Role of cholesterol in developing T-tubules: analogous mechanisms for T-tubule and caveolae biogenesis.

Recent work has suggested that caveolae biogenesis and transverse-tubule (T-tubule) formation in muscle cells share similar underlying features. We compared the properties of caveolin-1 (cav-1)-positive caveolae, in epithelial cells, with caveolin-3 (cav-3)-positive precursor T-tubules, in differentiating C2C12 muscle cells, using the cholesterol-binding drug, Amphotericin B (AmphB). Treatment of MDCK epithelial cells with acute high doses or chronic low doses of AmphB caused a loss of surface caveolae and the rapid redistribution of cav-1, and exogenously expressed cav-3, from the cell surface into modified endosomes. This effect was reversible and specific, as the GPI-anchored protein, alkaline phosphatase, was largely unaffected by the treatment unless it had been previously partitioned into caveolar domains. In differentiating C2C12 mouse myotubes, AmphB also caused a complete redistribution of cav-3 from precursor T-tubule elements into enlarged endosomes, morphologically very similar to those seen in MDCK cells. This was accompanied by redistribution of a T-tubule marker and a dramatic reduction in the extent of surface-connected tubular elements. We propose that cholesterol-enriched glycolipid 'raft' domains are involved in the formation and maintenance of diverse membrane systems including caveolae and the T-tubule system of muscle.

Molecular characterization of caveolin association with the Golgi complex: identification of a cis-Golgi targeting domain in the caveolin molecule.

Caveolins are integral membrane proteins which are a major component of caveolae. In addition, caveolins have been proposed to cycle between intracellular compartments and the cell surface but the exact trafficking route and targeting information in the caveolin molecule have not been defined. We show that antibodies against the caveolin scaffolding domain or against the COOH terminus of caveolin-1 show a striking specificity for the Golgi pool of caveolin and do not recognize surface caveolin by immunofluorescence. To analyze the Golgi targeting of caveolin in more detail, caveolin mutants were expressed in fibroblasts. Specific mutants lacking the NH2 terminus were targeted to the cis Golgi but were not detectable in surface caveolae. Moreover, a 32-amino acid segment of the putative COOH-terminal cytoplasmic domain of caveolin-3 was targeted specifically and exclusively to the Golgi complex and could target a soluble heterologous protein, green fluorescent protein, to this compartment. Palmitoylation-deficient COOH-terminal mutants showed negligible association with the Golgi complex. This study defines unique Golgi targeting information in the caveolin molecule and identifies the cis Golgi complex as an intermediate compartment on the caveolin cycling pathway.

Intracellular localization of phosphatidylinositide 3-kinase and insulin receptor substrate-1 in adipocytes: potential involvement of a membrane skeleton.

Phosphatidylinositide (PI) 3-kinase binds to tyrosyl-phosphorylated insulin receptor substrate-1 (IRS-1) in insulin-treated adipocytes, and this step plays a central role in the regulated movement of the glucose transporter, GLUT4, from intracellular vesicles to the cell surface. PDGF, which also activates PI 3-kinase in adipocytes, has no significant effect on GLUT4 trafficking in these cells. We propose that this specificity may be mediated by differential localization of PI 3-kinase in response to insulin versus PDGF activation. Using subcellular fractionation in 3T3-L1 adipocytes, we show that insulin- and PDGF-stimulated PI 3-kinase activities are located in an intracellular high speed pellet (HSP) and in the plasma membrane (PM), respectively. The HSP is also enriched in IRS-1, insulin-stimulated tyrosyl-phosphorylated IRS-1 and intracellular GLUT4-containing vesicles. Using sucrose density gradient sedimentation, we have been able to segregate the HSP into two separate subfractions: one enriched in IRS-1, tyrosyl-phosphorylated IRS-1, PI 3-kinase as well as cytoskeletal elements, and another enriched in membranes, including intracellular GLUT4 vesicles. Treatment of the HSP with nonionic detergent, liberates all membrane constituents, whereas IRS-1 and PI 3-kinase remain insoluble. Conversely, at high ionic strength, membranes remain intact, whereas IRS-1 and PI 3-kinase become freely soluble. We further show that this IRS-1-PI 3-kinase complex exists in CHO cells overexpressing IRS-1 and, in these cells, the cytosolic pool of IRS-1 and PI 3-kinase is released subsequent to permeabilization with Streptolysin-O, whereas the particulate fraction of these proteins is retained. These data suggest that IRS-1, PI 3-kinase, as well as other signaling intermediates, may form preassembled complexes that may be associated with the actin cytoskeleton. This complex must be in close apposition to the cell surface, enabling access to the insulin receptor and presumably other signaling molecules that somehow confer the absolute specificity of insulin signaling in these cells.

Transcriptional repression by the orphan steroid receptor RVR/Rev-erb beta is dependent on the signature motif and helix 5 in the E region: functional evidence for a biological role of RVR in myogenesis.

RVR/Rev-erb beta/BD73 is an orphan steroid receptor that has no known ligand in the "classical' sense. RVR binds as a monomer to an element which consists of an A/T-rich sequence upstream of the consensus hexameric half-site. However, RVR does not activate transcription and blocks transactivation of this element by ROR/RZR. The mechanism of RVR action remains obscure, hence we used the GAL4 hybrid system to identify and characterize an active transcriptional silencer in the ligand binding domain (LBD) of RVR. Rigorous deletion and mutational analysis demonstrated that this repressor domain is encoded by amino acids 416-449 of RVR. Furthermore, we demonstrated that efficient repression is dependent on the so-called LBD-specific signature motif, (F/W)AKxxxxFxxLxxxDQxxLL (which spans loop3-4 and helix 4) and helix 5 (H5; identified in the crystal structures of the steroid receptor LBDs). Although RVR is expressed in many adult tissues, including skeletal muscle, and during embryogenesis, its physiological function in differentiation and mammalian development remains unknown. Since other 'orphans', e.g. COUP-TF II and Rev-erbA alpha, have been demonstrated to regulate muscle and adipocyte differentiation, we investigated the expression and functional role of RVR during mouse myogenesis. In C2C12 myogenic cells, RVR mRNA was detected in proliferating myoblasts and was suppressed when the cells were induced to differentiate into post-mitotic, multinucleated myotubes by serum withdrawal. This decrease in RVR mRNA correlated with the appearance of muscle-specific markers (e.g. myogenin mRNA). RVR 'loss of function' studies by constitutive over-expression of a dominant negative RVR delta E resulted in increased levels of p21Cip1/Waf1 and myogenin mRNAs after serum withdrawal. Time course studies indicated that expression of RVR delta E mRNA results in the precocious induction and accumulation of myogenin and p21 mRNAs after serum withdrawal. In addition, we demonstrated that over-expression of the COUP-TF II and Rev-erbA alpha receptors in C2C12 cells completely blocked induction of p21 mRNA after serum withdrawal. In conclusion, our studies identified a potent transcriptional repression domain in RVR, characterized critical amino acids within the silencing region and provide evidence for the physiological role of RVR during myogenesis.

Constitutive expression of the orphan receptor, Rev-erbA alpha, inhibits muscle differentiation and abrogates the expression of the myoD gene family.

Rev-erbA alpha is an orphan steroid receptor that is expressed in skeletal muscle. Rev-erbA alpha binds to single/tandem copies of an AGGTCA motif, is transcribed on the noncoding strand of the c-erbA- alpha gene locus, and is postulated to modulate the thyroid hormone (T3) response. T3 induces terminal muscle differentiation and regulates fiber type composition via direct activation of the muscle-specific myoD gene family (e.g. myoD, myogenin). The myoD gene family can direct the fate of mesodermal cell lineages and activate muscle differentiation. Hence we investigated the expression and physiological role of Rev-erbA alpha during myogenesis. We observed abundant levels of Rev-erbA alpha mRNA in dividing C2C12 myoblasts, which were suppressed when the cells differentiated into postmitotic multinucleated myotubes. This decrease in Rev-erbA alpha mRNA correlated with the appearance of muscle-specific mRNAs (e.g. myogenin and alpha-actin). Constitutive overexpression of full length Rev-erbA alpha cDNA in the myogenic cells completely abolished differentiation, suppressed myoD mRNA levels, and abrogated the induction of myogenin mRNA. We then demonstrated that 1) GAL4-REV-erbA alpha chimeras that contain the 'AB' region and lack the 'E' region activated transcription of GAL4 response elements in the presence of 8-Br-cAMP and 2) the ligand-binding domain (LBD) contains an active transcriptional silencer. Overexpression of Rev-erbA alpha (delta AB) in myogenic cells had no impact on the ability of these cells to morphologically or biochemically differentiate. Furthermore, this orphan receptor 1) down-regulated thyroid hormone receptor (TR)/T3 mediated transcriptional activity from the myogenin promoter and thyroid hormone response element (TRE) an 2) disrupted TR homodimer and TR/retinoid X receptor (RXR) heterodimer formation on a number of TREs found in the myoD gene family. In conclusion, Rev-erbA alpha functions as a negative regulator of myogenesis by targeting the expression of the myoD gene family. The mechanism of action may involve inhibition of functional TR/RXR heterodimer formation on critical TREs and dominant trans-repression of gene expression.

Requirement for phosphoinositide 3-kinase in insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes.

Insulin stimulates glucose transport in muscle and fat cells by inducing the redistribution of a specific glucose transporter, GLUT4, from intracellular vesicles to the cell surface. Phosphoinositide (PI) 3-kinase has been implicated as a key intermediate in insulin-stimulated glucose transport by studies that have examined the effects of wortmannin and LY294002, which are thought to be specific inhibitors of this enzyme. However, the specificity of these compounds for PI 3-kinase has recently been questioned. Epidermal growth factor, which activates mitogen-activated protein kinase in mouse 3T3-L1 adipocytes, has now been shown to have no effect on PI 3-kinase activity or GLUT4 translocation in these cells. Furthermore, microinjection of a dominant negative mutant of the 85-kDa subunit of PI 3-kinase, which lacks a binding site for the catalytic 110-kDa subunit, inhibited GLUT4 translocation induced by insulin in 3T3-L1 adipocytes; microinjection of the wild-type protein had no effect. These observations indicate that PI 3-kinase is necessary for insulin-induced GLUT4 translocation and glucose transport in adipocytes.

Mutational analysis of phospholipase C-beta 2. Identification of regions required for membrane association and stimulation by guanine-nucleotide-binding protein beta gamma subunits.

Members of the beta isozyme subfamily of the phosphoinositide-specific phospholipases C (PLC beta) have recently been shown to be stimulated by both guanine-nucleotide-binding protein alpha and beta gamma subunits. The alpha subunits of the Gq class activate PLC beta isozymes in the order of PLC beta 1 > or = PLC beta 3 >> PLC beta 2, which is different from the order of PLC beta 3 > PLC beta 2 > PLC beta 1 for beta gamma subunit stimulation. The C-terminal region of PLC beta 1, in particular the sequence between Thr903 and Leu1142, has been shown to be involved in interacting with activated alpha q subunits and to contain a region required for efficient membrane association of PLC beta 1 [Park, D., Jhon, D.-Y., Lee, C.-W., Ryu, S. H. & Rhee, S. G. (1993) J. Biol. Chem. 268, 3710-3714, and Wu, D., Jiang, H., Katz, A. & Simon, M. I. (1993) J. Biol. Chem. 268, 3704-3709]. To examine the structure-function relationships of a PLC beta isozyme highly sensitive to beta gamma subunit stimulation, we have altered the cDNA of PLC beta 2 by site-directed mutagenesis and have examined the effects of these structural alterations on the functional properties of the mutant polypeptides. The results show that the C-terminal region of PLC beta 2 downstream of Phe818, which corresponds to Tyr816 of PLC beta 1, contains a region essential for membrane association, but is required neither for the interaction of PLC beta 2 with Ca2+ and the phospholipid substrate, nor for beta gamma subunit stimulation of PLC beta 2. These data suggest that PLC beta isozymes are activated by alpha q and beta gamma subunits via distinct domains.

Activation of phosphatidylinositol lipid-specific phospholipase C-beta 3 by G-protein beta gamma subunits.

A novel member of the inositol lipid-specific phospholipase C family, PtdIns-PLC beta 3, is shown to be activated by beta gamma subunits of the heterotrimeric GTP-binding protein, transducin. The activation is a direct effect since it is observed with the purified proteins. Furthermore, the activation is blocked by the GDP-liganded alpha subunit of transducin, confirming that the effect is due to free beta gamma subunits. The implications with respect to receptor-PtdIns-PLC coupling are discussed.

Identification, purification and characterization of a novel phosphatidylinositol-specific phospholipase C, a third member of the beta subfamily.

The partial sequence of a novel PtdIns-specific phospholipase C of the beta subfamily (PtdIns-PLC beta 3) is described. Based upon the predicted protein sequence, monospecific antibodies have been raised and used to identify a suitable source for purification of the protein. Fractionation of HeLa S3 cells revealed that immunoreactive PtdIns-PLC beta 3 is membrane associated; purification (approximately 1000-fold) from this fraction yielded a single immunoreactive protein of 158 kDa, with a specific activity of 136 mumol.min-1.mg-1, with PtdIns 4,5-bisphosphate as substrate. Substrate specificity and Ca2+ dependence of this purified PtdIns-PLC are characteristic of the PtdIns-PLC beta subfamily.

Isozyme-selective stimulation of phospholipase C-beta 2 by G protein beta gamma-subunits.

Hydrolysis by phospholipase C (PLC) of phosphatidylinositol 4,5-bisphosphate is a key mechanism by which many extracellular signalling molecules regulate functions of their target cells. At least eight distinct isozymes of PLC are recognized in mammalian cells. Receptor-controlled PLC is often regulated by G proteins, which can be modified by pertussis toxin in some cells but not in others. In the latter cells, PLC-beta 1, but not PLC-gamma 1 or PLC-delta 1, may be activated by members of the alpha q-subfamily of the G protein alpha-subunits. An unidentified PLC in soluble fractions of cultured human HL-60 granulocytes is specifically stimulated by G protein beta gamma subunits purified from retina and brain. Identification of a second PLC-beta complementary DNA (PLC-beta 2) in an HL-60 cell cDNA library prompted us to investigate the effect of purified G protein beta gamma subunits on the activities of PLC-beta 1 and PLC-beta 2 transiently expressed in cultured mammalian cells. We report here that PLC-beta 1 and PLC-beta 2 were stimulated by free beta gamma subunits and that PLC-beta 2 was the most sensitive to beta gamma stimulation. Thus stimulation of PLC by beta gamma subunits is isozyme-selective and PLC-beta 2 is a prime target of beta gamma stimulation. Activation of PLC-beta 2 by beta gamma subunits may be an important mechanism by which pertussis toxin-sensitive G proteins stimulate PLC.

A double-blind crossover comparison of flecainide and slow-release mexiletine in the treatment of stable premature ventricular complexes.

In 24 patients with stable premature ventricular contractions (PVCs) greater than or equal to 100/h, Lown class greater than or equal to 2 the relative anti-arrhythmic efficacy of flecainide 150 mg twice daily and slow-release mexiletine 360 mg twice daily was evaluated in a double-blind placebo-controlled randomized crossover study. All the patients had normal ventricular function. Criteria of efficacy were: reduction greater than or equal to 70% of PVCs or reduction greater than or equal to 50% with abolition of Lown class greater than 2 arrhythmias or suppression of non-sustained ventricular tachycardias (nSVT). Twenty-two patients completed the study protocol. The placebo phases showed comparable results and no carry over effect. The criteria of efficacy were fulfilled in 20 of the 22 patients (91%) on flecainide and in 12 of the 22 (55%) on mexiletine. The absolute reductions of PVCs, couplets and nSVT obtained on flecainide and mexiletine, in comparison to the placebo, were statistically significant (p less than 0.01 for flecainide, p less than 0.05 for mexiletine). Flecainide was superior to mexiletine in overall PVC reduction (p less than 0.05). In the 17 patients with couplets the reduction obtained with flecainide was superior to mexiletine (p less than 0.05). Both drugs were highly effective on nSVT. At steady state, the mean plasma levels of both drugs were within the range of clinical efficacy. The drugs were well tolerated and no patient withdrew because of side-effects. It was concluded that at the dosages employed flecainide was superior to mexiletine in reducing premature ventricular contractions and in abolishing couplets. The efficacy of both drugs for non-sustained ventricular tachycardias was comparable. Both drugs were highly effective by comparison with the placebo.

[Evaluation of the intestinal absorption of iron orally administered as chondroitin sulfate in normal subjects]. / Valutazione dell'assorbimento intestinale di ferro somministrato per via orale come condroitinsolfato in soggetti normali.

The behaviour of sideremia has been studied in order to assess the intestinal absorption of iron of a new compound, ferric chondroitin sulfate after oral administration in 12 normal volunteers. After administration of 90 mg of iron as ferric chondroitin sulfate, sideremia rose from a basal value of 88 +/- 27 micrograms/dl to a value of 128 +/- 22 micrograms/dl at the third hour. Variance analysis showed that the increases were statistically significant (F = 27.7; p less than 0.00001). In the same subjects, the test was carried out in accordance with a randomised crossover design in two periods after administration of 91 mg of ferritin iron: sideremia rose from a basal value of 92 +/- 27 micrograms/dl to a value at the third hour of 97 +/- 28 micrograms/dl, moderate increases but statistically significant (F = 3.2; P = 0.0354). Variance analysis by repeated measurements showed that increases in sideremia were significantly higher after iron administration as ferric chondroitin sulfate than after administration of ferritin iron (F = 13.18; p = 0.0042). This study documents the good bioavailability of the iron contained in ferric chondroitin sulfate.

Effect of a single daily dose treatment of fenofibrate on plasma lipoproteins in hyperlipoproteinaemia IIb.

The safety and efficacy of a single daily dose of fenofibrate (200 mg) have been evaluated in 12 Type IIB hyperlipidaemic patients in a three-month study. At the same time the pharmacokinetics was studied to check whether this new dosage schedule would give a therapeutic plasma levels of fenofibrate. At the single daily dose of 200 mg, fenofibrate was highly effective, very well tolerated, and it reached therapeutic plasma levels without accumulation. It appears that fenofibrate can usefully be employed at this dosage in hyperlipidaemia, especially since patient compliance is better when only one daily dose need be taken.

Bioavailability of rifampicin capsules.

In a 3-year bioavailability program 14 studies in 45 healthy volunteers were carried out to differentiate between experimental lots of rifampicin (RMP) capsules and marketed preparations of other manufacturers with lower bioavailability than Rifadin (RFD), used as standard reference drug. In each study single oral doses of 600 mg of 2-5 different RMP preparations were administered to 6-12 volunteers in fasting conditions according to a balanced crossover design. The pharmacokinetic parameters of RFD capsules were practically identical for the same batch tested at different times and for several batches tested in different groups of subjects. Variations in particle size, excipients, or manufacturing process of the experimental preparations or capsules marketed by other manufacturers produced a marked change in bioavailability of RMP. An additional study in four volunteers given 450 mg RMP confirmed that the absorption of RMP is less when the drug is taken with food. It is concluded that due to the wide variability in individual serum levels reported in the literature, some patients who absorb RMP poorly may be given ineffective therapy, especially when there are several concomitant unfavorable factors, such as a poor drug product or the effect of food.