Search

Urotensin-II (U-II), acting through its G-protein-coupled receptor, UT, is a possible contributor to hypertension. Variable functional responses to U-II, both within and between species studied to date, complicate the characterization of UT antagonists. In the cat, however, U-II causes systemic hypertension and constricts arterial segments isolated from several vascular beds. The purpose of this study was to clone and pharmacologically characterize cat recombinant UT to determine whether this system represents a model for characterizing UT antagonists. Cloned cat UT displayed 74% identity to primate UT, and 77% identity to rodent UT. [(125)I] hU-II bound in a saturable manner to a single site on recombinant cat UT with high affinity (K(D) 288+/-13pM) and high density (B(max) 747+/-66fmol/mg protein). U-II isopeptides displayed equipotent, high affinity binding to cat UT (K(i) 1.8-5.3nM). Cat UT was coupled to intracellular [Ca(2+)] release (EC(50) 0.6+/-0.2nM) and total inositol phosphate (IP) formation (EC(50) 0.4+/-0.1nM). Protein kinase C activation desensitized cat, but not human, UT-mediated IP formation. UT mRNA expression was detected in cat blood vessels, trachea, lung, and kidney, where the medulla (K(D) 815+/-34) and cortex and (K(D) 316+/-39pM) displayed high affinity binding for human U-II (hU-II). The cat urotensin-II receptor represents a suitable in vitro model to examine the role of the U-II/UT system in the etiology of hypertension, assisting in the evaluation of the UT antagonists to help treat cardiovascular disease.

Starting with a partial sequence from Genbank, polymerase chain reaction (PCR) was utilized to isolate the full-length cDNA for NK(3) receptor from mouse brain. The murine NK(3) receptor has a predicted sequence of 452 amino acids, sharing 96% and 86% identity to the rat and human NK(3) receptors, respectively. Binding affinities and functional potencies of tachykinin receptor agonists were similar in HEK (human embryonic kidney) 293 cells expressing murine NK(3) receptor and human NK(3) receptor, although substance P and neurokinin A were more potent stimulators of Ca(2+) mobilization in murine NK(3) receptor cells. NK(3) receptor-selective antagonists from two structural classes, had 10- to 100-fold lower binding affinities for murine NK(3) receptor compared to human NK(3) receptor, and about 5- to 10-fold reduced potency in the murine NK(3) receptor functional assay. The results demonstrate species differences in the potencies of tachykinin receptor antagonists in murine and human NK(3) receptors, and the lower potencies in the former should be taken into consideration when using murine disease models.

The contribution of the complement system to the exacerbation of cerebral ischemia/reperfusion injury was studied by comparing a group of rats with normal complement levels to another group that was complement depleted by cobra venom factor (CVF). The magnitude of reactive hyperemia was significantly greater in the complement depleted animals. There was also better preservation of somatosensory evoked potentials (SSEPs) in the complement depleted animals. These differences were not associated with changes in leukocyte infiltration as evidenced by myeloperoxidase and Leukotriene B4 activity. These data demonstrate that depleting the complement system can improve flow and outcome following cerebral ischemia with reperfusion.

Scalaradial, a marine natural product with anti-inflammatory activity, has been shown to be a selective inhibitor of 14 kDa type II phospholipase A2(PLA2). We have examined the inhibition by scalaradial (0.1 nM to 10 microM) of neutrophil function (degranulation) in response to receptor-mediated activation [N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), 30 nM; leuokotriene B4 (LTB4), 100 nM; platelet-activating factor (PAF), 100 nM] and non-receptor-mediated stimuli [A23187 (1 microM) and thapsigargin (100 nM)]. Furthermore, we evaluated the ability of scalaradial to inhibit the increase in intracellular Ca2+ in response to fMLP, LTB4, A23187, and thapsigargin as well as its ability to prevent either fMLP- or LTB4-mediated elevation in inositol phosphate production (InsP). Scalaradial was a potent inhibitor of both receptor- (IC50 = 50-200 nM) and non-receptor- (IC50 = 40-900 nM) mediated degranulation. Although scalaradial inhibited the mobilization of Ca2+ induced by fMLP, LTB4, and PAF, it did not affect the maximal Ca2+ levels attained with A23187 or thapsigargin. Neutrophil-binding studies with [3H]fMLP and [3H]LTB4 would suggest that the effect of scalaradial on agonist-induced degranulation and increase in intracellular Ca2+ was not at the receptor level because 50-fold higher concentrations were required to have a significant effect on the binding of these agonists. To determine if scalaradial affected phosphatidylinositol selective phospholipase C (PI-PLC) activity, assays were conducted to monitor fMLP- and LTB4-induced formation of InsPs using myo-[3H]inositol-labeled U-937 cells. In these cells, 2.5 to 9-fold higher concentrations of scalaradial were required to inhibit PI-PLC activity than to inhibit agonist-induced degranulation of neutrophils, suggesting that the effects of scalaradial on Ca2+ and degranulation are not the sole result of blocking receptor activation of PI-PLC. Results obtained with receptor-mediated stimuli suggest that scalaradial may have direct effects on Ca2+ channels and InsP turnover, but inhibition of intracellular Ca2+ levels was not required for scalaradial to block degranulation since scalaradial was capable of inhibiting degranulation produced by either A23187 or thapsigargin, without changing the maximal Ca2+ levels obtained with these two stimuli. These results demonstrate that scalaradial can inhibit degranulation in the presence of micromolar intracellular Ca2+ concentration, thus supporting the hypothesis that a 14 kDa PLA2 may be important in the regulation of neutrophil degranulation.

We have explored the role of phospholipase D (PLD) activation in leukotriene B4 (LTB4)-induced Ca2+ mobilization and degranulation of human neutrophils. Stimulation of [3H]alkyl-acyl-phosphatidylcholine-labeled neutrophils with LTB4 resulted in a rapid accumulation of [3H]alkyl-phosphatidic acid (PA) as well as a somewhat slower accumulation of [3H]alkyl-diglyceride (DG). In the presence of ethanol, PLD catalyzed a transphosphatidylation reaction in which LTB4 increased [3H]alkyl-phosphatidylethanol formation and simultaneously decreased LTB4-induced PA and DG accumulation. This pattern of lipid metabolism is consistent with the conclusion that LTB4 stimulates PLD activity in human neutrophils. Additional studies in which the extracellular and intracellular concentrations of Ca2+ were varied indicated that maximal LTB4-induced PLD activation was dependent upon Ca2+ and potentiated by inhibitors of protein kinase C. The time-course and concentration-response curves for LTB4-induced PLD activation were different from those for LTB4-induced Ca2+ mobilization, as measured by fura-2 fluorescence. On the other hand, the concentration-response curve for LTB4-induced PLD activation was similar to that for LTB4-induced degranulation. Preincubation of the cells with ethanol inhibited LTB4-induced PA and DG accumulation, as well as degranulation, suggesting that one or both of these metabolites were important for this response. In contrast, ethanol had no effect on LTB4-induced Ca2+ mobilization. Propranolol, an inhibitor of phosphatidate phosphohydrolase, abolished DG accumulation in response to LTB4 but had no effect on degranulation, suggesting that PA is more important than DG as a mediator of degranulation. Taken collectively, these data indicate that LTB4-induced activation of PLD in human neutrophils is mediated by a Ca(2+)-dependent mechanism, but not by protein kinase C. In addition, PLD activation in these cells may induce degranulation, but not Ca2+ mobilization.

SK&F 105809 [2-(4- methylsulfinylphenyl)-3-(4-pyridyl)-6,7-dihydro-[5H]-pyrrolo[1,2- a] imidazole] was determined to be a prodrug for the sulfide metabolite SK&F 105561 [2-(4- methylthiophenyl)-3-(4-pyridyl)-6,7-dihydro-[5H]-pyrrolo[1,2-a] imidazole] which inhibited interleukin-1 (IL-1) production in vitro and both 5-lipoxygenase (5-LO) and prostaglandin H (PGH) synthase activities in vitro and ex vivo. SK&F 105561 inhibited partially purified 5-LO with a half-maximal concentration (IC50) of 3 microM. This inhibition was reversible, independent of preincubation time, and dependent on the concentration of the substrate arachidonic acid. SK&F 105561 also inhibited purified PGH synthase with the potency dependent on the level of peroxidase activity. The IC50 was 100 microM in the absence of peroxidase activity, whereas an IC50 of 3 microM was observed in the presence of peroxidase activity. Using human monocytes, SK&F 105561 inhibited A23187-stimulated prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) production with IC50 values of 0.1 and 2 microM, respectively. In addition, IL-1 production by lipopolysaccharide-stimulated human monocytes was also inhibited (IC50 2 microM). Oral administration of SK&F 105809 to rats resulted in a dose-related generation of SK&F 105561 and in the inhibition of thromboxane B2 and LTB4 production ex vivo with a half-maximal dose (ED50) of 15 and 60 mg/kg, respectively. SK&F 105561 showed weak inhibitory activity on 12-lipoxygenase with an IC50 of greater than 200 microM. Neither SK&F 105561 nor SK&F 105809 inhibited the stimulated-turnover of arachidonic acid-containing phospholipids in human monocytes or the activity of cell-free phospholipases A2 and C. Moreover, neither SK&F 105561 nor SK&F 105809 antagonized the binding of LTB4 or leukotriene D4 to membrane receptors. From these results, SK&F 105561, the active principle of SK&F 105809, acts as an inhibitor of both inflammatory cytokine and eicosanoid production.

A sesquiterpene thioacetate, 15-acetylthioxy-furodysinin (SK&F 105900) has been isolated from the sponge Dysidea SP. This compound can bind specifically to the human peripheral blood polymorphonuclear leukocyte (PMN) and to the differentiated human monocytic leukemic U-937 cell membrane leukotriene B4 (LTB4) receptors with high-affinity. This compound can also promote a concentration-dependent chemotaxis in PMNs and an intracellular calcium mobilization in U-937 cells that can be blocked by the LTB4 receptor antagonist, LY-223982. Furthermore, the calcium mobilization induced by SK&F 105900 can specifically cross-desensitize with the LTB4-induced calcium mobilization. These observations indicate that SK&F 105900 is a novel and specific high-affinity agonist that can bind to the LTB4 receptors and activate the receptor-mediated signal transduction processes in human PMN and U-937 cells.

The effects of SK&F 86002 [5-(4-pyridyl)-6 (4-fluorophenyl)-2,3-dihydroimidazo (2,1-b) thiazole] on the generation of eicosanoids in vitro and on inflammatory responses in vivo are described and compared to other non-steroidal anti-inflammatory drugs. SK&F 86002 inhibited prostaglandin H2 (PGH2) synthase activity (IC50 120 microM) as well as prostanoid production by rat basophilic leukemia (RBL-1) cells (IC50 70 microM) and its sonicate (IC50 100 microM) and human monocytes (IC50 1 microM). In addition, SK&F 86002 inhibited the generation of dihydroxyeicosatetraenoic acid (diHETE) and 5-hydroxyeicosatetraenoic acid (5-HETE) by a high speed supernatant fraction of RBL-1 cells (IC50 10 microM). Cellular production of 5-lipoxygenase products was inhibited by SK&F 86002 as measured by leukotriene B4 (LTB4) generation from human neutrophils (IC50 20 microM), leukotriene C4 (LTC4) generation by human monocytes (IC50 20 microM), and 5-HETE production by RBL-1 cells (IC50 40 microM). The in vivo profile of anti-inflammatory activity of SK&F 86002 supports the dual inhibition of arachidonate metabolism as indicated by its activity in inflammation models that are insensitive to selective cyclooxygenase inhibitors. The responses of arachidonic-acid-induced edema in the mouse ear and rat paw, as well as the cell infiltration induced by carrageenan in the mouse peritoneum and by arachidonic acid in the rat air pouch, were inhibited by SK&F 86002 and phenidone but not by the selective cyclooxygenase inhibitors naproxen and indomethacin.

The effect of KC 2450 (racemic 3,5-cis-3-methylamino-2,3,4,5-tetrahydro-1-benzoxepine-5-ol hydrochloride) on lower esophageal sphincter pressure in pentobarbital-anesthetized dogs was determined and compared to the effect of metoclopramide. The ED20 value (i.e. the dose that increased lower esophageal sphincter pressure 20 mm Hg) was 0.72 (0.45-1.04) mg/kg i.v. for KC 2450, significantly different from 2.18 (1.30-3.42) mg/kg i.v. for metoclopramide (P less than 0.01). The superior potency of KC 2450 over metoclopramide also was demonstrated at a dose of 2 mg/kg i.v.; KC 2450 produced an increase in sphincter pressure of 43.2 +/- 4.4 mm Hg and metoclopramide produced an increase in sphincter pressure of only 28.5 +/- 5.4 mm Hg (P less than 0.05). Intraduodenally administered KC 2450 increased lower esophageal sphincter pressure at a threshold dose of 2 mg/kg with 10 mg/kg producing an increase in pressure of 53.2 +/- 9.9 mm Hg. KC 2450-induced increases in sphincter pressure were not affected by bilateral cervical vagotomy or ketanserin, but were eliminated by atropine and reduced by neuronal blockade using tetrodotoxin (TTX). KC 2450 effects also were determined in isolated circular strips of lower esophageal sphincter muscle. KC 2450 produced a concentration-related increase in canine (EC50 = 27 microM) and opossum (EC50 = 199 microM) sphincter muscle strip tension. The KC 2450 concentration-response curve was antagonized by atropine in canine and opossum sphincter muscle strips. Neuronal blockade of canine sphincter muscle with TTX antagonized the KC 2450 concentration-response curve in a non-competitive manner.(ABSTRACT TRUNCATED AT 250 WORDS)

A new peripheral dopamine agonist which causes dopaminergic renal vasodilation, was tested for central dopaminergic activity. SK & F 38393 stimulated the dopamine-sensitive adenylate cyclase in homogenates of rat caudate, as a partial agonist, and caused contralateral rotation in rats with unilateral 6-OHDA lesions of substantia nigra. Rotation was shown to be due to a direct effect on supersensitive dopamine receptors. Stimulation of cAMP formation and rotation were blocked by dopamine antagonists. In contrast to other dopamine agonists, SK & F 38393 did not cause stereotypy, emesis or inhibition of prolactin release, nor did SK & F 38393 affect dopamine turnover. The results suggest that SK & F 38393 may selectively stimulate supersensitive central dopamine receptors in vivo or may activate only a certain subclass of dopamine receptors including the receptor in the renal vasculature and the adenylate cyclase coupled postsynaptic receptor in the caudate.