Use of the Rabbit Intestine in Smooth Muscle Pharmacology ...

Chapter 1 Use of the Rabbit Intestine in Smooth Muscle Pharmacology Experiments: A New Approach Richard L. Walker and Charles C. Scott Department of Biological Sciences University of Calgary Calgary, Alberta, Canada T2N 1N4

Richard L. Walker received his B.S. degree in Biology from Alma College, Alma, Michigan in 1969 and his Ph.D. in Physiology from Michigan State University in 1975. He is the coordinator of the animal physiology teaching laboratories at the University of Calgary, a position which he has held since the Fall of 1975. He has lectured in General Biology, Introductory Zoology, Human Physiology and Mammalian and Compartive Physiology. His research interests include the respiratory and acid-based physiology of fish. Charles C. Scott received a B.Sc. degree in Zoology from the University of Calgary in 1968. He has been a laboratory instructor in Introductory Zoology, and Human and Mammalian Physiology. Currently, he is the laboratory technician for the animal physiology teaching laboratories.

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INTRODUCTION The i s o l a t e d i n t e s t i n a l smooth muscle p r e p a r a t i o n i s one o f t h e c l a s s i c a l p r e p a r a t i o n s i n p h y s i o l o g y and pharmacology f o r bioassays, o r t h e s t u d y o f d r u g a c t i o n and autonomic c o n t r o l o f m o t i l i t y . T h i s p r e p a r a t i o n i s i n c l u d e d i n many "in- house" l a b o r a t o r y manuals o f v a r i o u s c o l l e g e s and u n i v e r s i t i e s around N o r t h America, and i n some c o m m e r c i a l l y prepared manuals d e a l i n g w i t h p h y s i o l o g y and pharmacology (e.g. Nicpon- Marieb, 1981). B a s i c a l l y , t h e method p r e s e n t e d i n t h i s r e p o r t i s a m o d i f i c a t i o n o f t h e o r i g i n a l Finkleman p r e p a r a t i o n (Finkleman, 1930) f o r t h e s t u d y o f t h e autonomic c o n t r o l of i n t e s t i n a l m o t i l i t y . What i s d i f f e r e n t about o u r approach i s t h e method of mounting t h e p r e p a r a t i o n and t h e method o f s t i m u l a t i o n of t h e sympathetic nerve. The c l a s s i c way o f mounting t h e p i e c e o f i n t e s t i n e i s t o suspend it ve r t i c a l l y i n a muscle b a t h between an a e r a t i o n t u b e and a r e c o r d i n g l e v e r . The problems w i t h t h i s t e c h n i q u e a r e ( 1 ) s t r e s s p l a c e d on t h e i n t e s t i n e when t h e chamber i s emptied d u r i n g t h e process o f changing s o l u t i o n s , and (2) d i f f i c u l t y i n s t i m u l a t i n g t h e sympathetic nerve due t o t h e f a c t t h a t t h e p r e p a r a t i o n i s t o t a l l y submerged i n a p h y s i o l o g i c a l s a l i n e s o l u t i o n . W e h ave overcome t h e s e problems by mounting t h e segments of g u t h o r i z o n t a l l y i n a s h a l l o w muscle b a t h . As a r e s u l t , l e s s s t r e s s i s p l a c e d on t h e i n t e s t i n e d u r i n g changeover o f s o l u t i o n s , and it is e a s i e r t o m a n i p u l a t e and t o s t i m u l a t e t h e s y m p a t h e t i c n e r v e c o n t a i n e d w i t h i n t h e mesentery. A l s o s t u d e n t s f i n d i t much e a s i e r t o mount t h e p r e p a r a t i o n i n t h e h o r i z o n t a l b a t h and a r e l e s s l i k e l y t o s t r e t c h t h e muscle p r e p a r a t i o n i n t h e process. With these m o d i f i c a t i o n s we have improved t h e s t u d e n t success r a t e from 50-60% t o 90-100%. Large r e c o r d i n g s o f t h e muscle c o n t r a c t i o n , such as those shown i n Appendix A, a r e e a s i l y o b t a i n a b l e u s i n g a kymograph and s i m p l e l e v e r system. I n f a c t , a n o t h e r advantage o f t h i s e x e r c i s e i s t h a t it does n o t r e q u i r e expensive r e c o r d i n g equipment.

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INSTRUCTOR'S GUIDELINES Equipment and s u p p l i e s :

A l i s t o f t h e equipment, s u p p l i e s and s o l u t i o n s needed t o o p e r a t e t h i s l a b o r a t o r y e x e r c i s e i s g i v e n i n Appendix B. I n s t r u c t i o n s and dimensions f o r b u i l d i n g t h e muscle b a t h , s w i v e l assembly and s t i m u l a t i n g e l e c t r o d e s a r e g i v e n i n Appendix C. We recommend u s i n g a s i m p l e i s o t o n i c l e v e r system f o r r e c o r d i n g t h e muscle c o n t r a c t i o n because o f t h e ease w i t h which s t u d e n t s a r e a b l e t o comprehend and m a n i p u l a t e t h e r e c o r d i n g system t o o b t a i n e x c e l l e n t t r a c i n g s . Recordings of comparable q u a l i t y , however, a r e a l s o o b t a i n a b l e w i t h an i s o m e t r i c t r a n s d u c e r (e. g. a Physiograph F-60 f o r c e t r a n s d u c e r ) . P r e p a r a t i o n of i s o l a t e d segments of t h e r a b b i t i n t e s t i n e : The r a b b i t s h o u l d be s t a r v e d f o r 24 hours p r i o r t o use. Food i n t h e g u t w i l l r e s u l t i n a messy d i s s e c t i o n and w i l l n e c e s s i t a t e f l u s h i n g t o remove t h e g u t c o n t e n t s , a p r a c t i c e which c o u l d damage t h e i n t e s t i n e . Before s a c r i f i c i n g the r a b b i t :

1.

Prepare Tyrode's R i n g e r s o l u t i o n (see Appendix B ) and p l a c e about 250 mL o f t h i s s o l u t i o n i n a f l a s k on i c e .

2.

Cut s e c t i o n s of t h r e a d ( o r 4- 0 s i l k ) i n t o 5 cm l e n g t h s . w i l l be needed f o r each segment o f g u t .

3.

You w i l l need a p a i r o f f i n e ( i r i s ) s c i s s o r s , coarse s c i s s o r s , and curved f o r c e p s . Hemostats may a l s o be h e l p f u l i n r e t r a c t i n g t h e abdominal muscles.

4.

A l a r g e P e t r i p l a t e o r o t h e r c o n t a i n e r f o r h o l d i n g t h e segments o f g u t w i l l a l s o be necessary.

5.

Wash a l l i n s t r u m e n t s and glassware before use. hands a r e c l e a n b e f o r e h a n d l i n g t h e i n t e s t i n g .

One p i e c e

A l s o make s u r e y o u r

S a c r i f i c i n g t h e r a b b i t and removing t h e i n t e s t i n a l segments:

1.

Since a n a e s t h e t i c s can a f f e c t m o t i l i t y of t h e g u t , t h e animal s h o u l d be s a c r i f i c e d by c e r v i c a l d i s l o c a t i o n , w i t h o u t use o f a n a e s t h e t i c .

2.

Shave t h e abdomen and c a r e f u l l y vacuum t h e s u r f a c e t o remove any l o o s e o r c u t fur.

3.

Make a m i d - l i n e i n c i s i o n t h r o u g h t h e s k i n and abdominal muscle u s i n g a p a i r of coarse s c i s s o r s .

4.

Locate t h e ileum, a s e c t i o n l o c a t e d n e a r t h e end of t h e s m a l l i n t e s t i n e . Note t h e i n t e s t i n a l a r t e r i e s and v e i n s c o u r s i n g t h r o u g h t h e mesentery t o and f r o m t h e s m a l l i n t e s t i n e . I f a segment o f t h e i l e u m i s p i c k e d up w i t h b o t h hands, t h e i n t e s t i n a l v e s s e l s a r e e a s i l y seen spaced about 2 t o 3 cm a p a r t ( F i g . l a ) . I f y o u l o o k v e r y c a r e f u l l y , a f i n e w h i t e l i n e f o l l o w i n g t h e i n t e s t i n a l a r t e r y and v e i n i n t o each segment of t h e g u t i s v i s i b l e . The w h i t e l i n e i s a branch o f t h e sympathetic n e r v e a r i s i n g f r o m t h e p r e v e r t e b r a l g a n g l i o n .

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5

5.

6.

Using a p a i r o f f i n e curved forceps, c a r e f u l l y break through the mesentery on e i t h e r s i d e o f t h e i n t e s t i n a l vessel- sympathetic nerve complex about 4 cm away from t h e g u t (Fig. l b ) . I n s e r t a l e n g t h o f thread o r s i l k suture through t h e hole and t i e o f f the vessels and nerve. Follow t h e vessels down t o t h e gut. The vessels d i v i d e i n t o many branches j u s t above t h e surface o f t h e i n t e s t i n e . Select a 3-cm segment o f t h e i l e u m t h a t i s supplied by these vessels. Sever t h e g u t and remove t h i s segment along w i t h t h e mesentery c o n t a i n i n g t h e vessels and nerve. Place i n a P e t r i d i s h c o n t a i n i n g c o l d Tyrode's Ringer s o l u t i o n .

N.B. If t h e r a b b i t has been starved f o r 24 hours, t h e g u t segment should be f r e e o f chyme. However, if chyme i s present, it can be removed by c a r e f u l l y f l u s h i n g t h e segment w i t h Tyrode's. A s y r i n g e f i l l e d w i t h Tyrode's may be placed i n s i d e one end o f t h e segment, and u s i n g very l i t t l e pressure, t h e contents can be c a r e f u l l y f o r c e d out i n t o a waste container. Caution must be exercised because h i g h pressure w i l l severely damage t h e tissue. 7.

Repeat t h i s procedure u n t i l enough segments have been removed t o supply y o u r l a b o r a t o r y needs f o r t h e day. I f t h e e x t r a pieces of i l e u m a r e kept i n i c e c o l d Tyrode's, they w i l l s t a y v i a b l e f o r several hours. Do not s t o r e i n warm Tyrode's.

Suggestions: 1.

The i l e a l segments w i l l shorten when placed i n t o t h e c o l d Tyrode's s o l u t i o n and m o t i l i t y w i l l cease. However, when mounted i n t h e warm (37°C) Tyrode's s o l u t i o n i n s i d e t h e muscle bath, m o t i l i t y w i l l r e t u r n w i t h i n 5-10 min.

2.

As t h e muscle warms i n t h e bath, it w i l l r e l a x and segmental and p e r i s t a l t i c waves w i l l be evident.

3.

I t i s e s s e n t i a l t h a t t h e muscle baths be a t 37°C a t t h e s t a r t of class. T h i s means s t a r t i n g t h e c i r c u l a t i o n o f warm water through t h e baths a t l e a s t 1/2 hour b e f o r e class. Also, it i s e s s e n t i a l t h a t t h e e x t r a Tyrode's s o l u t i o n f o r t h e muscle baths be a t 37°C.

4.

I t i s n o t necessary t o warm t h e drug s o l u t i o n s used i n t h e experiment since they w i l l be added d i r e c t l y t o t h e muscle bath i n very small amounts (0.1 t o 1.0 mL o f drug/100 mL o f Tyrode's).

5.

One o f t h e advantages i n adding drug d i r e c t l y t o t h e bath i n concentrated form i s t h a t r a p i d changes i n muscle tonus can be c l e a r l y recorded. We recommend t h a t t h e Tyrode's s o l u t i o n be replaced between drug a d d i t i o n s so as n o t t o d i s t u r b t h e m o t i l i t y d i r e c t l y b e f o r e t h e a p p l i c a t i o n o f t h e drug. It may take 1-2 minutes f o r t h e rhythm t o s t a b i l i z e a f t e r r e p l a c i n g t h e s o l u t i o n i n t h e muscle bath.

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S t u d e n t ' s Guideline AUTONOMIC CONTROL OF THE VISCERAL SMOOTH MUSCLE OF THE RABBIT INTESTINE OBJECT: To show some of t h e autonomic p r o p e r t i e s o f smooth muscle as it occurs i n t h e r a b b i t ' s small i n t e s t i n e . EXPLANATION : A l l smooth muscle c e l l s have one t h i n g i n common: t h e a c t i n and myosin f i l a m e n t s o f t h e sarcomeres a r e n o t organized i n t o bands such as they a r e i n s k e l e t a l muscle. V i s c e r a l smooth muscle, such as t h e t y p e found i n t h e d i g e s t i v e t r a c t , i s a syncytium o f muscle c e l l s where i n t e r c e l l u l a r communication can occur v i a gap j u n c t i o n s . E l e c t r i c a l c u r r e n t i s easily conducted from one c e l l t o t h e n e x t v i a t h e gap j u n c t i o n s ; t h e r e f o r e , d e p o l a r i z a t i o n o f one muscle c e l l can q u i c k l y l e a d t o d e p o l a r i z a t i o n o f neighboring c e l l s . V i s c e r a l smooth muscle c o n t r a c t i o n occurs spontaneously, meaning t h a t muscle c e l l a c t i o n p o t e n t i a l s a r e generated w i t h o u t i n p u t from e i t h e r t h e motor o r autonomic nervous system. The muscle c e l l s undergo rhythmic o s c i l l a t i o n s i n membrane p o t e n t i a l which, o c c a s i o n a l l y , reach t h e t h r e s h o l d o f an a c t i o n p o t e n t i a l , and, thus, generate a spike. The a c t i o n p o t e n t i a l spreads v i a gap j u n c t i o n s f r o m muscle c e l l t o muscle c e l l , i n i t i a t i n g a wave o f muscle c o n t r a c t i o n i n i t s wake. V i s c e r a l smooth muscle c e l l s a l s o e x h i b i t muscle tonus, a s t a t e of long-term, steady c o n t r a c t i o n . The tonus i s v a r i a b l e , depending on t h e number o f muscle c e l l s t h a t p a r t i c i p a t e . The r h y t h m i c i t y and tonus i n h e r e n t i n t h e i n t e s t i n a l smooth muscle may be enhanced o r suppressed by two nerve plexuses found between t h e l a y e r s o f muscle and mucosa (Auerbach's and Meissner's plexuses). Known as t h e e n t e r i c nervous system o f t h e gut, t h e a c t i v i t y o f the plexuses can be m o d i f i e d by t h e autonomic nervous system. When a piece o f i n t e s t i n e i s removed f o r studv.. t h e plexuses remain v i a b l e and can be s t i m u l a t e d o r i n h i b i t e d u s i n g parasympathomimetic o r sympathomimetic drugs (mimicing t h e a c t i o n o f t h e parasympathetic and sympathetic nervous systems). The autonomic c o n t r o l o f t h e g u t i s very complicated. Figures 2 and 3 i l l u s t r a t e t h e parasympathetic and sympathetic input t o t h e e n t e r i c neurons o f Auerbach's and Meissner's plexuses.

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F i g u r e 2* (N)

NANC

Sympathetic s t i m u l i subdue t h e i n h e r e n t r h y t h m i c i t y o f t h e gut. Note t h a t t h e sympathetic i n p u t i s upon alpha- adrenergic receptors l o c a t e d on t h e axon t e r m i n a l s o f t h e e n t e r i c neurons, o r upon t h e axon t e r m i n a l s o f t h e p r e g a n g l i o n i c parasympathetic f i b e r s . Release o f norepinephrine from t h e sympathetic p o s t g a n g l i o n i c f i b e r s s t i m u l a t e s t h e alpha receptors, which prevent release o f a c e t y l c h o l i n e from t h e e n t e r i c neurons o r p r e g a n g l i o n i c parasympathetic f i b e r s . This a c t i o n i s known as p r e s y n a p t i c i n h i b i t i o n . There are alpha- and beta- adrenergic receptors on t h e smooth muscle c e l l s which will respond t o epinephrine and norepinephrine i n t h e c i r c u l a t i o n , as shown i n Figure 3. Both alpha and beta receptors i n h i b i t muscle tone and rhythmici t y . Figure 3*. ADRENAL MEDULLA

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INTRINSIC GANGLION

The parasympathetic c o n t r o l o f t h e g u t i s v i a c h o l i n e r g i c preganglionic f i b e r s which synapse w i t h e n t e r i c neurons. When a c t i v a t e d , t h e parasympathetic f i b e r s release a c e t y l c h o l i n e a t t h e e n t e r i c neurons, thus s t i m u l a t i n g t h e e n t e r i c neurons t o release a c e t y l c h o l i n e a t t h e i r neuro-muscular j u n c t i o n s . Enhanced smooth muscle tone and r h y t h m i c i t y result. *Reprinted from " I n n e r v a t i o n o f t h e G a s t r o i n t e s t i n a l T r a c t " i n A Guide t o G a s t r o i n t e s t i n a l M o t i l i t y , Christensen and Wingate (eds.), w i t h permission from John Wright Medical Publishers.

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The r e c e p t o r s o f t h e neuromuscular j u n c t i o n a r e m u s c a r i n i c type c h o l i n e r g i c receptors. They can be blocked by t h e a d d i t i o n o f a t r o p i n e t o t h e muscle preparation. I n t h e f o l l o w i n g l a b o r a t o r y e x e r c i s e you w i l l s t u d y t h e a c t i o n of sympathet i c nerve s t i m u l a t i o n , and t h e e f f e c t s o f v a r i o u s pharmacological agents on t h e r h y t h m i c i t y and tonus o f t h e r a b b i t g u t . The agents w i l l i n c l u d e sympatho- o r parasympathomimetic drugs, along w i t h adrenergic and c h o l i n e r g i c b l o c k i n g agents.

PROCEDURES: The r h y t h m i c a l a c t i v i t y o f the piece o f r a b b i t gut w i l l be recorded u s i n g a l e v e r system and kymograph. To m a i n t a i n i t s a c t i v i t y , t h e g u t w i l l be suspended i n a b a t h o f Tyrode's s o l u t i o n a t 37ºC w i t h adequate oxygen supply. ( A m i x t u r e o f 95% 02 - 5% CO2 w i l l be used). Wash y o u r hands thoroughly wi t h soap and w a t e r b e f o r e h a n d l i n g t h e gut. Body s a l t s , o i l a n d d i r t w i l l k i l l t h e g u t t i s s u e .

FIGURE 4.

Arrangement o f I n t e s t i n e i n Smooth Muscle Bath

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Preparation: The muscle chamber i s s e t up as i n F i g u r e 4. Warm water i s c i r c u l a t e d One end o f a through t h e o u t e r b a t h t o keep t h e muscle chamber a t 37°C. s e c t i o n o f i n t e s t i n e i s attached t o t h e anchor p i n i n t h e chamber u s i n g The o t h e r end i s connected v i a an S-hook t o a swivel which an S-hook. transposes h o r i z o n t a l c o n t r a c t i o n s and r e l a x a t i o n s o f t h e g u t i n t o v e r t i c a l movement of a r e c o r d i n g l e v e r . The movement o f the r e c o r d i n g l e v e r a r e t r a c e d upon a kymograph drum, o r c h a r t r e c o r d e r .

1.

A t t a c h two S-hooks t o the gut, on o p p o s i t e sides, a t t h e opposing ends. During t h i s operation, keep t h e g u t i n a P e t r i p l a t e c o n t a i n i n g Tyrode's s o l u t i o n . Do n o t a l l o w t h e t i s s u e t o dry.

2.

A t t a c h one hook t o the anchor p i n i n t h e muscle chamber. o t h e r hook t o t h e swivel.

3.

F i l l t h e chamber w i t h 100 mL o f warm Tyrode's s o l u t i o n and bubble 95% 02-5% CO2 gas i n t o t h e chamber f l u i d . There should be a f i n e stream o f bubbles, r a t h e r than l a r g e bubbles which w i l l d i s r u p t y o u r recordings.

4.

A t t a c h a t h r e a d from t h e muscle l e v e r t o t h e r e c o r d i n g l e v e r u s i n g p l a s t i c i n e . Remove slack from t h e t h r e a d by adding p l a s t i c e n e t o t h e r e c o r d i n g l e v e r t o a c t as a counter- balance. Keep t h e recording l e v e r horizontal.

Fasten t h e

Experiment: During t h e f o l l o w i n g experiment, check t h a t y o u r t r a c i n g s a r e l a b e l l e d c l e a r l y . Be sure t h a t a l l drug c o n c e n t r a t i o n s , washes and s o l u t i o n changes a r e l a b e l l e d so as n o t t o confuse a drug response w i t h an a r t i f a c t . The "normal" muscle r h y t h m i c i t y and tonus (i.e. p r i o r t o drug a p p l i c a t i o n ) w i l l v a r y throughout t h e experiment. Compare the drug e f f e c t t o the "normal" muscle c o n t r a c t i o n i n Tyrode's j u s t preceding t h e a d d i t i o n o f the drug.

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A.

A c e t y l chol i n e and N o r e p i n e p h r i n e : These two compounds a c t as p o s t g a n g l i o n i c n e u r o t r a n s m i t t e r s o f t h e parasympathetic and s y m p a t h e t i c nervous systems, r e s p e c t i v e l y . Apply t h e drugs i n t h e o r d e r l i s t e d below. A f t e r you have noted t h e e f f e c t of t h e drug, d r e i n t h e muscle b a t h , and r e f i l l w i t h 100 mL of f r e s h , warm Tyrode's s o l u t i o n . A f t e r t h e muscle c o n t r a c t i o n s have r e t u r n e d t o t h e normal r h y t h m i c i t y and tonus, you may add t h e n e x t drug. Add d r o p s t o t h e b a t h away from t h e g u t ; m i x i n g o c c u r s q u i c k l y by d i f f u s i o n . Check b a t h temperature f r e q u e n t l y .

B.

1.

10 - 7 M a c e t y l c h o l i n e : bath.

add 0.1

mL

2.

10 - 6 M acetylcholine: bath.

add 1.0

mL

3.

10 - 7 M n o r e p i n e p h r i n e : bath.

add 0.1

4.

10 - 6 M n o r e p i n e p h r i n e : bath.

add 1.0 mL o f 10 - 4 M n o r e p i n e p h r i n e t o t h e

mL

10 - 4 M a c e t y l c h o l i n e t o t h e

of

of

10 - 4 M acetylcholine t o t h e

o f 10 - 4 M n o r e p i n e p h r i n e t o t h e

Alpha- and B e t a - A d r e n e r g i c A g o n i s t s :

1.

D r a i n and r e f i l l t h e chamber w i t h f r e s h , warm T y r o d e ' s . A p p l y 1.0 mL o f 10 - 4 M p h e n y l e p h r i n e and n o t e t h e response. P h e n y l e p h r i n e i s an a l p h a - a d r e n e r g i c a g o n i s t ( s t i m u l a n t ) .

2.

D r a i n t h e chamber and wash t h e g u t w i t h Tyrode's. Refill the 4 chamber and add 1.0 mL o f 10 M isoproterenol, a beta- adrenergic agonist.

What can you conclude about t h e t y p e p r e s e n t i n t h e smooth muscle o f t h e g u t ?

C.

D.

of

adrenergic

receptors

Sympathetic Nerve S t i m u l a t i o n :

1.

Mount t h e s t i m u l a t i n g e l e c t r o d e on t h e s i d e o f t h e muscle chamber. C a r e f u l l y s l i d e t h e mesentery c o n t a i n i n g t h e sympathetic n e r v e i n t o t h e e l e c t r o d e s l e e v e v i a t h e t h r e a d a t t a c h e d t o t h e mesentery.

2.

S t i m u l a t e t h e n e r v e w i t h 30V (0.5 msec d u r a t i o n , 15 pps) f o r 10-20 Note t h e change i n seconds. If you do n o t see a response t r y 40V. tonus and r h y t h m i c i t y .

Alpha and Beta A d r e n e r g i c Blockade

1.

P l a c e 1.0 mL o f 10 - 4 M p h e n t o l a m i n e i n t h e chamber. minutes, t h e n t r y s t i m u l a t i n g t h e s y m p a t h e t i c n e r v e .

11

W a i t one o r two

2.

Add 1.0 mL o f 10 - 4 M phenylephrine. If the alpha receptors are blocked, effect.

3.

D r a i n and r e f i l l t h e chamber w i t h warm, Add 1.0 mL o f 10 - 4 M p r o p r a n o l o l . Wait two t o t h r e e minutes, nerve.

4.

f r e s h Tyrode's solution.

t h e n t r y s t i m u l a t i n g t h e sympathetic

Add 1.0 mL o f 10-4 M i s o p r o t e r e n o l t o t h e chamber. I f t h e beta receptors are blocked, effect.

E.

p h e n y l e p h r i n e s h o u l d have no

Atropine

-

isoproterenol

should have no

a c h o l i n e r g i c b l o c k i n g agent

D r a i n and r e f i l l t h e b a t h w i t h warm, f r e s h T y r o d e ' s s o l u t i o n . Add 1.0 A t r o p i n e b l o c k s t h e a c t i o n o f a c e t y l c h o l i n e and mL o f 10 - 4 M a t r o p i n e . i s , t h e r e f o r e , a p a r a s y m p a t h o l y t i c d r u g . W a i t two t o t h r e e m i n u t e s and t h e n add 1.0 mL o f 10 - 4 M a c e t y l c h o l i n e t o t h e b a t h . Does t h e a t r o p i n e successfully block the action o f ,acetylcholine?

12

APPENDIX A The c h a r t recordings of the m o t i l i t y o f an i s o l a t e d segment o f r a b b i t ileum, shown below and on t h e f o l l o w i n g pages, were c o l l e c t e d by a p a i r o f s t u d e n t s u s i n g a kymograph and i n k- r e c o r d i n g l e v e r system. The i n t e s t i n e was mounted h o r i z o n t a l l y i n a muscle b a t h c o n t a i n i n g Tyrode's s o l u t i o n a t 37°C. The p r e p a r a t i o n was aerated w i t h a m i x t u r e o f 5% C02-95% 02. Small volumes of concentrated drug s o l u t i o n s were added d i r e c t l y t o t h e muscle b a t h a t t h e p o i n t s i n d i c a t e d i n each record. The Tyrode's s o l u t i o n was r e p l a c e d between drug a d d i t i o n s and t h e m o t i l i t y was allowed t o s t a b i l i z e b e f o r e t h e n e x t a d d i t i o n o f drug. S t i m u l a t i o n of t h e sympathetic nerve was performed a t 15 pulses/sec, w i t h a p u l s e d u r a t i o n of 0.5 msec and s t r e n g t h o f 30-40 v o l t s (A.C.), f o r 20 seconds. See t e x t of t h e e x e r c i s e f o r o t h e r details.

Tyrode's S o l u t i o n

t Added 10-7 M A c e t y l c h o l i n e

paper speed = 1 mm/sec

Tyrode's s o l u t i o n

t

Added 10-6 M A c e t y l c h o l i n e paper speed = 1 mm/sec

13

14

15

APPENDIX B L i s t of equipment/group of s t u d e n t s : muscle b a t h and s w i v e l assembly clamp f o r muscle chamber d r a i n t u b e thermometer spool of t h r e a d 2-"S" hooks wax p e n c i l 7 30-mL beakers 250-mL beaker Petri dish 100-mL graduated c y l i n d e r p i p e t b u l b o r Pi-Pump 0.1-mL p i p e t 1-mL p i p e t P a s t e u r p i p e t and b u l b platinum wire electrodes s t i m u l a t o r (capable of d e l i v e r i n g 10-100 v o l t s A.C.) r e c o r d i n g d e v i c e (e.g. kymograph o r Physiograph ) stand f o r h o l d i n g transducer o r recording l e v e r plasticene M a t e r i a l s a t s u p p l y bench: extra plasticene r e c o r d i n g paper f o r c h a r t mover o r kymograph e x t r a Pasteur p i p e t s and b u l b s recording ink w a t e r b a t h a t 37ºC Ringer s o l u t i o n

c o n t a i n i n g 2-L b o t t l e s of T y r o d e ' s

carboy w i t h e x t r a T y r o d e ' s s o l u t i o n (make up 2 L/group o f s t u d e n t s ; we r u n through about 100 L/50 groups of s t u d e n t s ) . t h e f o l l o w i n g drugs made up i n T y r o d e ' s s o l u t i o n : each l a b * , and p r o t e c t f r o m l i g h t ) 10-4 M norepinephrine 10-4 M acetylcholine 1 0 - 44 M p h e n y l e p h r i n e 10- M isoproterenol

*

(make f r e s h p r i o r t o

10-4 M atropine 10-4 M propranolol 10 - 4 M phentolamine

see p.18 c o n c e r n i n g p r e p a r a t i o n o f t h e s e d i l u t i o n s .

16

Common Apparatus : Warm w a t e r must be r e c i r c u l a t e d t o each muscle b a t h . We use one w a t e r b a t h t o s u p p l y two muscle baths. Water i s pumped f r o m t h e c e n t r a l w a t e r b a t h t o each u n i t and back. Because o f t h e t e m p e r a t u r e d r o p en r o u t e , we s e t t h e c e n t r a l b a t h t h e r m o s t a t t o 40-45ºC, depending on t h e d i s t a n c e t h e w a t e r must t r a v e l and t h e v e l o c i t y o f f l o w . If you w i s h t o c o n s t r u c t such a system, a s t u r d y s u b m e r s i b l e pump and Tygon t u b i n g a r e necessary. There a r e commercial organ b a t h s a v a i l a b l e t h a t c o n t a i n b u i l t - i n h e a t i n g elements and a v e r t i c a l muscle chamber.

A system f o r d e l i v e r i n g a 5% C02-95% 0, gas m i x t u r e i s necessary. We use one 200 cu. ft. t a n k o f gas t o s u p p l y 10 s t a t i o n s . This provides enough gas f o r f i v e 3 - h r l a b o r a t o r y s e s s i o n s ( w i t h a 1 / 4 t a n k reserve). The gas stream t o each muscle b a t h need o n l y be enough t o p r o v i d e a s t e a d y stream of s m a l l bubbles. Tygon t u b i n g i s used t o d e l i v e r t h e gas t o each s t a t i o n . The g u t segments s u r v i v e much l o n g e r and t h e c o n t r a c t i o n s a r e more r e g u l a r u s i n g t h e 5% C02-95% 02 m i x t u r e t h a n w i t h a i r o r p u r e 0 2 . Tyrode's R i n g e r S o l u t i o n : NaCl

8.0 g

KCl

0.2 g

NaHCO3

1.0 g

MgCl2.6 H20

0.1 g

CaCl2.2 H20* NaH2PO4.H2O

Dextrose

*

0.1 g 0.05 g

1.0 g

d i s s o l v e t h e c a l c i u m c h l o r i d e s e p a r a t e l y and add l a s t

B r i n g up t o 1 L w i t h d i s t i l l e d water.

17

A d r e n e r g i c and Chol i n e r g i c Drugs:

A l l t h e drugs mentioned i n " M a t e r i a l s a t s u p p l y bench" a r e a v a i l a b l e f r o m Sigma Chemical Co., w i t h t h e e x c e p t i o n o f phentolamine, which u s u a l l y can The names and Sigma c a t a l o g u e be purchased t h r o u g h a l o c a l pharmacy. numbers a r e shown below: d r u g name

Sigma c a t . No.

( - ) norepinephrine (arterenol hydrochloride)

A 7381

L- phenylephrine hydrochloride

P 6126

(±)

I 5627

isop r o t e r e n o l h y d r o c h l o r i d e

acetylcholine chloride

A 6625

DL- propranolol hydrochloride

P 0884

atropine

A 0132

P r e p a r a t i o n o f 10-4 M d i l u t i o n s o f each d r u g To f a c i l i t a t e ease and a c c u r a c y i n p r e p a r a t i o n , 100 mL o f a 1 0 - 3 M s t o c k s o l u t i o n o f each d r u g i s p r e p a r e d a t t h e b e g i n n i n g o f t h e week and s t o r e d Just p r i o r t o i n a r e f r i g e r a t o r . A c i d i f y w i t h 2-3 drops o f 2 . 0 M H Cl. each l a b o r a t o r y s e s s i o n , a 1:10 d i l u t i o n o f t h e s t o c k s o l u t i o n i s made, u s i n g T y r o d e ' s , t o o b t a i n 10 - 4 M.

18

APPENDIX C Constructing the Muscle Bath F i g u r e 5 i l l u s t r a t e s t h e muscle bath used i n t h e measurement o f t h e m o t i l i t y of t h e r a b b i t i n t e s t i n e . As shown i n t h e diagram, the b a t h i s c o n s t r u c t e d of 1/4-inch and 1/8-inch a c r y l i c p l a s t i c . A water j a c k e t surrounds t h e muscle chamber and allows warm w a t e r t o be c i r c u l a t e d on t h r e e s i d e s of t h e chamber t o keep the g u t a t 37°C. An a e r a t i o n tube, made from P.E. 90 p o l y e t h y l e n e tubing, i s glued around t h e bottom o f t h e chamber. The tube i s perforated w i t h small h o l e s over i t s e n t i r e l e n g t h t o p r o v i d e maximum a e r a t i o n and m i x i n g o f t h e Tyrode's s o l u t i o n w i t h a f i n e stream of bubbles. The shank o f a 20 gauge needle i s attached t o t h e end o f t h e a e r a t i o n tube as a means of connection w i t h an a i r l i n e from a tank o f compressed 5% C02-95% 02 gas. A small p l a t f o r m a t t a c h e d t o one end o f t h e chamber c o n t a i n s a p i n hole. The p l a t f o r m serves as a h o l d e r f o r a push p i n , which i s used t o anchor one end o f t h e p r e p a r a t i o n i n p o s i t i o n . The g u t segment i s mounted h o r i z o n t a l l y and a t t a c h e d t o a swivel and t h e push p i n v i a " S " h ooks (see Fig. 4 ) . When a t t a c h e d t o a w r i t i n g s t y l u s o r a t r a n s d u c e r l e v e r w i t h thread, t h e swivel serves t o c o n v e r t h o r i z o n t a l movements o f t h e g u t i n t o v e r t i c l e movements o f t h e s t y l u s o r transducer l e v e r . A d r a i n i s attached near the bottom o f t h e muscle chamber. Except f o r t h e a e r a t i o n tube, which i s epoxyed t o t h e bottom of t h e chamber, t h e e n t i r e u n i t i s g l u e d t o g e t h e r w i t h dichloromethane. C o n s t r u c t i n g t h e Swivel Assembly Figures 6a and 6b show the dimensions and placement o f t h e s w i v e l assembly which attaches t o one s i d e o f t h e water j a c k e t . The assembly i s c o n s t r u c t e d from pieces o f 3/4, 1/2, 1/4 and 1/8-inch a c r y l i c p l a s t i c . There a r e t h r e e p a r t s t o t h e assembly; t h e swivel, t h e p a r t t h a t holds t h e s w i v e l , and a t r a c k which i s glued t o t h e s i d e o f t h e water j a c k e t . The s w i v e l i s made from 1/8-inch p l a s t i c . The arms, which f o r m a r i g h t angle, should be 5/16-inch wide and 2 inches on a side. A small h o l e i s d r i l l e d a t t h e end o f each arm f o r attachment o f t h e hooks o r t h r e a d t o t h e muscle and r e c o r d i n g l e v e r . A l t e r n a t i v e l y , t h e ends can be notched. A 3/32-inch h o l e i s d r i l l e d a t the i n t e r s e c t i o n o f t h e two arms t o a c t as a p i v o t . When placed i n t h e holder, t h e swivel i s h e l d i n p l a c e by a 1/16-inch r o d which a c t s as an axle, as shown i n F i g u r e 6b. Refer t o F i g u r e 4 f o r a s i d e view o f t h e swivel. As shown i n d e t a i l from p i e c e s of 1/4 1/4-inch groove as it snugly i n place. ( F i g . 6b) which i s

i n F i g u r e 6a, the h o l d e r f o r t h e swivel i s c o n s t r u c t e d and 3/4-inch p l a s t i c . The s w i v e l i s mounted i n a 5/8 x shown, w i t h t h e a x l e extending through holes which h o l d A b o l t two inches l o n g secures the h o l d e r onto a t r a c k glued t o t h e s i d e of t h e water j a c k e t .

The t r a c k i s made from a p i e c e o f 1/2-inch p l a s t i c which extends t h e f u l l l e n g t h o f t h e water j a c k e t . A 3/16-inch s l o t i s c u t i n t o t h e p l a s t i c as shown i n F i g u r e 6b. When h e l d i n the s l o t by t h e b o l t , t h e h o l d e r f o r t h e s w i v e l can be p o s i t i o n e d t o accommodate t h e l e n g t h o f t h e i n t e s t i n a l segment.

19

Constructing the Electrode The s t i m u l a t i n g e l e c t r o d e c o n s i s t s o f a p a i r o f p l a t i n u m w i r e s which a r e encased i n an epoxy c u f f ( s e e F i g . 7 ) . Two 2-cm p i e c e s o f 28-30 gauge p l a t i n u m w i r e a r e s o l d e r e d t o two I - m e t e r l e n g t h s of 20 gauge s t e e l w i r e , which have been b r a i d e d t o g e t h e r . The s o l d e r j o i n t s a r e i n s u l a t e d w i t h a l a y e r o f epoxy t o keep them f r o m making a s h o r t c i r c u i t . The p l a t i n u m w i r e s a r e t h e n l o o p e d t o f o r m a c i r c l e about 3 mm i n d i a m e t e r . The i n s i d e c u r v a t u r e o f t h e l o o p i s f i l l e d w i t h wax and then t h e t e r m i n a l 2- 3 cm o f t h e e l e c t r o d e i s d i p p e d i n epoxy s e v e r a l times t o f o r m a p r o t e c t i v e l a y e r around t h e p a t i n u m w i r e s . When t h e epoxy has hardened, t h e wax l a y e r on t h e i n s i d e c u r v a t u r e o f t h e e l e c t r o d e s i s scraped o r d i s s o l v e d away t o expose t h e b a r e p l a t i n u m w i r e .

20

21

22

23

ACKNOWLEDGEMENTS The a u t h o r s w i s h t o e x t e n d t h e i r g r a t i t u d e t o Dr. J. S. Davison and B. Greenwood of t h e Department o f M e d i c a l P h y s i o l o g y , U n i v e r s i t y o f C a l g a r y f o r t h e i r i n v a l u a b l e a s s i s t a n c e i n d e v e l o p i n g t h i s e x e r c i s e . Thanks a r e a l s o extended t o Mrs. F l o r e n c e Robertson f o r h e r a s s i s t a n c e i n p r o o f r e a d i n g t h e manuscript.

24

REFERENCES

A

Davison, J. S. 1983. I n n e r v a t i o n of t h e g a s t r o i n t e s t i n a l t r a c t . I n : Guide t o G a s t r o i n t e s t i n a l M o t i l i t y . Christensen and Wingate, (eds). Wright and Sons. B r i s t o l , U.K. Finkleman, B. 1930. On t h e n a t u r e o f i n h i b i t i o n i n t h e i n t e s t i n e . Physiol. 70: 145-157.

J.

Goodman, L. S. and A. Gilman. 1975. The Pharmacological Basis of Therapeutics. 5 t h ed. MacMillan and Co., New York. Nicpon-Marieb, E. 1981. Human Anatomy and Physiology. P u b l i s h i n g Co., Menlo Park, Ca.

Benjamin/Cummings

S t a f f o f Department o f Pharmacology, U n i v e r s i t y o f Edinburgh. 1970. Pharmacological Experiments on I s o l a t e d Preparations. E. And S. Livingstone, Edinburgh, U.K.

25