WaveGate: a versatile tool for temporal shaping of synchrotron beams.

We present a full performance characterization of a solid state pulse picker for hard x-ray pulses at synchrotrons. The device is called WaveGate. Specifically, we quantify its efficiency (>30 %), timing capabilities (switching times between 100 ns and ms), on-off contrast (>104) and influence on the coherence properties of the incident x-ray beam. In addition, we discuss the implementation of the WaveGate in an optical pump - x-ray probe setup. Even if single pulse selection is performed by external detector gating, the WaveGate drastically increases the efficiency of a measurement. Finally, we introduce advanced timing schemes that can be realized by modulating the time structure of the synchrotron beam.

Cytotoxic and apoptotic activity of essential oil from Ocimumviride towards COLO 205 cells.

We investigated the apoptosis inducing effect of essential oil (EO) from aerial parts of Ocimumviride in human colorectal adenocarcinoma cells (COLO 205 cell line). The COLO 205 cells were exposed to 0.0125-0.1 microl/ml of EO for 24, 48 and 72h. Growth inhibition was determined by sulphorhodamine B (SRB) assay. Double staining with acridine orange and ethidium bromide for nuclear changes was performed. Cell cycle analysis and change in mitochondrial membrane potential was quantified by flow cytometry. Subsequently, using annexin V/PI assay, the proportion of cells actively undergoing apoptosis was determined. Changes in DNA were observed by DNA ladder assay. Eventually the surface morphology of apoptotic cells was studied by scanning electron microscopy. EO is cytotoxic to COLO 205 cells in dose and time-dependent manner, as is evident by SRB assay. This observed cell death was due to apoptosis, as established by annexin V/PI assay, DNA ladder formation and scanning electron microscopy. Our results reveal that EO has apoptosis inducing effect against COLO 205 cells in vitro and is a promising candidate for further anti-cancer study.

Hypotensive function of the brain angiotensin-(1-7) in Sprague Dawley and renin transgenic rats.

Angiotensin-(1-7) (Ang-[1-7]) is present in the brain of normotensive Sprague Dawley (SD) rats, and its hypothalamic content is elevated in TGRmRen2(27) rats (TGR) with renin dependent transgenic hypertension. The purpose of the present study was to determine the role of intrabrain Ang-(1-7) in the regulation of cardiovascular functions in SD and TGR rats under resting conditions and during haemodynamic challenge produced by rapid bleeding. Two groups of experiments were performed on conscious SD and TGR rats that were chronically instrumented with a lateral cerebral ventricle (LCV) cannula and an intraarterial catheter. Blood pressure (MAP) and heart rate period (Hp=distance between two systolic peaks) were continuously monitored: 1) under resting conditions during an LCV infusion of either artificial cerebrospinal fluid (aCSF, 5 microl/hr) or Ang-(1-7) in aCSF (100 pmol/5 microl/hr), and 2) before and after haemorrhage performed during LCV infusion of either aCSF or Ang-(1-7) antagonist (A-779, 4 nmol/5 microl/hr). Cerebroventricular infusion of Ang-(1-7) did not affect baseline MAP in the SD rats but it caused a significant decrease in blood pressure in the TGR rats. In the control experiments, haemorrhage significantly reduced MAP in the SD and TGR rats and heart rate in the TGR rats. Cerebroventricular infusion of Ang-(1-7) antagonist eliminated posthaemorrhagic hypotension in both strains and bradycardia in the TGR rats. The results indicate that intrabrain Ang-(1-7) may contribute to posthaemorrhagic hypotension and bradycardia. Moreover, the manner in which it centrally regulates the cardiovascular functions in the SD and TGR rats may be considerably different.

Baroreflex improvement in shr after ace inhibition involves angiotensin-(1-7).

ACE inhibitors are extensively used in the treatment of hypertension mainly because of their efficiency in reducing blood pressure levels and decreasing vascular and cardiac hypertrophy. In addition, ACE inhibitors improve baroreceptor reflex control. Chronic inhibition of ACE produces (in addition to decreased angiotensin II levels) a severe increase in angiotensin-(1-7) [Ang-(1-7)] levels in several species. We have previously shown that Ang-(1-7) produces a facilitation of the baroreflex control of heart rate. In this study, we evaluated the participation of endogenous Ang-(1-7) in the improvement of baroreflex sensitivity in spontaneously hypertensive rats after central infusion of ramiprilat, an ACE inhibitor. Reflex changes in heart rate were elicited, in conscious rats, by bolus injections of phenylephrine (baroreflex bradycardia) before and after intracerebroventricular infusion of (1) saline (8 microL/h), 4 hours (n=5); (2) ramiprilat (14 microg/h), 4 hours (n=6); (3) ramiprilat for 2 hours, followed by ramiprilat combined with A-779 (4 microg/h), a selective Ang-(1-7) antagonist, for an additional 2 hours (n=6); and (4) A-779 for 2 hours, followed by A-779 combined with ramiprilat for an additional 2 hours (n=5). Intracerebroventricular infusion of ramiprilat produced an important increase ( approximately 40%) in baroreflex sensitivity (evaluated as the ratio between changes in heart rate and changes in mean arterial pressure) that was completely reversed by A-779. Furthermore, intracerebroventricular infusion of A-779 prevented the improvement of the baroreflex sensitivity produced by ramiprilat. Intracerebroventricular infusion of saline or A-779 alone did not significantly alter the baroreflex sensitivity. These results suggest that endogenous Ang-(1-7) is involved in the improvement of baroreflex sensitivity observed in spontaneously hypertensive rats during central ACE inhibition.

RasG regulates discoidin gene expression during Dictyostelium growth.

Activated rasG, rasG(G12T), was expressed in Dictyostelium cells under the control of the folate-repressible discoidin promoter (pVEII-rasG(G12T)) and found to have a unique pattern of expression when cells were transferred to folate-deficient media: an initial increase of RasG(G12T) resulting from the removal of folate, followed by a rapid decline while cells were still in the early exponential phase of growth. Discoidin levels were considerably lower and declined more rapidly in the pVEII-rasG(G12T) transformant than they did in the wild type, suggesting that RasG(G12T) represses discoidin expression. This was independently confirmed by placing the rasG(G12T) gene under the control of the ribonucleotide reductase (rnrB) promoter. Exposure of cells to 10 mM methyl methanesulfonate (MMS) rapidly generated RasG(G12T) and this was accompanied by an equally rapid decrease in discoidin mRNA levels. rasG null cells also contained decreased levels of discoidin under all conditions tested, indicating that RasG is essential for optimum discoidin expression. However, rasG null cells showed normal regulation of discoidin expression in response to PSF, CMF, folate, bacteria, and axenic media, indicating that RasG is not necessary for any of these responses. These results reveal a role for RasG in regulating discoidin gene expression and add a further level of complexity to the regulation of the discoidin promoter.

Expression of activated Ras during Dictyostelium development alters cell localization and changes cell fate.

There is now a body of evidence to indicate that Ras proteins play important roles in development. Dictyostelium expresses several ras genes and each appears to perform a distinct function. Previous data had indicated that the overexpression of an activated form of the major developmentally regulated gene, rasD, caused a major aberration in morphogenesis and cell type determination. We now show that the developmental expression of an activated rasG gene under the control of the rasD promoter causes a similar defect. Our results indicate that the expression of activated rasG in prespore cells results in their transdifferentiation into prestalk cells, whereas activated rasG expression in prestalk causes gross mislocalization of the prestalk cell populations.

Angiotensin-(1-7) regulates the levels of angiotensin II receptor subtype AT1 mRNA differentially in a strain-specific fashion.

Ang-(1-7) is an effector peptide of the renin-angiotensin system with several distinct actions that are likely mediated by a specific receptor. Regulatory effects of angiotensin (Ang) peptides, Ang-(1-7) and Ang II, on Ang receptor subtype 1 (AT1) mRNA expression were investigated in vascular smooth muscle cells (VSMC) from four University of Akron (Akr) rat strains (WKY, SHR and two backcross consomic lines SHR/y and SHR/a), and in SHR and WKY cells from Charles River Laboratories (Crl). In WKY/Akr and SHR/Akr, Ang-(1-7) treatment increased the levels of AT1 mRNA. This effect was inhibited by the specific Ang-(1-7) antagonist, A-779, in WKY/Akr but not SHR/Akr. Ang II had no effect in Akr cells, but it down-regulated AT1 mRNA in WKY/Crl and SHR/Crl VSMC. Ang-(1-7) did not affect AT1 mRNA levels in Crl lines. In conclusion, Ang-(1-7) regulates the AT1 receptor either directly or indirectly in a strain-specific fashion. The Ang-(1-7) antagonist, A-779, blocks the actions of Ang-(1-7) only in VSMC from WKY/Akr rats, suggesting either that the binding sites for Ang-(1-7) have different properties in SHR/Akr and WKY/Akr cell lines, or that some of the effects of Ang-(1-7) are not receptor mediated. Further, we found differences between Akr cells and Crl cells that are consistent with their genetic heterogeneity.

Dictyostelium RasD is required for normal phototaxis, but not differentiation.

RasD, a Dictyostelium homolog of mammalian Ras, is maximally expressed during the multicellular stage of development. Normal Dictyostelium aggregates are phototactic and thermotactic, moving towards sources of light and heat with great sensitivity. We show that disruption of the gene for rasD causes a near-total loss of phototaxis and thermotaxis in mutant aggregates, without obvious effects on undirected movement. Previous experiments had suggested important roles for RasD in development and cell-type determination. Surprisingly, rasD(-) cells show no obvious changes in these processes. These cells represent a novel class of phototaxis mutant, and indicate a role for a Ras pathway in the connections between stimuli and coordinated cell movement.

Functional overlap of the dictyostelium RasG, RasD and RasB proteins.

Disruption of the rasG gene in Dictyostelium discoideum results in several distinct phenotypes: a defect in cytokinesis, reduced motility and reduced growth. Reintroduction of the rasG gene restores all of the properties of the rasG(-) cells to those of the wild type. To determine whether the defects are due to impaired interactions with a single or multiple downstream effectors, we tested the ability of the highly related but non identical Dictyostelium ras genes, rasD and rasB, to rescue the defects. Introduction of the rasD gene under the control of the rasG promoter into rasG null (rasG(-)) cells corrected all phenotypes except the motility defect, suggesting that motility is regulated by a RasG mediated pathway that is different to those regulating growth or cytokinesis. Western blot analysis of RasD protein levels revealed that vegetative rasG(- )cells contained considerably more protein than the parental AX-3 cells, suggesting that RasD protein levels are negatively regulated in vegetative cells by RasG. The level of RasD was enhanced when the rasD gene was introduced under the control of the rasG promoter, and this increase in protein is presumably responsible for the reversal of the growth and cytokinesis defects of the rasG(- )cells. Thus, RasD protein levels are controlled by the level of RasG, but not by the level of RasD. Introduction of the rasB gene under the control of the rasG promoter into rasG(-) cells produced a complex phenotype. The transformants were extremely small and mononucleate and exhibited enhanced motility. However, the growth of these cells was considerably slower than the growth of the rasG(-) cells, suggesting the possibility that high levels of RasB inhibit an essential process. This was confirmed by expressing rasB in wild-type cells; the resulting transformants exhibited severely impaired growth. When RasB protein levels were determined by western blot analysis, it was found that levels were higher in the rasG(- )cells than they were in the wild-type parental, suggesting that RasG also negatively regulates rasB expression in vegetative cells. Overexpression of rasB in the rasG(- )cells also reduced the level of RasD protein. In view of the fact that alternate Ras proteins correct some, but not all, of the defects exhibited by the rasG(-) cells, we propose that RasG interacts with more than one downstream effector. In addition, it is clear that the levels of the various Ras proteins are tightly regulated in vegetative cells and that overexpression can be deleterious.

Modulation of the baroreflex control of heart rate by angiotensin-(1-7) at the nucleus tractus solitarii of normotensive and spontaneously hypertensive rats.

OBJECTIVES: In the present study, we evaluated the effect of angiotensin-(1-7) [Ang-(1-7)] and its selective antagonist, D-Ala7-Ang-(1-7) (A-779), at the nucleus tractus solitarii (nTS), in the modulation of the bradycardic component of the baroreceptor reflex. METHODS: Mean arterial pressure (MAP) and heart rate were continuously recorded. Reflex changes in heart rate elicited by bolus injection of graded doses of phenylephrine were evaluated before and after bilateral microinjection (glass micropipette) of Ang-(1-7) (10 pmol or 25 pmol), A-779 (50 pmol) or saline (vehicle) into the nTS of urethane anesthetized male Wistar rats or spontaneously hypertensive rats (SHR). The averaged ratio between reflex changes in heart rate and changes in MAP was used as index of baroreflex sensitivity. RESULTS: Microinjection of Ang-(1-7) into the nTS elicited significant decreases in MAP and heart rate in both Wistar and SHR. While the decrease in MAP was similar in both strains, the changes in heart rate were smaller in SHR. A-779 produced small changes in MAP and heart rate that were no different from those induced by saline. After microinjection of 10 pmol of Ang-(1-7) into the nTS of normotensive rats, there was a significant increase in baroreflex sensitivity. In SHR, only the microinjection of a higher dose (25 pmol) of Ang-(1-7) produced a significant increase in baroreflex sensitivity. A significant reduction inbaroreflex sensitivity was observed after microinjection of A-779 (50 pmol) in both strains. CONCLUSIONS: These results indicate that Ang-(1-7) exerts a tonic modulatory effect on the baroreflex control of heart rate at the nTS, probably through a non-AT1 non-AT2 receptor subtype. In addition, our data showed a reduced sensitivity to Ang-(1-7) at the nTS of SHR, that could be accounting, at least in part, for the decreased baroreflex sensitivity present in this model of hypertension.

Characterization of a local folding event of the Tetrahymena group I ribozyme: effects of oligonucleotide substrate length, pH, and temperature on the two substrate binding steps.

Binding of the Tetrahymena group I ribozyme's oligonucleotide substrate occurs in two steps: P1 duplex formation with the ribozyme's internal guide sequence which forms an "open complex" is followed by docking of the P1 duplex into tertiary interactions within the catalytic core which forms a "closed complex". By systematically varying substrate length, pH, and temperature, we have identified conditions under which P1 duplex formation, P1 docking, or the chemical cleavage step limits the rate of the ribozyme reaction. This has enabled characterization of the individual steps as a function of substrate length, pH, and temperature, leading to several conclusions. (1) The rate constant for formation of the open complex is largely independent of substrate length, pH, and temperature, analogous to that of duplex formation in solution. This extends previous results suggesting that open complex formation entails mainly secondary structure formation and strengthens the view that the second binding step, P1 docking, represents a simple transition from secondary to tertiary structure in the context of an otherwise folded RNA. (2) The temperature dependence of the rate constant for P1 docking yields a negative activation entropy, in contrast to the positive entropy change previously observed for the docking equilibrium. This suggests a model in which tertiary interactions are not substantially formed in the transition state for P1 docking. (3) Shortening the substrate by three residues decreases the equilibrium constant for P1 docking by 200-fold, suggesting that the rigidity imposed by full-length duplex formation facilitates formation of tertiary interactions. (4) Once docked, shortened substrates are cleaved at rates within 3-fold of that for the full-length substrate. Thus, all the active site interactions required to accelerate the chemical cleavage event are maintained with shorter substrates. (5) The rate constant of approximately 10(3) min(-1) obtained for P1 docking is significantly faster than the other steps previously identified in the tertiary folding of this RNA. Nevertheless, P1 docking presumably follows other tertiary folding steps because the P1 duplex docks into a core that is formed only upon folding of the rest of the ribozyme.

Haemorrhage increases the pressor effect of angiotensin-(1-7) but not of angiotensin II at the rat rostral ventrolateral medulla.

OBJECTIVE: To evaluate the effects of angiotensins acting at the rostral ventrolateral medulla (RVLM) on the cardiovascular adjustments following haemorrhage. DESIGN: Changes in mean arterial pressure (MAP) and heart rate (HR) produced by micro-injections of angiotensin II (Ang II) and angiotensin (Ang)-(1-7) and different angiotensin antagonists into the RVLM of anaesthetized rats submitted to haemorrhage, were determined. METHODS: Experiments were performed in 79 urethane-anaesthetized male Wistar rats. Ang-(1-7) (2.5 and 25 pmol), Ang II (25 pmol), [Sar1,Thr8]-Ang II (non-selective angiotensin antagonist, 0.2 nmol), A-779 (Ang-(1-7) antagonist, 0.1 nmol), losartan (AT1 Ang II receptor antagonist, 0.2 nmol) or vehicle (200 nl) were bilaterally micro-injected into the RVLM under basal conditions or 30 min after blood withdrawal (0.6 ml/100 g bodyweight). In additional groups, [Sar1,Thr8]-Ang II, A-779, losartan or vehicle were micro-injected into the RVLM 10 min before bleeding to uncover a possible role of endogenous peptides during haemorrhage. RESULTS: The pressor effect produced by Ang II micro-injection was not altered by haemorrhage. Conversely, haemorrhage significantly increased the magnitude and duration of the pressor effect of Ang-(1-7) at the RVLM. The fall in MAP induced by haemorrhage was similar after micro-injection of vehicle or A-779. However, micro-injection of [Sar1,Thr8]-Ang II significantly reduced the fall in MAP after haemorrhage. A similar finding was obtained with micro-injection of losartan. In addition, while RVLM micro-injection of [Sar1,Thr8]-Ang II or losartan 30 min after blood withdrawn produced MAP changes that were similar to that observed in control animals, micro-injection of A-779 did not significantly alter baseline MAP. CONCLUSIONS: These results suggest that changes in the RVLM reactivity to Ang-(1-7) but not Ang II may contribute to the haemodynamic adjustments triggered by acute reductions in blood volume. The data obtained with [Sar1,Thr8]-Ang II and losartan suggest a primary inhibitory role for endogenous Ang II at the RVLM during haemorrhage.

Potentiation of the hypotensive effect of bradykinin by angiotensin-(1-7)-related peptides.

In this study, we evaluated the bradykinin potentiating activity and ACE inhibitory activity of several Ang-(1-7)-related peptides: Ang-(2-7), Ang-(3-7), Ang-(4-7), Ang-(1-6), Ang-(1-5) and the selective antagonist of Ang-(1-7): D-[Ala7]Ang-(1-7) (A-779). In vivo experiments were performed in freely moving Wistar rats. ACE activity was evaluated by a fluorometric assay in rat plasma using Hip-His-Leu as a substrate. Intravenous injections of Ang-(1-7) (2.2 nmol) transformed the effect of a single dose of bradykinin (1 nmol) into the effect produced by a double dose. A similar bradykinin potentiating activity was demonstrated for Ang-(2-7) and Ang-(3-7). On the other hand, Ang-(1-5), Ang-(1-6), Ang-(4-7) and A-779 did not change the hypotensive effect of bradykinin in doses ranging from 8 up to 25 nmols. The hypotensive effect of bradykinin was increased by intravenous infusion (0.3 ng/min) of Ang-(1-7) > Ang-(2-7) > Ang-(3-7). Conversely, Ang-(1-5), Ang-(1-6), Ang-(4-7) or A-779 did not change the hypotensive effect of bradykinin. ACE inhibition with Ang-(1-7) related peptides occurred in the order: Ang-(2-7) > or = Ang-(3-7) > Ang-(1-7) [>>] Ang-(1-5) > Ang-(4-7) > or = Ang-(1-6) > or = A-779. A-779 in concentrations up to 10(-5) M did not change the ACE inhibitory activity of Ang-(1-7). These results suggest that Ang-(1-7), Ang-(2-7) and Ang-(3-7) can modulate bradykinin actions in vivo. More important, our data pointed out that alternative mechanisms besides interaction with ACE are required to explain the bradykinin potentiating activity of Ang-(1-7).

A mutation that separates the RasG signals that regulate development and cytoskeletal function in Dictyostelium.

The expression of an activated RasG, RasG-G12T, in vegetative cells of Dictyostelium discoideium produced an alteration in cell morphology. Cells underwent a transition between an extensively flattened form that exhibited lateral membrane ruffling to a less flattened form that exhibited prominent dorsal membrane ruffling. These rasG-G12T transformants exhibited a redistribution of F-actin at the cell periphery and did not undergo the rapid contraction upon refeeding that is characteristic of wild-type cells. These results suggest a role for RasG in regulating cytoskeletal rearrangement in D. discoideum. We had shown previously that expression of rasG-G12T inhibited starvation induced aggregation (M. Khosla et al., 1996, Mol. Cell. Biol. 16, 4156-4162). rasG-G12T genes containing secondary mutations were transformed into cells to test whether the effects of rasG-G12T were transmitted through a single downstream effector. Cells expressing rasG-G12T/T35S or rasG-G12T/Y40C (secondary mutations within the effector domain) exhibited normal morphology and underwent normal aggregation, suggesting that signaling through the effector domain was required for both the morphological and the development changes induced by rasG-G12T. In contrast, cells expressing rasG-G12T/T45Q (a secondary mutation in the effector distal flanking domain) exhibited normal aggregation but a morphology indistinguishable from that of rasG-G12T transformants. This result suggests that RasG regulates developmental and cytoskeletal functions by direct interaction with more than one downstream effector.

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 percenr 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 ñ 0.04 ml/min vs 1.45 ñ 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 ñ 0.34 ml/60 min and 3.39 ñ 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.

Diuresis and natriuresis produced by long term administration of a selective Angiotensin-(1-7) antagonist in normotensive and hypertensive rats.

In this study we evaluated the renal effects of chronic administration of the selective Angiotensin-(1-7)[Ang-(1-7)] antagonist, A-779, in normotensive and spontaneously hypertensive rats (SHR). Male adult SHR and Wistar rats were housed in metabolic cages with tap water and standard chow, for three-five days before starting infusion (Alzet osmotic mini-pumps) of A-779 (Wistar: 1 microg/h, n = 9; 2.5 microg/h, n = 6; SHR:2.5 microg/h, n = 6) or vehicle (0.9% NaCl - 1 microl/h, n = 7 and n = 10 for SHR and Wistar rats, respectively). Urine volume, water and food intake and urinary Na+ were measured daily. On the last day of infusions mean arterial pressure (MAP) was recorded and urine and blood samples were collected to determine renal function parameters. Chronic infusion of A-779 produced a sustained increase in diuresis in normotensive rats [seventh day values: 0.75+/-0.08 ml/h (1 microg/h) and 0.94+/-0.13 ml/h (2.5 microg/h) vs. 0.42 + 0.03 ml/h for the control group, P<0.05] associated to a dose-dependent increase in the creatinine clearance. In SHR, diuresis increased significantly after chronic infusion of A-779 (fifth day values: 0.44 + 0.06 ml/h vs. 0.25+/-0.04 ml/h for the control group, P<0.05), without changes in creatinine clearance. Infusion of A-779 in normotensive rats produced a decrease in water reabsorption. A-779 infusion also produced a dose-dependent increase in urinary Na+ excretion (1.49 + 0.14 mEq, 1 microg/h vs. 2.37+/-0.22 mEq, 2.5 microg/h, P<0.05), in Wistar rats, without modifying the fractional excretion of Na+. In SHR, urinary Na+ excretion was also increased by A-779 (2.21+/-0.46 mEq vs. 0.94+/-0.22 mEq for the control group, P<0.05). No significant changes in blood pressure were observed. These findings suggest that endogenous Ang-(l-7) participates in the control of hydroelectrolyte balance by modulating water excretion, acting at tubular and glomerular sites.

[7-D-ALA]-angiotensin 1-7 blocks renal actions of angiotensin 1-7 in the anesthetized rat.

Exogenous angiotensin (Ang) 1-7 affects renal function, but the receptor(s) involved in this response remain(s) to be determined. In an in vitro preparation of proximal tubules, Ang 1-7 was shown to act on Ang II AT1 receptors (minor component), but also on a non-AT1, non-AT2 Ang receptor (major component) to inhibit reabsorption. In brain, Ang 1-7 also exerts effects mediated by a non-AT1, non-AT2 binding site; these effects are inhibited, however, by the angiotensin analog [7-D-Ala]-Ang 1-7. Therefore we tested the effect of Ang II AT1-receptor antagonist losartan and [7-D-Ala]-Ang 1-7 on the renal response to exogenous Ang 1-7 in standard renal-clearance experiments in the anesthetized rat. We found that Ang 1-7 (100 pmol/kg/min, i.a.) increased glomerular filtration rate (GFR), urinary flow rate (UV), and urinary sodium excretion (UNaV) without affecting mean arterial blood pressure (MAP) or urinary potassium excretion (UKV), confirming previous reports. Losartan (10 mg/kg, i.v.) blocked the pressor effect of exogenous Ang II (100 pmol/kg/min, i.a.), but did not significantly affect the renal response to Ang 1-7. Conversely, pretreatment with [7-D-Ala]-Ang 1-7 (5 nmol/kg/min) did not affect the pressor effect of Ang II, but abolished the renal response to Ang 1-7. Application of [7-D-Ala]-Ang 1-7 in the absence of exogenous Ang 1-7 did not alter MAP or GFR, but increased UNaV (by 52%). Our data indicate that similar to the response in brain, the renal response to exogenous Ang 1-7 may be mediated predominantly by a distinct non-AT1 binding site, which is sensitive to blockade by [7-D-Ala]-Ang 1-7. Furthermore, ambient endogenous Ang 1-7 acting on this distinct binding site may not contribute significantly to control of MAP or GFR, but exerts an antinatriuretic influence in the anesthetized rat.

Negative influence of RasG on chemoattractant-induced ERK2 phosphorylation in Dictyostelium.

The Dictyostelium ERK2 protein is transiently activated when cells are treated with the chemotactic agents cAMP or folic acid. Activating phosphorylation is markedly inhibited in strains overexpressing the constitutively activated RasG protein. This is in marked contrast to mammalian cells where the highly related mitogen-activated protein kinases (MAPKs) are stimulated by Ras activation.

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.

Potentiation of the hypotensive effect of bradykinin by short-term infusion of angiotensin-(1-7) in normotensive and hypertensive rats.

In this study we evaluated the effect of angiotensin-(1-7) on the hypotensive action of bradykinin (BK) in normotensive rats, renal hypertensive rats (RHR), and spontaneously hypertensive rats (SHR). In addition, we evaluated the effect of angiotensin-converting enzyme (ACE) inhibition with enalaprilat treatment (10 mg/kg I.V.) on the BK-potentiating activity of Ang-(1-7). Renal hypertension was produced by aorta coarctation between the origin of renal arteries. Ang-(1-7) (0.3 pmol/min) or saline (0.9% NaCl, 5 microL/min) was infused intravenously in conscious male Wistar rats, adult SHR, or RHR. Intravenous bolus injections of BK (0.1 to 1.6 nmol in RHR and SHR; 0.625 to 5 nmol in Wistar rats) were made before and within 30 and 60 minutes of Ang-(1-7) infusion. Ang-(1-7) infusion did not change mean arterial pressure (MAP) of Wistar rats (MAP=97+/-3 mm Hg), RHR (MAP=173+/-3 mm Hg), or SHR (MAP=177+/-5 mm Hg). In Wistar rats, Ang-(1-7) increased the BK hypotensive effect by 24+/-6% within 60 minutes of infusion. No significant changes were observed at 30 minutes of infusion. In additional groups of rats, Ang-(1-7) (5 pmol/min, n=5) was infused alone or combined with its selective antagonist D-Ala7-Ang-(1-7) (A-779) (5 pmol/min, n=6). The bradykinin-potentiating activity of Ang-(1-7) was completely abolished by A-779. In SHR and RHR, Ang-(1-7) significantly increased the hypotensive effect of BK by 59+/-8% and 57+/-9.8%, respectively, within 60 minutes of infusion. No significant changes were observed with saline infusion. In Wistar rats, enalaprilat treatment increased the BK-potentiating activity of Ang-(1-7) transforming the effect of 0.3 pmol/min into that observed with a rate 16-fold higher (5 pmol/min). On the other hand, in SHR enalaprilat did not change the Ang-(1-7) effect, while it abolished the BK potentiation in RHR. Our data show that the BK-potentiating activity of Ang-(1-7) is preserved and even augmented in hypertensive rats. The finding that the BK-potentiating activity of Ang-(1-7) could be demonstrated at a very low infusion rate suggests that this angiotensin can act as an endogenous modulator of the vascular actions of kinins. ACE inhibition can influence differently the BK-potentiating activity of Ang-(1-7) in normotensive and hypertensive rats.