Atrial Natriuretic Factor Binding Research Articles

Cyclophilin A functions as an endogenous inhibitor for membrane-bound guanylate cyclase-A.

Cyclophilin A (CypA), a receptor for the immunosuppressive agent cyclosporin A, is a cis-trans-peptidyl-prolyl isomerase (PPIase). It accelerates the cis-trans isomerization of prolyl-peptide bonds. CypA binds and regulates the activity of a variety of proteins. Atrial natriuretic factor (ANF) and its receptor membrane-bound guanylate cyclase-A (GC-A) are involved in the regulation of blood pressure. We examined whether CypA affects the activation of GC-A by ANF. The results showed that CypA associated with GC-A. Interestingly, binding of ANF to GC-A released CypA. Transfection of CypA inhibited ANF-stimulated GC-A activity, indicating that CypA functions as an endogenous inhibitor for GC-A activation. CypA also inhibits the activity of guanylate cyclase-C (GC-c), the catalytic domain of GC-A, indicating that CypA interacts with the catalytic domain of GC-A. In contrast, transfection of CypA R55A, a CypA mutant expressing low PPIase activity, did not significantly attenuate the activity of GC-c and the activation of GC-A. Inhibition of PPIase activity of CypA with cyclosporin A also blocks the inhibitory effect of CypA on GC-c activity. These results demonstrate that PPIase activity is required for CypA to inhibit GC-c activity and GC-A activation by ANF. Furthermore, mutation of Pro 822, 902, or 958 in GC-c abolished its activity. Therefore, it is likely that CypA binds to GC-A and catalyzes the cis-trans isomerization of Pro 822, 902, or 958, which keeps GC-A in the inactive state, and that binding of ANF to GC-A alters the conformation of the catalytic domain that releases CypA from GC-A leading to enzyme activation.

Allosteric regulatory step and configuration of the ATP-binding pocket in atrial natriuretic factor receptor guanylate cyclase transduction mechanism

The atrial natriuretic factor (ANF) signal transduction mechanism consists of the transformation of the signal information into the production of cyclic GMP. The binding of ANF to its receptor, which is also a guanylate cyclase, generates the signal. This cyclase has been termed atrial natriuretic factor receptor guanylate cyclase, ANF-RGC. ANF-RGC is a single transmembrane-spanning protein. The ANF receptor domain resides in the extracellular region of the protein, and the catalytic domain is located in the intracellular region at the C-terminus of the protein. Thus, the signal is relayed progressively from the receptor domain to the catalytic domain, where it is converted into the formation of cyclic GMP. The first transduction step is the direct binding of ATP with ANF-RGC. This causes allosteric regulation of the enzyme and primes it for the activation of its catalytic moiety. The partial structural motif of the ATP binding domain in ANF-RGC has been elucidated, and it has been named ATP regulatory module (ARM). In this presentation, we provide a brief review of the ATP-regulated transduction mechanism and the ARM model. The model depicts a configuration of the ATP-binding pocket that has been experimentally validated, and the model shows that the ATP-dependent transduction process is a two- (or more) step event. The first step involves the binding of ATP with its ARM. This partially activates the cyclase and prepares it for the subsequent steps, which are consistent with its being phosphorylated and attaining the fully activated state.

Proteolytic activation of membrane-bound guanylate cyclase

Membrane-bound guanylate cyclase-A (GC-A), the receptor for atrial natriuretic factor (ANF), has been shown to be regulated by its kinase-like domain. To resolve the nature of this regulation, we measured the effects of various proteases on the activity of guanylate cyclase in rat lung membranes, and on the activity of the bacterial-expressed catalytic domain (GC-c) and on a recombinant peptide composed of both the kinase-like and catalytic domain (GC-kc) of guanylate cyclase. Pronase increased rat guanylate cyclase activity in a biphasic manner with a maximal effect at about 10–20 μg per assay tube. Thermolysin had effects similar to those of pronase on the activity of guanylate cyclase in rat lung membranes. In the case of bacterial-expressed proteins, pronase increased the activity of GC-kc, but not GC-c. These results indicate that GC-A contains an autoinhibitory site on its kinase-like domain, and that removal of the autoinhibitory site by limited proteolysis leads to enzyme activation. GC-A was poorly activated by ANF and ATP after the rat lung membrane was pretreated with pronase, suggesting that ANF/ATP and pronase activate guanylate cyclase through the same mechanism. It is suggested that the binding of ANF and ATP to GC-A may induce a conformational change of the receptor that releases the inhibitory constraint on enzyme activity leading to enzyme activation.

Three dimensional atomic model and experimental validation for the ATP-Regulated Module (ARM) of the atrial natriuretic factor receptor guanylate cyclase.

Atrial natriuretic factor (ANF) receptor guanylate cyclase (ANF-RGC) is a single chain transmembrane-spanning protein, containing both ANF binding and catalytic activities. ANF binding to the extracellular receptor domain activates the cytosolic catalytic domain, generating the second messenger cyclic GMP. Obligatory in this activation process is an intervening transduction step, which is regulated by the binding of ATP to the cyclase. The partial structural motif of the ATP binding domain of the cyclase has been elucidated and has been termed ATP Regulatory Module (ARM). The crystal structures of the tyrosine kinase domains of the human insulin receptor and haematopoietic cell kinase were used to derive a homology-based model of the ARM domain of ANF-RGC. The model identifies the precise configuration of the ATP-binding pocket in the ARM domain, accurately represents its ATP-dependent features, and shows that the ATP-dependent transduction phenomenon is a two-step mechanism. In the first step, ATP binds to its pocket and changes its configuration; in the second step, via an unknown protein kinase, it phosphorylates the cyclase for its full activation.

Atrial natriuretic factor binding to its receptor is dependent on chloride concentration: A possible feedback-control mechanism in renal salt regulation.

Although considerable evidence indicates a role for atrial natriuretic factor (ANF) in renal salt regulation, other studies have found a lack of natriuretic response to high-plasma ANF under certain physiological and pathophysiological conditions. The mechanism for this apparent insensitivity to ANF is unknown. In the present study, it was found that ANF binding to its receptor requires the presence of chloride and occurs in a chloride concentration-dependent manner. ANF binding was measured using the purified recombinant hormone-binding domain of the ANF receptor in the presence of 0.1 mol/L NaCl or other selected salt. High specific binding was detected in the presence of NaCl, KCl, or NH(4)Cl. However, binding was undetectable when the salt was replaced with NaHCO(3), CH(3)COONa, or CH(3)COONH(4), indicating that binding requires the presence of chloride. Chloride dependence was also found with the native receptor in bovine adrenocortical membrane preparations. ANF binding to the recombinant protein was chloride concentration-dependent over a range from 0.05 to 10 mmol/L, and a half-maximum binding was attained at approximately 0.6 mmol/L equivalent chloride concentration. Competitive-binding assays at several fixed concentrations of NaCl showed that lowering chloride concentration caused a decrease in maximum binding but did not alter K(d) values, suggesting that a loss of chloride turns off ANF binding rather than reducing affinity for ANF. Saturation-binding studies showed that excess ANF cannot overcome loss of binding caused by low chloride. Chloride-dependent ANF-receptor binding may function as a feedback-control mechanism regulating the ANF-receptor action and, hence, renal sodium excretion.

Regulation of Natriuretic Peptide Receptors by Thyrotropin in FRTL-5 Rat Thyroid Cells: Evidence for Nonguanylate Cyclase Atrial Natriuretic Factor-Binding Sites in Cells Lacking the Natriuretic Peptide Receptor C

Natriuretic peptide receptors (NPR) are expressed in thyroid-derived cells, including the rat FRTL-5 thyroid cell line. We have previously demonstrated that atrial natriuretic factor (ANF) binding consistent with the NPR-A receptor is significantly increased in FRTL-5 cells cultured in the presence of TSH. The purpose of the present study was to determine whether TSH treatment, therefore, results in higher levels of ANF-induced intracellular cGMP, and whether TSH elicits similar effects on cGMP signaling through the NPR-B receptor. We now show that contrary to expectation, long term exposure to 1 mIU/ml bovine TSH (6H medium) does not significantly alter maximal ANF-induced cGMP formation. Moreover, TSH treatment decreased C-type natriuretic peptide (CNP)-induced cGMP generation in FRTL-5 cells, suggesting a down-regulation of NPR-B. A similar effect of TSH on ANF- and CNP-induced cGMP was observed in FRTL cells, the precursor of the FRTL-5 cell line. Scatchard analysis of [125I]ANF binding in TSH-treated (6H) FRTL-5 cultures indicated a 5.6-fold increase in high affinity ANF-binding sites compared with TSH-deficient (5H) cultures [binding capacity (Bmax) of 6H cells, 227.2 +/- 33.7 fmol/mg protein; Bmax of 5H cells, 40.2 +/- 4.7 fmol/mg protein]. The effect of TSH on [125I]ANF binding was mimicked by forskolin and (Bu)2cAMP, indicating receptor up-regulation via a cAMP pathway. High affinity [125I]CNP-binding sites were present in much lower abundance (Bmax of 5H, 0.80 +/- 0.06 fmol/mg protein), and no effect of TSH treatment on them could be demonstrated. However, low affinity [125I]CNP binding was increased by TSH. RT-PCR confirmed the presence of both NPR-A and NPR-B transcripts in FRTL-5 cells and showed that TSH treatment significantly decreased NPR-B, but not NPR-A. NPR-C transcript was not detectable by RT-PCR in FRTL-5 cells cultured in high TSH medium, suggesting that the ANF-binding sites increased by TSH are not NPR-C. Both CNP and ANF transcript were also expressed in FRTL-5 cells, and CNP was increased by TSH. Together the data support the down-regulation of functional NPR-B and no change in functional NPR-A by TSH. The vast majority of ANF-binding sites in FRTL-5 cells, therefore, are not coupled to cGMP production and may represent a novel or altered form of NPR that is regulated by TSH independently of NPR-A and NPR-B.

Reduced vasodilator response to ANF in hypoxia-induced pulmonary hypertension in the newborn piglet.

Recent evidence suggests that, in adult animals with hypoxia-induced pulmonary hypertension, atrial natriuretic factor (ANF) may modulate pulmonary vascular tone and may have a protective effect. However, its role in the pathogenesis of pulmonary hypertension of the newborn is unknown. We hypothesized that, in the newborn, hypoxia-induced pulmonary hypertension would result in ANF receptor downregulation, resulting in decreased dilator response, favoring pulmonary vasoconstriction and vascular remodeling. Therefore, we studied, in 1-day-old piglets exposed to hypoxia (fraction of inspired O2 0.10) for 3 or 14 days to induce pulmonary hypertension, 1) ANF release by measuring circulating levels of ANF by radioimmunoassay in pulmonary artery and veins, 2) pulmonary vascular reactivity to ANF using isolated perfused lungs, and 3) binding characteristics by examining the concentration dependence of ANF binding and competitive binding of 125I-labeled ANF with ANF, brain natriuretic peptide, C-type natriuretic peptide, and the specific ligand for ANF clearance receptor on microsomes from pulmonary arteries (down to 100 microns). ANF circulating levels are increased after exposure to hypoxia compared with normoxia, reaching significance at 14 days (P < 0.005). The magnitude of ANF dilator response is diminished after exposure to hypoxia (P < 0.05). Saturation studies reveal that the number of ANF receptors is diminished in hypoxia after 3 days but reaches significance after 14 days (P < 0.01) compared with their respective normoxic control. At either condition, the majority of these receptors are of the functional type, whereas clearance receptors are virtually undectable. These results suggest that hypoxia increases circulating ANF and causes a decreased responsiveness of the pulmonary vasculature to ANF. Receptor down-regulation may explain part of the reduced dilator response, although the involvement of other mechanisms is not excluded.

Downregulation of renal atrial natriuretic factor receptors and receptor mRNAs during rat pregnancy.

Using renal glomeruli and papillae from virgin, pregnant (15- to 17-day), and postpartum (day 2) rats, we investigated whether the decrease in the renal effects of atrial natriuretic factor (ANF) during pregnancy is caused by a downregulation of ANF receptors. Pregnancy decreased the maximal binding of 125I-labeled ANF to guanylyl cyclase (GC)-linked ANF-GC receptors in glomeruli and papillae and increased the binding to clearance receptors (ANF-C) in glomeruli; ANF-C receptors were not detected in the papillae Ribonuclease protection assay detected mRNAs for all the three receptors in the papillae; pregnancy decreased GC-A and ANF-C but not GC-B-receptor mRNAs. Western blots revealed a decrease in GC-A receptors in the papillae of pregnant rats; GC-B-receptor protein was barely detectable. Effects of ANF on guanosine 3', 5'-cyclic monophosphate (cGMP) production by the glomeruli and papillae were decreased during pregnancy and returned to virgin levels during postpartum. It is concluded that a decrease in the renal effects of ANF during pregnancy is caused by a downregulation of renal ANF GC-A receptors and receptor-coupled cGMP production.

Immunocytochemical localization of atrial natriuretic factor and autoradiographic distribution of atrial natriuretic factor binding sites in the brain of the African lungfish, Protopterus annectens.

The localization of atrial natriuretic factor (ANF)-immunoreactive elements was investigated in the brain of the African lungfish, Protopterus annectens, by using antisera raised against rat and human ANF(1-28). Concurrently, the distribution of ANF binding sites was studied by autoradiography using radioiodinated human ANF(1-28) as a tracer. In general, there was a good correlation between the distribution of ANF-immunoreactive structures and the location of ANF binding sites in several areas of the brain, particularly in the ventral part of the medial subpallium, the rostral preoptic region, the preoptic periventricular nucleus, the caudal hypothalamus, the neural lobe of the pituitary, and the mesencephalic tectum. In contrast, mismatching was observed in the pallium (which contained a high density of binding sites and a low concentration of ANF immunoreactive elements) as well as in the lateral subpallium and the medial region of the ventral thalamus, in which a low concentration of binding sites but a high density of ANF-immunoreactive fibers were detected. The present data provide the first localization of ANF-related peptides in the brain of dipnoans and the first anatomical distribution of ANF binding sites in the brain of fish. The results show that the ANF peptidergic systems of P. annectens exhibit similarities with those previously described in the frog Rana ridibunda, supporting the existence of relationships between dipnoans and amphibians. The location of ANF-like immunoreactivity and the distribution of ANF binding sites suggest that ANF-related peptides may act as hypothalamic neurohormones as well as neurotransmitters and/or neuromodulators in the lungfish brain.

Modulation of ANF-R2/ANP-C receptors by angiotensin II in vascular smooth muscle cells

In the present studies, we have investigated the modulation of atrial natriuretic factor (ANF) receptor of R2 subtype (ANF-R2) coupled to adenylyl cyclase/cAMP signal transduction system by angiotensin II (AII). C-ANF 4–23 [C-ANF 4–23, [des (Gln 18, Ser 19, Gln 20, Leu 21, Gly 22) ANF 4–23NH 2] and AII inhibited adenylyl cyclase activity in control vascular smooth muscle cells (VSMC A-10) by about 40% and 30% respectively. Pretreatment of the cells with AII resulted in the attenuation of both C-ANF 4–23- and AII-mediated inhibition of adenylyl cyclase. Losartan treatment of the cells was able to partially block (∼50%) the AII- as well as C-ANF 4–23-mediated inhibitions of adenylyl cyclase that are completely lost by AII pretreatment. The pretreatment of the cells with AII alone or with losartan did not affect the [ 125I]-ANF binding to ANF receptors. However, AII treatment resulted in the augmentation of the levels of Giα2 and Giα3. On the other hand, staurosporine (a protein kinase C [PKC] inhibitor) treatment of cells before AII treatment was able to prevent the attenuation of both C-ANF 4–23 as well as AII-mediated inhibition of adenylyl cyclase elicited by AII. These results indicate that the AII modulation of ANF-R2 receptor-mediated inhibition of adenylyl cyclase is independent of ANF-R2 receptor density or the levels of Gi regulatory protein and may be due to the uncoupling of the ANF-R2 receptor from the Gi protein. This uncoupling may be associated with the phosphorylation of the Gi protein by PKC activated by AII.

High-yield affinity alkylation of the atrial natriuretic factor receptor binding site.

To facilitate characterization of the atrial natriuretic factor (ANF) receptor, we have developed an affinity labeling procedure, stepwise affinity labeling, which allows specific labeling of ANF binding sites in adrenal plasma membranes at high yields. An iodoacetyl (IAc-), bromoacetyl (BrAc-), or maleimidobenzoyl group was attached to the amino-terminal alpha-amino group of the ANF(4-28) peptide, and the peptide derivatives were radioiodinated at Tyr-28 to obtain affinity reagents, N4alpha-IAc-[125I]ANF(4-28), N4alpha-BrAc-[125I]ANF(4-28), and N4alpha-(maleimidobenzoyl)-[125I]ANF(4-28). Receptor labeling was carried out in a stepwise fashion as follows: (1) Membranes were treated with p-chloromercuriobenzenesulfonic acid (PCMBS) or N-ethylmaleimide to block sulfhydryl groups; (2) the affinity reagent was allowed to bind to the receptor at 0 degrees C for 1 h; and (3) the membranes were washed to remove unbound reagent and were incubated at room temperature to effect alkylation reaction. Sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) followed by autoradiography revealed specific labeling of a 130-kDa ANF receptor. On the basis of 125I-radioactivity incorporated, the labeling yields were estimated to be 70%, 52%, and 21% for the reactions with IAc-[125I]ANF(4-28), BrAc-[125I]ANF(4-28), and (maleimidobenzoyl)-[125I]ANF(4-28), respectively. The efficiency of receptor labeling by the stepwise procedure using IAc-[125I]ANF(4-28) was 27-fold greater than that obtained by photoaffinity labeling using N3Bz-[125I]ANF(4-28) and 63-fold greater than that by direct cross-linking using disuccinimidylsuberate and [125I]ANF(4-28) under comparable conditions. Digestion of the membrane protein labeled with IAc-[125I]ANF(4-28) by BrCN, endoproteinase Glu-C, and endoproteinase Lys-C gave single radiolabeled bands with apparent masses of 40, 18, and 29 kDa, respectively. Reversed-phase HPLC separation of the digests also gave single major peaks. The confinement of the affinity label to one major fragment in each digest suggests that the cross-linking occurred at a single or a limited number of sites. The stepwise affinity labeling with the high cross-linking yield and specificity may be useful for analyzing the ANF receptor binding site structure.

ATP Bimodal Switch That Regulates the Ligand Binding and Signal Transduction Activities of the Atrial Natriuretic Factor Receptor Guanylate Cyclase

Atrial natriuretic factor (ANF)-dependent guanylate cyclase (ANF-RGC) is a single-chain transmembrane-spanning protein, containing both ANF binding and catalytic cyclase activity. ANF binding to the extracellular receptor domain activates the cytosolic catalytic domain, generating the second messenger cyclic GMP. Obligatory in this activation process is an intervening step regulated by the ATP binding to the cyclase. This is a signal transduction step that bridges the events of ligand binding and cyclase activation. A defined structural motif (Gly 503-Xa-Gly 505-Xa-Xa-Xa-Gly 509), termed ATP regulatory module (ARM), is critical for this step. The present study shows that the ARM-Gly 505 residue acts as an ATP bimodal switch in regulating both the ligand binding and signal transduction activities of ANF-RGC, thus representing a critical site to turn the hormone signal on and off.

Transient downregulation of glomerular atrial natriuretic factor receptors in high output heart failure in the rat

The renal response to exogenous atrial natriuretic factor (ANF) is blunted in chronic heart failure. The aim of the present studies was to investigate whether renal ANF receptor regulation in chronic heart failure is a time related event. Glomerular ANF receptors were analysed in radioligand binding experiments at 0, 1, 2, 6, 12, 24, and 48 h, as well as at 1, 2, 4, and 8 weeks after the induction of an aortocaval shunt. Rats with aortocaval shunts had lower packed cell volume and body weight and higher relative heart weight than sham operated controls. Plasma ANF C and N terminal levels were increased in shunt rats as early as 5 min after establishment of the shunt. Right and left atrial ANF concentrations were decreased and ventricular ANF concentration was increased in shunt rats at 6 and 12 h respectively. Competitive inhibition of 125I-ANF binding showed that at 6 h the density (Bmax) of glomerular ANF receptors was significantly lower than in the controls [518(SEM 10) v 759(12) fmol.mg-1 protein] without differences in their affinity (Kd). The low Bmax in shunt animals persisted at 12, 24, and 48 h, even at 1 week [Bmax: 400(29) and 713(28) fmol.mg-1 protein; Kd: 80(2) and 70(4) pM, for AC rats and controls, respectively]. Bmax values were not significantly different at 2, 4, and 8 weeks. In 24 h animals, C-ANF displaced 65% of total binding, with both total and C ANF binding sites being 38% lower in shunt animals. Downregulation of glomerular ANF receptors is a transient event during the development of high output heart failure in the rat. Thus the blunted renal response to ANF during chronic heart failure is not likely to be due to a decrease in renal ANF receptor density or affinity.

Rat renal preglomerular vessels, glomeruli and papillae do not express detectable quantities of B-type natriuretic peptide receptor

Atrial natriuretic factor (ANF) receptor subtypes were quantitated by radioligand studies in three different rat renal isolated tissues: preglomerular vessels, glomeruli and papillae. In preglomerular vessels 100% of [125I]-ANF binding was displaced with high affinity by ANF. C-ANF (des-[Gln18,Ser19,Gly20,Leu21,Gly22]ANP(4- 23)) a specific ligand for ANP-C receptors, displaced 30% of total binding with a lower affinity than ANF. C-type natriuretic peptide (CNP) displaced [125I]-ANF binding in a biphasic manner, indicating that it binds to two sites with affinities three orders of magnitude apart. When CNP was incubated in the presence of 0.1 mumol/l C-ANF to saturate ANP-C receptors, the high-affinity binding site vanished and maximum binding decreased to 70%, suggesting that CNP binds with high affinity to ANP-C receptors. Since the ANP-A receptor has little or no avidity for CNP, it is probably the low-affinity binding site. CNP and C-ANF displaced most [125I]-[Tyr]CNP(1-22) binding with very close affinities, indicating that CNP binds primarily preglomerular vascular ANP-C receptors. In glomeruli CNP behaved similarly to C-ANF in its ability to displace approximately 85% of [125I]-ANF binding; the remaining 15% was completely displaced by ANF. C-ANF and CNP inhibited 100% of [125I]-[Tyr]CNP(1-22) binding. Both findings suggest that [125I]-[Tyr]CNP(1-22) binds ANP-C receptors exclusively. In renal papillae no displacement of [125I]-ANF by C-ANF was observed, and CNP was bound to ANP-A receptors with the same very low affinity as in preglomerular vessels. The absence of [125I]-[Tyr]CNP(1-22) binding to papillary membranes indicates that ANP-B receptors are not expressed in that tissue. Unlike ANF, CNP stimulated cGMP production in glomeruli and papillae only at extremely high, supraphysiological concentrations. The present results suggest that ANP-B receptors either are absent or are present in undetectable amounts in rat renal preglomerular vessels, glomeruli and papillae.

C-type natriuretic peptide (CNP) increases [ 125I]ANF binding to FRTL-5 rat thyroid cells by increasing ANF receptor affinity

To examine a possible role for C-type natriuretic peptide (CNP) in the thyroid, we studied the ability of this peptide to compete with atrial natriuretic factor (ANF) binding to FRTL-5 rat thyroid cells. Rather than competing for ANF binding, CNP significantly elevated [ 125I]ANF binding above control at both 23° and 2°C. The increase in ANF binding was due largely to a threefold increase in receptor affinity in the presence of CNP (control, K d = 8.7 nM ; 1 μ M CNP, K d = 3.1 nM ). Despite the failure to compete for ANF binding, CNP was almost as effective as ANF at inducing cGMP production in FRTL-5 cells. Competition binding studies using [ 125I]CNP indicated the presence of a relatively low-affinity site for CNP ( K d = 77 nM ) that bound ANF with equal affinity. These results show for the first time that ANF receptor binding can be positively regulated by the related natriuretic peptide, CNP.

Absence of C-type natriuretic peptide receptors in hamster glomeruli.

The distribution of atrial natriuretic peptide receptor B (ANPR-B) varies between tissues and species. The aim of this study is to determine whether ANPR-B is present in the hamster glomeruli. In vitro C-type natriuretic peptide (CNP)- and atrial natriuretic factor (ANF)-stimulated cGMP accumulation studies were performed in hamster glomeruli. Elevated cGMP accumulations were observed upon ANF addition. No cGMP response was seen with CNP. Competitive receptor-binding experiments were performed with 125I-CNP and 125I-ANF against their respective cold peptides in hamster glomeruli. Although no CNP binding was detected, positive ANF binding was found and two types of ANF receptor were demonstrated. The affinity (Kdl) and maximum binding capacity (Bmaxl) of the high-affinity ANF receptor were 0.014 +/- 0.001 nM and 60.4 +/- 10.2 fmol/mg protein, respectively. Those of the low-affinity receptor (Kd2 and Bmax2) were 45.7 +/- 6.2 nM and 28.3 +/- 6.3 pmol/mg protein, respectively. Similarly, saturation binding experiments also failed to show any CNP receptor binding in hamster glomeruli. This finding suggests that ANPR-B is not present in hamster glomeruli and CNP is not a direct physiological regulator of hamster renal function.

Atrial natriuretic factor (ANF) binds to thyrotropin-regulated receptors in FRTL-5 cells and increases thyroglobulin mRNA

Thyrotropin (TSH) regulation of atrial natriuretic factor (ANF) receptors was studied in the rat thyroid follicular cell line, FRTL-5. Exposure of FRTL-5 cells to 1 mU/ml TSH for 7 days resulted in a tenfold increase in ANF receptors ( B max = 188 fmol/mg protein) compared with control ( B max = 18 fmol/mg protein), without affecting binding affinity. An identical treatment of porcine thyrocytes with TSH resulted in a 50% decrease in ANF binding sites. Displacement binding studies indicated that > 80% of the ANF receptors in FRTL-5 cells belong to the ANF-R 1 (guanylate cyclase-coupled) receptor subtype. By contrast, > 98% of the ANF receptors in porcine thyrocytes were of the ANF-R 2, or clearance, receptor subtype. Intracellular cGMP content was increased thirty-sixfold in FRTL-5 cells by 1 μ M ANF, but only 2.5-fold in procine thyrocytes. cAMP levels were unaffected by ANF in either cell type. Northern blot analysis of poly A mRNA extracted from FRTL-5 cells incubated 2 days in the presence of 100 n M ANF indicated a twofold increase in thyroglobulin mRNA content compared with control. These findings suggest that the ANF-R 1 receptor, preferentially expressed in FRTL-5 cells and regulated by TSH, might play a role in regulating thyroid hormone production.

ANF binding sites in the heart of the quail ( Coturnix coturnix japonica)

The distribution pattern of rat [ 125I]-atrial natriuretic factor (ANF) binding sites in the cardiac regions of the Japanese quail was examined by in vitro quantitative autoradiography. Elevated ANF binding densities ( 519 ± 121 fmol/mg protein) were found in the posterior vena cava, while lower binding levels (between 40 and 50 fmol/mg protein) were found in sinus venosus, aortic bulb, and endomural vessels, with the ventricular wall having the lowest value ( 17.6 ± 8.8 fmol/mg protein). Scatchard analyses of the ANF binding characteristics ( K d , B max ) revealed both low ( 94 ± 55 fmol/mg protein) and high ( 1161 ± 69 fmol/mg protein) B max values. Receptors with higher K d values than those observed in other cardiac regions ( K d between 30 and 60 p M) were found in the vena cava and in the heart ventricle ( K d between 113.2 and 229 p M).

Receptors for Atrial Natriuretic Factor (ANF) in Kidney and Adrenal Tissue of Urodeles—Lack of Angiotensin II (A II) Receptors in these Tissues

Binding sites for atrial natriuretic factor (ANF) and angiotensin II (A II) were localized and quantified in renal and adrenal tissue of the Urodele Ambystoma mexicanum by quantitative in vitro autoradiography using 125I-ratANF(99-126) and 125I-[Val 5]-A II or 125I-human A II as labeled ligands. Specific 125 I-ratANF(99-126) binding was present in glomeruli, renal tubules, and adrenal tissue. ANF had heterogeneous binding sites exhibiting positive cooperativity with a half maximal binding concentration (EC 50) of 124 ± 12 p M in glomeruli and 118 ± 18 p M in adrenal tissue ( n = 8). The corresponding maximal binding capacities ( B max) were 0.84 ± 0.10 and 0.72 ± 0.18 femtomol/mm 2. 125I-ratANF(99-126) binding was competitively displaced by unlabeled ANF analogues with an intact disulfide bridge showing a lower affinity than the iodinated ligand. In in vitro perifusion experiments ANF inhibited basal as well as ACTH-stimulated steroid secretion whereas catecholamine release was not affected indicating that ANF binding in adrenal tissue is restricted to steroidogenic interrenal cells. In vivo experiments demonstrated that during acclimation to 1.5% salt water made using seawater salt the number of ANF binding sites in kidney glomeruli decreased after 12 hr and 7 days while no significant changes were observed in adrenal tissue. No A II binding sites were detected either in renal or adrenal tissue indicating a lack of A II receptors in these tissues, which was confirmed by in vitro perifusion experiments which showed that A II did not affect adrenal steroid and catecholamine secretion.

Inhibition of vasopressin and aldosterone release by atrial natriuretic peptide in conscious rabbits

To elucidate the regulatory role of atrial natriuretic factor (ANF) on vasopressin (AVP) and aldosterone release in conscious rabbits, ANF was administered systematically at a rate of 15 pmol min-1 (kg body wt)-1 for 15 min in two series of experimental animals in which AVP and/or aldosterone production was stimulated. In euhydrated rabbits (series I), systemic administration of angiotensin II (Ang II) (10 pmol min-1 (kg body wt)-1, 15 min) stimulated aldosterone release threefold from basal plasma concentrations (140 pg ml-1). The co-application of ANF inhibited the Ang II-induced release of aldosterone without influencing the non-stimulated AVP system. In dehydrated rabbits (series II) with elevated plasma osmolality and AVP concentration, exogenously applied ANF increased plasma ANF fourfold at marginally reduced arterial pressure. Plasma AVP concentrations were reduced by 3.4 pg ml-1 (25%) on average, and plasma aldosterone concentrations were lowered by 34 pg ml-1 (23%) at unchanged levels of plasma corticosterone. Receptor binding studies using [125I]ANF as radioligand revealed Ang II-independent high-affinity receptors for ANF in the zona glomerulosa of the adrenal gland. With regard to the hypothalamo-neurohypophyseal AVP system, ANF binding sites were localized to the median eminence and neurohypophysis, but not to the magnocellular nuclei. ANF receptors were also labelled in structures lacking a blood-brain barrier such as the subfornical organ and the choroid plexus.