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particularly up to date features, Gerald Karp's book includes a section in the Cancer chapter on angiogenesis, as does Lodish et al., but in Karp the information imparted extends to a scheme showing the effect of endostatin on mouse tumours, replete with photographs of mice from such an experiment. All of this has resulted in a book that is focused but highly informative and a delight to browse through. If it appears by now that your reviewer is suggesting you should dash out and buy all three of these books, I can only admit that I am delighted to have them on my shelf. If you have to choose, for fundamental biochemical science with a fair degree of breadth thrown in it has to be Lehninger. For a comprehensive molecular biology course Lodish et al. is the equal of anything else on the market. For a broad grounding in cell and molecular biology that will appeal especially to medical students go for Karp.

Latent transforming growth factor beta binding protein-1 (LTBP1) is important in regulating the localisation and activation of transforming growth factor beta(TGFbeta). Three forms of LTBP1 mRNA have previously been described, LTBP1L, LTBP1S and LTBPdelta53. Here, we have analysed the LTBP1 coding sequence and identified two other spliced forms, LTBP1delta55 and LTBP1delta41. LTBP1delta55 is a short form of LTBPIL which lacks 55 amino acids including two consensus N-glycosylation sites and LTBP1delta41 is a form of LTBP1 which lacks the 12th EGF-like repeat. Furthermore, sequencing of genomic clones showed that splicing to generate LTBP1L occurs using an intra-exonic 3' splice acceptor site in the first coding exon of LTBP1S and that LTBP1delta55 arises from the alternative use of an exonic 3' splice acceptor site at the end of the following intron. LTBP1delta41 arises from skipping the exon which encodes the 12th EGF-like repeat. LTBP1delta55 and LTBP1delta41 mRNA are expressed in a wide variety of human tissues but the proportions of each splice form vary in the tissues.

Latent transforming growth factor-beta binding protein-1 (LTBP-1), plays an important role in controlling localisation and activation of transforming growth factor-beta (TGF-beta). We show that alternative splicing generates a form of mRNA which lacks bases 1277-1435 (termed LTBP-1delta53). The 53 amino acids encoded by these bases include the eighth cysteine of the first cysteine repeat and a consensus heparin binding sequence. Sequencing of genomic clones showed that alternative splicing resulted from the use of an intra-exonic 3' splice acceptor site. The loss of the heparin binding site implies that LTBP-1delta53 will bind to the extracellular matrix less efficiently than LTBP-1.

Epidemiological studies have shown weak correlations between exposure to extremely low-frequency electromagnetic fields (ELF EMFs) and the incidence of several cancers, particularly childhood leukemias, although negative studies have also been reported. These observations have prompted a broad range of in vitro cellular studies in which effects of ELF EMFs have been observed. However, no reported response has been replicated widely in independent laboratories. One potentially important response is the rapid activation of proto-oncogenes and other genes in human leukemic (HL60) cells and a wide variety of other eukaryotic cells, because of the role of these genes in cell proliferation. We describe quantitative Northern analysis of MYC and beta-actin mRNAs from HL60 cells exposed to fields under conditions very similar to those reported previously to activate these genes, namely 60 Hz sinusoidal magnetic fields of 0.57, 5.7 or 57 microT for 20 min. In addition we have used a new design of field-exposure system and introduced a number of other modifications to the protocol to optimize any response. We have also developed a novel method providing enhanced accuracy for the quantitative measurement of mRNA. No significant effect of ELF EMFs on gene expression was observed using any of these systems and analytical methods.

The role of p21c-ras in the activation of DNA synthesis in quiescent Swiss 3T3 fibroblasts was examined by scrape loading or microinjecting the neutralizing monoclonal antibody Y13-259 into the cells. Y13-259 delayed but did not block both the serum-stimulated entry of the cells into S phase and the accumulation of cdc2 mRNA and protein at the G1-S boundary. Introduction of Y13-259 also stimulated expression of sufficient p21c-ras to neutralize the loaded antibody; this finding suggests that the delay of S phase is attributable to the time taken to synthesize an excess of p21c-ras over the antibody and implies autoregulation of c-ras expression. Y13-259 had no effect upon phosphoinositide-mediated responses ([Ca2+]i increase and activation of protein kinase C) to platelet-derived growth factor or bombesin, demonstrating that p21c-ras is not required for mitogen activation of protein kinase C. Y13-259 inhibited 5-bromo-2'-deoxyuridine incorporation in response to all of the combinations of mitogens used to stimulate quiescent cells (fetal calf serum, platelet-derived growth factor, and the combination of insulin with 12-O-tetradecanoylphorbol-13-acetate, bombesin, epidermal growth factor, or prostaglandin E1), indicating that p21c-ras functions on pathways common to all of the effective combinations of mitogens examined.

Hexamethylenebisacetamide (HMBA) selectively and reversibly inhibited proliferation of human and rat vascular smooth-muscle cells (VSMCs) compared with endothelial cells, fibroblasts or lymphocytes. Half-maximal inhibition of VSMC proliferation occurred at 2-5 mM-HMBA, and at 30- greater than 50 mM for other cell types. HMBA also prevented de-differentiation, defined by the loss of smooth-muscle-specific myosin heavy chain, of primary rat VSMCs and caused partial re-differentiation of subcultured cells. Other inhibitors of ADP-ribosyltransferase were also selective inhibitors of VSMC proliferation.

Vascular smooth-muscle cells (VSMCs) from rat aortae contained very little non-muscle myosin heavy chain (MHC) immediately after dispersal, and the protein did not accumulate if the cells were held in G0/G1 phase by withholding serum or were held in first S phase by the addition of bromodeoxyuridine (BrdU). However, non-muscle MHC accumulated by greater than 20-fold per cell during first M phase, when over 80% of the cells divided between 48 h and 72 h after addition of serum. Delaying the addition of serum caused a delay in the accumulation of the non-muscle MHC until the cells subsequently entered M phase. If the cells were held in M phase at the metaphase/anaphase boundary by nocadazole, the accumulation of non-muscle myosin still occurred, although division was blocked. When the cells were pulse-labelled with [35S]methionine, it was found that non-muscle MHC was one of the major proteins being made and that its synthesis occurred at similar rates throughout the cell cycle. This implied that the rate of degradation of the protein before first M phase was much faster than in M phase, when the protein accumulated rapidly. This was confirmed by direct measurements of the rate at which [35S]methionine-labelled non-muscle MHC disappeared from the cells, which gave a half-life for the protein of about 8 h before M phase but about 5 days in post-mitotic cells, i.e. an increase of approx. 15-fold. These data are consistent with the hypothesis that there is a mechanism in VSMCs which shortens the half-life of the protein before first M phase and that the accumulation of non-muscle MHC which results from the increase in half-life at first M phase may be necessary for division of these cells.

We have investigated whether any of the three isoforms of endothelin (ET) ET-1, ET-2 and ET-3 or the structurally similar peptide sarafotoxin S6b is mitogenic on its own for rat vascular smooth muscle cells in culture. DNA synthesis was determined by a peroxidase-linked double antibody technique to detect bromodeoxyuridine incorporation into the nucleus and stained nuclei were counted by image analysis. None of the ET peptides or sarafotoxin S6b (up to 100 nM) was capable of initiating DNA synthesis in the absence of platelet derived growth factor (PDGF) or fetal calf serum. All the peptides potentiated the mitogenic effect of low concentrations of PDGF. ET-1 and ET-2 (10 nM) caused a 2-fold increase in the number of stained nuclei induced by 5 nM and 10 nM PDGF, whereas ET-3 and sarafotoxin S6b were less potent. These findings demonstrate that ET is a co-mitogen for rat vascular smooth muscle cells. The release of ET at sites of endothelial injury may therefore enhance the mitogenic action of locally acting PDGF on vascular smooth muscle cells and potentiate the proliferative response.

Quiescent Swiss 3T3 fibroblasts stimulated with epidermal growth factor and insulin showed large transient increases in c-myc mRNA and c-myc protein accumulation which were maximal at about 2 h after addition of the co-mitogens. When the cells were loaded with 0.1 mM of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) by transient permeabilisation immediately before mitogenic stimulation, the increase in c-myc mRNA was similar to that observed in unloaded cells but the corresponding c-myc protein peak was reduced by at least 95%. The GTP gamma S completely blocked incorporation of [35S]methionine into cell proteins for 3-4 h after addition of the mitogens, but not thereafter, and caused a delay in the subsequent onset of DNA synthesis by the same period. The data show that less than 5% of the early increase in c-myc protein normally observed after mitogenic stimulation is required for its obligatory role in the progression of cells to S phase implied by other evidence.

The activation of protein synthesis by mitogens in quiescent (G0) mammalian cells is obligatory for progression from G0 through G1 to DNA synthesis in S phase. When the activation of the Na+/H+ antiporter which occurs in mitogen-stimulated Swiss 3T3 fibroblasts or murine fibroblasts is completely blocked by dimethylamiloride, there is little or no effect on the phosphorylation of the ribosomal protein S6 or the activation of protein synthesis assayed by [35S]methionine incorporation. Furthermore, the accumulation of the protein product of the activated c-myc gene is unaffected by dimethylamiloride in 3T3 fibroblasts. The data show that there is no requirement for activation of the Na+/H+ antiporter for the activation of S6 phosphorylation or protein synthesis by mitogens but do not preclude the possibility that activation of the antiporter is necessary for some other response(s) obligatory for DNA synthesis. These data are contrasted with previous reports for Chinese hamster lung fibroblasts that the increase in intracellular pH which results from activation of the Na+/H+ antiporter in bicarbonate-free media is necessary for S6 phosphorylation, protein synthesis, and hence, for subsequent DNA synthesis (Pouyssegur, J., Chambard, J. C., Franchi, A., Paris, S., and Van Obberghen-Schilling, E. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 3935-3939; Chambard, J.C., and Pouyssegur, J. (1986) Exp. Cell Res. 164, 282-294).

Phosphatidylinositol (PI) and its phosphorylated derivatives are rapidly broken down in 2H3 cells stimulated with antigen, with a time course which coincides with the generation of the Ca signal. Stimulated PI breakdown is absolutely dependent on Ca2+ in the medium with a concentration dependence similar to that of the Ca signal and histamine release described in the preceding paper. However, PI breakdown does not depend on the rise in free cytoplasmic Ca2+ concentration in stimulated cells over the range 100 nM to 1 microM. Thus, stimulation by the ionophore A23187 causes only a small increase in PI breakdown and the Ca signal stimulated by antigen can be selectively blocked with appropriate concentrations of Zn2+ (100 microM) or La3+ (10-100 microM) which have small or negligible effects on stimulated PI breakdown. Both PI breakdown and the Ca signal appear to depend on a common external Ca2+ site (or sites) with Km approximately equal to 0.4 mM, and the data are consistent with either independent activation of PI phosphodiesterase and the Ca signal after antigenic stimulation, or with PI breakdown as a component of the mechanism by which the Ca signal is generated.

1. The lipids composition of rat liver plasma membranes was substantially altered by introducing synthetic phosphatidylcholines into the membrane by the techniques of lipid substitution or lipid fusion. 40-60% of the total lipid pool in the modified membranes consisted of a synthetic phosphatidylcholine. 2. Lipid substitution, using cholate to equilibrate the lipid pools, resulted in the irreversible loss of a major part of the adenylate cyclase activity stimulated by F-, GMP-P(NH)P or glucagon. However, fusion with presonicated vesicles of the synethic phosphatidylcholines causes only small losses in adenylate cyclase activity stimulated by the same ligands. 3. The linear form of the Arrhenius plots of adenylate cyclase activity stimulated by F- or GMP-(NH)P was unaltered in all of the membrane preparations modified by substitution or fusion, with very similar activation energies to those observed with the native membrane. The activity of the enzyme therefore appears to be very insensitive to its lipid environment when stimulated by F- or gmp-p(nh)p. 4. in contrast, the break at 28.5 degrees C in the Arrhenius plot of adenylate cyclase activity stimulated by glucagon in the native membrane, was shifted upwards by dipalmitoyl phosphatidylcholine, downwards by dimyristoyl phosphatidylcholine, and was abolished by dioleoyl phosphatidylcholine. Very similar shifts in the break point were observed for stimulation by glucagon or des-His-glucagon in combination with F- or GMP-P(NH)P. The break temperatures and activation energies for adenylate cyclase activity were the same in complexes prepared with a phosphatidylcholine by fusion or substitution. 5. The breaks in the Arrhenius plots of adenylate cyclase activity are attributed to lipid phase separations which are shifted in the modified membranes according to the transition temperature of the synthetic phosphatidylcholine. Coupling the receptor to the enzyme by glucagon or des-His-glucagon renders the enzyme sensitive to the lipid environment of the receptor. Spin-label experiments support this interpretation and suggest that the lipid phase separation at 28.5 degrees C in the native membrane may only occur in one half of the bilayer.

The progression of Swiss 3T3 fibroblasts from the quiescent state (G0) through G1 to DNA synthesis in S phase generally requires the synergistic action of two mitogens. The main aim of this study was to compare systematically the early Ca2+ and pH responses in quiescent cells to all of the pair combinations of eight mitogens (bombesin, platelet-derived growth factor, vasopressin, prostaglandin F2 alpha, epidermal growth factor, 12-O-tetradecanoyl phorbol-13-acetate, insulin, 8-bromo-cAMP) with their subsequent effects on DNA synthesis. Each of the mitogens which caused inositol phosphate accumulation (bombesin, platelet-derived growth factor, vasopressin, prostaglandin F2 alpha) also activated Ca2+- and phospholipid-dependent protein kinase (protein kinase C) and generated both the Ca2+ and pH responses, although epidermal growth factor also generated the ionic responses without causing release of inositol phosphates or activation of protein kinase C. For sequential mitogen additions the ionic signals were measured in single cells as well as in cell populations to avoid ambiguities due to heterogeneity in the responses of the cells to the various mitogens. The modulating effects of the mitogens on the [Ca2+]i responses to subsequent mitogen additions varied widely, but detailed comparisons showed that the pattern of blocking effects could not be attributed solely to the effect of the first mitogen causing either maximal breakdown of phosphatidylinositol 4,5-bisphosphate or complete depletion of the intracellular Ca2+ pool or activation of protein kinase C. From these analyses it was concluded that the requirement for two mitogens for effective DNA synthesis could not be attributed to the summation to a critical threshold of either the ionic signals or phosphatidylinositol 4,5-bisphosphate breakdown, and that these responses are insufficient by themselves to cause the cells to progress to DNA synthesis in S phase.

Rat basophil leukemic (2H3) cells ( Siraganian , R.P., McGivney , A., Barsumian , E. L., Crews, F. T., Hirata , F., and Axelrod , J. (1982) Fed. Proc. 41, 30-34) loaded with fluorescent Ca2+ indicator quin 2 ( Tsien , R. Y. (1980) Biochemistry 19, 2396-2404) showed a rapid increase in free cytosol calcium concentration [( Ca]i) when histamine release was induced. Intracellular quin 2 concentrations up to 7 mM did not affect release of histamine in response to antigen (aggregated ovalbumin) or concanavalin A with cells primed with antigen-specific monoclonal IgE, or in response to Ca2+ ionophores. The [Ca]i increased from approximately 105 nM to a maximum of approximately 1200 nM within 2 to 3 min after antigenic stimulation and then declined slowly over 30 min toward the level in unstimulated cells. Histamine release was most rapid as [Ca]i reached the maximum value and then decreased continuously with [Ca]i over the subsequent 30 min. Neither the Ca signal nor histamine release was observed when the Ca2+ concentration in the medium [( Ca]o) was less than 50 microM, but both responses were restored on readdition of Ca2+ to 1 mM. The maximal Ca signal was obtained when [Ca]o was approximately greater than 1 mM and was half-maximal at [Ca]o congruent to 0.4 mM. In marked contrast [Ca]i in unstimulated cells varied very little with [Ca]o from 0.1 to 1 mM. Maintenance of the Ca signal required the continuous presence of stimulating ligand, external Ca2+, and the maintenance of cellular ATP; metabolic inhibitors blocked or reversed the Ca signal. La+ ions also caused a rapid and reversible block of the Ca signal and histamine release. The data are interpreted in a model in which the Ca signal is generated by a La3+-sensitive signal influx pathway that is functionally independent of the normal Ca2+ influx pathway in unstimulated cells, and that allows a 10-fold or greater increase in rate of Ca2+ entry. The Ca signal is maintained dynamically by the balance between the increased Ca2+ influx and active Ca2+ efflux across the plasma membrane.

In intact lymphocytes, linoleic acid acts as a typical mitochondrial uncoupler: it substantially decreases the mitochondrial membrane potential and the cellular ATP level, while stimulating oxygen consumption and glycolysis. Under these conditions the inhibition of cellular functions by linoleic acid cannot be attributed to its postulated effects on lipid domains in the plasma membrane.

When a cell suspension is formed by disruption of a pig lymph node into medium, large and transient increases in intracellular cyclic AMP occur. Similar effects are observed when pig lymphocytes are centrifuged and the cell pellets resuspended, or when the cells are subjected to rapid temperature changes. These observations define the conditions required to manipulate the cells while maintaining a stable cyclic AMP concentration. Under these conditions, neither concanavalin A nor ionophore A23187 at mitogenic concentrations have any early effect on cyclic AMP in pig lymphocytes, but small increases in cyclic AMP (less than 2-fold) were observed at supramitogenic concentrations of concanavalin A (50 microgram/ml) or A23187 (500nM). Mouse thymocytes show qualitatively similar but much smaller changes in cyclic AMP concentration in response to experimental manipulations, and no response to mitogenic or supramitogenic concentrations of concanavalin A below the cytotoxic value.

The inhibition of anti-immunoglobulin cap formation on lymphocytes by cis-unsaturated fatty acids, but not saturated or trans-unsaturated fatty acids, which recently been reported (Klausner, R. N., Bhalla, D. K., Dragsten, P., Hoover, R. L., and Karnovsky, M. J. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 437-441), can be accounted for quantitatively by the effects of the fatty acids on ATP levels in the intact cells. Only the cis-unsaturated fatty acids lowered the cellular ATP level sufficiently (to < 80%) to inhibit cap formation significantly. The profile for the dependence of cap formation on cellular ATP level obtained with the fatty acids is very similar to the corresponding profile described previously for several capping ligands and a range of metabolic inhibitors used to depress the cellular ATP levels. Oxygen electrode experiments indicate that the unsaturated, but not the saturated, fatty acids can uncouple lymphocyte mitochondria in intact cells, whereas both types of fatty acids can uncouple isolated rat liver mitochondria. The difference in the effect of the two types of fatty acids on ATP levels in the intact cells is attributed to the inability of the saturated fatty acids to penetrate in sufficient concentrations to the mitochondria to cause uncoupling. The protective action of external calcium against inhibition of capping by the free fatty acids is attributed to the effect of calcium in cross-bridging the anionic fatty acids and reducing their effective concentration. These interpretations account for all of the experimental data and are much simpler than the model proposed by Klausner et al.

Three aspects of the calcium hypothesis we have proposed previously [Metcalfe, Pozzan, Smith & Hesketh (1980) Biochem. Soc. Symp. 45, 1-26] for the control of mitogenic stimulation of lymphocytes are examined in studies on the mitogenic action of the Ca2+ ionophore A23187 and its effect on cap formation. (1) Pig lymphocytes that were mitogenically stimulated by continuous incubation with 3H-labelled A23187 for 48 h contained between 3 and 15 amol of ionophore per cell. Lymphocytes exposed to 3H-labelled A23187 for 2h before washing the cells and resuspending them in ionophore-free medium were only stimulated mitogenically at 48h if the residual ionophore associated with the cells after washing was in the concentration range 3-15 amol per cell. When the cells were washed repeatedly after 2h incubation with ionophore to reduce the cell-associated ionophore below the critical concentration range, no mitogenic stimulation occurred as a result of short-term exposure to any ionophore concentration. Re-addition of ionophore to within the indicated range of cell-associated concentrations restored mitogenic stimulation at 48h. We conclude that large, short-term Ca2+ fluxes into the cells induced by the ionophore cannot generate a mitogenic signal that commits the cells to enter the cell cycle. (2) Further experiments with the ionophore showed that detectable mitogenic stimulation at 48h required a minimum of 3h exposure to optimal ionophore concentrations, and that maximal stimulation required at least 20h exposure. This is consistent with the view that a prolonged increase in the free cytoplasmic calcium concentration is required to stimulate the maximum proportion of the cells into the cell cycle. (3) Mouse splenic lymphocytes treated for short periods with very high ionophore concentrations (30 microM) in the presence of various external Ca2+ concentrations showed significant inhibition of cap formation of surface immunoglobulin receptors in the range 1-10 microM-Ca2+ in normal or depolarizing medium. We conclude that mitogens at optimal concentrations for the stimulation of lymphocytes do not cause any early increase in the free cytoplasmic Ca2+ concentration above 10 microM.

Pure complexes of dipalmitoyllecithin (DPL, 16:0) which Ca2+, Mg2+ dependent ATPase from sarcoplasmic reticulum are unusual in retaining significant ATPase activity down to about 30 degrees C, well below the transition temperature of the pure lipid at 41 degrees C. A minimum of about 35 lipid molecules per ATPase is required to maintain maximal ATPase activity, but the complexes are progressively and irreversibly inactivated at lower lipid to protein ratios. Complexes containing more than the minimum lipid requirement show very similar temperature profiles of activity about 30 degrees C over a wide range of lipid to protein ratios, up to 1500:1. Spin-label studies indicate that, at lipid to protein ratios of less than about 30 lipids per ATPase, no DPL phase transition can be detected, but at all higher ratios, a phase transition occurs at about 41 degrees C. In all of these complexes there are breaks in the Arrhenius plots of ATPase activity at 27--32 degrees C and at 37.5--38.5 degrees C. Experiments with perturbing agents, such as cholesterol and benzyl alcohol which have well-defined effects on the DPL phase transition, indicate that these breaks in the Arrhenius plots of ATPase activity cannot be attributed to a depressed and broadened phase transition in the lipids near the protein molecules. These results are interpreted as evidence for a phospholipid annulus of at least 30 lipid molecules with interact directly with the ATPase and cannot undergo a phase transition at 41 degrees C. This structural interaction of the ATPase with the annular DPL molecules has a predominant effect in determining the form of the temperature-activity profiles. However, the perturbation of the DPL phase transition does not extend significantly beyond the annulus since a phase transition which starts at 41 degrees C can be detected as soon as extraannular lipid is present in the complexes. We suggest that it may be a general feature of membrane structure that penetrant membrane proteins interact with their immediate lipid environment so as to cause only a minimal perturbation of the lipid bilayer.

Stimulated histamine release was depressed at least tenfold in mitotic 2H3 rat basophilic cells when compared with interphase cells even though both contained comparable amounts of histamine. Antigen stimulation of IgE-sensitized interphase cells initiated an influx of Ca2+ that preceded secretion of histamine and a similar Ca2+ influx occurred in stimulated mitotic cells. This strongly suggests that during mitosis there is a dramatic inhibition of one or more of the steps on the pathway leading from elevated intracellular Ca2+ to the fusion of secretory granules with the plasma membrane.

We have replaced the lipid associated with a purified calcium transport protein with a series of defined synthetic dioleoyl phospholipids in order to determine the effect of phospholipid headgroup structure on the ATPase activity of the protein. At 37 degrees C the zwitterionic phospholipids (dioleoyl phosphatidylcholine and dioleoyl phosphatidylethanolamine) support the highest activity, while a phospholipid with two negative charges (dioleoyl phosphatidic acid) supports an activity which is at least twenty times lower. Dioleoyl phospholipids with a single net negative charge support at intermediate ATPase activity which is not affected by the precise chemical structure of the phospholipid headgroup. The protocol used to determine the phospholipid headgroup specificity of calcium transport protein is novel because it establishes the composition of the lipid in contact with the protein without the need to isolate defined lipid-protein complexes. This allows the lipid specificity to be determined using only very small quantities of test lipids. We also determined the ability of the same phospholipids to support calcium accumulation in reconstituted membranes. Two requirements had to be met. The phospholipid had to support the ATPase activity of the pump protein and it had to form sealed vesicles as determined by electron microscopy. Since a number of phospholipids met those requirements it is clear that in vitro the lipid specificity of the calcium-accumulating system is rather broad.

The early biochemical responses stimulated by the action of mitogens and growth factors on mouse thymocytes and 3T3 fibroblasts are analysed as part of a systematic attempt to define the mitogenic pathways from G0 to S phase in these cells. Although the primary response to each mitogen can be distinguished by the pattern of secondary responses they initiate, there is substantial overlap in these responses. The aim is therefore to determine whether there is early convergence on a common mitogenic pathway, defined by a sequence of responses obligatory for progression from G0 to S phase for different mitogens and cell types. The 'dual-signal' hypothesis for the mitogenic stimulation of thymocytes is a simple version of a common mitogenic pathway. It proposes that the T-cell receptor initiates the pathway via the breakdown of phosphatidylinositol (4,5)-bisphosphate to generate a Ca signal (from the release of inositol (1,4,5)-trisphosphate) and to activate protein kinase C (from the release of diacylglycerol). The rationale for this hypothesis lies in the co-mitogenic action of the Ca2+-ionophore, A23187, and the phorbol ester, 12-o-tetradecanoyl phorbol 13-acetate, which is assumed to activate specifically protein kinase C. However, detailed analysis of the coupling between some of the early responses, including the Ca and pH signals, phosphatidylinositol (4,5)-bisphosphate metabolism, c-myc gene activation and general metabolic stimulation, indicates clearly that the hypothesis is inadequate to account for the initiation of the normal mitogenic pathway in thymocytes.

The free cytoplasmic Ca2+ concentration [( Ca2+]i) in rat brain synaptosomes estimated by the indicator quin 2 is 104 +/- 8 nM (S.D.) in artificial cerebrospinal fluid (1.2 mM Ca2+), but decreases at lower Ca2+ concentrations in the medium. The presence of quin 2 in the synaptosomes does not affect either the spontaneous release of transmitter (gamma-aminobutyric acid) or the release induced by K+ depolarisation. In quin 2-loaded synaptosomes, depolarisation by K+ causes an abrupt increase in [Ca]i (less than 2-fold) that is approximately proportional to the extent of depolarisation, whereas depolarisation by veratrine alkaloids produces a slow rise in [Ca]i. The increase in [Ca]i produced by K+ depolarisation does not occur in the absence of Ca2+ in the medium. The data are consistent with a direct correlation between [Cai] and transmitter release in functional synaptosomes. The pH in synaptosomes estimated by the indicator quene 1 is 7.04 +/- 0.07 and is stable in media containing 5 mM bicarbonate. The pH in synaptosomes was decreased by protoveratrine but not by K+ depolarisation.

A technique of transient permeabilisation has been used to show that the introduction of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), a non-hydrolysable analogue of GTP, into intact Swiss 3T3 fibroblasts stimulates phosphoinositide hydrolysis, cyclic AMP accumulation and the activation of c-fos and c-myc proto-oncogenes. Of a number of nucleotide triphosphates introduced into the cells, only GTP and its non-hydrolysable analogues activated inositol phosphate release, suggesting that this response is mediated by guanine nucleotide regulatory (G) protein(s). The data demonstrate that transient permeabilisation provides a method of examining the involvement of G-proteins in nuclear activation.

The membrane potential of mouse spleen lymphocytes has been assessed with two fluorescent probes. 3,3'-Dipropylthiadicarbocyanine (diS-C3-(5)) was used for most of the experiments. Solutions with high K+ concentrations depolarised the cells. Valinomycin, an inophore which adds a highly K+-selective permeability membranes, slightly hyperpolarised cells in standard (6 mM K+) solution, and in 145 mM K+ solution produced a slight additional depolarisation. These findings indicate a membrane whose permeability is relatively selective for K+. Very small changes in potential were seen when choline replaced Na+, or gluconate replaced Cl-, supporting the idea of K+ selectivity. The resting potential could be estimated from the K+ concentration gradient at which valinomycin did not change the potential-the "valinomycin null point" - and under the conditions used the resting potential was approx.-60 mV. B cell-enriched suspensions were prepared either from the spleens of nu/nu mice or by selective destruction of T cells in mixed cell populations. The membrane potential of these cells was similar to that estimated for the mixed cells. In solution with no added K+, diS-C3-(5) itself appeared to depolarise the lymphocytes, in a concentration dependent manner. With the 100 nM dye normally used, the membrane potential in K+-free solution was around -45 mV, and 500 nM dye almost completely depolarised the cells. In standard solution quinine depolarised the cells. Valinomycin could still depolarise these cells indicating that depolarisation had not been due to dissipation of the K+ gradient. Since in K+-free solution diS-C3-(5) blocks the Ca2+-activated K+ channels in human red blood cell ghosts and quinine also blocks this K+ channel it is suggested that the resting lymphocyte membrane may have a similar Ca2+-activated K+ permeability channel. Because of the above mentioned effect of diS-C3-(5) and other biological side effects, such as inhibition of B cell capping, a chemically distinct fluorescent probe of membrane potential, bis(1,3-diethylthiobarbiturate)-trimethineoxonol was used to support the diS-C3-(5) data. This new probe proved satisfactory except that it formed complexes with valinomycin, ruling out the use of this ionophore. Results with the oxonol on both mixed lymphocytes and B cell-enriched suspensions gave confirmation of the conclusions from diS-C3-(5) experiments and indicated that despite its biological side effects, diS-C3-(5) could still give valid assessment of membrane potential.

The effects of inhibitors of mitochondrial ATP synthesis and the calcium ionophore, A23187, on the capping of surface immunoglobulin, concanavalin A receptors and theta antigen on mouse spleen or thymus cells have been examined. (i) For all of these capping ligands and inhibitors, the cellular ATP level must be above 80% of the normal level in resting lymphocytes for 90% of maximal cap formation to occur. Below 50% of the normal ATP level, less than 10% of maximal capping occurs. There is, therefore, a common dependence for all three capping systems on the cellular ATP level, irrespective of the metabolic inhibitor used. (ii) Inhibition of cap formation by A23187 follows the same profile for ATP dependence as the mitochondrial inhibitors, but in contrast to those inhibitors, A23187 requires extracellular calcium to decrease the ATP level and inhibit capping. Other agents can affect cap formation without reducing the ATP level. For example, concanavalin A inhibits its own cap formation and cytochalasin B reduces the rate of cap formation at concentrations which do not alter the cellular ATP level. (iii) From these and other data we conclude that there are cellular functions essential for cap formation, other than the maintenance of ionic gradients, that require a high concentration of cellular ATP. The possibility that high levels of ATP are required for the function of the cytoskeleton in lymphocytes is discussed.

Photometric fluorescence microscopy has been used to measure intracellular pH (pHi) and free calcium concentrations [( Ca]i) in individual mouse thymocytes and 2H3 rat basophil leukaemic cells containing indicators for pH (quene 1) or calcium (quin 2). The pHi and [Ca]i measurements in individual 2H3 cells and mouse thymocytes and their responses to various stimuli were consistent with the corresponding data obtained from suspensions of these cells measured in a spectrofluorimeter. Photometric fluorescence microscopy of these indicators in individual cells provides a sensitive and fast method of following pHi and [Ca]i responses in individual cells.

The effects of the lectins concanavalin A, succinylated concanavalin A, and wheat germ agglutinin on the free cytoplasmic Ca2+ concentration in mouse thymocytes were measured using the fluorescent Ca2+ indicator "quin 2" (Tsien, R. Y. (1980) Biochemistry 19, 2396-2404) and compared with the metabolic and mitogenic effects of the lectins on the cells. Within 1 min of adding each ligand, there is a dose-dependent increase in the free cytoplasmic Ca2+ concentration reported by quin 2. This response is selective for Ca2+, but it does not coincide closely with the subsequent mitogenic stimulation at 48 h by concanavalin A or succinyl concanavalin A. The nonmitogenic lectin wheat germ agglutinin also causes an increase in free cytoplasmic Ca2+ concentration and early metabolic stimulation of the cells, but stimulation is self-aborted before DNA synthesis occurs. At the intracellular concentrations of quin 2 required for measurement of the free Ca2+ concentration, the chelator causes early metabolic stimulation of the cells very similar to that produced by concanavalin A and the mitogenic Ca2+ ionophore A23187. Thus, phosphatidylinositol metabolism and lactate production are stimulated in mouse thymocytes and pig lymphocytes within 1 h of loading with quin 2 and significant increases in RNA synthesis occur after 8 h. Quin 2 causes mitogenic stimulation of pig lymphocytes measured as increased [3H]thymidine uptake at 48 h, that is variable but substantial in most experiments (up to 100% of the stimulation by A23187). The chelator itself has no significant activity as a Ca2+ ionophore, but the apparent free Ca2+ concentration in the cells increases both with the concentration of intracellular quin 2 and with the extracellular Ca2+ concentration. These data leave open the possibility that quin 2 itself affects the concentration of free Ca2+ or other cations in the cells.

An increase in the free cytoplasmic Ca2+ concentration in thymocytes can be detected by the fluorescent indicator quin 2 within a few seconds of the addition of concanavalin A and the response is quantified from the increased proportion of quin 2 in the cells chelated by Ca2+ ('% Ca-quin 2'). The % Ca-quin 2 in untreated cells is 53 +/- 6%, increases to 64 +/- 7% immediately after the addition of concanavalin A and declines spontaneously over 24 h back to the level in untreated cells (53 +/- 6%). The increase in % Ca-quin 2 in response to concanavalin A is completely blocked when 50 mM-alpha-methyl D-mannoside is added before concanavalin A and completely reversed when the competing sugar is added immediately after the mitogen. Addition of alpha-methyl D-mannoside at increasing intervals after concanavalin A addition causes a progressively smaller decrease in % Ca-quin 2 and has a negligible effect after 24 h, when the % Ca-quin 2 is the same as that in untreated cells. The decline in the calcium signal defined by these experiments has a similar time course to cap formation by concanavalin A on the cells. It is concluded that the calcium signal lasts only while concanavalin A is bound to the cell surface and is terminated either by capping or by the addition of alpha-methyl D-mannoside.

Concentrations of concanavalin A or the calcium ionophore A23187 that are optimal for the transformation of pig or mouse lymphocytes do not normally cause a measurable increase in calcium influx compared with unstimulated cells. If the cells are treated with the mitogens in conditions where a measurable increase in calcium influx occurs, no stimulation of the cells can occur while the flux is maintained. If an early influx of extracellular calcium is necessary for stimulation, then a much smaller increase in the total concentration of cellular calcium than reported previously is sufficient to allow the entry of lymphocytes into the cell cycle.

Quiescent 3T3 fibroblasts grown on microcarrier beads and loaded with the [Ca2+] indicator quin2 had a cytosolic free Ca2+ concentration ( [Ca2+]i) of 154 +/- 11 nM (SE; n = 32). Stimulation with the mitogens vasopressin, epidermal growth factor (EGF) or prostaglandin F2 alpha (PGF2 alpha) caused a very rapid increase in [Ca2+]i to a maximum of 200-500 nM after 60-90 s. [Ca2+]i declined thereafter to a level above that in quiescent cells which was maintained for at least 15 min. In contrast no immediate effects on [Ca2+]i were detected after the addition of the mitogens insulin or 12-O-tetradecanoylphorbol 13-acetate (TPA). These studies indicate that early changes in [Ca2+]i may be involved in the action on fibroblasts of some, but not all, mitogens.

The aim of this study was to determine whether the increase in cytosolic free Ca2+ concentration ([Ca2+]i) in response to antigen (aggregated ovalbumin) on IgE-primed 2H3 cells was sufficient to account for exocytosis. When the [Ca2+]i responses to antigen and the Ca2+ ionophore A23187 were compared, A23187 was much less effective at releasing histamine at equivalent [Ca2+]i increases, and little or no stimulated histamine release occurred with A23187 concentrations that matched the [Ca2+]i response to antigen concentrations that stimulated maximal histamine release. The [Ca2+]i response to antigen is not, therefore, sufficient to account for exocytosis, although extracellular Ca2+ is necessary to initiate both the [Ca2+]i response and histamine release: the antigen must generate an additional, unidentified, signal that is required for exocytosis. To determine whether this signal was the activation of protein kinase C, the effects of the phorbol ester 12-0-tetradecanoyl phorbol 13-acetate (TPA) on the responses to antigen were examined. TPA blocked the antigen-induced [Ca2+]i response and the release of inositol phosphates but had little effect on histamine release and did not stimulate exocytosis by itself. The unidentified signal from the antigen is therefore distinct from the activation of protein kinase C and is generated independently of the [Ca2+]i response or the release of inositol phosphates. Taken together with other data that imply that there is very little activation of protein kinase C by antigen when the rate of histamine release is maximal, it is concluded that the normal exocytotic response to antigen requires the synergistic action of the [Ca2+]i signal together with an unidentified signal that is not mediated by protein kinase C.

The interaction of the n-alkanols with lipid bilayers and excitable membranes shows that there is no simple correlation between conduction block and any of the perturbations of bilayer structure currently proposed as unitary mechanisms of local anaesthetic action. We propose instead that the n-alkanols act by direct interaction with target proteins to cause perturbations which depend directly on the precise structure of the alcohol.

The development is described of an Na+ chelator with appropriate properties for an indicator of intracellular free Na+ concentration ([Na+]i). The new indicator, FCryp-1, is a tribenzo derivative of the parent (2:2:1) cryptand structure, incorporating the same F-substituted dibenzo 19F-NMR reporter group as the free [Ca2+] indicator, 5FBAPTA (Smith, G.A., Hesketh, T.R., Metcalfe, J.C., Feeney, J. and Morris, P.G. (1983) Proc. Natl. Acad. Sci., USA 80, 7178-7182). FCryp-1 has appropriate affinity for Na+ (KNa = 10(1.3) M-1) and selectivity over other intracellular cations (KK; KCa; K Mg less than 10(-1) M(-1)) for a [Na]i indicator. There is an 19F-NMR chemical shift of 2.00 ppm between free FCryp-1 and the Na-FCryp-1 complex which provides a direct read out of free [Na+]. FCryp-1 carries four carboxylate groups to confer aqueous solubility which can be esterified with acetoxymethyl groups to render the indicator membrane permeant. Experiments on pig lymphocytes loaded with FCryp-1 gave an indicated [Na+]i of 13.8 +/- 1.8 mM (n = 4). The FCryp-1 structure can also be readily modified to provide fluorescent [Na+]i indicators.

Among the earliest responses to mitogens that have been detected in normal quiescent cells are ionic changes: we have described rapid increases in the cytosolic free Ca2+ concentration ([Ca]i) and in the intracellular pH (pHi) in mitogen-stimulated thymocytes and fibroblasts (Hesketh, T. R., Moore, J. P., Morris, J. D. H., Taylor, M. V., Rogers, J., Smith, G. A., and Metcalfe, J. C. (1985) Nature 313, 482-484). Here we investigate the relationship between these ionic signals and the subsequent expression of the c-fos and c-myc proto-oncogenes in murine thymocytes. We show that the plant lectin concanavalin A (ConA), the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) and the Ca2+-ionophore A23187 each causes a rapid increase in both c-fos and c-myc mRNAs. The activation of both genes is completely dependent on the extracellular Ca2+ concentration ([Ca]o) for A23187 and independent of [Ca]o for TPA. Activation of c-myc, but not c-fos, by ConA is partially dependent on [Ca]o. The pHi increases generated by ConA or TPA are not necessary for expression of mRNA from either gene in response to these mitogens. Exogenous 8-bromo-cyclic AMP (but not 8-bromo-cyclic GMP) inhibits the c-myc responses to ConA and TPA. The data also show that neither early c-fos nor c-myc expression is sufficient to commit the cells to DNA synthesis.

1. Activation of adenylate cyclase in rat liver plasma membranes by fluoride or GMP-P (NH)P yielded linear Arrheniun plots. Activation by glucagon alone, or in combination with either fluoride or GMP-P(NH)P resulted in biphasic Arrhenius plots with a well-defined break at 28.5 +/- 1 degrees C. 2. The competitive glucagon antagonist, des-His-glucagon did not activate the adenylate cyclase but produced biphasic Arrhenius plots in combination with fluoride or GMP-P(NH)P. The break temperatures and activation energies were very similar to those observed with glucagon alone, or in combination with either fluoride or GMP-P(NH)P. 3. It is concluded that although des-His-glucagon is a potent antagonist of glucagon, it nevertheless causes a structural coupling between the receptor and the catalytic unit.

Comparisons were made between transformed S49 T-lymphoma cells and normal murine thymocytes in their polyphosphoinositides, inositol polyphosphates and cytosolic free calcium concentrations ([Ca2+]i), and the effects of the T-cell mitogen concanavalin A (Con A) on these properties. 1. The ratios of the polyphosphoinositides to phosphatidylinositol in both exponential-phase S49 cells and mitogen-stimulated thymocytes (G1 phase) were greater than in quiescent (G0-phase) thymocytes. 2. In response to Con A, the amount of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) in S49 cells decreased slightly (17% in 30 min), and this was sufficient to account for the small amounts of inositol phosphates that accumulated. In contrast, it has been shown previously that Con A stimulates a rapid resynthesis of PtdInsP2 in thymocytes and the amounts of inositol phosphates released rapidly exceed the steady-state amount of the PtdInsP2 precursor [Taylor, Metcalfe, Hesketh, Smith & Moore (1984) Nature (London) 312, 462-465]. 3. The [Ca2+]i did not differ significantly in S49 cells and thymocytes before the addition of Con A, and the increases in [Ca2+]i in response to Con A were similar in both types of cell. 4. The [Ca2+]i increase in response to Con A was inhibited by similar concentrations of intracellular cyclic AMP (2-10 microM) in S49 cells and thymocytes, suggesting that similar regulatory mechanisms act on this response in both types of cell. The data demonstrate that the basal [Ca2+]i and phosphoinositide metabolism is similar in both the normal cells and their transformed counterparts. In addition, they suggest that the activated Con A receptors generate very similar signals in the two cell types, and that any perturbations of primary signal transduction to the secondary phosphoinositide and [Ca2+]i responses in the S49 phenotype are quantitative rather than qualitative.

Rat basophil leukaemic (2H3) cells, mast cells and mouse thymocytes respond to stimulation by specific ligands with an increase in the free cytosolic Ca2+ concentration. The time courses of these Ca signals and the biological responses have been compared with changes in phospholipid metabolism. Increased phosphoinositide metabolism coincides with the Ca signals and the responses in each cell system, whereas any increase in phospholipid methylation during the response is less than one molecule per receptor and at least 5-50-fold less than the increases reported previously. Furthermore, no significant changes were detected in the concentration of S-adenosylmethionine, the methyl-group donor in the synthesis of methylated phospholipids. The hypothesis that phospholipid methylation is obligatory for receptor-mediated Ca signals is not supported by these data and requires critical re-evaluation.

A new fluorescent intracellular pH indicator is described ("quene 1") which is related to the tetracarboxylate Ca2+ indicator based on the quinoline fluorophor ("quin 2"). Quene 1 has excitation and emission maxima at 390 and 530 nm, respectively, and shows a 30-fold increase in fluorescence between pH 5 and 9 with a pK alpha of 7.3. The fluorescence is insensitive to Ca2+ and Mg2+ at free concentrations up to 10(-4) M and to the proportions of Na+ and K+ at total concentrations of Na+ and K+ from 100 to 200 mM. The indicator is loaded into thymocytes using the tetraacetoxymethyl ester derivative which is hydrolyzed in the cells to give the tetracarboxylate anion. Intracellular pH can be measured at intracellular quene 1 concentrations of approximately 0.1 mM and quene 1 does not perturb glycolysis or the ATP level in resting cells at concentrations up to 0.8 mM. The intracellular pH of mouse thymocytes indicated by quene 1 is 7.15 +/- 0.04 and it is insensitive to the concentration of Ca2+ or Mg2+ in the extracellular medium. The intracellular pH decreased when the pH of the medium was lowered by addition of HCl, but was insensitive to NaOH at extracellular pH values up to 8.0. Rapid transient changes in intracellular pH are induced by NH4Cl, NaCO2CH3, or HCO3-/CO2. The thymocytes showed no early changes (within 30 min) in intracellular pH in response to mitogenic concentrations of lectins or 4 beta-phorbol-12-myristate-13-acetate.

We have recently described a cryptand structure, FCryp-1, with appropriate properties for an indicator of intracellular free Na+ concentration using the 19F-n.m.r. chemical shift of the incorporated 5FBAPTA [1,2-bis-(2-amino-5-fluorophenoxy)ethane-NNN'N'-tetra-acetic acid] reporter group to measure the free cytosolic Na+ concentration [( Na+]i) [Smith, Morris, Hesketh and Metcalfe (1986) Biochim. Biophys. Acta 889, 82-83]. FCryp-1 carries four carboxylate groups to confer aqueous solubility and the indicator is membrane-permeant when the carboxyls are esterified with acetoxymethyl ester groups. Here we describe the synthesis of FCryp-2 to provide a fluorescent indicator of [Na+]i. FCryp-2 retains the parent tribenzo (2:2:1) cryptand structure of FCryp-1, in which the benzenoid ring at C-21 in FCryp-1 is replaced by an indole derivative which acts as the fluorophor in FCryp-2. With excitation at 340 nm, FCryp-2 gives an emission maximum at 460 nm in the absence of Na+ which shifts to 395 nm when FCryp-2 is saturated with Na+, with an isosbestic point at 455 nm. The apparent dissociation constant of FCryp-2 in a buffer solution of 100 mM-KCl/20 mM-KH2PO4/K2HPO4, pH 7.0, at 37 degrees C is 6.0 mM and the free Na+ concentration can be measured either from the calibrated fluorescence intensity at 395 nm, which increases 25-fold when Na+ is bound to FCryp-2, or from the ratio of fluorescence intensities at 395 nm and 455 nm. The measurement of free [Na+] by either method is unaffected by K+, Ca2+ or Mg2+ in the normal intracellular concentration ranges. Free [Na+] measurements by the ratio method are unaffected by pH from 6.6 to 7.6.