The aim of this work was to describe the pharmacological profile of the Ascaris
muscle GABA receptor, and to compare its properties to those of GABA receptors in
vertebrates. The interest of such study is to show the existence of pharmacological
differences between Ascaris and vertebrate GABA receptors, which may be exploited in
antiparasitic drug research. A short review will be presented first on the anatomy,
physiology and pharmacology of the neuro-muscular system of Ascaris, and on the
pharmacological characteristics of GABA receptors in vertebrates and invertebrates. It
will be followed by a simplified presentation of the receptor theory and the subsequent
development of drug-receptor interaction theories.The effects of GABA and various GABA-agonists on the membrane input
conductance of Ascaris muscle cells were studied using a current-clamp technique.
Dose-response curves were obtained and described by a modified Hill equation for each
agonist tested. The relative potencies of GABA-agonists in Ascaris are compared to
those obtained at vertebrate GABA-A receptors. Despite some differences: inactivity of
sulphonic acid derivatives and the loss of relative potency of rigid analogues of GABA,
such as muscimol, THIP and isoguvacine, the agonist profile of Ascaris muscle
GABA receptor appears correlated with the agonist profile of vertebrate GABA-A
receptors.In contrast, the antagonist profile of Ascaris GABA receptors differs greatly from
the antagonist profile of the vertebrate GABA-A receptor. The two classical GABA-A
antagonists, picrotoxin and bicuculline, were found very weak or inactive at
antagonizing GABA responses in Ascaris. The steroid derivative, RU5135, a very
potent competitive antagonist in vertebrate (IC50=5nM, rat cuneate nucleus, Simmonds
and Turner, 1985), was found to hold back some activity in Ascaris , but in a non
competitive manner and with an IC50 of 117μM.The effects of the arylaminopyridazine GABA derivatives SR95103 and SR95531
on GABA responses were investigated on GABA-induced conductance changes using a
current-clamp technique. Arylaminopyridazine GABA derivatives are specific and
competitive antagonists at the vertebrate GABA-A receptor, SR95531 being the most
potent analogue. In Ascaris SR95103 was found more potent than SR95531 at
antagonizing GABA responses. This potency order contrasts with that at vertebrate
GABA-A receptors. The antagonism of SR95103 was associated with a parallel shift to
the right in the GABA dose-response relationship. A modified Schild plot was used to
describe the action of SR95103, the data is consistent with a competitive mechanism
involving two molecules of GABA but one molecule of antagonist interacting with the
receptor. The estimated KB for SR95103 is 64±13μM (mean±SE, n=14) and the
pA₂=4.The actions of SR95103 were further examined on GABA-activated single-channel
currents using an outside-out patch-clamp technique. The presence of SR95103 (30-
100μM) did not alter the GABA-activated channel main conductance, but reduced the
open-probability of the channels. No effect on the mean burst duration, corrected mean
open-time and the distribution of burst-durations, was observed with 30μM SR95103.
However, when a higher concentration was used, 100μM, mean open-time and mean
burst duration were reduced, and the proportion of short bursts (mainly represented by
single openings) was increased. The mode of action of SR95103 is discussed, and it is
suggested that most of the antagonism is competitive and produced by a single molecule
of SR95103 combining with the receptor-channel complex, as suggested by the
current-clamp data. However, an additional non-competitive component, possibly a
channel block, is detected at high concentrations. This non-competitive component
accounts only for a small proportion of the antagonism, since no reduction in the
maximal response was detected by the current-clamp technique, even when high
antagonist concentrations were used (ImM).Other arylaminopyridazine derivatives were tested on GABA response in Ascaris
using a current-clamp technique. The order of potency of these compounds on Ascaris
muscle GABA receptor, was found different from the order of potency at the vertebrate
GABA-A receptor. One analogue, SR95132, virtually inactive in vertebrate
preparations was found equipotent to SR95103 in Ascaris. When tested on GABA
dose-response curves, SR95132 (KB=65μM) and other potent analogues, displaced
GABA dose-response curves to the right without decrease in the maximal response.
The modified Schild plots for these compounds, were also consistent with a
competitive mechanism involving two molecules of GABA and only one molecule of
antagonist interacting with the receptor. Structure-activity relationships for this series of
compounds are examined in Ascaris and compared to the vertebrates. Substitution on
the pyridazine ring in 4-position, while detrimental for the antagonist potency at the
vertebrate GABA-A receptor, appears to be a prerequisite for antagonistic activity on
Ascaris muscle GABA receptor. Newly synthesized arylaminopyridazine derivatives
confirmed that the presence of a substituent in the 4-position is very important for the
antagonistic activity at the Ascaris GABA receptor. The ethyl-4-substituted derivative,
NCS247-90 with Kb=55μM, was found to be more potent than SR95103 and
SR95132. Benzyl- (NCS251-90) and the isopropyl-(NCS252-90) 4-substituted
derivatives also appeared to be potent antagonists. Besides the role of the 4-position, the length of the side chain and the acidic function were also investigated. A longer
side-chain (5C), as in NCS194-83, decreaseithe potency but does not produce a
complete loss of potency. Replacement of the carboxylic acid group by a sulphonic acid
group produceja total inactivation of the compound, as seen with NCS249-90 and
NCS250-90. This is in agreement with the lack of potency of sulphonic acid derivatives
as agonists. These results are discussed in terms of the accessory binding sites theory
of Ariens, and suggest the existence of different accessory sites responsible for the
binding of antagonists on Ascaris GABA receptor.In conclusion, the Ascaris muscle GABA receptor differs from vertebrate GABA-A
receptors in terms of antagonist properties, whereas its agonist properties are very
similar to those of vertebrate GABA-A receptors. Among GABA antagonists, the
exploration of the arylaminopyridazine GABA derivatives family may lead to the
discovery of a new generation of anthelminthics. Already, one analogue, SR95132,
shows an antagonistic activity in Ascaris but not in vertebrates
