Sucrose density gradient purified plasma membranes isolated from brown adipose tissue of cold-acclimated hamsters (4-10 weeks at 0-4 degreesC) were analysed for the content of the short (GsalphaS) and long (GsalphaL) variants of Gsalpha protein (the alpha subunit of the stimulatory G protein) and compared with the membranes isolated from control animals. The relative ratio between the two variants (GsalphaS/GsalphaL) decreased from 0.48 to 0.24 (P<0.01). This result, obtained by electrophoretic resolution of membrane proteins by standard SDS-PAGE and an immunoblot analysis with an antiserum oriented against an internal sequence of Gsalpha, was verified by resolution on urea-containing gels and an antiserum oriented against the C-terminus decapeptide of Gsalpha. Under these conditions, the GsalphaS/GsalphaL ratio was decreased from 0.41 to 0.31 (P<0.05). The total amount of both isoforms (GsalphaS plus GsalphaL) decreased to 83% (P<0.05) or 68% (P<0.01) by standard or urea SDS-PAGE respectively. These data demonstrate that cold-acclimation of hamster brown adipose tissue is associated with preferential decrease in the plasma membrane density of the short variant of the Gsalpha protein.%This decrease was paralleled by an increase in the other plasma membrane constituents, [3H]CGP12177 binding sites, [3H]ouabain binding sites and Na,K-ATPase activity to 147%, 212% and 191% respectively.
- MeSH
- Acclimatization physiology MeSH
- Cell Membrane metabolism MeSH
- Centrifugation, Density Gradient MeSH
- Adipose Tissue, Brown metabolism MeSH
- Enzyme Inhibitors metabolism pharmacology MeSH
- Cricetinae MeSH
- Mesocricetus physiology MeSH
- Membrane Proteins metabolism MeSH
- Mitochondria metabolism MeSH
- Molecular Weight MeSH
- Cold Temperature * MeSH
- Ouabain metabolism pharmacology MeSH
- Propanolamines metabolism MeSH
- Protein Isoforms deficiency isolation & purification physiology MeSH
- GTP-Binding Protein alpha Subunits, Gs deficiency isolation & purification physiology MeSH
- Sodium-Potassium-Exchanging ATPase antagonists & inhibitors metabolism MeSH
- Animals MeSH
- Check Tag
- Cricetinae MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- CGP 12177 MeSH Browser
- Enzyme Inhibitors MeSH
- Membrane Proteins MeSH
- Ouabain MeSH
- Propanolamines MeSH
- Protein Isoforms MeSH
- GTP-Binding Protein alpha Subunits, Gs MeSH
- Sodium-Potassium-Exchanging ATPase MeSH
Cartilaginous structures are at the core of embryo growth and shaping before the bone forms. Here we report a novel principle of vertebrate cartilage growth that is based on introducing transversally-oriented clones into pre-existing cartilage. This mechanism of growth uncouples the lateral expansion of curved cartilaginous sheets from the control of cartilage thickness, a process which might be the evolutionary mechanism underlying adaptations of facial shape. In rod-shaped cartilage structures (Meckel, ribs and skeletal elements in developing limbs), the transverse integration of clonal columns determines the well-defined diameter and resulting rod-like morphology. We were able to alter cartilage shape by experimentally manipulating clonal geometries. Using in silico modeling, we discovered that anisotropic proliferation might explain cartilage bending and groove formation at the macro-scale.
- Keywords
- BMP, GSalpha, Wnt/PCP, chondrocranium, developmental biology, facial cartilage growth, mathematical and material modelling, mouse, mouse mutants, scaling and shaping, stem cells,
- MeSH
- Models, Biological MeSH
- Cartilage embryology MeSH
- Mice MeSH
- Vertebrates embryology MeSH
- Computer Simulation MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Thyroid hormones influence a wide range of physiological responses and the heart is considered a major target organ for triiodothyronine action. In the present study we examined closely the presumed relationship between altered thyroid status in the newborn rat and maturation of the regulatory components of the myocardial hormone-sensitive adenylyl cyclase signaling system. Beta -adrenoceptors and the alpha subunits of the stimulatory (Gs) as well as inhibitory (Gi) G proteins were determined in ventricular myocardium of immature (21-day-old) hypo- or hyperthyroid rats and in adult (84-day-old) previously hypo- or hyperthyroid rats. The changes in receptor and G protein levels were correlated with basic parameters of cardiac function and inotropic responsiveness to isoproterenol. All results were compared with those obtained in age-matched controls. Hypothyroidism in immature rats was associated with markedly reduced myocardial beta-adrenoceptor density, lower content of the long isoform (Gsalpha-L) of the stimulatory G protein and increased amounts of Gialpha2 and Gialpha3. These changes were accompanied by substantially diminished sensitivity to the inotropic effect of isoproterenol. On the other hand, no change in beta-adrenoceptor number, an increased level of Gsalpha-L and decreased levels of Gialpha2 were found in hyperthyroid myocardium. Cardiac inotropic responsiveness to isoproterenol in immature hyperthyroid rats was not significantly altered. In adult hearts of previously hyper- or hypothyroid rats, beta-adrenoceptor density was decreased but G protein levels as well as functional response were comparable to those determined in control animals. It may be concluded that physiological level of thyroid hormones is an important modulator of the normal maturation of the beta-adrenergic system in the developing rat ventricular myocardium. In adult rats previously affected by altered thyroid status, the function of their myocardial beta-adrenergic signaling appears to be compensated despite a lower number of the receptors.
- MeSH
- Adenylyl Cyclases metabolism MeSH
- Adrenergic beta-Agonists pharmacology MeSH
- Receptors, Adrenergic, beta metabolism MeSH
- Hyperthyroidism metabolism physiopathology MeSH
- Hypothyroidism metabolism physiopathology MeSH
- Isoproterenol pharmacology MeSH
- Myocardial Contraction drug effects MeSH
- Rats MeSH
- Myocardium metabolism MeSH
- Rats, Wistar MeSH
- GTP-Binding Proteins metabolism MeSH
- Signal Transduction MeSH
- Heart Ventricles drug effects growth & development metabolism MeSH
- In Vitro Techniques MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Male MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Adenylyl Cyclases MeSH
- Adrenergic beta-Agonists MeSH
- Receptors, Adrenergic, beta MeSH
- Isoproterenol MeSH
- GTP-Binding Proteins MeSH
Low-density membrane-domain fractions were prepared from S49 lymphoma cells and clone e2m11 of HEK293 cells expressing a large number of thyrotropin-releasing hormone receptor (TRH-R) and G(11)alpha by flotation on sucrose density gradients. The intact cell structure was broken by detergent-extraction, alkaline-treatment or drastic homogenization. Three types of low-density membranes were resolved by two-dimensional electrophoresis and analyzed for G(s)alpha (S49) or G(q)alpha/G11) (e2m11) content. Four individual immunoblot signals of Gsalpha protein were identified in S49 lymphoma cells indicating complete resolution of the long G(s)alpha L+/-ser and short G(s)alpha S+/-ser variants of G(s)alpha. All these were diminished by prolonged agonist (isoprenaline) stimulation. In e2m11-HEK cells, five different immunoblot signals were detected indicating post-translational modification of G proteins of G(q)alpha/G(11)alpha family. The two major spots corresponding to exogenously (over)expressed G(11)alpha and endogenous G(q)alpha were reduced; the minor spots diminished by hormonal stimulation. Parallel analysis by silver staining of the total protein content indicated that no major changes in protein composition occurred under these conditions. Our data thus indicate that agonist-stimulation of target cells results in down-regulation of all different members of G(s) and G(q)/G(11) families. This agonist-specific effect may be demonstrated in crude membrane as well as domain/raft preparations and it is not accompanied by changes in overall protein composition.
- MeSH
- Electrophoresis, Gel, Two-Dimensional methods MeSH
- Isoproterenol pharmacology MeSH
- Cells, Cultured MeSH
- Kidney drug effects metabolism MeSH
- Humans MeSH
- Lymphoma metabolism MeSH
- Membrane Microdomains drug effects metabolism MeSH
- Cell Line, Tumor MeSH
- GTP-Binding Protein alpha Subunits metabolism MeSH
- Receptors, Thyrotropin-Releasing Hormone genetics metabolism MeSH
- Recombinant Proteins metabolism MeSH
- Dose-Response Relationship, Drug MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Isoproterenol MeSH
- GTP-Binding Protein alpha Subunits MeSH
- Receptors, Thyrotropin-Releasing Hormone MeSH
- Recombinant Proteins MeSH
