Laura Ribeiro

Comparison between uptake2 and rOCT1: effects of catecholamines, metanephrines and corticosterone

Abstract Active and specialized transmembrane transport systems are responsible for the functional inactivation of catecholamines. Uptake2, the classical extraneuronal uptake system, and rOCT1, a recently cloned organic cation transporter, share a number of properties. The present study was undertaken to investigate putative differences between these two transporters that might clarify their relative physiological roles.Uptake of [3H]MPP+ ([3H]1-methyl-4-phenylpyridi-nium) by Caki-1 cells (to study uptake2) and by primary cultured rat hepatocytes (to study rOCT1) was kinetically and pharmacologically characterized. In both cell types, [3H]MPP+ was avidly taken up and accumulated. All compounds tested (catecholamines, metanephrines and corticosterone) inhibited [3H]MPP+ uptake, albeit with different potencies. In Caki-1 cells, the ranking order of inhibitory potency was: (–)isoprenaline > (–)adrenaline >> (–)noradrenaline > dopamine. Metanephrine and normetanephrine were equipotent. Corticosterone had an IC50 of 102 nM. In cultured hepatocytes, the ranking order of inhibitory potency was: (–)isoprenaline > dopamine > (–)adrenaline >> (–)noradrenaline. Metanephrine was about seven times more potent than normetanephrine. Corticosterone had an IC50 of 72 μM, being about 700-fold less potent in inhibiting rOCT1 than uptake2.The results showed that uptake2 and rOCT1 can be clearly distinguished on a functional basis. On the one hand, uptake2 prefers adrenaline among the endogenous catecholamines, whereas rOCT1 has similar affinity for adrenaline and dopamine. On the other hand, corticosterone and normetanephrine are significantly more potent in inhibiting uptake2 than rOCT1. The results are compatible with a possible physiological role of corticosteroids in the modulation of adrenaline effects in tissues equipped with uptake2, without significant interference with the hepatic and renal excretion of catecholamines.

Brain natriuretic peptide as a marker of cardiac involvement in hypertension

Hypertensive patients with heart abnormalities have increased risk of cardiovascular events. Brain natriuretic peptide is a natriuretic peptide mainly of ventricular origin produced in response to pressure and stretch. We hypothesise that brain natriuretic peptide could be a useful marker of cardiac remodelling in hypertensive patients. We studied 36 consecutive community mild-to-moderate hypertensive patients and 11 well-matched normotensive controls with respect to clinical characteristics, brain natriuretic peptide, creatinine and echocardiography parameters (M-mode, 2-D arid transmitral pulsed Doppler). Brain natriuretic peptide levels were significantly higher in hypertensive patients than in controls [36.54 (IQR: 38.61) vs. 10.30 (IQR: 13.20) pg ml(-1), p<0.0001] and it was correlated with left ventricular mass index. Hypertensive patients with impairment of diastolic filling had significantly higher brain natriuretic peptide concentrations than patients with no abnormalities on echocardiography [61.16 (45.38) vs. 31.27 (18.10) pg ml(-1), p=0.001]. Multivariate analysis showed that only diastolic dysfunction and left ventricular mass index were significantly and independently related with brain natriuretic peptide concentrations in this population. In conclusion, impairment of diastolic function and left ventricular mass index are related to brain natriuretic peptide levels, thus giving the insight that this peptide can be a marker of ventricular remodelling in hypertensive patients.

arterial distensibility in subjects with white-coat hypertension with and without diabetes or dyslipidaemia: comparison with normotensives and sustained hypertensives

Background Arterial distensibility can be assessed by measuring pulse‐wave velocity (PWV). Objective To determine whether diabetes, smoking and dyslipidaemia were associated with greater than normal stiffness of aortic walls in subjects with white‐coat hypertension. Methods Arterial distensibility was assessed by automatic measurement of carotid‐femoral PWV in 35 healthy normotensives, 46 white‐coat hypertensives (WCH, clinic blood pressures > 140/90 mmHg, daytime blood pressures < 130/85 mmHg) and 81 ambulatory hypertensives (clinic blood pressures > 140/90 mmHg, daytime blood pressures ≥ 130 mmHg systolic or ≥ 85 mmHg diastolic, or both) all matched for age, sex and body mass index. Nineteen normotensives (subgroup A), 28 WCH (subgroup A) and 37 ambulatory hypertensives (subgroup A) had only one or no other major cardiovascular risk factor whereas 16 normotensives (subgroup B), 18 WCH (subgroup B) and 44 ambulatory hypertensives (subgroup B) had also some combination of non‐insulin‐dependent diabetes, a smoking habit and dyslipidaemia. Results Both for the WCH and for ambulatory hypertensives diabetes and dyslipidaemia (subgroups B) were associated with higher (P < 0.04) PWV (11.6 ± 0.3 and 12.8 ± 0.3 m/s, respectively) than for subgroups A (9.3 ± 0.5 and 10.9 ± 0.6 m/s, respectively). In contrast, PWV for WCH in subgroup A (9.3 ± 0.5 m/s) did not differ (P > 0.35) from those for the normotensive subgroups A (9.2 ± 0.3 m/s) and B (9.6 ± 0.4 m/s). PWV was not correlated to levels of glycaemia, glycosylated haemoglobin and cholesterolaemia. Conclusions These results suggest that, both for ambulatory hypertensives and for WCH, diabetes and dyslipidaemia are associated with an impairment of arterial distensibility that can entail a greater than normal cardiovascular risk, which might dictate a more than usually stringent treatment of concomitant risk factors and possibly of high blood pressure. In contrast, PWV in WCH of the subgroup A did not differ from those in normotensives, reinforcing the hypothesis that WCH is associated with a benign cardiovascular outcome in the absence of other cardiovascular risk factors.

Differences in behavior profile between normotensive subjects and patients with white-coat and sustained hypertension

It has been hypothesized that white-coat hypertensives (WCHs) have lower cardiovascular risk than sustained hypertensives (HTs), but higher emotional reactivity. We evaluated 92 HT patients (clinic and daytime BP>140/90 mmHg), 52 WCHs (clinic BP>140190 and ambulatory daytime BP<134/ 85 mmHg), and 74 normotensive subjects (NTs, clinic BP<140/90 and ambulatory daytime BP<134/85 mmHg), aged between 24 and 72 years, and matched for educational level, age, gender, and weight for depression, psychopathology, well-being, and quality of life. HTs showed worse scores than WCHs and NTs on most of the psychological variables; no differences were found between WCHs and NTs except on physical mobility. Daytime BP variability was HTs>WCHs>NTs, whereas nighttime BP variability was HTs>WCHs=NTs. We conclude that HTs have worse psychological profiles than the other two groups. WCHs and NTs have similar psychological profiles, although WCHs have a higher daytime BP variability, which is not associated with higher emotional reactivity.

Characterization of rat heart alkaline phosphatase isoenzymes and modulation of activity

Alkaline phosphatase (ALP) is important in calcification and its expression seems to be associated with the inflammatory process. We investigated the in vitro acute effects of compounds used for the prevention or treatment of cardiovascular diseases on total ALP activity from male Wistar rat heart homogenate. ALP activity was determined by quantifying, at 410 nm, the p-nitrophenol released from p-nitrophenylphosphate (substrate in Tris buffer, pH 10.4). Using specific inhibitors of ALP activity and the reverse transcription-polymerase chain reaction, we showed that the rat heart had high ALP activity (31.73 +/- 3.43 nmol p-nitrophenol.mg protein-1.min-1): mainly tissue-nonspecific ALP but also tissue-specific intestinal ALP type II. Both ALP isoenzymes presented myocardial localization (striated pattern) by immunofluorescence. ALP was inhibited a) strongly by 0.5 mM levamisole, 2 mM theophylline and 2 mM aspirin (91, 77 and 84%, respectively) and b) less strongly by 2 mM L-phenylalanine, 100 mL polyphenol-rich beverages and 0.5 mM progesterone (24, 21 to 29 and 11%, respectively). beta-estradiol and caffeine (0.5 and 2 mM) had no effect; 0.5 mM simvastatin and 2 mM atenolol activated ALP (32 and 36%, respectively). Propranolol (2 mM) tended to activate ALP activity and corticosterone activated (18%) and inhibited (13%) (0.5 and 2 mM, respectively). We report, for the first time, that the rat heart expresses intestinal ALP type II and has high total ALP activity. ALP activity was inhibited by compounds used in the prevention of cardiovascular pathology. ALP manipulation in vivo may constitute an additional target for intervention in cardiovascular diseases.

Cortisol: the villain in Metabolic Syndrome?

OBJECTIVE This article reviews the state of the art regarding the association between glucocorticoid actions and both obesity and insulin resistance, two main features of the metabolic syndrome. METHODS A methodological assessment of the literature on PubMed and SciELO databases was conducted by using the following terms: stress, metabolic syndrome, glucocorticoids, obesity, insulin resistance, hypothalamic-pituitary-adrenal-axis and 11β-hydroxysteroid dehydrogenase. RESULTS Chronic stress, mainly through hypothalamic-pituitary-adrenal axis dysregulation, promotes the accumulation of visceral fat. Reciprocally, obesity promotes a systemic low-grade inflammation state, mediated by increased adipokine secretion, which can chronically stimulate and disturb stress system. This vicious cycle, probably initiated by visceral adipose tissue dysfunction, might be the trigger for the development of metabolic syndrome. CONCLUSION Given the strong evidences linking glucocorticoid release, obesity and type 2 diabetes, better understanding of the mechanisms underlying this connection might be useful for prevention and treatment of the metabolic syndrome.

β-Adrenergic modulation of cancer cell proliferation: available evidence and clinical perspectives

PurposeIn this review, we aimed to present and discuss the available preclinical and epidemiological evidences regarding the modulation of cancer cell proliferation by β-adrenoceptors (β-AR), with a specific focus on the putative effects of β-blockers according to their pharmacological properties.MethodsA comprehensive review of the published literature was conducted, and the evidences concerning the involvement of β-AR in cancer as well as the possible role of β-blockers were selected and discussed.ResultsThe majority of reviewed studies show that: (1) All the cancer types express both β1- and β2-AR, with the exception of neuroblastoma only seeming to express β2-AR; (2) adrenergic agonists are able to increase proliferation of several types of cancers; (3) the proliferative effect seems to be mediated by both β1- and β2-AR; (4) binding to β-AR results in a cAMP transient flux which activates two major downstream effector systems: protein kinase A and EPAC and (5) β-blockers might be putative adjuvants for cancer treatment.ConclusionsOverall, the reviewed studies show strong evidences that β-AR activation, through several intracellular mechanisms, modulate tumor cell proliferation suggesting β-blockers can be a feasible therapeutic approach to antagonize β-adrenergic response or have a protective effect per se. This review highlight the need for intensifying the research not only on the molecular mechanisms underlying the β-adrenergic influence in cancer, but also on the implications of biased agonism of β-blockers as potential antitumor agents.

Antiproliferative effects of β-blockers on human colorectal cancer cells

Colon cancer is the fourth and third most common cancer, respectively in men and women worldwide and its incidence is on the increase. Stress response has been associated with the incidence and development of cancer. The catecholamines (CA), adrenaline (AD) and noradrenaline (NA), are crucial mediators of stress response, exerting their effects through interaction with α- and β-adrenergic receptors (AR). Colon cancer cells express β-AR, and their activation has been implicated in carcinogenesis and tumor progression. Interest concerning the efficacy of β-AR blockers as possible additions to cancer treatment has increased. The aim of this study was to investigate the effect of several AR agonists and β-blockers following cell proliferation of HT-29 cells, a human colon adenocarcinoma cell line. For this purpose, HT-29 cells were incubated in the absence (control) or in the presence of the AR-agonists, AD, NA and isoprenaline (ISO) (0.1-100 µM) for 12 or 24 h. The tested AR agonists revealed proliferative effects on HT-29 cells. In order to study the effect of several β-blockers following proliferation induced by AR activation, the cells were treated with propranolol (PRO; 50 µM), carvedilol (CAR; 5 µM), atenolol (ATE; 50 µM), or ICI 118,551 (ICI; 5 µM) for 45 min prior, and simultaneously, to incubation with each of the AR agonists, AD and ISO, both at 1 and 10 µM. The results suggested that adrenergic activation plays an important role in colon cancer cell proliferation, most probably through β-AR. The β-blockers under study were able to reverse the proliferation induced by AD and ISO, and some of these blockers significantly decreased the proliferation of HT-29 cells. The elucidation of the intracellular pathways involved in CA-induced proliferation of colon cancer cells, and in the reversion of this effect by β-blockers, may contribute to identifying promising strategies in cancer treatment.

Dopaminergic Receptors and Tyrosine Hydroxylase Expression in Peripheral Blood Mononuclear Cells: A Distinct Pattern in Central Obesity.

Background Dopamine (DA) may be involved in central obesity (CO), an inflammatory condition, through its role in the central nervous system and in periphery, where it may affect immune cell function through five different DA receptors (DR). Whether dopaminergic pathways in peripheral immune cells are implicated in the inflammatory condition linked to CO is however unknown. Methods In a cohort of blood donors with and without CO, categorized by waist circumference (WC) (CO: WC ≥0.80 m in women and ≥0.94 m in men), we studied the expression of DR and tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of DA, in peripheral blood mononuclear cells (PBMCs) and their relation with anthropometric and metabolic/endocrine and inflammatory parameters. DR D1-5 and TH expression was assessed by semi quantitative real-time PCR. As inflammatory markers we investigated the immunophenotype of monocyte subsets by flow cytometry, staining for CD14, CD16, CD11b and CD36. Results CO individuals showed higher plasma levels of leptin and higher inflammatory pattern of monocytes compared with non-CO. PBMC expression of DR D2, DR D4 and DR D5 as well as of TH were lower in CO in comparison with non-CO. DR D2, and DR D5 expression correlated with lower WC and weight, and with lower inflammatory pattern of monocytes, and TH expression correlated with lower WC. DR D4 expression correlated with lower plasma levels of glycosylated hemoglobin, and DR D2 expression correlated with lower CO. Conclusions Results show that CO is associated with peripheral inflammation and downregulation of dopaminergic pathways in PBMCs, possibly suggesting DR expressed on immune cells as pharmacological targets in obesity for better metabolic outcome.

Cardiovascular safety of type 2 diabetes medications: Review of existing literature and clinical implications.

Type 2 diabetes mellitus (T2DM), cardiovascular disease (CVD) and the cardiovascular effect of antidiabetic drugs are today critical medical issues, with the prevalence of T2DM in particular showing a steep increase worldwide, mainly due to unhealthy lifestyle habits. T2DM in association with obesity and other cardiovascular risk factors, results in the development of CVD, the leading cause of morbidity and mortality in patients with T2DM. Thus, treatment of T2DM is an individualized and complex challenge in which targeting cardiovascular risk factors is an important component in the decision making. Given the cardiovascular adverse events associated with rosiglitazone, both the Food and Drug Administration and the European Medicines Agency currently require the demonstration of cardiovascular safety of new antidiabetic drugs. Consequently, clinical trials to guarantee their cardiovascular safety are now obligatory. This review aims to summarize the available evidence on the cardiovascular effects and safety of the major drugs used in T2DM treatment and also to provide an overview of upcoming and ongoing clinical trials in this field. Our belief is that this review will be of substantial assistance to all medical doctors who are treating diabetic patients, namely primary care physicians, internal medicine doctors, endocrinologists, diabetologists and less well experienced personnel such as young doctors in training.