N-Carbamoyl Amino Acid Solid–Gas Nitrosation by NO/NO x : A New Route to Oligopeptides via α-Amino Acid N-Carboxyanhydride. Prebiotic Implications

α-N-Carbamoyl amino acid (CAA), whose conditions of formation in a prebiotic hydrosphere have been described previously (Taillades et al. 1998), could have been an important intermediate in prebiotic peptide synthesis through reaction with atmospheric NO _x . Nitrosation of solid CAA (glycine or valine derivative) by a 4/1 NO/O₂ gaseous mixture (1 atm) yields N-carboxyanhydride (NCA) quantitatively in less than 1 h at room temperature. The crude solid NCA undergoes quantitative oligomerization (from trimer to nonamer under the conditions we used) when treated with a (bi)carbonate aqueous buffer at pH 9. We therefore suggest that part of the prebiotic amino acid activation/polymerization process may have taken place in a dry phase (``drying-lagoon' scenario). Received: 23 June 1998 / Accepted: 7 December 1998     

Production of Guanine from NH4CN Polymerizations

The synthesis of adenine from the polymerization of concentrated ammonium cyanide solutions is well known. We show here that guanine is also produced by this reaction but at yields ranging from 10 to 40 times less than that of adenine. This synthesis is effective at both +80 and −20°C. Since high concentrations of NH₄CN are obtainable only by freezing, this prebiotic synthesis would be applicable to frozen regions of the primitive Earth, the Jovian satellite Europa and other icy satellites, and the parent body of the Murchison meteorite. Received: 18 September 1998 / Accepted: 31 March 1999     

Improved automated solid-phase microsequencing of peptides using DABITC

The methylated purines O⁶-methyl- and 7-methylguanine were isolated from mouse liver DNA hydrolysates by means of a column cleanup employing a Sep Pak C-18 reverse-phase cartridge. The purine bases were eluted from the cartridge with methanol, evaporated to dryness, and then dissolved in mobile phase for liquid chromatographic analysis by normalphase chromatography. The system consisted of a LiChrosorb Si 60 column with a watersaturated mobile phase of 20% methanol in chloroform containing 0.001% H₃PO₄. The two methylated bases eluted before adenine or guanine. For extremely low-level (<300 pmol) quantitation, the peaks corresponding to O⁶-methyl- and 7-methylguanine were collected and then analyzed by reverse-phase chromotography with a LiChrosorb RP-18 column and a mobile phase of 5% methanol in pH 7 phosphate buffer (for 7-methylguanine) or 9.5% methanol/buffer (for O⁶-methylguanine). Comparisons were made with fluorescence detection and with scintillation counting (in animal studies where [¹⁴C]dimethylnitrosamine was used). Minimum detectable levels at 254 nm were about 3 ng (3:1 signal to noise ratio) for each of the title compounds. As low as 10 pmol/mg of each could be detected in DNA hydrolysates. Recoveries of O⁶-methyl- and 7-methylguanine from DNA spiked at 750 pmol/mg were greater than 80%.     

Substrate-Directed formation of small biocatalysts under prebiotic conditions

One of the most debated issues concerning the origin of life, is how enzymes which are essential for existence of any living organism, evolved. It is clear that, regardless of the exact mechanism, the process should have been specific and reproducible, involving interactions between different molecules. We propose that substrate templating played a crucial role in maintaining reproducible and specific formation of prebiotic catalysts. This work demonstrates experimentally, for the first time, substrate-directed formation of an oligopeptide that possesses a specific catalytic activity toward the substrate on which it was formed. In our experiments we used the substrate o-nitrophenol-β-d-galactopyranoside (ONPG) as a molecular template for the synthesis of a specific catalyst that is capable of cleaving the same substrate. This was achieved by incubation of the substrate with free amino acids and a condensing agent (dicyandiamide) at elevated temperatures. A linear increase with time of the reaction rate (d[product]/d²t), pointed to an acceleration regime, where the substrate generates the formation of the catalyst. The purified catalyst, produced by a substrate-directed mechanism, was analyzed, and identified as Cys₂-Fe⁺². The mechanism of substrate-directed formation of prebiotic catalysts provides a solution to both the specificity and the reproducibility requirements from any prebiotic system which should evolve into the biological world. Received: 26 January 1996 / Accepted: 22 April 1997     

On the Origin of Metabolic Pathways

The heterotrophic theory of the origin of life is the only proposal available with experimental support. This comes from the ease of prebiotic synthesis under strongly reducing conditions. The prebiotic synthesis of organic compounds by reduction of CO₂ to monomers used by the first organisms would also be considered an heterotrophic origin. Autotrophy means that the first organisms biosynthesized their cell constituents as well as assembling them. Prebiotic synthetic pathways are all different from the biosynthetic pathways of the last common ancestor (LCA). The steps leading to the origin of the metabolic pathways are closer to prebiotic chemistry than to those in the LCA. There may have been different biosynthetic routes between the prebiotic and the LCAs that played an early role in metabolism but have disappeared from extant organisms. The semienzymatic theory of the origin of metabolism proposed here is similar to the Horowitz hypothesis but includes the use of compounds leaking from preexisting pathways as well as prebiotic compounds from the environment.     

Evidence for a Multi-ion Pore Behavior in the Plant Potassium Channel KAT1

KAT1 is a cloned voltage-gated K⁺ channel from the plant Arabidopsis thaliana L., which displays an inward rectification reminiscent of `anomalous' rectification of the i <sub>f</sub> pacemaker current recorded in animal cells. Macroscopic conductance of KAT1 expressed in Xenopus oocytes was 5-fold less in pure Rb<sup>+</sup> solution than in pure K<sup>+</sup> solution, and negligible in pure Na<sup>+</sup> solution. Experiments in different K<sup>+</sup>/Na<sup>+</sup> or K<sup>+</sup>/Rb<sup>+</sup> mixtures revealed deviations from the principle of independence and notably two anomalous effects of the K<sup>+</sup>/Rb<sup>+</sup> mole fraction (i.e., the ratio [K<sup>+</sup>]/([K<sup>+</sup>]+[Rb<sup>+</sup>])). First, the KAT1 deactivation time constant was both voltage- and mole fraction-dependent (a so-called `foot in the door' effect was thus observed in KAT1 channel). Second, when plotted against the K⁺/Rb⁺ mole fraction, KAT1 conductance values passed through a minimum. This minimum is more important for two pore mutants of KAT1 (T259S and T260S) that displayed an increase in P_Rb/P_K. These results are consistent with the idea that KAT1 conduction requires several ions to be present simultaneously within the pore. Therefore, this atypical `green' member of the Shaker superfamily of K⁺ channels further shows itself to be an interesting model as well for permeation as for gating mechanism studies. Received: 9 February 1998/Revised: 28 July 1998     

tRNA methylases from HeLa cells: purification and properties of an adenine-1-methylase and a guanine-N2-methylase

Two enzymes (methylases) that catalyze the transfer of methyl groups from S-adenosyl-l-methionine to tRNA (prepared from Escherichia coli) have been partially purified from extracts of HeLa cells. One catalyzes the methylation of adenine residues of the tRNA to give 1-methyladenine units and the other is responsible for the conversion of guanine residues to N²-methylguanine and N²,N²-dimethylguanine (and may be a mixture of two enzymes). Activities of these relatively unstable enzymes could be maintained by storage at ?20 °C in the presence of 50% glycerol. Substrate specificity studies have revealed that bacterial tRNA (E. coli, Bacillus subtilis) can be used as substrate, whereas tRNA of animal origin (HeLa cells, rat liver) cannot be used. Of the specific tRNA's tested, E. coli tRNA^fMet was used as substrate by both enzymes. E. coli tRNA^Tyr was used by the adenine-1-methylase but not by the guanine-N²-methylase. The adenine-1-methylase catalyzed the transfer of approximately one methyl group per mole of either tRNA^fMet or tRNA^Tyr offered as substrate; in the presence of the guanine-N²-methylase 1 mole of E. coli tRNA^fMet accepted 1 mole of methyl. Studies with the use of both enzymes established that enzymic methylation of the guanine site of E. coli tRNA^fMet did not interfere with subsequent methylation of an adenine residue and neither did prior methylation of adenine interfere with the subsequent methylation of a guanine residue. In the presence of both enzymes, approximately 2 moles of methyl groups were accepted by 1 mole of the E. coli tRNA^fMet.     

Suppression of Inward-Rectifying K+ Channels KAT1 and AKT2 by Dominant Negative Point Mutations in the KAT1 α-Subunit

The Arabidopsis thaliana cDNA, KAT1 encodes a hyperpolarization-activated K⁺ (K⁺ _in ) channel. In the present study, we identify and characterize dominant negative point mutations that suppress K⁺ _in channel function. Effects of two mutations located in the H5 region of KAT1, at positions 256 (T256R) and 262 (G262K), were studied. The co-expression of either T256R or G262K mutants with KAT1 produced an inhibition of K⁺ currents upon membrane hyperpolarization. The magnitude of this inhibition was dependent upon the molar ratio of cRNA for wild-type to mutant channel subunits injected. Inhibition of KAT1 currents by the co-expression of T256R or G262K did not greatly affect the ion selectivity of residual currents for Rb⁺, Na⁺, Li⁺, or Cs⁺. When T256R or G262K were co-expressed with a different K⁺ channel, AKT2, an inhibition of the channel currents was also observed. Voltage-dependent Cs⁺ block experiments with co-expressed wild type, KAT1 and AKT2, channels further indicated that KAT1 and AKT2 formed heteromultimers. These data show that AKT2 and KAT1 are able to co-assemble and suggest that suppression of channel function can be pursued in vivo by the expression of the dominant negative K ⁺ _in channel mutants described here. Received: 2 July 1998/Revised: 23 October 1998     

Involvement of Protein Tyrosine Kinase in Osmoregulation of Na+ Transport and Membrane Capacitance in Renal A6 Cells

Renal A6 cells have been reported in which hyposmolality stimulates Na⁺ transport by increasing the number of conducting amiloride-sensitive 4-pS Na⁺ channels at the apical membrane. To study a possible role of protein tyrosine kinase (PTK) in the hyposmolality-induced signaling, we investigated effects of PTK inhibitors on the hyposmolality-induced Na⁺ transport in A6 cells. Tyrphostin A23 (a PTK inhibitor) blocked the stimulatory action of hyposmolality on a number of the conducting Na⁺ channels. Tyrphostin A23 also abolished macroscopic Na⁺ currents (amiloride-sensitive short-circuit current, I _Na ) by decreasing the elevating rate of the hyposmolality-increased I _Na . Genistein (another type of PTK inhibitor) also showed an effect similar to tyrphostin A23. Brefeldin A (BFA), which is an inhibitor of intracellular translocation of protein, blocked the action of hyposmolality on I _Na by diminishing the elevating rate of the hyposmolality-increased I _Na , mimicking the inhibitory action of PTK inhibitor. Further, hyposmolality increased the activity of PTK. These observations suggest that hyposmolality would stimulate Na⁺ transport by translocating the Na⁺ channel protein (or regulatory protein) to the apical membrane via a PTK-dependent pathway. Further, hyposmolality also caused an increase in the plasma (apical) membrane capacitance, which was remarkably blocked by treatment with tyrphostin A23 or BFA. These observations also suggest that a PTK-dependent pathway would be involved in the hyposmolality-stimulated membrane fusion in A6 cells. Received: 6 October 1999/Revised: 4 February 2000     

Utilization of adenine and guanine as nitrogen sources by Chlamydomonas reinhardtii cells

Chlamydomonas reinhardtü Dangeard, adenine or guanine can be used as the sole nitrogen source for growth by means of an inducible system which is repressed by ammonia. Cells grown on either adenine or guanine were able to take up both purines, although the adenine uptake rate was always about 40% of the guanine uptake rate. Both adenine and guanine were taken up by an inducible system(s) exhibiting hyperbolic kinetics with identical apparent A, values of 3-2 mmol m^?3 for adenine and 3-2mmol m^?3 for guanine. Adenine and guanine utilization depended on pH, with similar optimal pH values of 7·3 and 7·4, respectively. Adenine and guanine each acted as a competitive inhibitor of the other's uptake, and their utilization was also inhibited by hypoxanthine, xanthine and urate. Inhibition of adenine uptake by guanine and hypoxanthine was competitive, with A′, values of 5·5 and 1. 6 mmol m^?3 respectively. Guanine uptake was also inhibited competitively by adenine (K₁= 1·3mmol m^?3) and hypoxanthine (K₁= 3. 3 mmol m^?3). Utilization of both adenine and guanine was inhibited by cyanide, azide, 3-(3,4-dichlorophenyl)-1,1-dimethyl urea, 2,4-dinitrophenol and carbonylcyanide m-chlorophenylhydrazone, and was also sensitive to p-hydroxymercuribenzoate and N-ethyl-maleimide. On the basis of these results, taken together, the possibility that adenine and guanine are translocated into Chlamydomonas by a common system is discussed.     

Effect of Adenosine on Na+ and Cl− Currents in A6 Monolayers. Receptor Localization and Messenger Involvement

The effect of adenosine regulation on sodium and chloride transport was examined in cultured A₆ renal epithelial cells. Adenosine and its analogue N⁶-cyclopentyladenosine (CPA) had different effects on short-circuit current (I _sc) depending on the side of addition. Basolateral CPA addition induced an approximately threefold increase of the I _sc that reached a maximum effect 20 min after addition and was completely inhibited by preincubation with either an A₂ selective antagonist, CSC, or the sodium channel blocker, amiloride. Apical CPA addition induced a biphasic I _sc response characterized by a rapid fourfold transient increase over its baseline followed by a decline and a plateau phase that were amiloride insensitive. The A₁ adenosine antagonist, CPX, completely prevented this response. This I _sc response to apical CPA was also strongly reduced in Cl⁻-free media and was significantly inhibited either by basolateral bumetanide or apical DPC preincubation. Only basolateral CPA addition was able to induce an increase in cAMP level. CPA, added to cells in suspension, caused a rapid rise in [Ca²⁺] _i that was antagonized by CPX, not affected by CSC and prevented by thapsigargin preincubation. These data suggest that basolateral CPA regulates active sodium transport via A₂ adenosine receptors stimulating adenylate cyclase while apical CPA regulates Cl⁻ secretion via A₁ receptor-mediated changes in [Ca²⁺] _i .     

Blood-Brain Barrier Permeation: Molecular Parameters Governing Passive Diffusion

53 compounds with clinically established ability to cross or not to cross the blood-brain barrier by passive diffusion were characterized by means of surface activity measurements in terms of three parameters, i.e., the air-water partition coefficient, K _aw , the critical micelle concentration, CMC _D , and the cross-sectional area, A _D . A three-dimensional plot in which the surface area, A _D , is plotted as a function of K ⁻¹ _aw and CMC _D shows essentially three groups of compounds: (i) very hydrophobic compounds with large air-water partition coefficients and large cross-sectional areas, A _D > 80 ?² which do not cross the blood-brain barrier, (ii) compounds with lower air-water partition coefficients and an average cross-sectional area, A _D ≅ 50 ?² which easily cross the blood-brain barrier, and (iii) hydrophilic compounds with low air-water partition coefficients (A _D < 50 ?²) which cross the blood-brain barrier only if applied at high concentrations. It was shown that the lipid membrane-water partition coefficient, K _lw , measured previously, can be correlated with the air-water partition coefficient if the additional work against the internal lateral bilayer pressure, π _bi = 34 ± 4 mN/m is taken into account. The partitioning into anisotropic lipid membranes decreases exponentially with increasing cross-sectional areas, A _D , according to K _lw =const. K _aw exp(−A _D π _bi /kT) where kT is the thermal energy. The cross-sectional area of the molecule oriented at a hydrophilic-hydrophobic interface is thus the main determinant for membrane permeation provided the molecule is surface active and has a pK _a > 4 for acids and a pK _a < 10 for bases. Received: 7 April 1998/Revised: 25 June 1998     

Chloride Currents Activated by Calcitonin and cAMP in Primary Cultures of Rabbit Distal Convoluted Tubule

Chloride (Cl⁻) conductances were studied in primary cultures of the bright part of rabbit distal convoluted tubule (DCTb) by the whole cell patch clamp technique. The bath solution (33°C) contained (in mm): 140 NaCl, 1 CaCl₂, 10 N-2-hydroxy-ethylpiperazine-N′-2-ethanesulfonic acid (HEPES), pH 7.4 and the pipette solution 140 N-methyl-d-glucamine (NMDG)-Cl, 5 MgATP, 1 ethylene-glycol-bis(b-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 10 HEPES, pH 7.4. We identified a Cl⁻ current activated by 10⁻⁵ m forskolin, 10⁻³ m 8-bromo adenosine 3′,5′-cyclic monophophosphate (8 Br-cAMP), 10⁻⁶ m phorbol 12-myristate 13-acetate (PMA), 10⁻³ m intracellular adenosine 3′,5′-cyclic monophophosphate (cAMP) and 10⁻⁷ m calcitonin. The current-voltage relationship was linear and the relative ion selectivity was Br⁻ > Cl⁻≫ I⁻ > glutamate. This current was inhibited by 10⁻³ m diphenylamine-2-carboxylate (DPC) and 10⁻⁴ m 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and was insensitive to 10⁻³ m 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS). These characteristics are similar to those described for the cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ conductance. In a few cases, forskolin and calcitonin induced an outwardly rectifying Cl⁻ current blocked by DIDS. To determine the exact location of the Cl⁻ conductance 6-methoxy-1-(3-sulfonatopropyl) quinolinium (SPQ) fluorescence experiments were carried out. Cultures seeded on collagen-coated permeable filters were loaded overnight with 5 mm SPQ and the emitted fluorescence analyzed by laser-scan cytometry. Cl⁻ removal from the apical solution induced a Cl⁻ efflux which was stimulated by 10⁻⁵ m forskolin, 10⁻⁷ calcitonin and inhibited by 10⁻⁵ m NPPB. In 140 mm NaBr, forskolin stimulated an apical Br⁻ influx through the Cl⁻ pathway. Forskolin and calcitonin had no effect on the basolateral Cl⁻ permeability. Thus in DCTb cultured cells, exposure to calcitonin activates a Cl⁻ conductance in the apical membrane through a cAMP-dependent mechanism. Received: 5 July 1995/Revised: 21 December 1995     

Sodium Recirculation and Isotonic Transport in Toad Small Intestine

Isolated small intestine of toad (Bufo bufo) was mounted on glass tubes for perfusion studies with oxygenated amphibian Ringer's solution containing glucose and acetate. Under open-circuit conditions (V _t =−3.9 ± 1.8 mV, N= 14) the preparation generated a net influx of ¹³⁴Cs⁺. The time course of unidirectional ¹³⁴Cs⁺-fluxes was mono-exponential with similar rate constants for influx and outflux when measured in the same preparation. The flux-ratio was time invariant from the beginning of appearance of the tracers to steady state was achieved. Thus, just a single pathway, the paracellular pathway, is available for transepithelial transport of Cs⁺. From the ratio of unidirectional Cs⁺-fluxes the paracellular force was calculated to be, 18.2 ± 1.5 mV (N= 6), which is directed against the small transepithelial potential difference. The paracellular netflux of cesium ions, therefore, is caused by solvent drag. The flux of ¹³⁴Cs⁺ entering and trapped by the cells was of a magnitude similar to that passing the paracellular route. Therefore, independent of the convective flux of ¹³⁴Cs⁺, every second ¹³⁴Cs⁺ ion flowing into the lateral space was pumped into the cells rather than proceeding, via the low resistance pathway, to the serosal bath. It is thus indicated that the paracellular convective flow of ¹³⁴Cs⁺ is driven by lateral Na⁺/K⁺-pumps. Transepithelial unidirectional ⁴²K⁺ fluxes did not reach steady state within an observation period of 70 min, indicating that components of the fluxes in both directions pass the large cellular pool of potassium ions. The ratio of unidirectional ²⁴Na⁺ fluxes was time-variant and declined from an initial value of 3.66 ± 0.34 to a significantly smaller steady-state value of 2.57 ± 0.26 (P < 0.001, N= 5 paired observations), indicating that sodium ions pass the epithelium both via the paracellular and the cellular pathway. Quantitatively, the larger ratio of paracellular Na⁺ fluxes, as compared to that of paracellular Cs⁺ fluxes, is compatible with convective flow of the two alkali metal ions through the same population of water-filled pores. With a new set of equations, the fraction of the sodium flux passing the basement membrane barrier of the lateral space that is recirculated through the cellular compartment is estimated. This fraction was, on average, 0.72 ± 0.03 (N= 5). It is concluded that isotonicity of the transportate can be maintained by producing a hypertonic fluid emerging from the lateral space combined with reuptake of salt via the cells. Received: 14 October 1998/Revised: 14 January 1999     

A Basolateral Chloride Conductance in Rat Lingual Epithelium

We used Ussing chamber measurements and whole-cell recordings to characterize a chloride conductance in rat lingual epithelium. Niflumic acid (NFA) and flufenamic acid (FFA), nonsteroidal anti-inflammatory aromatic compounds known to inhibit Cl⁻ conductances in other tissues, reduced transepithelial short-circuit current (I _sc ) in the intact dorsal anterior rat tongue epithelium when added from the serosal side, and reduced whole-cell currents in rat fungiform taste cells. In both Ussing chamber and patch-clamp experiments, the effect of NFA was mimicked by replacement of bath Cl⁻ with methanesulfonate or gluconate. In low Cl⁻ bath solution, the effect of NFA on whole-cell current was reduced. Replacement of bath Ca²⁺ with Ba²⁺ reduced the whole-cell Cl⁻ current. We conclude that a Ca²⁺-activated Cl⁻ conductance is likely present in the basolateral membrane of the rat lingual epithelium, and is present in the taste receptor cells from fungiform papillae. Further experiments will be required to identify the role of this conductance in taste transduction. Received: 8 September 1997/Revised: 27 March 1998     

Effects of Auxin Transport Inhibitors on Gibberellins in Pea

The effects of the auxin transport inhibitors 2,3,5-triiodobenzoic acid (TIBA), 9-hydroxyfluorene-9-carboxylic acid (HFCA), and 1-N-naphthylphthalamic acid (NPA) on gibberellins (GAs) in the garden pea (Pisum sativum L.) were studied. Application of these compounds to elongating internodes of intact wild type plants reduced markedly the endogenous level of the bioactive gibberellin A₁ (GA₁) below the application site. Indole-3-acetic acid (IAA) levels were also reduced, as was internode elongation. The auxin transport inhibitors did not affect the level of endogenous GA₁ above the application site markedly, nor that of GA₁ precursors above or below it. When plants were treated with [¹³C,³H]GA₂₀, TIBA reduced dramatically the level of [¹³C,³H]GA₁ recovered below the TIBA application site. The internodes treated with auxin transport inhibitors appeared to be still in the phase where endogenous GA₁ affects elongation, as indicated by the strong response to applied GA₁ by internodes of a GA₁-deficient line at the same stage of expansion. On the basis of the present results it is suggested that caution be exercised when attributing the developmental effects of auxin transport inhibitors to changes in IAA level alone. Received April 13, 1998; accepted April 14, 1998     

Discontinuities in the Temperature Function of Transmembrane Water Transport in Chara: Relation to Ion Transport

The NMR (nuclear magnetic resonance) method of Conlon and Outhred (1972) was used to measure diffusional water permeability of the nodal cells of the green alga Chara gymnophylla. Two local minima at 15 and 30°C of diffusional water permeability (P _d ) were observed delimiting a region of low activation energy (E _a around 20 kJ/mol) indicative of an optimal temperature region for membrane transport processes. Above and below this region water transport was of a different type with high E _a (about 70 kJ/mol). The triphasic temperature dependence of the water transport suggested a channel-mediated transport at 15–30°C and lipid matrix-mediated transport beyond this region. The K⁺ channel inhibitor, tetraethylammonium as well as the Cl⁻ channel inhibitor, ethacrynic acid, diminished P _d in the intermediate temperature region by 54 and 40%, respectively. The sulfhydryl agent p-(chloromercuri-benzensulfonate) the water transport inhibitor in erythrocytes also known to affect K⁺ transport in Chara, only increased P _d below 15°C. In high external potassium (`K-state') water transport minima were pronounced. The role of K⁺ channels as sensors of the optimal temperature limits was further emphasized by showing a similar triphasic temperature dependence of the conductance of a single K⁺ channel also known to cotransport water, which originated from cytoplasmic droplets (putatively tonoplast) of C. gymnophylla. The minimum of K⁺ single channel conductance at around 15°C, unlike the one at 30°C, was sensitive to changes of growth temperature underlining membrane lipid involvement. The additional role of intracellular (membrane?) water in the generation of discontinuities in the above thermal functions was suggested by an Arrhenius plot of the cellular water relaxation rate which showed breaks at 13 and 29°C. Received: 12 August 1998/Revised: 13 November 1998     

Potentiation by Isoproterenol on Carbachol-induced K+ and Cl− Currents and Fluid Secretion in Rat Parotid

Isoproterenol (IPR) and 8-(4-chlorophenylthio)-cyclic AMP (cpt-cAMP) enhanced carbachol (CCh)-induced fluid secretion from rat parotid glands, but had no effect by themselves. The enhancement by IPR was blocked by propranolol. In dispersed parotid acinar cells, IPR and cpt-cAMP potentiated CCh-induced K⁺ and Cl⁻ currents (I _K and I _Cl). IPR at the concentration of 0.1 μm significantly potentiated the CCh-induced increase in intracellular Ca²⁺ concentration ([Ca²⁺] _i ), but 1 mm cpt-cAMP did not. The incidence of the potentiation by IPR in CCh-induced Mn²⁺ entry was 31% and that by cpt-cAMP was 21%. The potentiation by IPR in the ionic currents and the [Ca²⁺] _i was suppressed by propranolol. These results suggest that the CCh-induced fluid secretion from rat parotid glands is enhanced by IPR through the potentiation of I _K and I _Cl mainly by the increased cyclic AMP level and partially by the potentiated Ca²⁺ influx and [Ca²⁺] _i increase, and that IPR is more effective than cpt-cAMP in the enhancement of the CCh-induced [Ca²⁺] _i increase. Received: 6 October 1997/Revised: 16 April 1998     

Effects of Mechano-Gated Cation Channel Blockers on Xenopus Oocyte Growth and Development

The putative role(s) of a mechanically gated (MG) cation channel in Xenopus oocyte growth, maturation, fertilization and embryogenesis has been examined. Using a pharmacological approach, we have tested the effects of the MG channel blockers, gadolinium, gentamicin and amiloride on the above developmental events. Our results indicate that oocyte maturation, fertilization and early embryogenesis (up to the free-swimming stage 45) can proceed normally in the presence of concentrations of agents that either completely abolish (i.e., ≥10 μm Gd³⁺) or partially block (i.e., 1 mm gentamicin) single MG channel activity as measured by patch-clamp recording. However, we also find that higher concentrations of Gd³⁺ (≥50 μm) can lead to an increased percentage (>20%) of axis-perturbed embryos compared with control (<1%) and that amiloride (0.5 mm) reduces the success of fertilization (from 100% to <50%) and increases mortality (by ∼75%) in developing embryos. Furthermore, we find that all three agents inhibit oocyte growth in vitro. However, their order of effectiveness (amiloride > gentamicin > Gd³⁺) is opposite to their order for blocking MG channels (Gd³⁺≫ gentamicin > amiloride). These discrepancies indicated that the drugs effects occur by mechanisms other than, or in addition to, MG channel block. Our results provide no compelling evidence for the idea that MG channel activity is critical for development in Xenopus. This could mean that there are other mechanisms in the oocyte that can compensate when MG channel activity is blocked or that the protein that forms the channel can undergo additional interactions that result in a function insensitive to MG channel blockers. Received: 27 March 1998/Revised: 10 June 1998     

The Role of Calcium in the Volume Regulation of Rat Lacrimal Acinar Cells

Earlier studies have suggested a role for Ca²⁺ in regulatory volume decrease (RVD) in response to hypotonic stress through the activation of Ca²⁺-dependent ion channels (Kotera & Brown, 1993; Park et al., 1994). The involvement of Ca²⁺ in regulating cell volume in rat lacrimal acinar cells was therefore examined using a video-imaging technique to measure cell volume. The trivalent cation Gd³⁺ inhibited RVD, suggesting that Ca²⁺ entry is important and may be via stretch-activated cation channels. However, Fura-2 loaded cells did not show an increase in [Ca²⁺] _i during exposure to hypotonic solutions. The absence of any changes in [Ca²⁺] _i resulted from the buffering of cytosolic Ca²⁺ by Fura-2 during hypotonic shock and therefore inhibition of RVD. The intracellular Ca²⁺ chelator, BAPTA, also inhibited the RVD response to hypotonic shock. An increase in [Ca²⁺] _i induced by either acetylcholine or ionomycin, was found to decrease cell volume under isotonic conditions in lacrimal acinar cells. Cell shrinkage was inhibited by tetraethylammonium ion, an inhibitor of Ca²⁺-activated K⁺ channels. On the basis of the presented data, we suggest an involvement of intracellular Ca²⁺ in controlling cell volume in lacrimal acinar cells. Received: 20 February 1998/Revised: 1 May 1998