Mechanism of inhibition of net ion transport across frog corneal epithelium by calcium channel antagonists

Summary In the isolated bullfrog cornea, three calcium channel antagonists had dose-dependent inhibitory effects on the Cl-originated short-circuit current (SCC). Their order of decreasing potency was bepridil, verapamil and diltiazem. One millimolar diltiazem inhibited the SCC by 98% and subsequent incubation with the calcium ionophore A23187 had no restorative effect. Increasing the bathing solution Ca concentration from 0.05 to 15mm, however, decreased diltiazem's inhibitory efficacy. This antagonist depolarized the intracellular potential differenceV _m from –54 to –18 mV (tear: reference) and the voltage divider ratioFR ₀ decreased from 0.58 to 0.30, suggesting an increase in basolateral membrane electrical resistance. Additional indication of a basolateral membrane effect by the drug was that preincubation with 10⁵ m amphotericin B in Cl-free Ringer's did not eliminate the inhibitory effect of the drug on the Na- and K-elicited SCC. In the absence of amphotericin B in Cl-free Ringer's (SCC=0), 1 ×10³ m diltiazem depolarized theV _m from –78 to –9 mV suggesting that the increase in basolateral membrane resistance was due to K channel blockade. Diltiazem (1×10³ m) significantly decreased cyclic AMP content; however, isoproterenol in the presence of the drug increased cyclic AMP fourfold without having any restorative effect on the inhibited SCC. Therefore, the inhibition of the Cl-originated SCC resulting from an increase in basolateral membrane K resistance is not caused by a decline in cyclic AMP content. In plasma membrane-enriched fractions prepared from broken cell preparations of bovine corneal epithelium, 1×10³ m diltiazem had no inhibitory effects on either Na,K-ATPase or Ca,Mg-ATPase activities. These latter effects further point to the selectivity of diltiazem as an inhibitor of K-channel activity, but do not preclude a Ca-channel blocker effect by the drug in the micromolar range.     

Effect of boundary layer conductance on the response of stomata to humidity

Abstract. Leaf conductance responses to leaf to air water vapour partial pressure difference (VPD) have been measured at air speeds of 0.5 and 3.0 ms⁻¹ in single attached leaves of three species in order to test the hypothesis that leaf conductance response to VPD is controlled by evaporation from the outer surface of the epidermis, rather than by evaporation through stomata. Total conductance decreased linearly with increassing VPD at both air speeds, but was decreased 1.6 3.0 times as much as by a given incrase in VPD at high than at low air speed. depending on species. In all species the relationship between leaf conductance and the gradient for evaporation from the epidermis was the same at both values of boundary layer conductance, supporting the hypothesis that direct epidermal evaporation controls stomatal guard cell behaviour in responses of stomata to VPD in these species.     

Oscillations in stomatal conductance of hybrid poplar leaves in the light and dark

Cycling of stomatal conductance in three hybrid poplar ( Populus sp.) cultivars was observed under a variety of conditions. Illumination of plants kept previously in the dark induced very large oscillations with a period of about 40 min and large oscillations with a shorter period (< 10 min) were superimposed on the longer cycles. During these oscillations, large changes in conductance could occur very rapidly (1.0 cm s⁻¹ in 3 min). Plants in constant light also displayed both long and short term cycles in conductance, but these were smaller in amplitude than those induced by sudden illumination. Stomatal oscillations were also observed in darkness and after darkening of previously illuminated plants. These oscillations had shorter (< 30 min) and less regular periods than those observed in the light. Such cycling in the dark is rare. Cycling of the two leaf surfaces was sometimes in synchrony in the light, and more so after a perturbation. Little synchrony between the two surfaces was observed in the dark. Stomatal movements of different leaves on a plant were usually relatively independent. Transient stomatal opening occurred following leaf excision in the light or dark, and often after sudden darkening of intact leaves. Also, stomata of intact leaves sometimes transiently closed following illumination.     

Regulation of the basolateral potassium conductance of theNecturus proximal tubule

Summary Two methods, the measurement of the response of the basolateral membrane potential (V _bl) of proximal tubule cells ofNecturus to step changes in basolateral K⁺ concentration, and cellular cable analysis, were used to assess the changes in basolateral potassium conductance (G _K) caused by a variety of maneuvers. The effects of some of these maneuvers on intracellular K⁺ activity (a _K ⁱ ) were also evaluated using double-barreled ion-selective electrodes. Perfusion with 0mm K⁺ basolateral solution for 15 min followed by 45 min of 1mm K⁺ solution resulted in a fall in basolateral potassium (apparent) transference number (t _K),V _bl anda _K ⁱ . Results of cable analysis showed that total basolateral resistance,R _b , rose. The electrophysiological effects of additional manipulations, known to inhibit net sodium reabsorption across the proximal tubular epithelium ofNecturus, were also investigated. Ouabain caused a fall int _K accompanied by large decreases ina _K ⁱ andV _bl. Lowering luminal sodium caused a fall int _K and a small reduction inV _bl. Selective reduction of peritubular sodium, a maneuver that has been shown to block sodium transport from lumen to peritubular fluid, also resulted in a significant decrease int _K. These results suggest thatG _K varies directly with rate of transport of the sodium pump, irrespective of the mechanism of change in pump turnover.Part of this material has been presented at the 10th International Conference on Biological Membranes (Cohen & Giebisch, 1984).     

Slow potential changes due to transport number effects in cells with unstirred membrane invaginations or dendrites

Summary Many neurones are extremely invaginated and possess branching processes, axons and dendrites. In general, they are surrounded by a restricted diffusion space. Many of these cells exhibit large, slow potential changes during the passage of current across their membranes. Whenever currents cross membranes separating aqueous solutions, differences in transport numbers of the major permeant ions give rise to local concentration changes of these ions adjacent to the membranes, which will result in various electrical and osmotic effects. These transport number effects are expected to be enhanced by the presence of membrane invaginations. Dendrites are equivalent to reversed invaginations and there should be significant changes in concentrations of permeant ions within them. In general, the effects of such changes on the electrical response of a cell will be greater when the concentration of a major permeant ion is low. The effects have been modelled in terms of two nondimensional parameters: the invagination transport number parameter and the relative area occupied by the invaginations A. If these two parameters are known, the magnitudes and time course of the slow potential changes can immediately be estimated and the time course converted to real time, if the length of the invaginations (l) and ionic diffusion coefficient (D) within them are also known. Both analytical and numerical solutions have been given and predictions compared. It is shown that in the case of large currents and potentials the analytical solution predictions will underestimate the magnitudes and rates of onset of the voltage responses. The relative magnitude of the transport number effect within the invaginations (or dendrites) and other transport number contributions to slow potential changes have also been assessed and order-of-magnitude values of these are estimated for some biological data.     

Direct evidence for Ca++-induced lateral phase separation in black membranes of lipid mixtures by the analysis of gramicidin A single-channels

Single-channel conductance fluctuations are analysed for gramicidin A incorporated into binary-mixed black lipid membranes of charged phosphatidic acid and neutral lecithin in different molar ratios. At very low Ca⁺⁺ concentrations in the electrolyte (i.e. in the presence of EDTA) homogeneous lipid mixtures are identified through their conductance and life time probability distributions for integral gramicidin pores. As for the pure lipid components, the conductance histograms each show a single maximum with regular width and for all channels a single mean lifetime is found.For Ca⁺⁺-levels (10^-6–10^-5 M) that are close to the critical demixing concentration (10^-4 M) unusually broad conductance distributions and reduced lifetimes are found provided the PC content, x, of the membrane is close to the critical mixture (x _crit0.5). We interpret this as a first example of the coupling of a membrane function (the transport of ions) to a lipid matrix with locally fluctuating composition close to a critical demixing point.For the conductance histogram of gramicidin A in an equimolar mixture of PA and PC shows two well-separated maxima. A correlation analysis between conductance and lifetime of the single pores shows that the two channel populations also differ significantly in their mean channel lifetime, ^*. This finding is interpreted as being direct evidence for Ca⁺⁺-induced lateral phase separation in black lipid membranes, as has been postulated recently.Abbreviations used HEPES N-2-hydroxyethyl-piperazine-N-2-ethane-sulfonic acid - EDTA ethylenediaminetetraacetic acid     

Rapid field determination of photosynthetic capacity of cut spruce twigs (Picea abies) at saturating ambient CO2

Summary Routine field determination of the parameters characterizing the activity of the photosynthetic apparatus is often difficult when attached branches of tall trees have to be used for gas exchange measurement. If severed twigs could be used, determining these parameters would be greatly facilitated. Because stomatal conductance changes when twigs or leaves are detached, CO₂ assimilation is usually altered. Thus, measurements made at ambient CO₂ concentration fail to accurately assess the activity of the photosynthetic apparatus because photosynthetic rates greatly depend on the supply of carbon dioxide. However, when photosynthetic carboxylation reactions are saturated by increased CO₂ partial pressure in the mesophyll, CO₂ assimilation rates no longer depend on instantaneous stomatal conductance, as shown by gas exchange measurements of spruce (Picea abies) twigs prior to and following detachment. Because net photosynthesis following detachment at saturating CO₂ remains constant for a minimum of 15 min, photosynthetic measurements of severed twigs may be reliable. This length of time is sufficient for detaching and recutting the twig, assembling a portable minicuvette system, re-establishing steady-state conditions with the gas analyser system, and reading the data over a reasonable period of time. The method described measures the maximal photosynthetic CO₂ assimilation of spruce needles of a single age-class from detached spruce twigs under the following conditions: saturating light, saturating external CO₂-partial pressure, standardized temperature and air humidity in the field. The method is applicable as a routine procedure to characterize the status of the photosynthetic apparatus of spruce trees that may be damaged in the process of forest decline.     

Effects of water stress on the chlorophyll content,nitrogen level and photosynthesis of leaves of two maize genotypes

The dynamics of leaf chlorophyll level, nitrogen content, photosynthesis and stomatal conductance were followed in detail in two cultivars of maize (Zea mays) during a short period of water stress, applied at tasseling, and during the subsequent recovery phase. Plants used in the experiment were grown in sand-nutrient solution culture under field weather conditions. Water stress reduced chlorophyll levels, stomatal conductance and photosynthesis, but the nitrogen content of the leaves was not affected. It is concluded that the stress-induced loss of chlorophyll is not mediated by a lack of nitrogen. Considerable differences were observed between genotypes in the rate of post-stress recovery of chlorophyll level. Recovery, upon rewatering, of stomatal conductance and photosynthesis preceded that of chlorophyll level. Losses of up to 40% of leaf chlorophyll content were insufficient to affect rates of photosynthesis measured at mid-day.     

Electrical tolerance (breakdown) of theChara corallina plasmalemma: II. Inductive property of membrane and effects of pH o and impermeable monovalent cations on breakdown phenomenon

Summary Changes in the chord conductanceG and the membrane electromotive forceE _m in the so-called breakdown region of large negative potential of theChara plasmalemma were analyzed in more detail. In addition to the increase inG, the voltage sensitivity of the change inG increased, which was the cause of marked inductive current in the breakdown region. The breakdown potential, defined as a critical potential at which both low and high slope conductances of theI–V _m relationship cross, almost coincided with the potential at which an inductive current began to appear. This breakdown potential level changed with pH _o in a range between 5 and 9. TheChara plasmalemma was electrically most tolerant around pH _o 7.In some cellsE _m shifted to a positive level as large as +50+70 mV during the breakdown phenomenon. Such a large positive shift ofE _m is caused mainly by the increase in conductance of Cl^– and partly Ca²⁺ and K⁺.