Chemical and stereochemical aspects of propranolol metabolism. Diastereomeric 1-(1-hydroxy-2-propylamino)-3-(1-naphthoxy)-2-propanols produced by rat liver microsomal omega-hydroxylation

A new metabolic pathway of terminal hydroxylation (omega-hydroxylation) of the N-isopropyl group of propranolol (1) was established. Selected ion-monitoring GC-MS analysis, based on use of the synthesized mixture of diastereoisomers of 1-(1-hydroxy-2-propylamino)-3-(1-naphthoxy)-2-propanol (2) as a standard, established formation of both diastereoisomers of 2 as metabolites of 1. These diastereoisomers were formed in unequal amounts when 1, its hexadeuterated analogue 8 or heptadeuterated analogue 9, were incubated in the presence of the rat liver microsomal fraction. Authentic (2R,2"S)-2, obtained from the amide formed from (2S)-3-(1-naphthoxy)-2-hydroxypropionic acid [(2S)-5] and (2S)-alaninol by diborane reduction, facilitated examination of stereochemical aspects of this process. From incubations of the enantiomers of 1 and pseudoracemic propranolol [equimolar (2R)-propranolol-3,3-d2 and (2S)-propranolol-d0] in the presence of the rat liver microsomal fraction, we established that the diastereomeric products were formed in the order (2S,2"S)-2 approximately equal to (2S,2"R)-2 greater than (2R,2"R)-2 greater than (2R,2"S)-2. (2S)-1, which was metabolized to 2 to a greater extent than (2R)-1, showed no stereoselectivity, affording about equal amounts of (2S,2"S)-2 and (2S,2"R)-2. (2R)-1, which was metabolized to 2 to a lesser extent, afforded considerably more (2R,2"R)-2 than (2R,2"S)-2. omega-Hydroxylation was a minor metabolic pathway in the microsomal incubation. About 2000X less 2 than 1-amino-3-(1-naphthoxy)-2-propanol (3), the product of N-dealkylation of 1, was formed.     

Characterization of calcium signaling by purinergic receptor-channels expressed in excitable cells

ATP-gated purinergic receptors (P2XRs) are a family of cation-permeable channels that conduct Ca(2+) and facilitate voltage-sensitive Ca(2+) entry in excitable cells. To study Ca(2+) signaling by P2XRs and its dependence on voltage-sensitive Ca(2+) influx, we expressed eight cloned P2XR subtypes individually in gonadotropin-releasing hormone-secreting neurons. In all cases, ATP evoked an inward current and a rise in [Ca(2+)](i). P2XR subtypes differed in the peak amplitude of [Ca(2+)](i) response independently of the level of receptor expression, with the following order: P2X(1)R < P2X(3)R < P2X(4)R < P2X(2b)R < P2X(2a)R < P2X(7)R. During prolonged agonist stimulation, Ca(2+) signals desensitized with different rates: P2X(3)R > P2X(1)R > P2X(2b)R > P2X(4)R > P2X(2a)R > P2X(7)R. The pattern of [Ca(2+)](i) response for each P2XR subtype was highly comparable with that of the depolarizing current, but the activation and desensitization rates were faster for the current than for [Ca(2+)](i). The P2X(1)R, P2X(3)R, and P2X(4)R-derived [Ca(2+)](i) signals were predominantly dependent on activation of voltage-sensitive Ca(2+) influx, both voltage-sensitive and -insensitive Ca(2+) entry pathways equally contributed to [Ca(2+)](i) responses in P2X(2a)R- and P2X(2b)R-expressing cells, and P2X(7)R operated as a nonselective pore capable of conducting larger amounts of Ca(2+) independently on the status of voltage-gated Ca(2+) channels. Thus, Ca(2+) signaling by homomeric P2XRs expressed in an excitable cell is subtype-specific, which provides an effective mechanism for generating variable [Ca(2+)](i) patterns in response to a common agonist.     

2-Aminoethoxydiphenyl borate directly inhibits store-operated calcium entry channels in human platelets

In this study, we examined 2-aminoethoxydiphenyl borate (2APB) as an inhibitor of Ca(2+) influx in human platelets. 2APB was found to inhibit thrombin-mediated intracellular Ca(2+) mobilization rapidly in platelets incubated in the absence of extracellular Ca(2+). This result supports an intracellular action of 2APB on inositol 1,4,5-trisphosphate (IP(3))-receptor Ca(2+) channels. 2APB was without effect on the ability of thapsigargin to mobilize intracellular Ca(2+). This result suggests that the efflux of Ca(2+) from the endoplasmic reticulum mediated by thapsigargin is not via IP(3) Ca(2+) channels. However, 2APB was able to prevent the entry of Ca(2+) and Sr(2+) through thapsigargin-activated, store-operated Ca(2+) channels (SOCC). This result supports a direct inhibitory effect of 2APB on SOCC. 2APB was also able to block the entry of Sr(2+), Ba(2+), and Mn(2+) entry into unstimulated platelets, which suggests that 2APB was inhibiting the Ca(2+) influx channels directly. The capacity of 2APB to prevent Ca(2+) influx and Sr(2+) influx was rapid because it occurred immediately upon addition to the platelets. The inhibition of Ca(2+) and Sr(2+) influx by 2APB was similar to that seen with the cell-impermeable nonselective Ca(2+)-channel blocker La(3+) or the Ca(2+) chelator EGTA. Diphenylboronic anhydride and 2,2-diphenyltetrahydrofuran, two compounds that are structurally similar to 2APB, also inhibited Ca(2+) influx. It was concluded that 2APB was a rapid and effective direct inhibitor of SOCC in human platelets; as such, it cannot be used to support the involvement of IP(3) receptors in the activation of SOCC.     

Dual effect of hydrogen peroxide on store-mediated calcium entry in human platelets

Redox regulation is important for the modulation of cytosolic Ca(2+) concentration. Hence, we have investigated the effect of H(2)O(2) on store-mediated Ca(2+) entry (SMCE). In fura-2-loaded human platelets treatment with H(2)O(2) resulted in a concentration-dependent increase in Ca(2+) release from intracellular stores, while the effect on Ca(2+) entry was biphasic. In addition, 1mM H(2)O(2) reduced SMCE induced by agonists. The inhibitory effect of 1mM H(2)O(2) was prevented by inhibition of actin polymerization with cytochalasin D. Consistent with this, we found that 10microM H(2)O(2) and store depletion by treatment with thapsigargin plus ionomycin induced a similar temporal sequence of actin reorganization, while exposure to 1mM H(2)O(2) shifted the dynamics between polymerization and depolymerization in favor of the former. One millimolar H(2)O(2)-induced polymerization was reduced by treatment with methyl 2,5-dihydroxycinnamate and farnesylthioacetic acid, inhibitors of tyrosine kinases and Ras superfamily proteins, respectively. Finally, exposure to 1mM H(2)O(2) significantly increased store depletion-induced p60(src) activation. We conclude that H(2)O(2) exerted a biphasic effect on SMCE. The inhibitory role of high H(2)O(2) concentrations is mediated by an abnormal actin reorganization pattern involving both Ras- and tyrosine kinases-dependent pathways.     

Effects of specific dosages of magnesium and zinc on the teratogenicity of cadmium, nickel, and cobalt in Xenopus embryos, as assessed by the FETAX test

The objective of this study was to determine if exposure to divalent cations, Cd(2+), Ni(2+), and Co(2+) would lead to malformations in Xenopus laevis embryos, and whether addition of Mg(2+) and Zn(2+); separately and in combination, would reduce their toxicity and teratogenicity on the embryos of Xenopus laevis as assessed by 96-h FETAX tests. Results indicate that exposure to Cd(2+), Ni(2+) or Co(2+) lead to an increase in toxicity and teratogenicity in embryos, whereas Mg(2+), Zn(2+), or a combination of them reduced the toxic and teratogenic effects of these divalent cations. Modulation of Cd(2+), Ni(2+) or Co(2+) toxicity and teratogenicity by Mg(2+) and Zn(2+), varied with the metal. Zn(2+) was observed to be a better suppressor of Co(2+) toxicity and teratogenicity than Mg(2+). In contrast, Ni(2+), and Cd(2+) teratogenicity was reduced more prominently by Mg(2+). On the other hand, combination of Mg(2+) and Zn(2+) showed potentialization effect on all divalent cation toxicity and teratogenicity. We concluded that Mg(2+) and Zn(2+) reduced the toxicity and teratogenicity of Cd(2+), Ni(2+), Co(2+).     

Ca(2+) signalling by recombinant human CXCR2 chemokine receptors is potentiated by P2Y nucleotide receptors in HEK cells

1. Human embryonic kidney (HEK)-293 cells expressing recombinant G alpha(i)-coupled, human CXC chemokine receptor 2 (CXCR2) were used to study the elevation of the intracellular [Ca(2+)] ([Ca(2+)](i)) in response to interleukin-8 (IL-8) following pre-stimulation of endogenously expressed P2Y1 or P2Y2 nucleotide receptors. 2. Pre-stimulation of cells with adenosine 5'-triphosphate (ATP) revealed a substantial Ca(2+) signalling component mediated by IL-8 (E(max)=83 +/- 8% of maximal ATP response, pEC(50) of IL-8 response=9.7 +/- 0.1). 3. 1 microM 2-methylthioadenosine 5'-diphosphate (2MeSADP; P2Y1 selective) and 100 microM uridine 5'-triphosphate (UTP; P2Y2 selective) stimulated equivalent maximal increases in [Ca(2+)](i) elevation. However, UTP caused a sustained elevation, whilst following 2MeSADP [Ca(2+)](i) rapidly returned to basal levels. 4. Both UTP and 2MeSADP increased the potency and magnitude of IL-8-mediated [Ca(2+)](i) elevation but the effects of UTP (E(max) of IL-8 response increased to 50 +/- 1% of the maximal response to ATP, pEC(50) increased to 9.8 +/- 0.1) were greater than those of 2MeSADP (E(max) increased to 36 +/- 2%, pEC(50) increased to 8.7 +/- 0.2). 5. 5. The potentiation of IL-8-mediated Ca(2+) signalling by UTP was not dependent upon the time of IL-8 addition following UTP but was dependent on the continued presence of UTP. Potentiated IL-8 Ca(2+) signalling was apparent in the absence of extracellular Ca(2+), demonstrating the release of Ca(2+) from intracellular stores. 6. Activation of P2Y1 and P2Y2 receptors also revealed Ca(2+) signalling by an endogenously expressed, G alpha(s)-coupled beta-adrenoceptor. 7. In conclusion, pre-stimulation of P2Y nucleotide receptors, particularly P2Y2, facilitates Ca(2+) signalling by either recombinant CXCR2 or endogenous beta-adrenoceptors.     

Partial agonist clonidine mediates alpha(2)-AR subtypes specific regulation of cAMP accumulation in adenylyl cyclase II transfected DDT1-MF2 cells

alpha2-Adrenergic receptor (alpha(2)-AR) activation in the pregnant rat myometrium at midterm potentiates beta(2)-AR stimulation of adenylyl cyclase (AC) via Gbetagamma regulation of the type II isoform of adenylyl cyclase. However, at term, alpha(2)-AR activation inhibits beta(2)-AR stimulation of AC. This phenomenon is associated with changes in alpha(2)-AR subtype expression (midterm alpha(2A/D)-AR > alpha(2B)-AR; term alpha(2B) >or =alpha(2A/D)-AR), without any change in ACII mRNA, suggesting that alpha(2A/D)- and alpha(2B)-AR differentially regulate beta(2)-cAMP production. To address this issue, we have stably expressed the same density of alpha(2A/D)- or alpha(2B)-AR with AC II in DDT1-MF2 cells. Clonidine (partial agonist) increased beta(2)-AR-stimulated cAMP production in alpha(2A/D)-AR-ACII transfectants but inhibited it in alpha(2B)-AR-ACII transfectants. In contrast, epinephrine (full agonist) enhanced beta(2)-stimulated ACII in both alpha(2A)- and alpha(2B)-ACII clonal cell lines. 4-Azidoanilido-[alpha-(32)P]GTP-labeling of activated G proteins indicated that, in alpha(2B)-AR transfectants, clonidine activated only Gi(2), whereas epinephrine, the full agonist, effectively coupled to Gi(2) and Gi(3). Thus, partial and full agonists selectively activate G proteins that lead to drug specific effects on effectors. Moreover, these data indicate that Gi(3) activation is required for potentiation of beta(2)-AR stimulation of AC by alpha(2A/D) and alpha(2B)-AR in DDT1-MF2 cells. This may reflect an issue of the amount of Gbetagamma released upon receptor activation and/or betagamma composition of Gi(3) versus Gi(2).     

O6-alkylguanine-DNA alkyltransferase has opposing effects in modulating the genotoxicity of dibromomethane and bromomethyl acetate

O(6)-Alkylguanine-DNA alkyltransferase (AGT) is a DNA repair protein that removes O(6)-alkylguanine adducts. The interaction of dibromomethane (CH(2)Br(2)) and bromomethyl acetate (BrCH(2)OAc) with AGT was studied in vitro, and the effect of AGT on their toxicity and mutagenicity was investigated using Escherichia coli strain TRG8 (lacking endogenous AGT) that expressed human AGT or its inactive C145A mutant. Both CH(2)Br(2) and BrCH(2)OAc reacted with AGT at its cysteine acceptor site, abolishing its DNA repair activity with the latter agent being much more potent. The formation of AGT-Cys(145)S-CH(2)OAc by BrCH(2)OAc was confirmed by mass spectral analysis, but the presumed AGT-Cys(145)S-CH(2)Br adduct from CH(2)Br(2) was too unstable for such characterization. In the presence of CH(2)Br(2), AGT was covalently cross-linked to an oligodeoxyribonucleotide, 5'-d(AG)(8)-3', but no cross-link was formed by BrCH(2)OAc. Survival of cells exposed to CH(2)Br(2) was reduced, and the number of mutants was greatly increased when wild-type AGT was present. The cytotoxicity of CH(2)Br(2) was similar to that of BrCH(2)CH(2)Br(2), but the mutagenicity was about four times less. Virtually all of the AGT-mediated mutants induced by CH(2)Br(2) in the rpoB gene were at G:C sites with equal numbers of transitions to A:T and transversions to T:A. In contrast, BrCH(2)OAc was more than 10-fold less genotoxic than CH(2)Br(2) and the survival of cells exposed to BrCH(2)OAc was not affected by AGT. The number of mutations (almost all G:C to A:T transitions) induced by BrCH(2)OAc was slightly reduced by the presence of wild-type AGT and substantially increased by the inactive C145A mutant. These results with CH(2)Br(2) are consistent with a mechanism in which reaction at the active site Cys145 residue followed by attack of AGT-Cys(145)S-CH(2)Br at guanine in DNA forms a covalent adduct, which leads to cytotoxicity and to mutagenicity. The results with BrCH(2)OAc suggest that it reacts directly with DNA to form O(6)-(CH(2)OAc)guanine, which, if unrepaired, causes G:C to A:T transitions. Our experiments reveal two novel pathways (direct inactivation of AGT and formation of AGT-Cys(145)S-CH(2)-DNA adducts) by which CH(2)Br(2) may cause damage to the genome in addition to the well-recognized pathway involving activation by GSTs.     

Pharmacological evaluation of some new 2-substituted pyridine derivatives

In the present study, a series of 2-substituted-pyridines were synthesized and characterized by IR, (1)H-NMR and Elemental Analysis. The compounds were assayed against seizures induced by maximal electro shock (MES) and pentylenetetrazole (scMet). Neurologic deficit was evaluated by the rotarod test. The decrease in the elevated motor activity by introceptive chemical stimuli (amphetamine antagonistic activity) was studied at the dose level of 25 and 50 mg/kg, antihistaminic and cardiac activity were also studied. All the compounds exhibited significant anticonvulsant activity. Compounds 2-(2'-piperazino-ethanoxy)pyridine, 2-(3'-morpholino-2'-hydroxypropyloxy)pyridine, 2-(3'-piperidino-2'-hydroxypropyloxy)pyridine and 2-(3'-piperazino-2'-hydroxypropyloxy)pyridine were most active of the series against MES-induced seizures. Compounds 2-(2'-piperazino-ethanoxy)pyridine, 2-(2'-phenylamino-ethanoxy)pyridine, 2-(3'-imidazolo-2'-hydroxypropyloxy)pyridine, 2-(3'-methylamino-2'-hydroxypropyloxy)pyridine and 2-(3'-piperidino-2'-hydroxypropyloxy)pyridine exhibited significant decrease in the elevated motor activity at the dose of 50 mg/kg. Remarkable sympathetic blocking activity was observed with 2-(3'-piperazino-2'-hydroxypropyloxy)pyridine, 2-(3'-piperidino-2'-hydroxypropyloxy)pyridine and 2-(3'-imidazolo-2'-hydroxypropyloxy)pyridine only. Compounds 2-(2'-morpholino-ethanoxy)pyridine, 2-(2'-piperidino-ethanoxy)pyridine, 2-(2'-piperazino-ethanoxy)pyridine, 2-(2'-imidazolo-ethanoxy)pyridine, 2-(2'-diphenylamino-ethanoxy)pyridine, 2-(2'-diethanolamino-ethanoxy)pyridine, 2-(2'-phenylamino-ethanoxy)pyridine and 2-(2'-(4"-hydroxy)phenylamino-ethanoxy)pyridine exhibited significant blocking of histamine induced contraction on guinea pig ileum.     

Molecular and functional characterization of human P2X(2) receptors

P2X receptors are a family of ATP-gated ion channels. Four cDNAs with a high degree of homology to the rat P2X(2) receptor were isolated from human pituitary and pancreas RNA. Genomic sequence indicated that these cDNAs represent alternatively spliced messages. Northern analysis revealed high levels of human P2X(2) (hP2X(2)) message in the pancreas, and splice variants could be detected in a variety of tissues. Two cDNAs encoded functional ion channels when expressed in Xenopus oocytes, a receptor structurally homologous to the prototype rat P2X(2) receptor (called hP2X(2a)) and a variant containing a deletion within its cytoplasmic C terminus (called hP2X(2b)). Pharmacologically, these functional human P2X(2) receptors were virtually indistinguishable, with the P2X receptor agonists ATP, 2-methylthio-ATP, 2' and 3'-O-(4-benzoylbenzoyl)-ATP, and ATP5'-O-(3-thiotriphosphate) being approximately equipotent (EC(50) = 1 microM) in eliciting extracellular Ca(2+) influx. The P2 receptor agonists alpha,beta-methylene ATP, adenosine, adenosine 5'-O-(2-thiodiphosphate), and UTP were inactive at concentrations up to 100 microM. Both hP2X(2a) and hP2X(2b) receptors were sensitive to the P2 receptor antagonist pyridoxal-5-phosphate-6-azophenyl-2', 4'-disulfonic acid (IC(50) = 3 microM). In contrast to the analogous rat P2X(2) and P2X(2b) receptors, the desensitization rates of the hP2X(2a) and hP2X(2b) receptors were equivalent. Both functional forms of the human P2X(2) receptors formed heteromeric channels with the human P2X(3) receptor. These data demonstrate that the gene structure and mRNA heterogeneity of the P2X(2) receptor subtype are evolutionarily conserved between rat and human, but also suggest that alternative splicing serves a function other than regulating the desensitization rate of the human receptor.     

A cytoplasm-sensitive peptide vector cross-linked with dynein light chain association sequence (DLCAS) enhances gene expression

We previously engineered a novel, non-viral, multifunctional gene vector (STR-CH(2)R(4)H(2)C) containing stearoyl (STR) and a block peptide consisting of Cys (C), His (H), and Arg (R). STR-CH(2)R(4)H(2)C forms a nano-complex with pDNA and is stabilized by electronic interactions and disulfide cross linkages. In blood, pDNA, a cytosol-sensitive gene vector, is released from the complex into the cytosol. The current study aimed to make STR-CH(2)R(4)H(2)C capable of active nuclear localization. The dynein light chain association sequence (DLCAS) was disulfide cross-linked to STR-CH(2)R(4)H(2)C/pDNA through disulfide linkages, and the gene expression ability of this DLCAS cross-linked gene vector was evaluated. We examined the gene transfection efficiency of S-180 cells transfected with the STR-CH(2)R(4)H(2)C/DLCAS/pDNA complex. STR-CH(2)R(4)H(2)C/DLCAS/pDNA showed significantly higher and faster gene expression compared with STR-CH(2)R(4)H(2)C/pDNA. We also evaluated the cellular uptake ability of STR-CH(2)R(4)H(2)C/DLCAS/Cy5-labeled pDNA complex. STR-CH(2)R(4)H(2)C/DLCAS/pDNA showed significantly lower cellular uptake compared with STR-CH(2)R(4)H(2)C/pDNA. This result indicates that high gene expression of STR-CH(2)R(4)H(2)C/DLCAS/pDNA does not facilitate its cellular uptake. In addition, the gene expression of DLCAS/STR-CH(2)R(4)H(2)C/pDNA in S-180 cells pretreated with the tubulin polymerization inhibitor, nocodazole (NCZ), was significantly lower than that in the absence of NCZ. These results indicate that the high transfection efficiency of DLCAS/STR-CH(2)R(4)H(2)C/pDNA is dependent on intra-cellular transport utilizing the microtubule motor protein, dynein. Taken together, our results suggest that DLCAS-modified STR-CH(2)R(4)H(2)C may be a promising gene delivery system.     

Tissue distribution and activity studies of 1,24-dihydroxyvitamin D2, a metabolite of vitamin D2 with low calcemic activity in vivo

The active vitamin D compound 1alpha,24(S)-dihydroxyvitamin D(2) (1,24(OH)(2)D(2)) is under development as a therapy for disorders including cancer and secondary hyperparathyroidism. 1,24(OH)(2)D(2) is a potent inhibitor of cell proliferation in vitro and, relative to calcitriol (1,25(OH)(2)D(3)), has reduced calcemic activity in vivo. To examine the mechanisms underlying this reduced calcemic activity, we studied the tissue distribution in rats of radiolabeled 1,24(OH)(2)D(2) or 1,25(OH)(2)D(3) over 24h. Serum levels of 1,24(OH)(2)D(2) were lower than those of 1,25(OH)(2)D(3) at all time points; however, tissue levels of radiolabeled compounds followed different patterns. In duodenum and kidney, 1,24(OH)(2)D(2) and 1,25(OH)(2)D(3) rose to similar levels at early time points; 1,24(OH)(2)D(2) levels then declined more rapidly. In bone marrow, 1,24(OH)(2)D(2) and 1,25(OH)(2)D(3) were present at similar levels at all time points. In liver, 1,24(OH)(2)D(2) levels were two-fold higher than 1,25(OH)(2)D(3) at 1h post-injection, declining to similar levels by 8h. In vitamin D-deficient rats, doses of 1,24(OH)(2)D(2) 30-fold higher than 1,25(OH)(2)D(3) were required to produce equal stimulation of intestinal calcium absorption. In the same deficient animals, 1,24(OH)(2)D(2) and 1,25(OH)(2)D(3) were nearly equipotent at stimulating bone calcium mobilization. In cultured bone cells, 1,24(OH)(2)D(2) and 1,25(OH)(2)D(3) were equipotent at stimulating osteoclast formation and bone resorption. In summary, the reduced calcemic activity of 1,24(OH)(2)D(2) may result from altered pharmacokinetics relative to 1,25(OH)(2)D(3), resulting in relatively rapid decreases in 1,24(OH)(2)D(2) levels and activity in target organs such as intestine. Further studies will be necessary to confirm these findings and to confirm the clinical utility of 1,24(OH)(2)D(2).     

Synthesis and cytotoxicity of some rigid derivatives of methyl 2,5-Dihydroxycinnamate

Eight rigid compounds designed as esterase-stable analogues of methyl 2,5-dihydroxycinnamate (1) were synthesized. These derivatives include 2-(2',5'-dihydroxybenzylidene)cyclopentenone (3a), 2-(2',5'-dihydroxybenzylidene)cyclohexanone (3b), 2,6-bis(2',5'-dihydroxybenzylidene)cyclohexanone (4b), 2,6-bis(2',5'-dihydroxybenzylidene)cyclopentenone (4a), (E)-3-(2',5'-dihydroxybenzylidene)pyrrolidin-2-one (5), (E)-5-(2',5'-dihydroxybenzylidene)-1,2-isothiazolidine-1,1-dioxide (6), 4-(2',5'-dihydroxyphenyl)-5H-furan-2-one (7), and 3-(2',5'-dihydroxyphenyl)cyclopent-2-ene-1-one (8). Among the eight compounds, the furanone 7 and cyclopentenone 8 showed the most potent cytotoxicity with IC50 values of 0.39-0.98 microg/mL. Compound 8 was further brominated, phenylated and methylated at the alpha position to give three corresponding analogues, including 2-bromo-3-(2',5'-dihydroxyphenyl)cyclopent-2-ene-1-one (24), 3-(2',5'-dihydroxyphenyl)-2-phenylcyclopent-2-ene-1-one (27), and 3-(2',5'-dihydroxyphenyl)-2-methylcyclopent-2-ene-1-one (28). Among the three, the most enhanced activity was observed with the phenylated compound 27.     

Comparison of H2O2-induced vasoconstriction in the abdominal aorta and mesenteric artery of the mouse

Hydrogen peroxide (H(2)O(2)) is generally perceived as an arterial vasodilator. Due to the emerging importance of H(2)O(2) as a possible vasoconstrictor, we examined whether H(2)O(2) constricts both the abdominal aorta and superior mesenteric artery and postulated that H(2)O(2) is a ubiquitous constrictor of quiescent mouse arteries. Moreover, we postulated that KCl depolarization discloses and/or exaggerates H(2)O(2)-induced constriction. Under quiescent conditions, H(2)O(2) constricted the mouse abdominal aorta but not the mesenteric artery. Vessel depolarization (a) exaggerated this constrictor response in the aorta, and (b) unmasked a contractile response in the mesenteric artery. Our final hypothesis tested whether tyrosine kinases, mitogen-activated protein kinases (MAPKs), and/or Rho-kinase are uniformly involved in H(2)O(2)-induced vasoconstriction. We observed a marked difference in the ability of tyrosine kinase inhibitor to block H(2)O(2)-induced vasoconstriction. p38 and ERK 1/2MAPK inhibitors reduced the maximal response to H(2)O(2), whereas JNK inhibitor had no effect. Finally, Rho-kinase inhibitor decreased the H(2)O(2) response in the mesenteric artery but not in the aorta. These data demonstrate a variable yet tightly regulated H(2)O(2) vasoconstrictor effect. Furthermore, we found that p38, ERK 1/2 and Rho-kinase play a role in H(2)O(2) constriction, which may be critical pathways involved in H(2)O(2)-induced constriction across vascular beds.     

Role of PGE(2) in protease-activated receptor-1, -2 and -4 mediated relaxation in the mouse isolated trachea

1. The potential mediator role of the prostanoid PGE(2) in airway smooth muscle relaxations induced by peptidic and proteolytic activators of PAR-1, PAR-2, PAR-3 and PAR-4 was investigated in carbachol-precontracted mouse isolated tracheal segments. 2. The tethered ligand domain sequences of murine PAR-1 (SFFLRN-NH(2)), PAR-2 (SLIGRL-NH(2)) and PAR-4 (GYPGKF-NH(2)), but not PAR-3 (SFNGGP-NH(2)), induced smooth muscle relaxation that was abolished by the non-selective cyclo-oxygenase (COX) inhibitor, indomethacin. The relative order for mean peak relaxation was SLIGRL-NH(2)>GYPGKF-NH(2) approximately amp; SFFLRN-NH(2)>SFNGGP-NH(2). 3. SFFLRN-NH(2), SLIGRL-NH(2) and GYPGKF-NH(2), but not SFNGGP-NH(2), induced significant PGE(2) release that was abolished by indomethacin. Like that for relaxation, the relative order for mean PGE(2) release was SLIGRL-NH(2)>GYPGKF-NH(2)>SFFLRN-NH(2)>SFNGGP-NH(2). 4. In dose-response studies, SLIGRL-NH(2) induced concentration-dependent increases in PGE(2) release (EC(50)=20.4 microM) and smooth muscle relaxation (EC(50)=15.8 microM). 5. The selective COX-2 inhibitor, nimesulide, but not the COX-1 inhibitor valeryl salicylate, significantly attenuated SLIGRL-NH(2)-induced smooth muscle relaxation and PGE(2) release. 6. Exogenously applied PGE(2) induced potent smooth muscle relaxation (EC(50)=60.3 nM) that was inhibited by the mixed DP/EP(1)/EP(2) prostanoid receptor antagonist, AH6809. SLIGRL-NH(2)-induced relaxation was also significantly inhibited by AH6809. 7. In summary, the results of this study strongly suggest that PAR-mediated relaxation in murine tracheal smooth muscle is dependent on the generation of the spasmolytic prostanoid, PGE(2). PAR-stimulated PGE(2) release appears to be generated preferentially by COX-2 rather than COX-1, and induces relaxation via activation of the EP(2) receptor.     

Solid-phase synthesis of 16 potent (selective and nonselective) in vivo antagonists of oxytocin

We describe the synthesis and some pharmacological properties of 16 new in vivo antagonists of oxytocin. These are based on modifications of three peptides: A, B, and C. A is our previously reported potent and selective antagonist of the vasopressor (V1 receptor) responses to arginine-vasopressin (AVP)/weak oxytocin antagonist, [1-(beta-mercapto-beta,beta-pentamethylenepropionic acid), 2-O-methyltyrosine]arginine-vasopressin (d(CH2)5[Tyr(Me)2]AVP. B reported here, the Ile3 analogue of A, is d(CH2)5[Tyr(Me)2]AVT (5 below) and C is our previously reported potent nonselective oxytocin antagonist/AVP V1 antagonist, [1-(beta-mercapto-beta,beta-pentamethylenepropionic acid),2-O- methyltyrosine,8-ornithine]vasotocin (d(CH2)5[Tyr(Me)2]OVT). The following substitutions and deletions, alone or in combination, were employed in A, B, and C: 1-deaminopenicillamine (dP); D-Tyr(Alk)2 (where Alk = Me or Et), D-Phe2; Val4, Thr4; delta 3-Pro7; Lys8, Cit8; desGly9, desGly-NH2(9), Ala-NH2(9); Leu-NH2(9); Arg-NH2(9). The 16 new analogues are (1) d(CH2)5[D-Tyr(Me)2]AVP, (2) d(CH2)5[D-Tyr(Me)2, Val4,delta 3-Pro7]AVP, (3) d(CH2)5[D-Tyr-(Et)2, Val4,Lys8]VP, (4) d(CH2)5[D-Tyr(Et)2,Val4,Cit8]VP, (5) d(CH2)5[Tyr(Me)2]AVT, (6) d(CH2)5[Tyr(Me)2,Lys8]VT, (7) dP[Tyr(Me)2]AVT, (8) dP[Tyr(Me)2,Val4]AVT, (9) d(CH2)5[D-Tyr(Me)2, Val4]AVT, (10) d(CH2)5[D-Phe2,Val4]AVT, (11) d(CH2)5[Tyr(Me)2,Thr4]OVT, (12) d(CH2)5[Tyr(Me)2,Thr4,Ala-NH2(9)]OVT, (13) d(CH2)5[Tyr(Me)2,Thr4,Leu-NH2(9)]OVT, (14) d(CH2)5[Tyr(Me)2,Thr4,Arg-NH2(9)]OVT, (15) desGly-NH2(9),d(CH2)5[Tyr(Me)2,Thr4]OVT, (16) desGly9,d(CH2)5[Tyr(Me)2,Thr4]OVT. 1-4 are analogues of A, 5-10 are analogues of B, and 11-16 are analogues of C. Their protected precursors were synthesized either entirely by the solid-phase method or by a combination of solid-phase and solution methods (1 + 8 or 8 + 1 couplings). All analogues were tested in rats for agonistic and antagonistic activities in oxytocic (in vitro, without and with Mg2+, and in vivo) assays as well as by antidiuretic and vasopressor assays. All analogues exhibit potent oxytocic antagonism in vitro and in vivo. With an in vitro pA2 (in the absence of Mg2+) = 9.12 +/- 0.09, dP[Tyr(Me)2]AVT is (7) one of the most potent in vitro oxytocin antagonists reported to date. Fifteen of these analogues (all but 6) appear as potent or more potent in vivo oxytocin antagonists than C (pA2 = 7.37 +/- 0.17). Analogues 1-9 and 14 are potent AVP V1 antagonists. Their anti-V1 pA2 values range from 7.92 to 8.45. They are thus nonselective oxytocin antagonists.(ABSTRACT TRUNCATED AT 400 WORDS)     

Polymorphisms in the mitochondrial aldehyde dehydrogenase gene (Aldh2) determine peak blood acetaldehyde levels and voluntary ethanol consumption in rats

Dependence on alcohol, a most widely used drug, has a heritability of 50-60%. Wistar-derived rats selectively bred as low-alcohol consumers for many generations present an allele (Aldh2(2)) of mitochondrial aldehyde dehydrogenase that does not exist in high-alcohol consumers, which mostly carry the Aldh2(1) allele. The enzyme coded by Aldh2(2) has a four- to five-fold lower affinity for NAD than that coded by Aldh2(1). The present study was designed to determine whether these polymorphisms account for differences in voluntary ethanol intake and to investigate the biological mechanisms involved. Low-drinker F0 Aldh2(2)/Aldh2(2) rats were crossed with high-drinker F0 Aldh2(1)/Aldh2(1) rats to obtain an F1 generation, which was intercrossed to obtain an F2 generation that segregates the Aldh2 alleles from other genes that may have been coselected in the breeding for each phenotype. Data show that, with a mixed genetic background, F2 Aldh2(1)/Aldh2(1) rats voluntarily consume 65% more alcohol (P<0.01) than F2 Aldh2(2)/Aldh2(2) rats. A major phenotypic difference was a five-fold higher (P<0.0025) peak blood acetaldehyde level following ethanol administration in the lower drinker F2 Aldh2(2)/Aldh2(2) compared to the higher drinker F2 Aldh2(1)/Aldh2(1) animals, despite the existence of identical steady-state levels of blood acetaldehyde in animals of both genotypes. Polymorphisms in Aldh2 play an important role in: (i) determining peak blood acetaldehyde levels and (ii) modulating voluntary ethanol consumption. We postulate that the markedly higher peak of blood acetaldehyde generated in Aldh2(2)/Aldh2(2)(2) animals is aversive, leading to a reduced alcohol intake in Aldh2(2)/Aldh2(2) versus that in Aldh2(1)/Aldh2(1) animals.     

Quantitation of catechol estrogens and their N-acetylcysteine conjugates in urine of rats and hamsters

A method for the analysis of N-acetylcysteine conjugates of catechol estrogens [catechol estrogen mercapturates (CE SRs)], which are likely to be urinary markers of estrogen-induced tumors, was established in this study. The characteristics of the method that was established were (1) cleanup of urine using the immunoaffinity column of CE SRs, (2) detection of catechol estrogens (CEs) and CE SRs by electrochemical detection, which provided the high specificity, and (3) stability of CE SRs through the cleanup. Using this method, the simultaneous quantitation of 2-hydroxy-17beta-estradiol (2-OHE(2)), 4-hydroxy-17beta-estradiol (4-OHE(2)), 2-hydroxyestrone (2-OHE(1)), 4-hydroxyestrone (4-OHE(1)), 2-hydroxyestrone 1-N-acetylcysteine thioether (2-OHE(1) 1SR), 2-hydroxyestrone 4-N-acetylcysteine thioether (2-OHE(1) 4SR), and 4-hydroxyestrone 2-N-acetylcysteine thioether (4-OHE(1) 2SR) in the range of 1-15 ng was performed. We first demonstrated the presence of CE SRs, 2-OHE(1) 1SR and 2-OHE(1) 4SR, in urine from rats treated intraperitoneally with 17beta-estradiol (E(2)) at a dose of 5 mg/kg. In female rats, the amount of 2-OHE(1) 1SR was several-fold greater than that of 2-OHE(1) 4SR, while the presence of 4-OHE(1) 2SR was not confirmed. The level of CEs and CE SRs in male rats was approximately (1)/(2)-(1)/(20) of that in female rats. The excretion rate following administration of 2-OHE(1) at 2 mg/kg and that following the administration of 4-OHE(1) at 2 mg/kg were different in female rats. In addition, 4-OHE(1) 2SR was present in the urine of male Syrian hamsters treated intraperitoneally with E(2), whereas it was absent in rats.     

Glycosylation and the activation of proteinase-activated receptor 2 (PAR(2)) by human mast cell tryptase

1. Human mast cell tryptase appears to display considerable variation in activating proteinase-activated receptor 2 (PAR(2)). We found tryptase to be an inefficient activator of wild-type rat-PAR(2) (wt-rPAR(2)) and therefore decided to explore the factors that may influence tryptase activation of PAR(2). 2. Using a 20 mer peptide (P20) corresponding to the cleavage/activation sequence of wt-rPAR(2), tryptase was as efficient as trypsin in releasing the receptor-activating sequence (SLIGRL.). However, in the presence of either human-PAR(2) or wt-r PAR(2) expressing cells, tryptase could only activate PAR(2) by releasing SLIGRL from the P20 peptide, suggesting that PAR(2) expressed on the cells was protected from tryptase activation. 3. Three approaches were employed to test the hypothesis that PAR(2) receptor glycosylation restricts tryptase activation. (a) pretreatment of wt-rPAR(2) expressing cells or human embryonic kidney cells (HEK293) with vibrio cholerae neuraminidase to remove oligosaccharide sialic acid, unmasked tryptase-mediated PAR(2) activation. (b) Inhibiting receptor glycosylation in HEK293 cells with tunicamycin enabled tryptase-mediated PAR(2) activation. (c) Wt-rPAR(2) devoid of the N-terminal glycosylation sequon (PAR(2)T25(-)), but not rPAR(2) devoid of the glycosylation sequon located on extracellular loop-2 (PAR(2)T224A), was selectively and substantially (>30 fold) more sensitive to tryptase compared with the wt-rPAR(2). 4. Immunocytochemistry using antisera that specifically recognized the N-terminal precleavage sequence of PAR(2) demonstrated that tryptase released the precleavage domain from PAR(2)T25(-) but not from wt-rPAR(2). 5. Heparin : tryptase molar ratios of greater than 2 : 1 abrogated tryptase activation of PAR(2)T25(-). 6. Our results indicate that glycosylation of PAR(2) and heparin-inhibition of PAR(2) activation by tryptase could provide novel mechanisms for regulating receptor activation by tryptase and possibly other proteases.     

Ca2+ permeability of the (alpha4)3(beta2)2 stoichiometry greatly exceeds that of (alpha4)2(beta2)3 human acetylcholine receptors

Human alpha4beta2 nicotinic acetylcholine receptors (AChRs) expressed in Xenopus laevis oocytes or transfected cell lines are present as a mixture of two stoichiometries, (alpha4)2(beta2)3 and (alpha4)3(beta2)2, which differ depending on whether a beta2 or alpha4 subunit occupies the accessory subunit position corresponding to beta1 subunits of muscle AChRs. Pure populations of each stoichiometry can be expressed in oocytes by combining a linked pair of alpha4 and beta2 with free beta2 to produce the (alpha4)2(beta2)3 stoichiometry or with free alpha4 to produce the (alpha4)3(beta2)2 stoichiometry. We show that the (alpha4)3(beta2)2 stoichiometry and the (alpha4)2(beta2)2beta3 and (alpha4)2(beta2)2alpha5 subtypes in which beta3 or alpha5occupy the accessory positions have much higher permeability to Ca2+ than does (alpha4)2(beta2)3 and suggest that this could be physiologically significant in triggering signaling cascades if this stoichiometry or these subtypes were found in vivo. We show that Ca2+ permeability is determined by charged amino acids at the extracellular end of the M2 transmembrane domain, which could form a ring of amino acids at the outer end of the cation channel. Alpha4, alpha5, and beta3 subunits all have a homologous glutamate in M2 that contributes to high Ca2+ permeability, whereas beta2 has a lysine at this position. Subunit combinations or single amino acids changes at this ring that have all negative charges or a mixture of positive and negative charged amino acids are permeable to Ca2+. All positive charges in the ring prevent Ca2+ permeability. Increasing the proportion of negative charges is associated with increasing permeability to Ca2+.