Lennart A. Ransnäs

Isoproterenol-induced subcellular redistribution of G-protein β subunits in S49 lymphoma cells demonstrated by a novel competitive ELISA

A novel competitive ELISA has been developed for the determination of levels of the beta subunit of guanine-nucleotide-binding protein (G-protein) using antipeptide antibodies directed against the amino terminus of the beta subunit. Because beta subunits form highly hydrophobic.heterodimeric complexes with gamma subunits of G-proteins, specific assay conditions were required. Optimal concentrations of antibodies, detergents, Mg2+ as well as ionic strength were determined. In addition, we found that an effective binding of the used antibodies to the beta subunit was ensured only after denaturation of the beta gamma complexes. Subsequently, this ELISA was used for quantitation of the beta subunit in subcellular fractions of S49 lymphoma cells during isoproterenol-mediated desensitization of beta-adrenergic controlled transmembrane signalling system. A 10 min as well as 60 min treatment of the cells with isoproterenol (1 nmol/ml) resulted in a significant shift of G-protein beta subunits (presumably as beta gamma complexes) from the plasma membrane fractions to low-density microsomal fractions. No significant change was detected after the hormone action in the distribution of plasma membrane constitutive enzymes. In conclusion, the developed ELISA helped us to reveal that beta-adrenergic stimulation can induce redistribution of the beta gamma dimer from plasma membranes to low-density microsomes.

3-Methylglutaconic aciduria in two infants

We studied two children who developed normally for the first 3-4 months of life and then displayed a failure-to-thrive syndrome, regression in psychomotor development, pronounced muscular hypotonia, and liver damage. At the age of about 1-2 years, optic atrophy and spastic parapareses were evident. One child died at the age of 2.5 years the other at an age of 4 years. Both children excreted 3-methylglutaconic acid, 0.1-0.4 mol/mol creatinine and 3-methylglutaric acid, 0.02-0.05 mol/mol creatinine. The excretion of 3-hydroxy-3-methylglutaric acid was not increased. One of the children was available for further biochemical studies. The activity of hydroxymethylglutaryl-CoA lyase (EC 4.1.3.4) was moderately reduced in leucocytes and fibroblasts. During a 21-h fast there was a normal formation of ketone bodies and we conclude that the cause of the syndrome is not a deficiency of hydroxymethylglutaryl-CoA lyase. Normal formation of 14CO2 from [1-14C]isovaleric acid and [2-14C]leucine in fibroblasts and leucocytes apparently excludes a deficiency of methylglutaconyl CoA-hydratase (EC 4.2.1.18).

Effects of malnutrition on rat myocardial beta-adrenergic and muscarinic receptors.

Malnutrition, as well as malignancy, induces alterations in heart metabolism and performance. Previous studies have implicated adrenergic mechanisms as the cause. The present study was undertaken to investigate if the adenylate cyclase system in the rat heart was affected by malnutrition. Three different animal groups with malnutrition were compared with a control group: rats with acute starvation for 14-percent; hours, rats with protein-calorie malnutrition for 2 weeks, and rats with tumors. Stimulation by β-adrenergic receptors and inhibition by muscarinic receptors of adenylate cyclase activity were not altered by malnutrition. However, conditions used for in vitro adenylate cyclase determinations were, of necessity, not physiological. Neither did the number of β-adrenergic and muscarinic receptors change. When competition-binding experiments were performed, differences comprising agonist affinity and affinity state distribution were noted among the groups. The myocardial β-adrenergic receptors formed a reduced number of high-affinity sites in all groups as compared with the control rats. All high-affinity sites displayed a more than 10-fold increase in affinity toward isoproterenol and an impaired sensitivity to guanine nucleotides except in heart membranes derived from rats starved less than 48 hours. While the protein-calorie restricted and the tumor-bearing rats had myocardial β-adrenergic receptors that were unresponsive to guanine nucleotides, after 48 hours of starvation the rats exhibited an attenuated guanine-nucleotide-induced affinity shift. No changes associated with malnutrition in myocardial membrane levels of the stimulatory guanine-nucleotide-binding protein were detected by cholera-toxin-induced ADP-ribosylation. In competition binding between quinudidinyl benzilate and carbachol, myocardial muscarinic receptors derived from malnourished rats exhibited a three-site affinity distribution whereas control rats displayed a two-site affinity distribution. The observations on myocardial /3- adrenergic and muscarinic receptors in malnourished rats are consistent with alterations in receptor and regulatory guanine-nucleotide-binding protein interaction. Our results point to the possibility of regulating cell functions by modifying coupling mechanisms. In addition, β-adrenergic receptors displayed a considerably increased affinity towards isoproterenol, which fact may contribute to the earlier findings of β-adrenergic hypersensitivity in malnutrition.

Muscarinic receptors in mammalian myocardium: effects of atrial and ventricular receptors on phosphatidylinositol metabolism and adenylate cyclase

Cat myocardium was used to investigate muscarinic receptor function in atria and ventricles. Carbachol and oxotremorine were used to determine agonist binding to the muscarinic receptors. It was found that carbachol was bound with almost the same characteristics in atria (KdH 1 microM; KdL 150 microM) as in ventricles (KdH 3 microM; KdL 150 microM). However, in the presence of guanylylimidodiphosphate Gpp(NH)p a difference was apparent so that the ventricular curve was shifted to a majority of low affinity sites whereas in atria two affinity sites remained even if the guanine nucleotide concentration was increased to 10 mM. Oxotremorine was bound with almost equal affinity in both atria and ventricles. The addition of Gpp(NH)p left all the receptors in the low affinity state irrespective of receptor localisation. The two agonists were also used to determine inhibition of adenylate cyclase activity. It was shown that the magnitude of adenylate cyclase inhibition was more pronounced in ventricles than in atria whether it was induced by oxotremorine or carbachol. When the effects of muscarinic agonists were determined on phosphatidylinositol metabolism it was shown that carbachol mediated a greater effect in atria than in ventricles. Almost no effect was seen with oxotremorine on phosphatidylinositol breakdown. Pirenzipine binding showed the presence of M1 receptors both in atria and ventricles. On the basis of diversity of muscarinic agonists on function and receptor occupancy it is suggested that heterogeneity exists for muscarinic receptors in both atria and ventricles.

Prolonged exposure of hamsters to cold changes the levels of G proteins in brown adipose tissue plasma membranes.

The levels of G proteins in plasma membranes prepared from brown adipose tissue of control and cold-exposed hamsters were determined by quantitative immunoblotting and competitive ELISA. Prolonged (four weeks) exposure of hamsters to cold decreased significantly the total content of the alpha subunits of the stimulatory (Gs alpha) as well as inhibitory (Gi alpha (1,2)) G proteins. Interestingly, the reduction in the Gs alpha content was solely due to a large reduction in the content of the short (45 kDa) isoform of Gs alpha, while the level of the long (52 kDa) isoform of Gs alpha remained unchanged. The level of the beta subunit of G protein was decreased comparably to the reduction in the total content of the alpha subunits. Cold-induced alterations in the G protein network associated with plasma membranes of brown adipose tissue were accompanied by changed characteristics of AlF(4-)-sensitive adenylyl cyclase activity.

Glucagon increases cellular uptake and plasma membrane binding of L-carnitine in S49 lymphoma cells

The carnitine carrier was investigated in S49 lymphoma cells, a murine cell type cultured in suspension culture and used widely in signal transduction studies. Carnitine uptake in S49 lymphoma cells was stimulated almost twofold by pretreatment of intact cells by 0.5 microM glucagon for 4 h. Plasma membranes derived from S49 lymphoma cells bound 556 +/- 81 pmol/mg protein whereas pretreatment by 0.5 microM glucagon for 4 h of cells, before cell harvesting and preparation of plasma membranes, increased the number of carnitine binding sites to 1196 +/- 52 pmol/mg protein. The glucagon pretreatment also altered the carnitine binding characteristics from a two site model to a single binding site. S49 lymphoma cells were further shown to contain 50.9 +/- 2.6 fmol glucagon receptors per 10(6) cells. We conclude that glucagon stimulated cellular uptake of carnitine by a mechanism that at least partially operated through increasing the number of available carnitine binding sites in plasma membranes.

Gsalpha-mediated regulation of the carnitine carrier in S49 lymphoma cells.

Carnitine is essential for mitochondrial oxidation of long-chain fatty acids. Peripheral cells rely on plasma transport of carnitine which is taken up by an active mechanism in the plasma membrane. This project investigated the plasma membrane bound carnitine carrier in cultured S49 lymphoma cells. We investigated wild-type cells and two mutant cells lines showing deficient activity of adenylyl cyclase, cyc- lacking and H21a containing a deficient Gsalpha. Plasma membranes derived from cyc- cells displayed six times more carnitine binding sites and a 1.35 times faster uptake rate than plasma membranes from wild-type cells. In vitro mixing of plasma membranes from cyc- and wild-type cells transferred a factor reducing the number of expected carnitine binding sites by about 30%. Cyclic AMP could not substitute for wild-type membranes as the inhibitor of carnitine binding to plasma membranes derived from cyc- cells. Cholera toxin induced ADP-ribosylation of Gsalpha causing activation of Gsalpha present in wild-type but not in cyc- cells, further reducing carnitine uptake and carnitine binding to plasma membranes. Our findings thus supported the notion that Gsalpha by a mechanism not involving cyclic AMP inhibited cellular uptake of carnitine by reducing the number of available carnitine binding sites in plasma membranes.

Activated Gsα but not Giα prevents the thermal inactivation of adenylyl cyclase in plasma membranes derived from S49 lymphoma cells

The thermal inactivation of adenylyl cyclase was studied in plasma membranes isolated from wild‐type and the mutant cell strain cyc− of S49 lymphoma. The half‐life of adenylyl cyclase activity at 30°C was decreased from 14.2 min to 3.4 min by the presence of detergents. ATP as well as forskolin prevented the adenylyl cyclase inactivation in a dose‐response manner independent of the utilized type of cell membranes. Activation of G‐proteins by GTPγS or by AlF− 4 in wild‐type membranes but not in cyc− membranes partially prevented adenylyl cyclase inactivation. Adenylyl cyclase activity in cyc− membranes was preserved in the presence of GTPγS or AlF4 from the observed detergent‐induced inactivation by complementation of these membranes with an extract from wild‐type membranes. ADP‐ribosylation of Giα in a cyc− membranes did not influence the kinetics of the inactivation process of adenylyl cyclase, whereas ADP‐ribosylated Gsα, protein protected adenylyl cyclase more effectively than non‐ribosylated Gsα in wild‐type plasma membranes when GTP was used as an activator.

Coxsackievirus B3 entry into the host cell interferes with G-protein-mediated transmembrane signalling

In the present work we used various cell lines in order to study the possible effect of coxsackievirus B3 (CVB3) entry on the adenylyl cyclase transmembrane signalling system. A significant decrease (by about 10–20%) was found in forskolin-augmented as well as in AlF4−- and GTPγS-sensitive adenylyl cyclase activity in plasma membranes isolated from HeLa, HEp-2, Vero and green monkey kidney cells shortly (up to 60 min) preincubated with CVB3 (5 PFU/cell). Moreover, the ability of G-proteins derived from plasma membranes of infected cells to reconstitute AC activity in the cyc− mutant of S49 cells was also reduced. Content of G-protein subunits, however, remained unchanged after CVB3 attachment. Functional alterations in the G-protein-mediated adenylyl cyclase signalling system were accompanied by a marked decrease (by about 20–40%) of intracellular cAMP levels in virus-affected cells. These findings demonstrate clearly that CVB3 may affect functioning of the G-protein regulated adenylyl cyclase transmembrane signalling system in virus-sensitive cells as early as during the first period of its contact with the cellular plasma membrane.