Comparison of 125I-Angiotensin III and 125I-Angiotensin II Binding to Rat Brain Membranes

The binding of 125I-angiotensin III (125I-ANG III) to rat brain membranes was examined and compared with that of 125I-angiotensin II (125I-ANG II). Degradation of each ligand, as monitored by HPLC, was effectively inhibited using fragments of ANG III and ANG II known to have little affinity for angiotensin binding sites. Three classes of 125I-ANG III-binding sites were observed based on affinity (KD = 0.13, 1.83, and 10.16 nM) and capacity (Bmax = 1.30, 18.41, and 67.2 fmol/mg protein, respectively). Two classes of 125I-ANG II-binding sites of high affinity (KD = 0.11 and 1.76 nM) and low capacity (Bmax = 1.03 and 18.86 fmol/mg protein, respectively) were also identified. Cross-displacement studies confirmed that the two highest-affinity 125I-ANG III-binding sites and the 125I-ANG II-binding sites were the same. On the other hand, the binding of 125I-ANG III to the low-affinity 125I-ANG III-binding site could not be inhibited with ANG II. These data imply that previously measured differences in the biological potency of cerebroventricularly applied ANG III and ANG II probably do not result from differential binding of these peptides to central angiotensin receptors.     

High affinity angiotensin II receptors in myocardial sarcolemmal membranes. Characterization of receptors and covalent linkage of 125I-angiotensin II to a membrane component of 116,000 daltons

High affinity receptors for angiotensin II have been identified on purified cardiac sarcolemmal membranes. Equilibrium binding studies were performed with 125I-labeled angiotensin II and purified sarcolemmal vesicles from calf ventricle. The curvilinear Scatchard plots were evaluated by nonlinear regression analysis using a two-site model which identified a high affinity site Kd1 = 1.08 +/- 0.3 nM and N1 = 52 +/- 10 fmol/mg of protein and a low affinity site Kd2 = 52 +/- 16 nM and N2 = 988 +/- 170 fmol/mg of protein. Monovalent and divalent cations inhibited the binding of 125I-angiotensin II by 50%. The affinity of angiotensin II analogs for the receptor was determined using competitive binding assays; sarcosine, leucine-angiotensin II (Sar,Leu-angiotensin II), Kd = 0.53 nM; angiotensin II, Kd = 2.5 nM; des-aspartic acid-angiotensin II, Kd = 4.81 nM; angiotensin I, Kd = 77.6 nM. There is a positive correlation between potency in inducing positive inotropic response in myocardial preparations reported by others and potency for the hormone receptor observed in the binding assays. Pseudo-Hill plots of the binding data showed that agonists display biphasic binding with Hill numbers around 0.65 while antagonists recognized a single class of high affinity receptors with Hill numbers close to unity. These data were confirmed using 125I-Sar,Leu-angiotensin II in equilibrium binding studies which showed that this antagonist bound to a single class of receptor sites; Kd = 0.42 +/- 0.04 nM and N = 1050 +/- 110 fmol/mg of protein. Competition-binding experiments with this 125I-peptide yielded monophasic curves with Hill numbers close to unity for both agonists and antagonists. Membrane-bound 125I-angiotensin II was covalently linked to its receptor by the use of bifunctional cross-linking reagents such as dithiobis(succinimidyl propionate) and bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone. Analysis of the membranes showed the labeling of a component with an apparent Mr = 116,000. The affinity labeled species showed characteristics expected of a functional component of the high affinity receptor. The affinity labeling of this membrane component was inhibited by nanomolar angiotensin II or Sar,Leu-angiotensin II. Together these data indicate that high affinity receptors exist for angiotensin II that most likely mediate the positive inotropic effects of this hormone on myocardial cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of angiotensin II receptor subtypes in rat liver

Radioligand binding studies identified two classes of 125I-angiotensin II-binding sites in rat liver membranes. High affinity binding sites (Kd = 0.35 +/- 0.13 nM, N = 372 +/- 69 fmol/mg of protein) were inactivated by dithiothreitol (0.1-10 mM) without any apparent change in low affinity binding sites (Kd = 3.1 +/- 0.8 nM, N = 658 +/- 112 fmol/mg of protein). Dithiothreitol inactivation was readily reversible but could be made permanent by alkylation of membrane proteins with iodoacetamide. Angiotensin II stimulation of glycogen phosphorylase in isolated rat hepatocytes (maximal stimulation 780%, EC50 = 0.4 nM) was completely inhibited by 10 mM dithiothreitol, a concentration which also abolished high affinity site binding; phosphorylase stimulation by glucagon and norepinephrine under these conditions was unaltered. Angiotensin II inhibition of glucagon-stimulated adenylate cyclase activity in hepatocytes required higher angiotensin II concentrations (EC50 = 3 nM) than phosphorylase stimulation and was not affected by dithiothreitol. Fractional occupancy of high affinity binding sites by 125I-angiotensin II correlated closely with angiotensin II-mediated phosphorylase stimulation, whereas occupancy of low affinity sites paralleled inhibition of adenylate cyclase activity. These data indicate that the physiologic effects of angiotensin II in rat liver are mediated by two distinct receptors, apparently not interconvertible, and provide the first evidence for angiotensin II receptor subtypes with differing biochemical features and mechanisms of action.     

[3H]N-Methylscopolamine Binding Studies Reveal M2 and M3 Muscarinic Receptor Subtypes on Cerebellar Granule Cells in Primary Culture

Saturation experiments with the muscarinic antagonist [3H]N-methylscopolamine ([3H]NMS) indicated that cerebellar granule cells in primary culture possess a high density of muscarinic acetylcholine receptors (mAChRs): Bmax = 1.85 +/- 0.01 pmol/mg of protein at 10 days in culture; KD = 0.128 +/- 0.01 nM. The selective M1 antagonist pirenzepine displaced [3H]NMS binding with a low affinity (Ki = 273 +/- 13 nM), whereas the M2/M3 muscarinic antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide competed with [3H]NMS with Ki values in the nanomolar range, a result suggesting that some of the mAChRs on cerebellar granule cells belong to the M3 subtype. Methoctramine, which discriminates between M2 and M3 subtypes with high and low affinity, respectively, displayed a high and low affinity for [3H]NMS binding sites (Ki(H) = 31 +/- 5 nM; Ki(L) = 2,620 +/- 320 nM). These results provide the first demonstration that both M2 and M3 mAChR subtypes may be present on cultured cerebellar cells. In addition, complete death of neurons induced by N-methyl-D-aspartate (100 microM for 1 h) reduced by 85% the specific binding of [3H]NMS, a result indicating that most mAChRs were associated with neuronal components. Finally, the evolution of the density of mAChRs, labeled by [3H]NMS, correlated with the neuronal maturation during the in vitro development of these cells.     

Identification and characterization of the catecholamine transporter in bovine chromaffin granules using [3H]reserpine

Characterization of the catecholamine transporter in chromaffin granule membranes has been hampered by the lack of a radioligand with high specific activity which binds selectively to the carrier with high affinity. We report here the identification of a high affinity binding site for [3H]reserpine on chromaffin granule membranes isolated from bovine adrenal gland which has the characteristics expected of the catecholamine transporter. [3H]Reserpine bound predominately to a high affinity site with a Kd for [3H]reserpine of 9 nM and a binding site density of 7.8 pmol/mg of protein. Comparison of the characteristics of the high affinity reserpine binding site to the characteristics of catecholamine transport indicated that (a) the Ki and rank order of potency for inhibition of [3H]reserpine binding by various biogenic amines was similar to their Ki for inhibition of catecholamine transport (b) both the inhibition of (-)-[3H]norepinephrine transport and inhibition of [3H]reserpine binding showed similar stereo-specificity, and (c) Kd for binding of reserpine to chromaffin granule membranes was similar to the Ki for reserpine inhibition of catecholamine transport. These results demonstrate that the high affinity binding site for [3H]reserpine on chromaffin granule membranes is associated with the catecholamine transporter.     

Specific, high-affinity binding sites for angiotensin II on Mycoplasma hyorhinis.

Mycoplasmataceae are known to express various proteins that are similar to those present in mammals. We report a strain of Mycoplasma hyorhinis isolated from opossum kidney cells with specific, high-affinity binding sites for human angiotensin II (Kd = 5.1 +/- 1.9 nM). In contrast, two strains of M. hominis revealed no specific binding. These binding sites resembled mammalian angiotensin II receptors by their high affinity and by their sensitivity to dithiothreitol. However, they are different from mammalian angiotensin II receptors in that they bind angiotensin I with high affinity (Kd = 1.6 +/- 0.29 nM) but not angiotensin III (Kd approximately 330,000 nM). [125I]-angiotensin II binding was not inhibited by angiotensin receptor subtype antagonists DuP 753 and CGP 42112A but it was sensitive to bacitracin and aprotinin. Positions Asp1, Ile5, His6 and Pro7 were essential for binding to M. hyorhinis as deletion of these residues led to a more than 10,000-fold decrease in affinity.     

ACE and non-ACE mediated effect of angiotensin I on intracellular calcium mobilization in rat glomerular arterioles

Because renin and angiotensin I (ANG I) level are high in the renal circulation, the conversion of ANG I is a critical step in the regulation of glomerular hemodynamics. We studied this conversion by investigating the effect of ANG I on intracellular Ca(2+) concentration ([Ca(2+)](i)) in rat juxtamedullary glomerular afferent and efferent arterioles (AA and EA, respectively). Two types of EA were considered, thin EA and muscular EA, terminating as peritubular capillaries and vasa rectae, respectively. In all arterioles, ANG I elicited [Ca(2+)](i) elevations. Maximal responses of 171 +/- 28 (AA), 183 +/- 7 (muscular EA), and 78 +/- 11 nM (thin EA) (n = 6), similar to those obtained with ANG II, were observed with 100 nM ANG I. The EC(50) values were 20 times higher for ANG I than for ANG II in AA (10.2 vs. 0.5) and muscular EA (6.8 vs. 0.4 nM) and 150 times higher in thin EA (15.2 vs. 0.1 nM). ANG I effect was blocked by losartan, indicating that AT(1) receptors were involved. The ANG-converting enyzme (ACE) inhibitor lisinopril inhibited the maximal response to ANG I in AA and muscular EA by 75 +/- 9% (n = 13) and 70 +/- 7% (n = 13), respectively, but had no effect in thin EA (n = 14). The serine protease inhibitor aprotinin, the chymase inhibitor chymostatin, and the cysteine protease inhibitors E64 and leupeptin had no effect on ANG I action. These data show that ANG I effects are mainly mediated by ACE in AA and muscular EA but not in thin EA. The lisinopril-insensitive response may be related to conversion by unknown enzyme(s) and/or to activation of AT(1) receptors by ANG I.     

Chronic administration of nitric oxide reduces angiotensin II receptor type 1 expression and aldosterone synthesis in zona glomerulosa cells

Acute nitric oxide (NO) inhibits angiotensin II (ANG II)-stimulated aldosterone synthesis in zona glomerulosa (ZG) cells. In this study, we investigated the effects of chronic administration of NO on the ANG II receptor type 1 (AT1) expression and aldosterone synthesis. ZG cells were treated daily with DETA NONOate (10(-4) M), an NO donor, for 0, 12, 24, 48, 72, and 96 h. Chinese hamster ovary (CHO) cells, stably transfected with the AT1B receptor, were used as a positive control. Western blot analysis indicated that AT1 receptor expression was decreased as a function of time of NO administration in both CHO and ZG cells. ANG II binding to its receptors was determined by radioligand binding. NO treatment of ZG cells for 96 h resulted in a decrease in ANG II binding compared with control. The receptor density was decreased to 1,864 +/- 129 fmol/mg protein from 3,157 +/- 220 fmol/mg protein (P < 0.005), but the affinity was not changed (1.95 +/- 0.22 vs. 1.88 +/- 0.21 nM). Confocal Raman microspectroscopy and immunocytochemistry both confirmed that the expression of AT1 receptors in ZG cells decreased with chronic NO administration. In addition, chronic NO administration also decreased the expression of cholesterol side-chain cleavage enzyme in ZG cells and inhibited ANG II- and 25-hydroxycholesterol-stimulated aldosterone synthesis in ZG cells. This study demonstrates that chronic administration of NO inhibits aldosterone synthesis in ZG cells by downregulation of the expression of both AT1 receptors and cholesterol side-chain cleavage enzyme.     

Parinaric acids as probes of binding domains in neutrophil elastase

Human neutrophil elastase has an extended hydrophobic substrate binding site which serves as a target for a number of hydrophobic inhibitors. We show here that the parinaric acids, fluorescent-conjugated tetraenoic fatty acids of plant origin, are inhibitors of neutrophil elastase. cis-Parinaric acid (cis-PA) interacts with the enzyme in two inhibitory modes. The high affinity interaction (Ki = 55 +/- 6 nM) results in partial noncompetitive inhibition of amidolytic activity, with 82% residual activity. A lower affinity interaction with cis-PA (Ki = 4 +/- 1 microM) results in competitive inhibition. trans-PA also acts as a high affinity partial noncompetitive inhibitor of elastase with a Ki equal to that for cis-PA but has no low affinity competitive inhibitory action. The endogenous fluorescence from the 3 tryptophan residues in elastase is partially quenched on binding cis- or trans-PA. Dependence of quenching of tryptophan fluorescence on PA concentration is consistent with binding to a single site with an apparent Kd of 26 +/- 3 nM, which may be equivalent to the high affinity partial noncompetitive inhibitory binding mode. Analysis of quenching according to the modified Forster theory of energy transfer developed by Snyder and Freire (Snyder, B., and Freire, E. (1982) Biophys. J. 40, 137-148) leads to an estimate of apparent closest indole-PA distance of 13 +/- 3 A. Fluorescence of either cis- or trans-PA is apparently unperturbed upon binding in the high affinity mode to elastase, but at micromolar cis-PA concentrations, binding to elastase results in a blue shift and 20% increase in intensity of PA emission, suggesting that the lower affinity competitive inhibitory binding mode of binding to elastase provides a hydrophobic environment for cis-PA.     

Angiotensin-(1-7) binds at the type 1 angiotensin II receptors in rat renal cortex.

Significant angiotensin (Ang) (1-7) production occurs in kidney and effects on renal function have been observed. The present study was undertaken to investigate binding characteristics of the heptapeptide to Ang II receptors present in rat renal cortex. [125I]-Ang II binding to rat glomeruli membranes was analyzed in the presence of increasing concentrations of Ang II, Ang-(1-7), DUP 753 and PD 123319. Linearity of the Scatchard plot of the [125I]-Ang II specific binding to rat glomeruli membranes indicated a single population of receptors, with a Kd value of 0.7 +/- 0.1 nM and a Bmax of 198 +/- 0.04 fmol/mg protein. DUP 753, an specific AT1 receptor antagonist, totally displaced the specific binding of [125I]-radiolabelled hormone with a Ki of 15.8 +/- 0.9 nM, while no changes were observed in the presence of the selective AT2 receptor antagonist, PD 123319. The specific [125I]-Ang II binding to rat glomerular membranes was displaced by Ang-(1-7) with high affinity (Ki = 8.0 +/- 3.2 nM). We conclude that radioligand binding assays in the presence of selective Ang II antagonists DUP 753 and PD 123319 suggest the unique presence of AT1, receptors in rat glomeruli and a possible role in the control of the biological renal effects of Ang-(1-7).     

Benzimidazole derivatives. Part 1: Synthesis and structure-activity relationships of new benzimidazole-4-carboxamides and carboxylates as potent and selective 5-HT4 receptor antagonists

New benzimidazole-4-carboxamides 1-16 and -carboxylates 17-26 were synthesized and evaluated for binding affinity at serotonergic 5-HT4 and 5-HT3 receptors in the CNS. Most of the synthesized compounds exhibited moderate-to-very high affinity (in many cases subnanomolar) for the 5-HT4 binding site and no significant affinity for the 5-HT3 receptor. SAR observations and structural analyses (molecular modeling, INSIGHT II) indicated that the presence of a voluminous substituent in the basic nitrogen atom of the amino moiety and a distance of ca. 8.0 A from this nitrogen to the aromatic ring are of great importance for high affinity and selectivity for 5-HT4 receptors. These results confirm our recently proposed model for recognition by the 5-HT4 binding site. Amides 12-15 and esters 24 and 25 bound at central 5-HT4 sites with very high affinity (Ki = 0.11-2.9 nM) and excellent selectivity over serotonin 5-HT3, 5-HT2A, and 5-HT1A receptors (Ki > 1000-10,000 nM). Analogues 12 (Ki(5-HT4) = 0.32 nM), 13 (Ki(5-HT4) = 0.11 nM), 14 (Ki(5-HT4) = 0.29 nM) and 15 (Ki(5-HT4) = 0.54 nM) were pharmacologically characterized as selective 5-HT4 antagonists in the isolated guinea pig ileum (pA2 = 7.6, 7.9, 8.2 and 7.9, respectively), with a potency comparable to the 5-HT4 receptor antagonist RS 39604 (pA2 = 8.2). The benzimidazole-4-carboxylic acid derivatives described in this paper represent a novel class of potent and selective 5-HT4 receptor antagonists. In particular, compounds 12-15 could be interesting pharmacological tools for the understanding of the role of 5-HT4 receptors.     

Effect of angiotensin II and III on inositol polyphosphate production in differentiated NG108-15 hybrid cells

Neuroblastoma x glioma hybrid cells (NG108-15), differentiated by treatment with 1.5% dimethyl sulfoxide (DMSO) and 0.5% fetal bovine serum, were used to measure the effect of angiotensin II and III (ANG II and ANG III) on the generation of inositol polyphosphates. ANG II increased the synthesis of inositol monophosphates (IP1), inositol diphosphates (IP2), and inositol trisphosphates (IP3) with maximal responses observed at 300, 120, and 30 sec, respectively. The percent increases above basal values at the maximal responses were 140% +/- 9% (IP1), 142% +/- 4% (IP2), and 132% +/- 4% (IP3). This effect was not attenuated by pretreatment of the cells with pertussis toxin. Furthermore, both ANG II and ANG III increased the production of inositol polyphosphates in a dose-dependent manner with ED50 values of 145 nM and 11 nM, respectively. We conclude that differentiated NG108-15 cells express an ANG III selective receptor that mediates phosphatidylinositol breakdown through a pertussis toxin insensitive G-protein.     

Angiotensin II attenuates myocardial interstitial acetylcholine release in response to vagal stimulation

Although ANG II exerts a variety of effects on the cardiovascular system, its effects on the peripheral parasympathetic neurotransmission have only been evaluated by changes in heart rate (an effect on the sinus node). To elucidate the effect of ANG II on the parasympathetic neurotransmission in the left ventricle, we measured myocardial interstitial ACh release in response to vagal stimulation (1 ms, 10 V, 20 Hz) using cardiac microdialysis in anesthetized cats. In a control group (n = 6), vagal stimulation increased the ACh level from 0.85 +/- 0.03 to 10.7 +/- 1.0 (SE) nM. Intravenous administration of ANG II at 10 microg x kg(-1) x h(-1) suppressed the stimulation-induced ACh release to 7.5 +/- 0.6 nM (P < 0.01). In a group with pretreatment of intravenous ANG II receptor subtype 1 (AT(1) receptor) blocker losartan (10 mg/kg, n = 6), ANG II was unable to inhibit the stimulation-induced ACh release (8.6 +/- 1.5 vs. 8.4 +/- 1.7 nM). In contrast, in a group with local administration of losartan (10 mM, n = 6) through the dialysis probe, ANG II inhibited the stimulation-induced ACh release (8.0 +/- 0.8 vs. 5.8 +/- 1.0 nM, P < 0.05). In conclusion, intravenous ANG II significantly inhibited the parasympathetic neurotransmission through AT(1) receptors. The failure of local losartan administration to nullify the inhibitory effect of ANG II on the stimulation-induced ACh release indicates that the site of this inhibitory action is likely at parasympathetic ganglia rather than at postganglionic vagal nerve terminals.     

Characterization of the alpha 1-adrenergic receptor subtype in a smooth muscle cell line

We have identified in the DDT1 smooth muscle cell line a [3H]dihydroergocryptine-binding site having the characteristics of an alpha 1-adrenergic receptor. Specific binding of [3H]dihydroergocryptine to DDT1 cells grown either in monolayer or suspension culture was reversible, saturable, and of high affinity, and the binding site demonstrated stereoselectivity. [3H]Dihydroergocryptine dissociation constants of 1.4 +/- 0.2 nM and 1.4 +/- 0.3 nM were observed for suspension and monolayer cells, respectively. However, the concentration of binding sites in suspension-cultured cells (65,100 +/- 8,300 sites/cell) was significantly greater (p less than 0.001) than that found in monolayer cells (27,900 +/- 4,300 sites/cell). The order of agonist competition for the binding site was epinephrine (Ki = 0.92 +/- 0.32 microM) greater than or equal to norepinephrine (Ki = 2.2 +/- 1.0 microM) greater than isoproterenol (Ki = 137 +/- 17 microM), consistent with an alpha-adrenergic interaction. Results of competition experiments with specific antagonists prazosin (alpha 1-selective) or yohimbine (alpha 2-selective) and a computer modeling technique indicated that the alpha-adrenergic receptor of the DDT1 cell was predominantly (greater than 95%) the alpha 1-subtype.     

Synthesis and pharmacological studies of new hybrid derivatives of fentanyl active at the mu-opioid receptor and I2-imidazoline binding sites

Two series of fentanyl-derived hybrid molecules bearing potent I2-imidazoline binding site (IBS) ligands (i.e., guanidine and BU224 moieties) linked with an aliphatic (m=2, 3, 4, 6, 7, 8, 9 and 12 methylene units) or aromatic spacer were prepared. Their affinities for the mu-opioid receptors and for the I2-IBS were determined through competition binding studies on human postmortem brain membranes. Whereas the BU224 hybrid molecules bound to the mu-opioid receptor and the I2-IBS in the micromolar to low micromolar range, the alkaneguanidine series exhibited remarkable affinities in the nanomolar range for both receptors. [35S]GTPgammaS functional assays were performed on human postmortem brain membranes with selected ligands from each series (4f and 8g) showing the highest dual affinity for the mu-opioid receptor and I2-IBS affinities. Both compounds displayed agonist properties: at the mu-opioid receptor for the alkaneguanidine derivative 4f (spacer: six methylene units) and at a G-protein coupled receptor (GPCR) which remains to be determined for 8g. The lack of analgesic properties of 4f in vivo (i.e., hot plate and writhing tests in mice), discordant with the good in vitro binding data (Ki mu=1.04+/-0.28 nM, Ki I2=409+/-238 nM), may possibly be due to the low intrinsic efficacy of the compound. Alternatively, a low access to the central nervous system for this kind of hybrid molecules cannot be ruled out. Two new compounds reported here (9f and 13), which were not dual acting, are worth mentioning for their outstanding binding affinities; 9f bound to the mu-opioid receptor with a picomolar affinity (Ki=0.0098+/-0.0033 nM), whereas 13 presented an I2-IBS affinity (Ki=18+/-11 nM) similar to the reference compound BU224.     

Solubilisation of the 5-Hydroxytryptamine3 Receptor from Pooled Rat Cortical and Hippocampal Membranes

5-Hydroxytryptamine3 (5-HT3) receptors have been identified in the rat brain using the radioligand [3H]Q ICS 205-930. We report here that these sites have been solubilised from membranes prepared from pooled rat cerebral cortex and hippocampus using various detergents. Of the six detergents tested (1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonate, 0.5% deoxycholate, 1% Lubrol, 0.5% digitonin, 1% Triton X-100, and 1% octyl glucoside), deoxycholate (0.5%) yielded the best solubilisation (54.6 +/- 6% of receptor, 70.5 +/- 4% of protein; n = 3). However, most detergents inhibited binding of [3H]Q ICS 205-930 in solution. Binding was found to be optimal after the receptor had been exchanged by gel filtration through Sephadex G-25 into the detergent Lubrol PX (0.05%). Binding of [3H]Q ICS 205-930 to these soluble sites was saturable and specific (Bmax = 46.1 +/- 6 fmol/mg of protein; KD = 0.33 +/- 0.09 nM; n = 4) and was similar to that observed in membranes. Kinetic studies of [3H]Q ICS 205-930 binding demonstrated it to be rapid, with equilibrium being achieved within 15 min at 4 degrees C. The KD determined from the rates of association and dissociation (0.38 nM) agreed well with that determined by saturation analysis. Various antagonists completed for the soluble receptors with a rank order of potency typical for binding at a 5-HT3 receptor site: zacopride (Ki = 0.26 nM) greater than quipazine (0.37 nM) = Q ICS 205-930 (0.33 nM) greater than ICS 205-930 (0.93 nM) greater than GR 38032F (2.2 nM) greater than BRL 24924 (4.1 nM) greater than MDL 72222 (23.4 nM) greater than ketanserin (6,000 nM). The agonists 5-HT and 2-methyl-5-HT also competed for [3H]Q ICS 205-930 binding with high affinity (39.6 and 55.6 nM, respectively). Therefore, we conclude that the 5-HT3 receptor of rat brain has been successfully solubilised, and this should provide a good starting point for purification of the receptor.     

Protein kinase C substrate and inhibitor characteristics of peptides derived from the myristoylated alanine-rich C kinase substrate (MARCKS) protein phosphorylation site domain.

The phosphorylation sites in the myristoylated alanine-rich C kinase substrate or MARCKS protein consist of four serines contained within a conserved, basic region of 25 amino acids, termed the phosphorylation site domain. A synthetic peptide comprising this domain was phosphorylated by both protein kinase C and its catalytic fragment with high affinity and apparent positive cooperativity. Tryptic phosphopeptides derived from the peptide appeared similar to phosphopeptides derived from the phosphorylated intact protein. The peptide was phosphorylated by cAMP- and cGMP-dependent protein kinases with markedly lower affinities. In peptides containing only one of the four serines, with the other three serines replaced by alanine, the affinities for protein kinase C ranged from 25 to 60 nM with Hill constants between 1.8 and 3.0. The potential pseudosubstrate peptide, in which all four serines were replaced by alanines, inhibited protein kinase C phosphorylation of histone or a peptide substrate with an IC50 of 100-200 nM with apparently non-competitive kinetics; it also inhibited the catalytic fragment of protein kinase C with a Ki of 20 nM, with kinetics of the mixed type. The peptide did not significantly inhibit the cAMP- and cGMP-dependent protein kinases. It inhibited Ca2+/calmodulin-dependent protein kinases I, II, and III by competing with the kinases for calmodulin. In addition, the peptide inhibited the Ca2+/calmodulin-independent activity of a proteolytic fragment of Ca2+/calmodulin protein kinase II, with an IC50 approximately 5 microM. Thus, the phosphorylation site domain peptide of the MARCKS protein is a high affinity substrate for protein kinase C in vitro; the cognate peptide containing no serines is a potent but not completely specific inhibitor of both protein kinase C and its catalytic fragment.     

Identification of B2 bradykinin binding sites in the guinea pig nasal turbinate

Specific high affinity BK binding sites in the nasal turbinate of the guinea pig have been demonstrated. Specific [3H]BK binding (10-330 pM) was saturable, and nonlinear least squares analysis indicated the presence of a high affinity binding site with a Kd value of 60 (50-78) pM and a Bmax value of 13.1 = 2.0 fmol/mg protein. In inhibition experiments, D-Phe7-BK (a B2 antagonist) inhibited [3H]BK binding with a Ki value of 23 nM, while des-Arg9[Leu8]-BK (a B1 antagonist) had no effect up to a concentration of 10 microM. These studies indicate the presence of B2 BK receptors in the guinea pig nasal turbinate.     

Characterization of an R-plasmid dihydrofolate reductase with a monomeric structure

A plasmid-encoded dihydrofolate reductase that originated in a clinical isolate of Salmonella typhimurium (phage type 179) moderately resistant to trimethoprim has been isolated and characterized. The dihydrofolate reductase (called type III) was purified to homogeneity using a combination of gel filtration, hydrophobic chromatography, and methotrexate affinity chromatography. Polyacrylamide gel electrophoresis under denaturing and nondenaturing conditions indicated that the enzyme is a 16,900 molecular weight monomeric protein. Kinetic analyses showed that trimethoprim is a relatively tight binding inhibitor (Ki = 19 nM) competitive with dihydrofolate. The enzyme is also extremely sensitive to methotrexate inhibition (Ki = 9 pM) and has a high affinity for dihydrofolate (Km = 0.4 microM). The sequence of the first 20 NH2-terminal residues of the protein shows 50% homology with the trimethoprim-sensitive chromosomal Escherichia coli dihydrofolate reductase and suggests that the two enzymes may be closely related. This is the first example of a plasmid encoding for a monomeric dihydrofolate reductase only moderately resistant to trimethoprim, and a resistance mechanism, dependent in part on the high dihydrofolate affinity of the type III enzyme, is proposed.     

Synthesis and binding affinities of methylvesamicol analogs for the acetylcholine transporter and sigma receptor

We synthesized methylvesamicol analogs 13-16 and investigated the binding characteristics of 2-[4-phenylpiperidino]cyclohexanol (vesamicol) and methylvesamicol analogs 13-16, with a methyl group introduced into the 4-phenylpiperidine moiety, to sigma receptors (sigma-1, sigma-2) and to vesicular acetylcholine transporters (VAChT) in membranes of the rat brain and liver. In competitive inhibition studies, (-)-o-methylvesamicol [(-)-OMV] (13) (Ki=6.7 nM), as well as (-)-vesamicol (Ki=4.4 nM), had a high affinity for VAChT. (+)-p-Methylvesamicol [(+)-PMV] (16) (Ki=3.0 nM), as well as SA4503 (Ki=4.4 nM), reported as a sigma-1 mapping agent for positron emission tomography (PET), had a high affinity for the sigma-1 receptor. The binding affinity of (+)-PMV (16) for the sigma-1 receptor (Ki=3.0 nM) was about 13 times higher than that for the sigma-2 (sigma-2) receptor (Ki=40.7 nM). (+)-PMV (16) (Ki=199 nM) had a much lower affinity for VAChT than SA4503 (Ki=50.2 nM) and haloperidol (Ki=41.4 nM). These results showed that the binding characteristics of (-)-OMV (13) to VAChT were similar to those of (-)-vesamicol and that (+)-PMV (16) bound to the sigma-1 receptor with high affinity. In conclusion, (-)-OMV (13) and (+)-PMV (16), which had a suitable structure, with a methyl group for labeling with 11C, may become not only a new VAChT ligand and a new type of sigma receptor ligand, respectively, but may also become a new target compound of VAChT and the sigma-1 receptor radioligand for PET, respectively.