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Vol. 294, Issue 1, 147-154, July 2000
Department of Pharmacology, College of Medicine, East Tennessee State University, Johnson City, Tennessee (Y.C., D.B.H., J.C.H.); and Department of Anatomy and Neurobiology, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada (F.M.S.)
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Abstract |
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 Top
 Abstract
 Introduction
Experimental Procedures
Results
Discussion
References
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Selective tachykinin agonists were used to identify cardiac and coronary responses mediated by specific tachykinin receptor subtypes in isolated, perfused guinea pig hearts. Receptor desensitization with selective agonists and blockade with selective antagonists were used to determine the role of specific subtypes in generating responses to neurokinin A (NKA). Dose-dependent cardiac and coronary effects were evoked by bolus injections of [Sar9,Met(O2)11]substance P ([Sar9,Met(O2)11]SP), GR64349, and [MePhe7]neurokinin B ([MePhe7]NKB) (selective agonists for NK1, NK2, and NK3 receptors, respectively). Each agonist caused bradycardia, but GR64349 was most effective (34 ± 4% decrease in heart rate with 32 nmol, n = 8). Prominent increases in ventricular contractility and perfusion pressure also occurred with 32 nmol of GR64349 (25 ± 6 and 33 ± 4%, respectively). [Sar9,Met(O2)11]SP was unique in having a high potency for decreasing ventricular contractility and perfusion pressure. Bolus injections of 25 nmol of NKA decreased rate (48 ± 2%, n = 51), increased contractility (26 ± 2%), and had biphasic effects on perfusion pressure (24 ± 1% decrease followed by 9.2 ± 1.4% increase). Desensitization with GR64349 or treatment with the NK2 antagonist SR48968 reduced the bradycardic response to NKA by greater than 75% and eliminated the positive inotropic response. The remaining bradycardia occurred through NK3 receptors. Desensitization with [Sar9,Met(O2)11]SP or NK1 blockade with FK888 eliminated the coronary relaxant action of NKA and enhanced the pressor response. It is concluded that three tachykinin receptor subtypes are present in the guinea pig heart and that each contributes to the overall response evoked by NKA.
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Introduction |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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The
tachykinins, substance P (SP) and neurokinin A (NKA), are colocalized
in a large subpopulation of primary afferent neurons that are capable
of exerting efferent functions through release of these bioactive
peptides from their peripheral processes (Maggi and Meli, 1988
; Otsuka
and Yoshioka, 1993). Many immunohistochemical studies have established
that such tachykinin-containing nerve processes are present in the
heart where they are localized to the intrinsic cardiac ganglia,
adventitia of coronary arteries, and, at a lower density, to many
myocardial sites (Wharton et al., 1981, 1988, 1990; Weihe et al.,
1984). Within the intrinsic cardiac ganglia, tachykinins are localized
to varicose nerve fibers that surround many of the principal neurons.
Collectively, these observations suggest that tachykinins may have
important effects on cardiac function through direct and neurally
mediated mechanisms.
Administration of SP to isolated guinea pig hearts has been reported to
cause bradycardia (Hoover and Hancock, 1988; Hoover, 1990), reduced
ventricular contractility (Chiao and Caldwell, 1995; Hoover et al.,
1998), and relaxation of coronary resistance vessels (Hoover and
Hancock, 1988; Hoover, 1990; Vials and Burnstock, 1992; Hoover and
Hossler, 1993). The negative chronotropic response to SP has been
attributed to stimulation of cholinergic neurons of the intrinsic
cardiac ganglia. Evidence supporting this conclusion includes the
autoradiographic identification of specific SP binding sites in guinea
pig cardiac ganglia (Hoover and Hancock, 1988) and the observations
that negative chronotropic responses to SP are attenuated by muscarinic
receptor blockade and potentiated by cholinesterase inhibition (Hoover,
1990; Chiao and Caldwell, 1995). There is also electrophysiological
evidence that SP can directly activate neurons of guinea pig
intracardiac ganglia in vitro (Konishi et al., 1985; Hardwick et al.,
1995, 1997).
A recent evaluation of responses to NKA in isolated guinea pig hearts
has revealed quantitative and qualitative differences compared with SP
(Hoover et al., 1998). NKA had a greater potency for evoking
bradycardia, and more than half of the negative chronotropic response
to NKA was unaffected by treatment with atropine. The dominant effect
of NKA on ventricular contractility was augmentation rather than
suppression. Last, NKA had a biphasic effect on coronary vascular tone,
whereas SP caused only relaxation. It is possible that some of these
differences may be attributed to the presence of more than one subtype
of tachykinin receptor in the heart.
Our goals in this study were: 1) to identify cardiac and coronary responses evoked by stimulation of specific tachykinin receptor subtypes, and 2) to determine the role of tachykinin receptor subtypes in generating responses to NKA in the isolated guinea pig heart. Subtype-selective agonists were used to address the first aim. Desensitization with selective agonists and administration of selective tachykinin antagonists were two approaches used to address the second aim.
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Experimental Procedures |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Isolated Heart Preparation.
Male Hartley guinea pigs
(350-450 g) were pretreated with 500 U of heparin. Approximately 20 min later, they were deeply anesthetized with sodium pentobarbital (75 mg/kg i.p.) and decapitated. The heart was rapidly removed and placed
in ice-cold perfusion buffer to enable cannulation of the ascending
aorta. After being flushed with 5 ml of cold buffer, the heart was
transferred to an isolated heart apparatus for perfusion by a
modification of the Langendorff technique (Broadley, 1979). The
perfusion solution was a modified Krebs-Ringer-bicarbonate buffer (pH
7.35-7.4) of the following composition (mM): NaCl, 127.2; KCl, 4.7;
CaCl2, 2.5;
KH2PO4, 1.2; NaHCO3, 24.9; MgSO4, 1.2;
sodium pyruvate, 2.0; dextrose, 5.5; and 0.1% BSA. A Masterflex
peristaltic pump (Cole-Parmer Instrument Co., St. Louis, MO) was used
to perfuse hearts at a constant rate of 8 ml
min
1. Buffer in the reservoir was continuously
gassed with 95% O2, 5%
CO2. The temperature of the buffer was maintained
at 37°C. Cardiac contractions were measured by attaching one end of a
silk suture to the apex of the heart and the other end to an isometric force transducer. Tension on the heart during diastole was adjusted to
approximately 1 g. Output from the force transducer was sent to a
Gould Universal amplifier and a Gould Biotach amplifier to monitor
ventricular contractions and heart rate, respectively, with a Gould
2400 recorder (Gould Instrument Systems, Valley View, OH).
Because the perfusion rate was held constant, perfusion pressure was
monitored as an indicator of coronary vascular resistance. This
parameter was recorded using a pressure transducer that was attached to
the sidearm of a three-way stopcock located at the proximal end of the
aortic cannula. Experiments were started after a 40-min stabilization period.
Preparation and Storage of Drugs.
NKA,
[Sar9,Met(O2)11]SP,
and GR64349 were dissolved in sterile saline.
[MePhe7]NKB, FK888, SR48968, and SR142801 were
dissolved in dimethyl sulfoxide (DMSO).
[MePhe7]NKB and FK888 were diluted further with
sterile saline to make stock solutions containing 15 and 60% DMSO,
respectively. Aliquots of tachykinin receptor agonists (3.2 mM) and
antagonists (1 mM) were stored at 
80°C. NKA and the selective
agonists were diluted with saline that contained 0.1% BSA.
Acetylcholine (ACh) was weighed and dissolved in saline before each experiment.
Drug Administration.
The selective tachykinin agonists, NKA,
and ACh were administered by bolus injection. Volumes of 100 µl were
given over ~3 s. For dose-response studies, the selective agonists
were given in order of ascending dose. Injections of the selective
agonists were separated by 20- to 40-min intervals to avoid
desensitization. In other experiments, we examined the effect of
desensitization with selective agonists on responses to NKA and ACh.
For these studies, a series of four bolus injections of selective
agonist (32 nmol/100 µl) were given at 1-min intervals. Either 25 nmol of NKA or 1 nmol of ACh was given 30 s after the last
injection of selective agonist. In experiments with selective
antagonists, responses to 25 nmol of NKA and 1 nmol of ACh were
determined in the absence of drug and again during exposure to the
antagonist or a combination of antagonists. The
NK1 receptor antagonist FK888 was diluted with
saline and given by infusion (8 nmol/50 µl
min1) through a short length of polyethylene 20 tubing that emptied into the perfusion buffer at a point near the
three-way stopcock attached to the aortic cannula. The infused solution
contained 10% DMSO. The final concentration of FK888 at the heart was
1 µM. The NK2 and NK3
receptor antagonists SR48968 and SR142801, respectively, were included
in the perfusion buffer when hearts were treated for 20 min before a
second challenge with NKA. In later experiments, the treatment interval
was extended to 60 min, and antagonists were given by infusion. The
concentration of DMSO in the buffer was 
0.4%.
Materials. NKA and selective tachykinin agonists were purchased from Peninsula Laboratories (Belmont, CA) or Research Biochemicals International (Natick, MA). FK888 was obtained from Research Biochemicals International. SR48968 (saredutant; (S)-N- methyl-N[4-(4-acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophenyl)butyl]benzamide) and SR142801 (osanetant; (R)-(N)-(1-(3-(1-benzoyl-3-(3,4-dichlorophenyl)piperidin-3-yl)propyl)-4-phenyl-piperidin-4-yl)-N-methylacetamide) were generously given by Dr. Xavier Emonds-Alt (Sanofi Recherche, Montpellier, France). ACh chloride was purchased from Sigma Chemical (St. Louis, MO).
Data Analysis. Heart rate (beats/min), diastolic perfusion pressure (mm Hg), and ventricular contractility (g) were measured before each injection and at the times of maximum responses. Changes were expressed as a percentage of baseline. Group data are presented as mean ± S.E. GraphPad Prism version 2.01 (GraphPad Software, San Diego, CA) was used for analysis of dose-response data and preparation of graphs. Statistical comparisons were made by a two-tailed, paired t test or ANOVA using GraphPad Prism or NCSS 97 software (NCSS, Kaysville, UT). In the case of significant F-values, post hoc comparisons following ANOVA were made using the Newman-Keuls procedure. A probability level of .05 or less was used to indicate statistical significance.
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Results |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Effects of Selective Agonists.
The presence and function
of tachykinin receptor subtypes in the isolated guinea pig heart was
evaluated using the NK1 agonists [Sar9,Met(O2)11]SP
and GR73632, NK2 agonists GR64349 and
[
Ala]NKA(4-10), and NK3 agonists
[MePhe7]NKB and senktide. For each receptor
subtype, both agonists produced similar responses, so data are
presented only for the drug that was most thoroughly studied in each class.
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Effect of Tachykinin Receptor Desensitization with Selective
Agonists on Responses to NKA.
NKA caused bradycardia, an increase
in the force of ventricular contractions, and either a decrease in
perfusion pressure or a biphasic coronary vascular response
(Figs. 3-6), as reported previously
(Hoover et al., 1998). Biphasic vascular responses consisted of an
initial decrease in perfusion pressure, followed by an increase above
the previous baseline; this type of response occurred in 30 of 51 hearts.
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Effect of Subtype-Selective Tachykinin Receptor Antagonists on
Responses to NKA.
Negative chronotropic responses to NKA were
attenuated by treatment with 100 nM SR48968 for 20 min (75% decrease;
Figs. 5 and 7A) or 100 nM SR142801 for 60 min (48% decrease; Fig. 5A). Treatment with SR142801 (100 or 320 nM)
for 20 min was ineffective (not shown). Combined treatment with
NK2 and NK3 antagonists
(100 nM each for 60 min) nearly eliminated the chronotropic response to
NKA without affecting the bradycardia evoked by 1 nmol of ACh (76 ± 1% decrease for control versus 73 ± 5% decrease in presence of antagonists; n = 4; P = .39). Minor
reductions in bradycardic responses to NKA (Fig. 5A) and ACh occurred
during treatment with 1 µM FK888. Positive inotropic responses to NKA
were abolished by 100 nM SR48968 and reduced by 100 nM SR142801 (Fig.
5B). Ventricular contractile responses to NKA were more variable in the
presence of 1 µM FK888 (Fig. 5B), so a reduction in the mean response
was not statistically significant.
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Discussion |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Results from this study provide the first evidence that all three subtypes of the tachykinin receptor are present in the guinea pig heart. The overall response evoked by administration of NKA to isolated hearts comprises elements generated through activation of each receptor subtype. Bradycardia occurs through activation of NK2 and NK3 receptors, whereas positive inotropic responses are mediated primarily by NK2 receptors. The dominant effect of NKA on coronary resistance vessels is NK1 receptor-mediated vasodilation, but a vasoconstrictor action becomes prominent following suppression of the vasodilator response. Last, this study provides the first evidence that NK2 receptors mediate coronary vasoconstriction.
We previously reported that NKA and SP cause a dose-dependent
bradycardia in the isolated guinea pig heart (Hoover and Hancock, 1988;
Hoover, 1990; Hoover et al., 1998). The present findings demonstrate
that bradycardia can occur through stimulation of each subtype of the
tachykinin receptor, but activation of NK2 receptors produces the most prominent decrease in heart rate. Two lines
of evidence suggest that negative chronotropic responses to NKA occur
primarily through stimulation of NK2 receptors.
First, desensitization with GR64349 caused an 80% decrease in the
magnitude of bradycardia evoked by NKA. Second, blockade of
NK2 receptors reduced the chronotropic responses
to NKA by 75%. Neither of these treatments affected the negative
chronotropic response to ACh. Results from analogous experiments using
[MePhe7]NKB and SR142801 also implicate
NK3 receptors in the chronotropic response to NKA
but suggest a smaller contribution. Neither desensitization with an
NK1 agonist nor blockade of
NK1 receptors with FK888 affected the bradycardic
response to NKA. Thus, NK1 receptors have no role in mediating this effect.
Stimulation of intracardiac cholinergic neurons is one mechanism by
which tachykinins can produce bradycardia. Binding sites for SP and NKA
are localized to intracardiac ganglia (Hoover and Hancock, 1988; Hoover
et al., 1998), and both peptides increase the excitability of
intracardiac neurons (Konishi et al., 1985; Hardwick et al., 1995,
1997). Negative chronotropic responses to SP are also attenuated by
treatment with atropine or hemicholinium-3 (Hoover, 1990; Chiao and
Caldwell, 1995). Electrophysiological evidence suggests that
intracardiac neurons express NK2 and
NK3 receptors (Hardwick et al., 1995), but the
slow depolarization evoked by SP occurs through the
NK3 subtype (Hardwick et al., 1997). Our previous
work established that more than half of the response to NKA occurs
through a noncholinergic mechanism because it is resistant to blockade
by atropine (Hoover et al., 1998). Accordingly, it is possible that
tachykinin receptors could be present at the sinoatrial node in a
concentration below the limit for detection by autoradiography.
Ventricular contractility was decreased by NK1
agonists and increased by NK2 agonists in this
study. Because tachykinin receptors are not present in the ventricular
myocardium (Hoover and Hancock, 1988; Hoover et al., 1998), such
inotropic responses must occur secondarily to other actions of these
drugs. In this regard, we found that the positive inotropic response to
NKA was replaced by a small inhibitory effect when isolated guinea pig
hearts were paced to prevent changes in heart rate (Hoover et al.,
1998). Based on this finding and our current results, we conclude that positive inotropic responses to NK2 agonists
probably occur secondarily to bradycardia. Other investigators have
presented evidence that SP can attenuate ventricular contractions by a
paracrine mechanism involving nitric oxide released from vascular
endothelial cells (Grocott-Mason et al., 1994; Paulus et al., 1995). It
is likely that this mechanism underlies negative inotropic responses to NKA in paced hearts and NK1 agonists in this
study because coronary vasodilator responses to NKA and SP are mediated
by nitric oxide (Vials and Burnstock, 1992; Hoover and Hossler, 1993).
The enhanced positive inotropic response to NKA in the presence of
FK888 could be explained by the loss of such a paracrine effect due to
blockade of endothelial NK1 receptors.
Previous studies implicated NK1 receptors in the
coronary vasodilator action of tachykinins based on the greater potency
of SP compared with NKA (Gulati et al., 1987; Hoover and Hossler, 1993). In accord with this concept, we observed that
NK1 agonists exhibited the highest potency for
decreasing perfusion pressure and that vasodilator responses to NKA
were eliminated by treatment with the NK1
antagonist FK888. Vasodilator responses to NK2
and NK3 agonists might be attributed to a
decrease in selectivity of these agents at higher doses. Alternatively,
they might occur indirectly through stimulation of
NK2 and NK3 receptors on
cholinergic neurons. This mechanism could also account for our
observation that vasodilator responses to NKA were attenuated after
treatment with an NK2 or
NK3 antagonist.
This study provides evidence that NK2 receptors mediate coronary vasoconstriction in the guinea pig heart. Support for this conclusion comes from observations that NK2-selective agonists caused a dose-dependent increase in perfusion pressure, whereas selective agonists for other tachykinin receptor subtypes only caused depressor responses. A majority of our data also indicates NK2 receptors can mediate coronary vasoconstrictor responses to administered NKA. Although NK1 receptor-mediated vasodilation normally dominates, vasoconstrictor responses to NKA were unmasked or enhanced during NK1 receptor blockade or receptor desensitization with an NK1 agonist. The only discordant result comes from experiments with SR142801 because this NK3 antagonist appeared to block pressor responses to NKA without affecting those evoked by the NK2 agonist GR64349. It is unclear at present whether this result is a real effect of the NK3 blocker or a consequence of the variability and small magnitude of the NKA-evoked pressor responses under normal conditions.
Although the compounds used to evaluate tachykinin receptor subtypes in
this study are classified as selective agonists or antagonists, it is
recognized that their selectivity is concentration-dependent (Advenier
et al., 1992; Fujii et al., 1992; Petitet et al., 1993; Regoli et al.,
1994; Wang et al., 1994b; Emonds-Alt et al., 1995; Patacchini and
Maggi, 1995). Furthermore, SR48968 and SR142801 can produce nonspecific
effects through binding to ion channels (Wang et al., 1994a). We have
attempted to minimize or monitor the influence of these factors in the
design of this study by the following procedures. Two selective
agonists for each tachykinin receptor subtype were evaluated, and
response profiles within each class were identical. Responses to NKA
were evaluated by two distinct but complementary approaches. The first
of these was to desensitize tachykinin receptors using selective
agonists. Although this approach undoubtedly affected more than a
single tachykinin receptor subtype, the results suggest that the
dominant effect was on the targeted receptor. The other approach was to block receptors with selective antagonists. Desensitization and receptor blockade produced similar results in most experiments. Last,
we determined responses to ACh before and after tachykinin receptor
desensitization or treatment with tachykinin receptor blockers and
observed that responses to ACh were generally unaffected.
In conclusion, this study has provided functional evidence for the
presence of NK1, NK2, and
NK3 receptors in the guinea pig heart and
identified receptor subtypes that mediate responses to the native
tachykinin NKA. Although this study has used pharmacologic doses of NKA
and synthetic tachykinins, it is likely that some or all of these
effects could likewise be triggered by endogenous tachykinins under
pathophysiological conditions. Local release of tachykinins from
cardiac afferents has been demonstrated in response to various
chemicals released within the myocardium during ischemia
(Franco-Cereceda et al., 1987, 1994; Geppetti et al., 1988).
Accordingly, these findings provide important insights regarding
effects that endogenous tachykinins might produce within the heart and
coronary vasculature during ischemic heart disease.
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Acknowledgments |
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We are grateful to Sanofi Recherche and Dr. Xavier Emonds-Alt for supplying SR48968 and SR142801.
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Footnotes |
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Accepted for publication March 14, 2000.
Received for publication December 22, 1999.
1 This study was supported by National Heart, Lung, and Blood Institute Grant HL-54633.
2 F.M.S. was a Research Scholar of the Heart and Stroke Foundation of Canada during part of this study.
Send reprint requests to: Dr. Donald B. Hoover, Department of Pharmacology, College of Medicine, East Tennessee State University, Johnson City, TN 37614-0577. E-mail: hoover{at}etsu.edu
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Abbreviations |
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SP, substance P; ACh, acetylcholine; DMSO, dimethyl sulfoxide; NKA, neurokinin A; NKB, neurokinin B.
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References |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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)
indicates significant difference from response to same dose of GR64349.
