Studies on the mechanism of action of guinea pig lymphotoxin. I. Membrane active substances prevent target cell lysis by lymphotoxin.

The effect of various pharamacologic agents on lysis of L cells by guinea pig lymphotoxin (LT) was studied. Among the many drugs tested, only those that affect plasma membrane functions were found to interfere with the cytolytic action of LT. Dimethyl sulfoxide or lidocaine potentiated L cell resistance against lysis. Stronger protection was provided by ouabain. Addition of ouabain to cells previously injured by LT was also effective in reducing cell death. Attempts to detect metabolic disturbances in cells before LT cytolysis were unsuccessful. The biosynthetic rate of DNA, RNA, or protein, and the total cellular content of ATP were not significantly changed in LT-treated cells. The results suggest that LT disturbs some plasma membrane functions of the target cell, perhaps ion transport systems, and consequently induces ionic imbalances between the intra-and extracellular milieu which eventually cause cell death.     

Calcium uptake studies of 1,4-dihydropyridine agonists into rabbit aortic smooth muscle cells in culture

The effects of the three dihydropyridine calcium channel agonists (+/-)BAY K 8644, (+)202-791 and (+/-)CGP 28392 on 45Ca++ uptake were studied in cultures of rabbit aortic smooth muscle cells. At 10(-7) M each agonist enhanced 45Ca++ uptake in 15-50 mM K+ but had no effect on the basal 45Ca++ uptake at 5 mM K+. At the uptake threshold of 15 mM K+ each agonist potentiated 45Ca++ uptake in a dose-dependent manner with half maximal effects at 2.4 nM for (+/-)BAY K 8644, 22 nM for (+)202-791 and 18 nM for (+/-)CGP 28392. The agonists showed no significant antagonistic activity. Responses were antagonized competitively by nifedipine and non-competitively by (+/-)D-600. The 45Ca++ uptake dose-response curves and the half maximal effects of the three agonists were over the same range of concentrations as their inhibition of [3H]nitrendipine binding to rat ventricular receptor membrane preparations. The data suggest that these cells mimic the calcium uptake by the intact aorta better than commercial vascular smooth muscle lines or cardiac cells.     

Effects of newly reported arachidonic acid metabolites on microsomal Ca++ binding, uptake and release

The 5,6-; 8,9-; 11,12- and 14,15-epoxyeicosatrienoic acids and their respective hydration products, the vic-diols, recently reported as metabolites of arachidonic acid in rat liver microsomes, were examined for effect on release of 45Ca from canine aortic smooth muscle microsomes. At 10(-6) M, the diols had no effect, but the 5,6-; 11,12- and 14,15-epoxyacids increased the loss of 45Ca. Further studies with the 14,15-epoxyacid demonstrated a dose-dependent decrease of Ca++ uptake (ATP present) in canine aortic microsomes in 0.03 mM Ca++, whereas Ca++ binding (ATP absent) was not affected. Ca++ uptake, binding and release in rat liver microsomes was similarly affected by the 14,15-epoxyacid, the major epoxyeicosatrienoic acid derivative produced by rat liver microsomal incubations. It is suggested that alterations in Ca++ metabolism might be a possible mechanism of action for these derivatives of arachidonic acid.     

Attractant-induced changes and oscillations of the extracellular Ca++ concentration in suspensions of differentiating Dictyostelium cells

We used a Ca++-sensitive electrode to measure changes in extracellular Ca++ concentration in cell suspensions of Dictyostelium discoideum during differentiation and attractant stimulation. The cells maintained an external level of 3-8 microM Ca++ until the beginning of aggregation and then started to take up Ca++. The attractants, folic acid, cyclic AMP, and cyclic GMP, induced a transient uptake of Ca++ by the cells. The response was detectable within 6 s and peaked at 30 s. Half-maximal uptake occurred at 5 nM cyclic AMP or 0.2 microM folic acid, respectively. The apparent rate of uptake amounted to 2 X 10(7) Ca++ per cell per min. Following uptake, Ca++ was released by the cells with a rate of 5 X 10(6) ions per cell per min. Specificity studies indicated that the induced uptake of Ca++ was mediated by cell surface receptors. The amount of accumulated Ca++ remained constant as long as a constant stimulus was provided. No apparent adaptation occurred. The cyclic AMP-induced uptake of Ca++ increased during differentiation and was dependent on the external Ca++ concentration. Saturation was found above 10 microM external Ca++. The time course and magnitude of the attractant-induced uptake of external Ca++ agree with a role of Ca++ during contraction. During development the extracellular Ca++ level oscillated with a period of 6-11 min. The change of the extracellular Ca++ concentration during one cycle would correspond to a 30-fold change of the cellular free Ca++ concentration.     

Effects of vasopressin and La3+ on plasma-membrane Ca2+ inflow and Ca2+ disposition in isolated hepatocytes. Evidence that vasopressin inhibits Ca2+ disposition.

Vasopressin caused a 40% inhibition of 45Ca uptake after the addition of 0.1 mM-45Ca2+ to Ca2+-deprived hepatocytes. At 1.3 mM-45Ca2+, vasopressin and ionophore A23187 each caused a 10% inhibition of 45Ca2+ uptake, whereas La3+ increased the rate of 45Ca2+ uptake by Ca2+-deprived cells. Under steady-state conditions at 1.3 mM extracellular Ca2+ (Ca2+o), vasopressin and La3+ each increased the rate of 45Ca2+ exchange. The concentrations of vasopressin that gave half-maximal stimulation of 45Ca2+ exchange and glycogen phosphorylase activity were similar. At 0.1 mM-Ca2+o, La3+ increased, but vasopressin did not alter, the rate of 45Ca2+ exchange. The results of experiments performed with EGTA or A23187 or by subcellular fractionation indicate that the Ca2+ taken up by hepatocytes in the presence of La3+ is located within the cell. The addition of 1.3 mM-Ca2+o to Ca2+-deprived cells caused increases of approx. 50% in the concentration of free Ca2+ in the cytoplasm [( Ca2+]i) and in glycogen phosphorylase activity. Much larger increases in these parameters were observed in the presence of vasopressin or ionophore A23187. In contrast with vasopressin, La3+ did not cause a detectable increase in glycogen phosphorylase activity or in [Ca2+]i. It is concluded that an increase in plasma membrane Ca2+ inflow does not by itself increase [Ca2+]i, and hence that the ability of vasopressin to maintain increased [Ca2+]i over a period of time is dependent on inhibition of the intracellular removal of Ca2+.     

Lectin- and ionophore-stimulated Ca2+ influx in murine lymphocytes: inhibition by disialoganglioside

Gangliosides suppress lymphocyte mitogenesis when added exogenously to the cells. On the premise that the mechanism of ganglioside action may be an interference with primary induction events, mitogen-induced 45Ca2+ influx in murine lymphocytes was studied. Disialoganglioside (GD1a) at physiopathological concentrations inhibits concanavalin A-induced 45Ca2+ uptake as well as blast transformation. The suppressive action of GD1a is both concentration dependent (50% suppression at 13 microM) and very rapid (within 1 min). GD1a is not cytotoxic nor does it significantly alter the rate of Ca2+ efflux. The uptake studies were extended to A23187, a compound with mitogenic and specific divalent cation ionophore activities. Ca2+ uptake by lymphoid cells from AKR/J, Swiss, and CBA mice is stimulated by A23187; and GD1a, in a dose-dependent manner, inhibits the ionophore-induced 45Ca2+ influx. Pretreatment of thymocytes with GD1a renders the cells greatly insensitive to the subsequent ionophore activity of A23187. The results suggest that exogenous gangliosides may function as an inhibitor of some of the mitogen-triggered early events, including Ca2+ metabolism, and thus influence the immunological behavior of intact lymphoid cells.     

Stimulus-secretion coupling in the developing exocrine pancreas: secretory responsiveness to cholecystokinin

We have studied the onset of secretory responsiveness to cholecystokinin (CCK) during development of the rat exocrine pancreas. Although acinar cells of the fetal pancreas (1 d before birth) are filled with zymogen granules containing the secretory protein, alpha- amylase, the rate of amylase secretion from pancreatic lobules incubated in vitro was not increased in response to CCK. In contrast, the rate of CCK-stimulated amylase discharge from the neonatal pancreas (1 d after birth) was increased four- to eightfold above that of the fetal gland. The postnatal amplification of secretory responsiveness was not associated with an increase in the number or cell surface expression of 125I-CCK binding sites. When 125I-CCK-33 binding proteins were analyzed by affinity crosslinking, two proteins of Mr 210,000 and 100,000-160,000 were labeled specifically in both fetal and neonatal pancreas. To determine if cell surface receptors for CCK in the fetal pancreas are functional and able to generate a rise in the cytosolic [Ca++], we measured 45Ca++ efflux from tracer-loaded lobules. 45Ca++ efflux from both fetal and neonatal pancreas was comparably increased by CCK, indicating CCK-induced Ca++ mobilization and elevated cytosolic [Ca++]. The Ca++ ionophore A23187 also stimulated the rate of 45Ca++ extrusion from pancreas of both ages. Increased amylase secretion occurred concurrently with A23187-stimulated 45Ca++ efflux in neonatal pancreas, but not in the fetal gland. A23187 in combination with dibutyryl cAMP potentiated amylase release from the neonatal gland, but not from fetal pancreas. Similarly, the protein kinase C activator, phorbol dibutyrate, did not increase the rate of secretion from the fetal gland when added alone or in combination with A23187 or CCK. We suggest that CCK-receptor interaction in the fetal pancreas triggers intracellular Ca++ mobilization. However, one or more signal transduction events distal to Ca++ mobilization have not yet matured. The onset of secretory response to CCK that occurs postnatally may depend on amplification of these transduction events.     

Transport and control of Ca2+ by pigeon erythrocytes. III. A 'paradoxical' expulsion of Ca2+ induced by a low dose of A23187 at 0 degrees C

Pigeon erythrocytes expelled preloaded 45Ca2+ in response to a low dose of A23187 at 0 degrees C. We call this phenomenon 'paradoxical' expulsion. Within the first minute, 1.85 +/- 0.38 mumol/l cell water was expelled; after that the internal 45Ca2+ began to rise. The rises in Ca2+ uptake with and without A23187 addition were essentially paralleled. No premonitory rise of 45Ca2+ upon the addition of A23187 was observed. Expulsion of 45Ca2+ in response to A23187 was probably by the action of the Ca2+ pump and not by Na+-Ca2+ exchange since vanadate inhibited, but K+ replacement of Na+ in the medium had no effect. Lysophosphatidylcholine (lysoPC) caused an abrupt increase in 45Ca2+ influx by cells at 0 degrees C and was dose dependent. However, a very low dose of lysoPC induced expulsion of preloaded 45Ca2+ similar to that by A23187, the response was fast and transitory, without any premonitory rise in 45Ca2+ uptake. The results lend support to the suggestion that the signal to which cells respond may be a sudden change in Ca2+ influx per se rather than a change in internal Ca2+ concentration. These features of 'paradoxical' 45Ca2+ expulsion induced by A23187 and lysoPC are not expected from mass-action equilibria but, instead, agree with the characteristics of an energy-dissipating control mechanism.     

Calcium permeability changes and neurotransmitter release in cultured rat brain neurons. I. Effects of stimulation on calcium fluxes

The permeability of neuronal membranes to Ca2+ is of great importance for neurotransmitter release. The temporal characteristics of Ca2+ fluxes in intact brain neurons have not been completely defined. In the present study 45Ca2+ was used to examine the kinetics of Ca2+ influx and efflux from unstimulated and depolarized rat brain neurons in culture. Under steady-state conditions three cellular exchangeable Ca2+ pools were identified in unstimulated cells: 1) a rapidly exchanging pool (t1/2 = 7 s) which represented about 10% of the total cellular Ca2+ and was unaffected by the presence of Co2+, verapamil, or tetrodotoxin; 2) a slowly exchanging pool (t1/2 = 360 s) which represented 42% of the total cellular Ca2+ and was inhibited by Co2+, but not by verapamil or tetrodotoxin; 3) a very slowly exchanging pool (t1/2 = 96 min) which represented 48% of the total cell Ca2+ was observed only in the prolonged efflux experiments. The rate of exchange of 45Ca2+ in the unstimulated cells was dependent on the extracellular Ca2+ concentration (half-saturation at 70 microM). Depolarization of the neurons with elevated K+ causes a rapid and sustained 45Ca2+ uptake. The cellular Ca2+ content increased from 56 nmol/mg protein in unstimulated cells to 81 nmol/mg protein during 5 min of depolarization. The kinetics of the net 45Ca2+ uptake by the stimulated neurons was consistent with movement of the ion with a first order rate constant of 0.0096 s-1 (t1/2 = 72 s) into a single additional compartment. The other cellular Ca2+ pools were apparently unaffected by stimulation. The stimulated 45Ca2+ uptake was inhibited by Co2+ and by the Ca2+ channel blocker verapamil but not by the Na+ channel blocker tetrodotoxin. Ca2+ uptake into this compartment was dependent on the extracellular Ca2+ concentration (half-saturation at 0.80 mM Ca2+). Predepolarization of the cells with high K+ for 10-60 s prior to the addition of the radioactive calcium did not alter the rate of 45Ca2+ incorporation into the stimulated cells. It is concluded that the rapidly exchanging, the slowly exchanging, and the depolarization-induced Ca2+ pools observed in intact brain neurons are physically as well as kinetically distinct from each other. In addition, the depolarization-induced component observed in stimulated cells represents movement of the Ca2+ ions through a single class of voltage-sensitive Ca2+ channels. These Ca2+ channels are inhibited by Co2+ ions and by verapamil and are not inactivated during depolarization of the brain neurons.     

Intracellular calcium pools and their metabolic dependence in normal versus simian virus 40-transformed human fibroblasts

Previous studies indicate that although normal and Simian virus (SV40)-transformed WI38 human fibroblasts have similar levels of intracellular Ca++ on a per mg protein basis, their ability to maintain this intracellular Ca++ against a low concentration of extracellular Ca++ differs markedly. The transformed but not the normal cells rapidly lose Ca++ when exposed to low extracellular Ca++, suggesting Ca++ transport and/or sequestration differ in the two cell types. In this study we have extended our investigations of Ca++ metabolism in the two cell types. We observe that normal WI38 cells, when exposed to metabolic inhibitors to deplete intracellular ATP, undergo a twofold increase in intracellular Ca++ levels. Under similar conditions and over the same time course, no comparable change in Ca++ level is observed in the SV40-transformed cell, despite the extensive depletion of ATP. 45Ca++ desaturation curves indicate that the bulk of the net increase in cell Ca++ following ATP depletion of the normal WI38 cell comes to reside in a slowly exchanging Ca++ pool. The data also indicate that glycolysis, and not oxidative phosphorylation, drives the active extrusion of Ca++ from these cells, an observation consistent with previous studies on the Na+-K+ pump in other cell types. Finally, the data indicate that in these cells mitochondria do not appear to be the major subcellular organelle responsible for regulation of at least the two cellular Ca++ pools measurable using isotope desaturation analysis. This is based on the inability of the respiratory inhibitor rotenone to alter significantly the size of either of these Ca++ pools. These pools compose 80-90% of total cell Ca++ in both cell types.     

Hepatic insulin effects independent of changes in Ca++ metabolism

The possibility of Ca++ acting as second messenger for insulin in rat liver was investigated using the net stimulation of 14C-glucose incorporation into glycogen by isolated hepatocytes as an index of insulin action. An insulin effect could be partially sustained in the virtual absence of Ca++ and Mg++ and a maximal insulin effect could be observed in the presence of either Ca++ or Mg++, suggesting that extracellular Ca++ is not required for insulin action. Inhibiting the activity of calmodulin, an intracellular mediator of Ca++ action, with trifluoperazine had little effect on insulin action. The efflux of 45Ca from prelabeled hepatocytes was not altered by the presence of insulin arguing against insulin-induced changes in Ca++ fluxes. Collectively, these results do not support the role of Ca++ as second messenger for insulin action in liver.     

Calcium regulation of growth and differentiation of mouse epidermal cells in culture

Modification of the ionic calcium concentration in the culture medium markedly alters the pattern of proliferation and differentiation in cultured mouse epidermal cells. When medium calcium is lowered to 0.05--0.1 mM, keratinocytes proliferate rapidly with a high growth fraction and do not stratify, but continue to synthesize keratin. The cells grow as a monolayer for several months and can be subcultured and cloned in low Ca++ medium. Ultrastructural examination of cells cultured under low Ca++ conditions reveals widened intercellular spaces, abundant microvilli and perinuclear organization of tonofilaments and cellular organelles. Desmosomes are absent. Epidermal cells growing as a monolayer in low Ca++ can be induced to terminally differentiate by adding calcium to the level normally found in the culture medium (1.2 mM). Cell-to-cell contact occurs rapidly and desmosomes form within 2 hr. The cells stratify by 1--2 days and terminally differentiate with cell sloughing by 3--4 days. After Ca++ addition, DNA synthesis decreases with a lag of 5--10 hr and is totally inhibited within 34 hr. In contrast, RNA and protein synthesis continue at 40--50% of the low Ca++ level at day 3, a time when many cells are detaching from the culture dish. Keratin synthesis is unaffected by the Ca++ switch.     

Interactions of Schwann cells with neurites and with other Schwann cells involve the calcium-dependent adhesion molecule, N-cadherin.

During embryogenesis, Schwann cells interact with axons and other Schwann cells, as they migrate, ensheath axons, and participate in organizing peripheral nervous tissues. The experiments reported here indicate that the calcium-dependent molecule, N-cadherin, mediates adhesion of Schwann cells to neurites and to other Schwann cells. Cell cultures from chick dorsal root ganglia and sciatic nerves were maintained in media containing either 2 mM Ca++ or 0.2 mM Ca++, a concentration that inactivates calcium-dependent cadherins. When the leading lamellae of Schwann cells encountered migrating growth cones in medium with 2 mM Ca++, they usually remained extended, and the growth cones often advanced onto the Schwann cell upper surface. In the low Ca++ medium, the frequency of withdrawal of the Schwann cell lamella after contact with a growth cone was much greater, and withdrawal was the most common reaction to growth cone contact in medium with 2 mM Ca++ and anti-N-cadherin. Similarly, when motile leading margins of two Schwann cells touched in normal Ca++ medium, they often formed stable areas of contact. N-cadherin and vinculin were co-concentrated at these contact sites between Schwann cells. However, in low Ca++ medium or in the presence of anti-N-cadherin, interacting Schwann cells usually pulled away from each other in a behavior reminiscent of contact inhibition between fibroblasts. In cultures of dissociated cells in normal media, Schwann cells frequently were aligned along neurites, and ultrastructural examination showed extensive close apposition between plasma membranes of neurites and Schwann cells. When dorsal root ganglia explants were cultured with normal Ca++, Schwann cells migrated away from the explants in close association with extending neurites. All these interactions were disrupted in media with 0.2 mM Ca++. Alignment of Schwann cells along neurites was infrequent, as were extended close apposition between axonal and Schwann cell plasma membranes. Finally, migration of Schwann cells from ganglionic explants was reduced by disruption of adhesive contact with neurites. The addition of antibodies against N-cadherin to medium with normal Ca++ levels had similar effects as lowering the Ca++ concentration, but antibodies against the neuronal adhesive molecule, L1, had no effects on interactions between Schwann cells and neurites.     

Ca++ and Na+ channels involved in neuronal cell death. Protection by flunarizine.

Flunarizine, a class IV Ca++ antagonist non-selective for slow Ca++ channels, has been shown to be beneficial in the prophylactic treatment of migraine, the treatment of vertigo, and as add-on treatment in therapy-resistant forms of epilepsy. Flunarizine protects the brain against functional and/or structural neuronal damage in various animal models of cerebral ischemia. In addition to its cerebrovascular effect, flunarizine has also direct neuroprotective actions. New data have emerged on flunarizine with regard to Ca++ and Na+ channels in neuronal cells. There are several possible mechanisms involved in the mode of action of flunarizine. Flunarizine may block Ca++ and Na+ channels, both of which may flux Ca++ as well as Na+. A decrease in Ca++ influx may prevent further release of glutamate, and activation of NMDA receptor gated Ca++ channels at physiological pH. A decrease in Na+ influx may prevent cytotoxicity secondary to a large gain in intracellular Ca++, by reverse operation of the Na+/Ca++ exchanger. This mechanism may be important when the glycolytic rate is increased with concomitant acidosis, and phospholipids are broken down as occurs typically during ischemia. Given the complexity of biochemical events leading to cell death, blocking exclusively one channel subtype is not likely to yield sufficient protection. Hence, it may be useful to develop anti-ischemic compounds which act on a series of pathways involved in Ca++ overload, rather than selectively block one such channel.     

Characterization of the cytolytic reaction mechanism of the human natural killer (NK) lymphocyte: resolution into binding, programming, and killer cell-independent steps

Various parameters of the cytolytic reaction mechanisms of the human natural killer (NK) lymphocyte were studied to characterize the lytic cycle. NK cytolysis was determined to occur in three definable steps. 1) Binding of PBL to the NK-sensitive targets Molt-4 or K562 was rapid (less than 1 min), occurred at temperatures below 37 degrees C, was Mg++3-dependent, Ca++3-independent, and was prevented by dispersion of the cells into 10% dextran. 2) Subsequent to binding, programming for lysis as determined by a Ca++ pulse method was more protracted, requiring up to 2 hr to occur and was strictly dependent on Ca++ for cytolysis to proceed. In standard cytotoxicity assays, however, programming for lysis was more rapid occurring in 10 to 30 min. Programming was inhibited by EDTA, EGTA/Mg++ and by temperatures below 37 degrees C. Furthermore, after binding but in the absence of initiation of programming for lysis, the frequency of target binding cells did not change and the NK cell did not lose its lytic potential. 3) Killer cell-independent cytolysis (KCIL) was determined by the addition of EDTA to "programmed" targets and dispersion of these cells into dextran-containing medium, which resulted in virtually 100% dissociation of conjugated cells. KCIL was Ca++ and Mg++-independent and was blocked at reduced temperatures only if the dextran was prechilled to 4 degrees C before addition. The kinetics of 51Cr release during KCIL was rapid and complete 30 min after dispersion. Interferon-activated NK cells expressed an increased rate of cytolysis in Ca++ pulse experiments. This was due to an increased rate of the Ca++-dependent step(s) during the programming events. The rate of the Ca++-independent steps, however, were similar with control and IFN-activated cells.     

Studies on alpha-adrenergic activation of hepatic glucose output. Studies on role of calcium in alpha-adrenergic activation of phosphorylase.

The role of Ca2+ ions in alpha-adrenergic activation of hepatic phosphorylase was studied using isolated rat liver parenchymal cells. The activation of glucose release and phosphorylase by the alpha-adrenergic agonist phenylephrine was impaired in cells in which calcium was depleted by ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid (EGTA) treatment and restored by calcium addition, whereas the effects of a glycogenolytically equivalent concentration of glucagon on these processes were unaffected. EGTA treatment also reduced basal glucose release and phosphorylase alpha activity, but did not alter the level of cAMP or the protein kinase activity ratio (-cAMP/+cAMP) or impair viability as determined by trypan blue exclusion, ATP levels, or gluconeogenic rates. The effect of EGTA on basal phosphorylase and glucose output was also rapidly reversed by Ca2+, but not by other ions. Phenylephrine potentiated the ability of low concentrations of calcium to reactivate phosphorylase in EGTA-treated cells. The divalent cation inophore A23187 rapidly increased phosphorylase alpha and glucose output without altering the cAMP level, the protein kinase activity ratio, and the levels of ATP, ADP, or AMP, The effects of the ionophore were abolished in EGTA-treated cells and restored by calcium addition. Phenylephrine rapidly stimulated 45Ca uptake and exchange in hepatocytes, but did not affect the cell content of 45Ca at late time points. A glycogenolytically equivalent concentration of glucagon did not affect these processes, whereas higher concentrations were as effective as phenylephrine. The effect of phenylephrine on 45Ca uptake was blocked by the alpha-adrenergic antagonist phenoxybenzamine, was unaffected by the beta blocker propranolol, and was not mimicked by isoproterenol. The following conclusions are drawn: (a) alpha-adrenergic activation of phosphorylase and glucose release in hepatocytes is more dependent on calcium than is glucagon activation of these processes; (b) variations in liver cell calcium can regulate phosphorylase alpha levels and glycogenolysis; (c) calcium fluxes across the plasma membrane are stimulated more by phenylephrine than by a glycogenolytically equivalent concentration of glucagon. It is proposed that alpha-adrenergic agonists activate phosphorylase by increasing the cytosolic concentration of Ca2+ ions, thus stimulating phosphorylase kinase.     

Inositol Hexakisphosphate (Phytic Acid) Enhances Ca2+Influx and D-[3H]Aspartate Release in Cultured Cerebellar Neurons

Inositol hexakisphosphate (InsP6) increased 45Ca2+ uptake in cultured cerebellar granule cells. This increase was concentration dependent (EC50 = 20 microM), exhibited slow kinetics, and was present after 5 days of cell maturation in culture. InsP6 also enhanced D-[3H]aspartate release in cerebellar granule cells at 11-12 days in vitro. Stimulation of 45Ca2+ uptake was also produced by inositol pentakisphosphate but not by inositol 1,3,4,5-tetrakisphosphate. The increase in 45Ca2+ influx induced by InsP6 was independent of extracellular Na+ and was only partially reduced by the organic calcium channel blocker nifedipine. The intrinsic action of InsP6 was not affected by competitive or noncompetitive glutamate receptor antagonists. In addition, stimulations of 45Ca2+ uptake by InsP6 and glutamate were additive. These data provide evidence that InsP6 directly activates a specific population of neurons in the CNS.     

Effect of in vivo and in vitro application of glucagon, insulin and epinephrine on Ca++-transport properties of liver mitochondria.

In vivo administration of glucagon, insulin or epinephrine, respectively, gives rise to an increase of Ca++-retention time as well as of the Ca++-uptake rate in subsequently isolated rat liver mitochondria. Whereas the changes of Ca++-transport properties after pretreatment with glucagon or epinephrine occur already 6--15 min after their administration, the effect of insulin is observed not earlier than 30 min after its application. Under diabetic and starving conditions the Ca++-retention time of isolated liver mitochondria is prolonged, whereas no alteration of the uptake rate occurs. Since alloxan as well as streptozotocin induced qualitatively similar changes, a specific action of alloxan on liver mitochondria can be ruled out. Application of insulin 60--90 min prior to decapitation normalizes the changes of mitochondrial Ca++-transport observed under chronic alloxan diabetic conditions. Cycloheximide abolishes the prolongation of Ca++-retention in mitochondria from alloxan diabetic rats, but has no influence on the changes induced by glucagon pretreatment.     

Standardization of a sensitive and rapid assay for lymphotoxin.

Actinomycin-treated mouse L cells and human HeLa cells are sensitive indicators of lymphotoxin activity in supernatant fluids of mitogen-stimulated lymphocytes. Without actinomycin, various strains of these cells are 10–200 times less sensitive. The concentration of actinomycin used amplifies the toxic effect of LT but is not itself cytotoxic. Actinomycin-treated indicator cells permit detection of LT activity where toxicity is not often found, as in supernatants of mixed lymphocyte cultures and of cell-mediated cytotoxicity reactions. This assay makes available to any investigator a sensitive indicator of LT activity.     

Mitogen-stimulated glucose transport in thymocytes. Possible role of Ca++ and antagonism by adenosine 3'':5''-monophosphate

The plant lectin, concanavalin A (Con-A), and the ionophore, A-23187 (specific for divalent cations), stimulated glucose transport in rat thymocytes. Con-A stimulation developed more slowly and was somewhat less extensive than that of stimulation developed more slowly and was somewhat less extensive than that of A-23187. Both responses showed saturation dose dependencies. The two responses were poorly additive, suggesting that A-23187 may saturate regulatory processes shared by the two stimulatory mechanisms. Doses of methylisobutylxanthine (MIX) and prostaglandin E2 which raised adenosine 3':5'-monophosphate (cAMP) levels in these cells also antagonized the Con-A stimulation of glucose transport but did not inhibit basal glucose transport or the A-23187 stimulation. Dibutyryl-cAMP and 8-bromo-cAMP also natagonized Con-A stimulation without inhibiting basal glucose transport. MIX antagonized high Con-A doses about as strongly as it did low Con-A doses, suggesting that MIX did not compete in the Con-A binding step or other process saturable by Con-A. [3H-A1Con-A binding was not affected by MIX. The stimulatory effects of Con-A and A-23187 were reduced by reduction of Ca++ in the medium. Both Con-A and A-23187 enhanced 45Ca++ influx and cellular Ca++ content. The A-23187 dose, which was saturating for glucose transport stimulation, enhanced Ca++ influx and cellular Ca++ content more than did the Con-A dose which was saturating for glucose transport stimulation. The dose fo MIX which specifically antagonized Con-A stimulation of glucose transport proved also to reduce Ca++ influx and cellular Ca++ in the presence of Con-A but not in the presence of A-23187. Thus, glucose transport correlates rather well with cellular Ca++. These results are compatible with the view that Ca++ in a cellular compartment can promote glucose transport, the Con-A's enhancement of Ca++ entry contributes to its stimulation of glucose transport, and the MIX antagonized Con-A action at least partly by reducing Ca++ entry. The action of MIX is apparently mediated by cAMP.