Caveolae are essential for angiotensin II type 1 receptor-mediated ANP secretion

Caveolae may act as mechanosensors and function as binding sites for calcium ions. The intracaveolar localization of atrial natriuretic peptide (ANP) derived from the direct interaction of atrial granules with caveolae has been demonstrated. The aim of this study was to define the effect of caveolae on ANP secretion induced by stretch and angiotensin II. The isolated perfused beating atria from Sprague-Dawley rats were used. To disrupt caveolae, 10 mM methyl-β-cyclodextrin (MbCD) was applied for 1 h and the number of caveoli were markedly decreased. MbCD increased basal ANP secretion and atrial diastolic pressure. The molecular profile of ANP in perfusate from control atria showed mainly one major peak corresponded to synthetic ANP whereas that from MbCD-treated atria showed two major immunoreactive peaks corresponded to synthetic rat ANP and proANP. High atrial stretch induced by elevating the height of outflow catheter from 5 cm H₂O to 7.5 cm H₂O increased atrial contractility and ANP secretion. The response of ANP secretion to high stretch was attenuated in MbCD-pretreated atria. Pretreatment with MbCD abolished angiotensin II-induced suppression and losartan-induced stimulation of ANP secretion. However, the effect of angiotenisin (1-7) on ANP secretion was not altered by MbCD treatment. The expression of angiotensin II type 1 receptor protein was reduced by MbCD treatment. These data suggest that caveolae are essential for angiotensin II type 1 receptor-mediated ANP secretion and relate to the processing of proANP.     

Suppression of high pacing-induced ANP secretion by antioxidants in isolated rat atria

Reactive oxygen species (ROS) are formed as a natural by-product of the normal metabolism of oxygen and have important roles in cell signaling. The aim of this study was to investigate direct effects of ROS on atrial hemodynamics and ANP secretion in isolated perfused beating rat atria with antioxidants. When atria were paced at 1.2 Hz, N-acetyl cystein (antioxidant, NAC), α-lipoic acid (antioxidant), tempol (superoxide dismutase mimic), and apocynin (NADPH oxidase inhibitor; NOX inhibitor) did not affect ANP secretion and atrial contractility. When pacing frequency was increased from 1.2 Hz to 4 Hz, the ANP secretion increased and atrial contractility decreased. H₂O₂ level was increased in perfusate obtained from atria stimulated by high pacing frequency. NAC, α-lipoic acid and tempol attenuated high pacing frequency-induced ANP secretion but apocynin did not. In contrast, pyrogallol (a superoxide generator) augmented high pacing frequency-induced ANP secretion. NOX-4 protein was increased by high pacing stimulation and in diabetic rat atria. In diabetic rat atria, high pacing frequency caused an increased ANP secretion and a decreased atrial contractility, that were markedly attenuated as compared to control rats. NAC and apocynin reduced high pacing frequency-induced ANP secretion in diabetic rat atria. These results suggest that intracellular ROS formation partly through an increasing NOX activity in response to high pacing frequency is associated with an increased ANP secretion in rat atria.     

Suppression of high pacing-induced ANP secretion by antioxidants in isolated rat atria

Reactive oxygen species (ROS) are formed as a natural by-product of the normal metabolism of oxygen and have important roles in cell signaling. The aim of this study was to investigate direct effects of ROS on atrial hemodynamics and ANP secretion in isolated perfused beating rat atria with antioxidants. When atria were paced at 1.2 Hz, N-acetyl cystein (antioxidant, NAC), α-lipoic acid (antioxidant), tempol (superoxide dismutase mimic), and apocynin (NADPH oxidase inhibitor; NOX inhibitor) did not affect ANP secretion and atrial contractility. When pacing frequency was increased from 1.2 Hz to 4 Hz, the ANP secretion increased and atrial contractility decreased. H₂O₂ level was increased in perfusate obtained from atria stimulated by high pacing frequency. NAC, α-lipoic acid and tempol attenuated high pacing frequency-induced ANP secretion but apocynin did not. In contrast, pyrogallol (a superoxide generator) augmented high pacing frequency-induced ANP secretion. NOX-4 protein was increased by high pacing stimulation and in diabetic rat atria. In diabetic rat atria, high pacing frequency caused an increased ANP secretion and a decreased atrial contractility, that were markedly attenuated as compared to control rats. NAC and apocynin reduced high pacing frequency-induced ANP secretion in diabetic rat atria. These results suggest that intracellular ROS formation partly through an increasing NOX activity in response to high pacing frequency is associated with an increased ANP secretion in rat atria.     

In vivo effects of propyl gallate,a novel Ca sensitizer,in a mouse model of dilated cardiomyopathy caused by cardiac troponin T mutation

Aims

We have previously demonstrated that propyl gallate has a Ca^2 + sensitizing effect on the force generation in membrane-permeabilized (skinned) cardiac muscle fibers. However, in vivo beneficial effects of propyl gallate as a novel Ca^2 + sensitizer remain uncertain. In the present study, we aim to explore in vivo effects of propyl gallate.

Main methods

We compared effects of propyl gallate on ex vivo intact cardiac muscle fibers and in vivo hearts in healthy mice with those of pimobendan, a clinically used Ca^2 + sensitizer. The therapeutic effect of propyl gallate was investigated using a mouse model of dilated cardiomyopathy (DCM) with reduced myofilament Ca^2 + sensitivity due to a deletion mutation ΔK210 in cardiac troponin T.

Key findings

Propyl gallate, as well as pimobendan, showed a positive inotropic effect. Propyl gallate slightly increased the blood pressure without changing the heart rate in healthy mice, whereas pimobendan decreased the blood pressure probably through vasodilation via inhibition of phosphodiesterase and increased the heart rate. Propyl gallate prevented cardiac remodeling and systolic dysfunction and significantly improved the life-expectancy of knock-in mouse model of DCM with reduced myofilament Ca^2 + sensitivity due to a mutation in cardiac troponin T. On the other hand, gallate, a similarly strong antioxidant polyphenol lacking Ca^2 + sensitizing action, had no beneficial effects on the DCM mice.

Significance

These results suggest that propyl gallate might be useful for the treatment of inherited DCM caused by a reduction in the myofilament Ca^2 + sensitivity.     

Endogenous angiotensin II suppresses stretch-induced ANP secretion via AT1 receptor pathway

Angiotensin II (Ang II) is released by stretch of cardiac myocytes and has paracrine and autocrine effects on cardiac myocytes and fibroblasts. However, the direct effect of Ang II on the secretion of atrial natriuretic peptide (ANP) is unclear. The aim of the present study is to test whether Ang II affects stretch-induced ANP secretion. The isolated perfused beating atria were used from control and two-kidney one-clip hypertensive (2K1C) rats. The volume load was achieved by elevating the height of outflow catheter connected with isolated atria from 5 cmH₂O to 7.5 cmH₂O. Atrial stretch by volume load caused increases in atrial contractility by 60% and in ANP secretion by 100%. Ang II suppressed stretch-induced ANP secretion and tended to increase atrial contractility whereas losartan stimulated stretch-induced ANP secretion. Neither PD123319 nor A779 had direct effect on stretch-induced ANP secretion. The suppressive effect of Ang II on stretch-induced ANP secretion was blocked by the pretreatment of losartan but not by the pretreatment of PD123319 or A779. In hypertrophied atria from 2K1C rats, stretch-induced ANP concentration attenuated and atrial contractility augmented. The response of stretch-induced ANP secretion to Ang II and losartan augmented. The expression of AT1 receptor protein and mRNA increased but AT2 and Mas receptor mRNA did not change in 2K1C rat atria. Therefore, we suggest that Ang II generated endogenously by atrial stretch suppresses stretch-induced ANP secretion through the AT1 receptor and alteration of Ang II effect in 2K1C rat may be due to upregulation of AT1 receptor.     

Suppression of ANP secretion by somatostatin through somatostatin receptor type 2

Somatostatin is a cyclic-14 amino acid peptide which mainly distributed in digestive system and brain. Somatostatin receptor (SSTR) is a G-protein coupled receptor and all five SSTR subtypes are expressed in cardiomyocytes. The aim of this study was to investigate the effect of somatostatin on atrial natriuretic peptide (ANP) secretion and its signaling pathway. Somatostatin (0.01 and 0.1 nM) decreased ANP secretion in isolated beating rat atrium in a dose-dependent manner. But atrial contractility and translocation of extracellular fluid were not changed. Somatostatin-induced decrease in ANP secretion was significantly attenuated by the pretreatment with CYN 154806 (SSTR type 2 antagonist; 0.1 μM), but not by BIM 23056 (SSTR type 5 antagonist; 0.1 μM) and urantide (urotensin II receptor antagonist; 0.1 μM). When pretreated with an agonist for SSTR type 2 (Seglitide, 0.1 nM) and SSTR type 5 (L 817818, 0.1 nM), only Seglitide reduced ANP secretion similar to that of somatostatin. The suppressive effect of somatostatin on ANP secretion was attenuated by the pretreatment with an inhibitor for adenylyl cyclase (MDL-12330A, 5 μM) or protein kinase A (KT 5720, 0.1 μM). In diabetic rat atria, the suppressive effect of somatostatin on ANP secretion and concentration was attenuated. Real time-PCR and western blot shows the decreased level of SSTR type 2 mRNA and protein in diabetic rat atria. These data suggest that somatostatin decreased ANP secretion through SSTR type 2 and an attenuation of suppressive effect of somatostatin on ANP secretion in diabetic rat atria is due to a down-regulation of SSTR type 2.     

Accentuation of ursolic acid on muscarinic receptor-induced ANP secretion in beating rabbit atria

Aims

Ursolic acid has recently been reported to increase both atrial natriuretic peptide (ANP) secretion and mechanical dynamics in rabbit atria.

Main methods

The present study was designed to clarify the regulatory effects of ursolic acid on the β-adrenergic or muscarinic receptor-mediated changes in ANP secretory and contractile function allowing measurement of atrial dynamics such as pulse pressure, stroke volume, and cAMP efflux in isolated perfused beating rabbit atria.

Key findings

Pretreatment with ursolic acid significantly attenuated the isoproterenol (β-adrenergic agonist)-induced decrease in ANP secretion and increases in cAMP levels and atrial dynamics. Interestingly, ursolic acid concentration-dependently accentuated the acetylcholine-induced increase in ANP secretion and decrease in pulse pressure in the presence of isoproterenol (p < 0.001). These findings indicate that acetylcholine-induced increase in ANP secretion is potentiated by ursolic acid; furthermore, acetylcholine-induced decrease in atrial dynamics is also potentiated by ursolic acid, suggesting that ursolic acid regulates muscarinic receptor-mediated secretory and contractile responses in perfused beating rabbit atria.

Significance

This implicates for the beneficial effects of ursolic acid in the regulation of cardiovascular and body fluid homeostasis.     

MAPK and PI3K pathways regulate hypoxia-induced atrial natriuretic peptide secretion by controlling HIF-1 alpha expression in beating rabbit atria

Mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) signaling pathways are pivotal and intensively studied signaling pathways in hypoxic conditions. However, the roles of MAPK and PI3K in the regulation of hypoxia-induced atrial natriuretic peptide (ANP) secretion are not well understood. The purpose of the present study was to investigate the mechanism by which the MAPK/ERK (extracellular signal-regulated kinase) and PI3K signaling pathways regulate the acute hypoxia-induced ANP secretion in isolated beating rabbit atria. An acute hypoxic perfused beating rabbit atrial model was used. The ANP levels in the atrial perfusates were measured by radioimmunoassay, and the hypoxia-inducible factor-1α (HIF-1α) mRNA and protein levels in the atrial tissue were determined by RT-PCR and Western blot. Acute hypoxia significantly increased ANP secretion and HIF-1α mRNA and protein levels. Hypoxia-induced ANP secretion was markedly attenuated by the HIF-1α inhibitors, rotenone (0.5 μmol/L) and CAY10585 (10 μmol/L), concomitantly with downregulation of the hypoxia-induced HIF-1α mRNA and protein levels. PD098059 (30 μmol/L) and LY294002 (30 μmol/L), inhibitors of MAPK and PI3K, markedly abolished the hypoxia-induced ANP secretion and atrial HIF-1α mRNA and protein levels. The hypoxia-suppressed atrial dynamics were significantly attenuated by PD098059 and LY294002. Acute hypoxia in isolated perfused beating rabbit atria, markedly increased ANP secretion through HIF-1α upregulation, which was regulated by the MAPK/ERK and PI3K pathways. ANP appears to be part of the protective program regulated by HIF-1α in the response to acute hypoxic conditions.     

Expression of atrial natriuretic peptide receptor-A antagonizes the mitogen-activated protein kinases (Erk2 and P38MAPK) in cultured human vascular smooth muscle cells

To understand the signaling mechanisms of atrial natriuretic peptide (ANP) receptor-A (NPRA), we studied the effect of the ANP/NPRA system on mitogen-activated protein kinases (MAPKs), with particular emphasis on the extracellular-regulated kinase (Erk2) and stress-activated protein kinase (p38^MAPK) in cultured human vascular smooth muscle cells (HVSMC). Angiotensin II (ANG II) and platelet-derived growth factor (PDGF) stimulated the immunoreactive Erk2 and p38^MAPK activities and their protein levels by 2–4 fold. The pretreatment of cells with ANP significantly inhibited the agonist-stimulated Erk2 and p38^MAPK activities and protein expression by 65–75% in HVSMC transiently transfected with NPRA, as compared with only 18–22% inhibition in vector-transfected cells. The pretreatment of cells with KT5823, an inhibitor of cGMP-dependent protein kinase (PKG), reversed the inhibitory effects of ANP on MAPK activities and protein expression by 90–95%. PD98059, which inhibits Erk2 by directly inhibiting the MAPK-kinase (MEK), and SB202192, a selective antagonist of p38^MAPK, blocked the Erk2 and p38^MAPK activities, respectively. Interestingly, ANP stimulated the MAPK-phosphatase-3 (MKP-3) protein levels by more than 3-fold in HVSMC over-expressing NPRA, suggesting that ANP-dependent inhibition of MAPKs may also proceed by stimulating the phosphatase cascade. These present findings provide the evidence that ANP exerts inhibitory effects on agonist-stimulated MAPKs (Erk2 and p38^MAPK) activities and protein levels in a 2-fold manner: by antagonizing the upstream signaling pathways and by activation of MKP-3 to counter-regulate MAPKs in a cGMP and PKG-dependent manner. Our results identify a signal transduction pathway in HVSMC that could contribute to vascular remodeling and structural changes in human hypertension.     

Leishmania amazonensis: Heme stimulates (Na + K)ATPase activity via phosphatidylinositol-specific phospholipase C/protein kinase C-like (PI-PLC/PKC) signaling pathways

In the present paper we studied the involvement of the phosphatidylinositol-specific PLC (PI-PLC)/protein kinase C (PKC) pathway in (Na⁺ + K⁺)ATPase stimulation by heme in Leishmania amazonensis promastigotes. Heme stimulated the PKC-like activity with a concentration of 50 nM. Interestingly, the maximal stimulation of the PKC-like activity promoted by phorbol ester was of the same magnitude promoted by heme. However, the stimulatory effect of heme is completely abolished by ET-18-OCH₃ and U73122, specific inhibitors of PI-PLC. (Na⁺ + K⁺)ATPase activity is increased in the presence of increased concentrations of heme, being maximally affected at 50 nM. This effect was completely reversed by 10 nM calphostin C, an inhibitor of PKC. Thus, the effect of 50 nM heme on (Na⁺ + K⁺)ATPase activity is completely abolished by ET-18-OCH₃ and U73122. Taken together, these results demonstrate that the heme receptor mediates the stimulatory effect of heme on the (Na⁺ + K⁺)ATPase activity through a PI-PLC/PKC signaling pathway.     

Stimulation of ANP by angiotensin-(1-9) via the angiotensin type 2 receptor

Aims

Angiotensin-(1-9) [Ang-(1-9)] and Ang-(1-7) are cleaved by Ang converting enzyme 2 forming Ang I and Ang II, respectively, and the truncated Angs play a role in regulating atrial natriuretic peptide (ANP) secretion. Previously, we found that Ang-(1-7) stimulates ANP secretion via the Mas receptor. However, the effect of Ang-(1-9) on ANP secretion is still unknown. The aim of the present study is to determine whether Ang-(1-9) stimulates ANP secretion and to characterize the signaling pathway involved in stimulating secretion.

Main methods

We examined the effects of Ang-(1-9) on ANP secretion and atrial contractility with and without inhibitors in isolated perfused atria.

Key findings

Ang-(1-9) stimulated ANP secretion and concentration without change in atrial contractility. Ang-(1-9)-induced-ANP secretion was increased from 5% to 60% by 3 μM Ang-(1-9) during the low-stretch state of the atrium. This stimulatory effect of Ang-(1-9) on ANP secretion was attenuated by pretreatment with an Ang II type 2 receptor (AT₂R) antagonist but not by AT₁R or Mas receptor antagonist. In addition, pretreatment with inhibitors of phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt), nitric oxide synthase (NOS) and soluble guanylyl cyclase (sGC) blocked Ang-(1-9)-induced ANP secretion. In the high-stretch atrial state, Ang-(1-9)-induced ANP secretion was increased more than in the low-stretch state following addition of 1 μM Ang-(1-9) (from 108% to 170%). In an in vivo experiment, acute infusion of Ang-(1-9) increased plasma ANP level without altering arterial blood pressure. This effect was attenuated by pretreatment with AT₂R antagonist but not by Mas receptor antagonist.

Significance

These results suggest that Ang-(1-9) stimulates ANP secretion via the AT₂R-PI3K-Akt-NO-cGMP pathway.     

p38/p53-Mediated Bax induction contributes to neurons degeneration in rotenone-induced cellular and rat models of Parkinson’s disease

Rotenone is an environmental neurotoxin that induces degeneration of dopaminergic (DA) neurons in substantia nigra pars compacta (SNpc), which ultimately results in parkinsonism, but the molecular mechanisms of selective degeneration of nigral DA neurons are not fully understood. In the present study, we investigated the induction of p38^MAPK/p53 and Bax in SNpc of Lewis rats after chronic treatment with rotenone and the contribution of Bax to rotenone-induced apoptotic commitment of differentiated PC12 cells. Lewis rats were subcutaneously treated with rotenone (1.5 mg/kg) twice a day for 50 days and the loss of tyrosine hydroxylase (THase), motor function impairment, and expression of p38^MAPK, P-p38^MAPK, p53, and Bax were assessed. After differentiated PC cells were treated with rotenone (500 nM) for 6–36 h, protein levels of p38^MAPK and P-p38^MAPK, p53 nuclear translocation, Bax induction and cell death were measured. The results showed that rotenone administration significantly reduced motor activity and caused a loss of THase immunoreactivity in SNpc of Lewis rats. The degeneration of nigral DA neurons was accompanied by the increases in p38^MAPK, P-p38^MAPK, p53, and Bax protein levels. In cultured PC12 cells, rotenone also induced an upregulation of p38^MAPK, P-p38^MAPK, p53 and Bax. Pharmacological inhibition of p38^MAPK with SB203580 (25 μM) blunted rotenone-induced cell apoptosis. Treatment with SB203580 prevented the p53 nuclear translocation and upregulation of Bax. Inhibition of p53 with pifthrin-alpha or Bax with siRNAs significantly reduced rotenone-induced Bax induction and apoptotic cell death. These results suggest that the p38^MAPK/p53-dependent induction of Bax contributes to rotenone’s neurotoxicity in PD models.     

Comparative study of cardiac electrophysiological effects of atrial natriuretic peptide

The effects of atrial natriuretic peptide (ANP) on action potential characteristics were studied in various (human, rabbit, guinea-pig) atrial and guinea-pig right ventricular papillary muscles. ANP (1–100 nM) did not modify the resting membrane potential nor the maximum rate of depolarization phase (V_max). Up to 10 nM, ANP dose-dependently decreased the action potential amplitude both in guinea-pig atrial and ventricular muscles, but it did not affect this parameter in the other atrial preparations. ANP caused a dose-dependent, marked decrease of action potential duration (APD) in practically every cardiac preparation studied (exception of guinea-pig left atrium). The strongest effect on APD can be observed in human atrial and guinea-pig ventricular fibers. The K⁺ channel blocker 4-aminopyridine (1 mM) and the ATP-dependent K⁺ channel inhibitor glibenclamide (10Nl) prevented the effect of ANP on APD in both ventricular atrial preparations. ANP prevented the appearance of isoprenaline (0.5 M) induced slow AP in K⁺ depolarized myocardium. The present data suggest that ANP may inhibit the slow inward Ca²⁺ channel activity and facilitate the K⁺ channel activity.     

Ceramide-activated protein kinases A and C zeta inhibit kidney proximal tubule cell Na-ATPase

The basolateral membranes of kidney proximal tubule cells have (Na⁺+K⁺)-ATPase and Na⁺-ATPase activities, involved in Na⁺ reabsorption. We showed that ceramide (Cer) modulates protein kinase A (PKA) and protein kinase C (PKC), which are involved in regulating ion transporters. Here we show that ceramide, promotes 60% inhibition of Na⁺-ATPase activity (I₅₀ ≈ 100 nM). This effect was completely reversed by inhibiting PKA but did not involve the classic PKC signaling pathway. In these membranes we found the Cer-activated atypical PKC zeta (PKCζ) isoform. When PKCζ is inhibited, Cer ceases to inhibit the Na⁺-ATPase, allowing the cAMP/PKA signaling pathway to recover its stimulatory effect on the pump. There were no effects on the (Na⁺+K⁺)-ATPase. These results reveal Cer as a potent physiological modulator of the Na⁺-ATPase, participating in a regulatory network in kidney cells and counteracting the stimulatory effect of PKA via PKCζ.     

Cardiodepressive effect elicited by the essential oil of Alpinia speciosa is related to L-type Ca current blockade

This study was undertaken to elucidate the effect of the essential oil from Alpinia speciosa (EOAs) on cardiac contractility and the underlying mechanisms. The essential oil was obtained from Alpinia speciosa leaves and flowers and the oil was analyzed by GC-MS method. Chemical analysis revealed the presence of at least 18 components. Terpinen-4-ol and 1,8-cineole corresponded to 38% and 18% of the crude oil, respectively. The experiments were conducted on spontaneously-beating right atria and on electrically stimulated left atria isolated from adult rats. The effect of EOAs on the isometric contractions and cardiac frequency in vitro was examined. EOAs decreased rat left atrial force of contraction with an EC₅₀ of 292.2 ± 75.7 μg/ml. Nifedipine, a well known L-type Ca²⁺ blocker, inhibited in a concentration-dependent manner left atrial force of contraction with an EC₅₀ of 12.1 ± 3.5 μg/ml. Sinus rhythm was diminished by EOAs with an EC₅₀ of 595.4 ± 56.2 μg/ml. Whole-cell L-type Ca²⁺ currents were recorded by using the patch-clamp technique. EOAs at 25 μg/ml decreased I_Ca,L by 32.6 ± 9.2% and at 250 μg/ml it decreased by 89.3 ± 7.4%. Thus, inhibition of L-type Ca²⁺ channels is involved in the cardiodepressive effect elicited by the essential oil of Alpinia speciosa in rat heart.     

Chloroplast-generated ROS dominate NaCl- induced K+ efflux in wheat leaf mesophyll

Mesophyll K⁺ retention ability has been recently reported as an important component of salinity stress tolerance in wheat. In order to investigate the role of ROS in regulating NaCl^-induced K⁺ efflux in wheat leaf mesophyll, a series of pharmacological experiments was conducted using MV (methyl viologen, superoxide radical inducer), DPI (an inhibitor of NADPH oxidase), H₂O₂ (to mimic apoplastic ROS), and EGCG ((−)-Epigallocatechin gallate, ROS scavenger). Mesophyll pre-treatment with 10 μM MV resulted in a significantly higher NaCl^-induced K⁺ efflux in leaf mesophyll, while 50 μM EGCG pre-treatment alleviated K⁺ leakage under salt stress. No significant change in NaCl^-induced K⁺ efflux in leaf mesophyll was found in specimens pre-treated by H₂O₂ and DPI, compared with the control. The highest NaCl^-induced H⁺ efflux in leaf mesophyll was also found in samples pre-treated with MV, suggesting a futile cycle between increased H⁺-ATPase activity and ROS-induced K⁺ leak. Overall, it is suggested that, under saline stress, K⁺ efflux from wheat mesophyll is mediated predominantly by non-selective cation channels (NSCC) regulated by ROS produced in chloroplasts, at least in bread wheat.     

Measuring Ca binding to short chain fatty acids and gluconate with a Ca electrode: Role of the reference electrode

Many organic anions bind free Ca²⁺, the total concentration of which must be adjusted in experimental solutions. Because published values for the apparent dissociation constant (K_app) describing the Ca²⁺ affinity of short chain fatty acids (SCFAs) and gluconate are highly variable, Ca²⁺ electrodes coupled to either a 3 M KCl or a Na⁺ selective electrode were used to redetermine K_app. All solutions contained 130 mM Na⁺, whereas the concentration of the studied anion was varied from 15 to 120 mM, replacing Cl⁻ that was decreased concomitantly to maintain osmolarity. This induces changes in the liquid junction potential (LJP) at the 3 M KCl reference electrode, leading to a systematic underestimation of K_app if left uncorrected. Because the Na⁺ concentration in all solutions was constant, a Na⁺ electrode was used to directly measure the changes in the LJP at the 3 M KCl reference, which were under 5 mV but twice those predicted by the Henderson equation. Determination of K_app either after correction for these LJP changes or via direct reference to a Na⁺ electrode showed that SCFAs do not bind Ca²⁺ and that the K_app for the binding of Ca²⁺ to gluconate at pH 7.4, ionic strength 0.15 M, and 23 °C was 52.7 mM.     

Cardiac activity of some peptide hormones in the frog,Rana tigrina

1. The cardiac responses of isolated frog (Rana tigrina) atria to peptide hormones were studied.2. Calcitonin gene-related peptide (CGRP), arginine vasotocin (AVT), bovine parathyroid hormone fragment (bPTH-(1–34)) and oxytocin (OXY) produced dose-related positive chronotropic and inotropic responses; atrial natriuretic peptide (ANP) was negative chronotropic and inotropic; cholecystokinin (CCK), vasoactive intestinal peptide (VIP) were without effects.3. The dose-related responses under bPTH-(1–34) stimulation but not CGRP or AVT were attenuated in the presence of ANP (300 ng/ml, ≈0.98 × 10⁻⁷ M). As expected ANP decreased the basal AR and AT responses of the isolated atria and the inhibitory effects were dose-dependent.4. As shown previously, propranolol blocked the atrial tension stimulated by bPTH (1–34) but did not alter the cardiac responses to CGRP and AVT.5. In the presence of β-adrenergic blocker (propranolol 10⁻⁷M) or ANP (10⁻⁷M), the AR and AT changes under ISO stimulation in the frog were also decreased.6. These cardiac changes suggest the cardiac inhibitory effects of ANP are related to β-adrenoceptor activity and ANP might be a β antagonist.     

Coupled gating modifies the regulation of cardiac ryanodine receptors by luminal Ca

Cardiac ryanodine receptors (RYR2s) infrequently exhibit coupled gating that is manifested by synchronous opening and closing. To better characterize this phenomenon, we investigated the regulation of coupled RYR2 channels by luminal Ca^2 + focusing on effects that are likely mediated by the true luminal activation mechanism. By reconstituting an ion channel into a planar lipid bilayer and using substantially lower concentration of luminal Ba^2 + (8 mM, the virtual absence of Ca^2 +) and luminal Ca^2 + (8 mM), we show that response of coupled RYR2 channels to caffeine at a diastolic cytosolic Ca^2 + (90 nM) was affected by luminal Ca^2 + in a similar manner as for the single RYR2 channel except the gating behavior. Whereas, the single RYR2 channel responded to luminal Ca^2 + by prolongation in open and closed times, coupled RYR2 channels seemed to be resistant in this respect. In summary, we conclude that the class of Ca^2 + sites located on the luminal face of coupled RYR2 channels that is responsible for the channel potentiation by luminal Ca^2 + is functional and not structurally hindered by the channel coupling. Thus, the idea about non-functional luminal Ca^2 + sites as a source of the apparent gating resistance of coupled RYR2 channels to luminal Ca^2 + appears to be ruled out.     

Angiotensin III stimulates high stretch-induced ANP secretion via angiotensin type 2 receptor

Angiotensin III (Ang III) is metabolized from Ang II by aminopeptidase (AP) A and in turn, Ang III is metabolized to Ang IV by APN. Ang III is known to have a similar effect to Ang II on aldosterone secretion, but the effect of Ang III on atrial natriuretic peptide (ANP) secretion from cardiac atria is not known. The aim of the present study is to define the effect of Ang III on ANP secretion and its receptor subtype using isolated perfused beating atria. The volume load was achieved by elevating the height of outflow catheter connected with isolated atria from 5 cmH₂O to 7.5 cmH₂O. Atrial stretch by volume load increased atrial contractility and ANP secretion. Ang III stimulated stretch-induced ANP secretion in a dose-dependent manner without change in atrial contractility. The stimulated effect of Ang III (1 μM) on stretch-induced ANP secretion was blocked by the pretreatment of Ang II type 2 (AT₂) receptor antagonist but not by AT₁ or Mas receptor antagonist. Pretreatment with inhibitor of phosphoinositide 3-kinase (PI3K), Akt, nitric oxide synthase, soluble guanylyl cyclase, or protein kinase G (PKG) attenuated Ang III-stimulated ANP secretion. When Ang III (40 nM) or Ang II (4 nM) was infused for 10 min into anesthetized rats, mean arterial pressure was increased about 10%. However, Ang III increased plasma ANP level by 35.81 ± 10.19% but Ang II decreased plasma ANP level by 30.41 ± 7.27%. Therefore, we suggest that Ang III, opposite to Ang II, stimulated stretch-induced ANP secretion through AT₂ receptor/PI3K/Akt/nitric oxide/PKG pathway.