cAMP-Dependent protein kinase inhibits the chloride conductance in apical membrane vesicles of hunman placenta

Summary The role of adenosine 3,5-monophosphate (cAMP) dependent protein kinase (PK-A) on the Cl^– conductance has been studied in the apical membrane vesicles purified from the chorionic villi of human placenta. In order to phosphorylate the cytosolic side of the membranes, vesicles have been hypotonically lysed, loaded with 100nm catalytic subunit of PK-A purified from human placenta and 1mm of the phosphatase resistant adenosine 5-thiotriphosphate (ATP-gamma-S) and resealed. Cl^– conductance has been measured by the quenching of the fluorescent probe 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ) at 23°C with membrane potential clamped at 0 mV. The actual volume of the resealed vesicles was measured in each experiment by trapping an impermeable radioactive molecule ([¹⁴C]-sucrose) and included in each Cl^– flux calculation. In 19 independent experiments, the mean Cl^– conductance in placental membranes in the absence of phosphorylation was 3.67±3.18 whereas with the addition of PK-A and ATP-gamma-S it was 1.97±1.75 nmol·sec^–1·(mg protein)^–1 (mean±sd). PK-A dependent phosphorylation reduced the Cl^– conductance in 14/19 experiments. The same protocol applied to the apical membranes of bovine trachea, where PK-A is known to activate the Cl^– channels, confirmed that the PK-A dependent phosphorylation increased the Cl^– conductance in 11/13 experiments, from 1.01±0.61 to 1.85±0.99 nmol·sec^–1·(mg protein)^–1(mean±sd). These studies indicate that the PK-A dependent phosphorylation inhibits one or more Cl^– channel(s) of the apical membranes of human placenta.     

Ion flow through membranes and the resting potential of cells

Summary A knowledge of the relationship between ion flow, both passive and active, ionic concentration, and membrane potential is essential to the understanding of cellular function. The problem has been analyzed on the basis of elementary physical and biophysical principles, providing a theoretical model of current flow and resting potential of cells, including those in epithelia. The model assumes that the permeability of the ion channets is not voltage dependent, but applies to gated channels when the gates are open. Two sources of nonlinearity of the current-voltage relationship are included in the analysis: ionic depletion and accumulation at the channels' mouths, and channel saturation at higher concentrations. The predictions of the model have been quantitative, validated by comparison with experiment, which has been limited to the only two cases in which adequate data was found. Application of the theory to the scala media of the mammalian cochlea has explained the source of its high positive potential and provided estimates of the Na⁺ and K⁺ permeabilities of the membranes of its marginel cess. This analysis provides a theoretically sound alternative to the widely used Goldman equation, the limited validity of which was emphasized by Goldman (D.E. Goldman, 1943,J. Gen. Physiol.27:37–60), as well as its derivatives, including the Goldman-Hodgkin-Katz equation for resting potentials.     

Electrogenic bicarbonate secretion in the turtle bladder: Apical membrane conductance characteristics

Summary We have recently shown that stimulation of electrogenic HCO ₃ ^– secretion is accompanied by a simultaneous increase in short-circuit current (I _sc, equivalent to HCO ₃ ^– secretion rate under these conditions), apical membrane capacitance (C _a , proportional to membrane area), and apical membrane conductance (G _a , proportional to membrane ionic permeability). The current experiments were undertaken to explore the ionic basis for the increase inG _a and the possibility that the rate of electrogenic HCO ₃ ^– secretion is regulated by changes inG _a . Membrane electrical parameters were measured using impedance-analysis techniques before and after stimulation of electrogenic HCO ₃ ^– secretion with cAMP in three solutions which contained different chloride concentrations. In another series of experiments, the effects of an anion channel blocker, anthracene-9-carboxylic acid (9-AA), were measured after stimulation of electrogenic HCO ₃ ^– secretion with cAMP. The major conclusions are: (i) a measurable apical Cl^– conductance exists in control hemibladders; (ii) the transport-associated increase inG _a includes a Cl^–-conductive component; (iii)G _a also appears to reflect a HCO ₃ ^– conductance; (iv) the relative magnitudes of the apical membrane conductances to Cl^– and HCO ₃ ^– are similar; (v) 9-AA reducesG _a andI _sc appear cAMP-stimulated hemibladders; and (vi) alterations inI _sc appear to be mediated by changes inG _a .     

Regulation of basolateral membrane potential after stimulation of Na+ transport in proximal tubules

Summary We have previously shown that stimulation of apical Na-coupled glucose and alanine transport produces a transient depolarization of basolateral membrane potential (V _bl) in rabbit proximal convoluted tubule (PCT. Sl segment). The present study is aimed at understanding the origin of the membrane repolarization following the intial effect of addition of luminal cotransported solutes. Luminal addition of 10–15mMl-alanine produced a rapid and highly significant depolarization ofV _bl (20.3±1.1 mV,n=15) which was transient and associated with an increase in the fractional K⁺ conductance of the basolateral membrane (t _K) from 8 to 29% (P<0.01,n=6). Despite the significant increase int _K, the repolarization was only slightly reduced by the presence of basolateral Ba²⁺ (2mM,n=6) or quinine (0.5 mM,n=5). The repolarization was greatly reduced in the presence of 0.1 mM 4-acetamino-4isothiocyamostilbene-2,2-disulfonic acid (SITS) and blunted by bicarbonate-free solutions. Intracellular pH (pH _i ) determined with the fluorescent dye 2, 7-bis-2-carboxyethyl-5(and-6)-carboxyfluorescein (BCECF), averaged 7.39±0.02 in control solution (n=9) and increased to 7.50±0.03 in the first 15 sec after the luminal application of alanine. This was followed by a significant acidification averaging 0.16±0.01 pH unit in the next 3 min. In conclusion, we believe that, contrary to other leaky epithelia, rabbit PCT can regulate its basolateral membrane potential not only through an increase in K⁺ conductance but also through a cellular acidification reducing the basolateral HCO ₃ ^– exit through the electrogenic Na-3(HCO₃) cotransport mechanism.     

松茶间作茶树叶片的解剖构造和气孔活动

本文利用光镜技术和MK-3型自动气孔计对松茶间作和单作茶园茶树叶片的解剖构造和气孔传导力进行了比较研究。研究表明,间作茶园茶树叶片的上表皮、栅拦组织和全叶均比单作茶树薄,分别为单作茶树的82.7％,78.2％和67.2％,叶质柔嫩。叶片气孔传导力比单作茶园低。嫩叶传导力>老叶;1芽5叶新梢按叶序3叶>2、4叶>1、5叶;按树冠垂直分布,冠上叶(0—5cm)>冠中叶(10—15cm)>冠下叶(30cm左右)。说明气孔传导力不仅受生态条件影响,与自身的叶龄、叶位等生理机能也有密切关系。     

Effects of electroconductive heat treatment and electrical pretreatment on thermal death kinetics of selected microorganisms

Suspensions of yeast cell (zygo Saccharomyces bailii) in a phosphate buffer solution were subjected to conventional (hot water) and ohmic (electric current) heating under identical temperature histories. Experiments were also conducted with cells of Escherichia coli to compare the lethal effect of combination of sublethal electrical preteatment and conventional heating with conventional heating. The kinetic parameters (D,Z,K and E(a)) were determined for both organisms during different treatments. There was no significant difference in the death rate of yeast cells during conventional and ohmic heating at the voltage range used in this study. Results of electrical pretreatment and conventional heating on E. coli indicated differences under certain conditions when compared with pure conventional heating. Thus it is concluded that microbial death during ohmic heating was due primarily to thermal effects with no significant effect of electric current per se. Sublethal electrical pretreatment appears to offer potential for increased bacterial inactivation in certain cases.     

Amiloride sensitivity of proton-conductive pathways in gastric and intestinal apical membrane vesicles

Summary Passive proton permeability of gastrointestinal apical membrane vesicles was determined. The nature of the pathways for proton permeation was investigated using amiloride. The rate of proton permeation (k _H ⁺ was determined by addition of vesicles (pH _i = 6.5) to a pH 8.0 solution containing acridine orange. The rate of recovery of acridine orange fluorescence after quenching by the acidic vesicles ranged from 4 × 10^–3 (gastric parietal cell stimulation-associated vesicles; SAV) and 5 × 10^–3 (duodenal brush-border membrane vesicles; dBBMV) to 11 × 10+^–3 sec^–1 (ileal BBMV; iBBMV). Amiloride, 0.03 and 0.1 mm, significantly reduced the rate of proton permeation in dBBMV and iBBMV, but not gastric SAV. The decreases in k _H ⁺ were proportionately greater in iBBMV as compared with dBBMV. The presence of Na⁺/H⁺ exchange was demonstrated in both dBBMV and iBBMV by proton-driven (pH _i < pH _o ) ²²Na⁺ uptake. Evidence was also sought for the conductive nature of pathways for proton permeation. Intravesicular acidification, again determined by quenching of acridine orange fluorescence, was observed during imposition of K⁺-diffusion potential ([K⁺] _i [K⁺ _o ). In dBBMV and iBBMV, intravesicular acidification was enhanced in the presence of the K⁺-ionophore valinomycin, indicating that the native K⁺ permeability is rate limiting. In the presence of valinomycin, the K⁺-diffusion potential drove BBMV intravesicular acidification to levels close to the electrochemical potential. In gastric SAV, acidification was not limited by the K⁺ permeability. Valinomycin was without effect, but the K⁺/H⁺ ionophore nigericin enhanced acidification in gastric SAV, illustrating the low proton permeability of these membranes. Amiloride, 0.03–1 mm, resulted in concentration-dependent reductions of K⁺-diffusion potential-driven acidification in dBBMV and iBBMV but not in gastric SAV. These data demonstrate that proton permeation in the three membrane types is rheogenic. The sensitivity of the proton-conductive pathways in intestinal BBMV to high concentrations of amiloride correlated with the presence of the Na⁺/H⁺ antiport and indicates that this transmembrane protein may represent a pathway for proton permeation.We thank Ruth Briggs for assistance with the Na/H exchange experiments. This work was supported by a grant from the Medical Research Council (G8418056CA).     

Carbon: terrestrial C4 plants

The carbon isotope composition of terrestrial C₄ plants depends on the primary carboxylation of phosphoenolpyruvate (PEP) and on the diffusion of CO₂ to the carboxylation sites, but is also influenced by the final carboxylation of ribulose-1,5-bisphosphate (RuBP). Several models have been used for reproducing this complex situation. In the present review, a particular model is applied as a means to interpret the effects of environmental and genetically determined factors on carbon isotope discrimination during C₄ photosynthesis. As a new feature, the model considers four types of limitation of the overall CO₂ assimilation rate. Both carboxylation reactions are assumed to be limited by either maximum enzyme activity or maximum substrate regeneration rate. The model is applied to experimental data on the effects of CO₂, irradiance and water stress on short-term discrimination by leaves of several C₄ species measured simultaneously with CO₂ gas exchange characteristics. In particular, different patterns of the influence of low irradiances on carbon isotope discrimination are interpreted as due to variations in that irradiance at which a transition from limitation by PEP regeneration rate and RuBP carboxylase activity to limitation by the regeneration rates of both substrates occurs. After discussing literature data on the effects of environmental conditions on carbon isotope discrimination by C₄ plants seasonal and developmental changes in carbon isotope composition, studies on the systematic and geographic distribution of C₄ plants, evolutionary and genetical aspects, and some ecological implications are reviewed.     

The thermal and energetic significance of clustering in the speckled mousebird,Colius striatus

Summary The effect of clustering behaviour on metabolism, body temperature, thermal conductance and evaporative water loss was investigated in speckled mousebirds at temperatures between 5 and 36°C. Within the thermal neutral zone (approximately 30–35 °C) basal metabolic rate of clusters of two birds (32.5 J·g^-1·h^-1) and four birds (28.5 J·g^-1·h^-1) was significantly lower by about 11% and 22%, respectively, than that of individuals (36.4 J·g^-1·h^-1). Similarly, below the lower critical temperature, the metabolism of clusters of two and four birds was about 14% and 31% lower, respectively, than for individual birds as a result of significantly lower total thermal conductance in clustered birds. Body temperature ranged from about 36 to 41°C and was positively correlated with ambient temperature in both individuals and clusters, but was less variable in clusters. Total evaporative water loss was similar in individuals and clusters and averaged 5–6% of body weight per day below 30°C in individuals and below 25°C in clusters. Above these temperatures total evaporative water loss increased and mousebirds could dissipate between 80 and 90% of their metabolic heat production at ambient temperatures between 36 and 39°C. Mousebirds not only clustered to sleep between sunset and sunrise but were also observed to cluster during the day, even at high ambient temperature. Whereas clustering at night and during cold, wet weather serves a thermoregulatory function, in that it allows the brrds to maintain body temperature at a reduced metabolic cost, clustering during the day is probably related to maintenance of social bonds within the flock.Abbreviations BMR basal metabolic rate - bw body weight - C _totab total thermal conductance - EWI evaporative water loss - M metabolism - RH relative humidity - T _a ambient temperature - T _b body temperature - T _ch chamber temperature - T _cl cluster temperature - TEWL total evaporative water loss - LCT lower critical temperature - TNZ thermal neutral zone