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Endocrinology Vol. 145, No. 11 5313-5321
Copyright © 2004 by The Endocrine Society

Participation of G Proteins in Natriuretic Peptide Hormone Secretion from Heart Atria

Michael Bensimon, Astra I. Chang, Mercedes L. Kuroski de Bold, Amalia Ponce, Daniel Carreras and Adolfo J. De Bold

Cardiovascular Endocrinology Laboratory, University of Ottawa Heart Institute, Ottawa, Ontario, Canada K1Y 4W7

Address all correspondence and requests for reprints to: Dr. Adolfo J. de Bold, Cardiovascular Endocrinology Laboratory, University of Ottawa Heart Institute, Ottawa, Ontario, Canada K1Y 4W7. E-mail: adebold{at}ottawaheart.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The involvement of G proteins in the mechanism underlying the increased atrial natriuretic factor (ANF) secretion observed after atrial muscle stretch (stretch-secretion coupling) was assessed using a combined pharmacological, immunocytochemical, and tissue fractionation approach. It was found that Gi/o inhibition by pertussis toxin (PTX) abolished stretch-secretion coupling without affecting baseline secretion through a mechanism that is independent of Gq signaling agonists. Mastoparan-7, a Gi/o agonist, significantly increased ANF secretion even in the absence of muscle stretch through a PTX-sensitive mechanism. By confocal and electron immunocytochemistry, ANF and Go partially colocalized, whereas ultracentrifugation analysis suggested the presence of two populations of granules, one of which was partially associated with Go, as demonstrated by Western blotting. PTX did not affect basal or endothelin-1-stimulated ANF secretion, in line with the view that endothelin-1 signals mainly through Gq. It is concluded there are at least two types of regulated secretory processes in atrial cardiocytes: one is acutely responsive to muscle stretch and is PTX sensitive, and the other is Gqmediated and PTX insensitive and may be responsible for changes in secretion after chronic changes in the neuroendocrine environment.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GENE EXPRESSION AND secretion of the atrial natriuretic factor (ANF) are influenced by both mechanical (atrial muscle stretch) and neurohumoral stimuli. The latter include ligands that typically signal through G protein-coupled receptors (1, 2). Comparatively little is known about the mechanism underlying ANF and brain natriuretic peptide (BNP) secretion elicited by stretch (stretch-secretion coupling) from adult atrial cardiocytes even though stretch-secretion coupling is deemed important in the regulation of hormone secretion (3, 4, 5, 6, 7). Whether stretch on its own could couple to secretion using G proteins is not known, but this appears mechanistically possible because pertussis toxin (PTX)-sensitive G proteins are known to mediate stretch-induced modulation of cell growth and cell functions, such as autacoid production (8) and because of the known associations of G protein with the cytoskeleton (9) and the secretory machinery (10). In addition, immunocytochemical evidence has localized Go immunoreactivity with the specific atrial granules, the site of storage for both ANF and BNP (11).

In the present study we used an isolated adult rat atria model (12) to investigate the effect of agents known to affect G protein function on ANF secretion and used a combined pharmacological, immunocytochemical, and tissue fractionation approach to further define the significance of the findings. Gi/o inhibition by PTX abolished stretch-secretion coupling without affecting baseline secretion through a mechanism that is independent of Gq signaling agonists. Mastoparan-7 (MAS-7), a Gi/o agonist, significantly increased ANF secretion even in the absence of muscle stretch through a PTX-sensitive mechanism. By confocal and by electron immunocytochemistry, ANF and Go partially colocalized whereas ultracentrifugation analysis suggested the presence of two populations of granules, one of which was partially associated with Go as demonstrated by Western blotting. We also show that PTX did not affect endothelin-1 (ET-1)-stimulated ANF secretion, in line with the fact that this agonist signals mainly through Gq.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
The experimental protocol was carried out according to the Canadian Council on Animal Care Guide to the Care and use of Experimental Animals after obtaining approval by the University of Ottawa animal care committee.

Isolated right atria
The atrial preparation has been previously described (12). In brief, right atria with an attached small portion of the right ventricle obtained from male Sprague Dawley rats (275–350 g; Charles River Laboratories, Wilmington, MA) were perfused at a rate of 3 ml/min with an oxygenated (95% O2-5% CO2) supplemented Krebs-Ringer bicarbonate buffer (KRBB). The flow of KRBB was from the superior vena cava into the right atrium and out through the tricuspid valve. The organ chamber contained 50 ml oxygenated KRBB, replenished at a rate of 2.5 ml/min. The basal intraatrial pressure was 0.5 mm Hg. In the stretch experiments, the outflow cannula was raised to generate an intraatrial pressure of 8 mm Hg. ANF secretion was quantified by RIA (13) in the perfusion medium collected for 5-min periods in siliconized glass tubes. The perfusion time required to observe the effect of experimental treatments was established in pilot experiments and were 360, 240, or 120 min depending on the stimulus as described below.

Drugs
PTX (Sigma-Aldrich Corp., St. Louis, MO) was reconstituted in 0.1 M sodium phosphate buffer, pH 7.4. Mastoparan-7 (BioSource International, Camarillo, CA; final concentration, 10–5 M) was reconstituted in distilled water. Stock endothelin-1 (ET-1; Peninsula Laboratories, Inc., San Carlos, CA) was dissolved in 1 M acetic acid at a concentration of 0.1 mg/100 µl.

Perfusion protocols
PTX and stretch.
PTX-catalyzed ADP ribosylation in vivo was carried out using previously described protocols (14, 15). Atria from rats with or without PTX treatment (25 or 40 µg/kg, ip) 48 h before death were perfused for 6 h after a 1-h equilibration period at basal intraatrial pressure (n = 5 each dose) or stimulated by stretch (n = 5 each dose).

MAS-7.
MAS-7 was incorporated into the perfusate at 10–5 M (n = 5) for 2 h after the equilibration period. A second group of rats (n = 5) were pretreated with PTX (40 µg/kg, ip) 48 h before a similar MAS-7 protocol.

ET-1.
Atria from untreated or PTX-treated rats were perfused for 2 h with ET-1 at 10–8 M (n = 4 each group) after the equilibration period.

ADP ribosylation assay
After perfusion, atria from control and PTX-treated animals were flash-frozen in liquid nitrogen and homogenized in sucrose buffer (20 m[scap]m HEPES, 1 mM EDTA, 255 mM sucrose, and 100 µM phenylmethylsulfonylfluoride) in a 1:4 (wt/vol) ratio using a chilled glass homogenizer. After centrifugation, the supernatant was collected and kept for determination of protein concentrations by spectrophotometry at 280 nm against BSA standards in the above buffer.

To determine the level of PTX-induced ADP-ribosylation of Gi/o protein in the perfused atria, a PTX-induced ADP ribosylation reaction was carried out by adding 20 µg protein from rat atria homogenate to 0.1 M Tris-HCl (pH 7.6), 20 mM dithiothreitol, 1.1 µM [32P]NAD, 0.1 mM ATP, and 1.5 µl PTX. After incubation for 1 h, the reaction was stopped by the addition of 12.5 µl sodium dodecyl sulfate-polyacrylamide sample buffer [62.5 mM Tris-HCl (pH 6.8), 25% glycerol, 2% sodium dodecyl sulfate, and 0.01% bromophenol blue] with ß-mecaptoethanol (710 mM) and boiled for 5 min at 100 C. Equal amounts of sample were loaded into 4–15% gradient sodium dodecyl sulfate-polyacrylamide gel. Broad range prestained SDS-PAGE standards (Bio-Rad Laboratories, Hercules, CA) were also run on each gel. The gel was fixed for 30 min in a solution of 7% glacial acetic acid in 10% methanol, stained with brilliant blue G-colloidal solution (Bio-Rad) for 2 h, and destained with 10% acetic acid in 25% methanol for 60 sec. The gel was air-dried overnight. Autoradiography was performed subsequently for 30 min on a PhosphorImager cassette. Radioactivity bound to the blot was quantified using a Bio-Rad personal molecular imager FX and Bio-Rad Quantity One software.

Antibodies
For ANF RIA, a specific antiserum against human ANF 99–126 (Peninsula Laboratories, Inc.) was used. For G protein immunocytochemistry, affinity-purified rabbit polyclonal antibodies were purchased from Santa Cruz Biotechnology, Inc. The G{alpha}i1 antibody (sc-391) was raised against a peptide mapping within a highly divergent domain of G{alpha}i1 of rat origin. sc-391 was described as reacting with G{alpha}i-1 and, to a lesser degree, G{alpha}i-2 and G{alpha}i-3 of mouse, rat, and human origins and was noncross-reactive with other G{alpha} subunit proteins. The G{alpha}i2 antibody (sc-7276) mapped within a highly divergent domain of G{alpha}i2 of human origin. sc-7276 was described as reacting with G{alpha}i-2 of mouse, rat, and human origin and was noncross-reactive with G{alpha}i1 or G{alpha}i3. The G{alpha}i3 antibody (sc-262) was raised against a peptide mapping at the carboxyl terminus of G{alpha}i-3 of rat origin. sc-262 was described as reacting with G{alpha}i1, G{alpha}i2, and G{alpha}i3 of mouse, rat, and human origin and was noncross-reactive with other G{alpha}-subunit proteins. A polyclonal rabbit antibody against purified Go{alpha} from bovine brain was purchased from MBL International Corp. (Woburn, MA). This antibody cross-reacts with rat and human Go{alpha}, but does not bind G{alpha}i2 and G{alpha}i3. Western blots were carried out using this antibody. Immunolabeling for ANF was performed using the RIA antibody.

RIA
RIA was performed using the double antibody technique as previously described by Sarda et al. (16). For determinations in plasma, trunk blood samples were collected from male Sprague Dawley rats in chilled EDTA-containing Vacutainer tubes (VWR International Ltd., Mississauga, Ontario, Canada). Plasma was immediately separated by centrifugation at 4 C and stored in cryovials at –80 C until assayed. Plasma samples were acidified by adding 100 µl 1.0 N HCl/ml plasma and extracted with Sep-Pak C18 cartridges (Millipore-Waters Corp., Milford, MA).

Immunochemistry
Atrial or ventricular tissue samples were fixed in 4% paraformaldehyde and embedded in paraffin for light microscopy or in Spurr’s resin for electron microscopy. Deparaffinized sections were washed for 5 min in tap water, followed by incubation in 3% hydrogen peroxide to quench endogenous peroxidase activity. The sections were then incubated for 20 min with 10% normal goat blocking serum and with one of the Gi{alpha}1–3 antibodies (1:50) or the Go{alpha} antibody (1:200) for 30 min. The immunostaining was performed using the avidin-biotin technique (Vector Laboratories, Inc., Burlingame, CA) according to the manufacturer’s instructions. G proteins were visualized using fluorescein-avidin and ANF using Texas Red-avidin. The sections were examined using a Bio-Rad 1024 confocal microscope. The green channel had an excitation of 488 nm and an emission of 525 nm. The red channel had an excitation of 594 nm and an emission 620 nm. Lack of cross-talk between the channels was established.

Spurr sections were mounted on nickel grids, etched with 8% aqueous sodium metaperiodate for 60 min, rinsed with water, and incubated in 10% normal goat serum for 10 min, followed by ANFR5B11 (an ANF-specific polyclonal antibody produced in our laboratory) diluted 1:1000 for 48 h. The grids were rinsed in PBS and incubated with 10 nm gold-labeled goat antirabbit IgG. After a rinse (three times, 10 min each time), the grids were incubated in the MLB International Corp. polyclonal anti-Go{alpha}-subunit at a 1:100 dilution for 48 h, rinsed in PBS, incubated with 15 nm gold-labeled goat antirabbit IgG, jet washed with water, and stained with lead citrate and uranyl acetate for 5 min.

Tissue fractionation by ultracentrifugation
Up to four atria or the equivalent weight of ventricular tissue were obtained from male Sprague Dawley rats (~350 g) after decapitation. The tissues were placed in ice-cold homogenization medium consisting of 0.25 M sucrose, 1 mM EDTA, and 10 mM Tris-HCl, pH 7.4. The tissues were finely chopped with a razor blade and then homogenized in 10 ml homogenization medium, first using a Polytron fitted with a PT30–35 probe at 70% power for 15 sec and then with four strokes in a Potter Elvehjem homogenizer. The atrial and ventricular homogenates so obtained were centrifuged at 1,900 x gmax for 10 min at 4 C on a Beckman J2-21 JM centrifuge using a JA-20 rotor, thus obtaining a nuclear and a postnuclear fraction. To 4 ml of a working density gradient solution consisting of 2 ml diluent solution (0.25 M sucrose, 10 mM EDTA, and 60 mM Tris-HCl, pH 7.4) and 10 ml OptiPrep (MJS Biolynx, Inc., Brockville, Ontario, Canada), 7.4 ml of the postnuclear fraction were added (final OptiPrep concentration, 17.5%). The mixture was then transferred to tubes fitting the NVT 65 rotor (Beckman Coulter, Fullerton, CA) and centrifuged for 5 h at 154,000 x g at 4 C to obtain a self-generated density gradient.

At completion of ultracentrifugation 23 fractions of 10 drops each were obtained by upward displacement of the tube’s content using a fraction recovery system (Beckman Fraction Recovery System). Fractions collected in polystyrene tubes were diluted with 1 ml distilled water and immediately stored at –80 C. Fifty microliters of this dilution were taken for protein determination using a commercial kit (bicinchoninic acid protein assay reagent; Pierce Chemical Co., Rockford, IL). An additional 500 µl were mixed with an equal volume of 1 M acetic acid, placed in a boiling water bath for 10 min, frozen, and freeze-dried for ANF RIA. The remainder of the diluted fractions was used for determination of the marker enzymes galactosyl transferase (Golgi) and succinate dehydrogenase (mitochondria) (17).

Western blotting
To 200 µl of the diluted density gradient fractions (see above), NaCl (final concentration, 1.0 M), sodium cholate (final concentration, 2%), and 1 µl protease inhibitor mixture (no. 8340, Sigma-Aldrich Corp.) were added, followed by centrifugation at 12,000 x g for 30 min at 4 C. Fifteen microliters of each processed fraction mix in 30 µl Laemmli sample buffer were loaded on a 10% sodium dodecyl sulfate-polyacrylamide gel using an electrode buffer made of 25 mM Tris, 192 mM glycine (pH 8.3), and 1% sodium dodecyl sulfate at 150 V. Proteins were transferred to a polyvinylidene difluoride membrane at 100 V for 1 h in transfer buffer (25 mM Tris and 192 mM glycine, pH 8.3), followed by blocking [Tris-buffered saline (TBS), 5% (wt/vol) nonfat milk, and 0.1% Tween 20] for 1 h at room temperature. Blots were incubated overnight at 4 C with G protein antibodies at 0.15 µg/ml in primary antibody dilution buffer (TBS, 5%BSA, and 0.1% Tween 20). After washing with TBS containing 0.1% Tween 20, blots were incubated in blocking buffer with horseradish peroxidase-conjugated secondary antibody (1:2,000) for 1 h at room temperature. Development of the blots was accomplished using LumiGLO (Cell Signaling Technology, Beverly, MA). Protein band densities were analyzed using a Kodak Image Station 440CF and the ID software (Eastman Kodak, Rochester, NY).

Statistical analysis
All data are reported as the mean ± SEM. Two-way ANOVA was performed to determine statistical significance between groups. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Degree of ADP ribosylation by PTX
To establish the extent of Gi/o binding inactivated via ADP ribosylation by PTX in vivo, an ADP ribosylation assay was used. The autoradiogram of the pretreated hearts showed weak bands at their expected molecular mass of approximately 39–41 kDa for the 25 µg/kg treated samples and no bands for the 40 µg/kg treated samples (Fig. 1AGo), indicating that no additional ribosylation could take place in vitro and that the degree of Gi/o-binding protein subunit ribosylation was dose dependent (Fig. 1BGo).



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  		FIG. 1. PTX blocks Gi/o proteins in vivo. A, Typical autoradiogram showing in duplicate the PTX-catalyzed incorporation of [32P]ADP-ribose from [32P]NAD into atrial homogenates from untreated and PTX-treated (25 or 40 µg/kg, ip, 48 h before assay) Sprague Dawley rats. PTX ADP-ribosylated Gi/o protein had a relative molecular mass of 39–41 kDa. B, The graph was obtained from duplicate determinations in four untreated and four PTX-treated rats. Values are the mean ± SEM (n = 4/group). **, P < 0.001, PTX vs. baseline.

 
Effect of PTX on stretch-secretion coupling
Atrial stretch (induced by increasing intraatrial pressure from 0.5 to 8 mm Hg) stimulated an immediate increase in ANF secretion that peaked at 20 min and decreased thereafter (Fig. 2Go). A statistically significant difference between the mean secretion rate at 140 min was observed between stretch vs. baseline [140 min, 570.9 ± 65.2 vs. 208 ± 32 pg/ml, respectively (P < 0.001; n = 5); 160 min, 430.9 ± 66.1 vs. 184.9 ± 21.9 pg/ml, respectively (P < 0.001; n = 5)]. Pretreatment with 20 µg/kg PTX, ip, injected 48 h before the perfusion experiments significantly decreased stretch-stimulated ANF secretory peak at 140 min from 570.9 ± 65.2 to 340.5 ± 32.5 pg/ml (P < 0.001; n = 5). Pretreatment with 40 µg/kg PTX, ip (n = 5), completely eliminated stretch-stimulated ANF secretion (Fig. 2Go). The plasma ANF concentration in the animals used for perfusion was significantly decreased by 40 µg/kg PTX, ip (25.7 ± 1.6 vs. 15.2 ± 2.6 pg/ml; P < 0.05; n = 5 each group) compared with that in untreated animals (Fig. 3Go).



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  		FIG. 2. Effect of PTX on stretch-stimulated ANF secretion. Isolated atria perfusions with were carried out in four experimental groups: 1) baseline ({square}), 2) stretch ({blacksquare}), 3) stretch plus PTX ({square}; 25 µg/kg, ip), and 4) stretch plus PTX ({diamondsuit}; 40 µg/kg, ip). Intraatrial pressure was changed from 0.5 to 8 mm Hg (stretch) at 120 min. Values are the mean ± SEM (n = 5 /treatment). *, P < 0.05, stretch vs. stretch plus PTX (by ANOVA).

 


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  		FIG. 3. In vivo effect of PTX on plasma ANF levels. Blood was collected from two groups of Sprague Dawley rats: 1) control animals injected with 0.9% saline, and 2) treated animals injected with PTX (40 µg/kg, ip). Injections were given 48 h before death. Values are the mean ± SEM (n = 5/group). **, P < 0.001, PTX vs. control.

 
ET-1-stimulated ANF secretion is not affected by PTX
PTX (40 µg/kg, ip, 48 h before perfusion) did not affect the expected secretory response to ET-1 (10–8 M; Fig. 4Go).



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  		FIG. 4. Effect of PTX on ET-1-stimulated ANF secretion. ET-1 (10–8 M) was infused (0.34 ml/min) for 2 h starting at 120 min in two groups of rats: 1) untreated ({blacksquare}), and 2) PTX-treated ({square}; 40 µg/kg, ip, 48 h before perfusion). Values are the mean ± SEM (n = 4/group). **, P < 0.001, ET-1 or ET-1 plus PTX vs.120 min.

 
MAS-7 toxin significantly increases ANF secretion
MAS-7 (10–5 M) stimulated ANF secretion (Fig. 5Go). PTX attenuated MAS-7-stimulated ANF secretion (P < 0.001; n = 5), suggesting that MAS-7 was acting via Gi/o proteins (Fig. 5Go). Lower doses of MAS-7 (10–6 and 10–8 M; n = 5, respectively) did not stimulate ANF secretion (data not shown).



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  		FIG. 5. Effect of PTX on MAS-7-stimulated ANF secretion. MAS-7 (10–5 M) was infused (0.34 ml/min) for 30 min starting at 120 min in two groups of rats: 1) untreated ({blacksquare}), and 2) PTX-treated ({square}; 40 µg/kg, ip, 48 h before perfusion). Values are the mean ± SEM (n = 5/group). *, P < 0.05, MAS-7 vs. MAS-7 plus PTX (by ANOVA).

 
Immunofluorescence and immunoelectron microscopy labeling of Go{alpha} and ANF
By confocal microscopy Go{alpha} colocalized to a significant extent with ANF in the paranuclear zones of atrial cardiocytes where the superimposition of the green color associated with Go{alpha} and the red color associated with ANF is visualized as yellow (Fig. 6AGo, arrow). Much of the ANF immunofluorescence present outside the paranuclear zones was not associated with Go{alpha} immunofluorescence. The latter appeared mainly localized along the sarcolemma. Ventricular cardiocytes did not display a similar staining pattern (Fig. 6BGo) for either Go{alpha} or ANF. Antibodies against Gi1, Gi2, and Gi3 did not stain paranuclear areas in atrial or ventricular cardiocytes (data not shown). By electron microscopy, several specific atrial granules showed ANF and Go{alpha} immunoreactivity. The latter tended to be found at the periphery of the atrial granules (Fig. 7Go).



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  		FIG. 6. A, Immunocytochemical demonstration of Go{alpha} protein (green) and ANF (red) in rat atria, showing their colocalization (yellow) in a paranuclear zone containing the specific atrial granules (single arrow). In addition, Go{alpha} staining is particularly strong along the sarcolemma (double arrow). B, Immunocytochemical staining of ventricular sections does not reveal an equivalent staining pattern. Bar units are in micrometers.

 


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  		FIG. 7. Immunoelectron microscopy demonstration of ANF and Go{alpha} protein, showing coexistence of both species in the specific atrial granules. Sections were stained with specific anti-ANF and anti-Go{alpha} antibodies and were revealed by anti-IgG labeled with gold (15-nm particle size; Go{alpha}) or 10 nm (ANF).

 
Ultracentrifugation analysis shows a bimodal distribution of ANF
Tissue ultracentrifugation analysis of the postnuclear fraction of atrial homogenates demonstrated a bimodal sedimentation for ANF in regions of the gradient similar, but not identical, to those for the mitochondrial marker succinate dehydrogenase and the Golgi marker galactosyl transferase. By Western blotting, Go{alpha} colocalized in part with the latter marker (Fig. 8Go).



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  		FIG. 8. Representative subcellular distribution of activities after density gradient centrifugation in OptiPrep of a postnuclear atrial homogenate fraction. Shown are ANF (A), Go{alpha} by Western blot and by densitometry reading relative to protein (B and C, respectively), and the marker enzymes glucose-6-phosphatase (endoplasmic reticulum), succinate dehydrogenase (mitochondria), and galactosyl transferase (Golgi).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study direct inhibition or activation of Gi/o proteins in the adult atria by pharmacological agents established the involvement of Gi/o proteins in stretch-stimulated ANF secretion. PTX, a pharmacological agent that prevents the G{alpha}-subunit from interacting with the receptor by ADP ribosylation of a Cys residue near the C terminus (18), resulting in the inhibition of G protein-mediated signal transduction, completely blocked stretch-secretion coupling. Activation of Gi/o protein by MAS-7, a peptide that stimulates GDP/GTP exchange in Gi/o proteins in a manner similar to that of agonists (19), potently stimulated ANF release. The latter effect was attenuated by PTX.

Although it is clear from the present studies that PTX-sensitive Gi/o proteins are involved in stretch-stimulated ANF secretion, the precise relationship between stretch and secretion was not identified. By extrapolation of findings from other investigations, some of the possible mechanisms include the KATP channel, the L-type, voltage-gated Ca2+ channel, adenylyl cyclase, and processes involved in the exocytotic release of hormone. Most of the literature concerning ANF release and possible targets of Gi/o protein activity is highly conflictive. The sulfonylurea tolbutamide and other KATP channel blockers have been reported to both increase and decrease stretch-stimulated ANF secretion (20, 21, 22, 23). Manipulations of the L-type Ca2+ channel have been reported to increase, decrease, or not affect ANF secretion (24, 25, 26). We previously reported, however, that Ca2+ tonically inhibits ANF secretion (27), a finding subsequently corroborated by others (24, 28). These results place ANF secretion from atrial cardiocytes in a similar relationship to calcium as the secretion of renin by renal juxtaglomerular cell, which is also stimulated by a low Ca2+ environment (29, 30). A considerable amount of research has been performed to establish the effect of manipulation of adenylyl cyclase activity and intracellular levels of cAMP on ANF secretion with highly variable results, suggesting that cAMP may both positively and negatively modulate ANF secretion depending on the intracellular levels (31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41).

It is possible that Gi/o directly interacts with a target participating in the exocytotic process of ANF-containing granules. The association of G proteins with secretory granules and their formation is well established (42, 43, 44, 45, 46). In the present studies, immunolabeling using the MLB International Corp. antibody demonstrated considerable colocalization of Go{alpha} proteins with ANF-storing granules by confocal and electron microscopy. The antibody used localizes Go{alpha} in the central nervous system, pituitary gland, and the membranes of neuroendocrine cells, including glandular cells in the anterior lobe of the pituitary gland, chromaffin cells in the medulla of the adrenal gland, the pancreas, and parafollicular cells in the thyroid (46, 47, 48). In our studies, a considerable amount of Go{alpha} was clearly associated with the sarcolemma in a fashion similar to the distribution seen for this protein in the plasma membrane of cells of the endocrine pancreas. These findings are in part similar to those reported by Wolf et al. (11), who described an association of Go{alpha} with the sarcolemma, intercalated disks, cavioli, and atrial granules, especially those found in the subsarcolemmal space. However, in the present studies we found that most of the Go{alpha} immunoreactivity that colocalized with ANF was present in the paranuclear zones of cardiocytes. This region of the cell also hosts most of the Golgi complex, which is highly developed in atrial cardiocytes. The confocal images thus tend to suggest that there is an association between ANF granules, Go{alpha}, and the Golgi complex.

The density gradient centrifugation analysis showed that ANF and Go{alpha} had bimodal distributions. The heavier ANF peak probably represent mature granules (49), whereas the lighter fraction, which sediments in the region of the Golgi marker galactosyl transferase, probably represents ANF immunoreactivity associated with Golgi complex components. However, the highest purification of Go{alpha} took place in a region of the gradient that is of lighter density than either the Golgi marker or ANF, thus suggesting that the association between ANF and Go{alpha} is not quantitatively as important as the association of Go{alpha} with lower density subcellular elements. Because a large amount of Go{alpha} immunoreactivity localized at the sarcolemma by light microscopy, it is possible that these lower density subcellular elements are sarcolemmal fragments.

The precise cellular location of the G protein on which PTX exerts its effect to prevent stretch-secretion coupling is not apparent from these studies. PTX can exert its effect on G proteins on membrane receptors or can be transported into intracellular sites (50, 51). Inhibitory G proteins have been localized in the trans-Golgi network (52, 53, 54) and in secretory granules (11, 43, 44, 46, 55, 56). Consistent with the view that in at least some instances PTX needs to access the intracellular milieu to exert its effect on G proteins, it has been shown that in chromaffin cells, secretion induced by high K+ is inhibited by mastoparan only if this peptide is injected intracellularly (10). Similar observations have been reached in studies using a pancreatic ß-cell model (45). In our experiments MAS-7 exerted a highly significant stimulation of ANF secretion without permeabilization, suggesting a localization of Gi/o directly accessible from the extracellular milieu.

In previous studies using double pulse-chase analysis, we found that stretch and ET-1 stimulated ANF secretion from a newly synthesized pool of hormone, and there was no change in the specific activity of the older, stored ANF pool that had been labeled from 2–5 h before stretch or ET-1 treatment (4, 57). Furthermore, monensin, an ionophore that impairs protein sorting and transport in the trans-Golgi network and inhibits vesicle formation without directly affecting protein synthesis, completely inhibited stretch- and ET-1-stimulated ANF secretion (4), but it did not affect baseline release. This occurs despite the fact that the cells have a large storage pool of hormone in the older storage compartment, suggesting that a constitutive-like release, which entails the regulated secretion of immature secretory granules (58), is the main release mechanism of stimulated hormone secretion operating in atrial cardiocytes. Agents that inhibit G proteins also inhibit the formation of constitutive secretory vesicles or immature secretory granules (54). These observations together with the observations presented in this report suggest that the effect of PTX could be exerted on immature secretory granules. However, had PTX affected immature granule formation, it should have affected not only stretch-stimulated secretion, but also ET-1-stimulated secretion, which displays constitutive-like characteristics. As shown in the present work, only stretch-stimulated secretion was affected, thus suggesting that PTX acts on a signal transduction pathway used exclusively by stretch-secretion coupling. There is indirect evidence of differences in signal transduction associated with ANF secretion when elicited by stretch or by Gq{alpha} signaling agonists, such as ET-1 or phenylephrine. Stretch-induced secretion results in a virtually instantaneous increase in the rate of ANF secretion from isolated atria. The secretion elicited by ET-1 or phenylephrine under the same circumstances requires several minutes to become fully established (4). These differences also suggest that ANF secretion elicited by stretch is not due to the action of locally released agonists such as ET-1.

It is noteworthy that baseline secretion of ANF was not affected by PTX. Similarly, monensin or the protein synthesis inhibitor cycloheximide did not alter baseline ANF secretion (4). These observations suggest that immature granules or newly synthesized ANF do not contribute to basal ANF secretion and point to the older, mature granules pool previously defined in our pulse-chase investigations (4, 57) as the source of basal hormone secretion.

In summary, the present study shows at least two types of regulated secretory processes in atrial cardiocytes: one is stretch stimulated and PTX sensitive, and the other is Gq mediated and PTX insensitive. Baseline ANF secretion is also PTX insensitive. It is conceivable that in vivo the first process mediates stimulated ANF secretion brought about by changes in central venous return and subsequent atrial muscle stretch as observed in head-out water immersion (7) or extracellular fluid volume expansion (59), whereas the second type of stimulation is brought about by sustained hemodynamic and neuroendocrine stimuli such as observed in deoxycorticosterone acetate-salt hypertension (60) and congestive heart failure (61).


   Footnotes
 
Abbreviations: ANF, Atrial natriuretic factor; ET-1, endothelin-1; KRBB, Krebs-Ringer bicarbonate buffer; MAS-7, mMastoparan-7; PTX, pertussis toxin; TBS, Tris-buffered saline.

Received June 1, 2004.

Accepted for publication August 2, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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