Potent Inhibitor Of Protein Kinase Research Articles

Identification of serine138 residue in the 4-residue segment K135K1361137S138 of LukS-I component of Staphylococcus intermedius leukocidin crucial for the LukS-I-specific function of staphylococcal leukocidin.

Luk-I produced by Staphylococcus intermedius was found to be a new member of the staphylococcal bi-component pore-forming toxin family, in which staphylococcal leukocidin, Panton-Valentine leukocidin, and gamma-hemolysin are included. Luk-I consists of LukS-I and LukF-I. From the deduced amino acid sequence of LukS-I, a 4-residue sequence, K135K1361137S138, at the root of the stem region was found to be identical with that of the phosphorylated segment of a protein phosphorylated by protein kinase A. A mutant of LukS-I (MLSI-SA), in which the Ser138 residue was replaced by an alanine residue, was created, purified, and assayed for its leukocytolytic and pore-forming activities with LukF-I. Both LukS-I and MLSI-SA formed a ring-shaped complex with LukF-I on rabbit erythrocytes and human polymorphonuclear leukocytes (HPMNLs) membrane. However, MLSI-SA showed no leukocytolytic activity with LukF-I. LukS-I was phosphorylated by protein kinase A in the presence of [gamma-32P] ATP in a cell-free system, but MLSI-SA was not phosphorylated significantly. A potent and selective inhibitor of protein kinase A (N-[2(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89)) showed 50% inhibition of the Luk-I-induced cell lysis at 0.5 nM. Thus, it is concluded that the phosphorylation of the Ser138 residue in the 4-residue segment K135K1361137S138 of LukS-I is important for the leukocytolysis of HPMNLs.

DNA-PK inhibitor wortmannin enhances DNA damage induced by bleomycin in V79 Chinese hamster cells.

The fungal metabolite wortmannin (WM) is a potent and irreversible inhibitor of the enzyme DNA-dependent protein kinase (DNA-PK), a nuclear serine-threonine kinase, member of the phosphaditylinositol-3 kinase related kinase family. WM has been used in the last few years as a promising radiosensitizer mainly throughout cell survival experiments. However, few studies have addressed the role of DNA-PK inhibition in the repair of DNA lesions generated by antitumor agents. Bleomycin (BLM) is an antitumor agent used in the treatment of various neoplasia with a unique genotoxicity profile that mimics the ionizing radiation effects. In this study, we evaluated the effect of different concentrations of WM on the DNA damage induced by BLM. The cytokinesis-block micronucleus assay (CBMN) in V79 Chinese hamster cells was used as the end-point. WM significantly increased the frequency of micronucleated cells (%MNBN) by about 2.2-fold, the number of micronuclei per binucleated cell (MN/BN) by about 2.4-fold, and also changed the pattern of the distribution of micronuclei induced by BLM. The frequency of micronucleated cells with 2 MN per cell and with > or = 3 MN per cell increased, whereas the frequency of micronucleated cells with 1 MN per cell decreased. WM was not genotoxic but decreased cell proliferation as assessed by the frequency of binucleated cells. Our results show that WM clearly enhances the efficacy of BLM in terms of DNA damage inflicted and therefore reinforces its use as a chemosensitizer.

A review of HNS-32: a novel azulene-1-carboxamidine derivative with multiple cardiovascular protective actions.

HNS-32 [N(1),N(1)-dimethyl-N(2)-(2-pyridylmethyl)-5-isopropyl-3,8-dimethylazulene-1- carboxamidine] (CAS Registry Number: 186086-10-2) is a newly synthesized azulene derivative. Computer simulation showed that its three dimensional structure is similar to that of the class Ib antiarrhythmic drugs, e.g., lidocaine or mexiletine. HNS-32 potently suppressed ventricular arrhythmias induced by ischemia due to coronary ligation and/or ischemia-reperfusion in dogs and rats. In the isolated dog and guinea pig cardiac tissues, HNS-32 had negative inotropic and chronotropic actions, prolonged atrial-His and His-ventricular conduction time and increased coronary blood flow. In the isolated guinea pig ventricular papillary muscle, HNS-32 decreased maximal rate of action potential upstroke (Vmax) and shortened action potential duration (APD). These findings suggest that HNS-32 inhibits inward Na+ and Ca2+ channel currents. In the isolated pig coronary and rabbit conduit arteries, HNS-32 inhibited both Ca2+ channel-dependent and -independent contractions induced by a wide variety of chemical stimuli. HNS-32 is a potent inhibitor of protein kinase C (PKC)-mediated constriction of cerebral arteries. It is likely to block both, Na+ and Ca2+ channels expressed in cardiac and vascular smooth muscles. These multiple ion channel blocking effects are largely responsible for the antiarrhythmic and vasorelaxant actions of HNS-32. This drug may represent a novel approach to the treatment of arrhythmias.

Coupling of L-type calcium channels to neurotransmitter release at mouse motor nerve terminals.

Previously, we have presented evidence for the presence of L-type voltage-dependent Ca2+ channels (VDCC) in 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, (acetoxymethyl)ester (BAPTA-AM)-incubated motor nerve terminals (MNTs) of the levator auris muscle of mature mice. The aim of the present work was to study the coupling of these L-type VDCC to neurotransmitter release by inhibiting protein phosphatases. We thus studied the effects of the protein phosphatase inhibitors okadaic acid (OA) and pervanadate on quantal content (QC) of transmitter release with the P/Q-type channels fully blocked. The QC was not significantly different under the three experimental conditions tested: incubation with dimethylsulphoxide (DMSO), ethylene-glycol-bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid, (acetoxymethyl)ester (EGTA-AM) and BAPTA-AM. After preincubation with OA (1 microM), but not with pervanadate, QC increased substantially in the BAPTA-AM-incubated (up to 400%) MNT, but not in those incubated with DMSO or EGTA-AM. The OA-induced increment of QC was attenuated greatly (approximately 95% reduction) by preincubation with either nitrendipine (10 microM) or calciseptine (300 nM). The effect of OA (1 microM) and pervanadate (0.1 mM) on spontaneous neurotransmitter release was also studied. After preincubation with OA, but not per-vanadate, miniature end-plate potential (MEPP) frequency increased only in the BAPTA-AM-incubated MNT (up to 700% increment). This response was attenuated (by approximately 80%) by nitrendipine (10 microM) or calciseptine (300 nM). In contrast, neither omega-agatoxin IVA (120 nM) nor omega-conotoxin GVIA (1 microM) affected this OA-induced increment significantly. We also evaluated the relationship between QC and extracellular [Ca2+] ([Ca2+]o) in BAPTA-AM-incubated MNT. Under conditions in which only P/Q-type VDCC were available to participate in neurotransmitter release, QC increased as [Ca2+]o was raised from 0.5 to 2 mM. However, when only L-type VDCC were available, QC increased when [Ca2+]o increased from 0.5 to 1 mM, but decreased significantly at 2 mM. The mean latency for P/Q-type VDCC-mediated EPP was 1.7-1.9 ms; for L-type VDCC-mediated EPP, 1.9-2.5 ms. The rise time of the L-type VDCC mediated EPP was significantly slower than that mediated by P/Q-type VDCC. Preincubation with H-7 (100 microM), a potent inhibitor of protein kinase C (PKC) and adenosine 3',5'cyclic monophosphate (cAMP)-dependent protein kinase (PKA), attenuated the OA-induced increment of both QC and MEPP frequency (50% and 70% decrement, respectively), suggesting the participation of at least these two protein kinases in the coupling of L-type VDCC. In summary, our results show coupling of L-type VDCC to neurotransmitter release when protein phosphatases are inhibited and intracellular [Ca2+] is buffered by the fast chelator BAPTA.

PKA-Induced Stimulation of ROMK1 Channel Activity is Governed by Both Tethering and Non-Tethering Domains of an A Kinase Anchor Protein

We have used the patch-clamp technique to explore the role of A Kinase Anchor Proteins (AKAP) in mediating the effect of cAMP on ROMK1 channels expressed in the Xenopus oocytes. Addition of membrane permeant cAMP analogs increased channel activity only in oocytes injected with ROMK1 and AKAP79 cRNA but had no effect on channel activity in oocytes injected with ROMK1 alone. Using the two-electrode voltage clamp technique, we determined that application of H89, a potent inhibitor of protein kinase A (PKA), abolished the stimulatory effect of cAMP/forskolin. To investigate the role of AKAP specificity in conferring cAMP responses to ROMK1 channels, we examined channel activity in oocytes expressing ROMK1 and either AKAP18, AKAP-KL or AKAP75. Addition of cAMP failed to increase channel current in oocytes expressing ROMK1 and either AKAP18 or AKAP-KL. In contrast, cAMP increased ROMK1 channel activity by 33% in oocytes coexpressing AKAP75, the bovine homologue of AKAP79. The effect of cAMP on ROMK1 in oocytes coexpressing AKAP75 is inhibited by H89. Since all three AKAPs bind PKAII, the results suggest that a unique structural domain in AKAP75/79 collaborates with the PKAII binding site and enables a productive association of PKA with ROMK1 channels. Deletion of either the membrane targeting region of AKAP75 (AKAP45) or PKAII binding domain of AKAP75 (AKAP75DC) abolished the effects of forskolin on ROMK1 channels. This suggests that the membrane targeting and the PKA binding domains of AKAP75 are essential for the effect of cAMP. However, the nature of the AKAP, that interacts with ROMK1 in the native tissue, remains to be determined because AKAP75/79 are not expressed in the kidney. We conclude that the regulation of ROMK1 channels by PKA requires the involvement of the cell membrane-directed AKAPs that are able to specifically link PKA to the target channel protein.

Signal Transduction in Eclosion Hormone-induced Secretion of Ecdysis-triggering Hormone

Inka cells of insect epitracheal glands (EGs) secrete preecdysis and ecdysis-triggering hormones (PETH and ETH) at the end of each developmental stage. Both peptides act in the central nervous system to evoke the ecdysis behavioral sequence, a stereotype behavior during which old cuticle is shed. Secretion of ETH is stimulated by a brain neuropeptide, eclosion hormone (EH). EH evokes accumulation of cGMP followed by release of ETH from Inka cells, and exogenous cGMP evokes secretion of ETH. The secretory responses to EH and cGMP are inhibited by the broad-spectrum kinase inhibitor staurosporine, and the response to EH is potentiated by the phosphatase inhibitor calyculin A. Staurosporine did not inhibit EH-evoked accumulation of cGMP. Changes in cytoplasmic Ca2+ in Inka cells during EH signaling were monitored via fluorescence ratioing with fura-2-loaded EGs. Cytoplasmic Ca2+ increases within 30-120 s after addition of EH to EGs, and it remains elevated for at least 10 min, corresponding with the time course of secretion. Secretion is increased in dose-dependent manner by the Ca2+-ATPase inhibitor thapsigargin, a treatment that does not elevate glandular cGMP above basal levels. The secretory response to EH is partially inhibited in glands loaded with EGTA, while cGMP levels are unaffected. These findings suggest that EH activates second messenger cascades leading to cGMP accumulation and Ca2+ mobilization and/or influx and that both pathways are required for a full secretory response. cGMP activates a staurosporine-inhibitable protein kinase. We propose that Ca2+ acts via a parallel cascade with a time course that is similar to that for cGMP activation of a cGMP-dependent protein kinase.

Preclinical pharmacokinetics of SB-203580, a potent inhibitor of p38 mitogen-activated protein kinase

1. SB-203580 (4-(4-fluorophenyl)-2-(4-methylsulphinylphenyl)-5-(4-pyridyl)imidazole) is a potent, selective inhibitor of p38 MAP kinase used extensively as a tool inhibitor in various pharmacological and toxicological models. This study was designed to evaluate the pharmacokinetics of SB-203580 in several preclinical species, both to assist with the interpretation of existing studies and to aid in the design of future studies with this inhibitor. 2. In vitro, SB-203580 was stable in mouse, rat, dog, monkey and human plasma over 24 h. However, species differences in plasma protein binding were observed; SB-203580 was 96-97% bound in human plasma and 78-92% bound in other species. These data suggest that protein binding may influence the results of in vitro studies using SB-203580, particularly when comparing results from different in vitro systems that incorporate plasma components. In vivo, SB-203580 demonstrated moderate to high clearance in all species tested, with non-linear elimination observed in the rat at plasma concentrations > 1000ng ml -1. Although good solution bioavailability was observed in non-rodents (78% in dog, 32% in monkey), lower and more variable bioavailability was observed in the rat and mouse (3-48%). 3. These interspecies differences in bioavailability, and the non-linear pharmacokinetics observed in rat, highlight the importance of monitoring SB-203580 systemic exposure in parallel with the pharmacological endpoint during in vivo pharmacology studies.

Gene transfer strategies for the physiologist

Foreign genes can be introduced into whole animals using methods of germline transgenesis and somatic gene delivery. While germline transgenesis can generate useful animal models for genetic studies, it can be costly, time-consuming and requires the use of a large number of animals. An alternative means of gene transfer is to deliver genes to somatic cells using non-viral and viral technologies. Non-viral methods such as naked DNA injection, electroporation and liposome/cation lipid-mediated gene transfer are relatively inefficient. In contrast, viruses are effective vehicles that carry foreign genes into a cell rapidly and efficiently. Here we illustrate the usefulness of adenoviral vectors to express a potent and specific inhibitor of cAMP-dependent protein kinase (PKA) to study the role of cyclic 3',5'-cyclic AMP (cAMP) in the osmotic regulation of the vasopressin gene in a transgenic rat model. The ability to modify endogenous systems within specific cells in a whole animal model allows gene effects to be studied with physiological relevance. The combination of molecular biology and integrative physiology is a powerful application that can aid in the elucidation of how gene function can translate into complex systems in an organism

Gene Transfer Strategies for the Physiologist

Foreign genes can be introduced into whole animals using methods of germline transgenesis and somatic gene delivery. While germline transgenesis can generate useful animal models for genetic studies, it can be costly, time-consuming and requires the use of a large number of animals. An alternative means of gene transfer is to deliver genes to somatic cells using non-viral and viral technologies. Non-viral methods such as naked DNA injection, electroporation and liposome/cation lipid-mediated gene transfer are relatively inefficient. In contrast, viruses are effective vehicles that carry foreign genes into a cell rapidly and efficiently. Here we illustrate the usefulness of adenoviral vectors to express a potent and specific inhibitor of cAMP-dependent protein kinase (PKA) to study the role of cyclic 3',5'-cyclic AMP (cAMP) in the osmotic regulation of the vasopressin gene in a transgenic rat model. The ability to modify endogenous systems within specific cells in a whole animal model allows gene effects to be studied with physiological relevance. The combination of molecular biology and integrative physiology is a powerful application that can aid in the elucidation of how gene function can translate into complex systems in an organism.

Photo-induced inactivation of protein kinase calpha by dequalinium inhibits motility of murine melanoma cells.

Dequalinium (DECA) is a potent antitumor agent and inhibitor of protein kinase C (PKC). Previously it was shown that PKCalpha activity in vitro could be irreversibly inhibited when treated with DECA at low micromolar concentrations and irradiated with 366 nm of light. This approach was used to probe the role of intracellular PKC activity in the motility of metastatic murine melanoma B16 F10 cells and as a target for DECA analogs with increasing PKC inhibitory potencies. Pretreatment of a monolayer of B16 F10 cells with 250 nM of a DECA analog in the presence of UV irradiation for 5 min resulted in 1) complete inhibition of cell motility for up to 4 h in a time-lapse motility assay and 40 to 60% inhibition of cell migration in a Boyden chamber, and 2) inhibition by 40 to 60% of intracellular phosphatidylserine/Ca(2+)-dependent PKC catalytic activity, signifying inactivation of a conventional PKC isoform. Because PKCalpha is the only conventional PKC isoform detected in B16 F10 cells, a stably transfected clone expressing a kinase-defective mutant of PKCalpha was developed that exhibited a substantial loss of adhesion and motility and was refractory to further inhibition by DECA. These findings identify PKCalpha catalytic activity both as a mechanistic component of cell motility and adhesion and as a critical intracellular target of DECA. These studies further suggest that the combined use of UV with nanomolar concentrations of DECA offers an effective chemotherapeutic approach to inhibit metastatic behavior of melanoma cells.

Indolocarbazoles exhibit strong antiviral activity against human cytomegalovirus and are potent inhibitors of the pUL97 protein kinase

We have analyzed a panel of protein kinase inhibitors (PKIs) and found that some indolocarbazoles (Gö6976, K252a, K252c) proved to be highly effective inhibitors of GCV-sensitive and -resistant human cytomegalovirus (HCMV) strains, but did not show any effect against herpes simplex virus. Antiviral activity was determined by focus reduction assays (IC 50 ranging from 0.009 to 0.4 μM). Other inhibitors of serine/threonine kinases (Gö6850, H-7, roscovitine) were found to be ineffective. Virus yield at 5 days after infection was reduced by three orders of magnitude with nanomolar concentrations of the indolocarbazoles. These compounds were fully effective when added up to 24 h post infection and showed reduced activity up to 72 h post infection. Cytotoxicity assays in proliferating and non-proliferating cells demonstrated that the effective antiviral concentration of these compounds was significantly lower than either antiproliferative (IC 50/CC 50 ranging from 6.5 to 390) or cytotoxic (IC 50/CC 50 ranging from 72.5 to 1000) doses. The effects of PKIs on the virus-encoded protein kinase pUL97 were studied using recombinant vaccinia viruses. Indolocarbazoles strongly inhibited both pUL97 autophosphorylation (IC 50 ranging from 0.0012 to 0.013 μM) and pUL97-dependent ganciclovir phosphorylation (IC 50 ranging from 0.05 to 0.26 μM). Other inhibitors of serine/threonine kinases showed only weak (Gö6850) or no (H-7, roscovitine) effect on these pUL97 functions, while oxoflavone tyrosine kinase inhibitors had no effect at all.

A role for amygdaloid PKA and PKC in the acquisition of long-term conditional fear memories in rats

Although there is great interest in the cellular mechanisms underlying Pavlovian conditioning, few studies have directly examined the contribution of intracellular signaling pathways in the amygdala to the acquisition and expression of conditional fear memories. In the present study, we examined this issue by infusing 1-(5′-isoquinolinesulfonyl)-2-methylpiperazine (H7), a potent inhibitor of both protein kinase C (PKC) and cAMP-dependent protein kinase (PKA), directly into the amygdala prior to fear conditioning or retention testing. We found that infusion of H7 prior to training attenuated long-term conditional fear in a dose-dependent manner (Experiment 1), but short-term fear memories were spared. The contribution of protein kinases to conditional fear was region-specific within the amygdala: infusion of H7 into the basolateral amygdala (BLA) but not the central nucleus of the amygdala (CEA) resulted in attenuated freezing (Experiment 2). Moreover, the deficits in fear conditioning produced by PKA/PKC inhibition were not modality-specific, insofar as intra-BLA H7 reduced both contextual and auditory fear. The effects of H7 on conditional freezing were not attributable to either state-dependency or performance deficits (Experiment 3). Together, these experiments suggest that amygdaloid PKA and PKC play an important role in the acquisition of fear memories.

Involvement of G protein-coupled receptor kinase-6 in desensitization of CGRP receptors

This investigation was undertaken to study the mechanisms of calcitonin gene-related peptide (CGRP)-mediated desensitization using recombinant porcine CGRP receptors stably expressed in human embryonic kidney (HEK-293) cells. Pretreatment of these cells with human αCGRP resulted in an ∼60% decrease in CGRP-stimulated adenylyl cyclase activity and an ∼10-fold rightward shift in the dose–response curve of CGRP. This effect was rapid ( t 1/2 ∼5 min) and was accompanied by a significant decrease in [ 125I]CGRP binding to membrane preparations from CGRP-pretreated cells. In contrast, CGRP pretreatment had no effect on isoproterenol- or forskolin-stimulated adenylyl cyclase activity in these cells. The potential involvement of protein kinase A or protein kinase C in CGRP-mediated desensitization was studied using selective inhibitors or activators of these kinases. Pretreatment of the cells with forskolin (adenylyl cyclase activator) or phorbol dibutyrate (protein kinase C activator) had no effect on CGRP-mediated adenylyl cyclase activity and did not influence CGRP-mediated desensitization. However, pretreatment of the cells with 2-(8-[(dimethylamino)methyl]-6,7,8,9-tetrahydropyrido[1,2-a]indol-3-yl]-3-(1-methylindol-3-yl)maleimide hydrochloride (Ro 32-0432) (a potent inhibitor of protein kinase C) resulted in significant attenuation of CGRP-mediated desensitization with an IC 50 ∼3 μM. To establish whether this effect might be due to inhibition of other protein kinases by Ro 32-0432, its effect was tested against several G protein-coupled receptor kinases (GRKs). Ro 32-0432 was found to inhibit GRK2, GRK5, and GRK6 with IC 50 values of 29, 3.6, and 16 μM, respectively, suggesting that its effect on CGRP-mediated desensitization might be a result of GRK inhibition. To further test this hypothesis, as well as the potential GRK specificity, the cells were treated with antisense oligonucleotides to GRK2, GRK5, and GRK6. While GRK2 and GRK5 antisense nucleotides had no effect on CGRP-mediated desensitization, the GRK6 antisense nucleotide treatment significantly reversed CGRP-mediated desensitization. These results suggest the involvement of GRK6 in CGRP-mediated desensitization in HEK-293 cells.

Importance of Protein Kinase C Targeting for the Phosphorylation of Its Substrate, Myristoylated Alanine-rich C-kinase Substrate

We visualized the translocation of myristoylated alanine-rich protein kinase C substrate (MARCKS) in living Chinese hamster ovary-K1 cells using MARCKS tagged to green fluorescent protein (MARCKS-GFP). MARCKS-GFP was rapidly translocated from the plasma membrane to the cytoplasm after the treatment with phorbol ester, which translocates protein kinase C (PKC) to the plasma membrane. In contrast, PKC activation by hydrogen peroxide, which was not accompanied by PKC translocation, did not alter the intracellular localization of MARCKS-GFP. Non-myristoylated mutant of MARCKS-GFP was distributed throughout the cytoplasm, including the nucleoplasm, and was not translocated by phorbol ester or by hydrogen peroxide. Phosphorylation of wild-type MARCKS-GFP was observed in cells treated with phorbol ester but not with hydrogen peroxide, whereas non-myristoylated mutant of MARCKS-GFP was phosphorylated in cells treated with hydrogen peroxide but not with phorbol ester. Phosphorylation of both MARCKS-GFPs reduced the amount of F-actin. These findings revealed that PKC targeting to the plasma membrane is required for the phosphorylation of membrane-associated MARCKS and that a mutant MARCKS existing in the cytoplasm can be phosphorylated by PKC activated in the cytoplasm without translocation but not by PKC targeted to the membrane.

NO-induced modulation of calcium-oscillations in pulmonary vascular smooth muscle

The effect of the nitric oxide (NO) donor sodium nitroprusside (SNP) on both [Ca2+]iand mechanical activity was studied in the rat isolated pulmonary artery (RPA). In freshly isolated myocytes loaded with 1 μM indo-lacetoxymethyl ester for 30 min, short (40–60 s) application of ATP (100 μM) or ET-1 (0.1 μM) induced 3–6 cyclic rises in [Ca2+]i(Ca-oscillations) of decreasing amplitude. Preincubation of cells with SNP (10–250 μM) for 10 min had no effect on the resting [Ca2+]ivalue, but progressively abolished the oscillations. A similar effect was obtained with 8-bromo-cGMP (100–500 μM). SNP (0.001–100 μM) concentration-dependently relaxed ATP (10 mM, n = 4) and ET-1 (0.1 μM, n = 4)-precontracted RPA. 1H-[1,2,4]oxadiazolol [4,3,-a]quinoxalin-1-one (ODQ, 10 μM), a potent inhibitor of the cytosolic guanylyl cyclase, fully reversed the effect of SNP on ATP-induced [Ca2+]ioscillations as well as on ATP-precontracted RPA. In contrast, N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide (H8, 10 μM), a potent inhibitor of cGMP-dependent protein kinase (PKG), did not alter the effect of SNP. Caffeine (5 mM) induced only one transient [Ca2+]i-increase (n = 24), the amplitude of which was altered neither by SNP nor by 8-bromo-cGMP. Our results show that the relaxing effect of NO in RPA is related, at least in part, to its action on the Ca-signalling pathway. NO interacts with inositol trisphosphate pathway without interacting with the ryanodine-sensitive receptor. Finally, the effect of NO involves an increase in cGMP but appears independent of activation of PKG.

Structure-Activity Relationship of Staurosporine Analogs in Regulating Expression of Endothelial Nitric-Oxide Synthase Gene

In human umbilical vein endothelial cells and in human umbilical vein endothelial cell-derived EA.hy 926 cells, staurosporine (Stsp) and its glycosidic indolocarbazole analogs 7-hydroxystaurosporine (UCN-01) and 4'-N-benzoyl staurosporine (CGP 41251) enhanced nitric-oxide synthase (NOS) III mRNA expression (analyzed by RNase protection assay), protein expression (determined by Western blot), and activity [measured by rat fetal lung fibroblast (RFL-6) reporter cell assay] in a concentration- and time-dependent manner. In contrast, the bisindolylmaleimide analogs GF 109203X, Ro 31-8220 and Gö 6983 had no effect on NOS III expression, and Gö 6976, a methyl- and cyanoalkyl-substituted nonglycosidic indolocarbazole derivative of Stsp, even reduced NOS III expression in a concentration-dependent fashion. The up-regulation of NOS III expression by Stsp and analogs appears to be a transcriptional event because Stsp, 7-hydroxystaurosporine, and CGP 41251 enhanced the activity of a 1.6-kb human NOS III promoter fragment transiently transfected into EA.hy 926 endothelial cells. Stsp and analogs did not affect the stability of the NOS III mRNA. Stsp is known as a potent protein kinase (PK) inhibitor. Data obtained with other kinase inhibitors (and stimulators) indicated, however, that the effect of Stsp and analogs on NOS III expression was unrelated to the activities of PKC, PKA, PKG, or tyrosine kinase(s). Stsp analogs such as CGP 41251 also counteracted the NOS III mRNA-decreasing effect of tumor necrosis factor-alpha. These findings demonstrate that Stsp analogs represent a new class of compounds positively interacting with the transcription of the endothelial NOS III gene. Such compounds may prove useful in the prophylaxis and therapy of vascular disease.

Cell membrane changes of structure and function in protein kinase inhibitor-induced polyploid cells.

Exogenous cyclic AMP has been thought to be a chemical without marked pharmacological effect until now, as it is not capable of penetrating the cell membrane in most eucaryotic cells. The present study obtained results consistent with those of most previous studies, showing that exogenous cyclic AMP itself did not interfere with the cell cycle even at the high dose of 100 microM. However, it was found that K252a, a potent inhibitor of protein kinases including protein kinase C, induced DNA re-replication, i.e. DNA synthesis at a elevated DNA ploidy in cells that had not undergone cytokinesis (leading to polyploidization), and that exogenous cyclic AMP markedly potentiated the K252a-induced polyploidization at a very low dose similar to the effective dose of membrane-permeable cyclic AMP analogue dibutyryl cyclic AMP. These findings suggested that the cell membrane changed during the formation of polyploid cells. This supposition was confirmed by scanning electron microscopy to observe structural changes and by determination of cellular attachment to investigate functional changes.

Antidiuretic effects of a factor in brain/corpora cardiaca/corpora allata extract on fluid reabsorption across the cryptonephric complex of Manduca sexta.

Extracts of the brain/corpora cardiaca/corpora allata (Br/CC/CA) complex of Manduca sexta larvae elicit an antidiuretic effect, measured by an increase in fluid reabsorption across the cryptonephric complex of larval M. sexta. Separation of the extract by reversed-phase liquid chromatography gave two fractions with antidiuretic effects. The more potent of these two factors was further characterized for its effects on the cryptonephric complex. Its antidiuretic effect is not inhibited by bumetanide, a drug that inhibits M. sexta diuretic hormone (Mas-DH)-stimulated fluid reabsorption. These data indicate that the mechanism of the antidiuretic effect of the factor is different from that of Mas-DH on the cryptonephric complex. The basal reabsorption of the cryptonephric complex is blocked when treated on the lumen side with bafilomycin A(1), an inhibitor of the H(+)-ATPase, or with amiloride, an inhibitor of the H(+)/K(+) antiporter. However, the antidiuretic-factor-stimulated fluid reabsorption is not affected by either bafilomycin A(1) or amiloride. The increase in reabsorption triggered by the semi-purified factor can be inhibited by Cl(-) channel blockers or by removing Cl(-) from the lumen side of the cryptonephric complex. It appears that this factor activates a Cl(-) pump associated with the cryptonephric complex. Forskolin mimics the effect of this factor on fluid reabsorption, and the effect of forskolin is not inhibited by bumetanide. A selective and potent inhibitor of protein kinase A, H-89, also inhibits antidiuretic-factor-stimulated fluid reabsorption. Addition of the factor to cryptonephric complexes maintained in vitro caused a significant increase in cyclic AMP levels extracted from these tissues compared with values for controls. These data suggest that the antidiuretic effect of the factor in Br/CC/CA extract is mediated by cyclic AMP.

Recent studies on the biofunctions and biotransformations of curcumin.

Curcumin is a major component of Curcuma species, which is commonly used as a yellow coloring and flavoring agent in foods. Curcumin has shown anti-carcinogenic activity in animals as indicated by its ability to block colon tumor initiation by azoxymethane and skin tumor promotion induced by phorbol ester TPA. Curcumin possesses anti-inflammatory activity and is a potent inhibitor of reactive oxygen-generating enzymes such as lipoxygenase/cyclooxygenase, xanthine dehydrogenase/oxidase and inducible nitric oxide synthase. Curcumin is also a potent inhibitor of protein kinase C, EGF-receptor tyrosine kinase and IkappaB kinase. Subsequently, curcumin inhibits the activation of NFkappaB and the expressions of c-jun, c-fos, c-myc and iNOS. It is proposed that curcumin may suppress tumor promotion through blocking signal transduction pathways in the target cells. Curcumin was first biotransformed to dihydrocurcumin and tetrahydrocurcumin and that these compounds subsequently were converted to monoglucuronide conjugates. These results suggest that curcumin-glucuronide, dihydro-curcumin-glucuronide, tetrahydrocurcumin-glucuronide and tetrahydrocurcumin are major metabolites of curcumin in mice.

REGULATION OF THE RENAL BASOLATERAL TRANSPORT SYSTEM FOR ORGANIC ANIONS BY CALCIUM CHANNEL BLOCKERS

Calcium channel blockers have been reported to exert multiple effects on renal tubular function. Therefore, the effects of the calcium channel blockers nifedipine and verapamil on the cellular uptake of tritiated para -aminohippuric acid (PAH) into microdissected non-perfused rabbit kidney S2 proximal tubule segments were investigated to study a possible influence of calcium channel blockers on renal PAH transport. Since the tubules were entirely collapsed, the accumulated radioactivity overwhelmingly reflects the transport across the basolateral membrane. Tubular PAH accumulation was increased by 1 and 10 μm verapamil, and by 1 μm nifedipine, but it was unaffected by lower or higher concentrations of these calcium channel blockers. The increase in PAH accumulation caused by 1 μm nifedipine or 10 μm verapamil was independent from changes in intracellular Ca2+and inhibited by staurosporine (1 and 10 n m) a potent inhibitor of protein kinase C (PKC). Our results indicate that the calcium channel blockers nifedipine and verapamil increase the transport of organic anions across the basolateral membrane of proximal tubules, probably by an activation of PKC. Furthermore, the increased tubular PAH transport may disturb the estimation of the renal PAH-clearance. 2000 Academic Press@p$hr