Picric acid-evoked release of [14C]acetylcholine from the isolated synaptosome of rat cerebral cortex

Picric acid stimulated, in a dose-dependent manner, the release of [14C]acetylcholine (ACh) from isolated synaptosomes of rat cerebral cortex pre-loaded with labelled choline. Radioactive ACh was separated for counting from choline in the synaptosomal supernatants by a liquid cation-exchange method. Neither the nicotinic antagonist (hexamethonium) nor the muscarinic antagonists (atropine and scopolamine) affected the effectiveness of picric acid, suggesting that the action of picric acid does not occur through a cholinoceptor-mediated mechanism. Moreover, oxotremorine, but not pilocarpine, inhibited ACh release in a concentration-dependent manner in either basal- or picric acid-evoked conditions, indicating the presence of muscarinic M2-receptors for auto-regulation of ACh release. The effect of picric acid was compared with high-K+ depolarization which also initiated a non-receptor-mediated release of ACh. Deletion of calcium ion from the medium negated the effects of both drugs. The ACh-releasing effect of picric acid was totally abolished, whereas high-K+ depolarization was reduced to some extent, when tetrodotoxin was added to the medium. These results indicate that picric acid acts as a releaser of ACh in the cerebrocortex of rat.     

The molluscan neuropeptide FMRFamide stimulates the release of [14C]acetylcholine from isolated ileal synaptosomal preparations of guinea-pig

FMRFamide stimulated, in a dose-dependent manner, the efflux of [14C]acetylcholine (ACh) from isolated synaptosomes of guinea-pig ileum preloaded with labelled choline. Participation of the cholinergic mechanism and/or substance P was ruled out by the finding that antagonists failed to affect the FMRFamide-induced release of ACh. The ACh-releasing action of FMRFamide was negated by the deletion of calcium ion from the bathing medium and it was also abolished by tetrodotoxin. The results obtained suggest that FMRFamide possesses the ability to induce the release of ACh from enteric synaptosomes of guinea-pig.     

Evoked release of [14C]norepinephrine from the rat hypothalamus during feeding.

The pattern of catecholamine release was studied at sites adjacent to the lateral ventricle or in the anterior, dorsomedial or ventromedial hypothalamus of the rat as it was feeding. Endogenous stores of norepinephrine (NE) were first labeled by the microinjection of [14C]NE into these circumscribed sites. Subsequently, [14C]NE release was examined by repeated perfusions of an artificial cerebrospinal fluid through a push-pull cannula at the rate of 23 mul/min for 5-10 min every 30 min. After successive control perfusates were collected, food or water was given to the animal. During an interval of feeding, a significant efflux of [14C]NE and its metabolites occurred reliably from midline structures of the hypothalamus at the level of the ventromedial nucleus. Although the feeding-related output of [14C]NE detected within the anterior hypothalamus was lower, [14C]NE was also released from this region when the rat pressed a lever to obtain food pellets. These results support the view that endogenous catecholamines underlie, at least partially, diencephalic mechanisms controlling food intake, including sensory, motor, or motivational components.     

A possible neuronal release of [14C] GABA from the rat cerebellum in vivo

Release patterns for exogenously applied [¹⁴C] labelled -amino-n-butyric-acid (GABA) have been investigated in rat cerebellar cortex in vivo. An increase in [¹⁴C] GABA release could be evoked by stimulating with high (40 mM) K⁺ or veratridine (10^–4 M) but not with direct electrical stimulation. Biphasic patterns for high K⁺ and possibly veratridine stimulated release of GABA suggest the existence of two separate anatomical sources of isotope which are sensitive to these depolarising stimuli. Both K⁺ and veratridine-evoked GABA release are calcium dependent. Studies involving partial replacement of Na⁺ with HEPES, (N-2-hydroxyethyl-piperazine-N-2-ethane-sulphonic acid), sucrose or choline chloride also reveal a sodium dependency of [¹⁴C] GABA release. These studies collectively indicate a neuronal source for evoked GABA release, a criterion for transmitter identification not previously satisfied in the cerebellar cortex.Supported in part by a grant from S.R.C. to N.D. and C.G.D.Supported by CAPES and FAPESP (Brazil)Supported by CNPq and FAPESP (Brazil)     

Lymphokine-induced uptake of [14C]glucosamine, [14C]glucose,and [3H]leucine by macrophages

Lymphokine-activated (LK⁺) and control (LK^–) macrophages were cultured for 66 h and then pulsed with [¹⁴C]glucosamine. Uptake of [¹⁴C]glucosamine was greater in LK⁺ than in LK^– cultures. If, after 66 h, the medium was replaced with fresh medium and then pulsed with either [¹⁴C]glucose or [¹⁴C]glucosamine, the uptake of isotope was greatly reduced compared to cultures with no change of medium. However, uptake of both radiolabeled substances was still found to be greater in LK⁺ cultures than in LK^– cultures. Although uptake of both substances was enhanced by lymphokines, the uptake kinetics of each isotope was different. Under similar conditions the uptake of [³H]leucine was not enhanced by lymphokine activation. These data are interpreted to mean that LK⁺ macrophages are metabolically stimulated and utilize more glucose and glucosamine. The difference in kinetics implies a different utilization by macrophages for each substance.     

In vitro autoradiography of [3H]acetylcholine binding in rat hind limb muscles

The morphological distribution of acetylcholine receptors in normal and denervated rat tibialis anterior and soleus muscles was studied by in vitro quantitative autoradiography with [3H]acetylcholine (ACh). In normal muscles [3H]ACh binding was restricted to the neuromuscular junction, while denervated muscles showed binding also in extrajunctional areas. After denervation there was also an increase in the junctional binding.     

The synthesis of lipids from [1-14C]acetate by isolated rat brain capillaries

Lipid biosynthesis was investigated in isolated cerebral microvessels obtained from adult Sprague-Dawley rats using [1-14C]acetate as precursor. All lipid classes were labelled by [1-14C]acetate. Neutral lipids incorporated about 50% of radiolabelled acetate, among which free fatty acids and triglycerids showed the highest level of incorporation. Moreover, about 4% of radioactivity was found in cholesterol fraction. In phospholipid fraction, phosphatidylcholine and phosphatidylethanolamine were the main radiolabelled phospholipids. [1-14C]acetate was also incorporated into sulphatides and cerebrosides. The presence of bovine serum albumin in incubation medium modified the percentage of incorporation in different lipid fractions.     

Evaluation of autoreceptor-mediated control of [3H]acetylcholine release in rat and human neocortex

In order to assess the autoinhibitory control of endogenous acetylcholine (ACh) in rat and human neocortex, slices of these tissues were prelabelled with [³H]choline, superfused continuously and stimulated electrically using various frequencies in the presence or absence of drugs. The autoinhibitory feedback control of [³H]ACh release was operative – despite the absence of blockers of ACh esterase – at stimulation frequencies ≥3 Hz in rat and ≥6 Hz in human neocortex tissue. At these frequencies the muscarinic antagonist atropine (0.1 μM) disinhibited the release of [³H]ACh in both species. Estimation of the biophase concentration of ACh near the autoreceptor in the rat neocortex from concentration-response curves of the muscarinic agonist oxotremorine revealed that at 3 Hz about 25% of the autoreceptors were activated by endogenously released ACh. This estimation is consistent with an increase in [³H]ACh release to about 120% of control values by complete blockade of autoreceptors with atropine. The observation that in human neocortical tissue presynaptic autoinhibition of [³H]ACh release is operative at stimulation frequencies ≥6 Hz suggests that selective blockade of autoinhibition may also increase ACh release in the cortex of Alzheimer’s disease patients, without additional blockade of the enzyme acetylcholinesterase. Received: 22 September 1998 / Accepted: 27 April 1999     

Comparison of the rate of absorption and proteolysis of [14C]choleragen and [14C]bovine serum albumin in the rat jejunum.

[14C]choleragen was used to study the rate of disappearance of choleragen enterotoxin from the jejunum of rats. [14C]bovine serum albumin (BSA) was studied in a similar manner. Almost one-third of the labeled toxin had disappeared from the intestine after 6 h. Its rate of disappearance was the same in germfree rats as in conventional rats. The rate of proteolysis of [14C]choleragen and [14C]BSA by intestinal mucodal lysosomal enzymes was also studied. Neither was significantly degraded by neutral proteases; however, heat-inactivated toxin was. They were all degraded by acid proteases; however, the rate of BSA proteolysis was only one-third of that of toxin. Soybean trypsin inhibitor had no effect on the in vivo disappearance of toxin nor on the acid proteases. It did inhibit the neutral protease digestion of heat-treated toxin. Aprotinin and protamine inhibited disappearance in loops of gut but had no effect to inhibit degradation rates. Gangliosides inhibited both rates of disappearance and proolysis of toxin. These agents had some different effects on disappearance rates and proteolysis of BSA. The data indicate that cholera enterotoxin is absorbed by intestinal mucosal cells and is degraded by acid proteases in the cells.     

Autoradiography of [14C]paraquat or [14C]diquat in frogs and mice: accumulation in neuromelanin

The herbicide paraquat has been suggested as a causative agent for Parkinson's disease because of its structural similarity to a metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which may induce a parkinsonism-like condition. MPTP as well as its metabolite 1-methyl-4-phenylpyridine have melanin affinity, and the parkinsonism-inducing potency of MPTP is much stronger in species with melanin in the nerve cells. Autoradiography of [3H]MPTP in experimental animals has shown accumulation in melanin-containing tissues, including pigmented neurons. In the present whole body autoradiographic study accumulation and retention was seen in neuromelanin in frogs after i.p. injection of [14C]paraquat or [14C]diquat. By means of whole body autoradiography of [14C]diquat in mice (a species with no or very limited amounts of neuromelanin) a low, relatively uniformly distributed level of radioactivity was observed in brain tissue. Accumulation of toxic chemical compounds, such as paraquat, in neuromelanin may ultimately cause lesions in the pigmented nerve cells, leading to Parkinson's disease.