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.

Myocardial reperfusion injury: reactive oxygen species vs. NHE-1 reactivation.

BACKGROUND/AIMS: Flow restoration to ischemic myocardium reduces infarct size (IS), but it also promotes reperfusion injury. A burst of reactive oxygen species (ROS) and/or NHE-1 reactivation were proposed to explain this injury. Our study was aimed to shed light on this unresolved issue. METHODS: Regional infarction (40 min-ischemia/2 hs-reperfusion) was induced in isolated and perfused rat hearts. Maximal doses of N-(2-mercaptopropionyl)-glycine (MPG 2mmol/L, ROS scavenger), cariporide (10µmol/L, NHE-1 inhibitor), or sildenafil (1µmol/L, phosphodiesterase5A inhibitor) were applied at reperfusion onset. Their effects on IS, myocardial concentration of thiobarbituric acid reactive substances (TBARS), ERK1/2, p90(RSK), and NHE-1 phosphorylation were analyzed. RESULTS: All treatments decreased IS ∼ 50% vs. control. No further protection was obtained by combining cariporide or MPG with sildenafil. Myocardial TBARS increased after infarction and were decreased by MPG or cariporide, but unaffected by sildenafil. In line with the fact that ROS induce MAPK-mediated NHE-1 activation, myocardial infarction increased ERK1/2, p90(RSK), and NHE-1 phosphorylation. MPG and cariporide cancelled these effects. Sildenafil did not reduce the phosphorylated ERK1/2-p90(RSK) levels but blunted NHE-1 phosphorylation suggesting a direct dephosphorylating action. CONCLUSIONS: 1) Reperfusion injury would result from ROS-triggered MAPK-mediated NHE-1 phosphorylation (and reactivation) during reperfusion; 2) sildenafil protects the myocardium by favouring NHE-1 dephosphorylation and bypassing ROS generation.

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.

Chronic NHE-1 blockade induces an antiapoptotic effect in the hypertrophied heart.

Na(+)/H(+) exchanger (NHE-1) inhibition was demonstrated to induce the regression of cardiac hypertrophy (CH) in several experimental models and to inhibit mitochondrial death pathway in "in-vitro" experiments. Since recent reports show that NHE-1 inhibition delays the transition from CH to failure, and apoptosis plays a key role in this process, we investigated the effect of chronic treatment with the NHE-1 blocker cariporide on CH and apoptosis in the SHR. One month of cariporide treatment (30 mg x kg(-1) x day(-1)) induced the regression of CH (cardiomyocyte cross-sectional area: 468 +/- 20 vs. 285 +/- 9 microm(2) in untreated and cariporide-treated spontaneously hypertensive rats; P < 0.05). Apoptosis was assessed by TUNEL staining, the expression of Bcl-2, Bax, and activation of caspase-3 and PARP-1 by immunoblot. Cariporide treatment decreased the TUNEL-positive cells, the Bax-to-Bcl-2 ratio (3.16 +/- 0.32 vs. 1.70 +/- 0.17, untreated and cariporide-treated, respectively; P < 0.05); caspase-3 and PARP-1 activation (465 +/- 62 vs. 260 +/- 22 and 2,239 +/- 62 vs. 1,683 +/- 85 AU, untreated and cariporide-treated, respectively; P < 0.05). Angiotensin II, a growth factor and apoptotic stimulus, was used to induce O(2)(-) production that activated the ERK1/2-p90(RSK) pathway, increasing NHE-1 phosphorylation. These effects were prevented by losartan, N-(2-mercaptopropionyl)-glycine, and cariporide. In conclusion, we present data demonstrating that chronic NHE-1 inhibition with cariporide decreases both hypertrophy and apoptosis susceptibility in the spontaneously hypertensive rat heart. The antiapoptotic effect would be the consequence of two different actions of cariporide: the prevention of cytosolic Na(+) and Ca(2+) overload due to the inhibition of the sarcolemmal NHE-1 and a direct mitochondrial effect preventing mitochondrial permeability transition pore opening.

Is cardiac hypertrophy in spontaneously hypertensive rats the cause or the consequence of oxidative stress?

The aim of this work was to assess the possible correlation between oxidative damage and the development of cardiac hypertrophy in heart tissue from young (40-d-old) and older (4-, 11- and 19-month-old) spontaneously hypertensive rats (SHR) in comparison with age-matched Wistar (W) rats. To this end, levels of thiobarbituric acid reactive substances (TBARS), nitrotyrosine contents, NAD(P)H oxidase activity, superoxide production, and the activities of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) were determined. Compared to age-matched normotensive rats, SHR showed a significant increase in systolic blood pressure from 40 d of age and left ventricular hypertrophy (LVH) was significantly evident from 4 months of age. W rats (11- and 19-month-old) also showed an increase in LVH with aging. TBARS and nitrotyrosine levels were similar in young rats from both strains and were significantly increased with age in both strains, with the values in SHR being significantly higher than those in age-matched W rats. NAD(P)H activity was similar in young SHR and W rats, whereas it was higher in aged SHR compared with age-matched W rats. Compared to W rats, superoxide production was higher in aged SHR, and was abolished by NAD(P)H inhibition with apocynin. CAT activity was increased in the hearts of 4-month-old SHR compared to age-matched W rats and was decreased in the hearts of the oldest SHR compared to the oldest W rats. SOD and GPx activities decreased in both rat strains with aging. Moreover, an increase in collagen deposition with aging was evident in both rat strains. Taken together, these data showed that aged SHR exhibited higher cardiac hypertrophy and oxidative damage compared to W rats, indicating that the two undesirable effects are associated. That is, oxidative stress appears to be a cause and/or consequence of hypertrophy development in this animal model.

Na+/H+ exchanger-1 inhibitors decrease myocardial superoxide production via direct mitochondrial action.

The possibility of a direct mitochondrial action of Na(+)/H(+) exchanger-1 (NHE-1) inhibitors decreasing reactive oxygen species (ROS) production was assessed in cat myocardium. Angiotensin II and endothelin-1 induced an NADPH oxidase (NOX)-dependent increase in anion superoxide (O(2)(-)) production detected by chemiluminescence. Three different NHE-1 inhibitors [cariporide, BIIB-723, and EMD-87580] with no ROS scavenger activity prevented this increase. The mitochondria appeared to be the source of the NOX-dependent ROS released by the "ROS-induced ROS release mechanism" that was blunted by the mitochondrial ATP-sensitive potassium channel blockers 5-hydroxydecanoate and glibenclamide, inhibition of complex I of the electron transport chain with rotenone, and inhibition of the permeability transition pore (MPTP) by cyclosporin A. Cariporide also prevented O(2)(-) production induced by the opening of mK(ATP) with diazoxide. Ca(2+)-induced swelling was evaluated in isolated mitochondria as an indicator of MPTP formation. Cariporide decreased mitochondrial swelling to the same extent as cyclosporin A and bongkrekic acid, confirming its direct mitochondrial action. Increased O(2)(-) production, as expected, stimulated ERK1/2 and p90 ribosomal S6 kinase phosphorylation. This was also prevented by cariporide, giving additional support to the existence of a direct mitochondrial action of NHE-1 inhibitors in preventing ROS release. In conclusion, we report a mitochondrial action of NHE-1 inhibitors that should lead us to revisit or reinterpret previous landmark observations about their beneficial effect in several cardiac diseases, such as ischemia-reperfusion injury and cardiac hypertrophy and failure. Further studies are needed to clarify the precise mechanism and site of action of these drugs in blunting MPTP formation and ROS release.

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.

Mitochondrial reactive oxygen species activate the slow force response to stretch in feline myocardium.

When the length of the myocardium is increased, a biphasic response to stretch occurs involving an initial rapid increase in force followed by a delayed slow increase called the slow force response (SFR). Confirming previous findings involving angiotensin II in the SFR, it was blunted by AT1 receptor blockade (losartan). The SFR was accompanied by an increase in reactive oxygen species (ROS) of approximately 30% and in intracellular Na(+) concentration ([Na(+)](i)) of approximately 2.5 mmol l(-1) over basal detected by H(2)DCFDA and SBFI fluorescence, respectively. Abolition of ROS by 2-mercapto-propionyl-glycine (MPG) and EUK8 suppressed the increase in [Na(+)](i) and the SFR, which were also blunted by Na(+)/H(+) exchanger (NHE-1) inhibition (HOE642). NADPH oxidase inhibition (apocynin or DPI) or blockade of the ATP-sensitive mitochondrial potassium channels (5HD or glybenclamide) suppressed both the SFR and the increase in [Na(+)](i) after stretch, suggesting that endogenous angiotensin II activated NADPH oxidase leading to ROS release by the ATP-sensitive mitochondrial potassium channels, which promoted NHE-1 activation. Supporting the notion of ROS-mediated NHE-1 activation, stretch increased the ERK1/2 and p90rsk kinases phosphorylation, effect that was cancelled by losartan. In agreement, the SFR was cancelled by inhibiting the ERK1/2 signalling pathway with PD98059. Angiotensin II at a dose that mimics the SFR (1 nmol l(-1)) induced an increase in .O(2)(-) production of approximately 30-40% detected by lucigenin in cardiac slices, an effect that was blunted by losartan, MPG, apocynin, 5HD and glybenclamide. Taken together the data suggest a pivotal role of mitochondrial ROS in the genesis of the SFR to stretch.

Phosphodiesterase 5A inhibition induces Na+/H+ exchanger blockade and protection against myocardial infarction.

Acute phosphodiesterase 5A inhibition by sildenafil or EMD360527/5 promoted profound inhibition of the cardiac Na(+)/H(+) exchanger (NHE-1), detected by the almost null intracellular pH recovery from an acute acid load (ammonium prepulse) in isolated papillary muscles from Wistar rats. Inhibition of phosphoglycerate kinase-1 (KT5823) restored normal NHE-1 activity, suggesting a causal link between phosphoglycerate kinase-1 increase and NHE-1 inhibition. We then tested whether the beneficial effects of NHE-1 inhibitors against the deleterious postmyocardial infarction (MI) remodeling can be detected after sildenafil-mediated NHE-1 inhibition. MI was induced by left anterior descending coronary artery ligation in Wistar rats, which were randomized to placebo or sildenafil (100 mg kg(-1) day(-1)) for 6 weeks. Sildenafil significantly increased left ventricular phosphoglycerate kinase-1 activity in the post-MI group without affecting its expression. MI increased heart weight/body weight ratio, left ventricular myocyte cross-sectional area, interstitial fibrosis, and brain natriuretic peptide and NHE-1 expression. Sildenafil blunted these effects. Neither a significant change in infarct size nor a change in arterial or left ventricular systolic pressure was detected after sildenafil. MI decreased fractional shortening and the ratio of the maximum rate of rise of LVP divided by the pressure at the moment such maximum occurs, effects that were prevented by sildenafil. Intracellular pH recovery after an acid load was faster in papillary muscles from post-MI hearts (versus sham), whereas sildenafil significantly inhibited NHE-1 activity in both post-MI and sildenafil-treated sham groups. We conclude that increased phosphoglycerate kinase-1 activity after acute phosphodiesterase 5A inhibition blunts NHE-1 activity and protects the heart against post-MI remodeling and dysfunction.

Endothelin-1 induced hypertrophic effect in neonatal rat cardiomyocytes: involvement of Na+/H+ and Na+/Ca2+ exchangers.

Endothelin-1 (ET-1) is a potent agonist of cell growth that also stimulates Na(+)/H(+) exchanger isoform 1 (NHE-1) activity. It was hypothesized that the increase in intracellular Na(+) ([Na(+)](i)) mediated by NHE-1 activity may induce the reverse mode of Na(+)/Ca(2+) exchanger (NCX(rev)) increasing intracellular Ca(2+) ([Ca(2+)](i)) which in turn will induce hypertrophy. The objective of this work was to test whether the inhibition of NHE-1 or NCX(rev) prevents ET-1 induced hypertrophy in neonatal rat cardiomyocytes (NRVMs). NRVMs were cultured (24 h) in the absence (control) and presence of 5 nmol/L ET-1 alone, or combined with 1 mumol/L HOE 642 or 5 mumol/L KB-R7943. Cell surface area, (3)H-phenylalanine incorporation and atrial natriuretic factor (ANF) mRNA expression were increased to 131 +/- 3, 220 +/- 12 and 190 +/- 25% of control, respectively (P < 0.05) by ET-1. [Na(+)](i) and total [Ca(2+)](i) were higher (8.1 +/- 1.2 mmol/L and 636 +/- 117 nmol/L, respectively) in ET-1-treated than in control NRVMs (4.2 +/- 1.3 and 346 +/- 85, respectively, P < 0.05), effects that were cancelled by NHE-1 inhibition with HOE 642. The rise in [Ca(2+)](i) induced by extracellular Na(+) removal (NCX(rev)) was higher in ET-1-treated than in control NRVMs and the effect was prevented by co-treatment with HOE 642 or KB-R7943 (NCX(rev) inhibitor). The ET-1-induced increase in cell area, ANF mRNA expression and (3)H-phenylalanine incorporation in ET-1-treated NRVM were decreased by NHE-1 or NCX(rev) inhibition. Our results provide the first evidence that NCX(rev) is, secondarily to NHE-1 activation, involved in ET-1-induced hypertrophy in NRVMs.

Involvement of AE3 isoform of Na(+)-independent Cl(-)/HCO(3)(-) exchanger in myocardial pH(i) recovery from intracellular alkalization.

Myocardial pH(i) recovery from intracellular alkalization results in part from the acid load (-J(H+)) carried by Cl(-)/HCO(3)(-) anion-exchangers (AE). Three AE isoforms, AE1, AE2 and AE3, have been identified in cardiac membranes, but the function of each isoform on pH(i) homeostasis is still under investigation. This work explored, by means of specific antibodies, the role of AE3 isoform in myocardial pH(i) regulation. We developed rabbit polyclonal antibodies against the extracellular "loops": one connecting the fifth to sixth and the other one the seventh to eighth transmembrane domains (loops 3 and 4, respectively) of AE3, and their effect on pH(i) regulation was studied in rat papillary muscles. The anti-AE3 loop 3 antibody decreased -J(H+) in response to myocardial alkalization (from a mean control value of 1.06+/-0.26 to 0.32+/-0.13 mmol/L/min, n=7, P<0.05) without affecting the baseline pH(i) (7.22+/-0.03 vs. 7.21+/-0.04). The anti-AE3 loop 4 antibody did not modify either pH(i) recovery or baseline pH(i). Under control conditions, endothelin-1 (ET-1) increased -J(H+) in response to myocardial alkalization from 1.30+/-0.18 to 2.01+/-0.33 mmol/L /min (n=5, P<0.05). This effect of ET-1 on -J(H+) was abolished by anti-AE3 loop 3 antibody. In addition, the MgATP-induced stimulation of AE activity was reduced by the anti-AE3 loop 3 antibody. These data support the key role of the AE3 isoform in myocardial pH(i) recovery from alkaline loads and also in the stimulatory effect of ET-1 on AE activity. To a lesser extent, it may also contribute to the effect of MgATP on pH(i).

Involvement of the Na+-independent Cl-/HCO3- exchange (AE) isoform in the compensation of myocardial Na+/H+ isoform 1 hyperactivity in spontaneously hypertensive rats.

Enhanced activity of Na+/H+ isoform 1 (NHE-1) and the Na+-independent Cl-/HCO3- exchange (AE) is a feature of the hypertrophied myocardium in spontaneously hypertensive rats (SHR). The present study explored the possibility that sustained intracellular acidosis due to increased myocardial acid loading through AE causes NHE-1 enhancement. To this aim, SHR were treated for 2 weeks with a rabbit polyclonal antibody against an AE3 isoform that was recently developed and proven to have inhibitory effects on myocardial AE activity. We then compared the AE activity in the left ventricle papillary muscles isolated from untreated SHR with antiAE3-treated SHR; AE activity was measured in terms of the rate of intracellular pH recovery after an intracellular alkali load was introduced. AE activity was diminished by approximately 70% in SHR treated with the antiAE3 antibody, suggesting that the AE3 isoform is a major carrier of acid-equivalent influx in the hypertrophied myocardium. However, the antibody treatment failed to normalize NHE-1 activity that remained elevated in the myocardium of normotensive rats. The data therefore rule out the possibility that NHE-1 hyperactivity in hypertensive myocardium was due to sustained intracellular acidosis induced by increased AE activity that characterizes SHR myocardial tissue.

Insulin resistance and GLUT-4 glucose transporter in adipocytes from hypertensive rats.

To investigate the mechanisms that cause insulin resistance in hypertension, experiments were performed to study the effect of insulin on glucose transport, GLUT-4 translocation from intracellular to plasma membranes and GLUT-4 phosphorylation in isolated adipocytes from normotensive Wistar (W) and spontaneously hypertensive rats (SHR). Glucose transport was measured in adipocytes incubated with 3-O-d[Methyl-(3)H] glucose with and without insulin (0.1 to 5 nmol/L). GLUT-4 protein was determined by Western blot immunoanalysis with GLUT-4 antibody. Phosphorylation of GLUT-4 was measured by immunoprecipitation with GLUT-4 antibody followed by immunoanalysis with phosphoserine or phosphothreonine antibodies. Compared with adipocytes from W, insulin-stimulated glucose transport was lower in the SHR (P <.05). GLUT-4 protein expression was similar in adipocytes from W and SHR. Insulin increased GLUT-4 translocation from intracellular to plasma membranes in both groups. This effect was lower in the SHR (P <.05). The effect of insulin on GLUT-4 serine phosphorylation showed no changes in plasma membranes from W and decreased in the SHR (P <.05). In intracellular membranes, insulin increased specific GLUT-4 serine phosphorylation in both groups (P <.05), but the increase was lower in the SHR (P <.05). The results suggest that a deficient GLUT-4 translocation to plasma membranes in response to insulin shown in adipocytes from SHR, which was accompanied by a decrease in GLUT-4 phosphorylation at serine site, could be one of the causes of insulin resistance in hypertension.

Upregulation of myocardial Na+/H+ exchanger induced by chronic treatment with a selective inhibitor.

Rats exposed to prolonged administration of the NHE-1 inhibitor cariporide showed enhanced activity of the exchanger in cardiac tissue, as assessed by the rise in the steady-state pHi value in the absence of bicarbonate (7.15+/-0.01 in control vs 7.49+/-0.06 and 7.41+/-0.05 in cariporide-treated for 1 or 2 months, respectively, P<0.05). In the presence of bicarbonate, the change in pHi was blunted due to a compensatory activation of acid loading pHi regulatory mechanisms. The enhancement of NHE activity disappeared after 1 week of the inhibitor withdrawal. The kinetic analysis of H+ fluxes after an acid load revealed an increased net H+ efflux (JH+) at any given pHi value and an alkaline shift of the apparent "set-point" of the exchanger (from 7.11+/-0.02 to 7.38+/-0.04,P <0.05) in treated rats. In the presence of the PKC inhibitor chelerythrine, the "set-point" of the exchanger was normalized in the cariporide-treated rats while JH+ at acidic pHi values persisted elevated. Cardiac NHE-1 mRNA levels and protein expression were increased in cariporide-treated rats. In addition to the increased protein expression after the treatment, the normalization of the augmented "set-point" by chelerythrine suggests an increased turnover rate of the units through a PKC dependent pathway. These data demonstrate that long-term treatment with the NHE-1 inhibitor cariporide enhances the antiporter activity in cardiac tissue through an increase of the number and turnover of functional units. This finding deserves further experimental and clinical evaluations to consider whether it would be advisable a gradual withdrawal of prolonged NHE inhibition to avoid an enhanced response when the exchanger is stimulated.

Positive lusitropic effect and diminished myofibrillar sensitivity to calcium produced by cAMP on toad (Bufo Arenarum Hensel) ventricle

En tiritas intactas de ventrículo de sapo, se estudió el efecto relajante o lusitrópico positivo de diferentes intervenciones qu aumentan el AMPc intracelular. El isoproterenol aumentó la tensión desarrollada (DT), la velocidad máxima de contracción (+T), y la velocidad máxima de relajación (-T aumentó proporcionalmente más que +T a concentraciones de isoproterenol desde 10-8 a 10-4M, por lo que la relación +T/-T disminuyó significativamente. Una dosis única de isoproterenol (3x10**-8M) aumentó significativamente los niveles de AMPc desde 0.174 ñ 0.022 a 0.329 ñ pmoles/mg peso húmedo y produjo un aumento en la contractilidad de 69 ñ 13% y una disminución de +T/-T de 18.5 ñ 4.55%. La administración de 10**-3M de dibutiril AMPc(dAMPc) aumentó significativamente DT y +T y disminuyó significativamente la relación +T/-T. Efectos similares produjo la administración de milrinona, un inhibidor específico de la fosfodiesterasa de AMPc. La papaverina, un inhibidor inespecífico de fosfodiesterasas, no produjo aumentos en +T, pero aumentó significativamente -T. En trabéculas desprovistas químicamente de membrana, la sensibilidad al calcio de las proteínas contráctiles aumentó significativamente por la administración de 10**-5M del inhibidor de fosfodiesterasa 3-isobutil-1-metil-xantina (IBMX). La administración de 10**-3M de dAMPc no afectó la sensibilidad al calcio de las trabéculas desprovistas de membrana. Sin embargo la misma concentración de dAMPc produjo una disminución en la sensibilidad al calcio de las proteínas contráctiles cuando se administró en presencia de IBMX o de papavarina. Los resultados indicarían que el efecto relajante del isoproterenol es mediado en el ventrículo de sapo por un aumento en los niveles de AMPc intracelular. Estos resultados sugieren además que la disminución de la sensibilidad al calcio de los miofilamentos podría ser un mecanismo por el que el AMPc produce su efecto relajante

Positive lusitropic effect and diminished myofibrillar sensitivity to calcium produced by cAMP on toad (Bufo Arenarum Hensel) ventricle

En tiritas intactas de ventrículo de sapo, se estudió el efecto relajante o lusitrópico positivo de diferentes intervenciones qu aumentan el AMPc intracelular. El isoproterenol aumentó la tensión desarrollada (DT), la velocidad máxima de contracción (+T), y la velocidad máxima de relajación (-T aumentó proporcionalmente más que +T a concentraciones de isoproterenol desde 10-8 a 10-4M, por lo que la relación +T/-T disminuyó significativamente. Una dosis única de isoproterenol (3x10**-8M) aumentó significativamente los niveles de AMPc desde 0.174 ñ 0.022 a 0.329 ñ pmoles/mg peso húmedo y produjo un aumento en la contractilidad de 69 ñ 13% y una disminución de +T/-T de 18.5 ñ 4.55%. La administración de 10**-3M de dibutiril AMPc(dAMPc) aumentó significativamente DT y +T y disminuyó significativamente la relación +T/-T. Efectos similares produjo la administración de milrinona, un inhibidor específico de la fosfodiesterasa de AMPc. La papaverina, un inhibidor inespecífico de fosfodiesterasas, no produjo aumentos en +T, pero aumentó significativamente -T. En trabéculas desprovistas químicamente de membrana, la sensibilidad al calcio de las proteínas contráctiles aumentó significativamente por la administración de 10**-5M del inhibidor de fosfodiesterasa 3-isobutil-1-metil-xantina (IBMX). La administración de 10**-3M de dAMPc no afectó la sensibilidad al calcio de las trabéculas desprovistas de membrana. Sin embargo la misma concentración de dAMPc produjo una disminución en la sensibilidad al calcio de las proteínas contráctiles cuando se administró en presencia de IBMX o de papavarina. Los resultados indicarían que el efecto relajante del isoproterenol es mediado en el ventrículo de sapo por un aumento en los niveles de AMPc intracelular. Estos resultados sugieren además que la disminución de la sensibilidad al calcio de los miofilamentos podría ser un mecanismo por el que el AMPc produce su efecto relajante (AU)

Correlacion entre la relajacion miocardica y la fosforilacion de la fosfolamban / Correlationship between myocardial relaxation and phospholamban phosphorylation

Se estudió la relación entre contractilidad relajación miocárdicas y fosforilación de la fosfolamban en corazones de rata latiendo a frecuencia cardíaca constante y perfundidos a flujo coronario constante con una solución de Ringer con 32Pi. De estos corazones se aislaron vesículas de membranas enriquecidas en retículo sarcoplásmico (RS), que fueron preparadas para ser corridas electroforéticamente en geles de poliacrilamida-SDS. La perfusión con isoproterenol (ISO) aumentó significativamente la tensión desarrolada (T) en 40 ñ 8% y la velocidad máxima de desarrollo de la tensión (+T) en 76 ñ 12%. La relación entre +T y la velocidad máxima de relajación (-T), +T/-T, disminuyó desde 1.65 ñ 0.04 a 1.23 ñ 0.04. El tiempo a la mitad de la relajación, t1/2, y la constante de tiempo de la relajación (Tau) disminuyeron significativamente en 27 ñ 2 y 6 ñ 1 ms respectivamente. Cuando el aumento en T y +T producido por ISO fue revertido a los valores controles por el agregado de nifedipina (ISO-NIFE) o la perfusión de bajo calcio (ISO-CA+2), +T/-T cayó de 1.63 ñ 0.07 a 1.47 ñ 0.07 y 1.66 ñ 0.06 a 1.41 ñ 0.06 respectivamente. t 1/2 y Tau disminuyeron en 16 ñ 2 y 3 ñ 1 ms con ISO-NIFE y 19 ñ 2 y 5 ñ 1 ms con ISO-Ca+2 respectivamente. Estas disminuciones fueron significativamente menores que las producidas por ISO. La perfusión con lato calcio aumentó significativamente +T y T, pero no alteró significativamente los parámetros de relajación. La fosforilación de la fosfolamban en pmoles 32pi/mg de proteí

Correlacion entre la relajacion miocardica y la fosforilacion de la fosfolamban / Correlationship between myocardial relaxation and phospholamban phosphorylation

Se estudió la relación entre contractilidad relajación miocárdicas y fosforilación de la fosfolamban en corazones de rata latiendo a frecuencia cardíaca constante y perfundidos a flujo coronario constante con una solución de Ringer con 32Pi. De estos corazones se aislaron vesículas de membranas enriquecidas en retículo sarcoplásmico (RS), que fueron preparadas para ser corridas electroforéticamente en geles de poliacrilamida-SDS. La perfusión con isoproterenol (ISO) aumentó significativamente la tensión desarrolada (T) en 40 ñ 8% y la velocidad máxima de desarrollo de la tensión (+T) en 76 ñ 12%. La relación entre +T y la velocidad máxima de relajación (-T), +T/-T, disminuyó desde 1.65 ñ 0.04 a 1.23 ñ 0.04. El tiempo a la mitad de la relajación, t1/2, y la constante de tiempo de la relajación (Tau) disminuyeron significativamente en 27 ñ 2 y 6 ñ 1 ms respectivamente. Cuando el aumento en T y +T producido por ISO fue revertido a los valores controles por el agregado de nifedipina (ISO-NIFE) o la perfusión de bajo calcio (ISO-CA+2), +T/-T cayó de 1.63 ñ 0.07 a 1.47 ñ 0.07 y 1.66 ñ 0.06 a 1.41 ñ 0.06 respectivamente. t 1/2 y Tau disminuyeron en 16 ñ 2 y 3 ñ 1 ms con ISO-NIFE y 19 ñ 2 y 5 ñ 1 ms con ISO-Ca+2 respectivamente. Estas disminuciones fueron significativamente menores que las producidas por ISO. La perfusión con lato calcio aumentó significativamente +T y T, pero no alteró significativamente los parámetros de relajación. La fosforilación de la fosfolamban en pmoles 32pi/mg de proteí