17 beta-estradiol enchances androgen-induced prostate growth in the castrate dog to a degree comparable to that seen in spontaneous prostatic hypertrophy. To investigate the mechanism of this synergism, cytosol androgen binding was measured by a density gradient technique in prostates of control and 17 beta-estradiol-treated castrate dogs. [3H]Dihydrotestosterone was bound principally to a moiety that averaged 8.6S in size. Approximately twofold enhancement of this binding by 17 beta-estradiol was demonstrable after 1 wk of treatment with 750 microgram/wk and after 3 wk with 75 microgram/wk. Under conditions in which binding in the 8S region was demonstrable with dihydrotestosterone and testosterone no binding of 3 alpha-androstanediol or progesterone was detectable. Thus, enhancement by 17 beta-estradiol of a prostate cytosol androgen-binding protein occurs under circumstances in which 17 beta-estradiol enhances androgen-mediated prostatic growth.
R J Moore, J M Gazak, J D Wilson
The idea that the gene(s) that cause diabetes mellitus can be expressed in extrapancreatic cells has been examined by tissue culture techniques. Skin biopsies were obtained from 25 normal subjects (N), 26 overt diabetics (D), 16 of juvenile onset (JOD) and 9 of maturity onset (MOD), and 21 subjects genetically predisposed to diabetes (P) on the basis of maturity-onset diabetes in both parents. Each biopsy was subdivided, multiple skin fragments were explanted in vitro, and several parameters of cellular outgrowth were monitored in primary and secondary cultures until cell division ceased because of senescence. In general, the rank order of growth vigor was N greater than P greater than D although differences were often marginal and statistically significant between N and JOD and(or) MOD. Outgrowth of epithelial cells was more vigorous in N explants in early stages, but later, JOD and MOD cells grew better than those of N. Outgrowth of fibroblast cells from N explants was more vigorous both at early and later stages and required less time to achieve maximum percent outgrowth. In secondary cultures, N cells grew faster than the other three groups so that fewer days elapsed between subcultures but significant differences were only seen between N and one or two of the other groups over some of the first seven subcultures. The onset of cellular senescence occurred earlier in P and JOD cultures both in mean population doublings and calendar time. N cultures had a higher percent surviving clones after picking than MOD, and a shorter recloning time than clones of JOD. The replicative life-spans of cultures (mean population doublings +/- SE) were N = 52.54 +/- 2.24, P = 47.84 +/- 2.43, JOD = 47.12 +/- 2.99, and MOD = 46.40 +/- 4.04, but differences did not reach significance for N vs the other three groups. The data demonstrate that cellular growth is impaired in both JOD and MOD types of cultures and to a generally lesser extent in P cultures. This is consistent with intrinsic genetic defects but the possibility that persistent deleterious effects of in vivo pathophysiology contribute alone or in combination cannot be ruled out. Therefore, the diabetic defect(s) can be expressed in extrapancreatic cells of mesenchymal origin. This system should prove useful in exploring the interplay between genetic and environmental factors in diabetes, the mechanisms(s) of hyperglycemia and other metabolic derangements, and the propensity that affected individuals have to develop degenerative diseases.
S Goldstein, E J Moerman, J S Soeldner, R E Gleason, D M Barnett
Recent observations indicate that in thyroparathyroidectomized (TPTX) rats fed a low (0.2 g/100 g) phosphorus diet, the tubular phosphaturic response to parathyroid hormone (PTH) remains markedly blunted even when it is assessed at normal or high plasma concentration and filtered load of inorganic phosphate (Pi). Because 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] decreases the tubular capacity to reabsorb Pi when chronically administered to TPTX rats, we have studied whether this vitamin D3 metabolite could specifically increase the phosphaturic response to PTH in phosphate-deprived animals. The results show that in Vitamin D-replete TPTX rats fed a low (0.2 g/100 g) phosphorus diet, 1,25(OH)2D3 (2 × 13 pmol/d i.p. for 7 d) markedly enhanced the acute tubular phosphaturic response to PTH (2.5 IU/h i.v.) without affecting the action of the peptide hormone on Ca reabsorption and cyclic-3′,5′-AMP excretion. The influence of 1,25(OH)2D3 on the phosphaturic response to PTH could not be ascribed to an increased plasma concentration and(or) filtered load of Pi during the administration of the peptide hormone. However, it could be, at least in part, related to the elevation in the basal level of plasma Pi which was observed in the 1,25(OH)2D3-treated animals. The results also indicate that 1,25(OH)2D3 significantly enhanced the calcemic response to PTH, which was blunted in these conditions of phosphate deprivation. Unlike 1,25-(OH)2D3, 25-hydroxyvitamin D3 did not unmask the phosphaturic effect of PTH in phosphate-depleted animals, even when given in doses 100 times larger. Thus, 1,25(OH)2D3 displays a selective and powerful activity in preventing the occurrence of tubular resistance to the phosphaturic action of PTH during Pi depletion. This finding suggests the existence of an important interaction between dietary Pi, 1,25(OH)2D3, and PTH in the homeostasis of phosphate.
H. J. Gloor, J-P. Bonjour, J. Caverzasio, H. Fleisch
It is unclear what factors control the secretion of pulmonary surface active material from alveolar type II cells in vivo. Other workers have suggested that cholinergic stimuli, adrenergic stimuli, and prostaglandins may all stimulate secretion. We isolated type II cells from the lungs of rats by treatment with elastase, discontinuous density centrifugation, and adherence in primary culture. β-Adrenergic agonists, but not cholinergic agonists, caused an increase in the release of [14C]disaturated phosphatidylcholine, the major component of surface-active material, from type II cells in culture. The β-adrenergic effect was stereo-selective, (−)-isoproterenol being 50 times more potent than (+)-isoproterenol. Terbutaline, 10 μM, a noncatecholamine β-2 adrenergic agonist, caused a release of 2.0±0.5 (mean±SD) times the basal release of [14C]disaturated phosphatidylcholine in 3 h; the concentration of terbutaline causing half maximal stimulation was 800 nM. The terbutaline effect was blocked by propranolol, a β-adrenergic antagonist (calculated Kd = 6 nM), but not by phentolamine, an α-adrenergic antagonist. Isobutylmethylxanthine, a phosphodiesterase inhibitor, and 8-Br cyclic AMP, but not 8-Br cyclic guanosine monophosphate, also stimulated release. We conclude that type II cells secrete disaturated phosphatidylcholine in response to treatment with adrenergic stimulation.
Leland G. Dobbs, Robert J. Mason
The effects of pregnancy and diabetes on systemic glucose production rates and the sources of glucose for the human fetus in utero were evaluated in five normal, four gestationally diabetic, and one insulin-dependent diabetic subject undergoing elective caesarean section at term gestation. Five normal nonpregnant women were studied for comparison. Systemic glucose production rates were measured with stable tracer [1-13C]glucose according to the prime-constant rate infusion technique. Even though the plasma glucose concentration during normal pregnancy had declined as compared with the nonpregnant subjects (P < 0.0005), the systemic glucose production rate was 16% greater, a rate sufficient to provide the glucose requirement of the fetus at term gestation. The decline in glucose concentration could be the result of an increase in apparent volume of distribution of glucose. Systemic glucose production rates in well-controlled, gestationally diabetic subjects were similar to those in normal pregnant subjects (2.07±0.53 vs. 2.42±0.51 mg/kg·min). The sources of glucose for the human fetus at term gestation were evaluated by comparing (a) natural variation in 13C:12C ratio of plasma glucose and (b) enriched 13C:12C ratio of plasma glucose during [1-13C]glucose infusion in maternal and fetal blood at delivery in both normal and diabetic subjects. These data showed that the fetal glucose pool was in equilibrium with the maternal glucose pool in both normal and diabetic subjects, indicating that a brief maternal fast did not initiate systemic glucose production in human fetus. A materno-fetal gradient was observed for betahydroxybutyrate.
Satish C. Kalhan, Larry J. D'Angelo, Samuel M. Savin, Peter A. J. Adam
The major goal of this investigation was to determine if activation of cardiac receptors during coronary artery occlusion could inhibit efferent renal sympathetic nerve activity. In nine chloralose anesthetized dogs with only carotid (n = 3) or with sinoaortic (n = 6) baroreceptors operative, anterior descending coronary artery (LAD) occlusion resulted in a small decrease in mean arterial pressure (−9.8±5.1 mm Hg, NS) and in a significant (P < 0.05) increase in renal nerve activity (24.0±4.1%). In these dogs, circumflex coronary artery (Cx) occlusion resulted in greater hypotension (−18.4±4.0 mm Hg), and yet no change (1.1±9%) in renal nerve activity was noted. Changes in left atrial pressure during LAD and Cx occlusion were not different. In seven dogs with carotid sinus denervation, coronary occlusions resulted in decreases both in arterial pressure and in renal nerve activity which were consistently greater during Cx occlusion. The responses to coronary occlusion in six dogs after sinoaortic deafferentation were similar to those observed with only carotid sinuses denervated. In all experiments, vagotomy abolished the difference in the blood pressure responses and the decreases in renal sympathetic nerve activity during Cx occlusion. Vagotomy also abolished the decrease in nerve activity during LAD occlusion in dogs with carotid or sinoaortic denervation. These data show that Cx occlusion and, to a lesser degree, LAD occlusion resulted in reflex withdrawal of renal sympathetic nerve activity mediated by left ventricular receptors with vagal afferents. The reflex withdrawal of renal nerve activity during Cx occlusion occurred in spite of hypotension and the presence of functioning sinoaortic baroreceptors.
Marc D. Thames, Francois M. Abboud
These investigations were designed to evaluate the effect of excess glucose and sodium chloride on lipolysis in the isolated adipocyte under normal and modelled pathological conditions simulating the hyperglycemic hyperosmolar syndrome.
B. Paul Turpin, William C. Duckworth, Solomon S. Solomon
Segments of superficial and juxtamedullary proximal convoluted tubules of the rabbit were perfused in vitro to examine the mechanisms responsible for net volume reabsorption. The very early postglomerular segments were not studied. Fluid reabsorptive rates and transepithelial potential differences were compared under various conditions: (a) with perfusate that simulated glomerular filtrate; (b) with perfusate that lacked glucose, amino acids, and acetate and that had HCO3 and Cl concentrations of 5 and 140 mM, respectively; (c) with perfusate that lacked glucose, amino acids, and acetate but with 20 meq of NaHCO3 replaced with 20 meq of Na cyclamate; (d) with the same perfusate as in b but in the presence of ouabain in the bath; (e) with ultrafiltrate of rabbit serum titrated with HCl to final HCO3 and Cl concentrations of 2 and 134 mM, respectively. Tubules were perfused with this titrated ultrafiltrate at 37°C, 21°C, and in the presence of 0.1 mM ouabain in the bath. Bath fluid in all experiments was regular rabbit serum. Under conditions a and b superficial proximal convoluted tubule (SFPCT) and juxtamedullary proximal convoluted tubule (JMPCT) behaved similarly with the exception that SFPCT exhibited a lumen-positive and JMPCT a lumen-negative electrical potential under condition b. However, under condition c SFPCT failed to exhibit net volume reabsorption, whereas reabsorption in JMPCT continued unchanged. Ouabain did not affect volume reabsorption in SFPCT under condition d, whereas neither ouabain nor hypothermia affected SFPCT under condition e. In contrast, ouabain and hypothermia totally inhibited volume reabsorption in JMPCT under conditions d and e. These studies document heterogeneous mechanisms responsible for volume reabsorption in the major portions of SFPCT and JMPCT with passive forces predominating in SFPCT and active forces in JMPCT.
Harry R. Jacobson, Howard L. Baer Jr.
To determine the mechanism underlying altered adrenal responsiveness in patients with essential hypertension, the renin-angiotensin-aldosterone axis was assessed in normotensive and hypertensive subjects using three pharmacological probes: SQ 20881, a converting enzyme inhibitor; saralasin, a competitive angiotensin antagonist with prominent agonist properties; and angiotensin itself. All subjects were studied while supine and in balance on a 10 meq Na/100 meq K intake. The decrement in plasma aldosterone with SQ 20881 in 26 hypertensive subjects (15±3 ng/dl) was normal (13±4 ng/dl), suggesting that the altered adrenal responsiveness in hypertensives is not because of a change in a postreceptor event or in the relative contribution of angiotensin to the control of aldosterone secretion.
Gordon H. Williams, Norman K. Hollenberg, Thomas J. Moore, Stephen L. Swartz, Robert G. Dluhy
Cultured choriocarcinoma (Be Wo) cells exist that share many of the morphologic and bio-synthetic properties of normal human trophoblasts. In an attempt to develop a model for the immunologic relationship between a sensitized mother and fetus, we mixed Be Wo cells with mitogen-activated cytotoxic lymphocytes in vitro. Be Wo cells were resistant to the cytolytic effects of the activated lymphocytes despite 24-h exposure and intimate cell-to-cell contact as determined by microscopy. Control target cells, a line of human hepatoma cells, were readily destroyed. Cytotoxicity was measured by determining residual radioactivity of [3H]thymidine-labeled target cells after exposure to activated lymphocytes. Employing the quantitative assay, we confirmed the morphologic results and showed that Be Wo and a number of other choriocarcinoma cell lines were resistant to the cytotoxic effects of lymphocytes activated by phytohemagglutinin, pokeweed mitogen, and allogeneic cells in mixed lymphocyte cultures. Moreover, Be Wo cells were resistant to injury over a wide range of killer to target cell ratios. Significant killing of the Be Wo cells occurred only after prolonged exposure (48 and 72 h) to the activated lymphocytes.
Charles S. August, Sheila T. Cox, Michael A. Naughton
C4-binding protein (bp), a glycoprotein with specific binding affinity for the activated form of C4 (C4b), has recently been isolated from human serum and partially characterized. This report demonstrates that C4-bp is incorporated into soluble immune complexes after complement activation in vitro. The reaction requires Ca++ ions and the presence of C4 in serum. Immunopathological studies of various forms of glomerulonephritis revealed intense C4-bp deposition in glomeruli from patients with immune-complex type of pathogenesis. C4-bp deposition was in close correlation with that of C4. These observations, together with the in vitro association of C4-bp to immune complexes, support the notion that the deposits in glomeruli represent the local accumulation of immune complexes.
J Scharfstein, E B Correa, G R Gallo, V Nussenzweig
The equilibrium between assembled and disassembled microtubules was studied in human platelets exposed to aggregating agents. Soluble and insoluble tubulin were “frozen” by addition of a glycerol-dimethyl sulfoxide-containing medium. The two pools were estimated by measuring the colchicine binding activities of total and polymerized tubulin. Resting platelets were found to contain an average of 56.2 μg tubulin/1 × 109 cells of which 56.7% was in polymerized form. Platelet aggregation induced by thrombin, ADP, epinephrine, or collagen produced a transient decrease in the pool of polymerized tubulin which was evident within 15 s after addition of the aggregating agent. A return to base-line values occurred within 1-4 min depending upon the specific aggregating agent used. Neither secretory release nor aggregation of platelets were found to be prerequisites for the temporary disturbance of the equilibrium between soluble and polymerized tubulin. With thrombin as the aggregating agent a clear threshold concentration could be demonstrated above with a dose-dependent dissociation response of microtubules was evident. We conclude that microtubules exist in a dynamic equilibrium between polymerized and depolymerized forms in human platelets, which is transiently disturbed by their interaction with aggregating agents.
M. Steiner, Y. Ikeda
The cyclotron-produced radionuclide, 13N, was used to label ammonia and to study its metabolism in a group of 5 normal subjects and 17 patients with liver disease, including 5 with portacaval shunts and 11 with encephalopathy. Arterial ammonia levels were 52-264 micron. The rate of ammonia clearance from the vascular compartment (metabolism) was a linear function of its arterial concentration: mumol/min = 4.71 [NH3]a + 3.76, r = +0.85, P less than 0.005. Quantitative body scans showed that 7.4 +/- 0.3% of the isotope was metabolized by the brain. The brain ammonia utilization rate, calculated from brain and blood activities, was a function of the arterial ammonia concentration: mumol/min per whole brain = 0.375 [NH3]a - 3.6, r = +0.93, P less than 0.005. Assuming that cerebral blood flow and brain weights were normal, 47 +/- 3% of the ammonia was extracted from arterial blood during a single pass through the normal brains. Ammonia uptake was greatest in gray matter. The ammonia utilization reaction(s) appears to take place in a compartment, perhaps in astrocytes, that includes less than 20% of all brain ammonia. In the 11 nonencephalopathic subjects the [NH3]a was 100 +/- 8 micron and the brain ammonia utilization rate was 32 +/- 3 mumol/min per whole brain; in the 11 encephalopathic subjects these were respectively elevated to 149 +/- 18 micron (P less than 0.01), and 53 +/- 7 mumol/min per whole brain (P less than 0.01). In normal subjects, approximately equal to 50% of the arterial ammonia was metabolized by skeletal muscle. In patients with portal-systemic shunting, muscle may become the most important organ for ammonia detoxification. Muscle atrophy may thereby contribute to the development of hyperammonemic encephalopathy with an associated increase in the brain ammonia utilization rate.
A H Lockwood, J M McDonald, R E Reiman, A S Gelbard, J S Laughlin, T E Duffy, F Plum
Classical glucocorticoid receptors (type II) have a high affinity for synthetic and natural glucocorticoids. We have previously demonstrated an additional binding site in kidney cytosol (type III) which has a high affinity for corticosterone but a low affinity for dexamethasone. In many ways, this binder resembles plasma corticosteroid-binding globulin (CBG). The first goal of this study was to determine the organ distribution of the type III binding sites. Cytosol was prepared from isolated cells to avoid plasma contamination. Of the tissues examined, type III sites were found only in liver and kidney; sites were absent from thymocytes, IM-9 lymphocytes, adipocytes, and bone cells. The second goal of this study was to ascertain whether CBG is synthesized in liver and kidney. Liver and kidney slices were incubated in vitro and the concentration of type III sites was seen to rise in hepatic cytosol and incubating medium but not kidney. To verify the impression that liver was synthesizing and secreting CBG, the following experiments were performed: (a) To demonstrate that type III sites were CBG, steroid-binding profiles and migration on polyacrylamide gel electrophoresis were shown to be identical for hepatic type III sites and serum. (b) To indicate that the rise in type III sites was dependent on protein synthesis, it was shown that cycloheximide blocked the appearance of new type III sites. (c) To establish that the type III sites were being secreted, in situ liver perfusion experiments showed time-dependent release of new sites into the perfusate. In conclusion, liver synthesizes and secretes type III sites, a finding previously suspected but never proved. The presence of type III sites in kidney remains to be explained.
Jeffrey N. Weiser, Yung-Shun Do, David Feldman
We have found immunoglobulin (Ig) G-containing material consistent with immune complexes in the sera of patients with Lyme arthritis. It was detected in 29 of 55 sera (55%) from 31 patients by at least one of three assays: 125I-C1q binding, C1q solid phase, or Raji cell. The presence of reactive material correlated with clinical aspects of disease activity; it was found early in the illness, was most prominent in sera from the sickest patients, was infrequent during remissions, and often fluctuated in parallel with changes in clinical status. The results in the two C1q assays showed a strong positive correlation (P<0.001). They were each elevated in 45% of the sera and were usually concordant (85%). In contrast, the Raji cell assay was less frequently positive and often discordant with the C1q assays. In sucrose density gradients, putative circulating immune complexes sedimented near 19S; they, too, were detected best by the two assays based on C1q binding. An additional 7S component was found in some sera by the 125I-C1q binding assay. Serum complement was often above the range of normal in patients with mild disease and normal in patients with severe disease but did not correlate significantly with levels of circulating immune complexes. IgM and IgG rheumatoid factors were not detectable.
John A. Hardin, Lesley C. Walker, Allen C. Steere, Thomas C. Trumble, Kenneth S. K. Tung, Ralph C. Williams Jr., Shaun Ruddy, Stephen E. Malawista
The effects of highly purified natural porcine cholecystokinin (CCK) and synthetic caerulein on the rate of flow of pancreatic juice, the rate of output of amylase, and the rate of release of immunoreactive insulin (IRI) and immunoreactive glucagon (IRG) were simultaneously investigated in the isolated perfused rat pancreas.
Makoto Otsuki, Choitsu Sakamoto, Hosai Yuu, Mitsuo Maeda, Soichiro Morita, Atsuhi Ohki, Noboru Kobayashi, Katsuhiro Terashi, Kuniyasu Okano, Shigeaki Baba
Fibrinogen survival and turnover were examined in 15 adult-onset diabetic patients. 125I-labeled fibrinogen was prepared from each patient during the period of poor carbohydrate control, or hyperglycemic period, and fibrinogen survival determined. Improved control was established in each patient and during this euglycemic period, fibrinogen survival was determined simultaneously with 125I-fibrinogen saved from the hyperglycemic period and 131I-labeled fibrinogen prepared from the patient during the euglycemic period. The results confirm reduced fibrinogen survival in hyperglycemic diabetic patients and demonstrate reversal of the fibrinogen abnormality when euglycemia is achieved. The results of the double-label experiments in the euglycemic period suggest that the fibrinogen molecule is not altered functionally and that an abnormal plasma or vascular environment is a more likely basis for reduced fibrinogen survival during hyperglycemia. Electrophoretic and chromatographic experiments demonstrated no gross chemical differences between the fibrinogens prepared from the hyperglycemic and euglycemic periods and normal fibrinogen. Fibrinogen survival gave a better correlation with serial glucose measurements than with correction of hemoglobin AIc levels indicating that the reduced fibrinogen survival noted in diabetics is a rapidly reversible phenomenon.
Robert L. Jones, Charles M. Peterson
Patients with nephrotic syndrome have low blood levels of 25 hydroxyvitamin D (25-OH-D) most probably because of losses in urine, and a vitamin D-deficient state may ensue. The biological consequences of this phenomenon on target organs of vitamin D are not known. This study evaluates one of these target organs, the bone. Because renal failure is associated with bone disease, we studied six patients with nephrotic syndrome and normal renal function. The glomerular filtration rate was 113±2.1 (SE) ml/min; serum albumin, 2.3±27 g/dl; and proteinuria ranged between 3.5 and 14.7 g/24 h. Blood levels of 25-OH-D, total and ionized calcium and carboxy-terminal fragment of immunoreactive parathyroid hormone were measured, and morphometric analysis of bone histology was made in iliac crest biopsies obtained after double tetracycline labeling. Blood 25-OH-D was low in all patients (3.2-5.1 ng/ml; normal, 21.8±2.3 ng/ml). Blood levels of both total (8.1±0.12 mg/dl) and ionized (3.8±0.21 mg/dl) calcium were lower than normal and three patients had true hypocalcemia. Blood immuno-reactive parathyroid hormone levels were elevated in all. Volumetric density of osteoid was significantly increased in three out of six patients and the fraction of mineralizing osteoid seams was decreased in all. Evidence for an increase in active lacunae (bone-osteoclast interface) occurred in three out of six patients and in inactive (Howship's lacunae) bone resorption in six out of six. The data indicate that the loss of 25-OH-D in urine of patients with nephrotic syndrome and normal renal function may result in a decrease of blood levels of ionized calcium, secondary hyperparathyroidism and enhanced bone resorption. In addition, the vitamin D-deficient state causes osteomalacia as evidenced by defective mineralization and increased osteoid volume.
Hartmut H. Malluche, David A. Goldstein, Shaul G. Massry
Electrochemical disturbances of skeletal muscle cells in untreated uremia are characterized by an increase in the intracellular sodium and chloride content, a decrease in intracellular potassium, and a low resting membrane potential. In this study, we have reexamined the foregoing and, in addition, have examined the effects of hemodialysis. Three groups of patients were studied. In the first group of 22 uncomplicated uremic patients, whose creatinine clearance (Ccr) ranged from 2 to 12 cm3/min per 1.73 m2, resting transmembrane potential difference (Em) of skeletal muscle cells was measured. In each of the nine patients whose Ccr ranged between 6.3 and 12 cm3/min, the Em was normal (i.e., −90.8±0.9 mV, mean±SEM). However, as Ccr dropped below 6.3 cm/min, the Em became progressively reduced and assumed a linear relationship with the Ccr.
James R. Cotton, Terry Woodard, Norman W. Carter, James P. Knochel
Studies were conducted to ascertain the in vitro effect of 3,5,3′-triiodothyronine (T3) on the accumulation of the glucose analogue, 2-deoxyglucose (2-DG), by thymocytes freshly isolated from weanling rats. At a concentration of 1 μM, T3 stimulated the 15-min uptake of 3H-2-DG after cells had been exposed to T3 for only 30 min. Significant stimulation of 2-DG accumulation was produced by 1 nM T3, with increasing stimulation at doses ranging up to 10 μM. T3 did not alter the fraction of accumulated 2-DG that was phosphorylated, and kinetic studies indicated that its effect was associated with a significant increase in the apparent Vmax of 2-DG accumulation, but not the apparent Km. T3 also enhanced the accumulation by thymocytes of the nonmetabolized glucose analogue, 3-O-methylglucose (3-O-MG), an effect that was evidently the result of an increase in 3-O-MG transport into the cell, because it was seen in cells incubated with 3H-3-O-MG for only 30 s. The proportionate increase in 2-DG accumulation produced by T3 was not altered by preincubating cells with concentrations of puromycin or cycloheximide sufficient to reduce [3H]-leucine incorporation by 95%, and T3 over a period of >2 h had no effect on [3H]leucine incorporation itself.
Joseph Segal, Sidney H. Ingbar
The role of nonprotein sulfhydryl groups (NPSH) in the decreased in vitro hepatic 3′,3,5-triiodothyronine (T3) generation from thyroxine (T4) in the starved, hypothyroid, fetal and 1- to 4-d-old neonatal rat and dwarf mouse was assessed. NPSH were measured in fresh 25% liver homogenates prepared in 0.1 M PO4/10 mM EDTA buffer. As compared with values in adult male rats, NPSH concentration was decreased in the 2-d-starved (1.1±0.04 (mean±SE) vs. 2.2±0.15 mmol/250 g wet liver weight, P < 0.001), fetal (1.0±0.04 vs. 3.2±0.08, P < 0.001), 1-d-old neonatal (1.1±0.03 vs. 2.1±0.04, P < 0.001), and hypothyroid (thyroidectomized 60 d) (1.4±0.06 vs. 2.2±0.15 P < 0.001) rat. NPSH were also decreased in the hypothyroid, hypopituitary dwarf mouse as compared with values in their normal litter mates (1.3±0.03 vs. 2.0±0.2, P < 0.01). Chronic administration of T3 (0.5 μg/100 g body wt per d) markedly increased hepatic T3 generation from T4 in the thyroidectomized rat and in the dwarf mouse to values similar to those observed in the normal rodent without affecting NPSH concentration. In contrast, T3 administration to the starved rat did not alter either hepatic T3 generation from T4 or NPSH. Reduced glutathione concentration was also markedly decreased in the starved rat (fed; 1.05±0.075 mmol/250 g wet tissue vs. starved 0.38±0.02, P < 0.001). Dithiothreitol (DTT), a thiol reducing agent, increased hepatic T3 generation from T4 in the normal adult male rat by 45±5% in six experiments. When compared to DTT-stimulated control homogenates, the addition of DTT completely restored hepatic T3 generation in starved rats, partially restored T3 generation in 1- and 4-d-old neonates, but had little or no effect in the fetal and hypothyroid rat and dwarf mouse. Liver homogenates stored for 6 mo at −20°C lost their capacity to generate T3 from T4. NPSH concentrations in the frozen homogenates decreased progressively with increasing storage and were absent by 6 mo. 5′-Deiodinase activity correlated with NPSH concentration in the stored homogenates (r = 0.95, P < 0.005). Addition of DTT partially restored hepatic T3 generation in the frozen homogenate. It is concluded that NPSH are important for the action of the liver 5′-deiodinase. The decreased hepatic T3 generation in the starved rat is associated with decreased NPSH but not with a decrease in the absolute quantity of 5′-deiodinase because provision of sulfhydryl groups restored hepatic T3 generation to normal. In contrast, the decreased hepatic T3 generation in the adult hypothyroid rodent and in the fetal rat is probably due to a decrease in the enzyme concentration per se. In the 1- and 4-d neonatal rat, the decrease in hepatic T3 generation is secondary to a decrease in NPSH and the deiodinating enzyme.
Arthur R. C. Harris, Shih-Lieh Fang, Lee Hinerfeld, Lewis E. Braverman, Apostolos G. Vagenakis
Because in the dog, the gastric fundus contains the largest amount of glucagon immunoreactivity (IRG), the IRG of mucosal scrapes of 105 canine stomachs was extracted by acid-ethanol and then precipitated by ether-ethanol. The IRG recovered was measured by antisera 30K, specific for glucagon and K-4023, which cross-reacts with glucagon-like immunoreactivity. Extracts of mucosa of stomach fundus were further purified by gel filtration on Bio-Gel P-30 in 3M acetic acid. One pooled fraction corresponding to marker pancreatic glucagon in its elution volume was then gel-filtered on Bio-Gel P-30 in 0.05 M NH4HCO3 and yielded one IRG peak, which, however, showed three immunoreactive components on polyacrylamide disc gel electrophoresis in urea. In addition, antiserum K-4023 reacted more strongly with that peak than antiserum 30K indicating the presence of glucagon-like immunoreactivity in this fraction. Subsequent ion-exchange column chromatography on DEAE-Sephadex A-25 and then CM-Bio-Gel A allowed purification to a single protein band on disc gel electrophoresis reacting equally to both antisera 30K and K-4023. 1.5 μg of purified gastric glucagon was obtained and its biological effects were compared to those of pancreatic glucagon in isolated rat hepatocytes. When immuno-equivalent amounts (300-2,500 pg/ml) of either type of glucagon were used, the same biological responses with respect to glycogenolysis and gluconeogenesis as well as urea, lactate, and pyruvate production were observed. Liver cyclic AMP was also raised to the same extent by either one of these hormones. We conclude that this moiety of gastric IRG is apparently identical to pancreatic glucagon because (a) their molecular weights, elution properties in ion exchange chromatography, and their electrophoretic mobility are indistinguishable and (b) both hormones elicited identical biological effects in isolated rat hepatocytes.
K. Doi, M. Prentki, C. Yip, W. A. Muller, B. Jeanrenaud, M. Vranic
Endothelial cells synthesize prostacyclin (PGI2), an unstable prostaglandin that inhibits platelet aggregation and serotonin release. Because cyclooxygenase, which is necessary for synthesis of PGI2, is inactivated by aspirin, we examined the effect of aspirin on PGI2 production by cultured human endothelial cells. Endothelial cells synthesize PGI2 (20.1±7.2 ng/106 cells, mean±SD) when stimulated with 20 μM sodium arachidonate for 2 min. PGI2 production is inhibited by low-dose aspirin (5 μM); the t½ of inactivation is 6.0±1.3 min (mean±SEM, n = 3). Thus, endothelial cell cyclooxygenase is as sensitive to aspirin as the enzyme in platelets. After 1 h incubation with aspirin, endothelial cell PGI2 production was inhibited 50% by 2.1±0.4 μM aspirin and was inhibited 90% by 6.2±0.9 μM aspirin (mean±SEM, n = 4). When endothelial cells were incubated with 100 μM aspirin, washed, and recultured, their ability to synthesize PGI2 returned to control levels in 35.6±1.0 h (mean±SEM, n = 4). Recovery of endothelial PGI2 production after aspirin depended on de novo protein synthesis because treatment with cycloheximide (3 μg/ml) inhibited recovery by 92%.
Eric A. Jaffe, Babette B. Weksler
Circulating antibodies that could be responsible for the suppressor thymus-derived (T)-cell dysfunction in active systemic lupus erythematosus (SLE) were investigated. Sera from 14 active and inactive SLE patients were compared with a pool of 22 normal sera. All sera were adsorbed with a pool of normal platelets to exclude antihistocompatibility leukocyte antigen antibodies; with AB erythrocytes to exclude isohemagglutinins; and with a pool of normal bone marrow-derived (B) lymphocytes, monocytes, and neutrophils to deplete anti-B-cell antibodies, Fc-receptor antibodies, and antibodies directed against neutrophils or monocytes. Sera from active SLE patients were capable of inhibiting the activation of normal, blood lymphocytes by concanavalin A to become suppressor cells. The latter were assayed by coculturing the concanavalin A-activated cells with autologous lymphocytes, which were then activated with either phytohemagglutinin for proliferative response or with pokeweed mitogen for B-cell immunoglobulin (Ig) synthesis and secretion. Specific incorporation of cultures with phytohemagglutinin showed a value of 67±13 (mean±SD) for suppressor cells treated with adsorbed, active SLE sera. This value was significantly different (P < 0.001) from that of cells treated with the inactive SLE sera or with the pool of normal sera. Similar findings were seen with respect to the B-cell target parameters. Cytoplasmic Ig and IgG in supernates of cultures with pokeweed mitogen showed values of 17±5% and 717±134 ng/culture, respectively, for suppressor cells treated with the adsorbed, active SLE sera. This was significantly different from those treated with the inactive SLE sera or with the pool of normal sera. The antisuppressor-cell factor was shown to be IgG, complement independent, not cytotoxic, active at 37°C and at room temperature, but not at 4°C, and adsorbable with T cells.
Akira Sagawa, Nabih I. Abdou
Platelet-associated fibronectin antigen has been identified by radioimmunoassay and immunofluorescent techniques. In radioimmunoassay, platelet fibronectin was immunochemically indistinguishable from plasma fibronectin. Platelet and plasma fibronectin were bound and eluted from gelatin-sepharose under similar conditions. The level of platelet fibronectin in detergent extracts of washed platelets from 12 healthy adults was 2.85 +/- 1.24 microgram/10(9) platelets. Immunofluorescence with F(ab')2 fragments of immunochemically purified antifibronectin showed that all platelets stained with a discrete punctate pattern. The identification of platelet fibronectin antigen raises the possibility that this protein may participate in platelet-platelet or platelet-surface interactions.
E F Plow, C Birdwell, M H Ginsberg