Calcium-mediated effects of calcitonin on cyclic AMP formation and lymphoblast proliferation in thymocyte populations exposed to prostaglandin E 1

The calcitonin (SCT) from salmon ultimobranchial bodies which (like mammalian calcitonins) lowers the plasma calcium concentration in mammals can also affect cyclic AMP (cyclic adenosine 3′,5′-monophosphate) metabolism and proliferation of lymphoblasts in normal and prostaglandin E₁ (PGE₁)-treated rat thymocyte populations in three different ways. In the first case, low concentrations (0.5–5.0 ng per milliliter) of SCT lower (by a calcium-mediated process) the ability of PGE₁ to transiently increase cyclic AMP synthesis, but the reduced surge of cyclic AMP production is still ample to stimulate lymphoblasts in the cell population to initiate deoxyribonucleic acid (DNA) synthesis. Secondly, these low SCT concentrations affect the eventual progression of the PGE₁-stimulated, DNA-synthesizing lymphoblasts into mitosis by a calcium-mediated process. Depending on the extracellular calcium concentration and the magnitude of the initial increment in the intracellular cyclic AMP content, SCT can either promote or inhibit the progression of the stimulated cells into mitosis. SCT's third action is a rapid (within 5 minutes), calcium-independent elevation of the cellular cyclic AMP content in otherwise untreated thymic lymphocyte populations exposed to a very high concentration (100 ng per milliliter) of the hormone. This early, transient rise in the cyclic AMP level is followed by a calcium-dependent increase in lymphoblast proliferation. An attempt is made to interrelate and explain the different actions of SCT on cyclic AMP metabolism and mitogenesis.     

Cyclic AMP-induced inhibition of collagen lattice contraction by fibroblasts may be attenuated by both cyclic AMP dependent and independent mechanisms

The contraction of collagen lattices made with forskin fibroblasts in medium containing 1% fetal bovine serum was inhibited by intracelluar cyclic AMP raising drugs including cholera toxin (CT), forskolin, and dibutyryl-cAMP. The inhibition by CT was attenuated by insulin, acidic fibroblast growth factor (aFGF), and transforming growth factor-β (TGF-β). All three peptide factors have previously been reported to promote collagen lattice contraction by arterial smooth muscle cells and/or fibroblasts. Incubation of cells suspended in collagen gels with CT and forskolin resulted in a transient rise of the intracellular cyclic AMP levels, which peaked at 2 hr and 30 min, respectively, after drug exposure. Cholera toxin-induced intracellular cyclic AMP increase was attenuated by TGF-β, but not by aFGF and insulin, when added simultaneously. Thus, TGF-β may attenuate CT's inhibition on collagen lattice contraction by attenuating CTinduced intracellualr cyclic AMP increse, whereas the attenuation by insulin and aFGF on the inhibition of lattice contraction may be mediated by a cyclic AMPindependent mechannism. © 1993 Wiley-Liss, Inc.     

Role of multipolar mitoses in the proliferation of multinucleate cells induced by cytochalasin B

A prolonged action of cytochalasin B results in the formation of numerous multipolar mitoses (26%) in Chinese hamster cell cultures. The transition to multipolar mitoses in the presence of cytochalasin B is not accompanied by K-mitotic delay. It is shown that a multipolar mitosis without cytoplasmic division is one of the main causes of multinucleation development in cytochalasin B-treated cultures. After stopping the drug action the cytochalasin B-induced multinucleate cells continue to divide by multipolar mitosis. In this case it completes with cytokinesis and, probably, leads to a decrease in the number of nuclei per cell. The origin of multipolar mitotic apparatus after the action of cytochalasin B is discussed in addition to the role of multipolar mitosis in formation and proliferation of multinucleate cells.     

The role of calcium in follicle-stimulating hormone signal transduction in Sertoli cells.

Sertoli cells are hormonally regulated by follicle-stimulating hormone (FSH) acting upon a G-protein-linked cell surface FSH receptor. FSH increases intracellular cyclic AMP but the involvement of other signal transduction mechanisms including intracellular calcium in FSH action are not proven. Using freshly isolated rat Sertoli cells we measured cytosolic free ionized calcium levels by dual-wavelength fluorescence spectrophotometry using the calcium-sensitive fluorescent dye Fura2-AM. The cytosolic calcium concentration in unstimulated Sertoli cells was 89 +/- 2 nM (n = 151 experiments) and was markedly increased by either calcium channel ionophores (ionomycin, Bay K8644) or plasma membrane depolarization consistent with the presence of voltage-sensitive and -independent calcium channel in Sertoli cell membranes. Ovine FSH stimulated a specific, sensitive (ED50, 5.0 ng of S-16/ml), and dose-dependent (maximal at 20 ng/ml) rise in cytosolic calcium commencing within 60 s to reach levels of 192 +/- 31 nM after 180 s and lasting for at least 10 min. The effect of FSH was replicated by forskolin, cholera toxin, and dibutyryl cyclic AMP, suggesting that cyclic AMP may mediate the FSH-induced rise in cytosolic calcium. The FSH-induced rise in cytosolic calcium required extracellular calcium and was abolished by calcium channel blockers specific for dihydropyridine (verapamil, nicardipine), nonvoltage-gated (ruthenium red) or all calcium channels (cobalt). Thus FSH action on Sertoli cells involves a specific, rapid, and sustained increase in cytosolic calcium which requires extracellular calcium and involves both dihydropyridine-sensitive, voltage-gated calcium channels and voltage-independent, receptor-gated calcium channels in the plasma membranes of rat Sertoli cells. The replication by cyclic AMP of the effects of FSH suggests that calcium may be a signal-amplification or -modulating mechanism rather than an alternate primary signal transduction system for FSH in Sertoli cells.     

Calcium Independence of Phosphoinositide Hydrolysis-Induced Increase in Cyclic AMP Accumulation in SK-N-SH Human Neuroblastoma Cells

Previous work has shown that stimulation of muscarinic receptors in various cell lines increases intracellular cyclic AMP (cAMP) levels. This unusual response has been hypothesized to be mediated by stimulation of calcium/calmodulin-sensitive adenylate cyclase, secondary to inositol trisphosphate (IP3)-mediated calcium mobilization. To test this hypothesis, we stimulated muscarinic receptors in SK-N-SH human neuroblastoma cells while blocking the IP3-mediated rise in intracellular calcium concentration using two different methods. Loading cells with the intracellular calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) abolished the carbachol-mediated intracellular calcium release without abolishing the carbachol-mediated increase in cAMP level. Similarly, in cells preexposed to carbachol, the agonist-induced change in intracellular calcium level was blocked, but the cAMP response was not. Thus, both of these methods failed to block the muscarinic receptor-mediated increase in cAMP level, thereby demonstrating that this cAMP level increase is not mediated by a detectable rise in intracellular calcium concentration.     

The influence of calcium on the cyclic AMP-mediated stimulation of DNA synthesis and cell proliferation by prostaglandin E 1

Prostaglandin type E₁ (PGE₁) rapidly stimulates cyclic AMP formation and the initiation of deoxyribonucleic acid (DNA) synthesis in rat thymic lymphocytes suspended in vitro by reactions which are not affected by wide variations in the extracellular calcium concentration. On the other hand, the operation of the associated reaction(s) responsible for the subsequent progression of the stimulated cells into mitosis is profoundly affected by the extracellular calcium level. If the maximum intracellular cyclic AMP concentration is in the lower range of stimulatory values (e.g., 150 × 10^?8 picomoles per cell as produced by an exposure to 0.5 μg of PGE₁ per milliliter of medium), an extracellular calcium concentration of 0.5 to 1.0 mM is needed to obtain maximum cell proliferation, but not the maximum stimulation of DNA synthesis. Contrariwise, if the cellular cyclic AMP content is raised to a much higher level (260 × 10^?8 picomoles per cell) by exposure to a greater PGE₁ concentration (5.0 μg per millilter), cell proliferation is maximally stimulated in calcium-free medium and increasing the extracellular calcium concntration above 0.2 mM actually prevents the stimulation of cell proliferation (but does not affect the stimulation of DNA synthesis). Thus, the ultimate translation of PGE₁'s early cyclic AMP-mediated reactions into increased cell proliferation is determined by both the intracellular cyclic AMP level and the extracellular calcium concentration.     

Influence of epidermal growth factor and cyclic AMP on growth and differentiation of palatal epithelial cells in culture

A serum-free, hormonally defined medium was developed which supports growth and differentiation in primary culture of epithelial cells from prefusion embryonic mouse palatal shelves. Using this culture system, medial epithelial programmed cell death was investigated. In the absence of EGF, medial epithelial cells undergo cell death and detach from the substratum by 24 hr of culture. The addition of EGF alone or in combination with various agents which increase intracellular cyclic AMP levels prevented medial epithelial cell death in both cell and organ culture. EGF appeared to exert its most dramatic effect in cell culture on growth and differentiation of the squamous oral epithelial cells. In addition, EGF and agents such as 8-bromo-cyclic AMP, dibutyryl cyclic AMP, or cholera toxin synergistically stimulated the appearance of a long-lived, rapidly proliferating cell type by Day 4 of culture. Our results suggest that both EGF and cyclic AMP together may be important in regulating proliferation of embryonic palatal epithelial cells.     

The effects of corticotrophin (ACTH1-24), cyclic AMP and TPA (12-O-tetradecanoyl phorbol-13-acetate) on DNA replication and proliferation of primary rabbit adrenocortical cells in a synthetic medium

ACTH1-24 stimulated the parenchymal cells in cultures of rat adrenal cortex in serum-free synthetic HiWoBa 2000 medium to replicate DNA, enter mitosis and divide. But ACTH's principal mediator, cyclic AMP, was not a complete mitogen: the adenylate cyclase-stimulating cholera toxin and dibutyryl cyclic AMP stimulated parenchymal cells to replicate DNA but not to enter mitosis. Thus, there must have been an additional mediator of the response to ACTH1-24 that enabled the parenchymal cells to enter mitosis. This additional mediator might have been protein kinase C because a protein kinase C activator and cyclic AMP elevator, TPA, stimulated the adrenocortical parenchymal cells to replicate DNA, enter mitosis and divide.     

Requirement and role of arachidonic acid in the differentiation of pre-adipose cells.

The terminal adipose differentiation of Ob1771 cells, characterized by glycerol-3-phosphate dehydrogenase activity and triacylglycerol accumulation, was studied in serum-free hormone-supplemented medium containing growth hormone, tri-iodothyronine, insulin, transferrin and fetuin. Arachidonic acid was able to substitute for a crude adipogenic fraction isolated from fetal bovine serum but not for growth hormone or tri-iodothyronine. Arachidonic acid was also able to increase in a rapid and dramatic manner cyclic AMP production; moreover it was able to amplify the adipose conversion promoted by other agents elevating cyclic AMP concentrations and to induce inositol phospholipid breakdown. Both phorbol 12-myristate 13-acetate, a protein kinase C activator and ionomycin, a Ca2+-mobilizing agent, showed potent synergy with agents elevating cyclic AMP concentrations for the promotion of adipose conversion, whereas 8-bromo cyclic GMP and 4 alpha-phorbol 12,13-dibutyrate were ineffective. The triggering of both the cyclic AMP and inositol phospholipid pathways was accompanied by a single round of cell division, and within a few days all the cells became differentiated. Similar results were obtained, after exposure to arachidonic acid, with preadipose 3T3-F442A cells and with rat adipose precursor cells in primary culture. The availability of arachidonic acid from intracellular stores and/or of exogenous origin should play a major role for the onset of critical mitoses leading to terminal differentiation in pre-adipose cells.     

CELL CLASSIFICATION AND KINETIC ASPECTS OF NORMOBLASTIC AND MEGALOBLASTIC ERYTHROPOIESIS

Mitotic indices and ³H-thymidine flash labelling indices have been determined in a total of 6000 erythroid cells from patients with megaloblastic anaemia (vitamin B₁₂ deficiency) and 4000 erythroid cells from patients with increased, normoblastic erythropoiesis. In the anaemic states there is a lack of mitoses in the more mature erythroid compartments relative to the number of mitoses in the early erythroid precursors; this must reflect skipped division and/or cell death in the later precursors. In order to further locate the deficit of mitoses, erythropoietic cells were subdivided in a way which aimed at stratifying them according to cell generations. It appears that there are four consecutive generations of recognizable proliferating red cell precursors. Balance considerations of mitotic figures suggest that in stressed normopoiesis all cells which enter generation III divide, whereas only about one-half of cells leaving generation III divide again in generation IV. In megaloblastic erythropoiesis it appears that only about one of three cells which leave generation III divide in generation IV. The data suggest that in megaloblastic anaemia, skipped division and/or cell death to a large extent take place in generation IV or at the transition from III to IV. In normoblastic erythropoiesis, the ratio labelled cells/mitotic cells is rather independent of cell maturation. In contrast, this ratio varies considerably in megaloblastic erythropoiesis, from 25:1 in early forms to 4-5:1 in late forms. As an explanation of the lack of mitoses, relative to cells in DNA-synthesis, in the early stages and the relative surplus of mitoses in the late stages it is proposed that cell cycle and cytological boundaries do not coincide in all cells. The present observations can be accounted for if a significant fraction of cells change their morphology (from basophilia to polychromasia) between their DNA-synthesis phase and the subsequent mitosis. It cannot be decided whether in addition there is a death function between DNA-synthesis and mitosis in the large basophilic megaloblasts, megaloblastic system could absorb a direct entry from the large basophilic cells amounting to perhaps about one-half of the flux through the S-pool of the large basophilic cells without being more dominated by very large cells than is actually the case; still, in large measure this will depend on the time from entry into the polychromatic pool until the subsequent mitosis or possible cell death. The alternative is a significant death function between S-phase and mitosis at the level of the large basophilic E1-E2 cells (generation I + II).     

Concanavalin A and the initiation of thymic lymphoblast DNA synthesis and proliferation by a calcium-dependent increase in cyclic GMP level

Exposure of a thymic lymphocyte population (suspended in serum-free synthetic medium) to the phytomitogen concanavalin A (Con A) causes brief (within the first 8 to 12 minutes) rises in the cellular contents of cyclic AMP and cyclic GMP. However, the rise in the cyclic GMP level is calcium (extracellular)-dependent, but the cyclic AMP rise is not. These changes are followed during the next hour by the initiation of DNA synthesis by a large fraction of the lymphoblast subpopulation which, like the preceding cyclic GMP rise, is calcium-dependent. The stimulated lymphoblasts eventually progress into mitosis. Additional observations indicate that Con A operates by sensitizing lymphoblasts to calcium ions which, in turn, cause the initiation of DNA synthesis by a process mediated by cyclic GMP, but not cyclic AMP.     

Cell density-dependent decrease in cytoskeletal actin and myosin in cultured osteoblastic cells: correlation with cyclic AMP changes.

During bone development, osteoblasts form a contiguous layer along recently deposited osteoid and their morphology changes from fibroblast-like to cuboidal. In culture, similar changes occur with increased cell density. We examined the possible role of cyclic AMP in this process since cyclic AMP was reported to increase in fibroblasts with increased cell density and similar shape changes were seen in response to parathyroid hormone, which also increases cellular cyclic AMP in osteoblastic cells. Osteoblast-enriched rat calvaria cells were seeded at increasing density. The distribution between Triton X-100 extractable and nonextractable actin and myosin was estimated by polyacrylamide gel electrophoresis. Intracellular cyclic AMP was estimated by prelabeling the cellular ATP pool with 3H-adenine, followed by extraction and separation of 3H-cAMP by high-performance liquid chromatography. We found that osteoblastic cells contain about 40 pg actin and 5.3 pg myosin per cell. Around 60% of the actin and 70% of the myosin were in the nonextractable (crosslinked) form at cell densities of 10,000 to 50,000 cells per cm2. Above 50,000 cells/cm2, there was a cell density-dependent reduction in crosslinked actin and myosin and a concomitant increase in cellular cyclic AMP. A comparable rise in cyclic AMP, produced by incubation with phosphodiesterase inhibitors, and treatment with other agents that increase cyclic AMP produced a similar decrease in the level of cytoskeletal actin and myosin. Cytochalasin B treatment, through its effect on actin polymerization, produced similar changes in cell shape and cytoskeletal actin. The findings suggest that an elevation in intracellular cyclic AMP may play a role in the density-dependent changes in cell shape and microfilament organization observed in osteoblasts.     

Cyclic AMP, calcium and control of cell growth

The role of cyclic AMP and calcium in the control of normal and tumour cell growth is considered in relation to the question whether cyclic AMP is a true mitogen or co-mitogen. It is proposed that cyclic AMP normally controls the cell cycle at a point in G1 phase only by virtue of its ability to exclude calcium required by cells to progress past this point into S phase. Therefore increased influx of calcium by other routes induced by various factors can bypass the inhibitory effect of cyclic AMP and stimulate growth. In these circumstances cyclic AMP or calcium may or may not facilitate further progress into S phase according to the metabolic requirements of individual cells. The relevance to cancer cells is considered.     

Cholera toxin and analogues of cyclic AMP stimulate the growth of cultured human mammary epithelial cells

The growth of human epithelial cells is stimulated by cholera toxin and analogues of cyclic AMP, while the growth of breast derived fibroblasts is inhibited. These compounds have little effect on DNA synthesis in the absence of other mitogens but show a synergistic effect with serum and/or EGF. The results suggest that high intracellular levels of cyclic AMP in human mammary epithelial cells increase the growth response of the cell to mitogens.     

Differentiation in Leaf Epidermis of Chlorophytum comosum Baker

The distribution of guard mother-cell formation has been studiedin developing abaxial epidermis in the basal meristem of theleaf of Chlorophytum comosum. It is concluded that, as tissueis displaced from the base of the leaf by growth, it passesthrough a proliferative zone in which only proliferative mitosesoccur, and then passes a boundary into a formative zone in whichformative mitoses occur, giving rise to guard mother cells,and proliferative mitoses are absent. Further distally, formativemitoses die out and in the next zone (the guard-cell zone) theonly mitoses which occur are those by which the guard mothercells give rise to the guard cells. Most distally there is azone with no mitotic activity. The probability of a cell undergoinga formative mitosis is highest at the proximal boundary of theformative zone. It is consequently suggested that the fate ofa cell on entering the formative zone depends partly on itsposition in the mitotic cycle; cells nearest to mitosis at entryare the most likely to undergo a formative mitosis during theirpassage through the formative zone. Similarly, guard mothercells which fail to undergo mitosis may be those which weremost distant from mitosis on entry into the guard cell zone.These suggestions may explain some of the elements of patternpreviously found in the mature epidermis. Chlorophytum comosum Baker, spider plant, leaf epidermis, stomata, pattern, development, formative mitosis, proliferative mitosis     

Chromatin diminution in early embryogenesis of Ascaris lumbricoides L. var. suum

The occurrence of chromatin diminution in early Ascaris lumbricoides L. embryos has been studied in detail, and it is shown that it is possible to preselect three characteristic types of mitoses: pre-diminution, diminution, and post-diminution mitosis. The first three embryonic mitotic divisions are of the pre-diminution type. Chromatin diminution occurs after the third mitosis, but there is a variation from embryo to embryo as to whether or not chromosomal diminution occurs during the fourth, fifth, and six divisions. However, the seventh embryonic division, which gives rise to an eight-cell embryo, always exhibits chromatin diminution. Subsequent mitoses of somatic cells already in the diminished state are of the post-diminution type of mitosis.     

Correlation between Intracellular Cyclic AMP Level and Cell Membrane Antigenicity of Normal and Malignant Cells

Tumour cells cultured with certain quantities of dbcAMP and theophylline are normalized with respect to cell morphology, cell volume and restoration of topo-inhibition. Raising the intracellular level of cyclic AMP by exogenous dbcAMP and/or theophylline also resulted in profound changes of the antigenic pattern of normal and transformed mouse cell surfaces. The expression of xenogeneic antigens is decreased, while at the same time the expression of embryonic and tumour-specific antigens is enhanced.
The results indicate that tumour-specific surface antigens are very consistent markers for the malignant state of a cell, even if the cell is phenotypically normalized by treatment with dbcAMP and theophylline. Furthermore, the antigenic pattern of the cell plasma membrane may well be influenced quantitatively by the intracellular cyclic AMP level.     

Control of morphogenesis in Arthrobacter crystallopoiets: effect of cyclic adenosine 3'',5''-monophosphate.

The intracellular levels of cyclic adenosine 3',5'-monophosphate (cyclic AMP) were measured at various intervals during growth and morphogenesis in Arthrobacter crystallopoietes. Cyclic AMP levels remained relatively constant throughout growth in spherical cells grown in glucose-based media. Immediately after inoculation of spheres from glucose- to succinate-containing media, a 30-fold increase in intracellular cyclic AMP was detected. This dramatic rise in cyclic AMP preceded the observed change in cellular morphology from spheres to rods. The cyclic AMP level in rod-shaped cells rapidly dropped to a relatively stable concentration during the exponential growth phase. At the onset of stationary phase and rod-to-sphere morphological transition, a second peak of cyclic AMP was observed. Neither of these two peaks was detectable in a morphogenetic mutant that grew only as spheres. The intracellular levels of cyclic AMP in this mutant remained constant throughout exponential growth and decreased slightly during stationary phase. Effects of exogenously added cyclic nucleotides and their derivatives to both parent and mutant cultures were investigated. The data presented indicate that dramatic changes in intracellular cyclic AMP levels occur just before the morphological transitions characteristic of the morphogenetic cycle in A. crystallopoietes. It is suggested that cyclic AMP is a contributing factor in the regulatory phenomenon associated with morphogenesis in this bacterium.     

Induction of 3T3 cell division at the monolayer stage : Early changes in macromolecular processes

We have previously demonstrated that 3T3 cells at the monolayer stage can be induced to divide by brief treatment with a variety of proteases. We have now used this system to investigate the macromolecular changes occurring in 3T3 cells induced to divide in this manner. Immediately after pronase treatment, 3T3 cells become agglutinable with concanavalin A. This is rapidly followed in order by a decrease in cyclic AMP concentration within the cell, and an increase in RNA synthesis and uridine transport. The increases in RNA synthesis and uridine transport are dependent on concomitant protein synthesis. Specific protein synthesis follows 1 h after protease treatment, with DNA synthesis occurring 24 h after treatment and mitosis 30 h after treatment. This work suggests that following alteration of the surface membrane a variety of intercellular macromolecular processes occur which eventually culminate in DNA synthesis and cell division. Such a series of events may occur during each cell cycle in non-confluent 3T3 cells.