Actin antagonists have previously been shown to alter responses of Commelina communis stomata to physiological stimuli, implicating actin filaments i n the control of guard cell volume changes (M. Kim, P.K. Hepler, 5-0. Eun, K.S. Ha, Y. Lee [1995] Plant Physiol 109: 1077-1084). Since K+ channels i n the guard cell play an important role i n stomatal movements, we examined the possible regulation of K+-channel activities by the state of actin polymerization. Agents affecting actin polymerization altered light-induced stomatal opening and inward K+-channel activities measured by patch clamping in Vicia faba. Cytochalasin D, which induces depolymerization of actin filaments, promoted light-induced stomatal opening and potentiated the inward K+ current in guard cell protoplasts. Phalloidin, a stabilizer of filamentous actin, inhibited both light-induced stomatal opening and inward K+ current. lnward K+-channel activities in outside-out membrane patches showed responses to these agents that support results at the whole-cell current levei, suggesting that cytochalasin D facilitates and phalloidin inhibits K+ influx in intact guard cells, thus resulting in enhancement and inhibition of stomatal opening, respectively. To our knowledge, this is the first report that provides evidence that actin filaments may regulate an important physiological process by modulating the activities of ion channels i n plant cells.The regulation of stomatal aperture is critica1 to a plant's ability to balance the need for a C source while avoiding the deleterious effects of water loss. The size of the stomatal opening is established through volume changes of stomatal guard cells under the concerted influence of light, temperature, CO,, and phytohormones (Assmann, 1993). Recently, Kim et al. (1995) showed that cortical actin filaments are distributed radially, fanning out from the stomatal pore site in mature guard cells of Commelinu communis. Moreover, fungal toxins that interfere with the polymerization or depolymerization of actin filaments brought about altered stomatal responses to physiological stimuli. Thus, dynamic changes in the actin cytoskeleton
In vivo evidence for the involvement of phospholipase A and protein kinase in the signal transduction pathway for auxin-induced com coleoptiie elongation Auxin-induced elongation of com coleoptiles is accompanied by cell wall acidification, which depends upon H"^-pump activity. We tested the hypothesis that phospholipase A and a protein kinase are involved in the pathway of auxin signal transduction leading to H^ secretion, and elongation of com coleoptiles. initiaOy. the pH of the bath solution at 50^100 pm from the surface of a coleoptiie segment (pH^,) ranged between 4.8 and 6.6 when measured with an H"-sensiti^'e microelectrode. Twenty or 50 ]iM lysophosphatidylcholine, 50 pM linolenic acid or 50 ]iM arachidonic acid induced a decline in pH^ by 0.3 to 2.1 units. The effect was blocked by 1 mM vanadate, suggesting that ]y.sophosphaEidylcholine or hnolenic acid induced acidification of the apoplast by activating the H^-pump. Lysophosphatidylcholine and linolenic acid also accelerated the elongation rate of the coleoptiles. While hnolenic acid and arachidonic acid, highly unsaturated fatty acids, promoted pH^^ decrease and coleoptiie elongation, linoleic acid, oleic acid, and stearic acid, fatty acids with a lesser extent of unsaturation, had no such effects. The effects of lysophosphatidylcholine, linolenic acid, and arachidonic acid on H* secretion were not additive to that of indoleacetic acid (IAA), suggesting that lysophosphoiipids, fatty acids and auxin use similar pathwaj^s for the activation of the H^-pump. The phospholipase Ai inhibitors, aristolochic acid and manoalide, inhibited the IAA-induced pH,^ decrease and coleoptiie elongation. The general protein kinase inhibitors., H-7 or staurosporine, blocked the IAA-or lysophosphatidylcholine-induced decrease in pH". H-7 also inhibited the eoleoptile elongation induced by LAA or lysophosphatidylcboline. These results support the hypothesis that phospholipase A is activated by auxin, and that the products of the enzvine, lysophosphoiipids and fatty acids, induce acidification of the apoplast by activating the H^pump through a mechanism involving a protein kinase, which in turn promotes com eoleoptile elongation.
The vast majority of human peripheral nerve injuries occur in the upper limb, whereas the most animal studies have been conducted using the hindlimb models of neuropathic pain, involving damages of the sciatic or lumbar spinal nerve(s). We attempted to develop a rat forelimb model of peripheral neuropathy by partial injury of the median and ulnar nerves. The halves of each nerve were transected by microscissors at about 5mm proximal from the elbow joint and behavioral signs of neuropathic pain, such as mechanical and cold allodynia, and heat hyperalgesia, were monitored up to 126 days following nerve injury. Mechanical allodynia was assessed by measuring the forepaw withdrawal threshold to von Frey filaments, and cold allodynia was evaluated by measuring the time spent in lifting or licking the forepaw after applying acetone to it. Heat hyperalgesia was also monitored by investigating the forepaw withdrawal latencies using the Hargreaves' test. After the nerve injury, the experimental animals exhibited long-lasting clear neuropathic pain-like behaviors, such as reduced forepaw withdrawal threshold to von Frey filaments, the increased response duration of the forepaw to acetone application, and the decreased withdrawal latency to radiant heat stimulation. These behaviors were significantly alleviated by administration of gabapentin (5 or 50mg/kg, i.p.) in a dose-dependent manner. Therefore, these abnormal sensitivities are interpreted as the signs of neuropathic pain following injury of the median and ulnar nerves. Our rat forelimb model of neuropathic pain may be useful for studying human neuropathic pain and screening for valuable drug candidates.
Auxin‐induced elongation of com coleoptiles is accompanied by cell wall acidification, which depends upon H+‐pump activity. We tested the hypothesis that phospholipase A and a protein kinase are involved in the pathway of auxin signal transduction leading to H+ secretion, and elongation of corn coleoptiles. Initially, the pH of the bath solution at 50–100 μm from the surface of a coleoptile segment (pHo) ranged between 4.8 and 6.6 when measured with an H+‐sensitive microelectrode. Twenty or 50 μM lysophosphatidylcholine, 50 μM linolenic acid or 50 μM arachidonic acid induced a decline in pHo by 0.3 to 2.1 units. The effect was blocked by 1 mM vanadate, suggesting that lysophosphatidylcholine or linolenic acid induced acidification of the apoplast by activating the H+‐pump. Lysophosphatidylcholine and linolenic acid also accelerated the elongation rate of the coleoptiles. While linolenic acid and arachidonic acid, highly unsaturated fatty acids, promoted pHo decrease and coleoptile elongation, linoleic acid, oleic acid, and stearic acid, fatty acids with a lesser extent of unsaturation, had no such effects. The effects of lysophosphatidylcholine, linolenic acid, and arachidonic acid on H+ secretion were not additive to that of indoleacetic acid (IAA), suggesting that lysophospholipids, fatty acids and auxin use similar pathways for the activation of the H+‐pump. The phospholipase A2 inhibitors, aristolochic acid and manoalide, inhibited the IAA‐induced pHo decrease and coleoptile elongation. The general protein kinase inhibitors, H‐7 or staurosporine, blocked the IAA‐ or lysophosphatidylcholine‐induced decrease in pHo. H‐7 also inhibited the coleoptile elongation induced by IAA or lysophosphatidylcholine. These results support the hypothesis that phospholipase A is activated by auxin, and that the products of the enzyme, lysophospholipids and fatty acids, induce acidification of the apoplast by activating the H+‐pump through a mechanism involving a protein kinase, which in turn promotes com coleoptile elongation.
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