Unilateral transfemoral amputees exhibit altered strength and dynamics of muscular co-activation modulated by visual feedback.

Objective.Somatosensory perception is disrupted in patients with a lower limb amputation. This increases the difficulty to maintain balance and leads to the development of neuromuscular adjustments. We investigated how these adjustments are reflected in the co-activation of lower body muscles and are modulated by visual feedback.Approach.We measured electromyography (EMG) signals of muscles from the trunk (erector spinae and obliquus external), and the lower intact/dominant leg (tibialis anterior and medial gastrocnemius) in 11 unilateral transfemoral amputees and 11 age-matched able-bodied controls during 30 s of upright standing with and without visual feedback. Muscle synergies involved in balance control were investigated using wavelet coherence analysis. We focused on seven frequencies grouped in three frequency bands, a low-frequency band (7.56 and 19.86 Hz) representing more sub-cortical and spinal inputs to the muscles, a mid-frequency band (38.26 and 62.63 Hz) representing more cortical inputs, and a high-frequency band (92.90, 129 and 170.90 Hz) associated with synchronizing motor unit action potentials. Further, the dynamics of changes in intermuscular coupling over time were quantified using the Entropic Half-Life.Main results.Amputees exhibited lower coherency values when vision was removed at 7.56 Hz for the muscle pair of the lower leg. At this frequency, the coherency values of the amputee group also differed from controls for the eyes closed condition. Controls and amputees exhibited opposite coherent behaviors with visual feedback at 7.56 Hz. For the eyes open condition at 129 Hz, the coherency values of amputees and controls differed for the muscle pair of the trunk, and at 170.90 Hz for the muscle pair of the lower leg. Amputees exhibited different dynamics of muscle co-activation at the low frequency band when vision was available.Significance.Altogether, these findings point to the development of neuromuscular adaptations reflected in the strength and dynamics of muscular co-activation.

Motor output complexity in Parkinson's disease during quiet standing and walking: Analysis of short-term correlations using the entropic half-life.

Parkinson's disease (PD) is associated with alterations in motor outputs such as center of pressure (CoP) adjustments during quiet standing and foot kinematics during walking. Previous research suggests that the complexity of motor outputs reflects the number of control processes stabilizing a specific movement, providing a measure that is linked to the neurological control of the movement. The Entropic Half Life (EnHL) represents a new method for assessing motor output complexity. We hypothesized that there will be a lack of neuromuscular control pathways for PD patients, resulting in a decrease in motor output complexity. We computed the EnHL of CoP adjustments during quiet standing and foot kinematics during walking of 70 PD patients and 33 age-matched controls. Patients with PD showed longer EnHL values compared to controls, suggesting a tighter motor control. Excluding vision led to a decrease of EnHL of CoP in both groups. EnHL was correlated with spatio-temporal gait parameters. We compared EnHL with the pull test and the timed up-and-go test. No significant differences were present in the pull test, yet motor output complexity was correlated with the timed up-and-go test. The results suggest a reduced complexity in motor outputs of PD patients affecting distinct motor functions.

Alterations in lower limb multimuscle activation patterns during stair climbing in female total knee arthroplasty patients.

Total knee arthroplasty (TKA) patients commonly experience neuromuscular adaptations that may affect stair climbing competence. This study identified multimuscle pattern (MMP) changes in postoperative female TKA patients during stair climbing with a support vector machine (SVM). It was hypothesized that TKA patients adopt temporal and spectral muscle activation characteristics indicative of muscle atrophy and cocontraction strategies. Nineteen female subjects [10 unilateral sex-specific TKAs, 62.2 ± 8.6 yr, body mass index (BMI) 28.2 ± 5.4 kg/m(2); 9 healthy control subjects, 61.4 ± 7.4 yr, BMI 25.6 ± 2.4 kg/m(2)] were recruited. Surface electromyograms (EMGs) were obtained for seven lower limb muscles of the affected limb of TKA subjects and a randomly assigned limb for control subjects during stair climbing. Stance phase (±30%) EMG data were wavelet transformed and normalized to total power. Data across all muscles were combined to form MMPs and analyzed with a SVM. Statistical analysis was performed with binomial tests, independent group t-tests, or independent group Mann-Whitney U-tests in SPSS (P < 0.05). SVM results indicated significantly altered muscle activation patterns in the TKA group for biceps femoris (recognition rate 84.2%), semitendinosus (recognition rate 73.7%), gastrocnemius (recognition rate 68.4%), and tibialis anterior (recognition rate 68.4%). Further analysis identified no significant differences in spectral activation characteristics between groups. Temporal adaptations, indicative of cocontraction strategies, were, however, evident in TKA MMPs. This approach may provide a valuable tool for clinical neuromuscular function assessment and rehabilitation monitoring.

Multi-muscle activation strategies during walking in female post-operative total joint replacement patients.

Dynamic knee joint function requires coordinated multi-muscle activation patterns (MMP) that may be adversely affected by total knee arthroplasty (TKA). This study identified MMP changes in post-operative female TKA patients using a Support Vector Machine (SVM). It was hypothesised that TKA patients can successfully be classified and display significant alterations in temporal and spectral muscle activation characteristics. 19 female subjects (10 unilateral gender-specific TKA, 62.2±8.6yrs, BMI 28.2±5.4; and 9 healthy controls, 61.4±7.4yrs, BMI 25.6±2.4) were recruited. Surface electromyograms (EMG) were obtained for 7 lower limb muscles during walking. Stance phase (±30%) EMG data were processed using a wavelet transform and normalized to total power. Data across all muscles were combined to form MMPs and analyzed using a SVM. Recognition rates for all subjects were computed for MMPs and individual muscles. A binomial test was used to establish statistical significance (p<0.05). The results supported the hypothesis indicating significantly altered muscle activations for vastus medialis (recognition rate ∼68.4%) and biceps femoris (recognition rate ∼73.7%). Further analysis identified distinct between group differences across temporal, spectral and intensity domains. Application of a combined SVM and MMP approach may be beneficial for the future assessment of post-surgical dynamic muscle function.

Motor strategy patterns study of diabetic neuropathic individuals while walking. A wavelet approach.

The aim of this study was to investigate muscle׳s energy patterns and spectral properties of diabetic neuropathic individuals during gait cycle using wavelet approach. Twenty-one diabetic patients diagnosed with peripheral neuropathy, and 21 non-diabetic individuals were assessed during the whole gait cycle. Activation patterns of vastus lateralis, medial gastrocnemius and tibialis anterior were studied by means of bipolar surface EMG. The signal׳s energy and frequency were compared between groups using t-test. The energy was compared in each frequency band (7-542 Hz) using ANOVAs for repeated measures for each group and each muscle. The diabetic individuals displayed lower energies in lower frequency bands for all muscles and higher energies in higher frequency bands for the extensors׳ muscles. They also showed lower total energy of gastrocnemius and a higher total energy of vastus, considering the whole gait cycle. The overall results suggest a change in the neuromuscular strategy of the main extensor muscles of the lower limb of diabetic patients to compensate the ankle extensor deficit to propel the body forward and accomplish the walking task.

[Muscle biomechanics in total ankle replacement]. / Muskuläre biomechanik in der sprunggelenkprothetik.

The purpose of this orthopaedic-biomechanical study was to evaluate the muscle function in total ankle replacement (TAR) patients 1 year after surgery. Ten patients underwent a combined clinical and muscle biomechanical assessment prior to implantation and at the 1-year follow-up. Pain score, American Orthopaedic Foot and Ankle Society (AOFAS) ankle score, ankle range of motion (ROM), and calf circumference difference between the affected leg and contralateral healthy leg were assessed. Biomechanically, isometric maximal voluntary torque for ankle dorsiflexion and plantar flexion was measured simultaneously with surface electromyography of four lower leg muscles. At follow-up, a significant improvement of the pain score (from 6.7 to 0.8 points), AOFAS ankle score (from 35.6 to 92.3 points), and ROM could be shown. Not significantly, the mean calf circumference difference between legs decreased from 2.2 to 1.4 cm. However, a significant increase was seen in the mean dorsiflexion (from 17.0 to 25.8 Nm) and plantar flexion torque (15.7 to 24.6 Nm) of the TAR-treated ankle. The mean EMG frequency content of the affected lower leg at TAR follow-up was lower than in the muscles of the contralateral healthy side. In contrast, the mean EMG intensity at TAR follow-up in side-comparison was statistically the same for all muscles. Ankle OA patients have better muscle function with TAR than under the arthritic condition, but they do not reach the normal level of the contralateral healthy leg 1 year after surgery.

Estimation of the interplay between groups of fast and slow muscle fibers of the tibialis anterior and gastrocnemius muscle while running.

Electromyograms recorded from the lower limbs of humans while running were submitted to a time/frequency analysis using wavelets. The results of the wavelet analysis yielded intensity spectra at every time point during the swing and the stance phase. It was previously shown that more or less high frequency components get activated during different periods of the movement. The purpose of this study was to test to what extent the spectra can be reconstructed by a linear superposition of two generating spectra that were associated to groups of fast and slow muscle fibers. The terms fast and slow do not only refer to the conduction velocity but also to the shape of the motor unit action potential and are used to characterize the groups in a broader sense. The principal component analysis of the spectra confirmed that a two dimensional spectral space was appropriate. A parametric spectral decomposition was used to extract the generating spectra within the two dimensional spectral space. The generating spectra were in turn used to compute the power with which the groups of muscle fibers contribute to the measured spectra and thus to the overall muscular activity. The power that was obtained for the different time points during the movement reflects the biomechanically important interplay between the groups of muscle fibers while running.

[Analysis of wavelet transformed electromyographic signals that were altered by wearing a knee brace]. / Analyse der Veränderungen von Wavelet-transformierten elektromyographischen Signalen, wie sie beim Tragen einer Kniebandage entstehen.

The comparison of electromyograms represents a challenge for data analysis. The aim of the project was to present a method that uses a minimal computational effort to resolve small but significant changes in the muscular activity that occur while walking with and without a knee brace. The wavelet transformed electromyograms were represented as intensity patterns that resolve the power of the signal in time and frequency. The intensity pattern of each electromyogram defines single points in a pattern space. The distance between these points in pattern space were used to detect and show the separation between the groups of electromyograms that were recorded while walking with and without a knee brace. The method proposes a distance versus angle representation to visually discriminate the intensity patterns. Once it has been shown that the differences are statistically significant, one can visualize the result in a difference intensity pattern that indicates at what time and at what frequency the electromyograms vary between the two conditions tested. It is to be expected that interventions that are more intrusive than a knee brace will reveal even more distinct differences.

Gender dependent EMGs of runners resolved by time/frequency and principal pattern analysis.

A promising approach for the analysis of surface electromyograms is to use wavelets to determine the spectral distribution of the signal intensity at any time. The authors have recently proposed using non-linearly scaled wavelets to obtain intensity patterns, which reflect the spectral distribution at any given time point. Further analysis of intensity-patterns is greatly facilitated by representing them as linear combinations of a base set of principal-patterns. The weight with which each principal-pattern contributes to the intensity-pattern can be represented on a set of orthogonal axes that span a previously introduced pattern space. The purpose of the present study was to show how to use pattern space to discriminate and classify male and female runners based on the electromyograms of five muscles of the limb. The results showed that there were significant gender specific differences, which allowed more than a 95% correct classification of the subjects as males or females. Classification was possible irrespective of the shod condition while running. Gender specific differences occurred at well-defined time periods during the movement. Common to both genders was that spectral changes did not parallel the changes in total signal intensity.

Muscle activity in the leg is tuned in response to ground reaction forces.

During walking and running, the human body reacts to its external environment. One such response is to the impact forces that occur at heel strike. This study tested previous speculation that the levels of muscle activity in the lower extremities are adjusted in response to the loading rate of the impact forces. A pendulum apparatus was used to deliver repetitive impacts to the heels of 20 subjects. Impact forces were of similar magnitude to those experienced during running, but the loading rate was varied by 13% using different materials in the subjects' shoes. Myoelectric patterns were measured in the tibialis anterior, medial gastrocnemius, vastus medialis, and biceps femoris muscles. Wavelet analysis was used to resolve intensity of the myoelectric patterns into time and frequency space. Substantial and significant differences in the myoelectric activity occurred between the impact conditions for the 50 ms before and the 50 ms after impact, reaching 3 ms in timing, 16% in wavelet number, and 154% in the intensity of the muscle activity.

Specified-resolution wavelet analysis of activation patterns from BOLD contrast fMRI.

Functional magnetic resonance (MR) MR imaging (fMRI) with blood-oxygenation-level-dependent (BOLD) contrast localizes neuronal processing of cognitive paradigms. As magnetic resonance signal responses are small, functional mapping requires statistical analysis of temporally averaged image data. Although voxels activating at the paradigm frequency can be identified from the Fourier power spectrum, such analyses collapse the temporal information that is useful to establish consistency of responses during the paradigm. The design of a set of nonorthogonal wavelets of specified frequency resolution within the power spectrum was investigated for extracting desired frequency responses from the noisy signal intensity of individual voxels. These wavelets separate the low-frequency cognitive response to the paradigm from the respiratory and cardiac responses at higher frequencies. The retention of the temporal information, possible by wavelet analysis, allows the MR signal changes to be compared to changes in behavioral responses over the duration of an entire paradigm. The amplitude and time delay of the wavelet specified by the paradigm identify quantitatively the size of the MR signal change and the temporal delay of the hemodynamic BOLD response, respectively. This specified-resolution wavelet analysis was demonstrated for individual voxels and maps through the frontal eye fields using a visually guided saccade paradigm.

Surface EMG shows distinct populations of muscle activity when measured during sustained sub-maximal exercise.

Muscle activity patterns were measured from the rectus femoris, biceps femoris, tibialis anterior and gastrocnemius medialis muscles using electromyography (EMG). Recordings were made from six runners while they ran for 30 mm around a track at a constant, self-selected pace. They were made from three sites along each muscle belly, and for ten consecutive steps on each 450 m lap completed. The intensity of the myoelectric signals was resolved into components in time and frequency space using wavelet analysis. Distinct populations of high- (170-220 Hz) and low-frequency (40-60 Hz) components could be seen in the frequency spectra. There was no significant effect of the electrode position on the rates of change of EMG intensity for any of the muscles, and there was no significant difference in these rates between a 150-ms window at the end of the swing phase and a 150-ms window during the stride phase of running. There were significant differences in the way in which each subject responded to the task, and the way in which the FMG intensities changed at the different frequency bands. There was a significant reduction in EMG intensity at low frequencies and a significant increase at high frequencies, and these changes were ubiquitous for all four muscles tested. The frequencies that showed the greatest changes coincided with the frequencies where distinct populations of activity occurred within the signal. These changes in muscle activity are different from those seen for maximal fatiguing contractions. However, they suggest that the patterns of muscle recruitment may change during sustained sub-maximal exercise.

Intensity analysis in time-frequency space of surface myoelectric signals by wavelets of specified resolution.

Surface myoelectric signals often appear to carry more information than what is resolved in root mean square analysis of the progress curves or in its power spectrum. Time-frequency analysis of myoelectric signals has not yet led to satisfactory results in respect of separating simultaneous events in time and frequency. In this study a time-frequency analysis of the intensities in time series was developed. This intensity analysis uses a filter bank of non-linearly scaled wavelets with specified time-resolution to extract time-frequency aspects of the signal. Special procedures were developed to calculate intensity in such a way as to approximate the power of the signal in time. Applied to an EMG signal the intensity analysis was called a functional EMG analysis. The method resolves events within the EMG signal. The time when the events occur and their intensity and frequency distribution are well resolved in the intensity patterns extracted from the EMG signal. Averaging intensity patterns from multiple experiments resolve repeatable functional aspects of muscle activation. Various properties of the functional EMG analysis were shown and discussed using model EMG data and real EMG data.

[Electromyographic measurement of human muscles depending on position to radiesthetically defined attraction zones compared with neutral zones]. / Standortabhängige elektromyographische Messungen an humanen Muskeln auf radiästhetisch definerten Reizzonen im Vergleich zu neutralen Zonen.

Experienced dowsers were able to localize radiesthetically defined zones with statistically highly significant accuracy in blind and double-blind experiments. The transition from neutral to active zones and vice-versa could be demonstrated electromyographically on various arm muscles of sensitive persons. Thus, there is most likely a relationship between an active zone and certain functions of the human body. This project deals with basic research, it does not examine possible causes of diseases. Nevertheless, the often postulated influence of active zones on health is part of the background of the project.

Activation of the respiratory burst in eosinophil leucocytes--a transduction sequence decoupled from cytosolic Ca2+ rise.

The activation of the respiratory burst by complement factor 5a (C5a), platelet-activating factor (PAF), formyl-Met-Leu-Phe (fMLP) and neutrophil-activating peptide IL-8 was explored in eosinophils from patients with the hypereosinophilic syndrome. The amplitude of the response increased with increasing concentrations of C5a and PAF, but the time for its induction was unaffected by the amount of stimulus applied. Respiratory burst activity resulting from phorbol 12-myristate, 13-acetate (PMA)-mediated activation of protein kinase C (PKC) produced longer onset times, which shortened with increasing PMA concentrations. Total inhibition of the C5a- and PMA-mediated burst could be achieved with the PKC inhibitor staurosporine at concentrations of 100 and 5nM, respectively. Calcium depletion abolished agonist-induced rises in cytosolic free calcium ([Ca2+]i) and respiratory burst activity, but not PMA-mediated NADPH-oxidase activation. While PMA reduced elevations in [Ca2+]i, it restored the burst response to agonists in Ca(2+)-depleted eosinophils. These results agree with the agonist-induced activation of the NADPH-oxidase via PKC, but suggest a parallel, Ca(2+)-, phospholipase C- and PKC-independent signal transduction pathway. Data obtained with B. pertussis toxin showed that the respiratory burst in eosinophils is blocked by ADP-ribosylation of G(i)-proteins, but that in the presence of PMA portions of the agonist response could be recovered.

Practical instructions for radioactively labeled ligand receptor binding studies.

This paper represents a practical guide to ligand receptor binding studies with special emphasis on the detailed characterization of radiolabeled ligands prior to receptor binding experiments. The effects due to labeled ligand inactivated by the labeling procedure are included. The inactive ligand may contribute as much as the active ligand to nonspecific binding. A method for measuring the fraction of active ligand in the initial sample and for determining the specific radioactivity and bindability is presented in detail. A straightforward procedure for extracting the concentration of bound and free active ligand from the measured crude binding data is then presented. The dissociation constant and the number of receptors are obtained by fitting the binding equation to the free and bound ligand concentrations. As a practical example, interleukin-8 binding to human neutrophils was used. The experiments are discussed with respect to possible errors, and specific conditions (concentrations of ligand and receptors) necessary for accurate determination of the respective binding parameters are given.

Monocyte chemotactic protein 3 is a most effective basophil- and eosinophil-activating chemokine.

CC chemokines constitute a novel class of cytokines that attract and activate monocytes and lymphocytes, as well as basophil and eosinophil leukocytes, with distinct target cell profiles, and are believed to be involved in the regulation of different types of inflammation. The action of the recently identified monocyte chemotactic protein 3 (MCP-3) on human basophil and eosinophil function was studied and compared with that of other CC chemokines. In basophils, MCP-3, MCP-1, RANTES, and macrophage inflammatory protein (MIP)-1 alpha all induced cytosolic-free calcium concentration ([Ca2+]i) changes and, with different efficacies, chemotaxis (RANTES = MCP-3 >> MCP-1 > MIP-1 alpha), histamine release (MCP-1 = MCP-3 >> RANTES > MIP-1 alpha), and leukotriene C4 formation, after IL-3 pretreatment (MCP-1 = MCP-3 >> RANTES > MIP-1 alpha). Thus, MCP-3 was as effective as MCP-1 as an inducer of mediator release, and as effective as RANTES as a stimulus of basophil migration. In contrast to MCP-1, MCP-3 was also a stimulus for eosinophils, and induced [Ca2+]i changes and chemotaxis as effectively as RANTES, which is the most potent chemotactic cytokine for these cells. Desensitization of the transient changes in [Ca2+]i was used to assess receptor usage. In basophils, stimulation with MCP-3 prevented responsiveness to MCP-1 and RANTES, but not to MIP-1 alpha. No single CC chemokine (except for MCP-3 itself) affected the response to MCP-3, however, which was prevented only when the cells were prestimulated with both MCP-1 and RANTES. In eosinophils, by contrast, cross-desensitization between RANTES and MCP-3 was obtained. RANTES and to a lesser extent MCP-3 also desensitized eosinophils toward MIP-1 alpha. The desensitization data suggest the existence of three chemokine receptors: (a) a MCP-1 receptor expressed on basophils but not eosinophils that is activated by MCP-1 and MCP-3; (b) a RANTES receptor in basophils and eosinophils that is activated by RANTES and MCP-3; and (c) a MIP-1 alpha receptor that is activated by MIP-1 alpha, RANTES and, more weakly, by MCP-3. This study shows that MCP-3 combines the properties of RANTES, a powerful chemoattractant, and MCP-1, a highly effective stimulus of mediator release, and thus has a particularly broad range of activities toward both human basophil and eosinophil leukocytes.

RANTES and related chemokines activate human basophil granulocytes through different G protein-coupled receptors.

Chemotactic cytokines related to interleukin-8 (IL-8; CXC-chemokines) or monocyte chemotactic protein-1 (MCP-1; CC-chemokines) have been shown to stimulate human basophils, and are considered important tissue-derived mediators of inflammation. We have studied the effects of four CC-chemokines and show that MCP-1, RANTES (regulated on activation, normal T expressed and secreted) and macrophage inflammatory protein-1 alpha (MIP-1 alpha) are potent basophil agonists inducing a rapid change of cytosolic free calcium ([Ca2+]i), the release of histamine and sulfido-leukotrienes, and chemotaxis. MCP-1 was the most potent stimulus of release, and the only chemokine that induced marked exocytosis in basophils without pretreatment with interleukin-3. RANTES was the strongest stimulus of chemotaxis, but only a moderate stimulus of release. MIP-1 alpha elicited relatively weak chemotaxis and release responses, but was effective at considerably lower concentrations than MCP-1 and RANTES. MIP-1 beta, by contrast, despite its high homology to MIP-1 alpha, was totally inactive. Normodense human eosinophils, tested for comparison, responded in a similar fashion to RANTES and MIP-1 alpha, but were unresponsive to MCP-1 and MIP-1 beta. All CC-chemokines except MIP-1 beta induced a similar rapid and transient rise of [Ca2+]i that was sensitive to pertussis toxin, indicating that they activate basophils via G-protein-coupled receptors. Cross-desensensitization experiments indicate that basophils bear different CC-chemokine receptors. Some interact selectively with MCP-1 or RANTES, while others are shared by RANTES and MIP-1 alpha.

Serine/threonine kinases in signal transduction in response to thrombin in human platelets. Use of 17-hydroxywortmannin to discriminate signals.

Although the importance of protein kinases in platelet activation, particularly protein kinase C (PKC), is well established there remain many problems regarding the various phosphorylation cascades, the role of phosphatases and the importance of other serine/threonine and tyrosine kinases. A particular problem is the mechanism of activation of the fibrinogen receptor, GPIIb/IIIa, a critical step in aggregation. Although GPIIIa is phosphorylated (on threonine) neither the stoichiometry nor the minor changes on activation seem adequate to explain the response. Relatively unspecific inhibitors of PKC such as staurosporine prevent PO4 incorporation into most kinase substrates but only inhibit platelet aggregation partially. However, staurosporine does induce activation and then inhibits several renaturable serine/threonine kinases, probably via phosphatases. Staurosporine did not, however, inhibit the platelet Ca2+ signal in response to thrombin but rather enhanced it. 17-Hydroxywortmannin (HWT), a fungal metabolite, has been shown to inhibit respiratory burst in neutrophils and causes haemorrhages. It was recently reported to be a myosin light chain kinase (MLCK) inhibitor and to inhibit PKC only at much higher concentrations. In platelets, HWT inhibits aggregation and partially inhibits phosphorylation of myosin light chain and P47 in thrombin-activated platelets. It also allows the discrimination of an early and a late phase in the cytoplasmic Ca2+ signal since at lower concentrations it only inhibits the late phase. The late phase of ATP release was also inhibited in a dose-dependent manner. The activation of most of the renaturable serine/threonine kinases was also inhibited by HWT. These results support earlier conclusions that the early phase of the Ca2+ signal is phospholipase C dependent but indicate that other mechanisms must be responsible for the late phase. The relative specificity of HWT for MLCK might indicate that this has an unexpected major role in controlling these late phase reactions including activation of GPIIb/IIIa or its clustering. However, staurosporine completely inhibits phosphorylation of myosin light chain by its kinase (as well as other kinases) and has the opposite effect on Ca2+ signals. Clearly, the interactions and feed-back mechanisms between these kinases are very complex but the results suggest that phosphatases acting together with their complementary kinases should also be considered as important platelet activation regulators. P47, long considered a major PKC substrate, may also be phosphorylated by MLCK.

Signal transduction for interleukin-3-dependent leukotriene synthesis in normal human basophils: opposing role of tyrosine kinase and protein kinase.

The intracellular signaling pathways regulating the synthesis of leukotrienes by myeloid cells are largely unknown. In addition, the signal transduction mechanisms utilized by the cytokine receptor family are still poorly understood. The fact that in mature human basophils the synthesis of leukotriene C4 (LTC4) induced by C5a is strictly dependent on a short preincubation with the cytokine interleukin-3 (IL-3), allowed us to investigate the metabolic requirements for LTC4 synthesis, and also to provide some information on early signal transduction mechanisms of IL-3 in these differentiated, non-dividing blood leukocytes. IL-3 itself does not alter intracellular free calcium concentration ([Ca2+]i) in basophils, whereas C5a induces a transient rise independent of IL-3 pretreatment, indicating that the priming effect of IL-3 cannot be explained by alterations in [Ca2+]i changes. The protein kinase C inhibitor staurosporine did not inhibit C5a-induced histamine release nor IL-3-dependent LTC4 formation in contrast to the IgE receptor-dependent basophil response. Activation of protein kinase C (PKC) by phorbol-12-myristate-13-acetate (PMA) induced histamine release without leukotriene formation. PMA-treated basophils did not produce LTC4 in response to C5a. Rather, PMA blocked the IL-3 effect on C5a-induced LTC4 synthesis. Only the C5a signal but not the IL-3 effect was pertussis toxin sensitive. Two unrelated tyrosine kinase inhibitors, tyrphostin RG-50864 and herbimycin A, were both very efficient blockers of IL-3-dependent lipid mediator formation whereas C5a-induced histamine release was preserved. Thus LTC4 formation does not require activation of a staurosporine-sensitive serine/threonine kinase. To the contrary, IL-3-dependent LTC4 formation appears to be regulated by serine/threonine and tyrosine phosphorylation in an antagonistic manner.