Atrial natriuretic peptide binds to ANP-R1 receptors in neuroblastoma cells or is degraded extracellularly at the Ser-Phe bond.

ANP-R1 receptors for atrial natriuretic peptide (ANP) showed the following rank order of affinity in intact human neuroblastoma cells NB-OK-1: human ANP-(99-126) approximately human ANP-(102-126) approximately rat ANP-(99-126) (K1 17-32 pM) > human ANP-(103-126) > porcine brain natriuretic peptide (BNP). Analogues truncated at the C-terminal extremity or devoid of a disulphide bridge, such as rat ANP-(103-123), rat C-ANP-(102-121), rat ANP-(111-126), rat ANP-(99-109) and rat [desCys105,Cys121]ANP-(104-126) and chicken C-type natriuretic peptide, were not recognized. The occupancy of these high affinity ANP-R1 receptors led to marked cyclic GMP accumulation in the presence of 3-isobutyl 1-methylxanthine. An ectoenzymic activity, partly shed in the incubation medium, provoked the stepwise release of Phe-Arg-[125I]Tyr, Arg-[125I]Tyr and [125I]Tyr from rat [125I]ANP-(99-126), at an optimal pH of 7.0. Its inhibition by 1,10-phenanthroline, EDTA and bacitracin but not by thiorphan suggests the contribution of at least one neutral metalloendopeptidase, distinct from EC 3.4.24.11, for which ANP showed high affinity.     

Stimulation of guanylate cyclase activity by irreversible binding of atrial natriuretic peptide to its receptor

We examined receptor binding profiles of atrial natriuretic peptide (ANP) in rat tissue using 125I-labeled alpha-rat ANP [( 125I]alpha-rANP). Specific [125I]alpha-rANP binding to its receptor was reversible following addition of unlabeled free alpha-rANP, but it became increasingly irreversible with time during incubation. Irreversible binding of alpha-rANP was observed both at 0 degrees and 25 degrees in homogenates of adrenal capsules and lungs, crude membranes of renal glomeruli, partially purified membranes of lung, solubilized membrane preparations from renal glomeruli, and intact renal glomeruli. Irreversible binding increased in a time- and temperature-dependent manner. HPLC analysis demonstrated that the irreversibly bound radioactivity, which was extracted by 1 N CH3COOH from both intact renal glomeruli and from partially purified membranes, was associated with intact [125I]alpha-rANP. Irreversibly bound alpha-rANP increased cGMP concentrations by activating guanylate cyclase activity. These findings suggest that the appearance of irreversible binding of alpha-rANP to its receptor is independent of its internalization, and may be involved in message transduction and subsequent biological responses.     

Multiple types of receptors for atrial natriuretic peptide

The binding of atrial natriuretic peptide (ANP) to olfactory bulb, pituitary anterior lobe and thymus gland membranes was examined. [125I]ANP (rat, 99-126) bound specifically to the three types of membranes. However, the affinity for ANP receptor in olfactory bulb was much higher than those in either pituitary or thymus gland. Competitive inhibition of cold ANP (rat, 99-126) with [125I]ANP binding sites on olfactory bulb membranes gave a value of 796 +/- 80 pM (mean +/- S.E.M., n = 4) as a dissociation constant (Kd) of cold ANP (rat, 99-126), while on pituitary and thymus membranes, the competitive curve gave a value of 9.3 +/- 0.4 nM (mean +/- S.E.M., n = 3) and 25.5 +/- 2.2 nM (mean +/- S.E.M., n = 6) as a Kd of cold ANP (rat, 99-126), respectively. Furthermore, a truncated ANP fragment (rat, 111-126) did not inhibit the [125I]ANP binding in olfactory bulb, while this peptide fragment inhibited the [125I]ANP binding in either pituitary or thymus gland with affinities only 2- to 4-fold less potent than ANP (rat, 99-126). These data indicate the possibility of the existence of multiple types of ANP receptors. We propose alpha-receptor in olfactory bulb and beta-receptor in either pituitary anterior lobe or thymus gland.     

Intrarenal localization of receptors for alpha-rat atrial natriuretic polypeptide: an autoradiographic study with [125I]-labeled ligand injected in vivo into the rat aorta

We examined intrarenal localization of receptors for alpha-rat atrial natriuretic polypeptide (alpha-rANP) by injecting [125I]-labeled ligand in vivo into the rat aorta. We found that the receptors for alpha-rANP are distributed also on the vasa recta of the outer and inner medulla in addition to the previously reported sites, i.e., the renal arteries, renal pelvis, glomeruli, and inner medullary tissues including collecting tubules. In the vascular bundle of the outer medulla, the majority of grains was preferentially localized on the arterial vasa recta. The electron microscopic autoradiography of the glomerulus showed that the binding sites were mainly localized on the foot process of the podocyte. Since alpha-rANP injected into the aorta under physiological conditions was bound to the glomerulus and vasa recta in the kidney, the effect of ANP on these binding sites may be important in the mechanism of natriuresis.     

Identification of structural requirements for analogues of atrial natriuretic peptide (ANP) to discriminate between ANP receptor subtypes

The structure-activity relationships for affinity and selective binding of atrial natriuretic peptide (ANP) and analogues to guanylate cyclase coupled (CC) and non-cyclase coupled (NC) receptors in rabbit lung membranes are described. We have designed a series of peptides to try to identify the minimal sequence involved in specific recognition of each receptor subtype. The affinity of the peptides was determined from competitive binding experiments. Several peptides derived from the rat ANP sequence, e.g., des-[Phe106, Gly107, Ala115, Gln116]ANP-(103-125)NH2 (4), des-[Cys105,121]ANP-(104-126) (5), and [Acm-Cys105]ANP-(105-114)NH2 (9) have high affinity and selectivity for the noncoupled site. Peptide 4 was the most selective ligand with an affinity superior to that of ANP-(103-126). This compound does not displace the radiolabeled ligand from the guanylate cyclase coupled receptor at the highest concentration tested (100 nM). The structure-activity relationship for affinity and selectivity is discussed. Comparison of the peptide sequences suggests that the structural feature responsible for recognition of the NC site resides in a single sequence of seven contiguous amino acids from the cyclic core of the hormone. The corresponding heptapeptide retains affinity to the guanylate cyclase uncoupled binding site and is proposed to encompass the minimal sequence for specific recognition of the non-guanylate cyclase coupled ANP receptor.     

The distribution of beta-adrenoceptors in dog kidney: an autoradiographic analysis

The distribution of beta-adrenoceptors in slide-mounted dog kidney sections was determined using the radioligand (-)-[125I]cyanopindolol ((-)-[125I]CYP) and autoradiography. Using conditions designed to prevent (-)-[125I]CYP binding to non-beta-adrenoceptor sites, biochemical studies revealed that (-)-[125I]CYP binding equilibrated within 150 min (K1 = 3.2 X 10(8) M-1 min-1), was saturable (KD = 30.72 +/- 2.96 pM; Bmax = 0.57 +/- 0.03 fmol/section, n = 4) and stereoselective with respect to the stereoisomers of propranolol and pindolol. Delineation of beta-adrenoceptor subtypes with the selective beta 1-adrenoceptor antagonist betaxolol and beta 2-adrenoceptor antagonist ICI 118,551 demonstrated that the proportions of beta 1-: beta 2-adrenoceptors was between 1:6 and 1:11. Autoradiographic studies showed that beta 1-adrenoceptors were localized on the juxtaglomerular apparatus and glomeruli, while beta 2-adrenoceptors were localized on medullary rays. The distribution of beta-adrenoceptors with respect to renal function in the dog kidney is discussed.     

Modulation of hypothalamic norepinephrine release by atrial natriuretic peptide: involvement of cyclic GMP.

The ability of atrial natriuretic peptide (ANP) to modulate K+-stimulated release of [3H]norepinephrine ([3H]NE) from rat hypothalamic slices was investigated. ANP-(1-28) significantly decreased K+-stimulated [3H]NE release in a concentration-dependent manner (maximal inhibition = 22% of control with 100 nM, ED50 = 70 pM). Pretreatment with pertussis toxin did not alter the response to ANP. 8Br-cGMP (10 microM), a cGMP analog, significantly decreased [3H]NE release and when combined with 10 nM ANP-(1-28), an additive effect was observed. Additionally, 3-isobutyl 1-methylxanthine (IBMX) (200 microM), a phosphodiesterase inhibitor, combined with ANP-(1-28) 10 nM, significantly decreased [3H]NE release. These results indicate that ANP-(1-28) modulated release of [3H]NE from rat hypothalamic slices and the effect is most likely mediated by elevation of intraneuronal cGMP.     

Suppression of atrial natriuretic peptide (ANP) receptor recovery from homologous down-regulation by 8-bromo-cGMP in endothelial cells.

In a previous study we reported that cyclic GMP (cGMP) selectively down-regulates the atrial natriuretic peptide clearance receptor (C-ANP receptor) in the cultured bovine pulmonary artery endothelial (CPAE) cell line. Our efforts in the current study are directed towards determining the effects of cGMP on C-ANP receptor recycling and de novo synthesis following homologous down-regulation by atriopeptin III (APIII, rat ANP 103-126). [125I]APIII binding to CPAE cells was decreased to 45.3 +/- 1.0% of control following the pretreatment with 100 nM APIII for 24 h. After the removal of APIII, the decreased [125I]APIII binding gradually recovered up to 68.8 +/- 1.8% of control for 24 h: a 'long-term recovery'. When CPAE cells were pretreated with 1 nM APIII for 30 min, [125I]APIII binding was also decreased to 62.6 +/- 2.6% of control. Following the removal of APIII, the decrease in [125I]APIII binding quickly recovered to 87.7 +/- 2.7% of control for 1 h: a 'short-term recovery'. 8-bromo-cGMP suppressed the long-term recovery of ANP receptor in a dose-dependent manner, while it had no effect on the short-term recovery. Both actinomycin D (1 ng/ml) and cycloheximide (10 ng/ml) significantly (P less than 0.01) suppressed the long-term recovery, but failed to affect the short-term recovery, whereas, the short-term recovery was significantly (P less than 0.01) inhibited by either 10 mM NH4Cl or 0.2 mM chloroquine which inhibits the recycling of internalized ANP receptor. These findings suggest that new ANP receptor synthesis is necessary for long-term but not for short-term recovery.(ABSTRACT TRUNCATED AT 250 WORDS)     

Discovery of a potent atrial natriuretic peptide antagonist for ANPA receptors in the human neuroblastoma NB-OK-1 cell line.

The effects of seven competitive atrial natriuretic peptide (ANP) receptor antagonists were compared on cultured human neuroblastoma NB-OK-1 cells expressing exclusively ANPA receptors, by evaluating their capacity to inhibit [125I]ANP binding and to suppress ANP-stimulated cyclic GMP elevation. In ANP analogues with a shortened Cys7-Cys18 bridge, Asp13 and a hydrophobic Tic residue at position 16 expressed antagonistic activity, while Ala16 provoked lower antagonistic potency and Phe16 induced receptor activation. The binding affinity of A71915 ([Arg6, Cha8]ANP-(6-15)-D-Tic-Arg-Cys-NH2), the most potent antagonist (with a pKi of 9.18 and a pA2 of 9.48) was only 22 times less lower than that of the agonist ANP-(1-28).     

Identification of renal natriuretic peptide receptor subpopulations by use of the non-peptide antagonist, HS-142-1.

1. The renal actions of natriuretic peptides are dictated by the distribution of guanylyl cyclase-linked (NPRA and NPRB) and non-guanylyl cyclase-linked (NPRC) receptors. Natriuretic peptide receptors have previously been distinguished on the basis of their differential affinity for peptide fragments and analogues; however, most of the available ligands are not fully selective. We have used the specific guanylyl cyclase-linked receptor antagonist, HS-142-1, to investigate the differential distribution of natriuretic peptide receptor subtypes in the human, bovine and rat kidney. 2. Specific, high affinity 3-([125I]-iodotyrosyl)-rat-ANP-(1-28)([125I]-rANP1-28) binding sites were identified in all three species, localized to glomeruli, inner medulla, intrarenal arteries and regions in the outer medulla corresponding to vasa recta bundles. Binding sites were also identified in the smooth muscle lining of the hilar region in the bovine and rat kidney. 3. In the rat, [125I]-rANP1-28 binding was inhibited by unlabelled peptide sequences with a rank order of potency (rANP1-28 > pCNP1-22 > C-ANP4-23). The glomeruli exhibited a heterogeneous population of binding sites, C-ANP4-23 and pCNP1-22 producing a significantly better fit to a two component inhibition curve compared to the single component curve for rANP1-28. 4. Competitive inhibition experiments with the receptor selective ligands, C-ANP4-23 and HS-142-1, suggested that, like the rat, human and bovine glomeruli possessed a heterogeneous population of binding sites, whilst those in the inner medulla and intrarenal arteries of all three species represented a homogeneous population.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neuropeptide Y (NPY) metabolism by endopeptidase-2 hinders characterization of NPY receptors in rat kidney.

1. Despite the observation of pharmacological responses to neuropeptide Y (NPY) in mammalian kidneys, there are species differences in the ease with which specific NPY binding sites can be demonstrated; we have investigated whether this can be explained by differential metabolism of NPY by a membrane-bound peptidase. 2. NPY receptors were identified on cell membranes isolated from the rabbit kidney (KD = 97 +/- 16 pM, Bmax = 290 +/- 30 fmol mg-1 protein), and this preparation did not degrade [125I]-NPY. However, a similar preparation of cell membranes from the rat kidney exhibited a much lower apparent receptor affinity (IC50 approximately 30 nM); these membranes rapidly degraded [125I]-NPY to fragments which did not bind NPY receptors in either tissue. 3. [125I]-NPY binding sites were revealed in the rat kidney when degradation was inhibited by insulin B chain. Chelating agents also inhibited degradation, but interfered with receptor binding. Binding sites could not be demonstrated in sections of rat kidney, even in the presence of insulin B chain. 4. The difference in degradative activity between rat and rabbit renal cell membranes, inhibition of degradation by chelating agents and insulin B chain, and insensitivity to phosphoramidon suggest that the enzyme responsible was endopeptidase-2, and this was confirmed by comparing the hydrolysis of [125I]-NPY by purified enzyme with rat renal tissue. Activity of this enzyme explains the difficulties encountered demonstrating receptors in the rat kidney. 5. Renal cell membranes from the mouse digested [125I]-NPY in a similar manner and this may be due to the closely related enzyme, meprin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization and regulation of atrial natriuretic peptide (ANP)-R1 receptors in the human neuroblastoma cell line NB-OK-1.

We characterized in membranes from the human neuroblastoma cell line NB-OK-1, an ANP-R1 receptor (Mr 130 kDa) for the atrial natriuretic peptide (ANP). This receptor recognized biologically active forms of ANP with high affinity but showed no affinity for truncated ANP forms. It was functional in that binding correlated with guanylate cyclase activation (a 2-fold increase in Vmax) with the following rank order of potency: rat ANP-(99-126) greater than human ANP-(99-126) greater than human ANP-(102-126) greater than porcine BNP (brain natriuretic peptide). The enzyme required free Mn2+ in addition to the Mn-GTP substrate (Km of about 0.3 mM for both basal and ANP-stimulated activity). In the presence of dithiothreitol, the dose-response curve of guanylate cyclase activation was shifted rightward by a factor of 30. ANP-R1 receptors were upregulated through protein synthesis in cells exposed to 1 mM carbamylcholine or 1 mM dibutyryl cyclic AMP for 8-24 h (ANP was ineffective).     

Atrial and brain natriuretic peptides share binding sites in the kidney and heart

We examined the distribution of binding sites for atrial natriuretic peptide (ANP) and the recently discovered brain natriuretic peptide (BNP) in rat kidney and heart by receptor autoradiography. In frozen kidney sections, both 125I-ANP and 125I-BNP exhibited localized binding to cortical glomeruli. The binding of each radiolabeled peptide was abolished by inclusion of either excess (1 microM) unlabeled ANP or excess unlabeled BNP, suggesting that ANP and BNP share cortical glomerular binding sites. In frozen cardiac sections, ANP and BNP binding sites were localized to the endothelium of the endomural channels and endocardium. As was the case for kidney, binding of each peptide at these sites was abolished by the presence of excess unlabeled ANP or BNP, suggesting that these natriuretic peptides share binding sites in the heart as well. To explore further the possibility that ANP and BNP utilize the same receptor(s), we performed competitive binding experiments using cultured pulmonary artery endothelial (CPAE) cells. ANP and BNP competitively displaced one another with equivalent IC50 values from CPAE cell binding sites. Furthermore, both ANP and BNP elevated the levels of cGMP, a putative second messenger for ANP, in these cells. These data are consistent with the observation that BNP, like ANP, causes a natriuresis and diuresis in rats, and suggest that BNP may possess other biological activities known for ANP.     

Suppression of atrial natriuretic peptide (ANP) receptor recovery from homologous down-regulation by 8-bromo-cGMP in endothelial cells

In a previous study we reported that cyclic GMP (cGMP) selectively down-regulates the atrial natriuretic peptide clearance receptor (C-ANP receptor) in the culture bovine pulmonary artery endothelial (CPAE) cell line. Our efforts in the current study are directed towards determining the effects of cGMP on C-ANP receptor recycling and de novo synthesis following homologous down-regulation by atriopeptin III (APIII, rat ANP 103–126). [¹²⁵I]APIII binding to CPAE cells was decreased to 45.3 ± 1.0% of control following the pretreatment with 100 nM APIII for 24 h. After the removal of APIII, the decreased [¹²⁵I]APIII binding gradually recovered up to 68.8 ± 1.8% of control for 24 h: a ‘long-term recovery’. When CPAE cells were pretreated with 1 nM APIII for 30 min, [¹²⁵I]APIII binding was also decreased to 62.6 ± 2.6% of control. Following the removal of APIII, the decrease in [¹²⁵I]APIII binding quickly recovered to 87.7 ± 2.7% of control for 1 h: a ‘short-term recovery’. 8-bromo-cGMP suppressed the long-term recovery of ANP receptor in a dose-dependent manner, while it had no effect on the short-term recovery. Both actinomycin D (1 ng/ml) and cycloheximide (10 ng/ml) significantly (P < 0.01) suppressed the long-term recovery, but failed to affect the short-term recovery, whereas, the short-term recovery was significantly (P < 0.01) inhibited by either 10 mM NH₄Cl or 0.2 mM chloroquine which inhibits the recycling of internalized ANP receptor. These findings suggest that new ANP receptor synthesis is necessary for long-term but not for short-term recovery. cGMP appears to suppress ANP receptor recovery from down-regulation by inhibiting ANP receptor synthesis without having any effect on ANP receptor recycling in cultured vascular endothelial cells.     

HS-142-1, a novel nonpeptide atrial natriuretic peptide (ANP) antagonist, blocks ANP-induced renal responses through a specific interaction with guanylyl cyclase-linked receptors.

HS-142-1, a novel microbial product, blocked 125I-labeled rat atrial natriuretic peptide (rANP) (= ANF(99-126)) binding to bovine adrenocortical membranes, where guanylyl cyclase-containing receptors are predominantly expressed. However, HS-142-1 only slightly inhibited [125I]rANP binding to bovine lung membranes where only a small portion of binding sites are coupled to guanylyl cyclase. Further, HS-142-1 only recognized the 135 kDa ANP receptor, which is considered to be the guanylyl cyclase-containing receptor based on the results obtained in affinity cross-linking studies with bovine adrenocortical and lung membranes. Under identical conditions, Atriopeptin I selectively recognized guanylyl cyclase-free receptors both in binding and affinity cross-linking experiments. When injected intravenously (1 mg/kg) to anesthetized rats, HS-142-1 abolished ANP-induced diuresis and natriuresis. These results suggest that HS-142-1 works in vivo through a specific interaction with the ANP functional receptor, and that HS-142-1 will be a powerful tool for understanding the physiological roles of ANP in distinction from its pharmacological effects.     

Induction of nitric oxide synthase regulates atrial natriuretic peptide receptors in vascular smooth muscle cells

In a previous study, we reported that cyclic GMP (cGMP) selectively down-regulates the atrial natriuretic peptide clearance receptor (ANP_C receptor) in the vascular endothelial cells. The aim of the present study is to determine whether nitric oxide (NO) regulates ANP receptor by accumulating intracellular cGMP in cultured rabbit aortic smooth muscle cells. Treatment with interleukin-1β (IL-1), a cytokine known to induce NO synthase, dose-dependently increased the basal level of intracellular cGMP with a concomitant increase of nitrite in the conditioned media. These effects of IL-1 were attenuated in the presence of N^ω-nitro-L-arginine. IL-1 (40 pM) significantly (P < 0.01) decreased [¹²⁵I]atriopeptin III binding to the cells, an effect that was inhibited by N^ω-nitro-L-arginine. Treatment with sodium nitroprusside (SNP) which releases NO also decreased [¹²⁵I]atriopeptin III binding to the cells. In addition, further decrease in [¹²⁵I]atriopeptin III binding following IL-1 or SNP treatment was observed in the presence of 0.1 mM zaprinast, a cGMP-specific phosphodiesterase inhibitor. Scatchard analysis of the binding data in the treated cells showed a decrease in B_max without a significant change in K_d. Affinity cross-linking of [¹²⁵I]atriopeptin III to the treated cells demonstrated a decrease in 70-kDa band corresponding to the ANP_C receptor. Meanwhile, intracellular cGMP response to atriopeptin III was significantly (P < 0.01) enhanced in the cells pretreated with IL-1 or SNP despite the decrease in receptor density. These findings suggest that NO down-regulates the ANP_C receptor by the accumulation of intracellular cGMP in cultured rabbit aortic smooth muscle cells. Enhanced cGMP response to atriopeptin III in the treated cells indicates that the membrane type guanylate cyclase-coupled receptor (ANP_B receptor) is regulated in a differential manner.     

Atrial natriuretic peptide modulators: Dissociation of receptor binding and particulate guanylate cyclase activity

Prazosin stimulated ¹²⁵l-rANP(102–126) binding to bovine adrenal zona glomerulosa membranes (BAZGM) allosterically by converting the lower-affinity state binding sites into the higher-affinity state. The modulatory effect could be mimicked by guanabenz and LY193422, and was concentration-dependent. SC₅₀ (concentration required to stimulate binding by 50%) values for guanabenz, prazosin, and LY193422 were 13, 25, and 1.8 μM, respectively. Prazosin (30 μM) enhanced ¹²⁵l-ANP(102–126) binding by decreasing the K_D from 251 ± 24 to 97.4 ± 8.3 pM without affecting the Bmax. Competitive inhibition of ¹²⁵l-ANP binding by ANP analogs was potentiated by prazosin (30 μM) and LY193422 (50 μM). The modulator-stimulated ¹²⁵l-rANP(102–126) binding was reversible by the addition of unlabelled ANP analogs during incubation. Previously, we also demonstrated that these modulators potentiated the ANP-mediated inhibition of ACTH-induced aldosterone synthesis in rat adrenal zona glomerulosa cells [Horng, J. S., Steinberg, M. I., Wyss, V. L., Wiest, S. A., Schirmer, E. W., McCowan, J. R., and Yu, M. J.: Atrial natriuretic peptide (ANP) receptor modulators: Effects of prazosin and guanabenz analogs on ANP receptor binding and in vitro biological activity. FASEB Journal 3:A733, 1989; Yu, M. J., McCowan, J. R., Steinberg, M. I., Wiest, S. A. Wyss, V. L., and Horng, J. S.: Atrial natriuretic peptide receptor modulators: Effect of disubstituted quinazolines on receptor binding and in vitro biological activity. Journal of Medicinal Chemistry 33:348–353, 1990]. However, neither prazosin nor LY193422 affected basal or ANP-induced particulate guanylate cyclase activity in rabbit glomeruli. This is the first study to our knowledge in which ANP modulators were demonstrated to potentiate ANP-mediated response independent of cGMP. Our studies suggest that potentiation of ANP-mediated inhibition of aldosterone synthesis by these analogs does not involve particulate guanylate cyclase and that these modulators induce a cyclase-uncoupled, high affinity ANP receptor in the adrenal gland, which might be different from cyclase-uncoupled ANP clearance receptor.     

Autoradiographic localisation of endothelin binding sites in kidney

[125I]Endothelin binding sites occurred in glomeruli of human, monkey, rat and dog kidney but not rabbit glomeruli. The renal medulla had more binding sites in all species, with the greatest densities of all in the rat and monkey renal papillas. Binding in rat kidney was inhibited by [Ala3,11]endothelin with lower potency than endothelin but not by unrelated peptides. These binding sites may represent the endothelin receptors and suggest actions for endothelin on renal function.     

Characterization of propranolol-resistant (-)-[125I]-cyanopindolol binding sites in rat soleus muscle.

1. The characteristics of a propranolol-resistant (-)-[125I]-cyanopindolol (CYP) binding site in rat soleus muscle were determined. 2. Saturation studies performed on homogenates of rat soleus muscle showed two phases of (-)-[125I]-CYP binding, a high affinity site (KD1 30.5 +/- 16.3 pM, Bmax 9.4 +/- 1.38 fmol mg-1 protein) and a lower affinity site (KD2 522.5 +/- 29.1 pM, Bmax 62.19 +/- 11.76 fmol mg-1 protein, n = 4). 3. In rat soleus muscle homogenates labelled with (-)-[125I]-CYP (500 pM), (-)-propranolol competition curves were biphasic with pKD values of 8.30 +/- 0.19, and 5.33 +/- 0.08, n = 7. 4. Competition between (-)-[125I]-CYP (500 pM) and (+/-)-tertatolol, (+/-)-nadolol, (+/-)-alprenolol, (+/-)-CYP, and (-) and (+)-pindolol showed that these compounds competed for binding at the propranolol-resistant site with affinities lower than those displayed at typical beta-adrenoceptors. The atypical beta-adrenoceptor agonists BRL 37344, SR58611A and ICI D7114 and the partial agonist (+/-)-CGP 12177 also competed for (-)-[125I]-CYP binding. 5. Stereoselectivity was demonstrated for the stereoisomers of alprenolol and tertalolol. The (-)-isomers of alprenolol and tertalolol had higher affinity than their corresponding (+)-isomers (3.1 and 2.6 fold respectively). These low stereoselectivity values are a characteristic of atypical beta-adrenoceptors. 6. The beta-adrenoceptor agonists, (-)-adrenaline, (-)-isoprenaline and (-)-noradrenaline, all showed lower affinity than the atypical beta-adrenoceptor agonists and competition curves appeared biphasic in nature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endothelin receptors in human coronary artery and aorta.

1. ETA and ETB-selective and non-selective ligands were used to define the endothelin receptors in the media (vascular smooth muscle layer) of human aorta and coronary artery. Saturation experiments with iodinated endothelin-1 (ET-1), endothelin-2 and sarafotoxin 6b (S6b) identified high affinity binding sites in aorta (KD [125I]-ET-1 0.33 +/- 0.02 nM (n = 9), KD [125I]-ET-2 1.04 +/- 0.23 nM (n = 5), KD [125I]-S6b 0.15 +/- 0.01 nM (n = 9 +/- s.e.mean)) and coronary artery (KD [125I]-ET-1 0.43 +/- 0.10 nM, KD [125I]-ET-2 0.71 +/- 0.17 nM, KD [125I]-S6b 0.27 +/- 0.03 nM (n = 3 +/- s.e.mean)). Hill coefficients (nH) approached unity in each case. 2. No specific binding was detectable with [125I]-ET-3 (4 pM-4 nM) in aorta. Unlabelled ET-3 competed monophasically with [125I]-ET-1 in aorta (KD, 8.21 +/- 1.62 nM, compared to unlabelled ET-1 KD, 0.60 +/- 0.20 nM) (n = 3 +/- s.e.mean). In coronary artery, the KD and Bmax values calculated from [125I]-ET-3 saturation experiments were 2.13 +/- 1.39 nM and 20.6 +/- 12.9 fmol mg-1 protein, respectively (n = 3 +/- s.e.mean). 3. ETA antagonists competed monophasically for [125I]-ET-1 (100 pM) binding sites with nanomolar or subnanomolar affinity in the aorta (KD BQ123, 0.47 +/- 0.13 nM; KD FR139317, 0.40 +/- 0.10 nM; KD PD151242, 2.09 +/- 0.48 nM) and coronary artery (KD FR139317, 0.41 +/- 0.13 nM; KD PD151242, 3.60 +/- 0.74 nM) (n = 3 +/- s.e.mean). However, two site fits were preferred on analysis of competition experiments with ETB-selective agonists versus [125I]-ET-1 in coronary artery (BQ3020: KDETA 0.96 +/- 0.14 microM, KD ETB 1.34 +/- 1.08 nM and sarafotoxin 6c: KD ETA 1.15 +/- 0.14 microM, KD ETB 1.77 +/- 0.72 nM) (n = 3 +/- s.e.mean). The selectivity of the agonists for ETB receptors (700 fold) was lower than reported in other species. 4. Sarafotoxin 6b (2 pM-2 microM) completely inhibited [125I]-ET-1 (100 pM) binding in aorta (KD 1.36 +/- 0.22 nM) (n = 3 +/- s.e.mean). The non-peptide compounds Ro462005 and bosentan, competed with [125I]-ET-1 binding in coronary artery with KD values of 0.19 +/- 0.04 microM and 2.94 +/- 0.95 nM, respectively (n = 3 +/- s.e.mean). 5. Inhibition of [125I]-ET-2 and [125I]-S6b binding by FR139317 was similar to the inhibition of [125I]-ET-1 binding in both arteries, being monophasic with KD values in the same range. 6. ETA receptors in coronary artery media were detected by [125I]-PD151242 (KD 0.23 +/- 0.04 nM, Bmax 10.1 +/- 1.2 fmol mg-1 protein) (n = 3 +/- s.e.mean). [125I]-BQ3020, an ETB-selective radioligand, indicated the presence of a smaller population of ETB receptors in this tissue (KD 0.60 +/- 0.31 nM, Bmax 4.5 +/- 2.1 fmol mg-1 protein) (n = 3 +/- s.e.mean). 7. Autoradiography with [125I]-PD151242 and [125I]-BQ3020 confirmed the predominance of ETA receptors in the media of both arteries. 8. The results of this study indicate that ETA receptors predominate in the vascular smooth muscle of human cardiac arteries, with a small and variable population of ETB receptors detectable in the coronary artery.