bluuebunny/crossref_metadata_2025 huggingface dataset:
A parameterised equation to estimate soil hydraulic conductivity in the field
Authors: Reichardt K. , Timm L. C. , Bacchi O. O. S. , Oliveira J. C. M. , Dourado-Neto D.
Published: May 2004

The description of soil water dynamics using the Darcy–Buckingham approach involves the determination and use of soil hydraulic conductivity K v. soil water content θ functions. Many of the methods developed for the measurement of K are based on simplifying assumptions, such as the unit gradient and the choice of fixed models for the K(θ) relation. The need of quick, simple, and inexpensive methods to measure K(θ) in the field using a large number of replicates has also led soil physicists to develop simple methods. This paper presents a procedure that makes use of parameters of equations used to explain the internal water drainage process, and that naturally leads to the exponential character of the K(θ) relation. Results show that the parameterised equation represents a more rigorous estimation of K(θ), compared with the methods that assume unit gradient.
Comparison of extraction techniques for measuring exchangeable cations in calcareous soils
Authors: Pierce C. G. , Morris S.
Published: May 2004

In this paper, 9 separate extraction techniques were undertaken to compare exchangeable cations for 25 calcareous soil samples taken from the central west of New South Wales. The variables evaluated included the means to extract (leach v. tumbler), the extraction solvents used to prewash soluble cations, the use of polyvinyl alcohol to reduce dispersion, and comparison with an unbuffered salt extraction. The time involved to conduct the extraction, along with correlations between methods, formed part of the assessment. The results provided verification that the concentration of the exchangeable cations extracted was based on the extraction technique, with each technique providing a slightly different result. Correlation showed that many of the extraction procedures yielded exchangeable cation results that showed a high level of agreement and demonstrated that the results from one extraction technique may be easily converted to another with a high degree of confidence. The end-over-end mixing method proposed by B. M. Tucker using ethylene glycol for a single soluble salt prewash and sequential ethanolic (60%) 1 M ammonium chloride extract was selected as the preferred method for this determination. Measuring ECEC using an unbuffered, low molarity salt solution was also found to be satisfactory. This assessment was based on the extraction efficiency for exchangeable sodium and potassium, agreement with other methods, time required to conduct the extraction and international acceptance.
Leptin modulates β cell expression of IL-1 receptor antagonist and release of IL-1β in human islets
Authors: Maedler Kathrin , Sergeev Pavel , Ehses Jan A. , Mathe Zoltan , Bosco Domenico , Berney Thierry , Dayer Jean-Michel , Reinecke Manfred , Halban Philippe A. , Donath Marc Y.
Published: May 2004

High concentrations of glucose induce β cell production of IL-1β, leading to impaired β cell function and apoptosis in human pancreatic islets. IL-1 receptor antagonist (IL-1Ra) is a naturally occurring antagonist of IL-1β and protects cultured human islets from glucotoxicity. Therefore, the balance of IL-1β and IL-1Ra may play a crucial role in the pathogenesis of diabetes. In the present study, we observed expression of IL-1Ra in human pancreatic β cells of nondiabetic individuals, which was decreased in tissue sections of type 2 diabetic patients.In vitro, chronic exposure of human islets to leptin, a hormone secreted by adipocytes, decreased β cell production of IL-1Ra and induced IL-1β release from the islet preparation, leading to impaired β cell function, caspase-3 activation, and apoptosis. Exogenous addition of IL-1Ra protected cultured human islets from the deleterious effects of leptin. Antagonizing IL-1Ra by introduction of small interfering RNA to IL-1Ra into human islets led to caspase-3 activation, DNA fragmentation, and impaired β cell function. Moreover, siIL-1Ra enhanced glucose-induced β cell apoptosis. These findings demonstrate expression of IL-1Ra in the human β cell, providing localized protection against leptin- and glucose-induced islet IL-1β.
Establishment of a normal medakafish spermatogonial cell line capable of sperm production
in vitro
Authors: Hong Yunhan , Liu Tongming , Zhao Haobin , Xu Hongyan , Wang Weijia , Liu Rong , Chen Tiansheng , Deng Jiaorong , Gui Jianfang
Published: May 2004

Spermatogonia are the male germ stem cells that continuously produce sperm for the next generation. Spermatogenesis is a complicated process that proceeds through mitotic phase of stem cell renewal and differentiation, meiotic phase, and postmeiotic phase of spermiogenesis. Full recapitulation of spermatogenesis
in vitro
has been impossible, as generation of normal spermatogonial stem cell lines without immortalization and production of motile sperm from these cells after long-term culture have not been achieved. Here we report the derivation of a normal spermatogonial cell line from a mature medakafish testis without immortalization. After 140 passages during 2 years of culture, this cell line retains stable but growth factor-dependent proliferation, a diploid karyotype, and the phenotype and gene expression pattern of spermatogonial stem cells. Furthermore, we show that this cell line can undergo meiosis and spermiogenesis to generate motile sperm. Therefore, the ability of continuous proliferation and sperm production in culture is an intrinsic property of medaka spermatogonial stem cells, and immortalization apparently is not necessary to derive male germ cell cultures. Our findings and cell line will offer a unique opportunity to study and recapitulate spermatogenesis
in vitro
and to develop approaches for germ-line transmission.
Proximal renal tubular acidosis in TASK2 K
+
channel-deficient mice reveals a mechanism for stabilizing bicarbonate transport
Authors: Warth Richard , Barrière Hervé , Meneton Pierre , Bloch May , Thomas Jörg , Tauc Michel , Heitzmann Dirk , Romeo Elisa , Verrey François , Mengual Raymond , Guy Nicolas , Bendahhou Saïd , Lesage Florian , Poujeol Philippe , Barhanin Jacques
Published: May 2004

The acid- and volume-sensitive TASK2 K
+
channel is strongly expressed in renal proximal tubules and papillary collecting ducts. This study was aimed at investigating the role of TASK2 in renal bicarbonate reabsorption by using the
task2
–/– mouse as a model. After backcross to C57BL6,
task2
–/– mice showed an increased perinatal mortality and, in adulthood, a reduced body weight and arterial blood pressure. Patch-clamp experiments on proximal tubular cells indicated that TASK2 was activated during

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transport. In control inulin clearance measurements,
task2
–/– mice showed normal NaCl and water excretion. During i.v. NaHCO
3
perfusion, however, renal Na
+
and water reabsorption capacity was reduced in –/– animals. In conscious
task2
–/– mice, blood pH,

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concentration, and systemic base excess were reduced but urinary pH and

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were increased. These data suggest that
task2
–/– mice exhibit metabolic acidosis caused by renal loss of

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. Both
in vitro
and
in vivo
results demonstrate the specific coupling of TASK2 activity to

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transport through external alkalinization. The consequences of the
task2
gene inactivation in mice are reminiscent of the clinical manifestations seen in human proximal renal tubular acidosis syndrome.
Catalysis-based enantioselective total synthesis of the macrocyclic spermidine alkaloid isooncinotine
Authors: Scheiper Bodo , Glorius Frank , Leitner Andreas , Fürstner Alois
Published: May 2004

A concise and efficient total synthesis of the spermidine alkaloid (-)-isooncinotine (
1
) incorporating a 22-membered lactam ring is outlined. The approach is largely catalysis-based, involving a selective iron-catalyzed alkyl–aryl cross-coupling reaction of a difunctionalized pyridine substrate, a heterogeneous asymmetric hydrogenation step to set the chiral center of the target, and a highly integrated ring-closing metathesis/hydrogenation sequence to forge the saturated macrocyclic edifice in a single operation.
T cell deficiency leads to cognitive dysfunction: Implications for therapeutic vaccination for schizophrenia and other psychiatric conditions
Authors: Kipnis Jonathan , Cohen Hagit , Cardon Michal , Ziv Yaniv , Schwartz Michal
Published: May 2004

The effects of the adaptive immune system on the cognitive performance and abnormal behaviors seen in mental disorders such as schizophrenia have never been documented. Here, we show that mice deprived of mature T cells manifested cognitive deficits and behavioral abnormalities, which were remediable by T cell restoration. T cell-based vaccination, using glatiramer acetate (copolymer-1, a weak agonist of numerous self-reactive T cells), can overcome the behavioral and cognitive abnormalities that accompany neurotransmitter imbalance induced by (+)dizocilpine maleate (MK-801) or amphetamine. The results, by suggesting that peripheral T cell deficit can lead to cognitive and behavioral impairment, highlight the importance of properly functioning adaptive immunity in the maintenance of mental activity and in coping with conditions leading to cognitive deficits. These findings point to critical factors likely to contribute to age- and AIDS-related dementias and might herald the development of a therapeutic vaccination for fighting off cognitive dysfunction and psychiatric conditions.
Morphometry of Hepatic Neoplasms and Altered Foci in the Mummichog, Fundulus heteroclitus
Authors: Stine Cynthia B. , Smith David L. , Vogelbein Wolfgang K. , Harshbarger John C. , Gudla Prabhakar R. , Lipsky Michael M. , Kane Andrew S.
Published: May 2004

The goal of this study was to intensively sample a small number of livers from a population of mummichog exposed to PAH-contaminated sediments and evaluate them for lesion pathology, distribution, shape, and volume, and the number of histological sections needed to adequately describe the extent of various lesions. Volumetric data for each lesion type from each step section was derived from digitized section images. The total number of hepatic alterations ranged from 10—125 per fish. Alterations included: eosinophilic, basophilic, and clear cell foci; hepatocellular carcinomas; hemangiopericytomas; and cholangiomas. Lesion volumes ranged from 0.00012—64 mm3 and represented 0.21%—67% of total liver volume. There was a tendency for the lesions to be more dorsal-ventrally compressed than spherical or ropelike when observed from longitudinal sections. Periodic subsampling of the data indicated that, on average, 6 evenly spaced, longitudinal histological sections were required to accurately estimate lesion volume and extent in our model population. These data provide a formulation for histological sampling techniques and methodological support for piscine and other cancer study models that observe lesion volume changes over time. Further, this study fosters the development of early quantitative endpoints, rather than using a large number of animals and waiting for tumor progression or death to occur.
Osteogenic Protein-1: Gene Expression and Treatment in the Rat Remnant Kidney Model
Authors: Dube Philip H. , Almanzar Maria M. , Frazier Kendall S. , Jones William K. , Charette Marc F. , Paredes Ana
Published: May 2004

Osteogenic Protein-1 (OP-1) is a bone morphogen involved in tissue repair and development. We have shown that OP-1 is downregulated during acute ischemic renal injury. Here we report the use of the rat remnant kidney model (RRKM) to evaluate changes in kidney OP-1 expression during chronic injury, and determine if treatment with recombinant human OP-1 (rhOP-1) aids in recovery from injury. Sprague—Dawley rats were subjected to kidney decapsulation (Cx) or 5/6 nephrectomy (Nx). Serum for BUN and creatinine and tissue for histology and mRNA analysis were collected at: 2, 10, and 12—14 wks post Nx. We show kidney OP-1 mRNA levels were downregulated at 2 and 12—14 wks post Nx. To determine the effect of rhOP-1 in the RRKM, rhOP-1 (0.25, 2.5 or 25 μg/kg) or vehicle (V) was injected in a second set of rats, 2 weeks after 2/3 left Nx for a total of six doses. Nx rats treated with rhOP-1 showed significantly increased tubular regeneration (increased mitotic figures, polyoid infolding, and tubular epithelial hyperplasia) in a dose dependent manner without changes in glomerular or tubular damage. rhOP-1 stimulates tubular epithelial cell regeneration, early in the repair process in a chronic renal failure model, before significant fibrosis is established.
Low myoplasmic Mg2+ potentiates calcium release during depolarization of frog skeletal muscle fibers.
Authors: Jacquemond V , Schneider M F
Published: May 2004

The role of intracellular free magnesium concentration ([Mg2+]) in modulating calcium release from the sarcoplasmic reticulum (SR) was studied in voltage-clamped frog cut skeletal muscle fibers equilibrated with cut end solutions containing two calcium indicators, fura-2 and antipyrylazo III (AP III), and various concentrations of free Mg2+ (25 microM-1 mM) obtained by adding appropriate total amounts of ATP and magnesium to the solutions. Changes in AP III absorbance were used to monitor calcium transients, whereas fura-2 fluorescence was used to monitor resting calcium. The rate of release (Rrel) of calcium from the SR was calculated from the calcium transient and found to be increased in low internal [Mg2+]. After correcting for effects of calcium depletion from the SR and normalization to SR content, the mean values of the inactivatable and noninactivatable components of Rrel were increased by 163 and 46%, respectively, in low Mg2+. Independent of normalization to SR content, the ratio of inactivatable to noninactivatable components of Rrel was increased in low internal [Mg2+]. Both observations suggest that internal [Mg2+] preferentially modulates the inactivatable component of Rrel, which is thought to be due to calcium-induced calcium release from the SR. This could also explain the observation that, in low internal [Mg2+], the time to the peak of the calcium transient for a 5-ms depolarizing pulse was not very different from the time to the peak of the delta [Ca2+] for a 10-ms pulse of the same amplitude. Finally, in low internal [Mg2+], the calcium transient elicited by a short depolarizing pulse was in some cases clearly followed by a very slow rise of calcium after the end of the pulse. The observed effects of reduced [Mg2+] on calcium release are consistent with a removal of the inhibition that the normal 1 mM myoplasmic [Mg2+] exerts on calcium release in skeletal muscle fibers.
Visual responses in teleosts. Electroretinograms, eye movements, and circadian rhythms.
Authors: McMahon D G , Barlow R B
Published: May 2004

We have recorded ocular potentials in response to brief flashes of light from two teleosts, the white perch (Roccus americana) and the green sunfish (Lepomis cyanellus). The animals were respired and maintained in an alert state for up to 2 d. Responses were recorded with corneal and transcleral electrodes. The responses of green sunfish were composed of electroretinogram (ERG) and eye movement potentials, whereas the responses in white perch contained only the ERG. Injection of curare abolished the sunfish eye movement potentials, unmasking the ERG. Observation under infrared illumination established a direct relationship between eye movements and the fast potentials which could be abolished by curare. We found no evidence of circadian changes in the amplitude of the ERG b-wave of either species. However, our results combined with those of a previous study of sunfish ocular potentials (Dearry, A., and B. Barlow, Jr. 1987. J. Gen. Physiol. 89: 745-770) suggest that the sunfish visual system exhibits rhythmic changes in oculomotor responses, which appear to be controlled by a circadian oscillator.
Is the mammalian cell plasma membrane a barrier to oxygen transport?
Authors: Subczynski W K , Hopwood L E , Hyde J S
Published: May 2004

Oxygen transport in the Chinese hamster ovary (CHO) plasma membrane has been studied by observing the collision of molecular oxygen with nitroxide radical spin labels placed in the lipid bilayer portion of the membrane at various distances from the membrane surface using the long-pulse saturation-recovery electron spin resonance (ESR) technique. The collision rate was estimated for 5-, 12-, and 16-doxylstearic acids from spin-lattice relaxation times (T1) measured in the presence and absence of molecular oxygen. Profiles of the local oxygen transport parameters across the membrane were obtained showing that the oxygen diffusion-concentration product is lower than in water for all locations at 37 degrees C. From oxygen transport parameter profiles, the membrane oxygen permeability coefficients were estimated according to the procedure developed earlier by Subczynski et al. (Subczynski, W. K., J. S. Hyde, and A. Kusumi. 1989. Proceedings of the National Academy of Sciences, USA. 86:4474-4478). At 37 degrees C, the oxygen permeability coefficient for the plasma membrane was found to be 42 cm/s, about two times lower than for a water layer of the same thickness as the membrane. The oxygen concentration difference across the CHO plasma membrane at physiological conditions is in the nanomolar range. It is concluded that oxygen permeation across the cell plasma membrane cannot be a rate-limiting step for cellular respiration. Correlations of the form PM = cKs between membrane permeabilities PM of small nonelectrolyte solutes of mol wt less than 50, including oxygen, and their partition coefficients K into hexadecane and olive oil are reported. Hexadecane: c = 26 cm/s, s = 0.95; olive oil: c = 23 cm/s, s = 1.56. These values of c and s differ from those reported in the literature for solutes of 50 less than mol wt less than 300 (Walter, A., and J. Gutknecht. 1986. Journal of Membrane Biology. 90:207-217). It is concluded that oxygen permeability through membranes can be reliably predicted from measurement of partition coefficients.
Modulation of rabbit ventricular cell volume and Na+/K+/2Cl- cotransport by cGMP and atrial natriuretic factor.
Authors: Clemo H F , Feher J J , Baumgarten C M
Published: May 2004

Previously we showed that atrial natriuretic factor (ANF) decreases cardiac cell volume by inhibiting ion uptake by Na+/K+/2Cl- cotransport. Digital video microscopy was used to study the role of guanosine 3',5'-monophosphate (cGMP) in this process in rabbit ventricular myocytes. Each cell served as its own control, and relative cell volumes (volume(test)/volume(control)) were determined. Exposure to 10 microM 8-bromo-cGMP (8-Br-cGMP) reversibly decreased cell volume to 0.892 +/- 0.007; the ED50 was 0.77 +/- 0.33 microM. Activating guanylate cyclase with 100 microM sodium nitroprusside also decreased cell volume to 0.889 +/- 0.009. In contrast, 8-bromo-adenosine 3',5'-monophosphate (8-Br-AMP; 0.01-100 microM) neither altered cell volume directly nor modified the response to 8-Br-cGMP. The idea that cGMP decreases cell volume by inhibiting Na+/K+/2Cl- cotransport was tested by blocking the cotransporter with 10 microM bumetanide (BUM) and removing the transported ions. After BUM treatment, 10 microM 8-Br-cGMP failed to decrease cell volume. Replacement of Na+ with N-methyl-D-glucamine or Cl- with methanesulfonate also prevented 8-Br-cGMP from shrinking cells. The data suggest that 8-Br-cGMP, like ANF, decreases ventricular cell volume by inhibiting Na+/K+/2Cl-cotransport. Evidence that ANF modulates cell volume via cGMP was also obtained. Pretreatment with 10 microM 8-Br-cGMP prevented the effect of 1 microM ANF on cell volume, and ANF suppressed 8-Br-cGMP-induced cell shrinkage. Inhibiting guanylate cyclase with the quinolinedione LY83583 (10 microM) diminished ANF-induced cell shrinkage, and inhibiting cGMP-specific phosphodiesterase with M&B22948 (Zaprinast; 100 microM) amplified the volume decrease caused by a low dose of ANF (0.01 microM) approximately fivefold. In contrast, neither 100 microM 8-Br-cAMP nor 50 microM forskolin affected the response to ANF. The effects of ANF, LY83583, and M&B29948 on cGMP levels in isolated ventricular myocytes were confirmed by 125I-cGMP radioimmunoassay. These data argue that ANF shrinks cardiac cells by increasing intracellular cGMP, thereby inhibiting Na+/K+/2Cl- cotransport. Basal cGMP levels also appear to modulate cell volume.
Kinetic and steady-state properties of Na+ channel and Ca2+ channel charge movements in ventricular myocytes of embryonic chick heart.
Authors: Josephson I R , Sperelakis N
Published: May 2004

Nonlinear or asymmetric charge movement was recorded from single ventricular myocytes cultured from 17-d-old embryonic chick hearts using the whole-cell patch clamp method. The myocytes were exposed to the appropriate intracellular and extracellular solutions designed to block Na+, Ca2+, and K+ ionic currents. The linear components of the capacity and leakage currents during test voltage steps were eliminated by adding summed, hyperpolarizing control step currents. Upon depolarization from negative holding potentials the nonlinear charge movement was composed of two distinct and separable kinetic components. An early rapidly decaying component (decay time constant range: 0.12-0.50 ms) was significant at test potentials positive to -70 mV and displayed saturation above 0 mV (midpoint -35 mV; apparent valence 1.6 e-). The early ON charge was partially immobilized during brief (5 ms) depolarizing test steps and was more completely immobilized by the application of less negative holding potentials. A second slower-decaying component (decay time constant range: 0.88-3.7 ms) was activated at test potentials positive to -60 mV and showed saturation above +20 mV (midpoint -13 mV, apparent valence 1.9 e-). The second component of charge movement was immobilized by long duration (5 s) holding potentials, applied over a more positive voltage range than those that reduced the early component. The voltage dependencies for activation and inactivation of the Na+ and Ca2+ ionic currents were determined for myocytes in which these currents were not blocked. There was a positive correlation between the voltage dependence of activation and inactivation of the Na+ and Ca2+ ionic currents and the activation and immobilization of the fast and slow components of charge movement. These complementary kinetic and steady-state properties lead to the conclusion that the two components of charge movement are associated with the voltage-sensitive conformational changes that precede Na+ and Ca2+ channel openings.
Voltage-dependent block of fast chloride channels from rat cortical neurons by external tetraethylammonium ion.
Authors: Sanchez D Y , Blatz A L
Published: May 2004

Tetraethylammonium ion (TEA) and its longer chain derivatives have been used extensively to block currents through K-selective ion channels. Substantial information has been gained about the structure and gating mechanisms of K and other cation channels from the analysis of the blocking interactions of TEA and other quaternary ammonium ions. We now present an analysis of blocking interactions between single Cl-selective ion channels from acutely dissociated rat cortical neurons and externally applied TEA. TEA applied to the extracellular membrane surface (TEAo) blocked Cl channels in a voltage-dependent manner, with hyperpolarizing potentials favoring block. The voltage dependence of block could be adequately fit assuming that TEA enters the channel pore and binds to a site located approximately 28% of the way through the membrane electrical field. The dose-response relationship between fractional current and [TEA]o at a fixed holding potential of -40 mV was well fit to a simple model with two blocking sites with dissociation constants (Kd) of approximately 2 and 70 mM. The dose-response relationship could also be fit by a mechanism where TEA only partially blocks the channels. At the bandwidth used in these experiments (1-2 kHz), both the mean open duration (composed of the open and blocked durations) and burst duration (composed of open, blocked, and short lifetime shut durations) increased with increased [TEA]o. This is expected if TEAo can bind and unbind only when the channel is in the open kinetic state. These results suggest that the structure of the permeability pathway of these anion-selective channels may be very similar to that of other channels that are blocked by TEA. Additionally, these results caution that a blocking effect by TEA cannot, by itself, be used as sufficient evidence for implicating the participation of K channels in a particular process.
Calcium current activated upon hyperpolarization of Paramecium tetraurelia.
Authors: Preston R R , Saimi Y , Kung C
Published: May 2004

Hyperpolarization of Paramecium tetraurelia under conditions where K+ currents are suppressed elicits an inward current that activates rapidly toward a peak at 25-80 ms and decays thereafter. This peak current (Ihyp) is not affected by removing Cl ions from the microelectrodes used to clamp membrane potential, or by changing extracellular Cl- concentration, but is lost upon removing extracellular Ca2+. Ihyp is also lost upon replacing extracellular Ca2+ with equimolar concentrations of Ba2+, Co2+, Mg2+, Mn2+, or Sr2+, suggesting that the permeability mechanism that mediates Ihyp is highly selective for Ca2+. Divalent cations also inhibit Ihyp when introduced extracellularly, in a concentration- and voltage-dependent manner. Ba2+ inhibits Ihyp with an apparent dissociation constant of 81 microM at -110 mV, and with an effective valence of 0.42. Ihyp is also inhibited reversibly by amiloride, with a dissociation constant of 0.4 mM. Ihyp is not affected significantly by changes in extracellular Na+, K+, or H+ concentration, or by EGTA injection. Also, it is unaffected by manipulations or mutations that suppress the depolarization-activated Ca2+ current or the various Ca(2+)-dependent currents of Paramecium. We suggest that Ihyp is mediated by a novel, hyperpolarization-activated calcium conductance that is distinct from the one activated by depolarization.
Divalent cation block and competition between divalent and monovalent cations in the large-conductance K+ channel from Chara australis.
Authors: Laver D R
Published: May 2004

The patch-clamp technique is used to investigate divalent ion block of the large-conductance K+ channel from Chara australis. Block by Ba2+, Ca2+, Mg2+, and Pt(NH3)4(2+) from the vacuolar and cytoplasmic sides is used to probe the structure of, and ion interactions within, the pore. Five divalent ion binding sites are detected. Vacuolar Ca2+ reduces channel conductance by binding to a site located 7% along the membrane potential difference (site 1, delta = 0.07; from the vacuolar side); it also causes channel closures with mean a duration of approximately 0.1-1 ms by binding at a deeper site (site 2, delta = 0.3). Ca2+ can exit from site 2 into both the vacuolar and cytoplasmic solutions. Cytoplasmic Ca2+ reduces conductance by binding at two sites (site 3, delta = -0.21; site 4, delta = -0.6; from the cytoplasmic side) and causes closures with a mean duration of 10-100 ms by binding to site 5 (delta = -0.7). The deep sites exhibit stronger ion specificity than the superficial sites. Cytoplasmic Ca2+ binds sequentially to sites 3-5 and Ca2+ at site 5 can be locked into the pore by a second Ca2+ at site 3 or 4. Ca2+ block is alleviated by increasing [K+] on the same side of the channel. Further, Ca2+ occupancy of the deep sites (2, 4, and 5) is reduced by K+, Rb+, NH4+, and Na+ on the opposite side of the pore. Their relative efficacy correlates with their relative permeability in the channel. While some Ca2+ and K+ sites compete for ions, Ca2+ and K+ can simultaneously occupy the channel. Ca2+ binding at site 1 only partially blocks channel conduction. The results suggest the presence of four K+ binding sites on the channel protein. One cytoplasmic facing site has an equilibrium affinity of 10 mM (site 6, delta = -0.3) and one vacuolar site (site 7, delta less than 0.2) has low affinity (greater than 500 mM). Divalent ion block of the Chara channel shows many similarities to that of the maxi-K channel from rat skeletal muscle.
Receptive field properties of rod-driven horizontal cells in the skate retina.
Authors: Qian H , Ripps H
Published: May 2004

The large receptive fields of retinal horizontal cells result primarily from extensive intercellular coupling via gap (electrical) junctions; thus, the extent of the receptive field provides an index of the degree to which the cells are electrically coupled. For rod-driven horizontal cells in the dark-adapted skate retina, a space constant of 1.18 +/- 0.15 mm (SD) was obtained from measurements with a moving slit stimulus, and a comparable value (1.43 +/- 0.55 mm) was obtained with variation in spot diameter. These values, and the extensive spread of a fluorescent dye (Lucifer Yellow) from the site of injection to neighboring cells, indicate that the horizontal cells of the all-rod retina of skate are well coupled electrically. Neither the receptive field properties nor the gap-junctional features of skate horizontal cells were influenced by the adaptive state of the retina: (a) the receptive field organization was unaffected by light adaptation, (b) similar dye coupling was seen in both dark- and light-adapted retinae, and (c) no significant differences were found in the gap-junctional particle densities measured in dark- and light-adapted retinas, i.e., 3,184 +/- 286/microns 2 (n = 8) and 3,073 +/- 494/microns 2 (n = 11), respectively. Moreover, the receptive fields of skate horizontal cells were not altered by either dopamine, glycine, GABA, or the GABAA receptor antagonists bicuculline and picrotoxin. We conclude that the rod-driven horizontal cells of the skate retina are tightly coupled to one another, and that the coupling is not affected by photic and pharmacological conditions that are known to modulate intercellular coupling between cone-driven horizontal cells in other species.
Flash photolysis of caged compounds in Limulus ventral photoreceptors.
Authors: Faddis M N , Brown J E
Published: May 2004

Rapid concentration jumps of Ins(1,4,5)P3 or ATP were made inside Limulus ventral photoreceptors by flash photolysis of the parent caged compounds. In intact ventral photoreceptors, the photolysis flash evokes a maximum amplitude light-activated current; therefore, a procedure was developed for uncoupling phototransduction by blocking two of the initial reactions in the cascade, rhodopsin excitation and G protein activation. Rhodopsin was inactivated by exposure to hydroxylamine and bright light. This procedure abolished the early receptor potential and reduced the quantum efficiency by 325 +/- 90-fold (mean +/- SD). G protein activation was blocked by injection of guanosine-5'-O-(2-thiodiphosphate) (GDP beta S). GDP beta S injection reduced the quantum efficiency by 1,881 +/- 1,153-fold (mean +/- SD). Together hydroxylamine exposure and GDP beta S injection reduced the quantum efficiency by 870,000 +/- 650,000-fold (mean +/- SD). After the combined treatment, photoreceptors produced quantum bumps to light that was approximately 10(6) times brighter than the intensity that produced quantum bumps before treatment. Experiments were performed with caged compounds injected into photoreceptors in which phototransduction was largely uncoupled. Photolysis of one compound, myo-inositol 1,4,5-triphosphate P4(5)-1-(2-nitrophenyl)ethyl ester (caged IP3), increased the voltage clamp current in response to the flashlamp by more than twofold without changing the latency of the response. The effect was not seen with photolysis of either adenosine-5'-triphosphate P3-1-(2-nitrophenyl)ethyl ester (caged ATP) or caged IP3 in cells preloaded with either heparin or (1,2-bis-(o-amino-phenoxy)ethane-N-N-N'-N' tetraacetic acid tetrapotassium salt (BAPTA). The results suggest that photoreleased IP3 releases calcium ions from intracellular stores and the resulting increase in [Ca2+]i enhances the amplification of the phototransduction cascade.
The nss mutation or lanthanum inhibits light-induced Ca2+ influx into fly photoreceptors.
Authors: Rom-Glas A , Sandler C , Kirschfeld K , Minke B
Published: May 2004

Ion-selective calcium microelectrodes were inserted into the compound eyes of the wild-type sheep blowfly Lucilia or into the retina of the no steady state (nss) mutant of Lucilia. These electrodes monitored light-induced changes in the extracellular concentration of calcium (delta[Ca2+]o) together with the extracellularly recorded receptor potential. Prolonged dim lights induced a steady reduction in [Ca2+]o during light in the retina of normal Lucilia, while relatively little change in [Ca2+]o was observed in the retina of the nss mutant. Prolonged intense light induced a multiphasic change in [Ca2+]o: the [Ca2+]o signal became transient, reaching a minimum within 6 s after light onset, and then rose to a nearly steady-state phase below the dark concentration. When lights were turned off, a rapid increase in [Ca2+]o was observed, reaching a peak above the dark level and then declining again to the dark level within 1 min. In analogy to similar studies conduced in the honeybee drone, we suggest that the reduction in [Ca2+]o reflects light-induced Ca2+ influx into the photoreceptors, while the subsequent increase in [Ca2+]o reflects the activation of the Na-Ca exchange which extrudes Ca2+ from the cells. In the nss mutant in response to intense prolonged light, the receptor potential declines to baseline during light while the Ca2+ signal is almost abolished, revealing only a short transient reduction in [Ca2+]o. Application of lanthanum (La3+), but not nickel (Ni2+), into the retinal extracellular space of normal Lucilia mimicked the effect of the nss mutation on the receptor potential, while complete elimination of the Ca2+ signal in a reversible manner was observed. The results suggest that La3+ and the nss mutation inhibit light-induced Ca2+ influex into the photoreceptor in a manner similar to the action of the trp mutation in Drosophila, which has been shown to block specifically a light-activated Ca2+ channel necessary to maintain light excitation.
Inhibition of heart calcium and chloride currents by sodium iodide. Specific attenuation in cAMP-dependent protein kinase-mediated regulation.
Authors: Walsh K B , Long K J
Published: May 2004

The enzymes cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) regulate the activity of cardiac ion channel proteins. In this study the whole-cell arrangement of the patch clamp technique was used to examine the effect of NaI on PKA-stimulated Cl- and Ca2+ channels in isolated guinea pig ventricular myocytes. Cl- currents (ICl) activated either by the beta-adrenergic agonist isoproterenol or the membrane-soluble cAMP analogue, 8-chlorphenylthio (8-CPT) cAMP, were greatly reduced in amplitude after substitution of an external solution containing 140 mM NaCl with a solution containing 140 mM NaI. This reduction was accompanied by a shift of -7 mV in the reversal potential (Erev) for ICl and could be reversed upon return to the NaCl external solution. Inhibition of ICl by NaI occurred in a concentration-dependent manner and was more pronounced for inward ICl (IC50 = 19 mM at -60 mV) than for outward ICl (IC50 = 60 mM at +60 mV). In contrast to ICl activated by PKA, ICl activated by PKC was slightly augmented in the presence of NaI and the Erev was found to shift by -15 mV. Based on these data, the relative permeability of I- to Cl- (PI/PCl) for this channel was calculated to be 1.79. NaI produced no change in the amplitude of inward calcium currents (ICa) recorded under basal conditions, but strongly inhibited ICa augmented by isoproterenol and 8-CPT cAMP, and during dialysis of cells with the catalytic subunit of PKA (CS). The in vitro incorporation of [gamma-32P]ATP into histone IIA and Kemptide, measured in the presence of PKA and cAMP, was not significantly different in assay mixtures containing salts of Cl- and I-. However, the ability of isoproterenol to augment basal ICa in whole-cell experiments was attenuated when experiments were carried out entirely in NaI external solution. Thus, the reduction in ICl and ICa observed in this study may result from a direct effect of I- on the phosphorylation/dephosphorylation of cardiac ion channel proteins or associated regulatory proteins.
Steady-state and dynamic properties of cardiac sodium-calcium exchange. Sodium-dependent inactivation.
Authors: Hilgemann D W , Matsuoka S , Nagel G A , Collins A
Published: May 2004

Sodium-calcium exchange current was isolated in inside-out patches excised from guinea pig ventricular cells using the giant patch method. The outward exchange current decayed exponentially upon activation by cytoplasmic sodium (sodium-dependent inactivation). The kinetics and mechanism of the inactivation were studied. (a) The rate of inactivation and the peak current amplitude were both strongly temperature dependent (Q10 = 2.2). (b) An increase in cytoplasmic pH from 6.8 to 7.8 attenuated the current decay and shifted the apparent dissociation constant (Kd) of cytoplasmic calcium for secondary activation of the exchange current from 9.6 microM to < 0.3 microM. (c) The amplitude of exchange current decreased synchronously over the membrane potential range from -120 to 60 mV during the inactivation, indicating that voltage dependence of the exchanger did not change during the inactivation process. The voltage dependence of exchange current also did not change during secondary modulation by cytoplasmic calcium and activation by chymotrypsin. (d) In the presence of 150 mM extracellular sodium and 2 mM extracellular calcium, outward exchange current decayed similarly upon application of cytoplasmic sodium. Upon removal of cytoplasmic sodium in the presence of 2-5 microM cytoplasmic free calcium, the inward exchange current developed in two phases, a fast phase within the time course of solution changes, and a slow phase (tau approximately 4 s) indicative of recovery from sodium-dependent inactivation. (e) Under zero-trans conditions, the inward current was fully activated within solution switch times upon application of cytoplasmic calcium and did not decay. (f) The slow recovery phase of inward current upon removal of cytoplasmic sodium was also present under the zero-trans condition. (g) Sodium-dependent inactivation shows little or no dependence on membrane potential in guinea pig myocyte sarcolemma. (h) Sodium-dependent inactivation of outward current is attenuated in rate and extent as extracellular calcium is decreased. (i) Kinetics of the sodium-dependent inactivation and its dependence on major experimental variables are well described by a simple two-state inactivation model assuming one fully active and one fully inactive exchanger state, whereby the transition to the inactive state takes place from a fully sodium-loaded exchanger conformation with cytoplasmic orientation of binding sites (E1.3Ni).
Steady-state and dynamic properties of cardiac sodium-calcium exchange. Secondary modulation by cytoplasmic calcium and ATP.
Authors: Hilgemann D W , Collins A , Matsuoka S
Published: May 2004

Dynamic responses of cardiac sodium-calcium exchange current to changes of cytoplasmic calcium and MgATP were monitored and analyzed in giant membrane patches excised from guinea pig myocytes. Secondary dependencies of exchange current on cytoplasmic calcium are accounted for in terms of two mechanisms: (a) The sodium-dependent inactivation process, termed I1 modulation, is itself strongly modulated by cytoplasmic calcium. Recovery from the I1 inactivated state is accelerated by increasing cytoplasmic calcium, and the calculated rate of entrance into I1 inactivation is slowed. (b) A second modulation process, termed I2 modulation, is not sodium dependent. As with I1 modulation, the entrance into I2 inactivation takes place over seconds in the absence of cytoplasmic calcium. The recovery from I2 inactivation is a calcium-dependent transition and is rapid (< 200 ms) in the presence of micromolar free calcium. I1 and I2 modulation can be treated as linear, independent processes to account for most exchange modulation patterns observed: (a) When cytoplasmic calcium is increased or decreased in the presence of high cytoplasmic sodium, outward exchange current turns on or off, respectively, on a time scale of multiple seconds. (b) When sodium is applied in the absence of cytoplasmic calcium, no outward current is activated. However, the full outward current is activated within solution switch time when cytoplasmic calcium is applied together with sodium. (c) The calcium dependence of peak outward current attained upon application of cytoplasmic sodium is shifted by approximately 1 log unit to lower concentrations from the calcium dependence of steady-state exchange current. (d) The time course of outward current decay upon decreasing cytoplasmic calcium becomes more rapid as calcium is reduced into the submicromolar range. (e) Under nearly all conditions, the time courses of current decay during application of cytoplasmic sodium and/or removal of cytoplasmic calcium are well fit by single exponentials. Both of the modulation processes are evidently affected by MgATP. Similar to the effects of cytoplasmic calcium, MgATP slows the entrance into I1 inactivation and accelerates the recovery from inactivation. MgATP additionally slows the decay of outward exchange current upon removal of cytoplasmic calcium by 2-10-fold, indicative of an effect on I2 inactivation. Finally, the effects of cytoplasmic calcium on sodium-calcium exchange current are reconstructed in simulations of the I1 and I2 modulation processes as independent reactions.
Steady-state and dynamic properties of cardiac sodium-calcium exchange. Ion and voltage dependencies of the transport cycle.
Authors: Matsuoka S , Hilgemann D W
Published: May 2004

Ion and voltage dependencies of sodium-calcium exchange current were studied in giant membrane patches from guinea pig ventricular cells after deregulation of the exchanger with chymotrypsin. (a) Under zero-trans conditions, the half-maximum concentration (Kh) of cytoplasmic calcium (Cai) for activation of the isolated inward exchange current decreased as the extracellular sodium (Nao) concentration was decreased. The Kh of cytoplasmic sodium (Nai) for activation of the isolated outward exchange current decreased as the extracellular calcium (Cao) concentration was decreased. (b) The current-voltage (I-V) relation of the outward exchange current with saturating concentrations of Nai and Cao had a shallow slope (twofold change in approximately 100 mV) and a slight saturation tendency at very positive potentials. The outward current gained in steepness as the Nai concentration was decreased, such that the Kh for Nai decreased with depolarization. The decrease of Kh for Nai with depolarization was well described by a Boltzmann equation (e alpha.Em/26.6) with a slope (alpha) of -0.06. (c) Voltage dependence of the outward current was lost as the Cao concentration was decreased, and the Kh for Cao increased upon depolarization with a Boltzmann slope of 0.26. (d) The I-V relation of the inward exchange current, under zero-trans conditions, was also almost linear (twofold change in approximately 100 mV) and showed some saturation tendency with hyperpolarization as the Cai concentration was decreased. The Kh for Cai decreased with depolarization (Boltzmann slope, -0.10). Voltage dependence of the inward current was decreased in the presence of a high (300 mM) Nao concentration. (e) In the presence of both Na and Ca on both membrane sides, the I-V relations with saturating Nai show sigmoidal shape and clear saturation at positive potentials. Measured reversal potentials were close to the equilibrium potential expected for a 3 Na to 1 Ca exchange. (f) Nai and Cai interacted competitively with respect to the outward current, but in a mixed competitive-noncompetitive fashion with respect to the inward current. (g) Cai inhibited the outward exchange current in a voltage-dependent manner. The half-effective concentration for inhibition (Ki) by Cai increased upon depolarization with a Boltzmann slope of 0.32 in 25 mM Nai and 0.20 in 100 mM Nai. (h) Nai also inhibited the inward exchange current voltage dependently. The Ki decreased upon depolarization (Boltzmann slope, -0.11 at 3 microM Cai and -0.10 at 1.08 mM Cai).(ABSTRACT TRUNCATED AT 400 WORDS)
Permeation and gating properties of the L-type calcium channel in mouse pancreatic beta cells.
Authors: Smith P A , Aschroft F M , Fewtrell C M
Published: May 2004

Ba2+ currents through L-type Ca2+ channels were recorded from cell-attached patches on mouse pancreatic beta cells. In 10 mM Ba2+, single-channel currents were recorded at -70 mV, the beta cell resting membrane potential. This suggests that Ca2+ influx at negative membrane potentials may contribute to the resting intracellular Ca2+ concentration and thus to basal insulin release. Increasing external Ba2+ increased the single-channel current amplitude and shifted the current-voltage relation to more positive potentials. This voltage shift could be modeled by assuming that divalent cations both screen and bind to surface charges located at the channel mouth. The single-channel conductance was related to the bulk Ba2+ concentration by a Langmuir isotherm with a dissociation constant (Kd(gamma)) of 5.5 mM and a maximum single-channel conductance (gamma max) of 22 pS. A closer fit to the data was obtained when the barium concentration at the membrane surface was used (Kd(gamma) = 200 mM and gamma max = 47 pS), which suggests that saturation of the concentration-conductance curve may be due to saturation of the surface Ba2+ concentration. Increasing external Ba2+ also shifted the voltage dependence of ensemble currents to positive potentials, consistent with Ba2+ screening and binding to membrane surface charge associated with gating. Ensemble currents recorded with 10 mM Ca2+ activated at more positive potentials than in 10 mM Ba2+, suggesting that external Ca2+ binds more tightly to membrane surface charge associated with gating. The perforated-patch technique was used to record whole-cell currents flowing through L-type Ca2+ channels. Inward currents in 10 mM Ba2+ had a similar voltage dependence to those recorded at a physiological Ca2+ concentration (2.6 mM). BAY-K 8644 (1 microM) increased the amplitude of the ensemble and whole-cell currents but did not alter their voltage dependence. Our results suggest that the high divalent cation solutions usually used to record single L-type Ca2+ channel activity produce a positive shift in the voltage dependence of activation (approximately 32 mV in 100 mM Ba2+).