Introduction

Summary Introduction Procedures Results Discussion References

Somatostatin-14 is a biologically active tetradecapeptide (1-3). To date, five distinct members of the somatostatin receptor family have been identified, and all are putative seven-transmembrane G protein-coupled receptors (4-8). The human and rat isoforms of sst₅ are the only somatostatin receptors claimed to show preferential affinity for somatostatin-28 compared with somatostatin-14 (8, 9). Somatostatin receptors have been demonstrated to couple to a multitude of transduction mechanisms, such as activation of potassium currents, inhibition of calcium currents, stimulation of phosphoinositide turnover, and interaction with protein phosphatase cascades, in addition to the inhibition of adenylate cyclase (10-14). The sst₅ receptor, like the other recombinant somatostatin receptor types, negatively couples to adenylate cyclase when recombinantly expressed in cell lines (8, 14-17). The human sst₅ receptor has also been shown to mediate activation of phosphoinositide metabolism and the accumulation of intracellular calcium (12, 18). Furthermore, there is evidence that this somatostatin receptor type can couple to both pertussis toxin-sensitive and -insensitive G proteins (12, 19, 20).

The aim of this work was to study the ability of a number of agonists to stimulate the binding of [³⁵S]GTPS to G proteins, mediated by the activation of the human recombinant sst₅ expressed in CHO-K1 cells. This technique relies on the receptor-stimulated exchange of GDP for GTP (or [³⁵S]GTPS) and therefore provides a quantitative measure of signal transduction close to the level of receptor activation. [³⁵S]GTPS binding has already been used in the study of other G protein-coupled receptors, such as human muscarinic receptors (21, 22) and adenosine A₁ receptors (23), to provide quantitative profiles of receptor/G protein interactions. Although sst₅ receptor-mediated decreases in cAMP levels and increases in inositol-1,4,5-trisphosphate production are both pertussis toxin sensitive, they are not necessarily mediated by the same pertussis toxin-sensitive G protein (12). The study of the initial step of the transduction pathway should provide a gross measure of receptor/G protein activation, independent of whether the activated G proteins form a heterogeneous or homogenous population. This approach has provided an opportunity to explore the pharmacology of the newly available, reportedly sst₅ receptor-selective ligands, L-362,855 and BIM-23056 (18, 19).

	Experimental Procedures

Summary Introduction Procedures Results Discussion References

Materials. [³⁵S]GTPS was purchased from DuPont (1000-1500 Ci/mmol; Bad Homburg, Germany). [¹²⁵I]-Tyr¹¹-somatostatin-14 (2000 Ci/mmol) was purchased from Amersham International (Buckinghamshire, UK). Bordetella pertussis toxin was purchased from Calbiochem (San Diego, CA). Bacitracin and phenylmethylsulfonyl fluoride were supplied by Sigma Chemical (Poole, Dorset, UK). Cell culture products were purchased from Life Technologies (Paisley, UK). Somatostatin-14 and somatostatin-28 were purchased from Peninsula Laboratories Europe (Merseyside, UK). BIM-23056 (D-Phe-Phe-Tyr-D-Trp-Lys-Val-Phe-D-Nal-NH₂), BIM-23027 (c[N-Me-Ala-Tyr-D-Trp-Lys-Abu-Phe]), and L-362,855 (c[Aha-Phe-Trp-D-Trp-Lys-Thr-Phe], where Nal is -(2-naphthyl)alanine, Abu is aminobutyric acid, and Aha is 7-amino-heptanoic acid) were custom-synthesized by Peptide and Protein Research Consultants (University of Exeter, Exeter, UK). All of the peptides, with the exception of BIM-23056, were initially dissolved in distilled water to produce a concentration of 1 mM and then divided into aliquots and stored frozen. BIM-23056 was initially dissolved in 10% dimethylsulfoxide.

Cell culture and membrane preparation. Production and culturing conditions of a CHO-K1 cell line stably expressing the human sst₅ receptor (CHOsst₅) have been previously described (18). To prepare a membrane fraction, cells were scraped into ice-cold phosphate-buffered saline and centrifuged for 20 min at 4° at 250 × g. The pellet was resuspended in a 10 mM HEPES buffer, pH 7.4, at 4° containing 0.1 mM EDTA, 0.1 mg/ml bacitracin, and 0.1 mM phenylmethylsulfonyl fluoride and homogenized in a Dounce ground glass homogenizer (20 strokes). After centrifugation for 30 min at 4° at 10,000 × g, the resultant membrane pellet was resuspended in the above buffer and stored at 80°.

[¹²⁵I]-Tyr¹¹-somatostatin-14 binding assays. To determine ligand affinities, ~2 µg of membrane protein was incubated with 0.03 nM [¹²⁵I]-Tyr¹¹-somatostatin-14 and a range of competing ligand concentrations in a 10 mM HEPES buffer, pH 7.4, containing 5 mM MgCl₂, 0.1 mM EDTA, and 0.2 mg/ml bacitracin for 120 min at 21°. Reactions were terminated by vacuum filtration onto 0.5% polyethylenimine-pretreated filters using a Brandell cell harvester, and radioactivity bound was quantified using a Cobra II -counter. Total [¹²⁵I]-Tyr¹¹-somatostatin-14 bound to the membranes was ~3000 dpm, with nonspecific binding of ~500 dpm, as defined with 1 µM somatostatin-14.

[³⁵S]GTPS binding assays. For studies of [³⁵S]GTPS binding, 2-6 µg of membrane protein was incubated for 120 min at 21° in a 10 mM HEPES buffer, pH 7.4, containing 50 or 150 mM NaCl, 5 mM MgCl₂, 0.1 mM EDTA, and 0.3 µM GDP with ligand, unless indicated otherwise. The protein concentration was the same within each set of experiments. To identify optimal conditions, experiments were performed to evaluate the effects of NaCl and GDP on [³⁵S]GTPS binding. Condition optimization studies were performed in 100 mM NaCl, 10 mM MgCl₂, and 1 µM GDP, except where one of these was under analysis, as indicated in the figure legend for the particular experiment. Steady state receptor occupation was achieved after 80-120 min in studies of [¹²⁵I]-Tyr¹¹-somatostatin-14 binding to the receptor. For this reason, in studies of [³⁵S]GTPS binding, ligands were preincubated with membranes for 90 min before the addition of 0.2 nM [³⁵S]GTPS to ensure the maximal possible agonist activation of the receptor. Under optimized conditions (0.3 µM GDP and 5 mM MgCl₂) and 150 mM NaCl and after preincubation, somatostatin-14-induced [³⁵S]GTPS binding was linear over the initial 50 min. A 30-min incubation period was routinely used to quantify the agonist-activated [³⁵S]GTPS binding. Reactions were terminated by vacuum filtration using a Packard Filtermate harvester. The filters were dried, and the amount of radioactivity bound was determined after the addition of 50 µl of Microscint-O (Packard) scintillation fluid by counting with a Canberra Packard Topcount Scintillation Counter. In 150 mM NaCl, this protocol resulted in basal [³⁵S]GTPS binding of ~2000 dpm, rising to 4000 dpm after incubation with 10 µM somatostatin-14. Nonspecific binding of [³⁵S]GTPS defined with 10 µM GTPS was ~100 dpm. This represented 3.8 ± 0.6% (n = 4) and 2.2 ± 0.2% (n = 4) of the total binding observed in the presence of 10 µM somatostatin-14 in 150 and 50 mM NaCl, respectively. Nonspecific binding was not subtracted from experimental data as it represented such a relatively small percentage of the overall binding, so all values represent the total levels of [³⁵S]GTPS binding that were observed.

Pertussis toxin treatment. Cells were pretreated with 100 ng/ml pertussis toxin for 18 hr before the cells were harvested for the production of a membrane fraction.

Data analysis. Competition binding data were analyzed by nonlinear least-squares regression using Prism (GraphPAD Software, San Diego, CA). The fitting of competition curves for [¹²⁵I]-Tyr¹¹-somatostatin-14 binding to membranes from CHOsst₅ cells by somatostatin-14 and the other ligands generated half-maximal inhibitory concentrations (IC₅₀ values). Hill slopes were close to unity, in most cases not significantly different from 1, and were constrained to unity for calculation purposes. To calculate K_D and B_max values from competition studies, the following equations were used: K_D = IC₅₀  [A], where K_D is the equilibrium dissociation constant of the radioligand, IC₅₀ is the half-maximal inhibitory concentration of somatostatin-14, and [A] is the concentration of [¹²⁵I]-Tyr¹¹-somatostatin-14 present in the assay medium (0.03 nM); and B_max = ([B] × IC₅₀)/[A], where B_max is the receptor density, and [B] is the concentration of specific [¹²⁵I]-Tyr¹¹-somatostatin-14 bound to the receptor in the absence of competing ligand.

	Results

Summary Introduction Procedures Results Discussion References

Binding of somatostatin-14 and its analogues to the sst₅ receptor. Somatostatin-14 displacement of [¹²⁵I]-Tyr¹¹-somatostatin-14 from its binding sites in membranes prepared from the CHOsst₅ cells gave an IC₅₀ value for somatostatin-14 of 0.21 ± 0.037 nM with an estimated B_max value of 3.01 ± 0.3 pmol/mg (n = 3). The receptor density remained constant over the time course of the study, with the corresponding B_max value being 2.91 ± 0.93 pmol/mg (n = 3) at the end of the series of experiments. No specific binding was detected in untransfected CHO-K1 cells (data not shown). Affinity estimates for a number of somatostatin-14 analogues in membranes from CHOsst₅ cells were determined from their abilities to compete with [¹²⁵I]-Tyr¹¹-somatostatin-14 for binding to the receptor. Their binding profiles are illustrated in Fig. 1, and the calculated parameter values are presented in Table 1. The data were fitted in each case to a one-site sigmoidal binding curve, although individual curves of L-362,855, BIM-23027, and BIM-23056 competition for [¹²⁵I]-Tyr¹¹-somatostatin-14 binding fitted better to a two-site model in a few cases. Somatostatin-28, somatostatin-14, and L-362,855 all bound with high affinity, in the subnanomolar range, with somatostatin-28 displaying an ~4-fold higher affinity than somatostatin-14 (K_i = 0.046 and 0.18 nM, respectively; Table 1). The following rank order of ligand affinity estimates (pK_i values) was observed: somatostatin-28 (10.34)  L-362,855 (10.09)  somatostatin-14 (9.74) > BIM-23056 (8.61) > BIM-23027 (7.89).

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Displacement of binding of [125I]-Tyr11-somatostatin-14 (0.03 nM) from membranes of CHOsst5 cells by somatostatin-14 (), somatostatin-28 (), L-362,855 (), BIM-23056 (), and BIM-23027 (). Assays were incubated at 21° until equilibrium was attained (2 hr) and were performed in the absence of sodium chloride (see Experimental Procedures for details regarding the buffer). Data are the mean ± standard error from three separate experiments performed in duplicate and are expressed as a percentage of specific [125I]-Tyr11-somatostatin-14 binding.

View this table: [in this window] [in a new window]   TABLE 1 Estimates of binding affinities and agonist potencies for stimulating [35S]GTPS binding by somatostatin sst5 receptor activation Ligand affinity estimates (dissociation equilibrium constants, Ki values) are compared for membranes prepared from CHOsst5 cells with potencies (EC50) and maximal effects for stimulating [35S]GTPS binding in the presence of 5 mM magnesium chloride, 0.3 µM GDP, and 50 or 150 mM sodium chloride. Hill slopes (nH) are also shown. The maximal [35S]GTPS binding response is expressed as a percentage of the response observed with somatostatin-28 (10 µM) in 150 mM NaCl. Values are mean ± standard error of nonlinear regression curves fitted to the experimental data (n = three to five). SRIF-28 and SRIF-14 refer to somatostatin-28 and somatostatin-14, respectively.

Conditions for [³⁵S]GTPS binding. Suitable conditions for somatostatin-14-induced increases in [³⁵S]GTPS binding to CHOsst₅ membranes were sought by attempting to maximize specific stimulation while reducing basal [³⁵S]GTPS binding. When GDP concentrations were increased from 0.1 nM to 0.3 mM, basal levels of binding were decreased with little effect on stimulated binding (Fig. 2). Optimal increases in somatostatin-14-induced binding of [³⁵S]GTPS were observed with 0.3 µM GDP due to the markedly decreased levels of basal binding. Hence, this concentration was used in further studies unless otherwise stated. In the presence of 0.3 µM GDP, basal [³⁵S]GTPS binding was 41.3 ± 4.9%, and somatostatin-14-stimulated (1 µM) levels were 59.6 ± 7.3% of that in the absence of GDP.

View larger version (13K): [in this window] [in a new window]   Fig. 2.   A, Effect of GDP concentration on binding of [35S]GTPS (0.2 nM) to membranes from CHOsst5 cells in the absence () and presence () of 1 µM somatostatin-14. Data are the mean ± standard error of the percentage of unstimulated [35S]GTPS binding in the absence of GDP. B, Effect of GDP on specific somatostatin-14-stimulated [35S]GTPS binding (). Data are the mean ± standard error and are expressed as a percentage of basal [35S]GTPS binding at each GDP concentration. Assays were carried out in triplicate in three separate experiments in 100 mM NaCl and 10 mM MgCl2. Basal levels of 1.51 ± 0.25 pmol/mg were observed in the absence of GDP, rising to 1.71 ± 0.27 pmol/mg with somatostatin-14.

View larger version (11K): [in this window] [in a new window]   Fig. 3.   A, Effect of sodium chloride on binding of [35S]GTPS (0.2 nM) to membranes from CHOsst5 cells in the absence () and presence () of 1 µM somatostatin-14. Data are the mean ± standard error and are expressed as a percentage of [35S]GTPS binding in the absence of sodium chloride. B, Effect of sodium chloride on specific 1 µM somatostatin-14 stimulated [35S]GTPS binding. Data are the mean ± standard error. Assays were carried out in triplicate in three separate experiments in 1 µM GDP and 10 mM MgCl2. In the absence of sodium chloride, basal levels of 0.72 pmol/mg were measured, increasing to 1.02 ± 0.36 pmol/mg in the presence of somatostatin-14.

Agonist potencies for stimulation of [³⁵S]GTPS binding in low and high sodium. The potency estimates and relative maximal effects of the peptide analogues studied are summarized in Table 1. All data were normalized to the maximal binding observed in 150 mM NaCl, which was produced by 10 µM somatostatin-28 because experiments in 50 and 150 mM NaCl were carried out simultaneously (Fig. 4). [³⁵S]GTPS binding in 150 mM NaCl and in the absence of any ligand was 47.8 ± 7.3% (n = 4), and in 50 mM NaCl, basal [³⁵S]GTPS binding was 98.94 ± 11.86% (n = 4). In high and low sodium, both somatostatin-14 and somatostatin-28 concentration-dependently increased [³⁵S]GTPS binding to give fitted curve maxima of 91 ± 4.5% and 102 ± 4.1% (high sodium) and 158 ± 5.04% and 161 ± 3.66% (low sodium), respectively (Fig. 4). Thus, somatostatin-28 (10 µM) increased [³⁵S]GTPS binding by 0.228 ± 0.027 pmol/mg in 150 mM NaCl and 0.204 ± 0.055 pmol/mg in 50 mM NaCl. The potency estimates (pEC₅₀ values) for somatostatin-14 were 6.90 in high sodium and 7.52 in low sodium, reflecting a 5-fold greater potency with reduced sodium; somatostatin-28 was 9-fold more potent in low sodium than in higher sodium (Table 1). BIM-23027 was relatively weak at stimulating [³⁵S]GTPS binding, causing an observable effect only at concentrations of >1 µM, in both low and high sodium (Table 1).

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Stimulation of [35S]GTPS binding in membranes from CHOsst5 cells by various concentrations of somatostatin-14 (), somatostatin-28 (), L-362,855 (), BIM-23056 (), and BIM-23027 () in the presence of 5 mM magnesium chloride, 0.3 µM GDP, and 50 mM sodium chloride (A) or 150 mM sodium chloride (B). Data are the mean ± standard error from four separate experiments performed in duplicate, in which 100% [35S]GTPS binding was the maximum response observed with 10 µM somatostatin-28 at 150 mM sodium chloride. For potency estimates, see Table 1. At 50 mM NaCl, basal binding was 0.51 ± 0.07 pmol/mg, and the net agonist effects at 10 µM were 0.20 ± 0.07, 0.20 ± 0.06, and 0.12 ± 0.09 pmol/mg for somatostatin-14, somatostatin-28, and L-362,855, respectively. At 150 mM NaCl, basal binding was 0.23 ± 0.04 pmol/mg, and 10 µM somatostatin-14, somatostatin-28, or L-362,855 produced 0.19 ± 0.04, 0.23 ± 0.03, or 0.03 ± 0.03 pmol/mg increases above basal levels, respectively.

Antagonist effects of L-362,855 and BIM-23056. In both 50 and 150 mM NaCl, L-362,855 stimulated [³⁵S]GTPS binding with high potency (pEC₅₀ = 8.03 and 7.77, n = 4, respectively; for corresponding EC₅₀ values, see Table 1). Although potent at stimulating [³⁵S]GTPS binding in the presence of 150 mM NaCl, L-362,855 stimulation of [³⁵S]GTPS binding reached a plateau at 30 nM, with a significantly lower curve maximum than those of somatostatin-14 and somatostatin-28 (unpaired t test, n = 4; see Table 1). When L-362,855, at concentrations of >30 nM, was coincubated with somatostatin-14, it was found to surmountably antagonize the responses to somatostatin-14 in a concentration-dependent manner (Fig. 5A). Fitting the data to the Black-Leff model provided an estimated pK_P value for L-362,855 of 7.44 ± 0.11, computed E_m value of 101.9 ± 0.8, n value of 0.75 ± 0.03, and _P value of 0.81 ± 0.1 (n = 11). In low sodium, the maximum of L-362,855 increased so it no longer significantly differed from those of somatostatin-14 and somatostatin-28 (unpaired t test, n = 4; see Table 1). In contrast to somatostatin-14 and somatostatin-28, there was no difference between the pEC₅₀ values for L-362,855 in low and high sodium (unpaired t test, n = 4; see Table 1).

View larger version (18K): [in this window] [in a new window]   Fig. 5.   A, Inhibition of somatostatin-14 stimulated [35S]GTPS binding was measured in the absence () or presence of increasing concentrations of L-362,855, 0.03 µM (), 0.1 µM (), and 0.3 µM (), in 150 mM sodium chloride. B, Inhibition of somatostatin-14 stimulated [35S]GTPS binding was measured in the absence () or presence of increasing concentrations of BIM-23056, 0.057 µM (), 0.24 µM (), and 1 µM (), in 150 mM sodium chloride. Data are the mean ± standard error of four separate experiments performed in duplicate and are expressed as percentage of the 10 µM somatostatin-14 response in each case. Basal levels of [35S]GTPS were 0.176 ± 0.016 and 0.179 ± 0.404 pmol/mg, and 10 µM somatostatin-14 raised this by 0.236 ± 0.019 and 0.225 ± 0.018 pmol/mg in A and B, respectively (n = 4).

Comparison of agonist affinity estimates with potencies for stimulating [³⁵S]GTPS binding. The different conditions used to determine agonist binding and functional measurements would be expected to influence the affinity estimates of the various ligands. To address this issue, attempts were made to perform both assays under similar conditions. Reducing the sodium content of [³⁵S]GTPS binding assays to only 5 mM resulted in very high basal [³⁵S]GTPS binding, above which agonist-stimulated [³⁵S]GTPS binding was not readily discernible (data not shown, n = 5). Therefore, the effects were examined of varying components of the GTPS assay on specific [¹²⁵I]-Tyr¹¹-somatostatin-14 binding to the sst₅ receptor. The addition of GTPS, GDP, or NaCl reduced specific binding of [¹²⁵I]-Tyr¹¹-somatostatin-14 (0.03 nM) to CHOsst₅ membranes: 150 mM NaCl by 59.5 ± 1.5%, 0.1 nM GTPS by 15.4 ± 0.8%, and 0.3 µM GDP by 41.4 ± 10.9% (n = 3). All three components together reduced specific [¹²⁵I]-Tyr¹¹-somatostatin-14 binding by 83.2 ± 3.5% (n = 3). The effects of GTPS and GDP were concentration dependent, with pEC₅₀ values of 9.00 ± 0.09 and 6.92 ± 0.40 and Hill slopes of 0.60 ± 0.07 and 0.51 ± 0.21, respectively (n = 3). Thus, under the conditions used for measurement of agonist-stimulated [³⁵S]GTPS binding, it was not possible to conduct filtration binding assays using [¹²⁵I]-Tyr¹¹-somatostatin-14.

Pertussis toxin sensitivity. An 18-hr pretreatment with 100 ng/ml pertussis toxin abolished [¹²⁵I]-Tyr¹¹-somatostatin-14 high affinity binding to the receptor. Somatostatin-14 (1 µM) stimulation of [³⁵S]GTPS binding was also profoundly reduced to only 3.4 ± 2% and 4.2 ± 1% of the stimulated binding in non-pertussis toxin-pretreated membranes in 50 and 150 mM NaCl, respectively. In comparison with untreated controls, reductions in the basal levels of [³⁵S]GTPS binding were also observed in membranes prepared from both pertussis toxin-pretreated CHOsst₅ and pertussis toxin-pretreated wild-type CHO-K1 cells. Basal [³⁵S]GTPS binding was reduced by the same extent by pertussis toxin pretreatment in CHOsst₅ and wild-type cells (62.8±5.3% and 75.3 ± 3.7%, and 20.0 ± 14.8% and 22.8 ± 12.1%) in the absence and presence of 150 mM NaCl, respectively (n = 4-5).

	Discussion

Summary Introduction Procedures Results Discussion References

The purpose of this study was to investigate agonist-stimulated [³⁵S]GTPS binding mediated by the human sst₅ receptor and compare the functional characteristics of some newly available, purportedly receptor-selective ligands. In addition, determination of agonist affinity estimates for the receptor allows comparison with equivalent estimates from functional studies. Because the literature contains some discrepancies in the absolute potencies of ligands for binding to the recombinant sst₅ receptor (see below), it was necessary to determine agonist affinity estimates for the receptor by radioligand binding in the same CHOsst₅ cells used in this study.

Somatostatin-28 bound to the sst₅ receptor with a greater affinity than somatostatin-14, with K_i values of 0.046 and 0.182 nM, respectively, confirming the preferential affinity of this receptor for somatostatin-28 (see introduction). Somatostatin-28 and somatostatin-14 bound to the human sst₅ with affinities very similar to those observed by O'Carroll et al. (17), who reported IC₅₀ values of 0.05 and 0.16 nM for somatostatin-28 and somatostatin-14, respectively. Similarly, the estimated dissociation constant for somatostatin-28 reported by Patel and Srikant (26) at the human sst₅ receptor (K_i = 0.07 nM) is very close to our finding. However, these authors (26), like Panetta et al. (16), found the affinity of somatostatin-14 to be lower, with K_D values of 0.9 and 2.24 nM, respectively (16). With regard to the somatostatin analogues, our results show the affinity of L-362,855, the putative sst₅-selective compound for the human sst₅ receptor, to be high (K_i = 0.082 nM), although somewhat less than previously reported (IC₅₀ = 0.016 nM; Ref. 17). The reportedly sst₂ and sst₃ receptor-selective compounds, BIM-23027 and BIM-23056, respectively, bound with lower affinity to this receptor. Patel and Srikant (26) have published an affinity estimate (K_D) for BIM-23056 at this receptor of 5.7 nM, which is in agreement with our observations, whereas BIM-23027 has been shown to be ~10-fold weaker than the value we determined (IC₅₀ = 176 nM; Ref. 17). Collectively, these values demonstrate a considerably lower affinity for BIM-23027 at the human sst₅ compared with the human sst₂ receptor for which BIM-23027 is selective (e.g., IC₅₀ = 0.04 nM; Ref. 27). Thus, our radioligand binding data provide a rank order of ligand affinities for the human sst₅ receptor of somatostatin-28  L-362,855  somatostatin-14 > BIM-23056 > BIM-23027, which is in broad agreement with those of other researchers.

In common with studies on other seven-transmembrane receptors, [³⁵S]GTPS binding was dependent on a number of variables, such as the concentrations of GDP, sodium chloride, and magnesium chloride (28). GDP reduced basal [³⁵S]GTPS binding, as did sodium chloride. In fact, sodium chloride was found to be obligatory to reduce basal binding to observe an agonist-dependent effect. Reduction of basal [³⁵S]GTPS binding by GDP is probably a consequence of its competition for the nucleotide binding site on all classes of G proteins. At concentrations of <1 µM, GDP preferentially reduced basal [³⁵S]GTPS binding compared with somatostatin-14-stimulated binding and resulted in an optimal increase in specific binding at 0.3 µM. At higher concentrations, GDP decreased the specific somatostatin-14 stimulation, presumably by competing with [³⁵S]GTPS for the G proteins that couple to sst₅ receptors. The GDP dependence of somatostatin-14-activated [³⁵S]GTPS binding is similar to that seen for other receptor systems (29). Interestingly, the rate of hydrolysis of GTP by G_o has previously been shown to correlate with the rate of release of GDP from G_o (Ref. 30 and references therein). From such studies, it has been suggested that GDP dissociation from the  subunit of G_o is rate limiting for receptor-mediated G protein activation. Thus, exogenously applied GDP may reduce the rate of GDP release and, therefore, the rate of G protein activation (31). Traynor and Nahorski (29) suggested that GDP-dependent [³⁵S]GTPS binding is a characteristic of receptors negatively coupled to adenylyl cyclase, such as the adenosine A₁ receptor and the µ-opioid receptor, and our observations are consistent with this proposal (25, 29). It should, however, be noted that norepinephrine-stimulated [³⁵S]GTPS binding to _2D-adrenoceptors in PC-12 cells has been shown to be GDP independent (32).

In 50 and 150 mM sodium chloride, both somatostatin-28 and L-362,855 were more potent than somatostatin-14 at stimulating [³⁵S]GTPS binding to the sst₅ receptor. The preferential potency of somatostatin-28 over somatostatin-14 for the human sst₅ receptor, evident in the functional measurements, correlated well with their relative affinity estimates for the receptor from binding studies. Indeed, the agonist profile of receptor activation was very similar to the profile of ligand binding to the receptor, showing the same rank order of agonist potencies, except that the potency estimates were 115-159-fold (low sodium) and 206-1130-fold (high sodium) weaker for stimulation of [³⁵S]GTPS binding. The low potency of somatostatin-14 to stimulate [³⁵S]GTPS binding compared with its affinity in [¹²⁵I]-Tyr¹¹-somatostatin-14 displacement binding studies suggests that in the former, the sst₅ receptors are primarily in a low affinity conformational state. Being necessarily restricted at present to the use of agonist radioligands it is not theoretically possible to detect binding to the receptor in its low affinity state. Indeed, it was not possible in practice to detect binding of [¹²⁵I]-Tyr¹¹-somatostatin-14 to the receptor under conditions identical to those used for the [³⁵S]GTPS assay (see Results).

It has been shown, when measuring the rate of extracellular acidification in CHOsst₅ cells, that L-362,855 produced a significantly lower maximal response compared with somatostatin-14, although it was more potent (EC₅₀ = 0.09 nM versus 0.54 nM, respectively; Ref. 19). In agreement with data from this study in high sodium, we have shown that L-362,855 potently increased [³⁵S]GTPS binding with a lower maximal response than either somatostatin-14, or somatostatin-28, suggesting that it behaved as a highly potent agonist with low efficacy. Consistent with its partial agonist nature, L-362,855 produced rightward shifts of concentration-effect curves to somatostatin-14, yielding an estimated dissociation equilibrium constant (pK_P) of 7.44. As would be expected for an agonist with low efficacy, which theoretically is required to occupy all the available receptors for a maximum response, this value was close to the pEC₅₀ value of 7.77 for L-362,855 for activation of the receptor in high sodium. Tallent et al. (33) recently reported the partial agonist nature of L-362,855 at murine sst₅ receptors for reducing calcium ion influx into an anterior pituitary cell line (AtT-20) and for inhibiting adenylate cyclase through the recombinant human sst₅ receptor expressed in CHO cells, although they provided no quantitative estimates of potency.

BIM-23056 exhibited a relatively high affinity for the sst₅ receptor in competition binding studies but was not able to stimulate [³⁵S]GTPS binding and was therefore tested as an antagonist. In high sodium, BIM-23056 concentration-dependently shifted the concentration-effect curve for somatostatin-14 rightward along the abscissa. BIM-23056 did not seem to decrease the curve maxima, which suggested it was acting in a competitive, or at least a surmountable, manner. Furthermore, the Gaddum-Schild analysis was consistent with competitive antagonism allowing calculation of the dissociation equilibrium constant for BIM-23056 in antagonizing somatostatin-14-induced [³⁵S]GTPS binding studies. Its pK_B value of 7.28 was lower than its estimated affinity for the sst₅ receptor from the binding data in the absence of sodium (pK_B = 8.61). An intermediate pK_B value was recently reported for BIM-23056 in antagonizing the effects of somatostatin-14-induced increases in intracellular calcium ion mobilization (8.0; Ref. 18). It is apparent that the estimates of the dissociation constant for BIM-23056 are dependent on the experimental conditions and the agonist response measured.

Costa et al. (34, 35) studied opioid receptor-stimulated GTPase activity and the effects of sodium on both ligand binding to the receptor and ligand-dependent GTPase activity. They showed that their experimental data could be fitted to the ternary complex model if they assumed that sodium increased the equilibrium dissociation constant for the receptor/G protein interaction. They also observed ligands with negative intrinsic activity, which was taken as evidence for the existence of constitutive receptor activation. In the absence of any known inverse agonist for the sst₅ receptor, evidence for constitutive receptor activity can only be provided by the ability of other agents, which disrupt receptor/G protein interaction, to reduce the apparent basal level of [³⁵S]GTPS binding. Increasing concentrations of GDP and sodium reduced basal and somatostatin-14-stimulated [³⁵S]GTPS binding with a profile close to that observed in other studies (25, 32). However, we have also shown that pertussis toxin pretreatment, which prevents [³⁵S]GTPS binding to activated G proteins, reduced basal [³⁵S]GTPS binding in membranes from wild-type and CHOsst₅ cells to a similar extent in a sodium-dependent manner. Thus, the presence of the sst₅ receptor does not seem to modify the amount of agonist-independent constitutive activity measured in these CHO-K1 cells, so it is unlikely that the sst₅ receptor possesses constitutive activity under these conditions. The cause of the constitutive activity observed here is unknown but has been demonstrated to be sodium sensitive and may be due to endogenously expressed G protein-coupled receptors in these cells.

According to the model used by Costa et al. (34,35), the efficacy of a ligand depends on its ability to induce coupling of the receptor to its G protein. Thus, factors such as the GDP or sodium chloride concentration, which alter the position of the binding equilibrium between the receptor and G protein, will affect the efficacy of a ligand in a manner inversely related to the efficacy of that ligand. A reduction in the sodium chloride concentration would be expected to increase the efficacy of a ligand. This prediction of their model seems to be borne out when studying the agonist activation of the sst₅ receptor in CHO-K1 cell membranes by quantification of [³⁵S]GTPS binding. At low sodium compared with high sodium, the partial agonist L-362,855 became a full agonist with no significant increase in EC₅₀ value, whereas somatostatin-14 and somatostatin-28 displayed the same maximal intrinsic activity while their EC₅₀ values decreased. Such profiles are consistent with the concept of differential behavior of partial and full agonists in the presence of changing receptor occupancy and/or receptor-effector coupling efficiency (e.g., Ref. 36).

The exact location of the site of sodium sensitivity remains to be determined; however, many sodium-sensitive G protein-coupled receptors possess a conserved aspartate residue in their second membrane-spanning domain (37-39). Mutation of this residue can lead to sodium-insensitive receptors and the perturbation of receptor/G protein interactions (38, 39). Therefore, the site of sodium sensitivity seems to be located on the receptor rather than the G protein. Somatostatin receptors also possess a conserved aspartate residue (amino acid 86 of the human sst₅ receptor), suggestive of a similar sodium-sensitive site. Agonist binding to the murine sst₂ receptor has been shown to be sensitive to sodium ions. whereas mutation of Asp89 to Asn89 rendered the receptor insensitive to sodium, but surprisingly it retained its sensitivity to pertussis toxin and GTPS (40). Further studies are needed to investigate the effects of sodium ions on the function of the different somatostatin receptor subtypes and the precise mechanism(s) involved.

In conclusion, we measured [³⁵S]GTPS binding to G proteins to directly study the activation of the recombinant human sst₅ receptor when stably expressed in CHO-K1 cells. The endogenous ligand somatostatin-14 is capable of stimulating human sst₅ receptor-mediated [³⁵S]GTPS binding in a concentration-dependent and pertussis toxin-sensitive manner. We also observed the negative influence of sodium on ligand potency and intrinsic activity such that the potencies of the agonists somatostatin-14, somatostatin-28, and BIM-23027 were increased as the sodium concentration decreased, whereas L-362,855, a partial agonist, exhibited full agonist activity when the sodium concentration was lowered. The modulation of intrinsic activity in this system by sodium chloride seems to provide a robust system in vitro to discriminate between full and partial agonists at human recombinant receptors, thereby generating information of potential clinical importance. However, the quantitative relationship of such data with that in more intact functional systems requires further study.