Intracellular mediators of potassium-induced aldosterone secretion

We have investigated the intracellular messengers of potassium in eliciting aldosterone secretion in calf adrenal glomerulosa cells since there were unresolved issues relating to the role of phosphoinositides, cAMP and protein kinases. We observed no evidence of hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) in 3H-inositol labeled alf adrenal cells or increase of cAMP in response to potassium. Addition of calcium channel blocker, nitrendipine after stimulating adrenal glomerulosa cells with potassium, markedly inhibited aldosterone secretion. A calmodulin inhibitor (W-7) produced greater reduction of aldosterone secretion than an inhibitor of protein kinase C (H-7). These results suggest that a rise in cytosolic free calcium concentration through voltage-dependent calcium channel and calmodulin are the critical determinants of aldosterone secretion stimulated by potassium.     

Short term and long term effects of beta-adrenergic effectors and cyclic AMP on nitrendipine-sensitive voltage-dependent Ca2+ channels of skeletal muscle

The effects of short term stimulation of beta-adrenergic receptors and elevations in intracellular cyclic AMP on nitrendipine-sensitive voltage-dependent Ca2+ channels of skeletal muscle cells in vitro has been studied using both the 45Ca2+ flux technique and [3H] nitrendipine-binding experiments. Isoproterenol increased the nitrendipine-sensitive 45Ca2+ influx under depolarizing conditions. The effects of isoproterenol were additive to those of depolarization and were antagonized by alprenolol. Half-maximal inhibition of 45Ca2+ influx induced both by depolarization and by isoproterenol occurred at a nitrendipine concentration of 1 nM. Treatments that resulted in an increased level of intracellular cyclic AMP, such as treatment with 1-methyl-3-isobutylxanthine, theophylline, dibutyryl cyclic AMP, or 8-bromocyclic AMP also resulted in an increased rate of 45Ca2+ entry via nitrendipine-sensitive Ca2+ channel. In contrast, long term treatment of myotubes in culture with isoproterenol and other compounds that increased intracellular cyclic AMP led to a large increase in the number of nitrendipine receptors. This increase was accompanied by a 4-10-fold decrease in the affinity of the receptors for nitrendipine. Alprenolol inhibited the long term effects of isoproterenol. In vivo treatment of 7-day-old chicks with reserpine and alprenolol produced a decrease in the number of skeletal muscle nitrendipine receptors. This decrease in receptor number was accompanied by an increase in the affinity of nitrendipine for its receptor by a factor of 4 to 5. These effects on the nitrendipine receptor were prevented by simultaneous injection of isoproterenol. The results are discussed in relation to the role of beta-adrenergic receptors and intracellular cyclic AMP in the regulation of skeletal muscle Ca2+ channels.     

CD4 and CD8 are positive regulators of T cell receptor signal transduction in early T cell differentiation.

Although cortical (CD4+CD8+) thymocytes mobilize intracellular calcium poorly when CD3/TCR is ligated, we have found that murine cortical thymocytes can transduce strong biochemical signals in response to ligation of the CD3/Ti TCR complex (CD3/TCR) and that the signals are regulated by CD4 and CD8 interactions with CD3/TCR. Striking increases in intracellular calcium were observed in cortical thymocytes from transgenic mice containing productively rearranged alpha and beta TCR genes, when CD3 or TCR was cross-linked with CD4 or CD8 using heteroconjugated mAb. However, in mature T cells derived from lymph nodes of these mice, identical stimuli elicited calcium responses that were significantly smaller in magnitude. A thymocyte cell line that expresses a low level of the transgenic TCR and has a phenotype characteristic of cortical thymocytes (CD4+CD8+J11d+Thy-1+) was established from a female alpha beta TCR transgenic mouse. Cross-linking of CD4 or CD8 molecules to CD3/TCR induced strong calcium responses in these cells. Responses were weak or absent when CD3 or TCR were aggregated alone. Heteroconjugates of Thy-1xCD3 did not increase the intracellular calcium concentration in transgenic thymocytes or in the thymocyte cell line, although Thy-1 is highly expressed on immature cells. Enhanced tyrosine phosphorylation was observed when CD3 or TCR was cross-linked with CD4 or CD8 on transgenic thymocytes or on the thymocyte cell line, in comparison with aggregation of CD3/TCR alone. Taken together, these data show that CD4 and CD8 molecules allow the weakly expressed CD3/TCR of cortical thymocytes to transduce strong intracellular signals upon receptor ligation. These signals may be involved in selection processes at the CD4+CD8+ stage of differentiation.     

Dihydropyridine-sensitive L-type Ca2+ channels in human foreskin fibroblast cells. Characterization of activation with the growth factor Lys-bradykinin.

We have previously characterized the calcium response of cultured human fibroblasts (HSWP cells) to stimulation by the mitogen Lys-bradykinin (BK). We have reported a biphasic response which includes a rapid rise to a peak that appears to result from mobilization of internal calcium, and a plateau phase, which is due to influx of external calcium (Byron, K., Babnigg, G., Villereal, M. L. (1992) J. Biol. Chem. 267, 108-118). In this paper we examine participation of L-type voltage operated calcium channels in the calcium entry phase of BK-stimulated HSWP cells. We show that there is an increase in 45Ca2+ uptake and an increase in intracellular free calcium concentration ([Ca2+]i) as measured by fura-2, when HSWP cells are stimulated with the L-channel agonist Bay K 8644 under depolarizing conditions. Furthermore, both of these effects are inhibited by low doses of the dihydropyridine antagonist nitrendipine. We also report that BK stimulation of 45Ca2+ uptake can be significantly inhibited by low doses of nitrendipine, while nitrendipine treatment has no effect on the BK-induced rise in [Ca2+]i, as measured by fura-2. These results suggest that under normal conditions the portion of the BK-stimulated Ca2+ influx which is mediated by a nitrendipine-sensitive entry pathway is invisible to the fura-2 technique used to measure [Ca2+]i. This suggest that the nitrendipine-sensitive influx pathway admits calcium preferentially into an intracellular store that is isolated from fura-2. This idea is supported by the observation that in media where calcium has been replaced by 2 mM Ba2+ nitrendipine inhibits most of the BK-stimulated Ba2+ influx.     

Involvement of CD2 and CD3 in galectin-1 induced signaling in human Jurkat T-cells

Galectin-1 (gal-1) a member of the mammalian beta-galactoside-binding proteins recognizes preferentially Galbeta1-4GlcNAc sequences of oligosaccharides associated with several cell surface glycoconjugates. In the present work, gal-1 has been identified to be a ligand for the CD3-complex as well as for CD2 as detected by affinity chromatography of Jurkat T-cell lysates on gal-1 agarose and by binding of the biotinylated lectin to CD3 and CD2 immunoprecipitates on blots. In CD45(+)Jurkat E6.1 cells, the lectin stimulates a sustained increase in the intracytoplasmic calcium concentration ([Ca(2+)](i)) consisting of both the release of calcium from intracellular stores and the calcium influx from the extracellular space. This effect of gal-1 on [Ca(2+)](i)is completely inhibited by lactose at 10 mM and was absent in CD45(-)Jurkat J45.01 cells. Preincubation of Jurkat E6.1 cells with cholera toxin or with the protein tyrosine kinase inhibitor herbimycin A reduced the gal-1 induced calcium response whereas the increase in [Ca(2+)](i)stimulated by CD2 or CD3 monoclonal antibodies (mAbs) was completely inhibited. Depolarization of E6.1 cells in a high-potassium buffer, a standard method to activate voltage-operated calcium channels, was without effect on [Ca(2+)](i). Membrane depolarization with gramicidin or by a high-potassium buffer was without effects on the lectin-mediated calcium release from intracellular stores but inhibited the gal-1 induced receptor-operated calcium influx. In Jurkat E6.1 cells the lectin stimulates the transient generation of inositol-1,4,5-trisphosphate and the tyrosine phosphorylation of phospholipase Cgamma1. The results suggest that the ligation of CD2 and CD3 by gal-1 induces early events in T-cell activation comparable with that elicited by CD2 or CD3 mAbs.     

Differential G protein-mediated coupling of D2 dopamine receptors to K+ and Ca2+ currents in rat anterior pituitary cells.

In anterior pituitary cells, dopamine, acting on D2 dopamine receptors, concomitantly reduces calcium currents and increases potassium currents. These dopamine effects require the presence of intracellular GTP and are blocked by pretreatment of the cells with pertussis toxin, suggesting that one or more G protein is involved. To identify the G proteins involved in coupling D2 receptors to these currents, we performed patch-clamp recordings in the whole-cell configuration using pipettes containing affinity-purified polyclonal antibodies raised against either Go alpha, Gi3 alpha, or Gi1,2 alpha. Dialysis with Go alpha antiserum significantly reduced the inhibition of calcium currents induced by dopamine, while increase of potassium currents was markedly attenuated only by Gi3 alpha antiserum. We therefore conclude that in pituitary cells, two different G proteins are involved in the signal transduction mechanism that links D2 receptor activation to a specific modulation of the four types of ionic channels studied here.     

The induction of T cell unresponsiveness by rapidly modulating CD3

The immunomodulatory effects of an IgM anti-CD3 mAb (38.1) were investigated. 38.1 was distinct from other anti-CD3 mAb, in that it was rapidly modulated from the cell surface in the absence of a secondary antibody. Although 38.1 induced an immediate increase in intracellular free calcium [Ca2+]i by highly purified T cells, it did not induce entry of the cells into the cell cycle in the absence of accessory cells (AC) or a protein kinase C-activating phorbol ester. Clearing of 38.1 from the surface of AC-depleted T cells, documented both by immunofluorescence and by functional activity, was rapid, with markedly reduced levels of initially bound mAb observed after a 1 to 2 h incubation at 37 degrees C and complete modulation noted after a 5-h incubation. Despite rapid modulation of 38.1, the T cells continued to express substantial amounts of surface CD3, suggesting there is a rapid rate of turnover of CD3 molecules on resting T cells. After modulation of 38.1 bound CD3, T cells were markedly inhibited in their capacity to respond to PHA. Inhibition could be overcome by culturing the cells with supplemental AC or IL-2. The inhibitory effects of 38.1 could be mimicked by briefly pulsing cells with the calcium ionophore, ionomycin, that had no effect on surface expression of CD3. 38.1- or ionomycin-pulsed cells were inhibited in their subsequent response to PHA even when exposures were carried out in the presence of EGTA to prevent increases in [Ca2+]i from extracellular sources. Inhibition could not be accounted for by an inability of the ionomycin-treated or 38.1-modulated T cells to increase [Ca2+]i in response to PHA. These studies demonstrate that a state of T cell nonresponsiveness can be induced by modulating CD3 with an anti-CD3 mAb in the absence of co-stimulatory signals. A brief increase in [Ca2+]i resulting from mobilization of internal calcium stores appears to be sufficient to induce this state of T cell nonresponsiveness.     

Insulin-like growth factor II stimulates calcium influx in competent BALB/c 3T3 cells primed with epidermal growth factor. Characteristics of calcium influx and involvement of GTP-binding protein

The action of insulin-like growth factor II (IGF-II) on calcium influx was studied in BALB/c 3T3 cells. IGF-II did not affect calcium influx rate in either quiescent or platelet-derived growth factor-treated "competent" cells. In contrast, IGF-II induced an approximately 2-fold sustained increase in calcium influx rate in competent cells briefly primed with epidermal growth factor ("primed competent" cells). The IGF-II-stimulated calcium influx was dependent on extracellular calcium and was inhibited by lanthanum, cobalt, and tetramethlin but not by nitrendipine. The IGF-II-stimulated [3H]thymidine incorporation was also dependent on extracellular calcium and was inhibited by cobalt and tetramethlin. A pharmacological stimulation of calcium influx by BAYK8644 resulted in an increase in [3H]thymidine incorporation in primed competent cells but not in either quiescent or competent cells. Pretreatment of primed competent cells with pertussis toxin completely abolished subsequent action of IGF-II on both calcium influx and [3H]thymidine incorporation. Inhibitory actions of pertussis toxin correlated well with toxin-induced ADP-ribosylation of a 41-kDa protein. The binding of 125I-IGF-II to membrane fraction was inhibited by guanosine 5'-O-(thiotriphosphate), and this inhibition was reversed by pretreatment of the cell with pertussis toxin. These results suggest that IGF-II stimulates calcium influx in primed competent BALB/c 3T3 cells by a mechanism involving G protein and that calcium influx may be a message of IGF-II action on cell proliferation.     

Time-dependent restoration of the trigger pool of calcium after termination of angiotensin II action in adrenal glomerulosa cells

In adrenal glomerulosa cells, angiotensin II causes an immediate release of calcium from an intracellular trigger pool (Kojima, I., Kojima, K., and Rasmussen, H. (1985) Am. J. Physiol. 247, E36-E43). The present study was conducted to determine how the trigger pool of calcium is restored after cessation of the agonist action. Upon termination of angiotensin II action, calcium influx rate decreased immediately while total cell calcium increased rapidly. The increase in total cell calcium is not affected by 1 microM nitrendipine, which blocks angiotensin II-stimulated calcium influx without inhibiting basal influx of calcium. In contrast, total cell calcium did not increase in medium containing 1 microM calcium, in which basal calcium influx is negligible. A rapid increase in total cell calcium after an addition of the antagonist was not accompanied by changes in cytoplasmic free calcium concentration. A second stimulation of cells with either angiotensin II or carbachol did not cause calcium release when the interval of two stimulations was shorter than 20 min. The longer the interval, the greater the magnitude of calcium release in response to the second stimulator. The maximum response was obtained when the interval was 40 min or more. When exogenous arachidonic acid, which mobilized calcium by acting directly on the inositol trisphosphate-sensitive pool, was employed as a second stimulator, the magnitude of the decrease in total cell calcium was also dependent on the interval. These results suggest that, upon termination of angiotensin II action, calcium is rapidly accumulated first in an intracellular pool which is insensitive to either inositol 1,4,5-trisphosphate or arachidonic acid and that the trigger pool is restored gradually thereafter.     

Effect of calcium channel modulators on isolated endothelial cells

Indirect evidence, using organic calcium channel modulators suggests that calcium channels exist in endothelial cells. Using freshly prepared and cultured bovine aortic endothelial cells, we have studied the effect of calcium channel modulators on Fura-2 fluorescence and have examined the binding of the dihydropyridine, (+)[3H]PN200-110. In both isolated primary and cultured cells, external calcium (0.5-2 mM) and bradykinin (10(-8) M) increased the intracellular calcium concentration. In cultured cells, the increase in calcium was not significantly attenuated by preincubation with nitrendipine (10(-8) M) or d-cis-diltiazem (10(-6) M). The calcium agonists (-)Bay k8644 and (+)202-791 had no effect on intracellular calcium concentration, but other agonists including ATP (10(-4) M) and thrombin (1.5 micrograms/ml) significantly increased the calcium concentration. Competition binding studies with (+)[3H]PN200-110 indicated specific binding of this ligand with a KD of 57 nM and a Bmax of 2.1 pmol/10(6) cells. While these data do not provide convincing evidence for the existence of calcium channels in cultured or fresh bovine aortic endothelial cells, explanations may yet reconcile our observations with the presence of calcium channels in these cells.     

CD5 antibodies increase intracellular ionized calcium concentration in T cells

The binding of a variety of monoclonal antibodies to the CD5 (T, gp67) pan T cell differentiation antigen has been shown to potentiate T cell proliferation. In this paper we show that CD5 monoclonal antibodies cause increased intracellular free calcium concentration ([Ca2+]i) in T cells. An increase in [Ca2+]i occurred within 1 min in indo-1-loaded PBMC after the addition of CD5 monoclonal antibodies and cross-linking with a second step anti-mouse kappa light chain antibody. Cross-linking of CD5 was effective when done directly on the cell surface or by the administration of preformed soluble complexes that contained CD5 antibodies. Calcium mobilization induced by suboptimal concentrations of CD3 antibodies was specifically augmented and sustained by CD5 antibodies, although the enhancement was modest in magnitude. When cell surface phenotype was correlated with calcium mobilization, it was found that the CD5 response was restricted to CD5+/CD3+ cells, and that approximately 90% of CD5+ cells had responded. CD5-induced calcium mobilization was found to differ from CD3 stimulation in that EGTA entirely ablated the CD5 response, whereas the CD3 response was resistant to EGTA, indicating that the CD5-induced increased [Ca2+]i is derived primarily or entirely from extracellular calcium. CD5-stimulated calcium mobilization also differed from CD3 in that the CD5 response was inhibited by pretreatment with phorbol myristate acetate, whereas the CD3 response was not, suggesting that depletion of protein kinase C causes an uncoupling of signal transduction between CD5 and calcium channels. Finally, experiments were done with T cells after antigenic modulation of the CD3 or CD5 molecules. Unexpectedly, both the CD5 and the CD3 responses were ablated on CD3-modulated cells, whereas only the CD5 response was ablated on CD5-modulated cells. In addition, several Cd5+/CD3- T cell leukemia lines also failed to respond to CD5 stimulation, providing further evidence which indicates that the CD5 response depends on the cell surface expression of CD3 or a CD3-associated structure. These findings suggest that one mechanism for CD5-induced augmentation of mitogen-stimulated T cell proliferation involves increased [Ca2+]i which is distinct from but interdependent with that induced by stimulation of the CD3 molecule.     

CD2/LFA-3 ligation induces phospholipase-C gamma 1 tyrosine phosphorylation and regulates CD3 signaling.

Activation of T cells through the TCR/CD3 receptor complex with either specific Ag or antibody results in tyrosine phosphorylation of intracellular protein substrates and phosphatidylinositol-phospholipase C (PLC) signaling, leading to the generation of PI breakdown products and the mobilization of intracellular calcium. Stimulation of the T cell surface receptor CD2 similarly propagates early signals through phosphatidylinositol-PLC activation. Previous reports have shown that CD3 activation leads to tyrosine phosphorylation of the PLC isozyme PLC gamma 1. In this report, we investigated the potential similarity between CD3-induced signaling through PLC gamma 1 and that induced by CD2. We show that stimulation of CD2 receptors on T cells caused tyrosine phosphorylation of PLC gamma 1. Cross-linking of CD2 with CD3 receptors augmented the phosphorylation of PLC gamma 1 on tyrosine, whereas ligation of the CD45 tyrosine phosphatase with CD2 receptors prevented PLC gamma 1 tyrosine phosphorylation. T cells stimulated by ligation of CD2 with its counter-receptor in the form of a soluble LFA-3/Ig fusion protein cross-linked on the cell surface, resulted in a low, but detectable level of PLC gamma 1 phosphorylation with prolonged kinetics, whereas that induced by cross-linking with anti-CD2 was stronger but transient. Co-ligation of LFA-3/Ig with suboptimal concentrations of anti-CD3 resulted in profound augmentation of PLC gamma 1 tyrosine phosphorylation, mobilization of intracellular calcium and T cell proliferation. To explore the relationship between CD3- and CD2-stimulated signaling, T cells were desensitized through 1 h incubation with anti-CD3. CD3 receptor modulation potently down-regulated CD2-induced PLC gamma 1 tyrosine phosphorylation and calcium mobilization. In contrast, PMA or ionomycin treatment did not alter CD2-stimulated tyrosine phosphorylation of PLC gamma 1, suggesting that tyrosine kinase inhibition by CD3 receptor modulation was not caused by signaling events downstream of PLC gamma 1. Taken together, these results support the hypothesis that CD2 provides a potent co-stimulatory signal for CD3-induced T cell activation that is associated with tyrosine kinase(s) and PLC gamma 1.     

Membrane depolarization selectively inhibits receptor-operated calcium channels in human T (Jurkat) lymphoblasts

Jurkat lymphoblasts were stimulated by a monoclonal antibody against the CD3 membrane antigen and the evoked calcium signal was followed by the intracellular fluorescent calcium indicator indo-1. The technique applied allowed us to separately investigate the stimulus-induced intracellular calcium release and the calcium-influx pathways, respectively. In the same cells membrane potential was estimated by the fluorescent dye diS-C3-(5). The resting membrane potential of Jurkat lymphoblasts under normal conditions was between -55 and -60 mV. Membrane depolarization, obtained by increasing external K+ concentration, removing external Cl-, or by increasing the Na+/K+ leak permeability with gramicidin or PCMBS, did not induce calcium influx in the resting cells and did not influence the CD3 receptor-mediated internal calcium release, while strongly inhibited the receptor-mediated calcium influx pathway. Half-maximum inhibition of this calcium influx was observed at membrane potential values of about -35 to -40 mV and this inhibition did not depend on the external calcium concentration varied between 5 and 2500 microM. Membrane hyperpolarization by valinomycin did not affect either component of the calcium signal. The observed selective inhibition of the receptor-operated calcium influx pathway by membrane depolarization is probably an important modulator of calcium-dependent cell stimulation.     

CD45 tyrosine phosphatase-activated p59fyn couples the T cell antigen receptor to pathways of diacylglycerol production, protein kinase C activation and calcium influx.

The role of the CD45 phosphotyrosine phosphatase in coupling the T cell antigen receptor complex (TCR) to intracellular signals was investigated. CD45- HPB-ALL T cells were transfected with cDNA encoding the CD45RA+B+C- isoform. The tyrosine kinase activity of p59fyn was found to be 65% less in CD45- cells than in CD45+ cells, whereas p56lck kinase activity was comparable in both sub-clones. In CD45- cells the TCR was uncoupled from protein tyrosine phosphorylation, phospholipase C gamma 1 regulation, inositol phosphate production, calcium signals, diacylglycerol production and protein kinase C activation. Restoration of TCR coupling to all these pathways correlated with the increased p59fyn activity observed in CD45-transfected cells. Co-aggregation of CD4- or CD8-p56lck kinase with the TCR in CD45- cells restored TCR-induced protein tyrosine phosphorylation, phospholipase C gamma 1 regulation and calcium signals. Receptor-mediated calcium signals were largely due (60-90%) to Ca2+ influx, and only a minor component (10-40%) was caused by Ca2+ release from intracellular stores. Maximal CD3-mediated Ca2+ influx occurred at CD3 mAb concentrations at which inositol phosphate production was non-detectable. These results indicate that CD45-regulated p59fyn plays a critical role in coupling the TCR to specific intracellular signalling pathways and that CD4- or CD8-p56lck can only restore signal transduction coupling in CD45- cells when brought into close association with the TCR.     

Spontaneous induction of superoxide release and degranulation of neutrophils in isotonic potassium medium: the role of intracellular calcium

When guinea pig peritoneal neutrophils were suspended in the isotonic medium of potassium, rubidium, and cesium ions at 37 degrees C, the cells released superoxide, while low activity was observed in the isotonic medium of sodium and lithium ions. The activity induced in the potassium medium was enhanced by potassium-ionophores, valinomycin, and gramicidin, and decreased by a potassium channel blocker, 4-aminopyridine. The superoxide-releasing activity was not affected by the presence or absence of extracellular calcium but was inhibited by an intracellular calcium antagonist-8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate(TMB-8) with the half-inhibition concentration of 50 microM. The release of granular enzymes, lysozyme and beta-glucuronidase, was also induced in the isotonic potassium medium in the absence of extracellular calcium and inhibited by TMB-8. A remarkable elevation of the intracellular free calcium concentration in neutrophils, which was monitored by quin-2 fluorescence, was found when the cells were added to the potassium medium without calcium. The elevation was inhibited by the addition of TMB-8. These observations suggest that calcium mobilization from intracellular storage sites, not an influx of calcium from the extracellular medium, causes the release of superoxide and the granular enzymes in isotonic potassium medium.     

Signal transduction during human natural killer cell activation: inositol phosphate generation and regulation by cyclic AMP

NK cells mediate both direct cytotoxicity against a variety of tumor cells and indirect (FcR-dependent) cytotoxicity against antibody-coated targets. When cloned human NK cells (CD16+/CD3-) were exposed to NK-sensitive targets for 30 min, the level of inositol phosphates rose two to five times above background. The rise in inositol phosphates induced by NK-sensitive targets was paralleled by an increase in intracellular free calcium concentration ([Ca2+]i). A panel of tumor cells that were resistant to NK cell lysis did not stimulate significant levels of inositol phosphate production and did not induce an elevation of intracellular free calcium. Ligation of the FcR (CD16) with the mAb 3G8 also triggered phosphoinositide turnover. Kinetic experiments demonstrated that stimulation by either susceptible target cells or by FcR ligation led to rapid (less than 1 min) generation of the Ca2+-mobilizing second messenger, inositol trisphosphate, with slower accumulation of inositol bisphosphate and inositol monophosphate. Previous studies have demonstrated that activation of the cAMP-dependent second messenger pathway strongly inhibits NK cell-mediated cytotoxic functions. Treatment of NK effector cells with forskolin to elevate intracellular cAMP levels resulted in a concentration-dependent inhibition of phosphoinositide hydrolysis induced by both NK-sensitive targets and 3G8-mediated FcR ligation. These results suggest that phosphoinositide turnover represents a critical early event in the human NK cell cytolytic process. Moreover, the potent inhibitory effect of cAMP on NK cell cytotoxicity may be explained by the uncoupling of NK receptors from phospholipase C-mediated phosphoinositide hydrolysis.     

Inhibitory and excitatory mechanisms of neurotensin action in canine intestinal circular muscle in vitro.

The effect of neurotensin on canine ileal circular muscle devoid of myenteric plexus was investigated using single and double sucrose gap techniques. Similar results were obtained with microelectrode techniques. Neurotensin caused a temperature-sensitive and dose-dependent biphasic response, an initial hyperpolarization associated with inhibition of contractile activity, followed by an excitatory phase, usually consisting of spike discharge and tonic and phasic contractions, for which depolarization was not required. Neither response was affected by tetrodotoxin, phentolamine, propranolol, or atropine. The hyperpolarization was associated with decreased membrane resistance, blocked by 10(-7) M apamin, and converted to tonic depolarization by apamin (10(-6) M). Tachyphylaxis to neurotensin occurred when the stimulation interval was less than 20 min. After Ca2+ depletion, depolarization was observed instead of the hyperpolarization; this depolarization was not affected by nitrendipine and was gradually abolished with repetitive stimulation at 20-min intervals. When Ca2+ was present, nifedipine did not alter the hyperpolarizing phase of the response but inhibited spiking and blocked all contractions. The excitatory phase of the response was enhanced by Bay K-8644. Neuromedin N elicited a response identical with that of neurotensin. The responses of the two peptides were completely cross tachyphylactic. Inhibitory junction potentials were not affected by neurotensin tachyphylaxis. It is concluded that neurotensin and neuromedin N activate apamin-sensitive, calcium-dependent potassium channels in circular muscle, causing membrane hyperpolarization and inhibition of muscle contraction. Release of intracellular calcium is involved in the activation of these potassium channels.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of tyrosine phosphorylation in signal transduction through surface Ig in human B cells. Inhibition of tyrosine phosphorylation prevents intracellular calcium release.

Cross-linking surface Ig on human B cells, or the TCR complex on T cells leads to the rapid appearance of newly tyrosine phosphorylated proteins. This is associated with inositol phospholipid turnover and a rise in intracellular calcium. Incubation of human B or T lymphocytes with the tyrosine kinase inhibitors, herbimycin and genistein, inhibits new tyrosine phosphorylation after receptor-linked activation. This is associated with complete abrogation of the increase in intracellular calcium in these lymphocytes and inhibition of inositol phospholipid turnover. Herbimycin- and genistein-treated lymphocytes are nevertheless still capable of responding to aluminum fluoride with a rise in intracellular calcium. These data support the contention that a B cell-associated protein tyrosine kinase regulates signal transduction via phospholipase C. CD45, the membrane associated protein tyrosine phosphatase, and PMA that activates protein kinase C, both inhibit the calcium response in B lymphocytes induced by receptor cross-linking. PMA and cross-linking CD45 both induced the appearance of tyrosine phosphorylated proteins in human B cells, although the pattern is quite distinct from that seen when surface lg is cross-linked. However, the induction of new tyrosine phosphorylation by anti-mu does not appear to be affected by these reagents. Although this may reflect an insensitivity of the tyrosine phosphorylation assay, it could indicate that regulation of the calcium response and regulation of the tyrosine kinase can be independent processes.     

Activation of human T4 cells by cross-linking class I MHC molecules

These studies examined whether cross-linking class I MHC molecules results in functional or biochemical responses in human T4 cells. The initial studies demonstrated that cross-linking class I MHC molecules either by culturing highly purified T4 cells with immobilized mAb to class I MHC Ag or reacting the T4 cells with mAb to class I MHC Ag and then cross-linking the mAb with goat antimouse Ig (GaMIg) enhanced T4 cell proliferation induced by an immobilized mAb to CD3, OKT3. More-over, immobilized but not soluble mAb to class I MHC Ag enhanced T4 cell proliferation induced by the combination of two mAb to CD2, OKT11, and D66.2. Finally, T4 cells reacted with mAb to CD3 and class I MHC Ag proliferated in the presence of IL-2 when cross-linked with GaMIg more vigorously than T4 cells reacted with either mAb alone. Cross-linking class I MHC molecules was also found to stimulate T4 cells directly. T4 cells reacted with mAb to class I MHC Ag or beta 2 microglobulin and cross-linked with GaMIg proliferated vigorously in the presence of IL-2 or PMA. In addition, it was demonstrated that cross-linking class I MHC molecules by culturing T4 cells with immobilized mAb to class I MHC Ag induced T4 cell proliferation in the presence of IL-2. T4 cell proliferation in the presence of IL-2 and PMA could also be induced by reacting the cells with specific mAb to polymorphic determinants on class I MHC molecules and cross-linking with GaMIg. Cross-linking mAb to CD4 or CD11a did not have a similar functional effect on T4 cells. Finally it was demonstrated that adding GaMIg to T4 cells reacted with mAb to class I MHC Ag but not CD11a resulted in an increase in intracellular calcium concentration. The data demonstrate that cross-linking class I MHC molecules results in the generation of at least one activation signal, a rise in intracellular calcium concentration, and, thereby, stimulates human T4 cells.     

A sequence-specific RNA binding complex expressed in murine germ cells contains MSY2 and MSY4

Small wounds (1.2 mm in diameter) made in the sheet of myoepithelial cells forming the "swimming" muscle of the jellyfish, Polyorchis penicillatus, were closed within 10 h by epithelial cells migrating centripetally to the wound center. Some 24 to 48 h later these cells redifferentiated into fully contractile muscle cells. Labeling with bromodeoxyuridine failed to reveal any cell proliferation during this process. Phenotype switching (within 1 h) from contractile muscle cells to migratory cells did not require synthesis of new protein as shown by treatment with 40 microM cycloheximide. Excitation-contraction coupling in undamaged muscle depended on entry of Ca(2+) through voltage-gated ion channels, as shown by a block of contractility by 40 microM nitrendipine and also on calcium released from intracellular stores since caffeine (10 mM) caused a 25% reduction in contractile force. In contrast, migratory cells did not require a source of extracellular calcium since migration was unimpeded by low (1 microM) free Ca(2+) or nitrendipine. Instead, modulatory calcium was derived from intracellular stores since caffeine (10 mM) and thapsigargin (10 microM) slowed migration. This lack of dependence on calcium influx in migratory cells was further confirmed by a dramatic down-regulation in voltage-gated inward current as shown by whole-cell patch recordings.