Mutation of aspartate residues in the third extracellular loop of the rat B2 bradykinin receptor decreases affinity for bradykinin.

Two aspartates in the third extracellular loop of the rat B2 bradykinin (BK) receptor have been implicated as important residues for agonist binding. Asp268 and Asp286 were mutated to alanine residues and changes in agonist and antagonist binding affinity were examined. The IC50 value for BK as a competitor of [3H] NPC 17731 binding to the rat wild type receptor was 1.1 nM, while the Ala268 and Ala286 receptor mutants exhibited IC50 values of 19 nM and 28 nM, respectively. The Ala268Ala268 receptor mutant exhibited an IC50 for BK of 500 nM. These mutations had little effect on binding affinity when NPC 17761, a BK antagonist, was used to compete [3H] NPC 17731 binding. Electrophysiological examination of Xenopus oocytes expressing wild type or Ala268 Ala286 receptors confirmed the importance of the Asp268 and Asp286 residues for BK recognition. BK activated the mutant receptor with comparable efficacy relative to the wild type receptor, but a 1750-fold reduction in potency was observed.

Pharmacological profile of NPC 17742 [2R,4R,5S-(2-amino-4,5-(1, 2-cyclohexyl)-7-phosphonoheptanoic acid)], a potent, selective and competitive N-methyl-D-aspartate receptor antagonist.

2R,4R,5S-(2-amino-4,5-(1,2-cyclohexyl)-7-phosphonoheptanoic acid) (NPC 17742), the most potent isomer of the mixture 2-amino-4,5-(1,2-cyclohexyl)-7-phosphonoheptanoic acid (NPC 12626), was evaluated for activity in tests associated with receptors for excitatory amino acids. In receptor binding assays, NPC 17742 was selective for the N-methyl-D-aspartate (NMDA) receptor with a potency comparable to that of D(-, -3-(2-carboxypiperazine-4-yl)propyl-1-phosphonic acid. Like (+/-)cis-4-phosphono-methyl-2-piperidine carboxylic acid (CGS 19755) and (+/-)(E)-2-amino-4-methyl-5-phosphono-3-penteneoic acid (CGP 37849), NPC 17742 competitively inhibited NMDA-induced enhancement of 1-[(2-thienyl)cyclohexyl]piperidine binding to the NMDA receptor ionophore and partially inhibited [3H]glycine binding to strychnine-insensitive sites. In contrast, NPC 17742 and CGP 37849 inhibited Mg(++)-stimulated 1-[(2-thienyl)cyclohexyl]piperidine binding in a noncompetitive fashion. In voltage-clamped Xenopus oocytes expressing excitatory amino acid receptors, NPC 17742 (pKB = 6.91) was equipotent with CGP 37849 (pKB = 7.17) in inhibiting NMDA-induced inward currents. Likewise, NPC 17742 (ED50 = 2.68 mg/kg) was equipotent with CGP 37849 and CGS 19755 in blocking NMDA-induced convulsions, but was less potent than these two compounds in the maximal electroshock test. Unlike CGP 37849 or CGS 19755, NPC 17742 potently antagonized seizures induced by pentylenetetrazol. In a model of global ischemia, low doses of NPC 17742 given either before or after ischemic result were effective in blocking damage to hippocampal CA1 neurons. The pharmacologic responses to NPC 17742 occurred at doses 30- to 300-fold lower than the acute lethal dose.(ABSTRACT TRUNCATED AT 250 WORDS)

Opposite effects of spermine and arcaine on responses of N-methyl-D-aspartate receptors expressed in Xenopus oocytes.

The ability of polyamines to modulate N-methyl-D-aspartate (NMDA) receptor function was investigated in Xenopus oocytes injected with rat brain mRNA. Whereas spermine and spermidine augmented NMDA/glycine-induced inwards currents, arcaine (1,4-diguanidinobutane) and 1,10-diaminodecane inhibited the response. The potency of arcaine to inhibit NMDA/glycine-induced currents was unaffected by spermine; similarly, arcaine did not influence the potency of spermine, but did reduce the maximal response to spermine. These findings demonstrate that polyamines exert both positive and negative modulatory control of the NMDA receptor expressed in Xenopus oocytes, and suggest that spermine and arcaine act at distinct sites in the NMDA receptor complex.

The bovine endothelin receptor has an apparent molecular weight of 43,000.

In crosslinking experiments, [125I]endothelin-1 was treated with N-hydroxysuccinimidyl-4-azidobenzoate, purified by HPLC, allowed to bind to bovine aortic membranes and then photoactivated. Autoradiography of sodium dodecyl sulfate polyacrylamide gel electrophoretograms of the products of this reaction showed that a component of apparent Mr = 42,000 was specifically labelled by endothelin-1 under reducing conditions. Under nonreducing conditions, a small amount of 125I-labelled endothelin-1 specifically labelled a component of apparent Mr = 45,900 in the absence of crosslinking agent. Non-radiolabelled endothelin analogues with a wide range of binding affinities inhibited specific labelling of the Mr = 42,000 and 45,900 components in parallel over the concentration ranges which inhibited binding of radiolabelled endothelin. Specific labelling of these components was also observed in parallel in membranes from bovine heart and kidney. The components labelled in the presence and absence of crosslinker appear to be the same, and the small difference in apparent Mr in the labelled components is likely due to a difference in conformational constraints arising from the two labelling processes, with a true, corrected Mr of 43,400. Since the specific labelling of this component is related to physiologically relevant binding in several bovine tissues, we conclude that it is a component of the bovine endothelin receptor.