The stimulation of chloride transport by prostaglandins and their interaction with epinephrine,theophylline, and cyclic AMP in the corneal epithelium

Summary Prostaglandins (E₁, E₂ and F₂) stimulated the chloride transport of the frog corneal epithelium with maximal effects at 10^–5 m in the aqueus side. This stimulation does not occur in Cl-free solutions and the net³⁶Cl flux increased proportionally to the short-circuit current. Polyphloretin phosphate (PPP) and diphloretin phosphate (DPP) inhibited the response if added within 3 min before PGE₁. The maximal response to epinephrine 10^–5 m and dibutyryl cyclic AMP 10^–3 m was not changed by further addition of prostaglandins, but these drugs produced their full effect when administered at the peak of the response of prostaglandins. The maximal response to theophylline 10^–5 m was increased by PGE₁. PPP and DPP did not modify the response to epinephrine. Prostaglandin stimulation of the chloride transport was accompanied by increased light transmission through partially opaque corneas. The known release of prostaglandins in the aqueous humor can be associated to a direct action on the corneal epithelium manifested in the activation described herein.     

Effects of calcium and lanthanum on phosphate efflux from nonmyelinated nerve fibers

Summary Phosphate efflux was measured as the fractional rate of loss of radioactivity from rabbit vagus loaded with radiophosphate. The effects of changes in extracellular calcium and of lanthanum have been investigated. In Locke solution with normal, 0.9mm, calcium and without phosphate, the fractional rate of loss was 1.62×10^–3 min^–1 at 120 min after the beginning of the washing period and fell slowly (9% hr^–1) during washing from 2 to 6 hr. Addition of calcium to the Locke solution produced a transient increase followed by a reversible maintained increase in phosphate efflux. The latter was 40 and 75% above efflux in normal calcium for 20 and 50mm calcium, respectively. Removal of calcium, with or without addition of EGTA, produced only a transient increase in phosphate efflux, with no subsequent maintained change. Addition of low concentrations of lanthanum produced a reversible inhibition of phosphate efflux. Half-maximal inhibition was at 3.5 m lanthanum and appeared to be due to binding of lanthanum to more than one, probably two, sites. Measurements of inhibition by lanthanum at different calcium concentrations did not indicate any competition between calcium and lanthanum. It is suggested that at least a part of phosphate efflux depends on internal calcium and that lanthanum acts by preventing release of phosphate from the phosphate transport mechanism.     

Receptor capping in mouse T-lymphoma cells: A Ca2+ and calmodulin-stimulated ATP-dependent process

Summary The roles that Ca²⁺, calmodulin, and ATP play in the redistribution of conconavalin A (Con A) binding sites on the surface of mouse T-lymphoma cells were examined. Membranes of cells labeled with fluorescein-conjugated Con A (Fl-Con A) were made permeable (skinned) to ions and proteins by incubation in a solution containing no added Ca²⁺, 7mm EGTA, and ATP. The intracellular ionic and protein concentrations could then be varied, and the degree of Con A receptor capping monitored simultaneously. A graded increase (9.0 to 30%) was found in the number of capped cells with increasing Ca²⁺ concentration from 10^–6–10^–4.9 m. Increasing concentrations of trifluoperazine, chlorpromazine, and promethazine (1.5×10^–6 to 1.0×10^–4 m) in cell suspensions containing 10^–4 m Ca²⁺ produced graded inhibition of capping in the same order that the drugs bind to calmodulin. Removal of extracellular Ca²⁺ dissociated (reversed) some of the caps into patches, thus reducing their number (12%). ATP was required for either capping or cap dissociation to occur. Addition of calmodulin (3.9×10^–8–6.3×10^–7 m) to the cell suspension increased the Ca²⁺ sensitivity. These results provide direct evidence that capping of Con A receptors is a reversible process (i) regulated by intracellular Ca²⁺ concentration, (ii) requiring ATP as an energy source, and (iii) susceptible to the influence of calmodulin. These findings are consistent with the hypothesis that the collection of surface receptor patches into cap structures is controlled by the interaction of actomyosin filaments, which in turn is regulated by a Ca²⁺-calmodulin-activated control system.     

Mechanism of inhibition of net ion transport across frog corneal epithelium by calcium channel antagonists

Summary In the isolated bullfrog cornea, three calcium channel antagonists had dose-dependent inhibitory effects on the Cl-originated short-circuit current (SCC). Their order of decreasing potency was bepridil, verapamil and diltiazem. One millimolar diltiazem inhibited the SCC by 98% and subsequent incubation with the calcium ionophore A23187 had no restorative effect. Increasing the bathing solution Ca concentration from 0.05 to 15mm, however, decreased diltiazem's inhibitory efficacy. This antagonist depolarized the intracellular potential differenceV _m from –54 to –18 mV (tear: reference) and the voltage divider ratioFR ₀ decreased from 0.58 to 0.30, suggesting an increase in basolateral membrane electrical resistance. Additional indication of a basolateral membrane effect by the drug was that preincubation with 10⁵ m amphotericin B in Cl-free Ringer's did not eliminate the inhibitory effect of the drug on the Na- and K-elicited SCC. In the absence of amphotericin B in Cl-free Ringer's (SCC=0), 1 ×10³ m diltiazem depolarized theV _m from –78 to –9 mV suggesting that the increase in basolateral membrane resistance was due to K channel blockade. Diltiazem (1×10³ m) significantly decreased cyclic AMP content; however, isoproterenol in the presence of the drug increased cyclic AMP fourfold without having any restorative effect on the inhibited SCC. Therefore, the inhibition of the Cl-originated SCC resulting from an increase in basolateral membrane K resistance is not caused by a decline in cyclic AMP content. In plasma membrane-enriched fractions prepared from broken cell preparations of bovine corneal epithelium, 1×10³ m diltiazem had no inhibitory effects on either Na,K-ATPase or Ca,Mg-ATPase activities. These latter effects further point to the selectivity of diltiazem as an inhibitor of K-channel activity, but do not preclude a Ca-channel blocker effect by the drug in the micromolar range.     

Activation of RAT erythrocyte phosphofructokinase by AMP and by non-physiological concentrations of Cyclic AMP

Summary Cyclic AMP (300µ m) activates phosphofructokinase from dialyzed haemolysates of mature rat erythrocytes. The main conclusions are: a) Cyclic AMP, at pH 7.1 and low concentrations of fructose-6-phosphate, is able to reverse the inhibition produced by different amounts of ATP (up to 1.5mm). b) The cyclic nucleotide is a positive allosteric effector of the enzyme as shown by the displacement of sigmoidal fructose-6-phosphate saturation curve to hyperbolic kinetics in the presence of inhibitory concentrations (1.5mm) of ATP. c) Cyclic AMP has no significant influence as deinhibitor of phosphofructokinase either at pH 7.1 and non-inhibitory levels (0.25mm) of ATP or at pH 8.1 and inhibitory (1.5mm) of non-inhibitory (0.25mm) concentrations of ATP. Similar conclusions were obtained with 300µ m AMP but not at a lower concentration (3µ m) with both nucleotides.The comparison of cyclic AMP results with those obtained under similar concentrations of AMP suggest that cyclic AMP is really only an in vitro modulator of the enzyme from rat erythrocytes, presumably at an AMP regulatory site, since non-physiological concentrations are required to act as deinhibitor.     

Electrochemical investigation of active malic acid transport at the tonoplast into the vacuoles of the CAM plantKalanchoë daigremontiana

Summary The membrane potential of cells in leaf slices of the CAM plantKalanchoë daigremontiana Hamet et Perrier in the light and in the dark is –200 mV on the average; it is reversibly depolarized by the metabolic inhibitors FCCP (5×10^–6 m) and CN^– (5×10^–3 m); it shows the light-dependent transient oscillations ubiquitously observed in green cells; it is independent of the amount of malic acid accumulated in the cells (in a tested range between 30 and 140mm); and it is considerably hyperpolarized by the fungal toxin fusicoccin (30×10^–6 m). Fusicoccin inhibits nocturnal malic acid accumulation in intact isolated phyllodia of the CAM plantKalanchoë tubiflora (Harv.) Hamet but does not affect remobilization of malic acid during the day.Electrochemical gradients for the various ions resulting from dissociation of malic acid, i.e., H⁺, Hmal^– and mal^2–, were calculated using the Nernst equation. With a very wide range of assumptions on cytoplasmic pH and malate concentration results of calculations suggest uphill transport of H⁺ and Hmal^– from the cytoplasm into the vacuole, while mal^2– might be passively distributed at the tonoplast. On the basis of the present data the most likely mechanism of active malic acid accumulation in the vacuoles of CAM plants appears to be an active H⁺ transport at the tonoplast coupled with passive movement of mal^2– possibly mediated by a translocator (catalyzed diffusion), with subsequent formation of Hmal^– (2 H⁺+mal^2–H⁺+Hmal^–) at vacuolar pH's.     

Fluctuation analysis of short-circuit current in a warm-blooded sodium-retaining epithelium: Site current,density, and interaction with triamterene

Summary Density and conductance of the Na-site in hen coprodeum were studied by employing fluctuation analysis of shortcircuit current at sodium concentrations from 26 to 130mm. Fluctuations of current in the frequency range 2–800 Hz were induced by triamterene, a reversible blocker of conducting epithelial Na-sites. At 130mm Na the site density was 5.8±1.0 m^–2 and the site conductance was 4 pS. This conductance is equal to that of the frog skin (W. Van Driessche and B. Lindemann, 1979,Nature (London) 282:519–520). Extrapolation of site density to zero sodium renders a total of 38±28 sites m^–2, which is compared with other estimates for the coprodeum. The site-triamterene association and dissociation constants were 9.5±0.4 rad sec^–1 m ^–1 and 255±20 rad sec^–1 and they were independent of external sodium concentration. An analysis of the affinity constant for triamterene based on the DC-short-circuit current was found to be unrelated to the external sodium concentration and identical to that obtained from fluctuation analysis indicating a noncompetitive interaction between sodium and triamterene. Due to the oxygen demand of the epithelium we have developed an experimental method using short data processing times. A new analytical approach using integration of the power density spectrum proved necessary because of low signal-to-noise ratios.     

Studies on the cation permeability of human red cell ghosts

Summary Net K movements in reconstituted human red cell ghosts and the resealing of ghosts to cations after osmotic hemolysis of red cells have been studied as functions of the free Ca ion concentration. The Ca-dependent specific increase in K permeability was shown to be mediated by a site close to the internal surface of the membrane with an apparent dissociation constant at pH 7.2 for Ca (K _D1) of 3–5×10^–7 m, for Sr of 7×10^–6 m. Ba and Mg did not increase the K-permeability of the membrane but inhibited the Ca-mediated permeability changes.K _D1 decreased in a nonlinear fashion when the pH was increased from 6.0 to 8.5. Two different pK values of this membrane site were found at pH 8.3 and 6.3. The Ca-activated net K efflux into a K-free medium was almost completely inhibited by an increase in intracellular Na from 4 to 70mm. Extracellular K antagonized this Na effect. Changes in the extracellular Na (0.1–140mm) or K(0.1–6mm) concentrations had little effect and did not changeK _D1. The Ca-stimulated recovery of a low cation permeability in ghost cells appeared to be mediated by a second membrane site which was accessible to divalent cations only during the process of hemolysis in media of low ionic strength. The apparent dissociation constant for Ca at this site (K _D2) varied between 6×10^–7 and 4×10^–6 m at pH 7.2. Mg, Sr, and Ba could replace Ca functionally. The selectivity sequence was Ca>Sr>Ba>Mg.K _D2 was independt on the pH value in the range between 6.0 and 8.0. Hill coefficients of 2 were observed for the interaction of Ca with both membrane sites suggesting that more than one Ca ion is bound per site. The Hill coefficients were affected neither by the ion composition nor by the pH values of the intra- and extracellular media. It is concluded that two different pathways for the permeation of cations across the membrane are controlled by membrane sites with high affinities for Ca: One specific for K, one unspecific with respect to cations. The K-specific channel has properties similar to the K channel in excitable tissues.     

Pyrimidine biosynthesis in Serratia marcescens: Polypeptide interactions of three nonsequential enzymes

Orotidine-5-monophosphate pyrophosphorylase (OMPppase, E.C. 2.4.2.10) and orotidylate decarboxylase (OMPdecase, E.C. 4.1.1.23) were purified from Serratia marcescens HY. These enzymes required physical association for maximal catalytic activities and formed a fragile complex with dihydroorotase (DHOase, E.C. 3.5.2.3.). OMPppase reversibly lost 50% of its activity upon separation from DHOase. The kinetic characteristics of OMPppase were modified by this separation. In the presence of DHOase, the K _ms for PRPP and orotate were stoichiometric: 2.3×10^–6 m and 2.6×10^–6 m, respectively. Following separation, the K _ms were significantly different: 1.3 × 10^–6 m for PRPP and 4.1×10^–6 m for orotate. OMPppase and OMPdecase could be reversibly separated by acrylamide gel electrophoresis, but the separation was accompanied by a loss of catalytic efficiency for both enzymes. DHOase readily associated into multiple molecular forms and could not be purified. The DHOase-OMPppase-OMPdecase interactions demonstrate that a weakly aggregated, multifunctional enzyme complex participates in the biosynthesis of pyrimidine nucleotides in S. marcescens. This unique association of nonsequential biosynthetic enzymes may represent a larger complex which provides a channeling or regulatory unit.This work was supported by grants from the National Science Foundation (NSF GB 5811) and the Office of Naval Research (Nonr 4413). One of us (J.W.) was a National Science Foundation Graduate Fellow.     

Noradrenaline induced secretion of nonelectrolytes through frog skin

Summary Addition of noradrenaline (4×10^–5 m) to the inner bathing fluid in the skin of the frogRana esculenta results in increased unidirectional fluxes of urea, thiourea, N-methyl-thiourea, N-N-dimethylthiourea and mannitol. Fluxes towards the external medium ( ₀) undergo a much greater increase than those moving in the opposite direction ( _i ). The effect of noradrenaline on ( ₀) is higher for urea and thiourea than mannitol, while its effect on ( ₀) thiourea derivatives is related to lipid solubility. This phenomenon does not occur for ( _i ) of the same molecules.FCCP (10^–6 m) pretreatment strongly inhibits the noradrenaline effect on ( ₀). In skin pretreated whith colchicine (2×10^–5 m) both urea fluxes are increased to the same extent by noradrenaline. Noradrenaline is concluded to exert two separate effects: (1) a change in permeability in both directions; (2) a secretion of nonelectrolytes towards the external fluid. Such secretion is most probably associated with the hormone-induced secretion of fluid and electrolytes, perhaps mediated by an exocytotic mechanism.     

Na+/K+/Cl− cotransport in cultured vascular smooth muscle cells: Stimulation by angiotensin II and calcium ionophores,inhibition by cyclic AMP and calmodulin antagonists

Summary The specific activity of the Na⁺/K⁺/Cl^– cotransporter was assayed by measuring the initial rates of furosemide-inhibitable⁸⁶Rb⁺ influx and efflux. The presence of all three ions in the external medium was essential for cotransport activity. In cultured smooth muscle cells furosemide and bumetanide inhibited influx by 50% at 5 and 0.2 m, respectively. The dependence of furosemide-inhibitable⁸⁶Rb⁺ influx on external Na⁺ and K⁺ was hyperbolic with apparentK _m values of 46 and 4mm, respectively. The dependence on Cl^– was sigmoidal. Assuming a stoichiometry of 112 for Na⁺/K⁺/Cl^–, aK _m of 78mm was obtained for Cl^–. In quiescent smooth muscle cells cotransport activity was approximately equal to Na⁺ pump activity with each pathway accounting for 30% of total⁸⁶Rb⁺ influx. Growing muscle cells had approximately 3 times higher cotransport activity than quiescent ones. Na⁺ pump activity was not significantly different in the gorwing and quiescent cultures. Angiotensin II (ANG) stimulated cotransport activity as did two calcium-transporting ionophores, A23187 and ionomycin. The removal of external Ca²⁺ prevented A23187, but not ANG, from stimulating the cotransporter. Calmodulin antagonists selectively inhibited⁸⁶Rb⁺ influx via the cotransporter. Beta-adrenoreceptor stimulation with isoproterenol, like other treatments which increase cAMP, inhibited cotransport activity. Cultured porcine endothelial cells had 3 times higher cotransport activity than growing muscle cells. Calmodulin antagonists inhibited cotransport activity, but agents which increase cAMP or calcium had no effect on cotransport activity in the endothelial cells.     

The nature of the neutral Na+−Cl−-coupled entry at the apical membrane of rabbit gallbladder epithelium: I. Na+/H+, Cl−/HCO 3 − double exchange and Na+−Cl− symport

Summary Cl^– influx at the luminal border of the epithelium of rabbit gallbladder was measured by 45-sec exposures to³⁶Cl^– and³H-sucrose (as extracellular marker). Its paracellular component was evaluated by the use of 25mm SCN^– which immediately and completely inhibits Cl^– entry into the cell. Cellular influx was equal to 16.7eq cm^–2 hr^–1 and decreased to 8.5eq cm^–2 hr^–1 upon removal of HCO ₃ ^– from the bathing media and by bubbling 100% O₂ for 45 min. When HCO ₃ ^– was present, cellular influx was again about halved by the action of 10^–4 m acetazolamide, 10^–5 to 10^–4 m furosemide, 10^–5 to 10^–4 m 4-acetamido-4-isothiocyanostilbene-2,2-disulfonate (SITS), 10^–3 m amiloride. The effects of furosemide and SITS were tested at different concentrations of the inhibitor and with different exposure times: they were maximal at the concentrations reported above and nonadditive. In turn, the effects of amiloride and SITS were not additive. Acetazolamide reached its maximal action after an exposure of about 2 min. When exogenous HCO ₃ ^– was absent, the residual cellular influx was insensitive to acetazolamide, furosemide and SITS. When exogenous HCO ₃ ^– was present in the salines, Na⁺ removal from the mucosal side caused a slow decline of cellular Cl^– influx; conversely, it immediately abolished cellular Cl^– influx in the absence of HCO ₃ ^– . In conclusion, about 50% of cellular influx is sensitive to HCO ₃ ^– , inhibitable by SCN^–, acetazolamide, furosemide, SITS and amiloride and furthermore slowly dependent on Na⁺. The residual cellular influx is insensitive to bicarbonate, inhibitable by SCN^–, resistant to acetazolamide, furosemide, SITS and amiloride, and immediately dependent on Na⁺. Thus, about 50% of apical membrane NaCl influx appears to result from a Na⁺/H⁺ and Cl^–/HCO ₃ ^– exchange, whereas the residual influx seems to be due to Na⁺–Cl^– contranport on a single carrier. Whether both components are simultaneously present or the latter represents a cellular homeostatic counterreaction to the inhibition of the former is not clear.     

Release of serotonin from nerve endings byl-5-hydroxytryptophan,α-methyl-m-tyramine,and elevated potassium ions

The release of [³H]5-hydroxytryptamine ([³H]5-HT) byl-5-hydroxytryptophan (L-5-HTP),-methyl-m-tyramine (-MMTA), and elevated levels of K⁺ was studied using crude synaptosomal preparations (P₂) isolated from the telencephalon of the rat and pigeon. Studies were conducted in vitro in the presence of either 2×10^–5 M tranylcypromine, which inhibited the MAO activity of both the extrasynaptosomal mitochondria and the mitochondria contained within the nerve endings (intrasynaptosomal mitochondria), or 2×10^–5 M nialamide, which inhibited the MAO activity of the extrasynaptosomal mitochondria under the experimental conditions used. In the P₂ fraction isolated from the rat, either 55 mM K⁺, 0.10 mMl-5-HTP, or 0.03 mM-MMTA significantly increased the release of [³H]5-HT above control levels, regardless of which MAO inhibitor was present in the medium. In the presence of tranylcypromine, this increased release by 55 mM K⁺ or 0.10 mMl-5-HTP was partially suppressed if Ca²⁺ was omitted from the medium. In the presence of nialamide, the release by 55 mM K⁺ was completely prevented if Ca²⁺ was omitted; the release byl-5-HTP was only partially affected. The release of [³H]5-HT by-MMTA did not appear to be markedly affected by removal of Ca²⁺, regardless of which MAO inhibitor was present. Very similar data were obtained in the presence of nialamide using the P₂ fraction isolated from the telencephalon of the pigeon, with the exception that 0.10 mMl-5-HTP caused an increase in the release of [³H]5-HIAA (which was not calcium-dependent) instead of [³H]5-HT. The data are discussed in     

Evidence for two compartments of exchangeable calcium in isolated rat liver mitochondria obtained using a45Ca exchange technique in the presence of magnesium,phosphate, and ATP at 37°C

Summary The distribution of calcium between isolated rat liver mitochondria and the extramitochondrial medium at 37°C and in the presence of 2mm inorganic phosphate, 3mm ATP, 0.05 or 1.1mm free magnesium and a calcium buffer, nitrilotriacetic acid, was investigated using a⁴⁵Ca exchange technique. The amounts of⁴⁰Ca in the mitochondria and medium were allowed to reach equilibrium before initiation of the measurement of⁴⁵Ca exchange. At 0.05mm free magnesium and initial extramitochondrial free calcium concentrations of between 0.15 and 0.5 m, the mitochondria accumulated calcium until the extramitochondrial free calcium concentration was reduced to 0.15 m. Control experiments showed that the mitochondria were stable under the incubation conditions employed. The⁴⁵Ca exchange data were found to be consistent with a system in which two compartments of exchangeable calcium are associated with the mitochondria. Changes in the concentration of inorganic phosphate did not significantly affect the⁴⁵Ca exchange curves, whereas an increase in the concentration of free magnesium inhibited exchange. The maximum rate of calcium outflow from the mitochondria was estimated to be 1.7 nmol/min per mg of protein, and the value ofK _0.5 for intramitochondrial exchangeable calcium to be about 1.6 nmol per mg of protein. Ruthenium Red decreased the fractional transfer rate for calcium inflow to the mitochondria while nupercaine affected principally the fractional transfer rates for the transfer of calcium between the two mitochondrial compartments. The use of the incubation conditions and⁴⁵Ca exchange technique described in this report for studies of the effects of agents which may alter mitochondrial calcium uptake or release (e.g., the pre-treatment of cells with hormones) is briefly discussed.     

Feeding rate inhibition in crowded Daphnia pulex

Feeding rates of Daphnia pulex fed a range of levels of the alga Chlamydomonas reinhardi of 15 °C are strongly density-dependent. At lower densities, Daphnia (30 1^–1) fed at higher rates than crowded (270 1^–1) Daphnia which manifest a relatively depressed saturation feeding response. At 30 individuals/liter, Daphnia consumed 8.5 – 15.7 × 10⁴ cells d^–1h^–1 (on a volume basis, 12.1 – 22.2 × 10⁶ m³), at 270 L^–1 3.7 – 3.9 × 10⁴ (5.2 – 5.5 = 10⁶ m³ cells d^–1h^–1 when feeding on algae at 80 000 cells ml^–1 (11.3 × 10⁶ m³ ml^–1). The feeding rate data best fit an Ivlev feeding function. An autoallelopath might be causing the repression. Water preconditioned with crowded Daphnia completely repressed feeding in uncrowded Daphnia after six hours.     

Re-evaluation of the specificity of adenylyl (beta,gamma-methylene)diphosphonate as a substrate for adenylate cyclase.

Summary The conversion of the ATP-analogue adenylyl(,-methylene)diphosphonate (AMPPCP) to cyclic AMP by adenylate cyclase of rat liver membranes was demonstrated using a radioimmunoassay for cyclic AMP. The conversion was only insignificantly lower than with adenylylimidodiphosphate (AMPPNP), another ATP-analogue which is usually used in the histochemical adenylate cyclase assay. The unspecific phosphate production was lower with AMPPCP as compared to AMPPNP. Therefore AMPPCP is considered to be a more suitable substrate for the histochemical assay.Unspecific phosphate deposition in the histochemical assay was due to ATP:pyrophosphatase activity and could be significantly inhibited by 1mm NAD. However, a residual phosphate deposition due to cleavage of NAD could not be suppressed. Adenylate cyclase activity could be markedly activated by 5×10^–5 m forskolin, an activator of the catalytic subunit of the enzyme, and inhibited by 1mm 25-dideoxyadenosine, a specific inhibitor of adenylate cyclase. Adenylate cyclase was localized predominantly in the sinusoidal part of the plasma membrane, while ATP-pyrophosphatase seemed to be restricted to the canalicular part. It is concluded that at least three parallel assays are necessary for routine histochemical demonstration of adenylate cyclase, namely (1) basal activity (2) activation by forskolin and (3) inhibition by 25-dideoxyadenosine, to demonstrate a specific enzyme reaction.     

Chloride-activated water permeability in the frog corneal epithelium

We have previously reported that the isolated frog corneal epithelium (a Cl^–-secreting epithelium) has a large diffusional water permeability (P_dw 1.8×10^–4 cm/s). We now report that the presence of Cl^– in the apical-side bathing solution increases the diffusional water flux, J_dw (in both directions) by 63% from 11.3 to 18.4 l min^–1 · cm^–2 with 60 mm [Cl] exerting the maximum effect. The presence of Cl^– in the basolateral-side bathing solution had no effect on the water flux. In Cl^–-free solutions amphotericin B increased J_dw by 29% but only by 3% in Cl^–-rich apical-side bathing solution, suggesting that in Cl^–-rich apical side bathing solution, the apical barrier is no longer rate limiting. Apical Br^– (75 mm) also increased J_dw by 68%. The effect of Cl^– on J_dw was observed within 1 min after its addition to the apicalside bathing solution. HgCl₂ (0.5 mm) reduced the Cl^–-increased P_dw by 31%. The osmotic permeability (P_f) was also measured under an osmotic gradient yielding values of 0.34 and 2.88 (x 10^–3 cm/s) in Cl^–-free and Cl^–-rich apical-side bathing solutions respectively. It seems that apical Cl^–, or Cl^– secretion into the apical bath could activate normally present but inactive water channels. In the absence of Cl^–, water permeability of the apical membrane seems to be limited to the permeability of the lipid bilayer.This work was supported by National Eye Institute grants EY-00160 and EY-01867.     

Thiamin transport by human erythrocytes and ghosts

Summary Thiamin transport in human erythrocytes and resealed pink ghosts was evaluated by incubating both preparations at 37 or 20°C in the presence of [³H]-thiamin of high specific activity. The rate of uptake was consistently higher in erythrocytes than in ghosts. In both preparations, the time course of uptake was independent from the presence of Na⁺ and did not reach equilibrium after 60 min incubation. At concentrations below 0.5 m and at 37°C, thiamin was taken up predominantly by a saturable mechanism in both erythrocytes and ghosts. Apparent kinetic constants were: for erythrocytes,K _m =0.12, 0.11 and 0.10 m andJ _max=0.01, 0.02 and 0.03 pmol·l^–1 intracellular water after 3, 15, and 30 min incubation times, respectively; for ghosts,K _m =0.16 and 0.51 m andJ _max=0.01 and 0.04 pmol·l^–1 intracellular water after 15 and 30 min incubation times, respectively. At 20°C, the saturable component disappeared in both preparations. Erythrocyte thiamin transport was not influenced by the presence ofd-glucose or metabolic inhibitors. In both preparations, thiamin transport was inhibited competitively by unlabeled thiamin, pyrithiamin, amprolium and, to a lesser extent, oxythiamin, the inhibiting effect being always more marked in erythrocytes than in ghosts. Only approximately 20% of the thiamin taken up by erythrocytes was protein-(probably membrane-) bound. A similar proportion was esterified to thiamin pyrophosphate. Separate experiments using valinomycin and SCN^– showed that the transport of thiamin, which is a cation at pH 7.4, is unaffected by changes in membrane potential in both preparations.     

Bacterial production in a mesohumic lake estimated from [14C]leucine incorporation rate

Incorporation of [¹⁴C]leucine into proteins of bacteria was studied in a temperate mesohumic lake. The maximum incorporation of [¹⁴C] leucine was reached at a concentration of 30 nm determined in dilution cultures. Growth experiments were used to estimate factors for converting leucine incorporation to bacterial cell numbers or biomass. The initially high conversion factors calculated by the derivative method decreased to lower values after the bacteria started to grow. Average conversion factors were 7.09 × 10¹⁶ cells mol^–1 and 7.71 × 10¹⁵ m³ mol^–1, if the high initial values were excluded. Using the cumulative method, the average conversion factor was 5.38 × 10¹⁵ m^–3 mol^–1 I . The empirically measured factor converting bacterial biomass to carbon was 0.36 pg C m^–3 or 33.1 fg C cell^–1. Bacterial production was highest during the growing season, ranging between 1.8 and 13.2 g C liter^–1 day^–1, and lowest in winter, at 0.2–2.9 g C liter^–1 day^–1. Bacterial production showed clear response to changes in the phytoplankton production, which indicates that photosynthetically produced dissolved compounds were used by bacteria. In the epilimnion bacterial production was, on average, 19–33% of primary production. Assuming 50% growth efficiency for bacteria, the allochthonous organic carbon could have also been an additional energy and carbon source for bacteria, especially in autumn and winter. In winter, a strong relationship was found between temperature and bacterial production. The measuring of [¹⁴C]leucine incorporation proved to be a simple and useful method for estimating bacterial production in humic water. However, an appropriate amount of [¹⁴C]leucine has to be used to ensure the maximum uptake of label and to minimize isotope dilution.