- Email: [email protected]
K+-ATPase isolated from human placenta of insulin-dependent pregnant women: A biochemical study
,ll~.~tra~t~" E P G and R T C. Belgtum 1995 NA+/K+-ATPASE ISOLATED FROM HUMAN PLACENTA OF INSULIN-DEPENDENT PREGNANT WOMEN: A BIOCHEMICAL STUDY.
N. Cester, 1 R. Staffolani2 (3. Zolese2, L. Mazz.antl2, Obstetrics 1 and Biochemistry Institute 2 Faculty of Medicine Ancona Umversity Ancona, Italy Na+/K+-ATPase ts the membrane-bound enzyme catalyzing the active transport of Na+ and K+ across the plasma membrane of ammal cells and spectfically inhibited by cardiac glycosides A reduced act=vity of Na+/K+-ATPase has been described in Insuhn-Dependent (IDDM) and =n Non Insuhn-Dependent (NIDDM) Diabetes Mellitus in a variety of cellular types The causes of the reduced acbwty of Na+/K+-ATPase In human dmbetes are sbll the object of controversms. The aim of this work was to investigate the mechamsm of =nh=blt=on using the Na+/K+-ATPase obtained from human placenta. We purified Na+/K+ATPase from term placentas of 6 healthy women and 6 age-matched IDDM women in good metabohc control The enzymatic achv=ty was reduced both in the mtcrosomal fraction and tn the Na+/K+-ATPase obtained from d=abetic women tissue, while no difference was found in the number of active molecules determined by anthroyl ouabam binding The Na+/K+-ATPase purified from IDDM women dtd not show any modification In the ouabain affinity or change rn the physlcochemlcal structure of the ouabain binding site investigated by dynam=c fluorescence nor alterataon =n lateral diffusions. The activatton energy above the trans~tzon temperature (being 15 3+0 9 kCal/mol for healthy women and 19 1+1 0 kCal/mol for IDDM) and the tryptophan accessibility (being 0 72_+004 for healthy and 0.98__.005 for IDDM) of the enzyme were increased m 1(3DMwomen The fluidity of the lipJds annulus of the enzyme was higher In IDDM than m control women as suggested by fluorescence polarization (p parameter) of 1-(4-tnmethyl-amlno-phenyl)-6-phenyl-1 3,5hexatnene (TMA-DPPI) (0 293±0 013 m the control and 0 223_+0 15 m IDDM). The present data indicate that placental Na+/K+-ATPase show subconformabonal changes in IDDM and suggest a relevant role of the lipid enwronment in this modtficatlon and that the hypothetLc inhtbitor must be bound to the complex protein-hpld annulus or cause a permanent modification of this complex. The present work was supported by a Grant of Regione Marche to N C. and of CNR to LM
IDENTIFICATION AND CHARACTERIZATION OF SOLUBLE VARIANTS OF VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) RECEPTORS IN THE HUMAN PLACENTA D. Stephen Chamock-Jones, Andrew M. Sharkey, Kate Day and Stephen K. Smith Reproductive Molecular Molecular Research Group. Department of Ob/Gyn University of Cambridge. The Rosle Matermty Hospttal. Cambridge. CB2 2SW Vascular endothehal growth factor (VEGF) is a potent stimulator of angiogenesis both in vwo and in wtro. It is also a potent reducer of increased vascular permeability. These actions are all mediated by endothelial cells and until recently the receptors for this factor were known to be localized solely on these cells The process of angmgenesis is clearly essential for placental development; it is therefore not surprising that the receptors for VEGF (both fit and KDR) are found in the placenta. However we have shown that these receptors are not restricted to the endothelial cells as might be expected. They are also highly expressed on the trophoblast cells themselves (Charnock-Jones et.al 1994). It is therefore probable that VEGF not only plays a role m endothelial development within the placenta but may also directly affect the trophoblast. Soluble variants of a vanety of tyrosine kinase receptors have been described and indeed variants of the fit receptor known to exist (Kendall et al 1993). We have also identified mRNAs encoding soluble fit receptors within ovarian carcinoma cell hnes (Boocock et al 1995). Soluble receptors are presumed to act as antagomsts for their hgand so we sought to determine whether there were soluble VEGF receptors present m the human placenta Therefore we c a m e d out northern blotting and PCR analysis of mRNA derived from human placenta Subsequent cloning revealed that the previously described variant (Kendall et al 1993) was present at h~gh levels m the placenta There were also several other variants which would be predicted to encode soluble VEGF receptors. Since the soluble receptors contain varying amounts of the extra cellular hgand binding domain of the classical receptor :t ts likely that they have different b~ochem~cal propemes Each of these may be VEGF antagomsts suggesting here is a complex mechanism for regulating the effects of VEGF both on endothehal cells and on trophoblast. This may be a fundamental tmportance in the regulation of vascular development and trophoblast functmn within the human placenta. D Stephen Chamock-Jones, Andrew M Sharkey, Christine A. Boocock, Aslf Ahmed, Robin Plevin, Napoleone Ferrara and Stephen K. Smith (1994). Btol Reprod 51 524-530 Kendall R.L and Thomas K A. (1993) Proc Natl Acad.Scl USA 90 (10705-10709) Christine A Boocock, D Stephen Charnock-Jones, Andrew M. Sharkey, John McLaren, Pat Barker, Karen Wright, Peter Twentyman and Stephen K Smtth. (1995)J.Natl.Cancer Inst. 87 (7)506-516
A.11
N. Cester, 1 R. Staffolani2 (3. Zolese2, L. Mazz.antl2, Obstetrics 1 and Biochemistry Institute 2 Faculty of Medicine Ancona Umversity Ancona, Italy Na+/K+-ATPase ts the membrane-bound enzyme catalyzing the active transport of Na+ and K+ across the plasma membrane of ammal cells and spectfically inhibited by cardiac glycosides A reduced act=vity of Na+/K+-ATPase has been described in Insuhn-Dependent (IDDM) and =n Non Insuhn-Dependent (NIDDM) Diabetes Mellitus in a variety of cellular types The causes of the reduced acbwty of Na+/K+-ATPase In human dmbetes are sbll the object of controversms. The aim of this work was to investigate the mechamsm of =nh=blt=on using the Na+/K+-ATPase obtained from human placenta. We purified Na+/K+ATPase from term placentas of 6 healthy women and 6 age-matched IDDM women in good metabohc control The enzymatic achv=ty was reduced both in the mtcrosomal fraction and tn the Na+/K+-ATPase obtained from d=abetic women tissue, while no difference was found in the number of active molecules determined by anthroyl ouabam binding The Na+/K+-ATPase purified from IDDM women dtd not show any modification In the ouabain affinity or change rn the physlcochemlcal structure of the ouabain binding site investigated by dynam=c fluorescence nor alterataon =n lateral diffusions. The activatton energy above the trans~tzon temperature (being 15 3+0 9 kCal/mol for healthy women and 19 1+1 0 kCal/mol for IDDM) and the tryptophan accessibility (being 0 72_+004 for healthy and 0.98__.005 for IDDM) of the enzyme were increased m 1(3DMwomen The fluidity of the lipJds annulus of the enzyme was higher In IDDM than m control women as suggested by fluorescence polarization (p parameter) of 1-(4-tnmethyl-amlno-phenyl)-6-phenyl-1 3,5hexatnene (TMA-DPPI) (0 293±0 013 m the control and 0 223_+0 15 m IDDM). The present data indicate that placental Na+/K+-ATPase show subconformabonal changes in IDDM and suggest a relevant role of the lipid enwronment in this modtficatlon and that the hypothetLc inhtbitor must be bound to the complex protein-hpld annulus or cause a permanent modification of this complex. The present work was supported by a Grant of Regione Marche to N C. and of CNR to LM
IDENTIFICATION AND CHARACTERIZATION OF SOLUBLE VARIANTS OF VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) RECEPTORS IN THE HUMAN PLACENTA D. Stephen Chamock-Jones, Andrew M. Sharkey, Kate Day and Stephen K. Smith Reproductive Molecular Molecular Research Group. Department of Ob/Gyn University of Cambridge. The Rosle Matermty Hospttal. Cambridge. CB2 2SW Vascular endothehal growth factor (VEGF) is a potent stimulator of angiogenesis both in vwo and in wtro. It is also a potent reducer of increased vascular permeability. These actions are all mediated by endothelial cells and until recently the receptors for this factor were known to be localized solely on these cells The process of angmgenesis is clearly essential for placental development; it is therefore not surprising that the receptors for VEGF (both fit and KDR) are found in the placenta. However we have shown that these receptors are not restricted to the endothelial cells as might be expected. They are also highly expressed on the trophoblast cells themselves (Charnock-Jones et.al 1994). It is therefore probable that VEGF not only plays a role m endothelial development within the placenta but may also directly affect the trophoblast. Soluble variants of a vanety of tyrosine kinase receptors have been described and indeed variants of the fit receptor known to exist (Kendall et al 1993). We have also identified mRNAs encoding soluble fit receptors within ovarian carcinoma cell hnes (Boocock et al 1995). Soluble receptors are presumed to act as antagomsts for their hgand so we sought to determine whether there were soluble VEGF receptors present m the human placenta Therefore we c a m e d out northern blotting and PCR analysis of mRNA derived from human placenta Subsequent cloning revealed that the previously described variant (Kendall et al 1993) was present at h~gh levels m the placenta There were also several other variants which would be predicted to encode soluble VEGF receptors. Since the soluble receptors contain varying amounts of the extra cellular hgand binding domain of the classical receptor :t ts likely that they have different b~ochem~cal propemes Each of these may be VEGF antagomsts suggesting here is a complex mechanism for regulating the effects of VEGF both on endothehal cells and on trophoblast. This may be a fundamental tmportance in the regulation of vascular development and trophoblast functmn within the human placenta. D Stephen Chamock-Jones, Andrew M Sharkey, Christine A. Boocock, Aslf Ahmed, Robin Plevin, Napoleone Ferrara and Stephen K. Smith (1994). Btol Reprod 51 524-530 Kendall R.L and Thomas K A. (1993) Proc Natl Acad.Scl USA 90 (10705-10709) Christine A Boocock, D Stephen Charnock-Jones, Andrew M. Sharkey, John McLaren, Pat Barker, Karen Wright, Peter Twentyman and Stephen K Smtth. (1995)J.Natl.Cancer Inst. 87 (7)506-516
A.11