mM For Cl Research Articles

Assessing water demand with remote sensing for two coriander varieties

The use of remote sensing to determine water needs has been successfully applied by several authors to different crops, maintaining, as an important basis, the relationship between the normalized difference vegetation index (NDVI) and biophysical variables, such as the fraction of coverage (fc) and the basal crop coefficient (Kcb). Therefore, this study quantified the water needs of two varieties of coriander (UNAPAL Laurena CL and UNAPAL Precoso CP) based on the response of fc and Kcb, using remote sensors and a water balance according to the FAO-56 methodology. A Campbell Scientific meteorological station, a commercial digital camera and a portable spectro radiometer were used to obtain information on the environmental conditions and the crop. By means of remote sensing associated with a water balance, it was found that the water demand was 156 mm for CL and 151 mm for CP until the foliage harvest (41 d after sowing); additionally, the initial Kcb was 0.14, the mean Kcb was 1.16 (approximately) and the final Kcb was 0.71 (approximately).

Novel light-guide-PMT geometries to reduce dead edges of a scintillation camera

Anger cameras based on monolithic NaI scintillators read out by an array of PMTs are predominant in planar gamma imaging and SPECT. However, position estimation of gamma interactions is usually severely degraded near the edges of the scintillator which can be extremely undesirable for applications like breast imaging. Here we propose a relatively cost-effective solution based on the use of scintillators with absorptive edges with an unconventional light-guide-PMT layout employing a maximum likelihood positioning algorithm. The basic design on which we aim to improve consists of a monolithic NaI(Tl) scintillator read out by 3 × 5 square PMTs (conventional layout, CL) that could be suitable for molecular breast imaging. To better detect gamma interactions near the crystal’s critical edge, we tried different set-ups: we replaced the 5 large PMTs near the edge by 11 smaller PMTs (small-sensor layout, SSL); we emulated rectangular PMTs along the critical edge by inserting a row of 5 rectangular light-guides that direct the light toward square PMTs placed behind (shifted layout, SL); we inserted rectangular light-guides alternatingly, such that the PMTs are in an interlocking pattern (alternating shifted layout, ASL). The performance of our designs was tested with Monte Carlo simulations. Results showed that SSL, SL, and ASL gave better spatial resolution near the critical edge than CL (3.4, 3.6, and 4.1 mm near the edge compared with 5.3 mm for CL), and thus resulted in a larger usable detector area. To conclude, for applications where small dead edges are crucial, our designs may be cost-effective solutions.

Population dynamics of a freshwater amphipod from South America (Crustacea, Amphipoda, Hyalellidae)

This study aimed to characterize the population dynamics of Hyalella bonariensis Bond-Buckup, Araujo & Santos, 2008 from headwater spring in a rural area of state of Rio Grande do Sul, Brazil. Four samples were collected in August (winter) and October (spring) of 2012 and January (summer) and April (autumn) of 2013. Ovigerous females and precopula pairs were separated from other individual in the field. A total of 5,266 specimens were sampled, being 1,878 males, 2,073 females (including 240 ovigerous females) and 1,315 juveniles. The frequency distribution of size classes (measured as cephalothorax length, CL) was polymodal and bimodal in males and females, respectively. Males reach larger size than females. Sexual maturity of males and females was estimated at 0.40 and 0.38 mm for CL, respectively. Total sex ratio favored females, and these were more frequent in intermediate size classes, while males were more frequent in larger size classes. Ovigerous females and couples were found in four seasons, but both were more abundant in winter. Juveniles were also found in all seasons, being more frequent in winter and spring. These results showed that this H. bonariensis population has similar dynamics to other species of Hyalella Smith, 1874 from Brazil, but present variations when compared to other freshwater amphipods.

Li fluxes reveal presence of Na‐Cl Cotransport (NCC) in Human Lens Epithelial Cells (LECs)

Na‐K‐2Cl cotransport (NKCC) moves RbCl/KCl+NaCl/LiCl and, in FHL124 LECs, is not inhibited by hypotonicity. Here, we studied Li fluxes through NKCC in hyposmotic high K media and Rb fluxes in hyposmotic high Na to understand this effect. Li and Rb uptake were measured by atomic absorption spectrophotometry to determine LiKCC or NRbCC through bumetanide‐sensitive (BS) and Cl‐dependent fluxes in Na/K or Na‐free/K‐free (N‐methyl‐D‐glucamine) and Cl or Cl‐free (sulfamate or nitrate) media ± ouabain (O) or [O+B], ± thiazides, at varying Rb, Li or Cl molar fractions (MF). Main findings are: Li influx in isosmotic (300 mOsM) K was 1/3 of Rb influx in Na, and was mainly mediated by a Cl‐dependent Li flux (LiKCC). In 200 mOsM high K, LiKCC was abolished, whereas in high Na, NRbCC remained active. LiKCC and BS‐Li influx (BS‐LiKCC) showed bell‐shape curves for 0.1–1 Li MF, maxing at ∼0.6 MF. BS‐LiKCC was ¼ of LiKCC. The difference, i.e. the B‐insensitive/Cl‐dependent‐Li influx, saturated with Li and Cl MFs. Kms for Li were 11 with and 7–8 mM without external K, respectively; and ∼40 mM for Cl. Neither furosemide (<100 μM) nor thiazide derivatives inhibited LiCC. RTPCR, Western blots and immunochemistry revealed NCC RNA and protein, with NCC‐specific antibodies, besides the expected NKCC1. Thus, a K‐independent/Cl‐dependent Li flux (LiCC) revealed the presence of NCC in FHL124 LECs.

Lithium Fluxes Indicate Presence of Na-Cl Cotransport (NCC) in Human Lens Epithelial Cells

During regulatory volume decrease (RVD) of human lens epithelial cells (hLECs) by clotrimazole (CTZ)–sensitive K fluxes, Na-K-2Cl cotransport (NKCC) remains active and K-Cl cotransport (KCC) inactive. To determine whether such an abnormal behavior was caused by RVD-induced cell shrinkage, NKCC was measured in the presence of either CTZ or in high K media to prevent RVD. NKCC transports RbCl + NaCl, and LiCl + KCl; thus ouabain-insensitive, bumetanide-sensitive (BS) or Cl-dependent (ClD) Rb and Li fluxes were determined in hyposmotic high NaCl media with CTZ, or in high KCl media alone, or with sulfamate (Sf) or nitrate as Cl replacement at varying Rb, Li or Cl mol fractions (MF). Unexpectedly, NKCC was inhibited by 80% with CTZ (IC₅₀ = 31 µM). In isosmotic (300 mOsM) K, Li influx was ñ 1/3 of Rb influx in Na, 50% lower in Sf, and bumetanide-insensitive (BI). In hypotonic (200 mOsM) K, only the ClD but not BS Li fluxes were detected. At Li MFs from 0.1-1, Li fluxes fitted a bell-shaped curve maxing at ñ0.6 Li MF, with the BS fluxes equaling ñ¼ of the ClD-Li influx. The difference, i.e. the BI/ClD Li influx, saturated with increasing Li and Cl MFs, with K_ms for Li of 11 with, and 7 mM without K, and of ñ46 mM for Cl. Inhibition of this K-independent Li influx by thiazides was weak whilst furosemide (<100 µM) was ineffective. Reverse transcription polymerase chain reaction and Western blots verified presence of both NKCC1 and Na-Cl cotransport (NCC). In conclusion, in hyposmotic high K media, which prevents CTZ-sensitive K flux-mediated RVD in hLECs, NKCC1, though molecularly expressed, was functionally silent. However, a K-independent and moderately thiazide-sensitive ClD-Li flux, i.e. LiCC, likely occurring through NCC was detected operationally and molecularly.

Green fluorescent protein-based halide indicators with improved chloride and iodide affinities

The green fluorescent protein YFP-H148Q is sensitive to halides by a mechanism involving halide binding and a shift in p K a. However, a limitation of YFP-H148Q is its low halide sensitivity, with K d>100 mM for Cl −. Indicators with improved sensitivities are needed for cell transport studies, particularly in drug discovery by high-throughput screening, and for measurement of Cl − concentration in subcellular organelles. YFP-H148Q libraries were generated in which pairs of residues in the vicinity of the halide binding site were randomly mutated. An automated procedure was developed to screen bacterial colonies for improved halide sensitivity. Analysis of 1536 clones revealed improved anion sensitivities with K d down to 2 mM for I − (I152L), 40 mM for Cl − (V163S), and 10 mM for NO 3 − (I152L). The anion-sensitive mechanism of these indicators was established and their utility in cells was demonstrated using transfected cells expressing the cystic fibrosis transmembrane conductance regulator chloride channel.

Comparative duodenal, jejunal and ileal responses to luminal saline load

Intestinal ionic exchanges were studied in rat duodenal, jejunal and ileal ligated loops in response to different luminal saline loads: NaCl concentration varied from 150-0 mM, solutions being made isoosmotic with mannitol. The contact delay was 60 min. An exponential relationship was found between water, Na and Cl movements and the initial saline concentration. Maximal absorption was obtained with 150 mM NaCl, and was significantly higher in the duodenum than in the jejunum and ileum. The NaCl concentration for which water, Na, and Cl movements were null was approximately 70 mM NaCl in the duodenum and jejunum, 41 mM for Na and 18 mM for Cl in the ileum. The water efflux induced by the 0-mM NaCl test solution was maximal in the duodenum (1.5 +/- 0.2 ml/h) and decreased in the jejunum (0.8 +/- 0.1 ml/h) and ileum (0.3 +/- 0.1 ml/h) as did sodium, chloride and non-chloride anion efflux. These data support the functional heterogeneity of the small intestine regulating the water and ion exchange in response to luminal saline load, the main difference being connected with the efflux capacity of the mucosa, decreasing from the duodenum to the jejunum and ileum.

Characteristics of the anion transport system in sea turtle erythrocytes

Erythrocytes of Kemp's ridley sea turtle (Lepidochelys kempi) contain a 100- to 105-kDa protein that is reactive with a monoclonal antibody to the membrane domain of human erythrocyte band 3. Based on inhibition of membrane HCO(3-)-Cl- exchange with 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), sea turtle erythrocytes were found to contain 4 x 10(6) copies of band 3 per cell. Unidirectional HCO3- transfer, specifically HCO3- out----in-Cl-in----out exchange, where subscript in----out represents transfer from inside to outside and subscript out----in represents transfer from outside to inside, was characterized by a maximal exchange rate of 1.0-1.1 nmol.cm-2.s-1, substrate affinity coefficients of 0.1-0.2 mM for HCO3- and 1.6 mM for Cl-, and an apparent inhibition constant for SITS of 0.6-1.0 microM (10 degrees C, pH 7.6). Under physiological conditions (30 degrees C, pH 7.4), the rate of net HCO3- transfer (i.e., the difference between HCO3- in----out-Cl-out----in and HCO3- out----in-Cl-in----out) was 1.13 nmol. cm-2.s-1 for cells subjected to a 5-mM decrement in CO2 content. This yields a rate coefficient for the "physiological" anion shift in sea turtle blood of 1.7 s-1, indicating that the anion shift may require 2.6 s to reach 99% completion in vivo. The erythrocyte anion shift appears to be a potential rate-limiting step for capillary CO2 exchange in these turtles.

Functional characterization of alpha- and beta-intercalated cell types in rabbit cortical collecting duct

Single-cell electrical measurements and spectrophotometric determinations of intracellular pH were used to determine unique features of alpha- and beta-intercalated cells (alpha-IC, beta-IC) in in vitro perfused rabbit cortical collecting ducts (CCD). pHi rose in alpha-IC and fell in beta-IC after bath Cl- removal. Luminal Cl- removal did not change pHi of alpha-IC, but pHi of beta-IC rose by 0.36 +/- 0.01 pH units. Cl- concentration-dependent recovery of beta-IC pHi revealed a Cl- Km of 18.7 mM for the luminal Cl(-) -HCO3- exchanger. Measurements of basolateral membrane voltage (Vbl) also showed two IC cell types. Removal of luminal Cl- did not change Vbl in alpha-IC, whereas Vbl hyperpolarized by a mean of 73.2 +/- 3.5 mV in beta-IC. Reducing bath Cl- depolarized both alpha- and beta-IC Vbl. In alpha-IC a large repolarization of 39.8 +/- 5.2 mV followed acute depolarization after bath Cl- removal. Reducing bath HCO3- (constant CO2) had little effect on beta-IC Vbl, whereas alpha-IC Vbl depolarized by 5.2 +/- 0.7 mV. Reducing luminal HCO3- in the absence of luminal Cl- produced a 17.6 +/- 1.8 mV depolarization in beta-IC. This change was independent of luminal Na+ and was not blocked by luminal 10(-4) M 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). In beta-IC, Vbl was not altered by either bath or lumen DIDS in the presence of luminal Cl-. However, when luminal Cl- was removed, luminal DIDS reversibly depolarized Vbl by 9.6 +/- 2.9 mV.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition by inorganic anions of glutathione S-transferases from insect and mammalian sources

Glutathione S-transferases 1-1, 3-3, 3-4 and 4-4 from rat liver and the major glutathione S-transferase from the wax moth (Galleria mellonella) are all inhibited by several simple inorganic anions. For each of 3-3, 3-4 and the insect enzyme, the order of inhibitory potency was ClO4- greater than or equal to SCN- greater than I- greater than NO3- greater than Br-. A more limited range of anions was tested on the isoenzymes 1-1 and 4-4, but the same trend was apparent. Values for Ki ranged from about 200 mM for Cl- to 6 mM for SCN- in the case of the insect enzyme and from 50 mM for Br- to 0.3 mM for SCN- for the rat isoform 3-3. Acetate, F-, SO4(2-) and PO4(3-) were not found to have significant inhibitory properties. The mode of inhibition was characterized as non-competitive in the case of the insect enzyme and rat transferase 1-1, whereas the mode of inhibition was partially non-competitive in the case of the rat isoforms 3-3, 3-4 and 4-4.

Osmotic and Ionic Regulation in the Aquatic CaecilianTyphlonectes compressicaudaand the Terrestrial CaecilianIchthyophis kohtaoensis

The terrestrial caecilian Ichthyophis kohtaoensis and the aquatic caecilian Typhlonectes compressicauda were comparedfor osmotic and ionic regulatory capabilities. The aquatic form has a lower rate of osmotic water uptake (0.09 vs. 0.24 μL/10 g/h), suggesting a lower osmotic permeability. The terrestrial Ichthyophis reduces its osmotic flux to 0.17μL/10 g/h) after 3 d of acclimation to water. The aquatic Typhlonectes produces a more dilute urine and has a lower glomerular filtration rate (GFR) (0.139 mL/10 g/h) and fractional water reabsorption (9.5%) than the terrestrial Ichthyophis (0.267 mL/10 g/h, 56.9%). Ichthyophis increases urine flow by reducing fractional water reabsorption to 32.3% when placed in water; GFR does not increase significantly. Analyses of net and unidirectional ionic fluxes and transcutaneous potential differences showed that both species actively transport Na⁺ and Cl⁻ across their body surfaces. The aquatic Typhlonectes has an apparent Km of 0.15 rmM for Na⁺ and 0.34 mM for Cl⁻, while the terrestrial Ichthyophis has an apparent Km of 0.65 for Na⁺ and 0.32 for Cl⁻. The apparent Km values did not differ significantly. The Vmax (capacity) was 1.97μeq/10 g/h for Na⁺ and 1.16μeq/10 g/h for Cl⁻ in Typhlonectes; the corresponding values for Ichthyophis were 3.00 and 2.50. The values did not difer significantly for the two species.

Characterization of the macula densa stimulus for renin secretion

These studies utilize the isolated perfused rabbit juxtaglomerular apparatus (JGA) to study the macula densa signal for renin secretion in the absence of the confounding influences of intravascular pressure and renal nerve activity. In the first experimental series, JGAs were perfused alternately with high- and low-NaCl solutions to determine the reversibility of the renin response to changes in NaCl concentration. Compared with high-NaCl controls, perfusion with a low-NaCl solution resulted in a fivefold increase in renin secretion rate (RSR) [2.1-10.0 nano-Goldblatt hog units (nGU)/min], and this response was largely reversible. When the solutions were presented in the reverse order, a similar inhibition by high NaCl was observed. In the second series, JGAs were perfused with high-, medium-, and low-NaCl solutions to determine the sensitive range of the renin response to NaCl concentration changes. The full renin response (3.2-16.6 nGU/min), similar in magnitude to that seen in series 1, was found to occur between 80 and 24 mM for Na+ and 61 and 7 mM for Cl-. In the third series, the NaCl concentration and flow rate of the perfusate were altered independently to separate the effects of flow rate, NaCl delivery, and NaCl concentration on RSR. Although a decrease in perfusate flow rate slightly increased RSR (3.4-8.1 nGU/min), a comparable decrease in NaCl concentration resulted in a much higher RSR (26.3 nGU/min). We conclude that in this preparation 1) RSR responds equally to both increases and decreases in macula densa NaCl concentration, and these changes are rapid and largely reversible, 2) the full renin response occurs within the concentration range normally occurring at the macula densa, i.e., below 80 mM Na+ and 61 mM Cl-, and 3) RSR responds with a larger change to alterations in NaCl concentration than in NaCl delivery or fluid flow rate.

The chloride-activated peroxidation of catechol as a mechanistic probe of chloroperoxidase reactions: Competitive activation as evidence for a catalytic chloride binding site on compound I

Abstract Chloride ion (Cl-) effects on chloroperoxidase (CPO)-catalyzed peroxidation of catechol were used to probe the involvement of Cl- in CPO reactions. High concentrations of Cl- inhibit catechol peroxidation by competing with hydrogen peroxide (KI = 370 mM). However, at lower concentrations, Cl- is a linear competitive activator versus catechol (KDC = 35 mM). Addition of good halogenation substrates to the peroxidatic reaction mixture converts Cl- from a competitive activator to a competitive inhibitor. The KI (10 mM) for this halogenation substrate promoted Cl- inhibition is equivalent to the KM (11 mM) for Cl- in CPO-catalyzed halogenation reactions. During this inhibition, the halogenation substrate is consumed and, at the point where its consumption is complete, Cl- again becomes an activator. Also, at 2.0 mM hydrogen peroxide, CPOs chlorination reaction and its Cl- -activated peroxidatic reaction have similar apparent kcat values. All data are consistent with a mechanism in which Cl- competes with catechol for binding to CPO Compound I. Catechol binding initiates the Cl- -independent path, in which Compound I acts as the oxidizing agent for catechol. When Cl- binds to Compound I, it reacts to yield the enzymatic chlorinating intermediate which is responsible for either the oxidation of catechol in the Cl- -dependent path or the chlorination of substrates in the halogenation pathway. Cl- activation of the peroxidatic reaction is due to a shift from the Cl- -independent pathway to the Cl- -dependent process. The mechanism is unique in that exclusion of the substrate from its primary binding site leads to an increase in the catalytic efficiency of the reaction. This catechol-Cl- system also offers further potential for probing the specificity and chemistry of the key enzymatic intermediates in haloperoxidase-catalyzed reactions.

Regulation of rabbit medullary collecting duct cell pH by basolateral Na+/H+ and Cl-/base exchange.

The collecting duct of the inner stripe outer medulla (OMCDi) is a major site of distal nephron acidification. Using the pH sensitive fluorescent dye 2'-7'-bis(carboxyethyl)-5,6,-carboxyfluorescein (BCECF) and quantitative spectrofluorometry to measure intracellular pH in isolated perfused OMCDi, we have characterized basolateral transport processes responsible for regulation of intracellular pH. Experiments suggesting the existence of basolateral Cl-/base exchange were performed. In HCO3- containing buffers, bath Cl- replacement resulted in reversible alkalinization of the OMCDi from 7.22 +/- 0.05 to 7.57 +/- 0.12. Similarly 0.1 mM bath 4',4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS) alkalinized the OMCDi from 7.14 +/- 0.09 to 7.34 +/- 0.09 and blocked further alkalinization by bath Cl- removal (delta = + 0.02 pH units). The concentration dependence kinetics of Cl-/base exchange revealed a K1/2 of 10 mM for external Cl- with a Vmax of 0.50 pH U/min. Experiments suggesting the existence of basolateral Na+/H+ exchange were also performed. Replacement of bath Na+ by tetramethylammonium resulted in reversible cell acidification (7.14 +/- 0.09 to 6.85 +/- 0.1). Tubules that were acidified by a brief exposure to NH4Cl displayed recovery of cell pH back to baseline at a rate that was highly dependent on bath Na+ concentration. Half maximal recovery rate was achieved at 7 mM bath Na+ and Vmax was 0.605 pH U/min. The Na+-dependent rate of cell pH recovery after acidification was blocked by 0.2 mM bath amiloride. These results suggest that intracellular pH in the OMCDi is regulated by parallel basolateral Na+/H+ exchange and Cl-/base exchange.

Energy‐dispersive X‐ray microanalysis of air‐dried microdroplets containing a macromolecular solute

In the preparation of microdroplets of biological fluids for X-ray microanalysis, we have found that incorporation of a macromolecular solute, dextran, to a final concentration of 1.5-2.5% retards crystal formation and produces sufficiently uniform deposits on thin films to allow droplets to be analysed without prior freeze-drying. Analyses have been carried out at 20 kV in a scanning electron microscope, using energy-dispersive spectrometry. Absorption of Na X-rays by the added solute can be significant but its effect is minimized by preparing droplets as thin as possible, and by using standards of similar composition. The minimum detectable concentrations are increased because of the extra background contribution, and for a single determination are about 6 mM for Na and 2 mM for Cl and K. These concentrations can be further reduced by measuring replicates. The reproducibility of analysis is significantly improved (to less than 5% for Na and K) over the use of calibration curves by calculating the element concentrations from a known element in the sample, chlorine. Under our analytical conditions loss of Cl did not occur. This method requires that the Cl is measured separately by microcoulometry, but eliminates the need for a range of standard droplets on the grid, and determination of the unknowns is then independent of droplet volume, beam current, counting time and magnification. We have compared, with biological samples, the results from using Cl as an internal standard with those obtained using an added standard element, cobalt. The reproducibility using Cl was approximately two-times better than that obtained with Co, probably because of unavoidable volumetric errors when the Co is pipetted separately.

Kinetic properties of the KCl transport at the secreting apical membrane of the oxyntic cell

We have previously reported marked structural and functional differencces between (H+/K+)-ATPase-rich vesicles obtained from resting fundic gastric mucosa and those obtained from secreting tissue. The functional differences seem to arise from activation of a permeability pathway for K+ salts in the vesicles originating from stimulated tissue. By providing the intravesicular K+ needed for the ATPase-mediated H+/K+ exchange, this pathway facilitates formation of large pH gradients (J.M. Wolosin & J.G. Forte.FEBS Lett.125:208–212, 1981). We have now expanded our observations on the characteristics of the K-salt transport. The dependence of intravesicular acidification on the concentration of various anions or of K+ was monitored using the rate of fluorescence quenching of the pH probe, acridine orange. Rates of acid accumulation followed the series I−>Br−>Cl−>NO3−>CH3SO3−>isethionate>gluconate. The effects of exogenous ionophores on pH gradients suggested that K+-salt transport is electroneutral. On the basis of the known properties of the H+/K+ pump, we devised a method by which the rates of unidirectional K+ salt influx can be monitored via the resulting rates of H+ accumulation. Accordingly, the dependence of these rates on the concentration of K+ and Cl− was studied and subjected to a kinetic analysis. It was found that the dependence follows that expected for an electroneutral K+ and Cl− transport system with apparent Michaelis constants equal to 15±2mm for K+ and 46±5mm for Cl−. It is concluded that intravesicular acid accumulation, believed to represent HCl secretion in whole tissue, results from the combined action of a KCl symport and the well known (H++K+)-ATPase. Though the calculated maximal transport capacity of the symport exceeds by several times the maximal transport capacity of the pump, at physiological concentrations of the ions both transport systems are expected to transport K+ in opposite directions at similar rates. Implications of these observations to HCl secretion by intact tissue are discussed.

Coupled NaCl entry into Necturus gallbladder epithelial cells.

NaCl entry into Necturus maculosus gallbladder epithelial cells was studied by determination of the rate of fluid movement into the cell when the Na+-K+-ATPase was inhibited by 10(-4) M ouabain in the serosal bathing solution. The cell swelling was due to continuing entrance of NaCl into the cell across the apical membrane, which increased the solute content of the cell; the resultant rise in cell osmolality induced water flow and cell swelling. The rate of swelling was 4.3% of the cell volume per minute, equivalent to a volume flow across the apical membrane of 1.44 x 10(-6) cm/s, similar in magnitude to the normal rate of fluid absorption by the gallbladder. We determined the mechanism of NaCl entry by varying the ionic composition of the mucosal bath; when most of the mucosal Na+ or Cl- was replaced, cell volume did not increase during pump inhibition. The rate of NaCl entry was a saturable function of Na+ or Cl- in the mucosal bathing solution with K1/2 values of 26.6 mM for Na+ and 19.5 mM for Cl-. The mode of NaCl entry was probably not the parallel operation of Na+-H+ and Cl(-)-HCO-3 exchangers because of the lack of effect of bicarbonate removal or of the inhibitors amiloride and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid. NaCl entry was reversibly inhibited by bumetanide in the mucosal bathing solution. Transepithelial NaCl and water absorption is the result of the coupled, carrier-mediated movement of NaCl into the cell across the apical membrane and the active extrusion of Na+ by the Na+-K+-ATPase in the basolateral membrane.

Comparison of digestive ?-amylases from two species of spiders (Tegenaria atrica andCupiennius salei)

1. Polyacrylamide gel electrophoreses of digestive fluids reveal the presence of two to three amylases (molecular mass about 58,000 Dalton) in the webbuilding spiderTegenaria atrica, and three enzymes (molecular mass about 63,000 Dalton) in the hunting spiderCupiennius salei. In either case, one amylase is largely predominant. 2. The dominating enzyme of each spider can be classified as an α-amylase (E.C.3.2.1.1) by the binding of and activation by chloride. TheK D is 1.6 mM for Cl− at 30°C. Chloride concentrations below the optimum (<10 mM) have neither effect on pH-optimum nor onK m , but lowerV max . 3. Tegenaria andCupiennius amylases show identical behaviour with respect to all kinetic parameters studied, except for temperature dependence. 4. The Michaelis constants are around 4.5 mg/ml for soluble starch and for glycogen as substrates. Values forV max are slightly higher with soluble starch than with glycogen. Total activity is in the order of 50 to 75 nkat reducing groups per mg of protein of the unfractionated digestive fluid. pH-optima are near the pH of the digestive fluid (pH 7.4). 5. Discontinuous Arrhenius plots show that both amylases exist in at least two temperature-dependent conformational states. Activation enthalpy values are about 30% lower inTegenaria than inCupiennius amylases. 6. The amylases appear to be calcium-dependent enzymes which are non-competitively inhibited by Hg++ and Cu++. 7. Other anions activate the amylases similar to chloride, the effect decreasing with increasing iondiameter. Fluoride does neither activate nor inhibit.