Figure 1.
Application of 10−4 M ACh evoked long lasting contraction.
Consecutive agonist application (arrows) did not show desensitization of the M3 muscle. The preparation was continuously perfused with physiological saline at a rate of approximately 1 ml/min. The values on the Y axis represent the force of contractions expressed in grams.
Figure 2.
A-Effect of nicotinic and muscarinic agonists on the flexor muscle.
Responses are expressed as % of the contraction elicited by 10−4 M ACh. Nicotinic agonist nicotine, carbachol (Carb), TMA, Succ and the α7- selective ChCl and PNU-282987 (PNU) applied at 10−4 M elicited muscle contraction comparable to that evoked by ACh. Muscarinic agonist Oxo and Arec applied at 10−4 M proved to be practically ineffective. n = number of muscles. B-Effect of α4 and α7 subunit specific agonists on flexor muscle applied at increasing concentrations (50 nM, 100 nM and 1 µM). RJR 2403 had no effect. 3-BrCy evoked muscle contraction in a concentration dependent manner. n = number of muscles.
Figure 3.
A-Relative effectiveness of nicotinic antagonists to block contractions of the flexor muscle evoked by 10−5 M ACh.
Antagonists were applied 5 min prior to ACh administration. B – Effect of MLA and DHβE at increasing concentrations (1 nM, 100 nM and 100 µM) on 10−6 M ACh-evoked contractions. n = number of muscles. Asterisks indicate a significant difference from the control value at *P<0.05, **P<0.01.
Figure 4.
The relative effect of specific α7-nAChR antagonists to block ACh (10−5 M) elicited responses.
A- αBgTx attenuated the ACh evoked contractions in a concentration dependent manner. The blocking effects of 50 nM and 100 nM toxin were statistically significant. The recovery is almost complete. B- The block by 50 nM αA-CTx PIVA was not significant, while the α-CTx ImI at 100 nM blocked almost completely the ACh elicited contraction. The recovery from block was partial. n = number of muscles. Asterisks indicate a significant difference from the control value: *P<0.01.
Figure 5.
Relative effectiveness of cys-loop receptor antagonists to block ACh (10−5 M) elicited contractions.
All antagonists were applied at 10−4 M 5 min prior to ACh administration. Orph, the specific mAChR antagonist proved to be ineffective. Atr is effective at all AChRs of mollusks. n = number of experiments. Asterisks indicate a significant difference from the control value: *P<0.01.
Figure 6.
Demonstration of nAChR subtypes by western blotting using different nAChR subunit specific antibodies.
A - Immunodetection of the α7 subunit in homogenates of central nervous system (CNS), flexor (FM) and columellar muscle (CM). The ab10096 recognized two bands (∼60 and 90 kDa) in the CNS which were blocked by the control peptide of the antibody (arrows). In the FM, two labeled bands (∼62 and 110 kDa) were present which could not be totally eliminated with the immunogen. In the CM, the ∼110 kDa band (but not the ∼60 kDa band) was blocked (arrow). B - Another α7 specific antibody, the ANC-007 labeled a single band in the CNS at ∼160 kDa, which was blocked with the immunogen (arrow). C – The α4 specific antibody ANC-004 labeled several bands between ∼80 and 110 kDa both in the CNS and FM, which were completely eliminated by the blocking peptide. Arrow shows the band of the size which is suggested by the producer.
Figure 7.
Distribution of ACh receptors containing the α4 subunit of nicotinic ACH receptor in the flexor muscles (A–C) and the cerebral ganglion (D).
The α4 immunoreactivity is confined to neuronal elements in both the flexor muscles and the ganglion. In the flexor muscle M1 (A) trunks of axons (thick arrows) run along the longitudinal axes of the muscles. In the nerve trunks (B–C) fine immunolabeled axons could be seen (thin arrows). In the cerebral ganglion (D) the majority of labeled cell bodies is located in the pleural lobe (pll) and is gathered in loose groups (empty asterisks). Compact groups of small neurons can also be observed (asterisk). mc- mesocerebrum, pc- procerebrum, cplc- cerebro-pleural connective, pn- perineurium. bar: 50 µm.
Figure 8.
Immunoreactivity revealed by α7 subunit Ab in the cerebral ganglion (A), M1, M3 flexor muscles (B–D), and the columellar muscle (E) as well as in the stem of tentacle (F–G).
In the cerebral ganglion (A) immunoreactivity can be seen in groups of small neurons (empty asterisks) in the mesocerebrum (mc) and procerebrum (pc). Additionally a few of immunolabeled varicose fibers (arrows) can also be observed. In the flexor muscles (B) immunoreactivity is distributed on thin muscle fiber trunks (arrows) and displayed dotted appearance (arrow heads) (B–C) which is a sign of the postsynaptic localization. In the columellar muscle (E) immunoreactivity is distributed on the surface of muscle fibers (black arrows). The immunoreactivity on the sarcolemma displays dotted appearance (insert, arrow heads). In the stem immunoreactivity (arrows) is located on the gland cells (gc) (F) and the wall of haemolymph vessels (v) (G). In both cases the immunoreactivity (arrow heads) shows dotted appearance (see inserts F, G). bar: 50 µm.
Figure 9.
PCR products of LnAChR A subunit expressed in different tissues of snail.
Left - DNA ladder, lane 1 - Lymnaea CNS, lane 2 - Helix CNS, lane 3 - Helix columellar muscle, lane 4 - Helix flexor muscle, loaded on ethidium bromide stained gel. In all lanes the calculated weight of the amplified product was ∼120 bp. DNA ladder lane shows bands at 100 bp increments. The template cDNS in the following amounts were added: 1 µl CNS cDNS, 2 µl CM cDNS and 4 µl FM cDNS in order to maintain the same volume for the PCR reaction. Accordingly, the amount of Master Mix was decreased proportionately.