Alteration of the Physicochemical Properties Of
Total Page：16
File Type：pdf, Size：1020Kb
Proc. Nati. Acad. Sci. USA Vol. 75, No. 2, pp.,784-788, February 1978 Biochemistry Alteration of the physicochemical properties of triphosphoinositide by nicotinic ligands (neuromuscular blockers/hydrophobicity/phosphatidylinositol bisphosphate) TAE MOOK CHO, JUNG SOOK CHO, AND HORACE H. LOH Langley Porter Neuropsychiatric Institute and Department of Pharmacology, University of California, San Francisco, California 94143 Communicated by Choh Hao Li, December 2,1977 ABSTRACT The concentrations of nicotinic drugs required from cerebral cortex by De Robertis et al. (7). Chemical de- to effect a 50% transfer of triphospho[3H]inositide from an terminations confirmed that P3inositide is present in this re- aqueous phase to a nonaqueous phase closely approximated ceptor fraction and suggested that P3inositide is the major their concentrations for both in vivo neuromuscular blocking activity and binding to purified nicotinic receptors, and corre- component responsible for the binding of nicotinic ligands lated well (correlation coefficient = 0.95) with their biological (6). activities measured by other workers in an Electrophorus The present study describes selective alterations in the electroplax preparation. The triphospho[3Hjinositide transfer physicochemical properties of P3inositide on interaction with induced by nicotinic ligands was dependent on the lipid con- various nicotinic ligands. The alterations have been shown to centration and was potentiated by Ca +. The affinity constants be related to the neuromuscular blocking activity of these li- of 45Ca2+ for triphosphoinositide were similar to those for the purified nicotinic receptor. These and other findings suggest gands. The results suggest that P3inositide may function as a the possibility that triphosphoinositide (phosphatidylinositol binding component of the nicotinic cholinergic receptor. is a site of the nicotinic bisphosphate) binding receptor. MATERIALS AND METHODS Recently, Cavallito (1) and others (2, 3) have reported that Chemicals. P3inositide was isolated by the method of Michell electrostatic bond formation between the cationic portion of et al. (8), further purified using triethylaminoethyl-cellulose nicotinic ligands and the anionic site of the cholinergic receptor column chromatography, and identified by the method of is essential for pharmacological activity. Cavallito further Gonzalez-Sastre and Folch-Pi (9), using thin-layer chroma- suggested that the highly charged phosphate macromolecules tography and visualization by iodine, which yielded a single may be more closely related to the nicotinic receptor than are spot with an RF value the same as the reported one. The P3- monovalent anions such as carboxylate, phosphate, and sulfate inositide spot was removed and the phosphate content was (4). From structural considerations of triphosphoinositide determined by Bartlett's method (10). On the basis of the (Psinositide; phosphatidylinositol bisphosphate), a membrane phosphate determination, the P3inositide was over 95% pure. acidic lipid with a maximum of five negative charges, it is P3[3H]inositide (specific activity 3 Ci/mmol) was prepared by theoretically possible that the molecule could interact with a C. T. Peng (School of Pharmacy, University of California, San variety of structurally unrelated nicotinic ligands. For example, Francisco) at the Lawrence Laboratories (Berkeley, CA). the.cationic nitrogen and carbonyl oxygen of a drug such as 45CaCl2 (20 Ci/g) was obtained from New England Nuclear carbachol could bind to an anionic oxygen atom in the phos- (Boston, MA). phate group and to a hydroxyl group of the inositol ring, re- The following chemicals were purchased from Sigma spectively. Similarly, the distance between the two cationic Chemical Co.: d-tubocurarine chloride, decamethonium nitrogen atoms in the hexamethonium molecule is similar to chloride, trimethylbenzylammonium chloride, succinylcholine the distance between two anionic oxygen atoms of the phos- chloride, hexamethonium chloride, carbachol chloride, pilo- phates in the 4 and 5 positions of the inositol ring. Furthermore, carpine, methacholine chloride, (+)-muscarine chloride, 5- the distance between the quaternary nitrogen atoms in cho- hydroxytryptamine, dopamine, epinephrine, norepinephrine, linergic ligands such as decamethonium, succinylcholine, gal- and histamine. Nicotine, 1,1-dimethylphenylpiperazonium lamine, and d-tubocurarine matches the distance between the iodide, and lobeline were purchased from K & K Laboratories; two anionic oxygen atoms of the phosphate group in positions gallamine triethiodide from Davis and Geck, American Cy- 1 and 4 or 1 and 5 of the inositol ring. These theoretical con- anamid Co.; and mecamylamine from Merck, Sharp and siderations are supported by experimental data showing that Dohme Research Laboratories. a-Bungarotoxin was a gift from P3inositide binds nicotinic agonists and antagonists with high C. C. Chang of National Taiwan University (Taipei, Republic affinities, and that the dissociation constants obtained are of China). comparable to those reported for the same agents binding to Preparation of P3inositide Micelles. P3[3H]inositide was purified nicotinic receptor (5). Of the membrane acidic lipids mixed with unlabeled P3inositide in a mixture of chloroform examined, only P3inositide selectively binds di[14C]methyl- and methanol (2:1) and the solution was then taken down to d-tubocurarine ([14CJDMTC), and the chromatographic be- dryness on a rotary vacuum evaporator, after which the dried havior of the [14C]DMTC-PNinositide complex on a Sephadex P,3inositide powder was suspended in 5 ml of Tris-HCI buffer LH-20 column (6) is identical to the behavior of the DMTC (2 mM, pH 7.4) and allowed to swell for an hour. The P3inosi- complex formed with the proteolipid receptor fraction isolated tide was sonicated for 1 min and then diluted with the same The costs of publication of this article were defrayed in part by the Abbreviations: P3inositide, triphosphoinositide (phosphatidylinositol payment of page charges. This article must therefore be hereby marked bisphosphate); DMTC, dimethyl-d-tubocurarine; IC50, concentration "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate required to induce a 50% transfer of P3inositide from aqueous to this fact. nonaqueous phase. 784 Downloaded by guest on September 26, 2021 Biochemistry: Cho et al. Proc. Natl. Acad. Sci. USA 75 (1978) 785 buffer to give an appropriate final P3inositide concentra- tion. 'or Interface P3[3H]inositide Transfer Studies. Five tenths milliliter of 9 the P3[3H]inositide micelle preparation was added to 0.5 ml of Tris buffer containing drug and/or cation at room temperature. 8 This aqueous solution was mixed with 1 ml of heptane on a Vortex mixer (speed setting at 5) for 1 min and the mixture was 0 7 centrifuged at 1500 X g for 10 min. A 0.5-ml aliquot of the x aqueous phase mixed with 5 ml of Scintiverse cocktail solution E6 (Fisher Scientific Co.) was assayed in a Beckman liquid scin- tillation counter to determine P3[3H]inositide activity. The 5 radioactivity in the nonaqueous phase (heptane plus interphase) 0. was determined by subtracting the radioactivity in the aqueous 0o phase from the total amount added. The percent of drug-in- I duced P3[3H]inositide transferred from the aqueous phase to 31 K\ie Heptane phase the nonaqueous phase was calculated by using the following 0 ° ~Oto. equation: at---la 2 F 4__hi % of P3[3H]inositide transferred I Aqueous phase radioactivity without - radioactivity with drug I I I I I I I I drug in aqueous phase in aqueous phase x 100 1 2 3 4 5 6 7 8 9 10 radioactivity without drug [Decamethoniuml, MM FIG. 1. Distribution of P3[3H]inositide in the presence and ab- in aqueous phase sence of decamethonium. The influence of various concentrations of 45Ca2+ Binding to P3inositide. One hundred milliliters of decamethonium on the distribution of P3[3H]inositide between the 45Ca2+ was mixed with 100 ml of the nonra- aqueous phase, the heptane phase, and the interface was determined (2 puM) prepared by measurement of the radioactivity in the buffer and heptane com- dioactive P3inositide micelles (20 jg/ml). An aliquot of the ponents. The amount ofP3[3H]inositide at the interface was indirectly mixture (0.5 ml) was added to a 0.5-ml solution containing deduced by calculations, subtracting the measured radioactivities various concentrations of unlabeled calcium or neuromuscular from the total. blocking agents and the aqueous solution was mixed with 1 ml of heptane on a Vortex mixer (speed at 5) for 1 min. The mix- K1 = 2.7 AtM, K2 = 25 MM, and K3 = 350 AM. These data ture was centrifuged at 1500 X g for 10 min and the radioac- suggest that in the presence of 100 AM calcium ion only the tivity of 45Ca2+ in the lower phase was determined by the same binding sites with the two lower dissociation constants (K1 and method described for the P3[3H]inositide transfer studies. 00 r- RESULTS Distribution of P3[3H]inositide. The radioactive P3inositide A'S in both the heptane and aqueous phases was determined in the presence and absence of decamethonium, and the amount of 8C /O labeled P3inositide at the interphase was obtained by sub- of. tracting the measured radioactivities from the total added. As shown in Fig. 1, the P3[3H]inositide in the aqueous phase de- 4O~0) creased with increasing concentration of the decamethonium (A added, while the radioactivity in the nonaqueous phases in- a) 60 creased, particularly in the interphase. c La 3+ ICa2t 0' Effects of Calcium and Lanthanum Ions on P3[3H]inosi- 'a (a tide Transfer. Fig. 2 shows that both calcium and lanthanum, *0 a calcium antagonist, promoted P3[3H]inositide transfer from 0 the aqueous to the nonaqueous phase in a concentration-de- 40I pendent manner. The concentration of lanthanum required to I- 0/ induce a 50% transfer from the aqueous to the nonaqueous 4 phase (IC50) was approximately 250 times less than that of calcium. The P3[3H]inositide transfer was also determined with 20 increasing concentrations of decamethonium and d-tubocu- 0 rarine in the presence and absence of 100 AM calcium ion, 0l which by itself did not significantly induce the transfer (Fig.