Development of different electrophysiological mechanisms for muscarinic inhibition of atria and ventricles

The negative inotropic effect of acetylcholine (ACh) in atrial muscle can be accounted for by a decrease of a voltage- and time-dependent slow inward current (Isi) carried by Ca2+/Na+ and an increase of outward time-dependent current carried by K+ (IK1) through inwardly rectifying channels. The negative inotropic effect of ACh in ventricular muscle is associated with a reduction of Isi; there is no important effect of ACh on IK1 in ventricular muscle. Because atrial and ventricular muscles display IK1 that is sensitive to Ba2+ and have similar numbers of muscarinic receptor sites, it is concluded that ventricular muscle lacks a metabolic link between the muscarinic receptor and inwardly rectifying K+ channels. Although there is much evidence for cyclic nucleotides as the mediator between muscarinic receptors and Isi channels, cyclic nucleotides do not seem to connect these receptors with inwardly rectifying K+ channels. According to this hypothesis, identification of a metabolic link between muscarinic receptors and IK1 channels should be demonstrable in atrial but not ventricular muscle.     

On the mechanism of basal and agonist-induced activation of the G protein-gated muscarinic K+ channel in atrial myocytes of guinea pig heart

Using the patch clamp technique, we examined the agonist-free, basal interaction between the muscarinic acetylcholine (m-ACh) receptor and the G protein (GK)-gated muscarinic K+ channel (IK.ACh), and the modification of this interaction by ACh binding to the receptor in single atrial myocytes of guinea pig heart. In the whole cell clamp mode, guanosine-5'-O-(3-thiotriphosphate) (GTP-gamma S) gradually increased the IK.ACh current in the absence of agonists (e.g., acetylcholine). This increase was inhibited in cells that were pretreated with islet-activating protein (IAP, pertussis toxin) or N-ethylmaleimide (NEM). In inside-out patches, even in the absence of agonists, intracellular GTP caused openings of IK.ACh in a concentration-dependent manner in approximately 80% of the patches. Channel activation by GTP in the absence of agonist was much less than that caused by GTP-gamma S. The agonist-independent, GTP-induced activation of IK.ACh was inhibited by the A promoter of IAP (with nicotinamide adenine dinucleotide) or NEM. As the ACh concentration was increased, the GTP-induced maximal open probability of IK.ACh was increased and the GTP concentration for the half-maximal activation of IK.ACh was decreased. Intracellular GDP inhibited the GTP-induced openings of IK.ACh in a concentration-dependent fashion. The half-inhibition of IK.ACh openings occurred at a much lower concentration of GDP in the absence of agonists than in the presence of ACh. From these results, we concluded (a) that the interaction between the m-ACh receptor and GK is essential for basal stimulation of IK.ACh, and (b) that ACh binding to the receptor accelerates the turnover of GK and increases GK's affinity to GTP analogues over GDP.     

Arachidonic acid metabolites alter G protein-mediated signal transduction in heart. Effects on muscarinic K+ channels

The muscarinic acetylcholine receptor (mAChR)-stimulated, inwardly rectifying K+ current (IK [ACh]) was examined in single bullfrog atrial cells using the whole-cell patch clamp technique. IK[ACh] was activated either by bath addition of 1 microM ACh or via activation of the G protein, Gk, with guanosine-gamma-thiotriphosphate (GTP gamma S). Arachidonic acid (AA) modulated IK[ACh] under both conditions. AA decreased mAChR-stimulated IK[ACh] and increased the rate of decay from the peak current (desensitization). In addition, AA affected GTP gamma S-activated IK[ACh] by modulation of Gk. The effects of AA and its metabolites on Gk were assessed by examining their effects on both the basal rate of Gk activation by GTP gamma S, and the mAChR-mediated increase in activation rate produced by nanomolar ACh. AA increased the basal rate of GTP gamma S-mediated IK[ACh] activation, but reduced the ACh-induced augmentation of this rate. All of the effects of AA on GTP gamma S-mediated IK[ACh] activation were produced by metabolites. A lipoxygenase inhibitor, nordihydroguaiaretic acid (NDGA), decreased the basal and ACh-enhanced rate of IK[ACh] activation in both the presence and absence of exogenous AA. In contrast, indomethacin (INDO), a cyclooxygenase inhibitor, increased the basal rate of IK[ACh] activation by GTP gamma S in both the presence and absence of exogenous AA, and reversed the effects of AA on the ACh-augmented basal rate. AA metabolites produced via lipoxygenase and cyclooxygenase pathways thus have opposing effects on the signal transduction pathway from mAChR to IK[ACh]. We directly tested a lipoxygenase pathway metabolite, LTC4, on GTP gamma S-mediated IK[ACh] activation and found that it not only overcame the inhibitory effects of NDGA, but also increased both the basal and ACh-augmented rate of IK[ACh] activation. From these data, we propose that AA metabolites modulate the function of Gk by altering its kinetic properties.     

Sphingosine 1-phosphate receptors

Sphingosine-1-phosphate (SPP) is a bioactive lipid produced from the metabolism of sphingomyelin. It is an important constituent of serum and regulates cell growth, survival, migration, differentiation and gene expression. Its mode of action has been enigmatic; however, recent findings have shown that a family of G-protein-coupled receptors (GPCR) of the endothelial differentiation gene (EDG) family serve as plasma membrane-localized receptors for SPP. Furthermore, the EDG receptors appear to be SPP receptor subtypes with distinct signaling characteristics. In vascular endothelial cells, SPP acts on EDG-1 and EDG-3 subtypes of receptors to induce cell survival and morphogenesis. Such pathways appear to be critical for SPP-induced angiogenic response in vivo. In addition, the EDG-1 gene is essential for vascular maturation in development. Moreover, developmental studies in Zebrafish have indicated that SPP signaling via the EDG-5 like receptor Miles Apart (Mil) is essential for heart development. These data strongly suggest that a physiological role of SPP is in the formation of the cardiovascular system. Despite these recent findings, much needs to be clarified with respect to the physiological role of SPP synthesis and action. This review will focus on the recent findings on SPP receptors and the effects on the cardiovascular system.     

Differential effects of pertussis toxin on the muscarinic regulation of Ca2+ and K+ currents in frog cardiac myocytes

The ability of acetylcholine (ACh) to inhibit beta-agonist stimulated calcium current was compared to its ability to activate the inwardly rectifying potassium current IK(ACh) in frog atrial myocytes. As suggested by previous studies, ACh inhibited the calcium current at concentrations (EC50 = 8 nM) significantly lower than those required for the activation of IK(ACh) (EC50 = 101 nM). The pharmacological profiles of the two responses suggest that despite the differences in agonist sensitivity, both are mediated by the same (m2) type of muscarinic receptors. Intracellular application of GDP beta S, an inhibitor of G protein function, completely abolished both responses, implying that both actions of ACh are coupled to effectors by G proteins. In contrast, intracellular application of pertussis toxin (PTX) shifted to higher concentrations (EC50 = 170 nM) but did not abolish inhibition of the calcium current by ACh even though the block of the IK(ACh) response was complete. Increasingly large PTX concentrations and/or prolonged PTX treatments revealed a limiting, PTX- resistant inhibitory component that appears to be mediated by a PTX- insensitive G protein distinct from that mediating IK(ACh). For the PTX- sensitive components, the different agonist dependencies of IK(ACh) activation and calcium current inhibition may imply that different G proteins mediate each response although alternate possibilities involving the same G protein either functionally sequestered and/or differentially affected by interactions with effectors, can not be ruled out.     

Sphingosine 1-phosphate receptors

Sphingosine-1-phosphate (SPP) is a bioactive lipid produced from the metabolism of sphingomyelin. It is an important constituent of serum and regulates cell growth, survival, migration, differentiation and gene expression. Its mode of action has been enigmatic; however, recent findings have shown that a family of G-protein-coupled receptors (GPCR) of the endothelial differentiation gene (EDG) family serve as plasma membrane-localized receptors for SPP. Furthermore, the EDG receptors appear to be SPP receptor subtypes with distinct signaling characteristics. In vascular endothelial cells, SPP acts on EDG-1 and EDG-3 subtypes of receptors to induce cell survival and morphogenesis. Such pathways appear to be critical for SPP-induced angiogenic response in vivo. In addition, the EDG-1 gene is essential for vascular maturation in development. Moreover, developmental studies in Zebrafish have indicated that SPP signaling via the EDG-5 like receptor Miles Apart (Mil) is essential for heart development. These data strongly suggest that a physiological role of SPP is in the formation of the cardiovascular system. Despite these recent findings, much needs to be clarified with respect to the physiological role of SPP synthesis and action. This review will focus on the recent findings on SPP receptors and the effects on the cardiovascular system.     

The multi-functional role of sphingosylphosphorylcholine

The sphingomyelin metabolite, sphingosylphosphorylcholine (SPC) has been the subject of much recent interest and controversy. Studies have indicated that SPC naturally occurs in plasma and a constituent of lipoproteins. Synthesis is also increased in some pathological conditions. Research has demonstrated that SPC is a potentially important lipid mediator of cell type specific functions in major tissues, such as heart, blood vessels, skin, brain and immune system. These effects are regulated via a number of different intracellular signalling cascades, also dependent upon cell type. Initial reports identifying high affinity SPC receptors at first appeared to reinforce the physiological relevance of this sphingolipid. However, these studies have now been retracted. Some SPC effects have been shown be occur via plasma membrane receptors for the related sphingolipid, sphingosine 1-phosphate (S1P). Despite a lack of well-defined receptor signal transduction mechanisms and sparse pharmacological data, several key characteristics of SPC are now emerging. SPC can act as a mitogen in several different cell types and in certain circumstances, may also be a pro-inflammatory mediator. In this review, these actions of SPC are discussed with a view to understanding the potential physiological relevance of this sphingolipid.     

A cellular mechanism for nitric oxide-mediated cholinergic control of mammalian heart rate

The biochemical signaling pathways involved in nitric oxide (NO)- mediated cholinergic inhibition of L-type Ca2+ current (ICa[L]) were investigated in isolated primary pacemaker cells from the rabbit sinoatrial node (SAN) using the nystatin-perforated whole-cell voltage clamp technique. Carbamylcholine (CCh; 1 microM), a stable analogue of acetylcholine, significantly inhibited ICa(L) after it had been augmented by isoproterenol (ISO; 1 microM). CCh also activated an outward K+ current, IK(ACh). Both of these effects of CCh were blocked completely by atropine. Preincubation of the SAN cells with L-nitro- arginine methyl ester (L-NAME; 0.2-1 mM), which inhibits NO synthase (NOS), abolished the CCh-induced attenuation of ICa(L) but had no effect on IK(ACh). Coincubation of cells with both L-NAME and the endogenous substrate of NOS, L-arginine (1 nM), restored the CCh- induced attenuation of ICa(L), indicating that L-NAME did not directly interfere with the muscarinic action of CCh on ICa(L). In the presence of ISO the CCh-induced inhibition of ICa(L) could be mimicked by the NO donor 3-morpholino-sydnonimine (SIN-1; 0.1 mM). SIN-1 had no effect on its own or after a maximal effect of CCh had developed, indicating that it does not inhibit ICa(L) directly. SIN-1 failed to activate IK(ACh), demonstrating that it did not activate muscarinic receptors. Both CCh and NO are known to activate guanylyl cyclase and elevate intracellular cGMP. External application of methylene blue (10 microM), which interferes with the ability of NO to activate guanylyl cyclase, blocked the CCh-induced attenuation of ICa(L). However, it also blocked the activation of IK(ACh), suggesting an additional effect on muscarinic receptors or G proteins. To address this, a separate series of experiments was performed using conventional whole-cell recordings with methylene blue in the pipette. Under these conditions, the CCh-induced attenuation of ICa(L) was blocked, but the activation of IK(ACh) was still observed. Methylene blue also blocked the SIN-1-induced decrease in ICa(L). 6-anilino-5,8-quinolinedione (LY83583; 30 microM), an agent known to decrease both basal and CCh-stimulated cGMP levels, prevented the inhibitory effects of both CCh and SIN-1 on ICa(L), but had no effect on the activation of IK(ACh) by CCh. In combination, these results show that CCh- and NO-induced inhibition of ICa(L) is mediated by cGMP.(ABSTRACT TRUNCATED AT 400 WORDS)     

Leukotriene C4 modulation of muscarinic K+ current activation in bullfrog atrial myocytes

The effects of leukotriene C4 (LTC4) on activation of muscarinic acetylcholine receptor (mAChR)-stimulated, inwardly rectifying K+ current (IK[ACh]) were examined in single bullfrog atrial myocytes using the whole-cell patch clamp technique. LTC4 produced a reversible, concentration-dependent increase in steady-state, guanosine-gamma- thiotriphosphate (GTP gamma S)-activated IK[ACh], with a K0.5 of 3.1 microM. LTC4 also increased the rate of GTP gamma S-mediated IK[ACh] activation, both in the absence and presence of 1 nM ACh, with comparable K0.5 values of 4.7 microM under basal conditions and 4.9 microM in the presence of 1 nM ACh. LTC4 did not alter the relative affinities of the G protein, Gk, for GTP gamma S and GTP. We hypothesize that all of the effects of LTC4 on the kinetics of Gk- mediated IK[ACh] activation are produced at a common site with a K0.5 of 3-5 microM. The effects of LTC4 on IK[ACh] activation are fully reversible in the presence of GTP gamma S. Under physiological conditions (i.e., intracellular GTP), 10 microM LTC4 increased the ACh- activated peak IK[ACh]. Inhibitors of cellular LTC4 production, including 5,8,11,14-eicosatetraynoic acid, baicalein, cinnamyl-3,4- dihydroxy-alpha-cyanocinnamate, and alpha-pentyl-4-(2- quinolinylmethoxy)-benzene methanol, greatly attenuated ACh-dependent IK[ACh] activation, preventing activation of peak, and producing a lower steady-state IK[ACh] (when compared with the control response in the same cell). Addition of exogenous LTC4 was able to overcome the effects of LTC4 synthesis inhibitors, restoring both the peak and steady-state IK[ACh] responses. Although the mechanism of LTC4-mediated modulation of IK[ACh] activation is not known, our results suggest that endogenously produced lipoxygenase metabolites of arachidonic acid, specifically LTC4, are involved in the physiological process of IK[ACh] activation.     

Molecular mechanism of doxorubicin-induced anticholinergic effect in guinea-pig atria

The molecular mechanisms of anticholinergic actions of doxorubicin were examined by electrophysiological methods in atria and myocytes isolated from guinea-pig heart. A direct anticholinergic action of doxorubicin was confirmed with antagonistic action on carbachol-induced negative inotropic effect in atria. Both carbachol and adenosine produced shortening of action potential duration in atria measured by a microelectrode method. Doxorubicin (10-100 microM) inhibited the carbachol-induced action potential shortening in a concentration-dependent manner. However, doxorubicin did not antagonize the shortening elicited by adenosine. The whole-cell voltage clamp technique was performed to induce the muscarinic acetylcholine-receptor-operated K+ current (IK.ACh) in atrial myocytes loaded with GTP or GTPgammaS, a nonhydrolysable analogue of GTP. Doxorubicin (1-100 microM) suppressed carbachol-induced IK.ACh in a concentration-dependent manner (IC50 = 5.6 microM). In contrast, doxorubicin (10 and 100 microM) suppressed neither adenosine-induced IK.ACh nor GTPgammaS-induced IK.ACh. These results indicate that doxorubicin produces a direct anticholinergic effect through the muscarinic receptors in atrial myocytes.     

Activation of Ca(2+)-dependent K+ current by acetylcholine and histamine in a human gastric epithelial cell line

The effects of acetylcholine (ACh) and histamine (His) on the membrane potential and current were examined in JR-1 cells, a mucin-producing epithelial cell line derived from human gastric signet ring cell carcinoma. The tight-seal, whole cell clamp technique was used. The resting membrane potential, the input resistance, and the capacitance of the cells were approximately -12 mV, 1.4 G ohms, and 50 pF, respectively. Under the voltage-clamp condition, no voltage-dependent currents were evoked. ACh or His added to the bathing solution hyperpolarized the membrane by activating a time- and voltage- independent K+ current. The ACh-induced hyperpolarization and K+ current persisted, while the His response desensitized quickly (< 1 min). These effects of ACh and His were mediated predominantly by m3- muscarinic and H1-His receptors, respectively. The K+ current induced by ACh and His was inhibited by charybdotoxin, suggesting that it is a Ca(2+)-activated K+ channel current (IK.Ca). The measurement of intracellular Ca2+ ([Ca2+]i) using Indo-1 revealed that both agents increased [Ca2+]i with similar time courses as they increased IK.Ca. When EGTA in the pipette solution was increased from 0.15 to 10 mM, the induction of IK.Ca by ACh and His was abolished. Thus, both ACh and His activate IK.Ca by increasing [Ca2+]i in JR-1 cells. In the Ca(2+)-free bathing solution (0.15 mM EGTA in the pipette), ACh evoked IK.Ca transiently. Addition of Ca2+ (1.8 mM) to the bath immediately restored the sustained IK.Ca. These results suggest that the ACh response is due to at least two different mechanisms; i.e., the Ca2+ release-related initial transient activation and the Ca2+ influx-related sustained activation of IK.Ca. Probably because of desensitization, the Ca2+ influx-related component of the His response could not be identified. Intracellularly applied inositol 1,4,5-trisphosphate (IP3), with and without inositol 1,3,4,5-tetrakisphosphate (IP4), mimicked the ACh response. IP4 alone did not affect the membrane current. Under the steady effect of IP3 or IP3 plus IP4, neither ACh nor His further evoked IK.Ca. Intracellular application of heparin or of the monoclonal antibody against the IP3 receptor, mAb18A10, inhibited the ACh and His responses in a concentration-dependent fashion. Neomycin, a phospholipase C (PLC) inhibitor, also inhibited the agonist-induced response in a concentration-dependent fashion. Although neither pertussis toxin (PTX) nor N-ethylmaleimide affected the ACh or His activation of IK,Ca, GDP beta S attenuated and GTP gamma S enhanced the agonist response.(ABSTRACT TRUNCATED AT 400 WORDS)     

Sphingosylphosphorylcholine induces endothelial cell migration and morphogenesis

Sphingosylphosphorylcholine (SPC) is one of the biologically active phospholipids that may act as extracellular messengers. Particularly important is the role of these lipids in the angiogenic response, a complex process involving endothelial cell migration, proliferation, and morphologic differentiation. Here we demonstrate that SPC and its hydrolytic product, sphingosine, induce chemotactic migration of human and bovine endothelial cells. The response is approximately equal to that elicited by vascular endothelial cell growth factor. The effect of SPC and sphingosine was associated with a rapid down-regulation of Edg1, a sphingosine 1-phosphate (SPP)-specific receptor involved in endothelial cell chemotaxis. Both SPC and sphingosine induced differentiation of endothelial cells into capillary-like structures in vitro. Thus, SPC and sphingosine join SPP among the biologically active lipids with angiogenic potential. Since neuronal abnormalities accompany pathological accumulation of SPC in brain tissue, it is possible that SPC is a modulator of angiogenesis in neural tissue upon its release from brain cells following trauma or neoplastic growth.     

Heterodimerization with Its Splice Variant Blocks the Ghrelin Receptor 1a in a Non-signaling Conformation: A STUDY WITH A PURIFIED HETERODIMER ASSEMBLED INTO LIPID DISCS*

Heterodimerization of G protein-coupled receptors has an impact on their signaling properties, but the molecular mechanisms underlying heteromer-directed selectivity remain elusive. Using purified monomers and dimers reconstituted into lipid discs, we explored how dimerization impacts the functional and structural behavior of the ghrelin receptor. In particular, we investigated how a naturally occurring truncated splice variant of the ghrelin receptor exerts a dominant negative effect on ghrelin signaling upon dimerization with the full-length receptor. We provide direct evidence that this dominant negative effect is due to the ability of the non-signaling truncated receptor to restrict the conformational landscape of the full-length protein. Indeed, associating both proteins within the same disc blocks all agonist- and signaling protein-induced changes in ghrelin receptor conformation, thus preventing it from activating its cognate G protein and triggering arrestin 2 recruitment. This is an unambiguous demonstration that allosteric conformational events within dimeric assemblies can be directly responsible for modulation of signaling mediated by G protein-coupled receptors.     

Differential pharmacological properties and signal transduction of the sphingosine 1-phosphate receptors EDG-1, EDG-3, and EDG-5.

Sphingosine 1-phosphate (SPP) is a potent lipid mediator released upon cellular activation. In this report, pharmacological properties of the three G-protein-coupled receptors (GPCRs) for SPP, EDG-1, -3, and -5 are characterized using a Xenopus oocyte expression system, which lacks endogenous SPP receptors. Microinjection of the EDG-3 and EDG-5 but not EDG-1 mRNA conferred SPP-responsive intracellular calcium transients; however, the EDG-5 response was quantitatively much less. Co-expression of EDG-1 receptor with the chimeric Galphaqi protein conferred SPP responsiveness. Galphaqi or Galphaq co-injection also potentiated the EDG-5 and EDG-3 mediated responses to SPP. These data suggest that SPP receptors couple differentially to the Gq and Gi pathway. All three GPCRs were also activated by sphingosylphosphorylcholine, albeit at higher concentrations. None of the other related sphingolipids tested stimulated or blocked SPP-induced calcium responses. However, suramin, a polycyclic anionic compound, selectively antagonized SPP-activated calcium transients in EDG-3 expressing oocytes with an IC50 of 22 microM, suggesting that it is an antagonist selective for the EDG-3 GPCR isotype. We conclude that the three SPP receptors signal differentially by coupling to different G-proteins. Furthermore, because only EDG-3 was antagonized by suramin, variations in receptor structure may determine differences in antagonist selectivity. This property may be exploited to synthesize receptor subtype-specific antagonists.     

乙酰胆碱对蛇运动神经末梢离子通道活动的反馈调节

由研究乙酰胆碱受体激动剂和阻断剂的作用提出,在脊椎动物运动神经末梢存在着对乙酰胆碱（ＡＣｈ）释放的反馈调节。神经末梢的离了通道在递质释放中有重要作用。本文是利用周膜下记录技术。研究ＡＣｈ对蛇运动神经末梢离子通道调节作用的报告。（１）２ｍｍｏｌ／ＬＡＣｈ明显抑制依钙Ｋ流（ＩＫ,Ｃａ）此效应与３ｍｍｏｌ／ＬＴＥＡ的相似。由于ｎＡＣｈＲ激动剂尼古丁（２ｍｍｏｌ／Ｌ） 不影响Ｉｋ,ｆ和ＩＫ,Ｃdisplay stat     

The bioactive lipid sphingosylphosphorylcholine induces differentiation of mouse embryonic stem cells and human promyelocytic leukaemia cells

Sphingosylphosphorylcholine (SPC) is the major component of high-density lipoproteins (HDL) in blood plasma. The bioactive lipid acts mainly via G protein coupled receptors (GPCRs). Similar to ligands of other GPCRs, SPC has multiple biological roles including the regulation of proliferation, migration, angiogenesis, wound healing and heart rate. Lysophospholipids and their receptors have also been implicated in cell differentiation. A potential role of SPC in stem cell or tumour cell differentiation has been elusive so far. Here we examined the effect of SPC on the differentiation of mouse embryonic stem (ES) cells and of human NB4 promyelocytic leukemia cells, a well established tumour differentiation model. Our data show that mouse embryonic stem cells and NB4 cells express the relevant GPCRs for SPC. We demonstrate both at the level of morphology and of gene expression that SPC induces neuronal and cardiac differentiation of mouse ES cells. Furthermore, SPC induces differentiation of NB4 cells by a mechanism which is critically dependent on the activity of the MEK-ERK cascade. Thus, the bioactive lipid SPC is a novel differentiation inducing agent both for mouse ES cells, but also of certain human tumour cells.     

Sphingosylphosphorylcholine is a ligand for ovarian cancer G-protein-coupled receptor 1

Sphingosylphosphorylcholine (SPC) is a bioactive lipid that acts as an intracellular and extracellular signalling molecule in numerous biological processes. Many of the cellular actions of SPC are believed to be mediated by the activation of unidentified G-protein-coupled receptors. Here we show that SPC is a high-affinity ligand for an orphan receptor, ovarian cancer G-protein-coupled receptor 1 (OGR1). In OGR1-transfected cells, SPC binds to OGR1 with high affinity (Kd = 33.3 nM) and high specificity and transiently increases intracellular calcium. The specific binding of SPC to OGR1 also activates p42/44 mitogen-activated protein kinases (MAP kinases) and inhibits cell proliferation. In addition, SPC causes internalization of OGR1 in a structurally specific manner.     

Dissociation of Calmodulin-Target Peptide Complexes by the Lipid Mediator Sphingosylphosphorylcholine: IMPLICATIONS IN CALCIUM SIGNALING*

Previously we have identified the lipid mediator sphingosylphosphorylcholine (SPC) as the first potentially endogenous inhibitor of the ubiquitous Ca²⁺ sensor calmodulin (CaM) (Kovacs, E., and Liliom, K. (2008) Biochem. J. 410, 427–437). Here we give mechanistic insight into CaM inhibition by SPC, based on fluorescence stopped-flow studies with the model CaM-binding domain melittin. We demonstrate that both the peptide and SPC micelles bind to CaM in a rapid and reversible manner with comparable affinities. Furthermore, we present kinetic evidence that both species compete for the same target site on CaM, and thus SPC can be considered as a competitive inhibitor of CaM-target peptide interactions. We also show that SPC disrupts the complex of CaM and the CaM-binding domain of ryanodine receptor type 1, inositol 1,4,5-trisphosphate receptor type 1, and the plasma membrane Ca²⁺ pump. By interfering with these interactions, thus inhibiting the negative feedback that CaM has on Ca²⁺ signaling, we hypothesize that SPC could lead to Ca²⁺ mobilization in vivo. Hence, we suggest that the action of the sphingolipid on CaM might explain the previously recognized phenomenon that SPC liberates Ca²⁺ from intracellular stores. Moreover, we demonstrate that unlike traditional synthetic CaM inhibitors, SPC disrupts the complex between not only the Ca²⁺-saturated but also the apo form of the protein and the target peptide, suggesting a completely novel regulation for target proteins that constitutively bind CaM, such as ryanodine receptors.     

Mechanism of acetylcholine-induced inhibition of Ca current in bullfrog atrial myocytes

The mechanism of the anti-beta-adrenergic action of acetylcholine (ACh) on Ca current, ICa, was examined using the tight-seal, whole-cell voltage clamp technique in single atrial myocytes from the bullfrog. Both isoproterenol (ISO) and forskolin increased ICa dose dependently. After ICa had been enhanced maximally by ISO (10(-6) M), subsequent application of forskolin (50 microM) did not further increase ICa, suggesting that ISO and forskolin increase ICa via a common biochemical pathway, possibly by stimulation of adenylate cyclase. ACh (10(-5) M) completely inhibited the effect of low doses of forskolin (2 x 10(-6) M), as well as ISO, but it failed to block the effects of high doses of forskolin (greater than 5 x 10(-5) M). Intracellular application of cyclic AMP (cAMP) also increased ICa. ACh (10(-5) M) failed to inhibit this cAMP effect, indicating that the inhibitory action of ACh occurs at a site proximal to the production of cAMP. ACh (10(-5) M) also activated an inwardly rectifying K+ current IK(ACh). Intracellular application of a nonhydrolyzable GTP analogue, GTP gamma S (5 X 10(-4) M), activated IK(ACh) within several minutes; subsequent application of ACh (10(-5) M) did not increase IK(ACh) further. These results demonstrate that a GTP-binding protein coupled to these K+ channels can be activated maximally by GTP gamma S even in the absence of ACh. Intracellular application of GTP gamma S also strongly inhibited the effect of ISO on ICa in the absence of ACh. Pertussis toxin (IAP) completely prevented both the inhibitory effect of ACh on ICa and the ACh-induced activation of IK(ACh). GTP gamma S (50 microM-1 mM) alone did not increase ICa significantly; however, when ISO was applied first, GTP gamma S (5 x 10(-4) M) gradually inhibited the ISO effect on ICa. These results indicate that ACh antagonizes the effect of ISO on ICa via a GTP-binding protein (Gi and/or Go). This effect may be mediated through a direct inhibition by the alpha-subunit of Gi which is coupled to the adenylate cyclase.