Rabbit antiidiotypic IgG directed against IgG F(ab')2 anti-tetanus toxoid (TT) antibodies ("idiotype") elicited a Prausnitz-Kustner reaction in normal skin sites sensitized 48 h earlier with the serum of the idiotype donor that contained IgE anti-TT antibodies. The serum moiety that caused the sensitization was heat sensitive (56 degrees C, 1 h), and was specifically removed by passage over immunosorbents containing rabbit antihuman IgE or TT antigen. The data obtained indicate that human IgG and IgE antibodies share idiotypic determinants and raise the possibility that idiotypic interactions may play a role in the regulation of the IgE antibody response in man.
R S Geha
Efforts to prevent Haemophilus influenzae type b (HIB) infections in infancy have been hampered by the low immunogenicity of capsular polysaccharide vaccines in children younger than 18 mos. In searching for alternate immunogens, we have studied the protective potential of polysaccharide-poor, lipid-rich endotoxin (LPS) core in experimental HIB infections. Because all gram-negative bacteria have similar LPS core structures, we were able to use as vaccine the J5 mutant of Escherichia coli 0111, the LPS of which consists only of core components, and thus to avoid problems in interpretation arising from vaccine contamination with non-LPS HIB immunogens. Mice were given graded inocula of HIB and developed lethal infection analogous to human HIB disease when virulence was enhanced with mucin and hemoglobin. After active immunization with heat-killed E. coli J5, 40/50 (80%) of infected mice survived, compared with 14/50 (28%) of saline-immunized controls (P less than 0.005). Passive immunization with rabbit antiserum against E. coli J5 prevented lethal HIB infection when administered 24 or 72 h before or 3 h after infection. This protection was abolished by adsorption of antiserum with purified J5 LPS, with survival reduced from 14/24 to 0/24 (P less than 0.005). Furthermore, rabbit antiserum to purified J5 LPS gave just as potent protection against death as antiserum to whole J5 cells. These studies demonstrate that immunity to core LPS confers protection against experimental murine HIB infection and provide the framework for a new approach to prevention of human disease from HIB.
M I Marks, E J Ziegler, H Douglas, L B Corbeil, A I Braude
Sex differences and steroid hormones are known to influence the vascular system as shown by the different incidence of atherosclerosis in men and premenopausal women, or by the increased risk of cardiovascular diseases in women taking birth control pills or men taking estrogens. However, the mechanisms for these effects in vascular tissues are not known. Since steroid actions in target tissues are mediated by receptors, we have looked for cytoplasmic steroid receptor proteins in vascular tissues of dogs. We find specific saturable receptors, sedimenting at 8S on sucrose density gradients for estrogens (measured with [3H]estradiol +/- unlabeled diethylstilbestrol), androgens (measured with [3H]R1881 +/- unlabeled R1881 and triamcinolone acetonide), and glucocorticoids (measured with [3H]dexamethasone +/- unlabeled dexamethasone); they are absent for progesterone (measured with [3H]R5020 +/- unlabeled R5020 and dihydrotestosterone). Progesterone receptors can, however, be induced by 1-wk treatment of dogs with physiological estradiol concentrations (100 pg/ml serum estrogen), indicating a functional estrogen receptor. Receptor levels range from 20 to 2,000 fmol/mg DNA. They are specific for each hormone; unrelated steroids fail to complete for binding. Low dissociation constants, measured by Scatchard analyses, show that binding is of high affinity. Steroid binding sites are in the media and/or adventitia since they persist when the intima is removed. Compared with the arteries, receptor levels are reduced 80% in inferior venae cavae of females, and are absent in the venae cavae of males. We hypothesize that steroid hormones can have direct effects on vascular tissues medicated by specific receptors present in arterial blood vessel walls.
K B Horwitz, L D Horwitz
Isolated cortical thick ascending limbs of Henle's loop were perfused in order to directly evaluate magnesium transport in this segment. Transepithelial potential difference was altered by varying the NaCl concentration in perfusate and bath and adding 50 microM furosemide to the perfusate. Perfusion under standard conditions with isotonic solutions resulted in a mean transepithelial potential difference of +8.8 +/- 0.7 mV and net magnesium absorption at a rate of 0.32 +/- 0.06 pmol/mm per min. Perfusion with a hypotonic solution significantly increased potential difference and the net absorptive rate of magnesium, calcium, and potassium. Conversely, reversal of the polarity of the potential difference with low NaCl bath and luminal furosemide produced net secretion of magnesium, calcium, and potassium. Parathyroid hormone in a bath concentration of 1.0 U/ml increased magnesium absorption from 0.32 +/- 0.06 to 0.63 +/- 0.06 pmol/mm per min (P less than 0.001) and calcium from 0.52 +/- 0.08 to 0.97 +/- 0.08 pmol/mm per min (P less than 0.001). Dibutyryl cyclic AMP produced similar effects on both calcium and magnesium absorption. Increasing bath calcium concentration twofold significantly inhibited net calcium absorption from 0.79 +/- 0.27 to 0.16 +/- 0.02 pmol/mm per min but magnesium transport was unaffected. Increasing bath magnesium concentration twofold significantly inhibited net magnesium absorption from 0.56 +/- 0.14 to -0.09 +/- 0.13 pmol/mm per min but had no effect upon net calcium transport. Net absorption of magnesium was significantly increased with increased concentration in the perfusate but calcium transport was unchanged. Similarly, increasing perfusate calcium concentration produced an increase in net calcium transport but did not alter magnesium transport. These data indicate that this segment of the loop of Henle is an important site for magnesium transport. Transport is influenced by luminal and bath concentration and is stimulated by parathyroid hormone and cyclic AMP. The data do not provide support for the concept of an interactive process between calcium and magnesium, and suggest that the positive transepithelial voltage is an important driving force for net reabsorption of magnesium, as well as calcium and potassium in this segment.
G R Shareghi, Z S Agus
The oppossum sphincter of Oddi (SO) exhibits peristaltic spike bursts with accompanying contraction waves that originate proximally in the sphincter of Oddi and propagate toward the duodenum. In this study we recorded myoelectrical activity of the opossum SO and upper gastrointestinal tract in six conscious animals using chronically implanted electrodes. Biopolar electrodes were implanted in the gastric antrum, duodenum, SO segment, jejunum, and ileum. During fasting the frequency of SO spike bursts, scored as number per minute, showed a cyclic pattern consisting of four phases (A to D). Phase A had a low spike burst frequency of ∼2/min that lasted ∼20 min. In phase B, the spike burst frequency increased progressively during a 40-45 min interval culminating in a short interval of phase C activity characterized by a maximal spike burst frequency of ∼5/min. During phase D, the spike bursts decreased over 15 min to merge with the low frequency of phase A and the cycle repeated. Cycle length of the interdigestive SO cycle, 87±11 SD min, was virtually identical with that of the interdigestive migrating myoelectric complex (MMC) of the upper gastrointestinal tract. The onset of phase C activity in the SO began 1-2 min before phase III of the MMC activity in the duodenum. Feeding abolished the cyclic pattern of spike burst activity in the SO as well as in the upper gastrointestinal tract. After feeding the SO spike bursts occurred at a frequency of 5-6/min for at least 3 h. We conclude that: (a) During fasting, the oppossum SO exhibits cyclic changes in its spike burst frequency; (b) Maximal spike burst frequency of the SO occurs virtually concurrent with passage of phase III MMC activity through the duodenum and; (c) Feeding abolishes the interdigestive cyclic spike burst pattern of the SO as well as that of the gastrointestinal tract.
Ryuichi Honda, James Toouli, Wylie J. Dodds, Sushil Sarna, Walter J. Hogan, Zen Itoh
Cardiovascular responses to intravenous administration of norepinephrine and the properties of alpha receptors on platelets were compared in normal human subjects and subjects with multiple system atrophy (MSA) and sympathetic degeneration. All the subjects with MSA had low plasma norepinephrine concentrations (in the supine position) (0.42 +/- 0.09 nM, normal 3.47 +/- 0.58 nM), which did not increase on tilt. The pressor sensitivity of subjects with MSA to norepinephrine infusion was increased 10- to 20-fold, demonstrating denervation supersensitivity to adrenergic agonists. Analysis of alpha receptors was by binding of [3H]dihydroergocryptine to platelets. Results are shown as mean +/- standard error of the mean. In the MSA subjects, the number of alpha receptors (1,712 +/- 699 fmol/10(8) platelets) was about sevenfold greater than in normal subjects (224 +/- 21 fmol/10(8) platelets), and the affinity, as measured by the equilibrium dissociation constant (Kd), was similar in both groups (MSA subjects, 9.6 +/- 4.3 nM; normal subjects, 4 +/- 0.5 nM). These observations suggest that an increase in alphaadrenergic receptor numbers may account for the denervation supersensitivity to infused norepinephrine in patients with sympathetic degeneration. All the subjects with MSA had low levels of the endogenous adrenergic transmitter norepinephrine: the simultaneous increase in alpha adrenergic receptors supports the theory of agonist regulation of receptor numbers.
B Davies, D Sudera, G Sagnella, E Marchesi-Saviotti, C Mathias, R Bannister, P Sever
Neutrophil turnover was studied in the blood and alveoli of normal rabbits. Blood neutrophil turnover was examined by two different methods. In the first method, donor rabbit neutrophils were labeled in vivo by injecting tritium-labeled thymidine intravenously. After 72 h recipient rabbits received blood from the donors. The decline of the specific radioactivity of blood neutrophils was used to determine that their half-life was 4.03 h. In the second method, rabbit peritoneal exudative neutrophils were elicited with oyster glycogen. These cells were labeled with 111Indium oxine and infused into the blood of recipient animals. By their decline in specific radioactivity, the half-life of the blood neutrophil was 4.08 h. These half-lives are not significantly different. Lung lavage was performed on the animals that received the 111Indium-labeled neutrophils and the turnover time of the lung neutrophil was found to be 2.63 h. The turnover of the alveolar neutrophil pool accounted for only 0.19% of the total turnover of the blood neutrophils. Therefore, the lung appears to contribute only minimally to the total capacity of the body to dispose of neutrophils.
A B Cohen, M Rossi, D Geczy, L Knight
The metabolism of thyroxine (T4) was studied in slices of rat pituitary gland and liver from the same animal incubated in vitro with [125I]T4 and 10 mM dithiothreitol. In the pituitary gland, generation of 125I-labeled 3,5,3′-triiodothyronine (T3), as well as overall T4 degradation, increased significantly at 24 h after thyroidectomy and by 2 wk were approximately five times control values. Conversely, following a single injection of T3 (1.5 μg/100 g body wt), values for both functions were significantly decreased at 4 h, and reached a nadir of ∼20% of control values at 12 and 24 h. Net T3-neogenesis accounted for ∼70% of T4 degradation in control pituitaries from intact rats. This proportion was increased by thyroidectomy and decreased by T3 replacement. Indirect evidence indicated that thyroidectomy decreased, and T3 administration increased, non-T3 generating pathways of T4 metabolism, probably 5-monodeiodination leading to formation of 3,3′5′-triiodothyronine (rT3). As judged from studies by others, the prompt changes in T4 metabolism that followed thyroidectomy or T3 administration could not be explained by changes in pituitary cell type. Changes in T3-neogenesis in liver were the converse of those in pituitary, and were much slower to occur.
Michiko Maeda, Sidney H. Ingbar
In this paper we show that the ferrochelatase defect in erythropoietic protoporphyria (EPP) can readily be identified in mitogen-stimulated lymphocytes since such cells from patients with EPP accumulate approximately twice as much protoporphyrin IX as cells from normal subjects when incubated with a porphyrin precursor, gamma-aminolevulinic acid (ALA). Treatment of cultures with ALA and with the iron chelator, CaMgEDTA significantly increased the level of protoporphyrin IX in mitogen-stimulated lymphocytes from normal subjects, while the same treatment failed to produce an increase in protoporphyrin IX in cell preparations from EPP patients. In contrast to the results with the chelator treatment, supplementation of the cultures with iron and ALA reduced the level of protoporphyrin IX in normal cells, but not in EPP cells. These findings are compatible with a partial deficiency of ferrochelatase in EPP lymphocytes. The gene defects of acute intermittent porphyria and hereditary coproporphyria have previously been identified using lymphocyte preparations from the gene carriers of these diseases. The present study demonstrates that EPP represents another form of human porphyria in which the gene defect of the disease can now be identified in lymphocyte preparations.
S Sassa, G L Zalar, M B Poh-Fitzpatrick, K E Anderson, A Kappas
Human cardiac ventricular myosins were prepared from autopsy samples from nine adults, seven infants, and from surgical specimens from seven patients undergoing left ventricular septal myectomy for obstructive hypertrophic cardiomyopathy. Infant myosin differed from adult myosin in two important characteristics: (a) ∼30% of the 27,000-dalton myosin light chain is replaced by a 28,000-dalton light chain, and (b) the actin-activated myosin MgATPase activity of infant myosin is significantly lower than that of adult myosin (64 nmol phosphate released/mg myosin per min vs. 124 nmol/mg per min at 37°C). The K+-EDTA ATPase activity of the myosin measured in 0.5M KCl is also lower in infants (1,210 nmol/mg per min vs. 620 nmol/mg per min at 37°C), but the Ca++-activated ATPase is not significantly different. There were no differences in enzymatic activity between the normal adult and cardiomyopathic myosins.
John J. Schier, Robert S. Adelstein
The dynamic equilibrium between in vivo occupied and unoccupied 1,25-dihydroxyvitamin D3[1,25(OH)2D3] receptors of the chick intestinal mucosa was investigated by the exchange assay previously reported [(1980). J. Biol. Chem.255: 9534-9537]. These parameters and their correlation to biological response, i.e., the levels of intestinal vitamin D-dependent calcium binding protein (CaBP), were assessed under different physiological conditions. After a single 1,25(OH)2D3 injection (3.25 nmol), occupied receptor levels increased sharply to a maximum between 1 and 2 h, followed by a rapid decline. A single dose of 1α-hydroxy-vitamin D3 [1α(OH)D3], an analog that requires 25-hydroxylation for biological activity, resulted in a protracted, albeit lower, response with maximal receptor occupancy at 6 h and half maximal levels 24 h after injection. The intestinal receptor occupancy patterns mirrored the serum 1,25(OH)2D3 levels after either 1,25(OH)2D3 or 1α(OH)D3 treatment. Additionally, time-course (half-life) of blood disappearance of 1,25(OH)2D3 and occupied receptor levels were similar (1.9 and 2.3 h, respectively), suggesting that the amount of occupied 1,25(OH)2D3 receptor is determined by a simple equilibrium between serum 1,25(OH)2D3 and unoccupied receptors. A dose-response study after intramuscular 1,25(OH)2D3 injection yielded a hyperbolic curve with an apparent plateau at 70% receptor occupancy, corresponding to 5 nmol 1,25(OH)2D3 injected. Half-maximal occupancy was reached after a dose of 1 nmol 1,25(OH)2D3, corresponding to 1.5 ng 1,25(OH)2D3/ml serum. From this value the apparent Kd in vivo is 3.7 nM, which is similar to that determined in vitro. A 10-fold increase in the 1α(OH)D3 dose resulted in less than a doubling of the levels of serum 1,25(OH)2D3, occupied 1,25(OH)2D3 receptors, or CaBP. Under all experimental conditions, there was a positive correlation between occupied receptor and CaBP levels; however, the slope of the lines depended on the times chosen for the assays due in part to the lag period for CaBP induction and its accumulation within the cell. Conversely, the correlation between serum 1,25-(OH)2D3 levels and occupied receptor levels yielded a single regression line independent of the observation time. Short and long-term treatment with different vitamin D metabolites, estrogen, progesterone, or cortisol did not affect the levels of total intestinal 1,25(OH)2D3 receptor. Under normal physiological conditions, only 10-15% of the total 1,25(OH)2D3 receptor population was occupied by ligand. These studies provide a basis for further investigations of physiological and biochemical parameters of the vitamin D endocrine system and their clinical applications.
Willi Hunziker, Marian R. Walters, June E. Bishop, Anthony W. Norman
Among 55 systemic lupus erythematosus patients having antibodies to Ro and/or La, two major groups were distinguished by titration of sera in counterimmunoelectrophoresis. The first group (30 patients) had antibodies to Ro alone. This was associated with a high incidence of antibodies to DNA (77%) and serious renal disease (53%). The second group (23 patients) had antibodies to Ro and La, and this was associated with a lower incidence of antibodies to DNA (30%) and a very low incidence of nephritis (9%). In this group a phenomenon of linkage of anti- Ro and anti-La titers was observed. Additionally two patients with only anti-La were found. Neither had clinically apparent renal disease. Thus, systemic lupus erythematosus patients with anti-Ro fall into two subgroups that differ considerably in their prevalence of anti-DNA and serious renal disease.
C A Wasicek, M Reichlin
Natural killer cells spontaneously lyse certain tumor cells and may defend against malignancy. We have previously shown that natural killing (NK) by human peripheral blood mononuclear cells (PBMC) is suppressed in vitro by phorbol diester tumor promoters, including 12-O-tetradecanoylphorbol-13-acetate (TPA). We here demonstrate that suppression of NK is mediated by monocytes or polymorphonuclear leukocytes (PMN) and that suppression is dependent on the generation of reactive forms of molecular oxygen (RO), particularly hydrogen peroxide (H2O2). NK was suppressed not only by TPA but also by opsonized zymosan (yeast cell walls), which, like TPA, was not toxic to PBMC. Both TPA and zymosan stimulated the production of superoxide anion (O2-) and H2O2 by PBMC. Production of RO correlated with suppression of NK. When PBMC were depleted of monocytes, the production of RO and the suppression of NK were both markedly reduced. Suppression could be restored by monocytes or PMN, both of which produced RO in response to TPA or zymosan. Suppression of NK was dependent on RO. Monocytes or PMN from a patient with chronic granulomatous disease, whose cells cannot generate RO, did not mediate suppression of NK. Suppression was also reduced in glucose-free medium, which did not support the generation of RO. Suppression of NK by TPA was inhibited by catalase. Bovine superoxide dismutase had a limited effect on suppression, even in high concentration, and tyrosine-copper (II) complex, which also enhances dismutation of O2- to H2O2, had almost no effect on suppression. When H2O2 was directly generated enzymatically from glucose oxidase and glucose, NK was suppressed and suppression was reversed by catalase. NK was also suppressed by the enzymatic generation of O2- from xanthine oxidase and xanthine, but suppression under these conditions was again inhibited by catalase and not by superoxide dismutase, indicating that suppression was due to the secondary formation of H2O2 from O2-. These results indicate that H2O2 is important in suppression of NK. Myeloperoxidase did not appear to play a role in suppression because inhibition of this enzyme by sodium azide, cyanide, or aminotriazole did not prevent suppression of NK. Suppression of NK was reversible; after exposure to zymosan, NK could be partially restored by the addition of catalase and superoxide dismutase or by the removal of zymosan. These studies demonstrate cellular regulation of NK by monocytes or polymorphonuclear leukocytes and indicate a role for RO in immunoregulation.
W E Seaman, T D Gindhart, M A Blackman, B Dalal, N Talal, Z Werb
A heat-stable neutrophil chemotactic factor (NCF) has been identified in the serum of 13 atopic asthmatic subjects after treadmill exercise. Peak activity was detected at 10 min and returned to prechallenge values by 1 h. No NCF activity was detected in the sera of three nonasthmatic atopic or four normal nonatopic individuals performing the same task. NCF produced by exercise (NCFEX) had a similar time-course of release to NCF provoked by specific antigen (NCFAG). The appearance of circulating NCFEX and NCFAG closely paralleled the fall in peak expiratory flow rate/forced expiratory volume in 1 s (PEFR/FEV1). Histamine challenge in atopic asthmatics at concentrations giving a comparable change in PEFR/FEV1 to that evoked by exercise or inhaled antigen was not associated with the appearance of circulating NCF. In seven subjects NCFEX release was inhibited by prior administration of disodium cromoglycate. NCFEX and NCFAG eluted as single peaks of activity when applied separately to columns of Sephadex G-200, and both were an estimated 750,000 daltons. NCFEX and NCFAG also eluted as single peaks of activity, at between 0.15 and 0.30 M NaCl, following anion exchange chromatography on DEAE-Sephacel (pH 7.8). The isoelectric points of NCFEX and NCFAG were virtually identical (between pH 6.0 and 6.5) as determined by chromatofocusing on Polybuffer Exchanger 94. The activities of NCFEX and NCFAG were substantially reduced, in both a time- and dose-dependent fashion, after incubation with trypsin and chymotrypsin. Partially purified NCFEX and NCFAG promoted both stimulated random migration (chemokinesis) as well as directional migration (chemotaxis). These experiments indicate that NCFEX and NCFAG might be identical substances and raise the possibility that mediators by hypersensitivity are released during exercise-induced asthma in atopic subjects.
T H Lee, L Nagy, T Nagakura, M J Walport, A B Kay
The presence and distribution of C3b receptors in normal human kidneys and in biopsies from 75 patients with renal disease were investigated by immunohistochemical techniques using monospecific rabbit antibody to the 205,000-mol wt glycoprotein that is the C3b receptor of human peripheral blood cells. Anti-C3b receptor bound exclusively to podocytes in normal renal cortex, and was homogeneously distributed on the plasma membrane of these cells. Biosynthesis of the receptor by the podocyte was suggested by the presence of antigenic activity in the Golgi apparatus. Although occupancy of receptor sites following the interaction of kidney sections with aggregated IgG preincubated with normal serum inhibited binding to glomeruli of C3b coated cells, the C3b receptor remained accessible to anti-C3b receptor antibody. No staining of podocytes was found in extra-capillary proliferating cells in rapidly progressive glomerulonephritis (GN). Segmental loss of staining was found in focal hyalinosis, nodular diabetic glomerulosclerosis, and amyloidosis while no detectable C3b receptor antigen was found in severe proliferative nephritis of systemic lupus erythematosus (SLE). Normal staining of podocytes was found in other nephropathies with endocapillary proliferation such as acute GN and mesangial GN and in renal diseases associated with immune deposits containing C3 such as mesangial proliferative and membranous SLE nephritis, idiopathic membranous GN, membranoproliferative GN types I and II, mesangial GN with IgA or C3 deposition and Henoch Schönlein's purpura. Loss of C3b receptor antigen in the diffuse proliferative nephritis of SLE distinguishes it both from nonproliferative lupus nephritis and other immunologically mediated proliferative GN.
M D Kazatchkine, D T Fearon, M D Appay, C Mandet, J Bariety
Mevinolin reduces cholesterol synthesis by inhibiting 3-hydroxy-3-methylglutaryl-coenzyme A reductase. The safety and effectiveness of this agent was evaluated in a double-blind, placebo-controlled study in 59 healthy men (serum cholesterol 3.88--7.76 mmol/liter) in five centers. Subjects maintained their usual diet and activities. Doses of 6.25, 12.5, 25, or 50 mg twice daily for 4 wk produced mean reductions of total serum cholesterol fo 23--27% [vs. placebo (4%), P less than 0.01]. Mean low density lipoprotein cholesterol fell 35--45%, while high density lipoprotein and very low density lipoprotein cholesterol, and triglycerides were not significantly affected. Mean apolipoprotein B fell 27--34%. 50 mg was not significantly more effective than 6.25 mg. Mevinolin was generally well tolerated, and no serious clinical or laboratory abnormalities occurred. One subject (12.5 mg) was withdrawn because of abdominal pain and diarrhea. These results suggest that if long-term safety can be demonstrated, inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase are likely to prove useful in the treatment of hypercholesterolemia.
J A Tobert ... Y S Chao, J A Bolognese
Radiolabeled, enzymatically active or chloromethyl ketone-inactivated porcine pancreatic elastase was endotracheally instilled into hamsters. Gel filtration of the bronchopulmonary lavage fluid revealed two major radioactive fractions: one, eluting at 780,000 daltons, corresponding to an alpha-macroglobulin-pancreatic elastase complex, and another, at 68,000 daltons, corresponding to an alpha-1-protease inhibitor-pancreatic elastase complex. Elastolytic activity was recovered in the bronchopulmonary lavage fluid up to 4 d after elastase instillation and was associated with the alpha-macroglobulin-pancreatic elastase complex. Small amounts of this complex were recovered 14 d after instillation. When less than 1% (1.5--1.7 micrograms) of the usual dose of elastase was instilled into hamsters, the major radioactive complex was alpha-1-protease inhibitor-pancreatic elastase complex, and little or no elastolytic activity was found in the lavage fluid. In contrast to the instillation of 220 micrograms of elastase, no disease or hemorrhagic reaction was detected with this low dose, and without hemorrhage only insignificant amounts of alpha-macroglobulin-pancreatic elastase complexes were recovered from the lungs. To study the interaction of circulating antiproteases with elastase, hamster plasma was allowed to interact directly with the radiolabeled elastase; alpha-macroglobulin bound much more of the elastase than alpha-1-protease inhibitor, confirming the findings in the lung lavage experiments. The hamster's susceptibility to pancreatic elastase-induced emphysema may depend on the preferential binding of elastase to alpha-macroglobulin, which protects the elastolytic potential, rather than to alpha-1-protease inhibitor, which inactivates elastase. We speculate that if even a fraction of the residual radioactivity found in the hamster lungs as long as 144 d after instillation of elastase represents enzymatically active alpha-macroglobulin-pancreatic elastase complex, this could serve as a source of persistent elastolytic activity, which might explain the progressive nature of the pulmonary lesion.
P J Stone, J D Calore, G L Snider, C Franzblau
An abnormal triglyceride-rich lipoprotein has been isolated from some patients with chronic renal failure or severe hypertriglyceridemia. The abnormal lipoprotein was characterized by an increased content of apolipoprotein (apo) C-III-2 (57.5% of total apo C-III peptides compared with 35.5% for controls, P less than 0.001) as characterized by isoelectric focusing and scanning densitometry. As determined by a substrate competition assay, the abnormal lipoprotein was a less efficient substrate for purified bovine milk lipoprotein lipase than control lipoproteins. Neuraminidase digestion of abnormal or control lipoprotein resulted in a reduction of the apo C-III-2 band with a corresponding increase in the region of apo C-III-0, which suggests that the increased content of apo C-III-2 in the abnormal is due to excessive sialylation of the C-III peptide. Limited incubation of the abnormal lipoproteins with neuraminidase caused a partial loss of sialic acid and resulted in a triglyceride-rich lipoprotein with a normal C-III-2:C-III-1 ratio. This preparation displayed normal substrate interaction with lipoprotein lipase. Three severely hypertriglyceridemic patients with the abnormal lipoprotein showed a marked reduction in serum triglyceride concentration, which is associated with a reversion to a normal C-peptide profile after dietary therapy. The results suggest that the extent of sialylation of the apo C-III peptide carried on triglyceride-rich lipoproteins may be critical for their interaction with lipoprotein lipase.
G Holdsworth, J Stocks, P Dodson, D J Galton
Patients with systemic lupus erythematosus (SLE) were found to have in their plasma antibodies specific for desialized T cells. Adsorption studies with intact or desialized T cells indicated that SLE anti-T cell antibodies consisted of two populations with different target cell specificities, one capable of recognizing unique determinants on desialized T cells and another able to bind to both intact and desialized T cells. Normal T cells did not remove the antibodies specific for desialized T cells. moreover, the antibodies to desialized T cells were not removed by adsorption with either desialized non-T cells or desialized erythrocytes. Thus, the antibodies to desialized T cells recognize a determinant that is unique to a T cell subset and also includes a sugar. Inhibition studies with various sugars indicated that lactose was the most potent inhibitor of antibody binding. The anti-desialized T cell antibody appears to recognize a T cell determinant which includes lactose, probably in the form of a beta-galactosyl residue, but which also includes additional T cell determinants. The antibodies to desialized T cells were found to bind preferentially to concanavalin A-induced autorosetting T cells, which had been already demonstrated to contain suppressor effector cells. Indeed, such antibodies were effective in eliminating suppressor effector function without interfering with T cells necessary for such activation (such as precursor or inducer cells). Finally, studies of patients with SLE yielded a highly significant correlation (r = 0.92) between impaired suppressor effector function of their cells and the presence of antibodies to desialized T cells in their plasma.
M Honda, T Sakane, A D Steinberg, H Kotani, T Tsunematsu, K Moriyama, M Fukase
The cofactor function of human Factor VIII in Factor X activation was investigated by an initial-rate assay of 3H-Factor X activation in the presence of human factor IXa, Ca2+, and either phospholipid or fresh washed human platelets. Purified Factor VIII that has not been activated by thrombin or Factor Xa supports Factor X activation after a lag of several minutes. A specific inhibitor of Factor Xa, which had no inhibitory activity against Factor IXa, markedly prolonged this lag, whereas specific thrombin inhibitors did not prolong the lag. These data support the conclusion that unactivated Factor VIII has no ability to support Factor X activation in a purified system until it is activated by Factor Xa feedback during the lag period. When Factor VIII was optimally preactivated by thrombin, the lag was completely abolished, regardless of the order of addition of the other reactants or the phospholipid source. These data indicate that there is no slow, time-dependent ordering of the reactants at the phospholipid or activated platelet surface if Factor VIII has been preactivated. Unactivated platelets did not support Factor X activation by Factors IXa and VIII. The effect of activated Factor VIII on the kinetics of bovine Factor X activation was primarily to increase the Vmax (54-fold), whereas with human Factor X, Factor VIII both increased the Vmax 56-fold and decreased the Km sixfold to 0.14 microM, similar to the plasma concentration of Factor X. Therefore, a change in the plasma factor X concentration would be expected to have a major effect on the rate of Factor X activation in vivo.
M B Hultin
Previous studies have suggested that a cationic bactericidal/permeability-increasing protein (BPI) present in both rabbit and human polymorphonuclear leukocytes is the principal O2-independent bactericidal agent of these cells toward several strains of Escherichia coli and Salmonella typhimurium (1978. J. Biol. Chem. 253: 2664--2672; 1979. J. Biol. Chem. 254: 11000--11009). To further evaluate the possible role of this protein in the killing of gram-negative bacteria by polymorphonuclear leukocytes, we have measured the bactericidal activity of intact rabbit peritoneal exudate leukocytes under aerobic or anaerobic conditions and of intact human leukocytes from a patient with chronic granulomatous disease. Anaerobic conditions were created by flushing the cells under a nitrogen stream. Effective removal of oxygen was demonstrated by the inability of nitrogen-flushed leukocytes to mount a respiratory burst (measured as increased conversion of 1-[14C]glucose leads to 14CO2 or by superoxide production) during bacterial ingestion. At a bacteria/leukocyte ratio of 10:1, killing of gram-positive, BPI-resistant, Staphylococcus epidermidis is markedly impaired in the absence of oxygen (76.4 +/- 3.3% killing in room air, 29.2 +/- 8.2% killing in nitrogen). Essentially all increased bacterial survival is intracellular. In contrast, both a nonopsonized rough strain (MR-10) and an opsonized smooth strain (MS) of S. typhimurium 395 are killed equally well in room air and nitrogen. A maximum of 70--80 MR-10 and 30--40 MS are killed per leukocyte either in the presence or absence of oxygen. There is no intracellular bacterial survival in either condition indicating that intracellular O2-independent bactericidal system(s) of rabbit polymorphonuclear leukocytes can at least match the leukocyte's ingestive capacity. Whole homogenates and crude acid extracts manifest similar bactericidal capacity toward S. typhimurium 395. This activity can be accounted for by the BPI content of these cell fractions and is virtually eliminated by immune (anti-BPI), but not by preimmune goat IgG-rich fractions. Opsonization of smooth MS, required for bacterial killing by intact leukocytes, does not alter bacterial sensitivity to BPI in crude or purified form. Leukocytes of a patient with chronic granulomatous disease killed ingested S. typhimurium 396 MS nearly as well as did normal leukocytes. The bactericidal activity toward E. coli (J5) of crude acid extracts of the CGD and normal human leukocytes was virtually the same and was nearly completely inhibited by anti-BPI IgG-rich fractions, but not by preimmune IgG-rich fractions. These findings suggest that the killing of gram-negative bacteria such as S. typhimurium by intact polymorphonuclear leukocytes may also be attributed to the action of BPI.
J Weiss, M Victor, O Stendhal, P Elsbach
In previous reports from this laboratory we have suggested that a reduction in medullary tonicity decreases the thin ascending loop of Henle sodium reabsorption and is in part responsible for the magnitude for the natriuresis accompanying 10% body weight Ringer loading. According to this postulate, one would expect that the medullary washout associated with water diuresis would also result in a natriuresis, but this does not occur. It is possible, however, that increased delivery from the proximal tubule is necessary to demonstrate an effect of medullary tonicity on urinary sodium excretion. Micropuncture studies were designed to test that possibility by increasing distal delivery by 2% Ringer loading in animals with and without reduced medullary tonicity. In an initial series of experiments the α-adrenergic agonist clonidine was used to induce a water diuresis. When given alone, this agent caused a marked decrease in urine osmolality and an increase in urine flow rate but had no effect on proximal reabsorption in either superficial or juxtamedullary nephrons, and did not alter urinary sodium excretion. Volume expansion with 2% body weight Ringer solution resulted in a significant fall in proximal reabsorption and a trivial increment in sodium excretion. When this same degree of volume expansion was conferred on animals undergoing a water diuresis, a marked increase in absolute and fractional sodium excretion occurred. In a second group of studies medullary tonicity was reduced in the left kidney only by removal of the left ureter 1 h before micropuncture. When these animals were infused with 2% body weight Ringer solution, proximal reabsorption was decreased in juxtamedullary nephrons, and a marked increase in sodium excretion was observed only from the left kidney. Finally, the effect of water diuresis on fractional sodium delivery to the early and late distal tubule of superficial nephrons during 2% Ringer loading was evaluated. Delivery to both of these sites was comparable after 2% Ringer loading alone and during 2% Ringer loading plus water diuresis.
H. John Reineck, Retta Parma
Rapid identification of Haemophilus influenzae and other bacillary meningitides was attempted by gas-liquid chromatography (GLC) of the metabolic by-products in broth cultures and in cerebrospinal fluid (CSF) samples obtained from experimental meningitis produced in New Zealand White male rabbits. These results were correlated with the GLC of CSF of meningitis patients. A major peak with retention time of succinic acid was found in the broth cultures of all bacilli tested including H. influenzae, Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae, Proteus mirabilis, Citrobacter freundii, Pseudomonas aeruginosa, and Listeria monocytogenes. Succinic acid was also found in the CSF of experimental meningitis and in the CSF of all patients with H. influenzae and Esch. coli meningitis. This peak was not detected in the blood samples of experimental animals. It was also absent in the broth cultures of all of the gram-positive and gram-negative cocci tested, such as Streptococcus pneumoniae and Neisseria meningitidis. Succinic acid, which appears to be a by product of fermentation, persisted as a clear cut marker in H. influenzae meningitis for at least 3 d after the initiation of treatment. In one patient, the succinic acid peak disappeared during treatment and reappeared with a clinical relapse. Clearly, the presence of succinic acid that can be rapidly detected by GLC in the CSF excludes pneumococcal or meningococcal meningitis and strongly suggests H. influenzae or other bacillary meningitides.
H Thadepalli, P K Gangopadhyay, A Ansari, G D Overturf, V K Dhawan, A K Mandal
Previous studies showed that deoxycholic acid (DCA) stimulated migrating action potential complexes (MAPC) in the colon. The aim of this study was to clarify the mechanism of DCA-stimulated colonic motility. Myoelectrical and contractile activity were measured in New Zealand White rabbits from a loop constructed in the proximal colon. During the control period, slow waves were present at a frequency of 10.8 +/- 0.5 cycle/min and there were 1.5 +/- 0.5 MAPC/ h. After adding DCA (16 mM) to the loop the slow wave activity was unchanged. However, MAPC increased to 15.1 +/- 2.4 MAPC/h (P less than 0.001). MAPC activity was not stimulated in the colonic smooth muscle outside the loop. The intraluminal addition of procaine or tetrodotoxin to the colonic loop inhibited the DCA-stimulated increase in MAPC activity (0.2 +/- 0.2 MAPC/h) (P less than 0.005). Intravenous administration of atropine or phentolamine also inhibited MAPC activity that had been stimulated by DCA (P less than 0.005). Pretreatment with 6-hydroxydopamine also inhibited an increase in MAPC activity. Propranolol, trimethaphan camsylate, or hexamethonium had no effect on DCA stimulation of MAPC activity. Although the concentration of bile salt increased in the mesenteric venous outflow from the colonic loop, the intravenous administration of bile salt did not stimulate colonic MAPC activity. These studies suggest: (a) the action of DCA on smooth muscle activity is a local phenomenon, (b) the increase in MAPC activity is dependent on intact cholinergic and alpha adrenergic neurons, and (c) an increase in the concentration of bile salts in the serum is not associated with an increase in colonic MAPC activity.
S J Shiff, R D Soloway, W J Snape Jr
5(S), 12(S)-Dihydroxy-cis-14,trans-6,8,10-eicosatetraenoate (compound I), 5(S),12(R)-dihydroxy-cis-14,trans-6,8,10-eicosatetraenoate (compound II), and 5(S),12(R)-dihydroxy-cis-6,14,trans-8,10-eicosatetraenoate (compound III) were prepared from rabbit peritoneal neutrophils challenged with arachidonic acid plus ionophore A23187. Each arachidonate metabolite caused rabbit neutrophils to aggregate and, in cells treated with cytochalasin B, release granule-bound enzymes. Compound III was 10- to 100-fold more potent than compounds II and I. When intravenously infused into rabbits at doses of 100--1,000 ng/kg, compound III induced abrupt, profound, transient neutropenia associated with a rapidly reversing accumulation of neutrophils in the pulmonary circulation. This in vivo action correlated closely with the ability of the fatty acid to activate neutrophils in vitro: neutropenia, aggregation, and degranulation occurred at similar doses of stimulus and the rapid, reversing kinetics of the neutropenic response paralleled the equally rapid, reversing formation of aggregates. The fatty acid did not alter the circulating levels of lymphocytes or platelets and did not aggregate platelets in vitro. At comparable doses (i.e., 100--1,000 ng/kg), compounds I and II did not cause neutropenia. Thus, compound III possesses a high degree of structural and target-cell specificity in stimulating neutrophils in vitro and in vivo. Clinical and experimental syndromes associating neutropenia with increased levels of circulating arachidonate metabolites may involve compound III as a mediator of neutrophil sequestration in lung.
J T O'Flaherty, M J Thomas, S L Cousart, W L Salzer, C E McCall
The survival of erythrocytes (RBC) is shortened in uremia, and it has been shown that calcium influx into RBC evoked crenation and increased their rigidity. The high blood levels of parathyroid hormone (PTH) may augment entry of calcium into RBC and hence affect their integrity. We examined the effect of PTH on osmotic fragility of human RBC and investigated the mechanisms through which PTH interacts with RBC.
Eitan Bogin, Shaul G. Massry, Joseph Levi, Meir Djaldeti, Greg Bristol, Jacqueline Smith
Systemic lupus erythematosus (SLE) is an autoimmune disease with multiple immune disturbances whose mechanisms remain unclear. We examined the interaction of antilymphocyte antibodies with cultured normal T lymphocytes. T cells were prepared by E-rosetting after petri-dish removal of adherent cells and cultured for 2-7 d in the presence of SLE sera or normal human sera. Cultured T cells were washed and sonicated, and the amount of cell-associated IgG was quantitated by radioimmunoassay or enzyme-linked immunoassay (ELISA) methods. T cells cultured with 27 of 39 SLE sera showed marked increments of associated immunoglobulin G (IgG) although this was not observed with sera from mixed connective tissue disease patients containing high titers of ribonucleoprotein antibody or normal donors. The effective factors for IgG association in SLE sera were absorbed with normal peripheral blood lymphocytes or T cells. Anti-T cell IgG cytotoxic activity strongly correlated with T cell IgG association (P less than 0.01). T cell-associated IgG was not removed by stripping of cell membrane IgG from living cells by acid buffer treatment; indirect immunofluorescence of cells fixed after 2-4 d of culture revealed cytoplasmic IgG staining. IgG anti-T cell antibodies appeared to associate inside the cell membrane or to penetrate into the cytoplasm of cells. T cell Fc receptor blocking by heat-aggregated IgG or anti-beta 2-microglobulin antibody did not alter IgG cell association. Since pepsin-digested SLE sera showed no T cell association activity, whole IgG antibody molecules appeared to be necessary for interaction with cultured T cells. In addition, reduction and alkylation of active SLE sera completely nullified T cell reactivity. When normal T cells were cultured with SLE sera showing marked IgG T cell association, viability of cultured T cells decreased rapidly after 4 d, which suggests that IgG anti-T cell antibodies were associated with cell destruction. IgG cell-associating antilymphocyte antibodies present in SLE sera may cause T cell disturbances in vivo and may be related to the lymphocytopenia present in SLE patients.
K Okudaira, R P Searles, K Tanimoto, Y Horiuchi, R C Williams Jr
We studied the effect of retinoic acid on the clonal proliferation of normal human early erythroid progenitor cells in vitro. Normal peripheral blood cells were cultured in methylcellulose with erythropoietin and the number of burst-forming units-erythroid (BFU-E) colonies were scored on day 12 of culture. All-trans retinoic acid increased the number of colonies in a dose-response fashion. Maximal stimulation occurred at 30 nM retinoic acid, which increased the number of BFU-E by a mean of 225 +/- 25% (+/- SE) over plates containing erythropoietin alone. Colony formation increased even in the presence of maximally stimulating concentrations of erythropoietin. The 13-cis retinoic acid stimulated BFU-E proliferation in a parallel manner as the trans analogue, while retinol (vitamin A) did not affect clonal growth. This data supports further the thesis that retinoic acid, in addition to its known effect on epithelial cells, may be involved in the growth of normal hematopoietic cells.
D Douer, H P Koeffler
When virulent group A streptococci of M type 24 were incubated in fresh heparinized whole blood or in blood reconstituted from cellular elements and plasma, little uptake by neutrophils occurred as determined by light microscopy. When fresh human serum (with or without added heparin) was substituted for plasma, uptake occurred promptly. Uptake in serum could be prevented by adding either plasma or purified human fibrinogen to the incubation mixtures, or by pretreating the organisms with plasma or fibrinogen. Fibrinogen solutions absorbed with purified homologous M protein and centrifuged to remove precipitates lost their inhibitory activity. Uptake in serum depended on heat-labile factors. Immunofluorescent staining of bacteria using fluorescein-labeled antibody to the third component of complement showed that streptococci incubated in fresh serum bound complement evenly over the entire cell surface, whereas streptococci incubated in fresh plasma or in serum plus fibrinogen fluoresced only at some of the cross-walls between adjacent daughter cocci and at occasional terminal cocci. In electron micrographs, the surface fibrillar layer of streptococci treated with plasma or fibrinogen lost its hairlike appearance and became a dark band that stained heavily with ferritin-labeled antifibrinogen. We conclude that the known antiopsonic effect of M protein derives in part from binding of fibrinogen.
E Whitnack, E H Beachey
The platelet-derived growth factor (PDGF) is shown to be chemotactic for monocytes and neutrophils. Maximum monocyte chemotaxis to PDGF is fully equal to that achieved with C5a and occurs at congruent to 20 ng/ml (congruent to 0.7 nM). Maximum neutrophil chemotaxis is congruent to 60% that achieved with C5A but occurs at congruent to 1 ng/ml (congruent to 32 pM). The chemotactic activity of PDGF is blocked by specific antisera to PDGF and by protamine sulfate, a competitive inhibitor of PDGF binding to cell surfaces. In contrast to PDGF, epidermal growth factor shows no chemotactic activity for inflammatory cells at 0.5-100 ng/ml. The high level of chemotactic activity of PDGF suggests that in addition to its role as a mitogen for smooth muscle cells and fibroblasts, PDGF may be involved in attracting inflammatory cells to sites of platelet release.
T F Deuel, R M Senior, J S Huang, G L Griffin
Hemoglobin E interacts with beta-thalassemia to produce a disorder of variable severity that is the most common form of symptomatic thalassemia in Southeast Asia. The beta E-globin gene acts as a mild thalassemia gene; there are low levels of beta E-messenger RNA (mRNA) in reticulocytes, and preliminary evidence had suggested that this might be due to instability of the beta E-mRNA. Analysis of beta E-mRNA levels in the nuclei and cytoplasm of bone marrow erythroblasts compared with reticulocytes has shown higher levels of beta E-mRNA in the former, providing direct evidence that this is the case.
J Traeger, P Winichagoon, W G Wood
Muscle glycogen stores are depleted during exercise and are rapidly repleted during the recovery period. To investigate the mechanism for this phenomenon, untrained male rats were run for 45 min on a motor-driven treadmill and the ability of their muscles to utilize glucose was then assessed during perfusion of their isolated hindquarters. Glucose utilization by the hindquarter was the same in exercised and control rats perfused in the absence of added insulin; however, when insulin (30-40,000 μU/ml) was added to the perfusate, glucose utilization was greater after exercise. Prior exercise lowered both, the concentration of insulin that half-maximally stimulated glucose utilization (exercise, 150 μU/ml; control, 480 μU/ml) and modestly increased its maximum effect. The increase in insulin sensitivity persisted for 4 h following exercise, but was not present after 24 h. The rate-limiting step in glucose utilization enhanced by prior exercise appeared to be glucose transport across the cell membrane, as in neither control nor exercised rats did free glucose accumulate in the muscle cell.
Erik A. Richter, Lawrence P. Garetto, Michael N. Goodman, Neil B. Ruderman
Factor XIa is a plasma protease that, by activating Factor IX, plays an important role in the early phase of the intrinsic pathway of blood coagulation. Four plasma protease inhibitors, α1-protease inhibitor, antithrombin III, C1-inhibitor, and α2-plasmin inhibitor, have been reported to inactivate human Factor XIa, but their quantitative contribution to the inactivation of Factor XIa in plasma has not been fully assessed. Using purified systems, we observed that the second-order rate constants for the reaction of Factor XIa with α1-protease inhibitor, antithrombin III, and CI-inhibitor were 4.08, 10, and 14.6 M−1 min−1 × 103, respectively. The pseudo-first-order rate constants, at plasma concentration of the inhibitors, were 1.86 × 10−1, 4.68 × 10−2, and 2.4 × 10−2 min−1, respectively. These kinetic data predict that α1-protease inhibitor should account for 68%, antithrombin III for 16%, and C1-inhibitor and the equipotent α2-plasmin inhibitor each for 8% of the total inhibitory activity of plasma against Factor XIa. The rate of inactivation of Factor XIa in various plasma samples specifically deficient in inhibitors was consistent with these predictions.
Cheryl F. Scott, Marc Schapira, Harold L. James, Allen B. Cohen, Robert W. Colman
The transferrin receptor, present on reticulocytes and nucleated cells in tissue culture, has been measured with both immunoassay techniques and transferrin binding studies. The total cellular immunoreactive receptor is rapidly lost from erythrocytes during the process of reticulocyte maturation (from as many as 400,000 molecules to <20,000 molecules/reticulocyte). This event parallels the loss of cell surface transferrin binding sites and RNA content, and correlates with previous studies that have measured the decline in hemoglobin synthesis.
Janet L. Frazier, Jennifer H. Caskey, Mark Yoffe, Paul A. Seligman
We have recently proposed that “down regulation” of the insulin receptor may be one of the many biological responses of a cell to insulin. In an attempt to further explore this hypothesis we have studied insulin action, binding, and degradation in freshly isolated hepatocytes from rats rendered insulin resistant by the administration of dexamethasone, 1.0 mg/kg every other day, for 1 and 4 wk, and in dexamethasone-treated (0.1 μM for 24 h) primary cultures of hepatocytes from normal rats.
Jose F. Caro, John M. Amatruda
Production of active lysosomal enzymes may involve limited proteolysis of inactive high molecular weight precursors. Precursor processing potentially regulates lysosomal enzyme activity. To test whether rabbit cardiac cathepsin D is first synthesized as a precursor and whether prolonged fasting (a condition affecting both cathepsin D and total cardiac protein turnover) influences precursor processing, rates of cathepsin D synthesis and processing were compared in left ventricular slices of control and 3-d-fasted rabbits incubated in vitro with [35S]methionine. 35S-labeled cathepsin D was isolated by butanol-Triton X-100 extraction, immunoprecipitation, and dodecyl sulfate-polyacrylamide gel electrophoresis. Total cardiac protein synthesis was measured by tracer incorporation and normalized for differences in precursor pool size by direct measurement of [35S]aminoacyl-tRNA-specific radioactivity. Relative cathepsin D synthetic rates were obtained by comparing 35S incorporation into cathepsin D with 35S incorporation into all cardiac proteins. Enzyme processing was assessed in pulse-chase experiments and assayed by autoradiography. The results indicate that (a) rabbit cardiac cathepsin D is synthesized as a precursor (53,000 mol wt) that is processed to a 48,000-mol wt form, (b) rates of both cathepsin D and total cardiac protein synthesis are similar in control and fasted rabbits, suggesting that decreased enzyme degradation rather than increased synthesis is responsible for the elevated levels of cardiac cathepsin D in starvation, and (c) cathepsin D processing in hearts of fasted animals is incomplete, with accumulation of the precursor during pulse-chase experiments of 6 h duration. Based upon these results, a three-stage model for the regulation of cathepsin D activity in rabbit heart is proposed.
Allen M. Samarel, Edward A. Ogunro, Alan G. Ferguson, Patricia Allenby, Michael Lesch
The effects of dopamine on active intestinal ion transport have been evaluated. An epithelial sheet preparation of rabbit ileum was used in vitro with the Ussing chamber-voltage clamp technique. Dopamine, in the presence of 1 mM ascorbic acid, added to the serosal bathing solution caused a dose-dependent decrease in short-circuit current, with a half-maximal effect at 1.2 μM and maximal effect of −50 μA/cm2 at 50 μM; dopamine decreased the potential difference, and increased the conductance and net Na and net Cl absorption. There was no effect on the residual ion flux. Dopamine did not alter the change in short-circuit current caused by mucosal glucose (10 mM) or serosal theophylline (10 mM). Mucosal dopamine had no effect. The effect of dopamine on short-circuit current was inhibited by the dopamine antagonists haloperidol and domperidone and the α2-adrenergic antagonist yohimbine; there was no effect of the α1-antagonist prazosin and the β-antagonist propranolol. In addition, the α2-adrenergic agonist clonidine, but not the α1-agonist methoxamine caused a dose-dependent decrease in short-circuit current. The ileal effects of dopamine did not occur via conversion into norepinephrine or release of norepinephrine from the peripheral nerves since “peripheral sympathectomy” with 6-hydroxydopamine did not alter the dopamine-induced change in ileal short-circuit current. The dopamine effects were not associated with a change in basal ileal cyclic AMP content but were associated with a decrease in total ileal calcium content as measured by atomic absorption spectrometry and as estimated by 45Ca++ uptake. The decrease in calcium content could be attributed to a dopamine-induced decrease in 45Ca++ influx from the serosal surface. Because of the presence of dopamine in ileal mucosa and these effects on ileal electrolyte transport, it is possible that dopamine may be involved in the physiologic regulation of active intestinal electrolyte absorption.
Mark Donowitz, Sheila Cusolito, Laurie Battisti, Ronald Fogel, Geoffrey W. G. Sharp
Copyright © 2014 American Society for Clinical Investigation