In vitro receptor autoradiography was performed on rat brain and kidney sections stored frozen at -20°C for extended time periods (17, 40, 64, 121, 183, 251, and 333 days). The results indicate that prolonged tissue storage has a differential effect upon125I sar1ile8 angiotensin II binding to AT1 and AT2 receptor sites. Binding at AT1 receptor rich tissues studied (renal medulla, renal cortex, anterior pituitary, ventral hippocampus, spinal trigeminal nucleus, and nucleus of the solitary tract) shows a first order exponential decay pattern. The logarithmic linear regression slope (log(e) specific binding versus time), is significantly different from zero (p<0.05) in all AT1 rich tissues except for nucleus of the solitary tract (p=0.086). There is no detected loss of 125I sar1ile8 angiotensin II binding at the AT2 prominent regions in the superior colliculus, medial geniculate nucleus, and the inferior olivary nucleus. The half lives of AT1 receptors are highly variable, ranging from 36 days in the anterior pituitary to 442 days in the nucleus of the solitary tract, and this might be related to variable stability of AT(1A) and AT(1B) receptors. These observations should be taken into account when assessing and comparing AT1 and AT2 receptor subtype densities.

Myocardial infarction (MI) is associated with remodeling of the heart and neurohumoral control systems. The objective of this study was to define time-dependent changes in intrinsic cardiac (IC) neuronal excitability, synaptic efficacy, and neurochemical modulation following MI. MI was produced in guinea pigs by ligation of the coronary artery and associated vein on the dorsal surface of the heart. Animals were recovered for 4, 7, 14, or 50. days. Intracellular voltage recordings were obtained in whole mounts of the cardiac neuronal plexus to determine passive and active neuronal properties of IC neurons. Immunohistochemical analysis demonstrated an immediate and persistent increase in the percentage of IC neurons immunoreactive for neuronal nitric oxide synthase. Examination of individual neuronal properties demonstrated that afterhyperpolarizing potentials were significantly decreased in both amplitude and time course of recovery at 7. days post-MI. These parameters returned to control values by 50. days post-MI. Synaptic efficacy, as determined by the stimulation of axonal inputs, was enhanced at 7. days post-MI only. Neuronal excitability in absence of agonist challenge was unchanged following MI. Norepinephrine increased IC excitability to intracellular current injections, a response that was augmented post-MI. Angiotensin II potentiation of norepinephrine and bethanechol-induced excitability, evident in controls, was abolished post-MI. This study demonstrates that MI induces both persistent and transient changes in IC neuronal functions immediately following injury. Alterations in the IC neuronal network, which persist for weeks after the initial insult, may lead to alterations in autonomic signaling and cardiac control.

The beneficial effects of angiotensin II type 1 (AT1) receptor blockers (ARB) in cerebrovascular disease have been shown in clinical trials. However, the effects of ARBs vary based on their unique pharmacologic properties. In this study, we focused on telmisartan, a fat-soluble ARB with selective peroxisome proliferator-activated receptor-γ (PPARγ) agonist activity, and investigated its effects on ischemic injury in cerebral vasculature using murine models of both transient and permanent focal ischemia. Analysis by triphenyltetrazolium-staining revealed that pre-treatment of mice with telmisartan reduced stroke volume 72 h after the transient ischemic insult in a dose-dependent manner, though such treatment did not reduce stroke volume due to permanent ischemia. Transient ischemia induced pro-inflammatory adhesion molecules, such as ICAM-1 and P-selectin in the ischemic region, and treatment with telmisartan diminished the expression of these adhesion molecules with diminished infiltration of inflammatory cells. The beneficial effect of telmisartan was attenuated, in part, by administration of a PPARγ antagonist. Treatment with valsartan (an ARB without PPARγ agonist activity) also decreased ischemic injury after transient ischemia, though to a lesser extent than telmisartan. Our findings indicate that telmisartan has a beneficial effect in a murine model of ischemia/reperfusion injury through blockade of AT1 receptors, and, in addition, due to a positive effect via its specific anti-inflammatory PPARγ agonist activity.

Recent studies show that angiotensin II (AngII) can act from within the cell, possibly via intracellular receptors pharmacologically different from typical plasma membrane AngII receptors. The role of this intracellular AngII (AngIIi) is unclear. Besides direct effects of AngIIi on cellular processes one could hypothesise a possible role of AngIIi in modulation of cellular responses induced after heterologous receptor stimulation. We therefore examined if AngIIi influences [Ca2+]i in A7r5 smooth muscle cells after serotonin (5HT) or UTP receptor stimulation. Application of AngIIi using liposomes, markedly inhibited 45Ca2+ influx after receptor stimulation with 5HT or UTP. This inhibition was reversible by intracellular administration of the AT1-antagonist losartan and not influenced by the AT2-antagonist PD123319. Similar results were obtained in single cell [Ca2+]i measurements, showing that AngIIi predominantly influences Ca2+ influx and not Ca2+ release via AT1-like receptors. It is concluded that AngIIi modulates signal transduction activated by heterologous receptor stimulation.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18159号 / 医博第3879号 / 新制||医||1003(附属図書館) / 31017 / 京都大学大学院医学研究科医学専攻 / (主査)教授 横出 正之, 教授 木村 剛, 教授 柳田 素子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第18924号 / 薬科博第38号 / 新制||薬||5(附属図書館) / 31875 / 京都大学大学院薬学研究科医薬創成情報科学専攻 / (主査)教授 岡村 均, 教授 中山 和久, 教授 竹島 浩 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DGAM

Cardiovascular disease (CVD) is the leading cause of death in the United States, and hypertension has been recognized as a major contributor to its manifestation and progression. Vascular smooth muscle cells control the tone and elasticity of vessels and their dysfunction in hypertension contributes to arterial remodeling and subsequent end organ damage. Evidence has indicated that the Endoplasmic Reticulum (ER) Unfolded Protein Response (UPR) may be compromised in hypertension, while the contribution of protein aggregate formation (a main driver of UPR activation) is undefined. Glucose regulated protein-78 (GRP78), a residential ER chaperone, acts to aid in the proper folding of nascent peptides during translation, while also acting as the primary signal transducer for UPR. We hypothesize overexpression of GRP78 can protect against Angiotensin II induced protein aggregation in vascular smooth muscle cells (VSMCs) to reduce pathological ER UPR signaling and hypertensive vascular remodeling. To test this hypothesis, we investigated protein aggregate induction by Ang II stimulation as well as ER UPR activation, and if overexpression of the ER-resident chaperone glucose regulated protein 78 (GRP78) could protect against these as well as VSMC remodeling markers: hypertrophy, collagen production and inflammation. Utilizing pre-amyloid oligomer (PAO) immunofluorescence staining to identify Ang II induction of amyloid in VSMCs, we found amyloid accumulation was maximal at 48h post stimulation, which could be prevented with adenoviral overexpression of GRP78. Ang II significantly induced ER stress markers p-IRE1α, p-PERK and cleaved ATF6 in VSMCs. Overexpression of GRP78 was able to attenuate these ER stress responders. Interestingly, shotgun proteomic analysis of triton X-100 insoluble aggregate fractions revealed proteostasis machinery enriched in Ang II treated VSMC aggregates (HSP70, VCP, CryAB), which were attenuated with GR78 overexpression. To investigate pathological VSMC remodeling markers, we found that Ang II induced VSMC collagen production, immune cell adhesion, VCAM-1 expression, and hypertrophy (via protein synthesis) which was attenuated by GRP78 overexpression. Utilizing a VSMC-promoter derived GRP78 overexpression mouse model (GRP78TG SM22α Cre-/- or GRP78TG SM22α Cre+/-), we investigated the effect of ER stress inhibition on Ang II induced vascular remodeling. Importantly, hypertrophy and fibrosis in the aorta and the cardiac vasculature were assessed by Masson’s Trichrome and Sirius red staining and found to be increased in Cre-/- mice, while Cre+/- were significantly protected. These effects were accompanied with a significant reduction in Ang II-induced aortic amyloid burden (PAO) and ER UPR signaling. Blood pressure was monitored via tail cuff which revealed GRP78 Cre+/- mice were not protected against Ang II induced hypertension. Overall, these findings indicate that VSMC protein aggregation activates the ER stress response and contribute to hypertensive vascular remodeling. Furthermore, therapeutically targeting this mechanism via overexpression of GRP78 may elude new pharmacological interventions for arterial stiffness, while addressing fundamental questions about the mechanisms involved. / Biomedical Sciences

This study was designed to characterize the distribution of angiotensin II (AII) binding sites in the hamster brain. Brain sections were incubated with [125I][sar1, ile8]-angiotensin II in the absence and presence of angiotensin II receptor subtype selective compounds, losartan (AAT, subtype) and PD123177 (AT2 subtype). Binding was quantified by densoritometric autoradiograms and localized by comparison with adjacent thionin stained sections. The distribution of AII binding sites was similar to that found in the rat, with some exceptions. [125I][sar1, ile8]-angiotensin II binding was not evident in the subthalamic nucleus and thalamic regions, inferior olive, suprachiasmatic nucleus, and piriform cortex of the hamster, regions of prominent binding in the rat brain. However, intense binding was observed in the interpeduncular nucleus and the medial habenula of the hamster, nuclei void of binding in the rat brain. Competition with receptor subtype selective compounds revealed a similar AII receptor subtype profile in brain regions where binding is evident in both species. One notable exception is the medial geniculate nucleus, predominately AT1 binding sites in the hamster but AT2 in the rat. Generally, the AII binding site distribution in the hamster brain parallels that of the other species studied, particularly in brain regions associated with cardiovascular and dipsogenic functions. Functional correlates for AII binding sites have not been elucidated in the majority of brain regions and species mismatches might provide clues in this regard.

Angiotensin II (AII) immunoreactivity in the mesopontine area of the rat brain is distributed through several areas where co-localization of AII receptors has not been established. The current in vitro receptor autoradiography study re-examined the distribution of AII binding using 125I-Sar1, Ile8-AII ([125I]SIAII). When incubations were conducted without sulfhydryl reducing agents, [125I]SIAII binding was observed in the locus coeruleus, inferior colliculus, superior colliculus and the central gray in agreement with previous reports. Novel [125I]SIAII binding sites were detected in the parabrachial nucleus, pedunculopontine tegmental nucleus and the caudal linear raphe nucleus, corresponding with previously reported localization of AII immunoreactivity in these nuclei. [125I]SIAII binding was also found in the paragenual nucleus where the peptide has not been detected. Thus, the observation of novel AII receptors which are sensitive to sulfhydryl reducing agents, resolves several AII-AII receptor mismatches.

The non-peptide angiotensin II (AII) receptor subtype selective antagonist, DuP 753, was used to characterize AII receptor binding sites in the rat brain. DuP 753 competed for specific 125I-[Sar1, Ile8]AII (125I-SIAII) binding in many brain nuclei (IC50 = 20-30 nM), but was a weak competitor at remaining sites (IC50 > 10-4 M). DuP 753 sensitive binding sites (designated AIIα subtype) correspond with areas where binding is inhibited by sulfhydryl reducing agents, whereas DuP 753 insensitive sites (AIIβ) correspond with areas where binding is not inhibited by sulfhydryl reducing agents.