G(i-1)/G(i-2)-dependent signaling by single-transmembrane natriuretic peptide clearance receptor

Single-transmembrane natriuretic peptide clearance receptor (NPR-C), which is devoid of a cytoplasmic guanylyl cyclase domain, interacts with pertussis toxin (PTx)-sensitive G proteins to activate endothelial nitric oxide synthase (eNOS) expressed in gastrointestinal smooth muscle cells. We examined the ability of NPR-C to activate other effector enzymes in eNOS-deficient tenia coli smooth muscle cells; these cells expressed NPR-C and NPR-B but not NPR-A. Atrial natriuretic peptide (ANP), the selective NPR-C ligand cANP-(4-23), and vasoactive intestinal peptide (VIP) inhibited (125)I-ANP and (125)I-VIP binding to muscle membranes in a pattern indicating high-affinity binding to NPR-C. Interaction of VIP with NPR-C was confirmed by its ability to inhibit (125)I-ANP binding to membranes of NPR-C-transfected COS-1 cells. In tenia muscle cells, all ligands selectively activated G(i-1) and G(i-2); VIP also activated G(s) via VIP(2) receptors. All ligands stimulated phosphoinositide hydrolysis, which was inhibited by ANP-(1-11), PTx, and antibodies to phospholipase C-beta3 (PLC-beta3) and Gbeta. cANP-(4-23) contracted tenia muscle cells; contraction was blocked by U-73122 and PTx and by antibodies to PLC-beta3 and Gbeta in intact and permeabilized muscle cells, respectively. VIP and ANP contracted muscle cells only after inhibition of cAMP- and cGMP-dependent protein kinases. ANP and cANP-(4-23) inhibited forskolin-stimulated cAMP in a PTx-sensitive fashion. We conclude that NPR-C is coupled to activation of PLC-beta3 via betagamma-subunits of G(i-1) and G(i-2) and to inhibition of adenylyl cyclase via alpha-subunits.     

Receptor-specific ligands distinguish natriuretic peptide receptors-A and -C in primate tissues

Systemic clearance of atrial natriuretic peptide (ANP) is in part due to neutral endopeptidase (NEP) proteolysis and natriuretic peptide receptor-C (NPR-C) mediated endocytosis. Biological responses to ANP are primarily mediated by the membrane guanylyl cyclase-A/natriuretic peptide receptor-A (NPR-A). Analogs of ANP selective for NPR-A and/or resistant to NEP may have increased activity in those tissues where NPR-C and NEP are coexpressed with NPR-A. The analog of ANP termed vANP; [(R3D, G9T, R11S, M12L, G16R)ANP] is selective for human NPR-A with at least 10,000 fold reduction in affinity for human NPR-C. We report that rat NPR-A is insensitive to 10 nM vANP, demonstrating the limitations of this species in evaluating human therapeutic candidates. As an alternative approach we tested the binding and potency of receptor-selective and NEP-resistant ANP analogs in rhesus monkey tissues. Competition binding studies with a simplified version of vANP, sANP [(G9T, R11S, G16R)rANP], in rhesus monkey kidney and lung membrane preparations shows displacement of ¹²⁵I-ANP from only a fraction of the total ANP receptor population, 30 and 85%, respectively. The remaining ANP binding sites can be occupied with the NPR-C selective ligand cANP(4-23). These data strongly suggest that only two classes of ANP receptor are present in these membrane preparations, NPR-A and NPR-C. The NEP resistant sANP derivative called sANP(TAPR) was 8 fold more potent (ED₅₀ = 0.6 nM) than rANP (ED₅₀ = SnM) in stimulating cGMP production in the lung membrane preparation. Our results demonstrate that the rhesus monkey natriuretic peptide receptors reflect the pharmacology of the human receptors, and that this species may be suitable to determine the role of NPR-C and NEP in peptide clearance and attenuating functional responses.     

Dendroaspis natriuretic peptide binds to the natriuretic peptide clearance receptor

Dendroaspis natriuretic peptide (DNP) is a newly-described natriuretic peptide which lowers blood pressure via vasodilation. The natriuretic peptide clearance receptor (NPR-C) removes natriuretic peptides from the circulation, but whether DNP interacts with human NPR-C directly is unknown. The purpose of this study was to test the hypothesis that DNP binds to NPR-C. ANP, BNP, CNP, and the NPR-C ligands AP-811 and cANP(4-23) displaced [(125)I]-ANP from NPR-C with pM-to-nM K(i) values. DNP displaced [(125)I]-ANP from NPR-C with nM potency, which represents the first direct demonstration of binding of DNP to human NPR-C. DNP showed high pM affinity for the GC-A receptor and no affinity for GC-B (K(i)>1000 nM). DNP was nearly 10-fold more potent than ANP at stimulating cGMP production in GC-A expressing cells. Blockade of NPR-C might represent a novel therapeutic approach in augmenting the known beneficial actions of DNP in cardiovascular diseases such as hypertension and heart failure.     

Binding of Brain and Atrial Natriuretic Peptides to Cultured Mouse Astrocytes and Effect on Cyclic GMP

125I-Porcine brain natriuretic peptide (125I-pBNP) bound to mouse astrocytes in primary culture in a time-dependent manner (t1/2 = 4.5 min), similar to 125I-human atrial natriuretic peptide (125I-hANP) (t1/2 = 5 min). Binding was saturable and reached equilibrium after 90 min at 22 degrees C for both radioligands. Scatchard analysis suggested a single class of binding sites for pBNP with a binding affinity and capacity (KD = 0.08 nM; Bmax = 78.3 fmol/mg of protein) similar to those of hANP1-28 (KD = 0.1 nM; Bmax = 90.3 fmol/mg of protein). In competition binding studies, pBNP or human/rat atrial natriuretic peptide (ANP) analogues [hANP1-28, rat ANP1-28 (rANP1-28), and rANP5-28] displaced 125I-hANP, 125I-pBNP, and 125I-rANP1-28 completely, all with IC50 values of less than nM (0.14-0.83 nM). All four peptides maximally stimulated cyclic GMP (cGMP) production by 10 min at 22 degrees C at concentrations of 1 microM with EC50 values ranging from 50 to 100 nM. However, maximal cGMP induction by brain natriuretic peptide (BNP) (25.9 +/- 2.1 pmol/mg of protein) was significantly greater than that by hANP1-28 (11.5 +/- 2.2 pmol/mg of protein), rANP1-28 (16.5 +/- 2.0 pmol/mg of protein), and rANP5-28 (15.8 +/- 2.2 pmol/mg of protein). These studies indicate that BNP and ANPs act on the same binding sites and with similar affinities in cultured mouse astrocytes. BNP, however, exerts a greater effect on cGMP production. The difference in both affinity and selectivity between binding and cGMP production may indicate the existence of receptor subtypes that respond differentially to natriuretic peptides despite similar binding characteristics.     

Downregulation of atrial natriuretic peptide ANP-C receptor is associated with alterations in G-protein expression in A10 smooth muscle cells

Atrial natriuretic peptide (ANP) receptors A and B are guanylyl cyclase receptors, whereas ANP-C receptors are coupled to adenylyl cyclase through inhibitory guanine nucleotide (Gi) protein. ANP has been shown to downregulate ANP-A and -B receptors and cGMP response in various tissues. In the present studies, we have examined the regulation of ANP-C receptor-adenylyl cyclase signal transduction by ANP and [des(Gln(18),Ser(19),Gln(20),Leu(21), Gly(22))ANP(4-23)-NH(2)](C-ANP(4-23)) that interacts specifically with ANP-C receptor in A10 smooth muscle cells (SMC). Treatment of the cells with C-ANP(4-23) for 24 h resulted in a reduction in ANP receptor binding activity. [(125)I]ANP(99-126) bound to control and C-ANP(4-23)-treated cell membranes at a single site with dissociation constants of 33.7 +/- 6 and 35.0 +/- 4.5 pM and B(max) of 74.0 +/- 5.0 and 57.6 +/- 4.0 fmol/mg of protein, respectively. C-ANP(4-23) inhibited adenylyl cyclase activity in a concentration-dependent manner in control cells. A maximal inhibition observed was about 30-40% with an apparent K(i) of about 1 nM; however, this inhibition was completely attenuated in cells pretreated with ANP(99-126) or C-ANP(4-23) (10(-7) M). However, the inhibition of adenylyl cyclase by 17-amino acid peptide (RRNHQEESNIGKHRELR) (R17A) of cytoplasmic domain of ANP-C receptor was attenuated by about 50% but was not completely abolished by C-ANP(4-23) treatment. The attenuation of C-ANP(4-23)-mediated inhibition of adenylyl cyclase was dependent on the concentration and time of pretreatment of the cells with C-ANP(4-23). In addition, angiotensin II- (Ang II-) mediated inhibition of adenylyl cyclase ( approximately 30%) was also abolished by C-ANP(4-23) treatment, indicating that the desensitization elicited by ANP was heterologous. In addition, C-ANP(4-23) treatment decreased the expression of Gialpha-2 and Gialpha-3 proteins by about 40 and 60%, respectively, and their mRNA by 40%. However, the levels of Gi proteins were not altered when the cells were treated for shorter period of time (2-4 h) or with lower concentrations of C-ANP(4-23) (10(-10) M). On the other hand, the levels of Gsalpha but not of Gbeta were increased by about 35% by C-ANP(4-23) treatment. Furthermore, the stimulations exerted by GTPgammaS, isoproterenol, FSK, and NaF on adenylyl cyclase were also augmented in cells treated with C-ANP(4-23). These results indicate that C-ANP(4-23) treatment of A10 cells desensitizes ANP-C receptor-mediated inhibition of adenylyl cyclase which may be due to the downregulation of ANP-C receptor and decreased expression of Gialpha proteins to which these receptors are coupled.     

Natriuretic peptide receptor-C signaling and regulation

The natriuretic peptides (NP) are a family of three polypeptide hormones termed atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP). ANP regulates a variety of physiological parameters by interacting with its receptors present on the plasma membrane. These are of three subtypes NPR-A, NPR-B, and NPR-C. NPR-A and NPR-B are guanylyl cyclase receptors, whereas NPR-C is non-guanylyl cyclase receptor and is coupled to adenylyl cyclase inhibition or phospholipase C activation through inhibitory guanine nucleotide regulatory protein (Gi). ANP, BNP, CNP, as well as C-ANP(4-23), a ring deleted peptide that specifically interacts with NPR-C receptor inhibit adenylyl cyclase activity through Gi protein. Unlike other G-protein-coupled receptors, NPR-C receptors have a single transmembrane domain and a short cytoplasmic domain of 37 amino acids, which has a structural specificity like those of other single transmembrane domain receptors. A 37 amino acid cytoplasmic peptide is sufficient to inhibit adenylyl cyclase activity with an apparent Ki similar to that of ANP(99-126) or C-ANP(4-23). In addition, C-ANP(4-23) also stimulates phosphatidyl inositol (PI) turnover in vascular smooth muscle cells (VSMC) which is attenuated by dbcAMP and cAMP-stimulatory agonists, suggesting that NPR-C receptor-mediated inhibition of adenylyl cyclase and resultant decreased levels of cAMP may be responsible for NPR-C-mediated stimulation of PI turnover. Furthermore, the activation of NPR-C receptor by C-ANP(4-23) and CNP inhibits the mitogen-activated protein kinase activity stimulated by endothelin-3, platelet-derived growth factor, phorbol-12 myristate 13-acetate, suggesting that NPR-C receptor might also be coupled to other signal transduction system or that there may be an interaction of the NPR-C receptor and some other signaling pathways. In this review article, NPR-C receptor coupling to different signaling pathways and their regulation will be discussed.     

Platelet receptors for atrial natriuretic peptide in man

The aim of our study was to characterize the receptor binding of human alpha-atrial natriuretic peptide (ANP) to human blood cells. Whereas no receptors were detected on red cells, on mononuclear cells and on granulocytes, we found ANP-receptors on human platelets. The binding studies were performed by incubating 40 X 10(6) platelets with 125I-ANP and with the competing ligand, when used, in a total incubation volume of 1 ml. Centrifugation was used to separate bound from free hormone. Specific binding of 125I-ANP was rapid, saturable and reversible. A steady state was achieved within 90 minutes. Scatchard analysis of saturation and competition experiments demonstrated the existence of one class of high affinity binding sites for ANP with a Kd of 8-16pM and 10-26 receptors per cell. The Kd obtained in our binding studies was in the range of physiological ANP concentrations in human plasma (8-20pM). Although characterization of platelet ANP receptors has the inherent disadvantage that there are only few of them, they could be a useful model to investigate the ANP receptor-status under different physiological and pathological conditions in man.     

Specific endothelin binding sites in renal medullary collecting duct cells: lack of interaction with ANP binding and cGMP signalling.

The diverse biological actions of endothelins (ET) appear to be mediated by specific cell-surface receptors. Autoradiography and membrane binding studies have shown abundant ET binding sites in the kidney. However, their expression in specific types of renal cells is unclear. We studied the binding of 125I-labelled endothelin-1 in freshly isolated cell suspensions from canine inner medullary collecting duct. Competition binding experiments revealed the presence of specific high-affinity binding sites: unlabelled ET-1 and ET-2 compared with the radioligand with an IC50 of 135 and 83 pM, respectively, while the IC50 of ET-3 and big ET-1 were 2 and 4 orders of magnitude higher, indicating the presence of ETA-type receptor. Angiotensin II, vasopressin, and atrial natriuretic peptide (ANP) did not compete for ET binding even at a concentration of 10(-6) M. Saturation binding experiments showed a single class of binding sites of high density (Bmax = 56.7 +/- 10.3 fmol/10(6) cells) and high affinity (Kd = 69.8 +/- 10 pM). In contrast, ANP receptors in the same cell preparations appeared as two classes of binding sites with widely different affinity and density. The high-affinity ANP site (Kd = 311 +/- 48 pM) was compatible with ANP-B (guanylate cyclase-coupled) receptor. ET-1 did not compete for this receptor. ET-1 (10(-7) M) did not alter ANP-induced cGMP generation in these cells (3.8-fold increase at 10(-7) M ANP), nor basal levels of cGMP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of ANP-C receptor signaling by arginine-vasopressin in A-10 vascular smooth muscle cells: role of protein kinase C

We have previously shown that pretreatment of A-10 vascular smooth muscle cells (VSMC) with angiotensin II (Ang II) attenuated atrial natriuretic peptide receptor-C (ANP-C)-mediated inhibition of adenylyl cyclase without altering [125I]ANP binding. In the present studies, we have investigated the modulation of ANP-C receptor signaling by arginine-vasopressin (AVP). Pretreatment of A-10 VSMC with AVP for 24h resulted in a reduction in ANP receptor binding activity by about 50% (B(max); control cells, 22.9+/-2.5 fmol/mg protein, AVP-treated cells, 11.4+/-1.2 fmol/mg protein). In addition, the expression of ANP-C receptor as determined by immunoblotting was also decreased by about 50% by AVP treatment, which was prevented by GF109203X, an inhibitor of protein kinase C (PKC). The decreased expression of ANP-C receptor was reflected in an attenuation of ANP-C receptor-mediated inhibition of adenylyl cyclase. C-ANP(4-23) [des(Gln(18),Ser(19),Gln(20),Leu(21),Gly(22))ANP(4-23)-NH(2)], a ring deleted peptide of ANP that interacts specifically with ANP-C receptor, inhibited adenylyl cyclase activity by about 30% in control cells, which was completely attenuated in AVP-treated cells. This attenuated inhibition was significantly restored by GF 109203X. In addition, AVP treatment augmented the levels of Gialpha-2 and Gialpha-3 proteins; however, the Gi functions were completely attenuated. The increased expression of Gialpha proteins induced by AVP was inhibited by GF109203X as well as by actinomycin D treatments. In addition, AVP treatment also enhanced the expression of Gsalpha protein and Gsalpha-mediated stimulation of adenylyl cyclase by GTPgammaS, N-ethylcarboxamide adenosine (NECA), and forskolin (FSK), whereas the levels of Gbeta were not altered by AVP treatment. These results indicate that AVP-induced PKC signaling may be responsible for the down-regulation of ANP-C receptor that results in the attenuation of C-ANP(4-23)-mediated inhibition of adenylyl cyclase activity, and suggest a cross-talk between vasopressin V(1) and ANP-C receptor-mediated signaling pathways.     

Action of atrial natriuretic peptide and angiotensin II on the myocardium: studies in isolated rat ventricular cardiomyocytes

Isolated calcium-tolerant rat ventricular cardiomyocytes were used to characterize the effects of atrial natriuretic peptide (ANP), Angiotensin II (AII) and their interaction on the myocardial contraction-/relaxation pattern free of interference from other types of cardiac cells. Binding of 125I-ANP showed a KD of 12 pM and approximately 600 binding sites per cell. At 37 degrees C (rate 140 bpm) ANP decreased the contraction maximum with an EC50 of about 70 pM, maximal decrease was 35%. ANP (10(-7) M) raised cellular cyclic-GMP from 0.76+/-0.12 to 1.32+/-0.13 pmole/10(6) cells (73%, p less than 0.05). Angiotensin II increased contractility by a maximum of 32% at 10(-7) M; the EC50 was 8 x 10(-10) M. AII markedly delayed relaxation (reduction of maximum relaxation velocity from 0.092 to 0.063 mm/s; p less than 0.05). ANP (10(-7) M) increased the effect of AII (10(-8) M) on contractility by 66% without changing relaxation parameters significantly. This unexpected interaction may be relevant in pathological conditions where both AII and ANP are stimulated, such as heart failure or secondary hypertension.     

Functional expression of components of the natriuretic peptide system in human ocular nonpigmented ciliary epithelial cells

The expression of the natriuretic peptide system in the human ocular ciliary epithelium (CE) and in cultured nonpigmented (NPE) ciliary epithelial cells was examined. By RT-PCR and DNA sequencing, we demonstrated that the CE and NPE cells express mRNA for (i) ANP; (ii) BNP; (iii) NPR-A, NPR-B, and NPR-C receptors; and (iv) the neutral endopeptidase 24.11. Radioimmunoassay results indicate that BNP is secreted by cultured NPE cells at much higher levels than ANP. NPR-A and NPR-B receptors elicited a cGMP response to ANP, BNP, and CNP, in a rank order of potency (CNP > ANP >/= BNP), indicative that the NPR-B receptor is predominant in NPE cells. A71915, an inhibitor of NPR-A activity, attenuated (65-75%) cGMP response to ANP and BNP, but not to CNP. C-ANP4-23 elicited an inhibitory effect (30-37%) on basal levels of cAMP in NPE cells and on forskolin NPE-treated cells, indicative that the NPR-C receptor is functional in these cells. PMA induced, in NPE cells, a long-term downregulation (75-85%) of NPR-C receptor mRNA, but not of NPR-A or NPR-B receptor mRNA, suggesting a differential regulation of NPR-C receptor mRNA via activation of PKC. Collectively, our data provide molecular evidence that all the components of the natriuretic peptide system with the exception of CNP are coexpressed in the ocular NPE ciliary epithelial cells, where they may function as local autocrine/paracrine modulators to influence eye pressure.     

Atrial Natriuretic Peptide Modulates Amiloride-Sensitive Na+ Transport Across the Blood-Brain Barrier

We obtained evidence that amiloride specifically potentiates 125I-labeled alpha-rat atrial natriuretic peptide (1-28) [atrial natriuretic peptide (ANP)-(99-126); rANP] binding to cerebral capillaries isolated from the rat cerebral cortex. The binding parameters, KD of 173 pM and Bmax of 159 fmol/mg of protein, became 33 pM and 88 fmol/mg of protein, respectively, when 10(-4) M amiloride was added to the incubation medium. When the effect of rANP was investigated on in vitro 22Na+ uptake into isolated cerebral capillaries, 10(-7) M rANP significantly inhibited the uptake in the presence of 1.0 mM ouabain, 1.0 mM furosemide, and 2.0 mM LiCl in the uptake buffer, a finding suggesting a specific inhibitory effect of rANP on amiloride-sensitive Na+ transport. Thus, the possibility that ANPs control amiloride-sensitive Na+ transport at the blood-brain barrier by interacting with specific receptors has to be considered.     

Renal atrial natriuretic peptide receptors binding properties and function are resistant to DOCA-salt-induced hypertension in rats

Atrial natriuretic peptide receptor types A (NPR-A) and C (NPR-C) binding properties and functional characteristics in renal glomeruli have been investigated in deoxycorticosterone acetate (DOCA)-treated hypertensive Wistar-Kyoto (WKY) rats and their respective controls. We found that DOCA administration had no significant effect on the maximum binding capacity or the affinity of renal NPR-A and NPR-C. NPR-C is involved in the regulation of cAMP production. Our results indicate that the cAMP production by NPR-C is not altered in DOCA-induced hypertension, since ANP(1-28), CNP(1-22) and C-ANP, which specifically bind to NPR-C, show a similar inhibitory effect on cAMP production stimulated by the physiological agonist histamine in glomeruli from DOCA-treated rats and controls. Finally, we have found that DOCA-induced hypertension does not modify NPR-A or NPR-C expression in rat glomerular membranes. These findings indicate that NPR-A and NPR-C binding properties and NPR-C-mediated inhibition of cAMP generation remain unaltered in DOCA-treated rats.     

Extracellular domain-IgG fusion proteins for three human natriuretic peptide receptors. Hormone pharmacology and application to solid phase screening of synthetic peptide antisera.

The natriuretic peptide receptors (NPRs) are a family of three cell surface glycoproteins, each with a single transmembrane domain. Two of these receptors, designated NPR-A and NPR-B, are membrane guanylyl cyclases that synthesize cGMP in response to hormone stimulation. The third receptor, NPR-C, has been reported to function in the metabolic clearance of ligand and in guanylyl cyclase-independent signal transduction. We engineered three chimeric proteins consisting of the natriuretic peptide receptor extracellular domains fused to the Fc portion of human IgG-gamma 1. These molecules provide material for detailed studies of the human receptor's extracellular domain structure and interaction with the three human natriuretic peptides, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and type-C natriuretic peptide (CNP). The homodimeric fusion proteins, designated A-IgG, B-IgG, and C-IgG, were secreted from Chinese hamster ovary cells and purified by protein-A affinity chromatography. We present here the primary characterization of these fusion proteins as represented by the intrinsic hormone affinities measured by saturation binding and competition assays. The dissociation constant of 125I-ANP for A-IgG was 1.6 pM and for C-IgG, 1.2 pM. The dissociation constant of 125I-Y0-CNP (CNP with addition of tyrosine at the amino terminus) for B-IgG was 23 pM. The rank order of potency in competitive binding for A-IgG was ANP greater than BNP much greater than CNP, whereas for B-IgG the ranking was CNP much greater than ANP greater than BNP. For C-IgG, we observed ANP greater than CNP greater than or equal to BNP. These data demonstrate that the receptor-IgG fusion proteins discriminate among the natriuretic peptides in the same manner as the native receptors and provide a basis for future structural studies with these molecules. The purified fusion proteins have a variety of potential applications, one of which we illustrate by a solid phase screening assay in which rabbit sera from a series of synthetic-peptide immunizations were titered for receptor reactivity and selectivity.     

Structural determinants of natriuretic peptide receptor specificity and degeneracy

Cardiovascular homeostasis and blood pressure regulation are reliant, in part, on interactions between natriuretic peptide (NP) hormones and natriuretic peptide receptors (NPR). The C-type NPR (NPR-C) is responsible for clearance of NP hormones from the circulation, and displays a cross-reactivity for all NP hormones (ANP, BNP, and CNP), in contrast to other NPRs, which are more restricted in their specificity. In order to elucidate the structural determinants for the binding specificity and cross-reactivity of NPR-C with NP hormones, we have determined the crystal structures of the complexes of NPR-C with atrial natriuretic peptide (ANP), and with brain natriuretic peptide (BNP). A structural comparison of these complexes, with the previous structure of the NPR-C/CNP complex, reveals that NPR-C uses a conformationally inflexible surface to bind three different, highly flexible, NP ligands. The complex structures support a mechanism of rigid promiscuity rather than conformational plasticity by the receptor. While ANP and BNP appear to adopt similar receptor-bound conformations, the CNP structure diverges, yet shares sets of common receptor contacts with the other ligands. The degenerate versus selective hormone recognition properties of different NPRs appears to derive largely from two cavities on the receptor surfaces, pocket I and pocket II, that serve as anchoring sites for hormone side-chains and modulate receptor selectivity.     

Binding sites of atrial natriuretic peptide in tree shrew adrenal gland

Adrenal gland binding sites for atrial natriuretic peptide-(99-126) (ANP) were quantitated in tree shrew (Tupaia belangeri) by incubation of adrenal sections with (3-[125I]-iodotyrosyl28) atrial natriuretic peptide-(99-126), followed by autoradiography with computerized microdensitometry. In the adrenal glands, there are three types of ANP binding sites. One is located in the zona glomerulosa (BMax 84 +/- 6 fmol/mg protein; Kd 122 +/- 9 pM); the second in the zona fasciculata and reticularis (BMax 29 +/- 2 fmol/mg protein; Kd 153 +/- 6 pM) and the third in the adrenal medulla (BMax 179 +/- 1 fmol/mg protein; Kd 70 +/- 2 pM). Besides the influence of ANP on the regulation of adrenocortical mineralcorticoid and glucocorticoid secretion our findings raise the possibility for a local site of action of atrial natriuretic peptide in the regulation of adrenomedullary catecholamines in the tree shrew, primates and man.     

Involvement of the atrial natriuretic peptide in the reduction of arterial pressure induced by swimming but not by running training in hypertensive rats

The aim of this study was to compare, under resting conditions, the influence of chronic training in swimming or running on mean arterial pressure (MAP) and the involvement of the natriuretic peptide system in this response. Two-month-old male spontaneously hypertensive rats (SHR) were divided into three groups—sedentary (SD), swimming (SW) and running (RN)—and were trained for eight weeks under regimens of similar intensities. Atria tissue and plasma atrial natriuretic peptide (ANP) concentrations were measured by radioimmunoassay. ANP mRNA levels in the right and left atria as well as the natriuretic peptide receptors (NPR), NPR-A and NPR-C, mRNA levels in the kidney were determined by real-time PCR. Autoradiography was used to quantify NPR-A and NPR-C in mesenteric adipose tissue. Both training modalities, swimming and running, reduced the mean arterial pressure (MAP) of SHR. Swimming, but not running, training increased plasma levels of ANP compared to the sedentary group (P < 0.05). Expression of ANP mRNA in the left atrium was reduced in the RN compared to the SD group (P < 0.05). Expression of NPR-A and NPR-C in the kidneys of the SW group decreased significantly (P < 0.05) compared to the SD group. Although swimming increased ¹²⁵I-ANP binding to mesenteric adipose tissue, displacement by c-ANF was reduced, indicating a reduction of NPR-C. These results suggest that the MAP reduction induced by exercise in SHR differs in its mechanisms between the training modalities, as evidenced by the finding that increased levels of ANP were only observed after the swimming regimen.     

Modulation of ANP-C receptor signaling by endothelin-1 in A-10 smooth muscle cells

We have previously shown that pretreatment of A-10 smooth muscle cells (SMC) with angiotensin II (Ang II) attenuated atrial natriuretic peptide (ANP) receptor-C (ANP-C)-mediated inhibition of adenylyl cyclase without altering (125)I-ANP binding. In the present studies, we have investigated the modulation of ANP-C receptor signaling by endothelin-1 (ET-1). Pretreatment of A-10 SMC with ET-1 for 24 h attenuated the expression of ANP-C receptor by about 60% as determined by immunoblotting which was reflected in attenuation of ANP-C-receptor-mediated inhibition of adenylyl cyclase. C-ANP(4-23) [des(Gln(18),Ser(19),Gln(20),Leu(21),Gly(22))ANP(4-23)-NH(2)], a ring-deleted peptide of ANP that interacts specifically with ANP-C receptor, inhibited adenylyl cyclase activity in a concentration-dependent manner with an apparent K(i) of about 1 nM in control cells. The maximal inhibition observed was about 30% which was almost completely attenuated in ET-1-treated cells. In addition, Ang II- and oxotremorine-mediated inhibitions of adenylyl cyclase were also attenuated by ET-1 treatment; however, the expression of Gialpha-2 and Gialpha-3 proteins and not of Gsalpha and Gbeta proteins was augmented by such treatment. The increased expression of Gialpha-2 and Gialpha-3 proteins by ET-1 treatment was inhibited by actinomycin D treatment (RNA synthesis inhibitor). On the other hand, the Gsalpha-mediated effects of some agonists on adenylyl cyclase activity were significantly decreased by ET-1 treatment. These results suggest that ET-1-induced downregulation of ANP-C receptor and not the overexpression of Gi proteins may be responsible for the attenuation of C-ANP(4-23)-mediated inhibition of adenylyl cyclase activity. From these studies it may be suggested that the downregulation of ANP-C receptors by increased levels of endothelin in vivo may be one of the possible mechanisms for the pathophysiology of hypertension.     

IL-1 binds to high affinity receptors on human osteosarcoma cells and potentiates prostaglandin E2 stimulation of cAMP production

IL-1 is a potent bone resorbing agent. Its mechanism of action is unknown, but the presence of osteoblasts was shown to be necessary for IL-1 stimulation of bone resorption by isolated osteoclasts. This study examines the presence of IL-1R and IL-1 effects in osteoblastic cells from a clonal human osteosarcoma cell line, Saos-2/B-10. We found that the binding affinity and the number of binding sites increases substantially during the postconfluent stage. Scatchard and curve-fitting analysis revealed one class of high affinity binding sites, with Kd/Ki's of 40 +/- 17 pM (mean +/- SD) for IL-1 alpha (n = 5) and 9 +/- 7 pM for IL-1 beta (n = 5) and 2916 +/- 2438 (n = 6) receptors/cell. Incubation of the cells with 125I-IL-1 alpha (100 pM) at 4 degrees C, followed by incubation at 37 degrees C up to 4 h, revealed internalization of receptor-bound IL-1 alpha. Chemical cross-linking studies showed that the IL-1R in Saos-2/B-10 cells had a molecular mass of approximately 80 kDa. To assess the biologic effect of IL-1 in Saos-2/B-10 cells, we determined PGE2 content and adenylate cyclase activity. Although IL-1 had no effect on PGE2 synthesis, both IL-1 alpha and IL-1 beta enhanced PGE2 stimulation of adenylate cyclase two- to four-fold in a dose-dependent manner. The half-maximal effect for IL-1 alpha was seen at 8 to 10 pM and for IL-1 beta at 0.6 to 1.8 pM. IL-1 did not enhance basal adenylate cyclase or stimulation by parathyroid hormone, isoproterenol, or forskolin. IL-1 enhancement of PGE2-stimulated adenylate cyclase was detected between 1 to 2 h, was maximal at 4 to 5 h, was not prevented by cycloheximide treatment, and was seen in membranes from IL-1 pretreated cells. These data show effects of IL-1 on a human osteoblast-like cell line that are mediated by high affinity receptors. These IL-1 effects could contribute to the biologic action of IL-1 on bone.     

Cyclic AMP increases bradykinin receptor binding affinity in human endothelial cells

We demonstrated bradykinin receptors in human endothelial cells and studied whether bradykinin receptors might be regulated by cyclic AMP. Messenger RNA for bradykinin B(1) and B(2) receptors was detected with real-time PCR and B(2) receptor protein was confirmed by immunoblotting. Saturation binding experiments with increasing concentrations of (125)I-[Tyr(8)]-bradykinin (25-700 pM) were made to determine maximal binding capacity and dissociation constant. However, saturation binding experiments suggested one class of binding sites, maximal binding capacity of 39.3 +/- 1.3 fmol/mg protein and dissociation constant of 352 +/- 27 pM. Competition studies with bradykinin B(1) and B(2) receptor antagonists showed that binding was competed by a B(1) antagonist, and when internalization was inhibited with hypertonic buffer, by both B(1) and B(2) antagonists. Stimulating cells with dibutyryl-cAMP, cholera toxin and forskolin for 24 h increased (125)I-[Tyr(8)]-bradykinin (90 pM) binding with approximately 50%. Saturation binding experiments with dibutyryl-cAMP stimulated cells showed, that the dissociation constant was altered from 352 +/- 27 pM in non-stimulated cells, to 203 +/- 18 pM (P < 0.001) in stimulated cells, while maximal binding capacity remained unchanged. Binding was competed similarly by the B(1) antagonist in stimulated and control cells. These results suggest, that the dibutyryl-cAMP stimulated increase in (125)I-[Tyr(8)]-bradykinin binding is probably due to increased B(1) receptor affinity with no change in receptor capacity. In conclusion, bradykinin B(1) and B(2) receptor mRNA was shown in human endothelial cells. Binding studies suggest that bradykinin receptors are competable with bradykinin antagonists. Adenylate cyclase activators probably increase bradykinin B(1) receptor affinity, without changing capacity, and thus increase bradykinin binding.