6beta-Acyloxy(nor)tropanes: affinities for antagonist/agonist binding sites on transfected and native muscarinic receptors.

A series of esters of 6beta-hydroxynortropane and the N-methyl analogue 6beta-tropanol were synthesized and screened versus binding of an antagonist (quinuclidinyl benzilate) and an agonist (oxotremorine-M) at sites on human m(1)-, m(2)-, m(3)-, and m(4)-muscarinic receptors in transfected cell membranes and on native M(1)-muscarinic receptors in rat brain membranes and native M(2)-muscarinic receptors in rat heart membranes. Most 6beta-acyloxy(nor)tropanes had higher affinity versus oxotremorine-M binding compared to quinuclidinyl benzilate binding at transfected m(1)- and native M(1)-receptors, indicative of agonist activity. 6beta-Acetoxynortropane had K(i) values versus oxotremorine-M binding at m(1)-, m(2)-, and m(4)-receptors ranging from 4 to 7 nM. N-Methylation reduced affinity greatly as did increasing the size of the acyl moiety. The affinity of 6beta-benzoyloxynortropane and other analogues with larger acyl moieties was little affected by N-methylation or in some cases was increased. 6beta-Acyloxy(nor)tropanes and classical muscarinic agonists, such as muscarine and oxotremorine, had higher affinity versus oxotremorine-M binding compared to quinuclidinyl benzilate binding at native M(2)-muscarinic receptors of heart, but not at transfected m(2)-muscarinic receptors. Antagonist/agonist binding ratios were not obtained for transfected m(3)-receptors, since significant oxotremorine-M binding could not be detected. 6beta-Acyloxy(nor)tropane, two other (nor)tropanes, and the classical muscarinic agonists had higher affinity versus agonist binding compared to antagonist binding for transfected m(4)-receptors. The antagonist/agonist binding ratio method is clearly not always reliable for predicting agonist activity at muscarinic receptors.

Syntheses of (R)- and (S)-2- and 6-fluoronorepinephrine and (R)- and (S)-2- and 6-fluoroepinephrine: effect of stereochemistry on fluorine-induced adrenergic selectivities.

Several routes to the enantiomers of fluoronorepinephrines (1) and fluoroepinephrines (2) were explored. A catalytic enantioselective oxazaborolidine reduction and a chiral (salen)Ti(IV) catalyzed asymmetric synthesis of silyl cyanohydrins proved efficacious in the key stereo-defining steps of two respective routes. Binding studies of the catecholamines with alpha(1)-, alpha(2)-, beta(1)-, and beta(2)-adrenergic receptors were examined. The assays confirmed that fluorine substitution had marked effects on the affinity of (R)-norepinephrine and (R)-epinephrine for adrenergic receptors, depending on the position of substitution. Thus, a fluoro substituent at the 2-position of (R)-norepinephrine and (R)-epinephrine reduced activity at both alpha(1)- and alpha(2)-receptors and enhanced activity at beta(1)- and beta(2)-receptors, while fluorination at the 6-position reduced activity at the beta(1)- and beta(2)-receptors. The effects of fluorine substitution on the S-isomers were less predictable.

6beta-Acetoxynortropane: a potent muscarinic agonist with apparent selectivity toward M2-receptors.

A series of tropane derivatives, related in structure to baogongteng A (1), an alkaloid from a Chinese herb, were synthesized. 6beta-Acetoxynortropane (5) had weak affinity (Ki 22 microM) for central (M1) muscarinic receptors in a [3H]quinuclidinyl benzilate binding assay but had extremely high affinity (Ki 2.6 nM) and selectivity for M2-muscarinic receptors expressed in CHO cells. It had 13-fold lower affinity for M4-receptors, 260-fold lower affinity for M3-receptors, and 8200-fold lower affinity for M1-receptors expressed in CHO cells. The 6beta-carbomethoxy analogue (14) of baogongteng A had only weak affinity for M2-muscarinic receptors, as did 6beta-carbomethoxynortropane (13) and 6beta-acetoxytropane (4). In transfected CHO cells, 6beta-acetoxynortropane (5) was an agonist at M2-receptors, based on a GTP-elicited decrease in affinity, and a full agonist with an IC50 of 11 nM at M4-receptors, based on inhibition of cyclic AMP accumulation, while being a full agonist at M1-receptors with an EC50 of 23 nM and a partial agonist at M3-receptors with an EC50 of 3.6 nM, based in both cases on stimulation of phosphoinositide breakdown. All of the 16 tropane derivatives had weak affinities for central alpha4beta2-nicotinic receptors with 6beta-carbomethoxynortropane (13) having the highest affinity, which was still 150-fold less than that of nicotine. 6beta-Acetoxynortropane (5) represents a potent muscarinic agonist with apparent selectivity toward M2-receptors.

Absence of tetrodotoxins in a captive-raised riparian frog, Atelopus varius.

Bufonid frogs of the genus Atelopus contain two classes of skin toxins, namely the steroidal bufadienolides and the water-soluble tetrodotoxins. Frogs of the Panamanian species Atelopus varius have now been raised in captivity and levels in skin extracts of bufadienolides and of tetrodotoxin-like compounds assessed, using inhibition of [3H]ouabain binding and inhibition of [3H]saxitoxin binding, respectively. Levels of ouabain equivalents, corresponding to bufadienolides, were comparable to those found in wild-caught frogs from the same population in Panama, while tetrodotoxin-like activity was undetectable. The results strongly implicate environmental factors, perhaps symbiotic microorganisms, in the genesis of tetrodotoxins in the skin of frogs of the genus Atelopus, while indicating that the frog itself produces the skin bufadienolides.

Pseudophrynaminol: a potent noncompetitive blocker of nicotinic receptor-channels.

(+/-)-Pseudophrynaminol inhibited carbamylcholine-elicited sodium-22 influx with an IC50 value of about 0.3 microM in both rat pheochromocytoma PC12 cells (ganglionic-type nicotinic receptor) and human medulloblastoma TE671 cells (neuromuscular-type nicotinic receptor). The inhibition in both cell lines appeared to be noncompetitive in nature. In rat cerebral cortical membranes, pseudophrynaminol had only low affinity (Ki 35 microM) for the agonist site on central nicotinic receptors at which [3H]nicotine binds. Pseudophrynaminol, at 10 microM, had marginal effects on a variety of other central receptors, and even at 100 microM inhibited batrachotoxin-elicited sodium-22 influx in a synaptoneurosomal preparation by only 40%. It had no effect at 30 microM on acetylcholinesterase and was a weak inhibitor of butyrylcholinesterase. Thus, pseudophrynaminol appears to be a potent, rather specific, noncompetitive inhibitor of ganglionic and neuromuscular nicotinic receptor-channels.

Synthesis and nicotinic activity of epiboxidine: an isoxazole analogue of epibatidine.

Synthetic (+/-)-epiboxidine (exo-2-(3-methyl-5-isoxazolyl)-7-azabicyclo[2.2.1]heptane) is a methylisoxazole analog of the alkaloid epibatidine, itself a potent nicotinic receptor agonist with antinociceptive activity. Epiboxidine contains a methylisoxazolyl ring replacing the chloropyridinyl ring of epibatidine. Thus, it is also an analog of another nicotinic receptor agonist, ABT 418 ((S)-3-methyl-5-(1-methyl-2-pyrrolidinyl)isoxazole), in which the pyridinyl ring of nicotine has been replaced by the methylisoxazolyl ring. Epiboxidine was about 10-fold less potent than epibatidine and about 17-fold more potent than ABT 418 in inhibiting [3H]nicotine binding to alpha 4 beta 2 nicotinic receptors in rat cerebral cortical membranes. In cultured cells with functional ion flux assays, epiboxidine was nearly equipotent to epibatidine and 200-fold more potent than ABT 418 at alpha 3 beta 4(5) nicotinic receptors in PC12 cells. Epiboxidine was about 5-fold less potent than epibatidine and about 30-fold more potent than ABT 418 in TE671 cells with alpha 1 beta 1 gamma delta nicotinic receptors. In a hot-plate antinociceptive assay with mice, epiboxidine was about 10-fold less potent than epibatidine. However, epiboxidine was also much less toxic than epibatidine in mice.

Ibogaine: a potent noncompetitive blocker of ganglionic/neuronal nicotinic receptors.

Ibogaine noncompetitively blocked (IC50 approximately 20 nM) 22NaCl influx through ganglionic-type nicotinic receptor channels of rat pheochromocytoma PC12 cells. The major metabolite O-des-methylibogaine was 75-fold less active, and O-t-butyl-O-des-methylibogaine was 20-fold less active. Ibogaine was relatively weak as a blocker (IC50 approximately 2000 nM) of the neuromuscular-type nicotinic receptor channels in human medulloblastoma TE671 cells. The blockade of nicotinic responses by ibogaine was only partially reversible in PC12 cells. In vivo, ibogaine at 10 mg/kg completely blocked epibatidine-elicited antinociception in mice, a response that is mediated by central nicotinic receptor channels. There was no significant blockade of the epibatidine response at 24 hr after the administration of 40 mg/kg ibogaine. The blockade of nicotinic channels could contribute to the antiaddictive properties of ibogaine.

Spiropyrrolizidines: a new class of blockers of nicotinic receptors.

The spiropyrrolizidine oximes 236 and 222 and a related spiropyrrolizidine alkaloid, nitropolyzonamine, block nicotinic receptor channels in rat pheochromocytoma PC12 cells and in human medulloblastoma TE671 cells. In PC12 cells with an alpha 3 beta 4(5)-nicotinic receptor, both the spiropyrrolizidine oxime 236 and nitropolyzonamine had IC50 values of about 1.5 microM, while spiropyrrolizidine oxime 222 had an IC50 value of 2.6 microM versus carbamylcholine-elicited sodium-22 influx. In TE671 cells with an alpha 1 beta 1 gamma delta nicotinic receptor, the spiropyrrolizidine oximes 236, 222, and nitropolyzonamine had IC50 values of 9.5, 14, and 67 microM, respectively. The inhibitions by the spiropyrrolizidine oxime 236 and nitropolyzonamine appeared to be noncompetitive in nature in both cell lines. In rat cerebral cortical membranes, binding of [3H]nicotine to alpha 4 beta 2 nicotinic receptors was not inhibited significantly by 10 microM concentrations of the spiropyrrolizidine oxime 236, or by nitropolyzonamine, as expected for a noncompetitive blocker. Both compounds at 10 microM had marginal effects on a variety of central receptors, but did inhibit binding of [3H]1,3-di(2-tolyl) guanidine to sigma receptors in mouse brain membranes with IC50 values of about 0.5 microM. The spiropyrrolizidine oxime 236 at 10 microM had no effect on batrachotoxin-elicited sodium influx in guinea pig cerebral cortical synaptoneurosomes or on ATP-elicited calcium influx in PC12 cells. Such spiropyrrolizidines represent a new structural class of blockers of nicotinic receptor channels with selectivity for ganglionic-type receptors.

Tetrahydrobenzothiophenone derivatives as a novel class of adenosine receptor antagonists.

A novel class of non-nitrogen-containing heterocycles, the tetrahydrobenzothiophenones, was found to bind to adenosine receptors as antagonists in the micromolar range. Affinity was determined in radioligand-binding assays at rat brain A1 and A2a receptors. A structure-activity analysis indicated that a 3-thioether group is favored and affinity at A2a, but not at A1, receptors is highly dependent on this thioether substituent. A carboxylic acid-derived substituent is required at the 1-position of the thiophene ring, with esters being more potent in binding at A1 receptors than the corresponding carboxyl hydrazide or carboxylic acid derivatives. The methyl (15) and ethyl (16) esters are about equipotent at A1 but not at A2a receptors. A 4-keto group on the saturated ring is favored for receptor affinity. Dimethyl substitution at the 6-position of the saturated ring is allowed. One of the most potent derivatives was the nonselective compound ethyl 3-(benzylthio)-4-oxo-4,5,6,7-tetrahydrobenzo[c] thiophene-1-carboxylate (BTH4, 7; Figure 1), which antagonized adenosine agonist-induced inhibition of adenylyl cyclase in rat adipocyte membranes with a KB value of 1.62 +/- 0.73 microM and adenosine agonist-induced stimulation of adenylyl cyclase in pheochromocytoma cell membranes with a KB value of 9.19 +/- 0.98 microM. Displacement of radioligand binding by BTH4 (7) at cloned human A3 receptors was negligible but one slightly A3 selective compound (11, 3.9-fold over A1 and >7.5-fold over A2a) was found. A 1-methylpropyl thioether (17) was 29-fold selective for A1 and A2a receptors. BTH4 (7) alone, at 10 mg/kg, stimulated locomotor activity in mice but paradoxically acted, under certain circumstances, synergistically with an A1 selective agonist to depress locomotor activity. A pharmacophore model relating structural features of xanthine and non-xanthine adenosine antagonists to BTH4 (7) suggests a high degree of similarity in electrostatic surfaces, assuming that the thiophene ring superimposes the region of the uracil ring of xanthines.

Maitotoxin-elicited calcium influx in cultured cells. Effect of calcium-channel blockers.

Maitotoxin elicited a marked influx of 45Ca2+ into NIH 3T3 fibroblast cells. The influx was blocked by imidazoles (econazole, miconazole, SKF 96365, clotrimazole, calmidazolium) with IC50 values from 0.56 to 3 microM. Phenylalkylamines (verapamil, methoxyverapamil) and nitrendipine were less potent, and diltiazem was very weak. Among other calcium blockers, the diphenylbutylpiperidines fluspirilene and penfluridol, the diphenylpropylpiperidine loperamide, and the local anesthetic proadifen were quite active with IC50 values of 2-4 microM. The pattern of inhibition of maitotoxin-elicited calcium influx did not correspond to the ability of the agents to block elevation of calcium that ensues through calcium-release activated calcium (CRAC) channels after activation of phosphoinositide breakdown by ATP in HL-60 cells. The imidazoles did block CRAC channels, but fluspirilene, penfluridol, loperamide and proadifen were ineffective. Loperamide actually appeared to enhance influx of calcium via the activated CRAC channels. The imidazoles, in particular calmidazolium, caused an apparent influx of calcium and caused a stimulation of phosphoinositide breakdown in HL-60 cells.

Inhibition of platelet aggregation by adenosine receptor agonists.

2-(Ar)alkoxyadenosines, which are agonists selective for the A2AAR in PC 12 cell and rat striatum membranes, are also agonists at the A2AR coupled to adenylate cyclase (AC) that mediates the inhibition of platelet aggregation. A panel of twelve well-characterized adenosine analogues stimulated human platelet AC and inhibited ADP-induced platelet aggregation at sub- to low-micromolar concentrations with a potency ranking CGS 21680 > adenosine > R-PIA. There were significant correlations between the EC50 of anti-aggregatory activity and either the EC50 of stimulation of platelet and PC 12 cell AC (r2 = 0.66 and 0.67, respectively) or the Ki of inhibition of [3H]NECA binding to the rat striatum membranes (r2 = 0.75). Likewise, platelet AC stimulation correlated well with stimulation of PC 12 cell AC and with [3H]NECA binding (r2 = 0.94 and 0.91, respectively). Ten 2-(ar)alkoxyadenosines stimulated platelet AC at EC50s ranging between 0.16 and 2.3 microM and inhibited platelet aggregation at EC50s ranging between 2 and 30 microM. There were no correlations between the EC50s of anti-aggregatory activity and either the EC50s of the stimulation of platelet or PC 12 AC (r2 = 0.08 and 0.06, respectively) or with the Ki of the inhibition of [3H]NECA binding to the A2aAR in rat striatum (r2 = 0.02). The EC50s of the stimulation of platelet AC correlated with those of the stimulation of PC 12 AC (r2 = 0.48), and also with the Ki of [3H]NECA binding (r2 = 0.71). Each of the 23 adenosines completely inhibited platelet aggregation and thus, functionally, all behaved as full agonists.(ABSTRACT TRUNCATED AT 250 WORDS)

8-(3-Chlorostyryl)caffeine (CSC) is a selective A2-adenosine antagonist in vitro and in vivo.

An adenosine antagonist, 8-(3-chlorostyryl)caffeine (CSC), was shown previously to be 520-fold selective for A2a-adenosine receptors in radioligand binding assays in the rat brain. In reversing agonist effects on adenylate cyclase, CSC was 22-fold selective for A2a receptors in rat phenochromocytoma cells (Kb 60 nM) vs. A1 receptors in rat adipocytes (Kb 1.3 microM). Administered i.p. in NIH mice at a dose of 1 mg/kg, CSC shifted the curve for locomotor depression elicited by the A2a-selective agonist APEC to the right (ED50 value for APEC shifted from 20 micrograms/kg i.p. to 190 micrograms/kg). CSC had no effect on locomotor depression elicited by an ED50 dose of the A1-selective agonist CHA. CSC alone at a dose of 5 mg/kg stimulated locomotor activity by 22% over control values. Coadministration of CSC and the A1-selective antagonist CPX, both at non-stimulatory doses, increased activity by 37% (P < 0.001) over CSC alone, suggesting a behavioral synergism of A1- and A2-antagonist effects in the CNS.

Structure-activity relationships for 2-substituted adenosines at A1 and A2 adenosine receptors.

A series of 55 2-alkyloxy-, 2-aryloxy- and 2-aralkyloxy-adenosines was screened as inhibitors of the binding of [3H]R-phenyl-isopropyladenosine to A1 adenosine receptors in rat cerebral cortical membranes, and of the binding of [3]N-ethylcarboxamidoadenosine to A2 adenosine receptors in rat striatal membranes and as agonists at A2 adenosine receptors coupled to adenylate cyclase in rat pheochromocytoma PC12 cell membranes. The activities are consonant with a hydrophobic binding site in the A2 receptors at a distance from the 2-position of the adenine ring corresponding to a spacer chain of -O-CH2-CH2-. These is little lateral steric tolerance in the region occupied by the spacer chain. Interaction with the hydrophobic binding site is greatest in the 2-alkyloxy series for 2-cyclohexylethoxy-, 2-cyclohexylpropoxy- and 2-cyclohexylbutoxyadenosines and in the 2-aralkoxy series for 2-phenylethoxy-, 2-(4-methylphenyl)ethoxy-, 2-(4-chlorophenyl)ethoxy-, and 2-naphthylethoxy-adenosine. The affinities of the 2-substituted adenosines for the rat cerebral cortical A1 receptors are not as markedly altered by structural changes and are in almost all cases two- to hundredfold less than the affinity of the 2-substituted adenosine for the rat striatal A2 receptor. There is excellent correspondence of the present data on rat A2 receptors with reported potencies of these 2-substituted adenosines as coronary vasodilators in guinea pig heart preparations.

Interaction of pumiliotoxin B with an "alkaloid-binding domain" on the voltage-dependent sodium channel.

The alkaloid pumiliotoxin B (PTX-B) "activates" voltage-dependent sodium channels in synaptoneurosomes and neuroblastoma cells. It appears that PTX-B activates sodium channels by interacting with a site that is allosterically coupled to other sites on the sodium channel, namely two scorpion toxin sites and the brevetoxin site. In guinea pig cortical synaptoneurosomes, alpha-scorpion toxin, beta-scorpion toxin, and brevetoxin induce a dose-dependent potentiation of PTX-B-induced 22Na+ influx. The synergism with beta-scorpion toxin differentiates PTX-B from the alkaloid veratridine, which induces an activation of sodium channels that is not affected by beta-scorpion toxin. PTX-B does not inhibit [3H]batrachotoxinin-A benzoate ([3H]BTX-B) binding to the alkaloid site on sodium channels. On the other hand, aconitine, which activates sodium channels and inhibits [3H]BTX-B binding, induces a 22Na+ influx that, like PTX-B-induced 22Na+ influx, is potentiated by alpha-scorpion toxin, beta-scorpion toxin, and brevetoxin. Inhibition of [3H]BTX-B binding by aconitine is reduced in the presence of PTX-B. Both a type I pyrethroid (allethrin) and a type II pyrethroid (fenvalerate) inhibit PTX-B- and PTX-B/alpha-scorpion toxin-mediated 22Na+ influx. Allethrin and fenvalerate also inhibit aconitine-mediated 22Na+ flux but not BTX-mediated 22Na+ influx. It is proposed that on the sodium channel there is an "alkaloid-binding domain" at which alkaloids exert stimulatory actions. However, depending on the region on the domain to which the binding occurs, different allosteric interactions with other sites can be observed. PTX-B is proposed to interact with a part of the alkaloid-binding domain that is shared by aconitine but not by batrachotoxin or veratridine, whereas aconitine interacts with a part of the domain shared by PTX-B and by batrachotoxin/veratridine.

Agonist activity of 2- and 5'-substituted adenosine analogs and their N6-cycloalkyl derivatives at A1- and A2-adenosine receptors coupled to adenylate cyclase.

The activity of N6-cycloalkyl derivatives of adenosine, 2-chloroadenosine, 5'-chloroadenosine and N-ethylcarboximidoadenosine (NECA) and of 2-fluoroadenosine and 5-methylthioadenosines were compared at the A1-adenosine receptor inhibitory to adenylate cyclase in rat fat cell membranes and at the A2A-adenosine receptors stimulatory to adenylate cyclase in rat PC12 cell membranes. The N6-cycloalkyl derivatives in all cases were more potent (4- to 23-fold) than the parent compound at the A1 receptor, and were less potent (1.6- to 11-fold) than the parent compound at the A2A receptor. N6-Cyclopentyl-5'-chloroadenosine was the most selective agonist (900-fold) for the A1 receptor, while 2-fluoroadenosine was the only agonist with some selectivity (4.8-fold) for the A2A receptor. 5'-Methylthioadenosine was a weak agonist at both adenosine receptors. A 2-fluoro derivative of 5'-methylthioadenosine was somewhat more potent. Affinities of these analogs for inhibition of binding of radioligands to rat brain A1 and A2A receptors are presented.

A2A adenosine receptors from rat striatum and rat pheochromocytoma PC12 cells: characterization with radioligand binding and by activation of adenylate cyclase.

Binding assays and assays of activation of adenylate cyclase with the agonists 5'-N-ethylcarboxyamidoadenosine (NECA) and CGS21680 have been used to compare adenosine receptors in rat pheochromocytoma PC12 cells and in rat striatum. The [3H]NECA binding showed two components, whereas [3H]CGS21680 bound to one component in both tissues. The Kd value for the high affinity site labeled with [3H]NECA in PC12 cell membranes (2.3 nM) was lower than that in striatum (6.5 nM). The [3H]CGS21680 binding site showed a Kd value of 6.7 nM and 11.3 nM in PC12 cells and striatum, respectively. In the presence of GTP the KD values of [3H]NECA and [3H]CGS21680 for the high affinity site were increased severalfold, whereas the low affinity sites for [3H]NECA were no longer detected with filtration assays. A comparison of the ability of a series of agonists and antagonists to inhibit high affinity binding of [3H]NECA to A2 receptors in PC12 cell and striatal membranes indicated that agonists had higher affinities and antagonists had lower affinities in PC12 cells, compared with affinities in striatal membranes. Analysis of activation of adenylate cyclase in PC12 cell membranes suggested that the dose-dependent stimulation by NECA involved two components, whereas CGS21680 stimulated via one component. The maximal stimulation by NECA significantly exceeded that caused by CGS21680. In intact PC12 cells, NECA caused a greater accumulation of AMP than did CGS21680, as was the case in membranes. In striatal membranes, NECA and CGS21680 showed similar maximal stimulations of adenylate cyclase. Both NECA and CGS21680 were more potent in PC12 cell membranes than in striatal membranes, in agreement with binding data. However, in contrast to binding data, antagonists were not less potent versus stimulation of adenylate cyclase by NECA or CGS21680 in PC12 cell membranes, compared with striatal membranes. In toto, the results suggest that A2A receptors in striatum are virtually identical to the A2A receptors in PC12 cells. But, in addition to an A2A receptor, it appears that a lower affinity functional receptor, probably an A2B receptor, is present in PC12 cells and PC12 cell membranes, whereas such a functional low affinity receptor is not detectable in striatal membrane.

Incorporation of 2-fluorohistidine in murine protein in vivo.

The tissue distribution and time course of incorporation into acid insoluble (bound) and acid soluble (free) fractions of [3H]2-fluorohistidine is compared to that of U[14C]Histidine in mouse tissues in vivo. The cycloheximide-sensitive incorporation of 2-FHis is between 9 and 17 percent of that of His. Unlike [14C]His a major fraction, approximately 90% at 72 hrs, of isotope derived from [3H]2-FHis remains in tissues for a prolonged period in an acid soluble form. The excretion of isotope derived from [14C]His (T1/2 = 5 hr) is more rapid than from [3H]2-FHis (T1/2 = 11.4 hrs). 2-FHis, at doses from 100 to 250 mg/kg produce a reversible inhibition of growth in mice.

Effects of the amphiphilic peptides melittin and mastoparan on calcium influx, phosphoinositide breakdown and arachidonic acid release in rat pheochromocytoma PC12 cells.

Two amphiphilic peptides from hymenopterid insects, melittin and mastoparan, stimulate secretion in a variety of cell types. In PC12 cells, both peptides stimulate calcium influx with melittin some 20-fold more potently than mastoparan. Melittin stimulates both breakdown of phosphoinositides (Pl) by phospholipase C to yield inositol phosphates and hydrolysis of phospholipids by phospholipase A2 to release arachidonic acid (AA). Mastoparan stimulates Pl breakdown, but has no effect on AA release. Maximal stimulation of Pl breakdown occurs at 1 to 2.5 micrograms/ml melittin and 30 micrograms/ml mastoparan, whereas maximal stimulation of AA release occurs at 2 to 5 micrograms/ml melittin. Organic calcium channel blockers (nifedipine, verapamil, diltiazem) have little or no effect on responses to the peptides. The influx of calcium elicited by melittin or mastoparan is completely or nearly completely blocked by inorganic calcium channel blockers (Co++, Mn++, Cd++). Mn++ and Cd++ inhibit melittin-induced Pl breakdown and AA release and mastoparan-induced Pl breakdown. Co++ has no effect on melittin-induced Pl breakdown and potentiates mastoparan-induced Pl breakdown. Pertussis toxin has no effect on the Pl breakdown induced by either peptide. The responses to melittin and mastoparan in PC12 cells are compared to those reported for maitotoxin.

Decahydroquinoline alkaloids: noncompetitive blockers for nicotinic acetylcholine receptor-channels in pheochromocytoma cells and Torpedo electroplax.

In pheochromocytoma PC12 cells, (+)-cis-decahydroquinoline 195A (5-methyl-2-propyl-cis-decahydroquinoline) and (+)-perhydro-cis-decahydroquinoline 219A (2,5-dipropyl-cis-decahydroquinoline) inhibit carbamylcholine-elicited sodium flux with IC50 values of 1.0 and 1.5 microM, respectively. Both of these decahydroquinolines appear to enhance desensitization, although apparent lack of complete removal of (+)-perhydro-cis-219A by washing complicates interpretation of the effects of that agent. A series of cis- and trans-decahydroquinolines with substituents in the 2- and 5-position also exhibit structure-dependent inhibition of carbamylcholine-elicited sodium flux in PC12 cells and all of the decahydroquinolines inhibit binding of the noncompetitive blocking agent [3H]perhydrohistrionicotoxin to muscle-type nicotinic acetylcholine receptor-channels in membranes from Torpedo electroplax. The Ki values in electroplax membranes range from 1.4 to 7.9 microM, making these alkaloids comparable in potencies to the histrionicotoxins. Potencies are increased 2- to 3-fold in the presence of an agonist, carbamylcholine. The profile of activities are similar in PC12 cells and electroplax membranes. The cis- and trans-decahydroquinolines represent another class of noncompetitive blockers for acetylcholine receptor-channels with similar activity for both muscle-type and ganglionic type nicotinic receptors.

5,8-disubstituted indolizidines: a new class of noncompetitive blockers for nicotinic receptor-channels.

A series of 8-methyl-5-substituted indolizidines inhibit binding of the noncompetitive blocking agent [3H]perhydrohistrionicotoxin to muscle-type nicotinic acetylcholine receptor-channels in membranes from Torpedo electroplax. The Ki values range from 0.16 to 1.12 microM, making these alkaloids among the most potent ligands for this site. Unlike most noncompetitive blockers, the potencies of the 8-methyl-5-substituted indolizidines are reduced in the presence of carbamylcholine. Indolizidine 205A (8-methyl-5-(4-pentynyl)indolizidine) is unique in enhancing binding of [3H]perhydrohistrionicotoxin by 1.5-fold. The enhancement is at a maximum at 0.01 to 0.1 microM, followed by progressive inhibition with an IC50 of about 20 microM. In the presence of carbamylcholine, which itself enhances binding of [3H]perhydrohistrionicotoxin, indolizidine 205A causes only an inhibition of binding with an IC50 of about 10 microM. Indolizidines with a hydroxy substituent on the 8-methyl group have very low activity. None of the indolizidines affect binding of [125I]alpha-bungarotoxin to acetylcholine recognition sites. In pheochromocytoma PC12 cells, indolizidine 205A has no agonist activity, but only inhibits carbamylcholine-elicited 22Na+ influx. The profile of potencies for the 8-methyl-5-substituted indolizidines is similar in electroplax membranes and PC12 cells. Indolizidines 205A and 209B (8-methyl-5-pentylindolizidine) have no apparent effect on desensitization of receptors in PC12 cells. The 5,8-disubstituted indolizidines appear to represent an atypical and potent class of noncompetitive blockers for muscle-type and ganglionic nicotinic receptor-channels.