The myocardial responsiveness of conscious, instrumental dogs to exogenously administered isoproterenol and norepinephrine was investigated in neonatal, 6-wk-old, and adult animals. Comparable base-line values for peak left ventricular derivative of pressure with respect to time were observed in all age categories. However, when compared with adult responses, the sympathomimetic amine-induced increases in neonatal left ventricular dP/dt were significantly blunted at each concentration of adrenergic agonist examined, whereas the 6-wk-old puppies displayed an intermediate inotropic response. To investigate the cellular mechanisms of this blunted neonatal response, we correlated physiologic and biochemical measurements of the myocardial responses to catecholamines in each age category. When compared with adult myocardial membrane preparations, neonatal cardiac membranes were characterized in vitro by an increased density of beta-adrenergic binding sites, comparable affinity for adrenergic agonists and antagonists, and an enhanced coupling of adenylate cyclase activation to receptor occupancy. Simultaneous changes in either the serum catecholamine concentration or the membrane content of other intrinsic proteins failed to account for the observed neonatal increase in beta-adrenergic receptor density. These findings are most consistent with a compensatory mechanism of the cardiac cell membrane, whereby an inherent depression in the adrenergic responsiveness of the immature myocardium appears to induce the increase in receptor density and activation of adenylate cyclase.
S G Rockson, C J Homcy, P Quinn, W T Manders, E Haber, S F Vatner
Patterns of bone loss in the axial and the appendicular skeleton were studied in 185 normal volunteers (105 women and 82 men; age range, 20--89 yr) and in 76 women and 9 men with vertebral fractures due to osteoporosis. Bone mineral density was measured in vivo at the lumbar spine (predominantly trabecular bone) by dual photon absorptiometry and at the midradius (greater than 95% cortical bone) and distal radius (75% cortical and 25% trabecular bone) by single photon absorptiometry. In normal women, bone diminution from the vertebrae began in young adulthood and was linear. In the appendicular skeleton, bone diminution did not occur until age 50 yr, was accelerated from aged 51 to 65 yr, and then decelerated somewhat after age 65 yr. Overall bone diminution throughout life was 47% for the vertebrae, 30% for the midradius, and 39% for the distal radius. In normal men, vertebral and appendicular bone diminution with aging was minimal or insignificant. Mean bone mineral density was lower in patients with osteoporosis than in age- and sex-matched normal subjects at all three scanning sites, although spinal measurements discriminated best; however, there was considerable overlap. By age 65 yr, half of the normal women (and by age 85 yr, virtually all of them) had vertebral bone mineral density values below the 90th percentile of women with vertebral fractures and, thus, might be considered to have asymptomatic osteoporosis. For men, the degree of overlap was less. The data suggest that disproportionate loss of trabecular bone from the axial skeleton is a distinguishing characteristic of spinal osteoporosis.
B L Riggs, H W Wahner, W L Dunn, R B Mazess, K P Offord, L J Melton 3rd
Contents obtained from jejunum of normal controls, self-emptying and self-filling blind loop rats were analyzed for the presence of glycoprotein-degrading glycosidases. The blind loop syndrome was documented by the increased fat excretion and slower growth rate of self-filling blind loop rats 6 wk after surgery. With p-nitrophenylglycosides as substrate, the specific activity of α-N-acetylgalactosaminidase, a potential blood group A destroying glycosidase, was 0.90±0.40 mU/mg of protein. This level was 23-fold higher than the specific activity of normal controls. In partially purified self-filling blind loop contents, the activity of α-N-acetylgalactosaminidase was 9- to 70-fold higher than activities of self-emptying and normal controls. Antibiotic treatment with chloromycetin and polymyxin decreased 24-fold the glycosidase levels in self-filling blind loops. In experiments with natural substrate, the blood group A titer of a20,000g supernate from normal jejunal homogenates decreased 128-fold after 24-h incubation with blind loop contents. Normal contents failed to diminish the blood group reactivity of the natural substrate. Furthermore, blind loop contents markedly decreased the blood group A titer of isolated brush borders. Incubation between blind loop bacteria and mucosal homogenates or isolated brush borders labeled with d-[U-14C]glucosamine revealed increased production of labeled ether extractable organic acids. Likewise, intraperitoneal injection of d-[U-14C]glucosamine into self-filling blind loop rats resulted in incorporation of the label into luminal short chain fatty acids. These results suggest that glycosidases may provide a mechanism by which blind loop bacteria obtain sugars from intestinal glycoproteins. The released sugars are used and converted by bacteria into energy and organic acids. This use of the host's glycoproteins would allow blind loop bacteria to grow and survive within the lumen independent of exogenous sources.
Previous studies from our laboratory indicated that both beta-adrenergic and cholinergic agents stimulate in vivo secretion by rat bronchiolar Clara cells. Those studies also provided support for an in-series beta-adrenergic-cholinergic stimulation of secretion. To further explore the regulation of secretion in Clara cells, and to do it in the absence of systemic influences, we have used the isolated ventilated perfused rat lung. We have again used morphometry and electron microscopy to assess secretion by measuring the volume density (fraction of cell volume) of the secretory granules of bronchiolar Clara cells. We found that in the isolated perfused lung, as in the intact animal, isoproterenol stimulated secretion in Clara cells and that this effect was blocked by the beta-adrenergic antagonist propranolol. Pilocarpine, unlike its action in the intact animal, did not stimulate secretion in the isolated lung; rather it inhibited the secretory effect of isoproterenol. Increased tidal-volume ventilation stimulated secretion; propranolol did not block this effect. Analogs of cyclic (c)AMP and of cGMP also stimulated secretion by Clara cells. These findings indicate that there are at least two mechanisms by which Clara cells can be stimulated to secrete. One seems to be beta-adrenergic-cAMP mediated but the triggering event is unknown. The other is initiated by increased tidal volume and cGMP may be involved in the intracellular mediation of this stimulatory event. Finally, we found evidence of beta-adrenergic (stimulatory) -cholinergic (inhibitory antagonism in the regulation of secretion in Clara cells.
G D Massaro, C M Fischman, M J Chiang, C Amado, D Massaro
During phagocytosis, neutrophils take oxygen from the surrounding medium and convert it to superoxide anion (O2-) and hydrogen peroxide (H2O2). Hydroxyl radical (.OH), a particularly potent oxidant, is believed to be produced by interaction between O2- and H2O2 in the presence of iron, according to the Haber-Weiss reactions. Production of .OH by whole human neutrophils, by particulate fractions from human neutrophils disrupted after stimulation, and by a xanthine oxidase system was measured by conversion of alpha-keto-gamma-methiol butyric acid to ethylene. FeCl3 or ferric EDTA enhanced ethylene production in all three systems by 155--406% of base line at a concentration of 50--100 microM. Iron-saturated human milk lactoferrin, 100 nM, increased ethylene generation by 127--296%; and purified human neutrophil lactoferrin, 10 nM, enhanced ethylene production by 167--369%. Thus, iron bound to lactoferrin was approximately 5,000 times more effective in producing an enhancement in ethylene generation than iron derived from FeCl3 or ferric EDTA. O2- and H2O2 were required for ethylene production in the presence of lactoferrin, since superoxide dismutase inhibited ethylene formation in the three systems by 76--97% and catalase inhibited by 76--98%. Ethylene production in the presence of lactoferrin was inhibited by the .OH scavengers mannitol, benzoate, and thiourea by 43--85, 45--94, and 76--96%, respectively. Thus, most of the ethylene production could be attributed to oxidation of alpha-keto-gamma-methiol butyric acid by .OH. The ability of neutrophil lactoferrin to provide iron efficiently to the oxygen radical-generating systems is compatible with a role for lactoferrin as regulator of .OH production. As such, lactoferrin may be an important component in the microbicidal activity of neutrophils.
D R Ambruso, R B Johnston Jr
Clinical studies on the minor hemoglobins (hemoglobin A1a-c) have suggested that a novel adduct may form in people abusing alcohol. Such patients were found to have an elevated concentration of minor hemoglobins, but normal or subnormal amounts of glycosylated hemoglobin (hemoglobin A1c) as determined by radioimmunoassay, Acetaldehyde, a reactive metabolite of ethanol, was postulated to form adducts with hemoglobin A that change its chromatographic properties. At physiological concentrations, acetaldehyde was found to form adducts with hemoglobin that were stable to extensive dialysis for several days. The amount of hemoglobin adducts formed were a function of the concentration and number of exposures to acetaldehyde. The reaction of acetaldehyde with hemoglobin A produced chromatographic variants, some of which migrated in the hemoglobin A1a-c region. Analysis of stable acetaldehyde-hemoglobin adducts demonstrated that valine, lysine, and tyrosine residues of globin were sites of reaction. The acetaldehyde-modified amino acid residues appear to exist in interconvertible conformations, only one of which is reducible by sodium borohydride. The amount of these adducts was found to be significantly elevated in hemoglobin from alcoholics as compared with normal volunteers.
V J Stevens, W J Fantl, C B Newman, R V Sims, A Cerami, C M Peterson
Premature lambs were treated with 50 mg/kg of natural surfactant lipid by tracheal instillation either at birth or shortly thereafter when respiratory failure was documented. All lambs were delivered by cesarean section and supported on infant ventilators with 100% oxygen under conditions to mimic the care of human infants with the respiratory distress syndrome. The natural surfactant used for therapy was recovered by lavage from sheep lung. Six 120-d gestational age lambs treated at birth had an initial mean oxygen pressure (pO2) value of 270 +/- 35 mm Hg; this fell within 3 h to less than 100 mm Hg. By 8.3 +/- 0.3 h after birth the lambs were in severe respiratory failure with a mean pH less than 7.1 and a mean pCO2 greater than 70 mm Hg. Six untreated lambs had pH values below 7.0 within 40 min of life despite more intensive respiratory support than was given the treated animals. Treatment with natural surfactant of 17 lambs of 120 and 130 d gestational age after early respiratory failure resulted in a prompt increase in pO2 values from about 35 mm Hg to values over 200 mm Hg and a fall in pCO2 values to normal levels in the majority of animals. This response lasted only approximately 3 h, and a second treatment was less predictably effective.
A Jobe, M Ikegami, T Glatz, Y Yoshida, E Diakomanolis, J Padbury
Guinea pigs, actively sensitized to ovalbumin, were inoculated by nasal insufflation with parainfluenza 3 or virus growth medium 4 d before performing in vitro pharmacological studies on tracheal and bronchial smooth muscle. In each airway segment, cumulative dose-response effects of ovalbumin were obtained in the absence and presence of a maximally effective concentration of a beta adrenergic receptor agonist, sulfonterol. Sulfonterol shifted the dose-response curve to the right and reduced the maximum smooth muscle contractile response to ovalbumin. Virus infection did not alter the dose-response effects of ovalbumin. However, the magnitude of the inhibitory effects of sulfonterol was smaller in segments taken from animals inoculated with virus. Blockade by virus infection of the inhibitory effect of sulfonterol was reversed when the concentrations of beta agonist were increased. Sulfonterol did not alter the dose-response effects of histamine at any of the concentrations that markedly antagonized the effects of ovalbumin. Virus infection did not alter the sensitivities to sulfonterol or papaverine in producing relaxation in either airway segment. The magnitude of relaxation produced by papaverine was significantly larger in bronchial rings taken from animals infected with virus for 4 d, but there was no alteration by virus of the dose-response effects of histamine or carbachol. In experiments measuring antigen-induced release of slow reacting substance of anaphylaxis and histamine from minced lung, virus infection did not alter the sensitivity or the maximum effects of ovalbumin. Also, the ability of sulfonterol to inhibit the release of slow reacting substance of anaphylaxis and histamine was not affected by virus infection.
C. K. Buckner, D. E. Clayton, A. A. Ain-Shoka, W. W. Busse, E. C. Dick, P. Shult
The pathogenesis of liver disease in protoporphyria has been presumed to result from the hepatic deposition of protoporphyrin. To examine the effects of protoporphyrin on hepatic bile flow and histopathology, studies were performed employing an isolated, in situ, rat liver perfusion system. Rat livers in the control group were perfused with 0-80 μmol sodium taurocholate/h. Rat livers in the experimental group were perfused with sodium taurocholate and (a) sufficient quantities of protoporphyrin to produce maximal canalicular secretion and (b) perfusate protoporphyrin concentrations of 0.01, 0.1, and 1 μM.
Dennis L. Avner, Randall G. Lee, Malcolm M. Berenson
In mongrel dogs, the effect of end-to-side portacaval shunt on plasma, cerebrospinal fluid (CSF) and brain tyramine, tyrosine, dopamine, norepinephrine, and epinephrine were studied. It was found that the level of tyramine in plasma, CSF, and selected brain regions increased steadily after the construction of the shunts. These elevations became more pronounced when the dogs manifested symptoms of hepatic encephalopathy. In postshunted dogs with stage II and III hepatic encephalopathy, tyramine concentration in corpus striatum (1,312 +/- 371), hypothalamus (400 +/- 67.0), and midbrain (660 +/- 78.7 ng/g) was significantly (P less than 0.05) higher than the level in dogs with stage 0 and I hepatic encephalopathy and sham-operated dogs serving as controls (corpus striatum, 831 +/- 140; hypothalamus, 167 +/- 40.0; and midbrain, 132 +/- 37.4 ng/g). This was followed by a concomitant depletion of dopamine and norepinephrine in these brain regions (postshunt: dopamine 104 +/- 20.0, 3,697 +/- 977, and 105 +/- 14.1; norepinephrine 521 +/- 71.6, 81.6 +/- 13.7, and 218 +/- 31.7 ng/g; vs. sham group: dopamine 532 +/- 83.1, 8,210 +/- 1,126, and 192 +/- 35.0; norepinephrine 1,338 +/- 425, 124 +/- 21.3, and 449 +/- 89.7 ng/g) of encephalopathic dogs with portacaval shunt. Furthermore, tyramine, tyrosine, dopamine, and norepinephrine levels in plasma and CSF increased markedly as clinical features in the dogs' behavior characteristic of hepatic encephalopathy occurred, including hypersalivation, ataxia, flapping tremor, somnolence, and coma. Cerebral hypertyraminemia and a defect in sympathetic neurotransmission may contribute to the development of hepatic encephalopathy of liver disease.
B A Faraj, V M Camp, J D Ansley, J Scott, F M Ali, E J Malveaux
We have found complement-dependent cytotoxic antibodies in the serum of 8 of 24 patients with insulin-dependent diabetes mellitus using a 51Cr cytotoxicity assay with monolayers of cloned rat islet cells (clones RINm 5F and RINm 14B). In contrast, complement-dependent cytotoxicity with 51Cr release greater than 24% was found with sera from 34 controls or from 5 patients with polyglandular failure without diabetes, and was present in only 1 serum our of 12 from patients with insulin-independent diabetes mellitus. The prevalence of antibodies appears to decrease with duration of insulin-dependent diabetes, and in one patient studied, cytotoxic antibodies were present at the time of diagnosis of diabetes. Cytotoxicity is independent of insulin synthesis, as evidenced by the linear correlation of cytotoxicity of sera for the insulin-producing clone RINm 5F and the somatostatin-producing clone RINm 14B. The present study identifies nonspecies-specific cytotoxic antibodies in the serum of patients with diabetes mellitus, and the assay used should facilitate studies of humoral immunity in the pathogenesis of diabetes mellitus.
G S Eisenbarth, M A Morris, R M Scearce
Severe pulmonary edema sometimes develops despite normal pulmonary capillary wedge pressure (Ppw). The equation describing net transvascular flux of lung liquid predicts decreased edema when hydrostatic pressure is reduced or when colloid osmotic pressure is increased in the pulmonary vessels. We tested these predictions in a model of pulmonary capillary leak produced in 35 dogs by intravenous oleic acid. 1 h later, the dogs were divided into five equal groups and treated for 4 h in different ways: (a) not treated, to serve as the control group (Ppw = 11.1 mm Hg); (b) given albumin to increase colloid osmotic pressure by 5 mm Hg (Ppw = 10.6 mm Hg); (c) ventilated with 10 cm H2O positive end-expiratory pressure (Peep) (transmural Ppw = 10.4 mm Hg); (d) phlebotomized to reduce Ppw to 6 mm Hg; (e) infused with nitroprusside, which also reduced Ppw to 6 mm Hg. Phlebotomy and nitroprusside reduced the edema in excised lungs by 50% (P< 0.001), but Peep and albumin did not affect the edema. Pulmonary shunt decreased on Peep and increased on nitroprusside, and lung compliance was not different among the treatment groups, demonstrating that these variables are poor indicators of changes in edema. Cardiac output decreased during the treatment period in all but the nitroprusside group, where Ppw decreased and cardiac output did not. We conclude that canine oleic acid pulmonary edema is reduced by small reductions in hydrostatic pressure, but not by increased colloid osmotic pressure, because the vascular permeability to liquid and protein is increased. These results suggest that low pressure pulmonary edema may be reduced by seeking the lowest Ppw consistent with adequate cardiac output enhanced by vasoactive agents like nitroprusside. Further, colloid infusions and Peep are not helpful in reducing edema, so they may be used in the lowest amount that provides adequate circulating volume and arterial O2 saturation on nontoxic inspired O2. Until these therapeutic principles receive adequate clinical trial, they provide a rationale for carefully monitored cardiovascular manipulation in treating patients with pulmonary capillary leak.
R. M. Prewitt, J. McCarthy, L. D. H. Wood
Other investigators have demonstrated that concentrations of immunoreactive somatostatin (IRS) are higher in blood from the hepatic portal vein or its tributaries than in blood from the hepatic or peripheral systemic veins of man and animals. This suggests that there is hepatic extraction of IRS from the portal system in vivo. In the rat, portal vein plasma IRS is reported to be heterogeneous and to contain, in part, a 1,600 mol wt form of IRS which is immunochemically similar to synthetic somatostatin and not significantly bound to high molecular weight plasma protein. Our study was undertaken to determine directly whether unbound synthetic cyclic somatostatin was cleared by the rat liver perfused through the hepatic portal vein in vitro with a recirculating, plasma-free, erythrocyte-containing perfusate.
Harold Sacks, L. Cass Terry
The effects of acute volume loading were examined on indices of left ventricular (LV) function in conscious, unrestrained and intact, tranquilized baboons. Experiments were conducted 1-3 mo after implantation of ultrasonic transducers to measure LV internal diameter and wall thickness, and miniature LV pressure gauges and aortic and left atrial catheters. In 10 intact, tranquilized baboons, rapid volume loading with saline increased LV end-diastolic pressure by 23.7±2.6 mm Hg, LV end-diastolic diameter by 7.8±1.5%, LV stroke work by 37.5±7.8%, while mean arterial pressure and peak LV wall stress did not change significantly. Despite the increase in preload and activation of the Frank-Starling mechanism, LV dP/dtmax and the maximum velocity of myocardial fiber shortening (LV dD/dtmax) did not change. Volume loading after β-adrenergic or combined β-adrenergic and cholinergic blockades or volume loading with blood instead of saline also failed to augment LV dP/dtmax and LV dD/dtmax despite the increase in preload. In order to volume load the baboons in the conscious state, a radiofrequency (RF) interrogator system was devised, which upon receipt of a radio command, activated a battery operated pump to infuse 1,000 ml of saline i.v. to the baboons. In these experiments, preload rose, i.e., LV end-diastolic diameter increased by 13.9±2.1% and the Frank-Starling mechanism could be demonstrated, i.e., stroke work rose by 42.8±7.4%, but LV dP/dtmax and LV dD/dtmax did not change. After preload was depressed by hemorrhage, the rapid infusion of either blood or saline increased LV dP/dtmax by 92.7±18.5% and LV dD/dtmax by 64.3±10.1%. Thus, acute volume loading in the conscious baboons increased LV end-diastolic size and even stroke work substantially. However, preload dependency of LV dP/dtmax and the maximum velocity of myocardial fiber shortening was only encountered at low levels of LV preload.
Michael Zimpfer, Stephen F. Vatner
Reports from several laboratories, showing extensive hepatic extraction of circulating parathyroid hormone, led us to examine the effect of near-total hepatectomy on the metabolism of the hormone to circulating fragments, and on its clearance from plasma. The rate of disappearance of 125I-labeled and unlabeled bovine parathyroid hormone from plasma, and the appearance, disappearance, and chemical and immunochemical characteristics of circulating fragments were examined by gel filtration and either sequence-specific radioimmunoassays or sequence analysis using the Edman reaction. Results from awake rats subjected to near-total hepatectomy were compared with those found in sham-treated, nephrectomized, and short-term uremic rats (studied 2 d after nephrectomy). When compared with the sham-treated group, all other groups clear 125I-labeled hormone more slowly; after hepatectomy, however, the clearance rate is most strikingly decreased.
Gino V. Segre, Pierre D'Amour, Anne Hultman, John T. Potts Jr.
Data from several laboratories indicate that hepatic mechanisms may have a distinctive role in the metabolism of intact hormone after secretion, a process that accounts, at least partly, for the heterogeneity of circulating parathyroid hormone. Accordingly, we studied the proteolysis of intact hormone by isolated rat Kupffer cells and hepatocytes. Kupffer cells (106 cells/ml) and hepatocytes (107 cells/ml) were incubated with unlabeled and 125I-labeled bovine parathyroid hormone at 37°C for periods ranging up to 2 h. When incubated with Kupffer cells, intact hormone disappeared with a t½ of 12±4 min. Radio-immunoassays using sequence-specific antisera showed that the dominant hormonal fragments recovered in the medium have an apparent molecular weight of ∼6,000, lack amino-terminal antigenic determinants, and react in assays that specifically recognize determinants in the carboxy-terminal portion of the intact hormone. Amino-terminal fragments also were detected in high concentrations, particularly after short incubation periods. Radioiodinated fragments resulting from incubation of 125I-labeled bovine parathyroid hormone with Kupffer cells had the same apparent size as fragments derived from the metabolism of unlabeled, intact hormone; when analyzed by Edman degradation, positions 34 and 37 of the intact hormone sequence were the amino-terminal amino acids of these dominant carboxy-terminal fragments. Hepatocytes did not hydrolyze the hormone. Thus, metabolism of parathyroid hormone by Kupffer cells results in the appearance of fragments in the media that are immunochemically indistinguishable from, and chemically identical with, those found in plasma when intact hormone is injected intravenously. This indicates that the proteolysis observed in vitro accurately reflects the metabolism of the hormone in vivo. The detection of amino-terminal fragments in concentrations nearly equal to those of carboxy-terminal fragments indicates that cleavage of intact hormone is, initially, by an endopeptidase(s).
Gino V. Segre, Archibald S. Perkins, Lee A. Witters, John T. Potts Jr.
This investigation was designed to define the cellular level at which the gamma to beta globin switch is established in the developing simian fetus in order to determine whether the switch is controlled by environmental influences within differentiating erythroid precursors or predetermined by the genetic program of erythroid progenitors. Samples of marrow and liver were obtained from rhesus fetuses throughout the switch period, and marrow was obtained from adult rhesus monkeys. Globin chain synthesis was then measured in differentiated erythroblasts and in erythroid progenitor-derived colonies grown in semisolid media. The relative rates of synthesis of gamma and beta chains were determined by the uptake of [3H]leucine into the respective chains separated by Triton gel electrophoresis and in some cases by urea carboxymethyl cellulose chromatography. Four periods of the switch were defined during fetal development. In the preswitch period both erythroblasts and progenitor-derived colonies produced <5% beta globin. In the early switch erythroblasts produced 5-15% beta globin, while progenitor-derived colonies produced 10-35% beta globin. In mid-switch erythroblasts synthesized 50% beta globin, whereas progenitor-derived colonies produced only 15-35% beta. At the completion of the switch erythroblasts produced 100% beta globin, while progenitor-derived colonies produced as little as 40% beta chains. We conclude that the program of globin synthesis that characterizes the fetal switch is established at the level of erythroid progenitors. Fetal erythroid burst-forming units (BFU-E) dominate the marrow prior to the switch. The early switch period is heralded by the appearances of adult erythroid burst-forming units programmed to express increasing beta chain synthesis in colonies. By mid-switch a second class of adult erythroid progenitors capable of giving rise to fetal and adult hemoglobin synthesis in in vitro colonies becomes apparent. These shifting populations of erythroid progenitors with unique globin synthesis programs give rise to the erythroblasts that create the sigmoid pattern of the fetal to adult hemoglobin switch in the developing simian fetus.
Blanche P. Alter, Benjamin T. Jackson, Jeffrey M. Lipton, George J. Piasecki, Pamela L. Jackson, Michele Kudisch, David G. Nathan
Three types of adrenergic receptors, beta, alpha-1, and alpha-2, were identified in human adipocytes, isolated from properitoneal adipose tissue, using both the binding of radioactive ligands and the effects of adrenergic agents on receptor-specific biochemical responses. Adrenergic binding studies showed the following results: [3H]dihydroalprenolol binding (beta adrenergic) Bmax 280 fmol/mg protein, KD 0.38 nM; [3H]para-aminoclonidine binding (alpha-2 adrenergic) Bmax 166 fmol/mg protein, KD 0.49 nM; [3H]WB 4101 binding (alpha-1 adrenergic) Bmax 303 fmol/mg protein, KD 0.86 nM. In adipocytes from subcutaneous adipose tissue, [3H]dihydroergocryptine binding indicated the presence of alpha-2 but not alpha-1 receptors.
Thomas W. Burns, Paul E. Langley, Boyd E. Terry, David B. Bylund, Brian B. Hoffman, Michael D. Tharp, Robert J. Lefkowitz, J. Adolfo García-Saínz, John N. Fain
Female B/W mice spontaneously develop an autoimmune disease that is similar to systemic lupus erythematosus. Antibodies to doublestranded DNA (dsDNA) and antinuclear antibodies develop in aging animals; death from immune complex-mediated glomerulonephritis occurs from 8 to 12 mo of age. It has been reported that prostaglandin (PG)E1 treatment of such mice prolongs survival. In the present study, four groups of female B/W mice were studied beginning at 6-11 wk of age on the following regimens: (a) a synthetic diet that contained 20% safflower oil, (b) a standard laboratory chow diet, (c) a standard diet together with injections of PGE1, and (d) an essential fatty acid-deficient synthetic diet that contained 20% coconut oil. All animals were tested monthly for antinuclear antibodies and anti-dsDNA. Kidney tissue was obtained for light and immunofluorescence microscopy when animals were dying. All disease manifestations were altered strikingly in the essential fatty acid (EFA)-deficient animals. Intermediate benefit was seen in PGE1-treated animals. 7% of the control animals and 18% of safflower oil-fed animals survived to 10 mo. In contrast, the PGE1-treated and EFA-deficient mice had a similar survival rate (78-88%). At age 16 mo, 78% of EFA-deficient mice and 45% of PGE1-treated mice were alive. 25% of the PGE1-treated and 55% of the EFA-deficient animals survived to 20 mo. Serum anti-dsDNA appeared at age 5 mo in safflower oil-fed and control animals, but not until 9 and 12 mo for PGE1-treated and EFA-deficient animals, respectively. All kidneys from 7- to 9-mo-old safflower oil-fed and control animals and the majority of kidneys from PGE1-treated animals were abnormal by light and immunofluorescence microscopy. Kidneys from EFA-deficient animals were essentially normal at 10 mo. At 13 mo, all PGE1-treated animals examined had significant kidney involvement, whereas none of the EFA-deficient animals had glomerulonephritis. These findings demonstrate that an EFA-deficient diet has a beneficial effect on murine lupus erythematosus.
Eric R. Hurd, John M. Johnston, Janice R. Okita, Paul C. MacDonald, Morris Ziff, James N. Gilliam
Vascular refractoriness to the systemic pressor effects of angiotension II (AII) develops normally during human pregnancy. To ascertain if the ewe might provide a suitable animal model to study the mechanisms responsible for this response (unique to pregnancy) we studied this phenomenon in unanesthetized, chronically instrumented nonpregnant and pregnant sheep, 68-143 d gestation. In these studies dose-response curves were established for changes in both mean arterial pressure and uterine blood flow. The pressor response to continuous infusions of AII increases as a function of the dose of AII in both nonpregnant and pregnant animals (P less than 0.001), R = 0.943 and 0.879, respectively. However, the pregnant animals were refractory to the pressor effects of AII, requiring 0.016 microgram of AII/min per kg to elicit a 20 mm HG rise in mean arterial pressure, in contrast to 0.009 for nonpregnant animals. The slope and intercept for the regression lines are different at P less than 0.001. In pregnant animals the dose-response curve for uterine blood flow was also determined. Increases in uterine blood flow were observed at doses of AII less than 0.016 microgram/min per kg, while larger doses resulted in a progressively greater reduction in blood flow. It appears likely that the ewe may serve as an animal model suitable for the further study of the unique pregnancy-modified systemic and uteroplacental vascular responses elicited by AII.
C R Rosenfeld, N F Gant Jr
The effects of dietary cholesterol on plasma lipoproteins and cholesterol homeostasis in blood mononuclear cells have been examined in healthy adults. Addition of 1,500 mg of cholesterol to the daily diet of 37 subjects for 14 d was associated with a wide range of response of plasma total cholesterol concentration (from −6 to +75 mg/dl; mean change, +29 mg/dl; P < 0.001). Increases in plasma cholesterol reflected increased cholesterol concentrations in intermediate density lipoprotein (IDL; 1.006-1.019 g/ml), low density lipoprotein (LDL; 1.019-1.063 g/ml), and the HDL2 subclass (1.063-1.125 g/ml) of high density lipoprotein, which on average accounted for 20, 58, and 22%, respectively, of the total increment. Similar responses occurred in 14 other subjects given 750 mg cholesterol per day for 28 d. Plasma apolipoprotein B concentrations in IDL and LDL also increased.
P. Mistry, N. E. Miller, M. Laker, W. R. Hazzard, B. Lewis
Prostacyclin (PGI2) is a powerful inhibitor of platelet aggregation, but its role in the pathogenesis of arterial thrombosis is uncertain. We have studied the thrombogenic effect of inhibiting PGI2 production by aspirin (ASA) in carotid arteries of rabbits given 0, 3, 10, or 100 mg ASA/kg either 1, 3, 6, or 20 h beforehand. Platelet accumulation onto injured carotid arteries was enhanced with ASA in a dose of 10 mg/kg. A higher dose of ASA (100 mg/kg) had no further effect. The enhanced thrombogenic effect of ASA persisted for at least 20 h and was associated with a decrease in vessel wall PGI2 production. There was a strong inverse correlation (r = 0.55, P less than 0.01) between PGI2 production and platelet accumulation. The findings suggest that the margin of safety in obtaining an antithrombotic effect of ASA and producing a potential thrombotic effect in arteries may not be as large as predicted by studies using cultured endothelial cells or experimentally induced thrombosis in veins.
M R Buchanan, E Dejana, M Gent, J F Mustard, J Hirsch
Responses in the mixed lymphocyte culture (MLC) are traditionally evaluated by measurement of DNA synthesis or blast transformation. However, these events occur too late in the MLC to permit prospective matching for cadaveric renal transplantation. Presentation of allogeneic cells to the T lymphocyte within the MLC results in the emergence of an insulin receptor pharmacokinetically similar to that on other tissues such as fat, liver, and muscle. Intrafamilial MLC were studied by simultaneous assessment of DNA synthesis and insulin receptor binding. In 68 studies from seven families that provide examples of two haplotype identical matches, haplo-identical matches and total haplo mismatches, the presence of an insulin receptor correlated in every case with a positive MLC as estimated by [3H]thymidine incorporation. A quantitative relationship existed between the strength of the MLC and the amount of receptor binding. Based on analysis of cells from several families in which crossover events were known to have occurred, the appearance of an insulin receptor always corresponded with a mismatch at the portion of histocompatibility leukocyte antigen (HLA) chromosome bearing the D region. Finally, it was demonstrated in each of 30 cultures that insulin receptor emergence occurred significantly before detectable DNA synthesis, as early as 24 h after the initiation of the MLC, well within the time-constraint limitations for renal preservation. Appearance of the insulin receptor on activated lymphocytes may be a more rapid measure of mixed lymphocyte responses, and should permit prospective matching for cadaveric renal transplantation.
J H Helderman, T B Strom, M R Garovoy
To investigate mechanisms of pulmonary edema in respiratory failure, we studied unanesthetized sheep with vascular catheters, pleural balloons, and chronic lung lymph fistulas. Animals breathed either a hypercapnic-enriched oxygen (n = 5) or a hypercapnic-hypoxic (n = 5) gas mixture for 2 h. Every 15 min blood gases, pressures, cardiac output, lymph flow (Qlym), plasma and lymph albumin (mol wt, 70,000), IgG (mol wt, 150,000), IgM (mol wt, 900,000), and blood bradykinin concentrations were determined. In both groups, cardiac output and pulmonary arterial pressures increased, whereas left atrial pressures were unchanged. Acidosis alone (arterial pH = 7.16, PaCO2 = 81 mm Hg, PaO2 = 250 mm Hg) resulted in a doubling of lymph flow, a small increase in protein flux, and a decrease in lymph to plasma protein concentration (L/P) ratio for all three proteins. Acidotic-hypoxic animals (arterial pH = 7.16, PaCO2 = 84 mm Hg, PaO2 = 48 mm Hg) tripled Qlym. In these animals the increase in lymphatic flux of albumin, IgG, and IgM was significantly (P < 0.05) greater than that seen in either the acidosis alone group or in animals where left atrial pressures were elevated (n = 5; P < 0.05). Also, their percent increase in flux of the large protein (IgM) was greater than for the small protein (albumin) (P < 0.05). With acidosis alone, only pulmonary arterial bradykinin concentration increased (1.27±0.25 ng/ml SE), whereas acidosis plus hypoxia elevated both pulmonary arterial bradykinin concentrations (4.83±1.14 ng/ml) and aortic bradykinin concentration (2.74±0.78 ng/ml). These studies demonstrate that hypercapnic acidosis stimulates in vivo production of bradykinin. With superimposed hypoxia, and therefore decreased bradykinin degradation, there is an associated sustained rise in Qlym with increased lung permeability to proteins.
Hugh M. O'Brodovich, S. Alex Stalcup, Leila Mei Pang, Joel S. Lipset, Robert B. Mellins
Using a panel of monoclonal antibodies and rabbit heteroantisera, we have studied the cell surface markers of peripheral blood (PB) Sezary cells from six patients with mycosis fungoides or Sezary syndrome, disease grouped within the spectrum of cutaneous T cell lymphomas (CTCL). Furthermore, we have studied two cell lines (Hut 78 and Hut 102) derived from malignant Sezary T cells from CTCL patients. The monoclonal antibody 3A1 defines a major human PB T cell subset (85% of PB T cells) while the antigen defined by the monoclonal antibody 4F2 is present on a subset (70%) of activated PB T cells and on circulating PB monocytes.
Barton F. Haynes, Paul Bunn, Dean Mann, Charles Thomas, George S. Eisenbarth, John Minna, Anthony S. Fauci
Membrane rigidity has been widely accepted as the dominant cause of reduced deformability both of ATP-depleted erythrocytes and erythrocytes containing excess calcium (Ca). However, recent studies have shown normal membrane deformability in ATP-depleted erythrocytes. In addition, Ca accumulation causes massive ion and water loss, and it has been shown that extensive dehydration causes an increase in intracellular viscosity with attendant loss of whole cell deformability. To obtain a detailed understanding of the processes accompanying ATP depletion and/or Ca accumulation that limit cell deformability, we have used a viscodiffractometric method to identify the cellular factors contributing to reduced whole cell deformability.
Margaret R. Clark, Narla Mohandas, Claude Feo, Mark S. Jacobs, Stephen B. Shohet
An analysis of prostaglandin-stimulated adenosine 3',5'-cyclic monophosphate (cyclic AMP) accumulation in cultured human umbilical vein endothelial cells showed prostacyclin (PGI2) to be the most potent agonist followed by prostaglandin (PG)H2, which was more potent than PGE2, while PGD2 was essentially inactive. The endothelial cells studied apparently have a high rate of cyclic AMP phosphodiesterase activity because significant PGI2-mediated increases in cyclic AMP could not be shown in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine (MIX). Endoperoxide PGH2-stimulation of cyclic AMP accumulation was inhibited 75--80% by the prostacyclin synthetase inhibitors 12-hydroperoxyeicosatetraenoic acid or 9,11-azoprosta-5,13-dienoic acid. These data indicate that the PGH2-stimulation is due primarily to conversion to PGI2. The beta-adrenergic agonist L-isoproterenol stimulated cyclic AMP accumulation in the endothelial cells. This accumulation was completely blocked by propranolol. However, stimulation of cyclic AMP accumulation by the beta-adrenergic agent did not equal that induced by PGI2. Furthermore, the PGI2 response could not be blocked by propranolol. Thrombin-stimulated PGI2 biosynthesis was attenuated by PGE1 or isoproterenol in the presence of MIX. MIX alone was less effective than a combination of PGE1 or isoproterenol plus MIX. These data suggest two potential effects of PGI2 biosynthesis by endothelial cells: first, the PGI2 can elevate cyclic AMP in platelets, and second, endothelial cell cyclic AMP can be elevated as well, so that subsequent PGI2 synthesis will be attenuated.
N K Hopkins, R R Gorman
It is well established that viral infections may precipitate or worsen attacks of bronchial asthma. Furthermore, in symptomatic atopic subjects, the local accumulation of basophils and the production of a basophil chemotactic factor have been reported. We have investigated the effect of cell-free supernates from viral stimulated cultures of human mononuclear cells on the in vitro migration of human basophils. Our results show the presence of a factor in these culture supernates that enhances the migration of basophils toward two separate chemoattractants, a peptide from C5 and a lymphokine. The enhancing activity, while affecting basophil migration, did not change the response of monocytes. The enhancing activity resembled viral-induced interferon when (a) pH 2 stability, (b) heat resistance, (c) trypsin sensitivity, and (d) species-specificity were compared. Finally, the enhancing activity for basophil chemotaxis and the interferon titer were highly correlated in preparations with a 10(4)-fold difference in interferon specific activity. Our studies show that viral-induced interferon can augment the in vitro chemotactic response of basophils. Because mediators present in basophils may be involved in the pathogenesis of immediate hypersensitivity, the modulation of basophil movement by interferon suggests a possible mechanism for the association between viral infections and atopic disorders.
M A Lett-Brown, M Aelvoet, J J Hooks, J A Georgiades, D O Thueson, J A Grant
We measured the effects of seven consecutive daily infusions of alpha-ketoisocaproate (the alpha-keto analogue of leucine) or leucine itself on urinary urea and total nitrogen excretion during fasting. Two study protocols were undertaken. In protocol I, subjects underwent three separate 14-d fasts: one during which 34 mmol/d of leucine were infused on days 1--7; a second during which 34 mmol/d of alpha-ketoisocaproate were infused on days 1--7; and a third control fast during which no infusions were given. Infusions of alpha-ketoisocaproate significantly reduced daily urine urea nitrogen excretion compared with both the control fasts and the fasts in which leucine was infused (P less than 0.001). This nitrogen-sparing effect of alpha-ketoisocaproate persisted during days 8--14 even though no further infusions were given. Daily urinary urea nitrogen excretion during fasts when leucine was administered did not differ from values observed during control fasts. In protocol II, subjects were starved on two occasions for 14 d. During one fast, infusions of 11 mmol/d of alpha-ketoisocaproate were given on days 1--7; during the control fast, no infusions were given. Daily urine urea nitrogen excretion was lower (P less than 0.001) on days 1--7 and also on days 8--14 of the fast during which alpha-ketoisocaproate was given. The nitrogen-sparing effect of alpha-ketoisocaproate could not be related to changes in circulating levels of amino acids, ketone bodies, or insulin in either protocol. We conclude that alpha-ketoisocaproate infusions decrease the nitrogen wasting of starvation, whereas leucine, studied under identical conditions, does not.
W E Mitch, M Walser, D G Sapir
Tissue sensitivity to insulin was examined with the euglycemic insulin clamp technique in 17 chronically uremic and 36 control subjects. The plasma insulin concentration was raised by approximately 100 microU/ml and the plasma glucose concentration was maintained at the basal level with a variable glucose infusion. Under these steady-state conditions of euglycemia, the glucose infusion rate is a measure of the amount of glucose taken up by the entire body. In uremic subjects insulin-mediated glucose metabolism was reduced by 47% compared with controls (3.71 +/- 0.20 vs. 7.38 +/- 0.26 mg/kg . min; P less than 0.001). Basal hepatic glucose production (measured with [3H]-3-glucose) was normal in uremic subjects (2.17 +/- 0.04 mg/kg . min) and suppressed normally by 94 +/- 2% following insulin administration. In six uremic and six control subjects, net splanchnic glucose balance was also measured directly by the hepatic venous catheterization technique. In the postabsorptive state splanchnic glucose production was similar in uremics (1.57 +/- 0.03 mg/kg . min) and controls (1.79 +/- 0.20 mg/kg . min). After 90 min of sustained hyperinsulinemia, splanchnic glucose balance reverted to a net uptake which was similar in uremics (0.42 +/- 0.11 mg/kg . min) and controls (0.53 +/- 0.12 mg/kg . min). In contrast, glucose uptake by the leg was reduced by 60% in the uremic group (21 +/- 1 vs. 52 +/- 8 mumol/min . kg of leg wt; P less than 0.005) and this decrease closely paralleled the decrease in total glucose metabolism by the entire body. These results indicate that: (a) suppression of hepatic glucose production by physiologic hyperinsulinemia is not impaired by uremia, (b) insulin-mediated glucose uptake by the liver is normal in uremic subjects, and (c) tissue insensitivity to insulin is the primary cause of insulin resistance in uremia.
R A DeFronzo, A Alvestrand, D Smith, R Hendler, E Hendler, J Wahren
Differences in the growth hormone (GH) responses to primary and to secondary stimulation with triiodothyronine (T3) were studied in rats deprived of thyroid hormone from birth. Neonatal hypothyroidism was induced in pups by feeding pregnant rats an iodine-deficient, propylthiouracil-containing diet. T3 stimulation was carried out in pups by subcutaneous injection of a single dose of 50 μg T3/100 g body wt. Pituitary GH content, rate of GH synthesis in vitro, and GH messenger (m)RNA activity in a cellfree translation system were measured.
Hisao Seo, Caryn Wunderlich, Gilbert Vassart, Samuel Refetoff
Previous studies are in conflict over the effect of infusing mixed fibrinogen-fibrin degradation products on fibrinogen synthesis, as determined by changes in fibrinogen concentration or by incorporation of labeled amino acids into fibrinogen. We have injected purified homologous fragments D1 and E into rats and measured their fibrinogen and albumin synthetic rates by the [14C]carbonate technique, a method that provides quantitative estimates of hepatic secretory protein synthesis. Fibrinogen fractional synthetic rates were increased 2.5 times in animals injected with fragment D1, compared with saline-injected controls. No increase were observed in fragment E-injected animals. Neither fragment produced changes in albumin synthesis. Fragment D increased plasma fibrinogen concentration, but did not raise plasma haptoglobin levels. These results suggest that fragment D is a regulator of fibrinogen synthesis.
J J Franks, R E Kirsch, L O Frith, L R Purves, W T Franks, J A Franks, P Mason, S J Saunders
C4 allotyping 13 homozygous C2-deficient individuals demonstrated 23 of 25 haplotypes to be of the relatively rare type C4A4 B2. This is of the same magnitude as the association of C2Q0 with HLA-DW2/DR2.
Z L Awdeh, D D Raum, D Glass, V Agnello, P H Schur, R B Johnston Jr, E W Gelfand, M Ballow, E Yunis, C A Alper
We have developed a technique for growing endothelial monolayers on micropore filters. These monolayers demonstrate confluence by phase and electron microscopy and provide a functional barrier to passage of radiolabeled albumin. Neutrophils readily penetrate the monolayer in response to chemotaxin, whereas there is little movement in the absence of chemotaxin. This system offers unique advantages over available chemotaxis assays and may have wider applications in the study of endothelial function.
R F Taylor, T H Price, S M Schwartz, D C Dale