We tested the hypothesis that prostacyclin (PGI2), 6-keto-prostaglandinF1 alpha(6-keto-PGF1 alpha), and several E series prostaglandins (PG) may affect the activity of cholesteryl ester (CE) hydrolase since our previous experiments indicated that smooth muscle cells (SMC) in neointima of injured rabbit aorta (a) acquire the capacity to produce PGI2 and (b) have increased lysosomal CE hydrolytic (acid cholesteryl ester hydrolase [ACEH])activity. Using cultured SMC from rabbit thoracic aorta, we demonstrated that PGI2, 6-keto-PGF1 alpha, and 6-keto-PGE1 enhanced ACEH activity fourfold. No significant effects on ACEH activity were observed with PGE1 or PGE2. Preincubation of SMC with an inhibitor of adenylate cyclase activity (dideoxyadenosine) abolished the effect of these PG on CE hydrolytic activity. Addition of dibutyryl cAMP to these SMC significantly increased ACEH activity. Although concentrations of PGI2 used significantly increased cAMP levels, proliferation of these SMC was not observed. In related experiments, we determined if the addition of PGI2, 6-keto-PGF1 alpha, or 6-keto-PGE1 to cultured aortic SMC would enhance the egress of unesterified cholesterol and CE from these SMC. A significant loss of total cholesterol from PG-treated SMC was observed at the end of 14 d. Results suggest that increased synthesis of PGI2 by neointimal SMC in the injured aortic wall may, at least in part, explain the changes in CE catabolism and accumulation following injury. These PG may also be important in CE metabolism and accumulation in human arteries.
D P Hajjar, B B Weksler, D J Falcone, J M Hefton, K Tack-Goldman, C R Minick
Nuclear DNA from individuals belonging to nine different families in which two sibs were affected with isolated growth hormone deficiency type I were studied by restriction endonuclease analysis. By using 32P-labeled human growth hormone or the homologous human chorionic somatomammotropin complementary DNA (cDNA) sequences as a probe, the growth hormone genes of affected individuals from all families yielded normal restriction patterns. Polymorphic restriction endonuclease sites (HincII and MspI), which are closely linked to the structural gene for growth hormone on chromosome 17, were used as markers in linkage analysis of DNA of family members. Of the nine affected sib pairs two were concordant, three were possibly concordant, and four were discordant for both linked markers. Since only concordant sib pairs would have inherited the same growth hormone alleles, further studies to identify mutations of the growth hormone genes should be limited to this subgroup. It is unlikely that the discordance observed in four of the sib pairs is due to recombination, because the polymorphic HincII site is only 116 base-pairs from the -26 codon of the growth hormone gene. Thus, in at least four of the nine families, the mutation responsible for isolated growth hormone deficiency is not within or near the structural gene for growth hormone on chromosome 17.
J A Phillips 3rd, J S Parks, B L Hjelle, J E Herd, L P Plotnick, C J Migeon, P H Seeburg
We studied the effect of triiodothyronine (T3) on mammalian growth-plate cartilage in vitro. Growth-plate cartilages from fetal pigs scapulae were incubated for 3 to 7 d in serum-free medium alone or medium containing T3. Alkaline phosphatase activity, a marker of hypertrophied chondrocytes, was increased in T3 (10 nM)-treated growth-plate cartilage 152 +/- 36% above that of cartilage incubated in medium alone after 3 d of incubation, and 324 +/- 47% after 7 d of incubation. There was a dose-response increase in alkaline phosphatase activity to T3 over the range of 0.01-10 nM. The rise in alkaline phosphatase activity was specific for T3 since growth-plate cartilage alkaline phosphatase activity was not increased by cortisol, insulin, parathyroid hormone, or 5% fetal calf serum. Histological studies of growth-plate cartilage showed that T3 in a concentration-dependent manner increased the width of the zone of maturation (hypertrophied chondrocytes). Histochemical staining for alkaline phosphatase activity demonstrated that T3 caused the recruitment of new cells into the zone of maturation. T3 also stimulated incorporation of L-[3H]leucine into protein and 35SO4 into proteoglycan in growth-plate cartilage. In contrast, T3 did not increase alkaline phosphatase activity or radiolabeled precursor incorporation into nongrowth-plate scapular cartilage. These studies demonstrate that T3 directly stimulates maturation and, to a lesser degree, growth-related processes in fetal mammalian growth-plate cartilage.
W M Burch, H E Lebovitz
To characterize the transport mechanisms responsible for formation of canalicular bile, we have examined the effects of ion substitution on bile acid-dependent and bile acid-independent bile formation by the isolated perfused rat liver. Complete replacement of perfusate sodium with choline and lithium abolished taurocholate-induced choleresis and reduced biliary taurocholate output by greater than 70%. Partial replacement of perfusate sodium (25 of 128 mM) by choline reduced bile acid-independent bile formation by 30% and replacement of the remaining sodium (103 mM) by choline reduced bile acid-independent bile formation by an additional 64%. In contrast, replacement of the remaining sodium (103 mM) by lithium reduced bile acid-independent bile formation by only an additional 20%, while complete replacement of sodium (128 mM) by lithium reduced bile formation by only 17%, and lithium replaced sodium as the predominant biliary cation. Replacement of perfusate bicarbonate by Tricine, a zwitterionic amino acid buffer, decreased bile acid-independent bile formation by greater than or equal to 50% and decreased biliary bicarbonate output by approximately 60%, regardless of the accompanying cation. In separate experiments, replacement of sodium by lithium essentially abolished Na,K-ATPase activity measured either as ouabain-suppressible ATP hydrolysis in rat liver or kidney homogenates, or as ouabain-suppressible 86Rb uptake by cultured rat hepatocytes. These studies indicate that bile acid(taurocholate)-dependent bile formation by rat liver exhibits a specific requirement for sodium, a finding probably attributable to the role(s) of sodium in hepatic sodium-coupled taurocholate uptake and/or in maintenance of Na,K-ATPase activity. The surprising finding that bile acid-independent bile formation was substantially unaltered by complete replacement of sodium with the permeant cation lithium does not appear to be explained by Na,K-ATPase-mediated lithium transport. Although alternative interpretations exist, this observation is consistent with the hypothesis that much of basal bile acid-independent bile formation is attributable to an ion pump other than Na,K-ATPase, which directly or indirectly mediates bicarbonate transport.
R W Van Dyke, J E Stephens, B F Scharschmidt
Gelatinase is a metallo-proteinase that acts specifically on denatured collagen. In human neutrophils, this enzyme is localized in small, morphologically still unidentified storage organelles that are resolved from the specific and the azurophil granules upon subcellular fractionation by differential sedimentation. When neutrophils isolated from freshly drawn blood are exposed to soluble stimuli such as N-formyl-methionyl-leucyl-phenylalanine, zymosan-activated serum, phorbol myristate acetate, or the calcium ionophore A 23187, or are induced to phagocytose opsonized zymosan, they rapidly release gelatinase in large amounts (30-70% of the cellular content in 10 min). When neutrophils from donor blood, which had been stored for 24 h at 4°C are used, extensive release even occurs without added stimuli by simply warming to 37°C.
Beatrice Dewald, Ursula Bretz, Marco Baggiolini
Human coagulation Factors VII and VIIa bind with equal affinity to monocytes stimulated with endotoxin. Equilibrium binding studies performed at 0 degrees C using 125I-labeled Factor VII and VIIa showed the dissociation constant (Kd) to be congruent to 82 pM with congruent to 3,600 binding sites/monocyte. Ca++ was required for Factor VII and VIIa interaction with monocytes (optimal CaC12 concentration greater than or equal to 2.5 mM) and binding was reversed by the addition of EDTA. The rate of conversion of Factor X to Xa in mixtures containing Factor VIIa and monocytes was directly related to the quantity of Factor VIIa bound to the monocyte surface. Thus the monocyte binding sites appear to represent tissue factor. Competition experiments showed that Factor VII and VIIa bind to the same monocyte sites and further, that unlabeled Factor VII and VIIa have the same affinity for the binding sites as the 125I-labeled proteins.
G J Broze Jr
The participation of prostaglandins in the physiologic alterations of endotoxin shock has been well established with the aid of prostaglandin synthetase inhibitors. Our study was designed to investigate the potential of ibuprofen, a highly specific cyclooxygenase inhibitor, to reverse the hemodynamic and acid base abnormalities of canine endotoxin shock. Mean blood pressure fell to 49.8 +/- 6.6 mm Hg in dogs given endotoxin by 5 min after injection, and remained below 83 mm Hg for the duration of the 120-min observation period. In animals given endotoxin followed by ibuprofen, a similar initial drop of systemic blood pressure was seen, but it subsequently recovered to 150.2 +/- 4.1 mm Hg by 120 min (P less than 0.001). Cardiac index increased in animals given ibuprofen (2.3 +/- 0.28 liter/m2 per min) compared with animals given endotoxin alone (1.0 +/- 0.09 liter/m2 per min) by termination of the experiment. The arterial pH dropped in endotoxin treated animals to 7.18 +/- 0.03 by 120 min. Ibuprofen prevented the acidosis, the final pH in ibuprofen and endotoxin treated animals measuring 7.36 +/- 0.01. We conclude that ibuprofen protects against the hypotension, acidosis, and depression of cardiac index of canine endotoxin shock.
E R Jacobs, M E Soulsby, R C Bone, F J Wilson Jr, F C Hiller
Using an enzyme-linked immunosorbent assay, we have demonstrated that purified human fibrinogen forms a complex with adsorbed platelet thrombospondin. The formation of the fibrinogen-thrombospondin complex was specific, saturable, and partially inhibited by mannosamine, glucosamine, and arginine. These same inhibitors have been previously shown to block thrombin-induced platelet lectin activity and platelet thrombospondin lectin activity. Adsorbed thrombospondin also formed a complex with fibronectin, although the extent of complex formation was significantly less than the extent of formation of the fibrinogen-thrombospondin complex. Platelet membrane glycoproteins IIb and IIIa, which have been previously shown to bind fibrinogen, did not inhibit the formation of the fibrinogen-thrombospondin complex. The present study supports the hypothesis that the interaction of fibrinogen with thrombospondin on the activated platelet surface may be an important step in the platelet aggregation process.
L L Leung, R L Nachman
Detailed quantitative studies were performed on the generation and utilization of energy by resting and phagocytosing human neutrophils. The ATP content was 1.9 fmol/cell, was constant during rest, and was not influenced by the presence or absence of glucose in the medium. The intracellular content of phosphocreatine was less than 0.2 fmol/cell.
Niels Borregaard, Troels Herlin
This report describes a novel method of immunochemotherapy; the active immunization to the drug 5-fluorouracil (5-FU) with enhanced antitumor activity resulting from its subsequent systemic administration. Two metastasizing carcinomas in the Fischer strain (F344) rat have been used: a chemically induced bladder carcinoma (FBCa) and a spontaneous mammary adenocarcinoma (MACa). Both tumors grow rapidly and result in 100% mortality within 10 wk of implantation. Neither tumor is sensitive to systemic 5-FU alone. Intradermal sensitization to 5-FU before FBCa tumor implantation, followed by 5-FU administered systemically, resulted in significant tumor regression and improvement in survival with eradication of all tumor and cure in 20% of animals. A similar antitumor effect was observed with the MACa. A comparable drug effect was observed when methotrexate sensitization was given before FBCa implantation followed by systemic MTX. Specificity to the sensitizing drug was demonstrated by the lack of effect of sensitization with either 5-FU or MTX unless followed by systemic therapy with the requisite sensitizing agent. Sensitization to 5-FU has also been assessed after FBCa implantation followed by resection of the local tumor. Resection was performed after distant tumor metastases had occurred, and was followed by systemic 5-FU therapy. Whereas tumor resection alone failed to cure any animal, sensitization to 5-FU increased cure rate fourfold over animals receiving systemic 5-FU alone. Antibody to 5-FU in the sera of sensitized animals has been suggested by an immunoenzymatic staining technique and its specificity confirmed in a radioimmunoassay. It is postulated that a combination of the systemic agent and the antibody elicited to it by sensitization produces the significant antitumor effect observed. The antitumor effect observed with this new approach to immunochemotherapy warrants further experimental and clinical study.
Rudolf E. Falk, Mark Hardy, Leonard Makowka, Julita Teodorczyk-Injeyan, Judith A. Falk
The expression of Ia-like antigens on human colony forming units-granulocyte macrophage (CFU-GM) is related to S-phase of the cell cycle, and associated with the control of normal granulocyte and macrophage production by prostaglandin E and acidic isoferritins in vitro. Ia-antigen expression by CFU-GM is lost within 3-6 h of culture at 37 degrees C and occurs simultaneously with loss of responsiveness to inhibition by these factors. Culture of bone marrow CFU-GM in a limited exposure suspension culture with 1 microM-1pM prostaglandin E (PGE1 or PGE2), but not prostaglandin F2 alpha or dibutyryl-cyclic-3'-5'-AMP results in the detection of CFU-GM Ia-antigen after 24 h. Antigen expression is associated with an absolute increase in total and S-phase CFU-GM, and restoration of responsiveness to inhibition by prostaglandin E and acidic isoferritins. The detection of Ia-antigen on CFU-GM after suspension culture with prostaglandin E results both from Ia-antigen reexpression as well as stimulation of noncycling cells to enter S-phase, express Ia-antigen and give rise to CFU-GM sensitive to inhibition by prostaglandin E and acidic isoferritins. The sensitivity of CFU-GM to inhibition by these factors after suspension culture with prostaglandin E is identical to that of the same cells tested prior to the suspension culture. These studies provide evidence for a direct regulatory association between Ia-antigen expression and control of myeloid progenitor cell differentiation, and suggest a role for prostaglandin E in the control of CFU-GM cell cycle, Ia-antigen expression, and growth regulation.
L M Pelus
Addisonian patients can maintain potassium homeostasis despite the absence of mineralocorticoid. The present in vitro microperfusion studies examine what role the cortical collecting tubule might play in this process. All studies were performed on tubules harvested from adrenalectomized rabbits, which were maintained on 0.15 M NaCl drinking water and dexamethasone 50 μg/d. Perfusion and bath solutions were symmetrical Ringer's bicarbonate with [K] of 5 meq/liter. Initial studies on cortical collecting tubules from adrenalectomized animals ingesting a high potassium chow (9 meq K/kg body wt) demonstrated net potassium secretion against an electrochemical gradient (mean collected fluid [K] 16.5±2.6 meq/liter with an observed transepithelial voltage of −6.3±4.1 mV; predicted voltage for passive distribution of potassium being −28.2 mV). To examine whether this active potassium secretion could be modulated by dietary potassium, independent of mineralocorticoid, two diets identical in all respects except for potassium content were formulated. Potassium secretion was compared in cortical collecting tubules harvested from adrenalectomized animals on low (0.1 meq K) and high (10 meq K) potassium intake.
Charles S. Wingo, Donald W. Seldin, Juha P. Kokko, Harry R. Jacobson
F1 hybrid offspring of New Zealand Black mothers and New Zealand White fathers [(NZB X NZW)F1] female mice develop antibodies to single-stranded (ss) and native DNA, immune complex glomerulonephritis, massive proteinuria, and premature death with renal failure. By a series of matings, congenic (NZB X NZW)F1 . xid/xid mice were prepared. These mice were different from (NZB X NZW)F1 mice in having the X chromosome-linked immune deficiency gene, xid, in homozygous form. Such congenic (NZB X NZW)F1 . xid/xid females failed to develop antibodies to single-stranded or native DNA. They also failed to develop fatal renal disease as measured by proteinuria, glomerular histology, glomerular immunofluorescence, and survival. To control for unknown genetic factors, studies were performed with littermates that were derived by mating NZB . xid/+ females with NZW . xid/Y males such that the resulting offspring were either (NZB X NZW)F1 . xid/xid (and therefore "defective") or (NZB X NZW)F1 . xid/+ [phenotypically like (NZB X NZW)F1]. In these and in additional studies, mice were housed in the same cages and identified by ear tagging so as to avoid possible environmental variations from cage to cage. In these studies, xid/xid mice failed to develop the characteristic signs of autoimmunity, whereas the controls did. Similar results were also obtained with (NZW X NZB)F1 xid/xid mice compared with (NZW X NZB)F1 xid/+ mice. The effect of xid/xid upon (NZB X NZW)F1 mice was further investigated by assessing responses to immunization and polyclonal B cell activation in vivo. The xid/xid mice failed to produce anti-ssDNA following immunization with ssDNA complexed to a protein carrier in fluid form or even emulsified in adjuvant. Finally, the xid/xid mice failed to produce antiDNA in response to multiple injections of the polyclonal activator, bacterial lipopolysaccharide (LPS), or the polyclonal activator, polyribose inosinic acid . polyribose cytidylic acid. However, the xid/xid mice were neither generally hyporesponsive nor unable to recognize LPS because they made normal antibody responses following immunization with LPS to which multiple trinitrophenyl groups were chemically attached. We conclude from these studies that xid/xid, which is known to cause the deletion of a B cell subset, has a profound affect upon (NZB X NZW)F1 mice, rendering them insusceptible to the naturally occurring autoimmune disease characteristic of (NZB X NZW)F1 mice, and preventing them from producing antibodies to DNA despite purposeful immunization and polyclonal B cell activation. These results force a reevaluation of previous concepts regarding the mechanisms by which xid/xid might interfere with the development of autoimmunity, and a consideration of therapeutic implications.
B J Steinberg, P A Smathers, K Frederiksen, A D Steinberg
The model hydrogen peroxide-myeloperoxidase-chloride system is capable of generating the powerful oxidant hypochlorous acid, which can be quantitated by trapping the generated species with the β-amino acid, taurine. The resultant stable product, taurine chloramine, can be quantitated by its ability to oxidize the sulfhydryl compound, 5-thio-2-nitro-benzoic acid to the disulfide, 5,5′-dithiobis(2-nitroben-zoic acid) or to oxidize iodide to iodine. Using this system, purified myeloperoxidase in the presence of chloride and taurine converted stoichiometric quantities of hydrogen peroxide to taurine chloramine. Chloramine generation was absolutely dependent on hydrogen peroxide, myeloperoxidase, and chloride and could be inhibited by catalase, myeloperoxidase inhibitors, or chloride-free conditions. In the presence of taurine, intact human neutrophils stimulated with either phorbol myristate acetate or opsonized zymosan particles generated a stable species capable of oxidizing 5-thio-2-nitrobenzoic acid or iodide. Resting cells did not form this species. The oxidant formed by the stimulated neutrophils was identified as taurine chloramine by both ultraviolet spectrophotometry and electrophoresis. Taurine chloramine formation by the neutrophil was dependent on the taurine concentration, time, and cell number. Neutrophil-dependent chloramine generation was inhibited by catalase, the myeloperoxidase inhibitors, azide, cyanide, or aminotriazole and by chloride-free conditions, but not by superoxide dismutase or hydroxyl radical scavengers. Thus, it appears that stimulated human neutrophils can utilize the hydrogen peroxide-myeloperoxidase-chloride system to generate taurine chloramine. Based on the demonstrated ability of the myeloperoxidase system to generate free hypochlorous acid we conclude that neutrophils chlorinate taurine by producing this powerful oxidant. The biologic reactivity and cytotoxic potential of hypochlorous acid and its chloramine derivatives suggest that these oxidants play an important role in the inflammatory response and host defense.
Stephen J. Weiss, Roger Klein, Adam Slivka, Maria Wei
Using electron microscopy and morphometric methods to assess secretion, we previously found that two times tidal volume ventilation of isolated perfused rat lung stimulates secretion by bronchiolar Clara cells; this effect is not prevented by β-adrenergic blockade (J. Clin. Invest. 1981. 67: 345-351.). In this study we used the isolated perfused rat lung and the anesthetized mechanically ventilated rat, to further study the mechanism by which large tidal volumes stimulate secretion by Clara cells. With the perfused lung we found (a) α-adrenergic inhibition did not block the secretory effect of ventilation at two times normal tidal volume; (b) indomethacin completely blocked the secretory action of two times tidal volume ventilation; (c) medium previously used to perfuse lungs ventilated at two times tidal volume, but not medium previously used to ventilate lungs at normal tidal volume, stimulated secretion by Clara cells when used to perfuse fresh lungs ventilated at tidal volume; (d) addition of prostacyclin to the fresh perfusate increased secretion by Clara cells of lungs ventilated at normal tidal volume. In anesthetized mechanically ventilated rats, sighs stimulated secretion by Clara cells; this increased secretion was inhibited by indomethacin but not by cholinergic blockade (bilateral vagotomy). These studies indicate that increased volume ventilation stimulates secretion by Clara cells in vivo and in vitro; they provide evidence that chemical nonadrenergic, noncholinergic mechanisms are involved in this secretion, and that prostaglandins may be the chemical messenger coupling the mechanico-secretory events.
Gloria D. Massaro, Ceferina Amado, Linda Clerch, Donald Massaro
Fibroblasts are known to have chemotactic responses to two components of the extracellular matrix, collagen and fibronectin. To extend these observations to other extracellular connective tissue macromolecules and their proteolytic fragments, fibroblasts from adult human skin and from late-gestation (270 d), fetal bovine ligaments were studied for chemotactic responsiveness to tropoelastin and elastin-derived peptides. Bovine ligament tropoelastin and elastin-derived peptides, generated from either human aortic elastin with human neutrophil elastase or from bovine ligament elastin with pancreatic elastase, elicited chemotactic responses that were maximal at 0.2 micrograms/ml (3 X 10(-9) M) and 0.5-2.0 micrograms protein/ml, respectively. Fractionation of the elastin-derived peptides by gel filtration (Bio-Gel P-10) indicated that comparable levels of chemotactic activity were present in all fractions, and amino acid analysis of the fractions showed no relationship between chemotactic activity and desmosine concentration. Taken in conjunction with the observations on tropoelastin, it appears that fibroblast chemotaxis to elastin components does not involve the cross-links of elastin. These results demonstrate that the influences of the connective tissue matrix upon fibroblast migration might include elastin precursors and fragments of elastin.
R M Senior, G L Griffin, R P Mecham
Congenital hypoplastic anemia (Diamond-Blackfan syndrome) is thought to involve the erythropoietic cell line alone. In this study, the evaluation of lymphocyte function in five patients with this syndrome revealed a number of abnormalities. Peripheral blood T lymphocyte percentages as assessed by monoclonal antibodies were decreased in three patients. T-helper/T-suppressor cell (OKT4:OKT8) ratios were almost unity in four of the five patients. We usually find a ratio of 2:1 in normal populations. Studies of lymphocyte-mediated suppression of lymphoproliferation demonstrated an inability to generate concanavalin A-induced suppressor cells in the same four patients and impaired prostaglandin-mediated suppression in two patients. Co-culture studies revealed a T lymphocyte-mediated suppression of erythropoiesis in a single patient, who also showed suppression of the mixed lymphocyte reaction. The four remaining patients showed no excessive suppressor effects either upon erythropoiesis or lymphoproliferation. These studies demonstrate that in congenital hypoplastic anemia, the cellular defect is not restricted to the erythroid progenitor cells, but extends to the lymphocytes.
J L Finlay, N T Shahidi, S Horowitz, W Borcherding, R Hong
Autodigestion of activated Hageman factor (HFa) yields a 40,000-mol wt activated enzyme as well as Hageman factor fragment (HFf); HFf consists of two molecular weight species of 28,500 and 30,000. We have investigated the structure of these active fragments and demonstrate that upon reduction, each possesses a heavy chain of 28,000. The associated light chains were identified by subjecting iodinated proteins to two-dimensional slab gel electrophoresis in which the second dimension is run reduced. The 40,000-dalton enzyme has a light chain of 15,000, the 30,000-dalton form of HFf has a light chain of 2,000 and we have suggestive evidence of a light chain associated with the 28,500-dalton form of HFf (putative mol wt approximately 500). We also demonstrate that the 30,000-dalton form of HFf precedes the 28,500 form. These data indicate that digestion of native HF to form HFa precedes cleavages that fragment the molecule and diminish its molecular weight. The 28,500-dalton light chain of HFa becomes the heavy chain of each of the fragmentation products while cleavage at different points along the heavy chain of HFa determines which fragments will be produced. In contrast to autoactivation, kallikrein digestion of HFa yields primarily HFf; however, the 40,000-dalton enzyme may be seen when prekallikrein-deficient (Fletcher trait) plasma is activated.
J T Dunn, A P Kaplan
Following exposure to hypotonic media, human peripheral blood lymphocytes swell initially but restore their isotonic volume within minutes. In contrast, tonsillar lymphocytes demonstrate a similar initial phase of swelling but fail to restore their isotonic volume. We have studied the ionic basis for this second or regulatory volume decrease (RVD) phase using lymphocytes from peripheral blood, tonsil, and thymus. RVD was characterized by 86Rb efflux and a decrease in K+ content. The increase in K+ permeability in response to hypotonic challenge was characteristic for T lymphocytes obtained from peripheral blood, tonsil, or thymus. B lymphocytes showed only a modest increase in K+ permeability and consequently little RVD. The data confirm that the response of peripheral blood and tonsillar lymphocytes to a hypotonic environment can be accounted for by differences in the proportions of T and B cells, and the differential behaviour of B and T lymphocytes is based on differences in membrane permeability to K+ upon swelling.
R K Cheung, S Grinstein, E W Gelfand
The effect of luminal and peritubular HCO3(-) concentrations and PCO2 on HCO3(-) reabsorption was examined in rabbit proximal convoluted tubules perfused in vitro. Increasing luminal HCO3(-) concentration from 25 to 40 mM without changing either peritubular HCO3(-) concentration or PCO2, stimulated HCO3(-) reabsorption by 41%. When luminal HCO3(-) concentration was constant at 40 mM and peritubular HCO3(-) concentration was increased from 25 to 40 mM without changing peritubular PCO2, a 45% reduction in HCO3(-) reabsorption was observed. This inhibitory effect of increasing peritubular HCO3(-) concentration was reversed when peritubular pH was normalized by increasing PCO2. Passive permeability for HCO3(-) was also measured and found to be 1.09 +/- 0.17 X 10(-7) cm2 s-1. Using this value, the passive flux of HCO3(-) could be calculated. Only a small portion (less than 23%) of the observed changes in net HCO3(-) reabsorption can be explained by the passive HCO3(-) flux. We conclude that luminal and peritubular HCO3(-) concentrations after HCO3(-) reabsorption by changing the active H+ secretion rate. Analysis of these data suggest that both luminal and peritubular pH are major determinants of HCO3(-) reabsorption.
S Sasaki, C A Berry, F C Rector Jr
Copper-zinc superoxide dismutase (SOD) is present in relatively high concentrations in the β-cells of human islets. The activity of the extracted enzyme is partially inhibited upon incubation with the diabetogenic drugs alloxan, streptozotocin, or Vacor. The role of this enzyme in protecting β-cells against chemically induced diabetes was further investigated.
Samuel E. Gandy, Maria G. Buse, Rosalie K. Crouch
We have produced a murine monoclonal antibody (F43 A1D2) that binds to the cell surface of both rat islet tumor cells (RINm clone 5F and RINm clone 14B) and normal rat islet cells. This antibody is cytotoxic in the presence of complement for RIN tumor cells as well as A, B, and D pancreatic polypeptide rat islet cells. Antibody A1D2 does not bind to rat thymus cells, pancreatic acinar cells, or fibroblasts. Antigen A1D2 (termed ICSAn-1 [islet cell surface antigen 1]) is a trypsin-sensitive glycoprotein with a molecular mass of approximately 24,000 daltons. In addition to purification of its islet cell antigen, antibody A1D2 can be used to identify and isolate living islet cells from pancreatic digests.
M A Crump, R Scearce, M Dobersen, W Kortz, G S Eisenbarth
Potassium is known to enhance the aldosterone-stimulating action of angiotensin II. Such a synergistic interaction of potassium with angiotensin II could represent an action by angiotensin II to potentiate potassium as a stimulus. To examine for this effect of angiotensin II on potassium, plasma aldosterone levels were measured before and after an infusion of potassium chloride (15 meq i.v.) into dogs without and with prevention of angiotensin II formation by captopril, an angiotensin converting-enzyme inhibitor. In addition, responses to potassium were measured in a group of dogs receiving angiotensin II plus captopril. After potassium infusion, control dogs showed an increase of 7.7 +/- 1.9 (SEM) ng/dl (P less than 0.001) in the level of plasma aldosterone. In contrast, captopril-treated dogs showed no change in plasma aldosterone concentration in response to potassium. When angiotensin II was administered to captopril-treated dogs responsiveness to potassium administration was restored (plasma aldosterone concentration increased by 7.4 +/- 2.1 ng/dl, P less than 0.002). ACTH stimulated aldosterone secretion despite captopril treatment (P less than 0.001), however, ACTH produced a greater increase in the plasma aldosterone concentration in controls than in captopril-treated animals. It is evident from these results that stimulation of aldosterone secretion by potassium is considerably enhanced by angiotensin II. There appears to exist an important interdependence of these stimuli in the regulation of aldosterone secretion.
J H Pratt
Autopsy findings suggest that lung surfactant is damaged in the adult respiratory distress syndrome. In the present study 225 bronchoalveolar lavage specimens (78 from 36 patients, 1-78 yr old with respiratory failure, 135 from another 128 patients with other respiratory disease, and 12 from healthy controls) were assayed for the lung profile [lecithin/sphingomyelin (L/S) ratio, saturated lecithin, phosphatidylinositol, and phosphatidylglycerol]. Bronchoalveolar lavage fluid was further analyzed for phospholipids and for phosphatidic acid phosphohydrolase, phospholipase A2, and phosphatidylinositol phosphodiesterase activities. A lipid-protein complex was isolated and analyzed for surface activity, and plasma was measured for myoinositol. There were only small differences seen in the recovery of total phospholipid between respiratory failure patients and normal controls. However, in respiratory failure, phospholipids in bronchoalveolar lavage were qualitatively different from those recovered either from normal controls or from patients with other lung disease: the LO/S ratio, phosphatidylglycerol, and disaturated lecithin were low, whereas sphingomyelin and phosphatidylserine were prominent. These abnormalities were present early in respiratory failure and tended to normalize during recovery. Low L/S ratio (less than 2), and low phosphatidylglycerol (1% or less of glycerophospholipids) in bronchoalveolar lavage was always associated with respiratory failure. Abnormal lavage phospholipids were not due to plasma contamination. The phospholipase studies revealed little evidence of increased catabolism of phospholipids. In respiratory failure, the lipid-protein complexes from lung lavage were not surface active, whereas that from healthy controls had surface properties similar to lung surfactant. Phospholipids from patients with respiratory failure were similar to those from respiratory distress syndrome in the newborn. However, the latter condition is characterized by fast recovery of surfactant deficiency and by high plasma myoinositol that suppresses the synthesis of surfactant phosphatidylglycerol and increases phosphatidylinositol (Pediatr. Res. 1981. 15: 720). On the other hand, in adult respiratory distress syndrome, the abnormality in surfactant phospholipids may last for weeks and in most cases is associated with low phosphatidylinositol, low phosphatidylglycerol, and low plasma myoinositol.
M Hallman, R Spragg, J H Harrell, K M Moser, L Gluck
The effect of corticosteroids on angiotensin converting enzyme was investigated in endothelial cell cultures and intact rat lung. Cultured endothelial cells from bovine aorta showed net production of angiotensin converting enzyme (ACE) over 2 d culture in serum-free medium. Dexamethasone (DM) increased cell ACE activity six- to sevenfold at 100 nM with a threshold effect at 0.3 nM. The effect of DM on ACE production was completely inhibited by actinomycin D or cycloheximide. Deoxycorticosterone (DOC) and aldosterone were markedly less active, with a threshold near 100 nM and significant (two to threefold) stimulation of ACE activity at 1 μM. In cells incubated in the presence of 10 nM DM, DOC (10 μM) significantly inhibited ACE production compared with 10 nM DM alone, suggesting that DOC is a partial agonist/partial antagonist in this enzyme system. Protein content of cells or medium was unchanged by steroids at all doses used.
F. A. O. Mendelsohn, C. J. Lloyd, C. Kachel, J. W. Funder
The photohemolysis of normal erythrocytes incubated with protoporphyrin is reduced in the presence of albumin. When globin is added to normal erythrocytes loaded with protoporphyrin, protoporphyrin is bound to globin. During irradiation protoporphyrin moves from globin to the erythrocyte membrane and photohemolysis is initiated. Erythrocytes in patients with erythropoietic protoporphyria contain large amounts of protoporphyrin bound to hemoglobin. Upon irradiation of these cells in the absence of albumin, 40% of protoporphyrin and 80% of hemoglobin is released after 240 kJ/m2. The released protoporphyrin is hemoglobin bound. In contrast, when albumin is present only 8% of hemoglobin is released whereas protoporphyrin is released to 76%. The released protoporphyrin is albumin bound. A hypothesis for the release of erythrocyte protoporphyrin in erythropoietic protoporphyria without simultaneous hemolysis is proposed. Upon irradiation protoporphyrin photodamages its binding sites on hemoglobin, moves through the plasma membrane, and is bound to albumin in plasma.
S Sandberg, A Brun