Increased calcium permeability is not responsible for the rapid lethal effects of amphotericin B on Leishmania sp

The mode of action of the polyene antibiotic amphotericin B (AmB), the drug of choice for the treatment of systemic fungal infections and visceral leishmaniasis, is still unclear. An increase in intracellular Ca2+ concentration [( Ca2+]i), toxic in many cases, has been postulated as a possible lethal mechanism for AmB. Cell permeabilization to ethidium bromide (EB) was used as a criterion of viability. Kinetics of the DNA-EB fluorescent complex formation was studied in ergosterol-containing Leishmania promastigotes. Intracellular Ca2+ concentration was measured using quin-2 fluorescence in parallel aliquots. It is shown in this work that AmB can act as an efficient Ca2+ ionophore. However, the rapid permeabilization effect induced by AmB on these cells was not dependent on an increase in [Ca2+]i. On the contrary, it was found that leishmanicidal effect of AmB was enhanced in the absence of external calcium. Furthermore, A23187 a Ca2+ ionophore did not provoke cell permeabilization to EB.     

Surface density of calcium ions and calcium spikes in the barnacle muscle fiber membrane

The effects of various divalent cations in the external solution upon the Ca spike of the barnacle muscle fiber membrane were studied using intracellular recording and polarizing techniques. Analysis of the maximum rate of rise of the spike potential indicates that different species of divalent cations bind the same membrane sites competitively with different dissociation constants. The overshoot of the spike potential is determined by the density of Ca (Sr) ions in the membrane sites while the threshold membrane potential for spike initiation depends on the total density of divalent cations. The order of binding among different divalent and trivalent cations is the following: La+++, UO2++ > Zn++, Co++, Fe++ > Mn++ > Ni++ > Ca++ > Mg++, Sr++.     

Divalent cation-dependent structure in the platelet membrane glycoprotein Ia-IIa (VLA-2) complex

Recent studies have shown that the platelet membrane glycoprotein Ia-IIa (VLA-2) complex mediates the Mg(++)-dependent adhesion of platelets to collagen and that this adhesion is inhibited by Ca++ in a simple, linear, noncompetitive manner. These findings suggested that separate binding sites for Mg++ and Ca++ stabilize different divalent cation-dependent structures within the receptor complex. To provide evidence for the existence of such structures purified platelet Ia-IIa complex was subjected to limited proteolytic digestion in the presence of Mg++, Ca++, Mg++ and Ca++, or EDTA and the resulting peptides mapped by SDS-PAGE using both one and two-dimensional techniques. Unique patterns of tryptic peptides were produced under each of the conditions. The results indicate that Mg++ and Ca++ stabilize different structures within the Ia-IIa (VLA-2) complex and that these structures influence both the collagen binding activity and proteolytic susceptibility of the complex.     

Properties of different Ca2+ pools in permeabilized rat thymocytes

The regulation of free Ca2+ concentration by intracellular pools and their participation in the mitogen-induced changes of the cytosolic free Ca2+ concentration, [Ca2+]i, was studied in digitonin-permeabilized and intact rat thymocytes using a Ca2+-selective electrode, chlortetracycline fluorescence and the Ca2+ indicator quin-2. It is shown that in permeabilized thymocytes Ca2+ can be accumulated by two intracellular compartments, mitochondrial and non-mitochondrial. Ca2+ uptake by the non-mitochondrial compartment, presumably the endoplasmic reticulum, is observed only in the presence of MgATP, is increased by oxalate and inhibited by vanadate. The mitochondria do not accumulate calcium at a free Ca2+ concentration below 1 microM. The non-mitochondrial compartment has a greater affinity for calcium and is capable of sequestering Ca2+ at a free Ca2+ concentration less than 1 microM. At free Ca2+ concentration close to the cytoplasmic (0.1 microM) the main calcium pool in permeabilized thymocytes is localized in the non-mitochondrial compartment. Ca2+ accumulated in the non-mitochondrial pool can be released by inositol 1,4,5-triphosphate (IP3) which has been inferred to mediate Ca2+ mobilization in a number of cell types. Under experimental conditions in which ATP-dependent Ca2+ influx is blocked, the addition of IP3 results in a large Ca2+ release from the non-mitochondrial pool; thus IP3 acts by activation of a specific efflux pathway rather than by inhibiting Ca2+ influx. SH reagents do not prevent IP3-induced Ca2+ mobilization. Addition of the mitochondrial uncouplers, FCCP or ClCCP, to intact thymocytes results in no increase in [Ca2+]i measured with quin-2 tetraoxymethyl ester whereas the Ca2+ ionophore A23187 induces a Ca2+ release from the non-mitochondrial store(s). Thus, the data obtained on intact cells agree with those obtained in permeabilized thymocytes. The mitogen concanavalin A increases [Ca2+]i in intact thymocytes suspended in both Ca2+-containing an Ca2+-free medium. This indicates a mitogen-induced mobilization of an intracellular Ca2+ pool, probably via the IP3 pathway.     

Effect of metal ions on growth and sporulation of Clostridium perfringens in a synthetic medium.

All cells, whatever their origin, function in an essentially inorganic environment. In this environment, metal cations play an important role. Attempts were made in the present study to determine if different amounts of five metal ions (MG++, Fe++, Mn++, Zn++, Ca++) are needed for secondary metabolism of Clostridium perfringens than are needed for primary metabolism. Both the vegetative growth stage (primary metabolic stage) and spore stage (secondary metabolic stage) of Clostridium perfringens were studied. Endeavors were made to detect the effects of metal ions, if any, on growth and sporulation of the organisms. Mg++ was required for growth of vegetative cells and maintenance of normal cellular morphology, Fe++ was needed for sporulation as well as for vegetative growth, but the amount needed for spore formation was higher than that needed for growth. Ca++ was essential to refractility and heat-resistance of spores. Zn++ inhibited both growth and sporulation, if the Mg++ concentration was low. Mn++ was required for neither growth nor sporulation. For maximum heat-resistance of the spores, Ca++ plus unidentified organic substances were required.     

Uptake of Ca2+ and refilling of intracellular Ca2+ stores in Ehrlich-ascites-tumour cells and in rat thymocytes.

We have studied the uptake of Ca2+ and its redistribution between the cytoplasm and the intracellular stores in Ehrlich-ascites-tumour cells and rat thymocytes previously depleted of Ca2+ by incubation in Ca2(+)-free medium. Measurements included changes of the cytoplasmic Ca2+ concentration ([Ca2+]i), uptake of 45Ca2+ and uptake of Mn2+, a Ca2+ surrogate for Ca2+ channels. Refilling of the Ca2+ stores in thymocytes was very fast (half-filling time: 4 s at 37 degrees C) and very sensitive to temperature (10 times slower at 20 degrees C). It was always preceded by increase of [Ca2+]i. In the Ehrlich cell, both refilling and increase of [Ca2+]i were about one order of magnitude slower. The increase of [Ca2+]i and the refilling of the intracellular stores were both almost completely blocked by Ni2+ in thymocytes, but only partially in the Ehrlich cell. The rates of 45Ca2+ and Mn2+ uptake varied consistently with temperature and the kind of cell. These results suggest that the intracellular stores are refilled by Ca2+ taken up from the cytoplasm. We also find that filling of the Ca2+ stores decreases by about 90% the rate of Mn2+ uptake in thymocytes. This is direct evidence of modulation of the plasma-membrane Ca2+ entry by the degree of filling of the intracellular stores. This modulation occurs in the absence of agonists, suggesting some kind of signalling between the intracellular stores and the Ca2+ entry pathways of the plasma membrane.     

Permeability changes induced by polylysines in rat spermatids

High molecular weight (HMW, >15 kDa) but not low molecular weight (LMW, <15 kDa) polylysines (PLs) bound and induced permeability changes in rat spermatid plasma membranes, estimated by Mn2+ quenching of intracellular indo-1 fluorescence (K(1/2) = 3.3 +/- 0.5 microg/ml) and Co2+ quenching of intracellular calcein. The pharmacology of the Mn2+ entry pathway activated by HMW PL does not suggest that Ca2+ channels are involved in this phenomenon. Concentrations of HMW PL that induced divalent ion entry did not induce the entry of ethidium bromide, suggesting that HMW PL first bound and perturbed the plasma membrane structure inducing a non-specific increase in membrane permeability. High concentrations of HMW PL induced cell lysis (K(1/2) = 23 microg/ml). The binding of HMW PL, initially homogenous on the cell surface, subsequently progressed to a segregated pattern resembling a clustering phenomenon.     

Carbamylcholine, TRH, PGF2 alpha and fluoride enhance free intracellular Ca++ and Ca++ translocation in dog thyroid cells

Effects on Ca++ translocation and [Ca++]i were studied in dog thyro?d cell monolayers using both 45Ca++ efflux and the indicator quin-2. Carbamylcholine, a non hydrolysable analog of acetylcholine, through muscarinic receptors, and to a lesser extent TRH and PGF2 alpha increased both these parameters. [Ca++]i increased by 171, 100 and 75% respectively over a basal level of 66 +/- 17 nM (mean +/- SD). The response to carbamylcholine was biphasic. A transient increase in [Ca++]i was followed by a more sustained phase where the [Ca++]i was slightly higher than the basal level. Only the first phase was insensitive to extracellular Ca++ depletion. This phase is probably due to a release of Ca++ from an intracellular store. NaF also induced a sustained rise in [Ca++]i dependent on extracellular Ca++ and affected 45Ca++ efflux. Our data provide direct evidence of an implication of intracellular Ca++ in the response of dog thyro?d cells to all these agents.     

Calcium depletion blocks the maturation of rotavirus by altering the oligomerization of virus-encoded proteins in the ER.

Maturation of rotavirus occurs in the ER. The virus transiently acquires an ER-derived membrane surrounding the virus particle before the eventual formation of double-shelled particles. The maturation process includes the retention and selective loss of specific viral protein(s) as well as the ER-derived membrane during formation of the outer capsid of the mature virus. When infected cells were depleted of Ca++ by use of the ionophore A23187 in calcium-free medium, membrane-enveloped intermediates were seen to accumulate. When Mn++, an efficient Ca++ competitor, was used to replace Ca++ in the medium, the accumulation of the enveloped intermediate was again observed, pointing to an absolute requirement of Ca++ in the maturation process. It was previously demonstrated in this laboratory that a hetero-oligomeric complex of NS28, VP7, and VP4 exists which may participate in the budding of the single-shelled particle into the ER (Maass, D. R., and P. H. Atkinson, 1990. J. Virol. 64:2632-2641). The present study demonstrates that either in the absence of Ca++ or in the presence of tunicamycin, a glycosylation inhibitor, VP7 is excluded from these hetero-oligomers. In the presence of Mn++, VP4 was blocked in forming a hetero-oligomeric complex with NS28 and VP7. The electrophoretic mobility of the viral glycoproteins synthesized in the presence of the ionophore were found to be altered. This size difference was attributed to altered N-linked glycosylation and carbohydrate processing of the viral glycoproteins. These results imply a major role for calcium and the state of glycosylation of NS28 in the assembly and acquisition of specific viral protein conformations necessary for the correct association of proteins during virus maturation in the ER.     

Structure and stability of Z* DNA

The structure and stability of the left handed Z* DNA aggregate was examined by spectroscopic methods and by electron microscopy. Poly(dGdC), upon heating in the presence of Mn++, forms a large aggregate which may be sedimented at 12,000 X g, with a circular dichroism spectrum characteristic of left handed DNA. Aggregation gives rise to turbidity changes at visible wavelengths, providing a convenient means of monitoring the transition in solution. The wavelength dependence of turbidity is consistent with the scattering behavior of a long thin rod. Electron microscopy shows that Z* DNA is a large fibrous structure of indeterminant length, with a uniform diameter of approximately 20 nm. The results obtained in solution and under the requisite conditions for electron microscopy are mutually consistent. Poly(dGdC) preparations with average lengths of 60, 240, 500, and 2000 base pairs all form Z* DNA. Poly(dGm5dC) forms Z* DNA in the presence of Mn++ without heating, but poly(dAdC)-poly(dGdT) and calf thymus DNA cannot be induced to the Z* form under any conditions tried. Kinetic studies, monitored by turbidity changes, provide evidence that the formation of Z* DNA proceeds by a nucleated condensation mechanism. Dissolution of the Z* aggregate results from the chelation of Mn++ or by the addition of the intercalator ethidium bromide. The allosteric conversion of Z* DNA to an intercalated, right handed form by ethidium is demonstrated by kinetic studies, equilibrium binding studies and circular dichroism spectroscopy. Electron microscopy provides a striking visualization of the dissolution of the Z* aggregate by ethidium.     

Stimulation of 3H-norepinephrine release by trifluoperazine from rat pineal glands

Trifluoperazine (5-200 microM) stimulated the release of 3H-NE from isolated whole pineal glands in a dose dependent manner. Trifluoperazine-induced release was not dependent on extracellular Ca++, whereas 60 mM K+-evoked release was attenuated in the presence of EGTA and zero Ca++ Krebs. 60 mM K+ and 50 microM trifluoperazine produced an additive effect on 3H-NE release. Clonidine (5 microM) significantly reduced trifluoperazine-induced release by approximately 50% in the presence of Ca++, and in its absence, clonidine significantly attenuated the trifluoperazine response by 42%. Thus trifluoperazine may be acting upon the alpha 2 receptor or intracellular stores of Ca++. These intracellular interactions remain for further study.     

Cytosolic Ca2+ homeostasis in Ehrlich and Yoshida carcinomas. A new, membrane-permeant chelator of heavy metals reveals that these ascites tumor cell lines have normal cytosolic free Ca2+

The intracellularly trappable fluorescent Ca2+ indicator quin-2 was used to measure free cytosolic Ca2+, [Ca2+]i, in the two highly dedifferentiated tumor cell lines, Ehrlich and Yoshida ascites carcinomas. It was found that these carcinoma cells can trap quin-2 similarly to normal cells, but [Ca2+]i was apparently significantly lower than in any normal cell tested previously with this method. By using a new lipid-soluble heavy metal chelator TPEN (N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine), which crosses artificial and natural membranes, it was found that endogenous heavy metals are responsible for partially quenching quin-2 fluorescence trapped inside the cells. Although the quenching of intracellular quin-2 fluorescence is quantitatively more relevant in these ascites carcinomas, TPEN was effective also in normal cells like lymphocytes and granulocytes. Both in the normal and especially in the malignant cell lines [Ca2+]i can be grossly underestimated at low intracellular quin-2 concentrations. Endogenous heavy metal quenching is thus a potential source of artifact when [Ca2+]i is measured with quin-2. When corrected for quin-2 fluorescence quenching by intracellular heavy metals, [Ca2+]i and basic regulatory mechanisms of [Ca2+]i homeostasis in Ehrlich and Yoshida carcinomas are similar to those of nontransformed cells.     

Purification and general properties of pectin methyl esterase from Curvularia inaequalis NRRL 13884 in solid state culture using orange peels as an inducer

Pectin methyl esterase (PME) [E.C.3. 1.1.11] production by Curvularia inaequalis (Shear) Boedijn NRRL 13884 was investigated using solid-state culture. The highest level of extracellular pectin methyl esterase was detected with orange peels as an inducing substrate and as a sole carbon source. The enzyme was partially purified using Sephadex G-100 and DEAE-Cellulose column chromatography. It was purified about 40 fold with optimum activity at pH 4.4 and 45 degrees C. The enzyme was activated by Co++, Mg++, Na+, whereas it was slightly activated in the presence of Cu++, K+, Mn++, Zn++. On the other hand Ag++, Ca++ and Hg++ inhibited the activity of the enzyme. The Km was calculated to be 0.52 mM.     

Transduction persists in rod photoreceptors after depletion of intracellular calcium

We have examined the role of Ca++ in phototransduction by manipulating the intracellular Ca++ concentration in physiologically active suspensions of isolated and purified rod photoreceptors (OS-IS). The results are summarized by the following. Measurement of Ca++ content using arsenazo III spectroscopy demonstrates that incubation of OS-IS in 10 nM Ca++-Ringer's solution containing the Ca++ ionophore A23187 reduces their Ca++ content by 93%, from 1.3 to 0.1 mol Ca++/mol rhodopsin. Virtually the same reduction can be accomplished in 10 nM Ca++-Ringer's without ionophore, presumably via the plasma membrane Na/Ca exchange mechanism. Hundreds of photoresponses can be obtained from the Ca++-depleted OS-IS for at least 1 h in 10 nM Ca++-Ringer's with ionophore. The kinetics and light sensitivity of the photoresponse are essentially the same in the presence or absence of the ionophore in 10 nM Ca++. The addition of A23187 in 1 mM Ca++-Ringer's results in a Ca++ influx that rapidly suppresses the dark current and the photoresponse. This indicates that there is an intracellular site at which Ca++ can modulate the light-regulated conductance. Both the current and photoresponse can be restored if intracellular Ca++ is reduced by lowering the external Ca++ to 10 nM. During the transition from high to low Ca++, the response duration becomes shorter, which suggests that it can be regulated by a Ca++-dependent mechanism. If the dark current and the photoresponse are suppressed by adding A23187 in 1 mM Ca++-Ringer's, the subsequent addition of the cyclic GMP phosphodiesterase inhibitor isobutylmethylxanthine can restore the current and photoresponse. This implies that under conditions where the rod can no longer control its intracellular Ca++, the elevation of cyclic GMP levels can restore light regulation of the channels. The persistence of normal flash responses under conditions where intracellular Ca++ levels are reduced and perturbed suggests that changes in the intracellular Ca++ concentration do not cause the closure of the light-regulated channel.     

Depolarizing response of rat parathyroid cells to divalent cations

Membrane potentials were recorded from rat parathyroid glands continuously perfused in vitro. At 1.5 mM external Ca++, the resting potential averages -73 +/- 5 mV (mean +/- SD, n = 66). On exposure to 2.5 mM Ca++, the cells depolarize reversibly to a potential of -34 +/- 8 mV (mean +/- SD). Depolarization to this value is complete in approximately 2-4 min, and repolarization on return to 1.5 mM Ca++ takes about the same time. The depolarizing action of high Ca++ is mimicked by all divalent cations tested, with the following order of effectiveness: Ca++ greater than Sr++ greater than Mg++ greater than Ba++ for alkali-earth metals, and Ca++ greater than Cd++ greater than Mn++ greater than Co++ greater than Zn++ for transition metals. Input resistance in 1.5 mM Ca++ was 24.35 +/- 14 M omega (mean +/- SD) and increased by an average factor of 2.43 +/- 0.8 after switching to 2.5 mM Ca++. The low value of input resistance suggests that cells are coupled by low-resistance junctions. The resting potential in low Ca++ is quite insensitive to removal of external Na+ or Cl-, but very sensitive to changes in external K+. Cells depolarize by 61 mV for a 10- fold increase in external K+. In high Ca++, membrane potential is less sensitive to an increase in external K+ and is unchanged by increasing K+ from 5 to 25 mM. Depolarization evoked by high Ca++ may be slowed, but is unchanged in amplitude by removal of external Na+ or Cl-. Organic (D600) and inorganic (Co++, Cd++, and Mn++) blockers of the Ca++ channels do not interfere with the electrical response to Ca++ changes. Our results show remarkable parallels to previous observations on the control of parathormone (PTH) release by Ca++. They suggest an association between membrane voltage and secretion that is very unusual: parathyroid cells secrete when fully polarized, and secrete less when depolarized. The extraordinary sensitivity of parathyroid cells to divalent cations leads us to hypothesize the existence in their membranes of a divalent cation receptor that controls membrane permeability (possibly to K+) and PTH secretion.     

Calcium-independent pathway of tumor necrosis factor-mediated lysis of target cells

The role of Ca2+ in cell-mediated cytotoxicity has been the subject of many investigations and both Ca2+-dependent and -independent pathways have been reported. TNF was suggested to play a role in NK and macrophage cell-mediated cytotoxicity. We assumed that its role in target cell lysis might take place by a Ca2+-independent mechanism. This hypothesis was investigated in assays of rTNF-mediated lysis of tumor target cells. Extracellular Ca2+ depletion by the calcium chelator EGTA (2 mM and 5 mM) and blocking of intracellular Ca2+ mobilization by 8-(diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride did not inhibit TNF-mediated tumor cell lysis. Furthermore, blocking of Ca2+ influx in the presence of the Ca2+ channel blocker Verapamil did not inhibit TNF-mediated tumor cell lysis. Previous reports showed that lysis of sensitive tumor cells by TNF is preceded by binding of TNF to TNF receptors, internalization, and DNA degradation. These events were tested in the absence of Ca2+. Treatment with Ca2+ inhibitors did not affect binding of 125I-TNF to target cells. Also TNF induced the fragmentation of cellular DNA in target cells without extracellular or intracellular Ca2+. These findings demonstrate that the mechanism of TNF-mediated tumor cell lysis does not depend on intracellular or extracellular Ca2+ and that events associated with target cell lysis can also function in the absence of Ca2+. Thus, our findings support the contention of a Ca2+-independent lytic pathway in which secreted or membrane-bound TNF may interact with the target cells and ultimately result in DNA degradation and target cell lysis.     

Exogenous ATP enhances calcium influx in intact thymocytes

Recent observations have indicated that exogenous adenosine triphosphate (ATP) may influence lymphocyte functions such as proliferation and cytoxicity. Here we report a novel activity of extracellular ATP--it specifically increases Ca2+ uptake in murine lymphocytes. ATP added to thymocytes increases the rate of [45Ca2+] uptake by up to 20-fold. The increased rate is seen with ATP concentrations as low as 500 microM and is half-maximal at approximately 2 mM ATP. The magnitude of stimulation by ATP is dependent on Mg2+ concentration, and ATP-Mg2+ complex is probably the true activator. Of the high-energy phosphate-containing compounds tested, including deoxy-ATP, only GTP showed a modest stimulation of calcium uptake. ADP, AMP, cyclic AMP, and adenosine did not significantly increase calcium uptake. Cellular integrity as indicated by trypan blue exclusion and ethidium bromide/acridine orange staining was unaffected by ATP. Ca2+ influx is the major mode of action of ATP in raising intrathymocyte Ca2+ levels, because neither the Ca2+ efflux nor the [45Ca2+]-Ca2+ exchange was significantly altered in the presence of ATP. Verapamil, a Ca2+ channel blocking agent, could not prevent the ATP effect, suggesting that ATP may be acting by a mechanism other than the voltage-dependent Ca2+ channel. An analysis of intracellular and extracellular ATP levels by chemiluminescence assay indicated no significant ATP entry into intact lymphocytes. Also, ATP added to the medium containing thymocytes was destroyed (approximately 50% by 20 min). The nonhydrolyzable ATP analogs, AMPPCP and AMPPNP, were unable to stimulate a significant amount of Ca2+ uptake, suggesting the involvement of a cell surface phosphotransferase activity. This was supported by the demonstration of a threefold to fivefold increase in the labeling of protein and phospholipid fractions obtained from intact thymocytes exposed to [gamma 32P]ATP for 30 min. Ca2+ is believed to play an important role in a variety of lymphocyte functions, including mitogenesis and natural killer cell activity. The data herein thus provide a potential mechanism for the action of exogenous ATP on these lymphocyte functions.     

Characterization of calcium-activated bifunctional peptidase of the psychrotrophic Bacillus cereus

The protease purified from Bacillus cereus JH108 has the function of leucine specific endopeptidase. When measured by hydrolysis of synthetic substrate (N-succinyl-Ala-Ala-Pro-Leu-p-nitroanilide), the enzyme activity exhibited optimal activity at pH 9.0, 60 degrees C. The endopeptidase activity was stimulated by Ca++, Co++, Mn++, Mg++, and Ni++, and was inhibited by metal chelating agents such as EDTA, 1,10-phenanthroline, and EGTA. Addition of serine protease inhibitor, PMSF, resulted in the elimination of the activity. The endopeptidase activity was fully recovered from the inhibition of EDTA by the addition of 1 mM Ca++, and was partially restored by Co++ and Mn++, indicating that the enzyme was stabilized and activated by divalent cations and has a serine residue at the active site. Addition of Ca++ increased the pH and heat stability of endopeptidase activity. These results show that endopeptidase requires calcium ions for activity and/or stability. A Lineweaver-Burk plot analysis indicated that the Km value of endopeptidase is 0.315 mM and Vmax is 0.222 mmol of N-succinyl-Ala-Ala-Pro-Leu-p-nitroanilide per min. Bestatin was shown to act as a competitive inhibitor to the endopeptidase activity.     

Rat ovarian and adrenal prolactin receptors. Sizes and effects of divalent metal ions

Receptor fractions were prepared from follicle-rich ovaries (for FSH), luteal cell-rich ovaries (for LH and PRL), and adrenals (for PRL) of rats. Divalent metal ions, Mg++, Ca++, and Mn++ showed inhibitory effects on the binding of LH and FSH to their receptors. The binding of the former was more sensitive to these ions than the latter. On the other hand they showed bell-shaped promotive effects on PRL-ovarian receptor binding, the maximal effects being observed at 10-20 mM. Besides these ions, Ba++ also had a promotive effect, while other divalent metal ions such as Zn++, Cd++, Ni++, and Co++ showed inhibitory effects on PRL-ovarian receptor binding at 5 mM. Mg++ and Ca++ also promoted PRL-adrenal receptor binding, while Mn++ promoted the binding at 10 mM but inhibited it at higher concentrations. Association constant (Ka) and binding capacity (Bmax) of PRL receptors of the ovary and the adrenal were significantly different (ovary: Ka = 0.69 X 10(10) M-1, Bmax = 62 fmol/mg protein, adrenal: Ka = 0.21 X 10(10) M-1, Bmax = 99 fmol/mg protein). Ka of the ovarian PRL receptor was not influenced by these divalent ions, while that of the adrenal receptor was doubled by Ca and Mn ions, Bmax of the latter was also increased. A cooperative effect of Mg and Ca ions was observed on Ka and Bmax of the adrenal receptor. The sizes of the PRL binding sites of these organs revealed by affinity labelling were 17K and 40K in the ovary, and 40K and 110K in the adrenal. These results indicate the different properties of receptors in these different target organs.     

Ca++-ionophore-induced dissipation of intracellular proton gradients in rat thymocytes

Rat thymocytes incorporate large amounts of acridine orange at concentrations of approximately 10 microM in about 10 minutes at 37 degrees C. The addition of (NH4)2SO4 (at a final concentration of several mMolars) releases about 30% of the incorporated dye into the medium. The NH4+-releasable dye uptake is almost completely abolished by 20 minutes' incubation with 4 microM of the Ca++-ionophore A23187. Dye uptake is associated with an absorbance change at 492 mm and thus may be followed spectrophotometrically. However, NH4+ at the above concentrations or nigericin (0.5 mg/ml) completely annul this change in absorbance, indicating that it reflects only the accumulation of the dye within the acidic cellular compartments. In a Ca++-containing physiological saline, the addition of A23187 (at a final concentration of 8 microM) at the end of the dye uptake phase initiated a reversal in the absorbance change; in the absence of Ca++, reversal occurred at a much lower rate. Incubation of cells for 30 minutes with 2 microM A23187 in Ca++-containing saline completely abolished NH4+-sensitive dye uptake; less A23187 (0.5 microM) and like or a longer incubation period brought about a striking decrease in NH4+-sensitive dye uptake. Similar results were obtained with cells suspended in RPLM 1640 medium. In the absence of external Ca++, A23187 impaired cell capacity to incorporate dye in a delta pH-dependent manner, but a longer incubation time or higher concentrations of the ionophore were required to obtain a comparable effect. It is thus concluded that the ionophore dissipates intracellular pH gradients by an intracellular divalent cation (Ca++ or also Mg++)-H+ exchange.