Vasopressin increases 45Ca2+ influx in rat aortic smooth muscle cells

[Arg8]Vasopressin (AVP)-induced 45Ca2+ influx was examined in vascular smooth muscle cells derived from rat aorta. AVP stimulated the 45Ca2+ influx in a concentration-dependent manner. The effect was abolished in the presence of La3+. The dihydropyridine calcium channel antagonist darodipine did not affect the AVP-induced influx of 45Ca2+. These data suggest that AVP stimulates in these cultured aortic smooth muscle cells a receptor-operated channel (ROC) that is permeable to Ca2+.     

Platelet-activating factor mobilizes intracellular calcium in vascular smooth muscle cells

The effect of platelet-activating factor (PAF) on polyphosphoinositide metabolism and 45Ca2+ efflux was examined in a vascular smooth muscle cell line (A7r5). PAF stimulated a rapid but transient production of inositol trisphosphate and inositol bisphosphate which, in the presence of lithium, resulted in an accumulation of inositol monophosphate. In addition, PAF induced a rapid efflux of 45Ca2+ from preloaded cells, an effect which was concentration-dependent. These data suggest that PAF mobilizes intracellular Ca2+ via the production of inositol trisphosphate.     

A smooth muscle cell line suitable for the study of voltage sensitive calcium channels

A cell line originating from the fetal rat aorta has been studied with respect to 45Ca2+ uptake. Kinetic experiments showed an initial rapid uptake followed by a slow linear phase; both the initial rate and the maximum uptake were increased in the presence of 55 mM potassium chloride. The calcium channel antagonists, darodipine (PY 108-068) and verapamil, inhibited both the basal and the potassium chloride stimulated uptake. Neither tetrodotoxin nor furosemide affected either basal or depolarisation induced 45Ca2+ uptake. Blockade of the Na+/K+ ATPase by ouabain and of the Ca2+ ATPase by vanadate caused a net increase in cellular 45Ca2+ accumulation.     

Caldesmon-induced inhibition of ATPase activity of actomyosin and contraction of skinned fibres of chicken gizzard smooth muscle

Caldesmon induces inhibition of MG2+-ATPase activity of actomyosin and relaxation of skinned fibers of chicken gizzard smooth muscle without influencing the level of myosin light chain-1 phosphorylation. Both these effects are reversed by calmodulin at a high molar excess over caldesmon in the presence of Ca2+.     

A rapid and sensitive assay for arginase

An assay for arginase is described that uses l-[guanido-¹⁴C]arginine as substrate. Unhydrolyzed arginine is removed in a batch procedure with sulfonate resin and the [¹⁴C]urea product is determined quantitatively in the resin supernatant. The assay requires 5 min and is performed in one tube. The sensitivity is approximately 0.1 munits of arginase. Arginase activities in fetal calf serum and in murine macrophage extract have been determined and the bovine liver enzyme has been used as a reference.     

Thermodynamic functions of biopolymer hydration. II. Enthalpy–entropy compensation in hydrophilic hydration processes

The thermodynamic functions of biopolymer hydration were investigated by multitemperature vapor pressure studies. Desorption measurements were performed that allowed determination of reversible isotherms in the hydration range of 0.1 to 0.3–0.5 g H₂O/g dry polymer. These isotherms are accessible to thermodynamic interpretation and are relevant to the interaction of water with biopolymers in their solution conformation. The results obtained on a series of different biopolymers (lysozyme, α-chymotrypsin, apo-lactoferrin, and desoxyribonucleic acid), show the following common features of interest: (1) The differential excess enthalpies (ΔH^e ) and entropies (ΔS^e ) are negative, and exhibit pronounced anomalies in a well-defined low-humidity range (approx. 0.1 g H₂O/g dry polymer). These initial extrema are interpretable by structural changes, induced in the native biopolymer structures by water removal below a critical degree of hydration. (2) The ΔH^e and ΔS^e terms exhibit statistically significant linear enthalpy–entropy compensation effects in all biopolymer–water systems investigated. The compensation temperatures \documentclass{article}\pagestyle{empty}\begin{document}$ \hat \beta = \overline {\Delta H} ^e /\overline {\Delta S} ^e $\end{document} are approximately identical for all biopolymers, ranging from 360 to 500 K. The compensation effects are attributable to phase transitions of water molecules between the bulk liquid and the inner-sphere hydration shell of native biopolymers. (3) The negative excess free energies (ΔG^e ) decrease monotonically with increasing water content and are close to zero at 0.3 to 0.5 g H₂O/g polymer. This result indicates that only transitions between the bulk liquid and the inner-sphere hydration shell are associated with significant net free energy effects. The outer-sphere hydration water is thermodynamically comparable to bulk water. The importance of the proportionality factor \documentclass{article}\pagestyle{empty}\begin{document}$ \hat \beta $\end{document} in the control of the free energy term is discussed.     

Cofilin 1 is revealed as an inhibitor of glucocorticoid receptor by analysis of hormone-resistant cells

Significant knowledge about glucocorticoid signaling has accumulated, yet many aspects remain unknown. We aimed to discover novel factors involved in glucocorticoid receptor regulation that do not necessarily require direct receptor interaction. We achieved this by using a functional genetic screen: a stable cell line which cannot survive hormone treatment was engineered, randomly mutated, and selected in the presence of glucocorticoid. A hormone-resistant clone was analyzed by two-dimensional gel electrophoresis. Differentially expressed proteins were identified and tested as candidates for regulation of the glucocorticoid receptor. An unexpected candidate, cofilin 1, inhibited receptor activity. Cofilin is known to promote actin depolymerization and filament severing. Several experiments suggest that this feature of cofilin is involved in its inhibitory action. Both its actin depolymerization activity and its inhibitory action on the receptor are dependent on its phosphorylation state. Treatment of cells with a cytoskeleton-disrupting agent decreased receptor activity, as did overexpression of actin, particularly a mutant actin that does not polymerize. In addition, overexpression of cofilin and actin as well as chemical cytoskeleton disruption changed the subcellular receptor distribution and upregulated c-Jun, which could constitute the inhibitory mechanism of cofilin. In summary, cofilin represents a novel factor that can cause glucocorticoid resistance.     

Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive

The target of rapamycin (TOR) is a highly conserved protein kinase and a central controller of cell growth. In budding yeast, TOR is found in structurally and functionally distinct protein complexes: TORC1 and TORC2. A mammalian counterpart of TORC1 (mTORC1) has been described, but it is not known whether TORC2 is conserved in mammals. Here, we report that a mammalian counterpart of TORC2 (mTORC2) also exists. mTORC2 contains mTOR, mLST8 and mAVO3, but not raptor. Like yeast TORC2, mTORC2 is rapamycin insensitive and seems to function upstream of Rho GTPases to regulate the actin cytoskeleton. mTORC2 is not upstream of the mTORC1 effector S6K. Thus, two distinct TOR complexes constitute a primordial signalling network conserved in eukaryotic evolution to control the fundamental process of cell growth.