Expression of C-type Natriuretic Peptide and its Specific Guanylyl Cyclase-Coupled Receptor in Pig Ovarian Granulosa Cells

Background: C-type natriuretic peptide (CNP) was isolated from porcine brain and is a 22-amino acid peptide which belongs to the natriuretic peptide (NP) family. Even though this peptide shares structural similarity to other endogenous NPs including atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) its receptor selectivity is different from other NPs. The present study was undertaken to investigate the expression of C-type natriuretic peptide (CNP) and its specifi c guanylyl cyclase (GC)-coupled receptor in the granulosa cells of the pig ovarian follicle. Results: Specifi c I-[Tyr]-CNP(1-22) binding sites were localized in the granulosa cell layer of the ovarian follicle with an apparent dissociation constant (K d) and a maximal binding capacity (B max ) of 1.41±0.39 nM and 2.75±0.65 fmol/mm 2 respectively. Binding of I-[Tyr]-CNP(1-22) to these sites was also prevented by atrial natriuretic peptide (ANP(1-28)), brain natriuretic peptide (BNP(126)) and des[Gln 18 ,Ser 19 ,Gly 20 , Leu 21 ,Gly 22 ] ANP (4-23) (C-ANP). Production of 3’,5’-cyclic guanosine monophosphate (cGMP) by particulate GC in the granulosa cell membranes was stimulated by natriuretic peptides (NPs) with a rank order of potency of CNP(1-22)>>BNP(1-26)>ANP(1-28). HS-142-1, a selective antagonist of the two recognized GC-coupled NPRs, inhibited CNP(1-22)-stimulated cGMP production in granulosa cell membranes in a dose-dependent manner. Also mRNAs for all three recognized NPRs were detected in granulosa cells using reverse transcriptase-polymerase chain reaction (RT-PCR). Serial dilution curves of granulosa cell extracts were parallel to the standard curve of synthetic CNP. Conclusion: These results indicate that CNP and its specifi c receptor are expressed in the granulosa cells of the pig ovary, and suggest that CNP may be a local autocrine and/or paracrine regulator via activation of its specifi c GC-coupled receptor, NPR-B. Research Article Expression of C-type Natriuretic Peptide and its Specifi c Guanylyl Cyclase-Coupled Receptor in Pig Ovarian Granulosa Cells Soo Mi Kim1, Suhn Hee Kim1, Kyung Woo Cho1, Sun Young Kim1 and Sung Zoo Kim2* 1Department of Physiology and Institute for Medical Sciences, Chonbuk National University Medical School, Jeonju 54907, Republic of Korea 2Department of Physiology, Chonbuk National University Medical School, Gungiro 20, Jeonju 54907, Republic of Korea *Address for Correspondence: Sung Zoo Kim, Ph.D, Department of Physiology, Chonbuk National University Medical School, Gungiro 20, Jeonju 54907, Republic of Korea, Tel: +8263-270-3093; Fax: + 82-63-274-9892; Email: szkim@jbnu.ac.kr Submitted: 08 August 2018 Approved: 21 August 2018 Published: 22 August 2018 Copyright: 2018 Kim SM, et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited


Introduction
C-type natriuretic peptide (CNP) was isolated from porcine brain [1] and is a 22-amino acid peptide which belongs to the natriuretic peptide (NP) family. Even though this peptide shares structural similarity to other endogenous NPs including atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) [1][2][3], its receptor selectivity is different from other NPs. The guanylyl cyclase (GC)-coupled NP receptor subtype A (NPR-A) is activated solely by ANP and BNP, and exerts well-de ined biological functions via activation of particulate GC. On the other hand, the GC-coupled NP receptor subtype B (NPR-B) is selectively activated by CNP [4,5]. In contrast to ANP or BNP, which are circulating hormones, CNP is likely to be an autocrine or paracrine mediator because it was detectable only at very low levels in plasma [6]. Since NPR-B is relatively speci ic for CNP, the localization of NPR-B predicates the possible biological actions of CNP in target organs [7]. Although NPR-B is expressed widely in the vascular smooth muscle and induces smooth muscle relaxation [8], it is also expressed in neural tissues including fetal telencephalon and somites [9,10], hypothalamus [11] and pituitary gland [12], and in atrial myocytes [13]. In these tissues, it has been suggested that CNP may be involved in modulation of the embryonic growth, neural transmission, other hormone synthesis and/or secretion, and membrane Ca 2+ channel activity [14,15]. Therefore, CNP system may serve autocrine and/or paracrine functions rather than the well-de ined functions of ANP and BNP as circulating hormones.
Gonadotropins and the ovarian steroid hormones act together in the hypothalamicpituitary-gonadal axis to regulate follicular development and oocyte maturation, but local mediators within the ovary may also control follicular development. As one example, the intra-ovarian renin-angiotensin system has been proposed to control follicular development [16]. Another possible factor may be atrial natriuretic peptide (ANP), which is a functional antagonist to angiotensin II (Ang II) in other tissues [17]. ANP occurs in the ovary [18,19]. The identi ication of mRNA encoding ANP and the immunohistochemical localization of ANP in the granulosa cells of the pig ovary suggests that ovarian granulosa cells elaborate ANP [20]. ANP has been shown to stimulate [21][22][23][24] or inhibit [25] the secretion of pregesterone in the ovary. Moreover, the presence of speci ic binding sites for ANP has been found in several ovarian tissues of mammals; in the preovulatory follicles of human [26], in cultured human granulosalutein cells [21], in the bovine corpus luteum [18] and in porcine granulosa and theca externa cells [27]. Furthermore, ANP speci ically stimulates a particulate GC in bovine luteal cells [28]. In comparison to the intra-ovarian ANP system, the role of CNP in the ovaries remains to be studied. Recently, CNP has been identi ied in the rat ovary but its sites of action have not been elucidated [29]. The purpose of the present study was to de ine the intra-ovarian CNP system. We have identi ied a CNP-speci ic GC-coupled receptor and endogenous CNP synthesis, using quantitative in vitro autoradiography, activation of particulate GC by NPs, RT-PCR, radioimmunoassay (RIA). Thus, CNP and its receptor may mediate autocrine and/or paracrine effects in the ovary.

Collection and transportation of ovaries
For the separation of granulosa cells, ovaries from healthy young pigs were collected at a slaughterhouse within 10 min of slaughter, and immediately transported to the laboratory in a sterile container in ice-cold 0.9% NaCl solution. For in vitro receptor autoradiography, other ovaries were collected from pigs within 10 min of slaughter, and were immediately snap frozen in liquid nitrogen, and stored in sealed boxes at -70 o C prior to frozen sectioning. ]-CNP  (speci ic activity 1,700 Ci/mmol) was performed as previously reported [9,10]. Brie ly, the sections were washed with 150 mM NaCl-0.5% acetic acid (pH 5.0) at room temperature for 10 min in order to remove endogenous NPs, and then preincubated with 30 mM phosphate buffer (pH 7.2) containing 120 mM NaCl and 1 mM phenanthroline at room temperature for 10 min. As shown in Figure 1, acidic washing increases the speci ic binding capacity for ]-CNP  was assessed in serial sections which were incubated with increasing concentrations (0-750 pM) of the radioligand in the presence or absence of 1 μM CNP  . ]-CNP  was examined in consecutive sections by coincubating with various concentrations of unlabeled CNP  , ANP  , BNP  or des[Gln 18 , Ser 19 , Gly ]-CNP  binding, adjacent sections were incubated in the presence of the unrelated peptides, angiotensin II or arginine vasopressin (all 10 μM).

Computerized microdensitometry of autoradiograms
Autoradiographic images were generated by exposing sections to Hyper ilm-   in the ovary was analysed using the PRISM image program (Version 3. ]-CNP  bound per square millimeter, as described elsewhere [30]. The number of ligand binding sites of different af inities, their apparent dissociation constants (K d ) and inhibitory constants (K i ), and their maximal binding capacities (B max ) on particular structures were derived separately in each individual using the LIGAND iterative model-itting computer program [31].

Particulate GC activity of granulosa cells
Granulosa cells were homogenized at 4 o C in 30 mM phosphate buffer (pH 7.2) containing 120 mM sodium chloride and 1 mM phenanthroline by three 30-second bursts of 27,000 rpm using a Polytron homogenizer (Brinkmann Instruments, Westbury, NY). The homogenate was centrifuged at 1,500xg for 10 min at 4 o C, and the supernatant was recentrifuged at 40,000xg for 60 min at 4 o C. The membrane pellet was washed three times with 50 mM Tris-HCl (pH 7.4) and resuspended in this solution. Protein contents were determined by a bicinchoninic acid assay kit (Sigma Chemical Co, St. Louis, MO). Particulate GC activity was measured according to the method described elsewhere [9]. Aliquots of 10 μg of membrane protein of the suspension were incubated for 15 min at 37 o C in a inal volume of 125 μl of 50 mM Tris-HCl (pH 7.6) containing 1 mM isobutylmethylxanthine, 1 mM guanosine triphosphate, 0.5 mM adenosine triphosphate, 15 mM creatine phosphate, 80 μg/ml creatine phosphokinase, and 4 mM magnesium chloride, plus a range of concentrations of either CNP  , BNP  , or ANP  . To test the speci icity of GC-coupled NPR, aliquots were also incubated 1 μM CNP  plus HS-142-1 (0 to 1,000 μg/ml) [31,32]. Incubations were stopped by adding 375 μl of ice cold 50 mM sodium acetate (pH 5.8) and boiling for 5 min. Samples were then centrifuged at 10,000xg for 5 min at 4 o C.
Production of cGMP was measured in the supernatants by equilibrated radioimmunoassay (RIA). In brief, standards or samples were taken up in a inal volume of 100 μl of 50 mM sodium acetate buffer (pH 4.8), and then 100 μl of diluted cGMP antiserum (Calbiochem-Novabiochem Co, San Diego, CA) and iodinated cGMP (10,000 cpm/100 μl, Speci ic activity=2,200 Ci/mmole, Du Pont-New England Nuclear, Wilmington, DE) were added and incubated for 24 hr at 4 o C. The bound form was separated from the free form by charcoal suspension. RIA for cGMP was done on the day of experiments, and all samples in an experiment were analyzed in a single assay. Nonspeci ic binding was <2.5%. The 50% intercept was at 0.39±0.03 pmol/tube (n=15). The intra and interassay coef icients of variation were 6.7 (n=12) and 8.6% (n=9), respectively. Average results of determinations were expressed as picomoles of cGMP generated per milligram protein per minute.

RT-PCR of CNP mRNA
Total RNA was extracted from the granulosa cells using TRI reagent (MRC, Cincinnati, OH) according to the manufacturer ' s protocol. Total RNA concentrations were quantitated by UV spectrophotometry. Five hundred nanograms of total RNA were suspended in 20 μl buffer containing 10 mM Tris (pH 8. RT-PCR of NPR mRNAs for subtypes: One microgram of total RNA was reverse transcribed the same method as described above. Complementary DNA products were ampli ied by PCR using the following primers [4,34]: Photographs of gels were taken with Polaroid 665 ilm. PCR products were con irmed by sequence analysis (data not shown).

Statistical analysis
The igures show the results (means±SE values) of individual experiments involving three or more replicates. These are representative of results obtained in at least three similar experiments or show data from multiple experiments pooled as described in the igure legends. Comparisons of results were performed by paired Student ' s t-test and ANOVA with Duncan multiple range test, accepting P<0.05 as the criterion of signi icance.

Bindings:
As shown in Figure  ]-CNP  occurred in the granulosa layer of the follicles. A low density of binding sites was observed in the theca externa layer of the follicles, but not in the theca interna layer and the interstitial region of the ovary ( Figure 2C). In the presence of 1 μM unlabeled CNP  , binding to the granulosa and theca externa layers of follicles was completely displaced, but the diffuse background binding was not affected ( Figure 2B). Nonspeci ic binding was 0.59±0.05% of total binding in these structures. Ten micromolar unrelated peptides including angiotensin II or arginine vasopressin did not displace the binding of ]-CNP  binding to granulosa by heterologous ligands was also investigated ( Figure 3). Unlabeled 10 μM ANP  and BNP  , as more selective endogenous ligands for NPR-A, respectively inhibited 99. ]-CNP  to the granulosa. In the presence of 10 μM unlabeled C-ANP, a speci ic ligand for NPR-C, the speci ic binding to the granulosa was inhibited by 93.22±1.61%. ]-CNP  to the granulosa layers in preovulatory follicles by increasing concentrations of unlabeled CNP  was consistent with a single high af inity binding site for  (Table 1). Increasing concentrations of ANP  , BNP  and C-ANP also progressively inhibited the speci ic binding of 125 I-[Tyr 0 ]-CNP  to granulosa ( Figure 4B). The effects were consistent with sites of a single af inity for ANP  , BNP  , and C-ANP (Table 1). C-ANP competed with much less af inity than any of the other ligands, including CNP  , for the binding sites  (Table 1).     and other ligands.

Particulate GC activity
The rate of cGMP production by particulate GC activation of granulosa cell membranes was basally 26.30 0.80 pmol/mg protein/min. As shown in Figure 5A, CNP  , BNP  and ANP  each produced dose-dependent increases in cGMP production. At 1 μM concentration, the rates of cGMP production by CNP  , BNP  and ANP  were 77.13±3.36, 41.99 1.50 and 34.97±1.51 pmol/mg protein/min, respectively. CNP  caused the highest increment of cGMP production with half-maximal response (EC 50 ) of ∼10 nM. The maximum increase in cGMP, measured in response to 1 μM CNP  was ∼3 fold over basal levels. But the maximum cGMP production stimulated by BNP  or ANP  was less than that by CNP  (p< 0.01). HS-142-1, a selective antagonist for the GC-coupled NPR, inhibited CNP  -stimulated cGMP production in a dose-dependent manner ( Figure 5B). At a concentration of 1,000 μg/ml, HS-142-1 inhibited completely the CNP  -stimulated cGMP production by the granulosa cell membranes.

Detection of NPR-B mRNA by RT-PCR
The presence of NPR-B transcripts was tested by RT-PCR. In granulosa cells, NPR-B cDNA was found in ethidium bromide stained gels with the expected size of 692 base pairs (bp) (Figure 6, lane 4) after 50 cycles of ampli ication, and NPR-A transcripts with 451 bp were also detected after 40 cycles of ampli ication ( Figure 6, lane 2). RT-PCR data from the pituitary gland of rat, which express NPR-A and NPR-B transcripts [12], were also shown for comparison. NPR-C transcripts were also found ( Figure 6). No transcripts were observed in RNA samples incubated without Moloney leukemia virus reverse transcriptase. and **P<0.01 for comparison of CNP  only-treated group vs. CNP  plus HS-142-1-treated groups.

Discussion
The new indings in our present study are the characteristics of CNP speci ic GCcoupled receptors and CNP in the pig ovarian granulosa cells, providing evidence for the presence of CNP system in these cells. Intra-ovarian NP systems have already generated considerable interest. The presence of ANP in ovarian extracts from cattle [18] and rats [19], the identi ication of mRNA encoding ANP [20], and the secretion [20] of ANP by granulosa cells of the pig ovary have all established the ovary as a site of synthesis and secretion of ANP. Furthermore, speci ic ANP binding sites have been detected in ovarian tissues [21][22][23][24][25][26]. Other studies have suggested that ovarian ANP might be involved in the regulation of follicular growth [19,20], steroidogenesis [21][22][23][24][25], and ovulation [35,36]. Nevertheless, the role of NPs within the ovary is not well understood. Recently, the presence of CNP and NPR-B have been detected in the whole rat ovary [29]. Rat ovarian CNP levels and the binding activity of the GC-coupled receptor vary with the estrous cycle, suggesting its possible role in follicular development and in the maturation or function of preovulatory follicles. However, it still has questions about the site of function and preference for NP within ovarian structures.
Our results clearly provide direct evidence for the local CNP system in the granulosa cells. Autoradiographic results revealed that speci ic binding sites of 125 I-[Tyr 0 ]-CNP  were localized in the granulosa and theca externa cell layers of primary and maturing follicles, but binding was not observed in the theca interna layer or in the interstitial region of the ovary. Ovarian tissue sections were washed by an acidic solution before incubation with 125 I-[Tyr 0 ]-CNP  to remove any unmeasured endogenous NP occupying receptors. This acidic washing signi icantly increased the maximal speci ic 125 I-[Tyr 0 ]-CNP  binding densities of the granulosa cell layer by about 17%. This suggests that the NPRs in granulosa cells are indeed exposed to endogenous NPs.
Classically, the receptors for NPs have been classi ied as biological and clearance receptors. The biological receptors consist of two subtypes, designated NPR-A and NPR-B, with two monomeric proteins of molecular mass of 120-140 kDa containing an extracellular ligand-binding domain and an intracellular guanylate cyclase (GC) catalytic domain [4,5]. Moreover, the biological receptor subtypes have been characterized by their ligand selectivity with the rank order of potency for cGMP production; relative potencies for NPR-A and NPR-B are ANP>BNP>>CNP and CNP>>BNP>ANP, respectively [4,5,8]. Therefore, the predominant ligands for NPR-A are ANP and BNP, whereas NPR-B binds selectively to CNP. The clearance receptor, NPR-C, consists of a single cloned protein which is a disul ide-linked 60-70 kDa homodimer without an intracelluar GC catalytic domain [37]. This subtype is a unique protein that binds all of NPs including C-ANP, a synthetic peptide which does not bind to either NPR-A or NPR-B [37][38][39][40].
Since speci ic binding with 125 I-[Tyr 0 ]-CNP  , which is a selective ligand for NPR-B subtype rather than NPR-A [41], was displaced by ANP  , BNP  and C-ANP, it seems likely that NPR-B in the granulosa cells either does not bind the radioligand ef iciently or is too low to be detected by in vitro autoradiographic technique. As shown in Figure   6, RT-PCR products for NPRs clearly demonstrate that the transcripts for NPR-B as well as NPR-A and NPR-C are all present in the granulosa cells. We found that an excess   to its speci ic granulosa cell layer binding sites. In preliminary studies, we have found that speci ic bindings of 125 I-ANP  in these structures were also inhibited about 95% by 10 μM of C-ANP. This implies that a main population of total NPRs in this structure is NPR-C, and the remained binding sites are NPR-B and/or NPR-A.