The binding-site crevice of the D4 dopamine receptor is coupled to three distinct sites of allosteric modulation.

Most biogenic amine G protein-coupled receptors contain a conserved aspartic acid residue positioned near the intracellular side of the second transmembrane-spanning (TMS) domain that is the primary site of allosteric modulation by sodium ions and pH. Recently, zinc ions and amiloride derivatives were found to allosterically modulate antagonist binding to dopamine receptors. In the current study, the wild-type D4 dopamine receptor showed an 8-fold decrease in zinc affinity in the presence of 120 mM NaCl, but the binding of zinc to the neutral TMS2 D4-D77N mutant was completely sodium-insensitive. In contrast to zinc, methylisobutylamiloride (MIA) binding to the wild-type D4 receptor was virtually unaffected by sodium. In addition, the binding affinity for MIA was essentially unchanged in the presence of an IC(50) concentration of zinc and vice versa. Furthermore, MIA binding affinity was decreased 4-fold for the D4-D77N mutant and increased 30-fold for the TMS3 mutant D4-M107V, even though the binding affinity for zinc was similar to the wild-type D4 background for both mutants. These findings demonstrate for the first time the existence of three distinct sites of allosteric modulation within a G protein-coupled receptor.

Tandem sulfur-containing amino acids are epicritical determinants of dopamine D(2) receptor pharmacology.

The conserved aspartic acid that is required for ligand binding to the dopamine D(2) receptor is followed by three tandem sulfur-containing amino acids. While previous point mutation studies did not reveal any single one of these residues as being critical for ligand binding, we now show that simultaneously substituting all three with isovolumetric, non sulfur-containing amino acids results in large decreases in the binding affinity for dopamine, (-)-raclopride and 7-(-4(4-(2, 3-dichlorophenyl)-1-piperazinyl)butyloxy)-3, 4-dihydro-2(1H)-quinolinone (aripiprazole), but not for methylspiperone or allosteric modulators.

Nonconserved residues in the second transmembrane-spanning domain of the D(4) dopamine receptor are molecular determinants of D(4)-selective pharmacology.

The molecular determinants that govern selective ligand binding to the rat D(4) dopamine receptor were investigated by substituting D(2) dopamine receptor sequences into a D(4) dopamine receptor background. The resulting mutant D(4) dopamine receptors were then screened with a panel of 10 selective and nonselective ligands, which included two allosteric modulators as sensitive measures of protein conformational changes. Mutation of a phenylalanine at position 88 in the second transmembrane-spanning domain (TMS2) of the D(4) receptor to the corresponding valine in the D(2) receptor D(4)-F88V resulted in an approximately 100-fold decrease in the affinity of the highly D(4)-selective drug 3-([4-(4-iodophenyl) piperazin-1-yl]methyl)-1H-pyrrolo[2,3-b]pyridine (L-750,667) without substantially affecting the binding of the other ligands. Mutations at the extracellular side of D(4)-TMS3 produced moderate decreases in L-750,667 binding affinities with concomitant increases in binding affinity for the D(2)/D(3)-selective antagonist (-)-raclopride. However, the binding affinities of these same D(4)-TMS3 mutants for the allosteric modulator isomethylbutylamiloride also were an anomalous 6- to 20-fold higher than either wild-type receptor. In the combined D(4)-F88V/TMS3 mutants, L-750,667 binding affinity was further decreased, but this decrease was not additive. More importantly, the combined D(4)-F88V/TMS3 mutants had (-)-raclopride and isomethylbutylamiloride binding properties that reverted back to those of the wild-type D(4)-receptor. In contrast to the D(4)-F88V mutant, the adjacent D(4)-L87W mutant had an increased affinity for ligands with extended structures, but had essentially no effect on ligands with compact structures. These findings demonstrate that two residues near the extracellular side of D(4)-TMS2 are critical molecular determinants for the selective binding of L-750,667 and ligands with extended structures.

Interactions of the novel antipsychotic aripiprazole (OPC-14597) with dopamine and serotonin receptor subtypes.

OPC-14597 {aripiprazole; 7-(-4(4-(2,3-dichlorophenyl)-1-piperazinyl) butyloxy)-3,4-dihydro-2(1H)-quinolinone} is a novel candidate antipsychotic that has high affinity for striatal dopamine D2-like receptors, but causes few extrapyramidal effects. These studies characterized the molecular pharmacology of OPC-14597, DM-1451 (its major rodent metabolite), and the related quinolinone derivative OPC-4392 at each of the cloned dopamine receptors, and at serotonin 5HT6 and 5HT7 receptors. All three compounds exhibited highest affinity for D2L and D2S receptors relative to the other cloned receptors examined. Both OPC-4392 and OPC-14597 demonstrated dual agonist/antagonist actions at D2L receptors, although the metabolite DM-1451 behaved as a pure antagonist. These data suggest that clinical atypicality can occur with drugs that exhibit selectivity for D2L/D2S rather than D3 or D4 receptors, and raise the possibility that the unusual profile of OPC-14597 in vivo (presynaptic agonist and postsynaptic antagonist) may reflect different functional consequences of this compound interacting with a single dopamine receptor subtype (D2) in distinct cellular locales.

Zinc modulates antagonist interactions with D2-like dopamine receptors through distinct molecular mechanisms.

Recently, zinc has been shown to modulate antagonist drug interactions with the D1 dopamine receptor (Schetz and Sibley, 1997) and the dopamine transporter (Norregaard et al., 1998). We now demonstrate that zinc also reversibly and dose-dependently modulates the specific binding of the butyrophenone antagonist [3H]methylspiperone to all D2-like dopamine receptors: D2L, D3, and D4. The molecular mechanisms of zinc regulation of these D2-like receptor subtypes are distinct because zinc inhibition of [3H]methylspiperone binding to the D4 receptor is noncompetitive by both equilibrium and kinetic measures (lower Bmax and essentially no change in koff), whereas the corresponding inhibition of zinc at D2L and D3 receptors is primarily characterized by competitive allosterism (increases in KD and koff). Interestingly, thermodynamic measurements reveal that the macroscopic properties of zinc binding are entropy-driven for all receptor subtypes, despite their having distinct molecular mechanisms. Zinc also reduces the binding affinity of the D2L receptor for [3H]raclopride, a structurally different antagonist of the substituted benzamide class. Sodium ions negatively modulate zinc inhibition of both sodium-insensitive [3H]methylspiperone binding and sodium-sensitive [3H]raclopride binding. In addition to its demonstrated effects on antagonist binding in membrane preparations, zinc also retards the functional effects of antagonist at the D2L receptor in intact cells. These findings suggest that synaptic zinc may be a factor influencing the effectiveness of therapies that rely on dopamine receptor antagonists.

Dopamine D2-receptor isoforms expressed in AtT20 cells inhibit Q-type high-voltage-activated Ca2+ channels via a membrane-delimited pathway.

Dopamine D2 receptors both acutely and chronically inhibit high-voltage-activated Ca2+ channels (HVA-CCs). Two alternatively spliced isoforms, D2L (long) and D2S (short), are expressed at high levels in rat pituitary intermediate lobe melanotropes but are lacking in anterior lobe corticotropes. We stably transfected D2L and D2S into corticotrope-derived AtT20 cells. Both isoforms coupled to inhibition of Q-type calcium channels through pertussis toxin-sensitive G proteins. Thus, we have created a model system in which to study the kinetics of D2-receptor regulation of Ca2+ channels. Rapid inhibition of HVA-CCs was characterized using a novel fluorescence video imaging technique for the measurement of millisecond kinetic events. We measured the time elapsed (lag time) between the arrival of depolarizing isotonic 66 mM K+, sensed by fluorescence from included carboxy-X-rhodamine (CXR), and the beginning of increased intracellular Ca2+ levels (sensed by changes in indo 1 fluorescence ratio). The lag time averaged 350-550 ms, with no significant differences among cell types. Addition of the D2-agonist quinpirole (250 microM) to the K+/CXR solution significantly increased the lag times for D2-expressing cells but did not alter the lag time for AtT20 controls. The increased lag times for D2L- and D2S-transfected cells suggest that at least a fraction of the Ca2+ channels was inhibited within the initial 350-550 ms. As this inhibition time is too fast for a multistep second messenger pathway, we conclude that inhibition occurs via a membrane-delimited diffusion mechanism.

Adenosine receptor mediates motility in human melanoma cells.

Cell motility is an essential component of tumor progression and metastasis. A number of factors, both autocrine and paracrine, have been found to influence cell motility. In the present study, adenosine and adenine nucleotides directly stimulated chemotaxis of A2058 melanoma cells in the absence of exogenous factors. Three adenosine receptor agonists stimulated motility in the melanoma cells and two adenosine receptor antagonists strongly inhibited the chemotactic response to both adenosine and AMP. The chemotactic stimulation by adenosine and AMP was pertussis toxin sensitive. Otherwise unresponsive Chinese hamster ovary cells which were transfected with the adenosine A1 receptor cDNA acquired the direct, pertussis toxin sensitive, chemotactic response to adenosine, and this response was inhibited by adenosine receptor antagonists. These findings demonstrate that adenosine and adenine nucleotides are capable of stimulating chemotaxis of tumor cells mediated through an adenosine receptor, probably of the A1 subtype. The possibility of antimetastatic therapies based on inhibition of adenosine receptor activity is raised.

Zinc allosterically modulates antagonist binding to cloned D1 and D2 dopamine receptors.

Cations of various size and charge were used as atomic scale probes of D1 and D2 dopamine receptors. Those cations that perturbed the binding of D1- and D2-selective dopamine receptor antagonists were identified by screening at 5 mM cation. Pseudo-noble-gas-configuration d-transition metals, such as zinc, exerted a complete inhibition of specific binding, whereas most other cations had little or no effect. The nature of zinc's actions was characterized by measuring the radioligand binding properties of [3H]SCH-23390 and [3H]methylspiperone to cloned D1A and D2L dopamine receptors in either the presence or absence of Zn2+. Zinc exerts a low-affinity, dose-dependent, EDTA-reversible inhibition of the binding of subtype-specific antagonists primarily by decreasing the ligands' affinity for their receptors. The mechanism of zinc inhibition appears to be allosteric modulation of the dopamine receptor proteins because zinc increases the dissociation constant (K(D)) of ligand binding, Schild-type plots of zinc inhibition reach a plateau, and zinc accelerates antagonist dissociation rates. Here we demonstrate the effect of zinc on the binding of D1- and D2-selective antagonists to cloned dopamine receptors and show that the inhibition by zinc is through a dose-dependent, reversible, allosteric, two-state modulation of dopamine receptors.

Rapid in vivo metabolism of a methylether derivative of (+/-)-BW373U86: the metabolic fate of [3H]SNC121 in rats.

Activation of opioid delta receptors produces antinociception without some of the side-effects associated with activation of mu and kappa receptors. (+/-)-BW373U86 [(+/-)-4-[(alpha-R*)-alpha-((2S*,5R*)-4-allyl-2, 5-dimethyl-1-piperazinyl)-3-hydroxybenzyl]-N,N-diethylbenzamide] is a first generation, racemic nonpeptide, partially delta-selective opioid agonist that produces short-lived antinocioception. After systemic, but not central, administration, (+/-)-BW373U86 is also a naltrindole-reversible convulsant. SNC80 [(+)-4-[9-alpha-R)-alpha-((2S,5RO-4-allyl-2, 5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide] is a chiral methylether derivative of (+/-)-BW373U86 with decreased potency, but greater selectivity for the delta-opioid receptor. Like BW373U86, SNC80 produces brief, nonlethal seizures when administered peripherally, albeit at higher doses. Radiolabeling of SNC80 yields a compound with similar pharmacology named [3H]SNC121. [3H]SNC121 was investigated to determine the relationship between its time course of metabolism and the physiological actions of SNC80. The biotransformation of i.p. administered [3H]SNC121 was established in rats in vivo and in vitro via high-performance liquid chromatography analysis of extracted radioactive tissues and fluids. Radioactive equivalents were characterized by their high-performance liquid chromatography retention times and opioid binding activity in rat brain membranes. The kidney, and especially the liver (within 5 min), rapidly metabolize SNC121 to a metabolite with delta-opioid activity coeluting with BW373U86. Direct i.c.v. administration of [3H]SNC121 resulted in minimal metabolism after 1 hr. We conclude that i.p., but not i.c.v., administered [3H]SNC121 can be metabolized rapidly and substantially by the liver to a BW373U86-like compound. The in vivo time course of metabolism after i.p. administration of [3H]SNC121 is consistent with the duration of SNC80 antinociception, and the rapid formation of a BW373U86-like metabolite may also account, in part, for its convulsant properties.

Pharmacology of the high-affinity apamin receptor in rabbit heart.

UNLABELLED: Apamin is a potent blocker of calcium-activated small conductance potassium (SK) channels in neurons, liver, skeletal muscle and ileum smooth muscle, but not in cardiac muscle. Cardiac muscle is devoid of SK channels; however, in isolated, single ventricular myocytes apamin is an extremely potent blocker of the L-type calcium current, and the anti-arrhythmic drug quinidine reverses apamin block. OBJECTIVE: To characterize the receptor binding properties and pharmacology of the apamin receptor in heart. METHODS: The binding properties of the apamin receptor were determined by rapid filtration of purified rabbit heart membranes. RESULTS: Monoiodinated apamin binds to a labile, membrane-bound protein in heart membranes at a single, high-affinity site (KD = 8.07 +/- 2.14 pM and Bmax = 686 +/- 167 fmoles/mg protein, significant run test at P = 0.05 for a one site fit). 125I-apamin binding is dose-dependently inhibited by apamin, scyllatoxin, quinidine, amiloride, as well as a variety of di- and trivalent cations that are classical blockers of L-type calcium channels (e.g. Co2+, Cd2+, Mn2+, La3+, Gd3+). The cardiac apamin receptor is also critically dependent upon pH, temperature and KCl, and co-purifies in the same membrane fraction as L-type cardiac Ca2+ channels. CONCLUSIONS: The apamin receptor in rabbit heart P2 membranes has pharmacological and biochemical properties in common with both an SK channel and an L-type Ca2+ channel.

Distribution and pharmacokinetics of a potent peptide antagonist of parathyroid hormone and parathyroid hormone-related protein in the rat.

Humoral hypercalcemia of malignancy results from the production by cancer cells of parathyroid hormone related protein that activates receptors in bone. Peptide antagonists that block parathyroid hormone receptors in vivo would be instrumental in the clinical treatment of humoral hypercalcemia of malignancy. We report the in vivo whole body distribution and blood plasma pharmacokinetics of the parathyroid hormone receptor antagonist [Nle8,18,D-Trp12,monoiodinated Tyr34]bPTH(7-34)amide to determine parameters that are likely to affect its administration regimen. A single intravenously injected dose of [Nle8,18,D-Trp12,monoiodinated Tyr34]bPTH(7-34)amide was rapidly cleared from blood plasma. The plasma concentration reaches a maximum at 10 min (Cmax = 1.93 +/- 0.27% of total injected CPM/ml), and the intact PTH derivative was detectable in plasma by HPLC analysis at this time. In vivo binding to plasma proteins was noncovalent. The peptide was rapidly cleared from blood by the liver, and more slowly by the kidney. Radiolabel was detected in excreted feces at 8 hr, but the preferred route of excretion was renal as judged by significant counts in excreted urine. Absorption of labeled peptide by skin and bone was sustained. Strong and sustained absorption also occurred in the vas deferens, seminal vesicle and hypothalamus. Given the rapid clearance of antagonist, multiple or sustained dosing schemes might be necessary to achieve the desired pharmacological effect. The high counts in liver at early time points after i.v. injection suggest that other routes of administration that do not bypass the hepatic first-pass effect would result in very low blood levels of drug.

Glycosylation patterns of membrane proteins of the jellyfish Cyanea capillata.

Integral and membrane-associated proteins extracted from neuron-enriched perirhopalial tissue of the jellyfish Cyanea capillata were probed with a panel of lectins that recognize sugar epitopes of varying complexity. Of the 13 lectins tested, only concanavalin A, jacalin lectin and tomato lectin stained distinct bands on Western blots, indicating the presence of repeating alpha-1,6-mannoses, terminal Gal-alpha-1,6-GalNAc and repeating beta-1,4-linked GlcNAc, respectively. In whole-mounted perirhopalial tissue, jacalin lectin stained several cell types, including neurons, muscle, cilia and mucus strands. Tomato lectin stained secretory cells intensely, and neurons in a punctate fashion. Concanavalin A stained cytoplasmic epitopes in both ecto- and endodermal cells, and ectodermal secretory cells and the mucus strands emanating from them. With the exception of tomato lectin's sugar epitope, the other sugar epitopes identified in this study are "non-complex". This study suggests that while glycosylation of integral and membrane-associated proteins occurs in Cyanea, the sugars post-translationally linked to these proteins tend to be simple.

A Reevaluation of the Structure in the Pore Region of Voltage-Activated Cation Channels.

Members of the Voltage-Sensitive Cation Channel (VSCC) superfamily from highly selective voltage-gated pores in excitable membranes. These pores are thought to be formed from the extrucellulur loops thut interconnect transmembrane segments 5 und 6 in each of the four domains that constitute the channel. Each of these loops is currently modeled as consisting of two short segments, SS1 and SS2, that are linked by a hairpin turn to form an antiparallel structure. In this study, the hypothesized ß-turn in the S5-S6 loop of each of 80 domains from the VSCC superfumily (26 different channel isoforms) were identified and located on the basis of their significant local maxima for {beta}-turn propensity (Pbend). Significant ß-turns were identified in all 80 sequences, but they are shtifted, and lie in the region currently defined as the SS2 ß-strand. This location of the ß-turn is incompatible with an antiparallel ß-sheet structure of the pore. The region identified here as,forming the turn corresponds to the ion selective determinants in the pore, implying that the turn imparts some of the ionic selectivity of each channel.

Anti-Ig-stimulated B lymphoblasts can be restimulated via their surface Ig.

Engaging AgR (surface Ig) on B lymphocytes leads to rapid inositol phosphate turnover and elevation of intracellular [Ca2+]. Continuous receptor occupancy (greater than 18 h) by anti-Ig leads to transit of most B lymphocytes from G0 to G1 stage of the cell cycle (blast transformation); a fraction of cells continue into S phase but do not proliferate continuously in the absence of growth factors. Prolonged exposure to ligand can induce receptor desensitization of some receptors. We therefore investigated whether such desensitization occurs in B cells activated by insolubilized anti-Ig. Resting B cells and anti-Ig-activated blasts were examined for their potential to elevate [Ca2+]i, maintain viability, and synthesize DNA in response to reexposure to anti-Ig. B cells and anti-Ig blasts had similar basal [Ca2+]i levels. Anti-Ig blasts retained the capacity to increase [Ca2+]i in response to anti-Ig; the magnitude of the increase was equal to or greater than that observed with resting B cells and occurred in more than 90% of cells. Isolated anti-Ig blasts subcultured in the presence of T cell-derived growth factors for 3 to 5 days responded to restimulation by anti-Ig with an increase in [Ca2+]i similar to that observed in freshly isolated blasts. The B cell and B lymphoblast ion channels were found to be permeable to Ca2+ but impermeable to Mn2+. Finally, blasts restimulated by anti-Ig retained viability and incorporated low levels of [3H]thymidine for 24 h. These results suggest that AgR on activated B lymphocytes can remain functionally coupled to intracellular signaling pathways and can participate in immune responses subsequent to initial activation.

Detection and characterization of a B cell stimulatory factor (BSF-TC) derived from a bone marrow stromal cell line.

Stromal cell lines derived from murine bone marrow support the growth of immature pre-B cells and produce cytokines that affect the growth and differentiation of other hematopoietic precursors. Conditioned medium (CM) from one such line (TC-1) stimulated marked proliferation of B cells previously activated by anti-Ig (anti-Ig blasts). Proliferation of anti-Ig blasts was not induced by purified cytokines known to be produced by TC-1 (CSF-1, GM-CSF, or G-CSF) or by IL-1, IL-2, IL-3, IL-4, IL-5, or IL-6. Furthermore, IL-2, IL-4, and IL-5, alone or in combination, failed to support proliferation or differentiation of anti-Ig blasts. TC-1 CM enhanced proliferation of B cells that were co-cultured with LPS, anti-Ig, or dextran sulfate; co-stimulation with anti-Ig was unaffected by the presence of monoclonal anti-IL-4. Proliferation of low, but not high, density B cells isolated from spleen was directly stimulated by TC-1 CM. These results suggest that bone marrow stromal cells produce a novel B cell stimulatory factor (BSF-TC) that induces proliferation of activated B cells.

Clinical trial of brinase and anticoagulants as a method of treatment for advanced limb ischemia.

A randomized, single-blind trial of repeated intravenous infusion of Brinase was carried out in 70 petients with severe chronic limb ischemia, who were candidates for lumbar sympathectomy or amputation. The enzyme caused s significant increase in calf and ankle pressure index. At six months follow-up, the clinical results were statistically significant in favour of Brinase when all patients were considered, but not if 10 patients with Buerger's disease were omitted from the analysis. Patients treated with a combination of Brinase and coumarins had a better clinical outcome than patients receiving either treatment on its own.

Biochemical changes noted during intermittent administration of streptokinase.

An intermittent dosage scheme of streptokinase (standard initial dose 600,000 units SK infused over 30 min and repeated injections of 250,000 units SK at 24 hr intervals) was applied during 4 days in 9 patients with chronic obliterative arterial disease and in 8 patients with venous occlusion. Each dose of streptokinase produced an immediate fall in plasminogen to 17% (SEM 5.1) of the initial value, the level then rose to 50% (SEM 5.4) within 24 hr. Lowered levels of antiplasmin and fibrinogen (both less than 40% of the initial values) were maintained. This safe level of fibrinogen was maintained despite brief but high peaks of plasmin activity after each injection of SK. A parallel increase of the thrombin time and the fibrin (ogen) degradation products was obtained following each infusion. No bleeding was observed. The relative therapeutic effect of intermittent infusions of streptokinase has still to be compared with the continuous administration of streptokinase in controlled clinical trials.