Insulin-like growth factor I (IGF-I) receptor overexpression abolishes the IGF requirement for differentiation and induces a ligand-dependent transformed phenotype in C2 inducible myoblasts.

Insulin-like growth factors (IGFs) stimulate both proliferation and differentiation of myogenic cell lines, and these actions are mostly mediated through the type I IGF receptor (type I IGF-R). To further investigate the role of this receptor in phenotypic characteristics of C2 murine myoblasts, we overexpressed the human type I IGF-R in the inducible clone of C2 cells, which requires IGFs in the differentiation medium to undergo terminal differentiation. Inducible myoblasts were transfected with either the eukaryotic expression vector pNTK or pNTK containing the human type I IGF-R complementary DNA, and we isolated two clones named Ind-Neo and Ind-R, respectively. Binding and autophosphorylation experiments indicate that Ind-R cells express about 10 times as much type I IGF-R compared with Ind-Neo control cells and that the transfected type I IGF-R is functional in Ind-R cells. We show that overexpression of the human type I IGF-R makes inducible myoblasts able to differentiate spontaneously, as assessed by expression of the myogenic transcription factors MyoD and myogenin, detection of the muscle-specific protein troponin T, and myotube formation. Moreover, when exposed to IGF-I, Ind-R cells lose contact inhibition, grow in the presence of a low level of growth factors and form colonies in soft agar, which is characteristic of a ligand-dependent transformed phenotype. It emerges from this study that 1) the type I IGF-R is strongly involved in the phenotypic differences between inducible and permissive cells with respect to the differentiation program; and 2) overexpression causes this receptor to act as a ligand-dependent transforming protein in muscle cells. We suggest that type I IGF-R abundance and level of activation may determine the efficiency of the autocrine mode of action of IGFs and discriminate their biological functions.

Rabbit slow and fast skeletal muscle-derived satellite myoblast phenotypes do not involve constitutive differences in the components of the insulin-like growth factor system.

The insulin-like growth factor (IGF) system is actively involved in the control of proliferation and differentiation of several myogenic cell lines, and phenotypic differences between myoblasts are associated with modifications of the equilibrium of the components of the IGF system. To determine whether this observation is a physiologic feature that also concerns the phenotypes of ex vivo adult satellite myoblasts in primary cell culture, we investigated the IGF system in rabbit slow-twitch muscle-derived satellite myoblasts (SSM), which differ phenotypically from fast-twitch muscle-derived satellite myoblasts (FSM) by their proliferation and differentiation kinetics in vitro. The expression of IGF-I and IGF-II were similar in SSM and FSM as well as their concentrations measured in cell-conditioned media. Ligand blotting of conditioned media samples indicated the presence of five IGF binding protein (IGFBP) species of Mr 37-40, 32, 30-31, 28, and 24 kDa. The 30-31 kDa doublet was visible in SSM-conditioned medium only and associated with the presence of a 22-kDa protein, which may represent a proteolytic fragment. In contrast, the 32-kDa band was observed in FSM conditioned medium only. The other IGFBP moieties were present in both SSM- and FSM-conditioned media. Cross-linking experiments revealed the presence of the M6P/IGF-II receptor on both SSM and FSM membranes. We also observed an IGF-I receptor form bearing unusual high affinity for IGF-II: the binding of [125I]IGF-I on this receptor was preferentially displaced by IGF-I but that of [125I]IGF-II was mostly inhibited by IGF-II, suggesting that the two tracers did not bind on the same epitopes. [125I]IGF-II binding to this receptor was greater on SSM than on FSM membranes. Autophosphorylation of WGA-purified receptors revealed an approximately 400-kDa band after SDS-PAGE under nonreducing conditions, which corresponded to the alpha 2 beta 2 form of the IGF-I receptor, and two beta subunit moieties of Mr 101 and 105 kDa under reducing conditions in both SSM and FSM extracts. Phosphorylation of the 105-kDa moiety was more intensively increased than that of the 101-kDa protein after growth factor stimulation. Basal phosphorylation state of the two beta subunits was similarly stimulated by IGF-I and IGF-II and less by insulin. Since both insulin and IGF-I receptors were expressed in FSM and SSM, one of the two beta subunits may actually correspond to that of the insulin receptor. We conclude that the IGF system is not considerably affected by the phenotypes of SSM and FSM. The differences observed, which mostly concern IGFBP species, more likely appear as regulatory adaptations than as phenotypic changes targeting the components of the IGF system.

Characterization of a null mutation in ace-1, the gene encoding class A acetylcholinesterase in the nematode Caenorhabditis elegans.

Two genes (ace-1 and ace-2) encode two major classes (A and B) of acetylcholinesterase (AChE) in the nematode Caenorhabditis elegans. A null mutation in ace-1 (allele p1000) suppresses all acetylcholinesterase activity of class A. We have identified an opal mutation TGG (W99)-->TGA (Stop) as the only alteration in the mutated gene. This leads to a truncated protein (98 instead of 620 amino acids) with no enzymatic activity. The mutation also reduces the level of ace-1 transcripts to only 10% of that in wild-type animals. This most likely results from a destabilization of mRNA containing the nonsense message. In contrast, compensation of class B by class A AChE in the null mutant strain ace-2 takes place with unchanged ace-1 mRNA level and enzymatic activity similar to class A AChE.

ATP-modulated K+ channels sensitive to antidiabetic sulfonylureas are present in adenohypophysis and are involved in growth hormone release.

The adenohypophysis contains high-affinity binding sites for antidiabetic sulfonylureas that are specific blockers of ATP-sensitive K+ channels. The binding protein has a M(r) of 145,000 +/- 5000. The presence of ATP-sensitive K+ channels (26 pS) has been demonstrated by electrophysiological techniques. Intracellular perfusion of adenohypophysis cells with an ATP-free medium to activate ATP-sensitive K+ channels induces a large hyperpolarization (approximately 30 mV) that is antagonized by antidiabetic sulfonylureas. Diazoxide opens ATP-sensitive K+ channels in adenohypophysis cells as it does in pancreatic beta cells and also induces a hyperpolarization (approximately 30 mV) that is also suppressed by antidiabetic sulfonylureas. As in pancreatic beta cells, glucose and antidiabetic sulfonylureas depolarize the adenohypophysis cells and thereby indirectly increase Ca2+ influx through L-type Ca2+ channels. The K+ channel opener diazoxide has an opposite effect. Opening ATP-sensitive K+ channels inhibits growth hormone secretion and this inhibition is eliminated by antidiabetic sulfonylureas.

ATP/ADP binding sites are present in the sulfonylurea binding protein associated with brain ATP-sensitive K+ channels.

Covalent labeling of nucleotide binding sites of the purified sulfonylurea receptor has been carried out with alpha-32P-labeled oxidized ATP. The main part of 32P incorporation is in the 145-kDa glycoprotein that has been previously shown to be the sulfonylurea binding protein (Bernardi et al., 1988). ATP and ADP protect against this covalent labeling with K0.5 values of 100 microM and 500 microM, respectively. Non-hydrolyzable analogs of ATP also inhibit 32P incorporation. Interactions between nucleotide binding sites and sulfonylurea binding sites have then been observed. AMP-PNP, a nonhydrolyzable analog of ATP, produces a small inhibition of [3H]glibenclamide binding (20-25%) which was not influenced by Mg2+. Conversely, ADP, which also produced a small inhibition (20%) in the absence of Mg2+, produced a large inhibition (approximately 80%) in the presence of Mg2+. This inhibitory effect of the ADP-Mg2+ complex was observed with a K0.5 value of 100 +/- 40 microM. All the results taken together indicate that ATP and ADP-Mg2+ binding sites that control the activity of KATP channels are both present on the same subunit that bears the receptors for antidiabetic sulfonylureas.

Antidiabetic sulfonylureas: localization of binding sites in the brain and effects on the hyperpolarization induced by anoxia in hippocampal slices.

The distribution of antidiabetic sulfonylurea [( 3H]glibenclamide) binding sites is heterogeneous in rat brain. Pyramidal and extrapyramidal motor system contain the highest densities of sites, particularly in the substantia nigra and in the globus pallidus. Only low levels are present in the hypothalamic nuclei and the main medulla oblongata regions. In hippocampal formation the stratum lucidum and the stratum lacunosum moleculare of CA3 show an important density of glibenclamide binding sites. Electrophysiological studies with hippocampal slices show that glibenclamide blocks hyperpolarization induced by anoxia, suggesting the involvement of adenosine triphosphate-sensitive K+ channel in this early hyperpolarization event.

Molecular properties of potassium channels.

The paper describes the molecular pharmacology and biochemistry of three types of K+ channels, the calcium-activated potassium channels, ATP-regulated potassium channels and voltage-sensitive potassium channels.

Characterization, purification, and affinity labeling of the brain [3H]glibenclamide-binding protein, a putative neuronal ATP-regulated K+ channel.

Sulfonylurea and particularly glibenclamide are potent blockers of ATP-regulated K+ channels in insulin-secreting cells. A very good correlation exists between binding of sulfonylurea to brain and insulinoma cell membranes. The [3H]glibenclamide-binding component from pig brain microsomes was solubilized with digitonin with a complete retention of its properties of interaction with glibenclamide and other sulfonylureas. A four-step purification was achieved that used (i) hydroxylapatite chromatography, (ii and iii) affinity chromatographies on ADP-agarose and wheat germ agglutinin-agarose columns, and (iv) a final chromatographic step on a mixture of AMP-agarose/GMP-agarose/hydroxylapatite. This procedure led to a 2500-fold purification. NaDodSO4/polyacrylamide gel electrophoresis of the purified material in reducing and nonreducing conditions showed that the sulfonylurea-binding component is made of a single major polypeptide chain of Mr 150,000 +/- 10,000. Direct photoaffinity labeling of the receptor with [3H]glibenclamide at different steps of the purification also showed that radioactivity was specifically incorporated into a polypeptide of Mr 150,000 +/- 5000, thus confirming the subunit structure indicated by the purification.