A cta Physiologica S inica, February 25, 2003, 55(1) : ( PACAP) ; Neuro22a ;

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1 42 A cta Physiologica S inica, February 25, 2003, 55(1) : Neuro22a 1, 3, 2, 3 3, 1, ; 2 Department of Psychiatry, Yale University School of Medicine, Italy ; 3, : M TT,, (pituitary adenylate cyclase activating polypeptide 27, PACAP27) A Neuro22a, PACAP ( < mol/l) Neuro22a A, PACAP PACAP mol/ L A, 011 mol/ L PACAP, 10 min, PACAP 10 min A, PACAP,, : ; ( PACAP) ; Neuro22a ; ; : Q421 ; R338 Pituitary adenylate cyclase activating polypeptide protects neuro22a cells from amyloid protein cytotoxicity by modulating intracellular calcium GU I Lan2Run 1, ZHOU Yan 2, ZHAN G Bing2Lie 3, L I Wen2Bin 3 1 Instit ute of M icrobiology, Beijing M ilitary Medical College, Beijing ; 2 Depart ment of Psychia2 t ry, Yale U niversity School of Medicine, Italy ; 3 Instit ute of Gerontology, PL A General Hospital, Beiji ng Abstract : M TT analysis and intracellular calcium measurement by using confocal laser scanning mi2 croscopy were used to st udy t he possible mechanism of protective effect of pit uitary adenylate cyclase acti2 vating polypeptide 27 ( PACAP27) from amyloid protein ( A )2induced neurotoxicity The results showed that treatment with PACAP (less than 011 mol/ L) increased the survival and reproductive abil2 ity of neuro22a cells and protected the neuro22a cells from being injured by A The protective effect of PACAP27 was reversed by t he competitive PACAP receptor antagonist PACAP6227 An increase in in2 tracellular calcium was observed when the cells were challenged with A and PACAP But the calcium in2 crease induced by A kept stable for a long time while PACAP caused a transient rise in intracellular cal2 cium The intracellular calcium increase induced by A was blocked by pretreatment with PACAP for 10 min It is suggested that the neuroprotective effect of PACAP against neuronal damage induced by A may result from its role in inhibiting the sustained rise in intracellular calcium Key words : amyloid protein ; pituitary adenylate cyclase activating polypeptide ; neuro22a cells ; confocal laser scanning microscopy ; int racellular calcium Received Accepted Corresponding author Tel : ; E2mail :

2 : PACAP27 Neuro22a 43 M TT, 595 [1 ], nm 96,, M TT, M TT 5, DNA, Alzheimer s 4 (AD), 113 AD (A ) [ 7] Fluo23/ AM ( [2 ], A Molecular Probes Inc, DMSO, 1 AD g/ ml, - 20 ) ( PACAP) F2127 (, 25 % (secretin) (glucagon) DMSO ) Hank s 20 (vasoactive intestinal peptide, VIP) 96,37 30 min,,, Hank s D2Hank s ( ), 200 [3 ], l, ( Meridian) PACAP Plus 5 W, 490 [4 ] PACAP nm, 520 nm,, 2 h, 4 h PACAP38, % [5 ] PACAP,, Time Plot, PACAP A 114, mean SD [6 ],, t PACAP A, A Neuro22a 2 PACAP, 211 PACAP A Neuro22a PACAP A 1, PACAP Neuro22a 48 h, Neuro22a,, 011 mol/ L 1, 111 Neuro22a 150 Neuro22a, PACAP PACAP6227 Neuro22a / ml 37 5 % 48 h CO 2 2, Table 11 Changes in mean cell viability after the treat % 37 5 min, ment with PACAP and PACAP6227 for 48 h 1 4 : 93 % DMEM 5 % (M TT reduction rate %) 2 % N 2 ( Sigma ) 2 g/ L HEPES PACAP PACAP + PACAP mg/ L 100 U/ L PACAP (Log mol/ L) (100 mol/ L) 112 MTT Neuro22a Control h 5 : PACAP A PACAP + A PACAP + A PACAP6227, Sigma A mol/ L, PACAP mol/ L 48 h, l M TT ( 32( 4, 52dimethylthiazol22yl )22, 52 diphenytetrazolium bromide, Sigma) (5 mg/ ml) 10 l, 37 4 h,, 200 l M TT test, means SD, n = 5 in each group, t test, P 3 < 0105 compared to control

3 44 Acta Physiol Si n1, February 25, 2003, 55 (1) : PACAP PACAP6227 (100 mol/ L ) 1 2, 25 A, ( 1 C), 100 mol/ L PACAP6227 PACAP mol/ L A Neuro22a ( 1 B ), ( 1 D ) ( 1 A ), 100 mol/ L PACAP mol/ L PACAP ( 1 E) 1 Neuro22a A Fig1 1 Morphological changes of Neuro22a cells treated with different agents A : Control ; B : A ; C : PACAP + A ; D : PACAP + A + PACAP6227 ; E : PACAP6227 (100 mol/ L) Scale bar, 10 m

4 : PACAP27 Neuro22a 45 2 Neuro22a 48 h min, 25 mol/ L A Neuro22a, ( ) Table 21 Changes in the cell viability after treatment wit h different agent s ( M T T reduction rate %, the cell viability in A group is set at 50 %) PACAP (Log mol/ L) A + PACAP A + PACAP + PACAP M TT test, mean SD, n = 5 in each group, the concen2 3 tration of PACAP6227 is 100 mol/ L, t test, P < 0105 com2 pared to A group 212 A Neuro22a Ca mol/ L A Ca 2 + ( 2 A), Ca 2 +, s, 400 s Ca 2 + D2Hank s ( 2 B ), A Ca 2 +, A Ca 2 +, 213 PACAP Neuro22a Ca 2 + PACAP ( 011 mol/ L), 011 mol/ L ( 2 C), 4 : (1),, ; (2) A, Hank s D2Hank s, 2 Neuro22a Confocal, ; (3) PACAP Fig1 2 Changes in confocal images of Neuro22a cells after Hank s D2Hank s, 1 h PACAP,, L A, and Hank s was used as the extracellular fluid B : ; (4) 100 mol/ L PACAP Neuro22a cells were treated with 25 mol/ L A, and D2 PACAP min, PACAP were treated with 011 mol/ L PACAP, and the results were similar when the cells were treated by extracellular Hank s or 214 PACAP Neuro22a A Ca mol/ L PACAP Neuro22a s treatment with different agents ( time plot between two im2 ages is 100 s) A : Neuro22a cells were treated with 25 mol/ Hank s was used as the extracellular fluid C : Neuro22a cells D2Hank s

5 46 Acta Physiol Si n1, February 25, 2003, 55 (1) : Neuro22a, [ 1 ] Schanne FA, Kane AB, Young EE, Farber JL Calcium,, dependence of toxic death : a final common pathway M TT Science 1979 ; 206 (4419) : [2 ] Arispe N, Pollard HB, Rojas E Giant multilevel cation, AD, channels formed by Alzheimer s disease amyloid 2pro2 [8 ] [9 ], teins A 1240 in bilayer membranes Pro Natl Sci USA 1993 ; 90 (22) : , A [ 3 ] Vaudry D, Gonzalez BJ, Basille M, Anouar Y, Fournier Neuro22a A, Vaudry H Pituitary adenylate cyclase activating, polypeptide stimulates both c2fos gene expression and calls survival in rat cerebella granule neurons through ac2, tivation of the protein kinase A pathway Neuroscience, A PACAP 1998 ; 84 : [4 ] Yuhara A, Nishio C, Abiru Y, Hatanaka H, Takei N Neuro22a, PACAP has a neurotrophic effect on cultured basal fore2 A, brain cholinergic neurons from adults rats Brain Res Dev Brain Res 2001 ; 131(122) : 41 45, [5 ] Reglodi D, Somogyvari2Vigh A, Vigh S, Kozicz T, PACAP Arimura A Delayed systemic administration of, PACAP38 is neuroprotective in transient middle cerebral artery occlusion in the rat Stroke 2000 ; 31 : 1411,, 1417, ; ( > 1 [ 6 ] Gui L R ( ), Li WB, Zhang BL, Fang ZY Acti2 vation of PACAP receptor could protect cultured hip2 h) pocampal neurons against amyloid peptide induced neu2 ; PACAP 10 min A rotoxity Chin J Applied Physiol ( ) 2002 ; 18 : (in Chinese with English ab2 stract) PACAP A [7 ],, [6, ], 1997, [10 ] [8 ] Suuronen T, Kolehmainen P, Salminen A Protective, 5 min 1 nmol/ L effect of L2deprenyl against apoptosis induced by okadaic 011 pmol/ L PACAP 500 mol/ L acid in cultured neuronal cells Biochem Pharmacol 2000 ; 59 : , [ 9 ] Sudoh S, Hua G, Kawamura Y, Maruyama K, Komano H, Yanagisawa K Intracellular site of gamma2secretase cleavage for Abeta42 generation in neuro22a cells harbor2 PACAP, ing a presenilin 1 mutation Eur J Biochem 2000 ; 267 : PACAP, PACAP V IP ; PACAP [10 ] Dong Y ( ), Tang TS, Lu CL, He C, Dong JB, [11, PACAP V IP ] Huang XY, Sun FZ, Bao X Pituitary adenylate cyclase activating polypeptide ameliorates the damage and in2, PACAP PACAP6227 hibits the increase of intracellular calcium concentration in cultured hippocampal neurons induced by glutamate PACAP, Acta Physical Sin ( ) 2000 ; 52 : (in PACAP Chinese with English abstract) [12, 13 ] [11 ] Arimura A Perspectives on pituitary adenylate cyclase, : (1) activating polypeptide ( PACAP) in the neuroendocrine, A C camp/ A endocrine, and nervous systems Jpn J Physiol 1998 ; ; (2) 48 : IP 3 [ 12 ] Rawlings SR, Canny BJ, Leong DA Pituitary adenylate ; (3) IP 3 camp cyclase activating polypeptide regulates cytosolic Ca 2 + in ; (4) P KC IP 3 rat gonadotropes and somatotropes through different in2 tracellular mechanisms Endocrinology 1993 ; 132 : 1447, PACAP 1452, [13 ] O Farrell M, Marley PD Multiple calcium channels are required for pituitary adenylate cyclase activating polypeptide induced catecholamine secretion from bovine, cultured adrenal chromaffin cells Naunyn Schmiedebergs Arch Pharmacol 1997 ; 356 :