ALBERT

Notes: Summary The intestinal secretagogues ricinoleate and deoxycholate have been tested for a capacity to form complexes with Ca2+ ions and to affect the passive equilibration of Ca2+ ions across the jejunal brush border membrane. Both of these agents formed butanol-soluble Ca2+ complexes in a model phase distribution system. They also promote the passive uptake and efflux of Ca2+ across brush border vesicles in a concentrationdependent manner. The levels of ricinoleate and deoxycholate that increase the rate of transvesicular Ca2+ movement are in the 100 to 300 μm range. Concentrations as high as 1.0mm had no significant detergent effects in vesicles as measured by release of entrapped sorbitol. The kinetics of Ca2+ uptake and efflux are similar in brush border vesicles treated with A23187, ricinoleate, or deoxycholate. The influx rates observed in this study were high enough to cause the collapse of a Ca2+ gradient, which had been generated by Ca-Mg ATPase enzyme activity in the brush border membrane. Ricinoleate did not affect Ca-Mg ATPase activity at concentrations used in this study, but deoxycholate was inhibitory, indicating two potential modes for elevation of intracellular Ca2+ content by deoxycholate. When compared with the effects of the Ca2+ ionophore, A23187, it appears that both ricinoleate and deoxycholate could have significant intestinal secretory activity due to this Ca2+ ionophore property. It is also noteworthy that, at least in this model system, potential secretory effects are expressed at concentrations significantly below levels that have been associated with detergent effects or altered epithelial morphology.

Notes: Summary Using patch-clamp techniques, we have studied Ca2+-activated K+ channels in the basolateral membrane of freshly isolated epithelial cells from rabbit distal colon. Epithelial cell clusters were obtained from distal colon by gentle mechanical disruption of isolated crypts. Gigaohm seals were obtained on the basolateral surface of the cell clusters. At the resting potential (approximately −45 mV), with NaCl Ringer's bathing the cell, the predominant channels had a conductance of 131±25 pS. Channel activity depended on voltage as depolarization of the membrane increased the open probability. In excised inside-out patches, channels were found to be selective for K+ over Na+. Channel activity correlated directly with bath Ca2+ concentration in the excised patches. Channel currents were blocked by 5mm TEA+ and 1mm Ba2+. In cell-attached patches, after addition of the Ca2+ ionophore A23187, which increases intracellular Ca2+, open probability was markedly increased. Channel activity was also regulated by cAMP as addition of 1mm dibutyryl-cAMP in the bath solution in cell-attached patches increased channel open probability over 20-fold. Channels that had been activated by cAMP were further activated by Ca2+. We conclude that the basolateral membrane of epithelial cells from descending colon contains a class of potassium channels, which are regulated by intracellular Ca2+ and cAMP.

Notes: Summary The properties of Ca2+-activated K+ channels in the apical membrane of theNecturus choroid plexus were studied using single-channel recording techniques in the cell-attached and excised-patch configurations. Channels with large unitary conductances clustered around 150 and 220 pS were most commonly observed. These channels exhibited a high selectivity for K+ over Na+ and K+ over Cs+. They were blocked by high cytoplasmic Na+ concentrations (110mm). Channel activity increased with depolarizing membrane potentials, and with increasing cytoplasmic Ca2+ concentrations. Increasing Ca2+ from 5 to 500nm, increased open probability by an order of magnitude, without changing single-channel conductance. Open probability increased up to 10-fold with a 20-mV depolarization when Ca2+ was 500nm. Lowering intracellular pH one unit, decreased open probability by more than two orders of magnitude, but pH did not affect single-channel conductance. Cytoplasmic Ba2+ reduced both channel-open probability and conductance. The sites for the action of Ba2+ are located at a distance more than halfway through the applied electric field from the inside of the membrane. Values of 0.013 and 117mm were calculated as the apparent Ba2+ dissociation constants (K d (0 mV) for the effects on probability and conductance, respectively. TEA+ (tetraethylammonium) reduced single-channel current. Applied to the cytoplasmic side, it acted on a site 20% of the distance through the membrane, with aK d (0 mV)=5.6mm. A second site, with a higher affinity,K d (0 mV)=0.23mm, may account for the near total block of chanel conductance by 2mm TEA+ applied to the outside of the membrane. It is concluded that the channels inNecturus choroid plexus exhibit many of the properties of “maxi” Ca2+-activated K+ channels found in other tissues.

Notes: Summary The tight-seal whole-cell recording method has been used to studyNecturus choroid plexus epithelium. A cell potential of −59±2 mV and a whole cell resistance of 56±6 MΩ were measured using this technique. Application of depolarizing step potentials activated voltage-dependent outward currents that developed with time. For example, when the cell was bathed in 110mm NaCl Ringer solution and the interior of the cell contained a solution of 110mm KCl and 5nm Ca2+, stepping the membrane potential from a holding value of −50 to −10 mV evoked outward currents which, after a delay of greater than 50 msec, increased to a steady state in 500 msec. The voltage dependence of the delayed currents suggests that they may be currents through Ca2+-activated K_ channels. Based on the voltage dependence of the activation of Ca2+-activated K+ channels, we have devised a general method to isolate the delayed currents. The delayed currents were highly selective for K+ as their reversal potential at different K+ concentration gradients followed the Nernst potential for K+. These currents were reduced by the addition of TEA+ to the bath solution and were eliminated when Cs+ or Na+ replaced intracellular K+. Increasing the membrane potential to more positive values decreased both the delay and the half-times (t 1/2) to the steady value. Increasing the pipette Ca2+ also decreased the delay and decreasedt 1/2. For instance, when pipette Ca2+ was increased from 5 to 500nm, the delay andt 1/2 decreased from values greater than 50 and 150 msec to values less than 10 and 50 msec. We conclude that the delayed currents are K+ currents through Ca2+-activated K+ channels. At the resting membrane potential of −60 mV, Ca2+-activated K+ channels contribute between 13 to 25% of the total conductance of the cell. The contribution of these channels to cell conductance nearly doubles with membrane depolarization of 20–30 mV. Such depolarizations have been observed when cerebrospinal fluid (CSF) secretion is stimulated by cAMP and with intracellular Ca2+. Thus the Ca2+-activated K+ channels may play a specific role in maintaining intracellular K+ concentrations during CSF secretion.

Notes: Summary The calcium sensitivity of exocytosis from electroper-meabilized chromaffin cells is increased by activators of protein kinase C, such as TPA and certain phorbol esters, diacylglycerols, and mezerein. A range of putative inhibitors of protein kinase C block both the phorbol ester-sensitive component of secretion and also the underlying insensitive component. These inhibitors are also shown to inhibit medulla protein kinase C activity in vitro. The extent of secretion is reduced when electropermeabilized cells are exposed to Ca2+ levels much in excess of 50 μm. The onset of inhibition is faster than the relatively slow rate of Ca-dependent exocytosis and is insensitive to inhibitors of proteolysis. Adrenal medulla protein kinase C activity is also irreversibly inhibited by high Ca2+ concentrations. Both the secretory response and the protein kinase C activity in vitro have similar nucleotide and cation specificities. Although these data do not definitely establish an involvement of protein kinase C in exocytosis, none argue against it.

Notes: Summary The physiological relevance of an apparent ionophore activity of cholera toxin towards Ca2+ has been examined in several different systems designed to measure affinity, specificity, rates of ion transfer, and effects on intracellular ion concentrations. Half-maximal transfer rates across porcine jejunal brush-border vesicles were obtained at a concentration of 0.20 μM Ca2+. When examined in the presence of competing ions the transfer process was blocked by very low concentrations of La3+ or Cd2+. Sr2+, Ba2+ and Mg2+ were relatively inefficient competitors for Ca2+ transport mediated by cholera toxin. The relative affinities observed would be compatible with a selectivity for Ca2+ transfer at physiological ion concentrations, as well as an inhibition of this ionophore activity by recognized antagonists of cholera toxin such as lanthanum ions. Entry rates of Ca2+ into brush-border vesicles exposed to cholera toxin were large enough to accelerate the collapse of a Ca2+ gradient generated by endogenous Ca, Mg-ATPase activity. The treatment of isolated jejunal enterocytes with cholera toxin caused a significant elevation in cytosolic Ca2+ concentrations as measured by Quin-2 fluorescence. This effect was specifically prevented by prior exposure of the cholera toxin to excess ganglioside GM1. We conclude that cholera toxin has many of the properties required for promoting transmembranes Ca2+ movement in membrane vesicles and appears to be an effective Ca2+ ionophore in isolated mammalian cells.

Notes: Abstract The possible contribution of mitochondrial Ca2+ accumulation and release to contractile phenomena has been investigated. Two intracellular fractions of Ca2+ sequestration can be identified in cardiac myocytes, one ascribed to mitochondria. Two modes of Ca2+ transport exist within the mitochondrial fraction, one dependent upon mitochondrial respiration and the other upon extramitochondrial [Na+]. Experiments with trabeculae show that under appropriate conditions, the rate of relaxation and the amount of tension developed is dependent on these two modes of Ca2+ transport. A model is presented quantifying the contribution of the mitochondria to relaxation.

Notes: Summary Calcium-dependent exocytosis in ‘leaky’ bovine adrenal medullary cells has a requirement for Mg-ATP. One possibility is that exocytosis depends in some way on the operation of the ATP-dependent proton pump that serves to maintain the core of the secretory vesicles both acid and at a positive potential with respect to the cytosol. This possibility has been tested in ‘leaky’ cells by monitoring exocytosis under conditions where the secretory vesicle pH and potential gradients are measuredin situ. The results show rather clearly that exocytosis can persist, with unchanged Ca-activation kinetics, in the virtual absence both of a difference in pH between the cytosol and secretory vesicle core and also of a difference in potential across the vesicle membrane. The results do not, however, exclude a small modulating effect of vesicle pH or potential on exocytosis and shed no light on whether or not the plasma membrane potential, which is maintained close to zero in these experiments, influences exocytosis.

Notes: Summary By subjecting isolated adrenal medullary cells to intense electric fields of brief duration it is possible to gain access to the cell interior without impairing the ability of the cell to undergo exocytosis. After a single exposure to a field of 2 kV/cm, τ=200 μsec, adrenal medullary cells behave as if their plasma membrane contains two pores of effective radius 2 nm. At 37°C these ‘equivalent pores’ remain patent for up to 1 hr. The formation and stability of these ‘pores’ is not affected by the Ca content of the bathing solution. The ‘pores’ permit externally applied catecholamine and Ca-EGTA to equilibrate rapidly with the cell water. Cells rendered ‘leaky’ in K glutamate medium containing 5mm Mg-ATP and EGTA to give an ionized Ca close to 10−8 m release less than 1% of their total catecholamine. These same cells can release up to 30% of their catecholamine when exposed to 10−5 m Ca. This Ca-dependent release is unaffected by Ca-channel blockers such as D600. Catecholamine release in response to a calcium challenge only seems to occur during the first few minutes whilst the Ca concentration is changing, and the extent of release depends on the final Ca concentration achieved. Half-maximal release occurs at about 1 μm Ca, and this value is independent of the EGTA concentration used to buffer the ionized Ca. The relation between ionized Ca and catecholamine release is best fitted by a requirement for 2 Ca ions. Calcium-evoked release of catecholamine is associated with the release of dopamine-β-hydroxylase (DβH) but not lactate dehydrogenase. The ratio DβH/catecholamine released is the same as that in stimulated intact cells and perfused glands. The time course of appearance in the external medium of DβH and catecholamine is identical. Transmission electron microscopy of ‘leaky’ cells exposed to 10−8 m Ca reveals no marked differences from unstimulated intact cells. The cytoplasm of ‘leaky’ cells exposed to 10−5 m Ca contains large membrane-bounded vacuoles. When secretion is caused to take place in the presence of horseradish peroxidase, this marker is found within the vacuoles. Ca-dependent release of both catecholamine and DβH requires Mg-ATP. Cells equilibrated with Ca in the absence of Mg-ATP can be triggered to undergo exocytosis by the addition of Mg-ATP. In the absence of Mg, ATP alone is ineffective. Of a variety of other nucleotides tested, none is as effective as ATP. Mg-ATP affects the extent of exocytosis and not its apparent affinity for Ca. Replacement of glutamate as the major anion by chloride results in a marked reduction in Ca-dependent release of both catecholamine and DβH. Chloride causes a small increase in Ca-independent release of catecholamine, a large reduction in the extent of exocytosis, and a decrease in the apparent affinity of exocytosis for Ca. Of a variety of anions examined, their order of effectiveness at supporting Ca-dependent exocytosis is glutamate−〉acetate−〉Cl−〉Br−〉SCN−. Exocytosis is not obviously affected by replacing K by Na or sucrose or by altering the pH over the range pH 6.6 to 7.8. Raising the free Mg concentration reduces the extent of Ca-dependent exocytosis and also its apparent affinity for calcium. Calcium-dependent exocytosis in ‘leaky’ cells is largely unaffected by (i) a variety of agonists and antagonists of the nicotinic receptor; (ii) agents that disrupt microtubules and microfilaments; (iii) phalloidin; (iv) vanadate; (v) inhibitors of anion permeability; (vi) protease inhibitors; and (vii) agents that dissipate the vesicle pH gradient and potential. It is partially inhibited by (i) certain antipsychotic drugs; (ii) a rise in osmotic pressure, (iii) lowering the temperature below 20°C, and (iv) N-ethyl maleimide.