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Phosphoinositide hydrolysis increase by angiotensin-(1–7) in neonatal rat brain
Regulatory Peptides 140 (2007) 162 – 167 www.elsevier.com/locate/regpep
Phosphoinositide hydrolysis increase by angiotensin-(1–7) in neonatal rat brain Susana Pereyra-Alfonso a , Georgina Rodríguez de Lores Arnaiz a,b , Clara Peña c,⁎ a
Instituto de Biología Celular y Neurociencias “Prof. E. De Robertis”, Facultad de Medicina, Universidad de Buenos Aires, Paraguay 2155, (1121) Buenos Aires, Argentina b Cátedra de Farmacología, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, (1113) Buenos Aires, Argentina c IQUIFIB–CONICET, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, (1113) Buenos Aires, Argentina Received 20 July 2006; received in revised form 24 November 2006; accepted 1 December 2006 Available online 10 January 2007
Abstract Angiotensin (Ang)-(1–7) is an endogenous peptide hormone of the renin–angiotensin system which exerts diverse biological actions, some of them counterregulate Ang II effects. In the present study potential effect of Ang-(1–7) on phosphoinositide (PI) turnover was evaluated in neonatal rat brain. Cerebral cortex prisms of seven-day-old rats were preloaded with [3H]myoinositol, incubated with additions during 30 min and later [3H] inositol-phosphates (IPs) accumulation quantified. It was observed that PI hydrolysis enhanced 30% to 60% in the presence of 0.01 nM to 100 nM Ang-(1–7). Neither 10 nM [D-Ala7]Ang-(1–7), an Ang-(1–7) specific antagonist, nor 10 nM losartan, an angiotensin II type 1 (AT1) receptor antagonist, blocked the effect of 0.1 nM Ang-(1–7) on PI metabolism. The effect of 0.1 nM Ang-(1–7) on PI hydrolysis was not reduced but it was even significantly increased in the simultaneous presence of [D-Ala7]Ang-(1–7) or losartan. PI turnover enhancement achieved with 0.1 nM Ang-(1–7) decreased roughly 30% in the presence of 10 nM PD 123319, an angiotensin II type 2 (AT2) receptor antagonist. The antagonists alone also enhanced PI turnover. Present findings showing an increase in PI turnover by Ang-(1–7) represent a novel action for this peptide and suggest that it exerts a function in this signaling system in neonatal rat brain, an effect involving, at least partially, angiotensin AT2 receptors. © 2007 Elsevier B.V. All rights reserved. Keywords: Phosphoinositide turnover; Neonatal brain cortex; Angiotensin receptors; [D-Ala7]angiotensin-(1–7); Losartan; PD 123319
1. Introduction Angiotensin (Ang)-(1–7) is an important biologically active component of the renin–angiotensin system, formed by enzymatic processing of Ang I or Ang II. The heptapeptide exerts diverse biological actions, some of them similar but others markedly different from those displayed by Ang II [1]. For instance, it lacks the vasoconstrictor and aldosterone secretagogue [2] or dipsogenic [3] effects of Ang II, but mimics Ang II stimulation of vasopressin [4] and prostaglandin [5] release as well as peripheral norepinephrine outflow [6]. On the contrary, Ang-(1–7) causes natriuresis [7], diuresis [8] vasodi-
⁎ Corresponding author. Tel.: +54 11 4964 8292; fax: +54 11 4962 5457. E-mail address: [email protected] (C. Peña). 0167-0115/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2006.12.005
lation [9] and inhibits vascular smooth muscle cell growth [10], suggesting that in many cases, this peptide could act as an endogenous antagonist of Ang II. Our laboratory has reported among Ang-(1–7) actions, its ability to enhance phosphatidylcholine biosynthesis in the rat renal cortex through activation of Ang-(1–7) specific receptors [11]. Activation of several neurotransmitter (i.e. cholinergic, adrenergic, etc.) and/or neuropeptide receptors enhances phosphatidylinositol (PI) turnover in brain, an effect which proved higher in neonatal than in adult brain [12]. The involvement of high-affinity neurotensin receptor (NTS1) in PI turnover enhancement by cholinergic agonist carbachol was previously documented [13]. The aim of this study was to evaluate the potential effect of Ang-(1–7) on PI turnover in neonatal rat brain and to determine the possible involved receptors.
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2. Materials and methods 2.1. Chemicals Reagents were of analytical grade. Losartan was purchased from DuPont Merck (Wilmington, DE, USA); myoinositol and (1-[[4-(dimethylamino)-3-methylphenyl]methyl]-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazol[4,5c]pyridine-6-carboxylic acid ditrifluoroacetate) (PD 123319) were from SigmaAldrich (St. Louis, MO, USA). A Dowex anion exchange resin (AG 1-X8, 100–200 mesh, formate form) was from Bio-Rad Laboratories (Richmond, CA, USA). OptiPhase “Hisafe” 3 was purchased from Wallac Oy (Turku, Finland). [3H]myoinositol (20 Ci/mmol) was from New England Nuclear (Boston, MA, USA). Ang-(1–7) and [D-Ala7]Ang-(1–7) were synthesized in our laboratory by the Merrifield solid-phase procedure as described previously [11]. The purity of both peptides was verified by mass spectrometry. All other chemicals were of analytical grade and purchased from local suppliers. 2.2. Animals Wistar neonatal rats (6–7 days old) of either sex were used, considering “day 0” the day of birth. All studies described were performed in accordance with the Guide for Care and Use of Laboratory Animals provided by the National Institutes of Health, USA. 2.3. Determination of [3H]-inositol phosphates (IPs) A procedure based on described methods [14,15] with modifications, was performed using cerebral cortices of neonatal rats. For each experiment, tissues pooled from three rats were placed on ice on a Petri dish with gassed Krebs– Henseleit buffer containing (mM): NaCl, 120; KCl, 4.7; CaCl2, 1.3; KH2PO4, 1.2; MgSO4, 1.2; NaHCO3, 25 and glucose, 11.7, equilibrated to pH 7.4 with O2/CO2 (95:5). Tissue was lightly minced with a bistoury to obtain prisms of approximately 1 mm each dimension [16], suspended at 10% (w/v) in the same buffer and incubated in bulk at 37 °C for 1 h under gentle shaking with an intermediate change of buffer, followed by 60 min incubation with [3H]myoinositol (6 μCi/ml; final concentration 3 × 10− 7 M) and four washes with fresh buffer replaced every 5 min under O2/CO2 (95:5). Fifty microliters of packed prisms containing 1.0 ± 0.15 mg protein (mean values ± SEM, n = 10) was transferred to tubes with 0.24 ml of the same buffer which contained LiCl (7.5 mM final concentration, with NaCl isoosmotically reduced), and the indicated additions to 0.3 ml final volume. Prism samples preloaded with [3H]myoinositol were incubated for 30 min in the presence of 0.01 nM to 100 nM Ang-(1–7). To test the effect of antagonists, 0.1 nM Ang-(1–7) was employed in the presence or absence of [D-Ala 7 ]Ang(1–7), losartan or PD 123319 at 10 nM concentration. Incubation proceeded for 30 min under O2/CO2 (95:5) with shaking at 37 °C. Corresponding controls with redistilled water or antagonists alone were processed.
Fig. 1. The effect of Ang-(1–7) on [3H]inositol-phosphates accumulation in cerebral cortex prisms obtained from neonatal rats. Prisms preloaded with [3H] myoinositol were incubated in triplicate for 30 min in the presence of 0.01 nM to 100 nM Ang-(1–7) and IPs accumulation assayed. Samples were processed simultaneously (in triplicate) without additions to determine basal IPs level. Results are expressed as percentage of labeling taking as 100% values of basal IPs accumulation and are means (± SEM) of 5 experiments. ⁎ denotes P b 0.05 versus control without Ang-(1–7) (basal condition), by Student's t test.
Prism samples preloaded with [3H]myoinositol were processed throughout without additions (in triplicate) to determine basal IPs accumulation. Incubations were stopped by the addition of 940μl chloroform:methanol (1:2), followed by chloroform (310 μl) and redistilled water (310 μl) to separate phases. Tubes were vortexmixed for 15 s, then centrifuged at 1000×g for 10 min to facilitate phase separation. Radiolabeled IPs were separated from inositol by ion-exchange chromatography using small columns containing 0.5 ml of a 50% slurry AG 1-X8 resin in the formate form. Upper aqueous phase aliquots (750 μl) diluted to 3 ml with redistilled water were added to the resin suspension, centrifuged, and washed 4 times with 3 ml of 5 mM myoinositol. [3H]-IPs were eluted with 1 ml of 1 M ammonium formate/0.1 M formic acid and 800 μl of this eluate added to 10 ml of OptiPhase “Hisafe” 3 and counted in a Tracor Analytic scintillation spectrometer with 30% efficiency. In all experiments, aliquots containing the same amount of brain tissue (that is, similar intraexperimental protein content) were processed, which allowed the comparison between basal condition and treatment in the same sample. Results are expressed as [3H]-IPs accumulation taking as 100% basal value recorded in the absence of additions. The relatively long incubation (30 min) did not allow to measure the accumulation of the more polar hydrolysis products inositol-1,4-diphosphate and inositol-1,4,5-triphosphate, since changes in these compounds are detectable at very short times after addition of drugs to the incubation buffer [17]. However, since the concentration of ammonium formate used allows the recovery of all IPs, the term IPs instead of IP1 (inositol monophosphate) was used. 2.4. Protein measurement Protein concentration was evaluated by the method of Lowry et al. [18] using bovine serum albumin as standard.
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Fig. 2. IPs accumulation in cerebral cortex prisms obtained from neonatal rats in the presence of Ang-(1–7) and/or [D-Ala7]Ang-(1–7). Prisms preloaded with [3H]myoinositol were incubated in triplicate for 30 min in the presence of 0.1 nM Ang-(1–7) and/or 10 nM [D-Ala7]Ang-(1–7) and IPs accumulation assayed. Samples were processed simultaneously (in triplicate) without additions to determine basal IPs level. Results are expressed as percentage of labeling taking as 100% values of basal IPs accumulation and are means (±SEM) of 3–8 experiments. ⁎⁎P b 0.02, ⁎⁎⁎P b 0.01, by Student's t test.
Fig. 4. IPs accumulation in cerebral cortex prisms obtained from neonatal rats in the presence of Ang-(1–7) and/or PD 123319. Prisms preloaded with [3H] myoinositol were incubated in triplicate for 30 min in the presence of 0.1 nM Ang-(1–7) and/or 10 nM PD 123319 and IPs accumulation assayed. Samples were processed simultaneously (in triplicate) without additions to determine basal IPs level. Results are expressed as percentage of labeling taking as 100% values of basal IPs accumulation and are means (±SEM) of 3–4 experiments. ⁎P b 0.05,⁎⁎P b 0.02, by Student's t test.
2.5. Statistical analysis
protein (mean values ± SEM from 15 experiments). PI hydrolysis was assayed in the presence of 0.01 nM to 100 nM Ang(1–7) concentrations; it was observed 30 to 60% stimulation above the control (Fig. 1). In order to characterize the receptors by which this peptide promotes PI hydrolysis, experiments were carried out with 0.1 nM Ang-(1–7) plus 10 nM [D-Ala7]Ang-(1–7), losartan or PD 123319. IPs accumulation with 0.1 nM Ang-(1–7) was 161 ± 10.0%; with the simultaneous presence of 10 nM [D-Ala7]Ang(1–7), an (Ang)-(1–7) specific antagonist for various Ang-(1–7) effects [19], this value significantly increased to 213 ± 11.8%, a value which was significantly different from those of the control and the data recorded with Ang-(1–7) alone. The single presence of 10 nM [D-Ala7]Ang-(1–7) stimulated PI hydrolysis to 153 ± 3.0% (Fig. 2). IP accumulation reached 144 ± 12.4% with 0.1 nM Ang-(1– 7), a value which was not significantly modified in the simultaneous presence of 10 nM losartan, an angiotensin II type 1 receptor (AT1) antagonist (163 ± 12.8%). This antagonist alone was likewise able to significantly increase PI turnover (157 ± 17.3%) (Fig. 3). [3H]-IPs accumulation caused by 0.1 nM Ang-(1–7) (165 ± 7.1%) decreased approximately 30% in the presence of 10 nM PD 123319, an angiotensin II type 2 receptor (AT2) antagonist. It was observed that PD 123319 alone was also able to increase IPs accumulation to 131 ± 11.3% (Fig. 4).
All values are means ± SEM. Data were analyzed by Student's t-test. Probability values lower than 0.05 were considered significant. 3. Results In cerebral cortex prisms of neonatal rats basal [3H]-IPs accumulation in the aqueous phase was 2834 ± 584 dpm per mg
Fig. 3. IPs accumulation in cerebral cortex prisms obtained from neonatal rats in the presence of Ang-(1–7) and/or losartan. Prisms preloaded with [3H] myoinositol were incubated in triplicate for 30 min in the presence of 0.1 nM Ang-(1–7) and/or 10 nM losartan and IPs accumulation assayed. Samples were processed simultaneously (in triplicate) without additions to determine basal IPs level. Results are expressed as percentage of labeling taking as 100% values of basal IPs accumulation and are means (±SEM) of 4–6 experiments. ns: no significance, ⁎P b 0.05, ⁎⁎P b 0.02, by Student's t test.
4. Discussion The effect of peptide Ang-(1–7) on PI hydrolysis in neonatal rat brain prisms was studied. Results obtained show that Ang(1–7) significantly enhances PI turnover, an effect blocked by PD 123319, suggesting the involvement of AT2 receptors.
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Neither [D-Ala7]Ang-(1–7), which antagonizes biological actions of Ang-(1–7), nor losartan, which blocks AT1 receptors, was able to inhibit PI hydrolysis stimulation by Ang-(1–7). As far as we know, the ability of Ang-(1–7) to enhance PI turnover has not previously been described. On the contrary, no effect of Ang-(1–7) on PI hydrolysis was recorded in astrocyte culture from neonatal rat brain (one- to two-day-old pups) [20]. This discrepancy with present results is attributable to experimental differences, including rat strain and age, cell type employed (pure astrocytes or pieces of whole tissue which include neurons and astrocytes), peptide concentration, etc. [20,21]. It has been demonstrated that Ang-(1–7) increases in ovine plasma during fetal stage and circulates at much higher levels than those determined in the adult sheep [22]. After Ang-(1–7) infusion there is an up-regulation of gene expression for AT1 and AT2 receptors in the kidney of ovine fetuses [22], suggesting an opposing action of Ang-(1–7) to that of Ang II in the regulation of angiotensin receptors. In contrast, other studies indicate that Ang-(1–7) might oppose the action of Ang II through the downregulation or desensitisation of the AT1 receptors [23]. In present experiments, which were performed in brain tissue from neonatal rats (6–7 days old), Ang-(1–7) enhanced PI turnover, an effect which was not blocked by [D-Ala7] Ang-(1–7), a selective heptapeptide known to antagonize several biological actions of Ang-(1–7) [24–26]. Unexpectedly, [D-Ala7]Ang-(1–7) induced a response by itself, leading to PI turnover enhancement. The existence of a G protein-coupled receptor for Ang-(1–7) termed Mas, which is blocked by [D Ala7]Ang (1–7) has been described [27]. Present results indicated that Ang-(1–7)-elicited PI hydrolysis enhancement does not seem to be mediated by a [D-Ala7]Ang-(1–7) sensitive site, ruling out a direct interaction with the Mas receptor. Regarding other actions of Ang-(1–7), previous studies from our laboratory showed that it increases phosphatidylcholine biosynthesis, an effect not diminished by [D-Ala7]Ang-(1–7), which instead, exerts an agonist-like activity [11]. It was also reported that [D-Ala7]Ang-(1–7) failed to block completely the nitric oxide release induced by Ang-(1–7) in cultured bovine aortic endothelial cells [28]. It has been reported that [D-Ala7]Ang-(1–7) does not influence Ang-(1–7) inhibitory effects on noradrenaline release in rat kidney [29]. These findings led to the suggestion that whenever [D-Ala 7 ]Ang-(1–7) fails to prevent Ang-(1–7) actions, the Mas receptor is inactive or even unexpressed [30]. The biochemistry of the Mas receptor remains somewhat controversial, since this receptor is capable of interacting with Ang II receptors, may have independent signaling properties and may not depend on the binding of Ang-(1–7) for its actions [31]. The effects of AT1 and AT2 antagonists on Ang-(1–7) responses are not consistent, possibly due to organ and species differences in Ang II receptor subtype specificity [28]. Current data strongly suggest that an interaction between receptors for angiotensin peptides may play a role in biological effects of
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Ang-(1–7) [32,33]. Taken jointly, these findings may indicate that Ang-(1–7) interacts with multiple binding sites. As regards the relationship between Ang-(1–7) and AT1 receptor, it should be recalled that renal actions of Ang-(1–7) [8] and the release of [3H]norepinephrine from rat atria [6] are blocked by AT1 receptor antagonists. Furthermore, the suggestion that Ang-(1–7) may be an endogenous antagonist for AT1 receptor or that it may modulate the effect of Ang II via the AT1 receptor was advanced [34]. It has been demonstrated that Ang-(1–7) antagonizes Ang II-evoked vasoconstriction in human arteries by a non-competitive blockade of AT1 receptor [35]. In the kidney, Ang-(1–7) also acts as an antagonist of Ang II [36] and competes with high affinity for the losartan sensitive receptor [37]. Losartan was not able to block the enhancement of PI turnover by Ang-(1–7), suggesting that this effect is independent of AT1 receptor. However, losartan itself enhanced PI turnover and, coincubated with Ang-(1–7), induced an even higher IPs accumulation. For other angiotensin receptor antagonists, partial agonist action was likewise described [38]. Blockade of AT1 receptors is accompanied by elevation in both Ang I and Ang II levels, thus stimulating their conversion into Ang-(1–7) in incubated tissue [39]. Alternatively, the losartan-induced response is attributable either to the increased Ang-(1–7) level or to a drug specific effect. It should be recalled that losartan interacts with Ang-(1–7) receptors at cardiac level [40] and that losartan chronic hypotensive effects in normal rats are mediated in part through the actions of Ang-(1–7) [41]. In general, cell signaling pathways and physiological functions recorded for AT2 are unconventional [42,43]. Expression of AT2 receptors is highly dependent on age. In fetal tissues, the AT2 receptor is the dominating receptor subtype [44]. In the adult, healthy organism – no matter what species – AT2 receptors are only expressed in specific cell types and tissues such as vascular endothelial cells, distinct areas of the brain, the adrenal, selected cutaneous, renal and cardiac structures, myometrium and ovaries [45,46]. Most interestingly, in the adult, AT2 receptors are re-expressed under pathophysiological conditions such as mechanical injury [47] or ischemia [48]. Recent investigations have established a role for the AT2 receptor in cardiovascular, brain and renal function as well as in the modulation of several biological processes involved in development, cell differentiation, tissue repair and apoptosis [46]. The AT2 receptor antagonist PD 123319 caused a significant attenuation of the Ang-(1–7) stimulatory effect on PI turnover, while increasing PI hydrolysis by itself. Thus, we conclude that Ang-(1–7)-induced PI hydrolysis may reflect a combination of an effect at an AT2 receptor site that is being blocked by PD 123319 together with an action of Ang-(1–7) at another yet unidentified receptor. Another plausible hypothesis may imply a direct effect of Ang-(1–7) on the phosphoinositide phosphodiesterase system. To conclude, these results demonstrate that the peptide Ang(1–7) enhances PI metabolism in neonatal rat brain. This effect seems partly mediated by AT2 receptors but independent of AT1 receptors.
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Phosphoinositide hydrolysis increase by angiotensin-(1–7) in neonatal rat brain Susana Pereyra-Alfonso a , Georgina Rodríguez de Lores Arnaiz a,b , Clara Peña c,⁎ a
Instituto de Biología Celular y Neurociencias “Prof. E. De Robertis”, Facultad de Medicina, Universidad de Buenos Aires, Paraguay 2155, (1121) Buenos Aires, Argentina b Cátedra de Farmacología, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, (1113) Buenos Aires, Argentina c IQUIFIB–CONICET, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, (1113) Buenos Aires, Argentina Received 20 July 2006; received in revised form 24 November 2006; accepted 1 December 2006 Available online 10 January 2007
Abstract Angiotensin (Ang)-(1–7) is an endogenous peptide hormone of the renin–angiotensin system which exerts diverse biological actions, some of them counterregulate Ang II effects. In the present study potential effect of Ang-(1–7) on phosphoinositide (PI) turnover was evaluated in neonatal rat brain. Cerebral cortex prisms of seven-day-old rats were preloaded with [3H]myoinositol, incubated with additions during 30 min and later [3H] inositol-phosphates (IPs) accumulation quantified. It was observed that PI hydrolysis enhanced 30% to 60% in the presence of 0.01 nM to 100 nM Ang-(1–7). Neither 10 nM [D-Ala7]Ang-(1–7), an Ang-(1–7) specific antagonist, nor 10 nM losartan, an angiotensin II type 1 (AT1) receptor antagonist, blocked the effect of 0.1 nM Ang-(1–7) on PI metabolism. The effect of 0.1 nM Ang-(1–7) on PI hydrolysis was not reduced but it was even significantly increased in the simultaneous presence of [D-Ala7]Ang-(1–7) or losartan. PI turnover enhancement achieved with 0.1 nM Ang-(1–7) decreased roughly 30% in the presence of 10 nM PD 123319, an angiotensin II type 2 (AT2) receptor antagonist. The antagonists alone also enhanced PI turnover. Present findings showing an increase in PI turnover by Ang-(1–7) represent a novel action for this peptide and suggest that it exerts a function in this signaling system in neonatal rat brain, an effect involving, at least partially, angiotensin AT2 receptors. © 2007 Elsevier B.V. All rights reserved. Keywords: Phosphoinositide turnover; Neonatal brain cortex; Angiotensin receptors; [D-Ala7]angiotensin-(1–7); Losartan; PD 123319
1. Introduction Angiotensin (Ang)-(1–7) is an important biologically active component of the renin–angiotensin system, formed by enzymatic processing of Ang I or Ang II. The heptapeptide exerts diverse biological actions, some of them similar but others markedly different from those displayed by Ang II [1]. For instance, it lacks the vasoconstrictor and aldosterone secretagogue [2] or dipsogenic [3] effects of Ang II, but mimics Ang II stimulation of vasopressin [4] and prostaglandin [5] release as well as peripheral norepinephrine outflow [6]. On the contrary, Ang-(1–7) causes natriuresis [7], diuresis [8] vasodi-
⁎ Corresponding author. Tel.: +54 11 4964 8292; fax: +54 11 4962 5457. E-mail address: [email protected] (C. Peña). 0167-0115/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2006.12.005
lation [9] and inhibits vascular smooth muscle cell growth [10], suggesting that in many cases, this peptide could act as an endogenous antagonist of Ang II. Our laboratory has reported among Ang-(1–7) actions, its ability to enhance phosphatidylcholine biosynthesis in the rat renal cortex through activation of Ang-(1–7) specific receptors [11]. Activation of several neurotransmitter (i.e. cholinergic, adrenergic, etc.) and/or neuropeptide receptors enhances phosphatidylinositol (PI) turnover in brain, an effect which proved higher in neonatal than in adult brain [12]. The involvement of high-affinity neurotensin receptor (NTS1) in PI turnover enhancement by cholinergic agonist carbachol was previously documented [13]. The aim of this study was to evaluate the potential effect of Ang-(1–7) on PI turnover in neonatal rat brain and to determine the possible involved receptors.
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2. Materials and methods 2.1. Chemicals Reagents were of analytical grade. Losartan was purchased from DuPont Merck (Wilmington, DE, USA); myoinositol and (1-[[4-(dimethylamino)-3-methylphenyl]methyl]-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazol[4,5c]pyridine-6-carboxylic acid ditrifluoroacetate) (PD 123319) were from SigmaAldrich (St. Louis, MO, USA). A Dowex anion exchange resin (AG 1-X8, 100–200 mesh, formate form) was from Bio-Rad Laboratories (Richmond, CA, USA). OptiPhase “Hisafe” 3 was purchased from Wallac Oy (Turku, Finland). [3H]myoinositol (20 Ci/mmol) was from New England Nuclear (Boston, MA, USA). Ang-(1–7) and [D-Ala7]Ang-(1–7) were synthesized in our laboratory by the Merrifield solid-phase procedure as described previously [11]. The purity of both peptides was verified by mass spectrometry. All other chemicals were of analytical grade and purchased from local suppliers. 2.2. Animals Wistar neonatal rats (6–7 days old) of either sex were used, considering “day 0” the day of birth. All studies described were performed in accordance with the Guide for Care and Use of Laboratory Animals provided by the National Institutes of Health, USA. 2.3. Determination of [3H]-inositol phosphates (IPs) A procedure based on described methods [14,15] with modifications, was performed using cerebral cortices of neonatal rats. For each experiment, tissues pooled from three rats were placed on ice on a Petri dish with gassed Krebs– Henseleit buffer containing (mM): NaCl, 120; KCl, 4.7; CaCl2, 1.3; KH2PO4, 1.2; MgSO4, 1.2; NaHCO3, 25 and glucose, 11.7, equilibrated to pH 7.4 with O2/CO2 (95:5). Tissue was lightly minced with a bistoury to obtain prisms of approximately 1 mm each dimension [16], suspended at 10% (w/v) in the same buffer and incubated in bulk at 37 °C for 1 h under gentle shaking with an intermediate change of buffer, followed by 60 min incubation with [3H]myoinositol (6 μCi/ml; final concentration 3 × 10− 7 M) and four washes with fresh buffer replaced every 5 min under O2/CO2 (95:5). Fifty microliters of packed prisms containing 1.0 ± 0.15 mg protein (mean values ± SEM, n = 10) was transferred to tubes with 0.24 ml of the same buffer which contained LiCl (7.5 mM final concentration, with NaCl isoosmotically reduced), and the indicated additions to 0.3 ml final volume. Prism samples preloaded with [3H]myoinositol were incubated for 30 min in the presence of 0.01 nM to 100 nM Ang-(1–7). To test the effect of antagonists, 0.1 nM Ang-(1–7) was employed in the presence or absence of [D-Ala 7 ]Ang(1–7), losartan or PD 123319 at 10 nM concentration. Incubation proceeded for 30 min under O2/CO2 (95:5) with shaking at 37 °C. Corresponding controls with redistilled water or antagonists alone were processed.
Fig. 1. The effect of Ang-(1–7) on [3H]inositol-phosphates accumulation in cerebral cortex prisms obtained from neonatal rats. Prisms preloaded with [3H] myoinositol were incubated in triplicate for 30 min in the presence of 0.01 nM to 100 nM Ang-(1–7) and IPs accumulation assayed. Samples were processed simultaneously (in triplicate) without additions to determine basal IPs level. Results are expressed as percentage of labeling taking as 100% values of basal IPs accumulation and are means (± SEM) of 5 experiments. ⁎ denotes P b 0.05 versus control without Ang-(1–7) (basal condition), by Student's t test.
Prism samples preloaded with [3H]myoinositol were processed throughout without additions (in triplicate) to determine basal IPs accumulation. Incubations were stopped by the addition of 940μl chloroform:methanol (1:2), followed by chloroform (310 μl) and redistilled water (310 μl) to separate phases. Tubes were vortexmixed for 15 s, then centrifuged at 1000×g for 10 min to facilitate phase separation. Radiolabeled IPs were separated from inositol by ion-exchange chromatography using small columns containing 0.5 ml of a 50% slurry AG 1-X8 resin in the formate form. Upper aqueous phase aliquots (750 μl) diluted to 3 ml with redistilled water were added to the resin suspension, centrifuged, and washed 4 times with 3 ml of 5 mM myoinositol. [3H]-IPs were eluted with 1 ml of 1 M ammonium formate/0.1 M formic acid and 800 μl of this eluate added to 10 ml of OptiPhase “Hisafe” 3 and counted in a Tracor Analytic scintillation spectrometer with 30% efficiency. In all experiments, aliquots containing the same amount of brain tissue (that is, similar intraexperimental protein content) were processed, which allowed the comparison between basal condition and treatment in the same sample. Results are expressed as [3H]-IPs accumulation taking as 100% basal value recorded in the absence of additions. The relatively long incubation (30 min) did not allow to measure the accumulation of the more polar hydrolysis products inositol-1,4-diphosphate and inositol-1,4,5-triphosphate, since changes in these compounds are detectable at very short times after addition of drugs to the incubation buffer [17]. However, since the concentration of ammonium formate used allows the recovery of all IPs, the term IPs instead of IP1 (inositol monophosphate) was used. 2.4. Protein measurement Protein concentration was evaluated by the method of Lowry et al. [18] using bovine serum albumin as standard.
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Fig. 2. IPs accumulation in cerebral cortex prisms obtained from neonatal rats in the presence of Ang-(1–7) and/or [D-Ala7]Ang-(1–7). Prisms preloaded with [3H]myoinositol were incubated in triplicate for 30 min in the presence of 0.1 nM Ang-(1–7) and/or 10 nM [D-Ala7]Ang-(1–7) and IPs accumulation assayed. Samples were processed simultaneously (in triplicate) without additions to determine basal IPs level. Results are expressed as percentage of labeling taking as 100% values of basal IPs accumulation and are means (±SEM) of 3–8 experiments. ⁎⁎P b 0.02, ⁎⁎⁎P b 0.01, by Student's t test.
Fig. 4. IPs accumulation in cerebral cortex prisms obtained from neonatal rats in the presence of Ang-(1–7) and/or PD 123319. Prisms preloaded with [3H] myoinositol were incubated in triplicate for 30 min in the presence of 0.1 nM Ang-(1–7) and/or 10 nM PD 123319 and IPs accumulation assayed. Samples were processed simultaneously (in triplicate) without additions to determine basal IPs level. Results are expressed as percentage of labeling taking as 100% values of basal IPs accumulation and are means (±SEM) of 3–4 experiments. ⁎P b 0.05,⁎⁎P b 0.02, by Student's t test.
2.5. Statistical analysis
protein (mean values ± SEM from 15 experiments). PI hydrolysis was assayed in the presence of 0.01 nM to 100 nM Ang(1–7) concentrations; it was observed 30 to 60% stimulation above the control (Fig. 1). In order to characterize the receptors by which this peptide promotes PI hydrolysis, experiments were carried out with 0.1 nM Ang-(1–7) plus 10 nM [D-Ala7]Ang-(1–7), losartan or PD 123319. IPs accumulation with 0.1 nM Ang-(1–7) was 161 ± 10.0%; with the simultaneous presence of 10 nM [D-Ala7]Ang(1–7), an (Ang)-(1–7) specific antagonist for various Ang-(1–7) effects [19], this value significantly increased to 213 ± 11.8%, a value which was significantly different from those of the control and the data recorded with Ang-(1–7) alone. The single presence of 10 nM [D-Ala7]Ang-(1–7) stimulated PI hydrolysis to 153 ± 3.0% (Fig. 2). IP accumulation reached 144 ± 12.4% with 0.1 nM Ang-(1– 7), a value which was not significantly modified in the simultaneous presence of 10 nM losartan, an angiotensin II type 1 receptor (AT1) antagonist (163 ± 12.8%). This antagonist alone was likewise able to significantly increase PI turnover (157 ± 17.3%) (Fig. 3). [3H]-IPs accumulation caused by 0.1 nM Ang-(1–7) (165 ± 7.1%) decreased approximately 30% in the presence of 10 nM PD 123319, an angiotensin II type 2 receptor (AT2) antagonist. It was observed that PD 123319 alone was also able to increase IPs accumulation to 131 ± 11.3% (Fig. 4).
All values are means ± SEM. Data were analyzed by Student's t-test. Probability values lower than 0.05 were considered significant. 3. Results In cerebral cortex prisms of neonatal rats basal [3H]-IPs accumulation in the aqueous phase was 2834 ± 584 dpm per mg
Fig. 3. IPs accumulation in cerebral cortex prisms obtained from neonatal rats in the presence of Ang-(1–7) and/or losartan. Prisms preloaded with [3H] myoinositol were incubated in triplicate for 30 min in the presence of 0.1 nM Ang-(1–7) and/or 10 nM losartan and IPs accumulation assayed. Samples were processed simultaneously (in triplicate) without additions to determine basal IPs level. Results are expressed as percentage of labeling taking as 100% values of basal IPs accumulation and are means (±SEM) of 4–6 experiments. ns: no significance, ⁎P b 0.05, ⁎⁎P b 0.02, by Student's t test.
4. Discussion The effect of peptide Ang-(1–7) on PI hydrolysis in neonatal rat brain prisms was studied. Results obtained show that Ang(1–7) significantly enhances PI turnover, an effect blocked by PD 123319, suggesting the involvement of AT2 receptors.
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Neither [D-Ala7]Ang-(1–7), which antagonizes biological actions of Ang-(1–7), nor losartan, which blocks AT1 receptors, was able to inhibit PI hydrolysis stimulation by Ang-(1–7). As far as we know, the ability of Ang-(1–7) to enhance PI turnover has not previously been described. On the contrary, no effect of Ang-(1–7) on PI hydrolysis was recorded in astrocyte culture from neonatal rat brain (one- to two-day-old pups) [20]. This discrepancy with present results is attributable to experimental differences, including rat strain and age, cell type employed (pure astrocytes or pieces of whole tissue which include neurons and astrocytes), peptide concentration, etc. [20,21]. It has been demonstrated that Ang-(1–7) increases in ovine plasma during fetal stage and circulates at much higher levels than those determined in the adult sheep [22]. After Ang-(1–7) infusion there is an up-regulation of gene expression for AT1 and AT2 receptors in the kidney of ovine fetuses [22], suggesting an opposing action of Ang-(1–7) to that of Ang II in the regulation of angiotensin receptors. In contrast, other studies indicate that Ang-(1–7) might oppose the action of Ang II through the downregulation or desensitisation of the AT1 receptors [23]. In present experiments, which were performed in brain tissue from neonatal rats (6–7 days old), Ang-(1–7) enhanced PI turnover, an effect which was not blocked by [D-Ala7] Ang-(1–7), a selective heptapeptide known to antagonize several biological actions of Ang-(1–7) [24–26]. Unexpectedly, [D-Ala7]Ang-(1–7) induced a response by itself, leading to PI turnover enhancement. The existence of a G protein-coupled receptor for Ang-(1–7) termed Mas, which is blocked by [D Ala7]Ang (1–7) has been described [27]. Present results indicated that Ang-(1–7)-elicited PI hydrolysis enhancement does not seem to be mediated by a [D-Ala7]Ang-(1–7) sensitive site, ruling out a direct interaction with the Mas receptor. Regarding other actions of Ang-(1–7), previous studies from our laboratory showed that it increases phosphatidylcholine biosynthesis, an effect not diminished by [D-Ala7]Ang-(1–7), which instead, exerts an agonist-like activity [11]. It was also reported that [D-Ala7]Ang-(1–7) failed to block completely the nitric oxide release induced by Ang-(1–7) in cultured bovine aortic endothelial cells [28]. It has been reported that [D-Ala7]Ang-(1–7) does not influence Ang-(1–7) inhibitory effects on noradrenaline release in rat kidney [29]. These findings led to the suggestion that whenever [D-Ala 7 ]Ang-(1–7) fails to prevent Ang-(1–7) actions, the Mas receptor is inactive or even unexpressed [30]. The biochemistry of the Mas receptor remains somewhat controversial, since this receptor is capable of interacting with Ang II receptors, may have independent signaling properties and may not depend on the binding of Ang-(1–7) for its actions [31]. The effects of AT1 and AT2 antagonists on Ang-(1–7) responses are not consistent, possibly due to organ and species differences in Ang II receptor subtype specificity [28]. Current data strongly suggest that an interaction between receptors for angiotensin peptides may play a role in biological effects of
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Ang-(1–7) [32,33]. Taken jointly, these findings may indicate that Ang-(1–7) interacts with multiple binding sites. As regards the relationship between Ang-(1–7) and AT1 receptor, it should be recalled that renal actions of Ang-(1–7) [8] and the release of [3H]norepinephrine from rat atria [6] are blocked by AT1 receptor antagonists. Furthermore, the suggestion that Ang-(1–7) may be an endogenous antagonist for AT1 receptor or that it may modulate the effect of Ang II via the AT1 receptor was advanced [34]. It has been demonstrated that Ang-(1–7) antagonizes Ang II-evoked vasoconstriction in human arteries by a non-competitive blockade of AT1 receptor [35]. In the kidney, Ang-(1–7) also acts as an antagonist of Ang II [36] and competes with high affinity for the losartan sensitive receptor [37]. Losartan was not able to block the enhancement of PI turnover by Ang-(1–7), suggesting that this effect is independent of AT1 receptor. However, losartan itself enhanced PI turnover and, coincubated with Ang-(1–7), induced an even higher IPs accumulation. For other angiotensin receptor antagonists, partial agonist action was likewise described [38]. Blockade of AT1 receptors is accompanied by elevation in both Ang I and Ang II levels, thus stimulating their conversion into Ang-(1–7) in incubated tissue [39]. Alternatively, the losartan-induced response is attributable either to the increased Ang-(1–7) level or to a drug specific effect. It should be recalled that losartan interacts with Ang-(1–7) receptors at cardiac level [40] and that losartan chronic hypotensive effects in normal rats are mediated in part through the actions of Ang-(1–7) [41]. In general, cell signaling pathways and physiological functions recorded for AT2 are unconventional [42,43]. Expression of AT2 receptors is highly dependent on age. In fetal tissues, the AT2 receptor is the dominating receptor subtype [44]. In the adult, healthy organism – no matter what species – AT2 receptors are only expressed in specific cell types and tissues such as vascular endothelial cells, distinct areas of the brain, the adrenal, selected cutaneous, renal and cardiac structures, myometrium and ovaries [45,46]. Most interestingly, in the adult, AT2 receptors are re-expressed under pathophysiological conditions such as mechanical injury [47] or ischemia [48]. Recent investigations have established a role for the AT2 receptor in cardiovascular, brain and renal function as well as in the modulation of several biological processes involved in development, cell differentiation, tissue repair and apoptosis [46]. The AT2 receptor antagonist PD 123319 caused a significant attenuation of the Ang-(1–7) stimulatory effect on PI turnover, while increasing PI hydrolysis by itself. Thus, we conclude that Ang-(1–7)-induced PI hydrolysis may reflect a combination of an effect at an AT2 receptor site that is being blocked by PD 123319 together with an action of Ang-(1–7) at another yet unidentified receptor. Another plausible hypothesis may imply a direct effect of Ang-(1–7) on the phosphoinositide phosphodiesterase system. To conclude, these results demonstrate that the peptide Ang(1–7) enhances PI metabolism in neonatal rat brain. This effect seems partly mediated by AT2 receptors but independent of AT1 receptors.
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