This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Clemo, H. F. Articles by Baumgarten, C. M. Search for Related Content PubMed PubMed Citation Articles by Clemo, H. F. Articles by Baumgarten, C. M.

(Circulation Research. 1995;77:741-749.)
© 1995 American Heart Association, Inc.

Articles

cGMP and Atrial Natriuretic Factor Regulate Cell Volume of Rabbit Atrial Myocytes

Henry F. Clemo, Clive Marc Baumgarten

From the Departments of Internal Medicine and Physiology, Medical College of Virginia, Virginia Commonwealth University, Richmond.

Correspondence to Dr C.M. Baumgarten, Department of Physiology, Medical College of Virginia, Richmond, VA 23298-0551. E-mail baumgart@gems.vcu.edu.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abstract Atrial natriuretic factor (ANF) reduces the volume of atrial myocytes by inhibiting Na⁺/K⁺/2Cl^- cotransport. We determined the role of cGMP and cAMP in ANF-induced shrinkage by using digital video microscopy to measure cell volume; volumes are reported relative to control. ANF (1 µmol/L) reversibly reduced atrial cell volume from 1.0 to 0.915±0.005 (mean±SEM). This effect was mimicked by 10 µmol/L 8-bromo-cGMP (8-Br-cGMP), which decreased myocyte volume to 0.894±0.007 with an ED₅₀ of 0.99±0.05 µmol/L. In contrast, 100 µmol/L 8-bromo-cAMP (8-Br-cAMP) did not affect volume, and activating the cAMP pathway with 100 µmol/L 8-Br-cAMP did not alter the volume decrease caused by 8-Br-cGMP or ANF. Inhibition of Na⁺/K⁺/2Cl^- cotransport with bumetanide (1 µmol/L) also reduced cell volume and prevented further shrinkage on subsequent exposure to 8-Br-cGMP. Similarly, 8-Br-cGMP (10 µmol/L) prevented further shrinkage by ANF. Block of Na⁺-H⁺ exchange, a participant in volume regulation in other cells, did not alter the response to 8-Br-cGMP. More evidence implicating cGMP was obtained by altering its metabolism. LY83583 (10 µmol/L), a guanylate cyclase inhibitor, blocked ANF-induced cell shrinkage. Zaprinast (100 µmol/L), a cGMP-specific phosphodiesterase inhibitor, markedly potentiated the effect of a threshold concentration of ANF (0.01 µmol/L). The actions of ANF, LY83583, and zaprinast on cGMP levels were verified by radioimmunoassay. These data strongly support the idea that the cGMP cascade is the intracellular signaling pathway responsible for ANF-induced atrial cell shrinkage. ANF activates guanylate cyclase, and the subsequent rise in cGMP inhibits Na⁺/K⁺/2Cl^- cotransport. This reduces the uptake of osmolytes, and cell shrinkage follows. In addition, LY83583 caused a small swelling and zaprinast caused a small shrinkage in the absence of other agents. This suggests that physiological levels of cGMP partially inhibit Na⁺/K⁺/2Cl^- cotransport and help set isosmotic cell volume. It is proposed that ANF-induced elevation of atrial cGMP and the ensuing cell shrinkage serve as a negative-feedback mechanism limiting stretch-induced ANF secretion.

Key Words: atrial natriuretic peptide • cell volume • Na⁺/K⁺/2Cl^- cotransport • cGMP • phosphodiesterase

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Besides its well-known natriuretic, diuretic, and vasodilatory actions,^{1 2} ANF also manifests direct cardiac effects. Specific ANF binding sites have been identified in the atria,^{3 4} ventricles,^{3 4 5} and His-Purkinje system.⁶ At the myocyte level, ANF inhibits the Ca²⁺ current^{7 8 9} and a Ca²⁺-independent transient outward K⁺ current.⁹ Furthermore, we found that ANF inhibits Na⁺/K⁺/2Cl^- cotransport and causes an 8% reduction in cell volume in rabbit atrial and ventricular myocytes under isosmotic conditions.^{10 11}

The physiological relevance of the direct cardiac effects of ANF may be to regulate ANF secretion. Osmotic¹² and mechanical¹³ stretch of atrial and ventricular myocytes can more than double ANF release, and stretch-activated channels have been implicated in ANF secretion by intact atria.¹⁴ These data raise the possibility that ANF-induced cell shrinkage might act as a negative-feedback mechanism limiting further secretion of ANF.

In ventricle, ANF regulates Na⁺/K⁺/2Cl^- cotransport and cell volume by activating guanylate cyclase.¹¹ It is unclear, however, whether the same signaling pathway operates in the atria. ANF simultaneously increases cGMP^{6 11 15 16} and decreases cAMP^{3 6 16} in heart. Moreover, the regulation of Na⁺/K⁺/2Cl^- cotransport is tissue specific. cGMP inhibits Na⁺/K⁺/2Cl^- cotransport in flounder intestinal epithelium,¹⁷ cultured bovine vascular endothelium,¹⁸ cultured HeLa cells,¹⁹ and rabbit ventricle¹¹ but stimulates it in rat aortic vascular smooth muscle²⁰ and neuroblastoma NB-OK-1 cells.²¹ The erythrocyte cotransporter is controlled by cAMP instead of cGMP, and its response to cAMP is species dependent.^{22 23}

Another reason that the second messenger linking ANF to isosmotic cell volume regulation may differ in atria and ventricles is the need for the atria to control stretch-dependent ANF release while both the atria and ventricles carry out a constitutive ANF release. The processes underlying ANF secretion are complex, only partially defined, and influenced by numerous neurohumoral agonists, circulating peptides, and other pharmacological agents.²⁴ Of immediate relevance are findings that elevation of cGMP levels or treatment with membrane-permeant analogues inhibits ANF release,^{25 26 27} whereas there is disagreement whether elevation of cAMP or exposure to its membrane-permeant analogues inhibits^{26 28} or stimulates²⁷ ANF release.

The present experiments used digital video microscopy to identify the intracellular second messenger responsible for ANF-induced reduction of atrial cell volume. 8-Br-cGMP, a membrane-permeant hydrolysis-resistant analogue of cGMP, significantly reduced atrial cell volume. The decrease in cell volume appears to be due to inhibition of Na⁺/K⁺/2Cl^- cotransport by 8-Br-cGMP, because pretreatment of cells with bumetanide prevented this effect. Furthermore, when cells were pretreated with a saturating concentration of 8-Br-cGMP, ANF had no effect on cell volume. Inhibition of guanylate cyclase markedly attenuated the ability of ANF to shrink atrial myocytes, but inhibition of cGMP-specific phosphodiesterase potentiated it. In contrast, exposure to 8-Br-cAMP neither altered atrial cell volume by itself nor affected the response to 8-Br-cGMP or ANF. [¹²⁵I]cGMP radioimmunoassay confirmed that agents had the expected effects on atrial cGMP levels. Taken together, these data provide strong support for the hypothesis that ANF reduces atrial cell volume by increasing intracellular cGMP levels and inhibiting Na⁺/K⁺/2Cl^- cotransport. We postulate that ANF-induced elevation of cGMP may inhibit atrial ANF release at least in part by causing atrial cell shrinkage.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cell Isolation Procedure
Atrial myocytes were isolated from adult rabbits (New Zealand White, 1.5 to 2.5 kg) by a collagenase-pronase dispersion method.^{10 11} This procedure typically gave 60% rod-shaped, Ca²⁺-tolerant, viable cells. Myocytes were used within 6 hours of harvesting, and only quiescent cells without membrane blebs were selected for the experiments.

Solutions and Drugs
The basic Tyrode's solution contained (in mmol/L) NaCl 130, KCl 5, CaCl₂ 2.5, glucose 10, MgSO₄ 1.2, and HEPES 5 (pH 7.4) and was equilibrated with 100% O₂. For experiments investigating anisotonic conditions, NaCl was reduced to 65 mmol/L and held constant. Osmolarity was adjusted by adding 60 or 138 mmol/L mannitol to make either hyposmotic (0.8T, 244 mOsm/L) or isosmotic (1T, 308 mOsm/L) solutions, and solution osmolarity was routinely verified with a freezing-point depression osmometer (Osmette S, Precision Systems). Substitution of mannitol for NaCl in 1T solution does not significantly alter cell length, width, or volume.²⁹

Water-soluble compounds including [Ser¹⁰³,Tyr¹²⁶]ANF (rat atriopeptin III, Calbiochem), dimethylamiloride (Research Biochemicals), and 8-Br-cGMP and 8-Br-cAMP (Sigma Chemical Co) were dissolved in Tyrode's solution just before use. The amino acid sequence of rat ANF is identical to that from rabbit. Bumetanide (Hoffmann-La Roche), zaprinast (M&B22948, Rhône-Poulenc Rorer), and LY83583 (Eli Lilly) were prepared as stock solutions in dimethyl sulfoxide (Fluka). At the final concentrations in the tissue bath (bumetanide, 0.05% [vol/vol]; LY83583 and zaprinast, 0.1% [vol/vol]), dimethyl sulfoxide did not affect morphometric parameters.^{11 29}

Cells were superfused with bathing solution at 5 mL/min. Because of its expense, when ANF was used, bath flow was stopped after 1 minute, a time sufficient to change bath volume >10 times. In control experiments, 10 minutes of stopped flow in Tyrode's solution did not affect cell volume.¹⁰ All experiments were performed at room temperature (21°C to 22°C).

Determination of Atrial Myocyte Dimensions
Myocytes were placed in a poly-L-lysine–coated glass-bottomed chamber on the stage of an inverted microscope equipped with Hoffman modulation contrast optics (Nikon CF LWD 40x; numerical aperture, 0.55). By use of a high-resolution television camera mounted on the microscope, cell images were captured on-line by a Targa-M8 video frame grabber (Truevision) in an ISA bus computer. The area of the cell's image and an outline of the cell were obtained with JAVA image analysis software (Jandel). A program written in ASYST (Keithley-Asyst) was used to obtain the average of cell widths measured at 1-µm intervals. The axis for width measurements was chosen by rotating the cell outline to find the minimum average cell width, which is equivalent to measuring the width perpendicular to the long axis of the cell. Cell length was estimated as area divided by width.

Since changes in cell width and thickness upon exposure to test solutions were assumed to be proportional, relative cell volume was determined as follows:

where t and c refer to test (eg, ANF) and control solutions, respectively. Using three-dimensional cell analysis, we showed that changes in myocyte width and thickness are proportional after osmotic stress.²⁹ The present digital video microscopy method provides estimates of relative cell volume that are reproducible to <1%. More complete accounts of methodological details have appeared previously.^{10 11}

Determination of cGMP
To verify the effects of ANF, LY83583, and zaprinast, cGMP levels in atrial myocytes were determined by [¹²⁵I]cGMP radioimmunoassay using a commercially available kit (Amersham Life Sciences, RPA525). Details of the methods for extracting cGMP were described previously.¹¹ After the extract was dried, the residue was taken up in 0.25 mL of 50 mmol/L sodium acetate buffer (pH 4.7) containing 0.01% (wt/vol) sodium azide, and 0.1-mL aliquots were acetylated and assayed for cGMP in duplicate according to the kit instructions. Duplicate assays were averaged and corrected for cGMP recovery based on the recovery of [³H]cGMP.

Statistics
Data are reported as mean±SEM; n represents the number of cells. After ANOVA, multiple comparisons were made by the Bonferroni method. When only a single comparison was planned, Student's t test was used. The null hypothesis was rejected for P<.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
The length, width, and volume of atrial myocytes were measured after cells were equilibrated in Tyrode's solution for at least 15 minutes. The dimensions were as follows: width, 11.9±0.7 µm; length, 140.8±8.0 µm (n=8). Estimating cell volume as areaxwidth gave 20.5±2.4 pL. This calculation assumes that cells are brick shaped, with equal width and thickness. If cells are cylindrical instead of brick shaped, cell volume is overestimated by a factor of 1.27 (ie, 4/). To avoid uncertainties arising from the shape of the myocytes, each cell was used as its own control, and the remaining data are presented as relative cell volumes.

ANF and 8-Br-cGMP Reduce Atrial Cell Volume
Fig 1A demonstrates the effect of 1 µmol/L ANF on atrial myocytes. Relative cell volume significantly decreased from 1.0 under control conditions to 0.915±0.005 (n=4) within 2 minutes of adding ANF. The nearly 9% reduction in cell volume was stable throughout the 20-minute exposure to ANF, and cell volume promptly returned to its initial value on washout. Previously, we found that the ED₅₀ for ANF-induced myocyte shrinkage was 0.072 µmol/L, and the 1 µmol/L concentration applied here was a maximally effective dose.¹⁰ The magnitude and rapidity of the volume response suggest that the transport mechanisms mediating cell shrinkage have the capacity to generate a large net flux of osmolytes.

View larger version (16K): [in this window] [in a new window]   Figure 1. Time course of atrial myocyte shrinkage during a 20-minute exposure to 1 µmol/L ANF and 10 µmol/L 8-Br-cGMP. Relative cell volume is calculated as the ratio of volume under test conditions to the volume of the same cell in isosmotic control solution (1T, 308 mOsm/L). A, ANF induced a sustained decrease in relative cell volume. Myocyte volume fell from 1.0 in 1T control solution to 0.915±0.005 after 2 minutes in ANF and returned to its initial value after 5 minutes of washout (n=4 for each time point). B, 8-Br-cGMP also induced a sustained decrease in relative cell volume. Myocyte volume fell to 0.894±0.007 after 5 minutes and returned to its initial value after 10 minutes of washout (n=4 for each time point).

If cGMP functions as a second messenger for ANF in the process leading to atrial cell shrinkage, application of 8-Br-cGMP, a membrane-permeant, hydrolysis-resistant analogue, should mimic the action of ANF. This prediction is confirmed in Fig 1B, which shows the effects of a 20-minute exposure to 10 µmol/L 8-Br-cGMP. 8-Br-cGMP significantly reduced atrial cell volume to 0.894±0.007 (n=4) within 5 minutes, and thereafter, cell shrinkage was maintained at a nearly constant level. Upon washout of 8-Br-cGMP, volume returned to its control value after 10 minutes.

Several aspects of the effects of ANF and cGMP can be compared. Both ANF and 8-Br-cGMP decreased cell volume by reducing cell width, while cell length was unaffected. The maximum shrinkage induced by ANF, 0.912±0.005 at 10 minutes, was slightly less than that for 8-Br-cGMP, 0.892±0.011 at 10 minutes, but the difference was not significant (P=.148, two-tailed t test). In contrast, the kinetics of the cell-volume changes was noticeably slower with 8-Br-cGMP than with ANF. This difference is consistent with the time needed for 8-Br-cGMP to cross the sarcolemma, but the possibility that ANF activates additional mechanisms cannot be ruled out at this point.

A cumulative dose-response relation for 8-Br-cGMP is shown in Fig 2. Relative cell volume was significantly less than control at 0.01, 0.1, 1, 10, and 100 µmol/L 8-Br-cGMP, but the responses to 10 and 100 µmol/L 8-Br-cGMP were not significantly different from each other. Dose-response curves were constructed for each cell by assuming single-receptor occupancy. These gave an ED₅₀ of 0.99±0.05 µmol/L (n=4).

View larger version (12K): [in this window] [in a new window]   Figure 2. Cumulative dose-response curve for the reduction of cell volume by the membrane-permeant hydrolysis-resistant cGMP analogue 8-Br-cGMP. Cells were successively exposed to 0.01, 0.1, 1, and 10 µmol/L 8-Br-cGMP for 5-minute periods. The data for each cell were analyzed separately by assuming 1:1 binding, and the ED50 was 0.99±0.05 µmol/L. The solid curve is drawn from the average parameters. Because there are probably several steps between the binding of 8-Br-cGMP and the reduction of cell volume, the ED50 is likely to differ from the Kd for cGMP binding (n=4 for each dose).

Although cGMP may have a direct effect on atrial cell volume, the possibility that its action involves cAMP must be considered also. Cyclic nucleotide levels may be linked because cGMP and some of its analogues stimulate a phosphodiesterase that degrades cAMP.^{30 31} To test the possibility that a fall in cAMP is directly responsible for cell shrinkage, cAMP was "clamped" by pretreating cells with a high concentration of 8-Br-cAMP before challenging them with 8-Br-cGMP. As shown in Fig 3, 100 µmol/L 8-Br-cAMP alone had no effect on cell volume over a 10-minute period at room temperature. With the addition of 10 µmol/L 8-Br-cGMP, cell volume decreased from 1.003±0.010 to 0.920±0.012 (n=4, P<.01). The 8-Br-cGMP–induced cell shrinkage in the presence of 8-Br-cAMP was indistinguishable from that observed in the absence of 8-Br-cAMP (see Figs 1 and 2). These results indicate that a fall in cAMP is not required to obtain the effect of 8-Br-cGMP. Furthermore, they show that cell volume is unaffected by activation of the cAMP pathway.

View larger version (13K): [in this window] [in a new window]   Figure 3. 8-Br-cAMP alters neither atrial cell volume nor the response to 8-Br-cGMP. Atrial myocytes were exposed to 100 µmol/L 8-Br-cAMP for 10 minutes and then to 10 µmol/L cGMP in the continued presence of 8-Br-cAMP for an additional 10 minutes. Volume in 100 µmol/L 8-Br-cAMP, 1.003±0.010, was indistinguishable from control cell volume. Addition of 10 µmol/L 8-Br-cGMP to 8-Br-cAMP–treated cells significantly reduced cell volume to 0.920±0.012, a value indistinguishable from that observed with 8-Br-cGMP alone (see Fig 1). It is unknown whether 8-Br-cAMP affected the time course of shrinkage induced by 8-Br-cGMP (n=4 for each time point).

Na⁺/K⁺/2Cl^- Cotransport
Inhibition of Na⁺/K⁺/2Cl^- cotransport reduces atrial and ventricular cell volume,^{10 29} and cGMP inhibits Na⁺/K⁺/2Cl^- cotransport in rabbit ventricle,¹¹ teleost intestinal epithelium,¹⁷ and vascular endothelium.¹⁸ These findings suggest that 8-Br-cGMP may bring about atrial cell shrinkage by inhibiting Na⁺/K⁺/2Cl^- cotransport. If that is the case, inhibition of Na⁺/K⁺/2Cl^- cotransport by bumetanide³² should prevent the effect of 8-Br-cGMP. A test of this idea is presented in Fig 4A. As expected,²⁹ a 10-minute exposure to 10 µmol/L bumetanide decreased relative cell volume to 0.892±0.008 (n=4). However, addition of 10 µmol/L 8-Br-cGMP to the bumetanide-treated cells did not significantly reduce cell volume; relative to cell volume in 1T control solution (squares), cell volume in 8-Br-cGMP plus bumetanide was 0.888±0.012. To better illustrate the inability of 8-Br-cGMP to further shrink bumetanide-treated cells, cell volumes were recalculated relative to that in bumetanide alone (circles). Expressed in this manner, volume in bumetanide plus 8-Br-cGMP was 0.998±0.009 (n=4), which is indistinguishable from unity.

View larger version (22K): [in this window] [in a new window]   Figure 4. Bumetanide (BUM), an inhibitor of Na+/K+/2Cl- cotransport, prevents 8-Br-cGMP (8BrcG)–induced atrial myocyte shrinkage. In this and following figures, squares represent cell volumes relative to the control volume in 1T Tyrode's solution and describe volume changes over the course of the experiment; circles represent selected cell volumes relative to volumes in the preceding solution. A, BUM (10 µmol/L) significantly reduced cell volume to 0.892±0.008. The addition of 10 µmol/L 8-Br-cGMP to BUM-treated cells did not significantly alter myocyte volume. When compared with the volume of BUM-treated cells, 8-Br-cGMP plus BUM did not significantly alter cell volume (0.993±0.009, circles) (n=4 for each time point). B, Cell shrinkage alone cannot explain the block of the action of 8-Br-cGMP by BUM. The protocol illustrated in panel A was repeated in cells swollen in 0.8T. BUM (10 µmol/L) reduced volume of osmotically swollen cells from 1.170±0.012 to 1.029±0.014, which was slightly greater than control. The addition of 8-Br-cGMP to these cells did not significantly affect volume; volume was 0.997±0.008 relative to the volume of cells in BUM-0.8T solution (n=4 for each time point).

One may ask whether the elimination of the effect of 8-Br-cGMP on cell volume might have resulted from physical shrinkage rather than inhibition of Na⁺/K⁺/2Cl^- cotransport. To address this concern, the experiment was repeated in osmotically swollen cells (see Fig 4B). In 0.8T solution, atrial cells swelled to 1.170±0.012 (n=4), and 10 µmol/L bumetanide reduced cell volume to 1.029±0.014, a value slightly greater than in control. 8-Br-cGMP also was ineffective under these conditions. Expressed relative to the volume in control 1T solutions (squares), the volume of bumetanide-treated, osmotically swollen cells was 1.026±0.017 after a 10-minute exposure to 8-Br-cGMP. When volumes are normalized to those of osmotically swollen cells in bumetanide alone, the volume in bumetanide plus 8-Br-cGMP was 0.997±0.008 (n=4). These results argue that cell shrinkage by itself cannot account for the lack of effect of 8-Br-cGMP after Na⁺/K⁺/2Cl^- cotransport is inhibited by bumetanide.

An alternative model should be considered. ANF and cGMP have been reported to inhibit Na⁺-H⁺ exchange in vascular smooth muscle.³³ Because Na⁺-H⁺ exchange is responsible for significant Na⁺ entry in myocytes, its inhibition might contribute to cell shrinkage. To test this idea, the effect of 8-Br-cGMP was investigated in cells that had been pretreated with dimethylamiloride, an amiloride analogue that inhibits the cardiac isoform of the Na⁺-H⁺ exchanger with a K_i of 0.1 µmol/L. As shown in Fig 5A, a 10-minute exposure to 20 µmol/L dimethylamiloride under isosmotic conditions did not significantly affect cell volume (1.007±0.013, n=4). Addition of 8-Br-cGMP (10 µmol/L) to dimethylamiloride-treated cells reduced cell volume to 0.884±0.016. This shrinkage was indistinguishable from that induced by 8-Br-cGMP alone, as shown in Fig 1B (0.894±0.007) and confirmed in these cells immediately before blockade of Na⁺-H⁺ exchange (0.891±0.011; data not shown). Similar effects were seen in osmotically swollen cells (Fig 5B). These experiments rule out inhibition of Na⁺-H⁺ exchange as the mechanism of cGMP-dependent volume regulation.

View larger version (21K): [in this window] [in a new window]   Figure 5. Inhibition of Na+-H+ exchange with dimethylamiloride (DMA, 20 µmol/L) did not alter cell volume or the response to 8-Br-cGMP (8BrcG, 10 µmol/L). A, After a 10-minute exposure to DMA, cell volume was 1.007±0.013, not significantly different from that in 1T control solution. 8-Br-cGMP induced a shrinkage of DMA-treated cells to 0.878±0.013 relative to cell volume in DMA alone. This shrinkage was indistinguishable from that observed in these cells with 8-Br-cGMP before inhibiting Na+-H+ exchange (0.891±0.011; data not shown) and the 8-Br-cGMP–induced shrinkage illustrated in Fig 1 (n=4 for each time point). B, Similar results are shown for cells osmotically swollen in 0.8T (n=4 for each time point). Volumes denoted by squares are relative to 1T control solution, and volumes denoted by circles are relative to the preceding solution.

Effect of cGMP on ANF-Induced Atrial Myocyte Shrinkage
The hypothesis that cGMP is the second messenger for ANF predicts that pretreatment of cells with a large dose of 8-Br-cGMP should cause the maximum cell shrinkage attainable with ANF. Therefore, subsequent activation of guanylate cyclase by ANF binding should have no effect. This prediction was explored in the experiments depicted in Fig 6. First, myocytes were treated for 10 minutes with 10 µmol/L 8-Br-cGMP, and cell volume fell to 0.893±0.007 (n=4). Then, they were challenged with 1 µmol/L ANF. ANF did not significantly alter the volume of 8-Br-cGMP–treated cells. Relative to the control volume (squares), cell volume was 0.891±0.012 after 10 minutes in ANF plus bumetanide; this is 0.998±0.014 relative to the volume of myocytes exposed to 8-Br-cGMP alone (circles). As before, we repeated this experiment in osmotically swollen cells to prove that cell shrinkage caused by 8-Br-cGMP is not itself blocking the effect of ANF (Fig 6B). 8-Br-cGMP (10 µmol/L) reduced the volume of atrial cells in 0.8T solution from 1.168±0.012 to 1.041±0.008 (n=5). After osmotic swelling, 1 µmol/L ANF still failed to alter the volume of 8-Br-cGMP–treated cells. Relative to that in 1T solution (squares), cell volume in 8-Br-cGMP and ANF was 1.033±0.008, which was 0.993±0.012 of the cell volume in 8-Br-cGMP alone (circles).

View larger version (23K): [in this window] [in a new window]   Figure 6. 8-Br-cGMP prevents ANF-induced atrial cell shrinkage. A, In 1T solution, 10 µmol/L 8-Br-cGMP induced a significant cell shrinkage (squares). A 10-minute exposure to 1 µmol/L ANF did not significantly reduce the volume of 8-Br-cGMP–treated cells; cell volume was 0.998±0.014 relative to volume in 8-Br-cGMP alone (circles) (n=4 for each time point). B, Similar results were observed in 8-Br-cGMP–treated cells that were first swollen in 0.8T solution (n=5 for each time point). Volumes denoted by squares are relative to 1T control solution, and volumes denoted by circles are relative to the preceding solution.

We already have shown that cGMP does not affect cell volume by modulating cAMP levels, but that is not sufficient. Because ANF also inhibits adenylate cyclase and decreases cAMP in heart,^{3 6 16} it is important to determine whether the effect of ANF on cell volume could result from its ability to lower cAMP levels independent of cGMP. To evaluate this question, the effect of ANF was studied in cells pretreated with of 8-Br-cAMP to "clamp" the cAMP pathway (see Fig 7). After confirming that 100 µmol/L 8-Br-cAMP alone did not significantly affect cell volume (0.997±0.008, n=4), 1 µmol/L ANF was added to 8-Br-cAMP–treated cells. This maneuver reduced cell volume to 0.913±0.003, a shrinkage indistinguishable from that observed with 1 µmol/L ANF alone (0.915±0.005; see Fig 1). Failure of 8-Br-cAMP to alter the response to ANF implies that cAMP does not mediate the effect of ANF on atrial cell volume.

View larger version (14K): [in this window] [in a new window]   Figure 7. 8-Br-cAMP does not inhibit ANF-induced atrial cell shrinkage. In cells pretreated with 100 µmol/L 8-Br-cAMP, 1 µmol/L ANF reduced atrial cell volume to 0.913±0.003. This shrinkage was indistinguishable from that observed with ANF alone (see Fig 1) (n=4 for each time point).

Modulation of cGMP Metabolism
The present results argue that ANF reduces atrial cell volume by increasing intracellular cGMP levels. A consequence of this hypothesis is that inhibition of guanylate cyclase should prevent the elevation of cytoplasmic cGMP levels and block ANF-induced cell shrinkage. Fig 8 demonstrates the ability of 10 µmol/L LY83583, a specific inhibitor of guanylate cyclase,^{34 35} to block cell shrinkage. Exposure to LY83583 for 10 minutes caused a small but significant increase of cell volume to 1.021±0.003 (n=5, P<.01). When 1 µmol/L ANF was added to LY83583-treated cells, volume decreased slightly to 0.999±0.003 (squares). This shrinkage was to 0.978±0.005 (P<.01), relative to the volume of LY83583-treated cells (circles), but was much less than the shrinkage to 0.919±0.005 caused by 1 µmol/L ANF alone (P<.01). Thus, inhibition of guanylate cyclase markedly attenuates the effect of ANF. Control studies verified that the cell volume response to 1 µmol/L ANF alone did not decay with repeated applications.

View larger version (15K): [in this window] [in a new window]   Figure 8. LY83583, an inhibitor of guanylate cyclase, attenuated ANF-induced atrial cell shrinkage. Exposure of cells to 10 µmol/L LY83583 for 10 minutes increased cell volume slightly to 1.021±0.003 (n=5) (squares). Addition of 1 µmol/L ANF in the continued presence of LY83583 decreased cell volume to 0.978±0.005 relative to that in LY83583 alone (circles). This shrinkage was statistically significant, but it was significantly smaller than the decrease in volume to 0.919±0.005 caused by 1 µmol/L ANF alone (n=5 for each time point). Volumes denoted by squares are relative to 1T control solution, and volumes denoted by circles are relative to the preceding solution.

Another method of demonstrating that cGMP is the second messenger for ANF-induced cell shrinkage makes use of zaprinast, a cGMP-specific phosphodiesterase (type V) inhibitor.^{31 36} Slowing the degradation of cGMP is expected to potentiate the effect of very low concentrations of ANF. Fig 9 illustrates the effect of 0.01 µmol/L ANF on cell volume before and after inhibiting the phosphodiesterase. This is 1/100th of the concentration of ANF used in the preceding experiments and is much less than 0.072 µmol/L, the ED₅₀ of for ANF-induced myocyte shrinkage.¹⁰ Exposure to 0.01 µmol/L ANF for 10 minutes reduced atrial myocyte volume to 0.984±0.007 (n=4, P=.0532). After washout of ANF, 100 µmol/L zaprinast reduced cell volume from 1.008±0.008 to 0.973±0.008 (P<.02). Adding 0.01 µmol/L ANF to zaprinast-treated cells caused a further reduction of cell volume to 0.904±0.014. Expressed relative to the volume in zaprinast alone, 0.01 µmol/L ANF reduced volume to 0.922±0.009 (circles). This shrinkage was significantly greater than the shrinkage caused by ANF alone (P<.01) and the sum of the shrinkages caused by ANF alone and zaprinast alone (P<.028). Thus, inhibiting the degradation of cGMP potentiated the effect of ANF on cell volume.

View larger version (16K): [in this window] [in a new window]   Figure 9. Zaprinast, a cGMP-specific phosphodiesterase inhibitor, potentiated the effect of a low concentration of ANF. Cell volume decreased slightly to 0.984±0.007 on exposure to 0.01 µmol/L ANF (squares). After washout, cells were treated with 100 µmol/L zaprinast for 10 minutes. By itself, zaprinast reduced cell volume slightly, to 0.973±0.008. After zaprinast, however, 0.01 µmol/L ANF significantly decreased volume to 0.922±0.009 relative to that in zaprinast alone (circles). This volume decrease was significantly greater than that caused by 0.01 µmol/L ANF alone and also was greater than the additive effects of ANF and zaprinast (n=4 for each time point). Volumes denoted by squares are relative to 1T control solution, and volumes denoted by circles are relative to the preceding solution.

cGMP Levels in Atrial Myocytes
cGMP levels were measured in atrial myocytes by radioimmunoassay to confirm that the effects of ANF, LY83583, and zaprinast under the present experimental conditions were as expected from the literature (Fig 10). The protocols paralleled those used to measure cell volume. However, spontaneous ANF release into the bathing media may be much more important in the concentrated aliquot of cells used to measure cGMP levels than in the very dilute sample used to measure cell volume. After inhibiting guanylate cyclase with 10 µmol/L LY83583 for 10 minutes, cGMP was 0.11±0.01 pmol/10⁶ cells and was not significantly affected by a 10-minute exposure to 1 µmol/L ANF. In contrast, when guanylate cyclase was not inhibited, 5-, 10-, and 20-minute exposures to 1 µmol/L ANF increased cGMP to between 0.22±0.01 and 0.27±0.01 pmol/10⁶ cells. A much lower concentration of exogenous ANF, 0.01 µmol/L (10 minutes), did not significantly increase cGMP above that observed when guanylate cyclase was inhibited. This mirrors the lack of significant cell shrinkage with 0.01 µmol/L ANF. Finally, inhibition of cGMP-specific phosphodiesterase by 100 µmol/L zaprinast also had the expected effects. In the absence of added ANF, zaprinast increased cGMP to 0.20±0.01 pmol/10⁶ cells, nearly the same level as observed with 1 µmol/L ANF alone. Moreover, zaprinast significantly potentiated the effect of 0.01 µmol/L ANF; the combination elevated cGMP to 0.32±0.01 pmol/10⁶ cells.

View larger version (25K): [in this window] [in a new window]   Figure 10. cGMP levels in isolated atrial myocytes were determined by [125I]cGMP radioimmunoassay. Experiments were performed in triplicate by using aliquots of 0.4x106 cells per tube, and each cGMP assay was performed in duplicate, averaged, and corrected for recovery. The concentrations applied were as follows: ANF, 0.01 and 1.0 µmol/L; LY83583 (LY), 10 µmol/L; and zaprinast (ZAP), 100 µmol/L. Cells were exposed to the indicated agent for 10 minutes, except 5-, 10-, and 20-minute exposures were used for 1 µmol/L ANF. cGMP levels in cells treated with 1 µmol/L ANF were greater than after treatment with 0.01 µmol/L ANF or after inhibiting guanylate cyclase with LY83583. Inhibition of guanylate cyclase blocked the effect of ANF (LY+ANF), and inhibition of cGMP-specific (type V) phosphodiesterase with zaprinast increased cGMP levels in the absence of exogenous ANF and potentiated the effect of 0.01 µmol/L ANF (ZAP+ANF).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
These studies suggest that cGMP is the intracellular signal by which ANF modulates rabbit atrial cell volume under isosmotic conditions. We propose that activation of the cGMP pathway leads to inhibition of coupled Na⁺, K⁺, and Cl^- influx via the Na⁺/K⁺/2Cl^- cotransporter. Because influx and efflux of osmotic equivalents must balance exactly in the steady state, inhibition of this component of ion influx creates a net loss of osmolytes, and cell shrinkage occurs as water follows. An identical mechanism operates in rabbit ventricle,¹¹ and preliminary data suggest its operation in human ventricle as well.³⁷

Evidence for cGMP as a Second Messenger
Several lines of evidence point to the crucial role of cGMP in modulating ANF-induced cell shrinkage in rabbit atrial myocytes. First, ANF previously has been shown to increase cGMP levels in cardiac muscle.^{6 11 15 16} Second, the membrane-permeant analogue 8-Br-cGMP mimics the effect of ANF on cell volume and induces a comparable shrinkage (10%). Third, LY83583, an inhibitor of guanylate cyclase,^{34 35} markedly attenuated the ANF-induced reduction of cell volume. Fourth, zaprinast, an inhibitor of cGMP-specific (type V) phosphodiesterase,^{31 36} potentiated the shrinkage caused by a low concentration of ANF. Finally, [¹²⁵I]cGMP radioimmunoassay demonstrated that ANF increased cGMP in atrial myocytes and that LY83583 attenuated and zaprinast potentiated this action. The present radioimmunoassay data are in accord with a recent report that anantin and HS-142-1, ANF-A (R₁) receptor antagonists, decrease cGMP in cultured atrial myocytes.²⁵

The role of the cGMP system is not limited to responding to exogenous stimulation. Close examination of "control" experiments with LY83583 and zaprinast alone suggests that atrial cell volume and Na⁺/K⁺/2Cl^- cotransport may be modulated by varying cGMP around its physiological level. cGMP is continually produced. Blockade of cGMP degradation by zaprinast results in the predicted increase in cGMP, and an inhibition of Na⁺/K⁺/2Cl^- cotransport can explain the observed 3.5% cell shrinkage. On the other hand, inhibition of cGMP synthesis by LY83583 should lead to a fall in cGMP. We detected a small amount of cell swelling (2%) but failed to observe a fall in cGMP. Perhaps the unavoidable presence of damaged myocytes in cell aliquots used to measure cGMP levels masked a small effect. Nonetheless, the small swelling induced by LY83583 is consistent with a disinhibition of Na⁺/K⁺/2Cl^- cotransport and increased osmolyte uptake.

Besides its well-known effect to elevate cGMP levels, ANF also inhibits adenylate cyclase and reduces the cytoplasmic cAMP concentration in a number of tissues including the heart.^{3 6 16} Adenylate cyclase is inhibited via a pertussis toxin–sensitive signal activated by the ubiquitous "clearance" receptors, termed ANF-C (R₂).³⁸ Furthermore, cGMP may lower cAMP levels by stimulating a cAMP-specific phosphodiesterase.^{30 31} Although the ANF-C receptor and a fall in cytoplasmic cAMP have been implicated in certain physiological responses, the present findings show that cAMP is not involved in atrial cell volume regulation. This argument is based on experiments "clamping" the cAMP pathway in an activated state with 100 µmol/L 8-Br-cAMP, a membrane-permeant, hydrolysis-resistant analogue. Direct activation of the cAMP pathway should have interrupted mechanisms dependent on a fall of cAMP. Nevertheless, pretreatment with 8-Br-cAMP did not affect cell shrinkage induced by ANF or 8-Br-cGMP. Furthermore, 8-Br-cAMP did not affect cell volume by itself, arguing that cell volume also is not sensitive to increases in cAMP.

Role of Na^+/K+/2Cl^- Cotransport
If cGMP is the second messenger for ANF, it should act on the same processes. We established previously that ANF reduces the volume of rabbit atrial and ventricular cells by inhibiting Na⁺/K⁺/2Cl^- cotransport.¹⁰ The simplest explanation for the present finding that pretreatment with bumetanide fully prevented 8-Br-cGMP from modulating atrial cell volume is that cGMP, like ANF, acts by inhibiting Na⁺/K⁺/2Cl^- cotransport. This is consistent with observations that ANF inhibits Na⁺/K⁺/2Cl^- cotransport via cGMP diverse tissues,^{17 18} including rabbit ventricle.¹¹ Furthermore, the ED₅₀s are comparable to those in rabbit atria.

cGMP affects several membrane transport processes besides Na⁺/K⁺/2Cl^- cotransport, and the possibility that these contribute to cell-volume regulation must be considered. For example, ANF and cGMP inhibit Na⁺-H⁺ exchange in vascular smooth muscle,³³ and decreasing Na⁺ entry via Na⁺-H⁺ exchange also might cause cell shrinkage. Such a mechanism does not appear to be important in atria, however. The Na⁺-H⁺ exchange blocker dimethylamiloride failed to altered cell volume under both isotonic and hypotonic conditions and failed to attenuate cell shrinkage caused by 8-Br-cGMP. In addition, cGMP partially inhibits Ca²⁺ current in heart^{7 8 9} and opens Ca²⁺-activated K⁺ channels in other tissues.^{39 40 41} These are unlikely to explain cGMP-induced cell shrinkage because blocking Ca²⁺ entry by removing Ca²⁺ from the bathing medium and with verapamil does not affect cell volume.⁴² Finally, cGMP also activates cyclic nucleotide–gated channels, and a member of this family is expressed in heart.⁴³ Cyclic nucleotide–gated channels are activated by both cGMP and cAMP, whereas cAMP did not affect cell volume.

A more complex possibility is that cGMP might modulate cell volume via an efflux of K⁺ and Cl^- through separate conductive pathways linked by macroscopic electroneutrality, as occurs in certain instances during a regulatory volume decrease. In other preparations, cGMP enhances the opening of ATP-sensitive K⁺ channels^{41 44} and Cl^- channels,^{45 46} and in human atria, it enhances the opening of Ca²⁺-independent transient outward K⁺ channels.⁹ If cGMP-induces a parallel efflux of K⁺ and Cl^-, in order to explain the effect of bumetanide it would be necessary to postulate that cGMP opens a bumetanide-sensitive Cl^- channel or a K⁺ channel that is linked by macroscopic electroneutrality to a bumetanide-sensitive Cl^- channel. Although bumetanide is reported to block Cl^- channels in epithelia⁴⁷ and nerve,⁴⁸ such a scheme is contradicted by other observations. Block of K⁺ and Cl^- efflux by bumetanide should increase cell volume. To the contrary, bumetanide induces cell shrinkage.^{10 29} In addition, bumetanide decreases intracellular Cl^- activity.⁴⁹ The postulated block of Cl^- channels would be expected to increase intracellular Cl^- activity because the electrochemical Cl^- gradient is directed outward in cardiac cells.⁵⁰ Activation of ATP-sensitive K⁺ channels also cannot explain cell shrinkage because neither aprikalim nor glibenclamide alters cell volume.⁴²

Although it seems likely that inhibition of Na⁺/K⁺/2Cl^- cotransport by cGMP is the primary mechanism underlying cell shrinkage, we cannot exclude contributions of other transport processes, even those ruled out above as the primary mechanism, to the final outcome. Besides the direct effects of cGMP, modulation of Na⁺/K⁺/2Cl^- cotransport may indirectly affect other transport processes by altering ion gradients. Na⁺/K⁺/2Cl^- cotransport is a major route of Na⁺ entry, and its inhibition may secondarily affect the activity of the Na⁺-K⁺ pump and Na⁺-Ca²⁺ exchange. Perhaps the strongest argument for participation of other transport processes is the fact that cell volume rapidly attains a new steady state after inhibition of Na⁺/K⁺/2Cl^- cotransport. Stable cell volume implies a zero net osmolyte flux despite continued inhibition of Na⁺/K⁺/2Cl^- cotransport. Therefore, the osmotic consequences of block of ion uptake by Na⁺/K⁺/2Cl^- cotransport must be balanced by other transport processes. In effect, the volume setup point has been reset, at least over the time course of these studies.

Physiological Implications
The physiological release of ANF appears to depend on the opening of stretch-activated channels¹⁴ and is known to be under negative-feedback control. ANF decreases central venous pressure via its natriuretic, diuretic, and vasodilatory actions, thereby reducing atrial stretch and the stimulus for further ANF secretion.²⁴ The present results in combination with several recent reports define a second, more direct, negative-feedback pathway. Inhibition of atrial ANF-A receptors increases ANF secretion,²⁵ and activation of the cGMP cascade inhibits secretion.^{27 28} However, the basis for these effects has not been clear. It is proposed that ANF increases atrial cGMP levels and that the ensuing cell shrinkage inhibits ANF secretion by reducing the opening of stretch-activated channels. A fall in atrial contractility, either by inhibition of Na⁺/K⁺/2Cl^- cotransport, reduced Na⁺ entry, and reduction in cellular Ca²⁺ via Na⁺-Ca²⁺ exchange or by inhibition of Ca²⁺ current,^{7 8 9} also may contribute.

Other processes that affect atrial cGMP levels also may modulate ANF release. One important mechanism is activation of soluble guanylate cyclase by nitric oxide. Most myocytes are within 8 µm of a capillary, and endothelium-derived relaxing factor, nitric oxide or a closely related compound, is thought to alter cardiac cell function.⁵¹ Nitrovasodilators also activate soluble guanylate cyclase and reduce ventricular cell volume.¹¹ A cytokine-inducible nitric oxide synthase is expressed in rat cardiac myocytes.⁵² Endotoxin, interleukins, and tumor necrosis factor all increase ventricular cGMP via a nitric oxide synthase within cardiac myocytes.^{53 54 55} It remains to be seen, however, whether nitric oxide–dependent activation of soluble guanylate cyclase reduces atrial cell volume or ANF secretion.

The present results indicate that ANF decreases atrial cell volume by means of a cGMP-dependent inhibition of Na⁺/K⁺/2Cl^- cotransport. Furthermore, physiological cGMP levels appear to help determine cell volume in the absence of exogenous stimulation. cGMP is modulated by physiological and pathophysiological processes. Consequently, atrial cell volume may not be constant as often is assumed.

	Selected Abbreviations and Acronyms

 

ANF = atrial natriuretic factor dimethylamiloride = 5-(N,N-dimethyl)-amiloride-HCl LY83583 = 6-anilinoquinoline-5,8-quinone

	Acknowledgments

 
This study was supported in part by National Institutes of Health grants HL-46764 and HL-02798 and the P.D. and E.O. Sang Foundation. We thank Dr Joseph J. Feher for his participation in the radioimmunoassay experiments and Jude Maghirang for software development and preparation of the figures.

Received November 4, 1994; accepted June 23, 1995.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Brenner BM, Ballermann BJ, Gunning ME, Zeidel ML. Diverse biological actions of atrial natriuretic peptide. Physiol Rev. 1990;70:665-699. [Free Full Text]

2. de Zeeuw D, Janssen WM, de Jong PE. Atrial natriuretic factor: its (patho)physiological significance in humans. Kidney Int. 1992;41:1115-1133. [Medline] [Order article via Infotrieve]

3. Anand-Srivastava MB, Cantin M. Atrial natriuretic factor receptors are negatively coupled to adenylate cyclase in cultured atrial and ventricular cardiocytes. Biochem Biophys Res Commun. 1986;138:427-436. [Medline] [Order article via Infotrieve]

4. Rutherford RA, Wharton J, Gordon L, Moscoso G, Yacoub MH, Polak JM. Endocardial localization and characterization of natriuretic peptide binding sites in human and fetal adult heart. Eur J Pharmacol. 1992;212:1-7. [Medline] [Order article via Infotrieve]

5. Rugg EL, Aiton JF, Cramb G. Atrial natriuretic peptide receptors and activation of guanylate cyclase in rat cardiac sarcolemma. Biochem Biophys Res Commun. 1989;162:1339-1345. [Medline] [Order article via Infotrieve]

6. Anand-Srivastava MB, Thibault G, Sola C, Fon E, Ballak M, Charbonneau C, Haile-Meskel H, Garcia R, Genest J, Cantin M. Atrial natriuretic factor in Purkinje fibers of rabbit heart. Hypertension. 1989;13:789-798. [Abstract/Free Full Text]

7. Mery PF, Lohmann SM, Walter U, Fischmeister R. Ca²⁺ current is regulated by cyclic GMP-dependent protein kinase in mammalian cardiac myocytes. Proc Natl Acad Sci U S A. 1991;88:1197-1201. [Abstract/Free Full Text]

8. Tohse N, Sperelakis N. cGMP inhibits the activity of single calcium channels in embryonic chick heart cells. Circ Res. 1991;69:325-331. [Abstract/Free Full Text]

9. Le Grand B, Deroubaix E, Couetil JP, Coraboeuf E. Effects of atrionatriuretic factor on Ca²⁺ current and Ca_i-independent transient outward K⁺ current in human atrial cells. Pflugers Arch. 1992;421:486-491. [Medline] [Order article via Infotrieve]

10. Clemo HF, Baumgarten CM. Atrial natriuretic factor decreases cell volume of rabbit atrial and ventricular myocytes. Am J Physiol. 1991;260:C681-C690. [Abstract/Free Full Text]

11. Clemo HF, Feher JJ, Baumgarten CM. Modulation of rabbit ventricular cell volume and Na⁺/K⁺/2Cl^- cotransport by cGMP and atrial natriuretic factor. J Gen Physiol. 1992;100:89-114. [Abstract/Free Full Text]

12. Greenwald JE, Apkon M, Hruska KA, Needleman P. Stretch-induced atriopeptin secretion in isolated rat myocyte and its negative modulation by calcium. J Clin Invest. 1989;83:1061-1065.

13. Gardner DG, Wirtz H, Dobbs LG. Stretch-dependent regulation of atrial peptide synthesis and secretion in cultured atrial cardiocytes. Am J Physiol. 1992;263:E239-E244. [Abstract/Free Full Text]

14. Laine M, Arjamaa O, Vuolteenaho O, Ruskoaho H, Weckström M. Block of stretch-activated atrial natriuretic peptide secretion by gadolinium in isolated rat atrium. J Physiol (Lond). 1994;480:553-561. [Abstract/Free Full Text]

15. Cramb G, Banks R, Rugg EL, Aiton JF. Actions of atrial natriuretic peptide (ANP) on cyclic nucleotide concentrations and phosphatidylinositol turnover in ventricular myocytes. Biochem Biophys Res Commun. 1987;148:962-970. [Medline] [Order article via Infotrieve]

16. McCall D, Fried TA. Effect of atriopeptin II on Ca influx, contractile behavior, and cyclic nucleotide content of cultured neonatal rat myocardial cells. J Mol Cell Cardiol. 1990;22:201-212. [Medline] [Order article via Infotrieve]

17. O'Grady SM, Field M, Nash NT, Rao MC. Atrial natriuretic factor inhibits Na-K-Cl cotransport in teleost intestine. Am J Physiol. 1985;249:C531-C534. [Abstract/Free Full Text]

18. O'Donnell ME. Regulation of Na-K-Cl cotransport in endothelial cells by atrial natriuretic factor. Am J Physiol. 1989;257:C36-C44. [Abstract/Free Full Text]

19. Kort JJ, Koch G. The Na⁺, K⁺, 2Cl^--cotransport system in HeLa cells: aspects of its physiological regulation. J Cell Physiol. 1990;145:253-261. [Medline] [Order article via Infotrieve]

20. O'Donnell ME, Owen NE. Atrial natriuretic factor stimulates Na/K/Cl cotransport in vascular smooth muscle cells. Proc Natl Acad Sci U S A. 1986;83:6132-6136. [Abstract/Free Full Text]

21. Delporte C, Winand J, Poloczek P, Christophe J. Regulation of Na-K-Cl cotransport, Na,K-adenosine triphosphatase and Na/H exchanger in human neuroblastoma NB-OK-1 cells by atrial natriuretic peptide. Endocrinology. 1993;133:77-82. [Abstract/Free Full Text]

22. Garay R, Ciccone J. Inhibition of the Na⁺/K⁺ cotransport system by cyclic AMP and intracellular Ca²⁺ in human red cells. Biochim Biophys Acta. 1982;688:786-792. [Medline] [Order article via Infotrieve]

23. Palfrey HC, Rao MC. Na⁺-K⁺-2Cl^- cotransport and its regulation. J Exp Biol. 1983;106:43-54. [Abstract/Free Full Text]

24. Ruskoaho H. Atrial natriuretic peptide: synthesis, release, and metabolism. Pharmacol Rev. 1992;44:479-602. [Medline] [Order article via Infotrieve]

25. Nachshon S, Zamir O, Matsuda Y, Zamir N. Effects of ANP receptor antagonists on ANP secretion from adult rat cultured atrial myocytes. Am J Physiol. 1995;268:E428-E432. [Abstract/Free Full Text]

26. Page E, Goings GE, Power B, Upshaw-Earley J. Basal and stretch-augmented natriuretic peptide secretion by quiescent rat atria. Am J Physiol. 1990;259:C801-C818. [Abstract/Free Full Text]

27. Ruskoaho H, Toth M, Ganten D, Unger T, Lang RE. The phorbol ester induced atrial natriuretic peptide secretion is stimulated by forskolin and Bay k8644 and inhibited by 8-bromo-cyclic GMP. Biochem Biophys Res Commun. 1986;139:266-274. [Medline] [Order article via Infotrieve]

28. Iida H, Page E. Inhibition of atrial natriuretic peptide secretion by forskolin in noncontracting cultured atrial myocytes. Biochem Biophys Res Commun. 1988;157:330-336. [Medline] [Order article via Infotrieve]

29. Drewnowska K, Baumgarten CM. Regulation of cellular volume in rabbit ventricular myocytes: bumetanide, chlorothiazide, and ouabain. Am J Physiol. 1991;260:C122-C131. [Abstract/Free Full Text]

30. Beavo JA. Multiple isozymes of cyclic nucleotide phosphodiesterase. Adv Second Messenger Phosphoprotein Res. 1988;22:1-38. [Medline] [Order article via Infotrieve]

31. Weishaar RE, Kobylarz-Singer DC, Keiser J, Haleen SJ, Major TC, Rapundalo S, Peterson JT, Panek R. Subclasses of cyclic GMP-specific phosphodiesterase and their role in regulating the effects of atrial natriuretic factor. Hypertension. 1990;15:528-540. [Abstract/Free Full Text]

32. Schlatter E, Greger R, Weidtke C. Effect of `high ceiling' diuretics on active salt transport in the cortical thick ascending limb of Henle's loop of rabbit kidney: correlation of chemical structure and inhibitory potency. Pflugers Arch. 1983;396:210-217. [Medline] [Order article via Infotrieve]

33. Caramelo C, Lopez-Farre A, Riesco A, Olivera A, Okada K, Cragoe EJ Jr, Tsai P, Briner VA, Schrier RW. Atrial natriuretic peptide and cGMP inhibit Na⁺/H⁺ antiporter in vascular smooth muscle cells in culture. Kidney Int. 1994;45:66-75. [Medline] [Order article via Infotrieve]

34. MacLeod KM, Diamond J. Effects of the cyclic GMP lowering agent LY83583 on the interaction of carbachol with forskolin in rabbit isolated cardiac preparations. J Pharmacol Exp Ther. 1986;238:313-318. [Abstract/Free Full Text]

35. Schmidt MJ, Sawyer BD, Truex LL, Marshall WS, Fleisch JH. LY83583: an agent that lowers intracellular levels of cyclic guanosine 3',5'-monophosphate. J Pharmacol Exp Ther. 1985;232:764-769. [Abstract/Free Full Text]

36. Thompson WJ. Cyclic nucleotide phosphodiesterases: pharmacology, biochemistry and function. Pharmacol Ther. 1991;51:13-33. [Medline] [Order article via Infotrieve]

37. Clemo HF, Cohen NM, Baumgarten CM. ANF, bumetanide and 8-Br-cGMP reduce cell volume of human myocytes. J Mol Cell Cardiol. 1992;24(suppl III):S59. Abstract.

38. Anand-Srivastava MB, Trachte GJ. Atrial natriuretic factor receptors and signal transduction mechanisms. Pharmacol Rev. 1993;45:455-497. [Medline] [Order article via Infotrieve]

39. Archer SL, Huang JM, Hampl V, Nelson DP, Shultz PJ, Weir EK. Nitric oxide and cGMP cause vasorelaxation by activation of a charybdotoxin-sensitive K channel by cGMP-dependent protein kinase. Proc Natl Acad Sci U S A. 1994;91:7583-7587. [Abstract/Free Full Text]

40. Ganz MB, Nee JJ, Isales CM, Barrett PQ. Atrial natriuretic peptide enhances activity of potassium conductance in adrenal glomerulosa cells. Am J Physiol. 1994;265:C1357-C1365.

41. Kubo M, Nakaya Y, Matsuoka S, Saito Y, Kuroda Y. Atrial natriuretic factor and isosorbide dinitrate modulate the gating of ATP-sensitive K⁺ channels in cultured vascular smooth muscle cells. Circ Res. 1994;74:471-476. [Abstract/Free Full Text]

42. Suleymanian MA, Clemo HF, Cohen NM, Baumgarten CM. Stretch-activated channel blockers modulate cell volume in cardiac ventricular myocytes. J Mol Cell Cardiol. 1995;25:721-728.

43. Biel M, Zong X, Distler M, Bosse E, Klugbauer N, Murakami M, Flockerzi V, Hofmann F. Another member of the cyclic nucleotide-gated channel family, expressed in testis, kidney, and heart. Proc Natl Acad Sci U S A. 1994;91:3505-3509. [Abstract/Free Full Text]

44. Sakuta H, Okamoto K, Watanabe Y. Modification by cGMP of glibenclamide-sensitive K⁺ currents in Xenopus oocytes. Jpn J Pharmacol. 1993;61:259-262. [Medline] [Order article via Infotrieve]

45. Kunzelmann K, Kubitz R, Grolik M, Warth R, Greger R. Small-conductance Cl^- channels in HT29 cells: activation by Ca²⁺, hypotonic cell swelling and 8-Br-cGMP. Pflugers Arch. 1992;421:238-246. [Medline] [Order article via Infotrieve]

46. Lin M, Nairn AC, Guggino SE. cGMP-dependent protein kinase regulation of a chloride channels in T₈₄ cells. Am J Physiol. 1992;262:C1304-C1312. [Abstract/Free Full Text]

47. Landry DW, Reitman M, Cragoe EJ Jr, Al-Awqati Q. Epithelial chloride channel: development of inhibitory ligands. J Gen Physiol. 1987;90:779-798. [Abstract/Free Full Text]

48. Gallagher JP, Nakamura J, Shinnick-Gallagher P. The effects of temperature, pH, and Cl^- pump inhibitors on GABA responses recorded from cat dorsal root ganglia. Brain Res. 1983;267:249-259. [Medline] [Order article via Infotrieve]

49. Baumgarten CM, Duncan SWN. Regulation of Cl^- activity in ventricular muscle: Cl^-/HCO₃^- exchange and Na⁺-dependent Cl^--cotransport. In: Dhalla NS, Pierce GN, Beamish RE, eds. Heart Function and Metabolism. Boston, Mass: Martinus-Nijhoff; 1987:117-131.

50. Désilets M, Baumgarten CM. K⁺, Na⁺, and Cl^- activities in ventricular myocytes isolated from rabbit heart. Am J Physiol. 1986;251:C197-C208. [Abstract/Free Full Text]

51. Brady AJB, Warren JB, Poole-Wilson PA, Williams TJ, Harding SE. Nitric oxide attenuates cardiac myocyte contraction. Am J Physiol. 1993;265:H176-H182. [Abstract/Free Full Text]

52. Balligand J-L, Ungureanu-Longrois D, Simmons WW, Pimental D, Malinski TA, Kapturczak M, Taha Z, Lowenstein CJ, Davidoff AJ, Kelly RA, Smith TW, Michel T. Cytokine-inducible nitric oxide (iNOS) expression in cardiac myocytes. J Biol Chem. 1994;44:27580-27588.

53. Schulz R, Nava E, Moncada S. Induction and potential biological relevance of a Ca²⁺-independent nitric oxide synthase in the myocardium. Br J Pharmacol. 1992;105:575-580. [Medline] [Order article via Infotrieve]

54. Evans HG, Lewis MJ, Shah AM. Interleukin-1ß modulates myocardial contraction via dexamethasone sensitive production of nitric oxide. Cardiovasc Res. 1993;27:1486-1490. [Abstract/Free Full Text]

55. Tao S, McKenna TM. In vitro endotoxin exposure induces contractile dysfunction in adult rat cardiac myocytes. Am J Physiol. 1994;267:H1745-1752.[Abstract/Free Full Text]

This article has been cited by other articles:

				W. C De Mello Intracellular and extracellular renin have opposite effects on the regulation of heart cell volume. Implications for myocardial ischaemia Journal of Renin-Angiotensin-Aldosterone System, June 1, 2008; 9(2): 112 - 118. [Abstract] [PDF]

				R. C. Massaro, L. W. Lee, A. B. Patel, D. S. Wu, M.-J. Yu, B. N. Scott, D. A. Schooley, K. M. Schegg, and K. W. Beyenbach The mechanism of action of the antidiuretic peptide Tenmo ADFa in Malpighian tubules of Aedes aegypti J. Exp. Biol., July 15, 2004; 207(16): 2877 - 2888. [Abstract] [Full Text] [PDF]

				C. E. Davis, J. J. Rychak, B. Hosticka, S. C. Davis, J. E. John III, A. L. Tucker, P. M. Norris, and J. R. Moorman A novel method for measuring dynamic changes in cell volume J Appl Physiol, May 1, 2004; 96(5): 1886 - 1893. [Abstract] [Full Text] [PDF]

				J. G. Akar, T. H. Everett, R. Ho, J. Craft, D. E. Haines, A. P. Somlyo, and A. V. Somlyo Intracellular Chloride Accumulation and Subcellular Elemental Distribution During Atrial Fibrillation Circulation, April 8, 2003; 107(13): 1810 - 1815. [Abstract] [Full Text] [PDF]

				I. Kishimoto, K. Rossi, and D. L. Garbers A genetic model provides evidence that the receptor for atrial natriuretic peptide (guanylyl cyclase-A) inhibits cardiac ventricular myocyte hypertrophy PNAS, February 8, 2001; (2001) 51625598. [Abstract] [Full Text]

				H. Chusho, Y. Ogawa, N. Tamura, M. Suda, A. Yasoda, T. Miyazawa, I. Kishimoto, Y. Komatsu, H. Itoh, K. Tanaka, et al. Genetic Models Reveal That Brain Natriuretic Peptide Can Signal through Different Tissue-Specific Receptor-Mediated Pathways Endocrinology, October 1, 2000; 141(10): 3807 - 3813. [Abstract] [Full Text] [PDF]

				D Duan and J R Hume NO and the regulation of VSOACs J. Physiol., October 1, 2000; 528(1): 2 - 2. [Full Text] [PDF]

				J. R. Hume, D. Duan, M. L. Collier, J. Yamazaki, and B. Horowitz Anion Transport in Heart Physiol Rev, January 1, 2000; 80(1): 31 - 81. [Abstract] [Full Text] [PDF]

				J. M. Russell Sodium-Potassium-Chloride Cotransport Physiol Rev, January 1, 2000; 80(1): 211 - 276. [Abstract] [Full Text] [PDF]

				F. LANG, G. L. BUSCH, M. RITTER, H. VOLKL, S. WALDEGGER, E. GULBINS, and D. HAUSSINGER Functional Significance of Cell Volume Regulatory Mechanisms Physiol Rev, January 1, 1998; 78(1): 247 - 306. [Abstract] [Full Text] [PDF]

				A. S. Mihailidou, K. A. Buhagiar, and H. H. Rasmussen Na+ influx and Na+-K+ pump activation during short-term exposure of cardiac myocytes to aldosterone Am J Physiol Cell Physiol, January 1, 1998; 274(1): C175 - C181. [Abstract] [Full Text] [PDF]

				L. A Brown, R. A.D Rutherford, D. J.R Nunez, John Wharton, D. G Lowe, and M. R Wilkins Downregulation of natriuretic peptide C-receptor protein in the hypertrophied ventricle of the aortovenocaval fistula rat Cardiovasc Res, December 1, 1997; 36(3): 363 - 371. [Abstract] [Full Text] [PDF]

				C.-F. Wu, N. H. Bishopric, and R. E. Pratt Atrial Natriuretic Peptide Induces Apoptosis in Neonatal Rat Cardiac Myocytes J. Biol. Chem., June 6, 1997; 272(23): 14860 - 14866. [Abstract] [Full Text] [PDF]

				I. Kishimoto, K. Rossi, and D. L. Garbers A genetic model provides evidence that the receptor for atrial natriuretic peptide (guanylyl cyclase-A) inhibits cardiac ventricular myocyte hypertrophy PNAS, February 27, 2001; 98(5): 2703 - 2706. [Abstract] [Full Text] [PDF]

This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Clemo, H. F. Articles by Baumgarten, C. M. Search for Related Content PubMed PubMed Citation Articles by Clemo, H. F. Articles by Baumgarten, C. M.