The influence of the serum binding protein (DBP) for vitamin D and its metabolites on the concentration of its main ligands, 25-hydroxyvitamin D3 (25-OHD3) and 1,25-dihydroxyvitamin D3 (1,25-[OH]2D3) was studied. The concentration of both 1,25-(OH)2D3 and DBP in normal female subjects (45±14 ng/liter and 333±58 mg/liter, mean±SD, respectively; n = 58) increased during the intake of estro-progestogens (69±27 ng/liter and 488±90 mg/liter, respectively; n = 29), whereas the 25-OHD3 concentration remained unchanged. A positive correlation was found between the concentrations of 1,25-(OH)2D3 and DBP in these women.
Roger Bouillon, Frans A. Van Assche, Hugo Van Baelen, Walter Heyns, Pieter De Moor
The influence of insulin on unidirectional flux of glucose across the blood-brain barrier and on net uptake of glucose by the brain was investigated in seven fasting patients. The unidirectional extraction, E, of [14C]D-glucose was determined using 36Cl- as an intravascular reference, by the indicator dilution method. 0.4 U insulin/kg body wt was infused intravenously over 30 min while blood glucose was maintained constant by glucose infusion. Six determinations were made in each patient, two before, two during insulin infusion, and two after. In connection with each blood-brain barrier study, arterial and cerebral venous samples were taken for measurement of glucose, oxygen, insulin, K+, and phosphate. Cerebral blood flow (CBF) was measured in each patient. The main finding was an increased extraction of glucose from 14 to 21% and a highly significant increase in unidirectional flux (CBF X unidirectional extraction X arterial glucose concentration) from 0.46 to 0.66 mumol/g X min during insulin infusion (plasma insulin approximately 1,500 microU/ml). The net brain uptake of glucose (CBF X arterio-venous difference for glucose) as unaltered during the investigation period of 45 min, which is too short a time for insulin to penetrate the barrier. It follows that the backflux of glucose from the brain was increased during insulin application. The effect of insulin might be a speeding up of the glucose carrier in analogy to heart muscle.
M M Hertz, O B Paulson, D I Barry, J S Christiansen, P A Svendsen
Patients with active systemic lupus erythematosus (SLE) often have circulating antibodies to T cells. These patients also often have leukopenia and diminished numbers of T lymphocytes. In addition, certain T lymphocyte functions are frequently impaired in patients with SLE. It has been previously considered that a complement-dependent cytotoxic mechanism was responsible for the above observations. We now demonstrate that antibody-dependent cell-mediated cytotoxicity (ADCC), a cytotoxic reaction mediated by antibody and effector cells in the absence of complement, can also kill T cells from normal individuals as well as from patients with SLE. Moreover, this ADCC could be observed using the plasma, effector cells, and target cells all obtained from the same individual with SLE. Plasma of those patients with active SLE, and in whom anti-T cell antibodies could be demonstrated by the more classical complement-dependent cytotoxicity, was most often able to mediate such an ADCC reaction. The IgG fraction of the plasma was responsible for inducing ADCC, and aggregated IgG could block the reaction. The fact that the IgG fraction was often more effective than the unfractionated plasma suggested that immune complexes present in SLE plasma might partially block the expression of ADCC. Because a single SLE plasma could induce ADCC in T cells from several different unrelated individuals, it is unlikely that antibodies directed against particular human leukocyte antigens (HLA) or blood group antigens are involved.
S Kumagai, A D Steinberg, I Green
To study the role of gonadotropin-releasing hormone (GnRH) receptors in the regulation of gonadotropin secretion, we used D-125I-alanine6 des glycyl10 GnRH ethylamide (D-125I-Ala analog), a nondegradable, superagonist GnRH analog to assess GnRH receptors on rat pituitary membranes. Receptor affinity in intact adult rats was 5.0 X 10(9) M-1 and was unchanged after castration in both sexes. Castration of adult male and female rats produced a twofold increase in GnRH binding capacity by 7 d and binding capacity remained elevated for the subsequent 14 d. GnRH receptor number rose more rapidly after castration in males than females, and the time-course of receptor rise was similar to the increase in serum gonadotropin levels. The increase in GnRH binding capacity was prevented by gonadal steroid replacement at the time of castration in both sexes. Injections of the GnRH analog, D-Ser6 (TBu) des Gly10 GnRH ethylamide for 4 d produced a 70% increase in GnRH receptor number in intact male rats and testosterone-replaced castrates. The same regimen, however, failed to increase the elevated receptor numbers present after castration. Administration of rabbit anti-GnRH serum concomitant with castration inhibited the rise in both GnRH receptor number and luteinizing hormone. The changes in pituitary GnRH receptors parallel previously demonstrated changes in hypothalamic secretion of GnRH. Thus, GnRH probably regulates its own receptor in vivo and gonadal steroids may influence pituitary GnRH receptors by changing hypothalamic GnRH secretion.
M S Frager, D R Pieper, S A Tonetta, J A Duncan, J C Marshall
We have recently characterized two lymphocyte-associated membrane proteins which have been termed 225,000-dalton and 185,000-dalton macromolecular insoluble cold globulin (225-MICG and 185-MICG, respectively) to distinguish their major physicochemical properties. These proteins differ antigenically, structurally, and in their cellular distribution. T cells can be distinguished by the synthesis and presence in the plasma membrane of 225-MICG, Null cells by the appearance of 185-MICG, and B cells by the appearance of both 225- and 185-MICG. The characterization of these two proteins in the monoclonal B lymphocytes of chronic lymphocytic leukemia forms the basis of this report.
Mary A. Simmonds, Gloria Sobczak, Stephen P. Hauptman
Uterine production of PGI2 (prostacyclin) was quantitated in late pregnant dogs to evaluate if PGI2 could act as circulating vasodepressor substance in pregnancy. In five anesthetized, laparotomized dogs, the uterine venous plasma concentration of 6-keto PGF1 alpha (the in vitro hydrolysis product of PGI2) was 6.7 +/- 1.9 ng/ml and the arterial plasma concentration was 2.6 +/0 0.8 ng/ml. In four nonpregnant female dogs the arterial plasma concentration of 6-keto PGF1 alpha was consistently below 0.2 ng/ml. In eight pregnant dogs we also evaluated the ability of the pregnant uterus to inactivate PGI2 by comparing the hypotensive response to increasing doses of PGI2 infused into the uterine artery to the hypotensive response to increasing doses of PGI2 infused into the inferior vena cava. In addition, the effect of PGI2 infused into the uterine artery on uterine blood flow and intraamniotic fluid pressure was evaluated. The dose-response curves of intrauterine and intravenous PGI2 in causing systemic hypotension were identical suggesting that the pregnant uterus does not inactivate infused PGI2. Intrauterine PGI2 had no consistent effect on uterine hemodynamics although it did increase intraamniotic fluid pressure significantly. These data demonstrate that the pregnant uterus has the capacity to produce large quantities of PGI2 which is not inactivated in the uterus and therefore can appear in the arterial blood to exert a systemic vasodepressor effect.
J G Gerber, N A Payne, R C Murphy, A S Nies
The pathogenesis of the increased erythrocytosis and extramedullary erythropoiesis observed in infants of diabetic mothers (IDM) has been obscure. In the present studies, IDM were found to have elevated umbilical plasma erythropoietin (Ep) concentrations by radioimmunoassay. 22 of 61 IDM (36%) had levels above the range of 28 nonasphyxiated, appropriately grown normal infants. In 16 controls and 20 IDM, plasma Ep correlated directly with plasma insulin (P less than 0.001, r = 0.73). To investigate this relationship further, a chronic rhesus model was studied with continuous fetal hyperinsulinemia for 21 d in utero in the last third of pregnancy. In five experimental fetuses, plasma insulin levels averaged 4,210 microU/ml at delivery, whereas plasma Ep was above the range of six controls. In addition, the experimental fetuses had elevated reticulocyte counts in umbilical cord blood. The mechanism for the increased plasma Ep associated with hyperinsulinemia in the fetus is unexplained but may be mediated by fetal hypoxia.
J A Widness, J B Susa, J F Garcia, D B Singer, P Sehgal, W Oh, R Schwartz, H C Schwartz
Hydrogen produced by colonic bacteria and excreted in breath is a useful index of carbohydrate malabsorption. Since colonic contents are often acidic in individuals with carbohydrate malabsorption and in normal newborns, we determined the effect of colonic acidification on H2 production. Acidification of colonic contents by dietary means significantly reduced excess breath H2 excretion from 55.4 +/- 11.1 (SEM) to 12.2 +/- 3.1 ml/4 h (P less than 0.05) after administration of 0.3 g/kg of the nonabsorbable sugar lactulose to five normal adult subjects. Similarly, the breath H2 response to lactose was reduced or eliminated in two proven lactose malabsorbers after acidification. The correlation between pH and H2 production from carbohydrate was further investigated in adults and neonates, using an in vitro fecal incubation system. Glucose disappearance and H2 production were pH dependent and highly correlated (r = 0.94) in the pH range 5.5-7.6. Maximal production of H2 from glucose by fecal incubates occurred at pH 7.0-7.45. Inhibition of H2 production from carbohydrate occurred at acid pH. H2 per hour from glucose at pH 6.2 and 5.5 averaged 60.2% and 24.2%, respectively, of that produced at neutral pH. Rapid reversal of pH-induced inhibition by neutralization indicated a metabolic, rather than a bactericidal process. The observations indicate that the breath H2 response to malabsorbed carbohydrate is affected by colonic pH. It appears that the efficiency of bacterial carbohydrate metabolism in the colon is pH dependent.
J A Perman, S Modler, A C Olson
A radioimmunoassay was established for the human eosinophil granule major basic protein (MBP). The mean level of MBP in sera from 105 normal control patients was 454 ng/ml, whereas in a sample of 188 patients with various forms of diseases, including the hypereosinophilic syndrome, levels as high as 14,000 ng/ml were measured. Serum levels of MBP did not correlate with eosinophil counts in normal subjects, but a positive correlation was seen in patients with eosinophilia; the patients with eosinophil counts greater than 350/mm3 generally showed increased levels of MBP. Many patients with skin disease and normal eosinophil counts had elevated levels of serum MBP. Monomer MBP has a molecular weight of 9,300, but in sera of patients with eosinophilia, the MBP activity was of high molecular weight, greater than 50,000. Analyses of serum by Sephadex G-200 and by electrofocusing suggest that MBP is not simply polymerized, but rather is bound to a larger carrier molecule. Monomeric MBP can be isolated from serum by reduction of serum with dithiothreitol, alkylation with iodoacetamide, and acidification to pH 2 followed by fractionation on Sephadex G-50 at pH 2. Under these conditions, up to 80% of the MBP emerges in monomeric form. The results indicate that eosinophil granule proteins circulate in blood covalently bound to serum proteins, and that elevated concentrations of serum MBP are present in some diseases associated with eosinophilia.
D L Wassom, D A Loegering, G O Solley, S B Moore, R T Schooley, A S Fauci, G J Gleich
Calcium ion plays a major regulatory role in many hormone-stimulated systems. To determine the site of calcium's action in the toad urinary bladder, we examined the effect of trifluoperazine, a compound that binds specifically to the calcium binding protein, calmodulin, and thereby prevents activation of enzymes by the calcium- calmodulin complex. 10 microM trifluoperazine inhibited vasopressin stimulation of water flow, but did not alter vasopressin's effects on urea permeability or short-circuit current. Trifluoperazine also blocked stimulation of water flow by cyclic AMP and methylisobutylxanthine, implying a "postcyclic AMP" site of action. Consistent with these results, trifluoperazine did not decrease epithelial cyclic AMP content or the cyclic AMP-dependent protein kinase activity ratio. Assay of bladder epithelial supernate demonstrated calmodulin-like activity of 1.5 U/microgram protein. Morphologic studies of vasopressin-treated bladders revealed that trifluoperazine did not alter the volume density of cytoplasmic microtubules or significantly decrease the number of fusions between cytoplasmic, aggregate-containing, elongated vesicles and the luminal membrane. Nonetheless, the frequency of luminal membrane aggregates, structures that correlate well with luminal membrane water permeability, was decreased by greater than 50%. Thus, trifluoperazine appears to inhibit the movement of intramembranous particle aggregates from the fused intracellular membranes to the luminal membrane, perhaps by blocking an effect of calcium on microfilament function.
S D Levine, W A Kachadorian, D N Levin, D Schlondorff
The role of the insulin-receptor compartment in the pharmacokinetics of intravenously injected insulin in rats was studied. Since streptozotocin-diabetes in rats results in increased insulin binding to tissues in vitro, insulin pharmacokinetics in streptozotocin-diabetic rats were compared to controls, using semisynthetic [3H]insulin as the tracer. The initial distribution volume for [3H]insulin was elevated by 60% in diabetic rats. By contrast, no difference in initial distribution volume for [14C]inulin was observed, and the absolute values were lower than those found for [3H]insulin. The metabolic clearance rate of [3H]insulin was elevated by 44% in diabetic rats. That these differences were the result of increased binding of insulin to a specific receptor compartment in diabetic rats was shown by three additional experiments. The first involved receptor saturation by injection of 10 U native insulin 2 min before the tracer injection, resulting in identical [3H]insulin disappearance rates in the two groups of rats. The second consisted of displacing [3H]insulin from receptors by injecting 10 U unlabeled insulin 6 min after the tracer injection. Displacement of intact [3H]insulin from receptors and subsequent reappearance in the circulation occurred in both control and diabetic animals; however, such displacement was 25% greater in the diabetic rats. Finally, treatment of diabetic rats with insulin for 8 d normalized [3H]insulin clearance even though the tracer was injected at a time when the animals were again hyperglycemic and hypoinsulinemic. This suggests that down-regulation of insulin receptors had occurred during insulin therapy. These results confirm that a specific compartment for insulin exists (the insulin-receptor compartment) and that this compartment plays an important role in insulin clearance.
Jacques Philippe, Philippe A. Halban, Asllan Gjinovci, William C. Duckworth, Jurek Estreicher, Albert E. Renold
To elucidate the relationship between Epstein-Barr virus (EBV) and rheumatoid arthritis (RA), we measured antibodies to RA-associated nuclear antigen (anti-RANA) and three other EBV-related antigens in the sera of RA patients and controls. Our study groups consisted of 89 patients with definite or classical RA, mean age 56, male/female ratio 47:42; and 53 normal and osteoarthritis controls, mean age 51, male/female ratio 25:28. In addition to anti-RANA, we measured antibodies to viral capsid antigen (anti-VCA), early antigen (anti-EA) and EBV-associated nuclear antigen (anti-EBNA). Anti-RANA was detected in 71% of RA patients but in only 6% of controls. Elevated anti-VCA titers (greater than 1:160) were more common in RA patients than controls, 31% compared with 15%. The geometric mean titer of anti-VCA was significantly higher iun the RA group, 133 compared with 58. Anti-EA was present in 53% of RA patients but only 19% of controls. Anti-EA in elevated titers (greater than 1:20) was present in 26% of RA patients but only 7% of controls. Characterization of the anti-EA antibodies revealed that the RA patients reacted primarily with the diffuse component, whereas the majority of the controls reacted with the restricted component of the EA complex. In contrast, the frequencies, distributions, and geometric mean titers of anti-EBNA were not significantly different between the two groups. Correlative analysis of these antibodies showed highly significant relationships between anti-VCA and anti-EA, and anti-RANA and anti-EBNA in the RA group. These data are compatible with the interpretation that RA patients have either more active EBV infections than controls or an altered regulation of their immune response to this infectious agent.
P B Ferrell, C T Aitcheson, G R Pearson, E M Tan
Hepatotoxicity caused by acetaminophen can be prevented by enzyme-catalyzed conjugation of its reactive metabolite with glutathione (GSH). Since we have shown in previous studies that 6-N-propyl-2-thiouracil (PTU) can substitute for GSH as a substrate for the GSH S-transferases, we examined the possibility that PTU might also protect against acetaminophen hepatotoxicity by direct chemical interaction with the reactive metabolite of acetaminophen. In an in vitro system consisting of [3H]acetaminophen, liver microsomes from phenobarbital-pretreated rats, and an NADPH-generating system, we found that PTU had a dose-dependent additive effect with GSH on inhibition of acetaminophen covalent binding. PTU administration also resulted in a dose-dependent decrease in both GSH depletion and covalent binding in vivo in acetaminophen-treated mice. To examine the possible mechanisms by which PTU exerts its protective effect, we studied the action of PTU on both acetaminophen conjugation and metabolic activation. PTU had no effect upon acetaminophen pharmacokinetics in phenobarbital-pretreated rats, as examined by measuring acetaminophen concentration in bile, urine, and blood after an intraperitoneal dose, nor did it alter the total amount of polar conjugates formed. Microsomes from PTU-treated rats were unaltered in cytochrome P-450 concentrations and p-nitroanisole-O-demethylase, benzo-α-pyrene hydroxylase, and cytochrome c-reductase activities. Furthermore PTU did not decrease acetaminophen-GSH adduct formation in vitro, suggesting that there was no reduction in drug activation. However, in bile from [35S]PTU and [3H]acetaminophen treated rats, as well as in incubates of the two drugs with liver microsomes, a new 35S- and 3H-containing product could be identified. By both thin layer chromatography and high pressure liquid chromatography this new product, which co-eluted with [3H]acetaminophen, was separated from unreacted [35S]PTU. The formation of this product in vitro was a function of PTU concentration and reached a maximum of 0.06 μmol/min per mg protein at 0.5 mM PTU. In vivo, the total biliary excretion of this product over 4 h (116 nmol) equaled the net reduction in acetaminophen metabolite covalent binding in the liver of phenobarbital-pretreated rats (108 nmol). We conclude that PTU, independent of its antithyroid effect, diminishes hepatic macromolecular covalent binding of acetaminophen reactive metabolite both in vivo and in vitro, and it does so by detoxifying the reactive metabolite through direct chemical interaction in a manner similar to GSH. These observations may define the mechanism by which PTU is protective against liver injury caused by acetaminophen.
Tadataka Yamada, Shelly Ludwig, John Kuhlenkamp, Neil Kaplowitz
We have examined the basis for the recently reported, but unexplained deficiency of S-adenosylhomocysteine hydrolase (AdoHcyase) in the erythrocytes of patients with genetic deficiencies of purine nucleoside phosphorylase and hypoxanthine-guanine phosphoribosyltransferase. We found that a hemolysate from a patient with purine nucleoside phosphorylase deficiency had only 7% of control AdoHcyase activity, conforming the original observation. Of the purine nucleosides known to accumulate in nucleoside phosphorylase-deficient patients, inosine alone caused the phosphate-dependent, irreversible inactivation of purified human placental AdoHcyase, and of AdoHcyase in intact erythrocytes and cultured lymphoblastoid cells. Hypoxanthine did not inactivate purified AdoHcyase, but potentiated the effect of inosine in intact hypoxanthine-guanine phosphoribosyltransferase-deficient human lymphoblastoid cells. This presumably resulted from the ability of hypoxanthine to shift the equilibrium of the nucleoside phosphorylase reaction, preventing inosine breakdown. This could account for the partial AdoHcyase deficiency reported in hypoxanthine-guanine phosphoribosyltransferase-deficient patients. We have also demonstrated the AdoHycase-catalyzed synthesis of S-inosylhomocysteine from inosine and L-homocysteine, a reaction which may occur in nucleoside phosphorylase-deficient patients.
M S Hershfield
The roles of monocytes and T lymphocytes in the regulation of human peripheral blood erythroid burst-forming units (BFU-E) were studied in erythropoietin-containing plasma clot cultures of subpopulations of human blood mononuclear cells. BFU-E growth was decreased significantly after depletion of monocytes alone (mean 11% of expected, range 0 to 42% of expected) or depletion of both monocytes and T cells (mean 6.5% of expected, range 0.5 to 12% of expected) from mononuclear cells. T cell depletion did not impair BFU-E growth in vitro. Using 10(5) monocyte- and T lymphocyte-depleted mononuclear cells as target cells (less than 1% monocytes, less than 5% T cells), BFU-E growth was restored to 40% of expected by addition of 10(4) monocytes, and to 96% of expected by 10(5) monocytes alone. Addition of as many as 2 X 10(5) T cells but no monocytes resulted in stimulation to only 34% of expected BFU-E growth. Addition of 2 X 10(4) T cells, which alone did not affect BFU-E growth, could augment significantly the stimulatory effect of 5-20 X 10(3) monocytes on BFU-E growth. Thus, monocytes alone appear to be capable of stimulating BFU-E growth in vitro in the presence of erythropoietin. T cells also may make small quantities of BFU-E stimulators. However, it seems more likely that the most important role of T lymphocytes in BFU-E regulation in vitro is a result of interactions with monocytes and augmentation of monocyte production of stimulators of BFU-E growth.
K S Zuckerman
Alteration of the surface of human neutrophils with the nonpenetrating, protein-inactivating agent p-diazobenzenesulfonic acid (DASA) was found to prevent activation of the respiratory burst by some stimuli, but not others. Production of superoxide anion (O2-) stimulated by concanavalin A or the chemotactic peptide formyl-methionyl-leucyl-phenylalanine FMLP was inhibited by DASA pretreatment, whereas O2- production stimulated by phorbol myristate acetate (PMA), sodium fluoride. or the ionophore A23187 was not inhibited by DASA. Pretreatment with DASA inhibited oxygen uptake stimulated by FMLP, but not oxygen uptake stimulated by PMA. DASA reproducibly inhibited activities of two known surface enzymes Mg++-ATPase and alkaline phosphatase, by 45-55% and 60-70%, respectively. The inhibition by DASA of O2- production did not appear to be caused by interference with binding of the affected stimuli, since pretreatment with DASA did not inhibit release of the lysosomal enzymes lysozyme and myeloperoxidase induced by concanavalin A or FMLP. Membrane-rich particulate fractions from neutrophils have been shown to contain NADPH-dependent oxidative activity that is presumably responsible for the phagocytosis-associated respiratory burst of intact cells. The PMA-activated enzyme was susceptible to inhibition of directly exposed to DASA in this particulate fraction. These findings suggest that more than one mechanism exists for activation of the respiratory burst oxidase in human neutrophils, and that the neutrophil possesses at least one oxidase that is not an ectoenzyme.
L C McPhail, P M Henson, R B Johnston Jr
The hereditary of the human platelet alloantigen, PlA1, has been studied in Glanzmann's thrombasthenia. The PlA1 content of platelets from three patients, 20 kindred of these patients, including parents and siblings, and 15 unrelated normal individuals was determined using immunologic techniques based on the release of 51Cr from labeled platelets. The amount of membrane glycoproteins (GP) IIb and IIIa in the platelets of these individuals was determined by quantitative crossed immunoelectrophoresis of Triton X-100 soluble proteins using a multispecific rabbit antibody raised against normal platelets. Platelets from the three thrombasthenic patients contained neither detectable GP IIb and GP IIIa nor detectable PlA1 antigen. Platelets from seven kindred with normal amounts of GP IIb and GP IIIa contained PlA1 antigen levels identical to those detected in platelets of normal individuals. Platelets from 13 kindred, including each parent studied, were shown to contain an amount of GP IIb and GP IIIa equivalent to 53% of that amount detected on normal platelets. Platelets from the same individuals expressed amounts of PlA1 antigen that were either 54.0 +/- 4.1 (mean +/- SD) or 28.0 +/- 2.7% of that present on platelets of normal individuals homozygous for the Al allele. The results presented in this report provide evidence that the expression of the thrombasthenic glycoprotein abnormality and the inheritance of PlA1 antigen are controlled by different genes. These results further suggest that lack of expression of the PlA1 antigen on thrombasthenic platelets results from the decrease or absence of the glycoprotein carrier of the PlA1 determinant, previously shown to be GP IIIa.
T J Kunicki, D Pidard, J P Cazenave, A T Nurden, J P Caen
Terminal deoxynucleotidyl transferase was purified to homogeneity from the blasts of eight patients with leukemia and compared with purified transferase from normal human and calf thymus. In two cases phenylmethanesulfonylfluoride was added during purification to reduce proteolysis. Comparative kinetic analyses of the purified enzymes indicated no differences in catalytic properties. There was substantial variation in the molecular structure of terminal transferase on denaturing polyacrylamide gels: (a) a protein that migrated as a single polypeptide with Mr = 62,000 was isolated from two patients with acute lymphoblastic leukemia and from MOLT-4 cells; (b) a protein that migrated as a single polypeptide with Mr = 42,500 was isolated from two patients with acute lymphoblastic leukemia; (c) a protein that migrated as a single polypeptide with Mr = 42,500 was isolated from two patients with chronic myelogenous leukemia in blast crisis; (d) a protein that migrated as two non-identical subunits of Mr = 27,000 and 10,000, respectively, was isolated from two additional patients with chronic myelogenous leukemia in blast crisis. The subunit structure of d is characteristic of the homogeneous enzymes purified from human and calf thymus. Neutralizing and precipitating antibodies to terminal transferase from human lymphoblasts and calf thymus have been produced in rabbits and goats. Antisera directed against either human or calf antigens neutralize enzymatic activity and precipitate all forms of human terminal transferase. The multiple human forms give reactions of antigenic identity by immunodiffusion, but differ antigenically from the calf enzyme. The multiple forms of terminal transferase could represent physiological processing, artifactual degradation, or isozymes coded by several genes.
Martin R. Deibel Jr., Mary Sue Coleman, Karen Acree
Prostacyclin (PGI2) synthetic capacity was assayed at the surface of aortas at various intervals after removal of endothelium with a balloon catheter. Results were correlated with morphologic changes in the vessel wall seen by light microscopy, scanning and transmission electron microscopy. To assay PGI2 synthetic capacity, we applied an incubation chamber to the luminal surface of the aortas; after arachidonic acid stimulation we assayed the PGI2 synthesized with a bioassay and radioimmunoassay. PGI2 synthesis in de-endothelialized aortas was determined immediately after balloon-catheter injury and at intervals of 1 h and 2, 4, 15, 35, and 70 d. PGI2 synthesis was low at 1 h and increased over time with levels at 35 and 70 d reaching that of normal artery. Scanning and transmission electron microscopy of de-endothelialized areas showed persistent absence of endothelium with formation of a neointima composed of smooth muscle cells. De-endothelialized aorta was covered with adherent platelets shortly after injury, however several days later only a few platelets adhered to the denuded surface. Results indicated that (a) endothelium is responsible for nearly all PGI2 production at the luminal surface of the normal aorta, (b) de-endothelialized muscular neointima synthesized increasing quantities of PGI2 with time after injury, and (c) increase of PGI2 production at the luminal surface of de-endothelialized aorta correlates with formation of a neointima and with the acquired thromboresistance of the aorta.
A Eldor, D J Falcone, D P Hajjar, C R Minick, B B Weksler
An EDTA procedure was used to prepare isolated epithelial cells of human gallbladder devoid of endogenous vasoactive intestinal peptide (VIP) as measured by radioimmunoassay. Specific binding sites for VIP were characterized in these cells. At 37°C, the binding of 125I-labeled VIP reached a peak within 20 min and then declined rapidly. At 15°C, binding was stable between 90 and 180 min of incubation. Binding of the labeled peptide was inhibited by concentrations of native VIP of 30 pM-0.1 μM. Half-maximal inhibition was observed at 2 nM. Scatchard analysis indicated two functionally independent classes of receptor sites: 62,000 high affinity sites/cell with a dissociation constant (Kd) of 1.3 nM, and 510,000 low affinity sites/cell with a Kd of 16.2 nM. Secretin inhibited tracer binding but with a 1,000 times lower potency than native VIP. VIP strongly stimulated adenosine 3′:5′ monophosphate (cyclic AMP) production in human gallbladder epithelial cells. At 37°C, 0.1 nM and 10 nM VIP raised cyclic AMP levels 44 and 100 times above the basal level, respectively. Maximal values remained constant between 60 and 90 min at 15°C. The importance of the VIP-induced cyclic AMP rise was related, at least in part, to a low phosphodiesterase activity in human gallbladder epithelial cells. At equilibrium, during a 60-min incubation at 15°C, cyclic AMP production was noted at concentrations of VIP as low as 3 pM. Maximal and half-maximal stimulations were observed at 10 nM and 0.2 nM VIP, respectively. Secretin also stimulated cyclic AMP production but with a 10,000 lower potency than VIP.
Christophe Dupont, Jean-Pierre Broyart, Yvonne Broer, Bernard Chenut, Marc Laburthe, Gabriel Rosselin
The human inducer (T4+) and reciprocal cytotoxic/suppressor (T5+/T8+) subsets have been defined by monoclonal antibodies. In the present study, we examined the relationship of naturally occurring anti-T cell autoantibodies found in patients with active juvenile rheumatoid arthritis (JRA) to these subsets. In one approach, normal T cells were treated with anti-T4 or anti-T8 to eliminate the corresponding subset of cells and then analyzed for reactivity with JRA sera. It was found that JRA sera were reactive with only 15% of an enriched cytotoxic/suppressor population, whereas they reacted with 37% of an enriched inducer population. In reciprocal studies, JRA+ T cells were eliminated with JRA sera and complement and the residual T cells (JRA−) reacted with monoclonal antibodies and indirect immunofluorescence on a fluorescence-activated cell sorter. As expected, the JRA sera and complement treatment of unfractionated T cells markedly diminished the T4+ subset, whereas there was a concomitant increase in T cells reactive with anti-T5 and anti-T8. A similar diminution in T4+ T cells was found in the circulating peripheral T cell compartment of patients with active JRA who possessed the JRA antibody.
Chikao Morimoto, Ellis L. Reinherz, Yves Borel, Evangelia Mantzouranis, Alfred D. Steinberg, Stuart F. Schlossman
Nitroglycerin (NTG), the agent most commonly used to treat acute angina pectoris, is a vasodilator whose mechanism of action remains unknown. We hypothesized that NTG might induce endothelial cells to synthesize prostacyclin (PGI2), a known vasodilator and inhibitor of platelet aggregation. Therefore, cultured human endothelial cells were incubated with NTG at various concentrations for 1-3 min. PGI2 biologic activity in the endothelial cell supernates was assayed by inhibition of platelet aggregation in vitro. The concentration of 6-keto-PGF1α, the stable hydrolysis product of PGI2, was measured by specific radioimmunoassay.
Richard I. Levin, Eric A. Jaffe, Babette B. Weksler, Karen Tack-Goldman
Physiologic and pharmacologic effects of glucocorticoids on ileal ion transport were examined in vitro. Tissues were obtained from the three following groups of rabbits: (a) normal; (b) glucocorticoid deficient, which were treated with aminoglutethimide (AG), 100 mg twice daily for 3 d, with a resulting marked reduction in urinary cortisol excretion but no decrease in urinary aldosterone; and (c) methylprednisolone-treated (MP), 40 mg daily for 2 d. Transileal NaCl fluxes were measured with radioisotopes under short-circuit conditions, and the net HCO3 flux was assumed equal to that portion of the short-circuit current (Isc) not accounted for by Na and Cl. In NaCl Ringer's solution containing 25 mM HCO3 (pH 7.4), normals absorbed both Na and Cl and secreted HCO3; the Isc was greater in both AG and MP groups than in normals; in the AG group, no Na was absorbed, and Cl as well as HCO3 was secreted; in the MP group, more Na was absorbed and more HCO3 secreted than in normals. Addition of glucose to the luminal side caused similar increments in Isc in all three groups, suggesting similar rates of Na-coupled glucose absorption. Secretory response was assessed with a maximal secretory simulus (8-Br-cAMP) and also a submaximal, cGMP-related secretory stimulus (Escherichia coli heat-stable enterotoxin). After addition of 8-Br-cAMP, the rates of net Cl secretion were similar in all three groups, suggesting no effect of glucocorticoids on maximal secretory capacity. Because the AG group was already secreting Cl, however, the cAMP-induced change in net Cl flux was least in this group. After addition of heat-stable enterotoxin, there were similar changes in net Cl flux in all three groups. To examine specifically Cl-independent, electrogenic Na transport, we used a 10 mM HCO3, Cl-free SO4-Ringer (ph 7.2) in which net Na absorption was previously shown to be equal to the Isc. Under these conditions, Isc was greatest in the MP group and least in the AG group. In vitro addition of hydrocortisone, 50 μg/ml, to AG tissues had no effect on Cl fluxes or Isc over a 3.5-h period. No differences among groups were observed with respect to morphology, electrical resistance, or cGMP concentration. We conclude that (a) the effect of glucocorticoid deficiency is similar to that of a submaximal secretory stimulus in that Na absorption is inhibited and some Cl secretion develops; (b) electrogenic Na absorption is depressed in glucocorticoid deficiency and enhanced in glucocorticoid excess; (c) glucocorticoid excess increases HCO3 secretion; and (d) glucocorticoid status does not affect maximal secretory capacity.
Joseph H. Sellin, Michael Field
Multiple myeloma is a malignancy characterized by uncontrolled monoclonal B cell differentiation and immunoglobulin production. In most instances, there is concomitant reduction in polyclonal differentiation and immunoglobulin synthesis both in vivo and in vitro. In in vitro pokeweed mitogen-induced B cell differentiation assays, proliferation and polyclonal immunoglobulin secretion optimally requires T cell help and can be inhibited both by monocytes and suppressor T cells. Helper function and monocyte-mediated suppression are relatively radio-resistant whereas T suppressor function is sensitive to 2,000 rad x-irradiation. We have examined myeloma T cell subset function in this assay using recombinations of isolated patient and normal B cells, T cells, and T cell subsets. Monocytes were removed by a carbonyl iron ingestion technique, normal and myeloma T cells were fractionated on the basis of Fc receptors for immunoglobulin (Ig) G (Tγ) or IgM (Tμ or T non-γ), and proliferation and IgG secretion after co-culture determined by [3H]thymidine incorporation and radio-immunoassay, respectively. Myeloma B cells demonstrate quantitatively and qualitatively normal blastogenic responses and are appropriately regulated by either autologous or allogeneic T helper and suppressor subsets. Despite normal proliferation, however, myeloma B cells remain deficient in subsequent differentiation and immunoglobulin secretion even when co-cultured in the absence of monocytes or suppressor T cells and the presence of normal helper cells. Myeloma T cell populations, in contrast, are entirely normal in helper capacity over a range of T:B ratios but are markedly deficient in radiosensitive and concanavalin A-induced suppressor activity. T suppressor cell dysfunction in multiple myeloma is apparently due to a deficit in the T non-γ suppressor subset, whereas Tγ cells, although proportionately reduced, are functionally normal. This unique T suppressor deficit reflects the heterogeneity of suppressor mechanisms in this disease and may represent a compensatory response to the monoclonal proliferation or the involvement of regulatory T cells in the pathogenesis of the malignancy.
H. Ozer, T. Han, E. S. Henderson, A. Nussbaum, D. Sheedy
The prostatic fluid of two patients with Escherichia coli bacterial prostatitis was analyzed for evidence of a local immune response to bacterial infection. A solid-phase radioimmunoassay was modified to measure the immunoglobulin (Ig)A and IgG antigen-specific antibody responses to infecting bacteria in serum and prostatic fluid from patient. Formalin-fixed whole E. coli were used as antigen. In one patient with acute E. coli prostatic infection, measurements of antigen-specific antibody confirm the presence of a systemic and local immune response. However, in another patient with a chronic E. coli prostatitis, a primarily local immune response was demonstrated. The response measured in the prostatic fluid appears to be locally stimulated and specific for the infecting bacteria. Furthermore, IgA was the predominant immunoglobulin involved in the local prostatic immune response to infection. Although elevations of serum IgA antigen-specific antibody levels were short-liver after treatment of prostatic infection, local IgA antigen-specific antibodies were detected for as long as 1 yr after the initial infection in both patients studied.
L M Shortliffe, N Wehner, T A Stamey
Thyrotropin-releasing hormone immunoreactivity (IR-TRH) has been detected in the circulation of the neonatal rat. This immunoreactivity was demonstrated in purified ethanol extracts of plasma, and was indistinguishable from synthetic TRH using radioimmunoassay and chromatographic criteria. To determine the source of the circulating IR-TRH, tissue concentrations of TRH were analyzed during maturation of the rat. These studies revealed that during the first 10 d of life, the pancreas contained the greatest concentration of IR-TRH of any organ (pancreas, 289±35 pg/mg; hypothalamus, 13±3 pg/mg, day 5). Thereafter, pancreatic IR-TRH concentrations declined progressively while hypothalamic concentrations gradually increased (pancreas, 1.2±0.2 pg/mg; hypothalamus, 365±54 pg/mg, adult rat). IR-TRH was also found throughout the gastrointestinal tract but was not detected in the liver, spleen, kidney, or heart. IR-TRH from the pancreas and gastrointestinal tract gave radio-immunoassay binding displacement curves that were parallel to a curve generated with synthetic TRH, and co-migrated with synthetic TRH on Sephadex G-10 and high performance liquid chromatography. In addition, IR-TRH from purified pancreatic extracts was biologically active in that it released thyrotropin and prolactin from rat adenohypophysial cells maintained in monolayer culture. When a total pancreatectomy was performed on the 5th d of life of the rat, mean plasma TRH concentrations were significantly decreased 3 h afterwards (84±9 vs. 63±7 pg/ml, P < 0.05). Neither the TRH concentrations in the brain, hypothalamus, or gastrointestinal tract, nor the pituitary-thyroid axis were affected by the pancreatectomy. However, mean plasma TRH concentrations remained unaltered 3 h after removal of the hypothalamus and extrahypothalamic brain.
Dennis Engler, Maurice F. Scanlon, Ivor M. D. Jackson
Thrombin converts fibrinogen to fibrin in two steps. First fibrinopeptide A and fibrin I are formed and then fibrinopeptide B (B beta 1-14) and fibrin II. Since it is postulated that fibrin II is important in the genesis of thrombosis, it is of interest to measure fibrinopeptide B in peripheral blood samples. Previous difficulties in interpreting fibrinopeptide B immunoreactivity in plasma resulted from crossreaction of fibrinogen and of plasmin digest peptides B beta 1-42 and B beta 1-21 and from rapid loss of fibrinopeptide B immunoreactivity resulting from cleavage of arginine 14 by blood carboxypeptidase B. We have obviated these difficulties by removing fibrinogen from plasma by precipitation with ethanol and peptides B beta 1-21 and B beta 1-42 by adsorption on bentonite. Fibrinopeptide B is then converted to a desarginine fibrinopeptide B, which is measured in a new specific assay. Studies of the kinetics of fibrinopeptide cleavage showed that when whole blood was allowed to clot in vitro, fibrinopeptide A was cleaved more rapidly than fibrinopeptide B. In 18 patients on an acute care medical ward, desarginine fibrinopeptide B levels were lower than fibrinopeptide A levels and did not correlate with the levels of fibrinopeptide A or B beta 1-42. Desarginine fibrinopeptide B levels were less than 1 pmol/ml in all but two patients. In six patients receiving intraamniotic infusions of hypertonic saline to induce abortion, desarginine fibrinopeptide B levels increased 10-fold from the preinfusion mean level of 0.4 pmol/ml and then decreased. The pattern of changes resembled that of the fibrinopeptide A levels rather than of the B beta 1-42 levels. On the basis of these data it is suggested that plasma desarginine fibrinopeptide B levels reflect fibrin II formation in vivo.
T Eckhardt, H L Nossel, A Hurlet-Jensen, K S La Gamma, J Owen, M Auerbach
The purpose of this study was to determine if human platelet factor 4 (PF4) stimulates human leukocyte elastase (HLE) against lung elastin. Lung elastin was purified from hamster lungs and tritiated by reduction with NaB3H4. We found that HLE activity against this substrate is increased by concentrations of PF4 as low as 1.6 microgram/ml, and that this stimulation increased linearly with additional PF4. Lungs removed from hamsters and inflated with solutions containing buffer alone, low dose HLE, HLE plus PF4, or PF4 alone were incubated for 2 h at 37 degrees C. Whereas low-dose HLE failed to lower lung elastin when compared to control animals, HLE stimulated by PF4 lowered lung elastin by 20%. PF4 alone had no effect. Furthermore, low-dose HLE failed to alter the mechanical properties of hamster lungs as measured by pressure-volume curves in saline, although there was a significant loss of lung elasticity in the mid- and high-lung volume ranges in lungs treated with HLE and PF4. Morphologic studies revealed that low dose HLE resulted in a minimal emphysemalike lesion whereas HlE plus PF4 caused a significantly more severe lesion. PF4 is capable of stimulating HLE against lung elastin, and this effect may have a role in the pathogenesis of emphysema.
S A Lonky, H Wohl
The addition of bacterial lipopolysaccharide (LPS) from Salmonella minnesota R595 to rabbit plasma results in a marked reduction of the hydrated buoyant density of the parent R595 LPS, from 1.38 to less than 1.2 g/cm3. Using immunopurified anti-R595 LPS antibody covalently linked to Sepharose 4B, we were able to separate the altered R595 LPS (d less than 1.2 g/cm3) from the remainder of the plasma proteins by elution of the bound material with 2.5 M KSCN. The KSCN eluate was shown to have a d less than 1.2 g/cm3 and to contain both R595 LPS as well as protein and lipid characteristic of high density lipoprotein (HDL). The major protein in the KSCN eluate is a single polypeptide chain with an apparent molecular weight of 26,000 in sodium dodecyl sulfate and an amino acid composition essentially identical to that of apoprotein AI, the major protein of rabbit HDL. The lipid composition of the KSCN eluate is similar to that of HDL, although marked differences in the cholesterol ester/cholesterol ration and the phosphatidyl choline/phosphatidyl ethanolamine ratio were observed when the KSCN eluate and rabbit HDL were compared. The formation of this R595 LPS-protein-lipid complex in plasma accounts for the marked reduction in buoyant density found when LPS is added to plasma.
R J Ulevitch, A R Johnston, D B Weinstein
We have measured lipolytic activity in gastric aspirates obtained at birth in a group of 142 infants. The infants ranged in gestational age from 26 to 41 wk. Lipolytic activity, measured by the hydrolysis of long chain triglyceride ([tri-3H]oleate), and expressed as nanomoles FFA per milliliter gastric aspirate per minute was 333±66 in 55 small premature infants (gestational age 26-34 wk and body wt 750-2,000 g) and 558±45 in a group of 87 larger infants (gestational age 35-41 wk and body wt 2,020-4,000 g). No activity was detected in seven infants with an unusually low pH in the gastric aspirate, 2.88±0.44 (compared with a mean pH level of 5.59±0.22 in the other 135 infants).
Margit Hamosh, John W. Scanlon, Dvora Ganot, Melodie Likel, Kathleen B. Scanlon, Paul Hamosh
An assay procedure was developed in which phosphatidyl[2-3H]inositol was employed as substrate for the measurement of phosphatidylinositol-specific phospholipase C activity. Employing this assay, phosphatidylinositol-specific phospholipase C activity in human fetal membranes and uterine decidua was identified and characterized. The specific activity of this enzyme in amnion (4.4 μmol × mg−1 protein × h−1) was three times that in uterine decidua and more than five times that in chorion laeve. No difference was found between the specific activity of phosphatidylinositol-specific phospholipase C in placental amnion and that in reflected amnion. The products of phosphatidylinositol hydrolysis in short-term incubations were stoichiometric amounts of diacylglycerol and inositol-1,2-cyclic-phosphate plus inositol-1-phosphate. After longer periods of incubation, monoacylglycerol also was detected. Diacylglycerol lipase activity also was demonstrated in these tissues. More than 90% of phosphatidylinositol-specific phospholipase C activity of amnion tissue was recovered in the 105,000-g supernatant fraction, and optimal enzymatic activity in vitro was observed at pH 6.5-7.5 in the presence of Ca2+ (8 mM) and mercaptoethanol (4 mM). Phosphatidylinositol-specific phospholipase C activity was stimulated by fatty acids in low concentrations, but was inhibited by lysophosphatidylcholine and a variety of detergents. No effect of labor on the specific activity of phosphatidylinositol-specific phospholipase C in either fetal membranes or uterine decidua could be detected. The finding of an active phosphatidylinositol-specific phospholipase C activity in human fetal membranes and uterine decidua is complementary to our previous finding of a selective loss of arachidonic acid from phosphatidylinositol of human fetal membranes during labor. The action of phosphatidylinositol-specific phospholipase C, coupled to diacylglycerol lipase action, could provide a mechanism for the release of arachidonic acid for prostaglandin biosynthesis during parturition.
Gian Carlo Di Renzo, John M. Johnston, Takeshi Okazaki, Janice R. Okita, Paul C. MacDonald, John E. Bleasdale
The daily transport of human plasma apolipoproteins A-I and A-II, triglyceride, and total cholesterol from the thoracic duct lymph into plasma was measured in two subjects before and three subjects after renal transplantation. Lymph triglyceride transport was ∼83% of the daily ingested fat loads, whereas lymph cholesterol transport was consistently greater than the amount of daily ingested cholesterol. Lymph apolipoprotein transport significantly (P < 0.05) exceeded the predicted apolipoprotein synthesis rate by an average of 659±578 mg/d for apolipoprotein A-I and 109±59 mg/d for apolipoprotein A-II among the five subjects. It is estimated that 22-77% (apolipoprotein A-I) and 28-82% (apolipoprotein A-II) of daily total body apolipoprotein synthesis takes place in the intestine.
David W. Anderson, Ernst J. Schaefer, Thomas J. Bronzert, Frank T. Lindgren, Trudy Forte, Thomas E. Starzl, Gary D. Niblack, Loren A. Zech, H. Bryan Brewer Jr.
Lymphocytes obtained from nonimmuno deficient children treated with commercially available preparations of gammaglobulin failed to proliferate and to mature into plasma cells in vitro after stimulation with pokeweed mitogen. The influence of the treatment on lymphocyte functions varied according to the cell population considered. A T helper cell activity was detected in these patients but only in the cell subset bearing receptors for IgG after irradiation. T lymphocytes exerted a suppressive effect that disappeared after irradiation or incubation at 37°C. The suppressive cells were found among E rosette-forming cells depleted of leukocytes bearing receptors for IgG. Their suppressive effect was expressed only in the presence of normal radioresistant T lymphocytes that did not bear Fc receptors for IgG. Similar dysfunctions could be induced in vitro by incubation of normal T and B lymphocytes with gammaglobulin preparations. Because F(ab)′2 fragments or deaggregated preparations of gammaglobulin failed to activate T suppressor lymphocytes, this activation was likely triggered by attachment of Fc portion of denatured IgG to the corresponding membrane receptor. This activation step was prostaglandin E2-dependent, suggesting that activated monocytes were involved in the activation process. B lymphocyte responses appeared directly inhibited by attachment of denatured gammaglobulin on membrane Fc receptor. Our observations suggest that immunological effects of gammaglobulin therapy are not limited to antibody transfer, since it also induces subtle modifications of in vitro pokeweed mitogen-stimulated T and B cell responses. These modifications must be considered in interpreting results obtained in immunodeficient patients investigated under gamma-globulin therapy.
Anne Durandy, Alain Fischer, Claude Griscelli
By restriction endonuclease mapping, gene cloning, and DNA sequencing we have determined the region of DNA that is deleted in a family with gamma delta beta-thalassemia. The deletion removes the linked epsilon, gamma-, and delta-globin structural genes and terminates within the coding portion of the beta-globin gene. Since the extent of DNA deletion in this family differs from that reported in another family, we conclude that gamma delta beta-thalassemia is heterogeneous at the molecular level.
S H Orkin, S C Goff, D G Nathan
The plasma disappearance rate (metabolic clearance rate) of administered intact parathyroid hormone (intact PTH) was analyzed in awake dogs with indwelling hepatic and renal vein catheters. The metabolic clearance rate (MCR) of intact PTH was found to be very rapid, 21.6 ± 3.1 ml/min per kg in 11 normal dogs. The liver accounted for the greatest fraction of the MCR of intact PTH (61 ± 4%) by virtue of an arterial minus venous (a - v) difference across the liver of 45 ± 3%. The renal uptake of intact PTH accounted for 31 ± 3% of the MCR of intact PTH. The renal a - v difference for intact PTH of 29 ± 2% was significantly greater than the filtration fraction indicating renal uptake of intact PTH at sites independent of glomerular filtration. Together, the hepatic and renal clearances of intact PTH accounted for all but a small fraction of the MCR of intact PTH. The MCR of intact PTH, rendered biologically inactive by oxidation, was markedly decreased to 8.8 ± 1 ml/min per kg. The a - v difference of oxidized intact PTH was reduced both in the liver and kidney. These data suggested that the high uptake rates of intact PTH are dependent, at least in part, upon sites recognizing only biologically active PTH.
K. A. Hruska, A. Korkor, K. Martin, E. Slatopolsky
The objective of this study was to determine the direct actions of nitroprusside and nitroglycerin on the pulmonary vascular bed in the intactchest dog. These widely used nitrogen oxide-containing vasodilator agents decreased pulmonary arterial pressure and increased cardiac output without altering left atrial pressure. Reductions in pulmonary arterial pressure and pulmonary vascular resistance were small under resting conditions, but were enhanced when pulmonary vascular tone was elevated by infusion of a stable prostaglandin analog that increases pulmonary vascular resistance by constricting intrapulmonary veins and upstream segments. In studies in which pulmonary blood flow to the left lower lobe was maintained constant, nitroprusside and nitroglycerin caused small but significant reductions in lobar arterial and small-vein pressures without significantly affecting left atrial pressure. With constant blood flow, lobar vascular pressures that were reduced in response to the vasodilators were more greatly reduced when lobar vascular resistance was increased by infusion of the prostaglandin analog or serotonin. However, when lobar vascular pressures were elevated by passive obstruction of lobar venous outflow, vasodilator responses to nitroprusside and nitroglycerin were not enhanced. These data suggest that nitroprusside and nitroglycerin decrease pulmonary vascular resistance by dilating intrapulmonary veins and upstream segments. These responses were minimal under control conditions but were enhanced when vascular tone was increased. This vasodilator action is independent of passive factors such as changes in pulmonary blood flow or left atrial pressure and is not secondary to an effect of these agents on the systemic circulation. Pulmonary vasodilator responses to nitroprusside and nitroglycerin were, however, found to be dependent on the existing level of vasomotor tone in the pulmonary vascular bed.
Philip J. Kadowitz, Premanand Nandiwada, Carl A. Gruetter, Louis J. Ignarro, Albert L. Hyman
Acetyl glyceryl ether phosphorylcholine (AGEPC) induced dose-dependent platelet aggregation and release of [3H]serotonin and platelet factor 4 in citrated human platelet-rich plasma. ADP scavengers or indomethacin prevented irreversible platelet aggregation responses induced by 0.2 microM AGEPC but had no effect upon platelet secretion; prostacyclin inhibited AGEPC-induced aggregation and secretion. EDTA or EGTA inhibited AGEPC-induced aggregation but had no effect on platelet secretion.
L M McManus, D J Hanahan, R N Pinckard
The requirement of Factor VIII-related antigen (VIIIR:Ag) for platelet damage by quinine-and quinidine-dependent antibodies was studied in platelet-rich plasma (PRP) of four patients with severe von Willebrand's disease (vWd) (Factor VIII deficiency). Platelet factor 3 availability, platelet aggregation, and release of [14C]serotonin from labeled vWd-PRP by drug-dependent antibodies were significantly reduced in comparison with PRP from normal controls. Addition of purified VIIIR:Ag restored levels of platelet damage to that of normal PRP. In vWd-PRP, platelet damage by two human antiplatelet sera, not dependent on drugs, and by a rabbit antiplatelet serum did not differ from that in normal PRP. PRP from patients deficient in Factor VIII coagulant activity, Factor IX, or Factors II, VII, IX, and X behaved like normal PRP.
Sharron L. Pfueller, Pari K. Hosseinzadeh, Barry G. Firkin
The hypophosphatemic male mouse, an animal model for human vitamin D-resistant rickets, is characterized by low serum phosphorus concentration due to increased urinary phosphate excretion, rickets, osteomalacia, and dwarfism. Because phosphate administration can heal rickets but not osteomalacia in the human disease, we have compared the effect of phosphate supplementation on the epiphyseal and endosteal bone mineralization in the mutant animal. Phosphate was given in drinking water for 137 d and the biochemical and bone responses were assessed by analytical and histomorphometric methods. Treatment with phosphate normalized the endochondral calcification (vertebral growthplate thickness: 83 +/- 5 SD vs. controls [+/Y] 73 +/- 8 micrometers, NS), but did not correct the endosteal bone mineralization (mineralization front: 13.6 +/- 2.7 vs. +/Y 67.1 +/- 6.9% osteoid surface, P less than 0.001, endosteal mean osteoid seam thickness: 46.4 +/- 6.1 vs. +/Y 3.3 +/- 0.3 micrometers, P less than 0.001). In addition, both osteoblastic and osteoclastic recruitment and activity were stimulated, as a result of a probable increase in parathyroid hormone secretion following the phosphate induced fall in serum calcium. Our results show that in the hypophosphatemic mouse, phosphate supplementation can heal the epiphyseal, but not the endosteal defective bone mineralization. Then, the biochemical and skeletal response to phosphate therapy appear to be similar to what we have observed in the human disease, further stressing the interest of the animal model.
P J Marie, R Travers, F H Glorieux