C-type Natriuretic Peptide Synthesis Research Articles

Amino-terminal propeptide of C-type natriuretic peptide (NTproCNP) predicts height velocity in healthy children.

C-type natriuretic peptide (CNP) is a paracrine regulatory factor of the growth plate and plays a key role in endochondral growth. Its amino-terminal propeptide (NTproCNP) is an equimolar product of CNP biosynthesis and is easily measured in plasma. Preliminary studies suggest that NTproCNP levels correlate with height velocity in sheep and children. The objectives of the study were to correlate NTproCNP levels with height velocity and to define the reference range for plasma CNP and NTproCNP across childhood. This was a prospective, cross-sectional, observational study of healthy children. Participants were 258 healthy children between 2months and 20years of age. Anthropometrics were obtained and CNP and NTproCNP levels were determined by radioimmunoassay. For both sexes, CNP and NTproCNP levels were high in infancy, lower in early childhood, rising during puberty, then falling to low adult levels. Levels of NTproCNP peaked at 14·1years in boys and 11·9years in girls, coincident with the age of peak height velocity. Levels of NTproCNP varied with pubertal status, peaking at genital Tanner stage IV in boys and III in girls. There was a highly significant correlation between NTproCNP and height velocity. C-type natriuretic peptide plays an integral role in endochondral growth. We show here that CNP synthesis (as measured by NTproCNP levels in plasma) is closely related to linear growth in healthy children at all ages. We propose NTproCNP as a biomarker of linear growth.

Skeletal contributions to plasma CNP forms: Evidence from regional sampling in growing lambs

Unlike the cardiac circulating hormones, atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP), C-type natriuretic peptide (CNP) appears to be largely tissue-based and circulates at concentrations considered insufficient to affect organ function. Consistent with CNP's crucial role in regulating skeletal growth, serial studies in juveniles show that both plasma CNP and aminoterminal proCNP (NTproCNP) are highly correlated with growth velocity raising the possibility that skeletal tissues contribute to circulating concentrations of CNP forms during the growing period. Hypothesizing that venous blood draining from bone dense regions is relatively enriched in CNP, we have performed trans-organ regional blood sampling for measurement of CNP forms in 4-week-old lambs and compared the findings to simultaneous levels of ANP and BNP. Because bone growth and CNP synthesis are inhibited by glucocorticoids, identical studies were also undertaken in lambs pretreated with dexamethasone. Highly significant positive arterio-venous gradients of CNP were found across the head, heart, leg and foot. Dexamethasone significantly reduced the CNP arterio-venous gradient across the head and leg but not heart, liver or kidney. In contrast, there was no evidence of tissue secretion of ANP or BNP except across the heart, and no effect on these gradients from dexamethasone. These findings of CNP enrichment in samples from bone dense regions in growing lambs, and their selective reduction by dexamethasone, provide in vivo evidence linking plasma and skeletal tissue concentrations of CNP and further support the use of plasma CNP forms as markers of bone growth.

Effect of sex steroids on plasma C-type natriuretic peptide forms: stimulation by oestradiol in lambs and adult sheep

Although C-type natriuretic peptide (CNP) is crucial to post-natal endochondral growth, roles for the hormone in pubertal bone growth and the physiological effects of sex steroid substitution on CNP synthesis are not known. Using a plasma marker of CNP tissue synthesis (amino-terminal proCNP, NTproCNP), we have studied the effect of exogenous oestrogen (E(2)) or testosterone (T) on plasma CNP forms and bone alkaline phosphatase (bALP) in pre-pubertal lambs. Responses to E(2) in non-cycling adult ewes were also studied. In 15-week-old intact ewe lambs, E(2) promptly increased plasma NTproCNP and CNP (P<0.001) to peak on day 2, and bALP (P<0.001 peaking on day 7), whereas no significant stimulation in response to T was observed in male lambs. Linear bone growth and live weight were unaffected. In adult anoestrous ewes, basal concentrations of CNP forms and bALP were lower than in ewe lambs, in keeping with skeletal maturity, but adults responded similarly to E(2). Prompt and sustained increases in NTproCNP and CNP, and a later threefold rise in bALP (all P<0.001), were induced by E(2). Possible contributions to these increases in plasma CNP forms by reproductive tissues (a known site of E(2)-induced CNP expression) were excluded by showing similar E(2)-induced CNP responses in adult ewes after surgical removal of reproductive tissues. These results are the first to show that E(2) stimulates plasma CNP forms and bALP in lambs and adult sheep and raise the possibility that CNP also participates in bone formation in the mature skeleton.

Response of plasma CNP forms to acute anabolic and catabolic interventions in growing lambs

Using a novel marker of C-type natriuretic peptide (CNP) synthesis [amino-terminal pro-CNP (NT-proCNP)], we have recently shown that plasma NT-proCNP is strongly correlated with skeletal growth and markers of bone formation and is reversibly reduced by glucocorticoids. The effects on CNP of other catabolic or anabolic factors, known to affect skeletal growth, are unknown. Accordingly, we have studied the response of plasma CNP forms to acute catabolic (caloric restriction) and anabolic [growth hormone (GH) stimulation] interventions in lambs and related the findings to circulating IGF-I levels, growth velocity, and markers of bone formation. Lambs fed a reduced caloric intake (25% of normal) for 6 days exhibited reduced live weight, plasma urea, and IGF-I (P < 0.001 for all) compared with control lambs. Basal levels of NT-proCNP (40.1 +/- 0.9 pmol/l) fell promptly to a nadir (28.1 +/- 0.8 pmol/l, P < 0.001) on day 6, returning rapidly to basal levels upon refeeding. Although plasma alkaline phosphatase (ALP) fell (P < 0.001), reductions in metacarpal growth velocity were not significant within the 12-day period of study. In contrast to caloric restriction, long-acting bovine recombinant GH (2.5 mg/kg on days 0 and 6), as expected, increased plasma IGF-I more than twofold above control for 12 days (P < 0.001). Growth velocity did not differ during the 30 days of observation, and, consistent with unchanged growth velocity, plasma NT-proCNP and ALP were also unaffected. In conclusion, CNP synthesis and markers of bone formation are acutely sensitive to catabolism but unaffected by doses of GH that fail to stimulate skeletal growth.

Extremely high conservation in the untranslated region as well as the coding region of CNP mRNAs throughout elasmobranch species

C-type natriuretic peptide (CNP) is a crucial osmoregulatory hormone in elasmobranchs, participating in salt secretion and drinking. In contrast to teleosts and tetrapods in which the NP family is composed of a group of structurally related peptides, we have shown that CNP is the sole NP in sharks. In the present study, CNP cDNAs were cloned from four species of batoids, another group of elasmobranchs. The cloned batoid CNP precursors contained a plausible mature peptide of 22 amino acid residues that is identical to most shark CNP-22s, but five successive amino acids were consistently deleted in the prosegment compared with shark precursors, supporting the diphyletic classification of sharks and rays. In addition, molecular phylogenetic trees of CNP precursors were consistent with a diphyletic interpretation. Except for the deletion, the nucleotide and deduced amino acid sequences of the CNP cDNAs are extremely well-conserved among all elasmobranch species, even between sharks and rays. Surprisingly, high conservation is evident not only for the coding region, but also for the untranslated regions. It is most likely that the high conservation is due to the low nucleotide substitution rate in the elasmobranch genome, and high selection pressure. The 3′-untranslated region of the elasmobranch CNP cDNAs contained three to six repeats of the ATTTA motif that is associated with the regulation of mRNA stability and translation efficiency. Alternative polyadenylation sites were also found; the long 3′-untranslated region contains a core of ATTTA motifs while the short form has only one or no ATTTA motif, indicating that the post-transcriptional modification of mRNA is important for regulation of CNP synthesis. These characteristics in the 3′-untranslated region were conserved among all elasmobranch CNP cDNAs. Since CNP has been implicated as a fast-acting hormone to facilitate salt secretion from the rectal gland, the conserved 3′-untranslated region most likely contributes to rapid regulation of CNP synthesis in elasmobranchs in response to acute changes in internal and external environments.

Amino-Terminal proCNP: A Putative Marker of Cartilage Activity in Postnatal Growth

Recent evidence from rodents and humans shows that C-type natriuretic peptide (CNP) plays an essential role in endochondral bone growth. We recently identified a stable product of proCNP, amino-terminal proCNP (NT-proCNP), which unlike CNP is readily measurable in human and ovine plasma. Hypothesizing that plasma NT-proCNP concentrations reflect in part CNP synthesis within growth plates of rapidly growing cartilage, we studied levels of CNP forms in both children and lambs and related these to age, growth velocity, and biochemical markers of bone turnover. Plasma NT-proCNP levels were elevated at birth and fell progressively with age. Significant associations between plasma NT-proCNP and height velocity, alkaline phosphatase, and type 1 collagen C telopeptide were identified in children (aged 5-18 y). In longitudinal animal studies, elevated plasma concentration of NT-proCNP in 1-wk-old lambs fell progressively to mature adult levels at age 27 wk. Plasma NT-proCNP showed a highly significant association with alkaline phosphatase and metacarpal growth velocity. Glucocorticoids, a treatment known to inhibit cartilage proliferation, reduced metacarpal growth elongation in 4-wk-old lambs and markedly lowered circulating NT-proCNP levels during the treatment period. In summary, NT-proCNP levels in blood show a strong association with growth velocity and markers of bone formation and may well serve as a useful marker of growth plate activity in humans and other mammals.

Sequence, circulating levels, and expression of C-type natriuretic peptide in a euryhaline elasmobranch, Carcharhinus leucas

The present study has examined expression and circulating levels of C-type natriuretic peptide (CNP) in the euryhaline bull shark, Carcharhinus leucas. Complementary DNA and deduced amino acid sequence for CNP in C. leucas were determined by RACE methods. Homology of CNP amino acid sequence in C. leucas was high both for proCNP and for mature CNP when compared with previously identified elasmobranch CNPs. Mature CNP sequence in C. leucas was identical to that in Triakis scyllia and Scyliorhinus canicula. Levels of expression of CNP mRNA were significantly decreased in the atrium but did not change in either the brain or ventricle following acclimation to a SW environment. However, circulating levels of CNP significantly increased from 86.0 ± 7.9 fmol ml −1 in FW to 144.9 ± 19.5 fmol ml −1 in SW. The results presented demonstrate that changes in environmental salinity influences both synthesis of CNP from the heart and also circulating levels in C. leucas. Potential stimulus for release and modes of action are discussed.

C-type natriuretic peptide in reproduction, pregnancy and fetal development.

C-type natriuretic peptide (CNP) belongs to the natriuretic peptide family that consists of three structurally related peptides with a 17-amino acid ring linked by a disulfide bond. In contrast to atrial and brain natriuretic peptides that are mainly cardiovascular hormones, CNP acts predominantly in an autocrine/paracrine fashion, is commonly considered to be an endothelial hormone with antimitogenic properties, and is characterized as a regulator of endochondral ossification. Its biological effects are mediated by an intracellular cGMP accumulation via specific membrane-bound guanylyl cyclase B (GC-B) activation. There is growing evidence that this peptide is also involved in various reproductive processes as well as in embryonic and fetal development. In rodents, CNP and its receptor are highly expressed in the uterus and ovaries with specific regulation during the estrous cycle. During pregnancy, CNP mRNA is detectable in mice embryos and shows an organ-specific expression in maternal reproductive tIssues with the highest concentration in the placenta. This could indicate a defined biological function of the CNP/GC-B/cGMP axis in gestation e.g. antagonizing vasoconstrictive peptides like angiotensin II. In humans, besides a postulated fetal de novo synthesis of CNP, both the peptide and its receptor are expressed in the placenta and myometrium with opposite regulation of CNP in pregnancies complicated by pre-eclampsia or intrauterine growth retardation. Since the maternal plasma levels do not reflect these alterations, one can conclude that this part of the natriuretic peptide system acts locally suggesting that CNP-stimulated cGMP release exhibits organ-specific effects. Importantly, CNP has also become a peptide with a distinct role in male reproductive processes, since endocrine function of the testis and the regulation of penile erection are regulated by the CNP/GC-B axis. This review gives a comprehensive overview of the multiple functions of CNP in reproduction and pregnancy as well as in embryonic and fetal development.

Increased C-type natriuretic peptide mRNA expression in the kidney of diabetic rats.

To investigate the responsiveness of renal-synthesized C-type natriuretic peptide (CNP) to changes in water and electrolyte balance, we measured renal CNP mRNA levels, plasma CNP concentrations and urinary CNP excretion rates in streptozotocin-induced diabetic rats eating a normal (0.26% NaCl) or low (0.04% NaCl) salt diet. Using reverse transcription-PCR followed by Southern blot analysis, we found that renal cortical and medullary CNP mRNA levels were markedly enhanced in diabetic rats from the 14th day and remained enhanced with an accompanying elevation of urinary CNP excretion rates for the entire 60-day study period. All increases of renal CNP mRNA and urinary CNP excretion rates in diabetic rats were attenuated in low salt diet-treated diabetic rats as well as insulin-treated diabetic rats. These results demonstrate that renal CNP synthesis is enhanced in diabetic rats and the increase of renal CNP mRNA is ameliorated by salt restriction and insulin treatment. These results imply that renal-synthesized CNP is responsive to the alteration of water and electrolyte homeostasis in diabetic rats.

C-type natriuretic peptide (CNP) in the pituitary: is CNP an autocrine regulator of gonadotropes?

Natriuretic peptides act via receptors with intrinsic guanylate cyclase activity to stimulate cGMP production and are thought to be important regulators of neuroendocrine systems. C-Type natriuretic peptide (CNP) is of particular interest in this regard because the highest tissue concentrations of CNP occur in the anterior pituitary, where it is a highly potent stimulator of cGMP production. Here we show that pituitaries of rats and mice contain abundant CNP prohormone messenger RNA (mRNA), but no atrial natriuretic peptide or B-type natriuretic peptide prohormone mRNAs. Using reverse transcriptase-polymerase chain reaction, both A- and B-type natriuretic peptide receptor (GC-A and GC-B, respectively) transcripts were detected in rat and mouse pituitaries, although only the GC-B mRNA was measurable by Northern blotting. Immunohistochemistry revealed CNP-positive cells in the anterior, but not posterior, pituitaries of rats, and the vast majority of these cells were identified as gonadotropes by colocalization of CNP and LH immunoreactivities. Targeted toxicity using GnRH conjugated to the ricin-A chain was used to test whether gonadotropes are also direct targets for GnRH action. The conjugate dose dependently inhibited the proliferation of alpha T3-1 cells (gonadotrope-derived cells with GnRH receptors), but had no such effect on GH3 cells (which do not have GnRH receptors). Culture of rat pituitary cells with the conjugate caused comparable reductions in CNP-stimulated cGMP production, GnRH-stimulated LH release, and CA2+ ionophore (A23187)-stimulated LH release, but did not measurably alter cAMP production in response to pituitary adenylate cyclase-activating polypeptide. We conclude that CNP is synthesized in the pituitary, where it is located predominantly in gonadotropes, and GC-B receptors expressed in the pituitary mediate the direct effects of CNP in gonadotropes. Together with the recent demonstration of CNP synthesis and action in alpha T3-1 cells, the data suggest CNP to be a novel autocrine regulator of gonadotropes.

Synthesis and localization of C-type natriuretic peptide in mammalian kidney

We have previously demonstrated the synthesis of atrial natriuretic factor (ANF) in the distal cortical nephron of the rat kidney. We now report the synthesis of C-type natriuretic peptide (CNP) in the rat, mouse, and human nephron. CNP mRNA was initially detected in the rat and mouse kidney, as well as in three transformed cell lines isolated from the proximal and distal nephron, using reverse transcription-polymerase chain reaction (RT-PCR). Confirmation of the kidney PCR product was performed by nucleotide sequence analysis and Southern hybridization. Northern hybridization of rat brain CNP cDNA to human kidney polyadenylated RNA detected a 1.4-kb gene transcript. Rat nephron segments were microdissected and subjected to RT-PCR to localize CNP mRNA. CNP mRNA was detected in the proximal convoluted tubule, cortical collecting duct, medullary thick limbs, and inner medullary collecting ducts. CNP, as detected by immunohistochemistry, was found to colocalize with CNP mRNA.