Quantitative structure–activity relationship (QSAR) study of elastase substrates and inhibitors

One hundred Suc-X-Y-Ala-pNA peptides (SUC: succinyl, pNA: p-nitroanilide, X, Y: Gly, Ala, Val, Leu, Ile, Phe, Pro, x-aminobutyric acid, norvaline, norleucine) were synthesized and their reaction constants with porcine pancreatic elastase (K_m, K_cat and K_cat/K_m) were determined. These reaction constants were quantitatively analyzed using the Free–Wilson/Fujita–Ban method. The contribution of amino acid side chains to the reaction constants K_m, K_cat and K_cat/K_m), expressed logarithmically, was found to be additive. On the other hand, 19 elastase inhibitors of the general formula CF₃CO-X-Y-Ala-pNA (X,Y: ten amino acids) were synthesized, and their inhibition constants were compared with the Michaelis constant for the corresponding substrates and analyzed using free-energy-related substituent constants. In the analysis of amino acid side chains in the Y position, the K_i value of the inhibitor was generally correlated to the K_m value of the substrate, which corresponded to the inhibitor, thus confirming the validity of the equation This study may serve as a prototypical approach to unraveling structure–activity relationships of peptide substrates and inhibitors of medicinal or agricultural importance.     

Hydrophobic Contribution Constants of Amino Acid Residues to the Hydrophobicities of Oligopeptides

Purpose. The main purpose of this study is to explore the additive-constitutive nature of common amino acids in their contribution to the partition coefficients of small peptides. Methods. The Log P values and other physico-chemical parameters of the peptides studied are taken from the literature. The frequency of appearance (n_i) of each individual amino acid is calculated as the number of the amino acids in a given peptide. Results. The partition coefficients (Log P_(oct./buff.) at PH 7) of 87 N-acetyl-peptide-amides have been correlated with the frequency of appearance of amino acids. From the correlation obtained, the de novo hydrophobic contribution constants of 19 amino acid residues are derived for the first time. The contribution constants are extended to 59 unmodified regular peptides with the inclusion of the pk_a values of both N-terminal and C-terminal amino acids. The models thus obtained have been validated with additional 27 peptides (both N-acetyl-peptide-amides and unmodified). Conclusions. The Log P of oligopeptides is very well correlated with the de novo hydrophobic contribution constants of amino acids. The models we have derived are reasonably accurate in predicting the hydrophobicities of new oligopeptides (tetrapeptides) at a fixed pH (e.g., 7).     

Specific binding of 3H-adenosine to rat brain membranes

Summary The binding of ³H-adenosine to rat brain membranes was studied by a microcentrifugation technique. Specific binding of ³H-adenosine was rapid, reversible, saturable and dependent on pH and temperature. Scatchard plots of equilibrium binding data were nonlinear suggesting the existence of two different binding sites for adenosine. The dissociation constants (K _d) were 1.7 M and 13.6 M and the maximal number of binding sites (B _max) 31 and 165 pmol adenosine bound per mg of membrane protein. Ten adenosine derivatives were studied for their ability to compete with ³H-adenosine binding. The phosphorylated adenosine compounds 5-AMP, cyclic AMP and ATP were most potent in displacing ³H-adenosine from its binding sites and the IC₅₀-values ranged from 11–25 M. N⁶-Phenylisopropyladenosine produced only partial inhibition (30%) of ³H-adenosine binding and no stereospecific difference between the (–)-and (+)isomer was observed. Several methylxanthines known as adenosine antagonists competed for the ³H-adenosine binding sites parallel with their pharmacological potency. The results offer a first approach for the study of adenosine binding sites in brain membranes.     

Studies in the cyclobutanedicarboxylic acid field

Some dialkylaminoalkyl esters of-truxillic acid and their bis-quaternary salts have been synthesized, and their properties and pharmacological activity have been studied.Translated from Khimiko-Farmatsevticheskii Zhurnal, No. 4, pp. 3–8, April, 1967.     

Design, synthesis and cytotoxicity of a new series of isoxazolidine based nucleoside analogues

5‐Arylisoxazolidin‐3‐yl‐3‐diethoxyphosphonates have been synthesized from N‐methyl‐C‐diethoxyphosphorylnitrone and vinyl aryls in good yields and their transformation into the respective phosphonic acids has been accomplished via dealkylation procedure using trimethylsilyl bromide. Phosphonates having 1‐ and 2‐naphthyl substituents at C5 in the isoxazolidine ring as well as the respective phosphonic acids have been found cytotoxic to HeLa and K562 cells with IC₅₀ in the 0.1–0.3 mM range. Preliminary studies on mechanism of action imply that intercalation to DNA is not responsible for their cytotoxic properties.     

Kinetics of drug binding to human serum albumin: allosteric and competitive inhibition at the benzodiazepine binding site by free fatty acids of various chain lengths

Summary The inhibition of dansylsarcosine (DS) binding at the benzodiazepine binding site of human serum albumin has been studied in the presence of saturated and unsaturated free fatty acids (FFA) of various chain lengths (C6–C20, C18:1, C18:2). In order to determine the mechanism of displacement, velocity constants for association (k ₂) and dissociation (k _–2) and binding constants (K _A and K _A) have been measured using the stopped-flow method.The inhibitory effect of FFA on DS binding kinetics at site II is dependent of their structure. With increasing amounts of FFA the association velocity constant of DS binding decreases from 520 s^–1 (fatty acid free albumin) by a factor of 3–10 and affinity decreases according to FFA chain length. Inhibition is strongest in the presence of caprylic, capric and lauric acid (C8–C12) i.e. with more than one mole FFA per mole albumin, DS association could no longer be measured. Short chain caproic and the long chain FFA C14–C20 showed only a less inhibitory effect since in the presence of a twofold excess k ₂ ranged between 100 and 200 s^–1. Dissociation velocity of DS from the benzodiazepine binding site could be measured in relationship to FFA chain length using ibuprofene, another drug binding at site II. Dissociation velocity constants k _–2 remained constant up to 2 moles FFA per mole albumin (k _–2 = 16–18 s^–1). A rise in k _–2 to 70 s^–1 was seen, however, when 2–4 moles capric, lauric, myristic and palmitic (C10–C16) acid were bound, whereas no change was observed when increasing concentrations of caproic, caprylic, stearic and arachic acid. The more compact unsaturated FFA oleic and linoleic acids did not inhibit DS binding to the same extent as their saturated homologue stearic acid. k ₂ and k _–2 values resembled those when comparable amounts of myristic acid were bound indicating that chain length is the relevant parameter.It can be concluded that two different types of FFA inhibition of binding at the benzodiazepine binding site exist. Short-chain FFA (C6–C8) may be specifically bound at binding site II thereby competitively displacing DS. Longchain FFA (C12–C20), however, occupy their binding sites far from the benzodiazepine binding site. Inhibition occurs via an allosteric mechanism. Capric acid is unique in showing as well as competitive as well as allosteric inhibition characteristics.Abbreviations k ₂ Dissociation velocity constant - k ₂ association velocity constant - K _A affinity constant - K _A affinity constant of the intermediate complex - DS dansylsarcosine - FFA free fatty acids - HSA human serum albumin This work has been presented in part at the Spring Meeting of the German Pharmacological Society in Mainz, March 1988 (Menke et al. 1988) Send offprint request to N. Rietbrock at the above address     

An Improved Method for Determination of Acid Dissociation Constants of Peptides

Purpose. The method described here enables the proton dissociation constants of several amino acid residues of a peptide to be determined simultaneously in aqueous solution without prior knowledge of the exact concentration of the peptide. Methods. The method used here employs a non-linear fitting program, the BEST program, or a linear least-squares method in combination with the BEST program. These methods are discussed in detail with an emphasis on the quality of the potentiometric titration data that are obtained. Two representative peptides, one with two proton dissociation constants (K_al, K_a2) and the other with four proton dissociation constants (K_al–K_a4) were used to illustrate the advantages and the limitations of these two complementary methods. Results. The pK _a values of TVL, a schizophrenia-related tripeptide, were found to be 3.62 (±0.02) and 7.17 (±0.05); the pK _a values of ELTLQE, a hexapeptide, were found to be 2.32, 3.77,4.58 and 7.74. Conclusions. The methods reported here are generally applicable to a variety of peptides. The possibility of integrating these procedures into a preparative chromatographic system for the on-line assessment of the pKa values of peptides during the purification stage is an attractive and novel feature of this method.     

Calculation of the gastric absorption rate constants of 5-substituted barbiturates through theR m values or substituent ΔR m constants in reversed-phase partition chromatography

A linear correlation between the logarithm of the in situgastric absorption rate constants (k_a)of 5-substituted barbiturates and their R_m values in selected reversed-phase partition chromatographic systems is demonstrated, as well as between the log k_a and the R_m derived parameters. On the basis of the chromatographic behavior of 14 closely related compounds of this series, the substituent R_m constants for the main functional groups are calculated, so that the gastric absorption constants of nontested barbiturates can be predicted with close approximation. Predicted R_m and log k_a values show an excellent correlation with log 1/C values taken from pharmacological literature data. The migration mechanisms involved in reversed-phase partition chromatography are discussed in connection with the results obtained with other types of partition systems and with bulk-phase solvents. The possible relationships between chromatographic parameters and absorption rate constants found from absorption sites other than the stomach are outlined; as well as the advantages and limitations of the procedure.     

Kinetics of antagonism at histamine-H1 receptors in isolated rabbit arteries

Summary Kinetics of antagonist-induced decrease of histamine-H₁ receptor-mediated steady-state responses in isolated rabbit arteries were studied in the presence of histamine-H₂ receptor antagonist famotidine. Data were fitted using a model which describes competition kinetics at the receptor level. Estimated rate and equilibrium constants were evaluated for their dependence on tissue, agonist and antagonist concentrations, using (+)-brompheniramine as antagonist. In large arteries (thoracic and arcus aorta), rate constants were observed to be modified by agonist and/or antagonist concentrations, suggesting a diffusion-controlled process. In relatively small (common carotid and iliac) arteries, estimated equilibrium constants (and consequently the rate constants) were found to diverge despite the invariance of equilibration times between arteries, leading us to include the effects of spare receptors in our evaluation. A model describing the effects of receptor reserve on the estimated equilibrium dissociation constant was developed and stimulated and the results then compared with those that had been experimentally estimated. The reserve hypothesis was experimentally verified in common iliac artery (where EC₅₀ K _A) using the irreversible antagonist phenoxybenzamine. A rationalized rule for the optimization of experimental design for in-vitro disequilibrium-competition experiments was proposed. Common carotic artery was found to be favorable for the present design in view of its reserve properties. In addition, competition reaction seems to be the rate-determining step in this artery. Rate and equilibrium constants of mepyramine, (+)-brompheniramine, diphenhydramine and antazoline were therefore determined in the common carotid artery and were compared with those obtained from independent experiments. Results suggest that the estimated parameters reflect drug-receptor interaction.This work constitutes a part of the PhD thesis of H. O. Onaran Send offprint requests to T. A. Bökesoy at the above address     

Electrophysiologic actions of aprindine in rabbit atrioventricular node

Summary Aprindine hydrochloride is a potent antiarrhythmic agent against various atrial and ventricular tachyarrhythmias. To elucidate its pharmacological actions in the atrioventricular node, electrophysiologic experiments were conducted by applying microelectrode and voltage clamp methods to small preparations of the rabbit atrioventricular node.At a concentration 1 mol/l, aprindine decreased the spontaneous firing frequency, maximal rate of depolarization, action potential amplitude, and take-off potential (P < 0.05, n = 7). The spontaneous and rate-controlled action potential durations at 50 and 100% repolarization were prolonged by aprindine. Voltage-clamp experiments using the double microelectrode method revealed that aprindine blocked the slow inward current (I_si) in a voltage-dependent manner with a dissociation constant of 10 mol/l and Hill coefficient of 0.8. The steady-state inactivation curve for I_si was shifted toward more negative potentials by 2.5 ± 0.9 mV (P < 0.05, n = 5) without a significant change in the slope factor. This finding suggests that aprindine has a higher affinity for inactivated slow inward (or Ca²⁺) channels than for resting channels. Aprindine caused use-dependent block of I_si, a result consistent with the drug's slow dissociation from inactivated Ca²⁺ channels. The delayed rectifying K⁺ current (I_K) tail obtained on repolarization from +10 mV to –60 mV was significantly decreased from 15.4 ± 2.4 to 6.8 ± 1.4 nA (P < 0.01, n = 6) and the deactivation time constant significantly increased by 20.7% (P < 0.01, n = 6). The steady-state activation curve for I_K was shifted in the hyperpolarized direction by 6.9 ± 2.9 mV, suggesting a potent voltage-dependent block of this current by aprindine. The hyperpolarization-activated inward current (1_h) was decreased from 14.4 ± 5.4 to 12.0 ± 5.5 nA (P < 0.05, n = 5). The transient outward and inward currents induced by 1 mol/l acetylstrophanthidin were almost completely suppressed after the addition of 1 mol/l aprindine.These results suggest that aprindine exerts a negative chronotropic action both by slowing deactivation of I_K and by reducing Is; and Ih, and delays atrioventricular nodal conduction by reducing Is; and IK. These blocking actions of aprindine together with its inhibition of the transient outward and inward currents may explain its antiarrhythmic effects on the atrioventricular node. Send offprint requests to Yoshio Watanabe at the above address     

Binding characteristics of (+)-, (±)- and (-)-[125Iodo] cyanopindolol to guinea-pig left ventricle membranes

Summary [¹²⁵Iodo]cyanopindolol [(±)-ICYP], a potent and selective ligand for -adrenoceptors, exhibited complex biphasic dissociation kinetics. Consequently, in receptor binding studies, the pure (+)- and (-)-enantiomers of ICYP were synthesised and their equilibrium and kinetic binding characteristics were investigated on a membrane preparation of guinea pig left ventricle containing almost only ₁-adrenoceptors.All three ligands, i. e. (+)-, (-)- and (±)-ICYP, bind to -adrenoceptors as assessed by competition experiments with different -blocking agents; irrespective of the ligand used, the same dissociation constant was found for the competitor. In a first series of saturation binding experiments performed in a low concentration range of free ligand (0–250 pM), ICYP showed the following dissociation constants: K _D=93, 9 and 23 pM, and number of binding sites: B _max=40,128 and 124 fmoles/mg protein for (+)-, (-)-, and (±)-ICYP, respectively. Asexpected, (±)-ICYP showed the same B _max as (-)-ICYP, whereas its K _D was approximately two times higher than that of (-)-ICYP. Surprisingly, the B _max of (+)-ICYP represented only 30% of the B _max of (-)-ICYP. All three ligands bound apparently to a single class of binding sites.In dissociation experiments, the enantiomers of ICYP showed biphasic dissociation curves as observed for the racemic ligand. (+)-, (-)- and (±)-ICYP showed a rapidly dissociating (k _-1=0.488, 0.047 and 0.049 min^–1) and a slowly dissociating component (k _-2=0.0205, 0.0033 and 0.0025 min^–1). The ratio slow dissociating/fast dissociating component represented respectively for (+)-, (-)- and (±)-ICYP 40/60, 90/10 and 90/10. For all three ligands, the association rate constants were of the same order of magnitude (ca. 10⁹ M^–1 min^–1), typical for a diffusion controlled reaction.In contrast to equilibrium binding studies, the existence of multiple receptor affinity sites was evident from the biphasic dissociation behaviour observed especially with the nonracemic ligands (+)-ICYP and (-)-ICYP.Simulation of theoretical saturation curves performed with the ratios of high versus low affinity sites and the K _D-values suggested by kinetic analysis, indicated that the delineation into two affinity states might be visible in saturation experiments, under certain conditions.Therefore, equilibrium binding studies were repeated with an increased number of ligand concentrations covering a large concentration range of 0–800 pM. Simultaneous analysis of saturation curves from the same experiment using three different ligands, provided more accurate estimates of the ratio of high and low affinity sites, as well as the affinity constants of the ligand for each receptor affinity state, in good agreement with the results from kinetic analysis.The contribution of the (+)enantiomer in the binding of the racemic ligand under low receptor concentrations could be neglected since dissociation characteristics of (±)- and (-)-ICYP were identical. A model that explains the biphasic dissociation of (±)-ICYP by differential binding of both enantiomers could be rejected. Kinetic and equilibrium binding characteristics of the three radioligands were not influenced by the guanylnucleotide Gpp(NH)p (10^–4 M).The antagonist ICYP binds to -adrenoceptors in a high and low affinity state which are probably interconvertible.Abbreviations CYP cyanopinodolol - ICYP [¹²⁵Iodo]cyanopindolol - HYP (±)-hydroxybenzylpindolol - IHYP (±)-[¹²⁵Iodo]hydroxybenzylpindolol - ³H-DHA (-)-[³H]dihydroalprenolol - ³H-HBI (±)-[³H]-hydroxybenzylisoproterenol - ISA intrinsic sympathomimetic activity; Gpp(NH)p, guanyl-5yl-imidodiphosphate - FM 24 1-(2-exobicyclo[2,2,1]hept-2yl-phenoxy)-3-[(1-methylethyl)amino]-2-propranolol Part of this work has been presented at the Spring Meeting of the German Pharmacological Society, Mainz, March 10–13, 1981     

阿魏酸衍生物的合成

本文报道3种类型18个阿魏酸衍生物的化学合成:Ⅰ.氨基醇酯类化合物,Ⅱ.5-(烷胺基)一次甲基阿魏酸,Ⅲ.双分子阿魏酸的醚类化合物。药理筛选的初步结果表明:Ⅰ类化合物的体外抗凝作用比阿魏酸本身为强,Ⅱ类化合物作用很弱。     

Synthesis and ganglion-blocking activity of derivatives of 2,2,6,6-tetramethyl-Δ3-dehydropiperidine

Conclusions The synthesis of some aminoalkyl esters and amides of 1,2,2,6,6-pentamethyl- and 2,2,6,6-tetramethyl-³-dehydro-4-piperidylacetic acids and the products of the reduction of these amides have been synthesized, and their ganglion-blocking activity has been studied.Translated from Khimiko-Farmatsevticheskii Zhurnal, No. 11, pp. 29–32, November, 1968.     

Thermodynamics and kinetics of t-butylbicyclophosphorothionate binding differentiate convulsant and depressant barbiturate stereoisomers acting via GABAA ionophores

The temperature dependence of [³⁵S]-t- butylbicyclophosphorothionate (TBPS) binding to the convulsant sites of the GABA_A receptor complex was studied in membrane preparations of rat forebrain. Although specific [³⁵S]TBPS binding was maximal around 20° C, the rate constants of dissociation decreased monotonously between 37°C and 2° C. The displacing potencies of the convulsant S(+) enantiomer of 1-methyl-5-phenyl-5-propyl-barbituric acid (MPPB) (IC₅₀ = 1250 ± 30 M) and the depressant R(–) MPPB (IC_5O = 310 ± 5 M) did not show significant changes between 19° C and 37° C. Therefore barbiturate binding seems to be driven by entropic, rather than enthalpic changes. An excess of MPPB enantiomers elicited accelerated and polyphasic dissociations of [³⁵S]TBPS as compared to the monophasic dissociation by TBPS. Arrhenius analysis was applied to the measurable initial rate constants of dissociation. Arrhenius plots were linear between 2° C and 37° C. Activation parameters were similar when [³⁵S] TBPS dissociation was triggered by the convulsants TBPS and S(+) MPPB. It can be attributed to similar conformations of the closed ionophore complex. In contrast, the depressant R(–) MPPB strongly decreased the activation energy of TBPS dissociation from the open ionophore ternary complex.In whole-cell patch-clamp experiments R(–) MPPB, but not S(+) MPPB, elicited chloride currents in rat primary cortical cultures with an EC₅₀ value of 560 ± 30 M and a Hill coefficient of 2.9 ± 0.2. These currents were similar to those elicited by GABA and blocked by TBPS. A kinetic scheme is proposed for the dissociation of TBPS and to explain the different effects of MPPB enantiomers. Submillimolar R(–) MPPB is supposed to bind to (about three) barbiturate sites on GABA_A-ionophores and to open them in a cooperative manner to result in a decreased activation energy for accelerated displacement of convulsant binding.     

Peptide formation in the presence of metal ion protecting groups

A quantitative study of the degree of racemization induced by the [(NH₃)₅Co-(III)-] protecting group when bound to the C-terminal of the amino acids Leu, Phe, and His, as has been carried out. Racemization was determined by forming the diastereomeric cobalt dipeptides [(Leu)(AA)Co(III)(NH3)₅] where AA = L-Leu, L-Phe, and L-His; after cobalt removal (using NaBH₄), the peptide diastereomers were analyzed quantitatively using an amino acid analyzer. No racemization was observed within experimental error (0.3%) as a result of the substitution of the [(NH3)₅Co(III)-] group on the amino acids and peptides studied.     

Molecular Mobility in Mixtures of Absorbed Water and Solid Poly(vinylpyrrolidone)

Poly(vinylpyrrolidone) (PVP) was used as model system to examine molecular mobility in mixtures of absorbed water with solid amorphous polymers. Water vapor absorption isotherms were determined, along with diffusion and proton NMR relaxation measurements of absorbed water. Concurrently, measurements of glass transition temperatures (T _g) and carbon-13 NMR relaxation times for PVP were determined as a function of water content. Two water contents were used as reference points: W _m, obtained from the fit of water absorption isotherms to the BET equation, corresponding to the first shoulder in the sigmoid isotherm; and W _g, the amount of water necessary to depress T _g to the isotherm temperature. Translational diffusion coefficients of water, along with proton T ₁ relaxation time constants, show that both the translational and the rotational mobility of the water is hindered by the presence of the solid polymer and that the absorbed water is most likely represented by two or more populations of water with different modes or time scales of motion. The presence of "tightly bound or immobilized water at levels corresponding to W _m, however, is unlikely, since water molecules maintain a high degree of mobility, even at the lowest levels of water. Above W _g, water shows an increase in mobility with increasing water content, but it is always less mobile than bulk water. With increasing water content, carbon-13 T ₁ relaxation time constants for PVP, measured under the same conditions as above, indicate a major increase in the molecular mobility of carbon atoms associated with the pyrrolidone side chains.     

Correlation of Physicochemical Parameters to the Hydrophobic Contribution Constants of Amino Acid Residues in Small Peptides

Purpose. This paper attempts to correlate the hydrophobic contribution constants (f _aa) of 21 amino acids in small peptides with commonly used physicochemical parameters. These f _aa constants can then be used to predict hydrophobicity change in peptides when any one of the amino acid residue is substituted with another. Methods. Non-weighted least squares method was used in deriving regression equations with a BMDP program. A Hyperchem program for Windows was used to calculate the group dipole moments of the side chain. Results. A good correlation (r = 0.97) was obtained using a four parameter equation including molecular weight (log MW), hydrogen bond forming ability (HB), dipole moment (µ) and an indicator variable (I) to account for the presence of a free primary amine group in the side chain. Conclusions. This proposed model should be useful in predicting the hydrophobic contribution constants of other uncommon amino acids and in the estimation of log P values of numerous peptides containing different possible combinations of these amino acids, as well as log P values resulting from amino acid substitution as is done in site-directed mutagenesis.     

Dehydro-digitoxosides of digitoxigenin and digoxigenin: Binding to beef heart (Na++K+)-ATPase in relation to unchanged digitoxosides

Summary Dehydro-digitoxosides are metabolites of digitalis glycosides. In order to study their possible biological activity their affinity to (Na⁺+K⁺)-activated ATPase was determined and compared with unchanged glycosides. Based on the dissociation constants of glycoside-enzyme-complexes, the affinity of the dehydro-digitoxosides ranged in the same order of magnitude as that of the native glycosides. Comparing mono-, bis-, and tris-digitoxosides of digitoxigenin (dt-1, dt-2, dt-3) and of digoxin (dg-1, dg-2, dg-3) with the corresponding dehydrodigitoxosides (3-dehydro-dt-1, 9-dehydro-dt-2, 15-dehydro-dt-3, 3-dehydro-dg-1 and 9-dehydro-dg-2, respectively) the dehydro-digitoxosides had lower affinities to the enzyme. The highest dissociation constants (K _D)were found for 3-dehydro-dt-1 and 3-dehydro-dg-1. The half maximal inhibition of (Na⁺+K⁺)-ATPase activity (I₅₀) corresponded to affinity measurements in all but two cases: dehydro-dt-3 and dehydro-dt-2 showed very low I₅₀ values.     

Pharmacological characterizations of recombinant human 5-HT1D1Dα and 5-HT1Dβ receptor subtypes coupled to adenylate cyclase inhibition in clonal cell lines: apparent differences in drug intrinsic efficacies between human 5-HT1D subtypes

Recombinant human 5-HT_1D and 5-HT_1D receptor subtypes were stably expressed in NIH-3T3 fibroblasts (1D cell line) and Y-1 adrenocortical tumor cells (1D cell line), respectively, for pharmacological evaluations of serotonergic compounds to inhibit forskolin-stimulated CAMP accumulation (FSCA). [³H]LSD saturation studies indicated that 5-HT_1D receptor expression levels were slightly higher in the 1D cell line (B _max = 1334 ± 134 fmol/mg protein) than in the (1D) cell line (B _max = 900 ± 900 fmol/mg protein). 5-HT inhibited FSCA with similar potencies (EC₅₀ 2 nM) in both assay systems. The rank order of agonist potencies in both clonal cell lines matched their pharmacological profiles previously determined in binding studies: dihydroergotamine >- 5-carboxamidotryptamine (5-CT) > LSD >- 5-HT > sumatriptan > 1-naphthylpiperazine (1-NP) > yohimbine > 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH DPAT) > 1-(2,5-dimethoxy4-iodophenyl)-2-aminopropane (DOI), with K_i/EC₅₀ ratios greater than unity. Methiothepin acted as a silent antagonist at both human 5-HT_1D and 5-HT_1D receptors with apparent dissociation constants (K_b values) of 12 ± 1 nM and 3 ± 1 nM, respectively. Whereas GR 127,935, metergoline, DOI, and quipazine acted as full agonists in the 1D cell line, these compounds behaved as partial agonists in the 1D cell line.To determine whether high levels of receptor reserve might mask partial agonist activity in the two second messenger assay systems, studies were performed using the irreversible receptor alkylating agent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). The relationships between receptor occupancy and inhibition of FSCA were determined for 5-HT, sumatriptan, and 1-NP in both clonal cell lines after partial receptor inactivation using Furchgott analysis. Hyperbolic relationships between receptor occupancy and second messenger response were determined for 5-HT in both transfected cell lines. Steep hyperbolic relationships were also found for sumatriptan and 1-NP in the 1D cell line whereas nearly linear relationships were observed for these two compounds in the 1D cell line. Moreover, K_A/EC₅₀ ratios of these compounds were significantly larger in the (1D)(10–32) as compared to the 1D (0.9–2.5) cell line. These data are consistent with the hypothesis that the two heterologous expression systems contain a differential amount of receptor reserve. Despite the presence of an apparently larger receptor reserve in the 1D cell line, GR 127,935, metergoline, DOI, and quipazine behaved as partial agonists.Although the potencies (EC₅₀ values) of compounds matched their respective affinity constants (K_i values) for the closely-related 5-HT_1D subtypes, differences in intrinsic activities were observed for a few compounds between the two 5-HT_1D receptor expression systems. Since receptor reserve is dependent on the properties of both the assay system and drug, the observed variations in intrinsic activity, although influenced by the variable amounts of receptor reserve in the two transfected cell lines, reflect primarily system-independent differences in the intrinsic efficacy of the tested compounds at the two human 5-HT_1D receptors. Higher intrinsic efficacies of compounds at the human 5-HT_1D receptor relative to the human 5-HT_1D subtype may be responsible for the higher intrinsic activities observed in the (1D) cell line, even though receptor reserve is apparently lower in this system.Abbreviations CRC Concentration-response curve - FSCA forskolin-stimulated cAMP accumulation - KA pseudo-dissociation constants - 5-CT 5-carboxamidotryptamine - 5-HT 5-hydroxytryptamine - 5MeOT 5-methoxytryptamine - PAPP 1-[2-(4-aminophenyl) ethyl] -4-(3-trifluoromethylphenyl)-piperazine - 1-NP 1-(1-naphthyl) piperazine - 8-OH-DPAT 8-hydroxy-2-(di-n-propylamino) tetralin - DOI 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane - MK-462 (N,N-dimethyl-2-[5-(1, 2, 4-triazol-l-yl methyl)-1H-indole3-yl]ethylamine - GR 127,935 (2-methyl-4-(5-methyl-[1, 2, 4]oxadiazol-3-yl)-biphenyl-4-carboxylic acid [4-methoxy-3-(4-methyl-piperazin-1-yl)-phenyl]-amide) - GR 46611 5-[4-methoxybenzyl ethylene]-1H-indole3-yl]ethyl amine) - L-694,247 (2-[5-[3-(4-methylsulfonylamino)benzyl-1, 2, 4-oxadiazol-5-yl]-1H-indole3-yl]ethylamine)