Association of mitogen-activated protein kinases with microtubules in mouse macrophages.

Taxol, a microtubule-binding diterpene, mimics many effects of lipopolysaccharide (LPS) on mouse macrophages. The LPS-mimetic effects of taxol appear to be under the same genetic control as responses to LPS itself. Thus we have postulated a role for microtubule-associated proteins (MAP) in the response of macrophages to LPS. Stimulation of macrophages by LPS quickly induces the activation of mitogen-activated protein kinases (MAPK). MAPK are generally considered cytosolic enzymes. Herein we report that much of the LPS-activatable pool of MAPK in primary mouse peritoneal macrophages is microtubule associated. By immunofluorescence, MAPK were localized to colchicine- and nocodazole-disruptible filaments. From both mouse brain and RAW 264.7 macrophages, MAPK could be coisolated with polymerized tubulin. Fractionation of primary macrophages into cytosol-, microfilament-, microtubule-, and intermediated filament-rich extracts revealed that approximately 10% of MAPK but none of MAPK kinase (MEK1A and MEK2) was microtubule bound. Exposure of macrophages to LPS did not change the proportion of MAPK bound to microtubules, but preferentially activated the microtubule-associated pool. These findings confirm the prediction that LPS activates a kinase bound to microtubules. Together with LPS-mimetic actions of taxol and the shared genetic control of responses to LPS and taxol, these results support the hypothesis that a major LPS-signaling pathway in mouse macrophages may involve activation of one or more microtubule-associated kinases.

The site of origin of electrical responses in visual cells of the leech, Hirudo medicinalis.

In leech visual cells the presumed light-absorbing structures are microvilli arising from the membrane of what would seem to be a large intracellular vacuole. This vacuole, however, is an extracellular compartment, since it communicates with the intercellular spaces through narrow channels. Therefore, the membrane of the microvilli is-as in other invertebrate visual cells-a part of the cell membrane. Visual responses recorded with an electrode within the vacuole were compared with the intracellular recordings. Following illumination the vacuole becomes negative with respect to the outside fluid, while the cells are depolarized. This finding indicates that inward current penetrates the cell through the microvillar membrane. It is concluded, therefore, that the electrical response (receptor potential) originates as a result of changes in the properties of the light-absorbing membrane.

Adhesion-dependent protein tyrosine phosphorylation in neutrophils treated with tumor necrosis factor.

Human neutrophils (PMN) respond to tumor necrosis factor (TNF) by releasing their granules, reorganizing their cytoskeleton, and massively secreting hydrogen peroxide. This response is dependent on adhesion to extracellular matrix proteins and expression of CD11b/CD18 integrins (Nathan, C., S. Srimal, C. Farber, E. Sanchez, L. Kabbash, A. Asch, J. Gailit, and S. D. Wright. 1989. J. Cell Biol. 109:1341-1349). We investigated the role of tyrosine phosphorylation in the response of PMN to TNF. PMN adherent to protein-coated surfaces but not suspended PMN showed tyrosine phosphorylation of several proteins (approximately 150, approximately 115, approximately 75, and approximately 65 kD) in response to TNF. Tyrosine phosphorylation was evident 5 min after addition of TNF and lasted at least 2 h. The tyrosine kinase inhibitors K252a, genistein and ST638 suppressed tyrosine phosphorylation and blocked hydrogen peroxide production in a reversible manner at low concentrations. Tyrosine kinase inhibitors also blocked the spreading of PMN in response to TNF. Dihydrocytochalasin B did not inhibit tyrosine phosphorylation, but in its presence phosphorylation was rapidly reversed. By immunocytochemistry, the majority of tyrosine phosphoproteins were localized to focal adhesions. Thus TNF-induced tyrosine phosphorylation depends on adhesion of PMN to extracellular matrix proteins, and participates in the transduction of the signals that direct the cells to spread on a biological surface and undergo a respiratory burst.

Beta 2 integrin-dependent tyrosine phosphorylation of paxillin in human neutrophils treated with tumor necrosis factor.

The focal adhesion protein paxillin undergoes tyrosine phosphorylation in response to signals mediated by integrins, neuropeptides and oncogene products, possibly via activation of the focal adhesion-associated kinase, p125FAK. In the present work, tumor necrosis factor-alpha (TNF) stimulated tyrosine phosphorylation of paxillin in human neutrophils. Cell adhesion and participation of the beta 2 integrin CD18 were necessary, but not sufficient, for the response. Adherent neutrophils also tyrosine phosphorylated paxillin in response to phorbol ester, formylmethionyl-leucyl-phenylalanine and opsonized bacteria. In contrast, p125FAK was constitutively tyrosine phosphorylated in a manner unaffected by adherence and/or TNF. Thus, cytokines and microbial products are among the stimuli that can induce the tyrosine phosphorylation of paxillin, and kinases other than p125FAK may be responsible. This is the first identification of paxillin and p125FAK in human cells and neutrophils, and one of the few identifications of a specific protein that undergoes tyrosine phosphorylation in response to any agonist in neutrophils or in response to TNF in any cell.

Role of the tyrosine kinase pyk2 in the integrin-dependent activation of human neutrophils by TNF.

Secretion of inflammatory products from neutrophils can be induced by a combination of signals from ligated integrins and receptors for soluble, physiological agonists such as TNF. Here we identify pyk2 in primary human neutrophils; localize it to focal adhesions and podosomes; and demonstrate its tyrosine phosphorylation, activation, and association with paxillin during stimulation of adherent cells by TNF. Tyrphostin A9 emerged as the most potent and selective of 51 tyrosine kinase inhibitors tested against the TNF-induced respiratory burst. Tyrphostin A9 inhibited TNF-induced tyrosine phosphorylation of pyk2 without blocking the cells' bactericidal activity. Wortmannin, an inhibitor of phosphatidylinositol-3-kinase, potently blocked the TNF-induced respiratory burst and selectively inhibited tyrosine phosphorylation of pyk2. Thus, pyk2 appears to play an essential role in the ability of neutrophils to integrate signals from beta(2) integrins and TNF receptors.

Responses of photoreceptors in Hermissenda.

The five photoreceptors in the eye of the mollusc Hermissenda crassicornis respond to light with depolarization and firing of impulses. The impulses of any one cell inhibit other cells, but the degree of inhibition differs in different pairs. Evidence is presented to show that the interactions occur at terminal branches of the photoreceptor axons, inside the cerebropleural ganglion. Properties of the generator potential are examined and it is shown that the depolarization develops in two phases which are affected differently by extrinsic currents. Finally, it is shown that by enhancing the differences in the responses of individual cells to a variety of stimuli, the interactions may facilitate a number of simple discriminations.

Ceramide selectively inhibits early events in the response of human neutrophils to tumor necrosis factor.

Cell spreading and the respiratory burst of neutrophils responding to soluble, physiological agents and adherent to model biological surfaces are typically delayed in onset by 15 min or more. The lag period may be a physiologically important feature of the action of such agents on neutrophils in that it may allow for migration before secretion. However, the mechanism that interposes such a long delay between stimulus and response is unknown. Tumor necrosis factor alpha (TNF-alpha) mediates some of its actions by triggering sphingomyelinase to generate ceramide. In adherent human neutrophils, however, exogenous ceramide did not mimic TNF-alpha's ability to stimulate cell spreading, paxillin tyrosine phosphorylation, or the respiratory burst. On the contrary, ceramide suppressed each such response. Ceramide did so by extending the lag period in the cells' response to TNF-alpha. Ceramide extended the lag period whether it was added exogenously or generated endogenously by sphingomyelinase, and the effect was reversible. Remarkably, however, ceramide inhibited cell spreading or the respiratory burst only if added together with TNF-alpha or within the next few minutes. Neutrophils ignored ceramide if it was added later, even if the TNF-alpha-triggered respiratory burst had not yet commenced. These features suggest that an early, brief elevation of ceramide in response to TNF-alpha could mediate the lag period. By temporarily inhibiting tyrosine phosphorylation, cell spreading, and the respiratory burst, ceramide or a functionally similar mediator could serve as a phase coordinator of the neutrophil's response to soluble agonists.

The effect of verapamil on cellular uptake, organ distribution, and pharmacology of cyclosporine.

Verapamil has been shown to potentiate cyclosporine's effect in inhibiting lectin-stimulated proliferation of murine and human lymphocytes, and in prolonging graft survival in experimental heterotopic cardiac transplantation in rats. A series of experiments were designed to determine whether verapamil's effect occurred by increasing cyclosporine uptake or decreasing cyclosporine's clearance by lymphocytes utilizing human peripheral blood lymphocytes and radiolabeled cyclosporine. Verapamil had no effect. The distribution of radiolabeled cyclosporine was also studied in mice that had been given verapamil (10 mg/kg) 1 hr prior to cyclosporine injection. No significant changes in organ distribution occurred. Lectin-stimulated release of intracellular ionized calcium was studied using a flurometric technique (Quin-2 and Fura-2). Neither cyclosporine nor verapamil had any effect on either lectin-stimulated or phorbol ester-stimulated release of intracellular ionized calcium. Phorbol ester and subproliferative doses of lectin were used to determine the effect of cyclosporine and verapamil on protein kinase C-mediated lymphocyte activation. Cyclosporine inhibited phorbol ester stimulated proliferation and verapamil potentiated this inhibition. Verapamil does not change cell or organ uptake of cyclosporine, and it does not affect the initial increase in intracellular ionized calcium that occurs with lymphocyte activation. Verapamil potentiates cyclosporine in inhibiting protein kinase C-mediated events in lymphocyte activation.

Release of vasoactive intestinal peptide during hyperdynamic sepsis in dogs.

Vasoactive intestinal peptide (VIP) is a potent vasodilator that has been reported to be a mediator of the hemodynamic changes in endotoxin-induced hypodynamic septic shock. We investigated the release of VIP in a hyperdynamic model of sepsis in awake, conscious dogs similar to that of sepsis in human beings. Sepsis was induced by intraperitoneal implantation of a fibrin clot containing live Escherichia coli (0.9 +/- 0.2 X 10(9) organisms per kilogram of body weight). All dogs developed hyperdynamic sepsis with increased cardiac output and decreased systemic vascular resistance. During the first 24 hours of sepsis, VIP was released without a concomitant decrease in blood pressure, suggesting that during septic shock it was released by a direct mechanism rather than as a result of hypotension. During peak VIP release (2 to 4 hours after induction of sepsis) no decreases in systemic vascular resistance or mean arterial pressure were observed. This suggests that mediators other than VIP may be responsible for the vasodilation observed during sepsis. The precise role of VIP during sepsis is therefore yet to be clarified.

Effect of endothelin-1 and vasoactive intestinal contractor on blood flow and output of vasoactive intestinal polypeptide in the feline colon.

Injection of the structurally related peptides, endothelin-1 and vasoactive intestinal contractor (VIC), into a branch of the superior mesenteric artery in anesthetized cats caused dose-dependent reductions in blood flow in the portal vein and inferior mesenteric artery. The maximum effect occurred after 1 minute and was more prolonged in the portal vein. The effects of the two peptides were not significantly different. The colonic output of vasoactive intestinal polypeptide (VIP) into portal venous blood was decreased significantly by endothelin-1 and VIC, returning to baseline more rapidly than blood flow. When norepinephrine was injected to produce comparable reductions in blood flow, the output of VIP into portal venous blood was not altered significantly. These results suggest that inhibition of output of the vasodilator VIP contributes to the vasoconstrictor effects of endothelin-1 and VIC in the feline colonic vascular bed.

Interactions leading to horizontal cell responses in the turtle retina.

1. Small responses to large fields of dim monochromatic lights were recorded intracellularly from luminosity horizontal cells (L-cells), chromaticity horizontal cells (C-cells) and cones in the retinae of turtles, Pseudemys scripta elegans.2. Responses of cones to brief flashes applied over steady backgrounds were studied in order to interpret the corresponding responses of horizontal cells. Steady red or green backgrounds make the responses of red-sensitive cones smaller, faster and often diphasic. Green backgrounds have similar effects on the responses of green-sensitive cones to green flashes, but red backgrounds do not change them appreciably. Responses of double cones have properties intermediate between those of red and green cones.3. L-cells of both type I and type II are hyperpolarized by all visible wave-lengths, and their spectral sensitivity in the linear range resembles that of red cones. Their responses are not invariant with respect to colour, and their sensitivity to green relative to red stimuli increases during red backgrounds. These properties suggest that L-cells are activated mainly by red cones but also receive impingement from the red members of double cones.4. Spectral properties of red/green C-cells resemble those of green cones as modified by the recurrent action of L-cells. They can be explained assuming that red/green C-cells receive their principal impingement from green cones and subsidiary interactions from green/blue C-cells and the green members of double comes.5. The spectral sensitivity of the hyperpolarizing responses of green/blue C-cells is ascribed to impingement from blue cones. Their depolarizing responses have complex properties which suggest that they are brought about by the activity of both L-cells (probably through the blue cones) and red/green C-cells.6. It is concluded that the main properties of the responses of the horizontal cells can be explained by a simple circuit in which each horizontal cell is connected to a corresponding type of cone and the L-cells have a recurrent impingement on all cones. The scheme is modified by additional interactions which operate on the responses of each horizontal cell type.

Analysis of responses in visual cells of the leech.

1. Potentials were recorded from the cytoplasm and from the vacuole of leech photoreceptors. Since the vacuole is lined with microvilli and is connected to the outside by narrow channels, the potential drops between vacuole and outside measure the current through the microvillar membrane.2. In darkness, the potential of the cytoplasm with respect to the outside is about - 45 mV while the potential of the vacuole is approximately zero.3. Following illumination the negativity of the cytoplasm decreases and the vacuole becomes negative relative to the outside.4. For dim intensities, the response to a flash of light may grow proportionately more than the intensity of the flash. This is probably due to development of a depolarizing local response.5. The resistance from the cytoplasm to the outside was about 150 MOmega in darkness and decreased to approximately 40 MOmega at the peak of the response to a bright flash (on average). Corresponding measurements from the vacuole gave 50 MOmega in darkness and 35 MOmega at the peak of the response.6. Charging curves produced by steps of constant currents applied to the cytoplasm or to the vacuole include two time constants (about 5 and 50 msec on average). The longer time constant decreases greatly with bright illumination.7. The results are consistent with the interpretation that the response to light is brought about by an increase of conductance of the microvillar membrane.

Responses to single photons in virual cells of limulus.

1. A system proposed in a previous article as a model of responses of visual cells has been analysed with the purpose of predicting the features of responses to single absorbed photons.2. As a result of this analysis, the stochastic variability of responses has been expressed as a function of the amplification of the system.3. The theoretical predictions have been compared to the results obtained by recording electrical responses of visual cells of Limulus to flashes delivering only few photons.4. Experimental responses to single photons have been tentatively identified and it was shown that the stochastic variability of these responses is similar to that predicted for a model with a multiplication factor of at least twenty-five.5. These results lead to the conclusion that the processes responsible for visual responses incorporate some form of amplification. This conclusion may prove useful for identifying the physical mechanisms underlying the transducer action of visual cells.

Electrical responses of single cones in the retina of the turtle.

1. Intracellular recordings have been made from single photoreceptors in the retina of the turtle. Histological sections of the retina made after injection of dye through the recording electrode reveal dye in the inner segments of single cones.2. Following a brief flash of light the cone undergoes a hyperpolarization which is graded with the intensity of the flash.3. The excitatory receptive field of a receptor is probably as small as the cross-section of a single cone, but accurate measurements are rendered difficult by scattering of light within the retina.4. The voltage drop produced by a current injected into the cell is increased during the response to light. Steady hyperpolarizing currents increase the size of the response to light; depolarizing currents of increasing strength reduce and then reverse the response.5. The results are consistent with the hypothesis that light activates the visual cell by decreasing the permeability of membrane channels which in darkness act as a shunt of the membrane.

Responses of hair cells in the statocyst of Hermissenda.

Responses to mechanical stimulation were recorded from hair cells in the statocyst of Hermissenda crassicornis. The response to a brief stimulus is a depolarizing wave which reaches peak in about 25 msec and decays slowly. 2. Hyperpolarization by extrinsic currents increases the amplitude of the response; depolarization decreases it and eventually reverses its polarity. It is inferred from these results that the primary outcome of the transduction process is an increase of membrane conductance and that the voltage change (generator potential) follows as a secondary event. 3. The features of the conductance change were reconstructed from the time course of the generator potential and the passive properties of the membrane. It was found that the increase of membrane conductance develops slowly and is roughly proportional to the energy delivered by the stimulus. 4. The time course of the conductance change required to reproduce the generator potential is similar to the output of a model involving a sequence of transformations. 5. The generator potential is sensitive to temperature, becoming faster as temperature is raised. This effect is reproduced by the model if the transition rates are assumed to be temperature-dependent, with a Q10 of about 2. 6. It is concluded that a chain of temperature-sensitive processes is interposed between the stimulus and the increase of membrane conductance.

Colour-dependence of cone responses in the turtle retina.

1. Responses to monochromatic lights were recorded intracellularly from red cones, green cones, and luminosity horizontal cells (L-cells) in the retinae of turtles.2. Both types of cones responded to small fields of illumination with graded hyperpolarizations. Red cones were only moderately more sensitive to deep red (680 nm) than to green (550 nm) light while green cones were much more sensitive to the green light than to the red. L-cells produced small responses for flashes of either colour covering small fields.3. Stimulation of large fields with monochromatic lights of moderate or high intensity evoked large L-cell responses and composite responses in cones. These latter include the hyperpolarizing action of the light absorbed by the cone itself (direct response), its enhancement by illumination of the near surround, and the depolarizing effects of L-cell feed-back.4. L-cells respond primarily to the activity of red cones; with sufficient intensity of the light, however, their responses are influenced also by green cones. As a result, if a red and a green light stimulate red cones equally, the L-cell response is larger for the green stimulus.5. Green cones were depolarized by deep red lights of moderate intensity applied over large fields. These depolarizing responses include oscillations which follow closely oscillations in L-cells. Green light applied to the same large fields produced hyperpolarization of green cones.6. Red cones were hyperpolarized by red or green light covering large fields, but the time course of their responses differed for the two colours, reflecting a corresponding difference in L-cell activity.7. Red light in the form of an annulus produced large responses in central L-cells without eliciting direct responses in central green cones. In these conditions green cones developed depolarizing waves which included a large, sharp transient.8. It is concluded from these and other results that the direct response of each cone is modified by two interactions: enhancement only from nearby cones of the same colour and depression controlled (through L-cell feed-back) by cones of all colours. In this way the response of any cone will change as the proportion of responses in cones of different colours changes, this proportion being a function of the wave-length of the light.