Joern R. Steinert

Environmental estrogenic pollutants induce acute vascular relaxation by inhibiting L-type Ca2+ channels in smooth muscle cells

There is an ongoing scientific debate concerning the potential threat of environmental estrogenic pollutants to animal and human health (1–5). Pollutants including the detergents 4‐octylphenol and p‐nonylphenol and chlorinated insecticides have recently been reported to modulate sexual differentiation by interacting with nuclear steroid receptors (6–8). So far, the focus has been on reproductive organs, but sex steroids have far more widespread actions. The lower incidence of cardiovascular disease in women has been attributed to estrogens (9–14), yet no information is available on the vascular actions of environmental estrogenic pollutants. In the present study we have investigated the effects of acute exposure to 17β‐estradiol, the antiestrogen ICI 182,780, and estrogenic pollutants on coronary vascular tone as well as on intracellular Ca2+ levels ([Ca2+]i) and Ca2+ and K+ channel activity in vascular smooth muscle cells. We report here that 4‐octylphenol, p‐nonylphenol, o.p′‐DDT, and the antiestrogen ICI 182,780 inhibit L‐type Ca2+ channels in vascular smooth muscle cells and evoke a rapid and endothelium‐independent relaxation of the coronary vasculature similar to that induced by 17β‐estradiol. Thus, inhibition of Ca2+ influx via L‐type Ca2+ channels in vascular smooth muscle cells may explain the acute, nongenomic vasodilator actions of environmental estrogenic pollutants.

Early activation of the p42/p44MAPK pathway mediates adenosine-induced nitric oxide production in human endothelial cells: a novel calcium-insensitive mechanism

Adenosine is released from the myocardium, endothelial cells, and skeletal muscle in ischemia and is an important regulator of coronary blood flow. We have already shown that acute (2 min) activation of A2a purinoceptors stimulates NO production in human fetal umbilical vein endothelial cells (1) and now report a key role for p42/p44 mitogen‐activated protein kinases (p42/p44MAPK) in the regulation of the L‐arginine‐nitric oxide (NO) signaling pathway. Expression of mRNA for the A2a‐, A2b‐, and A3‐adenosine receptor subtypes was abundant whereas A1‐adenosine receptor mRNA levels were negligible. Activation of A2a purinoceptors by adenosine (10 fM) or the A2a receptor agonist CGS21680 (100 nM) resulted in an increase in L‐arginine transport and NO release that was not mediated by changes in intracellular Ca2+, pH, or cAMP. Stimulation of endothelial cells with adenosine was associated with a membrane hyperpolarization and phosphorylation of p42/p44MAPK. L‐NAME abolished the adenosine‐induced hyperpolarization and stimulation of L‐arginine transport whereas sodium nitroprusside activated an outward potassium current. Genistein (10 fM) and PD98059 (10 fM), an inhibitor of MAPK kinase 1/2 (MEK1/2), inhibited adenosine‐stimulated L‐arginine transport, NO production, and phosphorylation of p42/p44MAPK. We found no evidence for activation of eNOS via the serine/threonine kinase Akt/ PKB (protein kinase B) in adenosine‐stimulated cells. Our results provide the first evidence that adenosine stimulates the endothelial cell L‐arginine‐NO pathway in aCa2+‐insensitive manner involving p42/p44MAPK, with release of NO leading to a membrane hyperpolarization and activation of L‐arginine transport.—Wyatt, A. W., Steinert, J. R., Wheeler‐Jones, C. P. D., Morgan, A. J., Sugden, D., Pearson, J. D., Sobrevia, L., Mann, G. E. Early activation of the p42/p44MAPK pathway mediates adenosine‐induced nitric oxide production in human endothelial cells: a novel calcium‐insensitive mechanism. FASEB J. 16, 1584–1594 (2002)

Preeclampsia is associated with altered Ca2+ regulation and NO production in human fetal venous endothelial cells

Preeclampsia (PE) is a leading cause of maternal hypertension in pregnancy, fetal growth restriction, premature birth, and fetal and maternal mortality (1). Activation and dysfunction of the maternal and fetal endothelium in PE may be the consequence of increased oxidative stress associated with circulating lipid peroxides (2–4), and in cases of severe maternal hypertension, uterine and umbilical artery waveforms are abnormal (5). We have investigated PE‐associated abnormalities in the regulation of intracellular Ca2+ ([Ca2+]i) and cyclic guanosine monophosphate (cGMP) production (index of nitric oxide [NO]) in human fetal umbilical vein endothelial cells. Basal [Ca2+]i was slightly elevated in PE cells, whereas agonist‐stimulated Ca2+ entry was reduced in cells from PE compared with normal term or age‐matched preterm pregnancies. Furthermore, PE cells exhibited a decreased permeability to Ba2+ but an increased permeability to Mn2+ and Gd3+, suggesting that PE is associated with phenotypic alterations in fetal endothelial cation channel(s). Basal and histamine‐stimulated cGMP levels were elevated in PE compared with preterm or normal cells, implying an increased NO production in PE. However, immunoblots for endothelial NO synthase (eNOS) and soluble guanylyl cyclase (sGC) revealed reduced eNOS expression in PE and preterm cells, with negligible changes in sGC levels. This study provides important and novel insights into abnormalities of fetal endothelial cells isolated from women with PE, revealing an altered cation membrane permeability and activity of eNOS‐sGC pathway. As these changes are sustained in culture in vitro, this may reflect long‐term —programming“ of the fetal cardiovascular system.

Nitric Oxide-Mediated Posttranslational Modifications: Impacts at the Synapse.

Nitric oxide (NO) is an important gasotransmitter molecule that is involved in numerous physiological processes throughout the nervous system. In addition to its involvement in physiological plasticity processes (long-term potentiation, LTP; long-term depression, LTD) which can include NMDAR-mediated calcium-dependent activation of neuronal nitric oxide synthase (nNOS), new insights into physiological and pathological consequences of nitrergic signalling have recently emerged. In addition to the canonical cGMP-mediated signalling, NO is also implicated in numerous pathways involving posttranslational modifications. In this review we discuss the multiple effects of S-nitrosylation and 3-nitrotyrosination on proteins with potential modulation of function but limit the analyses to signalling involved in synaptic transmission and vesicular release. Here, crucial proteins which mediate synaptic transmission can undergo posttranslational modifications with either pre- or postsynaptic origin. During normal brain function, both pathways serve as important cellular signalling cascades that modulate a diverse array of physiological processes, including synaptic plasticity, transcriptional activity, and neuronal survival. In contrast, evidence suggests that aging and disease can induce nitrosative stress via excessive NO production. Consequently, uncontrolled S-nitrosylation/3-nitrotyrosination can occur and represent pathological features that contribute to the onset and progression of various neurodegenerative diseases, including Parkinsons, Alzheimers, and Huntingtons.

Abnormalities in intracellular Ca2+ regulation in fetal vascular smooth muscle in pre-eclampsia: enhanced sensitivity to arachidonic acid

Pre‐eclampsia (PE) is a leading cause of maternal and fetal mortality and morbidity. As free fatty acid metabolism is abnormally regulated in PE, we investigated the intracellular Ca2+ ([Ca2+]i) response to arachidonic acid (AA) in primary cultures of human umbilical artery smooth muscle cells (HUASMC). AA (50 μM) caused a significantly greater [Ca2+]i elevation in PE than in normal HUASMC, with many cells displaying a delayed secondary increase. The nonmetabolizable AA analog ETYA did not induce a response, suggesting that the augmented PE response depends on an AA metabolite. Inhibition of the AA metabolizing cyclooxygenase or lipoxygenase pathways did not affect the AA response of PE HUASMC but induced in normal cells the secondary rise of [Ca2+]i observed in PE cells. This potentiated response and the response in PE cells were blocked by inhibitors of the monooxygenase pathway, a third AA metabolizing pathway. We conclude that the [Ca2+]i response of HUASMC is elevated in PE because of an increased level of a monooxygenase metabolite that stimulates Ca2+ influx and that this can be mimicked in normal cells by blocking cyclooxygenase or lipoxygenase to divert AA to the monooxygenase. This and our work with fetal endothelial cells (FASEB J. 10.1096/fj.01‐0916fje) demonstrate phenotypic changes in the fetal vasculature in PE.

Growth inhibitory activity of indapamide on vascular smooth muscle cells

Abnormal vascular smooth muscle cell proliferation has a fundamental role in the pathogenesis of vascular diseases. Indapamide is an oral diuretic antihypertensive drug effective for patients with mild or moderate essential hypertension. We now investigated the effects of indapamide on the growth of aortic vascular smooth muscle cells (A10 cell line). Indapamide inhibited cell proliferation as measured by the tetrazolium salt XTT (sodium 3-[1-(phenylamino-carbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate) test. The increase in cell number was significantly reduced in the presence of indapamide 10(-6) and 5 x 10(-4) M (P < 0.05 n = 3 and P < 0.01, n = 3, respectively). Serum-induced DNA synthesis, determined as the incorporation of 5-bromo-2-deoxyuridine (BrdU), was concentration-dependently inhibited by indapamide. BrdU incorporation was 47.2+/-1.6% (10% foetal calf serum). Indapamide treatment markedly prevented BrdU incorporation (37.2+/-2.1%, 29.2+/-4.8%, 15.0+/-1.8%, 8.7+/-2.1%) indapamide 10(-6), 10(-5), 5 x 10(-5) and 5 x 10(-4) M, respectively. Cell-cycle progression was also evaluated. Flow cytometry analysis of DNA content in synchronised cells revealed blocking of the serum-inducible cell-cycle progression by indapamide. This inhibition was abolished when the drug was added 2 h after serum repletion, indicating that indapamide must act at the early events of a cell cycle to be fully effective against DNA synthesis. In addition, serum-induced intracellular Ca2+ movements and also p44/p42 mitogen-activated protein kinase (MAPK) phosphorylation were studied in the presence or absence of indapamide. Indapamide 10(-5) and 5 x 10(-5) M decreased significantly cytosolic free calcium, and the p44/p42 mitogen-activated protein kinase phosphorylation (5 x 10(-5) M) stimulated by 10% foetal calf serum. In accordance with this finding, indapamide (5 x 10(-4) M) caused a 95% to 99% decrease in the early elevation of c-fos expression as evaluated by northern blot analysis of mRNA induced after serum addition. In conclusion, our results indicate that indapamide reduces vascular smooth muscle cell proliferation by a mechanism which involves a decrease in the intracellular Ca2+ movements that might link with the mitogen-activated protein kinase (MAPK) pathway, altering cell-cycle progression.

Nitric Oxide: A Regulator of Cellular Function in Health and Disease

Nitric oxide (NO) is a gaseous messenger molecule synthesized from L-arginine and molecular oxygen by three different NO synthases, that is, neuronal (nNOS), endothelial (eNOS), and inducible (iNOS) form [1]. Since its discovery in the early 1980s by the three Nobel Laureates Furchgott, Ignarro & Murad [2], NO has been widely recognised as an important signalling molecule in many physiological processes. The initial identification of NO as an endothelium-derived relaxing factor (EDRF) [3] generated great interest in its function in vascular biology. Over the following years, however, the focus on NO research rapidly expanded from the vascular system to its role in immunity and inflammation, the nervous system, pregnancy, aging, and cell death. n nMany studies suggest that excessive or abnormal production of NO plays a crucial role in neuronal cell death associated with neurodegenerative disorders such as Alzheimers and Parkinsons diseases, as well as various conditions of vascular dysfunction. At physiological levels, NO is essential for neuronal function, differentiation and survival through activation of signalling pathways that include the cyclic guanosine monophosphate (cGMP)/soluble guanylyl cyclase pathway [4] and S-nitrosylation, in which NO reversibly binds to thiol groups of proteins [5]. The vast network of NO-associated signalling paradigms further includes acetylation/deacetylation and methylation/demethylation modifications and peroxynitrite formation (leading to nitrotyrosination of protein residues), as well as modulation of gene expression via epigenetic changes [5–8]. How controlled S-nitrosylation/nitrotyrosination of proteins and activation of the NO/cGMP signalling pathway promote cellular survival and induce epigenetic changes while uncontrolled signalling promotes cell death and dysfunction remains to be elucidated. The aim of this Special Issue is to gather information encapsulating the above signalling pathways. n nThe articles published in this Special Issue largely cover (1) NO signalling in neuronal function and disease as well as (2) vascular targets in endothelial function and dysfunction, both of which involve the broad range of actions of this signalling molecule. In order to understand the contribution of NO to neuronal dysfunction, one has to consider that NO is a crucial molecule in cellular physiology. Numerous studies show the involvement of NO in neuronal development, plasticity, excitability, and transmission [8–12]. However, the common mechanisms across several neurodegenerative disorders relate to the neurotoxicity of NO and its downstream reactive nitrogen species (RNS). Enhanced nitrotyrosine immunoreactivity is evident in brains from patients with Alzheimers and Parkinsons disease. Nitrated proteins are associated with β-amyloid deposition and nitrotyrosination of Tau protein and synaptophysin has been reported in brain samples from patients with Alzheimers disease. The potential downstream signalling pathways of these posttranslational modifications are discussed in the review by S. A. Bradley and J. R. Steinert in this Issue. The cellular roles of NO in neurodegenerative disease, such as Alzheimers, are reviewed by R. Balez and L. Ooi with a focus on neurotoxicity versus neuroprotection while the role of aberrant NO signalling in neurodevelopmental disorders such as Fragile X syndrome is investigated in the study by E. Lima-Cabello et al. n nOther topics of this issue focus on aspects of vascular NO signalling with particular interest in fetoplacental dysfunction caused by abnormal eNOS regulation (as discussed by A. Leiva et al.). Endothelial function and eNOS regulation are essential for healthy cardiovascular responses but are also critical for adaptations during pregnancy. Several diseases associated with vascular dysfunction such as atherosclerosis, diabetes mellitus, hypertension, or preeclampsia involve altered NO signalling [13–15]. n nThe paper by A. Leiva et al. reviews the mechanisms leading to abnormal NO signalling, which include reduced bioavailability of L-arginine (NOS substrate) and tetrahydrobiopterin (BH4), abnormal calcium-calmodulin signalling, and activation and inhibition of eNOS activity through phosphorylation of Ser1177 or Thr495, respectively [16]. NO has also been reported to play a crucial role in the transition of fetoplacental endothelial cells from a mitogenic to a metabolic phenotype in the macrocirculation, compared with a change from a metabolic to a mitogenic phenotype in the microcirculation in response to insulin in gestational diabetes mellitus [17]. Thus, the actions of NO are not only important during vascular adaptations to pregnancy and related dysfunctions but also essential during vascular signalling induced by physical training resulting in elevated nitrite and nitrate levels as reported in the study by A. M. Jacomini et al. n nTogether, this Special Issue highlights the tremendous diversity of NO signalling pathways with regard to its function in health and disease. All contributing publications have emphasised that NO represents an important signalling molecule and future research required in this field will extend the understanding of the broad actions of NO. n n nLuis Sobrevia n nLezanne Ooi n nScott Ryan n nJoern R. Steinert