Randall K. Packer

Vasopressin increases Na-K-2Cl cotransporter expression in thick ascending limb of Henle’s loop

To investigate whether the enhancement of thick ascending limb (TAL) NaCl transport in response to long-term increases in circulating vasopressin concentration is associated with increased expression levels of the apical Na-K-2Cl cotransporter in the rat TAL, we have carried out immunoblotting and immunofluorescence studies using affinity-purified, peptide-directed antibodies. Semiquantitative immunoblotting studies demonstrated a marked increase (193% of controls) in Na-K-2Cl cotransporter band density in response to restriction of water intake to 15 ml/day for 7 days. In contrast, the expression levels of two other apical proteins of the TAL (the type 3 Na/H exchanger and Tamm-Horsfall protein) were unchanged in the outer medulla. A 7-day subcutaneous infusion of the V2receptor-selective vasopressin analog, 1-desamino-[8-d-arginine]vasopressin (DDAVP), to Brattleboro rats also markedly increased Na-K-2Cl cotransporter expression in the outer medulla (183% of controls). Immunofluorescence localization in outer medullary tissue sections confirmed the increase in Na-K-2Cl cotransporter expression in response to DDAVP. We conclude that vasopressin strongly upregulates the expression of the Na-K-2Cl cotransporter of the TAL and that it is likely to play an important role in the long-term regulation of the countercurrent multiplication system.

The role of the thyroid gland in osmotic and ionic regulation in Fundulus heteroclitus acclimated to freshwater and seawater

Abstract 1. 1. Histological examination showed that treatment with thiourea deactivated the thyroid gland in both freshwater (FW) and seawater (SW)-acclimated fish. 2. 2. FW acclimated fish treated with thiourea survived and showed no significant changes in serum osmolality or [Na + ]. 3. 3. SW-acclimated fish treated with thiourea survived less than 3 weeks and showed significant increases in serum osmolality and [Na + ]. 4. 4. SW-acclimated fish treated with thiourea and throxine survived until sacrificed and showed no significant changes in serum osmolality or [Na + ] compared to controls. 5. 5. It appears that a functional thyroid is essential for maintenance of [Na + ] and osmotic balance and for survival of Fundulus in SW.

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

Abstract—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 &agr; 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 &agr;ENaC protein abundance in rat kidney cortex. The decrease in &agr;ENaC protein abundance in response to AT1 receptor blockade was associated with a fall in &agr;ENaC mRNA abundance (real-time RT-PCR), consistent with transcriptionally mediated regulation. The effect of AT1 receptor blockade on &agr;ENaC expression was not blocked by spironolactone, suggesting a direct role of the AT1 receptor in regulation of &agr;ENaC gene expression. Candesartan administration was also found to increase the abundances of the &bgr; and &ggr; subunits. The increase in &bgr; and &ggr;ENaC 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 &bgr; and &ggr;ENaC 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.

Seasonal differences in activity of perch (Perca flavescens) gill Na+/K+ ATPase

Abstract We measured Na + /K + ATPase activity in homogenates of gill tissue prepared from field caught, winter and summer acclimatized yellow perch, Perca flavescens . Water temperatures were 2–4°C in winter and 19–22°C in summer. Na + /K + ATPase activity was measured at 8, 17, 25, and 37°C. V max values for winter fish increased from 0.48±0.07 μ mol P mg −1 protein h −1 at 8°C to 7.21±0.79 μ mol P mg −1 protein h −1 at 37°C. In summer fish it ranged from 0.46±0.08 (8°C) to 3.86±0.50 (37°C) μ mol P mg −1 protein h −1 . The K m for ATP and for Na + at 8°C was ≈1.6 and 10 mM, respectively and did not vary significantly with assay temperature in homogenates from summer fish. The activation energy for Na + /K + ATPase from summer fish was 10 309 ( μ mol P mg −1 h −1 ) K −1 . In winter fish, the K m for ATP and Na + increased from 0.59±0.08 mM and 9.56±1.18 mM at 8°C to 1.49±0.11 and 17.88±2.64 mM at 17°C. The K m values for ATP and Na did not vary from 17 to 37°C. A single activation energy could not be calculated for Na/K ATPase from winter fish. The observed differences in enzyme activities and affinities could be due to seasonal changes in membrane lipids, differences in the amount of enzyme, or changes in isozyme expression.

Thyroid hormones stimulate in vivo oxygen consumption of adult Rana pipiens berlandieri at high environmental temperatures.

Abstract Thyroxine injected frogs ( Rana pipiens berlandieri ) showed a 25.2% increase in oxygen consumption after 17 days of injection. Frogs injected with thyroid powder showed a 36.8% increase after only 3 days of injection.

A structural and functional study of the role of the renal sac in ionic regulation in the tunicate Molgula manhattensis

1. 1. The tunicate Molgula manhattensis is an estuarine animal lacking protonephridia and tubular excretory organs but possessing a well-developed renal sac thought to be the primary osmoregulatory organ. 2. 2. Animals were acclimated to three different salinities (271 mM Na+, 218 mM Na+ and 125 mM Na+) after which sodium and potassium levels were measured in plasma and renal sac fluid. The results indicate regulation of the sodium and potassium content of renal sac fluid, but not plasma. 3. 3. Transmission electron microscopy reveals that the renal sac epithelium possesses structural features known to be associated in other animals with ion secretion and/or osmoregulation.

Hemolymph acid-base balance in the terrestrial crab, Gecarcinus ruricola, with changing environmental temperature

Abstract 1. 1. Crabs were acclimated to 20, 25 and 30°C and acid-base status was assessed by measuring hemolymph pH, pCO2, lactate and selected electrolyte concentrations. 2. 2. Constant relative alkalinity of hemolymph was not maintained over the range of temperatures studied. As temperature increased, hemolymph pCO2 and total CO2 content increased while hemolymph pH showed no significant change. 3. 3. The results of this study are compared with findings of other investigators who studied acid-base balance in four other species of tropical terrestrial crabs. Of the five species compared, three appear not to maintain constant relative alkalinity.

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.