Immunofluorescent localization of cyclic nucleotide-dependent protein kinases on the mitotic apparatus of cultured cells.

Cyclic nucleotides and cyclic nucleotide-dependent protein kinases have been implicated in the regulation of cell motility and division, processes that depend on the cell cytoskeleton. To determine whether cyclic nucleotides or their kinases are physically associated with the cytoskeleton during cell division, fluorescently labeled antibodies directed against cyclic AMP, cyclic GMP, and the cyclic nucleotide-dpendent protein kinases were used to localize these molecules in mitotic PtK1 cells. Both the cyclic GMP-dependent protein kinase and the type II regulatory subunit of the cyclic AMP-dependent protein kinase were localized on the mitotic spindle. Throughout mitosis, their distribution closely resembled that of tubulin. Antibodies to cyclic AMP, cyclic GMP, and the type I regulatory and catalytic subunits of the cyclic AMP-dependent protein kinase did not label the mitotic apparatus. The association between specific components of the cyclic neucleotide system and the mitotic spindle suggests that cyclic nucleotide-dependent phosphorylation of spindle proteins, such as those of microtubules, may play a fundamental role in the regulation of spindle assembly and chromosome motion.

Oocyte-follicle cell gap junctions in Xenopus laevis and the effects of gonadotropin on their permeability.

Junctions between Xenopus laevis oocytes and follicle cells have been identified as gap junctions by the passage of microinjected fluorescent dye from oocytes to follicle cells. The opening or assembly of these junctions, or both, appears to be regulated by gonadotropins.

Alterations in the expression and cellular localization of protein kinase C isozymes epsilon and theta are associated with insulin resistance in skeletal muscle of the high-fat-fed rat.

We have tested the hypothesis that changes in the levels and cellular location of protein kinase C (PKC) isozymes might be associated with the development of insulin resistance in skeletal muscles from the high-fat-fed rat. Lipid measurements showed that triglyceride and diacylglycerol, an activator of PKC, were elevated four- and twofold, respectively. PKC activity assays indicated that the proportion of membrane-associated calcium-independent PKC was also increased. As determined by immunoblotting, total (particulate plus cytosolic) PKC alpha, epsilon, and zeta levels were not different between control and fat-fed rats. However, the ratio of particulate to cytosolic PKC epsilon in red muscles from fat-fed rats was increased nearly sixfold, suggesting chronic activation. In contrast, the amount of cytosolic PKC theta was downregulated to 45% of control, while the ratio of particulate to cytosolic levels increased, suggesting a combination of chronic activation and downregulation. Interestingly, while insulin infusion in glucose-clamped rats increased the proportion of PKC theta in the particulate fraction of red muscle, this was potentiated by fat-feeding, suggesting that the translocation is a consequence of altered lipid flux rather than a proximal event in insulin signaling. PKC epsilon and theta measurements from individual rats correlated with triglyceride content of red gastrocnemius muscle; they did not correlate with plasma glucose, which was not elevated in fat-fed rats, suggesting that they were not simply a consequence of hyperglycemia. Our results suggest that these specific alterations in PKC epsilon and PKC theta might contribute to the link between increased lipid availability and muscle insulin resistance previously described using high-fat-fed rats.

Analysis of the calcium transient at NEB during the first cell cycle in dividing sea urchin eggs.

Accumulating evidence from several systems suggests that nuclear envelope breakdown (NEB) is triggered by an endogenous transient of free calcium. Using h- and f-semisynthetic aequorins as cytosolic calcium indicators, we have clearly and regularly visualized a single large, global calcium transient just before first NEB in normally developing, monospermic Lytechinus eggs. Although similar transients were not observed at NEB in subsequent cell cycles, microinjection of the calcium buffer BAPTA into one blastomere of the two-celled embryo resulted in the inhibition of NEB. The NEB transient in the first cell cycle was some five-fold smaller than the one associated with egg activation. Our data suggest that this transient takes the form of a calcium wave that spreads inwards from the periphery of the egg toward the nucleus. We confirmed that these NEB transients did not require extracellular Ca2+. In polyspermic eggs, NEB-associated transients were four-fold larger than in monospermic eggs and were periodically repeated. Examination of the distribution of fluorescein-conjugated aequorins with a laser scanning confocal microscope indicated that aequorin both enters the nucleus and is evenly distributed within the cytosol of the egg. The use of h- and f-aequorins did not reveal any NEB transients during subsequent cell cycles, nor did we detect transients associated with other cell cycle events. However, a complex train of calcium transients in the form of both localized pulses and propagated waves was detected from embryos beginning at about the morula-to-blastula transition and continuing through to hatching.

Intercellular junctions between the follicle cells and oocytes of Xenopus laevis.

During development, the oocytes of Xenopus laevis establish junctional contact with the follicle cells enveloping them. These junctions have alternatively been described as desmosomes and as gap junctions. In this paper the morphology of these junctions has been examined in gonadotropin-stimulated and unstimulated animals at all stages of development. Contact between the oocyte and follicle cell plasma membranes is visible in stage I oocytes as thickenings in the membranes, separated by intercellular spaces of 20nm or greater. By stage III in unstimulated oocytes and stage II in gonadotropin-stimulated oocytes, intermembrane spaces at these junctional contacts are often reduced to 2 to 7 nm in width. These narrow intermembrane spaces persist through early stage IV, with greater frequency of occurrence in oocytes taken from hormonally stimulated animals. The closeness of these junctional contacts, and the permeability of the junctional spaces to intercellular tracer substances, supports the evidence that these are gap junctions.

Differential uptake of mercury vapor by gramineous c(3) and c(4) plants.

The uptake of mercury vapor by six gramineous plant species was compared under uniform conditions using a whole-plant chamber and (203)Hg-labeled mercury at a low atmospheric concentration. Mean Hg uptake by leaves of the C(3) species oats (Avena sativa), barley (Hordeum vulgare), and wheat (Triticum aestivum) was 5 times greater than that by leaves of the C(4) species corn (Zea mays), sorghum (Sorghum bicolor), and crabgrass (Digitaria sanguinalis). Although there was a difference in resistances associated with vapor entry into the leaves, as shown by estimates of gas exchange, the differential uptake by C(3) and C(4) species was largely attributable to internal resistances to Hg vapor binding. The nature of the internal resistances and the site or sites of Hg vapor binding remain unspecified.

Toxicity of selenium to developing Xenopus laevis embryos.

Se in the form of sodium selenite is toxic to Xenopus laevis embryos and tadpoles continuously exposed to concentrations above 1 ppm. Concentrations of 2 ppm and above result in severe developmental abnormalities and increased mortality. Uptake and loss of radioactive Se from water are rapid, but depuration is not complete indicating that some Se can remain bound by the organism. The facts that Se is toxic at low levels to Xenopus embryos and tadpoles, can cause developmental abnormalities, and accumulates in tissues suggest that increased release of Se compounds into the environment poses a potential threat to aquatic organisms.

Estimation of whole-plant resistance to gaseous exchange independent of leaf temperature measurement.

For studies into the uptake of mercury vapor by wheat (Triticum aestivum), a simple theory and plant chamber were employed to estimate total leaf resistance of whole plants to water vapor exchange. The estimates were independent of leaf temperature, for which mean values were indirectly determined. The approach involved the measurement, at steady-state conditions, of the net change in water vapor flux per unit of leaf surface (Deltaq(v)) in response to a small induced change in absolute humidity (DeltaC(a)). Assuming that total leaf resistance (r(l)) was constant and that change in leaf temperature (T(l)) was negligible, total leaf resistance was calculated from the equation, [Formula: see text]While the assumptions concerning r(l) and T(l) may or may not be correct, evidence is presented which indicates that such assumptions did not significantly alter estimates of r(l) from their true values for changes in ambient relative humidity ranging from 0.011 to 0.074. Total leaf resistance of groups of whole plants estimated in this manner did not differ for ambient temperatures of 17, 25, and 33 C. Mean values of r(l) ranged from 83 sec cm(-1) in darkness to 2.4 sec cm(-1) at an illumination of 12.8 klux.

Uptake of mercury vapor by wheat: an assimilation model.

Using a whole-plant chamber and (203)Hg-labeled mercury, a quantitative study was made of the effect of environmental parameters on the uptake, by wheat (Triticum aestivum), of metallic mercury vapor, an atmospheric pollutant. Factors were examined in relation to their influence on components of the gas-assimilation model, [Formula: see text]where U(Hg) is the rate of mercury uptake per unit leaf surface, C(a)' is the ambient mercury vapor concentration, C(l)' is the mercury concentration at immobilization sites within the plant (assumed to be zero), r(l.Hg) is the total leaf resistance to mercury vapor exchange, and r(m.hg) is a residual term to account for unexplained physical and biochemical resistances to mercury vapor uptake.Essentially all mercury vapor uptake was confined to the leaves. r(l.hg) was particularly influenced by illumination (0 to 12.8 klux), but unaffected by ambient temperature (17 to 33 C) and mercury vapor concentration (0 to 40 mug m(-3)). The principal limitation to mercury vapor uptake was r(m.hg), which was linearly related to leaf temperature, but unaffected by mercury vapor concentration and illumination, except for apparent high values in darkness.Knowing C(a)' and estimating r(l.hg) and r(m.hg) from experimental data, mercury vapor uptake by wheat in light was accurately predicted for several durations of exposure using the above model.

The role of nerve cell density in the regulation of bud production in hydra.

The role of nerve cell density in the regulation of bud production in hydra was examined. Animals with different rates of bud production were produced by altering the temperature, population density and illumination of their cultures. When the distribution of cell types was examined in animals with different rates of bud production, the density of nerve cells in those animals was found to be correlated with their rate of bud production. Transfer of animals from one environment to another resulted in immediate changes in the rate of differentiation of large interstitial cells into nerve cells. This suggests that the density of nerve cells may play a role in regulating the rate of bud production in hydra.

Cross-talk between muscarinic- and adenosine-receptor signalling in the regulation of cytosolic free Ca2+ and insulin secretion.

The effects of A1-adenosine-receptor occupation on Ca2+ handling in the insulin-secreting RINm5F cell line were investigated. The selective A1-agonist N6-cyclopentyladenosine (CPA) had no effect itself on the cytosolic free Ca2+ concentration in cells loaded with Fura 2. However, CPA (1) attenuated the rise due to activation of voltage-gated Ca2+ channels with Bay K 8644, and (2) caused a secondary increase (EC50 approx. 300 nM) if added after the primary Ca(2+)-mobilizing agonists vasopressin or carbamoylcholine (carbachol). Prior addition of CPA (10 microM) also potentiated (by approx. 20%) the subsequent Ca2+ peak due to maximal (100 microM) carbachol, but did not alter the EC50 of the carbachol response. Detailed analysis of the secondary rise in Ca2+ revealed further features. First, it was due to mobilization from intracellular stores, since it persisted in the absence of extracellular Ca2+. Second, it was associated with a rapid (5-15 s) increase in phospholipase C (PLC) activity, as measured by h.p.l.c. analysis of Ins(1,4,5)P3. This increase was only apparent after prior stimulation with carbachol. Third, and unlike the response to carbachol, it was mediated by a pertussis-toxin-sensitive G-protein. Fourth, it was not secondary to a decrease in cyclic AMP. Fifth, it was absolutely dependent on continued occupation of the primary receptor, since it was abolished if carbachol was displaced with the antagonist atropine. This implies a dynamic cross-talk between the two receptor coupling systems, rather than covalent modification as a result of the prior activation of PLC. Sixth, it was not associated with any desensitization of the ability of CPA to inhibit forskolin-stimulated adenylate cyclase activity. Glyceraldehyde (10 mM)-induced insulin secretion was also potently inhibited by CPA > 10 nM, but the secretory response to 100 microM carbachol was unaffected up to 10 microM. The results suggest that, in vivo, adenosine would inhibit secretion due to carbohydrate nutrients much more effectively than that due to stimuli which activate PLC.

Cellular mechanisms of stimulation of bud production in Hydra by low levels of inorganic lead compounds.

Treatment of Hydra with subtoxic levels of inorganic lead compounds (lead nitrate and lead chloride) for perods ranging from 5 min to one hour causes a temporary increase in bud production as compared to untreated control animals. This effect can be inhibited by the addition of large amounts of calcium chloride to the culture medium. The increased rate of budding is preceded by a dramatic increase in the number of nerve cells per animal, which is first observed within six hours after lead treatment. This appears to be the result of an increased rate of mitosis in the undifferentiated interstitial cells and their subsequent differentiation into nerve cells. The total number of cells per animal also increases after exposure to lead compounds, suggesting that lead may act as a general mitotic stimulator of all dividing cell types in Hydra.

Effect of inhibition of the catalytic activity of cyclic AMP-dependent protein kinase on mitosis in PtK1 cells.

Evidence has suggested that cyclic AMP, acting through activation of the type II cyclic AMP-dependent protein kinase, may play a role in the regulation of interphase and mitotic microtubules. In order to examine the potential role of the type II cAMP-dependent kinase during mitosis, dividing PtK1 cells were microinjected with two specific inhibitors of the catalytic activity of the type II kinase. These inhibitors were a specific protein inhibitor of cAMP-dependent protein kinase (PKI) and an affinity-purified polyclonal antiserum (anti-C) directed against the catalytic subunit of the kinase. Both have been shown previously to inhibit kinase activity in vitro. Microinjection of PKI during early- to mid-prophase significantly delayed the progression of the cells through mitosis, with the greatest delay occurring in metaphase. PKI injected during prometaphase also delayed progression through mitosis but to a lesser extent. Microinjection of anti-C during early- to mid-prophase also caused a significant delay in the completion of mitosis, with many cells becoming "hung up" in prometaphase. Anti-C injected during prometaphase had little effect on subsequent progression through mitosis. Microinjection of either anti-C or PKI during metaphase had no discernible effect. No effect on anaphase movement of chromosomes was observed with any treatment. These results provide further evidence that cAMP-dependent phosphorylation may be involved in the regulation of mitosis, although whether it acts directly through regulation of mitotic spindle microtubules is unclear.

Characterization of two forms of protein kinase C alpha, with different substrate specificities, from skeletal muscle.

We have investigated protein kinase C (PKC) in skeletal muscle cytosol and demonstrated the presence of two major activities. These did not correspond to different PKC isoenzymes but seemed to represent two species of PKC alpha as deduced by: elution during hydroxyapatite chromatography at KH2PO4 concentrations expected of PKC alpha; detection of the two species by three specific but unrelated anti-(PKC alpha) antibodies; immunodepletion of both activities with anti-(PKC alpha) antibody; and demonstration of identical requirements of both Ca2+ ions and lipid for activation. These species, termed PKC alpha 1 and PKC alpha 2, phosphorylated the modified conventional PKC pseudosubstrate peptide (19-31, Ser-25) equally well. Importantly, however, the activities differed in that PKC alpha 1 phosphorylated histone IIIS, and also peptides derived from the EGF receptor and glycogen synthase, to a much greater extent than did PKC alpha 2. Similarly, incubation of crude muscle extracts with either PKC alpha 1 or alpha 2 gave rise to different protein phosphorylation patterns. The involvement of proteolysis, dephosphorylation or oxidative modification in the interconversion of PKC alpha 1 and PKC alpha 2 during preparation was ruled out. Although some PKC-binding proteins were detected in overlay assays, their presence did not explain the anomalous PKC alpha 2 activity. The results suggest that a modification of PKC alpha in situ limits its substrate specificity, and indicate an additional level of control of the kinase that may be a site for modulation of PKC-mediated signal transduction.

Inhibition of mitosis in fertilized sea urchin eggs by inhibition of the cyclic AMP-dependent protein kinase.

Inhibition of cAMP-dependent protein kinase activity by microinjection of a specific physiologic protein inhibitor into sea urchin eggs inhibits the first cleavage after fertilization. Inhibition apparently occurs at some time prior to or during formation of the mitotic spindle. Measurement of the total protein kinase activity of sea urchin egg homogenates after fertilization showed that cAMP-dependent phosphorylation increases after fertilization and then declines prior to or at the time of the first cleavage. It is concluded that a cAMP-dependent phosphorylation plays a significant role in events leading to regulation of mitotic spindle assembly.

Activated protein kinase C alpha associates with annexin VI from skeletal muscle.

We have previously detected a number of protein kinase C (PKC) alpha-binding proteins in skeletal muscle cytosol by blot overlay assay, and now identify the major, 69 kDa binding protein as annexin VI by immunoblotting and overlay assay of hydroxyapatite chromatography fractions. Annexin VI was also detected in immunoprecipitates of PKC alpha. Annexin VI and PKC alpha are both calcium-dependent phospholipid-binding proteins, and detection of the interaction was dependent on the presence of calcium and phosphatidylserine (PS). The association probably involves specific protein-protein interactions rather than mere bridging by lipid molecules: firstly, detection of PKC alpha-annexin VI complexes by overlay assay was not diminished when PS concentrations were increased over a 10-fold range, while that of other PKC alpha-binding protein complexes was reduced or abolished; secondly, the presence in the overlay assay of a PKC pseudosubstrate peptide, analogous to a PKC sequence previously found to be involved in PKC binding activity, reduced complex formation; thirdly, we were also able to detect annexin VI interaction with PKC beta by overlay of skeletal muscle cytosol, but not with PKC theta, the major novel PKC in this tissue, suggesting sequences specific to calcium-dependent PKC isoenzymes are involved. While other annexin isoforms may be PKC substrates or inhibitors, annexin VI phosphorylation by PKC alpha could not be detected after co-purification, while phosphorylation of subsequently-added histone IIIS was readily observed. Annexin VI is a major skeletal muscle protein and our data are consistent with a role for this isoform in the control of calcium-dependent PKC.

Synergistic interaction of Y1-neuropeptide Y and alpha 1b-adrenergic receptors in the regulation of phospholipase C, protein kinase C, and arachidonic acid production.

Neuropeptide Y (NPY) and norepinephrine, found colocalized in sympathetic neurons innervating blood vessels, exert synergistic responses on vasoconstriction. To examine the signaling mechanisms involved, free of complications associated with mixed receptor populations, we have established a stable Chinese hamster ovary cell line expressing both Y1-NPY and alpha 1b-adrenergic receptors. Occupation of either receptor species, with 100 nM peptide YY (PYY) or 10 microM phenylephrine (PE), respectively, resulted in a rapid increase in the cytoplasmic free calcium concentration ([Ca2+]i) as assessed with Fura-2/AM. The rise due to PYY, but not that due to PE, was abolished by pretreatment with pertussis toxin. Both responses were largely maintained in the absence of extracellular Ca2+, but abolished by prior depletion of intracellular Ca2+ pools with either thapsigargin or 2,5-di-(t-butyl)-1,4-benzohydroquinone. Using cells prelabeled with myo-[3H]inositol, PE promoted a rapid (5 s) rise in inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) as analyzed by anion-exchange high pressure liquid chromatography, whereas the response to PYY (first significant at > 15 s post-stimulation) was too slow to play a causative role in Ca2+ mobilization. Combination of PE and PYY resulted in increases in [Ca2+]i which were at best additive, whereas they promoted a clearly synergistic rise in Ins(1,4,5)P3 at both 15 and 60 s. Co-stimulation also resulted in a synergistic activation of both protein kinase C (PKC) and [3H]arachidonic acid release. In either instance PYY alone was without effect. The potentiation of arachidonic acid release was abolished by depletion of cellular PKC following chronic treatment with phorbol esters. It is suggested that the ability of PYY to mobilize Ca2+ in an Ins(1,4,5)P3-independent fashion minimizes the functional importance of the capacity to potentiate PE-stimulated Ins(1,4,5)P3 generation. Instead the major consequences of the synergistic activation of phospholipase C are mediated via PKC, the other route of the signaling pathway.

Structural and biochemical studies of human galanin: NMR evidence for nascent helical structures in aqueous solution.

The 30-residue human neuropeptide, galanin, was shown to bind to rat insulinoma RINm5F cells and to inhibit glyceraldehyde-stimulated insulin secretion from these cells in a manner quantitatively similar to that of porcine galanin. Neither human nor porcine galanin stimulated Ca2+ mobilization in cultured human small cell lung carcinoma cells. Sedimentation equilibrium analysis of human galanin showed that it was strictly monomeric in aqueous solution, indicating that the peptide interacts with its receptor(s) as a monomer. The monomeric nature of the peptide makes it especially suitable for structural studies using NMR. Nuclear Overhauser enhancement spectroscopy experiments performed on galanin dissolved in aqueous solution (150 mM KCl, pH 4) at both 33 and 3 degrees C indicate that certain regions of the peptide are capable of adopting detectable levels of short-range structure in rapid equilibrium with random coil. At 33 degrees C, the short-range structures include a nascent helix spanning residues 3-11 which incorporates a hydrophobic core from residues 6-11. Residues 14-18 and 22-30 display sequential NH-NH and C beta H-NH connectivities, indicating that these regions of the peptide adopt nonrandom conformations by significantly populating the alpha-region of conformational space. However, no medium-range dipolar connectivities indicative of nascent helix or turn conformations were observed. At 3 degrees C, almost all residues significantly populate the alpha-region of conformational space, and the nascent helix between residues 3 and 11, with its hydrophobic core, is retained.(ABSTRACT TRUNCATED AT 250 WORDS)

Reversal of chronic alterations of skeletal muscle protein kinase C from fat-fed rats by BRL-49653.

We have recently shown that the reduction in insulin sensitivity of rats fed a high-fat diet is associated with the translocation of the novel protein kinase C epsilon (nPKC epsilon) from cytosolic to particulate fractions in red skeletal muscle and also the downregulation of cytosolic nPKC theta. Here we have further investigated the link between insulin resistance and PKC by assessing the effects of the thiazolidinedione insulin-sensitizer BRL-49653 on PKC isoenzymes in muscle. BRL-49653 increased the recovery of nPKC isoenzymes in cytosolic fractions of red muscle from fat-fed rats, reducing their apparent activation and/or downregulation, whereas PKC in control rats was unaffected. Because BRL-49653 also improves insulin-stimulated glucose uptake in fat-fed rats and reduces muscle lipid storage, especially diglyceride content, these results strengthen the association between lipid availability, nPKC activation, and skeletal muscle insulin resistance and support the hypothesis that chronic activation of nPKC isoenzymes is involved in the generation of muscle insulin resistance in fat-fed rats.