Emília C. Monteiro

Carotid body denervation prevents the development of insulin resistance and hypertension induced by hypercaloric diets

Increased sympathetic activity is a well-known pathophysiological mechanism in insulin resistance (IR) and hypertension (HT). The carotid bodies (CB) are peripheral chemoreceptors that classically respond to hypoxia by increasing chemosensory activity in the carotid sinus nerve (CSN), causing hyperventilation and activation of the sympathoadrenal system. Besides its role in the control of ventilation, the CB has been proposed as a glucose sensor implicated in the control of energy homeostasis. However, to date no studies have anticipated its role in the development of IR. Herein, we propose that CB overstimulation is involved in the etiology of IR and HT, core metabolic and hemodynamic disturbances of highly prevalent diseases like the metabolic syndrome, type 2 diabetes, and obstructive sleep apnoea. We demonstrate that CB activity is increased in IR animal models and that CSN resection prevents CB overactivation and diet-induced IR and HT. Moreover, we show that insulin triggers CB, highlighting a new role for hyperinsulinemia as a stimulus for CB overactivation. We propose that CB is implicated in the pathogenesis of metabolic and hemodynamic disturbances through sympathoadrenal overactivation and may represent a novel therapeutic target in these diseases.

Hypoxic intensity: a determinant for the contribution of ATP and adenosine to the genesis of carotid body chemosensory activity

Excitatory effects of adenosine and ATP on carotid body (CB) chemoreception have been previously described. Our hypothesis is that both ATP and adenosine are the key neurotransmitters responsible for the hypoxic chemotransmission in the CB sensory synapse, their relative contribution depending on the intensity of hypoxic challenge. To test this hypothesis we measured carotid sinus nerve (CSN) activity in response to moderate and intense hypoxic stimuli (7 and 0% O(2)) in the absence and in the presence of adenosine and ATP receptor antagonists. Additionally, we quantified the release of adenosine and ATP in normoxia (21% O(2)) and in response to hypoxias of different intensities (10, 5, and 2% O(2)) to study the release pathways. We found that ZM241385, an A(2) antagonist, decreased the CSN discharges evoked by 0 and 7% O(2) by 30.8 and 72.5%, respectively. Suramin, a P(2)X antagonist, decreased the CSN discharges evoked by 0 and 7% O(2) by 64.3 and 17.1%, respectively. Simultaneous application of both antagonists strongly inhibited CSN discharges elicited by both hypoxic intensities. ATP release by CB increased in parallel to hypoxia intensity while adenosine release increased preferably in response to mild hypoxia. We have also found that the lower the O(2) levels are, the higher is the percentage of adenosine produced from extracellular catabolism of ATP. Our results demonstrate that ATP and adenosine are key neurotransmitters involved in hypoxic CB chemotransduction, with a more relevant contribution of adenosine during mild hypoxia, while vesicular ATP release constitutes the preferential origin of extracellular adenosine in high-intensity hypoxia.

An antagonistic interaction between A2B adenosine and D2 dopamine receptors modulates the function of rat carotid body chemoreceptor cells

We have previously demonstrated that adenosine controls the release of catecholamines (CA) from carotid body (CB) acting on A2B receptors. Here, we have tested the hypothesis that the control is exerted via an interaction between adenosine A2B and dopamine D2 receptors present in chemoreceptor cells. Experiments were performed in vitro in CB from 3 months rats. The effect of A2B adenosine and D2 dopamine agonists and antagonists applied alone or in combination were studied on basal (20%O2) and hypoxia (10%O2)‐evoked release of CA and cAMP content of CB. We have found that adenosine A2 agonists and D2 antagonists dose‐dependently increased basal and evoked release CA from the CB while A2 antagonists and D2 agonists had an inhibitory action. The existence of A2B‐D2 receptor interaction was established because the inhibitory action of A2 antagonists was abolished by D2 antagonists, and the stimulatory action of A2 agonists was abolished by D2 agonists. Further, A2 agonists increased and D2 agonist decreased cAMP content in the CB; their co‐application eliminated the response. The present results provide direct pharmacological evidence that an antagonistic interaction between A2B adenosine and D2 dopamine receptors exist in rat CB and would explain the dopamine‐adenosine interactions on ventilation previously observed.

Evidence for nevirapine bioactivation in man: searching for the first step in the mechanism of nevirapine toxicity.

Despite its efficacy, including in the prevention of vertical transmission, the antiretroviral nevirapine is associated with severe idiosyncratic hepatotoxicity and skin rash. The mechanisms underlying nevirapine toxicity are not fully understood, but drug bioactivation to reactive metabolites capable of forming stable protein adducts is thought to be involved. This hypothesis is based on the paradigm that drug reactive metabolites have the potential to bind to self-proteins, which results in drug-modified proteins being perceived as foreign by the immune system. The aim of the present work was to identify hemoglobin adducts in HIV patients as biomarkers of nevirapine haptenation upon bioactivation. The ultimate goal is to develop diagnostic methods for predicting the onset of nevirapine-induced toxic reactions. All included subjects were adults on nevirapine-containing antiretroviral therapy for at least 1month. The protocol received prior approval from the Hospital Ethics Committees and patients gave their written informed consent. Nevirapine-derived adducts with the N-terminal valine of hemoglobin were analyzed by an established liquid chromatography-electrospray ionization-tandem mass spectrometry method and characterized on the basis of retention time and mass spectrometric fragmentation pattern by comparison with adduct standards prepared synthetically. The nevirapine adducts were detected in 12/13 patient samples, and quantified in 11/12 samples (2.58±0.8 fmol/g of hemoglobin). This work represents the first evidence of nevirapine-protein adduct formation in man and confirms the ability of nevirapine to modify self-proteins, thus providing clues to the molecular mechanisms underlying nevirapine toxicity. Moreover, the possibility of assessing nevirapine-protein adduct levels has the potential to become useful for predicting the onset of nevirapine-induced adverse reactions.

Adenosine in Peripheral Chemoreception: New Insights into a Historically Overlooked Molecule – Invited Article

In the present article we review in a concise manner the literature on the general biology of adenosine signalling. In the first section we describe briefly the historical aspects of adenosine research. In the second section is presented the biochemical characteristics of this nucleoside, namely its metabolism and regulation, and its physiological actions. In the third section we have succinctly described the role of adenosine and its metabolism in hypoxia. The final section is devoted to the role of adenosine in chemoreception in the carotid body, providing a review of the literature on the presence of adenosine receptors in the carotid body; on the effects of adenosine at presynaptic level in carotid body chemoreceptor cells, as well as, its metabolism and regulation; and at postsynaptic level in carotid sinus nerve activity. Additionally, a review on the effects of adenosine in ventilation was done. This review discusses evidence for a key role of adenosine in the hypoxic response of carotid body and emphasizes new research likely to be important in the future.

Activation of nicotinic ACh receptors with α4 subunits induces adenosine release at the rat carotid body

The effect of ACh on the release of adenosine was studied in rat whole carotid bodies, and the nicotinic ACh receptors involved in the stimulation of this release were characterized. ACh and nicotinic ACh receptor agonists, cytisine, DMPP and nicotine, caused a concentration‐dependent increase in adenosine production during normoxia, with nicotine being more potent and efficient in stimulating adenosine release from rat CB than cytisine and DMPP. D‐Tubocurarine, mecamylamine, DHβE and α‐bungarotoxin, nicotinic ACh receptor antagonists, caused a concentration‐dependent reduction in the release of adenosine evoked by hypoxia. The rank order of potency for nicotinic ACh receptor antagonists that inhibit adenosine release was DHβE>mecamylamine>D‐tubocurarine>α‐bungarotoxin. The effect of the endogenous agonist, ACh, which was mimicked by nicotine, was antagonized by DHβE, a selective nicotinic receptor antagonist. The ecto‐5′‐nucleotidase inhibitor AOPCP produces a 72% inhibition in the release of adenosine from CB evoked by nicotine. Taken together, these data indicate that ACh induced the production of adenosine, mainly from extracellular ATP catabolism at the CB through a mechanism that involves the activation of nicotinic receptors with α4 and β2 receptor subunits.

Carotid body, insulin, and metabolic diseases: unraveling the links

The carotid bodies (CB) are peripheral chemoreceptors that sense changes in arterial blood O2, CO2, and pH levels. Hypoxia, hypercapnia, and acidosis activate the CB, which respond by increasing the action potential frequency in their sensory nerve, the carotid sinus nerve (CSN). CSN activity is integrated in the brain stem to induce a panoply of cardiorespiratory reflexes aimed, primarily, to normalize the altered blood gases, via hyperventilation, and to regulate blood pressure and cardiac performance, via sympathetic nervous system (SNS) activation. Besides its role in the cardiorespiratory control the CB has been proposed as a metabolic sensor implicated in the control of energy homeostasis and, more recently, in the regulation of whole body insulin sensitivity. Hypercaloric diets cause CB overactivation in rats, which seems to be at the origin of the development of insulin resistance and hypertension, core features of metabolic syndrome and type 2 diabetes. Consistent with this notion, CB sensory denervation prevents metabolic and hemodynamic alterations in hypercaloric feed animal. Obstructive sleep apnea (OSA) is another chronic disorder characterized by increased CB activity and intimately related with several metabolic and cardiovascular abnormalities. In this manuscript we review in a concise manner the putative pathways linking CB chemoreceptors deregulation with the pathogenesis of insulin resistance and arterial hypertension. Also, the link between chronic intermittent hypoxia (CIH) and insulin resistance is discussed. Then, a final section is devoted to debate strategies to reduce CB activity and its use for prevention and therapeutics of metabolic diseases with an emphasis on new exciting research in the modulation of bioelectronic signals, likely to be central in the future.

Intra-Individual Variability in Efavirenz Plasma Concentrations Supports Therapeutic Drug Monitoring Based on Quarterly Sampling in the First Year of Therapy

Intrapatient variability in drug plasma concentrations is critical to the use of therapeutic drug monitoring with efavirenz, a non-nucleoside reverse-transcriptase inhibitor. Marked intrapatient variability, particularly for concentrations near the minimal therapeutic concentration, could be a predictor of virologic failure, meaning that a single concentration is of limited value. Previous reports on efavirenz intra-individual variability were obtained only in follow-up periods of 3 to 12 months and do not provide a rationale for the periodicity of sample measurements needed in long-term therapy to identify patients with a large variability and increased risk of therapeutic failure. The aim of this work was to investigate intra-individual variability in efavirenz plasma concentrations over a long-term follow-up period to support therapeutic drug monitoring. In a case series study, clinical and laboratory data were collected from all HIV-positive adults at the immunodeficiency outpatient clinic who were on regimens containing efavirenz in 2002 and who gave their informed consent (n = 31). Efavirenz plasma concentrations were measured throughout a 3 year period, without dose adjustments. For each patient, 6 to 12 samples were obtained over the follow-up period with an interval of at least 3 months between each sample. Mean plasma concentrations (mg/L) in the first, second, and third year of follow-up were 2.20 ± 0.64, 2.17 ± 0.68, and 2.31 ± 0.57. Mean intra-individual variability throughout the first, second, and third year of study was 27%, 31%, and 25%, ranging from 12% to 63%. No differences in intrapatient variability in efavirenz plasma concentrations were found between females and males, HBV/HCV+ and HBV/HCV− patients, or age above/below 40 years. Mean values (intra-individual variability) in plasma concentrations (mg/L) found in 3 of 31 patients who experienced virologic failure were 1.78 (42%), 1.52 (16%), and 1.68 (45%). The high interindividual variability and low maintained values of intrapatient variability in plasma concentrations support therapeutic drug monitoring, which could be based on measurements taken quarterly during the first year of therapeutics. In patients presenting high values of intra-individual variability (eg, >40%) associated with low plasma concentrations (eg, <2 mg/L), more frequent measurements over longer periods (more than 1 yr) of controlled concentrations might be recommended, but this requires further investigation.

Adenosine increases the cAMP content of the rat carotid body in vitro.

The adenosine receptor involved in the excitatory effect of this nucleoside on respiration mediated through carotid body chemoreceptors has been characterised in in vivo experiments, and exhibits agonist (Monteiro & Ribeiro, 1987) and antagonist (Ribeiro & Monteiro, 1991) profiles which are compatible with the presence of an A2 adenosine receptor subtype. The A2 adenosine receptors have been defined on the basis of their ability to stimulate adenyl cyclase and cAMP production (Fredholm et al, 1994). The present work was undertaken to investigate in vitro the effect of adenosine on cAMP content of the rat carotid body.

Differences in nevirapine biotransformation as a factor for its sex-dependent dimorphic profile of adverse drug reactions

OBJECTIVES Nevirapine is widely used for the treatment of HIV-1 infection; however, its chronic use has been associated with severe liver and skin toxicity. Women are at increased risk for these toxic events, but the reasons for the sex-related differences are unclear. Disparities in the biotransformation of nevirapine and the generation of toxic metabolites between men and women might be the underlying cause. The present work aimed to explore sex differences in nevirapine biotransformation as a potential factor in nevirapine-induced toxicity. METHODS All included subjects were adults who had been receiving 400 mg of nevirapine once daily for at least 1 month. Blood samples were collected and the levels of nevirapine and its phase I metabolites were quantified by HPLC. Anthropometric and clinical data, and nevirapine metabolite profiles, were assessed for sex-related differences. RESULTS A total of 52 patients were included (63% were men). Body weight was lower in women (P = 0.028) and female sex was associated with higher alkaline phosphatase (P = 0.036) and lactate dehydrogenase (P = 0.037) levels. The plasma concentrations of nevirapine (P = 0.030) and the metabolite 3-hydroxy-nevirapine (P = 0.035), as well as the proportions of the metabolites 12-hydroxy-nevirapine (P = 0.037) and 3-hydroxy-nevirapine (P = 0.001), were higher in women, when adjusted for body weight. CONCLUSIONS There was a sex-dependent variation in nevirapine biotransformation, particularly in the generation of the 12-hydroxy-nevirapine and 3-hydroxy-nevirapine metabolites. These data are consistent with the sex-dependent formation of toxic reactive metabolites, which may contribute to the sex-dependent dimorphic profile of nevirapine toxicity.