Complementary improvement of the method for determining cholinergic activities in the small intestine and its application to experiments in vivo.

The concentration of acetylcholine (ACh) in rat jejunum that had been homogenized with an ultra-high-speed homogenizer (Biotron) was significantly higher than that in jejunum homogenized with a glass homogenizer. Rats were injected once or repeatedly for 10 days with a muscarinic agonist, pilocarpine (1 mg/kg), or a muscarinic antagonist, scopolamine (5 mg/kg). Animals were killed 20 min or 24 hr after single or consecutive injections, respectively, for determinations of cholinergic activities in the jejunum. Single treatment: Pilocarpine did not cause significant changes in the level of ACh, the activity of acetylcholinesterase (AChE), the binding of [3H]quinuclidinyl benzilate (QNB) or the contractile responses to ACh. Scopolamine reduced the level of ACh and binding of [3H]QNB without inducing significant changes in the activity of AChE and the contractile response. Consecutive treatment: Pilocarpine reduced the binding of [3H]QNB by changing the value of Bmax and reduced the contractile response without affecting the level of ACh or the activity of AChE. Scopolamine increased the binding of [3H]QNB without any effects on the level of ACh, the activity of AChE or the contractile response. In summary, it is possible to determine the level of ACh in a tissue as hard as intestine by homogenization with a Biotron and to assess the cholinergic situation in the intestine of animals that have been poisoned with various agents by estimating cholinergic activities.

Effects of single or repeated administration of a carbamate, propoxur, and an organophosphate, DDVP, on jejunal cholinergic activities and contractile responses in rats.

Wistar rats were injected once or repeatedly for 10 days with dichlorvos (DDVP, 5 mg kg-1), propoxur (10 mg kg-1), oxotremorine (0.1 mg kg-1) or atropine (5 mg kg-1). Animals were killed 20 min or 24 h after single or consecutive injections, respectively, for determinations of cholinergic activities and contractile responses to acetylcholine (ACh) of the jejunum. Single treatments: while DDVP and propoxur decreased acetylcholinesterase (AChE) activity, oxotremorine and atropine did not. Although DDVP, propoxur and oxotremorine increased levels of ACh, atropine decreased them. Contractile responses to ACh were enhanced by DDVP and reduced by oxotremorine and atropine. The Bmax value of binding of [3H]quinuclidinyl benzylate (QNB) to muscarinic ACh receptors was decreased by atropine. Consecutive treatments: DDVP and oxotremorine decreased AChE activity markedly and slightly, respectively. Although DDVP and oxotremorine increased levels of ACh, propoxur decreased them. Without affecting the contractile responses, DDVP caused a reduction and propoxur and atropine caused an increase in the Bmax value for binding of [3H]QNB. Both the contractile responses and the value of Bmax for binding of [3H]-QNB were decreased by oxotremorine. In summary, propoxur and DDVP showed similar effects mainly through their anticholinesterase properties in the case of single injection, but DDVP had similar effects to those of oxotremorine and propoxur had similar effects to those of atropine in the case of repeated injection.

Effects of organotins on the cholinergic system in the chicken brain in vitro.

The effects of two organotins, trimethyltin chloride (TMT) and tributyltin chloride (TBT), on the cholinergic system in the chicken brain were investigated in vitro. Both compounds, at concentrations below 10(-4)m, had almost no effect on acetylcholinesterase activity in cortical homogenate. By contrast, TMT and TBT inhibited non-competitively the activity of choline acetyltransferase with K(i) values of 1.5 x 10(-5) and 0.99 x 10(-5)m, as well as the high-affinity uptake of choline with K(i) values of 35 x 10(-6) and 2.5 x 10(-6)m, respectively. These inhibitory effects were not reversed in the presence of cysteine. TMT and TBT inhibited the low-affinity uptake of choline with K(i) values of 2.6 x 10(-4) and 1.25 x 10(-4)m, respectively. Although both compounds inhibited the depolarized release of acetylcholine (ACh) from slices of brain, only TBT at 10(-4)m suppressed the synthesis of ACh. TBT, but not TMT, inhibited the binding of [(3)H]quinuclidinyl benzilate, which is an indicator of muscarinic ACh receptors, at 10(-5) and 10(-4)m. It is suggested that cholinergic transmission, in particular the synthesis of ACh and the release of ACh, is susceptible to trialkylin neurotoxicity.

Effects of triethyl lead on various cholinergic parameters in the rat brain in vitro.

The effects of triethyl lead acetate (triethyl Pb) on the cholinergic system in the brain of the rats were investigated in vitro. Triethyl Pb, at concentrations below 10(-4) M, inhibited the depolarized release of acetylcholine (ACh) from slices of cortex and they synthesis of ACh in such slices, while it potentiated in a dose-dependent manner the non-depolarized release of ACh. In contrast, lead inhibited noncompetitively the high-affinity uptake of choline into synaptosomes with a Ki of 4.03 X 10(-6) M and the activity of choline acetyltransferase with a Ki of 4.07 X 10(-5) M. Triethyl Pb has an inhibitory effect (IC50 not equal to 5 X 10(-5) M) on the binding of [3H]quinuclidinyl benzilate to muscarinic ACh receptors. Triethyl Pb inhibited acetylcholinesterase activity slightly at 5 X 10(-5) and 10(-4) M. It is suggested that ACh transmission, in particular the synthesis of ACh and the release of ACh, is susceptible to organolead neurotoxicity.

Effects of carbamate, BPMC, on the central cholinergic functions and behavior of mice.

O-sec-butylphenyl methylcarbamate (BPMC), an anticholinesterase carbamate, was injected once (100 mg/kg, s.c.) or repeatedly (50 mg/kg/day for 10 days) into mice. Animals were examined for their behavior and for parameters of cholinergic activity in the forebrain. Mice that received only a single injection exhibited reduced ambulation, hypothermia, and impairment of rotarod performance for up to 3 hr after a single injection. BPMC increased levels of acetylcholine (ACh) in the forebrain for up to 6 hr, and decreased acetylcholinesterase (AChE) activity for up to 24 hr. Both high-affinity choline uptake (HACU) and binding of [3H]quinuclidinyl benzilate (QNB) were reduced 20 min after a single injection without any effect on choline acetyltransferase (ChAT) activity. In behavioral tests conducted 10 min prior to the daily injections, rotarod performance and ambulation were slightly impaired for a few days before and after cessation of injection. Repeated treatment decreased HACU and binding of [3H]QNB for 24 hr after the final injection without any changes in levels of ACh content, AChE activity and ChAT activity. BPMC may reversibly impair cholinergic functions through effects not only on AChE activity but also on HACU and binding of [3H]QNB.

Effect of single or chronic injection with a carbamate, propoxur, on the brain cholinergic system and behavior of mice.

Propoxur, an anticholinesterase carbamate, was injected singly (10 mg/kg, s.c.) or chronically (5 mg/kg/day, s.c., for 10 days) into mice. Animals were examined for effects on the cholinergic system in brain tissue and on behavior. Single injection caused an increase in brain acetylcholine (ACh) content at 10 and 60 min; and it caused decreases in acetylcholinesterase (AChE) activity at 10, 60 and 180 min, high-affinity choline uptake into synaptosomes at 10 and 60 min, and [3H]quinuclidinyl benzilate (QNB) binding at 10 min without causing any change in choline acetyltransferase (ChAT) activity. Open-field behavior and rotarod performance were depressed at 10 min and rectal temperature was decreased at 10, 60 and 120 min after a single injection. Chronic treatment caused decreases in high-affinity choline uptake and [3H]QNB binding 24 hr after the final injection without any changes in ACh content, AChE activity or ChAT activity. In behavioral tests conducted 10 min prior to daily administration, rotarod performance was slightly suppressed only during the period of injection. Although propoxur is considered to have effects similar to those of organophosphorus compounds, the changes observed in the present experiments seemed to be reversible.

Effects of arsenic on cholinergic parameters in brain in vitro.

The effects of sodium arsenite (arsenite) on the cholinergic system in the brain of the mouse were investigated in vitro and compared with those of N-ethylmaleimide (NEM) and iodoacetate, both of which are alkylating sulfhydryl reagents. Arsenite, at concentrations greater than 10(-4) M, inhibited depolarized and nondepolarized release of acetylcholine (ACh) from cerebral slices, the synthesis of ACh in the slices, high-affinity uptake of choline into synaptosomes and activity of acetylcholinesterase (AChE). On the other hand, arsenite potentiated dose-dependently the activity of choline acetyltransferase (ChAT). N-Ethylmaleimide and iodoacetate showed inhibitory effects similar to those of arsenite. However, some exceptions were that N-ethylmaleimide did not have any effect on the nondepolarized release of ACh while iodoacetate had no effect on high affinity uptake of choline and activity of AChE. In contrast to arsenite, N-ethylmaleimide and iodoacetate inhibited the activity of ChAT. Neither of arsenite, N-ethylmaleimide nor iodoacetate showed any effect on the binding of [3H]quinuclidinyl benzilate to muscarinic ACh receptors. Although arsenite is thought to inhibit the cholinergic system in brain in vivo, its potentiating effect on ChAT and inhibition of AChE may reduce this harmful effect.

Cholinergic system of brain tissue in rats poisoned with the organophosphate, 0,0-dimethyl 0-(2,2-dichlorovinyl) phosphate.

The cholinergic system of the brain was investigated in rats acutely poisoned with the organophosphate, 0,0-dimethyl 0-(2,2-dichlorovinyl) phosphate (DDVP), (6 mg/kg, sc, with saline as a control). The amounts of three fractions of acetylcholine (ACh)--free (extraterminal), labile-bound (intraterminal/cytoplasmic), and stable-bound (intraterminal/vesicular)--increased in the rats over a period of 5 to 60 min after injection of DDVP, showing peaks which were 2.45, 1.82, and 1.4 times as high as the respective control amounts. No difference was seen in the amount of any fraction of ACh between treated and control rats killed 3 and 24 hr after injection. Acetylcholinesterase (AChE) activity decreased to between 12 and 43% of the control over a period of 5 to 180 min and recovered almost completely within 24 hr after injection. No appreciable changes were seen in either spontaneous or potassium-induced ACh release in brain tissue slices obtained from rats treated with DDVP. ACh synthesis in slices was suppressed significantly 20 min, but not 24 hr, after injection of DDVP. In the brain crude synaptosomal preparation, high-affinity choline uptake, which is generally thought to be a rate-limiting step for ACh synthesis, was suppressed 20 min after DDVP. No appreciable changes were seen in high-affinity choline uptake at 24 hr low-affinity choline uptake, and choline acetyltransferase activity after injection of DDVP. These results suggest that ACh synthesis and high-affinity choline uptake may be in a suppressed state when ACh concentration, especially intraterminal ACh, is increased and AChE activity is decreased in the brain cholinergic system of rats poisoned with DDVP. The increase in the intraterminal ACh may be due to an inhibition of AChE activity at this site and/or a re-uptake of ACh in the synaptic cleft, not to an inhibition of ACh release or an increase in ACh synthesis.

Effects of insecticidal carbamates on brain acetylcholine content, acetylcholinesterase activity and behavior in mice.

Mice showed no toxic signs after a single injection of o-sec-butylphenyl methylcarbamate (BPMC, 10 mg/kg) or 2-isopropoxyphenyl-N-methylcarbamate (propoxur, 2 mg/kg). Each dose of BPMC or propoxur caused an increase in acetylcholine content and a decrease in acetylcholinesterase activity in the forebrain of mice at 10 min, followed by an almost complete recovery in the content at 60 min. Spontaneous motor activity was depressed 10 min after and recovered 60 min after, injection of BPMC or propoxur. Neither rotarod performance nor rectal temperature showed any change after injection of BPMC or propoxur. Spontaneous motor activity may therefore be a simple method for assessing small changes in the cholinergic system.

Effects of organophosphorus compounds, O,O-dimethyl O-(2,2-dichlorovinyl)phosphate (DDVP) and O,O-dimethyl O-(3-methyl 4-nitrophenyl)phosphorothioate (fenitrothion), on brain acetylcholine content and acetylcholinesterase activity in Japanese quail.

Effects of 2 organophosphorus compounds, O,O-dimethyl O-(2,2-dichlorovinyl)phosphate (DDVP) and O,O-dimethyl O-(3-methyl 4-nitrophenyl)phosphorothioate (fenitrothion), on the brain cholinergic system were investigated in Japanese quail. Cholinergic signs, such as salivation and convulsions in legs and wings, were seen 7-15 min after administration with DDVP (3-4 mg/kg) or 6-120 min after administration with fenitrothion (250-350 mg/kg). In the DDVP-treated quail (10 min after dosage of 3 mg/kg), free acetylcholine (ACh), labile-bound ACh, increased significantly and acetylcholinesterase (AChE) decreased to 28% of the value determined in untreated quail. In the fenitrothion-treated group (60 min after dosage of 300 mg/kg), only free ACh increased and AChE activity decreased to 20% of the control value. In vitro, DDVP and fenitrothion inhibited AChE activity in brain homogenate with an I50 of 10(-8) M and 10(-5) M, respectively. It appeared that both organophosphorus compounds might have essentially the same effect on the brain cholinergic system. There were only small differences in the effect on various fractions of ACh between the 2 compounds, although there was a hundred-fold range in dose.

Reversible effects of monothiol (D-penicillamine) and dithiol (dimercaptosuccinic acid) chelating compounds on methylmercury-inhibited choline acetyltransferase activity and high affinity choline uptake.

The effects of thiol compounds on methylmercury chloride (MMC)-inhibited choline acetyltransferase (ChAT) activity and MMC-inhibited high affinity choline uptake of rat brain tissue were studied in vitro. D-penicillamine (D-Pc) and dimercaptosuccinic acid (DMS) reversed the MMC-inhibited ChAT activity dose-dependently. Equilibrium dialysis of MMC-inhibited ChAT against the buffer containing 10(-3) M D-Pc reversed the ChAT activity almost completely. The reversal effect of D-Pc (monothiol compound) on MMC-inhibited ChAT was significantly more potent than that of DMS (dithiol compound). D-Pc and DMS almost equally reversed the MMC-inhibited high affinity choline uptake by synaptosomes in a dose dependent fashion. Washing with a solution containing D-Pc or DMS equally reversed the MMC-inhibited high affinity choline uptake in a dose-dependent fashion. Neither D-Pc nor DMS could reverse the hemicholinium-3-inhibited high affinity choline uptake.

Neuropharmacological effect of methylmercury in mice with special reference to the central cholinergic system.

Effect of methylmercury chloride (MMC) on behavior was studied in male ICR-JCL mice. In order to clarify the causal relationship between the potent suppressing action of MMC on the central cholinergic system and toxic manifestations, behavioral changes induced by MMC were compared with those induced by the two reference drugs, hemicholinium-3 (HC-3) and 3'-chloro-4-stilbazole (CS; a potent choline acetyltransferase inhibitor). When administered intraperitoneally, daily in a dose of 5 mg Hg/kg/day, MMC caused a decrease in spontaneous motor activity, rotarod dysfunction, and hypothermia before an abrupt loss in body weight and the appearance of overt signs. These behavioral changes were similar to some extent to those induced by HC-3 or CS. A single intracerebral injection of HC-3 (51 or 100 micrograms/kg) caused hypothermia and rotarod dysfunction over a period of 40-250 min. A single intraperitoneal administration of CS (100 or 200 mg/kg) induced a decrease in spontaneous motor activity, hypothermia, and rotarod dysfunction over a period of 1-5 hours after injection. These results suggest that the prior toxic behavioral changes caused by MMC may be related to suppression of the cholinergic system.

Effects of acute and chronic administration of DDVP (dichlorvos) on distribution of brain acetylcholine in rats.

DDVP (dichlorvos), an irreversible cholinesterase (ChE) inhibitor, was administered acutely and chronically to rats in order to investigate effects on the distribution of brain acetylcholine (ACh). In acutely treated animals (4 mg/kg, singly), cholinergic signs were evident and accompanied with a 100, 146, 113, and 61 per cent increase in total, free, labile-bound and stable-bound ACh content of the brain, respectively, and a 66 per cent decrease in acetylcholinesterase (AChE) activity 20 min after injection. In no animals treated chronically with a low dose (0.2 mg/kg/day for 9 or 90 days, or 1 mg/kg/day for 9 or 30 days), any overt sign was shown during the experimental period, and the stable-bound ACh content of the brain was not altered. In the group given 0.2 mg/kg for 90 days and that given 1.0 mg/kg for 9 or 30 days, free ACh content increased slightly but significantly, and AChE activity decreased to 58 per cent. Total ACh content and labile-bound ACh content increased only in a group given 1 mg/kg of DDVP for 30 days. These results suggest that acute, as well as chronic, exposure to organophosphate may induce alteration in mobilization and storage of ACh in the central cholinergic nerves.

Effects of methylmercury chloride on various cholinergic parameters in vitro.

The effects of methylmercury chloride and other mercury compounds on cholinergic parameters were studied in vitro. Methylmercury chloride (MMC) and phenylmercury acetate inhibited choline acetyltransferase (ChA) with 20 microM of I50, and mercury nitrate (MN) with 100 microM of I50. All the three compounds had little effect on cholinesterase activity. MMC inhibited a high affinity choline uptake with 41 microM of Ki, as well as a low affinity choline uptake with 250 microM of Ki. MMC did not affect a spontaneous and potassium-stimulated ACh release from brain tissue slices incubated in eserinized Krebs-Ringer's solution up to the concentration of 100 microM. It was shown that the organic mercury compounds, such as methylmercury, were potent inhibitors of the choline uptake systems, as well as ChA activity.