Čedomil LucuCenter for Marine ResearchInstitute Ruđer BoškovićRovinj, Istria, Croatia

PHYSIOLOGICAL ADAPTIVE MECHANISMS OF ORGANISMS IN MARINE AND BRACKISH WATER ENVIRONMENT.

Na+ K+ ATPase AND CARBONIC ANHYDRASE

Adriatic Sea 36 -39 pptBrackish waters < 30 pptFreshwater < 0.5 ppt

European lobster Homarus gammarus (Linnaeus, 1758.)

Table 1.

Osmolarity and ion concentrations of the haemolymph of intermoult lobsters

Homarus gammarus acclimated to 38 ppt SW and 20 ppt DSW. Medium

concentration in which lobsters were acclimated are indicated. Each point is

given as the mean ± SD. In abbreviations are numbers of tested individuals.

OSMOLARITY Na Cl

(mOsmol/l) (mmol/ l)

SEAWATER

1126±58 (6) 528±53 (6) 532±21 (5)

HAEMOLYMPH

1168±68 (6) 538±19 (6) 575±20 (5)

DILUTE SEAWATER

548.3±17.7 (6) 274±13 (6) 306±14 (6)

HAEMOLYMPH

701±30 (6) 334±6 (6) 334±16 (6)

e

i

vt

k

e

c

c

pc

hl

e

Branchiostegite epithelium

c = cuticle

e = epithelium

pc = pillar cells

hl = haemolymph lacuna

i = ionocite

DILUTE SEAWATER ACCLIMATION

Morphological and ultrastructural changesIn the branchial cavity of homarid lobsters tissues i.e. branchiostegite and epipoditeincrease in tisssue thickness, basolateral infoldings, apical microvilli, increase in numbers of mitochondria, microvesicular bodies.

Biochemical and physiologica changesIncreased oxydation rates of glucose and amino acids –36% of total CO2 released contribution of alanine oxidation (Pressley and Graves, 1983)

increase of cytochrome c oxidase (Welcomme and Devos, 1988)

Increased oxygen consumption rate in the gills in vitro (Lucu and Pavičić, 1995)

Olfactory function sustained (Gleeson et al.,1997)

EXTERNAL 3Na+ 2K+ 3NaE2P ↑↔ E2P ↓↔ 2KE2P β β (3Na)E1P (2K)E2 β→ADP ATP→β 3 Na E1ATP ↑↔ E1ATP ↓↔ 2KE1ATP 3Na+ 2K+ CYTOPLASMIC

0

20

40

60

80

100

% O

F IN

HIB

ITIO

N

-5 -4 -3 -2-log Ouabain (mol/l)

0.0

0.2

0.4

0.6

0.8

( u

mol

Rb/

h/m

g pr

otei

n)

980 mosmol/l

650 mosmol/l

**

**

**

µ

-3 -2 -1 010

15

20

25

A

µl cDNA Template (log10)Th

resh

old

Cyc

le±

SD

0.0 4.0 8.00.00

0.25

0.50

0.75

1.00

1.25

1.50

Days of Acclimation to 5 ppt Salinity

Rel

ativ

eα

-Sub

unit

mR

NA

Expr

essi

on±

SE

B

Callinectes : MADTGRTDSYRHATDRNIPDDNRTVKGDPKSKKKNVKGKRKGEKEKDMDNLKQELELDEHKVPIEELFQRLSVNPDTGLTQAEARRRLERDGPNALTPPK : 100Homarus : MADTGRTDSYRHATDRTIPDDNRTVKGDPKSTKKMPKAKAK-KGEKDMDNLKRELELDEHKVPIEELFQRLSVNPDSGLTQAEALRRTERDGPNALTPPK : 99Artemia1 : ------------------------------------MAKGKQKKGKDLNELKKELDIDFHKIPIEECYQRLGSNPETGLTNAQARSNIERDGPNCLTPPK : 64Gallus3 : -----------------------------MGDKGEKESPKKGKGKRDLDDLKKEVAMTEHKMSIEEVCRKYNTDCVQGLTHSKAQEILARDGPNALTPPP : 71Gallus2 : ---------------------MDGREYSPAATTSENGGGRRKQKEKELDELKKEVNLDDHKLSLDELGRKYQVDLSRGLSNARAAEVLAQDGPNALTPPP : 79Gallus1 : ------------------MGKGAGRDKYEPTATSEHGTKKKKAKERDMDELKKEISMDDHKLSLDELHRKYGTDLSRGLTTARAAEILARDGPNTLTPPP : 82Artemia2 : ---------------------------------------MGKKQGKQLSDLKKELELDQHKIPLEELCRRLGTNTETGLTSSQAKSHLEKYGPNALTPPR : 61 Callinectes : QTPEWVKFCKNLFGGFSLLLWIGAILCFIAYSIEAAS-EEEPNNDNLYLGIVLTAVVIITGIFSYYQESKSSRIMESFKNLVPQYAIVIREGEKLNVQAE : 199Homarus : QTPEWVKFCKNLFGGFSLLLWIGAILCFIAYSIEAVS-EEEPNNDNLYLGIVLTAVVIITGVFSYYQENKSSRIMESFKNMVPQYAIVVREGEKQNVQAE : 198Artemia1 : TTPEWIKFCKNLFGGFALLLWTGAILCFLAYGIEASSGNEDMLKDNLYLGIVLATVVIVTGIFSYYQENKSSRIMDSFKNLVPQYALALREGQRVTLKAE : 164Gallus3 : TTPEWVKFCRQLFGGFSILLWIGAILCFLAYGIQAGT-EDEPSNDNLYLGIVLAAVVIITGCFSYYQEAKSSKIMESFKNMVPQQALVIREGEKMQLNAE : 170Gallus2 : TTPEWVKFCRQLFGGFSILLWIGAILCFLAYGIQAAM-EDEPSNDNLYLGVVLAAVVIVTGCFSYYQEAKSSKIMDSFKNMVPQQALVIREGEKIQINAE : 178Gallus1 : TTPEWVKFCRQLFGGFSLLLWIGSLLCFLAYGITSVM-EGEPNSDNLYLGVVLAAVVIITGCFSYYQEAKSSKIMESFKNMVPQQALVVRNGEKMSINAE : 181Artemia2 : TTPEWIKFCKQLFGGFQMLLWIGSILCFIAYTMEKYK-NPDVLGDNLYLGLALLFVVIMTGCFAYYQDHNASKIMDSFKNLMPQFAFVIRDGKKIQLKAE : 160 Callinectes : ELCIGDIIDVKFGDRIPADMRVIEARGFKVDNSSLTGESEPQSRSPEFTSENPLETKNLAFFSTNAVEGTCKGIVINIGDNTVMGRIAGLASGLETGETP : 299Homarus : ELCIGDIIEVKFGDRIPADVRVIEARGFKVDNSSLTGESEPQSRSPEFTSENPLETKNLAFFSTNAVEGTARGIVINIGDNTVMGRIAGLASGLETGETP : 298Artemia1 : ELTMGDIVEVKFGDRVPADLRVLEARSFKVDNSSLTGESEPQARSPEFTNDNPLETKNLAFFSTNAVEGTMRGIVIGIGDNTVMGRIAGLASGLDTGETP : 264Gallus3 : EVVVGDLVEVKGGDRVPADLRIISAHGCKVDNSSLTGESEPQTRSPDCTHDNPLETRNITFFSTNCVEGTARGVVIATGDRTVMGRIATLASGLEVGKTP : 270Gallus2 : NVVVGDLVEVKGGDRVPADMRIISSHGCKVDNSSLTGESEPQTRSPEFTHENPLETRNICFFSTNCVEGTARGIVISTGDRTVMGRIASLASGLEVGRTP : 278Gallus1 : GVVVGDLVEVKGGDRIPADLRIISAHGCKVDNSSLTGESEPQTRSPDFSNENPLETRNIAFFSTNCVEGTAVGIVISTGDRTVMGRIASLASGLEGGKTP : 281Artemia2 : EVTVGDLVEVKFGDRIPADIRITSCQSMKVDNSSLTGESEPQSRSTECTNDNPLETKNLAFFFTNTLEGTGRGIVINVGDDSVMGRIACLASSLDSGKTP : 260 Callinectes : IAKEISHFIHIITGVAVFLGVTFFVIAFIMGYHWLDAVVFLIGI-IVANVPEGLLATVTVCLTLTAKRMAAKNCLVKNLEAVETLGSTSTICSDKTGTLT : 398Homarus : IAKEISHFIHIITGVAVFLGVTFFVIAFILGYHWLDAVVSLIGYHCKPNVPEGLLATVTVCLTLTAKRMAAKNCLVKNLEAVETLGSTSTICSDKTGTLT : 398Artemia1 : IAKEIAHFIHIITGVAVFLGVTFFIIAFVLGYHWLDAVVFLIGI-IVANVPEGLLATVTVCLTLTAKRMASKNCLVKNLEAVETLGSTSTICSDKTGTLT : 363Gallus3 : IAVEIEHFIQLITGVAVFLGISFFVLSLILGYTWLEAVIFLIGI-IVANVPEGLLATVTVCLTLTAKRMARKNCLVKNLEAVETLGSTSTICSDKTGTLT : 369Gallus2 : IAMEIEHFIRLITGVAVFLGLSFFILSLILGYTWLEAVIFLIGI-IVANVPEGLLATVTVCLTLTAKRMARKNCLVKNLEAVETLGSTSTICSDKTGTLT : 377Gallus1 : IAMEIEHFIHLITGVAVFLGVSFFILSLILEYTWLEAVIFLIGI-IVANVPEGLLATVTVCLTLTAKRMARKNCLVKNLEAVGTLGSTSTICSDKTGTLT : 380Artemia2 : IAREIEHFIHIITAMAVSLAAVFAVISFLYGYTWLEAAIFMIGI-IVAKVPEGLLATVTVCLTLTAKRMAKKNCLVRNLEAVETLGSTSTICSDKTGTLT : 359 Callinectes : QNRMTVAHMWFDNTIIEADTSEDQSGCQYDKTSDGWKALSRIAALCNRAEFKTGQEDVPILKREVNGDASEAALLKCVELAIGDVRGWRSRNKKVCEIPF : 498Homarus : QNRMTVAHMWFDNTIIEADTSEDQSGCQYDKTSEGWKTLSRIAALCNRAEFKTGQEEVAILKREVNGDASEAALLKCVELAVGDVKGWRARNKKVCEVPF : 498Artemia1 : QNRMTVAHMWFDGTITEADTTEDQSGAQFDKSSAGWKALVKIAALCSRAEFKPNQSTTPILKREVTGDASEAAILKCVELTTGETEAIRKRNKKICEIPF : 463Gallus3 : QNRMTVAHMWFDNQIHEADTTEDQSGTSFDKSSATWVALSHIAGLCNRAVFKGGQENVPILKRDVAGDASESALLKCIELSSGSVKVMRERNKKVAEIPF : 469Gallus2 : QNRMTVAHMWFDNQIHEADTTEDQSGATFDKRSPTWAALSRIAGLCNRAVFKPGQENISISKRDTAGDASESALLKCIQLSCGSVKKMRDKNPKVTEIPF : 477Gallus1 : QNRMTVAHMWFDNQIHEADTTENQSGASFDKSSATWLALSRIAGLCNRAVFQANQENVPILKRAVAGDASESALLKCIELCCGSVKEMRERYPKVVEIPF : 480Artemia2 : QNRMTVAHMWFDQKIVTADTTENQSGNQLYRGSKGFPELIRVASLCSRAEFKTEHAHLPVLKRDVNGDASEAAILKFAEMSTGSVMNIRSKQKKVSEIPF : 459 Callinectes : NSTNKYQVSIHETQDKNDLRYLLVMKGAPERILERCSTIFMNGEEKPLDEEMKESFNNAYLELGGLGERVLGFCDYVLPSDKYPLGYPFDADAVNFPVHG : 598Homarus : NSTNKYQVSIHDTEDKNDPRYLLVMKGAPERILERCSTIYINGEEKPLDEELKESFNNAYLELGGLGERVLGFCDYILPSDKYPLGYPFDSDNPNFPVHG : 598Artemia1 : NSANKFQVSIHENEDKSDGRYLLVMKGAPERILERCSTIFMNGKEIDMTEELKEAFNNAYMELGGLGERVLGFCDYLLPLDKYPHGFAFNADDANFPLTG : 563Gallus3 : NSTNKYQLSIHETEDPNDNRYLLVMKGAPERILDRCSTILLQGKEQPLDEEMKEAFQNAYLELGGLGERVLGFCHFYLPEEQYPKGFAFDCDDVNFATDN : 569Gallus2 : NSTNKYQLSIHEREEDPQG-HILVMKGAPERILERCSRILLQGQEVPLDEEMKEAFQNAYLELGGLGERVLGFCHLYLPPDKFPRGFRFDADEVNFPTSD : 576Gallus1 : NSTNKYQLSIHKNANAGESRHLLVMKGAPERILDRCDSILIHGKVQPLDEEIKDAFQNAYLELGGLGERVLGFCHLALPDDQFPEGFQFDTDEVNFPVEK : 580Artemia2 : NSANKYQVSVHEREDKSG--YFLVMKGAPERILERCSTILIDGTEIPLDNHMKECFNNAYMELGGMGERVLGFCDFELPSDQYPRGYVFDADEPNFPISG : 557 Callinectes : LRFVGLMSMIDPPRAAVPDAVAKCRSAGIKVIMVTGDHPITAKAIAKSVGIISEGNETVEDIAQRLNIPIKEVDPREAKAAVVHGSELRDMTSEQLDDVL : 698Homarus : LRFVGLMSMIDPPRAAVPDAVAKCRSAGIKVIMVTGDHPITAKAIAKSVGIISEGNETVEDIAQRLNIPIKEVDPREAKAAVVHGSELRDMTSEQLDDIL : 698Artemia1 : LRFAGLMSMIDPPRAAVPDAVAKCRSAGIKVIMVTGDHPITAKAIAKSVGIISEGNETVEDIAARLNIPVSEVNPRDAKAAVVHGGELRDITPDALDEIL : 663Gallus3 : LCFVGLMSMIDPPRAAVPDAVGKCRSAGIKVIMVTGDHPITAKAIAKGVGIISEGNETVEDIAARLNIPVSQVNPRDAKACVIHGTDLKDMSSEQIDEIL : 669Gallus2 : LCFVGLMSMIDPPRAAVPDAVGKCRSAGIKVIMVTGDHPITAKAIAKGVGIISEGNETVEDIAARLNIPVSQVNPREAKACVVHGSDLKDMTAEQLDEIL : 676Gallus1 : LCFVGLMSMIDPPRAAVPDAVGKCRSAGIKVIMVTGDHPITAKAIAKGVGIISDGNETVEDIAARLNIPVSQVNPRDAKACVVHGSDLKDMTSEQLDDIL : 680Artemia2 : LRFVGLMSMIDPPRAAVPDAVSKCRSAGIKVIMVTGDHPITAKAIARQVGIISEGHETVDDIAARLNIPVSEVNPRSAQAAVIHGNDLKDMNSDQLDDIL : 657 Callinectes : IHHTEIVFARTSPQQKLIIVEGCQRMGAIVAVTGDGVNDSPALKKADIGVAMGIAGSDVSKQAADMILLDDNFASIVTGVEEGRLIFDNLKKSIAYTLTS : 798Homarus : IHHTEIVFARTSPQQKLIIVEGCQRMGAIVAVTGDGVNDSPALKKADIGVAMGIAGSDVSKQAADMILLDDNFASIVTGVEEGRLIFDNLKKSIAYTLTS : 798Artemia1 : RHHPEIVFARTSPQQKLIIVEGCQRQGAIVAVTGDGVNDSPALKKADIGVAMGIAGSDVSKQAADMILLDDNFASIVTGVEEGRLIFDNLKKSIVYTLTS : 763Gallus3 : QNHTEIVFARTSPQQKLIIVEGCQRQGAIVAVTGDGVNDSPALKKADIGVAMGIRGSDVSKQAADMILLDDNFASIVTGVEEGRLIFDNLKKSIAYTLTS : 769Gallus2 : RNHTEIVFARTSPQQKLIIVEGCQRQGAIVAVTGDGVNDSPALKKADIGIAMGIAGSDVSKQAADMILLDDNFASIVTGVEEGRLIFDNLKKSIAYTLTS : 776Gallus1 : LHHTEIVFARTSPQQKLIIVEGCQRQGAIVAVTGDGVNDSPALKKADIGVAMGIAGSDVSKQAADMILLDDNFASIVTGVEEGRLIFDNLKKSIAYTLTS : 780Artemia2 : RHYREIVFARTSPQQKLIIVEGVQRQGEFVAVTGDGVNDSPALKKADIGVAMGIAGSDVSKQAADMILLDDNFASIVTGVEEGRLIFDNIKKSIAYTLTS : 757 Callinectes : NIPEISPFLFFMIASVPLPLGTVTILCIDLGTDMVPAISLAYEEAESDIMKRQPRNPFTDKLVNERLISMAYGQIGMIQALAGFYVYFVIMAENGFLPPV : 898Homarus : NIPEISPFLFFMIASVPLPLGTVTILCIDLGTDMVPAISLAYEEAESDIMKRQPRNPFTDKLVNERLISMAYGQIGMIQALAGFFVYFVIMAENGFLPPK : 898Artemia1 : NIPEISPFLLFILFDIPLPLGTVTILCIDLGTDMVPAISLAYEEAESDIMKRRPRNPVTDKLVNERLISLAYGQIGMIQASAGFFVYFVIMAECGFLPWD : 863Gallus3 : NIPEITPFLLFIMANIPLPLGTITILCIDLGTDMVPAISLAYEAAESDIMKRQPRNPRSDKLVNERLISMAYGQIGMIQALGGFFSYFVILAENGFLPSC : 869Gallus2 : NIPEITPFLLFIIANIPLPLGTVTILCIDLGTDMVPAISLAYEAAESDIMKRQPRNPRTDKLVNERLISMAYGQIGMIQALGGFFTYFVILAENGFLPAR : 876Gallus1 : NIPEITPFLIFIIANIPLPLGTCTILCIDLGTDMVPAISLAYEQAESDIMKRQPRNPKTDKLVNERLISMAYGQIGMIQALGGFFTYFVIMAENGFLPSG : 880Artemia2 : KIPELSPFLMYILFDLPLAIGTVTILCIDLGTDVVPAISMAYEGPEAD--PRKPRDPVKEKLVNERLISMAYGQIGVMQAFGGFFTYFVIMGECGFLPNR : 855 Callinectes : LFGIREQWDSKAINDLEDYYGQEWTYHDRKILEYTCHTAFFVAIVVVQWADLIICKTRRNSILHQGMKNMVLNFGLCFETTLAAFLSYTPGMDKGLRMYP : 998Homarus : LFGIREKWDSKAINDLEDHYGQEWTYYDRKVLEYTCHTAFFVSIVIVQWADLIVCKTRRNSIVHQGMKNMVLNFGLCFETTLAAFLSYTPGMDKGLRMYP : 998Artemia1 : LFGLRKHWDSRAVNDLTDSYGQEWTYDARKQLESSCHTAYFVSIVIVQWADLIISKTRRNSVFQQGMRNNILNFALVFETCLAAFLSYTPGMDKGLRMYP : 963Gallus3 : LVGIRLSWDDRTINDLEDSYGQQWTYEQRKVVEFTCHTAFFVSIVVVQWADLIICKTRRNSVFQQGMKNKILIFGLFEETALAAFLSYCPGMDVALRMYP : 969Gallus2 : LLGVRLAWDDRSTNDLEDSYGQEWTYEQRKVVEFTCHTAFFASIVVVQWADLIICKTRRNSVFQQGMKNKILIFGLLEETALAAFLSYCPGMGVALRMYP : 976Gallus1 : LVGIRLQWDDRWINDVEDSYGQQWTFEQRKIVEFTCHTAFFVSIVVVQWADLIICKTRRNSVFQQGMKNKILIFGLFEETALAAFLSYCPGMDVALRMYP : 980Artemia2 : LFGLRKWWESKAYNDLTDSYGQEWTWDARKQLEYTCHTAFFISIVIVQWTDLIICKTRRLSLFQQGMKNGTLNFALVFETCVAAFLSYTPGMDKGLRMYP : 955 Callinectes : LKFYWWLPPLPFSLLIFVYDECRRFVLRRNPGGWVEMETYY : 1039Homarus : LKFYWWLPALPFSLLIFVYDECRRFILRRNPGGWMEMETYY : 1039Artemia1 : LKINWWFPALPFSFLIFVYDEARKFILRRNPGGWVEQETYY : 1004Gallus3 : LKPSWWFCAFPYSFLIFVYDEIRKLILRRNPGGWVEKETYY : 1010Gallus2 : LKVTWWFCAFPYSLLIFAYDEVRKLILRRYPGGWVEKETYY : 1017Gallus1 : LKPTWWFCAFPYSLLIFLYDEIRKLIIRRNPGGWVERETYY : 1021Artemia2 : LKIWWWFPPMPFSLLILVYDECRKFLMRRNPGGFLERETYY : 996

0

10

20

30

40

50

Na,

K-A

TPas

e ( u

mol

Pi/h

/g )

HOMARUS PALINURUS

SW

DSW

SWDSW

-100 0 100 200 300mmol Na/l (haemolymph - medium)

0

10

20

30

40

Na,

K-A

TPas

e (

mol

Pi/h

per

mg

prot

ein)

9

9

118 8

8

7

6

6

5

5

5

4

4

10

133

71

2

2

6

4

4

HYPEROSMOREGULATORSWEAK STRONG

OSMOCONFORMERSMODERATE

10

9

9

11

12

12

µ

8.00 12.00 16.00 20.00 24.00cAMP content (pmol/mg prot.)

12.00

16.00

20.00

24.00

28.00

V (

m

olP

i/h/

mg

prot

.)µ

Y = - 0.721x + 32.92r = 0.91; P<0.001

Short-circuit current ( ISC) and conductance in epipodite preparation from lobster Homarus gammarus acclimated in seawater (980 mosmol/l; ISC in SW saline) and dilute seawater (600 mosmol/l; ISC in DSW).

ISC CONDUCTANCE (µA cm-2 ) (mS cm-2 )

SW acclimated -138.2±43.2 (7) 94.5±28.3 (7)

DSW acclimated -341.3±76.3 (14)** 56.5±12.1 (12)*

0.0

0.4

0.8

1.2

1.6

( m

ol R

b c

m

h

)CONTROL

2mM OUABAIN

µ-2

-1

* **

***

8.0 10.0 12.0 14.0 Isc ( mol cm h )

0.8

1.2

1.6

Rb;

J

----

-> (

m

ol c

m

h )

6.0 8.0 10.0 12.0 Isc ( mol cm h )

0.3

0.6

0.9

Rb;

a--

-->

b (

mol

cm

h

)µ

-1-2

µ -2 -1

µ -1-2

Jb

aµ

- 2

-1

Y = 0.027 x + 0.122r = 0.896 ; P< 0.05

Y = 0.132 x - 0.203r = 0.90; P< 0.01

0 40 80 120 160TIME (Minutes)

0

200

400

600

SC

C (

A

cm

)

-2

CsCl; basolateral<−−−

A

B

µ

50 mS/cm2

50 mS/cm____

2

______

0.0

0.4

0.8

1.2

1.6

( m

ol R

b cm

h

)

Rb; a--->b

Rb; b---->a

Rb; netb--->a

µ-2

-1 * **

Na

Na Na

K

Na

K

K

K

Cl2Cl

APICAL BASO

--------------

->

_____

_____< Ca

SPECIFIC ACTIVITY OF Na+ ,K+ -ATPase INCREASE IN DILUTE SEAWATER (DSW). INCREASE OF AMOUNT OF Na+,K+-ATPase PROTEIN (WESTERN BLOT)

A NEGATIVE CORRELATION BETWEEN Na,K-ATPase and cAMP IN VIVO ENZYME ACTIVITY REGULATION THROUGH cAMP –DEPENDENT PROTEIN KINASE A

SHORT CIRCUITED EPIPODITE EPITHELIUM OF LOBSTER GENERATE -341 µA/cm2 AND CONDUCTIVITY OF 57 mS/cm2.

UNIDIRECTIONAL K (86Rb) SECRETIVE FLUXES WERE 2.5 –FOLD LARGER THAN INFLUXES

OUABAIN SENSITIVE PORTION OF SECRETIVE K(Rb) EFFLUXES WAS BLOCKED BY POTASSIUM CHANNEL BLOCKER CsCl.

APICALLY LOCATED K CHANNEL IS Na+ , K+ -ATPase REGULATED MODULATING UPREGULATION OF K CONDUCTANCE

1E-9 1E-8 1E-7 1E-6 1E-5ACETAZOLAMIDE ( M)

0

20

40

60

80

100

CA

(%

of

cont

rol)

CA (U/mg protein) Purification GILLS Seawater H 7.2±2.6 (6) V 35.8±18.9 (5) 5.2 ± 0.9 (5) C 1.2± 0.3 (3) 0.3 (3)

Dilute seawater

H 8.0±3.0 (8) V 37.2±15.7 (8) 5.1±1.3 (8) C 2.1±0.4 (4)* 0.3±0.1 (4)

EPIPODITES

Sea water H 9.9±4.6 (7) V 18.0±7.9 (4) 1.9±1.4 (4) C 9.0±2.8 (7) 0.9±0.1 (7)

Dilute seawater H 46.9±16.2 (8)*** V 111.1±24.9 (5)*** 2.7±1.2 (4)

C 72.0±14.6 (6)*** 1.1±0.4 (8)

BRANCHIOSTEGITE Seawater H 0.8±0.4 (8)

V 4.6±1.9 (4) 6.1±1.6 (4) C 1.4±1.2 (3) 0.4±0.1 (3)

Dilute seawater

H 10.5±5.0 (11)*** V 27.2±4.5 (7)*** 3.6±0.5 (5)∗ C 9.1±1.9 (3)** 0.6±0.3 (3) Specific activities of carbonic anhydrase (U/mg protein) in homogenates (H),vesicles (V) and cytosolic fraction (C). Purification = ratio of specific activity of partially purified vesicles or cytosolic fraction to that of homogenates (V/H; C/H). *P<0.05; ** P<0.02; ***P<0.001

0

40

80

120

160

CA

(U

/mg

prot

ein)

GILL EPIPODITE BRANCHIOSTEGITE

SW DSW

SW

DSW

SWDSW

N M V C0

40

80

% O

F C

A A

CTI

VIT

Y

N M V C0

40

80

%

OF

CA

AC

TIV

ITY

0

40

80

% O

F C

A A

CTI

VIT

Y

N M V C

BRANCHIOSTEGITE

EPIPODITE

GILL

ACETAZOLAMIDE INHIBITS DOSE DEPENDENTLY CA IN HOMOGENATES OF EPIPODITE (IC50 = 0.12µM; MEMBRANE VESICLES AND CYTOSOL) UPON ADAPTATION TO DSW, CA IN BRANCHIOSTEGITE AND EPIPODITE HOMOGENATES, MEMBRANE FRACTION AND CYTOSOL WAS INCREASED SEVERAL FOLD OVER THESE IN SW FRACTIONS THE MAIN PROPORTION OF CA (EPIPODITE AND BRANCHIOSTEGITE) 60 –76% IN CYTOSOL . MAMBRANE FRACTION CONTAINED 7 – 14 %. REMAINDER IN MITOCHONDRIAL AND NUCLEAR FRACTIONS

Lucu Č.,Wendelaar Bonga.S. and Flik,G. Potassium secretion modulated by Na+,K+-ATPase in the epipodite of American lobster Homarus americanus. J. Physiol. (Lond.) in press

Lucu, Č. and Towle, D. Na+,K+-ATPase in Aquatic Crustacea. Comp. Biochem. Physiol. review paper ; in press

Pavičić-Hamer,D., Devescovi,M.,Lucu,Č. 2003. Activation of carbonic anhydrase in branchial cavity tissues of lobsters (Homarus gammarus) by dilute seawater exposure. J. Exp. Mar. Biol. Ecol. 287, 79-92.

Lucu,Č., Devescovi,M.,Skaramuca,B.,Kožul, V. 2000 Gill Na+,K+-ATPase in the spiny lobster Palinurus elephas and other marine osmoconformers. Adaptivness of enzymes from osmoconformity to hyperregulation. J. Exp. Mar. Biol. Ecol. 246, 162-178

Lucu,Č. and Flik, G. 1999. Na+,K+-ATPase and Na/Ca exchange activity in gills of hyperregulating Carcinus maenas. Am. J. Physiol. 45, R490-R499