Yumiko Toyohira

Asymmetric Dimethylarginine Produces Vascular Lesions in Endothelial Nitric Oxide Synthase–Deficient Mice: Involvement of Renin-Angiotensin System and Oxidative Stress

Objective—Asymmetric dimethylarginine (ADMA) is widely believed to be an endogenous nitric oxide synthase (eNOS) inhibitor. However, in this study, we examined our hypothesis that the long-term vascular effects of ADMA are not mediated by inhibition of endothelial NO synthesis. Methods and Results—ADMA was infused in wild-type and eNOS-knockout (KO) mice by osmotic minipump for 4 weeks. In wild-type mice, long-term treatment with ADMA caused significant coronary microvascular lesions. Importantly, in eNOS-KO mice, treatment with ADMA also caused an extent of coronary microvascular lesions that was comparable to that in wild-type mice. These vascular effects of ADMA were not prevented by supplementation of l-arginine, and vascular NO production was not reduced by ADMA treatment. Treatment with ADMA caused upregulation of angiotensin-converting enzyme (ACE) and an increase in superoxide production that were comparable in both strains and that were abolished by simultaneous treatment with temocapril (ACE inhibitor) or olmesartan (AT1 receptor antagonist), which simultaneously suppressed vascular lesion formation. Conclusions—These results provide the first direct evidence that the long-term vascular effects of ADMA are not solely mediated by simple inhibition of endothelial NO synthesis. Direct upregulation of ACE and increased oxidative stress through AT1 receptor appear to be involved in the long-term vascular effects of ADMA in vivo.

Asymmetric Dimethylarginine Causes Arteriosclerotic Lesions in Endothelial Nitric Oxide Synthase-Deficient Mice. Involvement of Renin-Angiotensin System and Oxidative Stress

Objective—Asymmetric dimethylarginine (ADMA) is widely believed to be an endogenous nitric oxide synthase (eNOS) inhibitor. However, in this study, we examined our hypothesis that the long-term vascular effects of ADMA are not mediated by inhibition of endothelial NO synthesis. Methods and Results—ADMA was infused in wild-type and eNOS-knockout (KO) mice by osmotic minipump for 4 weeks. In wild-type mice, long-term treatment with ADMA caused significant coronary microvascular lesions. Importantly, in eNOS-KO mice, treatment with ADMA also caused an extent of coronary microvascular lesions that was comparable to that in wild-type mice. These vascular effects of ADMA were not prevented by supplementation of l-arginine, and vascular NO production was not reduced by ADMA treatment. Treatment with ADMA caused upregulation of angiotensin-converting enzyme (ACE) and an increase in superoxide production that were comparable in both strains and that were abolished by simultaneous treatment with temocapril (ACE inhibitor) or olmesartan (AT1 receptor antagonist), which simultaneously suppressed vascular lesion formation. Conclusions—These results provide the first direct evidence that the long-term vascular effects of ADMA are not solely mediated by simple inhibition of endothelial NO synthesis. Direct upregulation of ACE and increased oxidative stress through AT1 receptor appear to be involved in the long-term vascular effects of ADMA in vivo.

Pituitary Adenylate Cyclase‐Activating Polypeptide Causes Ca2+ Release from Ryanodine/Caffeine Stores Through a Novel Pathway Independent of Both Inositol Trisphosphates and Cyclic AMP in Bovine Adrenal Medullary Cells

Abstract: Pituitary adenylate cyclase‐activating polypeptide (PACAP) causes both Ca2+ release and Ca2+ influx in bovine adrenal chromaffin cells. To elucidate the mechanisms of PACAP‐induced Ca2+ release, we investigated expression of PACAP receptors and measured inositol trisphosphates (IP3), cyclic AMP, and the intracellular Ca2+ concentration in bovine adrenal medullary cells maintained in primary culture. RT‐PCR analysis revealed that bovine adrenal medullary cells express the PACAP receptor hop, which is known to couple with both IP3 and cyclic AMP pathways. The two naturally occurring forms of PACAP, PACAP38 and PACAP27, both increased cyclic AMP and IP3, and PACAP38 was more potent than PACAP27 in both effects. Despite the effects of PACAP on IP3 production, the Ca2+ release induced by PACAP38 or by PACAP27 was unaffected by cinnarizine, a blocker of IP3 channels. The potencies of the peptides to cause Ca2+ release in the presence of cinnarizine were similar. The Ca2+ release induced by PACAP38 or by PACAP27 was strongly inhibited by ryanodine and caffeine. In the presence of ryanodine and caffeine, PACAP38 was more potent than PACAP27. PACAP‐induced Ca2+ release was unaffected by Rp‐adenosine 3′,5′‐cyclic monophosphothioate, an inhibitor of protein kinase A. Ca2+ release induced by bradykinin and angiotensin II was also inhibited by ryanodine and caffeine, but unaffected by cinnarizine. Although IP3 production stimulated by PACAP38 or bradykinin was abolished by the phospholipase C inhibitor, U‐73122, Ca2+ release in response to the peptides was unaffected by U‐73122. These results suggest that PACAP induces Ca2+ release from ryanodine/caffeine stores through a novel intracellular mechanism independent of both IP3 and cyclic AMP and that the mechanism may be the common pathway through which peptides release Ca2+ in adrenal chromaffin cells.

Vascular neuronal NO synthase is selectively upregulated by platelet-derived growth factor: involvement of the MEK/ERK pathway.

Objective—We demonstrated recently that neuronal NO synthase (NOS) is expressed in arteriosclerotic lesions and exerts important vasculoprotective effects in vivo. In this study, we examined the molecular mechanism(s) for vascular neuronal NOS (nNOS) expression. Methods and Results—In cultured rat aortic smooth muscle cells, treatment with platelet-derived growth factor (PDGF) selectively upregulated nNOS expression but not inducible NOS (iNOS) or endothelial NOS (eNOS) expression. Treatment with PDGF also significantly caused activation of mitogen-activated protein kinase (MAPK) family, including extracellular signal-regulated kinase (ERK), p38MAPK, and c-Jun N-terminal kinase (JNK). ERK kinase (MAPK kinase [MEK]) inhibitors inhibited PDGF-induced nNOS expression, whereas a p38MAPK inhibitor or JNK inhibitor was without effects. Importantly, gene transfer of MEK per se elicited nNOS induction, and gene transfer of dominant-negative MEK abolished PDGF-induced nNOS expression. In isolated aortas of wild-type, eNOS−/−, and iNOS−/− mice, but not in those of nNOS−/− mice, treatment with PDGF significantly enhanced nNOS expression and nitrite plus nitrate production, both of which were again attenuated by a MEK inhibitor. Conclusions—These results provide the first evidence that vascular nNOS expression is upregulated selectively in response to PDGF through the MEK/ERK pathway. Upregulated nNOS may play an important compensatory role under arteriosclerotic/inflammatory conditions associated with eNOS dysfunction to maintain vascular homeostasis.

Regulation of Catecholamine Synthesis by Leptin

Abstract: Obesity is often associated with cardiovascular and metabolic disorders such as hypertension and hyperglycemia. Leptin, a protein product of the obese gene, regulates satiety and energy expenditure through its receptors in the hypothalamus. Recent studies have shown that leptin has extrahypothalamic and peripheral actions. The presence of leptin receptors has been reported in the adrenal medulla. In the present study, we examined the effects of leptin on catecholamine synthesis in cultured bovine adrenal medullary cells. Leptin (3‐30 nM) caused a significant increase in 14C‐catecholamine synthesis from [14C] tyrosine, but not from [14C] DOPA. Incubation of cells with leptin resulted in an activation and phosphorylation of tyrosine hydroxylase. Leptin caused a transient activation of mitogen‐activated protein kinases (MAPKs). U0126, an inhibitor of MAPK kinase, abolished the effect of leptin on 14C‐catecholamine synthesis. High concentrations of leptin (10‐100 nM) produced an increase in intracellular Ca2+ concentration, which was blocked by Cd2+, an inhibitor of voltage‐dependent Ca2+ channels. Concurrent treatment of cells with leptin (10 nM) and acetylcholine (0.3 mM) potently enhanced the stimulatory effect of acetylcholine on 14C‐catecholamine synthesis. Leptin, however, failed to enhance the stimulatory effect of acetylcholine on the phosphorylation and activity of tyrosine hydroxylase. Acetylcholine (0.3 mM) decreased the intracellular pH (pHi). Leptin (10 nM) affected neither the basal pHi nor the acetylcholine‐induced fall in pHi. These findings suggest that leptin phosphorylates and activates tyrosine hydroxylase and subsequently stimulates catecholamine synthesis through MAPK and probably Ca2+ pathways in the adrenal medulla.

Sites of positive allosteric modulation by neurosteroids on ionotropic γ-aminobutyric acid receptor subunits

Neurosteroids are known as allosteric modulators of ionotropic γ‐aminobutyric acid (GABA) receptors. Here, we investigated sites of positive allosteric modulation by allotetrahydrodeoxycorticosterone (5α‐THDOC) at GABA receptors using the technique of chimeragenesis and the Xenopus oocyte expression system. Our findings have demonstrated that the region from transmembrane segment (TM) 4 to the C‐terminus of the GABAA receptor α1 subunit is crucial for the action of 5α‐THDOC, but insufficient for the action of another neurosteroid allopregnanolone, suggesting that a specific region critical for neurosteroid action at GABA receptors exists in the domain between TM4 and the C‐terminus of GABA receptor subunits.

Inhibitory effects of clozapine and other antipsychotic drugs on noradrenaline transporter in cultured bovine adrenal medullary cells

Abstract The effects of clozapine and other antipsychotic drugs on noradrenaline (NA) transport were examined in cultured bovine adrenal medullary cells and in transfected Xenopus laevis oocytes expressing the bovine NA transporter. Incubation of adrenal medullary cells with clozapine (30–1000 ng/ml) inhibited desipramine (DMI)-sensitive uptake of [3H]NA in a concentration-dependent manner (IC50=110 ng/ml or 336 nM). Other antipsychotic drugs such as haloperidol, chlorpromazine, and risperidone also decreased [3H]NA uptake (IC50= 144, 220, and 210 ng/ml or 383, 690, and 512 nM, respectively). Eadie-Hofstee analysis showed that clozapine reduced Vmax of uptake of [3H]NA and increased Km. Furthermore, clozapine inhibited specific binding of [3H]DMI to plasma membranes isolated from bovine adrenal medulla (IC50=48 ng/ml or 146 nM). Scatchard plot analysis of [3H]DMI binding revealed that clozapine decreased both Bmax and Kd. Other antipsychotic drugs, including haloperidol, chlorpromazine, and risperidone, also reduced [3H]DMI binding to the membranes. In transfected Xenopus oocytes expressing the bovine NA transporter, clozapine inhibited [3H]NA uptake in a concentration-dependent manner similar to that observed in adrenal medullary cells. These results suggest that clozapine and haloperidol directly inhibit transport of NA by acting on the site of an NA transporter that influences both substrate transport and binding of tricyclic antidepressants.

Tramadol inhibits norepinephrine transporter function at desipramine-binding sites in cultured bovine adrenal medullary cells.

Tramadol is a widely used analgesic, but its mode of action is not well understood. To study the effects of tramadol on norepinephrine transporter (NET) function, we assayed the effect of tramadol on [3H]-norepinephrine ([3H]-NE) uptake and [3H]-desipramine binding to plasma membranes isolated from bovine adrenal medulla. We then characterized [14C]-tramadol binding in cultured bovine adrenal medullary cells. Tramadol inhibited the desipramine-sensitive uptake of [3H]-NE by the cells in a concentration-dependent manner (50% inhibitory concentration = 21.5 ± 6.0 &mgr;M). Saturation analysis revealed that tramadol increased the apparent Michaelis constant of [3H]-NE uptake without changing the maximal velocity, indicating that inhibition occurred via competition for the NET (inhibition constant, Ki = 13.7 &mgr;M). Tramadol inhibited the specific binding of [3H]-desipramine to plasma membranes. Scatchard analysis of [3H]-desipramine binding revealed that tramadol increased the apparent dissociation constant (Kd) for binding without altering maximal binding, indicating competitive inhibition (Ki = 11.2 &mgr;M). The binding of [14C]-tramadol to the cells was specific and saturable, with a Kd of 18.1 ± 2.4 &mgr;M. These findings indicate that tramadol competitively inhibits NET function at desipramine-binding sites.

Vascular Neuronal NO Synthase Is Selectively Upregulated by Platelet-Derived Growth Factor. Involvement of the Mitogen-Activated Protein Kinase Kinase/Extracellular Signal-Regulated Kinase Pathway

Objective—We demonstrated recently that neuronal NO synthase (NOS) is expressed in arteriosclerotic lesions and exerts important vasculoprotective effects in vivo. In this study, we examined the molecular mechanism(s) for vascular neuronal NOS (nNOS) expression. Methods and Results—In cultured rat aortic smooth muscle cells, treatment with platelet-derived growth factor (PDGF) selectively upregulated nNOS expression but not inducible NOS (iNOS) or endothelial NOS (eNOS) expression. Treatment with PDGF also significantly caused activation of mitogen-activated protein kinase (MAPK) family, including extracellular signal-regulated kinase (ERK), p38MAPK, and c-Jun N-terminal kinase (JNK). ERK kinase (MAPK kinase [MEK]) inhibitors inhibited PDGF-induced nNOS expression, whereas a p38MAPK inhibitor or JNK inhibitor was without effects. Importantly, gene transfer of MEK per se elicited nNOS induction, and gene transfer of dominant-negative MEK abolished PDGF-induced nNOS expression. In isolated aortas of wild-type, eNOS−/−, and iNOS−/− mice, but not in those of nNOS−/− mice, treatment with PDGF significantly enhanced nNOS expression and nitrite plus nitrate production, both of which were again attenuated by a MEK inhibitor. Conclusions—These results provide the first evidence that vascular nNOS expression is upregulated selectively in response to PDGF through the MEK/ERK pathway. Upregulated nNOS may play an important compensatory role under arteriosclerotic/inflammatory conditions associated with eNOS dysfunction to maintain vascular homeostasis.

Characterization and functional role of leptin receptor in bovine adrenal medullary cells.

We report here the characterization and functional roles of the leptin receptor (ObR) in bovine adrenal medullary cells. The plasma membranes isolated from bovine adrenal medulla showed a single class of specific binding sites of (125)I-leptin with an apparent K(d) of 6.6 nM and B(max) of 62 fmol/mg protein. ObRa but not ObRb mRNA was detected in bovine adrenal medulla by reverse transcriptase-polymerase chain reaction. Incubation of cultured adrenal medullary cells with leptin (3-30 nM) for 20 min resulted in a significant increase in [(14)C]catecholamine synthesis from [(14)C]tyrosine without any change in catecholamine secretion. These findings suggest that leptin stimulates catecholamine synthesis through its receptors in bovine adrenal medullary cells.