Sargachromenol, a novel nerve growth factor-potentiating substance isolated from Sargassum macrocarpum, promotes neurite outgrowth and survival via distinct signaling pathways in PC12D cells

We previously found that the methanol extract of a marine brown alga, Sargassum macrocarpum showed marked nerve growth factor (NGF)-dependent neurite outgrowth promoting activity to PC12D cells. The active substance purified was elucidated to be sargachromenol. The median effective dose (ED50) was 9 microM against PC12D cells in the presence of 10 ng/ml NGF, although it showed no neurotrophic effect on its own. Pretreatment of cells with protein kinase A (PKA) inhibitor or U0126 substantially suppressed the sargachromenol-enhanced neurite outgrowth from PC12D cells, suggesting that the activation of cyclic AMP-mediated protein kinase and mitogen-activated protein (MAP) kinase 1/2 was apparently required for the action of sargachromenol. On the other hand, sargachromenol significantly promoted the survival of neuronal PC12D cells at 0-50 ng/ml NGF in serum-free medium. Neither PKA inhibitor nor U0126 could inhibit the survival supporting effect of sargachromenol, whereas wortmannin significantly blocked the sargachromenol-induced survival supporting effect on neuronal PC12D cells, suggesting that sargachromenol rescued neuronal PC12D cells by activating phosphatidylinositol-3 kinase. These results demonstrate that sargachromenol promotes neuronal differentiation of PC12D cells and supports the survival of neuronal PC12D cells via two distinct signaling pathways.     

Induction of neurite outgrowth in PC12 cells by the medium-chain fatty acid octanoic acid

It has been shown that polyunsaturated fatty acids such as arachinonic and docosahexanoic acids but not monounsaturated and saturated long-chain fatty acids promote basal and nerve growth factor (NGF)-induced neurite extension of PC12 cells, a line derived from a rat pheochromocytoma. On the other hand, short-chain fatty acids and valproic acid (2-propylpentanoic acid) enhance the growth of neurite processes of the cells only in the presence of inducers. In this study, we demonstrated that straight medium-chain fatty acids (MCFAs) at millimolar concentrations alone potently induced neuronal differentiation of PC12 cells. Hexanoic, heptanoic and octanoic acids dose-dependently induced neurite outgrowth of the cells: their maximal effects determined 2 days after addition to the culture medium were more marked than the effect of NGF. PC12 cells exposed to octanoic acid expressed increased levels of the neuronal marker beta-tubulin isotype III. Nonanoic, decanoic, and dodecanoic acids also induced growth of neurite processes, but their maximal effects were less marked than that of octanoic acid. In contrast, the polyunsaturated fatty acid linoleic acid and short-chain fatty acids had only slight or almost no effects on neurite formation in the absence of NGF. The effect of octanoic acid was synergistic with or additive to the effects of NGF and dibutyryl cyclic AMP. Octanoic acid upregulated phosphorylation of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK), critical signaling molecules in neuronal differentiation, but not phosphorylation of Akt, a signaling molecule downstream of phosphatidylinositol 3-kinase (PI3K). Moreover, growth of neurites induced by octanoic acid was potently inhibited by treatment of cells with the p38 MAPK inhibitor SB203580 and the ERK kinase inhibitor PD98059 but not inhibited and only slightly inhibited by the JNK inhibitor SP600125 and the PI3K inhibitor wortmannin, respectively. Taken together, our results indicate that MCFAs, including octanoic acid, induced neurite outgrowth of PC12 cells in the absence of NGF and suggest that the activation of p38 MAPK and ERK pathways is involved in this process.     

Dorsomorphin stimulates neurite outgrowth in PC12 cells via activation of a protein kinase A-dependent MEK-ERK1/2 signaling pathway

In this study, we investigated the effect of dorsomorphin, a selective inhibitor of bone morphogenetic protein (BMP) signaling, on rat PC12 pheochromocytoma cell differentiation. PC12 cells can be induced to differentiate into neuron-like cells possessing elongated neurites by nerve growth factor, BMP2, and other inducers. Cells were incubated with BMP2 and/or dorsomorphin, and the extent of neurite outgrowth was evaluated. Unexpectedly, BMP2-mediated neuritogenesis was not inhibited by co-treatment with dorsomorphin. We also found that treatment with dorsomorphin alone, but not another BMP signaling inhibitor, LDN-193189, induced neurite outgrowth in PC12 cells. To further understand the mechanism of action of dorsomorphin, the effects of this drug on intracellular signaling were investigated using the following signaling inhibitors: the ERK kinase (MEK) inhibitor U0126; the tropomyosin-related kinase A inhibitor GW441756; and the protein kinase A (PKA) inhibitor H89. Dorsomorphin induced rapid and sustained ERK1/2 activation; however, dorsomorphin-mediated ERK1/2 activation and neuritogenesis were robustly inhibited in the presence of U0126 or H89, but not GW441756. These findings suggest that dorsomorphin has the potential to induce neuritogenesis in PC12 cells, a response that requires the activation of PKA-dependent MEK-ERK1/2 signaling.     

The heat shock protein inhibitor KNK437 induces neurite outgrowth in PC12 cells

The nervous system is highly sensitive to various environmental stresses, such as ischemia. Stress response mechanisms that result in neuroprotection, including the induction of heat shock proteins (HSP), are not well understood. We examined the effect of KNK437, a compound that inhibits the synthesis of inducible heat shock proteins, on neuronal differentiation in rat pheochromocytoma PC12 cells. KNK437 decreased the expression of HSP70, and induced the neurite outgrowth of PC12 cells in the absence of stress stimulation, although with lower efficacy than nerve growth factor (NGF). Neurite outgrowth stimulated by KNK437 and NGF was blocked by inhibitors of ERK mitogen-activated protein (MAP) kinase, p38 MAP kinase, and glycogen synthase kinase 3β signaling pathways. NGF, and not KNK437, induced acetylcholine esterase (AChE) activity, a functional differentiation marker, indicating that KNK437 utilizes a mechanism distinct from that of NGF. KNK437 enhanced the activity of low dose NGF treatment on neurite outgrowth induction and ERK phosphorylation in PC12 cells, a finding that identifies KNK437 as a possible nerve regeneration agent. This compound may be a useful tool for the investigation of neuronal differentiation and neuroprotection against environmental stress.     

ERK在NGF诱导PC12 细胞分化中的作用

目的探讨细胞外信号调节激酶(ERK)在NGF。诱导的PC12细胞分化中的作用机制。方法以NGF处理PC12细胞建立分化模型,运用免疫印迹检测不同浓度不同作用时间时NGF对ERK1／2蛋白和磷酸化ERK1／2蛋白水平的影响,并观察MAPK／ERK激酶(MEK)抑制剂U0126对NGF诱导的细胞形态学改变的影响。结果ERK1／2蛋白的磷酸化呈现NGF剂量和时间依赖性。NGF作用细胞5min即可观察到明显的ERK1／2蛋白磷酸化,持续1h左右,2h时降低到初始水平,而细胞形态的改变出现在NCF作用12h以后。倒置相差显微镜观察可见PC12细胞分化的程度与ERK1／2活化持续的时间正相关,U0126可完全即时抑制ERK1／2的活化,而ERK1／2活化的抑制可完全阻断。NGF诱导的PC12细胞分化。结论ERK1／2的活化是PC12细胞发生分化的必需事件,其活化时间的长短对分化具有决定作用。     

Neurite outgrowth of PC12 mutant cells induced by orange oil and d-limonene via the p38 MAPK pathway

We studied the effects of natural essential oil on neurite outgrowth in PC12m3 neuronal cells to elucidate the mechanism underlying the action of the oils used in aromatherapy. Neurite outgrowth can be induced by nerve growth factor (NGF), where ERK and p38 MAPK among MAPK pathways play important roles in activating intracellular signal transduction. In this study, we investigated whether d-limonene, the major component of essential oils from oranges, can promote neurite outgrowth in PC12m3 cells, in which neurite outgrowth can be induced by various physical stimulations. We also examined by which pathways, the ERK, p38 MAPK or JNK pathway, d-limonene acts on PC12m3 cells. Our results showed that neurite outgrowth can be induced when the cells are treated with d-limonene. After treatment with d-limonene, we observed that p38 MAPK is strongly activated in PC12m3 cells, while ERK is weakly activated. In contrast, JNK shows little activity. A study using an inhibitor of p38 MAPK revealed that neurite outgrowth in PC12m3 cells is induced via the activation of p38 MAPK by d-limonene. The results thus indicate that d-limonene may promote neural cell differentiation mainly via activation of the p38 MAPK pathway.     

Differences in neuritogenic response to nitric oxide in PC12 and PC12h cells

We have demonstrated that a natural iridoid compound, genipin, induces neurite outgrowth through the nitric oxide (NO)-cGMP-protein kinase G signaling pathway in PC12h cells. PC12 cells, the parental cell line of PC12h cells, have been shown to carry out neurite extension that accompanies NO production in response to nerve growth factor (NGF). This neurite outgrowth was significantly inhibited by NG-nitro-L-arginine methyl ester (L-NAME), an NO synthase inhibitor, in both PC12 and PC12h cells, suggesting that the neuritogenesis is NO-dependent in both cells. In this report, we investigated whether genipin also induces neurite outgrowth in PC12 cells in order to determine the NO-dependent neurotrophic action of genipin in more than just one cell type. Genipin induced marked neurite outgrowth in PC12h cells but not in PC12 cells. The genipin-induced neurite outgrowth was significantly inhibited by L-NAME in PC12h cells. An NO donor, NOR4, also significantly induced neurite outgrowth in a concentration-dependent manner in PC12h cells but not in PC12 cells. On the other hand, NGF-primed PC12 cells exhibited significant neurite extension, which was inhibited by L-NAME, in response to genipin. Interestingly, NGF-primed PC12 cells responded to NOR4 extending neurites and expressed detectable neuronal NO synthase protein which is not detected in naive PC12 cells. These results suggest that genipin exerts a neuritogenic action on neuronal cells which are responsive to NO itself. Furthermore, the results also suggest that PC12h cells are more suitable for the study of NO-dependent neuronal function than PC12 cells which were not responsive to NO.     

Multiple kinase pathways mediate the early sciatic ligation-associated activation of CREB in the rat spinal dorsal horn

Phosphorylation of the cyclic AMP response element-binding protein (CREB) in the spinal dorsal horn may critically contribute to chronic pain following peripheral nerve injury. We employed inhibitors and activators of protein kinase A (PKA), protein kinase C (PKC), extracellular signal-regulated kinase 1 and 2 (ERK1/2) and calcium/calmodulin-dependent kinase II (CaMKII) to examine whether these kinases individually or in concert mediate the increase in CREB phosphorylation that is evident as early as 2 h after loose ligation of the sciatic nerve. Specific inhibitors of each kinase significantly attenuated the ligation-associated CREB phosphorylation when compared to saline-treated animals. Combined application of the ERK1/2 and CaMKII inhibitors also attenuated the ligation-associated CREB activation but not to a greater extent than either inhibitor alone. Specific activators of PKA, PKC and ERK1/2 elicited significant increases in CREB phosphorylation 2 h after drug application in the spinal dorsal horn of control, peripherally uninjured animals. Pre-treatment of animals with the ERK1/2 inhibitor abolished the increases elicited by either the PKA or the PKC activator. Significant increases in ERK1/2 phosphorylation were also detected 2 h after sciatic ligation confirming a role for the ERK pathway in injury-related responses in the dorsal horn. Each kinase inhibitor significantly attenuated the ligation-associated activation of ERK1/2 as well. These data suggest that early, sciatic ligation-elicited phosphorylation of CREB in the spinal dorsal horn is mediated by multiple kinase pathways, and that PKA, PKC and CaMKII activate CREB at least in part by way of the ERK pathway.     

Inhibition of NGF-induced neurite outgrowth of rat pheochromocytoma cells (PC12) following administration of dioxyamphetamine

Amphetamine analogs are known to induce not only neurotoxicity at serotonergic axon terminals but also neocortical neuronal degeneration. However, a much less studied aspect involves the impact of amphetamine exposure on neuronal development. The present study investigated whether pretreatment of PC12 cells with dioxyamphetamine (DA) alters differentiation of PC12 cells by NGF and, if so, which components of the Ras/Raf/MEK/ERK pathway known to be involved in the differentiation response to NGF are particularly affected. Though exposure of PC12 cells to DA 1 h prior to NGF treatment resulted in apopotosis, several PC12 cells survived. However, neurite outgrowth of these NGF-responsive cells was repressed. Immunoblots of whole cell extracts revealed a strong induction rather than inhibition of ERK phosphorylation up to 48 h after DA/NGF treatment. Our results indicate that NGF-mediated neurite outgrowth was inhibited by pretreatment with DA, and this blockage of NGF-induced neuritogenesis was not due to an inhibition of ERK phosphorylation.     

Signalling pathways involved in the sensitisation of mouse nociceptive neurones by nerve growth factor

Nerve growth factor (NGF) causes a rapid sensitisation of nociceptive sensory neurones to painful thermal stimuli owing to an action on the heat and capsaicin receptor TRPV1 (formerly known as VR1). We have developed a new technique to study this rapid sensitisation of TRPV1 by monitoring the effects of NGF on the increase in intracellular calcium concentration ([Ca²⁺]_i) following exposure to capsaicin. Brief applications of capsaicin caused a rise in [Ca²⁺]_i, and NGF was found to enhance this rise in 37 % of capsaicin-responsive neurones within 2 min. Pathways responsible for transducing the sensitisation of TRPV1 by TrkA, the NGF receptor, were characterised by observing the effects of inhibitors of key members of NGF-activated second messenger signalling cascades. Specific inhibitors of the ras/MEK (mitogen-activated protein and extracellular signal-regulated kinases) pathway and of phospholipase C did not abolish the NGF-induced sensitisation, but wortmannin, a specific inhibitor of phosphatidylinositol-3-kinase (PI3K), totally abolished the effect of NGF. Pharmacological blockade of protein kinase C (PKC) or calcium-calmodulin-dependent protein kinase II (CaMK II) activation also prevented NGF-induced sensitisation, while blockade of protein kinase A (PKA) was without effect. These data indicate that the crucial early pathway activated by NGF involves PI3K, while PKC and CaMK II are also involved, probably at subsequent stages of the NGF-activated signalling pathway.     

Hypoxia induces neurite outgrowth in PC12 cells that is mediated through adenosine A2A receptors

Development of the nervous system is a complex process, involving coordinated regulation of diverse cellular processes including proliferation, differentiation and synaptogenesis. Disturbances to brain development such as pre- and perinatal hypoxia have been linked to behavioural and late onset of neurological disorders. This study examines the effect of hypoxia on neurite outgrowth in PC12 cells. Hypoxia not only caused a rapid induction of neurite outgrowth, but also synergistically enhanced nerve growth factor (NGF)-induced neurite outgrowth up to 24 h. Transactivation of TrkA receptors was ruled out since the TrkA inhibitor K252a did not block hypoxia-induced neurite outgrowth. Adenosine deaminase prevented hypoxia-induced neurite outgrowth indicating that the effect is mediated by adenosine. Use of the specific adenosine A2A receptor agonist CGS21680 and antagonist 8-3(chlorostyryl)caffeine demonstrated that activation of this receptor is critical for hypoxia-induced neurite outgrowth. Hypoxia-induced neurite outgrowth was blocked by the adenylate cyclase inhibitor, MDL-12,330A, indicating a role for activation of this enzyme in the pathway. Hypoxia was further shown to cause a decrease in growth-associated protein (GAP)-43 levels and a lack of induction of betaIII tubulin, in contrast to NGF treatment which resulted in increased cellular levels of both of these proteins. These findings suggest that hypoxia induces neurite outgrowth in PC12 cells via a pathway distinct from that activated by NGF. Thus, exposure to hypoxia at critical stages of development may contribute to aberrant neurite outgrowth and could be a factor in the pathogenesis of certain delayed developmental neurological disorders.     

Extracellular guanosine 5' triphosphate enhances nerve growth factor-induced neurite outgrowth via increases in intracellular calcium

Extracellular guanosine 5' triphosphate (GTP) enhances nerve growth factor-dependent neurite outgrowth from rat pheochromocytoma (PC12) cells; cultures of PC12 cells exposed to GTP and nerve growth factor together contain significantly more neurite-bearing cells than do those exposed to either nerve growth factor or GTP alone [Gysbers J. W. and Rathbone M. P. (1996) Int. J. devl Neurosci. 14, 19-34]. PC12 cells contain specific cell surface binding sites for extracellular GTP, which do not bind ATP or uridine 5' triphosphate. Exposure of PC12 cells to extracellular GTP (300microM) produced a robust and sustained increase in intracellular Ca(2+) ([Ca(2+)](i)), different from the transient response to the addition of ATP. The GTP-induced [Ca(2+)](i) increase was blocked by the L-type calcium channel inhibitor, nifedipine. The L-type Ca(2+) channel inhibitors, nifedipine or verapamil, also inhibited the enhancement of neurite outgrowth by GTP, but did not affect neurite outgrowth stimulated by nerve growth factor alone. Pre-treatment of PC12 cells with ryanodine (0.5-50microM) depleted calcium from internal stores and prevented the further release of calcium by GTP. Similarly, pre-treatment of PC12 cells with thapsigargin (an inhibitor of internal store Ca(2+)/ATPase) or dantrolene (which blocks Ca(2+) release from some of these stores) also reduced the enhancement of neurite outgrowth by GTP. Therefore, Ca(2+)-induced Ca(2+) release from specific stores, present in PC12 cells, is involved in the enhancement of nerve growth factor-induced neurite outgrowth by GTP, possibly acting at specific binding sites on the cell surface. GTP is proving to be an important extracellular trophic modulator in the central nervous system. These studies show that the neuritogenic actions of GTP involve moderate but sustained increases in intracellular Ca(2+) which are likely due to activation of L-type Ca(2+) channels and Ca(2+)-induced Ca(2+) release from intracellular stores. These effects of extracellular GTP are likely mediated at the cell surface and may be related to specific GTP binding sites which are distinct from G-proteins and from hitherto described purine nucleotide (P2) receptors.These data indicate a mechanism whereby the neuritogenic effects of GTP are mediated and emphasize the importance of considering GTP as a neurotrophic mediator.     

Involvement of BREK, a serine/threonine kinase enriched in brain, in NGF signalling

We identified AATYK2 (Apoptosis-Associated Tyrosine Kinase 2) through a database search as a kinase specifically expressed in the brain. After characterization, we renamed it BREK (Brain-Enriched Kinase). Mouse BREK mRNA is expressed predominantly in brain, especially in olfactory bulb, olfactory tubercle, hippocampus, striatum, cerebellum, and cerebral cortex. Levels of expression and phosphorylation of BREK were high at 0-2 weeks after birth, suggesting that BREK is involved in neural development and functions during the early postnatal period. Phosphoamino acid analysis following in vitro kinase reaction revealed that BREK is a catalytically active, serine/threonine kinase. In PC12 cells, BREK was phosphorylated rapidly upon stimulation with nerve growth factor (NGF) in a protein kinase C-dependent pathway. In differentiated PC12 cells, BREK was enriched in cell bodies and growth cones, and also present along neurites. Introduction of a kinase-defective mutant of BREK into PC12 cells enhanced both ERK phosphorylation and neurite outgrowth in response to NGF, suggesting that BREK is a negative regulator of NGF-induced neuronal differentiation. Thus, we conclude that BREK is a new member of the family of protein serine/threonine kinases and that it plays important roles in NGF-TrkA signalling.     

Ca(2+)-dependent inhibition of inwardly rectifying K(+) channel in opossum kidney cells

The effect of intracellular Ca(2+) on the activity of the inwardly rectifying ATP-regulated K(+) channel with an inward conductance of about 90 pS was examined by using the patch-clamp technique in opossum kidney proximal tubule (OKP) cells. The activity of the inwardly rectifying K(+) channel rapidly declined with an application of ionomycin (1 microM) in the presence of 10(-6) M Ca(2+) in cell-attached patches. The application of 10 microM phorbor-12-myristate-acetate (PMA) with 10(-6) M Ca(2+) reduced the K(+) channel activity. Although the channel activity was not influenced by an increase of bath Ca(2+) from 10(-7.5) to 10(-6) M, the activity was inhibited by protein kinase C (PKC, 1 U/ml) with 10(-6) M Ca(2+) in inside-out patches. The inhibitory effect of Ca(2+) with ionomycin on the channel activity was diminished by the pretreatment with a specific PKC inhibitor, GF 109203X (5 microM), in cell-attached patches. By contrast, the application of Ca(2+)/calmodulin kinase II (CaMK II, 300 pM) dramatically increased this channel activity in inside-out patches. In cell-attached patches, the addition of both GF 109203X and cyclospolin A (5 microM), a potent inhibitor of protein phosphatase 2B (calcineurin), instead stimulated the K(+) channel activity with ionomycin and 10(-6) M Ca(2+). The addition of protein phosphatase 2B (calcineurin) (2 U/ml) to the bath with calmodulin (1 microM) and Ni(2+) (10 microM) to stimulate calcineurin inhibited the channel activity in inside-out patches. Furthermore, the inhibitory effect of PKC or calcineurin on this channel activity was abolished by a removal of Ca(2+) from bath solution. These results suggest that Ca(2+)-dependent inhibitory effect on the inwardly rectifying K(+) channel in OKP cells was mainly mediated by Ca(2+)-PKC-mediated phosphorylation, and that the Ca(2+)-calmodulin-dependent phosphorylation process may be counterbalanced by the Ca(2+)-calmodulin-dependent dephosphorylation process.     

Cooperation in signal transduction of extracellular guanosine 5' triphosphate and nerve growth factor in neuronal differentiation of PC12 cells

Guanosine 5' triphosphate (GTP), acting synergistically with the nerve growth factor (NGF), enhances the proportion of neurite-bearing cells in cultures of PC12 rat pheochromocytoma cells. We studied the transduction mechanisms activated by GTP in PC12 cells and found that addition of GTP (100 microM) increased intracellular calcium concentration ([Ca(2+)](i)) in cells that were between 60 and 70% confluent. Addition of GTP also enhanced activation of NGF-induced extracellular regulated kinases (ERKs) and induced Ca(2+) mobilization. This mobilization, due to the activation of voltage-sensitive and ryanodine-sensitive calcium channels, as well as pertussis toxin-sensitive purinoceptors, modulates Ca(2+)-activated K(+) channels not involved in activation of ERKs. The results presented here indicate that GTP-triggered [Ca(2+)](i) increase may be a key event in GTP signal transduction, which can modulate activity of ERKs. The physiological importance of the GTP effect lies in its capacity to interact with the NGF-activated pathway to enhance neurite outgrowth from PC12 cells.     

Sweet taste transduction in hamster: role of protein kinases

Two different second-messenger pathways have been implicated in sweet taste transduction: sugars produce cyclic AMP (cAMP), whereas synthetic sweeteners stimulate production of inositol 1,4, 5-tris-phosphate (IP(3)) and diacylglycerol (DAG). Both sugars and sweeteners depolarize taste cells by blocking the same resting K(+) conductance, but the intermediate steps in the transduction pathways have not been examined. In this study, the loose-patch recording technique was used to examine the role of protein kinases and other downstream regulatory proteins in the two sweet transduction pathways. Bursts of action currents were elicited from approximately 35% of fungiform taste buds in response to sucrose (200 mM) or NC-00274-01 (NC-01, 200 microM), a synthetic sweetener. To determine whether protein kinase C (PKC) plays a role in sweet transduction, taste buds were stimulated with the PKC activator PDBu (10 microM). In all sweet-responsive taste buds tested (n = 11), PDBu elicited burst of action currents. In contrast, PDBu elicited responses in only 4 of 19 sweet-unresponsive taste buds. Inhibition of PKC by bisindolylmaleimide I (0.15 microM) resulted in inhibition of the NC-01 response by approximately 75%, whereas the response to sucrose either increased or remained unchanged. These data suggest that activation of PKC is required for the transduction of synthetic sweeteners. To determine whether protein kinase A (PKA) is required for the transduction of sugars, sweet responses were examined in the presence of the membrane-permeant PKA inhibitor H-89 (10 and 19 microM). Surprisingly, H-89 did not decrease responses to either sucrose or NC-01. Instead, responses to both compounds were increased in the presence of the inhibitor. These data suggest that PKA is not required for the transduction of sugars, but may play a modulatory role in both pathways, such as adaptation of the response. We also examined whether Ca(2+)-calmodulin dependent cAMP phosphodiesterase (CaM-PDE) plays a role in sweet taste transduction, by examining responses to sucrose and synthetic sweeteners in the presence of the CaM-PDE inhibitor W-7 (100 microM). Inhibition resulted in an increase in the response to sucrose, whereas the response to NC-01 remained unchanged. These data suggest that the pathways for sugars and sweeteners are negatively coupled; the Ca(2+) that is released from intracellular stores during stimulation with synthetic sweeteners may inhibit the response to sucrose by activation of CaM-PDE.     

Serotonergic modulation of the hyperpolarizing spike afterpotential in rat jaw-closing motoneurons by PKA and PKC.

Intracellular recordings were obtained from rat jaw-closing motoneurons (JCMNs) in slice preparations to investigate the effects of serotonin (5-HT) on the postspike medium-duration afterhyperpolarization (mAHP) and an involvement of protein kinases in the effects. Application of 50 microM 5-HT caused membrane depolarization and increased input resistance in the most cells without affecting the mAHP, whereas not only membrane depolarization and an increase in input resistance, but also the suppression of the mAHP amplitude was induced by higher dose of 5-HT (100 or 200 microM). On the other hand, when the mAHP amplitude was increased by raising [Ca(2+)](o) from 2 to 6 mM, 5-HT-induced attenuation of the mAHP amplitude was enhanced, and even 50 microM 5-HT reduced the mAHP amplitude. This 5-HT-induced suppression of the mAHP could be mimicked by application of membrane-permeable cAMP analogue 8-Bromo-cAMP, potentiated by the cAMP-specific phosphodiesterase inhibitor Ro 20-1724 and antagonized by protein kinase A (PKA) inhibitor H89. The enhancement of the mAHP attenuation induced by 50 microM 5-HT under raised [Ca(2+)](o) was blocked by a protein kinase C (PKC) inhibitor chelerythrine, suggesting an involvement of PKC in this enhancement. On the other hand, the attenuation of the mAHP induced by PKC activator phorbol 12-myristate 13-acetate was blocked almost completely by H89, suggesting that the PKC action on the mAHP requires PKA activation. Neither 5-HT(1A) antagonist NAN-190 or 5-HT(4) antagonist SB 203186 blocked 5-HT-induced attenuation of the mAHP. We conclude that 5-HT induces dose-dependent attenuation of the mAHP amplitude through cAMP-dependent activation of PKA and that PKC-dependent PKA activation is also likely to be involved in the enhancement of 5-HT-induced attenuation of the mAHP under raised [Ca(2+)](o). Because the slope of the linear relationship between firing frequency and injected current was increased only when the mAHP amplitude was decreased by 5-HT, it is suggested that the relation between incoming synaptic inputs and firing output in JCMNs varies according to serotonergic effects on JCMNs and calcium-dependent modulation of its effects.     

Screening of natural medicines that efficiently activate neurite outgrowth in PC12 cells in C2C12-cultured medium

We have studied the effects of natural medicines on neurite outgrowth in PC12D cells in a cultured medium of C2C12 cells. Derived from mouse myoblasts, the C2C12 cells secrete neurotrophic factors including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3). The secretion of these neurotrophins from C2C12 cells stimulate neurite outgrowth in PC12D cells. We have screened a total of 120 samples and found five natural medicines: Trichosanthes Root, Asiasarum Root, Lycium Bark, Sinomenium Stem, and Dictamni radicis Cortex, that enhance the activity of C2C12-cultured medium to stimulate neurite outgrowth in PC12D cells. These natural medicines promoted not only neurite outgrowth but also stabilized the neurite formation in PC12D cells for several days. RT-PCR analysis showed that NGF was significantly increased with Trichosanthes and Lycium Bark. However, BDNF was slightly decreased with Lycium Bark, Sinomenium Stem, and Dictamni radicis Cortex. NT-3 was increased slightly by all of these natural medicines except Sinomenium Stem. All these five natural medicines significantly increased the number and length of neurites in PC12D cells in co-culture with C2C12 cells.