Angel Cogolludo

Serotonin Inhibits Voltage-Gated K+ Currents in Pulmonary Artery Smooth Muscle Cells: Role of 5-HT2A Receptors, Caveolin-1, and KV1.5 Channel Internalization

Multiple lines of evidence indicate that serotonin (5-hydroxytryptamine [5-HT]) and voltage-gated K+ (KV) channels play a central role in the pathogenesis of pulmonary hypertension (PH). We hypothesized that 5-HT might modulate the activity of KV channels, therefore establishing a link between these pathogenetic factors in PH. Here, we studied the effects of 5-HT on KV channels present in rat pulmonary artery smooth muscle cells (PASMC) and on hKV1.5 channels stably expressed in Ltk− cells. 5-HT reduced native KV and hKV1.5 currents, depolarized cell membrane, and caused a contraction of isolated pulmonary arteries. The effects of 5-HT on KV currents and contraction were markedly prevented by the 5-HT2A receptor antagonist ketanserin. Incubation with inhibitors of phospholipase C (U73122), classic protein kinase Cs (Gö6976), or tyrosine kinases (genistein and tyrphostin 23), the cholesterol depletion agent β-cyclodextrin or concanavalin A, an inhibitor of endocytotic processes, also prevented the effects of 5-HT. In homogenates from pulmonary arteries, 5-HT2A receptors and caveolin-1 coimmunoprecipitated with KV1.5 channels, and this was increased on stimulation with 5-HT. Moreover, KV1.5 channels were internalized when cells were stimulated with 5-HT, and this was prevented by concanavalin A. These findings indicate that activation of 5-HT2A receptors inhibits native KV and hKV1.5 currents via phospholipase C, protein kinase C, tyrosine kinase, and a caveolae pathway. KV channel inhibition accounts, at least partly, for 5-HT-induced pulmonary vasoconstriction and might play a role in PH.

Thromboxane A2-Induced Inhibition of Voltage-Gated K+ Channels and Pulmonary Vasoconstriction

Abstract— Voltage-gated K+ channels (KV) and thromboxane A2 (TXA2) play critical roles in controlling pulmonary arterial tone under physiological and pathological conditions. We hypothesized that TXA2 might inhibit KV channels, thereby establishing a link between these two major pathogenic pathways in pulmonary hypertension. The TXA2 analogue U46619 inhibited IK(V) (Emax=56.1±3.9%, EC50=0.054±0.019 &mgr;mol/L) and depolarized pulmonary artery smooth muscle cells via activation of TP receptors. In isolated pulmonary arteries, U46619 simultaneously increased intracellular Ca2+ concentration and contractile force, and these effects were inhibited by nifedipine or KCl (60 mmol/L). U46619-induced contractions were not altered by the inhibitors of tyrosine kinase genistein or Rho kinase Y-27632 but were prevented by the nonselective protein kinase C (PKC) inhibitors staurosporine and calphostin C. Furthermore, these responses were sensitive to Gö-6983 but insensitive to bisindolylmaleimide I and Gö-6976. Based on the specificity of these drugs, we suggested a role for an atypical PKC in U46619-induced effects. Thus, treatment with a PKC&zgr; pseudosubstrate inhibitor markedly prevented the vasoconstriction, the inhibition of IK(V), and the depolarization induced by U46619. Western blots showed a transient translocation of PKC&zgr; from the cytosolic to the particulate fraction on stimulation with U46619. These results indicate that TXA2 inhibits IK(V), leading to depolarization, activation of L-type Ca2+ channels, and vasoconstriction of rat pulmonary arteries. We propose PKC&zgr; as a link between TP receptor activation and KV channel inhibition.

Activation of neutral sphingomyelinase is involved in acute hypoxic pulmonary vasoconstriction

Aims The mechanisms involved in hypoxic pulmonary vasoconstriction (HPV) are not yet fully defined. The aim of the study was to determine the role of protein kinase C ζ (PKCζ) and neutral sphingomyelinase (nSMase) in HPV. Methods and results Ceramide content was measured by immunocytochemistry and voltage-gated potassium channel (KV) currents were recorded by the patch clamp technique in isolated rat pulmonary artery smooth muscle cells (PASMC). Contractile responses were analysed in rat pulmonary arteries mounted in a wire myograph. Pulmonary pressure was recorded in anesthetized open-chest rats. Protein and mRNA expression were measured by western blot and RT–PCR, respectively. We found that hypoxia increased ceramide content in PASMC which was abrogated by inhibition of nSMase, but not acid sphingomyelinase (aSMase). The hypoxia-induced vasoconstrictor response in isolated pulmonary arteries and the inhibition of KV currents were strongly reduced by inhibition of PKCζ or nSMase but not aSMase. The nSMase inhibitor GW4869 prevented HPV in vivo. The vasoconstrictor response to hypoxia was mimicked by exogenous addition of bacterial Smase and ceramide. nSMase2 mRNA expression was ∼10-fold higher in pulmonary compared with mesenteric arteries. In mesenteric arteries, hypoxia failed to increase ceramide but exogenous SMase induced a contractile response. Conclusion nSMase-derived ceramide production and the activation of PKCζ are early and necessary events in the signalling cascade of acute HPV.

Reactive oxygen species signaling in pulmonary vascular smooth muscle.

In recent years, it has become evident that reactive oxygen species (ROS) play a critical role in the regulation of several physiological and pathophysiological processes. Herein we review the main sources, targets and pathophysiological roles of ROS in pulmonary vascular smooth muscle. Mitochondria and NADPH oxidases represent the major sources of ROS in vascular cells. In addition, ROS can be produced by different pathways of arachidonic acid metabolism, endothelial NO synthase (eNOS) and xantine oxidase. There is increasing evidence for the role of ROS, specially hydrogen peroxide, as signaling moieties to induce increase in intracellular calcium concentration ([Ca2+]i) and contraction in pulmonary artery smooth muscle cells (PASMC) through the modulation of a variety of targets, such as Rho kinases (ROCK), protein kinase C (PKC), voltage-gated potassium K+ (Kv) channels and ryanodine receptors (RyR). Thus, an increase in ROS has been reported to contribute to the responses induced by different vasoconstrictor stimuli, including hypoxia. Finally, results from recent studies highlighting the involvement of ROS in the development of pulmonary hypertension are discussed in the present paper.

Relaxant Effects of Carbon Monoxide Compared with Nitric Oxide in Pulmonary and Systemic Vessels of Newborn Piglets

Nitric oxide (NO) has been implicated in a number of diverse physiologic processes, including regulation of vascular tone. Carbon monoxide (CO) is another endogenously generated diatomic gas that may play an important physiologic role in vascular smooth muscle homeostasis. The purpose of this study was to compare the responses to exogenous NO and CO in isolated vessels (pulmonary arteries, pulmonary veins, and mesenteric arteries) from 12- to 24-h-old and 2-wk-old piglets. Vessels precontracted with the thromboxane A2 mimetic U46619 (10−7 M) relaxed in response to CO (2 × 10−6 to 2 × 10−4 M) and NO (2 × 10−9 to 2 × 10−7 M); these effects were not affected by endothelium removal but were completely abolished by the soluble guanylate cyclase inhibitor ODQ (10−5 M). In pulmonary arteries, the maximal relaxation to NO increased with postnatal age from 33 ± 4% of the precontraction value to 56 ± 5%, in 12- to 24-h-old and 2-week-old piglets, respectively (p < 0.01), but the response to CO decreased from 25 ± 3% to 12 ± 1%, respectively (p < 0.01). The maximal response to CO was greater in pulmonary veins than in pulmonary or mesenteric arteries for both age groups (p < 0.01). Vasorelaxation induced by endogenous NO (stimulated by acetylcholine) was also greater in pulmonary veins when compared with pulmonary arteries and increased with postnatal age in both vessels. In contrast, no age-related differences were observed in the vasorelaxation induced by the cGMP analog 8-bromo cGMP in pulmonary arteries. When the response to NO was analyzed under three different extracellular O2 concentrations (Po2 4.51 ± 0.03, 19.32 ± 0.17, and 86 ± 0.62, kPa), no significant differences were found. However, in the presence of superoxide dismutase (100 U/mL). the response to CO remained unchanged, and the response to NO improved in pulmonary arteries from 2-week-old but not from newborn piglets. In conclusion, both NO and CO relaxed neonatal vessels through soluble guanylate cyclase activation. However, when compared with NO, CO exhibited a poor vasorelaxant activity. Pulmonary vasorelaxation induced by NO increased with postnatal age, whereas that induced by CO decreased. Changes in extracellular oxygen concentration did not alter the pulmonary vascular response to NO. However, the presence of superoxide dismutase improved the response to NO, indicating that oxidant activity limits the vasorelaxant response to NO but not to CO.

Activation of peroxisome proliferator-activated receptor-β/-δ (PPARβ/δ) prevents endothelial dysfunction in type 1 diabetic rats.

Endothelial dysfunction plays a key role in the pathogenesis of diabetic vascular disease. Herein, we have analyzed if the peroxisome proliferator-activated receptor-β/-δ (PPARβ/δ) agonist GW0742 exerts protective effects on endothelial function in type 1 diabetic rats. The rats were divided into 4 groups: control, control-treated (GW0742, 5 mg kg(-1)day(-1) for 5 weeks), diabetic (streptozotocin injection), and diabetic-treated. GW0742 administration in diabetic rats did not alter plasma glucose, systolic blood pressure, or heart rate, but reduced plasma triglyceride levels. The vasodilatation induced by acetylcholine was decreased in aortas from diabetic rats. GW0742 restored endothelial function, increasing eNOS phosphorylation. Superoxide production, NADPH oxidase activity, and mRNA expression of prepro endothelin-1, p22(phox), p47(phox), and NOX-1 were significantly higher in diabetic aortas, and GW0742 treatment prevented these changes. In addition, GW0742 prevented the endothelial dysfunction and the upregulation of prepro endothelin-1 and p47(phox) after the in vitro incubation of aortic rings with high glucose and these effects were prevented by the PPARβ/δ antagonist GSK0660. PPARβ/δ activation restores endothelial function in type 1 diabetic rats. This effect seems to be related to an increase in nitric oxide bioavailability as a result of reduced NADPH oxidase-driven superoxide production and downregulation of prepro endothelin-1.

Diabetes induces pulmonary artery endothelial dysfunction by NADPH oxidase induction

Recent data suggest that diabetes is a risk factor for pulmonary hypertension. The aim of the present study was to analyze whether diabetes induces endothelial dysfunction in pulmonary arteries and the mechanisms involved. Male Sprague-Dawley rats were randomly divided into a control (saline) and a diabetic group (70 mg/kg(-1) streptozotocin). After 6 wk, intrapulmonary arteries were mounted for isometric tension recording, and endothelial function was tested by the relaxant response to acetylcholine. Protein expression and localization were measured by Western blot and immunohistochemistry and superoxide production by dihydroethidium staining. Pulmonary arteries from diabetic rats showed impaired relaxant response to acetylcholine and reduced vasoconstrictor response to the nitric oxide (NO) synthase inhibitor L-NAME, whereas the response to nitroprusside and the expression of endothelial NO synthase remained unchanged. Endothelial dysfunction was reversed by addition of superoxide dismutase or the NADPH oxidase inhibitor apocynin. An increase in superoxide production and increased expression of the NADPH oxidase regulatory subunit p47(phox) were also found in pulmonary arteries from diabetic rats. In conclusion, the pulmonary circulation is a target for diabetes-induced endothelial dysfunction via enhanced NADPH oxidase-derived superoxide production.

Role of Reactive Oxygen Species in Kv Channel Inhibition and Vasoconstriction Induced by TP Receptor Activation in Rat Pulmonary Arteries

Abstract:  Voltage‐gated potassium channels (Kv) and thromboxane A2 (TXA2) have been involved in several forms of human and experimental pulmonary hypertension. We have reported that the TXA2 analog U46619, via activation of TP receptors and PKCζ, inhibited Kv currents in rat pulmonary artery smooth muscle cells (PASMC), increased cytosolic calcium, and induced a contractile response in isolated rat and piglet pulmonary arteries (PA). Herein, we have analyzed the role of reactive oxygen species (ROS) in this signaling pathway. In rat PA, U46619 increased dichlorofluorescein fluorescence, an indicator of intracellular hydrogen peroxide, and this effect was prevented by the NADPH oxidase inhibitor apocynin and by polyethyleneglycol‐catalase (PEG‐catalase, a membrane‐permeable form of catalase). U46619 inhibited Kv currents in native PASMC and these effects were strongly inhibited by apocynin. The contractile responses to U46619 in isolated PA were inhibited by PEG‐catalase and the NADPH oxidase inhibitors diphenylene iodonium (DPI) and apocynin. A membrane permeable of hydrogen peroxide, t‐butyl hydroperoxide, also inhibited Kv currents and induced a contractile response. Activation of NADPH oxidase and the subsequent production of hydrogen peroxide are involved in the Kv channel inhibition and the contractile response induced by TP receptor activation in rat PA.

Postnatal Maturation of Phosphodiesterase 5 (PDE5) in Piglet Pulmonary Arteries: Activity, Expression, Effects of PDE5 Inhibitors, and Role of the Nitric Oxide/Cyclic GMP Pathway

After birth and during the first days of extrauterine life, pulmonary arterial pressure is progressively reduced to reach the adult values. We hypothesized that changes in PDE5 activity might be involved in the pulmonary postnatal maturation of the nitric oxide (NO)/cGMP pathway. The PDE5 inhibitor sildenafil produced vasorelaxant responses in isolated pulmonary arteries. These effects were similar in newborn (3–18 h) and 2-wk-old piglets, unchanged by endothelium removal, and markedly inhibited by the soluble guanylyl cyclase inhibitor ODQ. The peak of the transient vasorelaxant response to NO gas increased with postnatal age but was unaffected by PDE inhibition. However, the duration of the response to NO was significantly increased. The vasorelaxant response to sodium nitroprusside was potentiated by sildenafil in both age groups. The PDE5 inhibitors dipyridamole and zaprinast, produced qualitatively similar effects but with lower potency. Both total and PDE5-dependent cGMP hydrolytic activity and PDE5 protein expression increased with postnatal age. All these results suggest that PDE5 is a key regulator of NO-induced vasodilation in the postnatal pulmonary arteries. PDE5 inhibition is able to produce pulmonary vasodilation even in the absence of a functional endothelium and potentiates the vasorelaxant response to exogenous NO and nitroprusside. However, PDE5 is not responsible for the maturational increase of NO bioactivity during the first days of extrauterine life.

Neutral Sphingomyelinase, NADPH Oxidase and Reactive Oxygen Species. Role in Acute Hypoxic Pulmonary Vasoconstriction

The molecular mechanisms underlying hypoxic pulmonary vasoconstriction (HPV) are not yet properly understood. Mitochondrial electron transport chain (ETC) and NADPH oxidase have been proposed as possible oxygen sensors, with derived reactive oxygen species (ROS) playing key roles in coupling the sensor(s) to the contractile machinery. We have recently reported that activation of neutral sphingomyelinase (nSMase) and protein kinase C ζ (PKCζ) participate in the signalling cascade of HPV. Herein, we studied the significance of nSMase in controlling ROS production rate in rat pulmonary artery (PA) smooth muscle cells and thereby HPV in rat PA. ROS production (analyzed by dichlorofluorescein and dihydroethidium fluorescence) was increased by hypoxia in endothelium‐denuded PA segments and their inhibition prevented hypoxia‐induced voltage‐gated potassium channel (KV) inhibition and pulmonary vasoconstriction. Consistently, H2O2, or its analogue t‐BHP, decreased KV currents and induced a contractile response, mimicking the effects of hypoxia. Inhibitors of mitochondrial ETC (rotenone) and NADPH oxidase (apocynin) prevented hypoxia‐induced ROS production, KV channel inhibition and vasoconstriction. Hypoxia induced p47phox phosphorylation and its interaction with caveolin‐1. Inhibition of nSMase (GW4869) or PKCζ prevented p47phox phosphorylation and ROS production. The increase in ceramide induced by hypoxia (analyzed by immunocytochemistry) was inhibited by rotenone. Exogenous ceramide increased ROS production in a PKCζ sensitive manner. We propose an integrated signalling pathway for HPV which includes nSMase‐PKCζ‐NADPH oxidase as a necessary step required for ROS production and vasoconstriction. J. Cell. Physiol. 226: 2633–2640, 2011.