Nitza M. Cintron

Regulation of the Synthesis and Hydrolysis of ATP in Biological Systems: Role of Peptide Inhibitors of H+-ATPases

Publisher Summary This chapter presents the role of peptide inhibitors of H+-ATPases in the regulation of the synthesis and hydrolysis of ATP in biological systems. H+-ATPases are found throughout the phylogenetic scale—in mitochondria of eukaryotic cells, in chloroplasts of plant cells, and in both aerobic and anaerobic bacteria. H+-ATPases of aerobic organisms are bifunctional and can work in the reverse direction by hydrolyzing rather nonspecifically ATP, ITP, and GTP. H+-ATPases are inhibited by oligomycin, DCCD, aurovertin, and a number of other antibiotics or covalent labeling agents. F 1 -ATPase inhibitor peptides may be one of the five different subunits of F 1 as in chloroplast and some bacterial systems or they may be a sixth subunit distinct from the five different F 1 subunits, as in bovine heart. Their in vivo location may be between the F 1 and F 0 moieties of the complete H+-ATPase complex. This location in some bacteria and chloroplasts seems quite certain because the ɛ or binding subunit of F 1 has been identified as an ATPase inhibitor protein. There are substances that mimic the action of F 1 -ATPase peptide inhibitors and substances that oppose the action of such inhibitors. It would be of great help in elucidating the mode of action of ATPase peptide inhibitors to know whether ATP synthesis and NTP-dependent functions are catalyzed by pathways that involve the same site(s), overlapping sites, or completely separate catalytic sites on H+-ATPases

Alterations in Renal Stone Risk Factors after Space Flight

Exposure to the microgravity environment of space produces a number of physiological changes of metabolic and environmental origin that could increase the potential for renal stone formation. Metabolic, environmental and physicochemical factors that influence renal stone risk potential were examined in 24-hour urine samples from astronauts 10 days before launch and on landing day to provide an immediate postflight assessment of these factors. In addition, comparisons were made between male and female crewmembers, and between crewmembers on missions of less than 6 days and those on 6 to 10-day missions. Results suggest that immediately after space flight the risk of calcium oxalate and uric acid stone formation is increased as a result of metabolic (hypercalciuria, hypocitraturia, pH) and environmental (lower urine volume) derangements, some of which could reflect residual effects of having been exposed to microgravity.

Pharmacokinetics and Oral Bioavailability of Scopolamine in Normal Subjects

The pharmacokinetics and bioavailability of scopolamine were evaluated in six healthy male subjects receiving 0.4 mg of the drug by either oral or intravenous administration. Plasma and urine samples were analyzed using a radioreceptor binding assay. After iv administration, scopolamine concentrations in the plasma declined in a biexponential fashion, with a rapid distribution phase and a comparatively slow elimination phase. Mean and SE values for volume of distribution, systemic clearance, and renal clearance were 1.4 ± 0.3 liters/kg, 65.3 ± 5.2 liters/hr, and 4.2 ± 1.4 liters/hr, respectively. Mean peak plasma concentrations were 2909.8 ± 240.9 pg/ml following iv administration and 528.6 ± 109.4 pg/ml following oral administration. Elimination half-life of the drug was 4.5 ± 1.7 hr. Bioavailability of the oral dose was variable among subjects, ranging between 10.7 and 48.2%. The variability in absorption and poor bioavailability of oral scopolamine indicate that this route of administration may not be reliable and effective.

The endocrine system in space flight

Hormones are important effectors of the bodys response to microgravity in the areas of fluid and electrolyte metabolism, erythropoiesis, and calcium metabolism. For many years antidiuretic hormone, cortisol and aldosterone have been considered the hormones most important for regulation of body fluid volume and blood levels of electrolytes, but they cannot account totally for losses of fluid and electrolytes during space flight. We have now measured atrial natriuretic factor (ANF), a hormone recently shown to regulate sodium and water excretion, in blood specimens obtained during flight. After 30 or 42 h of weightlessness, mean ANF was elevated. After 175 or 180 h, ANF had decreased by 59%, and it changed little between that time and soon after landing. There is probably an increase in ANF early inflight associated with the fluid shift, followed by a compensatory decrease in blood volume. Increased renal blood flow may cause the later ANF decrease. Erythropoietin (Ep), a hormone involved in the control of red blood cell production, was measured in blood samples taken during the first Spacelab mission and was significantly decreased on the second day of flight, suggesting also an increase in renal blood flow. Spacelab-2 investigators report that the active vitamin D metabolite 1 alpha, 25-dihydroxyvitamin D3 increased early in the flight, indicating that a stimulus for increased bone resorption occurs by 30 h after launch.

Metabolic changes observed in astronauts

Study of metabolic alterations that occur during space flight can provide insight into mechanisms of physiologic regulation. Results of medical experiments with astronauts reveal rapid loss of volume (2 L) from the legs and a transient early increase in left ventricular volume index. These findings indicate that, during space flight, fluid is redistributed from the legs toward the head. In about 2 days, total body water decreases 2 to 3%. Increased levels of plasma renin activity and antidiuretic hormone while blood sodium and plasma volume are reduced suggest that space flight‐associated factors are influencing the regulatory systems. In addition to fluid and electrolyte loss, Skylab astronauts lost an estimated 0.3 kg of protein. Endocrine factors, including increased cortisol and thyroxine and decreased insulin, are favorable for protein catabolism. The body appears to adapt to weightlessness at some physiologic cost. Readaptation to Earths gravity at landing becomes another physiologic challenge.

[52] Purification of an ATPase inhibitor peptide fraction from rat liver mitochondria

Publisher Summary This chapter describes an inhibitor peptide of the mitochondrial ATPase from beef heart. It discusses the suppression of the hydrolytic activity of purified and membrane-bound F 1 ATPase. It has been suggested that the function of this inhibitor is that of a vectorial regulator in respiratory-chain-linked energy transfers. The chapter indentifies a trypsin-sensitive inhibitor activity in the rat liver mitochondrial system and describes the procedure for its purification. It describes analytical procedures, which include measurement and ATPase inhibitor activity and measurement of protein. It also discusses purification procedures, which includes isolation of mitochondria, alkaline extraction, ammonium sulfate precipitation, trichloroacetic acid precipitation, concentration by Sephadex solvent absorption, heat treatment, and Sephadex G-75 gel filtration affinity chromatography. The chapter provides an overview on the general properties of the inhibitor protein fraction. It examines the differences among the various inhibitors.

Immunoreactive prohormone atrial natriuretic peptides 1-30 and 31-67; existence of a single circulating amino-terminal peptide.

Sep-Pak C18 extraction of human plasma and radioimmunoassay using antibodies which recognize atrial natriuretic peptide (99-128) and the prohormone sequences 1-30 and 31-67 resulted in mean values from 20 normal subjects of 26.2 (+/- 9.2), 362 (+/- 173) and 368 (+/- 160) pg/ml, respectively. A high correlation coefficient between values obtained using antibodies recognizing prohormone sequences 1-30 and 31-67 was observed (R = 0.84). Extracted plasma immunoreactivity of 1-30 and 31-67 both eluted at 46% acetonitrile. In contrast, chromatographic elution of synthetic peptides 1-30 and 31-67 was observed at 48 and 39% acetonitrile, respectively. Data suggest that the radioimmunoassay of plasma using antibodies recognizing prohormone sequences 1-30 and 31-67 may represent the measurement of a unique larger amino-terminal peptide fragment containing antigenic sites recognized by both antisera.

Acute effects of head-down tilt and hypoxia on modulators of fluid homeostasis

In an effort to understand the interaction between acute postural fluid shifts and hypoxia on hormonal regulation of fluid homeostasis, the authors measured the responses to head‐down tilt with and without acute exposure to normobaric hypoxia. Plasma atrial natriuretic peptide (ANP), cyclic guanosine monophosphate (cGMP), cyclic adenosine monophosphate (cAMP), plasma aldosterone (ALD), and plasma renin activity (PRA) were measured in six healthy male volunteers who were exposed to a head‐down tilt protocol during normoxia and hypoxia. The tilt protocol consisted of a 17° head‐up phase (30 minutes), a 28° head‐down phase (1 hour), and a 17° head‐up recovery period (2 hours, with the last hour normoxic in both experiments). Altitude equivalent to 14,828 ft was simulated by having the subjects breathe an inspired gas mixture with 13.9% oxygen. The results indicate that the postural fluid redistribution associated with a 60‐minute head‐down tilt induces the release of ANP and cGMP during both hypoxia and normoxia. Hypoxia increased cGMP, cAMP, ALD, and PRA throughout the protocol and significantly potentiated the increase in cGMP during head‐down tilt. Hypoxia had no overall effect on the release of ANP, but appeared to attenuate the increase with head‐down tilt. This study describes the acute effects of hypoxia on the endocrine response during fluid redistribution and suggests that the magnitude, but not the direction, of these changes with posture is affected by hypoxia.

Isolation and quantitative analysis of hydroxylysine glycosides

SummaryMethods are described for the isolation of galactosyl hydroxylysine and glucosylgalactosyl hydroxylysine from collagen and for the quantitative analysis of these hydroxylysine glycosides. The isolation procedure, based upon gel filtration and preparative ionpaired reversed-phase high-performance liquid chromatography, is simple and rapid in comparison with existing methods, allowing the production of tens of milligrams of each glycoside in a single day. Quantitative analysis of the hydroxylysine glycosides is effected by reversed-phase high-performance liquid chromatography with precolumn derivatization (usingo-phthal-dialdehyde) and fluorescence detection.

The ATP synthesizing system of liver mitochondria.

Pathological states of the liver are numerous with prolonged alcohol consumption resulting in perhaps one of the most frequent. It seems quite appropriate, therefore, that a basic science workshop designed to review the current state of knowledge of liver metabolism includes a paper on the ATP synthesizing system of this tissue. Our laboratories were the first to isolate the ATP synthesizing system of liver (Catterall and Pedersen, 1971). A graduate student, William A. Catterall (now an Associate Professor of Pharmacology at the University of Washington, and an already well-known neurochemist), played a major role in these early studies (Catterall and Pedersen, 1971; Catterall and Pedersen, 1972; Catterall et al., 1973; Catterall and Pedersen, 1974). In the same year (1971) Henry Lardy’s laboratory also reported the isolation of the ATP synthesizing system of liver (Lambeth and Lardy, 1971). Since that time much additional information concerning the structure, function, and regulation of the ATP synthesizing system of liver mitochondria has accumulated in our laboratories. It is this information that I to summarize briefly today.