Regulation by CAPE, an NF-kB inhibitor, and MBCD, a Cholesterol Scavenger of Large-Conductance Ca2+-Activated K+ Channels in Pituitary Tumor (GH3) Cells

• Main Authors: Ming-wei Lin, 林明瑋
• Other Authors: Sheng-Nan Wu
• Format: Others
• Language: en_US
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/24984297359122581927

博士 === 國立成功大學 === 基礎醫學研究所 === 97 === Large-conductance Ca2+-activated K+ (BKCa) channels differ from most of other K+ channels in that their activation is under dual control, i.e., activated by either an increase in intracellular Ca2+ or by membrane depolarization. These channels, which are widely distributed in a variety of cells, can control Ca2+ influx as well as a number of Ca2+-dependent physiological processes. In neurons or neuroendocrine cells, BKCa channels are believed to play an important role in controlling hormonal secretion by altering the duration and frequency of action potentials. Experimental observations have revealed that a variety of components can directly modulate BKCa channel activity. Caffeic acid phenethyl ester (CAPE), a phenolic antioxidant derived from the propolis of honeybee hives, is known to be an inhibitor of the activation of nuclear transcript factor NF-κB. The molecule of this compound has the juxtaposition of two aromatic rings, the unique structure of which is similar to those of some BKCa channel openers, such as nordihydroguaiaretic acid and resveratrol. Since functional expression of BKCa channel has been demonstrated in pituitary GH3 cells, the effects of CAPE on ion currents have been investigated in these cells. In our study, this compound increased Ca2+-activated K+ current (IK(Ca)) in a concentration-dependent manner with an EC50 value of 14 ± 2 uM. However, the magnitude of CAPE-induced stimulation of IK(Ca) was attenuated in GH3 cells preincubated with the oxidants, 2,2’-azo-bis-(2-amidinopropane) hydrochloride (100 uM) or t-butyl hydroperoxide (1 mM). CAPE (50 uM) slightly suppressed voltage-dependent L-type Ca2+ current. In inside-out configuration, CAPE (20 uM) applied to the intracellular face of the detached patch enhanced the activity of BKCa channels with no modification in single-channel conductance. After BKCa channel activity was increased by CAPE (20 uM), subsequent application of nordihydroguaiaretic acid (20 uM) did not increase the channel activity further. CAPE-stimulated BKCa channel activity was dependent on membrane potential. Results suggested that CAPE could also increase Ca2+ sensitivity of BKCa channels in these cells. Additionally, its increase in the open probability could primarily involve a decrease in the mean closed time. In current-clamp conditions, CAPE hyperpolarized the membrane potential and reduced the firing of action potentials. Moreover, CAPE also attenuated the spontaneous [Ca2+]i oscillations in GH3 cells. Therefore, the stimulatory action of CAPE on BKCa-channel activity should be noted with caution in relation to its increasing use as an inhibitor of activation of the NF-kB. Lipid rafts are specialized cholesterol-enriched microdomains of the cellular membrane. They participate actively in signal transduction and cellular adaptation to changing environments. Several reports have provided evidence that certain ion channels physically associate with the lipid rafts. Therefore, changes in the amount of membrane cholesterol may directly modify the activity of ion channels. In this study, depletion of membrane cholesterol by pituitary GH3 cells to methyl-β-cyclodextrin (MβCD), an oligosaccharide, resulted in disruption of lipid raft and an increase in the density of IK(Ca). However, no significant change in IK(Ca) density was demonstrated in GH3 cells treated with a mixture of MβCD and cholesterol. Cholesterol depletion with MβCD (1.5 mg/ml) slightly suppressed the density of voltage-dependent L-type Ca2+ current. In inside-out patches recorded from MβCD-treated cells, the activity of BKCa channels was enhanced with no change in single-channel conductance. In MβCD-treated cells, voltage-sensitivity of BKCa channels was increased; however, no change in Ca2+-sensitivity could be demonstrated. In inside-out patches from MβCD-treated cells, dexamethasone applied to the intracellular surface did not increase BKCa-channel activity, although CAPE and cilostazol still opened its probability effectively. However, in MβCD-treated cells, the protein expression of BKCa channels remained unchanged. Current-clamp recordings demonstrated that the cholesterol depletion maneuver with MβCD reduced the firing of action potentials. Therefore, it can be concluded that the increase in BKCa-channel activity induced by membrane lipid raft disruption may, to some extent, influence the functional activities of neurons or neuroendocrine cells. The increased activity of BKCa channels may contribute to the underlying mechanisms by which it alters neuronal or neuroendocrine excitability. Therefore, with the increased understanding of the regulation of BKCa channel will open new therapeutic perspectives in various states of abnormal neuron activity.