A concentration-dependent localization of octopamine-sensitive adenylate cyclase activity in locust skeletal muscle

A concentration-dependent localization of octopamine-sensitive adenylate cyclase activity has been demonstrated in skeletal muscle of the locust, Schistocerca gregaria, using an histochemical technique. In the intermediate speed contracting muscle fibres from the fan region of the extensor-tibiae muscle of the locust hindleg, low concentrations of DL-octopamine (10(-8) M) induce reaction product preferentially in the sarcoplasmic reticular component of the dyads. At slightly higher concentrations (10(-7) and 10(-6) M) lower amounts of diffuse reaction product are also found in the non-dyad sarcoplasmic reticulum and at the sarcolemmal membrane, with occasional amounts of a less diffuse, punctuate product in the transverse tubule (T-tubule) component of the dyads. At higher concentrations (10(-5) and 10(-3) M) the predominant product is the dense, plaque-like accumulations of reaction product in the T-tubule component of the dyads. The results are discussed in terms of the likely physiological significance of the accumulation of reaction product in these different locations.     

Histochemical localization of octopamine- and proctolin-sensitive adenylate cyclase activity in a locust skeletal muscle

Summary A histochemical technique for the localization of adenylate cyclase activity has been applied to the extensortibiae muscle of the hindleg of the locust, Schistocerca gregaria to localise the sites of action of the modulatory compounds octopamine and proctolin. Octopamine-sensitive adenylate cyclase activity can be demonstrated in fast and intermediate type muscle fibres but not in the limited number of purely slow muscle fibres (3–6) in the fan region at the proximal end of the muscle. In contrast the latter fibres are the only ones in the muscle to exhibit proctolinsensitive adenylate cyclase activity. In both cases the bulk of the reaction product is localised in the sarcoplasmic reticulum component of the dyads, with lesser amounts occurring beneath the sarcolemmal membrane, in the non-dyad sarcoplasmic reticulum and in the T-tubule system. The results are consistent with physiological data suggesting that proctolin, but not octopamine, mediates its effects on the myogenic rhythm of contraction and relaxation in this muscle by changing the levels of cyclic AMP in the small group of slow muscle fibres which act as the pacemaker for this rhythm.     

Histochemical localization of octopamine- and proctolin-sensitive adenylate cyclase activity in a locust skeletal muscle

A histochemical technique for the localization of adenylate cyclase activity has been applied to the extensor-tibiae muscle of the hindleg of the locust, Schistocerca gregaria to localise the sites of action of the modulatory compounds octopamine and proctolin. Octopamine-sensitive adenylate cyclase activity can be demonstrated in fast and intermediate type muscle fibres but not in the limited number of purely slow muscle fibres (3-6) in the fan region at the proximal end of the muscle. In contrast the latter fibres are the only ones in the muscle to exhibit proctolin-sensitive adenylate cyclase activity. In both cases the bulk of the reaction product is localised in the sarcoplasmic reticulum component of the dyads, with lesser amounts occurring beneath the sarcolemmal membrane, in the non-dyad sarcoplasmic reticulum and in the T-tubule system. The results are consistent with physiological data suggesting that proctolin, but not octopamine, mediates its effects on the myogenic rhythm of contraction and relaxation in this muscle by changing the levels of cyclic AMP in the small group of slow muscle fibres which act as the pacemaker for this rhythm.     

The effect of 2-chloro, 6-(trichloromethyl) pyridine on the chemoautotrophic metabolism of nitrifying bacteria

Studies were conducted to elucidate the mechanism of action of 2-chloro-6-(trichloromethyl)pyridine or Technical N-SERVE on the nitrification process brought about byNitrosomonas europaea. The growth ofNitrosomonas was completely inhibited in the presence of 0.2 ppm N-SERVE while 1.0 ppm of the chemical was effective in the complete inhibition of ammonia oxidation by fresh cell suspensions. Cells stored at 4 C for a period of three days required somewhat higher concentrations (1.5 ppm) of N-SERVE for the complete inhibition of their ammonia oxidizing ability while the cytochrome oxidase of these cells was inhibited to the extent of 65 to 70 percent in the presence of a corresponding amount of N-SERVE. A 45 – 70 percent reversal of the inhibition of ammonia oxidation caused by N-SERVE was obtained by the addition of 6×10^–4 M Cu⁺⁺. An equivalent concentration of Cu⁺⁺ was also effective for the complete reversal of the inhibition of cytochrome oxidase present in whole cells.Hydroxylamine oxidation by intactNitrosomonas cells was not affected by levels of N-SERVE ranging from 1 – 3 ppm. The cytochrome oxidase effective in hydroxylamine oxidation and present in cell-free extracts was not inhibited by even 100 ppm N-SERVE. Likewise, the hydroxylamine activating enzyme hydroxylamine cytochromec reductase was also not inhibited by such levels of the chemical. Raising the concentration to 170 ppm N-SERVE, however, caused a 90 percent inhibition of the enzyme.Although a 5×10^–6 M concentration of allylthiourea completely inhibited ammonia oxidation byNitrosomonas cells, concentrations up to 10^–3 M of this compound did not affect the cytochrome oxidase activity of whole cells or cell-free extracts. The inhibition of ammonia oxidation caused by 5×10^–6 M allythiourea, unlike the inhibition by N-SERVE, could not be reversed by the addition of 6×10^–4 M Cu⁺⁺.Evidence is presented that the action of N-SERVE is on that component of cytochrome oxidase which is involved in ammonia oxidation.     

Neuropharmacological analysis of synaptic transmission in the Lorenzinian ampulla of the skate Raja clavata

Summary Dissected ampullae of Lorenzini of the skate (Raja clavata) were studied with the aim of determining the synaptic transmitter between electroreceptor cell and afferent fibre. Resting activity and stimulus-evoked activity in response to electrical pulses were recorded in single afferent units at constant perfusion with normal and test solutions containing different putative neurotransmitters. Presynaptic transmitter release was blocked by Mg²⁺ (up to 50 mM) to investigate the effects of the test substances upon the postsynaptic membrane. l-Glutamate (l-GLU) and l-aspartate (l-ASP), both at concentrations between 10^-7 and 10^-3 M, enlarged strongly resting and stimulus-evoked discharge frequency in the afferent fibre. If transmission was blocked by high Mg²⁺, resting discharge frequency could be restored by l-GLU or l-ASP. The glutamate agonists quisqualate (10^-8–10⁵ M) and N-methyl-D-aspartate (10^-5–10^-3 M) enlarged spontaneous activity in the afferent fiber. The same was found for kainic acid (10^-9–10^-5 M). Taurine at concentrations between 10^-5 and 10^-3 M caused a concentration-dependent decrease in afferent activity. The same was found for gammaaminobutyric acid (GABA; 10^-5–10^-4 M), and for the catecholamines adrenaline and noradrenaline, both in concentrations between 10^-5 and 10^-3 M. Serotonine (10^-5–10^-3 M) and dopamine (10^-5-10^-3 M) had no effect on resting or evoked activity in the Lorenzinian ampulla afferents. Acetylcholine (ACh; 10^-4 M) enlarged discharge frequency in those units with initial rates lower than 22–25 Hz, but diminished discharge frequency in fibres with initial activity higher than 25 Hz. When synaptic transmission was blocked by high Mg²⁺ solution, perfusion with additional ACh did not restore resting activity in the afferent fibre. The results suggest that the most probable transmitter in the afferent synapse of the ampullae of Lorenzini is l-GLU or l-ASP, or a substance of similar nature.Abbreviations ACh acetylcholine - GABA gamma aminobutyric acid - KA kainic acid - l-ASP l-aspartate - l-GLU l-glutamate - NMDA N-methyl-D-aspartate - Q quisqualate - n.s. normal solution     

Histochemical demonstration of carbonic anhydrase activity

Summary Freeze-dried frozen sections are floated on the surface of the freshly prepared incubation mixture (CoSO₄ 1.75 × 10^–3 M, H₂SO₄ 5.3 × 10^–2 M, NaHCO₃ 1.57 × 10^–2 M and KH₂PO₄ 1.17 to 11.7 × 10^–3 M; demonstration of weak activity requires high phosphate). A compound containing cobalt and phosphorous precipitates at carbonic anhydrase sites and is converted to CoS. Adequate staining requires only 2–10 minutes of incubation. Actazolamide inhibits the staining reaction in specific concentrations. Actazolamidein vivo, 20 mg/kgi.v. to mice 30 minutes before sacrifice also inhibited the staining. The proportion phosphorous in the specific precipitate increases with KH₂PO₄ of the medium (shown by the addition of⁶⁰Co and³²P). An explanation of the reaction mechanism is given, based on the catalyzed loss of CO₂ in the surface layer. The inclusion of phosphate in the medium makes this modification ofHäusler's method so sensitive that it shows carbonic anhydrase activity in for instance stratum spinosum of the skin.This investigation was supported by grants from the Medical Faculty, University of Uppsala and from the U.S. National Institutes of Health (Grant NB 3060 to E.Bárány).     

Swelling-activated Chloride and Potassium Conductance in Primary Cultures of Mouse Proximal Tubules. Implication of KCNE1 Protein

Volume-sensitive chloride and potassium currents were studied, using the whole-cell clamp technique, in cultured wild-type mouse proximal convoluted tubule (PCT) epithelial cells and compared with those measured in PCT cells from null mutant kcne1 –/– mice. In wild-type PCT cells in primary culture, a Cl^– conductance activated by cell swelling was identified. The initial current exhibited an outwardly rectifying current-voltage (I-V) relationship, whereas steady-state current showed decay at depolarized membrane potentials. The ion selectivity was I^– > Br^– > Cl^– >> gluconate. This conductance was sensitive to 1 mM 4,4-Diisothiocyanostilbene-2,2-disulfonic acid (DIDS), 0.1 mM 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and 1 mM diphenylamine-2-carboxylate (DPC). Osmotic stress also activated K⁺ currents. These currents are time-independent, activated at depolarized potentials, and inhibited by 0.5 mM quinidine, 5 mM barium, and 10 µM clofilium but are insensitive to 1 mM tetraethylammonium (TEA), 10 nM charybdotoxin (CTX), and 10 µM 293B. In contrast, the null mutation of kcne1 completely impaired volume-sensitive chloride and potassium currents in PCT. The transitory transfection of kcne1 restores both Cl^– and K⁺ swelling-activated currents, confirming the implication of KCNE1 protein in the cell-volume regulation in PCT cells in primary cultures.     

Detection and localization of triadin in rat ventricular muscle

Summary Dyads (transverse tubule—junctional sarcoplasmic reticulum complexes) were enriched from rat ventricle microsomes by continuous sucrose gradients. The major vesicle peak at 36% sucrose contained up to 90% of those membranes which possessed dihydropyridine (DHP) binding sites (markers for transverse tubules) and all membranes which possessed ryanodine receptors and the putative junctional foot protein (markers for junctional sarcoplasmic reticulum). In addition, the 36% sucrose peak contained half of the vesicles with muscarine receptors. Vesicles derived from the nonjunctional plasma membrane as defined by a low content of dihydropyridine binding sites per muscarine receptor and from the free sarcoplasmic reticulum as defined by the M_r 102K Ca²⁺ ATPase were associated with a diffuse protein band (22–30% sucrose) in the lighter region of the gradient. These organelles were recovered in low yield. Putative dyads were not broken by French press treatment at 8,000 psi and only partially disrupted at 14,000 psi. The monoclonal antibody GE4.90 against skeletal muscle triadin, a protein which links the DHP receptor to the junctional foot protein in skeletal muscle triad junctions, cross-reacted with a protein in rat dyads of the same M_r as triadin. Western blots of muscle microsomes from preparations which had been treated with 100mm iodoacetamide throughout the isolation procedure showed that cardiac triadin consisted predominantly of a band of Mr 95 kD. Higher molecular weight polymers were detectable but low in content, in contrast with the ladder of oligomeric forms in rat psoas muscle microsomes. Cardiac triadin was not dissolved from the microsomes by hypertonic salt or Triton X-100, indicating that it, as well as skeletal muscle triadin, was an integral protein of the junctional SR. The cardiac epitope was localized to the junctional SR by comparison of its distribution with that of organelle markers in both total microsome and in French press disrupted dyad preparations. Immunofluorescence localization of triadin using mAb GE4.90 revealed that intact rat ventricular muscle tissue was stained following a well-defined pattern of bands every sarcomere. This spacing of bands was consistent with the interpretation that triadin was present in the dyadic junctional regions.     

Anomalous resistance changes following application of the neurotransmitterl-Glutamate in insect muscle

Summary Bath application ofl-glutamate, to larval dipteran muscle, at concentrations between 10^–6 and 10^–4 M will cause a depolarisation of the muscle membrane potential associated with an increase in muscle input resistance. At concentrations above 10^–4 M there is usually a transient decrease in input resistance preceding the resistance increase. l-aspartate at concentrations above 10^–5 M causes membrane depolarisations which are always associated with an increase in muscle input resistance.The pharmacology of the receptors regulating membrane depolarisations associated with increases in input resistance has been compared with the receptors gating synaptically activated cation channels and found to show significant differences in sensitivity to various ligands.     

Malate enzyme fromPseudomonas putida: Some kinetic properties and function in glucose metabolism

Pseudomonas putida was grown on glucose and gluconate under different conditions with limiting amounts of carbon and nitrogen. The activities of some enzymes were determined in the periplasmic and intracellular fractions. The results indicate that malate enzyme (l-malate: NADP⁺ oxidoreductase, oxalacetate-decarboxylating EC 1.1.1.40) may function either as an NADPH-generating system or one of intracellular hydrogen transport. For determination of the effect of NADPH and the probable reaction mechanism by which NADPH produces this effect, kinetic studies with the purified enzyme were carried out. Malate enzyme showed hyperbolic saturation curves with respect to both substrates, malate and NADP, with Km values of 7.73 (±1.8)×10^–2 mM and 1.08 (±0.3) mM for NADP andl-malate, respectively, obtained by double reciprocal plots.     

The kinetics of anion equilibrium exchange across the red blood cell membrane as measured by means of35S thiocyanate

Summary Up to a SCN^– concentration of about 110mm, the concentration dependence of SCN^– equilibrium exchange in human red cell ghosts can be represented by the superimposition of two flux components. One component shows saturation kinetics, the other does not. The saturable component has an activation enthalpy of 105 kJ/mole, exhibits arans acceleration by Cl^– and can be inhibited by H₂DIDS. The nonsaturable component has a much lower activation enthalpy of 33 kJ/mole, is slightly reduced intrans acceleration experiments with Cl^– and insensitive to H₂DIDS but susceptible to inhibition by phloretin. At SCN^– concentrations exceeding 110mm, the saturable component undergoes irreversible self inhibition while the nonsaturable component remains unaltered.The half saturation concentration of the saturable flux component increases with decreasing pH from 3.0mm at pH 7.4 to 13.3mm at pH 6.0. Over this pH range, the maximal flux is only slightly increased from 19×10^–12 to 22×10^–12 moles×cm^–2×sec^–1. The nonsaturable flux component also increases slightly.In accordance with previous observations of Wieth (J. Physiol. (London) 207:563–580, 1970), we find that SCN^– increases K⁺ and Na⁺ permeability. The induced cation-permeability is considerably smaller than the SCN^– exchange and the latter does not show the paradoxical temperature dependence that is known to pertain to the former.     

Isolation of halophilic and halotolerant bacteria from a Japanese salt field and comparison of the partial 16S rRNA gene sequence of an extremely halophilic isolate with those of other extreme halophiles

Halophilic and halotolerant bacteria were isolated from soil samples of a Japanese salt field, an environment where salt concentrations vary annually. From 1 g of each of the five samples collected, over 1×10³ bacterial colonies (colony forming units (cfu)g^-1) grew on agar medium containing 2M Na⁺. In contrast, 0–4 bacterial colonies (cfu g^-1) were observed on agar medium containing 4M Na⁺. Two of the five samples contained numerous bacteria (10²–10³ cfu g^-1) capable of growth on a 2M Na⁺ alkaline (pH=9.5) medium, while few bacterial colonies were observed from the other three samples. Only one of the five samples was shown to contain bacteria capable of growth on a 4M Na⁺ alkaline medium. Most of the bacteria isolated on 4M Na⁺ agar were eubacteria, but one extreme halophile (TR-1, already described as Haloarcula japonica JCM7785) was also isolated. The 16S rRNA sequence of TR-1 was determined and shows high homology (94.4–98.5%) to Ha. marismortui and Ha. sinaiiensis. These results suggested that: 1) environments with seasonally varying salinity can harbour halotolerants as well as halophiles and, 2) closely related halophiles can be isolated from geographically distant habitats.     

Some effects of trinitrocresolate and valinomycin on Na and K transport across thin lipid bilayer membranes: A steady-state analysis with simultaneous tracer and electrical measurements

Summary This paper describes the effect of trinitrocresolate anions (TNC^–) on the electrical conductance (G _m ), and tracer-measured unidirectional Na and K fluxes (M _Na andM _K) across bilayers formed from sheep red cell lipids dissolved in decane. In the absence of TNC^–, typical low conductances were observed, while the cation fluxes were too low to measure by our techniques (<10^–12 moles cm^–2 sec^–1). In the presence of TNC^– (10^–2 m),G _m increased and TNC^– was the main charge carrier in the system. The cationic fluxes were also much increased, but the membranes showed no significant selectivity between K and Na. Furthermore, the Na and K fluxes were at least two orders of magnitude larger than the ionic fluxes calculated fromG _m . Thus, almost all of the K and Na transport across the membrane in the presence of TNC^– is electrically silent and is probably carried out as KTNC and NaTNC ion pairs.In the presence of valinomycin (10^–6 m) and no TNC^–, both the ion fluxes andG _m were 10³ times larger in KCl than in NaCl, thus exhibiting the characteristic high selectivity of valinomycin for K over Na. In the presence of both valinomycin (10^–6 m) and TNC^– (10^–2 m), this selectivity disappeared in that bothG _m andM _Na in the NaCl system were similar to the respective values in the KCl system. Even under these conditions, most of the Na is still transported by a process which does not carry charge.BothG _m andM _x increased alike and monotonically with increasing temperature over the range 7 to 30°C. In the absence of TNC^– the enthalpies of activation were invariably higher in KCl than in NaCl. Addition of TNC^– produced equal enthalpies of activation for both Na and K containing systems suggesting a common, temperature-dependent, ratedetermining step in charge transfer and the electrically silent cation fluxes.     

Control of K+ conductance by cholecystokinin and Ca2+ in single pancreatic acinar cells studied by the patch-clamp technique

Summary The effect of cholecystokinin (CCK) and internal Ca²⁺ on outward K⁺ current in isolated pig pancreatic acinar cells has been investigated using the patch-clamp method for whole-cell current recording under voltage-clamp conditions. CCK (2 × 10^–10 M) applied to the bath evoked a marked increase in the outward K⁺ current associated with depolarizing voltage steps, and this effect was fully reversible and acutely dependent on the presence of external Ca²⁺. When strongly buffered Ca²⁺-EGTA solutions were used inside the cells CCK failed to evoke an effect. Increasing the internal Ca²⁺ concentration ([Ca²⁺] _i ) from 5 × 10^–10 M to 10^–7 and 5 × 10^–7 M mimicked the effect of CCK. It would appear therefore that CCK controls K⁺ conductance in the acinar cells via changes in the internal free ionized Ca²⁺ concentration.     

Michaelis constant (K m ) of acid phospatase as affected by montmorillonite,illite, and kaolinite clay minerals

The influence of Ca homoionic clay minerals (montmorillonite, illite, and kaolinite) on the activity,K _m , andV _m values of acid phosphatase was examined in model experiments. At each substrate (p-nitrophenyl phosphate) level tested, the addition of increasing amounts of clays (50, 100, and 150 mg, respectively) decreased the activity and increased theK _m value from 1.43×10^–3 m PNP (in the soluble state) to 82.3×10^–3M (montmorillonite), 8.02×10^–3 m (kaolinite), and 7.65×10^–3 m (illite) at the 150 mg level. The maximum enzyme reaction velocity (V _m ) remained nearly constant at different amounts of kaolinite and illite, but increased remarkably with rising quantities of montmorillonite. Apparently, the substrate affinity of sorbed acid phosphatase is significantly lower with montmorillonite than with kaolinite or illite. This may be ascribed to an intensive sorption of both substrate and enzyme to the surface as well as to interlattice sites of montmorillonite.     

A silver-reducing component in rat striated muscle

Summary In previous work on rat striated muscle cells a sliver-reducing component was found selectively localized at the terminal cistern/transverse tubule system (Tandler and Pellegrino de Iraldi 1989). To further investigate that problem we performed the Hg–Ag argentaffin reaction on a sarcoplasmic reticulum fraction from rat skeletal muscle. Circular profiles corresponding to vesicular structures were found outlined by silver grains. The number of silver stained vesicles were less than the total number vesicles stained by conventional procedures. The correlation between argentaffinities in the intact muscle fiber and their subcellular organelles indicated that the Hg–Ag reactive vesicles must be those derived from the terminal cisternae of the sarcoplasmic reticulum. The silver-reducing constituent aggregates in the presence of 1 mM CaCl₂ or 0.5 M K cacodylate. The state of aggregation induced by Ca²⁺ was not affected by incubation with 0.5% Triton X-100 or by 2 mM EDTA, thus suggesting a localization at or near the membrane of the terminal cistern vesicle facing the junctional gap. In Laemmli SDS-acrylamide gels the Hg–Ag reaction stained all proteins in a manner similar to Coomasie blue. It is suggested that the selective histochemical staining is the result of differential reactivities due to steric requirements of the chemical reaction.     

Comparative studies of fructose 1,6-diphosphate aldolase fromEscherichia coli 518 andLactobacillus casei var.rhamnosus ATCC 7469

A comparative study has been carried out with FDP aldolases fromEscherichia coli 518 andLactobacillus casei ATCC 7469, which had been purified 17.6- and 65-fold, respectively. The aldolase ofL.casei was stable only in the presence of mercaptoethanol, whereas that ofE.coli was strongly inhibited at low (1.0×10^–4 m) and activated at high concentrations (2.0×10^–1 m) of the same compound.p-Chloromercuric benzoic acid inhibited both aldolases, with 40% inhibition at 2×10^–5 m withE.coli aldolase against at 2×10^–4 m withL.casei aldolase. Significant differences were also observed in pH optima and Km values.E.coli aldolase exhibited a maximal activity at pH 9.0 and gave a Km value of 1.76×10^–3 m FDP with strong substrate inhibition above 7×10^–3 m, against pH 6.8–7.0 and a Km of 7.04×10^–3 m FDP forL.casei aldolase. Strong resistance ofL.casei aldolase against inhibition by EDTA, Ca²⁺ and Mn²⁺ was observed compared with complete inhibition at concentrations of 20mm, 40mm and 20mm, respectively, withE. coli aldolase. Polyacrylamide gel electrophoresis did not reveal any differences between the two enzyme preparations.The differences of the properties of FDP aldolases from different bacterial genera are discussed in relation to other Class II aldolases.     

Alpha and beta adrenergic agonists stimulate water absorption in the rat proximal tubule

Summary Simultaneous capillary and luminal microperfusion studies were performed in the rat proximal tubule to determine the effects of the beta agonist isoproterenol and the alpha agonist phenylephrine on water absorption. Capillary and luminal perfusion solutions were composed such that organic solutes were not present, no bicarbonate was present in the lumen, and no chloride gradient was imposed. Under such conditions, water absorption (Jv) averaged 0.36±0.11 nl·min^–1·mm^–1. The addition of isoproterenol to the capillary solution in concentrations of 10^–6 and 10^–4 m resulted in significantly higherJv's of 0.68±0.10 and 0.71±0.11 nl·min^–1·mm^–1, respectively. The enhancing effect of isoproterenol was inhibited by the beta blocker propranolol (10^–4 m), but not by the alpha blocker phentolamine (10^–7 m). The addition of phenylephrine (10^–6 m) to the capillary perfusion solution also resulted in a significantly higherJv of 0.84±0.14 nl·min^–1·mm^–1, an effect inhibited by phentolamine (10^–7 m), but not by propranolol (10^–4 m). Neither phentolamine nor propranolol alone in the concentrations indicated had an effect on water absorption. These experiments indicate that both alpha and beta agonists stimulate water absorption in the superficial proximal tubule of the rat. This effect appears to be relatively specific for each class of agonist, as demonstrated by the effects of the specific antagonists.     

Response of chloride efflux from skeletal muscle ofRana pipiens to changes of temperature and membrane potential and diethylpyrocarbonate treatment

Summary Efflux of³⁶Cl^– from frog sartorius muscles equilibrated in two depolarizing solutions was measured. Cl^– efflux consists of a component present at low pH and a pH-dependent component which increases as external pH increases.For temperatures between 0 and 20°C, the measured activation energy is 7.5 kcal/mol for Cl^– efflux at pH 5 and 12.6 kcal/mol for the pH-dependent Cl^– efflux. The pH-dependent Cl^– efflux can be described by the relationu=1/(1+10n(pK _a -pH)), whereu is the Cl^– efflux increment obtained on stepping from pH 5 to the test pH, normalized with respect to the increment obtained on stepping from pH 5 to 8.5 or 9.0. For muscles equilibrated in solutions containing 150mm KCl plus 120mm NaCl (internal potential about –15 mV), the apparent pK _a is 6.5 at both 0 and 20°C, andn=2.5 for 0°C and 1.5 for 20°C. For muscles equilibrated in solutions containing 7.5mm KCl plus 120mm NaCl (internal potential about –65 mV), the apparent pK _a at 0°C is 6.9 andn is 1.5. The voltage dependence of the apparent pK _a suggests that the critical pH-sensitive moiety producing the pH-dependent Cl^– efflux is sensitive to the membrane electric field, while the insensitivity to temperature suggests that the apparent heat of ionization of this moiety is zero. The fact thatn is greater than 1 suggests that cooperativity between pH-sensitive moieties is involved in determining the Cl^– efflux increment on raising external pH.The histidine-modifying reagent diethylpyrocarbonate (DEPC) applied at pH 6 reduces the pH-dependent Cl^– efflux according to the relation, efflux=exp(–k·[DEPC]·t), wheret is the exposure time (min) to DEPC at a prepared initial concentration of [DEPC] (mm). At 17°C,k ^–1=188mm·min. For temperatures between 10 and 23°C,k has an apparent Q₁₀ of 2.5. The Cl^– efflux inhibitor SCN^– at a concentration of 20mm substantially retards the reduction of the pH-dependent Cl^– efflux by DEPC. The findings that the apparent pK _a is 6.5 in depolarized muscles, that DEPC eliminates the pH-dependent Cl^– efflux, and that this action is retarded by SCN^– supports the notion that protonation of histidine groups associated with Cl^– channels is the controlling reaction for the pH-dependent Cl^– efflux.     

Na−K−2Cl cotransport in winter flounder intestine and bovine kidney outer medulla: [3H] bumetanide binding and effects of furosemide analogues

Summary The effects of several sulfamoyl benzoic acid derivatives on Na–K–Cl cotransport were investigated in winter flounder intestine. The relative efficacy (IC₅₀ values) and order of potency of these derivatives were benzmetanide, 5×10^–8 m> bumetanide 3×10^–7 m>piretanide 3×10^–6 m>furosemide 7×10^–6 m> amino piretanide 1×10^–5 3-amino-4-penoxy-5-sulfamoyl benzoic acid. Binding of [³H] bumetanide was studied in microsomal membranes from winter flounder intestine and compared to that in bovine kidney outer medulla. Binding was also studied in brush-border membranes from winter flounder intestine. The estimated values forK _d and number of binding sites (n) were: bovine kidney,K _d =1.6×10^–7,n=10.5 pmol/mg protein; winter flounder intestine,K _d 1.2×10^–7,n=7.3 pmol/mg protein, and brush-border membranes from winter flounder,K _d =5.3×10^–7,n=20.4 pmol/mg protein. The estimatedK _d for bumetamide binding to winter flounder brush-border membranes derived from association and dissociation kinetics was 6.8×10^–7 m. The similarity in magnitudes of IC₅₀ andK _d for bumetanide suggests that the brush-border cotransporter is ordinarily rate-limiting for transmural salt absorption and that bumetanide specifically binds to the cotransporter. Measurement of bumetanide binding at various concentrations of Na, K and Cl showed that optimal binding required all three ions to be present at about 5mm concentrations. Higher Na and K concentrations did not diminish binding but higher Cl concentrations (up to 100mm Cl) inhibited bumetanide binding by as much as 50%. Still higher Cl concentrations (500 and 900mm) did not further inhibit bumetanide binding. Scatchard analysis of bumetanide binding at 5 and 100mm Cl concentrations showed that bothK _d andn were lower at the higher Cl concentration (5mm Cl:K _d =5.29×10^–7 m,n=20.4 pmol/mg protein; 100mm Cl:K _d =2.3×10^–7 m,n=8.8 pmol/mg protein). These data suggest two possibilities: that bumetanide and Cl binding are not mutually exclusive (in contrast to pure competitive inhibition) and that they each bind to separate sites or that two distinct bumetanide binding sites exist, only one of which exhibits Cl inhibition of binding. This inhibition would then be consistent with a competitive interaction with Cl.