Kinetics of calcium efflux and exchange from Rana pipiens oocytes immediately following reinitiation of the first meiotic division: Comparison of various meiotic agonists and antagonists

The reinitiation of the meiotic divisions and the release of ⁴⁵Ca from the $\text{Rana}$$\text{pipiens}$ oocyte has been studied as a function of meiotic agonists and antagonists. Each of the meiotic agonists tested (progesterone, insulin, D-600, La³⁺) caused a decreased ⁴⁵Ca uptake and an increased efflux during the first 15 min after exposure. The effects of progesterone, D-600, and La³⁺ are not additive and progesterone will not release additional ⁴⁵Ca in oocytes pretreated with D-600 or La³⁺. Tetracaine inhibits both progesterone-induced release of ⁴⁵Ca and an early step in meiosis (nuclear membrane breakdown). [Tetracaine]_o required for 50% inhibition of nuclear breakdown decreases with decreasing [progesterone]_o suggesting competitive inhibition. The Ca, Mg-ionophore A23187 shows a similar competitive inhibition of progesterone-induced nuclear breakdown and stimulates a rapid release of ⁴⁵Ca within the first 1–3 minutes after exposure to the ionophore. Unlike progesterone, insulin, D-600, or La³⁺, the ionophore A23187 stimulates both uptake and efflux of ⁴⁵Ca by oocytes. These results suggest that both a reduced influx and a selective release of calcium from specific membrane sites is essential for steroid reinitiation of the meiotic divisions in $\text{R.}$$\text{pipiens}$ oocytes.     

DNA polymerase alpha, beta and gamma activities in rabbit spleen cell populations stimulated by various doses of concanavalin A

Adherence and phagocytosis of ⁵¹chromium labeled sheep red blood cells ([⁵¹Cr]-sRBC) by P388 D₁ cells in tissue culture were studied under various conditions and were found to possess certain requirements including opsonization, temperature, microfilaments and cyclic nucleotide levels. Exogenous administration of 10^?2 M N⁶, O²-dibutyryl adenosine 3′–5′ cyclic monophosphoric acid (db-cAMP) or adenosine 3′–5′ cyclic monophosphoric acid (cAMP) inhibited phagocytosis of opsonized [⁵¹Cr]-sRBC by 36 and 42%, respectively. Aminophylline potentiated the inhibitory response to both cAMP and db-cAMP. The measurement of endogenous cyclic nucleotide levels during phagocytosis of opsonized sRBC showed a rise in guanosine 3′–5′ cyclic monophosphate (cGMP) during the first 5 min with a gradual decline to control levels at 45 min and a rise in cAMP levels reaching a peak at 30 min which remained above control values for the duration of the experiment. As the rate of phagocytosis decreased the ratio of $\text{cAMP}\text{cGMP}$ increased. These observations emphasize the importance of metabolic functions and cyclic nucleotides during phagocytosis by the P388 D₁ cells and strengthen the usefulness of the P388 D₁ cells as a model in evaluating various macrophage activities.     

Metabolism of 25-hydroxy-vitamin D3 by primary cultures of chick kidney cells

The primary culture of kidney cells from vitamin D deficient chicks is described. After four days in culture the cells reach confluency and retain their ability to metabolize 25-hydroxyvitamin D₃ to 1,25-dihydroxyvitamin D₃. Addition of one unit of bovine parathyroid hormone to the culture medium for 48 hours prior to assay had no effect on the cells' ability to produce 1,25-dihydroxy vitamin D₃, whereas after 24 hours in the presence of 5×10^?8$\text{M}$ 1,25-dihydroxyvitamin D₃ the cells produced not this metabolite, but 24,25-dihydroxyvitamin D₃. This cell culture system will allow the investigation of the regulation of renal 25-hydroxyvitamin D₃ metabolism under controlled $\text{in vitro}$ conditions.     

Evidence for aqueous soluble vitamin D-like substances in the calcinogenic plant, Tristetum flavescens

A crude aqueous extract of the leaves of $\text{T. flavescens}$ when administered orally to vitamin D-deficient chicks produced significant increases in plasma phosphate but had little effect on plasma calcium. When chicks, fed a high strontium diet to inhibit endogenous 1,25(OH)₂ vitamin D₃ production and intestinal calcium transport, were dosed with the extract or synthetic 1,25(OH)₂D₃⁴⁵Ca absorption from the duodenum $\text{in vivo}$ was stimulated, whereas vitamin D₃ was ineffective. Partial purification of the crude extract on a Sephadex GH25 column yielded two factors, one of which mimicked 1,25 (OH)₂D₃ activity in chicks fed the high strontium diet while the other produced a significant increase in plasma phosphate. The presence of these substances, together with previously demonstrated organic solvent soluble vitamin D-type activity, may account for the calcinogenic nature of the plant.     

The actions of leukotrienes C4 and D4 in the porcine renal vascular bed

The kidney of anaesthetised pigs was perfused $\text{in situ}$ with carotid arterial blood. Renal blood flow and perfusion pressure were recorded. Close intra-arterial injection of leukotriene (LT) C₄, D₄ or noradrenaline (NA) caused a dose-related increase in vascular resistance. Both LTs were more active than NA by one to two orders of magnitude. Systemically-administered indomethacin potentiated the effect of all three agonists. Incubation of renal artery tissue with calcium ionophore A23187 in the presence of indomethacin resulted in the generation of LT-like material which, when assayed on guinea-pig ileum, was indistinguishable from LTD₄. The results show that pig renal vessels produce LT-like material and suggest that the potent vasoconstriction induced by exogenous NA and LTs is modulated $\text{in vivo}$ by a vasodilator cyclo-oxygenase product.     

Determination of the rates of synthesis and degradation of vitamin D-dependent chick intestinal and renal calcium-binding proteins

Vitamin D₃ and its biologically active metabolite 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] are shown to induce in the chick intestine and kidney the biosynthesis of a calcium binding protein (CaBP). In vitamin D₃-replete chickens raised under adequate dietary calcium (Ca) and phosphorus (P) conditions, the steady-state level of intestinal CaBP (30–50 g/mg protein) is 5- to 20-fold greater than that of renal CaBP. Whereas dietary phosphorus restriction is known to elevate both intestinal and renal CaBP levels, dietary calcium restriction elevates only intestinal CaBP. The present study reports the rates of biosynthesis in vivo and in vitro, and of biodegradation in vivo, of both intestinal and renal CaBP after administration of vitamin D₃ or 1,25(OH)₂D₃ to rachitic chicks. The apparent rate constant of degradation for intestinal CaBP was 0.024 h^?1 ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 29}\text{h}$) and that for renal CaBP was 0.019 h^?1 ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 36}\text{h}$) while total cellular soluble protein in the intestine and kidney had half-lives of 43 and 70 h, respectively. The time course of induction of the synthesis of CaBP was determined in intestine and kidney after administration of a physiological dose of 1,25(OH)₂D₃ to rachitic chicks. Intestinal CaBP synthesis was detectable by 3 hours, reached a maximal rate by 10 hours, and sharply decayed by 16–20 hours. The time course of induction of renal CaBP synthesis was very similar, although the rate of renal CaBP synthesis was readily detectable at the initial time of administration of 1,25(OH)₂D₃. The relative rates of synthesis of CaBP in the intestine and kidney under a variety of dietary Ca and P conditions in the vitamin D₃-replete chick exactly paralleled the steady-state level of CaBP in these two tissues. These results are consistent with a model in which the steady-state levels of intestinal and renal CaBP are solely determined by their respective rates of biosynthesis; the CaBP biosynthetic capability, in turn, is regulated by the availability of 1,25(OH)₂D₃ to each target organ.     

Regulation of 24,25-dihydroxyvitamin D-3 production by 1,25-dihydroxyvitamin D-3 and synthetic human parathyroid hormone fragment 1–34 in a cloned monkey kidney cell line (JTC-12)

Regulation of 25-hydroxyvitamin D-3 24-hydroxylase by 1,25-dihydroxyvitamin D-3 and synthetic human parathyroid hormone fragment 1–34 (PTH_1–34) was investigated using a cloned monkey kidney cell line, JTC-12. Treatment of the cells with 1,25-dihydroxyvitamin D-3 markedly enhanced the conversion of [³H]-25-hydroxyvitamin D-3 into a more polar metabolite. The metabolite was identified as 24,25-dihydroxyvitamin D-3 by normal phase and reverse phase high-performance liquid chromatography and periodate oxidation. The 24-hydroxylae activity appeared to follow Michaelis-Menten kintics, and 1,25-dihydroxyvitamin D-3 treatment increased the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ of 24-hydroxylase from 33 to 95 pmol/h per 10⁶ cells without affecting the apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value of the enzyme (220 nM in control vs. 205 nM in 1,25-dihydroxyvitamin D-3 treated cells). The enzyme activity reached a maximum between 4 and 8 h of treatment with 1,25-dihydroxyvitamin D-3. The dose of 1,25-dihydroxyvitamin D-3 required to cause a half-maximal stimulation was about 3 · 10^?10 M. The 1,25-dihydroxyvitamin D-3-induced increase in 24-hydroxylase was almost completely inhibited by the presence of 1 μM cycloheximide. Treatment of the cells with PTH_1–34 caused a dose-dependent increase in cyclic AMP production. Half-maximal stimulation of cyclic AMP production was obtained at about 5 · 10^?9 M PTH_1–34. When 2.4 · 10^?9 M PTH_1–34 was added after 1,25-dihydroxyvitamin D-3 treatment, the 1,25-dihydroxyvitamin D-3-stimulated 24-hydroxylase was inhibited to $\text{70.7 ± 2.9\%}$ of control. Higher concentrations of PTH_1–34 caused less inhibition of the enzyme activity. When cyclic AMP was added instead of PTH_1–34, the enzyme activity was also suppressed significantly. These results indicate that, in JTC-12 cells, 1,25-dihydroxyvitamin D-3 stimulates 24-hydroxylase in a dose- and time-dependent manner by increasing the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ of the enzyme through a mechanism dependent upon new protein synthesis, and suggest that PTH_1–34 inhibits the 1,25-dihydroxyvitamin D-3-induced stimulation of 24-hydroxylase through its effect on cyclic AMP production.     

On the mechanism of stimulation of H+ transport in gastric mucosa by Ca++ ionophore A23187: I. Pathways for cytosolic calcium increase

The pathways for cytosolic Ca⁺⁺ increase under A23187 stimulation of H⁺ secretion were studied in the isolated gastric mucosa of the toad $\text{Bufo marinus}$. A23187 produced a more potent stimulation of secretion when added to the mucosal side which did not contain calcium. Measurements of ionophore incorporation by fluorometric methods indicated that A23187 incorporates into oxyntic cells intracellularly. The presence of divalent cations inhibited incorporation. This may be the reason for a more potent action when A23187 was added from the mucosal side. With-drawal of calcium from serosal solution largely inhibited the secretory response to A23187 added to the mucosal side. Reintroduction of calcium into the serosal side in the presence of ionophore elicited H⁺ secretion. The results are consistent with an uptake of A23187 from the mucosal side into cellular organelles and basolateral membranes. Calcium entry through the serosal side may be responsible for triggering secretion. Although A23187 likely releases calcium from intracellular stores, its rate of release may not be sufficient to bring about a full stimulation of secretion in serosal-Ca⁺⁺-free conditions.     

Studies on the mode of action of calciferol. X. 24-Nor-25-hydroxyvitamin D3, an analog of 25-hydroxyvitamin D3 having "anti-vitamin" activity.

24-Nor-25-hydroxyvitamin D₃, an analog of 25-hydroxyvitamin D₃, has been chemically synthesized in six steps. This steroid was tested in chicks, $\text{in vivo}$, for its ability to generate the classic vitamin D mediated responses of stimulation of intestinal calcium transport and bone calcium mobilization. Although the 24-nor-25-OH-vitamin D₃ itself exhibited no biological activity in these assays, the analog was found to inhibit the normal responses produced by a physiological dose of vitamin D₃. These results suggest that 24-nor-25-OH-vitamin D₃ may satisfy certain requirements expected of a calciferol “anti-vitamin.”     

Synthesis and bioassay of 3-deoxy-1α-hydroxyvitamin D3, an active analog of 1α,25-dihydroxyvitamin D3

Two synthetic routes to 3-deoxy-1α-hydroxyvitamin D₃, an analog of 1α,25-dihydroxyvitamin D₃, are described. One involved the six-step conversion of 1α,2α-epoxy-6,6-ethylenedioxy-5α-cholestan-3- one to 1α-acetoxycholest-5-ene, whereas, in the second, the same intermediate was prepared from 1α-hydroxycholesterol. Conversion of the Δ⁵-sterol to the required 5,7-diene was accomplished most efficiently via 7-keto and 7-tosylhydrazone intermediates. Bioassay of 3-deoxy-1α-hydroxyvitamin D₃ in the rat establishes that the analog can fulfill all common vitamin D functions including stimulation of intestinal calcium transport, mobilization of calcium and phosphate from bone, stimulation of growth, and calcification of bone. Direct comparison indicates the compound to have $\text{1}\text{20}$ to $\text{1}\text{50}$ of the activity of 1α-hydroxyvitamin D₃, but it acts with a time course indistinguishable from the latter.     

Role of calcium ions in the stimulatory actions of luteinizing hormone in isolated ovarian cells: Studies with divalent-cation ionophores

The divalent-cation ionophores A23187 and ionomycin exert dose-dependent suppressive effects on the stimulatory actions of luteinizing hormone in ovarian cells $\text{in}\text{vitro}$. Micromolar concentrations of both A23187 and ionomycin can inhibit the production of progesterone and the stimulation of ornithine decarboxylase activity. Inhibitory concentrations of these ionophores deplete total cell content of calcium, and also seem to suppress protein synthesis.     

Genome-independent effects of 1,25-dihydroxy vitamin D-3 on membrane potential

Cell membrane potential, $\text{V}{}_{\mathrm{<mtext>m</mtext>}}$, was monitored in rabbit hypertrophic cartilage metatarsals, amphibian proximal tubule and muscle cells during application of 1,25-dihydroxy vitamin D-3, 25-hydroxy vitamin D-3 or cholesterol (10^?10M). 1,25-Dihydroxy vitamin D-3 elicited quick variations of $\text{V}{}_{\mathrm{<mtext>m</mtext>}}$ (in less than 1 min) in proximal tubular cells (whether injected in the lumen or in peritubular capillaries) and in cartilage. The precursor 25-hydroxy vitamin D-3 and cholesterol produced a small shift of $\text{V}{}_{\mathrm{<mtext>m</mtext>}}$ in proximal tubule only when applied from the luminal side, but this change was significantly smaller than that observed with 1,25-dihydroxy vitamin D-3. Muscle cells were unresponsive to both metabolites and cholesterol. It is concluded that rapid effects of 1,25-dihydroxy vitamin D-3 on $\text{V}{}_{\mathrm{<mtext>m</mtext>}}$, in target cells, are specific, most likely due to permeability changes and not related to nuclear protein synthesis; they may contribute to early modulation of cell function.     

The role of calcium in transferrin and iron uptake by reticulocytes

The possible role of calcium in the uptake of transferrin and iron by rabbit reticulocytes was investigated by altering cellular calcium levels through the use of the chelating agents EDTA and $\text{ethyleneglycol-bis}\text{-(3-}\text{aminoethylether}\text{)-N,N′-}\text{tetraacetic}$ acid (EGTA) and the ionophores, A23187 and X537A. Incubation of reticuloyctes with EDTA or EGTA at 4°C had no effect on transferrin and iron uptake but incubation at 37°C resulted in an irreversible inhibition associated with decreased adsorption of transferrin to the cells and evidence of inactivation or loss of the transferrin receptors. Transferrin and iron uptake were also inhibited when the cells were incubated with A23187 or X537A. In the case of A23187 the action was primarily exerted on the temperature-sensitive stage of transferrin uptake and was associated with loss of cellular K⁺ and decrease in cell size. The effect was greater when Ca²⁺ was added to the incubation medium than its absence. X537A produced relatively greater inhibition of iron uptake than of transferrin uptake, associated with a reduction in cellular ATP concentratio. The action of X537A was unaffected by the presence of Ca²⁺ in the incubation medium.The results obtained with EDTA and EGTA indicate that cell membrane Ca²⁺ is required for the integrity or binding of transferrin receptors to the reticulocyte membrane. No evidence was obtained from the experiments with ionophores that an increase of cellular Ca²⁺ affects transferrin and iron uptake directly. The inhibition caused by A23187 was mainly due to a reduction in cell size resulting from increased membrane permeability to K⁺ and that caused by X537A appeared to result from an inhibition of energy metabolism and ATP production.     

Synthesis of an ion-binding peptide (DECYL-2) more selective for calcium than ionophore A23187.

The synthesis of the cation-binding cyclic octapeptide, cyclo(Glu-Sar-Gly-(N-decyl)Gly)₂ is reported. This peptide, containing two ionizable Glu carboxyl side chain protons per molecule, can form neutral cation complexes with divalent ions via protonmetal exchange. Solubilized in chloroform solution, the peptide has been found to extract calcium from an aqueous phase (pH 8.5, 100 mM Tris) generally on a $\text{1}\text{1}$ molar basis. By contrast, under comparable conditions but with other metal chlorides, the peptide does not extract magnesium, sodium, or potassium. That the extraction proceeds via proton-metal exchange was demonstrated by the absence of (radioactive) chloride ion from the organic phase. Parallel sets of experiments performed with the naturally-occurring ionophore A23187 reaffirmed that the latter substance extracts calcium and magnesium with nearly equal propensity.     

Calcium fluxes and intracellular distribution in clonal osteosarcoma cell lines

Calcium efflux patterns were investigated in two clonal osteosarcoma cell lines, ROS $\text{17}\text{2}$ and ROS $\text{2}\text{3}$. Efflux was measured after equilibrating the cells with ⁴⁵calcium in medium containing 1% or 10% serum. The results of ⁴⁵calcium efflux were analyzed by fitting them to a model of three exponential terms, using a computer program based on the non-linear least square method. The results indicated the presence of three exchangeable calcium pools which differ in their rate constants of calcium efflux: a “very fast turnover” (S₁) a “fast turnover” (S₂) and a “slow turnover” (S₃). Washing the labelled cells with sucrose solutions, at pH 7.8 and pH 4, removed most of the calcium localized in S₁, indicating that this calcium is membrane bound. The parameters of calcium efflux in ROS cells were found to be different from those measured previously in cultured bone cells: (1) There was no difference between efflux patterns in cells incubated in medium containing 1% and 10% serum; (2) Rates of calcium fluxes were much lower in ROS cells than those in bone cells; and (3) The amount of calcium in S₃ was very small.     

Viomycin resistance: alterations of either ribosomal subunit affect the binding of the antibiotic to the pair subunit and the entire ribosome becomes resistant to the drug.

Subcellular localization of $\text{[}{}^3\text{H]1α,24(R)-dihydroxyvitamin}$ D₃ and $\text{[}{}^3\text{H]1α,24(S)-dihydroxyvitamin}$ D₃ in rat intestinal mucosa was investigated in comparison with the $\text{[}{}^3\text{H]1α-hydroxyvitamin}$ D₃. The 24(R) and 24(S) isomers of 1α,24-dihydroxyvitamin D₃ were gradually transformed to 1α,24(R)25-trihydroxyvitamin D₃ and 1α,24(S)25-trihydroxyvitamin D₃, and the plasma concentrations of these metabolites were 10.30 and 1.36 pmol/ml, respectively. The major portions of the administered compounds distributed in the nuclear fraction of the intestinal mucosa remained unchanged, and the amounts of 1α,24(R)-dihydroxyvitamin D₃ and 1α,24(S)-dihydroxyvitamin D₃ were 4.25 and 0.306 pmol/g intestinal mucosa, respectively. No detectable amount of the metabolites, 1α,24(R)25-trihydroxyvitamin D₃ and 1α,24(S)25-trihydroxyvitamin D₃ were found in the same nuclear fractions. In the case with the $\text{[}{}^3\text{H]1α-hydroxyvitamin}$ D₃, however, the compound was rapidly metabolized to 1α,25-dihydroxyvitamin D₃.The metabolite, 1α,25-dihydroxyvitamin D₃, was seen in the nuclear fraction of the intestinal mucosa at a concentration of 2.44 pmol/g intestinal mucosa.     

Effects of lighting programs on onset of the ovulatory season in mares

Using 240 pony mares, lighting regimens were tested for their efficiency in hastening the onset of the ovulatory season. The mean number of days from January 1 to first ovulation was used as the end point. No advantage was gained by beginning a fixed lighting regimen ($\text{15.5L}\text{8.5D}$, hours light/hours dark) November 1 (66 ±8) versus December 1 (65 ±9), but beginning on January 1 was less efficient (98 ±8; controls, 132 ±5; P<0.05). In another experiment, daily three-hour interruptions of either the light phase (67 ±10) or the dark phase (71 ±11) did not significantly retard the effectiveness of a fixed regimen of $\text{15L}\text{9D}$ (54 ±5; controls, 142 ±6). A $\text{15L}\text{9D}$ regimen every other day (natural day length on alternate days) resulted in an interval (85 ±7) that was shorter (P<0.05) than for the controls and longer (not significant) than for the daily $\text{15L}\text{9D}$ regimen. When used with natural day length, a one-hour pulse of light in the evening (15 hours after sunrise) was not effective (141 ±6); a one-hour pulse in the morning 9.5 hours after sunset) was only partially effective (117 ±6). In another experiment, the interval was reduced (P<0.05) in a group with one hour of light fixed at 4:00 a.m. with natural day length (85 ±8; $\text{15L}\text{9D}$, 75 ±7; controls, 126 ±9). Results indicated that a fixed one-hour pulse of light at 4 a.m., used with natural day length, may provide an acceptable level of stimulation.     

Activities of enzymes that metabolize platelet-activating factor (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) in neutrophils and eosinophils from humans and the effect of a calcium ionophore

Enzymatic systems in human blood cells are described for the activation and inactivation of a biologically active phospholipid (1-alkyl-2-acetyl-$\text{sn}$-glycero-3-phosphocholine) with hypotensive, platelet-aggregating, and inflammatory properties. The results document the presence of alkyldihydroxyacetone-phosphate synthase (forms the $\text{O}$-alkyl linkage in lipids), 1-alkyl-2-lyso-$\text{sn}$-glycero-3-phosphocholine:acetyl-CoA acetyltransferase (produces the biologically active molecule), and 1-alkyl-2-acetyl-$\text{sn}$-glycero-3-phosphocholine: acetylhydrolase (destroys the biological activity) in human neutrophils and eosinophils. Both the acetyltransferase and acetylhydrolase activities are increased severalfold after treatment of normal neutrophils with ionophore A23187; however, alkyldihydroxyacetone-phosphate synthase activity is not influenced by the ionophore. Eosinophils isolated from patients with eosinophilia have significantly greater activities of all the enzymes studied than the eosinophils isolated from normal individuals. Our results indicate the acetyltransferase responsible for 1-alkyl-2-acetyl-$\text{sn}$-glycero-3-phosphocholine synthesis may serve an important role in human blood cells that release this biologically active phospholipid. Moreover, the acetyltransferase activity was found to be dramatically influenced by calcium flux.     

The site of 1α,25-dihydroxyvitamin D3 production in pregnancy

Metabolism of 25-hydroxyvitamin D₃ (25-OH-D₃) in pregnancy was investigated $\text{in}\text{vitro}$ in New Zealand White rabbits fed a rabbit chow. Kidney homogenates from pregnant mothers and fetuses were separately incubated with [³H]-25-OH-D₃. The homogenates from fetuses produced significant amounts of $\text{[}{}^3\text{H]-1α,25-dihydroxyvitamin}$ D₃ [1α,25-(OH)₂-D₃] from its precursor, while those from mothers predominantly produced [³H]-24,25-dihydroxyvitamin D₃ [24,25-(OH)₂-D₃]. The identity of the radioactive metabolites produced from [³H]-25-OH-D₃ was established by periodate cleavage and comigration with synthetic 1α,25-(OH)₂-D₃ or 24,25-(OH)₂-D₃ on high pressure liquid chromatography. These results clearly indicate that the fetal kidney is at least one of the sites of 1α,25-(OH)₂-D₃ synthesis in pregnancy.