Rab Proteins: Insights into Intracellular Trafficking in Endometrium.

Rab proteins belong to the Ras superfamily of small monomeric GTPases. These G proteins are the main controllers of vesicular transport in every tissue, among them, the endometrium. They are in charge of to the functional subcellular compartmentalization and cargo transport between organelles and the plasma membrane. In turn, intracellular trafficking contributes to endometrial changes during the menstrual cycle, secretion to the uterine fluid, and trophoblast implantation; however, few reports analyze the role of Rab proteins in the uterus. In general, Rab proteins control the release of cytokines, growth factors, enzymes, hormones, cell adhesion molecules, and mucus. Further, the secretion of multiple compounds into the uterine cavity is required for successful implantation. Therefore, alterations in Rab-controlled intracellular transport likely impair secretory processes to the uterine fluid that may correlate with abnormal endometrial development and failed reproductive outcomes. Overall, they could explain recurrent miscarriages, female infertility, and/or assisted reproductive failure. Interestingly, estrogen (E2) and progesterone (P) regulate gene expression of Rab proteins involved in secretory pathways. This review aims to gather information regarding the role of Rab proteins and intracellular trafficking in the endometrium during the different menstrual phases, and in the generation of a receptive stage for embryo implantation, modulated by E2 and P. This knowledge might be useful for the development of novel reproductive therapies that overcome low implantation rates of assisted reproductive procedures.

Cardiac hypertrophy reduction in SHR by specific silencing of myocardial Na(+)/H(+) exchanger.

We examined the effect of specific and local silencing of sodium/hydrogen exchanger isoform 1 (NHE1) with a small hairpin RNA delivered by lentivirus (L-shNHE1) in the cardiac left ventricle (LV) wall of spontaneously hypertensive rats, to reduce cardiac hypertrophy. Thirty days after the lentivirus was injected, NHE1 protein expression was reduced 53.3 ± 3% in the LV of the L-shNHE1 compared with the control group injected with L-shSCR (NHE1 scrambled sequence), without affecting its expression in other organs, such as liver and lung. Hypertrophic parameters as LV weight-to-body weight and LV weight-to-tibia length ratio were significantly reduced in animals injected with L-shNHE1 (2.32 ± 0.5 and 19.30 ± 0.42 mg/mm, respectively) compared with L-shSCR-injected rats (2.68 ± 0.06 and 21.53 ± 0.64 mg/mm, respectively). Histochemical analysis demonstrated a reduction of cardiomyocytes cross-sectional area in animals treated with L-shNHE1 compared with L-shSCR (309,81 ± 20,86 vs. 424,52 ± 21 µm(2), P < 0.05). Echocardiography at the beginning and at the end of the treatment showed that shNHE1 expression for 30 days induced 9% reduction of LV mass. Also, animals treated with L-shNHE1 exhibited a reduced LV wall thickness without changing LV diastolic dimension and arterial pressure, indicating an increased parietal stress. In addition, midwall shortening was not modified, despite the increased wall tension, suggesting an improvement of cardiac function. Chronic shNHE1 expression in the heart emerges as a possible methodology to reduce pathological cardiac hypertrophy, avoiding potentially undesired effects caused from a body-wide inhibition of NHE1.

Endogenous endothelin 1 mediates angiotensin II-induced hypertrophy in electrically paced cardiac myocytes through EGFR transactivation, reactive oxygen species and NHE-1.

Emerging evidence supports a key role for endothelin-1 (ET-1) and the transactivation of the epidermal growth factor receptor (EGFR) in angiotensin II (Ang II) action. We aim to determine the potential role played by endogenous ET-1, EGFR transactivation and redox-dependent sodium hydrogen exchanger-1 (NHE-1) activation in the hypertrophic response to Ang II of cardiac myocytes. Electrically paced adult cat cardiomyocytes were placed in culture and stimulated with 1 nmol l(-1) Ang II or 5 nmol l(-1) ET-1. Ang II increased ~45 % cell surface area (CSA) and ~37 % [(3)H]-phenylalanine incorporation, effects that were blocked not only by losartan (Los) but also by BQ123 (AT1 and ETA receptor antagonists, respectively). Moreover, Ang II significantly increased ET-1 messenger RNA (mRNA) expression. ET-1 similarly increased myocyte CSA and protein synthesis, actions prevented by the reactive oxygen species scavenger MPG or the NHE-1 inhibitor cariporide (carip). ET-1 increased the phosphorylation of the redox-sensitive ERK1/2-p90(RSK) kinases, main activators of the NHE-1. This effect was prevented by MPG and the antagonist of EGFR, AG1478. Ang II, ET-1 and EGF increased myocardial superoxide production (187 ± 9 %, 149 ± 8 % and 163.7 ± 6 % of control, respectively) and AG1478 inhibited these effects. Interestingly, Los inhibited only Ang II whilst BQ123 cancelled both Ang II and ET-1 actions, supporting the sequential and unidirectional activation of AT1, ETA and EGFR. Based on the present evidence, we propose that endogenous ET-1 mediates the hypertrophic response to Ang II by a mechanism that involves EGFR transactivation and redox-dependent activation of the ERK1/2-p90(RSK) and NHE-1 in adult cardiomyocytes.

Myocardial mineralocorticoid receptor activation by stretching and its functional consequences.

Myocardial stretch triggers an angiotensin II-dependent autocrine/paracrine loop of intracellular signals, leading to reactive oxygen species-mediated activation of redox-sensitive kinases. Based on pharmacological strategies, we previously proposed that mineralocorticoid receptor (MR) is necessary for this stretch-triggered mechanism. Now, we aimed to test the role of MR after stretch by using a molecular approach to avoid secondary effects of pharmacological MR blockers. Small hairpin interference RNA capable of specifically knocking down the MR was incorporated into a lentiviral vector (l-shMR) and injected into the left ventricular wall of Wistar rats. The same vector but expressing a nonsilencing sequence (scramble) was used as control. Lentivirus propagation through the left ventricle was evidenced by confocal microscopy. Myocardial MR expression, stretch-triggered activation of redox-sensitive kinases (ERK1/2-p90(RSK)), the consequent Na(+)/H(+) exchanger-mediated changes in pHi (HEPES-buffer), and its mechanical counterpart, the slow force response, were evaluated. Furthermore, reactive oxygen species production in response to a low concentration of angiotensin II (1.0 nmol/L) or an equipotent concentration of epidermal growth factor (0.1 µg/mL) was compared in myocardial tissue slices from both groups. Compared with scramble, animals transduced with l-shMR showed (1) reduced cardiac MR expression, (2) cancellation of angiotensin II-induced reactive oxygen species production but preservation of epidermal growth factor-induced reactive oxygen species production, (3) cancellation of stretch-triggered increase in ERK1/2-p90(RSK) phosphorylation, (4) lack of stretch-induced Na(+)/H(+) exchanger activation, and (5) abolishment of the slow force response. Our results provide strong evidence that MR activation occurs after myocardial stretch and is a key factor to promote redox-sensitive kinase activation and their downstream consequences.

Mineralocorticoid receptor activation is crucial in the signalling pathway leading to the Anrep effect.

The increase in myocardial reactive oxygen species after epidermal growth factor receptor transactivation is a crucial step in the autocrine/paracrine angiotensin II/endothelin receptor activation leading to the slow force response to stretch (SFR). Since experimental evidence suggests a link between angiotensin II or its AT1 receptor and the mineralocorticoid receptor (MR), and MR transactivates the epidermal growth factor receptor, we thought to determine whether MR activation participates in the SFR development in rat myocardium. We show here that MR activation is necessary to promote reactive oxygen species formation by a physiological concentration of angiotensin II (1 nmol l(-1)), since an increase in superoxide anion formation of ~50% of basal was suppressed by blocking MR with spironolactone or eplerenone. This effect was also suppressed by blocking AT1, endothelin (type A) or epidermal growth factor receptors, by inhibiting NADPH oxydase or by targeting mitochondria, and was unaffected by glucocorticoid receptor inhibition. All interventions except AT1 receptor blockade blunted the increase in superoxide anion promoted by an equipotent dose of endothelin-1 (1 nmol l(-1)) confirming that endothelin receptors activation is downstream of AT1. Similarly, an increase in superoxide anion promoted by an equipotent dose of aldosterone (10 nmol l(-1)) was blocked by spironolactone or eplerenone, by preventing epidermal growth factor receptor transactivation, but not by inhibiting glucocorticoid receptors or protein synthesis, suggesting non-genomic MR effects. Combination of aldosterone plus endothelin-1 did not increase superoxide anion formation more than each agonist separately. We found that aldosterone increased phosphorylation of the redox-sensitive kinases ERK1/2-p90RSK and the NHE-1, effects that were eliminated by eplerenone or by preventing epidermal growth factor receptor transactivation. Finally, we provide evidence that the SFR is suppressed by MR blockade, by preventing epidermal growth factor receptor transactivation or by scavenging reactive oxygen species, but it is unaffected by glucocorticoid receptor blockade or protein synthesis inhibition. Our results suggest that MR activation is a necessary step in the stretch-triggered reactive oxygen species-mediated activation of redox-sensitive kinases upstream NHE-1.

Aldosterone stimulates the cardiac Na(+)/H(+) exchanger via transactivation of the epidermal growth factor receptor.

The use of antagonists of the mineralocorticoid receptor in the treatment of myocardial hypertrophy and heart failure has gained increasing importance in the last years. The cardiac Na(+)/H(+) exchanger (NHE-1) upregulation induced by aldosterone could account for the genesis of these pathologies. We tested whether aldosterone-induced NHE-1 stimulation involves the transactivation of the epidermal growth factor receptor (EGFR). Rat ventricular myocytes were used to measure intracellular pH with epifluorescence. Aldosterone enhanced the NHE-1 activity. This effect was canceled by spironolactone or eplerenone (mineralocorticoid receptor antagonists), but not by mifepristone (glucocorticoid receptor antagonist) or cycloheximide (protein synthesis inhibitor), indicating that the mechanism is mediated by the mineralocorticoid receptor triggering nongenomic pathways. Aldosterone-induced NHE-1 stimulation was abolished by the EGFR kinase inhibitor AG1478, suggesting that is mediated by transactivation of EGFR. The increase in the phosphorylation level of the kinase p90(RSK) and NHE-1 serine703 induced by aldosterone was also blocked by AG1478. Exogenous epidermal growth factor mimicked the effects of aldosterone on NHE-1 activity. Epidermal growth factor was also able to increase reactive oxygen species production, and the epidermal growth factor-induced activation of the NHE-1 was abrogated by the reactive oxygen species scavenger N-2-mercaptopropionyl glycine, indicating that reactive oxygen species are participating as signaling molecules in this mechanism. Aldosterone enhances the NHE-1 activity via transactivation of the EGFR, formation of reactive oxygen species, and phosphorylation of the exchanger. These results call attention to the consideration of the EGFR as a new potential therapeutic target of the cardiovascular pathologies involving the participation of aldosterone.

Silencing of NHE-1 blunts the slow force response to myocardial stretch.

Myocardial stretch induces a biphasic force response: a first abrupt increase followed by a slow force response (SFR), believed to be the in vitro manifestation of the Anrep effect. The SFR is due to an increase in Ca²âº transient of unclear mechanism. We proposed that Na⁺/H⁺ exchanger (NHE-1) activation is a key factor in determining the contractile response, but recent reports challenged our findings. We aimed to specifically test the role of the NHE-1 in the SFR. To this purpose small hairpin interference RNA capable of mediating specific NHE-1 knockdown was incorporated into a lentiviral vector (l-shNHE1) and injected into the left ventricular wall of Wistar rats. Injection of a lentiviral vector expressing a nonsilencing sequence (scramble) served as control. Myocardial NHE-1 protein expression and function (the latter evaluated by the recovery of pH(i) after an acidic load and the SFR) were evaluated. Animals transduced with l-shNHE1 showed reduced NHE-1 expression (45 ± 8% of controls; P < 0.05), and the presence of the lentivirus in the left ventricular myocardium, far from the site of injection, was evidenced by confocal microscopy. These findings correlated with depressed basal pH(i) recovery after acidosis [(max)dpH(i)/dt 0.055 ± 0.008 (scramble) vs. 0.009 ± 0.004 (l-shNHE1) pH units/min, P < 0.05], leftward shift of the relationship between J(H⁺) (H⁺ efflux corrected by the intrinsic buffer capacity), and abolishment of SFR (124 ± 2 vs. 101 ± 2% of rapid phase; P < 0.05) despite preserved ERK1/2 phosphorylation [247 ± 12 (stretch) and 263 ± 23 (stretch l-shNHE1) % of control; P < 0.05 vs. nonstretched control], well-known NHE-1 activators. Our results provide strong evidence to propose NHE-1 activation as key factor in determining the SFR to stretch.

Phosphodiesterase 5A inhibition decreases NHE-1 activity without altering steady state pH(i): role of phosphatases.

BACKGROUND/AIMS: This study aimed to identify the signaling pathway for the proposed link between phosphodiesterase-5A (PDE5A) inhibition and decreased cardiac Na(+)/H(+) exchanger (NHE-1) activity. METHODS: NHE-1 activity was assessed in rat isolated papillary muscles by the Na(+)-dependent initial pH(i) recovery from a sustained acidosis (ammonium prepulse). ERK1/2, p90RSK and NHE-1 phosphorylation state during acidosis was determined. RESULTS: PDE5A inhibition (1 µmol/L sildenafil, SIL) did not modify basal pH(i) but significantly blunted pH(i) recovery after sustained acidosis. Although preventing ERK1/2- p90RSK signaling pathway (10 µmol/L U0126) mimicked SIL effect, SIL did not blunt the acidosis-mediated increase in kinases activation. SIL+U0126 did not show additive effect on NHE-1 activity. Then, we hypothesized that SIL could be activating phophasatases (PP1 and/or PP2A) to directly dephosphorylate NHE-1 despite preserved ERK1/2-p90RSK activation. Non-specific phosphatases inhibition (1 µmol/L okadaic acid) canceled SIL effect on pH(i) recovery from acidosis. Same result was observed by inhibiting PP2A either with a lower dose of okadaic acid (1 nmol/L) or, more specifically, with 100 µmol/L endothall. Consistently, NHE-1 phosphorylation at Ser703 increased after acidosis, SIL prevented this effect and PP2A inhibition (endothall) reverted SIL effect. CONCLUSION: We suggest that PDE5A inhibitors decrease NHE-1 phosphorylation and activity through a mechanism that involves PP2A activation.

Decreased activity of the Na+/H+ exchanger by phosphodiesterase 5A inhibition is attributed to an increase in protein phosphatase activity.

The beneficial effect of phosphodiesterase 5A inhibition in ischemia/reperfusion injury and cardiac hypertrophy is well established. Inhibition of the cardiac Na(+)/H(+) exchanger (NHE-1) exerts beneficial effects on these same conditions, and a possible link between these therapeutic strategies was suggested. Experiments were performed in isolated cat cardiomyocytes to gain insight into the intracellular pathway involved in the reduction of NHE-1 activity by phosphodiesterase 5A inhibition. NHE-1 activity was assessed by the rate of intracellular pH recovery from a sustained acidic load in the absence of bicarbonate. Phosphodiesterase 5A inhibition with sildenafil (1 µmol/L) did not affect basal intracellular pH; yet, it did decrease proton efflux (J(H); in millimoles per liter per minute) after the acidic load (proton efflux: 6.97±0.43 in control versus 3.31±0.58 with sildenafil; P<0.05). The blockade of both protein phosphatase 1 and 2A with 100 nmol/L of okadaic acid reverted the sildenafil effect (proton efflux: 6.77±0.82). In contrast, selective inhibition of protein phosphatase 2A (1 nmol/L of okadaic acid or 100 µmol/L of endothall) did not (3.86±1.0 and 2.61±1.2), suggesting that only protein phosphatase 1 was involved in sildenafil-induced NHE-1 inhibition. Moreover, sildenafil prevented the acidosis-induced increase in NHE-1 phosphorylation without affecting activation of the extracellular signal-regulated kinase 1/2-p90(RSK) pathway. Our results suggest that phosphodiesterase 5A inhibition decreases NHE-1 activity, during intracellular pH recovery after an acidic load, by a protein phosphatase 1-dependent reduction in NHE-1 phosphorylation.

The Anrep effect requires transactivation of the epidermal growth factor receptor.

Myocardial stretch elicits a biphasic contractile response: the Frank-Starling mechanism followed by the slow force response (SFR) or Anrep effect. In this study we hypothesized that the SFR depends on epidermal growth factor receptor (EGFR) transactivation after the myocardial stretch-induced angiotensin II (Ang II)/endothelin (ET) release. Experiments were performed in isolated cat papillary muscles stretched from 92 to 98% of the length at which maximal twitch force was developed (L(max)). The SFR was 123 +/- 1% of the immediate rapid phase (n = 6, P < 0.05) and was blunted by preventing EGFR transactivation with the Src-kinase inhibitor PP1 (99 +/- 2%, n = 4), matrix metalloproteinase inhibitor MMPI (108 +/- 4%, n = 11), the EGFR blocker AG1478 (98 +/- 2%, n = 6) or the mitochondrial transition pore blocker clyclosporine (99 +/- 3%, n = 6). Stretch increased ERK1/2 phosphorylation by 196 +/- 17% of control (n = 7, P < 0.05), an effect that was prevented by PP1 (124 +/- 22%, n = 7) and AG1478 (131 +/- 17%, n = 4). In myocardial slices, Ang II (which enhances ET mRNA) or endothelin-1 (ET-1)-induced increase in O(2)() production (146 +/- 14%, n = 9, and 191 +/- 17%, n = 13, of control, respectively, P < 0.05) was cancelled by AG1478 (94 +/- 5%, n = 12, and 98 +/- 15%, n = 8, respectively) or PP1 (100 +/- 4%, n = 6, and 99 +/- 8%, n = 3, respectively). EGF increased O(2)() production by 149 +/- 4% of control (n = 9, P < 0.05), an effect cancelled by inhibiting NADPH oxidase with apocynin (110 +/- 6% n = 7), mKATP channels with 5-hydroxydecanoic acid (5-HD; 105 +/- 5%, n = 8), the respiratory chain with rotenone (110 +/- 7%, n = 7) or the mitochondrial permeability transition pore with cyclosporine (111 +/- 10%, n = 6). EGF increased ERK1/2 phosphorylation (136 +/- 8% of control, n = 9, P < 0.05), which was blunted by 5-HD (97 +/- 5%, n = 4), suggesting that ERK1/2 activation is downstream of mitochondrial oxidative stress. Finally, stretch increased Ser703 Na(+)/H(+) exchanger-1 (NHE-1) phosphorylation by 172 +/- 24% of control (n = 4, P < 0.05), an effect that was cancelled by AG1478 (94 +/- 17%, n = 4). In conclusion, our data show for the first time that EGFR transactivation is crucial in the chain of events leading to the Anrep effect.

Endurance training in the spontaneously hypertensive rat: conversion of pathological into physiological cardiac hypertrophy.

The effect of endurance training (swimming 90 min/d for 5 days a week for 60 days) on cardiac hypertrophy was investigated in the spontaneously hypertensive rat (SHR). Sedentary SHRs (SHR-Cs) and normotensive Wistar rats were used as controls. Exercise training enhanced myocardial hypertrophy assessed by left ventricular weight/tibial length (228+/-7 versus 251+/-5 mg/cm in SHR-Cs and exercised SHRs [SHR-Es], respectively). Myocyte cross-sectional area increased approximately 40%, collagen volume fraction decreased approximately 50%, and capillary density increased approximately 45% in SHR-Es compared with SHR-Cs. The mRNA abundance of atrial natriuretic factor and myosin light chain 2 was decreased by the swimming routine (100+/-19% versus 41+/-10% and 100+/-8% versus 61+/-9% for atrial natriuretic factor and myosin light chain 2 in SHR-Cs and SHR-Es, respectively). The expression of sarcoplasmic reticulum Ca(2+) pump was significantly augmented, whereas that of Na(+)/Ca(2+) exchanger was unchanged (93+/-7% versus 167+/-8% and 158+/-13% versus 157+/-7%, sarcoplasmic reticulum Ca(2+) pump and Na(+)/Ca(2+) exchanger in SHR-Cs and SHR-Es, respectively; P<0.05). Endurance training inhibited apoptosis, as reflected by a decrease in caspase 3 activation and poly(ADP-ribose) polymerase-1 cleavage, and normalized calcineurin activity without inducing significant changes in the phosphatidylinositol 3-kinase/Akt pathway. The swimming routine improved midventricular shortening determined by echocardiography (32.4+/-0.9% versus 36.9+/-1.1% in SHR-Cs and SHR-Es, respectively; P<0.05) and decreased the left ventricular free wall thickness/left ventricular cavity radius toward an eccentric model of cardiac hypertrophy (0.59+/-0.02 versus 0.53+/-0.01 in SHR-Cs and SHR-Es, respectively; P<0.05). In conclusion, we present data demonstrating the effectiveness of endurance training to convert pathological into physiological hypertrophy improving cardiac performance. The reduction of myocardial fibrosis and calcineurin activity plus the increase in capillary density represent factors to be considered in determining this beneficial effect.

Early signals after stretch leading to cardiac hypertrophy. Key role of NHE-1.

The enhanced activity of the cardiac Na+/H+ exchanger (NHE-1) after myocardial stretch is considered a key step of the intracellular signaling pathway leading to the slow force response to stretch as well as an early signal for the development of cardiac hypertrophy. We propose that the chain of events triggered by stretch begins with the release of small amounts of Angiotensin II (Ang II)/endothelin (ET) and ends with the increase in intracellular Ca2+ concentration ([Ca2+]i) through the Na+/Ca2+ exchanger in reverse mode (NCX(rev)), which triggers cardiac hypertrophy by activation of widely recognized Ca2+-dependent intracellular signaling pathways.