Adrenomedullin in the rostral ventrolateral medulla increases arterial pressure and heart rate: roles of glutamate and nitric oxide

This study was done to investigate the effects of microinjections of adrenomedullin (ADM), a vasoactive neuropeptide, in the rostral ventrolateral medulla (RVLM) on mean arterial pressure (MAP) and heart rate (HR) in urethane-anesthetized rats, and to assess the potential roles of glutamate and nitric oxide (NO) in these effects. Unilateral injections of ADM (0.01 or 0.1 pmol) into the RVLM significantly increased MAP and HR in a dose-dependent manner, whereas ADM at 0.001 pmol was ineffective. Microinjections of ADM (0.01 pmol) outside the RVLM had no effects on MAP or HR. Coinjections of a putative ADM receptor antagonist, ADM(22-52) (0.01 pmol), abolished the increases in MAP and HR evoked by ADM (0.01 pmol). The vasopressor effects of ADM (0.01 pmol) in the RVLM were abolished by coinjections of either dizocilpine hydrogen maleate (a selective NMDA glutamate receptor antagonist, 500 pmol) or 6-cyano-7-nitroquinoxaline-2,3-dione (a selective non-NMDA glutamate receptor antagonist, 50 pmol). The ADM-induced vasopressor effects were also abolished by coadministration of either 7-nitroindazole sodium salt (a selective neuronal NO synthase inhibitor, 0.05 pmol) or methylene blue (a soluble guanylyl cyclase inhibitor, 100 pmol). These results suggest that ADM in the RVLM stimulates increases in MAP and HR through ADM receptor-mediated mechanisms. These effects are mediated by glutamate via both NMDA and non-NMDA receptors. NO, derived from neuronal NO synthase, also contributes to the ADM-induced vasopressor effects via a soluble guanylyl cyclase-associated signaling pathway.     

Decrease in arterial pressure induced by adrenomedullin in the hypothalamic paraventricular nucleus is mediated by nitric oxide and GABA

We tested the hypothesis that the decrease in arterial pressure induced by adrenomedullin (ADM) in the hypothalamic paraventricular nucleus (PVN) is mediated by nitric oxide (NO) and/or GABA. Unilateral microinjections of ADM into the PVN of anesthetized rats caused a significant decrease in mean arterial pressure (MAP). The ADM-induced decrease in MAP was significantly attenuated by pretreatment with N(psi)-nitro-L-arginine methyl ester (L-NAME, a non-selective NOS inhibitor), 7-nitroindazole sodium salt (7-NiNa, a selective neuronal NOS inhibitor), N5-(1-Iminoethyl)-L-ornithine (L-NIO, a selective endothelial NOS inhibitor) or bicuculline methiodide, but pretreatment with S-methylisothiourea (SMIT, a selective inducible NOS inhibitor) had no effect on this ADM-induced effect. In addition, coronal sections of rat brains were processed for combined NADPH-diaphorase (a marker of neuronal NOS-containing neurons) histochemistry and in situ hybridization for the receptor-activity-modifying protein 2 (a specific ADM receptor component). Double-labeled neurons were found in both parvocellular and magnocellular subdivisions of the PVN, confirming that NO-producing neurons in the PVN are capable of mediating ADM's effects. Thus, our data provide evidence that the ADM-induced decrease in MAP in the PVN is mediated by NO from neuronal and endothelial NOS, and by GABA.     

Activation of lateral parabrachial nucleus neurons restores blood pressure and sympathetic vasomotor drive after hypotensive hemorrhage

Lesions of the lateral parabrachial nucleus (LPBN) impair blood pressure recovery after hypotensive blood loss (Am J Physiol Regul Integr Comp Physiol 280: R1141, 2001). This study tested the hypothesis that posthemorrhage blood pressure recovery is mediated by activation of neurons, located in the ventrolateral aspect of the LPBN (VL-LPBN), that initiates blood pressure recovery by restoring sympathetic vasomotor drive. Hemorrhage experiments (16 ml/kg over 22 min) were performed in unanesthetized male Sprague-Dawley rats prepared with bilateral ibotenate lesions or guide cannulas directed toward the external lateral subnucleus of the VL-LPBN. Hemorrhage initially decreased mean arterial pressure (MAP) from approximately 100 mmHg control to 40-50 mmHg, and also decreased heart rate. In animals with sham lesions, MAP returned to 84 +/- 4 mmHg by 40 min posthemorrhage, and subsequent autonomic blockade with hexamethonium reduced MAP to 53 +/- 2 mmHg. In contrast, animals with VL-LPBN lesions remained hypotensive at 40 min posthemorrhage (58 +/- 4 mmHg) and hexamethonium had no effect on MAP, implying a deficit in sympathetic tone. VL-LPBN lesions did not alter the renin response or the effect of vasopressin V1 receptor blockade after hemorrhage. Posthemorrhage blood pressure recovery was also significantly delayed by VL-LPBN infusion of the ionotropic glutamate receptor antagonist kynurenic acid. Both VL-LPBN lesions and VL-LPBN kynurenate infusion caused posthemorrhage bradycardia to be significantly prolonged. Bradycardia was reversed by hexamethonium or atropine, but did not contribute to posthemorrhage hypotension. Taken together, these data support the hypothesis that stimulation of VL-LPBN glutamate receptors mediates spontaneous blood pressure recovery by initiating restoration of sympathetic vasomotor drive.     

Nitric oxide inhibits depolarization-evoked glutamate release from rat cerebellar granule cells.

Nitric oxide (NO) modulates the release of various neurotransmitters, some of these are considered to be involved in neuronal plasticity that includes long-term depression in the cerebellum. To date, there have been no reports on the modulation of the exocytotic release of neurotransmitters in the cerebellar granule cells (CGCs) by NO. The aim of this study was to investigate the effects of NO on the exocytotic release of glutamate from rat CGCs. Treatment with NO-related reagents revealed that NO inhibited high-K(+)-evoked glutamate release. Clostridium botulinum type B neurotoxin (BoNT/B) attenuated the enhancement of glutamate release caused by NO synthase (NOS) inhibition; this indicates that NO acts on the high-K(+)-evoked exocytotic pathway. cGMP-related reagents did not affect the high-K(+)-evoked glutamate release. NO-related reagents did not affect Ca(2+) ionophore-induced glutamate release, suggesting that NO inhibits Ca(2+) entry through voltage-dependent Ca(2+) channels (VDCC). Monitoring of intracellular Ca(2+) revealed that NO inhibited high-K(+)-evoked Ca(2+) entry. L-type VDCC blockers inhibited glutamate release and NO did not have an additive effect on the inhibition produced by the L-type VDCC blocker. The inhibition of the high-K(+)-evoked glutamate release by NO was abolished by a reducing reagent; this suggested that NO regulates the high-K(+)-evoked glutamate release from CGCs by redox modulation.     

Nicotine-induced phosphorylation of Akt through epidermal growth factor receptor and Src in PC12h cells

Nicotine treatment triggers calcium influx into neuronal cells, which promotes cell survival in a number of neuronal cells. Phosphoinositide (PI) 3-kinase and downstream PI3-kinase target Akt have been reported to be important in the calcium-mediated promotion of survival in a wide variety of cells. We investigated the mechanisms of nicotine-induced phosphorylation of Akt in PC12h cells, in comparison with nicotine-induced ERK phosphorylation. Nicotine induced Akt phosphorylation in a dose-dependent manner. A nicotinic acetylcholine receptor (nAChR) alpha7 subunit-selective inhibitor had no significant effect on nicotine-induced Akt phosphorylation, while a non-selective nAChR antagonist inhibited the phosphorylation. L-type voltage-sensitive calcium channel (VSCC) antagonists, calmodulin antagonist, and Ca2+/calmudulin-dependent protein kinase (CaM kinase) inhibitor prevented the nicotine-induced Akt phosphorylation. Three epidermal growth factor receptor (EGFR) inhibitors prevented the nicotine-induced phosphorylation of both extracellular signal-regulated protein kinase (p42/44 MAP kinase, ERK) and Akt. In contrast, an inhibitor of the Src family tyrosine kinase prevented the nicotine-induced Akt phosphorylation but not ERK phosphorylation. These results suggested that nicotine induces the activation of both PI3-kinase/Akt and ERK pathways via common pathways including non-alpha7-nAChRs, L-type VSCC, CaM kinase II and EGFR in PC12h cells, but Src family tyrosine kinases only participate in the pathway to activate Akt.     

血管紧张素—（1—7）在大鼠延髓头端腹外侧通过氨基酸类递质调节血压的作用

采用微量注射、微透析、高效液相色谱－荧光测定等技术和方法,观察和 血管紧张素－（1－7）[Ang-(1-7)]在延髓头端腹外侧（RVLM）与氨基酸类递质释放之间的关系,在麻醉大鼠RVLM注射Ang-(1-7)可引起血压升高,同时伴RVLM兴奋性氨基酸（EAA）释放增多;在RVLM注射Ang-(1-7)选择性受体拮抗剂Ang779可引起血压降低,同时伴RVLM EAA释放减少和抑制性氨基酸（IAA）释放增多,Ang-(1-7)的升压作用和Ang779的降压作用均可被相应的氨基酸受体拮抗剂部分阻娄。结果提示,Ang-(1-7)在RVLM的升压效庆可能部分是通过EAA释放增多所致;而Ang779在 RVLM的降压效应可能部分是通过EAA释放减少、IAA释放增多所致。     

HIV-1 Tat through phosphorylation of NMDA receptors potentiates glutamate excitotoxicity

Toxic effects of HIV-1 proteins contribute to altered function and decreased survival of select populations of neurons in HIV-1-infected brain. One such HIV-1 protein, Tat, can activate calcium release from IP3-sensitive intracellular pools, induce calcium influx in neural cells, and, as a result, can increase neuronal cell death. Here, we provide evidence that Tat potentiates excitatory amino acid (glutamate and NMDA) triggered calcium flux, as well as glutamate- and staurosporine-mediated neurotoxicity. Calcium flux in cultured rat hippocampal neurons triggered by the transient application of glutamate or NMDA was facilitated by pre-exposure to Tat. Facilitation of glutamate-triggered calcium flux by Tat was prevented by inhibitors of ADP-ribosylation of G(i)/G(o) proteins (pertussis toxin), protein kinase C (H7 and bisindolymide), and IP3-mediated calcium release (xestospongin C), but was not prevented by an activator of G(s) (cholera toxin) or an inhibitor of protein kinase A (H89). Facilitation of NMDA-triggered calcium flux by Tat was reversed by inhibitors of tyrosine kinase (genestein and herbimycin A) and by an inhibitor of NMDA receptor function (zinc). Tat increased 32P incorporation into NMDA receptor subunits NR2A and NR2B and this effect was blocked by genestein. Subtoxic concentrations of Tat combined with subtoxic concentrations of glutamate or staurosporine increased neuronal cell death significantly. Together, these findings suggest that NMDA receptors play an important role in Tat neurotoxicity and the mechanisms identified may provide additional therapeutic targets for the treatment of HIV-1 associated dementia.     

Hemodynamic actions of endothelin in conscious and anesthetized dogs

The newly described endogenous peptide, endothelin, was administered to five chronically instrumented conditioned dogs. Endothelin produced significant and simultaneous increases in both heart rate (HR) and mean arterial pressure (MAP) in conscious dogs. Endothelin also produced significant increases in MAP in anesthetized animals. Ganglionic suppression induced by hexamethonium (10 mg/kg) and atropine (0.1 mg/kg) blocked HR responses and markedly inhibited the pressor responses to endothelin in conscious animals. These results suggest that endothelin in part acts to elevate blood pressure and heart rate through modification of autonomic nervous system tone. When endothelin and angiotensin II were administered in mole equivalent doses, angiotensin II produced a pressor response of greater magnitude than did endothelin in conscious animals.     

Protein Kinase C Modulates Calcium Sensitivity of Nitric Oxide Synthase in Cerebellar Slices

Abstract: The possible modulation of nitric oxide (NO) synthase (NOS) activity by protein kinase C (PKC) was investigated. Incubation of rat cerebellar slices with the specific metabotropic glutamate receptor agonist, (±)-1-aminocyclopentane- trans -1,3-dicarboxylate ( trans -ACPD) increased cyclic GMP concentration two-fold. The increase was dose-dependently blocked by the protein kinase inhibitors staurosporine and calphostin C. Phorbol 12-myristate 13-acetate (PMA), a PKC activator, increased cyclic GMP concentration without glutamate receptor activation. The cyclic GMP increases induced by PMA and trans -ACPD were independent of extracellular calcium blocked by N ^ω-nitro- l -arginine, a specific NOS inhibitor, and were not additive. Measurement of citrulline formation in cerebellar slices confirmed that NOS was activated by trans -ACPD and the activation was blocked by calphostin C. These results suggest that metabotropic glutamate receptor activates NOS through PKC. The calcium dependency of NOS activation was assessed in slices incubated with PMA and okadaic acid. NOS in both PMA-treated and untreated slices had similar activities at 100 n M free calcium, whereas at 25–70 n M free calcium, NOS in PMA-treated slices was more active than that in untreated slices. These results suggest that PKC regulates NO release in resting neurons by modulating the sensitivity of NOS at low calcium concentrations.     

Involvement of ERK in NMDA receptor-independent cortical neurotoxicity of hydrogen sulfide

Hydrogen sulfide (H₂S), a gasotransmitter, exerts both neurotoxicity and neuroprotection, and targets multiple molecules including NMDA receptors, T-type calcium channels and NO synthase (NOS) that might affect neuronal viability. Here, we determined and characterized effects of NaHS, an H₂S donor, on cell viability in the primary cultures of mouse fetal cortical neurons. NaHS caused neuronal death, as assessed by LDH release and trypan blue staining, but did not significantly reduce the glutamate toxicity. The neurotoxicity of NaHS was resistant to inhibitors of NMDA receptors, T-type calcium channels and NOS, and was blocked by inhibitors of MEK, but not JNK, p38 MAP kinase, PKC and Src. NaHS caused prompt phosphorylation of ERK and upregulation of Bad, followed by translocation of Bax to mitochondria and release of mitochondrial cytochrome c, leading to the nuclear condensation/fragmentation. These effects of NaHS were suppressed by the MEK inhibitor. Our data suggest that the NMDA receptor-independent neurotoxicity of H₂S involves activation of the MEK/ERK pathway and some apoptotic mechanisms.     

Multiple calcium channels and kinases mediate alpha7 nicotinic receptor neuroprotection in PC12 cells

alpha7 Nicotinic receptors are calcium permeant and provide neuroprotection against many insults. We investigated the roles of intracellular calcium ions and downstream calcium channels in this protection. The alpha7 agonist GTS-21 prevented pheochromocytoma cell death induced by nerve growth factor + serum deprivation over a 3-day interval. This effect was blocked by the intracellular calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid in a manner that did not appear to involve changes in receptor density. 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid blocked GTS-21-induced protein kinase C activation, a necessary process for protection. The insositol triphosphate calcium-channel blocker xestospongin C and the phospholipases C inhibitor U-73122 blocked protection, ryanodine partially attenuated protection, but the L-type channel antagonist nifedipine had no effect. ERK1/2 but not JNK and p38 were activated by GTS-21, and the ERK phosphorylation inhibitors PD98059 and U0126 blocked protection.     

Ionotropic glutamate receptor activation increases intracellular calcium in prolactin-releasing cells of the adenohypophysis

Endocrine cells of the anterior pituitary are controlled by the central nervous system through hormonal interactions and are not believed to receive direct synaptic connections from the brain. Studies suggest that some pituitary cells may be modulated by the neurotransmitter glutamate. We investigated prolactin (PRL)-releasing cells of the anterior pituitary of a euryhaline fish, the tilapia (Oreochromis mossambicus), for the presence of possible glutamate receptors (GluRs). Fura-2 imaging addressed the ability of glutamate to increase intracellular calcium. We observed a dose-dependent increase in intracellular calcium with transient perfusion (1-2 min) of glutamate (10 nM to 1 mM) in two-thirds of imaged cells. This increase was attenuated by the ionotropic GluR antagonist kynurenic acid (0.5-1.0 mM). The increase was also blocked or attenuated by antagonists of L-type voltage-gated calcium channels. The GluR agonist alpha-amino-3-hydroxy-5-methylisoxazole propionic acid (AMPA; 100 microM) produced intracellular calcium increases that were reversibly blocked by the selective AMPA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). In contrast, the selective agonist N-methyl-D-aspartate (NMDA; 100 microM to 1 mM in magnesium-free solution with 10 microM glycine) had no effect on intracellular calcium. Radioimmunoassays demonstrated that glutamate stimulated PRL release. CNQX but not the NMDA receptor antagonist 2-amino-5-phosphonovaleric acid blocked this release. Antibodies for mammalian AMPA- and NMDA-type GluR produced a similar punctate immunoreactivity in the periphery of PRL cells. However, the NMDA antibody recognized a protein of a different molecular mass in PRL cells compared with brain cells. These results clearly indicate the presence of GluRs on tilapia PRL cells that can stimulate PRL release.     

Muscarinic and beta-adrenergic regulation of heart rate, force of contraction and calcium current is preserved in mice lacking endothelial nitric oxide synthase

Nitric oxide (NO) is an ubiquitous signaling molecule produced from L-arginine by NO synthase (NOS). In the vasculature, NO mediates parasympathetic endothelium-dependent vasodilation. NO may also mediate the parasympathetic control of myocardial function. This is supported by the observations that NOS3, the endothelial constitutive NOS, is expressed in normal cardiac myocytes from rodents and human, and NOS and/or guanylyl cyclase inhibitors antagonize the effect of muscarinic agonists on heart rate, atrio-ventricular conduction, contractility and L-type calcium current. Here we examine the autonomic regulation of the heart in genetically engineered mice deficient in NOS3 (NOS3-KO). We show that the chronotropic and inotropic responses to both beta-adrenergic and muscarinic agonists were unaltered in isolated cardiac tissue preparations from NOS3-KO mice, although these mice have a defective parasympathetic regulation of vascular tone. Similarly, beta-adrenergic stimulation and muscarinic inhibition of the calcium current did not differ in cardiac myocytes from NOS3-KO mice and those from wild-type mice. RT-PCR did not demonstrate upregulation of other NOS isoforms. Similarly, Gi/Go proteins and muscarinic receptor density were unaltered. These data refute the idea that NOS3 is obligatory for the normal autonomic control of cardiac muscle function.     

垂体腺苷酸环化酶激活肽减轻谷氨酸对海马神经元的损伤并抑制胞内钙升高

对原代培养７～９ｄ的海马神经元给予谷氨酸处理,２４ｈ后,神经元的存活率降低。预先给予垂体腺苷酸环化酶激活肽（ＰＡＣＡＰ）能显著减少谷氨酸引起的海马神经元死亡。谷氨酸呈剂量依赖性增加海马神经元细胞内钙离子含量,ＰＡＣＡＰ能抑制谷氨酸引起的海马神经元细胞内钙离子浓度的升高,特异性ＰＡＣＡＰ Ⅰ型受体拮抗剂ＰＡＣＡＰ ６－３８能完全阻断ＰＡＣＡＰ减轻谷氨酸所致海马神经元损伤及降低谷氨酸所致神经元细胞内钙     

Bis(7)-tacrine attenuates beta amyloid-induced neuronal apoptosis by regulating L-type calcium channels

Beta amyloid protein (Abeta) and acetylcholinesterase (AChE) have been shown to be closely implicated in the pathogenesis of Alzheimer's disease. In the current study, we investigated the effects of bis(7)-tacrine, a novel dimeric AChE inhibitor, on Abeta-induced neurotoxicity in primary cortical neurons. Bis(7)-tacrine, but not other AChE inhibitors, elicited a marked reduction of both fibrillar and soluble oligomeric forms of Abeta-induced apoptosis as evidenced by chromatin condensation and DNA specific fragmentation. Both nicotinic and muscarinic receptor antagonists failed to block the effects of bis(7)-tacrine. Instead, nimodipine, a blocker of L-type voltage-dependent Ca2+ channels (VDCCs), attenuated Abeta neurotoxicity, whereas N-, P/Q- or R-type VDCCs blockers and ionotropic glutamate receptor antagonists did not. Fluorescence Ca2+ imaging assay revealed that, similar to nimodipine, bis(7)-tacrine reversed Abeta-triggered intracellular Ca2+ increase, which was mainly contributed by the extracellular Ca2+ instead of endoplasmic reticulum and mitochondria Ca2+. Concurrently, using whole cell patch-clamping technique, it was found that bis(7)-tacrine significantly reduced the augmentation of high voltage-activated inward calcium currents induced by Abeta. These results suggest that bis(7)-tacrine attenuates Abeta-induced neuronal apoptosis by regulating L-type VDCCs, offers a novel modality as to how the agent exerts neuroprotective effects.     

Involvement of angiotensin-(1-7) in the hypothalamic hypotensive effect of captopril in sinoaortic denervated rats

The role of anterior hypothalamic angiotensin-(1-7) (Ang-(1-7)) on blood pressure regulation was studied in sinoaortic denervated (SAD) rats. Since angiotensin-converting enzyme inhibitors increase endogenous levels of Ang-(1-7), we addressed the involvement of Ang-(1-7) in the hypotensive effect induced by captopril in SAD rats. Wistar rats 7 days after SAD or sham operation (SO) were anaesthetized and the carotid artery was cannulated for monitoring mean arterial pressure (MAP). A needle was inserted into the anterior hypothalamus for drug administration. Intrahypothalamic administration of Ang-(1-7) (5 pmol) was without effect in SO rats but reduced MAP in SAD rats by 15.5+/-3.2 mm Hg and this effect was blocked by 250 pmol [D-Ala(7)]-Ang-(1-7), a Mas receptor antagonist. Angiotensin II (Ang II) induced an increase in MAP in both groups being the effect greater in SAD rats (DeltaMAP=15.8+/-1.4 mm Hg) than in SO rats (DeltaMAP=9.6+/-1.0 mm Hg). Ang-(1-7) partially abolished the pressor response caused by Ang II in SAD rats. Whilst the captopril intrahypothalamic injection did not affect MAP in SO animals, it significantly reduced MAP in SAD rats (DeltaMAP=-13.3+/-1.9 mm Hg). Either [D-Ala(7)]-Ang-(1-7) or an anti-Ang-(1-7) polyclonal antibody partially blocked the MAP reduction caused by captopril. In conclusion, whilst Ang-(1-7) does not contribute to hypothalamic blood pressure regulation in SO normotensive animals, in SAD rats the heptapeptide induces a reduction of blood pressure mediated by Mas receptor activation. Although Ang-(1-7) is not formed in enough amount in the AHA of SAD animals to exert cardiovascular effects in normal conditions, our results suggest that enhancement of hypothalamic Ang-(1-7) levels by administration of captopril is partially involved in the hypotensive effect of the ACE inhibitor.     

Activation of NMDA receptors induces rapid dephosphorylation of the cytoskeletal protein MAP2

Hippocampal slices were preincubated with 32P-orthophosphate and used to study the effect of glutamate analogs on protein phosphorylation. NMDA induced a rapid, 70% decrease in the phosphorylation of the microtubule-associated protein MAP2, with no change in the total amount of MAP2. Both competitive and noncompetitive NMDA antagonists blocked the effect of NMDA, but a glutamate antagonist acting at non-NMDA receptors did not. Kainate and quisqualate were less potent than NMDA in stimulating dephosphorylation of MAP2. Other forebrain regions (necortex, striatum, and olfactory bulb) also showed dephosphorylation of MAP2 in response to NMDA. These and other results suggest that NMDA receptor activation induces the dephosphorylation of MAP2 by stimulating a protein phosphatase, possibly the calcium/calmodulin-dependent protein phosphatase calcineurin. Moreover, they indicate that alteration in the properties of a microtubule-associated protein may account for some of the effects of glutamate on postsynaptic neurons.     

Dehydroepiandrosterone stimulates nitric oxide release in vascular endothelial cells: evidence for a cell surface receptor

Dehydroepiandrosterone (DHEA) improves vascular function, but the mechanism of this effect is unclear. Since nitric oxide (NO) regulates vascular function, we hypothesized that DHEA affects the vasculature by increasing endothelial NO production. Physiological concentrations of DHEA stimulated NO release from intact bovine aortic endothelial cells (BAEC) within 5min. This effect was mediated by activation of endothelial nitric oxide synthase (eNOS) in BAEC and human umbilical vein endothelial cells (HUVEC). Dehydroepiandrosterone increased cyclic GMP (cGMP) levels in BAEC, consistent with its effect on NO production. Albumin-conjugated DHEA also stimulated NO release, suggesting that DHEA stimulates eNOS by a plasma membrane-initiated signal. Tamoxifen blocked estrogen-stimulated NO release from BAEC, but did not inhibit the DHEA effect. Pertussis toxin abolished the acute effect of DHEA on NO release. Dehydroepiandrosterone had no effect on intracellular calcium fluxes. However, inhibition of tyrosine kinases or the mitogen-activated protein (MAP) kinase kinase (MEK) blocked NO release and cGMP production in response to DHEA. These findings demonstrate that physiological concentrations of DHEA acutely increase NO release from intact vascular endothelial cells, by a plasma membrane-initiated mechanism. This action of DHEA is mediated by a steroid-specific, G-protein coupled receptor, which activates eNOS in both bovine and human cells. The release of NO is independent of intracellular calcium mobilization, but depends on tyrosine- and MAP kinases. This cellular mechanism may underlie some of the cardiovascular protective effects proposed for DHEA.     

Angiotensin II type 2 receptor-coupled nitric oxide production modulates free radical availability and voltage-gated Ca2+ currents in NTS neurons

The medial region of the nucleus tractus solitarius (mNTS) is a key brain stem site controlling cardiovascular function, wherein ANG II modulates neuronal L-type Ca(2+) currents via activation of ANG II type 1 receptors (AT(1)R) and production of reactive oxygen species (ROS). ANG II type 2 receptors (AT(2)R) induce production of nitric oxide (NO), which may interact with ROS and modulate AT(1)R signaling. We sought to determine whether AT(2)R-mediated NO production occurs in mNTS neurons and, if so, to elucidate the NO source and the functional interaction with AT(1)R-induced ROS or Ca(2+) influx. Electron microscopic (EM) immunolabeling showed that AT(2)R and neuronal NO synthase (nNOS) are coexpressed in neuronal somata and dendrites receiving synapses in the mNTS. In the presence of the AT(1)R antagonist losartan, ANG II increased NO production in isolated mNTS neurons, an effect blocked by the AT(2)R antagonist PD123319, but not the angiotensin (1-7) antagonist D-Ala. Studies in mNTS neurons of nNOS-null or endothelial NOS (eNOS)-null mice established nNOS as the source of NO. ANG II-induced ROS production was enhanced by PD123319, the NOS inhibitor N(G)-nitro-l-arginine (LNNA), or in nNOS-null mice. Moreover, in the presence of losartan, ANG II reduced voltage-gated L-type Ca(2+) current, an effect blocked by PD123319 or LNNA. We conclude that AT(2)R are closely associated and functionally coupled with nNOS in mNTS neurons. The resulting NO production antagonizes AT(1)R-mediated ROS and dampens L-type Ca(2+) currents. The ensuing signaling changes in the NTS may counteract the deleterious effects of AT(1)R on cardiovascular function.