Thermodynamic analysis of ligands at cholecystokinin CCK2 receptors in rat cerebral cortex.

BACKGROUND AND PURPOSE: Several studies using radioligand binding assays, have shown that measurement of thermodynamic parameters can allow discrimination of agonists and antagonists (Weiland et al., 1979; Borea et al., 1996a). Here we investigate whether agonists and antagonists can be thermodynamically discriminated at CCK(2) receptors in rat cerebral cortex. EXPERIMENTAL APPROACH: The pK(L) of [(3)H]-JB93182 in rat cerebral cortex membranes was determined at 4, 12, 21 and 37 degrees C in 50 mM Tris-HCl buffer (buffer B pH 6.96; containing 0.089 mM bacitracin). pK(I) values of ligands of diverse chemical structure and with differing intrinsic activity (alpha), as defined by the lumen-perfused rat and mouse stomach bioassays, were determined in buffer B at 4, 12, 21 and 37 degrees C. KEY RESULTS: [(3)H]-JB93182 labelled a homogeneous population of receptors in rat cerebral cortex at 4, 12, 21 and 37 degrees C and the pK(L) and B(max) were not altered by incubation temperature. [(3)H]-JB93182 binding reached equilibrium after 10, 50, 90 and 220 min at 37, 21, 12 and 4 degrees C, respectively. pK(I) values for R-L-365,260, R-L-740,093, YM220, PD134,308 and JB95008 were higher at 4 degrees C than at 37 degrees C. There was no effect of temperature on pK(I) values for pentagastrin, CCK-8S, S-L-365,260, YM022, PD140,376 and JB93242. CONCLUSIONS AND IMPLICATIONS: CCK(2) receptor agonists and antagonists at rat CCK(2) receptors cannot be discriminated by thermodynamic analysis using [(3)H]-JB93182 as the radioligand.

Histamine H3-receptor agonists and imidazole-based H3-receptor antagonists can be thermodynamically discriminated.

BACKGROUND AND PURPOSE: Studies suggest that measurement of thermodynamic parameters can allow discrimination of agonists and antagonists. Here we investigate whether agonists and antagonists can be thermodynamically discriminated at histamine H(3) receptors. EXPERIMENTAL APPROACH: The pK(L) of the antagonist radioligand, [(3)H]-clobenpropit, in guinea-pig cortex membranes was estimated at 4, 12, 21 and 30 degrees C in 20 mM HEPES-NaOH buffer (buffer A), or buffer A containing 300 mM CaCl(2), (buffer A(Ca)). pK(I)' values for ligands with varying intrinsic activity were determined in buffer A and A(Ca) at 4, 12, 21 and 30 degrees C. KEY RESULTS: In buffer A, the pK(L) of [(3)H]-clobenpropit increased with decreasing temperature while it did not change in buffer A(Ca). The Bmax was not affected by temperature or buffer and n (H) values were not different from unity. In buffer A, pK(I)' values for agonists remained unchanged or decreased with decreasing temperature, while antagonist pK(I) values increased with decreasing temperature; agonist binding was entropy-driven while antagonist binding was enthalpy and entropy-driven. In buffer A(Ca), temperature had no effect on antagonist and agonist pK(I) values; both agonist and antagonist binding were enthalpy and entropy-driven. CONCLUSIONS AND IMPLICATIONS: The binding of H(3)-receptor agonists and antagonists can be thermodynamically discriminated under conditions where agonist pK(I)' values are over-estimated (pK(I)' not = pK(app)). However, under conditions when agonist pK(I) approximately pK(app), the thermodynamics underlying the binding of agonists are not different to those of antagonists.

Correlation of apparent affinity values from H3-receptor binding assays with apparent affinity (pKapp) and intrinsic activity (alpha) from functional bioassays.

BACKGROUND AND PURPOSE: Agonist apparent affinities (pK(I)') in histamine H(3)-receptor binding assays were higher than expected from apparent affinity values (pK(app)) estimated in bioassay. Here, we investigate whether the degree of pK(I)' overestimation is related to agonist intrinsic efficacy, by studying the effect of buffer composition on the pK(I)' of ligands with varying intrinsic activity. EXPERIMENTAL APPROACH: In the guinea-pig ileum bioassay, intrinsic activity (alpha) was determined from the maximal inhibition of the contraction produced by increasing agonist concentration. pK(app) values were estimated using the method of Furchgott. The pK(L) of [(3)H]clobenpropit in guinea-pig cerebral cortex was estimated by saturation analysis in 20 mM HEPES-NaOH buffer (buffer B(0,0,0)), or buffer B(0,0,0) containing 70 mM CaCl(2), 100 mM NaCl and 100 mM KCl (buffer B(0.07,0.1,0.1)). PK(I) values were determined in competition studies in both buffers. KEY RESULTS: [(3)H]clobenpropit saturation isotherms had n (H) values of unity in both buffers. In buffer B(0.07,0.1,0.1), agonist pK(I)' values were closer to pK(app) values than in buffer B(0,0,0) but were associated with n (H) values <1. A two-site analysis of agonist data in buffer B(0.07, 0.1, 0.1) provided a better fit than a one-site fit and low affinity values (pK(IL)) were comparable to pK(app). Differences between the pK(I)' in buffer B(0,0,0) and pK(IL) values in buffer B(0.07,0.1,0.1) (DeltapK) were correlated with alpha. CONCLUSIONS AND IMPLICATIONS: H(3)-receptor binding assays conducted in buffer B(0,0,0) and buffer B(0.07,0.1,0.1) can provide a measure of ligand affinity (pK(app)) and intrinsic efficacy. The assay predicts that some ligands previously classified as H(3)-receptor antagonists may possess residual intrinsic efficacy.

Pharmacological rescue of noise induced hearing loss using N-acetylcysteine and acetyl-L-carnitine.

Despite the use of hearing protection devices (HPDs) and engineering changes designed to improve workspaces, noise-induced hearing loss continues to be one of the most common and expensive disabilities in the US military. Many service members suffer acoustic trauma due to improper use of HPDs, sound levels exceeding the protective capacity of the HPDs, or by unexpected, injurious exposures. In these cases, there is no definitive treatment for the hearing loss. This study investigated the use of the pharmacological agents N-acetylcysteine and acetyl-L-carnitine after acoustic trauma to treat cochlear injury. N-Acetylcysteine is an antioxidant and acetyl-L-carnitine a compound that maintains mitochondrial bio-energy and integrity. N-Acetylcysteine and acetyl-L-carnitine, respectively, significantly reduced permanent threshold shifts and hair cell loss compared to saline-treated animals when given 1 and 4 h post-noise exposure. It may be possible to obtain a greater therapeutic effect using these agents in combination or at higher doses or for a longer period of time to address the secondary oxidative events occurring 7-10 days after acute noise exposure.

Pharmacological comparison of the alternatively spliced short and long CCK2 receptors.

(1) The alternatively spliced, short and long cholecystokinin receptors (CCK2S and CCK2L) were expressed in NIH3T3 cells, and compared using radioligand-binding assays with identical buffer and incubation conditions. (2) As judged by a saturation analysis, the selective CCK2-receptor antagonist radioligand [3H]-JB93182 did not discriminate between the CCK2S or CCK2L receptors. (3) A global analysis of competition studies, using a range of structurally diverse, CCK-receptor selective ligands, provided further evidence that these receptor subtypes were pharmacologically indistinguishable. However, when analysed individually a number of small, yet significant differences were observed with some of the compounds. (4) These data are consistent with previous study that suggested a possible pharmacological difference between these isoforms, at least in terms of the CCK2-receptor antagonist, L-365,260. However, it would appear that the pharmacological profile of these compounds is not consistent with their affinity at the putative G1/G2 receptors previously described by Harper et al.

Cardiac-specific overexpression of human beta2 adrenoceptors in mice exposes coupling to both Gs and Gi proteins.

1. Left atrial strips from transgenic (TG4) mice with cardiac-specific overexpression ( approximately 200-fold) of the beta(2) adrenoceptor (beta(2)AR) were isolated, and their isometric force of contraction (F(c)) in response to electrical stimulation was measured. 2. The betaAR agonist isoprenaline elicited negative inotropic responses in all left atrial strips; in 6/11 preparations, it also had a small positive inotropic effect. This 'up-phase' was observed from 0.1 to 10 nM, with the 'down-phase' occurring at higher concentrations. Both phases were mediated by beta(2)AR, as shown by their sensitivity to the beta(2)AR antagonist ICI-118,551 (100 nM; pA(2) 8.60+/-0.07, 8.45+/-0.19, for 'up-phase' and 'down-phase,' respectively), but not the beta(1)AR antagonist CGP-20712A (100 nM). Conversely, nontransgenic littermate preparations responded to isoprenaline treatment solely by an increase in F(c), which was beta(1)AR-mediated. 3. Pretreatment of left atrial strips with either 10 nM isoprenaline or 1 mM 8-bromo-cAMP significantly attenuated the TG4 'up-phase', while having no effect on either the TG4 'down-phase' or the littermate controls' responses. B. pertussis toxin treatment of the animals prevented isoprenaline's negative inotropic effects in TG4 preparations, but had no effect in littermate controls. 4. The findings imply that the responses of TG4 left atrium to isoprenaline are because of beta(2)AR coupling to G(s) and G(i) proteins, consistent with the model of Daaka et al., in which protein kinase A phosphorylation of the beta(2)AR causes a switch from G(s) to G(i) protein coupling.

Pharmacological evidence for putative CCK(1) receptor heterogeneity in human colon smooth muscle.

1. The pharmacology of the cholecystokinin CCK(1) receptors endogenously expressed in human gallbladder and human ascending colon smooth muscle tissue was compared using radioligand binding assays. 2. Saturation analysis of the interaction between the radiolabelled, selective CCK(1)-receptor antagonist, [(3)H]-L-364,718, and enriched gastrointestinal tissue membranes suggested the presence of multiple binding sites in human colon but not human gallbladder. 3. Competition studies, using a range of structurally diverse, CCK-receptor selective ligands provided further evidence for CCK(1) receptor heterogeneity in human colon tissue (n(H) values significantly less than unity for SR27897=0.77+/-0.07, 2-NAP=0.73+/-0.03, YM220=0.70+/-0.09 and PD-134,308=0.83+/-0.01). Moreover, the competition data for SR27897, 2-NAP and YM220 were consistent with the interaction of these compounds at two binding sites. In contrast, in the human gallbladder assay, a single binding site model provided a good fit of the competition curve data obtained with all the CCK receptor selective compounds. 4. The data obtained are consistent with the presence of a single CCK(1) receptor binding site in the gallbladder but not in the colon. A two-site analysis of the colon data, indicated that one of the two sites was indistinguishable from that characterized in the gallbladder. The molecular basis of the apparent receptor heterogeneity in the colon remains to be established.

Design, synthesis, and structure-activity relationships of novel non-imidazole histamine H(3) receptor antagonists.

Novel, potent, and selective non-imidazole histamine H(3) receptor antagonists have been prepared based on the low-affinity ligand dimaprit (pK(I) 7.32 +/- 0.12, pK(B) 5.93 +/- 0.17). Detailed structure-activity studies have revealed that N-(4-chlorobenzyl)-N-(6-pyrrolidin-1-ylhexyl)guanidine (pK(I) 8.38 +/- 0.21, pK(B) 8.39 +/- 0.13), 30, and N-(4-chlorobenzyl)-N-(7-pyrrolidin-1-ylheptyl)guanidine (pK(I) 8.78 +/- 0.12, pK(B) 8.38 +/- 0.10), 31, exhibit high affinity for the histamine H(3) receptor. Antagonists 30 and 31 demonstrate significant selectivity over the other histamine, H(1) and H(2), receptor subtypes and a 100-fold selectivity in the sigma(1) binding assay. Compounds 30and 31 are the most potent, selective non-imidazole histamine H(3) receptor antagonists reported in the literature to date.

Characterization of the binding of [3H]-clobenpropit to histamine H3-receptors in guinea-pig cerebral cortex membranes.

1 We have investigated the binding of a novel histamine H3-receptor antagonist radioligand, [3H]- clobenpropit ([3H]-VUF9153), to guinea-pig cerebral cortex membranes. 2 Saturation isotherms for [3H]-clobenpropit appeared biphasic. Scatchard plots were curvilinear and Hill plot slopes were significantly less than unity (0.63+/-0.03; n = 12+/-s.e.mean). The radioligand appeared to label two sites in guinea-pig cerebral cortex membranes with apparent affinities (pKD') of 10.91+/-0.12 (Bmax = 5.34+/-0.85 fmol mg(-1) original wet weight) and 9.17+/-0.16 (Bmax = 23.20+/-6.70 fmol mg(-1)). 3 In the presence of metyrapone (3 mM) or sodium chloride (100 mM), [3H]-clobenpropit appeared to label a homogeneous receptor population (Bmax=3.41+/-0.46 fmol mg-1 and 3.49+/-0.44 fmol mg(-1), pKD' = 10.59+/-0.17 and 10.77+/-0.02, respectively). Scatchard plots were linear and Hill slopes were not significantly different from unity (0.91+/-0.04 and 0.99+/-0.02, respectively). Granisetron (1 microM), rilmenidine (3 microM), idazoxan (0.3 microM), pentazocine (3 microM) and 1,3-di-(2-tolyl)guanidine (0.3 microM) had no effect on the binding of [3H]-clobenpropit. 4 The specific binding of [3H]-clobenpropit appeared to reach equilibrium after 25 min at 21+/-3 degrees C and remained constant for >180 min. The estimated pKD' (10.27+/-0.27; n = 3+/-s.e.mean) was not significantly different from that estimated by saturation analysis in the presence of metyrapone. 5 A series of histamine H3-receptor ligands expressed affinity values for sites labelled with [3H]-clobenpropit which were not significantly different from those estimated when [3H]-R-alpha-MH was used to label histamine H3-receptors in guinea-pig cerebral cortex membranes.

Evidence that histamine homologues discriminate between H3-receptors in guinea-pig cerebral cortex and ileum longitudinal muscle myenteric plexus.

1. The binding of the selective histamine H3-receptor agonist ([3H]-R-alpha-methylhistamine) to sites in guinea-pig cerebral cortex and ileum longitudinal muscle myenteric plexus has been characterized and a comparison made of the apparent affinities of a series of H3-receptor ligands. 2. Saturation analysis suggested that [3H]-R-alpha-methylhistamine labelled a homogeneous population of histamine H3-receptors in guinea-pig cerebral cortex (pKD=9.91+/-0. 07; nH=1.07+/-0.03; n=5) and ileum longitudinal muscle myenteric plexus (pKD=9.75+/-0.21; nH=0.97+/-0.02; n=5). There was no significant difference in the estimated affinity of [3H]-R-alpha-methylhistamine in the two tissues. The cerebral cortex had a significantly higher receptor density (3.91+/-0.37 fmol mg-1 tissue) than the ileum longitudinal muscle myenteric plexus (0. 39+/-0.11 fmol mg-1). 3. Overall, the apparent affinities of compounds, classified as H3-receptor ligands, in cerebral cortex and ileum longitudinal muscle myenteric plexus were well correlated (r=0. 91, P<0.0001) and consistent with the cerebral cortex and ileum longitudinal muscle myenteric plexus expressing histamine H3-receptor population(s) that are pharmacologically indistinguishable by the majority of histamine H3-receptor ligands. However, it was evident that the homologues of histamine within this group of compounds could discriminate between the receptor populations in the two tissues. Thus, the estimated affinity of five imidazole unbranched alkylamines (histamine, homohistamine, VUF4701, VUF4732 and impentamine) were significantly higher in the guinea-pig cerebral cortex than in the ileum longitudinal muscle myenteric plexus assay.

From histamine to imidazolylalkyl-sulfonamides: the design of a novel series of histamine H3-receptor antagonists.

Histamine was converted to a selective histamine H3-receptor antagonist by capping the primary amine with 2-naphthalenesulfonyl chloride. Higher receptor affinity and lower variability in the data from the various bioassays were achieved with the 2-naphthalensulfonamides of histamine homologues.

Analysis of the behaviour of selected CCKB/gastrin receptor antagonists in radioligand binding assays performed in mouse and rat cerebral cortex.

1. The previously described complex behaviour of the CCKB/gastrin receptor antagonist, L-365,260, in radioligand binding assays could be explained by a variable population of two binding sites. We have investigated whether other CCKB/gastrin receptor ligands (PD134,308, PD140,376, YM022 and JB93182) can distinguish between these sites. 2. In the mouse cortex assay, Hill slopes were not different from unity and the ligand pKI values did not differ when either [125I]-BH-CCK-8S or [3H]-PD140,376 was used as label as expected for a single site (G2). 3. In the rat cortex, where previous analysis of replicate (n=48) L-365,260 data indicated the presence of two CCKB/gastrin sites (G1 and G2), the competition data for PD134,308, PD140,376, YM022 and JB93182 could be explained by a homogeneous population of CCKB/gastrin sites because the Hill slope estimates were not significantly different from unity. However, the estimated affinity values for JB93182 and YM022 were significantly higher and that for PD134,308 was significantly lower than those obtained in the mouse cortex when the same radioligand was used. In view of our previous data obtained with L-365,260, the rat cortex data were also interpreted using a two-site model. In this analysis, SR27897 expressed approximately 9 fold, PD134,308 approximately 13 fold and PD140,376 approximately 11 fold selectivity for the G2 site. In contrast, JB93182 expressed approximately 23 fold and YM022 approximately 4 fold selectivity for the G1 site. If the two-site interpretation of the data is valid then, because of its reverse selectivity to L-365,260, JB93182 has been identified as a compound which if radiolabelled could provide a test of this receptor subdivision.

Characterization of the binding of a novel radioligand to CCKB/gastrin receptors in membranes from rat cerebral cortex.

1. We have investigated the binding of a novel radiolabelled CCKB/gastrin receptor ligand, [3H]-JB93182 (5[[[(1S)-[[(3,5-dicarboxyphenyl)amino]carbonyl]-2-phenylethyla mino]-carbonyl]-6-[[(1-adamantylmethyl) amino]carbonyl]-indole), to sites in rat cortex membranes. 2. The [3H]-JB93182 was 97% radiochemically pure as assessed by reverse-phase HPLC (RP-HPLC) and was not degraded by incubation (150 min) with rat cortex membranes. 3. Saturation analysis indicated that [3H]-JB93182 labelled a homogeneous population of receptors in rat cortex membranes (pKD=9.48+/-0.08, Bmax=3.61+/-0.65 pmol g(-1) tissue, nH=0.97+/-0.02, n=5). The pKD was not significantly different when estimated by association-dissociation analysis (pKD=9.73+/-0.11; n=10). 4. In competition studies, the low affinity of the CCKA receptor antagonists, L-364,718; SR27897 and 2-NAP, suggest that, under the assay conditions employed, [3H]-JB93182 (0.3 nM) does not label CCKA receptors in the rat cortex. 5. The affinity estimates obtained for reference CCKB/gastrin receptor antagonists were indistinguishable from one of the affinity values obtained when a two site model was used to interpret [125I]-BH-CCK8S competition curves obtained in the same tissue (Harper et al., 1999). 6. This study provides further evidence for the existence of two CCKB/gastrin sites in rat cortex. [3H]-JB93182 appears to label selectively sites previously designated as gastrin-G1 and therefore it may be a useful compound for the further discrimination and characterization of these putative receptor subtypes.

Analysis of variation in L-365,260 competition curves in radioligand binding assays.

1. For several years, we have used the cholecystokinin (CCK)B/gastrin receptor selective antagonist, L-365,260, as a reference compound in a variety of studies in CCKB/gastrin receptor radioligand binding assays. Here, we have analysed the competition curve data sets obtained between L-365,260 and [125I]-BH-CCK8S in guinea-pig gastric gland and mouse and rat cerebral cortex preparations. 2. Competition curves obtained for L-365,260 in the mouse cortex assay were not different from rectangular hyperbolae (slope = 1.01 +/- 0.02) implying the presence of a single population of binding sites (pKI = 8.41 +/- 0.01; data from 47 experiments, slope constrained to unity). However, in the rat cortex and guinea-pig gastric gland assays, the mean slope of the competition curves was significantly less than one and the mean apparent pKI significantly lower than that obtained in the mouse cortex (slope = 0.85 +/- 0.03, 0.90 +/- 0.03; apparent pKI = 7.98 +/- 0.05, 8.07 +/- 0.05; 48 and 45 experiments, in rat and guinea-pig, respectively). The distribution of the individual pKI and slope estimates of the competition curves in these two assays was consistent with expectations for the variable expression (in terms of absolute number and proportion) of two binding sites. The two sites were characterized by pKI values for L-365,260 of 8.50 +/- 0.04 and 8.48 +/- 0.04 for the high affinity site and 7.32 +/- 0.04 and 7.22 +/- 0.06 for the low affinity site in guinea-pig and rat, respectively. 3. The affinity estimates for L-365,260, although obtained on different tissues, are consistent with data obtained from the analysis of L-365,260 antagonism of pentagastrin-stimulated responses in mouse and rat stomach (acid secretion) and guinea-pig gastric muscle (isotonic contraction) assays. To this extent, these data suggest the existence of two CCKB/gastrin receptor subtypes.

Analysis of the variation in the action of L-365,260 at CCKB/gastrin receptors in rat, guinea-pig and mouse isolated gastric tissue assays.

1. Since L-365,260 was first described as a selective antagonist at cholecystokinin (CCK)B/gastrin receptors, we have used it periodically as a reference compound in isolated tissue assays of guinea-pig gastric muscle and lumen-perfused stomachs from mouse and immature rat. L-365,260 behaved as a surmountable antagonist and produced parallel rightward shifts of pentagastrin concentration-effect curves' in each of the replicate experiments. The experiments were performed by several different experimenters in the same laboratories over a five year period. 2. In the isolated, lumen-perfused, immature rat stomach assay, L-365,260 behaved as a simple competitive antagonist (Schild plot slope = 1.00 +/- 0.10, pKB = 7.54 +/- 0.03 from a global analysis of the data) acting at a homogeneous population of receptors in five separate, highly-reproducible, experiments. In contrast, the replicate data sets obtained from the interaction in the isolated, lumen-perfused mouse stomach and guinea-pig gastric muscle assays, over the same period, were not consistent with the presence of a single receptor population. The guinea-pig gastric muscle data were relatively reproducible between experiments but some individual Schild plot slopes and the slope estimated from a global analysis of all the data were significantly less than unity (slope = 0.80 +/- 0.07, pA2 = 8.56 +/- 0.05 from the global analysis). The data obtained in the mouse stomach were significantly more variable than that obtained in the same assay, during the same period, from the interaction between histamine and the H2-receptor antagonist, famotidine. The individual Schild plot slopes ranged from being very flat (0.20) to being not significantly different from unity (1.23) and the pA2 values ranged from 7.68 to 8.70. 3. Overall, the data could be accounted for by assuming the variable expression of two receptor subtypes across the assays. The rat stomach appeared to express a single receptor characterized by a low affinity constant for L-365,260 (pKB approximately 7.5). The guinea-pig gastric muscle and mouse stomach data could be explained by the presence of this receptor and a second one characterized by a high affinity constant for L-365,260 (pKB approximately 8.6). The activity of the two proposed receptor subtypes was consistent between experiments in the guinea-pig and the high affinity receptor appeared to be predominant. In contrast, the mouse stomach data could only be simulated by assuming that the proportion and absolute number of each subtype varied significantly between the replicate experiments. 4. The L-365,260 affinity estimates at the inferred receptor subtypes were indistinguishable from those obtained in a corresponding analysis of the behaviour of L-365,260 in CCKB/gastrin receptor radioligand binding experiments in guinea-pig gastric gland and mouse and rat cerebral cortex preparations.

2-Naphthalenesulphonyl L-aspartyl-(2-phenethyl)amide (2-NAP)--a selective cholecystokinin CCKA-receptor antagonist.

1. The in vitro pharmacological characterization of the sodium salt of 2-naphthalenesulphonyl 1-aspartyl-(2-phenethyl)amide [2-NAP], a hydrophilic compound derived from the C-terminal aspartate-phenylalanine dipeptide of cholecystokinin (CCK), is described. 2. 2-NAP behaved as a competitive antagonist of sulphated cholecystokinin octapeptide (CCK-8) at CCKA-receptors in both intact tissue bioassays (guinea-pig gall bladder, pancreas and ileum, human and rabbit gall bladder) and a radioligand displacement assay (guinea-pig pancreatic cells). The mean pKB, over assays, was 6.5. 3. Compared to the other assays, the rabbit gall bladder assay gave a significantly higher pKB estimate [7.0] for 2-NAP and a significantly lower estimate [8.9] for devazepide (formerly L-364,718 and MK-329), a well-characterized CCKA-receptor antagonist; these anomalous results suggest that a different class of CCKA-receptors may be involved. 4. 2-NAP, was found to be highly selective, having at least 300 fold greater affinity for CCKA-receptors than for 50 other pharmacological loci, including gastrin/CCKB, as estimated by bioassay or radioligand displacement.