Involvement of inhibition of RhoA/Rho kinase signaling in simvastatin-induced amelioration of neuropathic pain.

Small molecular G-protein plays a key role in several diseases. This study was designed to reveal the role of RhoA signaling in the pathophysiology of neuropathic pain in mice. Partial sciatic nerve injury caused thermal hyperalgesia, mechanical allodynia, and increased plasma membrane translocation of RhoA in the lumber spinal cord. GFAP-immunoreactivity (ir), Iba-1-ir, and Rho kinase 2 (ROCK2-ir) was also increased in the ipsilateral spinal dorsal horn of nerve-ligated mice. Moreover, partial nerve ligation increased the expression of phosphorylated myristoylated alanine-rich protein kinase C substrate (MARCKS)-ir in the ipsilateral spinal dorsal horn. Daily intrathecal administration of simvastatin, beginning 3days before nerve injury, completely blocked all these changes in nerve-ligated mice. Pharmacological inhibition of ROCK also attenuated the increased expression of GFAP-ir and phosphorylated MARCKS-ir. Together, it is suggested that astrogliosis initiated by the activation of RhoA/ROCK signaling results in MARCKS phosphorylation in nerve terminals, which leads to hyperalgesia in neuropathic pain. Furthermore, simvastatin exerts antihyperalgesic and antiallodynic effects through the inhibition of spinal RhoA activation.

Angiotensin II, as well as 5-hydroxytriptamine, is a potent vasospasm inducer of saphenous vein graft for coronary artery bypass grafting in patients with diabetes mellitus.

Diabetes mellitus (DM) is an important risk factor for adverse outcomes of coronary artery bypass grafting. The bypass grafts harvested from patients with DM tend to go into spasm after their implantation into the coronary circulation. To clarify the contribution of 5-hydroxytriptamine (5-HT) and angiotensin II (AngII) in the bypass graft spasm, we examined the contractile reactivity to 5-HT or AngII of isolated human endothelium-denuded saphenous vein (SV) harvested from DM and non-DM patients. The 5-HT-induced constriction of the SV was significantly augmented in the DM group than in the non-DM group, which is similar to our previous report. AngII-induced constriction of the SV was also significantly augmented in the DM group than the non-DM group. Especially in the non-DM group, the AngII-induced maximal vasoconstriction was markedly lower than the 5-HT-induced one. Meanwhile, the increasing rates of AngII-induced vasoconstriction in the DM group to the non-DM group were significantly greater than those of 5-HT-induced vasoconstriction. These results indicate that 5-HT is a potent inducer of SV graft spasm in both DM and non-DM patients, while AngII is a potent inducer of SV graft spasm only in patients with DM. Furthermore, the protein level of AngII AT1 receptor (AT1R), but not the protein level of 5-HT2A receptor, in the membrane fraction of the SV smooth muscle cells of DM patients was significantly increased as compared with that of the non-DM patients. These results suggest that the mechanism for hyperreactivity to AngII in the SV from DM patients is due to, at least in part, the increase in the amount of AT1R on membrane of the SV smooth muscle cells.

Involvement of renal sympathetic nerve activation on the progression of ischemic acute kidney injury in the mouse.

Renal ischemia produces renal sympathoexcitation that is responsible for the development of ischemic acute kidney injury. The present study examined changes in the sympathetic nerve function in mice. Ischemic acute kidney injury was induced by occlusion of both renal pedicles. Renal ischemia/reperfusion increased blood urea nitrogen and plasma creatinine and expression of tyrosine hydroxylase, a rate-limiting enzyme for the biosynthesis of noradrenaline, in the kidney. Renal immunoreactivity of tyrosine hydroxylase was observed along with vessel and tubular structure both in the sham-operated and the ischemic acute kidney injury mice. The prominent morphological change was that tyrosine hydroxylase immunoreactivity was observed in the glomeruli of the ischemic acute kidney injury mice, whereas there are almost no tyrosine hydroxylase immunoreactivity signals in the glomeruli of the sham-operated mice. This tyrosine hydroxylase immunoreactivity in the glomeruli is colocalized with synapsin I immunoreactivity in the ischemic acute kidney injury mice. Intraperitoneal pretreatment with DSP-4 (50 mg/kg) attenuated these changes induced by renal ischemia/reperfusion. These results suggest that morphological and functional changes of glomerulus adrenergic nerve terminal are involved in the pathophysiology of ischemia/reperfusion-induced ischemic acute kidney injury.

Involvement of protein isoprenylation in neuropathic pain induced by sciatic nerve injury in mice.

Isoprenylation is crucial step for activating many intracellular signaling. The present study examined whether inhibition of the protein isoprenylation could affect neuropathic pain in partial sciatic nerve-ligated mice. Intrathecal treatment with a geranylgeranyl transferase I inhibitor GGTI-2133, but not with a farnesyl transferase inhibitor FTI-277, dose-dependently blocked the thermal hyperalgesia in partial sciatic nerve-ligated mice. Intrathecal treatment with GGTI-2133 also attenuated the mechanical allodynia in partial sciatic nerve-ligated mice. Phosphorylated MARCKS expression was increased in the ipsilateral side of the spinal cord dorsal horn in partial sciatic nerve-ligated mice, and this increase was attenuated by GGTI-2133 but not by FTI-277. These results suggest that protein isoprenylation by geranylgeranyl transferase I is involved in the neuropathic pain.

5-hydroxytryptamine receptors as targets for drug therapies of vascular-related diseases.

5-hydroxytryptamine (5-HT) is involved in regulation of both physiological and pathophysiological conditions in tissues throughout the body. 5-HT induces vascular smooth muscle constriction in most vessels. The vasoconstrictive effects of 5-HT are mediated by 5-HT1B and 5-HT2A receptors located on the membrane of smooth muscle cells, except in the intracranial arteries which constrict only through 5-HT1B receptors. 5-HT also acts as vasodilator because it releases nitric oxide from endothelial cells. This response is dominantly mediated by 5-HT1B receptors but not by 5-HT2A receptors. In this review, we focus on the action of 5-HT via G protein-coupled 5-HT receptors involved in some vascular-related pathophysiological responses. Furthermore, we describe the possibilities of 5-HT receptors as targets for drug therapy against saphenous vein grafts diseases (especially in patients with diabetes mellitus), migraine and pulmonary arterial hypertension.

Effect of naloxone on ischemic acute kidney injury in the mouse.

Renal ischemia produces sympathoexcitation, which is responsible for the development of ischemic acute kidney injury. Stimulation of central opioid receptors activates the renal sympathetic nerve. The present study examined the effect of an opioid receptor antagonist naloxone on the ischemia/reperfusion-induced renal dysfunction in mice. Blood urea nitrogen (BUN) and plasma creatinine increased 24 h after the renal ischemia/reperfusion. Intraperitoneal or intracerebroventricular, but not intrathecal, pretreatment with naloxone suppressed the renal ischemia/reperfusion-induced increases in BUN and plasma creatinine. This effect of naloxone was reversed by subcutaneous pretreatment with morphine. Selective MOP receptor antagonist ß-funaltrexamine (FNA) also suppressed the renal ischemia/reperfusion-induced increases in BUN and plasma creatinine. Moreover, tyrosine hydroxylase expression in the renal tissue increased 24 h after renal ischemia/reperfusion, which was abolished by intraperitoneal or intracerebroventricular pretreatment with naloxone and FNA. Immunohistochemical experiments revealed a significant increase in the number of the Fos family proteins (c-Fos, FosB, Fra-1, and Fra-2) positive cells in the paraventricular nucleus of hypothalamus and supraoptic nucleus 24 h after the renal ischemia/reperfusion. Intracerebroventricular pretreatment with naloxone attenuated the renal ischemia/reperfusion-induced increase in the number of the Fos family proteins positive cells in these areas. Finally, we observed that i.c.v. pretreatment with antiserum against ß-endorphin also suppressed the increased blood urea and plasma creatinine. These results suggest that the blockade of central opioid receptors can attenuate the ischemic acute kidney injury through the inhibition of renal sympathoexcitation. The central opioid receptors may thus be a new target for the treatment of ischemic organ failures.

Effect of spinally administered simvastatin on the formalin-induced nociceptive response in mice.

Clinical and experimental observations indicated that 3-hydroxy-3-methylglutaryl CoA reductase inhibitor statins have pleiotropic effects. The present study determined the antinociceptive property of centrally administered simvastatin on the formalin-induced nociception in the mouse. Intrathecal administration of simvastatin at doses of 0.5 - 50 nmol dose-dependently attenuated the second, but not the first, phase of the formalin-induced nociception, which was partially reversed by mevalonate (5 µmol). Intracerebroventricular injection of simvastatin (50 nmol) did not affect the formalin-induced nociception. These results suggest that simvastatin-induced antinociception is mediated by attenuation of the sensitization of spinal nociceptive transmission.

Carnosine has antinociceptive properties in the inflammation-induced nociceptive response in mice.

Carnosine is a biologically active dipeptide that is found in fish and chicken muscle. Recent studies have revealed that carnosine has neuroprotective activity in zinc-induced neural cell apoptosis and ischemic stroke. In the present study, we examined the expression of carnosine in the spinal cord, and the antinociceptive potency of carnosine in a mouse model of inflammation-induced nociceptive pain. Immunohistochemical studies with antiserum against carnosine showed an abundance of carnosine-immunoreactivity in the dorsal horn of the mouse spinal cord. Double-immunostaining techniques revealed that carnosine was expressed in the neurons and astrocytes in the spinal cord. Oral administration of carnosine attenuated the number of writhing behaviors induced by the intraperitoneal administration of 0.6% acetic acid. Treatment with carnosine also attenuated the second phase, but not the first phase, of the nociceptive response to formalin. Moreover, intrathecal, but not intraplanter, administration of carnosine attenuated the second phase of the nociceptive response to formalin. Our immunohistochemical and behavioral data suggest that carnosine has antinociceptive effects toward inflammatory pain, which may be mediated by the attenuation of nociceptive sensitization in the spinal cord.

Insulin induces internalization of the plasma membrane 5-hydroxytryptamine2A (5-HT2A) receptor in the isolated human endothelium-denuded saphenous vein via the phosphatidylinositol 3-kinase pathway.

The aim of this study was to investigate the relaxant effect of insulin on the 5-hydroxytryptamine (5-HT)-induced constriction of the human endothelium-denuded saphenous vein (SV) and its signal transduction pathway. During the 5-HT-induced sustained constriction of vessels, insulin induced vasorelaxation in a concentration-dependent manner. This insulin-induced vasorelaxation was partially attenuated by L-NAME, a nitric oxide synthase (NOS) inhibitor, and was abolished by wortmannin, a phosphatidylinositol 3-kinase (PI3-K) inhibitor. Insulin increased the Ser(473) phosphorylation of Akt. Endothelial NOS and inducible NOS protein expressions were observed in SV smooth muscle when insulin induced relaxation of SV vessels preconstricted with 5-HT. Although insulin did not affect the total protein level of 5-HT(2A) receptors, it decreased the particulate protein level and reciprocally increased the soluble protein level of 5-HT(2A) receptors in a concentration-dependent manner. These results demonstrate that insulin can induce the internalization of 5-HT(2A) receptors from the plasma membrane to the cytoplasm. The insulin-induced internalization of 5-HT(2A) receptors was abolished by wortmannin but was not affected by L-NAME. These results suggest that the relaxant effect of insulin on 5-HT-induced vasoconstriction is mediated in part by the internalization of plasma membrane 5-HT(2A) receptors and the production of nitric oxide via the PI3-K/Akt pathway.

The defective protein level of myosin light chain phosphatase (MLCP) in the isolated saphenous vein, as a vascular conduit in coronary artery bypass grafting (CABG), harvested from patients with diabetes mellitus (DM).

We examined the contractile reactivity to 5-hydroxytryptamine (5-HT) in isolated human saphenous vein (SV), as a vascular conduit in coronary artery bypass grafting (CABG), harvested from patients with diabetes mellitus (DM) and non-DM (NDM). Vascular rings of endothelium-denuded SV were used for functional and biochemical experiments. The vasoconstrictions caused by 5-HT were significantly greater (hyperreactivity) in the DM group than in the NDM group. RhoA/ROCK pathway is activated by various G-protein-coupled receptor agonists and consequently induces phosphorylation of myosin phosphatase target subunit 1 (MYPT1), a subunit of myosin light chain phosphatase (MLCP), which inhibits MLCP activity. In the resting state of the vessels, total tissue protein levels of 5-HT(2A) receptor, 5-HT(1B) receptor, RhoA, ROCK1, and ROCK2 did not differ between NDM and DM groups. However, the total protein level of MYPT1 was significantly lower in the DM group than in the NDM group. Furthermore, the ratio of P(Thr(696))-MYPT1 to total MYPT1 was significantly higher in the DM group than in the NDM group. These results suggest that the hyperreactivity to 5-HT in the SV smooth muscle of patients with DM is due to not only enhanced phosphorylation of MLCP but also defective protein level of MLCP. Thus, we reveal for the first time that the defective protein level of MLCP in the DM group can partially explain the poor patency of SV graft harvested from patients with DM.

Relative contributions of 5-hydroxytryptamine (5-HT) receptor subtypes in 5-HT-induced vasoconstriction of the distended human saphenous vein as a coronary artery bypass graft.

It is established that the segment of saphenous vein (SV) that is widely used as a conduit vessel in coronary artery bypass graft (CABG) surgery is distended with high pressure to check for leaks and to increase the patency before implantation into coronary arterial circulation. The aim of the present study was to elucidate the relative contributions of 5-hydroxytryptamine (5-HT) receptor subtypes responsible for 5-HT-induced vasoconstriction of the distended human SV. Whereas about half of the 5-HT-induced vasoconstriction still remained in the presence of supramaximum concentration of sarpogrelate or of SB224289 (5-HT(2A) and 5-HT(1B) receptor antagonists, respectively), simultaneous treatment with sarpogrelate and SB224289 almost completely inhibited the 5-HT-induced vasoconstriction. Immunopositive staining for 5-HT(2A) and 5-HT(1B) receptors was detected in smooth muscle cells of the distended human SV and there was no significant difference between the immunopositive areas of 5-HT(2A) and 5-HT(1B) receptors. These results demonstrate that 5-HT(2A) and 5-HT(1B) receptors similarly contribute to 5-HT-induced vasoconstriction in human distended SV. Thus, when the SV is used as a CABG conduit, a combination of 5-HT(2A) and 5-HT(1B) receptor antagonists would appear to be most useful to prevent 5-HT-induced spasm.

Rho-kinase, but not protein kinase C, is involved in generation of the spontaneous tone in the resting phase of the isolated pig iris sphincter muscle.

PURPOSE: The purpose of the present study was to clarify the role of Rho-kinase and/or protein kinase C in the resting tension of the isolated pig iris sphincter muscle. MATERIALS AND METHODS: The motor activity of the isolated pig iris sphincter muscle was measured isometrically. RESULTS: EGTA, a chelator of extracellular Ca(2+), significantly reduced the resting tension. Y27632, a Rho-kinase inhibitor, significantly reduced the resting tension in a concentration-dependent manner. The resting tension diminished by Y27632 was significantly recovered by the addition of calyculin A, a myosin light chain phosphatase (MLCP) inhibitor, in a concentration-dependent manner. GF109203X, a protein kinase C inhibitor, had no effect on the resting tension. CONCLUSION: These results suggest that, in the isolated pig iris sphincter muscle, Rho-kinase plays an important role in the generation of spontaneous tone in the resting phase via the inhibition of MLCP activity.

Both 5-hydroxytryptamine 5-HT2A and 5-HT1B receptors are involved in the vasoconstrictor response to 5-HT in the human isolated internal thoracic artery.

1. The 5-hydroxytryptamine (5-HT, serotonin) receptor subtypes that mediate vasoconstriction in the human internal thoracic artery (ITA), which is frequently used as an arterial graft, remain unclear. The aim of the present study was to elucidate the 5-HT receptor subtypes responsible for 5-HT-induced contraction of the human ITA. 2. The contractile responses to 5-HT of endothelium-denuded human ITA obtained from patients undergoing coronary bypass surgery were examined. In addition, we investigated the effects of sarpogrelate and SB224289, antagonists of 5-HT(2A) and 5-HT(1B) receptors, respectively, on the 5-HT-induced vasoconstriction. Finally, 5-HT(2A) and 5-HT(1B) receptors in the human ITA were immunolabelled. 3. 5-Hydroxytryptamine (1 nmol/L-10 micromol/L) caused vasoconstriction in a concentration-dependent manner. Both sarpogrelate (1 micromol/L) and SB224289 (1 micromol/L) significantly, but not completely, inhibited 5-HT-induced vasoconstriction. 4. Conversely, simultaneous pretreatment with supramaximum concentrations (1 micromol/L for both) of sarpogrelate and SB224289 almost completely inhibited the 5-HT-induced vasoconstriction. 5. Immunopositive staining for 5-HT(2A) and 5-HT(1B) receptors was detected in smooth muscle cells of the human ITA. 6. These results demonstrate that, in human ITA, 5-HT-induced vasoconstriction is mediated by activation of both 5-HT(2A) and 5-HT(1B) receptors. Thus, when the human ITA is used as an arterial graft, a combination of 5-HT(2A) and 5-HT(1B) receptor antagonists would appear to be most useful to prevent 5-HT-induced vasospasm.

Mevalonate sensitizes the nociceptive transmission in the mouse spinal cord.

Isoprenylation is crucial for the biological activation of small molecular G proteins. Activation of Rho/Rho kinase (ROCK) signaling has been reported to be involved in the initiation and maintenance of hyperalgesia caused by nerve injury and inflammation. The present study was then undertaken to examine whether the protein isoprenylation could affect thermal nociceptive threshold in the mouse spinal cord. Intrathecal administration of mevalonate (0.05-5.0 micromol) dose-dependently decreased the paw-withdrawal latencies for the thermal stimulation, indicating that mevalonate induces thermal hyperalgesia. Intrathecal pretreatment with a geranylgeranyl transferase I inhibitor GGTI-2133 (0.001-1.0 nmol) or a ROCK inhibitor Y27632 (0.001-1.0 nmol) completely blocked the mevalonate-induced thermal hyperalgesia. On the other hand, mevalonate-induced thermal hyperalgesia was only slightly attenuated by a farnesyl transferase inhibitor FTI-277 (0.01-1.0 nmol). Intrathecal injection of mevalonate increased the amount of geranylgeranylated RhoA in the spinal cord, which was completely blocked by intrathecal pretreatment with GGTI-2133. Intrathecal injection of mevalonate also produced RhoA translocation from cytosol to plasma membrane. This mevalonate-induced RhoA translocation was also blocked by intrathecal pretreatment with GGTI-2133, indicating that the RhoA translocation is triggered by RhoA geranylgeranylation. Moreover, inhibition of mevalonate synthesis by HMG-CoA reductase inhibition with simvastatin attenuated the second phase, but not the first phase, of nociceptive response to formalin. Our present results suggest that mevalonate sensitizes the spinal nociceptive transmission, which is mediated by the activation of ROCK following the RhoA geranylgeranylation.

Cytoprotective effect of 3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate (DY-9760e) against ischemia/reperfusion-induced injury in rat heart involves inhibition of fodrin breakdown and protein tyrosine nitration.

We here assessed the effects of 3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate (DY-9760e), a novel calmodulin antagonist, on infarct size in the rat heart subjected to ischemia/reperfusion. Rats were subjected to a 30-min coronary occlusion followed by a 24-h reperfusion. DY-9760e was intravenously infused for 20 min, starting at 20 min after coronary occlusion. Treatment with DY-9760e (10 mg/kg) significantly reduced the infarct size in the risk area assessed by Evans Blue/TTC (triphenyltetrazolium chloride) staining. DY-9760e treatment also ameliorated contractile dysfunction of the left ventricle 72 h after reperfusion. DY-9760e significantly inhibited fodrin breakdown and caspase-3 activation. The inhibitory effect of DY-9760e on the fodrin breakdown was prominent in the rim rather than in the center of the risk area. DY-9760e also blocked protein tyrosine nitration associated with infarction. These results suggest that the cardioprotective effect of DY-9760e involved inhibition of calpain/caspase activation and protein tyrosine nitration.

Facilitatory role of NO in neural norepinephrine release in the rat kidney.

We examined modulation by nitric oxide (NO) of sympathetic neurotransmitter release and vasoconstriction in the isolated pump-perfused rat kidney. Electrical renal nerve stimulation (RNS; 1 and 2 Hz) increased renal perfusion pressure and renal norepinephrine (NE) efflux. Nonselective NO synthase (NOS) inhibitors [N(omega)-nitro-L-arginine methyl ester (L-NAME) or N(omega)-nitro-L-arginine], but not a selective neuronal NO synthase inhibitor (7-nitroindazole sodium salt), suppressed the NE efflux response and enhanced the perfusion pressure response. Pretreatment with L-arginine prevented the effects of L-NAME on the RNS-induced responses. 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO), which eliminates NO by oxidizing it to NO(2), suppressed the NE efflux response, whereas the perfusion pressure response was less susceptible to carboxy-PTIO. 8-Bromoguanosine cGMP suppressed and a guanylate cyclase inhibitor [4H-8-bromo-1,2,4-oxadiazolo(3,4-d)benz(b)(1,4)oxazin-1-one] enhanced the RNS-induced perfusion pressure response, but neither of these drugs affected the NE efflux response. These results suggest that endogenous NO facilitates the NE release through cGMP-independent mechanisms, NO metabolites formed after NO(2) rather than NO itself counteract the vasoconstriction, and neuronal NOS does not contribute to these modulatory mechanisms in the sympathetic nervous system of the rat kidney.

Role of K+ channels in the PACAP-induced catecholamine secretion from the rat adrenal gland.

We eluciated whether K+ channels modulate adrenal catecholamine secretion induced by pituitary adenylate cyclase-activating polypeptide (PACAP) in the isolated perfused rat adrenal gland. PACAP (100 nM) increased adrenal epinephrine output. The PACAP-induced responses were enhanced by treatment with apamin (10-100 nM) in a concentration-dependent manner. In the presence of nifedipine (3 microM), apamin (1 microM) did not enhance the PACAP-induced responses. Charybdotoxin (1-100 nM) had little influence on the PACAP-induced responses. These results suggest that small-conductance Ca2+-activated K+ channels interfere with L-type voltage-dependent Ca2+ channels to counteract the PACAP-induced adrenal catecholamine secretion.