Water and carbon dioxide loss from the cockroach Periplaneta americana (L.) measured using radioactive isotopes.

Tritiated water (THO) loss from Periplaneta americana is used to measure cuticular permeability (Pd). In dry air, following various periods of predesiccation, the values of Pd lie around 0.5 x 10(-9) ms-1, with the lowest values below 0.3 x 10(-9) ms-1, close to the lowest permeabilities previously reported. There is no obvious relationship between permeability and initial mass of the insect. A significant lowering of Pd was found after 72 h of predesiccation. A marked sudden decrease in permeability occurred when the airstream flowing over an insect was changed from humid air to dry air. Labelling the bicarbonate pool using 14CO3(2-) enabled the output of CO2 to be estimated. An average output of about 390 microliters g-1 h-1 was found. Simultaneous measurement of THO and 14CO2 loss showed that tracheal water loss is only a small component of total water loss, with an average value of 3.8%. The total water loss can therefore be used to make close estimates of cuticular permeability in Periplaneta americana. 2min sampling periods clearly show the pulsatile nature of 14CO2 release but, even when the effect is most discernible, the periodic release of CO2 has only a small effect on water loss.

The measurement of exchangeable pools of zinc using the stable isotope 70Zn.

The present study was designed to assess the feasibility of using small doses of a stable isotope of Zn to follow plasma kinetics over a 10 d period and, hence, make deductions about Zn turnover and body pool sizes. At the beginning of the 10 d metabolic balance, two adults, consuming their habitual diet, were given an intravenous injection of 70Zn. There was a fourfold difference in the administered dose between the two subjects (0.445 and 2.078 mg). Blood samples were taken at regular intervals and plasma enrichment with 70Zn measured by thermal ionization mass spectrometry. Urine and faeces were collected and analysed for Zn and 70Zn. Kinetic analysis of the plasma 70Zn decay by several different methods was undertaken. It was apparent from both deconvolution analysis of the short-term (0-90 min) decay data and four-compartment modelling of the longer-term (0-24 h) data that isotopic Zn very rapidly equilibrates with the plasma Zn and with a rapidly exchanging non-plasma pool, probably located within the liver. This latter pool appears to contain less than 10 mg Zn and the peak of isotope enrichment occurs at about 20 min post injection. The later decay of plasma Zn enrichment appears to be dictated by exchange with a much larger pool of approximate size 350 mg.

pH regulation in tissue-cultured bovine lens epithelial cells.

The intracellular pH (pHi) of tissue-cultured bovine lens epithelial cells was measured in small groups of 6 to 10 cells using the trapped fluorescent dye 2',7'-bis-(2-,carboxyethyl)-5 (and 6)carboxyfluorescein (BCECF). When perifused at 35 degrees C with artificial aqueous humour solution (AAH) containing 16 mM HCO3- and 5% CO2, pH 7.25, pH(i) was 7.19 +/- 0.02 (SEM, n = 95). On removing HCO3- and CO2 there was an initial transient alkalinization followed by a fall in pH to a steady value of 6.97 +/- 0.03 (SEM, n = 54). Addition of 0.25 mM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) to AAH containing HCO3- and CO2 led to a rapid and pronounced fall in pH. Exposure to Na(+)-free AAH again led to a marked fall in pH(i), but in this case the addition of DIDS did not produce a further fall. Substitution of the impermeant anion gluconate for Cl- in the presence of HCO3- led to a rise in pHi, while substitution in the absence of HCO3- led to a fall in pHi. The above data indicate a significant role for a sodium-dependent Cl(-)-HCO3- exchange mechanism in the regulation of pHi. Addition of 1 mM amiloride to control AAH in both the presence and absence of HCO3- led to a marked fall in pH(i), indicating that a Na+/H+ exchange mechanism also has a significant role in the regulation of pHi.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for a Na(+)-Cl(-)-H(+)-HCO3- exchange system in the mammalian lens.

36Cl- efflux was studied in the isolated rat lens under two conditions that are known to decrease internal pH. The first follows exposure to a pulse of ammonium chloride (50 mM) and the second accompanies exposure to an acidified propionate (20 mM) solution. Under acidifying conditions, a stimulation in 36Cl- efflux was observed, that was abolished on removing external Na+ and also on removing external Cl- and HCO3-. In the absence of external Cl-, the presence of HCO3- (16 mM) resulted in an increase in 36Cl- efflux during internal acidification. In the absence of internal acidification, the addition of 0.1 mM dibutyrylcAMP or 0.5 mM IBMX to the external medium produced a rapid increase in 36Cl- efflux. This stimulation was reduced by 0.2 mM SITS. Neither cAMP or IBMX had any significant effect on the electrical resistance of the lens membranes. It is suggested that a coupled SITS-sensitive, Na(+)-Cl(-)-H(+)-HCO3- exchange mechanism is activated when the lens internal pH falls and further that cAMP may play a role in regulating this mechanism.

Internal acidification modulates membrane and junctional resistance in the isolated lens of the frog Rana pipiens.

The normal internal pH (pHi) of the amphibian lens, measured using ion-sensitive microelectrodes, is 7.1 (pHo = 7.4) and the membranes appear to be relatively impermeable to hydrogen ions. Perifusing the lens with 100% CO2 appeared to be the most efficient way of decreasing pHi, which fell to 6.3 after an exposure lasting 30 min. Accompanying this acidification, there was a rapid depolarization of membrane potential (Em), a decrease in membrane resistance (Rm) and increase in internal or bulk resistance (Ri). These changes did not occur if the external pH alone was decreased. All changes were reversible, although the time course of Ri recovery was faster than the others. The decrease in membrane resistance could be prevented if the chloride concentration in the external solution was reduced, suggesting that internal acidification opens chloride channels in the amphibian lens. Since chloride ions are normally close to equilibrium across amphibian lens membranes, it is suggested that the pH-induced depolarization is due to a decrease in potassium conductance. The increase in internal resistance on perifusing with CO2 is most likely due to a closing of gap junctions between the fibre cells. The relationship between internal conductance and pHi was very similar to that obtained in other tissues and could be fitted by the Hill equation with n = 6 and pK = 6.9. Fibre junctional conductance seems sensitive to small changes in hydrogen ion concentration around the resting pH. Two agents, aspirin and cyanate, that are believed to influence cataract development, slowed the recovery of Em, Rm and Ri during recovery from an acid load.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of rat lens 45Ca2+ fluxes: evidence for Na(+)-Ca2+ exchange.

The influx and efflux kinetics of 45Ca2+ were studied in the rat lens in vitro. Both data sets could be fitted by a multi-compartment mathematical model and were interpreted in terms of extracellular, cytosolic and slowly-exchanging (bound) components. At the end of a 16-hr influx period, when uptake into the extracellular and cytosolic compartments is complete, the 45Ca2+ exchanged fraction is less than 20% of the total calcium determined by atomic absorption. The bound compartment is therefore by far the largest in the lens. The efflux rate constant determined from the model for the cytosolic compartment was approximately 8 x 10(-3) min-1 and its origin was confirmed by its sensitivity to temperature, absence of external sodium and presence of the amiloride-analogue, dichlorobenzamil. A 55% reduction in efflux was obtained in sodium-free solution, indicating that Na(+)-Ca2+ exchange is responsible for a large proportion of calcium movement from the lens against its electrochemical gradient. This was confirmed in influx studies where, reduction of the lens sodium gradient by either exposure to sodium-free medium or 0.1 mM ouabain significantly elevated the 45Ca2+ content of the lens relative to the control level. Exposure to sodium-free conditions also rendered the lens opaque, which did not occur in the absence of external calcium. These experiments suggest a critical role for Na(+)-Ca2+ exchange in maintaining a low internal Ca2+ and hence transparency.

Efflux of chloride from the rat lens: influence of membrane potential and intracellular acidification.

The efflux of 36C1 from perifused rat lenses consisted of two components: a fast (extracellular) component and a slow (cellular) component. The 36C1 efflux rate constant of the cellular component was 5.7 X 10(-3) min-1. The 36C1- efflux was sensitive to changes in lens potential induced by treatment with high-K+ solutions. The decrease in the 36C1- efflux rate constant caused by high-K+ solutions was consistent with the Goldman model, indicating that, under normal conditions, the majority of the 36C1 efflux is by diffusion. The 36C1- efflux rate constant corresponds to a C1 permeability of 1.3 X 10(8) ms 1. The C1- channel inhibitor anthracene-9-carboxylate (A-9-C), however, caused a relatively small reduction in the efflux rate constant. The anion-exchange inhibitor 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonate (SITS) has little effect on the 36C1 efflux under control conditions. Intracellular acidification, induced by pre-treatment with NH1+, leads to a rapid stimulation of 36C1- efflux. This increased 36C1- efflux is blocked by SITS. Thus, it appears that at low intracellular pH (pH), a normally quiescent, SITS-sensitive, anion-exchange mechanism is activated. The possible role of this exchange mechanism in regulating pH, is discussed.

Membrane conductance and potassium permeability of the rat lens.

The membrane potential, electrical impedance and 86Rb+ efflux rate constants were measured in the rat lens perifused at 35 degrees C. The membrane conductance was obtained from the difference between the magnitude of the impedance at low (less than 0.1 Hz) and high (greater than 100 Hz) frequencies. Values of the rubidium permeability coefficients (PRb) were obtained from the rate constant and potential data. The values for the membrane potential and conductance in control solution (5 mM potassium) were -69.6 mV and 5.5 X 10(-4) S respectively, while the computed permeability was 2.9 X 10(-8) m.s-1. On perifusing with 35 mM potassium, the membrane depolarized by 25 mV and the conductance and rubidium permeability increased considerably. These increases could be blocked by quinine (0.3 mM), tetraethylammonium (30 mM) and 4-aminopyridine (10 mM). The latter agent was more effective at alkaline pH (8.3). It is suggested that there are voltage-gated potassium channels that are inhibited by these three agents. After the initial depolarization in high potassium, there was little further change in membrane potential with any of the inhibitors. All three agents, however, produced a marked depolarization when applied in control solution. This was accompanied by a decrease in conductance and rubidium permeability, suggesting that, in the rat lens, some voltage-gated potassium channels are activated at the resting potential.

Potassium and rubidium permeability and potassium conductance of the beta-cell membrane in mouse islets of Langerhans.

The efflux of 42K+ and 86Rb+ has been studied in collagenase-isolated normal mouse islets of Langerhans. In double-labelled experiments, the ratio of efflux rate constants of Rb+ and K+ (kRb/kK) was 0.80 in 5 mM-K+ and 0 glucose. The ratio was unaffected by glucose concentrations up to 22.2 mM. In the presence of 50 mM-K+, 0 glucose, the ratio increased to 0.91 and in 50 mM-Rb+, 0 glucose and 0 K+, the ratio was 1.06. With these limitations, the results indicate that 86Rb+ is an acceptable isotope for K+. Using the Goldman model, K+ and Rb+ permeability coefficients and K+ slope and chord conductances were computed. The permeabilities decreased in glucose and in high K+ or high Rb+. In the case of high external K+, the K+ conductances increased. Also there may be more than one type of K+ channel with differing selectivities to K+ and Rb+. The addition of glucose in the presence of 50 mM-K+ had no further effect on Rb+ permeability. It is suggested that there are about ten small K+ channels open in the resting beta-cell and that progressive closure of these channels is involved in the depolarization of the cell membrane that initiates spike activity.

Electrophysiological evidence for the inhibition of potassium permeability in pancreatic beta-cells by glibenclamide.

The effects of glibenclamide on the electrical activity of the beta-cells of the islets of Langerhans of normal mice have been investigated in the absence and presence of glucose (11.1 mM). Glibenclamide depolarized the cell membrane and this has been interpreted in terms of an increase in the ratio of the Na+ and K+ permeabilities, PNa/PK. This ratio increased from 0.05 to 0.24 in the presence of 4 microM glibenclamide and zero glucose. The input resistance of the beta-cells also increased. These observations indicate a decrease in K+ permeability. The effect is only slowly reversed after removal of glibenclamide. Uncouplers of oxidative phosphorylation do not reverse the depolarization induced by glibenclamide. It is suggested that glibenclamide is acting directly to inhibit the [Ca2+]i-gated K+ permeability in the beta-cell membrane.

Ultrastructure of the branchial epithelium of an amphibious brackish-water crab.

The ultrastructure of the branchial epithelium of the amphibious brackish-water crab Uca mordax (Smith) was investigated in relation to adaptation to the salinity of the medium. No distinct differences were observed in the epithelial structure of animals adapted to either 100% sea water or to 1% sea water. Thus any interpretation of the significance of particular structures in relation to specific transport processes should be regarded with caution. Apart from strict epithelial cells, pillar cells and glycogen (presumed) storage cells were found. The epithelial cells showed very well-developed apical microvilli or lamellae and basal interdigitations with adjacent cells. Well-developed junctional complexes were seen (band desmosomes, septate desmosomes, gap junctions). The cells are extremely rich in mitochondria. Microtubules, peroxisome-like bodies, multivesicular bodies and near-nuclear Golgi complexes were present.