Structural and Functional Rearrangements of Mesophyll as a Probable Basis for the Cytokinin-Dependent Assimilate Translocation in Detached Leaves

The effects of benzyladenine (BA) on the mesophyll functioning, such as osmotic potential (), the effect of the inhibitors of ⁺-ATPase on the influx of ¹⁴C-sucrose, the direction of carbon metabolism, and the rate of dark respiration, were followed in the detached leaves of pumpkin (Cucurbita pepo L.) and broad beans (Vicia faba L.). BA elevated and established a gradient of (_p) between the treated and untreated leaf regions. The inhibitors of H⁺-ATPase did not affect the BA-induced influx of ¹⁴C-sucrose. The changes were accompanied with the elevated synthesis of starch and other polymeric compounds and the diminished synthesis of the substances of relatively low molecular weight. The stimulation of dark respiration was short and inconsiderable. The author concludes that the BA-induced transport was a passive process related to a increase. Leaf expansion accompanied by the synthesis of high-molecular-weight substances essential for cell growth and by starch synthesis apparently increased the sink capacity of the BA-treated detached leaves. The diminished efflux from the leaf blade was probably related to a lowered level of the transportable carbon compounds restricting their entry into the phloem. The influx induction could result from the activation of growth and metabolic processes, the decline in the number of organic molecules per cell volume unit, and the development of _p between the source and sink leaf regions.     

Effects of water stress on the gas exchange,the activities of some enzymes of carbon and nitrogen metabolism,and on the pool sizes of some organic acids in maize leaves

Water-stressed maize (Zea mays L.) leaves showed a large decrease in leaf conductance during photosynthesis. Net CO₂ uptake and evaporation declined fast at mild stress (=–0.6 to –1.0 MPa) and slower at more severe stress (=–1.0 to -1.2 MPa), whereas the CO₂ concentration in the intercellular spaces (C_i) did not drop to the CO₂ compensation point. The activities of the enzymes of photosynthetic carbon metabolism tested in this study dropped by approx. 30% at =-1.2 MPa. Glutamine synthetase activity was unaffected by water stress, whereas the activity of nitrate reductase was almost completely inhibited. The decline of enzyme activities in relation to was correlated with a concomitant decrease in the content of total soluble protein of the stressed leaves. The total leaf pools of malate, pyruvate and oxaloacetate decreased almost linearly in relation to , thus obviously contradicting the almost constant C_i. In comparison to the controls (=0.6 MPa) the content of citrate and isocitrate increaed markedly at =-0.9 MPa and decreased again at =-1.2 MPa.Abbreviations PCR photosynthetic carbon reduction cycle - PCO photosynthetic carbon oxidation cycle - PEP phosphoenolypyruvate - RuBP ribulose-1,5-bisphosphate     

Histochemical study of isolated neurons in culture from chick embryo sympathetic ganglia

Summary The dissociated sympathetic neurons maintained in culture without direct contact with glia cells keep up some enzymatic activities, like those of carboxylic esterases, of succinic-dehydrogenase (SDH), glutamic-dehydrogenase (GDH), monoamine oxydase (MAO), lactic-dehydrogenase (LDH) and alcoholic dehydrogenase (ADH) for more or less long periods.Nerve growth factor (NGF) promotes the maintenance of mitochondrial enzymes, of acetylcholinesterase (AChE) and non-specific cholinesterases (n. s. ChE), of LDH in sympathetic neurons. In absence of NGF, the more archaic enzymes, like ADH, or the less specific, like -naphtol esterases still are kept up, whereas the enzymatic mitochondrial and cholinesterasic activities are no more detected.With the technical assistance of Miss E. Darcel.This communication is part of the Doctorat de Biologie Humaine thesis.     

The effect of pinealectomy on entrainment of the locomotor activity rhythm in sparrows maintained on various short days

Summary Pinealectomy of house sparrows on 3L:21D (3 h light per 24 h) resulted in a significant increase in the time between the onset of perch-hopping activity and lights on (on) as well as the time between the offset of activity and lights of (off). The daily variance in on and off was also increased following the removal of the pineal gland. On longer light cycles (i.e., 5L:19D; 7L:17D), neither on or off, nor the variance of on or off was different between sham-pinealectomized and pinealectomized sparrows. Upon returning the birds to an ultrashort light cycle, 1L:23D, off, as well as the variance in on and off were again found to be significantly larger in the pinealectomized birds when compared to sham-operated controls. These results indicate that the effects of pinealectomy on the entrained rhythm of locomotor activity are most pronounced when birds are exposed to a weak entraining agent, such as an ultrashort light: dark cycle. In view of the observation that pinealectomy can alter the phase relationship between activity onset and offset, it is suggested that the pineal gland may be involved in the coupling of the oscillators that regulate activity onset and offset.     

Chloroplast volume: cell water potential relationships and acclimation of photosynthesis to leaf water deficits

Studies were undertaken to determine if there is an association between nonstomatally-mediated acclimation of photosynthesis to low water potential (w) and the maintenance of chloroplast volume during water stress. Spinach plants either kept well watered throughout their growth (non-acclimated), or subjected to water stress such that leaf w dropped to -1.5 megapascals (MPa) and then were rewatered (acclimated) were subjected to drought episodes. During these stress periods, photosynthesis was maintained to a greater extent in acclimated plants as compared to non-acclimated plants at w below -1 MPa.Estimates of internal leaf [CO₂] suggested that photosynthetic acclimation to low w was not primarily due to altered stomatal response. As w dropped from initial values, a decline in steady state levels of ribulose 1,5-bisphosphate (RuBP) occurred in both non-acclimated and acclimated plants. RuBP decline was less severe in acclimated plants.Low w effects on chloroplast volume in non-acclimated and acclimated plants were estimated by measuring the volume of intact chloroplasts isolated from plants in solutions which were made isotonic to declining leaf osmotic potential during the drought episodes. Chloroplast volume was maintained to a greater extent at low w in acclimated, as compared with non-acclimated plants. Although substantial osmotic adjustment occurred in both non-acclimated and acclimated plants, the extent of osmotic adjustment was the same. These data were interpreted as supporting the hypothesis that cellular-level acclimation to low w is associated with chloroplast volume maintenance, and this physiological acclimation is correlated with enhanced photosynthetic capacity of the leaf at low w.Abbreviations [CO₂]_i internal leaf CO₂ concentration - s osmotic potential - RWC relative water content - RuBP ribulose 1,5-bisphosphate - w water potential     

Temperature and moisture effects on C and N mineralization from surface applied clover residue

A better understanding of the effect of temperature (T) and moisture on soil microbial activity should improve our ability to predict N mineralization from soil organic matter and crop residues. The objective of this study was to evaluate the effects of water potential () and T on C and N mineralization from unamended Cecil loamy sand soil (clayey, kaolinitic, thermic Typic Kanhapludult) and from crimson clover (Trifolium incarnatum L.) residues applied on the soil surface. Cecil soil was packed into acrylic plastic cylinders, adjusted to -5.0, -1.5, -0.03, or -0.003 MPa, treated with clover residues on the surface or left unamended, and incubated at 10, 20, 28, or 35°C for 21 d. Headspace gas samples for CO2 and N2O determinations were taken periodically and NH3 evolved was trapped. Inorganic N in soil and residue extracts was analyzed after 21 d. When increased from -5.0 to -0.003 MPa, total CO2 evolved from unamended soil increased linearly with ln(-), whereas total CO2 evolved from clover residue increased exponentially with . In both cases the effect of was enhanced as T increased. Two-dimensional (T, ) equations were developed to describe these effects. Apparent net mineralized N from the clover residue increased with until it reached a maximum between -0.5 and -0.03 Mpa.     

Leaf water potential,component potentials and relative water content in a xeric grass,Agropyron dasystachyum (Hook.) Scribn.

Summary Leaf water potential ( _l ), osmotic potential ( _s ), pressure potential ( _p , turgor pressure), relative water content (R) and their interrelationships were determined for a xeric grass (Agropyron dasystachyum) found in the grasslands of Canada. Thermocouple psychrometers were used to measure _l and _s ; _p was obtained by subtraction. _l dropped from near 0 bars to about-28 bars as R went from 90% to 75%. R greater than 90% was not observed, perhaps because of a systematic error in determination of turgid water content. R remained relatively high in A. dasystachyum, even at low _l . The slope of the _l -R relationship was similar to other species which are generally considered to be drought tolerant. _p as high as 14 bars was observed. Most of the decrease in _l was accounted for by a decline in _p . The ability of A. dasystachyum to adjust to fluctuating water stress over the growing season is probably as much related to changes in tissue structure and turgor relationships as to simple changes in osmotic potential.     

Ecophysiological analysis of woody species in contrasting temperate communities during wet and dry years

This study employed an intensive sampling regime in which leaf gas exchange and tissue-water relations were measured simultaneously on the same leaf at midday on 19 tree species from three distinct forest communities during wet (1990) and dry (1991) growing seasons. The study sites were located on a xeric barrens, a misic valley floor, and a wet-mesic floodplain in central Pennsylvania, United States. The xeric, mesic, and wetmesic sties had drought-related decreases in gravimetric soil moisture of 53, 34 and 27%, respectively. During the wet year, xeric and mesic communities had high seasonal mean photosynthetic rates (A) and stomatal conductance of water vapor (g _wv) and low midday leaf water potential (), whereas the wet-mesic community had low A and g _wv and high midday . The mesic and wet-mesic communities had dry year decreases in predawn , g _wv and A with the greatest drought effect occurring in the mesic community. Regression analysis indicated that species from each site that exhibited high wet-year A and g _wv tended to have low midday . This trend was reversed only in the mesic community in the drought year. Despite differences in midday , all three communities had similar midday leaf turgor pressure (_p) in the wet year attributable to lower osmotic potential at zero turgor ( ⁰ ) with increasing site droughtiness. Lower wet year ⁰ in the xeric community was due to low symplast volume rather than high solute content. Species with the lowest ⁰ in the wet year often did not have the lowest ¹⁰⁰ possibly related to differences in tissue elasticity. Moreover, increased elasticity during drought may have masked osmotic adjustment in ¹⁰⁰ but not in ⁰ , via dilution of solutes at full hydration in some species. Despite the sampling regime used, there were no relationships between gas exchange and osmotic and elastic parameters that were consistently significant among communities or years. This result questions the universal, direct effect of osmotic and elastic adjustments in the maintenance of photosynthesis during drought. By including a large number of species, this study provided new insight to the ecophysiology of contrasting forest communities, and the community-wide impact of drought on contrasting sites.     

Water relations and growth of shrubs before and after fire in a semi-arid woodland

Summary Plant water relations and shoot growth rate of shrubs resprouting after fire or unburnt were measured in a semi-arid poplar box (Eucalyptus populnea) shrub woodland of eastern Australia. In vegetation unburnt for about 60 years, the dawn xylem water potential (_x) of the dominant shrub species was about-1.0 MPa when the soil was wet and-8.0 MPa when the soil was very dry. At any one time, the dominant shrub species,Eremophila mitchellii, E. sturtii, Geijera parviflora andCassia nemophila, were similar in _x butAcacia aneura andDodonaea viscosa were consistently higher in _x than this group when the soil was moist and lower when the soil was dry. The dominant tree species,Eucalyptus populnea andE. intertexta, appeared to have access to additional water beneath the hardpan which is located 60–80 cm below the surface. When shrubs were under extreme water stress (_x of-8 MPa), the trees had a _x of-3 to-3.6 MPa. Following a fire, both _x and leaf stomatal conductance (g _s) of resprouting shrubs were higher for about 5 years than comparable-aged unburnt vegetation, with relative differences in _x increasing with drought stress. Elongation rate of resprouts was positively linked to prefire shrub height in 3 of 4 species. However, shrubs resprouting after high intensity fires had substantially higher rates of shoot elongation than after low intensity fires which were in turn higher than for foliar expansion of unburnt shrubs. It is concluded that the growth rate of resprouting shrubs is primarily determined by physiological/ morphological factors associated with plant size but is also assisted by greater availability of water and possibly nutrients for a period after fire.     

The mitochondrial membrane potential (deltapsi(m)) in apoptosis; an update

Mitochondrial dysfunction has been shown to participate in the induction of apoptosis and has even been suggested to be central to the apoptotic pathway. Indeed, opening of the mitochondrial permeability transition pore has been demonstrated to induce depolarization of the transmembrane potential (_m), release of apoptogenic factors and loss of oxidative phosphorylation. In some apoptotic systems, loss of _m may be an early event in the apoptotic process. However, there are emerging data suggesting that, depending on the model of apoptosis, the loss of _m may not be an early requirement for apoptosis, but on the contrary may be a consequence of the apoptotic-signaling pathway. Furthermore, to add to these conflicting data, loss of _m has been demonstrated to not be required for cytochrome c release, whereas release of apoptosis inducing factor AIF is dependent upon disruption of _m early in the apoptotic pathway. Together, the existing literature suggests that depending on the cell system under investigation and the apoptotic stimuli used, dissipation of _m may or may not be an early event in the apoptotic pathway. Discrepancies in this area of apoptosis research may be attributed to the fluorochromes used to detect _m. Differential degrees of sensitivity of these fluorochromes exist, and there are also important factors that contribute to their ability to accurately discriminate changes in _m.     

On Mechanism of Interaction of Organophosphorus Inhibitors with Cholinesterases of Different Origin

A study is carried out as a development of A.P. Brestkin's concept of mechanism of irreversible inhibition of cholinesterases (ChE) by organophosphorus inhibitors (OPI) with taking into account reversibility of the first stage of this reaction, which has made it possible to determine individual constants of separate stages of the process. For the first time, a comparative study is performed on horse blood serum BuChE, human erythrocyte AChE, and ChE of optical ganglia of Pacific squid Todarodes pacificus. Besides, the OPI set is enlarged essentially due to use of some highly specific inhibitors of each of the enzymes. To evaluate the cholinesterase activity, chromogenic indophenol esters are used as substrates. For each of the studied ChE, differences in sensitivity to the studied OPI are realized only in values of the kinetic constant of formation of the enzyme-inhibitor complex (k ₅), whereas the rate constants of dissociation of this complex to initial components (ChE and OPI) (k _–5) and of process of its transformation into phosphorylated ChE (k ₆) are close to each other by the values, values of these constants k _–5 and k ₆ for different enzymes also being similar. Some statements about the molecular mechanism of the cholinesterase catalysis are formulated. It is suggested that the revealed elements of similarity of different ChE are realized in the work of the catalytic machine of active centers of the enzymes.     

Autumn stomatal closure in six conifer species of the Central Rocky Mountains

Summary Environmental and water relations parameters during fall were monitored for six conifer tree species common to the central Rocky Mountains growing naturally at the same location (Pinus contorta, Pinus ponderosa, Pinus flexilus, Pseudotsuga menziesii, Abies lasiocarpa, Picea engelmannii). Subsequent to what appeared to be the beginning of seasonal stomatal closure, leaf conductance to water vapor declined sharply following the onset of freezing air temperatures at night. A coincident rapid decline in morning xylem pressure potentials (_p) also occurred which resulted in values that were considerably below afternoon _p. Continuing decreases in maximum leaf conductance during the day were highly correlated with corresponding decreases in minimum nocturnal air temperatures of the preceding night. By mid-December, morning _p returned to values very near afternoon _p and were only slightly lower than before the onset of subfreezing nights. A preliminary model is proposed which interprets the qualitative interaction between air and soil temperatures, soil and plant water potentials, and leaf conductance during seasonal stomatal closure in fall.     

Shoot water relations and gas exchange of western hemlock and western red cedar seedlings during establishment on a reforestation site

Summary Shoot water relations, summer gas exchange response and morphological development of western hemlock [Tsuga heterophylla (Raf.) Sarg.] and western red cedar (Thuja plicata Donn) seedlings were monitored over the first growing season on a coastal reforestation site in British Columbia. In March, osmotic potential (_s) at saturation [_s(sat)] was –1.98 MPa and turgor loss point [_s(tlp)] –2.38 MPa for western hemlock, while western red cedar had –1.45 MPa _s(sat) and –1.93 MPa _s(tlp). Seasonally _s increased through June and then decreased through September, with western hemlock –0.15 to –0.50 MPa lower than western red cedar. Maximum bulk modulus of elasticity (_max) for western hemlock was 29.3 MPa in March, decreased to 15.0 MPa in June and increased to 25.0 MPa from July through September, while western red cedar _max was 10.6 MPa in March and around 8.0 MPa thereafter. Utilized turgor (T _util) for western hemlock was <40% from March through May, 69 to 78% from June through August and 96% in September, while western red cedar T _util was 68 to 73% during March and April, 84 to 96% from May through August and 100% in September. Maximum CO₂ assimilation rate (A) of western red cedar was more than double western hemlock, and for both species A declined in a linear fashion with increasing vapour pressure deficit (D). Maximum foliage conductance (g _wv) declined in a concave manner as D increased in both species, with western red cedar values 50 to 67% greater than western hemlock. Maximum daily g _wv declined in a concave manner as predawn shoot water potential (_pd) decreased, with maximum daily g _wv 1.8 to 3.6 times greater in western red cedar than western hemlock, when _pd was –0.25 and –1.4 MPa, respectively. Western red cedar, compared to western hemlock, had a greater increase in A as g _wv increased. Eight months after planting, western red cedar seedlings had twice the root growth, measured as root dry weight and root number, of western hemlock.     

The irreversibility of inner mitochondrial membrane permeabilization by Ca2+ plus prooxidants is determined by the extent of membrane protein thiol cross-linking

We have previously shown that mitochondrial membrane potential () drop promoted by prooxidants and Ca²⁺ can be reversed but not sustained by ethylene glycol-bis(-aminoethylether)-N,N,N,N-tetraacetic acid (EGTA) unless dithiothreitol (DTT), a disulfide reductant, is also added [Valle, V. G. R., Fagian, M. M., Parentoni, L. S., Meinicke, A. R., and Vercesi, A. E. (1993).Arch. Biochem. Biophys. 307, 1–7]. In this study we show that catalase or ADP are also able to potentiate this EGTA effect. When EGTA is added long after (12 min) the completion of swelling or elimination, no membrane resealing occurs unless the EGTA addition was preceded by the inclusion of DTT, ADP, or catalase soon after was collapsed. Total recovery by EGTA is obtained only in the presence of ADP. The sensitivity of the ADP effect to carboxyatractyloside strongly supports the involvement of the ADP/ATP carrier in this mechanism. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized membrane proteins shows that protein aggregation due to thiol cross-linkage formed during drop continues even after is already eliminated. Titration with 5,5-dithio-bis(2-nitrobenzoic acid) supports the data indicating that the formation of protein aggregates is paralleled by a decrease in the content of membrane protein thiols. Since the presence of ADP and EGTA prevents the progress of protein aggregation, we conclude that this process is responsible for both increased permeability to larger molecules and the irreversibility of drop. The protective effect of catalase suggests that the continuous production of protein thiol cross-linking is mediated by mitochondrial generated reactive oxygen species.     

The control by atmospheric factors and water stress of midday stomatal closure in Arbutus unedo growing in a natural macchia

Summary Midday closure of stomata of well-watered ( between-10 and-25 bar) or moderately stressed ( between-25 and-35 bar) Arbutus unedo plants occurs when midday leaf temperatures increase above 30°C and vapor pressure difference between leaf air spaces and the external air increases above approximately 30 mbar/bar. Moderate water stress decreases maximum conductance and may result in greater sensitivity to high leaf temperature and vapor pressure dificit, which results in earlier closure and later reopening of stomata. Severe water stress ( of-50 bar) changes the form of the daily pattern observed for leaf conductance. A single morning peak in conductance occurs followed by decrease in conductance over the remainder of the day. Morning fog in Portugal during the dry season may facilitate stomatal opening and may allow improvement of carbon balances of the leaves for short periods, but contributes little to improvement of plant water balances over the longer term.     

Relationships of leaf diffusion resistance of Populus clones to leaf water potential and environment

Summary Leaf diffusion resistance (r ₁) of the upper and lower leaf surfaces of several Populus clones was related to leaf water potential (₁), light intensity, vapor pressure deficit (VPD), and temperature by intrinsicallylinear, logarithmic multiple regression analyses. Regression equations accounted for up to 80% of variation in r ₁ data. Light intensity and VPD varied among clones in importance in influencing r ₁. Pronounced sensitivity of r ₁ of certain clones to VPD was related to drought resistance in their parentage. Increasing r ₁ was significantly positively correlated with ₁, in apparent contradiction to prevailing concepts of stomatal response to water status, and this relationship was probably attributable to effects of other environmental variables on ₁ and r ₁. Leaf resistance decreased after a storm characterized by winds in excess of 160 km·h^-1. Cuticular disruption and altered stomatal response may have been responsible for the storminduced r ₁ decrease.     

Effect of preganglionic sympathectomy on the cholinesterase activity in the superior cervical ganglia of rats and hamsters

Summary By employing biochemical assay and histochemical enzyme techniques the effect of preganglionic sympathectomy on the cholinesterase (ChE) activity in the superior cervical ganglia of rats and hamsters was investigated. Biochemical assays indicate that the ChE activity in the superior cervical ganglia of adult rats and hamsters is 57.19 and 28.63 respectively (expressed in u moles acetylcholine hydrolyzed per min per g of tissue); two weeks after preganglionic denervation, about 50% and 60% of ChE activity are lost respectively. Histochemical enzyme examination reveals that in the rat superior cervical ganglion, the majority of the neurons are adrenergic with weak to moderate acetylcholinesterase (AChE) reaction and the minority of the neurons are cholinergic with strong AChE activity, while only one type of adrenergic neurons exhibits a weak AChE activity in the hamster superior cervical ganglion. The AChE activity is localized in the perinuclear area, in the cisternae of the rough surfaced endoplasmic reticulum, in the Golgi complex and on the plasma membrane of the hamster's neurons; it is mainly localized in the cisternae of the rough surfaced endoplasmic reticulum of the rat's neurons. AChE reaction product is also detected on the axolemmal membranes of the preganglionic nerve fibers in the sympathetic ganglia of rats and hamsters.After preganglionic sympathectomy, the AChE activity in the adrenergic neurons and in the preganglionic unmyelinated nerve fibers is markedly reduced, whereas the cholinergic neurons and preganglionic myelinated nerve fibers remain unchanged. On the basis of these results two conclusions have been reached: (1) The fact that strong AChE activity localized in the cholinergic neurons and preganglionic myelinated fibers is not influenced by denervation, suggests that these structures are able to produce AChE. (2) The reduction of AChE activity in the rat and hamster superior cervical ganglia two weeks after preganglionic denervation, observed by histochemical examination, can be correlated with a concomitant measurable reduction determined by biochemical assays.Supported in part by a grant from the National Science Council, Republic of China. The author wishes to express his gratitude to the Department of Pharmacology, College of Medicine, National Taiwan University, for the use of its equipment for biochemical assays     

Effects of water deficit on N2(C2H2) fixation in cowpea and groundnut

The effect of water deficit on nodulation, N₂ fixation, photosynthesis, and total soluble sugars and leghemoglobin in nodules was investigated in cowpea and groundnut. Nitrogenase activity completely ceased in cowpea with a decrease in leaf water potential ( _leaf) from –0.4 MPa to –0.9 MPa, while in groundnut it continued down to –1.7 MPa. With increasing water stress, the acetylene reduction activity (ARA) declined very sharply in cowpea, but ARA gradually decreased in groundnut. Even with mild water stress ( _leaf of 0.2 MPa), nodule fresh weight declined 50% in cowpea partly due to a severe nodule shedding whereas nodule fresh weight declined in groundnut only when _leaf decreased by 1.0 MPa. No nodule shedding was noticed even at a higher stress level in groundnut. Photosynthesis and stomatal conductance were also more stable in groundnut than in cowpea under water stress. There was a sharp increase in total soluble sugars and leghemoglobin in the nodules of groundut with water stress, but no definite trend could be found in cowpea.     

Studies on chloride permeability of the skin ofLeptodactylus ocellatus: III. Na+ and Cl− effect on electrical phenomena

Summary During their flux through the skin of the frogLeptodactylus ocellatus, Na⁺ and Cl^– interact with each other. This interaction gives rise to electrical phenomena which are studied in the present paper. The skin is mounted in Na₂SO₄ Ringer's with 115 mM Na⁺ on the inside, and a variety of outer solutions,. The osmolarity of all solutions is kept constant at 237.8 mosmol by adding sucrose. When the main anion used on the outside is SO ₄ ⁼ the electrical potential difference () rises steadily with the concentration of sodium (Na⁺)_o up to 87 mV, which is reached at about 20mm. Thereafter remains constant. When the main anion is Cl^– it is observed that rises steadily with (NaCl)_o with a slope similar to the curve obtained with SO ₄ ⁼ (37 mV per decade), but with a lower intercept attributed to an inward Cl pumping which is characteristic of this frog species. At 2–9 mM (NaCl)_o a Cl-specific channel is activated. Further increases of (NaCl)_o produce a decrease of . The specificity of the activation of this site by monovalent cations and its use by monovalent anions is also studied.     

Apical sodium entry in split frog skin: Current-voltage relationship

Summary Apical Na⁺ entry into frog skin epithelium is widely presumed to be electrodiffusive in nature, as for other tight epithelia. However, in contrast to rabbit descending colon andNecturus urinary bladder, the constant field equation has been reported to fit the apical sodium current (N _Na)-membrane potential (^mc) relationship over only a narrow range of apical membrane potentials or to be inapplicable altogether. We have re-examined this issue by impaling split frog skins across the basolateral membrane and examining the current-voltage relationships at extremely early endpoints in time after initiating pulses of constant transepithelial voltage. In this study, the rapid transient responses in ^mc were completed within 0.5 to 3.5 msec. Using endpoints to 1 to 25 msec, the Goldman equation provided excellent fits of the data over large ranges in apical potential of 300 to 420 mV, from approximately –200 to about +145 mV (cell relative to mucosa). Split skins were also studied when superfused with high serosal K⁺ in order to determine whether theI _Na-^mc relationship could be generated purely by transepithelial measurements. Under these conditions, the basolateral membrane potential was found to be –10±3 mV (cell relative to serosa, mean±se), the basolateral fractional resistance was greater than zero, and the transepithelial current was markedly and reversibly reduced. For these reasons, use of high serosal K⁺ is considered inadvisable for determining theI _Na-^mc relationship, at least in those tissues (such as frog skin) where more direct measurements are technically feasible. Analysis of theI _Na-^mc relationships under baseline conditions provided estimates of intracellular Na⁺ concentration and of apical Na⁺ permeability of 9 to 14mm and of 3 × 10^–7 cm · sec^–1, respectively, in reasonable agreement with estimates obtained by different techniques.