B M Babior
Ammonia production was measured directly in 10 segments of the rat nephron to determine the relative importance of the segments as sites of renal ammonia production. Tubules were microdissected from normal rats and rats drinking 0.28 M NH4Cl or 0.28 M NaHCO3 for 3-8 d. The segments were incubated in vitro with and without 2 mM glutamine. Ammonia concentrations in the incubation fluid were measured by microfluorometry to determine ammonia production rates. All segments produced ammonia from glutamine. In normal rats, production with glutamine was highest (greater than 5 pmol/min per mm) in the proximal convoluted (S-1), proximal straight (S-3), and distal convoluted tubules, and lowest (less than or equal to 2) in cortical and medullary collecting ducts and thin descending limbs. Metabolic acidosis increased production by 60% in the S-1 segment of the proximal convoluted tubule and by 150% in the S-2 segment of the proximal straight tubule without significant effect in any other segment. Bicarbonate loading decreased production by S-1 but had no effect on S-2 or S-3. Thus, acid-base changes altered production only in specific segments of the proximal tubule. We infer that the bulk of ammonia production occurs in the proximal tubules and that production by collecting ducts can account for only a few percent of renal ammonia production and excretion in the rat.
D W Good, M B Burg
Vasculitis in systemic lupus erythematosus (SLE) is associated with the deposition of IgG and complement in blood vessel walls. However, it is not known whether immune injury to endothelial cells is a part of this process. Therefore, we used a solid phase radioimmunoassay to study the ability of IgG from normal human sera and sera from patients with SLE to bind to endothelial cells. In this assay, cultured human umbilical venous endothelial cells were sequentially incubated with normal or SLE sera, goat anti-human IgG, and 125I-labeled staphylococcal protein A (*SPA). After exposure to normal sera, 2.5 +/- 0.5% (mean +/- SD) of the added *SPA bound to the cells, whereas after exposure to SLE sera 13.8 +/- 7.6% of the added *SPA bound to these cells. This difference in binding was highly significant (P less than 0.001). Binding was partially reduced when SLE sera were preincubated with B-lymphocytes or monocytes, but not after exposure to erythrocytes, platelets, or T lymphocytes. Incubation of endothelial cells with the 7S fraction of SLE sera or with the F(ab')2 fragment of SLE-IgG resulted in the deposition of greater than 80% as much IgG as was deposited on endothelial cells by whole serum. However, since higher molecular weight fractions (greater than 7S) of SLE sera were also active, we tested the capacity of endothelial cells to bind IgG complexes. Endothelial cells bound heat-aggregated IgG (HA-IgG) in a saturable manner at one log concentration below the binding of normal monomeric IgG. Binding of HA-IgG to endothelial cells was markedly enhanced by preincubation with a serum source of complement. Both HA-IgG and SLE-IgG also bound to freshly obtained endothelial cells in suspension, as detected by automated fluorescence flow cytometry. Binding of SLE-IgG and HA-IgG to endothelium initiated complement activation, deposition of the third component of complement, and disruption of the monolayer. In addition, SLE-IgG and HA-IgG caused endothelial cells to secrete prostacyclin and caused the adherence of platelets, confirmed by scanning electron microscopy. These studies demonstrate that IgG anti-endothelial antibodies are present in the sera of patients with active SLE. These sera may also contain IgG complexes that are capable of binding to endothelial cells. The association of IgG and complement with endothelial cells may initiate vascular injury in SLE and other human disorders.
D B Cines, A P Lyss, M Reeber, M Bina, R J DeHoratius
Extracellular Ca2+ is required for platelet aggregation and secretion in response to ADP or epinephrine. Recently, we reported that the platelet surface contains two classes of high affinity binding sites for extracellular Ca2+. To identify these sites and clarify their role in platelet function, we have now (a) studied platelets congenitally deficient in surface membrane glycoproteins and (b) examined the effect of removing surface-bound Ca2+ on platelet responses to ADP and epinephrine. Unstimulated normal platelets contained 86,000 Ca2+-binding sites/platelet with a dissociation constant (Kd) of 9 nM and 389,000 sites with a Kd of 400 nM. In contrast, thrombasthenic platelets, which lack glycoproteins IIb and IIIa, exhibited a 92% reduction in the number of higher affinity Ca2+-binding sites and a 63% reduction in the number of lower affinity sites. Bernard-Soulier platelets, which lack glycoprotein Ib, were not deficient in Ca2+-binding sites. After stimulation with ADP, both normal and thrombasthenic platelets developed approximately 138,000 new Ca2+-binding sites/platelet (Kd = 400 nM), while the larger Bernard-Soulier platelets developed 216,000 new sites. These data suggest that IIb and IIIa represent the major Ca2+-binding glycoproteins on unstimulated platelets, while neither these glycoproteins nor Ib represent the new Ca2+-binding sites on stimulated platelets. Removal of Ca2+ from the platelet surface inhibited platelet function. Despite the presence of 1 mM Mg2+, ADP- and epinephrine-induced aggregation and [14C]serotonin release were markedly decreased at free Ca2+ concentrations less than 7 nM, a value similar to the Kd of the higher affinity Ca2+-binding sites. Moreover, gadolinium, a lanthanide that competed for these Ca2+-binding sites, also inhibited aggregation and serotonin release. These studies demonstrate, therefore, that the binding of extracellular Ca2+ to glycoproteins IIb/IIIa on unstimulated platelets or to additional membrane proteins on stimulated platelets is necessary for maximal platelet responses to ADP and epinephrine. Thus, the requirement for extracellular Ca2+ during platelet activation by these agonists may actually represent a requirement for surface-bound Ca2+.
L F Brass, S J Shattil
After intravenous glucose/insulin infusion there is an increase in oxygen consumption and energy expenditure that has been referred to as thermogenesis. To examine the contribution of the beta and alpha adrenergic nervous system to this thermogenic response, 12 healthy volunteers participated in three studies: (a) euglycemic insulin (plasma insulin approximately 100 microunits/ml) clamp study (n = 12); (b) insulin clamp study after beta adrenergic blockade with intravenous propranolol for 1 h (n = 12); (c) insulin clamp study after alpha adrenergic blockade with phentolamine for 1 h (n = 5). During the control insulin clamp study total glucose uptake, glucose oxidation and nonoxidative glucose uptake averaged 7.85 +/- 0.47, 2.62 +/- 0.22, and 5.23 +/- 0.51 mg/kg X min. After propranolol infusion, insulin-mediated glucose uptake was significantly reduced, 6.89 +/- 0.41 (P less than 0.02). This decrease was primarily the result of a decrease in glucose oxidation (1.97 +/- 0.19 mg/kg X min, P less than 0.01) without any change in nonoxidative glucose metabolism. Phentolamine administration had no effect on total glucose uptake, glucose oxidation, or nonoxidative glucose disposal. The increments in energy expenditure (0.10 +/- 0.01 vs. 0.03 +/- 0.01 kcal/min) and glucose/insulin-induced thermogenesis (4.9 +/- 0.5 vs. 1.5 +/- 0.5%) were reduced by 70% during the propranolol/insulin clamp study. The increments in energy expenditure (0.12 +/- 0.03 kcal/min) and thermogenesis (5.0 +/- 1.5%) were not affected by phentolamine. These results indicate that activation of the beta adrenergic receptor plays an important role in the insulin/glucose-mediated increase in energy expenditure and thermogenesis. In contrast, the alpha adrenergic receptor does not appear to participate in this response.
R A DeFronzo, D Thorin, J P Felber, D C Simonson, D Thiebaud, E Jequier, A Golay
Since calcium solubility is a prerequisite to calcium absorption, and since solubility of calcium is highly pH-dependent, it has been generally assumed that gastric acid secretion and gastric acidity play an important role in the intestinal absorption of calcium from ingested food or calcium salts such as CaCO3. To evaluate this hypothesis, we developed a method wherein net gastrointestinal absorption of calcium can be measured after ingestion of a single meal. A large dose of cimetidine, which markedly reduced gastric acid secretion, had no effect on calcium absorption in normal subjects, and an achlorhydric patient with pernicious anemia absorbed calcium normally. This was true regardless of the major source of dietary calcium (i.e., milk, insoluble calcium carbonate, or soluble calcium citrate). Moreover, calcium absorption after CaCO3 ingestion was the same when intragastric contents were maintained at pH 7.4 (by in vivo titration) as when intragastric pH was 3.0. On the basis of these results, we conclude that gastric acid secretion and gastric acidity do not normally play a role in the absorption of dietary calcium. Other possible mechanisms by which the gastrointestinal tract might solubilize ingested calcium complexes and salts are discussed.
G W Bo-Linn, G R Davis, D J Buddrus, S G Morawski, C Santa Ana, J S Fordtran
It has been suggested that beta-adrenergic responsiveness is reduced in hypertension. To evaluate a possible alteration in human beta-receptors that might account for diminished beta-adrenergic responsiveness, we studied leukocytes from hypertensive and normotensive subjects after an overnight rest supine, and then after being ambulatory, a maneuver that increases plasma catecholamines approximately twofold. In supine samples, beta-receptor affinity for the agonist isoproterenol was significantly reduced in hypertensives and was associated with a reduction in the proportion of beta-receptors binding agonist with a high affinity from 42 +/- 6% in normotensive subjects to 25 +/- 2% in hypertensives (P less than 0.05). Alterations in beta-adrenergic-mediated adenylate cyclase activity parallelled the differences seen in the beta-receptor affinity for agonist. In normotensive subjects, beta-receptor density and the proportion of receptors binding agonist with high affinity were reciprocally correlated with plasma catecholamines. However, in the hypertensive subjects these correlations were not evident. Thus, our data suggest an alteration in leukocyte beta-receptor interactions in hypertensive subjects, and may represent a generalized defect in beta-receptor function in hypertension.
R D Feldman, L E Limbird, J Nadeau, D Robertson, A J Wood
A human hepatocellular carcinoma line, HepG2, was found to secrete coagulation Factor V. Factor V activity in HepG2 culture fluid increased nearly linearly during a 20-h time course (5 ng Factor Va/h per 10(6) cells). Thrombin treatment increased Factor V activity in HepG2 culture medium six- to ninefold, indicating that the medium accumulates a mixture of Factors V and Va. To demonstrate de novo synthesis of Factor V, HepG2 cells were incubated in culture medium containing [35S]methionine. Labeled Factor V was immunoprecipitated from the medium and was shown to co-migrate with purified plasma Factor V upon sodium dodecyl sulfate polyacrylamide gel electrophoresis and fluorography. When medium was treated with thrombin before immunoprecipitation and fluorography, the 330,000-Mr [35S]methionine-labeled Factor V was converted to Factor Va. Factor Va coagulant activities from HepG2 cells and human plasma were inhibited in parallel by anti-Factor V antibody, indicating that HepG2 and plasma Factor Va have the same intrinsic activity. If normal hepatocytes synthesize Factor V at the same rate as HepG2 cells, then hepatocyte secretion can account for the total Factor V present in plasma. The production of Factor V by cultured human umbilical vein endothelial cells was also examined. Spent culture medium from endothelial cells contained only Factor Va and the amount was less than 1% of the activity found in medium from HepG2 cells under comparable conditions. The amount of Factor V activity in endothelial cell culture fluid did not change with time in culture.
D B Wilson, H H Salem, J S Mruk, I Maruyama, P W Majerus
To elucidate the mechanism of vectorial translocation of bile acids in the liver, taurocholate transport was studied in isolated liver canalicular membrane vesicles by a rapid filtration method. The membrane vesicles revealed temperature-dependent, Na+-independent transport of taurocholate into an osmotically reactive intravesicular space. In the absence of sodium, taurocholate uptake followed saturation kinetics (apparent Km for taurocholate = 43 microM and Vmax = 0.22 nmol/mg protein X 20 s at 37 degrees C) and was inhibited by cholate and probenecid. Transstimulation by unlabeled taurocholate was also demonstrated. When the electrical potential difference across the membranes was altered by anion replacement, a more positive intravesicular potential stimulated, and a more negative potential inhibited, transport of taurocholate by the vesicles. Valinomycin-induced K+-diffusion potential (vesicle inside-positive) enhanced the rate of taurocholate uptake that was not altered by imposed pH gradients. These results indicate that rat liver canalicular plasma membrane contains a sodium-independent taurocholate transport system that translocates the bile acid as an anion across the membrane. In intact hepatocytes, the electrical potential difference across the canalicular membrane probably provides the driving force for taurocholate secretion. The contribution of nonionic diffusion to taurocholate secretion appears to be minimal.
M Inoue, R Kinne, T Tran, I M Arias
The present study was designed to quantitate the interaction between the decrease in target tissue insulin action seen in subjects with Type II diabetes and the mass action effect of glucose exerted via the prevailing hyperglycemic state. To this end, euglycemic glucose clamp studies were performed in 26 control subjects using insulin infusion rates of 15, 40, 120, 240, and 1,200 mU/M2 per min and in 10 Type II diabetic subjects using insulin infusion rates of 120 and 1,200 mU/M2 per min. The results of these euglycemic studies indicated that insulin-stimulated peripheral glucose disposal was decreased in the Type II diabetics due to a combined receptor (rightward shift in the dose-response curve) and postreceptor defect in insulin action (decreased maximal response), whereas the decrease in insulin-mediated suppression of hepatic glucose output (HGO) was consistent with a defect in insulin binding (rightward shift in dose-response curve). Hyperglycemic glucose clamp studies were also performed in the Type II diabetics at their respective fasting serum glucose levels (mean [+/- SE] 280 +/- 17 mg/dl) employing insulin infusion rates of 15, 40, 120, and 1,200 mU/M2 per min. In the presence of their basal level of hyperglycemia, the noninsulin-dependent diabetes mellitus (NIDDM) subjects exhibited rates of overall glucose disposal that were similar to those observed in control subjects studied at euglycemia at similar steady state insulin concentrations. This suggests that in Type II diabetics, the mass action effect of glucose partially compensates for the marked decrease in insulin-stimulated glucose uptake observed under euglycemic conditions. However, even in the presence of hyperglycemia, insulin levels below 100 microU/ml had little effect and maximally effective insulin levels increased peripheral glucose disposal only 2.8-fold (142 +/- 7-413 +/- 47 mg/M2 per min) above basal in the Type II diabetics, compared with a sixfold increase (75 +/- 4-419 +/- 34 mg/M2 per min) in the control subjects studied at euglycemia. Thus, the severe insulin resistance that is a characteristic feature of NIDDM remains apparent. Basal HGO was elevated in the NIDDM subjects (157 +/- 6 vs. 76 +/- 4 mg/M2 per min for controls) and a high degree of correlation was found between the basal rate of HGO and the fasting glucose level (r = 0.80, P less than 0.01). The presence of hyperglycemia augmented insulin-mediated suppression of HGO, but did not restore it to normal. We concluded that: (a) in the presence of basal hyperglycemia, physiologic insulin levels exerts a diminished effect to suppress HGO and stimulate peripheral glucose disposal in NIDDM; (b) basal HGO is elevated in untreated Type II diabetics, and this may serve to maintain the level of hyperglycemia required to compensate for the decrease in peripheral insulin action; and (c) fasting hyperglycemia exerts a suppressive effect on HGO but does not completely compensate for the decrease in hepatic insulin action in Type II diabetics.
R R Revers, R Fink, J Griffin, J M Olefsky, O G Kolterman
The antilipolytic effect of insulin was studied in 9 obese and 10 age- and sex-matched subjects of normal weight. Isolated fat cells were taken before and 1 h after an 100 g oral glucose load. Insulin inhibition of basal and isoprenaline-induced rates of lipolysis were determined by using a sensitive bioluminescent glycerol assay. When compared with the controls, the obese group showed a lower glucose tolerance, a higher insulin secretion, and a lower specific insulin receptor binding per adipocyte surface area, which would suggest an insulin-resistant state. Before oral glucose, however, the sensitivity of the antilipolytic effect of insulin was enhanced 10-fold in obesity (P less than 0.01), but the maximum antilipolytic effect was not altered. Glucose ingestion induced a 10-25-fold increase in insulin sensitivity (P less than 0.01) and a 10% but not significant increase in specific adipocyte insulin receptor binding in the nonobese group. In the obese group, however, neither the insulin binding nor the antilipolytic effect of the hormone was increased by oral glucose. After oral glucose, insulin sensitivity was similar in the two groups. The concentration of the hormone which produced a half maximum effect was about 1 microU/ml. Similar results were obtained with insulin inhibition of basal and isoprenaline-stimulated glycerol release. It is concluded that, after an overnight fast, the sensitivity of the antilipolytic effect of insulin is markedly enhanced in adipocytes of "insulin-glucose resistant" obese subjects, presumably because of alterations at postreceptor levels of insulin action. In obesity, the antilipolytic effect of insulin seems normal after glucose ingestion. Furthermore, in adipocytes of subjects of normal weight, oral glucose rapidly stimulates the sensitivity of the antilipolytic effect of insulin, apparently because of changes at postreceptor sites. This short-term regulation of insulin action following the ingestion of glucose does not seem to be present in obesity.
P Arner, J Bolinder, P Engfeldt, J Hellmér, J Ostman
Sera from patients with first episode primary genital herpes infections who were treated with the antiviral drug acyclovir were studied to determine the effect of therapy on the immune response to herpes simplex virus (HSV) glycoproteins and polypeptides. 63 patients were evaluated, 35 patients received acyclovir: 11 intravenously, 12 orally, and 12 topically, while 28 received placebo. Topical application of acyclovir had no effect on the immune response to HSV infection. However, both oral and intravenous acyclovir were associated with later development of antibodies to two glycoproteins (of 80,000 and 60,000 mol wt [IIg80 and gD, respectively]) and one nonglycosylated polypeptide of 66,000 mol wt (vp66). Antibody to IIg80 was present in convalescent phase serum in 13/23 systemic acyclovir recipients vs. 18/19 placebo recipients (P = 0.01) and antibody to gD was detected in 8/23 oral or intravenous acyclovir recipients vs. 11/19 placebo recipients (P = 0.06). The mean time to seroconversion to IIg80 (39.0 d) and gD (55.5 d) was significantly longer for systemic acyclovir recipients than for the placebo controls, 23.4 and 18.5 d, respectively (P less than 0.05 for each comparison). 7 (30%) of 23 systemic acyclovir recipients compared with 100% of the placebo recipients had antibody to vp66 by 30 d after onset of the primary episode (P less than 0.001). Subsequent untreated recurrences of genital herpes were associated with seroconversion to gD, IIg80, and vp66. Patients who lacked antibody to both gD and vp66 in sera taken before their first clinical recurrence of disease experienced a longer duration of the recurrent episode (10.8 d) than those who possessed antibody to both vp66 and gD (6.3 d) (P less than 0.05). In addition, the mean duration of lesions, number of lesions, and mean lesion area were greater in patients who lacked antibody to vp66 but had anti gD, as compared with those who had anti-p66 but lacked anti-gD; suggesting that antibody to vp66 correlated more closely with subsequent disease severity than did antibody to gD. Acyclovir therapy appears to influence the frequency and time of development of antibody to a number of different HSV-specific polypeptides. Further studies of the effects of antiviral therapies on the immune response to these proteins may help clarify the role of these polypeptides in the pathogenesis of disease.
R L Ashley, L Corey
Previous studies have shown that patients with systemic lupus erythematosus (SLE) had differing T cell T4+/T8+ ratios and that the ratio correlated with clinical features of the disease. In the present study, we wished to determine whether the peripheral blood T cell subsets in these patients were related to the specificity of anti-T cell antibodies found in their plasma. Plasma from 24 SLE patients that reacted with greater than 20% of normal T cells were analyzed for their effect on in vitro pokeweed mitogen-stimulated immunoglobulin synthesis and for their reactivity with human T4+ and T8+ cells. Anti-T cell antibodies found in SLE patients have a spectrum of reactivities. We concentrated upon antibodies that interfere with suppressor function. One group of SLE anti-T cell antibodies reacts preferentially with the T8+ suppressor effector cell whereas another is reactive with T4+ suppressor inducer subsets. SLE patients with high T4+/T8+ ratios had anti-T cell antibodies predominantly reactive with the T8+ suppressor effector cells. Patients with low T4+/T8+ ratios, on the other hand, had anti-T cell antibodies reactive with either the T4+ suppressor inducer or with both the T4+ suppressor inducer and T8+ suppressor effector cells. In addition, a fourth group was defined whose anti-T cell antibodies were neither reactive with a functional T4+ suppressor inducer nor a functional T8+ suppressor effector cells. There was a significant correlation between the circulating T4+/T8+ ratio of peripheral T cells in these patients and the relative ability of their anti-T cell antibodies to kill T8+ cells vs. T4+ cells (gamma = 0.666, P less than 0.001). These results support the notion that in SLE different cellular defects in the immunoregulatory circuit underlie the development of autoimmune reactions and that the anti-T cell antibodies may cause numerical and functional deficiencies in T cell subsets.
C Morimoto, E L Reinherz, J A Distaso, A D Steinberg, S F Schlossman
The NADPH-dependent O2-.-generating oxidase in subcellular fractions from the neutrophils of three male patients with chronic granulomatous disease was compared with the corresponding preparations from normal neutrophils. The oxidase from normal neutrophils contained flavin adenine dinucleotide in an approximately 0.9:1 molar ratio with cytochrome b559. Each of the three chronic granulomatous disease patients had decreased amounts of the flavoprotein component of the oxidase fraction. The oxidase from two chronic granulomatous disease patients had undetectable amounts of cytochrome b559 whereas the third patient had a normal content of cytochrome b559, which was spectrally indistinguishable from the normal. The intrinsic cytochrome b559 in the oxidase fraction from stimulated neutrophils of the latter chronic granulomatous disease patient was not reduced by NADPH under anaerobic conditions, in distinction with the previously reported reduction of the normal cytochrome b559 under identical conditions. We conclude that the flavoprotein component of the oxidase may mediate transfer of electrons from NADPH to the cytochrome b559 in normal neutrophils, and that deficiency of this flavoprotein is associated with the chronic granulomatous disease phenotype in the three patients studied.
T G Gabig, B A Lefker
We have examined the role of the glutathione redox cycle as an antioxidant defense mechanism in cultured bovine and human endothelial cells by disrupting the glutathione redox cycle at several points. Endothelial glutathione reductase was selectively inhibited with 1,3-bis(chloroethyl)-1-nitrosourea (BCNU). Cellular stores of reduced glutathione were depleted by reaction with diethylmaleate (DEM) or 1-chloro-2,4-dinitrobenzene (CDNB) or by inhibition of glutathione synthesis with buthionine sulfoximine (BSO). Whereas several strains of untreated bovine and human endothelial cells were resistant to lysis by enzymatically generated hydrogen peroxide, BCNU-treated cells were readily lysed in a time- and dose-dependent manner. Glucose-glucose oxidase-mediated lysis of BCNU-treated bovine endothelial cells was catalase-inhibitable and directly related to BCNU concentration and endogenous glutathione reductase activity. Pretreatment of bovine endothelial cells with BCNU did not potentiate lysis by distilled water, calcium ionophore, lipopolysaccharide, or hypochlorous acid. Depletion of cellular reduced glutathione by reaction with DEM or CDNB or by inhibition of glutathione synthesis by BSO also potentiated endothelial lysis by enzymatically generated hydrogen peroxide. Inhibition of endothelial glutathione reductase by BCNU or depletion of reduced glutathione by BSO increased endothelial susceptibility to lysis by hydrogen peroxide generated by phorbol myristate acetate-activated neutrophils. We conclude that the glutathione redox cycle plays an important role as an endogenous antioxidant defense mechanism in cultured endothelial cells.
J M Harlan, J D Levine, K S Callahan, B R Schwartz, L A Harker
Familial hyperproinsulinemia, a hereditary syndrome in which individuals secrete high amounts of 9,000-mol wt proinsulin-like material, has been identified in two unrelated cohorts. Separate analysis of the material from each of the two cohorts had suggested that the proinsulin-like peptide was a conversion intermediate in which the C-peptide remained attached to the insulin B-chain in one case, whereas it was a conversion intermediate in which the C-peptide remained attached to the insulin A-chain in the other. To reinvestigate this apparent discrepancy, we have now used chemical, biochemical, immunochemical, and physical techniques to compare in parallel the structures of the immunoaffinity chromatography-purified, proinsulin-like peptides isolated from the serum of members of both families. Our results show that affected individuals in both cohorts secrete two-chained intermediates of proinsulin conversion in which the COOH-terminus of the C-peptide is extended by the insulin A-chain and from which the insulin B-chain is released by oxidative sulfitolysis. Analysis of the conversion intermediates by reverse-phase high-performance liquid chromatography using two different buffer systems showed that the proinsulin-related peptides from both families elute at a single position very near that of the normal intermediate des-Arg31, Arg32-proinsulin. Further, treatment of these peptides with acetic anhydride prevented trypsin-catalyzed cleavage of the C-peptide from the insulin A-chain, a result demonstrating the presence of Lys64 and the absence of Arg65 in both abnormal forms. We conclude that individuals from both cohorts with familial hyperproinsulinemia secret very similar or identical intermediates of proinsulin conversion in which the C-peptide remains attached to the insulin A chain and in which Arg65 has been replaced by another amino acid residue.
D C Robbins, S E Shoelson, A H Rubenstein, H S Tager
To assess the role of the purine nucleotide cycle in human skeletal muscle function, we evaluated 10 patients with AMP deaminase deficiency (myoadenylate deaminase deficiency; MDD). 4 MDD and 19 non-MDD controls participated in an exercise protocol. The latter group was composed of a patient cohort (n = 8) exhibiting a constellation of symptoms similar to those of the MDD patients, i.e., postexertional aches, cramps, and pains; as well as a cohort of normal, unconditioned volunteers (n = 11). The individuals with MDD fatigued after performing only 28% as much work as their non-MDD counterparts. Muscle biopsies were obtained from the four MDD patients and the eight non-MDD patients at rest and following exercise to the point of fatigue. Creatine phosphate content fell to a comparable extent in the MDD (69%) and non-MDD (52%) patients at the onset of fatigue. Following exercise the 34% decrease in ATP content of muscle from the non-MDD subjects was significantly greater than the 6% decrease in ATP noted in muscle from the MDD patients (P = 0.048). Only one of four MDD patients had a measurable drop in ATP compared with seven of eight non-MDD patients. At end-exercise the muscle content of inosine 5'-monophosphate (IMP), a product of AMP deaminase, was 13-fold greater in the non-MDD patients than that observed in the MDD group (P = 0.008). Adenosine content of muscle from the MDD patients increased 16-fold following exercise, while there was only a twofold increase in adenosine content of muscle from the non-MDD patients (P = 0.028). Those non-MDD patients in whom the decrease in ATP content following exercise was measurable exhibited a stoichiometric increase in IMP, and total purine content of the muscle did not change significantly. The one MDD patient in whom the decrease in ATP was measurable, did not exhibit a stoichiometric increase in IMP. Although the adenosine content increased 13-fold in this patient, only 48% of the ATP catabolized could be accounted for by the combined increases of adenosine, inosine, hypoxanthine, and IMP. Studies performed in vitro with muscle samples from seven MDD and seven non-MDD subjects demonstrated that ATP catabolism was associated with a fivefold greater increase in IMP in non-MDD muscle. There were significant increases in AMP and ADP content of the muscle from MDD patients following ATP catabolism in vitro, while there was no detectable increase in AMP or ADP in non-MDD muscle. Adenosine content of MDD muscle increased following ATP catabolism, but there was no detectable increase in adenosine content of non-MDD muscle following ATP catabolism in vitro. These studies demonstrate that AMP deaminase deficiency leads to reduced entry of adenine nucleotides into the purine nucleotide cycle during exercise. We postulate that the resultant disruption of the purine nucleotide cycle accounts for the muscle dysfunction observed in these patients.
R L Sabina, J L Swain, C W Olanow, W G Bradley, W N Fishbein, S DiMauro, E W Holmes
The monocyte factor, interleukin 1, or other factors homologous with interleukin 1, modulates functions of a variety of cells, including T and B lymphocytes, synovial cells, and chondrocytes. We have reported that a human monocyte cell line, U937, produces interleukin 1 when incubated with a soluble factor from lectin-stimulated T lymphocytes. We have also shown that U937 cells have a specific cytosolic receptor for 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25[OH]2D3). We now report that 1 alpha,25(OH)2D3(10(-11)-10(-10) M) induces maturational changes in the U937 cells similar to those produced by conditioned medium from lectin-stimulated T lymphocytes (increase in Fc receptors and OKM1 binding and decrease in proliferation), but does not induce monokine production as measured by mononuclear cell factor activity. 1 alpha,25(OH)2D3 is 200-300-fold more effective than 25-hydroxyvitamin D3, which is consistent with the known biological potency of these vitamin D3 metabolites. 1 alpha,25(OH)2D3 and the lymphokine together markedly augment maturational effects and, in addition, augment monokine production. The specificity of the interaction is further demonstrated by the lack of augmentation of monokine production with 1 beta,25-dihydroxyvitamin D3 in the presence of lymphokine. These interactions of a classical hormone and the hormonelike product(s) of the immune system with U937 cells serve as a model for human monocyte/macrophage differentiation and suggest a role for these interactions in some aspects of inflammation.
E P Amento, A K Bhalla, J T Kurnick, R L Kradin, T L Clemens, S A Holick, M F Holick, S M Krane
To characterize the type of alpha adrenergic receptor, the effects of specific alpha adrenergic agonists and antagonists on antidiuretic hormone [( Arg8]-vasopressin [AVP])-induced water absorption were evaluated in cortical collecting tubules isolated from the rabbit kidney and perfused in vitro. In the presence of AVP (100 microU/ml), net fluid volume absorption (Jv, nanoliters per minute per millimeter) was 1.39 +/- 0.09 and osmotic water permeability coefficient (Pf, X 10(-4) centimeters per second) was 150.2 +/- 15.0. The addition of 10(-6) M phenylephrine (PE), an alpha adrenergic agonist, resulted in a significant decrease in Jv and Pf to 0.72 +/- 0.11 (P less than 0.005) and 69.9 +/- 10.9 (P less than 0.005). The addition of 10(-4) M prazosin (PZ), an alpha adrenergic antagonist, did not cause any significant change in Jv and Pf, which were 0.71 +/- 0.09 (P = NS vs. AVP + PE) and 67.8 +/- 9.5 (P = NS vs. AVP + PE), respectively. In a separate group of tubules, in the presence of AVP (100 microU/ml) and PE (10(-6) M), Jv and Pf were 0.78 +/- 0.17 and 76.1 +/- 18.0, respectively. The addition of 10(-6) M yohimbine (Y), an alpha 2 adrenergic antagonist, resulted in a significant increase in Jv to 1.46 +/- 0.14 (P less than 0.01) and Pf to 157.5 +/- 22.3 (P less than 0.005). Y (10(-4) M) or PZ (10(-4) M) alone did not significantly affect Jv and Pf in the presence of AVP )100 microU/ml). The effect of the natural endogenous catecholamine norepinephrine (NE) on Jv and Pf in the presence of AVP and propranolol (PR) was next examined. Jv and Pf were 1.53 +/- 0.07 and 176.3 +/- 5.2, respectively, in the presence of AVP (100 microU/ml) and PR (10(-4) M). The addition of NE (10(-8) M) resulted in a significant decrease in Jv to 1.19 +/- 0.11 (P less than 0.05) and Pf to 127.0 +/- 11.3 (P less than 0.02). Increasing the concentration of NE to 10(-6) M resulted in a further decrease in Jv and Pf to 0.70 +/- 0.10 (P less than 0.01 vs. NE 10(-8) M) and 68.5 +/- 10.6 (P less than 0.01 vs. NE 10(-8) M), respectively. The inhibitory effect of NE on AVP-induced water absorption was blocked by Y, but not by PZ. The effect of the alpha 2 adrenergic agonist clonidine (CD) on Jv and Pf was also examined. In the presence of AVP (10 microU/ml) Jv and Pf were 1.65 +/- 0.04 and 175.1 +/- 13.1, respectively. The addition of CD (10(-6) M) resulted in a significant decrease in Jv to 1.08 +/- 0.12 (P < 0.01) and Pf to 108.1 +/- 15.4 (P < 0.01). Increasing the concentration of CD to 10(-4) M resulted in a further significant decrease in Jv and Pf to 0.57 +/- 0.13 (P < 0.02 vs. CD 10(-6) M) and 54.7 +/- 13.8 (P < 0.01 vs. CD 10(-6) M), respectively. Similar results were obtained in the presence of AVP (100 microU/ml). The inhibitory effect of CD on AVP-induced water absorption was blocked by Y. CD did not significantly affect Jv and Pf in the presence of 8-bromo adenosine 3',5'-cyclic monophosphate. These studies indicate that alpha adrenergic agonists directly inhibit AVP-mediated water absorption at the level of renal tubule, an effect that can be blocked by specific alpha2 adrenergic antagonists, but not by specific alpha1 adrenergic antagonists. Alpha2 adrenergic stimulation directly inhibits AVP-mediate water absorption at the level of the tubule, an effect that can be blocked by a specific alpha2 adrenergic antagonist. This effect appears to be exerted at the level of activation of adenylate cyclase, since it is absent in the present of cyclic AMP.
R K Krothapalli, W N Suki
A randomized comparison trial of two very low calorie weight reduction diets was carried out for 5 or 8 wk in 17 healthy obese women. One diet provided 1.5 g protein/kg ideal body weight; the other provided 0.8 g protein/kg ideal body weight plus 0.7 g carbohydrate/kg ideal body weight. The diets were isocaloric (500 kcal). Amino acid metabolism was studied by means of tracer infusions of L-[1-13C]leucine and L-[15N]alanine. After 3 wk of adaptation to the diets, nitrogen balance was zero for the 1.5 g protein diet but -2 g N/d for the 0.8 g protein diet. Postabsorptive plasma leucine and alanine flux decreased from base line by an equal extent with both diets by approximately 20 and 40%, respectively. It was concluded that protein intakes at the level of the recommended dietary allowance (0.8 g/kg) are not compatible with nitrogen equilibrium when the energy intake is severely restricted, and that nitrogen balance is improved by increasing the protein intake above that level. Basal rates of whole body nitrogen turnover are relatively well maintained, compared with total fasting, at both protein intakes. However, turnover in the peripheral compartment, as evidenced by alanine flux, may be markedly diminished with either diet.
L J Hoffer, B R Bistrian, V R Young, G L Blackburn, D E Matthews
We have reported previously that arginine-induced insulin secretion was impaired in the vitamin D-deficient rat pancreas, and that it was improved by dietary vitamin D repletion (Norman, A. W., B. J. Frankel, A. M. Heldt, and G. M. Grodsky, 1980, Science [Wash. DC]. 209:823-825). In this study, we evaluate in the perfused rat pancreas system whether the effects of vitamin D and its metabolites on insulin secretion are direct in action on the pancreas and limited to the secretagogue arginine, or whether they are secondary to the hypocalcemia or reduced caloric and calcium intake associated with vitamin D deficiency. In an experiment where vitamin D-replete (+D) rats were pair-fed to D-deficient (-D) rats fed ad lib., the secretion of insulin in response to arginine infusion in the +D perfused rat pancreas was threefold higher than in the -D control. In a second experiment, the serum calcium level was elevated from the characteristic hypocalcemic level of -D rats (4.9 +/- 0.1 mg/dl) to a normal calcemic level (10.0 +/- 0.3 mg/dl) by feeding the rats a -D diet with dietary calcium levels ranging from 0.4 to 4%. In these -D rats, the pancreatic perfusion study with the secretagogue arginine showed a similar blunted insulin secretion response in all groups in spite of the significant differences of serum calcium levels. In a third experiment, insulin secretion in response to the separate administration of arginine (10 mM), glucose (16.9 mM), and tolbutamide (0.37 mM) was found to be significantly higher in pair-fed, normocalcemic +D rats than in -D rats with normal calcium levels. These results indicate that vitamin D or its metabolites are essential for normal insulin secretion and that the dietary intake of calcium and the resulting serum calcium levels play a lesser role than vitamin D availability in mediating insulin secretion.
S Kadowaki, A W Norman
Overall characteristics and kinetics of tubular absorption of albumin (Alb) were studied in isolated perfused proximal convoluted tubules of the rabbit. The fate of absorbed Alb was determined in tubules perfused with low [Alb]. Alb was labeled with tritium by reductive methylation ( [3H3C]Alb). At [Alb] = 0.03 mg/ml, approximately 80% of the absorbed [3H3C]Alb was released to the peritubular bathing solution as catabolic products. Transcellular transport of intact [3H3C]Alb was negligible. Iodoacetate (IAA, 4 mM) inhibited albumin absorption (JAlb) by greater than 95% and fluid reabsorption (JV) by 55%. At [Alb] = 0.1 mg/ml the absorption rate of a derivatized cationic Alb (pI = 8.4) was fivefold greater (P less than 0.01) than that of anionic Alb. Higher cationic [Alb] had deleterious effects on tubular functions. Overall Alb absorption was of high capacity and low affinity (JmaxAlb = 3.7 ng/min per mm tubule length, apparent Michaelis constant (Km) = 1.2 mg/ml). A low capacity system that saturates at near physiological loads was also detected (JmaxAlb = 0.064 ng/min per mm, apparent Km = 0.031 mg/ml). High [Alb] did not alter the rate of endocytic vesicle formation as determined by the tubular uptake of [14C]inulin. Results show that Alb absorption is a saturable process that is inhibited by high IAA concentrations and is affected by the charge of the protein. Absorbed Alb is hydrolyzed by tubular cells and catabolic products are readily released to the peritubular side. The dual kinetics of Alb absorption may be due to a combination of adsorptive endocytosis (low capacity system) and fluid endocytosis of albumin aggregates (high capacity system). Results indicate that albuminuria occurs much before albumin absorption is saturated. The kinetic characteristics of the process of tubular absorption of albumin helps to explain the concomitance of albuminuria, increased renal catabolic rates of albumin, and renal cell deposition of protein absorption droplets in severe glomerular proteinurias.
C H Park, T Maack
Patients with glutaric aciduria (GA) have greatly increased urinary excretion of glutarate. Their leukocyte and fibroblast sonicates have deficient ability to produce 14CO2 from [1,5-14C]glutaryl-CoA, an enzymatic process with two sequential reaction steps, dehydrogenation and decarboxylation. In normal individuals, it is not known whether these two reaction steps require one or two enzymes, and currently it is assumed that a single enzyme, glutaryl-CoA dehydrogenase (GDH), carries out these two reactions. Since GA patients also excrete increased amounts of 3-hydroxyglutarate and glutaconate in urine, it was thought that glutaryl-CoA in these patients may be dehydrogenated but not decarboxylated. We developed a new assay specific for glutaryl-CoA dehydrogenation which measures enzyme-catalyzed tritium release from [2,3,4-3H]glutaryl-CoA, and we studied the glutaryl-CoA dehydrogenating activity in cultured normal human fibroblasts and those from patients with GA. The Michaelis constant (Km) of normal human fibroblast GDH for [2,3,4-3H]glutaryl-CoA was 5.9 microM, and activity was severely inhibited by (methylenecyclopropyl)acetyl-CoA at low concentrations. Sonicates from all five GA fibroblast lines examined showed 2-9% of control glutaryl-CoA dehydrogenating activity, corresponding to the deficient 14CO2 releasing activity. These results indicate either that the conversion of glutaryl-CoA to crotonyl-CoA is accomplished by two enzymes, and patients with GA are deficient in the activity of the first component, or alternatively, that this process is carried out by a single enzyme which is deficient in these patients. It is unlikely that urinary glutaconate and 3-hydroxyglutarate in GA patients are produced via GDH.
D B Hyman, K Tanaka
The effects of glucagon deficiency and excess on plasma concentrations of 21 amino acids were studied in six normal human subjects for 8 h. During glucagon deficiency, produced by intravenous infusion of somatostatin (0.5 mg/h) and insulin (5 mU/kg per h), amino acid concentration (sum of 21 amino acids) rose from 2,607 +/- 76 to 2,922 +/- 133 microM after 4 h (P less than 0.025). The largest increases occurred in lysine (+26%), glycine (+24%), alanine (+23%), and arginine (+23%) concentrations. During glucagon excess produced by intravenous infusion of somatostatin (0.5 mg/h), insulin (5 mU/kg per h), and glucagon (60 ng/kg per h), amino acid concentration decreased from 2,774 +/- 166 to 2,388 +/- 102 microM at 8 h (P less than 0.01). The largest decreases occurred in citrulline (-37%), proline (-32%), ornithine (-30%), tyrosine (-23%), glycine (-20%), threonine (-21%), and alanine (18%) concentrations. Urinary urea nitrogen and total nitrogen excretions were lower during glucagon deficiency than during glucagon excess (3.1 +/- 0.2 vs. 6.3 +/- 2.3 g/8 h, P less than 0.05 and 4.8 +/- 1.0 vs 7.0 +/- 2.6 g/8 h, respectively, P less than 0.05). Biostator-controlled euglycemic glucagon deficiency was produced in four normal subjects for 4 h to eliminate possible effects of changes in glucose concentration on amino acids. Amino acid concentration (sum of 18 amino acids) increases occurred in arginine (+42%), alanine (+28%), glutamine (+25%), and glycine (+16%) concentrations. The data show that small changes (-66 pg/ml and +50 pg/ml) in basal glucagon concentrations cause plasma amino acid concentrations to change in opposite directions. The finding that urinary excretion of nitrogen and urea nitrogen was greater during glucagon excess than during glucagon deficiency suggested alterations in the rate of gluconeogenesis from amino acids as one mechanism by which glucagon controls blood amino acid levels.
G Boden, I Rezvani, O E Owen
By using a combination of a heterologous antiserum to GPIb/glycocalicin and a radiolabeled monoclonal antibody to GPIb/glycocalicin, we were able to develop a sensitive and specific radioimmunoelectrophoretic assay that can distinguish small amounts of glycocalicin from GPIb. Normal plasmas were found to contain glycocalicin, even in samples treated with protease inhibitors and centrifuged extensively to remove platelets and platelet fragments. Confirmation that the plasma antigen had a relative molecular weight similar or identical to glycocalicin was obtained from studies employing gel chromatography and affinity chromatography. An immunoradiometric assay was developed to quantify plasma glycocalicin, and normal plasma was found to contain approximately 1-3 micrograms/ml. The plasma of a patient with severe thrombocytopenia due to aplastic anemia had less than 12.5% of the normal level of glycocalicin, whereas two patients with thrombocytopenia due to diseases of increased platelet destruction (idiopathic thrombocytopenic purpura and hemolytic-uremic syndrome) had normal levels. Thus, there appears to be ongoing catabolism of platelet GPIb in vivo, and we postulate that the plasma level of glycocalicin reflects a complex function of factors, including platelet count, platelet turnover, and the site of platelet destruction.
B S Coller, E Kalomiris, M Steinberg, L E Scudder
Previous studies have left unanswered whether human obesity, independent of glucose intolerance, is associated with a "postreceptor" defect in insulin action. We have studied the relationship between the degree of obesity (as estimated by underwater weighing) and the maximal insulin-stimulated glucose disposal rate (M) in vivo in 52 glucose-tolerant Pima Indian males. The relationship was examined independently of differences in age and maximal oxygen uptake (an estimate of "physical fitness"). The maximal insulin-stimulated glucose transport rate (MTR) was also measured in isolated abdominal adipocytes from the same subjects to determine whether differences in M could be explained by differences in glucose transport. The results showed that there was a large variance in M and MTR among these glucose-tolerant subjects. M was better correlated with glucose storage rates than with oxidation rates, as estimated by indirect calorimetry. The most obese subjects had only a 20% lower mean M and 30% lower MTR than the most lean subjects. The lower M in the obese subjects was due to both lower glucose oxidation and storage rates. There was no significant, independent correlation between age or degree of obesity and M or MTR. The maximal oxygen uptake (VO2 max) appeared to independently account for 20% of the variance observed in M. MTR was only weakly correlated with M (r = 0.36, P less than 0.02). We concluded that differences in M in these glucose-tolerant subjects must be explained by factor(s) other than maximal oxygen uptake, age, maximal insulin-stimulated glucose transport in vitro, or degree of adiposity per se.
C Bogardus, S Lillioja, D Mott, G R Reaven, A Kashiwagi, J E Foley
Fibrin deposition is prominent in the histopathology of a number of inflammatory lung diseases. Plasmin, activated locally in the lung, can degrade not only this fibrin but potentially structural proteins important to normal lung architecture. Because alveolar macrophages are prominent in inflammatory processes of the lung, we examined the plasminogen activator (PA) activity of human alveolar macrophages. Intact alveolar macrophages from each of 10 healthy subjects expressed PA activity. There was no difference in activity between smoking and nonsmoking individuals. The activator activity was largely cell-associated, but under certain culture conditions, macrophages released a soluble activator into the culture medium. The membrane-bound activator had an apparent molecular mass of 52-55 kD in nonreduced sodium dodecyl sulfate (SDS) gels, and monospecific antibody to urokinase neutralized the enzyme activity. Immunoprecipitation of [35S]methionine-labeled cells showed that human alveolar macrophages actually synthesize the PA in vitro. SDS-gel analysis of the immunoprecipitated material revealed the predominant species of PA to be structurally similar to reduced, active urokinase. We also examined the role of PA in the degradation of both insoluble fibrin and elastin matrices by live macrophages. Cells degraded an insoluble fibrin matrix in the presence of plasminogen whether or not the macrophages contacted the fibrin as long as proteinase inhibitors were not in the culture medium. In the presence of serum proteinase inhibitors, macrophages still degraded a fibrin matrix, but only if they were in contact with the fibrin. Live macrophages also degraded insoluble elastin only when in contact with the elastin but could do so even in the presence of serum proteinase inhibitors. In matrices containing a mixture of fibrin and elastin, cells did not degrade elastin unless plasminogen was added to the medium. These results indicate that normal alveolar macrophages synthesize and express, probably at the cell surface, a PA. The PA is physically and immunochemically similar to urokinase but is membrane bound. The PA is critical to the degradation of fibrin matrices by normal alveolar macrophages. Under tissue conditions where elastin is embedded within other structural proteins, the activator may be rate-limiting in elastin degradation as well. The findings also suggest that live macrophage proteolytic activity is relatively insensitive to the presence of serum proteinase inhibitors, suggesting a mechanism for proteolytic lung injury even in the presence of proteinase-proteinase inhibitor balance in the soluble phase.
H A Chapman Jr, O L Stone, Z Vavrin
Two major species of human apolipoprotein (apo) B have been identified, apo B-48 and apo B-100, which are the predominant forms in chylomicrons and very low density lipoproteins (VLDL), respectively. Due to defective hepatic clearance, apo B-48 containing lipoproteins accumulate in the plasma of subjects with type III hyperlipoproteinemia. In the present study, we have used immunoaffinity chromatography to separate type III VLDL into a nonretained (apo B-48 VLDL) and a retained (apo B-100 VLDL) fraction. To achieve complete separation, as determined by electrophoresis and radioimmunoassay, it was necessary to employ two different insolubilized anti-apo B-100 monoclonal antibodies because of immunochemical heterogeneity within the apo B-100 VLDL fraction. The ability to separate apo B-100 VLDL from apo B-48 VLDL shows that the two apo B species are found on different particles. The apo B-48 VLDL had an electrophoretic mobility similar to chylomicrons, whereas the apo B-100 VLDL migrated similarly to total type III VLDL. Both fractions showed a concentration of particles with diameters approximately 100 nm, with apo B-48 VLDL being somewhat more heterogeneous in particle size. The two fractions were qualitatively similar in apolipoprotein composition but apo B-48 VLDL was enriched in apo E, relative to apo B-100 VLDL. Apo B-48 VLDL was enriched in cholesterol esters and deficient in triglycerides and phospholipids when compared with apo B-100 VLDL. The existence of immunochemical heterogeneity in the apo B-100 VLDL may reflect different functional subpopulations of particles within this fraction.
R W Milne, P K Weech, L Blanchette, J Davignon, P Alaupovic, Y L Marcel
To study the relationship between vasopressin and the renal kallikrein-kinin system we measured the rate of excretion of kinins into the urine of anesthetized rats during conditions of increased and decreased vasopressin level. The excretion of immunoreactive kinins in Brattleboro rats with hereditary diabetes insipidus (DI) (24 +/- 3 pg min-1 kg-1) was lower than in the control Long Evans (LE) rats (182 +/- 22 pg min-1 kg-1; P less than 0.05). The DI rats also exhibited negligible urinary excretion of immunoreactive vasopressin, reduced urine osmolality, and increased urine flow and kininogenase excretion. In LE rats, volume expansion by infusion of 0.45% NaCl-2.5% dextrose to lower vasopressin secretion reduced (P less than 0.05) kinin excretion, vasopressin excretion, and urine osmolality to 41, 26, and 15% of their respective control values, while increasing (P less than 0.05) urine flow and kininogenase excretion. On the other hand, the infusion of 5% NaCl, which promotes vasopressin secretion, increased (P less than 0.05) the urinary excretion of kinins and vasopressin to 165 and 396% of control, while increasing (P less than 0.05) urine flow and kininogenase excretion. Infusion of vasopressin (1.2 mU/h, intravenous) enhanced (P less than 0.05) kinin excretion by two to threefold in DI rats and in LE rats during volume expansion with 0.45% NaCl-2.5% dextrose, while decreasing urine flow and increasing urine osmolality. This study demonstrates that the urinary excretion of immunoreactive kinins varies in relation to the urinary level of vasopressin, irrespective of urine volume and osmolality and of the urinary excretions of sodium and kininogenase. The study suggests a role for vasopressin in promoting the activity of the renal kallikrein-kinin system in the rat.
M L Kauker, J T Crofton, L Share, A Nasjletti
Immunoprecipitable double-stranded (dsDNA) was previously shown to persist in the circulation of a clinically recognizable subgroup of patients with systemic lupus erythematosus (SLE). Plasma from 10 such patients was subjected to a DNA isolation procedure that used a combination of proteolysis, phenol extraction, and hydroxylapatite adsorption and elution in the presence of urea. The isolated dsDNA was radiolabeled by nick translation and then characterized by isopyknic ultracentrifugation in CsCl under both neutral and alkaline conditions, as well as after digestion with S1-endonuclease. These experiments demonstrated essential identity in nucleotide base composition between the plasma-derived DNA and human genomic DNA. The presence of specific human base sequences in the plasma DNA was demonstrated by finding that authentic human genomic DNA accelerated the renaturation of plasma DNA when compared with the effect of nonhuman, control DNA. The proportion of such sequences in plasma DNA was estimated by attempting to renature the plasma DNA in the presence of human DNA under conditions shown to result in complete renaturation of human DNA in model experiments. In this way, a minimum of 47% of plasma DNA base sequences could be shown also to be present in human genomic DNA. However, an average of 10-20% of the plasma-derived DNA failed to renature under these conditions, a result that was further confirmed by comparing the renaturation of the tritium-labeled plasma DNA specimens, in double-label experiments, with internal controls consisting of 14C-labeled authentic human DNA. Attempts to drive the reaction to completion with human DNA led to a similar conclusion. The relative nonrenaturability of this fraction of plasma DNA did not appear to be attributable to extensive chain breakage, although adequate analysis of this DNA subfraction was limited by reagent availability. It was therefore concluded that, in this group of SLE patients, persistently circulating DNA consisted largely of base sequences also found in human genomic DNA. The additional presence in plasma of a DNA subfraction that differed in its renaturation behavior from human genomic DNA was recognized, although its significance could not be established with certainty.
C R Steinman
The role of Mycoplasma pneumoniae-generated superoxide and hydrogen peroxide in inducing host cell injury was studied in normal and trisomy 21 human cells. As a result of M. pneumoniae infection, catalase activity in infected normal skin fibroblasts and ciliated epithelial cells decreased by 74-77% as compared with uninfected controls. Addition of superoxide dismutase to the infected cultured cells totally prevented the inhibition whereas addition of catalase or catalytically inactivated superoxide dismutase had no protective effect. Trisomy 21 erythrocytes and cultured skin fibroblasts in which CuZn-superoxide dismutase content is 50% greater than in normal cells were infected by M. pneumoniae. The inhibition of catalase activity in these cells was 7-33% and 0-20.5%, respectively, as compared with 65-72% and 48-68% inhibition in normal infected controls. Following M. pneumoniae infection, the levels of malonyldialdehyde, an indicator for membrane lipid peroxidation were raised in trisomy 21 cultured fibroblasts by 10-32% while in normal cells malonyldialdehyde increased by 140-870%. Externally added superoxide dismutase, but not catalase, reduced the extent of lipid peroxidation in normal infected cells. Lactate dehydrogenase release from normal infected cells was time correlated with the increase in their malonyldialdehyde formation. It is suggested that superoxide generated during M. pneumoniae infection is involved in the inhibition of host cell catalase activity. The inactivation of this cellular antioxidative defense mechanism results in progressive oxidative damage to the M. pneumoniae-infected cells.
M Almagor, I Kahane, S Yatziv
Prematurely delivered lambs were treated with radiolabeled natural surfactant by either tracheal instillation at birth and before the onset of mechanical ventilation, or after 23 +/- 1 (+/- SE) min of mechanical ventilation. Right ventricular blood flow distributions, left ventricular outputs, and left-to-right ductal shunts were measured with radiolabeled microspheres. After sacrifice, the lungs of lambs receiving surfactant at birth inflated uniformly with constant distending pressure while the lungs of lambs treated after a period of ventilation had aerated, partially aerated, and atelectatic areas. All lungs were divided into pieces which were weighed and catalogued as to location. The amount of radiolabeled surfactant and microsphere-associated radioactivity in each piece of lung was quantified. Surfactant was relatively homogenously distributed to pieces of lung from lambs that were treated with surfactant at birth; 48% of lung pieces received amounts of surfactant within +/- 25% of the mean value. Surfactant was preferentially recovered from the aerated pieces of lungs of lambs treated after a period of mechanical ventilation, and the distribution of surfactant to these lungs was very nonhomogeneous. Right ventricular blood flow distributions to the lungs were quite homogeneous in both groups of lambs. However, in 8 of 12 lambs, pulmonary blood flow was preferentially directed away from those pieces of lung that received relatively large amounts of surfactant and toward pieces of lung that received less surfactant. This acute redirection of pulmonary blood flow distribution may result from the local changes in compliances within the lung following surfactant instillation.
A Jobe, M Ikegami, H Jacobs, S Jones
The human primary carnitine deficiency syndromes are potentially fatal disorders affecting children and adults. The molecular etiologies of these syndromes have not been determined. In this investigation, we considered the hypothesis that these syndromes result from defective transport of carnitine into tissues, particularly skeletal muscle. The problem was approached by mathematical modeling, by using the technique of kinetic compartmental analysis. A tracer dose of L-[methyl-3H]carnitine was administered intravenously to six normal subjects, one patient with primary muscle carnitine deficiency (MCD), and four patients with primary systemic carnitine deficiency (SCD). Specific radioactivity was followed in plasma for 28 d. A three-compartment model (extracellular fluid, muscle, and "other tissues") was adopted. Rate constants, fluxes, pool sizes, and turnover times were calculated. Results of these calculations indicated reduced transport of carnitine into muscle in both forms of primary carnitine deficiency. However, in SCD, the reduced rate of carnitine transport was attributed to reduced plasma carnitine concentration. In MCD, the results are consistent with an intrinsic defect in the transport process. Abnormal fluctuations of the plasma carnitine, but of a different form, occurred in MCD and SCD. The significance of these are unclear, but in SCD they suggest abnormal regulation of the muscle/plasma carnitine concentration gradient. In 8 of 11 subjects, carnitine excretion was less than dietary carnitine intake. Carnitine excretion rates calculated by kinetic compartmental analysis were higher than corresponding rates measured directly, indicating degradation of carnitine. However, we found no radioactive metabolites of L-[methyl-3H]carnitine in urine. These observations suggest that dietary carnitine was metabolized in the gastrointestinal tract.
C J Rebouche, A G Engel
Study of cobalamin-binding proteins revealed seminal plasma to be the most concentrated site of transcobalamin II in man. The next richest normal fluid, blood, has approximately one-tenth its concentration. Normal seminal unsaturated cobalamin-binding capacity averaged 15,030 +/- 7,290 pg/ml, of which 11,550 +/- 6,660 pg/ml was transcobalamin II. Transcobalamin II levels were markedly diminished in subjects lacking seminal vesicles (1520-1660 pg/ml), but not after vasectomy. This suggests that seminal vesicles are the chief source of this protein in semen. R binder concentration was increased in postvasectomy subjects (9,970 +/- 4,900 pg/ml vs. 2,980 +/- 1,370 pg/ml in normals) and varied in other patients. The endogenous cobalamin content of semen was only 88-699 pg/ml, and was carried largely by R binder rather than by transcobalamin II. The function of the unusually large seminal transcobalamin II pool in reproduction is unknown, but seems unlikely to be related solely to cobalamin transport needs, at least within the male reproductive tract itself.
R Carmel, G S Bernstein
A diazo-positive fraction of serum bilirubin that is irreversibly bound to albumin has been shown to accumulate in serum of patients with cholestasis. In the present study, a cholestatic animal model was used to determine the chemical nature of the bilirubin species involved in its formation. The data indicate that conjugated bilirubin is the precursor of "albumin-bound bilirubin" and that the presence or absence of light does not affect its formation. An albumin-bound bilirubin-complex indistinguishable from the complex detected in cholestatic sera from patients or in bile duct-ligated Sprague-Dawley rats can be formed in vitro in sera enriched in conjugated bilirubin at 37 degrees C, pH 7.4.
A Gautam, H Seligson, E R Gordon, D Seligson, J L Boyer
We studied the effect of phorbol myristate acetate (PMA) on the plasma membrane ATP-dependent calcium pump in neutrophils. Plasma membrane-enriched fractions ("podosomes") from PMA-stimulated guinea pig neutrophils exhibited a twofold stimulation of ATP-dependent calcium transport when compared with control podosomes. The stimulatory effect was rapid (beginning less than 2 min after exposure to PMA) and reached maximal values within 5 min. PMA increased the maximum velocity but not the affinity of the calcium pump for Ca++. Pump activation was not preceded by a rise in cytosolic free calcium concentration [Ca++]i, as assessed by the intracellularly trapped fluorescent calcium indicator Quin 2, but instead slightly lowered [Ca++]i and prevented the rise in [Ca++]i normally induced by the chemotactic peptide formyl-methionyl-leucyl-phenylalanine. These results suggest that the calcium pump in the plasma membrane of neutrophils may be stimulated by calcium-independent pathways, and that this activation could be one of the earliest events mediating some of the effects of phorbol esters.
H Lagast, T Pozzan, F A Waldvogel, P D Lew