Michelle A. Clark

Angiotensin III: A physiological relevant peptide of the renin angiotensin system

The renin angiotensin system (RAS) is a peptide hormone system that plays an important role in the pathophysiology of various diseases, including congestive heart failure, hypertension, myocardial infarction, and diabetic nephropathy. This has led researchers to focus extensively on this system, leading to the discovery of various peptides, peptidases, receptors and signal transduction mechanisms intrinsic to the RAS. Angiotensinogen (AGT), angiotensin (Ang) II, Ang III, Ang IV, and Ang-(1-7) are the main biologically active peptides of RAS. However, most of the available studies have focused on Ang II as the likely key peptide from the RAS that directly and indirectly regulates physiological functions leading to pathological conditions. However, data from recent studies suggest that Ang III may produce physiologically relevant effects that are similar to those produced by Ang II. Hence, this review focuses on Ang III and the myriad of physiological effects that it produces in the body.

Angiotensin II induces proliferation of cultured rat astrocytes through c-Jun N-terminal kinase.

Previously we showed that tyrosine kinases and ERK1/2 mediate angiotensin II (Ang II)-induced cellular proliferation in astrocytes. In the current study, we investigated whether Ang II activates c-Jun N-terminal kinase (JNK) and determined if JNK mediates Ang II-induced astrocyte growth in cultured brainstem astrocytes. Ang II activated JNK in a time- and concentration-dependent manner. Maximal stimulation of 14-fold over basal occurred with 100 nM Ang II and was significantly apparent 5 min after treatment. Ang II-induced JNK phosphorylation was abolished by co-treatment with the AT(1) receptor antagonist, losartan, but not by PD123319 (an AT(2) receptor antagonist). The JNK inhibitor, SP600125 (10 microM), markedly inhibited Ang II-induced JNK phosphorylation (by 84%) and astrocyte proliferation (by over 90%). Pretreatment of astrocytes with 10 microM PP2 (Src inhibitor) inhibited Ang II stimulation of JNK (by 90%) whereas, the protein kinase C (PKC) inhibitor, Go6976, failed to inhibit Ang II-mediated JNK phosphorylation. In conclusion, we showed for the first time that JNK mediates Ang II-specific astrocyte proliferation. In addition, Ang II activation of the JNK pathway is mediated by Src and not through PKC activation. This study is the first to show that JNK mediates Ang II effects in astrocytes and may provide a better understanding of the functions of Ang II in astrocytes from brainstem in particular and of glial cells in general.

Src and Pyk2 mediate angiotensin II effects in cultured rat astrocytes.

Angiotensin II (Ang II)-induced proliferation of rat astrocytes is mediated by multiple signaling pathways. In the present study, we investigated the role of non-receptor tyrosine kinases on Ang II-signaling and proliferation of astrocytes cultured from neonatal rat pups. Ang II stimulated astrocyte growth, ERK1/2 phosphorylation and the phosphorylation of Src and proline-rich tyrosine kinase-2 (Pyk2), in astrocytes obtained from brainstem and cerebellum. Pretreatment with 10 microM PP2, a selective Src inhibitor, inhibited Ang II stimulated ERK1/2 phosphorylation by 59% to 91% both in brainstem and cerebellum astrocytes. PP2 also inhibited Ang II induction of brainstem (76% inhibition) and cerebellar (64% inhibition) astrocyte growth. Similarly, pretreatment with 25 microM dantrolene, the Pyk2 inhibitor, attenuated ERK1/2 activity in brainstem (62% inhibition) and in cerebellum astrocytes (44% inhibition). Interestingly, inhibition of Pyk2 inhibited Ang II-induced Src activation suggesting that these two non-receptor tyrosine kinases may be acting in concert to mediate Ang II effects in astrocytes. In summary, we found that Ang II stimulates the non-receptor tyrosine kinases Src and Pyk2 which mediate Ang II-induced ERK1/2 activation leading to stimulation of astrocyte growth. In addition, these two tyrosine kinases may be interacting to regulate effects of the peptide in these cells.

Angiotensin II stimulates rat astrocyte mitogen-activated protein kinase activity and growth through EGF and PDGF receptor transactivation

We showed that the intracellular tyrosine kinases src and pyk2 mediate angiotensin II (Ang II) stimulation of growth and ERK1/2 mitogen-activated protein (MAP) kinase phosphorylation in astrocytes. In this study, we investigated whether the membrane-bound receptor tyrosine kinases platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) receptors mediate Ang II stimulation of ERK1/2 and astrocyte growth. Ang II significantly stimulated PDGF and EGF receptors in a dose- and time-dependent manner. The PDGF receptor and the EGF receptor were maximally stimulated with 100 nM Ang II (0.98+/-0.18- and 4.4+/-1.4-fold above basal, respectively). This stimulation occurred as early as 5 min, and was sustained for at least 15 min for both receptor tyrosine kinases. Moreover, 1 microM AG1478 and 0.25 microM PDGFRInhib attenuated Ang II stimulation of the EGF and PDGF receptors, respectively. Ang II-induced phosphorylation of ERK1/2 and astrocyte growth was mediated by both PDGF and EGF receptors. This report also provides novel findings that co-inhibiting EGF and PDGF receptors had a greater effect to decrease Ang II-induced ERK1/2 (90% versus 49% and 71% with PDGF receptor and EGF receptor inhibition, respectively), and astrocyte growth (60% versus 10% and 32% with PDGF receptor and EGF receptor inhibition, respectively). In conclusion we showed in astrocytes that the PDGF and the EGF receptors mediate Ang II-induced ERK1/2 phosphorylation and astrocyte growth and that these two receptors may exhibit synergism to regulate effects of the peptide in these cells.

Angiotensin III stimulates ERK1/2 mitogen-activated protein kinases and astrocyte growth in cultured rat astrocytes

Angiotensin (Ang) III is a biologically active metabolite of Ang II with similar effects and receptor binding properties as Ang II. Most Ang III studies delineate physiological effects of the peptide but, the intracellular pathways leading to the actions are unknown and are a focus of these studies. We investigated in cultured brainstem and cerebellum rat astrocytes whether Ang III stimulates ERK1/2 mitogen activated protein (MAP) kinases and astrocyte growth. Ang III significantly stimulated ERK1/2 MAP kinases in a dose- and time-dependent manner. The maximal stimulation occurred with 100 nM Ang III (2.8±0.3 and 2.3±0.1-fold over basal, in brainstem and cerebellum astrocytes, respectively). This stimulation occurred as early as 1 min, and was sustained for at least 15 min. Moreover, inhibition of the ERK1/2 MAP kinase pathway by 10 μM PD98059 attenuated Ang III-induced ERK1/2 phosphorylation. Ang III induction of ERK1/2 occurred via stimulation of the Ang AT(1) receptor since pretreatment with 10 μM Losartan, a selective AT(1) receptor blocker, prevented Ang III-induced ERK1/2 phosphorylation. The selective AT(2) Ang receptor blocker PD123319 was ineffective. Comparable to Ang II, Ang III also stimulated astrocyte growth in a concentration-dependent manner, an effect that occurred via activation of the AT(1) receptor as well. These findings suggest that Ang III has similar effects as Ang II in astrocytes since it rapidly stimulates the phosphorylation of the ERK1/2 MAP kinases and induces astrocyte proliferation through activation of the AT(1) receptor. These studies are important in establishing signaling pathways for Ang III and provide validation of the central role of Ang III.

Angiotensin II induces interleukin-6 expression in astrocytes: Role of reactive oxygen species and NF-κB

Previously, we showed that the bio-peptide angiotensin (Ang) II induces interleukin-6 (IL-6) in cultured astrocytes; however, the mechanism(s) involved in this effect were unknown. In the current study, we determined in brainstem and cerebellum astrocytes from the spontaneously hypertensive rat (SHR), the effect of Ang II to induce IL-6 as well as reactive oxygen species (ROS) generation. Results from this study showed that Ang II significantly induced the differential expression of IL-6 mRNA and protein levels in astrocytes from both regions of Wistar and SHRs. There were differences in the ability of Ang II to induce IL-6 mRNA and protein levels, but these differences were not apparent at all time points examined. Ang II also induced ROS generation, but there were no significant differences between ROS generation in SHR samples as compared to the Wistar samples. Ang II-induced IL-6 levels were mediated via the AT1/Nuclear Factor Kappa beta/ROS pathway. Overall, our findings suggest that there may be dysregulation in IL-6 production from astrocytes, contributing to differences observed in SHRs versus its normotensive control. Elucidating the mechanisms involved in Ang II pro-inflammatory effects in the central nervous system may lead to the development of novel therapeutic strategies that can be harnessed not just to treat hypertension, but other Ang II-mediated diseases as well.

Distinct Molecular Effects of Angiotensin II and Angiotensin III in Rat Astrocytes

It is postulated that central effects of angiotensin (Ang) II may be indirect due to rapid conversion to Ang III by aminopeptidase A (APA). Previously, we showed that Ang II and Ang III induced mitogen-activated protein (MAP) kinases ERK1/2 and stress-activated protein kinase/Jun-terminal kinases (SAPK/JNK) phosphorylation in cultured rat astrocytes. Most importantly, both peptides were equipotent in causing phosphorylation of these MAP kinases. In these studies, we used brainstem and cerebellum astrocytes to determine whether Ang IIs phosphorylation of these MAP kinases is due to the conversion of the peptide to Ang III. We pretreated astrocytes with 10 μM amastatin A or 100 μM glutamate phosphonate, selective APA inhibitors, prior to stimulating with either Ang II or Ang III. Both peptides were equipotent in stimulating ERK1/2 and SAPK/JNK phosphorylation. The APA inhibitors failed to prevent Ang II- and Ang III-mediated phosphorylation of the MAP kinases. Further, pretreatment of astrocytes with the APA inhibitors did not affect Ang II- or Ang III-induced astrocyte growth. These findings suggest that both peptides directly induce phosphorylation of these MAP kinases as well as induce astrocyte growth. These studies establish both peptides as biologically active with similar intracellular and physiological effects.

Angiotensin II induces cell growth and IL-6 mRNA expression through the JAK2-STAT3 pathway in rat cerebellar astrocytes

The pleiotrophic effects of angiotensin II (Ang II) play important roles in astrocyte growth and inflammatory responses. We investigated whether Ang II induces astrocyte growth and interleukin-6 (IL-6) mRNA expression in rat cerebellar astrocytes through Janus kinase 2-signal transduction activator of transcription (JAK2-STAT3). Ang II increased JAK2 and STAT3 phosphorylation in a time- and a dose-dependent manner. One hundred nanomolar Ang II induced maximal phosphorylation of both JAK2 and STAT3 between 15 min and 30 min. The Ang II-mediated phosphorylation of both JAK2 and STAT3 was blocked by AG490, a selective JAK2 inhibitor. Losartan, a selective AT1 receptor antagonist, inhibited Ang II-mediated JAK2 and STAT3 phosphorylation, while pretreatment with an AT2 receptor blocker, PD123319, was ineffective. Ang II increased the mRNA expression of IL-6 in a concentration-and time-dependent manner. Maximal IL-6 mRNA expression occurred with 100 nM Ang II, and the peak effect occurred in a biphasic manner at 3 h and between 12 and 24 h. Moreover, pretreatments with AG490 attenuated Ang II-induced IL-6 mRNA levels, and Ang II-induced astrocyte growth. This study has demonstrated that Ang II induced the phosphorylation of both JAK2 and STAT3 via the AT1 receptor in cerebellar astrocytes. In addition, our results suggest that JAK2 and STAT3 are upstream signals that mediate Ang II-induced IL-6 mRNA expression and astrocyte growth. These findings represent a novel non-classical mechanism of Ang II signaling in cerebellar astrocytes.

Regulation of angiotensinogen expression by angiotensin II in spontaneously hypertensive rat primary astrocyte cultures.

BACKGROUND Angiotensin (Ang) II, a bio-peptide of the renin-angiotensin system (RAS), plays a pivotal role in biological systems. It has been well established that in the brain, astrocytes are the predominant source for angiotensinogen (AGT), which is the precursor molecule for Ang II. The primary objective of this study was to determine the effect of Ang II on AGT mRNA and protein expression levels in primary cultures of astrocytes isolated from the brainstem and cerebellum regions of spontaneously hypertensive rats (SHRs) and normotensive Wistar rats. METHODS Astrocytes were treated with 100nM Ang II and the effect of time and the receptors involved in AGT mRNA and protein expression were measured using qPCR, and western blotting techniques, respectively. RESULTS Ang II significantly downregulated AGT mRNA levels and upregulated AGT protein levels in both SHR and Wistar rat astrocytes. Basal AGT mRNA levels in SHR samples were significantly lower as compared to Wistar astrocytes isolated from brainstem and cerebellum. There was no difference in the basal AGT protein levels when SHR and Wistar samples were compared. There was a tendency for higher Ang II-induced AGT protein levels in SHR samples compared to normotensive controls, but the difference was not significant. Ang II tended to decrease AGT mRNA levels of Wistar samples to a greater degree than SHR samples. The Ang AT1 receptor mediated the actions of Ang II on AGT protein and mRNA levels. CONCLUSION These findings highlight the complexity of AGT regulation and show that AGT protein and mRNA levels are responsive to Ang II in both SHR and Wistar astrocytes. Most importantly, our findings suggest that this peptide can induce its own synthesis by positively regulating AGT protein synthesis, an effect that was more robust in SHR astrocytes. Thus, dysregulation of this system may be important in preserving the hypertensive phenotype in this model.

Heterologous regulation of the cannabinoid type 1 receptor by angiotensin II in astrocytes of spontaneously hypertensive rats.

Brainstem and cerebellar astrocytes have critical roles to play in hypertension and attention‐deficit hyperactivity disorder, respectively. Angiotensin (Ang) II, via the astroglial Ang type 1 receptor (AT1R), has been demonstrated to elevate pro‐inflammatory mediators in the brainstem and the cerebellum. The activation of astroglial cannabinoid type 1 receptor (CB1R), a master regulator of homeostasis, has been shown to neutralize inflammatory states. Factors that drive disease progression are known to alter the expression of CB1Rs. In this study, we investigated the role of Ang II in regulating CB1R protein and mRNA expression in astrocytes isolated from the brainstem and the cerebellum of spontaneously hypertensive rats (SHRs). The results were then compared with their normotensive counterpart, Wistar rats. Not only was the basal expression of CB1R protein and mRNA significantly lower in SHR brainstem astrocytes, but treatment with Ang II resulted in lowering it further in the initial 12 h. In the case of cerebellum, Ang II up‐regulated the CB1R protein and mRNA in SHR astrocytes. While the effect of Ang II on CB1R protein was predominantly mediated via the AT1R in SHR brainstem; both AT1R‐ and AT2R‐mediated Ang IIs effect in the SHR cerebellum. These data are strongly indicative of a potential new mode of cross‐talk between components of the renin angiotensin system and the endocannabinoid system in astrocytes. The consequence of such a cross‐talk could be a potential reduced endocannabinoid tone in brainstem in hypertensive states, but not in the cerebellum under the same conditions.