The modulation of the induction of ornithine decarboxylase by spermine,spermidine and diamines

Extremely low concentrations of putrescine, spermidine and spermine added to the extracellular medium of cultures of mammalian cells inhibit the induction of ornithine decarboxylase activity despite 100- to 1,000-fold greater intracellular polyamine concentrations. The diamines, 1,2-diaminoethane, 1,3-diaminopropane, 1,5-diaminopentane, 1,7-diaminoheptane, 1,10-diaminodecane, 1,12-diaminododecane also inhibit ornithine decarboxylase at all concentrations tested (greater than 10^?6 M). In contrast, 10^?6 M to 10 ^?3 M 1,8-diaminooctane, the alkyl analog of spermidine, enhances ornithine decarboxylase activity. The concentraton of putrescine required to inhibit the activity of ornithine decarboxylase by 50% is a characteristic of each cell line; however, it varies by as much as 1,000-fold among the five cell lines we have tested (L1210 leukemic, H35 hepatoma, N18 neuroblastoma, W256 carcinosarcoma and 3T3 fibroblasts). The antizyme to ornithine decarboxylase can be induced in all these cells by high (di)(poly)amine concentrations. Based on these and other experiments we suggest a working hypothesis: that the polyamines regulate ornithine decarboxylase activity through two different sites that may be interrelated; a sensitive membrane-mediated site that responds to minute fluctuations of extracellular polyamine levels and a coarse site which may be intracellular or membrane associated that responds to larger fluctuations of intracellular polyamine levels. The consequences of such a control mechanism operating within the whole organism are discussed.     

Properties of γ-aminobutyric acid synthesis by rat renal cortex

Substantial synthesis of γ-aminobutyric acid occurs in rat renal cortex. Renal glutamate decarboxylase activity (24.3±2.9 (S.E.) nmols/mg protein per h) is 15% of that in brain; renal γ-aminobutyric acid content (39.5±5.3 (S.E.) nmols/g wet wt.) is 5% of the whole brain concentration. Properties of glutamate decarboxylase were studied in homogenates of rat renal cortex and rat brain under conditions for which γ-aminobutyric acid formation from [2,3-³H]glutamate and CO₂ release from [1-¹⁴C]glutamate were equal. Several properties of renal glutamate decarboxylase distinguish it from the corresponding brain enzyme: (1) renal glutamate decarboxylase is selectively inhibited by cysteine sulfinic acid (K_i = 5·10^?5 M) ; (20 renal glutamate decarboxylase is less sensitive (K_i = 3–5·10^?5 M)_to inhibition by aminooxyacetic acid than is the brain enzyme (K_i = 1·10^?6 M); (3) brain but not renal glutamate decarboxylase activity can be substantially stimulated in vitro by the addition of exogenous pyridoxal 5′-phosphate; (4) renal glutamate decarboxylase is significantly decreased in renal cortex from rats on a low-salt diet. Proximal tubules are enriched in glutamate decarboxylase compared to the activity in whole renal cortex or glomeruli (42, 22 and 14 nmols/mg protein per h, respectively). We speculate that renal γ-aminobutyric acid synthesis does not reflect the presence of GABAergic renal nerves, but may serve a function in proximal tubular cells.     

Effect of oxygen radicals and hyperoxia on rat heart ornithine decarboxylase activity

Rat heart ornithine decarboxylase activity from isoproterenol-treated rats was inactivated in vitro by reactive species of oxygen generated by the reaction xanthine/xanthine oxidase. Reduced glutathione, dithiothreitol and superoxide dismutase had a protective effect in homogenates and in partially purified ornithine decarboxylase exposed to the xanthine/xanthine oxidase reaction, while diethyldithiocarbamate, which is an inhibitor of superoxide dismutase, potentiated the damage induced by O₂^? on enzyme activity. Dithiothreitol at concentrations above 1.25 mM had an inhibitory effect oupon supernatant ornithine decarboxylase activity, while at 2.5 mM it was most effective in the recovery of ornithine decarboxylase activity, after the purification of the enzyme by the ammonium sulphate precipitation procedure. The ornithine decarboxylase inactivated by the xanthine/xanthine oxidase reaction showed a higher value of K_m and a reduction of V_max with respect to control activity. The exposure of rates to 100% oxygen for 3 h reduced significantly the isoproterenol-induced heart ornithine decarboxylase activity. The injection with diethyldithiocarbamate 1 h before hyperoxic exposure further reduced heart ornithine decarboxylase activity.     

Purification and functional properties of the DCCD-reactive proteolipid subunit of the H+-translocating ATPase from Mycobacterium phlei

Interaction of N,N′-dicyclohexylcarbodiimide (DCCD) with ATPase of Mycobacterium phlei membranes results in inactivation of ATPase activity. The rate of inactivation of ATPase was pseudo-first order for the initial 30–65% inactivation over a concentration range of 5–50 μM DCCD. The second-order rate constant of the DCCD-ATPase interaction was k = 8.5·10⁵ M^?1·min^?1. The correlation between the initial binding of [¹⁴C]DCCD and 100% inactivation of ATPase activity shows 1.57 nmol DCCD bound per mg membrane protein. The proteolipid subunit of the F₀F₁-ATPase complex in membranes of M. phlei with which DCCD covalently reacts to inhibit ATPase was isolated by labeling with [¹⁴C]DCCD. The proteolipid was purified from the membrane in free and DCCD-modified form by extraction with chloroform/methanol and subsequent chromatography on Sephadex LH-20. The polypeptide was homogeneous on SDS-acrylamide gel electrophoresis and has an apparent molecular weight of 8000. The purified proteolipid contains phosphatidylinositol (67%), phosphatidylethanolamine (18%) and cardiolipin (8%). Amino acid analysis indicates that glycine, alanine and leucine were present in elevated amounts, resulting in a polarity of 27%. Cysteine and tryptophan were lacking. Butanol-extracted proteolipid mediated the translocation of protons across the bilayer, in K⁺-loaded reconstituted liposomes, in response to a membrane potential difference induced by valinomycin. The proton translocation was inhibited by DCCD, as measured by the quenching of fluorescence of 9-aminoacridine. Studies show that vanadate inhibits the proton gradient driven by ATP hydrolysis in membrane vesicles of M. phlei by interacting with the proteolipid subunit sector of the F₀F₁-ATPase complex.     

5′-Haloacetamido-5′-deoxythymidines: novel inhibitors of thymidylate synthase

5′-Bromoacetamido-5′-deoxythymidine (BAT), 5′-iodoacetamido-5′-deoxythymidine (IAT), 5′-chloroacetamido-5′-deoxythymidine (CAT) and [¹⁴C]BAT were synthesized and their interactions with thymidylate synthase purified from L1210 cells were invesatigated. The inhibitory effects of these compounds on thymidylate synthase were in the order BAT > IAT > CAT, which is in agreement with their cytotoxic effects in L1210 cells. In the presence of substrate during preincubation, the concentration required for 50% inhibition of the enzyme activity by these inhibitors was 4–8 fold higher than it was in the absence of dUMP. The I₅₀ values for BAT were 1·10⁻⁵ M and 1.2·10⁻⁶ M in the presence and absence, respectively, of dUMP during preincubation. These results were in agreement with the observed inhibition of thynmidylate synthase by BAT in intact L1210 cells. A Lineweaver-Burk plot revealed that BAT behaved as a competitive inhibitor. The K_m for the enzyme was 9.2 μM, and the K_i determined for competitive inhibition by BAT was 5.4 μM. Formation of a tight, irreversible compledx is referred from the finding that BAT-inactivation of thymidylate synthase was not reversible on prolonged dialysis and that the enzyme-BAT complex was nondissociable by gel filtration through a Sephadex G-25 column or by TSK-125 column chromatography. Incubation of thymidylate synthase with BAT resulted in time-dependent, irreversible loss of enzyme activity by first-order kinetics. The rate constant for inactivation was 0.4 min⁻¹, and the steady-state constant of inactivation, K_i, was estimated to be 6.6 μM. The 5′-haloacetamido-5′-deoxythymidines provide specific inhibitors of thymidylate synthase that may also serve as reagents for studying the enzyme mechanism.     

The induction of ornithine decarboxylase by tumor promoting phorbol ester analogues in Reuber H35 hepatoma cells

The induction of ornithine decarboxylase activity was studied in a rat hepatoma cell line (Reuber H35) incubated with a group of structurally-related phorbol ester analogues. A single application of 1.6 μM of tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) to H35 cells caused a dramatic increase in the activity of ornithine decarboxylase. The stimulation of the enzyme activity was rapid but transient, peaking at 4 to 5 hr with a value which was 116-fold greater than control and then declining to the basal level after 8 hr. In addition, the increase in ODC activity was dependent upon the concentration of TPA added to the culture medium and the EC₅₀ was estimated to be about 2.63 × 10^?7 M. Our studies of the effect of various phorbol ester analogues on the H35 ODC activity indicated an apparent correlation between the ability of phorbol ester derivatives to induce ODC activity in the H35 cells and their activity to promote papilloma formation in the mouse skin in that the various derivatives possessed the following relative abilities to increase ODC activity: TPA > PDB > PDA > 4 α-P > 4 α-PDD. Concurrent addition of either actinomycin D or cycloheximide abolished the increase in ODC activity after TPA treatment. Changes of intracellular concentrations of polyamines, particularly putrescine, were in good agreement with the increase in ODC activity in response to TPA: a 10-fold increase in putrescine over the control level was observed at 6 hr. Our data suggest that cultured Reuber H35 hepatoma cells exhibit a marked and specific response to the phorbol ester tumor promoters and may be of great value in studying the biochemical mechanism of ODC induction by these agents.     

Valinomycin inhibition of insulin release and alteration of the electrical properties of pancreatic B cells

The effects of valinomycin on insulin release and rubidium efflux from perifused isolated rat islets were investigated and correlated with its effects on the electrical properties of mouse B cells studied with microelectrode techniques. Valinomycin produced a (1 · 10^?9–1 · 10^?6 M) dose- and time-dependent inhibition of (10–15 mM) glucose-stimulated insulin release but did not affect basal secretion. This inhibitory effect rapidly followed addition of the ionophore and equally affected the two phases of glucose-stimulated secretion. It was not reversible by simple washing of the islets, but could be reversed transiently by tetraethylammonium or high extracellular potassium ion levels. At low or high glucose, valinomycin rapidly augmented the rate of ⁸⁶Rb⁺ efflux from preloaded islets. Amplitude and rapidity of this effect were dose-dependent and it was antagonized by tetraethylammonium. Glucose metabolism by islet cells was reduced only slightly (15%) by 1 · 10^?7 M valinomycin. During the first 6 to 8 min of valinomycin addition, the membrane potential of B cells augmented slowly but the typical bursts of spikes disappeared rapidly. Later on, B cells hyperpolarized more quickly to a stable value of approx. ?70 mV. Increasing extracellular K⁺ immediately depolarized B cells and the linear relationship found between the logarithm of K⁺ concentration and the membrane potential was characterized by a slope of 58 mV for a ten-fold increase in extracellular K⁺.These results suggest that valinomycin interferes with the insulin releasing effect of glucose by increasing the potassium permeability of the B cell membrane.     

Action of aldosterone on frog skin in the presence and absence of in vitro molting effects

The molting which occurs in frog skin following exposure to high concentrations of aldosterone interferes with the interpretation of physiological measurements. Exposure of skins from frogs maintained in standard smooth tanks to 5 · 10^?7 M aldosterone caused within a few hours erratic responses in short-circuit current I₀ and conductance κ followed by sustained stimulation of I₀ and κ; 10^?8 M aldosterone caused only stimulation of I₀ and κ. Storage of frogs in “rough tanks” eliminated in vitro molting on exposure to 5 · 10^?7 M aldosterone. I₀ and κ were then superimposable for 3 h, after which I₀ increased far more rapidly than κ. These results are consistent with an early effect on permeability of the active pathway and later effects on metabolism, either a direct effect on the pump or enhanced interaction between transport and metabolism.     

Permeability and Electrical Properties of Thin Lipid Membranes

We present and discuss the permeability and electrical properties of thin lipid membranes, and the changes induced in these properties by several agents added to the aqueous phases after the membranes have formed. The unmodified membrane is virtually impermeable to ions and small "hydrophilic" solutes, but relatively permeable to water and "lipophilic" molecules. These properties are consistent with those predicted for a thin film of hydrocarbon through which matter is transported by dissolving in the membrane phase and then diffusing through it. The effect of cholesterol in reducing the water and "lipophilic" solute permeability is attributed to an increase of the "viscosity" of the hydrocarbon region, thus reducing the diffusion coefficient of molecules within this phase. The selective permeability of the membrane to iodide (I^-) in the presence of iodine (I₂) is attributed to the formation of polyiodides (perhaps I₅ ^-), which are presumed to be relatively soluble in the membrane because of their large size, and hence lower surface charge density. Thus, I₂ acts as a carrier for I^-. The effects of "excitability-inducing material" and the depsipeptides (particularly valinomycin) on ion permeability are reviewed. The effects of the polyene antibiotics (nystatin and amphotericin B) on ion permeability, discussed in greater detail, are the following: (a) membrane conductance increases with the 10th power of nystatin concentration; (b) the membrane is anion-selective but does not discriminate completely between anions and cations; (c) the membrane discriminates among anions on the basis of size; (d) membrane conductance decreases extraordinarily with increasing temperatures. Valinomycin and nystatin form independent conductance pathways in the same membrane, and, in the presence of both, the membrane can be reversibly shifted between a cation and anion permeable state by changes in temperature. It is suggested that nystatin produces pores in the membrane and valinomycin acts as a carrier.     

Properties of enzyme activities involved in protein phosphorylatino-dephosphorylation of the thyroid plasma membranes

Bovine thyroid tissue exhibited cAMP-dependent and Ca²⁺-dependent protien kinase activities as well as a basal (cAMP- and Ca²⁺-independent) one, and phosphoprotein phosphatase activity. Although the former two protein kiniase activities were not clearly demonstrated using endogenous protein as substrate, they were clearly shown in soluble, particulate and plasma membrane fractions using exogenous histones as substrate. The highest specific activities were in the plasma membrane. The apparent K_m values of cAMP and Ca²⁺ for the membrane-bound protein kinase were 5·10^?8 M and 8.3·10^?4M (in the presence of 1 mM EGTA), respectively. The apparent K_m values of Mg²⁺ were 7·10^?4 M (without cAMP and Ca²⁺, 5·10^?4 M (with cAMP) and 1.3·10^?3 M (with Ca²⁺), and those ATP were 3.5·10^?5 M (with or without cAMP) and 8.5·10^?5 M (with Ca²⁺). The Ca²⁺-dependent protein kinase could be dissociated from the membrane by EGTA-washing. The enzyme activity so released was further activated by added phospholipid (phosphatidylserine/1,3-diolein), but not by calmodulin. Phosphoprotein phosphatase activity was also clearly demonstrated in all of the fractions using ³²P-labeled mixed histones as substrate. The activity was not modified by either cAMP or Ca²⁺, but was sitmulated by a rather broad range (5–25 mM) of Mg²⁺ and Mn²⁺. NaCl and substrate concentrations also influenced the activity. Pyrophosphate, ATP, inorganic phosphate and NaF inhibited the activity in a dose-dependent manner. Trifluoperazine, chlorpromazine, dibucaine and Triton X-100 (above 0.05%, w/v) specifically inhibited the Ca²⁺-dependent protein kinase in plasma membranes. Repetitive phosphorylation of intrinsic and extrinsic proteins by the membrane-bound enzyme activities clearly showed an important co-ordination of them at the step of protein phosphorylation. These findings suggest that these enzyme activities in plasma membranes may contribute to regulation of thyroid function in response to external stimuli.     

3,5-Di-tert-butyl-4-hydroxybenzylidenemalononitrile. Effects of pH on its binding to liposomes and evidence for formation of a ternary complex with valinomycin and potassium ion

1. The properties of 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile (SF 6847) were studied chemically and spectroscopically. Two molecular species of SF6847 were identified: the undissociated form (SFH; ?₃₆₃, 10 mM^?1) and the dissociated form (SF^?; ?₄₅₄, 35 mM^?1). The pK_a value of the molecule was determined to be 6.9.2. On the basis of these properties the interactions of SF6847 with liposomes and valinomycin · K⁺ were studied. The partition constants of SFH (Kⁿ_p and SF^? (K^?_p) to liposomes were determined separately; Kⁿ_p was 56 mM^?1 and was independent of the pH of the medium, whereas K^?_p dependend greatly on the pH, being 1.2 mM^?1 at pH 7.0 and 2.9 mM^?1 at pH 8.0. Using these values, the partition constant of total SF6847 (K_p) was calculated and found to be essentially the same as that calculated from the kinetics of proton uptake. It was concluded that the amount of SF^? bound to liposomes is rate limiting for proton uptake.3. The effects of membrane potential on partition constants were studied. The K^?_p decreased greatly upon generation of a membrane potential negative inside the liposomes but increased upon generation of a membrane potential positive inside the liposomes.4. The interaction of SF6847 with valinomycin in aqueous solution and in liposomes was demonstrated only in the presence of potassium ion. Potassium ion could not be replaced by sodium ion. Evidence was obtained for the formation of the ternary complex valinomycin · K⁺ · SF^? in liposomes and in hexane. It was concluded that SF^? became more soluble in the liposomal membranes on formation of this ternary complex. All these results support our proposed mechanism for the proton uptake cycle (Yamaguchi, A. and Anraku, Y. (1978) Biochim. Biophys. Acta 501, 136–149).     

The appearance of an ornithine decarboxylase inhibitory protein upon the addition of putrescine to cell cultures

Quiescent, contact inhibited H-35 rat hepatoma cell cultures maintained in minimal essential medium contain a very low level of ornithine decarboxylase activity. However, 2 h after the addition of 10% fetal aclf serum to the culture medium, the enzyme activity increases by approx. 100-fold. This increase can be completely inhibited by the simultaneous additionof 10^?2 M putrescine. The presence of putrescine elicits the appearance of an intracellular inhibitor of ornithine decarboxylase. This inhibitor of ornithine decarboxylase has a molecular weight of 26500, is sensitive to the action of chymotrypsin and its noncompetitive with respect to ornithine. The intracellular appearance of this inhibitor is sensitive to cycloheximide but is only partially inhibited by actinomycin D.     

M3 cholinoreceptors alter electrical activity of rat left atrium via suppression of L-type Ca<Superscript>2+</Superscript> current without affecting K<Superscript>+</Superscript> conductance

Electrophysiological effects produced by selective activation of M3 cholinoreceptors were studied in isolated left atrium preparations from rat using the standard sharp glass microelectrode technique. The stimulation of M3 receptors was obtained by application of muscarinic agonist pilocarpine (10^?5 M) in the presence of selective M2 antagonist methoctramine (10^?7 M). Stimulation of M3 receptors induced marked reduction of action potential duration by 14.4 ± 2.4% and 16.1 ± 2.5% of control duration measured at 50 and 90% of repolarization, respectively. This effect was completely abolished by selective M3 blocker 4-DAMP (10^?8 M). In isolated myocytes obtained from the rat left atrium, similar pharmacological stimulation of M3 receptors led to suppression of peak L-type calcium current by 13.9 ± 2.6% of control amplitude (measured at +10 mV), but failed to affect K⁺ currents I _to, I _Kur, and I _Kir. In the absence of M2 blocker methoctramine, pilocarpine (10^?5 M) produced stronger attenuation of I _CaL and induced an increase in I _Kir. This additive inward rectifier current could be abolished by highly selective blocker of K_ir3.1/3.4 channels tertiapin-Q (10^?6 M) and therefore was identified as I _KACh. Thus, in the rat atrial myocardium activation of M3 receptors leads to shortening of action potentials via suppression of I _CaL, but does not enhance the major potassium currents involved in repolarization. Joint stimulation of M2 and M3 receptors produces stronger action potential shortening due to M2-mediated activation of I _KACh.     

Comparison of the effects of prostaglandin I2 and prostaglandin E2 stimulation of the rat kidney adenylate cyclase-cyclic AMP systems

Prostacyclin (Prostaglandin I₂) effects on the rat kidney adenylate cyclase-cyclic AMP system were examined. Prostaglandin I₂ and prostaglandin E₂, from 8 · 10^?4 to 8 · ^?7 M stimulated adenylate cyclase to a similar extent in cortex and outer medulla. In inner medulla, prostaglandin I₂ was more effective than prostaglandin E₂ at all concentrations tested. Both prostaglandin I₂ and prostaglandin E₂ were additive with antidiuretic hormone in outer and inner medulla. Prostaglandin I₂ and prostaglandin E₂ were not additive in any area of the kidney, indicating both were working by similar mechanisms. Prostaglandin I₂ stimulation of adenylate cyclase correlated with its ability to increase renal slice cyclic AMP content. Prostaglandin I₂ and prostaglandin E₂ (1.5 · 10^?4 M) elevated cyclic AMP content in cortex and outer medulla slices. In inner medulla, with Santoquin® (0.1 mM) present to suppress endogenous prostaglandin synthesis, prostaglandin I₂ and prostaglandin E₂ increased cyclic AMP content. 6-Ketoprostaglandin F_1α, the stable metabolite of prostaglandin I₂, did not increase adenylate cyclase activity or tissue cyclic AMP content. Thus, prostaglandin I₂ activates renal adenylate cyclase. This suggests that the physiological actions of prostaglandin I₂ may be mediated through the adenylate cyclase-cyclic AMP system.     

Effect of hormonal and neuronal agents on adenylate cyclase from smooth muscle of the gastric antrum

Smooth muscle adenylate cyclase of a membrane preparation of canine gastric antrum has been characterized, and the effect of hormonal and neuronal agents examined. The enzyme is active in the presence of Mg²⁺ or Mn²⁺, but is inhibited by Ca²⁺. The K_m is 0.5 mM ATP, similar to the K_m of skeletal muscle adenylate cyclase. The enzyme is activated by isoproterenol but not norepinephrine, consistent with a β₂-catecholamine receptor-adenylate cyclase interaction. Secretin activates the enzyme in concentrations as low as 1 · 10^?11 M, while glucagon was effective only at 1 · 10^?6 M. Prostaglandin E₁ and E₂ have a biphasic effect with activation of adenylate cyclase at 1 · 10^?5 M and a small but significant inhibition of enzyme activity at 1 · 10^?11 M.     

Effects of acetylated polyamines on ornithine decarboxylase in rat HTC cells

N-Monoacetylputrescine and N⁸-monoacetylspermidine, metabolites of the naturally occurring polyamines, activate the enzyme ornithine decarboxylase (ODC). When added to cultures of hepatoma (HTC) cells growing in log phase, in concentrations of 5×10^?5M and 2.5×10^?7M respectively, these substances cause a 3 to 5-fold increase in the activity of ODC with a peak effect at one hour. This previously undescribed stimulating effect is in sharp contrast to the well established suppressing effects of nonacetylated polyamines on ODC activity.     

Amide derivatives with pyrazole carboxylic acids of 5-amino-1,3,4-thiadiazole 2-sulfonamide as new carbonic anhydrase inhibitors: Synthesis and investigation of inhibitory effects

Pyrazole carboxylic acid amides of 5-amino-1,3,4-thiadiazole-2-sulfonamide were synthesized from 4-benzoyl-1,5-diphenyl-1H-pyrazole-3-carbonyl chloride and 4-benzoyl-1-(3-nitrophenyl)-5-phenyl-1H-pyrazole-3-carbonyl chloride. Carbonic anhydrase isoenzymes (hCA-I and hCA-II) were purified from human erythrocyte cells by the affinity chromatography method. The inhibitory effects of 5-amino-1,3,4-thiadiazole-2-sulfonamide 1, acetazolamide 2 and new synthesized amides on these isozymes have been studied in vitro. The I₅₀ concentrations (the concentration of inhibitor producing a 50% inhibition of CA activity) against hydratase activity ranged from 1.2 to 2.2 nM for hCA-I and from 0.4 to 2 nM for hCA-II. The I₅₀ values against esterase activity ranged from 1.4 to 8 nM for hCA-I and from 1.3 to 6 nM for hCA-II. The K_i values were observed between 8.2·10^{? 5} to 6.2·10^{? 4} M for hCA-I and between 2.9·10^{? 4} to 8.2·10^{? 4} M for hCA-II. The comparison of new synthesized amides to 5-amino-1,3,4-thiadiazole-2-sulfonamide 1, acetazolamide 2 indicated that the new synthesized compounds (18–23) inhibit CA activity more potently than the parent compounds.     

Measurement and significance of the membrane potential in Methanoba cterium bryantii

The membrane potential (Δψ) of Methanobacterium bryantii was 133–142 mV as measured from the distribution of ⁸⁶Rb⁺ in valinomycin-treated cells, and was considerably higher than that obtained using triphenylmethylphosphonium in the presence of tetraphenylboron. The Δψ measured using the Rb⁺/valinomycin method was sensitive to certain ionophores including gramicidin, nigericin, carbonyl cyanide m-chlorophenylhydrazone and 3,3′,4′,5-tetrachlorosalicylanilide. It was also dissipated by 1 mM tetraphenylphosphonium and was abolished in heat-treated or permeabilized cells. The Δψ could be varied by adjusting the extracellular potassium concentration in valinomycin-treated cells. Monensin-treated cells possessed a significantly increased Δψ, as monitored by the Rb⁺ / valinomycin method. Tetraphenylphosphonium cation (1 mM) abolished methane synthesis, intracellular ATP and Δψ, supporting a role for Δψ in ATP and CH₄ synthesis. However, lower concentrations of the lipophilic cation (50 μM) greatly elevated both the intracellular ATP concentration and Δψ but decreased the rate of CH₄ synthesis by almost 50%. Thus, tetraphenylphosphonium cation exerts a primary inhibitory effect on CH₄ synthesis which cannot be attributed to the loss of Δψ or ATP.     

Amiloride-induced stimulation of HCO3− reabsorption in turtle bladder

Amiloride in the mucosal fiuid (at concentrations of 5 · 10^?6 M to 10^?4 M) reversibly stimulates the HCO₃^?-dependent moiety of the short-circuiting current (I_sc) in ouabain-treated turtle bladders bathed by Na-free Ringer solutions with or without Cl_?.This effect is uniquely different from the known inhibitory effect of this agent on Na⁺ transport. Thus, any comprehensive hypothesis on the action of amiloride over a wide dosage-response fange should take into account its effect on HCO₃^? transport.