Abstracts / Comparative Biochemistry and Physiology, Part A 126 (2000) S1-$163 $89

ERYTHROCYTE K-CL COTRANSPORT AND BEYOND: IMPLICATIONS OF IMMUNO- CHEMICAL EVIDENCE OF TWO KCC ISOFORMS IN HK AND LK SHEEP Lauf P.K. l, Zhang J. t, Delpire E. 2, Fyffe R.E.W. 3, Mount D.B. 2 and Adragna N.C. 4

• 4 D e p a r t m e n t s o f P h y s i o l o g y & B i o p h y s i c s 1, A n a t o m y 3, and P h a r m a c o l o g y & T o x i c o l o g y , W r i g h t S ta te

U n i v e r s i t y , D a y t o n , O H , and V a n d e r b i l t U n i v e r s i t y 2 M e d i c a l Cen te r , N a s h v i l l e , T N Over the past twenty years we have characterized some of the thermodynamic, kinetic and regulatory properties of K-C1 cotransport (COT) in an animal model, the high and low K (HK and LK, respectively) sheep red blood cell (SRBC). In LK SRBCs, K-C1 COT is electroneutral using the chemical energy in the gradient of one ion to cotransport the other against its gradient ~0 Kinetically, K-Cl COT is asymmetric, with C1 binding outside prior to K, and both ions inside at random b) N- elhylmaleimide (NEM) treatment revealed presence of at least two thiol groups involved in regulation, one probably inhibiting a kinase causing transport stimulation, and another acting at and inhibiting the transport molecule c). K-C1 COT is also volume-sensitive a) The intriguing but unexplained fact is that, under isotonic conditions, LK but not HK SRBCs display K-C1 COT responding to NEM, kinase inhibitors (Mg removal, staurosporine, genistein) and swelling. This could mean that in contrast to LK sheep, HK SRBCs l) lack one or some of the recently discovered four KCC isoforms ~-~ carrying out K-C1 COT, 2) have different ratios of differently regulated KCC isoforms, or 3) do not have part of or the complete regulatory pathway. We have used two polyclonal rabbit antisera, one against a cytoplasmic C-terminal epitope of rat KCCI (anti-CtdKCC 1), and the other against a cytoplasmic N-terminal epitope (anti-NtdKCC3) of human KCC3 to test for the presence of the two isoforms in HK and LK SRBCs. In Western blots of SDS-PAGEL, anti-Ctd consistently detected a major -180kDa band, and anti-Ntd a smaller -140kDa band in ghosts prepared from homozygous HK and homo- and heterozygous LK cells. Immunofluorescence analysis by laser confocal microscopy revealed diffuse membrane staining of both HK and LK ghosts and of inside-out vesicles with anti-CtdKCCl, which was abolished by previous absorption with the immunogenic Ctd-maltose-binding fusion protein. Both, HK and LK SRBCs, therefore, posses the KCC 1 and KCC3 isoforms. The differences in molecular weight could be due to different glycosylation levels or additional peptides associated with KCC1. The functional differences between HK and LK SRBC K-C1 COT then maybe due to different regulation of either or both KCC1 and KCC3 proteins, or to mutations within them. References: a) Lauf, P.K. & N.C. Adragna (1996) J.Gen. Physiol.108, 341-350; b) Delpire, E. & P.K. Lauf, (1991) J. Gen. Physiol. 97:173-193; c) Lauf, P.K. & Adragna, N.C. (1995) Am.J.Physiol. 269:C 1167-C 1175; d) Dunham, P.B. & J.C. Ellory (1981) J. Physiol. 318:511- 530; e) Gillen et al. (1966) J. Biol.Chem. 217: 16237-16244; f) Mount et a1.(1999) J.Biol.Chem.274:16355-16362.(Supp. By NIH, WSU c~-grant, OHIO Research Challenge & AHA).

CALCIUM BALANCE IN DEVELOPING LARVAE OF FRESHWATER FISH

L e e T .H . , L in H .C . and H w a n g P.P.

D e p a r t m e n t o f Z o o l o g y , N a t i o n a l C h u n g - H s i n g U n i v e r s i t y , T a i c h u n g ; D e p a r t m e n t o f B i o l o g y , T u n g -

H a l U n i v e r s i t y , T a i c h u n g ; and Ins t i tu te o f Z o o l o g y , A c a d e m i a Sin ica , Ta ipe i , T a i w a n It has been well documented that larval stage is a critical period in the life history of fish. Fish Larvae, whose organ systems are still poorly -developed, have to perform biochemical and physiological activities in order to survive in the variable environments as the adult fishes do. Some recent studies have being focused on the development of ion- and osmoregulation mechanisms in larval stages.

Following development, tilapia larvae increase the content of ions (Na*, K +, and Ca 2+) and water, and the increase of water content accounts for most of the increase of body wet weight. The changes in ion and water contents are the result of increasing uptake of ion and water following larval development• Both Ca 2+ influx and efflux significantly increased following larval development, however, the extent of increase was much higher in influx than in efflux (15 fold vs. 2 fold). Upon 48 hours after fertilization, tilapia eggs were incubated respectively in 0.02 mM (low-Ca 2+) or 1.0 mM (high-Ca 2+) Ca z" artificial water for 8 days. The larvae showed similar hatching rates and wet weights in either high- or low-calcium medium, indicating neither the development nor the growth in tilapia larvae was affected by the environmental calcium levels. The body calcium content in low-Ca 2+ groups was about 90-95 % that of high-Ca 2÷ groups. Larvae of low-Ca 2+ group had a lower Ca 2÷ efflux, but they had a similar influx as the high-Ca 2+ group. Maintenance of the increasing uptake of ions and water appears to be critical fbr the survival of developing larvae. Moreover, the developing fish larvae, differently than adults, acclimate to low-Ca 2+ environments via declining passive Ca 2+ effiux, which is more advantageous from an energy standpoint. Ion content and uptake rate can be used as a sensitive indicator to monitor the impact of environmental factors on fish larvae.