Presence of C-type natriuretic endothelial cells and plasma
peptide in cultured
human
ANDREW J. STINGO, ALFRED0 L. CLAVELL, DENISE M. HEUBLEIN, CHI-MING WEI, MARK R. PITTELKOW, AND JOHN C. BURNETT, JR. Cardiorenal Research Laboratory, Divisions of Cardiovascular Diseases, Nephrology, and Dermatology, Mayo Clinic and Foundation, Rochester, Minnesota 55905 L. Clavell, Denise M. Stingo, Andrew J., Alfred0 Heublein, Chi-Ming Wei, Mark R. Pittelkow, and John C. Burnett, Jr. Presence of C-type natriuretic peptide in cultured human endothelial cells and plasma. Am. J. Physiol. 263 (Heart Circ. Physiol. 32): H1318H1321, 1992.-The present study was undertaken to investigate the presence of C-type natriuretic peptide (CNP) immunoreactivity in cultured human vascular endothelial cells and in human plasma. CNP immunoreactivity was present in cultured human aortic endothelial cells by both immunohistochemical staining and by radioimmunoassay. With the utilization of gel permeation chromatography, this immunoreactivity proved to be consistent with the higher molecular weight CNP-53. CNP immunoreactivity was also present in human plasma (n = 22) at low picogram concentrations (6.5 & 0.2 pg/ml) by specific radioimmunoassay. This immunoreactivity was consistent with the lower molecular weight CNP-22 by gel permeation chromatography. These findings suggest that the vascular endothelium may be the site of CNP production. The isolation of different molecular forms of CNP in tissue and plasma may be consistent with a storage form of the peptide in endothelial cells CNP-53, while CNP-22 circulates in plasma. In summary, the present study is consistent with CNP being a peptide of endothelial cell origin. natriuretic peptides; endothelium; tochemistry; radioimmunoassay
vascular
tone; immunohis-
C-TYPE NATRIURETIC PEPTIDE (CNP) is a 22-aminoacid peptide, which shares structural similarity to the cardiac hormones atria1 natriuretic peptide (ANP) and brain natriuretic peptide (BNP) (1). While ANP, BNP, and CNP function via activation of guanosine 3’,5’-cyclic monophosphate (cGMP), Koller et al. (6) have demonstrated that CNP is the specific ligand for a guanylate cyclase-linked receptor termed the ANPR-B receptor, which is separate from the receptor that binds ANP and BNP. Suga et al. (12) have further characterized the ANPR-B receptor and demonstrated that it is highly expressed in vascular smooth muscle cells. Studies by Furuya et al. (2) have demonstrated that CNP is a potent stimulator of cGMP in cultured vascular smooth muscle cells but not cultured endothelial cells. Furthermore, CNP via cGMP is a potent inhibitor of vascular smooth muscle cell proliferation (3). We have recently reported that in vivo the biological actions of CNP are characterized by a marked hypotensive response associated with activation of systemic cGMP (13). Studies suggest that CNP messenger RNA encodes for a 126-amino acid peptide thought to be the preprohormone (11, 15). Though the processing of the putative preprohormone to the biologically active form is not well H1318
036%6135/92
$2.00
Copyright
characterized, recent studies in the porcine brain have suggested that CNP in tissue exists as CNP-22 and as an NH2-terminal extended form CNP-53 (10, 11). While ANP and BNP are known to be of cardiac origin, localization of CNP continues to emerge. Recent studies have demonstrated CNP immunoreactivity in tissue homogenates from brain, kidney, and intestines of numerous species including humans (7). Recently, our laboratory reported the presence of CNP immunoreactivity in human vascular endothelial cells in paraffin-embedded sections of breast tissue (5). Based on the report of CNP immunoreactivity in human breast endothelial cells and the known localization of CNP receptors to vascular smooth muscle cells, the present study was undertaken to determine whether CNP immunoreactivity is detectable in human cultured vascular endothelial cells and, if present, to characterize the molecular form. Second, we sought to determine whether CNP is present in normal human plasma. METHODS
Cell culture. Human aortic endothelial cells (HAEC, EndoPack-AO, Clonetics, San Diego, CA) were grown in 75-ml culture flasks in a culture medium (EGM-UV, Clonetics) containing epidermal growth factor (10 rig/ml), hydrocortisone (1 pg/ml), fetal bovine serum (2%), gentamicin (0.05 mg/ml), and amphotericin-B (0.05 pg/ml). Cells were incubated at 37”C, 5% CO,-95% 0, in a humidified atmosphere. Cells were cultured at an initial density of 3 x 10:‘/cm2 and reached confluence in -4-6 days. Cell extracts were obtained by gently scraping the cells from the culture flask into phosphate buffer containing aprotinin 0.12 TIU/ml. The cells were then subjected to sonication, and the resulting suspension was centrifuged at 12,000 rpm for 5 min. The supernatants were pipetted and stored at -20°C until the time of assay. All cells were studied between the fourth and sixth passages. Immunohistochemical staining. For immunohistochemical staining, HAEC were grown to near confluence on glass cover slips as described above and fixed in 10% buffered Formalin. Slides were then incubated with 0.6% hydrogen peroxide in methanol for 20 min at room temperature to block endogenous peroxidase activity, and 5% normal goat serum was then used to block nonspecific protein binding sites before antibody was applied. Sections were placed in a moist chamber for 18-24 h at room temperature with the primary antibody (also utilized in the radioimmunoassay, rabbit anti-CNP, Peninsula Laboratory, Belmont CA) at a dilution of 1: 1,600. Control slides were treated with normal dilute rabbit serum. Sections were incubated with the secondary antibody-horseradish peroxidase conjugate and 3-amino-9-ethylcarbazole substrate for pen cidase visualization and were counterstained with hematoxylin.
0 1992 the American
Physiological
Society
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (129.215.017.188) on January 6, 2019.
CNP
IN ENDOTHELIAL
Radioimmunoussuy (RIA). For the determination of CNP immunoreactivity in cultured HAEC, cells were grown to near confluence and cell extracts obtained as described above. The values of CNP immunoreactivity in HAEC represent the mean of three samples obtained from separate subcultures. For each subculture, duplicate samples of cell culture medium and buffer were examined as a control. The samples from the cell extracts, cell culture media, and buffer were assayed nonextracted. For the determination of CNP immunoreactivity in human plasma, venous blood samples from normal male and female volunteers (n = 22) were collected in chilled ethylenediaminetetraacetic acid (EDTA) tubes and centrifuged at 4°C and 2,500 rpm for 10 min. Plasma was separated and stored at -20°C until the time of assay. For the RIA of plasma, samples were extracted by the Vycor glass technique modified from the method of Guktowska et al. (4). Briefly 1 ml of plasma was gently mixed with 0.5 ml of Vycor glass (Corning Glass Works, Corning, NY) suspension for 1 h at 4°C. The Vycor was washed with water, and the CNP was eluted from the Vycor with 60% acetone in 0.05 molar HCl. Eluates were concentrated on a savant and pellets were resuspended in assay buffer for RIA. CNP immunoreactivity was then determined utilizing a double-antibody RIA. A specific antibody to human CNP-22 was used in the assay (Peninsula Laboratory, Belmont CA). Recovery of CNP was 72 t 6% as determined by addition of synthetic CNP to plasma. The lower limit of detection was 2 pg/tube. Intra-assay variability was determined to be 5.2%. Cross-reactivity of the CNP-22 antibody with CNP-53 was established by addition of synthetic CNP-53 (Peninsula Laboratory) to the CNP-22 assay at concentrations ranging from 2 to 500 pg. The cross-reactivity was determined to be 97 t 6%. There is no reported cross-reactivity between the specific CNP-22 antibody and ANP, BNP, or endothelin. This was verified by addition of synthetic ANP, BNP, and endothelin (Peninsula Laboratory) to the CNP assay at concentrations ranging from 0.5 to 500 pg with no detectable immunoreactivity. Gel permeation chromatography. CNP was characterized from cell extracts of HAEC and nonextracted plasma by a P-6 (Bio-Rad Laboratories, Richmond CA) gel filtration column (1 x 13 cm). One-hundred microliters of cell extract or 1 ml of plasma were applied to the column and eluted with 0.5 M acetic acid buffer. Fractions of 0.5 ml were collected and dried on a savant. The concentration of CNP in each fraction was determined by RIA as described above. The P-6 column was calibrated with synthetic l”sI-labeled CNP-22 and nonlabeled CNP-53 (Peninsula Laboratory). Figure 1 represents the calibration of the P-6 column with CNP-22 and CNP-53. CNP-53 was eluted from fractions 5 to 9 (peak fraction at 7) and ?labeled CNP-22 from fractions 7 to 17 (peak fraction at 11). The mean column recovery of total CNP was 86%. CNP-53
CNP-22
JI
JI - 12000
3--=--9 0
1 10
5
u
I 15
-10000
s -2
-8000
2
! 0 20
Fraction
Fig. 1. Calibration peptide (CNP)-22
of P-6 column and nonlabeled
with ‘“‘)I-labeled CNP-53.
C-type
natriuretic
CELLS
AND
PLASMA
HI319
Total protein in the cell extracts was determined by the Lowry method (9). Concentrations of CNP immunoreactivity in human plasma and cell extracts are expressed as means t SE. RESULTS
Figure 2 illustrates the immunohistochemical staining for CNP in HAEC. In Fig. 2A, low magnification (x200) revealed positive immunohistochemical staining in all cells visualized. Figure 2B at high-power magnification (x400) and Fig. 2C (X 1,000) revealed positive immunohistochemical staining, which was predominately perinuclear cytoplasmic staining with granularity in all cells. Figure 20 illustrates the nonimmune staining of HAEC was negative. Measurement of CNP concentration in the cell extracts of HAEC revealed a mean concentration of 3.l+ - 1.7 pg/g total protein. In the absence of HAEC there was no detectable CNP immunoreactivity in the cell culture medium or buffer controls. Figure 3 illustrates a representative gel permeation chromatography (GPC) profile for the CNP immunoreactivity measured in HAEC. GPC revealed a peak from fractions 5 to 8 with peak activity at fraction 6. This profile corresponds with the profile of CNP-53 as illustrated in Fig. 1. CNP immunoreactivity was detectable in the plasma of all normal subjects. Plasma CNP concentrations ranged from 5.4 to 8.1 pg/ml. The mean plasma concentration for CNP in human plasma was 6.5 t 0.2 pg/ml. Figure 4 illustrates a representative GPC profile for CNP immunoreactivity in human plasma. GPC revealed a peak from fractions 10 to 15 with peak activity at fraction 13. This profile corresponds with the profile of 1251-labeled CNP-22 as illustrated in Fig. 1, although there is a slight shift to the right with human plasma compared with GPC utilizing iodinated CNP-22. This may be in part due to the differential handling of endogenous CNP-22 in plasma and iodinated CNP-22 in buffer. DISCUSSION
The present study demonstrates that CNP immunoreactivity is present in cultured human endothelial cells by both immunohistochemistry and RIA. This immunoreactivity corresponds to the higher molecular weight form CNP-53. The current study also demonstrates for the first time that CNP immunoreactivity is detectable in human plasma and circulates in low picogram concentrations. This immunoreactivity corresponds to the low molecular weight peptide CNP-22. The current study importantly extends previous reports by documenting the existence of CNP immunoreactivity in human tissue outside of the central nervous system. CNP was originally localized exclusively to the brain (1, 10, 14, 16), yet recent investigations have documented CNP immunoreactivity in tissue homogenates from the kidneys and intestines of rats (7). The current study clearly demonstrates the presence of CNP in cultured HAEC, which taken together with our previous report of CNP in endothelial cells from human tissue (5), is consistent with the vascular endothelium being a site for CNP production. While the current investigations localize CNP immunoreactivity to HAEC by immunohistochemistry, they also indicate that the principal intracellular form corresponds to CNP-53.
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (129.215.017.188) on January 6, 2019.
A
B
C
D
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (129.215.017.188) on January 6, 2019.
CNP
IN ENDOTHELIAI
CELLS
AND
H1321
PLASMA
findings are consistent with CNP representing a vasoactive, antimitogenic peptide of endothelial origin.
CNP-53
This work was supported by National Heart, Lung, and Blood Institute (NHLBI) Grants HL-36634 and HL-07111. A. L. Clavell was supported by a NHLBI training grant in cardiovascular disease (HL07111). J. C. B urnett, Jr. is an Established Investigator of The American Heart Association. Address for reprint requests: A. J. Stingo, Cardiorenal Research Laboratory, Mayo Clinic and Foundation, 200 First St., SW, Rochester, MN 55905. Received
17 June
1992; accepted
in final
form
31 July
1992.
REFERENCES Fig. 3. Representative extract.
gel permeation
chromatography
curve
for HAEC
CNP-22
0
Fig. 4. Representative plasma.
5
gel permeation
10 Fraction
15
20
chromatography
curve
for human
The present study also demonstrates the existence of CNP immunoreactivity in human plasma at low picogram concentrations. Although this may be consistent with CNP functioning as a circulating hormone, the high expression of CNP receptors in vascular smooth muscle (12) and the localization of CNP immunoreactivity to endothelial cells is also consistent with CNP functioning as a paracri ne factor in the regulation of vascular tone and smooth muscle cell growth (3, 13). An analogy would be the vasoconstrictor peptide of endothelial origin, endothelin, which like CNP, circulates at low picogram concentrations (8). In contrast to HAEC, CNP-22 appears to be the principal form of the peptide in human plasma. This observation has several important implications. One could speculate that CNP-53 is an intermediate storage form of CNP-22. If CNP-53 represents a prohormone, its conversion to CNP -22 may involve cleavage by a putative protease resu lting in release of a vasoactive peptide of endothelial origin into the circulation. Indeed, the presence of two distinct molecular forms of CNP in porcine brain has been reported by several investigators (10, 11). The present study, taken together with previ .ous reports, supports the existence of a complex system of structurally related peptides consisting of two circulating hormones of cardiac origin, ANP and BNP, and a paracrine substance of endothelial origin CNP. Together, these three peptides may serve in the integrated regulation of renal and cardiovascular homeostasis. In conclusion, this study demonstrates the presence of CNP immunoreactivity in cultured human endothelial cells and human plasma. Furthermore, these studies provide further insight by characterizing the molecular forms of CNP present in endothelial cells and plasma. These
1. Arimura, J. J., N. Minamino, K. Kangawa, and H. Matsuo. Isolation and identification of C-type natriuretic peptide in chicken brain. Biochem. Biophys. Res. Commun. 174: 142-148, 1991. 2. Furuya, M., M. Takehisa, Y. Minamitake, Y. Kitajima, and Y. Hayashi. Novel natriuretic peptide,CNP, potently stimulates cyclic GMP production in rat cultured vascular smooth muscle cells. Biochem. Biophys. Res. Commun. 170: 201-208, 1990. 3c . Furuya, M., M. Yoshida, Y. Hayashi, N. Ohnuma, N. Minamino, K. Kangawa, and H. Matsuo. C-type natriuretic peptide is a growth inhibitor of rat vascular smooth muscle cells. Biochem. Biophys. Res. Commun. 177: 927-931, 1991. 4. Guktowska, J., K. Horky, C. Lachance, K. Racz, R. Garcia, G. Thibault, 0. Kuchel, J. Genest, and M. Cantin. Atria1 natriuretic factor in spontaneously hypertensive rats. Hypertension Dallas 8, Suppl. I: 1-137-I-140, 1986. 5. Heublein, D. M., A. L. Clavell, A. J. Stingo, A. Lerman, L. Wold, and J. C. Burnett, Jr. C-type natriuretic peptide immunoreactivity in human breast vascular endothelial cells. Peptides In press. 6. Koller, K. J., D. G. Lowe, G. L. Bennett, N. Minamino, K. Kangawa, H. Matsuo, and D. V. Goeddel. Selective activation of the B-natriuretic peptide receptor by C-type natriuretic peptide (CNP). Science Wash. DC. 252: 120-123, 1991. 7. Komatsu, Y., K. Nakao, S. Suga, Y. Ogawa, M. Mukoyama, H. Arai, G. Shirakami, K. Hosoda, 0. Nakagawa, and N. Hama. C-type natriuretic peptide in rats and humans. Endocrinology 129: 1104-l 106, 1991. 8. Lerman, A., B. Edwards, J. Hallet, D. Heublein, S. Sandberg, and J. C. Burnett, Jr. Circulating and tissue endothelin immunoreactivity in advanced atherosclerosis. N. Engl. J. Med. 325: 997-1001, 1991. 9. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275, 1951. 10. Minamino, N., K. Kangawa, and H. Matsuo. N-terminally extended form of C-type natriuretic peptide CNP-53 identified in porcine brain. Biochem. Biophys. Res. Commun. 170: 973-979, 1990. 11. Ogawa, Y., K. Nakao, 0. Nakagawa, Y. Komatsu, K. Hosoda, S. Suga, H Arai, K. Nagata, N. Yoshida, and H. Imura. Human C-type natriuretic peptide: characterization of the gene and peptide. Hypertension Dallas 19: 809-813, 1992. 12. Suga, S., K. Nakao, K. Hosoda, M. Mukoyama, Y. Ogawa, G. Shirakami, H. Arai, Y. Saito, Y. Kambayashi, K. Inouye, and H. Imura. Receptor selectivity of natriuretic peptide family, atria1 natriuretic peptide, brain natriuretic peptide, and C-type natriuretic peptide. Endocrinology 130: 229-239, 1992. 13. Stingo, A. J., A. Clavell, L. Aarhus, and J. C. Burnett, Jr. Cardiovascular and renal actions of C-type natriuretic peptide. Am. J. Physiol. 262 (Heart Circ. Physiol. 31): H308-H312, 1992. 14. Sudoh, T., N. Minamino, K. Kangawa, and H. Matsuo. C-type natriuretic peptide (CNP) a new member of the natriuretic peptide family identified in porcine brain. Biochem. Biophys. Res. Commun. 168: 863-870, 1990. 15. Tawaragi, Y., K. Fuchimura, S. Tanaka, N. Minamino, K. Kangawa, and H. Matsuo. Gene and precursor structures of human C-type natriuretic peptide. Biochem. Biophys. Res. Commun. 175: 645-651, 1991. 16. Ueda, S., N. Minamino, M. Aburaya, K. Kangawa, S. Matsukura, and H. Matsuo. Distribution and characterization of immunoreactive porcine C-type natriuretic peptide. Biochem. Biophys. Res. Commun. 175: 759-767, 1991.
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (129.215.017.188) on January 6, 2019.
peptide in cultured
human
ANDREW J. STINGO, ALFRED0 L. CLAVELL, DENISE M. HEUBLEIN, CHI-MING WEI, MARK R. PITTELKOW, AND JOHN C. BURNETT, JR. Cardiorenal Research Laboratory, Divisions of Cardiovascular Diseases, Nephrology, and Dermatology, Mayo Clinic and Foundation, Rochester, Minnesota 55905 L. Clavell, Denise M. Stingo, Andrew J., Alfred0 Heublein, Chi-Ming Wei, Mark R. Pittelkow, and John C. Burnett, Jr. Presence of C-type natriuretic peptide in cultured human endothelial cells and plasma. Am. J. Physiol. 263 (Heart Circ. Physiol. 32): H1318H1321, 1992.-The present study was undertaken to investigate the presence of C-type natriuretic peptide (CNP) immunoreactivity in cultured human vascular endothelial cells and in human plasma. CNP immunoreactivity was present in cultured human aortic endothelial cells by both immunohistochemical staining and by radioimmunoassay. With the utilization of gel permeation chromatography, this immunoreactivity proved to be consistent with the higher molecular weight CNP-53. CNP immunoreactivity was also present in human plasma (n = 22) at low picogram concentrations (6.5 & 0.2 pg/ml) by specific radioimmunoassay. This immunoreactivity was consistent with the lower molecular weight CNP-22 by gel permeation chromatography. These findings suggest that the vascular endothelium may be the site of CNP production. The isolation of different molecular forms of CNP in tissue and plasma may be consistent with a storage form of the peptide in endothelial cells CNP-53, while CNP-22 circulates in plasma. In summary, the present study is consistent with CNP being a peptide of endothelial cell origin. natriuretic peptides; endothelium; tochemistry; radioimmunoassay
vascular
tone; immunohis-
C-TYPE NATRIURETIC PEPTIDE (CNP) is a 22-aminoacid peptide, which shares structural similarity to the cardiac hormones atria1 natriuretic peptide (ANP) and brain natriuretic peptide (BNP) (1). While ANP, BNP, and CNP function via activation of guanosine 3’,5’-cyclic monophosphate (cGMP), Koller et al. (6) have demonstrated that CNP is the specific ligand for a guanylate cyclase-linked receptor termed the ANPR-B receptor, which is separate from the receptor that binds ANP and BNP. Suga et al. (12) have further characterized the ANPR-B receptor and demonstrated that it is highly expressed in vascular smooth muscle cells. Studies by Furuya et al. (2) have demonstrated that CNP is a potent stimulator of cGMP in cultured vascular smooth muscle cells but not cultured endothelial cells. Furthermore, CNP via cGMP is a potent inhibitor of vascular smooth muscle cell proliferation (3). We have recently reported that in vivo the biological actions of CNP are characterized by a marked hypotensive response associated with activation of systemic cGMP (13). Studies suggest that CNP messenger RNA encodes for a 126-amino acid peptide thought to be the preprohormone (11, 15). Though the processing of the putative preprohormone to the biologically active form is not well H1318
036%6135/92
$2.00
Copyright
characterized, recent studies in the porcine brain have suggested that CNP in tissue exists as CNP-22 and as an NH2-terminal extended form CNP-53 (10, 11). While ANP and BNP are known to be of cardiac origin, localization of CNP continues to emerge. Recent studies have demonstrated CNP immunoreactivity in tissue homogenates from brain, kidney, and intestines of numerous species including humans (7). Recently, our laboratory reported the presence of CNP immunoreactivity in human vascular endothelial cells in paraffin-embedded sections of breast tissue (5). Based on the report of CNP immunoreactivity in human breast endothelial cells and the known localization of CNP receptors to vascular smooth muscle cells, the present study was undertaken to determine whether CNP immunoreactivity is detectable in human cultured vascular endothelial cells and, if present, to characterize the molecular form. Second, we sought to determine whether CNP is present in normal human plasma. METHODS
Cell culture. Human aortic endothelial cells (HAEC, EndoPack-AO, Clonetics, San Diego, CA) were grown in 75-ml culture flasks in a culture medium (EGM-UV, Clonetics) containing epidermal growth factor (10 rig/ml), hydrocortisone (1 pg/ml), fetal bovine serum (2%), gentamicin (0.05 mg/ml), and amphotericin-B (0.05 pg/ml). Cells were incubated at 37”C, 5% CO,-95% 0, in a humidified atmosphere. Cells were cultured at an initial density of 3 x 10:‘/cm2 and reached confluence in -4-6 days. Cell extracts were obtained by gently scraping the cells from the culture flask into phosphate buffer containing aprotinin 0.12 TIU/ml. The cells were then subjected to sonication, and the resulting suspension was centrifuged at 12,000 rpm for 5 min. The supernatants were pipetted and stored at -20°C until the time of assay. All cells were studied between the fourth and sixth passages. Immunohistochemical staining. For immunohistochemical staining, HAEC were grown to near confluence on glass cover slips as described above and fixed in 10% buffered Formalin. Slides were then incubated with 0.6% hydrogen peroxide in methanol for 20 min at room temperature to block endogenous peroxidase activity, and 5% normal goat serum was then used to block nonspecific protein binding sites before antibody was applied. Sections were placed in a moist chamber for 18-24 h at room temperature with the primary antibody (also utilized in the radioimmunoassay, rabbit anti-CNP, Peninsula Laboratory, Belmont CA) at a dilution of 1: 1,600. Control slides were treated with normal dilute rabbit serum. Sections were incubated with the secondary antibody-horseradish peroxidase conjugate and 3-amino-9-ethylcarbazole substrate for pen cidase visualization and were counterstained with hematoxylin.
0 1992 the American
Physiological
Society
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (129.215.017.188) on January 6, 2019.
CNP
IN ENDOTHELIAL
Radioimmunoussuy (RIA). For the determination of CNP immunoreactivity in cultured HAEC, cells were grown to near confluence and cell extracts obtained as described above. The values of CNP immunoreactivity in HAEC represent the mean of three samples obtained from separate subcultures. For each subculture, duplicate samples of cell culture medium and buffer were examined as a control. The samples from the cell extracts, cell culture media, and buffer were assayed nonextracted. For the determination of CNP immunoreactivity in human plasma, venous blood samples from normal male and female volunteers (n = 22) were collected in chilled ethylenediaminetetraacetic acid (EDTA) tubes and centrifuged at 4°C and 2,500 rpm for 10 min. Plasma was separated and stored at -20°C until the time of assay. For the RIA of plasma, samples were extracted by the Vycor glass technique modified from the method of Guktowska et al. (4). Briefly 1 ml of plasma was gently mixed with 0.5 ml of Vycor glass (Corning Glass Works, Corning, NY) suspension for 1 h at 4°C. The Vycor was washed with water, and the CNP was eluted from the Vycor with 60% acetone in 0.05 molar HCl. Eluates were concentrated on a savant and pellets were resuspended in assay buffer for RIA. CNP immunoreactivity was then determined utilizing a double-antibody RIA. A specific antibody to human CNP-22 was used in the assay (Peninsula Laboratory, Belmont CA). Recovery of CNP was 72 t 6% as determined by addition of synthetic CNP to plasma. The lower limit of detection was 2 pg/tube. Intra-assay variability was determined to be 5.2%. Cross-reactivity of the CNP-22 antibody with CNP-53 was established by addition of synthetic CNP-53 (Peninsula Laboratory) to the CNP-22 assay at concentrations ranging from 2 to 500 pg. The cross-reactivity was determined to be 97 t 6%. There is no reported cross-reactivity between the specific CNP-22 antibody and ANP, BNP, or endothelin. This was verified by addition of synthetic ANP, BNP, and endothelin (Peninsula Laboratory) to the CNP assay at concentrations ranging from 0.5 to 500 pg with no detectable immunoreactivity. Gel permeation chromatography. CNP was characterized from cell extracts of HAEC and nonextracted plasma by a P-6 (Bio-Rad Laboratories, Richmond CA) gel filtration column (1 x 13 cm). One-hundred microliters of cell extract or 1 ml of plasma were applied to the column and eluted with 0.5 M acetic acid buffer. Fractions of 0.5 ml were collected and dried on a savant. The concentration of CNP in each fraction was determined by RIA as described above. The P-6 column was calibrated with synthetic l”sI-labeled CNP-22 and nonlabeled CNP-53 (Peninsula Laboratory). Figure 1 represents the calibration of the P-6 column with CNP-22 and CNP-53. CNP-53 was eluted from fractions 5 to 9 (peak fraction at 7) and ?labeled CNP-22 from fractions 7 to 17 (peak fraction at 11). The mean column recovery of total CNP was 86%. CNP-53
CNP-22
JI
JI - 12000
3--=--9 0
1 10
5
u
I 15
-10000
s -2
-8000
2
! 0 20
Fraction
Fig. 1. Calibration peptide (CNP)-22
of P-6 column and nonlabeled
with ‘“‘)I-labeled CNP-53.
C-type
natriuretic
CELLS
AND
PLASMA
HI319
Total protein in the cell extracts was determined by the Lowry method (9). Concentrations of CNP immunoreactivity in human plasma and cell extracts are expressed as means t SE. RESULTS
Figure 2 illustrates the immunohistochemical staining for CNP in HAEC. In Fig. 2A, low magnification (x200) revealed positive immunohistochemical staining in all cells visualized. Figure 2B at high-power magnification (x400) and Fig. 2C (X 1,000) revealed positive immunohistochemical staining, which was predominately perinuclear cytoplasmic staining with granularity in all cells. Figure 20 illustrates the nonimmune staining of HAEC was negative. Measurement of CNP concentration in the cell extracts of HAEC revealed a mean concentration of 3.l+ - 1.7 pg/g total protein. In the absence of HAEC there was no detectable CNP immunoreactivity in the cell culture medium or buffer controls. Figure 3 illustrates a representative gel permeation chromatography (GPC) profile for the CNP immunoreactivity measured in HAEC. GPC revealed a peak from fractions 5 to 8 with peak activity at fraction 6. This profile corresponds with the profile of CNP-53 as illustrated in Fig. 1. CNP immunoreactivity was detectable in the plasma of all normal subjects. Plasma CNP concentrations ranged from 5.4 to 8.1 pg/ml. The mean plasma concentration for CNP in human plasma was 6.5 t 0.2 pg/ml. Figure 4 illustrates a representative GPC profile for CNP immunoreactivity in human plasma. GPC revealed a peak from fractions 10 to 15 with peak activity at fraction 13. This profile corresponds with the profile of 1251-labeled CNP-22 as illustrated in Fig. 1, although there is a slight shift to the right with human plasma compared with GPC utilizing iodinated CNP-22. This may be in part due to the differential handling of endogenous CNP-22 in plasma and iodinated CNP-22 in buffer. DISCUSSION
The present study demonstrates that CNP immunoreactivity is present in cultured human endothelial cells by both immunohistochemistry and RIA. This immunoreactivity corresponds to the higher molecular weight form CNP-53. The current study also demonstrates for the first time that CNP immunoreactivity is detectable in human plasma and circulates in low picogram concentrations. This immunoreactivity corresponds to the low molecular weight peptide CNP-22. The current study importantly extends previous reports by documenting the existence of CNP immunoreactivity in human tissue outside of the central nervous system. CNP was originally localized exclusively to the brain (1, 10, 14, 16), yet recent investigations have documented CNP immunoreactivity in tissue homogenates from the kidneys and intestines of rats (7). The current study clearly demonstrates the presence of CNP in cultured HAEC, which taken together with our previous report of CNP in endothelial cells from human tissue (5), is consistent with the vascular endothelium being a site for CNP production. While the current investigations localize CNP immunoreactivity to HAEC by immunohistochemistry, they also indicate that the principal intracellular form corresponds to CNP-53.
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (129.215.017.188) on January 6, 2019.
A
B
C
D
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (129.215.017.188) on January 6, 2019.
CNP
IN ENDOTHELIAI
CELLS
AND
H1321
PLASMA
findings are consistent with CNP representing a vasoactive, antimitogenic peptide of endothelial origin.
CNP-53
This work was supported by National Heart, Lung, and Blood Institute (NHLBI) Grants HL-36634 and HL-07111. A. L. Clavell was supported by a NHLBI training grant in cardiovascular disease (HL07111). J. C. B urnett, Jr. is an Established Investigator of The American Heart Association. Address for reprint requests: A. J. Stingo, Cardiorenal Research Laboratory, Mayo Clinic and Foundation, 200 First St., SW, Rochester, MN 55905. Received
17 June
1992; accepted
in final
form
31 July
1992.
REFERENCES Fig. 3. Representative extract.
gel permeation
chromatography
curve
for HAEC
CNP-22
0
Fig. 4. Representative plasma.
5
gel permeation
10 Fraction
15
20
chromatography
curve
for human
The present study also demonstrates the existence of CNP immunoreactivity in human plasma at low picogram concentrations. Although this may be consistent with CNP functioning as a circulating hormone, the high expression of CNP receptors in vascular smooth muscle (12) and the localization of CNP immunoreactivity to endothelial cells is also consistent with CNP functioning as a paracri ne factor in the regulation of vascular tone and smooth muscle cell growth (3, 13). An analogy would be the vasoconstrictor peptide of endothelial origin, endothelin, which like CNP, circulates at low picogram concentrations (8). In contrast to HAEC, CNP-22 appears to be the principal form of the peptide in human plasma. This observation has several important implications. One could speculate that CNP-53 is an intermediate storage form of CNP-22. If CNP-53 represents a prohormone, its conversion to CNP -22 may involve cleavage by a putative protease resu lting in release of a vasoactive peptide of endothelial origin into the circulation. Indeed, the presence of two distinct molecular forms of CNP in porcine brain has been reported by several investigators (10, 11). The present study, taken together with previ .ous reports, supports the existence of a complex system of structurally related peptides consisting of two circulating hormones of cardiac origin, ANP and BNP, and a paracrine substance of endothelial origin CNP. Together, these three peptides may serve in the integrated regulation of renal and cardiovascular homeostasis. In conclusion, this study demonstrates the presence of CNP immunoreactivity in cultured human endothelial cells and human plasma. Furthermore, these studies provide further insight by characterizing the molecular forms of CNP present in endothelial cells and plasma. These
1. Arimura, J. J., N. Minamino, K. Kangawa, and H. Matsuo. Isolation and identification of C-type natriuretic peptide in chicken brain. Biochem. Biophys. Res. Commun. 174: 142-148, 1991. 2. Furuya, M., M. Takehisa, Y. Minamitake, Y. Kitajima, and Y. Hayashi. Novel natriuretic peptide,CNP, potently stimulates cyclic GMP production in rat cultured vascular smooth muscle cells. Biochem. Biophys. Res. Commun. 170: 201-208, 1990. 3c . Furuya, M., M. Yoshida, Y. Hayashi, N. Ohnuma, N. Minamino, K. Kangawa, and H. Matsuo. C-type natriuretic peptide is a growth inhibitor of rat vascular smooth muscle cells. Biochem. Biophys. Res. Commun. 177: 927-931, 1991. 4. Guktowska, J., K. Horky, C. Lachance, K. Racz, R. Garcia, G. Thibault, 0. Kuchel, J. Genest, and M. Cantin. Atria1 natriuretic factor in spontaneously hypertensive rats. Hypertension Dallas 8, Suppl. I: 1-137-I-140, 1986. 5. Heublein, D. M., A. L. Clavell, A. J. Stingo, A. Lerman, L. Wold, and J. C. Burnett, Jr. C-type natriuretic peptide immunoreactivity in human breast vascular endothelial cells. Peptides In press. 6. Koller, K. J., D. G. Lowe, G. L. Bennett, N. Minamino, K. Kangawa, H. Matsuo, and D. V. Goeddel. Selective activation of the B-natriuretic peptide receptor by C-type natriuretic peptide (CNP). Science Wash. DC. 252: 120-123, 1991. 7. Komatsu, Y., K. Nakao, S. Suga, Y. Ogawa, M. Mukoyama, H. Arai, G. Shirakami, K. Hosoda, 0. Nakagawa, and N. Hama. C-type natriuretic peptide in rats and humans. Endocrinology 129: 1104-l 106, 1991. 8. Lerman, A., B. Edwards, J. Hallet, D. Heublein, S. Sandberg, and J. C. Burnett, Jr. Circulating and tissue endothelin immunoreactivity in advanced atherosclerosis. N. Engl. J. Med. 325: 997-1001, 1991. 9. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275, 1951. 10. Minamino, N., K. Kangawa, and H. Matsuo. N-terminally extended form of C-type natriuretic peptide CNP-53 identified in porcine brain. Biochem. Biophys. Res. Commun. 170: 973-979, 1990. 11. Ogawa, Y., K. Nakao, 0. Nakagawa, Y. Komatsu, K. Hosoda, S. Suga, H Arai, K. Nagata, N. Yoshida, and H. Imura. Human C-type natriuretic peptide: characterization of the gene and peptide. Hypertension Dallas 19: 809-813, 1992. 12. Suga, S., K. Nakao, K. Hosoda, M. Mukoyama, Y. Ogawa, G. Shirakami, H. Arai, Y. Saito, Y. Kambayashi, K. Inouye, and H. Imura. Receptor selectivity of natriuretic peptide family, atria1 natriuretic peptide, brain natriuretic peptide, and C-type natriuretic peptide. Endocrinology 130: 229-239, 1992. 13. Stingo, A. J., A. Clavell, L. Aarhus, and J. C. Burnett, Jr. Cardiovascular and renal actions of C-type natriuretic peptide. Am. J. Physiol. 262 (Heart Circ. Physiol. 31): H308-H312, 1992. 14. Sudoh, T., N. Minamino, K. Kangawa, and H. Matsuo. C-type natriuretic peptide (CNP) a new member of the natriuretic peptide family identified in porcine brain. Biochem. Biophys. Res. Commun. 168: 863-870, 1990. 15. Tawaragi, Y., K. Fuchimura, S. Tanaka, N. Minamino, K. Kangawa, and H. Matsuo. Gene and precursor structures of human C-type natriuretic peptide. Biochem. Biophys. Res. Commun. 175: 645-651, 1991. 16. Ueda, S., N. Minamino, M. Aburaya, K. Kangawa, S. Matsukura, and H. Matsuo. Distribution and characterization of immunoreactive porcine C-type natriuretic peptide. Biochem. Biophys. Res. Commun. 175: 759-767, 1991.
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (129.215.017.188) on January 6, 2019.
