Alterations in Messenger RNA Encoding Atrial Natriuretic Hormone Receptor A and C Subtypes During

Hepatic Regeneration

TARUN B. PATEL, BIPIN G. NAIR, EKAMBARAM PADMINI, HANI M. RASHED, AND HuI SUN

Previously, we demonstrated that, 48 hours after par- tial hepatectomy, in the regenerating liver the number of both atrial natriuretic hormone (ANF) receptor sub- types, the guanylyl eyclase-l inked and ANF-C receptors, is increased twofold. Subsequently, we demonstrated that activation of ANF-C receptors inhibits growth of hepatocytes. Therefore, studies were performed to de- termine whether, during hepatic regeneration, the in- crease in ANF receptor subtypes is accompanied by an increase in their respective transcripts. Our data dem- onstrate that in the normal and regenerating rat liver, the predominant guanylyl cyclase-l inked ANF receptor is of the ANF-A subtype. Moreover, messenger RNA (mRNA) encoding the ANF-A and ANF-C receptors are transiently increased after surgery; the levels of mRNA encoding both receptor subtypes remain unchanged in livers of sham-operated animals. ANF.A receptor mRNA is maximally increased 12 hours after partial hepatec- tomy, whereas the maximal increase in ANF-C receptor mRNA is observed between 0.5 hour and 4 hours after hepatectomy. The increase in ANF-C receptor transcript is accompanied by increased expression of protein, 4 hours after hepatectomy. However, the ANF-C receptor protein is also elevated 48 hours after partial hepatec- tomy when ANF-C receptor mRNA levels are not differ- ent from controls. Likewise, although ANF-A receptors are increased when hepatic levels of mRNA encoding the protein are maximally elevated, the maximal increase in ANF-A receptor protein occurs at times when transcript levels are low and similar to those in sham-operated con- trols. These findings demonstrate differential regulation in the expression of ANF-A and ANF-C receptors and are illustrative of regulation of expression of both receptors at the translational or posttranslational levels. (HEPA- TOLOGY 1995;21:1682-1689.)

Abbreviations: ANF, atrial natriuretic hormone; cGMP, cyclic guanosine monophosphate; CNP, C-type natriuretic peptide; mRNA, messenger RNA; cDNA, complementary DNA.

From the Department of Pharmacology, University of Tennessee, Memphis, Memphis, TN.

Received September 12, 1994; accepted January 13, 1995. Supported by National Institutes of Health grant DK35713, Bethesda, MD. Address reprint requests to: Tarun B. Patel, PhD, Department of Pharma-

cology, University of Tennessee, Memphis, The Health Science Center, 874 Union Ave, Memphis, TN 38163.

Copyright © 1995 by the American Association for the Study of Liver Diseases.

0270-9139/95/2106-002853.00/0

Before the realization that the particulate (mem- brane-associated) guanylyl cyclases are also receptors for ligands such as natriuretic peptides and heat-stable enterotoxin, 1 studies from Murad's laboratory 2 demon- strated that after partial hepatectomy, in the regener- ating rat liver, the activity of the particulate guanylyl cyclase was increased. The activity of hepatic par- ticulate guanylyl cyclase was maximally increased (twofold) 48 hours after hepatectomy2; the membrane- associated guanylyl cyclase activity in livers of sham- operated animals remained unchanged after surgery. 2 The demonstration that membrane-associated guany- lyl cyclase is also the receptor for atrial natriuretic hor- mone (ANF) 1'3'4 prompted us to investigate the hypoth- esis that after partial two-thirds hepatectomy, in the regenerating rat liver, the number of guanylyl cyclase- linked ANF receptors is also increased. Indeed, em- ploying three different approaches, namely, binding of [125I]ANF, affinity cross-linking, and the ability of ANF to st imulate guanylyl cyclase activity in hepatic mem- branes, we demonstrated that, 48 hours after two- thirds hepatectomy, the number of guanylyl cyclase- linked ANF receptors increased by twofold. 5 Besides confirming the findings of Kimura and Murad 2 that membrane-associated guanylyl cyclase activity in liv- ers is increased during hepatic regeneration, these data from our laboratory ~ also demonstrated that the in- crease in guanylyl cyclase activity is due to an upregu- lation of guanylyl cyclase- l inked ANF receptors.

Three forms of guanylyl cyclase-linked receptors (GC- A, GC-B, and GC-C) have been described. 6'7 The GC-A and GC-B isoforms belong to the family of the natriuretic peptide receptors 6s and are also referred to as ANF-A and ANF-B receptors. The other isoform of membrane- associated guanylyl cyclase, GC-C, is the receptor for gua- nylin and heat-stable enterotoxin 9'~° and is predomi- nantly present in intestinal epithelia. 9'1° The ANF-A and ANF-B receptor subtypes can be differentiated by the ability of different ligands to induce cyclic guanosine mo- nophosphate (cGMP) production, s'11'~2 For instance, ANF stimulates cGMP production after binding the ANF-A receptor but is not very effective at stimulating cGMP production by the ANF-B receptor. ~'12 Similarly, the C- type natriuretic peptide (CNP) produced in the brain 1.'14 stimulates cGMP production by the ANF-B receptor but not through activation of the ANF-A receptors. 11'12

1682

HEPATOLOGY Vol. 21, No. 6, 1995 PATEL ET AL 1683

Bes ides b ind ing to the guany ly l c y c l a s e - l i n k e d re- ceptors , in a v a r i e t y of t i ssues , A N F also b inds to the ANF-C receptors , s'15'1~ The ANF-C recep tors sha re s t r u c t u r a l s imi l a r i ty to the ANF-A recep tors ~7 bu t h a v e a t r u n c a t e d c a r b o x y - t e r m i n u s a n d the re fo re do not ex- p ress guany ly l cyclase act ivi ty, s'~5-17 Thus , b ind ing of A N F to the ANF-C recep to r s does not inc rease cGMP conten t of cells a n d tissues.~7'~s In the r e g e n e r a t i n g r a t liver, 48 hour s a f t e r pa r t i a l h e p a t e c t o m y , as mon i to red by d i s p l a c e m e n t of [~25I]ANF by a l inea r des-cys te ine ana log of A N F (analog I) t h a t select ively b inds only the ANF-C receptor , is and W e s t e r n ana lys i s wi th a n t i - ANF-C recep to r an t ibody, we d e m o n s t r a t e d t h a t the n u m b e r of ANF-C recep tors is also inc reased by two- fold. 5 More recent ly , 19 we h a v e d e m o n s t r a t e d t h a t acti- r a t i o n of ANF-C recep tors in hepa tocy t e s inh ib i t s cellu- la r g rowth and prol i fera t ion. An i m p o r t a n t impl ica t ion of the l a t t e r f indings is t h a t inc rease in ANF-C recep- tors in the r e g e n e r a t i n g l iver p lays a p ivota l role in r e g u l a t i n g hepa t i c g rowt h a f t e r pa r t i a l hepa t ec tomy . Therefore , to f u r t h e r u n d e r s t a n d the mechan i sm(s ) by which A N F recep to r s ub t ypes a re u p r e g u l a t e d du r ing hepa t i c r e g e n e r a t i o n a f t e r h e p a t e c t o m y , the objective of the c u r r e n t s t udy was to d e t e r m i n e w h e t h e r the in- c rease in the two A N F recep to r s ub t ypes was accompa- n ied by an inc rease in t r a n s c r i p t s encoding these recep- tors. In th is r epo r t we d e m o n s t r a t e t h a t the p r e d o m i n a n t guany ly l c y c l a s e - l i n k e d A N F recep tors in the l iver a re of the ANF-A subtype . F u r t h e r , a f t e r pa r t i a l two- th i rds h e p a t e c t o m y , the m e s s e n g e r RNAs (mRNAs) encoding ANF-A and ANF-C recep tors a re i nc reased t r a n s i e n t l y such t h a t m a x i m a l inc reases in ANF-A and ANF-C recep to r m R N A s occur 12 hour s and 0.5 to 4 hour s a f t e r h e p a t e c t o m y , respect ively . Moreover , whi le ANF-C recep tors a re inc reased 4 hour s and 48 hou r s a f t e r two- th i rds h e p a t e c t o m y , m a x i m a l inc rease in ANF-A recep tors occurs 24 hou r s and 48 hour s a f t e r pa r t i a l h e p a t e c t o m y w h e n the ANF-A re- ceptor m R N A levels a re low and s imi la r to those in controls. These f indings d e m o n s t r a t e d i f ferent ia l regu- la t ion in the express ion of ANF-A and ANF-C recep tors and are i l lus t ra t ive of r egu la t ion of express ion of bo th recep tors a t the t r a n s l a t i o n a l or p o s t t r a n s l a t i o n a l lev- els.

MATERIALS AND METHODS

Animals, Surgery, and Isolation of Hepatic Mem- branes. Male rats of the Sprague-Dawley strain (180 to 200 g body weight) maintained on standard laboratory chow and allowed free access to water were used in all of the studies. After anesthesia, hepatectomy and sham-operations (con- trols) were performed as described in our previous publica- tion. 5 At various times after surgery, the animals were killed and liver membranes were isolated by the method of Neville 2° as described previously. 5 All animals received humane care in compliance with the guidelines outlined in Guide for the Care and Use of Laboratory Animals by the National Insti- tutes of Health (publication No. 86-23, revised 1985). The Institutional Animal Care and Use Committee reviews all of the experimental protocols pertaining to use of animals be-

fore granting permission for the implementation of the proto- col on a given species.

Perfusion of Livers and Measurement of cGMP Produc- tion. At various times after surgery (sham and hepatectomy), rats were anesthetized by intraperitoneal administration of pentobarbital sodium. Livers were perfused with hemoglobin- free Krebs Henseleit bicarbonate buffer in a nonrecirculating mode at a constant flow rate of 35 mL/min as described pre- viously. 21 After an equilibration period of 30 minutes, perfu- sate samples were collected at 0.5-minute intervals for a pe- riod of 10 minutes to monitor the basal rate of cGMP production. Thereafter, either ANF or CNP was infused to attain a constant concentration of 20 nmol/L for a period of 10 minutes. The rates of cGMP production in perfusate collected at 0.5-minute intervals were monitored. Livers could not be perfused immediately after hepatectomy, and therefore, the zero time point for regenerating livers is not presented in Fig. 2. Data presented represent the maximal, steady-state rates of cGMP production in the presence of the peptides. 21 The cGMP in perfusate samples was measured by the radioimmunoassay method described previously. 5'21

Isolation of Liver RNA and Solution Hybridization. Total cellular RNA was isolated from livers by the method of Chom- czynski and Sacchi 22 as modified by Cinna/Biotecx Labora- tories International Inc., Houston, TX (RNAzol method). After checking the purity of the RNA (ratio of absorbance at 260 nm/absorbance at 280 nm > 1.9), ensuring the integrity of the RNA by gel fractionation, and after quantitation, 10 #g liver RNA was employed in the solution hybridization assays to monitor changes in ANF-A and ANF-C receptor mRNA content.

Solution hybridization assays to monitor ANF-A and ANF- C receptor mRNA were performed employing in vitro tran- scribed anti-sense mRNA for the two respective receptor sub- types. Essentially, the plasmids pBluescript-KS+/- and pGEM-3 containing the ANF-A (nt 1-1185) and ANF-C (nt 1- 2095) receptor complementary DNAs (cDNAs) were linear- ized by incubation with the restriction enzyme Pvu II. Anti- sense mRNAs corresponding to the two receptors were transcribed in the presence of [a-8~P]CTP with the T3 poly- merase (pBluescript-KS+/-) and T7 polymerase (pGEM-3) according to the methods of Melton et a123 as modified by Promega. The 3~P-labeled anti-sense mRNA probes were era- ployed in solution hybridization assays as described by Quarless and Heinrich. 24 Essentially, aliquots of this anti- sense mRNA (50,000 dpm) were mixed with total cell RNA (10 #g) and 50 #g salmon sperm DNA in a final volume of 20 #L and overlaid with 25 #L mineral oil. The mixtures were heated to 85°C for 15 minutes and incubated at 60°C for 5 hours (ANF-A receptor mRNA) or 18 hours (ANF-C receptor mRNA). Hybridization was terminated by the addition of 160 #L diethyl pyrocarbonate-treated water, 20 #L S1 nuclease buffer (300 mmol/L sodium acetate, i mol/L sodium chloride, 20 mmoFL ZnSO4, 50% glycerol, pH 4.6), 100 units of $1 nuclease and incubation at 37°C for I hour. The $1 nuclease- treated samples were mixed with trichloroacetic acid (final concentration = 7.5%) and after standing on ice for 20 min- utes, filtered through GF/C filters (Whatman, Hillboro, OR) soaked with 7.5% trichloroacetic acid. The filters were counted for ~2p content. Sense mRNAs were also transcribed in vitro from the Pvu II linearized plasmids by the aforemen- tioned method except that the polymerases employed were T7 (ANF-A receptor mRNA) and SP6 (ANF-C receptor mRNA). After transcription, the sense mRNAs were em- ployed as standards to quantitate the mRNA encoding the receptors in liver RNA. Under the conditions described above,

1684 PATEL ET AL HEPATOLOGY June 1995

sRNA liver RNA SI+a-sRNA a-sRNA

Hybrid -I~

41- Probe

sRNA: Sense mRNA a-sRNA: Anti-sense mRNA $ 1 : $ 1 Nuclease

FIG. 1. Hybridization of in vitro transcribed ANF-A receptor anti- sense mRNA with in vitro transcribed sense mRNA and total tissue RNA. ANF-A receptor anti-sense mRNA (50,000 dpm) was hybrid- ized with in vitro transcribed sense mRNA (1.46 ng) and liver RNA (10 #g). The incubations were then subjected to Sl nuclease treat- ment for 1 hour at 37°C, phenol/chloroform extraction, and separa- tion through a G-50 column. The RNA recovered from these proce- dures was subjected to electrophoresis on 6.5% polyacrylamide gels. An autoradiograph of the gel is presented. The arrow on left denotes the migration of the double-stranded sense and anti-sense RNA. The arrow on right denotes the migration of anti-sense RNA.

the assays were linear up to 3.9 ng ANF-A receptor sense mRNA and 3.13 ng ANF-C receptor mRNA. Control incuba- tions with anti-sense mRNA alone in the presence and ab- sence of $1 nuclease treatment were also performed to deter- mine the efficiency of $1 nuclease digestion. Moreover, by employing different concentrations of total cell RNA, ]inear- ity of the $1 nuc]ease protection assay was also determined to ensure that the anti-sense mRNA was not limiting in the incubations. Under the conditions employed, the solution hy- bridization assay was linear up to 20 #g total hepatic RNA. All assays were performed in triplicate, employing total cell RNA isolated from three different experiments.

To ensure that the solution hybridization assays were func- tioning appropriately, $1 nuclease digests of 32P-labeled anti- sense mRNA alone, and 82P-labeled anti-sense mRNA hybrid- ized to in vitro transcribed sense mRNA and liver RNA were subjected to 6.5% polyacrylamide gel electrophoresis. The gels were dried and exposed to Kodak X-OMAT film. As dem- onstrated in Fig. 1, on Sl nuclease treatment, the ANF-A receptor anti-sense mRNA could not be detected on the gel. Moreover, the S 1 nuclease-resistant hybrids of ANF-A recep- tor anti-sense mRNA and liver RNA or ANF-A receptor anti- sense mRNA and in vitro transcribed ANF-A receptor sense mRNA migrated more slowly than the single-stranded anti- sense mRNA. The finding that hybrids of anti-sense mRNA and in vitro transcribed sense mRNA or liver RNA migrated to the same position on the gel (Fig. 1) shows that the liver ANF-A receptor mRNA was being appropriately recognized and monitored by the technique. Similar results were also

observed with the in vitro transcribed ANF-C receptor sense and anti-sense mRNAs (not shown).

Slot Blot Analysis. Various amounts of liver RNA (2.5 #g, 5.0 #g, and 10.0 #g) were applied to nitrocellulose membranes by means of a slot blot apparatus as described by Kafatos et alY After overnight pre-hybridization in medium containing 5× SSC, 50% formamide, 5× Denhardt's solution, 100 #g/mL salmon sperm DNA, 50 mmol/L sodium phosphate, pH 7.0, the slot blots were hybridized to ANF-A receptor cDNA ex- cised by Eco R1 digestion of the pBluescript-KS+/- men- tioned above. The cDNA was labeled with [~-32P]dCTP by the nick translation method. 2~ The blots were washed once in medium containing 2 × SSC plus 0.1% sodium dodecyl sulfate at 60°C and twice at room temperature with 0.1× SSC, 0.1% sodium dodecyl sulfate, and 0.1% (wt/vol) sodium pyrophos- phate, and exposed to Kodak X-OMAT film.

Western Blotting and Detection of ANF-C Receptor. Liver plasma membrane proteins (50 #g each) were separated un- der reducing conditions on 7.5% polyacrylamide gels as de- scribed by Laemm]iY The proteins were electrophoretica]ly transferred onto nitrocellulose membranes, and after blocking with 10% fetal bovine serum for 1 hour, the blots were exposed to polyclonal anti-ANF-C receptor antibody 2320 B (1:500 dilution). After washing and exposing the nitro- cellulose membranes to goat anti-rabbit immunoglobulin G conjugated to peroxidase, the Western blots were developed with the Amersham (Arlington Heights, IL) ECL system as described previously. 2s

Measurements of Guanylyl Cyelase Activity. Maximal gua- nylyl cyclase activity in aliquots (80 #g protein) of liver mem- branes was estimated in the presence of manganese ac- cording to the protocol of Chinkers et al, 29 and cGMP formation in the incubations was determined by radioimmu- noassay as described previously. ~'21 The anti-cGMP antibod- ies employed (Biomedical Technologies Inc., Cambridge, MA) do not cross-react with cyclic adenosine monophosphate. All assays were performed in duplicate.

Materials. Restriction enzymes Pvu II and Eco R1 were purchased from Stratagene Inc (La Jolla, CA). $1 nuclease, RNA polymerases T3, T7, and SP6, in vitro transcription buffer, and nucleotides were obtained from Promega (Madi- son, WI). RNAse-free DNAse was purchased from Boehringer Mannheim (Indianapolis, IN). Molecular biology grade rain- eral oil, salmon sperm DNA, MOPS, HEPES, and diethyl pyrocarbonate were purchased from Sigma Chemical Co. (St. Louis, MO). Rat ANF and CNP were obtained from Peninsula Laboratories Inc. (Belmont, CA) [125I]ANF, [a-32P]dCTP, and [a-82P]CTP were purchased from Du Pont (Wilmington, DE). The ANF-A receptor cDNA cloned into pBluescript-KS+/- was the generous gift from Dr David L. Garbers, University of Texas Southwestern Medical School. Similarly, the ANF-C receptor cDNA cloned into pGEM-3 and anti-ANF-C receptor antibody (2320 B) were kindly provided by Dr J. Gordon Por- ter of California Biotechnology Inc. Rat ceruloplasmin cDNA was obtained from Dr Jonathan Gitlin, Washington Univer- sity School of Medicine, St. Louis, MO. All other chemicals and reagents were of the highest purity commercially avail- able.

R E S U L T S A N D D I S C U S S I O N

Employ ing the na t r iu re t ic peptides, A N F and CNP, which selectively act ivate the ANF-A and ANF-B recep- tors, respectively, s'11'12 init ial exper iments were per- formed to de te rmine the subtypes of guanyly l cyclase receptors t ha t are p resen t in control and regenera t ing

(D m

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E Q. c- O

o m

"1o ,9o g. 13.

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• Basal Sham [ ] CNP Sham [ ] ANF Sham [ ] Basal Hep [ ] CNP Hep [ ] ANF Hep

1

0 0

I i

12 24 48

HEPATOLOGY Vol. 21, No. 6, 1995 PATEL ET AL 1685

Hours After Surgery

FIG. 2. Cyclic GMP production from perfused livers derived from sham-operated and hepatectomized rats at different times after sur- gery. After operation (sham or hepatectomy), livers of rats were per- fused in a nonrecirculating mode with hemoglobin-free Krebs Hensel- eit bicarbonate buffer as described in Materials and Methods. After a 30-minute equilibration period, perfusate samples were collected for 10 minutes of control period to monitor basal cGMP production and then in the presence of the peptides for a further 10 minutes. The maximal steady states of cGMP production are presented as the mean ± SEM. Livers were not perfused immediately after hepatec- tomy because of practical considerations (leaks from the liver), and therefore, data for 0 hours of hepatectomy are not presented.

rat livers. In these studies, perfused livers were ex- posed to ANF or CNP, and cGMP production was moni- tored in the perfusate because alterations in perfusate (or medium) cyclic nucleotides have been demonstrated to be a more sensitive index of changes in these nucleo- tides occurring within tissues or cells. 21'3°'3~ As illus- t rated by the data in Fig. 2, at various times after surgery, in livers of sham-operated animals the ANF- B receptor ligand, CNP, did not alter cGMP production. However, ANF, which activates the ANF-A receptor, s t imulated cGMP production from livers of sham-oper- ated rats (Fig. 2). These data show that in control livers from sham-operated rats, the predominant guanylyl cy- clase-l inked ANF receptors are of the ANF-A subtype. Similarly, the largest increase in cGMP production in regenerating livers from hepatectomized rats was ob- served with ANF and not CNP (Fig. 2). However, as compared with sham-operated controls, in regenerat- ing livers, 24 and 48 hour's after hepatectomy, CNP caused a modest increase in cGMP production, indicat- ing that the ANF-B receptor expression is increased during hepatic regeneration after partial hepatectomy. As demonstrated in our previous study, 5 48 hours after hepatectomy, in regenerating livers, ANF-mediated cGMP production was approximately twofold greater than that observed in livers of sham-operated controls

(Fig. 2). Also as described before, 5 the basal cGMP pro- duction was greater in regenerating livers as compared with controls (Fig. 2). Thus, the fold-stimulation of cGMP production in response to ANF was not very dif- ferent between the two groups. Because the most pre- dominant alterations in cGMP production in control and regenerating livers were induced by the ANF-A receptor ligand, ANF, our studies were focused on de- termining the alterations in mRNA levels encoding this receptor and the ANF-C receptor that is important in regulation of hepatic growth. 19

To determine the temporal changes in ANF-A recep- tor mRNA content during hepatic regeneration after partial hepatectomy, the amount of ANF-A receptor mRNA was monitored by the solution hybridization method. The data in Fig. 3 demonstrate that in livers of sham-operated (control) animals, the levels of ANF- A receptor mRNA do not change after surgery. How- ever, in the regenerating rat liver, 8 hours after partial (two-thirds) hepatectomy, the amount of ANF-A recep- tor mRNA was significantly increased. Maximal in- crease (3.5-fold) in the ANF-A receptor mRNA content was observed 12 hours after partial hepatectomy and thereafter the levels of ANF-A receptor mRNA declined to control values such that at 24 hours and subsequent times after surgery no differences between regenerat- ing livers and normal livers were observed. The tran-

200

~0 / \ Hepatectomy

100 • - - *

~ 5o Sham-operated

Ot . . . . . . . . . 0 10 20 30 40 50

HOURS AFTER SURGERY

FIG. 3. Steady-state levels of ANF-A receptor mRNA in livers of sham-operated and hepatectomized rats as monitored by solution hybridization. Ten micrograms total liver RNA was employed in hy- bridization incubations with ANF-A receptor anti-sense mRNA (50,000 dpm). Varying amounts of in vitro transcribed sense mRNA were also employed to construct a s tandard curve, which was linear up to 3.9 ng of sense mRNA. After S1 nuclease t reatment of the incubations, the reactions were terminated by the addition of 7.5% trichloroacetic acid and filtration through GF/C filters. The filters were counted for t ~ r 3~p content. Each value is the mean ± SEM of triplicate determinations on three separate RNA preparations. *P < .001, Student 's unpaired t-test analysis.

1686 PATEL ET AL HEPATOLOGY June 1995

Slot Blot of liver RNA with ANF-A receptor cDNA

0.5s 0.5h 4s 4h 8s 8h 12s 12h 18s I8h 24s 24h 48s 48h

Numbers denote hours after surgery "s" and "h" indicate sham-operation and hepatectomy, respectively

0.7

O

0.6.

0.5-

0.4

0.3

0.2

0.1 - ~ S h a m - o p e r a t i o n

10 20 30

Hours after Surgery

i

40 50

FIG. 4. Slot blot analysis of the ANF-A receptor mRNA in control and regenerating rat livers with ANF-A receptor cDNA. Varying amounts (2.5 #g, 5.0 #g, and 10.0 #g) of liver RNA isolated from sham-operated or hepatectomized rats at different t imes after sur- gery were applied to nitrocellulose membranes by means of a slot blot apparatus. The blot was developed with 8~P-labeled ANF-A receptor cDNA. The top panel represents 5-#g amounts of liver RNA hybrid- ized with ANF-A receptor cDNA. The bottom panel is a densitometric scan of the top panel.

the current study the membrane-associated guanylyl cyclase activity was employed to detect the ANF-A re- ceptors. In these experiments (Fig. 5), the membrane- associated guanylyl cyclase was monitored in the pres- ence of Mn 2+ ions. 29 The data in Fig. 5 show that in membranes derived from sham-operated animals, the guanylyl cyclase activity did not change with time after surgery. However, in livers of partially hepatectomized rats, the membrane-associated particulate guanylyl cy- clase activity increased such that at 15 hours and 24 hours after hepatectomy, the activity was increased by twofold and threefold, respectively. Thereafter, the guanylyl cyclase activity declined somewhat so that 48 hours after hepatectomy the membrane-associated guanylyl cyclase activity was twice tha t in sham-oper- ated controls (Fig. 5). These data demonstrate that in the rat liver, 24 hours after hepatectomy the ANF-A receptors are maximally (threefold) increased and 48 hours after surgery, in confirmation of our previous findings, ~ the ANF-A receptors are elevated twofold. Although the membrane-associated guanylyl cyclase activity is elevated 15 hours after partial hepatectomy (Fig. 5), the response of the perfused liver to ANF or CNP is not altered at 12 and 24 hours after hepatec- tomy (Fig. 2). This lack of an enhanced response to ANF or CNP, at the early points after hepatectomy, despite the increase in membrane-associated guanylyl cyclase activity (Fig. 5) at 15 hours and 24 hours after hepatectomy, may be explained by either one or the combination of the following. First, the ability of ANF or CNP to stimulate ANF-A and ANF-B receptors is dependent on the presence of ATp.1'6'29 Therefore, if the

1200

sient increase in mRNA encoding the ANF-A receptors in livers during hepatic regeneration was also con- firmed by the quantitative slot blot method (Fig. 4). As a control, the effect of hepatectomy on the ceruloplas- min mRNA levels was monitored by Northern blot analyses. As reported previously by Milland et al, 32 the levels of ceruloplasmin mRNA did not change with t ime after hepatectomy (data not shown).

Interestingly, 48 hours after partial hepatectomy, when the number of ANF-A receptors is increased by twofold, ~ the ANF-A receptor mRNA content in regen- erat ing livers was not different from that in controls. Because in the previous study with ANF-A receptors ~ we had not monitored the differences in ANF-A recep- tor expression at the early points after hepatectomy, experiments were performed to determine if the en- hanced mRNA content encoding the ANF-A receptors in regenerat ing livers at the early times after hepatec- tomy was also accompanied by an increase in ANF-A receptors. Previously, we have shown that affinity cross-linking, [125I]ANF binding, and guanylyl cyclase activity measurements can all be used to detect alter- ations in the rat liver ANF-A receptors. 5 Therefore, in

| t 6"e

>,E

1000

800

600

400

200

0 10 20 30 40 50

Hours After Surgery

FIG. 5. Membrane-associated hepatic guanylyl cyclase activity in sham-operated and hepatectomized rat livers. Liver plasma mem- branes from sham-operated and hepatectomized rats were isolated at the indicated times after surgery. Guanylyl cyclase activity was monitored in 80 #g membranes by the method of Chinkers et al is and cGMP formation was monitored as described previously. 5 The mean _+ SEM of three determinations in duplicate are presented. *P < .001 as assessed by Student 's unpaired t-test analysis.

HEPATOLOGY Vol. 21, No. 6, 1995

5

'~ 4 Z

0 ¢~ 3

z rr o} Q. lit

0

Hepatectomy

4 Sham-operated

I I

2o 30 so

H O U R S A F T E R S U R G E R Y

FIG. 6. Steady-state levels of C-ANF receptor mRNA in livers of sham-operated and hepatectomized ra t s a t various t imes after sur- gery. Ten micrograms RNA isolated from livers of ra ts a t various t imes after surgery was hybridized with radiolabeled anti-sense mRNA (50,000 dpm) t ranscr ibed from ANF-C receptor cDNA cloned in pGEM-3. In vitro t ranscr ibed sense mRNA was employed to con- s t ruct a s tandard curve for quant i ta t ion purposes. The $1 nuclease- res i s tan t hybrids was separated from the incubation by te rmina t ing reactions with 7.5% trichloroacetic acid and fil tration through GF/C filters. The mean _+ SEM of triplicate determinat ions in three differ- ent preparat ions of liver RNA are presented. *P < .001, Student ' s unpaired t-test analysis.

ATP levels of livers were limited during the first 24 hours after partial hepatectomy, then the responses to ANF or CNP could be compromised. Second, ANF- mediated stimulation of the ANF-A receptor requires the phosphorylation of the receptor. 33 Thus, if after par- tial hepatectomy, the expression or activity of the kinase and phosphatase that regulates the receptor phosphorylation were decreased and increased, respec- tively, then the responses to ANF could also be compro- mised. Because 48 hours after hepatectomy, the re- sponse to ANF is normal (Fig. 2), the aforementioned changes must revert to normal at this point. The mech- anism(s) involved remain to be elucidated, however.

To determine the temporal changes in mRNA encod- ing the ANF-C receptor, the experiments depicted in Fig. 6 were performed. As assessed by solution hybrid- ization, the amount of the ANF-C receptor mRNA in livers of both control and hepatectomized animals was markedly lower than that of the ANF-A receptor mRNA (cf Figs. 3 and 6). Moreover, as observed for the ANF- A receptor mRNA, the ANF-C receptor mRNA content did not change in livers of sham-operated rats. How- ever, similar to our observations with the ANF-A recep- tor mRNA (Figs. 3 and 4), the ANF-C receptor mRNA content in regenerating livers was also transiently ele- vated. Maximal increase (-50%) in mRNA encoding the ANF-C receptors was observed between 0.5 hour

PATEL E T A L 1687

and 4 hours after hepatectomy, and by 8 hours after surgery it was the same as controls (Fig. 6). These data demonstrate that in the regenerating rat liver, the in- crease in ANF-C receptor mRNA precedes the increase in ANF-A receptor mRNA (cf Figs. 3, 4, and 6). To investigate the changes in ANF-C receptor protein ex- pression in regenerating livers after partial hepatec- tomy, the amount of ANF-C receptor protein in hepatic membranes was monitored by Western blot analysis employing the anti-ANF-C receptor antibody 2320 B as described previously, s The data in Fig. 7 show that the 66-kd ANF-C receptor protein did not change with time in membranes derived from livers of sham-oper- ated animals. However, in membranes from regenerat- ing livers, the expression of ANF-C receptor protein was elevated at the 4-hour and 48-hour points after hepatectomy (Fig. 7), but not at the intermediate points (Fig. 7).

The findings described above demonstrate that ex- pression of the ANF-C receptor protein in regenerating rat livers at early time (4 hours) after partial hepatec- tomy is accompanied by an increase in the mRNA en- coding this receptor (Figs. 6 and 7). Conversely, al- though ANF-A receptor mRNA content is maximally elevated 12 hours after partial hepatectomy, maximal increase in ANF-A receptor is not observed until 24 hours after hepatectomy, when hepatic contents of ANF-A receptor mRNA in hepatectomized and sham- operated animals are similar. Additionally, 48 hours after hepatectomy, when the hepatic content of mRNA

k D a

4 c 4 r 1 5 c 1 5 r 2 4 c 2 4 r 4 8 c 4 8 r

9 7 . 4 -

6 6 . 2 -

4 5 . 0 -

3 1 . 0 -

2 1 . 5 -

FIG. 7. Western blot analysis of the ANF-C receptor protein in liver plasma membranes of hepatectomized and sham-operated con- trol rats. Membrane proteins (50 #g) were separated by sodium dode- cyl sulfate polyaerylamide gel electrophoresis on 7.5% polyacryl- amide gels and electrophoretically t ransferred onto nitrocellulose membranes . The Western blot was developed with anti-ANF-C recep- tor antibody employing the Amersham ECL system. The numbers above lanes indicate the t ime (in hours) after surgery. The let ters "c" and "r" denote membranes from livers of sham-operated and hepatectomized rats, respectively.

1688 PATEL ET AL HEPATOLOGY June 1995

encoding the ANF-A receptor is not elevated (Figs. 3 and 4), the ANF-A receptor protein is still twice that in controls (Fig. 5). 5 Similarly, the ANF-C receptor pro- tein is also increased 48 hours after hepatectomy, when the ANF-C receptor mRNA content is not different from controls. These observations demonstrate that the in- creases in ANF-A and ANF-C receptors observed 48 hours after hepatectomy are not the manifestation of increased levels of transcripts encoding these proteins. Therefore, it would appear that 48 hours after hepatec- tomy, in the regenerating rat liver, the expression of the two subtypes of ANF receptor proteins is regulated by enhanced translation efficiency or increased protein half-life. 34'3~ Whatever the regulatory mechanism(s), the data from our studies demonstrate that while an increase in mRNAs encoding the two ANF receptor sub- types is accompanied by elevated receptor protein ex- pression, the lack of a change in mRNA for either recep- tor subtype does not necessarily correspond to a lack of change in their respective proteins, since the latter increase in the absence of any change in the levels of their corresponding transcripts.

Although the liver expresses the two ANF receptor subtypes, the role of these receptors in regulation of hepatic function has received little attention. In this respect, recent studies from our laboratory have shown that in the liver, atrial natriuretic peptides, via activa- tion of the ANF-A receptors and consequent increase in cellular cGMP content, inhibit glycolysis and stimulate gluconeogenesis. 21 Thus it is tempting to speculate that in the regenerating liver the role of the enhanced ex- pression of ANF-A receptors is to stimulate gluconeo- genesis so that the reduced liver mass is capable of maintaining normal glucose homeostasis. Similarly, because ANF-C receptor activation decreases growth of Hep G2 cells 19 and other cells, 36'~7 it is possible that the upregulation of ANF-C receptors is linked to control of hepatic growth during regeneration. However, the significance of the biphasic increase in ANF-C receptor expression in regulating growth or any other biological process remains unclear. It is possible that if ANF-C receptor activation is involved in regulating the growth of the regenerating liver, in addition to elevation in cell surface ANF-C receptors, the levels of circulating ANF may also be modulated. However, it is not known if the levels of ANF or related peptides in the circulation are altered after hepatectomy, and therefore this possibil- ity forms the subject of further studies.

In summary, our data demonstrate that in the regen- erating rat liver, transcripts encoding the ANF-A and ANF-C receptors are transiently elevated; the increase in ANF-C receptor mRNA precedes the enhanced levels observed for the ANF-A receptor mRNA. Although the increase in ANF-C and ANF-A receptor mRNAs is ac- companied by increased expression of the correspond- ing receptors, the elevation in ANF-A and ANF-C re- ceptors 48 hours after partial hepatectomy is not the manifestation of increases in mRNAs encoding these proteins. We conclude that increases in ANF-A and ANF-C receptor mRNA content in the liver may indi-

care increases in their proteins. However, the lack of a change in transcripts encoding the ANF-A and ANF-C receptors does not necessarily reflect the lack of alter- ations in expression of their respective proteins.

Acknowledgment: We are greatly indebted to Dr David L. Garbers, Howard Hughes Institute, Univer- sity of Texas Southwestern Medical School, for the gift of ANF-A receptor cDNA. We also thank Dr J. Gordon Porter of California Biotechnology for his generous gift of the ANF-C receptor cDNA and the anti-ANF-C re- ceptor antibody 2320 B. We are also grateful to Dr Jonathan Gitlin, Washington University, for the gift of rat ceruloplasmin cDNA.

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