Lung (1992) 170:85-93
©
SpriLg~ N e w York Inc, 1992
Calcitonin Gene-Related Peptide Potentiates Substance P-Induced Plasma Extravasation in the Rat Trachea James J. Brokaw and Gary W. White Department of Anatomy, Indiana University School of Medicine, 1800 Lincoln Avenue, Evansville, IN 47722, USA
Abstract. Antidromic stimulation of vagal sensory nerves is known to produce plasma extravasation in the rat trachea. This neurogenic inflammation is thought to be mediated by substance P or other tachykinins released from sensory nerve endings. We sought to determine whether calcitonin generelated peptide (CGRP), which is also released from sensory nerve endings, can potentiate substance P-induced plasma extravasation in the rat trachea. To accomplish this, we measured the amounts of Evans blue dye extravasated into the trachea after intravenous injections of substance P alone and combined with CGRP. We found that when substance P and CGRP were injected together, the amount of plasma extravasation produced in the trachea was substantially greater than the amount produced when substance P was injected alone. This potentiation was critically dependent on the dosage of CGRP and was not observed when relatively high dosages were used. We also found that CGRP had a potent hypotensive effect and speculate that reduced blood pressure may account for the lack of potentiation observed at the higher CGRP dosages. Based on these findings, we conclude that CGRP can potentiate substance P-induced plasma extravasation in the rat trachea and may therefore play a role in modulating neurogenic inflammation of the airways. Key words: Neurogenic inflammation--Plasma extravasation--Substance P--Calcitonin gene-related peptide. Introduction Antidromic stimulation of vagal sensory nerves causes extravasation of plasma proteins into the tracheal mucosa of the rat [ 13] and guinea pig [ 1! ]. This increase Offprint requests to: J.J. Brokaw
86
J.J. Brokaw and G.W. White
in vascular permeability is known as neurogenic inflammation [9] and is thought to be mediated by tachykinins, such as substance P, released from the peripheral terminals of sensory axons [ 16]. Although tachykinins are considered the principal mediators of neurogenic inflammation, other sensory neuropeptides may contribute to the response. Recent attention has focused on calcitonin generelated peptide (CGRP), which is stored with tachykinins in airway sensory nerves and is likewise released during antidromic stimulation [15]. Unlike tachykinins, however, CGRP does not produce plasma extravasation in the airways [12]. Nevertheless, because CGRP is a potent vasodilator [3], it might potentiate the effects of tachykinins on plasma extravasation. In support of this notion, some studies have shown that when substance P and CGRP are injected together intradermally, the amount of plasma extravasation produced in rat skin is greater than the amount produced when substance P is injected alone [2, 8]. This potentiation has yet to be demonstrated in the airways. The aim of the present study was therefore to determine whether CGRP can potentiate substance P-induced plasma extravasation in the rat trachea. To accomplish this, we measured the amounts of Evans blue dye extravasated into the trachea after intravenous injections of substance P alone and combined with CGRP. To help explain our findings, we also measured the effects of these neuropeptides on arterial blood pressure.
Methods
Animals The experiments were performed on female Long-Evans rats (190-370 g) purchased from Charles River Breeding Laboratories (Wilmington, MA). The protocol was approved by the Animal Care and Use Committee of the Indiana University School of Medicine.
Drugs and Chemicals The following drugs and chemicals were used: sodium methohexital (Eli Lilly and Company, Indianapolis, IN); sodium heparin (Organon, Inc., W. Orange, N J); Evans blue dye (Sigma Chemical Company, St. Louis, MO); substance P (Sigma); rat CGRP (Sigma); and formamide (Sigma). Evans blue dye was dissolved at a concentration of 30 mg/ml in normal saline (Abbott Laboratories, North Chicago, IL). Substance P and CGRP were each dissolved at a Concentration of 50/xg/ml in normal saline containing 0.1 N acetic acid and stored at - 80°C until use. Subsequent dilutions were made in normal saline.
Effects of Neuropeptides on Plasma Extravasation The extravasation of Evans blue dye, which binds to serum albumin [10], was used to assess plasma extravasation in the trachea [5, 19]. This method has been shown to correlate well with the extravasation of radiolabeled albumin [18]. Rats were anesthetized with intraperitoneal sodium methohexital (75 mg/kg) and given an injection of Evans blue dye through the external jugular vein (30 mg/kg). The dye injection was followed by an intravenous injection of substance P (0.25-3 nmol/
Neurogenic Inflammation of the Airways
87
kg), CGRP (0.25-2 nmol/kg), or both neuropeptides together (a single dose of substance P [0.5 nmol/kg] coinjected with different dosages of CGRP [0.25-2 nmol/kg] or a single dose of CGRP [0.5 nmol/kg] coinjected with different dosages of substance P [0.25-3 nmol/kg]). All rats received the same injection volume (2 ml/kg), regardless of whether the neuropeptides were administered alone or in combination. Control rats were given only the diluent (2 ml/kg). Five minutes later, the dye was flushed out of the circulation by the perfusion of 100 ml of 0.05 M citrate buffer (pH 3.5) containing 1% paraformaldehyde through the aorta. After being removed and weighed, each trachea was incubated overnight in 2 ml of formamide at 60°C to extract the dye. The concentration of dye in the extracts was determined spectrophotometrically by measuring the absorbance at 620 nm (Perkin-Elmer Model 35 spectrophotometer; Oak Brook, IL). Calculations were based on known concentrations of dye in formamide. Results are expressed in nanograms of Evans blue dye per milligram of tracheal wet weight.
Effects of Neuropeptides on Blood Pressure Arterial blood pressure was measured in a separate group of rats to assess the vasodilatory potencies of substance P and CGRP. The right common carotid artery of anesthetized rats was exposed and a PE-20 catheter was inserted into the lumen. A small volume of heparinized saline (10 U/ml) was flushed through the catheter before it was connected to a Statham pressure transducer (Model P23DC; Hato Rey, PR). Mean blood pressure (ram Hg) was recorded on a Grass polygraph (Model 79D; Quincy, MA). After a baseline trace was established, the rats were given an intravenous injection of substance P (0.25-2 nmol/kg), CGRP (0.25-2 nmol/kg), or both together (a single dose of substance P [0.5 nmol/kg] coinjected with different dosages of CGRP [0.25-2 nmol/kg]), All rats received the same injection volume (2 ml/kg), regardless of whether the neuropeptides were administered alone or in combination. Control rats were given only the diluent (2 ml/kg). No rats were given more than 1 dose of any substance. Tracings were recorded for 5 min and maximal changes in blood pressure noted. Changes in blood pressure are expressed as a percentage of the corresponding baseline values.
Analysis of Data All values are presented as the mean -+ SEM. Differences between group means were statistically evaluated with the unpaired t-test or 1-way analysis of variance and Dunnett's test [24]. Differences were considered significant when p < 0.05.
Results
Effects of Neuropeptides on Plasma Extravasation W h e n a single d o s e o f C G R P w a s c o i n j e c t e d w i t h d i f f e r e n t d o s a g e s o f s u b s t a n c e P, t h e a m o u n t s o f E v a n s b l u e d y e e x t r a v a s a t e d into t h e t r a c h e a w e r e g r e a t e r t h a n w h e n s u b s t a n c e P w a s i n j e c t e d a l o n g (Fig. 1). A t a s u b s t a n c e P d o s a g e o f 3 n m o l / k g , f o r e x a m p l e , t h e a m o u n t o f d y e e x t r a v a s a t i o n p r o d u c e d in c o m b i n a t i o n w i t h C G R P (196.7 -+ 16.8 ng d y e / m g t is s u e , m e a n - S E M ; n = 6) w a s 39% g r e a t e r (p < 0.01) t h a n t h e a m o u n t p r o d u c e d b y s u b s t a n c e P a l o n e (141.6 --- 7.8 ng d y e / m g t i s s u e ; n = 6). H o w e v e r , at s u b s t a n c e P d o s a g e s less t h a n 2 n m o l / kg, t h e a m o u n t s o f d y e e x t r a v a s a t i o n p r o d u c e d in c o m b i n a t i o n w i t h C G R P w e r e n o t s i g n i fi can t l y d i f f e r e n t f r o m t h o s e p r o d u c e d b y s u b s t a n c e P a l o n e .
88
J.J. Brokaw and G.W. White 250" • ---0--
SP + CGRP (0,5 nmol/kg)
Fig. 1. Extravasation of Evans blue dye produced in the rat trachea by substance P (SP) alone and combined with calcitonin gene-related peptide (CGRP). Rats were given an intravenous injection of dye and a single dose of CGRP combined with different dosages of SP. Other rats received SP without CGRP. Five minutes later, fixative was perfused through the aorta and the tracheas were removed and assayed for dye content. Each point represents the mean -+ SEM of 6 rats. **p < 0.01 compared to response produced without CGRP (unpaired t-test).
SP alone
200"
150'
8
100'
,.-., 50
I,-
1 SP
2 Dose
3
(nmol/kg)
140 ~
•
CGRP + SP (0.5 nmol/kg)
120 "
100-
"0
.
8 e'~
401
~ p-
20" O
CGRP
,
i
1
2
Dose
(nmo|/kg)
Fig. 2. Extravasation of Evans blue dye produced in the rat trachea by calcitonin gene-related peptide (CGRP) alone and combined with substance P (SP). Rats were given an intravenous injection of dye and a single dose of SP combined with different dosages of CGRP. Other rats received CGRP without SP. Five minutes later, fixative was perfused through the aorta and the tracheas were removed and assayed for dye content. Each point represents the mean -+ SEM of 6 rats. *p < 0.05, **p < 0.01 compared to response produced without CGRP (analysis of variance).
When a single dose of substance P was coinjected with different dosages of CGRP, the amounts of Evans blue dye extravasated into the trachea were greater than when substance P was injected alone (Fig. 2). At a CGRP dosage of 0.5 nmol/kg, for example, the amount of dye extravasation produced in combination with substance P (114.8 -+ 13.7 ng dye/mg tissue; n = 6) was approximately 3 times greater (p < 0.01) than the amount produced by substance P alone (39.0 -+ 4.2 ng dye/mg tissue; n = 6). However, at higher CGRP dosages,
Neurogenic Inflammation of the Airways
89
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©
SpriLg~ N e w York Inc, 1992
Calcitonin Gene-Related Peptide Potentiates Substance P-Induced Plasma Extravasation in the Rat Trachea James J. Brokaw and Gary W. White Department of Anatomy, Indiana University School of Medicine, 1800 Lincoln Avenue, Evansville, IN 47722, USA
Abstract. Antidromic stimulation of vagal sensory nerves is known to produce plasma extravasation in the rat trachea. This neurogenic inflammation is thought to be mediated by substance P or other tachykinins released from sensory nerve endings. We sought to determine whether calcitonin generelated peptide (CGRP), which is also released from sensory nerve endings, can potentiate substance P-induced plasma extravasation in the rat trachea. To accomplish this, we measured the amounts of Evans blue dye extravasated into the trachea after intravenous injections of substance P alone and combined with CGRP. We found that when substance P and CGRP were injected together, the amount of plasma extravasation produced in the trachea was substantially greater than the amount produced when substance P was injected alone. This potentiation was critically dependent on the dosage of CGRP and was not observed when relatively high dosages were used. We also found that CGRP had a potent hypotensive effect and speculate that reduced blood pressure may account for the lack of potentiation observed at the higher CGRP dosages. Based on these findings, we conclude that CGRP can potentiate substance P-induced plasma extravasation in the rat trachea and may therefore play a role in modulating neurogenic inflammation of the airways. Key words: Neurogenic inflammation--Plasma extravasation--Substance P--Calcitonin gene-related peptide. Introduction Antidromic stimulation of vagal sensory nerves causes extravasation of plasma proteins into the tracheal mucosa of the rat [ 13] and guinea pig [ 1! ]. This increase Offprint requests to: J.J. Brokaw
86
J.J. Brokaw and G.W. White
in vascular permeability is known as neurogenic inflammation [9] and is thought to be mediated by tachykinins, such as substance P, released from the peripheral terminals of sensory axons [ 16]. Although tachykinins are considered the principal mediators of neurogenic inflammation, other sensory neuropeptides may contribute to the response. Recent attention has focused on calcitonin generelated peptide (CGRP), which is stored with tachykinins in airway sensory nerves and is likewise released during antidromic stimulation [15]. Unlike tachykinins, however, CGRP does not produce plasma extravasation in the airways [12]. Nevertheless, because CGRP is a potent vasodilator [3], it might potentiate the effects of tachykinins on plasma extravasation. In support of this notion, some studies have shown that when substance P and CGRP are injected together intradermally, the amount of plasma extravasation produced in rat skin is greater than the amount produced when substance P is injected alone [2, 8]. This potentiation has yet to be demonstrated in the airways. The aim of the present study was therefore to determine whether CGRP can potentiate substance P-induced plasma extravasation in the rat trachea. To accomplish this, we measured the amounts of Evans blue dye extravasated into the trachea after intravenous injections of substance P alone and combined with CGRP. To help explain our findings, we also measured the effects of these neuropeptides on arterial blood pressure.
Methods
Animals The experiments were performed on female Long-Evans rats (190-370 g) purchased from Charles River Breeding Laboratories (Wilmington, MA). The protocol was approved by the Animal Care and Use Committee of the Indiana University School of Medicine.
Drugs and Chemicals The following drugs and chemicals were used: sodium methohexital (Eli Lilly and Company, Indianapolis, IN); sodium heparin (Organon, Inc., W. Orange, N J); Evans blue dye (Sigma Chemical Company, St. Louis, MO); substance P (Sigma); rat CGRP (Sigma); and formamide (Sigma). Evans blue dye was dissolved at a concentration of 30 mg/ml in normal saline (Abbott Laboratories, North Chicago, IL). Substance P and CGRP were each dissolved at a Concentration of 50/xg/ml in normal saline containing 0.1 N acetic acid and stored at - 80°C until use. Subsequent dilutions were made in normal saline.
Effects of Neuropeptides on Plasma Extravasation The extravasation of Evans blue dye, which binds to serum albumin [10], was used to assess plasma extravasation in the trachea [5, 19]. This method has been shown to correlate well with the extravasation of radiolabeled albumin [18]. Rats were anesthetized with intraperitoneal sodium methohexital (75 mg/kg) and given an injection of Evans blue dye through the external jugular vein (30 mg/kg). The dye injection was followed by an intravenous injection of substance P (0.25-3 nmol/
Neurogenic Inflammation of the Airways
87
kg), CGRP (0.25-2 nmol/kg), or both neuropeptides together (a single dose of substance P [0.5 nmol/kg] coinjected with different dosages of CGRP [0.25-2 nmol/kg] or a single dose of CGRP [0.5 nmol/kg] coinjected with different dosages of substance P [0.25-3 nmol/kg]). All rats received the same injection volume (2 ml/kg), regardless of whether the neuropeptides were administered alone or in combination. Control rats were given only the diluent (2 ml/kg). Five minutes later, the dye was flushed out of the circulation by the perfusion of 100 ml of 0.05 M citrate buffer (pH 3.5) containing 1% paraformaldehyde through the aorta. After being removed and weighed, each trachea was incubated overnight in 2 ml of formamide at 60°C to extract the dye. The concentration of dye in the extracts was determined spectrophotometrically by measuring the absorbance at 620 nm (Perkin-Elmer Model 35 spectrophotometer; Oak Brook, IL). Calculations were based on known concentrations of dye in formamide. Results are expressed in nanograms of Evans blue dye per milligram of tracheal wet weight.
Effects of Neuropeptides on Blood Pressure Arterial blood pressure was measured in a separate group of rats to assess the vasodilatory potencies of substance P and CGRP. The right common carotid artery of anesthetized rats was exposed and a PE-20 catheter was inserted into the lumen. A small volume of heparinized saline (10 U/ml) was flushed through the catheter before it was connected to a Statham pressure transducer (Model P23DC; Hato Rey, PR). Mean blood pressure (ram Hg) was recorded on a Grass polygraph (Model 79D; Quincy, MA). After a baseline trace was established, the rats were given an intravenous injection of substance P (0.25-2 nmol/kg), CGRP (0.25-2 nmol/kg), or both together (a single dose of substance P [0.5 nmol/kg] coinjected with different dosages of CGRP [0.25-2 nmol/kg]), All rats received the same injection volume (2 ml/kg), regardless of whether the neuropeptides were administered alone or in combination. Control rats were given only the diluent (2 ml/kg). No rats were given more than 1 dose of any substance. Tracings were recorded for 5 min and maximal changes in blood pressure noted. Changes in blood pressure are expressed as a percentage of the corresponding baseline values.
Analysis of Data All values are presented as the mean -+ SEM. Differences between group means were statistically evaluated with the unpaired t-test or 1-way analysis of variance and Dunnett's test [24]. Differences were considered significant when p < 0.05.
Results
Effects of Neuropeptides on Plasma Extravasation W h e n a single d o s e o f C G R P w a s c o i n j e c t e d w i t h d i f f e r e n t d o s a g e s o f s u b s t a n c e P, t h e a m o u n t s o f E v a n s b l u e d y e e x t r a v a s a t e d into t h e t r a c h e a w e r e g r e a t e r t h a n w h e n s u b s t a n c e P w a s i n j e c t e d a l o n g (Fig. 1). A t a s u b s t a n c e P d o s a g e o f 3 n m o l / k g , f o r e x a m p l e , t h e a m o u n t o f d y e e x t r a v a s a t i o n p r o d u c e d in c o m b i n a t i o n w i t h C G R P (196.7 -+ 16.8 ng d y e / m g t is s u e , m e a n - S E M ; n = 6) w a s 39% g r e a t e r (p < 0.01) t h a n t h e a m o u n t p r o d u c e d b y s u b s t a n c e P a l o n e (141.6 --- 7.8 ng d y e / m g t i s s u e ; n = 6). H o w e v e r , at s u b s t a n c e P d o s a g e s less t h a n 2 n m o l / kg, t h e a m o u n t s o f d y e e x t r a v a s a t i o n p r o d u c e d in c o m b i n a t i o n w i t h C G R P w e r e n o t s i g n i fi can t l y d i f f e r e n t f r o m t h o s e p r o d u c e d b y s u b s t a n c e P a l o n e .
88
J.J. Brokaw and G.W. White 250" • ---0--
SP + CGRP (0,5 nmol/kg)
Fig. 1. Extravasation of Evans blue dye produced in the rat trachea by substance P (SP) alone and combined with calcitonin gene-related peptide (CGRP). Rats were given an intravenous injection of dye and a single dose of CGRP combined with different dosages of SP. Other rats received SP without CGRP. Five minutes later, fixative was perfused through the aorta and the tracheas were removed and assayed for dye content. Each point represents the mean -+ SEM of 6 rats. **p < 0.01 compared to response produced without CGRP (unpaired t-test).
SP alone
200"
150'
8
100'
,.-., 50
I,-
1 SP
2 Dose
3
(nmol/kg)
140 ~
•
CGRP + SP (0.5 nmol/kg)
120 "
100-
"0
.
8 e'~
401
~ p-
20" O
CGRP
,
i
1
2
Dose
(nmo|/kg)
Fig. 2. Extravasation of Evans blue dye produced in the rat trachea by calcitonin gene-related peptide (CGRP) alone and combined with substance P (SP). Rats were given an intravenous injection of dye and a single dose of SP combined with different dosages of CGRP. Other rats received CGRP without SP. Five minutes later, fixative was perfused through the aorta and the tracheas were removed and assayed for dye content. Each point represents the mean -+ SEM of 6 rats. *p < 0.05, **p < 0.01 compared to response produced without CGRP (analysis of variance).
When a single dose of substance P was coinjected with different dosages of CGRP, the amounts of Evans blue dye extravasated into the trachea were greater than when substance P was injected alone (Fig. 2). At a CGRP dosage of 0.5 nmol/kg, for example, the amount of dye extravasation produced in combination with substance P (114.8 -+ 13.7 ng dye/mg tissue; n = 6) was approximately 3 times greater (p < 0.01) than the amount produced by substance P alone (39.0 -+ 4.2 ng dye/mg tissue; n = 6). However, at higher CGRP dosages,
Neurogenic Inflammation of the Airways
89
120• --'--~
"50
///~ ........
"[ L4s
¢.
100" • 40
"0
80c
oc
"35
,
