A New Isoxazolic Compound Acts as a7 Nicotinic ReceptorAgonist in Human Umbilical Vein Endothelial Cells

Magdalena P. Cortéso,§,x, Rocío Alvarezb,§, Evelyn Sepúlvedau, Felipe Jiménez-Aspeea,Luis Astudillo", Gabriel Vallejosd, and Margarita Gutiérrezc,x

a Departamento de Bioquímica, Facultad de Farmacia, Universidad de Valparaíso, Gran Bretaña

1093, Valparaíso, Chile. Fax *5632-250811.1 . E-mai1: magdalena.cortes@uv.c1b Depaftamento de Ciencias Farmacéuticas, Facultad de Farmacia, Universidad de Valparaíso,

Gran Breraña 1093, Valparaíso, Chilec Laboratorio de Síntesis Orgánica, Instituto de Química de Recursos Naturales, Universidad de

Talca, Casilla 747,Talca, Chile. Fax: +5671-200448. E-mai1: mgutienez@utalca.cld Laboratorio de Bioorganica, Instituto de Química, Facultad de Ciencias, Universidad Austral

de Chile, Valdivia, Chile

* Authors for correspondence and reprint requests

Z. Naturforsch . 69 c, 29 1 - 299 (201 4) / DOI: 1 0.5560 IZNC.2012-017 6

Received September 25,2012 / February 3,2014 / published online July 9,2014

Dedicated to the memot} of Dr. Luis Astudillo

Recent evidence suggests that the o7 nicotinic acetylcholine receptors (a7 nAChRs) participate inthe development of angiogenesis and could be a new endothelial target for revascularization in ther-apeutic angiogenesis. It has been shown that in human umbilical vein endothelial cells (HUVECs)a7 nAChR agonists increase the intracellular calcium concentration ([Ca2+]t), thus inducing prolif-eration and vessel formation which are impofiant stages of angiogenesis. In the present study weevaluated the effect of new isoxazole compounds on the cytosolic Ca2+ signal in HUVECs usingthe fluorescent Ca2+ indicator Fluo-3AM an<l probing the involvement of a7 nAChR by means ofpharmacological tools. HUVECs expressed mainly o7 nAChR, since there was no significant dif-ference in the increase in [Ca2+]1 induced by nicotine, a non-selective nicotinic agonist, in relationto choline, a selective a7 nAChR agonist. The increase in [Ca2+]1 induced by 1 mrr,t choline wasinhibited significantly (p - 0.014) in cells which had been pre-incubated for 15 min with methylly-caconitine (MLA), a selective a7 nAChR antagonist. The studied compounds 1,2, and 3 induced an

increase in [Ca2+]t in a dose-dependent manner. Compound 1 at 1 0 ¿iM induced a greater increase in[Cu2+]i than compounds 2 and 3. The increase in [Ca2+]i induced by compound 1 was significantlyinhibited by MLA (p : 0.013) and completely inhibited by mecamylamine, a non-selective nAChRantagonist, indicating that the isoxazolic compound 1 acts as an a7 nAChR agonist.

Key n-ords: a7 Nicotinic Receptor, Cytosolic Calcium Signal, Isoxazole

Introduction

Nicotinic acetylcholine receptors (nAChRs) are ex-pressed in non-neuronal tissues, such as epithelialcells, endothelial cells (ECs), and immune cells,thereby proving the existence of the cholinergic sys-tem beyond neurons (Wessler and Kirkpatrick, 2008).Of all the nAChRs identif,ed, the homomeric a] hasbeen an important target of investigations due to its

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Th"r.-rrhors contributed equally to the paper.

@ 2014 Verlag der Zeitschrift für Naturforschung, Tübingen .hftp:llznafurforsch.com

high permeability to calcium ions (Ca2+) (Castro andAlbuquerque, 1995).

In ECs. the a7 homomeric nAChRs are believedto predominate as the mediators of angiogenesis (Pil-lai and Chellapan, 2012; Heeschet et a1.,2002). Thisprocess is defined'as the dynamic process of neovas-cularization, representing the formation of new bloodvessels from pre-existing ones (Risau, 1997; Folk-man, 2006). Angiogenesis is a complex process thatis regulated by a balance between pro- and antian-giogenic molecules. The complex interactions among

292

these molecules and how they affect the vascular struc-ture and function in different environments are the ob-jects of research (Carmeliet and Jain, 2000).

By acting on nAChRs, acetylcholine (ACh) can reg-ulate vascularization and remodeling (Cooke, 2007;Arias e¡ a1.,2009).In the same way, nicotine has beendefined as a proangiogenic factor (Costa and Soares,2008), which modulates angiogenesis yia a7 nAChRsin several types of human ECs (Wu et a1.,2009), andpromotes EC migration, proliferation, survival, andtube formation in vitro (Kiuchi eral., 2008). Nico-tine has proved to be experimentally angiogenic in hu-man umbilical vein endothelial cells (HUVECs) by in-creasing the intracellulat Ca2+ concentration ([Cu2+]t)(Wang et a|.,2006).

Indeed, the a7 nAChRs play an important role incholinergic angiogenesis, and thus may be a target fortherapeutic modulation in disorders of pathological orinsufflcient angiogenesis (Carmeliet and Jain, 2000).Currently, there is an intensive search for such specificagonists as potential agents in a process called "thera-peutic angiogenesis". This process aims at the stimula-tion and growth of new blood vessels from pre-existingones in order to re-supply blood flow to affected is-chemic tissues (Deveza et a\.,2012).

Compounds with the isoxazole ring exhibit sev-eral important biological activities such as antibacte-rial (Ali et al., 20ll'), antiplatelet (Yrzheshch et al.,7994), and antioxidant (Kalirajan et al., 2012). Theisoxazole analogue of Nicotine, ABT-418, has becomea standard nAChR ligand (Briggs et al., 1995). Sim-ilarly, PNU-120596 has an isoxazole-based structurethat has proven to be a positive allosteric modula-tor of uJ nAChRs (Hurst e/ al., 2005). Many meth-ods have been employed in the synthesis of isox-azoles (Melo, 2005), including sequential (3 + 2)cycloaddition/silicon-based cross-coupling reactions(Denmark and Kallemeyn, 2005), reactions of hydrox-ylamine with aldehydes in acid medium (Kaffy et al.,2004), arñ reactions of 2-alkyn- 1- one-ortho-methyloximes with ICl, 12, Br2, or PhSeBr via electrophiliccyclization (Waldo and Larock, 2001). In this paper,we present the synthesis of three new isoxazolic com-pounds and the in vitro evaluation of their effects on[Ca2+]i in a primary culture of HUVECs.

Materials and Methods

General aspects

1H and 13C NMR spectra were recorded on a BrukerAM-400 spectrometer (Rheinstetten, Germany), using

M. P. Cortés et al. .Isoxazoles as Agonists of a7 Nicotinic Receptor

CDC13 as solvent. Tetramethylsilane (TMS) was usedas an internal standard. Chemical shifts (6) and "I val-ues are reported in ppm andHz, respectively, relativeto the solvent peak - CHCI3 in CDCI3 af 1 .24 ppm forprotons andll ppm for carbon atoms. Signals are des-ignated as follows: s, singlet; d, doublet; dd, doubletof doublets; t, triplet; m, multiplet; br.s, broad singlet.In DEPll35 spectra, the signals of CH3, CH2, andCH carbon atoms are shown as positive (*), negative(-), and positive (*), respectively. Quaternary carbonatoms are not shown.

ESI-MS (MS) data were collected using a high-resolution hybrid quadrupole (Q) and orthogonal time-of-flight (TOF) mass spectrometer (Q-Tof; Micromass,Milford, MA, USA) with constant nebulizer temper-ature of 100'C. The ESI source and the mass spec-trometer were operated in the positive ion mode; thecone and extractor potentials were set to 40 and 5 V,respectively, with a scan range of mf z 80 - 1000. Sam-ples were infused into the ESI source at flow rates ofca.5 ¡Lf min via a microsyringe pump. ESI-MS/MSexperiments were carried out by selection of a specificion in Q1 and by performing its collision-induced dis-sociation (CID) with argon in the collision chamber.The values expressed are average masses and core-spond to the [M + H]+ ions.

Reaction progress was monitored by means of thin-layer chromatography (TLC) using Merck silica gel60(Darmstadt, Germany). All reagents, including nico-tine, methyllycaconitine (MLA), and mecamylaminewere purchased from either Merck or Sigma Aldrich(St. Louis, MO, USA) and used without further pu-rification. Final purification of all products for anal-ysis was carried out by recrystallization. Acetonitrilewas distilled from CaH and dried over 4-Á molecularsieves.

Chemical synthesis

Isoxazoles 1 and 3 were obtained using the methoddescribed by Hansen et al. (2005), starting from thealdehyde (20 mmol), hydroxylamine hydrochloride(21 mmol), chloramine-T trihydrate (5.9 g, 21 mmol),and the respective alkyne (21 mmol). Compound 2 wasobtained by hydrogenation of L.

3 -(4-Nitrophenyl)-5-phenylisoxazole (1): Yellowpowder. - M.p. 225 "C. - Yield 197o. - ltt Ntr¿R(400 MHz, CDC13): ó : 8.37 (2H, d, J :8.84H2),8.01 (2H, d, J : 8.84 Hz), 1.87 (2H, dd, -r : 7.83and l.ll Hz),7.53 (3H, m), 6.92 (lH, s). - 13C NMR(101 MHz, CDCI3): 6 : 761.17, 157.10, 148.61,

'?'

M. P. Cortés et al. . lsoxazoles as Agonists of a7 Nicotinic Receptor

13s.20, (+) 130.74, (+) 129.19, (+) 121.69, (+)t25.95, (+) t24.28, (+) 91 .45. - MS (EI): mlz - 267

[M+ 1]+. - C15H1oN2O3: calcd. C 67.61, H 3.19,N 10.52, O 18.03; found C 66.52, H 3.80, N 10.63,o 18.12.

I - ( 3 -( B enTo I d ] [ l, 3 ] dioxol- 5 -yl )is oxazol- 5 -y I ) - N, N -

dimethylmethanamine (3): Amorphous solid. - Yield48vo. - rH NtrrtR (400 MHz, CDC13): s :1.33 (lH,s),7 .27 (1H, d, -/ : 8.0 Hz),6.87 (1H, d, "r

: 8.0 Hz),6.42 (tH, s), 6.02 (2H, s), 3.66 (2H, s ), 2.35 (6H, s).

- 13c NMR (101 MHz, cDCl3): 6 : ri0.46, 162.05,t49.t6,148.30, 123.24, (+) t21.20, (+) 108.69, (+)t01.06, (-) 101.s4, (+) 100.99, ( ) s4.s3, (+) 4s.32.

- MS (EI): mf 7 - 247 l}d+ 1l+. - C13H1aN2O3:calcd. C 63.40,H 5.73, N 11.38, O 19.49; found C63.52,H 5.65, N 11.42,O 19.41.

1 - ( 5 - Phenylisoxazol- 3 -yl ) aniline (2) : Amoryhoussolid. - Yield987o. -1u NlrtR (400MH2, CDC1:):ó : 7.83 (2H, dd, "/ - 8.08 ard 1.26 Hz),7 .68 (2H, d,

-i: 8.40 Hz),7.49 (3H, m), 1.16 (2H, d, J :7 .60 Hz),6.15 (tH, s),3.89 (2H, s, -NHz).- MS (EI): mlz:231 lld+ ll+. - C15H12N2O: calcd. C 14.91,H 6.11,N 11.66, O 6.66; found C 74.99, H 6.67, N 11.68,o 6.66.

Primary culture and íncubation conditions

A11 umbilical cords were collected after deliveryfrom full-term normal pregnancies of 40 weeks(Ethics Committee's approval and informed patientconsent were obtained at Hospital Carlos Van Büren,Valparaiso, Chile). The ECs were isolated as describedby Jaffe et al. (7973) and were identified by acetylatedlow-density lipoprotein (Voyta et al., 1984) and vonWillebrand factor (Jaffe et al., 1973') according to thevalidated methodologies developed in our laboratory(Cortés et al., 2013). In summary, endothelium wasisolated by collagenase-I (0.5 mg/ml) digestion fromhuman umbilical veins and cultured in a 199 medium,supplemented with 2.5 mtr,l L-glutamine, 14 mMHepes, 200lillL penicillin, 400 UIIL streptomycin,10Vo fetal calf serum, l0% newborn calf serum, atpH 7.42. Experiments were performed in confluentprimary cultures (at a density of 25.104/plate) ondays three to flve of culture.

Determination of [CaZ+ ]¡As described for other ECs (Vinet et al., 2009),

HUVECs were washed twice with phosphate-bufferedsaline (PBS) (.pH 1.4) and subsequently loaded for20 min with 7 pu Fluo-3AM (Molecular Probes, Eu-

gene, OR, USA) and 0.094Vo pluronic acid in Locke'ssolution consisting of (in mu): NaCl (135), KCI (5.6),CaCl2.2H2O (2.5), Hepes (10), MgCl2.6H2O (1.2),o-glucose (5.5), pH 7.4. Light emitted by Fluo-3AM,resulting from changes in [Ca2+]i in a group of 6- 10

cells placed in a superfusion chamber of an epifluo-rescence microscope (Nikon Eclipse E600 FN; NikonCorp., Tokyo, Japan), was continuously measured witha photomultiplier (Hamamatsu Photonics, Hamamatsu,Japan) and appropriate f,lters (excitation, 490 nm;emission, 530 nm). Data were digitalized at 3 Hz byan analog converter using the Axotape software (AxonInstruments, Foster City, CA, USA), analysed, andplotted by the OriginPro software (Originlab Corp.,Northampton, MA, USA). The magnitude of the fluo-rescent signal was presented as a ratio according to theequation LF lFb : @ - n) lFb, where .fl is the fluo-rescent value of Fluo-3AM at time / and F6 is the basalfluorescence, as described by Takahashi et al. (1999).

In each experiment, HUVECs were acutely stim-ulated with the respective agonist (30 s), and thenwashed with Locke's solution. At the end of each ex-periment, a pulse of AIP was applied to verify thefunctional state of cells through Ca2+ influx via P2Xareceptors (Yamamoto et al., 2000). Experiments thatincluded antagonists were done according to the fol-lowing protocol: HUVECs were loaded with Fluo-3AM and incubated for 15 min with the respectiveantagonist at room temperature. Then, the cells werewashed with Locke's solution, and Ca2+ measure-ments were immediately recorded.

Statistical treatment

Results are given as means t standard error (mean+ SEM), where ¡z represents the number of glassslips examined per experiment. For statistical analy-sis, Satistix software for Windows (Analytical Sofrware, Tallahassee, FL, USA) was used. Statistical anal-ysis comparing multiple groups was performed byANOVA. For comparison between two groups, the un-paired Student's /-test was applied. p < 0.05 was con-sidered statistically signifi cant.

Results

Synthesis and characterization of isoxazoles

The isoxazolic compounds 1 and 3 were synthe-sized according to the procedure described by Hansenet al. (2005). After conversion of an aldehyde tothe corresponding aldoxime via the reaction with

293

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hydroxylamine hydrochloride, the aldoxime wastransformed into the nitrile oxide with chloramine-Ttrihydrate, and in the presence of a catalytic amountof in sítu copper(I) (comproportionation), finallyin the 3,5-disubstituted isoxazole. The isoxazole 2was prepared from a mixture of isoxazole 1 with70Vo PdlC as catalyst and ethyl acetate (EtOAc),with stirring under H2 (1 atm) at room temperature.The reaction mixture was flltered, concentrated lnvacuo, and then purified by column chromatography(silica gel, petroleum ether/EtOAc). The schematicprocedure is presented in Fig. 1.

The isoxazoles were characterizedby 1H NMR, 13C

NMR, and mass specÍa. 1tt Xl,tR spectra of the syn-thesized isoxazoles showed the characteristic signal ofa singlet near 6.5 ppm, coffesponding to a proton ofthe isoxazolic ring. The mass spectra showed similarfragmentation patterns among the compounds, alwaysshowing the molecular ion [M + H]+.

Biological activity

In HUVECs loaded with Fluo-3AM, nicotine in-creased [Ca2+]i after l0 s of acute perfusion, and

[Ca2+]i reached its maximum 10 s after the beginningof the response. In the wash-out period, the responseinduced by nicotine decreased rp fo 507o of the max-imum and no late response was observed. The maxi-mum effect was achieved at 100 pu nicotine (Fig. 2A).

M. P. Cofiés et al. .lsoxazoles as Agonists of a7 Nicotinic Receptor

We selected the choline concentration accordingto data obtained for chromaffin cells (Maneu et al.,2002), starting at 1 mM. After basal stabilization, HU-VECs were acutely perfused with choline during 30 s.

Choline induced a [Ca2+]i increase in a concentration-dependent manner with a rapid onset at 10 s, and inthe wash-out period [Ca2+]1 decreased to 507o of themaximum, which was reached at l0 mtr¿ (Fig. 2B).

Figure 2C shows the respective maximum [Ca2+]tpeak in response to each agonist concentration (E),standardized with regard to the mean higher value of[Ca2+]i among the evaluated concentrations (E,,ur). Itis important to note that nicotine reached the maximumeffect at lower concentrations than choline.

In order to demonstrate the implication of o7nAChRs in the cholinergic agonist-induced [Ca2+]1 re-sponse, we used MLA, a selective a7 nAChR antag-onist (Astles et a1.,2002). Preincubation of HUVECswith MLA signiflcantly inhibited the [Ca2+]1 increaseinduced by choline (p < 0.005) and nicotine and par-tially inhibited the response to ACh (Fig. 3).

We analysed the effect of the isoxazolic compoundson [Ca2+]i following the same protocol as describedfor the agonists, i. e., the compounds were f,rst ex-amined at l0 pM and then, according to the observedresponse, higher or lower concentrations were em-ployed. The three compounds induced an increase in[Ca2+]i in a concentration-dependent manner, high-est in the case of compound 1 (Fig.4). To exam-

[.,

R2R1

Fig. 1. Reagents and conditions for the synthesis of isoxazoles. a) NaOH, NH2OH.HCI. b) TsN(Cl)Na.3H2O. c) Alkyne-R2Cu/CuSO4 (catalyst) in r-BuOlVH2O (1 : 1), with ultrasound.

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M. P Cortés et al. .lsoxazoles as Agonists of a7 Nicotinic Receptor

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duced by either cbmpound 2 and 3 in the presence

of the antagonist, was blocked neither by MLA norby mecamylamine, while hexamethonium (HEXA),a non-selective nAChR antagonist, completely inhib-ited their effect (data not shown). This indicates thatthe elevation of [Ca2+]1 in response to compounds2 ard 3 is not due to their agonistic action ona7 nAChRs.

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ine whether the effects of the isoxazole compoundswere metliated by nAChR, we incubated HUVECsin individual experiments with MLA (selective a7nAChR antagonist) or mecamylamine (non-selectivenAChR antagonist), following the protocol describedabove. The action of compound 1 (100 nM) wasblocked by both MLA (p : 0.013) and mecamyl-amine. On the other hand, the increase in [Ca2+]1, in-

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Fig.2. Cytosolic Ca2+ increment in HUVECs in response to acute stimulation with nicotine (Nic) (A and C) or choline (Col)(B and C). (A, B) Original recordings of cytosolic Ca2+ signals from four individual experiments. The cel1s were loaded withFluo-3AM during 20 min at 37 'C. The agonists nicotine (non-selective nAChR agonist) or choline (selective o7 nAChRagonist) were applied after a 30-s wash with Locke's solution. At least three experiments were done for each agonist at

a dehned concentration. (C) The bar graph shows comparative increments of cytosolic Car+ induced by the two agonists.Values are means :t SEM, and lr represents the number of experiments at each concentration. Nicotine at 1 mM inducesdesensitization of nAChR. n.s., statistically not significant.

0.3

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M. P. Cortés et al. .Isoxazoles as Agonists of a7 Nicotinic Receptor

Discussion

The results obtained in this study demonstrate thatthe isoxazolic compound 1 behaves as an agonist ofa7 nAChR. Precisely, based on the observation thatit induced a concentration-dependent increment in[Ca2+]i, and furthermore, that tñe response induced at100 nn¿ concentration was inhibited by MLA, 1 is a se-lective a7 nAChR antagonist.

The study also validates the functionality ofnAChRs in HUVECs obtained from primary culturesand grown in small groups. Our results indicate thesame behaviour that was observed by Heeschen et al.(2002), who, by using immunohistochemistry, foundthat nAChRs were expressed in HUVECs - af 50Voconfluence preferentially the a'l type. Under simi-lar conditions of confluence in our experiments, the[Ca2+]i increment induced by nicotine ánd by cholinewas inhibited by MLA, an a7 nAChRs antagonist, sug-gesting the preferential presence of a7 nAChRs.

The flrst phenomenon to take into account at thebeginning of the investigation was the rapid desen-sitization of nAChRs, in particular of a7 nAChRs,in response to nicotinic modulators that had previ-

Compound 3

lAgonistj

Fig. 3. Effect of cholinergic agonists on [Ca2+]¡ in the pres-ence or absence of MLA (selective a7 nAChR antagoñist).Cells were loaded with Fluo-3AM as in Fig. 2 and thenacutely stimulated by the agonist with or without a prior 15_min incubation with 10 nu MLA. The bars correipond tothe mean of the maximum response obtained in the individ-ual experiments. Values ¿re means + SEM. p < 0.05, statis-tically significant; n.s., statistically not signiñcant.

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Fig.4. Effect of isoxazolic compounds on cy-tosolic Ca2+signal in HUVEC; in the pres-ence and absence ol pharm-acological tools. 1A)Original recordings of lCaz I l¡ increase in HU-VECs loaded with Fluo-3AM. induced bv com-pounds t (100 nu, 10 pu), 2(tO pw,10"0 ¡rrvr),and 3 (10 iiM, 100 iiM). (B) To determine rhespecific effect on a7 nAChRs. the isoxazoliccompounds were acutely applied in the pres-ence of MLA (selective a7 nAChR antagonist)or mecamylamine (non-selective nAChR antae-onist). Values are means + SEM of at least"3experiments; n represents the number of experi-ments for each treatment. p < 0.05, statisticaliysignilicant; n.s., statistically not significant.

Col 1 mM ACh 100 pM Nic t0 i¡M N¡c 100 pM

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M. P. Cortés et al. -Isoxazoles as Agonists of a7 Nicotinic Receptor

ously been investigated in HUVECs by Xiao andWang (2006). These authors measured [Ca2+]i byloading the cells with Fluo-3AM and using confocalmicroscopy. They investigated the effects of nicotine,choline, mecamylamine, and a-bungarotoxin, a selec-fiye u7 nAChR antagonist, at different concentrationson intracellular Ca2+ and observed that acute stimu-lation of HUVECs with 10-12 tvt nicotine increased

[Ca2+]i, whereas a second nicotine stimulus of 10 ll,10-10, or 10-e v, respectively, applied after the firststimulus, had no effect on [Ca2+]i. Because of thischaracteristic of nicotinic receptors, we developedprotocols with unique concentrations per experimentwith single groups of cells. Under these conditions,we observed a sub-maximal response of the [Ca2+]1increase only at 1 mu nicotine (see Figs. 2A, 2C).This effect has been also seen in electrophysiologicalstudies in bovine chromaffin cells in which nicotine(1 mu) induced a higher current inactivation thanother agonists (Rojo, 2006).

In our experiments, nicotine, a non-selectivenicotinic agonist, and choline, a selective o7 nAChRagonist, induced similar [Ca2+]i increments. In bothcases, the basal [Ca2+]i signal was restored afterwashing out of the agonist. These results agree withthose of Xiao and Wang (2006), who stimulatedHUVECs with choline and attributed the increase in[Cu2+]i to the activation of o7 nAChRs. Additionally,the response induced by nicotine in HUVECs issimilar to that described by Wang et al. (2006), whoused Fura-2 to determine fCaz+fi. These authorsconcluded that nicotine mainly affects a7 nAChRs inHUVECs, which agrees with our conclusions fromthe experiments in which nicotine was applied in thepresence of l0 ntr¿ MLA given that the increment of[Ca2+]i was completely inhibited at 10 ¡rna nicotineand85o/o inhibited at 100 plr,t nicotine (see Fig. 3). Theresponse induced by 1 mu choline was significantlyinhibited by 10 nu MLA as we1l.

Compound 1 induced a concentration-dependent in-crement of [Ca2+]1. The response induced by 100 nrr¿ 1

was strongly inhibited by l0 ntlr MLA, demonstrating

Ali M. 4., Ismail R., Choon T. S., Pandian S., and HassanAnsari M. Z. (2011), Antimycobacterial activity: syn-thesis of novel 3-(substituted phenyl)-6,7-dimethoxy-3a,4-dihydro-3/1-indeno[1,2-c]isoxazole analogues. J.

Enzyme Inhib. Med. Chem. 26, 598-602.Arias H., Richards V. E., Ghafoori M., Le V., and Mousa

S. A. (2009), Role ofnon-neuronal nicotinic acetylcholine

297

the agonistic effect of I on aJ nAChRs. Compounds 2and 3 both increased [Cu2+]i without significant differ-ences between responses at 10 ¡ru and 100 ¡ru. In ad-dition, the magnitude of the [Ca2+]i response inducedby these compounds was minor in comparison to thatof 1 at the same concentration (10 pu), and, contraryto the response to 1, these responses were unaffectedby MLA. Thus, elevation of [Ca2+]i must have beenmediated by receptors other than o7 nAChRs.

According to a review by Grochot-Przeczek et al.(2013), therapeutic angiogenesis in peripheral arterydisease based on stimulation of collateral vesselformation seems to be a good altemative for peripheralartery disease therapy. This implies that studies of newcompounds, such as isoxazoles, should be conductedto develop a treatment for angiogenesis-associateddiseases.

Conclusion

The isoxazolic compound L appears to act as ana7 nAChR agonist, given that it induced an increase in[Ca2*]i in a concentration-dependent manner, whichwas inhibited by MLA. In our experiments, nicotineand choline exhibited similar responses which wereboth blocked by MLA, suggesting a preferential ex-pression of o7 nAChRs in HUVECs.

Acknowledgement

We gratefully thank Dr. Carlos Bennett, Head ofObstetrics and Gynecology Department, and LucíaMadrid, Hospital Carlos Van Büren, Valparaíso, Chile,for supplying umbilical cords and for cooperation. Weare grateful to Rachael Lange for her help with thetranslation, and to Dirección de Investigación y Desar-rollo (DID) of Universidad Austral de Chile for a grant(DID S-2010-21) to G. V. This work was supported bya FONDECYT project (1100481) to L. A. and DIUVproject (5912009) to M. P. C. from Universidad de Val-paraíso, Valparaíso, Chile.

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