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J R López-López

Publications and source records attributed to J R López-López.

At least 19 recordsLinked to original sources
PubMed2005

Comparative gene expression profile of mouse carotid body and adrenal medulla under physiological hypoxia.

The carotid body (CB) is an arterial chemoreceptor, bearing specialized type I cells that respond to hypoxia by closing specific K+ channels and releasing neurotransmitters to activate sensory axons. Despite having detailed information on the electrical and neurochemical changes triggered by hypoxia in CB, the knowledge of the molecular components involved in the signalling cascade of the hypoxic response is fragmentary. This study analyses the mouse CB transcriptional changes in response to low PO2 by hybridization to oligonucleotide microarrays. The transcripts were obtained from whole CBs after mice were exposed to either normoxia (21% O2), or physiological hypoxia (10% O2) for 24 h. The CB transcriptional profiles obtained under these environmental conditions were subtracted from the profile of control non-chemoreceptor adrenal medulla extracted from the same animals. Given the common developmental origin of these two organs, they share many properties but differ specifically in their response to O2. Our analysis revealed 751 probe sets regulated specifically in CB under hypoxia (388 up-regulated and 363 down-regulated). These results were corroborated by assessing the transcriptional changes of selected genes under physiological hypoxia with quantitative RT-PCR. Our microarray experiments revealed a number of CB-expressed genes (e.g. TH, ferritin and triosephosphate isomerase) that were known to change their expression under hypoxia. However, we also found novel genes that consistently changed their expression under physiological hypoxia. Among them, a group of ion channels show specific regulation in CB: the potassium channels Kir6.1 and Kcnn4 are up-regulated, while the modulatory subunit Kcnab1 is down-regulated by low PO2 levels.

Adrenal Medulla↗
PubMed2001

O(2) modulates large-conductance Ca(2+)-dependent K(+) channels of rat chemoreceptor cells by a membrane-restricted and CO-sensitive mechanism.

Hypoxic inhibition of large-conductance Ca(2+)-dependent K(+) channels (maxiK) of rat carotid body type I cells is a well-established fact. However, the molecular mechanisms of such inhibition and the role of these channels in the process of hypoxic transduction remain unclear. We have examined the mechanisms of interaction of O(2) with maxiK channels exploring the effect of hypoxia on maxiK currents recorded with the whole-cell and the inside-out configuration of the patch-clamp technique. Hypoxia inhibits channel activity both in whole-cell and in excised membrane patches. This effect is strongly voltage- and Ca(2+)-dependent, being maximal at low [Ca(2+)] and low membrane potential. The analysis of single-channel kinetics reveals a gating scheme comprising three open and five closed states. Hypoxia inhibits channel activity increasing the time the channel spends in the longest closed states, an effect that could be explained by a decrease in the Ca(2+) sensitivity of those closed states. Reducing maxiK channels with dithiothreitol (DTT) increases channel open probability, whereas oxidizing the channels with 2,2'-dithiopyridine (DTDP) has the opposite effect. These results suggest that hypoxic inhibition is not related with a reduction of channel thiol groups. However, CO, a competitive inhibitor of O(2) binding to hemoproteins, fully reverts hypoxic inhibition, both at the whole-cell and the single-channel level. We conclude that O(2) interaction with maxiK channels does not require cytoplasmic mediators. Such interaction could be mediated by a membrane hemoprotein that, as an O(2) sensor, would modulate channel activity.

Animals↗
PubMed2000

Viral gene transfer of dominant-negative Kv4 construct suppresses an O2-sensitive K+ current in chemoreceptor cells.

Hypoxia initiates the neurosecretory response of the carotid body (CB) by inhibiting one or more potassium channels in the chemoreceptor cells. Oxygen-sensitive K(+) channels were first described in rabbit CB chemoreceptor cells, in which a transient outward K(+) current was reported to be reversibly inhibited by hypoxia. Although progress has been made to characterize this current with electrophysiological and pharmacological tools, no attempts have been made to identify which Kv channel proteins are expressed in rabbit CB chemoreceptor cells and to determine their contribution to the native O(2)-sensitive K(+) current. To probe the molecular identity of this current, we have used dominant-negative constructs to block the expression of functional Kv channels of the Shaker (Kv1.xDN) or the Shal (Kv4.xDN) subfamilies, because members of these two subfamilies contribute to the transient outward K(+) currents in other preparations. Delivery of the constructs into chemoreceptor cells has been achieved with adenoviruses that enabled ecdysone-inducible expression of the dominant-negative constructs and reporter genes in polycistronic vectors. In voltage-clamp experiments, we found that, whereas adenoviral infections of chemoreceptor cells with Kv1.xDN did not modify the O(2)-sensitive K(+) current, infections with Kv4.xDN suppressed the transient outward current in a time-dependent manner, significantly depolarized the cells, and abolished the depolarization induced by hypoxia. Our work demonstrate that genes of the Shal K(+) channels underlie the transient outward, O(2)-sensitive, K(+) current of rabbit CB chemoreceptor cells and that this current contributes to the cell depolarization in response to low pO(2).

Adenoviridae↗
PubMed1999

Kvbeta1.2 subunit coexpression in HEK293 cells confers O2 sensitivity to kv4.2 but not to Shaker channels.

Voltage-gated K+ (KV) channels are protein complexes composed of ion-conducting integral membrane alpha subunits and cytoplasmic modulatory beta subunits. The differential expression and association of alpha and beta subunits seems to contribute significantly to the complexity and heterogeneity of KV channels in excitable cells, and their functional expression in heterologous systems provides a tool to study their regulation at a molecular level. Here, we have studied the effects of Kvbeta1.2 coexpression on the properties of Shaker and Kv4.2 KV channel alpha subunits, which encode rapidly inactivating A-type K+ currents, in transfected HEK293 cells. We found that Kvbeta1.2 functionally associates with these two alpha subunits, as well as with the endogenous KV channels of HEK293 cells, to modulate different properties of the heteromultimers. Kvbeta1.2 accelerates the rate of inactivation of the Shaker currents, as previously described, increases significantly the amplitude of the endogenous currents, and confers sensitivity to redox modulation and hypoxia to Kv4.2 channels. Upon association with Kvbeta1.2, Kv4.2 can be modified by DTT (1,4 dithiothreitol) and DTDP (2,2'-dithiodipyridine), which also modulate the low pO2 response of the Kv4.2+beta channels. However, the physiological reducing agent GSH (reduced glutathione) did not mimic the effects of DTT. Finally, hypoxic inhibition of Kv4.2+beta currents can be reverted by 70% in the presence of carbon monoxide and remains in cell-free patches, suggesting the presence of a hemoproteic O2 sensor in HEK293 cells and a membrane-delimited mechanism at the origin of hypoxic responses. We conclude that beta subunits can modulate different properties upon association with different KV channel subfamilies; of potential relevance to understanding the molecular basis of low pO2 sensitivity in native tissues is the here described acquisition of the ability of Kv4. 2+beta channels to respond to hypoxia.

2,2'-Dipyridyl↗
PubMed1998

Effects of almitrine bismesylate on the ionic currents of chemoreceptor cells from the carotid body.

Almitrine is a drug used in the treatment of hypoxemic chronic lung diseases such as bronchitis and emphysema because it is a potent stimulant of the carotid bodies in human and different animal species that produces a long-lasting enhancement of alveolar ventilation, ameliorating arterial blood gases. However, the mechanism of action of almitrine remains unknown. We investigated the effect of almitrine on ionic currents of chemoreceptor cells isolated from the carotid body of rat and rabbits by using the whole-cell and inside-out configurations of the patch-clamp technique. Almitrine at concentrations up to 10 microM did not affect whole-cell voltage-dependent K+, Ca2+, or Na+ currents in rat or rabbit cells. However, this concentration of almitrine significantly inhibited the Ca2+-dependent component of K+ currents in rat chemoreceptor cells. This effect of almitrine on the Ca2+-dependent component of K+ currents was investigated further at the single-channel level in excised patches in the inside-out configuration. In this preparation, almitrine inhibited the activity of a high-conductance (152 +/- 13 pS), Ca2+-dependent K+ channel by decreasing its open probability. The IC50 value of the effect was 0. 22 microM. The inhibitory effect of almitrine on Ca2+-dependent K+ channels also was observed in GH3 cells. We conclude that almitrine inhibits selectively the Ca2+-dependent K+ channel and that in rat chemoreceptor cells, this inhibition could represent an important mechanism of action underlying the therapeutic actions of the drug.

Almitrine↗
PubMed1997

Properties of ionic currents from isolated adult rat carotid body chemoreceptor cells: effect of hypoxia.

1. The electrical properties of chemoreceptor cells from neonatal rat and adult rabbit carotid bodies (CBs) are strikingly different. These differences have been suggested to be developmental and/or species related. To distinguish between the two possibilities, the whole-cell configuration of the patch-clamp technique was used to characterize the ionic currents present in isolated chemoreceptor cells from adult rat CBs. Since hypoxia-induced inhibition of O2-sensitive K+ currents is considered a crucial step in O2 chemoreception, the effect of hypoxia on the adult rat chemoreceptor cell currents was also studied. 2. Outward currents were carried mainly by K+, and two different components could be distinguished: a Ca(2+)-dependent K+ current (IK(Ca)) sensitive to Cd2+ and charybdotoxin (CTX), and a Ca(2+)-insensitive, voltage-dependent K+ current (IK(V)). IK(V) showed a slow voltage-dependent activation (time constant (tau) of 87.4 ms at -20 mV and 8.8 ms at +60 mV) and a very slow inactivation, described by the sum of two exponentials (tau 1 = 684 +/- 150 ms and tau 2 = 4.96 +/- 0.76 s at + 30 mV), that was almost voltage insensitive. The kinetic and pharmacological properties of IK(V) are typical of a delayed rectifier K+ channel. 3. Voltage-dependent Ca2+ currents (ICa) were present in nineteen of twenty-seven cells. TTX-sensitive Na+ currents were also observed in about 10% of the cells. 4. Low PO2 (< 10 mmHg) reduced the whole outward current amplitude by 22.17 +/- 1.96% (n = 27) at +20 mV. This effect was absent in the presence of Cd2+. Since low PO2 did not affect ICa, we conclude that hypoxia selectively blocks IK(Ca). 5. The properties of the currents recorded in adult rat chemoreceptor cells, including the specific inhibition of IK(Ca) by hypoxia, are similar to those reported in neonatal rat CB cells, implying that the differences between rat and rabbit chemoreceptor cells are species related.

Animals↗
PubMed1997

Carbon monoxide inhibits hypoxic pulmonary vasoconstriction in rats by a cGMP-independent mechanism.

Hypoxia activates erythropoietin-producing cells, chemoreceptor cells of the carotid body and pulmonary artery smooth muscle cells (PSMC) with a comparable arterial PO2 threshold of some 70 mmHg. The inhibition by CO of the hypoxic responses in the two former cell types has led to the proposal that a haemoprotein is involved in the detection of the PO2 levels. Here, we report the effect of CO on the hypoxic pulmonary vasoconstriction (HPV). Pulmonary arterial pressure (PAP) was measured in an in situ, blood-perfused lung preparation. PAP in normoxia (20% O2, 5% CO2) was 15.2+/-1.8 mmHg, and hypoxia (2% O2, 5% CO2) produced a DeltaPAP of 6.3+/-0.4 mmHg. Addition of 8% or 15% CO to the hypoxic gas mixture reduced the DeltaPAP by 88.3+/-2.7% and 78.2+/-6.1% respectively. The same levels of CO did not affect normoxic PAP nor reduced the DeltaPAP produced by angiotensin II. The effect of CO was studied after inhibition of the NO-cyclic guanosine monophosphate (cGMP) cascade with N-methyl-l-arginine (5.10(-5) M) or methylene blue (1.4.10(-4) M). It was found that both inhibitors more than doubled the hypoxic DeltaPAP without altering the effectiveness of CO to inhibit the HPV. In in vitro experiments we verified the inhibition of guanylate cyclase by measuring the levels of cGMP in segments of the pulmonary artery. Cyclic GMP levels were 1.4+/-0.2 (normoxia), 2.5+/-0.3 (hypoxia) and 3.3+/-0.5 pmole/mg tissue (hypoxia plus 8% CO); sodium nitroprusside increased normoxic cGMP levels about fourfold. Methylene blue reduced cGMP levels to less than 10% in all cases, and abolished the differences among normoxic, hypoxic and hypoxic plus CO groups. It is concluded that CO inhibits HPV by a NO-cGMP independent mechanism and it is proposed that a haemoprotein could be involved in O2-sensing in PSMC.

Animals↗
PubMed1995

Local calcium transients triggered by single L-type calcium channel currents in cardiac cells.

Excitation-contraction coupling was studied in mammalian cardiac cells in which the opening probability of L-type calcium (Ca2+) channels was reduced. Confocal microscopy during voltage-clamp depolarization revealed distinct local transients in the concentration of intracellular calcium ions ([Ca2+]i). When voltage was varied, the latency to occurrence and the relative probability of occurrence of local [Ca2+]i transients varied as predicted if Ca2+ release from the sarcoplasmic reticulum (SR) was linked tightly to Ca2+ flux through L-type Ca2+ channels but not to that through the Na-Ca exchanger or to average [Ca2+]i. Voltage had no effect on the amplitude of local [Ca2+]i transients. Thus, the most efficacious "Ca2+ signal" for activating Ca2+ release from the SR may be a transient microdomain of high [Ca2+]i beneath an individual, open L-type Ca2+ channel.

Animals↗
PubMed1995

Calcium signalling in cardiac muscle cells.

In heart cells, several distinct kinds of transient spatial patterns of cytoplasmic calcium ion concentration ([Ca2+]i) can be observed: (1) [Ca2+]i waves, in which regions of spontaneously increased [Ca2+]i propagate at high velocity (100 microns/s) through the cell; (2) Ca2+ 'sparks', which are spontaneous, non-propagating changes in [Ca2+]i that are localized in small (approximately 2 microns) subcellular regions; and (3) evoked [Ca2+]i transients that are elicited by electrical depolarization, in association with normal excitation-contraction (E-C) coupling. In confocal [Ca2+]i images, evoked [Ca2+]i transients appear to be nearly spatially uniform throughout the cell, except during their rising phase or during small depolarizations. In contrast to [Ca2+]i waves and spontaneous Ca2+ sparks, evoked [Ca2+]i transients are triggered by L-type Ca2+ channel current and they are 'controlled', in the sense that stopping the L-type Ca2+ current stops them. Despite their different characteristics, all three types of Ca2+ transient involve Ca(2+)-induced release of Ca2+ from the sarcoplasmic reticulum. Here, we address the question of how the autocatalytic process of Ca(2+)-induced Ca2+ release, which can easily be understood to underlie spontaneous regenerative ('uncontrolled'), propagating [Ca2+]i waves, might be 'harnessed', under other circumstances, to produce controlled changes in [Ca2+]i, as during normal excitation-contraction coupling, or changes in [Ca2+]i that do not propagate. We discuss our observations of Ca2+ waves, Ca2+ sparks and normal Ca2+ transients in heart cells and review our results on the 'gain' of Ca(2+)-induced Ca2+ release. We discuss a model involving Ca2+ microdomains beneath L-type Ca2+ channels, and clusters of Ca(2+)-activated Ca2+ release channels in the sarcoplasmic reticulum which may form the basis of the answer to this question.

Animals↗
PubMed1994

Local, stochastic release of Ca2+ in voltage-clamped rat heart cells: visualization with confocal microscopy.

1. Confocal microscopy and the fluorescent Ca2+ indicator fluo-3 (K+ salt) were used to measure cytosolic free calcium ion concentration ([Ca2+]) during excitation-contraction (E-C) coupling in single, voltage-clamped, rat cardiac ventricular cells. 2. Local [Ca2+]i transients were measured nearly simultaneously in different, separate, subcellular volumes of approximately 2.0 microns 3. During depolarization, local [Ca2+]i transients were distinctly different from each other and from whole-cell [Ca2+]i transients. These differences were particularly apparent during small depolarizations, and were substantially reduced by ryanodine. 3. Components of the local [Ca2+]i transients, particularly those evoked by small depolarizations, were closely similar, in time course and amplitude, to spontaneous local [Ca2+]i transients, or 'sparks' (which have been shown previously to be Ca2+ released from sarcoplasmic reticulum). 4. Analysis of local [Ca2+]i transients in the spatial frequency domain (power spectrum) revealed that high power at spatial frequencies of 0.05-0.2 microns-1 was always associated with spontaneous calcium 'sparks' and with local [Ca2+]i transients evoked by small depolarizing pulses (e.g. to -31 mV). Evoked local [Ca2+]o transients in the presence of ryanodine, and those evoked by depolarization to very positive clamp-pulse potentials (+45 mV), were associated with considerably lower power at this frequency. 5. The results suggest that whole-cell [Ca2+]i transients evoked by voltage-clamp depolarization, and thus by L-type Ca2+ current, are comprised of local [Ca2+]i transients that are similar to the spontaneous calcium 'sparks'. At very positive clamp-pulse potentials, however, the electrically evoked local [Ca2+]i transients may be smaller, perhaps as a result of smaller unitary L-type Ca2+ current.

Aniline Compounds↗
PubMed1994

Inhibition of [3H]catecholamine release and Ca2+ currents by prostaglandin E2 in rabbit carotid body chemoreceptor cells.

Basal release of [3H]catecholamine ([3H]CA) from rabbit carotid bodies (CBs), previously incubated in the presence of [3H]tyrosine, was not significantly modified by prostaglandin E2 (PGE2). On the contrary, PGE2 (3-300 nM) produced a dose-dependent inhibition of the low PO2-evoked release of [3H]CA. The inhibition was greatest (55%) at a low intensity of hypoxic stimulation (incubating solution PO2 approximately 66 mmHg) and decreased with increasing intensities of hypoxia. Chronic denervation of the CB did not modify the response to PGE2. The release of [3H]CA induced by incubating the CBs in a hypercapnic-acidic solution (PCO2 approximately 132 mmHg; pH = 6.60) and by dinitrophenol (100 microM) was not significantly modified by 300 nM PGE2. PGE2 (300 nM) inhibited the release of [3H]CA elicited by incubating the CBs in a high K+ (35 mM)-containing solution. The release response elicited by high K+ (25 mM) was strongly augmented by a dihydropyridine agonist of Ca2+ channels, Bay K 8644, at a concentration of 1 microM. The Bay K 8644 effect was partly inhibited by PGE2 (300 nM). Using whole-cell recordings in freshly dispersed or short-term cultured chemoreceptor cells from adult rabbits it was found that Ca2+ currents (ICa) were reversibly inhibited by bath application of PGE2. A good parallelism exits between the dose-response curves for PGE2 inhibition of ICa in isolated chemoreceptor cells and high extracellular [K+]- or hypoxia-evoked release of [3H]CA from the whole CB.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1994

Local control of excitation-contraction coupling in rat heart cells.

1. Cytosolic free calcium ion concentration ([Ca2+]i) and whole-cell L-type Ca2+ channel currents were measured during excitation-contraction (E-C) coupling in single voltage-clamped rat cardiac ventricular cells. The measurements were used to compute the total cellular efflux of calcium ions through sarcoplasmic reticulum (SR) Ca2+ release channels (FSR,rel) and the influx of Ca2+ via L-type Ca2+ channels (FICa). 2. FSR,rel was elicited by depolarizing voltage-clamp pulses 200 ms in duration to membrane potentials from -30 to +80 mV. Over this range, peak FSR,rel had a bell-shaped dependence on clamp pulse potential. In all cells, the 'gain' of the system, measured as the ratio, FSR,rel(max)/FICa(max), declined from about 16, at 0 mV, to much lower values as clamp pulse voltage was made progressively more positive. We named this phenomenon of change in gain as a function of membrane potential, 'variable gain'. At clamp pulse potentials in the range -30 to 0 mV, the gain differed from cell to cell, being constant at about 16 in some cells, but decreasing from high values (approximately 65) at -20 mV in others. 3. At clamp pulse potentials at which Ca2+ influx (FICa) was maintained, FSR,rel also had a small maintained component. When macroscopic Ca2+ influx was brief (1-2 ms, during 'tails' of FICa), FSR,rel rose rapidly to a peak after repolarization and then declined with a half-time of about 9 ms (typically). 4. The rising phase of [Ca2+]i transients could be interrupted by stopping Ca2+ influx rapidly (by voltage clamp). We therefore termed this phenomenon 'interrupted SR Ca2+ release'.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Properties of a transient K+ current in chemoreceptor cells of rabbit carotid body.

1. Adult rabbit carotid body chemoreceptor cells, enzymatically dispersed and short-term cultured, exhibit an inactivating outward K+ current that is reversibly inhibited by low PO2. In the present work we have characterized the biophysical and pharmacological properties of this current using the whole-cell voltage clamp recording technique. 2. Inactivating current was recorded after blockage of Ca2+ currents with extracellular Co2+, Cd2+, or after complete washing out of Ca2+ channels. 3. The threshold of activation of this inactivating current was about -40 mV. Current activated very quickly (mean rise time 4.8 +/- 0.42 ms at +60 mV) but inactivated more slowly. Inactivation was well fitted by two exponentials with time constants of 79.7 +/- 6.6 and 824 +/- 42.8 ms (at +40 mV). The inactivation process showed a little voltage dependence. 4. The steady-state inactivation was well fitted by a Boltzman function. Inactivation was fully removed at potentials negative to -80 mV and was complete at voltages near -10 mV; 50% inactivation occurred at -41 mV. 5. Recovery from inactivation had several components and was voltage dependent. Initial recovery was fast, but full recovery, even at -100 mV, required more than 30 s. 6. Inactivating current was selectively blocked by 4-aminopyridine (4-AP), in a dose-dependent manner (IC50, 0.2 mM). The duration of chemoreceptor cells action potentials was augmented by 1 mM 4-AP from 2.3 +/- 0.36 to 7.0 +/- 0.25 ms at 0 mV. Tetraethylamonium (TEA), at concentrations above 5 mM, blocked inactivating and non-inactivating components of the whole K+ current. 7. Inactivating current was modulated by cyclic AMP (cAMP). Bath application of 2 mM dibutyryl cAMP reduced peak amplitude by 18.7 +/- 2.9% (at +30 mV) and slowed down the rise time of the current. The effect was not voltage dependent. Forskolin (10-20 microM) also affected inactivating current, by accelerating the inactivation process. In the same preparations neither dibutyryl cAMP nor forskolin affected Ca2+ currents. 8. It is concluded that modulation of K+ channels by cAMP might play a physiological role potentiating the low PO2 inhibition of K+ channels.

4-Aminopyridine↗