Efectos vasculares del levamisol y su interacción con la cocaína

Alrededor del 30% de las visitas al servicio de urgencias por abuso de drogas están relacionadas con complicaciones inducidas por la cocaína sobre el sistema cardiovascular, principalmente como consecuencia de un aumento del tono simpático. El levamisol, fármaco antihelmíntico limitado al uso veterinario por sus efectos adversos en humanos, es el adulterante más frecuente de la cocaína. Al igual que la cocaína, el levamisol bloquea de forma aguda la la recaptación de aminas biógenas, pero sus efectos vasculares no son bien conocidos. Por lo tanto, el objetivo central de este estudio fue evaluar los efectos directos del levamisol y la cocaína en tres arterias diferentes. Se montaron segmentos de arterias carótida, renal y aorta de 3 mm de longitud, procedentes de 30 conejos sanos, en un sistema de baño de órganos que contenía solución fisiológica (solución de Krebs-Henseleit) para el registro de tensión isométrica. Además, se midió la expresión proteica de los receptores adrenérgicos (α1 y α2), de la eNOS y de marcadores de estrés oxidativo (SOD1, SOD2 y Nox4) mediante la técnica de Western Blot. El levamisol, al igual que la cocaína, no modificó el tono vascular basal. Sin embargo, el levamisol (10-3 M) produjo un ligero aumento en la arteria carótida. En vasos contraídos con noradrenalina, el levamisol, pero no la cocaína, indujo una relajación dependiente de la concentración. La administración conjunta de levamisol y cocaína no alteró la respuesta relajante máxima inducida por el levamisol. El levamisol (10-4-10-3 M) redujo la respuesta contráctil a la fenilefrina en todos los lechos estudiados, mientras que la cocaína (10-5 M) potenció esta respuesta en la arteria carótida. La administración conjunta de ambas sustancias no modificó la respuesta producida por el levamisol. Tanto el levamisol (10-6-10-4 M) como la cocaína (10-6-10-4 M) evocaron una potenciación dependiente de la concentración del estímulo nervioso simpático. Este efecto aumentó aún más con la combinación de levamisol (10-4 M) y cocaína (10-4 M) en las arterias carótida y renal. Sin embargo, una mayor concentración de levamisol (10-3 M) anuló la respuesta contráctil al estímulo simpático solo o en presencia de cocaína en todos los lechos vasculares. La respuesta relajante a la acetilcolina se redujo en presencia de levamisol, mientras que la cocaína solo la disminuyó en las arterias renal y aorta. Una menor relajación dependiente del endotelio inducida por ambas sustancias se ha relacionado con un aumento del estrés oxidativo que conduciría a disminuir la biodisponibilidad de NO. Ninguna de estas sustancias modificó la relajación inducida por el nitroprusiato sódico. La exposición aguda al levamisol y cocaína también produjo cambios a nivel molecular en los vasos estudiados. El levamisol y la cocaína a bajas concentraciones disminuyeron la expresión proteica de los receptores α2-adrenérgicos, mientras que los receptores α1-adrenérgicos solo fueron infra expresados en presencia de altas concentraciones de levamisol. La cocaína redujo la expresión proteica de los receptores α1 adrenérgicos en la arteria renal. Por otro lado, el levamisol disminuyó la expresión proteica de la eNOS en la aorta, mientras que la cocaína solo lo hizo en las arterias renal y aorta. Tanto el levamisol como la cocaína aumentaron la expresión proteica de la enzima prooxidante Nox4 en la arteria renal. Paradójicamente, el levamisol infra expresó esta enzima en la arteria carótida. La expresión de las enzimas antioxidantes SOD1 y SOD2 se redujo en presencia de levamisol o cocaína tanto en la arteria renal como en la aorta. Estos resultados indican que: 1) dependiendo de la concentración el levamisol se comporta como un antagonista no selectivo de los receptores adrenérgicos α1 y α2, 2) el levamisol a bajas concentraciones potencia los efectos de la cocaína a nivel presináptico, lo que se traduce en un sinergismo, 3) el levamisol disminuye la respuesta relajante a la acetilcolina de forma concentración-dependiente en los tres vasos estudiados, mientras que la cocaína solo afecta a las arterias renal y aorta. La combinación de levamisol y cocaína no altera la respuesta producida por el levamisol, 4) la disfunción endotelial inducida por ambas sustancias puede estar relacionada con un aumento de estrés oxidativo.About 30% of Emergency Department visits for drug abuse are related to cocaine cardiovascular toxicity, mainly secondary to an increased sympathetic tone. Levamisole, a veterinary anthelmintic drug, is the most used cocaine adulterant. Like cocaine, levamisole acutely blocks noradrenaline reuptake, but its vascular effects are not well known. Therefore, the main purpose of this study was to evaluate the direct effects of levamisole and cocaine in three different arteries. Rings (3 mm long) of carotid and renal arteries and the aorta from 30 healthy rabbits were mounted for isometric tension recording in organ bath chambers containing Krebs-Henseleit solution. α1 and α2 -adrenergic receptors, eNOS, SOD1 and SOD2, and Nox4 protein expression was measured by Western Blot. Only levamisole at the highest concentration (10 3 M) slightly increased basal vascular tone, effect limited to carotid artery. In vessels contracted with noradrenaline, only levamisole induced a concentration-dependent relaxation. Levamisole (10-4-10-3 M) reduced the contractile response to phenylephrine, while cocaine (10-5 M) enhanced this response in the carotid artery. Co-administration of both substances did not modify levamisole response. Both levamisole (10-6-10-4 M) and cocaine (10-6 -10-4 M) produced a concentration-dependent potentiation of the electrical field stimulation. This effect was further enhanced by the combination of levamisole (10-4 M) and cocaine (10 4 M) in the carotid and renal arteries. However, levamisole (10-3 M) abolished adrenergic neurotransmission alone or in the presence of cocaine. The relaxing response to acetylcholine was reduced by levamisole, while cocaine decreased relaxation only in the renal artery and aorta. Impaired relaxation induced by both substances was associated with increased oxidative stress. Neither cocaine nor levamisole modified the relaxation induced by sodium nitroprusside. Levamisole and cocaine also induced vessels changes at molecular level. Lower concentrations of each substance reduced α2 adrenergic receptor protein expression whereas α1 expression was only reduced by higher concentrations of levamisole. Cocaine decreased α1 expression only in the renal artery. Furthermore, cocaine reduced eNOS protein expression in both renal artery and aorta, whereas levamisole did it only in the aorta. Both cocaine and levamisole increased Nox4 protein expression in the renal artery. However, levamisole reduced its expression in the carotid artery. SOD1 and SOD2 expression was reduced by both substances in the renal artery and aorta. In conclusion, 1) depending on concentration, levamisole acts as a non-selective antagonist of α1 and α2 adrenergic receptors; 2) lower concentrations of levamisole enhances the effects of cocaine at presynaptic level, resulting in synergism; 3) levamisole decreases the relaxation to acetylcholine in a concentration dependent manner in the three vessels while cocaine does it only in the renal artery and aorta. 4) The endothelial dysfunction induced by both substances may be related to increased oxidative stress

Action of low doses of Aspirin in Inflammation and Oxidative Stress induced by aβ1-42 on Astrocytes in primary culture

Aspirin has been used as anti-inflammatory and anti-aggregate for decades but the precise mechanism(s) of action after the presence of the toxic peptide Aβ1-42 in cultured astrocytes remains poorly resolved. Here we use low-doses of aspirin (10-7 M) in astrocytes in primary culture in presence or absence of Aβ1-42 toxic peptide. We noted an increase of cell viability and proliferation with or without Aβ1-42 peptide presence in aspirin treated cells. In addition, a decrease in apoptosis, determined by Caspase 3 activity and the expression of Cyt c and Smac/Diablo, were detected. Also, aspirin diminished necrosis process (LDH levels), pro-inflammatory mediators (IL-β and TNF-α) and NF-ᴋB protein expression, increasing anti-inflammatory PPAR-γ protein expression, preventing Aβ1-42 toxic effects. Aspirin inhibited COX-2 and iNOS without changes in COX-1 expression, increasing anti-oxidant protein (Cu/Zn-SOD and Mn-SOD) expression in presence or absence of Aβ1-42. Taken together, our results show that aspirin, at low doses increases cell viability by decreasing inflammation and oxidative stress, preventing the deleterious effects of the Aβ1-42 peptide on astrocytes in primary culture. The use of low doses of aspirin may be more suitable for Alzheimer's disease

Neuronal effects of Sugammadex in combination with Rocuronium or Vecuronium.

Rocuronium (ROC) and Vecuronium (VEC) are the most currently used steroidal non-depolarizing neuromuscular blocking (MNB) agents. Sugammadex (SUG) rapidly reverses steroidal NMB agents after anaesthesia. The present study was conducted in order to evaluate neuronal effects of SUG alone and in combination with both ROC and VEC. Using MTT, CASP-3 activity and Western-blot we determined the toxicity of SUG, ROC or VEC in neurons in primary culture. SUG induces apoptosis/necrosis in neurons in primary culture and increases cytochrome C (CytC), apoptosis-inducing factor (AIF), Smac/Diablo and Caspase 3 (CASP-3) protein expression. Our results also demonstrated that both ROC and VEC prevent these SUG effects. The protective role of both ROC and VEC could be explained by the fact that SUG encapsulates NMB drugs. In BBB impaired conditions it would be desirable to control SUG doses to prevent the excess of free SUG in plasma that may induce neuronal damage. A balance between SUG, ROC or VEC would be necessary to prevent the risk of cell damag

PPARγ as an indicator of vascular function in an experimental model of metabolic syndrome in rabbits

Background and aims: Underlying mechanisms associated with vascular dysfunction in metabolic syndrome (MetS) remain unclear and can even vary from one vascular bed to another. Methods: In this study, MetS was induced by a high-fat, high-sucrose diet, and after 28 weeks, aorta and renal arteries were removed and used for isometric recording of tension in organ baths, protein expression by Western blot, and histological analysis to assess the presence of atherosclerosis. Results: MetS induced a mild hypertension, pre-diabetes, central obesity and dyslipidaemia. Our results indicated that MetS did not change the contractile response in either the aorta or renal artery. Conversely, vasodilation was affected in both arteries in a different way. The aorta from MetS showed vascular dysfunction, including lower response to acetylcholine and sodium nitroprusside, while the renal artery from MetS presented a preserved relaxation to acetylcholine and an increased sensitivity to sodium nitroprusside. We did not find vascular oxidative stress in the aorta from MetS, but we found a significant decrease in PPARγ, phospho-Akt (p-Akt) and phospho-eNOS (p-eNOS) protein expression. On the other hand, we found oxidative stress in the renal artery from MetS, and PPARγ, Akt and p-Akt were overexpressed. No evidence of atherosclerosis was found in arteries from MetS. Conclusions: MetS affects vascular function differently depending on the vessel. In the aorta, it decreases both the vasodilation and the expression of the PPARγ/Akt/eNOS pathway, while in the renal artery, it increases the expression of PPARγ/Akt signalling pathway without decreasing the vasodilation

WIN 55,212-2, agonist of cannabinoid receptors, prevents amyloid β1-42 effects on astrocytes in primary culture

Alzheimer´s disease (AD), a neurodegenerative illness involving synaptic dysfunction with extracellular accumulation of Aβ1-42 toxic peptide, glial activation, inflammatory response and oxidative stress, can lead to neuronal death. Endogenous cannabinoid system is implicated in physiological and physiopathological events in central nervous system (CNS), and changes in this system are related to many human diseases, including AD. However, studies on the effects of cannabinoids on astrocytes functions are scarce. In primary cultured astrocytes we studied cellular viability using MTT assay. Inflammatory and oxidative stress mediators were determined by ELISA and Westernblot techniques both in the presence and absence of Aβ1-42 peptide. Effects of WIN 55,212-2 (a synthetic cannabinoid) on cell viability, inflammatory mediators and oxidative stress were also determined. Aβ1-42 diminished astrocytes viability, increased TNF-α and IL-1β levels and p-65, COX-2 and iNOS protein expression while decreased PPAR-γ and antioxidant enzyme Cu/Zn SOD. WIN 55,212-2 pretreatment prevents all effects elicited by Aβ1-42. Furthermore, cannabinoid WIN 55,212-2 also increased cell viability and PPAR-γ expression in control astrocytes. In conclusion cannabinoid WIN 55,212-2 increases cell viability and anti-inflammatory response in cultured astrocytes. Moreover, WIN 55,212-2 increases expression of anti-oxidant Cu/Zn SOD and is able to prevent inflammation induced by Aβ1-42 in cultured astrocytes. Further studies would be needed to assess the possible beneficial effects of cannabinoids in Alzheimer's disease patients

Astrocytes protect neurons from Aβ1-42 peptide-induced neurotoxicity increasing TFAM and PGC-1 and decreasing PPAR-γ and SIRT-1.

One of the earliest neuropathological events in Alzheimer¿s disease is accumulation of astrocytes at sites of Aβ1-42 depositions. Our results indicate that Aβ1-42 toxic peptide increases lipid peroxidation, apoptosis and cell death in neurons but not in astrocytes in primary culture. Aβ1-42-induced deleterious neuronal effects are not present when neurons and astrocytes are mixed cultured. Stimulation of astrocytes with toxic Aβ1-42 peptide increased p-65 and decreased IκB resulting in inflammatory process. In astrocytes Aβ1-42 decreases protein expressions of sirtuin 1 (SIRT-1) and peroxisome proliferator-activated receptor γ (PPAR-γ) and over-expresses peroxisome proliferator-activated receptor γ coactivator 1 (PGC-1) and mitochondrial transcription factor A (TFAM), protecting mitochondria against Aβ1-42-induced damage and promoting mitochondrial biogenesis. In summary our data suggest that astrocytes may have a key role in protecting neurons, increasing neural viability and mitochondrial biogenesis, acquiring better oxidative stress protection and perhaps modulating inflammatory processes against Aβ1-42 toxic peptide. This might be a sign of a complex epigenetic process in Alzheimer¿s disease development

Changes in Chemokines and Chemokine Receptors Expression in a Mouse Model of Alzheimer's Disease

The amyloid precursor protein plus presenilin-1 (APP/PS1) mice are a frequently-used model for Alzheimer's disease studies (AD). However, the data relevant to which proteins are involved in inflammatory mechanism are not sufficiently well-studied using the AD mouse model. Using behavioral studies, quantitative RT-PCR and Western-blot techniques, significant findings were determined by the expression of proteins involved in inflammation comparing APP/PS1 and Wild type mice. Increased GFAP expression could be associated with the elevation in number of reactive astrocytes. IL-3 is involved in inflammation and ABDF1 intervenes normally in the transport across cell membranes and both were found up-regulated in APP/PS1 mice compared to Wild type mice. Furthermore, CCR5 expression was decreased and both CCL3 and CCL4 chemokines were highly expressed indicating a possible gliosis and probably an increase in chemotaxis from lymphocytes and T cell generation. We also noted for the first time, a CCR8 increase expression with diminution of its CCL1 chemokine, both normally involved in protection from bacterial infection and demyelination. Control of inflammatory proteins will be the next step in understanding the progression of AD and also in determining the mechanisms that can develop in this disease

Effects of ranolazine on astrocytes and neurons in primary culture

Ranolazine (Rn) is an antianginal agent used for the treatment of chronic angina pectoris when angina is not adequately controlled by other drugs. Rn also acts in the central nervous system and it has been proposed for the treatment of pain and epileptic disorders. Under the hypothesis that ranolazine could act as a neuroprotective drug, we studied its effects on astrocytes and neurons in primary culture. We incubated rat astrocytes and neurons in primary cultures for 24 hours with Rn (10−7, 10−6 and 10−5 M). Cell viability and proliferation were measured using trypan blue exclusion assay, MTT conversion assay and LDH release assay. Apoptosis was determined by Caspase 3 activity assay. The effects of Rn on proinflammatory mediators IL-β and TNF-α was determined by ELISA technique, and protein expression levels of Smac/Diablo, PPAR-γ, Mn-SOD and Cu/Zn-SOD by western blot technique. In cultured astrocytes, Rn significantly increased cell viability and proliferation at any concentration tested, and decreased LDH leakage, Smac/Diablo expression and Caspase 3 activity indicating less cell death. Rn also increased anti-inflammatory PPAR-γ protein expression and reduced pro-inflammatory proteins IL-1 β and TNFα levels. Furthermore, antioxidant proteins Cu/Zn-SOD and Mn-SOD significantly increased after Rn addition in cultured astrocytes. Conversely, Rn did not exert any effect on cultured neurons. In conclusion, Rn could act as a neuroprotective drug in the central nervous system by promoting astrocyte viability, preventing necrosis and apoptosis, inhibiting inflammatory phenomena and inducing anti-inflammatory and antioxidant agents

The Role of Aryl Hydrocarbon Receptor in the Endothelium : A Systematic Review

Activation of the aryl hydrocarbon receptor (AhR) has been shown to be important in physiological processes other than detoxification, including vascular homeostasis. Although AhR is highly expressed in the endothelium, its function has been poorly studied. This systematic review aims to summarise current knowledge on the AhR role in the endothelium and its cardiovascular implications. We focus on endogenous AhR agonists, such as some uremic toxins and other agonists unrelated to environmental pollutants, as well as studies using AhR knockout models. We conclude that AhR activation leads to vascular oxidative stress and endothelial dysfunction and that blocking AhR signalling could provide a new target for the treatment of vascular disorders such as cardiovascular complications in patients with chronic kidney disease or pulmonary arterial hypertension

Cerium dioxide nanoparticles modulate antioxidant defences and change vascular response in the human saphenous vein

Nanoparticles have a promising future in biomedical applications and knowing whether they affect ex vivo vascular reactivity is a necessary step before their use in patients. In this study, we have evaluated the vascular effect of cerium dioxide nanoparticles (CeONPs) on the human saphenous vein in response to relaxing and contractile agonists. In addition, we have measured the protein expression of key enzymes related to vascular homeostasis and oxidative stress. We found that CeONPs increased expression of both SOD isoforms, and the consequent reduction of superoxide anion would enhance the bioavailability of NO explaining the increased vascular sensitivity to sodium nitroprusside in the presence of CeONPs. The NOX4 reduction induced by CeONPs may lead to lower HO synthesis associated with vasodilation through potassium channels explaining the lower vasodilation to bradykinin. In addition, we showed for the first time, that CeONPs increase the expression of ACE2 in human saphenous vein, and it may be the cause of the reduced contraction to angiotensin II. Moreover, we ruled out that CeONPs have effect on the protein expression of eNOS, sGC, BKca channels and angiotensin II receptors or modify the vascular response to noradrenaline, endothelin-1 and TXA analogue. In conclusion, CeONPs show antioxidant properties, and together with their vascular effect, they could be postulated as adjuvants for the treatment of cardiovascular diseases.This work was supported by Carlos III Health Institute (PI22/00424) and the European Regional Development Fund (ERDF ‘‘A way to build Europe’’ grant PI19/00838), by the Ministry of Health of the Valencian Regional Government (PROMETEO/2019/027), and by Universitat de València (UV-INV-AE-1544052)