scholarly journals Organic anions stabilize the reactivated motility of sperm flagella and the latency of dynein 1 ATPase activity.

1985 ◽  
Vol 101 (4) ◽  
pp. 1281-1287 ◽  
Author(s):  
B H Gibbons ◽  
W J Tang ◽  
I R Gibbons
Keyword(s):  
Atpase Activity ◽  
Organic Anion ◽  
Beat Frequency ◽  
Organic Anions ◽  
Transport Systems ◽  
Bend Angle ◽  
Crossbridge Cycle ◽  
Vanadate Inhibition ◽  
Increased Sensitivity ◽  
Motility Of Sperm

Substitution of any of a variety of organic anions, including acetate, propionate, lactate, gluconate, and succinate, for chloride in the reactivation medium improves the motility of demembranated sperm of Tripneustes gratilla. At the optimum concentration of 0.20 N, all of these anions improve the duration of motility, with lactate and gluconate being the best. The Michaelis constant for beat frequency (Kmf) is lower (0.11-0.14 mM at 22 degrees C) in most of the organic anions than it is in Cl- (0.20 mM), and the minimum ATP concentration required to support oscillatory beating is reduced from 10 microM in chloride to 2 microM in acetate, which together indicate a greater affinity of the axonemal ATPase for MgATP2- in the organic anions media. The maximal beat frequency, fmax, is as high as 42 Hz in 0.2 N succinate compared to 31 Hz in Cl-, whereas the mean bend angle averages 2.8 rad in acetate compared to 2.4 rad in Cl-; these values give a calculated average velocity of tubule sliding of approximately 15 micron/s in acetate and succinate, which is approximately 30% greater than the value of 11 micron/s observed in chloride. The reactivated sperm are sixfold more sensitive to vanadate inhibition in 0.2 M acetate than they are in 0.15 M Cl-. The specific ATPase activity of soluble dynein 1, which increases more than 15-fold between 0 and 1.0 N Cl-, undergoes only a twofold activation over the same range of organic anion concentration, and, like the reactivated motility, is up to 50-fold more sensitive to vanadate. This greater apparent mechanochemical efficiency and the increased sensitivity to vanadate inhibition in the organic anions suggest that they, unlike chloride, do not promote the spontaneous dissociation of ADP and PO4(3-) from the dynein-ADP-PO4 kinetic intermediate in the dynein crossbridge cycle. The use of organic anion media may lead to significant improvements in reactivation of other motile and transport systems.

Increased sensitivity of frog skeletal muscle to procaine in the presence of organic anions

1978 ◽  
Vol 56 (5) ◽  
pp. 739-746 ◽  
Author(s):  
J. G. Foulks ◽  
F. A. Perry
Keyword(s):  
Action Potentials ◽  
Time Course ◽  
Organic Anion ◽  
Organic Anions ◽  
Depressant Effect ◽  
Alkyl Sulfonates ◽  
Carboxylate Anions ◽  
Increased Sensitivity ◽  
Local Anaesthetic Action ◽  
Anaesthetic Action

In solutions containing an organic anion in place of chloride, frog toe muscles displayed increased sensitivity to the local anaesthetic action of procaine. Twitch inhibition by procaine in all media was accompanied by suppression of action potentials without change in membrane resting potentials. The twitch depressant effect of procaine was greater in solutions with carboxylate anions than with alkyl sulfonates. The intensity and the rapidity of onset of the effects of organic anions was related to the size of their hydrophobic component.Procaine accentuated acetate-induced reductions in the [K]0 required to produce K contractures and in the time course of submaximum K contractures. These effects were not shared by benzocaine. They were antagonized by increased [Ca]0.The results indicate that separate agents can exert mutually enhancing actions from opposite surfaces of the sarcolemma to facilitate the inactivation of depolarization-induced excitation–contraction coupling as well as that of the potential-dependent sodium channel.

scholarly journals Properties of an antiserum against native dynein 1 from sea urchin sperm flagella.

1977 ◽  
Vol 73 (1) ◽  
pp. 182-192 ◽  
Author(s):  
K Ogawa ◽  
D J Asai ◽  
C J Brokaw
Keyword(s):  
Atpase Activity ◽  
Sea Urchin ◽  
Strongylocentrotus Purpuratus ◽  
Beat Frequency ◽  
Bend Angle ◽  
Tryptic Fragment ◽  
Effective Inhibition ◽  
Dynein Arms ◽  
Sea Urchin Sperm ◽  
Anthocidaris Crassispina

Effects of an antiserum against native dynein 1 from sperm flagella of the sea urchin Strongylocentrotus purpuratus were compared with effects of an antiserum previously obtained against an ATPase-active tryptic fragment (fragment 1A) of dynein 1 from sperm flagella of the sea urchin, Anthocidaris crassispina. Both antisera precipitate dynein 1 and do not precipitate dynein 2. Only the fragment 1A antiserum precipitates fragment 1A and produces a measurable inhibition of dynein 1 ATPase activity. Both antisera inhibit the movement and the movement-coupled ATP dephosphorylation of reactivated spermatozoa. The inhibition of movement by the antiserum against dynein 1 is much less than by the antiserum against fragment 1A, suggesting that a specific interference with the active ATPase site may be required for effective inhibition of movement. Both antisera reduce the bend angle as well as the beat frequency of reactivated S. purpuratus spermatozoa, suggesting that the bend angle may depend on the activity of the dynein arms which generate active sliding.

scholarly journals Inhibitors of membrane transport system for organic anions block fura-2 excretion from PC12 and N2A cells

1988 ◽  
Vol 256 (3) ◽  
pp. 959-963 ◽  
Author(s):  
F Di Virgilio ◽  
C Fasolato ◽  
T H Steinberg
Keyword(s):  
Cell Line ◽  
Pc12 Cells ◽  
Transport System ◽  
Free Acid ◽  
Organic Anion ◽  
Cell Types ◽  
Organic Anions ◽  
Transport Systems ◽  
Fura 2 ◽  
N2a Cells

The neuroblastoma-like cell line N2A and the pheochromocytoma-like cell line PC12 excrete about 20-25% of the intracellular fluorescent Ca2+ indicator fura-2 during 10 min of incubation at 37 degrees C. The drug probenecid, known to inhibit membrane systems for the transport of organic anions [Cunningham, Israili & Dayton (1981) Clin. Pharmacol. 6, 135-151], inhibited fura-2 excretion in both cell types. However, probenecid also had untoward effects on intracellular Ca2+ homeostasis in N2A and PC12 cells. We therefore tested the drug sulphinpyrazone, another known inhibitor of organic-anion transport systems. Sulphinpyrazone fully inhibited excretion of fura-2 at 250 microM, a concentration one order of magnitude lower than that of probenecid. At this concentration and for incubation times up to 20 min, sulphinpyrazone had no untoward effects on cell viability and metabolic functions. Fura-2 was also loaded into the cytoplasm of N2A cells by permeabilization of the plasma membrane with extracellular ATP. In this case as well, the dye was rapidly released from the cells and the efflux was blocked by sulphinpyrazone. These findings suggest that N2A and PC12 cells possess a membrane system for the transport of the free-acid form of fura-2. This transport system is probably responsible for the excretion of fura-2 from these cells. Incubation of N2A and PC12 cells with sulphinpyrazone may help overcome problems arising in the investigation of [Ca2+]i homeostasis in these cell types.

Biliary secretion in elasmobranchs. II. Hepatic uptake and biliary excretion of organic anions

1976 ◽  
Vol 230 (4) ◽  
pp. 974-981 ◽  
Author(s):  
JL Boyer ◽  
J Schwarz ◽  
N Smith
Keyword(s):  
Biliary Excretion ◽  
Binding Proteins ◽  
Organic Anion ◽  
Sodium Taurocholate ◽  
Hepatic Clearance ◽  
Hepatic Uptake ◽  
Organic Anions ◽  
Transport Systems ◽  
Raja Erinacea ◽  
Low Concentrations

[35S]Bromosulfophthalein ([35C]BSP), [14C]sodium taurocholate ([14C]NaTC), AND 10 MG OF UNLABELED BSP.and of phenol-3,6-dibromophthalein disulfonate (DBSP) per kilogram body weight were injected in the caudal artery of free-swimming dogfish sharks (Squalus acanthias) and small skates (Raja erinacea). Twenty-four hours later, 85.8 +/- 15.7% of [35S]BSP was recovered in bile and liver in dogfish and 78.4 +/- 9.9% in skates. Similar results were obtained for [14C]NaTC. Unlabeled BSP or DBSP (10 mg/kg body wt) were also selectively excreted in bile over a 4-day period and at comparable rates in both species. More than 85% of [35S]BSP, BSP, and DBSP in bile was in unconjugated form. Selective hepatic clearance of BSP occurred despite nonselective binding to liver homogenates and very low concentrations of binding proteins in liver cytosol. Analysis of the organic anion plasma disappearance curves suggest that the clearance of anions into bile in elasmobranchs is delayed disproportionately relative to hepatic uptake. Albumin-BSP infusions did not prevent selective hepatic uptake of [35S]BSP, although biliary excretion was delayed further. These studies demonstrate that transport systems for biliary excretion of organic anions evolved prior to migration of marine life from the sea and relatively independently of intrahepatic conjugation and organic anion-binding proteins.

Modification of flagellar waveform and adenosine triphosphatase activity in reactivated sea-urchin sperm treated with N-ethylmaleimide

1983 ◽  
Vol 60 (1) ◽  
pp. 231-249
Author(s):  
M.P. Cosson ◽  
W.J. Tang ◽  
I.R. Gibbons
Keyword(s):  
Atpase Activity ◽  
Sea Urchin ◽  
Atp Hydrolysis ◽  
Beat Frequency ◽  
Prolonged Treatment ◽  
Bend Angle ◽  
The Asymmetry ◽  
Bend Angles ◽  
The Waves ◽  
Sea Urchin Sperm

Treatment of demembranated sea-urchin sperm for 1–2 min with 10 microM-N-ethylmaleimide (Mal-NEt) at pH 8.0 prior to reactivation with 1 mM-ATP causes the asymmetry of the flagellar waveform to become desensitized to the presence or absence of Ca2+ in the reactivating medium. In such sperm, changes in concentration of free Ca2+ between 10(−7) M and 10(−3) M have no effect on the asymmetry of the waveforms as measured by the turning rate of the sperm in radians per beat cycle, while the beat frequency and the propulsive efficiency of the waves remain unchanged from the values observed in control preparations not treated with MalNEt. A somewhat more prolonged treatment with MalNEt causes a progressive decrease in the bend angles of the flagellar waves, while the beat frequency and the wavelength still remain largely unchanged. Further extension of the treatment with MalNEt causes complete loss of motility. Little ATP-induced sliding of the doublet tubules is observed upon treatment with trypsin of sperm flagella that have been rendered non-motile with MalNEt. However, the preparations of solubilized dynein 1 obtained by 0.6 M-NaCl extraction of axonemes treated with MalNEt appear almost identical to those obtained from untreated axonemes, both in terms of the amount solubilized and in the specific ATPase activities of their latent and Triton-activated forms. These preparations also appear capable of restoring much of the beat frequency of dynein-1-depleted flagella. These results suggest that the observed desensitization to Ca2+ and decrease in bend angle result from the reaction of MalNEt with axonemal polypeptides that are not part of the dynein 1 particle extracted with 0.6 M-NaCl. The rate of ATP hydrolysis by demembranated sperm rendered non-motile with MalNEt remains relatively high, and it decreases about 50% when the flagella are broken by brief homogenization. This ‘homogenizer-sensitive’ ATPase activity appears to be derived from some flagellar regulatory mechanism, which controls the ATPase activity of intact non-motile axonemes.

Interactions of organic anions with the organic cation transporter in renal BBMV

1988 ◽  
Vol 254 (1) ◽  
pp. F56-F61 ◽  
Author(s):  
P. H. Hsyu ◽  
L. G. Gisclon ◽  
A. C. Hui ◽  
K. M. Giacomini
Keyword(s):  
Proximal Tubule ◽  
Organic Cation ◽  
Organic Anion ◽  
Organic Cation Transporter ◽  
Organic Anions ◽  
Renal Proximal Tubule ◽  
Ph Gradient ◽  
Transport Systems ◽  
Organic Cations ◽  
Initial Uptake

It is generally assumed that the organic cation transport system in the renal proximal tubule is specific for organic cations and the transport of organic cations is not affected by organic anions. However, there are also data in the literature demonstrating that probenecid, a classical inhibitor of organic anion transport systems, inhibits the transport of an organic cation, cimetidine, in the renal proximal tubule. In this study we investigated the effects of probenecid and furosemide on the transport of N'-methylnicotinamide (NMN) the classical substrate of the organic cation transporter, in brush-border membrane vesicles prepared from rabbit renal cortex. In the presence of a pH gradient, both probenecid (10 mM) and furosemide reduced the initial uptake of NMN. Probenecid reduced the initial uptake of NMN to 12.1% of the control values (1.19 +/- 0.26 pmol/mg) and furosemide reduced the initial uptake of NMN to 39.2%. Probenecid (10 mM) also decreased the initial transport of NMN in the absence of a pH gradient. Inhibition of the transport of NMN by probenecid was concentration dependent, with the concentration of probenecid resulting in 50% inhibition of the transport of NMN equal to 2.31 +/- 1.18 mM in the presence of a pH gradient. Probenecid appeared to be a competitive inhibitor of NMN transport. The apparent Km (mean +/- SE) of NMN transport (2.01 +/- 0.78 mM) was increased to 18.7 +/- 10 mM (P less than 0.05) by probenecid (10 mM), whereas the Vmax was not changed (125 +/- 19.2 pmol.s-1.mg-1 vs. 186 +/- 94 pmol.s-1.mg-1, P greater than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Contraluminal transport systems in the proximal renal tubule involved in secretion of organic anions

1988 ◽  
Vol 254 (4) ◽  
pp. F453-F462 ◽  
Author(s):  
K. J. Ullrich ◽  
G. Rumrich
Keyword(s):  
Transport System ◽  
Driving Forces ◽  
Organic Anion ◽  
Organic Anions ◽  
Transport Systems ◽  
Exchange System ◽  
Ionic Charge ◽  
Anion Transporters ◽  
Directional Movement ◽  
Partial Charges

The transport of organic anions in the proximal tubule occurs primarily through the epithelial cells. This process involves movement across both the luminal and contraluminal membranes via specialized transport systems. Although some of the organic anions are taken up into the cell from the lumen, they can also be accumulated in tubule cells from the interstitial compartment by a variety of transporters. The relative affinities of anions for the different luminal and contraluminal transporters in concert with their conjugate driving forces determine the net directional movement, i.e., organic anion absorption or secretion. By use of the approach of stopped-flow microperfusion, it has been possible to characterize the contraluminal anion transporters in the rat. The following three different systems have been identified: 1) an exchange system for sulfate and oxalate; 2) a cotransport system for Na+ and dicarboxylates; and 3) an exchange system (the so-called p-aminohippuric acid transport system) for hydrophobic anions and long-chain fatty acids. By use of a wide variety of different analogues, the substrate specificities for these different systems were determined. Substrates with two negative ionic charges or with one negative ionic charge and one or more negative partial charges interact with all three systems, depending on the distance between the two charged groups. Polyhalogenated substrates are preferred by the dicarboxylate system. Those substrates which interact only with the p-aminohippurate transport system possess a hydrophobic area and one negative ionic charge or two negative partial charges.

Phosphorus deficiency enhances plasma membrane H+-ATPase activity and citrate exudation in greater purple lupin (Lupinus pilosus)

2004 ◽  
Vol 31 (11) ◽  
pp. 1075 ◽  
Author(s):  
Ayalew Ligaba ◽  
Mineo Yamaguchi ◽  
Hong Shen ◽  
Takayuki Sasaki ◽  
Yoko Yamamoto ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Atpase Activity ◽  
Phosphorus Deficiency ◽  
Organic Anion ◽  
Organic Anions ◽  
Al Toxicity ◽  
Channel Blockers ◽  
P Deficiency ◽  
Citrate Exudation ◽  
Lupinus Pilosus

The response of greater purple lupin (Lupinus pilosus L.) to a combination of phosphorus (P) deficiency and aluminium (Al) toxicity is unknown, and the mechanisms involved in the exudation of organic anions from greater purple lupin have not been reported. Therefore, plants grown with (+P) or without (–P) 250 µm P were exposed to 0 or 50 µm AlCl3 and the amount of organic anions exuded and the activities of plasma membrane H+-ATPase (E.C 3.6.3.6) and H+-pumps were investigated. Twenty days of P deficiency resulted in significantly reduced shoot growth and increased proteoid root formation. Exposure to 50 µm AlCl3 did not induce citrate exudation but did induce some malate exudation in –P plants. In contrast, P deficiency did induce exudation of citrate. Enhanced citrate exudation was attributed to the large increase in the activity of plasma membrane H+-ATPase and associated H+ transport. This was shown by the inhibitory effect of vanadate on plasma membrane H+-ATPase activity in vitro and on citrate exudation in vivo. However, vanadate did not suppress the exudation of malate. During 9 h of Al exposure, exudation of citrate showed a continuing increase for both –P and +P plants, while malate exudation increased only during the first 3 h, after which it rapidly declined. The total amount of organic anion exudation was significantly higher for –P plants. In the presence of 50 µm anion channel blockers [anthracene-9-carboxylic acid (A-9-C), niflumic acid (NIF) and phenylglyoxal (PG)], the exudation of citrate and malate was reduced by 25–40%. It was concluded that P deficiency induces citrate exudation by enhancing the activity of plasma membrane H+-ATPase and H+ export. In L. pilosus, exudation of organic anions occurs primarily in response to P deficiency but not Al toxicity. This contrasts with previous results obtained in Brassica napus L.

Effect of imposed head vibration on the stability and waveform of flagellar beating in sea urchin spermatozoa

1991 ◽  
Vol 156 (1) ◽  
pp. 63-80 ◽  
Author(s):  
C. Shingyoji ◽  
I. R. Gibbons ◽  
A. Murakami ◽  
K. Takahashi
Keyword(s):  
Sea Urchin ◽  
Beat Frequency ◽  
Distal Region ◽  
Proximal Region ◽  
Bend Angle ◽  
Vibration Frequencies ◽  
Sliding Velocity ◽  
Additional Energy ◽  
The Stability ◽  
Cyclic Changes

The heads of live spermatozoa of the sea urchin Hemicentrotus pulcherrimus were held by suction in the tip of a micropipette mounted on a piezoelectric device and vibrated either laterally or axially with respect to the head axis. Within certain ranges of frequency and amplitude, lateral vibration of the pipette brought about a stable rhythmic beating of the flagella in the plane of vibration, with the beat frequency synchronized to the frequency of vibration [Gibbons et al. (1987), Nature 325, 351–352]. The sperm flagella, with an average natural beat frequency of 48 Hz, showed stable beating synchronized to the pipette vibration over a range of 35–90 Hz when the amplitude of vibration was about 20 microns or greater. Vibration frequencies below this range caused instability of the beat plane, often associated with irregularities in beat frequency. Frequencies above about 90 Hz caused irregular asymmetrical flagellar beating with a marked decrease in amplitude of the propagated bends and a skewing of the flagellar axis towards one side; the flagella often stopped in a cane shape. In flagella that were beating stably under imposed vibration, the wavelength was reduced at higher frequencies and increased at lower frequencies. When the beat frequency was equal to or lower than the natural beat frequency, the apparent time-averaged sliding velocity of axonemal microtubules, obtained as twice the product of frequency and bend angle, decreased with beat frequency in both the proximal and distal regions of the flagella. However, at vibration frequencies above the natural beat frequency, the sliding velocity increased with frequency only in the proximal region of the flagellum and remained essentially unchanged in more distal regions. This apparent limit to the velocity of sliding in the distal region may represent an inherent limit in the intrinsic velocity of active sliding, while the faster sliding observed in the proximal region may be a result of passive sliding or elastic distortion of the microtubules induced by the additional energy supplied by the vibrating pipette. Axial vibration with frequencies either close to or twice the natural beat frequency induced cyclic changes in the waveform, compressing and expanding the bends in the proximal region, but did not affect bends in the distal region or alter the beat frequency.

Intracellular compartmentation of organic anions within renal cells

1993 ◽  
Vol 264 (5) ◽  
pp. R882-R890 ◽  
Author(s):  
D. S. Miller ◽  
D. E. Stewart ◽  
J. B. Pritchard
Keyword(s):  
Organic Anion ◽  
Basolateral Membrane ◽  
Organic Anions ◽  
Epifluorescence Microscopy ◽  
Renal Cells ◽  
Bladder Epithelium ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Intracellular Compartments ◽  
Energy Dependent

Epifluorescence microscopy and video-image analysis were used to measure the distribution of the monovalent organic anion fluorescein (FL) within the cells of three organic anion-secreting renal epithelia: crab urinary bladder (a proximal tubule analogue), opossum kidney (OK) cells in culture, and intact teleost proximal tubules. In all three preparations the intracellular FL distribution was nonuniform. Two distinct intracellular compartments were detected, one being diffuse and cytoplasmic and the other punctate. With low FL concentrations in the medium (1 microM and below) dye accumulation in the punctate compartment exceeded that of the cytoplasm. In crab bladder epithelium FL uptake into both compartments was inhibited by external probenecid, p-aminohippurate (PAH), and LiCl and stimulated by 10-50 microM external glutarate, suggesting that the punctate compartment loaded by a two-step mechanism: transport into the cytoplasm at the basolateral membrane, followed by accumulation at specific intracellular sites. Experiments in which FL was microinjected into OK cells directly demonstrated movement of FL from the cytoplasmic to the punctate compartment. Accumulation in the latter was specific, i.e., inhibitable by coinjected PAH and probenecid, and energy dependent. Together, these findings indicate that during secretion organic anions are sequestered within renal cells. The role of sequestration in overall transport remains to be determined.

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