Hemodynamic Response to Rapid Saline Infusion Compared with Exercise in Healthy Participants Aged 20-80 Years.

BACKGROUND AND OBJECTIVE: Rapid saline infusion and exercise has been proposed as methods to unmask cardiovascular disease. However, the normal hemodynamic response to rapid saline infusion has not been compared to exercise nor is it known whether the responses are age-dependent.We assessed the hemodynamic response to rapid saline infusion in healthy participants over a wide age-range and compared it to exercise in the same participants. METHODS AND RESULTS: Fifty healthy participants (young <40 years, n = 16, middle-aged 40-59 years, n = 15, elderly 60-80 years, n = 19) underwent right heart catheterization at rest, during semisupine ergometer exercise at three exercise levels (25%, 50%, and 75% of peak VO2) and after rapid saline infusion (10 ml/kg at a rate of 150 ml/min). Rapid saline infusion significantly increased pulmonary capillary wedge pressure (PCWP) similarly across all age groups (∆PCWP 6 ±â€¯2; 7 ±â€¯2; 6 ±â€¯4 mmHg for the young, middle-aged and elderly respectively) with no correlation between age and ∆PCWP (r = 0.05; p = 0.74). However, there was a negative correlation between age and ∆stroke volume (SV) as elderly participants had a lower increase in SV following rapid saline infusion (r = 0.44; p = 0.002). On the contrary, exercise-induced significantly larger and age-dependent increases in PCWP (r = 0.58; p < 0.0001). Exercise also caused a larger increase in SV compared with rapid fluid loading (p = 0.0003) CONCLUSION: Unlike exercise, rapid saline infusion caused an age-independent increase in PCWP in healthy adults. Suggesting that age-related impairments beyond passive stiffness have a greater impact on exercise-induced increase in PCWP. CLINICAL TRIAL REGISTRATION: https://clinicaltrials.gov/ct2/show/NCT01974557.

Effect of cholesterol on phospholipid, RNA, and protein synthesis in Mycoplasma capricolum.

The regulatory role of cholesterol on phospholipid, RNA and protein synthesis was investigated in Mycoplasma capricolum. The addition of 2 micrograms/ml of cholesterol to cells growing slowly on lanosterol (10 micrograms/ml) caused an early stimulation of unsaturated phospholipid synthesis followed by a later stimulation of RNA and protein synthesis. Epicoprostanol, a cholesterol antagonist, sequentially inhibited these three processes. Phospholipid served as the acyl donor for the acylation of membrane proteins. Membrane-bound cholesterol correlated with a decrease in the amount of [3H]amino acids and an increase in the amount of [3H]oleate found in two membrane proteins (MW 78 kDa and 26 kDa). Taken together, the results suggest that a small amount of membrane-associated cholesterol serves as a signal for membrane biogenesis and, in turn, macromolecular synthesis and cell growth.

Coordinate regulation of unsaturated phospholipid, RNA, and protein synthesis in Mycoplasma capricolum by cholesterol.

The effect of cholesterol, epicoprostanol, and phosphatidylcholine on phospholipid, RNA, and protein synthesis was investigated in the sterol auxotroph Mycoplasma capricolum. Cells growing poorly on lanosterol were stimulated to grow more rapidly by supplementing the medium with either 2 micrograms of cholesterol or 2.2 micrograms of egg phosphatidylcholine per ml. In such cells cholesterol caused a sequential stimulation of phospholipid, RNA, and protein synthesis. Enhanced oleate incorporation into phospholipid occurred early; the rates of RNA and protein synthesis increased later. In cells supplemented with phosphatidylcholine only RNA and protein syntheses were enhanced. The addition of 2 micrograms of epicoprostanol per ml to cells growing on lanosterol promptly inhibited the rate of unsaturated phospholipid synthesis and subsequently the rate of growth. Inhibition of both processes was relieved by supplying 2 micrograms of cholesterol or 2.2 micrograms of phosphatidylcholine per ml along with the inhibitory sterol. The results suggest that cholesterol in small amounts exerts a positive regulatory effect and epicoprostanol exerts a negative one on unsaturated phospholipid synthesis and, in turn, that RNA and protein synthesis are coordinately controlled with phospholipid synthesis. The previously reported phenomenon of sterol synergism and the postulated novel role of sterols in membranes.

Stimulation of cell proliferation and polyphosphoinositide metabolism in Saccharomyces cerevisiae GL7 by ergosterol.

The effect of ergosterol on cell division and phospholipid metabolism was investigated in Saccharomyces cerevisiae strain GL7, a sterol and unsaturated fatty acid auxotroph. Cells growing poorly on cholesterol were stimulated to grow more rapidly by supplementing the medium with 100 ng of ergosterol per ml. Within 10 min after ergosterol addition to cells prelabeled with 32Pi or [3H]inositol the isotope content of the polyphosphoinositides increases markedly followed by an equally striking and rapid decrease. Subsequently upon continuous labeling, 32P incorporation into phosphatidylinositol and, to a lesser degree, other phospholipids increased. Finally 3h after ergosterol addition the growth rate increased. Only stimulation of the first process, i.e. polyphosphoinositide metabolism, upon ergosterol addition is resistant to inhibition by cycloheximide.

Phospholipids as acyl donors to membrane proteins of Mycoplasma capricolum.

Mycoplasma capricolum, a procaryotic sterol and fatty acid auxotroph, contains a large number of membrane proteins covalently modified by both saturated and unsaturated fatty acids (Dahl, C.E., Dahl, J.S., and Bloch, K. (1983) J. Biol. Chem. 258, 11814-11818). Pulse-chase experiments show that the radioactivity in the fatty acid moieties of the acyl proteins increases rather than decreases during a 4.5-h incubation period suggesting that a large intracellular pool of metabolites such as phospholipid serves as the donor for protein acylation. We find that cells incubated for 4 h in a growth medium containing [3H]palmitate-labeled phosphatidylglycerol or 2-[3H]palmitoyl dipalmitoylphosphatidylcholine show the same labeling pattern as cells incubated for 4 h in a complete growth medium with [3H]palmitate. Exogenously added phospholipids are not hydrolyzed to free fatty acid during the labeling period. Acylation of proteins is inhibited in cells treated with chloramphenicol showing that there is no pool of proacyl protein in the cell. Labeling of membrane proteins also occurs with [3H]glycerol. Glycerol is incorporated primarily into the same proteins as oleate suggesting that acylation by unsaturated fatty acid may involve a protein bound diglyceride moiety. Palmitate, on the other hand, appears to bind to other sites along the polypeptide chain in addition to the diglyceride moiety.

Proteolipid formation in Mycoplasma capricolum. Influence of cholesterol on unsaturated fatty acid acylation of membrane proteins.

Mycoplasma capricolum, a procaryotic sterol and fatty acid auxotroph was grown on media supplemented with [3H]palmitate or [3H]oleate. The isolated bacterial membranes were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Of the more than 50 membrane polypeptides revealed by Coomassie blue staining, approximately 25 were labeled with [3H]palmitate and only about 6 were labeled with [3H]oleate. Exhaustive delipidation of the membranes with chloroform:methanol did not alter the labeling pattern. Treatment of delipidated membranes by mild alkaline hydrolysis released up to 71% of the [3H]palmitate and 93% of the [3H]oleate. The data suggest that numerous membrane proteins of M. capricolum are covalently modified by acylation with saturated and unsaturated fatty acids. Cerulenin, a specific inhibitor of fatty acid synthesis had no effect on the labeling of mycoplasma membrane proteins by either [3H]palmitate or [3H]oleate. A small amount of membrane-associated cholesterol previously shown to stimulate sequentially the synthesis of unsaturated phospholipid, RNA, and protein (Dahl, J. S., and Dahl, C. E. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 692-696) specifically enhances the acylation of certain proteolipids by oleate but not by palmitate.

Effect of alkyl-substituted precursors of cholesterol on artificial and natural membranes and on the viability of Mycoplasma capricolum.

Various alkyl-substituted sterols and stanols representative of the intermediates in cholesterol biosynthesis from lanosterol have been compared with respect to (a) their effect on the physical state of lecithin vesicles, (b) their efficacy as growth factors for the sterol auxotroph Mycoplasma capricolum, and (c) their effect on the physical state of the respective mycoplasma membranes. By all three criteria, sterol effectiveness progresses in the order lanosterol less than 4,4-dimethylcholestanol less than or equal to 4 beta-methylcholestanol less than 4 alpha-methylcholestanol less than cholestanol less than cholesterol. Since the corresponding steps in cholesterol biosynthesis occur in the same order, we conclude that the nuclear modifications of the lanosterol structure by oxidative demethylation serve to improve the membrane function of the sterol molecule.

Role of membrane sterols in Mycoplasma capricolum.

A low level of cholesterol in lanosterol-rich membranes of Mycoplasma capricolum exerts a synergistic effect on growth while leaving the bulk physical state of the membrane unchanged. This observation has led to the proposal that cholesterol may serve a dual role in membranes: one as a bulk component and another of a more specialized nature involving cholesterol-specific interactions. Lanosterol, while competent as a bulk membrane sterol, cannot effectively perform the second function. The fact that a low level of cholesterol in lanosterol-rich cells specifically alters the ability of these cells to incorporate unsaturated fatty acids suggests that this more specialized role for cholesterol may be concerned with fatty acid transport or phospholipid biosynthesis.

Effect of cholesterol on macromolecular synthesis and fatty acid uptake by Mycoplasma capricolum.

The rates of protein and lipid synthesis of Mycoplasma capricolum were essentially synchronous during growth and depended on the sterol supplement in the media increasing in the order cholesterol (0.5 microgram/ml) < lanosterol (10 microgram/ml) < lanosterol (10 microgram/ml) + cholesterol (0.5 microgram/ml) < cholesterol (10 microgram/ml). The effect of lanosterol plus low cholesterol on macromolecular synthesis was synergistic. Whereas protein and lipid synthesis were brought virtually to a halt by cholesterol starvation, DNA synthesis continued for about 8 h. Increasing the palmitate and elaidate concentrations 4-fold in the lanosterol-supplemented media raised the growth rate even in the absence of the small amount of cholesterol (0.5 microgram/ml) needed otherwise for the synergistic effect on growth. Studies of the kinetics of fatty acid uptake by resting cells showed that the apparent Km (17 microM) of oleate uptake in lanosterol-grown cells was specifically lowered to 3 microM, a value equal to that seen in cholesterol-grown cells, by the inclusion of a synergistic amount of cholesterol in the growth media. By contrast, the apparent Km for palmitate uptake was the same (2 microM) for all three cell types. The results are consistent with the membrane cholesterol serving in a dual role, one as a bulk component and another more specific function involving the regulation of unsaturated fatty acid uptake and thereby phospholipid biosynthesis.

New obligate methylotroph.

A new and unique obligate methylotroph was isolated from enrichment cultures with methanol as the sole source of carbon and energy. The organism grows only on methanol and methylamine and not on methane. It does not have a complex intracellular membrane system. (14)C-acetate was assimilated by growing cultures and cell suspensions but was incorporated into only a limited number of cell constituents. (14)C-acetate incorporation was strictly dependent on the oxidation of methanol or methylamine as a source of energy. Extracts had relatively low levels of enzymes of the tricarboxylic acid cycle, and alpha-ketoglutarate dehydrogenase was not detected. Comparisons were made with a facultative methylotroph isolated from the same enrichment cultures. The new obligate methylotroph contained hexose phosphate synthetase, a key enzyme in the ribose phosphate cycle of methyl metabolism.

Acylated proteins in Acholeplasma laidlawii.

The covalent modification of membrane proteins by long-chain fatty acids was determined in two strains of Acholeplasma laidlawii by one-dimensional gel electrophoresis of radiolabeled membranes. Of the more than 50 membrane polypeptides detected, approximately 30 were labeled with [3H]palmitate, whereas covalent binding of [3H]oleate to membrane proteins could not be demonstrated. We suggest that in these wall-less bacteria, membrane protein acylation with saturated fatty acids may serve to ensure the structural integrity of the membrane.

Interaction of a coupling factor from Rhodospirillum rubrum with coupling factor deficient chromatophores.

A coupling factor necessary for the photophosphorylation and Mg2+-ATPase activities in Rhodospirillum rubrum chromatophores has been separated from these particles. Although the redox potential of coupling factor deficient chromatophores is slightly more oxidized than of the control, the addition of the coupling factor for reconstitution does not alter the redox potential. Phenazine methosulfate cannot restore or significantly enhance the photophosphorylation activities of uncoupled or reconstituted chromatophores compared to the control. The coupling factor can bind to coupling factor deficient membranes without addition of magnesium ions and thus restore the photophosphorylation and Mg2+-ATPase activities of these vesicles. The Ca2+-ATPase in the coupling factor preparation shows binding characteristics similar to those of the coupling factor.