The molecular pathology of the porphobilinogen (PBG)-deaminase deficiency in heterozygotes for acute intermittent porphyria (AIP) was investigated by means of biochemical and immunologic techniques. The stable enzyme-substrate intermediates (A, B, C, D, and E) of PBG-deaminase were separated by anion-exchange chromatography of erythrocyte lysates from heterozygotes for AIP and normal individuals. In normal lysates, the intermediates eluted in a characteristic pattern with decreasing amounts of activity (A > B > C > D > E), the combined A and B intermediates representing >75% of total recovered activity. In contrast, two different profiles were observed in lysates from heterozygotes for AIP. In most heterozygotes, the elution profile was similar to that of normal individuals, but each intermediate was reduced ∼50%. A second profile in which the C intermediate had disproportionately higher activity than the A or B intermediates was observed in asymptomatic heterozygotes with high urinary levels of PBG (>5 μg/ml) as well as in heterozygotes during acute attacks. These findings suggested that the C intermediate (the dipyrrole-enzyme intermediate) may be rate limiting in the stepwise conversion of the monopyrrole, PBG, to the linear tetrapyrrole, hydroxymethylbilane. To investigate further the nature of the enzymatic defect in AIP, sensitive immunotitration and immunoelectrophoretic assays were developed with the aid of a rabbit anti-human PBG-deaminase IgG preparation produced against the homogeneous enzyme. Equal amounts of erythrocyte lysate activity from 32 heterozygotes for AIP from 22 unrelated families and 35 normal individuals were immunoelectrophoresed. There were no detectable differences in the amounts of cross-reactive immunologic material (CRIM) in lysates from the normal individuals and 25 heterozygotes from 21 of the 22 unrelated families with AIP. In contrast, when equal enzymatic activities were coimmunoelectrophoresed, all seven heterozygotes from one family had ∼ 1.6 times the amount of CRIM compared with that detected in normal lysates. Consistent with these findings, immunotitration studies also demonstrated similar quantities of noncatalytic CRIM in lysates from this AIP family. When equal activities of the individual A, B, C, and D enzyme-substrate intermediates from normal and CRIM-positive erythrocytes were immunoelectrophoresed, increased amounts of immunoreactive protein were observed for each intermediate, B > A ≃ C ≃ D, from the CRIM-positive AIP variants. On the basis of these findings, it is hypothesized that the enzymatic defect in the CRIM-positive AIP family resulted from a mutation in the structural gene for PBG-deaminase which altered the catalytic as well as a substrate binding site. These studies of the enzymatic defect provide the first demonstration of genetic heterogeneity in AIP.
Peter M. Anderson, Raman M. Reddy, Karl E. Anderson, Robert J. Desnick
Inhibition of complement-mediated granulocyte aggregation has recently been proposed as a mechanism of action of high-dose corticosteroids in shock states. Postulating that such inhibition might be effected through alteration of receptors function, we examined the effect of methylprednisolone (MP), hydrocortisone (HC), and dexamethasone (DEX) on the extent and kinetics of binding of the synthetic chemotaxin f-methionine-leucine-phenylalanine (FMLP) to its specific receptor on the granulocyte surface. Dose-dependent inhibition of binding was observed at corticosteroid concentrations paralleling plasma levels achieved with 30 mg/kg intravenous bolus therapy; the order of potency was MP greater than HC greater than DEX. Receptor number was unaffected by steroid exposure, but the steroids effected a decrease in association rate constant for the FMLP-receptor interaction (35% of N for 0.2 mg/ml MP), leading to decreased receptor-ligand affinity. Dissociation kinetics, as examined by cold-chase experiments, were unaltered by the corticosteroids. Furthermore, in addition to the inhibition of aggregation previously reported, aggregated granulocytes were found to disaggregate upon addition of corticosteroids; the order of potency was again MP greater than HC greater than DEX, with an MP concentration of approximately 2-3 mg/ml required to effect complete disaggregation. We conclude that corticosteroids can displace FMLP from the granulocyte surface by slowing association while allowing dissociation to proceed; altered kinetics of receptor-FMLP interaction may explain both the inhibition of granulocyte aggregation and granulocyte disaggregation. If these observations also hold for physiologic stimuli (such as C5adesarginine, which behaves similarly with respect to aggregation, inhibition, and disaggregation), such kinetic changes may be important in the clinical effects of very high-dose corticosteroids such as are administered in shock.
K M Skubitz, P R Craddock, D E Hammerschmidt, J T August
To assess the adequacy of oxygen availability and utilization within the cerebral cortex in vivo, we have measured the partial pressure of oxygen in tissue (PtO2), as well as the reduction oxidation state of cytochrome c oxidase (cyt aa3) during shock induced by slow or rapid hemorrhage in anesthetized cats. PtO2 was measured with pyrenebutyric acid-generated fluorescence in cerebral cortical cells. Cyt aa3 redox state was measured by the absorption of monochromatic light at 605 nm absorption peak of the enzyme reflected from the same cortical field. The PtO2 remained within the normal range until either 30 +/- 1.5 ml blood/kg was removed or the mean arterial pressure fell by 70 +/- 5% of base line. Beyond either point, the PtO2 fell rapidly to a low value approximating zero. By contrast, the reduction of cyt aa3 began early when as little as 5 ml blood/kg was removed. Thereafter, the shift toward reduction was progressive and continuous with a slow rate at first and a rapid rate later. This accelerated rate of cyt aa3 reduction preceded the rapid fall of PtO2. We concluded that, under these experimental conditions, cyt aa3 reduction is a much earlier and more sensitive indicator of perturbed intracellular aerobic metabolism due to hemorrhage that is PtO2.
K Kariman, F G Hempel, F F Jöbsis, S R Burns, H A Saltzman
The diminished cardiac output response to exercise with advancing age may be attributable to intrinsic inability of the old ventricle to respond appropriately and/or to an additional loading imposed upon the ventricle by the aged vascular system. The steady (resistance) and pulsatile (characteristic impedance) load components together comprise the vascular load faced by the ejecting ventricle. To study the effect of exercise on both vascular components of load, the aortic input impedance was measured in chronically instrumented young and old beagle dogs during graded treadmill exercise before and after beta blockade. Ascending aortic flow was measured by a cuff electromagnetic flow probe, and pressure was measured by a high-fidelity semiconductor transducer. At low levels of exercise the old animals demonstrated a striking 20% increase in characteristic impedance and a 28% decrease in peripheral resistance with no increase in stroke volume. This vascular loading and limitation in stroke volume persisted across the higher exercise levels. In contrast, the young group demonstrated no increase in characteristic impedence, a progressive decrease in peripheral resistance, and a progressive increase in stroke volume across the same exercise levels. These age differences in vascular response and ventricular output were abolished by beta blockade. The groups did not demonstrate a difference in heart rate response, but the young had a greater increase in external left ventricular power than the old across exercise. These data demonstrated a profound difference in the response of young and old vasculature to exercise. At low and intermediate exercise levels the pulsatile vascular load appeared to be a major factor in the limitation of stroke volume in old dogs. At high levels of exercise, the limited exercise response in the old dog may be caused in part by a diminished inotropic responsiveness as well as by the vascular loading.
F C Yin, M L Weisfeldt, W R Milnor
Varicocele had been repeatedly implicated as a cause of infertility in selected men, although neither a causal relationship nor a mechanism has been documented. The purpose of this investigation was to create a varicocele model in animals and to study the subsequent alterations in testicular physiology. Secondary dilatation of the left internal spermatic vein was achieved either by partial ligation of the left renal vein in rats and dogs or by surgical destruction of the valve of the left testicular vein in a second group of dogs. 1 mo after partial ligation in the rats and 3 mo after partial ligation or valve destruction in the dogs, testicular blood flow was measured using Strontium 85 (SR-85)-labeled microspheres (15 +/- 1.1 micrometer). Intratesticular temperature was measured with a Bailey needle probe thermometer and biopsies were obtained for histologic sections. Mean testicular blood flow in milliliters per minute per 100 g was significantly greater in the partially ligated rats; right testis control 26 +/- 2, left testis control 24 +/- 2 compared to right testis experimental 35 +/- 3, left testis experimental 35 +/- 4 (p less than 0.02). Dogs undergoing either partial vein ligation or valve destruction showed similar increases in mean testicular blood flow; right testis control 8 +/- 1, left testis control 8 +/- 1 vs. right testis experimental 16 +/- 3, left testis experimental 18 +/- 4 (p less than 0.01). The mean difference between intratesticular and intraperitoneal temperature in control rats was significantly higher (4.02 +/- 0.25 degrees C right testis, 3.77 +/- 0.14 degrees C left testis), than rats who underwent partial vein ligation (right testis 2.14 +/- 0.09 degrees C, left testis 2.34 +/- 0.12 degrees C) (p less than 0.001). Control dogs also had a significantly higher mean difference between intratesticular and rectal temperatures; (right testis control 3.61 +/- 0.42 degrees C, left testis control 3.60 +/- 0.40 degrees C) than the partially ligated or valve destruction dogs (right testis 2.31 +/- 0.17 degrees C, left testis 2.67 +/- 0.32 degrees C) (p less than 0.05). In addition, histologic evaluation revealed abnormalities in spermatogenesis in some of the animals. Thus, venous dilatation secondary to partial vein ligation or testicular vein valve obliteration is followed by large bilateral increases in testicular blood flow in these two species. A consequence of this increased flow is an elevation in bilateral testicular temperature, which it is postulated, leads to impaired spermatogenesis in some of the animals. In selected men varicocele may impair spermatogenesis by a similar physiologic mechanism.
D C Saypol, S S Howards, T T Turner, E D Miller Jr
An enzyme-linked differential antibody immunosorbent assay has been developed for the quantification of alpha2-plasmin inhibitor-plasmin and alpha2-macroglobulin-plasmin complexes. In this method the inhibitor-plasmin complex is bound to a surface by an inhibitor-specific antibody, and the plasmin bound to the inhibitor is quantified by a second antibody, rabbit antiplasminogen F(ab')2, labeled with alkaline phosphatase. The hydrolysis of p-nitrophenyl phosphate by the alkaline phosphatase is expressed in femtomoles of plasminogen per milliliter, by reference to a standard plasminogen curve. Inhibitor-enzyme complexes were generated in plasma by the addition of plasmin or of urokinase. The concentration of plasmin added was well below the plasma concentration of alpha2-plasmin inhibitor (1 microM) or of alpha2-macroglobulin (3.5 microM), so that neither inhibitor would be fully saturated with enzyme. Under these conditions increasing amounts of plasmin generated an increase in both alpha2-plasmin inhibitor-plasmin and alpha2-macroglobulin-plasmin complexes. Varying amounts of plasmin were incubated with each of the purified inhibitors in the concentration found in plasma, and the complexes. Varying amounts of plasmin were incubated with each of the purified inhibitors in the concentration found in plasma, and the complexes that formed were quantified by immunoassay. These studies made it possible to quantify the distribution of plasmin between the two inhibitors in plasmin or urokinase-treated plasma. In plasmin-treated plasma, 10% or less of the plasmin bound to both inhibitors was in complex with alpha2-macroglobulin. In contrast, between 19 and 51% of the plasmin generated in urokinase-activated plasma was bound to alpha2-macroglobulin. Thus, major changes in the distribution of plasma were observed, according to whether plasmin was added to plasma or whether plasminogen was activated endogenously. The pattern of inhibitor plasmin complexes generated in vivo by the therapeutic infusion of urokinase was similar to that found for urokinase-activated plasma. 23 normal individuals had low levels of alpha2-plasmin inhibitor-plasmin complexes, whereas six patients with laboratory evidence for disseminated intravascular coagulation demonstrated a 16- to 35-fold increase in he concentration of these complexes. These data indicated that a useful new probe for the study of the fibrinolytic enzyme system had been developed.
P C Harpel
Colony-stimulating activities (CSA) are potent granulopoietic stimulators in vitro. Using clonogenic assay techniques, we analyzed the degree to which mononuclear phagocytes and T lymphocytes cooperate in the positive (production/release of CSA) and feedback (inhibition of CSA production/release) regulation of granulopoiesis. We measured the effect of lactoferrin (a putative feedback regulator of CSA production) on CSA provision in three separate assay systems wherein granulocyte colony growth of marrow cells from 22 normal volunteers was stimulated by (a) endogenous CSA-producing cells in the marrow cells suspension, (b) autologous peripheral blood leukocytes in feeder layers, and (c) medium conditioned by peripheral blood leukocytes. The CSA-producing cell populations in each assay were varied by using cell separation techniques and exposure of isolated T lymphocytes to methylprednisolone or to monoclonal antibodies to surface antigens and complement. We noted that net CSA production increased more than twofold when a small number of unstimulated T lymphocytes were added to monocyte cultures. Lactoferrin's inhibitory effect was also T lymphocyte dependent. The T lymphocytes that interact with monocytes and lactoferrin to inhibit CSA production are similar to those that augment CSA production because their activities are neither genetically restricted not glucocorticoid sensitive, and both populations express HLA-DR (Ia-like) and T3 antigens but not T4 or T8 antigens. These findings are consistent with results of our studies on the mechanism of lactoferrin's inhibitory effect with indicate that mononuclear phagocytes produce both CSA and soluble factors that stimulate T lymphocytes to produce CSA, and that lactoferrin does not suppress monocyte CSA production, but does completely suppress production or release by monocytes of those factors that stimulate T lymphocytes to produce CSA. We conclude that mononuclear phagocytes and a subset of T lymphocytes exhibit important complex interactions in the regulation of granulopoiesis.
G C Bagby Jr, V D Rigas, R M Bennett, A A Vandenbark, H S Garewal
Acetylcholine produces venoconstriction of isolated vein strip preparations. However, the effect of acetylcholine on overall vascular capacity is not known. To investigate this effect and to elucidate the mechanisms involved, 38 anesthetized dogs were placed on total cardiopulmonary bypass, splenectomized, and given intraarterial infusions of acetylcholine. Almost all of the effect on vascular volume was found to be in the splanchnic circulation, because in four eviscerated animals there was no significant change in capacity. In the animals in which the mesenteric arteries were cannulated to provide constant inflow, and the hepatic vein was cannulated to measure splanchnic venus outflow, acetylcholine infusion for 5 and 21 min increased splanchnic vascular capacity in all animals by 107 +/- 28 (SEM)ml (p less than 0.01) and 291 +/- 132 ml (p less than 0.05), respectively. This increase in splanchnic vascular volume was associated with a rapid and sustained increase in transhepatic resistance to portal blood flow for the duration of the infusions (p less than 0.01). In the animals in which the portal vein was vented proximal to the liver, no significant volume change occurred in the splanchnic vasculature with acetylcholine infusion. Increasing hepatic venous pressure to elevate portal venous pressure to the same level as that achieved with acetylcholine resulted in a similar increase in splanchnic vascular volume. Atropine, but not adrenergic blockade, blocked the acetylcholine-induced volume retention, indicating that the effect of acetylcholine was direct. Substantial volume retention was also achieved by stimulation of the distal ends of the sectioned cervical vagi. Thus, acetylcholine administration directly increases transhepatic resistance and is associated with a pooling of volume in the splanchnic vasculature that would, in the intact animal, result in a decrease in venous return to the heart.
E W Supple, W J Powell Jr
The current study was conducted to determine if physical activity and/or weight control could influence the age-related decrease in beta cell insulin response noted in earlier studies. As such, virgin, male Sprague-Dawley rats were maintained in our facility for 1 yr on three differential experimental programs; in the first group, control rats lived under standard laboratory conditions; the second group of rats ran several miles a day in exercise wheels, and the third group was given a calorie-restricted diet designed to keep the rats weight-matched with the exercising rats. The results showed that the 12-mo-old sedentary control rats weighed an average of 800g. From the time these rats were 4 mo old, they were significantly hyperinsulinemic, with mean (+/- SEM) serum insulin levels of 55 +/- 6 microU/ml. Morphological studies on the pancreas of these rats at the end of the year revealed enlarged, multilobulated, fibrotic islets. After collagenase digestion, the most normal-appearing islets from the 12-mo-old controls were used for insulin secretion studies, these islets showed significantly reduced glucose-induced insulin release (0.83 microU insulin/min per volume islet) compared with islets from young rats (1.80 microU insulin/min per volume islet). In contrast, 12-mo-old exercised or calorie-restricted rats weighed approximately 500 g and did not show the changes in serum insulin levels or pancreas pathology exhibited by the sedentary control animals. However, islets from the calorie-restricted group functioned in vitro no better than islets from he sedentary control group. Islets from the exercised rats were somewhat improved in this regard. In summary, we believe exercise and weight control diminishes the animals' need for insulin-resulting in youthful-appearing islets after a year's time. However, these regimes do not appear able to correct the beta cell decline in function previously described.
E P Reaven, G M Reaven
The mechanism(s) by which the oral sulfonylurea, tolazamide, exerts its extrapancreatic hypoglycemic effects was studied using rat epididymal adipose tissue maintained 20-44 h in the presence or absence of the drug. Insulin binding, hexose transport and glucose metabolism were compared in adipocytes isolated from the cultured tissue. In contrast to earlier reports that suggested that sulfonylureas alter the binding of insulin, neither receptor number nor affinity were changed by tolazamide treatment. The uptake of the glucose analogs 2-deoxyglucose and 3-0-methylglucose in the absence of insulin (i.e., basal) was also unchanged. However, exposure to tolazamide resulted in a potentiation of the stimulatory effects of insulin by approximately 30% at each hormone concentration assayed (0.4-40 ng/ml). This potentiation was dependent on the tolazamide concentration (0.003-0.30 mg/ml), with a maximal effect observed at therapeutic levels. A tolazamide analog hypoglycemic activity in vivo was found not to enhance either basal or insulin-stimulated uptake in vitro. Conversion of 0.1-5.0 mM glucose to CO2 and total lipids in the presence of insulin was also potentiated by tolazamide treatment. The inability of the drug to directly stimulate basal glucose uptake was paralleled by its lack of effect on glucose metabolism. At 50 mM glucose, where transport is no longer rate-limiting, tolazamide did not potentiate metabolism in the absence or the presence of insulin. These studies demonstrate that tolazamide in vitro alters postreceptor insulin action without influencing the receptor, and suggests insulin-stimulated hexose transport as the cellular process responsible for the hypoglycemic effect of sulfonyureas in adipose tissue.
B L Maloff, D H Lockwood
To clarify conflicting reports concerning the effects of ischemia on left ventricular chamber stiffness, we compared the effects of hypoxia at constant coronary perfusion with those of global ischemia on left ventricular diastolic chamber stiffness using isolated, perfused rabbit hearts in which the left ventricle was contracting isovolumically. Since chamber volume was held constant, increases in left ventricular end diastolic pressure (LVEDP) reflected increases in chamber stiffness. At a control coronary flow rate (30 ml/min), 2 min of hypoxia and pacing tachycardia (4.0 Hz) produced major increases in postpacing LVEDP (10±1 to 24±3 mm Hg, P < 0.01) and the relaxation time constant, T, (40±4 to 224±37 ms, P < 0.001), while percent lactate extraction ratio became negative (+ 18±2 to −48±15%, P < 0.001). Coronary perfusion pressure decreased (72±5 to 52±3 mm Hg, P < 0.01), and since coronary flow was held constant, the fall in coronary perfusion pressure reflected coronary dilation and a decrease in coronary vascular resistance. Following an average of 71±6s reoxygenation and initial heart rate (2.0 Hz), LVEDP and relaxation time constant T returned to control. Hypoxia alone (without pacing tachycardia) produced similar although less marked changes (LVEDP, 10±1 to 20±3 mm Hg; and T, 32±3 to 119±22 ms; P < 0.01 for both) and there was a strong correlation between LVEDP and T (r = 0.82, P < 0.001).
Takashi Serizawa, W. Mark Vogel, Carl S. Apstein, William Grossman
The present experiments were designed to explore the mechanism whereby 3,5,3'-triiodothyronine (T3) stimulates the uptake of 2-deoxy-D-glucose (2-DG) in rat thymocytes in vitro. Addition of T3 evoked a transient, dose-related increase in cellular cyclic (c) AMP concentrations, evident within 5 min. followed soon by an increase in 2-DG uptake. The effects of T3 on both cAMP concentration and 2-DG uptake were dependent upon the presence of extracellular calcium. Epinephrine also induced a sequential increase in thymocyte cAMP concentration and 2-DG uptake. These responses were more prompt than those to T3, but were calcium independent. As with their combined effects on 2-DG uptake, T3 and epinephrine produced synergistic or additive effects on cellular cAMP concentration. Dibutyryl cAMP also stimulated 2-DG uptake, an effect that was more prompt than that of epinephrine, and like that of epinephrine, was calcium independent. Prior or simultaneous addition of L-alprenolol (10 microM), which, we have previously shown, blocks the effect of both T3 and epinephrine on 2-DG uptake, also blocked the increase in thymocyte cAMP concentration induced by these agents. In contrast, L-alprenolol failed to block the increase in 2-DG uptake produced by dibutyryl cAMP. On the basis of these observations we suggest that T3 increases 2-DC uptake in the rat thymocyte by increasing the cellular concentration of cAMP, which then acts to enhance sugar transport. The increase in 2-DC uptake induced by epinephrine is also mediated by an increase in cAMP concentration. The greater response of cellular cAMP concentration to T3 and epinephrine when added together than to either agent added alone may explain their synergistic action to increase 2-DG uptake. We suggest that these actions of T3 and epinephrine are both initiated at the level of the plasma membrane.
J Segal, S H Ingbar
From a single cell fusion, five stable hybridomas secreting antiapolipoprotein E (apo E) were obtained. The immunoglobulin (Ig)G subclasses containing the respective monoclonal antibodies were isolated and were used as the antibody component in a solid-phase radioimmunoassay. The binding of 125I-apo E to the insolubilized antibody was inhibited by unlabeled apo E but not by unlabeled apoproteins A-I, A-II, C-II, and C-III, or by low density lipoprotein immunodepleted of endogenous apo E. Competition curves were obtained with lipoprotein subfractions that had the same shape as those obtained with purified apo E. Apo E levels in normal and hyperlipidemic plasma were well correlated when measured by the five monoclonal antibodies and polyclonal anti-apo E, although differences in absolute values were observed. In normal subjects 34, 10, 20, and 36% of apo E was recovered in the very low density lipoprotein, low density lipoprotein, high density lipoprotein, and the d greater than 1.21-gl/ml fractions, respectively, whereas these values were 34, 7, 12, and 47%, respectively, in type III patients. All antibodies indicated the same subfraction distribution of apo E. The monoclonal antibodies reacted with all of the isomorphs of apo E. One of the antibodies could be clearly distinguished by its reactivity with chemically modified very low density lipoprotein.
R W Milne, P Douste-Blazy, Y L Marcel, L Retegui
Inheritance of the gene for betaE-globin is associated with hypochromia and microcytosis, reminiscent of typical heterozygous beta-thalassemia. Patients with hemoglobin (Hb)E-beta-thalassemia exhibit clinical phenotypes of severe beta-thalassemia, a circumstance not encountered in other compound heterozygous states for structural beta-chain mutations and beta-thalassemia. We have analyzed the kinetics of globin synthesis and the levels of globin messenger (m) RNA accumulation in patients with Hb E-beta-thalassemia and Hb E trait. The initial rate of beta-globin synthesis (betaE/alpha=0.20-0.34) was less than expected on the basis of gene dosage, or comparable studies of other compound heterozygous states for beta-thalassemia and structurally abnormal beta-chains. betaE-globin synthesis was not only reduced during short-term incubations (1-5 min), but also remained relatively unchanged during long-term pulse or chase incubations up to 5h. Analysis of globin mRNA by cell-free translation and molecular hybridization confirmed that the unexpectedly low levels of betaE-globin synthesis were associated with comparable reduction in the levels of beta-globin mRNA. In Hb E-beta-thalassemia the betaA + betaE (alpha globin nRNA ratio observed were substantially lower than those obtained from reticulocytes of patients with heterozygous beta-thalassemia, or Hb S-betaO-thalassemia, while in Hb E trait, the betaA + betaE/alpha mRNA ratio was in the ranged observed for beta-thalassemia trait. The betaE-globin gene specifies reduced accumulation of betaE-globin mRNA, a property characteristic of other forms of beta-thalassemia. The beta-thalassemia phenotype associated with inheritance of Hb E is thus determined at the level of beta-globin mRNA metabolism.
E J Benz Jr, B W Berman, B L Tonkonow, E Coupal, T Coates, L A Boxer, A Altman, J G Adams 3rd
We have previously reported that mannitol strikingly increases blood flow to rat kidneys hypoperfused at 35-40mm Hg. This vasodilator effect is not due to volume expansion or alterations in plasma osmolality. We have tested the hypothesis that the vasodilatory effect of mannitol in the ischemic rat kidney is mediated by one of the vasoactive renal hormone systems: renin-angiotension, kallikrein-kinin, or prostaglandins. Rats were infused with 5% mannitol in 0.9% saline to 3-5% of body weight. In agreement with our previous studies, RBF increased 1.3 +/- 0.1 ml/min despite maintenance of perfusion pressure at 35-40 mm Hg. The cyclooxygenase inhibitors, meclofenamate and indomethacin had no effect on renal blood flow (RBF) in hypoperfused kidneys. However, in rats pretreated with these inhibitors, expansion with mannitol increased RBF by only 0.37 +/- 0.02 ml/min, 28% of the response in the untreated group (p less than 0.001). Infusion of prostacyclin (PGI2) into the renal artery during reduced perfusion resulted in an increase in RBF of 1.0 +/- 0.1 ml/min. Subsequent expansion with mannitol increased RBF by only 0.5 +/- 0.1 ml/min more, less than one-half of the effect of mannitol in a concurrent group of rats not treated with PGI2. Unlike PGI2 prostaglandin E2 had only a minimal vasodilator effect during hyperperfusion. Imidazole, an inhibitor of thromboxane synthesis, did not alter RBF or renal vascular resistance during hypoperfusion. Treatment of rats during hypoperfusion. with the angiotensin-converting enzyme (kininase II) inhibitor teprotide increased RBF by 1.1 +/- 0.3 ml/min. However, teprotide did not alter the vascular response to mannitol: RBF increased 1.2 +/- 0.1 ml/min more when mannitol was infused into teprotide-treated rats. The renal vascular response to mannitol was not altered by treatment with aprotinin, an inhibitor of the kallikrein-kinin system. Aprotinin was ineffective whether given before or after the vascular response to mannitol was established. We conclude that the vasodilator response to mannitol in the ischemic rat kidney is mediated in large part by increased prostaglandin (PGI2) activity. The failure of converting enzyme inhibition and aprotinin to block the vasodilator response to mannitol is evidence against a role for the renin-angiotension or kallikreinkinin systems in mediating the vasodilator response.
P A Johnston, D B Bernard, N S Perrin, N G Levinsky
Radiolabeled photobilirubins, prepared in vitro by anaerobic illumination of [34C]bilirubin, were injected intravenously into homozygous jaundiced Gunn rats with an external bile fistula. With the animals kept in darkness, the labeled photobilirubins were excreted rapidly in bile. Photobilirubins IA and IB were excreted primarily as unconjugated bilirubin, whereas photobilirubin II was excreted primarily as photobilirubin II and not converted into bilirubin. Bile of Gunn rats given no exogenous pigments, but undergoing phototherapy, contained a large proportion of photobilirubin II and, if collected in liquid nitrogen, traces of photobilirubins I; neither was found in bile when these rats were kept in the dark. Because there is prior evidence that these rats were kept in the dark. Because there is prior evidence that these photobilirubins are isomers of bilirubin, these experiments indicate that the major mechanism of phototherapy is photoisomerization of bilirubin. Photobilirubin II is the unidentified major photoderivative described previously, whereas formation of photobilirubins IA and IB, and their reversion to bilirubin-IXalpha, account for the remarkably increased output of the parent pigment during phototherapy.
M S Stoll, E A Zenone, J D Ostrow
Studies were conducted to determine whether the direction of hepatic carbohydrate and lipid metabolism in the rat could be switched simultaneously from a "fasted" to a "fed" profile in vitro. When incubated for 2 h under appropriate conditions hepatocytes from fasted animals could be induced to synthesize glycogen at in vivo rates. There was concomitant marked elevation of the tissue malonyl-coenzyme A level, acceleration of fatty acid synthesis, and suppression of fatty acid oxidation and ketogenesis. In agreement with reports from some laboratories, but contrary to popular belief, glucose was not taken up efficiently by the cells and was thus a poor substrate for eigher glycogen synthesis or lipogenesis. The best precursor for glycogen formation was fructose, whereas lactate (pyruvate) was most efficient in lipogenesis. In both case the addition of glucose to the gluconeogenic substrates was stimulatory, the highest rates being obtained with the further inclusion of glutamine. Insulin was neither necessary for, nor did it stimulate, glycogen deposition or fatty acid synthesis under favorable substrate conditions. Glucagon at physiological concentrations inhibited both glycogen formation and fatty acid synthesis. Insulin readily reversed the effects of glucagon in the submaximal range of its concentration curve. The following conclusions were drawn. First, the fasted-to-fed transition of hepatic carbohydrate and lipid metabolism can be accomplished in vitro over a time frame similar to that operative in vivo. Second, reversal appears to be a substrate-driven phenomenon, in that insulin is not required. Third, unless an unidentified factor (present in protal blood during feeding) facilitates the uptake of glucose by liver it seems unlikely that glucose is the immediate precursor for liver glycogen or fat synthesis in vivo. A likely candidate for the primary substrate in both processes is lactate, which is rapidly formed from glucose by the small intestine and peripheral tissues. Fructose and amino acids may also contribute. Fourth, the requirement for insulin in the reversal of the fasting state of liver metabolism in vivo can best be explained by its ability to offset the catabolic actions of glucagon.
M E Boyd, E B Albright, D W Foster, J D McGarry
During tuberculosis, exposure of monocytes to circulating factors may induce the suppressor activity observed in some anergic patients. To explore this possibility, we examined the effects of plasma pooled from 28 untreated tuberculosis (TB) patients and the mycobacterial cell wall polysaccharide D-arabino-D-galactan (AG) on the in vitro function of peripheral blood mononuclear cells (PBMC) from healthy donors. In the [3H] thymidine incorporation assay, stimulated responses of PBMC incubated in culture medium supplemented with TB plasma or co-cultured with 3.0 microgram/ml AG were depressed significantly when compared with control responses. Cytotoxicity and altered kinetics of stimulated DNA synthesis did not contribute to the observed suppression. TB plasma and AG-induced suppression of the PBMC response to purified protein derivative was monocyte dependent and indomethacin reversible. In addition, TB plasma and AG directly inhibited the phytohemagglutinin-stimulated responses of T lymphocytes. In a quantitative assay of monocyte attachment to plastic, both TB plasma and AG significantly increased monocyte adherence from basal levels. These effects on monocyte adherence were reversed with indomethacin or antibody to mycobacterial polysaccharide. In addition, TB plasma passed over an immunoabsorbent column of Sepharose-linked antibody to mycobacterial polysaccharide was depleted of the suppressive and monocyte-adherence augmenting factors. 3.0 microgram/ml AG stimulated a fivefold increase in prostaglandin E2 production by cultured mononuclear cells. Our data suggest that AG circulating alone or bound in immune complexes may account for the observed effects of TB plasma. Similar in vivo exposure may contribute to the cell-mediated suppression of lymphocyte responses in tuberculosis.
M E Kleinhenz, J J Ellner, P J Spagnuolo, T M Daniel
We studied release of angiotensin-converting enzyme (ACE) by the lung after acute injury associated with an increase in pulmonary vascular permeability. In eight adult sheep with chronic lung lymph fistulas, we measured lymph flow (QL), and both ACE activity and total protein content in lymph and plasma under base-line conditions and during 24 h after an infusion of live pseudomonas organism. Under base-line conditions, ACE activity in plasma was 4.93 +/- 0.43 U/ml (mean +/- SEM). The [lymph]/[plasma] ([L]/[P]) ratio for ACE was 0.93 +/- 0.18, compared with a ratio of 0.79 +/- 0.08 for albumin (mean +/- SD). We estimated the molecular weight of ovine ACE to be 145,000 by gel chromatography. Predicted [L]/[P] ratio for a molecule this size is 0.51. Thus, a substantial fraction of ACE activity detected lung lymph under base-line conditions (11.1 +/- 6.2 U/h; mean +/- SD) originated in the lung, and did not diffuse passively from plasma. After pseudomonas infusion, endothelial injury was demonstrated by a rise in pulmonary vascular clearance for total protein (Crp = QL X [L]/[P]). Crp = 3.1 +/- 0.6 ml/h before pseudomonas bacteremia, and rose to 6.7 +/- 1.2 ml/h by 2 h after onset of the infusion (means +/- SEM, p less than 0.05). Crp remained significantly elevated for at least 10 h after the infusion. Release of ACE into lung lymph doubled after acute lung injury and equaled 22.3 +/- 13.8 U/h at 4 h after onset of the infusion. ACE secretion into lung lymph had returned to baseline levels by 24 h after bacteremia. We did not observe a significant rise in plasma ACE activity after acute lung injury. Pseudomonas bacteremia in sheep results in acute, reversible lung injury associated with increased pulmonary vascular permeability, and increased release of ACE by the lung. Failure to detect a rise in plasma ACE content might result from dilution in the large vascular pool or rapid catabolism of the enzyme at some site distant from the lung.
A B Gorin, G Hasagawa, M Hollinger, J Sperry, J Zuckerman
Cultured mononuclear cells (MNC) from individuals homozygous or heterozygous for the defective gene causing the inherited disease cystic fibrosis (CF) synthesize three unusual "mediators" termed ciliary dyskinesia substances (CDS), which markedly affect tracheal mucociliary systems in vitro. MNC cultures from normal healthy controls do not accumulate any CDS, whereas MNC cultures from non-CF patients controls with pulmonary disease synthesized at least one CDS. The possible involvement of the CDS in pulmonary disease is being investigated. In this study, we sought to determine whether the CDS could be chemoattractants for polymorphonuclear neutrophils (PMN), since they have characteristics in common with known chemoattractants generated by alveolar macrophages. Our analyses of crude MNC culture supernates indicated that cultures from both CF genotypes accumulate significantly higher levels of PMN chemoattractants than do analogous cultures from normal healthy controls. CF homozygote MNC also generated more activity than MNC from patient controls with chronic pulmonary disease. Fractionation of MNC culture supernates by gel permeation chromatography and characterization of active fractions demonstrated six distinct PMN chemoattractants in cultures from CF genotypes; five were also present in patient control and four in normal healthy control cultures. The excessive chemoattractant activity in MNC cultures from CF genotypes and patient controls was due to several different substances produced by monocytes: (a) two components of 1,000-3,500 mol wt. (b) two fragments of C5, and (c) a fragment of C3. One C5 fragment had ciliary dyskinesia activity, the other did not. The C3 fragment chemoattractant also had ciliary dyskinesia activity and was not found in MNC cultures from patient controls. A third CDS, Which is CF-specific (5,000 mol wt), was neither chemotactic not chemokinetic and did not inhibit random PMN migration; however, fractions containing this CF-specific CDS completely inhibited PMN chemotaxis in response to three different chemoattractants. We conclude that all of the CDS can potentially play a role in the pathophysiology of lung disease, as judged by their effects on PMN movement in vitro.
G B Wilson, H H Fudenberg, M T Parise, E Floyd
The metabolic clearance rate (MCR) and renal clearance rate (RCR) of human chorionic gonadotropin (hCG) were measured in healthy young men and women using techniques of continuous intravenous infusion and rapid intravenous injection of unlabeled, highly purified hCG. Seven subjects received 4 d of infusion at a rate of 0.2 microgram/min, followed by an additional 4 d of infusion at 0.8 microgram/min. Mean serum levels of hCG established at these rates of infusion were 61.1 +/- 3.3 and 237 +/- 16 ng/ml, respectively (mean +/- SEM). The MCR determined at the low infusion rate was not significantly different from that determined at the higher infusion rate (1.83 +/- 0.09 vs. 1.95 +/- 0.14 ml/min per m2). The mean MCR for all subjects was 1.88 +/- 0.08 ml/min per m2. The MCR was not significantly different between men amd women (2.04 +/- 0.13 vs. 1.76 +/- 0.07 ml/min per m2). The RCR also did not vary between low and high infusion rates (0.40 +/- 0.03 vs. 0.40 +/- 0.04 ml/min per m2). The mean RCR for all subjects was 0.40 +/- 0.02 ml/min per m2. There was no difference in RCR between men and women (0.42 +/- 0.05 vs. 0.39 +/- 0.03 ml/min per m2). Six subjects were given 1.0 mg of highly purified hCG by rapid intravenous injection. Initial serum levels of hCG were 300-400 ng/ml, and the subsequent disappearance curve was multiexponential over 8-10 d. The disappearance curve of hCG in each subject was fitted to a biexponential equation. The rapid component t1/2 was 5.97 +/- 0.63 h and the slow component t1/2 was 35.6 +/- 8.0 h. We conclude that the MCR of purified hCG in man is about 2 ml/min per m2 and the RCR is 0.4 ml/min per m2; these parameters are concentration independent and do not differ significantly between healthy young men and women.
R E Wehmann, B C Nisula
Adenosine levels in oxygen-deprived myocardium can rise to 10- 100 microM concentrations known to cause atrioventricular (AV) conduction delay and block. We reported that the AV conduction delay and block caused by hypoxia is markedly attenuated by 10 microM aminophylline, and adenosine competitive antagonist. THe purpose of the present study was to investigate adenosine's role in ischemic AV conduction disturbances. Dogs were anesthetized and instrumented for His bundle and surface electrogram recordings. The total AV conduction time was subdivided in to atrial-His bundle (AH) and His bundle-ventricle intervals. The atrioventricular node artery (AVNA) was cannulated for selective injection of drugs in the AV node region. Adenosine (10 to 100 microgram), as a 2-ml bolus injection, rapidly produced a dose-dependent, transient increase in the AH interval; a 1,000-microgram dose caused second degree AV block. The duration of the increase in AH interval was also dose-dependent. Dipyridamole, and inhibitor of nucleoside transport, potentiated the negative dromotropic effects of adenosine, whereas aminophylline attenuated them. In some dogs, after cannulation of the AVNA, first and second degree AV block occurred spontaneously or were induced by rapid atrial pacing. Injection of the aminophylline (5 mg/kg, i.e.) or theophylline (100-1,000 microgram) into the AVNA rapidly reversed the AV blocks. Upon washout of the drugs the AV blocks recurred. We conclude that endogenously released adenosine may account for a major fraction of the AV conduction delay and block associated with impaired blood supply to the AV node, and the theophylline and aminophylline reverse the AV conduction defect by antagonizing the effects of adenosine.
L Belardinelli, E C Mattos, R M Berne
To study possible physiologic relationships between somatostatin and the gastric interdigestive contractions (GIC), gastric motor activity, and plasma somatostatin-like immunoreactivity (SLI) concentration were determined simultaneously in four conscious dogs, each of which was studied on two separate occasions. Plasma SLI level was highest during the GIC period and lowest 60 and 80 min after the cessation of the GIC; the mean difference in plasma SLI was 41 +/- 6 pg/ml. When synthetic motilin, a known stimulus of GIC, was infused at a physiologic rate during the period in which plasma SLI levels were low, SLI rose to approximately the same values observed during the contraction period and GIC similar to those that occur spontaneously were observed. When synthetic somatostatin, a known inhibitor of endogenous motilin release, was infused at a rate that raised the plasma SLI to approximately the levels observed during the contraction period (0.1 microgram/kg per h), the appearance of the subsequent GIC was significantly delayed. These results are consistent with a physiological role for somatostatin in the regulation of GIC in dogs and suggest an interrelationship between motilin and somatostatin.
I Aizawa, Z Itoh, V Harris, R H Unger
To explore the possibility that the behavior of immune complexes can, under some circumstances, be directed by the antigen, we have studied the behavior of complexes of identical size made with the glycoproteins, orosomucoid (OR), and ceruloplasmin: or with their desialylated derivatives, asialo-orosomucoid (ASOR) and asialo-ceruloplasmin. Such desialylated proteins are rapidly removed from the circulation by a hepatic cell receptor for galactose, the sugar exposed upon removal of sialic acid. Mixtures of 125I-goat anti-ASOR with either ASOR or OR and mixtures of 125I-rabbit anti-OR with either ASOR or OR form complexes identically. The complexes were separated by density gradient centrifugation and injected intravenously into C3H mice. Blood clearance and hepatic uptake of the OR complexes and ASOR complexes were markedly different. T 1/2 for the goat OR complexes exceeded 300 min, whereas that for the ASOR complexes was 15 min. More detailed studies using rabbit complexes of various sizes revealed that light rabbit complexes behaved similarly to the goat complexes. The light rabbit OR complexes were cleared slowly, with only 18% found in the liver at 60 min, whereas the light rabbit ASOR complexes were cleared much more rapidly, with 62% found within the liver by 30 min. This rapid clearance was completely suppressed by a prior injection of a blocking dose of ASOR, which implies uptake by a galactose-mediated mechanism on hepatocytes. As the size of the rabbit complexes increased, so did the rate of Fc receptor-mediated clearance. Heavy rabbit OR complexes were cleared more rapidly than light OR complexes but not so rapidly as heavy ASOR complexes. The clearance and hepatic uptake of the heavy OR complexes were markedly suppressed by a prior injection of heat-aggregated gamma globulin, a known Fc receptor-blocking agent (45% hepatic uptake without and 6% with aggregated gamma globulin). The heavy rabbit ASOR complexes exhibited inhibition of blood clearance and hepatic uptake by both galactose receptor-blocking and Fc receptor-blocking agents. A blocking dose of ASOR reduced the hepatic uptake at 30 min from 75 to 49%, and heat-aggregated gamma globulin reduced it from 75 to 39%, which suggests that these heavy complexes were removed from the circulation by receptors both for the immunoglobulin and for the antigen. Cell separation studies and autoradiographs confirmed that those complexes cleared primarily by galactose-mediated mechanism were within hepatocytes, and those cleared by Fc receptors were within the nonparenchymal cells of the liver. It seems probable, therefore, the some antigen-antibody complexes may be removed from the circulation via receptors not only for immunoglobulin but also for antigen.
D S Finbloom, D B Magilavy, J B Harford, A Rifai, P H Plotz
Although numerous agents have been shown experimentally to protect ischemic myocardium, a critical unanswered question is whether function is preserved in the salvaged tissue. Accordingly, 38 openchest dogs had measurements of percent segment length shortening (%SS) and velocity of segment length shortening either in midmyocardial or subepicardial and subendocardial ischemic segments before and after 60 min of left anterior descending coronary artery occlusion during 5 h of reperfusion; 10 additional dogs were subjected to 3 h of coronary occlusion followed by 72 h of reperfusion. 15 min after coronary artery occlusion, radiolabeled microspheres were injected into the left atrium for measurement of regional myocardial blood flow, and dogs were treated with 1 mg/kg i.v. (n = 23) of an anti-inflammatory drug, flurbiprofen or an equal volume of saline (n = 25). The ischemic myocardium-at-risk for necrosis was determined by injecting methylene blue dye into the left atrium with the coronary artery reoccluded at the end of the reperfusion period, slicing the left ventricle into thin transverse sections, and measuring the areas of each slice that were not perfused (pink unstained tissue) by methylene blue. The quantity of necrotic tissue in each transverse section was measured by planimetry after incubation of the slices in triphenyltetrazolium chloride, and by direct histological examination in dogs with 72 h of reperfusion.
John R. Darsee, Robert A. Kloner, Eugene Braunwald
The roles of liver, kidney, and gut in maintaining fuel homeostasis were studied in 28 patients with severe hepatic cirrhosis, 25 of whom had alcohol-induced cirrhosis. Hepatic, portal, and renal blood flow rates were measured and combined with substrate concentration differences across liver, gut, and kidney to calculate the net flux of free fatty acids, ketone bodies, triglycerides, and glucose with selected glucose precursors, including glycerol, lactate, pyruvate, and amino acids. Data from the catheterization studies were related to hepatic histology, glycogen content, and activities of gluconeogenic enzymes and compared with data obtained from control patients. The effects of food deprivation on net flux of fuels across the liver, gut, and kidney were assessed after overnight and after 3d of fasting. Activities of gluconeogenic enzymes were normal, but hepatic glycogen content was diminished in cirrhotic livers, probably as a consequence of extensive hepatic fibrosis. Extrahepatic splanchnic tissues (gut) had only a small influence on total splanchnic flux rates of carbohydrates, lipids and, amino acids. In cirrhotic patients, there was no mean renal glucose contribution to the bloodstream after an overnight or after a 3-d fast. After an overnight fast hepatic glucose production in patients with cirrhosis was diminished as a result of low-rate glycogenolysis. Hepatic gluconeogenesis and ketogenesis were increased. This pattern of hepatic metabolism mimics that seen in "normal" patients after more advanced stages of starvation. After 3 d of starvation, patients with hepatic cirrhosis have hepatic gluconeogenic and ketogenic profiles comparable to those of normal patients undergoing starvation of similar duration. Nevertheless, the total number of caloric equivalents derived from ketone bodies plus glucose corrected for recycled lactate and pyruvate added to the bloodstream by the cirrhotic livers that could be terminally oxidized by peripheral tissues was less than the contributions made by the normal livers, both after and overnight and after a 3-d fast.
O E Owen, F A Reichle, M A Mozzoli, T Kreulen, M S Patel, I B Elfenbein, M Golsorkhi, K H Chang, N S Rao, H S Sue, G Boden
Cystic fibrosis (CF), a genetic disease characterized by abnormalities of exocrine gland and mucociliary function, has recently been shown to be associated with abnormal adrenergic and cholinergic physiologic responses in addition to decreased beta adrenergic-induced cyclic AMP generation in human leukocytes. In this study we have attempted to elucidate the nature of this hyporesponsiveness by assessing beta adrenergic receptor number and affinity (KD) in the intact neutrophil using the antagonist ligand [3H] dihydroalprenolol and cyclic AMP responses to isoproterenol in addition to histamine, and prostaglandin E1 in CF subjects, CF obligate heterozygotes (CFH), and normal control subjects. CF patients had significantly less (p less than 0.025) cyclic AMP stimulation above basals levels with isoproterenol (0.1 microM to 0.1 mM), compared with control values, but no consistent differences between groups were noted with histamine or PGE1. CF neutrophils had significantly fewer (p less than 0.005) beta adrenergic receptors per neutrophil (398.0 +/- 54.2 vs. 819.4 +/- 67.2) compared with control neutrophils, but the KD (0.740 +/- 0.11 vs. 0.630 +/- 0.05 nM) did not differ significantly (p greater than 0.05). There was no correlation between clinical severity and either cyclic AMP generation or dihydroalprenolol binding (r = 0.27 and 0.24, respectively, p greater than 0.05). The CFH group had approximately 50% of the cyclic AMP stimulation compared with controls, but the number (909.8 +/- 89.3) and KD (0.710 +/- 0.09 nM) of their beta adrenergic receptors were indistinguishable from control subjects. These findings suggest "down regulation" of the beta receptor in the CF patient. The cause of this remains unknown. Although the etiology of the decreased cyclic AMP responses in CFH was not due to decreased beta adrenergic receptors as assessed by antagonist ligand binding, further studies inthe CFH group to include agonist binding, receptor-adenylate cyclase coupling, intrinsic adenylate cyclase activity, and catecholamine metabolism may help determine the basic cause of beta adrenergic hyperesposiveness in both CFH and CF.
S P Galant, L Norton, J Herbst, C Wood
Neutrophils are a characteristic feature of the alveolitis of idiopathic pulmonary fibrosis (IPF). a chronic disorder limited to lung. One mechanism by which neutrophils may be selectively attracted to lung and not other tissues is via the secretion of the neutrophil-specific chemotactic factor by alveolar macrophages. To evaluate the role of alveolar macrophages in modulating the migration of neutrophils to he lung in IPF, alveolar macrophages, obtained by bronchoalveolar lavage of patients with IPF, were evaluated for their ability to release a chemotactic factor for neutrophils. Unstimulated alveolar macrophages from normal individuals did not release the factor. In patients with IPF, there was a significant correlation between the proportions of neutrophils in lavage fluid and the release of a chemotactic factor for neutrophils by alveolar macrophages (p less than 0.001). The chemotactic factor released by IPF alveolar macrophages was of low molecular weight (400-600), at least partially lipid in nature, and preferentially attracted neutrophils compared with monocytes. Several lines of evidence suggested that immune complexes in the lung stimulated alveolar macrophages of patients with IPF to release the chemotactic factor. First, immune complexes stimulated normal macrophages to release the factor.Second, there was a significant correlation between the release of the chemotactic factor by IPF alveolar macrophages and the levels of immune complexes in bronchoalveolar lavage fluid. Third, bronchoalveolar lavage fluid containing immune complexes stimulated normal macrophages to release the factor. Fourth, IPF alveolar macrophages that released large amounts of the chemotactic factor had an apparent suppression of their immunoglobulin (Ig)G Fc receptor function, suggesting that immune complexes were bound to their surface. In contrast, the IgG Fc receptor function of IPF alveolar macrophages that released only small amounts of the factor was similar to that of normal macrophages. These studies suggest that neutrophils are attracted to the lung in patients with IPF by a potent chemotactic factor released by alveolar macrophages that have been stimulated, in vivo, via their IgG Fc receptor by immune complexes.
G W Hunninghake, J E Gadek, T J Lawley, R G Crystal
The role of calcium gating in cholinergic stimulation of the function of parietal cells was studied using cells isolated from canine fundic mucosa by treatment with collagenase and EDTA and enriched by velocity separation in an elutriator rotor. Monitoring the accumulation of [14C[ aminopyrine as an index of parietal cell response, stimulation by carbachol, but not by histamine, was highly dependent upon the concentration of extracellular calcium. Incubation of parietal cells in 0-.1 mM calcium, rather than the usual 1.8 mM concentration, reduced the response to 100 microM carbachol by 92 +/- 2%, whereas histamine stimulation was impaired by 28 +/- 5%. A similar reduction in extracellular calcium suppressed the response to gastrin (100 nM) by 67 +/- 7%. The impairment of cholinergic stimulation found at low extracellular calcium concentrations was rapidly reversed with the readdition of calcium. Lanthanum, which blocks calcium movement across membranes, caused a similar pattern of effects on secretagogue stimulation of aminopyrine accumulation, with 100 microM lanthanum suppressing carbachol stimulation by 83 +/- 2%. This concentration of lanthanum suppressed gastrin stimulation by 40 +/- 7% and histamine stimulation by only 12 +/- 9%. Carbachol, but not histamine nor gastrin, stimulated 45Ca++ uptake. The magnitude of carbachol-stimulated calcium uptake correlated with the parietal cell content of the fractions examined (r = 0.88), and was dose responsive over carbachol concentrations from 1 microM to 1 mM. Atropine (100 nM) caused surmountable inhibition, and these effects of carbachol and atropine on calcium uptake correlated with their effects on oxygen consumption (r = 0.93) and [14C]-aminopyrine accumulation (r = 0.90). Cells preloaded with 45Ca++ lost cellular calcium in a time-dependent fashion; however, this rate of egress was not accelerated by treatment with histamine, gastrin, or carbachol, thus failing to implicate mobilization of intracellular calcium as primary mechanism for activation of parietal cell function. These data indicate a close link between stimulation of parietal cell function and enhancement of calcium influx by cholinergic agents.
A H Soll
Recessive congenital methemoglobinemia (RCM) is due to the homozygous deficiency of NADH-cytochrome b5 reductase (EC 22.214.171.124.). In type I disease, in which the patients are only methemoglobinemic, the enzyme defect is fully expressed in the erythrocytes, whereas the leukocytes are much less affected. In type II disease, in which the patients are, in addition, mentally retarded, the defect is generalized to all the tissues including cultured fibroblasts. In the present study we have investigated Epstein-Barr virus (EBV) transformed lymphoid cell lines (LCL) derived from patients with both types of cytochrome b5 reductase deficiency and from nondeficient individuals. The total cytochrome b5 reductase activity of the control LCL was found to be similar whatever the LCL origin, except for one lymphoma line (Daudi). The enzyme from the control LCL (c 252/B 95) was found to be immunologically related to the human soluble erythrocyte cytochrome b5 reductase, indicating that it is the product of the same gene: the DIA1 (diaphorase) locus. The LCL derived from one patient with the type I disease and two patients with the type II disease were investigated.l In the former the defect was expressed to a lesser degree than in the cases with mental retardation in which the defect was much pronounced, and involved both the mitochondrial and the microsomal fraction. This indicated that all the subcellular forms of the cytochrome b5 reductase are under the same genetic control. Altogether, these data show that the LCL are a favorable material for studying both types of cytochrome b5 reductase deficiency and for investigating in depth the molecular aspects of this metabolic disease.
D Lostanlen, G Lenoir, J C Kaplan
To increase our understanding of the pathophysiology of reflux esophagitis, we sought the early sequence of changes in mucosal structure and function in acutely acid-damage rabbit esophagus. Using a perfused catheter technique esophageal potential difference (PD) profiles were obtained in anesthetized rabbits before, during, and after perfusion of the lower one-half of the esophagus with phosphate-buffered saline or 80 mM NaCl. When acid perfusion reduced the lower esophageal PD by 40-50% or 80-100% of the initial values, the esophagus was removed, sectioned, and the mucosa studied with light microscopy, transmission electron microscopy, and Ussing chamber technique for evaluation of sodium and mannitol transport. The earlier stage of acid damage (PD 40-50%) was associated with reduced mucosal resistance fom 2,180 +/- 199 to 673 +/- 157 ohm cm2 and increased passive transport of sodium (0.10 +/- 0.06 to 1.82 +/- 0.48 microeq/h.cm2) and mannitol (0.008 +/- 0.003 to 0.051 +/- 0.012 microM/h.cm2) (p less than 0.05). There was no significant change in shirt circuit current (0.35 +/- 0.05 to 0.35 +/- 0.04) or net sodium transport (0.32 +/- 0.06 to 0.37 +/- 0.12) at this stage, and the only morphologic finding was dilated intercellular spaces on electron microscopy. The later stage of acid damage (PD 80-100%) exhibited a further reduction in resistance to 299 +/- 65 ohm.cm2 (p less than 0.05), a finding now accompanied by a reduction in short circuit current (0.35 +/- 0.05 to 0.21 +/- 0.04 microeq/h.cm2) and complete inhibition of net sodium transport (0.32 +/- 0.06 to 0.01 +/- 0.13) (p less than 0.05). Morphologic studies at this time revealed cellular necrosis, edema, and vesicle formation in the stratum spinosum. Both gross mucosal changes and transmural necrosis were notably absent. When esophageal perfusion was performed with a combination of acid (80 mM HCl-80 mM NaCl) and pepsin (100 microgram/ml), the morphologic and physiologic findings were essentially the same as with acid alone; however, the time of perfusion to reach either the 50 or 100% reduction in PD was shortened. The findings in this model can be explained on an initial increase in cellular and/or paracellular permeability followed by inhibition of active sodium transport. The resulting loss of osmolar regulation leads to cell necrosis in the stratum spinosum.
R C Orlando, D W Powell, C N Carney
The noncytotoxic immunosuppressive substance detected in crude extracellular products of Streptococcus intermedius (CEP-SI) was fractionated by two steps of preparative isoelectric focusing in sucrose gradients using ampholytes of pH range from 3.5 to 6 and 4 to 5, respectively. The in vitro and in vivo suppressor effects of the most highly purified fraction of CEP-Si, designated fraction 3' (F3'EP-Si), corresponded well with those of the original CEP-Si. F3'EP-Si was sensitive to the effects of alpha, gamma, and delta chymotrypsin, trypsin, and heating. It contained approximately 1% of the total amount of protein found in the original CEP-Si, corresponding to a single band on analytical isoelectric focusing, stainable by Coomassie Blue and of isoelectric point of 4.25. The absorption spectrum of F3'EP-Si had a maximum at 260 nm but its biological activity was resistant to deoxyribonuclease and ribonuclease A and it did not contain material stainable by methylene blue. It was also resistant to neuraminidase and did not contain material stainable by periodic acid schiff. We conclude that the substance responsible for the suppressor activity of CEP-Si is a protein of molecular weight approximately 90,000, which adheres to Sephadex of cellulose acetate and forms complexes with other, nonactive constituents of CEP-Si.
M P Arala-Chaves, M T Porto, P Arnaud, M J Saraiva, H Geada, C C Patrick, H H Fudenberg
HbE is a β-chain mutant frequently found among inhabitants of Southeast Asia and surrounding territories. We find that Plasmodium falciparum multiplies more slowly in erythrocytes from individuals homozygous for HbE than in cells from HbA individuals. In contrast, this parasite grows normally in erythrocytes heterozygous for HbE. This is the first direct evidence that suggests what has been suspected on the basis of circumstantial data, that HbE-containing erythrocytes might be advantageous to the carrier in regions with endemic malaria.
Ronald L. Nagel, Carmen Raventos-Suarez, Mary E. Fabry, Herbert Tanowitz
Metabolically active tissues from second trimester human fetuses were examined for their ability to synthesize the placental hormones chorionic gonadotropin and chorionic somatomammotropin. During short-term incubation studies both placenta and fetal kidney were found to synthesize and secrete the beta-subunit of chorionic gonadotropin, whereas its synthesis was not observed in fetal liver, lung or muscle. In addition, chorionic somatomammotropin synthesis and secretion was demonstrated with placental tissue but could not be detected in any of the fetal tissues examined. These observations constitute the first evidence that the genome of a fetal tissue directs the synthesis of what is considered a placental hormone.
W G McGregor, W J Raymoure, R W Kuhn, R B Jaffe
We have tried to elucidate the mechanism of phagosome acidification in human neutrophils. Assuming that phenomena occurring at the plasma membrane reflect reactions in the phagocytic vacuoles, we have stimulated human neutrophils with agents that induce a "respiratory burst," and we have measured the release of protons into the extracellular medium. Phorbol myristate acetate, N-formyl-methionyl-leucyl-phenylalanine and serum-opsonized zymosan particles each caused a rapid release of protons, concomitant with the increase in oxygen consumption. The stimulated release of protons was strictly coupled to the increase respiration of the cells, because inhibition of the respiration of either anaerobiosis, chlorpromazine, or glycolytic inhibitors also inhibited the release of protons. Also, in the presence of the above-mentioned stimulating agents, neutrophils from three patients with chronic granulomatous disease enhanced neither respiration not proton release. In normal cells, the ratio of deltaH+/-deltaO2 was 1.04 +/- 0.19 (mean +/ SD, n = 13). The mechanism of this proton release is not clear. The amount of lactic and carbonic acid produced by stimulated neutrophils was inadequate to explain the amount of protons released. Perhydroxyl radicals were also ruled out as the source of the protons. Because the cells did not release measurable amounts of phosphate ions, a phosphate-hydroxyl-ion antiport was also excluded. Finally, the lack of any effect of uncouplers renders it unlikely that a respiration-driven proton gradient is built up across the plasma membrane.
R van Zwieten, R Wever, M N Hamers, R S Weening, D Roos
The close anatomical relationships betaeen pancreatic alpha and beta cells makes possible their interaction at a local (paracrine) level. To demonstrate this in vivo, we have compared the acute glucagon response to intravenous arginine in the basal state and after beta cell suppression by infusions of insulin. The plasma glucose concentration was maintained by the glucose clamp technique. In six normal weight nondiabetics, infusion of insulin at 0.2 mU/kg per min (rate 1) raised the mean +/- SEM plasma insulin levels from 10 +/- 3 to 32 +/- 4 mU/liter and at 1 mU/kg per min (rate 2) raised plasma insulin to 84 +/- 8 mU/liter. This resulted in beta cell suppression, as shown by a diminution in the acute insulin response (incremental area under the insulin response curve, 0-10 min): basal = 283 +/- 61, 199 +/- 66 (rate 1) and 143 +/- 48 mU/liter per 10 min (rate 2) and a fall in prestimulus C-peptide from 1.05 +/- 0.17 to 0.66 +/- 0.15 and to 0.44 +/- 0.15 mM/liter (all P less than 0.01). This beta cell suppression was associated with increased glucagon responses to arginine: 573 +/- 75 (basal), 829 +/- 114 (rate 1), and 994 +/- 136 ng/liter per 10 min (rate 2) and increased peak glucagon responses 181 +/- 11 (basal), 214 +/- 16 (rate 1), and 259 +/- 29 ng/liter (rate 2) (all P less than 0.01). In all subjects, there was a proportional change between the rise in he acute glucagon response to arginine and the fall in the prearginine C-peptide level. To demonstrate that augmented glucagon response was due to betw cell suppression, and not to the metabolic effect of infused insulin, similar studies were performed in C-peptide-negative-diabetics. Their acute glucagon response to arginine was inhibited by the insulin infusion: 701 +/- 112 (basal), 427 +/- 33 (rate 1), and 293 +/- 67 ng/liter per 10 min (rate 2) as was their peak glucagon response: 268 +/- 69, 170 +/- 36, and 115 +/- 33 ng/liter (all P less than 0.01). Thus, hyperinsulinemia, within the physiological range achieved by insulin infusion, inhibits beta cell secretion which, via a paracrine mechanism, potentiates glucagon secretion.
C M Asplin, T L Paquette, J P Palmer