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Effects of anoxia on functionality of Chamelea gallina haemocytes
Abstracts / Comparative Biochemisto" and Physiology, Part A 126 (2000) $1-S163
EFFECTS OF ANOXIA O N F U N C T I O N A L I T Y
OF CHAMELEA
S I 15
GALLINA HAEMOCYTES
P a m p a n i n D . M . , C a r o t e n u t o L., B a l l a r i n L. a n d M a r i n M . G . D e p t . o f B i o l o g y , U n i v e r s i t y o f P a d o v a , v i a U. B a s s i 5 8 / B , 3 5 1 0 0 P a d o v a , I T A L Y
Chamelea gallina is a veneridae clam fairly common in the Mediterranean Sea; it is subject to intense fishing effort due to its economical relevance. Since catched clams remain exposed to air for several hours before being re-immersed in seawater for storage before marketing, we studied the effects of anoxic stress on functionality of clam haemocytes. Referring to literature data regarding survival on air of C. gallina (LTs0 values at 18°C is 4 days), we arranged clams in boxes with 100% humidity for 0-1-2-3 days. When clams were exposed to air they closed valves and were unable to feed, the tissues became anoxic and the metabolism processes switched from aerobic to anaerobic. Clams were considered dead a hen an external stimulus did not cause any shell closing; dead clams were removed immediately. Chosen stress indices uere closely related to some haemocyte functions, with particular reference to immune functions. The haematocrit value (concentration of circulating haemocytes giving an indication of haemopoietic activity) decreased significantly after the first day of air exposure (p<0.01, t-test) and it continued to decrease until the last day. Other two indices referred to the percentage of haemocytes positive for two lytic enzymes, acid phosphatase and [3-glucuronidase. Acid phosphatase activity decreased significantly from the first day of air exposure (p<0.001, G-test). Conversely, ~-glucuronidase activity had an opposite course, significantly increasing after the first day (p<0.001, G-test), probably due to a role of the enzyme in anaerobic metabolism. In general, the above stress indices were able to return to control values after one day of anoxia. Two-day exposed clams attempted to recover in the three days following anoxia, this behaviour being reversed in the fourth day. After three days of anoxia clams did not recover. The phagocytic index (percentage of haemocytes having ingested test particles) decreased significantly already after the first day (p<0.001, G-test). As the adhesion index (percentage of cells able to adhere to the glass slides) is concerned, a significant decrease of adherent cells was observed after the first day (p<0.01, ANOVA test). It was probably the result of two effects: decrease of haematocrit value and decrease of ability to adhere. As the percentage of three different type of cells (spreading, round and apoptotic cells) is referred, a significant increase of apoptotic cells was evident after the first day (p<0.001, ANOVA test), whereas the number of spreading and round cells decreased, that of spreading cells more rapidly than round cells.
DIFFERENT ROLE OF PROSTAGLANDIN E2 IN REGULATION OF W A T E R AND UREA TRANSPORT IN AMPHIBIAN URINARY B L A D D E R P a r n o v a R., B a c h t e e v a V. a n d L a v r o v a E. S e c h e n o v I n s t i t u t e o f E v o l u t i o n a r y P h y s i o l o g y & B i o c h e m i s t r y o f the R u s s i a n A c a d e m y o f S c i e n c e s , Saint-Petersburg, Russia The role of prostaglandin E2 (PGE2) in regulation of water reabsorption in osmoregulatory epithelium is well established. Since water channels and urea transporters are involved in the urinary concentrating process, and regulation of both of ~vater and urea reabsorption by antidiuretic hormone is mediated by cAMP through the same type of receptor, it may be expected that PGE2 should control the urea and water transport by common mechanism. To analyze this hypothesis, the role of PGE2 in regulation of urea transport was studied in isolated frog (Rana temporaria L.) urinary bladders. Hemibladders were filled with Ringer solution containing 0.01 /aCu/ml of [~4]C-urea. The urea permeability (Pu) was determined from the rate of appearance of isotope in the serosal solution. The water permeability was measured in separate experiments in the presence of an osmotic gradient. It was found that PGE2 (10 nM-1 pM) from the serosal side caused an increase in Pu. The time required to reach a maximum was 1-1.5 h after PGE2 addition. The highest stimulation was observed under 0.5 laM PGE2 (6.5_1.5 vs. 1.0+0.2 cm/s x 10.0 in control, p
EFFECTS OF ANOXIA O N F U N C T I O N A L I T Y
OF CHAMELEA
S I 15
GALLINA HAEMOCYTES
P a m p a n i n D . M . , C a r o t e n u t o L., B a l l a r i n L. a n d M a r i n M . G . D e p t . o f B i o l o g y , U n i v e r s i t y o f P a d o v a , v i a U. B a s s i 5 8 / B , 3 5 1 0 0 P a d o v a , I T A L Y
Chamelea gallina is a veneridae clam fairly common in the Mediterranean Sea; it is subject to intense fishing effort due to its economical relevance. Since catched clams remain exposed to air for several hours before being re-immersed in seawater for storage before marketing, we studied the effects of anoxic stress on functionality of clam haemocytes. Referring to literature data regarding survival on air of C. gallina (LTs0 values at 18°C is 4 days), we arranged clams in boxes with 100% humidity for 0-1-2-3 days. When clams were exposed to air they closed valves and were unable to feed, the tissues became anoxic and the metabolism processes switched from aerobic to anaerobic. Clams were considered dead a hen an external stimulus did not cause any shell closing; dead clams were removed immediately. Chosen stress indices uere closely related to some haemocyte functions, with particular reference to immune functions. The haematocrit value (concentration of circulating haemocytes giving an indication of haemopoietic activity) decreased significantly after the first day of air exposure (p<0.01, t-test) and it continued to decrease until the last day. Other two indices referred to the percentage of haemocytes positive for two lytic enzymes, acid phosphatase and [3-glucuronidase. Acid phosphatase activity decreased significantly from the first day of air exposure (p<0.001, G-test). Conversely, ~-glucuronidase activity had an opposite course, significantly increasing after the first day (p<0.001, G-test), probably due to a role of the enzyme in anaerobic metabolism. In general, the above stress indices were able to return to control values after one day of anoxia. Two-day exposed clams attempted to recover in the three days following anoxia, this behaviour being reversed in the fourth day. After three days of anoxia clams did not recover. The phagocytic index (percentage of haemocytes having ingested test particles) decreased significantly already after the first day (p<0.001, G-test). As the adhesion index (percentage of cells able to adhere to the glass slides) is concerned, a significant decrease of adherent cells was observed after the first day (p<0.01, ANOVA test). It was probably the result of two effects: decrease of haematocrit value and decrease of ability to adhere. As the percentage of three different type of cells (spreading, round and apoptotic cells) is referred, a significant increase of apoptotic cells was evident after the first day (p<0.001, ANOVA test), whereas the number of spreading and round cells decreased, that of spreading cells more rapidly than round cells.
DIFFERENT ROLE OF PROSTAGLANDIN E2 IN REGULATION OF W A T E R AND UREA TRANSPORT IN AMPHIBIAN URINARY B L A D D E R P a r n o v a R., B a c h t e e v a V. a n d L a v r o v a E. S e c h e n o v I n s t i t u t e o f E v o l u t i o n a r y P h y s i o l o g y & B i o c h e m i s t r y o f the R u s s i a n A c a d e m y o f S c i e n c e s , Saint-Petersburg, Russia The role of prostaglandin E2 (PGE2) in regulation of water reabsorption in osmoregulatory epithelium is well established. Since water channels and urea transporters are involved in the urinary concentrating process, and regulation of both of ~vater and urea reabsorption by antidiuretic hormone is mediated by cAMP through the same type of receptor, it may be expected that PGE2 should control the urea and water transport by common mechanism. To analyze this hypothesis, the role of PGE2 in regulation of urea transport was studied in isolated frog (Rana temporaria L.) urinary bladders. Hemibladders were filled with Ringer solution containing 0.01 /aCu/ml of [~4]C-urea. The urea permeability (Pu) was determined from the rate of appearance of isotope in the serosal solution. The water permeability was measured in separate experiments in the presence of an osmotic gradient. It was found that PGE2 (10 nM-1 pM) from the serosal side caused an increase in Pu. The time required to reach a maximum was 1-1.5 h after PGE2 addition. The highest stimulation was observed under 0.5 laM PGE2 (6.5_1.5 vs. 1.0+0.2 cm/s x 10.0 in control, p