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Biomedical subjects

S W Walker

Publications and source records attributed to S W Walker.

66 records · Page 4Linked to original sources
PubMed1984

Calmodulin activation of cyclic AMP phosphodiesterase in the B16 mouse melanoma.

Mouse B16 melanoma extracts of both cultured cells and tumour tissue contain cyclic AMP phosphodiesterase activity, with 95% present in the soluble fraction. Although activation of the enzyme by added calmodulin did not occur, it was found that endogenous calmodulin was present at a level sufficient to activate fully the enzyme. The ability of Ca-calmodulin to stimulate cyclic AMP phosphodiesterase in this tissue was shown by the inhibitory effect of N-(6-aminohexyl)-5-chloronaphthalenesulphonamide (W7), a known calmodulin antagonist; by the activation of the enzyme with exogenous calmodulin observed in supernatants depleted of endogenous calmodulin by passage over fluphenazine-Sepharose 6B in the presence of Ca2+; by the Ca-dependent binding of the enzyme to calmodulin-agarose and its activation by Ca-calmodulin after elution from the column with EGTA-containing buffer. It was calculated that about 50% of the total cyclic AMP phosphodiesterase activity was calmodulin-activated in this tissue.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1984

Isolation, purification and cell-free synthesis of calmodulin from the pig anterior pituitary gland.

Calmodulin was extracted and purified from pig anterior pituitary gland. The protein was characterized by its migration on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis in the presence of Ca2+ or EGTA, its U.V. spectrum between 240 and 290 nm and the activation of calmodulin-deficient cyclic AMP phosphodiesterase. The yield was 370 mg/kg wet wt. mRNA was also extracted from the same tissue and translated in a wheat-germ cell-free translation system. Translated calmodulin was identified by its heat-stability, its co-migration with authentic anterior-pituitary calmodulin on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, its acidic isoelectric point (4.15) on flat-bed isoelectric focusing, its Ca2+-dependent binding to fluphenazine-Sepharose 6B, and its co-elution from this gel with authentic unlabelled calmodulin with EGTA buffer. Calmodulin was not translated as a precursor form. In this tissue it was calculated that calmodulin accounted for 0.5-1% of the total translated protein.

Animals↗
PubMed1984

Calmodulin in human serum and the specific release of calmodulin from calmodulin-rich platelets.

Calmodulin (CaM)-like activity was detected in human serum and foetal calf serum, with an activity 10 times more than that detectable in plasma. Serum CaM was largely accounted for by release from human platelets as confirmed by both radioimmunoassay and sodium-dodecyl-sulphate/polyacrylamide-gel electrophoresis of CaM partially purified from platelet releasate obtained in response to thrombin. Lactate dehydrogenase release was unaffected by thrombin. Platelet CaM content was very variable (1.3 to 11.3 micrograms/mg protein; n = 15). It is suggested that intact platelets are rich in CaM and that release of CaM during preparation explains the variation in CaM content reported here and in the literature.

Adult↗
PubMed1984

Effects of extracellular calmodulin and calmodulin antagonists on B16 melanoma cell growth.

Two drugs known to inhibit the action of calmodulin, prochlorperazine offP) and N-(6-aminohexyl)-5-chloro-1-napthalene sulfonamide (W7), were investigated for their ability to control cell proliferation in murine B16 melanoma cells in culture. PCP and W7 inhibited [3H]thymidine uptake in these cells, 50% inhibition occurring with 13 microM PCP and 40 microM W7. In the presence of relatively high concentrations of fetal calf serum (FCS), cells withstood high concentrations of both drugs (100 microM PCP and 200 microM W7) and showed increased pigment production. Drug-inhibited DNA synthesis could be reversed by the addition of fresh medium containing FCS or by the addition of exogenous pure calmodulin. Extracellular calmodulin itself stimulated DNA synthesis. FCS was found to contain calmodulin-like activity at concentrations that may be relevant to the stimulation of [3H]thymidine uptake by cells in culture.

Animals↗
PubMed1983

A possible role for calmodulin in human thyroid cell metabolism.

A protein which shared several characteristics with authentic calmodulin was extracted from human thyroid homogenates. The protein bound to fluphenazine--Sepharose and could be specifically eluted using EGTA. The eluted protein had a u.v. spectrum characteristic of calmodulin and migrated like authentic calmodulin with a calcium-dependent shift on sodium dodecyl sulphate polyacrylamide-gel electrophoresis. Calmodulin in thyroid cell extracts was shown to be biologically active, measured by its ability to activate a calmodulin-deficient cyclic GMP phosphodiesterase; this activation could be inhibited by trifluoperazine. A possible role for calmodulin in the action of TSH on the thyroid was demonstrated by studying the effects of phenothiazines and the naphthalene sulphonamide, W7, a more specific calmodulin inhibitor, on TSH-stimulated cyclic AMP levels in cultured thyroid cells. The phenothiazines and W7 were found to inhibit the accumulation of cyclic AMP in response to TSH in a concentration-dependent manner although low concentrations of W7 enhanced TSH-stimulated cyclic AMP accumulation.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMedDate not supplied

Acetylcholine induces oscillations in intracellular calcium in isolated bovine adrenal zona fasciculata/reticularis cells.

The effects of acetylcholine (ACh) on intracellular free calcium were studied in primary cultures of purified bovine adrenal zona fasciculata/reticularis (ZFR) cells. In fura-2 loaded single cells, concentrations of ACh which stimulated cortisol secretion and phosphoinositol production were found to promote an increase in free cytosolic calcium ([Ca2+]i). This response was heterogeneous, showing either (i) an initial increase in [Ca2+]i followed by a fall to a level above that in unstimulated cells, or (ii) an initial increase followed by oscillations about the original resting level or a higher resting level. The frequencies of [Ca2+]i oscillations to ACh showed a dose-dependent trend. The sustained [Ca2+]i oscillations were abolished by the muscarinic antagonist atropine, or by removal of extracellular Ca2+. These data demonstrate for the first time in adrenocortical cells that: (i) ACh can induce [Ca2+]i oscillations in single ZFR cells; (ii) these oscillations occur in a dose-dependent manner; (iii) the sustained oscillatory phase is dependent on influx of extracellular Ca2+. Thus, like cells of the zona glomerulosa, bovine ZFR cells are also capable of sustained dose-dependent oscillatory responses to agonists which activate phosphoinositidase C.

Acetylcholine↗
PubMedDate not supplied

The expression of steroidogenic acute regulatory protein (StAR) in bovine adrenocortical cells.

StAR protein may facilitate rapid transfer of cholesterol from the outer to the inner mitochondrial membrane, the site at which cholesterol is converted to pregnenolone by the cholesterol side chain cleavage complex. We have studied the effect of ACTH treatment on StAR mRNA and protein levels in bovine adrenocortical cells in primary culture. Cells were initially cultured for 3 days after isolation, and then treated with ACTH (10(-8) M) for various times up to 24 hours. Northern analysis of total BAC mRNA, using a [alpha32P]-labelled cDNA probe encoding a 5' region of bovine StAR mRNA, revealed two principal hybridising species of 1.6 and 3.0 kb. Western immunoblot analysis revealed a principal band at 30 kDa. Levels of both StAR mRNA and protein showed an increase at 1 hour, reached a maximum at around 6 hours and declined to basal levels at 24 hours. Cortisol secretion (measured by RIA) showed a similar change over the same period. From these results it appears that StAR mRNA and protein levels in BAC are acutely regulated in concert with ACTH-stimulated cortisol secretion.

Adrenal Cortex↗
PubMedDate not supplied

Dopaminergic stimulation of cortisol secretion from bovine zfr cells occurs through nonspecific stimulation of adrenergic beta-receptors.

Previous reports have suggested a possible dopaminergic inhibition of the actions of AII on aldosterone secretion via adenylate-cyclase inhibitory 'D2' receptors. Others suggest a possible stimulation of aldosterone secretion via a stimulatory 'D1' receptor/cAMP pathway. We have examined the actions of dopamine on basal and AII-stimulated cortisol secretion by cultured bovine zfr cells. Dopamine alone caused a dose-dependent increase in cortisol secretion at doses >10(-5) M, and also enhanced steroidogenic output in response to submaximal (10(-10) M) but not maximal (10(-8) M) stimulatory doses of AII. The stimulatory action of dopamine alone on cortisol secretion was not, however, reproduced by the 'D1' agonist fenoldopam, and was fully blocked by propranolol. Dopamine had neither a stimulatory effect on basal phosphoinositol production nor an inhibitory effect on AII-stimulated phosphoinositol production. Our findings are therefore inconsistent with the activation of a 'D1' or 'D2' class receptor, and suggest the stimulation of cortisol secretion occurred nonspecifically through a beta-adrenergic receptor.

Angiotensin II↗