Leukemia inhibitory factor (LIF) potentiates antinociception activity and inhibits tolerance induction of opioids.

BACKGROUND: The efficacy of opioids typically decreases after long-term use owing to the development of tolerance. Glial activation and the upregulation of proinflammatory cytokines are related to the induction of tolerance. We investigated the effect of leukemia inhibitory factor (LIF) on morphine analgesia and tolerance. METHODS: LIF concentrations in rat spinal cords were measured by polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) after morphine administration. LIF distribution was examined using confocal microscopy. To evaluate the effects of LIF on morphine analgesia and tolerance, LIF was intrathecally administered 30 min before morphine injection. The analgesic effect of morphine was evaluated by measuring tail-flick latency. Human LIF concentrations from the cerebrospinal fluid (CSF) of opioid tolerant patients were also determined by specific ELISA. RESULTS: Chronic morphine administration upregulated LIF concentrations in rat spinal cords. Intrathecal injection of LIF potentiated the analgesic action of morphine. Patch clamp recording of spinal cord slices showed that LIF enhanced DAMGO ([D-Ala2, N-MePhe4, Gly-ol]-enkephalin)-induced outward potassium current. The development of tolerance was markedly suppressed by exogenous LIF, whereas neutralizing the endogenously released LIF with anti-LIF antibodies accelerated the tolerance induction. Moreover, LIF concentrations in the CSF of opioid-tolerant patients were higher than those in the opioid-naive controls. CONCLUSIONS: Intrathecal administration of LIF potentiated morphine antinociceptive activity and attenuated the development of morphine tolerance. Upregulation of endogenously released LIF by long-term use of opioids might counterbalance the tolerance induction effects of other proinflammatory cytokines. LIF might be a novel drug candidate for inhibiting opioid tolerance induction.

[An investigation for standardized diagnosis and treatment of idiopathic normal-pressure hydrocephalus].

OBJECTIVE: To investigate some important issues for diagnosis and treatment of idiopathic normal-pressure hydrocephalus (iNPH), such as standardized pre-operative assessment, initial pressure value of diverter pump, and pressure regulation during follow-up. METHODS: Twenty six iNPH patients (21 males) who treated in Department of Neurosurgery of 2nd Affiliated Hospital of Zhejiang University School of Medicine from 2011 to 2015 were analyzed retrospectively. The average age was 60.5 year. The analysis focused on the treatment process of iNPH, initial pressure value of diverter pump, choice of diverter pump, and pressure regulation during follow-up. RESULTS: As a result, 24 cases (92.3%) had a good prognosis based on their imaging and clinical manifestations. Based on the literature and their clinical experiences, this department established a diagnosis and treatment procedure of iNPH and a pressure regulation procedure for the follow-up of iNPH. Moreover, it is proposed that choosing an anti-gravity diverter pump and making an initial pressure value 20 mmH2O less than pre-surgical cerebrospinal pressure may be beneficial for the prognosis. CONCLUSION: This standardized diagnosis and treatment procedure for iNPH is practical and effective.

Hepatitis C virus infection: a risk factor for Parkinson's disease.

Recent studies found that hepatitis C virus (HCV) may invade the central nervous system, and both HCV and Parkinson's disease (PD) have in common the overexpression of inflammatory biomarkers. We analysed data from a community-based integrated screening programme based on a total of 62,276 subjects. We used logistic regression models to investigate association between HCV infection and PD. The neurotoxicity of HCV was evaluated in the midbrain neuron-glia coculture system in rats. The cytokine/chemokine array was performed to measure the differences of amounts of cytokines released from midbrain in the presence and absence of HCV. The crude odds ratios (ORs) for having PD were 0.62 [95% confidence interval (CI), 0.48-0.81] and 1.91 (95% CI, 1.48-2.47) for hepatitis B virus (HBV) and HCV. After controlling for potential confounders, the association between HCV and PD remained statistically significant (adjusted OR = 1.39; 95% CI, 1.07-1.80), but not significantly different between HBV and PD. The HCV induced 60% dopaminergic neuron death in the midbrain neuron-glia coculture system in rats, similar to that of 1-methyl-4-phenylpyridinium (MPP(+) ) but not caused by HBV. This link was further supported by the finding that HCV infection may release the inflammatory cytokines, which may play a role in the pathogenesis of PD. In conclusion, our study demonstrated a significantly positive epidemiological association between HCV infection and PD and corroborated the dopaminergic toxicity of HCV similar to that of MPP(+) .

Strengthened antioxidant capacity improves photosynthesis by regulating stomatal aperture and ribulose-1,5-bisphosphate carboxylase/oxygenase activity.

ABA is important for plant growth and development; however, it also inhibits photosynthesis by regulating the stomatal aperture and ribulose-1,5-bisphosphate carboxylase/oxygenase activity. Noteworthy, this negative effect can be alleviated by antioxidants including ascorbic acid (AsA) and catalase (CAT), but the underlying mechanism remains unclear. Two rice cultivars, Zhefu802 (recurrent parent) and its near-isogenic line, fgl were selected and planted in a greenhouse with 30/24 °C (day/night) under natural sunlight conditions. Compared to fgl, Zhefu802 had significantly lower net photosynthetic rate (PN) and stomatal conductance (Cond) as well as significantly higher ABA and H2O2 contents. However, AsA and CAT increased PN, Cond, and stomatal aperture, which decreased H2O2 and malondialdehyde (MDA) levels. In this process, AsA and CAT significantly increased the ribulose-1,5-bisphosphate carboxylase activity, while they strongly decreased the ribulose-1,5-bisphosphate oxygenase activity, and finally caused an obvious decrease in the ratio of photorespiration (Pr) to PN. Additionally, AsA and CAT significantly increased the expression levels of RbcS and RbcL genes of leaves, while H2O2 significantly decreased them, especially the RbcS gene. In summary, the removal of H2O2 by AsA and CAT can improve the leaf photosynthesis by alleviating the inhibition on the stomatal conductance and ribulose-1,5-bisphosphate carboxylase capacity caused by ABA.

Water solution of onion crude powder inhibits RANKL-induced osteoclastogenesis through ERK, p38 and NF-kappaB pathways.

UNLABELLED: Onion powder has been reported to decrease the ovariectomy-induced bone resorption of rats. However, the molecular mechanism of onion powder on the bone cells has not been reported. Here, we report that water solution of onion crude powder decreases the osteoclastogenesis from co-cultures of bone marrow stromal cells and macrophage cells. Additionally, water solution of onion crude powder inhibits the RANKL-induced ERK, p38 and NF-kappaB activation in macrophages. In summary, our data showed that onion powder may benefit bone through an anti-resorption effect on the osteoclasts. INTRODUCTION: A nutritional approach is important for both prevention and treatment of osteoporosis. Onion has been reported to decrease the ovariectomy-induced bone resorption. However, the functional effects of onion on the cultured osteoclasts and osteoblasts remain largely unknown. Here, we found that water solution of onion crude powder markedly inhibited the receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclastogenesis through ERK, p38 and NF-kappaB pathways. Other studies were also designed to investigate the potential signaling pathways involved in onion-induced decrease in osteoclastogenesis. METHODS: The osteoclastogenesis was examined using the TRAP staining method. The MAPKs and NF-kappaB pathways were measured using Western blot analysis. A transfection protocol was used to examine NF-kappaB activity. RESULTS: Water solution of onion crude powder inhibited the RANKL plus M-CSF-induced osteoclastic differentiation from either bone marrow stromal cells or from RAW264.7 macrophage cells. Treatment of RAW264.7 macrophages with RANKL could induce the activation of ERK, p38 and NF-kappaB that was inhibited by water solution of onion crude powder. On the other hand, it did not affect the cell proliferation and differentiation of human cultured osteoblasts. CONCLUSIONS: Our data suggest that water solution of onion crude powder inhibits osteoclastogenesis from co-cultures of bone marrow stromal cells and macrophage cells via attenuation of RANKL-induced ERK, p38 and NF-kappaB activation.

ATP potentiates spontaneous transmitter release at developing neuromuscular synapses.

Extracellular application of ATP, a substance co-stored and co-released with acetylcholine in peripheral nervous systems, potentiates the spontaneous secretion of acetylcholine at developing neuromuscular synapses in Xenopus cell culture, as shown by a marked increase in the frequency of spontaneous synaptic currents recorded in the postsynaptic muscle cell. The effect of ATP is apparently mediated by the activation of cytosolic protein kinases and requires the influx of Ca2+ through the plasma membrane. Since spontaneous acetylcholine release is known to regulate the development of contractile properties of the postsynaptic muscle cell, extracellular ATP may serve as a positive trophic factor at developing neuromuscular synapses.

Cranioplasty for patients developing large cranial defects combined with post-traumatic hydrocephalus after head trauma.

BACKGROUND: Large cranial defects combined with hydrocephalus after decompressive craniectomy are a common, harsh reality among patients with head trauma. Typically, a shunt is first used to relieve the hydrocephalus. However, subsequently the patients may develop a severe sinking scalp flap over the skull defect before cranioplasty, which would make the procedure difficult. METHODS: This problem was overcome by temporarily adjusting the shunt pressure using a programmable ventriculoperitoneal shunt tube, which allowed expansion of the depressed scalp flap and facilitated the subsequent cranioplasty. This study describes two patients who were treated for this problem after severe head trauma. RESULTS: When performing a titanium mesh cranioplasty after a shunt, this new method facilitated the separation of the scalp from the underlying muscle or dura and obliterated the dead space between the titanium mesh and the underlying tissue. Both patients had satisfactory outcomes without complications. CONCLUSIONS: This method is easy and safe and it facilitates the cranioplasty, reducing the potential complications, including intracranial haematoma, effusions and infection, and thereby improving the patient outcome.

YC-1 attenuates LPS-induced proinflammatory responses and activation of nuclear factor-kappaB in microglia.

BACKGROUND AND PURPOSE: An inflammatory response in the central nervous system mediated by the activation of microglia is a key event in the early stages of the development of neurodegenerative diseases. LPS has been reported to cause marked microglia activation. It is very important to develop drugs that can inhibit microglia activation and neuroinflammation. Here, we investigated the inhibitory effect of YC-1, a known activator of soluble guanylyl cyclase, against LPS-induced inflammatory responses in microglia. EXPERIMENTAL APPROACH: To understand the inhibitory effects of YC-1 on LPS-induced neuroinflammation, primary cultures of rat microglia and the microglia cell line BV-2 were used. To examine the mechanism of action of YC-1, LPS-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production, iNOS, COX-2 and cytokine expression were analyzed by Griess reaction, ELISA, Western blotting and RT-PCR, respectively. The effect of YC-1 on LPS-induced activation of nuclear factor kappa B (NF-kappaB) was studied by NF-kappaB reporter assay and immunofluorocytochemistry. KEY RESULTS: YC-1 inhibited LPS-induced production of NO and PGE2 in a concentration-dependent manner. The protein and mRNA expression of iNOS and COX-2 in response to LPS application were also decreased by YC-1. In addition, YC-1 effectively reduced LPS-induced expression of the mRNA for the proinflammatory cytokines, TNF-alpha and IL-1beta. Furthermore, YC-1 inhibited LPS-induced NF-kappaB activation in microglia. CONCLUSIONS AND IMPLICATIONS: YC-1 was able to inhibit LPS-induced iNOS and COX-2 expression and NF-kappaB activation, indicating that YC-1 may be developed as an anti-inflammatory neuroprotective agent.

ARHGEF10 knockout inhibits platelet aggregation and protects mice from thrombus formation.

Essentials ARHGEF10 single-nucleotide polymorphism provides risk of ischemic and atherothrombotic stroke. The role of ARHGEF10 in platelet function was examined using ARHGEF10 knockout mice. ARHGEF10 deficiency inhibits platelet function and arterial thrombus formation. ARHGEF10 knockout protects mice from stroke-induced infarction. SUMMARY: Background ARHGEF10, a member of the Rho guanine nucleotide exchange factor (GEF) family, stimulates Rho GTPases. Rho GTPases have been reported to regulate a variety of cellular behaviors, such as cell polarity, cytoskeletal organization, and gene transcription. ARHGEF10 single-nucleotide polymorphisms are linked to the risk of ischemic stroke. However, the role of ARHGEF10 in platelet function remains unknown. Objective To examine the role of ARHGEF10 in platelet function. Methods ARHGEF10-/- were generated. We examined the in vitro and in vivo effects of ARHGEF10 knockout on platelet function and arterial thrombosis formation. Results ARHGEF10-/- mice had normal platelet counts, but showed altered aggregation in response to thrombin, collagen, ADP, protease-activated receptor-4 peptide, and U46619 stimulation. ARHGEF10 knockout influenced platelet spreading on fibrinogen-coated surfaces, and caused the platelets to show less lamellipodia-like extension than wild-type platelets. ARHGEF10 knockout also inhibited platelet clot retraction induced by thrombin stimulation. ARHGEF10 knockout resulted in prolonged tail bleeding time and inhibited the stable thrombus formation induced by FeCl3 in the carotid artery. Conclusions ARHGEF10 serves as an important regulator in platelet shape change, spreading, and aggregation. Moreover, ARHGEF10 also plays an important role in arterial thrombosis formation.

Regulatory role of ATP at developing neuromuscular junctions.

Neuronal factors co-released with neurotransmitters may play an important role in synaptic development and function. Extracellular application of adenosine 5'-triphosphate (ATP), a substance co-stored and co-released with acetylcholine (ACh) in peripheral nervous systems, potentiated the spontaneous secretion of ACh at developing neuromuscular synapses in Xenopus 1-day-old cell cultures, as shown by a marked increase in the frequency of spontaneous synaptic currents recorded in the post-synaptic muscle cell. ATP also increased the frequency of miniature endplate potentials in the isolated tails of 2-week-old Xenopus tadpoles, with much smaller effect than that observed in cell cultures. The potentiation effect of ATP on ACh release in Xenopus cell cultures was inhibited by L-type Ca2+ channel blockers, suggesting that the L-type Ca2+ channel is responsible for the positive regulation of spontaneous ACh secretion by ATP at the developing neuromuscular synapses. The frequency of spontaneous synaptic events was found to vary greatly from cell to cell in the culture, over two orders of magnitude. Synapses with high frequency events are probably under the influence of endogenously released ATP. In addition, ATP was shown to potentiate the responses of isolated myocytes to iontophoretically-applied ACh. Local application of ATP to one region of the elongated myocyte surface resulted in potentiated ACh responses only at the ATP-treated region. Single channel recording showed that ATP specifically increased the open time and opening frequency of embryonic-type, low conductance ACh channels. Pharmacological experiments suggest that ATP exerted both its pre- and post-synaptic effects by binding to P2-purinoceptors and activating protein kinase C. Moreover, the potentiation effects of ATP were restricted to the early stages of embryos. Taken together, these results suggest that ATP co-released with ACh or released from stimulated myocytes may promote synaptic development by potentiating pre-synaptic ACh release and post-synaptic ACh channel activity during the early phase of synaptogenesis.

Porous tantalum seeded with bone marrow mesenchymal stem cells attenuates steroid-associated osteonecrosis.

OBJECTIVE: Bone marrow mesenchymal stem cells (BMMSCs) have been widely applied in osteonecrosis. However, lack of biomechanical support limited application of BMMSCs. And porous tantalum (PTA) has been identified as a cell-friendly scaffold for bone regeneration. Herein, we aimed to investigate the efficacy of PTA seeded with BMMSCs in the treatment of osteonecrosis. MATERIALS AND METHODS: After the production of PTA seeded with BMMSCs, MTT and GFP were performed to identify the proliferation and adhesion of BMMSCs respectively, which was further examined by scanning electron microscopy (SEM). And real-time PCR was also used to determine mRNA level of osteogenic markers, including Alp, OCN, OPN, Col I and Runx-2 in BMMSCs. Nineteen adult rabbits were applied for building steroid-associated osteonecrosis (SAON) models. Bone formation rate (BFR) and mineral apposition rate (MAR) were determined. And Goldner Trichrome Staining was used in these SAON models, which further confirmed the efficacy of PTA seeded with BMMSCs in SAON. RESULTS: PTA seeded with BMMSCs showed excellent biocompatibility. Additionally, SEM assay showed that BMMSCs adhered tightly and spread fully in the pores of PTA. Next, the expression of ALP and OPN mRNA in BMMSCs were significantly (p < 0.05) higher in the PTA-treated group compared to those in the PTA-untreated group. Furthermore, compared to those treated by only PTA, the dynamic bone formation in rabbits treated by PTA seeded with BMMSCs was significantly increased (p < 0.001) at both week 3rd and week 6th. CONCLUSIONS: The product, PTA seeded with BMMSCs, was successfully produced, and was determined as high efficacy for treatment of steroid-associated osteonecrosis. PTA seeded with BMMSCs may afford a promising option for treating osteonecrosis.

Morphological changes induced by prostaglandin E in cultured rat osteoblasts.

Prostaglandin E (PGE)-induced morphological changes of osteoblasts and its possible mechanisms were investigated in cultured calvaria and isolated osteoblasts from long bone fragments of neonatal rats. The control osteoblasts, either on the calvaria or isolated from the long bone fragments, were flat, polygonal in shape, and arranged in a monolayer under scanning electron microscopy (SEM) or phase contrast microscopy. Treatment with 1 mumol/L of prostaglandin E2 (PGE2, 2 h) caused these bone cells to contract a soma, whereas 10 and 100 mumol/L PGE2 (2 h) caused 18%-30% of the bone cells to elongate and expose the undersurface. Incubation of the cultured osteoblasts with PGE2 at different time periods showed a bell-shaped pattern with the optimal response at 2 h of incubation. A similar reaction can be induced by treatment with prostaglandin E1 (PGE1) or dibutyryl cyclic adenosine monophosphate (DBcAMP) in combination with 3-isobutyl-1-methylxanthine (IBMX). Furthermore, we assessed the percentage of responsive isolated bone cells to investigate interactions with other agents. The morphological changes induced by PGEs were inhibited by H-8, a protein kinase inhibitor. On the other hand, elevated intracellular calcium enhanced the PGE-induced morphological changes. Fluorescence labeling showed that PGEs caused the breakdown of the actin microfilaments, but spared the microtubules and vimentin filaments in the isolated osteoblast-like cells. These results suggest that the morphological changes of osteoblasts induced by PGEs may be related to the intracellular cAMP and calcium levels.

Antagonistic action of uranyl nitrate on presynaptic neurotoxins from snake venoms.

Uranyl ion (UO2+2) antagonized the neuromuscular blocking action and phospholipase A2 activity of neurotoxins which act presynaptically [beta-bungarotoxin (beta-BuTX) and crotoxin] but did not affect the action of alpha-bungarotoxin and tetrodotoxin. On the basis of the kinetic analysis of the UO2+2 and strontium ion (Sr2+) antagonism of muscle paralysis induced by beta-bungarotoxin, it was found that they inhibited both the binding of the toxin and the steps following binding that brought about the neuromuscular blocking action of beta-bungarotoxin. Uranyl ion was about 50 times more potent than Sr2+ in antagonizing beta-bungarotoxin. High Ca2+ (10 mM) abolished but low Ca2+ (0.25-1.25 mM) medium enhanced the antagonizing action of UO2+2 and Sr2+. In low Ca2+ medium, UO2+2 markedly potentiated the amplitude of the twitch, subsequent addition of beta-bungarotoxin produced three phases of effects on the twitches, e.g. an initial depression, followed by the second facilitation and finally a rapid depression of twitches; however, approx. 70 min after beta-bungarotoxin the small twitches reached a steady state which persisted for more than 350 min. Therefore, it is evident that UO2+2 is the most potent antagonist of beta-bungarotoxin so far tested.

Presynaptic action of uranyl nitrate on the phrenic nerve-diaphragm preparation of the mouse.

The contraction of the diaphragm of the mouse, induced by nerve stimulation, can be potentiated by uranyl nitrate [UO2(NO3)2, 0.2-0.8 mM]. At concentrations greater than 0.8 mM, uranyl nitrate also directly enhanced the contractions induced by electrical stimulation of muscle. The effects of uranyl nitrate in potentiating the twitch were augmented by low calcium (0.25-0.5 mM) and antagonized by high levels of calcium (5-10 mM). Electrophysiological studies on the effects of uranyl nitrate on the diaphragm in the mouse have revealed that the frequency of miniature end-plate potentials (MEPP) but not the amplitude was increased; the amplitude and quantal content of end-plate potential (EPPs) were also markedly increased by uranyl nitrate. The most peculiar phenomenon induced by uranyl nitrate was that repetitive end-plate potentials, as well as repetitive action potentials, of the diaphragm of the mouse were triggered by single nerve stimulation in the presence of uranyl nitrate. The duration of muscle action potentials was significantly prolonged. Recordings of the evoked compound action potentials from nerve axons showed that uranyl nitrate not only prolonged the duration of the axonal compound action potentials but also induced antidromic activity with a single stimulation. This latter effect of uranyl nitrate was blocked by d-tubocurarine. All of these findings suggest that uranyl nitrate potentiated the contractions of the diaphragm of the mouse, possibly through the activation of stimulus-activated repetitive discharges which resulted in the repetitive end-plate potentials and muscle action potentials.

Effects of sodium selenite on neuromuscular junction of the mouse phrenic nerve-diaphragm preparation.

The effects of sodium selenite on the neuromuscular junction of the phrenic nerve-diaphragm of the mouse were studied. Nerve-evoked twitches of the diaphragm of the mouse, the frequency of miniature endplate potentials, the quantal content of endplate potentials and the compound action potentials of the axon were measured. Sodium selenite induced a slight increase of the amplitude of the twitch, followed by twitch depression. The amplitude of the twitch, increased by selenite, became more prominent after the suppression of the twitch induced by cadmium ions, d-tubocurarine or magnesium ions. It appeared that the increased amplitude of twitch was due to the facilitation of transmitter release, since selenite significantly increased the frequency of miniature endplate potentials, and the amplitude and quantal content of endplate potentials; the amplitude and half decay time of miniature endplate potentials were unaffected. Twitch depression induced by selenite was enhanced by ammonium ions, high potassium and low magnesium and attenuated by high calcium. During the period of gradual depression of the twitch, selenite decreased the amplitude of compound action potentials of the phrenic nerve axon and caused the disappearance of endplate potentials. Ammonium ions enhanced the blockade of axonal conduction induced by selenite. Moreover, the depolarizing agents, ammonium and high potassium also induced an initial increase of twitch amplitude followed by depression of the twitch. These findings indicate that selenite probably alters the release of the transmitter by depolarizing the nerve membrane. The effects of selenite were antagonized by glutathione and cyanide, suggesting that the binding of selenite to sulfhydryl groups of the membrane was essential for inducing its pharmacological actions.

Studies on the neuromuscular blocking action of MPTP in the mouse phrenic nerve-diaphragm.

The peripheral neurotoxicity of 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP) in the mouse phrenic nerve-diaphragm preparation was studied. The inhibition of nerve-evoked twitches was dependent on the concentrations of MPTP, ranging from 1 to 200 microM, which only slightly increased the amplitude of the twitch evoked by direct stimulation of the muscle. Pretreatment with pargyline did not prevent this inhibitory action of MPTP. The inhibition of the twitch by MPTP was reversible and could be partially reversed by anticholinesterase agents such as neostigmine and physostigmine, as well as ecothiophate. Pretreatment with either 0.7 microM d-tubocurarine or 2.2 microM succinylcholine, did not affect the twitches individually but markedly potentiated the effects of MPTP, by shifting the concentration-inhibition curve of MPTP to the left. MPTP not only directly inhibited acetylcholine (0.1 mM)-induced contracture of the denervated mouse diaphragm but also protected the muscle from the inhibitory action of alpha-bungarotoxin. Furthermore, the specific binding of [125I]alpha-bungarotoxin to the mouse diaphragm was also inhibited by MPTP. Electrophysiological studies revealed that MPTP inhibited the frequency and amplitude of the m.e.p.p., as well as the amplitude of the e.p.p. All of these findings indicate that MPTP exerts a neuromuscular blocking action through a curare-like action, by binding to the nicotinic acetylcholine receptors of the mouse diaphragm. The inhibitory action of MPTP on the contraction of the diaphragm is considered to play a role in the respiratory failure and muscle paralysis of intoxication with MPTP.

Blockade by MPTP of the nicotinic acetylcholine receptor channels in embryonic Xenopus muscle cells.

The effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on nicotinic acetylcholine (ACh) receptor channels were studied in cultured myocytes of 1-day-old Xenopus embryos. The amplitude and decay time of iontophoretic ACh-induced currents were reduced by MPTP in a concentration-dependent manner. The inhibitory action of MPTP was frequency-dependent, being enhanced by higher frequencies of stimulation at 7 and 30 Hz. Neither pargyline nor tranylcypromine affected the inhibitory effects of MPTP on ACh-induced currents. Single channel recordings showed that MPTP decreased the opening frequency, mean open time as well as the conductance of both low- and high-conductance ACh channels. These results suggest that the inhibitory actions of MPTP on ACh receptor channels in skeletal muscle may contribute to the depression of the nerve-evoked contraction of the mouse diaphragm as previously reported.

Neuromuscular actions of sodium selenite on chick biventer cervicis nerve-muscle preparation.

Sodium selenite was found to be toxic to chicks, with an LD50 of 8.5 micrograms/g, which was increased to 16.3 micrograms/g by NaCN. The major symptoms of chicks, treated with selenite, were sedation and then dyspnea and paralysis. The cause of death by selenite was apparently due to the respiratory failure. The possible mechanism of toxicity was explored in the isolated chick biventer cervicis nerve-muscle preparation. Selenite initially increased the amplitude of the twitch, reversed the suppression of the twitch caused by d-tubocurarine, Mg2+, Cd2+ or Mn2+ and significantly increased the quantal content and amplitude of endplate potentials. Subsequently, selenite depressed the amplitude of the twitch, blocked the axonal conduction and inhibited excitatory postsynaptic potentials. Both NH4+ and K+ enhanced the action of selenite in depressing the twitches. In addition, selenite induced a sustained contracture of the muscle, which was partially inhibited by removal of external Ca2+ and markedly blocked by EGTA. Entry of Ca2+ and release of the internal Ca2+ were considered to be responsible for inducing contracture by selenite. Pretreatment with trypsin, glutathione (GSH) and cyanide profoundly inhibited the effects of selenite, indicating that the site of action of selenite was on the outer membrane and the binding of selenite to the sulfhydryl groups of membrane proteins was proposed to be an essential step for selenite-induced contracture and neuromuscular action. These findings suggest that neuromuscular blockade and tetanic spasm, produced by selenite in chicks, may play a role in causing respiratory failure in vivo.

L-type Ca2+ channel is involved in the regulation of spontaneous transmitter release at developing neuromuscular synapses.

Involvement of an L-type Ca2+ channel in the regulation of spontaneous transmitter release was studied in Xenopus nerve-muscle cultures. The frequency of spontaneous synaptic currents, which reflects impulse-independent acetylcholine release from the nerve terminals, showed a marked increase in high-K+ medium or after treatment with a phorbol ester, 12-O-tetradecanoyl-phorbol 13-acetate, a drug that activates protein kinase C and depolarizes the presynaptic neuron. The potentiation effect of high K+ and 12-O-tetradecanoyl-phorbol 13-acetate requires Ca2+ influx through the L-type Ca2+ channel in the plasma membrane, since it was significantly reduced by the presence of nifedipine, verapamil or diltiazem and enhanced by Bay K 8644, an L-type Ca2+ channel agonist. It was shown recently that adenosine 5'-triphosphate markedly potentiates the spontaneous acetylcholine release at these synapses through the binding of P2-purinoceptors and the activation of protein kinase C. We found in the present study that potentiation effects of adenosine 5'-triphosphate are inhibited by L-type Ca2+ channel blockers, suggesting that the L-type Ca2+ channel is responsible for the positive regulation of spontaneous acetylcholine secretion by adenosine 5'-triphosphate at the developing neuromuscular synapses. Our data suggest that modulation of the L-type Ca2+ channel in embryonic motor nerve terminals is important for the regulation of spontaneous transmitter release.