Atrial natriuretic peptide--cyclic GMP coupling and urinary sodium excretion during acute volume expansion in man

The present study examines hormonal and renal responses to acute volume expansion in normal man, with particular emphasis on the atrial natriuretic peptide (ANP)--cyclic GMP coupling. Two liters of isotonic saline were infused into eight normotensive male subjects over a 1-h period. Plasma and urinary measurements were made before, during, and up to 300 min after the start of the saline infusion. With the initial increase in urinary sodium excretion there were increases in plasma ANP and plasma cyclic GMP, which reached maximum levels at 15 min after the end of the saline infusion. Urinary cyclic GMP increased gradually during saline infusion up to approximately 60 min after the end of the infusion. Plasma ANP and plasma and urinary cyclic GMP excretion gradually declined thereafter. By contrast, urinary sodium excretion remained elevated up to the end of the observation period. The saline infusion was associated with marked reductions in plasma renin activity and aldosterone, which persisted up to the end of the study. These results suggest a coupling between the increases in plasma ANP, the production of cyclic GMP, and urinary sodium excretion, in particular during the initial renal response to acute volume expansion. However, other mechanisms including the suppression of the renin--angiotensin--aldosterone system may become increasingly important in the later natriuretic response to acute volume expansion.     

Droplet freezing of antibody-linked indicator red cells of sheep, ox, and human origin

A droplet freezing technique for the cryopreservation of indicator red cells is described. Recovery was crucially dependent on the composition of the solution in which the cells were suspended. Preliminary experiments to determine the relative importance of sucrose, glucose, sodium chloride and hydroxyethyl starch (HES) in determining the survival of trypsin-treated sheep red cells showed that the addition of sucrose or HES or both to isotonic sodium chloride solution increased recovery, whereas the additional inclusion of glucose was detrimental. It was shown that glucose penetrated the cells whereas sucrose did not. The optimum combination of sucrose and sodium chloride concentration, in the presence of 6 g/dl HES, was 7 g/dl sucrose plus 0.3 g/dl sodium chloride. Recovery was increased by increasing the concentration of HES, and maximal recovery was obtained by thawing the frozen droplets in phosphate-buffered saline at 40 °C. Trypsintreated ox and human cells gave much lower recovery than sheep cells when HES was used in the freezing mixture but the substitution of dextran (10 g/dl) for HES gave greater than 80% recovery with all three species. Ten different antibody-coupled reagent cells all gave >83% recovery. The effects of hematocrit, incubation time, and storage temperature are described. The preservation technique described is simple and convenient, and will make it possible to extend the use of immunoassay procedures using antibody-coupled red cells.     

Study of the viability of cultured human cells in suspensions

The success of cell therapy is directly related to the viability of cells used for transplantation. The cells used for transplantation are in some cases injected in suspension. However, the optimal conditions for the preservation of cell viability upon the preparation and storage of cell suspensions for transplantation have not been defined yet. The aim of the present work consisted in the selection of optimal conditions for the storage of suspensions of human submandibular salivary gland cells, differentiated cells of the submandibular salivary gland, and dermal fibroblasts in biocompatible solutions. Standard procedures of cell isolation and cultivation were used in the study. An automatic cell counter from BioRad was used to count the cells, and viability of the cells was assessed using staining with 4% Trypan Blue. The biocompatible solutions tested included phosphate-buffered saline, physiological saline for injections, and a 2% solution of human albumin in phosphate-buffered saline. The study showed that the human cells under investigation remained viable in suspension at both +4°С and +25°С for at least 24 hours, regardless of the carrier solution used. The highest content of viable cells of the salivary gland (more than 50%) at both temperatures examined was observed when cells were suspended in phosphate-buffered saline. However, the adhesive and proliferative properties of the salivary gland cells were better preserved at +4°С in case of 24 hours of incubation under the conditions described above. Fibroblasts maintained in physiological saline formed a homogeneous single-cell suspension that remained stable for 30 hours at +4°С; virtually no loss of cell viability was observed. The addition of 2% albumin resulted in a decrease of the viability of fibroblasts. Thus, storage and transportation in phosphate- buffered saline at +4°С can be recommended for suspensions of cells of the human submandibular salivary gland, whereas human fibroblast suspensions should be maintained at +4°С in physiological saline.     

Opposite effects of different hexachlorocyclohexane (lindane) isomers on cerebellar cyclic GMP: relation of cyclic GMP accumulation to seizure activity

A number of different depressant and convulsant agents have been shown to alter accumulation of cerebellar cyclic GMP. Since the different hexachlorocyclohexane (HCH) isomers elicit different pharmacological responses in mammals, we examined their effects on the accumulation of cerebellar cyclic GMP. Mice received one of the HCH isomers and were sacrificed for determination of cyclic GMP concentrations one hour later. Gamma-HCH increased cyclic GMP while alpha and delta-HCH decreased it. In addition, alpha and delta-HCH prevented the increase in cyclic GMP due to the gamma isomer. Picrotoxin increased cyclic GMP in a manner similar to that of gamma-HCH while strychnine produced only a small increase. All three HCH isomers inhibited the binding of 3H-TBOB (a ligand for the GABA-A-receptor linked chloride channel) in mouse cerebellum. It is concluded that the different HCH isomers can have different effects on cerebellar cyclic GMP accumulation and that these effects may be mediated through actions at the GABA-A receptor linked chloride channel.     

Dog Red Blood Cells : Adjustment of salt and water content in vitro

Dog red blood cells (RBC) lack a ouabain-sensitive sodium pump, and yet they are capable of volume regulation in vivo. The present study was designed to find in vitro conditions under which dog RBC could transport sodium outward, against an electrochemical gradient. Cells were first loaded with sodium chloride and water by preincubation in hypertonic saline. They were then incubated at 37°C in media containing physiologic concentrations of sodium, potassium, chloride, bicarbonate, glucose, and calcium. The cells returned to a normal salt and water content in 16–20 h. Without calcium in the medium the cells continued slowly to accumulate sodium. Removal of glucose caused rapid swelling and lysis, whether or not calcium was present. The net efflux of sodium showed a close relationship to medium calcium over a concentration range from 0 to 5 mM. Extrusion of salt and water was also demonstrated in fresh RBC (no hypertonic preincubation) when calcium levels in the media were sufficiently raised. The ion and water movements in these experiments were not influenced by ouabain or by removal of extracellular potassium. Magnesium could not substitute for calcium. It is concluded that dog RBC have an energy-dependent mechanism for extruding sodium chloride which requires external calcium and is quite distinct from the sodium-potassium exchange pump.     

Lysis of Yeast Cells Showing Low Susceptibility to Zymolyase

Lysis of commercial baker’s yeast cells was examined using Zymolyase. The lysis was stimulated by the addition of sodium sulfite or potassium chloride or both. The effect of potassium chloride was less than that of sodium sulfite, but the two compounds acted synergistically. The cells were effectively lysed by Zymolyase in the presence of 0.1 M sodium sulfite and 0.8 M potassium chloride. The extent of lysis was similar to that of brewery yeast cells obtained from a brewhouse. Cells pretreated with sodium sulfite did not show much of an increase in susceptibility to Zymolyase, but were effectively lysed by the enzyme in the presence of potassium chloride. Potassium chloride stimulated lysis only in the presence of Zymolyase. Yeast cells treated with cupric ions in the presence of sodium sulfite became highly susceptible to Zymolyase, suggesting irreversible destruction of the sodium sulfite-sensitive and potassium chloride-sensitive structure of the cell wall.

Cells of Saccharomyces cerevisiae prepared under various culture conditions were completely lysed by Zymolyase in the presence of sodium sulfite or potassium chloride or both.     

Role of hydrogen peroxide in the cytotoxicity of the xanthine/xanthine oxidase system.

1. The survival of mammalian epithelial cells exposed in vitro to the xanthine/xanthine oxidase system in phosphate-buffered saline (PBS) or serum-containing medium (SCMEM) was investigated. 2. The cytotoxic effect observed depended on the composition of the medium in which the enzymic reaction was carried out; a surviving fraction of 5 x 10(-5) was found for cells exposed in PBS and 5.2 x 10(-1) for those in SCMEM. 3. The cytotoxic product(s) formed by the xanthine/xanthine oxidase system was relatively stable in PBS; survival of cells incubated after completion of the enzymic reaction was always less than that found for cells exposed during the reaction in the same system. 4. Superoxide dismutase or mannitol present during the enzymic reaction did not inhibit the cytotoxic effect. 5. NaN3 (a single-oxygen quencher and a catalase inhibitor) added to the system in SCMEM caused a reduction in survival to the level observed for cells exposed to the enzymic reaction in PBS. 6. Catalase completely protected cells, but no protection was observed when both catalase and NaN3 were present in the reaction mixture. 7. A similar cytotoxic effect was produced when cells were treated with H2O2 alone. 8. The rate of H2O2 decomposition in medium was accelerated by the presence of serum, but this was completely inhibited by NaN3. 9. It is concluded that H2O2 is the major cytotoxic product formed by the xanthine/xanthine oxidase system.     

Modifications of liver content of cyclic nucleotides in the rat poisoned with white phosphorus]

In rat liver following white phorphorus poisoning a biphasic increase in cyclic AMP concentration was observed. After a lag period of 1 hour the cyclic AMP content rose to a first peak at 4 hours and to a second peak at 12 hours of intoxication. The cyclic AMP level fell to normal after 24 hours, by which time the cyclic nucleotide concentration was approaching control values. On the contrary, cyclic GMP content was found to the normal level during the different stages of intoxication. Only at 36 hours the cyclic GMP amount appeared significantly increased above the control values. Serum activity of alanine- and aspartate-amino transferases was found changed from 8 hours to 24 hours after poisoning. The serum level of the two enzymes was overlapping the control values after 36 hours. These results are discussed in relation to hepatocyte necrosis following white phosphorus intoxication.     

FACTORS INFLUENCING THE RESPIRATION OF ERYTHROCYTES : I. PRIMITIVE AVIAN ERYTHROCYTES

1. The oxygen consumption of normal and "primitive red cells" of fowls'' blood has been determined at intervals in the course of an anemia produced by the injection of phenylhydrazine. The "primitive red cells" have an oxygen consumption at least twenty to twenty-five times greater than the normal red cells. 2. Suspension of the cells derived from the blood in anemia in sodium chloride solutions of various concentrations has comparatively little effect upon the oxygen consumption of the cells. 3. The red cells from anemic blood are sensitive to variations in the reaction of the medium in which they are suspended. The maximum oxygen consumption, after addition of a saline solution containing variable amounts of acid to the blood, took place at pH 7.75. They appeared somewhat more sensitive to variations on the acid side of this reaction than on the alkaline. 4. Addition of glucose to the medium increased the oxygen consumption of the cells. Their metabolism in a physiological saline solution containing 0.6 per cent of glucose was 15 per cent higher than in one in which no glucose was present. 5. Certain amino acids in low concentrations had little effect on oxygen consumption, though at higher concentrations some of them definitely diminished it.     

Morphological characterization of cultured bovine aortic endothelial cells and the effects of atriopeptin II and sodium nitroprusside on cellular and extracellular accumulation of cyclic GMP

Primary, first and second passaged endothelial cells from bovine aorta were grown in plastic culture dishes or on glass coverslips. The cells were characterized by their monolayer cobblestone appearance at confluence, their immunofluorescent staining for factor VIII-related antigen, their specific uptake of low density lipoprotein and by their ultrastructure. Following stimulation of the cells by atriopeptin II or sodium nitroprusside, both cellular and extracellular cyclic GMP levels were measured. Cellular cyclic GMP content was increased greatly by atriopeptin II in a time-dependent manner while sodium nitroprusside was essentially without effect. Increases in tissue cyclic GMP levels were associated with a time-dependent accumulation of the nucleotide in the extracellular compartment. Zaprinast, a specific inhibitor of cyclic GMP phosphodiesterases, did not significantly affect either basal or atriopeptin II-stimulated increases in cyclic GMP content, nor extracellular accumulation of the nucleotide. It is concluded that the cyclic GMP content of endothelial cells is not solely dependent on degradation by phosphodiesterases but also involves release of cyclic GMP into the extracellular compartment.     

Studies on the role of cyclic guanosine 3':5'-monophosphate and extracellular Ca2+ in the regulation of glycogenolysis in rat liver cells.

Catecholamines increased guanosine 3':5'-monophosphate (cyclic GMP) accumulation by isolated rat liver cells. The increases in cyclic GMP due to 1.5 muM epinephrine, isoproterenol, or phenylephrine were blocked by phenoxybenzamine but not by propranolol. The possibility that cyclic GMP is involved in the glycogenolytic action of catecholamines seems unlikely since cyclic GMP accumulation is also elevated by carbachol, insulin, A23187, and to a lesser extent by glucagon. Furthermore, carbachol had little effect on glycogenolysis while insulin actually inhibited hepatic glycogenolysis. The rise in cyclic GMP due to carbachol was abolished by atropine and that due to all agents was markedly reduced by the omission of extracellular calcium. However, the glycogenolytic action of glucagon and catecholamines was only slightly inhibited by the omission of calcium. The only agent which was unable to stimulate glycogenolysis in calcium-free buffer was the divalent cation ionophore A23187. There was a drop in ATP content of liver cells during incubation in calcium-free buffer which was accompanied by an inhibition of glucagon-activated adenosine 3':5'-monophosphate (cyclic AMP) accumulation. The presence of calcium inhibited the rise in adenylate cyclase activity of lysed rat liver cells due to glucagon or isoproterenol but not that due to fluoride. These results suggest that the stimulation by catecholamines and glucagon of glycogenolysis is not mediated through cyclic GMP nor does it depend on the presence of extracellular calcium. Cyclic GMP accumulation was increased in liver cells by agents which either inhibit, have little affect, or accelerate glycogenolysis. The significance of elevations of cyclic GMP in rat liver cells remains to be established.     

Differential Lytic Response of Enterococci Associated with Addition Order of Lysozyme and Anions

Suspensions of Streptococcus faecium, prepared by washing with and resuspending in water, were lysed slowly if sodium chloride was added prior to lysozyme; however, if brief incubation with lysozyme was followed by addition of sodium chloride, lysis was immediate and extensive. Relatively lysozyme-resistant strains of S. faecalis could be lysed readily by adding lysozyme first. The primary addition of lysozyme apparently resulted in a "sensitized" cell with a damaged wall, as evidenced by N-acetylhexosamine release. Anionic detergents could replace sodium chloride in lysing these sensitized cells. The difference in activity associated with the order of addition probably involved a competition for reactive sites on the cell surface.     

Intake of water and sodium chloride solution by the rats after central administration of litorin and immunization with litorin-bovine serum albumin conjugate

Central injection of litorin suppressed water and sodium chloride solution intake, and also decreased the preference of saline of unrestrained rats during 1 hour test. Active immunization of animals with litorin-bovine serum albumin conjugate led to the augmentation of their daily saline preference. Immunized rats increased also avidity to salt after deprivation, though the consumption volume of liquids decreased. The data, obtained on the immunized animals prove the importance of definite level of endogenous bombesin-like peptide in the salt appetite of rats. The central effect of litorin is discussed in the aspect of changing by it the evaluation of gustatory information.     

Proteins of the human erythrocyte membrane as modified by pronase

Pronase degrades proteins on the outer surface of the human erythrocyte membrane which run in polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulfate at a molecular weight of approximately 125,000. Carbohydrate and sialic acid are removed, but fragments of molecular weight 50,000 to 100,000 remain attached to the membrane. The most prominent fragment, one of molecular weight about 73,000, can be labeled with a membrane-impermeable reagent (sulfanilic acid diazonium salt), so it is still accessible from the outside of the cell. Pronase rapidly inactivates membrane-bound acetylcholinesterase, but it has relatively little effect on the facilitated diffusion of glucose; both are inhibited by the diazonium salt. Extensive digestion leads to potassium loss and osmotic lysis. Ghosts prepared in 15 mosm-Tris (pH 7.6) are extensively degraded by pronase: essentially all the protein shifts to low molecular weight. Pronase is even more potent in 3% sodium dodecyl sulfate. Ghosts prepared from intact cells which have been treated with the enzyme hydrolyze when dissolved in the detergent unless steps are taken to inhibit proteolysis.     

Renal and systemic effects of synthetic atrial natriuretic factor

A synthetic peptide corresponding to a sequence of 26 amino acids contained in endogenous rat atrial natriuretic factor (ANF), was infused into one renal artery of anesthetized dogs for a comprehensive in vivo evaluation of the renal and systemic effects of pure ANF. The results proved conclusively that ANF acted directly on the kidney since urine volume and fractional excretion of sodium, potassium, chloride and calcium were elevated in a dose-related manner in the ANF-treated kidney, but were not significantly affected in the contralateral saline-infused organ. The maximum effects achieved with the synthetic ANF were higher than any reported following intravenous administration of crude extracts of rat atria and were similar to those produced by thiazide diuretics. In four of the five dogs studied, renal vascular resistance fell progressively as doses of ANF were increased. Glomerular filtration rate was not significantly elevated during ANF infusion, but was correlated with sodium excretion rates. Even though mean arterial pressure was progressively reduced, there was no significant change in heart rate and no stimulation of renin secretion. Arterial cyclic GMP concentration was higher in the basal state and rose more rapidly than did renal venous levels, indicating that increases in circulating concentrations of arterial cyclic GMP originated from an extrarenal source. Dose-related elevations in urinary cyclic GMP excretion could be explained by increased cyclic GMP filtration, by enhanced production in tubular cells, or by renal tubular secretion. Especially in the saline-infused kidney, there was a clear dissociation between excretion of cyclic GMP and fractional sodium excretion. We conclude that the synthetic ANF increased electrolyte excretion via a direct renal action which was not solely dependent upon changes in renal vasculature, renin secretion or cyclic GMP levels.     

Effects of hetastarch and mannitol on prolonging survival in stable hypothermia in rats

In rats, prolonged stable hypothermia ( approximately 24 h at body temperature of 19 degrees C) is characterized by a time-dependent increase in hematocrit, plasma osmolality, and red blood cell fragility and a decrease in plasma volume. These changes impede tissue microcirculation and could limit survival. As a countermeasure, we used plasma volume expanders of both long (hetastarch)- and short-lasting (mannitol) characteristics to improve microcirculation and hopefully hypothermia survival. Infusion of 6% hetastarch at hour 3 in hypothermia significantly (P < 0.05) enhanced survival over saline control (33.5 vs. 23.8 h); a significant delay in the increases of hematocrit and cell fragility was also observed compared with those in saline controls. Treating the animal with 6% hetastarch at hour 20 during hypothermia caused a similar but less-effective improvement in survival. In contrast, treating the rats with 6% mannitol at hour 3 or 20 during hypothermia failed to enhance survival over saline control, although transient improvement in plasma volume was observed. Our results indicate that by using a long-lasting volume expander, which tends to better maintain plasma volume and rheological parameters governing microcirculation than does saline or a short-lasting volume expander, hypothermia survival can be significantly improved.     

Digestion of host immunoglobulin and activity of midgut proteases in the buffalo fly Haematobia irritans exigua

The digestion of blood by the buffalo fly (Haematobia irritans exigua) was monitored for 6h at 33 degrees C after a single meal. Following the meal, the concentration of soluble protein within the midgut increased to a peak at 2 hours then decreased steadily over the next 4h. The magnitude of the increase in soluble protein at 2h indicated a release of protein from another source; most likely from lysed red blood cells. The immunoglobulin (IgG) fraction of the blood meal was digested rapidly (50% within one hour of feeding) and fully digested within 4h. This is indicative of its accessibility to digestive enzymes within the midgut. In contrast, when flies had continuous access to blood, the concentration of IgG in the midgut remained at a more constant level. The loss of antigen-binding activity of a specific antibody was more rapid than complete degradation of the IgG, with 70% of binding activity lost within one hour of feeding. The level of trypsin activity in the midgut increased from pre-feeding levels to reach a peak at 2h before returning to basal levels after 6h. The pattern of trypsin activity follows closely that of the concentration of soluble protein in the midgut (r=0.88). The activity of leucine aminopeptidase in the midgut also increased immediately after feeding and remained elevated for 4 h before declining to a basal level after 6h. The rapid digestion of IgG and subsequent loss of antibody activity suggests that for a specific anti-buffalo fly antibody to be effective it would need to be able to either evade the digestive system or induce a rapid response.     

Effects of circulating red cell mass on diet-induced atrial thrombosis in mice

Atrial thrombosis is a common lesion in female Taconic Swiss mice fed a high-fat (28%), low-protein (8%), hypolipotropic diet for 10 wk or longer. After the third week of such feeding the mice studied here were injected with either erythropoietin, washed, packed red blood cells, lysed red blood cells, plasma or physiological saline.In mice receiving injections of lysed red cells, plasma or saline, respectively 75, 54 and 82% of those surviving for 10 wk had developed atrial thrombosis. Hematocrits were 9.3% or below in these groups. Hematocrits were maintained at an average of 33.0% in the erythropoietin group and 32.4% in the transfused (packed erythrocytes) group. Only one of the erythropoietin injected animals and none of the transfused animals developed atrial thrombosis. The evidence indicates that the anemia induced by the experimental diet results from lack of erythropoietin production or activity and that the hypoxia of anemia plays a role in the development of atrial thrombosis.     

Azoindoxyl methods for the investigation of hydrolases. IV. Suitability of various diazonium salts (author's transl)]

Using fresh frozen, freeze-dried or cryostate sections from aldehyde fixed rat tissues 13 diazonium salts were tested as simultaneous coupling reagents for the localization of acid, neutral and alkaline hydrolases with azo indoxyl methods. Hexazotized new fuchsine and/or Fast blue B are the diazonium salts of choice for the demonstration of acid beta-galactosidase, neuraminidase, beta-N-acetylglucosaminidase, acid phosphatase, and non-specific esterase followed by hexazotized p-rosaniline. Fast blue VB, BB and RR and Fast violet B are recommended for the investigation of alkaline phosphatase and lactase, Fast garnet GBC for acid beta-galactosidase, glucosaminidase and lactase. Fast red B, RC, RL and TR and Fast black K can only be employed for lactase studies. The exact concentration of the coupling reagent depends on the activity of the enzyme and the organ imvestigated. On the average 0.01-0.02 ml unstable diazonium salt/ml and 0.3--1 microgram stable diazonium salt/ml are sufficient for the correct localization of these hydrolases. Freeze-dried cryostat sections yield the best results in the demonstration of lactase and alkaline phosphatase independent on the coupling reagent used. Sections from formaldehyde or glutaraldehyde fixed organs are superior for the localization of the other hydrolases; an exception is the investigation of acid beta-galactosidase and glucosaminidase with Fast garnet GBC. Then, excellent results are obtained also with freeze-dried material. Fresh frozen sections are suitable for the localization of lactase with hexazotized new fuchsine or p-rosaniline and of alkaline phosphatase with Fast blue VB and BB or violet B. The total activity of acid, neutral and alkaline hydrolases can be investigated using semipermeable membranes in combination with all unstable and stable diazonium salts of choice. Reliable osmification of the azoindoxyl dye is only possible if hexazotized p-rosaniline is employed for coupling; without further posttreatment all azoindoxyl dyes are extracted by ethanol, isopropanol or xylol. 7 incubation media are given for the demonstration of hydrolases with azoindoxyl methods at the level of light microscopy for routine studies and typical examples for the application of these methods are presented. A modified procedure is described for the freeze-drying of cryostat sections with the Edwards-Pearse tissue dryer EPD3.     

In vitro evaluation and study of the survival of erythrocytes preserved in a saline-adenine-glucose-mannitol medium for 35 days

The saline-adenine-glucose-mannitol (SAGM) solution for resuspension of red cells was evaluated on 30 blood units tested over 42 days and compared to 5 red cell concentrates collected on the conventional CPD medium. Total and extra-cellular hemoglobin, potassium, pH, ATP and DPG concentrations, osmotic fragility, schizocyte formation, and red cell antigenicity were studied through the storage period. Chromium survival studies of autologous donated red cells were performed in 10 donors. Red cell concentrates resuspended in SAGM solution showed at the 35th day of conservation at 4 degrees C, a mean storage hemolysis of only 0.66%, an ATP concentration of 67% of the initial value, a schizocyte proportion of less than 1.5%, a mean 24 hour posttransfusion viability of 88.33% and a mean red cell T 1/2 survival of 25 days 10 hours. No alteration of common blood group antigens could be found after storage of red cells for 42 days.