Adaptation of renal function to hypotonic medium in the winter flounder (Pseudopleuronectes americanus)

Summary The kidneys of winter flounders transferred to hypotonic medium were investigated for glomerular and tubular handling of fluid and electrolytes and for the urinary excretion of proteins. Media were sea water (925 mosm·kg^–1) and brackish water (70 mosm·kg^–1).In sea water, the urine was hypertonic to the plasma in 7 fish of this study. Urine flow rate was correlated with the GFR. After adaptation to brackish water a delay of 1 to 3 days was observed until the kidneys switched from fluid retention to the excretion of large amounts of dilute urine. GFR and urine flow rate were increased from 0.61±0.08 to 1.58±0.29 ml·h^–1·kg^–1 and from 0.14±0.02 to 0.68±0.08 ml·h^–1·kg^–1, respectively . With increased filtered load the tubular reabsorption of fluid decreased from 74±2.4% to 45±11.2%. The excretion rates of sodium and potassium were increased due to decreased fractional sodium and potassium reabsorption. The urinary excretion of divalent cations, however, was reduced because the net tubular reabsorption of calcium was increased and the net secretion of magnesium was diminished.Both the urinary total protein concentration and the protein pattern showed no significant change, but the rate of protein excretion was increased from 0.21±0.04 to 0.60±0.05 mg·h^–1·kg^–1. The comparison of protein patterns obtained from urine and serum samples revealed that high molecular weight (HMW) proteins prevail in the serum whereas low molecular weight (LMW) proteins dominate in the urine. The diminished quantity of the HMW-protein fraction in the urine thus may reflect size selectivity of the glomerular filtration barrier for serum proteins also in the winter flounder.Abbreviations BW brackish water - SW sea water - GFR glomerular filtration rate - HMW heigh molecular weight - LMW low molecular weight     

Chloride cells and chloride exchange in the skin of a sea-water teleost,the shanny (Blennius pholis L.)

Summary The fine structure of the skin and its importance in chloride outfluxes were investigated in a sea-water teleost, the shanny (Blennius pholis L.).The epidermis is composed of three cells types: epithelial cells, mucous cells and chloride cells. These chloride cells typically contain a great number of mitochondria and an extensive agranular reticulum extending through the whole cell body. They open at the surface of the epidermis into an apical pit. An undifferentiated small cell is often observed near these chloride cells and probably corresponds to the adjacent chloride cell.The values of chloride outfluxes through the skin and the gills are respectively 5333±884 Eq·h^–1·kg^–1 and 4479±2521 Eq·h^–1·kg ^–1; n=6; t=13±0.5°C. Thus the ratio between skin chloride outflux and total chloride outflux is 64.7±9.3%.     

Secretion of electrolytes,protein and urea by the mandibular gland of the common wombat (Vombatus ursinus)

Saliva was collected from the mandibular glands of anaesthetized common wombats (Vombatus ursinus) to ascertain maximal flow rates, salivary compostion and possible adaptations, particularly PO₄ ^3- secretion, to assist digestion. After temporary catheterization of the main duct through its oral opening, salivary secretion was evoked at flow rates ranging from 0.02±0.002 (±SEM) ml·min^-1 (0.7±0.07 l·min^-1·kg body weight^-1) to 0.4±0.05 ml·min^-1(14±1.9 l·min^-1·kg body weight^-1) by ipsilateral intracarotid infusion of acetylcholine. The [Na⁺] (15±5.1 to 58±8.6 mmol·l^-1) and [HCO₃ ^-] (35±1.9 to 60±1.9 mmol·l^-1) were positively correlated with salivary flow rate. The [K⁺] (58±5.2 to 30±2.4 mmol·l^-1), [Ca²⁺] (10.4±1.67 to 4.1±0.44 mmol·l^-1), [Mg²⁺] (0.94±0.137 to 0.17±0.032 mmol·l^-1), [Cl^-] (71±9.2 to 45±6.0 mmol·l^-1), [urea] (9.3±0.79 to 5.1±0.54 mmol·l^-1), H⁺ activity (29±1.6 to 17±1.6 nEq·l^-1) and amylase activity (251±57.4 to 92±23.3 kat·l^-1) were negatively correlated with flow. Both concentration and osmolality fell with increasing flow at the lower end of the flow range but osmolality always increased again by maximal flow whereas the relation between protein and flow was not consistent at the higher levels of flow and stimulation. Salivary [PO₄ ³⁺] was not correlated with flow and at 3–14% of the plasma concentration was extremely low. Thus, in contrast to its nearest relative, the koala (Phascolarctos cinereus), the wombat secretes little PO₄ ³⁺ presumably because it does not need high levels of PO₄ ³⁺ in its saliva to facilitate microbial digestion of plant fibre.Abbreviations bw body weight - ww wet weight     

Mucosal acidification and an acid microclimate in the hen colon in vitro

Experiments were performed on isolated, stripped colonic epithelia of low-salt-adapted hens (Gallus domesticus) in order to characterize acid secretion by this tissue. With symmetric, weak buffer solutions, colonic epithelia acidified both mucosal and serosal sides. Titration measurements of the mucosal acidification rate (pH-stat technique) averaged 1.63±0.25 Eq·cm^-2·h^-1. Mucosal acidification was also evident in colons from high-salt-adapted birds and in low-salt-adapted coprodeum, but was completely abolished in the high-salt coprodeum. Mucosal acidification by low-salt-adapted colonic epithelium was unaffected by sodium replacement, mucosal amiloride (10^-3 mol·l^-1), and serosal ouabain (5x10^-4 mol·l^-1), although all three treatments significantly reduced or reversed the short-circuit current. Acetazolamide (10^-3 mol·l^-1, serosal) reduced mucosal acidification by 15% and simultaneously increased short-circuit current by a similar amount. Colonic epithelia incubated in glucose-free solutions had significantly lower acidification rates (0.59±0.13 Eq·cm^-2·h^-1, P<0.002 versus controls) and addition of glucose (15 mmol·l^-1), but not galactose, partially restored acidification to control levels. Anoxia (N₂ gassing) completely inhibited short-circuit current, but reduced acidification by only 30%. A surface microclimate pH, nearly 2 pH units more acidic than the bath pH of 7.1–7.4 was measured in low-salt-adapted colon and coprodeum. The acid microclimate of both tissues was partially attenuated by adaptation to a high-salt diet. Colonic microclimate pH was dependent on the presence of glucose and sensitive to the bath pH. Histochemical staining for carbonic anhydrase localized this enzyme to cytoplasm and lateral margins of one subfraction of colonic cells, and to cytoplasm in a second subpopulation Intense staining was also evident in subepithelial capillaries. These results suggest that a large part of mucosal acidification and maintenance of the acid microclimate in hen colon may be dependent on glycolysis and metabolic acid production, although a smaller, electrogenic and acetazolamidesensitive component also appears to exist. This latter component may become more prominent under conditions of cellular acidification.Abbreviations CA carbonic anhydrase - I _SC short circuit current - NFM N-ethylmaleimide - PD transepithelial potential - SCFA short chain fatty acids     

Phosphorus and nitrogen excretion rates of zooplankton from the eutrophic Loosdrecht lakes,with notes on other P sources for phytoplankton requirements

Phosphorus and nitrogen excretion rates by zooplankton communities from two eutrophic and shallow Dutch lakes were measured in laboratory. The variations in excretion rates in the lakes (May–October) were caused mainly by fluctuation in zooplankton biomass. Mean summer excretion rates (June–September) were 2.4 and 0.9 µg PO₄P·1^–1·d^–1 in Lake Loosdercht and Lake Breukeleveen, respectively. This difference between the lakes was caused mainly by the lower zooplankton biomass in Lake Breukeleveen. The excretion of 2.4 µg PO₄P·1^–1·d^– compared with the calculated P-demand of phytoplankton of 8.0 µg PO₄P·1^–1·d^–1 is substantial in the summer (June–September) and far more important than the external P-supply of 0.4 µg P·1^–1·d^–1 and sediment release of 0.5 µg P·1^–1·d^–1. Both temperature and composition of zooplankton affected the weight specific excretion rates of the zooplankton community. The weight specific community excretion rates of P and N increased with temperature (exponential model); 1–8 g PO₄P·mg^–1 zooplankton-C·d^–1 and 5–42 µg NH₃N·mg^–1 zooplankton-C·d^–1 (10°C–20°C).     

Function and regulation of the avian caecal bulb: influence of dietary NaCl and aldosterone on water and electrolyte fluxes in the hen (Gallus domesticus) perfused in vivo

Summary The function of the caecal bulb, and its adaptation to chronic high- or low-Na⁺ intake, was investigated by in vivo perfusion of anaesthetised birds. Effects of acute aldosterone injection (125 g·kg^–1 body mass) were also measured.Evidence was found for primary active net absorption of Na⁺, inducing parallel Na-linked absorption of water and Cl^– and secretion of K⁺. Around 20–35% of total Cl^– absorption and K⁺ secretion were independent of Na⁺ fluxes, and these components appear to be driven by passive processes with apparent conductances of 6.3×10^–3 (G _Cl) and 1.1×10^–3 (G _K) S·cm^–2.Acetate (40mM) stimulated Na⁺ fluxes (8.5–9.9 Eq·cm^–2·h^–1) and Na-linked water fluxes (27–44 l·cm^–2·h^–1). Increased coupling ratios (2.9–4.6 l·Eq^–1) and other data indicate that these effects may be due to increased osmotic permeabilities of barriers involved in the Na-linked water transfer pathway.Low-Na⁺ maintenance enhanced EPD (49–69 mV, serosa positive) and all net fluxes:J _Na (6.8–11.6);J _K (–3.2––4.3);J _Cl (4.3–5.6 Eq·cm serosal area^–2·h^–1);J _v (28–43 l·cm^–2·h^–1) (mucosal-serosal fluxes positive).Acute aldosterone enhancedJ _Na (10.8–14.0 Eq·cm^–2·h^–1) and EPD (54–66 mV) by 3 h after injection, but had no effect on the Na-linked components ofJ _K orJ _Cl.Abbreviations ECPD, EPD Electrochemical or electrical potential difference - G _Cl ,G _K ionic conductances (Cl^–, K⁺) - J _v ,J _ion net volume or ion flux rate, mucosa-serosa positive;P _d (Cl) diffusive permeability coefficient (of Cl^–) - SEDM standard error of difference between means     

Excretion of photosynthetically fixed organic carbon by metalimnetic phytoplankton

The effects of light intensity, oxygen concentration, and pH on the rates of photosynthesis and net excretion by metalimnetic phytoplankton populations of Little Crooked Lake, Indiana, were studied. Photosynthetic rates increased from 1.42 to 3.14 mg C·mg^–1 chlorophylla·hour^–1 within a range of light intensities from 65 to 150E·m^–2·sec^–1, whereas net excretion remained constant at 0.05 mg C·mg^–1 chlorophylla·hour^–1. Bacteria assimilated approximately 50% of the carbon released by the phytoplankton under these conditions. Excreted carbon (organic compounds either assimilated by bacteria or dissolved in the lake water) was produced by phytoplankton at rates of 0.02–0.15 mg C·mg^–1 chlorophylla·hour^–1. These rates were 6%–13% of the photosynthetic rates of the phytoplankton. Both total excretion of carbon and bacterial assimilation of excreted carbon increased at high light intensities whereas net excretion remained fairly constant. Elevated oxygen concentrations in samples incubated at 150E· m^–2·sec^–1 decreased rates of both photosynthesis and net excretion. The photosynthetic rate increased from 3.0 to 5.0 mg C·mg^–1 chlorophylla· hour^–1 as the pH was raised from 7.5 to 8.8. Net excretion within this range decreased slightly. Calculation of total primary production using a numerical model showed that whereas 225.8 g C·m^–2 was photosynthetically fixed between 12 May and 24 August 1982, a maximum of about 9.3 g C·m^–2 was released extracellularly.     

Nitrogen distribution and excretion during embryonic post-yolk sac development inZoarces viviparus

In the viviparous teleostZoarces viviparus (L.) embryonic post-yolk sac development in the ovary is characterized by significant increases in dry weight and nitrogen per embryo, thus indicating an extensive matrotrophic relationship. In the ovarian fluid surrounding the embryos during their intraovarian development, sources of nitrogen were shown to derive from amino acids, urea, ammonia and various cellular components. The level of urea in the ovarian fluid increased significantly from 3.68±0.25 mol urea-N·ml^-1 during early post-yolk sac embryonic development to 6.14±0.44 in late development. The corresponding level of ammonia-N in the ovarian fluid increased from 0.25 to 0.45 mol·ml^-1. An estimation of embryonic nitrogen loss was made by measuring urea and ammonia-N excretion in vitro by post-yolk sac embryos or larvae (i.e. seawater-acclimated embryos). Urea-N constituted an average of 65% of the total nitrogen excreted by the embryos into the ambient medium during a 5-h time-course compared to only 35% in the larvae. Urea-N was excreted at maximum rates during the first hour of the experiment, 0.54±0.09 mol N·g^-1·h^-1 by embryos and 0.35±0.02 by larvae, and then declined to lower levels in both embryos and larvae. A decline after 1 h was also observed for excretion rates of ammonia. In conclusion, the capacity for urea excretion by post-yolk sac embryos ofZ. viviparus may be of adaptive significance for their prolonged stay in ovario. The capacity for excretion of urea seems to decrease after acclimation to sea water.Abbreviations aa amino acid(s) - bm body mass - dw dry weight - NPS ninhydrin-positive substances - sw sea water - ww wet weight     

Feasibility study on the use of hyperosmolar medium for improved antibody production of hybridoma cells in a long-term,repeated-fed batch culture

To test the feasibility of using hyperosmolar medium for improved antibody production in a long-term, repeated fed-batch culture, the influence of various culture conditions (serum concentration and cultivation method) on the hybridoma cells' response to hyperosmotic stress resulting from sodium chloride addition was first investigated in a batch culture. The degree of cell growth depression resulting from hyperosmotic stress was dependent on serum concentrations and cultivation methods (static and agitated cultures). Depression of cell growth was most significant in agitated cultures with low serum concentration. However, regardless of serum concentrations and cultivation methods used, the hyperosmotic stress significantly increased specific antibody productivity (q _MAb). Increasing osmolality from 284 to 396 mOsm kg^–1 enhanced the q_MAb in agitated cultures with 1% serum by approximately 124% while the similar osmotic stress enhanced the q _MAb in static cultures with 10% serum by approximately 153%. Next, to determine whether this enhanced q_MAb resulting from hyperosmotic stress can be maintained after adaptation, long-term, repeated-fed batch cultures with hyperosmolar media were carried out. The cells appeared to adapt to hyperosmotic stress. When a hyperosmolar medium (10% serum, 403 mOsmkg^–1) was used, the specific growth rate improved gradually for the first four batches and thereafter, remained constant at 0.040±0.003 (average ± standard deviation) hr^–1 which is close to the value obtained from a standard medium (10% serum, 284 mOsmkg^–1) in the batch culture. While the cells were adpating to hyperosmotic stress, the q_MAb was gradually decreased from 0.388×10^–6 to 0.265×10^–6 g cell hr^–1 and thereafter, remained almost constant at 0.272±0.014× 10^–6 g cell^–1 hr^–1. However, this reduced q _MAb after adaptation is still approximately 98% higher than the q_MAb obtained from a standard medium in the batch culture.The authors would like to thank Dr.M. Kaminski for providing the hybridoma cell line used in this study. This work was supported by the Korea Science and Engineering Foundation.     

Mechanisms of fluid and ion secretion by the parotid gland of the kangaroo,Macropus rufus,assessed by administration of transport-inhibiting drugs

Possible mechanisms of primary fluid formation by macropodine parotid glands were investigated in anaesthetized red kangaroos using ion transport inhibitors. Carotid plasma amiloride concentrations of 0.05–0.5 mmol·l^-1 progressively reduced a stable acetylcholine-evoked half-maximal flow rate of 2.0±0.04 to 0.22±0.024 ml·min^-1 (mean±SEM). Concurrently, saliva bicarbonate concentration and secretion fell (135±1.6 to 67±1.7 mmol·l^-1 and 272±7.6 to 15±2.6 mol·min^-1, respectively); [phosphate], [chloride] and [sodium] rose and [potassium] and osmolality were unaltered. High-rate cholinergic stimulation did not increase saliva flow beyond 11±1.0% of that for equivalent pre-amiloride stimulation. Equipotent levels of amiloride and methazolamide given concurrently were no more effective at blocking flow and bicarbonate secretion than when given separately. Furosemide (up to 2 mmol·l^-1), bumetanide (up to 0.2 mmol·l^-1) and ethacrynate (1 mmol·l^-1) in carotid plasma had no effect on salivary flow or ion concentrations. During methazolamide blockade, furosemide did not curtail the concurrent increase in salivary [chloride]. Chlorothiazide at 0.25–1.0 mmol·l^-1 caused progressive depression of saliva flow and [bicarbonate], and elevation of [chloride]. 4-acetamido-4-isothiocyanatostilbene-2,2 disulphonic acid at 0.1 mmol·l^-1 was without effect, whereas at 0.5 mmol·l^-1 it stimulated fluid secretion and increased saliva [protein], [sodium], [potassium], [bicarbonate] and osmolality. Concurrently, mean arterial blood pressure and pulse pressure fell and heart rate, haematocrit and carotid artery plasma flow rose. These responses were absent if saliva flow was kept constant by reduction in cholinergic stimulation during 4-acetamido-4-isothiocyanatostilbene-2,2 disulphonic acid administration. It is concluded that secretion of primary fluid by the kangaroo parotid is initiated mainly (>90%) by secretion of bicarbonate which is formed in the endpiece cells from CO₂ delivered by the circulation. No evidence was found for initiation of fluid secretion by chloride transport involving basolateral Na⁺-K⁺-2Cl^- symports, Na⁺-Cl^- symports or Cl^-/HCO ₃ ^- antiports.Abbreviations CA carbonic anhydrase - CAI carbonic anhydrase inhibitors - MAP mean arterial blood pressure - PAH p-aminohippurate - SITS 4-acetamido-4-isothiocyanatostilbene-2,2 disulphonic acid     

Beta-adrenergic control of trans-cutaneous evaporative cooling mechanisms in birds

Summary The decreasing effect of -adrenergic blockade on skin resistance to vapor diffusion and the onset of cutaneous water evaporation in the pigeon (Columba livia) was investigated. Oral administration of 1, 2.3 and 5 mg propranolol to pigeons (268±53 g) initiated intensive trans-cutaneous water evaporation (CWE) up to 29.1 mg H₂O·cm^–2·h^–1 in resting birds at 30°C air temperature (Ta), but had only a slight effect on CWE of birds exposed to 50 °C Ta.After 7 h of effective -adrenergic blockade (oral administration of 5 mg propranolol), skin and body temperature stabilized at 39.0±0.5 °C and 41.0±0.7 °C, compared to 40.2±0.8 °C and 41.9±0.6 °C in the control group, respectively. A slight hypothermia was accompanied by feather fluffing.Intradermal injection of 0.001, 0.01 and 0.12 mg propranolol also caused intensive CWE. Local -adrenergic blockade in relatively low blocker doses (0.001 and 0.01 mg propranolol) decreased skin resistance from a high value of 44.5 s·cm^–1 to about 6.0 s·cm^–1, and caused a sharp increase in CWE from a control value of about 4 to a high of 26.4 mg H₂O·cm^–2·h^–1 during the first two hours of exposure to 30°C Ta.The possible role of -adrenergic blockade in regulation of trans-cutaneous water evaporation of latent heat dissipation is discussed.     

Kidney and bladder function in a uricotelic treefrog (Phyllomedusa sauvagei)

Summary Phyllomedusa sauvagei, a xeric adapted treefrog, excretes large amounts of nitrogen as urate when fed insects, even when deprived of additional water. Most terrestrial anurans produce urea which they do not excrete when they are deprived of water. We investigated the differences in renal function underlying the unusual excretory capacities ofP. sauvagei. Glomerular filtration rates (GFR) were measured inP. sauvagei in water and when deprived of water, except that in food, for up to 27 days. For comparison a toad (Bufo boreas) was studied in water and during water deprivation. In water both species produced 30–40 ml urine kg^–1 h^–1 and resorbed only ca. 50% of the filtrate. With water deprivation, GFR rapidly approached zero inB. boreas, but remained high (20–40 ml kg^–1 h^–1) inP. sauvagei despite reductions in urine production of up to 100-fold. During water deprivation inP. sauvagei, urate excretion was between 250–300 moles kg^–1 h^–1 and 90% of this reflects net tubular secretion. Urate clearances were similar to those of para-amino hippurate, indicating effective removal of urate from the peritubular circulation. Urea, sodium and chloride showed net fractional resorptions of 98–99%, and 85% of the potassium was resorbed. At low rates of urine production, urine to plasma (U/P) ratios for inulin in bladder urine were 20–100 whereas those for ureteral urine were ca. 10. The urinary bladder also functions as a water reserve during dehydration.     

Time course of vasopressin-induced formation of microvilli in granular cells of toad urinary bladder

Summary Vasopressin-induced transformation of ridges to microvilli on the surface of granular cells of toad urinary bladder occurs in conjunction with induced alterations in the water permeability of the luminal membrane. This study was designed to establish the relationship between the time course for induction of microvilli and the time course for induction of increased water permeability after vasopressin stimulation. Hemibladders were examined at 2.5, 5, 10, 20 and 30 min following exposure to 20 mU/ml of vasopressin and at 5, 10, 20, 30, 40, 50 and 60 min after washout of vasopressin. Within 2.5 min, vasopressin initiated complete transformation of ridges to microvilli on approximately 13% of the granular cells, while osmotic water flow (Jv) was 0.31±0.10 l·min^–1·cm^–2. Five minutes following vasopressin stimulation, microvilli were present on approximately 30% of granular cells andJv was 2.27±0.13 l·min^–1·cm^–2. At 10 minJv was maximum at 4.03±0.15 l·min^–1·cm^–2 and 50% of the granular cells were covered with microvilli. This percentage increased to 70% at 20 min and was maintained at 30 min, althoughJv decreased to 3.9±0.35 l·min^–1·cm^–2 at 30 min. Five minutes following vasopressin washout, ridges interspersed with microvilli reappeared asJv fell to 1.10±0.30 l·min^–1·cm^–2. At 10 min after vasopressin washout,Jv approached basal levels, but the reversal of microvilli to ridges remained incomplete. At 60 min after vasopressin washout, the granular cells had regained their original ridgelike surface structures. Thus, these studies establish a temporal relationship between the induction and reversibility of vasopressin-induced microvillous formation and alterations in the osmotic water permeability of the apical plasmalemma.     

Continuous monitoring of cellulase action on microcrystalline cellulose

Summary Cellobiose oxidase from Phanerochaete chrysosporium was used for continuous monitoring of cellulase action on microcrystalline cellulose (Avicel). Two protocols are described, the parameter monitored being either the decline in electrode potential as ferricyanide is reduced or consumption of dioxygen. Most experiments used a commercial cellulase preparation from Trichoderma reesei and ferricyanide as acceptor. Within 1 min of an addition of cellulase, ferricyanide reduction reached a steady rate. This was converted into a rate of production of substrate for celobiose oxidase, in mol·min^–1. Experiments were conducted either with a constant concentration of cellulase and increasing Avicel, or with constant Avicel and increasing cellulase. Kinetic analysis of the experiments with constant cellulase indicated a K _mof 4.8 ± 1.0 (g cellulose)·1^–1, which was close to the value predicted from binding studies. The specific activity of the cellulase was measured as 375±25 mol·(g cellulase)^–1·min^–1 in experiments with a high cellulose concentration, but was less than half this value when the cellulose was saturated with cellulase. The maximal rate of cellulose degradation was 9.6±1.3 mol·(g cellulose)^–1·min^–1.     

Dynamics of production oftrans-zeatin andtrans-zeatin riboside by immobilized cytokinin-autonomous and cytokinin-dependent tobacco cells

Two strains of cultured tobacco cells (Nicotiana tabacum L. cv. Wisconsin 38) differing in their requirement for exogenous cytokinins (cytokinin-dependent and cytokinin-autonomous) were immobilized on polyphenylenoxide (Sorfix) activated with glutaraldehyde. Columns packed with immobilized cells were continually eluted with diluted Murashige and Skoog's medium lacking or supplemented with synthetic cytokinin (6-benzylaminopurine; BA). Purified samples of column eluates were fractionated by HPLC, andtrans-zeatin (t-Z) andtrans-zeatin riboside (t-ZR) content was estimated by enzyme immunoassay. Both cytokinin-autonomous and cytokinin-dependent tobacco cells produced and excretedt-Z and its riboside, and there were significant quantitative differences between the strains. The steady-state excretion rate oft-Z was 19.8 ng · g^–1 dw · h^–1 and 4 ng · g^–1 dw · h^–1, respectively, and that oft-ZR 4 ng · g^–1 dw · h^–1 and 1 ng · g^–1 dw · h^–1, respectively. Exposure of cytokinin-dependent cells to BA after 72 h of starving for this synthetic cytokinin caused temporary increase in excretion of both zeatin and its riboside. After the application of 5 M BA for 24 h, the excretion rate oft-ZR reached 5 ng · g^–1 dw · h^–1 (5-fold increase), and that oft-Z achieved 12 ng · g^–1 dw · h^–1 (3-fold increase). The elevation oft-Z excretion was delayed about 13 h compared witht-ZR excretion, which started increasing almost immediately after BA application. A pulse of BA in lower concentration (1.5 M for 30 h) provoked lower response.     

Functional Characterization of Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> Exchangers in Primary Cultures of Prairie Dog Gallbladder

Gallbladder Na⁺ absorption is linked to gallstone formation in prairie dogs. We previously reported Na⁺/H⁺ exchanger (NHE1-3) expression in native gallbladder tissues. Here we report the functional characterization of NHE1, NHE2 and NHE3 in primary cultures of prairie dog gallbladder epithelial cells (GBECs). Immunohistochemical studies showed that GBECs grown to confluency are homogeneous epithelial cells of gastrointestinal origin. Electron microscopic analysis of GBECs demonstrated that the cells form polarized monolayers characterized by tight junctions and apical microvilli. GBECs grown on Snapwells exhibited polarity and developed transepithelial short-circuit current, I_sc, (11.6 ± 0.5 µA · cm^–2), potential differences, V_t (2.1 ± 0.2 mV), and resistance, R_t (169 ± 12 · cm²). NHE activity in GBECs assessed by measuring dimethylamiloride-inhibitable ²²Na⁺ uptake under a H⁺ gradient was the same whether grown on permeable Snapwells or plastic wells. The basal rate of ²²Na⁺ uptake was 21.4 ± 1.3 nmol · mg prot^–1 · min^–1, of which 9.5 ± 0.7 (~45%) was mediated through apically-restricted NHE. Selective inhibition with HOE-694 revealed that NHE1, NHE2 and NHE3 accounted for ~6%, ~66% and ~28% of GBECs total NHE activity, respectively. GBECs exhibited saturable NHE kinetics (V_max 9.2 ± 0.3 nmol · mg prot^–1 · min^–1; K_m 11.4 ± 1.4 mM Na⁺). Expression of NHE1, NHE2 and NHE3 mRNAs was confirmed by RT-PCR analysis. These results demonstrate that the primary cultures of GBECs exhibit Na⁺ transport characteristics similar to native gallbladder tissues, suggesting that these cells can be used as a tool for studying the mechanisms of gallbladder ion transport both under physiologic conditions and during gallstone formation.     

The effect of carbonic anhydrase inhibitors on secretion by the parotid and mandibular glands of red kangaroos Macropus rufus

Summary The effects of carbonic anhydrase inhibitors on secretion by macropodine parotid and mandibular glands were investigated using anaesthetized red kangaroos. In the parotid gland, acetazolamide (500 mol·l^-1) reduced a stable acetylcholine-evoked, half-maximal flow rate of 2.02±0.034 to 0.27±0.023 ml·min^-1 (87% reduction). Concurrently, salivary bicarbonate concentration and secretion fell (129.4±1.46 to 80.9±1.63 mmol·l^-1 and 264.8±7.96 to 22.3±2.30 mol·min^-1, respectively), phosphate and chloride concentrations rose (14.0±0.79 to 27.6±0.85 mmol·l^-1 and 5.6±0.25 to 27.5±1.32 mmol·l^-1, respectively), sodium concentration and osmolality were unaltered, and potassium concentration fell (8.8±0.33 to 6.4±0.29 mmol·l^-1). High-rate cholinergic stimulation during acetazolamide blockade was unable to increase salivary flow beyond 11±0.9% of that for equivalent unblocked control stimulation. However, superimposition of isoprenaline infusion on the acetylcholine stimulation caused a three-fold increase in the blocked flow rate. These treatments were accompanied by small increases in salivary phosphate and chloride concentrations but not bicarbonate concentration. Methazolamide infusion caused similar changes in parotid secretion. In the mandibular gland, acetazolamide infusion had no effect on salivary flow rate during either low- or high-level acetylcholine stimulation. Acetazolamide caused no alterrations in salivary electrolyte secretion at low flow rates, but curtailed the rise in bicarbonate concentration associated with high-level acetylcholine stimulation. Acetazolamide administration did not affect the increase in salivary flow rate associated with isoprenaline infusion, but did block the concomitant increase in bicarbonate concentration and secretion substantially. It was concluded that neither cholinergic nor adrenergic stimulation of mandibular fluid secretion depends on secretion of bicarbonate derived from catalysed hydration of CO₂, but a substantial proportion of the increase in bicarbonate secretion during isoprenaline administration, which is probably ductal in origin, is so dependent. In contrast to other salivary glands, including the ovine parotid, fluid secretion by the kangaroo parotid gland during cholinergic stimulation is largely dependent (about 90%) on secretion of bicarbonate derived from hydration of CO₂ catalysed by glandular carbonic anhydrase. Fluid secretion during adrenergic stimulation is not bicarbonate dependent.Abbreviations b.w. body weight - PAH p-aminohippurate - PCO₂ partial pressure carbon dioxide - PCO₂ partial pressure of oxygen     

The sodium balance of the euryhaline marine loggerhead turtle,Caretta caretta

Summary The efflux of sodium ions from the loggerhead turtle,Caretta caretta, was investigated when animals were acclimated to seawater, freshwater, and during the acclimation period after transfer from seawater to freshwater. The rate of sodium loss in animals acclimated to seawater was found to be 3 M Na · g^–1 · hr^–1. Cannulation of the cloaca showed that only 5% of the gross efflux of sodium was via the cloaca and it was calculated that 60% of the gross efflux was via nasal gland secretion and 35 % via integumentary diffusional loss. Cannulation experiments indicated that a significant amount of sodium may enter the body via the cloaca. Transfer of animals from seawater to freshwater resulted in a decline of sodium efflux by 90–99 % within 6 hr, and a further decline to only approximately 0 01 M Na · g^–1· hr^–1 after 1–2 days in freshwater. Cannulation experiments of individuals acclimated to freshwater indicated that the role of the cloaca in sodium loss in freshwater was minimal. It was calculated that sodium loss in freshwater is so small that survival in freshwater for at least 20 days is possible without active extraction of sodium from the medium. No evidence could be found for active uptake of sodium ions from freshwater baths byCaretta.This research was supported by NSF grant GB 16839.     

Kinetic studies on the stimulation of Na+−H+ exchange activity in renal brush border membranes isolated from thyroid hormone-treated rats

Summary Na⁺–H⁺ exchange activity in renal brush border membrane vesicles isolated from hyperthyroid rats was increased. When examined as a function of [Na⁺], treatment altered the initial rate of Na⁺ uptake by increasingV _m (hyperthyroid, 18.9±1.1 nmol Na⁺ · mg^–1 · 2 sec^–1; normal, 8.9±0.3 nmol Na⁺ · mg^–1 · 2 sec^–1), and not the apparent affinityK _Na ⁺ (hyperthyroid, 7.3±1.7mm; normal, 6.5±0.9mm). When examined as a function of [H⁺] and at a subsaturating [Na⁺] (1mm), hyperthyroidism resulted in the proportional increase in Na⁺ uptake at every intravesicular pH measured. A positive cooperative effect on Na⁺ uptake was found with increased intravesicular acidity in vesicles from both normal and hyperthyroid rats. When the data were analyzed by the Hill equation, it was found that hyperthyroidism did not change then (hyperthyroid, 1.2±0.06; normal, 1.2±0.07) or the [H⁺]_0.5 (hyperthyroid, 0.39±0.08 m; normal, 0.44±0.07 m) but increased the apparentV _m (hyperthyroid, 1.68±0.14 nmol Na⁺ · mg^–1 · 2 sec^–1; normal 0.96±0.10 nmol Na⁺ · mg^–1 · 2 sec^–1). The uptake of Na⁺ in exchange for H⁺ in membrane vesicles from normal and hyperthyroid animals was not influenced by membrane potential. H⁺ translocation or debinding was rate limiting for Na⁺–H⁺ exchange since Na⁺–Na⁺ exchange activity was greater than Na⁺–H⁺ exchange activity. Hyperthyroidism caused a proportional increase and hypothyroidism caused a proportional decrease in Na⁺–Na⁺ and Na⁺–H⁺ exchange. We conclude that hyperthyroidism leads to either an increase in the number of functional exchangers in the membrane or exactly proportional increases in the rate-limiting steps for Na⁺–Na⁺ and Na⁺–H⁺ exchange activity.     

Secretion by the mandibular gland of the red kangaroo (Macropus rufus) during isoprenaline infusion

Summary Intracarotid infusion of isoprenaline, either alone or in combination with acetylcholine infusion was used to stimulate salivation by the mandibular glands of anaesthetized red kangaroos. Isoprenaline alone (0.20–1.25 mol·kg^–1·min^–1) elicited flow rates ranging from 0.014 to 0.239 ml·min^–1 (1.21–28.1 l·g gland^–1·min^–1). Salivary concentrations of sodium, chloride, phosphate and urea were negatively correlated with flow, whereas potassium, calcium, magnesium, hydrogen ion, bicarbonate, protein, and osmolality were poorly correlated with flow. Relative to cholinergic saliva produced at equivalent flow rates, isoprenaline-evoked saliva had higher osmolality, saliva/plasma urea ratios and concentrations of protein, potassium, magnesium, bicarbonate, and phosphate, but lower sodium, chloride and hydrogen ion levels. At a steady salivary flow (0.5 ml·min^–1), superimposition of isoprenaline infusion (0.15 mol·kg^–1·min^–1) on a pre-existing acetylcholine infusion reduced the rate of acetylcholine administration necessary to maintain flow, increased osmolality and the concentrations of protein, urea, potassium, calcium, magnesium, bicarbonate and phosphate and decreased sodium, chloride and hydrogen ion in the saliva. Salivary amylase activity was low and highly variable and the amylase activity/protein ratio fell substantially during isoprenaline stimulation. These results support the conclusion that the enzyme is of extrinsic origin. The response of the kangaroo mandibular gland to isoprenaline stimulation was very similar to that reported for rat mandibular gland, suggesting that the same ion transport phenomena underlie mandibular secretion in both species and probably in therian mammals generally.