Prostanoids Inhibit Release of Endogenous Norepinephrine from Rat Isolated Trachea 1- 4
KURT RACKE, JOACHIM BAHRING, CHRISTIANE LANGER, MATTHIAS BRAUTIGAM, and IGNAZ WESSLER
Introduction
As demonstrated in several species, the airways are innervated by adrenergic nerve fibers (see references 1 and 2). Stimulation of these fibers has been shown in guinea pig and calf tracheae to cause relaxation of the airway smooth muscle tone (3, 4). On the other hand, there are large species differences with regard to direct adrenergic innervation of the tracheobronchial smooth muscle. Thus, in rats, as in monkeys and humans, the adrenergic innervation of the tracheobronchial system appears to be mainly associated with the vasculature and the submucosal glands (5-9). In these species, one of the major functions of the sympathetic innervation of the airways may be the regulation of the local blood flow (see references 8-10), but effects on cholinergic neurotransmission (11) and paracrine and exocrine functions of the airways may also be of significance (see references 8 and 9). Recently we demonstrated that the release of endogenous norepinephrine from isolated, in vitro incubated rat trachea can be measured by high-performance liquid chromatography (HPLC) with electrochemical detection (12). Moreover, it was shown that mechanical removal of the epithelium resulted in a marked reduction in the release of norepinephrine evoked by electrical field stimulation (12). Endogenous eicosano ids, particularly of epithelial origin, appear to modulate numerous functions within the airways, such as smooth muscle tone, ion transport, and mucus secretion (for a recent review see reference 13). Eicosanoids may also participate in the local control of the release of neurotransmitters within the airways. Thus, the release of acetylcholine from canine (14) and guinea pig trachea (15) was facilitated by indomethacin, whereas prostaglandin E2 (PGE2 ) inhibited the release of acetylcholine (14).The aim ofthe present experiments was to test whether inhibition 1182
SUMMARY Prostanolds, of epithelial origin, are known as modulators of several processes In the airways. The present study examined whether prostanolds are Involved in the toeal control of sympathetic neurotransmlsslon. The release of endogenous norepinephrine from rat Isolated tracheae was evoked by electrical field stimulation (3 Hz, 540 pulses) In the presence of yohimbine, desipramine, and tyrosine. In different series of experiments, indomethacin (3 ~moIlL) Increased the evoked release of endogenous norepinephrine by 70 to 80%. In the presence of Indomethacin, prostaglandin E2 (PGE2) and several prostanoid receptor agonists Inhibited the evoked release of norepinephrine In a concentratlon-dependent manner, maximany by 60 to 70%. According to the concentration producing 35% Inhibition of norepinephrine release (ha"-maxlmal effect), the fonowlng rank order of potencies was observed (EC3 S) : nocloprost (8 nmoIlL), sulprostone (30 nmoIlL), PGE2 (308 nmoIlL), itoprost (2 ~moIlL), and U46619 (> 10 ~moIlL). The EP, receptor antagonist AH 6809 (3 ~moIlL) had no effect on the evoked norepinephrine release and did not affect the inhibitory effect of 1 ~ollL of sulprostone. In the absence of Indomethacin, the Inhibitory effect of PGE2 was similar to that observed In the presence of indomethacin. After removal of the epithelium, the evoked norepinephrine release was markedly reduced. However, no slgnmcant effect of Indomethacin was observed in eplthellum-denuded tracheae. In conclusion, norepinephrine release in the rat trachea Is Inhibited via prostaglandin receptors that have the pharmacologic characteristics of the EP3 subtype. Endogenous elcosanolds, most likely of epithelial origin, are Involved in the local control of the release of norepinephrine. AM REV RESPIR DIS 1992; 146:1182-1186
of cyclooxygenase by indomethacin or application of exogenous prostanoids may modulate the release of norepinephrine from the isolated rat trachea. It was of particular interest to see whether endogenous prostanoids are involved in the epithelium-dependent modulation of norepinephrine release. A preliminary account of part of the present results has been given to the Physiological Society (16) and the German Society of Pharmacology and Toxicology (17). Methods Preparation and Incubation of the Trachea Female Sprague-Dawley rats weighing 190 to 210g (Charles River Wiga, Sulzfeld, Germany) wereused. The animals were kept at a constant temperature (210 C) and a regular light (0630 to 1930) and dark (1930 to 0630) cycle with food and water ad libitum for at least 1 wk before use. As described in detail previously (12), the animals were killed by stunning followed by exsanguination and the entire trachea was dissected, opened by a cut along the ventral side, fixed between two platinum wire field electrodes, and finally incubat-
ed in 1.7 ml Krebs-HEPES solution of the following composition (mmol/L): NaCI, 118.5; KCI, 5.7; CaCb, 1.25; MgCI2, 1.2; sodium EDTA, 0.03; (+ )-ascorbic acid, 0.06; HE PES, 20.0 (adjusted to pH 7.4 using NaOH); and D-glucose, 11.1. The medium contained in addition the neuronal uptake inhibitor desipramine (1 umol/L), the norepinephrine precursor tyrosine (10umol/L) and the a2-adrenoceptor antagonist yohimbine (1 umol/L) (see reference 12). A muscarine receptor antagonist was not added since previous experiments showedthat scopolamine did not affect the release of norepinephrine un-
(Received in original form February 14, 1992 and in revised form June 1, 1992) 1 From the Departments of Pharmacology, University of Frankfurt and University of Mainz, and the Research Laboratory, Schering AG, Berlin, Germany. 2 Contains part of the Dr. med. thesis by Joachim Bahring and Christiane Langer. 3 Supported by Grant No. Ra 400/3-1 from the Deutsche Forschungsgemeinschaft. 4 Correspondence and requests for reprints should be addressed to K. Racke, Department of Pharmacology, University of Frankfurt, TheodorStem-Kai 7, D-6000-Frankfurt, Germany.
1183
PROSTANOID INHIBITION OF TRACHEAL NOREPINEPHRINE RELEASE
der the present in vitro conditions (12). The incubation medium, kept at 37° C and continuously gassed with 100010 Os, was changed every 10 min. The medium was collected into glass tubes that contained 50 J,J.I of a solution containing EUfA (2.3%, vol/vol) and NazSOa (2.8%) and 100 J,J.I of 1 mol/L HCI04 to protect the released norepinephrine (resulting in a total sample volume of 1.85 ml). In the respective experiments, indomethacin (3 umol/L) was added to the incubation medium either 30 min before the second period of electrical stimulation (S2, see below) or from the onset of incubation. The prostanoid receptor agonists (PGEz, sulprostone, nocloprost, iloprost, and U46619) were added 10 min before S2. If not stated otherwise, the effects of these agonists were studied in the presence of indomethacin, that is, after inhibition of the endogenous prostanoid production. The EP 1receptor antagonist AH 6809 was tested in the absence or presence of indomethacin. AH 6809 was added 10min before S2 to study its own effect. In interaction experiments with sulprostone, AH 6809 was present from the onset of incubation and sulprostone was added 10 min before S2. In some experiments the epithelium was removed before the start of incubation by gently rubbing the luminal surface of the trachea with a pipe cleaner. Removal of the epithelium was confirmed by light microscopy after staining with hematoxylin and eosin. At the end of incubation each trachea was blotted, weighed, minced, and extracted in 2 ml of 0.4 mol/L HCI0 4 for 1 to 2 h at o to 4 0 C. The extracts were centrifuged at 20,000 x g for 1 min, and the supernatants were stored at 0 to 4 0 C until analyzed later the same day. The mean weight of epitheliumcontaining tracheae was 44.7 ± 1.1mg (n = 221)and that of epithelium-denuded tracheae 40.2 ± 1.8 mg (n = 25).
Stimulation of Norepinephrine Release Electrical field stimulation (SI and S2) was carried out after 60 and 110 min of incubation, respectively (figure 1). Biphasic pulses were delivered from a Grass S6 stimulator. During each period of stimulation, pulses of 0.4 ms, 250 rnA, 3 Hz, wereapplied three times for 1 min at l-min intervals. The current flow was determined by the voltage drop across a resistance by oscilloscope. Analytic Procedure Norepinephrine in the samples and tissue extracts was determined as described previously (12, 18). Briefly,after extraction over alumina, norepinephrine and dihydroxybenzylamine (DHBA, internal standard) were separated on a reversed-phase column (length 250 mm, inner diameter 4.6 mm, prepacked with Shandon ODS-HypersilllD , 5 urn) using as mobile phase 0.1 mol/L of phosphate buffer (adjusted to pH 3.0), which contained octane sulfonic acid sodium salt (250 mg/L), sodium EDTA (0.3 mmol/L), and methanol (4%, vol/vol). Quantitation was carried out with an electrochemical detector (Waters 460)
equipped with a glassy carbon working electrode and an Ag/AgCI referenceelectrode. The potential was set at 0.70 V. The limit of detection was about 15and 50 fmol for norepinephrine and DHBA, respectively. The recovery of the extraction procedure, determined using DHBA as internal standard, was between 70 and 85%. The values given under RESULTS were corrected for the recoveries obtained in the individual analyses.
Calculations and Statistical Analysis The overflow of norepinephrine is expressed as pmol per g wet weight of tissue and per collection period (i,e., pmol g-1 10min-I). The evoked release of norepinephrine was calculated by summing the overflow of norepinephrine observed in the two samples collected during and immediately after the stimulation and by subtracting twice the spontaneous overflow determined immediately before the respective stimulation. The evoked release of norepinephrine is expressed in absolute terms (pmol g-1 stimulation"), as the ratio S2/S1 or as the percentage of tissue norepinephrine determined at the end of the incubation experiments. Mean values of n observations are given ± SEM. The significance of differences was evaluated by Student's t test. For comparison of one control with severalexperimental groups, the significance of differences was evaluated by the modified t test according to Bonferroni (see reference 19). Drugs and Special Chemicals These were AH 6809 (6-isopropoxy-9-oxoxanthene-2-carboxylic acid; Glaxo, Hamburg, Germany; dissolved in 1% NaHCOa in 0.9010 saline, 10 mmol/L); desipramine hydrochloride (Serva, Heidelberg, Germany; dissolved in H 20, 10 mmol/L); iloprost (Schering AG, Berlin, Germany; dissolved in ethanol, 0.1 mg/ml); indomethacin (Merck Sharp & Dohme, Munich, Germany; dissolved in ethanol, 10 mmol/L); nocloprost-Bvcyclodextrin-clathrate (Schering; dissolved in ethanol and dimethylsulfoxide, 50:50 vol/vol,
3 mmol/L); prostaglandin E, (Sigma, Munich, Germany; dissolved in ethanol, 10mmol/L); sulprostone (Schering; dissolved in ethanol, 10mmol/L); tyrosine (Sigma; dissolved in 0.1 mol/L of HCI, 10 mmol/L); U46619 (SPIBIO, Gif sur Yvette, France; dissolved in methyl acetate, 10 mg/ml); and yohimbine hydrochloride (Boehringer Ingelheim, Ingelheim, Germany; dissolved in 0.1 mol/L of HCI, 10mmol/L). None of the solvents alone significantly affected the spontaneous outflow and stimulation-evoked release of norepinephrine when tested in the concentration that was applied with the highest concentration of the respective test drug (n = 3 each, data not shown).
Results
Norepinephrine Release from Tracheae with Intact Epithelium The spontaneous overflow of norepinephrine in the presence of desipramine, yohimbine, and tyrosine was 3.4 ± 0.25 pmol g'" 10 min- t (n = 95). It was not altered when indomethacin was additionally present (3.82 ± 0.30 pmol g'" 10 min-I, n = 126). Likewise, none of the other drugs tested in the present study significantly altered the spontaneous overflow of norepinephrine (not shown). In the absence of indomethacin, the release of norepinephrine evoked by the first electrical stimulation (81) amounted to 39 pmol g-t, corresponding to 2.9070 of the tissue norepinephrine determined at the end of the incubation experiments (table 1). In control experiments, a second stimulation (82) caused a similar release of norepinephrine (figure 1). When indomethacin (3 umol/L) was added 30 min before 82, the ratio 82/81 of the evoked release of norepinephrine was enhanced to 1.48 ± 0.12 (n = 6); that is, compared with the respective control ex-
TABLE 1 EFFECTS OF INDOMETHACIN ON THE ELECTRICALLY EVOKED RELEASE OF NOREPINEPHRINE FROM EPITHELIUM-CONTAINING AND EPITHELIUM-DENUDED RAT TRACHEAE* Norepinephrine Release
Epithelium intact Control Indomethacin Epithelium denuded Control Indomethacin
pmol 9- 1 stimulation"
%
n
39 ± 2.0 65 ± 2.8t
2.9 ± 0.18 4.9 ± 0.18t
95 126
11 ± 1.2* 8 ± 1.5*
0.87 ± 0.08* 0.83 ± 0.16*
17 6
• The isolated tracheae were incubated and stimulated electrically as described in figure 1. When indicated, the epithelium was removed mechanically before the start of the incubation period. In the respective experiments, indomethacin (3 J.1moI/L) was present in the medium from the onset of incubation. Values are mean ± SEM of n experiments of the release of norepinephrine evoked by the first stimulation (51, see figure 1), expressed either as pmol g-1stimulation-1 or as a percentage of the tissue norepinephrine determined at the end of the experiments. t Significantly different from the corresponding value in the absence of indomethacin, p < 0.01. :j: Significantly different from the corresponding value with intact epithelium, p < 0.01.
1184
RACKE, BAHRING, LANGER, BRAuTIGAM, AND WESSLER
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periments (82/81, 0.81 ± 0.06, n = 12), the release of norepinephrine was increased by about 80070. When indomethacin was present from the onset of incubation, norepinephrine releaseevoked by 81 was also markedly enhanced (table 1). In experiments in which indomethacin was present throughout, PGEz and several prostanoid receptor agonistssulprostone, noeloprost, iloprost, and U46619(each added 10min before 82)inhibited the evoked release of norepinephrine in a concentration-dependent manner, maximally by about 60 to 70070 (figure 2). Based on the concentration that caused an inhibition of the evoked norepinephrine release by 35% (EC 3 S ) , that is, the concentration that caused a half-maximal effect, the following rank order of potencies was observed: noeloprost (8 nmol/L), sulprostone (30 nmol/L), PGEz (308 nmoI/L), iloprost (2 umol/L), and U46619 (> 10umol/L). The inhibitory effect of 1 umol/L of sulprostone was not affected by the selective EP I receptor antagonist AH 6809
Fig. 1. Effects of electrical stimulation on the overflow of norepinephrine from rat epithelium-containing tracheae incubated in vitro. The incubation medium contained 1 J1rTloIIL of desipramine, 1 IlmollL of yohimbine, and 10 J,lmollL of tyrosine (hatched area of the columns, n = 9) or, in addition, 3 umort, of indomethacin (total height of the columns, n = 12) from the onset of incubation. Two periods of electrical field stimulation (Sl and S2) were carried out commencing at 60 and 110min, respectively. Abscissa: time after onset of incubation. Ordinate: overflow of norepinephrine (NE), expressed as pmol g-l 10 min-I, mean ± SEM. Significance of differences from the corresponding value in the absence of indomethacin: * p
KURT RACKE, JOACHIM BAHRING, CHRISTIANE LANGER, MATTHIAS BRAUTIGAM, and IGNAZ WESSLER
Introduction
As demonstrated in several species, the airways are innervated by adrenergic nerve fibers (see references 1 and 2). Stimulation of these fibers has been shown in guinea pig and calf tracheae to cause relaxation of the airway smooth muscle tone (3, 4). On the other hand, there are large species differences with regard to direct adrenergic innervation of the tracheobronchial smooth muscle. Thus, in rats, as in monkeys and humans, the adrenergic innervation of the tracheobronchial system appears to be mainly associated with the vasculature and the submucosal glands (5-9). In these species, one of the major functions of the sympathetic innervation of the airways may be the regulation of the local blood flow (see references 8-10), but effects on cholinergic neurotransmission (11) and paracrine and exocrine functions of the airways may also be of significance (see references 8 and 9). Recently we demonstrated that the release of endogenous norepinephrine from isolated, in vitro incubated rat trachea can be measured by high-performance liquid chromatography (HPLC) with electrochemical detection (12). Moreover, it was shown that mechanical removal of the epithelium resulted in a marked reduction in the release of norepinephrine evoked by electrical field stimulation (12). Endogenous eicosano ids, particularly of epithelial origin, appear to modulate numerous functions within the airways, such as smooth muscle tone, ion transport, and mucus secretion (for a recent review see reference 13). Eicosanoids may also participate in the local control of the release of neurotransmitters within the airways. Thus, the release of acetylcholine from canine (14) and guinea pig trachea (15) was facilitated by indomethacin, whereas prostaglandin E2 (PGE2 ) inhibited the release of acetylcholine (14).The aim ofthe present experiments was to test whether inhibition 1182
SUMMARY Prostanolds, of epithelial origin, are known as modulators of several processes In the airways. The present study examined whether prostanolds are Involved in the toeal control of sympathetic neurotransmlsslon. The release of endogenous norepinephrine from rat Isolated tracheae was evoked by electrical field stimulation (3 Hz, 540 pulses) In the presence of yohimbine, desipramine, and tyrosine. In different series of experiments, indomethacin (3 ~moIlL) Increased the evoked release of endogenous norepinephrine by 70 to 80%. In the presence of Indomethacin, prostaglandin E2 (PGE2) and several prostanoid receptor agonists Inhibited the evoked release of norepinephrine In a concentratlon-dependent manner, maximany by 60 to 70%. According to the concentration producing 35% Inhibition of norepinephrine release (ha"-maxlmal effect), the fonowlng rank order of potencies was observed (EC3 S) : nocloprost (8 nmoIlL), sulprostone (30 nmoIlL), PGE2 (308 nmoIlL), itoprost (2 ~moIlL), and U46619 (> 10 ~moIlL). The EP, receptor antagonist AH 6809 (3 ~moIlL) had no effect on the evoked norepinephrine release and did not affect the inhibitory effect of 1 ~ollL of sulprostone. In the absence of Indomethacin, the Inhibitory effect of PGE2 was similar to that observed In the presence of indomethacin. After removal of the epithelium, the evoked norepinephrine release was markedly reduced. However, no slgnmcant effect of Indomethacin was observed in eplthellum-denuded tracheae. In conclusion, norepinephrine release in the rat trachea Is Inhibited via prostaglandin receptors that have the pharmacologic characteristics of the EP3 subtype. Endogenous elcosanolds, most likely of epithelial origin, are Involved in the local control of the release of norepinephrine. AM REV RESPIR DIS 1992; 146:1182-1186
of cyclooxygenase by indomethacin or application of exogenous prostanoids may modulate the release of norepinephrine from the isolated rat trachea. It was of particular interest to see whether endogenous prostanoids are involved in the epithelium-dependent modulation of norepinephrine release. A preliminary account of part of the present results has been given to the Physiological Society (16) and the German Society of Pharmacology and Toxicology (17). Methods Preparation and Incubation of the Trachea Female Sprague-Dawley rats weighing 190 to 210g (Charles River Wiga, Sulzfeld, Germany) wereused. The animals were kept at a constant temperature (210 C) and a regular light (0630 to 1930) and dark (1930 to 0630) cycle with food and water ad libitum for at least 1 wk before use. As described in detail previously (12), the animals were killed by stunning followed by exsanguination and the entire trachea was dissected, opened by a cut along the ventral side, fixed between two platinum wire field electrodes, and finally incubat-
ed in 1.7 ml Krebs-HEPES solution of the following composition (mmol/L): NaCI, 118.5; KCI, 5.7; CaCb, 1.25; MgCI2, 1.2; sodium EDTA, 0.03; (+ )-ascorbic acid, 0.06; HE PES, 20.0 (adjusted to pH 7.4 using NaOH); and D-glucose, 11.1. The medium contained in addition the neuronal uptake inhibitor desipramine (1 umol/L), the norepinephrine precursor tyrosine (10umol/L) and the a2-adrenoceptor antagonist yohimbine (1 umol/L) (see reference 12). A muscarine receptor antagonist was not added since previous experiments showedthat scopolamine did not affect the release of norepinephrine un-
(Received in original form February 14, 1992 and in revised form June 1, 1992) 1 From the Departments of Pharmacology, University of Frankfurt and University of Mainz, and the Research Laboratory, Schering AG, Berlin, Germany. 2 Contains part of the Dr. med. thesis by Joachim Bahring and Christiane Langer. 3 Supported by Grant No. Ra 400/3-1 from the Deutsche Forschungsgemeinschaft. 4 Correspondence and requests for reprints should be addressed to K. Racke, Department of Pharmacology, University of Frankfurt, TheodorStem-Kai 7, D-6000-Frankfurt, Germany.
1183
PROSTANOID INHIBITION OF TRACHEAL NOREPINEPHRINE RELEASE
der the present in vitro conditions (12). The incubation medium, kept at 37° C and continuously gassed with 100010 Os, was changed every 10 min. The medium was collected into glass tubes that contained 50 J,J.I of a solution containing EUfA (2.3%, vol/vol) and NazSOa (2.8%) and 100 J,J.I of 1 mol/L HCI04 to protect the released norepinephrine (resulting in a total sample volume of 1.85 ml). In the respective experiments, indomethacin (3 umol/L) was added to the incubation medium either 30 min before the second period of electrical stimulation (S2, see below) or from the onset of incubation. The prostanoid receptor agonists (PGEz, sulprostone, nocloprost, iloprost, and U46619) were added 10 min before S2. If not stated otherwise, the effects of these agonists were studied in the presence of indomethacin, that is, after inhibition of the endogenous prostanoid production. The EP 1receptor antagonist AH 6809 was tested in the absence or presence of indomethacin. AH 6809 was added 10min before S2 to study its own effect. In interaction experiments with sulprostone, AH 6809 was present from the onset of incubation and sulprostone was added 10 min before S2. In some experiments the epithelium was removed before the start of incubation by gently rubbing the luminal surface of the trachea with a pipe cleaner. Removal of the epithelium was confirmed by light microscopy after staining with hematoxylin and eosin. At the end of incubation each trachea was blotted, weighed, minced, and extracted in 2 ml of 0.4 mol/L HCI0 4 for 1 to 2 h at o to 4 0 C. The extracts were centrifuged at 20,000 x g for 1 min, and the supernatants were stored at 0 to 4 0 C until analyzed later the same day. The mean weight of epitheliumcontaining tracheae was 44.7 ± 1.1mg (n = 221)and that of epithelium-denuded tracheae 40.2 ± 1.8 mg (n = 25).
Stimulation of Norepinephrine Release Electrical field stimulation (SI and S2) was carried out after 60 and 110 min of incubation, respectively (figure 1). Biphasic pulses were delivered from a Grass S6 stimulator. During each period of stimulation, pulses of 0.4 ms, 250 rnA, 3 Hz, wereapplied three times for 1 min at l-min intervals. The current flow was determined by the voltage drop across a resistance by oscilloscope. Analytic Procedure Norepinephrine in the samples and tissue extracts was determined as described previously (12, 18). Briefly,after extraction over alumina, norepinephrine and dihydroxybenzylamine (DHBA, internal standard) were separated on a reversed-phase column (length 250 mm, inner diameter 4.6 mm, prepacked with Shandon ODS-HypersilllD , 5 urn) using as mobile phase 0.1 mol/L of phosphate buffer (adjusted to pH 3.0), which contained octane sulfonic acid sodium salt (250 mg/L), sodium EDTA (0.3 mmol/L), and methanol (4%, vol/vol). Quantitation was carried out with an electrochemical detector (Waters 460)
equipped with a glassy carbon working electrode and an Ag/AgCI referenceelectrode. The potential was set at 0.70 V. The limit of detection was about 15and 50 fmol for norepinephrine and DHBA, respectively. The recovery of the extraction procedure, determined using DHBA as internal standard, was between 70 and 85%. The values given under RESULTS were corrected for the recoveries obtained in the individual analyses.
Calculations and Statistical Analysis The overflow of norepinephrine is expressed as pmol per g wet weight of tissue and per collection period (i,e., pmol g-1 10min-I). The evoked release of norepinephrine was calculated by summing the overflow of norepinephrine observed in the two samples collected during and immediately after the stimulation and by subtracting twice the spontaneous overflow determined immediately before the respective stimulation. The evoked release of norepinephrine is expressed in absolute terms (pmol g-1 stimulation"), as the ratio S2/S1 or as the percentage of tissue norepinephrine determined at the end of the incubation experiments. Mean values of n observations are given ± SEM. The significance of differences was evaluated by Student's t test. For comparison of one control with severalexperimental groups, the significance of differences was evaluated by the modified t test according to Bonferroni (see reference 19). Drugs and Special Chemicals These were AH 6809 (6-isopropoxy-9-oxoxanthene-2-carboxylic acid; Glaxo, Hamburg, Germany; dissolved in 1% NaHCOa in 0.9010 saline, 10 mmol/L); desipramine hydrochloride (Serva, Heidelberg, Germany; dissolved in H 20, 10 mmol/L); iloprost (Schering AG, Berlin, Germany; dissolved in ethanol, 0.1 mg/ml); indomethacin (Merck Sharp & Dohme, Munich, Germany; dissolved in ethanol, 10 mmol/L); nocloprost-Bvcyclodextrin-clathrate (Schering; dissolved in ethanol and dimethylsulfoxide, 50:50 vol/vol,
3 mmol/L); prostaglandin E, (Sigma, Munich, Germany; dissolved in ethanol, 10mmol/L); sulprostone (Schering; dissolved in ethanol, 10mmol/L); tyrosine (Sigma; dissolved in 0.1 mol/L of HCI, 10 mmol/L); U46619 (SPIBIO, Gif sur Yvette, France; dissolved in methyl acetate, 10 mg/ml); and yohimbine hydrochloride (Boehringer Ingelheim, Ingelheim, Germany; dissolved in 0.1 mol/L of HCI, 10mmol/L). None of the solvents alone significantly affected the spontaneous outflow and stimulation-evoked release of norepinephrine when tested in the concentration that was applied with the highest concentration of the respective test drug (n = 3 each, data not shown).
Results
Norepinephrine Release from Tracheae with Intact Epithelium The spontaneous overflow of norepinephrine in the presence of desipramine, yohimbine, and tyrosine was 3.4 ± 0.25 pmol g'" 10 min- t (n = 95). It was not altered when indomethacin was additionally present (3.82 ± 0.30 pmol g'" 10 min-I, n = 126). Likewise, none of the other drugs tested in the present study significantly altered the spontaneous overflow of norepinephrine (not shown). In the absence of indomethacin, the release of norepinephrine evoked by the first electrical stimulation (81) amounted to 39 pmol g-t, corresponding to 2.9070 of the tissue norepinephrine determined at the end of the incubation experiments (table 1). In control experiments, a second stimulation (82) caused a similar release of norepinephrine (figure 1). When indomethacin (3 umol/L) was added 30 min before 82, the ratio 82/81 of the evoked release of norepinephrine was enhanced to 1.48 ± 0.12 (n = 6); that is, compared with the respective control ex-
TABLE 1 EFFECTS OF INDOMETHACIN ON THE ELECTRICALLY EVOKED RELEASE OF NOREPINEPHRINE FROM EPITHELIUM-CONTAINING AND EPITHELIUM-DENUDED RAT TRACHEAE* Norepinephrine Release
Epithelium intact Control Indomethacin Epithelium denuded Control Indomethacin
pmol 9- 1 stimulation"
%
n
39 ± 2.0 65 ± 2.8t
2.9 ± 0.18 4.9 ± 0.18t
95 126
11 ± 1.2* 8 ± 1.5*
0.87 ± 0.08* 0.83 ± 0.16*
17 6
• The isolated tracheae were incubated and stimulated electrically as described in figure 1. When indicated, the epithelium was removed mechanically before the start of the incubation period. In the respective experiments, indomethacin (3 J.1moI/L) was present in the medium from the onset of incubation. Values are mean ± SEM of n experiments of the release of norepinephrine evoked by the first stimulation (51, see figure 1), expressed either as pmol g-1stimulation-1 or as a percentage of the tissue norepinephrine determined at the end of the experiments. t Significantly different from the corresponding value in the absence of indomethacin, p < 0.01. :j: Significantly different from the corresponding value with intact epithelium, p < 0.01.
1184
RACKE, BAHRING, LANGER, BRAuTIGAM, AND WESSLER
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periments (82/81, 0.81 ± 0.06, n = 12), the release of norepinephrine was increased by about 80070. When indomethacin was present from the onset of incubation, norepinephrine releaseevoked by 81 was also markedly enhanced (table 1). In experiments in which indomethacin was present throughout, PGEz and several prostanoid receptor agonistssulprostone, noeloprost, iloprost, and U46619(each added 10min before 82)inhibited the evoked release of norepinephrine in a concentration-dependent manner, maximally by about 60 to 70070 (figure 2). Based on the concentration that caused an inhibition of the evoked norepinephrine release by 35% (EC 3 S ) , that is, the concentration that caused a half-maximal effect, the following rank order of potencies was observed: noeloprost (8 nmol/L), sulprostone (30 nmol/L), PGEz (308 nmoI/L), iloprost (2 umol/L), and U46619 (> 10umol/L). The inhibitory effect of 1 umol/L of sulprostone was not affected by the selective EP I receptor antagonist AH 6809
Fig. 1. Effects of electrical stimulation on the overflow of norepinephrine from rat epithelium-containing tracheae incubated in vitro. The incubation medium contained 1 J1rTloIIL of desipramine, 1 IlmollL of yohimbine, and 10 J,lmollL of tyrosine (hatched area of the columns, n = 9) or, in addition, 3 umort, of indomethacin (total height of the columns, n = 12) from the onset of incubation. Two periods of electrical field stimulation (Sl and S2) were carried out commencing at 60 and 110min, respectively. Abscissa: time after onset of incubation. Ordinate: overflow of norepinephrine (NE), expressed as pmol g-l 10 min-I, mean ± SEM. Significance of differences from the corresponding value in the absence of indomethacin: * p
