Extracellular Fluid Translocation Research Articles

Effects of endothelin family on ANP secretion

The endothelins (ET) peptide family consists of ET-1, ET-2, ET-3, and sarafotoxin (s6C, a snake venom) and their actions appears to be different among isoforms. The aim of this study was to compare the secretagogue effect of ET-1 on atrial natriuretic peptide (ANP) secretion with ET-3 and evaluate its physiological meaning. Isolated nonbeating atria from male Sprague-Dawley rats were used to evaluate stretch-activated ANP secretion in response to ET-1, ET-2, ET-3, and s6C. Changes in mean blood pressure (MAP) were measured during acute injection of ET-1 and ET-3 with and without natriuretic peptide receptor-A antagonist (A71915) in anesthetized rats. Changes in atrial volume induced by increased atrial pressure from o to 1, 2, 4, or 6cm H2O caused proportional increases in mechanically-stimulated extracellular fluid (ECF) translocation and stretch-activated ANP secretion. ET-1 (10nM) augmented basal and stretch-activated ANP secretion in terms of ECF translocation, which was blocked by the pretreatment with ETA receptor antagonist (BQ123, 1μM) but not by ETB receptor antagonist (BQ788, 1μM). ETA receptor antagonist itself suppressed stretch-activated ANP secretion. As compared to ET-1- induced ANP secretion (3.2-fold by 10nM), the secretagogue effects of ANP secretion by ET-2 was similar (2.8-fold by 10nM) and ET-3 and s6C were less potent (1.7-fold and 1.5-fold by 100nM, respectively). Acute injection of ET-1 or ET-3 increased mean blood pressure (MAP), which was augmented in the presence of natriuretic peptide receptor-A antagonist. Therefore, we suggest that the order of secretagogue effect of ET family on ANP secretion was ET-1≥ET-2>>ET-3>s6C and ET-1-induced ANP secretion negatively regulates the pressor effect of ET-1.

Angiotensin AT2 receptor agonist stimulates high stretch induced- ANP secretion via PI3K/NO/sGC/PKG/pathway

Angiotensin II (Ang II) type 1 receptor (AT1R) mediates the major cardiovascular effects of Ang II. However, the effects mediated via AT2R are still controversial. The aim of the present study is to define the effect of AT2R agonist CGP42112A (CGP) on high stretch-induced ANP secretion and its mechanism using in vitro and in vivo experiments. CGP (0.01, 0.1 and 1μM) stimulated high stretch-induced ANP secretion and concentration from isolated perfused rat atria. However, atrial contractility and the translocation of extracellular fluid did not change. The augmented effect of CGP (0.1μM) on high stretch-induced ANP secretion was attenuated by the pretreatment with AT2R antagonist or inhibitor for phosphoinositol 3-kinase (PI3K), nitric oxide (NO), soluble guanylyl cyclase (sGC), or protein kinase G (PKG). However, antagonist for AT1R or Mas receptor did not influence CGP-induced ANP secretion. In vivo study, acute infusion of CGP for 10min increased plasma ANP level without blood pressure change. In renal hypertensive rat atria, AT2R mRNA and protein levels were up-regulated and the response of plasma ANP level to CGP infusion in renal hypertensive rats augmented. The pretreatment with AT2R antagonist for 10min followed by CGP infusion attenuated an increased plasma ANP level induced by CGP. However, pretreatment with AT1R or Mas receptor antagonist unaffected CGP-induced increase in plasma ANP level. Therefore, we suggest that AT2R agonist CGP stimulates high stretch-induced ANP secretion through PI3K/NO/sGC/PKG pathway and these effects are augmented in renal hypertensive rats.

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.

Oxidative stress augments the secretion of atrial natriuretic peptide in isolated rat atria

Reactive oxygen species (ROS) play a role in cardiovascular diseases such as hypertension and heart failure. The objective of the present study was to investigate the role of endogenous ROS in atrial hemodynamics and ANP secretion in isolated perfused beating rat atria. Pyrogallol (a generator of superoxide anion, 0.1, 1mM) or hydrogen peroxide (0.1, 1, 10, 30mM) was perfused into atria paced at 1.2Hz. Pyrogallol and hydrogen peroxide stimulated ANP secretion and concentration in a dose-dependent manner and dramatically decreased atrial contractility and translocation of extracellular fluid. The stimulatory effect of pyrogallol and hydrogen peroxide on ANP secretion was attenuated by the pretreatment with ascorbic acid (an antioxidant; 1mM) and cariporide (an inhibitor of the Na(+)/H(+) exchanger; 1μM) but negative inotropic effect was not changed. U120 (a MAPK(erk) pathway inhibitor; 10μM) attenuated the stimulatory effect of hydrogen peroxide on ANP secretion. However, U120 augmented negative inotropic effect and stimulatory effect of ANP concentration induced by pyrogallol. Antioxidant such as N-acetyl cystein, gallate, propyl gallate, or ellagic acid did not cause any significant changes in atrial parameters. These results suggest that intracellular - formed ROS stimulates ANP secretion partly through activation of MAPK(erk) pathway and Na(+)/H(+) exchanger.

Urotensin II stimulates high frequency-induced ANP secretion via PLC-PI 3K-PKC pathway

Urotensin II (U-II) and its receptor are coexpressed in the heart and show various cardiovascular functions. However, the relationship between U-II and cardiac hormone atrial natriuretic peptide (ANP) is still unknown. The aim of the present study is to test whether U-II affects ANP secretion using in vitro perfusion experiments and in vivo studies. Human U-II (hU-II) (10−11, 5×10−11, 10−10, 5×10−10M) stimulated ANP secretion from isolated perfused rat atria paced with high frequency (6.0Hz). However, atrial contractility and translocation of extracellular fluid (ECF) did not change. An increase in ANP secretion by rat U-II was similar to that by hU-II; however, urotensin-related peptide showed no significant effect on ANP secretion. Pretreatment with urotensin receptor antagonist and inhibitor for phospholipase C (PLC), phosphoinositide 3-kinase (PI3K), or protein kinase C (PKC) attenuated hU-II-induced ANP secretion from atria paced with high frequency, but an inhibitor for inositol triphosphate did not. Intravenous infusion of hU-II at a dose of 2.5μM for 20min increased plasma ANP level, along with increased heart rate and pulse pressure in anesthetized rats. Therefore, we suggest that U-II stimulates high stimulation frequency-induced ANP secretion partly through the urotensin receptor and the PLC/PI3K/PKC pathway.

Stimulation of ANP secretion by 2-Cl-IB-MECA through A 3 receptor and CaMKII

Adenosine is a potent mediator of myocardial protection against hypertrophy via A 1 or A 3 receptors that may be partly related to atrial natriuretic peptide (ANP) release. However, little is known about the possible involvement of the A 3 receptor on ANP release. We studied the effects of the A 3 receptor on atrial functions and its modification in hypertrophied atria. A selective A 3 receptor agonist, 2-chloro-N 6-(3-iodobenzyl) adenosine-5′- N-methyluronamide (2-CI-IB-MECA), was perfused into isolated, beating rat atria with and without receptor modifiers. 2-CI-IB-MECA dose-dependently increased the ANP secretion, which was blocked by the A 3 receptor antagonist, but the increased atrial contractility and decreased cAMP levels induced by 30 μM 2-CI-IB-MECA were not affected. The 100 μM 2-(1-hexylnyl)- N-methyladenosine (HEMADO) and N 6-(3-iodobenzyl) adenosine-5′- N-methyluronamide (IB-MECA), A 3 receptor agonist, also stimulated the ANP secretion without positive inotropy. The potency for the stimulation of ANP secretion was 2-CI-IB-MECA ≫ IB-MECA = HEMADO. The inhibition of the ryanodine receptor or calcium/calmodulin-dependent kinase II (CaMKII) attenuated 2-CI-IB-MECA-induced ANP release, positive inotropy, and translocation of extracellular fluid. However, the inhibition of L-type Ca 2+ channels, sarcoplasmic reticulum Ca 2+-reuptake, phospholipase C or inositol 1,4,5-triphosphate receptors did not affect these parameters. 2-CI-IB-MECA decreased cAMP level, which was blocked only with an inhibitor of CaMKII or adenylyl cyclase. These results suggest that 2-CI-IB-MECA increases the ANP secretion mainly via A 3 receptor activation and positive inotropy by intracellular Ca 2+ regulation via the ryanodine receptor and CaMKII.

Attenuation of hypoosmotic stress-induced ANP secretion via ICl,swell in renal hypertensive rat atria

Cardiac hypertrophy, an adaptive process to an increased hemodynamic overload, includes not only an increase in cell size but also qualitative changes in constituent proteins. Although swelling-activated chloride channels ( I Cl,swell) chronically activate in hypertrophied atrial myocytes, the role of I Cl,swell in regulation of atrial natriuretic peptide (ANP) release is poorly understood. We investigated the effects of I Cl,swell on ANP release and contractility and its modification in hypertrophied rat atria. To stimulate I Cl,swell, hypoosmotic HEPES buffered solution (0.8T, 0.7T and 0.6T) was perfused into isolated perfused beating atria. The hypoosmotic HEPES buffered solution increased ANP release as compared to isoosmotic buffered solution (1T) in an osmolarity-reduction dependent manner. Atrial contractility and extracellular fluid translocation did not change. Exposure to hypoosmotic buffer (0.8T) containing low chloride (8 mM), tamoxifen or diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS) significantly attenuated hypoosmolarity-induced ANP release. The pretreatment with genistein, okdaic acid, U73122, GF109203x, and staurosporine attenuated hypoosmolarity-induced ANP release whereas orthovanadate augmented it significantly. In hypertrophied atria from renal hypertensive rats, hypoosmolarity-induced ANP release was markedly attenuated and DIDS-induced decrease in ANP release and negative inotropy were augmented as compared to sham-operated rat atria. Therefore, we suggest that I Cl,swell may partly participate hypoosmolarity-induced ANP release through protein tyrosine kinase and phospholipase C–protein kinase C pathway. The modification of responses of ANP release to hypoosmolarity and DIDS in hypertrophied atria may relate to changes in I Cl,swell activity by persistent high blood pressure.

Decreases in ANP secretion by lysophosphatidylcholine through protein kinase C.

Lysophosphatidylcholine (LPC) is an endogenous phospholipid released from the cell membrane during ischemia, and it has potent, local effects on cardiac tissues. LPC has been implicated in arrhythmogenesis during ischemia by increasing intracellular Ca2+. However, it is not known whether LPC influences atrial release of atrial natriuretic peptide (ANP). The aim of this study was to investigate the effect of LPC on ANP secretion from isolated, perfused, beating rat atria. LPC (10 and 30 micromol/L) caused decreases in ANP secretion in a dose-dependent manner, with slight increases in intra-atrial pressure and extracellular fluid (ECF) translocation. Therefore, the ANP secretion in terms of ECF translocation was markedly decreased by LPC. The order of the suppressive effect of ANP release was stearoyl-LPC>LPC>myristoyl-LPC=lauroyl-LPC. Staurosporine and wortmannin significantly attenuated suppression of the ANP release and an increase in intra-atrial pressure by LPC. High extracellular Mg2+ also attenuated the LPC-induced suppression of ANP release. However, other protein kinase C inhibitors such as chelerythrine, GF 109203X, and tamoxifen citrate did not affect LPC-induced suppression of ANP release. In single atrial myocytes, LPC caused increases in intracellular Ca2+ in a dose-dependent manner. The order of an increase in intracellular Ca2+ by LPC was stearoyl-LPC>LPC>myristoyl-LPC=lauroyl-LPC. An increase in intracellular Ca2+ by LPC was attenuated by staurosporine. These results suggest that LPC-induced suppression of ANP release through protein kinase C/Ca2+ and phosphoinositol-3-kinase might in part play an important role in the development of hypertension.

Stretch-activated atrial natriuretic peptide secretion in atria with heat shock protein 70 overexpression.

The mechanical overload of the heart is known to induce the expression of atrial natriuretic peptide (ANP) and heat-shock protein 70 (HSP70) in the heart. However, the relationship between these two substances remains unknown. In the present study, we characterized ANP secretion from mouse atria and examined a possible role that HSP70 may play in the regulation of ANP synthesis and secretion by using atria in transgenic mice in which HSP70 was overexpressed. We generated transgenic mice harboring the human HSP70gene under the transcriptional control of human myosin heavy-chain promoter. In these mice, the transgene was overexpressed in the heart. Both atrial ANP messenger RNA and its concentration in the HSP70 transgenic mice were measured; these were not significantly different from those in wild-type mice. In isolated perfused nonbeating atria, basal secretion of ANP was similar in both groups. When atrial volume was increased by changing atrial pressure, extracellular fluid (ECF) translocation and ANP secretion proportionately increased. Changes in atrial volume and ECF translocation and ANP secretion were positively correlated. However, these parameters did not significantly differ between the two groups. Endothelin-1 (ET-1), the strongest paracrine stimulus of ANP secretion, accentuated stretch-activated ANP secretion without significantly changing mechanically stimulated ECF translocation, as compared with that in the wild-type mice. The increased ANP secretion due to ET-1 in the transgenic mice was similar to that in the wild-type mice. The results suggest that both atrial stretching and ET-1 are important stimuli to ANP secretion from mouse atria, and the responsiveness of the ANP system to those stimuli are unlikely coupled to the pathway involving HSP70.

Postnatal changes in inhibitory effect of C-type natriuretic peptide on secretion of ANP.

To define developmental changes in atrial natriuretic peptide (ANP) secretion and in the cross talk between C-type natriuretic peptide (CNP) and ANP, we performed experiments in isolated perfused nonbeating cardiac atria isolated from rabbits between 1 and 8 wk of age. Changes in atrial pressure resulted in increases in atrial volume that rose with age and reached the peak value at 4 wk. A rise in volume change increased ANP secretion with concomitant translocation of extracellular fluid (ECF) into the atrial lumen, which increased with age and reached the peak value at 4 wk. The positive relationship between stretch-induced ANP secretion and ECF translocation shifted upward and leftward with age. CNP suppressed stretch-induced ANP secretion in the 8-wk-old group but not in the 2- and 4-wk-old groups without differences in ECF translocation and atrial volume. Therefore, the ANP secretion in terms of ECF translocation was markedly suppressed by CNP in the 8-wk-old group but not in the 2- and 4-wk-old groups. The production of cGMP by CNP in atrial tissue membranes was markedly attenuated in young rabbits. However, 8-bromo-cGMP suppressed stretch-induced ANP secretion in 2- and 8-wk-old groups. Natriuretic peptide receptor-B mRNA was similar in both groups. Therefore, we conclude that the inhibitory effect of CNP on atrial ANP secretion is developmentally regulated, being absent during normal cardiac development in young animals and intact in adult animals.

Mechanical control of extracellular space in rabbit atria: an intimate modulator of the translocation of extracellular fluid and released atrial natriuretic peptide.

We have previously shown that extracellular fluid (ECF) is translocated by atrial contraction. Following on from this finding we have proposed a two-step sequential mechanism for the regulation of stretch-activated secretion of atrial natriuretic peptide (ANP): myocytic release of ANP into the surrounding paracellular space followed by the translocation of ECF with the released ANP into the bloodstream. This latter step is induced by atrial contraction, and is therefore controlled by atrial workload. However, the mechanism that regulates the changes in translocation of the ECF has not been defined. To define the relationship between the atrial workload, ECF translocation, size of the extracellular space (ECS) and ANP secretion, experiments have been performed in isolated perfused beating rabbit atria. Atrial workload, transendocardial translocation of the ECF and the secretion of ANP were quantified. Changes in the size of the atrium and the ECS were determined by a newly developed methodology in the beating atria. Atrial workload determined the translocation of the ECF and released ANP with waning of the translocation at higher myocardial workloads. Atrial workload inversely determined the size of the atrium and the ECS. The latter directly determined the translocation of the ECF in terms of atrial workload. From these data we suggest that the size of the ECS is an intimate modulator of the translocation of the ECF and released ANP, and that the phenomenon of waning of the transendocardial translocation that appeared at higher atrial workloads is closely related to the shrinkage of the ECS.

Attenuation of negatively regulated ANP secretion by calcium in hypertrophied atria.

Abnormal intracellular Ca(2+)-handling has been described in various heart diseases associated with cardiac hypertrophy. The crucial role of Ca(2+) in the excitation-secretion coupling in atrial cardiomyocytes is not well established. To investigate modulation of atrial natriuretic peptide (ANP) secretion regulated by Ca(2+) in hypertrophied atria, responsiveness of stretch-induced ANP to Ca(2+) was studied using isolated perfused quiescent hypertrophied rat atria. Male Sprague-Dawley rats were given a single subcutaneous injection of 50 mg/kg monocrotaline (MCT) and were sacrificed at 5-6 weeks. In isolated perfused hypertrophied right atria from MCT rats, changes in atrial volume induced by increased atrial pressure caused proportional increases in mechanically stimulated extracellular fluid (ECF) translocation and stretch-induced ANP secretion. Stretch-induced ANP secretion was markedly increased by the depletion of extracellular Ca(2+). However, an accentuation of stretch-induced ANP secretion by Ca(2+) depletion was markedly attenuated in hypertrophied right atria, as compared to control right atria. Therefore, stretch-induced ANP secretion in terms of ECF translocation by Ca(2+) depletion in hypertrophied atria was significantly lower than in control right atria. However, no significant differences were observed between nonhypertrophied and control left atria. Depletion of extracellular Ca(2+) caused a decrease in intracellular calcium in single beating atrial myocytes, which was significantly attenuated in hypertrophied atrial myocytes. The results suggest that attenuation of Ca(2+)-induced negative regulation of ANP secretion in hypertrophied atria may be due to the disturbance of intracellular Ca(2+) regulation.

Attenuation of inhibitory effect of CNP on the secretion of ANP from hypertrophied atria.

It has been shown that atrial natriuretic peptide (ANP) influences proliferation of cardiac cells. To define the possible role of C-type natriuretic peptide (CNP) in cardiac hypertrophy, the influence of CNP on the secretion of ANP was studied with the use of perfused nonbeating atria from monocrotaline-treated rats. Increases in atrial volume caused proportional increases in ANP secretion that were markedly suppressed by CNP (10(-6) M) in nonhypertrophied left atria and control right atria but not in hypertrophied right atria. However, increases in atrial volume and mechanically stimulated extracellular fluid (ECF) translocation by CNP were similar to those in the control group. Therefore, the secretion of ANP in terms of ECF translocation was decreased by CNP in nonhypertrophied left and control right atria but not in hypertrophied atria. However, the inhibitory effect of 8-bromo-cGMP on the secretion of ANP was observed in both atria. The cGMP productions from perfused hypertrophied atria and their membranes exposed to CNP were significantly lower than those from nonhypertrophied atria. No significant difference in natriuretic peptide receptor-B transcript was found. Therefore, attenuation of the inhibitory effect of CNP on the ANP secretion in hypertrophied atria may be due to lack of cGMP production. The results showing the relief of CNP-induced negative inhibition of ANP secretion by atrial hypertrophy suggest that CNP may be a contributing factor to delay the development of cardiac hypertrophy.

Accentuation of ANP secretion to endothelin-1 in hypertrophied atria

To investigate modulation of ANP secretion by atrial hypertrophy, the secretion of ANP in response to stretch and endothelin-1 was studied using isolated perfused quiescent atria from rats treated with monocrotaline (MCT). Male Sprague-Dawley rats were given a single subcutaneous injection of 50 mg/kg MCT or saline and were sacrificed at 6 weeks. Rats with right heart hypertrophy showed an increase in ANP mRNA and decrease in tissue concentration of ANP in hypertrophied atria and a marked increase in plasma concentration of ANP. In isolated perfused hypertrophied right atria from MCT rats, changes in atrial volume induced by increased atrial pressure caused proportional increases in mechanically stimulated extracellular fluid (ECF) translocation and stretch-activated ANP secretion. Changes in atrial volume and mechanically stimulated ECF translocation in hypertrophied right atria were not different from those in control right atria. The stretch-activated ANP secretion was suppressed without significant difference in basal ANP secretion, as compared to control right atria. Therefore, the stretch-activated ANP secretion from hypertrophied right atria into the atrial lumen in relation to the ECF translocation (ANP concentration in the interstitium) was lower than that from control atria. A positive correlation between the stretch-activated ANP secretion in relation to the ECF translocation and tissue ANP content was found in control atria but not in hypertrophied atria. Endothelin-1 caused increases in stretch-activated ANP secretion in a dose-dependent manner, which were accentuated in hypertrophied right atria. Therefore, we suggest that atrial hypertrophy causes an attenuated response to stretch and accentuated response to endothelin-1 of ANP secretion.

Distinct roles for L- and T-type Ca(2+) channels in regulation of atrial ANP release.

Atrial secretion of atrial natriuretic peptide (ANP) has been shown to be regulated by atrial workload. Although modulating factors for the secretion of ANP have been reported, the role for intracellular Ca(2+) on the secretion of ANP has been controversial. The purpose of the present study was to define roles for L- and T-type Ca(2+) channels in the regulation of ANP secretion in perfused beating rabbit atria. BAY K 8644 (BAY K) increased atrial stroke volume and pulse pressure. BAY K suppressed ANP secretion and ANP concentration in terms of extracellular fluid (ECF) translocation concomitantly with an increase in atrial dynamics. BAY K shifted the relationship between ANP secretion and ECF translocation downward and rightward. These results indicate that BAY K inhibits myocytic release of ANP. In the continuous presence of BAY K, diltiazem reversed the effects of BAY K. Diltiazem alone increased ANP secretion and ANP concentration along with a decrease in atrial dynamics. Diltiazem shifted relationships between ANP secretion and atrial stroke volume or ECF translocation leftward. The T-type Ca(2+) channel inhibitor mibefradil decreased atrial dynamics. Mibefradil inhibited ANP secretion and ANP concentration in contrast with the L-type Ca(2+) channel inhibitor. These results suggest that activation of L- and T-type Ca(2+) channels elicits opposite effects on atrial myocytic release of ANP.

Glibenclamide suppresses stretch-activated ANP secretion: involvements of K+ATP channels and L-type Ca2+ channel modulation.

The mechanism by which glibenclamide regulates mechanically activated atrial natriuretic peptide (ANP) secretion was investigated using isolated perfused rat atria. A reduction in atrial pressure from an experimentally imposed distending pressure stimulated the secretion of ANP and caused concomitant translocation of extracellular fluid (ECF) into the atrial lumen. The activation of ANP secretion and ECF translocation were closely correlated with atrial volume changes and the increase in ANP secretion was a function of the ECF translocation. Glibenclamide (1, 10, 100 microM), an ATP-sensitive K+ (K+ATP) channel blocker, had no effect on the basal secretion of ANP, suppressed the stimulation of stretch-activated ANP secretion in a dose-dependent manner, but not the translocation of the ECF. Glipizide (100 microM) and tolbutamide (100 microM), other K+ATP channel blockers, had similar effects to those of glibenclamide. Suppression by glibenclamide (100 microM) of the stretch-induced ANP secretion was not observed in atria that had been pretreated with pinacidil (200 microM), an ATP-sensitive K+ channel opener: pinacidil alone had no effect on ECF translocation and ANP secretion. Furthermore, blocking Ca2+ influx by using the Ca2+ channel blocker diltiazem (10 nM), or a Ca2+-depleted medium prevented the suppression of stretch-induced ANP secretion by glibenclamide. In other experiments, atrial distension produced a slight membrane depolarization of cardiomyocytes; this was accentuated in the presence of glibenclamide. Furthermore, in single cardiomyocytes, glibenclamide increased the intracellular Ca2+ concentration ([Ca2+]i) in a dose-dependent manner. From these results, we suggest that glibenclamide suppresses atrial release of ANP by blocking K+ATP channels and increasing Ca2+ influx and that the K+ATP channels are associated with the regulation of the mechanically activated ANP secretion from the atria.

Mechanical basis of ANP secretion in beating atria: atrial stroke volume and ECF translocation.

To investigate the mechanism by which an increase in pacing frequency or distension increases the secretion of atrial natriuretic peptide (ANP), the changes in atrial volume during contraction (atrial stroke volume), transmural transport of the extracellular fluid (ECF), and ANP secretion were quantified in the beating perfused rabbit atria. The atrium was stimulated by transmural field stimulation or by atrial distension induced by an increase in intraatrial pressure. Atrial stretch and incremental increases in pacing frequency up to 2 Hz activated the secretion of ANP coincident with an increase in atrial stroke volume and the transendocardial translocation of the ECF. These results showed positive relationships between changes in the secretion of ANP and the atrial stroke volume or the translocation of the ECF. The translocation of the ECF was also positively correlated with the change in atrial stroke volume. The accentuated secretion of ANP and translocation of the ECF waned at higher stimulating rates to show a peak value. Even under this condition, the secretion of ANP was a function of the translocation of the ECF. These data suggest that the increases in atrial stroke volume and translocation of ECF are fundamental factors in the ANP stimulation in response to atrial stretch and increases in atrial rate.

Effect of extracellular calcium depletion on the two-step ANP secretion in perfused rabbit atria

Employing isolated perfused rabbit atrial model we have found that stretch-activated atrial natriuretic peptide (ANP) secretion takes place in two steps: release of ANP from myocytes into surrounding intercellular space, and then translocation of the released ANP into atrial lumen along with the extracellular fluid (ECF) translocated upon releasing the stretch. Ca 2+, one of the most important factors regulating secretory processes, has been reported by many workers to have influence on the ANP secretion, but at large variance. In the present study, therefore, we undertook to clarify the influence of Ca 2+ depletion on ANP secretion and further to define which of the two steps is affected by Ca 2+ removal. Extracellular Ca 2+ depletion resulted in increased basal secretion and in accentuated secretion of immunoreactive (ir) ANP in response to mechanical stimuli. The translocation of ECF increased in response to atrial stretch-and-release, but it was not affected by Ca 2+ depletion. The irANP concentration in the extracellular space calculated as the amount of irANP secreted in relation to the ECF translocated significantly increased when extracellular Ca 2+ was depleted. These results indicate that extracellular Ca 2+ depletion accentuates the stretch-induced ANP secretion through the augmentation of ANP release into the interstitium without changes in the ECF translocation.

Extracellular fluid translocation in perfused rabbit atria: implication in control of atrial natriuretic peptide secretion.

1. Transmural transport of 22Na+, 51Cr-EDTA, [3H]inulin and [14C]Dextran (57 kDa) was measured in perfused rabbit atria. The radiolabelled extracellular space (ECS) markers and [14C]Dextran were introduced into the pericardial space or atrial lumen. Atrial volume changes were induced by steps up and down in atrial pressure. 2. Basal rates of transmural transport of radiolabelled ECS markers across the atrial wall were relatively stable up to 70 min. Atrial stretch and release resulted in a rapid but transient, and reversible increase in the ECS fluid (ECF) translocation. The increased translocation of the ECF into the atrial lumen occurred within 15 s of the reduction of atrial distension and returned to the baseline level within 60 s. 3. Transmural transport of [3H]inulin across the atrial wall was bidirectional. 4. The clearance of radiolabelled ECS markers was molecular-size dependent. The transmural clearance of [3H]inulin was dependent on the distension-reduction volume changes induced by atrial stretch and release. Little transport of [14C]Dextran across the atrial wall was observed. 5. The ECF translocation across the atrial wall was not influenced by changes in external Ca2+ but was suppressed by low temperature. 6. Dynamic changes in the ECS of the atrium were observed in response to atrial distension and reduction. The ECS of the atrium increased on distension and decreased on reduction of atrial distension. 7. Reduction in atrial distension resulted in an increase in the secretion of immunoreactive atrial natriuretic peptide (ANP) which coincided with an increase in the translocation of the ECF. The secretion of immunoreactive ANP was a function of the translocation of the ECF. 8. It is suggested that atrial stretch and release may play a role in driving fluid flow within the interstitium and fluid translocation out of the interstitium. This fluid movement presumably leads to convective transport of released ANP into the atrial lumen.

Brain extracellular space in monkey fetuses subjected to prolonged partial asphyxia

Rhesus monkey fetuses, closely approaching term, were subjected to episodes of prolonged partial asphyxia while still in utero. Maternal blood presure was gradually decreased by increasing the concentration of inspired fluothane until a moderate degree of fetal compromise, indicated by a pH of 6.9–7.1 and a Po 2 of 15–18 mm Hg was sustained for 3–4 hr. Immediately after delivery by cesarian section, specimens of fetal cerebral cortex were frozen in liquid Freon-22, pre-chilled to —78C for 10 days. Electron micrographs of these specimens were prepared from which the relative volume of extracellular space was estimated stereometrically. A mean extracellular space of 4.1% in fetal animals subjected to prolonged partial asphyxia differed significantly from an extracellular space of 9.1% found in cerebral cortex from normal fetuses, similarly delivered. Additionally, mitochondria from asphyxiated fetuses were swollen and characterized by a dispersed, dilute-appearing matrix. These data suggested that the brain swelling concomitant with prolonged partial asphyxia is associated with a translocation of extracellular fluid, which appears to depend upon an altered mitochondrial metabolism and which results in a reduced volume of extracellular space.