Ca(2+)-independent fusion of synaptic vesicles with phospholipase A2-treated presynaptic membranes in vitro.

To clarify the mechanism of exocytosis in neurotransmitter release, the fusion of synaptic vesicles with presynaptic membranes prepared from rat brain synaptosomes and concomitant acetylcholine (ACh) release induced by fusion of them were studied in vitro. Fusion of the synaptic vesicles with presynaptic membranes was measured by a fluorescence-dequenching assay with octadecyl rhodamine B. Synaptic vesicles fused with presynaptic membranes which had been pretreated with porcine phospholipase A2 (PLA2) in the presence of 20 microM Ca2+ and released ACh, whereas synaptic vesicles did not interact with non-pretreated membranes. The fusion followed by ACh release depended (i) on the activity of PLA2 during the membrane pretreatment, (ii) on the amount of pretreated membrane and (iii) on the duration of the pretreatment. The presence of Ca2+ ions during the pretreatment was essential for inducing a fusogenic activity of the membranes, but Ca2+ ions were not required for the fusion itself because the fusion experiment was carried out in the presence of 5mM EGTA without added Ca2+. The presence of quinacrine, an antagonist of PLA2, during the membrane pretreatment inhibited their fusogenic activity, suggesting the importance of activation of PLA2. Presence of albumin during the pretreatment, which is an adsorbent of free fatty acids, also inhibited the fusogenic activity. Arachidonic acid, when added during the pretreatment, potentiated the fusogenic activity of the membrane. These findings suggest that the conformational change in the presynaptic membrane phospholipids induced by PLA2 and the presence of arachidonic acid produced by PLA2 are important in the process of fusion of synaptic vesicles with the presynaptic membranes of rat brain, and that the fusion process itself is independent of Ca2+.

Effects of H-89, an inhibitor of protein kinase A, on the acetylcholine release from myenteric plexus of guinea pig ileum.

In order to clarify the involvement of cyclic AMP-dependent protein kinase (protein kinase A) in acetylcholine (ACh) release from myenteric plexus of guinea pig ileum, the effect of H-89, a specific inhibitor of protein kinase A, on the ACh release was investigated. H-89 (0.1-10 microM) inhibited the spontaneous and nicotine-induced release of ACh in a concentration dependent manner. It at 1 microM decreased both kinds of release of ACh to almost half of the control, but it did not affect the ACh release evoked by electrical field stimulation and by 5-hydroxytryptamine. H-89 had no significant effect on the indomethacin (IND), an inhibitor of PG synthesis, -insensitive component of the spontaneous and nicotine-induced release of, ACh. OP-41483, an analog of PGI2 and forskolin, an activator nicotine-induced release of, ACh. OP-41483, an analog of PGI2 and forskolin, an activator of adenylate cyclase, reversed the inhibitory effect of IND on the ACh release. H-89 at 1 microM completely inhibited the reverse effects of OP-41483 and forskolin. These results suggest that activation of protein kinase A is essential for modulation of the nicotine-induced and spontaneous ACh release from myenteric plexus of guinea pig ileum and the activity of protein kinase A is regulated by endogenous PGs via intracellular cyclic AMP level.

The effect of a reduction in temperature on the quantal release of transmitter at the mouse neuromuscular junction.

1. The effects of a reduction in temperature were examined on evoked and spontaneous release of transmitter quanta and on presynaptic negative signals, blocked by Cd2+, measured externally at neuromuscular junctions in mouse diaphragm muscles in low-Ca2+, high-Mg2+ Krebs-Ringer solutions. 2. The evoked release was enhanced with lowering of the temperature, whereas the extent of spontaneous release was reduced. Cooperativity of Ca2+ in the evoked release was slightly reduced by lowering the temperature. 3. The presynaptic negative signals increased in duration with lowering of the temperature. 4. These results support the hypothesis that the effect of a reduction in temperature reflects the improved efficacy of the calcium-mediated mechanism of transmitter release, manifested as a prolongation of the inflow of Ca2+. The process involved in the evoked release is probably attributable to an almost passive mechanism.

Factors influencing the twin-pulse facilitation of the release of transmitter at the mouse neuromuscular junction.

1. The effects of several conditions and agents on the twin-pulse facilitation of the release of transmitter at the mouse neuromuscular junction in low-Ca2+ high-Mg2+ bathing solutions were examined. 2. Twin-pulses gave two endplate potential (epps) with first (m2) and second (m2) quantal contents. The ratio of m2/m1 was taken as a measure of the degree of facilitation. 3. The mean value of this ratio was > 1. Individual ratios fluctuated widely at junctions with smaller values of m1 but were focused around 1 at junctions with larger values of m1. Thus, some populations of junctions with smaller values of m1 contributed to an increment in the mean ratio. 4. The mean ratio was virtually constant irrespective of changes in the spontaneous and evoked release of transmitter at temperatures between 20 and 36 degrees C and at external concentrations of Ca2+ from 0.4 to 0.8 mM. 5. 4-Aminopyridine(4-AP) slightly but significantly increased this ratio with increases in m1 and m2 at temperatures of 24 and 36 degrees C. Ouabain slightly but significantly reduced the ratio, with increases in m1 and m2. The steadiness of the ratio was maintained in the presence of caffeine, high K+, neomycin or omega-conotoxin irrespective of changes in m1 and m2, except in the case of omega-conotoxin. 6. Spontaneous output at 36 degrees C increased in the presence of 4-aminopyridine, ouabain, caffeine, high K+ or neomycin. 7. These results indicate that maintenance of a stable value of the ratio of m2 to m1 is a dominant feature.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of prostacyclin in acetylcholine release from myenteric plexus of guinea-pig ileum.

The roles of metabolites of arachidonic acid in spontaneous and agonist-induced acetylcholine release from a longitudinal muscle preparation with myenteric plexus of guinea-pig ileum were studied. Indomethacin significantly decreased both spontaneous acetylcholine release and its release induced by nicotine and substance P. We had found that prostaglandin E2 (PGE2) partly reversed this inhibition. We now found that a stable prostacyclin analog, OP-41483 at 100 nM, completely reversed the inhibition of acetylcholine release by indomethacin. On the other hand, PGD2, PGF2 alpha and ONO-11113, a thromboxane A2 analog, did not have any significant effect on the inhibition by indomethacin. OP-41483 had no effect on acetylcholine release induced by nicotine or substance P in the absence of indomethacin. To confirm the modulatory role of endogenous prostaglandins on acetylcholine release, we also studied the release of 6-keto-PGF1 alpha, a metabolite of prostacyclin, and PGE2 from longitudinal muscle preparations. The preparations released appreciable amounts of 6-keto-PGF1 alpha continuously during the experiments. Indomethacin inhibited release, while nicotine did not affect it so significantly. Our results suggest that endogenous prostacyclin modulates acetylcholine release from cholinergic nerve terminals in the myenteric plexus of guinea-pig ileum.

Mediators of nonadrenergic, noncholinergic inhibition in the proximal, middle and distal regions of rat colon.

1. The mediators of non-adrenergic non-cholinergic (NANC) relaxation of the longitudinal muscle of rat proximal, middle and distal colon were examined in vitro. 2. Electrical transmural stimulation (TMS) of proximal, middle and distal segments of rat colon induced NANC relaxations which were inhibited by tetrodotoxin (1 microM), but not by atropine (1 microM) or guanethidine (4 microM). 3. In the proximal colon, L-nitro-arginine (N5-nitroamidino-L-2,5-diaminopentanoic acid) inhibited the TMS-induced NANC relaxation and L-arginine (1 mM) reversed this inhibition. Nitric oxide (0.3-10 microM) induced relaxation of the proximal segment. 4. NANC relaxation of the proximal segments was still evident after desensitization to vasoactive intestinal peptide (VIP). A VIP antagonist (VIP 10-28, 10 microM) had no effect on the TMS-induced NANC relaxation, which was also resistant to alpha-chymotrypsin (2 units ml-1) and a substance P antagonist ([D-Pro2, D-Trp7,9]substance P, 1 microM). 5. In the middle colon, L-nitro-arginine did not inhibit the TMS-induced NANC relaxation in 6 of 9 preparations tested and partially inhibited the relaxation in the other 3 preparations. L-Arginine did not reverse the partial inhibition. 6. Complete desensitization to VIP was not achieved in the middle colon. The VIP antagonist had no effect on the TMS-induced NANC relaxation. After alpha-chymotrypsin treatment of the segment, desensitization of the segments to substance P, or in the presence of the substance P antagonist, the TMS-induced NANC relaxation was augmented. 7. In the distal colon, L-nitro-arginine did not have any significant effect on the TMS-induced relaxation and nitric oxide did not induce relaxation. The VIP antagonist significantly inhibited TMS-induced NANC relaxation. Alpa-Chymotrypsin-treatment of the distal segments resulted in significant inhibition of NANC relaxation. No desensitization to substance P was achieved. Treatment with the substance P antagonist had no effect. 8. These results suggest that nitric oxide is the mediator of the NANC inhibitory response in the proximal region of rat colon; in the middle colon, substance P acts as an excitatory neurotransmitter, antagonizing the NANC relaxation caused by the mediator of the response, which is still uncertain. Our results suggest that that VIP is the most likely candidate as a NANC transmitter in the distal colon.

Role of cyclic AMP in prostaglandin-induced modulation of acetylcholine release from the myenteric plexus of guinea pig ileum.

Prostaglandins (PGs) have modulatory effects on spontaneous and nicotine-induced release of acetylcholine (ACh) from the myenteric plexus of guinea pig ileum. To determine whether cyclic AMP is involved in the mechanisms of these effects, we studied ACh release under conditions that inhibit PG synthesis. Indomethacin (IND), a cyclooxygenase inhibitor, inhibited ACh release concentration-dependently. The effect of the maximally inhibitory concentration of IND (2.8 microM) on nicotine-induced ACh release were reversed concentration-dependently by PGE2, forskolin, 3-isobutyl-1-methylxanthine (IBMX) and 8-bromo cyclic AMP. These compounds caused concentration-dependent reversal of the inhibition of spontaneous ACh release by IND, but their concentrations for restoration of spontaneous release were higher than those for restoration of nicotine-induced release. The effects of PGE2 and forskolin or IBMX were not additive in reversing the inhibition of nicotine-induced ACh release by IND. Neither forskolin nor 8-bromo cyclic AMP alone had any significant effect on either release. These results showed that increase in the level of cyclic AMP in myenteric cholinergic neurons restored ACh release from the tissue whose PG level had been lowered by IND and indicated that endogenous PGs may modulate the level of intraneuronal cyclic AMP.

Selective inhibitory effects of calcium channel antagonists on the two components of the neurogenic response of guinea pig vas deferens.

The effects of L-type calcium channel antagonists and omega-conotoxin on the contractile responses of guinea pig vas deferens were examined in vitro. Electrical stimulation of the postganglionic hypogastric nerve induced biphasic contraction consisting of rapid phasic and delayed tonic components. L-type calcium channel antagonists, such as diltiazem, verapamil and nicardipine, mainly inhibited the delayed tonic component, whereas omega-conotoxin mainly inhibited the rapid phasic component. Stimulations in the presence of prazosin and alpha, beta-methylene ATP induced rapid transient and delayed contraction, respectively, which were inhibited by omega-conotoxin and L-type calcium channel antagonists, respectively. Short-term stimulation with five pulses induced a small fast phasic contraction. This contraction, which could be desensitized by alpha, beta-methylene ATP, was inhibited by omega-conotoxin, but not by L-type calcium channel antagonists. At the concentrations used in the present study, none of the calcium channel antagonists inhibited the contractions induced by exogenously added ATP or norepinephrine. These findings suggest that L-type calcium channel antagonists and omega-conotoxin inhibit the neurotransmissions mediated by norepinephrine and ATP, respectively, from the postganglionic nerve to the vas deferens of the guinea pig. Inhibition of the voltage-dependent calcium channel is discussed in relation to the mechanism of cotransmission in this preparation.

Genetic analysis of susceptibility to radiation-induced apoptosis of thymocytes in mice.

Genetic control of thymocyte susceptibility to radiation-induced apoptosis was investigated in BALB/cHeA, STS/A and five other strains of mice by counting pyknotic cells in a selected area of thymic cortex on histological specimens after whole-body X-irradiation. Number of dead cells increased almost linearly with doses (range 0.25-0.75 Gy) in BALB/cHeA and STS/A mice. However, dead cell counts in BALB/cHeA mice were more than twice those in STS/A mice at each dose. Of five other strains of mice, C57BL/6N and B10.BR mice exhibited a sensitive phenotype similar to BALB/cHeA mice, while C3H/HeAMsNrs and NFS/N mice showed a resistant phenotype similar to STS/A mice. A/J mice seemed to be rather resistant. A sex difference was not recognized in BALB/cHeA and STS/A mice. Resistance was dominant over susceptibility in the progenies of reciprocal crosses between the two strains, indicating an autosomal inheritance and no maternal effect. Segregation ratio of susceptible phenotype to resistant one in the backcrosses of female (BALB/cHeA x STS/A)F1 mice with male BALB/cHeA mice was not significantly different from 1:1 and all backcrosses of female (BALB/cHeA x STS/A)F1 mice with male STS/A mice exhibited a resistant phenotype. Results suggested that thymocyte susceptibility to radiation-induced apotosis is controlled by one major autosomal allele.

Key roles of nitric oxide and cyclic GMP in nonadrenergic and noncholinergic inhibition in rat ileum.

Nonadrenergic and noncholinergic (NANC) inhibitory responses in circular and longitudinal muscles of the rat ileum were studied separately in vitro. Localized distension with a small balloon caused relaxation of the circular muscle on the anal side of the distended region. Nitro-arginine inhibited the relaxation and L-arginine counteracted the effect of nitro-arginine. Treatment of the preparation with superoxide dismutase (SOD) and methylene blue resulted in enhancement and inhibition, respectively, of the relaxation induced by distension. Nitric oxide caused relaxation of the circular muscle in a dose-dependent manner. 8-Bromo cyclic GMP (cGMP) caused relaxation of the circular muscle. Electrical transmural stimulation caused relaxation followed by a rebound contraction of the longitudinal muscle. Nitro-arginine inhibited the relaxation and L-arginine counteracted this inhibition. Similar results to those in the circular muscle were obtained in the longitudinal muscle with SOD, methylene blue, nitric oxide and 8-bromo cGMP. Electrical field stimulation increased the cGMP content of the longitudinal muscle preparation. Nitric oxide also increased the cGMP content of smooth muscle cells obtained from circular and longitudinal muscles of rat ileum. Preincubation of smooth muscle cells with methylene blue inhibited the effect of nitric oxide on the cGMP content. These results suggest a key role of cGMP in NANC inhibitory responses in rat ileum. The factors mediating the responses are discussed.

Diversity among mouse motor nerve terminals with respect to release transmitter quanta.

1. The aim of this work was to reexamine whether a positive correlation exists between the frequency (F, sec-1) of miniature endplate potentials (m.e.p.ps) and the quantal content (m) of endplate potentials (e.p.ps) or between quantal content, frequency and twin-pulse facilitation of transmitter release at a large number of neuromuscular junctions in the mouse. 2. The values of F and m were both measured intracellularly at endplates of mouse diaphragm in a high Mg2+/low Ca2+ bathing solution. 3. Values of both F and m varied from junction to junction. Smaller values of F were correlated with smaller values of m, and vice versa, resulting in a linear relationships. Histograms of F and m were skewed towards smaller values. 4. E.p.ps evoked by twin pulses gave the quantal contents of the first (m1) and second (m2) responses. 5. The ratio of m2 to m1 varied from junction to junction. A histogram of this ratio was skewed towards smaller values. 6. The ratio of m2 to m1 showed larger fluctuations at junctions with smaller values of F or m1 but was focused around 1 at junctions with larger values of F or m1. 7. The skewed parts of the histograms of F, m and m2/m1 accounted for the major population of junctions. 8. These results support the hypothesis that an intrinsic ability to release transmitter plays a role in regulation of the evoked output of transmitter at neuromuscular junctions in the mouse. 9. Such an ability is not correlated with the twin-pulse facilitation of transmitter release.

Role of PGE2 in neurotransmission from pre- to post-ganglionic hypogastric nerves of guinea pigs.

The hypogastric nerve to guinea pig vas deferens was stimulated pre- or post-ganglionically by adjusting the position of the suction electrode. Both stimulations induced a biphasic contraction consisting of a rapid transient phase and a delayed tonic phase. Indomethacin partially inhibited the contraction induced by pre-ganglionic stimulation, but did not inhibit that induced by post-ganglionic stimulation. Prostaglandin (PG) E2 counteracted the inhibitory effect of indomethacin. Mepacrine also inhibited the contraction induced by pre-ganglionic stimulation. Arachidonic acid and PGE2 both reversed the inhibition. The PGE2-receptor antagonist SC-19220 inhibited the contraction induced by pre-ganglionic, but not post-ganglionic nerve stimulation. These results suggested that endogenous PGE2 is important in neurotransmission in the pelvic ganglion of guinea pigs.

The different activation of int genes in mammary carcinomas developed in three mouse strains harboring mouse mammary tumor viruses derived from DD/Tbr.

RNA expressions of common integration site (int) genes and several oncogenes were investigated in mammary carcinomas spontaneously developed in different three strains of mice; DD/Tbr, NIH Swiss and BALB/c which harbor DD-MMTV derived from DD/Tbr mouse. Latter two strains of mice were designated NIH/Mtv+ and BALB/Mtv+, respectively. An increased expression of int-1 (wnt-1) and int-2 genes was observed in 56% (9/16) and 50% (8/16) of mammary carcinomas of DD/Tbr mice, respectively. Either int-1 or int-2 RNAs were expressed in 81% (13/16) of the carcinomas of DD/Tbr mice. IN NIH/Mtv+ mice, activation of int-1 and int-2 was observed in 41% (7/17) and 24% (4/17) of mammary carcinomas, respectively. Either int-1 or int-2 RNAs were expressed in 47% (8/17) of the carcinomas examined in this strain. In BALB/Mtv+ mice, on the other hand, either int-1 or int-2 gene were transcribed into RNAs at low frequency (33%: 3/9). These results suggest that the frequency of activation of int genes in mammary carcinomas induced by the same DD-MMTV in three strains of mice is genetically defined characteristics of these strains, and that the involvement of int-1 and int-2 genes in virus-induced mammary carcinogenesis may be influenced by genetic properties of animals. The activation of int-1 and int-2 genes did not clearly correlate with an increase in the expression of oncogenes examined; H-ras, K-ras, N-ras, myc, raf, fgr, fms, erB, mos, and src genes.

Prostaglandin E2 selectively affects purinergic transmission in guinea pig vas deferens.

The effect of prostaglandin E2 (PGE2) on the contractile response of the guinea pig vas deferens was examined. Postganglionic hypogastric nerve stimulation for 7 sec at 20 Hz induced a biphasic contractile response, consisting of fast phasic and delayed tonic components. Prostaglandin E2 delayed the onset and increased the maximum contractile responses. Stimulation in the presence of prazosin induced only a fast phasic contraction. Treatment with PGE2, in the presence of prazosin, delayed the onset of this response and increased its maximum. The delayed contraction, observed on stimulation in the presence of alpha,beta-methylene adenosine triphosphate (ATP), was enhanced moderately and concentration-dependently by PGE2. Short-term stimulation with 5 pulses induced a small fast phasic contraction. This contraction, which could be desensitized by alpha,beta-methylene ATP, was inhibited by PGE2 but not by prazosin. Prostaglandin E2 significantly enhanced the transient phasic contraction, induced by addition of exogenous ATP to the organ bath and had a similar but somewhat smaller effect on the tonic contraction induced by the addition of exogenous norepinephrine (NE). These findings suggest that PGE2 selectively delayed neurotransmission, mediated by ATP and enhanced contractions of the smooth muscle of guinea pig vas deferens, elicited by ATP or NE.

Strain difference in the susceptibility of thymocytes to radiation-induced apoptosis: in vitro study.

The susceptibility of thymocytes from STS/A to radiation-induced cell death was compared with that of thymocytes from BALB/cHeA. After in vitro exposure to 12 Gy X-ray, thymocytes were incubated at 37 degrees C for 8 h and then cell mortality was assessed by 0.02% erythrosin B exclusion. Cell death took place from 2 h in the incubation period, reaching a maximum at 6 h for both strains. The dose-effect on cell death at 4 h of incubation was examined after 1-24 Gy of X-irradiation. An increase in cell death was detectable even at 1 Gy in both strains. The number of dead cells in BALB/cHeA gradually increased with doses of more than 1 Gy, finally to a maximum (approximately 60%) in the dose range of 8-12 Gy, whereas the maximum cell death in STS/A was approximately 40%. The difference between the strains at maximum cell death was significant (P less than 0.005). The difference in the radiosensitivity of thymocytes between the two strains could not be attributed to a difference in the composition of their subpopulations because flow cytometric analysis based on the expression of CD4 and CD8 showed no intrinsic difference in the thymocyte subpopulations of BALB/cHeA and STS/A.

The differential contribution of endogenous prostaglandins to the release of acetylcholine from the myenteric plexus of the guinea-pig ileum.

1. Prostaglandin E (PGE) may be essential for maintaining the sensitivity of the myenteric plexus of guinea-pig ileum to nicotine. The contributions of prostaglandins to nervous activity evoked by different stimuli have now been investigated by measuring the amount of acetylcholine (ACh) released from the myenteric plexus of the guinea-pig ileum. 2. The amount of ACh released in response to dimethylphenylpiperazinium (DMPP) or substance P was depressed to about 40% of control by 2.8 microM indomethacin (Ind), whereas the release of ACh induced by 5-hydroxytryptamine (5-HT) was not affected. The inhibitory effects of Ind were overcome by 14.3 nM PGE2. 3. Mepacrine 5 microM, an inhibitor of phospholipase A2, depressed the release of ACh in response to DMPP and substance P to the same extent as Ind. These inhibitory effects of mepacrine were overcome by arachidonic acid (10 microM), but not by arachidonic acid plus Ind. The release of ACh evoked by 5-HT or electrical field stimulation (EFS) was also inhibited to about 60% of control by mepacrine but these inhibitions were overcome by arachidonic acid (10 microM) either in the absence or the presence of Ind. 4. The results suggest that endogenous prostaglandins and arachidonic acid contribute to the maintenance of the excitability of the myenteric plexus by DMPP and substance P. By contrast, the release of ACh induced by 5-HT and EFS may be regulated by arachidonic acid and not by prostaglandins.

Oxygen consumption stimulated by increases in permeability of Na+ of mouse diaphragm muscle in vitro.

1. The effects on oxygen consumption of agents that modify Na(+)-permeability were examined in mouse diaphragm muscle perfused with a bathing solution that contained (+)-tubocurarine in a flow-through mode. Twitch tension and levels of Na+ and K+ in the muscle were also measured. 2. Unstimulated preparations decreased the concentration of oxygen in the bathing solution, indicating a basal level of oxygen consumption. Electrical stimulation of the muscle further decreased the concentration of oxygen. Potassium cyanide eliminated both the basal and the stimulated consumption of oxygen. 3. Veratridine facilitated the effect of stimulation at 0.1 Hz on both the consumption of oxygen and the twitch tension. Ouabain antagonized those effects. 4. Twitch contractions were blocked in the presence of dantrolene sodium or by pretreatment with ethylene glycol. Electrical stimulation of such preparations still caused a residual but considerably decreased consumption of oxygen. Ouabain and tetrodotoxin reduced the residual consumption of oxygen. 5. Ouabain significantly increased the levels of Na+ in the tissue, while veratridine alone did not. The effect of ouabain was further potentiated by the simultaneous presence of veratridine. 6. These results indicate that the enhancement of the sarcolemmal permeability to Na+ increases the rate oxygen consumption. This concept supports the hypothesis that Na+ homeostasis depends on energy consumption.

Differences in control of descending inhibition in the proximal and distal regions of rat colon.

1. Descending inhibition in the proximal and distal portions of rat colon was studied separately, in vitro. 2. In the proximal colon, localized distension with a small balloon caused three types of response (contraction; relaxation; relaxation, then contraction) of the circular muscle on the anal side of the distended region. 3. Distension caused descending relaxation of circular muscle in all segments of the proximal colon, although for this prostaglandin F2 alpha (PGF 2 alpha) was necessary in some segments to increase muscle tone. 4. Atropine and guanethidine did not inhibit this descending relaxation, but tetrodotoxin did. 5. Hexamethonium inhibited the descending relaxation in 14 of 17 preparations of proximal colon tested, but not in the others. 6. In the distal colon, distension consistently caused an increase in the tone of the circular muscles. Descending relaxation was observed only after development of higher tone. Atropine and guanethidine did not inhibit the relaxation, but tetrodotoxin did. 7. Hexamethonium did not inhibit the descending relaxation in most of the preparations of distal colon examined. 8. AF64A, an inhibitor of choline uptake, inhibited the response mediated by cholinergic neurons in vitro to electrical transmural stimulation of the longitudinal muscle of proximal colon. 9. Treatment of colonic preparations with AF64A in vitro resulted in inhibition of descending relaxation in those of proximal, but not those of distal, colon. 10. The participation of intrinsic cholinergic neurones in the descending neuronal pathway is strongly suggested by the results in the proximal colon, but less so in the distal colon. 11. The tone and spontaneous contractile activity of colonic circular muscles are discussed in relation to their neuronal control.

The biochemical and ultrastructural examinations in central cholinergic damage of the rat induced by the intraperitoneal administration of AF64A.

Ethylcholine mustard aziridinium ion (AF64A), a synthesized cholinergic neurotoxin, was administered via intraperitoneal injection to the rat to study its effect on the central cholinergic nervous system. A single or consecutive daily injection of AF64A for 10 days resulted in a persistent reduction of acetylcholine (ACh) content in the several tested regions of the brain in the following order: hippocampus greater than cerebral cortex = striatum, the degree was the greatest in the hippocampus. Both resting and K(+)-stimulated release of ACh from the hippocampus were also significantly reduced 24 hr after a single injection of AF64A. Furthermore, daily injection of AF64A for 10 days induced a significant reduction of choline acetyltransferase (ChAT) activity in the homogenate obtained from the hippocampus but not from the cerebral cortex and striatum. ChAT activity in the crude synaptosomal fraction of the cerebral cortex was also significantly decreased. These results suggest that intraperitoneal administration of AF64A could induce cholinergic hypofunction more selectively in the nerve terminals. The high affinity choline uptake, which is located mainly on cholinergic nerve terminals, was not affected by the administration of AF64A. Any notable changes of ultrastructure in the cholinergic nerve terminals after the administration were not observed in all three regions examined. The present findings suggested that intraperitoneal administration of AF64A induces a specific damage of cholinergic nerve terminals by inhibiting ChAT activity. The cholinergic damage was most prominent in the hippocampus.