The augmentation of BK channel activity by nitric oxide signaling in rat cerebral arteries involves co-localized regulatory elements.

Large conductance, Ca2+-activated K+ (BK) channels control cerebrovascular tone; however, the regulatory processes influencing these channels remain poorly understood. Here, we investigate the cellular mechanisms underlying the enhancement of BK current in rat cerebral arteries by nitric oxide (NO) signaling. In isolated cerebral myocytes, BK current magnitude was reversibly increased by sodium nitroprusside (SNP, 100 µM) and sensitive to the BK channel inhibitor, penitrem-A (100 nM). Fostriecin (30 nM), a protein phosphatase type 2A (PP2A) inhibitor, significantly prolonged the SNP-induced augmentation of BK current and a similar effect was produced by sildenafil (30 nM), a phosphodiesterase 5 (PDE5) inhibitor. Using proximity ligation assay (PLA)-based co-immunostaining, BK channels were observed to co-localize with PP2A, PDE5, and cGMP-dependent protein kinase (cGKI) (spatial restriction < 40 nm); cGKI co-localization increased following SNP exposure. SNP (10 µM) reversibly inhibited myogenic tone in cannulated cerebral arteries, which was augmented by either fostriecin or sildenafil and inhibited by penitrem-A. Collectively, these data suggest that (1) cGKI, PDE5, and PP2A are compartmentalized with cerebrovascular BK channels and determine the extent of BK current augmentation by NO/cGMP signaling, and (2) the dynamic regulation of BK activity by co-localized signaling enzymes modulates NO-evoked dilation of cerebral resistance arteries.

Caveolin-1 limits the contribution of BKCa channel to MCF-7 breast cancer cell proliferation and invasion.

Increasing evidence suggests that caveolin-1 and large conductance Ca²âº-activated potassium (BKCa) channels are implicated in the carcinogenesis processes, including cell proliferation and invasion. These two proteins have been proven to interact with each other in vascular endothelial and smooth muscle cells and modulate vascular contractility. In this study, we investigated the probable interaction between caveolin-1 and BKCa in MCF-7 breast cancer cells. We identified that caveolin-1 and BKCa were co-localized and could be reciprocally co-immunoprecipitated in human breast cancer MCF-7 cells. siRNA mediated caveolin-1 knockdown resulted in activation and increased surface expression of BKCa channel, and subsequently promoted the proliferation and invasiveness of breast cancer cells. These effects were attenuated in the presence of BKCa-siRNA. Conversely, up-regulated caveolin-1 suppressed function and surface expression of BKCa channel and exerted negative effects on breast cancer cell proliferation and invasion. Similarly, these opposing effects were abrogated by BKCa up-regulation. Collectively, our findings suggest that BKCa is a critical target for suppression by caveolin-1 in suppressing proliferation and invasion of breast cancer cells. The functional complex of caveolin-1 and BKCa in the membrane microdomain may be served as a potential therapeutic target in breast cancer.

Effects of pulpectomy on the amount of root resorption during orthodontic tooth movement.

INTRODUCTION: Previous studies have revealed that orthodontic force affects dental pulp via the rupture of blood vessels and vacuolization of pulp tissues. We hypothesized that pulp tissues express inflammatory cytokines and regulators of odontoclast differentiation after excess orthodontic force. The purpose of this study was to investigate the effects of tensile force in human pulp cells and to measure inflammatory root resorption during tooth movement in pulpless rat teeth. METHODS: After cyclic tensile force application in human pulp cells, gene expression and protein concentration of macrophage colony-stimulating factor, receptor activator of nuclear factor kappa-B ligand, interleukin-1 beta, and tumor necrosis factor alpha were determined by real-time polymerase chain reaction and enzyme-linked immunoassay. Moreover, the role of the stretch-activated channel was evaluated by gadolinium (Gd(3+)) treatment. The upper right first molars of 7-week Wistar rats were subjected to pulpectomy and root canal filling followed by mesial movement for 6 months. RESULTS: The expression of cytokine messenger RNAs and proteins in the experimental group peaked with loading at 10-kPa tensile force after 48 hours (P < .01). Gd(3+) reduced the expression of these cytokine messenger RNAs and protein concentrations (P < .01). The amount of inflammatory root resorption was significantly larger in the control teeth than the pulpectomized teeth (P < .05). CONCLUSIONS: This study shows that tensile forces in the pulp cells enhance the expression of various cytokines via the S-A channel, which may lead to inflammatory root resorption during tooth movement. It also suggests that root canal treatment is effective for progressive severe inflammatory root resorption during tooth movement.

Positions of ß2 and ß3 subunits in the large-conductance calcium- and voltage-activated BK potassium channel.

Large-conductance voltage- and Ca(2+)-gated K(+) channels are negative-feedback regulators of excitability in many cell types. They are complexes of α subunits and of one of four types of modulatory ß subunits. These have intracellular N- and C-terminal tails and two transmembrane (TM) helices, TM1 and TM2, connected by an ∼100-residue extracellular loop. Based on endogenous disulfide formation between engineered cysteines (Cys), we found that in ß2 and ß3, as in ß1 and ß4, TM1 is closest to αS1 and αS2 and TM2 is closest to αS0. Mouse ß3 (mß3) has seven Cys in its loop, one of which is free, and this Cys readily forms disulfides with Cys substituted in the extracellular flanks of each of αS0-αS6. We identified by elimination mß3-loop Cys152 as the only free Cys. We inferred the disulfide-bonding pattern of the other six Cys. Using directed proteolysis and fragment sizing, we determined this pattern first among the four loop Cys in ß1. These are conserved in ß2-ß4, which have four additional Cys (eight in total), except that mß3 has one fewer. In ß1, disulfides form between Cys at aligned positions 1 and 8 and between Cys at aligned positions 5 and 6. In mß3, the free Cys is at position 7; position 2 lacks a Cys present in all other ß2-ß4; and the disulfide pattern is 1-8, 3-4, and 5-6. Presumably, Cys 2 cross-links to Cys 7 in all other ß2-ß4. Cross-linking of mß3 Cys152 to Cys substituted in the flanks of αS0-S5 attenuated the protection against iberiotoxin (IbTX); cross-linking of Cys152 to K296C in the αS6 flank and close to the pore enhanced protection against IbTX. In no case was N-type inactivation by the N-terminal tail of mß3 perturbed. Although the mß3 loop can move, its position with Cys152 near αK296, in which it blocks IbTX binding, is likely favored.

Functional and molecular characterization of maxi K+ -channels (BK(Ca)) in buffalo myometrium.

Large conductance potassium channels (BK(Ca) channels) play a central role in maintaining myometrial tone, thus activation of these channels proved to have therapeutic potential in preterm labor. Present study aims to unravel the presence of BK(Ca) (maxi-K) channels in buffalo myometrium. Tension experiments, mRNA and protein expression studies were done to characterize BK(Ca) channels in buffalo myometrium. Isolated myometrial preparations exhibited rhythmic spontaneity with regular pattern of amplitude and frequency. Selective blockers of BK(Ca) channels iberiotoxin (IbTx; 100nM) and tetraethylammonium (TEA; 1mM) produced excitatory effects as evidenced by increase in amplitude and frequency of myogenic activity. 1,3-Dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimi-dazol-2-one (NS-1619; 10(-7)-10(-4)M), a BK(Ca) channel opener, produced concentration-dependent relaxation of myometrium with pD(2) of 5.02±0.19 and R(max) of 31.35±3.5% (n=5). TEA significantly antagonized NS-1619-induced relaxation (pD(2) of 4.72±0.12 and R(max) of 22.72±1.78%; n=5). IbTx also significantly shifted the dose response curve of NS-1619 towards right (pD(2) of 3.98±0.16; n=4) without significant change in the per cent maximal response. Further, RT-PCR study detected mRNA encoding BK(Ca) α-subunit and Western blot analysis detected its protein expression in myometrium. Based on the results of the present investigation, it is suggested that BK(Ca) channels are present in the buffalo myometrium and are open in the resting state. Thus, their activation by potassium channel opener/ß(2)-adrenoceptor agonist (tocolytic drug) may lead to uterine relaxation in preterm labor.

Large-conductance calcium-activated potassium channels modulate vascular tone in experimental cirrhosis.

BACKGROUND: Large-conductance calcium-activated potassium (BK(Ca)) channels regulate vascular tone in different vascular systems. Moreover, activated hepatic stellate cells (HSC) contain BK(Ca) channels. The aim of this study was to evaluate the role of BK(Ca) channels in the regulation of vascular tone in control (CT) and carbon tetrachloride-cirrhotic (CH) rat livers. METHODS: Changes in intrahepatic vascular resistance were assessed by evaluating the portal perfusion pressure (PP) response to methoxamine (Mtx) in the presence of Iberiotoxin (Ibtx; a BK(Ca) channel blocker), NS1619 (a BK(Ca) channel opener), Ibtx plus the nitric oxide (NO) synthase inhibitor, N(G)-nitro-L-arginine (L-NNA) or L-NNA alone. In addition, in CH livers, PP dose-response curves to the NO donor, S-nitroso-N-acetyl-D,L-penicillamine (SNAP), were performed after pre-incubation with Ibtx or its vehicle. BK(Ca) mRNA expression was assessed in liver homogenates, and BK(Ca) protein expression in HSC isolated from CT and CH livers. RESULTS: In CH livers, Ibtx significantly increased baseline PP and exacerbated the PP response to Mtx. Conversely, NS1619 induced a mild nonsignificant decrease of baseline PP and attenuated the hyperresponse to Mtx. CH livers exhibited an upregulation of both mRNA and protein of the alpha-subunit of BK(Ca). CONCLUSION: Large-conductance calcium-activated potassium channels are overexpressed in CH livers and might represent a compensatory mechanism modulating the increased hepatic vascular tone of cirrhosis.

A Na+- and Cl- -activated K+ channel in the thick ascending limb of mouse kidney.

This study investigates the presence and properties of Na+-activated K+ (K(Na)) channels in epithelial renal cells. Using real-time PCR on mouse microdissected nephron segments, we show that Slo2.2 mRNA, which encodes for the K(Na) channels of excitable cells, is expressed in the medullary and cortical thick ascending limbs of Henle's loop, but not in the other parts of the nephron. Patch-clamp analysis revealed the presence of a high conductance K+ channel in the basolateral membrane of both the medullary and cortical thick ascending limbs. This channel was highly K+ selective (P(K)/P(Na) approximately 20), its conductance ranged from 140 to 180 pS with subconductance levels, and its current/voltage relationship displayed intermediate, Na+-dependent, inward rectification. Internal Na+ and Cl- activated the channel with 50% effective concentrations (EC50) and Hill coefficients (nH) of 30 +/- 1 mM and 3.9 +/- 0.5 for internal Na+, and 35 +/- 10 mM and 1.3 +/- 0.25 for internal Cl-. Channel activity was unaltered by internal ATP (2 mM) and by internal pH, but clearly decreased when internal free Ca2+ concentration increased. This is the first demonstration of the presence in the epithelial cell membrane of a functional, Na+-activated, large-conductance K+ channel that closely resembles native K(Na) channels of excitable cells. This Slo2.2 type, Na+- and Cl--activated K+ channel is primarily located in the thick ascending limb, a major renal site of transcellular NaCl reabsorption.

Membrane-delimited inhibition of maxi-K channel activity by the intermediate conductance Ca2+-activated K channel.

The complexity of mammalian physiology requires a diverse array of ion channel proteins. This diversity extends even to a single family of channels. For example, the family of Ca2+-activated K channels contains three structural subfamilies characterized by small, intermediate, and large single channel conductances. Many cells and tissues, including neurons, vascular smooth muscle, endothelial cells, macrophages, and salivary glands express more than a single class of these channels, raising questions about their specific physiological roles. We demonstrate here a novel interaction between two types of Ca2+-activated K channels: maxi-K channels, encoded by the KCa1.1 gene, and IK1 channels (KCa3.1). In both native parotid acinar cells and in a heterologous expression system, activation of IK1 channels inhibits maxi-K activity. This interaction was independent of the mode of activation of the IK1 channels: direct application of Ca2+, muscarinic receptor stimulation, or by direct chemical activation of the IK1 channels. The IK1-induced inhibition of maxi-K activity occurred in small, cell-free membrane patches and was due to a reduction in the maxi-K channel open probability and not to a change in the single channel current level. These data suggest that IK1 channels inhibit maxi-K channel activity via a direct, membrane-delimited interaction between the channel proteins. A quantitative analysis indicates that each maxi-K channel may be surrounded by four IK1 channels and will be inhibited if any one of these IK1 channels opens. This novel, regulated inhibition of maxi-K channels by activation of IK1 adds to the complexity of the properties of these Ca2+-activated K channels and likely contributes to the diversity of their functional roles.

Ca2+ -activated K+ channels of the BK-type in the mouse brain.

An antibody against the 442 carboxy-terminal amino acids of the BK channel alpha-subunit detects high immunoreactivity within the telencephalon in cerebral cortices, olfactory bulb, basal ganglia and hippocampus, while lower levels are found in basal forebrain regions and amygdala. Within the diencephalon, high density was found in nuclei of the ventral and dorsal thalamus and the medial habenular nucleus, and low density in the hypothalamus. The fasciculus retroflexus and its termination in the mesencephalic interpeduncular nucleus are prominently stained. Other mesencephalic expression sites are periaquaeductal gray and raphe nuclei. In the rhombencephalon, BK channels are enriched in the cerebellar cortex and in the locus coeruleus. Strong immunoreactivity is also contained in the vestibular nuclei, but not in cranial nerves and their intramedullary course of their roots. On the cellular level, BK channels show pre- and postsynaptic localizations, i.e., in somata, dendrites, axons and synaptic terminals.