European Journal of Pharmacology, 46 ( 1 9 7 7 ) 3 1 - - 3 9 © E l s e v i e r / N o r t h - H o l l a n d Biomedical P~'ess
31
RELEASE OF SPASMOGENS FROM RAT ISOLATED LUNGS BY TRYPTAMINES Y.S. B A K H L E a n d T E R E N C E W. S M I T H *
Dcpc~rtmenl of Pharmacology, Institute of Basic Medical Sciences, Royal College of Surgeons of England, Lincoln's Inn Fields, London, WC2A 3PN, U.K. Received 7 April 1977, revised MS received 5 J u l y 1977, a c c e p t e d 18 July ] 9 7 7
Y.S. i~AKIIT,E :ltl(t T.W. S~.~ITt-t, Release of spasmogens from rat isolated lungs by tryplamines, E u r o p e a n J. Pharmacol..16 (1977) 31--39. T r y p t a m i n e and 5 - h y d r o x y t r y p t a m i n e ( 5 - H T ) infused t h r o u g h t h e p u l m o n a r y c i r c u l a t i o n of rat isolated lungs released a s p a s m o g e n r e s e m b l i n g slow reacting s u b s t a n c e of a n a p h y l a x i s w h i c h we have d e n o t e d SRS-T a n d a PGE-like activity. SRS-T was n o t e x t r a c t a b l e f r o m Krebs s o l u t i o n b y several organic solvents at n e u t r a l or acid pH. It is t h e r e f o r e unlike o t h e r types o f SRS activity. T h e PGE-like release h a d a t h r e s h o l d at a b o u t 2 p g / m l o f t r y p t a m i n e or 5-HT a n d did n o t increase w i t h increasing doses ( u p to 10 p g / m l ) ; this release was a b o l i s h e d b y m e t h y s e r g i d e , BC 105 a n d BW 5 0 1 c 6 7 b u t n o t by m o r p h i n e . C o m p a r i s o n of agonist p o t e n c i e s of 5-HT a n d t r y p t a m i n e o n rat s t o m a c h strip a n d rat p u l m o n a r y a r t e r y a n d of a n t a g o n i s t p o t e n c i e s of m e t h y s e r g i d e , BC 105 a n d m o r p h i n e o n these t r y p t a m i n e r e c e p t o r s lead t o l h e c o n c l u s i o n t h a t the release r e c e p t o r s are u n l i k e e i t h e r o f the m y o t r o p i c receptors. In t e r m s of a n t a g o n i s t specificity t h e release r e c e p t o r s are closest to t h o s e in rat s t o m a c h strip. Slow r e a c t i n g s u b s t a n c e Tryptamine receptors
S p a s m o g e n .'elease Isolated lungs
Prostaglandins Tryptamines
1. Introduction The release of a mixture of spasmogens from the isolated perfused lungs of several species following infusion of vasoactive amines has been described earlier (Alabaster and Bakhle, 1970, 1976). Sandler (1972) has drawn analogies between spasmogen release from lungs induced by 5-hydroxytryptamine (5-HT) and migraine. In this paper we have investigated further the release of spasmogens from rat isolated lungs induced by 5-HT or tryptamine and have attempted to characterize the receptor mediating the release. A preliminary account of some of this work was given to the British Pharmacological Society (Bakhle and Smith, 1974a). * P r e s e n t address: D e p a r t m e n t of P h a r m a c o l o g y , W e l l c o m e R e s e a r c h L a b o r a t o r i e s , Langley C o u r t , Beckenham, Kent.
Methysergide Morphine
Migraine
2. Materials and methods
2.1. Isolated lungs Rat isolated lungs were perfused via a cannula positioned in the base of the pulmonary artery as described by Bakhle et al. (1969). In the present experiments, after perfusion of the isolated lungs sufficient to wash them free of blood, the lungs were disconnected from the perfusion apparatus and scarified by lightly drawing a needle over all the surfaces of the lobes. The scarified lungs were then resuspended in the lung chamber and perfusion through the pulmonary artery continued. Scarification improved the reproducibility of release without affecting the qualitative aspects of the release. The lung effluent superfused a bank of isolated assay tissues, usually comprising the rat stomach strip, chick rectum and guinea-
32 pig ileum. In all experiments, the tissues were made more specific for the assay of prostaglandin-like and slow-reacting substance-like materials by continued superfusion of the tissues with mixed antagonists. The mixed antagonists also served to block the effects on the assay tissues of the amines used to induce release of spasmogens from the lungs. The antagonists used and their final concentrations (as the base) in the Krebs solution were methysergide, 150 ng/ml; mepyramine, 100 ng/ml; hyoscine, 100 ng/ml; phenoxybenzamine, 100 ng/ml and propranolol, 2 pg/ml. In all experiments, isolated lungs were perfused at a rate of 8 ml/min with Krebs bicarbonate solution of the following composition (g/l): NaC1 6.9; KC1 0.35; CaC12" 6H20 0.55; KH2PO4 0.16; M g S O 4 " 7 H 2 0 0.29; glucose 1.0; NaHCO~ 2.1. The solution was gassed with a mixture of 95% oxygen and 5% carbon dioxide. Agonists were infused for 3 min at 0.1 ml/min either through the pulmonary circulation or directly to the assay tissues. Antagonists were infused continuously AL or DIR at a constant rate of 0.07--0.1 ml/min. Samples of BW 501 c67 and of BC 105 (Pizotifen) were generously supplied by Dr. O'Grady (Wellcome Foundation) and Dr. Salzmann (Sandoz) respectively.
2.2. Antagonist assays Antagonist studies on the rat stomach strip and pulmonary artery were carried out by obtaining dose--response relationships to bolus injections of at least 3 concentrations of agonist. Only tissues producing consistent dose--response relationships on repeated cycles of agonist administration were used. Antagonists were then infused and dose-response relationships to the agonists constructed. Details of this procedure and the preparation of rat pulmonary arterial strips have been described previously by Bakhle and Smith (1974b).
Y.S. BAKHLE, T.W. SMITH
2.3. Extraction methods Prostaglandin-like materials were extracted from acidified lung effluent (approximately pH 3) by extracting twice with either equal volumes of ethyl acetate or two volumes of diethyl ether. The combined ether fractions were washed by shaking with small volumes of distilled water. Back-extraction of the ether phase was carried out by shaking three times with a volume of ammonium hydroxide (pH 8.0) corresponding to the volume of the original lung effluent sample. In all extraction procedures, the final phase was evaporated to dryness and reconstituted in a small volume of saline for assay on the superfused isolated tissues. A simple ether-alkali procedure was carried out to separate slow-reacting substance of ~,naphylaxis (SRS-A) from prostaglandins in lung effluent. Lung effluent was extracted with diethyl ether and the ether phase evaporated to dryness. The dried material was dissolved in 5 ml of 0.05 N sodium hydroxide, incubated for l h at 40°C and, after acidification to pH 3, re-extracted with diethyl ether. The ether phase was then backextracted with NH4OH as described above.
2.4. Statistical methods Student's t-test for unpaired values was used to assess significance.
3. Results
3.1. The nature of spasmogens released by infusion of tryptamine through isolated rat lung The phenomenon of spasmogen release by tryptamine is illustrated by fig. 1. We had identified three components of the spasmogen mixture as PGE-like, SRS-A-like and, less frequently, a RCS-like c o m p o n e n t (Alabaster and Bakhle, 1970, 1976). All three components were released in the present experi-
TRYPTAMINE RECEPTORS FOR SPASMOGEN RELEASE FROM LUNG
10 fmin 1
RSS
cm
CR
GPI
ml lng tper
2~g 2ng
T
lng
AL Fig. 1. Release of spasmogens from isolated rat lungs by tryptamine. The record shows contractions of assay tissues superfused in series with the effluent f r o m rat lungs p e r f u s e d with K r e b s s o l u t i o n . In d e s c e n d i n g o r d e r , the assay tissues s h o w n are rat s t o m a c h strip (RSS), a c h i c k r e c t u m (CR) a n d a guinea-pig ileum (GPI). T h e assay tissues were continually s u p e r f u s e d with c o m b i n e d a n t a g o n i s t s . T r y p t a m i n e (T) infused directly to the tissues ( D I R ) h a d n o effect, b u t the same i n f u s i o n given t h r o u g h t h e lungs ( A L ) i n d u c e d the release o f s p a s m o g e n s causing all t h r e e tissues to c o n t r a c t . T h e tissue cont r a c t i o n s were c o m p a r e d with t h o s e p r o d u c e d b y d i r e c t i n f u s i o n s of p r o s t a g l a n d i n E2 (E2).
ments with scarified lungs. In fig. 1 the contractions of the rat stomach strip and the chick rectum are taken as indications of PGE-like activity. The SRS-A-like component, which we shall refer to as SRS-T (SRS-tryptamine), is disclosed by the contraction, later in onset and greater in duration, of the guinea-pig ileum. We wanted to separate these two activities so that they could be characterized further. 3.1.1. SRS-T Since we believed SRS-T to be related to SRS-A we had to develop a simple method of separating SRS-A from prostaglandins. We used as a model the effluent from isolated perfused lungs (from sensitised guinea pigs)
33
during anaphylaxis, which is known to contain both SRS-A and PGs {Piper and Vane, 1969). Extraction of this effluent with ethyl acetate at pH 3 removed both SRS-A activity (assayed on the mepyraminised guinea pig ileum) and PGs (assayed on rat stomach strip and chick rectum). This is in contrast to the findings of Gilmore et al. {1968} who were not able to extract SRS-A with ethyl acetate. On re-examination of their methods, however, we found that they had used partially purified SRS-A and not freshly released SRS-A. This probably accounts for the discrepancy between the two sets of results. Other organic solvents, e.g. ether, also extracted both components. Back-extraction of the organic phase by dilute ammonium hydroxide was also unsuccessful in separating PGs from SRS-A. However, SRS-A is stable to alkali {Orange and Austen, 1969), whereas PGs are readily converted to PGBs inactive on rat stomach strip and chick rectum. We therefore incubated the material extracted into ether at pH 3, with alkali (see Materials and methods) and found that the PG activity disappeared, whereas the SRS-A activity was unchanged. This simple ether-alkali procedure was then applied to the spasmogen mixture released by tryptamine infusions from rat lungs. No SRS-T activity (assayed on the mepyraminised guinea-pig ileum) was detected in the final product (5 experiments) and assays at each stage of the extraction procedure showed that SRS-T, unlike SRS-A, could not be extracted from Krebs solution into ether {2 experiments) or ethyl acetate (2 experiments) at acid pH. Extraction with methanol : chloroform (1 : 1, v/v; 4 experiments, Orange and Austen, 1969} of effluent at either acid or neutral pH was also unsuccessful in extracting SRS-T activity into the organic layer. Lung effluent containing SRS-T activity was evaporated to dryness and the residue extracted with ether (5 experiments), ethyl acetate (2 experiments), ethanol (5 experiments) or a mixture of water, chloroform and methanol ( 1 : 4 0 : 1 0 , v/v) (5
34
experiments). No SRS-T activity was found in any o f the organic extracts. T h e SRS-T activity was still present in the effluent when it was reperfused over the tissues after 2 h, at room temperature, at 4°C or 40°C (3 experiments each). The activity also withstood evaporation of the effluent and reconstitution with water to the original volume (5 experiments). In some experiments two pieces of guinea-pig ileum were used, set up in series and the lower one continuously superfused with indomethacin (2--10/~g/ml; 8 expts) or with hexam e t h o n i u m (362 ng/ml; 2 expts). The contractions p r o d u ced by SRS-T on the upper, untreated, tissue were not consistently different from those of the lower, treated, tissue.
3.1.2. PG-like activity As the SRS-T activity was not extracted by ether from the spasmogen containing effluent at pH 3, we expected the material which had been extracted to be PG-like. The extracted material was assayed on rat stomach strip, c h i c k rectum, gerbil colon, guinea-pig ileum and rat colon and its effects on these tissues were matched most closely by those of PGE2. We investigated the relation between the concentration of t r y p t a m i n e or 5-HT infused and the a m o u n t o f PG-like activity extracted from the effluent. In these experiments lung effluent was collected for 5 min periods before, during and after an infusion of the t r y p t a m i n e through the lung. The effluent samples were extracted and the activity in the organic phase assayed in terms of PGE2. The low basal release o f PG-like material from the lung increased during the amine infusion period and then returned to basal levels. No increase in PG-like activity in the effluent was caused by t r y p t a m i n e infusion direct to the assay tissues. The difference between basal and amineinduced o u t p u t of PG-like activity (net PGlike activity) was taken as the response and the variation o f this response with concentration of amine infused is shown in fig. 2. Five
Y.S. BAKHLE, T.W. SMITH
20
NS
p
31
RELEASE OF SPASMOGENS FROM RAT ISOLATED LUNGS BY TRYPTAMINES Y.S. B A K H L E a n d T E R E N C E W. S M I T H *
Dcpc~rtmenl of Pharmacology, Institute of Basic Medical Sciences, Royal College of Surgeons of England, Lincoln's Inn Fields, London, WC2A 3PN, U.K. Received 7 April 1977, revised MS received 5 J u l y 1977, a c c e p t e d 18 July ] 9 7 7
Y.S. i~AKIIT,E :ltl(t T.W. S~.~ITt-t, Release of spasmogens from rat isolated lungs by tryplamines, E u r o p e a n J. Pharmacol..16 (1977) 31--39. T r y p t a m i n e and 5 - h y d r o x y t r y p t a m i n e ( 5 - H T ) infused t h r o u g h t h e p u l m o n a r y c i r c u l a t i o n of rat isolated lungs released a s p a s m o g e n r e s e m b l i n g slow reacting s u b s t a n c e of a n a p h y l a x i s w h i c h we have d e n o t e d SRS-T a n d a PGE-like activity. SRS-T was n o t e x t r a c t a b l e f r o m Krebs s o l u t i o n b y several organic solvents at n e u t r a l or acid pH. It is t h e r e f o r e unlike o t h e r types o f SRS activity. T h e PGE-like release h a d a t h r e s h o l d at a b o u t 2 p g / m l o f t r y p t a m i n e or 5-HT a n d did n o t increase w i t h increasing doses ( u p to 10 p g / m l ) ; this release was a b o l i s h e d b y m e t h y s e r g i d e , BC 105 a n d BW 5 0 1 c 6 7 b u t n o t by m o r p h i n e . C o m p a r i s o n of agonist p o t e n c i e s of 5-HT a n d t r y p t a m i n e o n rat s t o m a c h strip a n d rat p u l m o n a r y a r t e r y a n d of a n t a g o n i s t p o t e n c i e s of m e t h y s e r g i d e , BC 105 a n d m o r p h i n e o n these t r y p t a m i n e r e c e p t o r s lead t o l h e c o n c l u s i o n t h a t the release r e c e p t o r s are u n l i k e e i t h e r o f the m y o t r o p i c receptors. In t e r m s of a n t a g o n i s t specificity t h e release r e c e p t o r s are closest to t h o s e in rat s t o m a c h strip. Slow r e a c t i n g s u b s t a n c e Tryptamine receptors
S p a s m o g e n .'elease Isolated lungs
Prostaglandins Tryptamines
1. Introduction The release of a mixture of spasmogens from the isolated perfused lungs of several species following infusion of vasoactive amines has been described earlier (Alabaster and Bakhle, 1970, 1976). Sandler (1972) has drawn analogies between spasmogen release from lungs induced by 5-hydroxytryptamine (5-HT) and migraine. In this paper we have investigated further the release of spasmogens from rat isolated lungs induced by 5-HT or tryptamine and have attempted to characterize the receptor mediating the release. A preliminary account of some of this work was given to the British Pharmacological Society (Bakhle and Smith, 1974a). * P r e s e n t address: D e p a r t m e n t of P h a r m a c o l o g y , W e l l c o m e R e s e a r c h L a b o r a t o r i e s , Langley C o u r t , Beckenham, Kent.
Methysergide Morphine
Migraine
2. Materials and methods
2.1. Isolated lungs Rat isolated lungs were perfused via a cannula positioned in the base of the pulmonary artery as described by Bakhle et al. (1969). In the present experiments, after perfusion of the isolated lungs sufficient to wash them free of blood, the lungs were disconnected from the perfusion apparatus and scarified by lightly drawing a needle over all the surfaces of the lobes. The scarified lungs were then resuspended in the lung chamber and perfusion through the pulmonary artery continued. Scarification improved the reproducibility of release without affecting the qualitative aspects of the release. The lung effluent superfused a bank of isolated assay tissues, usually comprising the rat stomach strip, chick rectum and guinea-
32 pig ileum. In all experiments, the tissues were made more specific for the assay of prostaglandin-like and slow-reacting substance-like materials by continued superfusion of the tissues with mixed antagonists. The mixed antagonists also served to block the effects on the assay tissues of the amines used to induce release of spasmogens from the lungs. The antagonists used and their final concentrations (as the base) in the Krebs solution were methysergide, 150 ng/ml; mepyramine, 100 ng/ml; hyoscine, 100 ng/ml; phenoxybenzamine, 100 ng/ml and propranolol, 2 pg/ml. In all experiments, isolated lungs were perfused at a rate of 8 ml/min with Krebs bicarbonate solution of the following composition (g/l): NaC1 6.9; KC1 0.35; CaC12" 6H20 0.55; KH2PO4 0.16; M g S O 4 " 7 H 2 0 0.29; glucose 1.0; NaHCO~ 2.1. The solution was gassed with a mixture of 95% oxygen and 5% carbon dioxide. Agonists were infused for 3 min at 0.1 ml/min either through the pulmonary circulation or directly to the assay tissues. Antagonists were infused continuously AL or DIR at a constant rate of 0.07--0.1 ml/min. Samples of BW 501 c67 and of BC 105 (Pizotifen) were generously supplied by Dr. O'Grady (Wellcome Foundation) and Dr. Salzmann (Sandoz) respectively.
2.2. Antagonist assays Antagonist studies on the rat stomach strip and pulmonary artery were carried out by obtaining dose--response relationships to bolus injections of at least 3 concentrations of agonist. Only tissues producing consistent dose--response relationships on repeated cycles of agonist administration were used. Antagonists were then infused and dose-response relationships to the agonists constructed. Details of this procedure and the preparation of rat pulmonary arterial strips have been described previously by Bakhle and Smith (1974b).
Y.S. BAKHLE, T.W. SMITH
2.3. Extraction methods Prostaglandin-like materials were extracted from acidified lung effluent (approximately pH 3) by extracting twice with either equal volumes of ethyl acetate or two volumes of diethyl ether. The combined ether fractions were washed by shaking with small volumes of distilled water. Back-extraction of the ether phase was carried out by shaking three times with a volume of ammonium hydroxide (pH 8.0) corresponding to the volume of the original lung effluent sample. In all extraction procedures, the final phase was evaporated to dryness and reconstituted in a small volume of saline for assay on the superfused isolated tissues. A simple ether-alkali procedure was carried out to separate slow-reacting substance of ~,naphylaxis (SRS-A) from prostaglandins in lung effluent. Lung effluent was extracted with diethyl ether and the ether phase evaporated to dryness. The dried material was dissolved in 5 ml of 0.05 N sodium hydroxide, incubated for l h at 40°C and, after acidification to pH 3, re-extracted with diethyl ether. The ether phase was then backextracted with NH4OH as described above.
2.4. Statistical methods Student's t-test for unpaired values was used to assess significance.
3. Results
3.1. The nature of spasmogens released by infusion of tryptamine through isolated rat lung The phenomenon of spasmogen release by tryptamine is illustrated by fig. 1. We had identified three components of the spasmogen mixture as PGE-like, SRS-A-like and, less frequently, a RCS-like c o m p o n e n t (Alabaster and Bakhle, 1970, 1976). All three components were released in the present experi-
TRYPTAMINE RECEPTORS FOR SPASMOGEN RELEASE FROM LUNG
10 fmin 1
RSS
cm
CR
GPI
ml lng tper
2~g 2ng
T
lng
AL Fig. 1. Release of spasmogens from isolated rat lungs by tryptamine. The record shows contractions of assay tissues superfused in series with the effluent f r o m rat lungs p e r f u s e d with K r e b s s o l u t i o n . In d e s c e n d i n g o r d e r , the assay tissues s h o w n are rat s t o m a c h strip (RSS), a c h i c k r e c t u m (CR) a n d a guinea-pig ileum (GPI). T h e assay tissues were continually s u p e r f u s e d with c o m b i n e d a n t a g o n i s t s . T r y p t a m i n e (T) infused directly to the tissues ( D I R ) h a d n o effect, b u t the same i n f u s i o n given t h r o u g h t h e lungs ( A L ) i n d u c e d the release o f s p a s m o g e n s causing all t h r e e tissues to c o n t r a c t . T h e tissue cont r a c t i o n s were c o m p a r e d with t h o s e p r o d u c e d b y d i r e c t i n f u s i o n s of p r o s t a g l a n d i n E2 (E2).
ments with scarified lungs. In fig. 1 the contractions of the rat stomach strip and the chick rectum are taken as indications of PGE-like activity. The SRS-A-like component, which we shall refer to as SRS-T (SRS-tryptamine), is disclosed by the contraction, later in onset and greater in duration, of the guinea-pig ileum. We wanted to separate these two activities so that they could be characterized further. 3.1.1. SRS-T Since we believed SRS-T to be related to SRS-A we had to develop a simple method of separating SRS-A from prostaglandins. We used as a model the effluent from isolated perfused lungs (from sensitised guinea pigs)
33
during anaphylaxis, which is known to contain both SRS-A and PGs {Piper and Vane, 1969). Extraction of this effluent with ethyl acetate at pH 3 removed both SRS-A activity (assayed on the mepyraminised guinea pig ileum) and PGs (assayed on rat stomach strip and chick rectum). This is in contrast to the findings of Gilmore et al. {1968} who were not able to extract SRS-A with ethyl acetate. On re-examination of their methods, however, we found that they had used partially purified SRS-A and not freshly released SRS-A. This probably accounts for the discrepancy between the two sets of results. Other organic solvents, e.g. ether, also extracted both components. Back-extraction of the organic phase by dilute ammonium hydroxide was also unsuccessful in separating PGs from SRS-A. However, SRS-A is stable to alkali {Orange and Austen, 1969), whereas PGs are readily converted to PGBs inactive on rat stomach strip and chick rectum. We therefore incubated the material extracted into ether at pH 3, with alkali (see Materials and methods) and found that the PG activity disappeared, whereas the SRS-A activity was unchanged. This simple ether-alkali procedure was then applied to the spasmogen mixture released by tryptamine infusions from rat lungs. No SRS-T activity (assayed on the mepyraminised guinea-pig ileum) was detected in the final product (5 experiments) and assays at each stage of the extraction procedure showed that SRS-T, unlike SRS-A, could not be extracted from Krebs solution into ether {2 experiments) or ethyl acetate (2 experiments) at acid pH. Extraction with methanol : chloroform (1 : 1, v/v; 4 experiments, Orange and Austen, 1969} of effluent at either acid or neutral pH was also unsuccessful in extracting SRS-T activity into the organic layer. Lung effluent containing SRS-T activity was evaporated to dryness and the residue extracted with ether (5 experiments), ethyl acetate (2 experiments), ethanol (5 experiments) or a mixture of water, chloroform and methanol ( 1 : 4 0 : 1 0 , v/v) (5
34
experiments). No SRS-T activity was found in any o f the organic extracts. T h e SRS-T activity was still present in the effluent when it was reperfused over the tissues after 2 h, at room temperature, at 4°C or 40°C (3 experiments each). The activity also withstood evaporation of the effluent and reconstitution with water to the original volume (5 experiments). In some experiments two pieces of guinea-pig ileum were used, set up in series and the lower one continuously superfused with indomethacin (2--10/~g/ml; 8 expts) or with hexam e t h o n i u m (362 ng/ml; 2 expts). The contractions p r o d u ced by SRS-T on the upper, untreated, tissue were not consistently different from those of the lower, treated, tissue.
3.1.2. PG-like activity As the SRS-T activity was not extracted by ether from the spasmogen containing effluent at pH 3, we expected the material which had been extracted to be PG-like. The extracted material was assayed on rat stomach strip, c h i c k rectum, gerbil colon, guinea-pig ileum and rat colon and its effects on these tissues were matched most closely by those of PGE2. We investigated the relation between the concentration of t r y p t a m i n e or 5-HT infused and the a m o u n t o f PG-like activity extracted from the effluent. In these experiments lung effluent was collected for 5 min periods before, during and after an infusion of the t r y p t a m i n e through the lung. The effluent samples were extracted and the activity in the organic phase assayed in terms of PGE2. The low basal release o f PG-like material from the lung increased during the amine infusion period and then returned to basal levels. No increase in PG-like activity in the effluent was caused by t r y p t a m i n e infusion direct to the assay tissues. The difference between basal and amineinduced o u t p u t of PG-like activity (net PGlike activity) was taken as the response and the variation o f this response with concentration of amine infused is shown in fig. 2. Five
Y.S. BAKHLE, T.W. SMITH
20
NS
p
