Permeation through store-operated CRAC channels in divalent-free solution: potential problems and implications for putative CRAC channel genes.

CRAC channels are key calcium conduits in both physiological and pathological states. Understanding how these channels are controlled is important as this will not only provide insight into a novel signal transduction pathway coupling intracellular stores to the channels in the plasma membrane, but might also be of clinical relevance. Determining the molecular identity of the CRAC channels will certainly be a major step forward. Like all Ca(2+)-selective channels, CRAC channels lose their selectivity in divalent-free external solution to support large monovalent Na(+) currents. This approach has provided new insight into channel permeation and selectivity, and identifies some interesting differences between CRAC channels and voltage-operated calcium channels (VOCCs). Studies in divalent-free solution are a double-edged sword, however. Electrophysiologists need to be wary because some of the conditions used to study I(CRAC) in divalent-free external solution, notably omission of Mg(2+)/Mg-ATP from the recording pipette solution, activates an additional current permeating through Mg(2+)-nucleotide-regulated metal ion current (MagNuM; TRPM7) channels. This channel underlies the large single-channel events that have been attributed to CRAC channels in the past and which have been used to as a tool to identify store-operated channels in native cells and recombinant expression systems.Are we any closer to identifying the elusive CRAC channel gene(s)? TRPV6 seemed a very attractive candidate, but one of the main arguments supporting it was a single-channel conductance in divalent-free solution similar to that for CRAC reported under conditions where MagNuM is active. We now know that the conductance of TRPV6 is approximately 200-fold larger than that of CRAC in native tissue. Moreover, it is unclear if TRPV6 is store-operated. Further work on TRPV6, particularly whether its single-channel conductance is still high under conditions where it apparently forms multimers with endogenous store-operated channels, and whether it is activated by a variety of store depletion protocols, will be helpful in finally resolving this issue.

[Disturbances of rhythm and atrio-ventricular conduction in digitalis overdose. Case reports]. / Zaburzenia rytmu i przewodzenia przedsionkowo-komorowego wystepujace przy przedawkowaniu naparstnicy. Opis przypadków.

Three cases of patients with symptoms of digitalis overdosage were presented. The principal manifestations included complex supraventricular dysrhythmias and atrio-ventricular conduction disturbances. In the discussion a special attention was paid to digitalis dosage. Multiple factors influencing plasma concentration of digitalis including pharmacokinetics, bioavailability and drug interactions with glycosides were described. Short review of toxic manifestations of digitalis was made and the treatment of digitalis intoxication was outlined.

Energized mitochondria increase the dynamic range over which inositol 1,4,5-trisphosphate activates store-operated calcium influx.

In eukaryotic cells, activation of cell surface receptors that couple to the phosphoinositide pathway evokes a biphasic increase in intracellular free Ca2+ concentration: an initial transient phase reflecting Ca2+ release from intracellular stores, followed by a plateau phase due to Ca2+ influx. A major component of this Ca2+ influx is store-dependent and often can be measured directly as the Ca2+ release-activated Ca2+ current (I(CRAC)). Under physiological conditions of weak intracellular Ca2+ buffering, respiring mitochondria play a central role in store-operated Ca2+ influx. They determine whether macroscopic I(CRAC) activates or not, to what extent and for how long. Here we describe an additional role for energized mitochondria: they reduce the amount of inositol 1,4,5-trisphosphate (InsP3) that is required to activate I(CRAC). By increasing the sensitivity of store-operated influx to InsP3, respiring mitochondria will determine whether modest levels of stimulation are capable of evoking Ca2+ entry or not. Mitochondrial Ca2+ buffering therefore increases the dynamic range of concentrations over which the InsP3 is able to function as the physiological messenger that triggers the activation of store-operated Ca2+ influx.

An examination of the secretion-like coupling model for the activation of the Ca2+ release-activated Ca2+ current I(CRAC) in RBL-1 cells.

One popular model for the activation of store-operated Ca2+ influx is the secretion-like coupling mechanism, in which peripheral endoplasmic reticulum moves to the plasma membrane upon store depletion thereby enabling inositol 1,4,5-trisphosphate (InsP3) receptors on the stores to bind to, and thus activate, store-operated Ca2+ channels. This movement is regulated by the underlying cytoskeleton. We have examined the validity of this mechanism for the activation of I(CRAC), the most widely distributed and best characterised store-operated Ca2+ current, in a model system, the RBL-1 rat basophilic cell line. Stabilisation of the peripheral cytoskeleton, disassembly of actin microfilaments and disaggregation of microtubules all consistently failed to alter the rate or extent of activation of I(CRAC). Rhodamine-phalloidin labelling was used wherever possible, and revealed that the cytoskeleton had been significantly modified by drug treatment. Interference with the cytoskeleton also failed to affect the intracellular calcium signal that occurred when external calcium was re-admitted to cells in which the calcium stores had been previously depleted by exposure to thapsigargin/ionomycin in calcium-free external solution. Application of positive pressure through the patch pipette separated the plasma membrane from underlying structures (cell ballooning). However, I(CRAC) was unaffected irrespective of whether cell ballooning occurred before or after depletion of stores. Pre-treatment with the membrane-permeable InsP3 receptor antagonist 2-APB blocked the activation of I(CRAC). However, intracellular dialysis with 2-APB failed to prevent I(CRAC) from activating, even at higher concentrations than those used extracellularly to achieve full block. Local application of 2-APB, once I(CRAC) had been activated, resulted in a rapid loss of the current at a rate similar to that seen with the rapid channel blocker La3+. Studies with the more conventional InsP3 receptor antagonist heparin revealed that occupation of the intracellular InsP3-sensitive receptors was not necessary for the activation or maintenance of I(CRAC). Similarly, the InsP3 receptor inhibitor caffeine failed to alter the rate or extent of activation of I(CRAC). Exposure to Li+, which reduces InsP3 levels by interfering with inositol monophosphatase, also failed to alter I(CRAC). Caffeine and Li+ did not affect the size of the intracellular Ca2+ signal that arose when external Ca2+ was re-admitted to cells which had been pre-exposed to thapsigargin/ionomycin in Ca2+-free external solution. Our findings demonstrate that the cytoskeleton does not seem to regulate calcium influx and that functional InsP3 receptors are not required for activation of I(CRAC). If the secretion-like coupling model indeed accounts for the activation of I(CRAC) in RBL-1 cells, then it needs to be revised significantly. Possible modifications to the model are discussed.

Sarcoplasmic/endoplasmic-reticulum-Ca2+-ATPase-mediated Ca2+ reuptake, and not Ins(1,4,5)P3 receptor inactivation, prevents the activation of macroscopic Ca2+ release-activated Ca2+ current in the presence of physiological Ca2+ buffer in rat basophilic leukaemia-1 cells.

Whole-cell patch-clamp experiments were performed to examine the mechanism underlying the inability of intracellular Ins(1,4,5)P(3) to activate the Ca(2+) release-activated Ca(2+) current (I(CRAC)) in rat basophilic leukaemia (RBL)-1 cells under conditions of weak cytoplasmic Ca(2+) buffering. Dialysis with Ins(1,4,5)P(3) in weak Ca(2+) buffer did not activate any macroscopic I(CRAC) even after precautions had been taken to minimize the extent of Ca(2+) entry during the experiment. Following intracellular dialysis with Ins(1,4,5)P(3) for >150 s in weak buffer, external application of the sarcoplasmic/endoplasmic-reticulum Ca(2+)-ATPase (SERCA) pump blocker thapsigargin activated I(CRAC), and the current developed much more quickly than when thapsigargin was applied in the absence of Ins(1,4,5)P(3). This indicates that the Ins(1,4,5)P(3) receptors had not inactivated much over this timecourse. When external Ca(2+) was replaced by Ba(2+), Ins(1,4,5)P(3) still failed to generate any detectable I(CRAC) even though Ba(2+) permeates CRAC channels and is not taken up into the intracellular Ca(2+) stores. In strong Ca(2+) buffer, I(CRAC) could be activated by muscarinic-receptor stimulation, provided protein kinase C (PKC) was blocked. In weak buffer, however, as with Ins(1,4,5)P(3), stimulation of these receptors with carbachol did not activate I(CRAC) even after inhibition of PKC. The inability of Ins(1,4,5)P(3) to activate macroscopic I(CRAC) in weak Ca(2+) buffer was not altered by inhibition of Ca(2+)-dependent phosphorylation/dephosphorylation reactions. Our results suggest that the inability of Ins(1,4,5)P(3) to activate I(CRAC) under conditions of weak intracellular Ca(2+) buffering is not due to strong inactivation of the Ins(1,4,5)P(3) receptors. Instead, a futile Ca(2+) cycle across the stores seems to be occurring and SERCA pumps resequester sufficient Ca(2+) to ensure that the threshold for activation of macroscopic I(CRAC) has not been exceeded.

Voltage-dependent conductance changes in the store-operated Ca2+ current ICRAC in rat basophilic leukaemia cells.

Tight-seal whole-cell patch-clamp experiments were carried out in order to investigate the effects of different holding potentials on the rate of development and amplitude of the Ca2+ release-activated Ca2+ current ICRAC in rat basophilic leukaemia (RBL-1) cells. ICRAC was monitored at -80 mV from fast voltage ramps, spanning 200 mV in 50 ms. At hyperpolarised potentials, the macroscopic CRAC conductance was lower than that seen at depolarised potentials. The conductance increased almost 5-fold over the voltage range -60 to +40 mV and was seen when the stores were depleted either by the combination of IP3 and thapsigargin in high Ca2+ buffer, or passively with 10 mM EGTA or BAPTA. The voltage-dependent conductance of the CRAC channels could not be fully accounted for by Ca2+-dependent fast inactivation, nor by other slower inhibitory mechanisms. It also did not seem to involve intracellular Mg2+ or the polycations spermine and spermidine. Voltage step relaxation experiments revealed that the voltage-dependent conductance changes developed and reversed slowly, with a time constant of several seconds at -60 mV. In the presence of physiological levels of intracellular Ca2+ buffers, ICRAC was barely detectable when cells were clamped at -60 mV and dialysed with IP3 and thapsigargin, but at 0 mV the current in low Ca2+ buffer was as large as that seen in high Ca2+ buffer. Our results suggest that CRAC channels exhibit slow voltage-dependent conductance changes which can triple the current amplitude over the physiological range of voltages normally encountered by these cells. The role of this conductance change and possible underlying mechanisms are discussed.

[The significance of lowered ejection fraction on prognosis after myocardial infarction ]. / Znaczenie obnizonej frakcji wyrzutowej w rokowaniu po zawale serca.

The subject of this study was a group of 757 patients hospitalized because of acute myocardial infarction in years 1992-1996, who survived the in-hospital course and were under observation for 2-6 years after the infarction. During the 14-18th day of the hospital stay they were made an echocardiographic test, including the ejection fraction (EF). The aim of this study was to define the influence of the ejection fraction value lowered below 40% on the long-term prognosis in patients after acute myocardial infarction. We compared two groups of patients; group I, consisting of 130 (17.2%) patients with EF lowered below 40% and group II, which included 627 patients with EF over 40%. To estimate the statistic significance we used the chi-square and t-Student test. The morbidity curves were made with the Kaplan-Meier method. The course of the myocardial infarction was much more grave in group I than in group II, what is confirmed by a more often anterior ventricular infarction and the quantity of dangerous complications which occurred during the in-hospital phase. The multi-factor regressive analysis showed that the ejection fraction lowered below 40% raises 2.47 times (95% confidence interval 1.50-4.07) (p < 0.001) the risk of death during the first year after myocardial infarction and nearly two times during the 5 year follow-up, compared to patients with a higher EF value. The influence of the EF value lowered below 40% on the creation of an infarction was not significant. The EF value lowered below 40% in patients after acute myocardial infarction was a significant risk factor in the long-term prognosis. More than 40% of deaths during the long-term prognosis in this group were caused by heart failure.

[The prognostic value of electrocardiographic exercise test in patients after acute myocardial infarction]. / Wartosc rokownicza elektrokardiograficznej próby wysilkowej u pacjentów po niepowiklanym zawale serca.

The subject of this trial were 243 patients with uncomplicated acute myocardial infarction, hospitalized in years 1992-1996, who were made an electrocardiographic exercise test in the second or third week of the in-hospital stay and whose further history in the 2-6 period after myocardial infarction (average follow-up time lasted for 4.0 +/- 1.9 years) was known. The aim of this trial was to determine the influence of the positive exercise test on the long-term prognosis after acute myocardial infarction in a group with uncomplicated acute myocardial infarction. The course of infarction and the frequency of cardiac events (cardiac death, reinfarction, revascularisation) occurrence in 78 patients with positive exercise test (group I) were compared with a group of 165 patients with negative exercise test (group II). Both groups were compared in respect to age, gender, history of myocardial infarction, risk factors and the course of the infarction in the in-hospital period. The multivariable logistic regression analyse showed that the positive exercise test did not have a statistically significant influence on the increase of post-hospital morbidity but it correlates with significantly more frequent use of the invasive treatment and reinfarction during the follow-up period in this group. Negative exercise test in patients with uncomplicated acute myocardial was a significant factor of the good long-term prognosis.

[Change of lifestyle as a relevant therapy after myocardial infarction]. / Zmiana stylu zycia jako istotna terapia po zawale serca.

The risk of ischemic heart disease is connected with the definite mode of life. Improper nourishment, smoking, alcohol abuse, sedentary lifestyle and excessive mental stress cause disturbances leading to development of atherosclerosis. The change of the lifestyle may prevent from coronary heart disease and may play a main role in secondary prevention, making the prognosis after myocardial infarction much better. The epidemiological and clinical studies have shown the significance of particular risk factors reduction on survival after myocardial infarction and allowed to create the optimal preventive mode of life. Therefore the change of lifestyle should become the priority in the postinfarction therapy.

[Diagnosis of pericarditis]. / Diagnostyka zapalenia osierdzia.

Acute pericarditis can be divided into primary pericarditis--of unknown etiology and secondary pericarditis--caused by earlier diagnosed disease. The diagnosis of acute pericarditis is based on clinical manifestations and accessory investigations, among which the most important is echocardiography. However, the etiologic diagnosis of pericarditis seems to be much more difficult, being ascertained in no more than 30%. It makes the causative treatment impossible in many cases. The good knowledge of epidemiology and symptoms of different pericardial diseases is important to make the proper choice of investigative procedures. At the beginning, usually less invasive investigations are planned to confirm etiologic diagnosis. Subsequently, more invasive procedures like pericardiocentesis, pericardiotomy and pericardial biopsy are reserved to explain the cause of pericarditis. The exceptions are cardiac tamponade and suspected purulent pericarditis, when pericardiocentesis and surgical drainage are made promptly in the therapeutic and diagnostic aim.