C.Y. Chiou

STUDIES ON THE MECHANISM OF ACTION OF A NEW Ca2+ ANTAGONIST, 8‐(N,N‐DIETHYLAMINO)OCTYL 3,4,5‐TRIMETHOXYBENZOATE HYDROCHLORIDE IN SMOOTH AND SKELETAL MUSCLES

1 The rabbit aortic strip, guinea‐pig ileum and rabbit skeletal muscle sarcoplasmic reticulum preparations were used to determine at which sites and in what manner 8‐(N,N‐diethylamino)‐octyl 3,4,5‐trimethoxybenzoate (TMB‐8) interferes with Ca2+ availability in smooth and skeletal muscles. 2 TMB‐8 (50 μM) significantly inhibited equivalent responses of the rabbit aortic strip to KCl and noradrenaline. 3 TMB‐8 (65 μM) produced no significant alteration in the extracellular space of the guinea‐pig ileum as measured with [3 H]‐sorbitol. 4 The resting cellular Ca2+ influx as well as the resting 45 Ca2+ efflux in the guinea‐pig ileum preparation were significantly inhibited by TMB‐8 (65 μM). 5 TMB‐8 (5 μM and 50 μM) had no significant effect on the uptake of 45 Ca2+ by the sarcoplasmic reticulum preparation of skeletal muscle; however, TMB‐8 (5 μM) did significantly inhibit the caffeine (20 μ)‐induced release of 45 Ca2+ from this preparation. 6 It is concluded that TMB‐8 reduces Ca2+ availability in smooth and skeletal muscles by stabilizing Ca2+ binding to cellular Ca2+ stores and thereby inhibits the release of this Ca2+ by contractile stimuli.

Pharmacological evaluation of a new Ca2+ antagonist, 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8): studies in smooth muscles.

Abstract The pharmacology of 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB-8) has been studied using guinea pig ileum and vas deferens preparations. TMB-8 inhibited responses to drugs that excite specific receptors (acetylcholine and norepinephrine) as well as to agents whose actions are not mediated via specific receptors (KCl and BaCl2) with ID50s of 3.8 × 10−6 to 1 × 10−4 M. TMB-8 inhibited responses to acetylcholine, norepinephrine, nicotine, dimethylphenylpiperazinium and KCl in an insurmountable manner in the guinea pig ileum, while responses to BaCl2 were inhibited in a competitive manner. Increasing Ca2+ concentrations of the bathing medium from 1.35 to 5.40 mM effectively antagonized the TMB-8 inhibition of responses to KCl in the guinea pig ileum and vas deferens preparations. These results indicate that TMB-8 may produce its inhibitory effects in smooth muscle by interfering with the availability of Ca2+ for muscle contraction by blocking the Ca2+ release from intracellular bound stores.

Effects of ganglionic blocking agents on the neuromuscular junction.

Abstract Effects of ganglionic blocking agents on neuromuscular transmission and on responses of nicotinic agents have been studied using the baby chick biventer cervicis nerve muscle preparation. Responses of nicotinic agents such as nicotine, tetramethylammonium, dimethylphenylpiperazinium and carbachol were markedly inhibited by 10 mg % of hexamethonium (C 6 ) and 100 mg % of tetraethylammonium (TEA) and almost completely abolished by 100 mg % of C 6 and 1 g% of TEA. However, responses induced by ACh were not significantly affected by these ganglionic blocking agents, indicating that nicotinic agents were blocked by these ganglionic blocking agents at a site other than the ACh receptor, probably at the motor nerve endings. Neuromuscular transmission was also blocked by C 6 and TEA without affecting ACh responses suggesting that C 6 and TEA might block the uptake of choline bynerve across the membrane and thus inhibit ACh synthesis and thereby block neuromuscular transmission. Initial facilitation and concomitant contraction of the biventer cervicis muscle preceded neuromuscular blockade induced by TEA indicating that part of the TEA effect might be due to the release of endogenous ACh.

Cholinergic receptor labelling specific irreversible binding of bromoacetylcholine to cholinergic receptors at neuromuscular junctions

This study shows that (a) the slopes of dose-response curves of bromoacetylcholine (BrACh) and acetylcoline (ACh) were the same; (b) the dose-response curves of BrACh to contract superfused chick biventer cervicis and frog rectus abdominis muscles were shifted to the right by d-tubocurarine in a parallel fashio; and (c) the responses of BrACh were not affected by triethylcholine, which paralyzes cholinergic neurons through inhibition of ACh synthesis. These results indicate that BrACh interacts with cholinergic receptors specifically and directly. When the incubation time was prolonged to 15 min or longer in the organ bath, BrACh (2×10−3 M) produced irreversible blockade of the neuromuscular transmission in chick biventer cervicis and rat diaphragm preparations. The latter blockade was prevented partially by ACh (2×10−2 M) and d-tubocurarine (3.4×10−6 M) (25–38%) and markedly by carbachol (1×10−4 M) and decamethonium (6.2×10−4 M)_(76–79%), indicating agani that BrACh binds specifically and irreversibly to the cholinergic receptor. Therefore, 14C-BrACh is recommended for use in mapping, estimation, and isolation of cholinergic receptors. In order to do such experiments, the biological preparations would have to be pretreated with cold organophosphorus compounds in order to prevent irreversible binding of BrACh to cholinesterases.

Effects of diethylcholine in two animal models of Parkinsonism tremors

(2-Hydroxyethyl) methyldiethylammonium iodide (diethylcholine; DEC) was tested against trihexyphenidyl for its ability to block tremors in two animal models of Parkinsonism tremors. Both DEC (75 mg/kg) and trihexyphenidyl (10 mg/kg) antagonized physostigmine tremors in mice. Both drugs also blocked tremors in rats which received intracaudate injections of carbachol. DEC was more efficacious than trihexyphenidyl in the rat model. No dose-related inhibition of tremors was seen for trihexyphenidyl (5--20 mg/kg) but inhibition by DEC was dose-related (25--50 mg/kg). The ED50 for tremor inhibition in the rat model by DEC was 33 mg/kg. DEC was also shown to cross the blood-brain barrier in mice. The probable mechanism of action of DEC is discussed.

Inhibition of choline acetyltransferase by tertiary alkylaminoethyl esters

Abstract Several dimethylaminoethyl esters were synthesized in order to develop a compound which would be better able to cross cell membranes and to inhibit choline acetyltransferase (ChAc) within the nerve terminal. A structure-activity relationship study demonstrated three requirements for ChAc inhibition by the alkylaminoethyl esters: (1) a terminal cationic head on the amine end of the molecule, (2) the ability to stabilize a partial negative charge on the acyl end, and (3) a leaving group on the α-carbon on the acyl end. Three tertiary amine esters which fulfilled these requirements were studied: N , N -dimethylaminoethylchloroacetate (Cl-DMA); N , N -dimethylaminoethyl bromoacetate (Br-DMA); a ChAc were 1.29–5.75 × 10 −4 M in both extracted cell-free ChAc system and cellular minced brain ChAc system. These compounds showed reversible inhibition and were uncompetitive in nature with respect to both substrates, choline and acetyl CoA. The interaction of Cl-DMA and acryl-DMA with acetylcholinesterase and butyrylcholinesterase was also investigated. Cl-DMA and acryl-DMA were 20–150-fold and 3–5-fold les potent, respectively, to inhibit cholinestyerases than to inhibit ChAc, demonstrating fair specificity toward ChAc inhibition, particularly with Cl-DMA. The hydrolysis of the ester linkage in either compound was not affected by these cholinesterases. Both compounds produced blockade of indirectly stimulated nerve-muscle preparations both in vitro and in vivo , and the blockade in the isolated preparation was reversible with washing. Therefore, Cl-DMA, in particular, would be a compound of interest to inhibit ChAc in the whole animal.

Choline and diethylcholine transport into a cholinergic clone of neuroblastoma cells

Abstract The transport of choline and diethylcholine has been investigated in a cholinergic clone (S20F 3 ) of mouse neuroblastoma cells. Choline transport was linear for the first 20 min of incubation and was temperature dependent at low concentrations ( −5 M). Diethylcholine transport was linear for the first 10 min of incubation and was also temperature dependent at low concentrations. High affinity ( K m −6 M) and low affinity ( K m > 1 × 10 −5 M) components of transport were found for both compounds. The transport system had a greater apparent affinity (lower K m ) for choline than for diethylcholine (3-fold), but maximal transport velocities were about equal. Each compound competitively inhibited the others high affinity transport. Hemicholinium (1 × 10 −5 M) slightly inhibited high affinity choline transport but triethylcholine (1 × 10 −6 −1 × 10 −4 M) did not. Choline transport was also found to be dependent on the pH and pCO 2 of the medium.

Effects of halogenoacetylcholines on the neuromuscular junction

Abstract The effect of halogenoacetylcholines (HAChs) has been studied on the chick biventer cervicis nerve muscle preparation. All of HACHs studied were shown to be neuromuscular blocking agents of a depolarizing type. Neither the nicotinic effect of HACHs or of ACh was affected by triethylcholine (TEC) and hexamethonium (C6) whereas that of nicotine, tetramethylammonium and dimethylphenylpiperazinium was abolished by TEC and C6. These results suggest that HAChs act directly on ACh receptor to produce nicotinic effects similar to ACh. The brief contact of HAChs with the biventer cervicis muscle using superfusion technique caused slow contraction of the muscle followed by an immediate relaxation. This transient reversible contracture can be blocked by d-tubocurarine, indicating that it is induced through depolarization of the muscle at the cholinergic receptor site. On the other hand, if the muscle was treated by bromoacetylcholine and iodoacetylcholine for 10 min or longer, it produced a persistent contracture which was not reversible after repeated washing. It was questioned whether the inhibition of 3-phosphoglyceraldehyde dehydrogenase in the Embden-Meyerhof pathway and consequential reduction in the availability of adenosine triphosphate to the muscle is the reason for the persistent muscle contracture in this case.

Pharmacological Studies of N,N-Dimethylaminoethyl Chloroacetate and N,N-Dimethylaminoethyl Acrylate as Inhibitors of Choline Acetyltransferase in Isolated Skeletal and Smooth Muscle Preparations

Two tertiary amine esters, N,N-dimethylaminoethyl chloroacetate (Cl-DMA) and N,N-dimethylaminoethyl acrylate (acryl-DMA), which have recently been shown to be inhibitors of choline acetyltransferase (ChAc) were investigated to determine their actions in isolated skeletal and smooth muscle preparations. Both compounds caused neuromuscular blockade in indirectly stimulated nerve-muscle preparations (ED50 values of Cl-DMA were 6.9 -42.0 X 10(-4) M and those of acryl-DMA were 1.2-5.8 X 10(-4) M). The blockade was completely or partially reversible after drug washout. A comparison of the ED50 values for neuromuscular blockade with the ID50 values for ChAc inhibition suggested that the acryl-DMA compound might not cause neuromuscular blockade via ChAc inhibition because the potency ratios (ED50/ID50) of Cl-DMA were higher than 1, whereas those of acryl-DMA were equal to or lower than 1. This was borne out by further experiments on isolated neuromuscular preparations which showed that the site of action for acryl-DMA was post-junctional, whereas that for Cl-DMA was prejunctional. In addition, the weak stimulating properties of Cl-DMA and acryl-DMA were investigated in isolated skeletal and smooth muscle. Cl-DMA was shown to be a partial cholinergic agonist, whereas acryl-DMA was a nonspecific stimulant not involving cholinergic receptors. Although both Cl-DMA and acryl-DMA are inhibitors of ChAc, only Cl-DMA appears to have sufficient specificity for use as a possible ChAc inhibitor in vivo.

Cholinolytic effects of 3,3-dimethylbutanol carbamate (DBC)

Abstract 3,3-Dimethylbutanol carbamate (DBC) was shown to block cholinergic responses of acetylcholine (ACh) on frog rectus abdominis muscle, guinea pig vas deferens and guinea pig ileum preparations. The blockade was reversed by washing and was non-competitive in nature. However, DBC did not block ACh responses on chick biventer cervicis preparation with concentrations up to 5.5 × 10 −3 M. In lower concentrations, DBC blocked neuromuscular transmission in the chick biventer cervicis nerve-muscle preparation, but in contrast to d-tubocurarine, it preserved completely the muscular responses to exogenously applied ACh. The neuroeffector transmission in the guinea pig nerve ileum preparation was also blocked by DBC. However, in contrast to atropine, which totally abolished responses to exogenous ACh during neuroeffector blockade, DBC preserved somewhat diminished responses to exogenous ACh. DBC (7 × 10 −6 M − 7 × 10 −5 M) incubated with minced guinea pig cerebral cortex caused a marked release of ACh. Since hemicholinium, hexamecholinium and tetrodotoxin inhibit ACh release from nerve endings, DBC does not appear to work like these compounds either.