Neuropeptide Y bioavailability is suppressed in the hindlimb of female Sprague-Dawley rats

Abstract

We recently reported that male, but not female, rats exhibit basal endogenous neuropeptide Y Y₁-receptor modulation of hindlimb vasculature. The lack of baseline endo-genous Y₁-receptor control in females was evident despite the expression of Y₁-receptors and neuropeptide Y in hindlimb skeletal muscle tissue. The following study addressed the hypothesis that neuropeptide Y bioavailability is blunted in female rats under baseline conditions. It was further hypothesized that enhanced prejunctional autoinhibitory neuropeptide Y Y₂-receptor expression and/or proteolytic processing of released neuropeptide Y may persist in female rats. Using western blot analysis, it was observed that females had greater overall neuropeptide Y Y₂-receptor expression in skeletal muscle compared to males (P < 0.05). To address the prevalence/impact of baseline endogenous Y₂-receptor activation on neuropeptide Y release in hindlimb vasculature, an arterial infusion of BIIE0246 (specific non-peptide Y₂-receptor antagonist; 170 μg kg⁻¹) was carried out on female and male rats. Y₂-receptor blockade resulted in a decrease in hindlimb vascular conductance in females and males (P < 0.05). However, the BIIE0246-induced decrease in vascular conductance was Y₁-receptor dependent in females, but not males (P < 0.05). In addition, compared to baseline, BIIE0246 infusion resulted in increased plasma neuropeptide Y concentration in females (P < 0.05), while there was no observable change in males. In a final experiment, systemic inhibition of proteolytic enzymes dipeptidylpeptidase IV (via 500 nm diprotin A) and aminopeptidase P (via 180 nm 2-mercaptoethanol) elicited a Y₁-receptor-dependent decrease in hindlimb vascular conductance in females (P < 0.05). It was concluded that our previously reported lack of basal endogenous Y₁-receptor activation in female hindlimb vasculature was (at least partially) due to prejunctional Y₂-receptor autoinhibition and proteolytic processing of neuropeptide Y.

In the periphery, arteriolar tone is modulated through neuronal release of noradrenaline, neuropeptide Y (NPY), and purines from sympathetic neurones. Classically, noradrenaline is considered the primary neurotransmitter involved in maintaining vascular tone under baseline conditions (Zukowska-Grojec, 1995) through activation of α-adrenoceptors (αR) on vascular smooth muscle cells. It has been well established that NPY exerts significant vasomotor control in resistance vessels via activation of postsynaptically located Y₁-receptors (Y₁R) (Zukowska-Grojec & Wahlestedt, 1993; Ekelund & Erlinge, 1997; Malmstrom, 1997). Importantly, synergistic vasoconstrictive effects can be observed with the coactivation of Y₁R and α₁R by NPY and noradrenaline, respectively (Zukowska-Grojec & Wahlestedt, 1993; Jackson et al. 2005). Furthermore, NPY can inhibit noradrenaline release as well as autoinhibit its own release via presynaptic NPY Y₂-receptors (Y₂R) (Zukowska-Grojec & Wahlestedt, 1993).

We have recently shown that under baseline conditions anaesthetized male (Jackson et al. 2004, 2005), but not female rats (Jackson et al. 2005), exhibit endogenous Y₁R modulation in hindlimb vasculature. The lack of baseline endogenous Y₁R activation in the female hindlimb was partially explained by 35% less total NPY and less overall Y₁R expression in skeletal muscle tissue homogenate. Despite these observed differences in ligand concentration and receptor expression, females still possessed the mechanism(s) for basal endogenous Y₁R control but did not express it functionally. The complete lack of endogenous Y₁R activation despite the existence of NPY and Y₁R in females suggests that the bioavailability of NPY may be limited under baseline conditions. In turn, limited bioavailability of NPY may be related to sex differences in the modulation of prejunctional control over NPY release and/or its post-release metabolism. The complete NPY (NPY_1–36) molecule binds and activates Y₁R. However, the conversion of vasoconstrictive NPY to non-vasoconstrictive NPY_3–36 or NPY_2–36 occurs through the effects of NPY-converting enzyme dipeptidyl peptidase IV (DPPIV) (Lee et al. 2003) or aminopeptidase P (Mentlein & Roos, 1996), respectively. Each of NPY_1–36 and its metabolites will activate prejunctional Y₂R-mediated inhibition of NPY release (Mentlein & Roos, 1996). Thus, sex differences in Y₂R expression/activation may be involved in modifying Y₁R vasomotor control. In addition, Glenn et al. (1997) concluded that NPY-converting enzymes (peptidases) may be more active in females versus males. One or both of these effects may reduce NPY availability for Y₁R binding and enhance Y₂R activation.

In the current study we provide evidence that Y₂R expression is affected by sex. Additionally, we tested the complementary hypotheses that basal endogenousY₁R modulation of hindlimb vascular conductance is blunted by Y₂R autoinhibition and/or NPY metabolism (via peptidases) in female Sprague-Dawley rats.

Methods

The Council on Animal Care at the University of Western Ontario approved the experimental protocol.

Animals

In total, 23 adult female (273 ± 96 g) and 11 adult male (354 ± 31 g) (mean ±s.d.), age-matched Sprague-Dawley rats (Charles River Laboratories Canada, Saint-Constant, Quebec) were used. The rats were housed in a light- (12 h cycle) and temperature- (22°C) controlled room in Plexiglas cages. Rats were allowed to eat (Prolab Rat chow, Mouse and Hamster 3000 Diet) and drink water ad libitum. Prior to surgery or tissue extraction animals were anaesthetized with an intraperitoneal injection of α-chloralose (80 mg kg⁻¹; Sigma-Aldrich) and urethane (500 mg kg⁻¹; Sigma-Aldrich). During the experiments, internal body (rectal) temperature was monitored continuously and was maintained at 37 ± 0.5°C with a water-perfused heating pad (mean ±s.d.).

For in vivo experiments, following surgery, a continuous intravenous infusion of α-chloralose (8–16 mg kg⁻¹ h⁻¹) and urethane (50–100 mg kg⁻¹ h⁻¹) was maintained to ensure a constant level of anaesthesia, which was verified by the stability of haemodynamic variables. Furthermore, animals showed no signs of pain or distress throughout the experiment, as adequate depth of anaesthesia was confirmed every half hour by the absence of flexor withdrawal reflex to a foot pinch.

Surgery

The trachea was intubated to facilitate spontaneous respiration, and end-tidal CO₂ (ET_CO2) measures were made from expired air at periods throughout the experiment. A polyethylene (PE50) cannula was inserted into the left common carotid artery to permit arterial blood pressure (ABP) recording from the amplified signal (ML118 Powerlab Quad Bridge Amplifier; ADInstruments, Colorado Springs, CO, USA) of a pressure transducer (MLT844; ADInstruments, Colorado Springs, CO, USA).

Through a midline abdominal incision, the gut was carefully moved aside and covered with sterile gauze moistened with sterile saline (0.9% NaCl). Using cotton swabs a small portion (∼1 cm) of the right iliac artery was exposed and a cannula (PE50) was precisely advanced to the bifurcation of the aorta using microscopic guidance. This cannula was used for drug delivery to the left hindlimb, such that the perfusion of drug was directed into the flow of blood travelling from the descending aorta to the left hindlimb. Immediately following these procedures, the gauze was removed and the contents of the gut were repositioned, and incisions were closed (Becton-Dixon, 9 mm stainless steel wound clips).

Femoral artery blood flow (Q_fem) was measured from the left hindlimb using a Transonic flowmeter (TS420 Perivascular Flowmeter Module; Transonic Systems Inc., Ithaca NY, USA) and Transonic flowprobe (0.7 mm; 0.7PSB acute model) placed approximately 3 mm distal to the femoral triangle. Specifically, with the animal placed on its back, a small ∼1 cm medial incision was made through the shaved skin of the left thigh. Under microscopic guidance, blunt dissection was used to clear the fascia and tissue overlying the femoral artery and vein; care was taken to ensure that nerves and vessels were not damaged during this procedure. With a small portion of the artery free from the nerve and vein, the vessel was carefully placed into the flowprobe using blunt microforceps. The probe and vessel were held in a natural anatomical position so as not to interfere with blood flow. Finally, innocuous water-soluble gel was spread over the opened area (∼1 cm²) of the hindlimb to maintain hydration of exposed tissue and quality of blood flow signal.

Upon completion of each experiment, animals were killed with a lethal dose of α-chloralose and urethane administered by intraperitoneal injection.

Experiments

Experiments 1 and 2 were carried out to examine the role of endogenous Y₂R activation on NPY release under baseline conditions in female skeletal muscle. Experiment 3 was performed to address the hypothesis that the proteolytic enzymes dipeptidyl peptidase IV (DPPIV) and aminopeptidase P (APP) limit NPY_1–36 bioavailability in female rats.

Experiment 1: assessment of Y₂R expression in skeletal muscle (n = 6 males, n = 6 females)

Using males as a comparison, this study was carried out to address the specific hypothesis that Y₂R are more abundant in female compared to male skeletal muscle. Western blot analyses were carried out on three different skeletal muscle groups known to contain differing expression of slow-twitch oxidative (SO), fast-twitch glycolytic (FG), and fast-twitch oxidative-glycolytic (FOG) fibre types. Skeletal muscle samples were taken from soleus (Sol; expressing SO > FOG fibres), white medial gastrocnemius (MG; expressing FG > FOG), and lateral gastrocnemius (LG; FOG > SO > FG) (Laughlin & Armstrong, 1983). The use of skeletal muscle groups expressing differing ratios of fibre types was based on early work by others illustrating that blood flow to such muscles is distributed differently at rest (Terjung & Engbretson, 1988) and during exercise (Armstrong & Laughlin, 1984; Terjung & Engbretson, 1988).

Y₂R western blotting

Approximately 70 mg of tissue was cut from the mid-belly of frozen muscle samples (from each of Sol, MG, and LG), immediately homogenized in 15 volumes of extraction buffer (25% glycerol, 0.42 m NaCl, 1.5 mm MgCl₂, 0.2 m EDTA, 20 mm Hepes, 10 μg ml⁻¹ aprotinin; pH = 7.5), and centrifuged at 16000 g for 20 min in order to collect the supernatant as the tissue extract. Sample homogenates were then stored at −70°C until the time of total protein concentration determination and electrophoresis. Total protein concentration was accomplished using the Bradford protein assay (Bradford, 1976). Equal amounts of total protein (60 μg) were run on a 12% acrylamide mini gel (Bio-Rad, Hercules, CA, USA) overlaid with a 4% acrylamide stacking gel. After electrophoresis, the proteins were transferred at constant voltage in cold transfer buffer (10% running buffer, 20% methanol in ddH₂O) to nitrocellulose membranes. The membranes were blocked in a 5% non-fat milk solution in Tris-buffered saline + 0.1% Tween 20 (TTBS) (80 mm Tris base, 0.5 m NaCl) overnight. Membranes were then incubated for 2 h in primary antibody specific to rat, human or mouse NPY Y₂-receptor (affinity purified rabbit antimouse NPY Y₂R immunoglobulin G (IgG), Catalogue number NPY2R11-A, Alpha Diagnostic International, San Antonio, TX, USA) at a dilution of 3 μg per ml in TTBS with 2% non-fat milk. Membranes were washed again in TTBS then incubated in secondary antibody (goat antirabbit antibody conjugated to horseradish peroxidase (HRP), BIO-RAD, Product no. 170–6518) in TTBS with 2% non-fat milk for 1 h. After washing, the blots were developed using enhanced chemiluminescent (ECL) western blotting detection reagents (Amersham, Product no. RPN2106) and exposed to Kodak BioMax Light film. The films were scanned and subjected to densitometric quantification (Scion Image analysis software).

NPY Y₂R expression data were analysed using two-way analysis of variance (ANOVA) with the two factors being sex and muscle group (Statistical Analysis System V.8.0.2, SAS Institute Inc., Cary, NC, USA). In the event of statistical significance (P < 0.05) Tukey's post hoc test was used to identify conditions that differed.

Experiment 2: pharmacological assessment of Y₂R contribution to hindlimb blood flow (n = 5 males, n = 10 females)

This study was carried out to address the specific hypothesis that endogenous Y₂R activation functionally restrains the release of NPY in females under baseline conditions, thus blunting the Y₁R contribution to basal vascular conductance.

Anaesthetized animals recovered for 1 h following surgery. Drugs were delivered to the left hindlimb by means of an intra-arterial drug cannula (see above). After recovery, a 10 s vehicle (0.9% saline) infusion (160 μl) was carried out followed by 10 min recovery. Baseline data were recorded for 5 min followed by a 10 s infusion of 170 μg kg⁻¹ (160 μl volume) BIIE0246 (S)-N²-[[1-[2-[4-[(R,S)-5,11-dihydro-6(6 h)-oxodibenz[b,e]azepin-11-yl]-1-piperazinyl]-2-oxoethyl]cyclopentyl]acetyl]-N-[2-[1,2-dihydro-3,5(4H)-dioxo-1,2-diphenyl-3H-1,2,4-triazol-4-yl]ethyl]-argininamide (specific non-peptide NPY Y₂-receptor antagonist; generously provided by Dr Henry N. Doods, Boehringer Ingelheim Pharma, Biberach, Germany).

After stabilization of BIIE0246 effects (approximately 30 min), 160 μl of 2 nm BIBP3226, N²-(diphenylacetyl)-N-[(4-hydroxyphenyl)methyl]-d-arginine amide (specific non-peptide NPY Y₁-receptor antagonist; Sigma-Aldrich) was infused over 10 s into the iliac arterial cannula. This intervention was included to verify Y₁R activation due to enhanced NPY release (from Y₂R blockade via BIIE0246).

The dose of BIBP3226 was chosen based on previous work that indicated as little as 100 μg kg⁻¹ BIBP3226 will completely block Y₁R in this preparation (Jackson et al. 2004, 2005). Since it was hypothesized that Y₂R blockade (via BIIE0246) would enhance NPY release, and knowing that BIBP3226 competes with NPY for binding on Y₁R, a large quantity of BIBP3226 was infused to ensure that Y₁R blockade prevailed. The concentration used within the current investigation represents approximately 600 μg kg⁻¹, which we have previously shown did not affect baseline hindlimb vascular conductance in females (Jackson et al. 2005).

In four males and four females, a 160 μl vehicle control infusion of saline was carried out instead of BIBP3226 to address the effects of vehicle infusion and time artefacts on recovery from BIIE0246. Saline had no effect on BIIE0246-induced vascular response. The infusion speed and volume of each drug infusion was held constant at 10 s for the 160 μl infusion. BIIE0246 is an experimental compound that is not yet commercially available. The quantity of infused BIIE0246 was chosen based on pilot work that indicated this dose elicited the greatest vascular response without affecting systemic haemodynamics (heart rate or blood pressure). In support, the amount of BIIE0246 delivered in the current study (170 μg kg⁻¹) was higher than that used by Malmstrom et al. (2001a, b) who have repeatedly shown that 100 μg kg⁻¹ BIIE0246 completely blocks Y₂R in vivo.

A Powerlab data acquisition system (ADInstruments, Colorado Springs, CO, USA) was used for real-time data collection. The pulsatile ABP signal was used to calculate heart rate (HR) and mean arterial pressure (MAP). Left hindlimb vascular conductance (VC) was calculated as the ratio of Q_fem/MAP. For all conditions, Q_fem, VC, MAP, and HR were calculated as a 5 min stable average during the baseline period (Baseline) and as a 5 min average upon stability of the drug response (BIIE0246 and/or BIBP3226).

Using a competitive immunoassay (Bachem Bioscience, King of Prussia, PA, USA), plasma NPY concentration was determined before (Baseline) and 30 min after Y₂R blockade (BIIE0246) in a group of five males and five females. Blood samples (500 μl; i.v.) were collected into chilled syringes, were slowly drawn to avoid an abrupt decrease in blood pressure and/or NPY release from platelet activation, and carefully transferred to chilled polypropylene tubes containing EDTA (0.5 mg; Sigma-Aldrich) and aprotinin (250 KIU; Sigma-Aldrich). Samples were centrifuged at 1600 g for 15 min at 0°C. Plasma was then transferred to fresh polypropylene tubes and immediately stored at −70°C until analysis.

NPY immunoassay

All samples and standards (constituted in charcoal stripped rat serum) were distributed in duplicate and were incubated at room temperature for 2 h in the provided 96-well immunoplate. The immunoplate was then washed five times with 300 μl per well of assay buffer. Subsequently, wells were incubated at room temperature with 100 μl of streptavidin-HRP for 1 h. The immunoplate was washed again five times with 300 μl per well of assay buffer. Following washing, 100 μl of a tetramethylbenzidine (TMB) peroxidase substrate solution was added to all wells. After a 1.5 h incubation at room temperature, the reaction was terminated by the addition of 100 μl 2 m HCl. Finally, the optical absorbance of each well was read at 450 nm (Bio-Rad Ultramark Microplate Imaging System, Bio-Rad, Hercules, CA, USA). Absorbance measures were converted to NPY concentration by comparison with the 10-point standard curve. The assay has a minimum detectable concentration of 0.04–0.06 ng ml⁻¹ or 2–3 pg well⁻¹ (manufacturer's data).

The functional vascular effects of BIIE0246 and subsequent BIBP3226 infusion were assessed independently (for males and females) using one-way repeated measures analysis of variance (ANOVA) (Statistical Analysis System V.8.0.2, SAS institute Inc., Cary, NC, USA). In the event of statistical significance (P < 0.05), Tukey's post hoc analysis was used to identify conditions that differed. Data are presented as mean (±s.e.m.). The effect of Y₂R blockade on NPY release (NPY immunoassay) was assessed independently in males and females using paired t tests. Data are presented as mean (±s.e.m.).

Experiment 3: effect of peptidase inhibition on baseline Y₁R contribution to hindlimb vascular conductance in female Sprague-Dawley rats (n = 7 females)

This study was carried out to address the hypothesis that the proteolytic enzymes dipeptidyl peptidase IV (DPPIV) and aminopeptidase P (APP) limit NPY_1–36 bioavailability, blunting Y₁R modulation of basal vascular conductance in females.

Anaesthetized animals recovered for 1 h following surgery. Baseline data were recorded for 5 min, followed by a slow 160 μl bolus intravenous infusion of a protease inhibitor cocktail consisting of 500 nm diprotin A (DPPIV inhibitor; Bachem Bioscience, King of Prussia, PA, USA) and 180 nm 2-mercaptoethanol (APP inhibitor; Sigma-Aldrich). Upon stabilization of hindlimb blood flow (approximately 20 min post protease inhibition), a 10 s vehicle (0.9% saline) control infusion (160 μl) was carried out followed by 5 min recovery. Subsequently, BIBP3226 (2 nm; 160 μl volume) was infused over 10 s into the arterial blood flow to the left hindlimb. This intervention was included to verify Y₁R activation via increased NPY bioavailability (from DPPIV and APP inhibition).

A Powerlab data acquisition system (ADInstruments, Colorado Springs, CO, USA) was used for real-time data collection. The pulsatile ABP signal was used to calculate heart rate (HR) and mean arterial pressure (MAP). Left hindlimb vascular conductance (VC) was calculated as the ratio of Q_fem/MAP. For all conditions, Q_fem, VC, MAP, and HR were calculated as a 5 min stable average during the baseline period (Baseline) and as a 3 min average upon stability of the drug response (Peptidase inhibition and/or BIBP3226).

The treatment effect was assessed using one-way repeated measures analysis of variance (ANOVA) (Statistical Analysis System V.8.0.2, SAS institute Inc., Cary, NC, USA). In the event of statistical significance (P < 0.05), Tukey's post hoc test was used to identify conditions that differed. Data are presented as mean (±s.e.m.).

Results

Experiment 1: assessment of Y₂R expression in skeletal muscle

As indicated by a significant sex effect, females had greater overall Y₂R expression in skeletal muscle tissue homogenate than males (Main effect, P < 0.05; Fig. 1).

Figure 1. Western blot analysis of NPY Y₂R from whole muscle tissue homogenate in male (n = 6) and female (n = 6) rats.

Female skeletal muscle homogenate contained greater overall Y₂R expression compared to male (main effect of sex, P < 0.05). Error bars indicate ±s.e.m.

Experiment 2: pharmacological assessment of Y₂R contribution to hindlimb blood flow (n = 5 males, n = 10 females)

Baseline heart rate (HR) and mean arterial pressure (MAP) were 350 ± 13 beats min⁻¹ and 98 ± 6 mmHg, respectively, for males and 348 ± 12 beats min⁻¹ and 96 ± 4 mmHg, respectively, for females. There were no changes in HR or MAP associated with BIIE0246 or BIBP3226 infusion (Table 1).

Table 1.

Haemodynamic measures at baseline and after each drug infusion in male and female animals

		Male	Female
HR (beats min−1)	Baseline	350 ± 13	348 ± 12
	BIIE0246	341 ± 17	342 ± 14
	BIBP3226	352 ± 15	340 ± 10
MAP (mmHg)	Baseline	98 ± 6	96 ± 4
	BIIE0246	100 ± 6	97 ± 5
	BIBP3226	106 ± 5	102 ± 4

Values represent mean ±s.e.m.

Y₂R blockade via BIIE0246 infusion resulted in a decrease in hindlimb blood flow (Q_fem) and vascular conductance (VC) from baseline for females (ΔQ_fem=−58 ± 11 μl min⁻¹; ΔVC =−0.6 ± 0.2 μl min⁻¹ mmHg⁻¹ or 36 ± 9%; P < 0.05 versus baseline; Fig. 2) and males (ΔQ_fem=−111 ± 14 μl min⁻¹; ΔVC =−1.2 ± 0.3 μl min⁻¹ mmHg⁻¹ or 38 ± 9%; P < 0.05 versus baseline; Fig. 3). The impact of BIIE0246 on VC was reversed with subsequent Y₁R blockade (2 nm BIBP3226; 160 μl volume) in females only. Additionally, Y₂R blockade resulted in an increase in plasma NPY in females (P < 0.05; Fig. 4), but not males.

Figure 2.

A, mean beat-by-beat vascular conductance data from a representative female under baseline conditions, in response to BIIE0246 infusion (170 μg kg⁻¹; 160 μl i.a.), and subsequent BIBP3226 infusion (2 nm; 160 μl i.a.). B, mean hindlimb blood flow (left) and hindlimb vascular conductance (right) in female rats (n = 10) under baseline conditions, after BIIE0246, and with subsequent BIBP3226 infusion. Bars indicate mean ±s.e.m.*Significant difference from baseline (P < 0.05).

Figure 3.

A, mean beat-by-beat vascular conductance data from a representative male under baseline conditions, in response to BIIE0246 infusion (170 μg kg⁻¹; 160 μl i.a.), and subsequent BIBP3226 infusion (2 nm; 160 μl i.a.). B, mean hindlimb blood flow (left) and hindlimb vascular conductance (right) in male rats (n = 5) under baseline conditions, after BIIE0246, and with subsequent BIBP3226 infusion. Bars indicate mean ±s.e.m.*Significant difference from baseline (P < 0.05).

Figure 4. Analysis of blood plasma NPY concentration in female (left; n = 5) and male (right; n = 5) rats using ELISA.

Plasma NPY increased from baseline with Y₂R blockade in females (P < 0.05) but not males. Bars indicate mean ±s.e.m.*Significant difference from baseline (P < 0.05).

Experiment 3: effect of peptidase inhibition on baseline Y₁R contribution to hindlimb vascular conductance in female Sprague-Dawley rats (n = 7 females)

Baseline HR and MAP were 352 ± 13 beats min⁻¹ and 93 ± 5 mmHg. There were no changes in HR or MAP associated with peptidase inhibition or BIBP3226 infusion (Table 2).

Table 2.

Haemodynamic measures at baseline and after each drug infusion for female animals

	HR (beats min−1)	MAP (mmHg)
Baseline	352 ± 13	93 ± 5
Peptidase inhibition	353 ± 11	89 ± 4
BIBP3226	372 ± 25	87 ± 4

Values represent mean ±s.e.m.

Y₁R-dependent vasoconstriction was observed after dipeptidyl peptidase IV (DPPIV) and aminopeptidase P (APP) blockade. Specifically, baseline Q_fem decreased 36 ± 7% and VC decreased 34 ± 7% from 221 ± 47 μl min⁻¹ and 2.5 ± 0.6 μl min⁻¹ mmHg⁻¹, respectively, to 147 ± 41 μl min⁻¹ and 1.7 ± 0.5 μl min⁻¹ mmHg⁻¹, respectively, following peptidase inhibition (P < 0.05; Fig. 5). This vasoconstriction was reversed by Y₁R blockade (2 nm BIBP3226; 160 μl volume) (Fig. 5).

Figure 5.

A, mean beat-by-beat vascular conductance data from a representative female, under baseline conditions, response to peptidase inhibition (500 nm diprotin A and 180 nm 2-mercaptoethanol; 160 μl i.v.), and subsequent BIBP3226 (2 nm; 160 μl i.a.) infusion. B, mean hindlimb blood flow (left) and hindlimb vascular conductance (right) in female rats (n = 7) under baseline conditions, after peptidase inhibition, and with subsequent BIBP3226 infusion. Bars indicate mean ±s.e.m.*Significant difference from baseline and BIBP3226 condition (P < 0.05).

Discussion

We had recently reported that hindlimb vasculature was under chronic Y₁R activation in males (Jackson et al. 2004, 2005) but not in females (Jackson et al. 2005). This could be partially explained by greater overall Y₁R expression and NPY concentration in male skeletal muscle tissue homogenate compared to females (Jackson et al. 2005). Thus, although female muscle tissue expressed the ‘cellular machinery’ for baseline Y₁R modulation of hindlimb vascular conductance, it did not exhibit this level of control. The current set of experiments provide evidence that baseline NPY bioavailability is blunted in females via endogenous Y₂R autoinhibition of NPY release coupled with enzymatic breakdown of NPY. Thus, endogenousY₁R activation in hindlimb vasculature is effectively minimized in female rats, at least under baseline conditions.

The concept of prejunctional autoinhibitory NPY-receptors was first introduced by Lundberg et al. (1982). Soon after, prejunctional autoinhibitory NPY-effects were suggested to be mediated by Y₂R (Wahlestedt et al. 1986). Recently, the development of a highly selective Y₂R antagonist, BIIE0246 (Doods et al. 1999) has led to the discovery of Y₂Rs' vasculature role in a limited number of species and tissues. Y₂Rs have been shown to regulate the release of NPY in porcine kidney (Malmstrom et al. 2002a, b), porcine spleen (Malmstrom et al. 2002b), and rat vas deferens (Smith-White et al. 2001).

In the present study we have provided the first quantitative account of sex-differentiated Y₂R expression in skeletal muscle (Fig. 1). In whole muscle tissue homogenate, females expressed greater overall Y₂R than males. As indicated above, Y₂Rs are generally presumed to be prejunctional and autoinhibitory. However, research has shown that postjunctional Y₂Rs can exist at low levels in pig spleen, causing vasoconstriction (Modin et al. 1991; Malmstrom, 2001a). Recently, moderate splenic vasodilation associated with Y₂R blockade was observed in the reserpine-treated pig (Malmstrom et al. 2002b). In the current study (experiment 2) there was no observable increase in vascular conductance after Y₂R blockade. In fact, a consistent and progressive decrease in vascular conductance ensued after infusion of BIIE0246 in males and females. Thus, the net phenotype of Y₂Rs in our model was prejunctional and/or autoinhibitory.

Even though a decrease in vascular conductance with Y₂R blockade occurred in males and females, the BIIE0246-induced vasoconstriction was completely reversed with Y₁R blockade in females only. Although modest in magnitude, these data suggest that the previously reported lack of endogenous Y₁R activation in female hindlimb vasculature (Jackson et al. 2005) was at least partially due to endogenous Y₂R autoinhibition of NPY release. This is supported by the finding that Y₂R blockade resulted in an increase in plasma NPY from baseline in females (Fig. 4). The lack of a full recovery in vascular conductance with Y₁R blockade (while under simultaneous Y₂R antagonism) in males suggests that baseline endogenous Y₂R activation inhibited transmitter(s) other than NPY in our model. This premise is supported by plasma NPY analysis where Y₂R blockade resulted in no change in NPY concentration in males (Fig. 4). Indeed, it has been demonstrated that prejunctional Y₂R activation inhibited both noradrenaline and NPY release in the male pig in vivo (Malmstrom et al. 2002a). In support of the current findings, Y₂R blockade did not alter baseline NPY levels in the male porcine kidney under control conditions (Malmstrom et al. 2002a). Thus, it is plausible that Y₂R blockade in males preferentially enhanced noradrenaline release in our preparation, leading to the observed decrease in vascular conductance.

Proteolytic processing of NPY leading to alterations in N-terminus residues abolishes its ability to bind to Y₁R. Therefore, all N-terminus truncated NPY fragments such as, NPY_3–36 or NPY_2–36 have little or no affinity for Y₁R. NPY_3–36 and NPY_2–36 exist endogenously in the presence of endothelial NPY-converting enzyme dipeptidyl peptidase IV (DPPIV) (Lee et al. 2003) or aminopeptidase P (APP) (Mentlein & Roos, 1996), respectively. These fragments are as equipotent as NPY in their binding affinity with Y₂R (Beck-Sickinger & Jung, 1995). It has been observed that NPY-converting enzymes (peptidases) may be more active in female rat tail arterial ring segments, compared to males (Glenn et al. 1997). However, until the present, the role of peptidases on endogenous NPY and vascular function had not been investigated. In experiment 3 we addressed the contribution of endogenous DPPIV and APP to NPY (NPY_1–36) bioavailability in females. The decrease in hindlimb vascular conductance with peptidase inhibition was reversed by BIBP3226 indicating that NPY bioavailability was limited by DPPIV and APP under baseline conditions.

In summary, we have provided mechanisms by which NPY bioavailability is blunted in female hindlimb vasculature under baseline conditions. First, female skeletal muscle has greater Y₂R expression compared to the male. Functionally, it appeared that endogenous prejunctional Y₂R activation limits NPY release in females but not males. This finding was supported by increased plasma NPY concentration after Y₂R blockade in females, but not males. Additionally, endogenous peptidases limit the potential for NPY to exert baseline Y₁R vascular effects, further decreasing the opportunity for Y₁R activation in females under baseline conditions. In conclusion, the current investigation provides evidence that the previously reported lack of basal endogenous Y₁R activation in female hindlimb vasculature (Jackson et al. 2005) was (at least partially) due to prejunctional Y₂R autoinhibition and proteolytic processing of NPY.