Analysis of Parathyroid Hormone and Its Fragments in Rat Tissues: CHEMICAL IDENTIFICATION AND MICROSCOPICAL LOCALIZATION

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Abstract

After intravenous injection of [125I]-iodo-parathyroid hormone in the rat, uptake of the hormone was greatest in the liver and kidneys. Uptake was rapid, reaching a maximal concentration by 4 and 8 min, respectively. Extracts, prepared from both these organs at intervals soon after the injection of intact hormone, showed three main radioactive peaks when samples were subjected to gel filtration under protein-denaturing conditions. The first peak coeluted with intact hormone. The second eluted at a position corresponding to the carboxy-terminal fragments previously described in plasma, and the last eluted at the salt volume of the column. Microsequence analysis of the radioiodinated fragments, a method that has proved valuable for chemically defining the circulating fragments resulting from metabolism of injected hormone, showed that extracts of liver and kidney, prepared at 4 and 8 min after injection of the intact hormone, contained different fragments. The radioiodinated fragments in liver extracts were identical to those previously reported in the plasma of rats and dogs, fragments resulting principally from proteolysis between positions 33 and 34, and 36 and 37 of the intact hormone. Although the same fragments were also present in the kidneys, they constituted less than 15% of the amount present in the liver. More than 50% of the labeled renal fragments consisted of a peptide whose amino-terminal amino acid was position 39 of the intact hormone, a fragment not present in plasma. The rate of appearance of radioiodinated fragments that were chemically identical to those in plasma was more rapid in the liver than in plasma. Correlation of these chemical analyses with studies of the localization of 125I by autoradiography showed that at the times when the intact hormone and the carboxy-terminal fragments comprised nearly all of the 125I-labeled moieties in the tissues, the proximal convoluted tubules of the kidney and sinusoidal lining cells of the liver, which probably are Kupffer cells, contained the highest concentration of 125I. Preferential localization of immunoreactive parathyroid hormone to these tissue sites also was shown by immunoperoxidase staining in studies with unlabeled hormone. Our results suggest that, unless multiple renal mechanisms are present for release of hormonal fragments, one of which releases the circulating fragments preferentially, the liver, rather than the kidney, is principally responsible for generating the carboxy-terminal fragments in plasma after injection of intact hormone, and the Kupffer cells may contain the enzymes that hydrolyze parathyroid hormone.

Authors

Pierre D'Amour, Gino V. Segre, Sanford I. Roth, John T. Potts Jr.

The Micellar Sink: A QUANTITATIVE ASSESSMENT OF THE ASSOCIATION OF ORGANIC ANIONS WITH MIXED MICELLES AND OTHER MACROMOLECULAR AGGREGATES IN RAT BILE

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Abstract

Although the importance of mixed micelles in the solubilization and biliary excretion of lipids is established, little is known about a possible role of mixed micelles in the excretion of other biliary solutes. Ultrafiltration and ultracentrifugation techniques were used to investigate the interaction between substances that are excreted in bile and biliary mixed micelles. Substances (urea, erythritol, sucrose) excreted in bile at concentrations equal to, or less than, that in plasma did not show an association with mixed micelles, whereas substances (indocyanine green, iopanoic acid, rose bengal, unconjugated and conjugated sulfobromophthalein, and conjugated bilirubin) excreted in bile at high concentration relative to plasma did. The percentage of these latter substances in bile associated with micelles varied from 26 to 93% and was relatively independent of concentration. In addition to their association with mixed micelles, these test solutes formed self-aggregates that were stabilized primarily by ionic bonds, and only a small percentage (range = 0-5%) of these solutes were present in bile in the form of monomer or complexes small enough to pass a 5,000-mol wt membrane.

Authors

Bruce F. Scharschmidt, Rudi Schmid

Antiherpesvirus Activity in Human Sera and Urines after Administration of Adenine Arabinoside: IN VITRO AND IN VIVO SYNERGY OF ADENINE ARABINOSIDE AND ARABINOSYLHYPOXANTHINE IN COMBINATION

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Abstract

The minimum inhibitory concentration (MIC) of adenine arabinoside (ara-A) in rabbit kidney microtiter tissue cultures (RK-13) to a prototype strain of herpes simplex virus, type 1 (E115) based upon inhibition of cytopathic effects is 1.5 μg/ml. In this system, the MIC of arabinosylhypoxanthine (ara-Hx), the major in vivo metabolic derivative of ara-A, is 75 μg/ml. Inhibition of cytopathic effects of herpes simplex virus, type 1 (HSV-1) in microtiter wells of RK-13 cells varies directly with the concentrations of ara-A or ara-Hx, and inversely with residual HSV-1. The MIC of ara-A for HSV-1 in RK-13 cells is 5-20 times lower than similar measures with vero renal, mouse embryo, or human foreskin cultures. With RK-13 tissue cultures in microtiter plates, an assay for “ara-A equivalents” in human body fluids was developed which compares in sensitivity with high pressure liquid chromatography and has the advantage of simultaneously measuring combined antiherpesvirus effects of ara-A and its major metabolic derivative, ara-Hx.

Authors

Keith J. Champney, Carl B. Lauter, Elizabeth J. Bailey, A. Martin Lerner

Serum Amyloid A: EVIDENCE FOR ITS ORIGIN IN POLYMORPHONUCLEAR LEUKOCYTES

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Abstract

In this study the presence of an amyloid A, antigenically related material was determined in four subpopulations of human leukocytes. Monocytes, granulocytes, thymus-derived lymphocytes, and bone marrow-derived and null lymphocytes were isolated from the peripheral blood of five apparently normal subjects, two patients with secondary amyloidosis, three patients with acute infections, and seven patients with metastatic cancer. Mononuclear leukocytes, isolated from the interface of a Ficoll-Hypaque gradient, were separated into monocytes, thymus-derived lymphocytes, and bone marrow-derived plus null lymphocytes by glass adherence and depletion of sheep erythrocyte rosette-forming lymphocytes. Granulocytes were isolated by sedimentation in 2% methyl cellulose from the erythrocyte-rich pellet formed at the bottom of the Ficoll-Hypaque gradient. The four isolated leukocyte subpopulations were cultured and, at varying intervals, the amyloid A content of the culture medium and of sonicated, 2 × 106 cells was determined by radioimmunoassay. Our results indicated a 2-14 times greater amount of amyloid A-related material in the sonicated granulocytes compared with the individuals' serum amyloid A levels. The mononuclear subpopulations showed a low or negligible amyloid A content. The amount of amyloid A antigenic material was further found to increase in cultured granulocytes, reaching a peak value between the 16th and 30th h of culture. The granulocytes of only two out of eight individuals tested released amyloid A antigenically related material into the culture medium. This release was found to be blocked by the presence of colchicine, vincristine, puromycin, or cycloheximide in the culture medium. In contrast, only the presence of puromycin or cycloheximide was shown to significantly inhibit the intracellular increase of amyloid A in the cultured granulocytes. Thus, it appears that among the circulating blood cells, the granulocytes produce amyloid A antigenically related material and could release it under conditions that remain to be further defined.

Authors

C. Julian Rosenthal, Lee Sullivan

Evidence of Incomplete Left Ventricular Relaxation in the Dog: PREDICTION FROM THE TIME CONSTANT FOR ISOVOLUMIC PRESSURE FALL

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Abstract

Although it has been proposed that incomplete relaxation explains certain increases in left ventricular end diastolic pressure relative to volume, there has been no clear demonstration that incomplete relaxation occurs in the intact working ventricle. To identify incomplete relaxation, left ventricular pressure-dimension relationships were studied in 10 canine right heart bypass preparations during ventricular pacing. The fully relaxed, exponential diastolic pressure-dimension line for each ventricle was first determined from pressure and dimension values at the end of prolonged diastoles after interruption of pacing. For 167 beats during pacing under widely varying hemodynamic conditions, diastolic pressure-dimension values encountered this line defining the fully relaxed state during the filling period indicating that relaxation was complete before end diastole. The time constant for isovolumic exponential pressure fall (T) was determined for all beats. For this exponential function, if no diastolic filling occurred, 97% of pressure fall would be complete by 3.5 T after maximal negative dP/dt. For the 167 beats the fully relaxed pressure-dimension line was always encountered before 3.5 T.

Authors

Myron L. Weisfeldt, James W. Frederiksen, Frank C. P. Yin, James L. Weiss

Modulation of Cyclic Nucleotides in Isolated Rat Glomeruli: ROLE OF HISTAMINE, CARBAMYLCHOLINE, PARATHYROID HORMONE, AND ANGIOTENSIN-II

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Abstract

Because glomerular functions are modulated by numerous humoral agents, probably acting through cyclic nucleotides, the effects of some polypeptide hormones and biogenic amines on cyclic AMP (cAMP) and cyclic 3′,5′-guanosine monophosphate (cGMP) were studied in glomeruli isolated from rat renal cortex. Glomeruli and cortical tubules were prepared by a combination of sieving and density-gradient centrifugation. Under basal conditions, the contents of cAMP and cGMP in glomeruli were significantly higher than in tubules and unfractionated renal cortical tissue.

Authors

Vicente E. Torres, Thomas E. Northrup, Richard M. Edwards, Sudhir V. Shah, Thomas P. Dousa

Metabolic Control of the Circulation: EFFECTS OF ACETATE AND PYRUVATE

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Abstract

Chloralose-anesthetized dogs were infused intravenously with either Tris-acetate or Tris-pyruvate at 0.0375, 0.075, and 0.15 mmol/kg per min successively, each for 20 min. Acetate infusion increased cardiac output, left ventricular dP/dt and dP/dt/P, and coronary blood flow, while pyruvate infusion did not. Infusions of either substance increased arterial blood and skeletal muscle concentrations of citrate and malate, but only acetate infusion increased the tissue AMP content and decreased the ATP:AMP ratio. The increase in cardiac output produced by acetate was accompanied by an increase in total body oxygen consumption and a decrease in the difference between arterial and mixed venous blood oxygen.

Authors

Chang-Seng Liang, John M. Lowenstein

Human Skin Aryl Hydrocarbon Hydroxylase: INDUCTION BY COAL TAR

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Abstract

Coal tar products, which are widely used in treating dermatologic disease, contain numerous polycyclic aromatic hydrocarbons, including 3,4-benzo[a]pyrene (BP). BP is among the most potent environmental chemical carcinogens and is known to evoke tumors in the skin of experimental animals and perhaps also of man. In this study the effect of cutaneous application of coal tar solution (U. S. Pharmacopeia) on aryl hydrocarbon hydroxylase (AHH) activity in the skin of patients usually treated with this drug was investigated. AHH, a cytochrome P-450 dependent carcinogen-metabolizing enzyme appears to play an important role in the activation of polycyclic hydrocarbons into reactive moieties that can bind to DNA and that may directly induce cancer. Application of coal tar solution to human skin caused a two to five-fold induction of cutaneous AHH in nine subjects. In further studies, the incubation of human skin with coal tar solution in vitro also caused variable induction of cutaneous AHH. Maximum responses in both systems occurred after 24 h and enzyme activity in vitro was time- and tissue- and substrate-concentration dependent. Studies in experimental animals showed that topical application of coal tar solution caused induction of AHH in skin and, after percutaneous absorption, in liver as well. Assay of several defined constituents of coal tar for AHH induction showed that BP was the most potent inducer of AHH tested. These studies indicate that topical application of coal tar solution in doses ordinarily used in treating dermatologic disease causes induction of AHH in human skin and suggest that such induced enzymatic activity could relate to carcinogenic responses to this agent in skin or, after percutaneous absorption, in other tissues as well.

Authors

David R. Bickers, Attallah Kappas

Receptor Specific Clearance by the Reticuloendothelial System in Chronic Liver Diseases: DEMONSTRATION OF DEFECTIVE C3b-SPECIFIC CLEARANCE IN PRIMARY BILIARY CIRRHOSIS

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Abstract

An approach to the assessment of reticuloendothelial function that quantitates clearance specifically mediated by membrane receptors for C3b and immunoglobulin (Ig)G has been applied in man. Clearance of isologous erythrocytes coated with IgM or C3b or coated with IgG were examined in patients with primary biliary cirrhosis (PBC), chronic hepatitis, or alcoholic cirrhosis and normal control subjects and compared with the clearance of aggregated human serum albumin. Clearance of these three types of particles varied independently. None of the patients studied had a defect in the clearance of aggregated albumin. No patient with PBC (0:6) had delayed clearance of IgG-coated erythrocytes; one of six patients with chronic hepatitis had delayed clearance of these cells. Indeed, four of six with PBC had increased rates of IgG-mediated clearance. In contrast, six out of six patients with PBC had an unequivocal defect in clearance mediated by C3b receptors. The patients with PBC varied widely in terms of duration of symptoms, degree of cholestasis, and histologic stage of disease. No defect of C3b-mediated erythrocyte clearance was found in the patients with chronic hepatitis or alcoholic cirrhosis. Furthermore, a patient with severe cholestasis secondary to large duct biliary obstruction exhibited normal C3b-mediated clearance. The defect in C3b-mediated clearance in PBC did not correlate with serum levels of individual complement components or inhibitors or with the presence of circulating immune complexes as measured by the Clq precipitation assay. Thus, measurements of receptor specific clearance, but not clearance of aggregated proteins, have revealed a highly specific defect in reticuloendothelial function in PBC.

Authors

Charles J. Jaffe, John M. Vierling, E. Anthony Jones, Thomas J. Lawley, Michael M. Frank

Low Serum Dopamine β-Hydroxylase Activity: A MARKER OF CONGESTIVE HEART FAILURE

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Abstract

To gain information about the nature of disturbances in sympathetic nervous system control in congestive heart failure, serum dopamine β-hydroxylase (DBH) activity was measured in 30 patients with heart failure of diverse etiologies and 29 healthy normotensive controls. The heart failure patients had been symptomatic for at least 6 wk and had elevated filling pressures, low cardiac indices, low ejection fractions, and wide arteriovenous oxygen differences. DBH activity was 47.1±4.7 (mean±SE) for the controls and 14.4±2.7 IU for the heart failure patients (P < 0.001). Sera from some patients with heart failure had potent inhibitory effects on DBH activity of normal sera. The inhibitor was heat stable and dialyzable and could be demonstrated despite presence of N-ethylmaleimide or Cu++ in the reaction mixture. However, some inhibitory activity was also present in sera of normal patients; this inhibitory property was not demonstrable in unheated normal serum, but was unmasked when DBH was heat inactivated. It is proposed that although the inhibitor may have been a factor in low serum DBH activity in some patients with heart failure, the major cause of the low activity in the heart failure group was a reduced rate of synthesis or release of the enzyme by sympathetic nerves. This may reflect a dissociation between rates of neural release of norepinephrine and release of DBH in chronic, severe heart failure. The observation of low serum DBH levels in patients with heart failure suggests that measurement of DBH levels may serve as a useful indicator of cardiac dysfunction.

Authors

Lawrence D. Horwitz, Victoria L. Travis