Structural relationships between colonic mucin species were assessed using a library of monoclonal antibodies (MAbs) directed against purified human colonic mucin (HCM). After immunization of mice with purified mucin from normal human colonic mucosa, 14% of 1,920 fusion products screened were positive for anti-HCM activity in a solid-phase assay. Patterns of selective binding by hybridomas to six discrete HCM species (I-VI) separated by DEAE-cellulose chromatography suggested the presence of both shared and species-specific antigenic determinants among HCM species I-VI. 23 anti-HCMs MAbs (7 IgM, 7 IgG1, and 9 IgG2) demonstrating a range of anti-HCM species specificities, were produced and used to study structural relationships between mucin species. Binding of various mucin species by individual anti-HCM MAbs was shown by competitive solid-phase radioimmunoassay to reflect the presence of identical epitopes on the different species. Adsorption of HCM species on a variety of affinity resins prepared with anti-HCM MAbs demonstrated that binding to multiple mucin species by a single MAb was related to intrinsic structural determinants. Four anti-HCM MAbs recognized protease-sensitive antigenic structures, which suggests that they may be directed to core HCM proteins. 12 of the anti-HCM MAbs were shown by solid-phase assay to recognize either complete (n = 5) or partial (n = 7) isolated colonic mucin oligosaccharide side chains of defined structure. Collectively, these data show the presence of both shared and unique antigenic structural determinants among colonic mucin species. Chromatographic heterogeneity of mucin glycoproteins seems to be related to the existence of biologically significant subclasses in the normal human colon.
D K Podolsky, D A Fournier, K E Lynch
We studied glycoprotein content of human colonic goblet cells, using a library of monoclonal antibodies (MAbs) directed against purified human colonic mucin (HCM). Using indirect immunofluorescence (IIF), we found that 17 of 23 anti-HCM MAbs stained some or all goblet cells of normal human colonic mucosa. We observed a variety of cellular staining patterns, including (a) diffuse (homogeneous) staining of intracellular mucin, (b) speckled (inhomogeneous) staining of mucin droplets, (c) peripheral staining of intracellular droplets, (d) cytoplasmic staining of goblet cells, and (e) apical (luminal) surface staining. Staining patterns were not associated with particular HCM species. In addition to variable patterns of IIF within individual cells, anti-HCM MAbs varied in the proportion of goblet cells stained. Some MAbs stained all goblet cells, while others stained a limited number of goblet cells. Although each goblet cell contained more than one type mucin, HCM species III, and IV and V appeared to exist in mutually exclusive goblet cell populations and it was possible to define at least seven subpopulations of goblet cells in colonic mucosa by their content of various combinations of HCM species. Anti-HCM MAbs stained goblet cells from other sites within the gastrointestinal tract to a varying extent. Anti-HCM MAbs also showed extensive cross-reactivity with rodent, rabbit, and monkey colonic mucosa. However, several anti-HCM MAbs stained only human colonic mucosa. These data show that human colonic mucosa contains discrete subpopulations of goblet cells that produce distinctive combinations of specific mucin glycoprotein species.
D K Podolsky, D A Fournier, K E Lynch
We have studied three afibrinogenemic patients, who had only trace amounts of plasma and platelet fibrinogen as measured by radioimmunoassay, and demonstrate here that the residual aggregation observed in their platelet-rich plasma is dependent upon von Willebrand factor (vWF) binding to the platelet membrane glycoprotein (GP)IIb/IIIa complex. The abnormality of aggregation was more pronounced when ADP, rather than thrombin, collagen, or the combination of ADP plus adrenaline was used to stimulate platelets. With all stimuli, nevertheless, the platelet response was completely inhibited by a monoclonal antibody (LJP5) that is known to block vWF, but not fibrinogen binding to GPIIb/IIIa. Addition of purified vWF to the afibrinogenemic plasma resulted in marked increase in the rate and extent of aggregation, particularly when platelets were stimulated with ADP. This response was also completely blocked by LJP5. Addition of fibrinogen, however, restored normal aggregation even in the presence of LJP5, a finding consistent with the knowledge that antibody LJP5 has no effect on platelet aggregation mediated by fibrinogen binding to GPIIb/IIIa. Two patients gave their informed consent to receiving infusion of 1-desamino-8-D-arginine vasopressin (DDAVP), a vasopressin analogue known to raise the vWF levels in plasma by two- to fourfold. The bleeding time, measured before and 45 min after infusion, shortened from greater than 24 min to 12 min and 50 s in one patient and from 16 min to 9 min and 30 s in the other. Concurrently, the rate and extent of ADP-induced platelet aggregation improved after DDAVP infusion. The pattern, however, reversed to baseline levels within 4 h. The concentration of plasma vWF increased after DDAVP infusion, but that of fibrinogen remained at trace levels. We conclude that vWF interaction with GPIIb/IIIa mediates platelet-platelet interaction and may play a role in primary hemostasis.
L De Marco, A Girolami, T S Zimmerman, Z M Ruggeri
Increases in extracellular calcium (Ca++) can alter vascular tone, and thus may result in increased blood pressure (Bp) and reduced renal blood flow (RBF). Ca++ can stimulate prostaglandin E2 (PGE2) and/or prostacyclin (PGI2) release in vitro, which may modulate Ca++ vascular effects. However, in man, the effect of Ca++ on PG release is not known. To study this, 14 volunteers received low-dose (2 mg/kg Ca++ gluconate) or high-dose (8 mg/kg) Ca++ infusions. The low-dose Ca++ infusion did not alter systemic or renal hemodynamics, but selectively stimulated PGI2, as reflected by the stable metabolite 6-keto-PGF1 alpha in urine (159 +/- 21-244 +/- 30 ng/g creatinine, P less than 0.02). The same Ca++ infusion given during cyclooxygenase blockade with indomethacin or ibuprofen was not associated with a rise in PGI2 and produced a rise in Bp and fall in RBF. However, sulindac, reported to be a weaker renal PG inhibitor, did not prevent the Ca++ -induced PGI2 stimulation (129 +/- 33-283 +/- 90, P less than 0.02), and RBF was maintained despite similar increases in Bp. The high-dose Ca++ infusion produced an increase in mean Bp without a change in cardiac output, and stimulated urinary 6-keto-PGF1 alpha to values greater than that produced by the 2-mg/kg Ca++ dose (330 +/- 45 vs. 244 +/- 30, P less than 0.05). In contrast, urinary PGE2 levels did not change. A Ca++ blocker, nifedipine, alone had no effect on Bp or urinary 6-keto-PGF1 alpha levels, but completely prevented the Ca++ -induced rise in Bp and 6-keto-PGF1 alpha excretion (158 +/- 30 vs. 182 +/- 38, P greater than 0.2). However, the rise in 6-keto-PGF1 alpha was not altered by the alpha 1 antagonist prazosin (159 +/- 21-258 +/- 23, P less than 0.02), suggesting that calcium entry and not alpha 1 receptor activation mediates Ca++ pressor and PGI2 stimulatory effects. These data indicate a new vascular regulatory system in which PGI2 modulates the systemic and renal vascular actions of calcium in man.
J L Nadler, M McKay, V Campese, J Vrbanac, R Horton
The effect of hyperglycemia per se on glucose uptake by muscle tissue was quantitated in six controls and six type II diabetics by the forearm technique, under conditions of insulin deficiency induced by somatostatin (SRIF) infusion (0.7 mg/h). Blood glucose concentration was clamped at its basal value during the first 60 min of SRIF infusion and then raised to approximately 200 mg/dl by a variable glucose infusion. Plasma insulin levels remained at or below 5 microU/ml during SRIF infusion, including the hyperglycemic period. No appreciable difference between controls and diabetics was present in the basal state as to forearm glucose metabolism. After 60 min of SRIF infusion and euglycemia, forearm glucose uptake fell consistently from 2.1 +/- 0.7 mg X liter-1 X min-1 to 1.0 +/- 0.6 (P less than 0.05) and from 1.7 +/- .2 to 0.4 +/- 0.3 (P less than 0.02) in the control and diabetic groups, respectively. The subsequent induction of hyperglycemia caused a marked increase in both the arterial-deep venous blood glucose difference (P less than 0.02-0.01) and forearm glucose uptake (P less than 0.01-0.005). However, the response in the diabetic group was significantly greater than that observed in controls. The incremental area of forearm glucose uptake was 276 +/- 31 mg X liter-1 X 90 min and 532 +/- 81 in the control and diabetic groups, respectively (P less than 0.02). In the basal state, the forearm released lactate and alanine both in controls and diabetic subjects at comparable rates. No increment was observed after hyperglycemia, despite the elevated rates of glucose uptake. It is concluded that (1) hyperglycemia per se stimulates forearm glucose disposal to a greater extent in type II diabetics than in normal subjects; and (2) the resulting increment of glucose disposal does not accelerate the forearm release of three carbon compounds. The data support the hypothesis that hyperglycemia per se may play a compensatory role for the defective glucose disposal in type II diabetes.
B Capaldo, D Santoro, G Riccardi, N Perrotti, L Saccà
We have used rat cortical collecting tubules perfused in vitro to study the effects of antidiuretic hormone (ADH) and desoxycorticosterone (DOCA) on the unidirectional fluxes of sodium. We found that in the basal state, lumen-to-bath flux (Jlb) and bath-to-lumen flux (Jbl) of 22Na were approximately equal, 39.5 +/- 3.9 and 41.8 +/- 11.0 pmol X min-1 X min-1, respectively, resulting in no net flux. Addition of 100 microU/ml ADH to the bath produced a stable increase in Jlb to 58.3 +/- 4.7 pmol X min-1 X mm-1. Pretreatment of the animal with DOCA for 4 to 7 d (20 mg/kg per d) increased baseline Jlb to 81.6 +/- 8.7 pmol X min-1 X mm-1. Addition of ADH to a tubule from a DOCA-pretreated rat caused an increase in Jlb to 144.1 +/- 12.0 pmol X min-1 X mm-1 X Neither hormone had an effect on Jbl X Thus ADH produced a greater absolute and fractional increase in Jlb when the animal was pretreated with DOCA, and the ADH-induced increase over baseline was greater than the DOCA-induced increase. Both the ADH-and DOCA-induced stimulation of Jlb were completely abolished by 10(-5) M luminal amiloride, suggesting that the route of sodium transport stimulated by both hormones involves apical sodium channels. However, ADH and DOCA have very different time courses of action; ADH acted within minutes, while aldosterone and DOCA are known to require 90-180 min. The facilitating action of ADH on DOCA-induced stimulation of sodium transport may be important for maximal sodium reabsorption and for the ability to achieve a maximally concentrated urine.
M C Reif, S L Troutman, J A Schafer
Factor D (D) is an essential component of the alternative complement pathway. To determine whether D is catabolized by the kidney and, if so, at what site, we studied the renal handling of human D by in vivo nephron microperfusion and in vitro perfusion of rat kidneys. Human D was purified and labeled with 125I. Individual nephrons were perfused in vivo at varying rates with perfusate that contained 125I-D and [14C]inulin. When nephrons were perfused from proximal sites with perfusate 125I-D in a concentration of 3.0 micrograms/ml, urinary recovery of 125I-D increased (P less than 0.05) from 57.7 +/- 5.0 to 74.4 +/- 2.5% as tubule fluid flow rate was increased from 10 to 40 nl/min; recovery of 125I-D was less than (P less than 0.001) [14C]inulin recovery at all perfusion rates. At 20 nl/min, an increase in perfusate 125I-D concentration from 1.5 to 3.0 micrograms/ml was associated with an increase (P less than 0.001) in urinary 125I-D recovery (42.1 +/- 4.0 vs. 65.8 +/- 2.6%). Similarly, the addition of unlabeled D, 30 micrograms/ml, to 125I-D, 3.0 micrograms/ml, increased urinary 125I-D recovery (95.3 +/- 2.1%) at 20 nl/min. When nephrons were perfused from early distal segments at 10 nl/min, 125I-D recovery (91.2 +/- 4.3%) did not differ from [14C]inulin recovery (95.8 +/- 1.3%). In the isolated perfused filtering kidney, the concentration of intact 125I-D in the perfusate declined 60.3 +/- 14.6% over 1 h. 83.4 +/- 6.3% of the decrement in 125I-D was catabolized by the kidney; the remainder was excreted in the urine as intact D. When glomerular filtration was prevented by increasing perfusate albumin concentration to 16 g/dl, perfusate intact (125I-D) remained unchanged over 1 h. These data show that human D is catabolized by the kidney via glomerular filtration and reabsorption by the proximal nephron. Reabsorption of D appears to be a saturable process.
P W Sanders, J E Volanakis, S G Rostand, J H Galla
Histidine-rich glycoprotein (HRGP) is a human plasma and platelet protein of apparently diverse biological functions. In this study a new interaction for HRGP is described. HRGP specifically interacts with fibrinogen as demonstrated by two independent systems. Using an enzyme-linked immunosorbent assay it was demonstrated that HRGP bound to adsorbed fibrinogen in a concentration-dependent and saturable manner, with an apparent dissociation constant (Kd) of 6.7 nM. The binding was specific, reversible, and not mediated by a conformationally altered adsorbed fibrinogen molecule. The interaction was divalent cation-dependent and ionic in nature. The HRGP-fibrinogen interaction was also demonstrated using rocket immunoelectrophoresis. The HRGP-fibrinogen interaction had an effect on the kinetics of conversion of fibrinogen to fibrin as demonstrated by a prolongation of the thrombin time. HRGP also became incorporated into fibrin clots in a concentration-dependent and saturable manner, with an apparent Kd of 0.25 microM. The incorporation of HRGP into fibrin clots occurred in a plasma milieu as demonstrated by the direct incorporation of radiolabeled HRGP into plasma clots and by a significant decrease in serum HRGP levels as compared with plasma levels. HRGP prolonged the lag time phase of fibrin gel formation, and decreased the rate of turbidity rise, as well as the final absorbance of fibrin gels. Since the extent of fibrin polymerization was not influenced by the presence of HRGP, these data suggest that fibrin is distributed over more, but thinner, fibrils in the presence of HRGP. In addition to its potential effect on fibrin polymerization, the HRGP-fibrin interaction may play a role in the cell-cell interactions of platelets and macrophages.
L L Leung
To determine the biochemical basis of the oxidant-induced injury of cells, we have studied early changes after exposure of P388D1 murine macrophages to hydrogen peroxide. Total intracellular NAD+ levels in P388D1 cells decreased with H2O2 concentrations of 40 microM or higher. Doses of H2O2 between 0.1 and 2.5 mM led to an 80% depletion of NAD within 20 min. With doses of H2O2 of 250 microM or lower, the fall in NAD and, as shown previously, ATP, was reversible. Higher doses of H2O2 that cause ultimate lysis of the cells, induced an irreversible depletion of NAD and ATP. Poly-ADP-ribose polymerase, a nuclear enzyme associated with DNA damage and repair, which catalyzes conversion of NAD to nicotinamide and protein-bound poly-ADP-ribose, was activated by exposure of the cells to concentrations of 40 microM H2O2 or higher. Activation of poly-ADP-ribose polymerase was also observed in peripheral lymphocytes incubated in the presence of phorbol myristate acetate-stimulated polymorphonuclear neutrophils. Examination of the possibility that DNA alteration was involved was performed by measurement of thymidine incorporation and determination of DNA single-strand breaks (SSB) in cells exposed to H2O2. H2O2 at 40 microM or higher inhibited DNA synthesis, and induced SSB within less than 30 s. These results suggest that DNA damage induced within seconds after addition of oxidant may lead to stimulation of poly-ADP-ribose polymerase, and a consequent fall in NAD. Excessive stimulation of poly-ADP-ribose polymerase leads to a fall in NAD sufficient to interfere with ATP synthesis.
I U Schraufstatter, D B Hinshaw, P A Hyslop, R G Spragg, C G Cochrane
A multitracer stable isotope study of lysine kinetics was carried out in fasted adult female volunteers to determine whether a multicompartmental model that partitions protein synthesis and breakdown into at least two types of tissue components can be constructed from plasma and breath data. Five female subjects, maintained on formula diets, received L-[13C1]lysine (27 mumol/kg) as an i.v. bolus and L-[15N2]lysine (27 mumol/kg) as an oral bolus 4 h postprandially. Plasma and breath samples were collected for 6 h. On an alternate day, subjects received NaH13CO3 (10 mumol/kg) as an i.v. bolus and breath samples were collected for 6 h. Plasma tracer lysine levels were determined by gas chromatography-mass spectrometry isotope ratiometry, and breath 13CO2 levels were measured by mass spectrometric gas isotope ratiometry. The tracer data could be fitted to a mammillary multicompartmental model that consisted of a lysine central compartment and slow- and fast-exchanging peripheral compartments containing 37, 38, and 324 mumol/kg, respectively. The rates of lysine oxidation, incorporation into protein, and release by protein breakdown were 21, 35, and 56 mmol/kg/h, respectively, in the fast-exchanging compartment, whereas the rates of protein synthesis and breakdown in the slow compartment were both 53 mmol/kg/min. These values corresponded to a whole-body lysine flux of 106 mmol/kg/h. The kinetic parameters were in excellent agreement with reported values obtained by constant-infusion methods. The measurements indicated that it will be possible to detect changes in amino acid pool sizes and protein synthesis and breakdown associated with the mobilization of protein stores from plasma and breath measurements in multitracer stable isotope experiments.
C S Irving, M R Thomas, E W Malphus, L Marks, W W Wong, T W Boutton, P D Klein