Ouabain prevents loss of autoregulation in rat pial arterioles caused by reoxygenation after a brief hypoxic episode

Pial arteriolar diameter changes inversely with changes in systemic arterial blood pressure. Such changes are consistent with autoregulatory functions. These responses are reduced by a brief period of hypoxia followed by reoxygenation. By using an open cranial window preparation we assessed the changes in pial arteriolar diameters during blood pressure changes in rats induced by hemorrhage and reinfusion of blood, before and after a brief period of hypoxia. The slopes of the changes in pial arteriolar diameter as a function of mean arterial blood pressure were -0.47 +/- 0.26 micron/mmHg (mean +/- SD; 1 mmHg = 133.3 Pa) before hypoxia and -0.11 +/- 0.23 micron/mmHg after hypoxia in the untreated rats. In ouabain-treated rats, corresponding slopes were -0.42 +/- 0.24 and -0.46 +/- 0.22 micron/mmHg. The observed protective effects of ouabain might be a blockade of the Na-K pump in the sarcolemma of the vascular smooth muscle.     

Ca2+ channels in chick neural retina cells characterized by 1,4-dihydropyridine antagonists and activators

The voltage-sensitive calcium channel in cultured chick neural retina cells was characterized by the actions of the enantiomers of Bay K 8644 and 202-791 and other 1,4-dihydropyridines. These cells showed time- and voltage-dependent Ca2+ uptake that was stimulated by K+ depolarization and blocked by the inorganic calcium channel blockers Cd2+ and Co2+. A small fraction only (15% maximum) of the uptake was inactivated by predepolarization of the cells with 80 mM K+. Ca2+ uptake was sensitive to the 1,4-dihydropyridine calcium channel antagonists and activators. (S)-Bay K 8644 and (S)-202-791 stimulated the Ca2+ uptake, and (R)-Bay K 8644 and (R)-202-791 as well as nitrendipine and PN 200-110 inhibited Ca2+ uptake stimulated by K+ depolarization or channel activators. The K+ depolarization-stimulated uptake was inhibited by 90%, but the activator-stimulated uptake was completely blocked by the 1,4-dihydropyridine antagonists. The potencies of these agents as inhibitors of Ca2+ uptake were significantly lower than the binding affinities in membrane preparations from the same cells or their binding and pharmacologic affinities in vascular smooth muscle. K+ depolarization or (S)-Bay K 8644 induced 45Ca2+ uptake was not observed in a glial cell culture. [3H]Nitrendipine and [3H]PN 200-110 bound to membrane preparations of the cells consistent with the presence of a single type of high affinity binding site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacology of L-660,711 (MK-571): a novel potent and selective leukotriene D4 receptor antagonist

L-660,711 (3-(3-(2-(7-chloro-2-quinolinyl)ethenyl)phenyl) ((3-dimethyl amino-3-oxo propyl)thio)methyl)thio)propanoic acid is a potent and selective competitive inhibitor of [3H]leukotriene D4 binding in guinea pig (Ki value, 0.22 nM) and human (Ki value, 2.1 nM) lung membranes but is essentially inactive versus [3H]leukotriene C4 binding (IC50 value in guinea pig lung, 23 microM). Functionally it competitively antagonized contractions of guinea pig trachea and ileum induced by leukotriene (LT) D4 (respective pA2 values, 9.4 and 10.5) and LTE4 (respective pA2 values, 9.1 and 10.4) and contractions of human trachea induced by LTD4 (pA2 value, 8.5). L-660,711 (5.8 x 10(-8)M) antagonized contractions of guinea pig trachea induced by LTC4 in the absence (dose ratio = 28) but not in the presence of 45 mM L-serine borate (dose ratio less than 2). L-660,711 (1.9 x 10(-5)M) did not block contractions of guinea pig trachea induced by histamine, acetylcholine, 5-hydroxytryptamine, PGF2 alpha, U-44069, or PGD2. In the presence of atropine, mepyramine, and indomethacin, L-660,711 (1.9 x 10(-5)M) inhibited a small component of the response to antigen on guinea pig trachea but completely blocked anti-IgE-induced contractions of human trachea. L-660,711 (i.v.) antagonized bronchoconstriction induced in anesthetized guinea pigs by i.v. LTC4, LTD4, and LTE4 but did not block bronchoconstriction to arachidonic acid, U-44069, 5-hydroxytryptamine, histamine, or acetylcholine. Intraduodenal L-660,711 antagonized LTD4 (0.2-12.8 micrograms/kg)-induced bronchoconstriction in guinea pigs, and p.o. L-660,711 blocked LTD4- and Ascaris-induced bronchoconstriction in conscious squirrel monkeys and ovalbumin-induced bronchoconstriction in conscious sensitized rats treated with methysergide (3 micrograms/kg). The pharmacological profile of L-660,711 indicates that it is a potent, selective, orally active leukotriene receptor antagonist which is well suited to determine the role played by LTD4 and LTE4 in asthma and other pathophysiologic conditions.     

The pharmacology of L-670,596, a potent and selective thromboxane/prostaglandin endoperoxide receptor antagonist

L-670,596 ((-)6,8-difluoro-9-rho-methylsulfonyl benzyl-1,2,3,4- tetrahydrocarbazol-1-yl-acetic acid) has been shown to be a potent receptor antagonist as evidenced by the inhibition of the binding of 125I-labeled PTA-OH to human platelets (IC50, 5.5 x 10(-9) M), inhibition of U-44069 induced aggregation of human platelet rich plasma (IC50, 1.1 x 10(-7) M), and competitive inhibition of contractions of the guinea pig tracheal chain induced by U-44069 (pA2,9.0). The compound was also active in vivo as shown by inhibition of arachidonic acid and U-44069 induced bronchoconstriction in the guinea pig (ED50 values, 0.04 and 0.03 mg/kg i.v., respectively), U44069 induced renal vasoconstriction in the pig (ED50, 0.02 mg/kg i.v.), and inhibition of ex vivo aggregation of rhesus monkey platelets to U-44069 (active 1-5 mg/kg p.o.). The selectivity of the compound was indicated by the failure to inhibit, first, ADP-induced human or primate platelet aggregation and, second, bronchoconstriction in the guinea pig in vivo and contraction of the guinea pig tracheal chain in vitro to a variety of agonists. It is concluded that L-670,596 is a potent, selective, orally active thromboxane A2/prostaglandin endoperoxide receptor antagonist.     

Electrophysiological assessment of putative antagonists of 5-hydroxytryptamine receptors: a single-cell study in the rat dorsal raphe nucleus

The intravenous administration of low doses of lysergic acid diethylamide (LSD) or of the selective 5-hydroxytryptamine1A (5-HT1A) receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) depresses the firing activity of dorsal raphe 5-HT-containing neurons, presumably via the activation of 5-HT1A receptors. The present studies were undertaken to determine the effect of different types of 5-HT receptor antagonists on this effect of LSD and 8-OH-DPAT. (-)-Propranolol (2 mg/kg i.v.), methiothepin (2 mg/kg i.p., twice daily for 4 days followed by an additional dose of 2 mg/kg i.p., prior to the experiment), pelanserine (0.5 mg/kg i.v.), and indorenate (125 micrograms/kg i.v.) failed to block the effects of either LSD or 8-OH-DPAT on the firing activity of 5-HT neurons of the dorsal raphe nucleus. However, spiperone (1 mg/kg i.v.) significantly reduced the effect of both LSD and 8-OH-DPAT. These results indicate that, among the five putative 5-HT receptor antagonists tested, only spiperone can antagonize the suppressant effect of 5-HT receptor agonists on the firing of dorsal raphe 5-HT neurons.     

Augmentation of the generation of cytotoxic T lymphocytes against syngeneic tumor cells by recombinant human tumor necrosis factor

In order to clarify the effect of recombinant human tumor necrosis factor (rHu-TNF) on the antitumor T cell immune response, we examined the effect of rHu-TNF on the generation of cytotoxic T cells (CTL) against syngeneic tumor cells. Spleen cells from X5563 plasmacytoma-transplanted mice were stimulated in vitro with mitomycin C-treated X5563 cells in the presence or absence of rHu-TNF. The generation of CTL was augmented in a dose-dependent manner by the addition of rHu-TNF. The augmenting effect of rHu-TNF was more marked when indomethacin was added to the culture. The augmenting effect was observed only when rHu-TNF was added at the early stage of the generation of CTL. The cell surface phenotype of CTL generated was L3T4- and Lyt2+. The augmentation was shown not only by the chromium-51 release assay but also by the Winn assay. As to the specificity, the augmentation of CTL generation was observed by the addition of rHu-TNF when responder-primed spleen cells were stimulated with the tumor cells in vitro. On the other hand, augmentation was not observed when responder spleen cells were not stimulated with the tumor cells in vitro, or when responder spleen cells were obtained from normal mice. The CTL generated was not cytotoxic against other tumor cells of the same haplotype. Thus, rHu-TNF augmented the generation of CTL against syngeneic tumor cells in an antigen-specific manner. The in vivo effect of rHu-TNF was examined by administering rHu-TNF into X5563-bearing mice. The spleen cells of rHu-TNF-injected mice generated a much higher CTL activity against X5563 cells in vitro than did the spleen cells of uninjected mice. From these results, a possibility can be considered that in some cases, rHu-TNF may exert its antitumor activity by stimulating the immune system.     

Human thymocytes do not respond to interleukin-2 after removal of mature "bright" CD5 positive cells

Interleukin-2 receptors (IL-2R) are expressed on minor populations of immature and mature human thymocytes. These studies were designed to determine if immature T cells could respond to the mitogen phytohemagglutinin (PHA-P) plus IL-2 in vitro by increasing the expression of IL-2R and by proliferation. Using monoclonal antibodies to CD5 and magnetic immunobeads we were able to remove all mature, "bright" CD5+ cells from nylon wool-purified thymocytes and to obtain less mature cells which consisted almost completely of cells with the CD4+CD8+ phenotype. These immature cells were mostly "dim" CD5+ and less than 5% CD5- and a small percentage expressed the IL-2R. After culture in serum-free medium with PHA-P, these cells showed only a slight increase in the percentage of IL-2R+ cells and the addition of IL-2 did not increase the percentage of IL-2R+ cells and no proliferation was observed. Unseparated, nylon wool-purified thymocytes contained 14% bright CD5+ cells. These bright CD5+ cells had a mature phenotype of CD4+CD8- (52%) and CD4-CD8+ (27%) cells. A small percentage of these cells were IL-2R+. These bright CD5+IL-2R+ cells were predominantly mature CD4+CD8- cells as measured by three-color flow cytometry. After culture with PHA-P and IL-2, the percentage of IL-2R+ cells increased and they were now found not only on CD4+CD8- but also on CD4-CD8+ and on CD4+CD8+ cells. IL-2 plus PHA-P increased proliferation of these cells as compared to those cultured in medium with PHA-P without IL-2. Thus, we show that human immature thymocytes in contrast to mature thymocytes are not responsive to IL-2 as measured by a lack of IL-2R expression and proliferation. These data indicate that mature thymocytes can express a functional high affinity receptor for IL-2 and suggest that immature thymocytes may not possess a (functional) p75 chain of the IL-2R.     

Synergistic induction of rat hepatic ornithine decarboxylase by multiple doses of cobalt chloride

The level of rat hepatic ornithine decarboxylase (ODC) induced by repetitive administration of Co2+ was determined by affinity labeling with [3H]difluoromethylornithine. Such a treatment with Co2+ ion induced ODC level to a 10-fold greater extent than single dose of the metal ion or well-known inducers of the enzyme, such as thioacetamide or carbon tetrachloride. The half life of ODC activity induced by repetitive treatment with Co2+ (95 min) was substantially increased to about 10-fold over the value obtained from the enzyme induced by single treatment with the metal ion (10 min). ODC activity induced by repetitive treatment with Co2+ was separated into two peaks by DEAE-Sepharose column chromatography. The two independently collected fractions of ODC peaks exhibited different affinity for pyridoxal 5'-phosphate in vitro and sensitivity to cycloheximide in vivo.     

Macrophage chemotactic factor (MCF) produced by a human T cell hybridoma clone

A human T cell hybridoma clone, D6-18, producing high levels of macrophage chemotactic factor (MCF) was established by the emetine-actinomycin D selection method. MCF was found to be present not only in the culture medium but also in the cell lysate of D6-18 cells. The secretion of the MCF from D6-18 cells was effectively inhibited by disodium cromoglycate, which is an inhibitor of the degranulation of mast cells, suggesting that MCF is stored in granules. The MCF of D6-18 cells was purified from the sonicated cell lysate by ion-exchange chromatographies and high-performance liquid chromatography. The amino acid sequence of the purified MCF was revealed to be WLGREDGSE or WLGRQDGSE. The synthetic peptide WLGREDGSE showed chemotactic activity against guinea pig macrophages and human monocytes at the concentration of about 10(-8) M.     

Pattern of ribonucleic acid synthesis in vitro in primary spermatocytes from mouse testis carrying an X-autosome translocation

In an attempt to elucidate the mechanism of sterility of X-autosome translocations in the mouse, we studied the distribution of [³H]-uridine incorporation in sterile males carrying the balanced X-16 reciprocal translocation. The results failed to show an overall reactivation of the X as has been postulated by Lifschytz and Lindsley (1972) but there was some spreading of X inactivation along the translocated and normal chromosome 16 in those regions that were close to the X breakpoint. We feel that this process could be responsible for metabolic disturbances leading to degeneration of primary spermatocytes and, therefore, to sterility.