardless of the source of the complex, both sites were almost equally phosphorylated until total phosphorylation was approximately 1 mol Pi/mol of alpha-subunit and the rate of inactivation was correlated with the rate of total, site 1, or site 2 phosphorylation. Phosphorylation beyond this amount was associated with greater site 2 than site 1 phosphorylation. alpha-Chloroisocaproate, a potent inhibitor of branched-chain alpha-ketoacid dehydrogenase kinase activity, greatly reduced total phosphorylation and inactivation; however, phosphorylation of site 2 was almost abolished and inactivation was directly correlated with phosphorylation of site 1. Thus, the complex isolated from different tissues and mammals had an apparent conservation of amino acid sequence adjacent to the phosphorylation sites. Both sites were phosphorylated to a similar extent temporally although site 1 phosphorylation was directly responsible for inactivation.     

Purification and characterization of a glycogen phosphorylase analog missing the amino-terminal segment

A glycogen phosphorylase analog missing only the amino-terminal 16 to 18 residues, which include the phosphorylation site, was produced by subtilisin Carlsberg cleavage of phosphorylase b in the presence of caffeine. The analog, named phosphorylase b's, was purified, and its enzymatic properties were compared with those of phosphorylase b. The KM's for glucose 1-phosphate are similar, but phosphorylase b's has a VM 43% higher than that of phosphorylase b. Also, phosphorylase b's is less sensitive to inhibition by glucose 6-phosphate and stimulation by sodium fluoride than is phosphorylase b. The subunit interactions in the two enzyme forms were also compared. The monomer-monomer interactions in phosphorylase b's are weaker than in phosphorylase b, as evidenced by a faster rate of resolution of the coenzyme, pyridoxal phosphate, from phosphorylase b's. The dimer-dimer interactions are also weaker in phosphorylase b's than in phosphorylase b, because phosphorylase b's does not form tetramers or crystals as readily as does phosphorylase b. Because removal of the amino-terminal segment changes the properties of the enzyme, this segment must be interacting with other parts of the protein. This statement conflicts with previous interpretation of X-ray crystallographic data that suggest that the amino-terminal region of phosphorylase b is freely mobile. Possible explanations for this contradiction are discussed.     

Characterization of human platelet basic protein, a precursor form of low-affinity platelet factor 4 and beta-thromboglobulin

Platelet basic protein (PBP) was purified from the supernatant of thrombin-stimulated, washed human platelets by ion-exchange, affinity, molecular sieve, and high-performance liquid chromatography (HPLC). The NH2-terminal amino acid sequence was determined by automated Edman degradation, revealing 9 unique residues followed by 10 residues of the established low-affinity platelet factor 4/beta-thromboglobulin (LA-PF4/beta TG) sequence. Among the nine were three basic residues, accounting for the high isoelectric point of PBP. Additional evidence for precursor status includes the immunological cross-reactivity of all three species and the ability of plasmin and trypsin to produce from PBP a species resembling beta TG in charge, hydrophobicity, and size. Tryptic peptide maps of PBP and LA-PF4 obtained by reverse-phase HPLC were very similar, and from each protein, a peptide was isolated which showed the amino acid composition predicted for the COOH-terminal tryptic peptide of beta TG. Normal platelets contained predominantly LA-PF4, with PBP ranging from 10% to 30% of total beta TG antigen. This was true even when fresh platelets were lysed with trichloroacetic acid in order to provide the most complete and rapid inhibition of proteolytic activity. beta TG itself was never detected in this situation or in the release supernatant of stimulated platelets, and only rarely in unprotected lysates. In agreement with earlier results, crude preparations of PBP were mitogenic for 3T3 cells, but highly purified preparations of PBP and LA-PF4 were free of this activity.     

Clonal variation in response to adrenocorticotropin in cultured bovine adrenocortical cells: relationship to senescence

During cellular senescence, non-clonal cultures of bovine adrenocortical cells show a continuous decline in the rate of production of cyclic AMP (cAMP) stimulated by adrenocorticotropin (ACTH), without changes in the rate of forskolin- or prostaglandin E1-stimulated cAMP production. We investigated the possible mechanisms for loss of response to ACTH by examining the properties of clones of bovine adrenocortical cells. ACTH-stimulated cAMP production rates were measured in clones immediately after isolation, during long-term growth following isolation, and after subcloning. ACTH-stimulated rates were compared with cAMP production in response to forskolin, which acts directly on the catalytic subunit of adenylate cyclase. The results show that cloning is not necessarily associated with a loss of response to ACTH, but that clones with high ACTH response can give rise to subclones with low response. Clones of adrenocortical cells, at the same approximate population doubling level (PDL), showed ACTH response levels that ranged from 12 to 135 pmol cAMP/10(6) cells/min, whereas mass cultures at this PDL showed approximately 50 pmol/10(6) cells/min. Forskolin-stimulated cAMP production rates in clones varied only over the range of 59-119 pmol/10(6) cells/min and showed no correlation with the ACTH-stimulated rates. All clones were adrenocortical cells, as shown by mitogenic response to angiotensin II and cAMP-inducible 17 alpha-hydroxylase activity. The replicative potential of clones varied widely, and there was no apparent correlation between ACTH response levels and growth potential. The level of ACTH response in each clone was stable during proliferation through at least 25 PD beyond the stage at which the clone was isolated. When clones were subcloned, a clone with a high ACTH response level produced sister subclones that had ACTH response levels ranging from 3% of that of the parent clone to a level slightly greater than that of the parent clone. The growth potential of sister subclones varied widely, as for the parent clones, and there was no obvious correlation between growth potential and ACTH response. Two subclones were cloned; in sub-subclones, levels of ACTH response were again different from each other and also from the parent subclone; in one case, the level of ACTH response was approximately eight-fold higher than that of the parent subclone. These experiments show that clonal variation in the extent of expression of a differentiated property may occur in a normal differentiated cell in culture. The loss of ACTH response and the loss of proliferative potential appear to be independent stochastic events.     

Properties of in vitro recombinant derivatives of pJV1, a multi-copy plasmid from Streptomyces phaeochromogenes

The 10.8 kb plasmid pJV1, isolated from Streptomyces phaeochromogenes, has a high copy number (about 150) and a broad host range among Streptomyces spp. Several pJV1 derivatives carrying the thiostrepton resistance gene (tsr) of S. azureus were made. One derivative, pWOR191, was shown to promote its own transfer and to mobilize chromosomal markers in S. lividans. Another derivative, pWOR109, was non-transmissible. Deletion in vitro of a segment of pWOR109 gave pWOR120 (5.6 kb), which has single BamHI and Bg/II sites shown to be capable of accepting 'foreign' DNA such as a previously cloned S. antibioticus DNA fragment encoding tyrosinase, giving vectors (pWOR125, pWOR126) with properties resembling the well-established multicopy vector pIJ702. Shuttle vectors capable of functioning in both S. lividans and Escherichia coli were also constructed. The region of pJV1 essential for replication and maintenance was localized to a 2.5 kb segment. Stable maintenance of pWOR109 and pWOR120 was observed in the presence of derivatives of pIJ101, the progenitor of pIJ702.     

Anti-Ig induces release of inositol 1,4,5-trisphosphate, which mediates mobilization of intracellular Ca++ stores in B lymphocytes

Evidence from a variety of laboratories indicates that crosslinking of B cell mIg induces a rapid increase in intracellular free calcium (Ca++i). This mobilized Ca++ appears to act in concert with diacylglycerol (DAG; also released upon mIg cross-linking) to optimally activate Ca++/phospholipid-dependent protein kinase C, which plays a pivotal role in B cell activation. Here we report analysis of the source of this mobilized calcium and the mechanism responsible for its release into the cytosol. We observed the cross-linking of mIg induces the release of inositol 1,4,5-trisphosphate (InsP3), presumably as a result of action of phospholipase C on plasma membrane phosphatidylinositol 4,5-bisphosphate (PtdInsP2). The release of InsP3 and the elevation of Ca++i are coincidental, suggesting that they may be causally related. Finally, we demonstrate that submicromolar doses of InsP3 induce release of Ca++ from permeabilized cells that had preaccumulated 45Ca++ in the endoplasmic reticulum. On the basis of these findings we suggest that mIg cross-linking leads to mobilization of Ca++, in part by causing hydrolysis of PtdInsP2, yielding InsP3, which in turn causes release of calcium from the endoplasmic reticulum.     

Mannose metabolism in corn and its impact on leaf metabolites, photosynthetic gas exchange, and chlorophyll fluorescence

When intact corn leaves were provided millimolar concentrations of d-mannose through the transpiration stream photosynthesis was inhibited; 5.7 millimolar resulted in a 50% inhibition of the carbon exchange rate. This inhibition was partially reversible by the addition of orthophosphate to the feeding solution. Mannose metabolism by corn leaves was limited in that it did not act as a resource for sucrose or starch synthesis. Mannose 6-phosphate accumulated in the leaf tissues and was slowly metabolized by a pathway involving mannose 1-phosphate. Correlated with the mannose-6-phosphate accumulation were decreases in ATP, orthophosphate, sucrose, and phosphoenolpyruvate and increases in starch and maltose. When provided in the transpiration stream mannose had access to both mesophyll and bundle sheath cells. Mannose feeding led to oscillations in steady state chlorophyll fluorescence emission (680 nanometers) and an elimination of the Kautsky effect during fluorescence induction. Pyridoxal 5-phosphate and 2,4-dinitrophenol were found to be inhibitors of CO₂ exchange when provided in the transpiration stream of intact corn leaves. However, Pyridoxal 5-phosphate induced a quenching of steady state fluorescence while 2,4-dinitrophenol led to an increase in fluorescence emission.     

Levels of Free and Conjugated Abscisic Acid in Developing Floral Organs of the Navel Orange (Citrus sinensi [L.] Osbeck cv Washington)

The levels of abscisic acid (ABA) and alkaline-hydrolyzable ABA-conjugate (putatively identified as the glucosyl ester, abscisyl-β-d-glucopyranoside) were determined by enzyme immunoassay in the organs of developing navel orange (Citrus sinensis [L.] Osbeck cv Washington) flowers. Although both compounds were detected in every tissue, developmentally related differences between organs in the total and relative contents were observed. The highest ABA levels were observed in the stigma/style shortly after anthesis (11.5 ± 0.6 nanomoles ABA per gram fresh weight and 4.8 ± 0.6 nanomoles ABA-conjugate per gram fresh weight); whereas, the highest ABA-conjugate levels were observed at the same time in the floral disc (hypogynous disc plus calyx; 3.5 ± 0.1 nmol nanomols ABA per gram fresh weight and 11.8 ± 0.9 nanomoles ABA-conjugate per gram fresh weight). These results suggest that differences in ABA content reflect tissue-specific variation in the facility for ABA conjugation. Increased ABA levels were observed in the stigma/style near anthesis; however, a relationship with pollination is discounted, since `Washington' navel orange flowers are male sterile and devoid of pollen.