Regulation of the functional activity of the human dopamine transporter by protein kinase C

The role of protein kinase C (PKC) was examined in the regulation of dopamine transport in C6 glioma cells stably expressing the human dopamine transporter. The PKC activating phorbol esters phorbol 12-myristate 13-acetate (PMA) and 4 beta-12,13-dibutyrate phorbol-ester (PDBu) inhibited [3H]dopamine uptake concentration dependently. These effects were attenuated by the PKC inhibitor staurosporine but were unaltered by another inhibitor, chelerythrine, or the phosphatase inhibitor okadaic acid. The potency of PMA in inhibiting [3H]dopamine uptake was similar to that in inhibiting the binding of 2 beta-carbomethoxy-3 beta-(4-fluorophenyl)tropane ([3H]WIN 35,428), and again staurosporine, but not chelerythrine, weakened the effect of PMA. The reduction in dopamine transporter activity by PMA was caused by a decrease in the Vmax value of [3H]dopamine uptake, opposed by a smaller reduction in the Km value, whereas the effect of PMA on [3H]WIN 35,428 binding was caused by a reduction in the Bmax value without a change in the Kd value. The lower Km value in the presence of PMA was accompanied by a higher IC50 of dopamine in inhibiting [3H]WIN 35,428 binding; the latter effect was attenuated by the co-presence of staurosporine. The results are discussed in the context of transporter loss from the cell surface, or a model with phosphorylation affecting the shared dopamine and WIN 35,428 binding domain on the transporter as well as affecting a part of the dopamine binding domain lying outside that for WIN 35,428.     

Effects of protein kinase inhibitors on in vitro protein phosphorylation and cellular differentiation of Streptomyces griseus

In vitro phosphorylation reactions using extracts of Streptomyces griseus cells and gamma-[32P]ATP revealed the presence of multiple phosphorylated proteins. Most of the phosphorylations were distinctly inhibited by staurosporine and K-252a which are known to be eukaryotic protein kinase inhibitors. The in vitro experiments also showed that phosphorylation was greatly enhanced by manganese and inhibition of phosphorylation by staurosporine and K-252a was partially circumvented by 10 mM manganese. A calcium-activated protein kinase(s) was little affected by these inhibitors. Herbimycin and radicicol, known to be tyrosine kinase inhibitors, completely inhibited the phosphorylation of one protein. Consistent with their in vitro effects the protein kinase inhibitors inhibited aerial mycelium formation and pigment production by S. griseus. All these data suggest that S. griseus possesses several protein kinases of eukaryotic type which are essential for morphogenesis and secondary metabolism. In vitro phosphorylation of some proteins in a staurosporine-producing Streptomyces sp. was also inhibited by staurosporine, K-252a and herbimycin, which suggests the presence of a mechanism for self-protection in this microorganism.     

Perivascular localization of nitric oxide synthase in the rat adenohypophysis: potential implications for function and cell-cell interaction

Effects of activation of protein kinase C (PKC) on N-methyl-D-aspartate) NMDA receptor function were analyzed by quantitative autoradiography using [3H]MK-801 in rat brain slices. The density of [3H]MK-801 binding was highest in hippocampus and high levels were found in cortex, striatum and thalamus. Levels in brainstem and molecular layer of cerebellum were low. The receptor binding was markedly decreased in almost all areas by addition of 2. 5 mM Mg2+. After activation of PKC by 100 nM phorbol-12, 13-dibutyrate (PDBu), [3H]MK-801 binding was increased in most areas, but binding levels were not changed in brainstem and cerebellum. The elevated [3H]MK-801 binding produced by PDBu was significantly inhibited by addition of Mg2+ except in inferior colliculus and cerebellum. These results suggest that activation of PKC potentiates NMDA receptor function in a region-specific manner in the rat brain.     

Tonic regulation of excitation-contraction coupling by basal protein kinase C activity in isolated cardiac myocytes

A high-speed imaging technique was used to investigate the effects of inhibitors and activators of protein kinase C (PKC) on the [Ca2+]i transients and contraction of fura-2 loaded rat ventricular cardiac myocytes. The amplitude of the [Ca2+]i transient was reduced following treatment with 100 nM phorbol 12,13-dibutyrate (PDBu), whereas the PKC inhibitors staurosporine (0.5 microM) and calphostin C (10 microM) increased [Ca2+]i transient amplitude, elevated basal [Ca2+]i and slowed the decay of the [Ca2+]i transient. These changes were paralleled by similar alterations in the rate and extent of cell shortening. The activity of nitrendipine-sensitive Ca2+ channels was monitored indirectly as the rate of Mn2+ quench of cytosolic fura-2 in electrically-paced cells. PDBu reduced Mn2+ influx by six-fold, whereas staurosporine and calphostin C increased the influx rate by eight-fold and seven-fold over basal quench, respectively. The caffeine releasable Ca2+ pool was reduced in the presence of PDBu and increased transiently in presence of staurosporine. The effects of PKC activation and inhibition on sarcoplasmic reticulum Ca2+ content may be secondary to alterations of sarcolemmal Ca2+ influx. However, the PKC inhibitors also decreased the rate of sarcoplasmic reticulum Ca2+ uptake in permeabilized myocytes, suggesting that a direct effect of PKC on the sarcoplasmic reticulum may contribute to the prolongation of the [Ca2+]i transient under these conditions. The present work demonstrates that basal PKC activity has a potent depressant effect, mediated primarily through inhibition of sarcolemmal Ca2+ influx, which may play a key role in setting the basal tone of cardiac muscle.     

High concentrations of cholecystokinin octapeptide suppress protein kinase C activity in guinea pig pancreatic acini

In pancreatic acini, calcium-mobilizing agents increase intracellular calcium and stimulate the production of diacylglycerol, and then activate protein kinase C (PKC). However, there are few studies which have examined the activation of PKC in intact acini. To examine the activation of PKC in intact acini by calcium-mobilizing agents, we measured the binding of [3H]phorbol-12,13-dibutyrate (PDBu) to intact acini. Acini were incubated with 10 nM [3H]PDBu at 25 degrees C with or without agents. The binding reactions were terminated by filtration. The filters were counted by a scintillation counter after washing. Acini possessed a single class of binding sites to PDBu, with Kd = 70 nM. CCK-8 and carbachol upregulated the binding affinity of PKC to PDBu in the acini. The ability of calcium-mobilizing agents to increase binding of [3H]PDBu to the acini had a close correlation to their ability to stimulate the amylase secretion from the acini, and higher concentrations of CCK-8 for amylase secretion suppressed binding of [3H]PDBu to the acini. 8Br-cAMP, 8Br-cGMP, and calcium ionophore did not inhibit the maximal activation of PKC induced by CCK-8. The calmodulin inhibitor W7 did not reverse the inhibitory effect of higher concentrations of CCK-8 on PKC activation. These results indicate that calcium-mobilizing agents upregulate the binding affinity of PKC to PDBu in intact acini, and that higher concentrations of CCK-8 for amylase secretion may activate the intracellular mechanism that inhibits PKC activity in acini. This inhibitory mechanism was mediated by some other mechanism other than cAMP-, cGMP-, calcium- and calmodulin-dependent mechanisms.     

Phospholipid and cholesteryl ester transfer activities in plasma from 14 vertebrate species. Relation to atherogenesis susceptibility

1. In this study, the underlying mechanism of stimulation of respiratory burst by kazinol B, a natural isoprenylated flavan, in rat neutrophils in vitro was investigated. 2. Kazinol B concentration-dependently stimulated the superoxide anion (O2*-) generation, with a lag but transient activation profile, in neutrophils but not in a cell-free system. The maximum response (13.2+/-1.4 nmol O2*- 10 min(-1) per 10(6) cells) was observed at 10 microM kazinol B. 3. Pretreatment of neutrophils with phorbol 12-myristate 13-acetate (PMA) or formylmethionyl-leucyl-phenylalanine (fMLP) significantly enhanced the O2*- generation following the subsequent stimulation of cells with kazinol B. 4. Cells pretreated with EGTA or a protein kinase inhibitor staurosporine effectively attenuated the kazinol B-induced O2*- generation. However, a p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 and a phosphoinositide 3-kinase (PI3K) inhibitor wortmannin had no effect on the kazinol B-induced response. 5. Kazinol B significantly stimulated [Ca2+]i elevation in neutrophils, with a lag and slow rate of rise activation profile, and this response was attenuated by a phospholipase C (PLC) inhibitor U73122. Kazinol B also stimulated the inositol bis- and trisphosphate (IP2 and IP3) formation with a 1 min lag time. 6. The membrane-associated PKC-alpha and PKC-theta but not PKC-iota were increased following the stimulation of neutrophils with kazinol B. It was more rapid and sensitive in the activation of PKC-theta than PKC-alpha by kazinol B. Kazinol B partially inhibited the [3H]phorbol 12,13-dibutyrate ([3H]PDB) binding to the neutrophil cytosolic PKC. 7. Neither the cellular mass of phosphatidic acid (PA) and phosphatidylethanol (PEt), in the presence of ethanol, nor the protein tyrosine phosphorylation were stimulated by kazinol B. In addition, the kazinol B-induced O2*- generation remained relatively unchanged in cells pretreated with ethanol or a tyrosine kinase inhibitor genistein. 8. Collectively, these results indicate that the stimulation of the respiratory burst by kazinol B is probably mediated by the synergism of PKC activation and [Ca2+]i elevation in rat neutrophils.     

Low-affinity thromboxane receptor mediates proliferation in cultured vascular smooth muscle cells of rats

We examined the binding properties and mitogenic effects of U46619, using cultured vascular smooth muscle cells (VSMCs), by ligand-binding assay, measuring [3H]thymidine and [3H]leucine incorporation, checking with flow cytometry, and counting the cell number. The U46619-activated mitogenic signal-transduction pathway was assessed by measuring formation of inositol monophosphate (IP); [Ca2+]i; mitogen-activated protein kinase (MAPK), MAPK kinase (MAPKK), and p74raf-1 activities; and GTP-bound Ras. [3H]U46619 bound to cultured VSMCs from Wistar-Kyoto (WKY) rats at a single class of site (Kd: 15.5 +/- 2.6 nmol/L). However, it bound to VSMCs from spontaneously hypertensive rats (SHRs) at two classes of sites (Kd: 2.3 +/- 0.6 nmol/L and 1.4 +/- 0.5 mumol/L). U46619 increased DNA and protein synthesis, cell number, IP formation, [Ca2+]i, and MAPK and MAPKK activities, with EC50 values close to its Kd value for the low-affinity binding site in VSMCs from SHR. Prostaglandin (PG) E2 and PGF2 alpha showed little of such mitogenic effects. All these effects of U46619 were inhibited by SQ29548, staurosporine, or pretreatment of VSMCs with phorbol 12-myristate 13-acetate for 24 hours. However, U46619 stimulation did not lead to a significant increase in the Ras-GTP complex or p74raf-1 activity. In conclusion, the mitogenic effect of U46619 appears to be mediated via the activation of low-affinity thromboxane binding sites that trigger phosphoinositide hydrolysis and activate the MAPK pathway, leading to DNA synthesis and cell proliferation.     

Cations affect [3H]mazindol and [3H]WIN 35,428 binding to the human dopamine transporter in a similar fashion

The present study addresses the possibility that there are different cocaine-related and mazindol-related binding domains on the dopamine transporter (DAT) that show differential sensitivity to cations. The effects of Zn2+, Mg2+, Hg2+, Li+, K+, and Na+ were assessed on the binding of [3H]mazindol and [3H]WIN 35,428 to the human (h) DAT expressed in C6 glioma cells under identical conditions for intact cell and membrane assays. The latter were performed at both 0 and 21 degrees C. Zn2+ (30-100 microM) stimulated binding of both radioligands to membranes, with a relatively smaller effect for [3H]mazindol; Mg2+ (0.1-100 microM) had no effect; Hg2+ at approximately 3 microM stimulated binding to membranes, with a relatively smaller effect for [3H]mazindol than [3H]WIN 35,428 at 0 degrees C, and at 30-100 microM inhibited both intact cell and membrane binding; Li+ and K+ substitution (30-100 mM) inhibited binding to membranes more severely than to intact cells; and Na+ substitution was strongly stimulatory. With only a few exceptions, the patterns of ion effects were remarkably similar for both radioligands at both 0 and 21 degrees C, suggesting the involvement of common binding domains on the hDAT impacted similarly by cations. Therefore, if there are different binding domains for WIN 35,428 and mazindol, these are not affected differentially by the cations studied in the present experiments, except for the stimulatory effect of Zn2+ at 0 and 21 degrees C and Hg2+ at 0 degrees C.     

Modulations of early and late secretory processes by activation of protein kinases in the rat adrenal medulla

Modulatory effects of the activation of either protein kinase C (PKC) by phorbol 12,13-dibutyrate (PDBu) or protein kinase A (PKA) by forskolin on stimulant-evoked secretory processes in the perfused rat adrenal medulla were studied. PDBu or forskolin was applied during repetitive stimulation (30 s each at 10-min intervals) with nicotine, bradykinin, muscarine or histamine, and changes in [Ca2+]i (fura-2 microfluorometry) and catecholamine secretions (electrochemical detection) were simultaneously measured. PDBu markedly potentiated the nicotine-evoked secretion without altering the [Ca2+]i response. PDBu partially inhibited the muscarine-evoked secretion and almost completely blocked the histamine-evoked secretion, concomitantly with extensive suppressions of the [Ca2+]i responses to these stimulants. The bradykinin-evoked secretion was enhanced by PDBu despite a slight attenuation of the [Ca2+]i response. PDBu reduced bradykinin-induced intracellular Ca2+ release in a Ca2+-free medium but enhanced the secretion associated with the released Ca2+. These results suggest that PDBu-activated PKC modulates secretory processes at, at least, two different stages. An early-stage modulation may downregulate receptor/G protein systems, which accounts for the inhibitory effect of PDBu on the muscarine- and histamine-evoked responses. A late-stage modulation may generally promote Ca2+-triggered exocytosis after elevation of [Ca2+]i, which explains the potentiation of the nicotine-evoked secretion by PDBu. The late-stage modulation may counteract the early-stage modulation in bradykinin-stimulated cells. Forskolin potentiated the secretory responses to the four secretagogues without increasing the [Ca2+]i responses. PKA may modulate secretory process at a step(s) distal to the rise in [Ca2+]i as is the case with the late-stage modulation by PKC.     

Pharmacological comparison of UTP- and thapsigargin-induced arachidonic acid release in mouse RAW 264.7 macrophages

1. Although stimulation of mouse RAW 264.7 macrophages by UTP elicits a rapid increase in intracellular free Ca2+ ([Ca2+]i), phosphoinositide (PI) turnover, and arachidonic acid (AA) release, the causal relationship between these signalling pathways is still unclear. In the present study, we investigated the involvement of phosphoinositide-dependent phospholipase C (PI-PLC) activation, Ca2+ increase and protein kinase activation in UTP-induced AA release. The effects of stimulating RAW 264.7 cells with thapsigargin, which cannot activate the inositol phosphate (IP) cascade, but results in the release of sequestered Ca2+ and an influx of extracellular Ca2+, was compared with the effects of UTP stimulation to elucidate the multiple regulatory pathways for cPLA2 activation. 2. In RAW 264.7 cells UTP (100 microM) and thapsigargin (1 microM) caused 2 and 1.2 fold increases, respectively, in [3H]-AA release. The release of [3H]-AA following treatment with UTP and thapsigargin were non-additive, totally abolished in the Ca2+-free buffer, BAPTA (30 microM)-containing buffer or in the presence of the cPLA2 inhibitor MAFP (50 microM), and inhibited by pretreatment of cells with pertussis toxin (100 ng ml(-1)) or 4-bromophenacyl bromide (100 microM). By contrast, aristolochic acid (an inhibitor of sPLA2) had no effect on UTP and thapsigargin responses. 3. U73122 (10 microM) and neomycin (3 mM), inhibitors of PI-PLC, inhibited UTP-induced IP formation (88% and 83% inhibition, respectively) and AA release (76% and 58%, respectively), accompanied by a decrease in the [Ca2+]i rise. 4. Wortmannin attenuated the IP response of UTP in a concentration-dependent manner (over the range 10 nM-3 microM), and reduced the UTP-induced AA release in parallel. RHC 80267 (30 microM), a specific diacylglycerol lipase inhibitor, had no effect on UTP-induced AA release. 5. Short-term treatment with PMA (1 microM) inhibited the UTP-stimulated accumulation of IP and increase in [Ca2+]i, but had no effect on the release of AA. In contrast, the AA release caused by thapsigargin was increased by PMA. 6. The role of PKC in UTP- and thapsigargin-mediated AA release was shown by the blockade of these effects by staurosporine (1 microM), Ro 31-8220 (10 microM), Go 6976 (1 microM) and the down-regulation of PKC. 7. Following treatment of cells with SK&F 96365 (30 microM), thapsigargin-, but not UTP-, induced Ca2+ influx, and the accompanying AA release, were down-regulated. 8. Neither PD 98059 (100 microM), MEK a inhibitor, nor genistein (100 microM), a tyrosine kinase inhibitor, had any effect on the AA responses induced by UTP and thapsigargin. 9. We conclude that UTP-induced cPLA2 activity depends on the activation of PI-PLC and the sustained elevation of intracellular Ca2+, which is essential for the activation of cPLA2 by UTP and thapsigargin. The [Ca2+]i-dependent AA release that follows treatment with both stimuli was potentiated by the activity of protein kinase C (PKC). A pertussis toxin-sensitive pathway downstream of the increase in [Ca2+]i was also shown to be involved in AA release.     

Binding profile of the novel 5-HT1B/1D receptor antagonist, [3H]GR 125,743, in guinea-pig brain: a comparison with [3H]5-carboxamidotryptamine

The aim of the research was to characterize muscarinic receptors of bovine ciliary muscle and to investigate the desensitization process. The role of protein kinase C was analyzed. The results show that muscarinic receptors of bovine ciliary muscle have the pharmacological characteristics of the M3 subtype. Acute exposure to phorbol esters (1 microM phorbol 12,13-dibutyrate, PDB, or 0.1 microM phorbol 12-myristate 13-acetate, PMA, for 15 and 5 min, respectively) resulted in antagonism of muscarinic receptor-mediated contraction. Long-term pretreatment (18 h) with PMA to down-regulate protein kinase C resulted in potentiation of carbachol-induced contraction, reduction of agonist-induced desensitization and loss of phorbol ester-induced desensitization. Staurosporine (3 microM) and H7 [1-(5-isoquinolinesulfonyl)-2-methyl-piperazine] (1 microM), protein kinase C inhibitors, produced a significant potentiation of the contractile effect of carbachol, reduced the desensitization produced by repeated addition of carbachol and suppressed that induced by phorbol esters. In vitro incubation with carbachol, PDB or PMA did not cause any modification of the binding of labeled [3H]quinuclidinyl benzilate. In vitro incubation with PDB and PMA produced, as expected, a significant translocation of protein kinase C from the cytosol to the membrane. The incubation of the ciliary muscle with carbachol, using the protocol of exposure that induced maximal desensitization of contractile responses, produced a significant redistribution of the enzyme from the cytosol to the membrane. These findings suggest that agonist-induced modulation of functional cholinergic sensitivity in ciliary muscle is correlated, at least partially, to the translocation of protein kinase C from the cytosol to the membrane. The desensitization by phorbol esters is completely due to protein kinase C activation; during the desensitization process, direct modification of the density and affinity of muscarinic receptors is not involved.     

Lysophosphatidylcholine potentiates vascular contractile responses by enhancing vasoconstrictor-induced increase in cytosolic free Ca2+ in rat aorta

We investigated the effects of palmitoyl-L-alpha-lysophosphatidylcholine on the contractile responses of the endothelium-denuded rat aorta to high K+, noradrenaline, UK14,304 (5-bromo-6-[2-imidazolin-2-ylamino]-quinoxaline) (a selective alpha2 adrenoceptor agonist) and phorbol 12-myristate 13-acetate (PMA). Lysophosphatidylcholine at concentrations from 10(-6) M to 10(-4) M did not contract aortic strips. However, lysophosphatidylcholine strongly potentiated the UK14,304-induced contraction. High K+ - and PMA-induced contractions were also potentiated. In contrast, the noradrenaline-induced contraction was only slightly potentiated by 10(-5) M lysophosphatidylcholine. In fura PE-3-loaded aortic strips, lysophosphatidylcholine (10(-5) M) markedly augmented the increase in both cytosolic free Ca2+ ([Ca2+]i) and contractile tension induced by UK14,304, high K+ and PMA. Nicardipine (10(-7) M) and 10(-6) M Ro-31-8220 (?1-[3-(amidinothio)propyl-1H-indoyl-3-yl]-3-(1-methyl-1H-++ +indoyl-3-yl)-maleimide-methane sulfate) strongly inhibited the increase in [Ca2+]i and contractile tension induced by UK14,304 and in the presence of these inhibitors, the enhancing effects of lysophosphatidylcholine were attenuated. However, the enhancing effect on high K+ -induced contraction was not affected by Ro-31-8220. These results suggest that lysophosphatidylcholine may cause an augmentation of the increase in [Ca2+]i induced by UK14,304 which response is depend on protein kinase C activation and in this way potentiate contractile responses in the rat aorta. Protein kinase C independent mechanisms may also be involved in the enhancing effect of lysophosphatidylcholine on smooth muscle contraction.     

Protein kinase C--catalyzed calponin phosphorylation in swine carotid arterial homogenate

Calponin, a thin filament-associated protein, inhibits actin-activated myosin ATPase activity, and this inhibition is reversed by phosphorylation. Calponin phosphorylation by protein kinase C and Ca2+/calmodulin-dependent protein kinase II has been shown in purified protein systems but has been difficult to demonstrate in more physiological preparations. We have previously shown that calponin is phosphorylated in a cell-free homogenate of swine carotid artery. The goal of this study was to determine whether protein kinase C and/or Ca2+/calmodulin-dependent protein kinase II catalyzes calponin phosphorylation. Ca2+-dependent calponin phosphorylation was not inhibited by calmodulin antagonists. In contrast, both Ca2+- and phorbol dibutyrate/1-oleoyl-2-acetyl-sn-glycerol dependent calponin phosphorylation were inhibited by the pseudosubstrate inhibitor of protein kinase C and staurosporine. Our results also demonstrate that stimulation with either Ca2+, phorbol dibutyrate, or 1-oleoyl-2-acetyl-sn-glycerol activates endogenous protein kinase C. We interpret our results as clearly demonstrating that the physiological kinase for calponin phosphorylation is protein kinase C and not Ca2+/calmodulin-dependent protein kinase II. We also present data showing that the direct measurement of 32P incorporation into calponin and the indirect measurement of calponin phosphorylation using nonequilibrium pH gradient gel electrophoresis provide similar quantitative values of calponin phosphorylation.     

Chronic effects of glucose on insulin signaling in A-10 vascular smooth muscle cells

To examine the effects of hyperglycemia on insulin signaling in A-10 vascular smooth muscle cells, cells were treated with extracellular D-glucose and effects of insulin were studied on the diacylglycerol-protein kinase C signaling system. A-10 cells specifically bound 125I-insulin, and insulin-like growth factor-I did not displace the label. 125I-insulin binding was unaltered under hyperglycemic conditions. To determine if insulin receptors were coupled to other insulin-regulated processes, diacylglycerol, protein kinase C, and glucose transport were evaluated. Insulin increased cellular diacylglycerol (DAG) levels which were also increased following glucose treatment and not further stimulated by insulin. The uptake of 2-[3H]deoxy-D-glucose (2-DOG) was stimulated by insulin and 12-O-tetradecanoyl phorbol 13-acetate (TPA). Insulin- and TPA-stimulated 2-[3H]DOG uptake was inhibited by a protein kinase inhibitor, staurosporine. Preincubation of cells with 500 nM TPA overnight resulted in the inhibition of insulin- and TPA-stimulated 2-[3H]DOG uptake. Protein kinase C activity was translocated from cytosolic to membrane fractions following insulin treatment. Overnight glucose (25 mM) treatment resulted in a 50% decrease in protein kinase C enzyme activity and > 90% decrease in protein kinase C beta immunoreactive levels. Protein kinase C activity and levels were not affected by osmotic control media containing mannitol. A-10 cells express GLUT4-type glucose transporters. Neither insulin-regulatable glucose transporter (GLUT4) mRNA nor GLUT4 protein levels were diminished by glucose. Significant decreases in insulin- and TPA-stimulated 2-[3H]DOG uptake occurred, however, with glucose. The down-regulation of protein kinase C beta and resultant inhibition of 2-[3H]DOG uptake by chronic glucose suggests a biochemical link between hyperglycemia and DAG-protein kinase C signaling in vascular smooth muscle cells.     

High affinity binding of 9-[3H]methyl-7-bromoeudistomin D to the caffeine-binding site of skeletal muscle sarcoplasmic reticulum

3H-Labeled 9-methyl-7-bromoeudistomin D ([3H] MBED), the most powerful inducer of Ca2+ release from sarcoplasmic reticulum (SR), was successfully prepared with a high specific activity of 10.2 Ci/mmol. [3H]MBED bound to terminal cisternae (TC) of skeletal muscle SR in a replacable and saturable manner, indicating the existence of its specific binding site. Caffeine inhibited the [3H]MBED binding to the TC-SR membranes from skeletal muscle with an IC50 value of 0.8 mM, in close agreement with a concentration that causes Ca2+ release from SR. Scatchard analysis gave values of KD = 40 nM and Bmax = 10 pmol/mg protein. The KD value was increased by caffeine, while that of Bmax was not changed, indicating a competitive mode of inhibition. Adenosine 5'-(beta, gamma-methylene)triphosphate enhanced [3H]MBED binding, but ryanodine and Ca2+ did not affect it. [3H]MBED binding to TC-SR membranes was inhibited by procaine, a representative blocker of Ca(2+)-induced Ca2+ release channels, whereas that was not changed by Mg2+, suggesting that procaine but not Mg2+ may exert its inhibitory effect on Ca(2+)-induced Ca2+ release by affecting the caffeine-binding sites. These results suggest that MBED shares the same binding site as that of caffeine in TC-SR. The [3H]MBED is the first radiolabeled ligand for caffeine-binding sites in Ca2+ release channels and thus may provide an essential biochemical tool for elucidating this site.     

Protein kinase C inhibits the Ca(2+)-activated K+ channel of cultured porcine coronary artery smooth muscle cells

The effect of protein kinase C (C-kinase) on the Ca(2+)-activated K+ channel (KCa-channel) was studied in cultured smooth muscle cells from porcine coronary artery by the patch-clamp technique. In cell-attached patches, bath application of phorbol 12-myristate 13-acetate (PMA, 1 microM), a C-kinase activator, significantly decreased the open probability of the activated KCa-channel in the presence of the calcium ionophore A23187 (20 microM), which increases intracellular Ca2+. This decrease in the open probability was reversed by subsequent application of staurosporine (1 nM), a C-kinase inhibitor. Application of 1-oleoyl-2-acetylglycerol (OAG, 30 microM) or 1,2-dioctanoylglycerol (DG8; 30 microM), activators of C-kinase, also inhibited KCa-channel activation by A23187, and these inhibitions were also reversed by staurosporine. PMA (1 microM) also inhibited KCa-channel activation by dibutylyl cyclic AMP (db-cAMP, 2 mM) or caffeine (30 mM). In inside-out patches, bath application of the C-kinase fraction from rat brain in the presence of ATP (1 mM) and PMA (1 microM) markedly inhibited the KCa-channel. These results indicate that activation of C-kinase inhibits the KCa-channel and may cause membrane depolarization and vascular contraction.     

Interference of S-alkyl derivatives of glutathione with brain ionotropic glutamate receptors

The effects of glutathione, glutathione sulfonate and S-alkyl derivatives of glutathione on the binding of glutamate and selective ligands of ionotropic N-methyl-D-aspartate (NMDA) and non-NMDA receptors were studied with mouse synaptic membranes. The effects of glutathione and its analogues on 45Ca2+ influx were also estimated in cultured rat cerebellar granule cells. Reduced and oxidized glutathione, glutathione sulfonate, S-methyl-, -ethyl-, -propyl-, -butyl- and -pentylglutathione inhibited the Na+-independent binding of L-[3H]glutamate. They strongly inhibited also the binding of (S)-2-amino-3-hydroxy-5-[3H]methyl-4-isoxazolepropionate [3H]AMPA (IC50 values: 0.8-15.9 microM). S-Alkylation of glutathione rendered the derivatives unable to inhibit [3H]kainate binding. The NMDA-sensitive binding of L-[3H]glutamate and the binding of 3-[(R)-2-carboxypiperazin-4-yl][1,2-(3)H]propyl-1-phosphonate ([3H]CPP, a competitive antagonist at NMDA sites) were inhibited by the peptides at micromolar concentrations. The strychnine-insensitive binding of the NMDA coagonist [3H]glycine was attenuated only by oxidized glutathione and glutathione sulfonate. All peptides slightly enhanced the use-dependent binding of [3H]dizocilpine (MK-801) to the NMDA-gated ionophores. This effect was additive with the effect of glycine but not with that of saturating concentrations of glutamate or glutamate plus glycine. The glutamate- and NMDA-evoked influx of 45Ca2+ into cerebellar granule cells was inhibited by the S-alkyl derivatives of glutathione. We conclude that besides glutathione the endogenous S-methylglutathione and glutathione sulfonate and the synthetic S-alkyl derivatives of glutathione act as ligands of the AMPA and NMDA receptors. In the NMDA receptor-ionophore these glutathione analogues bind preferably to the glutamate recognition site via their gamma-glutamyl moieties.     

Inhibition of nicotinic responses by cotinine in bovine adrenal chromaffin cells

We studied the effects of cotinine, the major metabolite of nicotine, on nicotine-induced increase in [3H]phorbol dibutyrate binding, activation of protein kinase C and [3H]noradrenaline release in primary cultured bovine adrenal chromaffin cells. Cotinine (1 mM, 15 min.) and nicotine (10 microM, 5 min.) increased the [3H]phorbol binding by 100% and 150%, respectively. Both a short-term (10 min.) and a long-term (24 hr) pretreatment with cotinine inhibited the effect of nicotine. A 24 hr pretreatment with cotinine (1 mM) also reduced the nicotine-induced increase in membrane-bound protein kinase C activity. Cotinine pretreatment (10 min.) dose-dependently inhibited the release of [3H]noradrenaline induced by nicotine and dimethylphenylpiperazinium. Cotinine pretreatment did not reduce the [3H]noradrenaline release induced by high extracellular potassium (56 mM) or veratrine (10 mg l-1). The results indicate that cotinine inhibits activation of protein kinase C and noradrenaline release induced by nicotinic agonists in primary cultures of bovine adrenal chromaffin cells. The results suggest that pre-existing cotinine could modify responses to acute exposure to nicotine in neural systems.     

Effect of protein kinase inhibitors on activity of mammalian small heat-shock protein (HSP25) kinase

The aim of this study was to investigate different protein kinase inhibitors (secondary metabolite-derived substances, synthetic compounds, and substrate-based peptides) for their potency to inhibit the mammalian small heat shock protein (HSP25) kinase (E.C. 2.7.1.37) isolated from Ehrlich ascites tumor cells. Among the secondary metabolite-derived inhibitors (staurosporine, K-252a, K-252b, KT5926, KT5720, erbstatin analog, and quercetin) and synthetic compounds (H-9, H-89, HA 1004, KN-62, ML-7, tyrphostin A25, and tyrphostin B42), KT5926, staurosporine, and K-252a inhibited HSP25 kinase most efficiently. Kinetic analysis revealed that inhibition by staurosporine (Ki = 32.4 nM) and K-252a (Ki = 13.7 nM) was competitive with ATP. Inhibition by KT5926 was competitive with the substrate peptide KKKALNRQLSVAA (Ki = 27.2 nM) and noncompetitive with respect to ATP (Ki = 38.8 nM). In comparison with other protein kinases, HSP25 kinase was relatively resistant to most of the inhibitors. KT5926 was the only tested inhibitor with certain preference for HSP25 kinase when compared with protein kinases A, C, and G. Among the tested substrate-based peptides, we identified one peptide (KKKALNRQLGVAA), which preferentially inhibited HSP25 kinase in comparison with protein kinases A and C and mitogen-activated protein kinase. This peptide inhibited HSP25 kinase competitively with the substrate peptide (Ki = 8.1 microM) and noncompetitively with ATP (Ki = 134 microM). A peptide (SRVLKEDKERWEDVK) derived from the putative autoinhibitory domain of the closely related human mitogen-activated protein kinase-activated protein kinase-2 did not inhibit HSP25 kinase activity, suggesting the existence of several species of HSP25 kinases. Furthermore, the data identified structural requirements for inhibitors of HSP25-kinase.