Aplysia Nervous Tissue Research Articles

Effects of hypotonic stress and ouabain on the apparent diffusion coefficient of water at cellular and tissue levels inAplysia

There is evidence that physiological or pathological cell swelling is associated with a decrease of the apparent diffusion coefficient (ADC) of water in tissues, as measured with MRI. However the mechanism remains unclear. Magnetic resonance microscopy, performed on small tissue samples, has the potential to distinguish effects occurring at cellular and tissue levels. A three-dimensional diffusion prepared fast imaging with steady-state free precession sequence for MR microscopy was implemented on a 17.2 T imaging system and used to investigate the effect of two biological challenges known to cause cell swelling, exposure to a hypotonic solution or to ouabain, on Aplysia nervous tissue. The ADC was measured inside isolated neuronal soma and in the region of cell bodies of the buccal ganglia. Both challenges resulted in an ADC increase inside isolated neuronal soma (+31 ± 24% and +30 ± 11%, respectively) and an ADC decrease at tissue level in the buccal ganglia (-12 ± 5% and -18 ± 8%, respectively). A scenario involving a layer of water molecules bound to the inflating cell membrane surface is proposed to reconcile this apparent discrepancy.

MALDI mass spectrometric imaging using the stretched sample method to reveal neuropeptide distributions in aplysia nervous tissue.

Neuropeptides are a diverse set of complex cell-cell signaling molecules that modulate behavior, learning, and memory. Their spatially heterogeneous distributions, large number of post-translational modifications, and wide range of physiologically active concentrations make their characterization challenging. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometric imaging is well-suited to characterizing and mapping neuropeptides in the central nervous system. Because matrix application can cause peptide migration within tissue samples, application parameters for MALDI typically represent a compromise between attaining the highest signal quality and preserving native spatial distributions. The stretched sample approach minimizes this trade-off by fragmenting the tissue section into thousands of spatially isolated islands, each approximately 40 mum in size. This inhibits analyte migration between the pieces and, at the same time, reduces analyte-salt adduct formation. Here, we present methodological improvements that enable the imaging of stretched tissues and reveal neuropeptide distributions in nervous tissue from Aplysia californica. The distributions of known neuropeptides are shown to correspond with previous immunohistochemical results, demonstrating that the stretched imaging method is well-suited for working with easily redistributed molecules and heterogeneous tissues and reduces adducts from physiological salts.

Identification of an 8-Lipoxygenase Pathway in Nervous Tissue of Aplysia californica

Arachidonic acid is converted to (8R)-hydroperoxyeicosa-5,9,11, 14-tetraenoic acid (8-HPETE) during incubations with homogenates of the central nervous system of the marine mollusc, Aplysia californica. 8-HPETE can be reduced to the corresponding hydroxy acid or be enzymatically converted to a newly identified metabolite, 8-ketoeicosa-5,9,11,14-tetraenoic acid (8-KETE). These metabolites were identified by high performance liquid chromatography, UV absorbance, and gas chromatography/mass spectrometry. Stereochemical analysis of the products demonstrate that the neuronal enzyme is an (8R)-lipoxygenase. Previously we have shown that the neurotransmitters, histamine and Phe-Met-Arg-Phe-amide, activate 12-lipoxygenase metabolism in isolated identified Aplysia neurons. We now show that acetylcholine activates the (8R)-lipoxygenase pathway within intact nerve cells. Thus, both (12S)- and (8R)-lipoxygenase co-exist in intact Aplysia nervous tissue but are differentially activated by several neurotransmitters. The precise physiological role of the 8-lipoxygenase products is currently under investigation, but by analogy to the well-described 12-lipoxygenase pathway, we suggest that (8R)-HPETE and 8-KETE may serve as second messengers in Aplysia cholinoceptive neurons.

Cloning and characterization of aplycalcin and Aplysia neurocalcin, two new members of the calmodulin superfamily of small calcium-binding proteins.

We report here the sequences of two new proteins from Aplysia, aplycalcin and Aplysia neurocalcin. These proteins belong to a family of calcium-binding proteins, found primarily in vertebrate brain and retina, that have been proposed to play a role in calcium-dependent regulation of enzymes in signal transduction pathways. Like other members of this family, the Aplysia proteins have consensus sequences for myristoylation, bind calcium, and translocate from cytosol to membrane when the calcium level is raised above the resting intracellular concentration. Both proteins are relatively enriched in Aplysia nervous system, but are also found to a significant degree in other tissues. The expression of mRNA for these proteins in Aplysia nervous tissue is regulated during development, roughly paralleling the reported emergence of several forms of synaptic plasticity. The messages are present at low levels in stage 11, show a large increase by late stage 12, and decline to a plateau of approximately 30% of the peak value afterward. On the basis of the properties of these proteins and by analogy with proposed functions of some of the retinal homologues, we suggest that these proteins may play a role in mediating calcium-dependent processes in neuronal function. The presence of both proteins in other tissues may suggest analogous roles for the proteins in other cell types.

Primary structure of a new neuropeptide, cerebral peptide 2, purified from cerebral ganglia of Aplysia.

We report the purification and characterization of a novel neuropeptide from Aplysia nervous tissue. The peptide was termed cerebral peptide 2 (CP2) because it was the larger of two peptides predominantly synthesized in the cerebral ganglia and transported to other regions of the central nervous system. The purification of CP2 from extracts of cerebral ganglia using three sequential modes of high-pressure liquid chromatography (HPLC) was followed using the [35S]methionine-labeled peptide obtained from transport experiments. The primary structure of CP2 was determined by automated Edman degradation of native CP2 and its proteolytic fragments in conjunction with mass spectrometry. CP2 is a 41 amino acid peptide with an amidated carboxyl terminal. A peptide with the proposed sequence of CP2 was synthetized and compared by HPLC with the native peptide. Chromatographic properties of the synthetic and native peptide labeled in vivo were found to be identical. CP2 does not appear to be a member of any previously identified peptide family.

Molecular Cloning, cDNA Sequence, and Localization of a Prohormone Convertase (PC2) from theAplysiaAtrial Gland

Neuropeptides and peptide hormones are synthesized as part of larger precursor proteins that are processed post-translationally by subtilisin-related calcium-dependent prohormone convertases (PCs), frequently at multiple basic sites, to generate biologically active peptides. The atrial gland of Aplysia californica produces large quantities of egg-laying hormone (ELH)-related peptides, providing a unique opportunity to study prohormone processing. We have screened an Aplysia atrial gland cDNA library using a Lymnaea stagnalis PC2 probe and have isolated an Aplysia PC2-related 4.6-kb cDNA partial clone that was truncated on the 5' end. The remaining 5' atrial gland PC2 nucleotide sequence was obtained by reverse transcription/polymerase chain reaction (RT-PCR). The composite cDNA structure (5.6 kb) was deduced from sequence analysis of the RT-PCR product combined with the sequence obtained from the cDNA clone. The deduced cDNA of Aplysia atrial gland PC2 encoded a putative preproendoprotease of 653 amino acids that was evolutionarily related to other eukaryotic PC2s, and showed the strongest sequence identity with recently reported Aplysia nervous tissue PC2 sequences. In situ hybridization demonstrated extensive expression of PC2 in atrial gland secretory cells. The cDNA clone contained a relatively long 3'untranslated region (3'-UTR) of 3,632 nucleotides. Strikingly, the 3'-UTR also contained several major nucleotide repeat sequences including the microsatellite repeats, (CA)n and (TG)n, and a TA-rich region comprised largely of the triplet repeat (TTA)n. The characterized Aplysia PC2 is a candidate endoprotease that may play an important role in the processing of ELH-related precursors in the atrial gland and represents the first example of PC2 expression in exocrine tissue.

Translocation of protein kinase Cs in Aplysia neurons: evidence for complex regulation

We examined the activation of protein kinase C (PKC) produced by phorbol esters in Aplysia nervous tissue. Translocation of PKC in intact ganglia requires higher concentrations of phorbol esters than would be expected from: (1) their affinity for Aplysia PKCs measured in vitro; (2) their physiological effects on cultured Aplysia neurons; and (3) their actions on PKC in synaptosomes. Although phorbol esters enter intact ganglia slowly, delayed access to neurons is insufficient to account for the high concentrations needed for translocation. Increasing accessibility to the neural components of ganglia increases the rate at which translocation occurs, but does not affect the concentration of phorbol ester required. We suggest that this might best be explained by the presence of a competitive inhibitor at the binding site for phorbol esters in PKC. An indication for an inhibitor is that the concentration of phorbol esters needed for translocation in homogenates of nervous tissue is markedly decreased by diluting the extract. Preliminary characterization shows that the inhibitory activity is unusual: in addition to being competitive with lipid activators, it is soluble and tissue-specific. This type of inhibitor may be an important regulator of protein phosphorylation by PKC in neurons.

Regulatory subunits of cAMP-dependent protein kinases are degraded after conjugation to ubiquitin: a molecular mechanism underlying long-term synaptic plasticity.

In Aplysia, behavioral sensitization of defensive reflexes and the underlying presynaptic facilitation of sensory-to-motor neuron synapses lasts for several minutes (short term) or days to weeks (long term). Short-term sensitization has been explained by modulation of ion-channel function through cAMP-dependent protein phosphorylation. Long-term facilitation requires additional molecular changes including protein synthesis. A key event is the persistent activation of the cAMP-dependent protein kinase at baseline concentrations of cAMP. This activation is due to selective loss of regulatory (R) subunits of PKA without any change in catalytic (C) subunits. To understand the molecular mechanisms that produce the loss of R subunits in long-term facilitation, we investigated how R subunits are degraded in extracts of Aplysia nervous tissue and in rabbit reticulocyte lysates. Degradation of Aplysia R subunits requires ATP, ubiquitin, and a particulate component that appears to be the proteasome complex. Degradation is blocked by hemin, which causes the accumulation of high molecular weight derivatives of R subunits that are likely to be ubiquitin conjugates of R subunits and intermediates in the degradative pathway. We also show that vertebrate RI and RII subunits can be degraded through the ubiquitin pathway. We suggest that degradation is initiated by cAMP, which causes the holoenzyme to dissociate and, further, that the altered R-to-C ratio in Aplysia sensory neurons is maintained in long-term facilitation by newly synthesized proteins that help target R subunits for accelerated degradation.

A signal sequence mediates the retrograde transport of proteins from the axon periphery to the cell body and then into the nucleus

The presynaptic terminal and axon of neurons can undergo structural changes in response to environmental signals. Since these changes require protein synthesis in the cell body, the needs of the periphery must somehow be communicated to the cell soma. To look for such a mechanism, we used artificial protein constructs with properties expected of a signal that is transported from the axon to the nucleus. One construct consisted of the nuclear import signal peptide (sp) of the SV40 large T antigen, coupled to human serum albumin (HSA) and rhodamine (r). When injected into the axoplasm of Aplysia californica neurons in vitro, the rHSA-sp was transported in the retrograde direction through the axon to the cell body and then into the nucleus. Little, if any, moved in the anterograde direction toward growth cones. The retrograde movement of injected rHSA-sp was rapid (greater than 25 mm/d) and depended upon intact microtubules. The sp portion of rHSA-sp provided access to both the retrograde transport system and the nuclear import apparatus. Thus, rHSA was not transported at all, but accumulated in organelles near the injection site. Also, rHSA-sp containing an sp with a Lys to Thr substitution, which is known to reduce nuclear import markedly, was transported only poorly. To look for endogenous molecules that use this system, we affinity-purified a rabbit polyclonal antibody to the signal sequence. The antibody recognized an 83 kDa polypeptide on Western blots of Aplysia nervous tissue. These data indicate that Aplysia neurons contain the machinery to convey macromolecules from the axon periphery to the nucleus.

Aplysia californica contains a novel 12-lipoxygenase which generates biologically active products from arachidonic acid.

Physiologic stimulation of identified neurons in ganglia of the marine mollusk, Aplysia californica, leads to the generation of arachidonic acid metabolites. Using various preparations of Aplysia nervous tissue, we have identified 12-lipoxygenase products including the inactive 12-hydroxyeicosatetraenoic acid (12-HETE) and the biologically active 12-ketoeicosatetraenoic acid (12-KETE) and 8-hydroxy-11(12)-epoxyeicosatrienoic acid (8-HEpETE). Each of these metabolites can be derived from the intermediate 12-hydroperoxyeicosatetraenoic acid (12-HPETE), which can itself activate several identified neurons in Aplysia. In spite of conflicting results in studies of mammalian brain 12-lipoxygenase, Aplysia nervous tissue clearly contains an enzymatic activity which generates stereochemically pure 12(S)-HETE. This activity is destroyed by boiling and is sensitive to nonspecific lipoxygenase inhibitors but not to agents specific for other lipoxygenases or the cyclooxygenase enzyme. The Aplysia 12-lipoxygenase is highly enriched in neural tissue and is almost completely absent in the neural sheath, which is composed primarily of connective tissue and muscle. Preliminary purification has shown that, in contrast to the previously characterized 12-lipoxygenases, the Aplysia enzyme is associated with membrane fractions and is not found in the cytosol. Further studies are in progress to determine the kinetic properties and to define the cellular and subcellular distribution of this novel lipoxygenase.

Products of heme-catalyzed transformation of the arachidonate derivative 12-HPETE open S-type K + channels in Aplysia

In Aplysia mechanosensory neurons, the neuropeptide FMRFamide increases the opening of the background S-K + channel. This action is mediated by activation of arachidonic acid metabolism. Arachidonic acid in Aplysia nervous tissue is transformed through the 12-li-poxygenase pathway to 12-HPETE, which undergoes further metabolism. In intact sensory cells, 12-HPETE simulates the FMRFamide response, raising the question of whether 12-HPETE is the messenger molecule ultimately acting on the S-K + channel. Here we show that in cell-free (inside-out) patches from sensory cells, 12-HPETE fails to modulate the S-K+ channel, but in the presence of hematin (which catalyzes 12-HPETE metabolism), it triggers sharp increases in the channel opening probability. We also found that SKF525A, an inhibitor of the cytochrome P 450, reduces the response to FMRFamide, arachidonic acid, and 12-HPETE in intact cells. We conclude that a heme-catalyzed transformation of 12-HPETE is necessary and sufficient to promote the opening of the S-K+ channel and a hemecontaining enzyme such as cytochrome P 450, might play this key role.

12‐Keto‐Eicosatetraenoic Acid A Biologically Active Eicosanoid in the Nervous System of Aplysia

The lipoxygenase product 12-hydroperoxy-5,8,10,14-eicosatetraenoic acid (12-HPETE), stimulates the synaptic response produced by the modulatory transmitter histamine and the neuroactive peptide Phe-Met-Arg-Phe-amide (FMRFamide) in identified neurons of the marine mollusk Aplysia californica. The 12-lipoxygenase pathway has not yet been fully characterized, but 12-HPETE is known to be metabolized further. Therefore, we began to search for other metabolites in order to investigate whether the actions of 12-HPETE might require its conversion to other active products. We have identified 12-keto-5,8,10,14-eicosatetraenoic acid (12-KETE) as a metabolite of 12-HPETE formed by Aplysia nervous tissue. 12-KETE was identified in incubations of the tissue with arachidonic acid using HPLC, UV spectrometry, and gas-chromatography/mass spectrometry. [3H]12-KETE is formed from endogenous lipid stores in nervous tissue, labeled with [3H]arachidonic acid upon stimulation by application of histamine. In L14 and L10 cells, identified neurons in the abdominal ganglion, applications of 12-KETE elicit changes in membrane potential similar to those evoked by histamine. Another metabolite of 12-HPETE, 12(s)-hydroxy-5,8,10,14-eicosatetraenoic acid [12(S)-HETE], is inactive. These results support the hypothesis that 12-HPETE and its metabolite, 12-KETE, participate in transduction of histamine responses in Aplysia neurons.

Sequence of pedal peptide: a novel neuropeptide from the central nervous system of Aplysia

We report the identification of a novel neuropeptide from Aplysia nervous tissue. The peptide was termed Pedal peptide (Pep) because it was predominantly synthesized in the pedal ganglia. Pep was purified and sequenced from pooled extracts of pedal ganglia. The following sequence was proposed: Pro-Leu-Asp-Ser-Val-Tyr-Gly-Thr-His-Gly-Met-Ser-Gly-Phe-Ala. Enzymatic hydrolysis procedures indicated that Pep had a free carboxyl terminal. A peptide with the proposed sequence was synthesized and compared with the native peptide. Chromatographic properties of the 2 peptides under 3 different conditioned were compared and found to be identical. Electrophysiological responses to the 2 peptides were compared on an identified neuron in the abdominal ganglia and found to be qualitatively and quantitatively very similar. Both peptides produced net inward currents that were associated with a decrease in membrane conductance. The results from these 2 procedures confirmed that the proposed Pep sequence was correct. Quantitative measurements of the incorporation of 35S-methionine into Pep suggest that cell bodies that synthesize Pep were present predominantly in the pedal ganglia but should also be found in other central ganglia as well. Pep-like immunoreactive neurons are found predominantly in the pedal ganglia and less frequently in the other ganglia (Pearson and Lloyd, 1989). Quantitatively, Pep constitutes one of the predominant peptides in the nervous system of Aplysia. Pep does not appear to be a member of any other previously identified invertebrate or vertebrate peptide family.

G proteins in Aplysia: biochemical characterization and regional and subcellular distribution

We studied G proteins and regulation of adenylate cyclase in nervous tissue and muscle Aplysia using bacterial toxin-catalyzed ADP-ribosylation. We identified G 5α, a M r 45,000 cholera toxin substrate, G 0α, a M r 40,000 pertussis toxin substrate, and G β (M r 37,000) by Western blot analysis with antisera specific for bovine brain G protein subunits. Partial proteolysis suggests that the neuronal pertussis toxin substrates are heterogenous. The concentration of these substrates in membranes from Aplysia ganglia is similar to that of rat, squid and Helix; in Aplysia nervous tissue, G protein subunits are most enriched in synaptosomes and neuropil. The stimulation of adenylate cyclase by serotonin (5-HT), low concentrations of GTP-γ-S, and cholera toxin, and the inhibition by high concentrations of GTP-γ-S that is blocked by pertussis toxin indicate that both a G s and a G i protein regulate that Aplysia enzyme. These results support the idea that G proteins in Aplysia are important in regulating synaptic function.

Formation and biological activity of 12-ketoeicosatetraenoic acid in the nervous system of Aplysia.

12-Hydroperoxy-5,8,10,14-eicosatetraenoic acid (12-HPETE), a lipoxygenase product, simulates the synaptic responses produced by the modulatory transmitter, histamine, and the neuroactive peptide, Phe-Met-Arg-Phe-amide (FMRFamide), in identified neurons of the marine mollusk, Aplysia californica (Piomelli, D., Shapiro, E., Feinmark, S. J., and Schwartz, J. H. (1987) J. Neurosci. 7, 3675-3886; Shapiro, E., Piomelli, D., Feinmark, S., Vogel, S., Chin, G., and Schwartz, J. H. (1988) Cold Spring Harbor Symp. Quant. Biol. 53, in press). The 12-lipoxygenase pathway has not yet been fully characterized, but 12-HPETE is known to be metabolized further. We therefore began to search for other metabolites in order to investigate whether the actions of 12-HPETE might require its conversion to other active products. Here we report the identification of 12-keto-5,8,10,14-eicosatetraenoic acid (12-KETE), a metabolite of 12-HPETE formed by Aplysia nervous tissue. This product was identified in incubations of the tissue with arachidonic acid using high performance liquid chromatography, UV spectrometry, and gas chromatography/mass spectrometry. [3H]12-KETE was formed from endogenous lipid stores in nervous tissue, labeled by incubation with [3H]arachidonic acid, when stimulated by application of histamine. In L14 and L10 cells, identified neurons in the abdominal ganglion, applications of 12-KETE elicit changes in membrane potential similar to those evoked by histamine. 12(S)-Hydroxy-5,8,10,14-eicosatetraenoic acid, another metabolite of 12-HPETE, is inactive. These results support the hypothesis that 12-HPETE and its metabolite, 12-KETE, participate in transduction of histamine responses in Aplysia neurons.

Abundant expression of ras proteins in Aplysia neurons.

We have cloned a DNA fragment from the marine mollusc Aplysia californica, which contains sequences homologous to mammalian ras genes, by screening a genomic library with a viral Ha-ras oncogene probe under conditions of low stringency hybridization. Nucleotide sequencing revealed a putative exon that encodes amino acids sharing 68% homology with residues 5 to 54 of mammalian p21ras polypeptides, and which therefore is likely to encode a ras-like Aplysia protein. The cloned locus, designated Apl-ras, is distinct from the Aplysia rho (ras-homologue) gene and appears to be more closely related to mammalian ras. We used a panel of monoclonal antibodies raised against v-Ha-ras p21 to precipitate an Mr 21,000 protein from extracts of Aplysia nervous tissue, ovotestis, and, to a much lesser degree, buccal muscle. Fluorescence immunocytochemistry revealed that ras-like protein is most abundant in neuronal cell bodies and axon processes, with staining most prominent at plasma membranes. Much less was present in other tissues. The prominence of ras protein in neurons, which are terminally differentiated and non-proliferating, indicates that the control of cell division is not the sole function of this proto-oncogene. The large identified neurons of Aplysia offer the opportunity to examine how ras protein might function in mature nerve cells.

Enkephalin-like substance in Aplysia nervous tissue and actions of leu-enkephalin on single neurons

Extract from the abdominal ganglia of Aplysia was fractionationed by high performance liquid chromatography. Several large uv absorbing peaks were found. One of these peaks from the ganglionic extract was analyzed by displacement assay with the use of Mytilus edulis membranes. The results revealed the substance in this peak was able to displace 3H-D-Ala2, met5-enkephalinamide. In electrophysiological studies of abdominal and cerebral neurons, depolarizing and hyperpolarizing responses were obtained from some neurons, including neurons in the identified cerebral B cell cluster. A smaller population of cells exhibited biphasic responses. Some of the responses could be depressed by prior naloxone treatment. In conclusion, an endogenous opioid system, using substance related to but distinct from the enkephalins, may exist in Aplysia .

Evidence for the inhibitory subunit of adenylate cyclase (N i) in nervous and heart tissue of Aplysia

Cyclic adenosine 3′,5′-monophosphate (cAMP) plays a critical role in modulating a variety of neuronal responses in Aplysia californica. Previous studies have focused on the neurotransmitter activation of adenylate cyclase, which presumably occurs via the guanosine 5′-triphosphate (GTP)-regulated excitatory subunit (N s). While adenylate cyclase has also been shown to be regulated by inhibitory neurotransmitters, coupled through the inhibitory GTP-regulated coupling protein N i in some systems, the effects of N i-mediated adenylate cyclase inhibition on neuronal processing in Aplysia have not previously been reported. In the present study N i is detected in Aplysia by both protein chemistry and enzymatic activity. A 40 kdalton substrate for the enzymatic activity of Bordetella pertussis toxin is observed. Incubation of Aplysia nervous tissue homogenates with pertussis toxin (IAP) and 32P-nicotinamide-adenine dinucleotide labels a single protein, assessed by polyacrylamide gel electrophoresis and autoradiography. Furthermore, crude membrane suspensions of this tissue demonstrate biphasic adenylate cyclase activity in response to increasing concentrations of GTP, showing N i and N s functional activities. These findings provide evidence that N i is present in Aplysia tissue. N i may serve as an important site for the regulation of cAMP synthesis and neuronal plasticity.

Structural studies on a family of cAMP-binding proteins in the nervous system of Aplysia.

Five major cAMP-binding proteins that differ in size and charge have been identified in neurons of Aplysia californica by photoaffinity labeling with [32P]8-N3cAMP. These proteins, which we believe are regulatory subunits of cAMP-dependent protein kinase, all differ from the major cAMP-binding protein of buccal muscle. We have compared the structures of these proteins by peptide mapping after chemical and proteolytic cleavage. These analyses indicate that the five binding proteins from nervous tissue and the major muscle protein are closely related to each other. For example, the three neuronal proteins that are most alike and the cAMP-binding protein from muscle have a similar, if not identical, Mr 20,000 domain that contains the 8-N3cAMP-binding site; beyond this domain they diverge. All six proteins appear to belong to a family in which homologous regions have been conserved to maintain common functions. We suggest that the regions of the molecules that differ mediate special functions such as ticketing to particular compartments of the cell. Evidence for regional assortment of the cAMP-dependent protein kinases according to structural type was afforded by subcellular fractionation of Aplysia nervous tissue; photoaffinity labeling of cytoplasm, cytoskeleton, and membrane fractions demonstrated a differential distribution of the five neuronal cAMP-binding proteins. Selective phosphorylation of specific substrates could be a consequence of the compartmentation of diverse cAMP-dependent kinases.

Stereoselective L-[3H]quinuclidinyl benzilate-binding sites in nervous tissue of Aplysia californica: evidence for muscarinic receptors

The muscarinic antagonist L-[3H]quinuclidinyl benzilate (L-[3H]QNB) binds with a high affinity (Kd = 0.77 nM) to a single population of specific sites (Bmax = 47 fmol/mg of protein) in nervous tissue of the gastropod mollusc, Aplysia. The specific L-[3H]QNB binding is displaced stereoselectively by the enantiomers of benzetimide, dexetimide, and levetimide. The pharmacologically active enantiomer, dexetimide, is more potent than levetimide as an inhibitor of L-[3H]QNB binding. Moreover, the muscarinic cholinergic ligands, scopolamine, atropine, oxotremorine, and pilocarpine are effective inhibitors of the specific L-[3H]QNB binding, whereas nicotinic receptor antagonists, decamethonium and d-tubocurarine, are considerably less effective. These pharmacological characteristics of the L-[3H]QNB-binding site provide evidence for classical muscarinic receptors in Aplysia nervous tissue. The physiological relevance of the dexetimide-displaceable L-[3H]QNB-binding site was supported by the demonstration of the sensitivity of the specific binding to thermal denaturation. Specific binding of L-[3H]QNB was also detected in nervous tissue of another marine gastropod, Pleurobranchaea californica. The characteristics of the Aplysia L-[3H]QNB-binding site are in accordance with studies of numerous vertebrate and invertebrate tissues indicating that the muscarinic cholinergic receptor site has been highly conserved through evolution.