Platelet-activating factor evokes Ca2+ transients after the blockade of ryanodine receptor by dantrolene in RAW 264.7 macrophages.

In the present study we studied platelet-activating factor (PAF)-, and ATP-induced increases in intracellular Ca2+ concentration ([Ca2+]i) using RAW 264.7 macrophages filled with fura-2/AM and imaged with fluorescence video microscopy. We found that the prevalence of detectable [Ca2+]i responses to PAF application was significantly higher in the presence of dantrolene. Dantrolene itself significantly decreased basal [Ca2+]i of macrophages compared to control cases after a 20-min incubation period. In the dantrolene-treated cells even the peak [Ca2+]i in response to PAF (as an average of all cells) was below the baseline of control suggesting that decreased [Ca2+]i plays a permissive role in the Ca2+ rise induced by PAF in macrophages. In contrast to the effect of PAF, neither the amplitude of response to ATP nor the frequency of responding cells changed significantly during dantrolene treatment in our experiments. These cells were able to respond to a standard immune stimulus as well: lipopolysaccharide (LPS) was able to increase [Ca2+]i. Our data indicate that the effectiveness of PAF to increase [Ca2+]i in RAW 264.7 macrophages depends on the resting [Ca2+]i. It has also been shown in this study that PAF and ATP differently regulate Ca2+ homeostasis in macrophages during inflammatory response and therefore they possibly differently modulate cytokine production by macrophages.

Hydrophilic, pro-drug analogues of T138067 are efficacious in controlling tumor growth in vivo and show a decreased ability to cross the blood brain barrier.

The novel anticancer compound T138067 is an irreversible inhibitor of tubulin polymerization. Amides 3-6 were synthesized using standard methodologies and determined to be significantly less lipophilic than T138067 based on logP calculations. Tubulin polymerization and [(3)H]-T138067 competition assays revealed that these amides are pro-drugs for parent aniline 2. Amides 3-5 showed no detectable signs of crossing the blood brain barrier, while amide 6 was found in extremely small amounts (12 ng/g of brain tissue). Aniline 2, which was formed in vivo from these amides, was found in significantly smaller amounts (approximately 20 to >5000 times) in the brain than when 2 was administered directly. The in vivo efficacy of amide 6 approached that of T138067 and was better tolerated when administered to athymic nude mice bearing MX-1 human mammary tumor xenografts.

Modulation of norepinephrine release by ATP-dependent K(+)-channel activators and inhibitors in guinea-pig and human isolated right atrium.

OBJECTIVE: The aim of this study was to show, whether ATP sensitive K+ channels (KATP channels), are involved in the modulation of norepinephrine (NE) release from the sympathetic nerves innervating the guinea-pig and human right atrium. METHODS: The resting and stimulation-evoked release of [3H]norepinephrine ([3H]NE) was measured from the isolated guinea-pig and human right atrium and the effect of activators and inhibitors of ATP sensitive K+ channels was studied. RESULTS: Cromakalim (30-300 microM), a KATP channel-agonist decreased concentration-dependently the stimulation-evoked release of NE from the guinea-pig atrium, an effect, antagonized by glibenclamide, a KATP channel-antagonist (30 microM). Diazoxide (30-300 microM), another activator of the KATP channels reduced the resting release of NE, and also attenuated the evoked release at a single concentration (100 microM), and this latter action was also counteracted by glibenclamide (30 microM). Pinacidil, increased dose-dependently the resting and stimulation-evoked release of NE in a glibenclamide-sensitive manner and reversed the inhibitory effect of cromakalim (100 microM), suggesting that it acts as an antagonist. Glibenclamide (30-300 microM), by itself enhanced the stimulation-evoked release of [3H]NE, and also increased the resting release of NE. On the other hand, 5-hydroxydecanoate, an ischemia-selective inhibitor of cardiac KATP channels did not change NE release. Adenosine, (30-300 microM), an A1-receptor agonist, clonidine (3 microM), an alpha 2-adrenoceptor agonist and oxotremorine, a muscarinic receptor agonist (30 microM) all reduced the evoked release of [3H]NE, but these effects were not modified by glibenclamide (300 microM), indicating that neuronal adenosine (A1), adrenergic (alpha 2) and muscarinic (M3) receptors do not act on KATP channels. In the human right atrium, cromakalim, and diazoxide did not affect significantly the release of [3H]NE. However, glibenclamide (30-300 microM) and pinacidil (30-300 microM) enhanced dose-dependently the evoked-release of NE, and pinacidil also augmented the resting release. CONCLUSIONS: Our results indicate that sympathetic nerve endings of the human and guinea-pig atrium are endowed with ATP-sensitive K+ channels. These channels responded to agonists and antagonists under the experimental conditions applied and they could modulate the release of NE thereby affecting the autonomic control of cardiac function under various physiological and pathophysiological conditions.

Selective, covalent modification of beta-tubulin residue Cys-239 by T138067, an antitumor agent with in vivo efficacy against multidrug-resistant tumors.

Microtubules are linear polymers of alpha- and beta-tubulin heterodimers and are the major constituents of mitotic spindles, which are essential for the separation of chromosomes during mitosis. Here we describe a synthetic compound, 2-fluoro-1-methoxy-4-pentafluorophenylsulfonamidobenzene (T138067), which covalently and selectively modifies the beta1, beta2, and beta4 isotypes of beta-tubulin at a conserved cysteine residue, thereby disrupting microtubule polymerization. Cells exposed to T138067 become altered in shape, indicating a collapse of the cytoskeleton, and show an increase in chromosomal ploidy. Subsequently, these cells undergo apoptosis. Furthermore, T138067 exhibits cytotoxicity against tumor cell lines that exhibit substantial resistance to vinblastine, paclitaxel, doxorubicin, and actinomycin D. T138067 is also equally efficacious in inhibiting the growth of sensitive and multidrug-resistant human tumor xenografts in athymic nude mice. These observations suggest that T138067 may be clinically useful for the treatment of multidrug-resistant tumors.

Differential mechanisms involved in the effect of nicotinic agonists DMPP and lobeline to release [3H]5-HT from rat hippocampal slices.

In the present study we investigated the effect of different nicotinic agonists (dimethylphenyl-piperazinium-iodide (DMPP), (-)nicotine, cytisine, (-)-lobeline, and (-)epibatidine) and antagonists (mecamylamine and dihydro-beta-erythroidine) on the release of [3H]5-HT from hippocampal slices. The nicotinic agonists DMPP and lobeline and electrical field stimulation, released [3H]5-HT from the hippocampus; other nicotinic agonists, such as (-)-nicotine, cytisine, and (-)-epibatidine had no effect. Unlike lobeline-induced release of [3H]5-HT, the effect of DMPP (10 and 40 microM) was antagonized by mecamylamine (20 and 10 microM). The effect of DMPP was [Ca2+]o-independent. In experiments carried out at 7 degrees C, i.e. the membrane carrier proteins are inhibited and the release by lobeline was abolished while the DMPP-induced release of 5-HT was rather potentiated. It is proposed that the effect of DMPP and lobeline, to enhance the release of [3H]5-HT from the hippocampus, was mediated by two different mechanisms. While DMPP-induced 5-HT release can be linked to a non-classical nAChR activation ([Ca2+]o-independence), the effect of lobeline was likely mediated by uptake carriers.

Protein-DNA interactions during phenotypic differentiation.

We have been studying the molecular mechanism of neuronal differentiation through which the multipotent precursor becomes limited to the final transmitter phenotype. Here we focused on the role of the 5' proximal regulatory cassette (-190; +53 bp) of the rat enkephalin (rENK) gene in the developmental regulation of the enkephalin phenotype. Several well characterized cis-elements, including AP2, CREB, NF1, and NFkB, reside on this region of the rENK gene. These motifs were sufficient to confer activity-dependent expression of the gene during neurodifferentiation when it was tested using transient transfection assays of primary developing spinal cord neurons treated with tetrodotoxin (TTX). This region was then used as a DNA probe in mobility shift assays, with nuclear proteins derived from phenotypically and ontogenetically distinct brain regions. Only a few low abundance protein-DNA complexes were detected and only with nuclear proteins derived from developing but not from adult brain. The spatiotemporal pattern of these complexes did not show correlation with enkephalin expression which was assessed by RT-PCR. We employed synthetic probes corresponding to consensus as well as ENK-specific sequences of the individual motifs to identify the nature of the observed bands. Although both consensus NF1 and enkCRE1(NF1) formed complexes with nuclear proteins derived from the striatum and cortex at various ages, the appearance of the bands was not correlated with ENK expression. Surprisingly, no complexes were detected if other ENK-specific motifs were used as probes. We also tested nuclear extracts derived from forskolin-induced and control C6 glioma cells, again using the whole proximal regulatory cassette as well as individual motifs. These experiments showed the formation of elaborate protein-DNA bands. There was no direct correlation between the appearance of bands and forskolin-induced ENK expression. Unexpectedly, all ENK-specific motifs formed specific and highly abundant protein-DNA complexes when nuclear extracts from the human tumor cell line (HeLa), which does not express ENK, were used. Based on these observations, we concluded that: 1. Interactions between the proximal regulatory cassette and additional probably far distant regions of the rENK gene and their binding proteins may be necessary to confer developmentally regulated, cell-specific expression of the ENK gene; and 2. Inducibility of the gene by common cis-elements can be governed by this region; however, the cell-specificity of the induction remains elusive.

[Measurement of the tensile force exerted on the flexor tendons of the hand]. / Die Messung der auf die Beugesehnen der Hand wirkenden Kraft.

Immediate postoperative active motion after flexor tendon repair is a recurring theme in hand surgery. In the present study the authors determined the tensile stress of the sutured tendon subjected to active motion by the use of cadaveric hands and a tensiometer. The force necessary to draw forth the proximal stump was also measured. A tendon suture which can resist two kilo-ponds tensile force seems to allow active motion of the reconstructed flexor tendon.

[Direct measurement of the pulling force affecting the flexor tendons of the hand]. / A kéz ujjhajlító inaira ható húzóerö direkt mérése.

A thought, returning again and again in hand surgery, is the immediate introduction of active motion therapy after the reconstructive operations of the flexor tendons. To the active motion an adequately strong tendon suture, to the definition of "adequately strong tendon suture" however the measurement of the pulling force, acting actually on the flexor tendons is necessary. Authors attempted to measure this pulling force during flexor tendon operations on cadaver's hand. On the basis of their measurements the maximal pulling force, that can be expected during postoperative active movement exercises with adequate circumspection, can be estimated as two kiloponds.

[Tendolysis after flexor tendon surgery]. / Tendolyse nach Beugesehnenoperationen.

This review concerns itself with experimental and clinical activities which will prevent adhesions. Reference is made to work which indicates the usefulness of tendolysis including among others, the work of BOYES, BUNNELL, DUPARC, FETROW, GREULICH, ISELIN, MICHON, NEHRING, NIGST, PULVERTAFT, VERDAN, WILHELM, WRAY. The rationale, timing, indications, operative technique, use of hydrocortisone, follow-up care, and the results are reported. The experience of the Hand Surgery Division of the National Institute for Traumatology in Budapest is analyzed. Between 1966 and 1975, 1886 patients with tendon injuries were treated. In 1469 flexor tendon injuries, 2060 operations on tendons were performed. The late results of 136 tendolyses were analyzed at different times primarily according to the zone of injury and to the type of primary operation. The results were graded according to the evaluation plan of BUCK-GRAMCKO. It is established that tendolysis is a difficult and big undertaking which should only be considered by surgeons with adequate experience in hand surgery. With these additional operations, the results of tendon repairs can be improved substantially.

Ultrastructural investigation of the human hand after tendon and motor nerve injury.

Specimens of the affected muscles obtained during reconstructive surgery 4 to 16 weeks after injury of the tendon or of the motor nerve of the human hand were studied. After tenotomy the most serious lesions were displayed by the contractile elements which were homogeneous brittle and atrophied, and disintegrated independently of the functional units. The number of mitochondria decreased, the sarcoplasmic reticulum accumulated and the red and white muscle fibres could not be distinguished. After motor nerve injury the motor end plates were completely destroyed and Wallerian degeneration appeared. The contractile elements atrophied and were brittle. The number of mitochondria and the sarcoplasmic reticulum decreased, the sarcoplasmic glycogen content was higher than normal, and often the cell nuclei occupied a central position.