- Email: [email protected]
Effect of substance P and eledoisin on K+ efflux, amylase release and cyclic nucleotide levels in slices of rat parotid gland
704
Biochimica et Biophysica A cta, 444 (1976) 704--711 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 28034
EFFECT OF SUBSTANCE P AND ELEDOISIN ON K+ EFFLUX, AMYLASE RELEASE AND CYCLIC NUCLEOTIDE LEVELS IN SLICES OF RAT PAROTID GLAND
LYNN RUDICH and F R E D R. BUTCHER
Division of Biological and Medical Sciences, Brown University, Providence, R.I. 02912
(U.S.A.) (Received March 23rd, 1976)
Summary The undecapeptides, substance P and eledoisin, caused a rapid, concentration~lependent increase in K ÷ efflux and amylase release from parotid tissue slices. The effects were not blocked by ~clrenergic, a-adrenergic, or cholinergic antagonists. Incubation buffer calcium was required for stimulation of K ÷ efflux and amylase release. The action of the undecapeptides was independent of any effects on parotid cyclic AMP or cyclic GMP levels. Since the actions of the undecapeptides were Ca 2+ dependent and no effects on cyclic nucleotide levels were discerned it was concluded that Ca 2+ plays a primary role in agonist regulation of K + efflux from the parotid.
Introduction In 1931~ while investigating the tissue distribution of acetylcholine, yon F~uler and Gaddum [1] found a substance in equine brain and intestine which caused hypotension and stimulated in vitro the contraction of intestinal tissue in rabbits. This material, substance P, was finally isolated in pure form from bovine hypothalamus in 1970 [2] and identified with a factor which was f o u n d to cause an increase in the fluid volume and amylase activity of rat salivary secretion in vivo [3]. Peptides such as physaelamin, isolated from the skin of the South American amphibian Physalaemus fuscumaculatus [4] and eledoisin, isolated from the posterior salivary gland of two molluscan species, Eledone moschata and Eledone androvandi [5] have been clauified with substance P by Erspamer [6] as tachychinins on the basis of similar biological and structural characteristics. In addition to causing atropine-resistant hypotension in the dog and rat [4], as a class they also increase capillary permeability in the rat, guinea pig and man,
705 and stimulate the contraction of isolated rabbit large intestine as well as the isolated ileum and large intestine of the guinea pig. They are also powerful stimulants of salivary and lacrimal secretions of the chicken, rat, dog and man [6,7]. The presence of substance P activity in nervous tissue and its specific distribution led Lembeck [8] to suggest that substance P is a transmitter released by primary sensory neurons. Much work has been directed toward verifying such a role [9,12]; however, on the basis of its relatively slow action, Henry et al. [13] have suggested that it functions as a modulator of sensory transmission. Mills et al. [14] have proposed the existence of "substance P-inergic" nerves which promote the release of renal kallikrein in dogs. Studies of the parotid have indicated that it is innervated by the parasympathetic system and sympathetic branches of the autonomic system [15]. Leeman and Hammerschlag [3] reported that the in vivo sialogenic activity of substance P in rats was not inhibited by atropine, phenoxybenzamine, or propranolol. Although the in vivo sialogenic activity of substance P was used to verify the success of its synthesis [16], it is not known with certainty that this was a direct effect on the salivary gland itself. An in vitro study of the action of these undecapeptides on rat parotid gland slices might serve as a model for their action. In addition the action of these undecapeptides on parotid gland might be exploited as tools in order to learn more about the regulation of parotid gland physiology by autonomic agents. Materials and Methods
Parotid glands from female Sprague-Dawley C D rats (Charles River Breeding Laboratories, Wilmington, Mass.), 7--9 weeks old fed adlibitum were used for all experiments. The glands were trimmed of connective tissue and slicedwith microscissors and maintained at 37°C in Krebs-Ringer bicarbonate buffer conraining 5 m M ~-hydroxybutyrate and 6 m M glucose with O2/CO2 (95 : 5, v/v) as the gas phase. Amylase was assayed by the method of Bernfeld [17] except that the assay was conducted at 37°C. Amylase content of the incubation buffer and that remaining in the tissue were both determined. The total amount of amylase was determined by summation of the amount in the buffer with the amount remaining in the tissue at the end of the incubation period. The percent of the total amylase which was released into the incubation buffer was then calculated. In our later experiments the amount of amylase released was determined by withdrawing aliquots of the incubation buffer and the amylase release data are expressed as units of amylase released into the incubation buffer. One unit of amylase was the amount of enzyme that caused the formation of I m g of maltose in 5 rain at 37°C. Potassium release studies were conducted in vials containing 1.0 ml of KrebsRinger bicarbonate buffer and the equivalent of one gland. Parotid slices contain a very active K ÷ uptake mechanism which can obscure effects of low agonist concentration or of weak against on K* efflux. In order to obviate this problem 1.0 mM ouabain was added to the incubation vials in all K ÷efflux experiments except those reported in Table II. Ouabain was omitted from these experiments because omission of Ca 2+ from the incubation buffer caised K÷ efflux
706 which was greatly magnified by ouabaln. The K* released was determined with an Instrumentation Laboratory model 143 flame photometer (Boston, Mass.). Incubations for the determination of cyclic nu¢leotides were stopped by adding HC104 to a final concentration of 0.5 M and rapidly homogenizing with a polytron homogenizer (Brinkman Instrument Co., Westbury, N.Y.). The cyclic nucleotides in the acid supernatant were purified by sequential column chromatography over neutral alumina and Dowex 1-X8 (formate form) according to Jakobs et at. [18] and Butcher et al. [19], respectively. Cyclic AMP was assayed by a protein binding assay [20] and cyclic GMP was assayed by the radioimmunoassay procedure of Steiner et al. [21] using the acetylation procedure of Harper and Brooker [22]. Protein was measured by the method of Lowry et al. [23]. Agonists were dissolved in glass distilled water to a concentration so that an a m o u n t no greater than 50 ~1 was added to each vial. Theophylline, atropine, sulfate, (±)-isoproterenol • HCI and ouabain were products of Sigma Chemical Co. (St. Louis, Mo.). Phentolamine methane sulfate was obtained from CIBA (Ardsley, N.Y.). (--)-Propranolol was a gift from Ayerst Laboratories, Inc. (New York, N.Y.). Synthetic bovine substance P was a product of Beckman Instruments, Inc. (Palo Alto, Calif.) and Eledoisin-950 was a gift of Sandoz Pharmaceuticals. Substance P and eledoisin are undecapeptides. Results
Effect of undecapeptides on amylase release. Both eledoisin and substance P cause a rapid, concentmtion~iependent increase in amylase release (Fig. 1). The ~mount of amylase release caused by the undecapeptides was small. Only 16% of the initial tissue amylase content was released after addition of the undecapeptides. The action of the undecapeptides on amylase release was not mediated through indirect effects of neurotransmitters since none of the pharmacological antagonists were inhibitory (Table I). The concentrations of the antagonists used for the studies in Table I were sufficient to completely inhibit the effects of saturating concentrations of the appropriate agonist. Effect of undecapeptides on K ÷efflux. In addition to stimulation of amylase release, the undecapeptides also promoted a rapid, concentration-dependent increase in K ÷ efflux (Fig. 2). Increased K ÷ efflux was detected at 30 s, the earliest time point examined. The time course for increased K* efflux caused by the undecapeptides was similar to that caused by cholinergic [ 2 4 ~ 5 ] and a~drenergic [19,26,27] agonists. The undecapeptides, however, are much more p o t e n t since increased K* effiux was detected at 10 nM while 100 rum was the lowest effective concentration for cholinergic agonists [25]. ~-Adrenergic agonists are less effective than chotinergic ago~Asts in promoting K ÷ efflux. Stimulation of K ÷ efflux by the undecapeptides appears pharmacologically distinct from ~-adrenergic, ~-ad~nergic, and cholinergic agonists since propranolol, phentolamine and atropine were n o t inhibitory. Calcium requirement for the effects of the undecapcp~ides. Incubation buffer calcium is r e q ~ for the effects of cholinorgic (Butcher, F.R., unpublished), ~-adrenergic [28] agonists, and the divalent cation ionophore [29,30] on
707
,~
J
•
ELEDOtSIN
AMYLASE
< 5,00
@
as
>
400 <[
Z 200 t_/
f
' °/""° [ J '.
t/.. .
;L O G2 :o C O N5,.0C E N TI 0R A T2 'o ;0 ION,riM
1.
1 0
l/II
20
3'0
MINUTES F i g . I . E f f e c t o f s u b s t a n c e P a n d e l e d o i s i n o n a m y l a s e r e l e a s e f r o m r a t p a r o t i d tissue slices. P a z o t i d slices f r o m t w o r a t s w e r e u s e d f o r each c o n e e n t ~ t i o n c u r v e a n d f o r t h e t l ~ e c o u r s e e x p e r l m e n t s . T h e v a l u e s plotted are the aventges of two sepmte paired experiments. Basal amylase Meue values, at 40 min, for the concentration curves were 2.2 ± 1.0 and 2.2 ~ 0.3% for substance P and eledols/n, respectively. The 5-, 1 0 . , 2 0 - a n d 4 0 . r a i n b a s a l a m y l a s e r s l e a m v a l u e s f o r t h e t / m e c o u r s e s t u d i e s w e r e 1 3 . 0 ± 8 . 4 , 1 5 . 3 ± 2.5, 20.1 + 4.1 and 25.1 ± 7.8 units/rag protein, respectively. The numbers 511, and 800 on the concent r a t i o n c u r v e r e f e r t o n M c o n c e n t r a t i o n s o f e l e d o i s l n a n d s u b s t a n c e P, r e ~ p e e t i v e l y . TABLE I EFFECT OF PHARMACOLOGICAL BLOCKING AGENTS ON THE STIMULATION RELEASE AND K+ EFFLUX BY ELEDOISIN AND SUBSTANCE P
OF AMYLASE
Tissue slices f r o m t w o r a t s w e r e u s e d in e a c h e x p e r i m e n t f o r d e t m n n l n a t i o n o f a m y l a s e M e M e . V a l u e s a r e the average of two expe]dments for each agonist. Basal amylase release was 4.6 + 2.2% for substance P ex• + p e r l m e n t s a n d 2 . 7 + 0 . 2 % f o r e l e d o h d n e x p e r i m e n t s . F o r t h e d e t e r m i n a t i o n o f K e f f l u x , tissue slices f r o m four rats were pooled for each experiment. Two experiments were performed for each agonist. Basal v a l u e s a f t e r 8 m l n o f i n c u b a t i o n i n t h e p r e s e n c e o f 1 . 0 m M o u a b a l n a l o n e w e r e 1 3 , 2 ± 1.7 a n d 1 8 . 3 ± 6 . 7 % po_t~_~m~__ume f f l u x f o r s u b . a n t e P a n d e i c d o i s l n e x p e r i m e n t s , r e s p e c t / v a l y . Additions
None Atropine, 10 #M Phentolamine, 5 pM (--)-Propranolol, 1 pM
Percent increase in amylase release above control Control
Substance P ( 3 7 riM)
Eledoisln ( 1 6 8 riM)
-23 + 7 (--) 22 ± Y (--) 5 ± 6
272 200 213 239
601 524 592 635
± ± ± ±
65 17 22 35
± ± ± ±
29 46 51 46
P e r c e n t i n c r e a s e in K + e f f l u x a b o v e c o n t r o l Control
None Atropine, 10#M Phentolamine, 5 #M (~)-Proprenolol, I #M
-15 + 48 14 + 7 16 ± 38
Substance P (149 nM)
Eledoisin
472 417 402 414
267 237 237 245
+ + + ±
65 20 14 20
(168 nM) + 2 + 15 ± 9 ± 16
708 I
K •~EFFLUX ,
& SUBSTANCEP
~)10(
&r
"•
•
z200
I0
20 S0 LOG CONCENTRATION
I00
5 MINUTES
nM
10
F i g . 2 . E f f e c t o f s u b s t a n c e P a n d e l e d o i s i n o n p o t a s s i u m e f f l u x f ~ m r a t p a r o t h l t / s a t e slices. P a r o t i d slices f r o m f o u r r a t s w e r e u s e d f o r e a c h c o n m m / n t i o n c u r v e s h o w n . P a r o ~ i d slices f r o m t w o r a t s w e r e u s e d f o r e a c h t i m e c o u r s e . T h e v a l u e s p l o t t e d a r e t h e a v e r a g e s o f t w o p a i r e d expez:lmonts. B a s a l p o t e s s / u m r e l e a s e v a l u e s f o r t h e c o n c e n t r a t i o n c u r v e s w e r e 1 0 . 1 ± 1 . 0 a n d 1 8 . 8 + 1.595 f o r 8 u b e t a n c e P a n d e l e d o i s i n , r e s p e c tively. The 0- and lO,m/n basal potmmlum release vslues for the t/me c ~ studies were 3.6 ± 0.1 and 1 5 . 3 ± 0.595 f o r e l e d o i s l n , r e s p ~ t i v e l ¥ , a n d t h o s e f o r s u b s t a n c e P w e r e 6 . 0 + 0 . 6 a n d 1 4 . 6 ± 0.495, t e e p e e Uvely. T h e c o n c e n t r a t i o n s o f s u b s t a n c e P a n d e l e d o i s i n w e r e 7 5 a n d 166 riM, r e s p e c t i v e l y .
amylase release, K ÷ efflux, and cyclic GMP accumulation. Calcium was also required for stimulation of a-amylase release and K ÷ effiux from the parotid slices by undecapeptides (Table If). TABLE H EFFECT OF CALCIUM ON THE STIMULATION ELEDOISIN AND SUBSTANCE P
OF AMYLASE
RELEASE
AND K+ EFFLUX
BY
T o d e t e r m i n e a m y l a s e r a l e ~ , t i s s u e slices f r o m t w o r a t s w e r e p o o l e d f o r e a c h o f t w o e x p e r i m e n t s , A v e r a g e b a s a l a m y l a s e release v a l u e w a s 4 . 9 + 1,395. F o r t h e d e t e r m i n a t i o n o f K ÷ e f f l u x , tissue slices f r o m f o u r rats were pooled for each experiment. Two experiments were performed for each qonist. Average basal values after incubation for 8 rain in the absence of 1.0 mM ouabain were 6.2 ± 0.3 and 10.2 ± 1.1% potassium efflux for substance P and aledoisin experiments, respectively. Additions
None Eledoisin, 168 nM S u b s t a n c e P, 3 7 n M
P e r c e n t i n c r e a s e i n ~-flnlylase r e l e ~ W i t h Ca ~
W i t h o u t C a 2+
-261 + 47 171 + 2
(--) 5 1 + (--) 20 ± (--) 41 +
above control
2 4 6
Percent increase in K + efflux above control None Eledoisin, 168 nM S u b s t a n c e P, 1 4 9 n M
-121 ± 33 97 + 39
(--) 0 ± 3 2 (--) 50 + 28 (--) 1 0 + 1 9
709
Effect of undecapeptides on parotid cyclic nucleotide levels. Considerable evidence exists which suggests that cyclic nucleotides might mediate agonist effects on their target tissues. The undecapeptides had no effect on either cyclic AMP or cyclic GMP levels in the parotid (Table III). Several time points and undecapeptide concentrations, in the presence and absence of theophylline or 1-methyl-3-isobutyl-xanthine, were examined, but still no effect on cyclic nucleotide levels could be detected (Table III, the data obtained in the absence of phosphodiesterase inhibitors is not shown). In other studies we have found that theophylline potentiates the stimulation of cyclic AMP accumulation by weak agonists [31] and that 1-methyl-3-isobutyl-xanthine enhanced the stimulation of cyclic GMP accumulation by low concentrations of various agonists [19,25,30]. The time points selected encompass both the time course for the effects of other agonists or parotid cyclic nucleotide levels and the time course for the effects of the undecapeptides on K ÷ efflux and amylase release from the parotid (see Figs. 1 and 2). Duffy et al. [32] have reported that substance P stimulated adenylate cyclase in broken cell preparations from rat and human brain. The undecapeptides did not inhibit stimulation of ~-amylase release or cyclic AMP accumulation by isoproterenol (not shown).
TABLE In T H E E F F E C T S U B S T A N C E P A N D E L E D O I S I N ON CYCLIC N U C L E O T I D E L E V E L S IN SLICES OF RAT PAROTID GLAND
Parotid ~ I c e s f r o m t h r e e r a t s were u s e d in each e x p e r i m e n t for cyclic AMP d e t e r m i n a t i o n s . T h e values f o r the cyciic A M P levels are the m e a n s + S.E., f r o m six e x p e r i m e n t s for c o n t r o l s a n d f r o m three experim e n t s f o r s u b s t a n c e P and ciedolsin. I n c u b a t i o n s w e r e carried o u t in the presence o f S 0 0 p M t h e o p h y l l i n e . Previous e x p e r i m e n t s h a v e i n d i c a t e d that this c o n c e n t r a t i o n o f t h e o p h y l l i n e greatly p o t e n t i a t e s the e f f e c t s o f i s o p r o t e r e n o l o n cyclic A M P a c c u m u l a t i o n in rat p a r o t i d tissue slices. F o r cyclic GMP d e t e r m i n a t i o n s , p a r o t i d slices f r o m f o u r rats w e r e u s e d in e a c h e x p e r i m e n t . T h e v a l u e s for the c y c l i c GMP levels are the m e a n s ±S.E. f r o m six e x p e ~ m e n t s f o r c o n s o l s a n d f r o m three e x p e r i m e n t s for substance P a n d eledolsin. I n c u b a t i o n s w e r e carried o u t in the presence o f 2 0 0 / ~ M m e t h y l - / s o b u t y l - x a n t h i n e , w h i c h is s u f f i c i e n t to p o t e n t i a t e the e f f e c t s o f e a r h a c h o l and p h e n y l e p h r i n e o n cyclic GMP a c c u m u l a t i o n in slices o f rat parotid gland. In all cases, t h e e x p e r i m e n t s to d e t e r m i n e the e f f e c t s o f substance P and ciedoiain o n c y c l i c n u e l e o tide levels were d o n e separately. T i m e (rain)
2.5 5.0 10.0 20.0
Cyclic A M P ( p m o l / m g protein)
Control
Substance P (149 nM)
Eledoisln ( 1 6 8 nM)
7.0 8.2 7.7 9.3
6.4 9.1 7.3 10.1
7.6 8.8 9.3 8.1
-+ 0 . 5 + 0.3 + 1.1 + 1.2
+ + ± ±
1.2 0.4 1.3 1.4
± 0.8 ~: 0 . 2 + 1.5 ± 0.1
Cyclic GMP (i~mol/mg protein) 0.5 1.0 2.0 5.0 10.0
275 235 375 427 539
+ 39 + 65 ± 55 + 21 -+ 8 2
298 265 338 397 581
+ 28 ± 51 -+ 37 ± 45 ± 71
311 301 385 452 490
+ 46 ~ 43 ± 22 + 45 + 111
710
Discussion These studies showed that the undecapeptides eledoisin and substance P caused the stimulation of amylase release and potassium efflux from parotid tissue slices. These effects were pharmacologically distinct from the effects of ~-adrenergic, ~-adrenergic, and cholinergic agonists since propranolol, phentolamine and atropine were not inhibitory. The concentrations of antagonists used would have blocked the effects of saturating concentrations of the respective agonists. Relative to the effect of ~ d r e n e r g i c agonists on amylase release [31] the action of the undecapeptides was slight. In this respect and because of the massive K ÷ efflux caused by the undecapeptides, the undecapeptides more closely resembled the effects of cholinergic and ~-adrenergic agonists on the parotid. As noted in Results, the undecapeptides are more potent at stimulating K + efflux than either carbachol or phenylephrine. Unlike the action of ~-adrenergic and cholinergic agonists, the undecapeptides had no effect on cyclic nucleotide levels in the parotid. We have previously shown that cholinergic and ~-adrenergic agonists inhibited increased accumulation of cyclic AMP caused by isoproterenol and that they increased cyclic GMP accumulation in the parotid [19,25]. Our findings suggest that the effects of the undecapeptides on both K ÷ and amylase release from the parotid were independent of effects on cyclic nucleotide levels. The similarity between the action of the undecapeptides, cholinergic and ~-adrenergic agonists also holds with respect to calcium requirement. In the present study we found that buffer calcium was required for the action of the undecapeptides on both amylase release and K ÷ efflux. A similar requirement for calcium was observed for ~-adrenergic [28] and cholinergic (Butcher, F.R., unpublished) agonist action. More recent experiments indicate that there might be some subtle differences between the requirement for calcium in the action of the undecapeptides and that for the action of either carbachol or phenylephrine. We found that D~00, a compound which blocks Ca2÷ flux, blocked the effect of phenylephrine and carbachol on K ÷ efflux while it did not inhibit the effects of the undecapeptides (Butcher, F.R., unpublished). A primary role for calcium in undecapeptide action is suggested since buffer calcium was required for increased K ÷ efflux and amylase release caused by the undecapeptides. The importance of calcium for agonist action is also underscored by the observations that the effects of phenylephrine [28], carbachol (Butcher., F.R., unpublished), and the ionophore A-23187 [29,30] on amylase release, K ÷ efflux and cyclic GMP accumulation are Ca 2÷ dependent. In this later instance Ca 2÷ was required for agonist action on both K ÷ efflux and cyclic GMP accumulation, which suggests that cyclic GMP might be involved in agonist effects on K ÷ efflux. However, the undecapeptides did not increase cyclic GMP accumulation but were more effective than carbachol, phenylephrine, or A-23187 at causing K ÷ efflux. We have also found that 1-methyl-3isobutyl-xanthine enhanced the effect of carbachol [25] and phenylephrine [19] on cyclic GMP accumulation but were without effect on increased K ÷ efflux caused by these agonists. These data suggest that cyclic GMP does not mediate the effects of the+ agonists on K ÷ efflux and that Ca 2÷ plays a major role in the regulation of K ÷ efflux from the parotid.
711 Acknowledgements This work was supported by a grant from the Cystic Fibrosis Research Foundation and by a U.S.P.H.S. Career Development Award to F.R.B. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
19 20 21 22 23 24 25 26 27 28 29 30 31 32
v o n Euler, U.S. a n d G a d d u m , J.H. (1931) J. Physiol. Lond. 72, 7 4 - - 8 7 Chang, M.M. a n d L e e m a n , S.E. (1970) Fed. Proc. 29, 202 L e e m a n , S.E. and Hammerschlag, R.E. (1967) Endocrinology 8 1 , 8 0 3 - - 8 1 0 Bertaccini, G., Cei, J.M. and Erspamer, V. (1965) Br. J. Pharmacol, 25, 363--879 Erspamer, V. a n d Falconieri Erspamer, G. (1962) Br. J. Pharmaeol. 1 9 , 3 3 7 - - 8 5 4 Erspamer, V. (1971) Ann. Rev. Pharmacol. 1 1 , 3 2 7 - - 3 5 0 Emmelin, N. and Lenninger, S. (1967) Br. J. Phm'maeol. 30, 6 7 6 - - 6 8 0 Lembeck, F. (1953) Naunyn-Schmiedeberg's Arch. Exp. Pathol. Pharmakol. 219, 197--213 Phillis, J.W. a n d Limaeher, J.J. (1974) Brain Res. 69, 158--163 Konashi, S. and Otsuka, M. (1974) Brain Res. 65, 3 9 7 - - 4 1 0 Takahashi, T., Kohishi, S., Powell, D., Leeman, S.E. and Otsuka, M. (1974) Brain Res. 73, 59---69 J o h n s o n , A.R, and Erdos, E.G. (1973) Proc. Soc. Exp. Biol. Med. 142, 1252--1256 Henry, J., Kinjevic, K. and Morris, M. (1974) Fed. Proe. 33, 548 Mills, I.H., Macfarlane, N.A.A. a n d Ward, P.E. (1974) Nature 247, 108--109 Burgen, A.S.V. (1967) H a n d b o o k of Physiology (Code, C., ed.), Section 6 (If); pp. 561--579, American Physiology Society, Washington, D.C. Treager, G.W., Nlall, H.D., Ports, Jr., J.T., Leeman, S.E. and Chang, M.M. (1971) Nat. New Biol. 232, 87~88 Bernfeld, P. (1955) (Colowick, S.P. and Kaplan, N.O., eds.), Methods in E n z y m o l o g y , VoL 1, pp. 149--154, Academic Press, New York Jakobs, K.H., B o h m e , E. and Schultz, G. (1976) Regulation of F u n c t i o n and Growth of Eukaryotie Ceils by Intracellular Cyclic Nueleotides ( D u m o n t , J.E., Butcher, R.W. and Brown, B., eds.), Plenum Press, New York Butcher, F.R., Rudieh, L., Emler, C. and Nemerovski, M. (1976) Mol. PharmacoL, in the press Brown, B.L., Albano, J.D,M., Ekins, R.P. and Sgherzi, A.M. (1971) Biochem. J. 121, 561--562 Steiner, A.L., Parker, C.W. and KipnJs, D.M. (1972) J. Biol. Chem. 247, 1106--1113 Harper, J.F. a n d Brooker, G. (1975) J. Cyclic Nucleotide Res. 1 , 2 0 7 - - 2 1 8 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265--275 Mangos, J.A., McSherry, N.R. and Barber, T. (1975) A m . J. Physiol. 229, 566---569 Butcher, F.R., McBride, P.A. and Rudich, L. (1976) Mol. Cell. Endocrin., in the press Batzri, S., selinger, Z., Schrernm, M. and Rabinovitch, M.R. (1973) J. Biol. Chem. 248, 361--~68 Mangos, J.A., MeSherry, N.R., Barber, T., Arvanitakis, S.N. and Wagner, V. (1975) A m . J. Physiol. 229, 560--565 Setinger, Z., Batzri, S., ELmer], S. and S c h r e m m , M. (1973) J. Biol. Chem. 248, 369--372 Selinger, Z., EimeH, S. a n d S c h r a m m , M. ( i 9 7 4 ) Proc. Natl. Aead. Sci. U.S. 71, 128--131 Butcher, F.R. (1975) Metabolism 24, 4 0 9 - - 4 1 8 Butcher, F.R., Goldman° J.A. a n d Nemerovsld, M. (1975) Biochim. Biophys. Aeta 392, 8 2 - - 9 4 Duffy, M.J., Wong, J. and Powell, D. (1974) Biochem. Soc. Trans. 2, 1262--1264
Biochimica et Biophysica A cta, 444 (1976) 704--711 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 28034
EFFECT OF SUBSTANCE P AND ELEDOISIN ON K+ EFFLUX, AMYLASE RELEASE AND CYCLIC NUCLEOTIDE LEVELS IN SLICES OF RAT PAROTID GLAND
LYNN RUDICH and F R E D R. BUTCHER
Division of Biological and Medical Sciences, Brown University, Providence, R.I. 02912
(U.S.A.) (Received March 23rd, 1976)
Summary The undecapeptides, substance P and eledoisin, caused a rapid, concentration~lependent increase in K ÷ efflux and amylase release from parotid tissue slices. The effects were not blocked by ~clrenergic, a-adrenergic, or cholinergic antagonists. Incubation buffer calcium was required for stimulation of K ÷ efflux and amylase release. The action of the undecapeptides was independent of any effects on parotid cyclic AMP or cyclic GMP levels. Since the actions of the undecapeptides were Ca 2+ dependent and no effects on cyclic nucleotide levels were discerned it was concluded that Ca 2+ plays a primary role in agonist regulation of K + efflux from the parotid.
Introduction In 1931~ while investigating the tissue distribution of acetylcholine, yon F~uler and Gaddum [1] found a substance in equine brain and intestine which caused hypotension and stimulated in vitro the contraction of intestinal tissue in rabbits. This material, substance P, was finally isolated in pure form from bovine hypothalamus in 1970 [2] and identified with a factor which was f o u n d to cause an increase in the fluid volume and amylase activity of rat salivary secretion in vivo [3]. Peptides such as physaelamin, isolated from the skin of the South American amphibian Physalaemus fuscumaculatus [4] and eledoisin, isolated from the posterior salivary gland of two molluscan species, Eledone moschata and Eledone androvandi [5] have been clauified with substance P by Erspamer [6] as tachychinins on the basis of similar biological and structural characteristics. In addition to causing atropine-resistant hypotension in the dog and rat [4], as a class they also increase capillary permeability in the rat, guinea pig and man,
705 and stimulate the contraction of isolated rabbit large intestine as well as the isolated ileum and large intestine of the guinea pig. They are also powerful stimulants of salivary and lacrimal secretions of the chicken, rat, dog and man [6,7]. The presence of substance P activity in nervous tissue and its specific distribution led Lembeck [8] to suggest that substance P is a transmitter released by primary sensory neurons. Much work has been directed toward verifying such a role [9,12]; however, on the basis of its relatively slow action, Henry et al. [13] have suggested that it functions as a modulator of sensory transmission. Mills et al. [14] have proposed the existence of "substance P-inergic" nerves which promote the release of renal kallikrein in dogs. Studies of the parotid have indicated that it is innervated by the parasympathetic system and sympathetic branches of the autonomic system [15]. Leeman and Hammerschlag [3] reported that the in vivo sialogenic activity of substance P in rats was not inhibited by atropine, phenoxybenzamine, or propranolol. Although the in vivo sialogenic activity of substance P was used to verify the success of its synthesis [16], it is not known with certainty that this was a direct effect on the salivary gland itself. An in vitro study of the action of these undecapeptides on rat parotid gland slices might serve as a model for their action. In addition the action of these undecapeptides on parotid gland might be exploited as tools in order to learn more about the regulation of parotid gland physiology by autonomic agents. Materials and Methods
Parotid glands from female Sprague-Dawley C D rats (Charles River Breeding Laboratories, Wilmington, Mass.), 7--9 weeks old fed adlibitum were used for all experiments. The glands were trimmed of connective tissue and slicedwith microscissors and maintained at 37°C in Krebs-Ringer bicarbonate buffer conraining 5 m M ~-hydroxybutyrate and 6 m M glucose with O2/CO2 (95 : 5, v/v) as the gas phase. Amylase was assayed by the method of Bernfeld [17] except that the assay was conducted at 37°C. Amylase content of the incubation buffer and that remaining in the tissue were both determined. The total amount of amylase was determined by summation of the amount in the buffer with the amount remaining in the tissue at the end of the incubation period. The percent of the total amylase which was released into the incubation buffer was then calculated. In our later experiments the amount of amylase released was determined by withdrawing aliquots of the incubation buffer and the amylase release data are expressed as units of amylase released into the incubation buffer. One unit of amylase was the amount of enzyme that caused the formation of I m g of maltose in 5 rain at 37°C. Potassium release studies were conducted in vials containing 1.0 ml of KrebsRinger bicarbonate buffer and the equivalent of one gland. Parotid slices contain a very active K ÷ uptake mechanism which can obscure effects of low agonist concentration or of weak against on K* efflux. In order to obviate this problem 1.0 mM ouabain was added to the incubation vials in all K ÷efflux experiments except those reported in Table II. Ouabain was omitted from these experiments because omission of Ca 2+ from the incubation buffer caised K÷ efflux
706 which was greatly magnified by ouabaln. The K* released was determined with an Instrumentation Laboratory model 143 flame photometer (Boston, Mass.). Incubations for the determination of cyclic nu¢leotides were stopped by adding HC104 to a final concentration of 0.5 M and rapidly homogenizing with a polytron homogenizer (Brinkman Instrument Co., Westbury, N.Y.). The cyclic nucleotides in the acid supernatant were purified by sequential column chromatography over neutral alumina and Dowex 1-X8 (formate form) according to Jakobs et at. [18] and Butcher et al. [19], respectively. Cyclic AMP was assayed by a protein binding assay [20] and cyclic GMP was assayed by the radioimmunoassay procedure of Steiner et al. [21] using the acetylation procedure of Harper and Brooker [22]. Protein was measured by the method of Lowry et al. [23]. Agonists were dissolved in glass distilled water to a concentration so that an a m o u n t no greater than 50 ~1 was added to each vial. Theophylline, atropine, sulfate, (±)-isoproterenol • HCI and ouabain were products of Sigma Chemical Co. (St. Louis, Mo.). Phentolamine methane sulfate was obtained from CIBA (Ardsley, N.Y.). (--)-Propranolol was a gift from Ayerst Laboratories, Inc. (New York, N.Y.). Synthetic bovine substance P was a product of Beckman Instruments, Inc. (Palo Alto, Calif.) and Eledoisin-950 was a gift of Sandoz Pharmaceuticals. Substance P and eledoisin are undecapeptides. Results
Effect of undecapeptides on amylase release. Both eledoisin and substance P cause a rapid, concentmtion~iependent increase in amylase release (Fig. 1). The ~mount of amylase release caused by the undecapeptides was small. Only 16% of the initial tissue amylase content was released after addition of the undecapeptides. The action of the undecapeptides on amylase release was not mediated through indirect effects of neurotransmitters since none of the pharmacological antagonists were inhibitory (Table I). The concentrations of the antagonists used for the studies in Table I were sufficient to completely inhibit the effects of saturating concentrations of the appropriate agonist. Effect of undecapeptides on K ÷efflux. In addition to stimulation of amylase release, the undecapeptides also promoted a rapid, concentration-dependent increase in K ÷ efflux (Fig. 2). Increased K ÷ efflux was detected at 30 s, the earliest time point examined. The time course for increased K* efflux caused by the undecapeptides was similar to that caused by cholinergic [ 2 4 ~ 5 ] and a~drenergic [19,26,27] agonists. The undecapeptides, however, are much more p o t e n t since increased K* effiux was detected at 10 nM while 100 rum was the lowest effective concentration for cholinergic agonists [25]. ~-Adrenergic agonists are less effective than chotinergic ago~Asts in promoting K ÷ efflux. Stimulation of K ÷ efflux by the undecapeptides appears pharmacologically distinct from ~-adrenergic, ~-ad~nergic, and cholinergic agonists since propranolol, phentolamine and atropine were n o t inhibitory. Calcium requirement for the effects of the undecapcp~ides. Incubation buffer calcium is r e q ~ for the effects of cholinorgic (Butcher, F.R., unpublished), ~-adrenergic [28] agonists, and the divalent cation ionophore [29,30] on
707
,~
J
•
ELEDOtSIN
AMYLASE
< 5,00
@
as
>
400 <[
Z 200 t_/
f
' °/""° [ J '.
t/.. .
;L O G2 :o C O N5,.0C E N TI 0R A T2 'o ;0 ION,riM
1.
1 0
l/II
20
3'0
MINUTES F i g . I . E f f e c t o f s u b s t a n c e P a n d e l e d o i s i n o n a m y l a s e r e l e a s e f r o m r a t p a r o t i d tissue slices. P a z o t i d slices f r o m t w o r a t s w e r e u s e d f o r each c o n e e n t ~ t i o n c u r v e a n d f o r t h e t l ~ e c o u r s e e x p e r l m e n t s . T h e v a l u e s plotted are the aventges of two sepmte paired experiments. Basal amylase Meue values, at 40 min, for the concentration curves were 2.2 ± 1.0 and 2.2 ~ 0.3% for substance P and eledols/n, respectively. The 5-, 1 0 . , 2 0 - a n d 4 0 . r a i n b a s a l a m y l a s e r s l e a m v a l u e s f o r t h e t / m e c o u r s e s t u d i e s w e r e 1 3 . 0 ± 8 . 4 , 1 5 . 3 ± 2.5, 20.1 + 4.1 and 25.1 ± 7.8 units/rag protein, respectively. The numbers 511, and 800 on the concent r a t i o n c u r v e r e f e r t o n M c o n c e n t r a t i o n s o f e l e d o i s l n a n d s u b s t a n c e P, r e ~ p e e t i v e l y . TABLE I EFFECT OF PHARMACOLOGICAL BLOCKING AGENTS ON THE STIMULATION RELEASE AND K+ EFFLUX BY ELEDOISIN AND SUBSTANCE P
OF AMYLASE
Tissue slices f r o m t w o r a t s w e r e u s e d in e a c h e x p e r i m e n t f o r d e t m n n l n a t i o n o f a m y l a s e M e M e . V a l u e s a r e the average of two expe]dments for each agonist. Basal amylase release was 4.6 + 2.2% for substance P ex• + p e r l m e n t s a n d 2 . 7 + 0 . 2 % f o r e l e d o h d n e x p e r i m e n t s . F o r t h e d e t e r m i n a t i o n o f K e f f l u x , tissue slices f r o m four rats were pooled for each experiment. Two experiments were performed for each agonist. Basal v a l u e s a f t e r 8 m l n o f i n c u b a t i o n i n t h e p r e s e n c e o f 1 . 0 m M o u a b a l n a l o n e w e r e 1 3 , 2 ± 1.7 a n d 1 8 . 3 ± 6 . 7 % po_t~_~m~__ume f f l u x f o r s u b . a n t e P a n d e i c d o i s l n e x p e r i m e n t s , r e s p e c t / v a l y . Additions
None Atropine, 10 #M Phentolamine, 5 pM (--)-Propranolol, 1 pM
Percent increase in amylase release above control Control
Substance P ( 3 7 riM)
Eledoisln ( 1 6 8 riM)
-23 + 7 (--) 22 ± Y (--) 5 ± 6
272 200 213 239
601 524 592 635
± ± ± ±
65 17 22 35
± ± ± ±
29 46 51 46
P e r c e n t i n c r e a s e in K + e f f l u x a b o v e c o n t r o l Control
None Atropine, 10#M Phentolamine, 5 #M (~)-Proprenolol, I #M
-15 + 48 14 + 7 16 ± 38
Substance P (149 nM)
Eledoisin
472 417 402 414
267 237 237 245
+ + + ±
65 20 14 20
(168 nM) + 2 + 15 ± 9 ± 16
708 I
K •~EFFLUX ,
& SUBSTANCEP
~)10(
&r
"•
•
z200
I0
20 S0 LOG CONCENTRATION
I00
5 MINUTES
nM
10
F i g . 2 . E f f e c t o f s u b s t a n c e P a n d e l e d o i s i n o n p o t a s s i u m e f f l u x f ~ m r a t p a r o t h l t / s a t e slices. P a r o t i d slices f r o m f o u r r a t s w e r e u s e d f o r e a c h c o n m m / n t i o n c u r v e s h o w n . P a r o ~ i d slices f r o m t w o r a t s w e r e u s e d f o r e a c h t i m e c o u r s e . T h e v a l u e s p l o t t e d a r e t h e a v e r a g e s o f t w o p a i r e d expez:lmonts. B a s a l p o t e s s / u m r e l e a s e v a l u e s f o r t h e c o n c e n t r a t i o n c u r v e s w e r e 1 0 . 1 ± 1 . 0 a n d 1 8 . 8 + 1.595 f o r 8 u b e t a n c e P a n d e l e d o i s i n , r e s p e c tively. The 0- and lO,m/n basal potmmlum release vslues for the t/me c ~ studies were 3.6 ± 0.1 and 1 5 . 3 ± 0.595 f o r e l e d o i s l n , r e s p ~ t i v e l ¥ , a n d t h o s e f o r s u b s t a n c e P w e r e 6 . 0 + 0 . 6 a n d 1 4 . 6 ± 0.495, t e e p e e Uvely. T h e c o n c e n t r a t i o n s o f s u b s t a n c e P a n d e l e d o i s i n w e r e 7 5 a n d 166 riM, r e s p e c t i v e l y .
amylase release, K ÷ efflux, and cyclic GMP accumulation. Calcium was also required for stimulation of a-amylase release and K ÷ effiux from the parotid slices by undecapeptides (Table If). TABLE H EFFECT OF CALCIUM ON THE STIMULATION ELEDOISIN AND SUBSTANCE P
OF AMYLASE
RELEASE
AND K+ EFFLUX
BY
T o d e t e r m i n e a m y l a s e r a l e ~ , t i s s u e slices f r o m t w o r a t s w e r e p o o l e d f o r e a c h o f t w o e x p e r i m e n t s , A v e r a g e b a s a l a m y l a s e release v a l u e w a s 4 . 9 + 1,395. F o r t h e d e t e r m i n a t i o n o f K ÷ e f f l u x , tissue slices f r o m f o u r rats were pooled for each experiment. Two experiments were performed for each qonist. Average basal values after incubation for 8 rain in the absence of 1.0 mM ouabain were 6.2 ± 0.3 and 10.2 ± 1.1% potassium efflux for substance P and aledoisin experiments, respectively. Additions
None Eledoisin, 168 nM S u b s t a n c e P, 3 7 n M
P e r c e n t i n c r e a s e i n ~-flnlylase r e l e ~ W i t h Ca ~
W i t h o u t C a 2+
-261 + 47 171 + 2
(--) 5 1 + (--) 20 ± (--) 41 +
above control
2 4 6
Percent increase in K + efflux above control None Eledoisin, 168 nM S u b s t a n c e P, 1 4 9 n M
-121 ± 33 97 + 39
(--) 0 ± 3 2 (--) 50 + 28 (--) 1 0 + 1 9
709
Effect of undecapeptides on parotid cyclic nucleotide levels. Considerable evidence exists which suggests that cyclic nucleotides might mediate agonist effects on their target tissues. The undecapeptides had no effect on either cyclic AMP or cyclic GMP levels in the parotid (Table III). Several time points and undecapeptide concentrations, in the presence and absence of theophylline or 1-methyl-3-isobutyl-xanthine, were examined, but still no effect on cyclic nucleotide levels could be detected (Table III, the data obtained in the absence of phosphodiesterase inhibitors is not shown). In other studies we have found that theophylline potentiates the stimulation of cyclic AMP accumulation by weak agonists [31] and that 1-methyl-3-isobutyl-xanthine enhanced the stimulation of cyclic GMP accumulation by low concentrations of various agonists [19,25,30]. The time points selected encompass both the time course for the effects of other agonists or parotid cyclic nucleotide levels and the time course for the effects of the undecapeptides on K ÷ efflux and amylase release from the parotid (see Figs. 1 and 2). Duffy et al. [32] have reported that substance P stimulated adenylate cyclase in broken cell preparations from rat and human brain. The undecapeptides did not inhibit stimulation of ~-amylase release or cyclic AMP accumulation by isoproterenol (not shown).
TABLE In T H E E F F E C T S U B S T A N C E P A N D E L E D O I S I N ON CYCLIC N U C L E O T I D E L E V E L S IN SLICES OF RAT PAROTID GLAND
Parotid ~ I c e s f r o m t h r e e r a t s were u s e d in each e x p e r i m e n t for cyclic AMP d e t e r m i n a t i o n s . T h e values f o r the cyciic A M P levels are the m e a n s + S.E., f r o m six e x p e r i m e n t s for c o n t r o l s a n d f r o m three experim e n t s f o r s u b s t a n c e P and ciedolsin. I n c u b a t i o n s w e r e carried o u t in the presence o f S 0 0 p M t h e o p h y l l i n e . Previous e x p e r i m e n t s h a v e i n d i c a t e d that this c o n c e n t r a t i o n o f t h e o p h y l l i n e greatly p o t e n t i a t e s the e f f e c t s o f i s o p r o t e r e n o l o n cyclic A M P a c c u m u l a t i o n in rat p a r o t i d tissue slices. F o r cyclic GMP d e t e r m i n a t i o n s , p a r o t i d slices f r o m f o u r rats w e r e u s e d in e a c h e x p e r i m e n t . T h e v a l u e s for the c y c l i c GMP levels are the m e a n s ±S.E. f r o m six e x p e ~ m e n t s f o r c o n s o l s a n d f r o m three e x p e r i m e n t s for substance P a n d eledolsin. I n c u b a t i o n s w e r e carried o u t in the presence o f 2 0 0 / ~ M m e t h y l - / s o b u t y l - x a n t h i n e , w h i c h is s u f f i c i e n t to p o t e n t i a t e the e f f e c t s o f e a r h a c h o l and p h e n y l e p h r i n e o n cyclic GMP a c c u m u l a t i o n in slices o f rat parotid gland. In all cases, t h e e x p e r i m e n t s to d e t e r m i n e the e f f e c t s o f substance P and ciedoiain o n c y c l i c n u e l e o tide levels were d o n e separately. T i m e (rain)
2.5 5.0 10.0 20.0
Cyclic A M P ( p m o l / m g protein)
Control
Substance P (149 nM)
Eledoisln ( 1 6 8 nM)
7.0 8.2 7.7 9.3
6.4 9.1 7.3 10.1
7.6 8.8 9.3 8.1
-+ 0 . 5 + 0.3 + 1.1 + 1.2
+ + ± ±
1.2 0.4 1.3 1.4
± 0.8 ~: 0 . 2 + 1.5 ± 0.1
Cyclic GMP (i~mol/mg protein) 0.5 1.0 2.0 5.0 10.0
275 235 375 427 539
+ 39 + 65 ± 55 + 21 -+ 8 2
298 265 338 397 581
+ 28 ± 51 -+ 37 ± 45 ± 71
311 301 385 452 490
+ 46 ~ 43 ± 22 + 45 + 111
710
Discussion These studies showed that the undecapeptides eledoisin and substance P caused the stimulation of amylase release and potassium efflux from parotid tissue slices. These effects were pharmacologically distinct from the effects of ~-adrenergic, ~-adrenergic, and cholinergic agonists since propranolol, phentolamine and atropine were not inhibitory. The concentrations of antagonists used would have blocked the effects of saturating concentrations of the respective agonists. Relative to the effect of ~ d r e n e r g i c agonists on amylase release [31] the action of the undecapeptides was slight. In this respect and because of the massive K ÷ efflux caused by the undecapeptides, the undecapeptides more closely resembled the effects of cholinergic and ~-adrenergic agonists on the parotid. As noted in Results, the undecapeptides are more potent at stimulating K + efflux than either carbachol or phenylephrine. Unlike the action of ~-adrenergic and cholinergic agonists, the undecapeptides had no effect on cyclic nucleotide levels in the parotid. We have previously shown that cholinergic and ~-adrenergic agonists inhibited increased accumulation of cyclic AMP caused by isoproterenol and that they increased cyclic GMP accumulation in the parotid [19,25]. Our findings suggest that the effects of the undecapeptides on both K ÷ and amylase release from the parotid were independent of effects on cyclic nucleotide levels. The similarity between the action of the undecapeptides, cholinergic and ~-adrenergic agonists also holds with respect to calcium requirement. In the present study we found that buffer calcium was required for the action of the undecapeptides on both amylase release and K ÷ efflux. A similar requirement for calcium was observed for ~-adrenergic [28] and cholinergic (Butcher, F.R., unpublished) agonist action. More recent experiments indicate that there might be some subtle differences between the requirement for calcium in the action of the undecapeptides and that for the action of either carbachol or phenylephrine. We found that D~00, a compound which blocks Ca2÷ flux, blocked the effect of phenylephrine and carbachol on K ÷ efflux while it did not inhibit the effects of the undecapeptides (Butcher, F.R., unpublished). A primary role for calcium in undecapeptide action is suggested since buffer calcium was required for increased K ÷ efflux and amylase release caused by the undecapeptides. The importance of calcium for agonist action is also underscored by the observations that the effects of phenylephrine [28], carbachol (Butcher., F.R., unpublished), and the ionophore A-23187 [29,30] on amylase release, K ÷ efflux and cyclic GMP accumulation are Ca 2÷ dependent. In this later instance Ca 2÷ was required for agonist action on both K ÷ efflux and cyclic GMP accumulation, which suggests that cyclic GMP might be involved in agonist effects on K ÷ efflux. However, the undecapeptides did not increase cyclic GMP accumulation but were more effective than carbachol, phenylephrine, or A-23187 at causing K ÷ efflux. We have also found that 1-methyl-3isobutyl-xanthine enhanced the effect of carbachol [25] and phenylephrine [19] on cyclic GMP accumulation but were without effect on increased K ÷ efflux caused by these agonists. These data suggest that cyclic GMP does not mediate the effects of the+ agonists on K ÷ efflux and that Ca 2÷ plays a major role in the regulation of K ÷ efflux from the parotid.
711 Acknowledgements This work was supported by a grant from the Cystic Fibrosis Research Foundation and by a U.S.P.H.S. Career Development Award to F.R.B. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
19 20 21 22 23 24 25 26 27 28 29 30 31 32
v o n Euler, U.S. a n d G a d d u m , J.H. (1931) J. Physiol. Lond. 72, 7 4 - - 8 7 Chang, M.M. a n d L e e m a n , S.E. (1970) Fed. Proc. 29, 202 L e e m a n , S.E. and Hammerschlag, R.E. (1967) Endocrinology 8 1 , 8 0 3 - - 8 1 0 Bertaccini, G., Cei, J.M. and Erspamer, V. (1965) Br. J. Pharmacol, 25, 363--879 Erspamer, V. a n d Falconieri Erspamer, G. (1962) Br. J. Pharmaeol. 1 9 , 3 3 7 - - 8 5 4 Erspamer, V. (1971) Ann. Rev. Pharmacol. 1 1 , 3 2 7 - - 3 5 0 Emmelin, N. and Lenninger, S. (1967) Br. J. Phm'maeol. 30, 6 7 6 - - 6 8 0 Lembeck, F. (1953) Naunyn-Schmiedeberg's Arch. Exp. Pathol. Pharmakol. 219, 197--213 Phillis, J.W. a n d Limaeher, J.J. (1974) Brain Res. 69, 158--163 Konashi, S. and Otsuka, M. (1974) Brain Res. 65, 3 9 7 - - 4 1 0 Takahashi, T., Kohishi, S., Powell, D., Leeman, S.E. and Otsuka, M. (1974) Brain Res. 73, 59---69 J o h n s o n , A.R, and Erdos, E.G. (1973) Proc. Soc. Exp. Biol. Med. 142, 1252--1256 Henry, J., Kinjevic, K. and Morris, M. (1974) Fed. Proe. 33, 548 Mills, I.H., Macfarlane, N.A.A. a n d Ward, P.E. (1974) Nature 247, 108--109 Burgen, A.S.V. (1967) H a n d b o o k of Physiology (Code, C., ed.), Section 6 (If); pp. 561--579, American Physiology Society, Washington, D.C. Treager, G.W., Nlall, H.D., Ports, Jr., J.T., Leeman, S.E. and Chang, M.M. (1971) Nat. New Biol. 232, 87~88 Bernfeld, P. (1955) (Colowick, S.P. and Kaplan, N.O., eds.), Methods in E n z y m o l o g y , VoL 1, pp. 149--154, Academic Press, New York Jakobs, K.H., B o h m e , E. and Schultz, G. (1976) Regulation of F u n c t i o n and Growth of Eukaryotie Ceils by Intracellular Cyclic Nueleotides ( D u m o n t , J.E., Butcher, R.W. and Brown, B., eds.), Plenum Press, New York Butcher, F.R., Rudieh, L., Emler, C. and Nemerovski, M. (1976) Mol. PharmacoL, in the press Brown, B.L., Albano, J.D,M., Ekins, R.P. and Sgherzi, A.M. (1971) Biochem. J. 121, 561--562 Steiner, A.L., Parker, C.W. and KipnJs, D.M. (1972) J. Biol. Chem. 247, 1106--1113 Harper, J.F. a n d Brooker, G. (1975) J. Cyclic Nucleotide Res. 1 , 2 0 7 - - 2 1 8 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265--275 Mangos, J.A., McSherry, N.R. and Barber, T. (1975) A m . J. Physiol. 229, 566---569 Butcher, F.R., McBride, P.A. and Rudich, L. (1976) Mol. Cell. Endocrin., in the press Batzri, S., selinger, Z., Schrernm, M. and Rabinovitch, M.R. (1973) J. Biol. Chem. 248, 361--~68 Mangos, J.A., MeSherry, N.R., Barber, T., Arvanitakis, S.N. and Wagner, V. (1975) A m . J. Physiol. 229, 560--565 Setinger, Z., Batzri, S., ELmer], S. and S c h r e m m , M. (1973) J. Biol. Chem. 248, 369--372 Selinger, Z., EimeH, S. a n d S c h r a m m , M. ( i 9 7 4 ) Proc. Natl. Aead. Sci. U.S. 71, 128--131 Butcher, F.R. (1975) Metabolism 24, 4 0 9 - - 4 1 8 Butcher, F.R., Goldman° J.A. a n d Nemerovsld, M. (1975) Biochim. Biophys. Aeta 392, 8 2 - - 9 4 Duffy, M.J., Wong, J. and Powell, D. (1974) Biochem. Soc. Trans. 2, 1262--1264