Toshie Kambe

Nitric oxide-mediated modulation of calcium/calmodulin-dependent protein kinase II.

The mechanisms of NO inhibition of CaMK [Ca(2+)/CaM (calmodulin)-dependent protein kinase] II activity were studied. In rat pituitary tumour GH3 cells, TRH [thyrotrophin (TSH)-releasing hormone]-stimulated phosphorylation of nNOS [neuronal NOS (NO synthase)] at Ser(847) was sensitive to an inhibitor of CaMKs, KN-93, and was enhanced by inhibition of nNOS with 7NI (7-nitroindazole). Enzyme activity of CaMKII following in situ treatment with 7NI was also increased. The in vitro activity of CaMKII was inhibited by co-incubation either with nNOS and L-arginine or with NO donors SNAP (S-nitroso-N-acetyl-DL-penicillamine) and DEA-NONOate [diethylamine-NONOate (diazeniumdiolate)]. Once inhibited by these treatments, CaMKII was observed to undergo full reactivation on the addition of a reducing reagent, DTT (dithiothreitol). In transfected cells expressing CaMKII and nNOS, treatment with the calcium ionophore A23187 further revealed nNOS phosphorylation at Ser(847), which was enhanced by 7NI and CaMKII S-nitrosylation. Mutated CaMKII (C6A), in which Cys(6) was substituted with an alanine residue, was refractory to 7NI-induced enhancement of nNOS phosphorylation or to CaMKII S-nitrosylation. Furthermore, we could identify Cys(6) as a direct target for S-nitrosylation of CaMKII using MS. In addition, treatment with glutamate caused an increase in CaMKII S-nitrosylation in rat hippocampal slices. This glutamate-induced S-nitrosylation was blocked by 7NI. These results suggest that inactivation of CaMKII mediated by S-nitrosylation at Cys(6) may contribute to NO-induced neurotoxicity in the brain.

Sp1 decoy oligodeoxynucleotide decreases angiotensin receptor expression and blood pressure in spontaneously hypertensive rats

The transcriptional factor Sp1 is associated with GC-rich promoters and involved in basal promoter activity. A GC-box-related sequence is located within the -58 to -34 base pair region of the angiotensin type 1 receptor gene promoter. We examined whether Sp1 in the hypothalamus was increased in spontaneously hypertensive rats (SHR) and whether inhibition of Sp1 binding sites suppressed angiotensin type 1 receptor expression and thus decreased blood pressure in SHR. Western blot analysis showed that Sp1 protein levels were increased in nuclear extracts of hypothalamus from SHR. Electrophoretic mobility shift assay (EMSA) using oligonucleotides containing Sp1 consensus sequence and -58 to -34 region sequence oligonucleotides showed that DNA-protein complexes were greater in nuclear extracts of hypothalamus from SHR than those of Wistar Kyoto rats (WKY). Sp1 decoy phosphorothioate oligodeoxynucleotides injected into the lateral ventricle produced a decrease in blood pressure in SHR, and decreased angiotensin type 1 receptor mRNA levels and number of angiotensin receptors in the hypothalamus of SHR. Pressor responses to angiotensin II but not to carbachol injected into the lateral ventricle were decreased in the Sp1 decoy-treated SHR. The results of the present study suggest that Sp1 levels in the hypothalamus of SHR are increased, and that inhibition of the binding of Sp1 to its binding sites decreases angiotensin type 1 receptor expression and blood pressure in SHR. The possibility cannot be ruled out that the Sp1 decoy oligodeoxynucleotides (ODN) also suppressed transcriptions of genes other than the angiotensin type 1 receptor gene.

Renin antisense injected intraventricularly decreases blood pressure in spontaneously hypertensive rats

Brain renin-angiotensin system plays an important role in blood pressure regulation and is suggested to play a role in the development and maintenance of hypertension. To test the hypothesis that brain renin may play a significant role in hypertension in spontaneously hypertensive rats (SHR), phosphorothioated antisense oligodeoxynucleotides targeted to renin mRNA were administered intracerebroventricularly in SHR. Administration of an antisense but not its sense oligodeoxynucleotide produced a prolonged duration of decrease in blood pressure. Intra-arterial administration of the antisense oligodeoxynucleotide at the same dose that decreased blood pressure when administered intraventricularly did not affect blood pressure. Furthermore, renin mRNA but not angiotensin AT1 receptor mRNA levels were decreased in the hypothalamus of the antisense oligodeoxynucleotide-treated rats. These results suggest that brain renin may play a significant role in hypertension in SHR.

Evidence that angiotensin II, endothelins and nitric oxide regulate mitogen-activated protein kinase activity in rat aorta

We measured the activity of mitogen-activated protein (MAP) kinases, enzymes believed to be involved in the pathway for cell proliferation, in rat aortic strips with or without endothelium, and examined effects of angiotensin receptor antagonists, endothelin receptor antagonists and nitric oxide (NO)-related agents. Endothelium removal produced an activation of MAP kinase activity in the strips, whereas the enzyme activity was not affected in the adventitia. The MAP kinase activation was inhibited by either the angiotensin AT1 receptor antagonist losartan or the endothelin ETA receptor antagonist BQ 123. The combination of both antagonists caused an additive inhibition. The angiotensin AT2 receptor antagonist PD 123,319 and the endothelin ETB receptor antagonist BQ 788 did not affect the MAP kinase activation. The NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) caused an activation of MAP kinase in the endothelium-intact aorta and the MAP kinase activation was inhibited by losartan or BQ123. The NO releaser nitroprusside inhibited the MAP kinase activation induced by endothelium removal or angiotensin II. These results suggest that even in isolated arteries, NO of endothelial origin tonically exert MAP kinase-inhibiting effects and endogenous angiotensin II and endothelins in the media are tonically released to cause MAP kinase-stimulating effects in medial smooth muscle.

Vascular mitogen-activated protein kinase activity is enhanced via angiotensin system in spontaneously hypertensive rats.

The vascular structural remodeling function may be altered in genetically hypertensive animals, spontaneously hypertensive rats (SHR). To examine this possibility, we measured the activity of mitogen-activated protein (MAP) kinases, enzymes believed to be involved in the pathway for cell proliferation, in rat aorta strips, and examined whether the endothelium removal-induced MAP kinase activation function is altered in SHR and whether vascular angiotensin and endothelin systems are responsible for the alteration of MAP kinase activation in SHR. Male 4-week-old SHR and age-matched Wistar Kyoto rats (WKY) supplied by Charles River Japan were used. Endothelium-denuded aorta strips were incubated at 37 degrees C in medium. MAP kinase activity after incubation was time-dependently increased in strips from SHR and WKY. MAP kinase activation was greater in SHR than in WKY aorta strips. Similarly, MAP kinase activation was enhanced in aorta strips from 4-week-old SHR and stroke prone SHR supplied by the Diseases Model Cooperative Research Association (Kyoto, Japan). In aorta strips from SHR and WKY, the angiotensin receptor antagonist, losartan, and the endothelin receptor antagonist, cyclo (D-alpha-aspartyl-L-prolyl-D-valyl-L-leucyl-D-tryptophyl)(BQ123), caused concentration-dependent inhibition of MAP kinase activation. The losartan-induced but not BQ123-induced inhibition of MAP kinase activation was greater in SHR than in WKY aorta strips. Angiotensin II caused a concentration-dependent increase in MAP kinase activity and the angiotensin II-induced MAP kinase activation was greater in SHR than in WKY aorta strips. These results indicate that endothelium removal-induced MAP kinase activation is enhanced in aorta strips from young SHR, suggesting that vascular structural remodeling function may be enhanced in SHR. It appears that the enhancement of MAP kinase activation results, at least in part, from enhanced function of vascular angiotensin system in SHR.

Inactivation of Ca2+/calmodulin-dependent protein kinase I by S-glutathionylation of the active-site cysteine residue

We show that Ca2+/calmodulin(CaM)‐dependent protein kinase I (CaMKI) is directly inhibited by its S‐glutathionylation at the Cys179. In vitro studies demonstrated that treatment of CaMKI with diamide and glutathione results in inactivation of the enzyme, with a concomitant S‐glutathionylation of CaMKI at Cys179 detected by mass spectrometry. Mutagenesis studies confirmed that S‐glutathionylation of Cys179 is both necessary and sufficient for the inhibition of CaMKI by diamide and glutathione. In transfected cells expressing CaMKI, treatment with diamide caused a reversible decrease in CaMKI activity. Cells expressing mutant CaMKI (179CV) proved resistant in this regard. Thus, our results indicate that the reversible regulation of CaMKI via its modification at Cys179 is an important mechanism in processing calcium signal transduction in cells.

Mitogen-activated protein kinase activity regulation role of angiotensin and endothelin systems in vascular smooth muscle cells

To examine whether angiotensin II and endothelins produced in vascular smooth muscle cells can play roles in the regulation of mitogen-activated protein (MAP) kinase activity in vascular smooth muscle cells, we measured the activity of MAP kinases in cultured vascular smooth muscle cells, and determined effects of renin-angiotensin and endothelin systems activators and inhibitors. Angiotensin II and endothelin-1 produced an activation of MAP kinase activity in vascular smooth muscle cells, whereas the angiotensin receptor antagonist, losartan and the endothelin receptor antagonist, cyclo (D-alpha-aspartyl-L-prolyl-D-valyl-L-leucyl-D-tryptophyl, BQ123) inhibited the enzyme activity. MAP kinase activity in vascular smooth muscle cells was also inhibited either by the renin inhibitor pepstatin A or by the angiotensin-converting enzyme inhibitor captopril. The degree of the inhibition of MAP kinase activity by pepstatin A, captopril and losartan was almost the same. Renin produced a considerable increase in MAP kinase activity and the renin-induced MAP kinase activation was inhibited by pepstatin A. The endothelin precursor big endothelin-1 produced an increase of MAP kinase activity in vascular smooth muscle cells, whereas the endothelin-converting enzyme inhibitor phosphoramidon inhibited the enzyme activity. These findings suggest that functional renin-angiotensin system and endothelin system are present in vascular smooth muscle cells and these systems tonically serve to increase MAP kinase activity. It appears that renin or renin-like substances play the determining role in the regulation of renin-angiotensin system in vascular smooth muscle cells.

Different activation of vascular mitogen-activated protein kinases in spontaneously and DOCA-salt hypertensive rats

Regulation mechanisms of the activity of vascular mitogen-activated protein (MAP) kinases, enzymes believed to be involved in the pathway for cell proliferation, may be altered in hypertension. To examine whether vascular MAP kinase activation mechanisms are altered in hypertension, we measured the activity of MAP kinases in rat aorta strips from spontaneously hypertensive rats (SHR) and from deoxycorticosterone acetate (DOCA)-salt hypertensive rats, and examined whether vascular angiotensin and endothelin systems are responsible for the alteration of MAP kinase activation in these hypertensive models. Endothelium-denuded aorta strips were incubated at 37 degrees C in medium. MAP kinase activity after incubation was increased in rat aorta strips. The MAP kinase activation was greater in 9- and 15-week-old SHR aorta strips than in age-matched Wistar Kyoto rats (WKY) aorta strips. Similarly, MAP kinase activation was enhanced in aorta strips from DOCA-salt hypertensive rats. In aorta strips from these kinds of rats, the angiotensin receptor antagonist, losartan, and the endothelin receptor antagonist, cyclo (D-alpha-aspartyl-L-prolyl-D-valyl-L-leucyl-D-tryptophyl) (BQ123), inhibited the MAP kinase activation. The losartan-induced, but not BQ123-induced, inhibition of MAP kinase activation was enhanced in 15-week-old SHR aorta strips, whereas the BQ123-induced, but not losartan-induced, inhibition of MAP kinase activation was enhanced in DOCA-salt hypertensive rat aorta strips. Angiotensin II-induced MAP kinase activation was enhanced in 15-week-old SHR aorta strips, whereas it was depressed in DOCA-salt hypertensive rat aorta strips. These results indicate that MAP kinase activation function is enhanced in aorta strips from both kinds of hypertensive rats. It appears that the enhancement of MAP kinase activation results partly from enhanced vascular angiotensin system in SHR and from enhanced vascular endothelin system in DOCA-salt hypertensive rats.

Transient receptor potential ankyrin 1 that is induced in dorsal root ganglion neurons contributes to acute cold hypersensitivity after oxaliplatin administration

BACKGROUND Peripheral cold neuropathic pain is a serious side effect of oxaliplatin treatment. However, the mechanism of oxaliplatin-induced cold hyperalgesia is unknown. In the present study, we investigated the effects of oxaliplatin on transient receptor potential ankyrin 1 (TRPA1) in dorsal root ganglion (DRG) neurons of rats. RESULTS Behavioral assessment using the acetone spray test showed that 3 and 6 mg/kg oxaliplatin (i.p.) induced acute cold hypersensitivity after 1, 2, 4, and 7 days. Real-time PCR showed that oxaliplatin (6 mg/kg) significantly increased TRPA1 mRNA expression in DRGs at days 1, 2, and 4. Western blotting revealed that oxaliplatin significantly increased TRPA1 protein expression in DRGs at days 2, 4, and 7. Moreover, in situ hybridization histochemistry revealed that most TRPA1 mRNA-labeled neurons in the DRGs were small in size. Oxaliplatin significantly increased co-localization of TRPA1 expression and isolectin B4 binding in DRG neurons. Oxaliplatin induced a significant increase in the percent of TRPA1 mRNA-positive small neurons in DRGs at days 1, 2, and 4. In addition, we found that intrathecal administration of TRPA1 antisense, but not TRPA1 mismatched oligodeoxynucleotides, knocked down TRPA1 expression and decreased oxaliplatin-induced cold hyperalgesia. Double labeling showed that p-p38 mitogen-activated protein kinase (MAPK) was co-expressed in TRPA1 mRNA-labeled neurons at day 2 after oxaliplatin administration. Intrathecal administration of the p38 MAPK inhibitor, SB203580, significantly decreased oxaliplatin-induced acute cold hypersensitivity. CONCLUSIONS Together, these results demonstrate that TRPA1 expression via activation of p38 MAPK in DRG neurons, at least in part, contributes to the development of oxaliplatin-induced acute cold hyperalgesia.

p90 RSK-1 associates with and inhibits neuronal nitric oxide synthase

Evidence is presented that RSK1 (ribosomal S6 kinase 1), a downstream target of MAPK (mitogen-activated protein kinase), directly phosphorylates nNOS (neuronal nitric oxide synthase) on Ser847 in response to mitogens. The phosphorylation thus increases greatly following EGF (epidermal growth factor) treatment of rat pituitary tumour GH3 cells and is reduced by exposure to the MEK (MAPK/extracellular-signal-regulated kinase kinase) inhibitor PD98059. Furthermore, it is significantly enhanced by expression of wild-type RSK1 and antagonized by kinase-inactive RSK1 or specific reduction of endogenous RSK1. EGF treatment of HEK-293 (human embryonic kidney) cells, expressing RSK1 and nNOS, led to inhibition of NOS enzyme activity, associated with an increase in phosphorylation of nNOS at Ser847, as is also the case in an in vitro assay. In addition, these phenomena were significantly blocked by treatment with the RSK inhibitor Ro31-8220. Cells expressing mutant nNOS (S847A) proved resistant to phosphorylation and decrease of NOS activity. Within minutes of adding EGF to transfected cells, RSK1 associated with nNOS and subsequently dissociated following more prolonged agonist stimulation. EGF-induced formation of the nNOS-RSK1 complex was significantly decreased by PD98059 treatment. Treatment with EGF further revealed phosphorylation of nNOS on Ser847 in rat hippocampal neurons and cerebellar granule cells. This EGF-induced phosphorylation was partially blocked by PD98059 and Ro31-8220. Together, these data provide substantial evidence that RSK1 associates with and phosphorylates nNOS on Ser847 following mitogen stimulation and suggest a novel role for RSK1 in the regulation of nitric oxide function in brain.