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Autoradiographic localization of human alpha-atrial natriuretic peptide (hANP) binding sites in human umbilical cord and placenta.
429
DISTRIBUTION
OF G A W K - L I K E
IMMUNOREACTIVITY
IN M A M M A L I A N
CNS
M J SALAHUDDIN, K SEKIYA, M A GHATEI and S R B L O O M D e p a r t m e n t of Medicine, P o s t g r a d u a t e Medical School H a m m e r s m i t h H o s p i t a l Du Cane Road L o n d o n W I 2 0 N N , U.K. The d i s t r i b u t i o n of G A W K ( c h r o m o g r a n i n - B 4 2 0 - 4 9 3 ) - i i k e i m m u n o r e a c t i v i t y (LI) was i n v e s t i g a t e d in the central nervous s y s t e m (CNS) of man, g u i n e a pig and rat, by u s i n g two d i f f e r e n t antibodies. In the h u m a n CNS, h i g h e s t GAWK-LI c o n c e n t r a t i o n s were found in the p i t u i t a r y gland, G A W K ( I - 1 7 ) - L I 74. 3~8. 5 (mean~SEM p m o l / g wet w e i g h t tissue) and G A W K ( 2 0 - 3 8 ) - L I 2017. 01~142. 3, f o l l o w e d by h y p o t h a l a m u s and s u b s t a n t i a nigrao In the spinal cord, the h i g h e s t c o n c e n t r a t i o n was found in the dorsal area. GAWK-LI was d e t e c t e d in h u m a n c e r e b r o s p i n a l fluid, G A W K ( I - 1 7 ) - L I 376. 7~ 77.9 and G A W K ( 2 0 - 3 8 ) - L I i174. 7~259. 3 pmol/litre. GAWK-LI was also found in the g u i n e a pig and rat CNS w i t h h i g h e s t in the p i t u i t a r y g l a n d s , b u t v e r y low GAWK-LI levels were p r e s e n t in o t h e r parts of the b r a i n of both species. C h r o m a t o g r a p h i c a n a l y s i s of d i f f e r e n t regions of h u m a n CNS s h o w e d m u l t i p l e GAWK-LI peaks. The p r o f i l e of p i t u i t a r y extracts was d i f f e r e n t from t h o s e of o t h e r parts of the CNSo S u b c e l l u l a r f r a c t i o n a t i o n of rat b r a i n s h o w e d h i g h e s t c o n c e n t r a t i o n in the s y n a p t o s o m e fractions. GAWK-LI r e l e a s e was s t i m u l a t e d from rat p i t u i t a r y cells by high K + concentrations. It is c o n c e i v a b l e that c h r o m o g r a n i n B or its fragments may p l a y a h o r m o n a l or n e u r o t r a n s m i t t e r role in the m a m m a l i a n CNS.
AUTORADIOGRAPHIC LOCALIZATION OF HUMANALPHA-ATRIAL NATRIURETIC PEPTIDE (hANP) BINDING SITES IN HUMANUMBILICAL CORDAND PLACENTA. S.P.SALAS1- R.F.POWER2, A.J.RUTHERFORD3, J.WHARTON2, J.BROWN4 and J.M.POLAK2. Depts. of IMedicine. 2Histochemistry and 3Obstetrics, RPMS. Du Cane Road, London W12 ONN and 4The Physiology Laboratory, Downing Street, Cambridge. Circulating hANP levels in human umbilical arterial blood are higher than maternal levels, suggesting that hANP may be a circulating hormone in the fetus. There is evidence for the existence of hANP binding sites in isolated membrane preparations of human placenta, indicating a possible role for hANP in regulating placental blood flow, but at present there is no information regarding their anatomical localization. In vitro autoradiographic techniques were employed to i n v e s t i g a ~ i s t r i b u t i o n of these sites in tissue sections. Normal, f u l l term placentae, including umbilical cords (n=6), were collected shortly after vaginal delivery (less than 1 hr.). Serial lO~m cryostat sections were incubated with [1251]-hANP (20-200pM) in phosphate buffer (pH 7.2), for 20 mins. at room temperature, with or without the addition of unlabelled peptide (10~7M). Autoradiograms were generated by exposing the sections to Hyperfilm ~H for 4-14 days. In the umbilical cord, specific hANP binding sites were observed on the intimal region of both arteries, with no specific labelling on the umbilical vein. In the placenta, binding sites were exclusively localized to the arterial tree. I t is possible that hANP may p l ~ a role in the regulation of fetal and placental blood flow acting via these specific binding sites. SPS is a Fellow of ODEPLAN, Chile. RFP is a Wellcome Trust Research Fellow.
DISTRIBUTION
OF G A W K - L I K E
IMMUNOREACTIVITY
IN M A M M A L I A N
CNS
M J SALAHUDDIN, K SEKIYA, M A GHATEI and S R B L O O M D e p a r t m e n t of Medicine, P o s t g r a d u a t e Medical School H a m m e r s m i t h H o s p i t a l Du Cane Road L o n d o n W I 2 0 N N , U.K. The d i s t r i b u t i o n of G A W K ( c h r o m o g r a n i n - B 4 2 0 - 4 9 3 ) - i i k e i m m u n o r e a c t i v i t y (LI) was i n v e s t i g a t e d in the central nervous s y s t e m (CNS) of man, g u i n e a pig and rat, by u s i n g two d i f f e r e n t antibodies. In the h u m a n CNS, h i g h e s t GAWK-LI c o n c e n t r a t i o n s were found in the p i t u i t a r y gland, G A W K ( I - 1 7 ) - L I 74. 3~8. 5 (mean~SEM p m o l / g wet w e i g h t tissue) and G A W K ( 2 0 - 3 8 ) - L I 2017. 01~142. 3, f o l l o w e d by h y p o t h a l a m u s and s u b s t a n t i a nigrao In the spinal cord, the h i g h e s t c o n c e n t r a t i o n was found in the dorsal area. GAWK-LI was d e t e c t e d in h u m a n c e r e b r o s p i n a l fluid, G A W K ( I - 1 7 ) - L I 376. 7~ 77.9 and G A W K ( 2 0 - 3 8 ) - L I i174. 7~259. 3 pmol/litre. GAWK-LI was also found in the g u i n e a pig and rat CNS w i t h h i g h e s t in the p i t u i t a r y g l a n d s , b u t v e r y low GAWK-LI levels were p r e s e n t in o t h e r parts of the b r a i n of both species. C h r o m a t o g r a p h i c a n a l y s i s of d i f f e r e n t regions of h u m a n CNS s h o w e d m u l t i p l e GAWK-LI peaks. The p r o f i l e of p i t u i t a r y extracts was d i f f e r e n t from t h o s e of o t h e r parts of the CNSo S u b c e l l u l a r f r a c t i o n a t i o n of rat b r a i n s h o w e d h i g h e s t c o n c e n t r a t i o n in the s y n a p t o s o m e fractions. GAWK-LI r e l e a s e was s t i m u l a t e d from rat p i t u i t a r y cells by high K + concentrations. It is c o n c e i v a b l e that c h r o m o g r a n i n B or its fragments may p l a y a h o r m o n a l or n e u r o t r a n s m i t t e r role in the m a m m a l i a n CNS.
AUTORADIOGRAPHIC LOCALIZATION OF HUMANALPHA-ATRIAL NATRIURETIC PEPTIDE (hANP) BINDING SITES IN HUMANUMBILICAL CORDAND PLACENTA. S.P.SALAS1- R.F.POWER2, A.J.RUTHERFORD3, J.WHARTON2, J.BROWN4 and J.M.POLAK2. Depts. of IMedicine. 2Histochemistry and 3Obstetrics, RPMS. Du Cane Road, London W12 ONN and 4The Physiology Laboratory, Downing Street, Cambridge. Circulating hANP levels in human umbilical arterial blood are higher than maternal levels, suggesting that hANP may be a circulating hormone in the fetus. There is evidence for the existence of hANP binding sites in isolated membrane preparations of human placenta, indicating a possible role for hANP in regulating placental blood flow, but at present there is no information regarding their anatomical localization. In vitro autoradiographic techniques were employed to i n v e s t i g a ~ i s t r i b u t i o n of these sites in tissue sections. Normal, f u l l term placentae, including umbilical cords (n=6), were collected shortly after vaginal delivery (less than 1 hr.). Serial lO~m cryostat sections were incubated with [1251]-hANP (20-200pM) in phosphate buffer (pH 7.2), for 20 mins. at room temperature, with or without the addition of unlabelled peptide (10~7M). Autoradiograms were generated by exposing the sections to Hyperfilm ~H for 4-14 days. In the umbilical cord, specific hANP binding sites were observed on the intimal region of both arteries, with no specific labelling on the umbilical vein. In the placenta, binding sites were exclusively localized to the arterial tree. I t is possible that hANP may p l ~ a role in the regulation of fetal and placental blood flow acting via these specific binding sites. SPS is a Fellow of ODEPLAN, Chile. RFP is a Wellcome Trust Research Fellow.