The major part of mast cell actin is Triton-soluble and behaves as a monomer in the DNase I inhibition assay. Thus, actin exists predominantly in monomeric or short filament form, though filamentous ...actin is clearly apparent in the cortical region after rhodamine-phalloidin (RP) staining. The minimum actin content is estimated to be ∼2.5 μ g/106 cells (cytosolic concentration ∼110 μM. After permeabilization of mast cells by the bacterial cytolysin streptolysin-O, ∼60% of the Triton-soluble actin leaks out within 10 min. However, the staining of the cortical region by RP remains undiminished, and the cells are still capable of exocytosis when stimulated by GTP-γ-S together with Ca2+. In the presence of cytochalasin E the requirement for Ca2+ is decreased, indicating that disassembly of the cytoskeleton may be a prerequisite for exocytosis. This disassembly is likely to be controlled by Ca2+-dependent actin regulatory proteins; their presence is indicated by a Ca2+-dependent inhibition of polymerization of extraneous pyrene-G-actin by a Triton extract of mast cells. The effect of cytochalasin E on secretion is similar to that of phorbol myristate acetate, an activator of protein kinase C; both agents enhance the apparent affinity for Ca2+ and cause variable extents of Ca2+-independent secretion. Exposing the permeabilized cells to increasing concentrations of Ca2+ caused a progressive decrease in F-actin levels as measured by flow cytometry of RP-stained cells. In this respect, both cytochalasin E and phorbol ester mimicked the effects of calcium. GTP-γ-S was not required for the Ca2+-dependent cortical disassembly. Thus, since conditions have not yet been identified where secretion can occur in its absence, cortical disassembly may be essential (though it is not sufficient) for exocytosis to occur.
Introducing non‐hydrolysable analogues of GTP into the cytosolic compartment of mast cells results in exocytotic secretion through the activation of GTP binding proteins. The identity and mechanism ...of action of these proteins are not established. We have investigated the effects of Rho GDP dissociation inhibitor (RhoGDI) on exocytosis induced by guanosine 5′‐O‐(3‐thiotriphosphate) (GTP‐gamma‐S) in rat mast cells, introducing the protein into cells by means of a patch pipette and recording the progress of exocytosis by monitoring cell capacitance. To allow time for the protein to enter the cells and find its correct location, stimulation was provided 5–10 min after patch rupture by photolysing caged GTP‐gamma‐S included in the pipette solution. When bovine RhoGDI was introduced into mast cells, exocytosis was inhibited at concentrations of 200–400 nM for native protein and 800 nM to 8 microM for the recombinant form. Protein denatured by heat or N‐ethylmaleimide treatment did not inhibit. In permeabilized cells, recombinant RhoGDI increased the rate at which cells lose their ability to respond to GTP‐gamma‐S. These data demonstrate that one or more small GTP binding proteins of the Rho family has a central role in the exocytotic mechanism in mast cells.
Widespread experience indicates that application of suboptimal concentrations of stimulating ligands (secretagogues) to secretory cells elicits submaximal extents of secretion. Similarly, for ...permeabilized secretory cells, the extent of secretion is related to the concentration of applied intracellular effectors. We investigated the relationship between the extent of secretion from mast cells (assessed as the release of hexosaminidase) and the degranulation (exocytosis) responses of individual cells. For permeabilized mast cells stimulated by the effector combination Ca2+ plus GTP-γ-S and for intact cells stimulated by the Ca2+ ionophore ionomycin, we found that exocytosis has the characteristics of an all-or-none process at the level of the individual cell. With a suboptimal stimulus, the population comprised only totally degranulated cells and fully replete cells. In contrast, a suboptimal concentration of compound 48/80 applied to intact cells induced a partial degree of degranulation. This was determined by observing the morphological changes accompanying degranulation by light and electron microscopy and also as a reduction in the intensity of light scattered at 90°, indicative of a change in the cell-refractive index. These results may be explained by the existence of a threshold sensitivity to the combined effectors that is set at the level of individual cells and not at the granule level. We used flow cytometry to establish the relationship between the extent of degranulation in individual rat peritoneal mast cells and the extent of secretion in the population (measured as the percentage release of total hexosaminidase). For comparison, secretion was also elicited by applying the Ca2+ ionophore ionomycin or compound 48/80 to intact cells. For permeabilized cells and also for intact cells stimulated with the ionophore, levels of stimulation that generate partial secretion gave rise to bimodal frequency distributions of 90° light scatter. In contrast, a partial stimulus to secretion by compound 48/80 resulted in a single population of partially degranulated cells, the degree of degranulation varying across the cell population. The difference between the all-or-none responses of the permeabilized or ionophore-treated cells and the graded responses of cells activated by compound 48/80 is likely to stem from differences in the effective calcium stimulus. Whereas cells stimulated with receptor-directed agonists can undergo transient and localized Ca2+ changes, a homogeneous and persistent stimulus is sensed at every potential exocytotic site in the permeabilized cells.
We have investigated the ATP-induced permeabilization of rat peritoneal mast cells using three different techniques: (a) by measuring uptake of fluorescent membrane and DNA marker dyes, (b) by ...voltage-clamp measurements using the patch-clamp technique, and (c) by measurements of exocytosis in response to entry of Ca2+ and GTP gamma S into permeabilized cells. In the absence of divalent cations cells become highly permeable at ATP concentrations as low as 3 microM. In normal saline containing 1 mM MgCl2 and 2 mM CaCl2, dye uptake and electric conductance are detectable at 100 microM ATP corresponding to 4 microM ATP4-. The permeabilization is half-maximal at an ATP4- concentration of 5-20 microM with a Hill coefficient near 2. The ATP-induced whole-cell conductance at saturating ATP concentrations was 35-70 nS, exhibiting only weak cation selectivity. The activation is very fast with a time constant less than or equal to 65 ms. Pores which are large enough to allow for permeation of substances of 300-900 D are expected to have a unit conductance of approximately 200-400 pS. However, in whole cells as well as outside-out patches, discrete openings and closings of channels could not be observed at a resolution of approximately 40 pS and the single-channel conductance obtained from noise analysis is approximately 2-10 pS. Entry of Ca2+ into cells permeabilized with ATP stimulates exocytosis at low but not at high ATP concentrations indicating loss of an essential intracellular component or components at a high degree of permeabilization. This inactivation is removed when GTP gamma S is provided in the medium and this leads to enhanced exocytosis. The enhancement only occurs at high ATP concentrations. These results strongly suggest that the ATP-induced pores are of variable size and can increase or decrease by very small units.
Pathophysiology of hypercortisolism in depression Carroll, B. J.; Cassidy, F.; Naftolowitz, D. ...
Acta Psychiatrica Scandinavica,
February 2007, Letnik:
115, Številka:
s433
Journal Article, Conference Proceeding
Recenzirano
Objective: The mechanisms mediating hypercortisolemia in depression remain controversial. Adopting the biomarker strategy, we studied adrenocorticotropin (ACTH) and cortisol dynamics in ...hypercortisolemic and non‐hypercortisolemic depressed in‐patients, and in normal volunteers.
Method: Deconvolution analysis of 24‐h pulsatile secretion, approximate entropy (ApEn) estimation of secretory regularity, cross‐ApEn quantitation of forward and reverse ACTH–cortisol synchrony, and cosine regression of 24‐h rhythmicity.
Results: Hypercortisolemia was strongly associated with melancholic and psychotic depressive subtypes. Hypercortisolemic patients had elevated ACTH and cortisol secretion, mediated chiefly by increased burst masses. Basal ACTH secretion was increased, ACTH half‐life was reduced, and mean 24‐h ACTH concentration was normal. Cortisol secretion was increased in a highly irregular pattern (high ApEn), with high ACTH → cortisol cross‐ApEn (impaired feedforward coupling). Cortisol‐mediated feedback on the secretory pattern of ACTH was normal. Hypercortisolemic depressed patients had normal programming of the central hypothalamo–pituitary–adrenal (HPA) axis pulse generator: ACTH pulse frequency, cortisol pulse frequency, circadian acrophases, and ApEn of ACTH secretion were normal. Responsiveness of the adrenal cortex to endogenous ACTH was normal. Non‐hypercortisolemic patients resembled hypercortisolemic patients on ACTH regulatory parameters but had low total cortisol secretion.
Conclusion: Increased ACTH secretion occurs in depressed in‐patients regardless of cortisolemic status, confirming central HPA axis overdrive in severe depression. Depressive hypercortisolemia results from an additional change in the adrenal cortex that causes ACTH‐independent, disorderly basal cortisol release, a sign of physiological stress in melancholic/psychotic depression.
We have used fast differential ramp voltammetry with carbon-fibre electrodes to monitor exocytotic secretion in single rat mast cells. The oxidation peak and other aspects of the electrochemical ...profile of the substance released were similar to those of 5-hydroxytryptamine (5-HT) and the signals were increased by preloading the secretory granules with exogenous 5-HT. Metabolic blockade inhibited both visible degranulation and the electrochemical signal. For comparison, quinacrine, which is fluorescent and accumulates in secretory vesicles, was used as an alternative means of detecting secretion in single cells. The amplitude of the electrochemical signals observed during degranulation correlated well with the loss of quinacrine fluorescence. Both methods were used to record successive rounds of secretion in single mast cells in response to repeated applications of compound 48/80.
Conjugation of the small ubiquitin-like modifier SUMO-1/SMT3C/Sentrin-1 to proteins in vitro is dependent on a heterodimeric E1 (SAE1/SAE2) and an E2 (Ubc9). Although SUMO-2/SMT3A/Sentrin-3 and ...SUMO-3/SMT3B/Sentrin-2 share 50% sequence identity with SUMO-1, they are functionally distinct. Inspection of the SUMO-2 and SUMO-3 sequences indicates that they both contain the sequence ψKXE, which represents the consensus SUMO modification site. As a consequence SAE1/SAE2 and Ubc9 catalyze the formation of polymeric chains of SUMO-2 and SUMO-3 on protein substrates in vitro, and SUMO-2 chains are detectedin vivo. The ability to form polymeric chains is not shared by SUMO-1, and although all SUMO species use the same conjugation machinery, modification by SUMO-1 and SUMO-2/-3 may have distinct functional consequences.
Clinical markers of cardiac autonomic function, such as heart rate and response to exercise, are important predictors of cardiovascular risk. Tetrahydrobiopterin (BH4) is a required cofactor for ...enzymes with roles in cardiac autonomic function, including tyrosine hydroxylase and nitric oxide synthase. Synthesis of BH4 is regulated by GTP cyclohydrolase I (GTPCH), encoded by GCH1. Recent clinical studies report associations between GCH1 variants and increased heart rate, but the mechanistic importance of GCH1 and BH4 in autonomic function remains unclear. We investigate the effect of BH4 deficiency on the autonomic regulation of heart rate in the hph-1 mouse model of BH4 deficiency.
In the hph-1 mouse, reduced cardiac GCH1 expression, GTPCH enzymatic activity, and BH4 were associated with increased resting heart rate; blood pressure was not different. Exercise training decreased resting heart rate, but hph-1 mice retained a relative tachycardia. Vagal nerve stimulation in vitro induced bradycardia equally in hph-1 and wild-type mice both before and after exercise training. Direct atrial responses to carbamylcholine were equal. In contrast, propranolol treatment normalized the resting tachycardia in vivo. Stellate ganglion stimulation and isoproterenol but not forskolin application in vitro induced a greater tachycardic response in hph-1 mice. β1-adrenoceptor protein was increased as was the cAMP response to isoproterenol stimulation.
Reduced GCH1 expression and BH4 deficiency cause tachycardia through enhanced β-adrenergic sensitivity, with no effect on vagal function. GCH1 expression and BH4 are novel determinants of cardiac autonomic regulation that may have important roles in cardiovascular pathophysiology.
We sought to examine changes in acute respiratory distress syndrome (ARDS) management over a 12-year period of two successive randomized trials.
Analyses included baseline data, from eligible ...patients, prior to influence of trial protocols, and daily study data, from randomized patients, of variables not determined by trial protocols. Mixed linear regressions examined changes in practice year-on-year.
A total of 2376 patients met the inclusion criteria. Over the 12-year period, baseline tidal volume index decreased (9.0 to 7.0 ml/kg, p < 0.001), plateau pressures decreased (30.8 to 29.0 cmH2O, p < 0.05), and baseline positive end-expiratory pressures increased (10.8 to 13.2 cmH2O, p < 0.001). Volume-controlled ventilation declined from 29.4 to 14.0% (p < 0.01). Use of corticosteroids increased (baseline: 7.7 to 30.3%; on study: 32.6 to 61.2%; both p < 0.001), as did neuromuscular blockade (baseline: 12.3 to 24.5%; on study: 55.5 to 70.0%; both p < 0.01). Inhaled nitric oxide use increased (24.9 to 65.8%, p < 0.05). We observed no significant change in prone positioning (16.2 to 18.9%, p = 0.70).
Clear trends were apparent in tidal volume, airway pressures, ventilator modes, adjuncts and rescue therapies. With the exception of prone positioning, and outside the context of rescue therapy, these trends appear consistent with the evolving literature on ARDS management.
•Evolution of ARDS management was studied in two successive trials over 12 years.•Over this time, tidal volume index and plateau pressures decreased; PEEP increased.•Use of volume-controlled ventilation reduced; use of other modes remained static.•Use of corticosteroids and neuromuscular blockade increased.•Inhaled nitric oxide and high frequency oscillatory ventilation use increased.
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