Identification of key amino acids in the gastrin-releasing peptide receptor (GRPR) responsible for high affinity binding of gastrin-releasing peptide (GRP)

The bombesin (Bn) receptor family includes the gastrin-releasing peptide (GRPR) and neuromedin B (NMBR) receptors, Bn receptor subtype 3 (BRS-3) and Bn receptor subtype 4 (BB(4)). They share 50% homology, yet their affinities for gastrin-releasing peptide (GRP) differ. The determinants of GRP high affinity for GRPR and BB(4), and low affinity for BRS-3 are largely unknown. To address this question we made an analysis of structural homologies in Bn receptor members correlated with their affinities for GRP to develop criteria to identify amino acids important for GRP selectivity. Fourteen differences were identified and each was mutated singly in GRPR to that found in hBRS-3. Eleven mutants had a loss of GRP affinity. Furthermore, three of four amino acids in the GRPR selected used a similar approach and previously reported to be important for high affinity Bn binding, were important for GRP affinity. Some GRPR mutants containing combinations of these mutations had greater decreases in GRP affinity than any single mutation. Particularly important for GRP selectivity were K101, Q121, A198, P199, S293, R288, T297 in GRPR. These results were confirmed by making the reverse mutations in BRS-3 to make GRP gain of affinity mutants. Modeling studies demonstrated a number of the important amino acids had side-chains oriented inward and within 6A of the binding pocket. These results demonstrated this approach could identify amino acids needed for GRP affinity and complemented results from chimera/mutagenesis studies by identifying which differences in the extracellular domains of Bn receptors were important for GRP affinity.     

Evidence that [Phe1 ψ(CH2-NH)Gly2]nociceptin-(1-13)-NH2, a peripheral ORL-1 receptor antagonist,acts as an agonist in the rat spinal cord

[Phe¹ ψ(CH₂-NH)Gly²]nociceptin-(1-13)-NH₂, a pseudopeptide analogue of nociceptin is an antagonist in peripheral assays. Here, using in vivo electrophysiological recordings of dorsal horn neurones, [Phe¹ ψ(CH₂-NH)Gly²]nociceptin-(1-13)-NH₂ appears to have agonist activity after spinal administration. The noxious evoked activity of the neurones was inhibited by [Phe¹ ψ(CH₂-NH)Gly²]nociceptin-(1-13)-NH₂, which was as potent as nociceptin itself.     

Influence of Fimasartan (a Novel AT1 Receptor Blocker) on Catecholamine Release in the Adrenal Medulla of Spontaneously Hypertensive Rats

The aim of this study was to determine whether fimasartan, a newly developed AT₁ receptor blocker, can affect the CA release in the isolated perfused model of the adrenal medulla of spontaneously hypertensive rats (SHRs). Fimasartan (5~50 µM) perfused into an adrenal vein for 90 min produced dose- and time-dependently inhibited the CA secretory responses evoked by ACh (5.32 mM), high K⁺ (56 mM, a direct membrane depolarizer), DMPP (100 µM) and McN-A-343 (100 µM). Fimasartan failed to affect basal CA output. Furthermore, in adrenal glands loaded with fimasartan (15 µM), the CA secretory responses evoked by Bay-K-8644 (10 µM, an activator of L-type Ca²⁺ channels), cyclopiazonic acid (10 µM, an inhibitor of cytoplasmic Ca²⁺-ATPase), and veratridine (100 µM, an activator of Na⁺ channels) as well as by angiotensin II (Ang II, 100 nM), were markedly inhibited. In simultaneous presence of fimasartan (15 µM) and L-NAME (30 µM, an inhibitor of NO synthase), the CA secretory responses evoked by ACh, high K⁺, DMPP, Ang II, Bay-K-8644, and veratridine was not affected in comparison of data obtained from treatment with fimasartan (15 µM) alone. Also there was no difference in NO release between before and after treatment with fimasartan (15 µM). Collectively, these experimental results suggest that fimasartan inhibits the CA secretion evoked by Ang II, and cholinergic stimulation (both nicotininc and muscarinic receptors) as well as by membrane depolarization from the rat adrenal medulla. It seems that this inhibitory effect of fimasartan may be mediated by blocking the influx of both Na⁺ and Ca²⁺ through their ion channels into the rat adrenomedullary chromaffin cells as well as by inhibiting the Ca²⁺ release from the cytoplasmic calcium store, which is relevant to AT₁ receptor blockade without NO release.     

The Structural and Functional Role of the B‐chain C‐terminal Arginine in the Relaxin‐3 Peptide Antagonist,R3(BΔ23‐27)R/I5

Relaxin‐3, a member of the insulin superfamily, is involved in regulating stress and feeding behavior. It is highly expressed in the brain and is the endogenous ligand for the receptor RXFP3. As relaxin‐3 also interacts with the relaxin receptor RXFP1, selective agonists and antagonists are crucial for studying the physiological function(s) of the relaxin‐3/RXFP3 pair. The analog R3(BΔ23‐27)R/I5, in which a C‐terminally truncated human relaxin‐3 (H3) B‐chain is combined with the INSL5 A‐chain, is a potent selective RXFP3 antagonist and has an Arg residue remaining on the B‐chain C‐terminus as a consequence of the recombinant protein production process. To investigate the role of this residue in the RXFP3 receptor binding and activation, the analogs R3(BΔ23‐27)R/I5 and R3(BΔ23‐27)R containing the B‐chain C‐terminal Arg as well as R3(BΔ23‐27)/I5 and R3(BΔ23‐27), both lacking the Arg, were chemically assembled and their secondary structure and receptor activity assessed. The peptides generally had a similar conformation but those with the extra Arg residue displayed a significantly increased affinity for the RXFP3. Interestingly, in contrast to R3(BΔ23‐27)R and R3(BΔ23‐27)R/I5, the peptide R3(BΔ23‐27) is a weak agonist. This suggests that the C‐terminal Arg, although increasing the affinity, alters the manner in which the peptide binds to the receptor and thereby prevents activation, giving R3(BΔ23‐27)R/I5 its potent antagonistic activity.     

The effects of a 5-HT1 receptor ligand isapirone (TVX Q 7821) on 5-HT synthesis and the behavioural effects of 5-HT agonists in mice and rats

The effects of 2-(4-(4-(2-pyrimidinyl)-1-piperazinyl)-butyl)-1,2-benzoisothiazol-3(2H)one-1,1-dioxide hydrochloride (isapirone, TVX Q 7821), a putative 5-HT₁ receptor antagonist, has been studied on various models of 5-HT receptor sub-type function. In mice TVX Q 7821 produced a dose-dependent inhibition of the hypothermia induced by 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) with an ED₅₀ of 5.3 mg/kg suggesting that TVX Q 7821 was an antagonist of the presynaptic (possibly somato-dendritic) 5-HT_1A receptor. TVX Q 7821 did not alter the locomotor response to the suggested 5-HT_1B agonist RU 24969. The rate of mouse brain 5-HT synthesis was accelerated by TVX Q 7821 (10 mg/kg). 5-HT₂ receptor-mediated head twitch behaviour induced by precursor loading with 5-HTP was unaffected by TVX Q 7821 (10 mg/kg) pretreatment 75 min earlier, but the head-twitch induced by the agonist 5-methoxy-N,N-dimethyltryptamine was enhanced by prior treatment with TVX Q 7821.In rats the hypothermia induced by 8-OH-DPAT was partially antagonised by TVX Q 7821 while the behavioural serotonin syndrome induced by 8-OH-DPAT (a possible post-synaptic 5-HT_1B-mediated effect) was unaffected by TVX Q 7821 as was the locomotion induced by RU 24969.The data suggest that TVX Q 7821 is a good presynaptic 5-HT_1A antagonist in mice, as indicated by the 8-OH-DPAT-induced hypothermia and 5-HT synthesis rate studies. It did not antagonise 5-HT_1B-mediated behaviour in mice or rats and appeared to have an antagonist action at pre- but not post-synaptic 5-HT_1A receptors in rats. Offprint requests to: G.M. Goodwin     

The colloidal thyroxine (T4) ring as a novel biomarker of perchlorate exposure in the African clawed frog Xenopus laevis.

The purpose of this study was to determine if changes in colloidal thyroxine (T(4)) immunoreactivity can be used as a biomarker of perchlorate exposure in amphibian thyroid tissue. Larval African clawed frogs (Xenopus laevis) were exposed to 0, 1, 8, 93, and 1131 microg perchlorate/l for 38 and 69 days to cover the normal period of larval development and metamorphosis. The results of this study confirmed the presence of an immunoreactive colloidal T(4) ring in thyroid follicles of X. laevis and demonstrated that the intensity of this ring is reduced in a concentration-dependent manner by perchlorate exposure. The smallest effective concentration of perchlorate capable of significantly reducing colloidal T(4) ring intensity was 8 microg perchlorate/l. The intensity of the immunoreactive colloidal T(4) ring is a more sensitive biomarker of perchlorate exposure than changes in hind limb length, forelimb emergence, tail resorption, thyrocyte hypertrophy, or colloid depletion. We conclude that the colloidal T(4) ring can be used as a sensitive biomarker of perchlorate-induced thyroid disruption in amphibians.     

Use of the A(2A) adenosine receptor as a physiological immunosuppressor and to engineer inflammation in vivo

Inflammation must be inhibited in order to treat, e.g., sepsis or autoimmune diseases or must be selectively enhanced to improve, for example, immunotherapies of tumors or the development of vaccines. Predictable enhancement of inflammation depends upon the knowledge of the "natural" pathways by which it is down-regulated in vivo. Extracellular adenosine and A(2A) adenosine (purinergic) receptors were identified recently as anti-inflammatory signals and as sensors of excessive inflammatory tissue damage, respectively (Ohta A and Sitkovsky M, Nature 2001;414:916-20). These molecules may function as an important part of a physiological "metabolic switch" mechanism, whereby the inflammatory stimuli-produced local tissue damage and hypoxia cause adenosine accumulation and signaling through cyclic AMP-elevating A(2A) adenosine receptors in a delayed negative feedback manner. Patterns of A(2A) receptor expression are activation- and differentiation-dependent, thereby allowing for the "acquisition" of an immunosuppressive "OFF button" and creation of a time-window for immunomodulation. Identification of A(2A) adenosine receptors as "natural" brakes of inflammation provided a useful framework for understanding how tissues regulate inflammation and how to enhance or decrease (engineer) inflammation by targeting this endogenous anti-inflammatory pathway. These findings point to the need of more detailed testing of anti-inflammatory agonists of A(2A) receptors and create a previously unrecognized strategy to enhance inflammation and targeted tissue damage by using antagonists of A(2A) receptors. It is important to further identify the contributions of different types of immune cells at different stages of the inflammatory processes in different tissues to enable the "tailored" treatments with drugs that modulate the signaling through A(2A) purinergic receptors.     

The effects of harman and norharman on the metabolism of benzo(a)pyrene in isolated perfused rat lung and in rat lung microsomes.

The effects of harman and norharman, nitrogen-containing pyrolysis products of amino acids present in cigarette smoke, on the metabolism of benzo(a)pyrene in rat lung microsomes in vitro and in isolated perfused rat lung were studied. In rat lung microsomes, both harman and norharman inhibited the metabolism of benzo(a)pyrene (BP) to dihydrodiols, phenols and quinones at concentrations over approximately 0.05 mM. The formation of BP-7, 8-dihydrodiol and BP-9, 10-dihydrodiol was inhibited more than that of BP-4, 5-dihydrodiol. No appreciable differences in inhibition were seen between microsomes from control or 3-methylcholanthrene-pretreated rats. In isolated perfused rat lung, 1 mM of harman in the perfusion fluid inhibited the formation of ethyl acetate-soluble metabolites of benzo(a)pyrene except BP-9, 10-dihydrodiol, and inhibited the total covalent binding of benzo(a)pyrene metabolites to lung tissue macromolecules. 0.03 mM of harman seemed to increase other metabolites than BP-7,8-dihydrodiol without changing the total covalent binding. These results suggest that at most concentrations both β-carboline derivatives, harman and norharman, inhibit benzo(a)pyrene metabolism and covalent binding both in lung microsomes in vitro and in isolated perfused rat lung.