Cytokine MIF Enhances Blood-Brain Barrier Permeability: Impact for Therapy in Ischemic Stroke.

Ischemic stroke is a devastating disease with limited therapeutic options. It is very urgent to find a new target for drug development. Here we found that the blood level of MIF in ischemic stroke patients is upregulated. To figure out the pathological role of MIF in ischemic stroke, both in vitro and in vivo studies were conducted. For in vitro studies, primary cortical neuron cultures and adult rat brain endothelial cells (ARBECs) were subjected to oxygen-glucose deprivation (OGD)/reoxygenation. Middle cerebral artery occlusion (MCAo) rodent models were used for in vivo studies. The results show that MIF exerts no direct neuronal toxicity in primary culture but disrupts tight junction in ARBECs. Furthermore, administration of MIF following MCAo shows the deleterious influence on stroke-induced injury by destroying the tight junction of blood-brain barrier and increasing the infarct size. In contrast, administration of MIF antagonist ISO-1 has the profound neuroprotective effect. Our results demonstrate that MIF might be a good drug target for the therapy of stroke.

Antagonism of proteasome inhibitor-induced heme oxygenase-1 expression by PINK1 mutation.

PTEN-induced putative kinase 1 (PINK1) is an integral protein in the mitochondrial membrane and maintains mitochondrial fidelity. Pathogenic mutations in PINK1 have been identified as a cause of early-onset autosomal recessive familial Parkinson's disease (PD). The ubiquitin proteasome pathway is associated with neurodegenerative diseases. In this study, we investigated whether mutations of PINK1 affects the cellular stress response following proteasome inhibition. Administration of MG132, a peptide aldehyde proteasome inhibitor, significantly increased the expression of heme oxygenase-1 (HO-1) in rat dopaminergic neurons in the substantia nigra and in the SH-SY5Y neuronal cell line. The induction of HO-1 expression by proteasome inhibition was reduced in PINK1 G309D mutant cells. MG132 increased the levels of HO-1 through the Akt, p38, and Nrf2 signaling pathways. Compared with the cells expressing WT-PINK1, the phosphorylation of Akt and p38 was lower in those cells expressing the PINK1 G309D mutant, which resulted in the inhibition of the nuclear translocation of Nrf2. Furthermore, MG132-induced neuronal death was enhanced by the PINK1 G309D mutation. In this study, we demonstrated that the G309D mutation impairs the neuroprotective function of PINK1 following proteasome inhibition, which may be related to the pathogenesis of PD.

CXCL12/CXCR4 Signaling Contributes to the Pathogenesis of Opioid Tolerance: A Translational Study.

BACKGROUND: Long-term opioid therapy for chronic pain may lead to analgesic tolerance, especially when administered intrathecally, thus preventing adequate pain relief. Discovering drug targets to treat opioid tolerance using a mechanism-based approach targeting opioid-induced neuroinflammation provides new therapeutic opportunities. In this study, we provide translational evidence that CXCL12/CXCR4 signaling contributes to the pathogenesis of opioid tolerance. METHODS: The CXCL12 levels in the cerebrospinal fluid of opioid-tolerant patients were compared with those of opioid-naive subjects. For further investigation, a rodent translational study was designed using 2 clinically relevant opioid delivery paradigms: daily intraperitoneal morphine injections and continuous intrathecal morphine infusion. We measured rats' tail flick responses and calculated the percentage of maximum possible effects (%MPE) to demonstrate opioid acute antinociception and the development of analgesic tolerance. The effects of exogenous CXCL12, CXCL12 neutralizing antibody, and receptor antagonist AMD3100 were investigated by intrathecal administration. Data were presented as mean ± SEM. RESULTS: CXCL12 was significantly upregulated in the cerebrospinal fluid of opioid-tolerant patients for 892 ± 34 pg/mL (n = 27) versus 755 ± 33 pg/mL (n = 10) in naive control subjects (P = .03). Furthermore, after 2 and 5 days of intrathecal morphine infusion, rat lumbar spinal cord dorsal horn CXCL12 messenger RNA levels were significantly upregulated by 3.2 ± 0.7 (P = .016) and 3.4 ± 0.3 (P = .003) fold, respectively. Results from the daily intraperitoneal morphine injection experiments revealed that administering an intrathecal infusion of CXCL12 for 24 hours before the first morphine injection did not decrease antinociception efficacy on day 1 but accelerated tolerance after day 2 (%MPE 49.5% vs 88.1%, P = .0003). In the intrathecal morphine coinfusion experiments, CXCL12 accelerated tolerance development (%MPE 9.4% vs 43.4% on day 1, P < .0001), whereas coadministration with CXCL12 neutralizing antibody attenuated tolerance (72.5% vs 43.4% on day 1, P < .0001; 47.6% vs 17.5% on day 2, P < .0001). Coadministration of receptor antagonist AMD 3100 can persistently preserve morphine analgesic effects throughout the study period (27.9% ± 4.1% vs 0.9% ± 1.6% on day 5, P = .03). CONCLUSIONS: The CXCL12/CXCR4 pathway contributes to the pathogenesis of opioid tolerance. Our study indicates that intervening with CXCL12/CXCR4 signaling has therapeutic potential for opioid tolerance.

Trends in major opioid analgesic consumption in Taiwan, 2002-2014.

BACKGROUND/PURPOSE: According to WHO guideline, the consumption of opioids is an important sign of national progress in cancer pain relief. However, precise data on the consumption of opioid analgesics consumption in Taiwan has not been published. We investigate opioid analgesic consumption in Taiwan between 2002 and 2014 compare the results with those in other countries to see what we could learn about other methods of pain management. To find out the different patterns between Taiwan and other country, improves the quality of pain management. METHODS: We extracted from the Controlled Drugs Management Information System (CDMIS) database, the consumption data of morphine, fentanyl, and pethidine, three strong opioids, and of codeine and buprenorphine, two weak ones. Data were presented as defined daily doses for statistical purposes per million inhabitants per day (S-DDD/m/d). The number of inhabitants was extracted from the Taiwan Ministry of Interior Statistics population database. RESULTS: During the thirteen studied years, the total consumption of opioids markedly increased in Taiwan. By category, the consumption of morphine, fentanyl and buprenorphine increased, but the use of pethidine and codeine decreased. Compared with the selected regions and countries, the use of opioid in Taiwan progressed in Asia, but it was still lower than in Western countries. CONCLUSION: Opioid analgesics are probable addictive; however, they can improve a patients' quality of life if properly used. The Taiwan FDA continuously introduces new opioid analgesics and educates physicians on how to use them correctly. These measures will improve the quality of pain management in Taiwan.

Hypoxic Preconditioning Suppresses Glial Activation and Neuroinflammation in Neonatal Brain Insults.

Perinatal insults and subsequent neuroinflammation are the major mechanisms of neonatal brain injury, but there have been only scarce reports on the associations between hypoxic preconditioning and glial activation. Here we use neonatal hypoxia-ischemia brain injury model in 7-day-old rats and in vitro hypoxia model with primary mixed glial culture and the BV-2 microglial cell line to assess the effects of hypoxia and hypoxic preconditioning on glial activation. Hypoxia-ischemia brain insult induced significant brain weight reduction, profound cell loss, and reactive gliosis in the damaged hemisphere. Hypoxic preconditioning significantly attenuated glial activation and resulted in robust neuroprotection. As early as 2 h after the hypoxia-ischemia insult, proinflammatory gene upregulation was suppressed in the hypoxic preconditioning group. In vitro experiments showed that exposure to 0.5% oxygen for 4 h induced a glial inflammatory response. Exposure to brief hypoxia (0.5 h) 24 h before the hypoxic insult significantly ameliorated this response. In conclusion, hypoxic preconditioning confers strong neuroprotection, possibly through suppression of glial activation and subsequent inflammatory responses after hypoxia-ischemia insults in neonatal rats. This might therefore be a promising therapeutic approach for rescuing neonatal brain injury.

Role of spinal CXCL1 (GROα) in opioid tolerance: a human-to-rodent translational study.

BACKGROUND: The pivotal role of glial activation and up-regulated inflammatory mediators in the opioid tolerance has been confirmed in rodents but not yet in humans. Here, the authors investigated the intraspinal cytokine and chemokine profiles of opioid-tolerant cancer patients; and to determine if up-regulated chemokines could modify opioid tolerance in rats. METHODS: Cerebrospinal fluid samples from opioid-tolerant cancer patients and opioid-naive subjects were compared. The cerebrospinal fluid levels of tumor necrosis factor-alpha, CXCL1, CXCL10, CCL2, and CX3CL1 were assayed. The rat tail flick test was utilized to assess the effects of intrathecal CXCL1 on morphine-induced acute antinociception and analgesic tolerance. RESULTS: CXCL1 level in cerebrospinal fluid was significantly up-regulated in the opioid-tolerant group (n = 30, 18.8 pg/ml vs. 13.2 pg/ml, P = 0.02) and was positively correlated (r = 0.49, P < 0.01) with opioid dosage. In rat experiment, after induction of tolerance by morphine infusion, the spinal cord CXCL1 messenger RNA was up-regulated to 32.5 ± 11.9-fold. Although CXCL1 infusion alone did not affect baseline tail-flick latency, the analgesic efficacy of a single intraperitoneal injection of morphine dropped significantly on day 1 to day 3 after intrathecal infusion of CXCL1. After establishing tolerance by intrathecal continuous infusion of morphine, its development was accelerated by coadministration of CXCL1 and attenuated by coadministration of CXCL1-neutralizing antibody or CXCR2 antagonist. CONCLUSIONS: CXCL1 is up-regulated in both opioid-tolerant patients and rodents. The onset and extent of opioid tolerance was affected by antagonizing intrathecal CXCL1/CXCR2 signaling. Therefore, the CXCL1/CXCR2 signal pathway may be a novel target for the treatment of opioid tolerance.

Targeted delivery of erythropoietin by transcranial focused ultrasound for neuroprotection against ischemia/reperfusion-induced neuronal injury: a long-term and short-term study.

Erythropoietin (EPO) is a neuroprotective agent against cerebral ischemia/reperfusion (I/R)-induced brain injury. However, its crossing of blood-brain barrier is limited. Focused ultrasound (FUS) sonication with microbubbles (MBs) can effectively open blood-brain barrier to boost the vascular permeability. In this study, we investigated the effects of MBs/FUS on extending the therapeutic time window of EPO and its neuroprotective effects in both acute and chronic phases. Male Wistar rats were firstly subjected to two common carotid arteries and right middle cerebral artery occlusion (three vessels occlusion, 3VO) for 50 min, and then the rats were treated with hEPO (human recombinant EPO, 5000 IU/kg) with or without MBs/FUS at 5 h after occlusion/reperfusion. Acute phase investigation (I/R, I/R+MBs/FUS, I/R+hEPO, and I/R+hEPO+MBs/FUS) was performed 24 h after I/R; chronic tests including cylinder test and gait analysis were performed one month after I/R. The experimental results showed that MBs/FUS significantly increased the cerebral content of EPO by bettering vascular permeability. In acute phase, both significant improvement of neurological score and reduction of infarct volume were found in the I/R+hEPO+MBs/FUS group, as compared with I/R and I/R+hEPO groups. In chronic phase, long-term behavioral recovery and neuronal loss in brain cortex after I/R injury was significantly improved in the I/R+hEPO+MBs/FUS group. This study indicates that hEPO administration with MBs/FUS sonication even at 5 h after occlusion/reperfusion can produce a significant neuroprotection.

Protection of dopaminergic neurons by 5-lipoxygenase inhibitor.

Neuroinflammation and oxidative stress are important factors that induce neurodegeneration in age-related neurological disorders. 5-Lipoxygenase (5-LOX) is the enzyme responsible for catalysing the synthesis of leukotriene or 5-HETE from arachidonic acid. 5-LOX is expressed in the central nervous system and may cause neurodegenerative disease. In this study, we investigated the effect of the pharmacological inhibition of 5-lipoxygenase on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)/MPP(+)-induced dopaminergic neuronal death in midbrain neuron-glia co-cultures and in mice. It was found that 5-LOX was over-expressed in astrocytes after the injection of MPTP into C57BL6 mice. MK-886, a specific inhibitor of 5-LOX activating protein (FLAP), significantly increased [(3)H]-dopamine uptake, a functional indicator of the integrity of dopaminergic neurons, in midbrain cultures or the SH-SY5Y human dopaminergic cell line following MPP(+) treatment. In addition, LTB4, one of 5-LOX's downstream products, was increased in the striatum and substantia nigra following MPTP injection in mice. LTB4 but not LTD4 and 5-HETE enhanced MPP(+)-induced neurotoxicity in primary midbrain cultures. MK-886 administration increased the number of tyrosine hydroxylase-positive neurons in the substantia nigra and the dopamine content in the striatum in MPTP-induced parkinsonian mice. Furthermore, the MPTP-induced upregulation of LTB4 in the striatum and substantia nigra was antagonised by MK-886. These results suggest that 5-LOX inhibitors may be developed as novel neuroprotective agents and LTB4 may play an important pathological role in Parkinson's disease.

Enhancement role of host 12/15-lipoxygenase in melanoma progression.

12/15-Lipoxygenase (12/15-LOX) is a non-haeme iron-containing dioxygenase that forms 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) or 15(S)-HETE. Several biological mediators including cytokines, growth factors and lipid metabolites released during tumour cell-endothelial cell adhesion are associated with malignant tumour progression. Here we found that HETEs released from the host organ played a critical role in tumour metastasis. Intravenous injection of B16F10 melanoma cells caused lung nodule formation, which was markedly attenuated in 12/15-LOX null mice. Co-injection of melanoma cells with 12(S)-HETE increased the lung homing activity of B16F10 melanoma cells. In vitro studies showed that 12(S)-HETE and 15(S)-HETE treatment resulted in a concentration-dependent increase of adhesion of B16F10 cells on collagen or fibronectin. The melanoma cell adhesion was then evaluated in pulmonary primary cell culture isolated from wild-type (WT) and 12/15-LOX knockout (KO) mice. It was found that the adhesion of melanoma cells on the epithelial cells isolated from 12/15-LOX null mice was reduced in comparison with those isolated from WT mice. Treatment of 12(S)-HETE increased the pFAK in melanoma cells adhering on collagen-coated slide. The enhancement of adherence elicited by 12(S)-HETE in B16F10 cells could be antagonised by focal adhesion kinase (FAK) inhibitor 14 (FAK inhibitor) or PD98059 (extracellular signal-regulated kinase (ERK) inhibitor). 12(S)-HETE increased the phosphorylation of FAK and ERK in adhering melanoma cells. The FAK phosphorylation induced by 12(S)-HETE was further inhibited by PD98059, indicating that FAK is the downstream target of ERK. The adhesion and lung metastasis of human melanoma cells of C32 in NOD/SCID mice were also potentiated by co-treatment with 12(S)-HETE. These results demonstrate that 12(S)-HETE/15(S)-HETE activates ERK and FAK signalling pathways, thereby upregulates the adhesion and metastatic potential of melanoma cells. The endogenous release of 12(S)-HETE/15(S)-HETE in the host organ may affect the metastatic potential of melanoma.

Increase of oxidative stress by a novel PINK1 mutation, P209A.

Mutation in the human PTEN-induced protein kinase 1 (PINK1) gene is responsible for the second most common form of recessive Parkinson disease (PD). We have identified a single heterozygous PINK1 mutation, P209A, from a cohort of 68 patients with early onset PD. From age 31, this patient developed an asymmetric bradykinesia with rigidity that was L-DOPA responsive. An [(18)F]-fluorodopa PET scan showed reduced DOPA uptake in the bilateral basal ganglia. The H2O2-induced cell death, ROS production, and caspase-3 activation in SH-SY5Y cells were enhanced by the transfection of the PINK1 P209A mutant. The heme oxygenase-1 (HO-1) induction in response to H2O2 and MPP(+) treatment was impaired by the overexpression of the PINK1 P209A mutant. In addition, SOD2 induction after TNFα treatment was also inhibited by the PINK1 P209A mutation. Akt and ERK are involved in HO-1 induction after oxidative stress. The phosphorylation of Akt and ERK after exposure to H2O2 or MPP(+) was also inhibited in PINK1 P209A mutant cells compared with empty-vector-transfected cells. These results indicate a novel pathway by which the P209A defect in the PINK1 kinase domain inhibits oxidative stress-induced HO-1 and SOD2 induction, which may accelerate the neurodegeneration in PD with PINK1 defect.

Impairment of oxidative stress-induced heme oxygenase-1 expression by the defect of Parkinson-related gene of PINK1.

Parkinson's disease (PD) is one of the most common neurodegenerative diseases. Mutation in the phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1) gene causes an autosomal recessive form of PD. However, the etiology related to PINK1 is still not clear. Here, we examined the effect of PINK1 on heme oxygenase (HO)-1 induction in SH-SY5Y neuronal cells following H(2)O(2) or 1-methyl-4-phenylpyridinium (MPP(+)) treatment. The HO-1 induction in response to H(2)O(2) and MPP(+) treatment was impaired by the expression of recombinant PINK1 G309D mutant. PINK1 G309D mutation increased the apoptosis of SH-SY5Y cells following H(2)O(2) treatment and cell survival was rescued by the over-expression of HO-1 using adenovirus (Ad) infection. In addition, knockdown of tumor necrosis factor receptor-associated protein-1 (TRAP1), which is the substrate of PINK1 kinase, in SH-SY5Y cells also inhibited the expression of HO-1 in response to oxidative stress. The up-regulation of TRAP1 expression following H(2)O(2) treatment was inhibited by the expression of recombinant PINK1 G309D mutant. The H(2)O(2)-induced HO-1 induction was Akt- and ERK-dependent. The phosphorylation of ERK and Akt but not p38 was inhibited in cells expressing the PINK1 G309D mutant and knockdown of TRAP1. These results indicate a novel pathway by which the defect of PINK1 inhibits the oxidative stress-induced HO-1 production. Impairment of HO-1 production following oxidative stress may accelerate the dopaminergic neurodegeneration in Parkinson patients with PINK1 defect.

Reversible blood-brain barrier disruption by repeated transcranial focused ultrasound allows enhanced extravasation.

The permeability of blood-brain barrier (BBB) for albumin can be enhanced by focused ultrasound (FUS) in a targeted region when this is applied in the presence of ultrasound contrast agent (UCA). In this study, we demonstrate that, using this noninvasive treatment, Evans Blue (EB) extravasation can be enhanced by repeated sonication. Sonications were applied at an ultrasound frequency of 1 MHz with a 5% duty cycle, and a repetition frequency of 1 Hz. The brains of male Sprague-Dawley rats were subjected to FUS exposure at the same targeted site. At the same acoustic power, the extravasation caused by leakage of EB into the brain was found to be dependent on the applied sonication time. In vivo, both single and repeated sonications increased the extravasation of the albumin binding EB, especially for the repeated sonication group. In the retreatment experiment, there was a nearly twofold increase in EB extravasation in groups with a second sonication compared with the single sonication group. BBB disruption can be prolonged by repeated FUS sonication and the duration is dependent on the time point of the resonication after the first sonication. Compared to a single sonication, the MR imaging analysis and histological examination of the affected brains indicated that the pattern of contrast enhancement was changed and that vacuolation occurred after repeated sonication. This noninvasive technology offers the possibility of controlling the extent of drug delivery by means of repeated treatment and adjusting the duration and interval between sonications.

Overexpression of heme oxygenase-1 protects dopaminergic neurons against 1-methyl-4-phenylpyridinium-induced neurotoxicity.

Heme oxygenase-1 (HO-1) is up-regulated in response to oxidative stress and catalyzes the degradation of pro-oxidant heme to carbon monoxide (CO), iron, and bilirubin. Intense HO-1 immunostaining in the Parkinsonian brain is demonstrated, indicating that HO-1 may be involved in the pathogenesis of Parkinsonism. We here locally injected adenovirus containing human HO-1 gene (Ad-HO-1) into rat substantia nigra concomitantly with 1-methyl-4-phenylpyridinium (MPP(+)). Seven days after injection of MPP(+) and Ad-HO-1, the brain was isolated for immunostaining and for measurement of dopamine content and inflammatory cytokines. It was found that overexpression of HO-1 significantly increased the survival rate of dopaminergic neurons; reduced the production of tumor necrosis factor alpha (TNF-alpha) and interleukin-1beta (IL-1beta) in substantia nigra; antagonized the reduction of striatal dopamine content induced by MPP(+); and also up-regulated brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) expression in substantia nigra. Apomorphine-induced rotation after MPP(+) treatment was also inhibited by Ad-HO-1. On the other hand, inhibition of HO enzymatic activity by zinc protoporphyrin-IX facilitated the MPP(+)-induced rotatory behavior and enhanced the reduction of dopamine content. HO-1 overexpression also protected dopaminergic neurons against MPP(+)-induced neurotoxicity in midbrain neuron-glia cocultures. Overexpression of HO-1 increased the expression of BDNF and GDNF in astrocytes and BDNF in neurons. Our results indicate that HO-1 induction exerts neuroprotection both in vitro and in vivo. Pharmacological or genetic approaches targeting HO-1 may represent a promising and novel therapeutic strategy in treating Parkinsonism.

Quantitative evaluation of focused ultrasound with a contrast agent on blood-brain barrier disruption.

The use of an ultrasound contrast agent (UCA) with focused ultrasound sonication has the potential to disrupt the blood-brain barrier (BBB) noninvasively and reversibly at target locations. This study investigated the effects of UCA dose and ultrasound pressure on BBB disruption. Sonications were applied at 1 MHz with a burst length of 10 ms, a 1% duty cycle and a repetition frequency of 1 Hz. The duration of the sonication was 30 s. In in vivo experiments, 16 male Wistar rats were sonicated in the presence of UCA at four doses (0, 30, 60 and 90 microL/kg). BBB integrity was evaluated by injecting Evans blue (EB) into the femoral vein of anesthetized rats. The relationship between UCA dose and the region of EB extravasation was evaluated at ultrasound pressures of 0.9 and 1.2 MPa. The BBB disruption, as quantified by the amount of EB extravasation, was significantly greater in rats injected with UCA at a dose of 60 or 90 microL/kg than at a dose of 0 or 30 microL/kg. The amount of EB extravasation increased monotonically with the quantity of UCA injected into the femoral vein before sonication. Furthermore, the BBB disruption could be enhanced in the focal region relative to the surrounding region with a higher dose of UCA (60 or 90 microL/kg). This study demonstrates that BBB disruption can be both increased and localized to the focal region by injecting an appropriate quantity of UCA before performing focused ultrasound sonications.

Mechanism of beta-bungarotoxin in facilitating spontaneous transmitter release at neuromuscular synapse.

The mechanism of the action of beta-bungarotoxin (beta-BuTx) in the facilitation of spontaneous transmitter release at neuromuscular synapse was investigated in Xenopus cell culture using whole-cell patch clamp recording. Exposure of the culture to beta-BuTx dose-dependently enhances the frequency of spontaneous synaptic currents (SSCs). Buffering the rise of intracellular Ca2+ with BAPTA-AM hampered the facilitation of SSC frequency induced by beta-BuTx. The beta-BuTx-enhanced SSC frequency was reduced when the pharmacological Ca2+ -ATPase inhibitor thapsigargin was used to deplete intracellular Ca2+ store. Application of membrane-permeable inhibitors of inositol 1,4,5-trisphosphate (IP3) but not ryanodine receptors effectively occluded the increase of SSC frequency elicited by beta-BuTx. Treating cells with either wortmannin or LY294002, two structurally different inhibitors of phosphatidylinositol 3-kinase (PI3K) and with phospholipase C (PLC) inhibitor U73122, abolished the beta-BuTx-induced facilitation of synaptic transmission. The beta-BuTx-induced synaptic facilitation was completely abolished while there was presynaptic loading of the motoneuron with GDPbetaS, a non-hydrolyzable GDP analogue and inhibitor of G protein. Taken collectively, these results suggest that beta-BuTx elicits Ca2+ release from the IP3 sensitive intracellular Ca2+ stores of the presynaptic nerve terminal. This is done via PI3K/PLC signaling cascades and G protein activation, leading to an enhancement of spontaneous transmitter release.

A rapid, nongenomic pathway facilitates the synaptic transmission induced by retinoic acid at the developing synapse.

We have previously shown that retinoic acid (RA), a factor highly expressed in spinal cord, rapidly and specifically enhances the spontaneous acetylcholine release at developing neuromuscular synapses in Xenopus cell culture, using whole-cell patch-clamp recording. We have now further investigated the underlying mechanisms that are involved in RA-induced facilitation on the frequency of spontaneous synaptic currents (SSCs). Buffering the rise of intracellular Ca2+ with BAPTA-AM hampered the facilitation of SSC frequency induced by RA. The prompt RA-enhanced SSC frequency was not abolished when Ca2+ was eliminated from the culture medium or there was bath application of the pharmacological Ca2+ channel inhibitor Cd2+, indicating that Ca2+ influx through voltage-activated Ca2+ channels are not required. Application of membrane-permeable inhibitors of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] or ryanodine receptors effectively blocked the increase of SSC frequency elicited by RA. Treating cells with either wortmannin or LY294002, two structurally different inhibitors of phosphatidylinositol 3-kinase (PI 3-kinase) and with the phospholipase Cgamma (PLCgamma) inhibitor U73122, abolished RA-induced facilitation of synaptic transmission. Preincubation of the cultures with pharmacological inhibitors, either genistein, a broad-spectrum tyrosine kinase inhibitor, or PP2, which predominantly inhibits the Src family of nonreceptor tyrosine kinase, completely abolished RA-induced synaptic facilitation. Taken collectively, these results suggest that RA elicits Ca2+ release from Ins1,4,5P3 and/or ryanodine-sensitive intracellular Ca2+ stores of the presynaptic nerve terminal. This is done via PLCgamma/PI 3-kinase signaling cascades and Src tyrosine kinase activation, leading to an enhancement of spontaneous transmitter release.

Both A chain and B chain of beta-bungarotoxin are functionally involved in the facilitation of spontaneous transmitter release in Xenopus nerve-muscle cultures.

In the present study, Xenopus nerve-muscle cultures were used to explore the functional roles of A chain (a phospholipase A(2) subunit) and B chain (a non-phospholipase A(2) subunit) of Bungarus multicinctus beta-bungarotoxin. It was found that beta-bungarotoxin induced an increment of the frequency of spontaneous synaptic currents (SSCs) in the nerve-muscle cultures. Modification of beta-bungarotoxin with pyridoxal-5'-phosphate or substitution of Ca(2+) with Ba(2+) in buffer abolished the phospholipase A(2) activity of beta-bungarotoxin and the facilitatory phase of SSC as well. Antibodies that were directed specifically against A chain or B chain effectively inhibited phospholipase A(2) activity, and as a consequence the SSC frequency was not greatly different from the control rate. These results suggest that both A and B chains are indispensable parts of beta-bungarotoxin for inducing the facilitation of SSC frequency with Xenopus nerve-muscle cultures.