Autotaxin-lysophosphatidic acid receptor 5 axis evokes endothelial dysfunction via reactive oxygen species signaling.

Lysophosphatidylcholine (LPC) is a bioactive lipid that has been shown to attenuate endothelium-dependent vasorelaxation contributing to endothelial dysfunction; however, the underlying mechanisms are not well understood. In this study, we investigated the molecular mechanisms involved in the development of LPC-evoked impairment of endothelium-dependent vasorelaxation. In aortic rings isolated from wild-type (WT) mice, a 20-min exposure to LPC significantly reduced the acetylcholine chloride (ACh)-induced vasorelaxation indicating the impairment of normal endothelial function. Interestingly, pharmacological inhibition of autotaxin (ATX) by GLPG1690 partially reversed the endothelial dysfunction, suggesting that lysophosphatidic acid (LPA) derived from LPC may be involved in the effect. Therefore, the effect of LPC was also tested in aortic rings isolated from different LPA receptor knock-out (KO) mice. LPC evoked a marked reduction in ACh-dependent vasorelaxation in Lpar1, Lpar2, and Lpar4 KO, but its effect was significantly attenuated in Lpar5 KO vessels. Furthermore, addition of superoxide dismutase reduced the LPC-induced endothelial dysfunction in WT but not in the Lpar5 KO mice. In addition, LPC increased H2O2 release from WT vessels, which was significantly reduced in Lpar5 KO vessels. Our findings indicate that the ATX-LPA-LPA5 receptor axis is involved in the development of LPC-induced impairment of endothelium-dependent vasorelaxation via LPA5 receptor-mediated reactive oxygen species production. Taken together, in this study, we identified a new pathway contributing to the development of LPC-induced endothelial dysfunction.

Intracellular signaling pathways of muscarinic acetylcholine receptor-mediated detrusor muscle contractions.

Acetylcholine plays an essential role in the regulation of detrusor muscle contractions, and antimuscarinics are widely used in the management of overactive bladder syndrome. However, several adverse effects limit their application and patients' compliance. Thus, this study aimed to further analyze the signal transduction of M2 and M3 receptors in the murine urinary bladder to eventually find more specific therapeutic targets. Experiments were performed on adult male wild-type, M2, M3, M2/M3, or Gαq/11 knockout (KO), and pertussis toxin (PTX)-treated mice. Contraction force and RhoA activity were measured in the urinary bladder smooth muscle (UBSM). Our results indicate that carbamoylcholine (CCh)-induced contractions were associated with increased activity of RhoA and were reduced in the presence of the Rho-associated kinase (ROCK) inhibitor Y-27632 in UBSM. CCh-evoked contractile responses and RhoA activation were markedly reduced in detrusor strips lacking either M2 or M3 receptors and abolished in M2/M3 KO mice. Inhibition of Gαi-coupled signaling by PTX treatment shifted the concentration-response curve of CCh to the right and diminished RhoA activation. CCh-induced contractile responses were markedly decreased in Gαq/11 KO mice; however, RhoA activation was unaffected. In conclusion, cholinergic detrusor contraction and RhoA activation are mediated by both M2 and M3 receptors. Furthermore, whereas both Gαi and Gαq/11 proteins mediate UBSM contraction, the activation at the RhoA-ROCK pathway appears to be linked specifically to Gαi. These findings may aid the identification of more specific therapeutic targets for bladder dysfunctions.NEW & NOTEWORTHY Muscarinic acetylcholine receptors are of utmost importance in physiological regulation of micturition and also in the development of voiding disorders. We demonstrate that the RhoA-Rho-associated kinase (ROCK) pathway plays a crucial role in contractions induced by cholinergic stimulation in detrusor muscle. Activation of RhoA is mediated by both M2 and M3 receptors as well as by Gi but not Gq/11 proteins. The Gi-RhoA-ROCK pathway may provide a novel therapeutic target for overactive voiding disorders.

Enhancement of Sphingomyelinase-Induced Endothelial Nitric Oxide Synthase-Mediated Vasorelaxation in a Murine Model of Type 2 Diabetes.

Sphingolipids are important biological mediators both in health and disease. We investigated the vascular effects of enhanced sphingomyelinase (SMase) activity in a mouse model of type 2 diabetes mellitus (T2DM) to gain an understanding of the signaling pathways involved. Myography was used to measure changes in the tone of the thoracic aorta after administration of 0.2 U/mL neutral SMase in the presence or absence of the thromboxane prostanoid (TP) receptor antagonist SQ 29,548 and the nitric oxide synthase (NOS) inhibitor L-NAME. In precontracted aortic segments of non-diabetic mice, SMase induced transient contraction and subsequent weak relaxation, whereas vessels of diabetic (Leprdb/Leprdb, referred to as db/db) mice showed marked relaxation. In the presence of the TP receptor antagonist, SMase induced enhanced relaxation in both groups, which was 3-fold stronger in the vessels of db/db mice as compared to controls and could not be abolished by ceramidase or sphingosine-kinase inhibitors. Co-administration of the NOS inhibitor L-NAME abolished vasorelaxation in both groups. Our results indicate dual vasoactive effects of SMase: TP-mediated vasoconstriction and NO-mediated vasorelaxation. Surprisingly, in spite of the general endothelial dysfunction in T2DM, the endothelial NOS-mediated vasorelaxant effect of SMase was markedly enhanced.

Influence of Vitamin D on the Vasoactive Effect of Estradiol in a Rat Model of Polycystic Ovary Syndrome.

We examined the vasoactive effect of estradiol in a rat model of early PCOS and the influence of vitamin D deficiency (VDD). We created a model of chronic hyperandrogenism and VDD in adolescent female Wistar rats (N = 46) with four experimental groups: vitamin D supplemented (T-D+), VDD (T-D-), hyperandrogenic and vitamin D supplemented (T+D+), and hyperandrogenic and VDD (T+D-). T+ groups received an 8-week-long transdermal Androgel treatment, D-animals were on vitamin D-reduced diet and D+ rats were supplemented orally with vitamin D3. Estrogen-induced vasorelaxation of thoracic aorta segments were measured with a wire myograph system with or without the inhibition of endothelial nitric oxide synthase (eNOS) or cyclooxygenase-2 (COX-2). The distribution of estrogen receptor (ER), eNOS and COX-2 in the aortic wall was assessed by immunohistochemistry. VDD aortas showed significantly lower estradiol-induced relaxation independently of androgenic status that was further decreased by COX-2 inhibition. COX-2 inhibition failed to alter vessel function in D+ rats. Inhibition of eNOS abolished the estradiol-induced relaxation in all groups. Changes in vascular function in VDD were accompanied by significantly decreased ER and eNOS staining. Short-term chronic hyperandrogenism failed to, but VDD induced vascular dysfunction, compromised estrogen-dependent vasodilatation and changes in ER and eNOS immunostaining.

Isoprostanes evoke contraction of the murine and human detrusor muscle via activation of the thromboxane prostanoid TP receptor and Rho kinase.

Local or systemic inflammation can severely impair urinary bladder functions and contribute to the development of voiding disorders in millions of people worldwide. Isoprostanes are inflammatory lipid mediators that are upregulated in the blood and urine by oxidative stress and may potentially induce detrusor overactivity. The aim of the present study was to investigate the effects and signal transduction of isoprostanes in human and murine urinary bladders in order to provide potential pharmacological targets in detrusor overactivity. Contraction force was measured with a myograph in murine and human urinary bladder smooth muscle (UBSM) ex vivo. Isoprostane 8-iso-PGE2 and 8-iso-PGF2α evoked dose-dependent contraction in the murine UBSM, which was abolished in mice deficient in the thromboxane prostanoid (TP) receptor. The responses remained unaltered after removal of the mucosa or incubation with tetrodotoxin. Smooth muscle-specific deletion of Gα12/13 protein or inhibition of Rho kinase by Y-27632 decreased the contractions. In Gαq/11-knockout mice, responses were reduced and in the presence of Y-27632 abolished completely. In human UBSM, the TP agonist U-46619 evoked dose-dependent contractions. Neither atropine nor the purinergic receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid decreased the effect, indicating that TP receptors directly mediate detrusor muscle contraction. 8-iso-PGE2 and 8-iso-PGF2α evoked dose-dependent contraction in the human UBSM, and these responses were abolished by the TP antagonist SQ-29548 and were decreased by Y-27632. Our results indicate that isoprostanes evoke contraction in murine and human urinary bladders, an effect mediated by the TP receptor. The G12/13-Rho-Rho kinase pathway plays a significant role in mediating the contraction and therefore may be a potential therapeutic target in detrusor overactivity.NEW & NOTEWORTHY Voiding disorders affect millions of people worldwide. Inflammation can impair urinary bladder functions and contribute to the development of detrusor overactivity. The effects and signal transduction of inflammatory lipid mediator isoprostanes were studied in human and murine urinary bladders ex vivo. We found that isoprostanes evoke contraction, an effect mediated by thromboxane prostanoid receptors. The G12/13-Rho-Rho kinase signaling pathway plays a significant role in mediating the contraction and therefore may be a potential therapeutic target.

Sphingosine-1-Phosphate Enhances α1-Adrenergic Vasoconstriction via S1P2-G12/13-ROCK Mediated Signaling.

Sphingosine-1-phosphate (S1P) has been implicated recently in the physiology and pathology of the cardiovascular system including regulation of vascular tone. Pilot experiments showed that the vasoconstrictor effect of S1P was enhanced markedly in the presence of phenylephrine (PE). Based on this observation, we hypothesized that S1P might modulate α1-adrenergic vasoactivity. In murine aortas, a 20-minute exposure to S1P but not to its vehicle increased the Emax and decreased the EC50 of PE-induced contractions indicating a hyperreactivity to α1-adrenergic stimulation. The potentiating effect of S1P disappeared in S1P2 but not in S1P3 receptor-deficient vessels. In addition, smooth muscle specific conditional deletion of G12/13 proteins or pharmacological inhibition of the Rho-associated protein kinase (ROCK) by Y-27632 or fasudil abolished the effect of S1P on α1-adrenergic vasoconstriction. Unexpectedly, PE-induced contractions remained enhanced markedly as late as three hours after S1P-exposure in wild-type (WT) and S1P3 KO but not in S1P2 KO vessels. In conclusion, the S1P-S1P2-G12/13-ROCK signaling pathway appears to have a major influence on α1-adrenergic vasoactivity. This cooperativity might lead to sustained vasoconstriction when increased sympathetic tone is accompanied by increased S1P production as it occurs during acute coronary syndrome and stroke.

[Preclinical and clinical investigation and development of electromagnetic oncological device - experience with solid tumors]. / Elektromágneses daganatterápiás készülék preklinikai és klinikai vizsgálatai, valamint mûszaki továbbfejlesztése: tapasztalatok szolid tumorokkal.

Our objective was to develop an electromagnetic tumor therapy device in a consortial cooperation between Semmelweis University and Oncotherm Ltd., to provide data and contribute to the development of the next generation of devices through preclinical, clinical and developmental modules via in vivo, in vitro studies, and patient treatments. Our numerous preclinical studies support the efficacy of mEHT. Clinical treatments were performed in 181 patients with inoperable and/or oligometastatic solid tumors. The protocols were developed, an international guideline was completed, and the planned steps of device development were realized. By optimizing previous selective RF techniques based on recent research findings, we can provide the most modern evidence-based treatment in the future.

NK2 receptor-mediated detrusor muscle contraction involves Gq/11-dependent activation of voltage-dependent Ca2+ channels and the RhoA-Rho kinase pathway.

Tachykinins (TKs) are involved in both the physiological regulation of urinary bladder functions and development of overactive bladder syndrome. The aim of the present study was to investigate the signal transduction pathways of TKs in the detrusor muscle to provide potential pharmacological targets for the treatment of bladder dysfunctions related to enhanced TK production. Contraction force, intracellular Ca2+ concentration, and RhoA activity were measured in the mouse urinary bladder smooth muscle (UBSM). TKs and the NK2 receptor (NK2R)-specific agonist [ß-Ala8]-NKA(4-10) evoked contraction, which was inhibited by the NKR2 antagonist MEN10376. In Gαq/11-deficient mice, [ß-Ala8]-NKA(4-10)-induced contraction and the intracellular Ca2+ concentration increase were abolished. Although Gq/11 proteins are linked principally to phospholipase Cß and inositol trisphosphate-mediated Ca2+ release from intracellular stores, we found that phospholipase Cß inhibition and sarcoplasmic reticulum Ca2+ depletion failed to have any effect on contraction induced by [ß-Ala8]-NKA(4-10). In contrast, lack of extracellular Ca2+ or blockade of voltage-dependent Ca2+ channels (VDCCs) suppressed contraction. Furthermore, [ß-Ala8]-NKA(4-10) increased RhoA activity in the UBSM in a Gq/11-dependent manner and inhibition of Rho kinase with Y-27632 decreased contraction force, whereas the combination of Y-27632 with either VDCC blockade or depletion of extracellular Ca2+ resulted in complete inhibition of [ß-Ala8]-NKA(4-10)-induced contractions. In summary, our results indicate that NK2Rs are linked exclusively to Gq/11 proteins in the UBSM and that the intracellular signaling involves the simultaneous activation of VDCC and the RhoA-Rho kinase pathway. These findings may help to identify potential therapeutic targets of bladder dysfunctions related to upregulation of TKs.

LPA1 receptor-mediated thromboxane A2 release is responsible for lysophosphatidic acid-induced vascular smooth muscle contraction.

Lysophosphatidic acid (LPA) has been recognized recently as an endothelium-dependent vasodilator, but several lines of evidence indicate that it may also stimulate vascular smooth muscle cells (VSMCs), thereby contributing to vasoregulation and remodeling. In the present study, mRNA expression of all 6 LPA receptor genes was detected in murine aortic VSMCs, with the highest levels of LPA1, LPA2, LPA4, and LPA6 In endothelium-denuded thoracic aorta (TA) and abdominal aorta (AA) segments, 1-oleoyl-LPA and the LPA1-3 agonist VPC31143 induced dose-dependent vasoconstriction. VPC31143-induced AA contraction was sensitive to pertussis toxin (PTX), the LPA1&3 antagonist Ki16425, and genetic deletion of LPA1 but not that of LPA2 or inhibition of LPA3, by diacylglycerol pyrophosphate. Surprisingly, vasoconstriction was also diminished in vessels lacking cyclooxygenase-1 [COX1 knockout (KO)] or the thromboxane prostanoid (TP) receptor (TP KO). VPC31143 increased thromboxane A2 (TXA2) release from TA of wild-type, TP-KO, and LPA2-KO mice but not from LPA1-KO or COX1-KO mice, and PTX blocked this effect. Our findings indicate that LPA causes vasoconstriction in VSMCs, mediated by LPA1-, Gi-, and COX1-dependent autocrine/paracrine TXA2 release and consequent TP activation. We propose that this new-found interaction between the LPA/LPA1 and TXA2/TP pathways plays significant roles in vasoregulation, hemostasis, thrombosis, and vascular remodeling.-Dancs, P. T., Ruisanchez, E., Balogh, A., Panta, C. R., Miklós, Z., Nüsing, R. M., Aoki, J., Chun, J., Offermanns, S., Tigyi, G., Benyó, Z. LPA1 receptor-mediated thromboxane A2 release is responsible for lysophosphatidic acid-induced vascular smooth muscle contraction.

Adaptation of the cerebrocortical circulation to carotid artery occlusion involves blood flow redistribution between cortical regions and is independent of eNOS.

Cerebral circulation is secured by feed-forward and feed-back control pathways to maintain and eventually reestablish the optimal oxygen and nutrient supply of neurons in case of disturbances of the cardiovascular system. Using the high temporal and spatial resolution of laser-speckle imaging we aimed to analyze the pattern of cerebrocortical blood flow (CoBF) changes after unilateral (left) carotid artery occlusion (CAO) in anesthetized mice to evaluate the contribution of macrovascular (circle of Willis) vs. pial collateral vessels as well as that of endothelial nitric oxide synthase (eNOS) to the cerebrovascular adaptation to CAO. In wild-type mice CoBF reduction in the left temporal cortex started immediately after CAO, reaching its maximum (-26%) at 5-10 s. Thereafter, CoBF recovered close to the preocclusion level within 30 s indicating the activation of feed-back pathway(s). Interestingly, the frontoparietal cerebrocortical regions also showed CoBF reduction in the left (-17-19%) but not in the right hemisphere, although these brain areas receive their blood supply from the common azygos anterior cerebral artery in mice. In eNOS-deficient animals the acute CoBF reduction after CAO was unaltered, and the recovery was even accelerated compared with controls. These results indicate that 1) the Willis circle alone is not sufficient to provide an immediate compensation for the loss of one carotid artery, 2) pial collaterals attenuate the ischemia of the temporal cortex ipsilateral to CAO at the expense of the blood supply of the frontoparietal region, and 3) eNOS, surprisingly, does not play an important role in this CoBF redistribution.

Endocannabinoids in cerebrovascular regulation.

The cerebral blood flow is tightly regulated by myogenic, endothelial, metabolic, and neural mechanisms under physiological conditions, and a large body of recent evidence indicates that inflammatory pathways have a major influence on the cerebral blood perfusion in certain central nervous system disorders, like hemorrhagic and ischemic stroke, traumatic brain injury, and vascular dementia. All major cell types involved in cerebrovascular control pathways (i.e., smooth muscle, endothelium, neurons, astrocytes, pericytes, microglia, and leukocytes) are capable of synthesizing endocannabinoids and/or express some or several of their target proteins [i.e., the cannabinoid 1 and 2 (CB1 and CB2) receptors and the transient receptor potential vanilloid type 1 ion channel]. Therefore, the endocannabinoid system may importantly modulate the regulation of cerebral circulation under physiological and pathophysiological conditions in a very complex manner. Experimental data accumulated since the late 1990s indicate that the direct effect of cannabinoids on cerebral vessels is vasodilation mediated, at least in part, by CB1 receptors. Cannabinoid-induced cerebrovascular relaxation involves both a direct inhibition of smooth muscle contractility and a release of vasodilator mediator(s) from the endothelium. However, under stress conditions (e.g., in conscious restrained animals or during hypoxia and hypercapnia), cannabinoid receptor activation was shown to induce a reduction of the cerebral blood flow, probably via inhibition of the electrical and/or metabolic activity of neurons. Finally, in certain cerebrovascular pathologies (e.g., subarachnoid hemorrhage, as well as traumatic and ischemic brain injury), activation of CB2 (and probably yet unidentified non-CB1/non-CB2) receptors appear to improve the blood perfusion of the brain via attenuating vascular inflammation.

Endocannabinoid-mediated modulation of Gq/11 protein-coupled receptor signaling-induced vasoconstriction and hypertension.

Activation of G protein-coupled receptors (GPCRs) can induce vasoconstriction via calcium signal-mediated and Rho-dependent pathways. Earlier reports have shown that diacylglycerol produced during calcium signal generation can be converted to an endocannabinoid, 2-arachidonoylglycerol (2-AG). Our aim was to provide evidence that GPCR signaling-induced 2-AG production and activation of vascular type1 cannabinoid receptors (CB1R) is capable of reducing agonist-induced vasoconstriction and hypertension. Rat and mouse aortic rings were examined by myography. Vascular expression of CB1R was demonstrated with immunohistochemistry. Rat aortic vascular smooth muscle cells (VSMCs) were cultured for calcium measurements and 2-AG-determination. Inhibition or genetic loss of CB1Rs enhanced vasoconstriction induced by angiotensin II (AngII) or phenylephrine (Phe), but not by prostaglandin(PG)F2α. AngII-induced vasoconstriction was augmented by inhibition of diacylglycerol lipase (tetrahydrolipstatin) and was attenuated by inhibition of monoacylglycerol lipase (JZL184) suggesting a functionally relevant role for endogenously produced 2-AG. In Gαq/11-deficient mice vasoconstriction was absent to AngII or Phe, which activate Gq/11-coupled receptors, but was maintained in response to PGF2α. In VSMCs, AngII-stimulated 2-AG-formation was inhibited by tetrahydrolipstatin and potentiated by JZL184. CB1R inhibition increased the sustained phase of AngII-induced calcium signal. Pharmacological or genetic loss of CB1R function augmented AngII-induced blood pressure rise in mice. These data demonstrate that vasoconstrictor effect of GPCR agonists is attenuated via Gq/11-mediated vascular endocannabinoid formation. Agonist-induced endocannabinoid-mediated CB1R activation is a significant physiological modulator of vascular tone. Thus, the selective modulation of GPCR signaling-induced endocannabinoid release has a therapeutic potential in case of increased vascular tone and hypertension.

Lysophosphatidic acid induces vasodilation mediated by LPA1 receptors, phospholipase C, and endothelial nitric oxide synthase.

Lysophosphatidic acid (LPA) has been implicated as a mediator of several cardiovascular functions, but its potential involvement in the control of vascular tone is obscure. Here, we show that both LPA (18:1) and VPC31143 (a synthetic agonist of LPA1-3 receptors) relax intact mouse thoracic aorta with similar Emax values (53.9 and 51.9% of phenylephrine-induced precontraction), although the EC50 of LPA- and VPC31143-induced vasorelaxations were different (400 vs. 15 nM, respectively). Mechanical removal of the endothelium or genetic deletion of endothelial nitric oxide synthase (eNOS) not only diminished vasorelaxation by LPA or VPC31143 but converted it to vasoconstriction. Freshly isolated mouse aortic endothelial cells expressed LPA1, LPA2, LPA4 and LPA5 transcripts. The LPA1,3 antagonist Ki16425, the LPA1 antagonist AM095, and the genetic deletion of LPA1, but not that of LPA2, abolished LPA-induced vasorelaxation. Inhibition of the phosphoinositide 3 kinase-protein kinase B/Akt pathway by wortmannin or MK-2206 failed to influence the effect of LPA. However, pharmacological inhibition of phospholipase C (PLC) by U73122 or edelfosine, but not genetic deletion of PLCε, abolished LPA-induced vasorelaxation and indicated that a PLC enzyme, other than PLCε, mediates the response. In summary, the present study identifies LPA as an endothelium-dependent vasodilator substance acting via LPA1, PLC, and eNOS.

Effects of vitamin D3 derivative--calcitriol on pharmacological reactivity of aortic rings in a rodent PCOS model.

BACKGROUND: The aim of this study was to examine the effects of the hyperandrogenic state in dihydrotestosterone (DHT)-induced polycystic ovary syndrome (PCOS), the vascular responses to different vasoactive agents, and the modulatory role of vitamin D3. METHODS: APCOS model was induced by DHT application in 20 female Wistar rats. Ten of the DHT treated rats simultaneously received calcitriol treatment. After 10 weeks, myographs were used to test the reactivity of isolated thoracic aortic rings to norepinephrine and acetylcholine. Thereafter, the vascular rings were incubated with the NO-synthase blocker (nitro-L-arginine methyl ester) or the cyclooxygenase inhibitor (indomethacin) for 20 min, and the effects of norepinephrine and acetylcholine were re-evaluated. RESULTS: Norepinephrine-induced vasoconstriction was enhanced after DHT treatment, but this effect was attenuated by calcitriol administration. Vasorelaxation of DHT-treated thoracic aortic rings was impaired, but this could be partly reversed by calcitriol application. Impaired NO-dependent vasorelaxation in DHT-treated animals was mostly reversed by concomitant calcitriol administration, but this effect was diminished by prostanoid-dependent vasoconstriction. CONCLUSIONS: These studies show that the enhanced sensitivity to vasoconstrictors and impaired NO-dependent vasorelaxation in hyperandrogenic PCOS rats could be partially reversed by calcitriol treatment.

Role of endocannabinoids and cannabinoid-1 receptors in cerebrocortical blood flow regulation.

BACKGROUND: Endocannabinoids are among the most intensively studied lipid mediators of cardiovascular functions. In the present study the effects of decreased and increased activity of the endocannabinoid system (achieved by cannabinoid-1 (CB1) receptor blockade and inhibition of cannabinoid reuptake, respectively) on the systemic and cerebral circulation were analyzed under steady-state physiological conditions and during hypoxia and hypercapnia (H/H). METHODOLOGY/PRINCIPAL FINDINGS: In anesthetized spontaneously ventilating rats the CB1-receptor antagonist/inverse agonist AM-251 (10 mg/kg, i.v.) failed to influence blood pressure (BP), cerebrocortical blood flow (CoBF, measured by laser-Doppler flowmetry) or arterial blood gas levels. In contrast, the putative cannabinoid reuptake inhibitor AM-404 (10 mg/kg, i.v.) induced triphasic responses, some of which could be blocked by AM-251. Hypertension during phase I was resistant to AM-251, whereas the concomitant CoBF-increase was attenuated. In contrast, hypotension during phase III was sensitive to AM-251, whereas the concomitant CoBF-decrease was not. Therefore, CoBF autoregulation appeared to shift towards higher BP levels after CB1-blockade. During phase II H/H developed due to respiratory depression, which could be inhibited by AM-251. Interestingly, however, the concomitant rise in CoBF remained unchanged after AM-251, indicating that CB1-blockade potentially enhanced the reactivity of the CoBF to H/H. In accordance with this hypothesis, AM-251 induced a significant enhancement of the CoBF responses during controlled stepwise H/H. CONCLUSION/SIGNIFICANCE: Under resting physiological conditions CB1-receptor mediated mechanisms appear to have limited influence on systemic or cerebral circulation. Enhancement of endocannabinoid levels, however, induces transient CB1-independent hypertension and sustained CB1-mediated hypotension. Furthermore, enhanced endocannabinoid activity results in respiratory depression in a CB1-dependent manner. Finally, our data indicate for the first time the involvement of the endocannabinoid system and CB1-receptors in the regulation of the cerebral circulation during H/H and also raise the possibility of their contribution to the autoregulation of CoBF.

Perivascular expression and potent vasoconstrictor effect of dynorphin A in cerebral arteries.

BACKGROUND: Numerous literary data indicate that dynorphin A (DYN-A) has a significant impact on cerebral circulation, especially under pathophysiological conditions, but its potential direct influence on the tone of cerebral vessels is obscure. The aim of the present study was threefold: 1) to clarify if DYN-A is present in cerebral vessels, 2) to determine if it exerts any direct effect on cerebrovascular tone, and if so, 3) to analyze the role of κ-opiate receptors in mediating the effect. METHODOLOGY/PRINCIPAL FINDINGS: Immunohistochemical analysis revealed the expression of DYN-A in perivascular nerves of rat pial arteries as well as in both rat and human intraparenchymal vessels of the cerebral cortex. In isolated rat basilar and middle cerebral arteries (BAs and MCAs) DYN-A (1-13) and DYN-A (1-17) but not DYN-A (1-8) or dynorphin B (DYN-B) induced strong vasoconstriction in micromolar concentrations. The maximal effects, compared to a reference contraction induced by 124 mM K(+), were 115±6% and 104±10% in BAs and 113±3% and 125±9% in MCAs for 10 µM of DYN-A (1-13) and DYN-A (1-17), respectively. The vasoconstrictor effects of DYN-A (1-13) could be inhibited but not abolished by both the κ-opiate receptor antagonist nor-Binaltorphimine dihydrochloride (NORBI) and blockade of G(i/o)-protein mediated signaling by pertussis toxin. Finally, des-Tyr(1) DYN-A (2-13), which reportedly fails to activate κ-opiate receptors, induced vasoconstriction of 45±11% in BAs and 50±5% in MCAs at 10 µM, which effects were resistant to NORBI. CONCLUSION/SIGNIFICANCE: DYN-A is present in rat and human cerebral perivascular nerves and induces sustained contraction of rat cerebral arteries. This vasoconstrictor effect is only partly mediated by κ-opiate receptors and heterotrimeric G(i/o)-proteins. To our knowledge our present findings are the first to indicate that DYN-A has a direct cerebral vasoconstrictor effect and that a dynorphin-induced vascular action may be, at least in part, independent of κ-opiate receptors.

Urothelial cells produce hydrogen peroxide through the activation of Duox1.

Hydrogen peroxide (H(2)O(2)) has important messenger and effector functions in the plant and animal kingdom. Phagocytes produce H(2)O(2) to kill pathogens, and epithelial cells of large airways have also been reported to produce H(2)O(2) for signaling and host defense purposes. In this report, we show for the first time that urothelial cells produce H(2)O(2) in response to a calcium signal. Using a gene-deficient mouse model we also demonstrate that H(2)O(2) is produced by the NADPH oxidase Duox1, which is expressed in the mouse urothelium. In contrast, we found no evidence for the expression of lactoperoxidase, an enzyme that has been shown to cooperate with Duox enzymes. We also found that specific activation of TRPV4 calcium channels elicits a calcium signal and stimulates H(2)O(2) production in urothelial cells. Furthermore, we detected altered pressure responses in the urinary bladders of Duox1 knockout animals. Our results raise the possibility that mechanosensing in epithelial cells involves calcium-dependent H(2)O(2) production similar to that observed in plants.

Parto Vaginal despues de dos o más cesáreas / Vaginal delivery after 2 or more cesarean sections

Se hace un análisis retrospectivo de 32 partos vaginales en pacientes con dos o más cesáreas anteriores. La revisión uterina demostró 26 segmentos indemnes (81,25//), 2 desuniones totales del segmento (6,25//) y 4 desuniones parciales (12,50//). Se requirió histerorrafia en 3 casos (9,37//). Se discute la ventaja de la atención del parto en pacientes con dos o más desáreas anteriores, como método alternativo a la realización de una cesárea iterativa