Large conductance Ca2+-activated K+ channel (BKCa) activating properties of a series of novel N-arylbenzamides: Channel subunit dependent effects

Large conductance calcium activated potassium channels (BK_Ca) are fundamental in the control of cellular excitability. Thus, compounds that activate BK_Ca channels could provide potential therapies in the treatment of pathologies of the cardiovascular and central nervous system. A series of novel N-arylbenzamide compounds, and the reference compound NS1619, were evaluated for BK_Ca channel opener properties in Human Embryonic Kidney (HEK293) cells expressing the human BK_Ca channel α-subunit alone or α + β1-subunit complex.Channel activity was determined using a non-radioactive Rb⁺ efflux assay to construct concentration effect curves for each compound. All N-arylbenzamide compounds and NS1619 evoked significant (p <0.05) concentration related increases in Rb⁺ efflux both in cells expressing α-subunit alone or α + β1-subunits. Co-expression of the β1-subunit modified the Rb⁺ efflux responses, relative to that obtained in cells expressing the α-subunit alone, for most of the N-arylbenzamide compounds, in contrast to NS1619. The EC₄₀ values of NS1619, BKMe1 and BKOEt1 were not significantly affected by the co-expression of the BK_Ca channel α + β1-subunits. In contrast, 5 other N-arylbenzamides (BKPr2, BKPr3, BKPr4, BKH1 and BKVV) showed a significant (p <0.05) 2- to 10-fold increase in EC₄₀ values when tested on the BK_Ca α + β1-subunit expressing cells compared to BK_Ca α-subunit expressing cells. Further, the E_max values for BKPr4, BKVV and BKH1 were lower in the BK_Ca channel α + β1-subunit expressing cells.In conclusion, the N-arylbenzamides studied, like NS1619, were able to activate BK_Ca channels formed of the α-subunit only. The co-expression of the β1-subunit, however, modified the ability of certain compounds to active the channel leading to differentiated pharmacodynamic profiles.     

Protection against cardiac injury by small Ca2 +-sensitive K+ channels identified in guinea pig cardiac inner mitochondrial membrane

We tested if small conductance, Ca^2 +‐sensitive K⁺ channels (SK_Ca) precondition hearts against ischemia reperfusion (IR) injury by improving mitochondrial (m) bioenergetics, if O₂‐derived free radicals are required to initiate protection via SK_Ca channels, and, importantly, if SK_Ca channels are present in cardiac cell inner mitochondrial membrane (IMM). NADH and FAD, superoxide (O₂^?), and m[Ca^2 +] were measured in guinea pig isolated hearts by fluorescence spectrophotometry. SK_Ca and IK_Ca channel opener DCEBIO (DCEB) was given for 10 min and ended 20 min before IR. Either TBAP, a dismutator of O₂^?, NS8593, an antagonist of SK_Ca isoforms, or other K_Ca and K_ATP channel antagonists, were given before DCEB and before ischemia. DCEB treatment resulted in a 2-fold increase in LV pressure on reperfusion and a 2.5 fold decrease in infarct size vs. non-treated hearts associated with reduced O₂^? and m[Ca^2 +], and more normalized NADH and FAD during IR. Only NS8593 and TBAP antagonized protection by DCEB. Localization of SK_Ca channels to mitochondria and IMM was evidenced by a) identification of purified mSK_Ca protein by Western blotting, immuno-histochemical staining, confocal microscopy, and immuno-gold electron microscopy, b) 2-D gel electrophoresis and mass spectroscopy of IMM protein, c) [Ca^2 +]‐dependence of mSK_Ca channels in planar lipid bilayers, and d) matrix K⁺ influx induced by DCEB and blocked by SK_Ca antagonist UCL1684. This study shows that 1) SK_Ca channels are located and functional in IMM, 2) mSK_Ca channel opening by DCEB leads to protection that is O₂^? dependent, and 3) protection by DCEB is evident beginning during ischemia.     

Flow-induced activation of TRPV5 and TRPV6 channels stimulates Ca2 +-activated K+ channel causing membrane hyperpolarization

TRPV5 and TRPV6 channels are expressed in distal renal tubules and play important roles in the transcellular Ca^2 + reabsorption in kidney. They are regulated by multiple intracellular factors including protein kinases A and C, membrane phospholipid PIP₂, protons, and divalent ions Ca^2 + and Mg^2 +. Here, we report that fluid flow that generates shear force within the physiological range of distal tubular fluid flow activated TRPV5 and TRPV6 channels expressed in HEK cells. Flow-induced activation of channel activity was reversible and did not desensitize over 2 min. Fluid flow stimulated TRPV5 and 6-mediated Ca^2 + entry and increased intracellular Ca^2 + concentration. N-glycosylation-deficient TRPV5 channel was relatively insensitive to fluid flow. In cells coexpressing TRPV5 (or TRPV6) and Slo1-encoded maxi-K channels, fluid flow induced membrane hyperpolarization, which could be prevented by the maxi-K blocker iberiotoxin or TRPV5 and 6 blocker La^3 +. In contrast, fluid flow did not cause membrane hyperpolarization in cells coexpressing ROMK1 and TRPV5 or 6 channel. These results reveal a new mechanism for the regulation of TRPV5 and TRPV6 channels. Activation of TRPV5 and TRPV6 by fluid flow may play a role in the regulation of flow-stimulated K⁺ secretion via maxi-K channels in distal renal tubules and in the mechanism of pathogenesis of thiazide-induced hypocalciuria.     

Ca2 + channels and praziquantel: A view from the free world

Targeting the cellular Ca^2 + channels and pumps that underpin parasite Ca^2 + homeostasis may realize novel antihelmintic agents. Indeed, the antischistosomal drug praziquantel (PZQ) is a key clinical agent that has been proposed to work in this manner. Heterologous expression data has implicated an action of PZQ on voltage-operated Ca^2 + channels, although the relevant in vivo target of this drug has remained undefined over three decades of clinical use. The purpose of this review is to bring new perspective to this issue by discussing the potential utility of free-living planarian flatworms for providing new insight into the mechanism of PZQ action. First, we discuss in vivo functional genetic data from the planarian system that broadly supports the molecular data collected in heterologous systems and the ‘Ca^2 + hypothesis’ of PZQ action. On the basis of these similarities we highlight our current knowledge of platyhelminth voltage operated Ca^2 + channels, their unique molecular pharmacology and the downstream functional PZQ interactome engaged by dysregulation of Ca^2 + influx that has potential to yield novel antischistosomal targets. Overall the broad dataset underscores a common theme of PZQ-evoked disruptions of Ca^2 + homeostasis in trematodes, cestodes and turbellarians, and showcases the utility of the planarian model for deriving insight into drug action and targets in parasitic flatworms.     

KMUP-1 ameliorates monocrotaline-induced pulmonary arterial hypertension through the modulation of Ca2+ sensitization and K+-channel

AimsThis study investigates the actions of KMUP-1 on RhoA/Rho-kinase (ROCK)-dependent Ca²⁺ sensitization and the K⁺-channel in chronic pulmonary arterial hypertension (PAH) rats.Main methodsSprague–Dawley rats were divided into control, monocrotaline (MCT), and MCT + KMUP-1 groups. PAH was induced by a single intraperitoneal injection (i.p.) of MCT (60 mg/kg). KMUP-1 (5 mg/kg, i.p.) was administered once daily for 21 days to prevent MCT-induced PAH. All rats were sacrificed on day 22.Key findingsMCT-induced increased right ventricular systolic pressure (RVSP) and right ventricular hypertrophy were prevented by KMUP-1. In myograph experiments, KCl (80 mM), phenylephrine (10 µM) and K⁺ channel inhibitors (TEA, 10 mM; paxilline, 10 µM; 4-AP, 5 mM) induced weak PA contractions in MCT-treated rats compared to controls, but the PA reactivity was restored in MCT + KMUP-1-treated rats. By contrast, in β-escin- or α-toxin-permeabilized PAs, CaCl₂-induced (1.25 mM, pCa 5.1) contractions were stronger in MCT-treated rats, and this action was suppressed in MCT + KMUP-1-treated rats. PA relaxation in response to the ROCK inhibitor Y27632 (0.1 μM) was much higher in MCT-treated rats than in control rats. In Western blot analysis, the expression of Ca²⁺-activated K⁺ (BK_Ca) and voltage-gated K⁺ channels (Kv2.1 and Kv1.5), and ROCK II proteins was elevated in MCT-treated rats and suppressed in MCT + KMUP-1-treated rats. We suggest that MCT-treated rats upregulate K⁺-channel proteins to adapt to chronic PAH.SignificanceKMUP-1 protects against PAH and restores PA vessel tone in MCT-treated rats, attributed to alteration of Ca²⁺ sensitivity and K⁺-channel function.     

Decreased expression of small-conductance Ca2+-activated K+ channels SK1 and SK2 in human chronic atrial fibrillation

AimsSmall-conductance Ca^2 +-activated K⁺ (SK) channels are recognized as new ion channel candidates in atrial fibrillation (AF), with pivotal implications as novel drug targets due to their atrial-selective distribution in humans. The purpose of this study was to investigate whether SK channels and the Ca^2 +-activated K⁺ current (I_K,Ca) are involved in electrical remodeling of human chronic AF (cAF) and whether they display the differential distribution between the right (RA) and left atria (LA).Main methodsThe right (RAA) and left atrial appendage (LAA) myocytes were obtained from 29 sinus rhythm (SR) and 22 cAF patients. The I_K,Ca and action potential (AP) were recorded using the patch-clamp technique. Three SK channel subtypes (SK1–3) expressions were assayed by western blot and real-time quantitative PCR analysis.Key findingsThe I_K,Ca was decreased and its role in AP repolarization was attenuated in cAF, concomitant with a significant decrease in protein and mRNA levels of SK1 and SK2. In either SR or cAF, there was no difference in the I_K,Ca density and protein and mRNA expression levels of SK1–3 between RAA and LAA myocytes.SignificanceOur results demonstrated that SK1 and SK2 are involved in electrical remodeling of cAF. SK1–3 and I_K,Ca do not display the inter-atrial differential distribution in SR or cAF. These findings provide a new insight into mechanisms of electrical remodeling of human cAF.     

Identification and characterization of Ca2+-activated K+ channels in granulosa cells of the human ovary

Background  

Granulosa cells (GCs) represent a major endocrine compartment of the ovary producing sex steroid hormones. Recently, we identified in human GCs a Ca²⁺-activated K⁺ channel (K_Ca) of big conductance (BK_Ca), which is involved in steroidogenesis. This channel is activated by intraovarian signalling molecules (e.g. acetylcholine) via raised intracellular Ca²⁺ levels. In this study, we aimed at characterizing 1. expression and functions of K_Ca channels (including BK_Ca beta-subunits), and 2. biophysical properties of BK_Ca channels.     

Exercise training changes the gating properties of large-conductance Ca2+-activated K+ channels in rat thoracic aorta smooth muscle cells

Large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels play a critical role in regulating the cellular excitability in response to change in blood flow. It has been demonstrated that vascular BK_Ca channel currents in both humans and rats are increased after exercise training. This up-regulation of the BK_Ca channel activity in arterial myocytes may represent a cellular compensatory mechanism of limiting vascular reactivity to exercise training. However, the underlying mechanisms are not fully understood. In the present study, we examined the single channel activities and kinetics of the BK_Ca channels in rat thoracic aorta smooth muscle cells. We showed that exercise training significantly increased the open probability (Po), decreased the mean closed time and increased the mean open time, and the sensitivity to Ca²⁺ and voltage without altering the unitary conductance and the K⁺ selectivity. Our results suggest a novel mechanism by which exercise training increases the K⁺ currents by changing the BK_Ca channel activities and kinetics.     

Magnolol inhibits colonic motility through down-regulation of voltage-sensitive L-type Ca2+ channels of colonic smooth muscle cells in rats

This study aimed to investigate the effect of magnolol (5,5′-diallyl-2,2′-biphenyldiol) on contraction in distal colonic segments of rats and the underlying mechanisms. Colonic segments were mounted in organ baths for isometric force measurement. Whole-cell voltage-sensitive L-type Ca²⁺ currents were recorded on isolated single colonic smooth muscle cells using patch-clamp technique. The spontaneous contractions and acetylcholine (ACh)- and Bay K 8644-induced contractions were inhibited by magnolol (3–100 μM). In the presence of Bay K8644 (100 nM), magnolol (10–100 μM) inhibited the contraction induced by 10 μM ACh. By contrast, tetrodotoxin (100 nM) and N_ώ-nitro-l-arginine methyl ester (l-NAME 100 μM) did not change the inhibitory effect of magnolol (10 μM). In addition, magnolol (3–100 μM) inhibited the L-type Ca²⁺ currents. The present results suggest that magnolol inhibits colonic smooth muscle contraction through downregulating L-type Ca²⁺ channel activity.     

Caveolin-1 Facilitates the Direct Coupling between Large Conductance Ca2+-activated K+ (BKCa) and Cav1.2 Ca2+ Channels and Their Clustering to Regulate Membrane Excitability in Vascular Myocytes

L-type voltage-dependent Ca²⁺ channels (LVDCC) and large conductance Ca²⁺-activated K⁺ channels (BK_Ca) are the major factors defining membrane excitability in vascular smooth muscle cells (VSMCs). The Ca²⁺ release from sarcoplasmic reticulum through ryanodine receptor significantly contributes to BK_Ca activation in VSMCs. In this study direct coupling between LVDCC (Cav1.2) and BK_Ca and the role of caveoline-1 on their interaction in mouse mesenteric artery SMCs were examined. The direct activation of BK_Ca by Ca²⁺ influx through coupling LVDCC was demonstrated by patch clamp recordings in freshly isolated VSMCs. Using total internal reflection fluorescence microscopy, it was found that a large part of yellow fluorescent protein-tagged BK_Ca co-localized with the cyan fluorescent protein-tagged Cav1.2 expressed in the plasma membrane of primary cultured mouse VSMCs and that the two molecules often exhibited FRET. It is notable that each BKα subunit of a tetramer in BK_Ca can directly interact with Cav1.2 and promotes Cav1.2 cluster in the molecular complex. Furthermore, caveolin-1 deficiency in knock-out (KO) mice significantly reduced not only the direct coupling between BK_Ca and Cav1.2 but also the functional coupling between BK_Ca and ryanodine receptor in VSMCs. The measurement of single cell shortening by 40 mm K⁺ revealed enhanced contractility in VSMCs from KO mice than wild type. Taken together, caveolin-1 facilitates the accumulation/clustering of BK_Ca-LVDCC complex in caveolae, which effectively regulates spatiotemporal Ca²⁺ dynamics including the negative feedback, to control the arterial excitability and contractility.     

Functional and molecular consequences of ionizing irradiation on large conductance Ca2+-activated K+ channels in rat aortic smooth muscle cells

AimsThe goal of this study was to evaluate the influence of γ-irradiation on Ca²⁺-activated K⁺ channel (BK_Ca) function and expression in rat thoracic aorta.Main methodsAortic cells or tissues were studied by the measurement of force versus [Ca²⁺]_i, patch-clamp technique, and RT-PCR.Key findingsStimulation of smooth muscle cells with depolarizing voltage steps showed expression of outward K⁺ currents. Paxilline, an inhibitor of BK_Ca channels, decreased outward K⁺ current density. Outward currents in smooth muscle cells obtained from irradiated animals 9 and 30 days following radiation exposure demonstrated a significant decrease in K⁺ current density. Paxilline decreased K⁺ current in cells obtained 9 days, but was without effect 30 days after irradiation suggesting the absence of BK_Ca channels. Aortic tissue from irradiated animals showed progressively enhanced contractile responses to phenylephrine in the post-irradiation period of 9 and 30 days. The concomitant Ca²⁺ transients were significantly smaller, as compared to tissues from control animals, 9 days following irradiation but were increased above control levels 30 days following irradiation. Irradiation produced a decrease in BK_Ca α- and β₁-subunit mRNA levels in aortic smooth muscle cells suggesting that the vasorelaxant effect of these channels may be diminished.SignificanceThese results suggest that the enhanced contractility of vascular tissue from animals exposed to radiation may result from an increase in myofilament Ca²⁺ sensitivity in the early post-irradiation period and a decrease in BK_Ca channel expression in the late post-irradiation period.     

Possible mechanisms underlying the biphasic regulatory effects of arachidonic acid on Ca2+ signaling in HEK293 cells

Arachidonic acid (AA), an endogenous lipid signal molecule released from membrane upon cell activation, modulates intracellular Ca^2 + ([Ca^2 +]_i) signaling positively and negatively. However, the mechanisms underlying the biphasic effects of AA are rather obscure. Using probes for measurements of [Ca^2 +]_i and fluidity of plasma membrane (PM)/endoplasmic reticulum (ER), immunostaining, immunoblotting and shRNA interference approaches, we found that AA at low concentration, 3 μM, reduced the PM fluidity by activating PKCα and PKCβII translocation to PM and also the ER fluidity directly. In accordance, 3 μM AA did not impact the basal [Ca^2 +]_i but significantly suppressed the thapsigargin-induced Ca^2 + release and Ca^2 + influx. Inhibition of PKC with Gö6983 or knockdown of PKCα or PKCβ using shRNA significantly attenuated the inhibitory effects of 3 μM AA on PM fluidity and agonist-induced Ca^2 + signal. However, AA at high concentration, 30 μM, caused robust release and entry of Ca^2 + accompanied by a facilitated PM fluidity but decreased ER fluidity and dramatic PKCβI and PKCβII redistribution in the ER. Compared with ursodeoxycholate acid, a membrane stabilizing agent that only inhibited the 30 μM AA-induced Ca^2 + influx by 45%, Gd^3 + at concentration of 10 μM could completely abolish both release and entry of Ca^2 + induced by AA, suggesting that the potentiated PM fluidity is not the only reason for AA eliciting Ca^2 + signal. Therefore, the study herein demonstrates that a lowered PM fluidity by PKC activation and a direct ER stabilization contribute significantly for AA downregulation of [Ca^2 +]_i response, while Gd^3 +-sensitive ‘pores’ in PM/ER play an important role in AA-induced Ca^2 + signal in HEK293 cells.     

Mechanism of dopamine D2 receptor-induced Ca2 + release in PC-12 cells

Intracellular Ca^2 + levels are tightly regulated in the neuronal system. The loss of Ca^2 + homeostasis is associated with many neurological diseases and neuropsychiatric disorders such as Parkinson's, Alzheimer's, and schizophrenia. We investigated the mechanisms involved in intracellular Ca^2 + signaling in PC-12 cells. The stimulation of NGF-differentiated PC-12 cells with 3 μM ATP caused an early Ca^2 + release followed by a delayed Ca^2 + release. The delayed Ca^2 + release was dependent on prior ATP priming and on dopamine secretion by PC-12 cells. Delayed Ca^2 + release was abolished in the presence of spiperone, suggesting that it is due to the activation of D2 dopamine receptors (D2R) by dopamine secreted by PC-12 cells. This was shown to be independent of PKA activation but dependent on PLC activity. An endocytosis step was required for inducing the delayed Ca^2 + release. Given the importance of calcyon in clathrin-mediated endocytosis, we verified the role of this protein in the delayed Ca^2 + release phenomenon. siRNA targeting of calcyon blocked the delayed Ca^2 + release, decreased ATP-evoked IP₃R-mediated Ca^2 + release, and impaired subsequent Ca^2 + oscillations. Our results suggested that calcyon is involved in an unknown mechanism that causes a delayed IP₃R-mediated Ca^2 + release in PC-12 cells. In schizophrenia, Ca^2 + dysregulation may depend on the upregulation of calcyon, which maintains elevated Ca^2 + levels as well as dopamine signaling.     

Bupivacaine inhibits large conductance,voltage- and Ca2+- activated K+ channels in human umbilical artery smooth muscle cells

Bupivacaine is a local anesthetic compound belonging to the amino amide group. Its anesthetic effect is commonly related to its inhibitory effect on voltage-gated sodium channels. However, several studies have shown that this drug can also inhibit voltage-operated K⁺ channels by a different blocking mechanism. This could explain the observed contractile effects of bupivacaine on blood vessels. Up to now, there were no previous reports in the literature about bupivacaine effects on large conductance voltage- and Ca²⁺-activated K⁺ channels (BK_Ca). Using the patch-clamp technique, it is shown that bupivacaine inhibits single-channel and whole-cell K⁺ currents carried by BK_Ca channels in smooth muscle cells isolated from human umbilical artery (HUA). At the single-channel level bupivacaine produced, in a concentration- and voltage-dependent manner (IC₅₀ 324 µM at +80 mV), a reduction of single-channel current amplitude and induced a flickery mode of the open channel state. Bupivacaine (300 µM) can also block whole-cell K⁺ currents (~45% blockage) in which, under our working conditions, BK_Ca is the main component. This study presents a new inhibitory effect of bupivacaine on an ion channel involved in different cell functions. Hence, the inhibitory effect of bupivacaine on BK_Ca channel activity could affect different physiological functions where these channels are involved. Since bupivacaine is commonly used during labor and delivery, its effects on umbilical arteries, where this channel is highly expressed, should be taken into account.     

Na+ and Ca2+ channel expression in cultured newt retinal pigment epithelial cells: Comparison with neuronal types of ion channels

We cultured retinal pigment epithelial (RPE) cells dissociated from adult newt eye and analyzed their voltage-gated ion channels during culture using whole-cell patch-clamp techniques. The results were compared with those of retinal neurons under identical experimental conditions. After 6–9 days in culture (early stage), > 60% of RPE cells developed voltage-gated Na⁺ and Ca²⁺ channels that were not observed in freshly dissociated RPE cells. The number of cells expressing Na⁺ channels and Na⁺ current density were high after 12–15 days in culture (intermediate stage), while the number of Ca²⁺ channel-expressing cells and Ca²⁺ current density were high after 20–30 days in culture (late stage). The activation voltage of the Na⁺ current in the RPE cells was similar to that in neurons. The voltage dependence of Na⁺ current inactivation was somewhat different between two cell types. The steepness of the inactivation curve tended to be less in cultured RPE cells than in neurons, and the half-inactivation voltage was about −54 mV for the RPE cells and −45 mV for neurons. The Ca²⁺ current expressed in cultured RPE cells was too small to detect without replacement of external Ca²⁺ with Ba²⁺. The Ba²⁺ current, like Ca²⁺ current in neurons, was enhanced by Bay-K 8644 and blocked by nicardipine. These results suggest that the RPE cells, like neurons, expressed L-type Ca²⁺ channels in culture. The possibility that the development of both Na²⁺ and Ca²⁺ channels in cultured RPE cells is a manifestation of the transdifferentiation of RPE cells into neurons is discussed. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 377–390, 1997.     

Enhanced charge-independent mitochondrial free Ca2 + and attenuated ADP-induced NADH oxidation by isoflurane: Implications for cardioprotection

Modulation of mitochondrial free Ca^2 + ([Ca^2 +]_m) is implicated as one of the possible upstream factors that initiates anesthetic-mediated cardioprotection against ischemia–reperfusion (IR) injury. To unravel possible mechanisms by which volatile anesthetics modulate [Ca^2 +]_m and mitochondrial bioenergetics, with implications for cardioprotection, experiments were conducted to spectrofluorometrically measure concentration-dependent effects of isoflurane (0.5, 1, 1.5, 2 mM) on the magnitudes and time-courses of [Ca^2 +]_m and mitochondrial redox state (NADH), membrane potential (ΔΨ_m), respiration, and matrix volume. Isolated mitochondria from rat hearts were energized with 10 mM Na⁺- or K⁺-pyruvate/malate (NaPM or KPM) or Na⁺-succinate (NaSuc) followed by additions of isoflurane, 0.5 mM CaCl₂ (≈ 200 nM free Ca^2 + with 1 mM EGTA buffer), and 250 μM ADP. Isoflurane stepwise: (a) increased [Ca^2 +]_m in state 2 with NaPM, but not with KPM substrate, despite an isoflurane-induced slight fall in ΔΨ_m and a mild matrix expansion, and (b) decreased NADH oxidation, respiration, ΔΨ_m, and matrix volume in state 3, while prolonging the duration of state 3 NADH oxidation, respiration, ΔΨ_m, and matrix contraction with PM substrates. These findings suggest that isoflurane's effects are mediated in part at the mitochondrial level: (1) to enhance the net rate of state 2 Ca^2 + uptake by inhibiting the Na⁺/Ca^2 + exchanger (NCE), independent of changes in ΔΨ_m and matrix volume, and (2) to decrease the rates of state 3 electron transfer and ADP phosphorylation by inhibiting complex I. These direct effects of isoflurane to increase [Ca^2 +]_m, while depressing NCE activity and oxidative phosphorylation, could underlie the mechanisms by which isoflurane provides cardioprotection against IR injury at the mitochondrial level.     

Involvement of plasma membrane Ca2+ channels,IP3 receptors,and ryanodine receptors in the generation of spontaneous rhythmic contractions of the cricket lateral oviduct

In the present study, the isolated cricket (Gryllus bimaculatus) lateral oviduct exhibited spontaneous rhythmic contractions (SRCs) with a frequency of 0.29 ± 0.009 Hz (n = 43) and an amplitude of 14.6 ± 1.25 mg (n = 29). SRCs completely disappeared following removal of extracellular Ca²⁺ using a solution containing 5 mM EGTA. Application of the non-specific Ca²⁺ channel blockers Co²⁺, Ni²⁺, and Cd²⁺ also decreased both the frequency and amplitude of SRCs in dose-dependent manners, suggesting that Ca²⁺ entry through plasma membrane Ca²⁺ channels is essential for the generation of SRCs. Application of ryanodine (30 μM), which depletes intracellular Ca²⁺ by locking ryanodine receptor (RyR)-Ca²⁺ channels in an open state, gradually reduced the frequency and amplitude of SRCs. A RyR antagonist, tetracaine, reduced both the frequency and amplitude of SRCs, whereas a RyR activator, caffeine, increased the frequency of SRCs with a subsequent increase in basal tonus, indicating that RyRs are essential for generating SRCs. To further investigate the involvement of phospholipase C (PLC) and inositol 1,4,5-trisphosphate receptors (IP₃Rs) in SRCs, we examined the effect of a PLC inhibitor, U73122, and an IP₃R antagonist, 2-aminoethoxydiphenyl borate (2-APB), on SRCs. Separately, U73122 (10 μM) and 2-APB (30–50 μM) both significantly reduced the amplitude of SRCs with little effect on their frequency, further indicating that the PLC/IP₃R signaling pathway is fundamental to the modulation of the amplitude of SRCs. A hypotonic-induced increase in the frequency and amplitude of SRCs and a hypertonic-induced decrease in the frequency and amplitude of SRCs indicated that mechanical stretch of the lateral oviduct is involved in the generation of SRCs. The sarcoplasmic reticulum Ca²⁺-pump ATPase inhibitors thapsigargin and cyclopiazonic acid impaired or suppressed the relaxation phase of SRCs. Taken together, the present results indicate that Ca²⁺ influx through plasma membrane Ca²⁺ channels and Ca²⁺ release from RyRs play an essential role in pacing SRCs and that Ca²⁺ release from IP₃Rs may play a role in modulating the amplitude of SRCs, probably via activation of PLC.     

Thapsigargin decreases the Na+- Ca2 + exchanger mediated Ca2 + entry in pig coronary artery smooth muscle

Na⁺- Ca^2 + exchanger (NCX) has been proposed to play a role in refilling the sarco/endoplasmic reticulum (SER) Ca^2 + pool along with the SER Ca^2 + pump (SERCA). Here, SERCA inhibitor thapsigargin was used to determine the effects of SER Ca^2 + depletion on NCX–SERCA interactions in smooth muscle cells cultured from pig coronary artery. The cells were Na⁺-loaded and then placed in either a Na⁺-containing or in a Na⁺-substituted solution. Subsequently, the difference in Ca^2 + entry between the two groups was examined and defined as the NCX mediated Ca^2 + entry. The NCX mediated Ca^2 + entry in the smooth muscle cells was monitored using two methods: Ca^2 +sensitive fluorescence dye Fluo-4 and radioactive Ca^2 +. Ca^2 +-entry was greater in the Na⁺-substituted cells than in the Na⁺-containing cells when measured by either method. This difference was established to be NCX-mediated as it was sensitive to the NCX inhibitors. Thapsigargin diminished the NCX mediated Ca^2 + entry as determined by either method. Immunofluorescence confocal microscopy was used to determine the co-localization of NCX1 and subsarcolemmal SERCA2 in the cells incubated in the Na⁺-substituted solution with or without thapsigargin. SER Ca^2 + depletion with thapsigargin increased the co-localization between NCX1 and the subsarcolemmal SERCA2. Thus, inhibition of SERCA2 leads to blockade of constant Ca^2 + entry through NCX1 and also increases proximity between NCX1 and SERCA2. This blockade of Ca^2 + entry may protect the cells against Ca^2 +-overload during ischemia–reperfusion when SERCA2 is known to be damaged.     

Involvement of store-operated Ca2 + entry in activation of AMP-activated protein kinase and stimulation of glucose uptake by M3 muscarinic acetylcholine receptors in human neuroblastoma cells

G_q/11-coupled muscarinic acetylcholine receptors (mAChRs) belonging to M₁, M₃ and M₅ subtypes have been shown to activate the metabolic sensor AMP-activated protein kinase (AMPK) through Ca^2 +/calmodulin-dependent protein kinase kinase-β (CaMKKβ)-mediated phosphorylation at Thr172. However, the source of Ca^2 + required for this response has not been yet elucidated. Here, we investigated the involvement of store-operated Ca^2 + entry (SOCE) in AMPK activation by pharmacologically defined M₃ mAChRs in human SH-SY5Y neuroblastoma cells. In Ca^2 +-free medium the cholinergic agonist carbachol (CCh) caused a transient increase of phospho-Thr172 AMPK that rapidly ceased within 2 min. Conversely, in the presence of extracellular Ca^2 + CCh-induced AMPK phosphorylation lasted for at least 180 min. The SOCE modulator 2-aminoethoxydiphephenyl borate (2-APB), at a concentration (50 μM) that suppressed CCh-induced intracellular Ca^2 + ([Ca^2 +]_i) plateau, inhibited CCh-induced AMPK phosphorylation. CCh triggered the activation of the endoplasmic reticulum Ca^2 + sensor stromal interaction molecule (STIM) 1, as indicated by redistribution of STIM1 immunofluorescence into puncta, and promoted the association of STIM1 with the SOCE channel component Orai1. Cell depletion of STIM1 by siRNA treatment reduced both CCh-induced [Ca^2 +]_i plateau and AMPK activation. M₃ mAChRs increased glucose uptake and this response required extracellular Ca^2 + and was inhibited by 2-APB, STIM1 knockdown, CaMKKβ and AMPK inhibitors, and adenovirus infection with dominant negative AMPK. Thus, the study provides evidence that SOCE is required for sustained activation of AMPK and stimulation of downstream glucose uptake by M₃ mAChRs and suggests that SOCE is a critical process connecting M₃ mAChRs to the control of neuronal energy metabolism.