Modulatory Role of Nitric Oxide on the Vasomotor Actions of NPY in Porcine Cerebral Arteries

ABSTRACT

Neuropeptide Y (NPY) is a sympathetic co‐transmitter that mediates vasoconstriction. However, there is evidence that it may also mediate dilation through a nitric oxide (NO)‐dependent mechanism.

Objective

We used a swine model to examine how NPY influences cerebral vascular regulation and hypothesized that NPY would elicit both vasoconstrictor and vasodilatory effects, and that such effects would be modulated partially by NO signaling.

Methods

Briefly, cerebral perfusion and blood pressure were monitored during intracarotid saline or NPY infusion (0.1 μg/kg) in the presence and absence of NO synthase (NOS) inhibition (N ^G‐nitro‐l‐arginine methyl ester; 0.35 mg/kg/min). Separately, Y1 receptor distribution (immunohistochemistry) and vasomotor responses to intra‐ and extraluminal NPY under control and NOS inhibition conditions were examined in isolated arteries.

Results

Intracarotid NPY infusions elicited transient dilation that was blocked by NOS inhibition. In isolated pial arteries, distinct populations of NPY‐Y1 receptors were observed on both the vascular smooth muscle (VSM) and endothelium. Extraluminal application of NPY elicited vasoconstriction, while intraluminal delivery elicited vasodilation. NOS inhibition enhanced the magnitude of vasoconstriction in isolated pial arteries. Endothelial denudation, Y1 receptor antagonism, and NOS inhibition each blunted NPY‐induced vasodilation.

Conclusion

These data suggest both vasoconstrictor and vasodilatory effects of NPY are modulated partially by NO signaling.

1. Introduction

In the brain, pial arteries are innervated extrinsically by sympathetic nerves that release the vasoactive neurotransmitters adenosine triphosphate, norepinephrine, and neuropeptide‐Y (NPY) [1]. NPY elicits vasoconstriction via Y1 peptidergic receptors located on the vascular smooth muscle and potentiates vasoconstriction induced by adenosine triphosphate or norepinephrine [1, 2, 3, 4]. In the skeletal muscle circulation, NPY‐induced constriction is greater in downstream compared with upstream arteries, suggesting greater receptor density distally [5]. Conversely, in the cerebral circulation, Y1 receptor content has been reported to be greater in upstream arteries [6]. Whether this translates to augmented or reduced vasoconstriction in downstream compared with upstream cerebral arteries remains to be elucidated.

In addition to mediating vasoconstriction, there are several lines of evidence from in vivo and ex vivo/in vitro experimental preparations that NPY also exerts an endothelial‐dependent vasodilatory influence. For example, Kobari and colleagues [7] reported that intracarotid NPY infusion in the cat induced a transient increase in cerebral blood flow that was abolished by inhibiting nitric oxide synthase (NOS). However, these findings are not uniform, as others have reported that arterial and intracerebral NPY infusion resulted in a dose‐dependent decrease in perfusion in the rat and canine brain without any observable dilatory effects [8, 9, 10]. Hallmark work from You and colleagues [11] demonstrated that extraluminal application of NPY caused vasoconstriction, whereas intraluminal application of NPY caused vasodilation in isolated rat middle cerebral arteries, suggesting perhaps divergent roles for NPY receptors located on the vascular smooth muscle compared with the endothelium. Supporting this interpretation, they further demonstrated that the dilatory actions of NPY were blunted by arterial denudation and NOS inhibition, implicating an endothelial‐dependent NO‐mediated mechanism. Similar observations have also been reported in isolated arteries from the renal circulation of rats [12], the penile circulation of horses [13], and the cutaneous circulation of humans [14]. Despite mounting evidence of an NPY‐dilator mechanism, the total number of observations and experimental continuity are limited. Thus, more studies are needed to validate findings, to determine what receptors may be involved, and whether vasomotor responses to NPY vary according to arterial diameter or branch order.

Using a porcine model, the purpose of this study was to further explore the role of NPY in modulating cerebrovascular regulation. It was hypothesized that (1) intracarotid NPY infusion would elicit a transient increase in perfusion and that such an effect would be abolished by NOS inhibition; (2) extraluminal and intraluminal application of NPY in isolated cerebral arteries would elicit Y1 receptor‐dependent vasoconstriction and dilation, respectively; (3) NPY‐induced constriction would be augmented, and dilation would be blunted by NOS inhibition; and (4) in line with decreased receptor density, countering observations in the skeletal muscle circulation [5], vasomotor responses to NPY would be attenuated in downstream compared with upstream arteries.

2. Methods

This animal protocol was approved by the University of Saskatchewan Animal Care Committee (#20190036). Female farm pigs (n = 40, age = 2.5 ± 1.0 months, mass = 25 ± 5 kg) were housed under temperature‐controlled conditions, with a 12‐h/12‐h light/dark cycle and consumed a standard commercially available chow diet with ad libitum access to water. Following an overnight fast, pigs were anesthetized with an intramuscular injection of ketamine (20–30 mg/kg) and xylazine (2 mg/kg) and anesthetic depth was maintained with inhaled isoflurane (1.5%–5%). Subsequently, pigs were utilized for either in vivo (protocol 1) or ex vivo (protocol 2) experiments.

2.1. Protocol 1

Pigs were intubated and tidal volume was maintained at 10 mL/kg. The ventilation rate was adjusted to maintain an end tidal CO₂ constant for each animal in a range of 35–45 mmHg. Prior to experimentation, pigs were slowly weaned off inhaled isoflurane and transferred to intravenous propofol anesthesia (8–20 mg/kg/h; Baxter, USA). While supine, heart rate (ECG), blood pressure (femoral artery catheter connected to pressure transducer; ADinstruments), and cerebral perfusion (laser Doppler needle probe positioned 1 cm anterior to the coronal suture and 1 cm lateral to the sagittal suture and inserted through burr hole 10 mm into the brain parenchyma; Fine Needle Probe MNP110XP; ADInstruments) were monitored during either NPY or saline in one set of animals or NOS inhibition (N ^G‐nitro‐l‐arginine methyl ester; L‐NAME, Cayman Chemical, USA) conditions in another set of animals. A catheter was advanced from a facial artery to the ascending pharyngeal artery which supplies the internal carotid artery and the brain [15]. Porcine NPY (Neuropeptide Y, Tocris bioscience, R&D systems, Canada) was infused for 1 min at 0.1 μg/kg/min through the catheter, the same dose and infusion protocol as Kobari et al. [7]. An isovolumetric saline infusion was also performed. For the NOS inhibition condition, L‐NAME was infused for ~15 min prior at 0.35 mg/kg/min and once a stable baseline was achieved, NPY was infused. Time‐aligned hemodynamic data were recorded using a PowerLab data acquisition system and extracted using Labchart 7 (ADInstruments). After a stable baseline was achieved, data were extracted pre‐NPY infusion (data averaged over 1 min) and every 2 min following infusion for 10 min (data averaged over 30 s). The conductance index was calculated as the quotient of cerebral perfusion and mean arterial pressure.

2.2. Protocol 2

Pigs were killed by exsanguination, the brain was removed, and a portion of the brain containing the middle cerebral artery and downstream branches was harvested for ex vivo vasomotor control and molecular experiments. The brain was placed in an ice‐cold physiological saline solution (PSS: NaCl 145 mM, KCl 4.7 mM, CaCl₂ 2.0 mM, and MgSO₄ 1.17 mM) with a pH of 7.4. Pial arteries along the middle cerebral arterial tree were harvested, transferred to a Plexiglass chamber filled with PSS and cannulated with two micropipettes filled with PSS. The chambers were relocated to the stage of an inverted microscope (Nikon Diaphot 200) attached to a video camera (Javelin Electronics, Los Angeles, CA, USA), video micrometer (Microcirculation Research Institute, Texas A&M University), and a Powerlab data acquisition system (ADInstruments), as previously described [16, 17, 18]. Fluid‐filled reservoirs were set at an intraluminal pressure of 60 mmHg and luminal diameter was monitored throughout the experiment.

Arteries were allotted 45–60 min to stabilize, at which point maximal arterial vasoconstriction in response to 80 mM KCl was measured. Vasomotor responses to intraluminal NPY (1e⁻⁹) were examined initially in control (vehicle), Y1 receptor blockade (BIBP3226; 30 μm), and arterial denudation (achieved by passing air bubbles through the lumen of the artery and confirmed by ≤ 10% vasodilation to 3e⁻¹² M bradykinin and ≥ 20% vasodilation to 1e⁻⁴ M sodium nitroprusside) conditions. Subsequently, vasomotor responses to intra‐ and extraluminal NPY were examined in control and NOS inhibition (L‐NAME; 3e⁻⁴ M) conditions. Vasomotor responses were expressed as ∆ diameter measured in micrometers (μm). A retrospective analysis was performed, data were grouped according to arterial branch order (1st = 1A; 2nd = 2A; and 3rd = 3A), and vasomotor responses were expressed as % change from baseline for vasodilatory responses or % KCl‐induced constriction for vasoconstrictor responses.

Intraluminal drug delivery was achieved and maintained by adjusting right and left pressure reservoirs in equal but opposite directions to establish a pressure gradient across the artery while maintaining mean pressure at the midpoint of the artery at 60 mmHg [17, 19]. The pressure gradient was maintained during data collection; therefore, vascular responses to intraluminal drug delivery were examined under flow conditions. The pressure gradients required to generate the desired flow rates for resistance matched pipettes were established prior to experimentation and confirmed throughout experimentation with a ball flowmeter (Omega Engineering). The flowmeter broke part‐way through the study; therefore, flow rates were confirmed following each subsequent experiment by direct measurement of fluid across reservoirs. The desired flow rates corresponded with 40 dynes/cm² of shear stress, which is considered physiological and produces constriction in intracranial cerebral arteries. For a detailed description of this, please consult this earlier work [17, 19]. Note, the mechanical properties of arteries included in this study were reported on previously [17].

2.3. Immunohistochemistry and Immunoblotting

1A, 2A, and 3A cerebral arteries were pinned in a 24‐well plate coated with Sylgard. For immunolabeling of NPY nerves (rabbit‐anti NPY primary, Abcam 1:500; goat‐anti‐rabbit AlexaFluor 488 secondary, ThermoFisher 1:500) arteries were prepared intact. For immunolabeling of Y1 receptors (rabbit‐anti‐Y1, Abcam, 1:500; goat‐anti‐rabbit AlexaFluor 488 secondary, ThermoFisher 1:500), arteries were prepared en face. Arteries for both studies were immunolabeled as previously described [20, 21]. Briefly, isolated 1A, 2A, or 3A cerebral arteries were fixed in 4% paraformaldehyde, blocked and permeabilized with PBS containing 1% BSA and 0.1% Triton X‐100, and incubated overnight in primary antibody. Arteries were then blocked again, incubated in secondary antibody, and mounted on slides using ProLong Gold. Slides were imaged using a Leica TCS SP8 confocal laser‐scanning microscope. NPY fluorescence was visualized using a Fluotor VISIR 25X water objective (NA = 0.95), with a 2× optical zoom and 1‐μm Z‐slices. Y1 fluorescence was visualized using an HC PL APO CS2 63X glycerol objective (NA = 1.3) with 2× optical zoom and 0.33 μm Z‐sections. Across replicates, arteries were imaged using similar laser power and gain settings. Representative images were prepared in FIJI [22] by generating maximum z‐projections. For NPY labeling studies, maximum z‐projections were used to quantify % fluorescent area, as previously described [20, 21] Maximum projections were threshold‐adjusted to eliminate background fluorescence, then converted to binary images. The percent labeled area was then quantified in FIJI, yielding the proportion of each image labeled for NPY. Protein expression was determined according to methods published previously [17, 18, 19, 23]. Polyvinylidene difluoride membranes were blocked in a 5% nonfat milk‐Tris‐buffered saline‐Tween 20 (TBST) solution and incubated overnight at 4°C with a primary antibody against the NPY‐Y1 receptor (55 kDa, 1:1000; Abcam, USA) and β‐actin (42 kDa; 1:2000; Sigma, USA). Subsequently, membranes were incubated with a horseradish peroxidase‐conjugated anti‐rabbit or anti‐mouse secondary antibody (Cell Signaling) in a 5% nonfat milk‐TBST solution. Blots were then incubated in Luminata Forte Western HRP (EMD Millipore, USA) substrate visualization reagent, and a Kodak image station (4000R) was used to visualize and quantify band densities. To contextualize branch order differences, data were normalized to β‐actin and expressed relative to the mean middle cerebral artery Y1 receptor protein content within each gel.

2.4. Statistics

Hemodynamic data were analyzed using either a repeated measures (condition × time; NPY vs. saline) or mixed model repeated measures ANOVA (condition × time; NPY vs. L‐NMAE + NPY) (GraphPad V. 11). Changes in arterial diameter between conditions were analyzed using a two‐tailed paired t‐test or one‐way ANOVA with a Dunnett's post hoc test to compare to control conditions. Fisher's exact test was used to compare the frequency of dilation to NPY under different experimental conditions. Branch order differences were examined using a one‐way ANOVA. Where necessary, a post hoc Tukey's test was used to determine the location of significance. The significance level was set at p ≤ 0.05. Where possible, individual data are presented; otherwise, data are presented as means ± SD.

3. Results

3.1. In Vivo Cerebrovascular Regulation

All in vivo cerebrovascular regulation data are presented in Figure 1. Two pigs were excluded because they did not have stable perfusion during saline infusion (> 15% change). Importantly, inclusion or exclusion of these data do not alter the statistical relationships. The final sample size was N = 8 for each condition. There was a significant time × condition interaction for cerebral perfusion (p ≤ 0.0105; Figure 1a). Pairwise comparisons indicate that perfusion increased compared to baseline in the NPY only group at 8‐min post‐infusion (p < 0.0001), but did not change in the saline (p ≤ 0.9620) or NOS inhibition group (p ≤ 0.1670). Further, it was greater than the saline and NOS inhibition group at 6‐, 8‐, and 10‐min post‐infusion (p ≥ 0.0166). Mean arterial pressure remained stable over time across all conditions (p ≥ 0.2344; Figure 1b). There was no difference in mean arterial pressure between NPY and saline conditions (p = 0.9402; Figure 1b). However, there was a main effect of condition, whereby mean arterial pressure was greater during NOS inhibition (p = 0.0046; Figure 1b). There was a significant time × condition interaction for the cerebral conductance index (p < 0.0001; Figure 1c). Pairwise comparisons indicate groups did not differ at baseline (p = 0.0913), but the NOS inhibition group had lower conductance at every other time point (p ≤ 0.0482). Further, conductance increased compared to baseline in the NPY only group from 6 to 10 min post‐infusion (p ≤ 0.0253; Figure 1c), but did not change in the saline group (p ≥ 0.9791) and reductions in the NOS inhibition group only approach significance (p ≥ 0.0501). Noting group differences in blood pressure, the conductance index data were subsequently normalized to the mean of baseline conductance within each condition. This approach removes potential group differences at baseline and enables a clearer comparison of time by group differences. There was a significant time × condition interaction for the normalized cerebrovascular conductance index (p ≤ 0.0039; Figure 1d). Pairwise comparisons indicate it increased compared to baseline in the NPY only group at 6‐, 8‐, and 10‐min post‐infusion (p ≤ 0.0262), it remained stable in the saline condition (p ≤ 0.9886) and decreased in the NOS inhibition condition at 6‐, 8‐ and 10‐min post‐infusion (p ≤ 0.0219). Further, the conductance index was greater in the NPY alone compared to the saline condition at 8 min post‐infusion (p = 0.0144) and was not different between NPY and NOS inhibition conditions (p ≥ 0.1451).

FIGURE 1.

Effects of intracarotid NPY infusion. Cerebral perfusion (a), mean arterial pressure (MAP; b), cerebrovascular conductance (c), and normalized vascular conductance index (d) following NPY (closed circles; n = 8) and saline infusion (open triangles; n = 8) or N^G‐nitro‐l‐arginine methyl ester (L‐NAME; open circles; n = 8) conditions. #p < 0.05, ##p < 0.01, ###p < 0.001; ####p < 0.0001 versus baseline; §p < 0.05, §§§§p < 0.0001 versus saline; *p < 0.05, **p < 0.01 versus NPY alone.

3.2. Ex Vivo/In Vitro Isolated Pial Arteries

The corresponding ex vivo/in vitro isolated pial artery data are presented in Figure 2. Extraluminal NPY caused constriction, and the magnitude of constriction was augmented by NOS inhibition (p = 0.0029; Figure 2a). Intraluminal NPY tended to cause dilation, and there was a significant effect of experimental condition on vasomotor responses to intraluminal NPY (p = 0.0014; Figure 2b). Pairwise comparisons reveal Y1 receptor antagonism and arterial denudation both decreased the ∆diameter following intraluminal delivery of NPY (p ≤ 0.0297; Figure 2b). Furthermore, NOS inhibition blunted dilation in response to intraluminal NPY (p = 0.0033; Figure 2c). Thus, NPY‐induced dilation was blocked independently by Y1 receptor antagonism, arterial denudation, and NOS inhibition. Overall, extraluminal NPY elicited ~8% ± 8% constriction, and intraluminal NPY elicited ~4% ± 20% dilation under control/untreated conditions, with the probability of observing dilation being greater with intraluminal NPY application (p < 0.0001; 2/20 arteries displayed dilation with extraluminal application vs. 20/25 arteries with intraluminal NPY application). Further, intraluminal NPY application during either denuded or NOS inhibition conditions elicited ~16% ± 19% constriction, with the probability of observing dilation being lower than control/untreated conditions (p < 0.0001; 20/25 arteries displayed dilation with intraluminal NPY application in control/untreated conditions vs. 2/25 arteries with denuded or L‐NAME conditions). Isolated arteries (n = 6) were examined for the presence of Y1 receptors, and they were documented in the vascular smooth muscle and endothelial cell layers of the porcine cerebral arteries (Figure 2d–f).

FIGURE 2.

Effects of extraluminal and intraluminal NPY administration in isolated pial arteries. Extraluminal NPY administration in control (n = 20) and N ^G ‐nitro‐l‐arginine methyl ester (L‐NAME; n = 20) conditions (a). Intraluminal NPY administration in control (n = 5), BIBP3226 (Y1 receptor antagonism; n = 5), and arterial denudation (endothelial removal; n = 5) conditions (b). Intraluminal NPY administration in control (n = 20) and N ^G ‐nitro‐l‐arginine methyl ester (L‐NAME; n = 20) conditions (c). Immunofluorescence imaging of Y1 receptors on the extraluminal and intraluminal aspects of a 1A cerebral artery, reflecting vascular smooth muscle (VSM) and endothelial cell (EC) Y1 receptors, respectively (d and e). Imaging of EC Y1 receptors in a denuded artery (f). Data for panels a and c were analyzed using an unpaired t‐test and data for panel b was analyzed using a one‐way ANOVA. *p < 0.05, **p < 0.01, and ***p < 0.001 significantly different between groups.

3.3. Branch Order Comparisons

Branch order did not affect vasomotor responses to intraluminal NPY in either absolute (p = 0.3587; Figure 3a) or relative (%∆) terms (p = 0.1432; Figure 3b). While the absolute ∆diameter in response to extraluminal NPY was similar (p = 0.2480; Figure 3c), the %∆ diameter was greater in 3A compared with 1A arteries (p = 0.0247; Figure 3d). Y1 receptor protein content was lower in 2A and 3A compared with 1A arteries (p ≤ 0.0002; Figure 3e) and perivascular NPY innervation was lower in 3A compared with 1A arteries (p = 0.0179; Figure 3f).

FIGURE 3.

Examining branch order effects of NPY and Y1 receptor distribution. Absolute change in diameter (n = 6–7 per branch order) and %change in diameter following either intraluminal (a, b) or extraluminal (c, d) NPY administration. Y1 receptor density (normalized to β‐actin and expressed relative to the middle cerebral artery Y1 receptor protein content within each gel; e) and perivascular NPY innervation (f) in 1A, 2A, and 3A branches of the middle cerebral artery. All data were analyzed using a one‐way ANOVA. *p < 0.05, ***p < 0.001, and ****p < 0.0001 significantly different between branch orders.

4. Discussion

The current study demonstrated that intracarotid NPY infusion can elicit a NOS‐dependent cerebral dilation in swine. Further, the isolated cerebral vessel data reveal that intraluminal NPY elicits vasodilation that is blunted independently by Y1 receptor blockade, arterial denudation, or NOS inhibition. In combination with the visual confirmation of endothelial Y1 receptors, these data suggest that NPY‐mediated cerebral vasodilation occurs via an endothelial Y1 receptor‐dependent NO‐mediated mechanism. In contrast to intraluminal NPY, extraluminal NPY elicited constriction that was enhanced by NOS inhibition. Thus, the dilation and constriction of NPY can be negatively and positively modulated through decreased NO signaling, respectively. Y1 receptor content and perivascular NPY nerve innervation were lower in 3A compared with 1A arteries, suggestive of a diminishing role for NPY in regulating vascular tone in the downstream circulation. Nevertheless, absolute vasomotor responses to NPY were similar across arterial branch orders, and relative constriction was greater in 3A arteries, highlighting the potential involvement of both upstream and downstream pial arteries in contributing to the effects of NPY on cerebral blood flow control.

In the present study, NPY infusion increased cerebral perfusion and the conductance index transiently, and such increases were blunted or abolished by NOS inhibition (Figure 1), indicating that NPY elicits a brief NO‐dependent vasodilation. Previous work in rats and canines has not reported transient dilatory actions of NPY [8, 9, 10]. Nevertheless, these data are consistent with those reported by Kobari and colleagues [7] who likewise observed a transient NPY‐induced NOS‐dependent increase in cerebral perfusion in the cat. They observed that intracarotid NPY infusion at the same dose increased cerebral perfusion ~30% and conductance ~20%. In the current study, increases in perfusion and conductance were less robust at ~10% and ~15%, respectively. The underlying cause of this discrepancy is unknown but may be attributable to several factors. The techniques used to assess perfusion were different, and species‐specific variation in the sensitivity to synthetic NPY may exist. Pigs were used in the current study, as they represent an ideal model for human biomedical research, including neuroscience research [23, 24, 25, 26]. Neither study assessed how much NPY was delivered to the site of measurement. Although the same dose was used in both studies, which was based originally on not eliciting systemic hemodynamic effects [7], it is possible that differences in vascular anatomy, probe placement, and sensitivity to synthetic NPY influenced results. Further, given NPY does not remain bound to endothelial Y receptors and crosses the blood–brain barrier [27], the response is highly transient and may depend on local brain metabolism. Altogether, to achieve comparable results between the two species, it is possible the dose or delivery method may need to be adjusted, and the measurement technique standardized.

Extending on the in vivo findings in the current study, it was also observed that luminal administration of NPY in isolated cerebral arteries induced dilation, and this response was abolished by NOS inhibition (Figure 2c). Further, the dilatory response was blunted by both Y1 receptor antagonism and endothelial denudation (Figure 2b). Sympathetic dilation or generation of endothelial NO may result from signal transmission between vascular smooth muscle and the endothelium through myoendothelial gap junctions [28, 29, 30]. However, another prospect is that because Y1 receptors were observed in the arterial endothelium (Figure 2e), together with the findings following intraluminal drug delivery, it is possible NPY elicits porcine cerebral vasodilation through an endothelial Y1 receptor‐mediated NO‐dependent mechanism. Of course, in the absence of co‐localization with the appropriate endothelial stain, the ratio of membrane bound to internalized Y1 receptor expression on the luminal aspect of the artery cannot be determined. Nonetheless, a similar Y1 receptor‐mediated NO‐dependent dilation was reported in isolated cutaneous arteries from humans, suggestive of functional membrane bound expression [14]. In contrast, data from You and colleagues [11] suggest that NPY‐induced NO‐dependent dilation in isolated middle cerebral arteries from rats is possibly Y1 or Y2 receptor‐mediated or attributed to a yet unknown Y receptor. The prospect of endothelial NPY signaling remains to be elucidated fully, but discrepant results may be related to differences in dipeptidyl peptidase‐4 activity, which cleaves NPY and alters receptor binding, drug specificity, species, sex, or maturation differences between studies [6, 11, 13, 23, 31, 32, 33]. Regardless of the receptor subtype, the evidence strongly supports that any NPY‐induced dilation is endothelial‐mediated and NO‐dependent [7, 11, 12, 14].

In addition to blunting NPY‐induced dilation in isolated arteries, NOS inhibition likewise enhanced cerebral vasoconstriction in response to extraluminal NPY (Figure 2a). These data suggest that NO signaling modulates both dilatory and constrictor actions of NPY. In the context of endogenous NPY, potentially any dilatory actions are mediated by the subpopulation of endothelial receptors responding to increases in circulating NPY during periods of heightened stress (i.e., enhanced NPY spillover) [34, 35, 36] and serve to buffer against excessive cerebral vasoconstriction [11]. In this regard, physiologically, impaired NO signaling would facilitate augmented constrictor actions of NPY. Dual cerebral dilatory and constrictor responses to adenosine triphosphate, norepinephrine, serotonin, angiotensin II, and endothelin analogues have also been observed, many of which can be modulated by NO, highlighting that similar counter regulatory mechanisms are common in the cerebral circulation [37, 38, 39, 40, 41, 42, 43, 44, 45].

Previous reports indicate that Y1 receptor content within the pial circulation is greater in larger arteries [6], suggestive perhaps of a decreased role for NPY in downstream compared with upstream arteries. In support, the current data show both Y1 receptor content and NPY perivascular innervation were lower in 3A compared with 1A branches of the middle cerebral artery. However, the functional significance of these findings are unclear, as absolute vasomotor responses to NPY were similar and relative constrictor responses were greater in 3A compared with 1A branches. Perhaps, fewer receptors are needed to initiate constriction of less vascular smooth muscle. A similar functional relationship among branch orders was documented by Al‐Khazraii and colleagues [5], who reported comparable absolute and augmented relative NPY‐induced constriction in 3A compared with 1A skeletal muscle arterioles. In porcine cerebral arteries, it was previously reported that basal NOS signaling was decreased in 3A compared with 1A arteries [17]. Thus, it is possible basal NO production may serve a role in buffering against NPY‐induced constriction. Altogether, the data suggest NPY innervation and Y1 receptor density decrease along the cerebrovascular tree, and this may contribute to varying roles for downstream compared with upstream arteries in mediating the effects of sympatho‐excitation and NPY on cerebrovascular regulation.

There are several limitations to consider when interpreting data from the present study. Only young, healthy female pigs were used, and vasomotor responses to NPY may be affected by age, disease status, sex, and sex hormones [23, 46]. Indeed, our group previously demonstrated that NPY‐induced cerebral artery constriction was greater in female pigs with experimental heart failure or following ovariectomy. Whether heightened constriction in certain conditions may be related to lower NPY‐induced dilatory input is a novel prospect that has not received much attention. Female pigs were used herein because they were easier to acquire from the supplier, but it should be noted that the observed responses may not be generalizable to broader populations. A major drawback of laser Doppler flowmetry is that it does not measure volumetric flow, and owing to the small volume of tissue captured by the laser, the signal can only be used to measure perfusion in a single area. Consequently, the results of the current study pertain exclusively to the cortical area measured and may underestimate the influence of NPY on gross cerebral perfusion. Further, the use of anesthetics may alter cerebrovascular regulation, potentially confounding observations. To minimize this potential effect, pigs were transitioned to intravenous propofol, which does not impair cerebral autoregulation at the doses used herein [47, 48]. Concerning the arterial experiments, although intraluminal and extraluminal effects of NPY were examined, it must also be noted that nerve stimulation was not performed in the present study. Therefore, while potential therapeutic applications of synthetic NPY may be gleaned, it cannot be concluded that NPY released from perivascular nerves mediates both constrictor and dilatory actions observed in the isolated artery studies. Further, the vasomotor responses to intraluminal NPY were examined under flow/shear stress conditions. Although flow/shear stress has been reported to cause constriction in cerebral arteries [17, 49, 50, 51, 52], the effects of intraluminal NPY and flow were not uncoupled herein. Thus, the physiological significance of these findings and the role of NPY release on vascular smooth muscle compared with that which spills over into the arterial lumen requires further study.

In conclusion, this study demonstrated that peak increases in conductance and cerebral perfusion following intracarotid NPY were blunted with NOS inhibition. Distinct populations of NPY‐Y1 receptors were observed on both the vascular smooth muscle and endothelium. In isolated pial arteries, extraluminal application of NPY, targeting the vascular smooth muscle, elicited vasoconstriction. In contrast, intraluminal delivery of NPY, targeting the endothelium, elicited vasodilation, and this effect was abolished with Y1 receptor blockade. Further, the magnitude of vasoconstriction was augmented, and vasodilation was abolished with NOS inhibition. Altogether, these data suggest the vasodilatory and vasoconstrictor effects of NPY‐Y1 receptors are modulated partially by NO signaling in the porcine cerebral circulation.

4.1. Perspectives

This study revealed that the sympathetic co‐transmitter NPY exerts both vasoconstrictor and vasodilatory actions. Inhibition of NOS enhances NPY‐induced vasoconstriction and blunts NPY‐induced vasodilation, highlighting that the vasomotor actions of NPY are modulated partially by NO. Functionally, an NPY‐mediated dilator mechanism may help buffer against excessive vasoconstriction in the cerebral circulation.

Author Contributions

T.D.O., M.H.L., C.A.E., E.M.B., C.R.T., G.D., J.P., and C.J.M. conceived the work. T.D.O., E.M.B., C.R.T., G.D., and C.J.M. assisted with data collection. T.D.O., E.M.B., G.D., and C.J.M. analyzed the data. All authors assisted with data interpretation. G.D. and T.D.O. drafted the manuscript. All authors edited the manuscript.

Delgado G., Morse C. J., Barlage B., et al., “Modulatory Role of Nitric Oxide on the Vasomotor Actions of NPY in Porcine Cerebral Arteries,” Microcirculation 32, no. 5 (2025): e70016, 10.1111/micc.70016.

Data Availability Statement

Data are available upon request from corresponding author.