The effects of manganese and barium on the cardiac pacemaker current,i f,in rabbit sino-atrial node myocytes

Summary The isolation of ionic fluxes contributing to electric currents through cell membranes often requires block of other undesired components which can be achieved, among others, by divalent cations. Mn²⁺ and Ba²⁺ are often used, for example, to block Ca and K currents. Here we have investigated the effects of these two cations on the properties of the hyperpolarization-activated pacemaker current i_f, in rabbit sino-atrial node myocytes, as obtained by voltage clamp analysis. We find that 2 mM Mn²⁺ shifts the i_f activation curve by 3.2±0.3 mV towards more positive values. However, when 1 mM Ba²⁺ is also added, the positive shift is more than halved (1.3±0.2 mV). We find, too, that in the absence of blocking cations the ACh-induced i_f inhibition is slightly higher than in their presence. These results indicate that the alteration of i_f kinetic properties by Ba²⁺ plus Mn²⁺-containing solutions is minimal.     

Manganese action potentials in mammalian cardiac muscle.

The membrane potential in guinea-pig's papillary muscles from right ventricle was recorded by glass microelectrodes and stimulation was effected by current pulses applied through a sucrose-gap. Action potentials with overshoot were recorded in the solution lacking Na+ and Ca++ but containing 2-95 mM Mn++. The overshoot was increased with the increase of [Mn++]o by about 30 mV/decade. Similar Mn++ dependent action potentials were also obtained in Na-free solution containing 0.6 mM Ca++. The results indicate that Mn inward current is sufficient to generate action potentials in cardiac muscle.     

Repolarization abnormalities in cardiomyocytes of dogs with chronic heart failure: role of sustained inward current

We previously showed that a canine model of chronic heart failure (HF) produced by multiple coronary microembolizations manifests ventricular arrhythmias similar to those observed in patients with chronic HF. In the present study, we used single canine cardiomyocytes isolated from the left ventricle (LV) of normal dogs (n = 13) and dogs with HF (n = 15) to examine the cellular substrate of these arrhythmias. Action potentials (APs) and ion currents were measured by perforated and whole cell patch clamp, respectively. We found prolonged APs and alterations of AP duration resulting in early afterdepolarizations (EADs) at the low pacing rates of 0.5 Hz and 0.2 Hz. Na+ channel blockers saxitoxin (STX, 100 nM) and lidocaine (90 microM) reduced AP duration dispersion and abolished EADs in HF cardiomyocytes. The steady-state current (Iss)-voltage relation, in the voltage range from -25 mV to 25 mV analogous to the AP plateau level, was significantly shifted inward in HF cardiomyocytes. STX and lidocaine shifted the Iss-voltage relationship in an outward direction. The shifts produced by both drugs was significantly greater in cardiomyocytes of dogs with HF, indicating an increase in inward current. In the experimental configuration in which K+ currents were blocked, the density of the steady-state Ca2+ current (ICa) was found to decrease in HF cardiomyocytes by approximately 33%. In contrast, the density of the steady-state Na+ current (INa) significantly (P < 0.01) increased in HF cardiomyocytes (0.17 +/- 0.06 pA/pF) compared with normal cells (0.08 +/- 0.02 pA/pF). The relative contribution of INa to the net inward current was greater in HF cardiomyocytes, as evident from the increased ratio of INa/ICa (from 0.22 to 0.68). These observation support a hypothesis that anomalous repolarization of HF cardiomyocytes is due, at least in part, to an increased steady-state inward Na+ current.     

High K+-induced release of somatostatin from the cortical preparation of rat brain.

Release of endogenous somatostatin (SRIF) from the rat cerebral cortical slices incubated in Krebs-bicarbonate buffer was increased from the basal rate of 3.4 +/- 0.6% of the total SRIF content in 15 min at [K+]o = 5.6 mM, to 13.1 +/- 1.6% upon raising the [K+]o to 56.6 mM. The high-K+ evoked SRIF release was absent when Ca++ in the medium was replaced by Mn++. The isolated synaptosomes from rat cerebral cortex contain 13.2 +/- 3.1 ng SRIF/mg protein compared to 0.33 +/- 0.01 ng/mg protein in the cortical tissue as a whole, suggesting that nerve terminals are the main source of the peptide released upon membrane depolarization.     

Calcium signalling: a historical account, recent developments and future perspectives

Ca2+ is a uniquely important messenger that penetrates into cells through gated channels to transmit signals to a large number of enzymes. The evolutionary choice of Ca2+ was dictated by its unusual chemical properties, which permit its reversible complexation by specific proteins in the presence of much larger amounts of other potentially competing cations. The decoding of the Ca2+ signal consists in two conformational changes of the complexing proteins, of which calmodulin is the most important. The first occurs when Ca2+ is bound, the second (a collapse of the elongated protein) when interaction with the targeted enzymes occurs. Soluble proteins such as calmodulin contribute to the buffering of cell Ca2+, but membrane intrinsic transporting proteins are more important. Ca2+ is transported across the plasma membrane (channel, a pump, a Na+/Ca2+ exchanger) and across the membrane of the organelles. The endoplasmic reticulum is the most dynamic store: it accumulates Ca2+ by a pump, and releases it via channels gated by either inositol 1,4,5-trisphosphate (IP3) and cyclic adenosine diphosphate ribose (cADPr). The mitochondrion is more sluggish, but it is closed-connected with the reticulum, and senses microdomains of high Ca2+ close to IP3 or cADPr release channels. The regulation of Ca2+ in the nucleus, where important Ca(2+)-sensitive processes reside, is a debated issue. Finally, if the control of cellular Ca2+ homeostasis somehow fails (excess penetration), mitochondria 'buy time' by precipitating inside Ca2+ and phosphate. If injury persists, Ca2(+)-death eventually ensues.     

Transmembrane potential of J774.2 mouse macrophage cells measured by microelectrode and ion distribution methods

The transmembrane potential (Em) of J774.2 macrophage cells measured by microelectrodes was -24.1 +/- 0.7 mV (mean +/- SEM). Em measured by lipophilic ion distribution was -35 +/- 2 mV or -40 +/- 2 mV, using a cation or anion, respectively. By any method, colchicine reduced Em by approximately 3 mV.     

Enzymatic characteristics of the catalytic subunit of the protein kinase complex of human lymphocytes]

In earlier reports we have shown the existence in human lymphocytes homogenate, of a cyclic-AMP dependent protein-kinase activity. We demonstrate by affinity chromatography that two subunits display respectively cyclic-AMP binding and phosphorylating properties. Divalent cations such as Ca++, Mg++ or Mn++ are required for enzymatic activity. ATP which is an obligatory cosubstrate acts as an inhibitor when its concentration is higher than 10(-6)M.     

Ambiquitous behavior of rabbit liver lactate dehydrogenase

Rabbit liver mitochondrial fraction shows lactate dehydrogenase activity. The enzyme can be released from particles by increasing the pH and the ionic strength of the medium. There is a narrow range of pH (6.8-7.4) and ionic strength (20-50 mM NaCl) in which the solubilization sharply increases. It has been shown that divalent anions (SO4(2-) and cations (Mg2+, Ca2+) are highly effective specific solubilizing agents. NADH (1.5 mM) and ATP (1.0 mM) were effective in solubilizing 50% of the enzyme bound, whereas the same concentrations of the analogs NAD+ and ADP had little effect. Cytosolic lactate dehydrogenase bound to the mitochondrial fraction and a saturation of particles by enzyme was observed in all experiments performed. The in vitro binding requires a short period of incubation between the enzyme and particles and the binding is independent of the temperature in the 0-37 degrees C range. Binding was prevented by 0.15 M NaCl. The bound enzyme is approximately 20% less active than the soluble one. The results described give support to the proposal that rabbit liver lactate dehydrogenase has an ambiquitous behavior, like other glycolytic enzymes, which have not a fixed intracellular localization.     

TRPM7 is regulated by halides through its kinase domain

Transient receptor potential melastatin 7 (TRPM7) is a divalent-selective cation channel fused to an atypical α-kinase. TRPM7 is a key regulator of cell growth and proliferation, processes accompanied by mandatory cell volume changes. Osmolarity-induced cell volume alterations regulate TRPM7 through molecular crowding of solutes that affect channel activity, including magnesium (Mg²⁺), Mg-nucleotides and a further unidentified factor. Here, we assess whether chloride and related halides can act as negative feedback regulators of TRPM7. We find that chloride and bromide inhibit heterologously expressed TRPM7 in synergy with intracellular Mg²⁺ ([Mg²⁺]_i) and this is facilitated through the ATP-binding site of the channel’s kinase domain. The synergistic block of TRPM7 by chloride and Mg²⁺ is not reversed during divalent-free or acidic conditions, indicating a change in protein conformation that leads to channel inactivation. Iodide has the strongest inhibitory effect on TRPM7 at physiological [Mg²⁺]_i. Iodide also inhibits endogenous TRPM7-like currents as assessed in MCF-7 breast cancer cells, where upregulation of SLC5A5 sodium-iodide symporter enhances iodide uptake and inhibits cell proliferation. These results indicate that chloride could be an important factor in modulating TRPM7 during osmotic stress and implicate TRPM7 as a possible molecular mechanism contributing to the anti-proliferative characteristics of intracellular iodide accumulation in cancer cells.     

Characterization of the (Na+-K+)-ATPase from endometrial plasma membranes of immature mammals]

The (Na+-K+)-ATPase in plasma membrane from Mammiferous endometrium is characterized by the Mg/ATP ratio equal to one, and by a distinct affinity for Na+ (1.3 mM) and K+ (2 mM). The activity is maximum for pH 7.4-7.5 in presence of Mg++ 2mM and ATP 2 mM, Na+ 140 mM and K+ 10 mM.     

Zinc ions block the intracellular calcium release induced by caffeine in guinea-pig taenia caeci

Zn²⁺ in low concentrations (0.005–0.1 mM) inhibited the transient contractions in response to caffeine (25 mM) in a dose-dependent manner in smooth muscle of intact guinea-pig taenia caeci. At Zn²⁺ concentrations higher than 0.1 mM, caffeine did not elicit any response. After saponin-treatment of the fibres, which leaves the Ca²⁺ storage sites intact, caffeine contraction was completely inhibited by Zn²⁺ at a relatively low concentration (0.03 mM). However, in Triton-X-100-treated fibres, in which the Ca²⁺ release sites are destroyed, the contraction could be induced in the presence of Zn²⁺ by an increase in Ca²⁺. In conclusion, Zn²⁺ can block the intracellular Ca²⁺ release from caffeine-sensitive release sites in taenia caeci.     

Ca<Superscript>2+</Superscript> signals,cell membrane disintegration,and activation of TMEM16F during necroptosis

Activated receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain like (MLKL) are essential components of the necroptotic pathway. Phosphorylated MLKL (pMLKL) is thought to induce membrane leakage, leading to cell swelling and disintegration of the cell membrane. However, the molecular identity of the necroptotic membrane pore remains unclear, and the role of pMLKL for membrane permeabilization is currently disputed. We observed earlier that the phospholipid scramblase and ion channel TMEM16F/anoctamin 6 cause large membrane currents, cell swelling, and cell death when activated by a strong increase in intracellular Ca²⁺. We, therefore, asked whether TMEM16F is also central to necroptotic cell death and other cellular events during necroptosis. Necroptosis was induced by TNFα, smac mimetic, and Z-VAD (TSZ) in NIH3T3 fibroblasts and the four additional cell lines HT₂₉, 16HBE, H441, and L929. Time-dependent changes in intracellular Ca²⁺, cell morphology, and membrane currents were recorded. TSZ induced a small and only transient oscillatory rise in intracellular Ca²⁺, which was paralleled by the activation of outwardly rectifying Cl^? currents, which were typical for TMEM16F/ANO6. Ca²⁺ oscillations were due to Ca²⁺ release from endoplasmic reticulum, and were independent of extracellular Ca²⁺. The initial TSZ-induced cell swelling was followed by cell shrinkage. Using typical channel blockers and siRNA-knockdown, the Cl^? currents were shown to be due to the activation of ANO6. However, the knockdown of ANO6 or inhibitors of ANO6 did not inhibit necroptotic cell death. The present data demonstrate the activation of ANO6 during necroptosis, which, however, is not essential for cell death.     

Sodium channels in cardiac Purkinje cells

Sodium (Na+) currents are responsible for excitation and conduction in most cardiac cells, but their study has been hampered by the lack of a satisfactory method for voltage clamp. We report a new method for low resistance access to single freshly isolated canine cardiac Purkinje cells that permits good control of voltage and intracellular ionic solutions. The series resistance was usually less than 3 omega cm2, similar to that of the squid giant axon. Cardiac Na+ currents resemble those of nerve. However, Na+ current decay is multiexponential. The basis for this was further studied with cell-attached patch clamp recording of single Na+ channel properties. A prominent characteristic of the single channels was their ability to reopen after closure. There was also a long opening state that may be the basis for a small very slowly decaying Na+ current. This rare long opening state may contribute to the Na+ current during the action potential plateau.     

The effect of shock on blood oxidation-reduction potential.

Oxidation-reduction (redox) potential measurements were made in the blood of rabbits subjected to hemorrhagic shock followed by treatment with a mild oxidizing agent (albumin). Control redox potential reading corrected for pH was -8.8 +/- 1.3 millivolts (mV) in arterial blood (A) and -18.0 +/- 2.0 mV in venous blood (V). This A-V difference indicated that hydrogen equivalents coming from muscle and other tissues were partially consumed in the lungs. A 20-mV drop on the V and a 13 mV on the A side was seen after shock. This did not fully return to control 2 h after return of the shed blood. Infusion of 2 g of albumin/kg/h raised the V redox potential to control, but it returned to untreated levels when the albumin was discontinued. The reductive load imposed on the animal by shock appeared to be large and not readily reversed by reperfusion or by the quantity of albumin given. Thus, it may be concluded that cellular respiration had not been adequately restored. This reductive load may impede recovery by suppression of cellular respiration and other cell and organ functions.     

Effects of barium on separately recorded fast and slow PIII responses in bullfrog retina

Investigation of Ba2+ effects on fast and slow PIII responses in isolated bullfrog retina revealed that Ba2+ suppressed slow PIII completely with little effect on fast PIII. A light-induced [K+]0 decrease in the photoreceptor layer was observed in spite of Ba2+ perfusion, indicating the suppressive action of Ba2+ on the K+ conductance of the Müller cell membrane.     

Contribution of the Na+/K+-pump to the membrane potential

The inward movement of sodium ions and the outward movement of potassium ions are passive and the reverse movements against the electrochemical gradients require the activity of a metabolism-driven Na+/K+-pump. The activity of the Na+/K+-pump influences the membrane potential directly and indirectly. Thus, the maintenance of a normal electrical function requires that the Na+/K+-pump maintain normal ionic concentrations within the cell. The activity of the Na+/K+-pump also influences the membrane potential directly by generating an outward sodium current that is larger when the Na+/K+-pump activity is greater. The activity of the Na+/K+-pump is regulated by several factors including the intracellular sodium concentration and the neuromediators norepinephrine and acetylcholine. The inhibition of the Na+/K+-pump can lead indirectly to the development of inward currents that may cause repetitive activity. Therefore, the Na+/K+-pump modifies the membrane potential in different ways both under normal and abnormal conditions and influences in an essential way many cardiac functions, including automaticity, conduction and contraction. Key words. Active transport of ions; cardiac tissues; electroneutral and electrogenic Na+/K/-pump; control of Na+/K+-pump; normal and abnormal electrical events.     

Net ATP synthesis by running the red cell calcium pump backwards.

Ca2+ loaded inside-out vesicles from human red blood cells, yielding C2+ into a Ca2+ free medium with 4 mM EGTA, 2 mM ADP and 10 mM phosphate, produced an excess of 14.9 pmoles . min-1 . (mg protein)-1 of ATP compared to controls in which the transmembrane Ca2+ gradient was abolished by the ionophore A 23 187.     

Caclium causes the biphasic dose-response curve for pancreatic amylase secretion

Summary High concentrations of bethanechol (10^–4 to 10^–3 M) were effective stimulants of amylase secretion from the mouse pancreas if incubations are performed in low [ca²⁺] (0.1 mM) solutions but not if normal Krebs solution (2.56 mM Ca²⁺) was used. This inhibitory effect of Ca²⁺ at high secretagogue concentrations did not appear to be mediated through the microtubules or microfilaments.     

Selective antagonism by Mg2+ of amino acid-induced depolarization of spinal neurones.

In the isolated frog or rat spinal cord, low concentrations of Mg2+ (0.5-1.00 mM) markedly depress, in a substantially Ca2+-independent manner, ventral root depolarizations produced by dorsal root stimulation and by certain amino acids (e.g. N-methyl-D-aspartate and L-homocysteate) but do not depress depolarizations produced by other excitatory amino acids (e.g. kainate and quisqualate). L-Aspartate-induced depolarizations are more sensitive to Mg2+ then are L-glutamate-induced depolarizations.     

Response of the ampullae of Lorenzini to static combined electric and thermal stimuli in Scyliorhinus canicula.

The effect of long-lasting electric currents on the Lorenzinian ampullae at constant temperatures between and 25 degrees C was investigated in the dogfish (Scyliorhinus canicula). Steady state neural impulse patterns in single afferent units were analyzed by plotting interval length histograms and computing mean values and standard deviations for currents between -100 and +100 nA. The mean values depended on temperature and on current strength; the relative standard deviations remained almost constant (ca. 20--30%). Negative currents, inserted at the orifice of the ampullary canal led to higher, and positive currents to lower, steady impulse rates in the whole temperature range investigated here. This static component of electrosensitivity again disappeared at higher currents (of 50 nA and more; electric overstimulation). The maximum static response was two orders of magnitude less than the maximum dynamic component of electroreception. The electrosensitivity depended on temperature: the ampullae were most sensitive to electric currents between 13 and 19 degrees C. The maximal neural activity at 19 degrees C was not shifted to higher or lower temperatures by electric stimulation. A constant equivalent of electric and thermal stimulation throughout the tested temperature and current range could not be found.