VIP and secretin receptors linked to an adenylate cyclase on central neuronal and glial cells in primary cultures

$23 E V O L U T I O N A R Y STUDIES ON THE VIP/SECRETIN F A M I L Y : THE ISOLATION AND CHARACTERISATION OF DOGFISH VIP. R. Dimaline and M.C. Thornd),ke. MRC Secretory Control Group, D e p a r t m e n t of Physiolog), University of L i v e r p o o l , P.O. Box 147, L i v e r p o o l , L69 3BX. D e p a r t m e n t of Zoology, Royal Holloway and Bedford Colleges, Egham Hill, Egham, Surre), TW20 OEX, U.K. The evolutionary relationships of VIP remain poor[), understood, even though it is clear that VIP-like peptides are present in a wide range of species. To examine the phylogenetic relationships in detail, we have undertaken the isolation and character[sat[on of VIP from intestine of a lower v e r t e b r a t e , the elasmobranch Sc) liorhinus canicula. Intestine (206g) from the dogfish, Sc),liorhinus, was boiled in water, acidified (0.SM acetic acid),homogenised and Hi Flo f i l t e r e d . The f i l t r a t e was adsorbed onto alginic acid, eluted with HCI and the eluate salted out twice. The final precipitate(1.82g) was dissolved in 1.6M acetic acid and desalted on Sephadex G25F (2.6xl00cm). The eluate containing the peak of absorbance was b ophilised (105mg), dissolved in 0.02M ammonium bicarbonate and applied to a column of CM22 cellulose equilibrated in the same. The VIP-like i m m u n o r e a c t i v i t y ( N - t e r m i n a l V[P antiserum L25) was eluted with 0.08M buffer and lyophilised. The lyophilisate was dissolved in 0.1% TFA and subjected to sequential reversed-phase HPLC on columns equilibrated in 0.1% TFA and eluted with a c e t o n i t r i l e . The m a t e r i a l was run on Techsil 511 C18 (twice), Vydac 101~ C18, and f i n a i l ) Vydac 51~ C18. In the final run the m a t e r i a l was eluted b) )0% a c e t o n i t r i l e and the i m m u n o r e a c t i v i t ) was associated with a single sharp peak of absorbance at 214nm, just before the elution position of synthetic porcine VIP. On 5ephadex GS0s. the peptide e[uted in a similar position to synthetic porcine VIP. Sc),liorhinus VIP diluted in parallel with synthetic VIP when an N - t e r m i n a l VIP antiserum was used, but did not cross react significant[) with a C - t e r m i n a l V[P antiserum. A similar pattern of VIP-like immunoreactivit), was seen in crude intestinal extracts of another dogfish 5qualus acanthius. Further structural studies are in progress. Conclusions. 1. The a m i n o - t e r m i n a l region of VIP has been well conserved during evolution. 2. There are distinct structural differences at the C-terminus of dogfish VIP, t h a t ma t well a f f e c t the biological properties of the molecule.

VIP AND SECRETIN RECEPTORS LINKED TO AN A D E N Y L A T E CYCLASE ON C E N T R A L N E U R O N A L AND G L I A L CELLS IN P R I M A R Y CULTURES. H. Chneiweiss~ J. P r 4 m o n t v J. Glowinski, C h a i r e de N e u r o p h a r m a c o log[e, INSERM U.II4, C o i l 6 g e de France, Paris, F r a n c e The p r e s e n c e of VIP r e c e p t o r s c o u p l e d to an a d e n y l a t e c y c l a s e was demonstrated on m e m b r a n e s of neurons or glial cells grown in p r i m a r y c u l t u r e s o r i g i n a t i n g from the c e r e b r a l cortex, s t r i a t u m and m e s e n c e p h a l o n of m o u s e embryos. A b i p h a s i c p a t t e r n of a c t i v a t i o n was o b s e r v e d in all cell types, i n v o l v i n g d i s t i n c t high and low a p p a r e n t a f f i n i t y m e c h a n i s m s . On neurons, the absence of a d d i t i v e e f f e c t s of VIP and d o p a m i n e (DA), i s o p r o t e r e n o l (ISO) and s e r o t o n i n (5-HT) suggests that the p e p t i d e r e c e p t o r s are c o - l o c a t e d w i t h each of the c o r r e s p o n d i n g amine receptors. The n o n - a d d i t i v i t y between VIP and ISO i n d u c e d r e s p o n s e s on g l i a l cells r e v e a l s a c o - l o c a t i o n of VIP- and @ - a d r e n e r g i c - s e n s i t i v e a d e n y l a t e c y c l a s e s on the same l cells. Secret[n, a structurally homologous peptide, has been shown to e l i c i t cAMP a c c u m u l a t i o n in b r a i n tissue. I r r e s p e c t i v e to the cell tested (neuron v e r s u s glial) or the b r a i n area u n d e r e x a m i n a t i o n , secretin induced never more than 10% of the VIP stimulation. Furthermore, when assayed simultaneously, s e c r e t i n did not alter the VIP p a t t e r n of activation. These results demonstrate that s e c r e t i n is not an a g o n i s t of VIP r e c e p t o r s in the CNS, and suggest that it acts on d i s t i n c t r e c e p t o r s linked to an a d e n y l a t e cyclase.