The biologically active leukotrienes. Biosynthesis, metabolism, receptors, functions, and pharmacology.

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Abstract

Authors

R A Lewis, K F Austen

Qualitative and quantitative differences in the pathways of extrathyroidal triiodothyronine generation between euthyroid and hypothyroid rats.

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Abstract

Propylthiouracil (PTU) in maximally inhibitory doses for liver and kidney iodothyronine 5'-deiodinase activity (5'D-I), reduces extrathyroidal T4 to T3 conversion by only 60-70% in euthyroid rats. A second pathway of T4 to T3 conversion (5'D-II) has been found in pituitary, central nervous system, and brown adipose tissue. 5'D-II is insensitive to PTU and increases in hypothyroidism, whereas 5'D-I decreases in hypothyroid rats. Thyroxine (T4) and triiodothyronine (T3) kinetics were assessed in euthyroid and thyroidectomized rats by noncompartmental analysis after injecting [125I]T4 and [131I]T3. Neither the volume of distribution nor the rate of fractional removal of plasma T4 was affected by the thyroid status, but the fractional removal rate of T3 was approximately 50% reduced in hypothyroid rats (P less than 0.001). Fractional T4 to T3 conversion was 22% in euthyroid and 26% in hypothyroid rats. In euthyroid rats, sufficient PTU to inhibit liver and kidney 5'D-I greater than 90% reduced serum [125I]T3 after [125I]T4 (results given as percent dose per milliliter X 10(-3) +/- SEM): 4 h, control 16 +/- 2 vs. PTU 4 +/- 1, P less than 0.005, and 22 h, control 6.4 +/- 0.4 vs. PTU 3.6 +/- 0.7, P less than 0.025. In thyroidectomized rats, the same dose of PTU also inhibited 5'D-I in liver and kidney, but had no effect on the generation of serum [125I]T3 from [125I]T4. Similarly, after 1 microgram T4/100 g bw was given to thyroidectomized rats, serum T3 (radioimmunoassay) increased by 0.30 +/- 0.6 ng/ml in controls and 0.31 +/- 0.09 ng/ml in PTU-treated rats. However, when the dose of T4 was increased to 2-10 micrograms/100 g bw, PTU pretreatment significantly reduced the increment in serum T3. T3 clearance was not affected by PTU in hypothyroid rats. The 5'D-II in brain, pituitary, and brown adipose tissue was reduced to less than or equal to 60% of control by 30 micrograms/100 g bw reverse T3 (rT3), an effect that lasted for at least 3 h after rT3 had been cleared. In rT3-pretreated thyroidectomized rats, the generation of [125I]T3 from tracer [125I]T4 was reduced in the serum: 6 +/- 1 vs. 12 +/- 1 X 10(-3)% dose/ml, P less than 0.01, during this 3-h period. We conclude that virtually all the T3 produced from low doses of exogenous T4 given to hypothyroid rats is generated via a PTU-insensitive pathway, presumably catalyzed by the 5'D-II. This is a consequence of the enhanced activity of this low Km enzyme together with the concomitant decrease in the hepatic and renal 5'D-I characteristic of the hypothyroid state. The results indicate that in some circumstances, 5D-II activity may contribute to the extracellular, as well as intracellular, T3 pool.

Authors

J E Silva, M B Gordon, F R Crantz, J L Leonard, P R Larsen

In vivo evidence of impaired solute transport by the thick ascending limb in potassium-depleted rats.

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Abstract

The objective of this investigation was to determine if thick ascending limb (TAL) solute removal is impaired in potassium-depleted rats, in vivo. We estimated TAL NaCl concentration by measuring in situ conductivity of tubular fluid presented to the early distal site after stop-flow periods of 10-60 s, during which a proximal equilibrium solution remained in contact with the reabsorbing epithelium. This allowed us to calculate the rate constant of the decrease in tubular fluid NaCl concentration and to determine equilibrium values for control, potassium-depleted, and potassium-repleted rats. After 60 s of stop-flow, NaCl concentration of TAL fluid decreased to 18.3 +/- 2.73 mM in control rats, while potassium-depleted rats had values almost twice as high (36.5 +/- 2.97 mM, P less than 0.01). The amount of NaCl remaining after 60 s of stop-flow in K-depleted rats was highly correlated with the plasma K concentration. Calculated rates of NaCl efflux from the TAL appeared to be normal in K-depleted rats while the concentration of NaCl achieved at equilibrium was nearly twice that measured in control rats. Acute systemic administration of KCl by gavage or infusion in K-depleted rats was associated with a decrease in TAL NaCl concentration to normal values. Addition of K to the perfusate, however, did not repair the defect. Our results can best be explained by assigning a special role to the peritubular K concentration. We suggest that the defect in TAL solute removal in K-depletion can be rapidly reversed, because decreases in peritubular K concentration limit Na efflux across the peritubular membrane by decreasing the activity of the Na-K-ATPase pump. We recognize that factors such as regional renal blood flow, local angiotensin II levels, and products of the cyclo-oxygenase enzyme system may play a role.

Authors

H U Gutsche, L N Peterson, D Z Levine

Mechanisms of glucagon secretion during insulin-induced hypoglycemia in man. Role of the beta cell and arterial hyperinsulinemia.

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Abstract

To elucidate the mechanisms controlling the response of glucagon to hypoglycemia, a vital component of the counterregulatory hormonal response, the role of intraislet insulin was studied in seven normal subjects and five subjects with insulin-dependent diabetes mellitus (IDDM) (of less than 15-mo duration). In the normal subjects, hypoglycemia (arterial plasma glucose [PG] 53 +/- 3 mg/dl) induced by an intravenous insulin infusion (30 mU/m2 X min for 1 h, free immunoreactive insulin [FIRI] 58 +/- 2 microU/ml) elicited a 100% fall in insulin secretion and an integrated rise in glucagon of 7.5 ng/ml per 120 min. When endogenous insulin secretion was suppressed by congruent to 50 or congruent to 85% by a hyperinsulinemic-euglycemic clamp (FIRI 63 +/- 1.5 or 147 +/- 0.3 microU/ml, respectively) before hypoglycemia, the alpha cell responses to hypoglycemia were identical to those of the control study. When the endogenous insulin secretion was stimulated by congruent to 100% (hyperinsulinemic-hyperglycemic clamp, FIRI 145 +/- 1.5 microU/ml, PG 132 +/- 2 mg/dl) before hypoglycemia, the alpha cell responses to the hypoglycemia were also superimposable on those of the control study. Finally, in C-peptide negative diabetic subjects made euglycemic by a continuous overnight intravenous insulin infusion, the alpha cell responses to hypoglycemia were comparable to those of normal subjects despite absent beta cell secretion, and were not affected by antecedent hyperinsulinemia (hyperinsulinemic-euglycemic clamp for 2 h, FIRI 61 +/- 2 microU/ml). These results indicate that the glucagon response to insulin-induced hypoglycemia is independent of the level of both endogenous intraislet and exogenous arterial insulin concentration in normal man, and that this response may be normal in the absence of endogenous insulin secretion, in contrast to earlier reports. Thus, loss of beta cell function is not responsible for alpha cell failure during insulin-induced hypoglycemia in IDDM.

Authors

G Bolli, P De Feo, G Perriello, S De Cosmo, P Compagnucci, F Santeusanio, P Brunetti, R H Unger

Role of volume in the regulation of vasopressin secretion during pregnancy in the rat.

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Abstract

We previously observed that osmoregulation and the osmotic threshold for antidiuretic hormone secretion were altered during pregnancy in Sprague-Dawley rats and the present study evaluated the influence of volume on arginine vasopressin (AVP) release during gestation in this species. Basal plasma osmolality (Posm) and intravascular volume were 297 +/- 3 mosmol/kg and 16.2 +/- 1.2 ml in virgin animals compared with 290 +/- 2 mosmol/kg and 20.2 +/- 2.3 ml in 14-d pregnant rats and 287 +/- 3 mosmol/kg and 25.2 +/- 2.3 ml in 21-d (near-term) pregnant rats (P less than 0.001, each pregnant group vs. virgin). Isosmotic volume depletion was produced by intraperitoneal polyethylene glycol. Volume decreased from 1 to 26% and blood pressure remained stable during decrements as high as 16%. Plasma AVP (PAVP) did not rise significantly in either group of pregnant animals or virgin controls until blood volume depletion reached 6-7%, after which levels rose in a similar exponential manner in virgin, 14-d, and 21-d pregnant animals. In terms of absolute changes, however, PAVP in gravid rats started to increase when intravascular volume was still considerably greater than basal blood volume in the nonpregnant controls. Other experiments, where Posm was increased by intraperitoneal hypertonic saline, reconfirmed that the osmotic threshold for AVP secretion was reduced congruent to 10 mosmol/kg during pregnancy and that AVP release was stimulated by increments in body tonicity as small as 1-2%. In parallel studies, blood volume contraction and increases in Posm were evoked by intraperitoneal polyethylene glycol dissolved in hypertonic saline and results compared with animals receiving intraperitoneal saline alone. Decrements in volume (congruent to 7%), which alone would increase PAVP minimally, increased the sensitivity of the secretory response to changes in osmolality two- to three-fold, an effect which was similar in virgin and gravid animals. Finally, restricting water intake of pregnant rats to that of virgins on days 16-20 of gestation led to suboptimal volume expansion, hypertonicity, and an exaggerated increase in PAVP. These results demonstrate that despite an intravascular space which at term is nearly twice that of virgin rats, pregnant animals secrete AVP in response to fractional volume depletion in a manner similar to nonpregnant controls; that is, the relationship between total blood volume and AVP secretion is altered during gestation such that the expanded blood volume is recognized as normal.

Authors

W M Barron, B A Stamoutsos, M D Lindheimer

Human Protein Z.

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Protein Z was purified from human plasma by a four-step procedure which included barium citrate adsorption, ammonium sulfate fractionation, DEAE-Sepharose chromatography, and blue agarose chromatography with a yield of 20%. It is a 62,000 mol wt protein with an extinction coefficient of 12.0. The concentration of Protein Z in pooled, citrated plasma is 2.2 micrograms/ml and its half-life in patients starting warfarin anticoagulation therapy is estimated to be less than 2.5 d. The NH2-terminal sequence is Ala-Gly-Ser-Tyr-Leu-Leu-(Gla)-(Gla)-Leu-Phe-(Gla)-Gly-Asn-Leu. Neither Protein Z nor its cleavage products, which were obtained by treatment of Protein Z with thrombin or plasmin, incorporated [3H]diisopropyl fluorophosphate. The physiological function of Protein Z remains unknown.

Authors

G J Broze Jr, J P Miletich

HLA-DR human histocompatibility leukocyte antigens-restricted lymphocyte-monocyte interactions in the release from monocytes of acidic isoferritins that suppress hematopoietic progenitor cells.

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Abstract

Acidic isoferritins, which under normal conditions are released from monocytes and macrophages, have a suppressive effect in vitro on granulocyte-macrophage, erythroid, and multipotential hematopoietic progenitor cells. Cell interactions modulating the release of acidic isoferritin-inhibitory activity (AIFIA) from human monocytes were investigated using the bone marrow granulocyte-macrophage progenitor cells as a target cell assay for assessing AIFIA. Monocytes, in the absence of T lymphocytes, released AIFIA when allowed to condition culture medium at 10(4) or higher concentrations of monocytes/ml. However, subpopulations of T lymphocytes modulated the release of AIFIA from monocytes. OKT8+- and OKT4+-T lymphocytes were obtained from E-rosette-positive lymphocytes by using T lymphocyte subset-specific monoclonal antibodies in either a complement-dependent cytotoxicity test to select negatively for the cells or by selection using a "panning" procedure. OKT8+-T lymphocytes suppressed completely and OKT4+-T lymphocytes enhanced the constitutive release of AIFIA from monocytes. OKT4+ lymphocytes also induced the release of AIFIA from concentrations of 10(3) monocytes/ml which did not release measurable amounts of AIFIA by themselves. The release of AIFIA from monocytes involved HLA-DR+-monocytes and -T lymphocytes. Pulsing monocytes with monoclonal antibodies to framework determinants on HLA-DR molecules, in the absence of complement, did not influence the constitutive release of AIFIA. Pulsing monocytes or T lymphocyte subpopulations with such antibodies, in the absence of complement, blocked the suppressing and inducing activities of the appropriate subpopulations of T lymphocytes. Monoclonal antibodies to common determinants shared by HLA-A, B, and C molecules did not block these cellular interactions. Treating monocytes and T lymphocytes in a complement-dependent cytotoxicity test with dilutions of the anti-HLA-DR antibodies that did not block the cellular interactions removed the populations of monocytes constitutively releasing AIFIA and the T lymphocyte subsets modulating this release. Modulation of the release of AIFIA from monocytes by T lymphocyte subpopulations required the use of autologous cells, cells from HLA-identical siblings, or unrelated donors matched for HLA-DR. Matching for only one HLA haplotype gave partial responses and this was seen in testing cells from related individuals as well as among unrelated test combinations. These cellular interactions were not detected with HLA-DR-incompatible cells differing for two HLA-DR antigens. Admixture of such HLA-DR- incompatible allogeneic cells did not interfere with the regulation of AIFIA release in the autologous cell interactions. Thus, release of AIFIA from monocytes is restricted genetically by HLA-DR at the level of T lymphocyte-monocyte interactions. The genetic determinants on the HLA-class II molecules that induce stimulation in vitro in mixed lymphocyte culture (i.e., HLA-D), however, were not involved in this effort.

Authors

H E Broxmeyer, L Juliano, L Lu, E Platzer, B Dupont

Cleavage of human high molecular weight kininogen markedly enhances its coagulant activity. Evidence that this molecule exists as a procofactor.

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Abstract

High molecular weight kininogen (HMW)-kininogen, the cofactor of contact-activated blood coagulation, accelerates the activation of Factor XII, prekallikrein, and Factor XI on a negatively charged surface. Although prekallikrein and Factor XI circulate as a complex with HMW-kininogen, no physical association has been demonstrated between Factor XII and HMW-kininogen, nor has the order of adsorption to surfaces of these proteins been fully clarified. In this report we explore the requirements for adsorption of HMW-kininogen to a clot-promoting surface (kaolin), in purified systems, as well as in normal plasma and plasma genetically deficient in each of the proteins of the contact system. The fraction of each coagulant protein associated with the kaolin pellet was determined by measuring the difference in coagulant activity between the initial sample and supernatants after incubation with kaolin, or by directly quantifying the amount of 125I-HMW-kininogen that was associated with the kaolin pellet. In normal plasma, the adsorption of HMW-kininogen to kaolin increased as the quantity of kaolin was increased in the incubation mixture. However, the HMW-kininogen in Factor XII-deficient plasma did not absorb appreciably to kaolin. Furthermore, the quantity of HMW-kininogen from prekallikrein-deficient plasma that adsorbed to kaolin was decreased as compared with normal plasma. These observations suggested that HMW-kininogen in plasma must be altered by a reaction involving both Factor XII and prekallikrein in order for HMW-kininogen to adsorb to kaolin, and to express its coagulant activity. Subsequently, the consequence of the inability of HMW-kininogen to associate with a negatively charged surface results in decreased surface activation. This assessment was derived from the further observation of the lack of prekallikrein adsorption and the diminished Factor XI adsorption in both Factor XII-deficient and HMW-kininogen-deficient plasmas, since these two zymogens (prekallikrein and Factor XI) are transported to a negatively charged surface in complex with HMW-kininogen. The percentage of HMW-kininogen coagulant activity that adsorbed to kaolin closely correlated (r = 0.98, slope = 0.97) with the amount of 125I-HMW-kininogen adsorbed, suggesting that adsorption of HMW-kininogen results in the expression of its coagulant activity. Since kallikrein, which is known to cleave HMW-kininogen, is generated when kaolin is added to plasma, we tested the hypothesis that proteolysis by kallikrein was responsible for the enhanced adsorption of HMW-kininogen to kaolin. When purified HMW-kininogen was incubated with purified kallikrein, its ability to absorb to kaolin increased with time of digestion until a maximum was reached. Moreover, (125)I-HMW-kininogen, after cleavage by kallikrein, had markedly increased affinity for kaolin than the uncleaved starting material. Furthermore, fibrinogen, at plasma concentration (3 mg/ml), markedly curtailed the adsorption of a mixture of cleaved and uncleaved HMW-kininogen to kaolin, but was unable to prevent fully cleaved HMW-kininogen from adsorbing to the kaolin. Addition of purified kallikrein to Factor XII-deficient plasma, which bypasses Factor XII-dependent contact-activation amplified the ability of its HMW-kininogen to adsorb to kaolin. These observations indicate that HMW-kininogen is a procofactor that is activated by kallikrein, a product of a reaction which it accelerates. This cleavage, which enhances its association with a clot-promoting surface in a plasma environment, is an event that is necessary for expression of its cofactor activity. These interactions would allow coordination of HMW-kininogen adsorption with the adsorption of Factor XII, which adsorbs independently of cleavage, to the same negatively charged surface.

Authors

C F Scott, L D Silver, M Schapira, R W Colman

Vasoactive intestinal polypeptide. A neurotransmitter for lower esophageal sphincter relaxation.

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Abstract

The effect of rabbit vasoactive intestinal polypeptide (VIP) antiserum on in vitro relaxation of the lower esophageal sphincter (LES) was studied in 10 cats. The stomach and esophagus were opened along the lesser curvature of the stomach and stripped of mucosa. Consecutive strips were cut and mounted in a 2.5-ml muscle chamber. They were perfused with Tyrode's solution and oxygenated continuously. After equilibration for 1 h, perfusion was stopped and one strip from the lower esophageal sphincter region was incubated in solution that contained 12-25 parts of VIP antiserum per 1,000 to Tyrode's solution, while a second strip was incubated in a solution of normal rabbit serum at the same concentration. A third strip was maintained in Tyrode's solution for the duration of the experiment. After a 1-h incubation, the strips were stimulated with 6-s square wave trains of 0.1-, 0.2-, 0.4-, and 0.8-ms pulses at 1, 2, and 5 Hz. These stimulation parameters produced LES relaxation that was completely blocked by tetrodotoxin but not by atropine or phentolamine. The strips incubated in Tyrode's solution or in normal serum relaxed reliably and consistently at all levels of stimulation. In the antiserum-treated strips, LES relaxation in response to all stimuli was significantly inhibited. Strips treated with normal serum were relaxed in a dose-dependent fashion by 10(-7) and 10(-6) M VIP, whereas the antiserum inhibited the relaxation induced by 10(-7) M, but not by 10(-6) M, VIP. Stimulation with two successive 15-min trains of electrical pulses (2 ms, 5 Hz) separated by 30 min of rest released increasing amounts of VIP into the bathing solution. VIP released during the second train of electrical stimulation was significantly (P less than 0.05) greater than in control conditions. In the cat LES, VIP antiserum inhibits the relaxation induced by exogenous VIP or by electric stimulation of nonadrenergic, noncholinergic inhibitory nerves at a level that causes the release of VIP. These findings are consistent with the hypothesis that VIP may be an inhibitory neurotransmitter responsible for LES relaxation.

Authors

P Biancani, J H Walsh, J Behar

Effects of thrombomodulin and coagulation Factor Va-light chain on protein C activation in vitro.

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Abstract

Protein C activation by thrombin is significantly accelerated by the endothelial cell surface protein thrombomodulin, Factor Va, or its light chain. In this study we have compared the activation of protein C in the presence of either cofactor and examined the possibility that thrombomodulin and Factor Va-light chain act together to regulate protein C activation by thrombin. At all concentrations of protein C used, thrombomodulin was 20 times more efficient than Factor Va-light chain in accelerating protein C activation by thrombin. Protein C treated with chymotrypsin to remove the amino-terminal 41 amino acids that contain the gamma-carboxyglutamyl residues was activated by the thrombin-thrombomodulin complex at an identical rate to native protein C, whereas the modified protein C was activated by Factor Va-light chain and thrombin at only 5% of the rate obtained by using native protein C. Increasing concentrations of Factor Va-light chain, greater than or equal to 30 nM, inhibited thrombin-thrombomodulin catalyzed protein C activation with complete inhibition observed at 90 nM Factor Va-light chain. On the other hand, increasing thrombomodulin concentrations did not inhibit protein C activation by Factor Va-light chain and thrombin. These reactions in solution mimic, in part, those obtained on endothelial cells where protein C lacking the gamma-carboxyglutamyl domain is activated poorly and Factor Va-light chain at concentrations greater than 50 nM inhibited the activation of native protein C. The results of this study suggest that thrombomodulin and Factor Va-light chain may act in concert to regulate protein C activation by thrombin.

Authors

H H Salem, N L Esmon, C T Esmon, P W Majerus