© 1996 American Heart Association, Inc.
Articles |
cGMP Formation in Rat Atrial Slices Is Ligand and Endothelin Receptor Subtype Specific
From the Laboratory of Neurobiochemistry, The George S. Wise Faculty of Life Sciences, Tel Aviv (Israel) University.
Correspondence to Dr Mordechai Sokolovsky, Laboratory of Neurobiochemistry, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel. E-mail sokol@post.tau.ac.il.
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Abstract |
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Abstract Involvement of a cGMP pathway in signal transduction stimulated by endothelins (ETs) and sarafotoxins (SRTXs) was examined in rat atrial slices. These peptides activated different receptor-binding sites (ET-1 and SRTX-b reacted with the picomolar binding sites of the ETA receptor, and ET-3 and SRTX-c reacted with the nanomolar binding sites of the ETB receptor) to produce cGMP. ET-1 and SRTX-b stimulated an increase in cGMP levels via a Ca2+-dependent NO pathway involving a pertussis toxin–insensitive G protein, whereas ET-3 and SRTX-c elevated cGMP levels via a Ca2+-independent CO pathway involving a pertussis toxin–sensitive G protein. These results can best be explained in terms of formation of different ligand-receptor–G-protein complexes. The ligands had no effect on ventricular slices, indicating that these signal transduction mechanisms are unique to the atria.
Key Words: endothelins • sarafotoxins • rat atria • signaling • cGMP
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Introduction |
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TopAbstractIntroductionMaterials and Methods Results Discussion References |
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The ETs are the most potent of the known mammalian vasoconstrictor peptides and exert their effects in various tissues and cells. They are remarkably similar in structure to the SRTXs present in the venom of the snake Atractaspis engaddensis (reviewed in Reference 1). Numerous studies have indicated a role for these peptides in the cardiovascular system, where, for example, they produce positive inotropic responses, are strongly mitogenic, and induce cellular hypertrophies.2 3 4
A number of studies have demonstrated the existence of two main ET receptor subtypes, ETA-R and ETB-R. The different peptides, at nanomolar concentrations, mediate the stimulation of phosphoinositide hydrolysis in various tissues and cell types.1 However, many tissues, cells, and cell lines possess another distinct receptor, which binds ETs at the picomolar concentration range and does not stimulate phospholipase C activity.5 6 7 We recently showed that this latter receptor elevates cAMP levels in rat atrial slices and cGMP levels in rat cerebellar slices.8 9 Therefore, we were interested in determining whether ETs and SRTXs induce changes in cGMP levels in rat atrial slices and whether this signaling pathway is mediated through a receptor with a binding affinity for picomolar ligand concentrations.
In this communication, we show that in rat atrial slices ET-1 and SRTX-b elevate cGMP levels in the picomolar range, whereas ET-3 and SRTX-c stimulate cGMP production in the nanomolar range. The complex formed between ET-1 or SRTX-b and the ETA-R coupled to a PT-insensitive G protein stimulates cGMP production via the NO pathway and is dependent on Ca2+. In contrast, the complex formed between ET-3 or SRTX-c and the ETB-R coupled to a PT-sensitive G protein stimulates cGMP production through the CO pathway and is independent of Ca2+. In addition, we show that the peptides have no effect in rat ventricular slices, indicating that this signaling process is unique to the atria.
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Materials and Methods |
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TopAbstractIntroductionMaterials and Methods Results Discussion References |
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Reagents
ET-1, ET-3, SRTX-b, SRTX-c, and Res-701-1 were purchased from American Peptide Co. cGMP was assayed by a radioimmunoassay kit (NEX-133, Du Pont–New England Nuclear). PT was purchased from List Biochemicals. BQ-123 was purchased from Novabiochem. IBMX, sodium nitroprusside, L-NNA, verapamil, and nifedipine were purchased from Sigma Chemical Co. Zn PP-9 was purchased from Porphyrin Products, Inc.
Tissue Preparations
Myocyte-enriched cultures (>95%) were
prepared from the
hearts of newborn (1-day-old) rats (CD strain) as described in
detail previously.10 Atria and ventricles dissected from
decapitated adult male rats were scissored into slices and dispersed in
Krebs' buffer (mmol/L: NaCl 123, KCl 5, KH2PO4
1.4, MgSO4 1.3, CaCl2 0.8, glucose 10, and
HEPES 20, bubbled with 5% CO2 and 95% O2 to
achieve pH 7.4).
cGMP Assay
Slices and cells were preincubated with 0.5 mmol/L
IBMX for 10
minutes in Krebs' buffer. The slices were transferred to vials
containing 0.5 mmol/L IBMX and the indicated ligands for 5 minutes,
then inactivated by boiling for 5 minutes in 4 vol
Tris-HCl, pH 8.4, and 5 mmol/L EDTA, and transferred to ice. The slices
were homogenized in a Brickmann polytron PT-10 (setting 7,
1 minute). Experiments with the myocyte cultures were carried out in
six-well plates. Ligands were added for 5 minutes, and the reaction
was terminated by the addition of boiling water for 5 minutes. The
cells were scraped and transferred to vials. All assays were performed
in triplicate. cGMP levels were determined by radioimmunoassay. Protein
concentrations were determined with bovine serum albumin used
as a standard. Results (mean±SEM) are expressed as the amount of cGMP
(picomoles) per milligram protein in the assay. Student's t
test was used for statistical analysis. Values of
P<.05 and P<.01 were considered
significant.
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Results |
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TopAbstractIntroductionMaterials and Methods Results Discussion References |
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Effect of ETs and SRTXs on cGMP Formation in Atrial Slices
Dose-response curves of cGMP production in rat atrial slices are depicted in Figs 1
and 2. As
shown in Fig 1, ET-1 (10-12 mol/L)
produced a rapid increase (2.8±0.24-fold) in cGMP levels. The effect
was maximal within 0.5 to 1 minute and then slowly decreased (a
representative time course for ET-1 is shown in Fig 3). Similar
results not shown were obtained with the
other ligands (ET-3, SRTX-b, and SRTX-c). For technical convenience, we
chose an incubation period of 5 minutes (after which the effect of the
ligand was 
90% of that obtained after 1 minute) in all subsequent
experiments, which were carried out in the presence of 0.5 mmol/L IBMX.
ET-1 at concentrations higher than 10-10
mol/L caused a dose-dependent inhibition of cGMP
production, which decreased to basal levels at concentrations
higher than 10-7 mol/L. We recently
described a similar behavior pattern for cGMP production in rat
cerebellar slices and for cAMP production in rat atrial
slices.8 9 SRTX-b showed a trend similar to that
produced
by ET-1 in stimulating cGMP production (2.27±0.14-fold
increase at 10-12 mol/L SRTX-b), as was
also observed previously in rat cerebellar slices.
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indicates
ET-1 (top) and SRTX-b (bottom); 
, basal value.
*P<.05, **P<.01.
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As shown in Fig
2
, ET-3 and SRTX-c exhibited different behavior. cGMP
production was stimulated by these ligands at the nanomolar
concentration range, with maximal effect at
10-5 mol/L. However, no cGMP was produced
when ET-3 and SRTX-c concentrations were in the picomolar range. Note
that these results are also similar to those previously obtained in
cerebellar slices, where ET-3 and SRTX-c were effective over the
nanomolar range.
Involvement of NO Pathway
The possibility that cGMP
accumulation induced by ETs and SRTXs is
mediated by an NO-dependent pathway (as demonstrated in Reference 9)
was investigated by using a specific inhibitor of NO
synthesis, L-NNA.11 L-NNA was maximally effective at a
concentration of 200 µmol/L (dose-response curve shown in Fig
4). This relatively high concentration is necessary
because nitroarginine acts by competing with endogenous
arginine, whose levels are rather high. As shown in Fig 5,
preincubation with L-NNA completely eliminated ET-1–
and SRTX-b–stimulated accumulation of cGMP in atrial slices. This
effect was reversed by L-Arg (1 mmol/L). However, L-NNA did not inhibit
cGMP accumulation induced by ET-3 and SRTX-c. These results suggest
that in atrial slices, ET-1 and SRTX-b increase cGMP levels by
stimulation of NO synthesis, which activates the guanylyl
cyclase pathway; however, ET-3 and SRTX-c stimulate cGMP
production via an L-Arg–independent pathway.
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Involvement of CO Pathway
In our earlier study carried out
with cerebellar slices, we showed
that the SRTXs stimulated an increase in cGMP levels via the CO
pathway, an alternative pathway of cGMP
production.12 To determine whether this is the
pathway of cGMP production stimulated by ET-3 and SRTX-c, we
used Zn PP-9, which inhibits the activity of heme
oxygenase, an enzyme that degrades heme to biliverdin with
the release of CO. We have tested several concentrations of Zn PP-9
(0.01, 0.1, 1, and 10 µmol/L); the maximal effect was obtained at 0.
1 µmol/L, and this concentration was used because it does not inhibit
guanylyl cyclase directly, as shown by its failure to affect cGMP
levels elevated by sodium nitroprusside (10 µmol/L). As shown in Fig
6, Zn PP-9 had no effect on cGMP production
stimulated by ET-1 and SRTX-b. However, it dramatically decreased cGMP
levels elevated by ET-3 and SRTX-c, indicating that these ligands act
most likely through the CO pathway in stimulating cGMP
production.
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Mediation by G Proteins
To determine whether the above
effects are mediated by G proteins,
using a previously described protocol,13 we examined
the effect of PT-catalyzed ADP ribosylation of G proteins on cGMP
production. Treatment of atrial slices with PT (1 µg/mL, 1
hour, 36°C) completely inhibited cGMP production
stimulated by ET-3 and SRTX-c but did not affect its stimulation by
ET-1 and SRTX-b (Fig 7). Therefore, it seems that ET-1
and SRTX-b stimulate cGMP formation through a PT-insensitive G protein
via the NO pathway, whereas ET-3 and SRTX-c stimulate cGMP formation
through a PT-sensitive G protein via the CO pathway. These observations
also support the notion that ET-1 and SRTX-b act through a different
signaling cascade than that mediating stimulation by ET-3 and
SRTX-c.
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Dependence on Ca2+
Previous studies have
shown that guanylyl cyclase activity
is dependent on the presence of
Ca2+.14 15 To
determine whether the observed rise in cGMP production
stimulated by the ligands is Ca2+ dependent, we incubated
the slices in medium without CaCl2 and in which
extracellular Ca2+ was further depleted by the addition
of 1 mmol/L EGTA. The slices were washed twice with
Ca2+-deficient EGTA-containing medium 10 minutes before the
experiment and remained in this medium for the duration of the
experiment (10 to 15 minutes). As shown in Fig 8,
exclusion of Ca2+ from the reaction mixture attenuated the
increase in cGMP production stimulated by ET-1 and SRTX-b.
However, this treatment had no effect on the cGMP production
stimulated by ET-3 and SRTX-c. These findings further support the
notion that ET-1 and SRTX-b act through a different pathway than that
of ET-3 and SRTX-c in stimulating the guanylyl cyclase pathway.
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To
determine whether Ca2+ channels play a role in the
stimulation of cGMP production by ET-1 and SRTX-b, we used the
Ca2+ channel blockers nifedipine and
verapamil. As shown in Fig 9, both
nifedipine and verapamil blocked the increase
in cGMP production stimulated by ET-1 and SRTX-b, indicating
that Ca2+ channels are important in the stimulation of cGMP
production by ET-1 and SRTX-b and suggesting that this
dependence on Ca2+ can be explained in terms of the
Ca2+ dependence of NO synthase activity. However, it should
be noted that the present data should be regarded as qualitative;
more quantitative studies, such as dose dependence of the above
blockers, are currently under way in our laboratory.
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ET Receptor Subtypes Involved in Stimulation of cGMP
Production by ETs and SRTXs
The different action mechanisms of ET-1
and SRTX-b versus ET-3 and
SRTX-c can be explained if we assume the involvement of different
receptor subtypes in the production of cGMP. To test this
assumption, we used the following ET receptor antagonists:
BQ-123 (an ETA-R–specific
antagonist)16 and Res-701-1 (an
ETB-R–specific antagonist).17 As
shown in Fig 10, preincubation with BQ-123 decreased,
in a dose-dependent manner, the production of cGMP induced
by 1 pmol/L ET-1. As depicted in Fig 11, BQ-123 blocked
both ET-1– and SRTX-b–induced stimulation of cGMP
production but had no effect on its stimulation by ET-3 and
SRTX-c. In contrast, the ETB-R antagonist
abolished cGMP production stimulated by ET-3 and SRTX-c but had
no significant effect on its stimulation by ET-1 and SRTX-b. Thus, it
seems that in atrial slices ET-1 and SRTX-b stimulate cGMP
production through binding to ETA-R, whereas ET-3
and SRTX-c stimulate it through binding to ETB-R.
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Effects of ETs and SRTXs on cGMP Levels in Primary Myocyte Cultures
and Ventricular Slices
Primary myocyte cultures prepared from the
hearts of newborn
rats18 19 provide a convenient cellular model for
studies
of cardiac receptors. Since this cell culture contains binding sites in
both the high-affinity (nanomolar) and the
super–high-affinity binding site (picomolar) concentration
ranges,6 it was of interest to determine whether the
ligands activate guanylyl cyclase in this preparation as well.
However, as shown in Fig 12, ET-1 at 1 pmol/L caused a
slight increase in cGMP levels (21% above control at 1 minute, 51%
above control at 5 minutes). At 1 µmol/L, ET-1 had no significant
effect on cGMP levels. It should be noted that sodium nitroprusside
(100 µmol/L) caused a twofold increase in cGMP levels, indicating
that this cell culture is capable of producing cGMP. The newborn heart
contains 80% to 90% ventricular cells. In an attempt
to resolve the discrepancy between the results in atrial slices and the
myocyte cell culture, we performed the same experiments on
ventricular slices. Like the atria, rat ventricles contain
a nanomolar and a picomolar binding site for ETs. As determined by
binding experiments with iodinated ET-1, the nanomolar
sites had a Kd of 2 nmol/L and Bmax
of 220 fmol/mg protein, whereas the picomolar sites had a
Kd of 80 pmol/L and Bmax of 100
fmol/mg protein. However, as shown in Fig 13, ET-1 had
no effect on cGMP levels in ventricular slices at any of
the concentrations tested, whereas it caused an increase in cGMP levels
in atrial slices from the same rats. It should be noted that sodium
nitroprusside caused an increase in cGMP levels in the
ventricular slices, confirming that these slices contain
the guanylyl cyclase machinery.
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Discussion |
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TopAbstractIntroductionMaterials and Methods Results Discussion References |
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The results of the present investigation can be summarized as follows: (1) In atrial slices, cGMP production by ET-1 and SRTX-b is stimulated at picomolar concentrations, and cGMP production by ET-3 and SRTX-c is stimulated at nanomolar concentrations. (2) ET-1 and SRTX-b stimulate cGMP production through an NO pathway, whereas ET-3 and SRTX-c stimulate it through a CO pathway. (3) The signal for ET-1 or SRTX-b stimulation is mediated through an ETA-R subtype coupled to a PT-insensitive G protein, whereas the ET-3 and SRTX-c signals are mediated by an ETB receptor subtype coupled to a PT-sensitive G protein. (4) Stimulation of cGMP production by ET-1 and SRTX-b is dependent on Ca2+, probably via Ca2+ channels. (5) The above signals are unique to the atria, as indicated by the failure of these ligands to affect cGMP levels in the ventricles.
Since the discovery of a picomolar binding site for the ET family, attempts have been made to elucidate its physiological role. Because the physiological effect of ET-1 at picomolar concentrations is vasodilation (as opposed to vasoconstriction at nanomolar and micromolar concentrations), its likely mediators are cAMP and cGMP. We have previously shown that ET-1 at picomolar concentrations increases cAMP levels in atrial slices8 and that ET-1 and SRTX-b at picomolar concentrations increase cGMP levels in cerebellar slices.9 ET-1 has also been shown to exert other distinct effects at picomolar concentrations, such as transcription of the c-fos gene and activation of mitogen-activated protein kinase.20 21 The present study confirms and emphasizes the fact that the guanylyl cyclase pathway is stimulated by low doses of ET-1 and SRTX-b. Interestingly, at higher doses of these ligands, cGMP production is inhibited. Dual effects of ET-1 have also been reported in other systems. Thus, for example, intravenous injection of ET-1 was shown to cause a transient dose-dependent vasodilation followed by sustained vasoconstriction.22 In arterial smooth muscle cells, 100 pmol/L ET-1 activated a Ca2+-dependent K+ channel but inhibited it at concentrations higher than 100 nmol/L.23 ET-1 also exhibits dual effects on neurite outgrowth induced by phorbol ester.24 The pattern of behavior of ET-1 and SRTX-b may be indicative of either a rapid desensitization process or the existence of a distinct receptor. Attempts are under way in our laboratory to clarify this point.
The soluble form of guanylyl cyclase is activated by vasodilatory agents such as NO, nitroprusside,25 and also, as recently shown, CO.12 The present study demonstrates that cGMP production is stimulated by ET-1 and SRTX-b via an NO pathway and by ET-3 and SRTX-c via an L-Arg–independent pathway, possibly a CO pathway. Since ET-1 and SRTX-b bind to an ETA-R, this appears to be the receptor-ligand complex that activates the NO signal. Binding of ET-3 or SRTX-c to ETB-R forms a different receptor-ligand complex, which presumably activates a different signal, the CO pathway. The observed difference in sensitivity of the complexes to PT further supports this notion, since it indicates that the various receptor-ligand complexes are coupled to different G proteins.
In our previous study in the cerebellum, cGMP production was stimulated by the binding of ligands to the ETA-R; in that preparation, the ETs functioned through NO, and the SRTXs functioned through CO. A particular ligand might exhibit preferential functional coupling to a specific G protein. We have suggested9 that the observed differences between ETs and SRTXs stem from the existence of specific ligand-receptor–G-protein complexes, in which the specific pathway is dependent on the nature of the G protein as well as on the ligand used (ET versus SRTX). The differences between the present results in the atrium and the previous results in the cerebellum could be explained in terms of either nonidentical receptor subtypes in different tissues (for example, the ETA-R in the cerebellum might be distinct from the ETA-R in the atrium) or different G proteins. The latter explanation is the more likely one, in view of the large number of known G proteins. This variety of potential combinations might provide an explanation for the difference in results obtained in the cerebellar and atrial preparations. Thus, for instance, in the cerebellum, ET-1 and ET-3 may form similar ligand-receptor–G-protein complexes, which differ from those formed in the atrium (because either the receptor or the G protein is different). Accordingly, the signaling pathways activated by ET-1 and ET-3 in the atrium will differ from those in the cerebellum. Similarly, SRTX-b and SRTX-c may activate identical signals in one tissue and different signals in another. Finally, one cannot rule out the possible existence of a factor that controls the formation of receptor–G-protein complexes and which, being tissue specific, leads to the formation of different complexes in different tissues.
The Ca2+ dependence of the stimulation of cGMP production by ET-1 and SRTX-b might be explained by the Ca2+ dependence of the NO synthase enzyme involved in their pathway. At least three distinct enzymes that catalyze the production of NO have been described: a 135-kD enzyme in endothelial cells that is activated by elevation of intracellular Ca2+,26 a 168-kD neuronal NO synthase that is responsible for the Ca2+-dependent release of NO from neurons and nonadrenergic noncholinergic nerves,27 and an inducible Ca2+-independent 130-kD NO synthase.28 NO is synthesized by Ca2+-dependent enzymes in endocardial endothelial cells as well as cardiomyocytes.29 30 ETs also activate a Ca2+/calmodulin–dependent NO synthase in endothelial cells,31 and ET-1 exhibits Ca2+ dependence in its stimulation of cGMP production in a neuronal cell line.14
The inability of ET-1 to stimulate cGMP in myocytes might be explained by the fact that our cell culture was derived from neonatal as opposed to adult rats. Age may be one of the reasons for variability in the activation of ET receptors; thus, for example, responses of adult rabbit myocytes were found to differ dramatically from those of embryonic or neonatal myocytes.32 However, as the present study demonstrates, the most likely reason for the limited stimulation of cGMP production in the myocyte cell cultures is that the ventricular fraction is unresponsive to such stimulation by ETs and SRTXs. Previous studies have shown that cAMP formation is inhibited in ventricular myocytes33 but is stimulated in the atria.8 Therefore, one might speculate that in the atria ETs and SRTXs have a positive effect on cGMP and cAMP formation, whereas in the ventricles they have a negative effect. Further studies are required to clarify the functional differences between ET ligand activity in the atria and the ventricles.
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Selected Abbreviations and Acronyms |
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-nitro-L-arginine
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Acknowledgments |
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We thank Shirley Smith for excellent editorial assistance.
Received June 19, 1995; accepted November 9, 1995.
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References |
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