arene C99 inhibits myosin atPase aCtivity and Changes the organization of ContraCtile filaments of myometrium

Calix[4]arenes are cup-like macrocyclic (polyphenolic) compounds, they are regarded as promising molecular "platforms" for the design of new physiologically active compounds. We have earlier found that сalix[4]arenе C-99 inhibits the AtPase activity of actomyosin and myosin subfragment-1 of pig uterus іn vitro. the aim of this study was to investigate the interaction of calix[4]arene C-99 with myosin from rat uterine myocytes. It was found that the AtPase activity of myosin prepared from pre-incubated with 100 mM of calix[4]arene C-99 myocytes was almost 50% lower than in control. Additionally, we have revealed the effect of calix[4]arene C-99 on the subcellular distribution of actin and myosin in uterus myocytes by the method of confocal microscopy. this effect can be caused by reorganization of the structure of the contractile smooth muscle cell proteins due to their interaction with calix[4]arene. the obtained results demonstrate the ability of calix[4]arene C-99 to penetrate into the uterus muscle cells and affect not only the myosin AtPase activity, but also the structure of the actin and myosin filaments in the myometrial cells. Demonstrated ability of calix[4]arene C-99 can be used for development of new pharmacological agents for efficient normalization of myometrial contractile hyperfunction.

Calix [4]arenes are cup-like macrocyclic (polyphenolic) compounds, they are regarded as promising molecular "platforms" for the design of new physiologically active compounds.We have earlier found that сalix [4]arenе C-99 inhibits the AtPase activity of actomyosin and myosin subfragment-1 of pig uterus іn vitro.the aim of this study was to investigate the interaction of calix [4]arene C-99 with myosin from rat uterine myocytes.It was found that the AtPase activity of myosin prepared from pre-incubated with 100 mM of calix [4]arene C-99 myocytes was almost 50% lower than in control.Additionally, we have revealed the effect of calix [4]arene C-99 on the subcellular distribution of actin and myosin in uterus myocytes by the method of confocal microscopy.this effect can be caused by reorganization of the structure of the contractile smooth muscle cell proteins due to their interaction with calix [4]arene.the obtained results demonstrate the ability of calix [4]arene C-99 to penetrate into the uterus muscle cells and affect not only the myosin AtPase activity, but also the structure of the actin and myosin filaments in the myometrial cells.Demonstrated ability of calix [4]arene C-99 can be used for development of new pharmacological agents for efficient normalization of myometrial contractile hyperfunction.k e y w o r d s: myosin, actin, myometrial myocytes, AtPase activity, calix [4]arene C-99, confocal microscopy.

C onsiderable attention has been recently
given to such compounds as сalixarenes in international biochemical publications.These are synthetic macrocyclic phenol oligo mers, which molecules have a cup-like structure and functionalizedwithdifferentchemicalgroups.Calixa renes, calix [4]arenes in particular, can affect biochemical processes in a cell, due to their ability to form supramolecular complexes with biologically important molecules and ions; correspondingly, they are considered as promising molecular "platforms" for the design of new physiologically active compounds [1,2].
The calix [4]arene C99 ability to inhibit ATPase activity of contractile myometrium proteins, as well as activity of Na + ,K + АТРaseofPM of the uterine smooth muscles may be further used for elaboration of new pharmacologic agents capable ofefficientrecoveryofthenormaluteruscontractile function under pathologic states of the myometrium.Sincetheaboveexperimentswerecarriedoutonisolated enzymes or on membrane vesicles, i. e., not on the cell level, it is very important to investigate the ability of calix [4]arene C99 to penetrate through PM inside the uterine smooth muscle cells.
The aim of our work was to study the interactionofcalix [4]areneC99withacontractilecomplex of smooth muscles on the model of isolated uterine myocytes.Sincetheisolateduterinesmoothmuscle cellswereforeseentobeusedasthemainobjectof researchitwasnecessary,firstofall,toadaptknown protocols of isolation and cultivation of the primarycultureofmyocytestoourinvestigations [5,6].Isolation of a homogeneous population of uteri ne smooth-muscle cells is considerably complicated becauseotheruterinecells,includingfibroblasts [6], which are visually similar to myocytes and contain nonmuscularmyosinandactin,areisolatedjointly withthesmoothmuscleuterinecells(myometrium).Thus, it was necessary to develop an approach, which could give a possibility to analyze localization ofcontractileproteinsexceptionallyinuterinemyocytes.Hence, we had proposed an approach, which consistedindualstainingofcellsampleswithfluorescently labeled antibodies against smooth muscle αactin and myosin II.It is known that myosin II isexpressednotonlyinsmoothmyocytes,butalso inothercells,whileαactinisaspecificmarkerof smoothmusclecells [7],thatiswhytheapproach of dual staining permitted us to analyze the effect ofcalix [4]areneC99onmyosinIIofonlysmooth muscle cells, which were stained simultaneously forαactin.Forthispurpose,wehavedevelo peda protocol for staining myosin and actin in the uterine smoothmusclecellswithfluorescentlylabeledanti-bodiesagainstmyosinIIandsmoothmuscleαactin under conditions of preincubation of native cells with calix [4]areneC99andincontrol(withoutC99),and their localization in myocyte was investigated by the method of confocal microscopy.
Cultivation of primary myocyte cells.All procedures for obtaining cultivated myocytes were performed in sterile conditions under a laminar flow using collagenase 1 of A type.The cells were cul-tivatedinRPMI1640orDMEMF12nutrientmediumwithmixofantibiotics(penicillin1000U/ml, streptomycin100µg/ml)andanantimycotic(ampho-tericinB0.25µg/ml),and20%FetalBovineSerumat37°Сat5%Со 2 concentration in the atmosphere (standard conditions).Cells were placed on cover glassesinthewellsofa24wellplateandcultivated in standard conditions for 1-2 days to ensure the attachment and spreading of the cells.The cells were washedwithPBSbeforeaddingcalix [4]areneC99.The medium was changed by serum-free one with 25or100µMcalix[4]areneC99.Afterthat,cells were cultivated for 1 h in standard conditions.The control cells were incubated in the same conditions butcalix [4]arenewasreplacedbyitsbufferfordissolving.
MTTassay was used to study the effect of calix [4]areneC99onviabilityoftheuterinecells.Itis based on the ability of mitochondrial dehydrogenas-es of respirating actively cells to convert water-solu-ble3(4,5dimethylthiazol2yl)2,5diphenyltetrazoliumbromide(МТТ)intoinsolublevioletformazan, whichiscrystallizedinsidethecell.Formazansolubilizationwiththeuseofdimethylsulfoxide(DMSO) and the further photometry permits us to compare exactchangesinthesolutionabsorptionastothe control with a change of the number of viable cells, and to estimate the cell death induced by one or anothercytotoxicagentincytotoxicresearch [8].

Study of calix[4]arene C-99 ability to penetrate into cells and to affect myosin localization.
To study calix [4]arene C99 ability to penetrate into cells, the suspension of native rat uterus myocytes were preincubatedwith100mMcalix [4]areneC99.The cells were thoroughly washed after incubation; myosin was isolated from them and its ATPase activity wasdetermined.Itappearedthatmyosinobtained

AtPase activity of myosin, isolated from the suspension of rat uterus cells, in control and in conditions of myocyte suspension preincubation with 100 µM calix[4]arene C-99 (М ± m, n =5 ). the value of activity in the absence of calix[4]arene (Р < 0,05) was taken as 100% (control)
MyosinIIincontrolcellsislocatednearthe cellPM,thatisagreedwithpublisheddata(Fig.4).We have found the differences in myosin structure in the uterus myocytes comparing the confocal photographsofthecontrolandexperimentalmyocytes.Myosin from the experimental cells preincubated withcalix [4]areneC99hadmorediscretelocation incontrasttothecellswithoutaddingcalix [4]arene C-99, in which stained myosin apparently has more integrated structure.The differences of subcellular distribution of myosin in uterine myocytes in conditionsofintactcellsincubationwithcalix [4]arene C-99 compared to the control may be caused by changes in the structure of the proteins of contractile apparatus of the smooth muscle cells as a result of its interactionwithcalix [4]arenepenetratingintocells.

Control Calix[4]arene C-99
the suspension that did not meet their physiological condition.At the same time, the results obtained do notguaranteeanyspecificityofcalix [4]areneeffect ontheSMcellmyosin,sincetheSMcellmarkerwas not used.Thatiswhy,thenextstagewastheinvestigation of calix [4]arene C99 influence on localization of contractile proteins on cultivated cells that allowed us to create conditions for cells adhesion, proliferationandgrowth.Inouropinion,theinvestigationon a model of cultivated cells permits the effectofcalix [4]areneC99onthestructureofactomyosincomplexoflivecellsintheconditionsoptimallyclosetophysiologicstate.Theexperiments were conducted on the culture of uterine myocytes, which were passaged no more than 5-6 times from the moment of isolation that was necessary to keep cellsinaphaseofexponentialgrowthandtoprevent their aging.The samples for confocal microscopy were prepared from myocytes adhered to cover glasses, as it was described for myocytes suspension.Actinwasvisualizedbyincubationoffixedandper-meabilizedcellswithFITClabeledmonoclonalantibodiesagainstsmoothmuscleαactin,whichisa markerofSMcells [7].
Partiallossofthefilamentstructureofactin and appearance of its dispersed coloring (Fig. 5) was noticeable in the experimental cells, which wereincubatedwith25µMcalix [4]areneC99.It was observed virtually complete relocalization of actin upon increasing of calix [4]arene C99 con-centrationto100µM,namelycompletelossofac-Fig. 5. Standard confocal images of fixed samples of rat uterine myocytes stained by monoclonal antibodies against smooth muscle α-actin and secondary FItC-labeled antibodies: in control and under preincubation of myocytes with 25 and 100 µM calix [4]arene C-99 tinfilamentsanddiffuse(discrete)coloring.Actin filaments,locatedmainlylengthwiseinmyocytes, were well noticed on confocal photographs in control cellswhichwerenotincubatedwithcalix [4]arene C-99.Thus, under previous incubation of myocytes withcalix [4]areneC99αactinundergoesstructural changes in a cell: it can be observed a gradual loss of integrityofαactinfilaments [5].
An analysis of confocal images of the fixed uterus myocytes, grown in the culture and preincubatedwithcalix [4]areneC99for1hat37°С,has also revealed a difference in distribution and structurization of myosin and αactin compared to the control.αActinandmyosinhavebeenvisualizedinaformoffilamentsinthecontrolcellsincubated withaddingabufferforcalix [4] ЕкСпЕРиМЕнТАльнІРобоТи myosinlosttheirfilamentstructureandlookedlike discreteparticlesinexperimentalmyocytespreincubatedwithcalix [4]areneC99(Fig.6).Itcouldbesupposedthatsynchronisminthe changesofαactinandmyosinlocalizationinuterine myocytes is a result of associated and coordinated function of two contractile proteins in the muscle cells.Thedifferences(comparedtocontrol)inthe subcellular distribution of myosin and actin in uterus myocytes in conditions of incubation of intact cell with calix [4]arene C99 have been found by the method of confocal microscopy.These differences maybearesultofcalix [4]arenepenetrationintoa cell and its interaction with contractile proteins.The abilityofcalix [4]arenestopenetrateinsidethecell wasshownbyotherauthorsusingfluorescentderivativesofsomecalix [4]arenes [11,12].Similarresultsconcerningtheeffectofsome compoundsontheintegrityofactomyosincomplex havebeendescribedinpublisheddata.Inparticular, theabilityoftapsigargin [13]andphorbolester [14] to affect actin and myosin association was shown by the method of confocal microscopy on the culture of А7r5SMcells;thatisaccompaniedbytransformationofactinfilamentsintodiscreteperipheralcorpusclesandbydiffuselocalizationofmyosin.Such effect is a result of penetration of these compounds to A7r5 cell cytoplasm.