The role of BKCa channels on hyperpolarization mediated by hyperosmolarity in human articular chondrocytes.

Chondrocytes, the only cell in cartilage, are subjected to hyperosmotic challenges continuously since extracellular osmolarity in articular cartilage increases in response to mechanical loads during joint movement. Hyperosmolarity can affect membrane transport, and it is possible that load modulates matrix synthesis through alterations in intracellular composition. In the present study, the effects of hyperosmotic challenges were evaluated using the whole-cell patch clamp technique, whole cell mode on freshly isolated human and bovine articular chondrocytes. In human chondrocytes, hypertonicity induced the activation of outward Ca(2+)-sensitive K(+) currents, which were inhibited by iberiotoxin and TEA-Cl. The current induced by hypertonic switching (osmolarity from 300 to 400 mOsm/l) caused cell hyperpolarization (from -39 mV to -70 mV) with a reversal potential of -96 ± 7 mV. These results suggest a role for Ca(2+)-activated K(+) channels in human articular chondrocytes, leading to hyperpolarization as a consequence of K(+) efflux through these channels. These channels could have a role in the articular chondrocyte's response to a hyperosmotic challenge and matrix metabolism regulation by load.

A calcium-induced calcium release mechanism supports luteinizing hormone-induced testosterone secretion in mouse Leydig cells.

Leydig cells are responsible for the synthesis and secretion of testosterone, processes controlled by luteinizing hormone (LH). Binding of LH to a G protein-coupled receptor in the plasma membrane results in an increase in cAMP and in intracellular Ca(2+) concentration ([Ca(2+)](i)). Here we show, using immunofluorescence, that Leydig cells express ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP(3)Rs). Measurements of intracellular calcium changes using the fluorescent calcium-sensitive dye fluo-3 and confocal microscopy show that both types of receptors are involved in a calcium-induced calcium release (CICR) mechanism, which amplifies the initial Ca(2+) influx through plasma membrane T-type calcium channels (Ca(V)3). The RyRs and IP(3)Rs are functional, as judged from both their activation by caffeine and IP(3) and block by ryanodine and 2-aminoethoxydiphenyl borate (2-APB), respectively. RyRs are the principal players involved in the release of Ca(2+) from the endoplasmic reticulum, as evidenced by the fact that global Ca(2+) changes evoked by LH are readily blocked by 100 muM ryanodine but not by 2-APB or xestospongin C. Finally, steroid production by Leydig cells is inhibited by ryanodine but not by 2-APB. These results not only broaden our understanding of the role played by calcium in Leydig cells but also show, for the first time, that RyRs have an important role in determining testosterone secretion by the testis.

Reactive oxygen species potentiate the P2X2 receptor activity through intracellular Cys430.

P2X receptor channels (P2XRs) are allosterically modulated by several compounds, mainly acting at the ectodomain of the receptor. Like copper, mercury, a metal that induces oxidative stress in cells, also stimulates the activity of P2X(2)R and inhibits the activity of P2X(4)R. However, the mercury modulation is not related to the extracellular residues critical for copper modulation. To identify the site(s) for mercury action, we generated two chimeras using the full size P2X(2) subunit, termed P2X(2a), and a splice variant lacking a 69 residue segment in the C terminal, termed P2X(2b), as the donors for intracellular and transmembrane segments and the P2X(4) subunit as the donor for ectodomain segment of chimeras. The potentiating effect of mercury on ATP-induced current was preserved in Xenopus oocytes expressing P2X(4/2a) chimera but was absent in oocytes expressing P2X(4/2b) chimera. Site-directed mutagenesis experiments revealed that the Cys(430) residue mediates effects of mercury on the P2X(2a)R activity. Because mercury could act as an oxidative stress inducer, we also tested whether hydrogen peroxide (H(2)O(2)) and mitochondrial stress inducers myxothiazol and rotenone mimicked mercury effects. These experiments, done in both oocytes and human embryonic kidney HEK293 cells, revealed that these compounds potentiated the ATP-evoked P2X(2a)R and P2X(4/2a)R currents but not P2X(2b)R and P2X(2a)-C430A and P2X(2a)-C430S mutant currents, whereas antioxidants dithiothreitrol and N-acetylcysteine prevented the H(2)O(2) potentiation. Alkylation of Cys(430) residue with methylmethane-thiosulfonate also abolished the mercury and H(2)O(2) potentiation. Altogether, these results are consistent with the hypothesis that the Cys(430) residue is an intracellular P2X(2a)R redox sensor.

Intracellular mechanisms of specific beta-adrenoceptor antagonists involved in improved cardiac function and survival in a genetic model of heart failure.

beta-blockers, as class, improve cardiac function and survival in heart failure (HF). However, the molecular mechanisms underlying these beneficial effects remain elusive. In the present study, metoprolol and carvedilol were used in doses that display comparable heart rate reduction to assess their beneficial effects in a genetic model of sympathetic hyperactivity-induced HF (alpha(2A)/alpha(2C)-ARKO mice). Five month-old HF mice were randomly assigned to receive either saline, metoprolol or carvedilol for 8 weeks and age-matched wild-type mice (WT) were used as controls. HF mice displayed baseline tachycardia, systolic dysfunction evaluated by echocardiography, 50% mortality rate, increased cardiac myocyte width (50%) and ventricular fibrosis (3-fold) compared with WT. All these responses were significantly improved by both treatments. Cardiomyocytes from HF mice showed reduced peak [Ca(2+)](i) transient (13%) using confocal microscopy imaging. Interestingly, while metoprolol improved [Ca(2+)](i) transient, carvedilol had no effect on peak [Ca(2+)](i) transient but also increased [Ca(2+)] transient decay dynamics. We then examined the influence of carvedilol in cardiac oxidative stress as an alternative target to explain its beneficial effects. Indeed, HF mice showed 10-fold decrease in cardiac reduced/oxidized glutathione ratio compared with WT, which was significantly improved only by carvedilol treatment. Taken together, we provide direct evidence that the beneficial effects of metoprolol were mainly associated with improved cardiac Ca(2+) transients and the net balance of cardiac Ca(2+) handling proteins while carvedilol preferentially improved cardiac redox state.

Regulation of intracellular calcium by bupivacaine isomers in cardiac myocytes from Wistar rats.

In this study we investigated the effects of a racemic mixture of bupivacaine (RS(+/-)bupivacaine) and its isomers (S(-)bupivacaine and R(+)bupivacaine) on the Ca2+ handling by ventricular myocytes from Wistar rats. Single ventricular myocytes were enzymatically isolated and loaded with the fluorescent Ca2+ indicator fura 2-am to estimate intracellular Ca2+ concentration during contraction and relaxation cycles. S(-)bupivacaine (10 muM) significantly increased peak amplitude and the rate of increase of Ca2+ transients in 155% +/- 54% (P < 0.05) and 194% +/- 94% (P < 0.01) of control. However, exposure to R(+)bupivacaine had no effect on either peak amplitude or rate of increase at any concentration tested. Saponin-skinned ventricular fibers were used to investigate the effect of bupivacaine on the intracellular Ca2+ regulation by sarcoplasmic reticulum (SR) and on the Ca2+ sensitivity of contractile system. S(-), R(+), and RS(+/-)bupivacaine induced Ca2+ release from SR (P < 0.01). In SR-disrupted skinned ventricular cells, bupivacaine and its isomers (5 mM) increased the sensitivity of contractile system to Ca(2+). S(-), RS(+/-), and R(+)bupivacaine significantly increased pCa50 from 5.8 +/- 0.1, 5.8 +/- 0.1, and 5.8 +/- 0.1, to 6.1 +/- 0.1 (P < 0.05), 6.0 +/- 0.1 (P < 0.05), and 6.1 +/- 0.1 (P < 0.05). Ca2+ release from SR through RyR2 activation could explain the increase of Ca2+ transients in cardiac cells. Increased intracellular Ca2+ in cardiac myocytes display a stereoselectivity to S(-)bupivacaine.

To be or not to be ... nicotinic acid adenine dinucleotide phosphate (NAADP): a new intracellular second messenger?

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent activator of intracellular Ca2+ release in several vertebrate and invertebrate systems. The role of the NAADP system in physiological processes is being extensively investigated at the present time. The NAADP receptor and its associated Ca2+ pool have been hypothesized to be important in several physiological processes including fertilization, T cell activation, and pancreatic secretion. However, whether NAADP is a new second messenger or a tool for the discovery of a new Ca2+ channel is still an unanswered question. Research developed over the last two years has provided some important clues to whether NAADP is or not a physiological cellular messenger. In this short review, I will discuss some of these new findings that are helping us to find an answer to the important question: Is NAADP a second messenger or not?

To be or not to be nicotinic acid adenine dinucleotide phosphate (NAADP): a new intracellular second messenger?

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent activator of intracellular Ca2+ release in several vertebrate and invertebrate systems. The role of the NAADP system in physiological processes is being extensively investigated at the present time. The NAADP receptor and its associated Ca2+ pool have been hypothesized to be important in several physiological processes including fertilization, T cell activation, and pancreatic secretion. However, whether NAADP is a new second messenger or a tool for the discovery of a new Ca2+ channel is still an unanswered question. Research developed over the last two years has provided some important clues to whether NAADP is or not a physiological cellular messenger. In this short review, I will discuss some of these new findings that are helping us to find an answer to the important question: Is NAADP a second messenger or not?.

Lymphangiogenesis in human dental pulp.

AIM: To investigate the impact of inflammation on lymphangiogenesis in human dental pulp. METHODOLOGY: Eleven samples of dental pulp without inflammation and 11 dental pulps with moderate to intense mononuclear cell inflammatory infiltrate associated with dentine caries were selected. The streptavidin-biotin complex stain was used to detect CD31, vascular endothelial growth factor receptor-3 (VEGFR-3) and alpha-smooth muscle actin. The number of lymphatic vessels was obtained by counting the number of vessels positive for CD31 and VEGFR-3 and negative for alpha-smooth muscle actin. RESULTS: The results demonstrated that the mean number (+/-SD) of vessels positive for CD31 and VEGFR-3 (lymphatic vessels) in the group with inflammation (6.09 +/- 1.81) was statistically higher (P = 0.0123) than the mean number in the group without inflammation (3.73 +/- 2.20). CONCLUSION: Increased co-immunostaining of CD31 and VEGF-3 in vessels associated with human dental pulp inflammation occurred, which suggests lymphangiogenesis.

Mechanism of action of volatile anesthetics: involvement of intracellular calcium signaling.

There have been extensive efforts to characterize the mechanism of action of volatile anesthetics, but their molecular and cellular actions are still a matter of debate. Volatile anesthetics act primarily on synaptic transmission in the central nervous system but proof of this as the predominant mechanism of action remains elusive. Changes in neurotransmitter release may relate to direct interaction of the anesthetic molecule with an ion channel protein or synaptic protein, but can also be a consequence of alterations in intracellular signaling. Calcium is one of the most important messengers in cells and its intracellular concentration may be modified by several agents including volatile anesthetics. Neuronal excitability is in part determined by calcium availability that is controlled by several mechanisms. Because voltage-gated calcium channels (VGCC) play a key role in controlling Ca2+ entry and in initiating cellular responses to stimulation through an elevation of intracellular calcium concentration ([Ca2+](i)), they are thought to be one of the targets for volatile anesthetics. However, [Ca2+](i) can also be altered without the participation of VGCC through receptor-mediated pathways. Indeed, calcium homeostasis is also controlled by plasma membrane Ca2+ -adenosine triphosphatase, sarcoplasmic-endoplasmic reticular Ca2+ -ATPase, the Na+ -Ca2+ exchanger, and mitochondrial Ca2+ sequestration. Alteration of any of those mechanisms that control [Ca2+](i) may lead to a change in presynaptic transmission or postsynaptic excitability. Here we will review some of the recent progress in identifying putative actions of volatile anesthetics, specifically the effect on intracellular calcium homeostasis in neurons.

Importance of timing of risk factors for cerebral oedema during therapy for diabetic ketoacidosis.

Cerebral oedema is the most common cause of mortality and morbidity during the first day of conventional treatment for diabetic ketoacidosis in paediatric patients. It is possible that therapy contributes to its development. Risk factors that predispose to cerebral oedema should lead to an expansion of the intracellular and/or the extracellular fluid compartment(s) of the brain because water normally accounts for close to 80% of brain weight. With respect to the intracellular fluid compartment, the driving force to cause cell swelling is a gain of effective osmoles in brain cells and/or a significant decline in the effective osmolality of the extracellular fluid compartment. Factors leading to an expansion of the intracerebral extracellular fluid volume can be predicted from Starling forces acting at the blood-brain barrier. Some of these risk factors have an early impact, while others have their major effects later during therapy for diabetic ketoacidosis. Based on a theoretical analysis, suggestions to modify current therapy for diabetic ketoacidosis in children are provided.

Líquidos y electrolitos en la nutrición parenteral en pediatría / Liquids and electrolyte in the parenteral nutrition in pediatric

El mayor componente de la nutrición parenteral total(NPT) se encuentra representado por el agua y los electrolitos dos elementos esenciales para la vida. El agua se encuentra distribuida en el cuerpo humano en dos grandes compartamientos: intracelular y extracelular, este último se divide en tres espacios: plasmático, intersticial-linfático y transcelular (líquido cefalorraquídeo, intestinal y renal). Contribuye al 80 por ciento del peso corporal total del prematuro, al 70 por ciento del recién nacido a término y aproximadamente al 60 por ciento en el adulto. La disminución se debe a la sustitución del agua por grasa a medida que crece el individuo. Así tenemos que la concentración del agua corporal total en el recién nacido sufre un descenso de 3 a 5 por ciento en los primeros tres a cinco días de vida, alcanzando los valores del adulto al año de edad

Nitric oxide and peroxynitrite-dependent aconitase inactivation and iron-regulatory protein-1 activation in mammalian fibroblasts.

The reaction of reactive oxygen and nitrogen species with the [4Fe-4S]2+ cluster of mitochondrial (m-) and cytosolic (c-) aconitases leads to loss of catalytic activity and, in the case of the c-aconitase, triggers total cluster disruption to yield the iron-regulatory protein-1 (IRP-1). Herein we have studied the relative contribution and interplay of reactive oxygen species (O and H2O2), nitric oxide (*NO), and peroxynitrite in the modulation of m- and c-aconitase and IRP-1 activities in V79-M8 mammalian fibroblasts, identifying key variables that control the various reactivities at the cellular level. Extracellular production of H2O2 led to inactivation of both m- and c-aconitase and IRP-1 activation, while extracellular had no effect. However, increased intracellular production of caused a loss in m- and c-aconitase activity and IRP-1 activation. Nitric oxide released from NOC-12 had a more complex effect on aconitase and IRP-1 activities. Mitochondrial aconitase was more sensitive than c-aconitase to *NO-mediated inactivation and minimal activation of IRP-1 was observed during a 30-min exposure to the *NO donor. The action of *NO was down- or upregulated by the presence of extra- or intracelular, respectively. Extracellular decreased the *NO-mediated inactivation of aconitases, due to the preferential extracellular decomposition and the lower diffusivity of peroxynitrite compared to *NO. On the other hand, *NO exposure concomitant with enhanced intracellular fluxes lead to intracellular peroxynitrite formation as evidenced by Western blot analysis of nitrated proteins, which increased the effects observed with *NO alone. Peroxynitrite-mediated aconitase inactivation, IRP-1 activation, and cellular protein nitration were more pronounced in cells with low GSH content such as V79-M8 glutathione-depleted cells as well as in pGSOD4 cells which contain 32% of the GSH of the parental strain. Mechanistically, our results imply that the differential actions of the studied reactive species toward cellular aconitases depend on at least three critical factors: (i) their reaction rates with aconitases, (ii) the cellular compartment where they are formed, and (iii) the intracellular status of glutathione.

Possible thiol group involvement in intracellular pH effect on low-conductance Ca(2+)-dependent K+ channels.

We have studied the effect of intracellular pH (pHi) shifts on the activity of Ca(2+)-dependent, inwardly rectifying K+ channels of HeLa cells. Recordings of macroscopic currents in symmetrical 145 mK K+ and internal pH of 7.4 gave moderate inward rectification of the current. At pH 6.4, inward rectification was more marked, whereas it switched to outward rectification at pH 8.2. In excised inside-out membrane patches, similar changes in pHi did not affect the single-channel conductance of the channels underlying the Ca(2+)-dependent K+ currents. At neutral pH, the open state probability (Po) was independent of voltage in the range from -70 to 70 mV. At alkaline pH, Po became voltage dependent, decreasing at negative potentials and increasing with depolarization compared with pH 7.4. These changes accounted for the pH-dependent changes in rectification of the macroscopic current. The possibility that voltage dependence might arise from the ionization of a thiol group was tested by using thiol-directed reagents. The decrease in Po produced by intracellular alkalinization at negative potential was reverted by treatment with N-ethylmaleimide, 5,5'-dithiobis(2-nitrobenzoic acid), and 2,2'-dithiodipyridine. The effect of intracellular alkalinization is speculated to occur through ionization of a cysteine group(s) within the field of the membrane affecting gating.

Volume regulation in NIH/3T3 cells not expressing P-glycoprotein. II. Chloride and amino acid fluxes.

The osmolyte function of amino acids and Cl in native NIH/3T3 cells not expressing the P-glycoprotein was examined by investigating the free amino acid concentration and the swelling-activated efflux of [3H]taurine, as representative of amino acids, and of 125I, as a tracer for Cl. Taurine and 125I efflux was activated by 20 and 30% hyposmotic solutions. At 50% hyposmotic solutions, the osmolyte pool was essentially depleted. The Cl channel blockers 5-nitro-2-(3-phenylpropyl-amino)benzoic acid, 1,9-dideoxyforskolin, dipyridamole, and niflumic acid inhibited the release of the two osmolytes by 80-95%. 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid (400 microM) decreased the efflux of taurine 80% without affecting that of 125I. Linolenic and arachidonic acids (5-20 microM) showed a concentration-dependent inhibitory effect on taurine and 125I fluxes. Omission of Ca decreased osmolyte fluxes by 16%. Verapamil inhibited the osmolyte release only at 500 microM. Nimodipine at 25 and 50 microM decreased the release of [3H]taurine and 125I by approximately 60 and 80%, respectively, but this effect was independent of the presence of extracellular Ca. These results indicate that amino acids and Cl function as osmolytes during regulatory volume decrease in native NIH/ 3T3 cells.

Sarco/endoplasmic reticulum Ca2+-ATPase isoforms: diverse responses to acidosis.

The effects of acidic pH on the kinetics of Ca2+-ATPase isoforms from intracellular membranes of skeletal muscle, cardiac muscle, cerebellum and blood platelets were studied. At neutral pH, all four Ca2+-ATPase isoforms exhibited similar Ca2+-concentration requirements for half-maximal rates of Ca2+ uptake and ATP hydrolysis. A decrease in the pH from 7.0 to 6.0 promoted a decrease in both the apparent affinity for Ca2+ [increasing half-maximal activation (K0.5)] and the maximal velocity (Vmax) of Ca2+ uptake. With skeletal muscle vesicles these effect were 5 to 10 times smaller than those observed with all the other isoforms. Acidification of the medium from pH 7.0 to 6.5 caused the release of Ca2+ from loaded vesicles and a decrease in the amount of Ca2+ retained by the vesicles at the steady state. With the vesicles derived from skeletal muscle these effects were smaller than for vesicles derived from other tissues. The rate of passive Ca2+ efflux from skeletal and cardiac muscle vesicles, loaded with Ca2+ and diluted in a medium containing none of the ligands of Ca2+-ATPase, was the same at pH 7.0 and 6.0. In contrast, the rate of Ca2+ efflux from cerebellar and platelet vesicles increased 2-fold after acidification of the medium. The effects of DMSO, Mg2+ with Pi and arsenate on the rate of Ca2+ efflux varied among the different preparations tested. The differences became more pronounced when the pH of the medium was decreased from 7.0 to 6.0. It is proposed that the kinetic differences among the Ca2+-ATPase isoforms may reflect different adaptations to cellular acidosis, such as that which occurs during ischaemia.