microM Alpha Research Articles

Antifibrotic effects of tocotrienols on human Tenon’s fibroblasts

To compare the antifibrotic effect of vitamin E isoforms alpha-, gamma-, and delta-tocotrienol on human Tenon's fibroblasts (hTf) to the antimetabolite mitomycin C. Antifibrotic effects of alpha- (40, 60, 80, 100, and 120 microM), gamma- (10, 20, 30, and 40 microM) and delta-tocotrienol (10, 20, 30, and 40 microM) on hTf cultures were evaluated by performing proliferation, migration and collagen synthesis assays. Whereas for vitamin E the exposure time was set to 7 days to mimic subconjunctival application, cultures were exposed only 5 min to mitomycin C 100 microg/ml to mimic intraoperative administration. Cell morphology (phase contrast microscopy) as an assessment for cytotoxicity and cell density by measuring DNA content in a fluorometric assay to determine proliferation inhibition was performed on day 0, 4, and 7. Migration ability and collagen synthesis of fibroblasts were measured. All tested tocotrienol isoforms were able to significantly inhibit hTf proliferation in a dose-dependent manner (maximal inhibitory effect without relevant morphological changes at day 4 for alpha-tocotrienol 80 microM with 36.7% and at day 7 for alpha-tocotrienol 80 microM with 42.6% compared to control). Degenerative cell changes were observed in cultures with concentrations above 80 microM for alpha- and above 30 microM for gamma- and delta-tocotrienol. The highest collagen synthesis inhibition has been found with 80 microM alpha-tocotrienol (62.4%) and no significant inhibition for mitomycin C (2.5%). Migration ability was significantly reduced in cultures exposed to 80 microM alpha- and 30 microM gamma-tocotrienol (inhibition of 82.2% and 79.5%, respectively, compared to control) and also after mitomycin C treatment (60.0%). Complete growth inhibition without significant degenerative cell changes could only be achieved with mitomycin C. In vitro, all tested tocotrienol isoforms were able to inhibit proliferation, migration and collagen synthesis of human Tenon's fibroblasts and therefore may have the potential as an anti-scarring agent in filtrating glaucoma surgery.

TNP-ATP-resistant P2X ionic current on the central terminals and somata of rat primary sensory neurons.

P2X receptors have been suggested to be expressed on the central terminals of A delta-afferent fibers innervating dorsal horn lamina V and play a role in modulating sensory synaptic transmission. These P2X receptors have been widely thought to be P2X2+3 receptors. However, we have recently found that P2X receptor-mediated modulation of sensory transmission in lamina V is not inhibited by trinitrophenyl-adenosine triphosphate (TNP-ATP), a potent antagonist of P2X1, P2X3 homomers, and P2X2+3 heteromers. To provide direct evidence for the presence of TNP-ATP-resistant P2X receptors on primary afferent fibers, we examined alpha,beta-methylene-ATP (alpha beta meATP)-evoked currents and their sensitivity to TNP-ATP in rat dorsal root ganglion (DRG) neurons. alpha beta meATP evoked fast currents, slow currents, and mixed currents that contained both fast and slow current-components. Fast currents and fast current components in the mixed currents were both completely inhibited by 0.1 microM TNP-ATP (n = 14). Both slow currents and slow-current components in the mixed currents showed broad spectrum of sensitivity to 1 microM TNP-ATP, ranging from complete block (TNP-ATP-sensitive) to little block (TNP-ATP-resistant). TNP-ATP-resistant currents evoked by 10 microM alpha beta meATP could be largely inhibited by 10 microM iso-pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid. Cells with P2X currents that were highly resistant to TNP-ATP were found to be insensitive to capsaicin. These results suggest that TNP-ATP-resistant P2X receptor subtypes are expressed on capsaicin-insensitive A delta-afferent fibers and play a role in modulating sensory transmission to lamina V neurons.

Different Sensitivities to .ALPHA.1 Adrenoceptor Blockade on Periarterial Sympathetic Nerve-Induced Constriction by Low and High Frequencies in Canine Isolated Splenic Arteries.

The periarterial nerve electrical stimulation at 4 and 10 Hz induced a monophasic vasoconstriction of the canine splenic artery in a pulse number-related manner (1-30 pulses). The responses at 4 Hz were not significantly affected by 0.1 microM prazosin, but abolished by 1 microM alpha, beta-methylene ATP. Prazosin (0.1 microM) partially but significantly inhibited responses at 10 Hz, and the remaining responses were blocked by 1 microM alpha, beta-methylene ATP. It indicates that the monophasic vasoconstrictor response to short pulses of stimulation at a low frequency is mediated by P2X-receptors, whereas the response at a high frequency may be due to activation of not only P2X-receptors but also alpha1 adrenoceptors.

Vitamin E inhibition of normal mammary epithelial cell growth is associated with a reduction in protein kinase C(alpha) activation.

Tocopherols and tocotrienols represent the two subclasses within the vitamin E family of compounds. However, tocotrienols are significantly more potent than tocopherols in suppressing epidermal growth factor (EGF)-dependent normal mammary epithelial cell growth. EGF is a potent mitogen for normal mammary epithelial cells and an initial event in EGF-receptor mitogenic-signalling is protein kinase C (PKC) activation. Studies were conducted to determine if the antiproliferative effects of specific tocopherol and tocotrienol isoforms are associated with a reduction in EGF-receptor mitogenic signalling and/or PKC activation. Normal mammary epithelial cells isolated from midpregnant BALB/c mice were grown in primary culture, and maintained on serum-free media containing 10 ng/mL EGF as a mitogen, and treated with various doses (0-250 microm) of alpha-, gamma-, or delta-tocopherol or alpha-, gamma-, or delta-tocotrienol. Treatment with growth inhibitory doses of delta-tocopherol (100 microm), alpha-tocotrienol (50 microm), or gamma- or delta-tocotrienol (10 microm) did not affect EGF-receptor levels, EGF-induced EGF-receptor tyrosine kinase activity, or total intracellular levels of PKC(alpha). However, these treatments were found to inhibit EGF-induced PKC(alpha) activation as determined by its translocation from the cytosolic to membrane fraction. Treatment with 250 microm alpha- or gamma-tocopherol had no affect on EGF-receptor mitogenic signalling or cell growth. These findings demonstrate that the inhibitory effects of specific tocopherol and tocotrienol isoforms on EGF-dependent normal mammary epithelial cell mitogenesis occurs downstream from the EGF-receptor and appears to be mediated, at least in part, by a reduction in PKC(alpha) activation.

Computational Approach for Determining the Spectrum of DNA Damage Induced by Ionizing Radiation

To study the characteristics of molecular damage induced by ionizing radiation at the DNA level, Monte Carlo track simulation of energetic electrons and ions in liquid water, a canonical model of B-DNA, and a comprehensive classification of DNA damage in terms of the origin and complexity of damage were used to calculate the frequencies of simple and complex strand breaks. A threshold energy of 17.5 eV was used to model the damage by direct energy deposition, and a probability of 0.13 was applied to model the induction of a single-strand break produced in DNA by OH radical reactions. For preliminary estimates, base damage was assumed to be induced by the same direct energy threshold deposition or by the reaction of an OH radical with the base, with a probability of 0.8. Computational data are given on the complexity of damage, including base damage by electrons with energies of 100-4500 eV and ions with energies of 0.3-4.0 MeV/nucleon (59-9 keV microm(-1) protons and 170-55 keV microm(-1) alpha particles). Computational data are presented on the frequencies of single- and double-strand breaks induced as a function of the LET of the particles, and on the relative frequencies of complex single- and double-strand breaks for electrons. The modeling and calculations of strand breaks show that: (1) The yield of strand breaks per unit absorbed dose is nearly constant over a wide range of LET. (2) The majority of DNA damage is of a simple type, but the majority of the simple single-strand breaks are accompanied by at least one base damage. (3) For low-energy electrons, nearly 20-30% of the double-strand breaks are of a complex type by virtue of additional breaks. The proportion of this locally clustered damage increases with LET, reaching about 70% for the highest-LET alpha particles modeled, with the complexity of damage increasing further, to about 90%, when base damage is considered. (4) The extent of damage in the local hit region of the DNA duplex is mostly limited to a length of a few base pairs. (5) The frequency of base damage when no strand breaks are present in the hit segment of DNA varies between 20-40% as a function of LET for protons and alpha particles.

Antiproliferative and apoptotic effects of tocopherols and tocotrienols on normal mouse mammary epithelial cells.

Studies were conducted to determine the comparative effects of tocopherols and tocotrienols on normal mammary epithelial cell growth and viability. Cells isolated from midpregnant BALB/c mice were grown within collagen gels and maintained on serum-free media. Treatment with 0-120 microM alpha- and gamma-tocopherol had no effect, whereas 12.5-100m microM tocotrienol-rich fraction of palm oil (TRF), 100-120 microM delta-tocopherol, 50-60 microM alpha-tocotrienol, and 8-14 microM gamma- or delta-tocotrienol significantly inhibited cell growth in a dose-responsive manner. In acute studies, 24-h exposure to 0-250 microM alpha-, gamma-, and delta-tocopherol had no effect, whereas similar treatment with 100-250 microM TRF, 140-250 microM alpha-, 25-100 microM gamma- or delta-tocotrienol significantly reduced cell viability. Growth-inhibitory doses of TRF, delta-tocopherol, and alpha-, gamma-, and delta-tocotrienol were shown to induce apoptosis in these cells, as indicated by DNA fragmentation. Results also showed that mammary epithelial cells more easily or preferentially took up tocotrienols as compared to tocopherols, suggesting that at least part of the reason tocotrienols display greater biopotency than tocopherols is because of greater cellular accumulation. In summary, these findings suggest that the highly biopotent gamma- and delta-tocotrienol isoforms may play a physiological role in modulating normal mammary gland growth, function, and remodeling.

Role of N-, P- and Q-type voltage-gated calcium channels in transmitter release from sympathetic neurones in the mouse isolated vas deferens.

1. N-type voltage-gated calcium channels are known to play an important role in transmitter release from autonomic neurones, and recent studies have demonstrated that non-N-type calcium channels are also involved. The calcium channels coupled to transmitter release from sympathetic neurones in the mouse isolated vas deferens were investigated in the present study. 2. Contractions of the mouse vas deferens were evoked by electrical stimulation at 1-50 Hz. The contractions were entirely nerve-mediated, since they were abolished by tetrodotoxin, and were used as an indirect measure of transmitter release. 3. The N-type calcium channel blocker, omega-conotoxin GVIA, inhibited contractions in a concentration-dependent manner, with a maximal effect at 30 nM. Contractions evoked by stimulation frequencies less than 10 Hz were abolished, and those evoked by 20 and by 50 Hz stimulation were decreased in amplitude by 51.3 +/- 13.9% and 9.3 +/- 2.6%, respectively. 4. The N-, P- and Q-type channel blocker, omega-conotoxin MVIIC, inhibited contractions in a concentration-dependent manner and caused greater maximum inhibition than omega-conotoxin GVIA, suggesting an action on P- and/or Q-type channels, in addition to N-type. 5. The P-type channel blocker, omega-agatoxin IVA, alone did not have a significant effect at concentrations up to 300 nM, but inhibited contractions in the presence of omega-conotoxin GVIA. Subsequent addition of omega-conotoxin MVIIC abolished the remaining contractions. Identical results were obtained when the three toxins were tested cumulatively on the purinergic and noradrenergic components of the contraction in the presence of (1.3 microM prazosin and following desensitization to 10 microM alpha, beta-methylene adenosine 5'-triphosphate (alpha, beta-NeATP), respectively. 6. The results suggest that N-, P- and Q-type channels are involved in the release of noradrenaline and ATP from sympathetic neurones in the mouse vas deferens.

Potentiation of purinergic transmission by angiotensin in prostatic rat vas deferens.

1. Angiotensin II (AII) elicited only a minute, if any, direct contractile response in smooth muscle cells of prostatic rat vas deferens, but it potentiated contractile responses to field stimulation. 2. Angiotensin-potentiated contractile response to field stimulation was concentration-dependent, and the order of potency was AII > AIII approximately AI. The EC50 of AII was 8.11 +/- 2.79 nM. 3. AII did not modify the contractile response of exogenous noradrenaline (NA) on non-stimulated prostatic vas deferens. Furthermore, the concentration-response curve for AII-potentiated contractile responses to field stimulation in reserpine-treated rats did not significantly differ from the control group. 4. Desensitization of purinoceptors with 30 microM alpha, beta-methylene-ATP almost completely abolished the potentiation of the contractile response to field stimulation by AII. 5. The response to AII in the prostatic rat vas deferens was blocked by the AT1 selective antagonist losartan, but not by the AT2 selective antagonist CGP 42112. Losartan acted as a competitive antagonist with a pA2 value of 8.75. 6. In conclusion, AII potentiated purinergic transmission in the prostatic rat vas deferens via the AT1 receptor.

An ATP-activated nonselective cation channel in guinea pig ventricular myocytes.

Extracellular ATP released from nerves onto vascular smooth muscle or released from damaged tissues during traumatic injury, shock, or ischemia profoundly alters cardiovascular physiology. We have used patch-clamp methods to investigate the effects of extracellular ATP on guinea pig ventricular myocytes because guinea pigs are a commonly used model for the study of cardiac electrophysiology. We have found that ATP activates a rapid, desensitizing, inward current. This inward current is activated by a P2 receptor that does not conform to published receptor subclasses. A concentration of 100 microM ATP activates more current than 100 microM alpha, beta-methyleneadenosine 5'-triphosphate, which in turn activates more current than 100 microM ADP. 2-Methylthioadenosine 5'-triphosphate (2-MeS-ATP) and adenosine 5'-O-(3-thiotriphosphate) are also effective agonists. Adenosine, AMP, guanosine 5'-triphosphate, and uridine 5'-triphosphate are ineffective at 100 microM. The inward conductance has a reversal potential near 0 mV and in ion-substitution experiments was found to be carried through nonselective cation channels rather than chloride channels. The conductance has inwardly rectifying current-voltage (I-V) relations. When ATP is used as the agonist, fluctuation analysis yields an apparent unitary conductance of 0.08 pA at a holding potential of -120 mV with sodium as the main charge-carrying ion. The combination of inwardly rectifying I-V relations, the efficacy of 2-MeS-ATP, and the very low conductance distinguish this conductance from other ATP-activated nonselective channels, including those recently cloned from rat vas deferens and PC-12 cells.

Contribution of P2‐purinoceptors to neurogenic contraction of rat urinary bladder smooth muscle

1. The contribution of P2-purinoceptors to neurogenic contraction was investigated in rat urinary bladder smooth muscle by measurement of isotonic tension. 2. Contraction of rat urinary bladder smooth muscle induced by electrical stimulation was decreased to 84.19 +/- 3.90% of the control (n = 16) in the presence of atropine (1 microM), which was further decreased to 38.80 +/- 2.75% of the control (n = 49) in the presence of both atropine and 10 microM alpha, beta-methylene adenosine 5'-triphosphate (alpha, beta-Me ATP). 3. The contractile response induced by electrical stimulation in the presence of atropine and alpha, beta-Me ATP was decreased to 27.81 +/- 4.07% (n = 23) and 26.63 +/- 5.01% (n = 15) of the control, by the addition of 100 microM cibacron blue 3GA and 100 microM suramin, respectively. The application of 100 microM adenosine 5'-o-2-thiodiphosphate (ADP beta S) in the presence of atropine and alpha, beta-Me ATP decreased the contractile response induced by electrical stimulations to 17.15 +/- 3.71% (n = 15) of the control. 4. Pretreatment of muscle strips with 100 microM ADP beta S significantly reduced the response to either 200 microM alpha, beta-methylene adenosine 5'-diphosphate or 200 microM ADP beta S. 5. Uridine 5'-triphosphate (100 microM to 1 mM) concentration-dependently contracted muscle strips, and this contraction was significantly antagonized by desensitization of P2-receptors with alpha, beta-Me ATP (10 microM), and completely antagonized by pretreatment of muscle strips with both alpha, beta-Me ATP and ADP beta S (100 microM). 6. Di(adenosine-5') tetraphosphate (30 and 100 microM) contracted muscle strips, whereas it failed to contract after desensitization of P2-receptors.7. It is suggested that about 20% of the neurogenic contraction of rat urinary bladder smooth muscle is mediated via ADP beta S-sensitive purinoceptors.

Discrimination between UTP- and P2-purinoceptor-mediated depolarization of rat superior cervical ganglia by 4,4'-diisothiocyanatostilbene-2,2'- disulphonate (DIDS) and uniblue A.

1. Using a grease-gap recording technique we have investigated the effects of some antagonists of P2-purinoceptors on the depolarization of the rat isolated superior cervical ganglion evoked by 100 microM alpha, beta-methylene-adenosine 5'-triphosphate (alpha,beta-MeATP) or uridine 5'-triphosphate (UTP). The effects of the putative P2Z-purinoceptor antagonist, coomassie brilliant blue G, putative P2X-purinoceptor antagonist, 4,4'-diisothiocyanatostilbene-2,2'-disulphonate (DIDS) and uniblue A (an analogue of the P2Y- and P2X-purinoceptor antagonist reactive blue 2) were investigated. 2. At the highest concentration examined uniblue A (300 microM) depressed alpha,beta-MeATP-induced depolarization and at 100 and 300 microM enhanced UTP-evoked depolarizations. Coomassie brilliant blue G (1 and 10 microM) did not affect depolarizations evoked by alpha,beta-MeATP or UTP. Depolarizations evoked by potassium (5 mM) or muscarine (100 nM) were unaltered by either coomassie brilliant blue G or uniblue A. Uniblue A (100 and 300 microM) produced a concentration-dependent depression of hyperpolarizations evoked by adenosine (100 microM) whereas coomassie brilliant blue G at up to 10 microM, did not alter adenosine-induced hyperpolarizations. 3. DIDS (30 and 100 microM) did not alter adenosine-evoked hyperpolarizations, or depolarizations evoked by potassium or UTP. DIDS at 100 microM did not alter depolarizations evoked by muscarine. In contrast DIDS produced a concentration-dependent depression of alpha,beta-MeATP-evoked depolarizations. 4. These results are consistent with the proposal that uniblue A and DIDS but not coomassie brilliant blue G are antagonists of P2-purinoceptors and that uniblue A is also an antagonist at P1-purinoceptors present on the rat superior cervical ganglion. 5. The ability of uniblue A and DIDS to distinguish between depolarizations evoked by UTP and alpha,beta-MeATP provides further justification for the proposal that these nucleotides activate separate receptors present on the rat superior cervical ganglion, i.e. pyrimidinoceptors and P2-purinoceptors respectively.

Transforming growth factor beta isoform 2-specific high affinity binding to native alpha 2-macroglobulin. Chimeras identify a sequence that determines affinity for native but not activated alpha 2-macroglobulin.

Transforming growth factor beta 2 (TGF-beta 2) is less potent than TGF-beta 1 in some endothelial cell proliferation assays due to the greater tendency of TGF-beta 2 to bind alpha 2-macroglobulin (alpha 2M). Substitution of TGF-beta 1 residues 40-47 into the TGF-beta 2 sequence yields a chimeric molecule that, like TGF-beta 1, expresses activity that is not substantially affected by serum alpha 2M (Burmester, J. K., Qian, S. W., Roberts, A. B., Huang, A., Amatayakul-Chantler, S., Suardet, L., Odartchenko, N., Madri, J. A., and Sporn, M. B. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 8628-8632). In this investigation, we studied the binding of TGF-beta chimeras, which contain TGF-beta 1 residues 40-47, to both major conformations of human alpha 2M under apparent equilibrium conditions. Native alpha 2M, the primary form of this protein in serum, bound TGF-beta 2/beta 1 (40-82) and TGF-beta 2/beta 1 (40-47) with low affinity. The apparent KD values for the two chimeras and native alpha 2M were 310 and 330 nM, respectively. These values were much higher than the KD determined for TGF-beta 2 and native alpha 2M (11 nM) and equivalent to the KD determined for TGF-beta 1 and native alpha 2M. By contrast, both TGF-beta chimeras bound alpha 2M-methylamine, an altered conformation of alpha 2M, with high affinity (16 and 19 nM), which is characteristic of TGF-beta 2 and not TGF-beta 1. Fetal bovine heart endothelial cell DNA synthesis was inhibited to a similar degree by TGF-beta 1, TGF-beta 2, TGF-beta 2/beta 1 (40-82), and TGF-beta 2/beta 1 (40-47) in the presence of dilute (0.2%) fetal bovine serum. When 0.07 microM alpha 2M-methylamine was added, the activities of TGF-beta 2, TGF-beta 2/beta 1 (40-82), and TGF-beta 2/beta 1 (40-47) were significantly counteracted while the activity of TGF-beta 1 was unchanged, as would be predicted by the equilibrium binding analyses. These studies indicate that the TGF-beta structural elements, which mediate binding to native alpha 2M and conformationally transformed alpha 2M, are not equivalent. Residues 40-47 are critical in determining affinity for native alpha 2M but are less important in determining affinity for alpha 2M-methylamine.

SAAT1 is a low affinity Na+/glucose cotransporter and not an amino acid transporter. A reinterpretation.

Recently a member of the Na+/glucose (SGLT1) gene family of cotransporters was isolated from a pig renal cell line and was thought to be the neutral amino acid transporter System A. This cDNA (Kong, C. T., Yet, S. F., and Lever, J. E. (1993) J. Biol. Chem. 268, 1509-1512) encodes a 660-amino acid protein with 76% identity to SGLT1. To confirm and extend the kinetic characterization of SAAT1, we have expressed this clone in Xenopus oocytes and measured transport using both radiotracer and electrophysiological techniques. SAAT1 did not stimulate either 50 microM 2-(methylamino)isobutyrate uptake or 2-(methylamino)isobutyrate-evoked inward Na+ currents, but instead stimulated 50 microM alpha MG (alpha-methyl-D-glucopyranoside) uptake 27-fold from 2 +/- 1 pmol.h-1/oocyte (n = 9) to 55 +/- 6 pmol.h-1/oocyte (n = 9) and alpha MG-evoked inward Na+ currents (I) by up to 1000 nA/oocyte. The apparent affinity constant for alpha MG (K alpha MG 0.5) was approximately 2 mM and was independent of membrane potential from -30 to -150 mV but was voltage-sensitive between -30 and +30 mV. The relative sugar specificity for the transporter was alpha MG > or = D-glucose >> D-galactose >>> 3-O-methyl-D-glucopyranose, L-glucose. The sugar-evoked currents were Na(+)-dependent (KNa 0.5 approximately 10 mM at -50 mV) and the Hill coefficient was 1. KNa 0.5 decreased with hyperpolarization of the membrane from -50 to -150 mV. Phlorizin inhibited the alpha MG-evoked current with apparent Ki of 18 microM at -50 mV. We conclude that the SAAT1 cDNA encodes a renal low affinity Na+(1)/glucose(1) cotransporter and propose that pig SAAT1 be renamed pSGLT2.

Interactions of alpha Ni3S2 with guinea pig alveolar macrophages and liberation of inflammatory mediators.

Our previous investigation presented evidence of interaction between alpha Ni3S2 and membranous and cellular lipids of lung cells, resulting in significant increases in linoleic, linolenic and arachidonic acids. The present work was designed to follow the metabolic fate of arachidonic acid in alpha Ni3S2-exposed guinea pig alveolar macrophages (GPAM) in culture (50 microM alpha Ni3S2 for 3 days). The metabolites of arachidonic acid were assessed by HPLC coupled with UV or electrochemical detection. The concentrations of malondialdehyde (MDA), hydroxyeicosatetraenoic acid (HETE), leukotrienes (LT) and reduced glutathione (GSH) were measured. In exposed cells a significant increase of MDA, a breakdown product of lipid peroxidation, was observed. In addition, the enzymatic reduction of 5-hydroperoxyeicosatetraenoic acid (5-HPETE) by the associated oxidation of GSH to GSSG increased 5-HETE in GPAM cells and decreased GSH. 5-Hydroperoxyeicosatetraenoic acid was furthermore converted to epoxides, such as leukotriene A4, and we also quantified in exposed cells a significant increase of its subsequent catabolites LTB4, LTC4 and LTE4. Direct measurements of MDA and other metabolites of arachidonic acid clearly show that exposure of GPAM cells to alpha Ni3S2 enhances lipid peroxidation. This lipid peroxidation is an autocatalytic free-radical process and could be responsible for DNA damage. Furthermore, alpha Ni3S2 intoxication induces the release of proinflammatory products, such as leukotrienes, and the decrease of glutathione.

Effect of alpha Ni3S2 on arachidonic acid metabolites in cultured human lung cells (L132 cell line).

Our previous investigations have shown evidence of an interaction between alpha Ni3S2 and membranous and cellular lipids of lung cells with a significant increase in the linoleic, linolenic and arachidonic acid pool. The present work is designed to follow the metabolic fate of arachidonic acid in alpha Ni3S2-exposed human embryonic pulmonary epithelial cells (L132) in culture (50 microM alpha Ni3S2 for 3 days). The metabolites of arachidonic acid were assessed by HPLC determination coupled with UV or electrochemical detection. We determined malondialdehyde (MDA), hydroxyeicosatetraenoic acid (HETE), leukotrienes (LT) and reduced glutathione (GSH). In exposed cells we observed a significant increase of MDA, which is a breakdown product of lipid peroxidation. In addition, we noted significant increases of 5-HETE and 15-HETE in L132 cells resulting from the enzymatic reduction of 5-HPETE and 15-HPETE respectively. There was also a simultaneous decrease of GSH--confirmed by a strong decrease of GSH in exposed cells with respect to controls. 5-HPETE is furthermore converted to epoxides such as leukotriene A4 and we also quantified in exposed cells a significant increase of its subsequent catabolites LTB4, LTC4 and LTE4. These investigations show clearly that exposure of L132 cells to alpha Ni3S2 enhances lipid peroxidation based upon direct measurements of MDA and other metabolites of arachidonic acid. This lipid peroxidation is an autocatalytic free-radical process and could be responsible for DNA damage.

Adenosine discriminates between the caffeine and adenine nucleotide sites on the sheep cardiac sarcoplasmic reticulum calcium-release channel.

Calcium-release channels of sheep cardiac sarcoplasmic reticulum were incorporated into phosphatidylethanolamine bilayers and single channel currents were recorded under voltage-clamp conditions. The effect of adenosine on single channel conductance and gating was investigated, as were the interactions between adenosine and caffeine and adenosine and alpha, beta-methylene ATP. Addition of adenosine (0.5-5 mM) to the cytosolic but not the luminal side of the membrane increased the open probability of single calcium-activated calcium-release channels by increasing the frequency and duration of open events, yielding an EC50 of 0.75 mM at 10 microM activating Ca2+. Addition of 1 mM caffeine potentiated the effects of adenosine at 10 or 100 microM-activating cytosolic calcium, but had no effect on the inability of adenosine to activate the channel at 80 pM calcium, suggesting discrete sites of action on the calcium-release channel for adenosine and caffeine. In contrast, addition of 100 microM alpha, beta-methylene-ATP decreased single channel open probability in the presence of adenosine, suggesting that these compounds act on the same site on the channel. Activation of single channel opening by adenosine, or by adenosine together with caffeine, had no effect on single channel conductance or the Ca2+/Tris+ permeability ratio. Channels activated by adenosine were characteristically modified by ryanodine and blocked by microM ruthenium red or mM magnesium. These results show that adenosine activates the sheep cardiac sarcoplasmic reticulum Ca(2+)-release channel by increasing the frequency and duration of open events in a Ca(2+)-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Intracellular formation and release of adenosine from rat hippocampal slices evoked by electrical stimulation or energy depletion.

In this study, the basal and evoked release of [3H]- and endogenous adenosine, inosine and hypoxanthine from rat hippocampal slices, labelled with [3H]adenine, was investigated. Evoked release was brought about by either electrical stimulation or energy depletion. The aim was to determine whether adenosine is formed intracellularly, and released as adenosine or extracellularly, from sequential extracellular hydrolysis of released ATP. All measurements were made in the presence of 5 microM erythro-9-(2-hydroxy-3-nonyl) adenosine (EHNA) to inhibit the enzyme adenosine deaminase. It was found that electrical field stimulation (5 min) increased the release of endogenous adenosine from hippocampal slices 10-fold and increased the proportion of [3H]-label associated with adenosine from approx 7% of the total released to 13% after the first stimulation and 20% after the second stimulation. Removal of oxygen and glucose from the superfusion medium (energy depletion) increased the release rate of endogenous adenosine 16-fold and increased the proportion of [3H]-label associated with [3H]adenosine from approx 10% of the total released to 50%. In order to prevent extracellular formation of adenosine, experiments were carried out in the presence of 50 microM alpha, beta-methylene ADP (AOPCP), an inhibitor of ecto-5'-nucleotidase. AOPCP was found to be without effect on either the basal or evoked release of adenosine. In contrast, L-homocysteine thiolactone (0.1-1.0 mM) which was used to "trap" intracellular adenosine reduced both the basal and evoked release of adenosine by 70-85%. This effect of L-homocysteine thiolactone also occurred in the presence of adenosine uptake inhibitors. It is concluded from these results that adenosine is formed predominantly intracellularly in hippocampal slices and is released as adenosine as a result of either tissue depolarisation or energy depletion. Furthermore, the finding that during energy depletion there is a proportionally greater release of adenosine than other ATP breakdown products, such as inosine and hypoxanthine, indicates that energy depletion is both a potent and selective stimulus for adenosine formation and release.

Binding of platelet-derived growth factor-BB and transforming growth factor-β 1 to α2-macroglobulin in vitro and in vivo: comparison of receptor-recognized and non-recognized α2-macroglobulin conformations

alpha 2-Macroglobulin (alpha 2M) undergoes a major conformational change when reacting with proteinases or primary amines. This conformational change has been referred to as the 'slow' to 'fast' transformation based on the increase in alpha 2M mobility shown by non-denaturing PAGE. Previous studies demonstrated that many cytokines, including transforming growth factor beta 1 (TGF-beta 1) and interleukin-1 beta, bind preferentially or exclusively to alpha 2M which has undergone conformational change. In this study, we demonstrate that platelet-derived growth factor-BB (PDGF-BB) also binds preferentially to conformationally transformed alpha 2M (alpha 2M-methylamine, alpha 2M-trypsin) in vitro. Purified 125I-PDGF-BB-alpha 2M-methylamine complex cleared rapidly from the circulation of mice via the alpha 2M receptor/low-density-lipoprotein-receptor-related protein (alpha 2M-R/LRP). In order to determine whether PDGF-BB or TGF-beta 1 binds to native alpha 2M, we defined the native conformation by lack of interaction with alpha 2M-R/LRP instead of electrophoretic mobility. 125I-PDGF-BB was incubated with 4.3 microM native alpha 2M and 0.47 microM alpha 2M-methylamine. The 125I-PDGF-BB distributed evenly between slow-form and fast-form alpha 2M without shifting the electrophoretic mobility of either species. When the mixed preparation was injected intravenously in mice, 125I-PDGF-BB-fast-form-alpha 2M cleared rapidly and selectively from the circulation; 125I-PDGF-BB which was bound to slow-form alpha 2M was stable in the blood (apparently not recognized by alpha 2M-R/LRP). Therefore, while conformationally transformed alpha 2M binds PDGF-BB preferentially in vitro, non-alpha 2M-R/LRP-recognized alpha 2M binds PDGF-BB as well. Binding of 125I-PDGF-BB and 125I-TGF-beta 1 to alpha 2M was demonstrated in vivo by injecting the free growth factors intravenously into mice. Plasma samples which were subjected to non-denaturing PAGE and autoradiography demonstrated binding of both growth factors exclusively to the slow-form of alpha 2M. Therefore, under normal physiological conditions, native alpha 2M (non-alpha 2M-R/LRP-recognized) is the primary form of the proteinase inhibitor functioning as a carrier of PDGF-BB and TGF-beta 1 in the blood.

αMSH but not Org 2766 induces expression of c-fos in cultured rat spinal cord cells

Alpha-melanocyte stimulating hormone (alpha MSH) and Org 2766 (an adrenocorticotrophic hormone (ACTH) MSH4-9 analogue) stimulate sprouting after nerve damage in vivo. In cultures of rat spinal cord cells only alpha MSH enhances neurite outgrowth. In order to analyse the mode of action of alpha MSH and to get a clue why its action differs from that of Org 2766, we studied whether expression of c-fos was induced in spinal cord cells by treatment with alpha MSH or Org 2766. We found a rapid and transient induction of both c-fos mRNA and protein after treatment with 100 microM alpha MSH. Treatment with Org 2766 (100 pM-100 microM) had no effect on c-fos expression. These results suggest that c-fos is involved in the signal transduction system of alpha MSH stimulated neuronal outgrowth in vitro.

Interaction between staphylokinase, plasmin(ogen), and alpha 2-antiplasmin. Recycling of staphylokinase after neutralization of the plasmin-staphylokinase complex by alpha 2-antiplasmin

Although the plasminogen activating equimolar complex of staphylokinase (STA) with human plasmin is very rapidly inhibited by alpha 2-antiplasmin, STA is a potent fibrinolytic agent in a human plasma milieu which contains 1 microM alpha 2-antiplasmin. In the present study, it was found that the complex of plasmin with recombinant STA (STAR), after neutralization with alpha 2-antiplasmin, retained the full plasminogen activating potential of STAR when added to a plasminogen solution (93 +/- 5% residual activity). When added to human plasma containing a 125I-fibrin-labeled plasma clot, equi-effective concentrations (causing 50% lysis in 2 h) were 17 +/- 3.0, 13 +/- 1.0, and 20 +/- 1.0 nM for STAR, equimolar plasmin-STAR mixtures, and plasmin-STAR mixtures neutralized by alpha 2-antiplasmin, respectively. Gel filtration of mixtures of plasmin(ogen) and STAR revealed elution as plasmin-STAR complex (Mr approximately 100,000), whereas after addition of alpha 2-antiplasmin, STAR eluted with an apparent Mr of 20,000. When mixtures of plasmin and STAR were adsorbed to lysine-Sepharose, STAR adsorbed quantitatively (96 +/- 1%) to the gel, whereas it was nearly quantitatively recovered in the unbound fraction (92 +/- 4%) after addition of alpha 2-antiplasmin to the mixture. Scatchard analysis of the binding of STAR to plasmin-Sepharose yielded a dissociation constant of 55 nM, whereas no specific binding of STAR to plasmin-alpha 2-antiplasmin-Sepharose could be demonstrated. These findings indicate that, both in purified systems and in a human plasma milieu containing a 125I-fibrin-labeled plasma clot, neutralization of the plasmin-STAR complex by alpha 2-antiplasmin results in dissociation of functionally active STAR from the complex and recycling of STAR to other plasminogen molecules. This dissociation-recycling process may explain the high fibrinolytic potency of STAR in a plasma milieu in the presence of high concentrations of alpha 2-antiplasmin.