| Ca2+ plays an important role in organisms.Ca2+ homeostasis affects a variety of molecular and cellular processes and eventually leads to many diseases.For mammals,the maintenance and absorption of Ca2+ occur in the small intestine,kidneys and bones.For fish,the absorption and exchange of Ca2+ occur mainly in the gill.Ca2+concentration fluctuate widely in the surface and deep water of the sea.But the calcium concentration in fish need to be kept within a narrow range.Marbled eels(Anguilla marmorata),a vital catadromous fish,need to adapt to different Ca2+concentration.In order to explore the stress mechanism of eels in the acute calcium concentration water environment,we measured the expression pattern of CACNBs,NCXs and PMCAs,the serum Ca2+ and Na+ concentration,serum osmotic pressure and five blood index(the number of white cells,the number of red blood cells,the concentration of glucose,the concentration of hemoglobin and the number of platelets)from the control to high and deficient calcium group.To provide theoretical basis for further studies on the molecular mechanism of calcium homeostasis in eels.Specific research results are as follows:(1)Expression analysis of CACNB genes of A.marmorataWe conducted the temporal expression verification of voltage-gated L-type Ca2+channels(CACNB 1,CACNB2 and CACNB3)in vivo and vitro using qRT-PCR,Western blot,immunohistochemistry and primary branchial cell culture techniques.Tissue distribution analysis of Western blot showed that all three subunits had a significantly high expression in gill while mRNA results showed the expressions of CACNB 1 and CACNB3 were higher in skin tissue and the CACNB2 was highly presented in intestine.With the increasing of exposure time,mRNA and protein expressions of CACNB 1 were up-regulated in high level of Ca2+(10 mM)and down-regulated in deficient Ca2+(0 mM)compared to control Ca2+(2 mM).However,the opposite results were observed in CACNB2 and CACNB3.On day 7,the CACNB2 expression was not significantly different among Ca2+ treatments.Thus,we speculate that CACNB 1 has an indicating effect on high calcium and CACNB2 has an indicating effect on calcium homeostasis effect,and CACNB3 has an indicating effect on lack calcium.(2)Expression analysis of NCXs and PMCAs genes of A.marmorataWe used qRT-PCR to explore the distribution of NCXs and PMCAs in thirteen different tissues(skin,brain,gill,liver,muscle,spleen,heart,swim ladder,front intestine,mid-intestine,back intestine,head kidney and hind kidney)and study the changes of the mRNA level in NCXs and PMCAs genes of the gill tissues.Results of tissue distribution showed that NCX2 and NCX3 were highly expressed in the skin,while NCX1,PMCA2 and PMCA4 were highly expressed in the kidney;Results of temporal expression showed that there were a distinct difference in NCX1 and PMCA4 with the change of days,while other subunits were not.The NCX1 expression gradually increased in high calcium condition and decreased in low calcium environment with acclimating days,which indicated a role in acute environment.While the PMCA4 expression gradually decreased in high calcium condition and increased in low calcium environment with acclimating days,which indicated a role in steady environment.These findings might revealed that NCX1 and PMCA4 played important roles in the molecular mechanism of A.marmorata under Ca2+ homeostasis.(3)The determination of serum and blood indexes of A.marmorataUsing atomic absorption spectrometry to detect the serum Ca2+ and Na+concentration,the results showed the serum Ca2+ concentration gradually reduced in high Ca2+ environment and increased in low Ca2+ environment with the growth of time.While there was no significant difference between each treat in calcium homeostasis on the seventh day.Serum Na+ concentration gradually rose in high calcium environment and reduced in low calcium environment.The Na+concentration in each group has no significant difference.And it had no steady state.Using vapor pressure osmometer to detect serum osmotic pressure,the results showed that serum osmotic pressure gradually decreased under the high calcium environment and it increased under low calcium environment.The serum osmotic pressure had no significant difference compared with the control group on the seventh day.It appeared the calcium homeostasis.Using automated blood analyzer to determine the number of red blood cells,the white blood cells and using a standard kit to determine the glucose concentration and hemoglobin concentration,the results showed that five index decreased under the high calcium environment with the growth of the time.The five index increased in the absence of calcium environment.And it showed no significant difference and a steady state was observed on the seventh day.Therefore,there is a calcium homeostasis regulation mechanism in the body of eel to regulate Ca2+homeostasis. |
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