Biochemical and functional responses stimulated by platelet-activating factor in murine peritoneal macrophages.

Platelet-activating factor (PAF) is a potent stimulant of leukocytes, including macrophages. To analyze the mechanisms of its effects upon macrophages, we determined whether macrophages bear specific surface receptors for PAF. By competitive radioactive binding assays, we determined two classes of specific receptors to be present on purified membranes derived from murine peritoneal macrophages (one having a Kd of approximately 1 X 10(-10) M and one a Kd of approximately 2 X 10(-9) M). When the macrophages were incubated with PAF, rapid formation of several inositol phosphates including inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate were observed. PAF also elevated intracellular levels of calcium to 290 +/- 27% of basal levels which were 82.7 +/- 12 nM. Increases in calcium were observed first in submembranous areas of the macrophages. PAF also led to increases of 1,2-diacylglycerol of approximately 200 pmol/10(7) cells. A characteristic pattern of enhanced protein phosphorylation, similar to that initiated by both phorbol 12,13-myristate and lipopolysaccharide, was observed and involved enhanced phosphorylation of proteins of 28, 33, 67, and 103 kD. The half-maximal dose of PAF for initiating all the above effects was approximately 5 X 10(-9) M. PAF also initiated significant chemotaxis of the cells; the half-maximal dose for this effect was approximately 1 X 10(-11) M. Taken together, these observations suggest that murine mononuclear phagocytes bear specific membrane receptors for PAF and that addition of PAF leads to generation of break-down products of polyphosphoinositides, subsequent changes in intracellular calcium and protein phosphorylation, and chemotaxis.

Role of Na+/H+ exchange by interferon-gamma in enhanced expression of JE and I-A beta genes.

The rapid transductional sequences initiated by interferon-gamma (IFN-gamma) on binding to its receptor regulate functional and genomic responses in many cells but are not well defined. Induction of macrophage activation is an example of such functional and genomic changes in response to IFN-gamma. Addition of IFN-gamma to murine macrophages, at activating concentrations, produced rapid (within 60 seconds) alkalinization of the cytosol and a concomitant, rapid influx of 22Na+. Amiloride inhibited the ion fluxes and the accumulation of specific messenger RNA for two genes induced by IFN-gamma (the early gene JE and the beta chain of the class II major histocompatibility complex gene I-A). The data indicate that IFN-gamma initiates rapid exchange of Na+ and H+ by means of the Na+/H+ antiporter and that these amiloride-sensitive ion fluxes are important to some of the genomic effects of IFN-gamma.

Differentiation of stem cells in the dental follicle.

The dental follicle (DF) differentiates into the periodontal ligament. In addition, it may be the precursor of other cells of the periodontium, including osteoblasts and cementoblasts. We hypothesized that stem cells may be present in the DF and be capable of differentiating into cells of the periodontium. Stem cells were identified in the DF of the rat first mandibular molar by Hoechst staining, alkaline phosphatase staining, and expression of side-population stem cell markers. These cells were shown to be able to differentiate into osteoblasts/cementoblasts, adipocytes, and neurons. Treating the DF cell population with doxorubicin, followed by incubation in an adipogenesis medium, suggested that the adipocytes originated from stem cells. Thus, a possibly puripotent stem cell population is present in the rat DF.

Bacterial toxins affect early events of T lymphocyte activation.

The effects of pertussis toxin and cholera toxin on early events of T lymphocyte activation were examined in the T lymphocyte cell line, Jurkat. Pertussis toxin treatment of these T cells increased inositol phosphates production and led to increases in intracellular free calcium concentration. These effects were produced by the isolated B (binding) subunit of pertussis toxin, alone. Inositol phosphates production resulting from perturbation of the T cell antigen receptor-CD3 complex by MAb was not affected by pertussis toxin treatment but was markedly inhibited by cholera toxin. This effect of cholera toxin paralleled elevations in cAMP content. However, forskolin, in concentrations equipotent for cAMP production, was a weaker inhibitor of inositol phosphates production. Cholera toxin inhibition of inositol phosphates production did not result from inhibition of baseline incorporation of inositol into phosphoinositide substrates of phospholipase C. These studies underline the complexity of toxin effects on cellular systems and suggest that other approaches will be required to implicate guanine nucleotide-binding regulatory proteins in control of the early events of T lymphocyte activation. However, the data presented here provide a molecular basis for the clinical observations of lymphocytosis and the in vitro observations of lymphocyte mitogenesis after pertussis toxin stimulation.

Correlation between erythromycin and acid phosphatase in mouse liver.

1. Whole liver homogenates obtained from mice 1h after an intraperitoneal injection of erythromycin lactobionate (343 mg/kg, 1/3 LD 50) were fractionated into nuclear (7000 times g min), mitochondrial (33000 times g min) and lysosomr-rich (250 000 times g min) fractions. 2. The resulting fractions, as well as the final supernatant, were analyzed for erythromycin, acid phosphatase and protein. 3. The highest relative specific activities (per cent total protein) of erythromycin and of acid phosphatase were exhibited by the lysosome-rich fraction. 4. It was of interest, therefore, to examine the effects of erythromycin upon the free activity of acid phosphatase in soluble form and on its in vitro and in vivo release from liver lysosomes. 5. Concentrations of 1.7 - 10-4 M, 3.4 - 10-4 M, 6.8 - 10-4 M and 13. 6 - 10-4 M of erythromycin lactobionate had no significant effect upon the free activity of acid phosphatase in soluble form but retarded the release of this enzyme from the liver lysosome-rich preparation. This effect of erythromycin lactobionate was dose- and time-dependent. 6. Treatment of mice with erythromycin lactobionate (343 mg/kg, 1/3 LD 50) iwtraperitoneally for 7 days significantly decreased the unsedimentable acid phosphatase activity expressed as per cent of total activity in whole liver homogenates. This indicated an in vivo diminished release of acid phosphatase from liver lysosomes by erythromycin. 7. Since erythromycin lactobionate is ionisable it could be possible that erythromycin basis as many other cationic molecules accumulates in lysosomes. 8. The in vitro and in vivo diminished release of acid phosphatase may suggest that erythromycin decreases permeability of lysosomal membrane.

Differential regulation of receptor-stimulated cyclic adenosine monophosphate production by polyvalent cations in MC3T3-E1 osteoblasts.

Extracellular cations have paradoxical trophic and toxic effects on osteoblast function. In an effort to explain these divergent actions, we investigated in MC3T3-E1 osteoblasts if polyvalent cations differentially modulate the agonist-stimulated cyclic adenosine monophosphate (cAMP) pathway, an important regulator of osteoblastic function. We found that a panel of cations, including gadolinium, aluminum, calcium, and neomycin, inhibited prostaglandin E1 (PGE)-stimulated cAMP accumulation but paradoxically potentiated parathyroid hormone (PTH)-stimulated cAMP production. In contrast, these cations had no effect on forskolin- or cholera toxin-induced increases in cAMP, suggesting actions proximal to adenylate cyclase and possible modulation of receptor interactions with G proteins. Phorbol 12-myristate 13-acetated (PMA) mimicked the effects of cations on PGE1- and PTH-stimulated cAMP accumulation in MC3T3-E1 cells, respectively, diminishing and augmenting the responses. Moreover, down-regulation of protein kinase C (PKC) by overnight treatment with PMA prevented gadolinium (Gd3+) from attenuating PGE1- and enhancing PTH-stimulated cAMP production, indicating involvement of PKC-dependent pathways. Cations, however, activated signal transduction pathways not coupled to phosphatidylinositol-specific phospholipase C (PI-PLC), since there was no corresponding increase in inositol phosphate formation or intracellular calcium concentrations. In addition, pertussis toxin treatment failed to prevent Gd(3+)-mediated suppression of PGE1-stimulated cAMP, suggesting actions independent of Gm. Thus, polyvalent cations may either stimulate or inhibit hormone-mediated cAMP accumulation in osteoblasts. These differential actions provide a potential explanation for the paradoxical trophic and toxic effects of cations on osteoblast function that occur in vivo under different hormonal conditions.

Age-dependent changes in beta-adrenergic receptor subtypes and adenylyl cyclase activation in adipocytes from Fischer 344 rats.

Epididymal adipocytes were isolated from Fischer 344 rats aged 3, 6, 12, and 24 months, to study the mechanisms responsible for age-dependent diminution in cellular adrenergic responsiveness. Messenger RNA (mRNA) levels for the beta 1-, beta 2-, and beta 3-adrenergic receptors (ARs) were compared across age groups and related to adenylyl cyclase activation by selective receptor agonists in adipocyte plasma membranes and activation of lipolysis in intact cells. mRNA levels for the beta 1-AR decreased by 60% between 3-6 months and remained at this reduced level through 12 and 24 months. A modest increase in beta 2-AR mRNA was noted between 3-12 months, but decreased between 12-24 months to levels seen in the 3-month-old group. mRNA for the beta 3-AR did not change between 3-6 months, but decreased by about 40% between 6-12 months, and by a further 50% between 12-24 months. Lipolytic responsiveness also diminished with age, and regardless of whether beta 3-selective or beta 1/beta 2-selective agonists were used, the maximal release of glycerol was most severely blunted in adipocytes from 24-month-old rats. The age-dependent changes in adenylyl cyclase activation by beta-adrenergic agonists mirrored the observed changes in lipolytic responsiveness with respect to diminished efficacy. These results together with the similar forskolin-stimulated adenylyl cyclase activity among the groups suggest age-dependent changes in activation of adenylyl cyclase at a prior step. This suggestion is also supported by the comparable inhibitory capacities of the alpha 2-adrenergic and A1-adenosine signaling systems among the age groups. In view of the similar levels of Gs alpha, the age-dependent decrease in adrenergic responsiveness in rat adipocytes appears to result primarily from specific decreases in the expression of both beta 3-AR and beta 1-ARs.

Regulation of a liver plasma membrane phosphoinositide phosphodiesterase by guanine nucleotides and calcium.

Rat liver plasma membranes are enriched in a Ca2+-dependent phosphodiesterase active on phosphatidylinositol 4,5-P2 and phosphatidylinositol 4-P, but not phosphatidylinositol. Inositol-P3 is the first product of the reaction, but is rapidly degraded. Micromolar concentrations of GTP and its nonhydrolyzable analogues stimulate the reaction, whereas GDP, GMP and other nucleoside triphosphates are inactive. GTP and its analogues decrease the requirement of the reaction for Ca2+ and also increase its activity at saturating Ca2+. These results support the hypothesis that guanine nucleotides and a guanine nucleotide binding regulatory protein are involved in coupling the receptors for Ca2+-mediated agonists to the breakdown of plasma membrane phosphatidylinositol 4,5-P2.

Inhibitors of ATP-sensitive potassium channels stimulate intestinal cholecystokinin secretion.

Recently, a role for adenosine 5'-triphosphate(ATP)-sensitive potassium channels in the regulation of cholecystokinin (CCK) secretion has been described in STC-1 cells, an intestinal CCK-secreting cell line. To examine whether a similar mechanism might participate in the regulation of hormone secretion from native CCK cells, the effects of two established inhibitors of ATP-sensitive potassium channels (e.g. glucose, disopyramide) were examined on CCK release from dispersed murine intestinal cells. Both glucose and disopyramide were found to stimulate CCK secretion. Furthermore, CCK release induced by glucose was inhibited by the calcium channel blocker diltiazem. It is concluded that, ATP-sensitive potassium channels may play a role in the regulation of intestinal CCK secretion.

Maturation in liver mitochondria of Ruthenium Red-sensitive calcium-ion-transport activity and the influence of glucagon administration in vivo and in utero.

The maturation of Ca(2+) transport in mitochondria isolated from rat liver was examined, from 5 days before birth. The mitochondria used were isolated from liver homogenates by centrifugation at 22000g-min. Ca(2+) transport by mitochondria isolated from foetal liver is energy-dependent and Ruthenium Red-sensitive. The transmembrane pH gradient in these mitochondria is higher by about 7mV and the membrane potential lower by about 20mV than in adult mitochondria. The inclusion of 2mm-P(i) in the incubation medium enhances the protonmotive force by approx. 30mV. The rate of Ca(2+) influx in foetal mitochondria measured in buffered KCl plus succinate is low until about 2-3h after birth, when it increases to about 60% of adult values; approx. 24h later it has reached near-adult values. Higher rates of Ca(2+) influx are observed in the presence of 2mm-P(i); 3-5 days before birth the rates are about one-third of adult values and decline slightly as birth approaches. By 2-3h post partum they have reached adult values. The inclusion of 12.5mum-MgATP with the P(i) stimulates further the initial rate of Ca(2+) influx in foetal mitochondria. The rates observed are constant over the prenatal period examined and are 50-60% of those observed in adult mitochondria. Mitochondria isolated from foetal livers 4-5 days before birth retain the accumulated Ca(2+) for about 50min in the presence of 2mm-P(i). In the period 2 days before birth to birth, this ability is largely lost, but by 2-3h after birth Ca(2+) retention is similar to that of adult mitochondria. The presence of 12.5mum-MgATP progressively enhances the Ca(2+) retention time as development proceeds until 2-3h after birth, when it becomes less sensitive to added MgATP. Glucagon administration to older foetuses in utero enhances both the rate of mitochondrial Ca(2+) influx assayed in the presence of 2mm-P(i) and the time for which mitochondria retain accumulated Ca(2+) in the presence of 12.5mum-MgATP and 2mm-P(i). Its administration to neonatal animals leads to an increase in mitochondrial Ca(2+) retention similar to that seen in adult mitochondria. The data provide evidence that the Ruthenium Red-sensitive Ca(2+) transporter is potentially as active in foetal mitochondria 5 days before birth as it is in adult mitochondria. They also show that foetal mitochondria have an ability to retain accumulated Ca(2+) reminiscent of mitochondria from tumour cells and from hormone-challenged rat liver.

Stable enhancement of calcium retention in mitochondria isolated from rat liver after the administration of glucagon to the intact animal.

1. Mitochondria isolated from rat liver by centrifugation of the homogenate in buffered iso-osmotic sucrose at between 4000 and 8000g-min, 1h after the administration in vivo of 30mug of glucagon/100g body wt., retain Ca(2+) for over 45min after its addition at 100nmol/mg of mitochondrial protein in the presence of 2mm-P(i). In similar experiments, but after the administration of saline (0.9% NaCl) in place of glucagon, Ca(2+) is retained for 6-8min. The ability of glucagon to enhance Ca(2+) retention is completely prevented by co-administration of 4.2mg of puromycin/100g body wt. 2. The resting rate of respiration after Ca(2+) accumulation by mitochondria from glucagon-treated rats remains low by contrast with that from saline-treated rats. Respiration in the latter mitochondria increased markedly after the Ca(2+) accumulation, reflecting the uncoupling action of the ion. 3. Concomitant with the enhanced retention of Ca(2+) and low rates of resting respiration by mitochondria from glucagon-treated rats was an increased ability to retain endogenous adenine nucleotides. 4. An investigation of properties of mitochondria known to influence Ca(2+) transport revealed a significantly higher concentration of adenine nucleotides but not of P(i) in those from glucagon-treated rats. The membrane potential remained unchanged, but the transmembrane pH gradient increased by approx. 10mV, indicating increased alkalinity of the matrix space. 5. Depletion of endogenous adenine nucleotides by P(i) treatment in mitochondria from both glucagon-treated and saline-treated rats led to a marked diminution in ability to retain Ca(2+). The activity of the adenine nucleotide translocase was unaffected by glucagon treatment of rats in vivo. 6. Although the data are consistent with the argument that the Ca(2+)-translocation cycle in rat liver mitochondria is a target for glucagon action in vivo, they do not permit conclusions to be drawn about the molecular mechanisms involved in the glucagon-induced alteration to this cycle.

Separation and assay of phosphodiesterase isoforms in murine peritoneal macrophages using membrane matrix DEAE chromatography and [32P]cAMP.

Cyclic nucleotide phosphodiesterases (PDE) are a family of exquisitely regulated enzymes which play a central role in regulating the biological half-life of cAMP and cGMP. Hormonal regulation of specific isoforms is the basis for crosstalk and antagonism between several well-described signaling pathways. In the present work improved methods are described which accelerate and simplify the separation and assay of PDE isoforms occurring in mouse peritoneal macrophages. The described method is equally applicable to other cell and tissue types, and is based on the mobilization of DEAE beads on a macroporous support structure which allows high flow rates, high resolution, and reproducible separations. The failure of the resin to undergo compression in this configuration prevents band spreading caused by diffusion into small pores and channeling associated with bed movement. Therefore, the PDE isoforms from a tissue extract can be resolved in a chromatographic run taking only 30-35 min. In addition, the column cartridge can be regenerated and fully reequilibrated in 10-15 min. After each chromatographic run, cAMP PDE activity in the fractions is characterized by incubating each fraction with [32P]cAMP, followed by quantitative conversion of [32P]AMP formed in the initial reaction to 32PO4 and adenosine. The unreacted [32P]-cAMP is then adsorbed onto charcoal and after centrifugation, the 32PO4 remaining in the supernatant is determined by counting an aliquot. The latter step replaces the need to use small columns to separate tritiated cAMP from tritiated adenosine as in previous assays, and provides the expected improvement in sensitivity associated with a high specific activity substrate.(ABSTRACT TRUNCATED AT 250 WORDS)

Vasopressin-, angiotensin II-, and alpha 1-adrenergic-induced inhibition of Ca2+ transport by rat liver plasma membrane vesicles.

A rapid method for isolating highly purified rat liver plasma membrane vesicles using isotonic medium and Percoll self-forming gradient centrifugation is described. The vesicles were characterized by enzyme markers and electron microscopy. The method also yielded a fraction rich in nuclei. The vesicles transported Ca2+ in an ATP-dependent manner and this was enhanced by oxalate. The Vmax for Ca2+ uptake was 0.65 +/- 0.08 nmol/mg X min, which was approximately 18-fold higher than for other liver plasma membrane preparations, and the Km for Ca2+ was 5.2 +/- 0.4 nM. Calcium uptake was inhibited by 40-50% in vesicles isolated from rat livers perfused for 3 min with 10(-7)M vasopressin. The half-maximally effective concentration of vasopressin was 5 X 10(-10)M which correlates with that for raising cytosolic Ca2+ and phosphorylase a. Inhibition was not significant in vesicles from livers perfused with vasopressin for only 1 min, indicating that inhibition of the Ca2+ pump may not be involved in the rise in cytosolic Ca2+ observed at 1-2 s with this hormone. Epinephrine (10(-5)M) and angiotensin II (10(-7)M) inhibited Ca2+ uptake by 31 +/- 10 and 26 +/- 5%, respectively, at 3 min. Glucagon (10(-7)M) had no effect. It is proposed that the inhibitory action of the Ca2+-dependent hormones on the plasma membrane Ca2+ pump plays an important role in the actions of these hormones by prolonging the elevation in cytosolic Ca2+.