cytochalasin-d and Acidosis

Hypothermia and acidosis were reported to influence coagulopathy in different clinical settings. We evaluated whole blood coagulation to determine the effects of hypothermia and/or acidosis on hemostasis.. Whole blood samples (3.000 microL) from 10 healthy volunteers (2 female, 8 male) were acidified by adding 40 microL of hydrochloric acid of increasing molarity to achieve a blood pH (alpha-stat) between 7.0 and 7.37, and coagulation was analyzed by rotational thromboelastometry after an incubation period of 30 min using both intrinsically (InTEM) and extrinsically (ExTEM) activated assays. To assess temperature-dependent effects, all tests were performed at blood/thromboelastometer temperatures of 30, 33, 36, and 39 degrees C, respectively. An additional extrinsically activated test with addition of cytochalasin D was performed to examine clot formation without platelet contribution.. Hypothermia at a normal pH produced an increased coagulation time [ExTEM: 65 s +/- 3.6 (36 degrees C) vs 85 +/- 4 (30 degrees C), P < 0.001; coagulation time, InTEM: 181 s +/- 10 (36 degrees C) vs 226 +/- 9, P < 0.001] and clot formation time [ExTEM: 105 s +/- 5 (36 degrees C) vs 187 +/- 6 (30 degrees C), P < 0.001]; clot formation time [InTEM: 101 s +/- 5 (36 degrees C) vs 175 +/- 7, P < 0.001], as well as decreased alpha angle [ExTEM: 65.6 +/- 1.8 (36 degrees C) vs 58 +/- 1.1, P < 0.01, P < 0.01; InTEM: 70.5 +/- 1.8 (36 degrees C) vs 60.2 +/- 1.5, P < 0.001]. Maximum clot firmness was significantly impaired only in InTEM assays [56.9 mm +/- 0.9 (36 degrees C) vs 52.7 +/- 0.9, P < 0.05]. In contrast, acidosis per se had no significant effects during normothermia. Acidosis amplified the effects of hypothermia, and synergistically impaired clotting times, alpha angle, and decreased maximum clot firmness, again in both extrinsically and intrinsically activated assays. Formation of a fibrin clot tested after abolition of platelet function by cytochalasin D was not impaired. Clot lysis decreased under hypothermic and/or acidotic conditions, but increased with hyperthermia.. In this in vitro study, hypothermia produced coagulation changes that were worsened by acidosis whereas acidosis without hypothermia has no significant effect on coagulation, as studied by thromboelastometry. This effect was mediated by the inhibition of coagulation factors and platelet function. Thus, thromboelastometry performed at 37 degrees C overestimated integrity of coagulation during hypothermia in particular in combination with acidosis.

Topics: Acidosis; Adult; Blood Coagulation; Blood Coagulation Tests; Blood Platelets; Cytochalasin D; Female; Fibrin; Humans; Hydrogen-Ion Concentration; Hypothermia; Male; Reproducibility of Results; Temperature

1. L-type Ca2+ channel currents (I(Ca)) were measured in guinea-pig ventricular myocytes (22 degrees C, 300 ms steps from -45 to +10 mV). Pulsing at 0.5 Hz reduced I(Ca) within 5 min to 92 +/- 3% (mean +/- S.E.M., n = 14) and within 10 min to 83 +/- 4 % ('run-down' with reference to I(Ca) after a 5 min equilibration period). 2. Bath-applied cytochalasin D (cytD, 10 microM) reduced I(Ca) to 75 +/- 4% within 5 min and to 61 +/- 4% within 10 min ('cytD reduction of I(Ca)') by reduction of maximal Ca2+ conductance (suggested by fits of time course and of current-potential (I-V) curves). 3. Preincubation with phalloidin (bath applied, 100 microM, 5 h) prevented the cytD reduction of I(Ca). Since phalloidin specifically blocks F-actin depolymerization, cytD reduction of I(Ca) is linked to depolymerization of F-actin. 4. CytD did not attenuate the beta-adrenergic stimulation of I(Ca) (30 nM isoproterenol), suggesting that A kinase anchoring proteins are unlikely to mediate the cytD reduction of I(Ca). The cytD reduction of I(Ca) was abolished by extra-/intracellular acidosis (pH(o) 6.9), by cell dialysis of 5 mM BAPTA, or by serine/threonine protein phosphatase inhibitors. 5. Actin-depolymerizing factor (ADF)/cofilin are proteins that bind to actin, mediate a pH-sensitive depolymerization of F-actin, and are activated by dephosphorylation. Western blots from hearts perfused with solutions containing zero or 10 microM cytD indicated that cytD reduces the ratio of phosphorylated to total ADF/cofilin content by 50%. 6. The data support the concept that cytD mediates dephosphorylation and activation of ADF/cofilin, leading to depolymerization of F-actin with a subsequent reduction of I(Ca).

Topics: Acidosis; Actin Depolymerizing Factors; Actins; Animals; Calcium; Calcium Channels, L-Type; Carrier Proteins; Chelating Agents; Cyclic AMP-Dependent Protein Kinases; Cytochalasin D; Egtazic Acid; Electric Conductivity; Guinea Pigs; Microfilament Proteins; Myocardium; Phalloidine; Phosphoprotein Phosphatases; Polymers

Microglia are equipped with a strong proton (H(+)) extrusion pathway, a voltage-gated H(+) channel, probably to compensate for the large amount of H(+) generated during phagocytosis; however, little is known about how this channel is regulated in pathological states. Because neural damage is often associated with intracellular and extracellular acidosis, we examined the regulatory mechanisms of the H(+) current of rat spinal microglia in acidic environments. More than 90% of round/amoeboid microglia expressed the H(+) current, which was characterized by slow activation kinetics, dependencies on both intracellular and extracellular pH, and blockage by Zn(2+). Extracellular lactoacidosis, pH 6.8, induced intracellular acidification and cell swelling. Cell swelling was also induced by intracellular dialysis with acidic pipette solutions, pH 5.5-6.8, at normal extracellular pH 7.3 in the presence of Na(+). The H(+) currents were increased in association with cell swelling as shown by shifts of the half-activation voltage to more negative potentials and by acceleration of the activation kinetics. The acidosis-induced cell swelling and the accompanying potentiation of the H(+) current required nonhydrolytic actions of intracellular ATP and were inhibited by agents affecting actin filaments (phalloidin and cytochalasin D). The H(+) current was also potentiated by swelling caused by hypotonic stress. These findings suggest that the H(+) channel of microglia can be potentiated via cell swelling induced by intracellular acidification. This potentiation might operate as a negative feedback mechanism to protect microglia from cytotoxic acidification and hence acidosis-induced swelling in pathological states of the CNS.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Acidosis; Acids; Adenosine Triphosphate; Alkalies; Animals; Cells, Cultured; Chloride Channels; Chlorides; Cytochalasin D; Hydrogen-Ion Concentration; Hypotonic Solutions; Intracellular Fluid; Lactic Acid; Membrane Potentials; Microglia; Nucleic Acid Synthesis Inhibitors; Patch-Clamp Techniques; Phalloidine; Proton Pumps; Proton-Translocating ATPases; Protons; Rats; Rats, Wistar; Sodium-Hydrogen Exchangers; Vacuolar Proton-Translocating ATPases; Zinc Compounds