Electrophysiological properties of the mitochondrial permeability transition pores: Channel diversity and disease implication.

The mitochondrial permeability transition pore (mPTP) is a channel that, when open, is responsible for a dramatic increase in the permeability of the mitochondrial inner membrane, a process known as the mitochondrial permeability transition (mPT). mPTP activation during Ca2+ dyshomeostasis and oxidative stress disrupts normal mitochondrial function and induces cell death. mPTP opening has been implicated as a critical event in many diseases, including hypoxic injuries, neurodegeneration, and diabetes. Discoveries of recent years indicate that mPTP demonstrates very complicated behavior and regulation, and depending on specific induction or stress conditions, it can function as a high-conductance pore, a small channel, or a non-specific membrane leak. The focus of this review is to summarize the literature on the electrophysiological properties of the mPTP and to evaluate the evidence that it has multiple molecular identities. This review also provides perspective on how an electrophysiological approach can be used to quantitatively investigate the biophysical properties of the mPTP under physiological, pharmacological, pathophysiological, and disease conditions.

Mechanical ventilation preserves diaphragm mitochondrial function in a rat sepsis model.

BACKGROUND: To describe the effect of mechanical ventilation on diaphragm mitochondrial oxygen consumption, ATP production, reactive oxygen species (ROS) generation, and cytochrome c oxidase activity and content, and their relationship to diaphragm strength in an experimental model of sepsis. METHODS: A cecal ligation and puncture (CLP) protocol was performed in 12 rats while 12 controls underwent sham operation. Half of the rats in each group were paralyzed and mechanically ventilated. We performed blood gas analysis and lactic acid assays 6 h after surgery. Afterwards, we measured diaphragm strength and mitochondrial oxygen consumption, ATP and ROS generation, and cytochrome c oxidase activity. We also measured malondialdehyde (MDA) content as an index of lipid peroxidation, and mRNA expression of the proinflammatory interleukin-1ß (IL-1ß) in diaphragms. RESULTS: CLP rats showed severe hypotension, metabolic acidosis, and upregulation of diaphragm IL-1ß mRNA expression. Compared to sham controls, spontaneously breathing CLP rats showed lower diaphragm force and increased susceptibility to fatigue, along with depressed mitochondrial oxygen consumption and ATP production and cytochrome c oxidase activity. These rats also showed increased mitochondrial ROS generation and MDA content. Mechanical ventilation markedly restored mitochondrial oxygen consumption and ATP production in CLP rats; lowered mitochondrial ROS production by the complex 3; and preserved cytochrome c oxidase activity. CONCLUSION: In an experimental model of sepsis, early initiation of mechanical ventilation restores diaphragm mitochondrial function.

The voltage-dependent anion channel (VDAC): function in intracellular signalling, cell life and cell death.

Research over the last decade has extended the prevailing view of mitochondria to include functions well beyond the critical bioenergetics role in supplying ATP. It is now recognized that mitochondria play a crucial role in cell signaling events, inter-organelle communication, aging, many diseases, cell proliferation and cell death. Apoptotic signal transmission to the mitochondria results in the efflux of a number of potential apoptotic regulators to the cytosol that trigger caspase activation and lead to cell destruction. Accumulating evidence indicates that the voltage-dependent anion channel (VDAC) is involved in this release of proteins via the outer mitochondrial membrane. VDAC in the outer mitochondrial membrane is in a crucial position in the cell, forming the main interface between the mitochondrial and the cellular metabolisms. VDAC has been recognized as a key protein in mitochondria-mediated apoptosis since it is the proposed target for the pro- and anti-apoptotic Bcl2-family of proteins and due to its function in the release of apoptotic proteins located in the inter-membranal space. The diameter of the VDAC pore is only about 2.6-3 nm, which is insufficient for passage of a folded protein like cytochrome c. New work suggests pore formation by homo-oligomers of VDAC or hetero-oligomers composed of VDAC and pro-apoptotic proteins such as Bax or Bak. This review provides insights into the central role of VDAC in cell life and death and emphasizes its function in the regulation of mitochondria-mediated apoptosis and, thereby, its potential as a rational target for new therapeutics.

The SR/ER-mitochondria calcium crosstalk is regulated by GSK3ß during reperfusion injury.

Glycogen synthase kinase-3ß (GSK3ß) is a multifunctional kinase whose inhibition is known to limit myocardial ischemia-reperfusion injury. However, the mechanism mediating this beneficial effect still remains unclear. Mitochondria and sarco/endoplasmic reticulum (SR/ER) are key players in cell death signaling. Their involvement in myocardial ischemia-reperfusion injury has gained recognition recently, but the underlying mechanisms are not yet well understood. We questioned here whether GSK3ß might have a role in the Ca(2+) transfer from SR/ER to mitochondria at reperfusion. We showed that a fraction of GSK3ß protein is localized to the SR/ER and mitochondria-associated ER membranes (MAMs) in the heart, and that GSK3ß specifically interacted with the inositol 1,4,5-trisphosphate receptors (IP3Rs) Ca(2+) channeling complex in MAMs. We demonstrated that both pharmacological and genetic inhibition of GSK3ß decreased protein interaction of IP3R with the Ca(2+) channeling complex, impaired SR/ER Ca(2+) release and reduced the histamine-stimulated Ca(2+) exchange between SR/ER and mitochondria in cardiomyocytes. During hypoxia reoxygenation, cell death is associated with an increase of GSK3ß activity and IP3R phosphorylation, which leads to enhanced transfer of Ca(2+) from SR/ER to mitochondria. Inhibition of GSK3ß at reperfusion reduced both IP3R phosphorylation and SR/ER Ca(2+) release, which consequently diminished both cytosolic and mitochondrial Ca(2+) concentrations, as well as sensitivity to apoptosis. We conclude that inhibition of GSK3ß at reperfusion diminishes Ca(2+) leak from IP3R at MAMs in the heart, which limits both cytosolic and mitochondrial Ca(2+) overload and subsequent cell death.

Transmembrane Na+ and Ca2+ electrochemical gradients in cardiac muscle and their relationship to force development.

Na+- and CA2+-sensitive microelectrodes were used to measure intracellular Na+ and Ca2+ activities (alpha iCa) of sheep ventricular muscle and Purkinje strands to study the interrelationship between Na+ and Ca2+ electrochemical gradients (delta muNa and delta muCa) under various conditions. In ventricular muscle, alpha iNa was 6.4 +/- 1.2 mM and alpha iCa was 87 +/- 20 nM ([Ca/+] = 272 nM). A graded decrease of external Na+ activity (alpha oNa) resulted in decrease of alpha iNa, and increase of alpha iCa. There was increase of twitch tension in low-alpha oNa solutions, and occasional increase of resting tension in 40% alpha oNa. Increase of external Ca2+ (alpha oCa) resulted in increase of alpha iCa and decrease of alpha iNa. Decrease of alpha oCa resulted in decrease of alpha iCa and increase of alpha iNa. The apparent resting Na-Ca energy ratio (delta muCa/delta muNa) was between 2.43 and 2.63. When the membrane potential (Vm) was depolarized by 50 mM K+ in ventricular muscle, Vm depolarized by 50 mV, alpha iNa decreased, and alpha iCa increased, with the development of a contracture. The apparent energy coupling ratio did not change with depolarization. 5 x 10(-6) M ouabain induced a large increase in alpha iNa ad alpha iCa, accompanied by an increase in twitch and resting tension. Under the conditions we have studied, delta muNa and delta muCa appeared to be coupled and n was nearly constant at 2.5, as would be expected if the Na-Ca exchange system was able to set the steady level of alpha iCa. Tension threshold was about 230 nM alpha iCa. The magnitude of twitch tension was directly related to alpha iCa.

3-Nitropropionic acid exacerbates N-methyl-D-aspartate toxicity in striatal culture by multiple mechanisms.

We examined the effects of 3-nitropropionic acid-induced succinate dehydrogenase inhibition on neuronal ATP content, N-methyl-D-aspartate-induced neuronal death, resting membrane potential, and N-methyl-D-aspartate-induced changes in cytosolic calcium concentration ([Ca2+]c) in cultured rat striatal neurons. Exposure of cultures to 3 mM 3-nitropropionic acid for 3 h did not cause overt toxicity, but reduced ATP content by 35%. Treatment with 3-nitropropionic, or removal of Mg2+ from the medium, enhanced subsequent N-methyl-D-aspartate toxicity, reducing the LC50 from 250 microM to 12 microM or 30 microM, respectively. Even after Mg2+ removal, enhancement of N-methyl-D-aspartate toxicity by 3-nitropropionic acid remained pronounced, with the LC50 further decreasing to 3 microM. The mean resting membrane potential of neurons treated with 3-nitropropionic acid was -37 mV, while that in control neurons was -61 mV. Treatment with 3-nitropropionic did not affect baseline [Ca2+]c as determined by fura-2 microfluorimetry. N-methyl-D-aspartate (30 microM) caused a rapid rise in [Ca2+]c, the initial magnitude of which was not affected by 3-nitropropionic acid. However, after a 1-h treatment, [Ca2+]c was dramatically higher in 3-nitropropionic acid-treated neurons. This increased calcium load was washed out slowly and only partially, although calcium in control neurons washed out rapidly and almost completely. These results suggest that in striatal neurons, the enhancement of N-methyl-D-aspartate toxicity caused by succinate dehydrogenase inhibition may be due to synergism between partial relief of the Mg2+ blockade of the N-methyl-D-aspartate receptor and other mechanisms, including disruption of neuronal calcium regulation. This synergism may be relevant to the neuronal death observed in neurodegenerative disorders.

Autonomic modulation of action potential and tension in guinea pig papillary muscles.

The effects of alpha 1-adrenoceptor and muscarinic acetylcholine receptor stimulation on action potential and tension were studied in guinea pig papillary muscles obtained from both right and left ventricles. Stimulation of muscarinic acetylcholine receptors with carbachol produced a reduction of the action potential duration and a positive inotropic effect in papillary muscles from both ventricles. Both effects were concentration dependent and atropine sensitive. However, differential responsiveness was found upon alpha 1-adrenoceptor activation in muscles obtained from left and right ventricles. In right side papillary muscles, the alpha 1-adrenoceptor agonist, methoxamine, decreased the action potential duration and produced a positive inotropic effect. In contrast, methoxamine decreased the action potential duration but failed to produce a positive inotropic effect in left side papillary muscles. All methoxamine effects were antagonized by prazosin. Responses to maximum concentration of carbachol and methoxamine on the action potential duration and contractility were additive in right side papillary muscles. Phorbol 12,13-dibutyrate (PDB), a direct protein kinase C activator, also decreased the action potential duration in a manner that was additive to both carbachol and methoxamine. However, PDB reversed the positive inotropic effect of carbachol and methoxamine. The methoxamine-induced shortening of the action potential duration was prevented by pretreatment with indomethacin and nordihydroguaiaretic acid, blockers of arachidonic acid metabolism, but not by the protein kinase C antagonist, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7).(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of cytosolic free calcium concentration by alpha 1-adrenoceptors in rat atrial cells.

The effects of alpha 1-adrenoceptor stimulation by phenylephrine (PE) and beta-adrenoceptor stimulation by isoprenaline (ISO) on Ca2+ current (ICa) and free intracellular Ca2+ concentration ([Ca2+]i) were studied in isolated atrial myocytes from rat hearts. PE did not significantly affect the magnitude of ICa, whereas large increases of peak ICa were observed in response to ISO. In electrically driven cells, PE evoked a concentration-dependent, gradual increase in diastolic [Ca2+]i and, initially, an increase in the height of peak [Ca2+]i transients. When the diastolic [Ca2+]i was increased to a greater extent, the amplitude of [Ca2+]i transients was decreased. Simultaneous measurements of [Ca2+]i and membrane potential showed that the increase in diastolic [Ca2+]i was associated with a depolarization of the membrane, and the greater amplitude of [Ca2+]i transients with a prolongation of the action potential (AP). The PE-induced increase in diastolic [Ca2+]i was eliminated when the cells were voltage-clamped at the original resting membrane potential (RP); under these conditions, an increase in [Ca2+]i transients was observed in response to PE. ISO usually caused larger increases in the amplitude of [Ca2+]i transients with only minor changes in diastolic [Ca2+]i. These results suggest that PE and ISO increase the amplitude of [Ca2+]i transients in rat atrium in different ways. The increase in [Ca2+]i transients in response to beta-adrenoceptor stimulation is commonly thought to be mediated by a greater conductance of voltage-dependent Ca2+ channels causing a greater Ca2+ influx and a release of more Ca2+ from the sarcoplasmic reticulum during the AP. The increase in diastolic [Ca2+]i in response to PE is probably a consequence of the depolarization of the membrane, possibly involving the voltage-dependent Na(+)-Ca2+ exchange mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

Does mammalian heart contain only the M2 muscarinic receptor subtype?

Five muscarinic acetylcholine receptor (mAChR) subtypes, m1-m5, have been cloned and sequenced to date. The question as to which mAChR subtypes exist in mammalian heart has been studied extensively and is still under considerable debate. We used the reverse transcriptase-polymerase chain reaction to amplify mRNA from adult rat ventricular myocytes, and found that these cells express mRNA for m1 and m2 mAChRs. Immunocytochemical analysis confirmed that m1 and m2, but not m3, mAChR proteins are present on the surface of these cells. Finally, the functional significance of these receptors was examined. Administration of the m1 mAChR antagonist pirenzepine inhibited the stimulatory effect of the muscarinic agonist carbachol on Ca transients. These findings are consistent with the presence of at least two mAChR subtypes in mammalian heart, m1 and m2, and suggest that activation of m1 mAChRs is involved in the stimulatory effects of muscarinic agonists in mammalian heart.

[3H]benzylpempidine, a new radioligand for probing a putative channel site on nicotinic cholinergic receptors.

A study was undertaken to assess the receptor binding characteristics of [3H]4-benzylpempidine to an allosteric site on calf brain membranes associated with nicotinic cholinergic receptors and to compare the binding affinity of novel arylpempidine analogs with their ability to antagonize the behavioral effects of nicotine in mice. Scatchard analysis of the binding yielded a K(d) of 20 nM and a B(max) of 330 fmols/mg membrane protein. [3H]4-benzylpempidine appears to be a more satisfactory ligand than [3H]mecamylamine, since it possessed a 50-fold greater affinity and its binding was far less sensitive to inorganic ions and Tris. Among the arylpempidine analogs 4-m-chlorobenzylidenepempidine and 4-benzylidenepempidine had the lowest K(i) values (1.4 nM and 5.0 nM, respectively) and were the most potent in antagonizing nicotine-induced seizures in mice. Although the K(i) values for pempidine and mecamylamine were 1-2 orders of magnitude greater than any of the arylpempidines, the dose required to antagonize nicotine-induced seizures in mice was comparable to the arylpempidines. One explanation for this apparent discrepancy in the correlation of binding affinity and nicotine antagonism is the lower brain penetration of arylpempidines compared to mecamylamine, following their systemic administration to mice.

Differences in ligand binding and phosphoinositide turnover between M1 muscarinic receptor gene transfected cells and mouse and rat brain membranes.

The present study describes some unexpected receptor mediated effects of N-methylcarbamylcholine on mouse M1 muscarinic receptor gene transfected cell line (M1Y1) that were not evident from biochemical studies with mouse and rat brain tissue where N-methylcarbamylcholine exhibited only nicotinic properties. Although N-methylcarbamycholine was devoid of muscarinic properties in mouse and rat brain preparations, as determined by phosphoinositide turnover and inhibition of [3H]QNB binding, it exhibited significant muscarinic characteristics in the transfected M1Y1 cell line. At a concentration of 10(-6) M or greater, N-methylcarbamycholine caused a transient increase in intracellular Ca2+ of 50 s duration that was reversible by atropine or pirezepine. The Ca(2+)-transient was not elicited by other nicotinic agents such as nicotine and N,N-dimethylcarbamylcholine, a close analogue of N-methylcarbamylcholine, with comparable affinity for nicotinic receptors and devoid of muscarinic activity. N-Methylcarbamylcholine also stimulated phosphoinositide turnover in M1Y1 cells with an estimated EC50 value 10 times greater than that of carbachol, and the effect was blocked by atropine. Both carbachol and N-methylcarbamycholine inhibited [3H]QNB binding in a concentration-dependent manner; however, the IC50 for carbachol was over two orders of magnitude greater than that observed in mouse and rat brain membranes. In considering possible explanations for the differential characteristics of N-methylcarbamylcholine in mouse and rat brain as compared to the transfected M1Y1 cells, it was concluded that the difference may be attributable to differences in the receptor-transduction coupling efficiency and the microenvironment of the muscarinic receptors.

Serum immunoglobulin E in primary IgA nephropathy.

The biosynthesis of human immunoglobulin E (IgE) is regulated by a complex network involving T and B lymphocytes. Diseases associated with high serum IgE (sIgE) levels are usually characterized by T cell disorders. Total sIgE level has been found to be of clinical relevance in minimal change nephrotic syndrome. However, the clinical significance has rarely been studied in primary IgA nephropathy (IgA N). We retrospectively studied 99 cases of primary IgA N. There were 59 males and 40 females with a mean age of 30.0 +/- 12.1 years. The mean follow-up duration was 45.9 +/- 31.1 months. Pathological grading was done according to the criteria of Meadow et al. Median sIgE for the entire group was 122.0 IU/ml (range: 2.8-5805 IU/ml) which was significantly higher than the healthy control group (median: 43,7 IU/ml, range: 5.0-1003 IU/ml, p < 0.001). However, when the IgA N cases were stratified into grades, only grade I (median: 514 IU/ml, range: 72.1-5805.0 IU/ml) and grade II (median: 229 IU/ml, range: 5.0-5464 IU/ml) patients had significantly higher sIgE than the control group (p < 0.0005 and p < 0.001 respectively). Patients with nephrotic ranged proteinuria (32 cases) were further classified into "stable" and "progressive" groups. The "stable" group had a significantly higher sIgE level (median: 922.0 IU/ml, range: 2.8-5805 IU/ml), compared to that of the "progressive" group (median: 55.3 IU/ml, range: 5.0-1600 IU/ml, p < 0.02). The effect of aggressive treatment (including corticosteroid and/or cyclophosphamide, cyclosporine) was also assessed.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytosolic sodium concentration regulates contractility of cardiac muscle.

The present chapter provides experimental evidence to show that intracellular Na+ concentration regulates cardiac contractility effectively by altering intracellular Ca2+ concentration via the Na-Ca exchange. This steep coupling between the Na+ and Ca2+ electrochemical gradients implies that a change in intracellular Na+ concentration is accompanied by a concomitant change in intracellular Ca2+ concentration (and, therefore, contractility). Under the physiologic conditions, each cardiac action potential alters intracellular Na+ concentration in a dynamic manner. Therefore, Na-Ca exchange can regulate cardiac contraction from a beat-to-beat basis.

Effects of forskolin on intracellular sodium activity in resting and stimulated cardiac Purkinje fibers from sheep.

In the present investigation, the effects of forskolin on intracellular sodium activity were studied in quiescent and electrically stimulated cardiac Purkinje fibers from sheep using Na+-sensitive microelectrodes. Also assessed, were the effects of this promoter of cytosolic cAMP production on resting membrane potential, action potential and twitch tension. In the quiescent fibers, forskolin (12 microM) caused intracellular sodium activity to decrease in the face of cellular depolarization. This cellular depolarization was occasionally accompanied by spontaneous firing of action potentials. In the stimulated fibers, forskolin (10 microM) also caused intracellular sodium activity to decrease. Moreover, it caused a marked acceleration of phase 4 pacemaker depolarization, an elevation of the plateau of the action potential and an increase in twitch tension. When the Na+ pump was inhibited by either strophanthidin (1 microM) or by 0 mM extracellular K+, forskolin had no effect on intracellular sodium activity. In summary, the results of the present study indicate that forskolin, presumably by increasing intracellular cAMP, causes the following to occur in cardiac Purkinje fibers from sheep: (a) a decrease in intracellular sodium activity when the Na+ pump is functioning normally; (b) a promotion of membrane depolarization in quiescent fibers; (c) an increase in the steepness of the pacemaker potential in electrically stimulated fibers, and (d) an increase in the force of contraction. Therefore, forskolin will be a useful tool for investigating the role of cAMP in physiological function of cardiac cells.

Effects of calcium on membrane potential and sodium influx in barnacle muscle fibers.

The influence of internal and external Ca2+ on membrane potential and 22Na influx were tested in internally perfused giant barnacle muscle fibers. The fibers depolarized by about 2-3 mV, and Na+ influx increased when external Ca2+ was removed. These effects were inhibited and reversed by adding 2 mM La3+ externally but not by tetrodotoxin (TTX). Ca2+ channel blockers did not prevent the depolarization. Increasing internal free Ca2+ ([Ca2+]i) from 10(-7) to 10(-5) M also stimulated Na+ influx and depolarized the fibers by a few millivolts. Neither external La3+ nor TTX prevented the effects of raising [Ca2+]i; however, internal tetrabutylammonium ions depolarized the fibers and attenuated the internal Ca2+-dependent effects. These data are consistent with the idea that removal of external Ca2+ activates a La3+-sensitive channel that is permeable to Na+; raising [Ca2+]i activates a La2+-insensitive, Na+-permeable channel that may be similar to the internal Ca2+-activated nonselective cation channels observed in cardiac muscle. The results demonstrate that all Na+ (and Ca2+) fluxes that do not contribute to Na-Ca exchange must be carefully identified before the exchange stoichiometry can be determined from Na+ and Ca2+ flux measurements.