The QRS complex during myocardial ischemia. An experimental analysis in the porcine heart.

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Abstract

Although ST segment deflections have been widely utilized as a means of assessing the degree of underlying ischemic injury, the relationship of QRS complex alterations to the ischemic process is poorly understood. In this study we made a beat-to-beat analysis of the QRS complex in terms of ventricular activation time (CT) and R wave voltage (V) in the acutely ischemic porcine myocardium and analyzed the relationship of these responses to changes in the area of ischemic involvement, altered myocardial energy demands, and plasma [K+]0 levels. With the onset of ischemia the QRS complex underwent a specific and reproducible biphasic sequence with an initial decrease in CT and V indicating a transient increase in the conduction velocity of the ischemic tissue. Subsequently both CT and V returned briefly to control and then increased dramatically, now indicating a marked decrease in conduction velocity. The time when CT first began to increase (Tc) was shortened by enlarging the area of ischemia or after an inotropic intervention and was lengthened by decreasing the area of ischemia or with administration of propranolol. Moreover Tc was found to be inversely proportional to plasma [K+]0 in the range 3.4-8.8 mM, above which the initial decrease in CT and V was no longer present. We conclude that this biphasic sequence of QRS alterations in early myocardial ischemia is attributable to a progressive leakage of potassium out of the ischemic cells which in turn alters both the time-course and transmural pathway of the activation process through the ischemic tissue. These changes are related to both inotropic state and the area of ischemic involvement.

Authors

R P Holland, H Brooks

Responsiveness to glucagon in fetal hearts. Species variability and apparent disparities between changes in beating, adenylate cyclase activation, and cyclic AMP concentration.

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Abstract

Previous studies of the ability of the immature heart to respond to glucagon have yielded conflicting results. To test the possibility that the apparent discrepancies might be explained in part by species variability, isolated hearts of fetal mice and rats (13-22 days' gestational age) were studied under identical conditions in vitro. Changes in atrial rate and ventricular contractility were measured in spontaneously beating hearts exposed to glucagon, and activation of adenylate cyclase was assayed in cardiac homogenates. In mice of 16 days' gestational age or less, there was no change in heart rate in response to glucagon; at 17-18 days, minimal responsiveness was present; and after 19 days, 10muM glucagon caused an increase in spontaneous atrial rate of 30 +/- 4% (SEM) (P less than 0.001). Measurement of the extent and speed of volume displacement of the isotonically contracting hearts with a specially constructed capacitance transducer revealed that ventricular inotropic responsiveness also appeared after 17-19 days. Cardiac stores of glycogen were reduced in older hearts exposed to glucagon, but not in those aged less than 16 days. In contrast, glucagon failed to activate adenylate cyclase in homogenates of hearts of fetal mice at any age. Furthermore, glucagon failed to elicit an increase in the concentration of cyclic AMP in spontaneously beating hearts that developed tachycardia. Responses in hearts of fetal rats were distinctly different from those in mouse hearts: at no age was there any change in heart rate, strength of contraction, glycogen content, or adenylate cyclase activation. Thus, there are major species differences in cardiac pharmacological maturation. Although the mouse heart develops the ability to increase its rate and strength of contraction and to undergo glycogenolysis in response to glucagon well before birth, the rat heart does not. In addition, there is an apparent disparity in late fetal mouse hearts between the ability of glucagon to induce functional responses and its ability to stimulate adenylate cyclase and increase cyclic AMP levels. It is impossible, of course, to rule out absolutely the possibility that localized increases in a critical cyclic AMP pool were present but too small to measure in the entire tissue. Nevertheless, the most obvious interpretation of our results is that they are compatible with the hypothesis that glucagon may exert some of its hemodynamic effects independently from the adenylate cyclase-cyclic AMP system in the late-fetal mouse heart.

Authors

K Wildenthal, D O Allen, J Karlsson, J R Wakeland, C M Clark Jr

Renal adenylate cyclase and the interrelationship between parathyroid hormone and vitamin D in the regulation of urinary phosphate and adenosine cyclic 3',5'-monophosphate excretion.

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Abstract

This study examined the role of cyclic AMP in the phosphaturic response to parathyroid hormone in vitamin D-deficient rats. Infusion of purified bovine parathyroid hormone (13.3 mug/h) into control, D-fed, or D-deficient, thyroparathyroidectomized rats produced a sixfold increase in renal phosphate and cyclic AMP excretion in D-fed rats, but only a two- to threefold increase in both parameters in D-deficient animals. Intravenous injection of parathyroid hormone over the dosage range from 1-50 mug/kg resulted in a dose-dependent increase in phosphate and cyclic AMP excretion with both D-fed and D-deficient thyroparathyroidectomized rats. However, the D-deficient rats responded to these injections of parathyroid hormone with a two- to threefold increase in both renal phosphate and cyclic AMP excretion at the highest dose of 50 mug/kg, whereas the D-fed animals' response was 35-fold and 11-fold over control excretion levels of phosphate and cyclic AMP, respectively. To directly examine the role of the renal cortical adenylate cyclase system in the blunted phosphaturic and urinary cyclic AMP responses to parathyroid hormone in D-deficient rats, we prepared a plasma membrane fraction enriched in this enzyme activity from the renal cortex of D-fed and D-deficient thyroparathyroidectomized rats. The renal cortical adenylate cyclase of D-deficient rats showed significantly (P less than 0.001) less activation by parathyroid hormone over the hormone concentration range from 0.3 to 7.0 mug/ml than was observed with the enzyme prepared from D-fed animals. Basal adenylate cyclase activity and the fluoride-stimulated enzyme activity were not altered by the state of D-deficiency. These experiments demonstrate that the blunted phosphaturic response to parathyroid hormone observed in D-deficient rats is associated with the reduced responsiveness of the renal cortical adenylate cyclase to the hormone. Moreover, the defect in the renal membrane adenylate cyclase system appears to be localized at the level of PTH binding to membrane receptors or, alternatively, at the level of transmission of the hormone-receptor binding signal to the catalytic moiety of this membrane enzyme.

Authors

L R Forte, G A Nickols, C S Anast

Heart muscle performance after experimental viral myocarditis.

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Abstract

As part of an inquiry into possible antecedents of idiopathic cardiomyopathy, acute experimental coxsackie virus myocarditis was studied for late structural and functional sequelae. Myocarditis was induced in 12- and 22-day-old hamsters by inoculation with coxsackie virus B3. Early viremia occurred, followed by virus replication in heart muscle. Maximum peak developed tension (Tpd) of isometrically contracting isolated heart muscle was depressed 17 and 43% in the animals inoculated at 12 days, and studied 18 and 90 days later, respectively, as compared to their uninoculated controls. In both infected groups, less muscle stretch was required to reach the length at which Tpd was produced. Animals studied 180 days after inoculation did not differ from controls. The muscles from animals inoculated at 22 days of age and studied 18 days later showed a 15% depression of Tpd compared to their controls. Glycerinated muscles from this infected group developed 50% less tension than their controls. The muscles of hamsters inoculated with virus at 22 days and studied 90 and 180 days later showed no change in Tpd. The data suggest that contractility and compliance of heart muscle are decreased 18 days after inoculation, but recover by 90 days if the animals are inoculated at age 22 days. However, if the animals are inoculated at a younger age (12 days), depression of myocardial performance persists for at least an additional 90 days. It is concluded that the inflammatory stage of experimental acute coxsackie virus B3 myocarditis in the Syrian golden hamster may be followed by residual alterations in contractile proteins and myocardial function.

Authors

C O Adesanya, A H Goldberg, W P Phear, K A Thorp, N A Young, W H Abelmann

Translation of ovalbumin mRNA in Xenopus laevis oocytes. Characterization of the system and effects of estrogen on injected mRNA populations.

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Abstract

Ovalbumin messenger RNA (mRNAov) purified from hen oviduct was injected into Xenopus laevis oocytes. The oocytes were incubated in culture medium containing [3H]leucine. Analysis of the oocyte cytosol on Sephadex G-15O columns demonstrated a peak of radioactivity which cochromatographed with authentic ovalbumin. Radioactive protein contained in this peak was precipitated by ovalbumin antiserum, coelectrophoresed with ovalbumin on sodium dodecyl sulfate (SDS) and urea gels at pH 8.7, and eluted with the protein at the same pH (4.8) on CM-cellulose chromatography. Injection of increasing amounts of mRNAov was found to elicit a linear response in terms of ovalbumin synthesis. Moreover, there was linear incorporation of radioactivity into microinjected oocytes over a minimum period of 91 h. Less than 1 ng mRNAov was detected in this system. Ovalbumin mRNA activity was present in RNA preparations from chicks treated with estrogen but was undetectable in animals withdrawn from the hormone. This study constitutes an initial demonstration of a steroid hormone-induced alteration in mRNA population as assayed in intact viable heterologous cells.

Authors

L Chan, P O Kohler, B W O'Malley

Reagin-mediated asthma in rhesus monkeys and relation to bronchial cell histamine release and airway reactivity to carbocholine.

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Abstract

Rhesus monkeys with persistent immediate-type cutaneous and respiratory responses (RR) to ascaris antigen (AA) were compared with rhesus monkeys with skin reactivity and no respiratory responses, and animals with no skin reactivity and no respiratory responses to inhaled antigen (NR). The RR group could not be distinguished from the nonresponding (NR) group by the cutaneous skin test titers, serum, or respiratory secretion IgE concentration. Leukocyte histamine (H) release due to anti-IgE was similar with peripheral blood leukocytes and bronchial lumen mast cells (MC) from RR and NR animals. The RR group of animals could be distinguished from the NR group by their degree of sensitivity to inhaled carbocholine and H release from respiratory MC exposed to AA. The RR group demonstrates consistent, persistent respiratory responses suitable for immunologic, pharmacologic, and physiologic studies. Finally, it was found that the IgE concentration in respiratory secretions of rhesus monkeys was comparatively higher than in serum, evidence for IgE as a secretory Ig in the respiratory tract of this species.

Authors

R Patterson, K E Harris, I M Suszko, M Roberts

A rabbit reticulocyte model for the role of hemin-controlled repressor in hypochromic anemias.

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Abstract

Hemin allows maximal protein synthesis in intact rabbit reticulocytes and their cell-free lysate preparations by retarding the formation of a translational repressor (HCR) found in the postribosomal supernate. In order to evaluate the role of HCR in the pathogenesis of hypochromic anemias, HCR was isolated and partially purified from intact rabbit reticulocytes incubated in vitro with either 0.1 mM alpha,alpha-dipyridyl (an iron-chelating agent) or 0.1 M ethanol. Both of these agents inhibit reticulocyte protein synthesis. Hemin (50 muM) protects against the inhibition by both agents. A ferrous iron-transferrin mixture, however, protects only against alpha,alpha-dipyridyl. Both alpha,alpha-dipyridyl and ethanol inhibit heme synthesis before the time that protein synthesis is affected, while neither lowers either ATP or GSH levels. These results indicate that while both agents inhibit heme synthesis, alpha,alpha-dipyridyl does so by inducing iron deficiency while ethanol works at a non-iron-requiring step. When HCR was isolated from intact cells and assayed in the reticulocyte cell-free systems, plus and minus hemin, premature appearance of HCR was found in cells incubated in vitro with alpha,alpha-dipyridyl or ethanol. When hemin was present in the intact cell incubation, the appearance of HCR was retarded. The HCR from alpha,alpha-dipyridyl ethanol-treated cells was partially purified and eluted at the same location on a Sephadex G-200 column (molecular weight approximately 3 x 10(5)) as that from postribosomal supernates incubated minus hemin. In addition rabbits with phenylhydrazine-induced hemolytic anemia were given intravenous ethanol in vivo at a dose of 0.4 ml/kg. This concentration of alcohol resulted in an inhibition of the rate of heme synthesis and protein synthesis as well as an acceleration of HCR formation in reticulocytes. The HCR from these in vivo treated rabbits was isolated, partially purified, and assayed in an identical fashion as the in vitro experiments. These in vivo experiments further support the physiological and pathophysiological role of HCR in reticulocytes. On the basis of these results a model for a role of HCR in some of the hypochromic anemias is proposed. In iron deficiency or chronic disease (where iron is not available to the erythroblast for heme synthesis) HCR appears prematurely and inhibits protein synthesis. When heme synthesis is inhibited by ethanol but there is sufficient intracellular iron, HCR appears prematurely and inhibits protein synthesis, iron accumulates in the erythroblast, and the end result is sideroblastic anemia.

Authors

M L Freedman, J Rosman

Fractionation of cells on a discontinuous Ficoll gradient. Study of subpopulations of human T cells using anti-T-cell antibodies from patients with systemic lupus erythematosus.

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Abstract

Patients with active systemic lupus erythematosus (SLE) had a decrease in a subpopulation of cells (fraction D) when peripheral blood lymphocytes were separated on a discontinuous Ficoll gradient. Preincubation of SLE cells at 37 degrees C for 30 min led to a marked decrease in this fraction, composed primarily of thymus-derived (T) cells. Supernates of such preincubations were found to cause a reduction in fraction D cells from normal humans. The active factor in the supernate was found to be an IgG antibody. Similarly, serum from patients with active SLE produced a reduction in fraction D cells from normal donors. This activity was also found in the IgG fraction, and could be absorbed with a pure T-cell population. Depletion of macrophages and complement did not reduce the SLE anti-T-cell antibody-mediated loss of cells from fraction D; however, heat-aggregated human gamma globulin led to impairment of the reaction. These findings suggest that antibody-dependent direct lymphocyte-mediated cytotoxicity may play a role in T-cell lymphopenia of SLE. It was further noted that the SLE anti-T-cell antibodies, in contrast to rabbit antihuman thymocyte serum, recognized fraction D cells but not fraction E cells from normals. Since both fractions are largely T cells, it appeared that the SLE serum was directed against cell-membrane antigenic determinants present on fraction D T cells, which were absent or reduced in quantity on fraction E T cells. Thus, evidence was presented indicating the presence of at least two subpopulations of cells in man. This was supported by differential absorption of the anti-T-cell sera with fractions D and E.

Authors

W Glinski, M E Gershwin, A D Steinberg

Degradation of cartilage proteoglycan by human leukocyte granule neutral proteases--a model of joint injury. I. Penetration of enzyme into rabbit articular cartilage and release of 35SO4-labeled material from the tissue.

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Abstract

The present work was undertaken to explore the effect of two purified neutral proteases derived from human peripheral blood polymorphonuclear leukocytes (PMN) on articular cartilage as a model of joint injury. Human leukocyte elastase and chymotrypsin-like enzyme, purified by affinity chromatography, released 32SO4 from labeled rabbit articular cartilage slices in vitro. Release of isotope was initially delayed, suggesting that either a lag in enzyme penetration occurs or that size of degradation fragments is a limiting factor in diffusion of label out of the tissue. The release of 35SO4 was inhibited by preincubation of elastase and chymotrypsin-like enzyme with human alpha 1-anti-trypsin, or with their specific chloromethyl ketone inactivators, and the action of elastase was also inhibited by a monospecific antiserum to PMN elastase, freed of major serum proteinase inhibitors. Immunohistochemical staining procedures revealed the presence of PMN elastase inside the matrix of cartilage slices after a 20-min exposure of tissue to either the pure enzyme or crude PMN granule extract. Serum alpha 1-antitrypsin failed to penetrate into the cartilage slices under identical in vitro conditions. In association with the results reported in the accompanying paper, these findings suggest a model of cartilage matrix degradation by PMN neutral proteases in which local protease-antiprotease imbalance, coupled with different rates of penetration of protease and antiprotease into target tissue, plays a key role in accounting for matrix damage.

Authors

A Janoff, G Feinstein, C J Malemud, J M Elias

Degradation of cartilage proteoglycan by human leukocyte granule neutral proteases--a model of joint injury. II. Degradation of isolated bovine nasal cartilage proteoglycan.

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Abstract

Extracts of human peripheral blood polymorphonuclear leukocyte granules, and two purified proteases derived from such extracts, an elastase and a chymotrypsin-like enzyme, degrade isolated bovine nasal cartilage proteoglycan at neutral pH. Viscosity studies indicate that the leukocyte granule extracts lack hyaluronidase activity and that their degradative effect on proteoglycan at physiological pH is due entirely to proteolytic action. Sepharose 4B gel chromatography and SDS-polyacrylamide gel electrophoresis of proteoglycan fractions treated with leukocyte granule enzymes at pH 7.0 indicate that they degrade one of the proteoglycan link proteins, release a fragment from the hyaluronic acid-binding portion of the proteoglycan subunit core protein, and break down the remainder of the proteoglycan subunit molecule into peptide fragments with varying numbers of chondroitin sulfate chains. Immunodiffusion studies indicate that the antigenic determinants of the proteoglycan subunit core protein and the link proteins survive treatment with granule proteases. Similar degradation of human articular cartilage proteoglycan by granule neutral proteases can be presumed to occur, in view of the similarity of structure of human articular and bovine nasal cartilage proteoglycans. The release of granule enzymes in the course of neutrophil-mediated inflammation can thus result in the degradation of cartilage matrix proteoglycan, leading to cartilage destruction and joint injury.

Authors

H Keiser, R A Greenwald, G Feinstein, A Janoff

Comparative properties of the Charcot-Leyden crystal protein and the major basic protein from human eosinophils.

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Abstract

Guinea pig eosinophil granules contain a protein, the major basic protein (MBP), which accounts for more than half of the total granule protein, has a high content of arginine, and displays a remarkable tendency to form disulfide-linked aggregates. In this study we have purified a similar protein from human eosinophil granules and have compared the human MBP to the protein comprising the Charcot-Leyden crystal (CLC). Eosinophils from patients with various diseases were purified and disrupted, and the granule fraction was obtained. Examination of the granule fraction by transmission electron microscopy showed numerous typical eosinophil granules. Analyses of granule lysates by gel filtration and by polyacrylamide gel electrophoresis revealed the presence of peroxidase and MBP with properties similar to that previously found in guinea pig eosinophil granules. The human MBP had a molecular weight of 9,200, contained less than 1% carbohydrate, was rich in arginine, and readily formed disulfide-bonded aggregates. CLC were prepared from eosinophil-rich cell suspensions by homogenization in hypotonic saline. The supernates following centrifugation of cell debris spontaneously formed CLC. Analysis of CLC revealed the presence of a protein with a molecular weight of 13,000 containing 1.2% carbohydrate. The protein displayed a remarkable tendency to aggregate even in the presence of 0.2 M acetic acid. Human MBP and CLC protein differed in their molecular weights, carbohydrate compositions, and amino acid analyses. Mixtures of the MBP and the CLC protein yielded two bands in polyacrylamide gel electrophoresis. Neither eosinophil protein increased vascular permeability in the guinea pig skin or contracted the guinea pig ileum. The results indicate that the human MBP and the CLC are distinct substances with properties such that one cannot be derived from the other.

Authors

G J Gleich, D A Loegering, K G Mann, J E Maldonado

The content and metabolism of cyclic adenosine 3', 5'-monophosphate and cyclic guanosine 3', 5'-monophosphate in adenocarcinoma of the human colon.

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Abstract

Data from cultured cells have suggested that cyclic AMP and cyclic GMP may be important determinants of cell growth and transformation. However, few studies have examined cyclic nucleotide content and metabolism in naturally occurring tumors of man. Accordingly, in the present study we compared cAMP and cGMP levels and metabolism in carcinomas of the human colon to those of the adjacent uninvolved mucosa after therapeutic resection of these tissues. The cAMP content of the tumors, determined in samples frozen 30 min after excision, was significantly lower than that of the adjacent mucosa, when expressed on the basis of tissue wet weight, protein, or DNA content. By contrast, the cGMP content of the tumors was higher than that of the surrounding mucosa if calculated on the basis of tissue wet weight, but this difference did not persist when correction was made for the higher protein or DNA content of the tumors. Incubation of slices of mucosa or tumor with or without theophylline in vitro increased tissue cAMP and cGMP content above levels observed in frozen samples of the same tissue. However, after such incubations cAMP levels in the tumors remained clearly below that of the mucosa, while cGMP content of the two tissues did not differ. The failure of theophylline to abolish differences in cAMP content and the comparable activities of high and low Km cAMP-phosphodiesterase in homogenates of the two tissues suggested that the lower cAMP content of the tumors was a consequence of diminished cAMP synthesis rather than enhanced degradation. This possibility was supported by the reduction in basal and maximal prostaglandin E1 (PGE1)-responsive adenylate cyclase activity found in tumor homogenates relative to those of mucosa, and the lower levels of cAMP in tumor slices after incubation of the tissues with a maximal dose of PGE1 and theophylline. Since NaF-responsive adenylate cyclase activity was not significantly reduced in the tumors, the lower basal and PGE1 activities may not be related to a deficiency of the catalytic unit of the cyclase complex in this tissue. The role of reduced activity of the adenylate cyclase-cAMP system and/or reduced tissue cAMP-to-cGMP ratios in the pathogenesis of colonic carcinoma is uncertain, but these changes might favor unregulated cellular proliferation.

Authors

F R DeRubertis, R Chayoth, J B Field

Morphology and metabolism of an aortic intima-media preparation in which an intact endothelium is preserved.

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Abstract

An in vitro preparation of rabbit aortic "intima-media" previously shown to exhibit stable rates of respiration and glucose metabolism and the high rate of aerobic glycolysis considered characteristic of the metabolism of this tissue was subjected to electron microscopic examination. In samples examined immediately after the aortae were dissected free of adipose tissue and adventitia, under conditions similar to those now in common use, marked and widespread alterations in endothelial cell structure were present, including loss of cell integrity. The vascular smooth muscle cells retained a normal electron microscopic (EM) appearance. During subsequent incubation with 5 mM glucose in Krebs-Ringer bicarbonate (KRB), pH 7.4, under the conditions usually employed in studies of this preparation, large zones of the luminal surface were rapidly denuded of endothelium, and the remaining endothelial cells exhibited a wide range of ultrastructural alterations. The smooth muscle cells, however, continued to maintain a normal EM appearance. A method was developed to prepare segments of rabbit aortic intima-media which retained an intact layer of endothelium resembling that observed in tissue fixed in situ. During a 1-h incubation with 5 mM glucose in KRB, pH 7.4, gas phase 5% CO2/95% O2, containing 6% bovine serum albumin, the intact aortic intima-media preparation retains an essentially unmodified EM appearance and exhibits linear rates of respiration. Under these conditions the intact aortic intima-media preparation exhibits significantly higher rates of O2 uptake and glucose uptake than those observed in our previous preparation or in other reported aortic intima-media preparations. The intact aortic intima-media does not exhibit the high rate of aerobic glycolysis during in vitro incubation that has been considered characteristic of the metabolism of rabbit, rat, and swine aortic intima-media. In addition, the magnitude of the Pasteur effect was far greater than that observed in other aortic intima-media preparations. The data suggest that component cells of the aortic intima-media may derive a major fraction of their energy requirements from respiration; they raise further questions concerning the significance of the high rate of aerobic glycolysis observed when aortic intima-media preparations are incubated in vitro, and they suggest that documentation of the EM appearance of the endothelium in such preparations is desirable.

Authors

A D Morrison, L Berwick, L Orci, A I Winegrad

Diaphragmatic blood flow and energy expenditure in the dog. Effects of inspiratory airflow resistance and hypercapnia.

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Abstract

To investigate the mechanisms which enable the diaphragm to preserve ventilation when the work of breathing is elevated, we measured diaphragmatic blood flow (Q di) and oxygen consumption (VO2 di) in lightly anesthetized dogs. The animals were studied when they breathed quietly, when they inhaled 5% CO2 in 21% or 14% O2, or when they inhaled these gas mixtures through moderate to severe inspiratory resistances. Q di was determined from the integrated diaphragmatic arteriovenous difference of krypton-85, by the Kety-Schmidt technique. VO2 di was calculated as the product of Q di and the diaphragmatic arteriovenous oxygen difference ([A-V]O2 di). Alteration in these parameters consequent to augmentation of ventilatory effort were compared with concomitant alterations in diaphragmatic electrical activity (EMG di) and an inspiratory pleural pressure-time index (PPTI). Addition of inspiratory resistances combined with inhalation of CO2 usually increased Q di and consistently increased VO2 di, EMG di, and PPTI, the maximum increases being approximately 400-1,600% above control levels. In individual animals, as inspiratory resistance was increased, VO2 di, EMG di, and PPTI rose in direct proportion to each other. In the group as a whole, during resistance breathing the oxygen requirements of the diaphragm were met by a combination of increased [A-V]O2 di and Q di. Unlike other skeletal muscles, oxygen extraction tended to plateau at peak loads, whereas blood flow continued to rise as PPTI and VO2 di increased. We conclude that augmentation of perfusion permits the diaphragm to sustain high levels of contractile effort when the work of breathing is increased.

Authors

D F Rochester, G Bettini

Erythroid colony growth in congenital hypoplastic anemia.

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Abstract

Four children with congenital hypoplastic anemia (Diamond-Blackfan syndrome) and 30 control children with normal erythropoiesis were studied by a cell culture method in which human marrow, grown in a plasma clot, responds to added erythropoietin (EPO) with the appearance of discrete colonies of nucleated erythroid cells. The colonies arise from EPO-responsive stem cells and are not related to the number of morphologically identifiable marrow erythroids plated. Results of studies on control marrow indicated that without EPO there was little or no colony formation. Increasing EPO doses or nucleated marrow cells per culture resulted in a linear increase in colony numbers. The optimal EPO concentration of 2.5 U/ml yielded a mean of 158 +/- 79 colonies/1 x 10(5) nucleated cells on day 7 of incubation. Even in the absence of recognizable erythroids, marrows of all four patients with anemia grew erythroid colonies. Two patients on no therapy had decreased colony numbers. The other two, on prednisone, had normal numbers. Sera from patients did not inhibit colony formation from either autologous or control marrow. In contrast, serum from an adult with acquired pure red cell aplasia produced striking inhibition of colony growth. It appears that the red cell failure in this disorder is not due to an absence of erythroid stem cells, and a serum inhibitor to erythropoiesis as seen in the acquired disease is unlikely.

Authors

M H Freedman, D Amato, E F Saunders

Parathyroid hormone secretion in vivo. Demonstration of a calcium-independent nonsuppressible component of secretion.

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Abstract

The response of normal bovine parathyroid glands to hypercalcemia was assessed in vivo by radioimmunoassay of immunoreactive parathyroid hormone concentrations in parathyroid effluent blood obtained by surgical cannulation of both anesthetized and nonanesthetized calves. Hypercalcemia was induced for periods of 0.3-35 h by intravenous infusion of a solution of calcium chloride. Assessment of immunoreactivity in effluent and peripheral blood included measurements of selected samples by use of a radioimmunoassay specific for a site residing in the biologically active portion of the hormone molecule. In all instances, the concentration of immunoreactive parathyroid hormone in hypercalcemic venous effluent from a superior parathyroid gland exceeded that of the peripheral blood. Failure of hypercalcemia to suppress completely secretion by normal parathyroids indicates that a portion of parathyroid hormone secretion occurs independent of blood calcium concentration. Consequently, continued parathyroid hormone secretion despite hypercalcemia can no longer be regarded as a unique feature of parathyroid neoplasia.

Authors

G P Mayer, J F Habener, J T Potts Jr

Phosphoinositide metabolism and insulin secretion from isolated rat pancreatic islets.

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Abstract

Since many cell types have been shown to respond to extracellular stimulation with a rapid increase in phosphatidylinositol turnover, the present studies were undertaken to determine whether carbohydrate-stimulated insulin secretion from the isolated rat pancreatic islet is accompanied by detectable alterations in the phosphatidylinositol metabolism of this tissue. We have demonstrated that rat pancreatic islets incubated with tritiated myo-inositol rapidly incorporate radioactivity into islet phosphatidylinositol. Incubation of prelabeled islets with elevated concentrations of carbohydrates which stimulate insulin secretion (D-glucose and D-mannose) results in a decrease in the recovery of lipid-bound radioactivity, whereas incubation with carbohydrates which do not stimulate insulin secretion (D-galactose and myo-inositol) has no effect upon the recovery of lipid-bound radioactivity. Within 2 min of exposure of prelabeled islets to elevated concentrations of D-glucose, a decrease in the recovery of [2-3H]myo-inositol-derived radioactivity in islet phosphatidylinositol can be demonstrated. When islets prelabeled with [2-3H]myo-inositol are perifused with elevated concentrations of D-glucose or D-mannose (but not D-galactose or myoinositol) a rapid and transient increase in the rate of extracellular release of water-soluble radioactivity is observed. Since a significant fraction of the radioactivity released under these conditions is in the form of myo-inositol phosphate, cyclic myo-inositol-1,2-phosphate, and glycerophosphorylmyo-inositol, it is presumably derived from the cleavage of labeled islet phosphatidylinositol. It is speculated that alterations in the metabolism of myo-inositol-containing phospholipids may have a functional role in the process of insulin secretion from the pancreatic beta cell.

Authors

R S Clements Jr, W B Rhoten

Extraosseous calcification. Evidence for abnormal pyrophosphate metabolism in uremia.

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Abstract

The inorganic constituents and crystalline features of extraosseous calcium-phosphate deposits obtained from dialyzed uremic and hypercalcemic patients were studied. Visceral calcification (heart, lung, and kidney) in hypercalcemic patients exhibited either an amorphous or apatitic X-ray diffraction pattern. Uremic visceral calcification consistently gave an amorphous diffraction pattern. Although the calcium content of uremic and hypercalcemic visceral deposits was similar, other inorganic constituents were different. The mean pyrophosphate was 11 +/- 11.8 and magnesium 4.91 +/- 3.86 mg/g in the uremic group as compared to 0.92 +/- 0.24 and 1.36 +/- 1.26 mg/g in the hypercalcemic group (P less than 0.025). After incineration hypercalcemic visceral deposits having an amorphous diffraction pattern were found to generate pyrophosphate supporting the presence of brushite in these deposits. The small amount of pyrophosphate in apatitic deposits from both uremic and hypercalcemic patients actually decreased after incineration and the pyrophosphate content of uremic visceral deposits was unchanged by incineration. It is concluded that in hypercalcemic patients the initial visceral deposit is brushite which is subsequently transformed to apatite. Arterial and tumoral calcium-phosphate deposits in uremic patients were also apatite. Uremic visceral calcium-phosphate deposits are an unique mineral high in magnesium with approximately 30% of the phosphorus present as pyrophosphate. The high pyrophosphate content of these deposits could alter their crystalline structure and prevent the transformation to apatite. The infrared features, high magnesium content of the deposit, and resistance of pyrophosphate in the deposit to hydrolysis by pyrophosphatase suggests that the pyrophosphate may be deposited as the magnesium salt.

Authors

A C Alfrey, C C Solomons, J Ciricillo, N L Miller

Bone pyrophosphate in uremia and its association with extraosseous calcification.

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Abstract

The mean bone pyrophosphate was 0.360 +/- 0.15 mg/g in 8 controls and 1.22 +/- 1.39 mg/g bone in 27 uremic patients (P less than 0.0025). 13 of the 27 uremic patients had bone pyrophosphate levels greater than 2 SD above control values. The ash content of uremic bones with increased pyrophosphate levels (group II) was 56 +/- 9% as compared to 64 +/- 2% in control bones (P less than 0.01) and 60 +/- 7% in uremic bones having normal pyrophosphate levels (P less than 0.1) (group I). The magnesium content of bones in group II was 338 +/- 47 as compared to 211 +/- 13 (P less than 0.0005) in the controls and 294 +/- 73 mmol/kg ash (P less than 0.05) in group I. In group II, but not group I, there was a significant inverse correlation between duration of dialysis and percent bone ash (r = -0.59) (P less than 0.05). A definite relationship existed between elevated bone pyrophosphate levels and soft tissue calcification. In group II the mean pulmonary calcium content was 530 +/- 459 as compared to 32 +/- 26 mmol/kg/ash in group I (P less than 0.0025). All patients with a bone pyrophosphate level greater than 1.4 mg/g bone had extensive pulmonary calcification. It is concluded that the excess bone pyrophosphate present in some uremic patients is either deposited in the apatite crystal in the transphosphorylated form or else as the magnesium salt since the pyrophosphate is resistant to pyrophosphatase and surface adsorption of pyrophosphate is not altered by the increased bone pyrophosphate levels. The excess bone pyrophosphate could disturb bone calcification mechanisms in uremic patients. The association between increased bone pyrophosphate and soft tissue calcification suggests that the disordered pyrophosphate metabolism may be important in the pathogenesis of extraosseous calcification.

Authors

A C Alfrey, C C Solomons

Distribution of extravascular fluid volumes in isolated perfused lungs measured with H215O.

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Abstract

The distributions per unit volume of extravascular water (EVLW), blood volume, and blood flow were measured in isolated perfused vertical dog lungs. A steady-state tracer technique was employed using oxygen-15, carbon-11, and nitrogen-13 isotopes and external scintillation counting of the 511-KeV annihilation radiation common to all three radionuclides. EVLW, and blood volume and flow increased from apex to base in all preparations, but the gradient of increasing flow exceeded that for blood and EVLW volumes. The regional distributions of EVLW and blood volume were almost identical. With increasing edema, lower-zone EVLW increased slightly relative to that in the upper zone. There was no change in the distribution of blood volume or flow until gross edema (100% wt gain) occurred when lower zone values were reduced. In four lungs the distribution of EVLW was compared with wet-to-dry ratios from lung biopsies taken immediately afterwards. Whereas the isotopically measured EVLW increased from apex to base, the wet-to-dry weight ratios remained essentially uniform. We concluded that isotopic methods measure only an "exchangeable" water pool whose volume is dependent on regional blood flow and capillary recruitment. Second, the isolated perfused lung can accommodate up to 60% wt gain without much change in the regional distribution of EVLW, volume, or flow.

Authors

T Jones, H A Jones, C G Rhodes, P D Buckingham, J M Hughes

Serum lipoproteins and apolipoproteins in rats with streptozotocin-induced diabetes.

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Abstract

The lipoproteins of rats fed a high sucrose diet and made diabetic by administration of 45 mg/kg of streptozotocin were studied. All lipoprotein classes were found to be present in increased concentrations. The apolipoprotein composition of the various lipoprotein fractions was studied by polyacrylamide-gel electrophoresis in the presence of 8 M urea, isoelectric focusing in the presence of 8 M urea, and sodium dodecyl sulfate gel electrophoresis in polyacrylamide gels. In the very low density lipoproteins (VLDL) of diabetic rats, there was a marked alteration in the relative amounts of C proteins by polyacrylamide-gel electrophoresis, and this was found by isoelectric focusing to be primarily a relative increase in C-III-3 apoprotein and a decrease in C-III-O. In addition, in the diabetic rats, the VLDL contained a protein of mol wt 46,000, the A-IV protein, which normally is only present in the high density lipoproteins. In the high density lipoproteins, (HDL) the same alterations in pattern of the C proteins seen in the VLDL were present. Furthermore, the arginine-rich and A-IV protein normally present in HDL could not be detected in the HDL, although the other apolipoproteins are present. Apolipoprotein concentrations were determined by quantitative immunoelectrophoresis. It was found that in the diabetic rats there was an increase in the total amount of apo-B in the plasma, with the increment divided proportionately between the VLDL and the low density lipoprotein (LDL). The total apo-C concentration of plasma increased minimally. The A-IV concentration of plasma increased by 27%; it decreased markedly in the HDL, but appeared in increased amounts in both VLDL and in the d greater than 1.21 fraction. The arginine-rich protein decreased by 63% in the plasma and decreased significantly in the HDL, but increased in VLDL, LDL, and in the d greater than 1.21 fraction. These alterations in apolipoprotein patterns in diabetic animals suggest that the apolipoproteins may play an important role in determining the concentration of various lipoprotein fractions, or may be the result of altered metabolism of the lipoproteins. These lipoproteins with altered apolipoprotein composition may have important biologic differences from normal lipoporteins. Nevertheless, the HDL, despite the fact that it is deficient in some of its major constituents, was unchanged in its cholesterol content.

Authors

H Bar-On, P S Roheim, H A Eder

Influence of uremia and hemodialysis on the turnover and metabolic effects of glucagon.

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Abstract

To evaluate the mechanism and role of hyperglucagonemia in the carbohydrate intolerance of uremia, 19 patients with chronic renal failure (12 of whom had undergone chronic hemodialysis for at least 11 mo) and 35 healthy control subjects were studied. Plasma glucagon, glucose, and insulin were measured in the basal state, after glucose ingestion (100 g), after intravenous alanine (0.15 g/kg), and during a 3-h continuous infusion of glucagon (3 ng/kg per min) which in normal subjects, raised plasma glucagon levels into the upper physiological range. Basal concentrations of plasma glucagon, the increment in glucagon after infusion of alanine, and post-glucose glucagon levels were three- to fourfold greater in uremic patients than in controls. The plasma glucagon increments after the infusion of exogenous glucagon were also two- to threefold greater in the uremics. The metabolic clearance rate (MCR) of glucagon in uremics was reduced by 58% as compared to controls. In contrast, the basal systemic delivery rate (BSDR) of glucagon in uremics was not significantly different from controls. Comparison of dialyzed and undialyzed uremics showed no differences with respect to plasma concentrations, MCR, or BSDR of glucagon. However, during the infusion of glucagon, the increments in plasma glucose in undialyzed uremics were three- to fourfold greater than in dialyzed uremics or controls. When the glucagon infusion rate was increased in controls to 6 ng/kg per min to produce increments in plasma glucagon comparable to uremics, the glycemic response remained approximately twofold greater in the undialyzed uremics. The plasma glucose response to glucagon in the uremics showed a direct linear correlation with oral glucose tolerance which was also improved with dialysis. The glucagon infusion resulted in 24% reduction in plasma alanine in uremics but had no effect on alanine levels in controls. It is concluded that (a) hyperglucagonemia in uremia is primarily a result of decreased catabolism rather than hypersecretion of this hormone; (b) sensitivity to the hyperglycemic effect of physiological increments in glucagon is increased in undialyzed uremic patients; and (c) dialysis normalizes the glycemic response to glucagon, possibly accounting thereby for improved glucose tolerance despite persistent hyperglucagonemia. These findings thus provide evidence of decreased hormonal catabolism contributing to a hyperglucagonemic state, and of altered tissue sensitivity contributing to the pathophysiological action of this hormone.

Authors

R S Sherwin, C Bastl, F O Finkelstein, M Fisher, H Black, R Hendler, P Felig

Vitamin E. An inhibitor of the platelet release reaction.

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Abstract

Formation of lipid peroxides rises sharply when platelets undergo the release reaction. In this study the in vitro effect of vitamin E on platelet aggregation was investigated. alpha-Tocopherol, an anitoxidant of known inhibitory action on lipid peroxidation, was added to platelet suspensions in concentrations up to 1.5 mM. A dose-dependent reduction in platelet aggregation was observed, with complete inhibition of the secondary wave of aggregation at greater than or equal to 0.9 mM alpha-tocopherol. The inhibitory effect of alpha-tocopherol on the platelet release reaction was further documented by the decrease in aggregation-induced release of [14C]5-hydroxytryptamine from prelabeled platelets and by the reduction of N-acetylglucosaminidase activity released into the medium. The sharp rise in lipid peroxides normally associated with platelet aggregation was markedly reduced by alpha-tocopherol and also by acetylsalicylic acid, a known inhibitor of the platelet release reaction. In vivo studies examined the effect of oral vitamin E administration (1,200-2,400 IU daily) on plasma and platelet levels of alpha-tocopherol. Up to 1,800 IU daily, increasing dosages of vitamin E resulted in increasing concentrations of alpha-tocopherol in plasma and platelets, but intake of vitamin E in excess of this dosage failed to show any further increase in plasma or platelet levels.

Authors

M Steiner, J Anastasi

Arylsulfatase B of human lung. Isolation, characterization, and interaction with slow-reacting substance of anaphylaxis.

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Abstract

Arylsulfatase B was separated from arylsulfatase A in extracts of human lung tissue by anion exchange chromatography and further purified by gel filtration and cation exchange chromatography. Arylsulfatase B of human lung was similar to that enzyme in other tissues and species, exhibiting an apparent mol wt of approximately 60,000, a pH optimum for cleavage of 4-nitrocatechol sulfate (pNCS) of 5.5-6.0, and a sensitivity to inhibition by phosphate ions and especially pyrophosphate in the presence of NaCl. Human lung arylsulfatase B inactivated slow-reacting substance of anaphylaxix (SRS-A) in a linear time-dependent reaction in which the rate was determined by the enzyme-to-substrate ratio. Cleavage of pNCS by human lung arylsulfatase B was competitively suppressed by SRS-A. The finding that human lung tissue contains predominately arylsulfatase B discloses a potential regulatory mechanism for inactivation of SRS-A at or near the site of its generation.

Authors

S I Wasserman, K F Austen

Altered thyroidal responsivity to thyrotropin induced by circulating thyroid hormones. A "short-loop" regulatory mechanism?

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Abstract

We studied the effect of thyroid hormone administration on responsivity of murine thyroid to exogenous thyrotropin (TSH) in order to explore the possibility that the thyroid gland might be directly inhibited by its own hormones. In the rat both L-thyroxine (T4) and 3,5,3'-L-triiodothyronine (T3) pretreatment inhibited TSH-induced thyroidal ornithine decarboxylase (ODC) activity in vivo in a dose-related manner (half-maximal inhibition, 1.7 mug/rat and 0.6 mug/rat, respectively). Other structurally related compounds exhibited the following inhibitory potencies compared to T4: T3, 283%; triiodothyroacetic acid, 40%; D-T4, 18%; 3,5-L-diiodothyronine, 9%. Monoiodotyrosine, diiodotyrosine, and iodide were not inhibitory. The full inhibitory effect of T4 or T3 was observed when thyroid hormone was administered from 96 to 12 h before TSH and was also seen in hypophysectomized animals. Pretreatment with T4 or T3 in divided doses over 2 1/2 days inhibited TSH-induced increase in [1-14C]glucose oxidation to 14C02 and [3H] leucine incorporation into protein in rat thyroid. In the mouse T4 or T3 pretreatment (0.25-25 mug daily) caused dose-related inhibition of both thyroidal ODC activity and 131I release induced by TSH in vivo. In mice on a low-iodine diet (LID) but not in animals on a regular diet (RD) NaI pretreatment also blunted TSH-induced thyroidal ODC activation and 131I release. When LID or RD mice were pretreated with 12.5-125 mug of T4 or T3 over 2 1/2 days, TSH-induced in vitro stimulation of thyroid cyclic 3',5'-adenosine monophosphate formation was inhibited in a dose-related manner; NaI pretreatment was inhibitory in the LID mouse only. Prior administration of exogenous TSH blunted the activation of thyroid ODC and thyroid hormone release induced by subsequent TSH administration in rat and mouse. These studies indicate altered thyroid responsivity to TSH under the influence of circulating thyroid hormones and suggest the existence of a "short-loop" negative feedback regulating thyroid function.

Authors

S Yu, Y Friedman, R Richman, G Burke

Adenosine deaminase activity in chronic lymphocytic leukemia. Relationship to B- and T-cell subpopulations.

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Abstract

The level, phenotypes, and isozyme distribution of adenosine deaminase (ADA) were determined in lymphocytes from patients with chronic lymphocytic leukemia (CLL). The ADA level in lymphocytes from patients with untreated CLL was consistently lower than in lymphocytes from normal subjects. No significant differences were found in the phenotype or isozyme distribution. In untreated patients, the ADA level was inversely correlated with the lymphocyte count and the percentage of bursa-equivalent (B) cells. After therapy, a diminution in the lymphocyte count was associated with an increase of ADA activity towards normal levels. The ADA levels were slightly higher in the thymus-derived (T) than in the B lymphocytes from normal subjects. The B cells had lower activity than T cells in patients with CLL. They also had a lower activity than the B cells from normal subjects. The ADA level was 2.3-fold higher in T cells from patients with CLL than in normal T cells. The decrease in ADA levels is an anomaly that is reversible and appears to be a reflection of the proliferation of abnormal B cells in this disorder.

Authors

R Tung, R Silber, F Quagliata, M Conklyn, J Gottesman, R Hirschhorn

Surface immunoglobulin on activated human peripheral blood thymus-derived cells.

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Abstract

Human peripheral blood lymphocytes grown in vitro were stimulated with nonspecific mitogens and in mixed lymphocyte culture. The presence of IgM and thymus (T) surface markers on large and small lymphocytes was investigated by immunofluorescence and correlated with spontaneous rosette formation. All stimulated large lymphocytes formed spontaneous rosettes and all except pokeweed mitogen (PWM)-stimulated large lymphocytes had IgM and T markers. IgG, IgA, and light chain determinants were only detected on PWM-induced large lymphocytes. Thus, surface markers expressed on activated human lymphocytes may differ for different mitogens. IgM was always present on large cells which had the T markers, and it cannot be used to identify a lymphocyte as a bone marrow-derived (B) cell. Due to the overlap of surface markers, the classification into B and thymus-derived (T) cells ought to be restricted to functional phenomena of antibody-production or cell-mediated immunity.

Authors

T L Whiteside, B S Rabin

Hemoglobin McKees Rocks (alpha2beta2145Tyr leads to Term). A human "nonsense" mutation leading to a shortened beta-chain.

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Abstract

The first example of the premature termination of a polypeptide chain in man appears to be Hb McKees Rocks, beta145 Tyr leads to Term, discovered in polycythemic members of a Caucasian family. Point mutation has apparently occurred at the codon for Tyr beta145 from UAU to a "nonsense" codon, UAA or UAG, resulting in a shortened polypeptide chain with Lys 144 as its carboxyl-terminal amino acid. Evidence for this structural conclusion is the absence of tryptic peptide betaT-15 from "fingerprints" of the abnormal beta-chain, the finding of C-terminal Lys, and the similarity between the functional properties of this variant hemoglobin and those of des Tyr (145)-His(146)beta hemoglobin resulting from carboxypeptidase-A digestion of normal human hemoglobin. Hb McKees Rocks has markedly abnormal properties: its oxygen affinity is the highest of the human variants described to date; its Bohr effect is reduced; it is devoid of subunit cooperativity; and it is unaffected by 2,3-diphosphoglyceric acid. These properties are probably the consequences of decreased stability of the T quaternary conformation and are partially restored in the presence of the strong allosteric effector inositol hexaphosphate.

Authors

R M Winslow, M L Swenberg, E Gross, P A Chervenick, R R Buchman, W F Anderson

Studies on the structural abnormality of fibrinogen Paris I.

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Abstract

The structural properties of an inherited fibrinogen abnormality designated fibrinogen Paris I were investigated. Dodecyl sulfate gel electrophoresis of unreduced samples revealed no discernible differences in molecular weight from normal; this implied that in fibrinogen Paris I, the normal fibrinogen architecture of six covalently linked chains per molecule is preserved. Examination of dithiothreitol reduced samples before and after treatment with Reptilase or thrombin revealed that the Aalpha- and Bbeta-chains could release the A and B peptides, respectively. A mutant chain (mol wt 52,500, termed gammaParis I) which replaces a large proportion of gamma-chains (mol wt 49,400) was shown, like normal gamma-chains, to lack thrombin- and Reptilase-sensitive sites. The gamma-chains and alpha-chains of Paris I fibrin underwent Factor XIIIa-catalyzed cross-linking slowly; this behavior was not attributable to an intrinsic abnormality of these chains themselves but rather to the inhibitory effect of the mutant gammaParis I chains on this process. Results of DEAE-cellulose gradient elution chromatography of Paris I fibrinogen preparations revealed the presence of small amounts of normal fibrinogen molecules and also indicated that the gammaParis I chains possessed structural overlap with gamma-chains. Unlike gamma-chains however, the gammaParis I chains did not incorporate dansylcadaverine in the prescence of Factor XIIIa, nor, as previously reported, did they undergo cross-linking. The observations indicate that the amine acceptor site found in the COOH-terminal region of the gamma-chain is either not present on the gammaParis I chain or is unavailable for cross-linking. Further support for localization of the abnormality in the COOH-terminal region of the molecule was obtained from the observation that during plasmic hydrolysis of Paris I fibrinogen, at least one unique form of core Fragment D (DParis I) was evolved, whereas Fragment E did not differ from normal.

Authors

M W Mosesson, D L Amrani, D Ménaché

A role for alpha-adrenergic receptors in abnormal insulin secretion in diabetes mellitus.

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Abstract

To determine whether endogenous alpha-adrenergic activity contributes to abnormal insulin secretion in nonketotic, hyperglycemic, diabetic patients, alpha-adrenergic blockade was produced in normal and diabetic subjects. The diabetics had a significantly (P less than 0.01) greater increase in circulating insulin 1 h after an intravenous phentolamine infusion than did the normal subjects. During the phentolamine infusion, there was also a significant augmentation of acute insulin responses to intravenous glucose (20 g) pulses in normal subjects (P less than 0.05) and diabetics (P less than 0.02); this augmentation was fivefold greater in the diabetics. Simultaneous treatment with the beta-adrenergic blocking agent, propranolol, did not alter these findings. Thus a role for exaggerated endogenous alpha-adrenergic activity in abnormal insulin secretion of the diabetic subjects is suggested. To determine whether this alpha-adrenergic activity might be related to elevated circulating catecholamines, total plasma-catecholamine levels were compared in normal and nonketotic diabetic subjects given intravenous glucose pulses. These levels were significantly greater (P less than 0.02) in the diabetic compared to the normal group before the glucose pulse, and increased significantly in both groups (P less than 0.02 and less than 0.001, respectively) after the pulse. These data suggest that excessive catecholamine secretion may lead to an abnormal degree of endogenous alpha-adrenergic activity, which contributes to defective insulin secretion in diabetic subjects.

Authors

R P Robertson, J B Halter, D Porte Jr

Function and structure in the diphenylamine-exposed kidney.

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Abstract

Standard micropuncture and microdissection techniques were used to examine the function and structure of nephrons in rats whose kidneys were made cystic by dietary exposure to diphenylamine. Heterogeneity characterized the lesion, with dilation and frank cyst formation occurring in 5-30% of nephrons. Elevated intraluminal hydrostatic pressures, occurring in the absence of increased glomerular filtration or decreased net water reabsorption, were recorded in dilated, but not in nondilated nephrons. Structural studies demonstrated communication of dilated nephrons with cysts, concretions of debris within tubular lumens, evidence of extrinsic pressure by cysts on adjacent tubules, and apparent luminal narrowing of some proximal tubules. These observations were used to explain prolonged loop of Henle transit times and occasional failure to detect [3H]inulin excretion after microperfusion into dilated tubules. It was concluded that the elevated hydrostatic pressures in the dilated nephrons of diphenylamine-exposed kidneys were the consequence of variably severe and frequently incomplete tubular occlusion. These findings support the hypothesis that cyst formation is a consequence of partial obstruction and elevated intratubular pressure in this model and perhaps in other susceptible mammalian kidneys.

Authors

K D Gardner Jr, S Solomon, W W Fitzgerrel, A P Evan

The biosynthesis of guanidinosuccinic acid by perfused rat liver.

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Abstract

The metabolic pathway for the synthesis of guanidinosuccinic acid was studied in the rat. Labeled guanidinosuccinic acid was isolated from the urine of animals given L-[guanidino-14C]arginine intraperitoneally but did not appear in the urine after administration of D,L-[guanidino-14C]canavanine. Radioactive arginine and nonradioactive aspartic acid and arginine were infused in the isolated, perfused rat liver. After 20 min, small amounts of both labeled and unlabeled guanidinosuccinic acid and large amounts of urea were detected in radiochromatograms of the perfusate. These results support the theory that guanidinosuccinic acid is formed in the liver from transamidination of arginine to aspartic acid.

Authors

G Perez, A Rey, E Schiff