José R. López-López

TRPA1 channels mediate acute neurogenic inflammation and pain produced by bacterial endotoxins.

Gram-negative bacterial infections are accompanied by inflammation and somatic or visceral pain. These symptoms are generally attributed to sensitization of nociceptors by inflammatory mediators released by immune cells. Nociceptor sensitization during inflammation occurs through activation of the Toll-like receptor 4 (TLR4) signalling pathway by lipopolysaccharide (LPS), a toxic by-product of bacterial lysis. Here we show that LPS exerts fast, membrane delimited, excitatory actions via TRPA1, a transient receptor potential cation channel that is critical for transducing environmental irritant stimuli into nociceptor activity. Moreover, we find that pain and acute vascular reactions, including neurogenic inflammation (CGRP release) caused by LPS are primarily dependent on TRPA1 channel activation in nociceptive sensory neurons, and develop independently of TLR4 activation. The identification of TRPA1 as a molecular determinant of direct LPS effects on nociceptors offers new insights into the pathogenesis of pain and neurovascular responses during bacterial infections and opens novel avenues for their treatment.

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.

Molecular identification of Kvα subunits that contribute to the oxygen-sensitive K+ current of chemoreceptor cells of the rabbit carotid body

Rabbit carotid body (CB) chemoreceptor cells possess a fast‐inactivating K+ current that is specifically inhibited by hypoxia. We have studied the expression of Kvα subunits, which might be responsible for this current. RT‐PCR experiments identified the expression of Kv1.4, Kv3.4, Kv4.1 and Kv4.3 mRNAs in the rabbit CB. There was no expression of Kv3.3 or Kv4.2 transcripts. Immunocytochemistry with antibodies to tyrosine hydroxylase (anti‐TH) and to specific Kv subunits revealed the expression of Kv3.4 and Kv4.3 in chemoreceptor cells, while Kv1.4 was only found in nerve fibres. Kv4.1 mRNA was also found in chemoreceptor cells following in situ hybridization combined with anti‐TH antibody labelling. Kv4.1 and Kv4.3 appeared to be present in all chemoreceptor cells, but Kv3.4 was only expressed in a population of them. Electrophysiological experiments applying specific toxins or antibodies demonstrated that both Kv3.4 and Kv4.3 participate in the oxygen‐sensitive K+ current of chemoreceptor cells. However, toxin application experiments confirmed a larger contribution of members of the Kv4 subfamily. [Ca2+]i measurements under hypoxic conditions and immunocytochemistry experiments in dispersed CB cells demonstrated the expression of Kv3.4 and Kv4.3 in oxygen‐sensitive cells; the presence of Kv3.4 in the chemoreceptor cell membrane was not required for the response to low PO2. In summary, three Kv subunits (Kv3.4, Kv4.1 and Kv4.3) may be involved in the fast‐inactivating outward K+ current of rabbit CB chemoreceptor cells. The homogeneous distribution of the Kv4 subunits in chemoreceptor cells, along with their electrophysiological properties, suggest that Kv4.1, Kv4.3, or their heteromultimers, are the molecular correlate of the oxygen‐sensitive K+ channel.

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.

Characterization of the Kv channels of mouse carotid body chemoreceptor cells and their role in oxygen sensing

As there are wide interspecies variations in the molecular nature of the O2‐sensitive Kv channels in arterial chemoreceptors, we have characterized the expression of these channels and their hypoxic sensitivity in the mouse carotid body (CB). CB chemoreceptor cells were obtained from a transgenic mouse expressing green fluorescent protein (GFP) under the control of tyrosine hydroxylase (TH) promoter. Immunocytochemical identification of TH in CB cell cultures reveals a good match with GFP‐positive cells. Furthermore, these cells show an increase in [Ca2+]i in response to low PO2, demonstrating their ability to engender a physiological response. Whole‐cell experiments demonstrated slow‐inactivating K+ currents with activation threshold around −30 mV and a bi‐exponential kinetic of deactivation (τ of 6.24 ± 0.52 and 32.85 ± 4.14 ms). TEA sensitivity of the currents identified also two different components (IC50 of 17.8 ± 2.8 and 940.0 ± 14.7 μm). Current amplitude decreased reversibly in response to hypoxia, which selectively affected the fast deactivating component. Hypoxic inhibition was also abolished in the presence of low (10–50 μm) concentrations of TEA, suggesting that O2 interacts with the component of the current most sensitive to TEA. The kinetic and pharmacological profile of the currents suggested the presence of Kv2 and Kv3 channels as their molecular correlates, and we have identified several members of these two subfamilies by single‐cell PCR and immunocytochemistry. This report represents the first functional and molecular characterization of Kv channels in mouse CB chemoreceptor cells, and strongly suggests that O2‐sensitive Kv channels in this preparation belong to the Kv3 subfamily.

Release of dopamine and chemoreceptor discharge induced by low pH and high PCO2 stimulation of the cat carotid body.

1. Cat carotid bodies were incubated with the precursor [3H]tyrosine to label the catecholamine deposits and then mounted in a superfusion chamber which allowed simultaneous collection of the released [3H]dopamine (DA) and recording of action potentials from the carotid sinus nerve. 2. Low pH (7.2‐6.6) superfusion of the carotid bodies for periods of 10 min produced a parallel increase in the release of [3H]DA and chemoreceptor discharge. 3. Carotid sinus nerve denervation of the carotid body 12‐15 days prior to the experiments did not modify the release of [3H]DA elicited by low pH. 4. Superfusion of the carotid bodies with Ca(2+)‐free, high‐Mg2+ (1.6 mM) media reduced basal release of [3H]DA and chemoreceptor discharge by about 30%. Release evoked by low pH was reduced by 82%. Peak and average chemoreceptor discharge recorded in response to low pH were reduced by 28%. 5. Solutions containing weak acids (sodium acetate, 10 mM), adjusted at pH 7.4, elicited release of [3H]DA and increased chemoreceptor discharge. 6. With HCO3‐CO2‐buffered superfusion media, a reduction of bicarbonate to 5.6 mM (pH 6.8), an increase in CO2 to 20% (pH 6.8), or a simultaneous increase in CO2 to 20% and bicarbonate to 90 mM (pH 7.4), resulted in all cases in a corresponding increase in [3H]DA release and chemoreceptor discharge. The most effective stimulus was 20% CO2‐pH 6.8 and the least effective 5% CO2‐5.6 mM‐HCO3‐pH 6.8. 7. Inhibition of carbonic anhydrase with acetazolamide while perfusing the carotid bodies with a 20% CO2‐equilibrated (pH 7.4) solution resulted in comparable reductions in the release of [3H]DA and chemoreceptor discharge. 8. It is concluded that the effective acidic stimulus at the carotid body chemoreceptors is an increase in hydrogen ion concentration in type I cells. It is also concluded that DA plays a critical role in the genesis of carotid sinus nerve discharges.

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

Abstract 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 ΔPAP of 6.3±0.4 mmHg. Addition of 8% or 15% CO to the hypoxic gas mixture reduced the ΔPAP by 88.3±2.7% and 78.2±6.1% respectively. The same levels of CO did not affect normoxic PAP nor reduced the ΔPAP 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 ΔPAP 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.

Contribution of Kv Channels to Phenotypic Remodeling of Human Uterine Artery Smooth Muscle Cells

Vascular smooth muscle cells (VSMCs) perform diverse functions that can be classified into contractile and synthetic (or proliferating). All of these functions can be fulfilled by the same cell because of its capacity of phenotypic modulation in response to environmental changes. The resting membrane potential is a key determinant for both contractile and proliferating functions. Here, we have explored the expression of voltage-dependent K+ (Kv) channels in contractile (freshly dissociated) and proliferating (cultured) VSMCs obtained from human uterine arteries to establish their contribution to the functional properties of the cells and their possible participation in the phenotypic switch. We have studied the expression pattern (both at the mRNA and at the protein level) of Kv&agr; subunits in both preparations as well as their functional contribution to the K+ currents of VSMCs. Our results indicate that phenotypic remodeling associates with a change in the expression and distribution of Kv channels. Whereas Kv currents in contractile VSMCs are mainly performed by Kv1 channels, Kv3.4 is the principal contributor to K+ currents in cultured VSMCs. Furthermore, selective blockade of Kv3.4 channels resulted in a reduced proliferation rate, suggesting a link between Kv channels expression and phenotypic remodeling.

Characterization of Ion Channels Involved in the Proliferative Response of Femoral Artery Smooth Muscle Cells

Objective—Vascular smooth muscle cells (VSMCs) contribute significantly to occlusive vascular diseases by virtue of their ability to switch to a noncontractile, migratory, and proliferating phenotype. Although the participation of ion channels in this phenotypic modulation (PM) has been described previously, changes in their expression are poorly defined because of their large molecular diversity. We obtained a global portrait of ion channel expression in contractile versus proliferating mouse femoral artery VSMCs, and explored the functional contribution to the PM of the most relevant changes that we observed. Methods and Results—High-throughput real-time polymerase chain reaction of 87 ion channel genes was performed in 2 experimental paradigms: an in vivo model of endoluminal lesion and an in vitro model of cultured VSMCs obtained from explants. mRNA expression changes showed a good correlation between the 2 proliferative models, with only 2 genes, Kv1.3 and Kv&bgr;2, increasing their expression on proliferation. The functional characterization demonstrates that Kv1.3 currents increased in proliferating VSMC and that their selective blockade inhibits migration and proliferation. Conclusion—These findings establish the involvement of Kv1.3 channels in the PM of VSMCs, providing a new therapeutical target for the treatment of intimal hyperplasia.

De novo expression of Kv6.3 contributes to changes in vascular smooth muscle cell excitability in a hypertensive mice strain

Essential hypertension involves a gradual and sustained increase in total peripheral resistance, reflecting an increased vascular tone. This change associates with a depolarization of vascular myocytes, and relies on a change in the expression profile of voltage‐dependent ion channels (mainly Ca2+ and K+ channels) that promotes arterial contraction. However, changes in expression and/or modulation of voltage‐dependent K+ channels (Kv channels) are poorly defined, due to their large molecular diversity and their vascular bed‐specific expression. Here we endeavor to characterize the molecular and functional expression of Kv channels in vascular smooth muscle cells (VSMCs) and their regulation in essential hypertension, by using VSMCs from resistance (mesenteric) or conduit (aortic) arteries obtained from a hypertensive inbred mice strain, BPH, and the corresponding normotensive strain, BPN. Real‐time PCR reveals a differential distribution of Kv channel subunits in the different vascular beds as well as arterial bed‐specific changes under hypertension. In mesenteric arteries, the most conspicuous change was the de novo expression of Kv6.3 (Kcng3) mRNA in hypertensive animals. The functional relevance of this change was studied by using patch‐clamp techniques. VSMCs from BPH arteries were more depolarized than BPN ones, and showed significantly larger capacitance values. Moreover, Kv current density in BPH VSMCs is decreased mainly due to the diminished contribution of the Kv2 component. The kinetic and pharmacological profile of Kv2 currents suggests that the expression of Kv6.3 could contribute to the natural development of hypertension.