María Teresa Pérez-García

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.

Effects of Different Types of Stimulation on Cyclic AMP Content in the Rabbit Carotid Body: Functional Significance

Abstract: Cyclic AMP levels in rabbit carotid bodies incubated under control conditions, 100% O2‐ or 95% O2/5% CO2‐equilibrated medium, are close to 1 pmol/mg wet tissue (range 0.4–2.43 pmol/mg). Isobutylmethylxanthine (0.5 mM) increases cyclic AMP levels by a factor of 14 and 8 in HEPES‐and CO2/CH3O−‐buffered medium, respectively. Forskolin (0.5–10 μM) applied during 30 min increases cyclic AMP levels in a dose‐dependent manner. Incubation of carotid bodies at low O2 tensions resulted in an elevation of cyclic AMP levels both in the absence and in the presence of isobutylmethylxanthine. In the latter conditions cyclic AMP increase was maximum at an O2 tension of 46 mm Hg and tended to decrease at extremely low Po2. In isobutylmethylxanthine‐containing Ca2+‐free medium, cyclic AMP increased linearly with decreasing Po2 from 66 to 13 mm Hg; the absolute cyclic AMP levels attained in Ca2+‐free medium were smaller than those observed in Ca2+‐containing medium at any Po2. The differences between Ca2+‐free and Ca2+‐containing media appear to be due to the action of released neurotransmitters in the latter conditions, because dopamine and norepinephrine, which are known to be released by hypoxia in a Ca2+‐dependent manner, increase cyclic AMP in the carotid body. Low pH/high Pco2 and high [K+]e increase cyclic AMP levels only in Ca2+‐containing medium. Forskolin potentiates the release of catecholamines induced by low Po2. These results suggest that cyclic AMP plays an important role in the modulation of the chemoreception process.

CELLULAR MECHANISMS OF OXYGEN CHEMORECEPTION IN THE CAROTID BODY

The carotid bodies (CB) are arterial chemoreceptors that by sensing changes of arterial PO2, PCO2 and pH can initiate and modify ventilatory and cardiovascular reflexes in order to maintain PO2, PCO2 and pH within physiological levels. It is now generally accepted that the glomus or type I cells of the CB are the transducers of hypoxic stimuli, and relay chemosensory information to the brainstem via neurotransmitter release at synaptic contacts with afferent terminals of the carotid sinus nerve. This article reviews the mechanisms of the O2-sensing process at the cellular level. We consider first the transduction of the hypoxic stimulus, in which most of the experimental evidence currently favors a mechanism involving modulation of the electrical properties of type I cells. The last part of the article deals with the transmission of the stimulus between type I cells and afferent nerve terminals, and we present an overview on the issue of neurotransmission in the CB, summarizing the actions of the main neurotransmitters present in the organ.

Cyclic AMP Modulates Differentially the Release of Dopamine Induced by Hypoxia and Other Stimuli and Increases Dopamine Synthesis in the Rabbit Carotid Body

We have investigated the effects of different treatments that increase cyclic AMP levels on the in vitro synthesis and release of catecholamines in the rabbit carotid body. We also measured the rate of 45Ca2+ efflux from previously loaded carotid bodies under different conditions. Forskolin produced a dose‐dependent increase in the release of [3H]dopamine elicited by a hypoxic stimulus of medium intensity (Po2= 33 mm Hg) without altering basal [3H]dopamine release (100% O2‐equilibrated medium). At a concentration of 5 × 10‐‐6M, forskolin increased the release of [3H]dopamine induced by hypoxic stimuli of different intensities; the increase was maximal (498%) at the lowest intensity of hypoxic stimuli (Po2= 66 mm Hg), averaged 260% for hypoxic stimuli of intermediate intensity and 2 × 10‐‐4M cyanide, and was 150% under anoxia. Dibutyryl cyclic AMP (2 mM) and 3‐isobutyl‐1‐methylxanthine (0.5 mM) mimicked forskolin effects under hypoxic stimulation. Forskolin (5 × 10‐‐6M) also increased (180%) the release of [3H]dopamine induced by 20% CO2/pH 6.6, 2.5 × 10‐‐4M dinitrophenol, and 3 × 10‐‐5M ionomycin. Forskolin and 3‐isobutyl‐1‐methylxanthine were without effect on the release of [3H]dopamine elicited by 30 mM extracellular K+. Forskolin (5 × 10‐‐6M) augmented significantly the rate of 45Ca2+ efflux induced by hypoxic stimuli (Po2 of 33 and 66 mm Hg) and 2 × 10‐‐4M cyanide and showed a tendency to increase (20%) the 45Ca2+ efflux induced by dinitrophenol and low pH and to decrease (21%) the efflux induced by 30 mM K+ without altering the rate of efflux under basal conditions. Finally, forskolin (10‐‐5M) produced a 40% increase in the rate of [3H]dopamine synthesis from [3H]tyrosine. From the present and previously published observations, two physiological roles emerge for cyclic AMP in the carotid body: (a) The release of dopamine elicited by natural stimuli (low Po2 and low pH/high Pco2) is positively modulated by the associated increase in cAMP levels. In the case of the hypoxic stimulus, cyclic AMP may act on the O2‐sensing machinery and the exocytotic process, whereas in the case of the acidic stimuli, it may affect only the exocytotic process. (b) The activation of dopamine synthesis observed during hypoxic stimulation may be produced. at least in part, by the concomitant increase in level of cyclic AMP. via a cyclic AMP‐dependent phosphorylation of tyrosine hydroxylase.

Presence of D1 receptors in the rabbit carotid body

Rabbit carotid bodies incubated in vitro in the presence of 5 x 10(-4) M 3-isobutyl 1-methylxanthine have cyclic AMP levels of 13.1 +/- 1.6 pmol/mg fresh tissue (means +/- S.E.M; n = 11). Dopamine (10(-6) and 10(-5) M) increased the cyclic AMP content to 24.4 +/- 2.85 and 40.6 +/- 3.29 pmol/mg tissue. The specific D1 agonist SKF38393 at 10(-6) M increased the cyclic AMP content to 23.9 +/- 2.3 pmol/mg fresh tissue and the specific D1 antagonist SCH23390 at 10(-6) M completely blocked the effect of 10(-5) M dopamine. dBcAMP (2 x 10(-3) M) potentiated the release of dopamine induced by a mild hypoxic stimulus and SKF38393 did not modify it. It is concluded that the carotid body has D1 receptors positively coupled to adenylate cyclase that seem to be located in the vasculature of the organ. This set of receptors, when activated by injected dopamine, produced vasodilatation leading to a decrease in carotid sinus nerve activity.

Functional Identification of Kvα Subunits Contributing to the O2-Sensitive K+ Current in Rabbit Carotid Body Chemoreceptor Cells

Carotid body (CB) chemoreceptors are the major contributors to the hyperventilatory responses to acute hypoxia. Low PO2 inhibits K channel activity of chemoreceptor cells, inducing a depolarization-mediated neurotransmitter release that elicits the ventilatory response (Gonzalez et al, 1994). Although O2-sensitive K+ currents were described in rabbit CB chemoreceptor cells several years ago (Lopez-Barneo et ai, 1988), the molecular identity of the underlying K channels remains unresolved.

Cinnamaldehyde inhibits L-type calcium channels in mouse ventricular cardiomyocytes and vascular smooth muscle cells

Cinnamaldehyde (CA), a major component of cinnamon, is known to have important actions in the cardiovascular system, including vasorelaxation and decrease in blood pressure. Although CA-induced activation of the chemosensory cation channel TRPA1 seems to be involved in these phenomena, it has been shown that genetic ablation of Trpa1 is insufficient to abolish CA effects. Here, we confirm that CA relaxes rat aortic rings and report that it has negative inotropic and chronotropic effects on isolated mouse hearts. Considering the major role of L-type Ca2+ channels in the control of the vascular tone and cardiac contraction, we used whole-cell patch-clamp to test whether CA affects L-type Ca2+ currents in mouse ventricular cardiomyocytes (VCM, with Ca2+ as charge carrier) and in mesenteric artery smooth muscle cells (VSMC, with Ba2+ as charge carrier). We found that CA inhibited L-type currents in both cell types in a concentration-dependent manner, with little voltage-dependent effects. However, CA was more potent in VCM than in VSMC and caused opposite effects on the rate of inactivation. We found these divergences to be at least in part due to the use of different charge carriers. We conclude that CA inhibits L-type Ca2+ channels and that this effect may contribute to its vasorelaxing action. Importantly, our results demonstrate that TRPA1 is not a specific target of CA and indicate that the inhibition of voltage-gated Ca2+ channels should be taken into account when using CA to probe the pathophysiological roles of TRPA1.

Neurotransmitters and second messenger systems in the carotid body.

It is well known that multiple cellular events are regulated by second messenger systems (Greengard and Costa, 1970; Sekar et al., 1986; Hockberger et al., 1987). Among second messengers, cAMP have been shown to modulate several ionic channels and neurotransmitter synthesis and release in different structures (Joh et al.,1978; Hockberger et al., 1987; Morita et al., 1987; Santiago et al., 1990). It has been reported that several stimuli of the carotid body (CB) like hypoxia, hypercapnic acidosis, high extracellular and cyanide, stimulate adenylate cyclase activity, and that hypoxia was the only one that induces cAMP increases in a calcium-free medium, this accumulation being proportional to the intensity of the hypoxic stimulation (Perez-Garcia et al., 1990). Therefore it was proposed a primary role for cAMP in CB chemotransduction (Perez-Garcia et al., 1990, 1991). It was further suggested that the specific activation of adenylate cyclase by hypoxia could reflect a direct effect of low pO2 on the cAMP biosynthetic machinery.

Proliferative role of Kv11 channels in murine arteries

K+ channels encoded by the ether-a-go-go related gene (ERG1 or KCNH2) are important determinants of the cardiac action potential. Expression of both cardiac isoforms (ERG1a and ERG1b) were identified in murine portal vein and distinctive voltage-gated K+ currents were recorded from single myocytes. The aim of the present study was to ascertain the expression and functional impact of ERG channels in murine arteries. Methods: Quantitative RT-PCR was undertaken on RNA extracted from a number of murine arteries. Immunofluorescence was performed on single vascular smooth muscle cells using antibodies against the ERG1 expression product (Kv11.1). Single cell electrophysiology was performed on myocytes from portal vein and several different arteries, complimented by isometric tension recordings. Proliferation assays were undertaken on smooth muscle cells isolated from femoral arteries. Results: ERG1 transcripts were detected in all murine blood vessels, and Kv11.1 immunofluorescence was observed in all smooth muscle cells. However, K+ currents with properties consistent with ERG channels were only recorded in portal vein myocytes. Moreover, ERG channel blockers (E4031 or dofetilide, 1 μM) failed to depolarize carotid arteries or produce contraction. Proliferation of arterial smooth muscle cells was associated with a marked increase in ERG1 expression and ERG blockers suppressed proliferation significantly. Conclusions: These data reveal that arterial blood vessels express ERG channels that appear to be functional silent in contractile smooth muscle but contribute to proliferative response.