In this thesis, the Ca2+ signalling pathway of H1-histaminoceptor stimulated DDT1 MF-2 smooth muscle cells was investigated. Further, the ATP sensitive receptor on the plasma membrane was characterized. In this last chapter, the results of the previous chapters will be summarized and evaluated. ...

Zie: Summary.

Zee, Lucie van der,,

Zee, Lucie van der

University of Groningen

   University of GroningenReceptor activated Ca2+ signalling processes in DDT1 MF-2 smooth muscle cellsZee, Lucie van derIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:1994Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Zee, L. V. D. (1994). Receptor activated Ca2+ signalling processes in DDT1 MF-2 smooth muscle cells.s.n.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 12-11-2019STIMMARYIn this thesis, the Ca2* signalling pathway of H,-histaminoceptor stimulatedDDT' MF-2 smooth muscle cells was investigated. Further, the ATP sensitivereceptor on the plasma membrane was characterized. In this last chapter, theresults of the previous chapters will be summarized and evaluated.Stimulation of certain receptor sites results in a pronounced enhancementof the intracellular Ca2* concentration, due to Ca2* mobilization from internalstores and Ca2+ entry from the extracellular space. The receptor activatedformation of inositol phosphates, in particular Ins(I,4,5)Pr, is assumed to beresponsible for the release of intracellular stored Ca2t. However, other inositolphosphates t lns(1.3.4.5)P4).  f te presence of GTP. or t}e Ca2* act ivated Ca: 'releasing process may also contribute to the rise in internal Car* upon receptorstimulation (chapter I t.Activation of H,-histaminoceptors of vas deferens derived DDTr MF-2smooth muscle cells resulted in a pronounced formation of Ins(1,3,4,5)P, witlrrespect to that of Ins(I,4,5)Pr, a rapid enhancement of the intracellular Car'concentration and a Ca2* dependent outward K* current. In chapter 2, the role ofthe inositol phosphates and the Ca2* induced Ca2* release mechanism wasinvestigated in penneabilized as well as histamine stimulated, intact, cells. Insaponin permeabilized DDTr MF-2 cells, it was found that both Ins(1,4,5)P, andIns( 1.3.4.5)Po sensit ive Ca2' s lores were preseut whi le a Ca" act ivated Car 'releasing mechanism could not be detected in this permeabilized cell systern. TheCal* releasing capacity of Ins(1,3,4,5)P4 was dependent on the simultaneouspresence of Ins(I,4,5)Pr, and the effect of both inositol phosphates was inhibitedby the Ins(l,4,5)P, receptor antagonist heparirr. Thus, the additional Car'released by Ins(1,3,4,5)P4 was mediated via Ins(1,4,5)Pr sensitive Car* channelsand it was concluded that the effect of Ins(1,3,4,5)P.t was mediated viaconnection of an Ins(1,4.5)Pr insensitive Car* store to the Ins(1,4,5)Pr sensitiveCa2* compartrnent.H,-histaminoceptor stimulation in intact cells induced a Car* dependentoutward K+ current, representing the intemal Ca2* release if Ca2* entry from theextracellular space was prevented. The histamine activated current was notdetected if the inositol phosphate induced Cart release was inhibited by heparin.This result indicated that both Ins(1,4,5)P. and Ins(1,3,4,5)P4 sensitive Car*stores could play a role in the rise in intennl Caz* upon stirnulation withhistamine and excluded Ltre contribution of the Ca2* induced Caz* releasemechanism to this process. Depletion of Ins(1,4,5)P. sensitive Car* stores inadvance reduced the K* current only to some extent. Flowever, when bothIns(1,4,5)P, and Ins(1.3,4,5)P4 sensit ive Cat* stores were depleted pr ior tohistamine stimulation, the Car* sensitive K* current could not be detectedanymore. These results denronstrated that Ins(1,3.4,5)Po sensitive Car*compar0nents were mainly responsible for the histarnine activated Ca2* release98Ifiom internal stores thereby showing the importance of Ins(1,3,4,5)P4 in the Ca2*releasing process of H,-histaminoceptor activated DDTr MF-2 cells, as depictedin Figure l.Apart from the mobilization of intracellular stored Caz*, the receptoractivated Ca2+ entry process is also of considerable importance upon histaminestimulation. As mentioned in the introduction (chapter 1.2), the mechanismunderlying the receptor activated Cat* influx is not established yet. but in mostcells it is different from the well documented voltage operated Ca2* channels.Atthough activation of receptor stimulated Caz' entry may be mediated viadifferent mechanisms, the capacitative Ca2+ entry, in which Ca2* entry is relatedto the filling state of the intracellular Car* stores and the formation of inositolphosphates, in particular Ins(1,3,4,5)Po, are considered to be the most importantmechanisms for activation of receptor operated Ca2* entry. The snrdy presentedin chapter 3 was carried out to identify the cellular component activating thehistamine stimulated Ca2* entry process in DDTr MF-2 cells. The resultsdemonstrated that the Caz* entry process was independent of the histamineactivated formation of the second messengers Ins(1,4,5)P3 or Ins(1,3,4,5)Po andwas still detected when depletion of intracellular Ca:] stores was prevented byheparin, thereby excluding activation of the capacitative or inositol phosphateinduced Ca2* entry. These results were therefore suggestive for anothercomponent involved in initiation of the H,-histaminoceptor activated Ca2+ entryprocess. It was observed that stirnulation with histamine resulted in a markedrelease of AA in DDT, MF-2 cells. This AA formation was biphasic in nature,consisting of a fast rise followed by a more sustained second phase.Exogeneously applied AA to these cells induced a concentration dependentincrease in intcrnal Ca} concentra(ion. due to activation of Car' entry from theextracellular space. This effect of AA was not mediated by its metabolites, sinceinhibition of lipoxygenase, cyclo-oxygenase or epoxygenase pathways did notaffect the AA induced Ca2" entry. Pre-activation of the Ca2* channels involvedby AA resulted in abolition of the histamine activated Ca2* entry, but not theinternal Ca2* mobilization. These observations howed that AA is functioning asa second Ínessenger to activate plasmambrane Ca:' channels, thereby promotingCa2n entry from the extracellular space in DDTr MF-2 cells, as is represented inFigure 1.Although several enzymatic pathways are reported to be responsible forthe mobilization of AA frorn nrembrane tipids, the most common pathway torelease receptor activated AA is via the action of phospholipase A2.Alternatively, PLC derived DAG can serve as a precursor for AA via activationof diglyceride lipase. Further, AA can be released from phosphatidic acid,derived frorn the hydrolysis of membrane lipids by PLD (chapter 1). In chapter 4the biochernical pathway involved in H,-histaminoceptor activated AA release ofDDT' MF-2 cells was studied. to understand the histzunine activated Ca2" entryprocess. For DD'[, MF 2 srnooth muscle cells it was demonstrated that thehistamine stimulated formation of AA was independent of dre PLA,, or PLDltil,d)eol! +ot.,rtht2 'of/asInndt''he)uS.edrels'rialve)ntIerotin.l - 'ithtseinIthto:edt - 'lse99activity, respectivery, but courd bc brocked by the pLC inhibitor neomycine.These results suggested that AA rerease occured through the action of pLC anddiglyceride lipase. In accord, it *u, iouno that inhibitioï "i;; digryceride lipaseactivity resulted in a marked accumuration of DAG, resurting in reduction of theAA release. Therefore, in .ont.uri fo'."c.pto. activation-in many other ceiltypes, the H,-histaminoceptor stirnurated rerease of AA in nór, MF_2 smoothmuscle cells is predominantly mediated via the sequentiar action of pLC anddiglyceride lipase (Figure t).Receptor activ-ated Ca2* signalring Tu.y be regulated by activation of pKC;a large famity of ubiquitousry Jistribuïed isozymá ra ir' reporteo that pKCmodulates channer activity *á plc *riui,y, resurting in the reduction of rheinositol phosphate format-ion and subsequ.ni curn ,.Ëor. rrl* int..r,al storesand may affect the recepror activated release gl AA (chapter l). In chapter 5, therote of pKC in the histamine stimuraiJ ca2* signàiil;-;.;á.rs, incruaing theÍbrmation of inositor phosphates unà-Àe ,.r.ur., ilr, ,tu"oiià. wrren the possibrerole of PKC regurati'g. cêilurar processes is considerJ,';;r; ro separare rwotypes of experimenrar^g:r.ign Fiist, exogeneous activation of pKC by rhe use ofphorbol esters like pMA, provides ,u'id"n." about the .*irr"n". of a pKCregulated feedback roop. Ho*eu"., to determine whether such a loop isphysiologicaily activated . upon- receptor stimuration, one has to rook at theconsequences of interruption of the ioop, which can be achieve<t by the use ofPKC inhibitors rike skurosporine or via^down reguration of pKC activity by rongrerïn prerreatment with,oMl . For DDT, MF-J cets it war shown that pKCactivation with pMA reduced the histamine stimurate<' formation of Ins(1,4,5)pr,Ins(1,3,4,5)p, and rhe concomio"i-.óui- mobirizariori ;;;; intenar srores.Moreover, the receptor acdvated Ca2n entry. was aborished, while the rerease ofAA was strongry reduced. Thus, these ."ruru show the .*irt.*. of a negativefeedback mechanism,berween .*og.n.árriy activare<I pKC and the fbrmation ofinositol phosphates, the subseque"ï *"uiiir"ion of ca2*, the release of AA aruithe ca2* entry. In contrast, inhibition of pKC activity 'ti' ,ruï.osporine or bylong-term pretreatmenr with "MA, ár,ry^un.ted the hisiamine activared ca2+entry, while the formation of inositor piosphates, trre ca2* -àoitiration o, tt.release of AA were not changed. *,esË lus..uations demonstrated that onry thefeedback circuir between pKó and a.r.-11q, was functionary activared uponiiËtsï:ïI;*.j';*'., resurting i  u pxc..ri*Àá i"tiuriiá" o, Caz+ enrryApparently different isoforms of pKC have to considered in these celrs, coupledto PLC activity, the release of AA *à ,r,. receptor ".ri""r"o ca2* entry,respectively. The exact nature of the pKC-isozymes remained to be identified.Thus, the results obtained from this and the previous studies, asrepresented by Figure l, indicate that stimulation of H,-nistaÁinàceptors on thesecells causes activation of pLC, resuttirt in hydrorysis of phosphatidyr inositol4'5 bisphosphate 'n,: ,1.t.ortarnin!' ËRc *à tr,. 'inárior phosphatesIns(1,4,5)P, and Ins(1,3,4,5)P1. The .à"tirJ phosphates uinc to *rei. receptors tor00mobilize Ca2n from intracellular stores and DAG is metabolized by diglyceridelipase to produce AA. In turr, AA activates Caz* channels in theplasmamembrane to allow Ca2* entry from the extracellular space. Besides itsfunctioning as a precursor for AA, the formation of DAG may serve to activateprotein kinase C (PKC). Since a functional inhibitory feedback loop betweenhistamine stimulated PKC and Car* entry, but not the formation of inositolphosphates and the release of AA was observed, an attractive hypothesis is thatreceptor stimulated DAG is first rapidly mekbolized to AA, thereby promotingCaz+ entry, which process is limited by a Ca2* sensitive PKC, activated by (asubsequent) release of DAG.In chapter 6, the effect of different nucleotides on the Ins(l,4,5)P,activated Ca2* release of saponin permeabilized cells was investigated. Althoughthe presence of ATP was not required to activate the Ins(l ,4,5)P3 induced Ca2*releasing process, the amount of Car* released by different concentrationsIns(I,4,5)P, was considerably enhanced upon addition of this nucleotide. Theamount of additional Ca2* released at a certain concentration of ATP wasindependent of the Ins( I ,4,5)P, concentration used and an Ins( I ,4,5)P,concentration-response curve with similar ECr,, values and other characteristics asobserved without ATP was obtained in the presence of ATP. Thus, even atmaximal effective Ins(1,4.5)P3, assumed to fully occupy the Ins(1,4,5)P.sensitive receptors and completely depleting the Ins(1,4,5)P, sensitive Ca2* store,the presence of ATP induced an additional release of CaZ*. The Pr"-purinoceptor agonist 2-methylthio-ATP and the non-hydrolysable ATP analogueATPTS enhanced the Ins(I,4,5)Pj activated Ca2* release to the same extent asATP. Less effective were GTP, ADP and AMP, while UTP and o,B-methyleneATP appeared to be ineffective. The rank order of potency was 2-methylthio-ATP : ATPTS : ATP > GTP > ADP > AMP ) a,B methylene ATP :UTP. These results demonstrated that the presence of these nucleotidesenhanced the total releasing capacity of the Ins(1,4,5)P, sensitive Ca2+ store viaan ATP sensitive binding site, without changing the affinity of the Ins(I,4,5)P,receptor for Ins(I,4.5)Pr. The mechanism by which this ATP activated bindingsite exerts its effect rnay be due by increasing the efficacy of the Ins(1,4,5)P,receptor. In view of the sensitivity for ATP, ATPTS and 2-methylthio ATP, itwas concluded that the effect observed is mediated by an internal binding sitewith comparable characteristics as an external Pr"-purinoceptor.Apart from the presence of an internal ATP sensitive binding site. intactDDTr MF-2 cells were previously described to possess external P2-purinoceptorslocated on the plasma membrane. Stimulation of these receptors with ATPcaused responses comparable to that obtained upon H,-histaminoceptorstimulation: a relative srnall increase in Ins I ,4,5)P, concentration and apronounced formation of Ins(1,3.4,5)P4 were accompanied by a rise in internalCa2* due to Ca2* mobilization from internal stores and Car* influx from theextracellular space. Furthermore, stimulation of these P"-purinoceptors resultedin a change in membrane current, consisting of a Ca'* dependent outward K*1 0 1kcurrent followed by a sustained, non-specit'ic inward current. The aim of theshrdy presented in chapter 7 was to characterize the A'l'P sensitive receptors ofDDTr MF-2 cells by investigating the cellular response to different nucleotides,as represented by the membrane current. It was found that these receptors couldnot be classified as P,-purinoceptors, since adenosine appeared to be inactive inmediating a change in membrane current. The cellular response was also notmediated via Pr.,.-, P,"-, and P,r-purinoceptors, since ADP, cv,B-methylene ATPand 2-methylthio-ATP, known to stimulate these receptors, respectively, did notchange the membrane current either. In contrast. it was observed that pyrimidinecontaining nucleotides, in particular UTP, caused a pronounced change inmembrane current with similar characteristics as observed with ATP. The rankorder of potency to evoke the response was ATP > LITP > TTP > CTP :GTP. The responses mediated by ATP and UTP were not additive, and could beblocked by the reversible Pr-purinoceptor antagonist suramin. In view of thesensitivity for purine as well as pyrimidine containing nucleotides, the externalATP sensitive receptor of DDT, MF-2 cells was characterized as a nucleotidereceptor (Pr,,-purinoceptor).In summary, this thesis described the receptor stimulated Caz* signallingprocess of DDT, MF-2 cells. It was shown that although Ins(l,4,5)P,,Ins(1,3,4,5)Po but no Ca2* sensitive Ca2* stores were present in these cells, theH,-histaminoceptor stimulated rise in internal Ca'* was predominantly derivedfrom Ins(l,3,4,5)P,r sensitive Ca2* stores.Furthermore, it was found that the histamine activated Ca2* entry process wasindependent of the state of depletion of internal stores or the fomration of inositolphosphates, but was activated by the release of AA generated by the orderedaction of PLC and diglyceride lipase. Inhibition of this Ca2* entry process wasshown to be mediated by a histunine activated PKC isozyme. Evidence about thepresence of PKC isozymes regulating PLC activity and AA release was alsoobtained, although involvement of these isozymes was not observed on histaminestimulation.The Ins(1,4,5)Pj activated Car* release was enhanced by the presence of adeninenucleotides via activation of an internal binding site. In particular ATP, ATPTSand the Pr"-purinoceptor agonist 2-methylthio-ATP considerably augmented theCa2* release induced by Ins(1.4,5)P3, without changing the affinity of theIns(1,4,5)P, receptor for Ins(1,4.5)Pj. On the other hamd, extracellular ATP wasshown to activate Ca2* signalling by interaction with a plasmamembrane locatednucleotide receptor, which was also sensitive for the pyrimidine containingnucleotide UTP. A model representing these receptor stimulated Ca2* signallingprocesses in DDT, MF-2 cells is presented in Figure 1.t02

Receptor activated Ca2+ signalling processes in DDT1 MF-2 smooth muscle cells

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   University of GroningenReceptor activated Ca2+ signalling processes in DDT1 MF-2 smooth muscle cellsZee, Lucie van derIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:1994Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Zee, L. V. D. (1994). Receptor activated Ca2+ signalling processes in DDT1 MF-2 smooth muscle cells.s.n.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 09-09-2023STIMMARYIn this thesis, the Ca2* signalling pathway of H,-histaminoceptor stimulatedDDT' MF-2 smooth muscle cells was investigated. Further, the ATP sensitivereceptor on the plasma membrane was characterized. In this last chapter, theresults of the previous chapters will be summarized and evaluated.Stimulation of certain receptor sites results in a pronounced enhancementof the intracellular Ca2* concentration, due to Ca2* mobilization from internalstores and Ca2+ entry from the extracellular space. The receptor activatedformation of inositol phosphates, in particular Ins(I,4,5)Pr, is assumed to beresponsible for the release of intracellular stored Ca2t. However, other inositolphosphates t lns(1.3.4.5)P4).  f te presence of GTP. or t}e Ca2* act ivated Ca: 'releasing process may also contribute to the rise in internal Car* upon receptorstimulation (chapter I t.Activation of H,-histaminoceptors of vas deferens derived DDTr MF-2smooth muscle cells resulted in a pronounced formation of Ins(1,3,4,5)P, witlrrespect to that of Ins(I,4,5)Pr, a rapid enhancement of the intracellular Car'concentration and a Ca2* dependent outward K* current. In chapter 2, the role ofthe inositol phosphates and the Ca2* induced Ca2* release mechanism wasinvestigated in penneabilized as well as histamine stimulated, intact, cells. Insaponin permeabilized DDTr MF-2 cells, it was found that both Ins(1,4,5)P, andIns( 1.3.4.5)Po sensit ive Ca2' s lores were preseut whi le a Ca" act ivated Car 'releasing mechanism could not be detected in this permeabilized cell systern. TheCal* releasing capacity of Ins(1,3,4,5)P4 was dependent on the simultaneouspresence of Ins(I,4,5)Pr, and the effect of both inositol phosphates was inhibitedby the Ins(l,4,5)P, receptor antagonist heparirr. Thus, the additional Car'released by Ins(1,3,4,5)P4 was mediated via Ins(1,4,5)Pr sensitive Car* channelsand it was concluded that the effect of Ins(1,3,4,5)P.t was mediated viaconnection of an Ins(1,4.5)Pr insensitive Car* store to the Ins(1,4,5)Pr sensitiveCa2* compartrnent.H,-histaminoceptor stimulation in intact cells induced a Car* dependentoutward K+ current, representing the intemal Ca2* release if Ca2* entry from theextracellular space was prevented. The histamine activated current was notdetected if the inositol phosphate induced Cart release was inhibited by heparin.This result indicated that both Ins(1,4,5)P. and Ins(1,3,4,5)P4 sensitive Car*stores could play a role in the rise in intennl Caz* upon stirnulation withhistamine and excluded Ltre contribution of the Ca2* induced Caz* releasemechanism to this process. Depletion of Ins(1,4,5)P. sensitive Car* stores inadvance reduced the K* current only to some extent. Flowever, when bothIns(1,4,5)P, and Ins(1.3,4,5)P4 sensit ive Cat* stores were depleted pr ior tohistamine stimulation, the Car* sensitive K* current could not be detectedanymore. These results denronstrated that Ins(1,3.4,5)Po sensitive Car*compar0nents were mainly responsible for the histarnine activated Ca2* release98Ifiom internal stores thereby showing the importance of Ins(1,3,4,5)P4 in the Ca2*releasing process of H,-histaminoceptor activated DDTr MF-2 cells, as depictedin Figure l.Apart from the mobilization of intracellular stored Caz*, the receptoractivated Ca2+ entry process is also of considerable importance upon histaminestimulation. As mentioned in the introduction (chapter 1.2), the mechanismunderlying the receptor activated Cat* influx is not established yet. but in mostcells it is different from the well documented voltage operated Ca2* channels.Atthough activation of receptor stimulated Caz' entry may be mediated viadifferent mechanisms, the capacitative Ca2+ entry, in which Ca2* entry is relatedto the filling state of the intracellular Car* stores and the formation of inositolphosphates, in particular Ins(1,3,4,5)Po, are considered to be the most importantmechanisms for activation of receptor operated Ca2* entry. The snrdy presentedin chapter 3 was carried out to identify the cellular component activating thehistamine stimulated Ca2* entry process in DDTr MF-2 cells. The resultsdemonstrated that the Caz* entry process was independent of the histamineactivated formation of the second messengers Ins(1,4,5)P3 or Ins(1,3,4,5)Po andwas still detected when depletion of intracellular Ca:] stores was prevented byheparin, thereby excluding activation of the capacitative or inositol phosphateinduced Ca2* entry. These results were therefore suggestive for anothercomponent involved in initiation of the H,-histaminoceptor activated Ca2+ entryprocess. It was observed that stirnulation with histamine resulted in a markedrelease of AA in DDT, MF-2 cells. This AA formation was biphasic in nature,consisting of a fast rise followed by a more sustained second phase.Exogeneously applied AA to these cells induced a concentration dependentincrease in intcrnal Ca} concentra(ion. due to activation of Car' entry from theextracellular space. This effect of AA was not mediated by its metabolites, sinceinhibition of lipoxygenase, cyclo-oxygenase or epoxygenase pathways did notaffect the AA induced Ca2" entry. Pre-activation of the Ca2* channels involvedby AA resulted in abolition of the histamine activated Ca2* entry, but not theinternal Ca2* mobilization. These observations howed that AA is functioning asa second Ínessenger to activate plasmambrane Ca:' channels, thereby promotingCa2n entry from the extracellular space in DDTr MF-2 cells, as is represented inFigure 1.Although several enzymatic pathways are reported to be responsible forthe mobilization of AA frorn nrembrane tipids, the most common pathway torelease receptor activated AA is via the action of phospholipase A2.Alternatively, PLC derived DAG can serve as a precursor for AA via activationof diglyceride lipase. Further, AA can be released from phosphatidic acid,derived frorn the hydrolysis of membrane lipids by PLD (chapter 1). In chapter 4the biochernical pathway involved in H,-histaminoceptor activated AA release ofDDT' MF-2 cells was studied. to understand the histzunine activated Ca2" entryprocess. For DD'[, MF 2 srnooth muscle cells it was demonstrated that thehistamine stimulated formation of AA was independent of dre PLA,, or PLDltil,d)eol! +ot.,rtht2 'of/asInndt''he)uS.edrels'rialve)ntIerotin.l - 'ithtseinIthto:edt - 'lse99activity, respectivery, but courd bc brocked by the pLC inhibitor neomycine.These results suggested that AA rerease occured through the action of pLC anddiglyceride lipase. In accord, it *u, iouno that inhibitioï "i;; digryceride lipaseactivity resulted in a marked accumuration of DAG, resurting in reduction of theAA release. Therefore, in .ont.uri fo'."c.pto. activation-in many other ceiltypes, the H,-histaminoceptor stirnurated rerease of AA in nór, MF_2 smoothmuscle cells is predominantly mediated via the sequentiar action of pLC anddiglyceride lipase (Figure t).Receptor activ-ated Ca2* signalring Tu.y be regulated by activation of pKC;a large famity of ubiquitousry Jistribuïed isozymá ra ir' reporteo that pKCmodulates channer activity *á plc *riui,y, resurting in the reduction of rheinositol phosphate format-ion and subsequ.ni curn ,.Ëor. rrl* int..r,al storesand may affect the recepror activated release gl AA (chapter l). In chapter 5, therote of pKC in the histamine stimuraiJ ca2* signàiil;-;.;á.rs, incruaing theÍbrmation of inositor phosphates unà-Àe ,.r.ur., ilr, ,tu"oiià. wrren the possibrerole of PKC regurati'g. cêilurar processes is considerJ,';;r; ro separare rwotypes of experimenrar^g:r.ign Fiist, exogeneous activation of pKC by rhe use ofphorbol esters like pMA, provides ,u'id"n." about the .*irr"n". of a pKCregulated feedback roop. Ho*eu"., to determine whether such a loop isphysiologicaily activated . upon- receptor stimuration, one has to rook at theconsequences of interruption of the ioop, which can be achieve<t by the use ofPKC inhibitors rike skurosporine or via^down reguration of pKC activity by rongrerïn prerreatment with,oMl . For DDT, MF-J cets it war shown that pKCactivation with pMA reduced the histamine stimurate<' formation of Ins(1,4,5)pr,Ins(1,3,4,5)p, and rhe concomio"i-.óui- mobirizariori ;;;; intenar srores.Moreover, the receptor acdvated Ca2n entry. was aborished, while the rerease ofAA was strongry reduced. Thus, these ."ruru show the .*irt.*. of a negativefeedback mechanism,berween .*og.n.árriy activare<I pKC and the fbrmation ofinositol phosphates, the subseque"ï *"uiiir"ion of ca2*, the release of AA aruithe ca2* entry. In contrast, inhibition of pKC activity 'ti' ,ruï.osporine or bylong-term pretreatmenr with "MA, ár,ry^un.ted the hisiamine activared ca2+entry, while the formation of inositor piosphates, trre ca2* -àoitiration o, tt.release of AA were not changed. *,esË lus..uations demonstrated that onry thefeedback circuir between pKó and a.r.-11q, was functionary activared uponiiËtsï:ïI;*.j';*'., resurting i  u pxc..ri*Àá i"tiuriiá" o, Caz+ enrryApparently different isoforms of pKC have to considered in these celrs, coupledto PLC activity, the release of AA *à ,r,. receptor ".ri""r"o ca2* entry,respectively. The exact nature of the pKC-isozymes remained to be identified.Thus, the results obtained from this and the previous studies, asrepresented by Figure l, indicate that stimulation of H,-nistaÁinàceptors on thesecells causes activation of pLC, resuttirt in hydrorysis of phosphatidyr inositol4'5 bisphosphate 'n,: ,1.t.ortarnin!' ËRc *à tr,. 'inárior phosphatesIns(1,4,5)P, and Ins(1,3,4,5)P1. The .à"tirJ phosphates uinc to *rei. receptors tor00mobilize Ca2n from intracellular stores and DAG is metabolized by diglyceridelipase to produce AA. In turr, AA activates Caz* channels in theplasmamembrane to allow Ca2* entry from the extracellular space. Besides itsfunctioning as a precursor for AA, the formation of DAG may serve to activateprotein kinase C (PKC). Since a functional inhibitory feedback loop betweenhistamine stimulated PKC and Car* entry, but not the formation of inositolphosphates and the release of AA was observed, an attractive hypothesis is thatreceptor stimulated DAG is first rapidly mekbolized to AA, thereby promotingCaz+ entry, which process is limited by a Ca2* sensitive PKC, activated by (asubsequent) release of DAG.In chapter 6, the effect of different nucleotides on the Ins(l,4,5)P,activated Ca2* release of saponin permeabilized cells was investigated. Althoughthe presence of ATP was not required to activate the Ins(l ,4,5)P3 induced Ca2*releasing process, the amount of Car* released by different concentrationsIns(I,4,5)P, was considerably enhanced upon addition of this nucleotide. Theamount of additional Ca2* released at a certain concentration of ATP wasindependent of the Ins( I ,4,5)P, concentration used and an Ins( I ,4,5)P,concentration-response curve with similar ECr,, values and other characteristics asobserved without ATP was obtained in the presence of ATP. Thus, even atmaximal effective Ins(1,4.5)P3, assumed to fully occupy the Ins(1,4,5)P.sensitive receptors and completely depleting the Ins(1,4,5)P, sensitive Ca2* store,the presence of ATP induced an additional release of CaZ*. The Pr"-purinoceptor agonist 2-methylthio-ATP and the non-hydrolysable ATP analogueATPTS enhanced the Ins(I,4,5)Pj activated Ca2* release to the same extent asATP. Less effective were GTP, ADP and AMP, while UTP and o,B-methyleneATP appeared to be ineffective. The rank order of potency was 2-methylthio-ATP : ATPTS : ATP > GTP > ADP > AMP ) a,B methylene ATP :UTP. These results demonstrated that the presence of these nucleotidesenhanced the total releasing capacity of the Ins(1,4,5)P, sensitive Ca2+ store viaan ATP sensitive binding site, without changing the affinity of the Ins(I,4,5)P,receptor for Ins(I,4.5)Pr. The mechanism by which this ATP activated bindingsite exerts its effect rnay be due by increasing the efficacy of the Ins(1,4,5)P,receptor. In view of the sensitivity for ATP, ATPTS and 2-methylthio ATP, itwas concluded that the effect observed is mediated by an internal binding sitewith comparable characteristics as an external Pr"-purinoceptor.Apart from the presence of an internal ATP sensitive binding site. intactDDTr MF-2 cells were previously described to possess external P2-purinoceptorslocated on the plasma membrane. Stimulation of these receptors with ATPcaused responses comparable to that obtained upon H,-histaminoceptorstimulation: a relative srnall increase in Ins I ,4,5)P, concentration and apronounced formation of Ins(1,3.4,5)P4 were accompanied by a rise in internalCa2* due to Ca2* mobilization from internal stores and Car* influx from theextracellular space. Furthermore, stimulation of these P"-purinoceptors resultedin a change in membrane current, consisting of a Ca'* dependent outward K*1 0 1kcurrent followed by a sustained, non-specit'ic inward current. The aim of theshrdy presented in chapter 7 was to characterize the A'l'P sensitive receptors ofDDTr MF-2 cells by investigating the cellular response to different nucleotides,as represented by the membrane current. It was found that these receptors couldnot be classified as P,-purinoceptors, since adenosine appeared to be inactive inmediating a change in membrane current. The cellular response was also notmediated via Pr.,.-, P,"-, and P,r-purinoceptors, since ADP, cv,B-methylene ATPand 2-methylthio-ATP, known to stimulate these receptors, respectively, did notchange the membrane current either. In contrast. it was observed that pyrimidinecontaining nucleotides, in particular UTP, caused a pronounced change inmembrane current with similar characteristics as observed with ATP. The rankorder of potency to evoke the response was ATP > LITP > TTP > CTP :GTP. The responses mediated by ATP and UTP were not additive, and could beblocked by the reversible Pr-purinoceptor antagonist suramin. In view of thesensitivity for purine as well as pyrimidine containing nucleotides, the externalATP sensitive receptor of DDT, MF-2 cells was characterized as a nucleotidereceptor (Pr,,-purinoceptor).In summary, this thesis described the receptor stimulated Caz* signallingprocess of DDT, MF-2 cells. It was shown that although Ins(l,4,5)P,,Ins(1,3,4,5)Po but no Ca2* sensitive Ca2* stores were present in these cells, theH,-histaminoceptor stimulated rise in internal Ca'* was predominantly derivedfrom Ins(l,3,4,5)P,r sensitive Ca2* stores.Furthermore, it was found that the histamine activated Ca2* entry process wasindependent of the state of depletion of internal stores or the fomration of inositolphosphates, but was activated by the release of AA generated by the orderedaction of PLC and diglyceride lipase. Inhibition of this Ca2* entry process wasshown to be mediated by a histunine activated PKC isozyme. Evidence about thepresence of PKC isozymes regulating PLC activity and AA release was alsoobtained, although involvement of these isozymes was not observed on histaminestimulation.The Ins(1,4,5)Pj activated Car* release was enhanced by the presence of adeninenucleotides via activation of an internal binding site. In particular ATP, ATPTSand the Pr"-purinoceptor agonist 2-methylthio-ATP considerably augmented theCa2* release induced by Ins(1.4,5)P3, without changing the affinity of theIns(1,4,5)P, receptor for Ins(1,4.5)Pj. On the other hamd, extracellular ATP wasshown to activate Ca2* signalling by interaction with a plasmamembrane locatednucleotide receptor, which was also sensitive for the pyrimidine containingnucleotide UTP. A model representing these receptor stimulated Ca2* signallingprocesses in DDT, MF-2 cells is presented in Figure 1.t02

English

   University of GroningenReceptor activated Ca2+ signalling processes in DDT1 MF-2 smooth muscle cellsZee, Lucie van derIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:1994Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Zee, L. V. D. (1994). Receptor activated Ca2+ signalling processes in DDT1 MF-2 smooth muscle cells.s.n.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 08-06-2022STIMMARYIn this thesis, the Ca2* signalling pathway of H,-histaminoceptor stimulatedDDT' MF-2 smooth muscle cells was investigated. Further, the ATP sensitivereceptor on the plasma membrane was characterized. In this last chapter, theresults of the previous chapters will be summarized and evaluated.Stimulation of certain receptor sites results in a pronounced enhancementof the intracellular Ca2* concentration, due to Ca2* mobilization from internalstores and Ca2+ entry from the extracellular space. The receptor activatedformation of inositol phosphates, in particular Ins(I,4,5)Pr, is assumed to beresponsible for the release of intracellular stored Ca2t. However, other inositolphosphates t lns(1.3.4.5)P4).  f te presence of GTP. or t}e Ca2* act ivated Ca: 'releasing process may also contribute to the rise in internal Car* upon receptorstimulation (chapter I t.Activation of H,-histaminoceptors of vas deferens derived DDTr MF-2smooth muscle cells resulted in a pronounced formation of Ins(1,3,4,5)P, witlrrespect to that of Ins(I,4,5)Pr, a rapid enhancement of the intracellular Car'concentration and a Ca2* dependent outward K* current. In chapter 2, the role ofthe inositol phosphates and the Ca2* induced Ca2* release mechanism wasinvestigated in penneabilized as well as histamine stimulated, intact, cells. Insaponin permeabilized DDTr MF-2 cells, it was found that both Ins(1,4,5)P, andIns( 1.3.4.5)Po sensit ive Ca2' s lores were preseut whi le a Ca" act ivated Car 'releasing mechanism could not be detected in this permeabilized cell systern. TheCal* releasing capacity of Ins(1,3,4,5)P4 was dependent on the simultaneouspresence of Ins(I,4,5)Pr, and the effect of both inositol phosphates was inhibitedby the Ins(l,4,5)P, receptor antagonist heparirr. Thus, the additional Car'released by Ins(1,3,4,5)P4 was mediated via Ins(1,4,5)Pr sensitive Car* channelsand it was concluded that the effect of Ins(1,3,4,5)P.t was mediated viaconnection of an Ins(1,4.5)Pr insensitive Car* store to the Ins(1,4,5)Pr sensitiveCa2* compartrnent.H,-histaminoceptor stimulation in intact cells induced a Car* dependentoutward K+ current, representing the intemal Ca2* release if Ca2* entry from theextracellular space was prevented. The histamine activated current was notdetected if the inositol phosphate induced Cart release was inhibited by heparin.This result indicated that both Ins(1,4,5)P. and Ins(1,3,4,5)P4 sensitive Car*stores could play a role in the rise in intennl Caz* upon stirnulation withhistamine and excluded Ltre contribution of the Ca2* induced Caz* releasemechanism to this process. Depletion of Ins(1,4,5)P. sensitive Car* stores inadvance reduced the K* current only to some extent. Flowever, when bothIns(1,4,5)P, and Ins(1.3,4,5)P4 sensit ive Cat* stores were depleted pr ior tohistamine stimulation, the Car* sensitive K* current could not be detectedanymore. These results denronstrated that Ins(1,3.4,5)Po sensitive Car*compar0nents were mainly responsible for the histarnine activated Ca2* release98Ifiom internal stores thereby showing the importance of Ins(1,3,4,5)P4 in the Ca2*releasing process of H,-histaminoceptor activated DDTr MF-2 cells, as depictedin Figure l.Apart from the mobilization of intracellular stored Caz*, the receptoractivated Ca2+ entry process is also of considerable importance upon histaminestimulation. As mentioned in the introduction (chapter 1.2), the mechanismunderlying the receptor activated Cat* influx is not established yet. but in mostcells it is different from the well documented voltage operated Ca2* channels.Atthough activation of receptor stimulated Caz' entry may be mediated viadifferent mechanisms, the capacitative Ca2+ entry, in which Ca2* entry is relatedto the filling state of the intracellular Car* stores and the formation of inositolphosphates, in particular Ins(1,3,4,5)Po, are considered to be the most importantmechanisms for activation of receptor operated Ca2* entry. The snrdy presentedin chapter 3 was carried out to identify the cellular component activating thehistamine stimulated Ca2* entry process in DDTr MF-2 cells. The resultsdemonstrated that the Caz* entry process was independent of the histamineactivated formation of the second messengers Ins(1,4,5)P3 or Ins(1,3,4,5)Po andwas still detected when depletion of intracellular Ca:] stores was prevented byheparin, thereby excluding activation of the capacitative or inositol phosphateinduced Ca2* entry. These results were therefore suggestive for anothercomponent involved in initiation of the H,-histaminoceptor activated Ca2+ entryprocess. It was observed that stirnulation with histamine resulted in a markedrelease of AA in DDT, MF-2 cells. This AA formation was biphasic in nature,consisting of a fast rise followed by a more sustained second phase.Exogeneously applied AA to these cells induced a concentration dependentincrease in intcrnal Ca} concentra(ion. due to activation of Car' entry from theextracellular space. This effect of AA was not mediated by its metabolites, sinceinhibition of lipoxygenase, cyclo-oxygenase or epoxygenase pathways did notaffect the AA induced Ca2" entry. Pre-activation of the Ca2* channels involvedby AA resulted in abolition of the histamine activated Ca2* entry, but not theinternal Ca2* mobilization. These observations howed that AA is functioning asa second Ínessenger to activate plasmambrane Ca:' channels, thereby promotingCa2n entry from the extracellular space in DDTr MF-2 cells, as is represented inFigure 1.Although several enzymatic pathways are reported to be responsible forthe mobilization of AA frorn nrembrane tipids, the most common pathway torelease receptor activated AA is via the action of phospholipase A2.Alternatively, PLC derived DAG can serve as a precursor for AA via activationof diglyceride lipase. Further, AA can be released from phosphatidic acid,derived frorn the hydrolysis of membrane lipids by PLD (chapter 1). In chapter 4the biochernical pathway involved in H,-histaminoceptor activated AA release ofDDT' MF-2 cells was studied. to understand the histzunine activated Ca2" entryprocess. For DD'[, MF 2 srnooth muscle cells it was demonstrated that thehistamine stimulated formation of AA was independent of dre PLA,, or PLDltil,d)eol! +ot.,rtht2 'of/asInndt''he)uS.edrels'rialve)ntIerotin.l - 'ithtseinIthto:edt - 'lse99activity, respectivery, but courd bc brocked by the pLC inhibitor neomycine.These results suggested that AA rerease occured through the action of pLC anddiglyceride lipase. In accord, it *u, iouno that inhibitioï "i;; digryceride lipaseactivity resulted in a marked accumuration of DAG, resurting in reduction of theAA release. Therefore, in .ont.uri fo'."c.pto. activation-in many other ceiltypes, the H,-histaminoceptor stirnurated rerease of AA in nór, MF_2 smoothmuscle cells is predominantly mediated via the sequentiar action of pLC anddiglyceride lipase (Figure t).Receptor activ-ated Ca2* signalring Tu.y be regulated by activation of pKC;a large famity of ubiquitousry Jistribuïed isozymá ra ir' reporteo that pKCmodulates channer activity *á plc *riui,y, resurting in the reduction of rheinositol phosphate format-ion and subsequ.ni curn ,.Ëor. rrl* int..r,al storesand may affect the recepror activated release gl AA (chapter l). In chapter 5, therote of pKC in the histamine stimuraiJ ca2* signàiil;-;.;á.rs, incruaing theÍbrmation of inositor phosphates unà-Àe ,.r.ur., ilr, ,tu"oiià. wrren the possibrerole of PKC regurati'g. cêilurar processes is considerJ,';;r; ro separare rwotypes of experimenrar^g:r.ign Fiist, exogeneous activation of pKC by rhe use ofphorbol esters like pMA, provides ,u'id"n." about the .*irr"n". of a pKCregulated feedback roop. Ho*eu"., to determine whether such a loop isphysiologicaily activated . upon- receptor stimuration, one has to rook at theconsequences of interruption of the ioop, which can be achieve<t by the use ofPKC inhibitors rike skurosporine or via^down reguration of pKC activity by rongrerïn prerreatment with,oMl . For DDT, MF-J cets it war shown that pKCactivation with pMA reduced the histamine stimurate<' formation of Ins(1,4,5)pr,Ins(1,3,4,5)p, and rhe concomio"i-.óui- mobirizariori ;;;; intenar srores.Moreover, the receptor acdvated Ca2n entry. was aborished, while the rerease ofAA was strongry reduced. Thus, these ."ruru show the .*irt.*. of a negativefeedback mechanism,berween .*og.n.árriy activare<I pKC and the fbrmation ofinositol phosphates, the subseque"ï *"uiiir"ion of ca2*, the release of AA aruithe ca2* entry. In contrast, inhibition of pKC activity 'ti' ,ruï.osporine or bylong-term pretreatmenr with "MA, ár,ry^un.ted the hisiamine activared ca2+entry, while the formation of inositor piosphates, trre ca2* -àoitiration o, tt.release of AA were not changed. *,esË lus..uations demonstrated that onry thefeedback circuir between pKó and a.r.-11q, was functionary activared uponiiËtsï:ïI;*.j';*'., resurting i  u pxc..ri*Àá i"tiuriiá" o, Caz+ enrryApparently different isoforms of pKC have to considered in these celrs, coupledto PLC activity, the release of AA *à ,r,. receptor ".ri""r"o ca2* entry,respectively. The exact nature of the pKC-isozymes remained to be identified.Thus, the results obtained from this and the previous studies, asrepresented by Figure l, indicate that stimulation of H,-nistaÁinàceptors on thesecells causes activation of pLC, resuttirt in hydrorysis of phosphatidyr inositol4'5 bisphosphate 'n,: ,1.t.ortarnin!' ËRc *à tr,. 'inárior phosphatesIns(1,4,5)P, and Ins(1,3,4,5)P1. The .à"tirJ phosphates uinc to *rei. receptors tor00mobilize Ca2n from intracellular stores and DAG is metabolized by diglyceridelipase to produce AA. In turr, AA activates Caz* channels in theplasmamembrane to allow Ca2* entry from the extracellular space. Besides itsfunctioning as a precursor for AA, the formation of DAG may serve to activateprotein kinase C (PKC). Since a functional inhibitory feedback loop betweenhistamine stimulated PKC and Car* entry, but not the formation of inositolphosphates and the release of AA was observed, an attractive hypothesis is thatreceptor stimulated DAG is first rapidly mekbolized to AA, thereby promotingCaz+ entry, which process is limited by a Ca2* sensitive PKC, activated by (asubsequent) release of DAG.In chapter 6, the effect of different nucleotides on the Ins(l,4,5)P,activated Ca2* release of saponin permeabilized cells was investigated. Althoughthe presence of ATP was not required to activate the Ins(l ,4,5)P3 induced Ca2*releasing process, the amount of Car* released by different concentrationsIns(I,4,5)P, was considerably enhanced upon addition of this nucleotide. Theamount of additional Ca2* released at a certain concentration of ATP wasindependent of the Ins( I ,4,5)P, concentration used and an Ins( I ,4,5)P,concentration-response curve with similar ECr,, values and other characteristics asobserved without ATP was obtained in the presence of ATP. Thus, even atmaximal effective Ins(1,4.5)P3, assumed to fully occupy the Ins(1,4,5)P.sensitive receptors and completely depleting the Ins(1,4,5)P, sensitive Ca2* store,the presence of ATP induced an additional release of CaZ*. The Pr"-purinoceptor agonist 2-methylthio-ATP and the non-hydrolysable ATP analogueATPTS enhanced the Ins(I,4,5)Pj activated Ca2* release to the same extent asATP. Less effective were GTP, ADP and AMP, while UTP and o,B-methyleneATP appeared to be ineffective. The rank order of potency was 2-methylthio-ATP : ATPTS : ATP > GTP > ADP > AMP ) a,B methylene ATP :UTP. These results demonstrated that the presence of these nucleotidesenhanced the total releasing capacity of the Ins(1,4,5)P, sensitive Ca2+ store viaan ATP sensitive binding site, without changing the affinity of the Ins(I,4,5)P,receptor for Ins(I,4.5)Pr. The mechanism by which this ATP activated bindingsite exerts its effect rnay be due by increasing the efficacy of the Ins(1,4,5)P,receptor. In view of the sensitivity for ATP, ATPTS and 2-methylthio ATP, itwas concluded that the effect observed is mediated by an internal binding sitewith comparable characteristics as an external Pr"-purinoceptor.Apart from the presence of an internal ATP sensitive binding site. intactDDTr MF-2 cells were previously described to possess external P2-purinoceptorslocated on the plasma membrane. Stimulation of these receptors with ATPcaused responses comparable to that obtained upon H,-histaminoceptorstimulation: a relative srnall increase in Ins I ,4,5)P, concentration and apronounced formation of Ins(1,3.4,5)P4 were accompanied by a rise in internalCa2* due to Ca2* mobilization from internal stores and Car* influx from theextracellular space. Furthermore, stimulation of these P"-purinoceptors resultedin a change in membrane current, consisting of a Ca'* dependent outward K*1 0 1kcurrent followed by a sustained, non-specit'ic inward current. The aim of theshrdy presented in chapter 7 was to characterize the A'l'P sensitive receptors ofDDTr MF-2 cells by investigating the cellular response to different nucleotides,as represented by the membrane current. It was found that these receptors couldnot be classified as P,-purinoceptors, since adenosine appeared to be inactive inmediating a change in membrane current. The cellular response was also notmediated via Pr.,.-, P,"-, and P,r-purinoceptors, since ADP, cv,B-methylene ATPand 2-methylthio-ATP, known to stimulate these receptors, respectively, did notchange the membrane current either. In contrast. it was observed that pyrimidinecontaining nucleotides, in particular UTP, caused a pronounced change inmembrane current with similar characteristics as observed with ATP. The rankorder of potency to evoke the response was ATP > LITP > TTP > CTP :GTP. The responses mediated by ATP and UTP were not additive, and could beblocked by the reversible Pr-purinoceptor antagonist suramin. In view of thesensitivity for purine as well as pyrimidine containing nucleotides, the externalATP sensitive receptor of DDT, MF-2 cells was characterized as a nucleotidereceptor (Pr,,-purinoceptor).In summary, this thesis described the receptor stimulated Caz* signallingprocess of DDT, MF-2 cells. It was shown that although Ins(l,4,5)P,,Ins(1,3,4,5)Po but no Ca2* sensitive Ca2* stores were present in these cells, theH,-histaminoceptor stimulated rise in internal Ca'* was predominantly derivedfrom Ins(l,3,4,5)P,r sensitive Ca2* stores.Furthermore, it was found that the histamine activated Ca2* entry process wasindependent of the state of depletion of internal stores or the fomration of inositolphosphates, but was activated by the release of AA generated by the orderedaction of PLC and diglyceride lipase. Inhibition of this Ca2* entry process wasshown to be mediated by a histunine activated PKC isozyme. Evidence about thepresence of PKC isozymes regulating PLC activity and AA release was alsoobtained, although involvement of these isozymes was not observed on histaminestimulation.The Ins(1,4,5)Pj activated Car* release was enhanced by the presence of adeninenucleotides via activation of an internal binding site. In particular ATP, ATPTSand the Pr"-purinoceptor agonist 2-methylthio-ATP considerably augmented theCa2* release induced by Ins(1.4,5)P3, without changing the affinity of theIns(1,4,5)P, receptor for Ins(1,4.5)Pj. On the other hamd, extracellular ATP wasshown to activate Ca2* signalling by interaction with a plasmamembrane locatednucleotide receptor, which was also sensitive for the pyrimidine containingnucleotide UTP. A model representing these receptor stimulated Ca2* signallingprocesses in DDT, MF-2 cells is presented in Figure 1.t02

Receptor activated Ca2+ signalling processes in DDT1 MF-2 smooth muscle cells

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