Effects of substance P on inositol triphosphate accumulation, on contractile responses and on arachidonic acid release and prostaglandin biosynthesis in rabbit iris sphincter muscle

Exp. E2/e Res. {198t~) 43, 21t'~-226

Effects of Substance P on Inositol Triphosphate Accumulation, on Contractile Responses and on Arachidonic Acid Release and Prostaglandin Biosynthesis in Rabbit Iris Sphincter Muscle S A R D A R Y. 1~. Y O U S U F Z A I , I ~ A S I t l D A. A K I t T A R AIqD A T A A. A B D E L - L A T I F

Department of Cell a~d 3]olecular ]3iology, Medical College of Georgia, Augusta, GA 30912, U . S . A . (Received 9 October 1985 and in revised f o r m 17 February 1986} Addition of substance P (10 -7 to 10-~ m) to rabbit iris sphincger muscle induee(t: (a) a rapid phosphodiest(;ratic hre~tkdown of phosl)hatidylinositol 4,5-bisphosphate (PIPz) into 1,2diaeylglycero} (])G), measured as phosphatidic acid, and in(mitol triphosphate (1Pa}, measured by anion-exchange ehromatogral)hy; (b) a rapid and strong contractile response, and (e) a rapid rt~le,~e of prostaglandin E z (PGE2), measured by radioimmtmoassay, and rapid release of I~C-labe~ed arachidonic acid, measured by radiochromatography. The.~e substance P actions are cot~c~entration and time-del~r~dent, and are blocked by substance P antagonist, ll>-]'ro ~, DTrpT'~]SP. The effects of sut)st~mee P on arachidonie acid release and PG synthesis are not mediated through the cyelo-oxygenase and lipoxygenase pathways. Substance P exerted little effect on PG Ea velea~ by the iris dilator muscle. IVe conclude that substance P, which is liberated t¥om the sensory nerves t h a t innerwtte the sphincter region of the iris and plays a role in miosis, may function as a Ca~+-mt~bilizing agonist in this tissue. Thus, a substance P=induced release of IPa and formation of DG, a source for arachidonic acid in PG synthesis, followed by Ca~+ mobilization could underlie the mechanism for the biological actions, such as muscle co~traction, of the neurolmptide reported ir~ the eye. However, the preei~ relationship remains to l)e established. Eey words: Iris sphincter; substance P; phoaphoinositides; arachidonic acid release; prostagland|as; inositol triphosphate; contraction.

I. I n t r o d u c t i o n

~NTerve fibers c o n t a i n i n g t h e u n d e e a p e p t i d e s u b s t a n c e P a r e w i d e l y d i s t r i b u t e d in mammalian tissues (Hokfelt, Johansson, Ljungdahl, Lundberg and Schultzberg, 1980); e s p e c i a l l y (lense s u b s t a n c e P n e t w o r k s a r e f o u n d i a t i s s u e s i n n e r v a t e d b y t h e t r i g e m i n a l n e r v e s y s t e m s u c h a s ttm e y e . S u b s t a n c e P - c o n t a i n i n g n e u r o n s a r e p r e s e n t in a u t o n o m i c g a n g l i a , b l o o d vessels a n d s m o o t h m u s c l e , a n d t h e n e u r o p e p t i d e h a s b e e n i m p | | c a r e d b o t h as a n e u r o t r a n s m i t t e r a n d a n e u r o h o r m o n e r e l e a s e d b y p r i m a r y s e n s o r y n e u r o n s (for r e v i e w see B a r k e r , 1976). S u b s t a n c e P h a s a w i d e s p e c t r u m o f b i o l o g i c a l action, s, i n c | u d i n g s t i m u l a t i o n o f s a l i v a r y seeretion, tmurotransmission o f p a i n , c o n t r a c t i o n o f s m o o t h m u s c l e a n d p e r i p h e r a l vasodilation r e s u l t i n g in a l o w e r i n g o f b l o o d p r e s s u r e (for r e v i e w see P e r n o w , 1983). J[n t h e e y e , s u b s t a n c e P is p r o b a b l y t h e p e r i l ) h e r a | s e n s o r y t r a n s m i t t e r s u b s t a n c e a f f e c t i n g a m i o s i s (Bill, S t j e r n s c h a n t z , M a n d a h l , B r o d i n a n d N i i s s o n , t 9 7 9 ; B u t l e r a n d H a m m o n d , 1980; C a m r a s a n d B i t o , I 9 8 0 ; M a n d a h l a n d Bill, 1981 ; B a n n o , I m a i z u m i a n d W a t a n a b e , 1985). S u b s t a n c e P is a s t r o n g m i o t i c , b o t h w h e n i n f u s e d i n t r a c a m e r a l l y o r w h e n a p p l i e d t o a n i s o l a t e d iris s p h i n c t e r p r e p a r a t i o n o b t a i n e d f r o m n o r m a l - o r s e n s o r i l y d e n e r v a t e d r a b b i t e y e s ( B u t l e r a n d H a m m o n d , 1980; M a n d a h l a n d Bill~ Address correspondence to D r A t a A. Abdel-Latif, Department of Cell and Molecular Biology, Medical College of Georgia, Augusta, Georgia 30912: : .... 0014-4835]86/080215 + 12 $ 0 3 . 0 0 / 0

(~) 1986 Academic Press Inc. (London) Limited

216

S.Y.K. YOUSUFZAI ET AL.

1981; Stjernschantz, Sears jand Stjernschantz, 1981; Bito, Nichols and Baroody, 1982; Zhang, Butler, Ohara and Cole, 1982, U n g e r and Tighe, 1984). F u r t h e r m o r e , in the eye, substance P elicits hyperemia, miosis and a breakdown of the b l o o d - a q u e o u s barrier (Bill et al., 1979; Holmdahl et al., 1981). Similar responses, which are characteristic of the inflammatory reponse to trauma, can also be elicited by antidromic stimulation of t h e sensory innervation to the eye. Intravitreal or topical application into the eye of the substance P antagonsist, [D-Pro ~, v-TrpT.g]SP, inhibited not only the irritant effects of exogenous substance P b u t also the inflammatory response to t h e t r a u m a induced by infrared irradiation of t h e eye (Holmdahl et al., 1981). The molecular mechanism underlying the biological actions of substance P in the eye (Bill, 1985) and o t h e r tissues (Pernow, 1983) has n o t y e t been elucidated. Isolated iris sphincter muscles from rabbit, pig and cow showed stron~ and consistent responses to substance P whilst those from t h e cat, dog, baboon and h u m a n eyes did n o t contract (Unger and Tighe, 1984). I t is generally assumed t h a t an increase in the intracellular [Ca ~+] is a prerequisite for t h e activation of the contractile apparatus. Thus in guinea-pig intestinal muscle, substance P increases intracelluar [Ca 2+] both by an increased influx of Ca 2+ (Bury a n d Mashford, 1976; Fujisawa and Ito, 1982; Holzer and Petsche, 1983) and a release of Ca 2+ from intracellular stores (Holzer and Lippe, 1984). Since agonists t h a t lead to Ca ~+ mobilization also cause a rapid breakdown of m e m b r a n e polyphosphoinositides, it has been suggested t h a t the breakdown of inositol phospholipids is a mechanism t h a t links receptor activation to Ca ~+ mobilization (for reviews see Berridge and Irvine, 1984; Exton, 1985; AbdelLatif, in press). Substance P has'been reported to cause an accumulation of inositol phosphates, indicative of phosphoinositide breakdown, in guinea-pig ileum (Watson and Downes, 1983) and intestinal smooth muscles (Holzer and Lippe, 1985). Activation of Ca~+-mobilizing receptors, such as muscarinic cholinergic a n d aladrenergic, in the rabbit iris muscle leads to cleavage of phosphatidylinositol 4,5-bisphosphate (PIP2) into 1,2-diacylglycerol (DG), measured as phosphatidic acid (PA), a n d inositol trisphosphate (IPa) (for reviews see Abdel-Latif, Smith a n d Akhtar, 1985; A k h t a r and Abdel-Latif, 1984). F u r t h e r m o r e , activation of the same type of receptors in the iris leads to the release of arachidonic acid for prostaglandin (PG) biosynthesis (Yousufzai and Abdel-Latif, 1983, 1984). More recently we have focused our efforts on the c0ncept t h a t the agonist-stimulated breakdown of P I P 2 in the iris muscle is a p r i m a r y (initial) e v e n t t h a t could couple activated Ca~+-mobilizing receptors, through mobilization of cellular Ca 2+, to muscle contraction (Akhtar and Abdel-Latif, 1985; Abdel-Latif, Howe and A k h t a r , in press) and to arachidonic acid release and PG synthesis (Yousufzai and Abdel-Latif, 1984; Abdel-Latif, Naderi and Yousufzai, in press). To shed more light on t h e molecular mechanism underlying the biological action of substance P in the eye we have investigated the effects of the neuropeptide on PIP~ breakdown, measured as IP3 release, on muscle contraction and on arachidonic acid release for PG synthesis in the r a b b i t iris sphincter muscle. We have found t h a t substance P provokes increases in IPa accumulation, in muscle contraction and in arachidonic acid release for PG synthesis in this tissue. 2. M a t e r i a l s a n d M e t h o d s Chemical8 Substance P and substance P antagonist, [D-Pro2, D-Trp~.9]SP, were purchased from Sigma Chemical Co., St Louis, MC~.PGE 2 [Sill RIA Kit was obtained from Seragen Inc., Boston, MA;

E F F E C T S OF SUBSTANCE P

217

and [l-14C]arachidonic acid (56"5 mCi mmo1-1) and myo-[SH]inositol (15"5 Ci mmo1-1) were purchased from Amersham Corp., Arlington Heights, IL. All other chemicals were of reagent grade.

Preparation of irides, sphincter and dilator muscles Albino rabbits of either sex weighing approximately 2 kg were used in the present work. The rabbits were killed by cervical dislocation. The eyes were enucleated immediately and irides were removed. The sphincter and dilator muscles were dissected and placed in pairs from the same animal in 2 ml of K r e b s - R i n g e r bicarbonate buffer t h a t contained 10 mM Vg l u c o s e . The pH of the K r e b s - R i n g e r was adjusted to 7"4 with Oz-CO 2 (97:3). Incubation of sphincter muscle for studies on the effects of ~ubstance P on PIP~ breakdown and IPa accumulation (a) Incubation with a~p and phospholipid analysis. The sphincter muscles were incubated singly (of the pair from a rabbit, one served as control and the other as experimental) for 60 rain in I ml of K r e b s - R i n g e r bicarbonate buffer (pH 7'4) containing 30 pCi *sPi. A t the end of incubation, substance P was added as indicated and incubation continued for an additional 10 rain. The reaction was stopped with 10 ?/o (w/v) trichloreacetic acid (TCA) and the phospholipids were extracted and analysed for radioactivity by one- and two-dimensional thin-layer c h r o m a t o g r a p h y (TLC) as previously described (Akhtar and Abdel-Latif, 1984). (b) Incubation with myo-[SH]inositol and analysis of I Pa accumulation. Paired sphincters (from the same rabbit) were incubated at 37 °C in 1 ml of K r e b s - R i n g e r bicarbonate buffer (pH 7.4) containing I0 pCi myo-[*H]inositol for 90 rnin. At this time the sphincters were washed four times with 3 ml of non-radioactive K r e b s - R i n g e r bicarbonate buffer and then suspended singly (of the paired sphincters, one was used as a control and the other as experimental) in 1 ml of the non-radioactive buffer. Ten-millimolar LiC1 (final concentration) was added to each incubation and 10 min later substance P was added as indicated and incubation continued for an additional 10 rain. The incubations were terminated with 10 ~/o (w/v) TCA and the radiolabeled myo-inositol phosphates were analysed by anion-exchange c h r o m a t o g r a p h y (Akhtar and Abdel-Latif, 1984). Incubation and assay of PGE~ release by radioimmunoassay In general, sphincter or dilator muscles were incubated (of the pair, one was used as control and the other as experimental) in 1 ml o f K r e b s - R i n g e r bicarbonate buffer a t 37 °C for 10 min or as indicated. Substance P and other pharmacological agents were a d d e d as indicated. The reactions were ended by acidification of the medium with 10 % formic acid to pH 3"5 and addition of ethyl acetate. The solvent was evaporated under N~. The residue was dissolved in chloroform-methanol (2:1, v / v ) and PG was determined by R I A as previously described (Yousufzai and Abdel-Latif, 1983). The a m o u n t of PG in each sample was determined b y interpolation from the s t a n d a r d curve. D a t a presented in the t e x t and Figures are means+s.~.(M.).

Prelabellng of iris sphincter with [14C]arachidonic acid and assay for the release of labeled arachidonate and PGs In general, paired sphincters from the same rabbit were preincubated in 2 ml o f K r e b s Ringer bicarbonate buffer t h a t contained 0-4 pCi of [14C]arachidonic acid a t 37 °C for I hr. The prelabeled muscles were washed three times with non-radioactive medium, then incubated (of the Pair one was used as control) in the absence and presence of t h e neuropeptide as indicated. In experiments where the effects o f substance P antagonist were investigated, the antagonist was added 5 rain prior to the addition of the agonist. A t the end of incubation the medium was analysed for radioactive arachidonie acid, PGE~ and P G F ~ . The medium was acidified "zith 10 ~/o formic acid to p H 3"5 and extracted three times with 3 ml of ethyl acetate. The solvent was evaporated under N v The residue was dissolved in chloroform-methanol (2:1, v/v), spotted on W h a t m a n precoated silica gel LI~6DF plates and developed in a solvent system (Hamberg and Samuelsson, 1966) of ethyl a c e t a t e - a c e t i c a e i d - t r i m e t h y l p e n t a n e - w a t e r (11:2:5:10, b y vo].). After visualization o f the PG standards b y exposure to 12 vapour, the radioactive P G spots were located b y a u t o r a d i o g r a p h y a n d

218

S. Y. K. Y O U S U F Z A | ET AL.

t h e r a d i o a c t i v e c o n t e n t s o f P G s a n d a r a c h i d o n i e acid were m e a s u r e d by c o u n t i n g in a B e c k m a n liquid-scintillation counter. Changes in t h e release o f PGE~ into the m e d i u m are presented as the a m o u n t o f PG in /tg g - l o f tissue, a n d changes in t h e release o f a r a c h i d o n a t e from prelabeled tissue into the m e d i u m are expressed as 14C-radioactivity d p m g - l tissue. T h e d a t a presented in t h e Figures are m e a n o f three or more e x p e r i m e n t s a n d the bars represent t h e S.E.(M.).

~leasurement of contraction response in iris sphincter muscle F o r m e a s u r e m e n t of c o n t r a c t i o n responses, the sphincter muscles were m o u n t e d in 25-ml organ b a t h s c o n t a i n i n g K r e b s - R i n g e r b i c a r b o n a t e buffer which was c o n t i n u o u s l y o x y g e n a t e d (97 ~/o O2-3 % CO~) a n d m a i n t a i n e d a t 37 °C. T h e tissues were allowed to equilibrate for 90 rain u n d e r a resting tension of 50 rag. D u r i n g this period the muscles were washed w i t h fresh o x y g e n a t e d K r e b s - R i n g e r buffer (pH 7"4) e v e r y 30 min. After equilibration, s u b s t a n c e P was a d d e d as indicated a n d isometric c o n t r a c t i o n s were recorded using a Grass F T - 0 3 t r a n s d u c e r a n d Grass DC amplifier. The effects o f different c o n c e n t r a t i o n s of s u b s t a n c e P on iris s p h i n c t e r were recorded as the increase in tension by the agonist. I n e x p e r i m e n t s where t h e effect o f s u b s t a n c e P a n t a g o n i s t was i n v e s t i g a t e d , t h e a n t a g o n i s t was a d d e d 5 rain prior to the a d d i t i o n o f the agonist. M a x i m u m response was defined as t h e m a x i m u m tension recorded with s u b s t a n c e P (plotted as 100%). 3. R e s u l t s

Effect of substance P on a2P-labelinq of phospholipids A d d i t i o n o f s u b s t a n c e P (1 pra) i n c r e a s e d t h e b r e a k d o w n o f t h e p o l y p h o s p h o i n o s i t i d e s ( P I P a n d P I P 2 ) b y 2 0 - 3 0 % a n d i n c r e a s e d t h e l a b e l i n g o f P I a n d P A b y 27- a n d 30 ~/o, r e s p e c t i v e l y ( T a b l e I). A t I0/~M, t h e n e u r o p e p t i d e i n c r e a s e d t h e l a b e l i n g o f P I TABLE I

Effect of substance P on asP-labeling ~,f phospholipids in iris sphincter muscle* s~P-Radioactivity in the phospholipids (epm × 10-2) Addition

PI

PIP

PIPt

PA

PC

None Substance P I p.~l 10 p,~l

114-t-8

83-t-2

188-t-8

20+ I

122-t-6

145__.12 17! + 17

65+2 67_.+3

134__.10 132=t=5

26-1-3 31 - t - 5

120-t-11 132-1-12

* The sphincter muscles were incubatet] singly (of the pair, one served as control and the other as experimental) for 60 rain in 1 ml of Krebs-Ringer bicarbonate buffer (pH 7-4) containing 30 pCi azPi. At the end of incubation, substance P was added as indicated and incubation continued for an additional 10 rain. The reaction was stopped with 10% (w/v) TCA and the phospholipids were extracted and analysed for radioactivity by one- and two.dimensional TLC. The data are means~s.l~.(M.) of three ~eparate determinations. PI, phosphatidylinositol; PIP, phosphatidylinositol 4-phosphate; PIPs, phosphatidy]inosit~l 4,5-hisphosphate; PA, phosphatidic acid; PC, phosphatidylcholine.

a n d P A b y 50- a n d 55 ~/o, r e s p e c t i v e l y . H o w e v e r , u n d e r t h e s a m e e x p e r i m e n t a l cond i t i o n s t h e l a b e l i n g o f p h o s p h a t i d y l c h o l i n e (PC) d i d n o t i n c r e a s e s i g n i f i c a n t l y , as expected, by the neuropeptide. These data suggest that activation of substance P ~..ceptors r e s u l t s in p o I y p h o s p h o i n o s i t i d e b r e a k d o w n in t h e iris s p b i n c t e r m u s c l e , a n action similar to that previously reported for muscarinic cholinergic- and ~l-adrenergic a g o n i s t s in t h e w h o l e iris m u s c l e ( f o r r e v i e w see A b d e l - L a t i f e t al., 1985).

EFFECTS 1O0

-

tll,--e tP3 accumtdal:on

c

80-

P

/0

'22:='0: '2Y' J

(1)

0

OF SUBSTANCE

219

/tl

/

¢1 co

~" eo E

.E_

4o

"6

2o o 8

7

6

--Log (Substance P), M Fro. 1. Dose--response effect of substance P on accumulation of inositoi triphosphate (IPa) and on muscle contraction in the iris sphincter. For experiments involving IPs accumulation, the paired sphincters (from the same rabbit) were incubated in 1 ml of Krebs-Ringer bicarbonate buffer (pH 7"4) containing 10 FCi myo-[3H]inositoi for 90 rain. At this time the sphincters were washed four times with 3 ml of non-radioactive Krebs-Ringer bicarbonate buffer and then suspended singly (of the paired sphincters, one was used as a control and the other as experimental) in 1 ml of the non-radioactive buffer. Ten millimolar LiCI was added to each incubation and 10 rain later different concentrations of substance P were added and incubation continued for an additional 10 rain. The incubations were terminated with 10% (w/v) TCA and the radio[.tbeled myo-inositol phosphates analysed by anion-exchange chromatography. The amounts of radioactivity recovered in IPa in the absence and presence of 10 p,~ substance P (supramaximal dose) were 2613-+156- and 4074=i:375cpm, respectively. For measuremvnt of contraction responses, the sphincter muscleswere mounted in 25-ml organ baths containing Krebs-Ringer bicarbonate buffer which was continuously oxygenated (97 % Os-3 % COs) and maintained at 37 °C. The tissues were allowed to equilibrate for 90 rain under a resting tension of 50 rag. During this period the muscles were washed with fresh oxygenated Krebs-Ringer buffer (pH 7"4) every 30 rain. After equilibration, different concentrations of substance P were added and isometric contractions were recorded using a Grass FT-03 transducer and Grass DC amplifier. A supramaximal dose (Sp,,'d) of substance P elicited a contractile response of l 1"9+ 0"5 mg tension mg -l tissue wet weight. In the figure, accumulation of IP3 and muscle contraction have been expressed as percentage of the corresponding maximal response to the agonist. TABLE II.

Effects of substance P and its antagonist, (D-Pro 2, D-TrpT,~),~P, on accumulation of myo-inositol phosphates and on contraction in the iris sphincter muscle*

IP

IPz

IPs

Increase in tension (rag per mg wet ~tissue)

442 -+ 26 761 + 8 7 552 -+28

65 ± 7 99-+ 16 79-+3

28-+ 2 45-+2 34-+ 1

m 12"56~0"76 3-03-+0"37

~05-+31

61 -+2

27-+2

1"15-+0"13

SH-radioactivity in myo-inositol phosphates (cpm × 10-=) Additions None S P (10 pM) SP (10 pM) ÷ (D-Pro ~, D-TrpT.9)-SP (0"! pM) S P (10 pM)+

(D-Pro~, D-TrpT,9)-SP (0"lpM) * All experime~tal details were the same as described in the legend to Fig.'1, except t h a t the tissues were incubated with the antagonist for 5 rain before the addition of the agoni~t. The incubation time with the agonist, was 10 rain. The d a t a are mean-+s.]~.(M.) of two separate experiments conducted in triplicate.

Effects of substance P and its antagonist on accumulation of myo-inositol phosphates and on contraction T o fihed m o r e l i g h t on t h e m e c h a n i s m o f a c t i o n o f s u b s t a n c e P in t h e iris s p h i n c t e r we h a v e i n v e s t i g a t e d t h e ' e f f e c t s o f t h e n e u r 0 p e p t i d e a n d its a n t a g o n i s t o n b o t h

220

S.Y.K. YOUSUFZAI ~

ET AL.

180 ':E

~

170

.~ 1 4 o -

,,o.

oc

~

lO0 0

0.1

1

Substance

5 10 P (pM)

25

FIG. 2. Dose response to substance P for release of p'rostaglandin E= (PGE=). Irides, sphincter or dilator muscles {of the pair, one was used as control and the other as experimental) were incubated in I ml of Krebs-Ringer bicarbonate buffer (pH 7-4) in the absence and presence of various concentrations of substance P for 10 rain at 37 °C. At the end of incubation prostaglandin (PG) in the medium was quantified by RIA. The amounts of PG released are expressed as pg per g of tissue. In general, after I0 rain of incubation, the basal release of PGE= (pg per g of tissue) for the iris, for the sphincter and for the dilator were l "7:J:(~03, (P90::t:0"03 and 1.5 :J::0"8, respectively. The effects of substance P on PGE= release are expressed as percentages of the control. Each point is the mean of values from four separate experiments conducted in triplicate. i n o s i t o l p h o s p h a t e s release a n d on m u s c l e c o n t r a c t i o n (Fig. I ; T a b l e I I ) . S u b s t a n c e P c a u s e d a r a p i d d o s e - d e p e n d e n t a c c u m u l a t i o n o f I P a (ECho, 1-3 × 10 -e M) a n d c o n t r a c t i o n (ECso, 2"2 × 10 -~ M) in t h e s p h i n c t e r m u s c l e (Fig. 1). B o t h s u b s t a n c e P - i n d u c e d inositol p h o s p h a t e s a c c u m u l a t i o n a n d m u s c l e c o n t r a c t i o n w e r e b l o c k e d b y 1 0 - 1 0 0 nM s u b s t a n c e P a n t a g o n i s t ( T a b l e I I ) , i n d i c a t i n g t h a t b o t h t h e b i o c h e m i c a l a n d p h y s i o l o g i c a l r e s p o n s e s a r e m e d i a t e d b y specific r e c e p t o r ( s ) .

Dose-response to substance P for PGE~ release P r e v i o u s l y , w e h a v e r e p o r t e d t h a t r a b b i t irides s p o n t a n e o u s l y r e l e a s e d P G E = i n t o the medium, w i t h o u t addition of exogenous precursor, and t h a t this increased with t i m e o f i n c u b a t i o n u p t o 15 rain, t h e n leveled off b e t w e e n 30- a n d 60 rain ( Y o u s u f z a i a n d A b d e l - L a t i f , 1983, 1984}. A s c a n b e seen f r o m Fig. 2, a d d i t i o n o f s u b s t a n c e P e i t h e r t o t h e w h o l e i r i s o r t~ t h e Bphincter r e s u l t e d in e n h a n c e d release o f P G E , in a d o s e - d e p e n d e n t m a n n e r . T h u s a d d i t i o n o f 0"1 a n d 10 pM o f s u b s t a n c e P t o t h e s p h i n c t e r i n c r e a s e d P G E = r e l e a s e b y 25- a n d 55 % , r e s p e c t i v e l y . I n c o n t r a s t , a d d i t i o n o f s u b s t a n c e P t o t h e d i l a t o r i n c r e a s e d P G E = release b y o n l y 6 - 9 ~/o o f t h a t o f t h e control.

Time-course for the effect of substance P on the release of PGE= T h e t i m e c o u r s e for t h e e f f e c t o f I-F~: s u b s t a n c e P on t h e r e l e a s e o f P G E = is s h o w n in Fig. 3. S i g n i f i c a n t i n c r e a s e in t h e r e l e a s e o f P G E = (20 % ) w a s o b s e r v e d w i t h i n 1 m i n a f t e r a d d i t i o n o f t h e n e u r o p e p t i d e a n d m a x i m a l i n c r e a s e in t h e a g o n i s t - s t i m u l a t e d release o f P G E ~ (43-587/o} w a s r e a c h e d b e t w e e n 5- a n d 10 m i n o f i n c u b a t i o n . T h e r e w a s a d e c r e a s e in t h e s t i m u l a t o r y effect o f t h e p e p t i d e o n P G E = release a f t e r 1 0 - 2 0 m i n o f i n c u b a t i o n (Fig. 3).

Dose-response to substance P for release of x4C-arachidonic acid, PGE~ and PGF~ T h e r e is g e n e r a l a g r e e m e n t n o w t h a t P G f o r m a t i o n m u s t b e p r e c e e d e d b y a l i p o l y t i c p r o c e s s t o release free a r a c h i d o n i c a c i d f r o m t h e tissue p h o s p h o l i p i d s . T o i n v e s t i g a t e t h e m e c h a n i s m o f a c t i o n o f s u b s t a n c e P o n a r a c h i d o n a t e release a n d P G s y n t h e s i s , t h e

EFFECTS

OF SUBSTANCE

P

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Time (Minutes)

FIo. 3. Time course for the effect o f substance P on the release of prostaglandin E= (PGE=) in the iris sphincter. Conditions of incubation were the same as described under Fig. 2 except that the experimental contained 1 FM substance P and the incubations were carried out for various time intervals as indicated. Each point is the mean of values from two separate experiments conducted in triplicate.

200 180 160

c

140120-

A

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0.1

1

Substance

5

10

25

P (~M)

FIo. 4. Dose-response to substance P for release, of 14C-labeled arachidonic acid, PGE= and PGF== in iris sphincter. Sphincter muscles (in pairs) were incubated for 1 hr in 2 ml of Krebs-Ringer bicarbonate buffer (pH 7"4) containing 0-4 pCi of [14C]arachidonic acid. The prelabeled muscles were washed three times with non-radioactive buffer, then incubated in the ab.~nce and presence of various concentrations of substance P as indicated for 10 rain at 37 °C. Extraction ofarachidonic acid and PG from the medium and analysis of radioactivity were as described in the Materials and Methods section. Typical control values (in absence of substance P) for radioactive arachidonic acid, PGEs and P G F ~ were (dpm per g tissue) 167 435 + 7126, 63 058 ± 5268 and 45555 + ! 774, respectively. The effects of substance P on araehidonic acid and prostaglandin (PG) release are expressed as percentages of their respective controls. Each point is the mean of values from three separate experiment~ run in triplicate.

sphincter muscles were prelabeled -frith [14C]-arachidonic acid a n d the effects of various concentrations of substance P on the release of radioactivity was d e t e r m i n e d by radiochromatography. As can be seen from Fig. 4, substance P increased the release of labeled arachidonate, PGE2 and P G F ~ in a dose-dependent manner. Thus, substance P at 0"1- and 5pM increased araehidonate release by 30- and 7 2 % , respectively.

Dose-response to substance P for release of 14C-labeled arachidonic acid in iris sphincter treated with indomethacin and nordihydroguaiaretic acid ( N D G A ) To show w h e t h e r the metabolites g e n e r a t e d from arachidonic, acid through the cyclo-oxygenase and the lipoxygenase p a t h w a y s are involved in the actions of

222

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S u b s t a n c e P [~M) Fro. 5. Dose response to substance P release of J4C-labeled araehidonie acid in iris sphincter muscles treated with indomethacin and nordihydrogucJaretic acid (lgDGA), Conditions of incubation were the same as described under Fig. 4 except that incubations were carried out in a medium containing 1"6t1,~ indomethacin or 1 p,vl NDGA, Control value (after 10 rain) for ['~C]arachidonie acid was 170525_+6233 dpm per g tissue. Each point is the mean of values from two separate experiments run in duplicate.

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Flt~. 6. Dose-response curves for inhibition of substan(~ P-induced rel*-,me of [a~C]arachidonic acid, [14C]prostaglandin E 2 (PGE~) and [14C]PGF2a by substance P antagonist in the iris sphincter. Sphincter muscles (in pairs) were pr~-incubated for 1 hr in 2 ml o f K r e b s - R i n g e r bicarbonate buffer (pH 7.4) containing 0.4/tCi of [zaC]arachidonie acid. The prelabeled musclea were washed three times with non-radioactive buffer, then incubated in the absenee and presence of substance P (I ttM) and various eoncentrations of substance P antagonist (added 5 rain prior to the ~tddition of the agoniat) for 10 rain at 37 °C. Extraction of araehidonie acid and prostaglandirm from the medium and analysis of radioaetivity were as described in the Materials and Methods section. The inhibitory effects of substance P antagonist on substance P-induced release of arachidonie acid. I~GE~ and PGF2a are expressed as percentages of their respective controls. Each point is the mean of values from three separate experiments run in triplicate.

substance P we have investigated the effects of indomethacin (a cyelo-oxygenase inhibitor) and N D G A (a ]ipoxygenase inhibitor) on substance P-stimulated release of labeled arachidonate (Fig. 5). Neither indomethacin nor N D G A had any inhibitory effect on the substance P actions. The increase in the radioactivity of arachidonie acid release b y the neuropeptide in the presence of these ir;hiL'itors is due to their known inhibitory effects on the eyclo-oxygenase and lipoxygenase pathways. T h e effects of various concentrations of substance P on [14C]araehidonie acid release in indornethaein-

EFFECTS OF SUBSTANCE

P

223

Membrane Phospholnosllldes

Phospholnoslttde-[ Specific Phosphol|pase C~ Diacylglycerol

Phclphoilpnso Ae

-F Inosltol Phosphates

Arachldonlc Acid + Lylophosphstldyl Inoellol

LIpases Arachldonic Acid 4- Fatty Acids + Glycerol

PGs

+

HETES + Leukolrlenes

-t- METES + Leukotrlenes Fro. 7. Scheme showing the two pathways by which arachidonic acid can be released from phosphoinositides: (I) phosphoinositides can be hydrolyzed by phospholipase C, an enzyme which hydrolyzes only phosphoinositides, followed by hydrolysis of diacylglycerol via iipases to liberate arachidonic acid. (II) Arachidonic acid can be released from phosphoinositides via phospholir ase Az. PGs

t re a t e d s phi nc t e r muscle (Fig. 5) were significantly higher t h a n those w h i c h were obtained in t h e absence of t he inhibitor (Fig. 4). Thus, a t 1-pM substance P increased the release o f [14C]arachidonic acid from control (Fig. 4) a n d i n d o m e t h a c i n - t r e a t e d (Fig. 5) muscles by 55- a n d 7 0 % , respectively. These d a t a indicate t h a t t h e metabolites o f araehidonic acid are n o t involved in t h e substance P - i n d u c e d release of a ra c hi d oni c acid in t h e iris sphincter muscle.

Effect of concentration of substance P antagonist on the substance P-induced releaze of 14C-labeled arachidonic acid, PGE 2 and P G F ~ To d e t e r m i n e w h e t h e r the substance P - i n d u c e d arachidonic acid release is receptor mediated, we h a v e investigated the effects o f various concentrations of s u b s t a n c e P a n t a g o n i s t on the release of 14C-arachidonic acid from the sphincter muscle. T h e a n t a g o n i s t inhibited t h e substance P - i n d u c e d arachidonic acid release a n d conseque nt l y t he biosynthesis of PGE~ and PGF2a in a d o s e - d e p e n d e n t m a n n e r (Fig. 6). T h e rates of subs t a nc e P - i n d u c e d ~ l e a s e of [14C]arachidonic acid, PGE~ a n d P G F ~ were half-maximally inhibited by 1.0 × 10 -7 M, 3"3 × 10 -~ M a n d 2"5 × 10 -~ M of s u b s t a n c e P antagonist, respectively (Fig. 6). These d a t a indicate t h a t substance P - i n d u c e d arachidonic acid release from m e m b r a n e phospholipids is receptor-mediated. 4. D i s c u s s i o n

There is general a g r e e m e n t t h a t the k ey step r e g u l a t i n g the p r o d u c t i o n ofeicosanoids in m a m m a l i a n tissues is the release :~f t h e p r e c u r s o r arach id o n ic acid from m e m b r a n e phospholipids t br ough t he action of phospholipases ( f o r rev'ew see I r v i n e , 1982). Phosphoinositides are enriched with this p o l y u n s a t u r a t e d f a t t y a c i d , which is esterS fled a l m o s t exclusively a t the second positio n of glycerol o f the phospholipid, from which it could be released either i n d i r e c t l y t h r o u g h th e action of phospholipase C, followed b y t h a t o f o t h e r lipases [Fig. 7(I)], or directly by t h e a c t i o n of phospholipase A~ [Fig. 7 (II)]. I t is well acoepted n o w t h a t a c t i v a t i o n o f Ca~+-mobilizing receptors, s u c h a s muscarinic cholinergic, a n d ~1-adrenergic, leads to th e formation o f DG, w h i c h can ~ be metabolized t~.release arachidonie acid :for P G s y n t h e s i s , a n d to th e release of IP8

224

S.Y.K. YOUSUFZA! ET AL.

which mobilizes Ca s+ from intracellular sites (for reviews see Berridge and Irvine, 1984; Extort, 1985; Abdel-Latif, in press). I n the present study, we have d e m o n s t r a t e d t h a t in the rabbit iris sphincter muscle substance P (10-v-10 -6 M) can: (a) provoke a rapid breakdown of t h e polyphosphoinositide PIP2 to release IPa and form DG, measured as PA, implying t h a t this neuropeptide behaves similar to other Ca s+mobilizing agonist in this tissue; (b) induce smooth-muscle contraction, which correlates well with I P 3 release in a d o s e - d e p e n d e n t m a n n e r (Fig. 1) and (c) induce a rapid release of PGEs, measured by RIA, and rapid release of 14C-labded arachidonic acid and PGs, measured by radiochromatography. The finding t h a t substance P had little effect on PGEs release by the iris dilator indicates t h a t this sympathetically innervated muscle, in contrast to the sphincter, is not innervated by the sensory nerves which liberate the neuropeptide. Substance P has been reported to increase the accumulation of I P in guinea-pig ileum muscle (Watson and Downes, 1983). I n guinea-pig intestinal s m o o t h muscle, in addition to IP, the IPs and to a lesser e x t e n t IP3 were also increased (Holzer and Lippe, 1985). I n the iris sphincter muscle, the substance P-induced IP3 release as well as muscle contraction were inhibited by substance P antagonist. The type o f substance P receptor mediating the phosphoinositide and contractile responses in s m o o t h muscle is still unresolved (Watson, 1984). The potency of substance P in causing IPa accumulation was lower t h a n t h a t in evoking muscle contraction (Fig. 1). I n guinea-pig ileum (Watson and Downes, 1983) and in guineapig intestinal muscle (Holzer and Lippe, 1985) the dose-response curve for inositol phosphates release by substance P was also found to be shifted to the right in relation to the dose-response curve for contraction. These authors have explained their findings by the assumption t h a t maximal agonist-induced breakdown of phosphoinositides requires full receptor occupancy whereas the biological effect (contraction) m a y be fully accomplished when only a small proportion of t h e available receptors is occupied. The d a t a presented on arachidonic acid release and PG synthesis (Fig. 2-5) indicate t h a t activation of substance P receptors can also lead to the release ofarachidonic acid from m e m b r a n e phospholipids. Previously, we have reported t h a t in the rabbit iris activation of muscarinic cholinergic and al-adrenergic receptors leads to the release of arachidonic acid for PG synthesis (¥ousufzai a n d Abdel-Latif, 1983, 1984). I t is also possible t h a t %he Ca s+ released by I P 3 could activate the phospholipase A s p a t h w a y to liberate arachidonic acid from m e m b r a n e phosphoinositides and other phospholipids. Furthermore, we have shown t h a t activation of receptors for the platelet-activating factor (PAF) in the rabbit iris leads to the release of arachidonic acid and PG synthesis, b u t in contrast to t h e activation of Ca ~+ mobilizing receptors, which is coupled to the phospholipase C p a t h w a y [Fig. 7 (I)], this occurs through the phospholipase As p a t h w a y [Fig. 7 (II)]; Yousufzai and Abdel-Latif, 1985). Many lines of evidence support t h e concept t h a t t h e sensory nerves, which i n n e r v a t e the sphincter region of the iris, m a y ialay a role in miosis probably by releasing substance P u n d e r certain conditions (see Introduction). The d a t a presented in this communication indicate t h a t substance P acts as a Ca2+-mobilizing agonist in this tissue, implying t h a t the agonist-stimulated b r e a k d o w n of PIPs is probably a t r a n s d u c t i o n mechanism t h a t links substanbe P receptor activation to contraction. :Furthermore, we have shown t h a t activation of substance P receptors leads to the release of arachidonic acid a n d consequently PG synthesis. PGs have been reported t o p l a y i m p o r t a n t regulat~ary roles in contraction and relaxation o f smooth muscle, in n e u r o t r a n s m i t t e r release, in permeability changes, and i n secretion (for reviews see

EFFECTS OF SUBSTANCE P

225

Samuelsson, 1981; Marcus, 1984), a n d in regulation of i n t r a o c u l a r pressure (for review see Bito, 1984). Thus, a substance P-in d u ced release of IPa and formation of DG, a source for arachidonic acid release in PG synthesis, followed by Ca ~+ mobilization could underlie t he mechanism for th e biological actions, such as muscle contraction, of the n e u r o p e p t i d e reported in t h e eye. However, th e precise relationship remains to be established. ACKNOWLEDGMENTS This work was supported by USPHS Grants EY-04387 and EY-04171 from the National Eye Institute. We are grateful to Lisa Latimer and Lynn Manning for technical assistance. This is contribution number 0944 from the Department of Cell and Molecular Biology, Medical College of Georgia, Augusta, Georgia. REFERENCES Abdel-Latif, A.A. (1986). Calcium-mobilizing receptors, polyphosphoinositides and the generation of second messengers. Pharmacol. Rev. (In press.) Abdel-Latif, A. A., Howe, P. H. and Akhtar, R. A. (1986). Polyphosphoinositides, phosphoproteins, and reoeptor function in rabbit iris smooth muscles. Proyress in Brain Res. (Symposium Proceedings). (In press.) Abdel-Latif, A. A., Naderi, S. and Yousufzai, S. Y. K. (1986). Pharmacological agents and the release of arachidonic acid and prostaglandin biosynthesis by rabbit iris muscle. In: Symposium on Phosopholipids in the Nervous System: Biochemical and Molecular Pharmac~loyy. (Eds Freysz, L., Horrocks, L.A. and Toffano, G.). Liviana Press: Padova, Italy. (In press.) Abdel-Latif, A.A., Smith, J . P . and Akhtar, R.A. (1985). Polyphosphoinositides and muscarinic cholinergic and ~l-adrenergic receptors in the iris smooth muscle. In Inositol and Phosphoinositides: Me~bolism and Regulation. (Eds Bleasdale, J. E., Eichberg, J. and Hauser, G.). Pp, 275-98. The Humana Press: Clifton, N.J. Akhtar, R.A. and Abdel-Latif, A.A. (1984). Carbachol causes rapid phosphodiesteratic cleavage of phosphatidylinositol 4,5-bis-phosphate and accumulation of inositol phosphates in rabbit iris muscle; prazosin inhibits noradrenaline- and ionophore A23187stimulated accumulation of inositol phosphates. Biochem. J. 224, 291-300. Akhtar, R. A. and Abdel-Latif, A. A. (i985). Surgical Sympathetic denervation increases ~l-adrenoceptor mediated accumulation of myo-inositol trisphosphate and muscle contraction in rabbit iris dilator smooth muscle. J. Neurochem. 46, 96-104. Banno, H., Imaizumi, Y. and Watanabe, M. (1985). Pharmaco-mechanical coupling in the response to acetylcholine and substance P in smooth muscle of the rat iris sphincter. Br. J. Pharma~l. 85, 905-1 I. Barker, J. L. (1976). Peptides: roles in neuronal excitability. Physiol. Rev. 56, 435-52. Berridge, M. J. and Irvine, R. F. (1984). Inositol trisphosphate, a novel secor~d messenger in cellular signal transduction. Nature (London), 312, 316-21. Bill, A. (1985). Some aspects of the ocular circulation. Invest. Ophthalmol. Vis. ,~ci. 26, 410--24. Bill, A., Stjernschantz, J., Mandahl, A., Brodin, E. and Nilsson, G. (1979). Substance P: release on trigeminal nerve stimulation, effects in the eye. ~lcta Physiol. 8cand. 106, 371-3. Bito, L.Z. (1984). Prostaglandins, other eicosanoids, and their derivatives as potential antiglaucoma agents. In Glaucoma: Applied Pharmacology in Medical Treatment. (Eds Drance, S. M. and Neufeld, A. H.). Pp. 477-505. Grune and Stratton, Inc.: New York. Bito, L.Z., Nichols, R . R . and Baroody, T. (1982). A comparison of the miotic and inflammatory effects of biologically active polypeptides and prostagl*andin E z on the rabbit eye. Exp. E y e Res. 34, 325-37. Bury, R . W . and Mashford, M.L. (1976). Interactions between local anaesthetics and spasmogens on the guinea-pig ileum. J. Pharmacol. Exp. Ther. 197, 633-40.

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Butler, J.M. and H a m m o n d , B.R. (1980). The effects of serlsory denervation on the responses of the rabbit eye to prostaglandin E I, bradykinin and substance P. Br. J. Pharmacol. 69, 495-502. Camras, C. B. and Bite, L. Z. (1980). The patbophysiological effects of nitrogen mustard on the rabbit eye. II. The inhibition of the initial hypertensive phase by capsaicin and the apparent role of substance P. Invest. Ophtl~lmol. Via. Sci. 19, 423-8. Exton, J. H. (1985). Mechanisms involved in cz-adrenergic phenomena. Am. J. Physiol. 248, E633-47. Fujisawa, K. and Ire, Y. (1982). The effect of substance P on smooth muscle cells and on neuro-effector transmission in the guinea-pig ileum. Br. J. Pharmacol. 76, 279-90. Hamberg, M. and Samuelsson, B. (1966). Prostaglandins in human seminal plasma. J. Biol. Chem. 241,257-63. Hokfelt, T., Johansson, O., Ljundahl, A., Lundberg, J. M. and Schultzberg, M. (1980). Peptidergie neurones, Nature (London) 284, 515-21. Holmdahl, G., Hakanson, R., Leander, S., Resell, S., Folkers, K., and Sundler, F. (1981). A substo~nce P antagonist [D-Pro ~, D-Trp~'~]SP inhibits inflammatory responses in the rabbit eye. Science 214, 1029-31. Holzer, P. and Lippe, I. Th. (1984). Substance P can contract the longitudinal muscle of the guinea-pig small intestine by releasing intracelluiar calcium. Br. J. Pharmacol. 82, 259-67. Holzer, P. and Lippe, I. Th. (1985). Substance P action on phosphoinositides in guinea-pig intestinal muscle: a possible transduction mechanism ? Naunyn-Schmiedd~erg's Arch. Pharmacol. 329, 50-5. Holzer, P. and Petsche, U. (1983). On the mechanism of contraction and desensitization induced by substance P in the intestinal muscle of the guinea-pig. J. _Physiol. 342, 549-68. ]trine, R: F. (1982). How is the level of free arachidonic acid controlled in mammalian cell ? Biochem. J. 204, 3-16. Mandahl, A. and Bill, A. (1981). Ocular response to antidromie trigeminal stimulation, intraocular prostaglandin E 1 and E 2, capsaicin and substance P. Acts Physiol. Sca~l. 112, 331-8. Marcus, A. J. (1984). The eicosanoids in biology and medicine. J. Lipid Res. 25, 1511-6. Pernow, B. (1983). Substance P. Pharmacol. Rev. 3S, 85-283. Samuelsson, B. (1981). Prostaglandins, thromboxanes, and leukotrienes: formation and biological roles. The Harvey Lectures, series 75, 1-40. Stjernschantz, J., Sears, M. and Stjernschantz, I. (1981). Intra~bular effects of substance P in the rabbit. Invest. Ophthalmol. Vis. Sci. 20, 53-60. Unger, W . G . and Tighe, J. (1984). The response o f the isolated iris sphincter muscle of various mammalian species to substance P. Exp. Eye Res. 39, 677-84. Watson, S. P. (I984). The action of substance P on contraction, inositol phospholipids and adenylate cyclase in ~'at small intestine. Biochem. Pharmac~l. 33, 3733-7. Watson, S . P . and Dowries, C . P . (I983). Substance P induced hydrolysis of inositol phospholipids in guinea-pig ileum and rat hypothalamus. Eur. J. Pharmacol. 93, 245-53. Yousufzai, S. Y. K. and Abdel-Latif, A . A . (1983). Effects of norepinephrine and other pharmacological agents on prostaglandin E~ release by rabbit and bovine irides. Exp. Eye Res. 37, 279-92. Yousufzai, S. Y. K. and Abdel-Latif, A . A . (1984). The effects of ~l-l-a~lrenergic and muscarinic cholinergic stimulation on prostaglahdin release by rabbit iris. Prostaglandin8 28, 399-415. Yousufzai, S. Y. K. and Abdel-I~atif, A. A. (1985). Effects ofplatelet-activating factor on the release of arachidonic acid and prostaglandins by rabbit iris smooth muscle: inhibition by calcium channel antagonists. Biochem. J. 228, 697-706. Zhang, S. A., Butler, J . M. Ohara, K. and Cole, D. F. (1982). Sensory neural mechanisms in contraction of the isolated sphincter pupillae: the role for substance P and the effects of sensory denervation on the response to miotic. Exp. Eye Res. 35, 43-54.