Familial amyloidotic polyneuropathy (FAP) is an autosomal dominant late-onset disorder characterized by the extracellular deposition of amyloid fibrils. In all cases studied these fibrils have been found to be composed of plasma prealbumin (transthyretin) containing a single amino acid substitution. Biochemical studies were conducted on amyloid from one patient and plasma prealbumin from his affected brother, both part of a large kindred from the Appalachian region of the United States. Sequence analysis of the amyloid subunit protein showed it to be prealbumin with about two-thirds of the molecules containing a substitution of alanine for threonine at position 60. Studies of the plasma prealbumin showed that the same substitution was present in 40-45% of the protein. Based on this substitution and the prealbumin cDNA sequence, a Pvu II restriction fragment length DNA polymorphism (RFLP) was predicted and demonstrated in DNA of both patients as well as other family members. This RFLP confirms the predicted DNA mutation responsible for the protein variant, and represents an accurate method for detection of this gene.
M R Wallace, F E Dwulet, P M Conneally, M D Benson
Human thrombomodulin significantly inhibited the rate of prothrombin conversion to thrombin by Factor Xa in the presence of phospholipid or platelets, calcium, and Factor Va. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of 125I-prothrombin activation revealed that thrombomodulin reduced the rate of prothrombin activation but did not alter the cleavage pattern. The inhibition was reversed by the inclusion of a highly specific rabbit antithrombomodulin antibody. If thrombomodulin was replaced by hirudin, the rate of thrombin generation was not decreased excluding the possibility that the inhibition by thrombomodulin was secondary to the binding of small amounts of thrombin formed early in the reaction and the prevention of feedback breakdown of prothrombin by thrombin. The inhibitory activity of thrombomodulin was overcome by increasing the concentration of Factor Xa and specific, saturable binding of thrombomodulin to Factor Xa was demonstrated. These results indicate that thrombomodulin binds to Factor Xa and thereby inhibits the activity of the prothrombinase complex.
E A Thompson, H H Salem
To determine if postexercise thermal events play a role in exercise-induced asthma (EIA), nine normal and eight asthmatic subjects on three occasions exercised while they inhaled frigid air. During the recovery period, either cold air, air at room temperature and humidity, or air at body conditions was administered in a random fashion. On a fourth occasion, body-condition air was given during exercise. Pulmonary mechanics were measured before and after each challenge. No changes in mechanics developed when air at body conditions was inhaled during exercise, however, increasing the heat content of the air during recovery produced progressively greater obstruction in both groups. On a separate occasion, seven asthmatics hyperventilated frigid air and either recovered spontaneously or had their ventilation slowly reduced. Controlling ventilation markedly attenuated the obstructive response. These data demonstrate that the severity of EIA is dependent not only on airway cooling but also upon the rapidity and magnitude of airway rewarming postchallenge.
E R McFadden Jr, K A Lenner, K P Strohl
Bone marrow from a normal male pig was transplanted into a related female pig with severe homozygous von Willebrand's disease (vWd). After engraftment the circulating leukocytes were of the male karyotype, and the platelets were strongly positive for von Willebrand factor (vWF) by indirect immunofluorescence. The average level of vWF was 1.96 U/dl and of ristocetin cofactor was 2.8 U/dl. The ear immersion bleeding time before transplantation was consistently more than 15 min and afterwards varied between 5 min and more than 15 min. Transfused vWF corrected the bleeding time at a level of 10 U/dl, which is lower than that required for a von Willebrand pig. We concluded that: the plasmatic compartment is only minimally replenished by the vWF from platelets and megakaryocytes; and the platelet vWF alone only partially corrects the abnormal tests of the hemostatic mechanism in severe vWd.
E J Bowie, L A Solberg Jr, D N Fass, C M Johnson, G J Knutson, M L Stewart, L J Zoecklein
To define the role of local synthesis of angiotensinogen in tissue angiotensin production, we have quantitated angiotensinogen messenger RNA (mRNA) levels in 17 different tissues of four groups of rats: control rats, nephrectomized rats, rats given dexamethasone, ethynylestradiol, and triiodothyronine, and nephrectomized rats given dexamethasone, ethynylestradiol, and triiodothyronine. Angiotensinogen mRNA was identified in 12 tissues: liver, kidney, brain, spinal cord, aorta, mesentery, atria, lung, adrenal, large intestine, stomach, and spleen. Angiotensinogen mRNA was not identified in pituitary, ventricle, testis, small intestine, or pancreas. When expressed per gram tissue wet weight, angiotensinogen mRNA levels of extrahepatic tissues were less than 4% of hepatic levels. However, when expressed per milligram total RNA, angiotensinogen mRNA levels of brain, spinal cord, aorta, and mesentery were 26-42% of hepatic levels. Regulation of angiotensinogen mRNA levels was tissue specific. This demonstration of a widespread tissue distribution of angiotensinogen mRNA may indicate a similarly widespread distribution of local angiotensin systems that are independent of the circulating renin-angiotensin system.
D J Campbell, J F Habener
Lymphocytes from many elderly individuals exhibit depressed proliferative responses to the plant phytohemagglutinin (PHA). We have previously reported that this proliferative defect is not due to a failure to generate a cytoplasmic activator of DNA replication (ADR). In the present study, we tested the DNA synthetic response of nuclei derived from aged cells to an exogenous source of ADR. We found that nuclei from aged lymphocytes exhibiting low PHA responses were impaired in their ability to synthesize DNA in responses to ADR, compared with nuclei from younger adult donors. In contrast, those aged cells maintaining intact PHA responses provided nuclei that were also unimpaired in their response to ADR. The relationship between PHA responsiveness of the intact cells and ADR responsiveness of nuclei derived from these was a linear one. These results suggest that the depressed cellular reactivity of aged lymphocytes to PHA (when seen) may be due to a failure of these nuclei to respond to cytoplasmic stimulatory signals induced by the mitogen.
J K Gutowski, J B Innes, M E Weksler, S Cohen
The transport of bile acid was studied in basolateral membrane vesicles isolated from rat small intestine. Taurocholate transport into an osmotically reactive intravesicular space was Na+ independent. The uptake of taurocholate in jejunal and ileal vesicles preloaded with sulfate was stimulated with respect to uptake in unpreloaded vesicles. Glycocholate inhibited the transstimulation of taurocholate uptake by sulfate. Sulfate and taurocholate uptake in ileal vesicles preloaded with bicarbonate was stimulated with respect to uptake in unpreloaded vesicles. Taurocholate inhibited the transstimulation of sulfate uptake by bicarbonate. When ileal vesicles were loaded with p-aminohippurate, an early transstimulation of taurocholate was found that exceeded equilibrium uptake, was insensitive to a K+ diffusion potential, and was cis-inhibited by taurocholate, glycocholate, pyruvate, p-aminohippurate, probenecid, chloride, sulfate, and bicarbonate. These data indicate the presence of an anion exchanger in intestinal basolateral membrane vesicles that may be involved in the exit of bile acids from the enterocyte.
S L Weinberg, G Burckhardt, F A Wilson
Human embryonic development involves transition from yolk sac (YS) to liver (L) hemopoiesis. We report the identification of pluripotent, erythroid, and granulo-macrophage progenitors in YS, L, and blood from human embryos. Furthermore, comprehensive studies are presented on the number of hemopoietic progenitors and precursors, as well as of other cell types, in YS, L, and blood at precisely sequential stages in embryos and early fetuses (i.e., at 4.5-8 wk and 9-10 wk postconception, respectively). Our results provide circumstantial support to a monoclonal hypothesis for human embryonic hemopoiesis, based on migration of stem and early progenitor cells from a generation site (YS) to a colonization site (L) via circulating blood. The YS----L transition is associated with development of the differentiation program in proliferating stem cells: their erythroid progeny shows, therefore, parallel switches of multiple parameters, e.g., morphology (megaloblasts----macrocytes) and globin expression (zeta----alpha, epsilon----gamma).
G Migliaccio, A R Migliaccio, S Petti, F Mavilio, G Russo, D Lazzaro, U Testa, M Marinucci, C Peschle
Alveolar macrophages from normal individuals and patients with interstitial lung diseases spontaneously expressed a 4.2-kilobase mRNA complementary to the c-sis gene, a proto-oncogene coding for one of the chains of platelet-derived growth factor (PDGF). Concomitantly, these cells released a mediator with the properties of PDGF, including: chemotactic factor for smooth muscle cells whose activity was resistant to heat and acid, but sensitive to reduction; mitogenic (competence) activity for fibroblasts; ability to compete with PDGF for its receptor; and precipitated by an anti-PDGF antibody. While blood monocytes did not contain c-sis mRNA transcripts, monocytes matured in vitro expressed c-sis, consistent with the concept that expression of c-sis occurs during the differentiation of monocytes into alveolar macrophages. Together with the known actions of PDGF, these observations suggest that the c-sis proto-oncogene and its PDGF product are part of the armamentarium available to the alveolar macrophages for normal lung defense and participation in lung inflammation.
J F Mornex, Y Martinet, K Yamauchi, P B Bitterman, G R Grotendorst, A Chytil-Weir, G R Martin, R G Crystal
Certain hormonal and nonhormonal binding systems such as the leukoagglutinin-lymphocyte model exhibit complex receptor-ligand interactions that result in nonlinear Scatchard plots. Such plots are interpreted as indicating either homogeneous negatively interacting binding sites or heterogeneous sites with different and fixed affinity. We assessed the validity of these interpretations in our system by conjugating the ligand to a photoactivated heterobifunctional agent and cross-linking the conjugate to a subset of receptors before studying the binding interactions of non-cross-linked sites. Conjugation did not qualitatively or quantitatively affect the binding properties of the ligand. Cross-linking was specific, efficient, and stable and had no effect on irrelevant surface receptors. Cross-linking of only 3% of the total receptors resulted in 50% decreased ligand binding to high affinity sites consistent with a calculated inactivation of 85% and 2% of high and low affinity sites, respectively. Such preferential inactivation of high affinity sites in an unequivocal demonstration of binding sites heterogeneity in this system and shows a clear rejection of the homogeneous cooperation model.
G B Faguet, D Beebe
Studies have been performed on the biochemical mechanism of platelet activation induced by the fibrinolytic protease plasmin. In washed human platelets, greater than or equal to 1.0 caseinolytic units (CU/ml plasmin induced aggregation. Platelet [14C]serotonin release was stimulated by 1.0 CU/ml plasmin to an extent comparable to that induced by 1.0 U/ml thrombin. A dose- and time-dependent phosphorylation of the platelet 47,000- and 20,000-kD proteins was noted in 32PO4-labeled platelets incubated with plasmin; phosphorylation was not affected by extracellular Ca2+, but was completely inhibited by an increase in platelet cyclic AMP. Phosphorylation of these platelet proteins suggested that plasmin may act on platelets by stimulating a rise in cytosolic calcium concentration ([Cai2+]) and activating inositol phospholipid-dependent phospholipase C and protein kinase C. Using both quin2 fluorescence and aequorin luminescence as indicators, plasmin was found to elevate platelet [Cai2+] in the presence or absence of extracellular Ca2+. Phospholipase C activation was shown by the generation of [3H]diglyceride in [3H]arachidonic acid-labeled platelets and [32P]phosphatidic acid in 32PO4 labeled platelets exposed to plasmin. Plasmin did not induce formation of thromboxane A2 (TXA2). Only small amounts of this eicosanoid were detected late in the time course after plasmin stimulation. Our results indicate that plasmin causes platelet aggregation and secretion associated with phosphorylation of the 47,000- and 20,000-kD proteins, Ca2+ mobilization, and phospholipase C and protein kinase C activation.
A I Schafer, A K Maas, J A Ware, P C Johnson, S E Rittenhouse, E W Salzman
Protein 4.1, a principal component of the erythrocyte membrane skeleton, is thought to be important in regulating membrane stability through its interaction with spectrin and actin. A key role for protein 4.1 has been indicated in studies in which deficiency of this protein was shown to result in marked instability of the membrane. In order to obtain direct evidence for the functional role of protein 4.1, we reconstituted protein 4.1-deficient membranes with purified protein 4.1 and showed restoration of membrane stability. Erythrocyte membranes totally and partially deficient in protein 4.1 were reconstituted by exchange hemolysis with various concentrations of purified protein 4.1, and their stability measured using an ektacytometer. Native erythrocyte membranes totally deficient in protein 4.1 were markedly unstable, while those partially deficient had intermediate reductions in membrane stability. Reconstitution with increasing concentrations of purified protein 4.1 resulted in progressive restoration of membrane stability. Near-normal membrane stability could be restored to both totally and partially protein 4.1-deficient membranes. In contrast, the addition of protein 4.1 to resealed membranes did not improve membrane stability. This implies that the added protein 4.1 must have access to the cell interior in order to affect membrane stability. Furthermore, in control experiments, the addition of protein 4.1 to normal membranes did not increase their stability. Also, the addition of purified spectrin and human serum albumin during resealing did not improve stability of protein 4.1-deficient membranes. These results provide direct evidence for the crucial role of protein 4.1 in regulating erythrocyte membrane stability.
Y Takakuwa, G Tchernia, M Rossi, M Benabadji, N Mohandas
The regional cerebral metabolic rate for glucose was measured in normal and portacaval shunted rats and the effects of unilateral carotid infusions of "threshold" amounts of ammonia were assessed. 8 wk after shunting the glucose metabolic rate was increased in all 20 brain regions sampled. Effects on subcortical and phylogenetically older regions of the brain were most pronounced with a 74% increase observed in the reticular formation at the collicular level. Increases in the cerebral cortex ranged from 12 to 18%. Unilateral infusions of ammonia did not affect behavior but altered the electroencephalogram and selectively increased the glucose metabolic rate in the thalamus, hypothalamus, and substantia nigra in half of the animals, a pattern similar to that seen after a portacaval shunt, suggesting hyperammonemia as the cause of postshunt increases in glucose metabolism. Visual inspection of autoradiograms, computed correlation coefficients relating interregional metabolism, and principal component analysis suggest that normal cerebral metabolic and functional interrelationships are altered by shunting. Ammonia stimulation of the hypothalamic satiety centers may suppress appetite and lead to cachexia. Reductions in the ammonia detoxification capacity of skeletal muscle may increase the probability of developing future episodes of hyperammonemia, perpetuating the process. Direct effects of ammonia on specific brain centers such as the dorsomedial hypothalamus and reticular activating system may combine with global disruptions of cerebral metabolic-functional relationships to produce the protean manifestations of portal-systemic encephalopathy.
A H Lockwood, M D Ginsberg, H M Rhoades, M T Gutierrez
Clinical and biochemical characteristics of familial hypercholesterolemia (FH) heterozygotes possessing an abnormally high molecular weight low density lipoprotein receptor (HMWR) are reported. The disorder is transmitted as an autosomal dominant trait and is not distinguishable from classic heterozygous FH on clinical grounds. The average plasma low density lipoprotein (LDL) level is 360 mg/dl and tendon xanthomata and early coronary disease are present. LDL receptor activity is higher than expected. In skin fibroblast cultures two types of functional LDL receptors are present, one with a normal apparent native molecular weight of 140,000, and the other of 176,000. When immobilized on nitrocellulose paper both receptors bind LDL. Maximum 125I-LDL binding capacity of fibroblast monolayers is reduced only 20%, compared with 50% in typical heterozygous FH. Affinity for 125I-LDL is increased and a 38% reduction in the Michaelis constant for LDL is observed. When autologous 125I-LDL was injected intravenously, the fractional catabolic rate of LDL was 205% and the LDL apoprotein B production rate was 328% of that found in a typical heterozygous FH subject. Thus, both in vitro and in vivo testing indicated only a modest deficiency of LDL receptor activity. Kindred members possessing the HMWR had an associated abnormality of cholesterol biosynthesis. Cholesterol balance studies in three individuals with the HMWR trait demonstrated elevated cholesterol biosynthesis of two to three times the mean of normal subjects. These findings suggest that increased LDL production and increased cholesterol production may assume a significant role in the pathologic manifestations of heterozygous FH. Functional abnormalities in LDL receptor activity as measured in fibroblast culture may be relatively small.
R A Levy, R E Ostlund Jr, C F Semenkovich, J L Witztum
Muscle capillary basement membrane width is a sensitive marker for the presence of diabetic microangiopathy. Studies have indicated that genetic factors and alterations in glucose metabolism influence muscle capillary basement membrane width. To define the role of these factors we have measured muscle capillary basement membrane thickness in controls, insulin dependent diabetics, and individuals with diabetes secondary to the ingestion of Vacor, a rat poison, which results in hyperglycemia. Hemoglobin A1 concentrations were increased in both diabetic groups, but hemoglobin A1 levels and the duration of diabetes were similar in the two diabetic groups. The muscle capillary basement membrane width was increased to a similar extent in the insulin-dependent diabetics (control, 1,781 +/- 46 vs. IDD, 2,287 +/- 144 A, P less than 0.001) and in the Vacor diabetic group (2,320 +/- 149 A, P less than 0.001). In the insulin-dependent diabetic group, 63% of the patients had a muscle capillary basement membrane width greater than two standard deviations above the mean of the controls, while in the Vacor diabetic group this figure was 56%. Despite the relatively short duration of diabetes (6.2 +/- 0.3 yr), 44% of the Vacor diabetic patients had retinopathy and 28% had proteinuria. The present study provides strong evidence that even in the absence of genetic diabetes mellitus, hyperglycemia or some other abnormality related to insulin lack can cause microvascular changes.
K R Feingold, T H Lee, M Y Chung, M D Siperstein
We reported a rat model of necrotizing enterocolitis by injecting platelet-activating factor (PAF) into the mesenteric vascular bed, and suggested that leukotrienes (LT) are secondary mediators. The present study, using isolated, buffer-perfused rat small intestine, shows: Isolated, perfused small intestine synthesizes LTs in response to PAF. Leukotriene C4 (LTC4) was the predominant LT released. The initial vasoconstriction after PAF injection was due to a transient release of LTC4 since FPL 55712 pretreatment abolished the vasoconstriction. The sustained rise in perfusion pressure was also blocked by FPL 55712, which suggests that other vasoconstrictors released are regulated by LTs. The vasoconstrictor(s) responsible for sustained rise in perfusion pressure is unknown, but is not thromboxane. Most of the LT was released from intestinal tissue rather than mesenteric arteries. Vasodilating prostaglandins (PGs) were also released, probably secondary to LTs. The complex interaction of these lipid mediators (PAF, LTs, and PGs) and their subtle balance may affect the course of the disease.
W Hsueh, F Gonzalez-Crussi, J L Arroyave
We evaluated the cellular immunity of 408 clinically stratified subjects at risk for acquired immune deficiency syndrome (AIDS), to define the role of interferon-alpha production deficits in the pathogenesis of opportunistic infections (OI). We followed 115 prospectively for up to 45 mo. Onset of OI was associated with, and predicted by, deficiency both of interferon-alpha generation in vitro, and of circulating Leu-3a+ cells. Interferon-alpha production is an index of the function of certain non-T, non-B, large granular lymphocytes (LGL) that are independent of T cell help. Leu-3a+ cell counts are a marker of T cell function. OI did not usually develop until both of these mutually independent immune functions were simultaneously critically depressed, leading to a synergistic interaction. These data suggest that the AIDS virus affects a subset of LGL, and that cytokine production by these cells is an important component of the host defense against intracellular pathogens that becomes crucial in the presence of severe T cell immunodeficiency.
F P Siegal, C Lopez, P A Fitzgerald, K Shah, P Baron, I Z Leiderman, D Imperato, S Landesman
We examined the effects of metabolic acidosis in vivo and reduced bath and luminal pH in vitro on total NH3 (NH3 + NH+4) production rates by isolated mouse proximal tubule segments. Midproximal tubule segments were obtained from mice with NH4Cl-induced metabolic acidosis and from nonacidotic controls. The segments were perfused with modified Krebs-Ringer bicarbonate (KRB) buffer, incubated in KRB buffer containing 0.5 mM L-glutamine and 1.0 mM sodium acetate, and gassed with 95% O2 and 5% CO2. Isolated unperfused and perfused proximal tubules from acidotic mice produced total NH3 at higher rates than corresponding tubules from nonacidotic mice. Perfusion of the tubular lumen stimulated total NH3 production by tubules from both acidotic and nonacidotic mice. In contrast, lowering the bath pH to 7.0 by lowering the HCO3- concentration increased total NH3 production rates by tubules from nonacidotic mice but not by tubules from acidotic mice. Reducing the HCO3- concentration of the bath buffer to 10 mM while maintaining a pH of 7.4 had no significant effect on total NH3 production by tubules from nonacidotic mice. Lowering the luminal fluid pH by reducing the perfusate HCO-3 from 25 mM to 10, 5, or 1.2 mM while maintaining a bath pH of 7.4 lowered collected luminal fluid pH but had no effect on total NH3 production by proximal tubules from nonacidotic mice. These observations demonstrated that metabolic acidosis in vivo stimulated total NH3 production in isolated mouse proximal tubule segments and that low peritubular pH and HCO-3 stimulated total NH3 production by proximal tubule segments from nonacidotic mice in vitro.
G T Nagami, C M Sonu, K Kurokawa
We have identified and purified a platelet integral membrane protein (140,000 mol wt), using the KC4 monoclonal antibody specific for activated platelets, that is internal in resting platelets but exposed on activated platelets (Hsu-Lin S.-C., C.L. Berman, B.C. Furie, D. August, and B. Furie, 1984, J. Biol. Chem. 259: 9121-9126.). The expression of the protein on the platelet surface is secretion-dependent. This protein has been named platelet activation-dependent granule-external membrane (PADGEM) protein. PADGEM protein is distinct from the surface glycoproteins of resting platelets, but identical to the S12 antigen, GMP-140. Using immunofluorescent staining, resting platelets failed to stain for PADGEM protein with the KC4 antibody, but after permeabilization showed a punctate staining of the cell interior. Thrombin-stimulated intact platelets stained with a peripheral rim pattern thus demonstrating the translocation of PADGEM protein from an internal location to the cell surface. PADGEM protein expression on the platelet surface at varying thrombin concentrations correlated with alpha granule release, as measured by the secretion of platelet factor 4. Further evidence for an alpha granule localization of PADGEM protein was provided by nitrogen cavitation of resting platelets followed by metrizamide density gradient centrifugation; PADGEM protein codistributed with platelet factor 4. Using immunoelectron microscopy, the protein was localized to the alpha granule in frozen ultrathin sections of resting platelets labeled using rabbit anti-PADGEM protein antibodies, whereas in thrombin-activated platelets, the plasma membrane was labeled. These studies indicate that PADGEM protein is a component of the alpha granule membrane of resting platelets and is incorporated into the plasma membrane upon activation and secretion.
C L Berman, E L Yeo, J D Wencel-Drake, B C Furie, M H Ginsberg, B Furie
The influence of endotoxin-induced elevated plasma levels of the fast-acting inhibitor of plasminogen activator (PA-inhibitor) on thrombolysis was investigated in rabbits with a jugular vein thrombus. Infusion of human tissue-type plasminogen activator (t-PA) produced similar degrees of thrombolysis in control and endotoxin-treated rabbits, although no free t-PA could be demonstrated in plasma of endotoxin-treated animals. Infusion of t-PA in an extracorporeal arteriovenous shunt resulted in loss of thrombolytic activity in endotoxin-treated animals but not in control animals. Blood clots superfused in vitro with mixtures of t-PA and normal plasma lysed in contrast to clots superfused with t-PA and PA-inhibitor-rich plasma. However, addition of rabbit lung slices to the plasma surrounding the blood clot, reversed the inhibition of thrombolysis by PA-inhibitor-rich plasma. This indicates that tissue-derived factor(s) are involved in the regulation of in vivo thrombolysis. These hypothetical factor(s) are, however, very unstable in plasma, which has thus far precluded their further characterization.
M Colucci, J A Paramo, J M Stassen, D Collen
A squamous cell carcinoma of 33-yr-old patient who developed marked leukocytosis and hypercalcemia was transplanted into nude mice in which more marked leukocytosis and hypercalcemia also developed. This tumor (LJC-1-JCK) produced a colony-stimulating factor (CSF) and formed a cyst in the tumor from which a CSF-producing cell line (T3M-1) was established. The CSF causes predominantly formation of granulocytic colonies in addition to macrophage colonies. Bone-resorbing activity (BRA) was detected in the cystic fluid and was eluted as two separate peaks with proteins of an apparent molecular weight of 30,000-50,000 and 10,000-20,000. Colony-stimulating activity (CSA) was eluted at an apparent 30,000 mol wt. The conditioned medium of the T3M-1 cells also contained a BRA with an apparent 14,000 mol wt, whereas CSA eluted at an apparent 30,000 mol wt. PTH, epidermal growth factor, transforming growth factor-alpha, prostaglandin Es, and vitamin D could not account for the powerful BRA. In contrast to CSA, BRA was not inactivated by trypsin and more stable at 70 degrees C. When T3M-1 cells were transplanted into nude mice, marked hypercalcemia developed in addition to granulocytosis. Our findings suggest that the tumor produces and secretes a powerful BRA in vivo and in vitro, which is different from CSA in terms of molecular weight, heat stability, and trypsin treatment. We speculate that the synergistic action of CSF that stimulates macrophage colony formation and recruits osteoclast precursors, and BRA, which stimulates mononuclear phagocytes and/or osteoclasts were responsible for a marked increase in osteoclastic bone resorption and humoral hypercalcemia in the patient.
K Sato, H Mimura, D C Han, T Kakiuchi, Y Ueyama, H Ohkawa, T Okabe, Y Kondo, N Ohsawa, T Tsushima
Leukocyte extracts contain enzymes that digest fibrinogen and release a fibrinopeptide A-containing fragment. This study was undertaken to identify the responsible proteinase and to characterize the fibrinopeptide A-containing fragment so that it could be used as an index of enzyme activity. Both the fibrinogenolytic activity and the release of the fibrinopeptide A-containing fragment mediated by the leukocyte extracts were shown to be due to human neutrophil elastase (HNE) by the following criteria: activity was completely blocked by a specific HNE inhibitor or by adsorbing HNE from the extracts with a monospecific antibody and reconstitution with purified HNE restored the ability to release the fibrinopeptide A-containing fragment. This fragment was not released by a variety of other proteinases or by HNE-inhibitor complexes indicating that, at least with respect to the enzymes tested, it is a specific product of HNE and its release requires the free enzyme. By separating the products of HNE digestion of fibrinogen using high performance liquid chromatography, identifying the immunoreactive fractions and subjecting them to amino acid analysis, the fragment was identified as A alpha 1-21, indicating an HNE cleavage site at the Val(A alpha 21)-Glu(A alpha 22) bond. The mean plasma A alpha 1-21 level was markedly higher in patients with alpha 1-proteinase inhibitor deficiency as compared to healthy controls (0.2 nM vs. 7.9 nM; P less than 0.0001), consistent with increased in vivo HNE activity in these individuals.
J I Weitz, S L Landman, K A Crowley, S Birken, F J Morgan
Functionally active A and B chains were separated from a two-chain form of recombinant tissue-type plasminogen activator after mild reduction and alkylation. The A chain was found to be responsible for the binding to lysine-Sepharose or fibrin and the B chain contained the catalytic activity of tissue-type plasminogen activator. An extensive reduction of two-chain tissue-type plasminogen activator, however, destroyed both the binding and catalytic activities. A thermolytic fragment, Fr. 1, of tissue-type plasminogen activator that contained a growth factor and two kringle segments retained its lysine binding activity. Additional thermolytic cleavages in the kringle-2 segment of Fr. 1 caused a total loss of the binding activity. These results indicated that the binding site of tissue-type plasminogen activator to fibrin was located in the kringle-2 segment.
A Ichinose, K Takio, K Fujikawa
Studies of plasma prekallikrein in a family with prekallikrein deficiency were made. Three children had no clotting activity but approximately 35% antigen levels, and the mother and five children had twice as much prekallikrein antigen as clotting activity, suggesting the presence of a dysfunctional molecule. A nonfunctional variant form of prekallikrein was purified that contained no prekallikrein clotting activity. The variant and normal molecules were both 80,000 mol wt, immunologically indistinguishable and complexed similarly with high molecular weight kininogen. Isoelectric focusing studies suggested a difference of one charged amino acid residue. The variant was cleaved by beta-Factor XIIa 200 times slower than the normal molecule, and no amidolytic activity was detected for the cleaved variant. These data and other observations suggest that an amino acid was substituted in the variant near the NH2-terminal end of the kallikrein light chain resulting in slower cleavage by beta-Factor XIIa and the absence of enzymatic activity.
B N Bouma, D M Kerbiriou, J Baker, J H Griffin
Titrimetric addition of hypochlorous acid (HOCl) or chloramine (NH2Cl) to suspensions of Escherichia coli decreases their ability to accumulate 14C-labeled glutamine, proline, thiomethylgalactoside, and leucine in a manner that approximately coincides with loss of cell viability; quantitative differences in cellular response are observed with the two oxidants. Inhibition of beta-galactosidase activity in E. coli ML-35, a strain lacking functional lactose permease, is complex and also depends upon the identity of the oxidant. Membrane proton conductivities and glycerol permeabilities are unchanged by addition of HOCl or NH2Cl in excess of that required for inactivation. The combined results are interpreted to indicate that the locus of HOCl attack is the cell envelope, that HOCl inactivation does not occur by loss of membrane structural integrity, that loss of transport function can be identified with either selective oxidative inhibition of the transport proteins or loss of cellular metabolic energy, and that different mechanisms of inactivation may exist for HOCl and NH2Cl.
J M Albrich, J H Gilbaugh 3rd, K B Callahan, J K Hurst
Adipocytes contain adenosine receptors, termed A1 receptors, which inhibit lipolysis by decreasing adenylate cyclase activity. The inhibition of lipolysis by adenosine agonists in vivo acutely suppresses the plasma concentrations of free fatty acids (FFA) and triglycerides. We have found that infusions of the adenosine receptor agonist phenylisopropyladenosine (PIA) initially decreases plasma FFA concentrations; however, with prolonged exposure (6 d), rats become very tolerant to the effects of the drug. Adipocytes isolated from epididymal fat pads from PIA-infused rats have altered lipolytic responses. When lipolysis is stimulated with a relatively high concentration of isoproterenol (10(-7) M), PIA does not inhibit lipolysis in adipocytes from the infused animals. However, PIA inhibits isoproterenol-stimulated cyclic AMP (cAMP) accumulation in adipocytes from the infused rats although with decreased sensitivity compared with controls. The explanation for the impaired antilipolytic effect appears to be due to the fact that isoproterenol-stimulated cAMP accumulation is markedly increased in cells from infused rats. Indeed, basal lipolysis and lipolysis stimulated with lower concentrations of isoproterenol (10(-9), 10(-8) M) are effectively inhibited by PIA. cAMP accumulation is greatly increased in adipocytes from infused rats when stimulated by isoproterenol, ACTH, and forskolin. The results have some striking analogies to changes induced in nerve cells by prolonged exposure to narcotics. These data suggest that tolerance to PIA develops in adipocytes as a consequence of enhanced cAMP accumulation.
B B Hoffman, H Chang, E Dall'Aglio, G M Reaven
To determine the effects of maternal hyperglycemia on fetal hemodynamic and cardiac function, a study was carried out on nine chronically catheterized fetal sheep. In six fetuses, glucose was infused intravenously with an initial dose of 5 mg/kg per min. Data were compared with controls. This dose was gradually increased to 16 mg/kg per min by the fifth day. The initial blood glucose was 14.7 +/- 3.0 mg/dl and increased to 54.6 +/- 16.4 mg/dl by the last day of the infusion period (P less than 0.001). The PO2 decreased from a baseline of 20.25 +/- 3.40 to 15.88 +/- 5.24 mmHg (P less than 0.01). Similarly significant decreases were also observed for the blood O2 content and O2 hemoglobin saturation: 8.5 +/- 1.7 to 6.4 +/- 2.2 ml/dl and 62.3 +/- 13.6 to 46.1 +/- 17.6%, respectively, during hyperglycemia (P less than 0.01). The duration of the preejection period (PEP) before the start of the experiment was 45 +/- 4 ms; a final value of 57 +/- 10 ms was obtained (P less than 0.01). However, the electromechanical delay and ejection time (ET) showed no significant variation. The ratio of the PEP/ET increased from 0.31 +/- 0.04 to 0.38 +/- 0.07 (P less than 0.01) during hyperglycemia. The reticulocytes increased from 1.4 +/- 1.8 to 3.1 +/- 2.9% (P less than 0.05) and the 2,3-diphosphoglycerate decreased from 4.4 +/- 1.1 to 2.8 +/- 1.2 mumol/g hemoglobin (P less than 0.005). This study demonstrated that fetal hyperglycemia depresses myocardial function in the fetal lamb. The changes in cardiac function could not be explained by the small drop in O2 saturation.
H Bard, J C Fouron, X De Muylder, G Ducharme, J S Lafond
To explore whether bacteremia potentiates gentamicin nephrotoxicity, we injected rats with either 1 X 10(9) Escherichia coli (E. coli), Pseudomonas aeruginosa, or Staphylococcus aureus, and then gave them gentamicin, 100 mg/kg. Renal injury was assessed over the next 24-48 h. Staphylococcus/gentamicin or gentamicin alone induced no renal injury. However, E. coli/gentamicin and Pseudomonas/gentamicin caused acute renal failure (severe azotemia; tubular necrosis; cast formation). This effect was not due to acute reductions in arterial blood pressure or renal blood flow, it could be reproduced by substituting nonviable for viable gram-negative organisms, and it was associated with increased renal gentamicin uptake. E. coli without gentamicin induced only mild azotemia and no tubular necrosis. Endotoxin-tolerant rats were significantly protected against the E. coli/gentamicin nephrotoxic interaction. We conclude that gram-negative bacteremia and gentamicin exert synergistic nephrotoxicities; and that this effect is mediated, at least in part, by endotoxin and in part by increased renal gentamicin uptake.
R A Zager, R B Prior
The multiple acyl-coenzyme A (CoA) dehydrogenation disorders (MAD) include severe (S) and mild (M) variants, glutaric aciduria type II (MAD:S) and ethylmalonic-adipic aciduria (MAD:M). Intact MAD:M mitochondria oxidized [1-14C]octanoate, [1-14C]palmityl-CoA, and [1,5-14C]glutarate at 20-46% of control levels; MAD:S mitochondria oxidized these three substrates at 0.4-18% of control levels. In MAD:M mitochondria, acyl-CoA dehydrogenase (ADH) activities were similar to control, whereas MAD:S ADH activities ranged from 38% to 73% of control. Electron transfer flavoprotein (ETF) activities in five MAD:M cell lines ranged from 29 to 51% of control (P less than 0.01); ETF deficiency was the primary enzymatic defect in two MAD:M lines. In four MAD:S patients, ETF activities ranged from 3% to 6% of control (P less than 0.001); flavin adenine dinucleotide addition increased residual ETF activity from 4% to 21% of control in a single MAD:S line (P less than 0.01). Three MAD:S patients had ETF activities ranging from 33 to 53% of control; other investigators found deficient ETF-dehydrogenase activity in these MAD:S and three of our MAD:M cell lines.
B A Amendt, W J Rhead
To define molecular signals elaborated by inducer populations supporting growth or differentiation of T8+ cells, we collected supernatants of pokeweed mitogen (PWM)-stimulated cultures depleted of T8+ cells. When added to purified T8+ cells, these supernatants caused significant proliferation. PWM plus interleukin 2 (IL-2) in amounts equivalent to those in the supernatant could not reconstitute the response caused by the supernatant. T8+ cells activated by supernatants obtained from PWM-pulsed T4+ cells suppressed fresh PWM cultures. Although exhibiting little proliferation, T8+ cells cultured for 6 d in PWM plus IL-2 still suppressed a fresh PWM response. The supernatants therefore contain an additional T suppressor cell growth factor (TsGF). Elaboration of TsGF required radiosensitive T4+Leu8+ cells. Molecular weight determination by high performance liquid chromatography gave a single peak of TsGF activity at approximately 8,000. Finally, whereas TsGF in the absence of IL-2 could not support the proliferation of T suppressor cells, it did cause T8+ cells to become strongly IL-2 receptor-positive.
E J Fox, R G Cook, D E Lewis, R R Rich
Abnormalities of intestinal calcium absorption and the vitamin D axis in the spontaneously hypertensive rat (SHR) are controversial. The present report documents a reduction in circulating 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in the 12-14-wk-old male SHR with evidence of its functional significance. Both plasma 1,25(OH)2D3 and mucosa-to-serosa duodenal calcium flux (Jm-s), measured by the Ussing chamber, were significantly lower (approximately 60% of value in Wistar-Kyoto rats [WKY]) in SHR on both normal (1%) and low (0.1%) calcium diets than in corresponding control WKY. Low dietary calcium increased both 1,25(OH)2D3 and Jm-s by approximately 80% in SHR and WKY, with levels of both parameters rising in the SHR to levels found in the WKY under baseline conditions. The latter fact suggests the improbability of intestinal resistance to the action of 1,25(OH)2D3 in the SHR. Plasma 25-hydroxyvitamin D3 (25(OH)D3) was not significantly different between the strains. Intraperitoneal 1,25(OH)2D3 increased Jm-s in 12-14-wk-old SHR to levels observed in equivalent WKY. In 20-24-wk-old SHR, calcium deprivation was associated with significantly reduced Jm-s compared with equivalent WKY. Bone density and bone calcium content in 20-30-wk-old SHR were significantly reduced. In summary, we provide evidence that the SHR was unable to sustain appropriate circulating levels of 1,25(OH)2D3, an impairment which resulted in reduced duodenal calcium absorption.
P A Lucas, R C Brown, T Drüeke, B Lacour, J A Metz, D A McCarron
In a chronic canine model of pulmonary emphysema, we studied the interaction between left ventricular (LV) mechanics and pulmonary disease during severe hypoxemia. The hypoxemia was similar to that which may occur during a severe exacerbation of chronic obstructive lung disease. In six dogs with papain-induced emphysema and in seven dogs without emphysema, LV mechanics were examined when a hypoxic gas mixture was inspired to reduce PO2 to about 35 mmHg (hypoxic study) and during nonhypoxic conditions (room air study). In both groups, LV diastolic compliance was reduced during the hypoxic study by a similar amount. This finding could not be explained in terms of ventricular interdependence. Our analysis suggested that hypoxia decreased diastolic compliance (i.e., increased LV diastolic stiffness) by impairing LV relaxation. The primary effect of hypoxia was to decrease the extent to which LV relaxation occurred for a given end-diastolic pressure, while the rate of LV relaxation was decreased just slightly. This study indicates that severe hypoxemia because of respiratory failure may impair myocardial relaxation leading to a decrease in LV filling.
A Gomez, S Mink
The carboxy-terminal cross-linking domain (NCl) of type IV procollagen was isolated from human placenta and used for the production of polyclonal and monoclonal antibodies. Purity of the antigen and specificity of the antibodies were verified by Western blotting and radioimmunoassays. A radioimmunoassay was developed using rabbit antiserum. Intra- and interassay coefficients of variation were 4.7% and 5.8%, respectively; recovery of NCl added to serum and bile was 95-105%. NCl concentration in sera of healthy volunteers was 6 +/- 2.9 ng/ml (mean +/- 2.5 SD) and was elevated up to 18 ng in sera of patients with autoimmune or metastatic tumor disease and up to 240 ng in sera of patients with fibrogenic liver disease. Substantial amounts of antigen were also found in bile, urine, and ascites. 67% of serum antigens eluted from an agarose A5M column with an apparent molecular weight of 60 kD and 23% with a molecular weight of 90 and 150 kD, well below the molecular weight of type IV procollagen (550 kD). Serum NCl is apparently derived from the degradation of basement membrane collagen. The time course of NCl concentrations in sera of patients with fibrogenic liver disease showed no correlation with the serum concentration of the amino-terminal procollagen type III peptide, a marker of hepatic collagen biosynthesis. A decline of serum NCl levels along with elevated serum procollagen type III peptides apparently indicates bad prognosis in fibrogenic liver disease. The radioimmunoassay for NCl is a useful tool for studying type IV collagen metabolism in conditions causing remodeling or breakdown of basement membranes.
D Schuppan, M Besser, R Schwarting, E G Hahn
We have developed a method to isolate insulin-responsive human hepatocytes from an intraoperative liver biopsy to study insulin action and resistance in man. Hepatocytes from obese patients with noninsulin-dependent diabetes were resistant to maximal insulin concentration, and those from obese controls to submaximal insulin concentration in comparison to nonobese controls. Insulin binding per cell number was similar in all groups. However, insulin binding per surface area was decreased in the two obese groups because their hepatocytes were larger. In addition, the pool of detergent-extractable receptor was further decreased in diabetics. Insulin receptors in all groups were unaltered as determined by affinity-labeling methods. However, insulin-stimulated insulin receptor kinase activity was decreased in diabetics. Thus, in obesity, decreased surface binding could explain resistance to submaximal insulin concentrations. In diabetes, diminished insulin-stimulated protein kinase activity and decreased intracellular pool of receptors could provide an explanation for postinsulin-binding defect(s) of insulin action in human liver.
J F Caro, O Ittoop, W J Pories, D Meelheim, E G Flickinger, F Thomas, M Jenquin, J F Silverman, P G Khazanie, M K Sinha
A canine sepsis model that simulates the human cardiovascular response to septic shock was produced in 10 conscious unsedated dogs by implanting an Escherichia coli-infected clot into the peritoneum, resulting in bacteremia. By employing serial, simultaneous measurements of radionuclide scan-determined left ventricular (LV) ejection fraction (EF) and thermodilution cardiac index (CI), the end-diastolic volume index (EDVI) was calculated (EDVI = stroke volume index divided by EF). By using three different methods of quantifying serial ventricular performance (EF, shifts in the Starling ventricular function curve using EDVI vs. stroke work index, and the ventricular function curve response to volume infusion), this study provides evidence (P less than 0.01) that septic shock produces a profound, but reversible, decrease in systolic ventricular performance. This decreased performance was not seen in controls and was associated with ventricular dilatation (P less than 0.01); the latter response was dependent on an adequate volume infusion. Further studies of EDVI and pulmonary capillary wedge pressure during diastole revealed a significant, though reversible, shift (P less than 0.001) in the diastolic volume/pressure (or compliance) relationship during septic shock.
C Natanson, M P Fink, H K Ballantyne, T J MacVittie, J J Conklin, J E Parrillo
Lysophosphatidylcholine (LPC) accumulates in ischemic tissue, and exogenous LPC (20-100 microM) induces electrophysiologic alterations in vitro. However, to determine whether compartmentalization is critical, intracellular pressure microinjection of LPC was performed with simultaneous recording of the transmembrane action potential. Intracellular LPC in concentrations as high as 500 microM (n = 18), calculated based on calibration of injectate volume and cellular volume, did not induce electrophysiologic alterations. The concentrations and efflux of phospholipids and lysophospholipids were assessed in lymph obtained from the supracardiac lymph vessel in anesthetized dogs to assess the extent of extracellular accumulation. Prior to ischemia, phosphatidylcholine (PC) was the major phospholipid in lymph (79 +/- 2%) with substantial quantities of sphingomyelin (11 +/- 2%) and LPC (6 +/- 1%). With ischemia, the concentration of LPC increased by 18%, and net efflux of LPC increased by 24% (P less than 0.01) with no net efflux of PC or other assayed phospholipids. The calculated concentration of LPC increased from 84 to 197 microM in lymph within the ischemic region, a concentration sufficient to induce electrophysiologic derangements.
H Akita, M H Creer, K A Yamada, B E Sobel, P B Corr
Substance P (SP), neurotensin (NT), bombesin (BB), serotonin (5HT), and carbamylcholine (CCH) transiently increase electrogenic anion secretion in chinchilla and chicken ileum. SP and CCH also transiently inhibit amiloride-sensitive Na/H exchange in isolated chicken enterocytes. Loperamide (LP) inhibits the short-circuit current responses caused by SP, NT, and BB, but not those caused by CCH, 5HT, Ca ionophore, or cyclic nucleotides. Similarly, LP inhibits the effects of SP, but not those of CCH, on Na/H exchange. LP inhibition of the SP effects was further studied in isolated chicken enterocytes. CCH and SP transiently increased cytosolic Ca activity by 20-50 nmol/liter, but only the response to SP was inhibited by LP (10(-5) M) and by the absence of extracellular Ca. We conclude SP and CCH effects on intestinal electrolyte transport are mediated by increasing enterocyte Ca activity and LP specifically inhibits peptide hormone-activated Ca entry by an opiate receptor-independent mechanism.
E B Chang, D R Brown, N S Wang, M Field
Synthetic human corticotropin-releasing factor (hCRF) stimulated ACTH secretion by human fetal pituitaries in superfusion and dispersed human fetal pituitary cells cultured on an extracellular matrix in static incubation from 14 to 23 wk gestational age. The action of hCRF in vitro was potentiated by arginine vasopressin (AVP) at all ages studied. 8-Br-cAMP induced a response similar to hCRF. The AVP effect on ACTH was synergistic with both CRF and 8-Br-cAMP. hCRF-mediated secretion of ACTH was noncompetitively inhibited by 24-h pretreatment, or by 3-h concomitant treatment, with dexamethasone. Neither oxytocin, catecholamines, prostaglandins, nor indomethacin exerted significant effects on ACTH secretion, either alone or in combination with hCRF or AVP during the gestational ages studied. These results support a physiologic role for CRF in the regulation of secretion by corticotropic cells as early as 14 wk gestation, by which time corticotropes and ability to secrete ACTH have been demonstrated.
Z Blumenfeld, R B Jaffe
Laser angioplasty, the in situ ablation of arterial obstructions with laser radiation, has been demonstrated in animal models and early clinical trials. A problem with this technique, however, is the possibility of thermal damage to adjacent or underlying normal tissues that also absorb the radiation. Using a spectrophotometer with an integrating sphere and a specially constructed tunable-dye laser-based spectrophotometer, we evaluated the transmittance and remittance of human cadaveric atheromas and adjacent normal aorta from 250 to 1,300 nm to identify wavebands where there is preferential light absorption by atheromas. Data were analyzed by both the Kubelka-Munk formalism and a Beer's law model. Both methods indicate that atheromas absorb more than normal aorta between 420 and 530 nm. At 470 nm the average Kubelka-Munk absorption coefficient of atheromas from 10 cadavers was 54 +/- 9 cm-1 compared with 26 +/- 6 cm-1 for normal aortic specimens from seven cadavers. Yellow chromophores responsible for the atheroma absorbance were extractable with xylenes. Thin-layer chromatography and absorption spectra identified the extracted chromophores as predominantly consisting of a mix of carotenoids, which are known constituents of atheromatous lesions. Preferential absorption of blue light by carotenoids in atheromas may permit selective ablation of atheromatous obstructions with appropriate pulses of laser radiation.
M R Prince, T F Deutsch, M M Mathews-Roth, R Margolis, J A Parrish, A R Oseroff
Retinoids were studied both to identify what skeletal components are important in the modulation of normal and leukemic human myeloid clonal proliferation and differentiation in vitro and to elucidate the mechanism by which retinoids modulate proliferation of hematopoietic cells. Retinoids with a derivatized terminal carboxyl group were significantly less active than all-trans-retinoic acid, and those with the addition of two methyl groups to the cyclohexenyl ring of retinoic acid or substitution of its beta-cyclogeranylidene group with a 1,1,3,3-5-indanyl ring system were markedly more active than all-trans-retinoic acid. Five of the retinoids strongly inhibited clonal growth of the HL-60 and KG-1 human leukemic cell lines (50% inhibition in the range of 3 X 10(-10)-1 X 10(-8) M) and markedly stimulated normal human myeloid colony formation (granulocyte-macrophage colony-forming cells [GM-CFC] 150% stimulation in the range of 3 X 10(-9)-3 X 10(-8) M). Further studies suggested that: Common structural requirements of the retinoids were important in the modulation of both normal and leukemic hematopoiesis. The retinoids were able to inhibit leukemic proliferation without induction of differentiation of the neoplastic cells. Studies on normal human GM-CFC suggested that the retinoids did not act by themselves as a colony-stimulating factor (CSF), or by stimulating accessory cells to produce CSF, but either required earlier progenitor cells to become GM-CFC or enhanced the sensitivity of GM-CFC to the action of CSF.
A Tobler, M I Dawson, H P Koeffler
Studies were performed to determine if the unstimulated platelet membrane has a site for high molecular weight kininogen (HMWK) binding. 125I-HMWK bound to unstimulated platelets. Zn++ was required for 125I-HMWK binding to unstimulated platelets and binding was maximal at 50 microM Zn++. Neither Mg++ nor Ca++ substituted for Zn++ in supporting 125I-HMWK binding to unstimulated platelets, and neither ion potentiated binding in the presence of 50 microM zinc. 125I-HMWK competed with equal affinity with HMWK for binding, and excess HMWK inhibited 125I-HMWK-platelet binding. Only HMWK, not prekallikrein, Factor XII, Factor XI, Factor V, fibrinogen, or fibronectin inhibited 125I-HMWK-platelet binding. 125I-HMWK binding to unstimulated platelets was 89% reversible within 10 min with a 50-fold molar excess of HMWK. Unstimulated platelets contained a single set of saturable, high affinity binding sites for 125I-HMWK with an apparent dissociation constant of 0.99 nM +/- 0.35 and 3,313 molecules/platelet +/- 843. These studies indicate that the unstimulated external platelet membrane has a binding site for HMWK that could serve as a surface to modulate contact phase activation.
E J Gustafson, D Schutsky, L C Knight, A H Schmaier
The Rice-500 Leydig cell tumor (LCT) in Fischer rats is a model of humoral hypercalcemia of malignancy (HHM). In this model, the elevation of plasma calcium (Ca) does not result merely from an increased bone resorption, but also from an enhanced tubular Ca reabsorption (TRCa). We investigated the hypocalcemic response to WR-2721 [S-2,2-(3-aminopropylamino)-, ethylphosphorothioic acid] in LCT-bearing Fischer rats. WR-2721 is a potent inhibitor of normal and aberrant parathyroid hormone (PTH) secretion. Moreover, it exerts a PTH-independent inhibitory effect on TRCa. In hypercalcemic LCT-bearing rats WR-2721 induced a fall in plasma Ca from 3.24 +/- 0.12 to 2.66 +/- 0.23 mmol/liter within 2 h after one single injection of 0.7 mmol/kg body wt. The decrement in plasma Ca was associated with a marked increase in urinary Ca excretion, indicating an inhibition of TRCa. The elevated urine cyclic AMP of LCT-bearing rats, however, was not altered by WR-2721 treatment. These results suggest that in this HHM model, WR-2721 can normalize calcemia through its PTH-independent inhibitory effect on TRCa. WR-2721 could therefore be an effective drug to treat human hypercalcemia of malignancy, particularly in those tumors wherein a markedly enhanced renal Ca reabsorption contributes to the elevation of the plasma Ca level.
S Hirschel-Scholz, J Caverzasio, R Rizzoli, J P Bonjour
In rats intravenous aspirin was only slightly more effective an inhibitor of platelet thromboxane B2 (TxB2) than of aorta 6-keto-prostaglandin (PGF)1 alpha generation (1.9 versus 2.1 mg/kg). In contrast, oral aspirin was about five times more effective on platelet than on aorta cyclooxygenase activity. The "biochemical selectivity" of aspirin as an inhibitor of platelet and vascular cyclooxygenase thus was not apparent after intravenous administration of the drug. However, this could be achieved by relatively low doses of oral (or intraduodenal) aspirin, on account of "presystemic" acetylation of platelet cyclooxygenase. Even in this condition, though, aspirin selectivity was relative to "systemic" peripheral vessels but not to the vessels of the enterohepatic circulation. Indeed after an oral or intraduodenal dose of 5 mg/kg aspirin, generation of portal vein 6-keto-PGF1 alpha was inhibited to much the same extent as platelet TxB2, while inferior vena cava 6-keto-PGF1 alpha formation was spared.
C Cerletti, M C Gambino, S Garattini, G de Gaetano