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Biomedical subjects

M J Wakelam

Publications and source records attributed to M J Wakelam.

At least 127 records · Page 7Linked to original sources
PubMed1987

L6 skeletal muscle cells have functional V1-vasopressin receptors coupled to stimulated inositol phospholipid metabolism.

The effects of vasopressin and related peptides upon the rat skeletal muscle cell line, L6, have been examined. No effects upon cellular cyclic AMP levels were found indicating that L6 cells possess no functional V2-vasopressin receptors. Vasopressin and its analogues did, however, stimulate the rapid and dose-dependent accumulation of inositol phosphates. This effect and the rank order of potency of vasopressin analogues demonstrate the presence of functional V1-vasopressin receptors upon L6 cells. These results suggest that the L6 line may be a useful model for vasopressin effects upon skeletal muscle metabolism.

Animals↗
PubMed1984

Prostaglandins and cyclic AMP stimulate creatine kinase synthesis but not fusion in cultured embryonic chick muscle cells.

Cyclic AMP levels have been measured in cultures derived from 12-day-old chick embryonic muscle. A rise in concentration was found after the onset of myoblast fusion. Cells cultured at a medium Ca2+ concentration of 0.1 microM did not fuse and exhibited only a small rise in cyclic AMP concentration during culture. Addition of 1.4 mM Ca2+ to these cells after 50 h in culture caused rapid, synchronous fusion with a concomitant rise in cyclic AMP levels. Indomethacin, an inhibitor of prostaglandin synthesis, did not inhibit fusion, but inhibited the rise in cyclic AMP concentration. Indomethacin-treated cultures exhibited lower creatine kinase levels, though no change in the ratio of the three isoenzymes was observed. Addition of prostaglandins E1 and E2 to indomethacin-treated cultures overcame this inhibition. We propose that prostaglandin synthesis is a consequence of the stimulation of myoblast fusion and that via cyclic AMP it stimulates protein synthesis.

Alprostadil↗
PubMed1984

Myoblast fusion and inositol phospholipid breakdown: causal relationship or coincidence?

The fusion of embryonic chick myoblasts has been examined in culture. Cells were prepared from 12-day-old chick embryonic breast muscle and cultured for 50 h at a Ca2+ concentration in the medium of 10(-7) M. During this period they attain fusion competence. Addition of 1.4 mM-Ca2+ to these cells elicits rapid fusion. Changes in the metabolism of myoblast phospholipids in response to the raised Ca2+ concentration have been examined. Only the inositol phospholipids are affected. Phosphatidylinositol, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate are rapidly broken down and 1,2-diacylglycerol and phosphatidic acid are synthesized. Myoblast fusion has also been found to be stimulated by a factor present in chick embryo extract, probably of neuronal origin. A receptor-mediated mechanism for myoblast fusion is proposed. This envisages the polyphosphoinositides acting as a fusion block, either themselves or by their binding to membrane proteins. The inositol phospholipid breakdown could result in a more fluid membrane and the breakdown products 1,2-diacylglycerol and phosphatidic acid, two known fusogens, could stimulate fusion.

Animals↗
PubMed1983

Inositol phospholipid metabolism and myoblast fusion.

The fusion of chick embryonic myoblasts has been studied in tissue culture. Myoblasts are maintained at 0.1 microM-Ca2+ for 50 h. During this time they achieve fusion competence. Fusion is initiated by raising the medium Ca2+ concentration to 1.4 mM. A rapid breakdown of the polyphosphoinositides was detected within 3 min of Ca2+ addition. Rapid synthesis of phosphatidic acid was also detected at this time. Breakdown of phosphatidylinositol and synthesis of 1,2-diacylglycerol were also detected. Other phospholipids were unaffected. Sr2+ could replace Ca2+ in this process but Mg2+ could not and also inhibited the Ca2+ effect. The Ca2+-ionophore A23187 stimulated further apparent polyphosphoinositide breakdown in the presence of Ca2+. 6. The results are discussed with respect to myoblast fusion.

Animals↗
PubMed1982

The control of glucose 1,6-bisphosphatase by Ca2+ and calmodulin.

1. The control of glucose 1,6-bisphosphatase activity, the enzyme that degrades glucose 1,6-bisphosphate, a metabolite that regulates hexose phosphate metabolism, has been examined in a rat muscle extract. 2. The enzyme has been found to be activated by physiological Ca2+ concentrations. Ca2+ can be replaced by Sr2+ and Mn2+, but the effects are not so pronounced. 3. The Ca2+ effect is inhibited by EGTA. Trifluoperazine also inhibits enzyme activity. This effect can be reversed by adding exogenous calmodulin.

Animals↗
PubMed1982

The control of glucose 1,6-bisphosphate by developmental state and hormonal stimulation in cultured muscle tissue.

1. The concentration of glucose 1,6-bisphosphate, a potent regulator of muscle glucose metabolism, was examined in embryonic muscle cells in culture. 2. The concentration in fused myotubes was twice that in unfused myoblasts. 3. The effect of various hormones and agonists on the glucose 1,6-bisphosphate concentration in both pre- and post-fusion muscle cells was examined. In pre-fusion cells no effect of adrenaline or cyclic AMP was observed, but stimulation by vasopressin, adrenaline + propranolol, ionophore A23187 and dibutyryl cyclic GMP significantly decreased glucose 1,6-bisphosphate. In post-fusion cells similar effects were observed, except that stimulation by adrenaline and by dibutyryl cyclic AMP significantly increased metabolite concentration. 4. All effects increased with time (over a 1 h period), except for that of vasopressin, which was transient. 5. The changes in glucose 1,6-bisphosphate concentration were accompanied by changes in the fructose 1,6-bisphosphate/fructose 6-phosphate ratio, implying an effect on phosphofructokinase activity.

Animals↗
PubMed1982

The breakdown of phosphatidylinositol in myoblasts stimulated to fuse by the addition of Ca2+.

1. The fusion of chick-embryo myoblasts to produce myotubes was studied. The myoblasts were grown for 50 h in medium containing 10--20 microM-Ca2+; during this period they achieve fusion competence. 2. A rapid breakdown of phosphatidylinositol is also observed on addition of 1.4 mM-Ca2+ to these cells. This Ca2+ concentration also stimulates rapid myoblast fusion. 3. The breakdown is complete within 15 min and shows the same dependence on Ca2+ concentration as the fusion process. 4. Fusion-incompetence myoblasts and cells where fusion is inhibited by sodium butyrate exhibit no phosphatidylinositol breakdown on Ca2+ addition. 5. The Ca2+ ionophore A23187 inhibits the Ca2+-stimulated breakdown by about 50%, but has no effect on fusion. 6. A concomitant increase in 1,2-diacylglycerol labelled and fall in phosphatidylinositol labelling was observed when the lipids were labelling with [14C]glycerol on increasing the Ca2+ concentration in the medium to 1.4 mM. 7. We propose that the breakdown of phosphatidylinositol with a resultant increase in 1,2-diacylglycerol content of the cell membrane promotes myoblast fusion.

Animals↗
PubMed1981

The separate roles of glucose and insulin in the induction of glucokinase in hepatocytes isolated from neonatal rats.

1. The specificity of the effect of glucose on the induction of glucokinase activity that occurs when hepatocytes freshly isolated from 13-day-old rats are incubated in Medium 199 together with insulin [Wakelam & Walker (1980) FEBS Lett. 111, 115-119] was examined. A pattern that is different from other known effects of glucose is found, and metabolism of this compound is not necessarily to account for this particular effect. 2. The effects of a raised glucose concentration and of insulin on the induction can be separated. The hexose initiates the process in the absence of insulin in a manner that is sensitive to actinomycin D but not to cycloheximide. The subsequent effect of insulin is dependent on the prior effect of glucose or other positive analogue, does not require the presence of glucose and is inhibited by cycloheximide but not by actinomycin D. 3. Induction of glucokinase in vitro in hepatocytes from neonatal animals is inhibited by adrenaline, glucagon and dibutyryl cyclic AMP, but not by vasopressin or angiotensin II. The inhibition by cyclic AMP is on the stage requiring insulin and is comparatively specific, because total protein synthesis is not apparently diminished. 4. The implications of these results are discussed with reference to possible mechanisms of induction and to the situation in vivo.

Animals↗
PubMed1979

Thyroid hormones and the precocious induction of hepatic glucokinase in the neonatal rat.

1. Oral intubation of glucose is more effective than intraperitoneal injection in inducing the premature appearance of hepatic glucokinase in suckling rats. 2. The inducing effect of glucose is enhanced by treatment of the animals 12 h or more earlier with 1 microgram triiodothyronine/g body weight. 3. Low but significant activities of glucokinase appear at the normal time of development in hypothyroid neonatal rats. Intubation of glucose into 13-day-old and 24-day-old hypothyroid results in the rapid appearance of glucokinase similar to that in normal animals treated likewise. 4. The enhancing effect of thyroid hormones on glucokinase induction by glucose does not necessarily mean that the normal postnatal increase in plasma thyroid hormones is essential for the normal appearance of glucokinase activity at the time of weaning. Other possible explanations are discussed.

Animals↗
PubMedDate not supplied

Transmitter-like action of ATP on patched membranes of cultured myoblasts and myotubes.

The concept of purinergic neurotransmission, first proposed by Burnstock, has been confirmed in various cell types. We show here, by the patch-clamp method, that external ATP in micromolar concentrations (1-100 microM) activates cation channels in the membranes of fusion-competent myoblasts and myotubes. In cell-attached membrane patches of myoblasts and myotubes the mean number of simultaneously activated channels increases with time after external ATP application. In myoblasts only one population of channels having a mean single-channel conductance of gamma=43 pS was found, while in myotubes two populations with gamma 1=48 pS and gamma 2=20 pS were observed. Treatment of myotube membranes with acetylcholine (ACh) or carbachol resulted in two populations of channels which had conductance values and voltage-dependent mean channel lifetimes similar to those produced in response to ATP. The results show that embryonic skeletal muscle cells contain cation channels sensitive to ATP and provide evidence for a neurotransmitter-like action of ATP on these cells.

Adenosine Triphosphate↗
PubMedDate not supplied

Activation of two signal-transduction systems in hepatocytes by glucagon.

The ability of glucagon to stimulate glycogen breakdown in liver played a key part in the classic identification of cyclic AMP and hormonally stimulated adenylate cyclase. But several observations indicate that glucagon can exert effects independent of elevating intracellular cAMP concentrations. These effects are probably mediated by an elevation of the intracellular concentration of free Ca2+ although the mechanism by which this occurs is unknown. We show here that glucagon, at the low concentrations found physiologically, causes both a breakdown of inositol phospholipids and the production of inositol phosphates. Indeed, we show that the glucagon analogue, (1-N-alpha-trinitrophenylhistidine,12-homoarginine)glucagon (TH-glucagon), which does not activate adenylate cyclase or cause any increase in cAMP in hepatocytes yet can fully stimulate glycogenolysis, gluconeogenesis and urea synthesis, stimulates the production of inositol phosphates. This stimulation of inositol phospholipid metabolism by low concentrations of glucagon provides a mechanism whereby glucagon can exert cAMP-independent actions on target cells. We suggest that hepatocytes possess two distinct receptors for glucagon, a GR-1 receptor coupled to stimulate inositol phospholipid breakdown and a GR-2 receptor coupled to stimulate adenylate cyclase activity.

Adenylyl Cyclases↗