Published online before print February 7, 2005, doi: 10.1161/01.HYP.0000153953.69799.f2
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(Hypertension. 2005;45:780.)
© 2005 American Heart Association, Inc.
Original Articles |
Central Neuropeptide Y Signaling Ameliorates N(
)-Nitro-L-Arginine Methyl Ester Hypertension in the Rat Through a Y1 Receptor Mechanism
From the Department of Physiology, Human Molecular and Genetic Center, Medical College of Wisconsin, Milwaukee.
Correspondence to Mieczyslaw Michalkiewicz, DVM, PhD, Department of Physiology, Medical College of Wisconsin, 8701 Watertown Plank Rd, PO Box 26509, Milwaukee, WI 53226-0509. E-mail mmichalk{at}mcw.edu
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Abstract |
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Neuropeptide Y is a potent inhibitory neurotransmitter expressed in the central neurons that control blood pressure. NO also serves as an inhibitory neurotransmitter, and its deficit causes sympathetic overactivity, which then contributes to hypertension. This study tested the hypothesis that neuropeptide Y functions as a central neurotransmitter to lower blood pressure, therefore its increased signaling ameliorates hypertension induced by NO deficiency. Conscious neuropeptide Y transgenic male rats, overexpressing the peptide under its natural promoter, and nontransgenic littermates (controls) were used in this study. Neuropeptide Y, Y1 receptor antagonist BIBP3226, or vehicle (saline) were administered continuously for 14 days into the cerebral lateral ventricle in unrestrained animals using osmotic pumps. Blood pressure was measured by radiotelemetry. Compared with control animals, transgenic overexpression of neuropeptide Y significantly ameliorated (by 9.7±1.5 mm Hg) NO deficiency hypertension (induced by administration of N(
)-nitro-L-arginine methyl ester in the drinking water). This hypotensive effect of neuropeptide Y upregulation was associated with reduced proteinuria and cardiac hypertrophy and fibrosis. Central administration of neuropeptide Y in nontransgenic rats also reduced (by 10.2±1.6 mm Hg) the NO deficiency hypertension, whereas a neuropeptide Y1 receptor antagonist centrally administered in the transgenic subjects during NO deficiency hypertension completely attenuated the depressor effect of neuropeptide Y upregulation. Thus, acting at the level of the central nervous system distinctively via a Y1 receptor–mediated mechanism, endogenous neuropeptide Y exerted a potent antihypertensive function, and its enhanced signaling ameliorated NO deficiency hypertension.
Key Words: hypotension • central nervous system • sympatholytics • catecholamines • rats, transgenic • nitric oxide
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Introduction |
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Neuropeptide Y (NPY) is extensively expressed in the central neural circuits, which control blood pressure (BP). Namely, NPY-containing neurons are present in the paraventricular nucleus (PVN) of the hypothalamus, the ventrolateral medulla (VLM), the nucleus of tractus solitari (NTS), the presynaptic bulbospinal neurons of the brain stem, and in the sympathetic fibers innervating blood vessels.1–5 This peptide elicits a wide variety of physiological effects, including stimulation of feeding, sodium secretion, and reduction of sympathetic activity and behavioral stress.5–10 Four subtypes of NPY receptors, Y1, Y2, Y4, and Y5, are expressed in the rat brain, but the density of the Y1 seems to predominate over the other subtypes.1–3
Considering the dense distribution of NPY within the cardiovascular neural centers, its colocalization with other excitatory neurotransmitters, including norepinephrine or glutamate, and inhibitory activity on the release of these neurotransmitters,11–13 we reasoned that this peptide is well positioned to play the physiological role of a buffering neurotransmitter within the central nervous system (CNS), and that its increased signaling will ameliorate chronic hypertension induced by NO deficiency.
We chose the N()-nitro-L-arginine methyl ester (L-NAME)–induced NO deficiency hypertension because it is a commonly used hypertension model because of (among others) overactivity of the central sympathetic nervous system14–17 and a close anatomic coexistence of NPY with NO signaling system in the sympathetic nuclei of the brain,1–4,14–17 indicating functional cooperation between NPY and NO signaling systems in BP regulation.18,19
The goal of this study was to elucidate the role of endogenous NPY in the long-term regulation of BP. Specifically, we aimed to: (1) determine the effect of transgenic upregulation of NPY on L-NAME hypertension in the rat; (2) establish whether this effect takes place in the CNS; and (3) identify the NPY receptor subtype involved in this effect.
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Materials and Methods |
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This study was reviewed and approved by the Medical College of Wisconsin committee on animal care and use.
Animals
NPY transgenic (NPY-tg) Sprague Dawley male rats carrying 5 copies of the genomic rat NPY transgene (line no. 400) and nontransgenic littermates (wild-type control)20–24 were used. Animals were individually housed in a temperature-controlled (21°C to 22°C) and light/dark cycle–controlled (lights on 6:00 AM to 6 PM) room and were provided with 5001 LabDiet chow and water ad libitum. All surgeries were done under anesthesia with intraperitoneal injections of ketamine (75 mg/kg–1) and xylazine (7.5 mg/kg–1).
Radiotelemetry
Mean arterial pressure (BP), heart rate (HR), and locomotor activity of the animals were measured by 24-hour radiotelemetry (Data Sciences, Inc.) as described previously.24 Telemetry transmitters (TA11PA-C40) were implanted subcutaneously (under anesthesia), and a catheter was inserted into the abdominal aorta via the femoral artery. Animals were allowed to recover for 7 to 9 days.
Experiment 1: Pressor Response to L-NAME in NPY-tg and Wild-Type Rats
After completion of a 3-day baseline recording, rats received L-NAME in drinking water ad libitum at a concentration of 100 mg/L for 7 days. Then, for the next 9 days, the dose of the NO inhibitor was increased to 200 mg/L. Subsequently, animals were transferred to metabolic cages for 2 days, while continuously receiving the higher dose of the NO inhibitor, to collect urine for protein assay. Urine protein concentrations were determined by the Bradford method (Bio-Rad Laboratories) using BSA as the standard. For histological assessment, hearts fixed in a 5% formalin solution were embedded in paraffin, sectioned, stained with Mason’s trichrome stain, and examined by light microscopy. Each heart was sectioned at its midpoint to ensure that sections were taken from an anatomically consistent site. The area of the left ventricular wall and the degree of its interstitial fibrosis were then measured using a digital imaging program.
Experiment 2: Effect of NPY and the Y1 Receptor Antagonist BIBP3226 on Pressor Response to L-NAME in the 2 Strains of Rats
A chronic cannula was implanted into the lateral cerebral ventricle (intracerebroventricularly) in an anesthetized rat using a Kopf stereotaxic apparatus according to procedures described previously.25 Briefly, a stainless-steel 12-mm 23-gauge guide cannula was implanted into the right intracerebroventricular on the bregma line, 1.4 mm lateral from the midline and 3 mm below the surface of the skull. At the same time, telemetry transmitters were implanted. After recovery and 3 days of baseline BP recording, a stainless 15.5-mm 30-gauge injection cannula was inserted into the guide cannula, and synthetic rat NPY (7.0 nmol/kg per day in saline), NPY receptor Y1 receptor antagonist BIBP3226 (63.3 nmol/kg per day in saline), or saline (vehicle) was continuously delivered for 14 days using a subcutaneously implanted Alzet osmotic pump (model 2002; infusion rate, 0.5 µL/hr). The doses of NPY and BIBP3226 were chosen on the basis of their effectiveness to increase or abolish food intake, respectively, in rat.26,27 Animals were given L-NAME in drinking water (200 mg/L) at the same time. The correct placement of each cannula was confirmed at the end of the experiment by methylene blue staining and microtome section of the brain.
Data Analysis
Data are presented as means±SE. The statistical methods used are indicated in each figure legend. A P-value <0.05 was considered statistically significant.
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Results |
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Transgenic Upregulation of NPY Ameliorated L-NAME Hypertension, Protecting the Heart and Kidney (Experiment 1)
In the control animals, BP increased in a dose-dependent manner in response to the increasing doses of L-NAME (Figure 1A). BP was significantly (P<0.05) raised (131.0±2.3 mm Hg) from the baseline measurement during administration of 100 mg/L of the inhibitor, and it gradually rose higher in response to doubling the dose of L-NAME, reaching a level of 155.3±3.3 mm Hg.
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Compared with the control subjects, the pressor response of the NPY-tg rats to 100 mg/L L-NAME was significantly (P<0.05) lower (122.7±2.2 mm Hg). Moreover, unlike the nontransgenic controls, the higher dose of L-NAME had no additional pressor effect on these subjects. In both groups, after cessation of drug administration, BP decreased gradually, reaching the basal level 4 to 5 days after stopping the treatment, confirming that the pressor response was specifically related to the treatment with NO synthase inhibitor.
HR was reduced during L-NAME treatment in both strains of rats (Figure 1B), but there was a statistically significant (P<0.05) difference between the genotypes. Although the bradycardia of the NPY-tg subjects remained steady during the treatment, the HR of the wild-type rats gradually increased, starting on day 4 of the higher dose. Like BP, the HR response completely reversed in both strains after stopping the treatment.
The heart weight and the left ventricular wall size of the NPY-tg rats treated with L-NAME were smaller (P<0.05) than those of the nontransgenic littermates (Table 1). In addition, NPY overexpression reduced the development of cardiac fibrosis and proteinuria because of L-NAME hypertension. In the basal state, these cardiac and renal indices were not different between the strains (Table 1). Body weight, food intake (Table 1), and locomotor activity (data not shown) did not differ between the 2 strains and were not affected by the L-NAME treatment.
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Central NPY Ameliorated the L-NAME Hypertension Involving a Y1 Receptor Mechanism (Experiment 2)
Treatment with L-NAME resulted in a significant increase in BP in all groups (Figure 2A). Consistent with the results of experiment 1, L-NAME hypertension was significantly reduced (P<0.05) in the NPY-tg subjects when compared with wild-type rats (122.7±1.5 versus 132.4±1.6 mm Hg, respectively). Likewise, centrally given NPY in wild-type rats reduced L-NAME hypertension compared with treatment with vehicle (122.2±1.7 versus 132.4±1.6 mm Hg, respectively; P<0.05). Interestingly, the magnitude of the reduction of L-NAME hypertension in response to intracerebroventricular administration of NPY was similar to the reduction of hypertension observed in the NPY-tg rats.
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In contrast, for the NPY-tg rats treated intracerebroventricularly with the NPY receptor Y1 antagonist BIBP3226, the L-NAME hypertension was significantly higher than in the NPY-tg animals treated intracerebroventricularly with vehicle (132.7±1.7 versus 122.7±1.5, respectively; P<0.05). In fact, the L-NAME hypertension in this group was as high as that observed in the wild-type animals treated intracerebroventricularly with vehicle. Thus, intracerebroventricular administration of a specific Y1 receptor antagonist completely attenuated the depressor effect of transgenic NPY upregulation in L-NAME hypertension.
Treatment with L-NAME produced substantial bradycardia (P<0.05) in all groups, which tended to decrease in the second week of treatment. Chronic intracerebroventricular administration of NPY temporarily reduced the HR in the wild-type rats (Figure 2B). This treatment also resulted in increased body weight gain and water intake and reduced locomotor activity (Table 2). The increase in water intake in this group, which also increased L-NAME intake, was most likely secondary to the increased food intake because of central NPY administration. However, this higher L-NAME intake did not prevent the depressor effect of intracerebroventricular NPY infusion because the BP was lowest in this group.
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Discussion |
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The novel observation from this study was that transgenic overexpression of NPY under its natural promoter in the rat significantly ameliorated L-NAME hypertension. A similar effect was demonstrated during long-term intracerebroventricular infusion of synthetic NPY in the wild-type rats. Furthermore, the present work demonstrated that the hypotensive effect of enhanced NPY signaling was mediated by the central Y1 receptor subtype because the ameliorating effect of transgenic overexpression was completely blocked by intracerebroventricular administration of the specific Y1 receptor antagonist BIBP3226. Thus, we can report that the Y1 receptor is a potent antihypertensive receptor of the CNS.
In the present study, we used transgenic rats overexpressing NPY under its natural promoter, telemetry for continuously monitoring BP in a conscious state, and chronic NPY receptor ligand delivery using osmotic pumps. This approach allowed us to assess the role of endogenous NPY in long-term control of BP under experimental conditions that excluded complications associated with anesthesia, restraint, and short-term drug administration.
The present finding is coherent with anatomic and functional reports that indicate potent sympatholytic and hypotensive functions of central NPY signaling. The peptide and its Y1 receptor subtype are expressed in the central neurons participating in the processing of cardiovascular signals, including the PVN, NTS, VLM, and in the presynaptic bulbospinal neurons.1–5 On the basis of its ability to inhibit the neuronal excitability and the release of excitatory neurotransmitters, NPY is considered a potent inhibitory neurotransmitter of the CNS.7–13,28 Microinjections of NPY into the PVN or intracerebroventricularly reduced the release of norepinephrine, peripheral sympathetic nerve activity, HR, and BP.7–10 Compared with wild-type rats, the concentrations of NPY in the CNS, including the hypothalamic nuclei, were significantly increased in the NPY-tg subjects used for the present study.21,22,24 The phenotypes of these NPY-tg rats reported previously, including reduced BP and sympathetic drive to the blood vessels, lower catecholamines, diminished behavioral and pressor responsiveness to acute stress, and enhanced vascular response to exogenous norepinephrine, point to a potent sympathoinhibitory effect of transgenic NPY upregulation.21–24 NO is also a potent sympatholytic and depressor neurotransmitter of the CNS.14–17,19 L-NAME, when administered systemically, crosses the blood–brain barrier and inhibits NO production in the brain.16 The resulting increased sympathetic outflow is an important component of L-NAME hypertension.14–17
Thus, it is very likely that in the present experiment, Y1 receptor–induced reduction of sympathetic outflow underlies the mechanism of ameliorated L-NAME hypertension by central NPY.
It has been demonstrated that NPY-producing neurons of the rat hypothalamus make synaptic contact with NO neurons that express the Y1 receptor,3 and that stimulation of this receptor enhances NO production in the rat hypothalamus and cerebral cortex.3,29,30 Thus, it seems reasonable to suggest that the depressor effect of NPY in the present experiment was a result of its direct interaction with the NO-producing neurons. NPY-induced NO production might be possible despite treatment with L-NAME because although this drug easily crosses the blood–brain barrier,16 it may not completely block the neuronal NO synthase in the brain. In addition, in the present experiment, increased NPY could compensate for NO deficiency in another way by inhibiting the activity of NO-receptive neurons in the circuits controlling sympathetic outflow. Such compensation seems possible because NO and NPY are inhibitory neurotransmitters and are synthesized together within the same neural circuits, perhaps affecting the same neurons.1,4,14,15,17
The disappearance of L-NAME–induced bradycardia in the wild-type but not in the NPY-tg rats (experiment 1) indicates that an increased sensitivity of the baroreceptor reflex in the transgenic animals might partially contribute to the attenuated L-NAME hypertension in this group. Indeed, treatment with NPY increased sensitivity of the baroreceptor reflex in the rat,5 and this could also explain the enhanced bradycardia in the wild-type animals treated intracerebroventricularly with synthetic NPY (experiment 2).
We also report that NPY overexpression reduced L-NAME–induced hypertrophy of the heart and diminished the extent of cardiac fibrosis. In addition to its cardioprotective effect, NPY also reduced urinary excretion of protein in the NPY-tg rats treated with the NO synthase inhibitor. These observations are very consistent with the increased longevity of these transgenic rats.24 It is likely that the lower BP contributed to the reduction of cardiac damage and proteinuria in the NPY-tg subjects treated with L-NAME.
Food intake and body weight were not affected by transgenic overexpression of NPY. This observation was in contrast to the known orexigenic effect of exogenous NPY6 and the increased body weight gain and water intake induced by infusion of synthetic NPY in the present study. The increased water intake in this group was most likely secondary to the increased food intake. The lack of body weight phenotype in this transgenic rat22,24 is consistent with the reported NPY and Y1 receptor knockout experiments in mice, in which body weight and appetite were also unaffected.31,32
In conclusion, this study demonstrates that increased NPY expression in the NPY-tg rat significantly ameliorated L-NAME hypertension and protected the heart and kidney from hypertension-induced left ventricular hypertrophy and proteinuria, respectively. Furthermore, we have established that the CNS is the major site of the antihypertensive function of endogenous NPY, and that this effect is specifically mediated by the Y1 receptor subtype. Thus, central Y1 receptor signaling emerges as an endogenous buffering mechanism responsible for reducing BP and conferring protection of the cardiovascular system and end organs during NO deficiency.
Perspectives
The results presented here suggest that endogenous NPY functions within the CNS to protect the cardiovascular system in situations of chronic hyperexcitation and hypertension. This buffering function could be particularly important in states of chronic hyperactivity and neural hypertension, including chronic psychosocial anxieties, NO deficiency, or heart failure. The mechanism underlying the antihypertensive and organ-protective activities of NPY may stem from the peptide neuroinhibitory properties and may involve interaction with other signaling pathways, including adrenergic or NO, leading to sympathoinhibition and lowering BP. In addition, there is a significant possibility that NPY may also directly stimulate NO production in the central neurons involved in cardiovascular regulation.
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Acknowledgments |
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This study was supported by National Institutes of Health grant HL-57921. We thank Drs Leon F. Tseng and Thom R. Feroah for help with chronic intracerebroventricular infusion, Glenn R. Slocum for assistance with digital microscopy, and Beverly Ventura for editing this manuscript.
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Footnotes |
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G.Z. and Z.J. contributed equally to this study.
Received October 12, 2004; first decision November 5, 2004; accepted December 9, 2004.
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