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The activity, immunolocalization, and mRNA expression of Na, K-ATPase were studied in gills of the hyper-/ hypoosmoregulating crab Pachygrapsus marmoratus acclimated to salinities of 10, 37, and 45 ppt. Enzyme activity was significantly higher in pooled posterior gills (7-9) than in anterior gills (1-6) and increased significantly upon acclimation to diluted medium, confirming previous observations with other crab species. Immunofluorescent analysis of gill sections indicated localization of Na, K-ATPase protein in basolateral membranes of branchial epithelium adjacent to the afferent blood vessel. Using degenerate primers based on Na, K-ATPase ALPHA subunit homologies published in GenBank, we amplified related sequences in cDNA transcribed from gill mRNA and employed the resulting PCR products for sequencing and mRNA expression analyses. Immunofluorescence and mRNA quantitation produced parallel results, showing high expression of Na, K-ATPase protein and mRNA in gills 6, 7, and 8 at low salinity. In gills 6 and 8, the abundance of Na, K-ATPase protein and mRNA increased dramatically with decreasing salinity. However, gill 7 showed substantial levels of Na, K-ATPase protein and mRNA even in 45 ppt seawater, suggesting that this gill may play an important role in hypoosmoregulation. Our results offer a molecular explanation for the previously observed changes in Na, K-ATPase activity in gills of euryhaline crabs adjusting to osmoregulatory demands in salinity extremes, namely the upregulation of Na, K-ATPase gene transcription and subsequent translation of Na, K-ATPase protein. Supported by NSF (IBN-9807539).

Many studies have shown that hyperosmoregulation in euryhaline crabs is accompanied by enhanced Na, K-ATPase activity in posterior gills, but it is not clear whether the response is due to activation of pre-existing enzyme or to increased gene transcription and mRNA translation. To address this question, the complete open reading frame and 3' untranslated region of the mRNA coding for the ALPHA subunit of Na, K-ATPase were amplified and sequenced by RT-PCR. The resulting 3,433-nucleotide cDNA encodes a putative 1,011-amino acid protein with a predicted molecular mass of 112.5 kDa. Hydrophobicity analysis of the amino acid sequence indicated eight membrane-spanning regions, in agreement with previously suggested topologies. The ALPHA subunit amino acid sequence is highly conserved among species, with the blue crab sequence showing 81-83% identity to other arthropods and 74-77% identity to vertebrate and non-arthropod invertebrate species. Semi-quantitative PCR showed high levels of ALPHA subunit mRNA in posterior gills 6-8 compared with anterior gills 3-5. Western blots of gill plasma membranes revealed a single Na, K-ATPase ALPHA subunit protein band of the expected size. Posterior gills contained a much greater level of ALPHA subunit protein compared with anterior gills, in agreement with historical measurements of enzyme activity. Transfer of blue crabs from 35 to 5 ppt salinity was accompanied by modest increases in ALPHA subunit mRNA and protein in posterior gills, providing a molecular explanation for the enhanced Na, K-ATPase enzyme activity that accompanies the hyperosmoregulatory response. Supported by NSF (IBN-9807539).