We established that acute adrenergic receptor stimulation in β-cells suppresses oxidative metabolism. This effect provides the basis for understanding how CAs reduce cell proliferation. Furthermore, the effects of acute CA on Min6 cells were distinguished from chronic CA culture using proteomics. Together, the RNAseq, qPCR and proteomic studies support a role for adrenergic receptor signaling in the regulation of proliferaton in β-cells. This work describes the genetic and proteomic profile underlying chronic adrenergic signaling and identifies CA independent suppression of β-cell growth and metabolism. Through the use of multiple models and comparative bioinformatics, we refined the list of molecular dysfunctions associated with the IUGR pathology to a set of specific and testable adrenergic targets.

The cAMP-second messenger hypothesis for β-adrenoceptor-induced relaxation of uterine smooth muscle was tested in high-K+ depolarized rat uterus. At 10⁻⁸ M concentration, Isoproterenol, a β -adrenergic agonist, could cause relaxation of the depolarized uterus without Increasing tissue cAMP levels. Further, although increases in cAMP levels were associated, in some cases, with -isoproterenol (10⁻⁸ M or 10⁻⁴ M)-Induced relaxation, there was no quantitative correlation between the Increases in cAMP and relaxation. Pretreatment of the tissue with a phosphodiesterase Inhibitor, RO 20-1724 (10⁻⁴ M), did not potentiate the relaxation response to Isoproterenol. These results suggested that there Is no simple cause and effect relationship between β -adrenoceptor-Induced Increases in cAMP levels and relaxation in uterine smooth muscle. The dissociation between cAMP and relaxation found ln the present study was also extended to cGMP, since no changes in cGMP levels were observed with isoproterenol-induced relaxation. It is generally accepted that the ionic environment of the cell affects the cellular responses of the tissue. It was demonstrated that hlgh-K*" depolarization of uterine smooth muscle caused an impairment of the ability of isoproterenol to induce cAMP accumulation. This was found to be related to Increased Ca++-Influx known to occur during depolarization. This Is because pretreatment of the tissue with 10⁻⁵M D-600, an Inhibitor of Ca++-lnflux, restored the stimulation of cAMP by Isoproterenol ln the depolarized muscle to a level similar to that observed ln non-depolarized muscle. Furthermore, there was an Inverse relationship between [ca++] ex in the depolarizing medium (range 0.9 to 7.2 mM) and increases in cAMP produced by isoproterenol (10⁻⁴ M). It was also found that exposure of the rat uterus to a Ca++-deficient solution (Ca++-free with 0.2 mM EGTA) accentuated the Increase of tissue cAMP content produced by isoproterenol (10⁻⁸ M). The studies on ionic interactions demonstrated that the presence of Na+(80 mM) or high Mg++(2.5 mM) in the depolarizing medium could overcome the blockade of lsoproterenol-induced increases in cAMP levels by high-K+ depolarization. The studies on the mechanism of this effect of Na+ on the cAMP response revealed that Na+ exerted this effect probably by reducing the Increase In Ca++-influx occurring during depolarization. A similar type of interaction between Mg++ and Ca++ was also observed. These studies have pointed out a possible regulatory role of Ca++ in isoproterenol-lnduced Increases in cAMP levels in uterine smooth muscle. Since it was also demonstrated that cAMP Is not an obligatory requirement In order for Isoproterenol to produce relaxation, these data have raised the question as to whether the Increases ln cAMP produced by β-adrenoceptor stimulation Is an event secondary to the changes in Ca++ movements produced by the agonist. The electrophysiological studies showed that isoproterenol (10 M) could inhibit spontaneous contractility of the rat uterus without causing hyperpolarlzation. In hlgh-K+ depolarized muscle, Isoproterenol (10⁻⁶M) produced relaxation without any change in membrane potential. These data suggested that hyperpolarlzation of cell membranes is not a prerequisite for β-adrenoceptor-med-lated relaxation of uterine smooth muscle. / Pharmaceutical Sciences, Faculty of / Graduate

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23468号 / 医博第4775号 / 新制||医||1053(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 竹内 理, 教授 山下 潤, 教授 戸口田 淳也 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

We have shown that the stimulation of β-adrenergic receptors (β-AR) increases apoptosis in adult rat ventricular myocytes (ARVMs). Integrins, a family of αβ-heterodimeric cell surface receptors, are postulated to play a role in ventricular remodeling. Here, we show that norepinephrine (NE) increases β1 integrins expression in ARVMs via the stimulation of α1-AR, not β-AR. Inhibition of ERK1/2 using PD 98059, an inhibitor of ERK1/2 pathway, inhibited α1-AR-stimulated increases in β1 integrins expression. Activation of β1 integrins signaling pathway using laminin (LN) inhibited β-AR-stimulated apoptosis as measured by terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL)-staining and flow cytometry. Likewise, ligation of β1 integrins with anti-β1 integrin antibodies prevented β-AR-stimulated apoptosis. Treatment of cells using LN or anti-β1 integrin antibodies activated ERK1/2 pathway. PD 98059 inhibited activation of ERK1/2 by LN, and prevented the anti-apoptotic effects of LN. Thus (1) stimulation of α1-AR regulates β1 integrins expression via the activation of ERK1/2, (2) β1 integrins signaling protects ARVMs from β-AR-stimulated apoptosis, (3) activation of ERK1/2 plays a critical role in the anti-apoptotic effects of β1-integrin signaling. These data suggest that β1 integrin signaling protects ARVMs against β-AR-stimulated apoptosis possibly via the involvement of ERK1/2.

Stimulation of β-adrenergic receptor (β-AR) induces cardiac myocyte apoptosis. Integrins, a family of cell-surface receptors, play an important role in the regulation of cardiac myocyte apoptosis and ventricular remodeling. Cleavage of extracellular domain of β1 integrin, also called integrin shedding, is observed during cardiac hypertrophy and progression to early heart failure. Here we show that stimulation of β-AR induces β1 integrin fragmentation in mouse heart. To examine the role of intracellular domain of β1 integrin in cardiac myocyte apoptosis, a chimeric receptor consisting of the cytoplasmic tail domain of β1A integrin and the extracellular/transmembrane domain of the interleukin-2 receptor (TAC-β1) was expressed in adult rat ventricular myocytes (ARVM) using adenoviruses. TAC-β1 increased the percentage of apoptotic ARVM as measured by TUNEL-staining assay. TAC-β1-induced apoptosis was found to be associated with increased cytosolic cytochrome c and decreased mitochondrial membrane potential. TAC-β1 increased caspase-8 activity. Z-IETD-FMK, a specific caspase-8 inhibitor, significantly inhibited TAC-β1-induced apoptosis. TAC-β1 expression also increased cleavage of Bid, a pro-apoptotic Bcl-2 family protein. These data suggest that shedding of β1 integrin may be a mechanism of induction of apoptosis during β-AR-stimulated cardiac remodeling.

Recent data suggest novel functional roles for cerebellar involvement in a number of neurologic diseases. Function of cerebellar neurons is known to be modulated by norepinephrine and adrenergic receptors. The distribution of adrenergic receptor subtypes has been described in experimental animals, but corroboration of such studies in the human cerebellum, necessary for drug treatment, is still lacking. In the present work we studied cell-specific localizations of α1 adrenergic receptor subtype mRNA (α1a, α1b, α1d), and α2 adrenergic receptor subtype mRNA (α2a, α2b, α2c) by in situ hybridization on cryostat sections of human cerebellum (cortical layers and dentate nucleus). We observed unique neuron-specific α1 adrenergic receptor and α2 adrenergic receptor subtype distribution in human cerebellum. The cerebellar cortex expresses mRNA encoding all six α adrenergic receptor subtypes, whereas dentate nucleus neurons express all subtype mRNAs, except α2a adrenergic receptor mRNA. All Purkinje cells label strongly for α2a and α2b adrenergic receptor mRNA. Additionally, Purkinje cells of the anterior lobe vermis (lobules I to V) and uvula/tonsil (lobules IX/HIX) express α1a and α2c subtypes, and Purkinje cells in the ansiform lobule (lobule HVII) and uvula/tonsil express α1b and α2c adrenergic receptor subtypes. Basket cells show a strong signal for α1a, moderate signal for α2a and light label for α2b adrenergic receptor mRNA. In stellate cells, besides a strong label of α2a adrenergic receptor mRNA in all and moderate label of α2b message in select stellate cells, the inner stellate cells are also moderately positive for α1b adrenergic receptor mRNA. Granule and Golgi cells express high levels of α2a and α2b adrenergic receptor mRNAs. These data contribute new information regarding specific location of adrenergic receptor subtypes in human cerebellar neurons. We discuss our observations in terms of possible modulatory roles of adrenergic receptor subtypes in cerebellar neurons responding to sensory and autonomic input signals, and review species differences in cerebellar adrenergic receptor expression.

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Th ocular bilateral hypotensive effect after unilateral topical administration of medetomidine and 4 analogs was demonstrated in a dose-response study (0.5%-2%) in NZW rabbits (bilateral IOP-lowering efficacy: medetomidine>detomidine and MPV-1440>MPV-1441 and MPV-305BIII).

Cardiovascular disease is responsible for the loss of one life every 38 seconds and accounts for 26.6 percent of all infants that die of congenital birth defects. Adrenergic hormones are critically important regulators of cardiovascular physiology in embryos and adults. They are key mediators of stress responses and have profound stimulatory effects on cardiovascular function, and dysregulation of adrenergic function has been associated with many adverse cardiac conditions, including congenital malformations, arrhythmias, ischemic heart disease, heart failure, and sudden cardiac death. Despite intensive study, the specific roles these hormones play in the developing heart is not well-understood. Further, there is little information available regarding how these important hormones mediate stress responses in adult females (before and after menopause) in comparison to males. My thesis thus has two major foci: (1) What role(s) do catecholamines play in the embryonic heart?, and (2) Do catecholamines differentially influence cardiac function in aging male and female hearts? Initially, we sought to uncover the roles of adrenergic hormones in the embryonic heart by utilizing an adrenergic-deficient (Dbh-/-) mouse model. We found that adrenergic hormones influence heart development by stimulating expression of the gap junction protein, connexin 43, facilitating atrioventricular conduction, and helping to maintain cardiac rhythm. As development progresses, cardiac energy demands increase substantially, and oxidative phosphorylation becomes vital. Adrenergic hormones regulate metabolism in adults, thus we hypothesized they may stimulate energy metabolism during the embryonic/fetal transition period. We examined ATP, ADP, oxygen consumption rate, and extracellular acidification rates and found these metabolic indices were significantly decreased in Dbh-/- hearts compared to Dbh+/+ controls. We employed transmission electron microscopy of embryonic cardiomyocytes and found the mitochondria were significantly larger in Dbh-/- hearts compared to controls, and had more branch points. Taken together, these results suggest adrenergic hormones play a major role mediating the shift from predominantly anaerobic to aerobic metabolism during the embryonic/fetal transition period. Since there are known differential cardiac responses due to sex, age, and menopause to stress, we used echocardiography to measure left ventricular (LV) function in adult (9, 18 and 21 month) male and female mice (pre and postmenopausal) in response to epinephrine, and immobilization stress to investigate the roles of these factors. My results show 9-month premenopausal female mice display significantly decreased LV responsiveness to epinephrine compared to males, and an increased response to epinephrine due to age, especially in the premenopausal females. Similar LV function was also observed between postmenopausal females and males, and this pattern persisted after immobilization stress. I also investigated anatomical differences in the distribution of adrenergic cells within the heart comparing age, sex, and menopausal status. Notably, the density of cells derived from an adrenergic lineage in the heart was significantly increased in postmenopausal mice compared to age-matched males and cycling females. The selective re-appearance of adrenergic cells in the heart following menopause may provide an explanation for the differential stress responses observed in our system, and could have important clinical ramifications for stress-induced cardiomyopathies.