Tissue culture of rat adult decapsulated adrenal glands

Summary A method of cultivation involving both repeated trypsinisations (at room temperature) and explantation of the tissue fragments on polythene discs has been shown to be apt to the growth in vitro of rat adult decapsulated adreno-cortical tissue. This is the first time that the successful cultivation of such a tissue is reported. The technique and its applications are discussed.The effects of _1–24 corticotrophin (ACTH_1–24) on the rat adult adrenal cultures have been examined by both electron microscopy and autoradiography. Zona fasciculata and reticularis cells grown in the absence of ACTH for long terms (15–16 days) survive and proliferate as dedifferentiated elements. If ACTH_1–24 is added to the cultures, adrenocortical cells will, within 2 days, simultaneously increase their proliferation rate and differentiate. After 7 days of treatment, cortical cells exhibit not only fully differentiated but even hypertrophic morphologic features. Significant stimulations of adrenal DNA, RNA and gross protein synthesis have been found to take place at different times after the starting of the ACTH_1–24 treatment. These data are discussed in relation to the findings previously reported in literature.Rat adult adrenal gland tissue cultures are proposed as a non-previously available tool for investigations into the physiopathology of the adrenal cells to be carried out in a carefully controlled environment.A Preliminary report on part of this material was given at the annual meeting of the Société Française de Microscopie Elécronique, Nantes, May 1972.Authors wish to thank Drs. P. G. Andreis and A. S. Belloni for their skilful assistance in the autoradiographic experiments. Thanks are also due to Mr. G. Gottardo for his excellent technical help in electron microscopic work. This work was partly supported by a contract with the CNR-Italy (No 69.0172/115.3439).     

Interaction of thymic peptide thymosinα 1 with vasoactive intestinal peptide (VIP) receptors

Thymic peptide thymosin ₁ (10^–9 to 3 x 1010^–7 M) is shown to inhibit the specific binding of [¹²⁵I]VIP to rat blood mononuclear cells and liver plasma membranes. Thymosin ₁ was 160 and 6250 times less potent that VIP at inhibiting [¹²⁵I]VIP binding to blood mononuclear cells and liver plasma membranes, respectively. Thymosin ₁ (10^–10 to 1010^–7 M) was weak in stimulating adenylate cyclase activity. Its efficacy is about 25 % and 27 % that of native VIP in blood mononuclear cells and liver plasma membranes, respectively. Thymosin ₁ may behave as a partial VIP agonist in rat.Abbreviations GRF growth hormone releasing factor - PHI porcine intestinal peptide having N-terminal histidine and C-terminal isoleucine amide - GIP gastric inhibitory polypeptide - VIP vasoactive intestinal peptide     

Immunocytochemical demonstration of proopiomelanocortin- and other opioid-related substances and a CRF-like peptide in the gut of the american cockroach,Periplaneta americana L.

Summary Using the peroxidase-antiperoxidase technique, we showed the presence of peptides which are immunologically resembling mammalian corticotropin releasing hormone (CRF)-, adrenocorticotropic hormone (ACTH)-, -endorphin (-END)-, -melanocyte stimulating hormone (-MSH)-, methionine-enkephaline (met-ENK)- and leucine enkephaline (leu-ENK)- like immunoreactivity in hundreds to thousands of endocrine cells and nerve fibers in the midgut of the American cockroach Periplaneta americana.In the cockroach hindgut no immunoreactive cell bodies could be observed, although nerve fibers were clearly noticed to be recognized by antisera to CRF, ACTH_1–24, ACTH_11–24 and -END.Nothing is exactly known as to the function(s) of the demonstrated materials, but one can speculate that these numerous immunoreactive cells, might have important paracrine and/or endocrine functions in the insect physiology.     

Grazing by a dominant rotifer Conochilus unicornis Rousselet in a mountain lake: in situ measurements with synthetic microspheres

Grazing rates of zooplankton were analysed in the summer of 1999 in Yellow Belly Lake, an oligotrophic system in the Sawtooth Mountains of Idaho (U.S.A.). The colonial rotifer Conochilus unicornis was a dominant species in the epilimnion, with densities reaching 20 colonies l^–1 (ca. 400 ind. l^–1). Clearance rates were measured with an in situ Haney Grazing chamber and synthetic microspheres 5, 9 and 23m in diameter. At epilimnetic temperatures of around 14 °C, mean clearance rates for 5m particles ranged from 30 to 65 l ind.^–1 h ^–1. Clearance rates were 2–9 times higher on the 5m spheres than on the 9 m spheres, and C. unicornis almost never fed on the 23 m spheres. Grazing rates did not change over the diel cycle. Clearance rates declined more than 10-fold as temperatures declined from 14 °C in the epilimnion to 7 °C in the metalimnion. In the epilimnion, grazing by C. unicornis was more important than grazing by crustaceans in the community, at least on particles 9m. The results show the importance of grazing by rotifers in lakes, and the significance of spatial variations that influence grazing rates.     

Interferometeric sensing of β-galactosidase released by recombinant E. coli responding to an endocrine disruptor, tributyltin

A recombinant E. coli ACV1003 releasing -galactosidase by a SOS regulon system when it is exposed to a DNA-damaging compound, has been used to detect endocrine disruptors such as tributyltin (TBT) and triphenyltin (TPT). Maximum response ratio by E. coli ACV 1003 (recA::lacZ) – indicating the maximum ratio of enzyme produced against an environmental toxicant to that produced in the absence of a toxicant – was estimated as 6.3 with 1.0 g TBT ml^–1 at 37 °C, which was considerably higher than those with other strains. Extracellular -galactosidase activity was 51 unit ml^–1, which was 5% of that obtained by the conventional Miller's enzyme assay using solvents. Such a low enzyme activity can be rapidly determined, not by the usual time-consuming and tedious enzyme assay, but by an alternative interferometric biosensor. Heavily-doped porous silicon to apply to an interferometer was fabricated by etching to produce a Fabry–Pérot fringe pattern, which caused the change in the refractive index of the medium including -galactosidase. The change in the effective optical thickness versus -galactosidase activity showed a sigmoid increase up to the concentration of 250 unit -galactosidase ml^–1.     

ACTH,cyclic nucleotides,and brain protein phosphorylation in vitro

Endogenous phosphorylation of proteins from rat brain synaptosomal plasma membranes was studied in vitro. Cyclic AMP (cAMP) markedly stimulated³²P incorporation in three protein bands with molecular weights of 75,000, 57,000, and 54,000, respectively. The effect of the behaviorally active peptide ACTH_1–24 on this endogenous phosphorylation in vitro was studied using peptide concentrations from 10^–10 to 10^–4 M. In a number of protein bands, a biphasic effect of ACTH_1–24 was observed: in concentrations of 10^–4–10^–5 M, a reduced amount of³²P was found; in concentrations of 10^–6–10^–7 M, hardly any effect could be detected, whereas consistently at concentrations around 10^–8 M, a significant decrease was again observed. The phosphoprotein bands affected by in vitro addition of ACTH_1–24 were of a smaller molecular weight than those affected by in vitro addition of cAMP.     

Distribution of selected phospholipid modifying enzymes in rat brain microsomal subfractions prepared by density gradient zonal rotor centrifugation

A rat brain P₃ fraction enriched in ER derived microsomes was centrifuged through a 20–40% linear sucrose gradient in a Beckman Ti-14 Zonal rotor and 11 fractions were obtained. The distribution of marker enzyme activities and protein were determined in these 11 subfractions. NADPH-Cytochrome C reductase, choline phosphotransferase were employed for endoplasmic reticulum, Na⁺, K⁺-ATPase, 5-nucleotidase, and acetylcholinesterase were employed for plasma membrane, 2, 3-cyclic nucleotide phosphohydrolase was employed for myelin. The bulk of the protein was recovered in the 24–34% sucrose fractions, Na⁺, K⁺-ATPase, 5-nucleotidase, and acetylcholinesterase were in the 22–38% sucrose fractions while NADPH-cytochrome C reductase and CNPase were enriched in the 20–22% sucrose fractions. The ethanolamine and the serine base exchange activities had a bimodal distribution, with highest specific activities in sucrose fractions 32–34% and 20–24%. Choline base exchange activity was nearly undetectable in all the fractions. The specific activities of CDP-choline phosphotransferase, and phospholipid-N-methyltransferase were highest in the 20–22% sucrose fraction. Phospholipid-N-methyltransferase activity was significantly stimulated in the presence of exogenous phospholipid acceptors as phosphatidylethanolamine or phosphatidylmonomethylethanolamine or phosphatidyldimethylethanolamine, however, the greatest response was with phosphatidylmonomethylethanolamine. The rat brain P₃ fraction yielded a population of a membrane at the light end of the sucrose gradient which has a buoyant density similar to myelin but seemed to be enriched with NADPN cytochrome C reductase and phospholipid modifying enzymes. This is in contrast to liver microsomes submitted to a similar fractionation.     

Studies on adenosine triphosphate transphosphorylases. XVIII. Synthesis and preparation of peptides and peptide fragments of rabbit muscle ATP-AMP transphosphorylase (adenylate kinase) and their nucleotide-binding properties

Two peptide fragments, derived from the head and tail of rabbit muscle myokinase, were found to possess remarkable and specific ligand-binding properties (Hamadaet al., 1979).By initiating systematic syntheses and measurements of equilibrium substrate-binding properties of these two sets of peptides, or portions thereof, which encompass the binding sites for (a) the magnesium complexes of the nucleotide substrates (MgATP^2– and MgADP^–) and (b) the uncomplexed nucleotide substrates (ADP^3– and AMP^2–) of rabbit muscle myokinase, some of the requirements for binding of the substrates to ATP-AMP transphosphorylase are being deduced and chemically outlined. One requirement for tight nucleotide binding appears to be a minimum peptide length of 15–25 residues. In addition, Lys-172 and/or Lys-194 may be involved in the binding of AMP.The syntheses are described as a set of peptides corresponding to residues 31–45, 20–45, 5–45, and 1–45, and a set of peptides corresponding to residues 178–192, 178–194, and 172–194 of rabbit muscle adenylate kinase. The ligand-binding properties of the first set of synthetic peptides to the fluorescent ligands: MgATP/ATP and MgADP/ADP are quantitatively presented in terms of their intrinsic dissociation constants (K_d) and values ofN (maximal number of moles bound per mole of peptide); and compared with the peptide fragment MT-I (1–44) obtained from rabbit muscle myokinase (Kubyet al., 1984) and with the native enzyme (Hamadaet al., 1979). In addition, the values ofN andK_d are given for the second set of synthetic peptides to the fluorescent ligands AMP and ADP as well as for the peptide fragments MT-XII(172–194) and CB-VI(126–194) (Kuby et al., 1984) and, in turn, compared with the native enzyme.A few miscellaneous dissociation constants which had been derived kinetically are also given for comparison (e.g., theK _i for AMP and the value of obtained for the native enzyme) (Hamada and Kuby, 1978), and theK'_d measured for Cr³⁺ and the synthetic peptide I_1–45 (Fryet al., 1985b).Paper XVII of this series is Kubyet al. (1983).     

n-3 PUFA productivity in chemostat cultures of microalgae

Eicosapentaenoic (EPA) and docosahexaenoic (DHA) acid productivities from chemostat cultures of an isolate of Isochrysis galbana have been studied. The productivities reached in the interval of dilution rates between 0.0295 h^–1 and 0.0355 h^–1 were 1.5mg·1^–1·h^–1 for lipids, 300 g·1^–1·h^–1 for EPA and 130g1·1^–1·h^–1 for DHA. Furthermore, light attenuation by mutual shading, and agitation speed influences on growth and fatty acid composition were analysed. A model relating steady-state dilution rates to internal average light intensity has been proposed, the parameter values of which obtained by non-linear regression were: maximum specific growth rate (_max)=0.0426 h^–1; the affinity of cells to light (I_k) = 10.92 W·m^–2; the exponent (n) = 5.13; regression coefficient (r ²)=0.9999. Correspondence to: E. Molina Grima     

ACTH-induced inhibition of endogenous rat brain protein phosphorylation in vitro: Structure activity

ACTH_1–24 inhibits the endogenous phosphorylation in vitro of distinct SPM protein bands. Using N-terminal fragments of ACTH, the structure-activity requirements for this effect were studied. A rather complex interaction of the ACTH fragments with endogenous SPM phosphorylation was observed. The effects were not only dependent on the primary structure of the peptide used, but also on the protein band studied and the ATP/SPM ratio used in the incubation system. ACTH_1–24 did not interfere with the ATP-hydrolyzing activity of the SPM preparation, nor did it influence the endogenous phosphatase activity. Therefore, a direct interaction of ACTH with SPM protein kinase(s) is likely to be responsible for its effect on phosphorylation.     

New Substrates for Enteropeptidase: I. Biologically Active Hepta-, Octa-, and Nonapeptides

Enteropeptidase (enterokinase, EC 3.4.21.9) hydrolyzes peptide bonds formed by carboxyl groups of Lys or Arg residue if less than four negatively charged amino acid residues are in positions P ₂–P ₅ of its substrate. We determined the kinetic parameters of three substrates of this type: human angiotensin II (AT) (DR VYIHPF) and the Hb(2–8) (LTAEEK A) and Hb(1–9) (MLTAEEK AA) peptides of the cattle hemoglobin -chain. The K _m values for all the substrates (10^–3 M) were one order of magnitude higher than those of the typical synthetic substrates of enteropeptidase or chimeric proteins with the –DDDDK– full-size linker (K _m 10^–4 M). The k _cat values for AT and Hb(2–8) were also close and low (30 min^–1). The general hydrolysis efficiency of such substrates is no more than 1% of the corresponding value for the typical peptide and protein substrates of the enteropeptidase. However, the elongation of Hb(2–8) peptide by one amino acid residue from both its N- and C-termini results in a dramatic increase in the catalytic efficiency of the hydrolysis: the k _cat value for Hb(1–9) is 1510 min^–1, which means that it is hydrolyzed only three times less effective than the chimeric protein with the full-size linker.     

Action of a cell-envelope proteinase (CEPIII-type) from Lactococcus lactis subsp. cremoris AM1 on bovine κ-casein

The specificity of the cell-envelope proteinase (CEP_III-type) from Lactococcus lactis subsp. cremoris AM₁ in its action on bovine -casein was studied. A 4-h digest (pH 6.2, 15°C) of -casein was made with the purified proteinase. The pH-4.6 soluble fraction, representing more than 95% of the whole hydrolysate, was ultrafiltered to obtain a high-molecular-mass (HMM) and a low-molecular-mass (LMM) fraction, which were separately further purified by electrophoretic and chromatographic techniques. Isolated HMM and LMM products were identified by amino acid analysis, end-group determination and mass spectrometry. On-line HPLC/mass spectrometry was also used for the separation of an LMM peptide mixture and the identification of its components. The HMM products formed were the fragments 1–160, 1–151, 1–95 and 1–79 of -casein, whereas the main LMM products found were the 161–169 and 152–160 fragments. The enzyme specificity was concluded to be primarily directed towards the C-terminal region of the substrate molecule by cleavage of the 160–161 and 151–152 peptide bonds. Two minor LMM products were identified as the fragments 96–104 and 103–106, indicating additional cleavage at positions 102–103, 104–105 and 106–107 of the sequence. Also several peptide bonds within the 161–169 sequence were found to be subject to secondary cleavage by the proteinase. From electrophoretic and identification data it is concluded that the lactococaal CEP_I, CEP_III and several mixed-type proteinases all act on the peptide bonds at positions 79–80 and 95–96. However, the C-terminal region of the -casein sequence is the exclusive target of the CEP_III-aand, to variable extents, of the mixed-type enzymes.     

Immunohistochemical detection of adenohypophyseal cells containing hormones in Rana ridibunda

Summary The pars distalis of the pituitary of Rana ridibunda captured throughout the spring and summer was examined with immunofluorescence techniques using antisera to mammalian pituitary hormones. On the basis of their immunoreactivity, four different cell types are recognized: 1) cells immunoreactive to anti-bovine LTH, 2) cells immunoreactive to anti-bovine STH, 3) cells immunoreactive to anti-ovine LH, and 4) cells immunoreactive to anti-synthetic ACTH (1–24). Their distribution and morphology as well as their staining characteristics (classical histological techniques) are reported in this study.     

A Dual Pathway for Acth Steroidogenic Action in Purified Adrenocortical Cells

Abstract

Isolated adrenal fasciculata cells were purified by centrifugation through a 0-50% hyperbolic gradient of Percoll^R. The dose-dependence and kinetics of both intracellular cyclic AMP accumulation and steroido-genesis in response to ACTH_1-39 and ACTH_5-24 (corticotropin-(1-39) and corticotropin-(5-24)-peptides) were determined using purified cells. The rate of intracellular cyclic AMP formation was maximal during the first five minutes after hormone addition and remained constant or fell thereafter. Therefore intracellular cyclic AMP accumulation, assessed after 5 min., was compared with steroid output after 20 min. Maximal steroidogenesis was elicited by ACTH^5-24 without discerning a significant stimulation of intracellular cyclic AMP accumulation. ACTH_6-24 (corticotropin-(6-24)-peptide) could completely inhibit the intracellular cyclic AMP accumulation elicited by ACTH_1-39 or by ACTH_5-24 at concentrations that only partially inhibited steroidogenesis.

It is possible that there are two pathways for the steroidogenic action of ACTH, one of which is obligatorily mediated by intracellular cyclic AMP, and another which involves a different mediator.     

Effect of temperature and surface potential on the electrogenic proton uptake in the QB site of the Rhodobacter sphaeroides photosynthetic reaction center: QA −. B −. → QAQBH2 transition

Direct electrometry was used to study the light-induced voltage changes in the Rhodobacter sphaeroides chromatophores adsorbed to a phospholipid-impregnated nitrocellulose film. After the second laser flash, a fast increase in the voltage associated with charge separation was followed by a slower increase attributed to the proton uptake in the Q_B site of the photosynthetic reaction centers. Kinetics and relative amplitudes of these voltage changes attributed to the Q_A ^–. _B ^–. Q_AQ_BH₂ transition, were measured as a function of pH and temperature between +4 and +40 °C. The kinetics can be approximated by a single exponent above +23 °C (100 µs at +25 °C, pH 7.2), whereas below this temperature, it was a good fit of two exponential approximation (65 µs and 360 µs with similar contributions at +10 °C, pH 7.2). The faster component diminished with an apparent pK 8.5, whereas the slower one was maintained at a constant level until pH 9.5 and then decreased. The calculated activation energy from the temperature dependence of the slower component (55 – 65 kJ/mol) was much higher than that of the faster component (< 10 kJ/mol). The two voltage components can be attributed to the transfer of the first (faster component) and the second (slower component) proton from the reaction center surface to Q_B. We suggested that higher activation energy of the slower component was due to a conformational change in the reaction center kinetically coupled to the second proton transfer to Q_BH^–.The faster component diminished in the presence of 1 M KCl, with an apparent pK 7.5. To explain this observation, we assume that: (i) the midpoint potential of the Q_A/Q_A ^–. redox pair was higher in 1 M KCl because of the reduced surface potential of chromatophores; (ii) the midpoint potential of the Q_B ^–./Q_BH^–. redox pair was insensitive to the surface potential change; (iii) the equilibrium constant of the reaction Q_A ^–.Q_B ^–. Q_AQ_BH^– decreased at high ionic strength.     

Experimental data on the inheritance of some taxonomic characters inHordeum spontaneum C. Koch emend. Bacht

Summary Hordeum spontaneum C. Koch emend. Bacht. varieties have been both intercrossed and crossed with two cultivated barley varieties ofH. vulgare (L.) emend Vav. et Bacht. with a view of eliciting the nature of inheriting the spikelet-pedicel of the lateral spikelets and the shape of their apex in the said wildgrowing barley. The investigations of F₁ and F₂ showed the inheritance of the spikelet-pedicel to have a dominating nature and to segregate in F₂ in conformity with the Mendelian monohybrid type. In the second case the forms with shorter awn-like formations, or their rudiments, were dominating.As a result ofH. spontaneum x H. vulgare hybridization along with already known forms, new formations were received, they have been conditionally named by the author:sessiliproskowetzii, proskowfertillum, ischnofertillum, and pallipodum.

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Zusammenfassung Im Rahmen größerer Untersuchungen über die Abstammung und Phylogenie der Gerste wurden mehrere Varietäten vonHordeum spontaneum C. Koch emend. Bacht. sowohl untereinander als auch mit zwei Varietäten der Kulturgerste,H. vulgare (L.) emend. Vav. et Bacht., gekreuzt. Es sollte geklärt werden, wie bei den genannten Wildgersten das Stielchen (pedicel) der Seitenährchen sowie die Ausbildung des Apex der Seitenährchen (d. h. ihre Begrannung) vererbt werden. Die Untersuchung der F₁ und F₂ zeigte, daß das Stielchen (gegenüber ungestielten Seitenährchen) dominant und gemäß einer monohybriden Mendelspaltung vererbt wird. Bezüglich der Ausbildung des Apex der Seitenährchen ergab sich im allgemeinen Dominanz der kürzeren oder rudimentären Grannen gegenüber längeren Grannen.Im Ergebnis der Hybridisation zwischenH. spontaneum undH. vulgare wurden, neben bereits bekannten, verschiedene neue Formen gefunden, die vom Autor vorläufig wie folgt benannt werden:sessiliproskowetzii, proskowfertillum, ischnofertillum, pallipodum.Die Ergebnisse werden im Zusammenhang mit Fragen der Abstammung der Kulturgerste diskutiert.

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With 4 figures     

Augmentation of H2 photoproduction in Rhodopseudomonas palustris by N-heterocyclic aromatic compounds

Increases of 23- (5.6 mmol acetylene reduced mg dry wt^–1) and 16- (4 mmol acetylene reduced mg dry wt^–1) fold in nitrogenase activity and 12- (671 l H₂ mg dry wt^–1 h^–1) and 6- (349 l mg dry wt^–1 h^–1) fold in H₂ photoproduction in Rhodopseudomonas palustris JA1 over 24 h were achieved with pyrazine 2-carboxylate (3 mM) and 3-picoline (3 mM), respectively, and were higher than earlier reports of enhancement (1.5 to 5- fold) in biological H₂ production using various alternative methods.     

Chloride distribution in the proximal convoluted tubule ofNecturus kidney

Summary To assess the mechanism(s) by which intraluminal chloride concentration is raised above equilibrium values, intracellular Cl^– activity ( _i ^Cl ) was studied in the proximal tubule ofNecturus kidney. Paired measurements of cell membrane PD (V _BL) and Cl-selective electrode PD (V _BL ^Cl ) were performed in single tubules, during reversible shifts of peritubular or luminal fluid composition. Steadystate _i ^Cl was estimated at 14.6±0.6 mmol/liter, a figure substantially higher than that predicted for passive distribution. To determine the site of the uphill Cl^– transport into the cell, an inhibitor of anion transport (SITS) was added to the perfusion fluid. Introduction of SITS in peritubular perfusate decreased _i ^Cl , whereas addition of the drug in luminal fluid slightly increased _i ^Cl ; both results are consistent with basolateral membrane uphill Cl^– transport from interstitium to the cell. TMA⁺ for Na⁺ substitutions in either luminal or peritubular perfusate had no effect on _i ^Cl . Removal of bicarbonate from peritubular fluid, at constant pH (a situation increasing HCO ₃ ^– outflux), resulted in an increase of _i ^Cl , presumably related to enhanced Cl^– cell influx: we infer that Cl^– is exchanged against HCO ₃ ^– at the basolateral membrane. The following mechanism is suggested to account for the rise in luminal Cl^– concentration above equilibrium values: intracellular CO₂ hydration gives rise to cell HCO ₃ ^– concentrations above equilibrium. The passive exit of HCO ₃ ^– at the basolateral membrane energizes an uphill entry of Cl^– into the cell. The resulting increase of _i ^Cl , above equilibrium, generates downhill Cl^– diffusion from cell to lumen. As a result, luminal Cl^– concentration also increases.C.N.R.S. Greco 24. Part of this work was presented at the 12^th annual meeting of the American Society of Nephrology, Boston, Mass. (Edelman et al., 1979).     

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     

Export of chloride after clear-cutting in the Hubbard Brook sandbox experiment

The objective of this study was to discern the source of higher than usual concentrations of chloride in drainage water collected from experimental forest plots after clear-cutting. When the sandbox experiments were initiated at the Hubbard Brook Experimental Forest Station three vegetation types were established: red pine, grass, and minimally vegetated (scattered lichens and bryophytes) as the bare control plot. After 15 years of growth the trees were cut down and above-ground biomass removed from the red pine sandbox. For several years prior to the cut, high concentrations ( 75 M) of dissolved Cl^– in drainage waters occurred in November/December. This is attributed to the buildup of rainfall-derived Cl^– due to evapotranspiration that depletes soil moisture to low levels resulting in a lack of drainage during this period. The excess Cl^– is quickly flushed out by subsequent drainage over a few weeks and Cl^– concentrations return to values characteristic of rainfall and throughfall. After the trees were removed in May, 1998, Cl^– continued to be leached from the system. The concentration of Cl^– peaked (175 M) in Sept. 1998 and did not return back to base level concentration until Dec. 1999. The Cl^– release pattern is distinctly different from that of dissolved NO₃ ^–, which peaked about one year later than Cl^–. An excess (over that of the control sandbox) of 78 g Cl^– was released in the 1.5 year period after clear-cut, showing that a large amount of leachable chloride is stored in the bulk soil/root/organic matter fraction. Lack of uptake by trees may be part of the reason for this chloride pulse. But an analysis of chloride content in roots and litter indicates that as much as 50% of the chloride leached from the sandbox may have come from the decaying roots and litter. Additional chloride may have been released from the soil organic matter by decomposition. The biochemical behavior of Cl^– in systems such as this should be evaluated before assuming Cl^– to be conservative for purposes of hydrological transport or soil weathering studies.