Quantitative assay for bradykinin in rat plasma by liquid chromatography coupled to tandem mass spectrometry.

An assay to quantify bradykinin in rat plasma has been developed and validated, using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Sar-D-Phe(8)-des-Arg(9)-bradykinin was used as internal standard. Aprotinin was added to rat plasma to inhibit the activity of proteinases. Recoveries for solid-phase extraction (SPE) on Strata X reversed phase were greater than 80%. Multiple reaction monitoring (MRM) on a triple quadrupole mass spectrometer equipped with an electrospray source (ESI), operating in the positive ion-mode, was used for detection. The assay was validated and stability was explored. Bradykinin (10-500 ng/mL) was quantified with accuracy values (% RE) below 10% and intra- and inter-day precisions (% RSD) below 12 and 16%, respectively, for all concentrations. The method was successfully applied to several plasma samples from low levels kallikrein rats (LKRs) compared with normal kallikrein rats (NKRs).

Brain renin-angiotensin system modifies the blood pressure response to intracerebroventricular cadmium in rats.

In order to elucidate the involvement of the brain renin-angiotensin system (RAS) in cadmium intracerebroventricular (ICV) hypertension, we evaluated the effects of a pretreatment with different drugs: clonidine, an alpha(2) adrenergic agonist, enalapril and captopril, both ACE inhibitors, and saralasin, a competitive nonselective AT(1) and AT(2) receptor antagonist. We used a rat strain with low levels of kallikrein (LKR) that was more sensitive to ICV cadmium hypertension, compared with normal kallikrein rats (NKRs), the control strain. The interplay between the kallikrein-kinin system and the RAS in the LKR strain caused various hemodynamic alterations, which we believe were the result of elevated RAS activity in these animals. Moreover, we suggest that the defective kallikrein-kinin system in LKR may also cause an alteration in the activation of brain RAS in these animals. The LKR displayed elevated concentrations of plasma AII, hypertrophy of the myocardium, and initial alterations in the renal glomerulotubular system. With the exception of clonidine, all of the other drugs showed greater antihypertensive effects of differing statistical significance in LKR, compared with NKR. Both ACE inhibitors were able to significantly reduce pressor response to cadmium ICV in LKR throughout the experiment, whereas in NKR, they were only able to reduce the hypertensive peak of cadmium. A significant protective effect was also observed in LKR pretreated with saralasin, while no effect was observed in NKR. These findings confirm the presence of brain RAS activation in LKR and its contribution to the central control of pressor response to cadmium ICV.

Gluten exorphin B5 stimulates prolactin secretion through opioid receptors located outside the blood-brain barrier.

Gluten exorphin B5 (GE-B5) is a food-derived opioid peptide identified in digests of wheat gluten. We have recently shown that GE-B5 stimulates prolactin (PRL) secretion in rats; this effect is abolished by preadministration of the opioid receptor antagonist naloxone. However, since the structure of naloxone allows it to cross the blood-brain barrier (BBB) and antagonize opioid effects centrally as well as peripherally, it could not established, on the basis of those data, if GE-B5-induced PRL release is exerted through sites located inside or outside the BBB. In this study, we sought to determine the site of action of GE-B5 on PRL secretion, by pretreating male rats with naloxone methobromide (NMB), an opioid antagonist that does not cross the BBB. Four groups of rats were given the following treatments: 1) intravenous vehicle; 2) intravenous GE-B5 (3 mg kg(-1) body weight); 3) intraperitoneal NMB (5 mg kg(-1) body weight), followed by vehicle; 4) NMB, followed by GE-B5. Blood samples for PRL were taken at intervals for 40 minutes after vehicle or GE-B5 administration. GE-B5 stimulated PRL secretion; the effect was statistically significant at time 20. NMB preadministration completely abolished PRL response. Our experiment indicates that GE-B5 stimulates PRL secretion through opioid receptors located outside the BBB. Since opioid peptides do not exert their effect on PRL secretion directly, but via a reduced dopaminergic tone, our data suggest that GE-B5 can modify brain neurotransmitter release without crossing the BBB.

Low urinary kallikrein rats: different sensitivity of verapamil on hypertensive response to central acute cadmium administration.

Essential hypertension is a common disease caused by a combination of genetic and environmental factors. Cadmium (Cd), an important environmental pollutant, is able to induce hypertension in humans. In rats, intracerebroventricular (icv) Cd administration causes a sustained increase in arterial blood pressure. The kallikrein-kinin system appears an important regulator of cardiovascular function and rats with low renal excretion of kallikrein differ from normal-kallikrein Wistar rats in their pressor response to icv Cd. To clarify these differences in pressor response, we evaluated the protective effect of a calcium antagonist following the administration of 10 microg Cd icv. Pre-treatment with increasing doses of verapamil (100, 150200 microg) in normal-kallikrein rats produced a blocking of the hypertensive effects of Cd, even at the lower doses. In low-kallikrein rats we observed a dose-dependent inhibition of hypertensive effects at 100 and 150 microg, while at 200 microg there was, paradoxically, an increase in pressor values. Our results suggest that a genetically-determined defect in urinary kallikrein excretion leads to different modulation of brain calcium channels antagonists in the hypertensive response to icv Cd. This different sensitivity of low-kallikrein rats suggests that the hypertensive effect of icv Cd is, at least in part, the result of blocking the calcium channels, but is also sensitive to a new hemodynamic equilibrium, such as that present in low kallikrein rats, and probably intervenes as a modulator at the central level in other as yet not well identified systems, also linked to the hypotensive pathways, which may be activated in certain conditions.

Intravenous administration of the food-derived opioid peptide gluten exorphin B5 stimulates prolactin secretion in rats.

Gluten Exorphin B5 (GE-B5) is a food-derived opioid peptide, identified in vitro in enzymatic digests of wheat gluten. It has been suggested that this peptide may play a regulatory role on pituitary secretion, since it stimulates prolactin (PRL) secretion when administered in the cerebral ventricles in rats. It is not known, however, if GE-B5 can exert this stimulatory action after peripheral administration. In order to clarify this aspect, we gave the following treatments to four groups of male rats: intravenous (i.v.) vehicle, GE-B5 3 mg/kg body weight i.v., naloxone intraperitoneally (i.p.) followed by vehicle i.v., naloxone i.p. followed by GE-B5 i.v. Blood samples for PRL were taken at intervals for 60 min after vehicle or GE-B5 administration. At the dose of 3 mg/kg body weight, GE-B5 induced a significant increase in PRL levels; naloxone completely abolished any effect of GE-B5 on PRL secretion. The present study indicates that GE-B5 stimulates PRL secretion after peripheral administration and that its action is mediated via classical opioid receptors; moreover, it identifies the minimum peptide dose which must reach the blood in order to exert its action on PRL secretion.

Prolactin and growth hormone response to intracerebroventricular administration of the food opioid peptide gluten exorphin B5 in rats.

Although it has long been known that opioid peptides cause marked changes of pituitary hormone secretion in both animals and humans, little is known about the possible effect(s) of food-derived opioids (exorphins) on pituitary function. In order to investigate the possible role of exorphins derived from wheat gluten on pituitary function, we gave the following treatments to four groups of male rats: intracerebroventricular (ICV) vehicle, Gluten Exorphin B5 (GE-B5) 200 microg ICV, naloxone intraperitoneally (IP) followed by vehicle ICV, naloxone IP followed by GE-B5 ICV. Blood samples for Prolactin (PRL) and Growth Hormone (GH) were taken at intervals for 90 minutes after vehicle or GE-B5 administration. GE-B5 strongly stimulated PRL secretion; its effect was completely abolished by naloxone administration. GH secretion was unaffected by GE-B5 under these experimental conditions. The present study shows for the first time that an opioid peptide derived from wheat gluten, GE-B5, has an effect on pituitary function when administered ICV; its mechanism of action appears to be mediated via classical opioid receptors.