Target-directed catalytic metallodrugs

Most drugs function by binding reversibly to specific biological targets, and therapeutic effects generally require saturation of these targets. One means of decreasing required drug concentrations is incorporation of reactive metal centers that elicit irreversible modification of targets. A common approach has been the design of artificial proteases/nucleases containing metal centers capable of hydrolyzing targeted proteins or nucleic acids. However, these hydrolytic catalysts typically provide relatively low rate constants for target inactivation. Recently, various catalysts were synthesized that use oxidative mechanisms to selectively cleave/inactivate therapeutic targets, including HIV RRE RNA or angiotensin converting enzyme (ACE). These oxidative mechanisms, which typically involve reactive oxygen species (ROS), provide access to comparatively high rate constants for target inactivation. Target-binding affinity, co-reactant selectivity, reduction potential, coordination unsaturation, ROS products (metal-associated vsmetal-dissociated; hydroxyl vs superoxide), and multiple-turnover redox chemistry were studied for each catalyst, and these parameters were related to the efficiency, selectivity, and mechanism(s) of inactivation/cleavage of the corresponding target for each catalyst. Important factors for future oxidative catalyst development are 1) positioning of catalyst reduction potential and redox reactivity to match the physiological environment of use, 2) maintenance of catalyst stability by use of chelates with either high denticity or other means of stabilization, such as the square planar geometric stabilization of Ni- and Cu-ATCUN complexes, 3) optimal rate of inactivation of targets relative to the rate of generation of diffusible ROS, 4) targeting and linker domains that afford better control of catalyst orientation, and 5) general bio-availability and drug delivery requirements.

Effects of sequential enzymatic hydrolysis on structural, bioactive and functional properties of Phaseolus lunatus protein isolate

Significant initiatives exist within the global food market to search for new, alternative protein sources with better technological, functional, and nutritional properties. Lima bean (Phaseolus lunatus L.) protein isolate was hydrolyzed using a sequential pepsin-pancreatin enzymatic system. Hydrolysis was performed to produce limited (LH) and extensive hydrolysate (EH), each with different degrees of hydrolysis (DH). The effects of hydrolysis were evaluated in vitro in both hydrolysates based on structural, functional and bioactive properties. Structural properties analyzed by electrophoretic profile indicated that LH showed residual structures very similar to protein isolate (PI), although composed of mixtures of polypeptides that increased hydrophobic surface and denaturation temperature. Functionality of LH was associated with amino acid composition and hydrophobic/hydrophilic balance, which increased solubility at values close to the isoelectric point. Foaming and emulsifying activity index values were also higher than those of PI. EH showed a structure composed of mixtures of polypeptides and peptides of low molecular weight, whose intrinsic hydrophobicity and amino acid profile values were associated with antioxidant capacity, as well as inhibiting angiotensin-converting enzyme. The results obtained indicated the potential of Phaseolus lunatus hydrolysates to be incorporated into foods to improve techno-functional properties and impart bioactive properties.

The long-term use of enalapril and hydrochlorothiazide in two novel mutations patients with Dent's disease type 1

Dent's disease type 1 is an X-linked tubular disease caused by mutations in the renal chloride channel CLCN-5, and it is characterized by low molecular weight proteinuria, hypercalciuria, nephrocalcinosis, and renal failure. Several cases have been described in which the only presenting symptoms were asymptomatic proteinuria, and focal segmental or global glomerulosclerosis. The renal failure in these patients may be caused by hypercalciuria and persistent proteinuria. Therefore, angiotensin converse enzyme inhibitor and thiazides could be useful. Our aim is to report the effects of these drugs in two novel mutations patients with Dent's disease type 1. In this report, no significant correlations between dosage of hydrochlorothiazide and calciuria and no significant correlations between proteinuria and dosage of enalapril were detected. This is important since these are polyuric patients and these drugs could be dangerous to their renal function.

Characterization of renin mRNA expression and enzyme activity in rat and mouse mesangial cells

Renin is an enzyme involved in the stepwise generation of angiotensin II. Juxtaglomerular cells are the main source of plasma renin, but renin activity has been detected in other cell types. In the present study we evaluated the presence of renin mRNA in adult male Wistar rat and mouse (C-57 Black/6) mesangial cells (MC) and their ability to process, store and release both the active and inactive forms of the enzyme. Active renin and total renin content obtained after trypsin treatment were estimated by angiotensinogen consumption analyzed by SDS-PAGE electrophoresis and quantified by angiotensin I generation by HPLC. Renin mRNA, detected by RT-PCR, was present in both rat and mouse MC under basal conditions. Active renin was significantly higher (P<0.05) in the cell lysate (43.5 ± 5.7 ng h-1 10(6) cells) than in the culture medium (12.5 ± 2.5 ng h-1 10(6) cells). Inactive prorenin content was similar for the intra- and extracellular compartments (9.7 ± 3.1 and 3.9 ± 0.9 ng h-1 10(6) cells). Free active renin was the predominant form found in both cell compartments. These results indicate that MC in culture are able to synthesize and translate renin mRNA probably as inactive prorenin which is mostly processed to active renin inside the cell. MC secrete both forms of the enzyme but at a lower level compared with intracellular content, suggesting that the main role of renin synthesized by MC may be the intracellular generation of angiotensin II.

Endogenous angiotensin II modulates nNOS expression in renovascular hypertension

Nitric oxide (NO) influences renal blood flow mainly as a result of neuronal nitric oxide synthase (nNOS). Nevertheless, it is unclear how nNOS expression is modulated by endogenous angiotensin II, an inhibitor of NO function. We tested the hypothesis that the angiotensin II AT1 receptor and oxidative stress mediated by NADPH oxidase contribute to the modulation of renal nNOS expression in two-kidney, one-clip (2K1C) hypertensive rats. Experiments were performed on male Wistar rats (150 to 170 g body weight) divided into 2K1C (N = 19) and sham-operated (N = 19) groups. nNOS expression in kidneys of 2K1C hypertensive rats (N = 9) was compared by Western blotting to that of 2K1C rats treated with low doses of the AT1 antagonist losartan (10 mg·kg-1·day-1; N = 5) or the superoxide scavenger tempol (0.2 mmol·kg-1·day-1; N = 5), which still remain hypertensive. After 28 days, nNOS expression was significantly increased by 1.7-fold in the clipped kidneys of 2K1C rats and by 3-fold in the non-clipped kidneys of 2K1C rats compared with sham rats, but was normalized by losartan. With tempol treatment, nNOS expression increased 2-fold in the clipped kidneys and 1.4-fold in the non-clipped kidneys compared with sham rats. The changes in nNOS expression were not followed by changes in the enzyme activity, as measured indirectly by the cGMP method. In conclusion, AT1 receptors and oxidative stress seem to be primary stimuli for increased nNOS expression, but this up-regulation does not result in higher enzyme activity.

Angiotensin II induces NF-κB, JNK and p38 MAPK activation in monocytic cells and increases matrix metalloproteinase-9 expression in a PKC- andRho kinase-dependent manner

Angiotensin II (ANG II), the main effector of the renin-angiotensin system, is implicated in endothelial permeability, recruitment and activation of the immune cells, and also vascular remodeling through induction of inflammatory genes. Matrix metalloproteinases (MMPs) are considered to be important inflammatory factors. Elucidation of ANG II signaling pathways and of possible cross-talks between their components is essential for the development of efficient inhibitory medications. The current study investigates the inflammatory signaling pathways activated by ANG II in cultures of human monocytic U-937 cells, and the effects of specific pharmacological inhibitors of signaling intermediates on MMP-9 gene (MMP-9) expression and activity. MMP-9 expression was determined by real-time PCR and supernatants were analyzed for MMP-9 activity by ELISA and zymography methods. A multi-target ELISA kit was employed to evaluate IκB, NF-κB, JNK, p38, and STAT3 activation following treatments. Stimulation with ANG II (100 nM) significantly increased MMP-9 expression and activity, and also activated NF-κB, JNK, and p38 by 3.8-, 2.8- and 2.2-fold, respectively (P < 0.01). ANG II-induced MMP-9 expression was significantly reduced by 75 and 67%, respectively, by co-incubation of the cells with a selective inhibitor of protein kinase C (GF109203X, 5 µM) or of rho kinase (Y-27632, 15 µM), but not with inhibitors of phosphoinositide 3-kinase (wortmannin, 200 nM), tyrosine kinases (genistein, 100 µM) or of reactive oxygen species (α-tocopherol, 100 µM). Thus, protein kinase C and Rho kinase are important components of the inflammatory signaling pathways activated by ANG II to increase MMP-9 expression in monocytic cells. Both signaling molecules may constitute potential targets for effective management of inflammation.

Renal actions of angiotensin-(1-7)

The heptapeptide angiotensin-(1-7) is considered to be a biologically active endproduct of the renin-angiotensin system. This angiotensin, which is devoid of the most known actions of angiotensin II such as induction of drinking behavior and vasoconstriction, has several selective effects in the brain and periphery. In the present article we briefly review recent evidence for a physiological role of angiotensin-(1-7) in the control of hydroelectrolyte balance

Effect of angiotensin-(1-7) on reperfusion arrhythmias in isolated rat hearts

There is increasing evidence that angiotensin-(1-7) (Ang-(1-7)) is an endogenous biologically active component of the renin-angiotensin system (RAS). In the present study, we investigated the effects of Ang-(1-7) on reperfusion arrhythmias in isolated rat hearts. Isolated rat hearts were perfused with two different media, i.e., Krebs-Ringer (2.52 mM CaCl2) and low-Ca2+ Krebs-Ringer (1.12 mM CaCl2). In hearts perfused with Krebs-Ringer, Ang-(1-7) produced a concentration-dependent (27-210 nM) reduction in coronary flow (25% reduction at highest concentration), while only slight and variable changes in contraction force and heart rate were observed. Under the same conditions, angiotensin II (Ang II; 27 and 70 nM) produced a significant reduction in coronary flow (39% and 48%, respectively) associated with a significant increase in force. A decrease in heart rate was also observed. In low-Ca2+ Krebs-Ringer solution, perfusion with Ang-(1-7) or Ang II at 27 nM concentration produced similar changes in coronary flow, contraction force and heart rate. In isolated hearts perfused with normal Krebs-Ringer, Ang-(1-7) produced a significant enhancement of reperfusion arrhythmias revealed by an increase in the incidence and duration of ventricular tachycardia and ventricular fibrillation (more than 30-min duration). The facilitation of reperfusion arrhythmias by Ang-(1-7) was associated with an increase in the magnitude of the decreased force usually observed during the post-ischemic period. The effects of Ang-(1-7) were abolished in isolated rat hearts perfused with low-Ca2+ Krebs-Ringer. The effect of Ang II (27 nM) was similar but less pronounced than that of Ang-(1-7) at the same concentration. These results indicate that the heart is a site of action for Ang-(1-7) and suggest that this heptapeptide may be involved in the mediation of the cardiac effects of the RAS

Effect of selective angiotensin antagonists on the antidiuresis produced by angiotensin-(1-7) in water-loaded rats

In the present study we evaluated the nature of angiotensin receptors involved in the antidiuretic effect of angiotensin-(1-7) (Ang-(1-7)) in water-loaded rats. Water diuresis was induced in male Wistar rats weighing 280 to 320 g by water load (5 ml/100 g body weight by gavage). Immediately after water load the rats were treated subcutaneously with (doses are per 100 g body weight): 1) vehicle (0.05 ml 0.9% NaCl); 2) graded doses of 20, 40 or 80 pmol Ang-(1-7); 3) 200 nmol Losartan; 4) 200 nmol Losartan combined with 40 pmol Ang-(1-7); 5) 1.1 or 4.4 nmol A-779; 6) 1.1 nmol A-779 combined with graded doses of 20, 40 or 80 pmol Ang-(1-7); 7) 4.4 nmol A-779 combined with graded doses of 20, 40 or 80 pmol Ang-(1-7); 8) 95 nmol CGP 42112A, or 9) 95 nmol CGP 42112A combined with 40 pmol Ang-(1-7). The antidiuretic effect of Ang-(1-7) was associated with an increase in urinary Na+ concentration, an increase in urinary osmolality and a reduction in creatinine clearance (CCr: 0.65 ± 0.04 ml/min vs 1.45 ± 0.18 ml/min in vehicle-treated rats, P<0.05). A-779 and Losartan completely blocked the effect of Ang-(1-7) on water diuresis (2.93 ± 0.34 ml/60 min and 3.39 ± 0.58 ml/60 min, respectively). CGP 42112A, at the dose used, did not modify the antidiuretic effect of Ang-(1-7). The blockade produced by Losartan was associated with an increase in CCr and with an increase in sodium and water excretion as compared with Ang-(1-7)-treated rats. When Ang-(1-7) was combined with A-779 there was an increase in CCr and natriuresis and a reduction in urine osmolality compared with rats treated with Ang-(1-7) alone. The observation that both A-779, which does not bind to AT1 receptors, and Losartan blocked the effect of Ang-(1-7) suggests that the kidney effects of Ang-(1-7) are mediated by a non-AT1 angiotensin receptor that is recognized by Losartan.

The hyperglycemia induced by angiotensin II in rats is mediated by AT1 receptors

We have shown that the renin-angiotensin system (RAS) is involved in glucose homeostasis during acute hemorrhage. Since almost all of the physiological actions described for angiotensin II were mediated by AT1 receptors, the present experiments were designed to determine the participation of AT1 receptors in the hyperglycemic action of angiotensin II in freely moving rats. The animals were divided into two experimental groups: 1) animals submitted to intravenous administration of angiotensin II (0.96 nmol/100 g body weight) which caused a rapid increase in plasma glucose reaching the highest values at 5 min after the injection (33% of the initial values, P<0.01), and 2) animals submitted to intravenous administration of DuP-753 (losartan), a non-peptide antagonist of angiotensin II with AT1-receptor type specificity (1.63 µmol/100 g body weight as a bolus, iv, plus a 30-min infusion of 0.018 µmol 100 g body weight-1 min-1 before the injection of angiotensin II), which completely blocked the hyperglycemic response to angiotensin II (P<0.01). This inhibitory effect on glycemia was already demonstrable 5 min (8.9 ± 0.28 mM, angiotensin II, N = 9 vs 6.4 ± 0.22 mM, losartan plus angiotensin II, N = 11) after angiotensin II injection and persisted throughout the 30-min experiment. Controls were treated with the same volume of saline solution (0.15 M NaCl). These data demonstrate that the angiotensin II receptors involved in the direct and indirect hyperglycemic actions of angiotensin II are mainly of the AT1-type.

Ligand-induced endocytosis and nuclear localization of angiotensin II receptors expressed in CHO cells

A construct (AT1R-NF) containing a "Flag" sequence added to the N-terminus of the rat AT1 receptor was stably expressed in Chinese hamster ovary cells and quantified in the cell membrane by confocal microscopy after reaction with a fluorescein-labeled anti-Flag monoclonal antibody. Angiotensin II bound to AT1R-NF and induced endocytosis with a half-time of 2 min. After 60-90 min, fluorescence accumulated around the cell nucleus, suggesting migration of the ligand-receptor complex to the nuclear membrane. Angiotensin antagonists also induced endocytosis, suggesting that a common step in the transduction signal mechanism occurring after ligand binding may be responsible for the ligand-receptor complex internalization.

Effect of intraperitoneally administered hydrolyzed whey protein on blood pressure and renal sodium handling in awake spontaneously hypertensive rats

The present study evaluated the acute effect of the intraperitoneal (ip) administration of a whey protein hydrolysate (WPH) on systolic arterial blood pressure (SBP) and renal sodium handling by conscious spontaneously hypertensive rats (SHR). The ip administration of WPH in a volume of 1 ml dose-dependently lowered the SBP in SHR 2 h after administration at doses of 0.5 g/kg (0.15 M NaCl: 188.5 ± 9.3 mmHg vs WPH: 176.6 ± 4.9 mmHg, N = 8, P = 0.001) and 1.0 g/kg (0.15 M NaCl: 188.5 ± 9.3 mmHg vs WPH: 163.8 ± 5.9 mmHg, N = 8, P = 0.0018). Creatinine clearance decreased significantly (P = 0.0084) in the WPH-treated group (326 ± 67 µL min-1 100 g body weight-1) compared to 0.15 M NaCl-treated (890 ± 26 µL min-1 100 g body weight-1) and captopril-treated (903 ± 72 µL min-1 100 g body weight-1) rats. The ip administration of 1.0 g WPH/kg also decreased fractional sodium excretion to 0.021 ± 0.019% compared to 0.126 ± 0.041 and 0.66 ± 0.015% in 0.15 M NaCl and captopril-treated rats, respectively (P = 0.033). Similarly, the fractional potassium excretion in WPH-treated rats (0.25 ± 0.05%) was significantly lower (P = 0.0063) than in control (0.91 ± 0.15%) and captopril-treated rats (1.24 ± 0.30%), respectively. The present study shows a decreased SBP in SHR after the administration of WPH associated with a rise in tubule sodium reabsorption despite an angiotensin I-converting enzyme (ACE)-inhibiting in vitro activity (IC50 = 0.68 mg/mL). The present findings suggest a pathway involving ACE inhibition but measurements of plasma ACE activity and angiotensin II levels are needed to support this suggestion.

Roles of mitogen-activated protein kinases and angiotensin II in renal development

Experimental and clinical evidence suggests that angiotensin II (AII) participates in renal development. Renal AII content is several-fold higher in newborn rats and mice than in adult animals. AII receptors are also expressed in higher amounts in the kidneys of newborn rats. The kidneys of fetuses whose mother received a type 1 AII receptor (AT1) antagonist during gestation present several morphological alterations. Mutations in genes that encode components of the renin-angiotensin system are associated with autosomal recessive renal tubular dysgenesis. Morphological changes were detected in the kidneys of 3-week-old angiotensin-deficient mice. Mitogen-activated protein kinases (MAPKs) are important mediators that transduce extracellular stimuli to intracellular responses. The MAPK family comprises three major subgroups, namely extracellular signal-regulated protein kinase (ERK), c-jun N-terminal kinases (JNK), and p38 MAPK (p38). Important events in renal growth during nephrogenesis such as cellular proliferation and differentiation accompanied by apoptosis on a large scale can be mediated by MAPK pathways. A decrease in glomerulus number was observed in embryos cultured for 48 and 120 h with ERK or p38 inhibitors. Many effects of AII are mediated by MAPK pathways. Treatment with losartan during lactation provoked changes in renal function and structure associated with alterations in AT1 and type 2 AII (AT2) receptors and p-JNK and p-p38 expression in the kidney. Several studies have shown that AII and MAPKs play an important role in renal development. However, the relationship between the effects of AII and MAPK activation on renal development is still unclear.

Toxic effects of mercury, lead and gadolinium on vascular reactivity

Heavy metals have been used in a wide variety of human activities that have significantly increased both professional and environmental exposure. Unfortunately, disasters have highlighted the toxic effects of metals on different organs and systems. Over the last 50 years, the adverse effects of chronic lead, mercury and gadolinium exposure have been underscored. Mercury and lead induce hypertension in humans and animals, affecting endothelial function in addition to their other effects. Increased cardiovascular risk after exposure to metals has been reported, but the underlying mechanisms, mainly for short periods of time and at low concentrations, have not been well explored. The presence of other metals such as gadolinium has raised concerns about contrast-induced nephropathy and, interestingly, despite this negative action, gadolinium has not been defined as a toxic agent. The main actions of these metals, demonstrated in animal and human studies, are an increase of free radical production and oxidative stress and stimulation of angiotensin I-converting enzyme activity, among others. Increased vascular reactivity, highlighted in the present review, resulting from these actions might be an important mechanism underlying increased cardiovascular risk. Finally, the results described in this review suggest that mercury, lead and gadolinium, even at low doses or concentrations, affect vascular reactivity. Acting via the endothelium, by continuous exposure followed by their absorption, they can increase the production of free radicals and of angiotensin II, representing a hazard for cardiovascular function. In addition, the actual reference values, considered to pose no risk, need to be reduced.

Angiotensin II type 1 receptor blockade partially attenuates hypoxia-induced pulmonary hypertension in newborn piglets: relationship with the nitrergic system

The objective of this study was to observe possible interactions between the renin-angiotensin and nitrergic systems in chronic hypoxia-induced pulmonary hypertension in newborn piglets. Thirteen chronically instrumented newborn piglets (6.3 ± 0.9 days; 2369 ± 491 g) were randomly assigned to receive saline (placebo, P) or the AT1 receptor (AT1-R) blocker L-158,809 (L) during 6 days of hypoxia (FiO2 = 0.12). During hypoxia, pulmonary arterial pressure (Ppa; P < 0.0001), pulmonary vascular resistance (PVR; P < 0.02) and the pulmonary to systemic vascular resistance ratio (PVR/SVR; P < 0.05) were significantly attenuated in the L (N = 7) group compared to the P group (N = 6). Western blot analysis of lung proteins showed a significant decrease of endothelial NOS (eNOS) in both P and L animals, and of AT1-R in P animals during hypoxia compared to normoxic animals (C group, N = 5; P < 0.01 for all groups). AT1-R tended to decrease in L animals. Inducible NOS (iNOS) did not differ among P, L, and C animals and iNOS immunohistochemical staining in macrophages was significantly more intense in L than in P animals (P < 0.01). The vascular endothelium showed moderate or strong eNOS and AT1-R staining. Macrophages and pneumocytes showed moderate or strong iNOS and AT1-R staining, but C animals showed weak iNOS and AT1-R staining. Macrophages of L and P animals showed moderate and weak AT2-R staining, respectively, but the endothelium of all groups only showed weak staining. In conclusion, pulmonary hypertension induced by chronic hypoxia in newborn piglets is partially attenuated by AT1-R blockade. We suggest that AT1-R blockade might act through AT2-R and/or Mas receptors and the nitrergic system in the lungs of hypoxemic newborn piglets.