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Expression of inositol 1,4,5-trisphosphate receptors in rat adrenocortical zones
J. SteroidBiochem. Molec. Biol. Vol. 57, No. 1/2, pp. 13-17, 1996 Copyright © 1996 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0960-0760/96 $15.00 + 0.00
Pergamon
Rapid Communication Expression of Inositol 1,4,5-Trisphosphate Receptors in Rat Adrenocortical Zones G. Szabadkai, A. Horvfith, T. Rohfics, L. Vimlfiti, A. Sprit* and P. Enyedi Department of Physiology, Semmelweis University of Medicine, P.O. Box 259, 1444, Budapest, Hungary P r e v i o u s l y we d e m o n s t r a t e d the presence o f InsP3R-l, -II and -IIl subtypes in the z o n a g l o m e r u l o s a . N o w we have e x a m i n e d the expression o f different subtypes o f inositol 1,4,5=trisphosphate receptor (InsP3R) in the inner, z o n e s o f rat adrenal cortex. R N A extracted from decapsulated adrenal tissue (zonae fasciculata=reticularis and the medulla) or from fasciculata-reticularis cells was reverse transcribed. Subsequ~ent p o l y m e r a s e c h a i n reaction revealed the presence o f InsP3R-I, -II and -III subtypes in decapsulated tissue but failed to d e m o n s t r a t e the expression o f any k n o w n subtypes o f InsP3R in fasciculata-reticularis cells. Accordingly, InsP3 receptors expressed in the decapsulated tissue are o f m e d u l l a r y origin. C o p y r i g h t © 1996 Elsevier S c i e n c e Ltd.
.7. SteroidBiochem. Molec. Biol., Vol. 57, No. 1/2, pp. 13-17, 1996
INTRODUCTION
P450 species [8,9,10], and to different regulation of expression of the respective genes by separate control systems [11,12]. Yet, there are no data available comparing the expression of the elements of signal transduction in these two cell types. Ca2+-mobilizing stimuli induce inositol 1,4,5-trisphosphate (InsP3) formation, which in turn releases Ca 2+ from intracellular stores in a wide variety of tissues [ 13]. InsP3 receptors (InsP3R) are expressed virtually in all nucleated animal cell types [14,15,16,17,18,19,20,21]. We have previously demonstrated the presence and measured the relative expression of InsP3R-I, -II and -III subtypes in rat glomerulosa cells [22]. In the present work, with the purpose of further analyzing zonal differences we designed a study to characterize the expression pattern of the InsP3R in rat fasciculatareticularis cells.
T h e adrenal cortex consists of concentric layers of morphologically and functionally diverse cell types. T h e oldest but still prevailing hypothesis for the development of these cells postulates that conversion of glomerulosa cells to fasciculata and later to reticularis cells takes place as cells migrate inward from the subcapsular layer toward the medulla [ 1,2]. T h e bulk of evidence supporting this assumption is provided by cell turnover rate measurements in the different layers [2,3] and by demonstration of labeled cell migration [1]. Further data also show' that under special conditions glomerulosa cells can transform to fasciculata cells either in vitro [4] or in vivo [5]. On the other hand, the results obtained with cell turnover rate measurements have recently been questioned [6]. In addition, the existence of a zone without steroid synthesizing activity between the zonae glomerulosa and fasciculata of the rat adrenal cortex has been proposed as progenitor cell zone [7]. In spite of the uncertainty concerning the ontogenesis of adrenocortical cells, there is considerable evidence that the main functional difference between the glomerulosa and fasciculata cells is due to the different expression pattern of cytochrome
EXPERIMENTAL
Materials Enzymes for molecular biological studies were purchased from Fermentas (Vilnius, Lithuania), Perkin Elmer Cetus (Norwalk, C T ) , Promega (Madison, WI) and Stratagene (La Jolla, CA). Molecular biological grade chemicals, where required, or analytical ones
*Correspondence to A. Sprit. Received 27 June 1995; accepted 5 Sept. 1995. 13
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were obtained from Fluka (Buchs, Switzerland) and Sigma (St Louis, MO). Cell isolation and culture
Fasciculata-reticularis cells were prepared from decapsulated adrenal tissue (containing the zonae fasciculata and reticularis and the medulla) of male Wistar rats ( ~ 2 5 0 g b.w.) according to a previously described method [23] with the following modifications: digestion with collagenase lasted for a 50 min. period and the cells were exposed to a brief osmotic shock (50 m M NaC1, 30 s) for reducing the red blood cell contamination. Electron microscopic studies demonstrated that the cell population was free of contaminating medullary and glomerulosa cells. Molecular biological studies
Total RNA was extracted from capsular tissue (containing the fibrous capsule and the zona glomerulosa), decapsulated tissue (containing fasciculata, reticularis and medullary cells) or from isolated fasciculata-reticularis cells with the guanidium isothiocyanate-phenol extraction method [24]. T h e subsequent reverse transcription of 1 pg of total RNA and polymerase chain reaction ( R T - P C R ) , as well as restriction enzyme mapping of the R T - P C R products were performed as previously described [22]. Primers were designed to amplify a variable segment of the ligand binding region of the first strand c D N A of InsP3R m R N A [22]. T h e conservative upstream primer A (GAGGATCCTG TCAATGCTGG GCAGCC) hybridizes with bases 885-904 of InsP3R-I [25], with bases 802-821 of InsP3R-II [26] (mismatching with base 807) and with bases 693-712 of InsP3R-III [19] (mismatching with bases 701 and 704). T h e conservative downstream primer E (TACCCA G G T G T T G G T A C A C A G G T G ) is complementary to bases 1503-1526 (mismatching with bases 1514 and 1526) of InsP3R-I, to bases 1417-1440 (mismatching with base 1434) of InsP3R-II and to bases 1305-1328 (mismatching with bases 1310,1326,1328) of InsP3RIII mRNA. As positive control a 658 bp segment of fl-actin m R N A was amplified by a primer pair obtained from Stratagene (La Jolla, CA; Cat. No. 302010). For estimating the sensitivity of P C R we used the same conditions as in R T - P C R except that the template was c D N A (Epg5, ref. 22) and M M L V - R T was not present. In restriction enzyme mapping studies we used Eco811 which cuts InsP3R-I at bases 1119 and 1405, PstI which cuts InsP3R-II at bases 1194 and 1370 and also cuts InsP3R-III at base 1258, HindIII which cuts InsP3R-III at base 912, as well as AccI which cuts the SI + variant of InsP3R-I at base 1282. RESULTS AND D I S C U S S I O N
T h e InsP3 receptor protein has been purified from brain and peripheral tissues [27,28], its c D N A has been cloned and sequenced from various sources
[18,19,25,26,29,30]. These molecular cloning studies also indicated that at least three different InsP3R coding genes exist and further receptor heterogeneity arises from alternative splicing [15,16,25]. We examined the presence of receptor m R N A in the different zones of the rat adrenal cortex by means of R T - P C R . Total RNA was extracted from capsular and decapsulated tissue, isolated fasciculata-reticularis cells and cultured glomerulosa cells of rat adrenal gland. Following reverse transcription of the extracted RNA conservative primers A and E, flanking a variable region of the binding domain of the receptor were used in P C R studies to amplify an approx. 650 bp fragment, present in each known subtype of InsP3R. T h e expression of InsP3R m R N A both in capsular (fibrous capsule and zona glomerulosa) and decapsulated (zonae fasciculatareticularis and medulla) tissues was indicated by the appearance of an appropriate band on agarose gel (Fig. 1). Two alternative splicing sites (SI and SII) of InsP3R-I were described previously for whole murine adrenal gland by means of N o r t h e r n blot [16]. After dissecting the rat adrenal gland, we demonstrated [22] that glomerulosa cells express InsP3R-I m R N A both with and without the SI segment. This zone also contains InsP3R-II and of InsP3R-III mRNAs, attaining ~ 20% and ~ 5% of total InsP3R mRNA, respectively. T h e presence of the different InsP3R messages from decapsulated adrenal gland was shown by restriction mapping of the amplified fragment. In the present experiments digestion of the amplification product with Eco81I, PstI, HindIII and AccI indicated that in this tissue the predominant receptor subtype is InsP3R-I (both S I - and SI + variants), and we could also detect InsP3R-II and InsP3R-III m R N A (Fig. 2).
676
517
i
~L~~ ! ~ i ~ ! ¸
--,,.-
A
B
Fig. 1. D e t e c t i o n o f lnsP3R e x p r e s s i o n b y m e a n s o f r e v e r s e transcription-polymerase c h a i n r e a c t i o n (RT-PCR). Total R N A e x t r a c t e d f r o m c a p s u l a r (A) a n d d e c a p s u l a t e d (B) t i s s u e was reverse transcribed and the cDNA obtained was amplified with P C R u s i n g c o n s e r v a t i v e p r i m e r s A a n d E ( s e e e x p e r i m e n t a l p r o t o c o l s ) . T h e P C R p r o d u c t s w e r e r u n on a 1.5% a g a r o s e gel s t a i n e d w i t h e t h i d i u m b r o m i d e . A r r o w s indicate the position of comigrating standards.
Expression of InsP3R in Rat Adrenocortical Zones
15
676 676 ----
517
517 ---350 ---222 ---5x107 5x106
126 --,.-
5x105
5x104
5x103
Number of template molecules
Eco81I
PstI
HindlII
AccI
Fig. 2. R e s t r i c t i o n e n z y m e m a p p i n g o f l n s P 3 R - I , I n s P 3 R - I I a n d InsP3R-IIl. Total RNA from decapsulated tissue (zonae fasciculata-reticularis and the medulla) was reverse transcribed and the cDNA obtained was amplified with conservative p r i m e r s A a n d E. T h e P C R p r o d u c t w a s p u r i f i e d , s u b j e c t e d to d i g e s t i o n w i t h Eco811, P s t I , a n d H i n d l I I , w h i c h e n z y m e s c u t s I n s P 3 R - l , In!~P3R-II a n d I n s P 3 R - I I I , r e s p e c t i v e l y . Accl cuts only the SI+ variant of InsP3R-l. The cleavage p r o d u c t s w e r e e l e c t r o p h o r e s e d o n 1.5% a g a r o s e gel s t a i n e d with ethidium bromide. Arrows indicate the position of comigr~ting standards.
N o R T - P C R product was generated using three separate suspensions of fasciculata-reticularis cells (Fig. 3), or using primary cultures of fasciculata-reticularis cells (n = 5, data not shown). Similarly, no product
glom
fasc
676 517
A
B
A
B
Fig. 3. D e t e c t i o n o f I n s P 3 R a n d ~ - a c t i n e x p r e s s i o n i n c u l t u r e d g l o m e r u l o s a (left p a n e l ) a n d i s o l a t e d f a s c i c u l a t a - r e t i c u l a r i s cells ( r i g h t p a n e l ) . T h e e x t r a c t e d t o t a l R N A w a s r e v e r s e transcribed and the cDNA obtained was amplified with PCR u s i n g c o n s e r v a t i v e p r i m e r s A a n d E f o r I n s P 3 R (A) a n d u s i n g ~ - a c t i n p r i m e r s (B) a s p o s i t i v e c o n t r o l . T h e P C R p r o d u c t s w e r e r u n o n a 1.5% a g a r o s e gel s t a i n e d w i t h e t h i d i u m b r o m i d e . Arrows indicate the position of comigrating standards.
Fig. 4. S e n s i t i v i t y o f I n s P 3 R d e t e c t i o n b y P C R . A s e r i e s o f 10-fold d i l u t i o n (5 × 107 to 5 x 103 m o l e c u l e s ) o f a p r e v i o u s l y s u b c l o n e d I n s P 3 R - P C R p r o d u c t ( E P g 5 , ref.[22]) w a s u s e d a s template in the PCR using the same conditions as in RT-PCR. The number of plasmid molecules in the starting reaction m i x t u r e is s h o w n b e l o w e a c h l a n e . T h e P C R p r o d u c t s w e r e r u n o n a 1.5% a g a r o s e g e l s t a i n e d w i t h e t h i d i u m b r o m i d e . A r r o w s indicate the position of comigrating standards.
was obtained when P C R was performed under low stringency conditions (50°C annealing temperature, n = 2, data not shown). These preparations, in contrast to decapsulated tissue, do not contain medullary cells. It follows that all three InsP3R subtypes in the decapsulated tissue are of medullary origin. T h e presence of m R N A with preserved structure in these preparations was demonstrated by the amplification of a 658 bp segment of fl-actin c D N A (Fig. 3). When InsP3R and fl-actin c D N A were amplified simultaneously, comparable amounts of fl-actin products were obtained from capsular tissue (n = 3, not shown), glomerulosa cell (n = 2, Fig. 3), decapsulated tissue (n = 3, not shown) and fasciculata-reticularis cell extracts ( n = 4 , Fig. 3), yet InsP3R c D N A was amplified only in the first three cases but not in fasciculata-reticularis cells. In two separate experiments R T - P C R was performed using 10-fold more (10 pg) total RNA. A hardly detectable band of ~ 6 5 0 bp appeared on the gel (n = 2, not shown). Even if this result is not due to contamination with another cell type, the biological significance of this level of expression is questionable. For the validity of the conclusion that InsP3Rs are not significantly expressed in fasciculata-reticularis cells, we tested the sensitivity of P C R for the amplification of the chosen receptor segment. Using a series of dilution of the previously subcloned InsP3R R T - P C R product (Epg5, Ref.22) as template we could detect the product from 5 x 104 recombinant plasmids (Fig. 4) Assuming that 102 cells contains ~ 1 /ag RNA [31] this result means that our method has a sensitivity of ~ 0 . 5 m o l e c u l e s InsP3R m R N A per fasciculata cell. In contrast, our previous quantitative P C R studies in glomerulosa cells [22] showed that using 0.5/~g adrenal capsular RNA the
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G. Szabadkai et al.
amount of InsP3R-I mRNA was e q u i v a l e n t to 6 3 . 6 _ 10.9 p g i n t e r n a l s t a n d a r d p l a s m i d . T h i s r e s u l t implies ~,300 InsP3R mRNA copies per glomerulosa cell. I n c o n c l u s i o n , z o n a l d i f f e r e n c e s in t h e rat a d r e n a l c o r t e x are n o t r e s t r i c t e d to s t e r o i d h y d r o x y l a s e s , w e c o u l d also d e t e c t d i f f e r e n t e x p r e s s i o n o f InsP3Rs. O u r data allow the localization of the different InsP3R m e s s a g e s w i t h i n t h e w h o l e a d r e n a l c o r t e x o f t h e rat. InsP3R-I (SI-/SI+), I n s P 3 R - I I a n d I n s P 3 R - I I I are e x p r e s s e d in t h e z o n a g l o m e r u l o s a , b u t n o n e o f t h e m are e x p r e s s e d in t h e d e e p e r c o r t i c a l z o n e s . It f o l l o w s t h a t t h e l o w a m o u n t o f I n s P 3 R - I I I m R N A o b s e r v e d in c a p s u l a r tissue [22] is n o t d u e to c o n t a m i n a t i o n w i t h f a s c i c u l a t a cells. As far as w e k n o w , t h e rat f a s c i c u l a t a - r e t i c u l a r i s cells c o n s t i t u t e t h e first k n o w n nucleated mammalian cell t y p e w i t h o u t s i g n i f i c a n t e x p r e s s i o n o f h i t h e r t o d e s c r i b e d i n t r a c e l l u l a r InsP3Rs. O u r P C R p r i m e r s w e r e d e s i g n e d for c o n s e r v a t i v e r e g i o n s in t h e N - t e r m i n a l l i g a n d b i n d i n g d o m a i n o f t h e InsP3R, so d o n o t n e c e s s a r i l y r e c o g n i z e t h e m e s s a g e o f the putative InsP3R-IV and InsP3R-V subtypes, of which only partial sequences of the hydrophobic C - t e r m i n a l r e g i o n are a v a i l a b l e [ 1 8 , 3 2 ] . O u r d a t a d o not rule out the expression of plasmalemmal InsP3R, of still u n k n o w n p r i m a r y s t r u c t u r e , d e s c r i b e d in J u r k a t and thymus T-lymphocytes [33,34]. The absence or v e r y l o w level o f i n t r a c e l l u l a r I n s P 3 R e x p r e s s i o n in rat f a s c i c u l a t a cells m a y a c c o u n t f o r t h e l a c k o f C a 2÷ r e l e a s e a n d t h e f a i l u r e to i n c r e a s e s t e r o i d p r o d u c t i o n in r e s p o n s e to a n g i o t e n s i n I I [ 3 5 , 3 6 ] . H o w e v e r , it s h o u l d b e e m p h a s i z e d t h a t s t e r o i d p r o d u c t i o n in h u m a n a n d b o v i n e f a s c i c u l a t a cells is e n h a n c e d b y a n g i o t e n s i n II, therefore the data presented here may be a specific p h e n o m e n o n for rat f a s c i c u l a t a - r e t i c u l a r i s cells. O u r d a t a also s u g g e s t t h a t a n g i o t e n s i n I I - i n d u c e d h y p e r t r o p h y o f t h e d e e p e r c o r t i c a l z o n e s o f rat a d r e n a l [37] m a y b e m e d i a t e d b y a m e s s e n g e r d i f f e r e n t f r o m InsP3. Acknowledgements--We are grateful to Dr Evelin Ors6 for electron
microscopic studies. The valuable advice of Dr L. Szilfigyi is greatly appreciated. We thank Mrs Ir6n Veres and Miss Erika Kovfics for their excellent technical help. This work was supported by grants No. 1111 and 5011 from the Hungarian National Science Foundation (OTKA), grant No. T 04 331/93 from the Hungarian Council for Medical Research and a grant (No. CIPA-CT92-3014/9) for "Cooperation in Science and Technology with Central and Eastern European Countries" of the European Union. REFERENCES
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32. De Smedt H., Missiaen L., Parys J. B., Bootman M. D., Mertens L., Van den Bosch L. and Casteels R.: Determination of relative amounts of inositol trisphosphate receptor mRNA isoforms by ratio polymerase chain reaction. ~. Biol. Chem. 269 (1994) 21,691-21,698. 33. Khan A. A., Steiner J. E and Snyder S. H.: Plasma membrane inositol 1,4,5-trisphosphate receptor of lymphocytes: selective enrichment in sialic acid and unique binding specificity. Proc. Nam. Acad. Sci. U.S.A. 89 (1992) 2849-2853. 34. Khan A. A., Steiner J. E, Klein M. G., Schneider M. F. and Snyder S. H.: IP3 receptor: localization to plasma membrane of T cells and cocapping with the T cell receptor. Science 257 (1992) 815-818. 35. Roskelley C. D., Baimbridge K. G., Leung P. C. K. and Auersperg N.: Divergent differentiation of rat adrenocortical cells is associated with an interruption of angiotensin II-mediated signal transduction. Molec. Cell. Endocr. 89 (1992) 79-89. 36. Whitley G. S., Hyatt P. J. and Tait J. E: Angiotensin IIinduced inositol phosphate production in isolated rat zona glomerulosa and fasciculata/reticularis cells. Steroids 49 (1987) 271-286. 37. Nussdorfer G. G., Robba C., Mazzocchi G. and Rebuffat P.: Effect of angiotensin II on the zona fasciculata of the rat adrenal cortex: an ultrastructural stereologic study. ~. Anat. 132 (1981) 235-242.
Pergamon
Rapid Communication Expression of Inositol 1,4,5-Trisphosphate Receptors in Rat Adrenocortical Zones G. Szabadkai, A. Horvfith, T. Rohfics, L. Vimlfiti, A. Sprit* and P. Enyedi Department of Physiology, Semmelweis University of Medicine, P.O. Box 259, 1444, Budapest, Hungary P r e v i o u s l y we d e m o n s t r a t e d the presence o f InsP3R-l, -II and -IIl subtypes in the z o n a g l o m e r u l o s a . N o w we have e x a m i n e d the expression o f different subtypes o f inositol 1,4,5=trisphosphate receptor (InsP3R) in the inner, z o n e s o f rat adrenal cortex. R N A extracted from decapsulated adrenal tissue (zonae fasciculata=reticularis and the medulla) or from fasciculata-reticularis cells was reverse transcribed. Subsequ~ent p o l y m e r a s e c h a i n reaction revealed the presence o f InsP3R-I, -II and -III subtypes in decapsulated tissue but failed to d e m o n s t r a t e the expression o f any k n o w n subtypes o f InsP3R in fasciculata-reticularis cells. Accordingly, InsP3 receptors expressed in the decapsulated tissue are o f m e d u l l a r y origin. C o p y r i g h t © 1996 Elsevier S c i e n c e Ltd.
.7. SteroidBiochem. Molec. Biol., Vol. 57, No. 1/2, pp. 13-17, 1996
INTRODUCTION
P450 species [8,9,10], and to different regulation of expression of the respective genes by separate control systems [11,12]. Yet, there are no data available comparing the expression of the elements of signal transduction in these two cell types. Ca2+-mobilizing stimuli induce inositol 1,4,5-trisphosphate (InsP3) formation, which in turn releases Ca 2+ from intracellular stores in a wide variety of tissues [ 13]. InsP3 receptors (InsP3R) are expressed virtually in all nucleated animal cell types [14,15,16,17,18,19,20,21]. We have previously demonstrated the presence and measured the relative expression of InsP3R-I, -II and -III subtypes in rat glomerulosa cells [22]. In the present work, with the purpose of further analyzing zonal differences we designed a study to characterize the expression pattern of the InsP3R in rat fasciculatareticularis cells.
T h e adrenal cortex consists of concentric layers of morphologically and functionally diverse cell types. T h e oldest but still prevailing hypothesis for the development of these cells postulates that conversion of glomerulosa cells to fasciculata and later to reticularis cells takes place as cells migrate inward from the subcapsular layer toward the medulla [ 1,2]. T h e bulk of evidence supporting this assumption is provided by cell turnover rate measurements in the different layers [2,3] and by demonstration of labeled cell migration [1]. Further data also show' that under special conditions glomerulosa cells can transform to fasciculata cells either in vitro [4] or in vivo [5]. On the other hand, the results obtained with cell turnover rate measurements have recently been questioned [6]. In addition, the existence of a zone without steroid synthesizing activity between the zonae glomerulosa and fasciculata of the rat adrenal cortex has been proposed as progenitor cell zone [7]. In spite of the uncertainty concerning the ontogenesis of adrenocortical cells, there is considerable evidence that the main functional difference between the glomerulosa and fasciculata cells is due to the different expression pattern of cytochrome
EXPERIMENTAL
Materials Enzymes for molecular biological studies were purchased from Fermentas (Vilnius, Lithuania), Perkin Elmer Cetus (Norwalk, C T ) , Promega (Madison, WI) and Stratagene (La Jolla, CA). Molecular biological grade chemicals, where required, or analytical ones
*Correspondence to A. Sprit. Received 27 June 1995; accepted 5 Sept. 1995. 13
14
G. Szabadkai et al.
were obtained from Fluka (Buchs, Switzerland) and Sigma (St Louis, MO). Cell isolation and culture
Fasciculata-reticularis cells were prepared from decapsulated adrenal tissue (containing the zonae fasciculata and reticularis and the medulla) of male Wistar rats ( ~ 2 5 0 g b.w.) according to a previously described method [23] with the following modifications: digestion with collagenase lasted for a 50 min. period and the cells were exposed to a brief osmotic shock (50 m M NaC1, 30 s) for reducing the red blood cell contamination. Electron microscopic studies demonstrated that the cell population was free of contaminating medullary and glomerulosa cells. Molecular biological studies
Total RNA was extracted from capsular tissue (containing the fibrous capsule and the zona glomerulosa), decapsulated tissue (containing fasciculata, reticularis and medullary cells) or from isolated fasciculata-reticularis cells with the guanidium isothiocyanate-phenol extraction method [24]. T h e subsequent reverse transcription of 1 pg of total RNA and polymerase chain reaction ( R T - P C R ) , as well as restriction enzyme mapping of the R T - P C R products were performed as previously described [22]. Primers were designed to amplify a variable segment of the ligand binding region of the first strand c D N A of InsP3R m R N A [22]. T h e conservative upstream primer A (GAGGATCCTG TCAATGCTGG GCAGCC) hybridizes with bases 885-904 of InsP3R-I [25], with bases 802-821 of InsP3R-II [26] (mismatching with base 807) and with bases 693-712 of InsP3R-III [19] (mismatching with bases 701 and 704). T h e conservative downstream primer E (TACCCA G G T G T T G G T A C A C A G G T G ) is complementary to bases 1503-1526 (mismatching with bases 1514 and 1526) of InsP3R-I, to bases 1417-1440 (mismatching with base 1434) of InsP3R-II and to bases 1305-1328 (mismatching with bases 1310,1326,1328) of InsP3RIII mRNA. As positive control a 658 bp segment of fl-actin m R N A was amplified by a primer pair obtained from Stratagene (La Jolla, CA; Cat. No. 302010). For estimating the sensitivity of P C R we used the same conditions as in R T - P C R except that the template was c D N A (Epg5, ref. 22) and M M L V - R T was not present. In restriction enzyme mapping studies we used Eco811 which cuts InsP3R-I at bases 1119 and 1405, PstI which cuts InsP3R-II at bases 1194 and 1370 and also cuts InsP3R-III at base 1258, HindIII which cuts InsP3R-III at base 912, as well as AccI which cuts the SI + variant of InsP3R-I at base 1282. RESULTS AND D I S C U S S I O N
T h e InsP3 receptor protein has been purified from brain and peripheral tissues [27,28], its c D N A has been cloned and sequenced from various sources
[18,19,25,26,29,30]. These molecular cloning studies also indicated that at least three different InsP3R coding genes exist and further receptor heterogeneity arises from alternative splicing [15,16,25]. We examined the presence of receptor m R N A in the different zones of the rat adrenal cortex by means of R T - P C R . Total RNA was extracted from capsular and decapsulated tissue, isolated fasciculata-reticularis cells and cultured glomerulosa cells of rat adrenal gland. Following reverse transcription of the extracted RNA conservative primers A and E, flanking a variable region of the binding domain of the receptor were used in P C R studies to amplify an approx. 650 bp fragment, present in each known subtype of InsP3R. T h e expression of InsP3R m R N A both in capsular (fibrous capsule and zona glomerulosa) and decapsulated (zonae fasciculatareticularis and medulla) tissues was indicated by the appearance of an appropriate band on agarose gel (Fig. 1). Two alternative splicing sites (SI and SII) of InsP3R-I were described previously for whole murine adrenal gland by means of N o r t h e r n blot [16]. After dissecting the rat adrenal gland, we demonstrated [22] that glomerulosa cells express InsP3R-I m R N A both with and without the SI segment. This zone also contains InsP3R-II and of InsP3R-III mRNAs, attaining ~ 20% and ~ 5% of total InsP3R mRNA, respectively. T h e presence of the different InsP3R messages from decapsulated adrenal gland was shown by restriction mapping of the amplified fragment. In the present experiments digestion of the amplification product with Eco81I, PstI, HindIII and AccI indicated that in this tissue the predominant receptor subtype is InsP3R-I (both S I - and SI + variants), and we could also detect InsP3R-II and InsP3R-III m R N A (Fig. 2).
676
517
i
~L~~ ! ~ i ~ ! ¸
--,,.-
A
B
Fig. 1. D e t e c t i o n o f lnsP3R e x p r e s s i o n b y m e a n s o f r e v e r s e transcription-polymerase c h a i n r e a c t i o n (RT-PCR). Total R N A e x t r a c t e d f r o m c a p s u l a r (A) a n d d e c a p s u l a t e d (B) t i s s u e was reverse transcribed and the cDNA obtained was amplified with P C R u s i n g c o n s e r v a t i v e p r i m e r s A a n d E ( s e e e x p e r i m e n t a l p r o t o c o l s ) . T h e P C R p r o d u c t s w e r e r u n on a 1.5% a g a r o s e gel s t a i n e d w i t h e t h i d i u m b r o m i d e . A r r o w s indicate the position of comigrating standards.
Expression of InsP3R in Rat Adrenocortical Zones
15
676 676 ----
517
517 ---350 ---222 ---5x107 5x106
126 --,.-
5x105
5x104
5x103
Number of template molecules
Eco81I
PstI
HindlII
AccI
Fig. 2. R e s t r i c t i o n e n z y m e m a p p i n g o f l n s P 3 R - I , I n s P 3 R - I I a n d InsP3R-IIl. Total RNA from decapsulated tissue (zonae fasciculata-reticularis and the medulla) was reverse transcribed and the cDNA obtained was amplified with conservative p r i m e r s A a n d E. T h e P C R p r o d u c t w a s p u r i f i e d , s u b j e c t e d to d i g e s t i o n w i t h Eco811, P s t I , a n d H i n d l I I , w h i c h e n z y m e s c u t s I n s P 3 R - l , In!~P3R-II a n d I n s P 3 R - I I I , r e s p e c t i v e l y . Accl cuts only the SI+ variant of InsP3R-l. The cleavage p r o d u c t s w e r e e l e c t r o p h o r e s e d o n 1.5% a g a r o s e gel s t a i n e d with ethidium bromide. Arrows indicate the position of comigr~ting standards.
N o R T - P C R product was generated using three separate suspensions of fasciculata-reticularis cells (Fig. 3), or using primary cultures of fasciculata-reticularis cells (n = 5, data not shown). Similarly, no product
glom
fasc
676 517
A
B
A
B
Fig. 3. D e t e c t i o n o f I n s P 3 R a n d ~ - a c t i n e x p r e s s i o n i n c u l t u r e d g l o m e r u l o s a (left p a n e l ) a n d i s o l a t e d f a s c i c u l a t a - r e t i c u l a r i s cells ( r i g h t p a n e l ) . T h e e x t r a c t e d t o t a l R N A w a s r e v e r s e transcribed and the cDNA obtained was amplified with PCR u s i n g c o n s e r v a t i v e p r i m e r s A a n d E f o r I n s P 3 R (A) a n d u s i n g ~ - a c t i n p r i m e r s (B) a s p o s i t i v e c o n t r o l . T h e P C R p r o d u c t s w e r e r u n o n a 1.5% a g a r o s e gel s t a i n e d w i t h e t h i d i u m b r o m i d e . Arrows indicate the position of comigrating standards.
Fig. 4. S e n s i t i v i t y o f I n s P 3 R d e t e c t i o n b y P C R . A s e r i e s o f 10-fold d i l u t i o n (5 × 107 to 5 x 103 m o l e c u l e s ) o f a p r e v i o u s l y s u b c l o n e d I n s P 3 R - P C R p r o d u c t ( E P g 5 , ref.[22]) w a s u s e d a s template in the PCR using the same conditions as in RT-PCR. The number of plasmid molecules in the starting reaction m i x t u r e is s h o w n b e l o w e a c h l a n e . T h e P C R p r o d u c t s w e r e r u n o n a 1.5% a g a r o s e g e l s t a i n e d w i t h e t h i d i u m b r o m i d e . A r r o w s indicate the position of comigrating standards.
was obtained when P C R was performed under low stringency conditions (50°C annealing temperature, n = 2, data not shown). These preparations, in contrast to decapsulated tissue, do not contain medullary cells. It follows that all three InsP3R subtypes in the decapsulated tissue are of medullary origin. T h e presence of m R N A with preserved structure in these preparations was demonstrated by the amplification of a 658 bp segment of fl-actin c D N A (Fig. 3). When InsP3R and fl-actin c D N A were amplified simultaneously, comparable amounts of fl-actin products were obtained from capsular tissue (n = 3, not shown), glomerulosa cell (n = 2, Fig. 3), decapsulated tissue (n = 3, not shown) and fasciculata-reticularis cell extracts ( n = 4 , Fig. 3), yet InsP3R c D N A was amplified only in the first three cases but not in fasciculata-reticularis cells. In two separate experiments R T - P C R was performed using 10-fold more (10 pg) total RNA. A hardly detectable band of ~ 6 5 0 bp appeared on the gel (n = 2, not shown). Even if this result is not due to contamination with another cell type, the biological significance of this level of expression is questionable. For the validity of the conclusion that InsP3Rs are not significantly expressed in fasciculata-reticularis cells, we tested the sensitivity of P C R for the amplification of the chosen receptor segment. Using a series of dilution of the previously subcloned InsP3R R T - P C R product (Epg5, Ref.22) as template we could detect the product from 5 x 104 recombinant plasmids (Fig. 4) Assuming that 102 cells contains ~ 1 /ag RNA [31] this result means that our method has a sensitivity of ~ 0 . 5 m o l e c u l e s InsP3R m R N A per fasciculata cell. In contrast, our previous quantitative P C R studies in glomerulosa cells [22] showed that using 0.5/~g adrenal capsular RNA the
16
G. Szabadkai et al.
amount of InsP3R-I mRNA was e q u i v a l e n t to 6 3 . 6 _ 10.9 p g i n t e r n a l s t a n d a r d p l a s m i d . T h i s r e s u l t implies ~,300 InsP3R mRNA copies per glomerulosa cell. I n c o n c l u s i o n , z o n a l d i f f e r e n c e s in t h e rat a d r e n a l c o r t e x are n o t r e s t r i c t e d to s t e r o i d h y d r o x y l a s e s , w e c o u l d also d e t e c t d i f f e r e n t e x p r e s s i o n o f InsP3Rs. O u r data allow the localization of the different InsP3R m e s s a g e s w i t h i n t h e w h o l e a d r e n a l c o r t e x o f t h e rat. InsP3R-I (SI-/SI+), I n s P 3 R - I I a n d I n s P 3 R - I I I are e x p r e s s e d in t h e z o n a g l o m e r u l o s a , b u t n o n e o f t h e m are e x p r e s s e d in t h e d e e p e r c o r t i c a l z o n e s . It f o l l o w s t h a t t h e l o w a m o u n t o f I n s P 3 R - I I I m R N A o b s e r v e d in c a p s u l a r tissue [22] is n o t d u e to c o n t a m i n a t i o n w i t h f a s c i c u l a t a cells. As far as w e k n o w , t h e rat f a s c i c u l a t a - r e t i c u l a r i s cells c o n s t i t u t e t h e first k n o w n nucleated mammalian cell t y p e w i t h o u t s i g n i f i c a n t e x p r e s s i o n o f h i t h e r t o d e s c r i b e d i n t r a c e l l u l a r InsP3Rs. O u r P C R p r i m e r s w e r e d e s i g n e d for c o n s e r v a t i v e r e g i o n s in t h e N - t e r m i n a l l i g a n d b i n d i n g d o m a i n o f t h e InsP3R, so d o n o t n e c e s s a r i l y r e c o g n i z e t h e m e s s a g e o f the putative InsP3R-IV and InsP3R-V subtypes, of which only partial sequences of the hydrophobic C - t e r m i n a l r e g i o n are a v a i l a b l e [ 1 8 , 3 2 ] . O u r d a t a d o not rule out the expression of plasmalemmal InsP3R, of still u n k n o w n p r i m a r y s t r u c t u r e , d e s c r i b e d in J u r k a t and thymus T-lymphocytes [33,34]. The absence or v e r y l o w level o f i n t r a c e l l u l a r I n s P 3 R e x p r e s s i o n in rat f a s c i c u l a t a cells m a y a c c o u n t f o r t h e l a c k o f C a 2÷ r e l e a s e a n d t h e f a i l u r e to i n c r e a s e s t e r o i d p r o d u c t i o n in r e s p o n s e to a n g i o t e n s i n I I [ 3 5 , 3 6 ] . H o w e v e r , it s h o u l d b e e m p h a s i z e d t h a t s t e r o i d p r o d u c t i o n in h u m a n a n d b o v i n e f a s c i c u l a t a cells is e n h a n c e d b y a n g i o t e n s i n II, therefore the data presented here may be a specific p h e n o m e n o n for rat f a s c i c u l a t a - r e t i c u l a r i s cells. O u r d a t a also s u g g e s t t h a t a n g i o t e n s i n I I - i n d u c e d h y p e r t r o p h y o f t h e d e e p e r c o r t i c a l z o n e s o f rat a d r e n a l [37] m a y b e m e d i a t e d b y a m e s s e n g e r d i f f e r e n t f r o m InsP3. Acknowledgements--We are grateful to Dr Evelin Ors6 for electron
microscopic studies. The valuable advice of Dr L. Szilfigyi is greatly appreciated. We thank Mrs Ir6n Veres and Miss Erika Kovfics for their excellent technical help. This work was supported by grants No. 1111 and 5011 from the Hungarian National Science Foundation (OTKA), grant No. T 04 331/93 from the Hungarian Council for Medical Research and a grant (No. CIPA-CT92-3014/9) for "Cooperation in Science and Technology with Central and Eastern European Countries" of the European Union. REFERENCES
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