ALBERT

Notes: Summary Miniature end-plate currents (MEPCs) and acetylcholine-induced current fluctuations were recorded in voltageclamped, glycerol-treated toad sartorius muscle fibers in control solution and in solutions with added divalent cations. In isosmotic solutions containing 20mm Ca or Mg, MEPCs had time constants of decay (τ D ) which were about 30% slower than normal. In isotonic Ca solutions (Na-free), greater increases in both τ D and channel lifetime were seen; the null potential was −34 mV, and single-channel conductance decreased to approximately 5 pS. Zn or Ni, at concentrations of 0.1–5mm, were much more effective in increasing τ D than Ca or Mg, although they did not greatly affect channel conductance. The normal temperature and voltage sensitivity of τ was not significantly altered by any of the added divalent cations. Surface potential shifts arising from screening of membrane fixed charge by divalent cations cannot entirely explain the observed increases in τ, especially when taken together with changes in channel conductance.

Notes: Summary Phosphate efflux was measured as the fractional rate of loss of radioactivity from rabbit vagus loaded with radiophosphate. The effects of changes in extracellular calcium and of lanthanum have been investigated. In Locke solution with normal, 0.9mm, calcium and without phosphate, the fractional rate of loss was 1.62×10−3 min−1 at 120 min after the beginning of the washing period and fell slowly (9% hr−1) during washing from 2 to 6 hr. Addition of calcium to the Locke solution produced a transient increase followed by a reversible maintained increase in phosphate efflux. The latter was 40 and 75% above efflux in normal calcium for 20 and 50mm calcium, respectively. Removal of calcium, with or without addition of EGTA, produced only a transient increase in phosphate efflux, with no subsequent maintained change. Addition of low concentrations of lanthanum produced a reversible inhibition of phosphate efflux. Half-maximal inhibition was at 3.5 μm lanthanum and appeared to be due to binding of lanthanum to more than one, probably two, sites. Measurements of inhibition by lanthanum at different calcium concentrations did not indicate any competition between calcium and lanthanum. It is suggested that at least a part of phosphate efflux depends on internal calcium and that lanthanum acts by preventing release of phosphate from the phosphate transport mechanism.

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 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.

Notes: Summary The extracellular Ca2+ requirement for antidiuretic hormone (ADH) stimulation of water permeability in the toad urinary bladder has been critically examined. The polarity of the tissue was maintained with 1mm Ca2+ in the mucosal bathing medium and a serosal bath nominally free of Ca2+. Under these condition, ADH-induced osmotic water flow was inhibited by more than 60% while enhancement of the diffusional permeability to water was unaffected. Structural studies revealed that low serosal Ca2+ led to parallel alterations in epithelial architecture that amounted to a significant distorition of the osmotic water pathway. Prevention of these alterations, or restoration of normal cell-cell contact showed that the reduction of serosal Ca2+ did not restrict hormonal action,per se, but that it resulted in a weakening of cell-cell junctions such that intercellular space distension during water flow occurred to a point where the geometric conditions for maintenance of osmotic flow were compromised. We conclude that extracellular Ca2+ is not a requirement for the molecular aspects of ADH action but that, in its absence, a direct measurement of ADH-induced osmotic flow proves to be an inaccurate index of the hormone-generated changes in epithelial transport characteristics. Under certain conditions the ADH-effect on the tissue's hydraulic permeability is probably best assessed by measurement of the diffusional permability to water; although accuracy in this determination is difficult, it is not as strongly dependent on tissue geometry.

Notes: Summary In many cell systems, the permeability of membrane junctions is modulated by the cytoplasmic level of free Ca++. To examine whether the calcium-dependent regulatory protein calmodulin is involved in this process, the ability of anticalmodulin drugs to influence the cell-to-cell passage of injected current and an organic tracer was tested using standard intracellular glass microelectrode techniques. Several antipsychotics and local anesthetics were found to block junctional communication in the epidermis of the beetleTenebrio molitor. Treatment of the epidermis with chlorpromazine (0.25 mM) raised intercellular resistance two- to threefold within 20 to 25 min; cell-to-cell passage of electrical current was abolished within 41±5 min. Loss of electrotonic coupling was accompanied by a block in the cell-to-cell movement of the organic tracer carboxyfluorescein. The reaction is fully reversible, with normal electrotonic coupling being restored within 2 to 4 hr. Other antipsychotics and local anesthetics had similar effects on cell coupling. The order of potency found was: trifluoperazine〉thioridazine〉 d-butaclamol〉chlorprothixine=chlorpromazine〉 l-butaclamol〉 dibucaine〉tetracaine. The relative uncoupling potencies of these drugs correlate well with their known ability to inhibit calmodulin-dependent phosphodiesterase activity. Other anesthetic compounds, procaine and pentobarbital, did not block cell-to-cell communication. Altering the extracellular Ca++ concentration did not affect the rate of uncoupling by antipsychotics, while chelation of extracellular Ca++ with EGTA raised electrotonic coupling. The effect of three metabolic inhibitors on coupling was also examined. Iodoacetate uncoupled the epidermal cells while DNP and cyanide did not. These results are discussed in terms of possible mechanisms by which calmodulin may control junctional communication in this tissue.

Notes: Abstract Sugarbeet cells from normal and habituated callus released peroxidases in liquid cultures, in proportion to their endogenous level. Calcium promoted this release more in the normal than in the habituated line. Treatment of the cells with sodium azide, sodium hydrogenarsenate or phenothiazine inhibited the calcium effect, which indicated a dependence on metabolic energy and on calmodulin regulation. The Ca ionophore Ro (bromolasalocid ethanolate) restricted peroxidase release.

Notes: Abstract The 40,000-dalton glycoprotein and 2000-dalton peptide inducing selective Ca2+-transport through bilayer lipid membranes were isolated from beef heart homogenate and mitochondria. Micromolar concentrations of these substances were found to increase the conductivity of membranes by 3–4 orders. Transmembrane Ca2+ gradient induces an electric potential difference whose magnitude is close to the theoretical for ideal Ca2+ selectivity. The inhibitor of mitochondrial Ca2+ transport, ruthenium red, abolishes both the glycoprotein-and peptide-induced Ca2+ transport in bilayer lipid membranes. Thiol groups essential for Ca2+ transport activity were revealed in the glycoprotein and peptide. Addition of these substances to rat liver mitochondria induces Ca2+-dependent inhibition of the state 3 respiration that can be released by uncouplers (oligomycin-like effect).

Notes: Abstract The lattice entropy derived from the measured heat capacity at intermediate and high temperatures is analyzed to yield a weakly temperature dependent entropy Debye temperature. An unusual temperature dependence of this quantity may be a sign of error in the heat capacity data. When this analysis is applied to heat capacity data recommended by Hultgren et al. (1973) for 20 nontransition metals, the result for fcc Ca stands out as anomalous. We have reconsidered heat capacity data of fcc Ca and find that measurements by Eastman et al. (1924), which were given little weight by Hultgren et al., are consistent with a normal behavior of the entropy Debye temperature up to 450 K.

Notes: Summary 1. An ultrastructural and cytochemical study of carotenoid-containing membrane structures (CMS) of molluscan neurons was made in order to elucidate the role of carotenoids in these cells. 2. CMS were found to be the modified parts of mitochondria in which the bulk of cellular calcium salts is accumulated. 3. These data, as well as previously reported subfraction studies, suggest that carotenoids participate in the membrane transfer of calcium.

Notes: Summary 1. Adenosine analogues inhibit calcium-dependent K+-evoked release of [3H]norepinephrine from guinea pig cerebral cortical and hippocampal vesicular preparations. Inhibition requires high concentrations (100µM) of the adenosine analogues and is abolished in the presence of high concentrations (2 mM) of calcium ions. The inhibitory effect of 2-chloroadenosine is blocked by theophylline. The structure activity profile (N 6-d-phenylisopropyladenosine ≥N 6-l-phenylisopropyladenosine ≥ 2-chloroadenosine 〉N 6-cyclohexyladenosine, adenosine 5′-cyclopropylcar-boxamide) is not that expected of either A1 (high-affinity) or A2 (low-affinity) adenosine receptors. 2. Calcium-dependent K+-evoked release of [3H]dopamine from guinea pig striatal vesicular preparations is inhibited by apomorphine. However, only 2-chloroadenoine causes an inhibition of K+-evoked release of [3H]dopamine. Other adenosine analogues such asd- andl-phenylisopropyladenosine and adenosine 5′-cyclopropylcar-boxamide cause a facilitation of K+-evoked release. The facilitation is abolished or reduced in the presence of high concentrations (2 mM) of calcium ions. The sites of action of adenosine analogues do not appear to have structural requirements identical to those expected of A1 (high-affinity) or A2 (low-affinity) adenosine receptors. 3. The results indicate that adenosine analogues can have either inhibitory or facilitory effects on K+-evoked release of catecholamines from central synaptic terminals.

Notes: Summary 1. The calcium-dependent K+-evoked release of [3H]norepinephrine from guinea pig cerebral cortical vesicular preparations is inhibited by norepinephrine, clonidine, and epinephrine. Isoproterenol has no effect and phentolamine prevents the inhibition by norepinephrine. The results indicate that anα-adrenergic receptor mediates an inhibitory input to the calcium-dependent release process. The inhibition by norepinephrine is prevented by high concentrations (3.0 mM) of calcium ions. 2. A cyclic AMP phosphodiesterase inhibitor, ZK 62771, slightly elevates [3H]cyclic AMP levels in the guinea pig cerebral cortical preparation and potentiates the marked elevation of [3H]cyclic AMP elicited by the adenylate cyclase activator, forskolin. 3. Neither ZK 62771 nor forskolin alone has significant effects on K+-evoked release of [3H]norepinephrine from the cerebral cortical vesicular preparation; however, a combination of ZK 62771 and forskolin inhibits K+-evoked release by as much as 60%. The inhibition is reversed by high concentrations (2.0 mM) of calcium ions. The results suggest that a marked accumulation of cyclic AMP elicited via both activation of adenylate cyclase and inhibition of phosphodiesterase can be inhibitory to neurotransmitter release from central synaptic terminals.

Notes: Summary 1. The calcium antagonists D-600 (1–10µM) and diltiazem (10–25µM) inhibit K+-evoked release of [3H]norepinephrine from guinea pig cerebral cortical vesicular preparations. The inhibition of release is partially reversed by increasing concentrations of calcium to 2 mM. Diltiazem at 100µM has no effect on K+-evoked release of [3H]norepinephrine at 0.15 mM calcium but does inhibit release at 2.0 mM calcium. 2. The calcium antagonist nifedipine and dantrolene, an agent purported to antagonize release of calcium from intracellular storage sites, have no effect on K+-evoked release of [3H]norepinephrine. 3. The calcium antagonists D-600 (1µM) and diltiazem (10µM) inhibit K+-evoked release of [3H]dopamine from guinea pig striatal vesicular preparations. Higher concentrations of drug, namely, 10µM for D-600 and 100µM for diltiazem, cause a potentiation rather than an inhibition of K+-evoked release. The potentiation is reduced in magnitude upon raising the extracellular calcium to 2.0 mM. Indeed, 10µM D-600 then inhibits K+-evoked release of [3H]dopamine. 4. The results indicate that putative calcium antagonists can have both inhibitory and facilitory effects on calcium-dependent K+-evoked release of catecholamines from central synaptic endings. Furthermore, certain peripheral calcium antagonists such as nifedipine and dantrolene may prove ineffective in central systems.

Notes: Summary 1. Spontaneous transmitter release was studied at the frog sartorius neuromuscular junction in the presence of a variety of cations before and after treatment with the specific presynaptic neurotoxin,β-bungarotoxin (β-BuTX). 2. Treatment withβ-BuTX produced a maintained increase in spontaneous release, as indicated by the miniature end-plate potential (m.e.p.p.) frequency. It was demonstrated that the m.e.p.p. frequency remained dependent on the extracellular calcium concentration. 3. A 30 mM increase in extracellular sodium chloride produced a reversible increase in frequency only afterβ-BuTX treatment, indicating thatβ-BuTX had increased the permeability of the presynaptic terminal. 4. Furthermore, several divalent cations other than calcium were shown to either maintain or greatly increase the m.e.p.p. frequency afterβ-BuTX treatment (before toxin treatment replacement of calcium by these divalent cations produced only small changes in frequency). The relative effectiveness of the divalent cations tested in increasing spontaneous transmitter release after toxin treatment was Co2+ ≃ Ni2+ 〉 Mg2+ 〉 Ca2+ ≃ Sr2+ 〉 Mn2+. The effect of cobalt, which increased the m.e.p.p. frequency 6.5 times after toxin treatment, was studied in detail. 5. It is proposed thatβ-BuTX, through its phospholipase activity, increases the ionic permeability of the terminal membrane and allows access to intracellular sites of relatively impermeant cations. This allowed us to demonstrate that several divalent cations other than calcium can influence transmitter release either directly at release sites or by altering internal calcium buffering.