Circulatory effect of TCS-80, a new imidazoline compound, in rats.

BACKGROUND: Synthesis and hypotensive properties of centrally acting imidazoline agents: 1-[(imidazolidin-2-yl)imino]-1H-indazole (Marsanidine) and 7-chloro-1-[(4,5-dihydro-1H-imidazol-2-yl)methyl]-1H-indazole (TCS-80) were tested in rats. We have recently synthesized two novel Marsanidine analogues which decrease blood pressure and heart rate in rats: 1-[(4,5-dihydro-1H-imidazol-2-yl)methyl]-1H-indole (TCS-54), and 7-chloro-1-[(4,5-dihydro-1H-imidazol-2-yl)methyl]-1H-indole (TCS-213). Among all these analogues, compound TCS-80 exhibits the highest affinity to I1-imidazoline receptors and the lowest α2/I1 selectivity ratio. The observed cardiovascular effects of the compounds might be mediated through α2-adrenergic and I1-imidazoline receptors and subsequent decrease of the symphathetic nerve activity. The present studies were performed to determine whether α2-adrenergic and/or I1-imidazoline receptors are involved in the decrease of blood pressure and heart rate induced by Marsanidine, TCS-54, TCS-80, and TCS-213 in rats. METHODS: Anesthetized rats were infused iv with the tested compounds and selective α2-adrenoceptor antagonist, RX821002, or nonselective α2-adrenergic/I1-imidazoline receptor antagonist, Efaroxan. The mean arterial blood pressure and heart rate were monitored directly and continuously throughout the experiment. RESULTS: Efaroxan inhibited the hypotensive effect of TCS-80 stronger than RX821002. The degree of inhibition of the hypotensive effect of the remaining compounds was similar for both antagonists. The presence of Efaroxan and RX821002 diminished the heart rate decrease induced by all compounds administration, though the influence on the maximal chronotropic effect was attenuated significantly in the TCS-80 and TCS-213 treated animals only. CONCLUSION: Our results indicate that hypotensive and negative chronotropic activities of all tested compounds are mediated by both the α2-adrenergic and I1-imidazoline receptors. Moreover, the circulatory effect of TCS-80 might be mediated to relatively higher degree by the I1-imidazoline receptors than by the α2-adrenergic ones.

The effect of a selective neuronal nitric oxide synthase inhibitor 3-bromo 7-nitroindazole on spatial learning and memory in rats.

Since the discovery of nitric oxide (NO) as a neuronal messenger, its way to modulate learning and memory functions is subject of intense research. NO is an intercellular messenger in the central nervous system and is formed on demand through the conversion of L-arginine to L-citrulline via the enzyme nitric oxide synthase (NOS). Neuronal form of nitric oxide synthase may play an important role in a wide range of physiological and pathological conditions. Therefore the aim of this study was to investigate the effects of chronic 3-bromo 7-nitroindazole (3-Br 7-NI), specific neuronal nitric oxide synthase (nNOS) inhibitor, administration on spatial learning and memory performance in rats using the Morris water maze (MWM) paradigm. Male rats received either 3-Br 7-NI (20mg/kg/day) or saline via intraperitoneal injection for 5days. Daily administration of the specific neuronal nitric oxide synthase (nNOS) inhibitor, 3-Br 7-NI impaired the acquisition of the MWM task. 3-Br 7-NI also impaired the probe trial. The MWM training was associated with a significant increase in the brain-derived neurotrophic factor (BDNF) mRNA expression in the hippocampus. BDNF mRNA expression in the hippocampus did not change after 3-Br 7-NI treatment. L-arginine significantly reversed behavioural parameters, and the effect of 3-Br 7-NI was found to be NO-dependent. There were no differences in locomotor activity and blood pressure in 3-Br 7-NI treated rats. Our results may suggest that nNOS plays a key role in spatial memory formation in rats.

Inhibition of sperm capacitation and fertilizing capacity by adjudin is mediated by chloride and its channels in humans.

STUDY QUESTION: Does adjudin disrupt chloride ion (Cl⁻) ion transport function in human sperm and impede sperm capacitation and fertilizing ability in vitro? SUMMARY ANSWER: In this study the results indicate that adjudin is a potent blocker of Cl⁻ channels: disrupting Cl⁻ ion transport function results in a decline in sperm capacitation and fertilizing ability in humans in vitro. WHAT IS KNOWN ALREADY: Although our previous studies have demonstrated that adjudin exerts its effect by disrupting sertoli-germ cell adhesion junctions, most notably apical ectoplasmic specialization by targeting testin and actin filament bundles that disrupts the actin-based cytoskeleton in sertoli cells, it remains unclear whether adjudin impedes Cl⁻ ion transport function in the human sperm. STUDY DESIGN, SIZE AND DURATION: Semen samples were obtained from 45 fertile men (aged 25-32). Spermatozoa were isolated from the semen in the human tube fluid (HTF) medium by centrifugation through a discontinuous Percoll gradient, and incubated with adjudin at 10 nM-10 µM and/or other reagents under capacitating conditions for 0-5 h. PARTICIPANTS/MATERIALS, SETTING, METHODS: We evaluated the effect of adjudin and different reagents on sperm functions with which they were incubated at 37 °C. Sperm motility and hyperactivation were analyzed by a computer-assisted sperm analysis (CASA) system. Sperm capacitation and the acrosome reaction were assessed by chlortetracycline fluorescence staining. Sperm fertilizing ability was evaluated by sperm penetration of zona-free hamster egg assay, and cellular cAMP levels in spermatozoa were quantified by the EIA kit. The proteins tyrosine, serine and threonine phosphorylation in the presence or absence of adjudin were analyzed by means of a immunodetection of spermatozoa, especially, compared the effect of adjudin on sperm hyperactivation and capacitation in the complete HTF medium with the Cl⁻-deficient HTF medium as well as the various Cl⁻ channel blockers. MAIN RESULTS AND THE ROLE OF CHANCE: Adjudin significantly inhibited sperm hyperactivation but not sperm motility. Adjudin-induced inhibition of sperm capacitation was reversible, and it was found to block the rhuZP3ß- and progesterone-induced acrosome reaction in a dose-dependent manner. Adjudin also blocked sperm penetration of zona-free hamster eggs, and significantly inhibited both forskolin-activated transmembrane adenylyl cyclase and soluble adenylyl cyclase activities leading to a significant decline in the cellular cAMP levels in human spermatozoa. Adjudin failed to reduce sperm protein tyrosine phosphorylation but it did prevent sperm serine and threonine protein phosphorylation. Interestingly, adjudin was found to exert its inhibitory effects on sperm capacitation and capacitation-associated events only in the complete Cl⁻-HTF medium but not Cl⁻-deficient medium, illustrating the likely involvement of Cl⁻. Adjudin inhibits the fertility capacity of human sperm is mediated by disrupting chloride ion and its transport function. LIMITATIONS, REASONS FOR CAUTION: This study has examined the effect of adjudin only on human sperm capacitation and fertilizing ability in vitro and thus has some limitations. Further investigations in vivo are needed to confirm adjudin is a potent male contraceptive. WIDER IMPLICATIONS OF THE FINDINGS: Our studies demonstrated that adjudin inhibition of capacitation is reversible and its toxicity is low, opening the door for the examination of adjudin as a mediator of male fertility control. Adjudin may be a safe, efficient and reversible male antifertility agent and applicable to initial clinical trials of adjudin as a male antifertility agent in humans. STUDING FUNDING/COMPETING INTEREST(S): This work was supported by the National Basic Research Program of China (2006CB504002), the Nature Science Foundation of China (Nos. 81000244 and 81170554), Zhejiang Project of Science and Technology (2011C23046), the Nature Science Fund of Zhejiang province (Nos.Y2100058 and Y2090236), the key Science and Technology Innovation Team of Zhejiang Province (No.2012R10048-07) and the National Institutes of Health (NICHD U54 HD029990 project 5), USA. The authors declare no conflict of interest.

Evidence for the involvement of neuronal nitric oxide synthase and soluble guanylate cyclase on cognitive functions in rats.

AIMS: The influence of 3-bromo-7-nitroindazole (3-Br 7-NI), a potent and selective neuronal nitric oxide synthase (nNOS) inhibitor, and [1H-[1,2,4]-oxadiazole[4,3a]-quinoxaline-1-one] (ODQ), a highly selective, irreversible inhibitor of soluble guanylate cyclase (sGC), on working and reference memory and emotional learning was investigated in rats. MAIN METHODS: The effects were assessed in the three-panel runway and step-down passive avoidance task, respectively. KEY FINDINGS: 3-Br 7-NI (5, 10, and 20mg/kg) and ODQ (5, 10, and 20mg/kg) significantly increased the number of errors and latency of both working and reference memory performance of rats and impaired retention for the passive avoidance task. The effect of 3-Br 7-NI was reversed by l-arginine (250 mg/kg). SIGNIFICANCE: Findings of the study supported the hypothesis that nNOS inhibition disrupts reference and working memory processes in terms of an impairment in the strategies used for solving learning tasks, and, according to these results, nNOS-sGC may be required for emotional learning and both reference and working memory.

Myricitrin, a nitric oxide and protein kinase C inhibitor, exerts antipsychotic-like effects in animal models.

Myricitrin is a nitric oxide (NO) and protein kinase C (PKC) inhibitor that has central nervous system activity, including anxiolytic-like action. Nitric oxide inhibitors blocked the behavioral effects of apomorphine, suggesting an antipsychotic-like effect. Furthermore, PKC inhibition reduced psychotic symptoms in acute mania patients and blocked amphetamine-induced hyperlocomotion, suggesting a potential antipsychotic-like effect. The present study evaluated the effects of myricitrin in animal models that assess antipsychotic-like effects (apomorphine-induced stereotypy and climbing and the paw test) and extrapyramidal side effects (catalepsy test and paw test). Olanzapine was used as a positive control. 7-Nitroindazole (7-NI), a NOS inhibitor, and l-arginine, a NO precursor, were used to evaluate nitrergic modulation, and tamoxifen was used to test the effect of PKC inhibition. In mice, myricitrin dose-dependently and olanzapine blocked the stereotypy and climbing induced by apomorphine at doses that did not induce catalepsy. 7-Nitroindazole also blocked apomorphine-induced stereotypy and climbing, which were reversed by l-arginine pretreatment. l-arginine only attenuated the effects of myricitrin on apomorphine's effects. Tamoxifen also blocked apomorphine-induced stereotypy and climbing. In the paw test in rats, myricitrin and olanzapine increased hindlimb retraction time at doses that did not affect forelimb reaction time, whereas haloperidol affected both parameters at the same dose. Myricitrin did not induce catalepsy in the bar test. Tamoxifen did not affect hindlimb retraction time or forelimb retraction time, whereas 7-NI significantly increased hindlimb reaction time. Thus, myricitrin exhibited an antipsychotic-like profile at doses that did not induce catalepsy, and this effect may be related to nitrergic action.

YC-1 enhances natural killer cell differentiation from hematopoietic stem cells.

NK cells play crucial roles in innate immunity and adaptive immunity. The detailed mechanisms, however, governing NK cell development remains unclear. In this study, we report that YC-1 significantly enhances NK cell populations differentiated from human umbilical cord blood hematopoietic stem cells (HSCs). NK cells increased by YC-1 display both phenotypic and functional features of fully mature NK (mNK) cells, but YC-1 does not affect the activation of mNK cells. YC-1 did not affect cGMP production and phosphorylation of STAT-5 which is essential for IL-15R signaling. On the other hand, YC-1 increased p38 MAPK phosphorylation during NK cell differentiation. Furthermore, p38 inhibitor SB203580 inhibited the differentiation of NK cells enhanced by YC-1. Taken together, these data suggest that YC-1 enhances NK cell differentiation through the activation of p38 MAPK which is involved in NK cell differentiation.

Effects of L-arginine on 7-nitroindazole-induced reference and working memory performance of rats.

BACKGROUND/AIMS: This study was performed in order to elucidate the effects of 7-nitroindazole (7-NI) and L-arginine on cognitive functions of rats in a three-panel runway task. METHODS: 7-NI (2.5, 5, 10 mg/kg) and L-arginine (200 mg/kg) were injected intraperitoneally into male Wistar rats 30 min before the test. RESULTS: 7-NI (5, 10 mg/kg) significantly increased the number of errors (pushes made on the 2 incorrect panels of the panel gate at 4 choice points) and prolonged the latency of reference memory in the three-panel runway task. The negative effects of 7-NI on reference memory was counteracted by L-arginine, a substrate for nitric oxide synthase. While having no effect on the number of working memory errors in working memory trials, 7-NI prolonged the latency from the 2nd to the 6th trial at the 5- and 10-mg/kg doses. Concurrent administration with L-arginine counteracted the 7-NI-induced prolongation of latency from the 2nd to the 6th trial. CONCLUSION: 7-NI impaired reference memory but not working memory in the three-panel runway test. Moreover, nitric oxide played an essential role in the development of reference memory and the negative effects of 7-NI on memory were counteracted by L-arginine.

Inhibitory effect of sulfobutyl ether beta-cyclodextrin on DY-9760e-induced cellular damage: In vitro and in vivo studies.

The effects of water-soluble beta-cyclodextrin derivatives (beta-CyDs), such as 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CyD) and sulfobutyl ether beta-cyclodextrin (SBE7-beta-CyD) on cytotoxicity of DY-9760e (3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate) toward human umbilical vein endothelial cells (HUVECs) in vitro and vascular damage of the auricular vein of rabbits by DY-9760e in vivo were investigated. The spectroscopic study revealed that of the four beta-CyDs SBE7-beta-CyD forms the most stable inclusion complex in phosphate-buffered saline, probably because of a synergetic effect of hydrophobic and electrostatic interactions. beta-CyDs inhibited DY-9760e-induced cell death toward HUVECs in an order of G(2)-beta-CyD < beta-CyD < HP-beta-CyD < SBE7-beta-CyD, which was consistent with the order of the magnitude of stability constants. When the DY-9760e solution was infused into the auricular vein of rabbits for 24 h, SBE7-beta-CyD suppressed a DY-9760e-induced irritation such as thrombus, desquamation of the endothelium vasculitis, and perivasculitis. The present data indicated that SBE7-beta-CyD formed an inclusion complex with DY-9760e in a buffer solution and possessed the protective effect on DY-9760e-induced cytotoxicity toward HUVECs and vascular damage in rabbits. These results suggested potential use of SBE7-beta-CyD as a parenteral carrier for DY-9760e.

Protective effect of sulfobutyl ether beta-cyclodextrin on DY-9760e-induced hemolysis in vitro.

The hemolytic behavior of a novel cytoprotective agent, DY-9760e (3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate) was investigated using rabbit erythrocytes. Further, the effects of water-soluble cyclodextrin derivatives, such as 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CyD) and sulfobutyl ether of beta-cyclodextrin (SBE-beta-CyD), on the hemolytic activity of DY-9760e were studied. DY-9760e induced hemolysis at concentrations >0.2-0.3 mM in phosphate buffered saline (PBS) of pH 4.0 and 6.0, where DY-9760e is predominantly in dicationic and monocationic forms, respectively. The hemolytic activity of the monocationic DY-9760e was higher than that of the dicationic species, and the hemolysis at pH 4.0 involved the formation of methemoglobin. DY9760e induced the morphological change of erythrocytes towards membrane invagination at both pH 4.0 and 6.0. SBE7-beta-CyD significantly suppressed the DY-9760e-induced hemolysis and morphological change at both pH 4.0 and 6.0, as well as the formation of methemoglobin at pH 4.0. On the other hand, HP-beta-CyD suppressed only the hemolysis, but neither the morphological change nor the formation of methemoglobin. In addition, the inhibitory effect of SBE7-beta-CyD on the hemolysis was greater than that of HP-beta-CyD. The superior inhibitory effect of SBE7-beta-CyD on the DY-9760-induced hemolysis, the morphological change, and the formation of methemoglobin may be attributable to the formation of a stable inclusion complex with DY-9760e and to the weaker hemolytic activity of SBE7beta-CyD than HP-beta-CyD. These results suggest potential use of SBE7-beta-CyD as a parenteral carrier for DY-9760e.

Chronic hyperammonemia in rats impairs activation of soluble guanylate cyclase in neurons and in lymphocytes: a putative peripheral marker for neurological alterations.

Chronic hyperammonemia impairs the glutamate-nitric oxide-cGMP pathway in rat brain in vivo. The aims of this work were to assess whether hyperammonemia impairs modulation of soluble guanylate cyclase, and to look for a peripheral marker for impairment of this pathway in brain. We activated the pathway at different steps using glutamate, SNAP, or YC-1. In control neurons these compounds increased cGMP by 7.4-, 9.7- and 7.2-fold, respectively. In ammonia-treated neurons formation of cGMP induced by glutamate, SNAP, and YC-1 was reduced by 50%, 56%, and 52%, respectively, indicating that hyperammonemia impairs activation of guanylate cyclase. This enzyme is also present in lymphocytes. Activation of guanylate cyclase by SNAP or YC-1 was impaired in lymphocytes from hyperammonemic rats. These results suggest that determination of the activation of soluble guanylate cyclase in lymphocytes could serve as a peripheral marker for impairment of the neuronal glutamate-nitric oxide-cGMP pathway in brain.

Impaired learning in rats in a 14-unit T-maze by 7-nitroindazole, a neuronal nitric oxide synthase inhibitor, is attenuated by the nitric oxide donor, molsidomine.

In previous experiments, it was demonstrated that systemic or central administration of the nitric oxide synthase (NO synthase) inhibitor, NG-nitro-L-arginine (N-Arg), produced dose-dependent learning impairments in rats in a 14-unit T-maze; and that sodium nitroprusside, a NO donor, could attenuate the impairment. Since N-Arg is not specific for neuronal NO synthase and produces hypertension, it is possible that effects on the cardiovasculature may have contributed to the impaired maze performance. In the present experiment, we have investigated the maze performance of 3-4 months old male Fischer-344 rats following treatment with 7-nitroindazole, a NO synthase inhibitor that is selective for neuronal NO synthase and does not produce hypertension. In addition, we examined the effects of the NO donor, molsidomine, which is much longer acting than sodium nitroprusside. Rats were pretrained to avoid footshock in a straight runway and received training in a 14-unit T-maze 24 h later. In an initial dose-response study, rats received intraperitoneal (i.p.) injections of either 7-nitroindazole (25, 50, or 65 mg/kg) or peanut oil 30 min prior to maze training. 7-nitroindazole produced significant, dose-dependent maze acquisition deficits, with 65 mg/kg producing the greatest learning impairment. This dose of 7-nitroindazole had no significant effect on systolic blood pressure. Following the dose-response study, rats were given i.p. injections of either 7-nitroindazole (70 mg/kg) plus saline, 7-nitroindazole (70 mg/kg) plus the NO donor, molsidomine (2 or 4 mg/kg), or peanut oil plus saline as controls. Both doses of molsidomine significantly attenuated the learning deficit induced by 7-nitroindazole relative to controls. These findings represent the first evidence that impaired learning produced by inhibition of neuronal NO synthase can be overcome by systemic administration of a NO donor.

The antineoplastic drug lonidamine interferes with the acidification mechanism of cell organelles.

The effect of the anticancer drug lonidamine (LND) on the pH of intracellular organelles was studied in isolated rat thymocytes by fluorimetric analysis of the (bafilomycin-nigericin sensitive) uptake of the acridine orange dye (AO) into acidic compartments. LND brought about a marked reduction (> 60%) in the above pH gradients with a half maximal decrease at a concentration of 0.25 mM. LND also caused a decrease, although to a lesser extent, in the ATP levels. In isolated rat liver lysosomes, 0.6 mM LND was found to inhibit ATP-driven organelle acidification by about 80%; minor inhibition of ATPase activity was observed in the same conditions. In addition, LND was able to promote proton efflux from isolated lysosomes. On the basis of our results it is suggested that the effect of LND on intracellular proton gradients may be due partially to the decrease in ATP levels, and mostly to a drug-induced increase in the ion (proton) permeability of the membranes.

Characterization of the novel nitric oxide synthase inhibitor 7-nitro indazole and related indazoles: antinociceptive and cardiovascular effects.

1. 7-Nitro indazole (7-NI, 10-50 mg kg-1), 6-nitro indazole and indazole (25-100 mg kg-1) administered i.p. in the mouse produce dose-related antinociception in the late phase of the formalin-induced hindpaw licking and acetic acid-induced abdominal constriction assays. The ED50 values (mg kg-1) were as follows: 7-NI (27.5 and 22.5), 6-nitro indazole (62.5 and 44.0) and indazole (41.0 and 48.5) in the two assays respectively. 3-Indazolinone, 6 amino indazole and 6-sulphanilimido indazole (all 50 mg kg-1) were without effect. With the exception of 5-nitro indazole (50 mg kg-1) which produced sedation, none of the other indazole derivates examined caused overt behavioural changes. 2. The antinociceptive effect of 7-NI (25 mg kg-1, i.p.) in the late phase of the formalin-induced hindpaw licking assay was partially (46.7 +/- 16.2%, n = 18) reversed by pretreatment with L- but not D-arginine (both 50 mg kg-1, i.p.). 3. The time course of 7-NI induced antinociception in the mouse was correlated with inhibition of brain (cerebellum) nitric oxide synthase (NOS) activity. Maximum antinociceptive activity and NOS inhibition was detected 18-30 min following i.p. administration. In contrast, no antinociceptive effect or inhibition of cerebellar NOS was detected 75 min post-injection. 4. 7-NI, 6-nitro indazole, indazole, 3-indazolinone and 6-amino indazole (all 50 mg kg-1) failed to influence mean arterial pressure (MAP) over the 45 min after i.p. administration in the anaesthetized mouse. Similarly, 7-NI (25 mg kg-1) administered i.v. in the anaesthetized rat did not increase MAP or influence the vasodepressor effect of i.v. injected acetylcholine (ACh) over the same period.5. 7-NI (100 microM) did not influence the vasorelaxant effect of ACh (IC50, 0.2 +/- 0.04 microM, cf. 0.16+/-0.06 microM, n = 6) in phenylephrine-precontracted rabbit aortic rings.6. These data provide further evidence that antinociception following administration of 7-NI in the mouse results from inhibition of central NOS activity and is not associated with inhibition of in vivo vascular endothelial cells NOS. Accordingly, 7-NI (or a derivative thereof) may provide an alternative approach to the development of novel antinociceptive drugs.

Heterogeneity of binding sites for ICI 198,615 in human lung parenchyma.

We have identified and characterized two different subclasses of binding site for the novel peptido-leukotriene (LT) antagonist, [3H]ICI 198,615, in membranes from human lung parenchyma using a receptor-ligand assay. This novel compound is representative of a new class of LT receptor antagonists and it has been demonstrated to be several orders of magnitude more potent and selective than most other LT antagonists described to date. The binding of [3H]ICI 198,615 is rapid, specific and saturable. Equilibrium was reached within 5-10 min. Non linear fitting of dissociation time courses has revealed the presence of two different components (K(off)1 = 8.3 +/- 6.8 x 10(-4) sec-1 and K(off)2 = 0.79 +/- 1.66 x 10(-3) sec-1) of the kinetic curves, suggesting heterogeneity of the binding sites. Computer analysis of equilibrium binding data obtained at 25 degrees results in a model with two classes of binding sites, a high affinity-low capacity class with Kd1 = 0.024 +/- 0.014 nM and Bmax1 = 0.015 +/- 0.004 pmol/mg protein and a low affinity-high capacity class with Kd2 = 6326 +/- 3859 nM and Bmax2 = 473 +/- 383 pmol/mg protein. In competition studies, LTD4 was also found to interact with two classes of binding site (Kd1 = 0.016 +/- 0.008 nM and Kd2 = 15195 +/- 8965 nM). On the contrary, LTE4 and LTC4 were found to interact with a homogeneous class of sites only with Kd = 7466 +/- 4629 nM and Kd = 428 +/- 73 nM, respectively. Furthermore, we have evaluated the effect of a number of LT antagonists on the binding of [3H]ICI 198,615. Ro 24-5913 (Kd = 3.0 +/- 2.1 nM), FPL55712 (Kd = 4945 +/- 2868 nM), LY171883 (Kd = 19628 +/- 12365 nM), SKF 104353 (Kd = 74.2 +/- 46 nM) and its enantiomer SKF 104373 (Kd = 13627 +/- 6813 nM) were found to interact with a single class of binding sites. The present studies indicate a heterogeneity of binding sites for ICI 198,615 in membranes from human lung parenchyma and that ICI 198,615 is a very potent and selective antagonist of LTD4 receptors in this tissue.

Leukotriene constriction of mouse pial arterioles in vivo is endothelium-dependent and receptor-mediated.

The diameters of pial arterioles of mice were monitored in vivo with an image-splitting technique and television microscopy. Concentrations of leukotriene C4 as low as 10(-7) M constricted the arterioles. The leukotriene C4-D4 receptor blocker ICI 198615 (10(-8) M) inhibited the response. Endothelial injury by helium-neon laser/Evans blue technique eliminated the constriction and unmasked a slight but consistent relaxation that was not inhibited by 10(-8) M ICI 198615. Since leukotrienes are produced by the brain and enter the cerebrospinal fluid in ischemia, head trauma, and subarachnoid hemorrhage, the possibility that leukotrienes C4 and D4 contribute to decreases in cerebral blood flow during these conditions should be considered. However, the present data makes such a possibility far less likely because the endothelium is frequently injured in these conditions, and therefore the ability of leukotrienes to constrict vessels would be severely curtailed.