INTRODUCTION

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Neuropeptide Y (NPY) is a 36 amino acid peptide colocalized with noradrenaline in many peripheral sympathetic nerve fibers. In the human nasal mucosa NPY immunoreactivity is mostly found in nerve fibers surrounding small arterial vessels and arteriovenous shunts, whereas venous vessels and glands are more sparsely innervated (1, 2). Intraarterially injected NPY and sympathetic nerve stimulation produces a long-lasting vasoconstriction resistant to adrenergic blockers in the pig nasal mucosa (3). The vasoconstrictive effect of NPY in the upper airways has also been shown using laser Doppler flowmetry (LDF) in the rabbit maxillary sinus (4). Indirect vasoactive effects by NPY have been demonstrated in humans using the nasal airway resistance (NAR), which reflects the vascular tone of venous sinusoids and arteriovenous shunts (5). The direct effect on nasal blood flow has not previously been measured in humans.

NPY can also suppress the release of transmitters from parasympathetic and sensory nerve endings in the airways (6- 9). Such prejunctional effects may explain recent findings that NPY inhibits the secretory response to vagal stimulation in the lower airways (10). In the human upper airways, NPY has been shown to reduce sneezing, itching, and secretion after allergen, capsaicin, or bradykinin challenge (5, 11, 12). These findings suggest that NPY receptor agonists may represent a novel therapeutic approach in the treatment of allergic and vasomotor rhinitis. NPY acts via multiple receptors, of which Y1 and Y2 appear to be the predominant receptor types in the peripheral nervous system (13).

Nitric oxide (NO) is produced at high concentrations in the paranasal sinuses (14). The role of NO is unclear but in vitro experiments have shown NO to be bacteriostatic (15). NO production in the nose is increased by allergen challenge (16) and by the NO donor sodium nitroprusside (17) but decreased by ₂-adrenoceptor agonists (18).

The aim of the present study was to examine dose-dependent effects of intranasal application of NPY on nasal mucosal blood flow, measured by LDF and nasal mucosal blood content measured by rhinomanometry. Mucosal biopsies were taken for investigation of Y1 and Y2 receptor messenger RNA (mRNA) expression, using the reverse transcriptase- polymerase chain reaction (RT-PCR). Furthermore the possible influence of NPY on intranasal NO production was examined.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Subjects

Nine healthy volunteers, 4 female and 5 male subjects, participated. Mean age was 26 yr (23 to 41 yr). They were evaluated by medical history and were excluded if they had any history of atopic disease, chronic sinusitis, perennial rhinitis, asthma, or previous surgery of the nose. The mucosa was normal as seen at anterior rhinoscopy. All stated that they had not suffered from respiratory infections within the past month. The study design was approved by the ethics committee of the medical faculty of the University of Lund.

Measurement of NAR

NAR was measured by computerized anterior rhinomanometry using Rhinocomp RHC-1 (Beta Data, Alingsås, Sweden). The mathemathical model of Broms was used to calculate NAR (19).

Nasal Mucosal Blood Flow

Nasal mucosal blood flow was measured using LDF (Periflux Pf2b; Perimed, Järfälla, Sweden). The probe had an angled tip of 90° and a fiber separation of 0.25 mm (PF 310S; Perimed, Järfälla, Sweden).The experiments were performed in the sitting position with the chin and forehead resting against a rigid stand. The probe was advanced into the nose under direct supervision with a 30° rigid endoscope. Local anesthesia was not used. The tip of the probe was directed toward the septal mucosa at the anterior level of the middle turbinate and positioned approximately 1 mm above the mucosa, taking care not to touch the mucosa. The gain setting was ×1, and the frequency limit set at 12 kHz. The LDF signal was continuously displayed by a penwrite recorder and also recorded by computer every 10 s.

Challenges were delivered after a stable baseline level was established during 3 to 5 min. Recordings ended 17 min after challenge.

Measurements of Nasal NO

NO was measured using a chemiluminiscence NO analyzer CLD 700 AL-med (EcoPhysics, Dürnten, Switzerland). The NO analyzer has a sampling flowrate of 0.66 L/min and a detection limit according to the manufacturer of 1 part per billion (ppb). The analyzer was calibrated using known concentrations of NO (20 and 700 ppb). All measurements were made at room temperature (21 to 24° C). Ambient NO varied between 6 and 13 ppb. The subjects were asked to breathe through the mouth and try not to close the soft palate. The tube from the NO analyzer, with an olive-shaped nasal adapter was held gently in contact with one of the nostrils. When the reading of the NO concentration had stabilized usually within 1 min, this plateau level was considered to be the nasal NO concentration. The nasal adapter was removed from the the nostril and after approximately 1 min when the NO levels had fallen to ambient air levels, the contralateral side was measured.

Administration of Test Solutions

Challenges were delivered by a commercially available nasal spray pump device. The spray bottle was weighed before and after challenges. One puff of the test solution averaged 100 µl if the pump mechanism was properly primed. Two puffs were administered without removing the LDF probe within a time period of 20 s into the nasal cavity with the highest NAR value. The subject held his or her breath during nebulization. An approximate total volume of 200 µl fluid was nebulized. A washout period of at least 48 h between experiments was allowed.

Drugs

Human NPY (Clinalfa AG, Leufelfingen, Switzerland) (50 µg) was dissolved in saline to a stock solution of 12 nmol. Further dilutions were also made in saline. Preparations of NPY were made the same day as the experiments. Before administration, the NPY solution was adjusted to 33° C (the temperature of the nasal mucosa) in a microincubator.

RT-PCR

From six healthy volunteers a mucosal biopsy was taken from the anterior edge of the inferior turbinate under local anesthesia. The tissue was immediately frozen (within 30 s) in liquid nitrogen. The samples were transported in liquid nitrogen to a freezer (80° C). In addition, the superior cervical ganglion was removed from two subjects who had died of coronary infarction (12 h postmortem).

Total cellular RNA was extracted usung the TRIzol reagent (GIBCO BRL, Paisley, Scotland), following the manufacturer's instructions. Frozen tissue was homogenized with 1 ml of TRIzol reagent until completely disrupted at room temperature, using the microprobe of a power homogenizer (Model PT 1200; Polytron Kinematica AG, Labora, Upplands-Väsby, Sweden) for 30 to 60 s. The homogenates were mixed with chloroform and centrifuged at 12,000 × g for 15 min at 4° C. The aqueous phase, containing RNA, was transferred to a fresh tube and the RNA precipitated by the addition of isopropanol. Samples were incubated at room temperature for 10 min and centrifuged at 12,000 × g for 10 min at 4° C.

The RNA pellet was finally washed with 70% ice-cold ethanol, air-dried, dissolved in 20 µl of diethylpyrocarbonate-treated water, and stored at 20° C until use. The purity and yield of total RNA were determined spectrophotometrically by measurement of the optical density of a portion at 260 nm and 280 nm using a DU-65 spectrophotometer (Beckman Instruments, Fullerton, CA) The ratio of absorption (260:280) was between 1.6 and 1.8. Finally, samples were subjected to gel electrophoresis and stained with ethidium bromide to prove the integrity of the 18 and 28 S ribosomal RNAs.

First-strand complementary DNA (cDNA) and subsequent PCR amplification were done with the GeneAmp RNA PCR kit reagents (Perkin-Elmer, Stockholm, Sweden) in a PCR DNA thermal cycler (Perkin-Elmer). DnAse-treated RNA samples were reverse-transcribed to cDNA in a 20-µl reaction volume in the presence of 1 × PCR buffer (50 mM KCl, 10 mM Tris-HCl, pH 8.3), 5 mM MgCl₂, 1 mM of each deoxyribonucleoside triphosphate (dNTP), 50 pmol of oligo dT9 primers, 50 units of Moloney murine leukemia virus (MMLV) reverse transcriptase. To determine if the the amplification product came exclusively from the RNA, a reverse transcriptase-negative reaction was run where the enzyme was replaced by ribonuclease (RNase)-free water. The samples were incubated at room temperature for 10 min, at 42° C for 15 min, heated to 99° C for 5 min, and chilled to 5° C for 5 min.

The primers for NPY receptors were as follows: hY1 receptor forward (hY1F: 5'-ATCATTCTTGGTGTCTCTG) and reverse (hY1R: 5'-TAGGCGTATATATATCTTGAAGT), generating a 563-bp product corresponding to a region in the vicinity of TM4 and TM7 of the hY1 receptor. The hY2 receptor, forward (hY2F: 5'-CTGCTCSCATCATCTTGCT-3) and reverse (hY2R: 5'-CTGGCTGTCAATGTCAAC-3'), generating a 718-bp product.

Resultant cDNA was amplified by PCR in a final volume of 100 µl following the standard PCR protocol GeneAmp RNA PCR kit and AmpliTaq DNA polymerase (Perkin Elmer, Foster City, CA) was used as the thermostable enzyme. The following amplifcation profile was used: 2 min at 95° 1 cycle, followed by 1 min at appropriate annealing temperature and 1 min at 72° C for 35 cycles, and finally 7 min at 72° C. The anealing temperatures for the different receptors were 61° C (NPY Y1) and 54° C (NPY Y2). The identity of all products was verified as described previously (20) and a blank containing water instead of reverse transcriptase was included in all experiments.

Electrophoresis analysis. From each PCR-amplified product 10 µl was electrophoresed in a 1.5% agarose gel (GIBCO) containing 0.5 g/ml ethidium bromide (Sigma E1510), in TBE buffer (89 mM Tris-Borate, 2 mM ethylenediaminetetraacetic acid [EDTA], pH 8.0) at 5 V/cm for 90 min. This analysis was performed in a 20 × 10 cm Midicell (Model EC 350; E-C Apparatus Corporation, Techtum Lab AB, Klippan, Sweden). A 100-bp DNA ladder (Promega SDS, Falkenberg, Sweden) was run in each of the outside lanes to confirm the molecular size of the amplification product.

Statistics

Blood flow changes were expressed as percentages of baseline LDF signal. Induced changes in NAR were calculated as percentages of NAR in the unchallenged nose. Results are expressed as means ± standard error of the mean. Pairwise comparisions were based on Wilcoxon matched pairs, signed rank test. The 95% confidence limits were used.

Experimental Procedures

Challenge of subjects. Rhinomanometry was performed approximately 10 min before challenge, followed by measurement of nasal NO. The subject was then seated in position for LDF measurements and the LDF probe was positioned within the nose and blood flow was registered until a stable signal was recorded for 3 min. Two nebulizations of the vehicle (NaCl 0.9%), NPY 0.12, 1.2, or 12 nmol at a total volume of 0.2 ml were introduced into the nasal cavity without removing the LDF probe. The healthy volunteers were exposed to vehicle and increasing doses of NPY, and each exposure was separated by a minimum of 2 d. Blood flow was measured continuously for at least another 17 min. After completed blood flow registration, rhinomanometry and nasal NO measurements were repeated.

Nasal biopsies. When the blood flow experiments were completed, after a 1-wk washout period, nasal biopsies were performed in six of the subjects (3 males, 3 females). Initially tetracaine 40 mg/ml was sprayed into the nostril, followed by an injection of 0.5 ml 0.5% carbocain (ASTRA Pharmaceuticals, Södertälje, Sweden) injected into the anterior edge of the inferior turbinate. A cutting forceps was used for the biopsy. The wound was cauterized and an anterior nasal packing was applied.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The term "blood flow" is used throughout to describe the signal registered by the LDF probe from the nasal septal mucosa. Intranasal application of NPY evoked a dose-dependent reduction of nasal mucosal blood flow. Maximal vasoconstriction, seen at 12 nmol was 37.5 ± 6.2%, p < 0.05 (n = 9) (Figure 1). Initially, there was a slight and transient increase in blood flow, followed by a long-lasting reduction of the mucosal blood flow. At 1.2 and 12 nmol, the vasoconstrictive effect developed within 2 to 4 min and lasted during the entire length of the registration, > 17 min, shown for 12 nmol in Figure 2). Intranasal spray of saline also induced a slight and transient increase in blood flow before returning to baseline level (Figure 2). However, three subjects did not respond in a dose-dependent manner. One subject only responded with a reduced blood flow at 12 nmol and in two subjects there was no response at 0.12 and 12 nmol, but at 1.2 nmol. Neither was there any decrease seen in NAR in the latter two subjects.

View larger version (20K): [in this window] [in a new window]   View larger version (22K): [in this window] [in a new window]   Figure 1.   The effect of NPY 0.12, 1.2, and 12 nmol on blood flow measured by LDF. The x-axis shows the log-dose in both graphs. The top graph (A) shows on the y-axis the response in percent from baseline levels, whereas in the bottom graph (B) the y-axis shows the area under the curve (%s) during a registration of 17 min. An asterisk denotes a statistically significant response, n = 9, p < 0.05. Responses are expressed as means ± SEM.

View larger version (29K): [in this window] [in a new window]   Figure 2.   Time-course curves showing the effects on human nasal blood flow of challenge with the vehicle (NaCl) and NPY 12 nmol (NPY), expressed as means ± SEM, n = 9.

For brevity, "ipsilateral" is used for the side of the challenge and "contralateral" for the unchallenged side. NPY evoked a dose-dependent reduction of NAR on the ipsilateral side (Figure 3). Maximal decrease was 24.0 ± 10.0% at 12 nmol, p < 0.05 (n = 9). There was no effect on NAR on the contralateral side. In four subjects NAR was followed for 60 min after challenge with NPY 12 nmol. The reduction in NAR remained during the entire time period. Two subjects did not respond to NPY with a reduction of NAR.

View larger version (43K): [in this window] [in a new window]   Figure 3.   The change of NAR measured on the ipsilateral side before (gray bars) and after challenge (checked bars) with the vehicle NaCl and NPY, n = 9. Asterisk denotes a statistically significant response, p < 0.05.

There was a significant decrease in nasal NO production on the ipsilateral side, but not on the contralateral side after application of NPY 12 nmol (7.4 ± 1.2%, p < 0.05, n = 8; from 242 ± 24.1 ppb to 224 ± 24.6 ppb) (Figure 4). NPY 0.12 to 1.2 nmol had no significant effect on the NO production.

View larger version (52K): [in this window] [in a new window]   Figure 4.   Nasal NO concentration on the ipsilateral side before and after challenge with the vehicle NaCl and NPY. NO was measured immediately before challenge (gray bars) and approximately 25 min after challenge (checked bars). n = 9 except at NPY 12 nmol, n = 8. Asterisk denotes a statistically significant response, p < 0.05.

Total RNA was extracted from the nasal mucosa. By using one forward and one backward primer in RT-PCR, the presence of mRNA for the human NPY Y1 receptor was shown (Figure 5). As a positive control the Y1 receptor expressed in the neuroblastoma cell line, SK-N-MC, was used. The PCR products were of the expected size (520 bp) in the nasal mucosa and in the SK-N-MC cell line, corresponding to mRNA encoding the Y1 receptor in all subjects. An additional small band was seen, probably representing a splice variant of the Y1 receptor (Figure 5). In negative controls, when reverse transcriptase enzyme was replaced by RNase-free water, no band was detected. No band containing the Y2 receptor mRNA was detected in the nasal mucosa (Figure 5). As a positive control, the Y2 receptor was seen to be expressed in the superior cervical ganglion (718 bp, data not shown).

View larger version (94K): [in this window] [in a new window]   Figure 5.   Gel electrophoresis of RT-PCR reaction products after 35 cycles of amplification of mRNA fragments corresponding to human NPY Y1 receptor transcripts in the human nasal mucosa (lane 2), but no signal was seen for NPY Y2 mRNA (lane 4). As a negative control no amplification occurred when reverse transcriptase was omitted in the first-strand cDNA reaction (lanes 1 and 3). A 100 bp DNA ladder was run to confirm the molecular size of the amplification product (lane M). As positive controls SK-N-MC (Y1) and human cervical ganglion (Y2) were examined separately (not shown).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

NPY is a well known vasoconstrictor in many vascular beds (3, 4, 21). Previous studies of vasoactive effects of NPY in the human nasal mucosa have been indirect, measuring the nasal decongestion using rhinomanometry (5, 11). The reduction of NAR reflects an effect on venous capacitance vessels (venous sinusoids) and/or an effect on the arteries or arteriovenous shunts regulating the blood flow into the sinusoids mainly of the turbinates (22).

The present study has shown that NPY evoked a dose- dependent vasoconstrictor response measured both directly by LDF and indirectly by rhinomanometry in the nasal mucosa of healthy volunteers, suggesting that NPY acts both on the venous sinusoids/arteriovenous shunts as well as on resistance vessels of the septum mucosa. The effect was powerful and potent because the maximal reduction in blood flow was approximately 37% at NPY 12 nmol. As a comparison, the maximal reductions in blood flow measured by LDF following xylometazoline 100 µg or cocaine 10 mg have in another study been shown to be 26% and 23%, respectively (23).

The vasoactive effect of NPY in the nasal mucosa was long-lasting. The decrease in blood flow by NPY lasted for the duration of the registration, i.e., > 17 min. In a subset of the subjects we observed that the effect by NPY 12 nmol as measured by NAR lasted for at least 60 min, which is in accordance with a previous study in humans (5). In addition, the vasoconstrictive effect of NPY has been shown to outlast that of noradrenaline in the pig (24).

Comparing the effects of NPY on NAR with other studies where the techniques and algorithms used for calculating NAR differs is difficult. Furthermore, the normal human nasal passages exhibit spontaneous 4- to 5-h cyclic changes in unilateral NAR (the nasal cycle) (25), which may influence the individual outcome of the NAR registration after challenges with vasoactive agents. However, all subjects in the present study served as their own control in order to reduce the possible effect of the nasal cycle. Previous studies have all shown a reduction of NAR by 57% following NPY 2.3 nmol (11), by 25% following 50 nmol, and by 32% following 100 nmol (5).

Three subjects in the present study did not respond dose-dependently to NPY challenge, possibly reflecting interindividual differences in the sensitivity to NPY. Alternatively, it may be speculated that NPY can induce histamine release from mast cells, thereby counteracting the vasoconstrictive effect of NPY, which has been shown in the rat (8, 26). Another possible explanation may be that degradation of NPY in the nasal mucosa by endopeptidase activity varies among individuals. The slight and transient increase in blood flow immediately after administration of NPY and vehicle observed in this and previous studies (5) is probably a result of the tactile stimulation of the spray droplets on the mucosa.

The lack of effect of NPY on the contralateral side on NAR suggests that the systemic absorption after intranasal application of NPY (0.12 to 12 nmol) is neglectable, which is in accordance with a previous observation that the systemic absorption of intranasally administered NPY in the dose range of 10, 50, and 100 nmol showed no effect on NPY plasma levels at 10 nmol. However, a significant increase of plasma NPY was seen at the dosages of 50 and 100 nmol (5).

NPY inhibited intranasal NO production at the highest concentration only (12 nmol). Other vasoconstrictors such as ₂-adrenoceptor agonists have been shown to reduce nasal NO production in humans (18, 27). The mechanism behind the reduction is not clear. Conceivably, the vasoconstriction per se reduces the availability of L-arginine for the synthesis of NO.

Nasal biopsies from the inferior turbinate in humans have shown both NPY-containing nerve fibers surrounding both small arteries and venous sinusoids and NPY binding sites (1). However, the type of NPY receptor has not been identified. The predominant NPY receptor type in blood vessels appears to be the Y1 type, although certain vascular beds have been shown to contain also Y2 receptors (28). Expression studies have not detected any Y4 and Y5 receptors in the vascular- endothelial systems (29, 30). The Y3 receptor has not yet been cloned and the Y6 receptor is a pseudogene in humans. NPY may also suppress parasympathetic nerve activity via prejunctional Y2 receptors and sensory C fibers activity via Y1 receptors (9, 28). In biopsies from the nasal mucosa we obtained a RT-PCR amplified band corresponding to Y1 receptor mRNA, but no expression of Y2 receptor mRNA. Thus, it is likely that the NPY-evoked vasoconstriction in the human nasal mucosa is mediated via Y1 receptors. The presence of an additional band in the present study may be a splice variant of the Y1 receptor. We have in subsequent studies sequenced this spliced variant and found that it is a short part of the Y1 segment TM1 to TM5 and hence report incomplete reading (unpublished). The number of Y2 receptors seems to be small compared with Y1 as no band corresponding to mRNA for the Y2 receptor could be detected. The finding of mRNA corresponding to the Y2 receptor in the superior cervical ganglion using the same primers confirms that the primers are functioning (4, 31).

In conclusion, NPY is a powerful and potent vasoconstrictor in the human nose reducing arterial and capillary blood flow, as well as the blood content of the mucosa. The effect is probably mediated via Y1 receptors. NPY receptor agonists may prove beneficial in the treatment of the congested nose in allergic or vasomotor rhinitis.