The Globin Fragment LVV-Hemorphin-7 Is an Endogenous Ligand for the AT4 Receptor in the Brain

Abstract: Angiotensin IV (Val-Tyr-Ile-His-Pro-Phe) has been reported to interact with specific high-affinity receptors to increase memory retrieval, enhance dopamine-induced stereotypy behavior, and induce c- fos expression in several brain nuclei. We have isolated a decapeptide (Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe) from sheep brain that binds with high affinity to the angiotensin IV receptor. The peptide was isolated using ¹²⁵I-angiotensin IV binding to bovine adrenal membranes to assay receptor binding activity. This peptide is identical to the amino acid sequence 30–39 of sheep β^A- and β^B-globins and has previously been named LVV-hemorphin-7. Pharmacological studies demonstrated that LVV-hemorphin-7 and angiotensin IV were equipotent in competing for ¹²⁵I-angiotensin IV binding to sheep cerebellar membranes and displayed full cross-displacement. Using in vitro receptor autoradiography, ¹²⁵I-LVV-hemorphin-7 binding to sheep brain sections was identical to ¹²⁵I-angiotensin IV binding in its pattern of distribution and binding specificity. This study reveals the presence of a globin fragment in the sheep brain that exhibits a high affinity for, and displays an identical receptor distribution with, the angiotensin IV receptor. This globin fragment, LVV-hemorphin-7, may therefore represent an endogenous ligand for the angiotensin IV receptor in the CNS.     

Identification and characterization of the beta-adrenergic receptor on neuroblastoma × glioma hybrid NG108-15 cells

1. Using [3H]DHA and unlabeled L-alprenolol, a substantial amount of over 64% specific binding of beta-adrenergic receptor has been identified on the neuroblastoma x glioma hybrid NG108-15 cell, which has been proven to display numerous functional characteristics of intact neurons. 2. Beta-adrenergic receptor binding on intact NG108-15 cells does not change significantly upon morphological differentiation, induced by 1 mM dibutyryl cyclic AMP (dBcAMP). 3. The [3H]DHA binding on intact NG108-15 cells is rapid, saturable, and reversible, having a t1/2 of 1.0 min for association and 3.5 min for dissociation. 4. The affinity constant (Kd) and maximum binding capacity (Bmax) for binding of [3H]DHA to beta-adrenergic receptors on NG108-15 cells have been estimated by Scatchard plot analysis to be 2.5 and 0.23 nM, respectively. Further analysis indicates a single class of receptors for [3HDHA binding on NG108-15 cells. 5. Studies on kinetic properties have revealed on-rate (K + 1) and off-rate (K - 1) constants of 0.7 X 10(-9) M min-1 and 0.19 min-1, respectively. Further, the IC50 value and inhibition constant (Ki) for unlabeled L-alprenolol to inhibit [3HDHA binding on NG108-15 cells have been estimated to be 10(-5) and 8.9 X 10(-6) M, respectively. 6. The rank-order potency of catecholamine agonists, (-)ISO greater than (+)ISO greater than EPI greater than NE, reveals the presence of type 2 receptor for the beta-adrenergic binding on both differentiated and undifferentiated NG108-15 cells. 7. The present study indicates that the clonal neuroblastoma x glioma hybrid NG108-15 cell line possesses substantial amounts of beta-adrenergic receptors with characteristics similar to those on neuronal cells.     

AT1 Angiotensin Receptors Mobilize Intracellular Calcium in a Subclone of NG108–15 Neuroblastoma Cells

The effects of angiotensin II (AII) and related peptides on the mobilization of internal Ca2+ were studied in a subclone of NG 108-15 cells. The subclone, C1, was prepared by fluorescence-activated cell cloning using a rapid response kinetics and a large response magnitude following stimulation by AII as the selection criteria. Angiotensin I, AII, and angiotensin III (AIII) stimulated Ca2+ mobilization in the C1 cells in a concentration-dependent manner (1 nM-100 microM), yielding EC50 values of 437 +/- 80 nM (n = 4; slope = 1.6 +/- 0.3), 57 +/- 8 nM (n = 12; slope = 1.5 +/- 0.3), and 36 +/- 5 nM (n = 7; slope = 1.4 +/- 0.3), respectively. AIII was significantly more potent than AII (p less than 0.05). In contrast, Des-Phe8-AII, AII-hexapeptide (AII 3-8), and p-NH2-Phe6-AII (1-10 microM) were inactive as agonists. Although the effects of AII and AIII in C1 and parent NG108-15 cells were totally inhibited by the AT1 receptor-selective nonpeptide antagonist, DUP-753 (0.3-1 microM), the AT2-selective antagonists, EXP-655 and CGP42112A (1-10 microM), failed to block the effects of AII. DUP-753 (0.3-100 nM) produced dextral shifts of the AII-induced concentration-response curves and yielded an estimated affinity constant (pA2) of 8.5 +/- 0.2 (n = 16) using single-point analysis involving different concentrations of DUP-753. These data compared well with those obtained for the inhibition of AII-induced aortic contractions by DUP-753 (pA2 = 8.5) reported previously by others.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characteristics of [14C]Guanidinium Accumulation in NG 108-15 Cell Exposed to Serotonin 5-HT3 Receptor Ligands and Substance P

Abstract: In the presence of substance P (SP; 10 μM), serotonin (5-HT; 1 μM) triggered a cation permeability in cells of the hybridoma (mouse neuroblastoma X rat glioma) clone NG 108-15 that could be assessed by measuring the cell capacity to accumulate [¹⁴C]guanidinium for 10-15 min at 37°C. In addition to 5-HT (EC₅₀, 0.33 μM), the potent 5-HT₃ receptor agonists 2-methyl-serotonin, phenylbiguanide, and m-chlorophenylbiguanide, and quipazine, markedly increased [¹⁴C]guanidinium uptake in NG 108-15 cells exposed to 10 μM SP. In contrast, 5-HT₃ receptor antagonists prevented the effect of 5-HT. The correlation (r= 0.97) between the potencies of 16 different ligands to mimic or prevent the effects of 5-HT on [¹⁴C]guanidinium uptake, on the one hand, and to displace [³H]zacopride specifically bound to 5-HT₃ receptors on NG 108-15 cells, on the other hand, clearly demonstrated that [¹⁴C]guanidinium uptake was directly controlled by 5-HT₃ receptors. Various compounds such as inorganic cations (La³⁺, Mn²⁺, Ba²⁺, Ni²⁺, and Zn²⁺), D-tubocurarine, and memantine inhibited [¹⁴C]guanidinium uptake in NG 108-15 cells exposed to 5-HT and SP, as expected from their noncompetitive antagonistic properties at 5-HT₃ receptors. However, ethanol (100 mM), which has been reported to potentiate the electrophysiological response to 5-HT₃ receptor stimulation, prevented the effects of 5-HT plus SP on [¹⁴C]guanidinium uptake. The cooperative effect of SP on this 5-HT₃-evoked response resulted neither from an interaction of the peptide with the 5-HT₃ receptor binding site nor from a possible direct activation of G proteins in NG 108-15 cells. Among SP derivatives, [D-Pro⁹]SP, a compound inactive at the various neurokinin receptor classes, was the most potent to mimic the stimulatory effect of SP on [¹⁴C]guanidinium uptake in NG 108-15 cells exposed to 5-HT. Although the cellular mechanisms involved deserve further investigations, the 5-HT-evoked [¹⁴C]guanidinium uptake appears to be a rapid and reliable response for assessing the functional state of 5-HT₃ receptors in NG 108-15 cells.     

Cyclic insulin-regulated aminopeptidase (IRAP)/AT4 receptor ligands.

The angiotensin IV receptor (AT4 receptor) is the insulin-regulated aminopeptidase enzyme (IRAP, EC 3.4.11.3). This membrane-spanning enzyme belongs to the M1 family of zinc-dependent metallo-peptidases. It has been proposed that AT4 receptor ligands exert their physiological effects by binding to the active site of IRAP and thereby inhibiting the catalytic activity of the enzyme. The biological activity of a large series of linear angiotensin IV analogs was previously disclosed. Herein, the synthesis and biological evaluation of a series of angiotensin IV analogs, encompassing macrocyclic ring systems of different sizes, are presented. It is demonstrated that disulfide cyclizations of angiotensin IV can deliver ligands with high IRAP/AT4 receptor affinity. One ligand, with an 11-membered ring system (4), inhibited human IRAP and aminopeptidase N (AP-N) activity with similar potency as angiotensin IV but was considerably more stable than angiotensin IV toward enzymatic degradation. The compound provides a promising starting point for further optimization toward more drug-like derivatives. The cyclic constrained analogs allowed us to propose a tentative bioactive conformation of angiotensin IV and it seems that the peptide adopts an inverse gamma-turn at the C-terminal.     

Evidence that the angiotensin IV (AT(4)) receptor is the enzyme insulin-regulated aminopeptidase.

Central infusion of angiotensin IV or its more stable analogues facilitates memory retention and retrieval in normal animals and reverses amnesia induced by scopolamine or by bilateral perforant pathway lesions. These peptides bind with high affinity and specificity to a novel binding site designated the angiotensin AT(4) receptor. Until now, the AT(4) receptor has eluded molecular characterization. Here we identify the AT(4) receptor, by protein purification and peptide sequencing, to be insulin-regulated aminopeptidase (IRAP). HEK 293T cells transfected with IRAP exhibit typical AT(4) receptor binding characteristics; the AT(4) receptor ligands, angiotensin IV and LVV-hemorphin 7, compete for the binding of [(125)I]Nle(1)-angiotensin IV with IC(50) values of 32 and 140 nm, respectively. The distribution of IRAP and its mRNA in the brain, determined by immunohistochemistry and hybridization histochemistry, parallels that of the AT(4) receptor determined by radioligand binding. We also show that AT(4) receptor ligands dose-dependently inhibit the catalytic activity of IRAP. We have therefore demonstrated that the AT(4) receptor is IRAP and propose that AT(4) receptor ligands may exert their effects by inhibiting the catalytic activity of IRAP thereby extending the half-life of its neuropeptide substrates.     

Characterization by [3H]Dihydroergocryptine Binding of Alpha-Adrenergic Receptors in Neuroblastoma × Glioma Hybrid Cells

[3H]Dihydroergocryptine ([3H]DHE) was shown to bind to sites in membranes from neuroblastoma X glioma hybrid cells (NG 108-15) that had the characteristics expected of alpha-adrenergic receptors. The binding was saturable with 0.3 pmol [3H]DHE bound per mg of protein and of high affinity, with an apparent dissociation constant (KD) of 1.8 nM. The specificity of the binding site for various ligands was more similar to that of alpha 2 receptors than to that of alpha 1. No specific binding of [3H]WB-4101 was found in the membranes derived from NG 108 cells. This finding also indicated that the [3H]DHE binding site in the cell is the alpha 2 receptor. GTP lowered the affinity of agonists for the [3H]DHE binding site, although the nucleotide hardly affected the affinity of antagonists including [3H]DHE.     

Angiotensin AT4 ligands are potent,competitive inhibitors of insulin regulated aminopeptidase (IRAP)

Angiotensin IV (Ang IV) exerts profound effects on memory and learning, a phenomenon ascribed to its binding to a specific AT4 receptor. However the AT4 receptor has recently been identified as the insulin-regulated aminopeptidase (IRAP). In this study, we demonstrate that AT4 receptor ligands, including Ang IV, Nle1-Ang IV, divalinal-Ang IV, and the structurally unrelated LVV-hemorphin-7, are all potent inhibitors of IRAP catalytic activity, as assessed by cleavage of leu-beta-naphthylamide by recombinant human IRAP. Both Ang IV and divalinal-Ang IV display competitive kinetics, indicating that AT4 ligands mediate their effects by binding to the catalytic site of IRAP. The AT4 ligands also displaced [125I]-Nle1-Ang IV or [125I]-divalinal1-Ang IV from IRAP-HEK293T membranes with high affinity, which was up to 200-fold greater than in the catalytic assay; this difference was not consistent among the peptides, and could not be ascribed to ligand degradation. Although some AT4 ligands were subject to minor cleavage by HEK293T membranes, none were substrates for IRAP. Of a range of peptides tested, only vasopressin, oxytocin, and met-enkephalin were rapidly cleaved by IRAP. We propose that the physiological effects of AT4 ligands result, in part, from inhibition of IRAP cleavage of neuropeptides involved in memory processing.     

Binding sites for alpha-bungarotoxin and the noncompetitive inhibitor phencyclidine on a synthetic peptide comprising residues 172-227 of the alpha-subunit of the nicotinic acetylcholine receptor.

The binding of the competitive antagonist alpha-bungarotoxin (alpha-Btx) and the noncompetitive inhibitor phencyclidine (PCP) to a synthetic peptide comprising residues 172-227 of the alpha-subunit of the Torpedo acetylcholine receptor has been characterized. 125I-alpha-Btx bound to the 172-227 peptide in a solid-phase assay and was competed by alpha-Btx (IC50 = 5.0 x 10(-8) M), d-tubocurarine (IC50 = 5.9 X 10(-5)M), and NaCl (IC50 = 7.9 x 10(-2)M). In the presence of 0.02% sodium dodecyl sulfate, 125I-alpha-Btx bound to the 56-residue peptide with a KD of 3.5 nM, as determined by equilibrium saturation binding studies. Because alpha-Btx binds to a peptide comprising residues 173-204 with the same affinity and does not bind to a peptide comprising residues 205-227, the competitive antagonist and hence agonist binding site lies between residues 173 and 204. After photoaffinity labeling, [3H]PCP was bound to the 172-227 peptide. [3H]PCP binding was inhibited by chlorpromazine (IC50 = 6.3 x 10(-5)M), tetracaine (IC50 = 4.2 x 10(-6)M), and dibucaine (IC50 = 2.7 x 10(-4)M). Equilibrium saturation binding studies in the presence of 0.02% sodium dodecyl sulfate showed that [3H]PCP bound at two sites, a major site of high affinity with an apparent KD of 0.4 microM and a minor low-affinity site with an apparent KD of 4.6 microM. High -affinity binding occurred at a single site on peptide 205-227 (KD = 0.27 microM) and was competed by chlorpromazine but not by alpha-Btx.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nitric oxide inhibits the expression of AT1 receptors in neurons

We have previously observed an increased of angiotensin II (ANG II) type 1 receptor (AT(1)R) with enhanced AT(1)R-mediated sympathetic outflow and concomitant downregulation of neuronal nitric oxide (NO) synthase (nNOS) with reduced NO-mediated inhibition from the paraventricular nucleus (PVN) in rats with heart failure. To test the hypothesis that NO exerts an inhibitory effect on AT(1)R expression in the PVN, we used primary cultured hypothalamic cells of neonatal rats and neuronal cell line NG108-15 as in vitro models. In hypothalamic primary culture, NO donor sodium nitroprusside (SNP) induced dose-dependent decreases in mRNA and protein of AT(1)R (10(-5) M SNP, AT(1)R protein was 10 ± 2% of control level) while NOS inhibitor N(G)-monomethyl-l-arginine (l-NMMA) induced dose-dependent increases in mRNA and protein levels of AT(1)R (10(-5) M l-NMMA, AT(1)R protein was 148 ± 8% of control level). Similar effects of SNP and l-NMMA on AT(1)R expression were also observed in NG108-15 cell line (10(-6) M SNP, AT(1)R protein was 30 ± 4% of control level while at the dose of 10(-6) M l-NMMA, AT(1)R protein was 171 ± 15% of the control level). Specific inhibition of nNOS, using antisense, caused an increase in AT(1)R expression while overexpression of nNOS, using adenoviral gene transfer (Ad.nNOS), caused an inhibition of AT(1)R expression in NG108 cells. Antisense nNOS transfection augmented the increase while Ad.nNOS infection blunted the increase in intracellular calcium concentration in response to ANG II treatment in NG108 cells. In addition, downregulation of AT(1)R mRNA as well as protein level in neuronal cell line in response to S-nitroso-N-acetyl pencillamine (SNAP) treatment was blocked by protein kinase G (PKG) inhibitor, while the peroxynitrite scavenger deforxamine had no effect. These results suggest that NO acts as an inhibitory regulator of AT(1)R expression and the activation of PKG is the required step in the regulation of AT(1)R gene expression via cGMP-dependent signaling pathway.     

Signals from the AT2 (angiotensin type 2) receptor of angiotensin II inhibit p21ras and activate MAPK (mitogen-activated protein kinase) to induce morphological neuronal differentiation in NG108-15 cells.

In a previous study, we had shown that activation of the AT2 (angiotensin type 2) receptor of angiotensin II (Ang II) induced morphological differentiation of the neuronal cell line NG108-15. In the present study, we investigated the nature of the possible intracellular mediators involved in the AT2 effect. We found that stimulation of AT2 receptors in NG108-15 cells resulted in time-dependent modulation of tyrosine phosphorylation of a number of cytoplasmic proteins. Stimulation of NG108-15 cells with Ang II induced a decrease in GTP-bound p21ras but a sustained increase in the activity of p42mapk and p44mapk as well as neurite outgrowth. Similarly, neurite elongation, increased polymerized tubulin levels, and increased mitogen-activated protein kinase (MAPK) activity were also observed in a stably transfected NG108-15 cell line expressing the dominant-negative mutant of p21ras, RasN17. These results support the observation that inhibition of p21ras did not impair the effect of Ang II on its ability to stimulate MAPK activity. While 10 microM of the MEK inhibitor, PD98059, only moderately affected elongation, 50 microM PD98059 completely blocked the Ang II- and the RasN17-mediated induction of neurite outgrowth. These results demonstrate that some of the events associated with the AT2 receptor-induced neuronal morphological differentiation of NG108-15 cells not only include inhibition of p21ras but an increase in MAPK activity as well, which is essential for neurite outgrowth.     

Human brain contains a novel non-AT1, non-AT2 binding site for active angiotensin peptides

AIMS: To determine whether the novel non-AT1, non-AT2 binding site for angiotensins recently discovered in rodent brains occurs in the human brain. MAIN METHODS: Radioligand binding assays of (125)I-sarcosine(1), isoleucine(8) angiotensin II binding were carried out in homogenates of the rostral pole of the temporal cortex of human brains containing 0.3 mM parachloromercuribenzoate (PCMB), 10 microM losartan to saturate AT1 receptors, 10 microM PD123319 to saturate AT2 receptors, with or without 10 microM angiotensin II to define specific binding. Competition binding assays employed a variety of angiotensin peptides, specific angiotensin receptor antagonists, several neuropeptides and an endopeptidase inhibitor to determine pharmacological specificity for this binding site. KEY FINDINGS: The novel non-AT1, non-AT2 binding site was present in similar amounts in female and male brains: Bmax 1.77+/-0.16 and 1.52+/-0.17 fmol/mg initial wet weight in female and male brains, respectively. The K(D) values, 1.79+/-0.09 nM for females, and 1.53+/-0.06 nM for males were also similar. The binding site shows pharmacological specificity similar to that in rodent brains: sarcosine(1), isoleucine(8) angiotensin II>angiotensin III>angiotensin II>angiotensin I'angiotensin IV>angiotensin 1-7. Shorter angiotensin fragments and non-angiotensin peptides showed low affinity for this binding site. SIGNIFICANCE: The presence in human brain of this novel non-AT1, non-AT2 binding site supports the concept that this binding site is an important component of the brain angiotensin system. The functional significance of this binding site, either as a novel angiotensin receptor or a highly specific angiotensinase remains to be determined.     

Characterization of the AT(4) receptor in a human neuroblastoma cell line (SK-N-MC)

Angiotensin IV (Ang IV), the 3-8 fragment of angiotensin II (Ang II), binds to a distinct receptor designated the AT(4) receptor. The peptide elicits a range of vascular and central actions including facilitation of memory retention and retrieval in several learning paradigms. The aim of this study was to characterize the AT(4) receptor in a human cell line of neural origin. Receptor binding studies indicate that the human neuroblastoma cell line SK-N-MC cells express a high-affinity Ang IV binding site with a pharmacological profile similar to the AT(4) receptor: (125)I]-Ang IV and (125)I]-Nle(1)-Ang IV bind specifically to the SK-N-MC cell membranes (K(d) = 0.6 and 0.1 nM) in a saturable manner (B(max) = 1.2 pmol/mg of protein). AT(4) receptor ligands, Nle(1)-Ang IV, Ang IV and LVV-haemorphin 7 (LVV-H7), compete for the binding of [(125)I]-Ang IV or [(125)I]-Nle(1)-Ang IV to the SK-N-MC cell membranes with rank order potencies of Nle(1)-Ang IV > Ang IV > LVV-H7 with IC(50) values of 1.4, 8.7 and 59 nM ([(125)I]-Ang IV) and 1.8, 20 and 168 nM ([(125)I]-Nle(1)-Ang IV), respectively. The binding of [(125)I]-Ang IV or [(125)I]-Nle(1)-Ang IV to SK-N-MC cell membranes was not affected by the presence of GTP gamma S. Both Ang IV and LVV-H7 stimulated DNA synthesis in this cell line up to 72 and 81% above control levels, respectively. The AT(4) receptor in the SK-N-MC cells is a 180-kDa glycoprotein; under non-reducing conditions a 250-kDa band was also observed. In summary, the human neuroblastoma cell line, SK-N-MC, expresses functional AT(4) receptors that are responsive to Ang IV and LVV-H7, as indicated by an increase in DNA synthesis. This is the first human cell line of neural origin shown to express the AT(4) receptor.     

Covalent crosslinking analysis of angiotensin receptors on differentiated NG108-15 cells

Neuroblastoma x glioma hybrid cells (NG108-15) were used as a model system to characterize neuronal-glial type angiotensin (ANG) receptors by covalent crosslinking analysis. After differentiation with 1.5% DMSO and 0.5% fetal bovine serum for four to five days, saturation analysis revealed a single high affinity site with a Kd = 1.35 +/- 0.42 nM and a Bmax = 468 +/- 106 fmol/mg protein. Using the homobifunctional crosslinking reagent bis(sulfosuccinimidyl) suberate (BS3), a site with an estimated Mr of 78 kDa was specifically labeled with 125I-ANG II as determined by SDS-polyacrylamide gel electrophoresis. Both ANG II and ANG III (10(-6) M) inhibited specific labeling. The Ki for ANG III binding was similar by both pharmacologic (Ki = 3.33 +/- 0.98 nM) and gel densitometric (Ki = 2.65 +/- 0.32 nM) analyses. We conclude that the 78 kDa protein represents a high affinity ANG binding site with similar affinities for both ANG II and ANG III.     

Insulin-regulated aminopeptidase: analysis of peptide substrate and inhibitor binding to the catalytic domain

Peptide inhibitors of insulin-regulated aminopeptidase (IRAP) accelerate spatial learning and facilitate memory retention and retrieval by binding competitively to the catalytic site of the enzyme and inhibiting its catalytic activity. IRAP belongs to the M1 family of Zn2+-dependent aminopeptidases characterized by a catalytic domain that contains two conserved motifs, the HEXXH(X)18E Zn2+-binding motif and the GXMEN exopeptidase motif. To elucidate the role of GXMEN in binding peptide substrates and competitive inhibitors, site-directed mutagenesis was performed on the motif. Non-conserved mutations of residues G428, A429 and N432 resulted in mutant enzymes with altered catalytic activity, as well as divergent changes in kinetic properties towards the synthetic substrate leucine beta-naphthylamide. The affinities of the IRAP inhibitors angiotensin IV, Nle1-angiotensin IV, and LVV-hemorphin-7 were selectively decreased. Substrate degradation studies using the in vitro substrates vasopressin and Leu-enkephalin showed that replacement of G428 by either D, E or Q selectively abolished the catalysis of Leu-enkephalin, while [A429G]IRAP and [N432A]IRAP mutants were incapable of cleaving both substrates. These mutational studies indicate that G428, A429 and N432 are important for binding of both peptide substrates and inhibitors, and confirm previous results demonstrating that peptide IRAP inhibitors competitively bind to its catalytic site.     

Cyclic AMP-independent involvement of Rap1/B-Raf in the angiotensin II AT2 receptor signaling pathway in NG108-15 cells

The angiotensin II (Ang II) type 2 (AT(2)) receptor is an atypical seven-transmembrane domain receptor. Controversy surrounding this receptor concerns both the nature of the second messengers produced as well as its associated signaling mechanisms. Using the neuronal cell line NG108-15, we have reported previously that activation of the AT(2) receptor induced morphological differentiation in a p21(ras)-independent, but p42/p44(mapk)-dependent mechanism. The activation of p42/p44(mapk) was delayed, sustained, and had been shown to be essential for neurite elongation. In the present report, we demonstrate that activation of the AT(2) receptor rapidly, but transiently, activated the Rap1/B-Raf complex of signaling proteins. In RapN17- and Rap1GAP-transfected cells, the effects induced by Ang II were abolished, demonstrating that activation of these proteins was responsible for the observed p42/p44(mapk) phosphorylation and for morphological differentiation. To assess whether cAMP was involved in the activation of Rap1/B-Raf and neuronal differentiation induced by Ang II, NG108-15 cells were treated with stimulators or inhibitors of the cAMP pathway. We found that dibutyryl cAMP and forskolin did not stimulate Rap1 or p42/p44(mapk) activity. Furthermore, adding H-89, an inhibitor of protein kinase A, or Rp-8-Br-cAMP-S, an inactive cAMP analog, failed to impair p42/p44(mapk) activity and neurite outgrowth induced by Ang II. The present observations clearly indicate that cAMP, a well known stimulus of neuronal differentiation, did not participate in the AT(2) receptor signaling pathways in the NG108-15 cells. Therefore, the AT(2) receptor of Ang II activates the signaling modules of Rap1/B-Raf and p42/p44(mapk) via a cAMP-independent pathway to induce morphological differentiation of NG108-15 cells.     

Porcine brain natriuretic peptide, another modulator of bovine adrenocortical steroidogenesis

Porcine brain natriuretic peptide (pBNP) significantly inhibited aldosterone production stimulated by an angiotensin II analog and ACTH-stimulated cortisol secretion, together with simultaneously increasing the formation of cGMP in dispersed bovine adrenocortical cells. Receptors for pBNP were identified in bovine adrenal gland using an in vitro receptor autoradiographic technique and studies of 125I-pBNP binding. In vitro receptor autoradiography demonstrated specific binding sites for 125I-pBNP in bovine adrenal cortex. Complete displacement of 125I-pBNP by unlabeled pBNP or human atrial natriuretic peptide (hANP) can take place at these sites. Analysis of 125I-pBNP binding to bovine adrenocortical membrane fractions showed that the adrenal cortex had high-affinity, low-capacity pBNP-binding sites, with a dissociation constant (Kd) of 2.32 +/- 0.33 x 10(-10) M (mean +/- SE) and a maximal binding capacity (Bmax) of 36.7 +/- 1.6 fmol/mg protein. Moreover, the specific binding sites for 125I-pBNP were completely displaced not only by unlabeled pBNP but also by unlabeled hANP. The hANP dose required for 50% inhibition of specific 125I-pBNP binding was almost identical to that for pBNP (IC50 values for hANP and pBNP: 8.5 x 10(-10) and 6.5 x 10(-10) M, respectively). These results suggest that pBNP exerts a suppressive effect on bovine adrenocortical steroidogenesis via a receptor which may be shared with ANP.     

The angiotensin AT2 receptor modulates T-type calcium current in non-differentiated NG108-15 cells.

We report here that angiotensin II (AII) and the AT2 receptor-selective ligand, CGP 42112, modulate the T-type calcium current in non-differentiated NG108-15 cells, which express only AT2 receptors. Both peptides decrease the T-type calcium current at membrane potentials above -40 mV and shift the current-voltage curve at lower potentials with maximal effect between 5 and 10 min after application. These data describe a new cellular response to AII and suggest that the AT2 receptor mediates certain neurophysiological actions of this hormone.     

Ouabain Binding, ATP Hydrolysis, and Na+,K+-Pump Activity During Chemical Modification of Brain and Muscle Na+,K+-ATPase

The effects of 16 group-specific, amino acid-modifying agents were tested on ouabain binding, catalytical activity of membrane-bound (rat brain microsomal), sodium dodecyl sulfate-treated Na+,K(+)-ATPase, and Na+,K(+)-pump activity in intact muscle cells. With few exceptions, the potency of various tryptophan, tyrosine, histidine, amino, and carboxy group-oriented drugs to suppress ouabain binding and Na+,K(+)-ATPase activity correlated with inhibition of the Na+,K(+)-pump electrogenic effect. ATP hydrolysis was more sensitive to inhibition elicited by chemical modification than ouabain binding (membrane-bound or isolated enzyme) and than Na+,K(+)-pump activity. The efficiency of various drugs belonging to the same "specificity" group differed markedly. Tyrosine-oriented tetranitromethane was the only reagent that interfered directly with the cardiac receptor binding site as its inhibition of ouabain binding was completely protected by ouabagenin preincubation. The inhibition elicited by all other reagents was not, or only partially, protected by ouabagenin. It is surprising that agents like diethyl pyrocarbonate (histidine groups) or butanedione (arginine groups), whose action should be oriented to amino acids not involved in the putative ouabain binding site (represented by the -Glu-Tyr-Thr-Trp-Leu-Glu- sequence), are equally effective as agents acting on amino acids present directly in the ouabain binding site. These results support the proposal of long-distance regulation of Na+,K(+)-ATPase active sites.     

Characterization and coupling of angiotensin-II receptor subtypes in cultured bovine adrenal fasciculata cells.

Angiotensin-II (A-II) receptor subtypes and their potential coupling mechanisms were investigated in bovine adrenal fasciculata cells (BAC) in culture, by the use of selective antagonists for AT1 (DUP 753 or Losartan) and AT2 (PD 123177 and CGP 42112A) sites. Competition for [125I]A-II specific binding with AT1 or AT2 selective ligands produced a biphasic displacement curve, suggesting two distinct A-II binding sites. In the presence of PD 123177 (10(-5) M), a concentration at which most of the AT2 sites were saturated, DUP 753 displaced [125I]A-II specific binding in a monophasic manner with an IC50 of 6.2 +/- 1.4 x 10(-7) M. In the presence of DUP 753 (10(-5) M), the displacement produced by CGP 42112A and PD 123177 was also monophasic, with IC50s of 8 +/- 3 x 10(-10) and 4.6 +/- 2.1 x 10(-7) M, respectively. The reducing agent dithio-1,4-erythritol inhibited the binding of [125I]A-II to AT1 (DUP 753 sensitive) sites, but increased its binding to AT2 sites 2-fold. The IC50 values for these two effects were about 0.5 and 3 mM, respectively. The biological effects of A-II in BAC, phosphoinositide hydrolysis and cortisol production, were inhibited in a dose-dependent manner by DUP 753, but not by AT2 antagonists. Similarly, the potentiating action of A-II on corticotropin-induced cAMP production was blocked by DUP 753, but not by AT2 antagonists. These data indicate that BAC contain both receptor subtypes, but that all the known effects of A-II in BAC were induced via the AT1 receptor subtype.