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The molluscicidal activity of abamectin were assessed compared with methomyl against the most two abundant snail species brown garden snail (BGS) Eobania vermiculata and white garden snail (WGS) Theba pisana under laboratory conditions. The results indicated that LC50 values of methomyl were almost equal LC50 values of abamectin indicating more molluscicidal potency of abamectin and methomyl. Results also revealed a complete consistency of correlation between the LC50 values and exposed time intervals. The high molluscicidal activity of abamectin against E. vermiculata and T. pisana compared to methomyl using topical application technique could be used practically in the field of land snails control program and overcome some drawbacks of using bait forms in this respect. Comparing the LD50 values indicate the significant variation of the molluscicidal activity between the two applications methods used in addition to the variation between the two species of chosen snails, the brown garden snail E. vermiculata were resistant species than the white garden snail T. pisana. The molluscicidal activity of abamectin were assessed compared with methomyl against the most two abundant snail species brown garden snail (BGS) Eobania vermiculata and white garden snail (WGS) Theba pisana under laboratory conditions. The results indicated that LC50 values of methomyl were almost equal LC50 values of abamectin indicating more molluscicidal potency of abamectin and methomyl. Results also revealed a complete consistency of correlation between the LC50 values and exposed time intervals. The high molluscicidal activity of abamectin against E. vermiculata and T. pisana compared to methomyl using topical application technique could be used practically in the field of land snails control program and overcome some drawbacks of using bait forms in this respect. Comparing the LD50 values indicate the significant variation of the molluscicidal activity between the two applications methods used in addition to the variation between the two species of chosen snails, the brown garden snail E. vermiculata were resistant species than the white garden snail T. pisana. Keywords: Molluscicides, Abamectin, Methomyl, Eobania vermiculata and Theba pisana

The purpose of this study was to investigate the effects of 9-amino-1,2,3,4-tetrahydroacridine (THA; Tacrine) on muscarinic-receptor-linked second-messenger systems in rat brain and to determine the selectivity and mechanisms of these effects. Both competitive and noncompetitive antagonism was revealed in saturation radioligand binding studies performed in cortical and striatal tissue, depending on THA concentration. Micromolar THA concentrations blocked muscarinic-receptor-mediated inhibition of cAMP formation and stimulation of phosphoinositide (PI) hydrolysis with poor selectivity between the two responses. While both responses were blocked in the same concentration range (4-60 mumol/l), non-competitive antagonism of PI hydrolysis occurred at THA concentrations greater than 10 mumol/l while competitive antagonism was displayed for the cAMP response at concentrations of THA up to 40 mumol/l. THA was equally effective at inhibiting PI hydrolysis stimulated by histamine, phenylephrine or oxotremorine-M, when these agonists were employed in concentrations equal to their EC50s for the response. THA did not antagonize PI hydrolysis mediated by the quisqualate receptor at any agonist concentration used. Furthermore, THA blocked carbachol- but not morphine-induced inhibition of forskolin-stimulated cAMP formation in the striatum.

The effects of the organophosphorus anticholinesterase paraoxon on the binding of radioactive ligands to the M3 subtype of the muscarinic receptor and receptor-coupled synthesis of second messengers in intact rat submaxillary gland (SMG) cells were investigated. The binding of [3H]quinuclidinyl benzilate ([3H]QNB) was most sensitive to atropine and the M3-specific antagonist 4-DAMP followed by pirenzepine and least sensitive to the cardioselective M2 antagonist AFDX116. This, and the binding characteristics of [3H]4-DAMP, confirmed that the muscarinic receptors in this preparation are of the M3 subtype. Activation of these muscarinic receptors by carbamylcholine (CBC) produced both stimulation of phosphoinositide (PI) hydrolysis and inhibition of cAMP synthesis, suggesting that this receptor subtype couples to both effector systems. Paraoxon (100 microM) reduced Bmax of [3H]4-DAMP binding from 27 +/- 4 to 13 +/- 3 fmol/mg protein with nonsignificant change in affinity, suggesting noncompetitive inhibition of binding by paraoxon. Like the agonist CBC, paraoxon inhibited the forskolin-induced cAMP formation in SMG cells with an EC50 of 200 nM, but paraoxon was greater than 500 fold more potent than CBC. However, while the inhibition by CBC was counteracted by 2 microM atropine, that by paraoxon was unaffected by up to 100 microM atropine. It suggested that this effect of paraoxon was not via binding to the muscarinic receptor. Paraoxon did not affect beta-adrenoreceptor function in the preparation, since it did not affect the 10 microM isoproterenol-induced cAMP synthesis, which was inhibited totally by 10 microM propranolol and partially by CBC. Paraoxon had a small but significant effect on CBC-stimulated PI metabolism in the SMG cells.(ABSTRACT TRUNCATED AT 250 WORDS)

The muscarinic receptor subtype present in the bovine adrenal medulla was characterized. Hybridization of RNA to highly specific m 1 –m 5 muscarinic receptor cDNA probes detected the presence of only m 4 subtype mRNA in this tissue. Muscarinic receptor binding studies using the non-selective ligand [ 3 H]N-methyl-scopolamine showed a single class of binding sites with a maximum density of 19.8 fmol/mg protein and a dissociation constant (K D ) of 220 pM in the adrenal medulla, while the M 1 selective ligand [ 3 H]telenzepine did not bind detectably. Competition of specific antagonists with [ 3 H]N-methyl-scopolamine for binding to the membranes produced a rank order of potencies with a profile that fitted either the cloned m 3 or m 4 receptor. In further comparative studies, the adrenal gland of the rat showed the presence of m 4 subtype mRNA in addition to the m 3 subtype.

The mechanism of action of the anticholinesterase paraoxon on the function of a muscarinic receptor subtype in rat brain striatum was investigated. Paraoxon inhibited binding of the muscarinic agonist cis -[ 3 H]methyldioxolane, which binds to a high-affinity population of muscarinic receptors, with K 0.5 of 80 n M , compared to a K 0.5 of 7 μ M for parathion. The inhibition was competitive, suggesting that paraoxon and CD bind to a common site. When this muscarinic receptor (possibly an M 4 subtype) is activated it inhibits cAMP synthesis. Thus, function of the paraoxon-sensitive receptor was assayed by the inhibition of the forskolin-activated [ 3 H]cAMP synthesis. Paraoxon inhibited cAMP synthesis in a dose-dependent manner as did the muscarinic agonist carbachol, and this inhibition was completely blocked by the muscarinic antagonist atropine. When low concentrations of carbachol and paraoxon were used together, there was additive inhibition of cAMP synthesis. However, there was no further increase when both paraoxon and carbachol were present in concentrations that individually produced maximal inhibition. The data suggest that paraoxon acts like carbachol, causing activation of the muscarinic receptor subtype in brain striatum and leading to inhibition of cAMP synthesis. Considering the high affinity that this muscarinic receptor has for paraoxon, it is suggested that this direct reversible action of paraoxon on the muscarinic receptor could affects its toxicity, expecially early on before most of the acetylcholinesterase is phosphorylated.

Muscarinic receptors in brain membranes from honey bees, houseflies, and the American cockroach were identified by their specific binding of the non-selective muscarinic receptor antagonist [3H]quinuclidinyl benzilate ([3H]QNB) and the displacement of this binding by agonists as well as subtype-selective antagonists, using filtration assays. The binding parameters, obtained from Scatchard analysis, indicated that insect muscarinic receptors, like those of mammalian brains, had high affinities for [3H]QNB (KD = 0.47 nM in honey bees, 0.17 nM in houseflies and 0.13 nM in the cockroach). However, the receptor concentration was low (108, 64.7, and 108 fmol/mg protein for the three species, respectively). The association and dissociation rates of [3H]QNB binding to honey bee brain membranes, sensitivity of [3H]QNB binding to muscarinic agonists, and high affinity for atropine were also features generally similar to muscarinic receptors of mammalian brains. In order to further characterize the three insect brain muscarinic receptors, the displacement of [3H]QNB binding by subtype-selective antagonists was studied. The rank order of potency of pirenzepine (PZ), the M1 selective antagonist, 11-[2-[dimethylamino)-methyl)1-piperidinyl)acetyl)-5,11- dihydro-6H-pyrido(2,3-b)-(1,4)-benzodiazepin-6 one (AF-DX 116), the M2-selective antagonist, and 4-DAMP (4-diphenylacetoxy-N-methylpiperidine methiodide) the M3-selective antagonist, was also the same as that of mammalian brains, i.e., 4-DAMP greater than PZ greater than AF-DX 116. The three insect brain receptors had 27-50-fold lower affinity for PZ (Ki 484-900 nM) than did the mammalian brain receptor (Ki 16 nM), but similar to that reported for the muscarinic receptor subtype cloned from Drosophila. Also, the affinity of insect receptors for 4-DAMP (Ki 18.9-56.6 nM) was much lower than that of the M3 receptor, which predominates in rat submaxillary gland (Ki of 0.37 nM on [3H]QNB binding). These drug specificities of muscarinic receptors of brains from three insect species suggest that insect brains may be predominantly of a unique subtype that is close to, though significantly different from, the mammalian M3 subtype.

Relationship between in vivo down-regulation of cardiac muscarinic receptors and changes in their encoding mRNA was investigated. Rats were treated either once or for ten days with an irreversible inhibitor of acetylcholinesterase, followed by measurements of cardiac acetylcholinesterase, the density and affinity of muscarinic receptors, and the concentration of mRNA coding for these receptors. mRNA was quantitated using the sensitive method of DNA-excess solution hybridization. Our data indicate that while short-term treatment resulted in a marked decrease in the density of cardiac muscarinic receptors by 34%, there was no accompanying significant change in the concentration of their mRNA. In contrast, long-term inhibition of acetylcholinesterase significantly decreased the concentration of both receptors and mRNA by 40% and 29%, respectively. These results are indicative of multiple mechanisms of down-regulation of cardiac muscarinic receptors, some of which might involve alterations at the transcriptional level.

We studied the effects of acute and chronic in vivo inhibition of acetylcholinesterase on both the density and function of brain muscarinic cholinergic receptors. Adult male rats were treated either once or multiple times over a period of 10 days with the irreversible acetylcholinesterase inhibitor diisopropylfluorophosphate (DFP). The concentration and affinity of muscarinic receptors in various brain regions were determined using radioligand binding techniques. Acute DFP treatment resulted in a significant reduction in receptor number only in the brain stem, while chronic treatment caused receptor down-regulation in the brain stem, cerebral cortex, and striatum. There was no change in ligand affinity in any of the brain regions. In sharp contrast, muscarinic receptor function was fully preserved, in terms of coupling of the receptors to increased phosphoinositide hydrolysis in the cerebral cortex, hippocampus, and striatum, or inhibition of cyclic AMP formation in the cerebral cortex or striatum. Therefore, there is a marked lack or correlation between DFP-induced muscarinic receptor down-regulation and receptor desensitization.

Our results demonstrate that phorbol esters and tetrodotoxin (TTX) partially inhibit muscarinic receptor-mediated increase in phosphoinositide (PI) hydrolysis in rat cerebral cortex cell aggregates; this inhibition was observed using several muscarinic agonists. While these effects were not accompanied by major changes in the total muscarinic receptor population, phorbol esters, but not TTX, reduced the relative concentration of the high affinity binding sites of the M1-selective ligands pirenzepine and telenzepine. In contrast, the binding of a muscarinic agonist to multiple receptor conformations was not influenced by either phorbol esters or TTX. Our data also show that the partial inhibition of the PI response by these agents is not due to a selective effect on the response mediated by a certain muscarinic receptor subtype or a receptor population which is more sensitive to agonist-induced desensitization. Evidence is provided that the effects of both phorbol esters and TTX might be mediated largely, although not entirely, by a common mechanism.

The effects of aging on the inhibition of forskolin-stimulated cyclic AMP formation by muscarinic receptors were investigated. There were no detectable changes in the magnitude of maximal inhibition by carbamylcholine or the potency of the agonist in inducing this response in either the striatum or hippocampus obtained from young or old Fisher 344 rats.

The reactivation of the rat brain muscarinic acetylcholine receptor (mACh-R) binding with dimercaptosuccinic acid (DMSA) after in vitro and in vivo inhibition by mercuric chloride (HgCl2) and methylmercuric chloride (MeHg) was investigated. Receptor binding was estimated by the potent and specific antagonist l -[3H]quinuclidinyl benzilate ([3H]QNB). Rat brain synaptosomal membranes were exposed to HgCl2 and MeHg. At 1 × 10−4 M. HgCl2 caused complete inhibition of the [3H]QNB binding. The inhibition of [3H]QNB binding by HgCl2 was still higher than 50% at 1 × 10−8 M. MeHg caused less inhibition of [3H]QNB binding than HgCl2. The inhibited receptors showed a significant degree of reactivation when treated with DMSA. The recovery was almost complete after MeHg inhibition or with the lower HgCl2 concentrations. Generally, the reactivation was dependent on the concentration of DMSA. When rats injected with either early or delayed doses of DMSA following administration with five consecutive daily doses (8 mg/kg body wt, Gavage method) of MeHg or HgCl2, the inhibition of [3H]QNB binding was less than untreated ones. The early treatment with DMSA decreased the inhibition of [3H]QNB binding due to MeHg or HgCl2 intoxication. However, DMSA was more effective in reducing HgCl2 inhibition than MeHg either in vitro or in vivo treatment. The ability of DMSA to reactivate the mACh-R after inhibition with the mercurials emphasizes the involvement of essential sulfhydryl groups in [3H]QNB binding sites, and also shows that these sulfhydryl groups are the primary target for the MeHg and HgCl2 inhibition of the rat brain muscarinic receptors.

The effects of various lysophospholipids on the calcium transport activity of sarcoplasmic reticulum (SR) from rabbit skeletal and canine cardiac muscles were examined. The lipids decreased calcium transport activity in both membrane types; the effectiveness being in the order lysoPC greater than lsyoPS, lysoPG greater than lysoPE. The maximum inhibition induced by lysoPC, lysoPG and lysoPS was greater than 85% of the normal Ca2+-transport rate. In cardiac SR lysoPE had a maximal inhibition of about 50%. Half maximal inhibition of calcium transport by lysoPC was achieved at 110 nmoles lysoPC/mg SR. At this concentration of lysoPC, the (Ca2+ + Mg2+)-ATPase and Ca2+-uptake activities were inhibited to the same extent (about 60%) in skeletal sarcoplasmic reticulum, while in cardiac sarcoplasmic reticulum, there was less than 20% inhibition of the Ca2+ + Mg2+-ATPase activity. Studies with EGTA-induced passive calcium efflux showed that up to 200 nmoles lysoPC/mg SR did not alter calcium permeability significantly in cardiac sarcoplasmic reticulum. In skeletal muscle membranes the lysophospholipid mediated decrease in calcium uptake correlated well with the increase in passive calcium efflux due to lysophosphatidylcholine. The difference in the lysophospholipid-induced effects on the sarcoplasmic reticulum from the two muscle types probably reflects variations in protein and other membrane components related to the respective calcium transport systems.