Release of calcitonin gene-related peptide in the pig nasal mucosa by antidromic nerve stimulation and capsaicin

Regulatory Peptides, 33 (1991) 251-262 © 1991 Elsevier Science Publishers B.V. 0167-0115/91/$03.50

251

REGPEP 01034

Release of calcitonin gene-related peptide in the pig nasal mucosa by antidromic nerve stimulation and capsaicin P. S t j ~ n e 1'2, J . S . Lacroix 2'3, A. ) ~ n g g ~ d 1"2 and J . M . L u n d b e r g 2 l Department of Oto-Rhino-Laryngology, Karolinska Hospital, Stockholm, 2Department of Pharmacology, Karolinska Institute, Stockholm (Sweden) and 3 Clinique d'Oto-Rhino-Laryngologie et de Chirurgie Cervico-Faciale, H6pital Cantonal Universitaire, Gen~ve (Switzerland) (Received 10 October 1990; revised version received and accepted 28 January 1991)

Key words: Nasal mucosa; Antidromic nerve stimulation; Capsaicin; C G R P release

Summary The overflow of calcitonin gene-related peptide like-immunoreactivity (CGRP-LI) in the nasal venous effluent upon antidromic stimulation of the maxillary division of the trigeminal nerve with 6.9 Hz for 3 min or upon capsaicin (0.3 #mol bolus injection) were analysed in the nasal mucosa of sympathectomized pentobarbital anaesthetized pigs. The overflow of CGRP-LI upon antidromic stimulation displayed a slower appearance in the venous effluent than the overflow upon bolus injection of capsaicin. The vascular effects as revealed by the arterial blood flow, the venous blood flow, the blood volume of the nasal mucosa, i.e., the filling of the capacitance vessels and the superficial mucosal blood flow as revealed by the laser-Doppler signal were also studied. Antidromic stimulation of the trigeminal nerve as well as capsaicin bolus injection induced a marked vasodilation which was parallel to the overflow of CGRP. However, capsaicin bolus injection also resulted in a marked increase in the mean arterial blood pressure which may be due to reflex activation of sympathetic fibers. In conclusion, we have demonstrated that chemical stimulation with capsaicin as well as antidromic stimulation of nasal sensory nerves in sympathectomized animals induces both vasodilation and overflow of CGRP-LI in vivo. This indicates that C G R P may contribute to the sensory regulation of the microcirculation in the nasal mucosa.

Correspondence." P. Stj~irne, Department of Oto-Rhino-Laryngology, Karolinska Hospital, S-10401 Stockholm, Sweden.

252

Introduction

Calcitonin gene-related peptide (CGRP) is a 37 amino acid neuropeptide [ 1,2], which has been shown to co-exist with substance P (SP) in the capsaicin sensitive sensory neurons in the nasal mucosa [3], as well as in other parts of the respiratory system [4,5]. Capsaicin, the pungent agent in hot pepper, has been demonstrated to release SP [6] as well as other tachykinins [7] and CGRP [8] from sensory nerves in vitro and in the eye in vivo [9]. However, CGRP rather than SP or other tachykinins mimics the capsaicin induced vasodilation, at least in the cat nasal and pig coronary vessels [ 3,10]. Furthermore, the capsaicin vasodilation seems to be endothelium independent [ 11 ] in contrast to the endothelium dependent relaxation by SP of the vascular smooth muscle [ 12]. The capsaicin sensitive unmyelinated afferent fibers transmit information to the CNS. However, through an axon-reflex arrangement these fibers may also play a significant efferent role in blood flow control of certain regions, like the skin [ 13]. Thus, release of neuropeptides from peripheral sensory C-fibre nerve endings induces the so called 'neurogenic inflammation', i.e., local vasodilation and protein extravasation [ 14,15 ]. We know from earlier studies using antidromic stimulation of a transected sensory nerve as a model for the axon-reflex that vasodilation as well as extravasation of macromolecules may be induced in the respiratory tract [16,17]. The difficulty in evaluating the responses is that most sensory nerves, like the vagus nerve, are mixed containing also parasympathetic fibers. In the nasal mucosa parasympathetic (Vidian nerve) and sensory (trigeminal nerve) inputs follow at least partly separate anatomical pathways [ 18]. However, we have earlier shown that the maxillary division of the trigeminal nerve in the pig contains an abundance of tyrosine hydroxylase positive nerves, presumably of sympathetic origin [3], which suggests that the vasodilatory response to electrical stimulation of the trigeminal nerve under control conditions may be restricted by a concomitant sympathetic activation. The aim of the present study was to investigate the possible release of CGRP-LI from the pig nasal mucosa in vivo upon antidromic stimulation of the maxillary portion of the trigeminal nerve as well as upon bolus injection of capsaicin in relation to the vascular effects induced by these stimuli.

Materials and Methods

Six pigs (17-30 kg body weight), which 14 days prior to the experiment had undergone unilateral removal of the left superior cervical sympathetic ganglion, to ensure degeneration of sympathetic fibers running in the trigeminal nerve [3,19], were fasted overnight and premedicated i.m. with ketamine (Ketalar, Parke Davies, U.S.A., 20 mg kg- 1) and atropine (ACO, Stockholm, Sweden, 0.05 mg kg- 1). After tracheotomy the pigs were intubated and artificially ventilated by a volume regulated respirator (EngstrOm, model 150). Anesthesia was maintained by continuous i.v. infusion of pentobarbital (3 mg kg- 1). Body temperature was maintained between 37-38 ° C using a heating lamp. For muscle relaxation, pancuronium bromide (Pavulon, Organon,

253 Netherlands, 0.25 mg k g - 1) was given intermittently. A catheter was introduced into a femoral vein for the administration of a Ringer solution (3 ml k g - l h - 1) and heparin (750 i.u. k g - 1). On the same side the femoral artery was cannulated for continuous recording o f systemic arterial blood pressure. All experiments and recordings were carried out unilaterally. Blood flow in the sphenopalatine (Fig. 1) was recorded using a BL-Pulsed-Logic Flow Meter (Biotronics Laboratory Inc., Silver Spring, U.S.A.) with a flow probe (diameter 1.5-2 mm) around the external carotid artery in the portion proximal to the superficial temporal artery. All branches of the external carotid artery distal to the flow probe, apart from the sphenopalatine and superficial temporal arteries, were ligated and cut. Blood flow increase was expressed as percentage of the basal flow, estimated by clamping the internal maxillary artery downstream to the flowprobe (100~o) [20]. The nasal vascular resistance was calculated by dividing the mean systemic arterial blood pressure by the nasal arterial blood flow. The superficial temporal artery, situated downstream from the flow meter probe, was cannulated with a polyethylene (PE 90) catheter and used for bolus injections. The facial and dorsal veins were ligated and cut, while the sphenopalatine vein was cannulated using a polyethylene (PE 50) catheter and the blood flow in the catheter was measured using a photo-cell drop counter (Fig. 1). The blood was continuously returned to the brachial vein, using a roller pump (Gilson, Minipuls II, L a m b d a AB, Stockholm, Sweden). At the end of the experiment a solution o f NaC1 containing Evans blue was

V nousb 7£ArtelooOow Anti~romic Stimulation

V. b.v.

Nasal volume

Superficial mucosal blood flow

Fig. 1. Schematic representation of the experimental set up. The sphenopalatine vein (v.) was cannulated and the blood flow measured using a photo-cell drop counter. Venous samples for RIA analysis were taken under the photo-cell. The blood was returned into the brachial vein (b.v.) using a roller pump. The blood flow in the sphenopalatine artery (a.) was recorded using a BL-Pulsed-Logicflow meter. The nasal volume was recorded by introducing a thin-walled balloon into the nasal cavity and connected to a reservoir. The system was filled with water and the reservoir was attached to a force displacement transducer. Finally, the laser-Doppler probe was introduced into the nasal cavity and positioned 1-2 mm from the nasal mucosa. A bipolar platinum electrode was carefully placed around the trigeminal nerve for the nerve stimulations. bolus injections of capsaicin were performed into the sphenopalatine artery.

254 injected retrogradely in the sphenopalatine vein and antegradely in the sphenopalatine artery and blue staining was initially observed only in the nasal mucosa. A thin-walled balloon was tied onto a polyethylene catheter (diameter 0.2 cm) and placed into the ipsilateral nostril. The system was filled with 5 ml of water and connected to a Grass FTO3C force displacement transducer. At steady state the volume of the transducer reservoir, placed at the same level as the nostril, was 5-8 ml. Calibration was performed by removing 1 ml from the reservoir. Variations in blood content of the nasal mucosa, which mainly reflects changes in the capacitance vessel function, were observed as changes in the volume of the balloon and, subsequently, the weight of the reservoir (Fig. 1) [20,21]. A laser-Doppler probe (diameter 1 ram, open) was introduced into the nasal cavity on the ipsilateral side between the balloon and the mucosa. The probe was positioned 1-2 mm from the nasal mucosa. It was fixated to the operating table and no headbar was used. The monochromatic and coherent laser light from the P E R I F L U X PF2 (Perimed KB, Stockholm, Sweden), with a known frequency (632.8 nm, He-Ne, 2 mW), undergoes a Doppler shift by the moving red blood cells and the backscattered changed frequencies are computed to a single number. This number is a product of the red cell body volume and their mean velocity and is expressed as a percentage of the maximal internal calibration signal (100 ~o). The signal probably mainly reflects superficial blood flow [22] and was not influenced by the filling of the balloon in the nasal cavity (Fig. 1). All modalities were continuously recorded using a Grass polygraph 7D. After the dissection a minimum of 1 h elapsed before the initiation of the experiments. Deep in the pterygopalatine fossa, the maxillary division of the trigeminal nerve was identified and transected and prepared for antidromic electrical stimulation (Fig. 1). The distal end was carefully placed on to a bipolar platinum electrode, connected to a Grass $88 stimulator and isolated with plastibase (Squibb, Moreton, U.K.). Nerve stimulations (15 V, 5 ms) at 6.9 Hz continuous frequency was performed for 3 min [20]. The catheter in the superficial temporal artery was used for bolus injections of capsaicin (Sigma, St. Louis, MO, U.S.A.) (0.3 #mol), given in a volume of 100/~1 followed by 0.5 ml NaC1. A systemic arterial blood sample was taken from the femoral artery, as well as a venous blood sample from the sphenopalatine vein immediately before stimulation of the trigeminal nerve and capsaicin bolus injection. Venous blood samples were thereafter taken 30 s, 3, 5, 8 and 15 min after the stimulation/injection and collected in test tubes containing EDTA (final concentration 10 mM). The samples were immediately submerged in ice water and at the end of the experiment, after centrifugation (1200 g, + 4 °C, 10 rain), the plasma was stored at - 70 °C. The plasma content of C G R P like immunoreactivity (CGRP-LI) in 0.5 ml plasma was determined by radioimmunoassay after acid ethanol extraction, using an antiserum raised against human CGRP-~ (Peninsula, CA, U.S.A.) [23]. The detection limit of the assay for CGRP-LI in 0.5 ml plasma sample was 3.9 pmol/1. The recovery for human CGRP-c~ (200 pM) added to plasma after the ethanol extraction procedure was 65 _+ 2~o. The CGRP-LI overflow has been expressed as the total integrated increase in the veno-arterial gradient concentration multiplied by the plasma flow at the time that each blood sample was collected. This value is given in fmol/min.

255 For characterization of plasma CGRP-LI, plasma from the nasal venous effluent was collected upon capsaicin administration. Plasma samples (5 ml) were desalted, using SEP-PAK C18 cartridges [24], lyophilized and redissolved in 0.3 ml of distilled watei" and centrifuged through a Millipore Ultrafree-MC filter. The samples (200 #1) were than injected onto a reverse-phase column (Super Pac Cartridge 4.0 x 250 mm, 5 #m) (Pharmacia, Sweden) through a Rheodyne 7125 injector. The mobile phase consisted of a 40 rain linear gradient of 20 to 60 ~o acetonitrile in 0.1 ~o trifluoroacetic acid (TFA) and was delivered at a flowrate of 1.0 ml min - i using a 2249 LKB Gradient Pump. The mobile phase was continuously degassed with helium. 0.5 ml fractions of the eluate were collected using a HeliRac fraction collector (Pharmacia, Sweden) evaporated in a Speedvac Concentrator (Savant Instruments Inc., U.K.) and stored at - 2 0 °C until being analysed with RIA using antiserum RAS 6009. For calibration separate runs were performed with synthetic human C G R P - e (100 fmol). The elution position of the standard was also determined by RIA. At the end of the experiments pieces of the nasal mucosa of both sides were taken at three different levels of the nasal cavity (anterior part of the inferior turbinate, septum and the posterior part of the inferior turbinate) and immediately frozen on dry ice. The tissue content of CGRP-LI was analysed by RIA as above and noradrenaline (NA) by high-performance liquid chromatography with electrochemical detection [25]. Experimentalprocedures. The experimental animals were carefully monitored during the pentobarbital anaesthesia. When the animals were considered to be in optimal condition regarding blood pressure as well as arterial blood gas analysis, antidromic trigeminal stimulation or capsaicin intraarterial injection was performed. The experiment was initiated with the trigeminal stimulation and the different parameters studied were observed until they returned to baseline values after which an additional 20 min was added before the capsaicin injection. The effects were calculated against the baseline value of the parameter studied just prior to the stimulation/injection. Data are given as mean + S.E.M., and statistical differences have been estimated using Student's t-test.

Results

Nasal CGRP-LI and NA content The relative content of CGRP-LI and Na was similar in the three regions of the nasal mucosa of six control pigs (Table I). 2 weeks after sympathectomy, the CGRP-LI levels were unchanged while the NA content was reduced by 99~o on the sympathectomized side (Table I). CGRP-LI oveqTow from the nasal mucosa Basal values of CGRP-LI in arterial and venous plasma were 4.5 + 0.3 and 5.9 + 0.7 fmol/min, respectively. Antidromic stimulation of the maxillary division of the trigeminal nerve with 6.9 Hz induced a clear cut increase of CGRP-LI in the venous effluent from the nasal mucosa to 70.0 + 12.2 fmol/min. Thus, CGRP-LI appeared in

256 TABLE I Pig nasal mucosa content of NA (nmol g ~) and CGRP-LI (pmol g ~) Tissue content of NA (nmol/g) and CGRP-LI (pmol/g) at three different levels of the pig nasal mucosa on the control side and on the operated side two weeks after removal of the superior cervical sympathetic ganglion. Data are given as means _+ S.E.M. (n = 6), *** P < 0.001, Student's t-test. Level

Ant Sept Post

Control side

After sympathectomy

NA

CGRP-LI

NA

CGRP-LI

16.1 _+ 1.4 15.0 + 1.7 19.2 + 3.0

9.6 _+0.9 9.2 + 1.3 7.7 _+0.6

0.12 + 0.03*** 0.08 + 0.02*** 0.06 + 0.02***

10.9 + 1.7 9.0 + 1.0 7.6 + 0.6

the venous effluent during the 3 min stimulation and p l a s m a C G R P - L I did not return to b a s a l levels until 15 min after the initiation o f the electrical stimulus (Fig. 2). The highest values o f overflow were registered at the very end o f the stimulation period. The total a m o u n t o f C G R P - L I in the nasal venous effluent recovered under a 15 min period was 378 fmol. This m e a n s that 0.3 fmol (378/1242) o f C G R P - L I overflow was detected per given impulse. During the stimulation there was also an intense reddening, i.e., flare reaction with a very long duration in the skin on the ipsilateral side o f the muzzle. Intraarterial bolus injection of capsaicin 0.3 # m o l also induced a m a r k e d increase in the overflow o f C G R P - L I from the nasal m u c o s a with a p e a k value in the venous effluent o f 75.3 + 8.0 fmol/min (Fig. 2). However, in c o n t r a s t to the electrical stimulation, the bolus injection o f capsaicin i n d u c e d a transient overflow with a high initial peak at 30 s, after which there was a progressive decrease to the 15 min sample (Fig. 2). The total calculated overflow by capsaicin was 295 fmol during the 15 min observation period. CGRP-LI (fmol/min) I00" -----o--- Trig Stim 6.9Hz for 3 min ~

--

Caps 0.3 gmol bolus inj.

5O

01 I 0

.

~ 300

sec 600

900

Fig. 2. Overflow of CGRP-LI upon antidromic stimulation of the trigeminal nerve with 6.9 Hz for 180 s (open squares) or bolus injection of capsaicin with 0.3 #mol (closed squares). Data are given as means + S.E.M. (n = 6).

257

Reserved-phase HPLC characterization of plasma CGRP-LI revealed a heterogeneous pattern with the main peak in the position of the synthetic human CGRP-ct (Fig. 3). Vascular responses in the nasal mucosa

Electrical stimulation of the maxillary division of the trigeminal nerve induced a small decrease in the mean systemic arterial blood pressure ( - 12.6 + 1.9 mmHg), but there was an approx. 40 °/o, long lasting increase in the nasal arterial blood flow (Fig. 4). The nasal vascular resistance decreased from 73.3 + 4.5 to 45.8 + 3.6 units. P < 0.01. There was also an increase of the venous blood flow (Fig. 4) and an increase in the volume of the nasal mucosa (Fig. 4). However, the changes in the laser-Doppler signal (Fig. 4) were positive in some of the experiments and negative in others, resulting in no consistent response. In contrast, when capsaicin was injected into the superficial temporal artery there was an increase (by 50.3 + 3.4 mmHg) in the mean systemic arterial blood pressure. HowCGRP standard -60

12 ¸

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Fraction number

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Fraction number Fig. 3. Reversed-phase HPLC characterization of CGRP-LI in the venous effluent from the nasal mucosa. The elution position for standard human CGRP
258 zx v (ml)

A ldf (%) 10 5 0

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-5 A vbf (ml/min)

A abf (%) 1001

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[] Trig Stim 6.9 Hz for 3 min

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Fig. 4. Summary of changes in the arterial blood flow (abf), venous blood flow (vbf), ml/min; nasal volume (v), ml; laser-Doppler signal (ldf), ~o ; upon stimulation of the trigeminal nerve with 6.9 Hz for 180 s (hatched bars) as well as upon local injection with capsaicin 0.3/~mol (filled bars). Data are given as means + S.E.M. (n = 6), ** P < 0.01, * P < 0.05, Student's t-test.

ever, there was also a clear cut increase in the nasal arterial blood flow (Fig. 4) and a decrease in nasal vascular resistance from 72.8 + 4.6 to 52.2 _ 3.7. P < 0.01. Furthermore, there was a substantial increase in the venous outflow and laser-Doppler signal upon capsaicin injection (Fig. 4) as well as a moderate increase in the mucosal volume (Fig. 4). The duration of the vascular effects upon trigeminal stimulation and capsaicin injection were similar in all parameters studied (Fig. 5). However, upon trigeminal stimulation the nasal volume response had a significantly longer duration than the sec

[] Trig Stim 6.9 Hz for 3 min

1000

•

Caps0.3 gmol bolus inj.

v

abf

vbf

ldf

v

Fig. 5. Duration of changes in arterial blood flow (abf), venous blood flow (vbf), laser-Doppler signal (ldf) and nasal volume (v). Data are given as means + S.E.M. (n = 6), *** P <0.001, Student's t-test.

259 capsalcin induced response. Furthermore, the arterial as well as the venous blood flow responses were significantly more long-lasting than the duration of the nasal volume and the laser-Doppler signal responses to both the trigeminal stimulation and the capsaicin injection (Fig. 5).

Discussion In this study we have shown that CGRP-LI is released into the pig nasal venous effluent upon antidromic stimulation of the maxillary division of the trigeminal nerve and upon local intraarterial bolus injection of capsaicin, in vivo. Furthermore, we have established that antidromic stimulation of the trigeminal nerve as well as bolus injections of capsalcin induces vasodilation concomitant to the overflow of CGRP-LI. It has earlier been demonstrated that antidromic stimulation of sensory nerves to the dental pulp [26], the skin [27] as well as of the nasal mucosa [ 19] induces a vasodilation that is atropine as well as hexamethonium resistant. Furthermore, we know that there is a depletion of the SP content in the dental pulp, suggesting release, upon prolonged stimulation of the inferior alveolar nerve [28]. It has also been shown in vitro that capsaicin releases SP-LI [6] and other tachykinins [7] as well as CGRP-LI [8,29]. The present data indicate that C G R P at least in part may be responsible for the vascular events of antidromic nerve stimulation and acute capsaicin administration, in vivo. This is also in accordance with the data that C G R P rather than SP mimics the vasodilatory effects of capsalcin in vitro [ 10] and in vivo, at least in cat nasal mucosa [3]. The H P L C characterization of plasma CGRP-LI indicated that the main peak was similar to human CGRP-~, although the immunoreactivity was heterogeneous. It is known that porcine C G R P is partly different from human CGRP-~ (in 6 out of 37 amino acids) [30]. However, in the present H P L C chromatogram, the main peak of porcine plasma CGRP-LI eluted in the same position as the human CGRP-~ although the gradient system used may not separate these different forms. Furthermore, the second largest peak which eluted sightly ahead of the C G R P standard may represent an oxidized form since porcine C G R P contains methionine in contrast to human CGRP-~ [30]. The overflow of CGRP-LI upon bolus injection of capsaicin reached the peak value at the 30 s sample after which there was an exponential decrease, while the overflow upon trigeminal stimulation reached the peak value at the 3 min sample. This may indicate that capsaicin induced release of C G R P is mediated via another mechanism than the release initiated by the electrically evoked nerve impulse propagation. This interpretation of the data is in accordance with the earlier findings that capsaicin induced local effects early undergo tachyphylaxis [7,31 ]. Furthermore, the concomitant sympathetic activation upon capsaicin injection may also influence peptide overflow from sensory nerves [32]. The tissue content of CGRP-LI in the nasal mucosa of the pig was between 8-10 pmol/g (Table I). Using careful surgical techniques we have removed the nasal mucosa from one nostril and estimated the weight to approx. 10 g. Stimulation with 6.9 Hz for 3 min resulted in a total overflow of 380 fmol CGRP-LI, which would be

260

approx. 4/°ooof the total tissue content in the nasal mucosa in one nostril (100 pmol). This implies a recovery of released C G R P in the nasal venous effluent per impulse (fractional release), that has escaped local degradation, of 0.3 • 10 5 of the total tissue content, which is in the same range as findings of release of other nasal neurotransmitters [33]. Trigeminal stimulation induced a fall in systemic mean arterial blood pressure and an increase in nasal arterial blood flow paralleled by a decrease in the nasal vascular resistance. In contrast, capsaicin injection induced an increase in the systemic mean arterial blood pressure, in accordance with earlier findings [32] which may contribute to the local increase in nasal arterial blood flow. It has been suggested that the blood pressure increase upon local nasal capsaicin application might be due to a sympathetic reflex since local anaesthesia inhibits the cardiovascular effects of topically applied capsaicin on to the guinea-pig nasal mucosa [33]. However, in the present experimental situation, where the pigs had been subjected to sympathectomy 14 days prior to the experiment, with a 99~o depletion of NA but normal tissue levels of CGRP, the nasal mucosa is supersensitive to the vasoconstrictor action of catecholamines [35]. An increase in circulating catecholamines induced by the capsaicin bolus injection would counteract local vasodilatory actions in the nasal mucosa and possibly also the release of peptides from sensory nerves [35]. It is striking that the laser-Doppler signal was not consistent upon the antidromic trigeminal stimulation in spite of clear cut increase in local arterial blood flow. Opposite changes in local arterial blood flow and the laser-Doppler recording are also obtained upon sympathetic stimulation with low frequencies [36]. However, capsaicin given in the sphenopalatine artery increased the laser-Doppler signal. Possibly the antagonistic changes in nasal superficial blood flow observed upon antidromic nerve stimulation and capsaicin may be related to the difference in distribution of the stimuli. Whereas the i.a. capsaicin was distributed to the nasal mucosa only, stimulation of the maxillary nerve activated many other structures, including the skin, which may change the nasal vascular reactions [37]. Although it is generally assumed that the laser-Doppler signal reflects superficial blood flow, it seems clear that further analysis of, e.g., shunt flow from superficial to deeper layers may be necessary to understand the basis for the findings in the pig nasal vascular bed. Venous sinusoids in the pig nasal mucosa are also surrounded by perivascular C G R P / S P containing nerves of presumably sensory origin [3]. The present data of increased volume of the nasal mucosa upon antidromic trigeminal nerve stimulation is in accordance with such an anatomical arrangement. It cannot be ruled out, however, that at least part of this increase of nasal volume is secondary to the concomitant changes in arterial blood flow although a direct coupling between nasal volume and arterial blood flow is not obligatory [36]. In conclusion, we have demonstrated overflow of CGRP-LI into the venous effluent upon stimulation of the maxillary division of the trigeminal nerve and upon bolus injection of capsaicin into the sphenopalatine artery in vivo.

261

Acknowledgements T h e p r e s e n t study was s u p p o r t e d by g r a n t s from the S w e d i s h M e d i c a l R e s e a r c h C o u n c i l (17x-5438), the Suiss N a t i o n a l F u n d for Scientific R e s e a r c h (32.25205.88), the A m e r i c a n C o u n c i l for T o b a c c o R e s e a r c h , the S w e d i s h T o b a c c o C o m p a n y , the S w e d i s h Work and Environmental Fund. The Swedish Environmental Protection Board, S 0 d e r b e r g s Stiftelse, W i b e r g s Stiftelse, A B D r a c o a n d f u n d s from the K a r o l i n s k a Institute. F o r expert technical a s s i s t a n c e we are grateful to M i s s A n e t t e H e m s 6 n , M i s s M a r g a r e t a S t e n s d o t t e r a n d M i s s C a r i n a SOderblom.

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