Neuropeptide Y overflow and metabolism in skeletal muscle arterioles

Non-technical summary

Neuropeptide Y (NPY) is involved in a number of vascular physiological processes that affect sympathetic neurotransmission and angiogenesis. While NPY is of physiological significance, very little is known regarding local overflow characteristics at specific levels of the vasculature. Through the use of a new technique, we were able to quantify NPY overflow from isolated skeletal muscle arterioles of female rats. We observed age-related differences in NPY overflow and its degradation via dipeptidyl peptidase IV. These results will provide insights into the release and breakdown of NPY at local levels of the vasculature.

Abstract

Abstract

The purpose of this study was to characterize neuropeptide Y (NPY) overflow and metabolism from isolated skeletal muscle arterioles of female rats. Gastrocnemius first-order arterioles were removed from young (2 months), young adult (6 months) and middle-aged (12 months) F344 female rats. Arterioles were isolated, cannulated and pressurized in a microvessel bath with field stimulation electrodes. NPY overflow from isolated arterioles was assessed at 0 s and 30 s post-field stimulation. Dipeptidyl peptidase IV (DPPIV) activity was quantified via fluorometric assay of whole vessel homogenate. In young adult and middle-aged rats, NPY overflow increased 0 s and 30 s following field stimulation. In young adult rats, DPPIV inhibition resulted in an increase in NPY overflow at 30 s, while middle-aged rats had no increase in NPY overflow with DPPIV inhibition (P < 0.05). DPPIV activity was influenced by factors such as age, vessel type, and endothelium (P < 0.05). The present data suggest that DPPIV plays a significant role in modulating the actions of NPY in arterioles of young adult females; however, this role appears to diminish with age.

Introduction

Neuropeptide Y (NPY) is a 36-amino acid polypeptide chain with ubiquitous expression in the central and peripheral nervous systems (Tatemoto et al. 1982). In the peripheral nervous system, NPY coexists with noradrenaline and adenosine triphosphate within the sympathetic end terminal where it participates in propagating sympathetically mediated vasoconstriction (Edvinsson et al. 1984; Ekblad et al. 1984; Stjarne et al. 1986; Buckwalter et al. 2004, 2005). NPY stimulates direct vasoconstriction and modulates the effects of other neurotransmitters through its post-junctional (Y₁) and pre-junctional (Y₂) receptors (Wahlestedt et al. 1990). In addition to neuronal sources, NPY is also present in blood elements (platelets) (Ericsson et al. 1987; Myers et al. 1988), and is stored and released from the adrenal medulla (Allen et al. 1983; Varndell et al. 1984). Thus, plasma NPY levels can exhibit relatively large fluctuations (Lundberg et al. 1986a) owing to the multiple sources of, and multiple stimuli for, NPY release. While studies of NPY release have focused on larger systems (adrenal release, arterial beds) (Allen et al. 1984; Lundberg et al. 1986b,c; Han et al. 1998), the characterization of NPY overflow at local levels of the vasculature has received little study.

Dipeptidyl peptidase IV (DPPIV; CD26) is a protease with an affinity for alanine or proline amino-acid residues in the penultimate position (Karl et al. 2003), such as the proline residue located near the N-terminus of NPY. In the vasculature, DPPIV is active as a homodimer (Chung et al. 2010) anchored to the plasma membrane of endothelial cells (Zukowska-Grojec et al. 1998) in addition to its presence in serum as a soluble enzyme (Durinx et al. 2000). The actions of NPY can be modulated by DPPIV via removal of the tyrosine–proline residues. This enzyme yields a truncated product (NPY_(3-36)) (Mentlein et al. 1993; Mentlein & Roos, 1996) that expresses little affinity for the post-junctional Y₁ receptor while maintaining affinity for the pre-junctional Y₂ receptor (Wahlestedt et al. 1986, 1990). Therefore, DPPIV would attenuate NPY-mediated vasoconstriction by decreasing the bioavailability of the Y₁ receptor agonist (NPY_(1-36)), while concurrently limiting further NPY_(1-36) release from the sympathetic end terminal by increasing the bioavailability of the Y₂ receptor agonist (NPY_(3-36)).

The physiological role of NPY in the arterial vasculature may depend on vascular level/region with respect to direct vasoconstriction and the degree to which NPY modulates the actions of other neurotransmitters (Hieble et al. 1988; Lacroix et al. 1988; Abel & Han, 1989; Clarke et al. 1991; Han et al. 1998; Malmstrom, 2000). In the femoral artery, direct application of NPY or a post-junctional Y₁-receptor agonist either failed to elicit vasoconstriction (Grundemar & Hogestatt, 1992; Tsurumaki et al. 2003; Kluess et al. 2006) or elicited vasoconstriction that was less in magnitude to that achieved with noradrenaline (Tessel et al. 1993a,b;). However, more recent studies on skeletal muscle conduit vessels using Y₁-receptor blockers educed changes in haemodynamics suggesting that NPY does indeed play a larger role in the skeletal muscle vasculature (Buckwalter et al. 2005; Jackson et al. 2005a). Specifically, vascular conductance and blood flow increased with blockade of the Y₁ receptor in male external iliac arteries. While females failed to demonstrate a similar response, they were sensitive to DPPIV and aminopeptidase P blockade (Jackson et al. 2005b) suggesting a larger role for the proteolytic enzymes involved in NPY metabolism in females.

There is evidence to support a physiological role for NPY in the regulation of small vessel diameter. NPY was either equipotent or more potent than noradrenaline in arterioles of cremaster (Joshua, 1991) and tenuissimus (Pernow et al. 1987) muscles. Little is known regarding the role or presence of NPY or its proteolytic enzyme, DPPIV, in skeletal muscle resistance vessels. This level of the vasculature plays a substantial role in blood flow regulation; therefore, it is prudent to examine NPY overflow along with proteolytic activity of the associated enzymes at this level to better understand NPY's role in sympathetic neurotransmission.

The current study represents a first step in the characterization of NPY overflow from isolated skeletal muscle first-order arterioles. The isolated microvessel preparation allows for direct measurement of NPY overflow in the absence of confounding elements such as platelet- and adrenal-derived NPY that are otherwise difficult to control. A supplementary interest of this study was to explore the efficacy of DPPIV in modulating the bioavailability of NPY in young and middle-aged females through the use of DPPIV inhibitors. A second purpose of this study was to directly assess DPPIV activity in skeletal muscle first-order arterioles, and to determine how this activity differed according to age, vessel type, and endothelial status (endothelium-intact and -denuded). It was our hypothesis that NPY is released from skeletal muscle arterioles following neural stimulation, and DPPIV blockade would increase the bioavailability of NPY in both age groups. In addition, we wanted to ascertain the impact of age and other vessel characteristics on DPPIV activity.

Methods

Animals

Female (2-month, 6-month, 12-month) and male (6-month) Fischer-344 rats were used in this study. Rats were housed at the university's animal care facility under static environmental conditions (22°C; 12:12 h light–dark cycle). Water and rat chow were provided ad libitum. Animal care and experimental protocols were approved by the university's Institutional Animal Care and Use Committee.

Vessel preparation

Rats were killed with an overdose of pentobarbital (i.p.; 40 mg kg⁻¹) followed by pneumothorax with the depth of anaesthesia determined through flexor withdrawal reflex in response to a foot pinch. Red gastrocnemius first-order arterioles were removed and placed in a cold (4°C) Krebs–Ringer physiological saline solution (mmol l⁻¹:119 NaCl, 4.7 KCl, 2.5 CaCl₂, 1.2 MgSO₄, 25 NaHCO₃, 1.2 KH₂PO₄, 5.5 glucose, 2 glycerol). Arterioles designated for DPPIV activity analysis were homogenized in 200 μl of warm (37°C) Krebs–Ringer physiological saline solution, centrifuged 2000g for 3 min, and the supernatant removed. In select vessels, the endothelium was removed by cannulating one side of the arteriole and slowly administering 12 ml of air through the lumen. All samples were flash-frozen and stored at −80°C.

NPY sampling

Arterioles for NPY analysis were transferred to a vessel chamber (Living Systems, Inc., Burlington, VT, USA) with the ends secured to micropipettes using 11–0 opthalmic suture. The vessel bath contained a Krebs–Ringer physiological saline solution (37°C, pH 7.4, bubbled with 5% CO₂–30% O₂), and the vessel was perfused with Krebs–Ringer physiological saline solution containing 1% albumin (37°C, pH 7.4) (Pourageaud & De Mey, 1998). A sampling port was placed within the vessel chamber immediately below the suspended vessel. The vessel chamber was transferred to the stage of an inverted microscope (Olympus CKX41, Melville, NY, USA). The micropipettes were connected to independent reservoir systems. Initial luminal pressure was set at 60 cmH₂O for 30 min. Luminal pressure was then increased to 90 cmH₂O, which is a pressure associated with normal in vivo conditions (Williams & Segal, 1993). The bath solution was replaced at 15 min intervals during equilibration. The arterioles were considered viable if they were able to constrict by 10% to phenylephrine (10 μmol l⁻¹) and dilate by 20% to acetylcholine (1 μmol l⁻¹) (Schneider et al. 1994).

Field stimulation was delivered via two parallel platinum electrodes placed on either side of the vessel. The electrical current was supplied using a DS3 isolated constant current stimulator (Digitimer Ltd, Welwyn Garden City, UK) interfaced with a Powerlab 16/30 with Chart software (version 5.2, ADInstruments, Colorado Springs, CO, USA). Appreciable NPY release from sympathetic nerves is dependent upon the magnitude of neural stimulus (Lundberg et al. 1986c, 1987; Lacroix, 1989; Donoso et al. 1997). Therefore, field stimulation was applied at 60 Hz, 32 mA and 200 impulses. Bath samples (200 μl) were taken before field stimulation (baseline), immediately following the cessation of field stimulation (0 s), and 30 s post-field stimulation. For experiments assessing the effects of DPPIV activity on NPY overflow, a DPPIV inhibitor, K579 (10 μmol l⁻¹; Tocris Bioscience, Ellisville, MO, USA), was added to the vessel bath and allowed to incubate for 20 min. K579 does not elicit sympathetic symptoms (mydriasis or piloerection) as assessed through modified Irwin's screening (personal communication, Dr Kotaro Takasaki, Fukuoka University, Fukuoka, Japan). Field stimulation and sampling time points were performed as previously stated. Samples were flash-frozen and stored at −80°C.

NPY analysis

A peptide enzyme immunoassay (S-1145; Bachem, King of Prussia, PA, USA) was used to determine NPY overflow. Samples and kit components were brought to room temperature. Samples and antiserum were incubated overnight (4°C) in a coated 96-well microplate. On day 2, the kit components and microplate were brought to room temperature, and a competitive biotinylated agent was added. After 2 h of incubation, the plate was washed five times with wash buffer, which was followed by the addition of an enzyme (streptavidin horseradish peroxidase). The enzyme incubated for 1 h, and the plate was washed as described previously. A substrate (tetramethylbenzidine peroxidase) was added and allowed to incubate for 30 min, which was followed by termination of the reaction using 2 n HCl. Absorbance was measured at 450 nm (KCJunior Software, Bio-Tek Instruments, Inc., Winooski, VT, USA) on a Powerwave XS (Bio-Tek Instruments, Inc., Winooski, VT, USA) microplate reader. Optical density values were converted to ng ml⁻¹ using a six-point standard curve spreadsheet provided by the manufacturer.

DPPIV activity analysis

DPPIV samples were brought to room temperature along with assay components. Incubation buffer (50 mmol l⁻¹ Tris-HCl, pH 8.3), substrate solution (20 mmol l⁻¹ glycyl-l-proline-4-methoxy-2-naphthylamide), and DPPIV sample were added to the sample wells of a black 96-well microplate, while standard solution (50 mmol l⁻¹ 4-methoxy-2-naphthylamine), ‘stopping’ solution (100 mmol l⁻¹ citrate, pH 4.0), substrate solution and incubation buffer were added to the standard wells. The microplate incubated on a heating block (37°C) for 30 min, and the reaction was terminated with stopping solution. Fluorescence was measured at 360 and 440 nm (excitation and emission, respectively) on a FLX800 Fluorometer (Biotek Instruments, Inc., Winooski, VT, USA). DPPIV activity was proportional to the production of the fluorophore, 4-methoxy-2-naphthylamine, via enzymatic cleavage of the gly-pro compound from glycyl-l-proline-4-methoxy-2-naphthylamide (Scharpe et al. 1988; Maes et al. 2005). Relative DPPIV activity was determined using the following equation:

Where F is the fluorescence of the sample minus the fluorescence of the sample blank, V_t is the total well volume, 1000 is the correction factor from millilitres to litres, C_st is the concentration of the standard (μmol l⁻¹), T is the incubation time, V_s is the sample volume, and F_st is the fluorescence of the standard minus the fluorescence of the standard blank.

Statistical analysis

Data were expressed as means ± SEM. NPY overflow mean data were derived using difference scores from baseline (Δ) NPY levels expressed in ng ml⁻¹. NPY overflow data during control and DPPIV inhibition conditions were analysed using a repeated measures design (α = 0.05; SAS 9.1.3, Cary, NC, USA). One-way analyses of variance (ANOVA) were performed in the event of overall significance by sampling time point or by age group. Differences in DPPIV activity were determined using independent samples t tests (α = 0.05). Cohen's d statistic was used to convey the magnitude of the difference between means.

Results

NPY overflow

Mean baseline NPY overflow data were 0.47 ± 0.09 ng ml⁻¹ and 0.16 ± 0.05 ng ml⁻¹ in 6- and 12-month-old animals, respectively, and pressurized vessel diameters did not differ between groups (6 months: 305.50 ± 11.77 μm; 12 months: 319.67 ± 6.95 μm). The isolated gastrocnemius 1A arteriole was exposed to field stimulation, which was followed by vessel bath sampling across multiple time points to elucidate the nature of NPY overflow at the arteriole level. NPY overflow (Δ baseline) increased immediately following, and 30 s after, termination of field stimulation (Fig. 1: n = 6) in 6-month-old females. A DPPIV inhibitor was added to the bath in order to ascertain the relevance of the enzyme in mitigating the bioavailability of NPY. The addition of the DPPIV inhibitor, K579, produced an increase in NPY overflow from baseline with the greatest quantity observed at 30 s post-stimulation (P < 0.05). A characteristic feature of the inhibition data was the increase in variability of NPY overflow that occurred following administration of the inhibitor. NPY overflow 30 s after field stimulation ranged from 0.00–0.13 ± 0.03 ng ml⁻¹ at control conditions to 0.00–0.37 ± 0.07 ng ml⁻¹ with DPPIV inhibition. A similar increase in variability occurred at 0 s post-field stimulation (control: 0.00–0.01 ± 0.02 ng ml⁻¹; DPPIV inhibition: 0.00–0.63 ± 0.11 ng ml⁻¹). While variability contributed to statistical volatility, the large magnitude of NPY overflow during DPPIV inhibition yielded substantial effects at 30 s (control vs. inhibition: d = 1.53; P = 0.08) and 0 s (d = 1.04; P = 0.18) following field stimulation. The peptide enzyme immunoassay possessed a minimal amount of variability as measured by the coefficient of variation (CV < 0.05).

Figure 1. NPY overflow in gastrocnemius first-order arterioles of 6-month-old female rats (n = 6) during control and DPPIV inhibition conditions.

NPY overflow was greater immediately following field stimulation (0 s) and 30 s after the termination of field stimulation. Arterioles produced substantial increases in NPY overflow following DPPIV inhibition (K579, hatched bars). Bars indicate mean and SEM. *Significant difference from baseline (P < 0.05).

DPPIV activity

In an effort to identify areas where the enzymatic degradation of NPY may differ, whole vessel homogenates were assayed to assess the relative activity of DPPIV according to age, vessel type and endothelial status. An age effect was detected as 6-month-old animals (n = 8) had greater DPPIV activity as compared to 2-month-old females (n = 9; Fig. 2: P < 0.05; d = 1.91). The resistance vessel (gastrocnemius first-order arteriole) exhibited greater DPPIV activity as compared to the conduit (femoral artery) vessel (Fig. 3A: n = 16/group; P < 0.05; d = 0.88). In 2-month-old females, removal of the endothelium significantly decreased DPPIV activity in gastrocnemius first-order arterioles (Fig. 3B: endothelium-intact (n = 9); endothelium-denuded (n = 5); P < 0.05; d = 1.31). The assay possessed minimal variability (CV < 0.05). The DPPIV inhibitors, K579 and diprotin A, fluoresce in response to the fluorometric parameters used for this assay. This fluorescent property precluded their use during DPPIV analysis.

Figure 2. DPPIV activity in gastrocnemius first-order arterioles of 2- (n = 9) and 6-month-old (n = 8) female rats.

DPPIV activity was greater in 6-month-old female arterioles. Bars indicate mean and SEM. *Significant difference between 2- and 6-month-old females (P < 0.05).

Figure 3. DPPIV activity according to vessel type (A) and endothelial status (B).

DPPIV activity was greater in gastrocnemius first-order arterioles (arteriole) as compared to femoral arteries (artery) (A: n = 8/group). DPPIV activity was less in endothelium-denuded arterioles (n = 5) as compared to endothelium-intact arterioles (n = 9; B). Bars indicate mean and SEM. *Significant difference from other condition(s) (P < 0.05).

NPY overflow by age

An older cohort was examined to uncover possible trends in NPY overflow that may occur with age. Similar to 6-month-old females, 12-month-old females experienced an increase in NPY overflow following field stimulation (Fig. 4: n = 6/group). However, NPY overflow of the 12-month-old group did not change with DPPIV inhibition. Twelve-month-old females registered significantly less NPY overflow at 30 s post-field stimulation (P < 0.05; d = 1.77) as compared to the 6-month-old females. The volatility (statistical variance) of NPY overflow that was observed in the 6-month-old group with DPPIV inhibition was absent in the 12-month-old group (0 s: 0.00–0.13 ± 0.03 ng ml⁻¹; 30 s: 0.00–0.06 ± 0.01 ng ml⁻¹).

Figure 4. NPY overflow in gastrocnemius first-order arterioles of 6- and 12-month-old female rats (n = 6/group) during control and DPPIV inhibition conditions.

NPY overflow was greater immediately following field stimulation (0 s) and 30 s after the termination of field stimulation for both age groups (6 months, open bars; 12 months, filled bars). In 6-month-old animals (thinly hatched bars), arterioles produced substantial increases in NPY overflow following DPPIV inhibition (K579), while 12-month-old animals (thickly hatched bars failed to exhibit a response to DPPIV inhibition. Bars indicate mean and SEM. *Significant difference from 12-month-old group (P < 0.05).

Discussion

The current paradigm of NPY release is based on early work (Allen et al. 1984; Lundberg et al. 1986b,c;) that described the release characteristics of NPY occurring in large systems (adrenal gland, arterial beds). A novel aspect of the present study was the measurement of NPY overflow using an isolated microvessel technique. This technique provided an opportunity to directly assess NPY overflow from a resistance vessel, while controlling for the influence of extraneous variables inherent in the study of larger systems. These findings suggest that NPY overflow appreciably occurs at this level of the vasculature with detectable overflow persisting 30 s after the termination of field stimulation. DPPIV is active in mitigating the bioavailability of NPY in young adult rats, although this effect was not observed in middle-aged rats. DPPIV activity was influenced by factors such as age, endothelial status, and vessel type. This is the first study to our knowledge examining NPY overflow along with DPPIV activity in isolated skeletal muscle arterioles.

NPY overflow

Prior studies of NPY release utilized perfused vascular beds (Lundberg et al. 1986b,c; Han et al. 1998) or peripheral arterial and venous sampling methods (Allen et al. 1984) with low to mid intensity frequency stimulation delivered over a series of time intervals. NPY release is proportional to stimulation frequency with larger frequencies eliciting the greatest concentrations. NPY coexists with noradrenaline within large dense-cored vesicles of sympathetic nerve terminals (Lundberg et al. 1983; Ekblad et al. 1984; Tainio et al. 1986). Low intensity sympathetic bursts cause the exocytosis primarily of small vesicles that contain noradrenaline (Thureson-Klein, 1983; Fried et al. 1985; Morris et al. 1986); thus, appreciable release of NPY requires a high intensity sympathetic stimulus in order to initiate the exocytosis of the large type vesicles (Lundberg et al. 1987). In the present study, an increase in NPY overflow was detected immediately following and 30 s after the application of high frequency field stimulation. The amount of NPY overflow ranged from approximately 10 pmol l⁻¹ observed during control conditions to 80 pmol l⁻¹ that occurred with DPPIV inhibition in the 6-month-old group.

The significance of proteolytic enzyme activity in mitigating the vasoconstrictive response to NPY has been documented in external iliac and tail arteries of the rat (Glenn et al. 1997; Jackson et al. 2005b). In females, the blockade of enzymes that metabolize NPY results in a change in blood vessel diameter or haemodynamics. In the present study, we observed an increase in NPY bioavailability between the control baseline and two of the time points sampled after DPPIV inhibition. We also observed a number of ‘spikes’ in NPY overflow in as many as half the animals across different time points. These spikes were of interest because they were present only with DPPIV inhibition. The statistical ramification of the spikes was the creation of statistical noise or volatility due to the increase in variability, which precluded statistically significant differences when comparing control versus inhibition conditions (0 s and 30 s, respectively). Therefore, statistical effects were calculated in order to provide a more comprehensive representation underlying the mean differences between control and DPPIV conditions. Effects that exceed 0.80 are considered as large statistical effects (Kirk, 1995), and the statistical effects at 0 and 30 s both exceeded 1.0. The results with DPPIV inhibition suggest that DPPIV is active in mitigating the bioavailability of NPY in skeletal muscle arterioles of mature adult animals. Thus, while this arteriole of this age group is capable of releasing large amounts of NPY with field stimulation, a substantial portion of NPY released by the nerve is diminished due to the activity of DPPIV under normal conditions.

DPPIV activity

The effects of age on DPPIV activity has yet to be fully understood. In the present study, 6-month-old females had greater DPPIV activity as compared to 2-month-old females. In a study of mice spleen, Kitlinska et al. (2002) noted a decrease in DPPIV mRNA in 18-month-old animals as compared to 2-month-old animals. However, the use of a senescent age group involves other age-related changes such as endothelial dysfunction, which would be a plausible effect of ageing since the enzyme is present in the endothelium among other sources (Zukowska et al. 2003). Virtually all 2-month-old animals experience normal oestrous cycles at this age (Haim et al. 2003), so it is difficult to draw a conclusion based on the present results that oestrogen was an underlying cause behind the differences. Sex steroid levels rise and fall during the different phases of the oestrous cycle; therefore, future studies should be aimed at controlling oestrogen levels to ascertain the role of oestrogen in modulating DPPIV activity in addition to elucidating the nature of age-related differences in DPPIV activity.

The available literature examining the vascular actions of DPPIV in NPY metabolism have utilized large arteries such as the external iliac or tail arteries. Our results indicate, perhaps, a larger role for DPPIV in the smaller resistance vessels. Skeletal muscle arterioles function as gatekeepers in a vascular sense as they possess a direct influence on blood flow into the capillary beds (Segal, 2005). Resistance vessels can differ from their conduit counterparts in the quantity and type of vascular protein expressed such as the differences in adrenergic receptor subtype involved in adrenergic vasoconstriction with respect to vessel size (Ohyanagi et al. 1991). While little information exists on the nature of NPY in skeletal muscle arterioles, even less exists for DPPIV. However, as NPY appears to be a substantial vasoconstrictor in small calibre vessels, it would be plausible that its enzyme could be of greater importance at this level.

NPY and DPPIV are present in vascular endothelial cells where they play major roles in angiogenic mechanisms (Kitlinska et al. 2003; Zukowska et al. 2003). Truncated forms of NPY such as NPY_(3-36) are considered angiogenic as they demonstrate an affinity for pro-angiogenic NPY receptors. Previous study of vascular smooth muscle cell culture of rat aortae failed to detect DPPIV activity (Mentlein & Roos, 1996). In the skeletal muscle arterioles of 2-month-old animals, the endothelium-denuded group had less DPPIV activity; however, there was enzymatic activity present. DPPIV activity on vascular smooth muscle cells of arterioles would support a proteolytic presence at or around the sympathetic end terminal, thus mitigating the post-junctional (Y₁) effects of NPY.

NPY overflow with age

Previous works examining the effects of age on the actions of NPY have concluded with mixed results. In rat adrenal gland, NPY immunoreactivity experienced a 40-fold increase with age (Higuchi & Yang, 1986). Contrary to this increase with age, Glenn & Duckles (1994) failed to observe differences in NPY content of rat tail artery with age; however, aged animals exhibited a greater contractile response to NPY as compared to young. While a 12-month-old animal is not considered senescent, this age does represent a middle-aged animal (Turturro et al. 1999) relative to life expectancy. In the present study, the 12-month-old group did not differ from the 6-month-old group during control conditions (no DPPIV inhibition) immediately following and 30 s after field stimulation. Interestingly, there were similar NPY overflow patterns with DPPIV inhibition as there were during control conditions in 12-month-old animals. This was in contrast to the increase in, and volatile nature of, NPY overflow of the 6-month-old animals with DPPIV inhibition as compared to control patterns. It would appear, based on the present data, that NPY overflow is greater in 6-month-old animals, but that the actions of DPPIV suppress the bioavailability of NPY to levels similar to those observed in the middle-aged animals. Whether the decrease in DPPIV functionality with age elicits a compensatory decrease in NPY overflow or vice versa is a question for future studies.

Functional significance

Preliminary studies in our lab assessing NPY-mediated vasoconstriction in 6-month-old females via a Y₁-receptor agonist, Leu³¹Pro³⁴NPY, elicited an EC₅₀ of approximately 2 nmol l⁻¹ (unpublished data). While the picomolar concentration of NPY overflow assessed using the present method may not be representative of NPY concentration at the synapse, it is difficult to surmise the functional meaningfulness of such a concentration in light of the possible required concentration to elicit vasoconstriction in these vessels. This is an area that merits more attention as NPY may be a potent vasoconstrictor in skeletal muscle first-order arterioles of females.

Limitations

This study represents an initial exploration in NPY overflow and its proteolytic processing in skeletal muscle arterioles. While the assay employed for NPY detection is quite sensitive (0.04–0.06 ng ml⁻¹), the amount of NPY detected using this particular preparation is near the assay's minimum range. Deviation away from the assay's IC₅₀ decreases sensitivity, thus increasing the difficulty in detecting small differences. It is also unclear how well the assay differentiates NPY_(1-36) from the metabolized, truncated forms of NPY. We would assert that an increase in NPY detection following DPPIV inhibition would be a function of increased NPY_(1-36) as it is this particular peptide that is acted upon by the enzyme. While we believe increases in the availability of the full-length peptide to be the underlying cause behind the increase in detectable NPY with DPPIV inhibition, the NPY concentration given by the assay may include a portion of truncated fragments.

DPPIV is an oft-studied proteolytic enzyme involved in NPY metabolism; however, there are other enzymes, most notably aminopeptidase P, which are involved in NPY degradation (Mentlein & Roos, 1996). Future studies in NPY overflow of skeletal muscle arterioles should include aminopeptidase P activity to better characterize its role in NPY metabolism at this level. Lastly, the present study did not measure DPPIV protein or any of its antecedent proteins (mRNA, etc.). It cannot be concluded whether the differences observed in DPPIV activity were actually a product of protein content, if they were due to true differences in enzymatic activity, or if they were a function of both protein content and enzymatic activity. Future studies should include protein expression to elucidate the sources of these differences. Lastly, future studies should examine multiple age groups in an effort to characterize the rate of change that occurs with age for both DPPIV activity and NPY overflow in these vessels.

Conclusions

NPY overflow of first-order arterioles can be directly assessed via an isolated microvessel technique. There is evidence to support a difference in NPY overflow with respect to age in females; however, this age difference may be mitigated through the actions of proteolytic enzymes such as DPPIV. DPPIV activity is influenced by many factors (age, vessel type), which could potentially impact the type of response (vasoconstriction, angiogenic mechanisms) elicited by NPY.

Author contributions

K.W.E. was involved in some concept development, data collection and analysis, and the drafting and revision of the manuscript. A.J.S was involved in some data collection and analysis, and in revising the manuscript. A.L.H. was involved in some concept development and some data collection. H.A.K. was involved in concept development, data collection and analysis, and in revising the manuscript. All authors approved the final version of the manuscript.