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In mammals, receptors for the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) are divided into three pharmacological classes, which are denoted GABAA, GABAB, and GABAC. GABAC receptors are defined by their insensitivity to the GABAA receptor antagonist bicuculline and the GABAB receptor agonist (-)-baclofen. GABAC receptors probably are a heterogeneous group of proteins. The most extensively studied mammalian GABAC receptors are those found in neurons of the outer retina. These receptors are GABA-gated Cl- channels comprised of p subunits, of which there are two subtypes. The physiological functions served by GABAC receptors are largely unknown; to determine the functions, it would be useful to have GABAC-selective ligands. In a previous study, we found that isoguvacine, a GABAA-selective agonist, and 3-aminopropyl-(methyl)phosphinic acid (3-APMPA), a GABAB-selective agonist, show affinity for retinal GABAC receptors. In particular, 3-APMPA is an antagonist with low micromolar potency (Kb approximately 1 microM). Here, we report the synthesis and pharmacological characterization of (1,2,5,6-tetrahydropyridine-4-yl)methylphosphinic acid (TPMPA), a hybrid of isoguvacine and 3-APMPA designed to retain affinity for GABAC receptors but not to interact with GABAA or GABAB receptors. Electrical assays show that TPMPA is a competitive antagonist of cloned human mu 1 GABAC receptors expressed in Xenopus laevis oocytes (Kb approximately 2 microM). TPMPA is100-fold weaker as an inhibitor of rat brain GABAA receptors expressed in oocytes (Kb approximately 320 microM) and has only weak agonist activity on GABAB receptors assayed in rat hippocampal slices (EC50 approximately 500 microM). TPMPA should be a useful pharmacological probe with which to investigate GABAC receptor function in the outer retina and in any other areas of the nervous system in which these types of receptor are present.

Pumiliotoxin B (PTX-B, 6-(6',7'-dihydroxy-2',5'-dimethyl-(E)-4'-octenylidene)-8-hydroxy-8 -methyl-1- azabicyclo-[4.3.0] nonane) increases the force of contractures of spontaneously beating guinea pig atrial strips by 3- to 5-fold with half-maximal effects at about 3 microM and increases rates of atrial contractions by 2- to 3-fold with half-maximal effects at about 6 microM. The presence of an axial 7-hydroxy substituent (PTX 339A) decreases the efficacy but not the potency of PTX-B as a positive inotropic agent while having only slight effects on activity as a positive chronotropic agent. The presence of an equatorial 7-hydroxy substituent (PTX 339B) greatly decreases efficacy and potency of PTX-B as a positive chronotropic and inotropic agent. Pumiliotoxin A which lacks the side-chain 7'-hydroxy group of PTX-B causes only a 2-fold increase in force of contracture at 54 microM while having minimal effects on rate. The presence of an axial 7-hydroxy substituent (PTX 323B' and 323B", epimeric at the 6'-hydroxy) markedly enhances positive inotropic and chronotropic effects of PTX-A. Another congener, PTX 251D with a 6-(2'-methylhexylidene) side chain, and a synthetic analogue with a 6-(6'-heptenylidene) side chain are cardiac depressants. Both lack hydroxyl groups in the side chain. The presence of an omega-1 hydroxy group in the side chain of PTX 251D yields an alkaloid (267C) with weak positive inotropic effects and minimal chronotropic effects. The presence of an axial 7-hydroxy group in the indolizidine ring of PTX 251D results in a compound (PTX 267A) with very weak positive inotropic effects while retaining the negative chronotropic effects of PTX 251D. A synthetic analogue with a 6-(7'-hydroxyheptylidene) side chain is a cardiac depressant even though it contains a side-chain hydroxyl corresponding in position to the 7'-hydroxyl of the side chain of PTX-B. The positive chronotropic and inotropic effects of pumiliotoxin B are reversed only by relatively high concentrations of the calcium channel blockers nifedipine and verapamil, suggesting that pumiliotoxin B may owe its cardiotonic activities to effects on internal mobilization of calcium.

Pumiliotoxin-C (PTX-CI), a cis-decahydroquinoline alkaloid, and synthetic analogues inhibited directly and indirectly elicited twitches in rat phrenic nerve diaphragm preparations. At 40 microM the most potent analogue, 2,5-di-n-propyl-cis-decahydroquinoline (PTX-CII), reversibly blocked the indirectly elicited twitch and markedly depressed the amplitude of end-plate potentials and miniature end-plate potentials without affecting muscle membrane potential. PTX-CII did not depress the directly elicited twitch in diaphragm muscle treated with alpha-bungarotoxin. At 40 microM, the compound blocked extrajunctional acetylcholine (ACh) sensitivity in chronically denervated soleus muscle of the rat. The peak amplitude of the end-plate current in frog sartorius muscle was depressed by PTX-CII at both positive and negative membrane potentials. Nonlinearity in the current-voltage relationship appeared at membrane potentials greater than -100 mV with concentrations of PTX-CII from 2.5 to 7.5 microM. The decay phase of the end-plate current was shifted to lower values such that it decayed faster at all membrane potentials but retained voltage sensitivity. PTX-CII, the most potent of the analogues, inhibited binding of [3H]perhydrohistrionicotoxin to Torpedo electroplax membranes with an IC50 value of 1.5 microM in the absence of carbamylcholine (Carb) and 0.1 microM in the presence of Carb. The other analogues were less potent, but all showed higher affinities in the presence of Carb. PTX-CI, PTX-CII, and PTX-CIII had no effect on the binding of [125I]alpha-bungarotoxin to ACh receptors in Torpedo membranes while slightly enhancing the binding of [3H]ACh, whereas PTX-CIV gave slight inhibition of both receptor ligands at concentrations greater than 10 microM. The pumiliotoxin-C class of alkaloids therefore appears to block neuromuscular transmission primarily via interactions with sites associated with the ion channel controlled by the ACh receptor.