(Hypertension. 1995;26:480-484.)
© 1995 American Heart Association, Inc.
Articles |
Neuropeptide Y Prevents Agonist-Stimulated Increases in Contractility
From the Division of Cardiovascular Sciences, St Boniface General Hospital Research Centre and Department of Anatomy, University of Manitoba, Winnipeg, Canada.
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Abstract |
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Abstract Neuropeptide Y has been shown to inhibit contractility in the rat heart. Although the reasons for this effect are not known, it is possible that postsynaptic adrenergic mechanisms involving neuropeptide Y may be responsible. To ascertain whether this neuromodulatory effect is possible for decreasing contractility, we investigated the effect of neuropeptide Y on agonist-stimulated contractility of the isolated rat myocardium. Receptor binding studies of purified cardiac membranes showed that incubating membrane in the presence of neuropeptide Y (10-7 mol/L) decreased the number of
-/ß-adrenoceptor binding sites
without affecting the affinity of these receptors. Isolated hearts
perfused with phenylephrine
(10-5 to
10-10 mol/L) or isoproterenol
(10-5 to
10-10 mol/L) in a nonrecirculating
Langendorff setup demonstrated a significant increase in
contractility over control values, whereas no change in
contractility was observed when the hearts were
perfused with neuropeptide Y (10-7
mol/L). However, in the presence of both agonist and neuropeptide Y the
increase in contractility previously seen with agonist
alone was not evident. Comparisons made with hearts taken from aortic
banded rats yielded similar results. Although neuropeptide Y itself was
ineffective in decreasing contractility, it prevented
the agonists from stimulating contractility when
perfused together. We conclude that neuropeptide Y does not directly
decrease contractility but prevents agonist-stimulated
increases in contractility through
-/ß-adrenoceptor pathways. This neuromodulatory effect of
neuropeptide Y is unchanged in situations of increased sympathetic
activity, such as hypertension.
Key Words: neuropeptide Y • receptors, adrenergic • models, cardiovascular • phenylephrine • isoproterenol
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Introduction |
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Neuropeptide Y (NPY) can modulate sympathetic responses by inhibiting the presynaptic release of norepinephrine,1 with which it coexists. Systemic application of NPY decreases cardiac contractility in the rat2 and decreases the contractile force of atrial and ventricular strips in rat hearts.3 However, the biochemical mechanisms responsible for this negative inotropic effect of NPY have not been elucidated. In addition, it is unclear whether this negative contractile effect is altered in a situation of increased sympathetic nerve activity, such as in hypertension.4 Recent studies have shown that NPY increases inositol phosphate formation in the femoral artery of normotensive rats5 while inhibiting forskolin-stimulated adenylate cyclase activity in cells of the vas deferens,6 pig spleen,7 and pulmonary artery smooth muscle.8 Furthermore, other studies have demonstrated that ventricular myocytes have receptors for NPY and that these receptors are linked to adenylate cyclase by an inhibitory guanylate binding protein.9 Thus, NPY may act on cardiac adrenergic receptors, modulating myocardial contractile behavior through this postsynaptic mechanism. We therefore examined whether NPY affects the expression of postsynaptic
-/ß-adrenoceptors in the rat myocardium, and if so
whether NPY affects -/ß-adrenoceptor–mediated inotropy
in the isolated rat heart of sham-operated and aortic banded
hypertensive rats. |
Methods |
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[3H]Dihydroalprenolol (DHA) and [3H]prazosin were obtained from DuPont–New England Nuclear. Phenylephrine hydrochloride was obtained from Research Biochemicals International. NPY, isoproterenol hydrochloride, phentolamine hydrochloride, propranolol, and aprotinin were purchased from Sigma Chemical Co. All other chemicals were of analytical grade.
The animals used in this study were healthy male Sprague-Dawley rats (250 to 300 g, n=48) handled according to procedures established by the University of Manitoba. Specifically, the rats were kept in a 12-hour day/night cycle and fed ad libitum. At the time of harvesting the rats were killed by quick decapitation; their hearts were trimmed of atria, right ventricle, and fat; and the left ventricle was processed according to the methods described below.
Cardiac membranes were prepared to a protein concentration of 0.5 mg/mL
according to the method of Ganguly et al.10 The
composition of the incubation buffer (mmol/L) was Tris-HCl 250, NaCl
600, KCl 25, MgCl2 5, and CaCl2 5, with 15 g of
aprotinin added per liter of buffer. For determination of the affinity
of -adrenoceptors, the membranes were incubated with increasing
concentrations of [3H]prazosin (0.5 to 10 nmol/L) in a
total volume of 0.5 mL for 20 minutes at 37°C in the presence and
absence of 10 µmol/L phentolamine hydrochloride.
ß-Adrenoceptor binding was analyzed in a manner similar to
the above except that the antagonist [3H]DHA
was used in the presence and absence of 10 µmol/L
propranolol. All incubations were terminated by rapid
vacuum filtration through Whatman GF/B glass fiber filters that were
washed in ice-cold buffer to reduce nonspecific binding. In a second
set of experiments NPY (10-7 mol/L)
dissolved in deionized water was included during incubation for
determination of its effect on the binding ability of
-/ß-adrenoceptors.
Isolated myocardium was attached to a nonrecirculating perfusion apparatus of Langendorff and perfused with Krebs-Henseleit solution.11 The flow rate was maintained at 10 mL/min throughout the experiments. The composition of the perfusion medium (nmol/L) was NaCl 118, KCl 4.7, CaCl2 1.25, MgSO4 1.2, NaHCO3 25, KH2PO4 1.2, glucose 7, sodium pyruvate 2, and mannitol 1.1. The perfusion solution was continuously oxygenated with a mixture of 95% O2/5% CO2 (pH 7.4) and maintained at a temperature of 37°C. The hearts were electrically driven by an electrode placed at the atrioventricular node with 2-millisecond pulses at four events per second and a voltage of 10% above the threshold. The hearts were vented near the apex, and a resting tension of 2 g was applied on start of the perfusion. After an equilibration period of 20 minutes the resting force was increased to 5 g. Contractile force was monitored on a Beckman Dynograph Recorder -R 511A by means of a Grass FT-03 force displacement transducer. Individually, phenylephrine (10-5 to 10-10 mol/L), isoproterenol (10-5 to 10-10 mol/L), and NPY (10-7 mol/L) were infused over a 30-minute period and their effects recorded. The contractility of the heart was allowed to return to baseline values before combination treatments were initiated.
A second set of hearts taken from rats whose aortas were constricted for 14 days12 also underwent the above-described protocol for Langendorff perfusion. Briefly, the rats assigned to the aortic banded group were anesthetized with intramuscular ketamine (40 mg)/xylazine (4 mg) and underwent a midline laparotomy with placement of a constricting ligature around the suprarenal abdominal aorta. A blunt 21-gauge needle was used as a guide. Sham controls underwent the same procedure except that the aorta was not banded. Once they had recovered, all rats were housed and fed under identical conditions.
Blood pressure assessments were carried out in a separate set of banded and sham-operated rats that were anesthetized with sodium pentobarbital (50 mg/kg). After tracheal intubation for maintenance of adequate ventilation the right carotid artery was exposed, and a microtip pressure transducer (model SPR-249, Millar Instruments) was introduced through a proximal arteriotomy. The catheter was carefully advanced through the lumen of the carotid artery and secured in place with a silk ligature tied around the artery. The catheter was connected to a Dynograph recorder (model RSHA, Beckman Instruments), and measurements of arterial pressure were carried out. Norepinephrine turnover and plasma catecholamines were measured as markers of sympathetic activity as previously described.4
All results are expressed as mean±SEM. Statistical differences between mean values for the two groups were evaluated by paired Student's t test. Analyses of saturation binding assays were performed according to the method of Scatchard13 and confirmed by the ligand program (Elsevier-Biosoft). Significance for all tests was set at a value of P<.05.
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Results |
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-/ß-Adrenoceptor BindingThe status of
-/ß-adrenoceptors from cardiac membrane
preparations was examined in the presence and absence of NPY
(10-7 mol/L). The addition of NPY altered
the binding of both [3H]prazosin and
[3H]DHA at varying concentrations. The specific binding
of [3H]prazosin was significantly decreased in the
presence of NPY regardless of the [3H]prazosin
concentration used (0.5 to 10 nmol/L) (Fig 1). Similarly, the specific binding of
[3H]DHA to ß-adrenoceptors was significantly decreased
in the presence of NPY throughout the tested range (0.5 to 10 nmol/L)
of [3H]DHA (Fig 2).
Scatchard analysis (Table) of the data revealed
that the Bmax for -adrenoceptors was 112±13
fmol/mg protein for controls and 53±12 when NPY
(10-7 mol/L) was included. For
ß-adrenoceptors the Bmax was 136±18 fmol/mg
protein for controls and 85±9 when NPY was included. However, the
binding affinity (Kd) of -/ß-adrenoceptors
remained unchanged when exposed to NPY (-adrenoceptors, 1.03±0.35
nmol/L for controls, 1.22±0.24 with NPY; ß-adrenoceptors, 2.01±0.24
nmol/L for controls, 2.17±0.31 with NPY).
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) and absence (
) of neuropeptide Y
(10-7 mol/L). Results are expressed as
disintegrations per minute (DPM) per milligram protein and
represent mean±SEM of six experiments. Significance between
groups was established at a value of P<.05.
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Contractility Studies on Isolated Perfused
Myocardium
As evident in Fig 3, phenylephrine
(10-5 to
10-10 mol/L) and isoproterenol
(10-5 to
10-10 mol/L) were both effective at
raising the contractility of the perfused isolated
myocardium from baseline in a dose-dependent fashion. This
agrees with data previously published from this
laboratory.14 However, the addition of NPY
(10-7 mmol/L) to the perfusate resulted
in a decrease in contractility in both circumstances.
Perfusion of NPY (10-6 to
10-10 mol/L) through the Langendorff
preparation resulted in a dose-dependent decrease in
contractility when either phenylephrine
(10-6 mmol/L) or isoproterenol
(10-6 mmol/L) was
simultaneously infused (Fig 4). NPY alone did not affect the basal
level of contractile activity (data not shown).
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A second set of hearts harvested from rats that underwent suprarenal aortic banding was also examined in the Langendorff setup. The contractility results using hearts taken from sham-operated controls demonstrated no statistical differences compared with hearts taken from unoperated controls (data not shown). For this section of the study comparisons were made between hearts taken from sham-operated controls and aortic banded rats. As seen in Fig 5 both phenylephrine and isoproterenol increased the contractility of the isolated hearts in a dose-dependent manner over baseline levels. However, this effect was negated when NPY (10-7 mmol/L) was included in the perfusate with either phenylephrine or isoproterenol. Contractility was unchanged when NPY alone was infused (data not shown).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The present results of the receptor binding studies demonstrate that NPY can nonselectively modulate
-/ß-adrenoceptor binding sites in vitro. Thus, it is possible that
NPY may modulate adrenergic transmission by decreasing the number of
-/ß-adrenoceptors. These findings are consistent with
other reports that there are negative contractile responses of
cardiomyocytes to NPY.1 2 3 Furthermore, this is
substantiated by the fact that NPY antagonizes the contractile response
of isolated rat cardiomyocytes stimulated by
isoproterenol15 and is abolished by treatment of cells
with pertussis toxin, an inhibitor of G protein function.
This indicates that NPY is coupled in an inhibitory way to
the adenylate cyclase via adrenoceptors and Gi
protein.
No significant changes in the Kd value of
myocardial -/ß-adrenoceptors in the presence of NPY were observed,
but NPY appeared to decrease the number of -/ß-adrenoceptors as
indicated by Bmax. Although many studies have
reported that NPY exerts a prejunctional inhibitory effect
on norepinephrine release from sympathetic nerve endings in
the heart,16 this is the first report showing that NPY can
modulate postsynaptic catecholamine receptors in the
myocardium. Although the mechanism of action presently
is unknown, the possibility exists that NPY may act directly on the
adrenoceptor or through its own receptor (subtype
Y1).17 In addition, the presence of a
second messenger after binding of NPY to surface receptors is possible
and has been demonstrated in feline cerebral blood
vessels.18 Furthermore, it is possible that NPY may
initiate changes in the membrane lipid bilayer. In this regard it has
been shown that antagonist binding to ß-adrenoceptors can
be either increased or decreased in response to alterations in the
membrane lipid content.19 In another study treatment of
frog erythrocyte membrane with phospholipase C or D decreased
[3H]DHA binding without changes in the dissociation
constant.20 The mechanism proposed to explain this result
is that the polar head groups of the phospholipids, which are
hydrolyzed by these phospholipases, may be related to recognition of
the ß-antagonists.
Considering these possible mechanisms, NPY may well attenuate the agonist effect by modifying the method in which the cell interprets agonist stimulation. By decreasing receptor number without changing affinity, it is possible that NPY may enhance internalization of the receptor complex. The receptor affinity, which is at a baseline level, remains unchanged, and the end result is a cell that is less responsive to agonist stimulation. Clearly, additional studies are needed to further define the precise mechanism by which NPY alters adrenergic receptors.
Agnati et al1 reported that NPY increased the number of
2-, but not 1-adrenergic, and
ß-adrenergic binding sites in rat brain membranes. Furthermore, these
investigators observed no changes in the affinity of adrenergic
receptors. However, Pernow et al21 observed that NPY did
not change the number of -adrenergic binding sites nor the affinity
of -adrenoceptors in the femoral arteries of rats. Therefore, it is
suggested that the observed changes in adrenergic receptors in our
study are specific to cardiac membranes.
The contractility studies carried out on isolated
myocardium support the above -/ß-adrenoceptor
findings. Although unable to decrease the basal level of contractile
activity by itself, NPY modulated the positive inotropic effects of
both phenylephrine and isoproterenol in a dose-dependent
fashion when infused simultaneously. The fact that NPY was
able to exert its effects in the hearts isolated from banded rats
indicates that the control mechanisms are intact in the hypertensive
rat. Since the aortic banded rat is known to have an increase in
sympathetic nerve activity,4 as well as an altered
-/ß-adrenoceptor number,22 it is possible that NPY
might be more functional, in terms of decreasing agonist-stimulated
contractility, in a situation in which higher
sympathetic tone exists. It must be emphasized that in our experiments
NPY failed to alter myocardial contractility by itself.
Without concurrent adrenergic stimulation or an increase in sympathetic
activity, NPY does not affect contractility, at least
not with the dose used. Furthermore, it has been shown that NPY has no
inotropic effects in the human
myocardium.23
In conclusion, NPY is able to modulate the characteristics of
myocardial -/ß-adrenoceptors and decrease the inotropic response
of rat myocardium to adrenergic agonists. This leads to the
possibility of a novel method of reducing cardiac
contractility associated with hypertension. The
development of an analogue of NPY that retains this
anticontractility effect without vasoconstrictor
properties24 would be an appropriate therapeutic
approach.
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Acknowledgments |
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This study was supported by a grant from the Heart and Stroke Foundation, Manitoba, Canada.
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Footnotes |
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Reprint requests to Pallab K. Ganguly, MD, Division of Cardiovascular Sciences, St Boniface General Hospital Research Centre, 351 Tache Ave, Winnipeg, Manitoba, Canada R2H 2A6.
Received April 20, 1995; first decision May 24, 1995; accepted May 31, 1995.
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