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Derek Lipton Changes in Heart Rate from Agonist and Antagonist Drug Interaction with Membrane Receptors on the

Heart of Daphnia Derek Lipton, BSCI330, Section 1104 TA: Sara Linden, November 6, 01 Abstract

Introduction C!an"e# in ce$$%$ar activit& are d%e to receptor protein# 'o%nd on t!e #%r'ace o' tar"et ce$$#( T!e#e receptor# are re#pon#ib$e 'or receivin" #i"na$# t!at provoke event# to occ%r t!at eit!er decrea#e or increa#e ce$$%$ar re#pon#e# )Carpenter, et a$, 011*( T!e#e #i"na$# are ca$$ed $i"and# and can come in contact +it! tar"et ce$$# 'o%r di''erent +a&#: contact,dependent, paracrine, #&naptic and endocrine( T!e met!od e-perienced in t!i# e-periment +a# #&naptic #i"na$in", +!ere #tim%$i ca%#e rapid e$ectrica$ imp%$#e# a$on" t!e a-on o' a ne%ron, re$ea#in" ne%rotran#mitter# to +!ic! ca%#e# an e$ectrica$ c!an"e in t!e po#t#&naptic ce$$ )A$bert#, et a$, 00.*( T!i# e-periment %#ed a compo%nd micro#cope, Detain #o$%tion and vario%# #o$%tion# o' a"oni#tic and anta"oni#tic dr%"#( T!e bio$o"ica$ #pecimen %#ed +a# Daphnia magna, a c$adoceran cr%#tacean 'o%nd in 're#!+ater( A$t!o%"! #ma$$er t!an !%man#, it# !eart i# re"%$ated ver& #imi$ar$& to !%man#/ )Carpenter, et a$, 011*( T!e !eart rate# o' t+e$ve di''erent Dap!nia +ere mea#%red at re#t and t!en a co%p$e min%te# $ater a'ter e-po#%re to one o' 'o%r di''erent dr%"#( T!i# e-periment +a# #i"ni'icant beca%#e it a$$o+ed vi#%a$i0ation o' +!at !appen# to t!e !%man !eart +!en e-po#ed to certain dr%"# 1#ince t!e Daphnia !eart act# #imi$ar$& to t!e !%man !eart(

Derek Lipton 2revio%# re#earc! !a# been done on a%tonomic nervo%# #&#tem )ANS* tar"etin" o' membrane receptor# in t!e !eart( T!i# re#earc! i# important 'or %nder#tandin" !o+ certain dr%"# can ind%ce vario%# cardiova#c%$ar condition#( Additiona$$&, )D0ia$o+#ki, 3(4(, et a$, 005* per'ormed re#earc! on t!e e''ect o' 6,b$ocker dr%"# on t!e !eart o' Daphnia( T!i# #!o+ed t!e #imi$aritie# bet+een t!eir !eart# and t!o#e !%man#, #ince t!e dr%"# #!o+ed #imi$ar re#%$t#, de#pite t!e $ack o' 6,adrener"ic receptor# in Daphnia !eart#( T!e t+o main c$a##e# o' receptor# in t!e vertebrate !eart tar"eted b& t!e a%tonomic nervo%# #&#tem are m%#carinic and 6,adrener"ic receptor# )S$otkin, T( A(, 1777*( In Daphnia, t!e main membrane receptor i# t!e m%#carinic 4 acet&$c!o$ine receptor )4 receptor* )8aid&a, A( 9(, et a$, 007*( T!ere'ore, t!e main ne%rotran#mitter re$ea#ed i# acet&$c!o$ine, +!ic! activate# t!e para#&mpat!etic nervo%# #&#tem %pon bindin" to t!e membrane receptor )4a$m#:;, 4(, et a$, 1777*( A# a re#%$t, dr%"# t!at bind to t!e 4 receptor and decrea#e t!e !eart rate are a"oni#t#1#ince t!e& mimic t!e e''ect t!at t!e ne%rotran#mitter !a#( Dr%"# increa#in" t!e !eart rate, t!ere'ore, are anta"oni#t#( The purpose of this experiment was to test the effect that acetylcholine, nicotine, ethanol and caffeine have on the heart rate of Daphnia and use that information to determine if the drug binds agonistically or antagonistically to the membrane receptor. The hypothesis for this experiment was that caffeine and nicotine would bind antagonistically, increasing the heart rate, while acetylcholine and ethanol would bind agonistically, decreasing the heart rate.

Derek Lipton Materials and Methods Daphnia #pecimen# +ere obtained 'rom t!e c%$t%re %#in" a tran#'er pipette, care'%$$& tr&in" to $imit t!e a##ociated #o$%tion t!at i# +it! t!e or"ani#m( A #ma$$ drop o' Detain #o$%tion +a# t!en p$aced on a depre##ion #$ide, in t!e +e$$, and t!e Daphnia +a# t!en p$aced in t!i# drop, +!ic! made it immobi$e( A di''erent Daphnia +a# %#ed 'or eac! tria$( T!e Daphnia +a# t!en e-amined at 4< and 10< ob:ective %#in" a compo%nd micro#cope( T!i# +a# done to $ocate and identi'& t!e dor#a$ !eart o' eac! Daphnia. D%e to t!e !eat o' t!e $amp, ob#ervation +a# done =%ick$& a# to not a''ect t!e or"ani#m( >nce t!e !eart +a# $ocated, t!e !eart rate o' Daphnia +a# mea#%red b& co%ntin" t!e amo%nt o' beat# over a 'i'teen #econd period( T!i# +a# done a tota$ o' t+e$ve time#1 t!ree tria$# 'or eac! o' t!e 'o%r dr%"# te#ted, %#in" a di''erent Daphnia 'or eac! time( ?or t!e Daphnia +it! 'a#ter !eart rate#, a penci$ +a# tapped on paper 'or eac! beat and t!en t!e dot# +ere co%nted )and m%$tip$ied b& 'o%r to "et beat# per min%te*( A'ter eac! tria$, t!e micro#cope +a# t%rned o'', a$$o+in" it to coo$ do+n( Ne-t, a beaker +a# 'i$$ed +it! di#ti$$ed +ater and #ome o' t!i# +ater +a# po%red into a #ma$$ +e$$ depre##ion c!amber( T!e Daphnia +a# $i'ted o%t o' t!e Detain re#in %#in" a rea"ent #pat%$a and t!en p$aced into t!e +ater( @!i$e +aitin" 'or t!e Daphnia to re"ain mobi$it& and recover, one mi$$i$iter o' acet&$c!o$ine +a# obtained +it! a pipet and added to one o' t!e +e$$# o' t!e c!amber( >nce t!i# +a# read&, t!e Daphnia +a# p$aced into t!i# dr%" #o$%tion 'or one min%te( T!e Daphnia +a# t!en taken o%t and p$aced into a #ma$$ drop o' Detain, #imi$ar to be'ore( T!e Daphnia +a# t!en p$aced %nder t!e micro#cope and it# !eart rate +a# mea#%red b& co%ntin" t!e n%mber beat# d%rin" a 'i'teen #econd period, t!e #ame +a& it +a# done 'or t!e ba#a$ !eart rate(

Derek Lipton T!i# entire proce## +a# t!en repeated t+o more time#, #tartin" +it! t!e mea#%rement o' ba#a$ !eart rate, %#in" a di''erent Daphnia eac! time( A'ter t!at, t!e proced%re +a# repeated a tota$ o' nine more time#1rep$acin" acet&$c!o$ine +it! nicotine, et!ano$ and ca''eine, per'ormin" t!ree tria$# +it! eac!( At t!e end, t!e avera"e ba#a$ !eart rate and avera"e dr%""ed !eart rate o' t!e t!ree tria$# +ere ca$c%$ated 'or eac! o' t!e 'o%r dr%"# )Carpenter, et a$, 011*(

Results T!e !eart rate# o' di''erent Daphnia +ere mea#%red )Tab$e 1*( ?or eac! Daphnia, t!e ba#a$ )re#tin"* !eart rate +a# mea#%red and t!en a co%p$e min%te# $ater t!e !eart rate o' t!e #ame Daphnia +a# mea#%red a'ter treatment +it! one mi$$i$iter o' acet&$c!o$ine, nicotine, et!ano$ or ca''eine( 3ac! tria$ )'or eac! dr%"* re'er# to a di''erent Daphnia( 3ac! Daphnia !ad a di''erent ba#a$ !eart rate and #!o+ed #ome time,dependent c!an"e in !eart rate a'ter dr%" e-po#%re( T!e avera"e# 'rom Tab$e 1 +ere t!en %#ed to create bar "rap!# comparin" t!e e''ect t!at eac! dr%" !a# on t!e !eart rate o' Daphnia )?i"%re 1*( Acet&$c!o$ine and et!ano$ bot! decrea#ed t!e !eart rate )?i"%re# 1a and 1c*( Nicotine and ca''eine bot! ca%#ed #$i"!t increa#e# in !eart rate )?i"%re# 1b and 1d*(

Table 1. Basal and Drugged Heart Rates of Daphnia Acet lcholine Trial 1 # $ AVG Basal . A 176 3 Drugged 15 160 14. 153 !icotine Basal .4 60 76 .0 Drugged 3 0 .0 316 305 "thanol Basa l Drugged 0. 1A6 31 0 176 15 37 1.3 Caffeine Basa l Drugged 3 04 4. 76 3 6. 3A 56

Derek Lipton Table 1. Compari#on o' Daphnia !eart rate# be'ore and a'ter e-po#%re to one o' 'o%r dr%"#(
Dr%"# %#ed +ere acet&$c!o$ine, nicotine, et!ano$ and ca''eine( T!ree di''erent Daphnia +ere te#ted 'or eac! dr%"( Beart rate# +ere mea#%red t!ro%"! ob#ervation %nder a compo%nd micro#cope and a #top+atc! 'or 'i'teen #econd#(

a%

b)

c%

d%

&igure 1. "ffect of Drug "'posure on the Heart Rate of Daphnia. T!e avera"e !eart rate
be'ore and a'ter treatment +it! a partic%$ar dr%" #o$%tion 'rom t!e t!ree tria$# i# compared( T!e $e't bar #!o+# t!e ba#a$ !eart rate and t!e ri"!t bar #!o+# t!e dr%""ed !eart rate( ?o%r dr%"# +ere %#ed, eac! !avin" a di''erent e''ect( a* Acet&$c!o$ine decrea#ed t!e !eart rate( b* Nicotine #$i"!t$& increa#ed !eart rate( c* 3t!ano$ $o+ered !eart rate( d* Ca''eine #$i"!t$& increa#ed t!e !eart rate(

Discussion

Derek Lipton T!e p%rpo#e o' t!i# e-periment +a# to te#t t!e e''ect di''erent dr%"# !ave on !eart rate( T!e !&pot!e#i# +a# t!at !eart rate +o%$d increa#e +!en Daphnia +a# e-po#ed to ca''eine and nicotine, and +o%$d decrea#e +!en e-po#ed to acet&$c!o$ine and et!ano$( A# evident in ?i"%re 1, t!i# !&pot!e#i# +a# correct( Nicotine ca%#ed an .(7C increa#e in !eart rate and ca''eine ca%#ed an .(0C increa#e1a# indicated b& t!e bar on t!e $e't bein" ta$$er, indicatin" a "reater ba#a$ !eart rate t!an dr%""ed( 4ean+!i$e, acet&$c!o$ine and et!ano$ !ad "reater e''ect# on !eart rate, b%t in t!e ot!er directionD acet&$c!o$ine ca%#ed a 34(1C decrea#e in !eart rate and et!ano$ ca%#ed a 3(4C decrea#e1a# indicated b& t!e #$i"!t "ro+t! o' t!e bar 'rom ba#a$ to dr%""ed !eart rate( T!ere'ore, a# t!e Daphnia +a# e-po#ed to nicotine and ca''eine, t!eir #&mpat!etic nervo%# #&#tem# o' t!e ANS +ere activatedD e-po#%re to acet&$c!o$ine and et!ano$ activated t!eir para#&mpat!etic nervo%# #&#tem# )Carpenter, et a$, 011*( T!e#e re#%$t# are #imi$ar to +!at occ%r# in t!e !%man bod&1e-cept t!e ba#a$ and dr%""ed !eart rate# are m%c! $o+er in !%man#( T!i# atte#t# to t!e Daphnia !eart !avin" t!e #ame circ%$ator& p%mp '%nction a# a !%man !eart )Carpenter, et a$, 011*( T!e main ne%rotran#mitter re$ea#ed b& t!e ANS, acet&$c!o$ine, i# part o' t!e para#&mpat!etic nervo%# #&#tem( T!ere'ore, +!en bo%nd to t!e 4 receptor, it decrea#e# t!e !eart rate( @!en ot!er #i"na$in" mo$ec%$e# are pre#ent, in t!i# ca#e dr%"#, t!e& bind to t!e 4 receptor a# +e$$, a$terin" it# '%nction( T!%#, t!e decrea#e in !eart rate 'rom acet&$c!o$ine and et!ano$ indicate# t!at t!e#e dr%"# are a"oni#t# to t!e m%#carinic 4 acet&$c!o$ine receptor, #ince t!e& mimic t!e !eart rate,decrea#in" e''ect# o' t!e ne%rotran#mitter( ?%rt!ermore, nicotine and ca''eine are anta"oni#t#, beca%#e t!e& b$ock t!e activit& o' acet&$c!o$ine, ca%#in" t!e !eart rate to increa#e(

Derek Lipton De#pite t!e po#itive re#%$t#, t!e activit& o' ca''eine +a# not entire$& con#i#tent +it! t!e prediction#( A# #een in Tab$e 1, t!e 'ir#t tria$ 'or ca''eine act%a$$& #a+ a decrea#e in !eart rate a'ter e-po#%re( T!ere i# no kno+n rea#on 'or t!i#, e#pecia$$& #ince t!e ot!er t+o tria$# #a+ an increa#e a# e-pected( ?%rt!er re#earc! #!o%$d be done to #ee i' t!ere i# a potentia$ "ene t!at ca%#e# t!i#, or i' certain environmenta$ condition# can create imm%nit& to ca''eine( Nevert!e$e##, Tab$e 1 and ?i"%re 1 #ti$$ prove t!at acet&$c!o$ine and et!ano$ are a"oni#t# to t!e 4 receptor and ca''eine and nicotine and anta"oni#t# to t!e receptor( T!e#e re#%$t# prove a #tron" re$ation bet+een !eart activit& in Daphnia and !%man#( T!i# provide# #i"ni'icant opport%nitie# 'or '%rt!er re#earc! on ot!er #&#tem# o' t!e bod&( Bio$o"i#t# can per'orm e-periment# on brain activit&, 'or e-amp$e, to #ee i' t!ere are more #imi$aritie#( T!i# re#earc! co%$d be a breakt!ro%"! 'or %nder#tandin" re$ation#!ip# in p!&#io$o"& acro## a$$ #pecie#(

Derek Lipton 9e'erence# A$bert#, B(, Eo!n#on, A(, Le+i#, E(, 9a'', 4(, 9obert#, F(, @a$ter, 2(, 00.( 4o$ec%$ar Bio$o"& o' t!e Ce$$, ?i't! 3d( Garland Science( D0ia$o+#ki, 3( 4(, T%rner, 2( F(, G Brook#, B( @(, 006( 2!&#io$o"ica$ and reprod%ctive e''ect# o' beta adrener"ic receptor anta"oni#t# in Dap!nia ma"na( Archives of environmental contamination and toxicology( 50(4: 503,510( 4a$m#:;, 4(, Ber"da!$, A(, H!ao, <( B(, S%n, <( I(, Bedner, T(, 3dvin##on, L(, and 3r$in"e, D(, 1777( 3n!anced acet&$c!o$ine and 2 I,receptor #tim%$ated va#c%$ar 3DB?,di$atation in con"e#tive !eart 'ai$%re( Cardiovascular research( 43(1: 00, 07( S$otkin, T( A(, 3pp#, T( A(, Sten"er, 4( L(, Sa+&er, F( E(, and Seid$er, ?( E(, 1777( C!o$iner"ic receptor# in !eart and brain#tem o' rat# e-po#ed to nicotine d%rin" deve$opment: imp$ication# 'or !&po-ia to$erance and perinata$ morta$it&( Developmental brain research( 113(1: 1,1 ( 8aid&a, A( 9(, J%ren$ian, L(, Brad&, L(, 9omero, N( 3(, and Fo!n, 9(, 007( Cardioactive e''ect# o' dip!en!&dramine and c%rc%min in Dap!nia ma"na( Impulse: an ndergraduate !ournal for "euroscience(