Simultaneous remediation of nutrients from liquid anaerobic digestate and municipal wastewater by the microalga Scenedesmus sp. AMDD grown in continuous chemostats.

AIMS: The primary aim of this study was to investigate the capacity of a microalga, Scenedesmus sp. AMDD, to remediate nutrients from municipal wastewater, either as the sole nutrient source or after blending with wastewater obtained from the anaerobic digestion of swine manure. A complimentary aim was to study and define the effects of these wastewaters on microalgal growth, biomass productivity and composition which have important implications for a commercial biofuels production system. METHODS AND RESULTS: A microalga, Scenedesmus sp. AMDD, was grown in continuous chemostats in municipal wastewater or wastewater supplemented with 1·6× or 2·4× higher levels of nitrogen (N) obtained through supplementation with anaerobic digestates. Biomass productivity increased with increasing nutrient supplementation, but was limited by light at high cell densities. Cellular quotas of carbon (C), nitrogen and phosphorus (P) all increased in direct proportion to their concentrations in the combined wastewaters. At higher cell densities, total carbohydrate decreased while protein increased. Fatty acid content remained relatively constant. Under high nutrient levels, the fatty acid profiles contained a higher concentration of polyunsaturated fatty acids at the expense of monounsaturated fatty acids. Chlorophyll a was 2·5 times greater in the treatment of greatest nutrient supplementation compared to the treatment with the least. Ammonium (NH4(+)) and phosphate (PO4(3-)) were completely removed by algal growth in all treatments and with maximal removal rates of 41·2 mg N l(-1) d(-1) and 6·7 mg P l(-1) d(-1) observed in wastewater amended with 2·4× higher N level. SIGNIFICANCE AND IMPACT OF THE STUDY: The study is the first to report stable, long-term continuous algal growth and productivity obtained by combining wastewaters of different sources. The study is supported by detailed analyses of the composition of the cultivated biomass and links composition to the nutrient and light availabilities in the cultures. Simultaneous remediation of these wastes by algal growth is discussed as a strategy for the valorization of the biomass.

Arylpropanolamines: selective beta3 agonists arising from strategies to mitigate phase I metabolic transformations.

Utilization of N-substituted-4-hydroxy-3-methylsulfonanilidoethanolamines 1 as selective beta(3) agonists is complicated by their propensity to undergo metabolic oxidative N-dealkylation, generating 0.01-2% of a very potent alpha(1) adrenergic agonist 2. A summary of the SAR for this hepatic microsomal conversion precedes presentation of strategies to maintain the advantages of chemotype 1 while mitigating the consequences of N-dealkylation. This effort led to the identification of 4-hydroxy-3-methylsulfonanilidopropanolamines 15 for which the SAR for the unique stereochemical requirements for binding to the beta adrenergic receptors culminated in the identification of the potent, selective beta(3) agonist 15f.

BMS-201620: a selective beta 3 agonist.

A series of N-(4-hydroxy-3-methylsulfonanilidoethanol)arylglycinamides were prepared and evaluated for their human beta3 adrenergic receptor agonist activity. SAR studies led to the identification of BMS-201620 (39), a potent beta3 full agonist (Ki = 93 nM, 93% activation). Based on its favorable safety profile, BMS-201620 was chosen for clinical evaluation.

Beta 3 agonists. Part 1: evolution from inception to BMS-194449.

Screening of the BMS collection identified 4-hydroxy-3-methylsulfonanilidoethanolamines as full beta 3 agonists. Substitution of the ethanolamine nitrogen with a benzyl group bearing a para hydrogen bond acceptor promoted beta(3) selectivity. SAR elucidation established that highly selective beta(3) agonists were generated upon substitution of C(alpha) with either benzyl to form (R)-1,2-diarylethylamines or with aryl to generate 1,1-diarylmethylamines. This latter subset yielded a clinical candidate, BMS-194449 (35).(1)

BMS-196085: a potent and selective full agonist of the human beta(3) adrenergic receptor.

A series of 4-hydroxy-3-methylsulfonanilido-1,2-diarylethylamines were prepared and evaluated for their human beta(3) adrenergic receptor agonist activity. SAR studies led to the identification of BMS-196085 (25), a potent beta(3) full agonist (K(i)=21 nM, 95% activation) with partial agonist (45%) activity at the beta(1) receptor. Based on its desirable in vitro and in vivo properties, BMS-196085 was chosen for clinical evaluation.

Molecular characterization, pharmacological properties and chromosomal localization of the human GALR2 galanin receptor.

The neuropeptide galanin mediates a diverse spectrum of biological activities by interacting with specific G protein-coupled receptors. We have used homology genomic library screening and polymerase chain reaction (PCR) techniques to isolate both genomic and cDNA clones encoding the human homolog of the recently cloned rat GALR2 galanin receptor. By fluorescence in situ hybridization, the gene encoding human GALR2 (GALNR2) has been localized to chromosome 17q25.3. The two coding exons of the human GALNR2 gene, interrupted by an intron positioned at the end of transmembrane domain III, encode a 387 amino acid G protein-coupled receptor with 87% overall amino acid identity with rat GALR2. In HEK-293 cells stably expressing human GALR2, binding of [125I]porcine galanin is saturable and can be displaced by galanin, amino-terminal galanin fragments and chimeric galanin peptides but not by carboxy-terminal galanin fragments. In HEK-293 cells, human GALR2 couples both to Galphaq/11 to stimulate phospholipase C and increase intracellular calcium levels and to Galphai/o to inhibit forskolin-stimulated intracellular cAMP accumulation. A wide tissue distribution is observed by reverse transcriptase (RT)-PCR analysis, with human GALR2 mRNA being detected in many areas of the human central nervous system as well as in peripheral tissues.

Identification of [3H]P1075 binding sites and P1075-activated K+ currents in ovine choroid plexus cells.

This study examined the pharmacological characteristics of binding sites for the potent K+ channel opener [3H]P1075, as well as the functional effects of P1075 on ionic currents and membrane potential, in ovine choroid plexus (OCP) cells. [3H]P1075 bound to OCP cells with a Kd of 26 +/- 4 nM and a Bmax of 10400 +/- 480 sites/cell. Labelled sites were stereoselective and inhibited by potassium channel openers with a rank order of potency: P1075 > BMS-182264, ((4-[[9cyanoimino)[(1,2,2-trimethylpropyl)amino]-methyl]amino]benz onitrile) > pinacidil >> nicorandil > diazoxide. The K(ATP) channel antagonist glyburide inhibited [3H]P1075 binding with a Ki of 2 microM. The presence of K(ATP) channels on OCP cells was examined by patch clamp and fluorescent (membrane-potential sensitive dye) techniques. In some cells, P1075 activated an outward potassium current which was blocked by glyburide. P1075 produced a glyburide-sensitive, concentration-dependent, hyperpolarization of OCP cells. Levcromakalim hyperpolarized more strongly than its 3R,4S enantiomer, BRL 38226 ((3R-trans)-3,4-dihydro-3-hydroxy-2,2-dimethyl-4-(2-oxo-1-pyrrolidinyl)- 2H-1-benzopyran-6-carbonitrile) indicating a stereoselective interaction. These data indicate that epithelial OCP cells contain glyburide-sensitive K(ATP) channels.

Nucleotide regulation and characteristics of potassium channel opener binding to skeletal muscle membranes.

[3H]P1075 binding to membrane preparations of rabbit skeletal muscle were observed in the presence of nucleotide triphosphates or diphosphates but not AMP, cAMP, adenosine, tripolyphosphate, or pyrophosphate. Nonhydrolyzable or poorly hydrolyzable ATP analogs inhibited MgATP-supported binding. The EC50 value for MgATP-supported binding (0.4 mM) was decreased approximately 10-fold in the presence of an ATP-regenerating system, and significant metabolism by membrane nucleotidases was confirmed by high performance liquid chromatographic analysis. [3H]P1075 bound to skeletal muscle with a Kd value of 37 +/- 3 nM and a Bmax value of 280 +/- 14 fmol/mg of protein. [3H]P1075 binding to subcellular fractions was highest in membranes enriched in T tubules. Specific binding was reversible, trypsin-sensitive, maximal at pH 8, and stereoselective for the (3S,4R)-enantiomer of cromakalim. Potassium channel openers exhibited a rank order of potency of P1075 > pinacidil > levcromakalim = BMS-180448 > nicorandil > diazoxide = BRL 38226. Fluorescein analogs (ethyleosin, phloxine B, and rose bengal) were relatively potent inhibitors of binding (Ki = 200-300 nM). The potassium channel openers cromakalim and BMS-180448 were competitive inhibitors of [3H]P1075 binding. In contrast, rose bengal and the ATP-regulated potassium channel antagonist glyburide increased the rate of [3H]P1075 dissociation in a manner consistent with noncompetitive interaction.

Beta 3-adrenergic receptor-mediated lipolysis and oxygen consumption in brown adipocytes from cynomolgus monkeys.

Primary adipocytes were isolated from axillary brown adipose tissue from adult cynomolgus monkeys. That this tissue contained brown adipocytes was verified by morphological examination and by demonstrating the presence of uncoupling protein messenger ribonucleic acid in the isolated adipocytes. The contributions of beta 1-, beta 2-, and beta 3-adrenergic receptors (AR) to lipolysis and oxygen consumption of isolated brown adipocytes were determined after agonist stimulation. Dose responses were determined using isoproterenol (a nonselective beta-AR agonist), denopamine (beta 1-AR agonist), procaterol (beta 2-AR agonist), and CGP12177A (beta 1- and beta 2-AR antagonist, beta 3-AR agonist). Isoproterenol, denopamine, and procaterol stimulated lipolysis with EC50 values of 4,500, and 83 nmol/L, respectively. Intrinsic activities (relative to isoproterenol maxima) were 100%, 74%, and 59%, respectively. The presence of beta 3-ARs coupled to lipolysis was demonstrated by the activity of CGP12177A (EC50 = 1.6 mumol/L; intrinsic activity = 62%). Isoproterenol stimulated oxygen consumption of brown adipocytes by 75-100% above the basal rate, with an EC50 of 1 mumol/L. Denopamine, procaterol, and CGP12177A stimulated oxygen consumption at a concentration of 100 mumol/L. These results demonstrate that all three beta-adrenergic receptor subtypes are coupled to lipolysis and oxygen consumption in brown adipocytes from cynomolgus monkeys.

Mutational analysis of the endothelin type A receptor (ETA): interactions and model of selective ETA antagonist BMS-182874 with putative ETA receptor binding cavity.

Endothelin (ET) receptor antagonism is a potential therapeutic intervention in the treatment of vascular diseases. To elucidate the mechanism of antagonist-ET receptor complex formation, the interactions of four chemically distinct antagonists were investigated using a combination of genetic and biochemical approaches. By site-specific mutagenesis we previously demonstrated that Tyr129 in the second transmembrane domain was critical for high-affinity, subtype-selective binding to the A subtype of ET (ETA) receptors [Krystek et al. (1994) J. Biol. Chem. 269, 12383-12386]. Affinities of the constrained cyclic pentapeptide BQ-123, the pyrimidinylbenzenesulfonamide bosentan, the indancarboxlic acid SB 209670, and the naphthalenesulfonamide BMS-182874 were decreased 20-1000-fold in Tyr129Ala, Tyr129Ser, and Tyr129His ETA receptor mutants. Substitution of Tyr129 with Phe or Trp did not alter the high-affinity binding of BQ-123, bosentan, or SB 209670. BMS-182874 binding affinity was decreased 10-fold in Tyr129Phe and Tyr129trp ET receptors. These data indicate a role of aromatic interactions in the binding of these antagonists to ETA receptors an, in the case of BMS-182874, also suggested a hydrogen bond with the tyrosine hydroxyl. This hypothesis was supported by structure-activity data with analogs of BMS-182874 that varied the C-5 dimethylamino substituent on the naphthalene ring. Mutation of Asp126 and Asp133 also altered binding of BMS-182874 and C-5 analogs. In all cases, naphthalenesulfonamide binding was more severely affected by mutation of Asp133 than by mutation of Asp126. Phosphoinositide hydrolysis and extracellular acidification rate studies demonstrated the importance of Tyr129 to ETA-mediated signal transduction. On the basis of these data, two plausible models of the docked conformation of BMS-182874 in the ETA receptor are proposed as a starting point for further delineation of interactions that underlie antagonist-ETA receptor complex formation.

BMS-180560, an insurmountable inhibitor of angiotensin II-stimulated responses: comparison with losartan and EXP3174.

1. This study compares the activity of BMS-180560 (2-butyl-1-chloro-1-[[1-[2-(2H-tetrazol-5-yl)phenyl]-1H-indol-4- yl]methyl]-1H-imidazole-5-carboxylic acid), an insurmountable angiotensin II (AII) receptor antagonist, with that of losartan and EXP3174 in functional and biochemical models of AII-receptor activation. 2. BMS-180560 selectively inhibited [125I]-Sar1Ile8AII ([125I]SI-AII) binding to rat aortic smooth muscle (RASM) cell and rat adrenal cortical AT1 receptors (Ki = 7.6 +/- 1.2 and 18.4 +/- 3.9 nM respectively) compared to adrenal cortical AT2 receptors (Ki = 37.6 +/- 1.3 microM). The Ki values of BMS-180560 and EXP3174, but not losartan, varied as a function of the BSA concentration used in the assays, indicating that the diacid drugs bound to albumin. 3. BMS-180560 (3-300 nM) increased the KD of SI-AII for RASM cell AT1 receptors. Only at high concentrations of BMS-180560 (300 nM) were Bmax values decreased. 4. BMS-180560 inhibited AII-stimulated contraction of rabbit aorta with a calculated KB = 0.068 +/- 0.048 nM and decreased maximal AII-stimulated contraction at 1 nM BMS-180560 by 75%. In the presence of 0.1% BSA, a higher KB value (5.2 +/- 0.92 nM) was obtained. Losartan behaved as a competitive antagonist with a KB = 2.6 +/- 0.13 nM. Contraction stimulated by endothelin-1, noradrenaline, KCl, or the TXA2 receptor agonist U-46619 were unaffected by BMS-180560 (1 nM). 5. AII stimulated the acidification rates of RASM cells as measured by a Cytosensor microphysiometer with an EC50 of 18 nM. Losartan (30 nM) shifted the AII concentration-effect curves in a competitive manner whereas BMS-180560 (0.01 and 0.1 nM) decreased the maximum responses by 60 and 75% respectively. Inhibition by losartan and BMS-180560 could be reversed following washout although recovery took longer for BMS-180560. 6. In [3H]-myoinositol-labelled RASM cells, losartan (30 and 200 nM), shifted the EC50 for AII-stimulated [3H]-inositol monophosphaste formation to higher values, with no change in the maximal response. By contrast, EXP3174 (0.1 to 1 nM) decreased the maximal response in a concentration-dependent manner (17-55%). BMS-180560 (3 and 10 nM) increased the EC50 for AII and decreased the maximum response by 30 and 80% respectively. The inhibition by EXP3174 and BMS-180560 could be reversed by inclusion of losartan (200 nM) indicating that the inhibition was not irreversible. 7. In conclusion, BMS-180560 is a potent, specific, predominantly competitive, reversible All receptor antagonist, which displays insurmountable receptor antagonism. At concentrations of BMS-180560 which have no effect on receptor number, BMS-180560 produced insurmountable antagonism of AII-stimulated second messenger formation, extracellular acidification, and smooth muscle contraction.

Quinoxaline N-oxide containing potent angiotensin II receptor antagonists: synthesis, biological properties, and structure-activity relationships.

A series of novel quinoxaline heterocycle containing angiotensin II receptor antagonist analogs were prepared. This heterocycle was coupled to the biphenyl moiety via an oxygen atom linker instead of a carbon atom. Many of these analogs exhibit very potent activity and long duration of effect. Interestingly, the N-oxide quinoxaline analog was more potent than the nonoxidized quinoxaline as in the comparison of compounds 5 vs 30. In order to improve oral activity, the carboxylic acid function of these compounds was converted to the double ester. This change did result in an improvement in oral activity as represented by compound 44.

Peptidases and smooth muscle cell angiotensin II receptor pharmacology.

Angiotensin (A) II receptors on rat aortic smooth muscle (RASM) cell membranes were characterized using the radioligand [125I][Sar1Ile8]AII ([125I]SI-AII). Angiotensin I, AII, and AIII inhibited specific [125I]SI-AII binding, and their rank order of potencies, and Ki values (nM) were: AII (3.7) > AI (32.5) > or = AIII (54.0), which differed from that observed for rat adrenal cortex: AII (0.85) > AIII (3.3) >> AI (100). Similar results were observed for RASM membranes in the presence of guanine nucleotides, and for intact cells in the absence or presence of an internalization inhibitor. Lowering the incubation temperature from 37 degrees C to 4 degrees C, or inclusion of PMSF (1 mM), and preparing membranes in the presence of EGTA (1 mM) altered the rank order of potencies and Ki values (nM) of the angiotensin peptides to: AII (1.1) > AIII (7.0) >> AI (144). [125I]Angiotensin I was metabolized completely over the course of 90 min to small (

Binding and signal transduction of the cloned vascular angiotensin II (AT1a) receptor cDNA stably expressed in Chinese hamster ovary cells.

The vascular angiotensin (A) II receptor cDNA (AT1a) was transfected into Chinese hamster ovary (CHO) cells to generate the stable cell line CHO-AT1a. This cell line was used to investigate the binding and signal transduction properties of the cloned vascular AT1 receptor. Specific binding of sarcosine1(-)[125I]tyrosine4-isoleucine8-AII ([125I]SI-AII) to CHO-AT1a membranes reached equilibrium after 1 h at 25 degrees C and was consistently greater than 95% of total binding. Saturation binding analyses demonstrated [125I]SI-AII bound to a saturable population of sites on membranes with an equilibrium dissociation constant (KD) of 0.7 nM and a binding site maximum of 1.2 pmol/mg protein. [125I]SI-AII binding to CHO cells was inhibited by the following compounds with a rank order of potency of SI-AII > AII > losartan > AI >> PD 123,177. AII (1 microM) treatment of CHO-AT1a cells caused an increase in inositol phosphates and intracellular calcium relative to basal levels. These responses were blocked by losartan but not by PD 123,177. AII (1 microM) did not effect adenylate cyclase activity in CHO-AT1a cells, whereas the agonist inhibited adenylate cyclase activity in rat liver cell membranes. These effects were blocked by 10 microM losartan. These results indicate that CHO-AT1a cells express functional AT1a receptors which stimulate phospholipase C activity but not adenylate cyclase activity. CHO-AT1a cells should provide a useful model for studies of AT1a receptor domains which are critical to signaling pathways.

Dihydropyrimidine angiotensin II receptor antagonists.

The discovery of the nonpeptide angiotensin II (AII) receptor antagonist losartan, previously called DuP 753, has stimulated considerable interest in the synthesis of novel analogs of this compound. Our efforts in this area have resulted in the discovery of dihydropyrimidines as potent AII receptor antagonists. The chemistry leading to this novel class of AII antagonists and their biological properties are reported in this publication. Structure-activity studies showed that a variety of substituents are tolerated on the dihydropyrimidine ring, indicating that the AII receptor is permissive in accepting this region of the nonpeptide antagonists. As reported for imidazole-based AII antagonists, the tetrazolyl dihydropyrimidine analogs were found to be more potent than the corresponding carboxylic acids. Our studies show that dihydropyrimidine analogs 2-butyl-4-chloro-1,6-dihydro-6-methyl-1-[[2'-(1H-tetrazol-5-yl)[1, 1'-biphenyl]-4-yl]methyl]pyrimidine-5-carboxylic acid, ethyl ester (Ki = 8.3 nM), 2-butyl-4-chloro-1,6-dihydro-6-methyl-1- [[2'-(1H-tetrazol-5-yl)[1,1'-biphenyl]-4-yl]methyl]-5- pyrimidinecarboxylic acid (Ki = 1.0 nM), and 2-butyl-6-chloro-1,4-dihydro-4,4-dimethyl-1-[[2'-(1H-tetrazol-5-yl )[1,1'- biphenyl]-4-yl]methyl]-5-pyrimidinecarboxylic acid, ethyl ester (Ki = 1.1 nM), display affinities for the AII receptor which are comparable to or better than losartan (Ki = 9.0 nM). One of these derivatives, 2-butyl-4-chloro-1,6-dihydro-6-methyl-1-[[2'-(1H-tetrazol-5- yl)[1,1'-biphenyl]-4-yl]methyl]pyrimidine-5-carboxylic acid, ethyl ester, showed antihypertensive activity on oral administration to spontaneously hypertensive rats. These results demonstrate that the imidazole of losartan can be successfully replaced with a dihydropyrimidine ring.

The endothelin receptor antagonist, BQ-123, inhibits angiotensin II-induced contractions in rabbit aorta.

The purpose of this study was to examine the specificity of the cyclic pentapeptide ET(A) receptor antagonist BQ-123. BQ-123 competitively antagonized endothelin-1-induced contractions in rabbit aorta, increases in inositol phosphates in cultured rat vascular smooth muscle A10 cells, and binding of [125I]endothelin-1 to the cloned ETA receptor cDNA expressed in Cos 7 cells. In contrast, BQ-123 was a weak antagonist of [125I]endothelin-3 binding to rat cerebellar membranes and to membranes from Cos 7 cells transfected with the cloned ETB receptor cDNA. BQ-123 shifted concentration-response curves in isolated rabbit aorta elicited by angiotensin II, but did not bind to angiotensin II receptors nor affect angiotensin II-induced increases in inositol phosphates. BQ-123 also did not affect contractions induced by KCl or norepinephrine. These data suggest that endothelin may play a role in angiotensin II-induced contractions of rabbit aorta.

Activation of T84 cell chloride channels by carbachol involves a phosphoinositide-coupled muscarinic M3 receptor.

Muscarinic agonists stimulate Cl- secretion across monolayers of the colon tumor epithelial cell line, T84. The muscarinic receptor has been characterized in T84 cell homogenates by radioligand binding using [3H]N-methylscopolamine ([3H]NMS). [3H]NMS bound to a single population of sites at 25 degrees C in 100 mM NaCl, 20 mM HEPES, 10 mM MgCl2, pH 7.4 buffer, with calculated Kd = 278 (+/- 44) pM and Bmax = 40 (+/- 6) fmol/mg protein (n = 4). Binding was reversible (diss. t1/2 = 18 +/- 3 min) and stereoselective (dexetimide Ki = 0.3 nM) much greater than levetimide (Ki = 8300 nM). Antagonists exhibited the following rank order of potencies and Ki values (nM): atropine (0.54) greater than 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP) (0.84) greater than dicyclomine (14) = hexahydrosiladifenidol (18) greater than pirenzepine (136) greater than AF-DX 116 (3610). The same sequence was observed for inhibition of carbachol-induced 125I efflux from T84 monolayers. This is indicative of an M3 'glandular' muscarinic receptor. Coupling to second messenger systems was examined by labelling monolayers with [14C]arachidonic acid (AA) or [3H]inositol. Carbachol (0.3 mM) did not release [14C]AA from labelled lipids, but ionomycin produced a dose-dependent increase in media [14C]AA. Carbachol (0.3 mM) elevated inositol monophosphate 14-fold. The results suggest that muscarinic agonists stimulate Cl- secretion by interacting with an M3 receptor coupled to inositide lipid hydrolysis.

Bombesin, neuromedin B and neuromedin C interact with a common rat pancreatic phosphoinositide-coupled receptor, but are differentially regulated by guanine nucleotides.

Bombesin (BB), neuromedin C (NMC) and neuromedin B (NMB) stimulated amylase secretion to similar maximum levels, with EC50 values (concentrations causing 50% of maximum effect) of 0.2, 0.3 and 2 nM respectively. Treatment of pancreatic acini with BB or NMB (10 nM) for 30 min resulted in cross-desensitization of secretory responses to subsequent BB and NMB, but not to acetylcholine, which suggests that NMB and BB activate the same receptor. BB, NMC and NMB stimulated production of similar maximum amounts of inositol mono-, bis- and tris-phosphates, with EC50 values of 3, 5 and 141 nM respectively. The bombesin receptor antagonist [Leu13-psi(CH2NH)Leu14]BB inhibited stimulation of amylase secretion and inositol phosphate formation by BB, NMC and NMB. Binding of 125I-labelled gastrin-releasing peptide (GRP; 200 pM) to rat pancreatic membranes at 22 degrees C was inhibited with relative potencies and IC50 (concn. causing 50% of maximal inhibition; nM) as follows: NMC (0.4) = BB (0.5) greater than NMB (1.8 = GRP (2.6). IC50 values for BB, NMC and NMB inhibition of 125I-GRP binding to intact acini were 5-, 19- and 68-fold higher than their respective values in membranes. The guanine nucleotide analogue guanosine 5'-[beta gamma-imido]triphosphate (Gpp[NH]p) produced rightward shifts of NMC and NMB competition curves by 3.5- and 16-fold respectively, but had little effect on the BB and GRP curves. Elevation of the temperature to 37 degrees C or inclusion of NaCl (40 mM) produced quantitatively similar effects to those of Gpp[NH]p. In the presence of both NaCl and Gpp[NH]p the affinities of peptides for membrane receptors were similar to those for intact cells. Modulation of NMB competition curves by Gpp[NH]p was not attenuated by prior treatment of acini with activated pertussis toxin. These results suggest that BB, NMB and NMC stimulate pancreatic secretion by interaction with a common phosphoinositide-linked receptor. Differences in guanine nucleotide regulation suggest that secretagogue-induced receptor-protein interactions may not be identical for NMB and BB.