In the present study, it was aimed to further identify the intracellular action mechanism of cromakalim and levcromakaliin in the porcine coronary artery. In intact porcine coronary arterial strips loaded with fura-2/AM, acetylcholine caused an increase in intracellular free Ca2+ ((Ca2+)-i) in association with a contraction in a concentration-dependent manner. Cromakalim (1 micrometer) caused a reduction in acetylcholine-induced increased (Ca2+)-i not only in the normal physiological salt solution (PSS) but also in Ca2+ -free PSS (containing 1mM EGTA). In the skinned strips prepared by exposure of tissue to 20 micrometer beta-escin, inositol 1,4,5-trisphosphate (IP-3) evoked an increase in (Ca2+)-i but it was without effect on the intact strips. The IP-3-induced increase in (Ca2+)-i was inhibited by cromakalim by 78% and levcromakalim by 59% (1 micrometer, each). Pretreatment with glibenclamide (a blocker of ATP-sensitive K+ channels, 10 micrometer and apamin (a blocker of small conductance Ca2+/-activated K+ channels, 1 micrometer strongly blocked the effect of cromakalim and levcromakalim. However, charybdotoxin (a blocker of large conductance Ca2+ -activated K+ channels, 1-micrometer) was without effect. In addition, cromakalim inhibited the GTP-gamma-S (100 micrometer, nonhydrolysable analogue of GTP)-induced increase in (Ca2+)-i. Based on these results, it is suggested that cromakalim and levcromakalim exert a potent vasorelaxation, in part, by acting on the K+ channels of the intracellular sites (e.g., sarcoplasmic reticulum membrane), thereby, resulting in decrease in release of Ca2+ from the intracellular storage site.
Acetylcholine ; Apamin ; Charybdotoxin ; Coronary Vessels* ; Cromakalim* ; Escin ; Glyburide ; Inositol 1,4,5-Trisphosphate ; Sarcoplasmic Reticulum ; Skin ; Vasodilation

Animals ; Escin ; pharmacology ; Intestines ; blood supply ; Lipid Peroxidation ; drug effects ; Male ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; physiopathology ; prevention & control

OBJECTIVETo establish a perforated patch-clamp technology with amphotericin B and beta-escin and to research the regulation of small conductance calcium-activated potassium channel SK2 currents by calcium ions.

METHODSSingle human atrial myocytes were enzymatically isolated from the right atrial appendage. Amphotericin B and / or beta-escin were used by perforated electrode liquid. The regulation of SK2 current by calcium ions in human atrial myocytes was performed with the perforated patch-clamp technique. The intracellular calcium changes were measured by the intracellular calcium test system.

RESULTSMixed perforated electrode liquid compared with 150 microg/ml amphotericin B or 6.88 microg/ml beta-escin alone, it was easy to seal cells and activate SK2 current by the former method. Moreover, the ration of F340/380 was consistent with the change of intracellular free calcium ion concentration increase after the formation of perforation. The ration of F340/380 was measured by intracellular calcium test system.

CONCLUSIONThe appropriate concentration of amphotericin B mixed with beta-escin can form a stable whole-cell patch recording technology that is appropriate for the research of SK2 current regulation by intracellular calcium.

Amphotericin B ; pharmacology ; Calcium ; metabolism ; Electric Conductivity ; Escin ; pharmacology ; Humans ; Myocytes, Cardiac ; physiology ; Patch-Clamp Techniques ; Potassium Channels, Calcium-Activated ; drug effects

OBJECTIVETo investigate the effects of intrathecal escin and clonidine, used alone or in combination, in the treatment of neuropathic pain in rats and the possible mechanism.

METHODSNinety-six male SD rats weighing 250-300 g were chronically implanted with lumbar intrathecal catheters. One week later, the left L5 and L6 spinal nerve roots were ligated to establish the model of spinal nerve ligation neuropathic pain (SNL). The rats were then randomly divided into 16 groups (n=6), including the control (saline), escin, clonidine, escin+clonidine, and the antagonist groups. Mechanical withdrawal threshold was assessed before and at 5, 10, 20, 30, 40, 50 and 60 min after intrathecal administration was evaluated. The maximal possible effect (MPE) was calculated and the effect of the treatments on the neuropathic pain. Isobolographic analysis was performed to characterize any potential interactions between the drugs.

RESULTSMPE was significantly higher in escin group (20, 40 microg), clonidine group (2, 5, 10 microg) and escin+clonidine group [1/4, 1/2 (escin ED(50)+clonidine ED(50))] than in the saline group (P<0.05). Intrathecal escin (5-40 microg) or clonidine (1-10 microg) alone dose-dependently alleviated neuropathic pain. Isobolographic analysis suggested a synergistic effect between escin and clonidine. Intrathecal pretreatment with yohimbine (20 microg) antagonized the effects of clonidine (P<0.01) and attenuated the action of the combination treatment with escin and clonidine (P<0.05).

CONCLUSIONIntrathecal escin and clonidine alone can dose-dependently relieve neuropathic pain. Escin and clonidine produce a synergistic effect for pain relief, which may involve the actions of alpha(2) receptor and Ca(2+) channel.

Analgesics ; administration & dosage ; Animals ; Clonidine ; administration & dosage ; Drug Synergism ; Escin ; administration & dosage ; Injections, Spinal ; Male ; Pain ; drug therapy ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Spinal Nerve Roots ; injuries

To elucidate the Ca2+ release mechanisms in the rabbit coronary artery, arterial preparations were permeabilized with beta-escin and changes in tension were measured under varying experimental conditions. Additionally, we investigated properties and distribution of two kinds of Ca2+ release mechanisms, Ca2+-induced Ca2+ release (CICR) and IP3-induced Ca2+ release (IICR). The results obtained were summarized as follows; 1. When a rabbit coronary artery was incubated in a relaxing solution containing 30 microM beta-escin for 40 min. sensitivity to externally added Ca2+ was much higher in beta-escin permeabilized muscle than in intact preparations. The contractile effect of IP3 in beta-escin permeabilized muscle was also demonstrated; 2. Caffeine and IP3 contracted coronary arteries were permeabilized with beta-escin, but the amplitude of contraction was much larger in the presence of caffeine than of IP3. 3. Intracellular heparin completely inhibited the contractions induced by IP3, but not those by caffeine. On the other hand, procaine inhibited the responses to caffeine, but not those to IP3. Ryanodine inhibited both the caffeine- and IP3-induced contractions. 4. The amplitude of contractile responses was much larger to the maximal stimulation of CICR by applying caffeine than to the maximal stimulation of IICR by applying IP3. After the maximal CICR stimulation by caffeine, the activation of IICR by IP3 without the reloading of Ca2+ could no longer evoke contraction. On the other hand, after the maximal IICR activation, the activation of CICR could still evoke contraction although the amplitude of the contraction was smaller when compared with the case without the initial IICR stimulation. 5. Acetylcholine contracted coronary artery smooth muscles were permeabilized with beta-escin. However, in the absence of added guanosine triphosphate (GTP), the responses were very small. Acetylcholine-induced contraction was inhibited by heparin, but not by procaine. From the above results, it may be concluded that there are two kinds of mechanisms of Ca2+ release, CICR and IICR, in the rabbit coronary artery smooth muscle cell. Also, whereas the CICR mechanism distributes on the membrane of the whole smooth muscle Ca2+ store, the IICR mechanism distributes only on a part of it.
Animal ; Arteries/metabolism ; Calcium/*metabolism ; Capillary Permeability/drug effects ; Coronary Vessels/drug effects/*metabolism ; Escin/pharmacology ; In Vitro ; Intracellular Membranes/*metabolism ; Rabbits ; Support, Non-U.S. Gov't ; Tissue Distribution

OBJECTIVE: To investigate the effect of escin in relieving chemotherapy-induced peripheral neuropathic pain in rats and explore and the underlying mechanism. METHODS: Eighteen SD rats were randomly divided into 3 groups ( RESULTS: The rats in both the escin preconditioning group and escin postconditioning group showed obviously increased thresholds of mechanical allodynia and thermal hyperalgesia as compared with those in the control group ( CONCLUSIONS Escin can alleviate chemotherapy-induced peripheral neuropathic pain in rats possibly by upregulating the expressions of autophagy-related proteins in the spinal cord.
Animals ; Antineoplastic Agents/therapeutic use* ; Autophagy ; Escin/therapeutic use* ; Hyperalgesia/drug therapy* ; Mice ; Neuralgia/drug therapy* ; Rats ; Rats, Sprague-Dawley ; Spinal Cord

It was hypothesized that NaF induces calcium sensitization in Ca2+-controlled solution in permeabilized rat mesenteric arteries. Rat mesenteric arteries were permeabilized with beta-escin and subjected to tension measurement. NaF potentiated the concentration-response curves to Ca2+ (decreased EC50 and increased E(max)). Cumulative addition of NaF (4.0, 8.0 and 16 mM) also increased vascular tension in Ca2+-controlled solution at pCa 7.0 or pCa 6.5, but not at pCa 8.0. NaF-induced vasocontraction and GTPgammaS-induced vasocontraction were not additive. NaF-induced vasocontraction at pCa 7.0 was inhibited by pretreatment with Rho kinase inhibitors H1152 or Y27632 but not with a MLCK inhibitor ML-7 or a PKC inhibitor Ro31-8220. NaF induces calcium sensitization in a Ca2+-dependent manner in beta-escin-permeabilized rat mesenteric arteries. These results suggest that NaF is an activator of the Rho kinase signaling pathway during vascular contraction.
Amides ; Animals ; Azepines ; Calcium ; Contracts ; Escin ; Indoles ; Mesenteric Arteries ; Naphthalenes ; Passive Cutaneous Anaphylaxis ; Pyridines ; Rats ; rho-Associated Kinases ; Sodium ; Sodium Fluoride

Many gastrointestinal muscles show electrical oscillation, so-called "slow wave", originated from interstitial cells of Cajal (ICCs). Thus, a technique to freshly isolate the cells is indispensable to explore the electrophysiological properties of the ICCs. To apply an enzyme solution on the serosal surface for cell isolation, the intestine was inverted and 0.02% trypsin solution and 0.04% collagenase solution were applied to serosal cavity. After the enzyme treatment, mucosal layer was removed and longitudinal muscle layer was gently separated from the rest of tissue. The thin layer was stretched in the recording chamber and mounted on an inverted microscope. Using beta-escine, perforated whole cell patch clamp technique was used. Under a microscope, the tissue showed smooth muscle cells and interstitial cells around the myenteric plexus. Under voltage clamp condition, three types of membrane potential were recorded. One group of interstitial cells, which were positive to methylene blue and CD34, showed spontaneous outward current. These cells had bipolar shape and were considered as fibroblast-like cells because of their peculiar shape and arrangement. Another group, positive to c-kit and methylene blue, showed spontaneous inward current. These cells had more rounded shape and processes and were considered as ICCs. The third, positive to c-kit and had granules containing methylene blue, showed quiet membrane potentials under the voltage-clamp mode. These cells appeared to be resident macrophages. Therefore, in the freshly isolated thin tissue preparation, methylene blue could easily identify three types of cells rather than morphological properties. Using this method, we were able to study electrical properties of fibroblast and residential macrophage as well as myenteric ICCs.
Cell Separation ; Collagenases ; Escin ; Fibroblasts ; Interstitial Cells of Cajal ; Intestine, Small* ; Intestines ; Macrophages ; Membrane Potentials ; Methylene Blue ; Muscles ; Myenteric Plexus ; Myocytes, Smooth Muscle ; Trypsin

AIMTo investigate the protective effects of sodium beta-aescin on cerebral ischemia-reperfusion injury in rats.

METHODSRats were pretreated with sodium beta-aesein for 7 d and then subjected to cerebral ischemia-reperfusion injury induced by a middle cerebral artery occlusion (MCAO). The neurological outcome was evaluated by the Longa's method; The infarct volume was assessed by hemmatoxylin-Eosin staining and the cerebral water content was measured by dry weight method. The activities of SOD, GSH-Px, CAT, Na+ -K+ -ATPase and the MDA content were measured in the cortex and hippocampus of ischemic and non-ischemic hemisphere.

RESULTSSodium beta-aescin significantly reduced the volume of cerebral infarct and water content, and ameliorated the neurological deficit (P < 0.05). In vehicle-treated rats, the activities of SOD, GSH-Px and Na+ -K+ -ATPase in the cortex and hippocampus of ischemic hemisphere were all decreased (P < 0.01) , while the CAT activity was slightly elevated and the MDA of content was significantly increased (P < 0.01) compared with the sham-operated group. After treated with sodium beta-aescin, the effects on recovery of SOD, GSH-Px, Na+ -K+ -ATPase activities were observed (P < 0.05), and the MDA content was reduced (P < 0.05).

CONCLUSIONThese results showed that pretreatment with sodium beta-aescin can attenuate brain injury and its antioxidant activity on rats which encountered cerebral ischemia-reperfusion.

Animals ; Brain ; metabolism ; Brain Ischemia ; metabolism ; pathology ; Catalase ; metabolism ; Escin ; pharmacology ; Glutathione Peroxidase ; metabolism ; Male ; Malondialdehyde ; metabolism ; Neuroprotective Agents ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; metabolism ; pathology ; Sodium-Potassium-Exchanging ATPase ; metabolism ; Superoxide Dismutase ; metabolism

BACKGROUND: Extracellular and intracellular pH (pHo and pHi), which can be changed in various pathological conditions such as hypoxia, affects vascular contractility. To elucidate the mechanism to alter vascular contractility by pH, the effects of pH on reactivity to vasocontracting agents, intracellular Ca2+ influx, and Ca2+ sensitivity in vascular smooth muscle were examined. MATERIAL AND METHOD: Isometric contractions in rat superior mesenteric arteries (SMA) were observed. Intracellular Ca2+ concentration ([Ca2+]i) was recorded by microfluorometer using Fura-2/acetoxylmethyl ester in muscle cells. pHo was increased from 7.4 to 7.8 or decreased to 6.9 or 6.4. pHi was decreased by applying NH4+ or propionic acid or modulated by changing pHo after increasing membrane permeability using beta-escin. RESULT: Decreases in pHo from 7.4 to 6.9 or 6.4 shifted concentration-response curve by norepinephrine (NE) or serotonin (SE) to the right and significantly increased half maximal effective concentration (EC50) to NE or SE. Increase in pHo from 7.4 to 7.8 shifted concentration-response curve by norepinephrine (NE) or serotonin (SE) to the left and significantly reduced EC50 to NE or SE. NE increased [Ca2+]i in cultured smooth muscle cells from SMA and the increased [Ca2+]i was reduced by decreases in pHo. NE-induced contraction was inhibited by NH4+, whereas the resting tension was increased by NH4+ or propionic acid. When the cell membrane of SMA was permeabilized using beta-escin, SMA was contracted by increasing extracellular Ca2+ concentration from 0 to 10micrometer and the magnitude of contraction was decreased by a decrease in pHo and vice versa. CONCLUSION: From these results, it can be concluded that a decrease in pHo might inhibit vascular contraction by reducing the reactivity of vascular smooth muscle to vasoactive agents, Ca2+ influx and the sensitivity of vascular smooth muscle to Ca2+.
Animals ; Anoxia ; Calcium ; Cell Membrane ; Contracts ; Diethylpropion ; Escin ; Hydrogen-Ion Concentration ; Isometric Contraction ; Membranes ; Mesenteric Artery, Superior ; Muscle Cells ; Muscle, Smooth, Vascular ; Myocytes, Smooth Muscle ; Norepinephrine ; Permeability ; Propionates ; Rats ; Serotonin