Spontaneous opening at zero membrane potential of sodium channels from eel electroplax reconstituted into lipid vesicles

Summary The voltage-dependent sodium channel from the eel electroplax was purified and reconstituted into vesicles of varying lipid composition. Isotopic sodium uptake experiments were conducted with vesicles at zero membrane potential, using veratridine to activate channels and tetrodotoxin to block them. Under these conditions, channel-dependent uptake of isotopic sodium by the vesicles was observed, demonstrating that a certain fraction of the reconstituted protein was capable of mediating ion fluxes. In addition, vesicles untreated with veratridine showed significant background uptake of sodium; a considerable proportion of this flux was blocked by tetrodotoxin. Thus these measurements showed that a significant subpopulation of channels was present that could mediate ionic fluxes in the absence of activating toxins. The proportion of channels exhibiting this behavior was dependent on the lipid composition of the vesicles and the temperature at which the uptake was measured; furthermore, the effect of temperature was reversible. However, the phenomenon was not affected by the degree of purification of the protein used for reconstitution, and channels in resealed electroplax membrane fragments or reconstituted, solely into native eel lipids did not show this behavior. The kinetics of vesicular uptake through these spontaneously-opening channels was slow, and we attribute this behavior to a modification of sodium channel inactivation.     

Antisera Against the Indolealkylamines: Tryptophan, 5-Hydroxytryptophan, 5-Hydroxytryptamine, 5-Methoxytryptophan, and 5-Methoxytryptamine Tested by an Enzyme-Linked Immunosorbent Assay Method

Antisera were raised against tryptophan, 5-hydroxytryptophan, 5-hydroxytryptamine, 5-methoxytryptophan, and 5-methoxytryptamine, by conjugating each molecule to bovine serum albumin and to human serum albumin via glutaraldehyde, in such a way as to preserve the original part. Antibody specificity was tested with the enzyme-linked immunosorbent assay method. The specificity of each anti-indolealkylamine-glutaraldehyde antibody was established with competition experiments by using an adsorbed immunogenic conjugate and indolealkylamines either free or conjugated with poly-L-lysine. The nonconjugated compounds were poorly recognized. In the same way, the nonreduced conjugates always appeared less immunoreactive than the reduced ones. Calculated from the specificity study of each antiserum, the cross-reactivity ratios were found to be smallest for the most immunoreactive conjugates. Thus, a specific immune response was defined for each compound belonging to the same metabolic pathway.     

Uptake and acylation of 2-acyl-lysophospholipids by Escherichia coli.

The efficiency of extracellular 2-acyl-lysophospholipid incorporation into Escherichia coli membranes and the acyl donor utilized to acylate the 2-acyl-lysophospholipid was determined. Exogenous 2-acyl-lysophospholipids were acylated via the acyl-acyl carrier protein synthetase/2-acylglycerophosphoethanolamine acyltransferase pathway. The maximum extent of 2-acyl-lysophospholipid incorporation into the membrane was approximately 2.5% of the normal phospholipid biosynthetic rate.     

Acetoacetyl-acyl carrier protein synthase. A target for the antibiotic thiolactomycin

The biochemical basis for the inhibition of fatty acid biosynthesis in Escherichia coli by the antibiotic thiolactomycin was investigated. A biochemical assay was developed to measure acetoacetyl-acyl carrier protein (ACP) synthase activity, a recently discovered third condensing enzyme from E. coli (Jackowski, S., and Rock, C.O. (1987) J. Biol. Chem. 262, 7927-7931). In contrast to the other two condensing enzymes in E. coli, acetoacetyl-ACP synthase (synthase III) condensed malonyl-ACP with acetyl-CoA, rather than with acetyl-ACP. The concentration dependence of thiolactomycin inhibition of fatty acid biosynthesis in vivo was the same as the inhibition of acetoacetyl-ACP synthase activity in vitro indicating that the two phenomena were related. A thiolactomycin-resistant mutant (strain CDM5) was isolated. The specific activity of acetoacetyl-ACP synthase in extracts from this mutant was 10-fold lower than in extracts from its thiolactomycin-sensitive parent resulting in a marked defect in the ability of strain CDM5 to incorporate acetyl-CoA into fatty acids in vitro. The residual acetoacetyl-ACP synthase activity in the resistant strain was refractory to thiolactomycin inhibition. In addition, acetyl-CoA:ACP transacylase activity in strain CDM5 was resistant to inactivation by thiolactomycin suggesting that the acetoacetyl-ACP synthase also catalyzes this transacylation reaction. These data point to acetoacetyl-ACP synthase as a target for thiolactomycin inhibition of bacterial fatty acid biosynthesis.     

Stimulation of phosphatidylinositol 4,5-bisphosphate phospholipase C activity by phosphatidic acid

Phosphatidic acid was a potent activator of the phosphatidylinositol 4,5-bisphosphate (PtdIns-P2) phospholipase C activity associated with human platelet membranes. Lysophosphatidic acid was half as active as phosphatidic acid, and shortening the fatty acid chain reduced the effectiveness of the corresponding phosphatidic acid. Compounds lacking either the phosphate group (diacylglycerol or phorbol ester) or the fatty acid (glycerol phosphate) were not activators. When the negative charge was contributed by a carboxyl group (fatty acid or phosphatidylserine), stimulation of phospholipase C was weak but detectable. Structural analogs of phosphatidic acid (lipopolysaccharide, lipid A, and 2,3-diacylglucosamine 1-phosphate) were less effective but also enhanced PtdIns-P2 hydrolysis. Phosphatidic acid potentiated the activation of phospholipase C by alpha-thrombin, chelators, and guanine nucleotides. Phosphatidylinositol 4-phosphate and PtdIns-P2 were also effective activators of PtdIns-P2 degradation. Other phospholipids were without effect. The production of inositol 1,4,5-trisphosphate and diacylglycerol via the activation of phospholipase C provides a rationale for the cellular responses evoked by phosphatidic acid and the ability of this phospholipid to potentiate and initiate hormonal responses.     

Responses of plasma human atrial natriuretic factor to high intensity submaximal exercise in the heat

No data exists regarding responses of human atrial natriuretic factor (ANF) to exercise in the heat. The purpose of this study was to examine the responses of plasma ANF to high intensity submaximal (71% +/- 0.9 VO2max) exercise in the heat over an eight day acclimation period. Fourteen healthy males volunteered to participate in the study. Subjects performed intermittent exercises on a treadmill (0% grade) during 50 min of each 100 min trial in an environmental chamber maintained at 41.2 +/- 0.5 degrees C, 39.0 +/- 1.7% relative humidity. Blood was obtained from an antecubital vein after standing 20 min in the heat prior to exercise, and immediately after exercise. Measures were compared on days 1, 4 and 8. ANF did not change pre- to post-exercise nor did it change over the eight day heat acclimation period despite other heat acclimation adaptations. Conversely, plasma aldosterone (ALDO), renin activity (PRA) and cortisol (COR) all increased (p less than 0.05) pre- to post-exercise on each day but again no changes were observed over the eight day period. These data support that ANF may not increase when ALDO and PRA increases are observed.     

Effects of arterial ligation and embolization on pulmonary vascular pressure distribution

The effects of embolization on the longitudinal distribution of pulmonary vascular pressures with respect to vascular compliance were determined by the vascular inflow and outflow occlusion technique in isolated blood-perfused pig lungs treated with papaverine to prevent vasomotor responses. Embolization with microspheres having mean diameters of 75, 200, and 550 microns and with barrier beads (2 X 3 X 3.5 mm) significantly increased the pressure gradient across the relatively compliant middle region (delta Pm) without increasing the gradients across the relatively noncompliant regions on the arterial (delta Pa) or venous (delta Pv) ends of the vasculature. In contrast ligation of several lobar arteries caused delta Pa to increase from 0.9 +/- 0.3 to 5.9 +/- 1.1 mmHg but did not change delta Pm or delta Pv. Assuming that delta Pa and delta Pv measured by vascular occlusion result from cessation of flow through resistances, these data suggest that in isolated pig lungs the vessels at the boundary between the arterial and middle regions defined by the occlusion technique are arteries greater than 2-3 mm diam and smaller than lobar arteries.