Blockade NF-kappaB activation prohibits TNF-alpha-induced cyclooxygenase-2 gene expression in ED27 trophoblast-like cells.

Among the many functions of trophoblast cells is the production of prostaglandins (PGs) for governing several fetoplacental vascular functions during gestation and the triggering of events leading to parturition. Recent evidence suggests that pro-inflammatory cytokines such as tumour necrosis factors (TNF-alpha) induce PG formation via cyclooxygenase-2 (COX-2), a highly inducible enzyme whose gene is regulated at least in part by inducible transcription factor NF-kappaB. To examine the mechanism by which COX-2-driven PG biosynthesis occurs in trophoblast cells, we utilized the immortalized trophoblast-like cell line ED(27). These cells exhibit many of the properties of villous or extravillous trophoblasts and produce large amounts of PGs in response to TNF-alpha. We demonstrated that challenge of ED(27)cells with TNF-alpha caused binding of the NF-kappaB complex to its kappaB site followed by increased accumulation of COX-2 transcripts. In addition, the inhibitor of NF-kappaB, IkappaB-alpha, became phosphorylated and was rapidly degraded in cytokine-treated cells; this process was abolished by co-incubation with the proteasome inhibitor, MG-132. Finally, when cells were pre-incubated with MG-132 and then challenged with TNF-alpha, PG formation was attenuated in a concentration-dependent manner. These data indicate that, in ED(27)trophoblast-like cells isolated from the first-trimester placenta, TNF-alpha treatment leads to activation of NF-kappaB and subsequent transcription of the COX-2 gene.

Biochemical activities of trimetoquinol analogs at human beta(1)- and beta(3)-adrenergic receptors.

The biochemical activities of trimetoquinol (TMQ) analogs were evaluated at the human beta(1)- and beta(3)-adrenergic receptor (AR) subtypes expressed in Chinese hamster ovary cells. In radioligand binding assays, the 1-benzyl iodine-substituted analogs exhibited higher binding affinities at both beta(1)- and beta(3)-AR subtype as compared to TMQ. In cAMP accumulation assays, these analogs exhibited high potencies at both beta(1)- and beta(3)-AR. The 3',5'-diiodo-4'-amino analog of TMQ was the most potent beta(3)-AR agonist, 17-fold more potent at the beta(3)-AR versus the beta(1)-AR. Masking of the 6,7-dihydroxy group of the catechol ring of 3',5'-diiodo-4'-acetamido analog of TMQ, a potent beta(1)- and beta(3)-AR agonist, abolished activity at both beta-AR subtypes. Furthermore, substitution of a strong electron withdrawing group such as the trifluoromethyl moiety at the 1-benzyl ring of TMQ dramatically decreased potency at beta(1)- and beta(3)-AR compared to TMQ. Replacement of the 1-benzyl ring of TMQ with a naphthalene ring did not alter affinity but reduced potency of resulting 1-naphthylmethyl and 2-naphthylmethyl analogs at beta(1)- and beta(3)-AR compared to TMQ. Our results define the structural and electronic properties of substituents on TMQ necessary for potent activation of beta(1)- and beta(3)-AR and suggest that further modifications of the 1-benzyl iodine-substituted analogs may yield potent beta(3)-AR agonists.

Biochemical and functional characterization of 1-benzyl substituted trimetoquinol affinity analogs on rat and human beta-adrenoceptors.

The site of interaction for the 1-(3',4',5'-trimethoxybenzyl) group of trimetoquinol (TMQ) with beta-adrenoceptors (beta-ARs) is important for the rational design of highly potent and beta3-AR-selective analogs. 1-Benzyl ring-substituted TMQ analogs were evaluated for binding affinities and biochemical activities (cyclic AMP accumulations) in Chinese hamster ovary (CHO) cells expressing the rat and human beta3-AR, and for functional activities on isolated rat tissues. Binding affinities (K1 approximately 0.055 to 1.5 microM) for the rat beta3-AR and potencies for adenylyl cyclase activation (K(act) approximately 0.43 to 2;5 nM) of the 3'-monoiodo or 3',5'-diiodo derivatives with 4'-isothiocyanato-, 4'-amino, 4'-acetamido, or 4'-alpha-haloacetamido substitutions were higher than those of (-)-isoproterenol, and comparable to those of BRL 37344 [(+/-)-(R*,R*-[4-[2-[[2-(3-chlorophenyl)-2-hydroxy-ethyl]amino]propyl]ph enoxy]-acetic acid sodium]. A similar rank order of binding affinities (K(i) approximately 0.11 to 2.5 microM) and potencies (K(act) approximately 0.45 to 9.5 nM) was obtained for TMQ analogs on the human beta3-AR. The 4'-acetamido and 4'-alpha-chloroacetamido analogs of 3',5'-diiodoTMQ were more potent than (-)-isoproterenol in rat atria (beta1-AR) and rat trachea (beta2-AR) and exhibited partial agonist activities, whereas full agonist activities were observed in rat esophageal smooth muscle (EC50 approximately 2-8 nM, beta3-AR). 4'-alpha-Chloroacetamido-3',5'-diiodoTMQ-mediated chronotropic responses in atria were sustained and resistant to washout. Further, the 4'-alpha-chloroacetamido and 4'-alpha-bromoacetamido analogs of 3',5'-diiodoTMQ demonstrated significant concentration-dependent irreversible binding to the rat beta3-AR. Reversible beta-AR agonists such as (-)-isoproterenol, BRL 37344, and 4'-acetamido-3',5'-diiodoTMQ or nucleophilic 1-amino acids (lysine, glutathione, cysteine) did not protect against this irreversible binding. Thus, the lipophilic 1-benzyl ring of TMQ analogs interacts with a hydrophobic region of the beta-AR that may represent an exo-site or an allosteric binding site.

Beta-adrenoceptor subtype activities of trimetoquinol derivatives: biochemical studies on human beta-adrenoceptors expressed in chinese hamster ovary cells.

The beta-adrenoceptor activities of trimetoquinol (TMQ) isomers and selected derivatives were evaluated on human beta-adrenoceptor subtypes expressed in Chinese hamster ovary cells. In cAMP accumulation assays, (-)-TMQ was 214-, 281-, and 776-fold more potent than (+)-TMQ at stimulating beta(1)-, beta(2)-, and beta(3)-adrenoceptor subtypes, respectively. In radioligand binding assays, (-)-TMQ exhibited 123-, 331-, and 5-fold greater affinity than (+)-TMQ for beta(1)-, beta(2)-, and beta(3)-adrenoceptor subtypes, respectively. (-)-TMQ and (+/-)-TMQ activated the human beta(3)-adrenoceptor with an 8.2- and 3.4-fold greater efficacy, respectively, than the reference beta-adrenoceptor agonist (-)-isoproterenol (efficacy = 1). The 3',5'-diiodo analogs of TMQ were partial agonists of the beta(2)-adrenoceptor relative to (-)-isoproterenol, and their potencies were 5- to 10-fold higher at the beta(3)-adrenoceptor as compared with beta(1)-adrenoceptors. Modification of the catechol (6,7-dihydroxy) nucleus, such as replacement of the 7-hydroxy group with a chloro group (7-chloroTMQ), ring fluorination (8-fluoro and 5,8-difluoro analogs), or preparation of bioisosteric tetrahydrothiazolopyridine (THP) derivatives of TMQ yielded compounds that displayed partial agonist activity (relative to (-)-isoproterenol) or were inactive at the beta(2)-adrenoceptor and exhibited beta(3)-adrenoceptor-selective stimulation compared with the beta(1)-adrenoceptor. Furthermore, the 3',5'-diiodo-4'-methoxybenzylTHP derivative of TMQ was 65-fold more potent than the corresponding 3',4',5'-trimethoxybenzylTHP at the human beta(3)-adrenoceptor. Our results indicate that 6, 7-dihydroxy-catechol-modified and 1-benzyl halogen-substituted derivatives of TMQ represent promising leads for the development of beta(3)-adrenoceptor-selective agonists.

Endogenous nitric oxide inhibits endothelin-1-induced chloride secretion in guinea pig colon.

Segments of guinea pig distal colon, stripped of the external muscle layers, were set up in flux chambers for measurement of short-circuit current (Isc) indicative of active, electrogenic ion transport. During neural blockade with tetrodotoxin, the nitric oxide scavenger, hemoglobin, and the nitric oxide synthase inhibitor, N omega-nitro-L-arginine (L-NNA), reduced Isc. The reduction in Isc in response to hemoglobin was reversed by L-arginine and blockers of chloride secretion, including bumetanide and diphenylamine-2-carboxylic acid, but not by the potassium channel blockers, barium and tetraethylammonium, nor by amiloride, an epithelial sodium channel blocker. The hemoglobin-induced reduction in Isc was not affected by blockade of prostaglandin synthesis with piroxicam. During neural blockade, the nitric oxide donors, sodium nitroprusside and NONOate, increased Isc which was abolished by piroxicam. Endothelin-1 (ET-1) also evoked an increase in Isc that was unaffected by amiloride and was inhibitable by bumetanide, chloride-free solutions, tetrodotoxin, piroxicam, and the ETA receptor antagonist, BQ123. The ETB receptor agonist, [Ala1,3,11,15]-endothelin-1, had no appreciable effect on Isc. Hemoglobin and L-NNA enhanced the ET-1-induced Isc response by about twofold without affecting prostaglandin E2 release or its secretory response. The results suggest that endogenous nitric oxide stimulates a low level of chloride secretion that is independent of prostaglandins, unlike nitric oxide donors which increase chloride secretion by releasing prostaglandins. In addition, endogenous nitric oxide suppresses ET-1-evoked chloride secretion by mechanisms that are unrelated to the release of prostaglandin E2 or its ability to stimulate epithelial cells. Endogenous nitric oxide may play an important role in modulating chloride secretion during ischemic challenge when endothelin levels are high.

Activation of the epidermal platelet-activating factor receptor results in cytokine and cyclooxygenase-2 biosynthesis.

Recent studies suggest that the lipid mediator platelet-activating factor (PAF) is involved in keratinocyte function and skin inflammation. Indeed, PAF is found in association with inflammatory skin diseases, intradermal injections of PAF induce inflammation, and keratinocytes express functional PAF receptors (PAF-R). One mechanism by which the keratinocyte PAF-R could contribute to epidermal functions and inflammatory states would be through the synthesis of inflammatory regulators, such as PAF, PGs, and cytokines. The ability of the epidermal PAF-R to induce the synthesis of these immunomodulators was tested using a model system created by transduction of the PAF-R-negative human epidermal cell line KB with the PAF-R. Activation of this epidermal PAF-R resulted in arachidonic acid release, and the biosynthesis of PAF and PGE2. In addition, the KB PAF-R triggered increased levels of mRNA and protein for the inducible isozyme of cyclooxygenase (COX-2) as well as IL-6 and IL-8, both of which have been implicated in skin inflammatory processes. Studies with the human keratinocyte-derived epidermal cell line HaCaT revealed that activation of the endogenous PAF-R led to the increased accumulation of COX-2, IL-6, and IL-8 mRNA similar to that seen with the KB PAF-R model system. Finally, treatment of HaCaT keratinocytes with IL-8 resulted in PAF biosynthesis, indicating the existence of a positive feedback loop between IL-8 and PAF in epidermal cells. These studies suggest involvement of PAF and the PAF-R in the epidermal cytokine network.

Interleukin-1 receptor antagonist blocks interleukin-1-induced expression of cyclooxygenase-2 in endometrium.

OBJECTIVE: Our purpose was to test the hypothesis that the interleukin-1 receptor antagonist can inhibit interleukin-1-induced prostaglandin production and de novo expression of the inducible cyclooxygenase-2 isoform in a human endometrial epithelial cell line. STUDY DESIGN: A continuous line of human endometrial epithelial cells was established from a hysterectomy specimen from a nonmalignant uterus. Cells were maintained as a monolayer culture in medium 199 supplemented with 10% fetal bovine serum and 50 micrograms/ml gentamicin. Cultures were treated with cytokines (interleukin-1 alpha or interleukin-1 beta, interleukin-1 receptor antagonist, or tumor necrosis factor-alpha), and media were collected for analysis of prostaglandin E2 and prostaglandin F2 alpha) by radioimmunoassay, whereas cells were harvested for ribonucleic acid and protein extractions and subsequent Northern blot or Western blot analyses, respectively. RESULTS: When endometrial cells were incubated with interleukin-1 alpha or interleukin-1 beta, each cytokine was shown to stimulate the production of prostaglandin E2 and prostaglandin F2 alpha in a time- and dose-dependent fashion, with interleukin-1 alpha being far more potent than interleukin-1 beta. Interleukin-1 receptor antagonist inhibited interleukin-1 alpha- and interleukin-1 beta-induced prostaglandin formation, with 50% inhibitory concentration values of 30 ng/ml for prostaglandin E2 and 90 ng/ml for prostaglandin F2 alpha. When Northern blots of interleukin-1 alpha-treated cells were probed with a complementary deoxyribonucleic acid fragment specific for either cyclooxygenase-1 or cyclooxygenase-2, rapid de novo induction of cyclooxygenase-2 messenger ribonucleic acid was observed; however, cyclooxygenase-1 expression was constant regardless of interleukin-1 alpha concentration or incubation time. Coincubation of cells with interleukin-1 alpha (10 ng/ml) and cycloheximide caused superinduction of cyclooxygenase-2 messenger ribonucleic acid but had no effect on the expression of cyclooxygenase-1 messenger ribonucleic acid. Actinomycin D completely abolished interleukin-1 alpha-induced cyclooxygenase-2 messenger ribonucleic acid expression, suggesting that the cytokine caused transcriptional activation of the cyclooxygenase-2 gene. Experiments were conducted to examine whether interleukin-1 receptor antagonist could suppress interleukin-1-induced cyclooxygenase-2 expression. Cells were preincubated for 30 minutes with interleukin-1 receptor antagonist and then challenged with interleukin-1 alpha. Northern and Western analyses revealed that interleukin-1 receptor antagonist blocked interleukin-1 alpha-induced expression of cyclooxygenase-2 messenger ribonucleic acid transcripts and the subsequent appearance of cyclooxygenase-2 protein. Interleukin-1 receptor antagonist had no effect on the constitutive expression of cyclooxygenase-1 messenger ribonucleic acid and protein. Interleukin-1 receptor antagonist failed to alter prostaglandin E2 formation in response to tumor necrosis factor-alpha, indicating that the antagonist is specific for interleukin-1 family cytokines. Finally, interleukin-1 receptor antagonist acted as a partial agonist in some experiments in that relatively high concentrations (> 100 ng/ml) caused a modest increase in prostaglandin E2 and F2 alpha production. CONCLUSIONS: These data indicate that interleukin-1 receptor antagonist is a potent inhibitor of interleukin-1-induced arachidonic acid metabolism and could possibly serve as an endogenous or exogenous modulator of interleukin-1 action in the endometrial epithelium.

Ganglioside GM1 activates the mitogen-activated protein kinase Erk2 and p70 S6 kinase in U-1242 MG human glioma cells.

Gangliosides are implicated in the regulation of cellular proliferation as evidenced by differences in ganglioside composition associated with malignant transformation and density of cells in culture, as well as their inhibitory effects when added to cells growing in culture. Exogenously added gangliosides have a bimodal effect on proliferation in U-1242 MG glioma cells, inhibiting DNA synthesis in growing cells and stimulating it in quiescent cells. We investigated the mechanisms involved in stimulation of DNA synthesis using [3H]thymidine incorporation and immune complex kinase assays to identify responsible signal transduction pathways. Treatment of quiescent U-1242 MG cells with GM1 caused activation of the mitogen-activated protein (MAP) kinase isoform Erk2. Pretreatment with the specific MAP kinase kinase inhibitor PD98059 prevented the GM1-stimulated Erk2 activation and GM1-stimulated DNA synthesis. GM1 treatment stimulated another distinct signaling pathway leading to activation of p70 S6 kinase (p70s6k), and this was prevented by pretreatment with rapamycin. Rapamycin also inhibited GM1-stimulated DNA synthesis. Activation of both pathways and stimulation of DNA synthesis were inhibited by forskolin treatment; however, GM1 had no effect on cyclic AMP levels. Platelet-derived growth factor also activated both Erk2 and p70s6k but did not cause DNA synthesis, suggesting that GM1 may stimulate additional cascades, which also contribute to GM1-mediated DNA synthesis.

Suppression of cAMP formation by adenosine in myenteric ganglia from guinea-pig small intestine.

Effects of the adenosine receptor agonist 2-chloro-N6-cyclopentyl-adenosine (CCPA) on stimulation of cAMP formation by histamine, 5-hydroxytryptamine, substance P and forskolin were determined for enzymatically dissociated ganglia from the myenteric plexus of guinea-pig small intestine. Each of the 4 substances stimulated cAMP production. CCPA blocked the stimulation of cAMP by histamine, but not by 5-hydroxytryptamine or substance P. CCPA marginally suppressed stimulation by forskolin. CCPA alone suppressed basal levels of cAMP. The adenosine receptor antagonist 8-cyclopentyl-1,3-dimethylxanthine (CPT) reversed the inhibitory action of CCPA on stimulation of cAMP formation by histamine. Exposure to adenosine deaminase or CPT increased cAMP in the ganglia. The results are consistent with a hypothesis that stimulation of adenylate cyclase and elevation of intraneuronal cAMP in enteric neurons are steps in the signal transduction cascade for the excitatory actions of 5-hydroxytryptamine, substance P and histamine. They are consistent also with an original hypothesis from electrophysiologic studies which states that stimulation of adenosine A1 receptors suppresses cAMP formation and thereby slow synaptic excitation in response to histamine, but not to 5-hydroxytryptamine or substance P. The results support evidence from intracellular microelectrode studies which suggested that endogenous adenosine accumulates to levels sufficient for tonic suppression of cAMP formation in myenteric ganglia in vitro.

An increase in intracellular cyclic AMP modulates nitric oxide production in IFN-gamma-treated macrophages.

Macrophages treated with IFN-gamma alone are stimulated to produce nitric oxide. The level of nitric oxide production can be enhanced significantly when IFN-gamma treatment is combined with other agents (e.g., LPS, TNF-alpha, IL-2, etc.). We tested the hypothesis that cAMP plays a role in the IFN-gamma-induced activation of macrophages. Our experiments indicate that factors that increase the concentration of cAMP in the murine macrophage cell line ANA-1 can also enhance IFN-gamma-induced production of nitric oxide. PGE2 and cholera toxin increased the production of nitrite (an indicator of nitric oxide production) in IFN-gamma-treated ANA-1 macrophages by at least twofold. These factors produced no increase in nitric oxide production in the absence of IFN-gamma treatment. The increase in nitric oxide production corresponded to an increase in the accumulation of nitric oxide synthase mRNA without a change in stability of mRNA. Dibutyryl cAMP and Sp-cAMPs (a selective activator of cAMP-dependent protein kinase I and II) also increased nitric oxide production in IFN-gamma-treated macrophages. However, at very high concentrations (i.e., >100 microM), the stimulatory effect was decreased. These studies indicate that elevation of intracellular cAMP causes a dose-dependent, biphasic alteration of IFN-gamma-induced nitric oxide production in murine macrophages. Moreover, they suggest that agents that affect nitric oxide synthesis may do so via modulation of the cAMP second messenger system.

Stimulation of formation of adenosine 3',5'-phosphate by histamine in myenteric ganglia isolated from guinea-pig small intestine.

Effects of histamine and related agonists and antagonists on formation of cAMP were determined for enzymatically dissociated ganglia from the myenteric plexus of the guinea-pig small intestine. Formation of cAMP was stimulated by histamine in both dose- and time-dependent manners. The stimulatory action of histamine was suppressed by the histamine H2 receptor antagonist, cimetidine. The histamine H1 receptor antagonists, tripelennamine or pyrilamine also suppressed the stimulatory action of histamine, but only at concentrations 3-4 orders higher than required for cimetidine. Formation of cAMP was stimulated dose-dependently by the histamine H2 receptor agonist, dimaprit. The histamine H1 receptor agonist, 2-methyl-histamine, also stimulated cAMP production, but required a threshold concentration 4-5 orders higher than dimaprit. We conclude that histamine acts at the histamine H2 receptor subtype to stimulate adenylate cyclase and the formation of cAMP in myenteric ganglia of the guinea-pig small bowel.

Pharmacological activities of trimetoquinol and 1-benzyl halogen-substituted analogues on rat beta-adrenoceptor subtypes.

The beta-adrenoceptor activity profile of trimetoquinol and its 1-benzyl halogen-substituted analogues was studied in rat tissues containing primarily beta 1 (atria)-, beta 2 (trachea)- and atypical beta/beta 3 (distal colon and brown adipose tissue)-adrenoceptors. Functional biological activity resided in the (-)-isomer of trimetoquinol which was 112-, 275-, 372- and 513-fold more potent than (+)-trimetoquinol in trachea, right atria, distal colon and brown adipose tissue, respectively. (+/-)-Trimetoquinol was equally or slightly less active than (-)-trimetoquinol. The 1-benzyl halogen-substituted analogues of trimetoquinol exhibited differential activation of beta-adrenoceptor subtypes. In functional assays, 3'-iodotrimetoquinol was a potent activator of all beta-adrenoceptor subtypes. 3',5'-Diiodotrimetoquinol was 10-fold more potent as an agonist in tissues containing atypical beta/beta 3-adrenoceptors than those tissues containing beta 1- and beta 2-adrenoceptor sites. Furthermore, this drug was a partial agonist as compared to (+/-)-trimetoquinol and 3'-iodotrimetoquinol on beta 1-adrenoceptors. Pharmacological properties of the compounds on rat beta 3-adrenoceptors expressed in Chinese hamster ovary (CHO) cells were consistent with results observed in functional assays. 3',5'-Diiodotrimetoquinol possessed the greatest potency for activation of adenylyl cyclase. Rank order of affinity for rat beta 3-adrenoceptor was 3'-iodotrimetoquinol = 3',5'-diiodotrimetoquinol > (+/-)-trimetoquinol > (-)-isoprenaline. These results suggest that 3',5'-diiodotrimetoquinol is a promising drug for further chemical modification in the development of selective beta 3-adrenoceptor ligands.

Genistein suppresses EGF-induced prostaglandin biosynthesis by a mechanism independent of EGF receptor tyrosine kinase inhibition.

This study demonstrated that genistein, a selective tyrosine kinase inhibitor, blocked PGE2 production in human A431 and WISH cells and murine 3T3 cells in response to epidermal growth factor and platelet-derived growth factor. Blockade of growth factor-induced PGE2 production was dose-dependent (IC50 approximately equal to 7-8 microM). Genistein also abolished PGE2 formation in response to calcium ionophores, A23187 and ionomycin, and the phorbol ester, phorbol myristate acetate. Moreover, genistein-treated A431 and WISH cells incorporated significantly less [3H]arachidonic acid into membrane phospholipids than control cells. Finally, genistein decreased the specific activity of prostaglandin H2 synthase prepared from A431 cells, WISH cells, and ram seminal vesicle. The IC50 of genistein for inhibition of prostaglandin H2 synthase specific activity extracted from A431 and WISH cells approximated that half-maximal inhibitory concentration in the whole cell assay. These data indicate that genistein may interfere with arachidonic acid metabolism at several key points by a mechanism(s) that is independent of its inhibitory action on receptor tyrosine protein kinases. Taken together, these results also suggest that caution should be exercised when drawing conclusions about the putative role of tyrosine kinases in signal transduction events using genistein as a pharmacological blocker.

Response of failing canine and human heart cells to beta 2-adrenergic stimulation.

BACKGROUND: Failing human hearts lose beta 1- but not beta 2-adrenergic receptors. In canine hearts with tachypacing failure, the ratio of beta 2- to beta 1-adrenergic receptors is increased. The present study was designed to determine whether heart failure increases sensitivity to beta 2-adrenergic stimulation in isolated canine ventricular cardiomyocytes and to verify that myocytes from failing human ventricles contain functional beta 2-adrenergic receptors. METHODS AND RESULTS: Myocytes from healthy dogs, dogs with tachypacing failure, and human transplant recipients were loaded with fura 2-AM and subjected to electric field stimulation in the presence of zinterol, a highly selective beta 2-adrenergic agonist. Zinterol significantly increased [Ca2+]i transient amplitudes in all three groups. The failing canine myocytes were significantly more responsive than normal to beta 2-adrenergic stimulation. We also measured isotonic twitches, indo-1 fluorescence transients, and L-type Ca2+ currents in healthy canine myocytes. Zinterol (10(-5) mol/L) elicited large increases in the amplitudes of simultaneously recorded twitches and [Ca2+]i transients. Zinterol also increased L-type Ca2+ currents in the normal canine myocytes; this augmentation was abolished by 10(-7) mol/L ICI 118,551. cAMP production by suspensions of healthy and failing canine myocytes was not increased by zinterol (10(-9) to 10(-5) mol/L), nor did 10(-5) mol/L zinterol elicit phospholamban phosphorylation. CONCLUSIONS: Failing human ventricular cardiomyocytes contain functional beta 2-adrenergic receptors. Canine myocytes also contain functional beta 2-adrenergic receptors. The canine ventricular response to beta 2-agonists is increased in tachypacing failure. Positive inotropic responses to beta 2-stimulation are not mediated by increases in cAMP or cAMP-dependent phosphorylation of phospholamban.

Biochemical and pharmacological characterization of high-affinity trimetoquinol analogs on guinea pig and human beta adrenergic receptor subtypes: evidence for partial agonism.

Radioligand binding assays were used to characterize the interaction of a series of trimetoquinol [1-(3',4',5'-trimethoxybenzyl)-6,7-dihydroxy-1,2,3,4-tetrahydroisoqui nol ine; TMQ] analogs with beta adrenergic receptors (beta-AR). The results indicated that TMQ analogs bound with similar affinities to guinea pig (heart, lung and skeletal muscle) and human (beta-AR in Escherichia coli) beta-1- and beta-2-AR subtypes. However, the isomers of TMQ and 8-fluoro-TMQ bound stereoselectively to beta-AR with the S-isomers having affinities at least 112- and 8-fold greater, respectively, than their corresponding R-isomers. In general, a direct relationship existed between TMQ analog binding to guinea pig beta-AR and functional activity on guinea pig right atria (beta-1) and trachea (beta-2). For selected halogenated TMQ analogs (3',5'-diiodo-TMQ, 3'-iodo-TMQ, 5,8-difluoro-TMQ and 5-iodo-TMQ) which had higher beta-AR affinities than TMQ, but were less potent beta-AR agonists than TMQ, this relationship was not seen. To explain this, the function of the TMQ analogs was analyzed at the level of the beta-AR-associated effector mechanism (i.e., G-protein and adenylyl cyclase). In Chinese hamster ovary cells expressing human beta-2-AR, TMQ and halogenated analogs bound to the receptor with high affinity (nanomolar range); however, they failed to effectively couple with beta-AR-associated G-protein and only partially activated receptor-associated adenylyl cyclase. Receptor occupancies of 0.14, 2 and 23% were required for (-)-isoproterenol, S-(-)-TMQ and 3'5'-diiodo-TMQ to produce equivalent cyclic AMP accumulations in human beta-2-AR Chinese hamster ovary cells. Thus, TMQ and halogenated TMQ derivatives bind stereoselectively to beta-AR with high affinity, and may be classified as partial beta-AR agonists.

Isomeric-activity ratios of trimetoquinol enantiomers on beta-adrenergic receptor subtypes: functional and biochemical studies.

Trimetoquinol [1-(3',4',5'-trimethoxybenzyl)-6,7-dihydroxy-1,2,3,4- tetrahydroisoquinoline , TMQ] exists as two enantiomers, and the (-)-(S)-isomer is a potent beta-adrenergic receptor (beta-AR) agonist. Experiments were conducted to examine the functional and biochemical potencies of the (S)-and (R)-enantiomers of TMQ for interaction with beta-AR subtypes in tissues, membrane fractions, and cell systems. The isomeric-activity ratios (IARs) of the TMQ isomers [(S)-isomer > > (R)-isomer] for stimulation of beta 1- and beta 2-AR of guinea pig right atria and trachea were 224 and 1585, respectively; these IARs were similar to those observed on atypical beta-AR systems of rat distal colon (575), rat brown adipocytes (398), but differed from that of rat esophageal smooth muscle (2884) in the presence of pindolol. In the absence of pindolol, the potencies for the TMQ enantiomers were slightly increased; however, the IARs remained unchanged in rat distal colon, rat brown adipocytes, and rat esophageal smooth muscle. Similarly, radioligand binding studies demonstrated that the TMQ isomer beta-AR affinities were stereoselective for the (-)-(S)-isomer in membranes of guinea pig left ventricle (beta 1) and lung (beta 2) giving IARs of 115 and 389, respectively; and in E. coli expressing human beta 1- and beta 2-AR giving IARs of 661 and 724, respectively. Corresponding IARs of receptor affinities and stimulation of cAMP accumulation in Chinese hamster ovary cells expressing human beta 2-AR and rat beta 3-AR were 331 and 282, and 118 and 4678, respectively. These results indicate that the (-)-(S)-isomer of TMQ exhibits high affinity, and is a potent and highly stereoselective agonist for each beta-AR subtype, that the isomers generally fail to differentiate between the beta-AR subtypes, and that, based upon differences in IAR within beta 3-AR containing systems, subtypes of atypical beta (or beta 3)-AR may exist in adipose tissue and smooth muscle.

Stimulation of formation of cAMP by 5-hydroxytryptamine in myenteric ganglia isolated from guinea pig small intestine.

Effects of 5-hydroxytryptamine (5-HT) and related agonists and antagonists on formation of cAMP were determined for enzymatically dissociated ganglia from the myenteric plexus of the guinea-pig small intestine. Formation of cAMP was stimulated by 5-HT in both dose- and time- dependent manners. The stimulatory action of 5-HT was suppressed by the 5-HT1P antagonist, renzapride, but not by the 5-HT3 antagonist, tropisetron (formerly ICS 205-930). Neither renzapride nor cisapride increased the levels of cAMP. Levels of cAMP were suppressed by concentrations of tropisetron greater than 1.0 microM. Levels of cAMP were unaffected by the 5-HT3 agonist, 2-methyl-5-hydroxytryptamine. A putative 5-HT4 receptor agonist, 5-methoxytryptamine, stimulated formation of cAMP, but to a lesser extent than 5-HT. We conclude that 5-HT acts to stimulate adenylate cyclase and the formation of cAMP in myenteric ganglia. The 5-HT1P receptor is the most likely subtype involved in 5-HT action on cAMP formation.

Tumor necrosis factor alpha, interleukin-1 alpha, interleukin-6, and prostaglandin E2 production in murine peritoneal macrophages infected with Ehrlichia risticii.

Ehrlichia risticii is a gram-negative obligate intracellular bacterium which primarily infects macrophages and crypt epithelial cells in the intestinal wall and is the etiologic agent of Potomac horse fever. To understand the pathogenesis of the disease, we tested whether E. risticii induces inflammation-associated products in thioglycolate-induced mouse peritoneal macrophages. Mouse peritoneal macrophages produced larger amounts of interleukin-1 alpha (IL-1 alpha) but lower levels of tumor necrosis factor alpha (TNF-alpha), IL-6, and prostaglandin E2 (PGE2) when exposed to live or killed E. risticii than when exposed to Escherichia coli lipopolysaccharide (LPS). Preincubation of macrophages with live or killed E. risticii suppressed TNF-alpha, IL-6, and PGE2 generation but not IL-1 alpha production in response to LPS. Murine gamma interferon treatment of macrophages did not influence TNF-alpha, IL-1 alpha, IL-6, or PGE2 production regardless of exposure to E. risticii. Intracellular cyclic AMP was significantly greater in E. risticii-infected macrophages than in uninfected macrophages. These results suggest that increased levels of IL-1 alpha but not TNF-alpha or PGE2 production by macrophages may be primarily involved in the pathogenesis of the disease caused by E. risticii. Increased intracellular concentration of cyclic AMP in infected macrophages may be chiefly responsible for the high level of IL-1 alpha and inhibition of TNF-alpha production in response to LPS.

Effects of obesity and hypertension on ventricular myocytes: comparison of cells from adult SHHF/Mcc-cp and JCR:LA-cp rats.

OBJECTIVE: The aim was to compare beta adrenergic receptors, cAMP production, and Ca2+ accumulation by the sarcoplasmic reticulum in ventricular cardiomyocytes from female SHHF/Mcc-cp and JCR:LA-cp rats. Whereas rats from both strains exhibit gross obesity when the animals are homozygous for the recessive "corpulent" gene, the SHHF rats, which are hypertensive, all develop heart failure during their second year of life. The normotensive JCR:LA-cp animals do not. METHODS: beta Adrenergic receptor number, ligand affinity, isoprenaline and forskolin stimulated cyclic AMP production, and ATP dependent, phosphate supported 45Ca2+ uptake by the sarcoplasmic reticulum were compared in ventricular cardiomyocytes isolated from 6 months old obese female SHHF/Mcc-cp and obese and lean female JCR:LA-cp rats. RESULTS: Bmax and Kd for (-)-[125iodo]-cyanopindolol (125ICYP) binding were each approximately 50% lower in SHHF/Mcc-cp v JCR:LA-cp myocytes. Cyclic AMP production in response to isoprenaline, isoprenaline plus isobutylmethylxanthine (IBMX), and forskolin plus IBMX was also significantly depressed in the SHHF/Mcc-cp cells. In addition, sarcoplasmic reticular 45Ca2+ uptake by SHHF/Mcc-cp cells was 35% lower than in lean or obese JCR:LA-cp myocytes. Isoprenaline stimulated cAMP production and sarcoplasmic reticular Ca2+ uptake by the lean JCR:LA-cp cells were comparable to that described previously for myocytes from normal Sprague-Dawley rats. By contrast, Bmax and Kd for 125ICYP binding by the JCR myocytes differed substantially from previously described results for normal Sprague-Dawley rats, whereas values for the SHHF cells did not. CONCLUSIONS: Declines in Ca sequestration by the sarcoplasmic reticulum of ventricular cardiomyocytes from obese, hypertensive SHHF rats are not related to their obesity. However, obesity may contribute to the decline in cAMP production. This may account, in part, for the exacerbation by obesity of cardiac dysfunction in essential hypertension.

Transforming growth factor-beta potentiates epidermal growth factor-induced prostaglandin E2 production in amnion cells.

Primary cultures of human amnion cells and the amnion-derived cell line WISH were used to evaluate the hypothesis that transforming growth factor-beta (TGF-beta) can modulate epidermal growth factor (EGF)- induced prostaglandin E2 (PGE2) production. Cells were preincubated for 1 hr with TGF-beta (0.0001-10 ng/ml) and then incubated in the presence or absence of EGF (10 ng/ml) for 4 hrs. TGF-beta alone did not stimulate PGE2 synthesis at any dose examined. However, when primary cultures of amnion cells or WISH cells were preincubated with TGF-beta and then challenged with EGF, there was a potentiation of PGE2 production that was much greater than the additive values of TGF-beta or EGF alone. These data suggest that EGF-induced PGE2 production by amnion cells can be modulated by low concentrations of TGF-beta.