The Characterization of AT<Subscript>1</Subscript> Expression in the Dorsal Root Ganglia After Chronic Constriction Injury

To clarify the role of Angiotensin II in the regulation of sensory signaling, we characterized the AT₁ expression in neuronal subpopulation of lower lumbar dorsal root ganglia under normal conditions and its alteration in neuropathic pain model. The characterization of AT₁ expression was done under control and after the chronic constriction injury induced by four loose ligatures of the sciatic nerve representing the model of posttraumatic painful peripheral neuropathy. Major Angiotensin II receptor type was expressed in approximately 43 % of small-sized and 62 % of large-sized neurons in control. The AT₁ overexpression after sciatic nerve ligation lasting 7 days was detected predominantly in small-sized AT₁ immunoreactive neurons (about 38 % increase). Chronic constriction injury caused a statistically marked increase in number of the small-sized peptidergic (CGRP immunoreactive) neuronal subpopulation expressing AT₁ (about 64 %). The subpopulations of AT₁-immunoreactive and nonpeptide-containing primary sensory neurons revealed by IB4 binding, tyrosine hydroxylase- and parvalbumin-immunoreactive neurons were not markedly changed. Our results indicate that: (1) the AT₁ overexpression after the chronic constriction injury is an important factor in Angiotensin II-potentiated pain perception; (2) Angiotensin II is involved in pathological mechanisms of neuropathic pain and this effect can be mediated perhaps in combination with other neuropeptides synthesized in the primary sensory neurons.     

Silymarin- and melatonin-mediated changes in the expression of selected genes in pesticides-induced Parkinsonism

Expression of angiotensin II (Ang II) and its receptors (AT₁/AT₂) is undetected in the mature microglia in normal brain. We report here that the immunoexpression of Ang II and AT₁/AT₂ was altered in activated microglia notably at 1 week in rats subjected to middle cerebral artery occlusion (MCAO). Immunolabeled activated microglia were widely distributed in the infarcted cerebral tissue after MCAO. By enzyme immunoassay, Ang II protein expression levels of the ischemic tissues were decreased drastically at 12 h after ischemia, then rose rapidly at 3 days and 1 week after MCAO when compared with the control. On the other hand, AT₁ and AT₂ receptor mRNA and protein levels were up-regulated after MCAO, peaking at 12 h, but declined thereafter. Expression of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) mRNA and protein levels was concomitantly increased. Edaravone significantly suppressed Ang II and AT₁/AT₂ receptor expression as well as that of TNF-α and IL-1β suggesting that microglia-derived Ang II can act through an autocrine manner via its receptor that may be linked partly to the production of proinflammatory cytokines. We conclude that neuroinflammation in MCAO may be attenuated by Edaravone which acts through suppression of expression of Ang II and its receptors and proinflammatory cytokines in activated microglia.     

Hypertrophic response to angiotensin II is mediated by protein kinase D-extracellular signal-regulated kinase 5 pathway in human aortic smooth muscle cells

Angiotensin II plays a critical role in hypertrophy of vascular smooth muscle cells, however, the molecular underpinnings remain unclear. The present study indicated that AT₁/PKC/PKD pathway was able to regulate downstream ERK5, affecting pro-hypertrophic responses to Ang II. Ang II-stimulated phosphorylation of ERK5 in a time- and dose-dependent manner in human aortic smooth muscle cells (HASMCs). The pharmacological inhibitors for AT₁ and PKCs significantly inhibited Ang II-induced ERK5 activation, suggesting the involvement of the AT₁/PKC pathway. In particular, PKD was critical for Ang II-induced ERK5 activation since silencing PKD by siRNA markedly inhibited Ang II-induced ERK5 activation. Consequently, we found that Losartan, Gö 6983 and PKD siRNA significantly attenuated ERK5 activated translocation and hypertrophy of HASMCs by Ang II. Taken together, we demonstrated for the first time that Ang II activates ERK5 via the AT₁/PKC/PKD pathway and revealed a critical role of ERK5 in Ang II-induced HASMCs hypertrophy.     

The role of Jak/STAT signaling in heart tissue renin-angiotensin system

The involvement of the Renin Angiotensin System (RAS) and the role of its primary effector, angiotensin II (Ang II), in etiology of myocardial hypertrophy and ischemia is well documented. In several animal models, the RAS is activated in cardiac cell types that express the receptor AT₁, and/or AT₂, through which the Ang II mediated effects are promoted. In this article, we briefly review recent experimental evidence on the critical role of a prominent signaling pathway, the Jak/Stat pathway in activation and maintenance of the local RAS in cardiac hypertrophy and ischemia. Recent studies in our laboratory document that the promoter of the prohormone angiotensinogen (Ang) gene serves as the target site for STAT proteins, thereby linking the Jak/Stat pathway to activation of heart tissue autocrine Ang II loop. Stat5A and Stat6, are selectively activated when the heart is subjected to ischemic injury, whereas activation of Stat3 and Stat5A is involved in myocardial hypertrophy. Blockage of RAS activation by treatment with specific inhibitor promotes a remarkable recovery in functional hemodynamics of the myocardium. Thus, activation of selective sets of Stat proteins constitutes the primary signaling event in the pathogenesis of myocardial hypertrophy and ischemia.     

Activation of satellite glial cells in trigeminal ganglion following dental injury and inflammation

Satellite glial cells (SGCs), a peripheral neuroglial cell, surround neurons and form a complete envelope around individual sensory neurons in the trigeminal ganglia (TG), which may be involved in modulating neurons in inflammation. The purpose of this study was to determine the effect of dental injury and inflammation on SGCs in the TG. Pulp exposure (PX) was performed on the first maxillary molar of 28 rats. The neurons innervating injured tooth in TG were labeled by the retrograde transport of fluoro-gold (FG). Specimens were collected at 1, 3, 7, 14, 21 and 28 days after PX and stained immunohistochemically for glial fibrillary acid protein (GFAP), a marker of SGCs activation, in the TG. We observed that GFAP-immunoreactivity (IR) SGCs enclosed FG-labeled neurons increased in a time-dependent manner after PX. The neurons surrounded by GFAP-IR SGCs were mainly small and medium in size. The GFAP-IR SGCs encircled neurons increased significantly in the maxillary nerve region of the TG at 7–28 days following PX. The results show that dental injury and inflammation induced SGCs activation in the TG. It indicates that activation of SGCs might be implicated in the peripheral mechanisms of pain following dental injury and inflammation.     

Venous endothelium reactivity to Angiotensin II: A study in primary endothelial cultures of rat vena cava and portal vein

The role of the vascular endothelium in modulating the arterial system has been widely investigated, but poorly explored at the venous site. In the present work, primary cultures of venous endothelium from rat Vena Cava (VC) and Portal Vein (PV) were established, characterized and analyzed according to their growth pattern and ability to produce nitric oxide (NO) and prostanoids (PGF_{2 α} and PGI₂), at basal state and after stimulation with Angiotensin II (Ang II, 1 μmol/L). Basal NO was detected in all examined cells in culture. Pre-incubation with Ang II increased NO production in cells from VC (but not in PV cultures), through activation of both AT₁ and AT₂ receptors. Both cultures exhibited detectable levels of PGF_{2 α} at resting conditions, which were similarly enhanced by Ang II. Basal PGI₂ levels were higher in PV, but increased after Ang II treatment in VC, with no further effect on PV cells. We conclude that endothelial cells from VC and PV exhibit important properties and react to Ang II, probably influencing the whole circulatory system. This experimental cell model gives support to further studies concerning intracellular pathways of the venous endothelium, analyzed in separate from the vascular smooth muscle wall.     

Losartan, an antagonist of AT1 receptor for angiotensin II, attenuates lipopolysaccharide-induced acute lung injury in rat

Angiotensin II (Ang II) plays an important role in inflammatory process. Acute lung injury (ALI), an inflammatory disorder of the lung, is commonly associated with endotoxemia; however, the mechanism that endotoxin (lipopolysaccharide, LPS) induces the inflammatory response in ALI is not well defined. Here, we showed, in LPS-induced ALI rat model, that Ang II and Ang II type 1 (AT₁) receptor were significantly increased in lung tissues, compared with those in controls. Meanwhile, nuclear factor (NF)-κB-DNA-binding activity, tumor necrosis factor (TNF)-α mRNA, and pneumocytic apoptosis were significantly increased. Moreover, pretreatment of rats with losartan, an antagonist of AT₁ receptor for Ang II, improved the inflammation, reduced the elevation of Ang II and AT₁ receptor, and inhibited NF-κB-DNA-binding activity, expression of TNF-α mRNA, and pneumocytic apoptosis. The data indicate that Ang II may mediate the inflammatory process in LPS-induced ALI through AT₁ receptor, which can be blocked by losartan.     

Angiotensin II and Angiotensin-(1-7) in Paraventricular Nucleus Modulate Cardiac Sympathetic Afferent Reflex in Renovascular Hypertensive Rats

Background

The enhanced cardiac sympathetic afferent reflex (CSAR) is involved in the sympathetic activation that contributes to the pathogenesis and progression of hypertension. Activation of AT₁ receptors by angiotension (Ang) II in the paraventricular nucleus (PVN) augments the enhanced CSAR and sympathetic outflow in hypertension. The present study is designed to determine whether Ang-(1-7) in PVN plays the similar roles as Ang II and the interaction between Ang-(1-7) and Ang II on CSAR in renovascular hypertension.

Methodology/Principal Findings

The two-kidney, one-clip (2K1C) method was used to induce renovascular hypertension. The CSAR was evaluated by the renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) responses to epicardial application of capsaicin in sinoaortic-denervated and cervical-vagotomized rats with urethane and α-chloralose anesthesia. Either Ang II or Ang-(1-7) in PVN caused greater increases in RSNA and MAP, and enhancement in CSAR in 2K1C rats than in sham-operated (Sham) rats. Mas receptor antagonist A-779 and AT₁ receptor antagonist losartan induced opposite effects to Ang-(1-7) or Ang II respectively in 2K1C rats, but losartan had no effects in Sham rats. Losartan but not the A-779 abolished the effects of Ang II, while A-779 but not the losartan blocked the effects of Ang-(1-7). PVN pretreatment with Ang-(1-7) dose-dependently augmented the RSNA, MAP, and CSAR responses to the Ang II in 2K1C rats. Ang II level, AT₁ receptor and Mas receptor protein expression in PVN increased in 2K1C rats compared with Sham rats but Ang-(1-7) level did not.

Conclusions

Ang-(1-7) in PVN is as effective as Ang II in enhancing the CSAR and increasing sympathetic outflow and both endogenous Ang-(1-7) and Ang II in PVN contribute to the enhanced CSAR and sympathetic outflow in renovascular hypertension. Ang-(1-7) in PVN potentiates the effects of Ang II in renovascular hypertension.     

Direct activation of Transient Receptor Potential Vanilloid 1(TRPV1) by Diacylglycerol (DAG)

Voltage-gated sodium channels play important roles in modulating dorsal root ganglion (DRG) neuron hyperexcitability and hyperalgesia after peripheral nerve injury or inflammation. We report that chronic compression of DRG (CCD) produces profound effect on tetrodotoxin-resistant (TTX-R) and tetrodotoxin-sensitive (TTX-S) sodium currents, which are different from that by chronic constriction injury (CCI) of the sciatic nerve in small DRG neurons. Whole cell patch-clamp recordings were obtained in vitro from L₄ and/or L₅ dissociated, small DRG neurons following in vivo DRG compression or nerve injury. The small DRG neurons were classified into slow and fast subtype neurons based on expression of the slow-inactivating TTX-R and fast-inactivating TTX-S Na⁺ currents. CCD treatment significantly reduced TTX-R and TTX-S current densities in the slow and fast neurons, but CCI selectively reduced the TTX-R and TTX-S current densities in the slow neurons. Changes in half-maximal potential (V_1/2) and curve slope (k) of steady-state inactivation of Na⁺ currents were different in the slow and fast neurons after CCD and CCI treatment. The window current of TTX-R and TTX-S currents in fast neurons were enlarged by CCD and CCI, while only that of TTX-S currents in slow neurons was increased by CCI. The decay rate of TTX-S and both TTX-R and TTX-S currents in fast neurons were reduced by CCD and CCI, respectively. These findings provide a possible sodium channel mechanism underlying CCD-induced DRG neuron hyperexcitability and hyperalgesia and demonstrate a differential effect in the Na⁺ currents of small DRG neurons after somata compression and peripheral nerve injury. This study also points to a complexity of hyperexcitability mechanisms contributing to CCD and CCI hyperexcitability in small DRG neurons.     

Discovery of tetrahydropyrido[4,3-d]pyrimidine derivatives for the treatment of neuropathic pain

A series of tetrahydropyridopyrimidine derivatives were synthesized and evaluated for neurotoxicity and peripheral analgesic activity followed by assessment of antiallodynic and antihyperalgesic potential in two peripheral neuropathic pain models, the chronic constriction injury (CCI) and partial sciatic nerve ligation (PSNL). Compounds (4b and 4d) exhibiting promising efficacies in four behavioral assays of allodynia and hyperalgesia (spontaneous pain, tactile allodynia, cold allodynia and mechanical hyperalgesia) were quantified for their ED₅₀ values (15.12–65.10 mg/kg). Studies carried out to assess the underlying mechanism revealed that the compounds suppressed the inflammatory component of the neuropathic pain and prevented oxidative and nitrosative stress.     

Na-ATPase in spontaneous hypertensive rats: Possible AT1 receptor target in the development of hypertension

Clinical and experimental data show an increase in sodium reabsorption on the proximal tubule (PT) in essential hypertension. It is well known that there is a link between essential hypertension and renal angiotensin II (Ang II). The present study was designed to examine ouabain-insensitive Na⁺-ATPase activity and its regulation by Ang II in spontaneously hypertensive rats (SHR). We observed that Na⁺-ATPase activity was enhanced in 14-week-old but not in 6-week-old SHR. The addition of Ang II from 10^− 12 to 10^− 6 mol/L decreased the enzyme activity in SHR to a level similar to that obtained in WKY. The Ang II inhibitory effect was completely reversed by a specific antagonist of AT₂ receptor, PD123319 (10^− 8 mol/L) indicating that a system leading to activation of the enzyme in SHR is inhibited by AT₂-mediated Ang II. Treatment of SHR with losartan for 10 weeks (weeks 4-14) prevents the increase in Na⁺-ATPase activity observed in 14-week-old SHR. These results indicate a correlation between AT₁ receptor activation in SHR and increased ouabain-insensitive Na⁺-ATPase activity. Our results open new possibilities towards our understanding of the pathophysiological mechanisms involved in the increased sodium reabsorption in PT found in essential hypertension.     

Vascular mechanisms involved in angiotensin II-induced venoconstriction in hypertensive rats

To investigate the venoconstrictor effect of angiotensin II (Ang II) in spontaneously hypertensive rats (SHR), we used preparations of mesenteric venular beds and the circular muscle of the portal veins. Vessels were tested with Ang II in the presence or absence of losartan, PD 123319, HOE 140, L-NAME, indomethacin, or celecoxib. In the mesenteric venular bed of SHR, the effect of Ang II (0.1 nmol) was nearly abolished by losartan and enhanced by HOE 140, indomethacin, and celecoxib, while PD123319 and L-NAME had no effect. In portal vein preparations, cumulative-concentration response curves (CCRC) to Ang II (0.1–100 nmol/L) exhibited a lower maximal response (E_max) in SHR compared to Wistar rats. AT₁ receptor expression was similar in the two strains, while AT₂ receptor levels were lower in SHR portal veins when compared to Wistar. In SHR portal veins, losartan shifted the CCRC to Ang II to the right, while indomethacin and HOE 140 increased the E_max to Ang II. PD 123319, celecoxib, and L-NAME had no effect. Taken together, our results suggest that Ang II-induced venoconstriction in SHR is mediated by activation of AT₁ receptors and this effect may be counterbalanced by kinin B₂ receptor and COX metabolites. Furthermore, our data indicate that there are different cellular and molecular mechanisms involved in the regulation of venous tonus of normotensive and hypertensive rats. These differences probably reflect distinct factors that influence arterial and venous bed in hypertension.     

N-type voltage-dependent Ca channel in non-excitable microglial cells in mice is involved in the pathophysiology of neuropathic pain

Peripheral nerve injury induces neuropathic pain which is characterized by tactile allodynia and thermal hyperalgesia. N-type voltage-dependent Ca²⁺ channel (VDCC) plays pivotal roles in the development of neuropathic pain, since mice lacking Ca_v2.2, the pore-forming subunit of N-type VDCC, show greatly reduced symptoms of both tactile allodynia and thermal hyperalgesia. Our study on gene expression profiles of the Ca_v2.2 knockout (KO) spinal cord after spinal nerve ligation (SNL)-injury revealed altered expression of genes known to be expressed in microglia, raising an odd idea that N-type VDCC may function in not only excitable (neurons) but also non-excitable (microglia) cells in neuropathic pain state. In the present study, we have tested this idea by using a transgenic mouse line, in which suppression of Ca_v2.2 expression can be achieved specifically in microglia/macrophage by the application of tamoxifen. We found SNL-operated transgenic mice exhibited greatly reduced signs of tactile allodynia, whereas the degree of thermal hyperalgesia was almost the same as that of control. Immunohistochemical analysis of the transgenic lumbar spinal cord revealed reduced accumulation of Iba1-positive cells (microglia/macrophage) around the injured neurons, indicating microglial N-type VDCC is important for accumulation of microglia at the lesion sites. Although the mechanism of its activation is not clear at present, activation of N-type VDCC expressed in non-excitable microglial cells contributes to the pathophysiology of neuropathic pain.     

Central angiotensin II stimulates cutaneous water intake behavior via an angiotensin II type-1 receptor pathway in the Japanese tree frog Hyla japonica

Angiotensin II (Ang II) stimulates oral water intake by causing thirst in all terrestrial vertebrates except anurans. Anuran amphibians do not drink orally but absorb water osmotically through ventral skin. In this study, we examined the role of Ang II on the regulation of water-absorption behavior in the Japanese tree frog (Hyla japonica). In fully hydrated frogs, intracerebroventricular (ICV) and intralymphatic sac (ILS) injection of Ang II significantly extended the residence time of water in a dose-dependent manner. Ang II-dependent water uptake was inhibited by ICV pretreatment with an angiotensin II type-1 (AT₁) receptor antagonist but not a type-2 (AT₂) receptor antagonist. These results suggest that Ang II stimulates water-absorption behavior in the tree frog via an AT₁-like but not AT₂-like receptor. We then cloned and characterized cDNA of the tree frog AT₁ receptor from the brain. The tree frog AT₁ receptor cDNA encodes a 361 amino acid residue protein, which is 87% identical to the toad (Bufo marinus) AT₁ receptor and exhibits the functional characteristics of an Ang II receptor. AT₁ receptor mRNAs were found to be present in a number of tissues including brain (especially in the diencephalon), lung, large intestine, kidney and ventral pelvic skin. When tree frogs were exposed to dehydrating conditions, AT₁ receptor mRNA significantly increased in the diencephalon and the rhombencephalon. These data suggest that central Ang II may control water intake behavior via an AT₁ receptor on the diencephalon and rhombencephalon in anuran amphibians and may have implications for water consumption in vertebrates.     

Angiotensin II enhances endothelin-1-induced vasoconstriction through upregulating endothelin type A receptor

Endothelin-1 (ET-1) is the most potent vasoconstrictor by binding to endothelin receptors (ET_AR) in vascular smooth muscle cells (VSMCs). The complex of angiotensin II (Ang II) and Ang II type one receptor (AT₁R) acts as a transient constrictor of VSMCs. The synergistic effect of ET-1 and Ang II on blood pressure has been observed in rats; however, the underlying mechanism remains unclear. We hypothesize that Ang II leads to enhancing ET-1-mediated vasoconstriction through the activation of endothelin receptor in VSMCs. The ET-1-induced vasoconstriction, ET-1 binding, and endothelin receptor expression were explored in the isolated endothelium-denuded aortae and A-10 VSMCs. Ang II pretreatment enhanced ET-1-induced vasoconstriction and ET-1 binding to the aorta. Ang II enhanced ET_AR expression, but not ET_BR, in aorta and increased ET-1 binding, mainly to ET_AR in A-10 VSMCs. Moreover, Ang II-enhanced ET_AR expression was blunted and ET-1 binding was reduced by AT₁R antagonism or by inhibitors of PKC or ERK individually. In conclusion, Ang II enhances ET-1-induced vasoconstriction by upregulating ET_AR expression and ET-1/ET_AR binding, which may be because of the AngII/Ang II receptor pathways and the activation of PKC or ERK. These findings suggest the synergistic effect of Ang II and ET-1 on the pathogenic development of hypertension.     

Angiotensin II induces the production of MMP-3 and MMP-13 through the MAPK signaling pathways via the AT1 receptor in osteoblasts

Angiotensin II (Ang II) plays an important role in the maintenance of bone mass and integrity by activation of the mitogen-activated protein kinases (MAPKs) and by modulation of balance between resorption by osteoclasts and formation by osteoblasts. However, the role of Ang II in the turnover of extracellular matrix (ECM) in osteoid by osteoblasts remains unclear. Therefore, we examined the effect of Ang II on the expression of matrix metalloproteinases (MMPs), plasminogen activators (PAs), and their inhibitors [i.e., tissue inhibitors of metalloproteinases (TIMPs) and PA inhibitor-1 (PAI-1)] using osteoblastic ROS17/2.8 cells. Treatment with Ang II strikingly increased the expressions of MMP-3 and -13 and promoted cell proliferation associated with reduced alkaline phosphatase activity as well as enhanced phosphorylated expression of extracellular signal-regulated kinase (ERK)1/2, p38 MAPK, and stress-activated protein kinases/c-jun N-terminal kinases (SAPK/JNK) in ROS17/2.8 cells. However, Ang II had no effect on the expression of MMP-2, -9, -14, urokinase-type PA, tissue-type PA, TIMP-1, -2, -3, and PAI-1 in cells. Losartan (AT₁ receptor blocker) blocked Ang II-induced expression of MMP-3 and -13, whereas PD123319 (AT₂ receptor blocker) did not completely block these responses. Losartan also blocked the Ang II-induced phosphorylation of ERK1/2, p38 MAPK, and SAPK/JNK. MAPK kinase 1/2 inhibitor PD98059 and JNK inhibitor SP600125 suppressed Ang II-induced expression of MMP-3 and -13. These results suggested that Ang II stimulated the degradation process that occurs during ECM turnover in osteoid by increasing the production of MMP-3 and -13 through MAPK signaling pathways via the AT₁ receptor in osteoblasts. Furthermore, our findings suggest that Ang II does not influence the plasminogen/plasmin pathway in osteoblasts.