Graded intracellular acidosis produces extensive and reversible reductions in the effective free energy change of ATP hydrolysis in a molluscan muscle

Phosphorus nuclear magnetic resonance spectroscopy was used to evaluate the impact of experimental reductions of intracellular pH on in vitro preparations of the radula protractor muscle of the marine gastropod, Busycon canaliculatum. The intracellular pH of radula refractor muscle bundles superfused with buffered artificial sea water (pH=7.8) was 7.29. It was possible to clamp muscle intracellular pH at various acidotic states by changing the superfusate to 5, 10, and 15 mmol·l^-1 5,5-dimethyl-oxazolidine-2,4-dione in buffered artifical sea water (pH=6.5). Consistent and temporally stable reductions of intracellular pH were achieved (intracellular pH=6.98, 6.79, and 6.62, respectively). During the acidotic transitions, arginine phosphate concentrations decreased and inorganic phosphate concentrations increased in a reciprocal manner and remained essentially constant after the intracellular pH stabilized. The extent of changes in arginine phosphate and inorganic phosphate was directly proportional to the magnitude of the imposed acidosis. Total adenosine triphosphate concentrations remained unchanged in all treatments. However, the magnesium adenosine triphosphate to total adenosine triphosphate ratio declined in direct relation to the extent of the acidosis. Intracellular free Mg²⁺ fell incrementally with reduced intracellular pH. All of the above effects were rapidly reversed when the 5,5-dimethyl-oxazolidine-2,4-dione was washed out by changing the superfusate to buffered artificial sea water (pH=7.8). Mg-adenosine diphosphate concentrations were calculated in all treatments using equilibrium constants for the arginine kinase reaction corrected for pH and intracellular free [Mg²⁺]. The metabolite, intracellular pH, and [Mg²⁺] data were used to estimate the effective free energy of hydrolysis of adenosine triphosphate (dG/d_ATP) under most experimental conditions. Experimental acidosis resulted in dramatic reductions in dG/d_ATP which were fully reversible upon wash-out of 5,5-dimethyl-dioxazolidine-2,4-dione and recovery to normal intracellular pH conditions. Acidosis resulted in net hydrolysis of arginine phosphate, likely via a complex mechanism involving enhancement of rate of adenosine triphosphate hydrolysis and/or inhibition of adenosine triphosphate synthesis.Abbreviations ABRM anterior byssus retractor muscle - ADP, ATP adenosine di- and triphosphate - AP arginine phosphate - BASW buffered artificial sea-water - CP creatine phosphate - DMO 5, 5-dimethyl-oxazolidine-2, 4-dione - G _o ^obs standard free energy change of ATP hydrolysis - dG/d_ATP effective free energy change of ATP hydrolysis - HEPES 4-(2-hydroxyethyl)-piperazine-1-ethanesulphonic acid - K _obs equilibrium constant under specified conditions - MES 2-[N-morpholino] ethanesulphonic acid - [Mg²⁺]_i intracellular free magnesium concentration - NMR nuclear magnetic resonance - pH _i intracellular pH·P _i inorganic phosphate - PIPES piperazine-N,N-bis-2-ethane sulphonic acid - RPM radula protractor muscle - SR sarcoplasmic reticulum     

Intracellular sodium homeostasis in relation to the effective free-energy change of ATP hydrolysis: studies of crayfish muscle fibers

We investigated the impact of reductions in the effective free-energy change of ATP hydrolysis (dG/d_ATP) on intracellular sodium homeostasis in bundles of fibers from the abdominal extensor muscle of the crayfish Procambarus clarkii. ³¹P nuclear magnetic resonance (NMR) spectroscopy was used to monitor high-energy phosphate levels and intracellular pH while interleaved ²³Na-NMR spectra were acquired to monitor changes in sodium. Previous work has shown that the bulk of intracellular Na⁺ is NMR visible (see Ivanics et al. 1994). The ²³Na-NMR spectra were diffusion-weighted which effectively filtered out signal contributions from extracellular sodium. The efficacy of this procedure was validated using a relatively non-toxic chemical shift reagent which allowed resolution of extracellular and intracellular Na⁺ signals. Metabolic inhibition (cyanide/iodoacetate) produced pronounced reductions in dG/d_ATP coincident with dramatic increases in intracellular Na⁺ levels ([Na⁺]_i). However, the increases in [Na⁺]_i occurred at dG/d_ATP values well above the threshold value of −46 kJ · mol⁻¹ required by the existing Na⁺ gradient and the membrane potential. These results suggest that the global dG/d_ATP value may not reflect the dG/d_ATP value in the vicinity of the pump. Alternatively, other factors, including low molecular modulators of Na⁺, K⁺-ATPase activity, may be important in this context. Accepted: 15 May 1997     

Golgi'S Dichromate-Silver Method 3. Chromating Fluids

Slices of cat brains that had been fixed in 10% aqueous formalin for various periods from 2 days to over a year were subjected to different types of chromation prior to impregnation with silver nitrate. Acid solutions of Al, Ba, Ca, Cd, Ce, Co, Cu, Fe, K, Ni, Sr and Zn chromates were tested for usefulness as chromating agents. The chromates of Cd, Co, K, Sr and Zn were found to be best; Ca, Ce and Ni gave positive results, but Al, Ba, Cu and Fe were quite unsatisfactory. Acetic acid was somewhat preferable to formic as the acidifying agent. A formula consisting of potassium chromate, 5% aq., 100 ml. and glacial acetic acid 6-8 ml. was found to be similar in action to comparable mixtures that contained the chromate of Cd, Co, Sr or Zn. Brain slices chromated 24-48 hours in these acidified chromates and silvered in 0.75-1.0% silver nitrate for 12-24 hours at 37-40° C. gave at least three times as many good preparations as similar specimens chromated with plain potassium dichromate solution.     

Evaluation of a modified high-performance liquid chromatography assay for acebutolol and its major metabolite

Extensive modification of an existing high-performance liquid chromatography assay for acebutolol and its major metabolite has markedly improved chromatographic stability eliminating the previous need for frequent adjustment of the eluent composition to accommodate continuous loss of column retention. The eluents now used and avoidance of the requirement for elevated column temperature may be significant factors in the ability to maintain column life over 8 months of continuous use with little decrease in retention As a result of the improved chromatographic stability full advantage can now be taken of automatic injection devices for the unattended processing of large numbers of samples. A significant modification of the work-up of blood samples has improved precision of the assay in whole blood. Nevertheless, it is recommended that plasma samples rather than whole blood be analyzed, since the plasma assay is faster and still more precise.     

DIFFERENTIATION AND PROLIFERATION OF EMBRYONIC MAST CELLS OF THE RAT

Histochemical reactions and radioautography were used to investigate the sequence of mast cell development in rat embryos. Mast cells arise ubiquitously in and are confined to the loose connective tissue in the embryo. The alcian blue-safranin reaction distinguishes between weakly sulfated and strongly sulfated mucopolysaccharides by a shift from alcian blue to safranin staining. Based on this reaction and morphologic characteristics, four stages were identified. Stage I mast cells are lymphocyte-like cells with cytoplasmic granules which invariably stain blue with the alcian blue-safranin reaction. In Stage II cells the majority of granules are alcian blue-positive, but some safranin-positive granules have appeared. Stage III mast cells are distinguished by a majority of safranin-positive cytoplasmic granules; some alcian blue-positive granules still remain. Stage IV cells contain only safranin-positive granules. Thymidine-H³ uptake and identification of mitotic figures indicates that mast cells in Stages I and II comprise a mitotic pool while those in Stages III and IV are mitotically inactive. The pattern of S³⁵O₄ incorporation and the sequence of appearance of histochemically identifiable mast cell constituents corroborates division of the proliferation and differentiation of embryonic mast cells into the four stages described above. The process of formation of mast cell granules is interpreted as reflecting the synthesis and accumulation of a heparin precursor in alcian blue positive granules followed by the synthesis and accumulation of highly N-sulfated heparin along with mast cell chymase and finally histamine in safranin-positive granules.     

Effects of probucol on plasma lipids, lipoproteins and parameters of high density lipoprotein metabolism.

Eight patients with primary hypercholesterolemia were treated with probucol for 17 weeks. Plasma total cholesterol, low density lipoprotein (LDL)-cholesterol, and high density lipoprotein (HDL)-cholesterol decreased by 16.6, 15.0 and 25.7%, respectively, in response to probucol treatment. Plasma levels of apolipoprotein B and apolipoprotein A-I also decreased, while apolipoprotein A-II concentrations were unchanged. The decrease in HDL-cholesterol levels was associated with a reduction in HDL particle size. No changes in the plasma lecithin:cholesterol acyltransferase activity or mass occurred in response to probucol treatment. In contrast, a significant 25% increase in plasma cholesteryl ester and triglyceride transfer activity occurred following probucol treatment. There was a positive correlation (R = 0.94) between cholesterol ester and triglyceride transfer. We propose that the increase in lipid transfer activity may in part explain the changes in HDL concentration and size, as well as the previously reported effect probucol has on reducing atherosclerosis in animal models.     

Reduction of endogenous TGF-beta does not affect phenotypic development of sympathoadrenal progenitors into adrenal chromaffin cells.

Adrenal chromaffin cells and sympathetic neurons are related, but phenotypically distinct derivatives of the neural crest. Molecular cues that determine the chromaffin cell phenotype have not yet been identified; in contrast to a widely held belief, glucocorticoid signaling is apparently not relevant (Development 126 (1999) 2935). Transforming growth factor-betas (TGF-betas) regulate various aspects of embryonic development and are expressed in the environment of sympathoadrenal (SA) progenitor cells. We have previously shown that neutralization of endogenous TGF-beta from E4 to E8 in the quail embryo significantly increases numbers of adrenal tyrosine hydroxylase-positive cells. Whether endogenous TGF-beta may also be involved in influencing phenotypic development of adrenal chromaffin cells and their SA progenitors has not been analyzed. We now demonstrate that neutralization of endogenous TGF-beta1, -beta2 and -beta3 with a pan-anti-TGF-beta antibody in quail embryos during distinct time windows does not alter phenotypic development of chromaffin cells. In situ hybridizations revealed unaltered expression of neurofilament (NF-160), synaptotagmin I and neurexin I in adrenal glands. Likewise, the NF-associated antigen 3A10, and polyphosphorylated NF epitopes (RT 97) were unaltered. Most importantly, the typical ultrastructure of adrenal chromaffin cells including their large chromaffin secretory granules, a hallmark of the neuroendocrine phenotype, which distinguishes them from sympathetic neurons, was not affected. We therefore conclude that neutralization of endogenous TGF-beta influences chromaffin cell proliferation, but does not interfere with the development of the typical chromaffin cell phenotype.     

NADH Enzyme-Dependent Fluorescence Recovery after Photobleaching (ED-FRAP): Applications to Enzyme and Mitochondrial Reaction Kinetics, In Vitro

NADH enzyme-dependent fluorescence recovery after photobleaching (ED-FRAP) was evaluated for studying enzyme kinetics in vitro and in isolated mitochondria. Mass, optical, and nuclear magnetic resonance spectroscopy data were consistent with the UV NADH photolysis reaction being NADH → NAD^· + H⁺ + e⁻. The overall net reaction was O₂ + 2NADH + 2H⁺ → 2NAD⁺ + 2H₂O, or in the presence of other competing electron acceptors such as cytochrome c, NADH + 2Cyt_ox → NAD⁺ + H⁺ + 2Cyt_red. Solution pH could differentiate between these free-radical scavenging pathways. These net reactions represent the photooxidation of NADH to NAD⁺. Kinetic models and acquisition schemes were developed, varying [NADH] and [NAD] by altering NADH photolysis levels, for extracting kinetic parameters. UV irradiation levels used did not damage mitochondrial function or enzymatic activity. In mitochondria, [NADH] is a high affinity product inhibitor that significantly reduced the NADH regeneration rate. Matrix NADH regeneration only slightly exceeded the net rate of NADH consumption, suggesting that the NADH regeneration process is far from equilibrium. Evaluation of NADH regeneration in active mitochondria, in comparison to rotenone-treated preparations, revealed other regulatory elements in addition to matrix [NADH] and [NAD] that have yet to be fully characterized. These studies demonstrate that the rapid UV photolysis of NADH to NAD is an effective tool in evaluating the steady-state kinetic properties of enzyme systems. Initial data support the notion that the NADH regeneration process is far from equilibrium in mitochondria and is potentially controlled by NADH levels as well as several other matrix factors.