Inhibitory activity of loratadine and descarboxyethoxyloratadine on histamine-induced activation of endothelial cells

BACKGROUND: The allergic inflammatory reaction is characterized by leucocyte adherence and infiltration processes which are controlled by the expression of adhesion molecules on the surface of vascular endothelium. One of the main mediators implicated in allergic reactions is represented by histamine. Histamine is a potent activator of endothelial cells (EC): it induces the expression of P-selectin on the surface of endothelium and the secretion of IL-6 and IL-8. OBJECTIVES: Loratadine (L), a histamine H1-antagonist, and one of its active metabolites, descarboxyethoxyloratadine (DCL), were studied at different concentrations for their ability to reduce the histamine-induced activation of human umbilical vein EC (HUVEC). METHODS: HUVEC were stimulated in the presence of histamine at 10(-6) M, 10(-5) M and 10(-4) M. We assessed by ELISA the expression of P-selectin on EC surface, as well as cytokine production in EC supernatants of 24 h culture. RESULTS: Our results showed that for a 10(-4) M-histamine stimulation, L and DCL have a similar inhibitory effect on P-selectin expression (IC50 = 13 x 10[-9] M and 23 x 10[-9] M, respectively). L and DCL inhibited significantly IL-6 and IL-8 secretion induced by histamine with a more powerful efficiency of the active metabolite. For the dose of 10(-4) M histamine, a 50% inhibition of IL-6 secretion was obtained for a dose of DCL equal to 2.6 x 10(-12) M whereas the same magnitude of effects were only reached for a higher concentration of L (0.3 x 10[-6] M). Similar results were obtained for IL-8 (IC50 = 0.2 x 10[-6] M for L and 10[-9] M for DCL). Analysis of IL-8 mRNA expression by RT-PCR was in accordance with these data. CONCLUSION: These results demonstrate that both L and DCL are active to reduce the histamine-induced activation of EC. Interestingly, DCL seems to be effective at lesser concentrations especially to inhibit cytokine secretion.     

Intracranial facial nerve neurinoma: surgical strategy of tumor removal and functional reconstruction

The effects of a new forskolin derivative, (13R)-spiroforskolin, on the ventricular cAMP-activated chloride current (I(Cl(cAMP))) and the atrial L-type calcium current (I(Ca,L)) were measured by means of whole-cell recording from isolated guinea-pig cardiac myocytes at 30 degrees C and 20-22 degrees C, respectively. In contrast to forskolin, the derivative contains a tetrahydrofuran rather than a tetrahydropyran moiety. (13R)-spiroforskolin activated I(Cl(CAMP)) in 58% of the ventricular myocytes studied. The concentration required for the half maximal effect (EC50 value) amounted to 9.6x10(-11) M and was lower than the EC50 value for forskolin (2.4x10(-8) M). (13R)-spiroforskolin evoked a smaller maximal I(Cl(cAMP)) amplitude than forskolin. The rundown of the (13R)-spiroforskolin-activated I(Cl(cAMP)) was faster than that of the forskolin-induced current. Neither forskolin nor (13R)-spiroforskolin in maximally effective concentrations increased I(Cl(cAMP)) in cells containing high concentrations of cAMP. Furthermore, as an activator of atrial I(Ca,L) (13R)-spiroforskolin displayed a smaller activation and a lower EC50 value (5.8x10(-10) M) than forskolin (EC50 value: 3.7x10(-7) M). The effect of (13R)-spiroforskolin was observed in only 30% of the atrial cells studied. None of the drugs exerted a stimulatory effect in atrial cells containing a high [cAMP]. The washout of the drug effect was significantly faster in (13R)-spiroforskolin- than in forskolin-treated atrial myocytes. We conclude that (13R)-spiroforskolin as a forskolin derivative displays unique characteristics. It is a more potent but less efficacious activator of cardiac ionic conductances than the parent compound. The results suggest that (13R)-spiroforskolin, like forskolin, most probably exerts its effects via stimulation of the adenylyl cyclase.     

Mode of action of site-directed irreversible folate analogue inhibitors of thymidylate synthase

5,8-Dideazafolate analogues are tight binding but not irreversible inhibitors of thymidylate synthase (TS). However, when a chloroacetyl (ClAc) group is substituted at the N10-position of 2-desamino-2-methyl-5,8-dideazafolate (DMDDF), the resulting compound, ClAc-DMDDF, although still a reversible inhibitor (KI = 3.4 x 10(-3) M), gradually inactivates thyA-TS irreversibly at a rate of 0.37 min-1. The corresponding iodoacetyl derivative alkylated the enzyme somewhat slower (k3 = 0.15 min-1 ) than ClAc-DMDDF but was bound more tightly (KI = 1.4 x 10(-5) M), resulting in a second-order rate constant (k3/KI) of inactivation that was 100-fold greater than that of ClAc-DMDDF. A tryptic digest of the ClAc-DMDDF-inactivated enzyme yielded a peptide on HPLC, which revealed that cysteine-146, the residue at the active site that is intimately involved in the catalytic process, had reacted with ClAc-DMDDF to form a covalent bond. This derivative was confirmed indirectly by Edman analysis and more directly by mass spectrometry. Deoxyuridine 5'-monophosphate, a substrate in the catalytic reaction, protected against inactivation. Similar to previously described Lactobacillus casei TS inhibition studies with sulfhydryl reagents [Galivan, J., Noonan, J., and Maley, F. (1977) Arch. Biochem. Biophys. 184, 336-345], the kinetics of inhibition suggested that complete inhibition occurs on reaction of only one of the two active site cysteines, although sequence and amino acid analysis revealed that iodoacetate and ClAc-DMDDF had reacted with both active site cysteines. These studies demonstrate that a sulfhydryl reactive compound that is directed to the folate binding site of TS may diffuse to the active site cysteine, and form a covalent bond with this residue. How this inhibition comes about is suggested in a stereoscopic view of the ligand when modeled to the known crystal structure of Escherichia coli TS.     

Adaptive survival trials

1. An orally active, nonpeptide bradykinin (BK) B2 receptor antagonist, FR173657 (E)-3-(6-acetamido-3-pyridyl)-N-[N-[2-4-dichloro-3-[(2-methyl-8-quinolin yl) oxymethyl]phenyl]-N-methylaminocarbonyl-methyl] acrylamide) has been identified. 2. This compound displaced [3H]-BK binding to B2 receptors present in guinea-pig ileum membranes with an IC50 of 5.6 x 10(-10) M and in rat uterus with an IC50 of 1.5 x 10(-9) M. It did not inhibit different specific radio-ligand binding to other receptor sites. 3. In human lung fibroblast IMR-90 cells, FR173657 displaced [3H]-BK binding to B2 receptors with an IC50 of 2.9 x 10(-9) M and a Ki of 3.6 x 10(-10) M, but did not reduce [3H]-des]Arg10-kallidin binding to B1 receptors. 4. In guinea-pig isolated preparations, FR173657 antagonized BK-induced contractions with an IC50 of 7.9 x 10(-9) M, but did not antagonize acetylcholine or histamine-induced contractions even at a concentration of 10(-6) M. FR173657 caused parallel rightward shifts of the concentration-response curves to BK at concentrations of 10(-9) M and 3.2 x 10(-9) M, and a little depression of the maximal response in addition to the parallel rightward shift of the concentration-response curve at a concentration of 10(-8) M. Analysis of the data yield a pA2 of 9.2 +/- 0.2 (n = 5) and a slope of 1.5 +/- 0.2 (n = 5). 5. In vivo, the oral administration of FR173657 inhibited BK-induced bronchoconstriction dose-dependently in guinea-pigs with an ED50 of 0.075 mg kg-1, but did not inhibit histamine-induced bronchoconstriction even at 1 mg kg-1. FR173657 also inhibited carrageenin-induced paw oedema with an ED50 of 6.8 mg kg-1 2 h after the carrageenin injection in rats. 6. These results show that FR173657 is a potent, selective, and orally active bradykinin B2 receptor antagonist.     

Enhancing communication with the Passy-Muir valve

Effective copper and cadmium concentrations which limited the growth of two chlorophytes by 50%, EC(50)s, after 96 h of static exposure were determined. EC(50)s were 5.94 microM copper and 4.55 microM cadmium for Dunaliella salina, and 0.78 microM copper and 0.025 microM cadmium for Chlamydomonas bullosa. The relationship of the two cations was synergistic towards the growth of both species. Chronic exposure to 4.5 x 10(-6) microM cadmium or 4.9 x 10(-4) microM copper increased the sensitivity of C. bullosa by 26% and 29% towards cadmium and copper, respectively. Changes in co-tolerance were not observed. Cd-treated D. salina was 50% more tolerant towards this cation, whereas Cu-treated cultures showed extreme sensitivity towards copper and "co-sensitivity" towards cadmium. Furthermore, the phylogenetic hypothesis, predictive of toxic response, failed to hold at the familial level.     

Dermatophilus dermatitis enzootic in deer in New York State and vicinity

Bis-(4-methyl-1-homopiperazinylthiocarbonyl)-disulphide (FLA-63) inhibits in vitro purified bovine adrenal medullary dopamine beta-hydroxylase (BDH) concentration-dependently; 50% inhibition is produced by 2 x 10(-6)M FLA-63. Spectrophotometric experiments show that FLA-63 is reduced by ascorbate very likely to the corresponding dithiocarbamate-derivative, which acts as a Cu++-chelator. Cu++-ions are able to completely abolish the inhibition of the DBH caused by FLA-63 in concentrations up to 2.5 x 10(-6)M. It is concluded that FLA-63 inhibits the DBH mainly in its reduced form via the formation of a chelate-complex with copper ions derived from the DBH.     

B2-kininergic action of linear and cyclic tryptophan6- and tyrosine6-kallidin

This study was undertaken to determine the importance of the central aromatic moiety in the kallidin and cyclokallidin molecules, using the relaxation of the isolated duodenum of the rat. Replacement in kallidin of the central phenylalanine by tryptophan increased the potency from an EC50 of 3 x 10(-10)M to 2 x 10(-12)M. Replacement by tyrosine decreased the potency to an EC50 of 8 x 10(-8)M. In cyclo-kallidin (EC50: 10(-8)M) the potencies were decreased: cyclo-Trp6-kallidin showed an EC50 of 10(-6)M and cyclo-Tyr6-kallidin of 3 x 10(-7)M. The relaxation of the rat duodenum by linear and cyclic kinins was potentiated by the bradykinin potentiating peptide BPP5a and antagonized by the B2 antagonist HOE-140. At a concentration of 10(-9)M, HOE-140 significantly decreased the potencies of bradykinin and cyclo-kallidin, but not of the B1 agonist desArg9-bradykinin.     

Chronic oxytocin pretreatment inhibits adenylyl cyclase activity in cultured rat myometrial cells

To determine whether chronic oxytocin pretreatment inhibits adenylyl cyclase, we compared adenylyl cyclase activity in membranes prepared from cultured, immortalized rat myometrial cells that were untreated or pretreated for 24 h with oxytocin. Chronic oxytocin pretreatment (1 x 10(-5) M for 24 h) attenuated basal, guanosine triphosphate (1 x 10(-5) M)-, isoproterenol (1 x 10(-4) M)-, forskolin (1 x 10(-5) M)-, MnCl2 (20 mM)- or NaF (1 x 10(-2) M)-stimulated adenylyl cyclase activity by 27 +/- 5% to 39 +/- 11% (n = 6, p < 0.05). Oxytocin pretreatment for 2 h (n = 5) did not produce a significant effect. To understand the mechanism by which oxytocin pretreatment decreased activity of the adenylyl cyclase pathway, we compared effects of pretreatment with either oxytocin or phenylephrine on adenylyl cyclase activity and determined the effects of Gi inhibition and protein kinase C (PKC) depletion. Chronic (24 h) phenylephrine pretreatment (1 x 10(-4) M) had effects similar to those of oxytocin pretreatment (1 x 10(-5) M). PKC depletion with phorbol 12-myristate 13-acetate (1 x 10(-6) M, 41 h) prevented attenuation of adenylyl cyclase activity by oxytocin pretreatment (1 x 10(-5) M for 24 h). Inhibition of Gi by pertussis toxin pretreatment (1.25 microg/ml, 41 h) had no significant effect. These findings suggest that chronic oxytocin pretreatment desensitizes the adenylyl cyclase pathway by a cross-regulatory mechanism that involves activation of Gq and PKC.     

Synthesis and structure-activity relationships of 2-pyrazinylcarboxamidobenzoates and beta-ionylideneacetamidobenzoates with retinoidal activity

The structure-activity relationships of two series of novel retinoids (2-pyrazinylcarboxamidobenzoates and beta-ionylideneacetamidobenzoates) have been investigated by evaluating their ability to induce differentiation in both human promyelocytic leukemia (HL60) cells and mouse embryonal carcinoma (P19) cells. The most active compound (ED50 = 8.3 x 10(-9) M) of the 2-pyrazinylcarboxamidobenzoates is 4-[2-(5,6,7,8-tetrahydro-5,5,8, 8-tetramethylquinoxalyl)carboxamido]benzoic acid (9u), while the most active analogue of the beta-ionylideneacetamidobenzoates is 4-[3-methyl-5-(2',6',6'-trimethyl-1'-cyclohexen-1'-yl)-(2E, 4E)-pentadienamido]benzoic acid (10a, ED50 = 3.2 x 10(-8) M). Our studies identify an absolute requirement for the carboxylic acid moiety on the aromatic ring to be para relative to the amide linkage for activity. Benzoate substitutions in the ortho position relative to the terminal carboxylate (9d,k,r) are well-tolerated; however, a methoxy substituent meta relative to the terminal carboxylate gives rise to only weakly active analogues (9x). Conformational studies (NMR, X-ray crystallography) of the 2-pyrazinylcarboxamidobenzoates indicate that the preferred conformation exhibits a trans-amide bond and an internal hydrogen bond between the quinoxaline N1 and HN amide which locks the torsional angle between C2 and CO in the s-trans conformation. N-Methylation (9y) results in loss of activity. Studies indicate that there is now a cis-amide bond present which redirects the carboxylate toward the pharmacophoric gem-dimethyl groups. The distance between the gem-dimethyl group and the terminal carboxylate appears to be too short to activate the retinoid receptor. N-Methylation in the beta-ionylideneacetamidobenzoate series (10c) also results in the formation of a cis-amide bond and loss of activity.     

Synthesis and calcium channel modulating effects of isopropyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(thienyl)-5-pyridinecarboxylates

A group of racemic isopropyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(thienyl)-5-pyridinecarboxylates++ + 7a-f were prepared using a modified Hantzsch reaction that involved the condensation of a thienylcarboxaldehyde 4a-f with isopropyl 3-aminocrotonate 5 and nitroacetone 6. In vitro calcium channel antagonist activities were determined using a guinea pig ileum longitudinal smooth muscle (GPILSM) assay. Compounds 7a-f exhibited weaker calcium channel antagonist activity (IC50 = 10(-5) to 10(-7) M range) than the reference drug nifedipine (IC50 = 1.43 x 10(-8) M). The point of attachment of the C-4 thienyl ring system was a determinant of antagonist activity [3-thienyl (7b) > 2-thienyl (7a)]. A 5-substituent in the 2-thienyl moiety influenced antagonist activity where the potency order was 5-bromo-2-thienyl 7f > or = 5-methyl-2-thienyl 7c > 2-thienyl 7a. Although the 5-methyl-2-thienyl 7c and 3-methyl-2-thienyl 7d isomers are equipotent antagonists, the 5-bromo-2-thienyl compound 7f appears to be marginally more active than the 4-bromo-2-thienyl isomer 7e. The 2-thienyl compound 7a, unlike the 3-thienyl isomer 7b, exhibited an agonist effect on GPILSM in the absence of the muscarinic agonist carbachol. Effects of the 2-thienyl 7a and 3-thienyl 7b isomers on the magnitude of calcium current were determined in guinea pig ventricular myocytes with voltage clamp techniques. Results showed that 2-thienyl 7a inhibited calcium current (antagonist) when voltage steps were made from a potential of -40 mV. However, when voltage steps were made from -60 mV, 7a enhanced calcium current (agonist). The 3-thienyl isomer 7b had little, if any, effect on calcium current.     

Interleukin-2 and -4 induce resistance of granulocyte-macrophage colony-stimulating factor to corticosteroids

In vitro pretreatment of human mononuclear blood cells with a combination of interleukin-2 and interleukin-4 decreases corticosteroid receptor affinity and reduces the anti-proliferative effects of corticosteroids. Similar abnormalities have been observed in mononuclear blood cells of steroid-resistant asthmatics. In vitro steroid resistance was induced by 48 h pretreatment of mononuclear blood cells from healthy individuals (n = 10) with interleukin-2 and interleukin-4 (500 Units (U)/ml). The effects of three structurally different corticosteroids (10(-7)-10(-11) M) on lipopolysaccharide-stimulated (10 ng/ml; 20 h) production of granulocyte-macrophage colony-stimulating factor (GM-CSF) were examined. GM-CSF production was efficiently inhibited by all three corticosteroids in the control cultures. Cortivazol was significantly more potent (IC50 = 3 x 10(-11) M) than budesonide and tipredane (IC50 = 2.5 x 10(-10) M and IC50 = 2 x 10(-10) M, respectively). However. interleukin-2 and interleukin-4 pretreatment counteracted the inhibitory effects of all three corticosteroids to a similar degree. The results highlight the importance of interleukin-2 and interleukin-4 in the induction of steroid resistance, since pretreatment of mononuclear blood cells with these cytokines impaired corticosteroid inhibition of GM-CSF production.     

Endothelin-1-induced in vitro cerebral venoconstriction is mediated by endothelin ETA receptors

The in vitro effects of endothelin-1 on cerebral veins were studied using cylindrical segments, 5 mm long, from dog pial veins. Isometric responses to endothelin-1 (10(-12)-10(-7) M) and to the endothelin ET(B) receptor agonist, IRL 1620 (Suc-[Glu9,Ala11,15]endothelin-1-(8-21), 10(-12) -10(-7) M), were recorded in veins under control conditions and pretreated with the endothelin ET(A) receptor antagonist, BQ-123 (cyclo-(D-Asp-Pro-D-Val-Leu-D-Trp), 10(-8) -10(-5) M), and the endothelin ETB receptor antagonist, BQ-788 (N-[N-[N-[(2,6-dimethyl-1-piperidinyl)carbonyl]-4-methyl-L-leucyl]-1-(me thoxycarbonyl)-D-tryptophyl]-D-norleucine monosodium, 10(-6) and 10(-5) M). The response to endothelin-1 was also recorded in veins pretreated with the nitric oxide synthesis inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME, 10(-4) M), or the cyclooxygenase inhibitor, meclofenamate (10(-5) M), and in veins without endothelium or placed in medium without Ca2+ but with EDTA (0.1 mM). In control veins, endothelin-1 produced a concentration-dependent contraction (EC50 = 2.0 x 10(-10) M; maximal contraction = 113 +/- 6 mg) and IRL 1620 induced no effects or a small contraction only with high concentrations (10(-8) - 10(-6) M) (EC50 = 1.5 x 10 (-8) M; maximal contraction = 9 +/- 3 mg). BQ-123 shifted the response to endothelin-1 to the right in a parallel, concentration-dependent way, whereas BQ-788, L-NAME or meclofenamate did not modify the response to endothelin-1. Compared with the control, veins in a medium without Ca2+ had similar EC50 values, but a lower maximal contraction induced by endothelin-1 (57 +/- 10 mg, P < 0.05), and veins without endothelium exhibited similar EC50 values. Thus, endothelin-1 produces marked cerebral venoconstriction that could be mainly mediated by activation of endothelin ETA receptors, may be dependent on extracellular Ca2+, and may be independent of endothelium, nitric oxide and prostanoids.     

Measurement of the kinetics of protein uptake by proximal tubular cells using an optical biosensor

BACKGROUND: The affinity and specificity of protein reabsorption by proximal tubular cells have been investigated using techniques for monitoring endocytosis, demonstrating a high capacity but low affinity process. It is not known whether uptake is through binding to a single binding site/receptor with differing affinities, or if there are several classes of binding sites receptors, each specific for differing proteins or groups, such as, high or low molecular weight proteins. METHODS: We have developed a novel technique for analyzing the kinetics of protein binding to tubular cells using a optical biosensor system. We have studied the binding of cultured LLCPK cells to albumin and RBP immobilized onto the sensor. By adding increasing concentrations of competing proteins [varying in molecular weight from 66,000 to 11,800 D and pI from 4.6 to 9.2 as represented by albumin, alpha1-microglobulin (alpha1M), retinol binding protein (RBP), cystatin C and beta2-microglobulin (beta2m)], specific and inhibitable cell binding was demonstrated. RESULTS: Equilibrium constants, KA, could be calculated from the reciprocal of the protein concentration causing 50% inhibition in binding rate. These were: albumin = 8.0 x 10(4) M(-1), alpha1M = 2.0 x 10(5) M(-1), RBP = 2.7 x 10(4) M(-1), cystatin C = 2.0 x 10(4) M(-1), beta2m = 4.2 x 10(3) M(-1). There were no significant differences between the measured KA's whether RBP or albumin were immobilized on the surface. CONCLUSIONS: All the proteins gave similar shaped inhibition profiles, suggesting that there is one binding site/receptor for all proteins studied, regardless of molecular weight or charge, but there are differing affinities for each protein.     

Biguanide derivatives: agonist pharmacology at 5-hydroxytryptamine type 3 receptors in vitro

The effects of 24 biguanide and four guanidine derivatives on 5-hydroxytryptamine (5-HT)3 receptors in N1E-115 neuroblastoma cells were examined using radioligand binding and whole-cell voltage-clamp techniques. Displacement of the selective 5-HT3 receptor antagonist [3H]BRL 43694 by phenylbiguanide (PBG) derivatives revealed Ki values ranging from 3.4 x 10(-4) to 4.4 x 10(-10) M. The rank order of potency of agonists was 2,3,5-trichloro-PBG > 2,3-dichloro-PBG = 2,5-dichloro-PBG = 3,5-dichloro-PBG > 3,4-dichloro-PBG = 3-chloro-PBG > 2-chloro-PBG = 4-chloro-PBG = 2-methyl-PBG = 2,4-difluoro-PBG > PBG = 2-trifluoro-5-chloro-PBG > 4-fluoro-PBG = 3-trifluoromethyl-PBG > 4-nitro-PBG = 1,5-bis-4-chloro-PBG = 3,5-ditrifluoromethyl-PBG > 4-ethoxy-PBG > 4-sulfonic acid-PBG. All of the benzylbiguanides and indanylbiguanide were inactive on [3H]BRL 43694 binding or displaced it only weakly. The four guanidine derivatives were quite inactive. In the PBG series, all antagonist competition curves were steep (pseudo-Hill coefficients ranging from 1.05 to 1.58), monophasic, and best fit with a one-site model. Among PBG derivatives, the chlorinated compounds exhibited a good degree of selectivity for 5-HT3 receptors versus other 5-HT receptor subtypes and other neurotransmitter binding sites. Electrophysiological studies showed that the PBG derivatives tested produced rapid inward currents, at a holding potential of -65 mV, that showed rapid desensitization. The current induced by the 2,3,5-trichloro-PBG derivative was inhibited by the specific 5-HT3 receptor antagonist ICS 205-930 but was unaffected by the 5-HT2 receptor antagonist ketanserin. Analysis of concentration-response curves for the PBG derivatives gave EC50 values ranging from 2.2 x 10(-5) to 2.7 x 10(-8) M and Hill slopes ranging from 1.02 to 2.10. The rank order of potency was similar to that obtained from the binding data, and a good correlation was found between Ki and EC50 values. It is concluded that the triple-chloro substitution yielded a compound that is 30-fold more potent than 3-chloro-PBG and approximately 10-fold more potent than dichloro-PBG derivatives, making 2,3,5-trichloro-PBG the most potent 5-HT3 agonist described thus far.     

Pharmacological characterization of a rat 5-hydroxytryptamine type3 receptor subunit (r5-HT3A(b)) expressed in Xenopus laevis oocytes

The present study has utilized the two electrode voltage-clamp technique to examine the pharmacological profile of a splice variant of the rat orthologue of the 5-hydroxytryptamine type 3A subunit (5-HT3A(b)) heterologously expressed in Xenopus laevis oocytes. At negative holding potentials, bath applied 5-HT (300 nM - 10 microM) evoked a transient, concentration-dependent (EC50 = 1.1+/-0.1 microM), inward current. The response reversed in sign at a holding potential of -2.1+/-1.6 mV. The response to 5-HT was mimicked by the 5-HT3 receptor selective agonists 2-methyl-5-HT (EC50= 4.1+/-0.2 microM), 1-phenylbiguanide (EC50=3.0+/-0.1 microM), 3-chlorophenylbiguanide (EC50 = 140+/-10 nM), 3,5-dichlorophenylbiguanide (EC50 = 14.5+/-0.4 nM) and 2,5-dichlorophenylbiguanide (EC50 = 10.2+/-0.6 nM). With the exception of 2-methyl-5-HT, all of the agonists tested elicited maximal current responses comparable to those produced by a saturating concentration (10 microM) of 5-HT. Responses evoked by 5-HT at EC50 were blocked by the 5-HT3 receptor selective antagonist ondansetron (IC50=231+/-22 pM) and by the less selective agents (+)-tubocurarine (IC50=31.9+/-0.01 nM) and cocaine (IC50 = 2.1+/-0.2 microM). The data are discussed in the context of results previously obtained with the human and mouse orthologues of the 5-HT3A subunit. Overall, the study reinforces the conclusion that species differences detected for native 5-HT3 receptors extend to, and appear largely explained by, differences in the properties of homo-oligomeric receptors formed from 5-HT3A subunit orthologues.     

Stereoselective differences in the vasorelaxing effects of S(+) and R(-) ketamine on rat isolated aorta

BACKGROUND: S(+) ketamine, because of its higher anesthetic potency and lower risk of psychotomimetic reactions, has been suggested to be superior to presently available racemic ketamine. The racemate is a direct vasodilator in vivo, and thus the authors investigated the vasorelaxing effects of ketamine enantiomers on rat aorta. METHODS: Rat isolated aortic rings with and without endothelium were contracted with 3 x 10(-7) M norepinephrine. Then 10(-5) to 3 x 10(-3) M S(+), R(-), or racemic ketamine were added cumulatively. Vascular responses to ketamine were further studied in rings pretreated with the nitric oxide synthase inhibitor N(omega)-nitro-L-arginine (NNLA), the adenosine triphosphate-sensitive K+ channel antagonist glibenclamide, and the L-type calcium channel blocking agent D888. RESULTS: Ketamine enantiomers and the racemate produced concentration-dependent vasorelaxation. The relaxing effect of S(+) ketamine was significantly weaker compared with R(-) ketamine and the racemate reflected by the half-maximum effective concentration (EC50) values of 11.6 x 10(-4), 4.8 x 10(-4), and 6 x 10(-4) M, respectively. Removal of the endothelium and NNLA or glibenclamide pretreatment did not significantly alter the vasorelaxing effect of ketamine. In contrast, D888 pretreatment significantly shifted the concentration-effect curves of both S(+) and R(-) ketamine rightward (EC50 values of 18.9 x 10(-4) and 8.5 x 10(-4) M, respectively), whereas the difference between the isomers was not affected. CONCLUSIONS: Vasorelaxation by ketamine enantiomers is quantitatively stereoselective: The effect of S(+)ketamine is significantly weaker compared with that of the racemate and R(-) ketamine. This stereoselective difference is not due to nitric oxide release, activation of adenosine triphosphate-sensitive potassium channels, or differential inhibition of L-type calcium channels.     

kappa-opioid agonist U50488 inhibits P-type Ca2+ channels by two mechanisms

The effects of U50488, kappa-opioid agonist on P-type Ca2+ channels, were studied. U50488 inhibited depolarization-induced Ca2+ uptake into rat brain synaptosomes, which was sensitive to omega-Agatoxin IVA (omega-AgaIVA; P-type Ca2+ channel blocker) and inhibited P-type Ca2+ channel currents recorded from rat cerebellar Purkinje neurons by the whole-cell patch clamp method. Dynorphin A also inhibited P-type Ca2+ channel currents. The inhibition by U50488 was biphasic; high affinity component (21%, IC50 = 8.9 x 10(-8) M) and low affinity component (79%, IC50 = 1.1 x 10(-5) M). At low concentrations of U50488 (10(-6) M), P-type Ca2+ channel current inhibition was attenuated by norbinartorphimine (nor-BNI), kappa-opioid antagonist, and by dialysis of cells with a pipette solution containing guanosine 5'-O-(2-thiodiphosphate) (GDP-beta S). At high concentrations of U50488 (10(-5) M), P-type Ca2+ channel current inhibition was frequency-dependent. Thus U50488-induced current inhibition is mediated by two mechanisms. Its high affinity component is produced by activation of kappa-opioid receptors, whereas the low affinity component is due to its direct action on the P-type Ca2+ channel.     

Acetylcholine-induced endothelium-independent relaxations in monkey isolated superior and inferior caval veins

1. We examined the effects of acetylcholine (ACh), isoprenaline (Isop) and Ca-ionophore, A23187 on monkey isolated superior (SCV) and inferior caval veins (ICV) with and without intact endothelium, which had been partially contracted by 2 x 10(-6)-5 x 10(-6) M prostaglandin F2 alpha (PGF2 alpha). 2. Low concentrations of ACh (10(-10)-10(-9) M) produced a dose-dependent relaxation in the precontracted venous segments with endothelium. ACh at concentrations more than 10(-7) M elicited a transient contraction followed by a relaxation in these segments. 3. An addition of 5 x 10(-7) M A 23187 induced about 60% of maximum relaxation produced by 10(-5) M sodium nitroprusside (SNP) in each venous segment with endothelium. 4. Isop (10(-10)-10(-5) M) caused a dose-related relaxation in the precontracted caval veins with intact endothelium. 5. Removal of endothelium caused no significant effect on the ACh-induced dual responses but a significant inhibition of the A23187-induced relaxation. 6. Pretreatment with atropine antagonized competitively the ACh-induced relaxations in the endothelium-intact and endothelium-denuded caval veins. The Schild plot analysis showed that the pA2 values of the segments with and without endothelium were 9.72 +/- 0.14 (n = 5) and 10.01 +/- 0.23 (n = 6) in the ICV; and 9.95 +/- 0.20 (n = 5) and 9.70 +/- 0.10 (n = 5) in the SCV, respectively. 7. Pretreatment with 5 x 10-5M aspirin, 3 x 10-5M N0-nitro-L-arginine methylester, 1 mM tetraethylammonium,or 3 x 10-6 M glibenclamide caused no significant effect on the basal tone, ACh induced transient contraction, and ACh;.induced relaxation in the precontracted venous segments with and without endothelium.8. Pretreatment with 10-5 M methylene blue produced a significant reduction of the ACh- and SNP induced relaxations in the precontracted venous segments with and without endothelium. The pretreatment with the same concentration of methylene blue, however, caused no significant effect on the Isop-induced relaxation in venous segments with endothelium.9. The results suggest that ACh acts directly on the venous smooth muscle cells via a high-affinity muscarinic receptor subtype to accumulate cellular cyclic GMP producing endothelium-independent relaxation in the monkey caval veins.     

Calcium binding to tandem repeats of EGF-like modules. Expression and characterization of the EGF-like modules of human Notch-1 implicated in receptor-ligand interactions

The Ca(2+)-binding epidermal growth factor (cbEGF)-like module is a structural component of numerous diverse proteins and occurs almost exclusively within repeated motifs. Notch-1, a fundamental receptor for cell fate decisions, contains 36 extracellular EGF modules in tandem, of which 21 are potentially Ca(2+)-binding. We report the Ca(2+)-binding properties of EGF11-12 and EGF10-13 from human Notch-1 (hNEGF11-12 and hNEGF10-13), modules previously shown to support Ca(2+)-dependent interactions with the ligands Delta and Serrate. Ca2+ titrations in the presence of chromophoric chelators, 5,5'-Br2BAPTA and 5-NBAPTA, gave two binding constants for hNEGF11-12, Kd1 = 3.4 x 10(-5) M and Kd2 > 2.5 x 10(-4) M. The high-affinity site was found to be localized to hNEGF12. Titration of hNEGF10-13 gave three binding constants, Kd1 = 3.1 x 10(-6) M, Kd2 = 1.6 x 10(-4) M, and Kd3 > 2.5 x 10(-4) M, demonstrating that assembly of EGF modules in tandem can increase Ca2+ affinity. The highest affinity sites in hNEGF11-12 and hNEGF10-13 had 10 to 100-fold higher affinity than reported for EGF32-33 and EGF25-31, respectively, from fibrillin-1, a connective tissue protein with 43 cbEGF modules. A model of hNEGF11-12 based on fibrillin-1 EGF32-33 demonstrates electronegative potential that could contribute to the higher affinity of the Ca(2+)-binding site in hNEGF12. These data demonstrate that the Ca2+ affinity of cbEGF repeats can be highly variable among different classes of cbEGF containing proteins.