Differential Ca2+ and Sr2+ regulation of intracellular divalent cations release in ventricular myocytes.

The regulation of the Ca2+ -induced Ca2+ release (CICR) from intracellular stores is a critical step in the cardiac cycle. The inherent positive feedback of CICR should make it a self-regenerating process. It is accepted that CICR must be governed by some negative control, but its nature is still debated. We explore here the importance of the Ca2+ released from sarcoplasmic reticulum (SR) on the mechanisms that may control CICR. Specifically, we compared the effect of replacing Ca2+ with Sr2+ on intracellular Ca2+ signaling in intact cardiac myocytes as well as on the function of single ryanodine receptor (RyR) Ca2+ release channels in panar bilayers. In cells, both CICR and Sr2+ -induced Sr2+ release (SISR) were observed. Action potential induced Ca2+ -transients and spontaneous Ca2+ waves were considerably faster than their Sr2+ -mediated counterparts. However, the kinetics of Ca2+ and Sr2+ sparks was similar. At the single RyR channel level, the affinities of Ca2+ and Sr2+ activation were different but the affinities of Ca2+ and Sr2+ inactivation were similar. Fast Ca2+ and Sr2+ stimuli activated RyR channels equally fast but adaptation (a spontaneous slow transition back to steady-state activity levels) was not observed in the Sr2+ case. Together, these results suggest that regulation of the RyR channel by cytosolic Ca2+ is not involved in turning off the Ca2+ spark. In contrast, cytosolic Ca2+ is important in the propagation global Ca2+ release events and in this regard single RyR channel sensitivity to cytosolic Ca2+ activation, not low-affinity cytosolic Ca2+ inactivation, is a key factor. This suggests that the kinetics of local and global RyR-mediated Ca2+ release signals are affected in a distinct way by different divalent cations in cardiac muscle cells.

Percutaneous toxicity of uranyl nitrate: its effect in terms of exposure area and time.

Different groups have undertaken research work focusing their attention on the biological effects of uranium and have described kidney and bone to be the main target organs in uranium poisoning. In this study we used the skin as the route of entry of uranium. We carried out two sets of experiments in adult rats: in one of them topical applications with uranyl nitrate (UN) over different areas were performed; in the other topical applications with UN on a given area over different times were carried out. In the latter experiment the exposure to UN was stopped by removing it from skin with soap and water. Kidney and bone samples were removed for histological studies. This work is based on the determination of the survival rate of the exposed animals and on the effects elicited in kidney and bone. There is a relation between the area of the surface exposed to uranium and the time of exposure and the subsequent percutaneous toxicity. There were no surviving animals following topical application of UN to an 8 cm2 area nor when the time of exposure was 24 h. The survival rate of the animals increased when either the topical area or the time of exposure to UN was reduced. Although the inhibition of bone formation in metaphysical bone has been previously described by our group as a result of UN poisoning, this is the first time that such an effect is found after percutaneous exposure for such short periods of time. The general toxic effects of UN, evidenced as kidney histological alterations, increased in severity as either one of the two variables studied increased. This is a condition that could be considered as hazardous for those workers engaged in uranium processing and purification. It is noteworthy that a simple method such as washing with soap and water is an effective method to reduce the lethality of UN percutaneous intoxication.

Increased intracellular Ca2+ and SR Ca2+ load contribute to arrhythmias after acidosis in rat heart. Role of Ca2+/calmodulin-dependent protein kinase II.

Returning to normal pH after acidosis, similar to reperfusion after ischemia, is prone to arrhythmias. The type and mechanisms of these arrhythmias have never been explored and were the aim of the present work. Langendorff-perfused rat/mice hearts and rat-isolated myocytes were subjected to respiratory acidosis and then returned to normal pH. Monophasic action potentials and left ventricular developed pressure were recorded. The removal of acidosis provoked ectopic beats that were blunted by 1 muM of the CaMKII inhibitor KN-93, 1 muM thapsigargin, to inhibit sarcoplasmic reticulum (SR) Ca(2+) uptake, and 30 nM ryanodine or 45 muM dantrolene, to inhibit SR Ca(2+) release and were not observed in a transgenic mouse model with inhibition of CaMKII targeted to the SR. Acidosis increased the phosphorylation of Thr(17) site of phospholamban (PT-PLN) and SR Ca(2+) load. Both effects were precluded by KN-93. The return to normal pH was associated with an increase in SR Ca(2+) leak, when compared with that of control or with acidosis at the same SR Ca(2+) content. Ca(2+) leak occurred without changes in the phosphorylation of ryanodine receptors type 2 (RyR2) and was blunted by KN-93. Experiments in planar lipid bilayers confirmed the reversible inhibitory effect of acidosis on RyR2. Ectopic activity was triggered by membrane depolarizations (delayed afterdepolarizations), primarily occurring in epicardium and were prevented by KN-93. The results reveal that arrhythmias after acidosis are dependent on CaMKII activation and are associated with an increase in SR Ca(2+) load, which appears to be mainly due to the increase in PT-PLN.

Peritubular fluid viscosity modulates H+ flux in proximal tubules through NO release.

We evaluated the effects of increasing the viscosity (eta) in peritubular capillary perfusates (PCP; 20 mM HNaPO4--Ringer, pH 7.4) on proximal convoluted tubule (PCT) acidification. Micropuncture experiments were performed with simultaneous luminal and peritubular perfusion. Changes in pH of a 20 mM HNaPO4--Ringer (pH 7.4 at t = 0) droplet placed in PCT lumen were measured with H+-sensitive microelectrodes. By adding neutral dextran (molecular wt 300,000-400,000) to the PCP, eta was increased. The effect of 10(-5) M ATP added to normal-eta PCP was evaluated. High eta increased H+ flux (85 and 97% when eta was increased 20 and 30%, respectively, above the control value). This increase was abolished by adding the nitric oxide antagonist N(omega)-nitro-L-arginine (L-NNA; 10(-4) M) or the purinoreceptor antagonists suramin (10(-4) M) and reactive blue 2 (3 x 10(-5) M). Addition of 5 x 10(-3) M L-arginine to the peritubular perfusate overcame the inhibitory effect of L-NNA on high-eta-induced increase in H+ flux. ATP increased H+ flux (80%), and this effect was blocked by L-NNA. These results suggest that changes in eta can modulate proximal H+ flux, at least in part, through ATP-dependent nitric oxide release from the endothelial cells of the peritubular capillaries.

Differential activation by Ca2+, ATP and caffeine of cardiac and skeletal muscle ryanodine receptors after block by Mg2+.

The block of rabbit skeletal ryanodine receptors (RyR1) and dog heart RyR2 by cytosolic [Mg2+], and its reversal by agonists Ca2+, ATP and caffeine was studied in planar bilayers. Mg2+ effects were tested at submaximal activating [Ca2+] (5 microM). Approximately one third of the RyR1s had low open probability ("LA channels") in the absence of Mg2+. All other RyR1s displayed higher activity ("HA channels"). Cytosolic Mg2+ (1 mM) blocked individual RyR1 channels to varying degrees (32 to 100%). LA channels had residual P(o) <0.005 in 1 mM Mg2+ and reactivated poorly with [Ca2+] (100 microM), caffeine (5 mM), or ATP (4 mM; all at constant 1 mM Mg2+). HA channels had variable activity in Mg2+ and variable degree of recovery from Mg2+ block with Ca2+, caffeine or ATP application. Nearly all cardiac RyR2s displayed high activity in 5 microM [Ca2+]. They also had variable sensitivity to Mg2+. However, the RyR2s consistently recovered from Mg2+ block with 100 microM [Ca2+] or caffeine application, but not when ATP was added. Thus, at physiological [Mg2+], RyR2s behaved as relatively homogeneous Ca2+/caffeine-gated HA channels. In contrast, RyR1s displayed functional heterogeneity that arises from differential modulatory actions of Ca2+ and ATP. These differences between RyR1 and RyR2 function may reflect their respective roles in muscle physiology and excitation-contraction coupling.