Application Of Flufenamic Acid Research Articles

Effects of San-Huang-Xie-Xin-tang, a traditional Chinese prescription for clearing away heat and toxin, on the pacemaker activities of interstitial cells of Cajal from the murine small intestine

Ethnopharmacological relevanceSan-Huang-Xie-Xin-Tang (SHXXT) is a traditional Chinese medicinal formula composed of Coptidis rhizoma (Coptis chinesis Franch), Scutellariae radix (Scutellaria baicalensis Georgi), and Rhei rhizoma (Rheum officinale Baill) and is widely used in Eastern Asia, especially to ameliorate the symptoms of gastrointestinal (GI) disorders related to gastritis, gastric bleeding, peptic ulcers, and abnormal GI motility Aim of the studyInterstitial cells of Cajal (ICCs) are pacemaker cells in the GI tract that generate rhythmic oscillations in membrane potentials known as slow waves. Because GI disorders, especially abnormal GI motility, are major lifelong problems, the authors investigated the effects of SHXXT on mouse small intestine ICCs, and sought to identify the receptors and the action mechanisms involved. Materials and methodsEnzymatic digestions were used to dissociate ICCs from small intestines, and the whole-cell patch-clamp configuration was used to record potentials generated by cultured ICCs. ResultsSHXXT produced membrane depolarization in current-clamp mode, and Y25130 (a 5-HT3 receptor antagonist) and RS39604 (a 5-HT4 receptor antagonist) blocked SHXXT-induced membrane depolarizations, whereas SB269970 (a 5-HT7 receptor antagonist) did not. However, during external Ca2+ free conditions or in the presence of thapsigargin, SHXXT did not exhibit membrane depolarization. Furthermore, the application of flufenamic acid (a nonselective cation channel (NSCC) blocker) or DIDS (a chloride channel blocker) abolished pacemaker potential generation and blocked SHXXT-induced membrane depolarizations. In addition, SHXXT-induced membrane depolarizations, which are dependent on G-protein, in ICCs were blocked by PD 98059 (a p42/44 mitogen-activated protein kinase (MAPK) inhibitor), SB203580 (a p38 MAPK inhibitor), and by a c-jun NH2-terminal kinase (JNK) II inhibitor. Regarding the components of SHXXT, Coptidis rhizome and Rhei rhizoma modulated ICC pacemaking activity, whereas Scutellariae radix did not. ConclusionSHXXT modulates pacemaker potentials via 5-HT3 and 5-HT4 receptor-mediated pathways, external Ca2+ influx, and Ca2+ release from internal stores. Furthermore, NSCCs and Cl- channels play important roles in the regulation of pacemaking activity in a MAPK dependent manner in ICCs. The regulation of pacemaking activity by SHXXT may be due to the activity of Coptidis rhizome and Rhei rhizome. The study shows SHXXT can modulate the pacemaking activity of ICCs in the GI tract, and thus, suggests SHXXT has potential pharmacological relevance for the treatment of GI motility disorders.

Effects of Samchulkunbi-tang in Cultured Interstitial Cells of Cajal of Murine Small Intestine

We studied the modulation of pacemaker activities by Samchulkunbi-tang (SCKB) in cultured interstitial cells of Cajal (ICC) from murine small intestine with the whole-cell patch-clamp technique. Externally applied SCKB produced membrane depolarization in the current-clamp mode. The pretreatment with Ca 2+ -free solution and thapsigargin, a Ca 2+ -ATPase inhibitor in endoplasmic reticulum, abolished the generation of pacemaker potentials and suppressed the SCKB-induced action. The application of flufenamic acid (a nonselective cation channel blocker) abolished the generation of pacemaker potentials by SCKB. However, the application of niflumic acid (a chloride channel blocker) did not inhibit the generation of pacemaker potentials by SCKB. In addition, the membrane depolarizations were inhibited by not only GDP-β-S, which permanently binds G-binding proteins, but also U-73122, an active phospholipase C inhibitor. These results suggest that SCKB modulates the pacemaker activities by nonselective cation channels and external Ca 2+ influx and internal Ca 2+ release via G-protein and phospholipase C-dependent mechanism. Therefore, the ICC are targets for SCKB and their interaction can affect intestinal motility.

Effects of Lizhong Tang on cultured mouse small intestine interstitial cells of Cajal

To investigate the effects of Lizhong Tang, an herbal product used in traditional Chinese medicine, on mouse small intestine interstitial cells of Cajal (ICCs). Enzymatic digestions were used to dissociate ICCs from mouse small intestine tissues. The ICCs were morphologically distinct from other cell types in culture and were identified using phase contrast microscopy after verification with anti c-kit antibody. A whole-cell patch-clamp configuration was used to record potentials (current clamp) from cultured ICCs. All of the experiments were performed at 30-32 °C. ICCs generated pacemaker potentials, and Lizhong Tang produced membrane depolarization in current-clamp mode. The application of flufenamic acid (a nonselective cation channel blocker) abolished the generation of pacemaker potentials by Lizhong Tang. Pretreatment with thapsigargin (a Ca²⁺-ATPase inhibitor in the endoplasmic reticulum) also abolished the generation of pacemaker potentials by Lizhong Tang. However, pacemaker potentials were completely abolished in the presence of an external Ca²⁺-free solution, and under this condition, Lizhong Tang induced membrane depolarizations. Furthermore, When GDP-β-S (1 mmol/L) was in the pipette solution, Lizhong Tang still induced membrane depolarizations. In addition, membrane depolarizations were not inhibited by chelerythrine or calphostin C, which are protein kinase C inhibitors, but were inhibited by U-73122, an active phospholipase C inhibitors. These results suggest that Lizhong Tang might affect gastrointestinal motility by modulating pacemaker activity in interstitial cells of Cajal.

Orexin-A excites pyramidal neurons in layer 2/3 of the rat prefrontal cortex

The arousal peptides, orexins, play an important role in regulating the function of the prefrontal cortex (PFC). Although orexins have been shown to increase the excitability of deep-layer neurons in the medial prefrontal cortex (mPFC), little is known about their effect on layer 2/3, the main intracortical processing layer. In this study, we investigated the effect of orexin-A on pyramidal neurons in layer 2/3 of the mPFC using whole-cell recordings in rat brain slices. We observed that orexin-A reversibly depolarized layer 2/3 pyramidal neurons through a postsynaptic action. This depolarization was concentration-dependent and mediated via orexin receptor 1. In voltage-clamp recordings, the orexin-A-induced current was reduced by the replacement of internal K+ with Cs+, removal of external Na+, or an application of flufenamic acid (an inhibitor of nonselective cation channels). A blocker of Na+/Ca2+ exchangers (SN-6) did not influence the excitatory effect of orexin-A. Moreover, the current induced by orexin-A reversed near Ek when the external solution contained low levels of Na+. When recording with Cs+-containing pipettes in normal external solution, the reversal potential of the current was approximately −25mV. These data suggest an involvement of both K+ channels and nonselective cation channels in the effect of orexin-A. The direct excitatory action of orexin-A on layer 2/3 mPFC neurons may contribute to the modulation of PFC activity, and play a role in cognitive arousal.

Effects of ginsenoside on pacemaker potentials of cultured interstitial cells of Cajal clusters from the small intestine of mice.

Ginsenoside, one of the active ingredients of Panax ginseng, has a variety of physiological and pharmacological actions in various organs. However, little is known about the effects of ginsenosides on gastrointestinal (GI) motility. We studied the modulation of pacemaker potentials by ginsenoside in the interstitial cells of Cajal (ICCs) using the whole-cell patch clamp technique in the current clamp mode. Among ginsenosides, we investigated the effects of ginsenoside Rb1, Rg3 and Rf. While externally applied Rb1 and Rg3 had no effects on pacemaker potentials, Rf caused membrane depolarization. The application of flufenamic acid or niflumic acid abolished the generation of pacemaker potentials and inhibited the Rf-induced membrane depolarization. Membrane depolarization induced by Rf was not inhibited by intracellular application of guanosine 5'-[β-thio]diphosphate trilithium salt. Pretreatment with a Ca(2+)-free solution, thapsigargin, a Ca(2+)-ATPase inhibitor of the endoplasmic reticulum, U-73122, a phospholipase C inhibitor, or 2-APB, an IP3 receptor inhibitor, abolished the generation of pacemaker potentials and suppressed Rfinduced actions. However, treatment with chelerythrine and calphostin C, protein kinase C inhibitors, did not block Rf-induced effects on pacemaker potentials. These results suggest that ginsenoside Rf modulates the pacemaker activities of ICCs and thereby regulates intestinal motility.

Effects of prostaglandin F2αon small intestinal interstitial cells of Cajal

To explore the role of prostaglandin F(2α) (PGF(2α))) on pacemaker activity in interstitial cells of Cajal (ICC) from mouse small intestine. In this study, effects of PGF(2α) in the cultured ICC cells were investigated with patch clamp technology combined with Ca(2+) image analysis. Externally applied PGF(2α) (10 μmol/L) produced membrane depolarization in current-clamp mode and increased tonic inward pacemaker currents in voltage-clamp mode. The application of flufenamic acid (a non-selective cation channel inhibitor) or niflumic acid (a Cl(-) channel inhibitor) abolished the generation of pacemaker currents but only flufenamic acid inhibited the PGF(2α)-induced tonic inward currents. In addition, the tonic inward currents induced by PGF(2α) were not inhibited by intracellular application of 5'-[-thio]diphosphate trilithium salt. Pretreatment with Ca(2+) free solution, U-73122, an active phospholipase C inhibitor, and thapsigargin, a Ca(2+)-ATPase inhibitor in endoplasmic reticulum, abolished the generation of pacemaker currents and suppressed the PGF(2α)-induced tonic inward currents. However, chelerythrine or calphostin C, protein kinase C inhibitors, did not block the PGF(2α)-induced effects on pacemaker currents. When recording intracellular Ca(2+) ([Ca(2+)](i)) concentration using fluo-3/AM, PGF(2α) broadly increased the spontaneous [Ca(2+)](i) oscillations. These results suggest that PGF(2α) can modulate pacemaker activity of ICC by acting non-selective action channels through phospholipase C-dependent pathway via [Ca(2+)]i regulation.

Induction of pacemaker currents by DA-9701, a prokinetic agent, in interstitial cells of Cajal from murine small intestine.

The interstitial cells of Cajal (ICC) are pacemaking cells required for gastrointestinal motility. The possibility of whether DA-9701, a novel prokinetic agent formulated with Pharbitis Semen and Corydalis Tuber, modulates pacemaker activities in the ICC was tested using the whole cell patch clamp technique. DA-9701 produced membrane depolarization and increased tonic inward pacemaker currents in the voltage-clamp mode. The application of flufenamic acid, a non-selective cation channel blocker, but not niflumic acid, abolished the generation of pacemaker currents induced by DA-9701. Pretreatment with a Ca2+-free solution and thapsigargin, a Ca2+-ATPase inhibitor in the endoplasmic reticulum, abolished the generation of pacemaker currents. In addition, the tonic inward currents were inhibited by U-73122, an active phospholipase C inhibitor, but not by GDP-beta-S, which permanently binds G-binding proteins. Furthermore, the protein kinase C inhibitors, chelerythrine and calphostin C, did not block the DA-9701-induced pacemaker currents. These results suggest that DA-9701 might affect gastrointestinal motility by the modulation of pacemaker activity in the ICC, and the activation is associated with the non-selective cationic channels via external Ca2+ influx, phospholipase C activation, and Ca2+ release from internal storage in a G protein-independent and protein kinase C-independent manner.

Persistent sodium current in preBötzinger neurons is not necessary for respiratory rhythm generation in the neonatal mouse?

Mammalian breathing is dependent upon the activity of a set of respiratory neurons in the preBotzinger complex (preBotC). Smith and colleagues identified a set of pacemaker neurons whose rhythmicity depends on the activation of a persistent sodium current (INa(P)), in the preBotC (Smith et al. 1991; Del Negro et al. 2002). Since then, many studies have focused on the role of INa(P) in respiratory rhythm generation. Still, it has not been clear whether INa(P) is obligatory for respiratory rhythm generation. INa(P) is widely distributed in neurons in the preBotC and also outside the preBotC (Ptak et al. 2005). Bath application of riluzole, a blocker of INa(P), to brain slices that include the preBotC inhibited the respiratory rhythm; however, this does not necessarily prove a role for INa(P) in the preBotC since riluzole could affect neurons outside preBotC too. The beauty of the new study by Pace et al. is that it directly tested the role of INa(P) in the preBotC in respiratory rhythm generation using carefully controlled drug microinjections (Pace et al. 2007). Muscimol, a GABAA receptor agonist, reversibly inhibits neurons. Microinjecting muscimol into the preBotC reversibly inhibited the respiratory rhythm within 1 min and Pace et al. (2007) used muscimol microinjection to find the appropriate position for microinjecting riluzole. Surprisingly, microinjecting riluzole into the preBotC had no effects on respiratory rhythm generation even when combined with bath application of flufenamic acid (FFA), a calcium-activated non-specific cation current (ICAN) blocker. These results differ from previous experiments using bath application of riluzole and FFA (Pena et al. 2004; Del Negro et al. 2005). Furthermore, Pace et al. (2007) found that the respiratory rhythm was still present after microinjecting another INa(P) blocker, a low concentration solution of TTX, into the preBotC together with bath application of FFA. The frequency of the respiratory rhythm was decreased, but this could be caused by a reduction of action potential amplitude by TTX. Together, these experiments suggest that INa(P) in preBotC neurons is not obligatory for respiratory rhythm generation in neonatal mice. What might explain these differing results on the role of INa(P) in respiratory rhythm generation? One hypothesis is that bath-applied riluzole, or a low concentration solution of TTX, exert their effects on neurons outside the preBotC to inhibit respiratory rhythm generation in the slice preparation. To explore which type of neuron might be the target, Pace et al. (2007) tested serotonergic neurons in the raphe nucleus. They found another surprising result, that microinjecting riluzole into the raphe obscurus with bath application of FFA abolished respiratory rhythm generation reversibly. Thus, it is no surprise that bath application of riluzole and FFA inhibit respiratory rhythm generation. However, this inhibition might not result from the block of INa(P) in preBotC neurons as suggested by previous studies (Pena et al. 2004; Del Negro et al. 2005). In conclusion, this study provides evidence to support the argument that INa(P) in preBotC neurons is not necessary for respiratory rhythm generation in the neonatal mouse. Like other good articles, this one raises many interesting new scientific questions. First, how does riluzole act in the raphe nucleus to abolish the respiratory rhythm? One possibility is that riluzole decreases the excitability of serotonergic neurons, and thus reduces the amplitude of the serotonergic input to the preBotC. Second, is the rhythmic activity of INa(P)-dependent pacemaker neurons in the preBotC inhibited by microinjecting riluzole into the raphe nucleus? If the answer is ‘no’, then the result would demonstrate that the INa(P)-dependent pacemaker activity in the preBotC is not obligatory for respiratory rhythm generation. Certainly, more experiments are needed to clarify these questions.

Effects of Ginseng Total Saponins on Pacemaker Currents of Interstitial Cells of Cajal from the Small Intestine of Mice

Although ginsenosides have a variety of physiologic or pharmacologic functions in various regions, there are only a few reports on the effects of ginsenosides on gastrointestinal (GI) motility. We studied the modulation of pacemaker activities by ginseng total saponins in the interstitial cells of Cajal (ICC) using the whole cell patch-clamp technique. Externally applied ginseng total saponins (GTS) produced membrane depolarization in the current-clamp mode and increased tonic inward pacemaker currents in the voltage-clamp mode. The application of flufenamic acid or niflumic acid abolished the generation of pacemaker currents, but only treatment with flufenamic acid inhibited the GTS-induced tonic inward currents. The tonic inward currents induced by GTS were not inhibited by the intracellular application of guanosine 5'-[beta-thio]diphosphate trilithium salt. Pretreatment with a Ca(2+)-free solution, with U-73122, an active phospholipase C inhibitor, and with thapsigargin, a Ca(2')-ATPase inhibitor of the endoplasmic reticulum, abolished the generation of pacemaker currents and suppressed the GTS-induced action. However, treatment with chelerythrine and calphostin C, protein kinase C inhibitors, did not block the GTS-induced effects on the pacemaker currents. These results suggest that ginsenosides modulate the pacemaker activities of the ICC, and the ICC can be targets for ginsenosides, and their interaction can affect intestinal motility.

Inhibition of gap junction hemichannels by chloride channel blockers.

Electrophysiological methods were used to assess the effect of chloride-channel blockers on the macroscopic and microscopic currents of mouse connexin50 (Cx50) and rat connexin46 (Cx46) hemichannels expressed in Xenopus laevis oocytes. Oocytes were voltage-clamped at -50 mV and hemichannel currents (ICx50 or ICx46) were activated by lowering the extracellular Ca2+ concentration ([Ca2+]o) from 5 mM to 10 microM. Ion-replacement experiments suggested that ICx50 is carried primarily (>95%) by monovalent cations (PK : PNa : PCl = 1.0 : 0.74 : 0.05). ICx50 was inhibited by 18beta-glycyrrhetinic acid (apparent Ki, 2 microM), gadolinium (3 microM), flufenamic acid (3 microM), niflumic acid (11 microM), NPPB (15 microM), diphenyl-2-carboxylate (26 microM), and octanol (177 microM). With the exception of octanol, niflumic acid, and diphenyl-2-carboxylate, the above agents also inhibited ICx46. Anthracene-9-carboxylate, furosemide, DIDS, SITS, IAA-94, and tamoxifen had no inhibitory effect on either ICx50 or ICx46. The kinetics of ICx50 inhibition were not altered at widely different [Ca2+]o (10-500 microM), suggesting that drug-hemichannel interaction does not involve the Ca2+ binding site. In excised membrane patches, application of flufenamic acid or octanol to the extracellular surface of Cx50 hemichannels reduced single channel-open probability without altering the single-channel conductance, but application to the cytoplasmic surface had no effect on the channels. We conclude that some chloride-channel blockers inhibit lens-connexin hemichannels by acting on a site accessible only from the extracellular space, and that drug-hemichannel interaction involves a high-affinity site other than the Ca2+ binding site.

The Induction of Ornithine Decarboxylase in Human Epidermis Is Independent of Lipoxygenase and Cyclo‐oxygenase Pathways

In vivo studies in rodents suggest that prostaglandins and/or leukotrienes are involved in the epidermal induction of ornithine decarboxylase (ODC). Recently, we have shown that, in human epidermis, prostaglandins are not involved in this process. Here we report the role of leukotrienes in epidermal ODC induction in human skin. Topical flufenamic acid (Dignodolin), vehicle, or nothing was applied under plastic occlusion to three sites on the backs of healthy volunteers. This was followed 1 h later by Sellotape stripping. After renewed application and occlusion for 8 h, biopsies were carried out for the estimation of ODC levels. There were no significant differences in the levels of ODC between the flufenamic acid treated and control sites. To confirm this finding, test sites were irradiated with 3 MED of UVB. This was immediately followed by the application of flufenamic acid, vehicle, or nothing to the three irradiated sites. After 8 h, biopsies were taken, and the levels of ODC were again similar in the flufenamic acid- and the vehicle-treated sites. The data indicate that, following Sellotape stripping or UVB irradiation, neither lipoxygenase not cyclooxygenase products contribute to the in vivo induction of ODC in human epidermis.