bioRxiv (Cold Spring Harbor Laboratory), Sep 28, 2023
Atmospheric CO 2 and temperature are rising concurrently, and may have profound impacts on the tr... more Atmospheric CO 2 and temperature are rising concurrently, and may have profound impacts on the transcriptional, physiological, and behavioral responses of aquatic organisms. Further, spring snow melt may cause transient increases of pCO 2 in freshwater systems. Lake sturgeon (Acipenser fulvescens) groups were raised in current and projected levels of warming and pCO 2. Following an overwintering period, lake sturgeon were exposed to a transient increase in pCO 2 , simulating a spring melt. Diverging transcriptional patterns were found in each group and metabolic rate was lower in the combined stressor group compared to others. Behavioral assays revealed no effect of environment on alarm cue responses or boldness, but there was a decrease in total activity following an acute CO 2 exposure. These results demonstrate compensatory and compounding mechanisms of pCO 2 and warming dependent on developmental conditions of a freshwater fish, and provide key information for responses to future climate change. .
Postprandial nitrogen and acid-base regulation in the seawater acclimated green crab, Carcinus maenas
Comparative Biochemistry and Physiology A-molecular & Integrative Physiology, May 1, 2022
The effects of feeding (meal of 3% of body mass) on acid-base and nitrogen homeostasis were inves... more The effects of feeding (meal of 3% of body mass) on acid-base and nitrogen homeostasis were investigated in the seawater acclimated green shore crab, Carcinus maenas. Feeding did not change gastric fluid pH (~pH 6); however, feeding was associated with a respiratory acidosis. Hemolymph HCO3- did not increase during this acidosis, although titratable and net acid efflux changed from an uptake to an excretion. Feeding affected the crabs' nitrogen homeostasis causing a substantial increase in hemolymph ammonia and urea concentrations after six hours. At this point, hemolymph urea accounted for ~1/3 of nitrogenous waste accumulated within the hemolymph, suggesting a potential role in ammonia detoxification. The postprandial increase in hemolymph ammonia coincided with an 18-fold increase in ammonia excretion rates that accounted for the majority of net acid excreted by the crabs. Urea excretion rates did not increase after feeding; however, branchial urease activity increased, implying that the gills may possess a mechanism to form excretable ammonia through the catabolism of urea. Our results demonstrate that despite an acidic gastric compartment, C. maenas does not experience a postprandial alkaline tide and that any feeding related acid-base challenges are primarily derived from metabolic acid production. Our findings also indicate that unlike the bicarbonate buffering acid-base compensatory response induced by hypercapnia and emersion, acid-base challenges upon feeding are compensated through changes in the excretion of acid equivalents, mainly in the form of ammonia.
American Journal of Physiology-regulatory Integrative and Comparative Physiology, Sep 15, 2015
Remarkably little is known about nitrogenous excretion in freshwater invertebrates. In the curren... more Remarkably little is known about nitrogenous excretion in freshwater invertebrates. In the current study, the nitrogen excretion mechanism in the carnivorous ribbon leech, Nephelopsis obscura, was investigated. Excretion experiments showed that the ribbon leech is ammonotelic, excreting 166.0 Ϯ 8.6 nmol•grams fresh weight (gFW) Ϫ1 •h Ϫ1 ammonia and 14.7 Ϯ 1.9 nmol•gFW Ϫ1 •h Ϫ1 urea. Exposure to high and low pH hampered and enhanced, respectively, ammonia excretion rates, indicating an acidlinked ammonia trapping mechanism across the skin epithelia. Accordingly, compared with body tissues, the skin exhibited elevated mRNA expression levels of a newly identified Rhesus protein and at least in tendency the Na ϩ /K ϩ-ATPase. Pharmacological experiments and enzyme assays suggested an ammonia excretion mechanism that involves the V-ATPase, Na ϩ /K ϩ-ATPase, and carbonic anhydrase, but not necessarily a functional microtubule system. Most importantly, functional expression studies of the identified Rh protein cloned from leech skin tissue revealed an ammonia transport capability of this protein when expressed in yeast. The leech Rh-ammonia transporter (NoRhp) is a member of the primitive Rh protein family, which is a sister group to the common ancestor of vertebrate ammonia-transporting Rh proteins. Exposure to high environmental ammonia (HEA) caused a new adjustment of body ammonia, accompanied with a decrease in NoRhp and Na ϩ /K ϩ-ATPase mRNA levels, but unaltered ammonia excretion rates. To our knowledge, this is only the second comprehensive study regarding the ammonia excretion mechanisms in a freshwater invertebrate, but our results show that basic processes of ammonia excretion appear to also be comparable to those found in freshwater fish, suggesting an early evolution of ionoregulatory mechanisms in freshwater organisms. primitive Rhesus proteins; cutaneous ammonia excretion; high environmental ammonia; ammonia trapping
Comparative Biochemistry and Physiology A-molecular & Integrative Physiology, Dec 1, 2010
In this study participation of cation/proton exchangers (NHE) in ammonia uptake in the medial mid... more In this study participation of cation/proton exchangers (NHE) in ammonia uptake in the medial midgut of Manduca sexta larvae was investigated employing a modified Ussing chamber. There was a mean inward net ammonia (NH 3 + NH 4 +) flux of 194 ± 17 nmol cm −2 h −1 across the isolated epithelium under conditions of 0.1 mmol L −1 ammonia on both sides of the tissue and a 100-fold inwardly directed P NH3-gradient (pH 8.5 luminal side, pH 6.5 hemolymphal side). Employing a 100-fold NH 4 + gradient amiloride applied to the luminal side inhibited the influx in a dose-dependent manner, with a maximal inhibition of 75% at 20 mmol L −1 and an estimated IC 50 = 2 mmol L −1. Amiloride also caused a dose-dependent but smaller decrease in the short-circuit current (I sc). No inhibition by apical or basal applied amiloride was noticed on cellular metabolic ammonia release, of which ca. 1/3 and 2/3 was secreted towards the apical and basal side, respectively. Using molecular methods full and partial sequence information of two putative cation/proton exchangers (MsNHE8, MsNHE7, 9) were obtained, both containing the characteristic amiloride binding motif. An mRNA expression analysis revealed ubiquitous expression patterns for both proteins, with similar expression levels for NHE8 in all tissues investigated and lower mRNA abundances for MsNHE7, 9 in the midgut sections of the caterpillar. In contrast, in this tissue high expression levels of the V-ATPase (D subunit) were detected, likely the sole pump responsible for energizing goblet cell K + excretion, but also involved in columnar cell ammonia uptake.
Cystic fibrosis transmembrane conductance regulator (CFTR) is a C1 channel that is defect in cyst... more Cystic fibrosis transmembrane conductance regulator (CFTR) is a C1 channel that is defect in cystic fibrosis, cAMPactivation of CFTR occurs via at least two distinct pathways: activation of CFTR molecules already present in the plasma membrane and PKA-mediated vesicular transport of new CFTR molecules to the plasma membrane and functional insertion into the membrane. We investigated the mechanisms that are responsible for these activation pathways. Oocytes of the South African clawed toad Xenopus laevis are widely used for the expression and characterization of foreign transport proteins. Oocyte size makes it easy to handle them and allows the application of several techniques on a single cell. We expressed CFTR and recorded continuously membrane current (Ira), conductance (Gin) and capacitance (C,,), which is a direct measure of membrane surface area. Expression of CFTR did not change plasma membrane surface area. Activation of CFTR with cAMP increased Ira, Gm and Cm while AF508-CFTR-expressing oocytes showed no response on cAMP. Inhibition of protein kinase A or buffering intracellular Ca 2+ abolished the cAMP-induced increase in Cm while increases of Im and G,,, were unaffected. >From these data we conclude that cAMP activates CFTR in at least two distinct pathways: activation of CFTR already present in the plasma membrane and exocytotic delivery of new CFTR molecules to the oocyte membrane and functional insertion into it. Exocytotic delivery of new CFTR to the plasma membrane could be prevented by agents that interfere with the cellular vesicle transporting machinery. Brefeldin A reversibly destroys one or more elements of the central vacuolar system and inhibited cocktail-mediated stimulation of CFTR in a time-dependent manner. Nocodazole, a drug that blocks microtubule polymerisation, also prevented complete activation of CFTR by our cocktail. Primaquine, an inhibitor of vesicle trafficking also abolished cocktail-induced stimulation of CFTR. All substances significantly reduced cocktail-mediated increases in I,,,, Gm and Cm These data show that functional integrity of the vesicle forming and transporting machinery of the cell is necessary for complete activation of CFTR expressed in oocytes. In another series of experiments we tested the effects of non-steroidal anti-inflammatory drugs on CFTR. Salicylic acid and ibuprofen inhibited about half of the CFTR current while acetylsalicylic acid (aspirin) had nearly no effect on CFTR. The anti-tumor drug tamoxifen and the anti-depressant compound fluoxetine (Prozac) had no significant influence on I,, and Gm Furthermore, we used two possible activators of CFTR namely genistein and 8-cyclopentyl-l,3dipropylxanthine (CPX) to stimulate CFTR. However, in contrast to some recent reports we found no activating effects of these compounds neither on wild-type CFTR nor on the disease-causing mutation AF508-CFTR.
Inorganic nitrogen is a key element for plant growth under salt stress. A comparative study inclu... more Inorganic nitrogen is a key element for plant growth under salt stress. A comparative study including physiological responses, ion content, transcript regulation of ammonium/nitrate transporters (AMTs/NRTs) as well as key enzymes for nitrogen assimilation was undertaken in wild salt-tolerant tomatoes (Solanum pennellii) and cultivated tomatoes (Solanum lycopersicon) exposed to 100 mM NaCl for 1 and 7 days. In comparison to S. lycopersicon, S. pennellii was more salt tolerant as evidenced by its higher survival rate, lower biomass reduction, and less salt injury (reduced electrolyte leakage and proline accumulation). In root tissues of both species, salt exposure (7 days) reduced the mRNA expression levels of low affinity nitrate transporters (NRT1.1 and NRT1.2). This was associated with a decline in both nitrate content and expression level of the nitrate reductase gene (NR). Salt-stressed root tissues of S. pennellii showed relatively higher mRNA expression of the high affinity ammonium transporters (AMT1.1 and AMT1.2) compared to S. lycopersicon. The root ammonium content was increased only in S. lycopersicon going hand in hand with a reduction in mRNA level of cytosolic glutamine synthetase (GS1) after 7 days of salt stress, whereas the expression level of GS1 was unchanged in S. pennellii, suggesting a lower salt-induced inhibition in ammonium assimilation in this species. Our comparative study demonstrated that the salttolerant and salt-sensitive tomato species show differential contribution of the nitrogen transporters and key genes associated with nitrogen assimilation under salt stress. While the reduction in the expression of key components of NO 3 − uptake (NRT1.1, NRT1.2) and assimilation (NR gene) in both species, likely contributed to the reduction in plant growth under salt stress, the observed salt tolerance for S. pennellii was associated with relative higher mRNA expression of ammonium uptake and assimilation genes. These results provide crucial knowledge for tomato breeding employing salt-tolerant wild species in salt-induced nitrogen-deficient environments.
Recent investigations on specialized epithelia suggest a role of the smooth endoplasmic reticulum... more Recent investigations on specialized epithelia suggest a role of the smooth endoplasmic reticulum Ca 2+-ATPase (SERCA) in epithelial Ca 2+-transport (Franklin et al., 2001; Hagedorn and Ziegler, 2002). These epithelia are involved in quick mineralisation processes in which epithelial Ca 2+-transport predominantly follows a transcellular route (transport through the cells) (Hubbard, 2000; Roer, 1980; Wheatly, 1997; Ziegler, 2002), rather than a paracellular pathway in which Ca 2+ would move extracellularly across the apical cell contacts and between the epithelial cells along its electrochemical gradient (Bronner, 1991). In the transcellular pathway, Ca 2+ enters the cells passively across the plasma membrane at one side of the cells and is actively extruded on the other side (Ahearn and Franco, 1993; Ahearn and Zhuang, 1996; Roer, 1980). The most critical step in transcellular epithelial Ca 2+-transport, however, is the Ca 2+ transport within the cells, through the cytoplasm from one side to the plasma membrane on the opposite side. Sustained elevated concentrations of Ca 2+ within the cytoplasm would interfere with the multiple regulatory functions of cytosolic free Ca 2+ signals. In addition, such elevated concentrations of ionized calcium can lead to cell damage and even cell death (Berridge, 1993). How such a toxic rise is prevented during epithelial Ca 2+-transport is still unknown. Simkiss (1996) proposed a model in which organelles, e.g. the smooth endoplasmic reticulum (SER), function as a transient calcium store allowing vectorial bulk flow through epithelial cells without toxic effects. Within cells the SER generally functions as source and sink for cytosolic Ca 2+ signals, with the inositol 1,4,5 trisphosphate (IP3)-receptors and/or ryanodine-receptors releasing Ca 2+ and the SERCA pumping Ca 2+ back in a regulated fashion (Hussain and Inesi, 1999). Previous investigations on the anterior sternal epithelium (ASE) of Porcellio scaber (Hagedorn and Ziegler, 2002; Ziegler, 2002) suggest that the SER contributes to transcellular calcium transport. Like most crustaceans, P. scaber has a calcified cuticle, which is moulted regularly to allow for growth of the animal. During premoult the ASE transports Ca 2+ , originating from the posterior cuticle to form large CaCO3 deposits located within the ecdysial gap of the first four anterior sternites (Messner, 1965
Comparative Biochemistry and Physiology A-molecular & Integrative Physiology, 2012
The present study has examined the role of the colon in regulating ammonia and urea nitrogen bala... more The present study has examined the role of the colon in regulating ammonia and urea nitrogen balance in two species of chondrichthyans, the ratfish, Hydrolagus colliei (a holocephalan) and the spiny dogfish, Squalus acanthias (an elasmobranch). Stripped colonic tissue from both the dogfish and ratfish was mounted in an Ussing chamber and in both species bi-directional urea flux was found to be negligible. Urea uptake by the mucosa and serosa of the isolated colonic epithelium through accumulation of 14 C-urea was determined to be 2.8 and 6.2 fold greater in the mucosa of the dogfish compared to the serosa of the dogfish and the mucosa of the ratfish respectively. Furthermore, there was no difference between serosal and mucosal accumulation of 14 C-urea in the ratfish. Through the addition of 2 mM NH 4 Cl to the mucosal side of each preparation the potential for ammonia flux was also examined. This was again found to be negligible in both species suggesting that the colon is an extremely tight epithelium to the movement of both urea and ammonia. Plasma, chyme and bile fluid samples were also taken from the agastric ratfish and were compared with solute concentrations of equivalent body fluids in the dogfish. Finally molecular analysis revealed expression of 3 isoforms of the urea transport protein (UT) and an ammonia transport protein (Rhbg) in the gill, intestine, kidney and colon of the ratfish. Partial nucleotide sequences of the UT-1, 2 and 3 isoforms in the ratfish had 95, 95 and 92% identity to the equivalent UT isoforms recently identified in another holocephalan, the elephantfish, Callorhinchus milii. Finally, the nucleotide sequence of the Rhbg identified in the ratfish had 73% identity to the Rhbg protein recently identified in the little skate, Leucoraja erinacea.
An experiment was conducted with piglets to determine the effect of dietary phytic acid supplemen... more An experiment was conducted with piglets to determine the effect of dietary phytic acid supplementation on performance, electrophysiological properties of jejunum mounted in Ussing chambers, sodium-dependent glucose transporter 1 (SGLT1) protein expression in jejunum, and plasma glucose and Na concentrations. Sixteen piglets with an average initial BW of 7.40 ± 0.36 kg were randomly assigned to 2 experimental diets with 8 piglets per diet. The diets were casein-cornstarch-based and were either unsupplemented or supplemented with 2% phytic acid (as Na phytate). The basal diet was formulated to meet the recommendation of NRC (1998) for energy, AA, minerals, and vitamins for piglets. The experiment lasted for 21 d, and at the end, BW gain and feed consumption were determined, and blood samples were collected for determination of plasma glucose and Na concentrations. The piglets were then euthanized to determine jejunal electrophysiological properties (transmural potential difference and short-circuit current) and SGLT1 protein expression. Phytic acid supplementation reduced ADG (P = 0.002), ADFI (P = 0.017), and G:F (P = 0.001) from 316.1 to 198.2 g, 437.4 to 360.3 g, and 0.721 to 0.539 g/g, respectively. Phytic acid supplementation also tended to reduce (P = 0.088) potential difference (−3.80 vs. −2.23 mV) and reduced (P = 0.023) short-circuit current from 8.07 to 0.1 µA/cm 2. However, phytic acid supplementation did not affect SGLT1 protein, and blood plasma glucose and Na concentrations. In conclusion, dietary phytic acid reduced growth performance and transmural short-circuit current in the jejunum of piglets. The reduced transmural short-circuit current in the jejunum by phytic acid implies reduced active Na transport in the jejunum by the phytic acid. Therefore, it seems that dietary phytic acid reduces growth performance of pigs partly through reduced capacity of the small intestine to absorb Na.
Ammonia transport by terrestrial and aquatic insects
Journal of Insect Physiology, Apr 1, 2012
Ammonia, an end product from amino acid and nucleic acid metabolism, is highly toxic for most ani... more Ammonia, an end product from amino acid and nucleic acid metabolism, is highly toxic for most animals. This review will provide an update on nitrogen metabolism in terrestrial and aquatic insects with emphasis on ammonia generation and transport. Aspects that will be discussed include metabolic pathways of nitrogenous compounds, the origin of ammonia and other nitrogenous waste products, ammonia toxicity, putative ammonia transporters as well as ammonia transport processes known in insects. Ammonia transport mechanisms in the mosquito Aedes aegypti, the tobacco hornworm Manduca sexta and the locust Schistocerca gregaria will be discussed in detail while providing additional, novel data.
The crustacean gill is a multi-functional organ, and it is the site of a number of physiological ... more The crustacean gill is a multi-functional organ, and it is the site of a number of physiological processes, including ion transport, which is the basis for hemolymph osmoregulation; acid-base balance; and ammonia excretion. The gill is also the site by which many toxic metals are taken up by aquatic crustaceans, and thus it plays an important role in the toxicology of these species. This review provides a comprehensive overview of the ecology, physiology, biochemistry, and molecular biology of the mechanisms of osmotic and ionic regulation performed by the gill. The current concepts of the mechanisms of ion transport, the structural, biochemical, and molecular bases of systemic physiology, and the history of their development are discussed. The relationship between branchial ion transport and hemolymph acid-base regulation is also treated. In addition, the mechanisms of ammonia transport and excretion across the gill are discussed. And finally, the toxicology of heavy metal accumulation via the gill is reviewed in detail.
Models of branchial transport in teleosts have been reshaped by the recent discovery of Rhesus (R... more Models of branchial transport in teleosts have been reshaped by the recent discovery of Rhesus (Rh) glycoproteins, a family of proteins that facilitate the movement of NH 3 across cell membranes. This study examines the effects of crowding and feeding on ammonia excretion in gulf toadfish (Opsanus beta) within the context of Rh glycoproteins and the ammonia-fixing enzyme, glutamine synthetase (GS). Four Rh isoforms (Rhag, Rhbg, Rhcg1 and Rhcg2) were isolated from toadfish. Tissue distributions showed higher levels of mRNA expression in the gills and liver, moderate levels in the intestine and lower levels in the stomach. Crowding significantly lowered branchial Rh expression and ammonia excretion rates in fasted toadfish. A comparison of Rh expression in the digestive tract revealed relatively low levels of Rhcg1 and Rhcg2 in the stomach and high mRNA abundance of Rhbg, Rhcg1 and Rhcg2 in the intestine of fasted, crowded toadfish. We speculate that these trends may reduce secretion and enhance absorption, respectively, to minimize the amount of ammonia that is lost through gastrointestinal routes. By contrast, these patterns of expression were modified in response to an exogenous ammonia load via feeding. Post-prandial ammonia excretion rates were elevated twofold, paralleled by similar increases in branchial Rhcg1 mRNA, gastric Rhcg1 mRNA and mRNA of all intestinal Rh isoforms. These changes were interpreted as an attempt to increase post-prandial ammonia excretion rates into the environment owing to a gradient created by elevated circulating ammonia concentrations and acidification of the digestive tract. Overall, we provide evidence that toadfish modulate both the expression of Rh isoforms and urea synthesis pathways to tightly control and regulate nitrogen excretion.
The production of secondary metabolites by aposymbiotic lichen-forming fungi in culture is though... more The production of secondary metabolites by aposymbiotic lichen-forming fungi in culture is thought to be influenced by environmental conditions. The effects of the environment may be studied by culturing fungi under defined growing parameters to provide a better understanding of the role of the large number of polyketide synthase (PKS) gene paralogs detected in the genomes of many fungi. The objectives of this study were to examine the effects of culture conditions (media composition and pH level) on the colony growth, the numbers of secondary products, and the expression of two PKS genes by the lichenforming fungus Ramalina dilacerata. Four types of growth media at four different pH levels were prepared to culture spore isolates of R. dilacerata. Colony diameter and texture were recorded. The number of secondary compounds were determined by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). Expression of two PKS genes (non-reducing (NR) and 6-MSAS-type PKS) were compared with expression of an internal control mitochondrial small subunit gene (mtSSU). The results showed that media containing yeast extracts produced the largest colony diameters and the fewest number of secondary metabolites. Colony growth rates also varied with different media conditions, and a significant negative relationship occurred between colony diameter and number of secondary metabolites. Expression of the NR PKS gene was significantly higher at pH 6.5 on the glucose malt agar than any other media, and expression of the 6-MSAS-type (partially-reducing) PKS gene was significantly higher at pH 8.5 on (malt agar) malt agar than on the other types of agar. Gene expression was correlated with the pH level and media conditions that induced the production of the larger number of secondary substances. This is the first study to examine secondary metabolite production in R. dilacerata by comparing the number of polyketides detected with quantitative polymerase chain reaction (qPCR) of two PKS genes under different culture conditions.
Most crustaceans live in sea water. In this medium, their haemolymph is nearly iso-osmotic with r... more Most crustaceans live in sea water. In this medium, their haemolymph is nearly iso-osmotic with respect to the environment. Thus, osmoregulation of the body fluid seems to be unnecessary. However, many species migrate to brackish or freshwater habitats. When confronted with these more dilute media, the osmotic concentration of the body fluids is hyperregulated by means of active salt uptake. For decapod crustaceans, it has been shown that this osmoregulatory ion uptake occurs predominantely in the thoracic gills (for reviews,
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Papers by Dirk Weihrauch