Differential permeabilization of membranes by saponin treatment of isolated rat hepatocytes. Release of secretory proteins.

Monolayer cultures of rat hepatocytes were treated with increasing concentrations of saponin (prepared from Gypsophila plants) for 30 min at 6 degrees C. Differential permeabilization of the intracellular membranes could be demonstrated: at 0.040 mg of saponin/ml the plasma membrane was permeabilized, as assessed by the release of 50% of the total cellular amount of lactate dehydrogenase, and at 0.20 mg/ml the endoplasmic reticulum was permeabilized, as measured by the release of 50% of pulse-35S-labelled albumin. The Golgi complex was permeabilized at an intermediate saponin concentration, as indicated by the release of homogeneously 35S-labelled albumin; about half the intracellular albumin is located in this organelle. At 1.0 up to 5.0 mg of saponin/ml 90-95% of the radioactively labelled albumin was released. Even at 5.0 mg/ml less than 10% of the membrane of the endoplasmic reticulum was solubilized, as judged by the degree of release of a membrane-bound enzyme specific for this organelle. These results demonstrate the usefulness of saponin as a tool for investigating the interior of different intracellular compartments.     

Some differences in the properties of carnitine palmitoyltransferase activities of the mitochondrial outer and inner membranes.

Recent evidence has shown that the outer, overt, malonyl-CoA-inhibitable carnitine palmitoyltransferase (CPTo) activity resides in the mitochondrial outer membrane [Murthy & Pande (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 378-382]. A comparison of CPTo activity of rat liver mitochondria with the inner, initially latent, carnitine palmitoyltransferase (CPTi) of the mitochondrial inner membrane has revealed that the presence of digitonin and several other detergents inactivates CPTo activity. The CPTi activity, in contrast, was markedly stimulated by various detergents and phospholipid liposomes. These findings explain why in previous studies, which used digitonin or other detergents to expose, separate and purify the CPT activities, the inferences were drawn that (a) the ratio of latent to overt CPT was quite high, (b) both the CPT activities could be ascribed to one active protein recovered, and (c) the observed lack of malonyl-CoA inhibition indicated possible loss/separation of a putative malonyl-CoA-inhibition-conferring protein. Although both CPTo and CPTi were found to catalyse the forward and the backward reactions, CPTo showed greater capacity for the forward reaction and CPTi for the backward reaction. The easily solubilizable CPT, released on sonication of mitoplasts or of intact mitochondria under hypo-osmotic conditions, resembled CPTi in its properties. When octyl glucoside was used under appropriate conditions, 40-50% of the CPTo of outer membranes became solubilized, but it showed limited stability and decreased malonyl-CoA sensitivity. Malonyl-CoA-inhibitability of CPTo was decreased also on exposure of outer membranes to phospholipase C. When outer membranes that had been exposed to octyl glucoside or to phospholipase C were subjected to a reconstitution procedure using asolectin liposomes, the malonyl-CoA-inhibitability of CPTo was restored. A role of phospholipids in the malonyl-CoA sensitivity of CPTo is thus indicated.     

Glycolipid anchors are attached to Thy-1 glycoprotein rapidly after translation.

The attachment of glycolipid anchors to the Thy-1 glycoprotein during biosynthesis was followed by the change of detergent-binding properties of biosynthetically labelled Thy-1 precursors upon phospholipase C treatment in the murine thymoma lines BW5147 and S1A. In S1A, 80% of the Thy-1 molecules were phospholipase-C-sensitive after a 2 min pulse with [35S]methionine, indicating that these molecules were already anchored via a glycolipid tail. In BW5147, 47% of the Thy-1 molecules had phospholipase-C-sensitive anchors attached after a 1.5 min labelling and, with longer pulses, this percentage rose to 76%. Tunicamycin did not block the addition of glycolipid anchors, and glycolipid attachment also occurred at 21 degrees C. The findings suggest that the attachment of glycolipid anchors occurs in the rough endoplasmic reticulum.     

Quantitative analysis of the cytosolic-free-Ca2+-dependency of aldosterone production in bovine adrenal glomerulosa cells. Different requirements for angiotensin II and K+.

Angiotensin II (AII) and K+ raise the cytosolic free Ca2+ concentration [( Ca2+]i) and stimulate aldosterone production in isolated bovine adrenal glomerulosa cells. The mechanisms leading to an elevation of [Ca2+]i were analysed with the fluorescent Ca2+ probe quin 2. (1) Whereas [Ca2+]i rose transiently and returned to basal values within 5 min in response to AII, the effect of K+ was sustained for at least 15 min. (2) AII released Ca2+ from intracellular stores, whereas the [Ca2+]i response to K+ depended solely on extracellular [Ca2+]. (3) When added after K+ stimulation, AII provoked a dramatic decrease in [Ca2+]i to below the resting value. The role of [Ca2+]i in stimulating steroidogenesis was determined by manipulating the concentration of this cation. (4) In a cell superfusion system, the aldosterone response to AII is biphasic. Suppressing the transient [Ca2+]i elevation triggered by AII resulted in the disappearance of the initial secretory peak, but the final production rate was similar to that of control cells. (5) Normal basal [Ca2+]i levels were, however, necessary to maintain continuous AII-induced steroidogenesis. (6) When added after AII, the antagonist analogue [Sar1,Ala8]AII suppressed steroidogenesis without affecting [Ca2+]i levels. (7) In contrast, continuously elevated [Ca2+]i values were required for the initiation and the maintenance of K+-stimulated aldosterone production. These results demonstrate important differences in the mechanisms through which AII and K+ activate the Ca2+ messenger system. Moreover, functional correlations have shown that K+, but not AII, depends solely on a sustained [Ca2+]i response for its steroidogenic effect. However, the AII-induced effect is also a Ca2+-requiring process: the initial [Ca2+]i transient accelerates the onset of steroidogenesis, which is subsequently extremely sensitive to [Ca2+]i decreases below normal basal levels.     

The derivation and interpretation of control coefficients.

1. Equations for control coefficients are derived by using a method that generates all the control coefficients for a system in a single procedure. This requires solving fewer simultaneous equations than an equivalent method based on 'control theorems'. 2. The interpretation of control coefficients is discussed: in particular, it is shown that these functions are unsatisfactory as measures of 'control' and are perhaps best used as a means of testing control theories (models).     

Fluoroaluminates mimic guanosine 5''-[gamma-thio]triphosphate in activating the polyphosphoinositide phosphodiesterase of hepatocyte membranes. Role for the guanine nucleotide regulatory protein Gp in signal transduction.

Fluoride and guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) both activate the hepatocyte membrane polyphosphoinositide phosphodiesterase (PPI-pde) in a concentration-dependent manner. AlCl3 enhances the fluoride effect, supporting the concept that [A1F4]- is the active species. Analysis of the products of inositol lipid hydrolysis demonstrate that phosphatidylinositol bisphosphate is the major lipid to be hydrolysed. Guanosine 5'-[beta-thio]diphosphate (GDP beta S) is an inhibitor of activation of PPI-pde by both fluoride and GTP gamma S. These observations suggest that the guanine nucleotide regulatory protein (termed Gp) bears a structural resemblance to the well-characterized G-proteins of the adenylate cyclase system and the cyclic GMP phosphodiesterase system in phototransduction.     

Differential control of proto-oncogene c-myc and c-fos expression in lymphocytes and fibroblasts.

In lymphocytes stimulated with the mitogen phytohaemagglutinin, an inhibitor of the enzyme ADP-ribosyltransferase (ADPRT) completely blocks the proliferative response and the increase in expression of the proto-oncogene c-myc without affecting c-fos significantly. Conversely, in fibroblasts the serum-induced growth is not affected by the ADPRT inhibitor, and both oncogenes are dramatically super-induced. Hence there are differences between lymphocyte and fibroblast early responses to mitogenic stimulation and also between regulation of c-fos and c-myc gene expression.     

Re-evaluation of the structural integrity of red-cell glycoproteins during aging in vivo and nutrient deprivation.

Results presented in this paper show that removal of white-cell contaminations from human red blood cells by filtration through cellulose [Beutler, West & Blume (1976) J. Lab. Clin. Med. 88, 328-333] is a necessity whenever red cells are incubated at elevated temperatures or haemolysed after density separation. Omission of this precaution results in proteolysis of sialoglycoproteins in membranes from less-dense (young), but not dense (old), subpopulations. This proteolytic damage occurs during haemolysis of the cytoplasmic domain of glycophorin. A different type of proteolysis occurs if white-cell-contaminated red cells are incubated in the absence of glucose at elevated temperatures. Red cells release sialoglycopeptides. This process is stimulated by Ca2+ ions and is accompanied by the release of vesicles that differ from spectrin-free vesicles [Lutz, Liu & Palek (1977) J. Cell Biol. 73, 548-560]. This sialoglycopeptide release is dependent on white-cell contamination and is not required for the release of spectrin-free vesicles.     

Glucose metabolism in isolated brown adipocytes under beta-adrenergic stimulation. Quantitative contribution of glucose to total thermogenesis.

To quantify the potential of brown adipose tissue as a target organ for glucose oxidation, O2 consumption and glucose metabolism in isolated rat brown adipocytes were measured in the presence and absence of insulin, by using the beta-agonists isoprenaline or Ro 16-8714 to stimulate thermogenesis. Basal metabolic rate (278 mumol of O2/h per g of lipid) was maximally stimulated with isoprenaline (20 nm) and Ro 16-8714 (20 microM) to 1633 and 1024 mumol of O2/h per g respectively, whereas insulin had no effect on O2 consumption. Total glucose uptake, derived from the sum of [U-14C]glucose incorporation into CO2 and total lipids and lactate release, was enhanced with insulin. Isoprenaline and Ro 16-8714 had no effect on insulin-induced glucose uptake, but promoted glucose oxidation while inhibiting insulin-dependent lipogenesis and lactate production. A maximal value for glucose oxidation was obtained under the combined action of Ro 16-8714 and insulin, which corresponded to an equivalent of 165 mumol of O2/h per g of lipid. This makes it clear that glucose is a minor substrate for isolated brown adipocytes, fuelling thermogenesis by a maximum of 16%.     

Dexamethasone modulates the metabolism of type IV collagen and fibronectin in human basement-membrane-forming fibrosarcoma (HT-1080) cells.

The effect of dexamethasone on the synthesis and degradation of type IV collagen was studied in human fibrosarcoma cells, HT-1080. A dexamethasone concentration as low as 0.1 microM markedly increased collagen synthesis in HT-1080 cells labelled with [14C]proline. The increase in type IV collagen synthesis was not specific, since total protein synthesis was also increased. Further studies indicated that part of the increase was due to an increase in the specific radioactivity of the intracellular proline pool, after dexamethasone treatment. In fact, with dexamethasone concentrations of 0.1-10 microM the relative collagen synthesis was decreased, indicating that synthesis of other protein was increased more than that of type IV collagen. This was also confirmed by measuring the relative amount of type IV collagen RNA by using recombinant plasmid cDNA specific for the human procollagen pro alpha l (IV) RNA. The results indicated that relative collagen synthesis and the relative amount of type IV collagen messenger RNA was decreased similarly, indicating that dexamethasone affected type IV collagen synthesis at the pre-translational level. The dexamethasone-induced effect on total protein and collagen synthesis was maximal after 12-24 h. Dexamethasone induced a marked accumulation of collagen into the cell layer, leading to diminished deposition of soluble collagen into the medium. Since bacterial-collagenase treatment of the cell layer drastically decreased the collagen content of the dexamethasone-treated cells, this indicates that dexamethasone caused an accumulation of collagen into the extracellular matrix of the cell layer. In contrast, the amount of fibronectin was markedly increased in the medium. Dexamethasone decreased the type IV collagen-degrading activity in HT-1080 cells. The HT-1080 cells contained glucocorticoid receptors, as demonstrated by two different methods: by a whole-cell binding assay and by using a cytosol-gel-filtration method. The number of specific binding sites was similar to that in human skin fibroblasts. In conclusion, glucocorticoids affect the metabolism of type IV collagen and fibronectin in HT-1080 cells, and, since these cells contain specific glucocorticoid receptors, the effects are apparently receptor-mediated.