Inhibitory Effect Of C-type Natriuretic Peptide Research Articles

Mechanism of CNP-mediated DG-PKC and IP4 signaling pathway in diabetic rats with gastric motility disorder.

In the precedent research conducted by the same team, it concluded that the activities in C-type natriuretic peptide (CNP)/cyclic guanosine monophosphate (cGMP)/cyclic adenosine monophosphate (cAMP)/β-type phospholipase C (PLCβ) pathways of rat antral smooth muscle were changed due to diabetes, which was the key pathogenetic mechanism for diabetic gastric dysmotility. As the follow-on step, this study was designed to probe into the downstream signaling pathway of CNP/PLCβ. The results showed that level of α-type protein kinase C (PKCα),cell membrane to cytoplasm ratio of PKCα, cell membrane to cytoplasmic ratio of βI-type protein kinase C (PKCβI) and level of Phosphor-PKCα (P-PKCα) were significantly reduced in diabetes rat antral smooth muscle samples. The content of tetraphosphate inositol (IP4) in gastric antral smooth muscle of diabetic rats reduced, and the content of diacyl-glycerol (DG) was unchanged. CNP significantly decreased the content of IP4 and DG, this effect was more obvious in diabetic rats. Subsequent to the addition of protein kinase A (PKA) blocker N-[2- (p-Bromocin-namylamino)ethyl]-5 -isoquinolinesulfonamide dihydrochloride (H-89) before CNP treatment, the inhibitory effect of CNP was reduced; subsequent to the addition of protein kinase G (PKG) blocker KT5823 before CNP treatment, the inhibitory effect of CNP was also reduced. With the addition of the combination of H-89 and KT5823 before CNP treatment, the inhibition by CNP could be offset. These results were concluded that CNP inhibited the activity of PKC family in rat smooth muscle and reduced the levels of IP4 and DG through the PKG/PKA-PLCβ pathways, causing inhibited muscular contractions, which may be a key pathogenetic factor for diabetic gastroparesis.

The role of CNP-mediated PKG/PKA-PLCβ pathway in diabetes-induced gastric motility disorder

Our previous work demonstrated that the C-type natriuretic peptide (CNP)/cyclic guanosine monophosphate (cGMP)/cyclic adenosine monophosphate (cAMP) pathway in gastric antrum smooth muscle of rats with diabetes was upregulated and played an important role in the development of diabetic gastric dysmotility. Our goal for this study was to explore the downstream signaling pathways of CNP. We found that the expressions of protein kinase G (PKG) and protein kinase A (PKA) in gastric smooth muscle tissue of rats with diabetes were significantly upregulated. The expressions of β-type phospholipase C 3(PLCβ3) and β-type phospholipase C 1(PLCβ1) protein were reduced, whereas Phosphor-PLCβ3Ser1105 (P-PLCβ3Ser1105) was increased. The inhibitory effect of CNP on gastric antral smooth muscle in diabetic rats was significantly greater than in the normal group. The content of trisphosphate inositol (IP3) in the gastric antral smooth muscle of rats with diabetes was significantly lower than that of the normal group. After blocking PKA with N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H-89, a blockage PKA), the effect of CNP on the production of IP3 was decreased, while blocking PKG with KT5823 (a blockage PKG) simultaneously, and CNP can no longer reduce the IP3 production. CNP promoted the phosphorylation of PLCβ3Ser1105, thereby inhibiting the activity of PLCβ3 in gastric smooth muscle tissue of rats with diabetes; this effect can be abolished by blocking PKA and PKG. These results suggested that CNP can decrease IP3 level in gastric smooth muscle cells and thus inhibit gastric smooth muscle contraction through PKG/PKA-PLCβ pathway, which may play an important role in the development of diabetic gastroparesis.

Forskolin Modulates the Inhibitory Effect of C-Type Natriuretic Peptide on Hypoxia-Induced Atrial Dynamics and Hypoxia Inducible Factor 1 Alpha Activity

Our study investigated effects of C-type natriuretic peptide (CNP) on atrial dynamics and hypoxia inducible factor 1 alpha (HIF-1α) activity in perfused beating rat atria, under hypoxic conditions. Hypoxia significantly increased the levels of HIF-1α, concomitant with decreased trial dynamics. CNP (0.1 μmol/L) further decreased atrial dynamics under hypoxia and suppressed hypoxia-induced stimulation of HIF-1α expression. An adenylylcyclase (AC) activator, forskolin (0.1 μmol/L), significantly up-regulated atrial phosphodiesterase subtype 3A (PDE 3A) protein without affecting hypoxia-induced dynamics. In the presence of forskolin, the inhibitory effects of CNP on hypoxia-induced atrial dynamics and HIF-1α levels were significantly attenuated. Forskolin also prevented hypoxia-induced downregulation of PDE3A protein. These findings suggested that CNP inhibited atrial dynamics and HIF-1α activity in the isolated perfused beating rat atria under hypoxic conditions. Furthermore, both effects were modulated by the AC activator forskolin, through activation of CNP-PDE 3A signaling.

CGMP–PDE3–cAMP signal pathway involved in the inhibitory effect of CNP on gastric motility in rat

In the present study, we investigated the mechanism of C-type natriuretic peptide (CNP)-induced inhibitory effect on spontaneous contraction of gastric antral smooth muscle to clarify CNP-NPR-B/pGC-cGMP downstream signal transduction pathway using organ bath and ELISA methods in rat. CNP significantly reduced the amplitude of the spontaneous contraction and increased the contents of cGMP and cAMP in the gastric antral smooth muscle tissue. In the presence of IBMX, a non-selective phosphodiesterase (PDE) inhibitor, the inhibitory effect of CNP on spontaneous contraction was significantly suppressed; however, the production of cGMP but not cAMP was still increased by CNP. EHNA, a PDE2 inhibitor, did not affect both CNP-induced inhibition of the contraction and CNP-induced increase of cGMP and cAMP generations in gastric smooth muscle tissue, while milrinone, a PDE3 inhibitor, similar to IBMX, attenuated the CNP-induced inhibitory effect on spontaneous contraction and increased the content of cGMP but not cAMP. The results suggest that cGMP–PDE3–cAMP signal pathway is also involved in the CNP-induced inhibition of gastric motility in rat.

Natriuretic Peptide-Dependent cGMP Signal Pathway Potentiated the Relaxation of Gastric Smooth Muscle in Streptozotocin-Induced Diabetic Rats

A common gastrointestinal complication of diabetes is gastroparesis, and patients with gastroparesis may present with early satiety, nausea, vomiting, bloating, postprandial fullness, or upper abdominal pain. However, the pathogenesis is not clear yet. A recent study indicated that atrial natriuretic peptide (ANP) was secreted from the gastric mucosa and the ANP family plays an inhibitory role in the regulation of gastrointestinal motility, but the effect of the natriuretic peptide signal pathway on diabetic gastroparesis has not been reported. The study investigated the effect of C-type natriuretic peptide (CNP) particulate guanylyl cyclase (pGC) cyclic guanosine monophosphate (cGMP) signaling on gastroparesis in streptozotocin (STZ)-induced diabetic rats. Male Sprague-Dawley rats were divided into two groups; group I: normal control rats; group II: STZ-induced diabetic rats; 4 weeks after induction, the experiments were performed. The spontaneous contraction of gastric smooth muscle strips was recorded by using physiographs in control and diabetic rats. The pGC activity in response to CNP and cGMP production in gastric smooth muscle were measured by using radioimmunoassay (RIA) in normal and diabetic rats. CNP induced a longer lasting relaxation of gastric antral circular smooth muscle strips in STZ-induced diabetic rats. The inhibitory effect of CNP on spontaneous contraction revealed a dose-dependency, and the inhibitory percentages were 25.5 +/- 1.7%, 43.6 +/- 3.2%, 85.1 +/- 2.5% in diabetic rats and 20.5 +/- 1.5%, 31.1 +/- 1.7%, 58.9 +/- 3.7% in the control group at the concentrations of 0.01, 0.03, and 0.1 mumol/l, respectively. The cGMP production and pGC activity in response to CNP in gastric muscle tissues were significantly potentiated in STZ-induced diabetic rats. Natriuretic peptide receptor type B (NPR-B) gene was expressed in the gastric smooth muscles of normal and diabetic rats, and the expression was increased in diabetic rats. The results suggest that natriuretic peptide-dependent pGC-cGMP signal is upregulated and may contribute to diabetic gastroparesis in STZ-induced diabetic rats.

C-type natriuretic peptide regulates blood–testis barrier dynamics in adult rat testes

In adult rat testes, the blood-testis barrier (BTB) in the seminiferous epithelium must "open" (or "disassemble") to accommodate the migration of preleptotene spermatocytes from the basal to the adluminal compartment that occurs at stage VIII of the epithelial cycle. However, the molecule(s) and/or mechanism(s) that regulate this event are unknown. In this report, C-type natriuretic peptide (CNP) was shown to be a regulator of BTB dynamics. Although Sertoli and germ cells contributed to the pool of CNP in the seminiferous epithelium, its receptor, natriuretic peptide receptor B, resided almost exclusively in Sertoli cells. CNP also expressed stage-specifically and localized predominantly at the BTB in the seminiferous epithelium at stage VIII of the epithelial cycle. A synthetic CNP-22 peptide, when added to Sertoli cell cultures, was shown to perturb Sertoli cell tight junction in vitro, causing disappearance of BTB-associated proteins (JAM-A, occludin, N-cadherin, and beta-catenin) from the cell-cell interface. This inhibitory effect of CNP on the tight junction was confirmed by transient overexpression of CNP in these cells, which was mediated, at least in part, by accelerating the internalization of BTB integral membrane proteins. To validate these in vitro findings, CNP-22 was administered to testes at a dose of 0.35 or 3.5 mug per testis, which was shown to perturb the BTB integrity In vivo when the barrier function was assessed by monitoring the diffusion of a small molecular probe across the BTB. In summary, CNP secreted by Sertoli and germ cells into the BTB microenvironment regulates BTB dynamics during spermatogenesis.

Pacemaker currents modulated by C-type natriuretic peptide in interstitial cells of Cajal from murine small intestine

Although the presence of C-type natriuretic peptide (CNP) in gastrointestinal tract has been demonstrated, the effect of CNP on interstitial cells of Cajal (ICC), pacemaker cells in gastrointestinal tract, is still unclear. This study was designed to investigate the effect of CNP on pacemaker currents of ICC and possible mechanisms. We used immunocytochemistry techniques to exhibit natriuretic peptide receptors (NPR) and recorded membrane currents by using whole-cell patch clamp technique on cultured ICC. Our experiment showed that NPR-A and NPR-B were expressed in ICC from murine small intestine. Whole cell recordings further showed that the amplitude of pacemaker currents in intestinal small networks of ICC was 322+/-22pA and the frequency was 16.25+/-0.95Hz. CNP significantly reduced the amplitude of pacemaker currents in small networks of ICC in a dose-dependent manner, and the amplitude was inhibited by 23.95%, 61.76% and 81.67%, the amplitude values in 329+/-28.0pA, 311.2+/-14.8pA and 295+/-26.5pA before treatment with CNP and 237.9+/-27.5pA, 119.6+/-18.5pA and 57.2+/-13.5pA after treatment with 0.01 micromolxL(-1), 0.1 micromolxL(-1) and 1pmolxL(-1) CNP, respectively. The frequencies of pacemaker currents were also significantly reduced from 16.25+/-0.95Hz of control to 13+/-0.9Hz, 12+/-0.8Hz and 3+/-0.2Hz by 0.01micromolxL 1, 0.1micromolxL(-1) and 1 micromol x L(-1) CNP, respectively. CNP also inhibited the amplitude of pacemaker currents in single ICC. The inhibitory effect of CNP was mimicked by 8-Br-cGMP, a membrane permeable cGMP analogue, which suggests that CNP could inhibit pacemaker currents via NPR-B-particulate guanylate cyclase (pGC)-cGMP signal pathway.

Inhibitory effect of C-type natriuretic peptide (CNP) on cultured cardiac myocyte hypertrophy: interference between CNP and endothelin-1 signaling pathways.

C-type natriuretic peptide (CNP) is known to play a role in the local regulation of vascular tone. We recently found that CNP is also produced by cardiac ventricular cells. However, its local effect on myocyte hypertrophy remains to be elucidated. The present study investigated the effects of CNP on cultured cardiac myocyte hypertrophy and the interaction between CNP and endothelin-1 (ET-1) signaling pathways. CNP attenuated basal and ET-1-augumented protein synthesis, atrial natriuretic peptide secretion, hypertrophy-related gene expression, GATA-4 and MEF-2 DNA binding activities, Ca(2+)/calmodulin-dependent kinase II activity, and ERK phosphorylation. CNP also inhibited ET-1-induced increase in intracellular Ca(2+) concentration. These effects of CNP were mimicked by a cGMP analog, 8-bromo cGMP. However, the inhibitory effects of CNP on the hypertrophic response of myocytes were significantly diminished at high concentrations of ET-1. Although CNP increased intracellular cGMP levels in myocytes, ET-1 suppressed CNP-induced cellular cGMP accumulation. A protein kinase C activator and Ca(2+) ionophore mimicked this suppressive effect of ET-1. We further examined the effect of CNP on the paracrine action of ET-1 secreted from cardiac nonmyocytes. CNP and 8-bromo cGMP significantly inhibited ET-1 secretion from nonmyocytes. Although nonmyocyte-conditioned medium increased the protein synthesis in myocytes through endogenous ET-1 action, this increase was significantly attenuated by pretreatment of nonmyocytes with CNP and 8-bromo cGMP. These findings demonstrate that CNP inhibits ET-1-induced cardiac myocyte hypertrophy via a cGMP-dependent mechanism, and conversely, ET-1 inhibits CNP signaling by a protein kinase C- and Ca(2+)-dependent mechanism, suggesting mutual interference between CNP and ET-1 signaling pathways.

NPR-C receptors are involved in C-type natriuretic peptide response on bile secretion

C-type natriuretic peptide (CNP) is a member of the natriuretic peptide family. Previous studies reported the presence of natriuretic peptide receptors and mRNA CNP in the liver. In the present work, we sought to establish the role of CNP in the regulation of bile secretion in the rat and the possible pathways involved. CNP diminished basal as well as bile salt-evoked bile flow and bile acid output in a dose-dependent manner. It also reduced the excretion of sodium, chloride, and potassium but did not modify bile pH or the excretion of phospholipids, total proteins, and glutathione. Neither parasympathetic nor sympathetic blockade abolished CNP inhibitory response on bile secretion. The selective NPR-C agonist, C-ANP-(4-23) amide, diminished bile flow and the co-administration of both peptides did not further decrease it. CNP did not alter mean arterial pressure or portal venous pressure at any given doses. CNP decreased bile acid-dependent flow without affecting bile acid-independent flow. The inhibitory effect of CNP did not involve the participation of the autonomic nervous system or hemodynamic changes. The participation of NPR-C receptors in CNP response is strongly supported by present findings. The present study shows that CNP modulates bile secretion in the rat, suggesting that CNP may be part of the large family of peptides involved in the regulation of gastrointestinal physiology.

Effects of adrenomedullin, C-type natriuretic peptide, and parathyroid hormone-related peptide on calcification in cultured rat vascular smooth muscle cells.

To clarify the regulating mechanism of vascular calcification, the investigators observed the effects of three vasoactive peptides, adrenomedullin (ADM), C-type natriuretic peptide (CNP), and parathyroid hormone-related peptide (PTHrP) on calcification in rat vascular smooth muscle cells (VSMCs). Beta-glycerophosphate stimulated growth and calcification in VSMCs. Adrenomedullin and CNP lowered beta-glycerophosphate-induced increase in VSMC growth. All three vasoactive peptides attenuated the increases of 45Ca accumulation, calcium content, and alkaline phosphatase activity in calcified VSMCs. As for comparing the inhibitory effects, the strongest was PTHrP. Both ADM and PTHrP increased cyclic adenosine monophosphate (cAMP) content in calcified VSMCs, but CNP upregulated cyclic guanosine monophosphate (cGMP) content. The PKA inhibitor PKAI completely reversed the inhibition of ADM on cell growth and all inhibitory effects of PTHrP on the parameters of calcification. The PKG inhibitor H8, however, strongly antagonized all the inhibitory effects of CNP on calcification. These data suggested that beta-glycerophosphate-induced calcification in VSMCs was inhibited by ADM, CNP, and PTHrP. Adrenomedullin and PTHrP inhibited VSMC calcification partially through the cAMP/PKA pathway, whereas CNP inhibited VSMC calcification through the cGMP/PKG pathway. This study could be of help in understanding the pathogenesis of vascular calcification, and providing new target for clinical treatment of cardiovascular diseases associated with vascular calcification.

Role of calcium-activated potassium currents in CNP-induced relaxation of gastric antral circular smooth muscle in guinea pigs.

To investigate ion channel mechanism in CNP-induced relaxation of gastric circular smooth muscle in guinea pigs. Spontaneous contraction of gastric smooth muscle was recorded by a four -channel physiograph. The whole cell patch-clamp technique was used to record calcium-activated potassium currents and membrane potential in the gastric myocytes isolated by collagenase. C-type natriuretic peptide (CNP) markedly inhibited the spontaneous contraction in a dose-dependent manner in gastric circular smooth muscle in guinea pigs. Ly83583, an inhibitor of guanylate cyclase, weakened CNP-induced inhibition on spontaneous contraction but Zaparinast, an inhibitor of cGMP sensitive phosphoesterase, potentiated CNP-induced inhibition in gastric circular smooth muscles. The inhibitory effects of CNP on spontaneous contraction were blocked by tetrathylammonium (TEA), a nonselective potassium channel blocker. CNP hyperpolarized membrane potential from -60.0 mV+/-2.0 mV to -68.3 mV+/-3.0 mV in a single gastric myocyte. CNP increased calcium-activated potassium currents (I(K(ca))) in a dose-dependent manner in gastric circular myocytes. CNP also increased the spontaneously transient outward currents (STOCs). Ly83583 partly blocked CNP-induced increase of calcium-activated potassium currents, but Zaparinast potented the effect. CNP inhibits spontaneous contraction, and potassium channel may be involved in the process in gastric circular smooth muscle of guinea pigs. CNP-induced increase of I(K(ca)) is mediated by a cGMP dependent pathway.

Postnatal changes in inhibitory effect of C-type natriuretic peptide on secretion of ANP.

To define developmental changes in atrial natriuretic peptide (ANP) secretion and in the cross talk between C-type natriuretic peptide (CNP) and ANP, we performed experiments in isolated perfused nonbeating cardiac atria isolated from rabbits between 1 and 8 wk of age. Changes in atrial pressure resulted in increases in atrial volume that rose with age and reached the peak value at 4 wk. A rise in volume change increased ANP secretion with concomitant translocation of extracellular fluid (ECF) into the atrial lumen, which increased with age and reached the peak value at 4 wk. The positive relationship between stretch-induced ANP secretion and ECF translocation shifted upward and leftward with age. CNP suppressed stretch-induced ANP secretion in the 8-wk-old group but not in the 2- and 4-wk-old groups without differences in ECF translocation and atrial volume. Therefore, the ANP secretion in terms of ECF translocation was markedly suppressed by CNP in the 8-wk-old group but not in the 2- and 4-wk-old groups. The production of cGMP by CNP in atrial tissue membranes was markedly attenuated in young rabbits. However, 8-bromo-cGMP suppressed stretch-induced ANP secretion in 2- and 8-wk-old groups. Natriuretic peptide receptor-B mRNA was similar in both groups. Therefore, we conclude that the inhibitory effect of CNP on atrial ANP secretion is developmentally regulated, being absent during normal cardiac development in young animals and intact in adult animals.

C-type natriuretic peptide system in rabbit oviduct

C-type natriuretic peptide (CNP), a third member of the natriuretic peptide family, is known to be distributed mainly in brain and vascular endothelium and is considered to act as a local regulator in many tissues. The purpose of this study was to determine the presence of CNP system and its biological function in rabbit oviduct. The serial dilution curve of tissue extracts was parallel to the standard curve of CNP (1–22) and a major peak of molecular profile of tissue extracts by HPLC was CNP (1–53). mRNA of CNP which was the same size as positive control was also detected by Southern blot analysis. CNP increased the production of 3′,5′-cyclic guanosine monophosphate (cGMP) in the purified membrane of oviduct, which was more in membranes derived from the isthmic portion than in the ampullar portion. The presence of mRNAs of natriuretic peptide receptor-A (NPR-A) and NPR-B was demonstrated by RT-PCR. Synthetic CNP (1–22) inhibited both frequency and amplitude of basal motility of oviduct in a dose-dependent manner. The inhibitory effect of CNP on the basal motility was more potent in the isthmic portion than in the ampullar portion. These results demonstrate the presence of CNP system in the oviduct and regional differences in motility inhibition by CNP between isthmic and ampullar portions. Therefore, these findings suggest the possible existence of a CNP system that may exert a local regulator of basal motility, either alone or in concert with other hormones.

C-Type natriuretic peptide suppresses arginine-vasopressin secretion from dissociated magnocellular neurons in newborn rat supraoptic nucleus

Central administration of C-type natriuretic peptide (CNP) affects various neuroendocrine systems. In the present study, we examined whether CNP acts directly on arginine-vasopressin (AVP) secretion from rat supraoptic nucleus (SON) neurons, using acute dissociated cell preparations. CNP inhibited the basal secretion of AVP in a dose-dependent manner (10 −11–10 −6 M). A- type natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) also suppressed the basal secretion of AVP, however, the effects were two-orders of magnitude less potent than CNP. CNP also suppressed AII-induced AVP secretion, however, the inhibitory effect of CNP was less than that of ANP or BNP. These findings suggest that CNP inhibits the basal secretion of AVP through natriuretic peptide receptor (NPR)-B and has a role in the body water and electrolyte homeostasis in the central nervous system.

C-Type natriuretic peptide is a growth inhibitor of rat vascular smooth muscle cells

C-type natriuretic peptide (CNP) which potently stimulates particulate guanylate cyclase activity in cultured rat vascular smooth muscle cells (VSMC) inhibited serum-induced DNA synthesis of the cells 10-fold more effectively than α-human atrial natriuretic peptide (α-hANP). The inhibitory effect of CNP was mimicked by 8-bromo-cGMP. The proliferation of VSMC was also suppressed by CNP more potently than α-hANP, while the peptide was less active for cGMP augmentation and for vasorelaxation than α-hANP in isolated rat aorta. These results suggest that CNP may be a growth regulating factor of VSMC rather than a vasodilator.