Lutz Pott

Sphingosylphosphocholine is a naturally occurring lipid mediator in blood plasma: a possible role in regulating cardiac function via sphingolipid receptors.

Blood plasma and serum contain factors that activate inwardly rectifying GIRK1/GIRK4 K+ channels in atrial myocytes via one or more non-atropine-sensitive receptors coupled to pertussis-toxin-sensitive G-proteins. This channel is also the target of muscarinic M(2) receptors activated by the physiological release of acetylcholine from parasympathetic nerve endings. By using a combination of HPLC and TLC techniques with matrix-assisted laser desorption ionization-time-of-flight MS, we purified and identified sphingosine 1-phosphate (SPP) and sphingosylphosphocholine (SPC) as the plasma and serum factors responsible for activating the inwardly rectifying K+ channel (I(K)). With the use of MS the concentration of SPC was estimated at 50 nM in plasma and 130 nM in serum; those concentrations exceeded the 1.5 nM EC(50) measured in guinea-pig atrial myocytes. With the use of reverse-transcriptase-mediated PCR and/or Western blot analysis, we detected Edg1, Edg3, Edg5 and Edg8 as well as OGR1 sphingolipid receptor transcripts and/or proteins. In perfused guinea-pig hearts, SPC exerted a negative chronotropic effect with a threshold concentration of 1 microM. SPC was completely removed after perfusion through the coronary circulation at a concentration of 10 microM. On the basis of their constitutive presence in plasma, the expression of specific receptors, and a mechanism of ligand inactivation, we propose that SPP and SPC might have a physiologically relevant role in the regulation of the heart.

A novel membrane receptor with high affinity for lysosphingomyelin and sphingosine 1-phosphate in atrial myocytes

Activation of IK(ACh) is the major effect of the vagal neutrotransmitter acetylcholine in the heart. We report that both lysosphingomyelin (D‐erythro‐sphingosyl‐phosphorylcholine; SPC) and sphingosine 1‐phosphate (SPP) activate IK(ACh) in guinea pig atrial myocytes through the same receptor with an EC50 of 1.5 and 1.2 nM, respectively. Pertussis toxin abolished the activation of IK(ACh) by either lipid. The putative receptor showed an exquisite stereoselectivity for the naturally occurring D‐erythro‐(2S,3R)‐SPC stereoisomer, the structure of which was confirmed by mass spectroscopy and NMR. These lipids caused complete homologous and heterologous desensitization with each other but not with ACh, indicating that both act on the same receptor. This receptor displays a distinct structure‐activity relationship: it requires an unsubstituted amino group because N‐acetyl‐SPC, lysophosphatidic acid and lysophosphatidylcholine were inactive. Because SPP and SPC are naturally occurring products of membrane lipid metabolism, it appears that these compounds might be important extracellular mediators acting on a family of bona fide G protein‐coupled receptors. Expression of these receptors in the heart raises the possibility that sphingolipids may be a part of the physiological and/or pathophysiological regulation of the heart. Based on their ligand selectivity we propose a classification of the sphingolipid receptors.

Activation of muscarinic K+ current in guinea‐pig atrial myocytes by sphingosine‐1‐phosphate.

1. Activation of muscarinic K+ current (IK(ACh)) by sphingosine‐1‐phosphate (Sph‐1‐P) was studied in isolated cultured guinea‐pig atrial myocytes using whole‐cell voltage clamp. 2. Sph‐1‐P caused activation of IK(ACh) with an EC50 of 1.2 nM. The maximal current that could be activated by Sph‐1‐P amounted to about 90% of the IK(ACh) caused by a saturating concentration of acetylcholine (ACh, 10 microM). Sphingosine (1 microM), which can mimic the signalling effects of Sph‐1‐P in other cells, failed to cause measurable activation of IK(ACh). 3. IK(ACh) activation by Sph‐1‐P was completely suppressed in cells treated with pertussis toxin. 4. Desensitization of muscarinic receptors by pre‐incubation of the cells with carbachol did not affect the response to Sph‐1‐P; likewise, pre‐incubation of the cells with Sph‐1‐P resulted in a reduced sensitivity to the phospholipid but not to ACh. In contrast, pre‐incubation with either Sph‐1‐P or a serum phospholipid previously described as activating atrial IK(ACh) resulted in reduced sensitivity to both phospholipids. 5. It is concluded that activation of IK(ACh) by Sph‐1‐P in atrial myocytes is induced by binding to a novel G protein‐coupled phospholipid receptor.

Removal of Ca current inactivation in dialysed guinea-pig atrial cardioballs by Ca chelators

Abstract1.Ca currents flowing during voltage clamp depolarizations were studied in cultured guinea-pig atrial cardioballs by means of single low resistance patch clamp pipettes.2.The pipettes were filled with solutions containing Cs+ as major cation in order to block K+ currents and high concentrations of various Ca chelating agents (EGTA, nitrilotriacetic acid, citrate, dipicolinic acid) to prevent rises of the intracellular Ca-activity by Ca-entry.3.Ca currents of myocytes loaded with 20 mM of either EGTA [(ethylenedioxy)-diethylenedinitrilo)tetra-acetic acid] or NTA (nitrilotriacetic acid) display a biphasic time course of inactivation at membrane potentials between −25 and +45 mV. The fast phase is reduced with increasingly positive membrane potentials.4.In cells loaded with either citrate or DPA (dipicolinic acid, pyridine-2,6-dicarboxylic acid) inactivation is negligible or absent for small depolarizations. In the range of membrane potentials where maximum current flows (0−+10 mV) a monophasic slow time course of inactivation is observed. At more positive membrane potentials inactivation is slowed. The amount of inactivation under this condition is related to the current density of the cell.5.Conditions, which for a given membrane potential reduce the amplitude ofICa such as extracellular application of blocking ions (Co2+, Cd2+), a conditioning depolarization, or ‘rundown’ of Ca-channels lead to a slowing or a complete removal of inactivation in cells dialysed with citrate or DPA respectively. Cells loaded with these Ca chelators did not show any symptom of voltage dependent inactivation ofICa.6.Under the conditions described action potentials were recorded in the current clamp mode. Upon dialysis with EGTA the typical ‘triangular shaped’ atrial action potential develops a plateau of 500 to 800 ms in duration. With citrate-containing pipette solutions the action potential duration usually is several seconds.7.The results for the first time demonstrate that inactivation of cardiacICa can be considerably slowed or even removed. They provide further strong support for the hypothesis that inactivation of this current depends on Ca entry rather than membrane potential. The fast phase of inactivation observed with EGTA (NTA) possibly reflets the slow kinetics of the binding reaction of this type of Ca chelators.

Simultaneous recording of Indo-1 flourescence and Na+/Ca2+ exchange current reveals two components of Ca2+-release from sarcoplasmic reticulum of cardiac atrial myocytes.

Simultaneous measurements of intracellular Ca2+‐concentration ([Ca2+]1) using Indo‐1 and the current generated by electrogenic Na1/Ca2+‐exchange (INa??? have been performed on atrial myocytes from hearts of adult guinea‐pigs. Whereas the flourescence‐measurements provide information on global[Ca2+]i, INa??? which is a linear function of Ca2+‐concentration, indicates subsarcolemmal [Ca2+]. Under conditions in which intracellular Ca2+‐transients due to Ca2+‐release from the sarcoplasmic reticulum (SR) have been artificially slowed, a deviation between the two different Ca2+‐signals can be found. During onset of release signals Ca2+‐concentration seen by the membrane is higher than global [Ca2+]1. Our results provide evidence that in atrial myocytes, lacking a T‐system, Ca2+‐release occurs first from a subsarcolemmal compartment of the SR. The resulting Ca2+‐transient serves to trigger Ca2+‐release from deeper SR‐compartments.

Pannexin 1 Constitutes the Large Conductance Cation Channel of Cardiac Myocytes

A large conductance (∼300 picosiemens) channel (LCC) of unknown molecular identity, activated by Ca2+ release from the sarcoplasmic reticulum, particularly when augmented by caffeine, has been described previously in isolated cardiac myocytes. A potential candidate for this channel is pannexin 1 (Panx1), which has been shown to form large ion channels when expressed in Xenopus oocytes and mammalian cells. Panx1 function is implicated in ATP-mediated auto-/paracrine signaling, and a crucial role in several cell death pathways has been suggested. Here, we demonstrate that after culturing for 4 days LCC activity is no longer detected in myocytes but can be rescued by adenoviral gene transfer of Panx1. Endogenous LCCs and those related to expression of Panx1 share key pharmacological properties previously used for identifying and characterizing Panx1 channels. These data demonstrate that Panx1 constitutes the LCC of cardiac myocytes. Sporadic openings of single Panx1 channels in the absence of Ca2+ release can trigger action potentials, suggesting that Panx1 channels potentially promote arrhythmogenic activities.

Type 10 adenylyl cyclase mediates mitochondrial Bax translocation and apoptosis of adult rat cardiomyocytes under simulated ischaemia/reperfusion

AIMS Apoptosis of cardiomyocytes significantly contributes to the development of post-ischaemic cardiomyopathy. Although mitochondria have been suggested to play a crucial role in this process, the precise mechanisms controlling the mitochondria-dependent apoptosis in cardiomyocytes under ischaemia/reperfusion are still poorly understood. Here we aimed to analyse the role of the soluble adenylyl cyclase (sAC). METHODS AND RESULTS Adult rat cardiomyocytes were subjected to simulated in vitro ischaemia (SI) consisting of glucose-free anoxia at pH 6.4. Apoptosis was detected by DNA laddering, chromatin condensation, and caspases cleavage. SI led to the translocation of sAC to the mitochondria and mitochondrial depolarization followed by cytochrome c release, caspase-9/-3 cleavage and apoptosis during simulated reperfusion (SR). Pharmacological inhibition of sAC during SI, but not during SR, significantly reduced the SI/SR-induced mitochondrial injury and apoptosis. Similarly, sAC knock-down mediated by an adenovirus coding for shRNA targeting sAC prevented the activation of the mitochondrial pathway of apoptosis. Analysis of the link between sAC and apoptosis revealed a sAC and protein kinase A-dependent Bax phosphorylation at Thr(167) and its translocation to mitochondria during SI, which subsequently caused mitochondrial oxygen radical formation followed by cytochrome c release and caspase-9 cleavage during SR. CONCLUSION These results suggest a key role of sAC in SI-induced mitochondrial Bax translocation and activation of the mitochondrial pathway of apoptosis in adult cardiomyocytes.

Spontaneous tension oscillations in guinea-pig atrial trabeculae

SummarySpontaneous tension oscillations have been recorded from intact guinea-pig auricular trabeculae bathed in Na-poor and/or Ca-rich solutions.The frequency of these oscillations and that of after-contractions (oscillations following an electrically induced contraction) evoked under identical experimental conditions was the same (33°C).The amplitude of the oscillations rose when the [Ca2+]0/[Na+]02-ratio or the intracellular Na-concentration was increased. When the increase of the [Ca2+]0/[Na+]02-ratio was relatively small, tension oscillations only occured after a period of electrical stimulation.The oscillation-frequency increased slightly in media containing 70 instead of 5.4 mM KCl.MnCl2 (3mM) did not affect either the amplitude or the frequency of the oscillations.Caffeine (0.5–2.5 mM) decreased the amplitude and enhanced the frequency of the oscillations. After-contractions were diminished and, at higher concentrations, abolished.It is demonstrated that the membrane potential does not participate in the process causing the tension oscillations. An increased [Ca2+]i is a prerequisite for the occurrence of these oscillations. Characteristics of intracellular Ca-movement probably determine the amplitude and frequency of the spontaneous oscillations of tension.

Downregulation of muscarinic M2 receptors linked to K+ current in cultured guinea-pig atrial myocytes.

1. Desensitization of muscarinic K+ current (IK(ACh)) was studied in cultured atrial myocytes from guinea‐pig hearts using whole‐cell voltage clamp. 2. Three different types of desensitization could be identified. A fast component which upon rapid superfusion with ACh resulted in a partial relaxation of IK(ACh) within a few seconds to a plateau which was maintained in the presence of ACh. Recovery from this type of desensitization paralleled the decay of IK(ACh) after washout of the agonist. A second type of desensitization was observed within minutes. This was reversed around 10 min after washout of ACh. Both types were heterologous with regard to the A1 receptor and the novel phospholipid (Pl) receptor, both of which activate IK(ACh) via the same signalling pathway. 3. A third type of desensitization (downregulation) occurred upon exposure of the cultures for 24‐48 h to the muscarinic agonist carbachol (CCh). The level of downregulation depended on the concentration of CCh (0.1 microM < or = [CCh] < or = 10 microM). No recovery was observed within 5 h after washout of CCh. Thereafter sensitivity to ACh slowly returned (half‐time (t1/2), approximately 20 h). 4. Downregulation by CCh (0.1‐5 microM) was characterized by an increase in EC50 for ACh with no reduction in maximum IK(ACh). With 5 microM CCh, EC50 was increased from 0.1 to 3.7 microM. At 10 microM CCh EC50 was increased to 15 microM and maximal current that could be evoked by ACh was reduced to 15%. 5. Downregulation by CCh was homologous with regard to A1 and Pl receptors. Maximum IK(ACh), assayed by a saturating concentration of Pl, was not reduced in downregulated cells, suggesting a mechanism localized at the M2 receptor. 6. The changes in the concentration‐response curves can be accounted for by assuming an excess of M2 receptors relative to the subsequent component of the signalling pathway. 7. As the intact heart is under tonic vagal control, downregulation is likely to contribute to controlling the sensitivity of the heart to vagal activity in situ.

Extracellular links in Kir subunits control the unitary conductance of SUR/Kir6.0 ion channels

Potassium (K+) channels are highly selective for K+ ions but their unitary conductances are quite divergent. Although Kir6.1 and Kir6.2 are highly homologous and both form functional K+ channels with sulfonylurea receptors, their unitary conductances measured with 150 mM extracellular K+ are ∼35 and 80 pS, respectively. We found that a chain of three amino acid residues N123–V124–R125 of Kir6.1 and S113–I114–H115 of Kir6.2 in the M1–H5 extracellular link and single residues M148 of Kir6.1 and V138 of Kir6.2 in the H5–M2 link accounted for the difference. By using a 3D structure model of Kir6.2, we were able to recognize two independent plausible mechanisms involved in the determination of single channel conductance of the Kir6.0 subunits: (i) steric effects at Kir6.2V138 or Kir6.1M148 in the H5–M2 link influence directly the diffusion of K+ ions; and (ii) structural constraints between Kir6.2S113 or Kir6.1N123 in the M1–H5 link and Kir6.2R136 or Kir6.1R146 near the H5 region control the conformation of the permeation pathway. These mechanisms represent a novel and possibly general aspect of the control of ion channel permeability.