Helder Pinheiro

Modulation of insulin transport in rat brain microvessel endothelial cells by an ecto‐phosphatase activity

The physiological function of alkaline phosphatase (ALP) remains controversial. It was recently suggested that this membrane‐bound enzyme has a role in the modulation of transmembranar transport systems into hepatocytes and Caco‐2 cells. ALP activity expressed on the apical surface of blood‐brain barrier cells, and its relationship with 125I‐insulin internalization were investigated under physiological conditions using p‐nitrophenylphosphate (p‐NPP) as substrate. For this, an immortalized cell line of rat capillary cerebral endothelial cells (RBE4 cells) was used. ALP activity and 125I‐insulin internalization were evaluated in these cells. The results showed that RBE4 cells expressed ALP, characterized by an ecto‐oriented active site which was functional at physiological pH. Orthovanadate (100 μM), an inhibitor of phosphatase activities, decreased both RBE4‐ALP activity and 125I‐insulin internalization. In the presence of l‐arginine (1 mM) or adenosine (100 μM) RBE4‐ALP activity and 125I‐insulin, internalization were significantly reduced. However, d‐arginine (1 mM) had no significant effect. Additionally, RBE4‐ALP activity and 125I‐insulin internalization significantly increased in the presence of the bioflavonoid kaempferol (100 μM), of the phorbol ester PMA (80 nM), IBMX (1 mM), progesterone (200 μM and 100 μM), β‐estradiol (100 μM), iron (100 μM) or in the presence of all‐trans retinoic acid (RA) (10 μM). The ALP inhibitor levamisole (500 μM) was able to reduce 125I‐insulin internalization to 69.1 ± 7.1% of control. Our data showed a positive correlation between ecto‐ALP activity and 125I‐insulin incorporation (r = 0.82; P < 0.0001) in cultured rat brain endothelial cells, suggesting that insulin entry into the blood‐brain barrier may be modulated through ALP. J. Cell. Biochem. 84: 389–400, 2002.

Involvement of G-protein βγ subunits on the influence of inhibitory α2-autoreceptors on the angiotensin AT1-receptor modulation of noradrenaline release in the rat vas deferens

The influence of alpha2-autoreceptors on the facilitation of [3H]-noradrenaline release mediated by angiotensin II was studied in prostatic portions of rat vas deferens preincubated with [3H]-noradrenaline. Angiotensin II enhanced tritium overflow evoked by trains of 100 pulses at 8 Hz, an effect that was attenuated by the AT1-receptor antagonist losartan (0.3-1 microM), at concentrations suggesting the involvement of the AT1B subtype. The effect of angiotensin II was also attenuated by inhibition of phospholipase C (PLC) and protein kinase C (PKC) indicating that prejunctional AT1-receptors are coupled to the PLC-PKC pathway. Angiotensin II (0.3-100 nM) enhanced tritium overflow more markedly, up to 64%, under conditions that favor alpha2-autoinhibition, observed when stimulation consisted of 100 pulses at 8 Hz, than under poor alpha2-autoinhibition conditions, only up to 14%, observed when alpha2-adrenoceptors were blocked with yohimbine (1 microM) or when stimulation consisted of 20 pulses at 50 Hz. Activation of PKC with 12-myristate 13-acetate (PMA, 0.1-3 microM) also enhanced tritium overflow more markedly under strong alpha2-autoinhibition conditions. Inhibition of Gi/o-proteins with pertussis toxin (8 microg/ml) or blockade of Gbetagamma subunits with the anti-betagamma peptide MPS-Phos (30 microM) attenuated the effects of angiotensin II and PMA. The results indicate that activation of AT1-receptors coupled to the PLC-PKC pathway enhances noradrenaline release, an effect that is markedly favoured by an ongoing activation of alpha2-autoreceptors. Interaction between alpha2-adrenoceptors and AT1-receptors seems to involve the betagamma subunits released from the Gi/o-proteins coupled to alpha2-adrenoceptors and protein kinase C activated by AT1-receptors.

Bioactive Peptides - Are There More Antihypertensive Mechanisms Beyond ACE Inhibition?

Diet has a high relevance in health. Hypertension is a major risk factor for cardiovascular diseases and has an important impact on public health, and consequently on countries economy. Scientific research gathered strong evidence about the role of several dietary factors either in etiology or in treatment/prevention of these diseases. Peptides from different food matrices have been studied, and indicated as compounds with particular interest in the context of hypertension. The classical approach involves the identification of peptides with an in vitro ACE inhibitory activity and the assumption that the observed in vivo effects are due to this enzyme blockade. However, in some cases the potency of ACE blockade does not correlate with the antihypertensive activity in vivo. This paper reviews the current literature that identifies mechanisms of action, other than ACE inhibition, that might explain antihypertensive effects of biologically active peptides from different food sources.

In vitro ACE-inhibitory peptide KGYGGVSLPEW facilitates noradrenaline release from sympathetic nerve terminals: Relationship with the lack of antihypertensive effect on spontaneous hypertensive rats

This study aimed to validate the antihypertensive activity of the angiotensin-converting enzyme (ACE)-inhibitor whey protein hydrolysate (WPH) obtained through the action of proteolytic enzymes from Cynara Cardunculus. The antihypertensive activity of WPH fractions containing peptides with molecular weight below 3kDa (Whey<3kDa) and 1kDa (Whey<1kDa) along with the antihypertensive activity of three potent ACE-inhibitory peptide sequences (DKVGINYW, DAQSAPLRVY and KGYGGVSLPEW), previously identified in WPH, were also investigated. In parallel, the influence of KGYGGVSLPEW (the most potent ACE-inhibitory peptide sequence) on AT1 receptors (a common pharmacological target of antihypertensive therapies beyond ACE), was evaluated. The effect of WPH and fractions (300mg/kg) and peptide sequences (5mg/kg) on systolic, diastolic and mean blood pressure was evaluated by telemetry on spontaneously hypertensive rats (SHR), after single oral administration. Despite their ACE-inhibitory effect in vitro, neither WPH, Whey <3kDa, Whey <1kDa or peptide sequences exhibited antihypertensive activity. In addition, KGYGGVSLPEW was not only devoid of AT1 receptor antagonism but, on the contrary, had a similar effect to that of Ang II by facilitating the noradrenaline release from sympathetic nerve terminals. In vitro ACE blockade does not always correlate with antihypertensive activity and food-derived peptides cannot be classified as antihypertensive agents based exclusively on in vitro assays. The absence of an antihypertensive effect may also be a result of the interaction of these compounds with other components of the systems involved in the blood pressure control.

Optical Fiber Bioanalyzer Based on Enzymatic Coating Matrix for Catecholamines and Their Metabolites Assessment in Patients With Down Syndrome

The urinary levels of catecholamines [adrenaline (AD), noradrenaline (NA), and dopamine (DA)] and their metabolites [L-3,4-dihydroxyphenylalanine (L-DOPA), and 3,4-dihydroxyphenylacetic acid (DOPAC)], as indicators of physiological stress, were assessed in 40 patients with Down syndrome (DS). The analysis was performed by an optical fiber (OF) bioanalyzer; which was constituted by two main components: a miniaturized chromatographic system and a detection system based on an OF coated with an enzymatic matrix. In this study some working conditions such as, number of fibers in the miniaturized chromatographic column, number of dips for sensitive coating deposition, temperature and time of cure of the sensitive matrix, were optimized in order to achieve higher analytical performance. After tested for calibration the bioanalyzer was applied to urine samples analysis of catecholamines and their metabolites, comparing the results with those obtained by both, a classical analytical method, namely high performance liquid chromatography coupled to an electrochemical detector (HPLC-ED), and an OF biosensor based on a sensitive cladding of laccase (LacOF biosensor). The results of catecholamines in patients with DS revealed that 15% and 22.5% of the analyzed subjects showed DA and AD concentrations, respectively, above the pathological levels. In the determinations of NA, the 40 samples showed concentration values below the normal levels, while in the determination of catecholamines metabolites 5% of the urine samples showed values above 118 and 1681 for L-DOPA and DOPAC, respectively.

Can wheat germ have a beneficial effect on human health? A study protocol for a randomised crossover controlled trial to evaluate its health effects

Introduction Cardiovascular diseases (CVD) are the leading cause of mortality worldwide and diet is an important contributor to CVD risk. Thus, several food derivatives are being investigated for their beneficial impact on reducing cardiometabolic risk factors, either in risk groups or in healthy population as a preventive measure. Wheat germ is a food by-product with high nutritional value, especially as a concentrated source of dietary fibre and essential fatty acids, but its incorporation into the diet has been rare up to now. Previous studies do not clarify the hypothesised potential causal relationship between the consumption of wheat germ and benefits for human health. Methods and analysis We are conducting a randomised, double-blinded, crossover, placebo-controlled clinical trial designed to assess the physiological effects of daily consumption of wheat germ-enriched bread (containing 6 g of wheat germ) compared with non-enriched bread, over a 4-week period with a 15-week follow-up, in a healthy human population. A total of 55 participants (healthy volunteers, aged 18–60) have been recruited from the Porto metropolitan area in northern Portugal. Our aim is to evaluate the health effects of wheat germ on blood cholesterol and triglycerides, postprandial glycaemic response, gastrointestinal function and discomfort, and changes in intestinal microbiota and insulin resistance as secondary outcomes. The study follows the best practices for evaluating health claims in food according to the European Food Safety Authority (EFSA) scientific opinion, namely random allocation, double blinding, reporting methods to measure and maximise compliance, and validated outcomes with beneficial physiological effects as recommended by EFSA. Ethics and dissemination The study has been approved by the Health Ethics Committee of São João Hospital Centre (156-15) and the Ethics Committee of Faculty of Medicine of the University of Porto (PCEDCSS-FMUP07/2015). Results will be disseminated through peer-reviewed publications and presentations at international scientific meetings. Trial registration number NCT02405507; pre-results.