The purpose of this study was to determine the receptor subtype and the underlying mechanisms involved in the relaxant effect to leptin in mid- and late-pregnant mouse uterus. We determined the relative mRNA expression of receptor subtypes, eNOS, and BKCa channel by quantitative PCR and also the overall receptor expression by immunohistochemistry. Isometric tension studies were conducted to evaluate the effects of leptin and to delineate its mechanisms. A selective siRNA for the ObRb receptor was used to determine the participation of the receptor subtype in biochemical and molecular effects of leptin. The relaxant response to leptin was greater in mid-pregnancy compared to late pregnancy and was mediated by the activation of BKCa channels by eNOS-derived nitric oxide in an ObRb receptor-dependent manner. In comparison to mid-pregnancy, expression of short forms (mainly ObRa receptor) of the receptor was significantly increased in late pregnancy, whereas ObRb receptor expression was similar in both phases. The results of the study suggest that ObRb receptor mediates leptin-induced increase in eNOS expression and NO synthesis. Leptin-induced eNOS expression and activation cause cGMP-independent stimulation of BKCa channels causing uterine relaxation. Increased short forms of the receptors and reduced BKCa channels exert a negative effect on uterine relaxation in late pregnancy. Leptin may have a physiological role in maintaining uterine quiescence in mid-pregnancy and its reduced relaxant response in late gestation may facilitate labor. Further, ObRb receptor agonists may be useful in the management of preterm labor.

Plasma membrane large-conductance calcium-activated potassium (BKCa) channels are important players in various physiological processes, including those mediated by epithelia. Like other cell types, human bronchial epithelial (HBE) cells also express BKCa in the inner mitochondrial membrane (mitoBKCa). The genetic relationships between these mitochondrial and plasma membrane channels and the precise role of mitoBKCa in epithelium physiology are still unclear. Here, we tested the hypothesis that the mitoBKCa channel is encoded by the same gene as the plasma membrane BKCa channel in HBE cells. We also examined the impact of channel loss on the basic function of HBE cells, which is to create a tight barrier. For this purpose, we used CRISPR/Cas9 technology in 16HBE14o- cells to disrupt the KCNMA1 gene, which encodes the α-subunit responsible for forming the pore of the plasma membrane BKCa channel. Electrophysiological experiments demonstrated that the disruption of the KCNMA1 gene resulted in the loss of BKCa-type channels in the plasma membrane and mitochondria. We have also shown that HBE ΔαBKCa cells exhibited a significant decrease in transepithelial electrical resistance which indicates a loss of tightness of the barrier created by these cells. We have also observed a decrease in mitochondrial respiration, which indicates a significant impairment of these organelles. In conclusion, our findings indicate that a single gene encodes both populations of the channel in HBE cells. Furthermore, this channel is critical for maintaining the proper function of epithelial cells as a cellular barrier.

Despite many migraine-specific treatments that became available over the past 5 years, many patients still suffer from debilitating migraine. Emerging and future directions of migraine research and treatment should consider different aspects including revising the headache diagnostic criteria to reflect disease burden and prognosis, developing biomarkers, including genetic, serum, imaging, and deep phenotyping biomarkers to facilitate personalized medicine for headache treatment. Additionally, research should also emphasize identifying novel treatment targets for drug development. In this chapter, we provide an overview of current studies and controversies in the diagnosis of migraine and available research on potential migraine biomarkers. We also discuss potential treatment targets for migraine, including CGRP, PACAP, orexin, non-μ opioid receptors, nitric oxide, BKCa channel, KATP channel, amylin, TRP channels, prolactin, PAR-2, and other potential targets.

Hypothyroidism is associated with elevated levels of serum thyrotropin (TSH), which have been shown to promote abnormal proliferation of vascular smooth muscle cells and contribute to the development of atherosclerosis. However, the specific mechanisms underlying the TSH-induced abnormal proliferation of vascular smooth muscle cells remain unclear. The objective of this study was to investigate the role of TSH in the progression of atherosclerosis. Our research findings revealed that hypothyroidism can trigger early atherosclerotic changes in the aorta of Wistar rats. In alignment with our in vitro experiments, we observed that TSH induces abnormal proliferation of aortic smooth muscle cells by modulating the expression of α and β1 subunits of large conductance Ca2+-activated K+ (BKCa) channels within these cells via the cAMP/PKA signaling pathway. These results collectively indicate that TSH acts through the cAMP/PKA signaling pathway to upregulate the expression of α and β1 subunits of BKCa channels, thereby promoting abnormal proliferation of arterial smooth muscle cells. These findings may provide a basis for the clinical prevention and treatment of atherosclerosis caused by elevated TSH levels.

The effect of lipopolysaccharide (LPS)-based neuroinflammation following cerebral ischemia/reperfusion (I/R) on the genotypic transformation of reactive astrocytes and its relationship with endogenous hydrogen sulfide (H2S) were investigated in present study. We found that LPS promoted the cerebral I/R-induced A1 astrocytes proliferation in mouse hippocampal tissues and deteriorated the reduction of hydrogen sulfide (H2S) content in mouse sera, H2S donor NaHS could inhibit A1 astrocytes proliferation. Similarly, knockout of cystathionine γ-lyase (CSE), one of endogenous H2S synthases, likewise up-regulated the cerebral I/R-induced A1 astrocytes proliferation, which could also be blocked by NaHS. Besides, supplement with H2S promoted the A2 astrocytes proliferation in hippocampal tissues of CSE knockout (CSE KO) mice or LPS-treated mice following cerebral I/R. In the oxygen glucose deprivation/reoxygenation (OGD/R) model of astrocytes, H2S also promoted the transformation of astrocytes into A2 subtype. Moreover, we found that H2S could up-regulate the expression of α-subunit of large-conductance Ca2+-activated K+ (BKCa) channels in astrocytes, and the channel opener BMS-191011 likewise promoted the transformation of astrocyte into A2 subtype. In conclusion, H2S inhibits the proliferation of A1 astrocytes induced by LPS-based neuroinflammation following cerebral I/R and promotes the transformation of astrocytes into A2 subtype, which may be related to up-regulation of BKCa channels.

The large-conductance Ca2+-activated K+ channel (BKCa channel) is involved in repolarizing the membrane potential and has a variety of cellular functions. The BKCa channel is highly expressed in bladder smooth muscle and mediates muscle relaxation. Compounds that activate the BKCa channel have therapeutic potential against pathological symptoms associated with the overactivity of bladder smooth muscle. In this regard, we screened a chemical library of 9938 compounds to identify novel BKCa channel activators. A cell-based fluorescence assay identified a structural family of compounds containing a common tricyclic quinazoline ring that activated the BKCa channel. The most potent compound TTQC-1 (7-bromo-N-(3-methylphenyl)-5-oxo-1-thioxo-4,5-dihydro[1,3]thiazolo[3,4-a]quinazoline-3-carboxamide) directly and reversibly activated the macroscopic current of BKCa channels expressed in Xenopus oocytes from both sides of the cellular membrane. TTQC-1 increased the maximum conductance and shifted the half activation voltage to the left. The apparent half-maximal effective concentration and dissociation constant were 2.8 μM and 7.95 μM, respectively. TTQC-1 delayed the kinetics of channel deactivation without affecting channel activation. The activation effects were observed over a wide range of intracellular Ca2+ concentrations and dependent on the co-expression of β1 and β4 auxiliary subunits, which are highly expressed in urinary bladder. In the isolated smooth muscle cells of rat urinary bladder, TTQC-1 increased the K+ currents which can be blocked by iberiotoxin. Finally, oral administration of TTQC-1 to hypertensive rats decreased the urination frequency. Therefore, TTQC-1 is a BKCa channel activator with a novel structure that is a potential therapeutic candidate for BKCa channel-related diseases, such as overactive bladder syndrome.

In the present study, we report that neolignan1 (Diethyl-4,4\'-dihydroxy-8,3\'-neolign-7,7\'-dien-9,9\'-dionate) relaxes the superior mesenteric artery in a concentration dependent manner (pD2 value 5.392 ± 0.04; n = 8 for endothelium intact and 5.204 ± 0.03; n = 8 for endothelium denuded mesenteric rings, respectively). The relaxation response of neolignan1 was found to be endothelium independent and sensitive to 1H-[1,2,4] oxadiazolo [4,3-a]quinoxalin-1-on (ODQ; 1 μM) and tetraethyl ammonium (TEA; 1 mM). In-silico studies showed good LibDock score (92.66) of neolignan1 with BKCa channel and are in well corroboration with ex-vivo study. Further, neolignan1 significantly decreased the systolic blood pressure, diastolic blood pressure and mean arterial pressure in the Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME; 50 mg/kg) treated Wistar rats at the dose of 30 and 100 mg/kg given once orally for 15 days. In addition, neolignan1 is well tolerated up to 100 mg/kg when given as a repeated dose, once orally for 28 days in Swiss albino mice. Neolignan1 was well absorbed from oral route, reached peak at 4 h and eliminated below detection level by 12 h after administration. Our present study concludes that neolignan1 produced relaxation in superior mesenteric artery by opening of BKCa channel and produced significant antihypertensive activity in L-NAME treated Wistar rats and was well tolerated by the experimental animal.

Excitation of dorsal root ganglion (DRG) neurons by interleukin 1β (IL-1β) is implicated in the onset of neuropathic pain. To understand its mechanism of action, isolectin B4 positive (IB4+) DRG neurons were exposed to 100pM IL-1β for 5-6d. A reversible increase in action potential (AP) amplitude reflected increased TTX-sensitive sodium current (TTX-S INa). An irreversible increase in AP duration reflected decreased Ca2+- sensitive K+ conductance (BK(Ca) channels). Different processes thus underlie regulation of the two channel types. Since changes in AP shape facilitated Ca2+ influx, this explains how IL-1β facilitates synaptic transmission in the dorsal horn; thereby provoking pain.

BACKGROUND: Fetal origin of adult cardiovascular disease is one of the most pressing public concerns and economic problem in modern life. Maternal cigarette smoking/nicotine abuse increases the risk of cardiovascular disease in offspring. However, the underlying mechanisms and theranostics remain unclear. We hypothesized that fetal and neonatal nicotine exposure enhances microRNA-181a (miR-181a) which targets large-conductance Ca2+-activated K+ (BKCa) channels, resulting in increased coronary vascular tone in adult offspring.
METHODS: Nicotine or saline was administered to pregnant rats via subcutaneous osmotic minipumps from gestational day 4 until postnatal day 10. Experiments were conducted in adult (~6 month old) male offspring.
RESULTS: Nicotine enhanced pressure-induced coronary vascular tone, which was abrogated by BKCa channel blocker. Nicotine selectively attenuated coronary BKCa β1 but not α subunit expression. Functionally, nicotine suppressed BKCa current density and inhibited BKCa activator NS1619-induced coronary relaxations. Furthermore, activation of BKCa increased coronary flow and improved heart ischemia/reperfusion-induced infarction. Nicotine selectively enhanced miR-181a expression. MiR-181a mimic inhibited BKCa β1 expression/channel current and decreased NS1619-induced coronary relaxation. Antioxidant eliminated the difference of BKCa current density between the saline and nicotine-treated groups and partially restored NS1619-induced relaxation in nicotine group. MiR-181a antisense decreased vascular tone and eliminated the differences between nicotine exposed and control groups.
CONCLUSIONS: Fetal and neonatal nicotine exposure-mediated miR-181a overexpression plays an important role in nicotine-enhanced coronary vascular tone via epigenetic down-regulation of BKca channel mechanism, which provides a potentially novel therapeutic molecular target of miR-181a/BKca channels for the treatment of coronary heart ischemic disease.

This study investigated the effect of the β2 receptor agonist terbutaline on the single channel activity of BKCa channel. The effects of racemate and two isomers of terbutaline were all assessed. β2 adrenoceptors were stably overexpressed on HEK293 cells by lentiviral transduction method and chicken BKCa channels were transiently expressed on normal HEK293 cell line or HEK293 cells overexpressing β2 receptors. Data showed that terbutaline significantly increased the single channel open probability of BKCa channel within 10min. The channel activating effects of terbutaline are stereoselective and mainly stay with the R-enantiomers. The opening probability of BKCa channel at 10min after drug application normalized to that just before drug application (Po10/Po0s) for R- and S-terbutaline were 7.85±3.20 and 1.06±0.45 respectively at 1μM concentration, corresponding to 28.37±9.96 and 2.68±1.09 at the higher concentration of 10μM. ICI 118551 blocked the effect of R- but not S-terbutaline (10μM), whereas atropine blocked the channel activating effects of S-terbutaline of higher concentration. In addition, the muscarinic receptor agonist carbachol increased the BKCa channel activity in an atropine-sensitive manner as an positive control experiment, which indicate the involvement of M receptor in the channel activating effect of S-terbutaline.