Acute and chronic temperature dependence of Na+/H+ exchange activity of Pimephales promelas gills.

Na+/H+ exchangers (NHE) mediate at least part of Na+ entry into gill epithelia via Na+/NH4+ exchange. For homeostasis, Na+ entry into and exit via Na+/K+ ATPase from gill epithelia must balance. Na+/K+ ATPase activity is reduced in cold- compared to warm-acclimated freshwater temperate fish. We hypothesized gill NHE activity is greater in warm- than cold-acclimated fish when measured at acclimation temperatures, and NHE activity displays a temperature dependence similar to Na+/K+ ATPase. Since NHE mRNA expression does not differ, we measured the Na+-dependence of pH-induced Na+ fluxes in gill vesicles from warm- and cold-acclimated fathead minnows at 20o and 7 °C, and calculated maximum transport rates (Vmax) and Na+ K1/2s. We also measured NH4+-induced Na+ fluxes and Na+-induced H+ fluxes. In vesicles from warm-acclimated fish, NHE Vmaxs were 278 ± 33 and 149 ± 23 arbitrary unit/s (au/s) and Na+ K1/2s were 12 ± 4 and 6 ± 4 mmol/l when assayed at 20o and 7 °C (p < 0.004), respectively. In vesicles from cold-acclimated fish, Vmaxs were 288 ± 35 and 141 ± 13 au/s and Na+ K1/2s 17 ± 5 and 7 ± 2 mmol/l when assayed at 20o and 7 °C (p < 0.002), respectively. Na+-induced H+ fluxes were 98 ± 8 and 104 ± 26 au/s in warm- and cold-acclimated fish assayed at 20 °C, respectively. Na+/NH4+ exchange was 120 ± 11 and 158 ± 13 au/s in warm- and cold-acclimated fish, respectively. Conclusions: Gill NHE activity was greater in warm- than cold-acclimated fish assayed at acclimation temperatures. The temperature dependence of NHE activity was similar in both groups, but differed from that reported for Na+/K+ ATPase suggesting complex mechanisms to maintain Na+ homeostasis.

Activity changes in gill ion transporter enzymes in response to salinity and temperature in fathead minnows (Pimephales promelas).

Fathead minnows, Pimephales promelas, are found throughout the continental United States in waters in which salinity can change with tides and temperatures vary seasonally. They have been used extensively in studies of environmental toxicology and are commercially important. In a very recent study in our labs RNA Seq was used to assemble transcriptomes from the gills of fatheads acclimated to either 5° or 22 °C. By comparison with published genomes, transcripts were identified for a number of ion transporters, ion channels, and signal molecule receptors, as well as enzymes that generate ammonia. H-ATPase and Na/K-ATPase activities were measured in supernatants of gill homogenates from fish acclimated to water sodium concentrations of 1.6, 3.1 or 124 mM sodium. As the water sodium concentration increased, in vitro activities of Na/K-ATPase activity and gill glutamate dehydrogenase activity decreased while H-ATPase activity increased. In a second series of experiments minnows were acclimated to 5 °C, 12.5 °C or 22 °C. In vitro activity of Na/K-ATPase decreased but activities of H-ATPase and glutamate dehydrogenase increased as temperature increased in gill membranes. These data do not support a primary role for apical H-ATPase in sodium influx under all conditions but do suggest a role for glutamate dehydrogenase production of ammonium to act as a counter-ion for sodium uptake by NHE-3.

Transcriptomic analysis of changes in gene expression of immune proteins of gill tissue in response to low environmental temperature in fathead minnows (Pimephales promelas).

In the face of ongoing climate change, it is imperative to understand better the effects of temperature on immune function in freshwater teleosts. It is unclear whether previously observed changes were caused by temperature per se. We studied changes in the gill transcriptome of fathead minnows (Pimephales promelas) at low temperature to understand better the effects of temperature on immune function. De novo assembly of the transcriptome using Trinity software resulted in 73,378 assembled contigs. Annotation using the Trinotate package yielded 58,952 Blastx hits (accessions). Expression of 194 unique mRNA transcripts changed in gill tissue of fathead minnows acclimatized to 5° compared to controls at 22 °C. At 5 °C mRNAs coding for proteins involved in innate immune responses were up-regulated. Those included proteins that block early-stage viral replication and macrophage activation. Expression of mRNAs coding for pro-inflammatory molecules and mucus secretion were also enhanced. Messenger RNAs coding for proteins associated with adaptive immune responses were down-regulated at 5 °C. Those included antigen-presenting proteins and proteins involved in immunoglobin production. Messenger RNAs coding for proteins that stimulate the cell cycle were also down-regulated at 5 °C. Histological comparison revealed that gills of cold acclimated fish had fewer mucus cells but cells contained larger mucus droplets. We conclude that decreased temperature modifies the immune systems of freshwater teleosts, leading to genome-wide upregulation of innate immunity and down regulation of adaptive immunity. Such acclimation likely evolved as an adaptive strategy against seasonal changes in infectious insults.

Increased renal alpha-ENaC and NCC abundance and elevated blood pressure are independent of hyperaldosteronism in vasopressin escape.

Previously, we demonstrated that rats undergoing vasopressin escape had increased mean arterial blood pressure (MAP), plasma and urine aldosterone, and increased renal protein abundance of the alpha-subunit of the epithelial sodium channel (ENaC), the thiazide-sensitive Na-Cl cotransporter (NCC), and the 70-kDa band of gamma-ENaC (Song J, Hu X, Khan O, Tian Y, Verbalis JG, and Ecelbarger CA. Am J Physiol Renal Physiol 287: F1076-F1083, 2004; Ecelbarger CA, Knepper MA, and Verbalis JG. J Am Soc Nephrol 12: 207-217, 2001). Here, we determine whether changes in these renal proteins and MAP require elevated aldosterone levels. We performed adrenalectomies (ADX) or sham surgeries on male Sprague-Dawley rats. Corticosterone and aldosterone were replaced to clamp these hormone levels. MAP was monitored by radiotelemetry. Rats were infused with 1-deamino-[8-D-arginine]-vasopressin (dDAVP) via osmotic minipumps (5 ng/h). At day 3 of dDAVP infusion, seven rats in each group were offered a liquid diet [water load (WL)] or continued on a solid diet (SD). Plasma aldosterone and corticosterone and urine aldosterone were increased by WL in sham rats. ADX-WL rats escaped, as assessed by early natriuresis followed by diuresis; however, urine volume and natriuresis were somewhat blunted. WL did not reduce the abundance or activity of 11-beta-hydroxsteroid dehydrogenase type 2. Furthermore, the previously observed increase in renal aldosterone-sensitive proteins and escape-associated increased MAP persisted in clamped rats. The densitometry of immunoblots for NCC, alpha- and gamma-70 kDa ENaC, respectively, were (% sham-SD): sham-WL, 159, 278, 233; ADX-SD, 69, 212, 171; ADX-WL, 116, 302, 161. However, clamping corticosteroids blunted the rise at least for NCC and gamma-ENaC (70 kDa). Overall, the increase in aldosterone observed in vasopressin escape is not necessary for the increased expression of NCC, alpha- or gamma-ENaC or increased MAP associated with "escape."

Long-term regulation of ENaC expression in kidney by angiotensin II.

We carried out semiquantitative immunoblotting of kidney to identify apical sodium transporter proteins whose abundances are regulated by angiotensin II. In NaCl-restricted rats (0.5 mEq Na/200 g BW/d), the type 1 angiotensin II receptor (AT1 receptor) antagonist, candesartan, (1 mg/kg of body weight per day SC for 2 days) markedly decreased the abundance of the alpha subunit of the epithelial sodium channel (ENaC). This subunit has been shown to be rate-limiting for assembly of mature ENaC complexes. In addition, systemic infusion of angiotensin II increased alphaENaC protein abundance in rat kidney cortex. The decrease in alphaENaC protein abundance in response to AT1 receptor blockade was associated with a fall in alphaENaC mRNA abundance (real-time RT-PCR), consistent with transcriptionally mediated regulation. The effect of AT1 receptor blockade on alphaENaC expression was not blocked by spironolactone, suggesting a direct role of the AT1 receptor in regulation of alphaENaC gene expression. Candesartan administration was also found to increase the abundances of the beta and gamma subunits. The increase in beta and gammaENaC protein abundance was not associated with a significant increase in the renal abundances of the corresponding mRNAs, suggesting a posttranscriptional mechanism. Immunocytochemistry confirmed the increase in beta and gammaENaC protein abundance and demonstrated candesartan-induced ENaC internalization in collecting duct cells. The results support the view that the angiotensin II receptor regulates ENaC abundance, consistent with a role for angiotensin II in regulation of collecting duct function.