The effect of parathyroid hormone and atrial natriuretic peptide on cyclic nucleotides production and proliferation of avian epiphyseal growth plate chondroprogenitor cells

Cells derived from avian tibia epiphyseal growth plate were cultured in vitro. The cells which exhibited a polygonal phenotype and are termed chondroprogenitor cells, developed in culture as a monolayer with a doubling time of 40-48 h in 5% fetal calf serum. Production of cAMP by the chondroprogenitor cells was stimulated by human and bovine native (1-84) PTH. The effect of PTH on cAMP production could be blocked by the (3-34) PTH analog, suggesting interaction with specific receptors. cAMP production by avian chondroprogenitor cells was also stimulated by cholera toxin, forskolin, and prostaglandin E2 but not by ACTH or prostaglandin F2 alpha. PTH, cholera toxin, and forskolin also stimulated proliferation of the chondroprogenitor cells. In contrast, neither cAMP production nor proliferation of avian skin fibroblasts was affected by PTH. Human (1-28) and rat (5-28) atrial natriuretic peptide stimulated cGMP production by avian chondroprogenitor cells and also by skin fibroblasts. Atrial natriuretic peptide inhibited the basal and PTH-stimulated [3H]thymidine incorporation into DNA of chondroprogenitor cells, but did not affect avian skin fibroblast proliferation. These results suggest that the proliferation of avian epiphyseal growth plate chondroprogenitor cells is modulated by opposing mechanisms induced by PTH and ANP, probably mediated by cAMP and cGMP, respectively.     

Atrial natriuretic factor receptor heterogeneity and stimulation of particulate guanylate cyclase and cyclic GMP accumulation

Since the seminal discovery by deBold that atria contain factors that produce diuresis and natriuresis, the biologic effects attributed to ANF have expanded to the point where the name "atrial natriuretic factor" seems inappropriate. In addition to promoting diuresis and natriuresis, ANF has been shown to produce vascular smooth muscle relaxation and to inhibit the secretion of aldosterone from the adrenal cortex, renin from the juxtaglomerlular apparatus, vasopressin from the hypothalamus, and salt and water intake after central administration. ANF also promotes intestinal secretion and stimulates testosterone synthesis in Leydig cells. However, the cellular mechanisms whereby ANF elicits these diverse effects are poorly understood. ANF has been reported to inhibit adenylate cyclase in a number of tissues. However, the significance of ANF inhibition of adenylate cyclase is unknown. This effect cannot be associated with vascular relaxation since decreased cyclic AMP would be expected to promote contraction rather than relaxation. ANF inhibition of adenylate cyclase may mediate the inhibitory effects of ANF on hormone secretion from the anterior pituitary gland. The inhibition of adenylate cyclase could also explain the inhibitory effect of ANF on aldosterone synthesis, since agents that stimulate cyclic AMP increase aldosterone synthesis. However, ANF also inhibits the dibutyryl-cyclic AMP-induced stimulation of aldosterone secretion, suggesting that an inhibition of adenylate cyclase cannot account fully for the inhibitory effects of ANF on aldosterone synthesis. There is no evidence to support a role for cyclic AMP in the diuretic and natriuretic action of ANF. An inhibition of membrane phosphoinositide breakdown by ANF and the subsequent formation of IP3 and intracellular calcium release could explain the inhibitory effects of ANF on vascular contraction and steroid synthesis. However, there is very little evidence to suggest that ANF regulates phosphoinositide metabolism, while some recent studies suggest that ANF may regulate calcium fluxes in vascular tissue. Clearly, cyclic GMP has emerged as the most likely intracellular mediator of ANF effects. ANF increases cyclic GMP in a wide range of tissues by selectively activating particulate guanylate cyclase. However, it is not known which effects of ANF are mediated by cyclic GMP. The discovery that ANF increases cyclic GMP in vascular tissue clearly suggests that cyclic GMP mediates the vascular relaxation effect of ANF, since other classes of vasodilators also increase cyclic GMP. There is preliminary evidence that cyclic GMP may inhibit renin secretion and sodium transport in kidney cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Epidermal growth factor receptor gene expression in avian epiphyseal growth-plate cartilage cells: effect of serum, parathyroid hormone and atrial natriuretic peptide.

Avian chondrocytes and fibroblasts, derived from epiphyseal growth-plate and skin, respectively, were cultured in vitro. In chondrocytes, epidermal growth factor (EGF) caused a dose-dependent stimulation of proliferation. EGF receptor mRNA was not detected with the v-erb B probe in chondrocytes cultured in the presence of 5% fetal calf serum (FCS). In the absence of FCS in the medium, a time-dependent increase in the level of EGF receptor mRNA was observed. Parallel changes were also observed in the level of EGF receptor, as demonstrated by immunofluorescence using antibodies directed against avian EGF receptor. In avian fibroblasts, EGF receptor mRNA and EGF receptor levels were not affected by FCS. Furthermore, FCS did not affect the level of thyroid hormone receptor mRNA (using v-erb A as a probe) in either chondrocytes or fibroblasts. Parathyroid hormone (PTH), which acts as a mitogen in avian chondrocytes attenuated--whereas atrial natriuretic peptide (ANP), a suppressor of chondrocyte proliferation, enhanced--EGF receptor mRNA. The present results show that avian growth-plate chondrocytes respond to EGF and bear EGF receptors. The levels of EGF mRNA and EGF receptor are inversely related to cell proliferation. The results also support previous suggestions that PTH and ANP play important roles in chondrocyte proliferation, possibly through their effect on the synthesis of the EGF receptor.     

Atrial natriuretic peptide: binding and cyclic GMP response in cultured vascular smooth muscle cells from spontaneously hypertensive rats

Atrial natriuretic peptide (ANP) is vasodilatory and natriuretic, but whereas increased plasma ANP levels occur in spontaneously hypertensive rats, their elevated vascular resistance suggests inappropriate target tissue responsiveness to ANP. This study examines ANP-receptor binding properties (at 25 degrees C and 4 degrees C) in cultured vascular aortic smooth muscle cells from spontaneously hypertensive (SHR) and control Wistar-Kyoto (WKY) rats. [I125]-human ANP saturation (0.0625-12.0 nmol) profiles were analyzed using nonlinear regression (LIGAND). Vascular smooth muscle cells from WKY possessed both high affinity (KD1 0.3 nmol; R1 33 fmol/10(5) cells) and low affinity (KD2 15 nmol; R2 400 fmol/10(5) cells) binding sites for ANP. In contrast, for smooth muscle cells from SHR, two receptor forms could not be resolved using identical analytical protocols. Parameter estimates at 25 degrees C and 4 degrees C were not different for either SHR or WKY. The number of receptors for SHR (Bmax approximately 100 fmol/10(5) cells) was lower than the total number of receptors for WKY (high plus low affinity approximately 430 fmol/10(5) cells). The intermediary KD value (approximately 1.0 nmol) for ANP binding in SHR suggests an ANP-receptor interconversion from high affinity to low affinity in smooth muscle cells from SHR. Competition-binding experiments also revealed a decreased affinity for ANP in SHR-derived smooth muscle cells. The cyclic GMP response (intracellular accumulation and extracellular levels) was decreased in SHR smooth muscle cells compared to WKY, although this difference was evident only after prolonged (one hour) stimulation with ANP. Our data indicate a reduced sustained vascular responsiveness to ANP in hypertension.     

Effects of alpha-human atrial natriuretic polypeptide, sodium nitroprusside and dibutyryl cyclic GMP on aldosterone production in bovine zona glomerulosa cells

When bovine adrenal zona glomerulosa cells were incubated with alpha-human atrial natriuretic polypeptide (alpha-hANP), the basal aldosterone production in the cells was hardly affected, although the angiotensin II- or K+-stimulated production was completely inhibited. alpha-hANP was found to cause the generation of cyclic GMP in the cells. When the cells were incubated with sodium nitroprusside, the drug inhibited the angiotensin II- or K+-stimulated aldosterone production, and also generated cyclic GMP in the cells. In contrast, dibutyryl cyclic GMP was found to be a stimulator of the aldosterone response rather than an inhibitor. The results obtained in this study cast doubt on the role of cyclic GMP as an intracellular second messenger for the action of ANP on aldosterone secretion.     

Specific binding of atrial natriuretic peptide increases cyclic GMP levels in human astrocytoma cells

Specific high-affinity binding sites (dissociation constant 100 pmol/l) for atrial natriuretic peptide (ANP) have been identified in the clone D384 derived from the human astrocytoma cell line G-CCM. Unrelated peptides such as angiotensin II, vasopressin and bradykinin did not compete for these sites. Of the atrial natriuretic peptides studied, both the human and rat ANP competed equally, while peptides with either C- or N-terminal residue missing or with no internal -S-S-bond either competed less effectively or did not compete at all. Human ANP stimulated the cells to increase their intracellular level of cyclic GMP in a time- and dose-dependent manner with maximum stimulation being approached but not reached at concentrations of 1 mumol/l. These results support both the notion that ANP has an important functional role within the brain and the concept of neurotransmitter/neuromodulator communication between neurones and glia.     

Control of lipolysis by natriuretic peptides and cyclic GMP

Human fat cell lipolysis was, until recently, thought to be mediated exclusively by a cAMP-dependent protein kinase (PKA)-regulated pathway under the control of catecholamines and insulin. We have shown that atrial- and B-type natriuretic peptides (ANP and BNP respectively) stimulate lipolysis in human fat cells through a cGMP-dependent protein kinase (PKG) signaling pathway independent of cAMP production and PKA activity. Pharmacological or physiological (exercise) increases in plasma ANP levels stimulate lipid mobilization in humans. This pathway becomes important during chronic treatment with beta-adrenoceptor antagonists, which inhibit catecholamine-induced lipolysis but enhance cardiac ANP release. These findings have metabolic implications and point to potential problems when natriuretic peptide secretion is altered or during therapeutic use of recombinant BNP.     

Atrial natriuretic peptide during the elevation in blood pressure induced by sodium restriction.

This study examined the effects of dietary sodium restriction combined with unilateral nephrectomy on systolic blood pressure (SBP), heart rate, plasma renin activity (PRA) and immunoreactive atrial natriuretic peptide (iANP) in the conscious rat. SBP and heart rate, measured by photoelectric tail-cuff, were elevated in both one- and two-kidney, sodium-restricted rats compared with one- and two-kidney rats maintained on a normal-sodium intake. In addition, the SBP of one-kidney, low-sodium rats was significantly elevated compared with two-kidney, low-sodium rats on days 10 and 14 postnephrectomy. PRA was significantly elevated two- to threefold in one- and two-kidney, low-sodium rats compared with rats fed the normal-sodium chow. Plasma iANP levels in rats fed the normal-sodium diet averaged 291 +/- 45 and 277 +/- 35 pg/ml in one- and two-kidney rats, respectively. Plasma iANP levels were significantly lower in the one- and two-kidney, low-sodium rats and averaged 165 +/- 15 and 182 +/- 22 pg/ml, respectively. These results indicate that dietary sodium restriction can elevate blood pressure in the rat and that this response can be augmented by unilateral nephrectomy. In addition, the exacerbation of the hypertension by unilateral nephrectomy in sodium-restricted rats is not attributable to differences in PRA or plasma levels of iANP between one- and two-kidney, sodium-restricted rats.     

Stimulation of testosterone production by atrial natriuretic peptide in isolated mouse Leydig cells results from a promiscuous activation of cyclic AMP-dependent protein kinase by cyclic GMP.

The aim of this study was to examine the possibility that atrial natriuretic peptide-stimulated testosterone production by mouse Leydig cells results from an activation of cAMP-dependent protein kinase (kinase A) by cGMP. In these cells, both 8Br-cGMP and 8Br-cAMP could stimulate testosterone production, though the latter was approximately 50-fold more potent. Following the stimulation of the cells with the atrial peptide, a dose-related decrease in the cellular protein-bound cAMP accompanied by a concomitant increase in the protein-bound cGMP was observed. The steroidogenesis stimulated by both human chorionic gonadotrophin (hCG) and atrial peptide was inhibited in a dose-dependent manner by a cAMP antagonist, adenosine 3',5'-cyclic monophosphothioate, Rp-isomer (RpcAMPS). In a cell-free [3H]cAMP binding assay, we have shown that unlabelled cGMP and RpcAMPS could competitively inhibit the [3H]cAMP binding, confirming that cAMP, RpcAMPS and cGMP could bind to the same binding protein. Finally, in a cell-free kinase A assay system, we have demonstrated that in lysates prepared from either atrial peptide or hCG-stimulated cells, the cellular kinase A was activated to an equal extent. We conclude from the data obtained that cGMP can bind to the cAMP-binding sites of kinase A and thereby brings about a promiscuous activation of this kinase. This appears to be an underlying mechanism by which atrial peptide hormone is able to stimulate the steroidogenesis in mouse Leydig cells.     

Roles for both cyclic GMP and cyclic AMP in the inhibition of collagen-induced platelet aggregation by nitroprusside

In studies on human platelets, nitroprusside (NP) alone at 1-10 micromol/l increased platelet cyclic AMP (cAMP) by 40-70%, whereas increases in cyclic GMP (cGMP) were much larger in percentage though not in concentration terms. Collagen enhanced these increases in cAMP up to fourfold, without affecting cGMP. This effect was partly prevented by indomethacin or aspirin, indicating that platelet cyclo-oxygenase products acted synergistically with NP to increase cAMP. ADP released from the platelets by collagen tended to restrict this cAMP accumulation. Addition of 2',5'-dideoxyadenosine (DDA), an inhibitor of adenylyl cyclase, decreased both the inhibition of collagen-induced platelet aggregation by NP and the associated accumulation of cAMP without affecting cGMP, indicating that cAMP mediates part of the inhibitory effect of NP. Unlike DDA, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of guanylyl cyclase, blocked all increases in both cGMP and cAMP caused by NP, as well as the inhibition of platelet aggregation, suggesting that cAMP accumulation was secondary to that of cGMP. Human platelet cGMP-dependent protein kinase (PKG) coelectrophoresed with the purified bovine type Ibeta isoenzyme. An inhibitor of this enzyme (Rp)-beta-phenyl-1,N2-etheno-8-bromoguanosine 3',5'-cyclic-monophosphorothioate, diminished the inhibition of collagen-induced platelet aggregation by NP, but had little additional effect when DDA was present. This showed that both PKG and cAMP participate in the inhibition of collagen-induced platelet aggregation by NP. Moreover, selective activators of PKG and cAMP-dependent protein kinases had supra-additive inhibitory effects, suggesting that an optimal inhibitory effect of NP requires simultaneous activation of both enzymes.     

Cyclic AMP and cyclic GMP accumulation by isolated hamster granulosa cells stimulated by LH and FSH.

Cyclic AMP and cyclic GMP accumulation in hamster granulosa cells as a function of gonadotrophin dose (LH or FSH) and time (0-30 min) was determined. The pattern of acute cyclic AMP accumulation was similar in LH and FSH stimulated granulosa cells, except that the cells were more sensitive to FSH than LH. There was a positive dose response relationship of cyclic AMP accumulation in LH and FSH stimulated cells. LH appeared to partially inhibit FSH stimulated cyclic AMP synthesis. Cyclic GMP accumulation was distinctly different in LH and FSH stimulated cells. An inverse dose response relationship of cyclic GMP to dose LH was observed, with only the lowest dose of LH (0.005 IU/ml) stimulating cyclic GMP synthesis. FSH at 0.005 IU/ml did not stimulate cyclic GMP synthesis, but at higher doses generated cyclic GMP in a positive dose-related manner. The results suggest that specificity of hormone action in granulosa cells may be governed in part by differential on cyclic AMP and cyclic GMP in these cells.     

Atrial natriuretic peptide inhibits renin release from juxtaglomerular cells by a cGMP-mediated process.

We have examined the effect of a synthetic analogue of human alpha-atrial natriuretic peptide (ANP), APII, on renin release in cultured renal juxtaglomerular cells (JGA cells). Using cell cultures containing 80-90% renal juxtaglomerular cells, we found that ANP (10(-13)-10(-9) M) strongly inhibited renin release from the cells in a dose-dependent fashion (ki, 10 pM) to about 10% of control. Inhibition of renin release by ANP was paralleled by an increase in cellular cGMP levels; while in the presence of the cGMP-phosphodiesterase inhibitor M&B 22948 (1 mM), concentrations of ANP lower by a factor of 100 were required to obtain the same effects on renin release and cGMP levels. The guanylate cyclase inhibitor methylene blue (10 microM), on the other hand, shifted the dose-response curves for renin release and cGMP levels to 100-fold higher concentrations of ANP. Neither the influx of 45Ca into the cells nor the intracellular quin-2 signal, which is a measure for changes of intracellular Ca concentration, was in any way altered by ANP. Our results suggest that ANP inhibits renin release from juxtaglomerular cells by a cGMP-dependent process that does not involve changes in intracellular calcium.     

新活素与硝普钠治疗顽固性心衰的临床观察

目的观察新活素（重组人脑利钠肽,rhBNP）与硝普钠治疗顽固性心力衰竭的疗效与安全性。方法将62例顽固性心力衰竭患者随机分为治疗组32例,对照组30例,在常规抗心衰治疗基础上分别给予新活素和硝普钠,连续用药3d后观察疗效。结果两组治疗后临床症状好转率比较差异有统计学意义,治疗组显著优于对照组（P〈0．05）,左室射血分数、每搏输出量、心输出量、心指数及尿量改善治疗组均优于对照组。结论新活素能明显改善顽固性心力衰竭患者的血流动力学,其疗效优于硝普钠,安全性与硝普钠类似。     

Atrial natriuretic peptide inhibits transforming growth factor beta-induced Smad signaling and myofibroblast transformation in mouse cardiac fibroblasts

This study tested the hypothesis that activation of atrial natriuretic peptide (ANP)/cGMP/protein kinase G signaling inhibits transforming growth factor (TGF)-beta1-induced extracellular matrix expression in cardiac fibroblasts and defined the specific site(s) at which this molecular merging of signaling pathways occurs. Left ventricular hypertrophy and fibrosis, collagen deposition, and myofibroblast transformation of cardiac fibroblasts in response to pressure overload by transverse aortic constriction were exaggerated in ANP-null mice compared with wild-type controls. ANP and cGMP inhibited TGF-beta1-induced myofibroblast transformation, proliferation, collagen synthesis, and plasminogen activator inhibitor-1 expression in cardiac fibroblasts isolated from wild-type mice. Following pretreatment with cGMP, TGF-beta1 induced phosphorylation of Smad3, but the resultant pSmad3 could not be translocated to the nucleus. pSmad3 that had been phosphorylated with recombinant protein kinase G-1alpha was analyzed by use of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and ion trap tandem mass spectrometry. The analysis revealed phosphorylation of Ser309 and Thr388 residues, sites distinct from the C-terminal Ser423/425 residues that are phosphorylated by TGF-beta receptor kinase and are critical for the nuclear translocation and down-stream signaling of pSmad3. These results suggest that phosphorylation of Smad3 by protein kinase G is a potential molecular mechanism by which activation of ANP/cGMP/protein kinase G signaling disrupts TGF-beta1-induced nuclear translocation of pSmad3 and downstream events, including myofibroblast transformation, proliferation, and expression of extracellular matrix molecules in cardiac fibroblasts. We postulate that this process contributes to the antifibrogenic effects of the natriuretic peptide in heart.     

Modulating effects of atrial natriuretic peptide on vascular permeability and blood pressure by inhibition of cyclic phosphodiesterase GMP in the rat]

Atrial natriuretic peptide lowers arterial pressure and increases hematocrit through reduction in plasma volume caused by a transcapillary shift of plasma fluid and protein toward the interstitium. Cyclic GMP, the second messenger of atrial natriuretic peptide is catabolized by cGMP-phosphodiesterase; therefore we examined the consequences of inhibition of the phosphodiesterase on these responses using the specific cGMP inhibitor M&B 22.948. In anesthetized, bilaterally nephrectomized rats, a 45-min infusion of atrial natriuretic peptide (1 microgram/kg/min) reduced arterial pressure by 7.6 +/- 1.5% and increased hematocrit by 9 +/- 0.6% (both p < 0.01), leading to a calculated decrease in plasma volume of 14.4 +/- 0.9%. Infusion of M&B 22.948 (0.68 mg/kg/min) did not affect hematocrit and lowered arterial pressure by 8.1 +/- 0.5% (p < 0.01), an effect similar to that observed following administration of sodium nitroprusside (10 micrograms/kg/min). Simultaneous infusion of atrial natriuretic peptide and M&B 22.948 had additive arterial pressure lowering effects (-15.9 +/- 1.1%; p < 0.01 vs atrial natriuretic peptide or M&B 22.948 alone), while the increase in hematocrit of 9.4 +/- 0.7% was identical to that seen with atrial natriuretic peptide alone. Thus, M&B 22.948 amplified atrial natriuretic peptide effects on arterial pressure, but not on vascular permeability. These findings indicate differential regulation of atrial natriuretic peptide effects by inhibition of the cGMP-phosphodiesterase.     

Effect of a tumour-promoting phorbol ester on atrial peptide-induced testosterone production and cyclic GMP accumulation by isolated mouse Leydig cells

The objective of this study was to investigate the effects of 4 beta-phorbol 12-myristate 13-acetate (4 beta-PMA)--a potent activator of protein kinase C--on the responsiveness of mouse Leydig cells to stimulation with rat atriopeptin II (rAP-II). We report that, in these cells, the stimulation of testosterone production by rAP-II could be inhibited in a dose-dependent manner by 4 beta-PMA (1-200 nM). In contrast, the basal steroidogenesis was stimulated 2-fold by 4 beta-PMA. There was no inhibition of testosterone production when the cells were stimulated with 8-bromo cyclic GMP (8Br-cGMP) in the presence of 4 beta-PMA. Furthermore, addition of 4 beta-PMA resulted in a marked reduction in the amount of cGMP accumulated in response to rAP-II stimulation. 4 alpha-Phorbol 12-myristate 13-acetate (4 alpha-PMA) was found to have no effect at all. The inhibitory effect of 4 beta-PMA on steroidogenesis could be completely reversed by the addition of 0.25 mM 3-isobutyl 1-methylxanthine (IBMX), a phosphodiesterase inhibitor. Also, the 4 beta-PMA-induced lowering of cGMP content could be partially reversed by IBMX. Membrane fractions from cells treated with 4 beta-PMA or 4 alpha-PMA did not differ in their contents of either basal or rAP-II-stimulated guanylate cyclase activities. We conclude that the 4 beta-PMA-mediated inhibition of testosterone production by Leydig cells stimulated with rAP-II results from an activation of a phosphodiesterase enzyme, hypothetically through an activated protein kinase C. This leads to a reduction in the cellular cGMP content through an increased metabolic removal of cGMP formed in response to rAP-II stimulation.     

Atrial natriuretic peptide alters neither capillary filtration nor vascular compliance of both skin and skeletal muscle of humans

Summary We studied the effects of hANP 99-126 on capillary filtration and venous compliance in both the calf (mainly skeletal muscle) and foot (mainly skin) of humans. Six healthy mean received ANP (intravenous injection of 25 g followed by continuous infusion of 0.1 g·kg^–·min^–1) for 30 min. Another six men served as time controls.Capillary filtration coefficient, venous compliance, and also volume and blood flow of both calf and foot were measured repeatedly by occlusion plethysmography before, during, and after ANP. Additionally, we determined hematocrit, central venous pressure, venous pressure in the foot, arterial pressure, and heart rate.Filtration coefficients, venous compliance, blood flow of both calf and foot as well as arterial blood pressure did not change systematically during the infusion of ANP, and yet leg volume and central venous pressure (3.1±0.8 cm H₂O) decreased while both hematocrit (3.1±1.0%) and heart rate (17±11 min^–1) increased.Thus, the ANP-evoked decrease in central venous pressure and increase in hematocrit are unrelated to blood pooling or increases in capillary filtration in skeletal muscle and skin of healthy humans.