In vitro experiments on the accumulation and release of 14C-histamine by snail (Helix pomatia) nervous tissue.

When isolated snail ganglia were incubated at 25° in a medium containing ¹⁴C-histamine, tissue:medium ratios of about 4 were obtained after only 5 min. Metabolism of the accumulated amine is rapid, for even within this short incubation period, 20 per cent of the substance was metabolised. The process responsible for this accumulation showed properties of an active transport system: it was temperature-sensitive, sodium dependent, and particularly inhibited by ouabain, phenoxybenzamine, chlorpromazine and desimipramine. The accumulation of ¹⁴C-histamine showed saturation kinetics typical of a carrier-mediated process and can be divided into sodium-sensitive and -insensitive components. Kinetic analysis of the data revealed similar K_m values for both sodium-sensitive and sodium-insensitive plus -sensitive components, i.e. 10^?5 M.A rapid efflux of radioactivity from tissue loaded with ¹⁴C-histamine was observed when exposed to 70 mM KCl. This release was inhibited when the calcium content in the medium was replaced by sucrose. Moreover, cobalt ions added to the incubation medium counteracted the effect of KCl.The discussion of the present results is based on the hypothesis that histamine has a transmitter function in the snail CNS.     

The in vitro uptake of biogenic amines by snail (Heliz pomatia) nervous tissue.

The uptake of [³H]5-HT, [³H]dopamine, [³H]noradrenaline and [³H]octopamine into the suboesophageal ganglia of the snail, Helix pomatia, was studied. When tissues were incubated at 25° in mediums containing radioactive amines, tissue: medium ratios of about 30:1, 18: l, 4:1 and 5:1 for 5-HT, dopamine, noradrenaline and octopamine respectively were obtained after 20–30 min incubation. Tissues incubated at 25° in mediums containing radioactive amines for 20–30 min showed that 90% of the radioactivity was present as unchanged [³H]5-HT, [³H]dopamine, [³H]noradrenaline or [³H]octopamine. The high tissue : medium ratios for 5-HT and dopamine, but not for noradrenaline and octopamine, demonstrated saturation kinetics which were dependent upon temperature and sodium ions. From the Lineweaver-Burk plots, two uptake mechanisms for 5-HT at 25° were resolved, the high affinity uptake process having a K_m1valueof 8.48 × 10^?8 M and V_max1 value of 0.077 nmole/g per min, while the lower affinity process had a K_m2 value of 1.8 × 10^?6 M and a V_max2 value of 0.66 mole/g per min. At 0° a single uptake mechanism for 5-HT occurred which gave a K_m value of 0.152 × 10^?8 M and a V_m value of 0.0203 nmole/g per min. In the case of dopamine, the Lineweaver-Burk plot of 25° showed a single uptake process with values for K_m and V_max of 1.02 × 10^?7 M and 0.0673 nmole/g per min respectively. This process did not function at 0°. The effects of various agents and ions upon the accumulation processes for all amines were also studied, and the findings indicate that the same neurons may well accumulate more than one amine type. It is concluded that 5-HT and dopamine uptake in the snail ganglia is a mechanism for inactivating these substances at 25° and that an uptake mechanism for 5-HT also functions at 0°. The present results are discussed from the point of view of the monoamines having transmitter functions in the snail CNS.     

Effects of 5,7-dihydroxytryptamine on an identified 5-hydroxytryptamine-containing neurone in the central nervous sytem of the snail Helix pomatia.

1. The effect of 5,7-dihydroxytryptamine (5,7-DHT) on an identified 5-hydroxytryptamine (5-HT)-containing neurone in the CNS of the snail was studied by histochemical, biochemical and electrophysiological methods. 2. Low concentrations of 5,7-DHT decreased the endogenous 5-HT content of the neurone without affecting the amino acids, while relatively large amounts of the drug proportionately lowered 5-HT and in addition slightly decreased the tryptophan and methionine content of the cell. 3. 5,7-DHT blocked the uptake of [3H]-5-HT into the neurone; the close analogue 5,6-DHT was more potent. 4. As well as slightly influencing the accumulation of [3H]-tryptophan by the neurone 5,7-DHT inhibited the metabolism of this amino acid to form 5-HT, probably by affecting the enzyme tryptophan-hydroxylase. 5. 5,7-DHT produced a postsynaptic blockade of transmission from the neurone by blocking the 5-HT receptors of the follower neurones. This effect appeared to be specific for 5-HT receptors. 6. The data support the idea that 5,7-DHT is neurotoxic for indoleamine-containing neurones.     

Properties of slow early potassium current in neurons of snail Helix pomatia

• 1.1. In isolated neurons of visceral ganglia of snail Helix pomatia a slow early outward current (IAs) was studied using a two-microelectrode voltage clamp technique.
• 2.2. The time of activation and inactivation of I_As at −40 mV were 90–120 msec and 3–5 sec respectively. The removal of inactivation at −120 mV took 2–5 min.
• 3.3. The reversal potential of the I_As was about −80mV in normal saline and was sensitive to the external potassium concentration, changing about 35 mV per fivefold change in potassium over the range from 4 to 20 mM. The results suggest that I_As were due to K⁺.
• 4.4. The I_As persisted in Ca²⁺-free medium and in the presence of Ca²⁺-channels blockers, e.g., Cd²⁺.
• 5.5. The I_As were blocked by 1–10 μM extracellular 4-aminopyridine, 1 mM of tetraethylammonium ions, 1 mM of Ba²⁺, but one was resistant to 1 mM Cs⁺.
• 6.6. 4-aminopyridine had a dual effect on the I_As. It blocked the normal current, and then appeared to increase the inactivated currents.

     

大鼠丘脑酪氨酸羟化酶调控丙泊酚诱导的镇静作用

目的通过定量磷酸化蛋白质组学研究丙泊酚诱导大鼠丘脑镇静作用的新型调节机制。方法①采用同位素标记相对和绝对定量(iTRAQ)技术对丙泊酚(100 mg·kg-1,ip)诱导镇静模型SD大鼠丘脑差异磷酸化蛋白质进行标记和定量分析;基因本体(GO)富集分析差异磷酸化蛋白质的生物进程、细胞组分及分子功能;Western印迹法检测大鼠丘脑酪氨酸羟化酶(TH)第19位丝氨酸磷酸化表达水平。②利用TH抑制剂甲基酪氨酸(20 mg·kg-1,提前30 min ip给药),考察其对丙泊酚诱导C57BL/6J小鼠翻正反射消失的诱导时间、持续时间和翻正反射消失率的影响。结果质谱结果显示,与正常对照组相比,丙泊酚组SD大鼠丘脑中共92个磷酸化蛋白质表达水平发生改变,其中TH第19位丝氨酸磷酸化水平显著下调(P<0.01);与Western印迹法结果一致(P<0.05)。丙泊酚(70~120 mg·kg-1)剂量依赖性地增加小鼠的翻正反射消失率,TH抑制剂甲基酪氨酸预处理可使丙泊酚诱导小鼠翻正反射消失率的半数有效剂量(ED50)由92.32 mg·kg-1(95%CI:90.60~94.08,R2=0.9969)降至85.38 mg·kg-1(95%CI:81.30~89.67,R2=0.9768),量效曲线左移。与单用丙泊酚组比较,甲基酪氨酸预处理组可显著延长丙泊酚(90,100和120 mg·kg-1)诱导的C57BL/6J小鼠翻正反射消失持续时间(P<0.05)。结论本研究筛选出大鼠丘脑中多种磷酸化蛋白质参与调控丙泊酚诱导的镇静效应,抑制TH的活性可显著增强丙泊酚的镇静作用。     

Potassium channels in nervous tissue.

There is a multiplicity of potassium channels in nervous tissue. These have been characterized on the basis of their electrophysiological actions but more information is required on their structures and on the functions of the different subtypes of channel in different parts of the nervous system. We currently also lack drugs which are specific in opening or closing individual subtypes of channel. However, when more is known about the structure and function of these channels and when more specific modulators of their activity are available, it is likely that the use of such compounds may be of great value in the treatment of a variety of conditions affecting the nervous system, including epilepsy, the damage due to cerebral anoxia, neurodegenerative disorders and demyelinating disorders.     

Tyrosine hydroxylase activity in rat brain regions after chronic treatment with +/--propranolol.

Rats were injected twice daily with +/--propranolol (6 mg kg-1 day-1) for 14 days and killed 16 h after the final injection. Tyrosine hydroxylase activity was measured in both soluble and particle-bound forms in various brain regions. The activity of the soluble enzyme was not significantly altered by propranolol treatment in any of the brain regions studied. The tyrosine hydroxylase activity in the particulate fraction was significantly increased in corpus striatum and unchanged in other brain regions. The propranolol concentrations in the various brain regions in this chronic study were far lower than necessary to produce a significant change in tyrosine hydroxylase activity in acute experiments. It was concluded that chronic propranolol treatment produces a persistent increase in bound tyrosine hydroxylase activity in rat corpus striatum.     

Tyrosine hydroxylase:--examination of conditions influencing activity in pheochromocytoma, adrenal medulla and striatum.

Soluble tyrosine hydroxylase from human pheochromocytoma, bovine adrenal medulla and rat striatum was studied with regard to factors influencing enzyme activity. The pH optimum was different for tyrosine hydroxylase from the three tissues examined: pheochromoeytoma, pH 7.8; adrenal medulla, pH 6.8; and striatum, pH 6.0. In preparations of tyrosine hydroxylase from each tissue, the presence of Mg²⁺. ATP and cyclic AMP resulted in a significant activation of the enzyme. On the other hand, addition of catecholamines to the reaction mixture produced a marked inhibition which became more pronounced with increasing pH. In all three tissues, the pH for maximum per cent stimulation by Mg²⁺, ATP and cyclic AMP was different from the pH optimum for tyrosine hydroxylase. In addition, the magnitude of this stimulation, as well as the basal activity, was dependent on the buffer present in the reaction mixture.     

Presynaptic modulation of synaptic transmission in Helix pomatia: the effects of serotonin and dopamine antagonists

Some presynaptic cells in Helix pomatia possess serotonin and dopamine receptors, which are involved in the modulation of synaptic transfer. The serotonin antagonists tryptamine, 7-methyltryptamine, morphine, atropine, and methysergide decreased transmitter mobilization. 5-Methoxy-gramine, which is a selective inhibitor of hyperpolarizing serotonin responses, had no effect. The dopamine antagonists curare, chlorpromazine and haloperidol decreased transmitter mobilization, while ergometrine, flupenthixol, promethazine and 6-OH dopamine induced a small increase. The mechanisms are discussed and the properties of the presynaptic receptors are compared to the postsynaptic receptors. The usefulness of cell F76 in Helix brain as a model system for vertebrate brain function is discussed.     

Tyrosine hydroxylase activity increases in pineal sympathetic nerves after depletion of neuronal serotonin

Summary The injection of p-chlorophenylalanine (PCPA) acutely reduced serotonin in the pineal gland of the rat and selectively elevated the noradrenaline (NA) content during the subsequent 24 h. The activity of tyrosine hydroxylase (TH) also increased in intact glands during the first 6 h after PCPA injection but returned to normal at 24 h. This enhancement of enzyme activity was only observed in the presence of a non-saturating concentration of the cofactor. Serotonin depletion by PCPA cannot directly account for the increased enzyme activity, because the amine does not modify TH activity. Moreover, this increase is restricted to the pineal, since in other sympathetically innervated organs, such as the atria, PCPA produced an acute but transient reduction in TH activity. The elevation described here is not due to a net increase in the amount of enzymatic protein, because TH activity is similar in pineal homogenates from treated and control rats when a saturating concentration of the cofactor was used. The availability of storage sites in pineal nerve vesicles due to serotonin depletion, seems to release TH activity from the negative control exerted by cytoplasmic catecholamines. Enzyme activity in the pineal is acutely enhanced until a new steady state is reached at a higher concentration of endogenous NA.     

Influence of Helix pomatia enzyme preparations on the oxidative conversion of some clostebol acetate metabolites in urine

Clostebol acetate (4-chloro-testosterone acetate) is an anabolic steroid used for fattening purposes in cattle breeding. To safeguard public health, its use has been prohibited by the European Commission since 1986. Screening for its urinary metabolites is therefore an important tool for the control of possible violations. Because those metabolites appear conjugated to glucuronic acid or sulfate, deconjugation prior to analysis is necessary. This work describes the variability in results seen with the use of various commercial preparations of Helix pomatia (SHP) for enzymatic hydrolysis of the conjugates. A simultaneous oxidative side reaction was observed, converting metabolites with a 3-OH-4-ene structure into a 3-oxo-4-ene structure. This was not observed when samples were incubated without enzyme or in the presence of heat-inactivated SHP. GC-MS analysis revealed oxidation of some metabolites of clostebol acetate, 4-chloro-4-androsten-3alpha-ol-17-one and 4-chloro-4-androsten-3alpha,17beta-diol, changing them into other metabolites, 4-chloro-4-androsten-3,1 7-dione and clostebol (4-chloro-testosterone), respectively. Based on the difference in cross-reactivities of the antibodies for these metabolites, comparative analysis in enzyme immunoassay, following enzymatic hydrolysis, confirmed this transformation. This oxidative conversion phenomenon could be of great importance when considering the choice or target analytes for screening bovine urine.     

Lectins in drug delivery: a study of the acute local irritancy of the lectins from Solanum tuberosum and Helix pomatia.

Lectins are proteins or glycoproteins of non-immune origin capable of binding to one or more specific sugar residues. The potential for using lectins as a means of 'anchoring' a drug delivery system to the mucosal surfaces of the eye has been investigated in previous work, with the lectins from Solanum tuberosum and Helix pomatia showing particular promise. In this study the acute local dermal irritancy of these lectins, in terms of their potential to cause inflammation and tissue necrosis, was investigated. After an initial study in terminally anaesthetised animals (to ensure no gross toxicity was evident), five male New Zealand white rabbits from the same litter were briefly anaesthetised and Evans blue injected intravenously as a marker of inflammation. Sterile lectin solutions in normal saline at a range of concentrations from 50 to 500 microg ml(-1) were prepared and 50-microl volumes injected intradermally at 18 sites across a shaved area of each rabbit's back. The rabbits were then allowed to regain consciousness. There was no evidence of tissue necrosis, oedema or Evans blue infiltration with any of the lectin solutions administered. The rabbits did not display any signs of discomfort such as scratching or continued grooming throughout the experiment. Histological examination of the injection sites revealed little sign of any inflammation, such as heterophil migration, oedema or tissue damage. It was concluded that these lectins demonstrate minimal acute irritancy, and will, therefore, be taken forward for formulation and in vivo studies.