- Email: [email protected]
Lung surfactant protein A (SP-A) interaction with alveolar macrophages
Cell Biology International
Reports, Vol. 14, Abstracts Supplement
ASSTFIACT: “INVOLVEMENT OF TWO MECHANISMS IN THE PDGF INDUCED UPREGUIATION OF IL10 RECEPTOR.” Glorgio Ferratf and Domenico Rotilio. lstituto di Ricerche Famlogiihe Mario Negri, Consorzio Mario Negri Sud, via Nazionale. 68030 S. Maria lmbaro (Chieti). Biochemical events elicited by intetfeukin 1 I3 (ILlO) in 3T3 NIH cells are mediated by a specific transmsmbrane receptor. The IL1 Oreceptor complex formation triggers phosphorylation of protein substrates and the activation of second messenger mediating nuclear events. lt is known that an early event induced by PDGF is the modification of actin mlcrottlaments and the activation of PKC. Pretreatment with PDGF of 3T3 NIH results in a rapid increase of lLll3 binding sites (after 5-10 min. PDGF treatment). Phorbol ester (PMA) partially mimiced the PDGF early effect on IL1 I3 receptor. Desensitization of PKC. (18 h treatment with PMA), or addition of a specific PKC inhibitor (staurosporfne) resulted in a loss of this early effect. The sams was obtained with the depolymetfzation of actin microfilaments caused by cylochalasin 6 (30 min. pretreament). When exposure to PDGF was longer than 2 h, the 125l-ILIB binding to 3T3 NIH cells was further increased. Such delayed effect was inhib4ted by cicloheximids and actinomycin D with no ef?ecf on the early event, suggesting a neosynthesis of receptors. The use of Scatchatd plot analysis indicates a 30% increase in the number of the cellular binding sites without any change in the affinity of IL1 I3for its receptor (300pM). The functional involvement of the induced receptor was tested by assaying mitogenic activity in long term pulse of 3H-Thimidine and prostaglandin production. The results showed a 200% increase of ILlI induced growth response and a 2-3 fold increase in PGEP production in respect to controls. We can thus propose that PDGF increases some biobgiial effects due to lLll3 through the induction or better availability of specific receptors. Such rearrangement may be mediated by PKC and actin microfilament redistributiin as early event, and by neosynthesis of IL1 8 receptors as delayed event.
P241
~243
1990
139
DOPAMINE MODULATION OF ION CURRENTS IN CHROMAFFIN CELLS Jean-Marie Sontae. *Pamela Sanderson, *Michel Kletmer , Dominique Am&&Kenneth Takeda & Marie-France dr;der, INSERM U-44 & *URA 600 du CNRS, STRASBOURG, FRANCE. The presence of D2 dopaminergic receptors has been characterized in adrenal chromaffin cells. In order to elucidate the mechanisms by which dopaminergic agonists inhibit catecholamine release in these cells, we have analyzed the effect of D2 agonists on Ca2+ and Na+ currents evoked by different secretagogues.Catecholamine secretion and 45Ca2+ influx were similarly inhibited and whole-cell Ca2+currents were reduced by apomorphine treatment. These inhibitory effects of D2 agonists depended on the secretagogue used, being much more pronounced for nicotine-evoked responses compared to high K+ stimulation, indicating another possible site of action of apomorphine upstream of Ca2+ entry. We examined the possibility that apomorphine might modulate voltage-dependent sodium channels and/or nicotinic receptor associatedchannels. Action potentials recorded in whole-cell current clamp were blocked by apomorphme when they were triggered by nicotinic depolarization but not by direct electrical stimulation. Apomorphine also caused reductions of inward whole-cell nicotinic current evoked by acetylcholine and nicotine. Inhibition of nicotine-evoked secretion and 22Na+ influx by apomorphine were not affected by tetrodotoxin, and voltage-dependent, whole-cell Na+ currents were unaltered by apomorphine. These results suggest that apomorphine inhibits catecholamine secretion from chromaffin cells by reducing calcium entry through voltage-gated calcium channels and also by decreasing current through nicotinic receptor associatedchannels.
P242
~~msa$ct~h~~e
(SP,;“’ inter Phag Jutta Schlepper - Schiifer, Heiderose Manz - Keinke, Christine Egcn hofer, Helmut Plattner, Faculty of Biology, University of Konstanz, POX: 5560, D - 7750 Konstanz, Federal Republic of Germany SP-A is the main protein component of hmg surfactant, which lines the alveolar surface and maintains lung stability. We tested the interaction of SP - A with rat alveolar macrophages (hi+). Glyco syiated recombiit human SP - A (Mw: 32 - 36 kDa) was from Dr. K. P. Schjifer (Byk Gulden Pharmazeutika, Konstanz, Fed. Rep. Germany). MQ were freshly isolated by lung lavage. SP - A was ad sorbed onto 5, 10 or 17 nm gold partides (SP - A - Au) and incubated with MI$ at 37’ C for different time periods. The uptake ’ ‘on electron microscopy. SP- A - Au route was studied by traDSrmSSl binding and uptake follows the coated pit/vesicle pathway and par tides are transported to secondary lysosomes. Bindii and uptake of SP - A is dependent on Ca++, as shown by inhibition with EGTA/Mg++. SP -A-Au uptake is inhiited by an excess of SPA as well as by mannosyl bovine serum albumin (ManeSA) but not by gaJactosyl BSA or BSA. Mannose dependency of interaction has recently heen shown with SP - A - Au and human monocytes and macropbages (1). Preincubation of MI$ with ManBSA - Au for 10 min at 37 C reduced biding of ManBSA - Au but not binding of SP A-Au to the cell surface of the MQ. Therefore, Man specific re ceptor 011the cdl surface of the MI$I seems not to be involved in SP-A-AU binding. SP-A binding to M$ was also studied in the light microscope with FITC labellcd SP - A as well as with uniabclled SP -A visualized subsequently with anti-SP -A antiserum from rabbit (Byk Gulden Pharmazutika) and protein A-FITC. Specilic binding of SP - A and its dependency on Ca*+ ions thus could be. demonstrated. SP - A - FITC binding was inhibited completely by an excess of SP - A but not by ManBSA or by Clq. We condude that SP-A interacts specitically with the cell surface of alveolar macrophages and that interaction of SPA-Au includes mannosc spccitic recognition. Supported by a grant of the DFG and by Byk Guldcn Pbarmazeutika,Konsta. (1) E. Wintergerst, H. Manz - Kciie, H. Plattner and J. Schlepper -Schtier (1989) Eur. J. Cell Biol. 50, 291-298.
Icsg-tim kale marrow dzmgeaftermiceirradiaticn~bsl~ tentforapmlmgedperiod andbe reflectedintme marrow cell flmticm. h%5kraw tramfeminmzeptors play anessential roleinthe tmrqmrtofim into the cells. Ircnmtakmlimis iqmrtznt becausetheEarelrariy ir-m-cmtaininpproteinsand~~~ ssaries to the cell fimkicn. The aim of the present vmk was to sb.@ the effect of 5 Gy irradiaticncntransferrinreceptcosalcng~yearpostirradiaticnpericxl. lhis &&has kenperfomedin tw cellularpqx+ latims: txxe rl?armvcellsand~ccytes&tainedfmlcs7gtermcul~,establishedac~~toDexter~~~s.~f~ r-rinbindingassays havebeen camiedoutusing I-ix.snsferrin. Irlbcne IEUTW cells of atit mice (3 months old) data are in agnmnsntwithamdelintichtwo diffemtzscciaticncm+ tants (highandlm~affinity) cculdbe defined. Hwever, inmice 15 mths old, cnly cne asscciaticn ccnstant is cixerved which correspond to those of hi& affinity. Atone yearpastirradiatim (15 mmths old) an unique asscciatim cmstant is present in bone IBTW cells which has a value hi&er than those of sgwmtchedccntxwlanimls.Fkthem~~,a dezxzemimnsfeminbin dingcapacityparalleltoageingis afealm-eckserved inmt&Z andi.madiatimice. Tramferrinbindingcapxityhasbeenobservedingmnulocytes beirgsligbtlyhigher incells fmirmdiatedmiceUanccntm1 Ones. The imxmsed value in the association constant inbmemrruv cellsandin~~~stsansferrinb~capacityasa~querxe ofimadiaticm auld be related to praricus results (1.2) aboutthehi&r r&akolic activit;y in these cells. l.-CXienllasetal(1989)XIXthMeetingFEK,Rane.Abs.J)l. Z.- cLienl.lsa et al (1929) Exp. Hemtol. 17, 529.
Reports, Vol. 14, Abstracts Supplement
ASSTFIACT: “INVOLVEMENT OF TWO MECHANISMS IN THE PDGF INDUCED UPREGUIATION OF IL10 RECEPTOR.” Glorgio Ferratf and Domenico Rotilio. lstituto di Ricerche Famlogiihe Mario Negri, Consorzio Mario Negri Sud, via Nazionale. 68030 S. Maria lmbaro (Chieti). Biochemical events elicited by intetfeukin 1 I3 (ILlO) in 3T3 NIH cells are mediated by a specific transmsmbrane receptor. The IL1 Oreceptor complex formation triggers phosphorylation of protein substrates and the activation of second messenger mediating nuclear events. lt is known that an early event induced by PDGF is the modification of actin mlcrottlaments and the activation of PKC. Pretreatment with PDGF of 3T3 NIH results in a rapid increase of lLll3 binding sites (after 5-10 min. PDGF treatment). Phorbol ester (PMA) partially mimiced the PDGF early effect on IL1 I3 receptor. Desensitization of PKC. (18 h treatment with PMA), or addition of a specific PKC inhibitor (staurosporfne) resulted in a loss of this early effect. The sams was obtained with the depolymetfzation of actin microfilaments caused by cylochalasin 6 (30 min. pretreament). When exposure to PDGF was longer than 2 h, the 125l-ILIB binding to 3T3 NIH cells was further increased. Such delayed effect was inhib4ted by cicloheximids and actinomycin D with no ef?ecf on the early event, suggesting a neosynthesis of receptors. The use of Scatchatd plot analysis indicates a 30% increase in the number of the cellular binding sites without any change in the affinity of IL1 I3for its receptor (300pM). The functional involvement of the induced receptor was tested by assaying mitogenic activity in long term pulse of 3H-Thimidine and prostaglandin production. The results showed a 200% increase of ILlI induced growth response and a 2-3 fold increase in PGEP production in respect to controls. We can thus propose that PDGF increases some biobgiial effects due to lLll3 through the induction or better availability of specific receptors. Such rearrangement may be mediated by PKC and actin microfilament redistributiin as early event, and by neosynthesis of IL1 8 receptors as delayed event.
P241
~243
1990
139
DOPAMINE MODULATION OF ION CURRENTS IN CHROMAFFIN CELLS Jean-Marie Sontae. *Pamela Sanderson, *Michel Kletmer , Dominique Am&&Kenneth Takeda & Marie-France dr;der, INSERM U-44 & *URA 600 du CNRS, STRASBOURG, FRANCE. The presence of D2 dopaminergic receptors has been characterized in adrenal chromaffin cells. In order to elucidate the mechanisms by which dopaminergic agonists inhibit catecholamine release in these cells, we have analyzed the effect of D2 agonists on Ca2+ and Na+ currents evoked by different secretagogues.Catecholamine secretion and 45Ca2+ influx were similarly inhibited and whole-cell Ca2+currents were reduced by apomorphine treatment. These inhibitory effects of D2 agonists depended on the secretagogue used, being much more pronounced for nicotine-evoked responses compared to high K+ stimulation, indicating another possible site of action of apomorphine upstream of Ca2+ entry. We examined the possibility that apomorphine might modulate voltage-dependent sodium channels and/or nicotinic receptor associatedchannels. Action potentials recorded in whole-cell current clamp were blocked by apomorphme when they were triggered by nicotinic depolarization but not by direct electrical stimulation. Apomorphine also caused reductions of inward whole-cell nicotinic current evoked by acetylcholine and nicotine. Inhibition of nicotine-evoked secretion and 22Na+ influx by apomorphine were not affected by tetrodotoxin, and voltage-dependent, whole-cell Na+ currents were unaltered by apomorphine. These results suggest that apomorphine inhibits catecholamine secretion from chromaffin cells by reducing calcium entry through voltage-gated calcium channels and also by decreasing current through nicotinic receptor associatedchannels.
P242
~~msa$ct~h~~e
(SP,;“’ inter Phag Jutta Schlepper - Schiifer, Heiderose Manz - Keinke, Christine Egcn hofer, Helmut Plattner, Faculty of Biology, University of Konstanz, POX: 5560, D - 7750 Konstanz, Federal Republic of Germany SP-A is the main protein component of hmg surfactant, which lines the alveolar surface and maintains lung stability. We tested the interaction of SP - A with rat alveolar macrophages (hi+). Glyco syiated recombiit human SP - A (Mw: 32 - 36 kDa) was from Dr. K. P. Schjifer (Byk Gulden Pharmazeutika, Konstanz, Fed. Rep. Germany). MQ were freshly isolated by lung lavage. SP - A was ad sorbed onto 5, 10 or 17 nm gold partides (SP - A - Au) and incubated with MI$ at 37’ C for different time periods. The uptake ’ ‘on electron microscopy. SP- A - Au route was studied by traDSrmSSl binding and uptake follows the coated pit/vesicle pathway and par tides are transported to secondary lysosomes. Bindii and uptake of SP - A is dependent on Ca++, as shown by inhibition with EGTA/Mg++. SP -A-Au uptake is inhiited by an excess of SPA as well as by mannosyl bovine serum albumin (ManeSA) but not by gaJactosyl BSA or BSA. Mannose dependency of interaction has recently heen shown with SP - A - Au and human monocytes and macropbages (1). Preincubation of MI$ with ManBSA - Au for 10 min at 37 C reduced biding of ManBSA - Au but not binding of SP A-Au to the cell surface of the MQ. Therefore, Man specific re ceptor 011the cdl surface of the MI$I seems not to be involved in SP-A-AU binding. SP-A binding to M$ was also studied in the light microscope with FITC labellcd SP - A as well as with uniabclled SP -A visualized subsequently with anti-SP -A antiserum from rabbit (Byk Gulden Pharmazutika) and protein A-FITC. Specilic binding of SP - A and its dependency on Ca*+ ions thus could be. demonstrated. SP - A - FITC binding was inhibited completely by an excess of SP - A but not by ManBSA or by Clq. We condude that SP-A interacts specitically with the cell surface of alveolar macrophages and that interaction of SPA-Au includes mannosc spccitic recognition. Supported by a grant of the DFG and by Byk Guldcn Pbarmazeutika,Konsta. (1) E. Wintergerst, H. Manz - Kciie, H. Plattner and J. Schlepper -Schtier (1989) Eur. J. Cell Biol. 50, 291-298.
Icsg-tim kale marrow dzmgeaftermiceirradiaticn~bsl~ tentforapmlmgedperiod andbe reflectedintme marrow cell flmticm. h%5kraw tramfeminmzeptors play anessential roleinthe tmrqmrtofim into the cells. Ircnmtakmlimis iqmrtznt becausetheEarelrariy ir-m-cmtaininpproteinsand~~~ ssaries to the cell fimkicn. The aim of the present vmk was to sb.@ the effect of 5 Gy irradiaticncntransferrinreceptcosalcng~yearpostirradiaticnpericxl. lhis &&has kenperfomedin tw cellularpqx+ latims: txxe rl?armvcellsand~ccytes&tainedfmlcs7gtermcul~,establishedac~~toDexter~~~s.~f~ r-rinbindingassays havebeen camiedoutusing I-ix.snsferrin. Irlbcne IEUTW cells of atit mice (3 months old) data are in agnmnsntwithamdelintichtwo diffemtzscciaticncm+ tants (highandlm~affinity) cculdbe defined. Hwever, inmice 15 mths old, cnly cne asscciaticn ccnstant is cixerved which correspond to those of hi& affinity. Atone yearpastirradiatim (15 mmths old) an unique asscciatim cmstant is present in bone IBTW cells which has a value hi&er than those of sgwmtchedccntxwlanimls.Fkthem~~,a dezxzemimnsfeminbin dingcapacityparalleltoageingis afealm-eckserved inmt&Z andi.madiatimice. Tramferrinbindingcapxityhasbeenobservedingmnulocytes beirgsligbtlyhigher incells fmirmdiatedmiceUanccntm1 Ones. The imxmsed value in the association constant inbmemrruv cellsandin~~~stsansferrinb~capacityasa~querxe ofimadiaticm auld be related to praricus results (1.2) aboutthehi&r r&akolic activit;y in these cells. l.-CXienllasetal(1989)XIXthMeetingFEK,Rane.Abs.J)l. Z.- cLienl.lsa et al (1929) Exp. Hemtol. 17, 529.