Antagonist Affinity Estimates Research Articles

Muscarinic receptor M3 mediates human gallbladder contraction through voltage-gated Ca2+ channels and Rho kinase

Objective. Muscarinic receptors mediate contraction of the human gallbladder through unclear receptor subtypes. The aim of the present study was to characterize muscarinic acetylcholine receptors mediating contraction of the human gallbladder. Materials and methods. Contraction of human gallbladder muscle strips caused by agonists carbachol and muscarine was measured and the inhibition of carbachol-induced contraction by muscarinic receptor antagonists was evaluated. Reverse transcription polymerase chain reaction was performed to determine the existence of muscarinic receptor subtypes. Results. Carbachol and muscarine caused concentration-dependent contraction of gallbladder strips. Four receptor antagonists, including atropine, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), methoctramine, and pirenzepine, inhibited the carbachol-induced contraction. The relative inhibitory potency of these receptor antagonists was atropine > 4-DAMP > methoctramine > pirenzepine. The antagonist affinity estimates (pA2 values) correlated with the known affinities at M3, M4, and M5 muscarinic receptors. In addition, the M4-selective antagonist muscarinic toxin 3 did not inhibit and the M5-selective positive allosteric modulator VU0238429 did not potentiate carbachol-induced gallbladder contraction. This suggests that M3 muscarinic receptors mediate the muscarinic response predominantly. The contractile response of carbachol was attenuated by the voltage-gated Ca2+ channel inhibitor nifedipine and Rho-kinase inhibitor H-1152, but not affected by protein kinase C inhibitor chelerythrine. This implies the involvement of voltage-gated Ca2+ channel and Rho kinase but not protein kinase C. Conclusions. These results suggest a major role of M3 muscarinic receptors mediating the human gallbladder contraction through voltage-gated Ca2+ channels and Rho kinase. M3-selective muscarinic receptor antagonists could be of therapeutic importance in the treatment of biliary motility disorders.

Analysis of the pharmacokinetic/pharmacodynamic relationship of a small molecule CXCR3 antagonist, NBI‐74330, using a murine CXCR3 internalization assay

Pharmacokinetic/pharmacodynamic (PK/PD) models are necessary to relate the degree of drug exposure in vivo to target blockade and pharmacological efficacy. This manuscript describes a murine agonist-induced CXCR3 receptor internalization assay and demonstrates its utility for PK/PD analyses. Activated murine DO11.10 cells were incubated with agonist in the presence or absence of a CXCR3 antagonist and changes in surface CXCR3 expression were detected by flow cytometry. For PK/PD analysis, mice were dosed with a small molecule CXCR3 antagonist, NBI-74330, (100 mg kg(-1)) orally or subcutaneously and plasma samples taken at specified timepoints for the CXCR3 internalization assay. Surface CXCR3 expression was specifically decreased in response to CXCL9, CXCL10 and CXCL11. CXCL11 was the most potent CXCR3 agonist in buffer (pA50=9.23+/-0.26) and the pA50 for CXCL11 was unaltered in murine plasma (pA50=9.17+/-0.15). The affinity of a small molecule CXCR3 antagonist, NBI-74330, was obtained in the absence or presence of plasma (buffer pA2 value: 7.84+/-0.14; plasma pKB) value 6.36+/-0.01). Administration of NBI-74330 to mice resulted in the formation of an N-oxide metabolite, also an antagonist of CXCR3. Both antagonists were detectable up to 7 h post oral dose and 24 h post subcutaneous dose. Measurement of CXCR3 internalization demonstrated significant antagonism of this response ex vivo, 24 h following subcutaneous administration of NBI-74330. The CXCR3 receptor internalization assay provides a robust method for determining agonist potency orders, antagonist affinity estimates and PK/PD analyses, which discriminate between dosing regimens for the CXCR3 antagonist NBI-74330.

Multiple GPCR conformations and signalling pathways: implications for antagonist affinity estimates

Antagonist affinity measurements have traditionally been considered important in characterizing the cell-surface receptors present in a particular cell or tissue. A central assumption has been that antagonist affinity is constant for a given receptor–antagonist interaction, regardless of the agonist used to stimulate that receptor or the downstream response that is measured. As a consequence, changes in antagonist affinity values have been taken as initial evidence for the presence of novel receptor subtypes. Emerging evidence suggests, however, that receptors can possess multiple binding sites and the same receptor can show different antagonist affinity measurements under distinct experimental conditions. Here, we discuss several mechanisms by which antagonists have different affinities for the same receptor as a consequence of allosterism, coupling to different G proteins, multiple (but non-interacting) receptor sites, and signal-pathway-dependent pharmacology (where the pharmacology observed varies depending on the signalling pathway measured).

α 1L-Adrenoceptors mediate contractions of the isolated mouse prostate

The subtype of α 1-adrenoceptor mediating noradrenaline-induced contractile responses in isolated mouse prostate glands was investigated. Adrenoceptor agonists were able to produce concentration-dependent contractions with the following rank order of potency: adrenaline ≥ noradrenaline ≥ clonidine = phenylephrine > dopamine ≥ isoprenaline. Concentration–response curves to noradrenaline of the prostatic smooth muscle were antagonised by prazosin, N-[2-(2-cyclopropylmethoxyphenoxy)ethyl]-5-chloro-α, α-dimethyl-1 H-indole-3-ethanamine (RS-17053), 2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane (WB 4101), tamsulosin and yohimbine with mean antagonist affinity estimates (p A 2 or apparent p K B) of 8.12 ± 0.10, 6.56 ± 0.11, 8.38 ± 0.06, 10.14 ± 0.19 and 7.38 ± 1.36 respectively. Propranolol (1 μM) had no antagonist activity ( P = 0.994, n = 6). Yohimbine (0.01, 0.1, 1 μM) had no antagonist activity in the presence of prazosin (0.1 μM) ( P ≥ 0.059). The results obtained indicate that α 1-adrenoceptors mediate the contractile response in isolated preparations of the mouse prostate. Furthermore, the particular subtype of α 1-adrenoceptor mediating the response to exogenously administered noradrenaline corresponds to the α 1L-subtype, the same subtype as that which has been shown to mediate noradrenaline-induced contractile activity in the human prostate.

Pharmacological characterization of cholecystokinin receptors mediating contraction of human gallbladder and ascending colon

Cholecystokinin (CCK) produces contractions of gallbladder and colon in a number of different species. Although the effects of CCK on the human gallbladder are relatively well documented, the CCK receptors in the human colon have not been clearly characterised. Therefore, in this study, the CCK receptors in the human gallbladder and colon were compared using pharmacological techniques. Contraction of specimens of the human tissue was measured using in vitro organ bath bioassay. The effect of selective concentrations of CCK 1 and CCK 2 receptor antagonists (L-364,718 and JB93182, respectively) was determined on agonist concentration–effect ( E/[ A]) curves obtained by cumulative dosing with sulphated CCK. The CCK 1 antagonist L-364,718 produced a rightward shift of the CCK-8S [ E/[ A] curve in the human gallbladder (p A 2=9.15±0.26) and ascending colon (p A 2=9.20±0.33). In both tissues, the CCK 2 receptor antagonist, JB93182, had no effect on the CCK E/[ A] curves. In addition, in the colon, pentagastrin responses were inhibited by L-364,718 but unaffected by JB93182. These data indicate that the CCK-induced contraction of the human colon and gallbladder smooth muscle is mediated solely through the CCK 1 receptor subtype, and the antagonist affinity estimates are consistent with those previously obtained in experiments on animal tissue.

Characterization of an atypical muscarinic cholinoceptor mediating contraction of the guinea‐pig isolated uterus

In many smooth muscle tissues a minor M3-muscarinic acetylcholine (mACh) receptor population mediates contraction, despite the presence of a larger M2-mACh receptor population. However, this is not the case for guinea-pig uterus where radioligand binding and functional studies exclude a dominant role for M3-mACh receptors. Using tissue from animals pre-treated with diethylstilboestrol, estimates of antagonist affinity were made before and after selective alkylation procedures, together with estimates of agonist affinity to characterise the mACh receptor population mediating carbachol-induced contraction of guinea-pig isolated uterus. Antagonist affinity estimates made at 'protected' receptors were not significantly different from those made in untreated tissues. However all estimations were significantly different from those reported in guinea-pig ileum and atria. The rank order of affinities were atropine>zamifenacin=tripitramine> methoctramine. Carbachol-induced contractions were insensitive to the M4-selective muscarinic toxin MTx-3, or PD102807 (0.1 microM) ruling out a role for M4-mACh receptors. The agonist affinity value for L-660,863, a putative 'M2-selective' agonist of 5.44+/-0.30 (n=6) was significantly different from that reported in guinea-pig atria. In contrast, the pKA value for carbachol (4.22+/-0.17 n = 8) agrees with that reported for guinea-pig ileum. Carbachol-induced contractions were insensitive to pertussis toxin although carbachol-induced inhibition of forskolin-stimulated cyclic AMP production was attenuated, ruling out the involvement of Gi-proteins in contraction. Radioligand binding studies revealed a KD for N-[3H]-methylscopolamine of 0.12+/-0.05 nM and a Bmax of 147+/-18 fmol mg protein(-1). Antagonist affinity estimates made using competition binding studies supported previous data suggesting the presence of a homogenous population of M2-mACh receptors. These data suggest a small population of mACh receptors with an atypical operational profile which can not be distinguished using radioligand binding studies may mediate carbachol-induced contraction of guinea-pig isolated uterus.

Pharmacological classification of adenosine receptors in the sinoatrial and atrioventricular nodes of the guinea-pig.

1. The effects of seven agonist and three antagonist adenosine receptor ligands were compared on the guinea-pig sinoatrial (SA) node (isolated right atrium) and atrioventricular (AV) node (perfused whole heart). Single agonist concentration-effect curves were obtained to 5'-N-ethylcarboxamidoadenosine (NECA), R(-)-N6-(2-phenylisopropyl)adenosine (R-PIA), N6-cyclohexyladenosine (CHA), 2-chloroadenosine (CADO),),S(+)-N6-(2-phenylisopropyl)adenosine (L-PIA), 2-phenylaminoadenosine (CV 1808) and N6-aminoadenosine (MeAdo). Adenosine and/or NECA curves were obtained in the absence and presence of the antagonists 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), 9-chloro-2 (2-furanyl)-5,6-dihydro-1,2,4-triazolo[1,5-c]quinazolin-5-imine (CGS15943) and N6-(endonorbornan-2-yl)-9-methyladenine (N-0861). 2. A formal comparison of the agonist and antagonist potency data was made by fitting the data to a straight line using a least squares procedure based on principal components analysis to account for the variance on both axes. The antagonist affinity estimates made on the two assays did not deviate significantly from the line of identity. 3. The agonist p[A]50 data obtained on the two assays did not deviate from the line of identity, indicating that there were no significant differences in potencies between the two assays. The p[A]50 ratio of R-PIA and S-PIA was 1.24+/-0.09 in the SA node and 1.36+/-0.11 in the AV node, indicating no difference in the stereoselectivity of the PIA isomers between the two tissues. 4. The agonist potency and antagonist affinity data obtained are consistent with previous findings showing that the AV and SA node data are pharmacologically indistinguishable and belong to the adenosine A1-receptor class. No evidence for the reported A3-receptor was found.

Characterization of alpha1-adrenoceptor subtypes mediating contractions to phenylephrine in rat thoracic aorta, mesenteric artery and pulmonary artery.

1. The subtype of alpha1-adrenoceptor mediating contractions to phenylephrine of the rat thoracic aorta, mesenteric artery and pulmonary artery were investigated by use of antagonists which show selectivity between the cloned alpha1-adrenoceptor subtypes in binding studies. 2. Cumulative concentration-contraction curves for phenylephrine were competitively antagonized in the rat thoracic aorta by prazosin (pA2 9.9), WB4101 (pA2 9.6), 5-methylurapidil (pA2 8.1), benoxathian (pA2 9.2) and indoramin (pA2 7.4). These compounds were also competitive antagonists in the mesenteric and pulmonary arteries (except for 5-methylurapidil in the pulmonary artery), (prazosin pA2 9.9 and 9.7; WB4101 pA2 9.8 and 9.6; 5-methylurapidil pA2 7.9 and pK(B) estimate 8.0; benoxathian pA2 8.8 and 9.3; indoramin pA2 7.2 and 7.5, respectively). 3. RS 17053 was not a competitive antagonist in any blood vessel as Schild plot slopes were greater than unity. The pK(B) estimates for RS 17053 were 7.1 in aorta, 7.0 in the mesenteric artery and 7.7 in the pulmonary artery. 4. The alpha1D-subtype selective antagonist BMY 7378 appeared to be non-competitive with shallow Schild plot slopes. The data were better fitted with two lines in all tissues, with Schild plot slopes that were no longer different from unity, except in the pulmonary artery. The higher affinity site for BMY 7378 in the aorta had a pA2 of 9.0, while it was 8.8 and 8.9 in the mesenteric and pulmonary arteries, respectively. 5. MDL73005EF acted in a non-competitive manner in all three blood vessels, with shallow Schild plot slopes. The pK(B) estimates for MDL73005EF were 8.4 in aorta, 7.5 in the mesenteric artery and 8.0 in the pulmonary artery. 6. In all three blood vessels the functionally determined antagonist affinity estimates correlated best with published pKi values for their displacement of [3H]-prazosin binding on membranes expressing cloned alpha1d-adrenoceptors compared with alpha1a- or alpha1b-adrenoceptors. The antagonist affinity estimates in the aorta, mesenteric and pulmonary arteries correlated highly with their previously published pA2 values in rat aorta (alpha1D) and less well with those for alpha1A- and alpha1B-adrenoceptors mediating contraction of the rat epididymal vas deferens and rat spleen, respectively. 7. The results of this study suggest that the contraction to phenylephrine of the rat thoracic aorta, mesenteric artery and pulmonary artery are mediated in part via the alpha1D-subtype of adrenoceptor. The data for both BMY 7378 and MDL73005EF in all three blood vessels are consistent with receptor heterogeneity. However, the identity of the second site is unclear.

α2 D-Adrenoceptors modulate renal noradrenaline release in normotensive and spontaneously hypertensive rats

The aim of the present study was to investigate α-adrenoceptor modulation of neurotransmission in kidneys of spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rat (WKY) and to classify the prejunctional α 2-adrenoceptor subtype involved. Kidneys of SHR (12–14 weeks) and age-matched WKY were isolated and the sympathetic nerves were stimulated electrically. The renal nerve stimulation-induced outflow of endogenous noradrenaline was measured by high-pressure liquid chromatography with electrochemical detection (HPLC-ECD). In the presence of uptake blockade by cocaine and corticosterone, the renal nerve stimulation-induced outflow of endogenous noradrenaline was significantly greater in SHR than in WKY at 1 Hz (688 ± 42 pg/g vs. 525 ± 46 pg/g) and 4 Hz (5265 ± 495 pg/g vs. 3544 ± 245 pg/g). At a stimulation frequency of 1 Hz the α 2-adrenoceptor agonist UK 14304 (0.01-1 μM) potently inhibited the renal nerve stimulation-induced outflow of noradrenaline with a pEC 50 of 7.49 (7.32–7.68) and a maximum of 91% in SHR and with a pEC 50 of 7.46 (7.30–7.74) and a maximum of 92% in WKY. α-Adrenoceptor antagonist affinity estimates (p K B values) against UK 14304 at the prejunctional α 2-autoreceptors were determined. The rank order of affinities (phentolamine > rauwolscine > 2-(2,6-dimethoxyphenoxyethyl) aminomethyl-1,4-benzodioxane HCl (WB 4101) > prazosin) was identical in SHR and WKY, and comparison with data from radioligand binding, molecular cloning and functional studies in other tissues and cell lines indicates that prejunctional α 2-autoreceptors of SHR and WKY kidneys are of the α 2 D subtype.

Pharmacology characterization of guanine nucleotide exchange reactions in membranes from Cho cells stably transfected with human muscarinic receptors m1–m4

We have studied muscarinic agonist stimulated [ 35S]GTPγS binding and [γ 32P]GTP hydrolysis (GTPase) in membranes from CHO cells stably transfected with human muscarinic m1–m4 receptors, ‘Full’ agonists were at least 10-fold more potent at m2 & m4 receptors at m1 & m3. This pattern was less marked with ‘partial” agonists, which has a greater maximal effect at m2 & m4 than at m1 & m3. McN-A343 uniquely was more potent and efficacious at m4 than at m2 receptors. Antagonist affinity constanats were estimated by fitting the data from inhibition curves directly to the Schild model. Antagonist affinity estimates were very similar to those measured earlier in binding studies using animal tissues, and confirmed a small degree of m4 selectively for tropicamide and secoverine. The receptor subtypes activated more than one G-protein subtype; m2 & m4 receptors activated only pertussis (PTX) sensitive G-protein, while m1 & m3 couples to both PTX sensitive and insensitive G-protein. Acetylcholine (ACh) was more potent in stimulating guanine nucleotide exchange in PTX-treated m1 cells than in control.