Phosphodiesterase Activities for Cyclic Nucleotides in Nerve Endings from the Bovine Posterior Pituitary Gland

Abstract: The cyclic nucleotide phosphodiesterase (PDE) activities were studied in a nerve ending fraction from bovine neural lobes. Most of the activity was particulate and unaffected by calcium. Lineweaver-Burk plots for this fraction showed negative cooperativity with apparent K _m values for cyclic AMP of 11 μ M and for cyclic GMP of 4 μ M . The soluble activities for both cyclic nucleotides were activated by calcium and inhibited by calmodulin-binding drugs (trifluoperazine and calmidazolium). The apparent K _m values were 50 μ M for cyclic AMP and 20 μ M for cyclic GMP for the soluble activities. Sucrose density gradients resolved the soluble activities into two peaks. The activity with the higher sedimentation rate (MW 122,000 daltons) hydrolysed both cyclic nucleotides and was calcium-calmodulin-dependent. The other peak (MW 47,000 daltons) had a higher affinity for cyclic AMP than for cyclic GMP and was calcium-independent. Solubilized particulate activities gave two main peaks on the density gradient, both calcium-independent. One was mainly for cyclic AMP (MW 47,000 daltons) and the other mainly for cyclic GMP (MW 133,000 daltons). The function of PDEs in relation to secretion was discussed.     

Uniform Staining of Nerve Endings in Skeletal Muscle with Gold Chloride

Skeletal muscle, teased into strips several millimeters long and 1 mm or less thick, is fixed in a 3:1 mixture of lemon juice and formic acid until transparent. The tissue is compressed between folds of absorbent material to remove excess fixative and impregnated for 10 min in a volume of 1% gold chloride equal to the volume of muscle. After removing excess gold chloride, reduction is effected. Method a. Exposure to 25% formic acid in the dark for 6 hr. When intramuscular nerves are visible, the muscle is further teased with glass instruments. Reduction is continued for an additional 18 hr in 25% formic acid, and the tissue is cleared in glycerol for 24 hr. Method b. Exposure to intense artificial visible light while the tissue is just immersed in distilled water at 37° C. Colour develops in the nerve endings as in a photographic print. When nerves only are stained (approximately 0.5 hr), the lamp is removed and microdissection at room temperature commenced. When nerve endings in muscle spindles are just visible (approximately 1 hr), the distilled water is replaced by glycerol. After either method of reduction, areas of motor end-plates and individual sensory receptors are isolated by microdissection. Preparations are mounted in a small drop of glycerol, considerable pressure is applied to the cover slip, and the cover ringed with a sealing fluid.     

Effect of Tetanus Toxin on Oxytocin and Vasopressin Release from Nerve Endings of the Neurohypophysis

The effect of tetanus toxin on neuropeptide hormone release from isolated nerve endings of the neural lobe of rat pituitaries (neurosecretosomes) was measured in a perfusion system. Tetanus toxin inhibited depolarization-evoked release of oxytocin and vasopressin in a time- and dose-dependent manner. At 1 microgram/ml, tetanus toxin blocked stimulated release by 85%. Tetanus toxin that was preincubated with a neutralizing monoclonal antibody or heated to 100 degrees C had no effect on hormone release. The ionophores A23187 and ionomycin were potent stimulators of hormone release in control nerve endings, but were not able to overcome the effect of tetanus toxin in intoxicated nerve endings. 8-Bromo-cyclic GMP, which has been reported to reverse the action of tetanus toxin in PC12 cells, had no effect on the action of tetanus toxin in neurosecretosomes. Neurosecretosomes are the first system in which tetanus toxin has been shown to block release from peptidergic nerve terminals. They appear to be a valuable in vitro system for studying the biochemical mechanism of tetanus toxin action.     

Effects of Peptides of the Secretin-Glucagon Family and Cyclic Nucleotides on Tyrosine Hydroxylase Activity in Sympathetic Nerve Endings

Previous studies have shown that certain peptides of the secretin-glucagon family stimulate tyrosine hydroxylase activity in sympathetic neurons of the superior cervical ganglion and three of its end organs, i.e., the iris, pineal gland, and submaxillary gland. To determine whether a similar regulation occurs in other sympathetic neurons, the effects of two of these peptides, secretin and vasoactive intestinal peptide, were examined in the right cardiac ventricle of the rat, a tissue innervated primarily by the middle and inferior cervical ganglia. Both peptides stimulated tyrosine hydroxylase activity, measured in situ, in this tissue. In addition, several second messenger systems were investigated as possible mediators of this peptidergic stimulation of tyrosine hydroxylase activity in autonomic end organs. 8-Bromoadenosine 3',5'-cyclic monophosphate and forskolin elevated tyrosine hydroxylase activity in slices of both the right ventricle and the submaxillary gland. 8-Bromoguanosine 3',5'-cyclic monophosphate also stimulated tyrosine hydroxylase activity in both tissues, whereas nitroprusside stimulated activity only in the submaxillary slices. Furthermore, the phosphodiesterase inhibitors 3-isobutyl-1-methylxanthine and/or Ro 20-1724 potentiated the stimulation by secretin, as well as the stimulations by forskolin and nitroprusside. Phorbol 12,13-dibutyrate also stimulated tyrosine hydroxylase activity in cardiac and submaxillary slices; however, no potentiation of these effects was seen following addition of either phosphodiesterase inhibitor. These data, taken together with those of previous studies, suggest a role for a cyclic nucleotide, probably adenosine 3',5'-cyclic monophosphate, in the peptidergic stimulation of tyrosine hydroxylase activity in sympathetic nerve terminals.     

A Bioluminescent γ-Aminobutyrate Assay for Monitoring Its Release from Inhibitory Nerve Endings

Abstract: A bioluminescent GABA assay is described. The principle of the procedure is based on the action of GABASE (GABA-aminotransferase plus succinic semialdehyde dehydrogenase), coupled to the detection of succinic semialdehyde and NADH, using Photobacterium luciferase. The method was used for monitoring GABA release from depolarized brain slices.     

Sensory Nerve Endings Shown by Nitro Blue Tetrazolium in Isolated Muscle Spindles of the Cat

Muscle spindles were isolated from freshly removed cat lumbrical muscles in oxygenated Ringer's solution and placed in a solution containing 10 ml of 0.1% nitro blue tetrazolium and 10 ml of 0.2 M phosphate buffered sodium succinate, pH 7.6. Spindles were incubated in this solution at 37 C for 4-12 hr, returned to Ringer's for 30 min at room temperature, fixed in 10% formal-Ringer's for 30 min, and stored indefinitely in distilled water. With this technique the patterns of sensory innervation can be clearly visualized by the deposition of diformazan. The stained preparations may be mounted in glycerol and teased further for whole mount inspection or they may be embedded in Epon and serially sectioned for more detailed study.     

Activation and Inactivation of Oxytocin and Vasopressin Release from Isolated Nerve Endings (Neurosecretosomes) of the Rat Neurohypophysis

Neurosecretory terminals (neurosecretosomes, NSS) were isolated from rat neurohypophyses. High [K⁺]_oor veratridine stimulated secretion of vasopressin and oxytocin by up to ～ 100-fold. Stimulated secretion was dependent on calcium and temperature, and could be elicited from NSS maintained in culture for 4 days. After overnight culture of the NSS, secretion was still inhibited by calcium channel blockers (cobalt, dihydropyridines, ω-conotoxin, D 600) and K opiates (dynorphin and U50488). Ionomycin evoked dose and calcium-dependent hormone release, with a Hill coefficient for calcium of 1.74. High [K⁺]_o enhanced the 5 μMionomycin-induced secretion, apparently through calcium entry rather than depolarization, as the increase in secretion was abolished by 100 μM D 600. During prolonged depolarization the hormone secretion peaked within 2 min, then declined to near basal levels. Depolarization for 25 min without calcium neither activated secretion nor prevented subsequent secretion on readdition of calcium, suggesting that the decline in secretion was not due to membrane depolarization. Indeed, the rates of decline in secretion were similar for different levels of depolarization (0.070 ± 0.003 and 0.081 ± 0.003 min^?1 for 25 and 45 mM [K⁺]_o, respectively). Four minutes after the onset of continuous depolarization (45 mM[K⁺]_o) in the presence of calcium, the declining secretion was still dependent on voltage-activated calcium influx through channels sensitive to D 600 and nitrendipine. The results presented here suggest that the decline in secretion during prolonged depolarizing stimuli may be due to exhaustion, inactivation, or desensitization of a calcium-triggered event.     

Ang II Enhances Noradrenaline Release from Sympathetic Nerve Endings Thus Contributing to the Up-Regulation of Metalloprotease-2 in Aortic Dissection Patients' Aorta Wall

Object

To test the hypothesis that angiotensin II (Ang II) could enhance noradrenaline (NA) release from sympathetic nerve endings of the aorta thus contributing to the up-regulation of matrix metalloproteinase 2 (MMP-2) during the formation of aortic dissection (AD).

Methods

Ang II, NA, MMP-2, MMP-9 of the aorta sample obtained during operation from aortic dissection patients were detected by High Performance Liquid Chromatography and ELISA and compared with controls. Isotope labelling method was used to test the impact of exogenous Ang II and noradrenaline on the NA release and MMP-2, MMP-9 expression on Sprague Dawley (SD) rat aorta rings in vitro. Two kidneys, one clip, models were replicated for further check of that impact in SD rats in vivo.

Results

The concentration of Ang II, MMP-2, 9 was increased and NA concentration was decreased in aorta samples from AD patients. Exogenous Ang II enhanced while exogenous NA restrained NA release from aortic sympathetic endings. The Ang II stimulated NA release and the following MMP-2 up-regulation could be weakened by Losartan and chemical sympathectomy. Beta blocker did not influence NA release but down-regulated MMP-2. Long term in vivo experiments confirmed that Ang II could enhance NA release and up-regulate MMP-2.

Conclusions

AD is initiated by MMP-2 overexpression as a result of increased NA release from sympathetic nervous endings in response to Ang II. This indicates an interaction of RAS and SAS during the formation of AD.     

Neurofilament Proteins Are Synthesized in Nerve Endings from Squid Brain

Abstract: It is generally believed that the proteins of the nerve endings are synthesized on perikaryal polysomes and are eventually delivered to the presynaptic domain by axoplasmic flow. At variance with this view, we have reported previously that a synaptosomal fraction from squid brain actively synthesizes proteins whose electrophoretic profile differs substantially from that of the proteins made in nerve cell bodies, axons, or glial cells, i.e., by the possible contaminants of the synaptosomal fraction. Using western analyses and immunoabsorption methods, we report now that (a) the translation products of the squid synaptosomal fraction include neurofilament (NF) proteins and (b) the electrophoretic pattern of the synaptosomal newly synthesized NF proteins is drastically different from that of the IMF proteins synthesized by nerve cell bodies. The latter results exclude the possibility that NF proteins synthesized by the synaptosomal fraction originate in fragments of nerve cell bodies possibly contaminating the synaptosomal fraction. They rather indicate that in squid brain, nerve terminals synthesize NF proteins.     

A Modified Pyridine-Silver Stain for Teased Preparations of Motor and Sensory Nerve Endings in Skeletal Muscle

This technique has been developed especially to stain sensory receptors which have been localised intramuscularly by electrophysiological means. Rat intertransverse caudal muscles, removed immediately after death, are fixed for 24 hr in a freshly prepared mixture of absolute ethyl alcohol, 4.5 ml; distilled water, 5 ml; and concentrated HNO_a, 0.1 ml. After a further 24 hr in 10 ml of absolute ethyl alcohol containing 0.1 ml of ammonia solution (sp. gr. 0.88), the muscles are washed in distilled water for 30 min and placed in full strength pyridine for 2 days. They are then washed for 24 hr in distilled water (changed 5-8 times) and left in 2% AgNO₃, in the dark for 3 days at 25 C. Following reduction in 10 ml of 5% formic acid containing 0.4 gm of pyrogallol for 6-24 hr, the specimens are washed briefly in distilled water and stored in pure glycerol. The nerve endings can then be teased out and mounted in glycerol, under cover glasses ringed with a waterproof cement. The advantage of this method is that it gives consistently good staining of receptors and motor end-plates in small muscles of the rat     

Neonatal Hypothyroidism Affects the Adenine Nucleotides Metabolism in Astrocyte Cultures from Rat Brain

Neonatal hypothyroidism is associated with multiple and severe brain alterations. We recently demonstrated a significant increase in hydrolysis of AMP to adenosine in brain of hypothyroid rats at different ages. However, the origin of this effect was unclear. Considering the effects of adenine nucleotides to brain functions and the harmful effects of neonatal hypothyroidism to normal development of the central nervous system, in this study we investigated the metabolism of adenine nucleotides in hippocampal, cortical and cerebellar astrocyte cultures from rats submitted to neonatal hypothyroidism. ATP and AMP hydrolysis were enhanced by 52 and 210%, respectively, in cerebellar astrocytes from hypothyroid rats. In hippocampus of hypothyroid rats, the 47% increase in AMP hydrolysis was significantly reverted when the astrocytes were treated with T3. Therefore, the imbalance in the ATP and adenosine levels in astrocytes, during brain development, may contribute to some of the effects described in neonatal hypothyroidism.Elizandra Braganhol and Alessandra Nejar Bruno are first authors.     

The Dimensions of the Extracellular Space in Sartorius Muscle

A survey has been made of the amount of muscle water available to inulin, sucrose, and radioiodinated human serum albumin (RISA). The percentage spaces available to the three molecules are of the same order of magnitude, but the sucrose space > inulin space > albumin space. The kinetics of influx and efflux of RISA have been studied, and it appears that a small part of the albumin may be adsorbed in the extracellular phase. Nevertheless the albumin space would appear to give the best index of the extracellular volume. The scatter in values found for the extracellular space by all methods is very great, ranging from 8 to 40 per cent and renders invalid the use of a mean value for the calculation of intracellular concentrations. The variation within paired muscles is less than between pairs, provided the tissue has undergone no volume change. Increase in total muscle volume when the muscle is placed in a hypotonic solution leads to a decrease in the size of the extracellular space.     

MICROTUBULE PROTEIN : Identification in and Transport to Nerve Endings

The subunit protein of microtubules, tubulin, has been demonstrated to be present in isolated nerve endings by gel electrophoresis, amino acid composition, and peptide mapping. The tubulin constitutes approximately 28% of the soluble protein of the nerve endings. The transport of tubulin to the nerve endings has been demonstrated and its relationship to slow transport is discussed.     

烟碱型乙酰胆碱受体在面神经支配的口轮匝肌和躯体神经支配的腓肠肌中的表达差异研究

目的:研究烟碱型乙酰胆碱受体在在面神经支配的口轮匝肌和躯体神经支配的腓肠肌运动终板处的表达差异及可能的原因。方法:分离SD大鼠的口轮匝肌和腓肠肌,通过免疫共沉淀技术计算口轮匝肌和腓肠肌肌肉特异性激酶(Muscle Specific Kinase,MuSK)的表达以及MuSK磷酸化水平。对MuSK的上游信号通路中能使其发生磷酸化的集聚蛋白Agrin、低密度脂蛋白受体相关蛋白4(low-density lipoprotein receptor-related protein 4,Lrp4)以及表皮生长因子家族受体ErbB2、ErbB3和ErbB4(epidermal growth factor receptor)免疫荧光染色,计算这两条不同通路中的蛋白在运动终板处的表达水平。结果:口轮匝肌中MuSK磷酸化水平显著高于腓肠肌(P<0.05)。口轮匝肌与腓肠肌的运动终板处的Agrin和Lrp4表达没有显著差异(P>0.05)。口轮匝肌ErbB2、ErbB3、ErbB4的表达显著高于其在腓肠肌运动终板处的表达(P<0.01)。结论:口轮匝肌和腓肠肌运动终板ErbB、ErbB3、ErbB4的差异表达造成MuSK磷酸化水平不同,可能是两种肌肉运动终板处烟碱型乙酰胆碱亚基表达量不同的原因。     

The Pattern of Activation in the Sartorius Muscle of the Frog

The development of isometric twitch tension has been compared with the redevelopment of isometric tension in the fully active frog sartorius muscle following release. At 0°C the rate of rise of isometric twitch tension is the same as that for the muscle in the fully active state at the same tension but not until about 40 msec. after the stimulus and then only for a few milliseconds. The rates of rise of tension in the twitch and in the redevelopment of tension in the fully active muscle appear to be nearly the same at low tensions. Substitution of nitrate for chloride in the Ringer's solution bathing the muscle retards the development of tension during the early part of the contraction phase of the twitch and the effect reaches a maximum within 3 minutes after changing the solutions. These observations have been discussed in connection with some possible patterns of activation and the hypothesis has been advanced that the rate of activation of a sarcomere is determined mainly by the rate at which the transverse component of the link between excitation and contraction is propagated inwards from the periphery to the center of the fiber. This hypothesis has been discussed in relation to others concerning the nature of excitation-contraction coupling.     

Postnatal Changes in Enzyme Activities in Synaptosomes Isolated from Hamster Optic Nerve Endings

Subsynaptosomal fractions isolated from optic terminal nuclei of adult and neonatal hamsters exhibited developmental changes in specific density, mitochondrial activity, and K+-stimulated, ouabain-inhibited p-nitrophenylphosphatase (K-pNPPase) activity around the time of eye opening. The specific activity of K-pNPPase was six- to sevenfold higher after eye opening (14-16 days postnatal). A significant proportion of high-specific- activity K-pNPPase was recovered from the lightest subsynaptosomal fraction at all ages. This fraction contained very little external membrane by galactose oxidase - NaB3H4 labeling, suggesting that it may represent an internal pool, possibly the axonally transported form of the enzyme. Synaptic mitochondrial cytochrome c. oxidase activity also approximately doubled in the period between 12 and 16 days. The specific density of the external membrane increased very slowly, banding at 1.0 M sucrose at 12 and 16 days, and at 1.2 M in adults. These maturational events may reflect increased energetic needs for optic nerve endings following eye opening.     

Submicroscopic Changes of the Nerve Endings in the Adrenal Medulla after Stimulation of the Splanchnic Nerve

The nerve endings of the adrenal medulla of the rabbit were studied under the electron microscope in the normal condition and after prolonged electrical stimulation of the splanchnic nerve. With a stimulus of 100 pulses per second for 10 minutes, there is an increase in the number of synaptic vesicles in the nerve ending. The mean number is of 82.6 vesicles per square micron in the normal and of 132.7 per square micron in the stimulated glands. With a stimulus of 400 pulses per second for 10 minutes, there is a considerable depletion of synaptic vesicles and other changes occur in the nerve endings. The mean number of vesicles is of 29.2 per square micron. These results are interpreted as indicative of an increased activity of the ending in one case, and as a diminished activity and fatigue of the synaptic junction in the other.     

Inhibition of Catecholamine Secretion from Bovine Chromaffin Cells by Adenine Nucleotides and Adenosine

ATP, ADP, and adenosine have been found to inhibit acetylcholine-stimulated secretion from isolated cells of bovine adrenal medulla (chromaffin cells). Maximal inhibition is approximately 30% under the conditions studied; half-maximal inhibition occurs at nucleotide concentration in the micromolar range. Cells must be incubated with ATP for approximately 90 s for maximal inhibition, but inhibition by adenosine occurs much faster, an observation suggesting the possibility that ATP and ADP exert their effect after being converted to adenosine. Experiments with cells preloaded with the fluorescent calcium chelator quin 2 indicate that external ATP can diminish the rise in cytosolic Ca2+ concentration that follows stimulation by acetylcholine.