Endothelin-dependent vasoconstriction in human uterine artery: application to preeclampsia

Background

Reduced uteroplacental perfusion, the initiating event in preeclampsia, is associated with enhanced endothelin-1 (ET-1) production which feeds the vasoconstriction of uterine artery. Whether the treatments of preeclampsia were effective on ET-1 induced contraction and could reverse placental ischemia is the question addressed in this study. We investigated the effect of antihypertensive drugs used in preeclampsia and of ET receptor antagonists on the contractile response to ET-1 on human uterine arteries.

Methodology/Principal Findings

Experiments were performed, ex vivo, on human uterine artery samples obtained after hysterectomy. We studied variations in isometric tension of arterial rings in response to the vasoconstrictor ET-1 and evaluated the effects of various vasodilators and ET-receptor antagonists on this response. Among antihypertensive drugs, only dihydropyridines were effective in blocking and reversing the ET-1 contractile response. Their efficiency, independent of the concentration of ET-1, was only partial. Hydralazine, alpha-methyldopa and labetalol had no effect on ET-1 induced contraction which is mediated by both ET_A and ET_B receptors in uterine artery. ET receptors antagonists, BQ-123 and BQ-788, slightly reduced the amplitude of the response to ET-1. Combination of both antagonists was more efficient, but it was not possible to reverse the maximal ET-1-induced contraction with antagonists used alone or in combination.

Conclusion

Pharmacological drugs currently used in the context of preeclampsia, do not reverse ET-1 induced contraction. Only dihydropyridines, which partially relax uterine artery previously contracted with ET-1, might offer interesting perspectives to improve placental perfusion.     

Modulation by endothelin-1 of spontaneous activity and membrane currents of atrioventricular node myocytes from the rabbit heart

Background

The atrioventricular node (AVN) is a key component of the cardiac pacemaker-conduction system. Although it is known that receptors for the peptide hormone endothelin-1 (ET-1) are expressed in the AVN, there is very little information available on the modulatory effects of ET-1 on AVN electrophysiology. This study characterises for the first time acute modulatory effects of ET-1 on AVN cellular electrophysiology.

Methods

Electrophysiological experiments were conducted in which recordings were made from rabbit isolated AVN cells at 35–37°C using the whole-cell patch clamp recording technique.

Results

Application of ET-1 (10 nM) to spontaneously active AVN cells led rapidly (within ∼13 s) to membrane potential hyperpolarisation and cessation of spontaneous action potentials (APs). This effect was prevented by pre-application of the ET_A receptor inhibitor BQ-123 (1 µM) and was not mimicked by the ET_B receptor agonist IRL-1620 (300 nM). In whole-cell voltage-clamp experiments, ET-1 partially inhibited L-type calcium current (I_Ca,L) and rapid delayed rectifier K⁺ current (I_Kr), whilst it transiently activated the hyperpolarisation-activated current (I_f) at voltages negative to the pacemaking range, and activated an inwardly rectifying current that was inhibited by both tertiapin-Q (300 nM) and Ba²⁺ ions (2 mM); each of these effects was sensitive to ET_A receptor inhibition. In cells exposed to tertiapin-Q, ET-1 application did not produce membrane potential hyperpolarisation or immediate cessation of spontaneous activity; instead, there was a progressive decline in AP amplitude and depolarisation of maximum diastolic potential.

Conclusions

Acutely applied ET-1 exerts a direct modulatory effect on AVN cell electrophysiology. The dominant effect of ET-1 in this study was activation of a tertiapin-Q sensitive inwardly rectifying K⁺ current via ET_A receptors, which led rapidly to cell quiescence.     

Endothelin-1 in paraventricular nucleus modulates cardiac sympathetic afferent reflex and sympathetic activity in rats

Background

Cardiac sympathetic afferent reflex (CSAR) is a positive-feedback, sympathoexcitatory reflex. Paraventricular nucleus (PVN) is an important component of the central neurocircuitry of the CSAR. The present study is designed to determine whether endothelin-1 (ET-1) in the PVN modulates the CSAR and sympathetic activity, and whether superoxide anions are involved in modulating the effects of ET-1 in the PVN in rats.

Methodology/Principal Findings

In anaesthetized Sprague–Dawley rats with cervical vagotomy and sinoaortic denervation, renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) were recorded. The CSAR was evaluated by the responses of the RSNA and MAP to epicardial application of capsaicin. Microinjection of ET-1 into the bilateral PVN dose-dependently enhanced the CSAR, increased the baseline RSNA and MAP. The effects of ET-1 were blocked by PVN pretreatment with the ET_A receptor antagonist BQ-123. However, BQ-123 alone had no significant effects on the CSAR, the baseline RSNA and MAP. Bilateral PVN pretreatment with either superoxide anion scavenger tempol or polyethylene glycol-superoxide dismutase (PEG-SOD) inhibited the effects of ET-1 on the CSAR, RSNA and MAP. Microinjection of ET-1 into the PVN increased the superoxide anion level in the PVN, which was abolished by PVN pretreatment with BQ-123. Epicardial application of capsaicin increased superoxide anion level in PVN which was further enhanced by PVN pretreatment with ET-1.

Conclusions

Exogenous activation of ET_A receptors with ET-1 in the PVN enhances the CSAR, increases RSNA and MAP. Superoxide anions in PVN are involved in the effects of ET-1 in the PVN.     

Male-Female Differences in Upregulation of Vasoconstrictor Responses in Human Cerebral Arteries

Background and purpose

Male-female differences may significantly impact stroke prevention and treatment in men and women, however underlying mechanisms for sexual dimorphism in stroke are not understood. We previously found in males that cerebral ischemia upregulates contractile receptors in cerebral arteries, which is associated with lower blood flow. The present study investigates if cerebral arteries from men and women differ in cerebrovascular receptor upregulation.

Experimental approach

Freshly obtained human cerebral arteries were placed in organ culture, an established model for studying receptor upregulation. 5-hydroxtryptamine type 1B (5-HT_1B), angiotensin II type 1 (AT₁) and endothelin-1 type A and B (ET_A and ET_B) receptors were evaluated using wire myograph for contractile responses, real-time PCR for mRNA and immunohistochemistry for receptor expression.

Key results

Vascular sensitivity to angiotensin II and endothelin-1 was markedly lower in cultured cerebral arteries from women as compared to men. ET_B receptor-mediated contraction occurred in male but not female arteries. Interestingly, there were similar upregulation in mRNA and expression of 5-HT_1B, AT₁, and ET_B receptors and in local expression of Ang II after organ culture.

Conclusions and Implications

In spite of receptor upregulation after organ culture in both sexes, cerebral arteries from women were significantly less responsive to vasoconstrictors angiotensin II and endothelin-1 as compared to arteries from men. This suggests receptor coupling and/or signal transduction mechanisms involved in cerebrovascular contractility may be suppressed in females. This is the first study to demonstrate sex differences in the vascular function of human brain arteries.     

Cigarette smoke upregulates rat coronary artery endothelin receptors in vivo

Background

Cigarette smoking is a strong cardiovascular risk factor and endothelin (ET) receptors are related to coronary artery diseases. The present study established an in vivo secondhand smoke (SHS) exposure model and investigated the hypothesis that cigarette smoke induces ET receptor upregulation in rat coronary arteries and its possible underlying mechanisms.

Methodology/Principal Findings

Rats were exposed to SHS for 200 min daily for 8 weeks. The coronary arteries were isolated and examined. The vasoconstriction was studied by a sensitive myograph. The expression of mRNA and protein for receptors was examined by real-time PCR, Western blot and immunofluorescence. Compared to fresh air exposure, SHS increased contractile responses mediated by endothelin type A (ET_A) and type B (ET_B) receptors in coronary arteries. In parallel, the expression of mRNA and protein for ET_A and ET_B receptors of smoke exposed rats were higher than that of animals exposed to fresh air, suggesting that SHS upregulates ET_A and ET_B receptors in coronary arteries in vivo. Immunofluorescence staining showed that the enhanced receptor expression was localized to the smooth muscle cells of coronary arteries. The protein levels of phosphorylated (p)-Raf-1 and p-ERK1/2 in smoke exposed rats were significantly higher than in control rats, demonstrating that SHS induces the activation of the Raf/ERK/MAPK pathway. Treatment with Raf-1 inhibitor GW5074 suppressed SHS-induced enhanced contraction mediated by ET_A receptors, and inhibited the elevated mRNA and protein levels of ET_A and ET_B receptors caused by SHS. The results of correlation and regression analysis showed that phosphorylation of Raf and ERK1/2 were independent determinants to affect protein expression of ET_B and ET_A receptors.

Conclusions/Significance

Cigarette smoke upregulates ET_B and ET_A receptors in rat coronary artery, which is associated with the activation of the Raf/ERK/MAPK pathway.     

Cerebrospinal Fluid from Patients with Subarachnoid Haemorrhage and Vasospasm Enhances Endothelin Contraction in Rat Cerebral Arteries

Introduction

Previous studies have suggested that cerebrospinal fluid from patients with subarachnoid hemorrhage (SAH) leads to pronounced vasoconstriction in isolated arteries. We hypothesized that only cerebrospinal fluid from SAH patients with vasospasm would produce an enhanced contractile response to endothelin-1 in rat cerebral arteries, involving both endothelin ET_A and ET_B receptors.

Methods

Intact rat basilar arteries were incubated for 24 hours with cerebrospinal fluid from 1) SAH patients with vasospasm, 2) SAH patients without vasospasm, and 3) control patients. Arterial segments with and without endothelium were mounted in myographs and concentration-response curves for endothelin-1 were constructed in the absence and presence of selective and combined ET_A and ET_B receptor antagonists. Endothelin concentrations in culture medium and receptor expression were measured.

Results

Compared to the other groups, the following was observed in arteries exposed to cerebrospinal fluid from patients with vasospasm: 1) larger contractions at lower endothelin concentrations (p<0.05); 2) the increased endothelin contraction was absent in arteries without endothelium; 3) higher levels of endothelin secretion in the culture medium (p<0.05); 4) there was expression of ET_A receptors and new expression of ET_B receptors was apparent; 5) reduction in the enhanced response to endothelin after ET_B blockade in the low range and after ET_A blockade in the high range of endothelin concentrations; 6) after combined ET_A and ET_B blockade a complete inhibition of endothelin contraction was observed.

Conclusions

Our experimental findings showed that in intact rat basilar arteries exposed to cerebrospinal fluid from patients with vasospasm endothelin contraction was enhanced in an endothelium-dependent manner and was blocked by combined ET_A and ET_B receptor antagonism. Therefore we suggest that combined blockade of both receptors may play a role in counteracting vasospasm in patients with SAH.     

Endothelin-1 Inhibits Prolyl Hydroxylase Domain 2 to Activate Hypoxia-Inducible Factor-1α in Melanoma Cells

Background

The endothelin B receptor (ET_BR) promotes tumorigenesis and melanoma progression through activation by endothelin (ET)-1, thus representing a promising therapeutic target. The stability of hypoxia-inducible factor (HIF)-1α is essential for melanomagenesis and progression, and is controlled by site-specific hydroxylation carried out by HIF-prolyl hydroxylase domain (PHD) and subsequent proteosomal degradation.

Principal Findings

Here we found that in melanoma cells ET-1, ET-2, and ET-3 through ET_BR, enhance the expression and activity of HIF-1α and HIF-2α that in turn regulate the expression of vascular endothelial growth factor (VEGF) in response to ETs or hypoxia. Under normoxic conditions, ET-1 controls HIF-α stability by inhibiting its degradation, as determined by impaired degradation of a reporter gene containing the HIF-1α oxygen-dependent degradation domain encompassing the PHD-targeted prolines. In particular, ETs through ET_BR markedly decrease PHD2 mRNA and protein levels and promoter activity. In addition, activation of phosphatidylinositol 3-kinase (PI3K)-dependent integrin linked kinase (ILK)-AKT-mammalian target of rapamycin (mTOR) pathway is required for ET_BR-mediated PHD2 inhibition, HIF-1α, HIF-2α, and VEGF expression. At functional level, PHD2 knockdown does not further increase ETs-induced in vitro tube formation of endothelial cells and melanoma cell invasiveness, demonstrating that these processes are regulated in a PHD2-dependent manner. In human primary and metastatic melanoma tissues as well as in cell lines, that express high levels of HIF-1α, ET_BR expression is associated with low PHD2 levels. In melanoma xenografts, ET_BR blockade by ET_BR antagonist results in a concomitant reduction of tumor growth, angiogenesis, HIF-1α, and HIF-2α expression, and an increase in PHD2 levels.

Conclusions

In this study we identified the underlying mechanism by which ET-1, through the regulation of PHD2, controls HIF-1α stability and thereby regulates angiogenesis and melanoma cell invasion. These results further indicate that targeting ET_BR may represent a potential therapeutic treatment of melanoma by impairing HIF-1α stability.     

Autoantibodies to angiotensin and endothelin receptors in systemic sclerosis induce cellular and systemic events associated with disease pathogenesis

Introduction

Vasculopathy, inflammatory fibrosis and functional autoantibodies (Abs) are major manifestations of systemic sclerosis (SSc). Abs directed against the angiotensin II type 1 receptor (AT₁R) and endothelin-1 type A receptor (ET_AR) are associated with characteristic disease features including vascular, inflammatory, and fibrotic complications indicating their role in SSc pathogenesis. Therefore, the impact of anti-AT₁R and anti-ET_AR Abs on initiation of inflammation and fibrosis was analyzed.

Methods

Anti-AT₁R and anti-ET_AR Ab-positive immunoglobulin G (IgG) from SSc patients (SSc-IgG) was used for experiments. Healthy donor IgG served as a normal control, and AT₁R and ET_AR activation was inhibited by antagonists. Protein expression was measured with ELISA, mRNA expression with real time-PCR, endothelial repair with a scratch assay, and collagen expression with immunocytochemistry. Transendothelial neutrophil migration was measured with a culture insert system, and neutrophil ROS activation with immunofluorescence. Neutrophils in bronchoalveolar lavage fluids (BALFs) were analyzed microscopically after passive transfer of SSc-IgG or NC-IgG into naïve C57BL/6J mice. KC plasma levels were quantified by a suspension array system. Histologic analyses were performed by using light microscopy.

Results

Anti-AT₁R and anti-ET_AR Ab-positive SSc-IgG induced activation of human microvascular endothelial cells (HMEC-1). Elevated protein and mRNA levels of the proinflammatory chemokine interleukin-8 (IL-8, CXCL8) and elevated mRNA levels of the vascular cell adhesion molecule-1 (VCAM-1) were induced in HMEC-1. Furthermore, activation of HMEC-1 with SSc-IgG increased neutrophil migration through an endothelial cell layer and activation of reactive oxygen species (ROS). SSc-IgG decreased HMEC-1 wound repair and induced type I collagen production in healthy donor skin fibroblasts. Effects of migration, wound repair, and collagen expression were dependent on the Ab-levels. Passive transfer of anti-AT₁R and anti-ET_AR Ab-positive SSc-IgG into naïve C57BL/6J mice increased neutrophil BALF counts. In parallel, increased levels of the murine functional IL-8 homologue, chemokine KC, were found in the plasma of SSc-IgG-treated mice as well as structural alterations of the lungs.

Conclusions

We conclude that angiotensin and endothelin-receptor activation via anti-AT₁R and anti-ET_AR Abs mediate pathogenic effects, indicating their contribution to pathogenesis of SSc. Therefore, anti-AT₁R and anti-ET_AR Abs could provide novel targets for therapeutic intervention in the treatment of SSc.     

Intestinal epithelial stem/progenitor cells are controlled by mucosal afferent nerves

Background

The maintenance of the intestinal epithelium is of great importance for the survival of the organism. A possible nervous control of epithelial cell renewal was studied in rats and mice.

Methods

Mucosal afferent nerves were stimulated by exposing the intestinal mucosa to capsaicin (1.6 mM), which stimulates intestinal external axons. Epithelial cell renewal was investigated in the jejunum by measuring intestinal thymidine kinase (TK) activity, intestinal ³H-thymidine incorporation into DNA, and the number of crypt cells labeled with BrdU. The influence of the external gut innervation was minimized by severing the periarterial nerves.

Principal Findings

Luminal capsaicin increased all the studied variables, an effect nervously mediated to judge from inhibitory effects on TK activity or ³H-thymidine incorporation into DNA by exposing the mucosa to lidocaine (a local anesthetic) or by giving four different neurotransmitter receptor antagonists i.v. (muscarinic, nicotinic, neurokinin1 (NK1) or calcitonin gene related peptide (CGRP) receptors). After degeneration of the intestinal external nerves capsaicin did not increase TK activity, suggesting the involvement of an axon reflex. Intra-arterial infusion of Substance P (SP) or CGRP increased intestinal TK activity, a response abolished by muscarinic receptor blockade. Immunohistochemistry suggested presence of M3 and M5 muscarinic receptors on the intestinal stem/progenitor cells. We propose that the stem/progenitor cells are controlled by cholinergic nerves, which, in turn, are influenced by mucosal afferent neuron(s) releasing acetylcholine and/or SP and/or CGRP. In mice lacking the capsaicin receptor, thymidine incorporation into DNA and number of crypt cells labeled with BrdU was lower than in wild type animals suggesting that nerves are important also in the absence of luminal capsaicin, a conclusion also supported by the observation that atropine lowered thymidine incorporation into DNA by 60% in control rat segments.

Conclusion

Enteric nerves are of importance in maintaining the intestinal epithelial barrier.     

Cardiovascular agents affect the tone of pulmonary arteries and veins in precision-cut lung slices

Introduction

Cardiovascular agents are pivotal in the therapy of heart failure. Apart from their action on ventricular contractility and systemic afterload, they affect pulmonary arteries and veins. Although these effects are crucial in heart failure with coexisting pulmonary hypertension or lung oedema, they are poorly defined, especially in pulmonary veins. Therefore, we investigated the pulmonary vascular effects of adrenoceptor agonists, vasopressin and angiotensin II in the model of precision-cut lung slices that allows simultaneous studies of pulmonary arteries and veins.

Materials and Methods

Precision-cut lung slices were prepared from guinea pigs and imaged by videomicroscopy. Concentration-response curves of cardiovascular drugs were analysed in pulmonary arteries and veins.

Results

Pulmonary veins responded stronger than arteries to α₁-agonists (contraction) and β₂-agonists (relaxation). Notably, inhibition of β₂-adrenoceptors unmasked the α₁-mimetic effect of norepinephrine and epinephrine in pulmonary veins. Vasopressin and angiotensin II contracted pulmonary veins via V_1a and AT₁ receptors, respectively, without affecting pulmonary arteries.

Discussion

Vasopressin and (nor)epinephrine in combination with β₂-inhibition caused pulmonary venoconstriction. If applicable in humans, these treatments would enhance capillary hydrostatic pressures and lung oedema, suggesting their cautious use in left heart failure. Vice versa, the prevention of pulmonary venoconstriction by AT₁ receptor antagonists might contribute to their beneficial effects seen in left heart failure. Further, α₁-mimetic agents might exacerbate pulmonary hypertension and right ventricular failure by contracting pulmonary arteries, whereas vasopressin might not.     

Pharmacology of endothelins: vascular preparations for studying ETA and ETB receptors

Three rabbit vessels, the carotid and pulmonary arteries and the jugular vein were investigated to identify vascular monoreceptor systems (either ET_A or ET_B) to be used in structure-activity studies on endothelins and their antagonists. The RbCA has been found to behave as a monoreceptor ETA preparation, since it shows much greater sensitivity to ET-1 than to ET-3 and is insensitive to IRL 1620. The contractile response of the RbCA to ET-1 is reduced in the presence of BQ-123 but is not influenced by BQ-788. The RbPA behaves as a pure ET_B system when stimulated with the ET_B selective agonist IRL 1620. The contractile effect of IRL 1620 is reduced in the presence of BQ-788 but is not influenced by BQ-123. The RbJV responds to ET_A and to IRL 1620 with contractions that are reduced by both BQ-123 and BQ-788, respectively. The RbJV appears to be a mixed ET_A and ET_B system in which the two functional sites play an equivalent role in the stimulatory contractile response.Thus, contractile ET_A and ET_B receptors have been found in arterial and venous vessels of the rabbit and some of these vessels provide sensitive and selective (either ET_A or ET_B) preparations that appear to be adequate for pharmacological studies on ET receptor agonists or antagonists.     

The effects of different exercise modes for preventing endothelial dysfunction of arteries and bone loss in ovariectomized rats

[Purpose]

Several epidemiological studies have demonstrated that there are positive correlations between vascular disorders and bone loss in postmenopausal women. The aim of the present study was to examine the effect of different types of exercise (e.g., climbing and swimming) for preventing endothelial dysfunction of arteries and bone loss in ovariectomized rats.

[Methods]

Twenty Sprague-Dawley female rats were randomly divided into three groups: ovariectomy (OVX) plus treatment with vitamin D₃ and nicotine (VDN) (control rats [Con], n = 7), which is an animal model for endothelial dysfunction and bone loss; voluntary climbing resistance exercise with OVX plus VDN (climbing rats [Clim], n = 6), and swimming exercise with OVX plus VDN (swimming rats [Swim], n = 7). The period of exercise training was 8 weeks.

[Results]

The endothelin-1 (ET-1) protein levels were significantly lower in the Clim and Swim groups than in the Con. The endothelial nitric oxide synthase protein levels were significantly higher in the Swim group than in the Con, but they did not differ between the Clim and Con groups. The cortical bone mineral density in the tibia and breaking energy of the femur were significantly higher in the Clim group than in the Con, but this positive effect was not seen in the Swim group.

[Conclusion]

Voluntary climbing exercise decreased arterial ET-1 protein levels and prevented bone loss in a postmenopause-model rat combining OVX and VDN. Conversely, swimming suppressed endothelial dysfunction of the arteries but did not prevent bone loss. Thus, the type of exercise should be cautiously chosen for enhancing vascular function and bone status, especially in females after menopause.     

Endothelin-1/Endothelin A receptor-mediated biased signaling is a new player in modulating human ovarian cancer cell tumorigenesis

The endothelin-1 (ET-1)/endothelin A receptor (ET_AR, a G protein-coupled receptor) axis confers pleiotropic effects on both tumor cells and the tumor microenvironment, modulating chemo-resistance and other tumor-associated processes by activating Gαq- and β-arrestin-mediated pathways. While the precise mechanisms by which these effects occur remain to be elucidated, interference with ET_AR signaling has emerged as a promising antitumor strategy in many cancers including ovarian cancer (OC). However, current clinical approaches using ET_AR antagonists in the absence of a detailed knowledge of downstream signaling have resulted in multiple adverse side effects and limited therapeutic efficacy. To maximize the safety and efficacy of ET_AR-targeted OC therapy, we investigated the role of other G protein subunits such as Gα_s in the ET_AR-mediated ovarian oncogenic signaling. In HEY (human metastatic OC) cells where the ET-1/ET_AR axis is well-characterized, Gαs signaling inhibits ET_AR-mediated OC cell migration, wound healing, proliferation and colony formation on soft agar while inducing OC cell apoptosis. Mechanistically, ET-1/ET_AR is coupled to Gαs/cAMP signaling in the same ovarian carcinoma-derived cell line. Gαs/cAMP/PKA activation inhibits ET_AR-mediated β-arrestin activation of angiogenic/metastatic Calcrl and Icam2 expression. Consistent with our findings, Gαs overexpression is associated with improved survival in OC patients in the analysis of the Cancer Genome Atlas data. In conclusion, our results indicate a novel function for Gαs signaling in ET-1/ET_AR-mediated OC oncogenesis and may provide a rationale for a biased signaling mechanism, which selectively activates Gαs-coupled tumor suppressive pathways while blocking Gαq-/β-arrestin-mediated oncogenic pathways, to improve the targeting of the ET_AR axis in OC.     

Opposing Actions of Sevoflurane on GABAergic and Glycinergic Synaptic Inhibition in the Spinal Ventral Horn

Background

The ventral horn is a major substrate in mediating the immobilizing properties of the volatile anesthetic sevoflurane in the spinal cord. In this neuronal network, action potential firing is controlled by GABA_A and glycine receptors. Both types of ion channels are sensitive to volatile anesthetics, but their role in mediating anesthetic-induced inhibition of spinal locomotor networks is not fully understood.

Methodology/Principal Findings

To compare the effects of sevoflurane on GABAergic and glycinergic inhibitory postsynaptic currents (IPSCs) whole-cell voltage-clamp recordings from ventral horn interneurons were carried out in organotypic spinal cultures. At concentrations close to MAC (minimum alveolar concentration), decay times of both types of IPSCs were significantly prolonged. However, at 1.5 MAC equivalents, GABAergic IPSCs were decreased in amplitude and reduced in frequency. These effects counteracted the prolongation of the decay time, thereby decreasing the time-averaged GABAergic inhibition. In contrast, amplitudes and frequency of glycinergic IPSCs were not significantly altered by sevoflurane. Furthermore, selective GABA_A and glycine receptor antagonists were tested for their potency to reverse sevoflurane-induced inhibition of spontaneous action potential firing in the ventral horn. These experiments confirmed a weak impact of GABA_A receptors and a prominent role of glycine receptors at a high sevoflurane concentration.

Conclusions

At high concentrations, sevoflurane mediates neuronal inhibition in the spinal ventral horn primarily via glycine receptors, and less via GABA_A receptors. Our results support the hypothesis that the impact of GABA_A receptors in mediating the immobilizing properties of volatile anesthetics is less essential in comparison to glycine receptors.     

Role of calcitonin gene-related peptide in bone repair after cyclic fatigue loading

Background

Calcitonin gene related peptide (CGRP) is a neuropeptide that is abundant in the sensory neurons which innervate bone. The effects of CGRP on isolated bone cells have been widely studied, and CGRP is currently considered to be an osteoanabolic peptide that has effects on both osteoclasts and osteoblasts. However, relatively little is known about the physiological role of CGRP in-vivo in the skeletal responses to bone loading, particularly fatigue loading.

Methodology/Principal Findings

We used the rat ulna end-loading model to induce fatigue damage in the ulna unilaterally during cyclic loading. We postulated that CGRP would influence skeletal responses to cyclic fatigue loading. Rats were fatigue loaded and groups of rats were infused systemically with 0.9% saline, CGRP, or the receptor antagonist, CGRP_8–37, for a 10 day study period. Ten days after fatigue loading, bone and serum CGRP concentrations, serum tartrate-resistant acid phosphatase 5b (TRAP5b) concentrations, and fatigue-induced skeletal responses were quantified. We found that cyclic fatigue loading led to increased CGRP concentrations in both loaded and contralateral ulnae. Administration of CGRP_8–37 was associated with increased targeted remodeling in the fatigue-loaded ulna. Administration of CGRP or CGRP_8–37 both increased reparative bone formation over the study period. Plasma concentration of TRAP5b was not significantly influenced by either CGRP or CGRP_8–37 administration.

Conclusions

CGRP signaling modulates targeted remodeling of microdamage and reparative new bone formation after bone fatigue, and may be part of a neuronal signaling pathway which has regulatory effects on load-induced repair responses within the skeleton.     

Antihypertensive treatment differentially affects vascular sphingolipid biology in spontaneously hypertensive rats

Background

We have previously shown that essential hypertension in humans and spontaneously hypertensive rats (SHR), is associated with increased levels of ceramide and marked alterations in sphingolipid biology. Pharmacological elevation of ceramide in isolated carotid arteries of SHR leads to vasoconstriction via a calcium-independent phospholipase A₂, cyclooxygenase-1 and thromboxane synthase-dependent release of thromboxane A₂. This phenomenon is almost absent in vessels from normotensive Wistar Kyoto (WKY) rats. Here we investigated whether lowering of blood pressure can reverse elevated ceramide levels and reduce ceramide-mediated contractions in SHR.

Methods and Findings

For this purpose SHR were treated for 4 weeks with the angiotensin II type 1 receptor antagonist losartan or the vasodilator hydralazine. Both drugs decreased blood pressure equally (SBP untreated SHR: 191±7 mmHg, losartan: 125±5 mmHg and hydralazine: 113±14 mmHg). The blood pressure lowering was associated with a 20–25% reduction in vascular ceramide levels and improved endothelial function of isolated carotid arteries in both groups. Interestingly, losartan, but not hydralazine treatment, markedly reduced sphingomyelinase-induced contractions. While both drugs lowered cyclooxygenase-1 expression, only losartan and not hydralazine, reduced the endothelial expression of calcium-independent phospholipase A₂. The latter finding may explain the effect of losartan treatment on sphingomyelinase-induced vascular contraction.

Conclusion

In summary, this study corroborates the importance of sphingolipid biology in blood pressure control and specifically shows that blood pressure lowering reduces vascular ceramide levels in SHR and that losartan treatment, but not blood pressure lowering per se, reduces ceramide-mediated arterial contractions.     

Milrinone Relaxes Pulmonary Veins in Guinea Pigs and Humans

Introduction

The phosphodiesterase-III inhibitor milrinone improves ventricular contractility, relaxes pulmonary arteries and reduces right ventricular afterload. Thus, it is used to treat heart failure and pulmonary hypertension (PH). However, its action on pulmonary veins (PVs) is not defined, although particularly PH due to left heart disease primarily affects the pulmonary venous bed. We examined milrinone-induced relaxation in PVs from guinea pigs (GPs) and humans.

Material and Methods

Precision-cut lung slices (PCLS) were prepared from GPs or from patients undergoing lobectomy. Milrinone-induced relaxation was studied by videomicroscopy in naïve PVs and in PVs pre-constricted with the ET_A-receptor agonist BP0104. Baseline luminal area was defined as 100%. Intracellular cAMP was measured by ELISA and milrinone-induced changes of segmental vascular resistances were studied in the GP isolated perfused lung (IPL).

Results

In the IPL (GP), milrinone (10 µM) lowered the postcapillary resistance of pre-constricted vessels. In PCLS (GP), milrinone relaxed naïve and pre-constricted PVs (120%) and this relaxation was attenuated by inhibition of protein kinase G (KT 5823), adenyl cyclase (SQ 22536) and protein kinase A (KT 5720), but not by inhibition of NO-synthesis (L-NAME). In addition, milrinone-induced relaxation was dependent on the activation of K_ATP-, BK_Ca ²⁺- and K_v-channels. Human PVs also relaxed to milrinone (121%), however only if pre-constricted.

Discussion

Milrinone relaxes PVs from GPs and humans. In GPs, milrinone-induced relaxation is based on K_ATP-, BK_Ca ²⁺- and K_v-channel-activation and on cAMP/PKA/PKG. The relaxant properties of milrinone on PVs lead to reduced postcapillary resistance and hydrostatic pressures. Hence they alleviate pulmonary edema and suggest beneficial effects of milrinone in PH due to left heart disease.