Induction of G alpha i2-specific antisense RNA in vivo inhibits neonatal growth.

Guanosine triphosphate-binding regulatory proteins (G proteins) are key elements in transmembrane signaling and have been implicated as regulators of more complex biological processes such as differentiation and development. The G protein G alpha i2 is capable of mediating the inhibitory control of adenylylcyclase and regulates stem cell differentiation to primitive endoderm. Here an antisense RNA to G alpha i2 was expressed in a hybrid RNA construct whose expression was both tissue-specific and induced at birth. Transgenic mice in which the antisense construct was expressed displayed a lack of normal development in targeted organs that correlated with the absence of G alpha i2. The loss of G alpha i2 expression in adipose tissue of the transgenic mice was correlated with a rise in basal levels of adenosine 3',5'-monophosphate (cAMP) and the loss of receptor-mediated inhibition of adenylylcyclase. These data expand our understanding of G protein function in vivo and demonstrate the necessity for G alpha i2 in the development of liver and fat.

Regulation of the differentiation of teratocarcinoma cells into primitive endoderm by G alpha i2.

The amount of the heterotrimeric G protein subunit G alpha i2 decreases after the induction of F9 teratocarcinoma cells to become primitive endoderm in the presence of retinoic acid (RA). The reduction of the G alpha i2 protein in F9 cells by antisense RNA expression was associated with (i) loss of receptor-mediated inhibition of adenylyl cyclase; (ii) decreased cell doubling time; (iii) induction of a primitive, endoderm-like phenotype in the absence of RA; and (iv) production of the differentiation marker tissue-type plasminogen activator. Expression of a constitutively active, mutant G alpha i2 blocked RA-induced differentiation. These data suggest the involvement of G alpha i2 in the control of stem cell differentiation and provide insight into the involvement of G proteins in growth regulation.

G protein signaling from activated rat frizzled-1 to the beta-catenin-Lef-Tcf pathway.

The frizzled receptors, which mediate development and display seven hydrophobic, membrane-spanning segments, are cell membrane-localized. We constructed a chimeric receptor with the ligand-binding and transmembrane segments from the beta2-adrenergic receptor (beta2AR) and the cytoplasmic domains from rat Frizzled-1 (Rfz1). Stimulation of mouse F9 clones expressing the chimera (beta2AR-Rfz1) with the beta-adrenergic agonist isoproterenol stimulated stabilization of beta-catenin, activation of a beta-catenin-sensitive promoter, and formation of primitive endoderm. The response was blocked by inactivation of pertussis toxin-sensitive, heterotrimeric guanine nucleotide-binding proteins (G proteins) and by depletion of Galphaq and Galphao. Thus, G proteins are elements of Wnt/Frizzled-1 signaling to the beta-catenin-lymphoid-enhancer factor (LEF)-T cell factor (Tcf) pathway.

Protein kinase C is differentially stimulated by Wnt and Frizzled homologs in a G-protein-dependent manner.

In studies of developmental signaling pathways stimulated by the Wnt proteins and their receptors, Xenopus Wnt-5A (Xwnt-5A) and a prospective Wnt receptor, rat Frizzled 2 (Rfz2), have been shown to stimulate inositol signaling and Ca2+ fluxes in zebrafish [1] [2] [3]. As protein kinase C (PKC) isoforms can respond to Ca2+ signals [4], we asked whether expression of different Wnt and Frizzled homologs modulates PKC. Expression of Rfz2 and Xwnt-5A resulted in translocation of PKC to the plasma membrane, whereas expression of rat Frizzled 1 (Rfz1), which activates a Wnt pathway using beta-catenin but not Ca2+ fluxes [5], did not. Rfz2 and Xwnt-5A were also able to stimulate PKC activity in an in vitro kinase assay. Agents that inhibit Rfz2-induced signaling through G-protein subunits blocked Rfz2-induced translocation of PKC. To determine if other Frizzled homologs differentially stimulate PKC, we tested mouse Frizzled (Mfz) homologs for their ability to induce PKC translocation relative to their ability to induce the expression of two target genes of beta-catenin, siamois and Xnr3. Mfz7 and Mfz8 stimulated siamois and Xnr3 expression but not PKC activation, whereas Mfz3, Mfz4 and Mfz6 reciprocally stimulated PKC activation but not expression of siamois or Xnr3. These results demonstrate that some but not all Wnt and Frizzled signals modulate PKC localization and stimulate PKC activity via a G-protein-dependent mechanism. In agreement with other studies [1] [2] [3]. [6] [7] these data support the existence of multiple Wnt and Frizzled signaling pathways in vertebrates.

3,3',5-triiodothyronine administration in vivo modulates the hormone-sensitive adenylate cyclase system of rat hepatocytes.

The ability of 10 muM epinephrine or isoproterenol to stimulate cyclic AMP accumulation was decreased in hepatocytes isolated from hyperthyroid (triiodothyronine treated) as compared to euthyroid rats. In the presence of methylisobutylxanthine, epinephrine or isoproterenol-stimulated cyclic AMP accumulation was approximately 65% lower in hyperthyroid as compared with euthyroid rat hepatocytes. The ability of glucagon to stimulate a cyclic AMP response was also decreased in the hyperthyroid state, when assayed in either the absence or presence of a methyl xanthine. The character of the catecholamine-stimulated cyclic AMP response was beta adrenergic in both the hyperand euthyroid states. No evidence for an alpha(2) adrenergic mediated component of catecholamine action on cyclic AMP levels was noted. Cyclic AMP phosphodiesterase activity of hepatocyte homogenates was not altered in the hyperthyroid state. Hormone-stimulated, guanine nucleotide- and fluoride-activatable adenylate cyclase activity was reduced in subcellular fractions obtained from hyperthyroid as compared with euthyroid rat hepatocytes. Beta adrenergic receptor binding was reduced approximately 35% and glucagon receptor binding reduced approximately 50% in the hyperthyroid as compared with euthyroid rat hepatocyte membrane fractions. The status of the regulatory components of adenylate cyclase were examined by in vitro treatment of subcellular fractions with cholera toxin. The ability of cholera toxin to modulate adenylate cyclase was not altered by hyperthyroidism. Cholera toxin catalyzed AD[(32)P]ribosylation of hyperthyroid and euthyroid rat hepatocyte proteins separated electrophoretically displayed nearly identical autoradiograms. Studies of the reconstitution of adenylate cyclase activity of S49 mouse lymphoma cyc(-) mutant membranes by detergent extracts from rat hepatocyte membranes, indicated that hyperthyroidism was associated with a reduced capacity of regulatory components to confer fluoride, but not guanine nucleotide activatability to catalytic cyclase. Thyroid hormones regulate the hormone-sensitive adenylate cyclase system of rat hepatocytes at several distinct loci of the system.

Hyperexpression of mitogen-activated protein kinase in human breast cancer.

Mitogen-activated protein (MAP) kinases act as transducers of extracellular signaling via tyrosine kinase-growth factor receptors and G-protein-linked receptors to elements regulating transcription. The activity, abundance, and localization of MAP kinase was investigated in normal and malignant neoplasia of the breast. In carcinoma of the breast, MAP kinase was heavily phosphorylated on tyrosyl residues and its activity elevated 5-10-fold over benign conditions, such as fibroadenoma and fibrocystic disease. By in situ reverse transcription-polymerase chain reaction, hyperexpression of MAP kinase mRNA can be localized to malignant, epithelial cells. Metastatic cells within involved lymph nodes of patients with breast cancer also display hyperexpression of MAP kinase. In spite of persistent activation via phosphorylation, MAP kinase expression is upregulated 5-20-fold and this hyperexpression may be a critical element to initiation as well as the metastatic potential of various forms of human breast cancer.

Effect of thyroid status on insulin action in rat adipocytes and skeletal muscle.

Isolated adipocytes and soleus muscles prepared from mature rats, rendered hypothyroid by a low iodine diet and propylthiouracil, markedly resisted the ability of insulin to increase glucose utilization. In adipocytes, the sum of basal d-(1-(14)C)-glucose conversion to CO(2), glyceride-glycerol, and fatty acid was unaltered by hypothyroidism, although conversion to fatty acid was decreased. The response of each of these metabolic pathways to insulin at all concentrations tested was greatly diminished in hypothyroid rat adipocytes. 3-O-Methylglucose transport rates in the presence of insulin were not significantly different in adipocytes from hypothyroid as compared with euthyroid rats, although basal transport rates were significantly higher in the hypothyroid state. Lipolysis and cyclic AMP accumulation in adipocytes from hypothyroid rats in response to theophylline were markedly diminished compared with euthyroid controls, but insulin was about as effective in inhibiting lipolysis in these cells as in those derived from euthyroid animals. The binding of (125)I-insulin to adipocytes at several hormone concentrations was also shown to be unaffected by hypothyroidism. In soleus muscle, basal glucose conversion to H(2)O and glycogen was unaltered in the hypothyroid state, whereas insulin action on these pathways was markedly inhibited. The decrease in muscle insulin responsiveness was less marked than that observed in adipocytes. Uptake of either 2-deoxyglucose or l-arabinose in the presence or absence of insulin was similar in soleus muscles derived from euthryoid vs. hypothyroid rats. Similarly, insulin action on the conversion of soleus muscle glycogen synthase D to the I form in the absence of glucose was unaltered by hypothyroidism. We conclude that (a) hypothyroidism in mature rats leads to a marked decrease in the responsiveness of glucose metabolism in adipocytes and skeletal muscle to insulin; (b) no detectable impairment of the membrane insulin effector systems that mediate the regulation of adipocyte hexose transport and glycogen synthase is caused by hypothyroidism in this animal model; and (c) the cellular defect that leads to apparent insulin resistance of adipocyte and soleus muscle glucose utilization resides at the level of one or more intracellular enzymes involved in glucose catabolism.

Regulation of receptor expression by agonists: transcriptional and post-transcriptional controls.

The molecular cloning of certain members of several distinct classes of receptors has opened up new avenues by which the regulation of signal-transducing proteins can be investigated. The information derived from molecular cloning permits not only studies of functional domains via mutagenesis, but also the study of gene regulation and the cell biology of these receptors by the use of molecular probes. All receptors are bifunctional, possessing domains for ligand binding as well as for signal propagation through binding to other protein(s), DNA or ion channels. Regulation of receptor expression and function in response to agonist stimulation is a central feature of receptor biology. In this article, Drs Hadcock and Malbon focus on the regulation of receptor expression by agonists at the transcriptional and post-transcriptional levels. Regulation of the expression of three classes of receptors central to neurobiology is highlighted: namely, steroid hormone receptors (estrogen receptor), G protein-coupled receptors (beta2-adrenergic receptor) and tyrosine kinase receptors (epidermal growth factor receptor). The emerging idea of cross regulation between receptors is also discussed in order to demonstrate the complexities of studying receptor expression.

Regulating expression and function of G-protein-linked receptors.

G-protein-linked receptors constitute a populous family of heptahelical, membrane-localized receptors for hormones, drugs and neurotransmitters that activate a diverse and smaller subset of effectors, including adenylylcyclases, phospholipases and various ion channels. The expression and functional status of G-protein-linked receptors is highly regulated. Expression is controlled largely by activation or repression of the genes encoding the receptors, balanced by post-transcriptional mechanisms such as destabilization of receptor mRNA. Agonist-induced down-regulation of receptors involves both transcriptional and post-transcriptional controls. Gene structure reveals details of promoters as well as determinants for mRNA stability. Post-translational regulation of G-protein-linked receptors is dominated by protein phosphorylation. G-protein-linked receptors are substrates not only for protein kinase A, protein kinase C and receptor-specific kinases, but also for growth factor receptors with intrinsic tyrosine kinase activity. Recent advances in the study of beta-adrenergic receptors (beta ARs) illuminate new dynamic features of receptor regulation, central to our understanding of neurobiology.

Beta-adrenergic activity of (+/-)-hydroxybenzylisoproterenol in isolated rat fat cells and hepatocytes.

The pharmacology of (+/-)-hydroxybenzylisoproterenol with respect to stimulation of cyclic AMP accumulation by isolated rat fat cells and liver cells was examined. (+/-)-Hydroxybenzylisoproterenol was found to be a full agonist and twice as potent as (-)-isoproterenol in liver cells, and equipotent to (-)-isoproterenol in fat cells with regard to stimulating cyclic AMP accumulation. A study of the ability of this catecholamine to stimulate adenylate cyclase activity of broken-cell preparations revealed that (+/-)-hydroxybenzylisoproterenol was equipotent to (-)-isoproterenol in liver cell homogenates, while 3- to 4-fold more potent than (-)-isoproterenol in fat cell ghost membranes. (+/-)-Hydroxybenzylisoproterenol was also found to be as potent as (-)-isoproterenol in stimulating cyclase activity of S49 mouse lymphoma cell membranes. Competition studies of specific [125I]iodohydroxybenzylpindolol binding to liver cell membranes revealed a Kd of 10 nM for (+/-)-hydroxybenzylisoproterenol and 25 nM for (-)-isoproterenol binding to the liver beta-adrenergic receptor. Competition studies of specific (-)-[3H]dihydroalprenolol binding to fat cell membranes indicated a similar affinity of these sites for both (+/-)-hydroxybenzylisoproterenol and (-)-isoproterenol. The guanyl nucleotide Gpp(NH)p induced a shift in the curve for competition of (-)-[3H]dihydroalprenolol binding by (-)-isoproterenol to the right, but failed to do so when (+/-)-hydroxybenzylisoproterenol was the competing agonist. Properties of (+/-)-[3H]hydroxybenzylisoproterenol binding to fat cell or liver cell membranes were inconsistent with those expected of adenylate cyclase coupled beta-adrenergic receptors.

Peptide mapping of fat cell membrane substrates for cholera toxin-catalyzed ADP-ribosylation.

Incubating rat fat cell membranes with [32P]NAD+ and cholera toxin results in ADP-ribosylation of three distinct components with approximate molecular weights of 42,000, 46,000 and 48,000. Partial proteolytic peptide maps of the Mr = 46,000 and 48,000 toxin-specific substrates generated by elastase, alpha-chymotrypsin, or Staphylococcus aureus V-8 protease were nearly identical, while those of the Mr = 42,000 target lacked several peptides common to both of the larger molecular weight targets. In addition, peptide maps generated from the Mr = 42,000 target displayed a number of peptides which were absent from the maps generated from either the Mr = 46,000 or 48,000 targets. These data suggest that the Mr = 46,000 and 48,000 substrates are closely related proteins, however the relationship between the Mr = 42,000 toxin-specific substrate and the larger peptides remains to be established. The relative patterns of fat cell membrane labelling by cholera toxin in the presence of [32P]NAD+ were identical in hypothyroid as compared to euthyroid rat fat cells.

ADP-ribosylation of membrane proteins and activation of adenylate cyclase by cholera toxin in fat cell ghosts from euthyroid and hypothyroid rats.

Incubation of fat cell ghosts with activated cholera toxin, nucleoside triphosphate, cytosol, and NAD results in increased adenylate cyclase activity and the transfer of ADP-ribose to membrane proteins. The major ADP-ribose protein comigrates on sodium dodecyl sulfate-polyacrylamide gels with the putative GTP-binding protein of pigeon erythrocyte membranes (Mr 42 000), which is also ADP-ribosylated by cholera toxin. The treatment with cholera toxin enhances the stimulation of the fat cell membrane adenylate cyclase by GTP, but the stimulation by guanyl-5'-yl imidodiphosphate is unaltered. Subsequent stimulation of fat cell adenylate cyclase by 10 micrometers epinephrine is not particularly affected. These changes were qualititatively the same for membranes isolated from fat cells of hypothyroid rats. Although the cyclase of these membranes has a reduced response to epinephrine, guanyl-5'-yl imidodiphosphate or GTP, as compared to euthyroid rat fat cell membranes, the defect is not rectified by toxin treatment and cannot be explained by a deficiency in the cholera toxin target.

Evaluation of the negative cooperativity model for fat cell beta-adrenergic receptors.

Cooperative site-to-site interactions among beta-adrenergic receptors of fat cell membranes are probed with the potent beta-adrenergic antagonist (-)-[3H]dihydroalprenolol according to the kinetic method of De Meyts et al. (De Meyts, P., Roth, J., Neville, Jr., D.M., Gavin, III, J.R., and Lesniak, M.A. (1973) Biochem. Biophys. Res. Commun. 55, 154--161). Dissociation of specific (-)-[3H]dihydroalprenolol binding from fat cell membranes following a 100-fold dilution was rapid at 37 degrees C; only 40% of the initial equilibrium binding remained 30 s after dilution. Dissociation of (-)-[3H]dihydroalprenolol bound under conditions yielding approximately 20% initial occupancy was performed in the absence and in the presence of a large molar excess of beta-adrenergic agonist ((-)-isoproterenol) or beta-adrenergic antagonist ((-)-alprenolol or (-)-propanalol). Neither agonists nor antagonists influenced the rate of (-)-[3H]dihydroalprenolol dissociation from fat cell membranes performed at 4, 22 or 37 degrees C. Although analysis of the steady-state binding of (-)-[3H]dihydroalprenolol to fat cell membranes yields Hill coefficients, nH, less than 1.0, the present study indicates that these fat cell beta-adrenergic receptors display no cooperative site-to-site interactions.

G-protein subunit mRNA levels in rat heart, liver, and adipose tissues: analysis by DNA-excess solution hybridization.

The steady-state levels of mRNAs for the G-proteins Gi alpha 2, Go alpha, and the G beta-subunits common to each were established in rat adipose, heart and liver. Uniformly-radiolabeled, single-stranded antisense probes were constructed from cDNAs or assembled from oligonucleotides. Direct comparison of the steady-state levels of the G-protein mRNAs was performed under identical assay conditions, and on a molar basis. In adipose, liver and heart, Gs alpha mRNA was more abundant than mRNA for Go alpha, Gi alpha, and G beta. In adipose tissue, mRNA levels were as follows: 19.4, 7.6, 7.0, and 2.3 amol mRNA per micrograms total cellular RNA for Gs alpha, G beta, Gi alpha 2, and Go alpha, respectively. In heart Gs alpha mRNA was less abundant than in adipose, but the relative trend among the G-protein subunits was the same. In liver, G beta mRNA was more abundant than either Go alpha or Gi alpha 2. Go alpha mRNA levels ranged from 1.2 to 2.3 amol/micrograms total RNA in liver and adipose, respectively. The present work demonstrates the many advantages of this strategy when applied to the study of a family of homologous, low-abundance proteins and establishes for the first time the molar levels of Gi alpha 2, Gs alpha, Go alpha, and G beta-subunit mRNAs in several mammalian tissues.

Immunological approaches for probing receptor structure and function.

Molecular cloning has revealed the primary sequence of numerous membrane receptors, and this information catalysed two important efforts: modeling of receptor structure by hydropathy analysis and generating sequence-specific immunological probes with which these models can be tested experimentally. Craig Malbon and his colleagues outline the recent advances that illustrate how anti-peptide antibodies raised to synthetic sequences of membrane receptor have generated new information on the topology, functional domains and cellular localization of transmembrane signaling elements. They focus on two examples, the G protein-linked beta-adrenoceptor, and the nicotinic acetylcholine receptor, an intrinsic ion channel receptor. These two classes of receptor provide templates for the analysis of topographical models of membrane proteins with immunological probes, especially anti-peptide antibodies, and demonstrate how these results complement those obtained from molecular, biochemical and biophysical techniques. Although this powerful strategy is not without faults, it is likely to continue to be applied successfully to the analysis of the structure and function of receptors, ion channels and other membrane proteins.

G-protein-linked receptors as tyrosine kinase substrates: new paradigms in signal integration.

Understanding how cells integrate signals from a variety of chemically diverse information-containing molecules into complex, orchestrated responses such as cell proliferation, differentiation and apoptosis is an overarching goal of cell biology. The ligand molecules that act upon cell surface receptors include those mediating proximal aspects of signal transduction through two major pathways: those that are G protein linked and those that are tyrosine kinase linked. G-protein receptors in the hundreds operate by means of less populous groups of heterotrimeric G proteins and the effectors regulated by G proteins. Growth factor receptors with intrinsic tyrosine kinase activity constitute a relatively large group of receptors, which share several downstream signalling elements with the G-protein-linked receptors. Integration between these two dominant pathways has been observed at several levels. The most proximal and intimate interaction possible--that between G-protein-linked receptors and tyrosine kinase receptors--has been discovered. Emerging data reveal new paradigms in which phosphorylation of G-protein-linked receptors on specific tyrosyl residues by tyrosine kinases enable G-protein-linked receptors to interact with adaptor molecules and enzymes previously thought to be restricted only to the signalling derivative of tyrosine kinase receptors.

Serum and insulin induce a Grb2-dependent shift in agonist affinity of beta-adrenergic receptors.

Beta-adrenergic receptors transduce catecholamine binding to activation of adenylylcyclase, a response counter-regulated by insulin. Insulin stimulates tyrosine phosphorylation of Tyr 350/354, which abolishes the catecholamine response. Phosphorylation of Try 350/354 creates a Src homology 2 (SH2) domain on the beta2-adrenergic receptor and the binding of adaptor protein Grb2 to this SH2 domain of the beta-adrenergic receptor takes place in an insulin-dependent manner. In membranes from serum-deprived S49 mouse lymphoma cells, GTPgammaS yields the well-known agonist-specific shift in agonist affinity for beta2-adrenergic receptors. The agonist-specific shift is observed in cell membranes either in the absence or in the presence of exogenously added purified Grb2. In membranes for serum-fed cells, in contrast, the addition of Grb2 induces an agonist-specific shift in receptor affinity that mimics addition of GTPgammaS to the membranes. The ability of the Grb2 to induce an agonist-specific shift in the membranes from serum-fed cells was abolished equally effectively either by competition with phosphopeptide harbouring the (p)YVNV motif or by disruption of the SH2 domain of added Grb2. Challenging Chinese hamster ovary cells with insulin (100 nM) for 30 min enabled Grb2 to induce an agonist-specific shift in agonist affinity for beta2-adrenergic receptors, suggestive of uncoupling of the receptors from G proteins. The insulin-dependent Grb2 effect on receptor-G-protein coupling was sensitive to competition by the pYVNY phosphopeptide and to disruption of the SH2 domain of Grb2. These data provide a biochemical link between the ability of insulin to counter-regulate catecholamine stimulation of cyclic AMP accumulation and the phosphorylation of the beta-adrenergic receptor, consequent biding of the adaptor molecule Grb2 and disruption of receptor-G-protein coupling.

Conditional, tissue-specific expression of Q205L G alpha i2 in vivo mimics insulin action.

Deficiency of the G protein subunit G alpha i2 that is known to mediate the inhibitory control of adenylylcyclase impairs insulin action [11]. Using the promoter for the phosphoenolpyruvate carboxykinase gene, conditional tissue-specific expression of the constitutively active mutant form (Q205L) of G alpha i2 was achieved in mice harboring the transgene. Expression of Q205L G alpha i2 was detected in liver and adipose tissue of transgenic mice. Whereas the G alpha i2 deficient mice displayed blunted glucose tolerance, the Q205L G alpha i2 expressing mice displayed enhanced glucose tolerance. Hexose transport and the recruitment of GLUT4, but not GLUT1, transporters to the membrane were elevated in adipocytes from Q205L G alpha i2 expressing mice in the absence of insulin. Additionally, hepatic glycogen synthase was found to be activated in Q205L G alpha i2 expressing mice, in the absence of the administration of insulin. Serum insulin levels in transgenic mice fasted overnight were equivalent to those of their control littermates. These data demonstrate that much as G alpha i2 deficiency leads to insulin resistance, expression of Q205L constitutively active G alpha i2 mimics insulin action in vivo, reflecting a permissive role of G alpha i2 in signaling via this growth factor receptor tyrosine kinase linked pathway.

Integration of transmembrane signaling Cross-talk among G-protein-linked receptors and other signal transduction pathways.

The heart is a point of convergence for many transmembrane signal transduction pathways and, as such, exemplifies the complexities of sorting out their interactions. In the heart and cardiovascular system, hormone and neurotransmitter signals are propagated via G-protein-and non-G-protein-linked receptors. The receptors, in turn, are coupled to many effectors, including adenylylcyclases, phospholipases, and ion channels, as well as protein kinases and phosphatases. Integrating the information from numerous signaling pathways requires "cross-talk" among the different pathways. This article provides a perspective and attempts to describe in some detail what is known about a few of these interactions. Cross-talk from stimulatory to inhibitory adenylylcyclase pathways, from inhibitory to stimulatory adenylylcyclase pathways, from the adenylylcyclase to the phospholipase-C pathway, and from tyrosine kinase to G-protein-coupled receptors is described. Because of its central importance in the regulation of the cardiovascular system, and due to the availability of good model systems, the powerful responses mediated by the ß-adrenergic receptor pathway serve as an excellent model to approach this subject.

Thyroid hormone administration in vivo regulates the activity of hepatic glycogen phosphorylase phosphatase.

Short term (48 h) administration in vivo of either T4 or T4, but not the biologically inactive D-isomer of T3, was associated with a decrease in basal glycogen phosphorylase alpha activity and an increase in phosphorylase alpha phosphatase activity of rat hepatocytes. This influence of thyroid hormones on hepatic phosphorylase alpha and phosphorylase phosphatase activities was shown to be dose dependent. As little as 0.0025 mg T3/kg BW administered in vivo at 48, 24, and 3 h before liver excision increased the phosphatase activity by 20%. The administration of 0.25 mg T3/kg BW on the same schedule increased the phosphatase activity by nearly 100%.