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Lipocalin-2 is not only a sensitive biomarker, but it also contributes to the pathogenesis of renal injuries. The present study demonstrates that adipose tissue–derived lipocalin-2 plays a critical role in causing both chronic and acute renal injuries. Four-week treatment with aldosterone and high salt after uninephrectomy (ANS) significantly increased both circulating and urinary lipocalin-2, and it induced glomerular and tubular injuries in kidneys of WT mice. Despite increased renal expression of lcn2 and urinary excretion of lipocalin-2, mice with selective deletion of lcn2 alleles in adipose tissue (Adipo-LKO) are protected from ANS- or aldosterone-induced renal injuries. By contrast, selective deletion of lcn2 alleles in kidney did not prevent aldosterone- or ANS-induced renal injuries. Transplantation of fat pads from WT donors increased the sensitivity of mice with complete deletion of Lcn2 alleles (LKO) to aldosterone-induced renal injuries. Aldosterone promoted the urinary excretion of a human lipocalin-2 variant, R81E, in turn causing renal injuries in LKO mice. Chronic treatment with R81E triggered significant renal injuries in LKO, resembling those observed in WT mice following ANS challenge. Taken in conjunction, the present results demonstrate that lipocalin-2 derived from adipose tissue causes acute and chronic renal injuries, largely independent of local lcn2 expression in kidney.

The purpose of this study was to assess, in the murine kidney, the mechanisms underlying the endothelium-dependent control of vascular tone and whether or not, in a severe model of hypertension and renal failure, K-Ca channels contribute to its regulation. Wild-type (BL) and double-transgenic female mice expressing human angiotensinogen and renin (AR) genes received either control or a high-salt diet associated to a nitric oxide (NO) synthase inhibitor treatment (BLSL and ARSL). Changes in renal perfusion pressure (RPP) were measured in isolated perfused kidneys. BLSL and AR were moderately hypertensive without kidney disease while ARSL developed severe hypertension and renal failure. In the four groups, methacholine induced biphasic endothelium-dependent responses, a transient decrease in RPP followed by a cyclooxygenase-dependent increase in RPP. In the presence or not of indomethacin, the vasodilatations were poorly sensitive to NO synthase inhibition. However, in the presence of cyclooxygenase and NO synthase inhibitors, apamin, and/or TRAM-34, blockers of K(Ca)2.3 and K(Ca)3.1, respectively, abolished the decrease in RPP in response to either methacholine or the two activators of K(Ca)2.3/K(Ca)3.1, NS309, and SKA-31. Thus, K(Ca)2/3 channels play a major role in the regulation of murine kidney perfusion and this mechanism is maintained in hypertension, even when severe and associated with kidney damage.

m/s n° 2, vol. 30, fevrier 2014 DOI : 10.1051/medsci/20143002007 9. Alderton GK, Galbiati L, Griffith E, et al. Regulation of mitotic entry by microcephalin and its overlap with ATR signalling. Nat Cell Biol 2006 ; 8 : 725-33. 10. Quintyne NJ, Reing JE, Hoffelder DR, et al. Spindle multipolarity is prevented by centrosomal clustering. Science 2005 ; 307 : 127-9. 11. Ganem NJ, Godinho SA, Pellman D. A mechanism linking extra centrosomes to chromosomal instability. Nature 2009 ; 460 : 278-82. 12. Thompson SL, Compton DA. Proliferation of aneuploid human cells is limited by a p53-dependent mechanism. J Cell Biol 2010 ; 188 : 369-81. 13. Holland AJ, Fachinetti D, Zhu Q, et al. The autoregulated instability of Polo-like kinase 4 limits centrosome duplication to once per cell cycle. Genes Dev 2012 ; 26 : 2684-9. 2. Habedanck R, Stierhof YD, Wilkinson CJ, Nigg EA. The Polo kinase Plk4 functions in centriole duplication. Nat Cell Biol 2005 ; 7 : 1140-6. 3. Nigg EA. Origins and consequences of centrosome aberrations in human cancers. Int J Cancer 2006 ; 119 : 2717-23. 4. Bettencourt-Dias M, Hildebrandt F, Pellman D, et al. Centrosomes and cilia in human disease. Trends Genet 2011 ; 27 : 307-15. 5. Thornton GK, Woods CG. Primary microcephaly: do all roads lead to Rome? Trends Genet 2009 ; 25 : 501-10. 6. Kaindl AM, Passemard S, Kumar P, et al. Many roads lead to primary autosomal recessive microcephaly. Prog Neurobiol 2010 ; 90 : 363-83. 7. Marthiens V, Rujano MA, Pennetier C, et al. Centrosome amplification causes microcephaly. Nat Cell Biol 2013 ; 15 : 731-40. 8. Hussain MS, Baig SM, Neumann S, et al. A truncating mutation of CEP135 causes primary microcephaly and disturbed centrosomal function. Am J Hum Genet 2012 ; 90 : 871-8. developpement du cerveau est le processus le plus perturbe. ‡ From centrosomes to microcephaly: follow the link

BACKGROUND AND PURPOSE The purpose of the study was to investigate renal endothelium-dependent vasodilatation in a model of severe hypertension associated with kidney injury. EXPERIMENTAL APPROACH Changes in perfusion pressure were measured in isolated, perfused kidneys taken from 18-week-old Wistar–Kyoto rat (WKY), spontaneously hypertensive rats (SHR) and SHR treated for 2 weeks with Nω-nitro-L-arginine methyl ester in the drinking water (L-NAME-treated SHR, 6 mg·kg−1·day−1). KEY RESULTS Acetylcholine caused similar dose-dependent renal dilatation in the three groups. In vitro administration of indomethacin did not alter the vasodilatation, while the addition of Nw-nitro-L-arginine (L-NA) produced a differential inhibition of the vasodilatation, (inhibition in WKY > SHR > L-NAME-treated SHR). Further addition of ODQ, an inhibitor of soluble guanylyl cyclase, abolished the responses to sodium nitroprusside but did not affect the vasodilatation to acetylcholine. However, the addition of TRAM-34 (or charybdotoxin) inhibitors of Ca2+-activated K+ channels of intermediate conductance (KCa3.1), blocked the vasodilatation to acetylcholine, while apamin, an inhibitor of Ca2+-activated K+ channels of small conductance (KCa2.3), was ineffective. Dilatation induced by an opener of KCa3.1/KCa2.3 channels, NS-309, was also blocked by TRAM-34, but not by apamin. The magnitude and duration of NS-309-induced vasodilatation and the renal expression of mRNA for KCa3.1, but not KCa2.3, channels followed the same ranking order (WKY < SHR < L-NAME-treated SHR). CONCLUSIONS AND IMPLICATIONS In SHR kidneys, an EDHF-mediated response, involving activation of KCa3.1 channels, contributed to the mechanism of endothelium-dependent vasodilatation. In kidneys from L-NAME-treated SHR, up-regulation of this pathway fully compensated for the decrease in NO availability.

Endothelium-dependent contractions contribute to endothelial dysfunction in various animal models of aging, diabetes and cardiovascular diseases. In the spontaneously hypertensive rat, the archetypal model for endothelium-dependent contractions, the production of the endothelium-derived contractile factors (EDCF) involves an increase in endothelial intracellular calcium concentration, the production of reactive oxygen species, the predominant activation of cyclooxygenase-1 (COX-1) and to a lesser extent that of COX-2, the diffusion of EDCF towards the smooth muscle cells and the subsequent stimulation of their thromboxane A2-endoperoxide TP receptors. Endothelium-dependent contractions are also observed in various models of hypertension, aging and diabetes. They generally also involve the generation of COX-1- and/or COX-2-derived products and the activation of smooth muscle TP receptors. Depending on the model, thromboxane A2, PGH2, PGF2α, PGE2 and paradoxically PGI2 can all act as EDCFs. In human, the production of COX-derived EDCF is a characteristic of the aging and diseased blood vessels, with essential hypertension causing an earlier onset and an acceleration of this endothelial dysfunction. As it has been observed in animal models, COX-1, COX-2 or both isoforms can contribute to these endothelial dysfunctions. Since in most cases, the activation of TP receptors is the common downstream effector, selective antagonists of this receptor should curtail endothelial dysfunction and be of therapeutic interest in the treatment of cardiovascular disorders. LINKED ARTICLES This article is part of a themed issue on Vascular Endothelium in Health and Disease. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2011.164.issue-3

The present review first summarizes the complex chain of events, in endothelial and vascular smooth muscle cells, that leads to endothelium-dependent relaxations (vasodilatations) due to the generation of nitric oxide (NO) by endothelial nitric oxide synthase (eNOS) and how therapeutic interventions may improve the bioavailability of NO and thus prevent/cure endothelial dysfunction. Then, the role of other endothelium-derived mediators (endothelium-derived hyperpolarizing (EDHF) and contracting (EDCF) factors, endothelin-1) and signals (myoendothelial coupling) is summarized also, with special emphasis on their interaction(s) with the NO pathway, which make the latter not only a major mediator but also a key regulator of endothelium-dependent responses.

BACKGROUND AND PURPOSE The purpose of the present study was to determine whether a stimulator of soluble guanylyl cyclase, BAY 41-2272, inhibits platelet aggregation and to clarify its interaction with nitric oxide (NO). EXPERIMENTAL APPROACH Blood was collected from anaesthetized Wistar Kyoto rats. The aggregation of washed platelets was measured and the production of cAMP and cGMP was determined. KEY RESULTS In adenosine 5'-diphosphate (ADP)-induced platelet aggregation, the anti-aggregating effects of BAY 41-2272, nitroglycerin, sodium nitroprusside and DEA-NONOate were associated with increased levels of cGMP while that of beraprost, a prostacyclin analogue, was correlated with an increase in cAMP. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) prevented the effects of BAY 41-2272 and that of nitroglycerin and sodium nitroprusside, but only inhibited the increase in cGMP produced by of DEA-NONOate. Hydroxocobalamin, an NO scavenger, inhibited the effects of the three NO donors and BAY 41-2272 but did not affect those of beraprost. ADP-induced aggregation and the effects of BAY 41-2272 were not affected by L-nitroarginine. A positive interaction was observed between BAY 41-2272 and the three NO donors. BAY 41-2272 potentiated also the anti-aggregating effects of beraprost, and again this potentiation was inhibited by hydroxocobalamin. CONCLUSIONS AND IMPLICATIONS Inhibition of platelet aggregation by BAY 41-2272 requires the reduced form of soluble guanylyl cyclase and the presence of NO. The positive interaction observed between BAY 41-2272 and various NO donors is qualitatively similar whatever the mechanism involved in NO release. Furthermore, a potent synergism is observed between BAY 41-2272 and a prostacyclin analogue, but only in the presence of NO.

In cardiovascular diseases and during aging, endothelial dysfunction is due in part to the release of endothelium-derived contracting factors that counteract the vasodilator effect of the nitric oxide. Endothelium-dependent contractions involve the activation of endothelial cyclooxygenases and the release of various prostanoids, which activate thromboxane prostanoid (TP) receptors of the underlying vascular smooth muscle. The stimulation of TP receptors elicits not only the contraction and the proliferation of vascular smooth muscle cells but also diverse physiological/pathophysiological reactions, including platelet aggregation and activation of endothelial inflammatory responses. TP receptor antagonists curtail endothelial dysfunction in diseases such as hypertension and diabetes, are potent antithrombotic agents, and prevent vascular inflammation.

The endothelium can evoke relaxations (dilatations) of the underlying vascular smooth muscle, by releasing vasodilator substances. The best characterized endothelium-derived relaxing factor (EDRF) is nitric oxide (NO). The endothelial cells also evoke hyperpolarization of the cell membrane of vascular smooth muscle (endothelium-dependent hyperpolarizations, EDHF-mediated responses). Endothelium-dependent relaxations involve both pertussis toxin-sensitive G(i) (e.g. responses to serotonin and thrombin) and pertussis toxin-insensitive G(q) (e.g. adenosine diphosphate and bradykinin) coupling proteins. The release of NO by the endothelial cell can be up-regulated (e.g. by oestrogens, exercise and dietary factors) and down-regulated (e.g. oxidative stress, smoking and oxidized low-density lipoproteins). It is reduced in the course of vascular disease (e.g. diabetes and hypertension). Arteries covered with regenerated endothelium (e.g. following angioplasty) selectively loose the pertussis toxin-sensitive pathway for NO release which favours vasospasm, thrombosis, penetration of macrophages, cellular growth and the inflammatory reaction leading to atherosclerosis. In addition to the release of NO (and causing endothelium-dependent hyperpolarizations), endothelial cells also can evoke contraction (constriction) of the underlying vascular smooth muscle cells by releasing endothelium-derived contracting factor (EDCF). Most endothelium-dependent acute increases in contractile force are due to the formation of vasoconstrictor prostanoids (endoperoxides and prostacyclin) which activate TP receptors of the vascular smooth muscle cells. EDCF-mediated responses are exacerbated when the production of NO is impaired (e.g. by oxidative stress, ageing, spontaneous hypertension and diabetes). They contribute to the blunting of endothelium-dependent vasodilatations in aged subjects and essential hypertensive patients.

The three subtypes of calcium-activated potassium channels (K(Ca)) of large, intermediate and small conductance (BK(Ca), IK(Ca) and SK(Ca)) are present in the vascular wall. In healthy arteries, BK(Ca) channels are preferentially expressed in vascular smooth muscle cells, while IK(Ca) and SK(Ca) are preferentially located in endothelial cells. The activation of endothelial IK(Ca) and SK(Ca) contributes to nitric oxide (NO) generation and is required to elicit endothelium-dependent hyperpolarizations. In the latter responses, the hyperpolarization of the smooth muscle cells is evoked either via electrical coupling through myo-endothelial gap junctions or by potassium ions, which by accumulating in the intercellular space activate the inwardly rectifying potassium channel Kir2.1 and/or the Na(+)/K(+)-ATPase. Additionally, endothelium-derived factors such as cytochrome P450-derived epoxyeicosatrienoic acids and under some circumstances NO, prostacyclin, lipoxygenase products and hydrogen peroxide (H(2)O(2)) hyperpolarize and relax the underlying smooth muscle cells by activating BK(Ca). In contrast, cytochrome P450-derived 20-hydroxyeicosatetraenoic acid and various endothelium-derived contracting factors inhibit BK(Ca). Aging and cardiovascular diseases are associated with endothelial dysfunctions that can involve a decrease in NO bioavailability, alterations of EDHF-mediated responses and/or enhanced production of endothelium-derived contracting factors. Because potassium channels are involved in these endothelium-dependent responses, activation of endothelial and/or smooth muscle K(Ca) could prevent the occurrence of endothelial dysfunction. Therefore, direct activators of these potassium channels or compounds that regulate their activity or their expression may be of some therapeutic interest. Conversely, blockers of IK(Ca) may prevent restenosis and that of BK(Ca) channels sepsis-dependent hypotension.

Background and purpose: C-type natriuretic peptide (CNP) has been proposed to make a fundamental contribution in arterial endothelium-dependent hyperpolarization to acetylcholine. The present study was designed to address this hypothesis in the guinea-pig carotid artery. Experimental approach: The membrane potential of vascular smooth muscle cells was recorded in isolated arteries with intracellular microelectrodes. Key results: Acetylcholine induced endothelium-dependent hyperpolarizations in the presence or absence of NG-nitro-L-arginine, indomethacin and/or thiorphan, inhibitors of NO-synthases, cyclooxygenases or neutral endopeptidase, respectively. Acetycholine hyperpolarized smooth muscle cells in resting arteries and produced repolarizations in phenylephrine-stimulated arteries. CNP produced hyperpolarizations with variable amplitude. They were observed only in the presence of inhibitors of NO-synthases and cyclooxygenases and were endothelium-independent, maintained in phenylephrine-depolarized carotid arteries, and not affected by the additional presence of thiorphan. In arteries with endothelium, the hyperpolarizations produced by CNP were always significantly smaller than those induced by acetylcholine. Upon repeated administration, a significant tachyphylaxis of the hyperpolarizing effect of CNP was observed, while consecutive administration of acetycholine produced sustained responses. The hyperpolarizations evoked by acetylcholine were abolished by the combination of apamin plus charybdotoxin, but unaffected by glibenclamide or tertiapin. In contrast, CNP-induced hyperpolarizations were abolished by glibenclamide and unaffected by the combination of apamin plus charybdotoxin. Conclusions and implications: In the isolated carotid artery of the guinea-pig, CNP activates KATP and is a weak hyperpolarizing agent. In this artery, the contribution of CNP to EDHF-mediated responses is unlikely. British Journal of Pharmacology (2008) 153, 57–65; doi:10.1038/sj.bjp.0707476; published online 1 October 2007

Background and Purpose: Experiments were designed to assess whether or not the intracellular concentration of calcium and reactive oxygen species (ROS) increase in endothelial cells of the rat thoracic aorta in response to releasers of endothelium-derived contracting factor (EDCF) and if so, whether or not a difference exists between spontaneously hypertensive (SHR) and normotensive (WKY) rats. Experimental approach: Calcium and ROS were measured by confocal microscopy, using Fura-red in combination with Fluo-4 and dichlorodihydrofluorescein diacetate, respectively. Key results: Acetylcholine caused a rapid increase in cytosolic calcium concentration in endothelial cells of both SHR and WKY, which was significantly more pronounced in aortae of the former strain. This rise of calcium was not affected by indomethacin (an inhibitor of cyclooxygenase) or Tiron plus diethyldithiocarbamate acid (DETCA) (membrane permeable antioxidants). In the presence of a nitric oxide synthase blocker, acetylcholine also caused a rapid increase in ROS in endothelial cells of SHR but not in those of WKY. The burst of ROS was prevented by indomethacin or Tiron plus DETCA. Conclusions and implications: These experiments show that endothelial cells of SHR are more prone to calcium and ROS overload upon stimulation with acetylcholine. The abnormal accumulation of calcium is a prerequisite to initiate the release of EDCF and can be mimicked using the calcium ionophore A23187. The sequence of events occurring during endothelium-dependent contractions firstly requires the accumulation of calcium, which then activates cyclooxygenase and produces ROS along with EDCF that in turn stimulates TP-receptors, resulting in EDCF-mediated contractions. British Journal of Pharmacology (2007) 151, 15–23. doi:10.1038/sj.bjp.0707190

Background and purpose: To study the importance of endothelium-derived contracting factors (EDCFs) in arteries of rats with type I diabetes. Experimental approach: Rat femoral arteries were collected four or twelve weeks after induction of diabetes with streptozotocin. Rings, with or without endothelium, were suspended in organ chambers for isometric tension measurement. COX protein levels were determined by Western blotting. Key results: Four weeks after the injection of streptozotocin, the endothelium-dependent relaxations (during contractions to phenylephrine) to A23817 were attenuated, but the endothelium-dependent contractions (quiescent preparations) to the ionophore were augmented. Indomethacin and S18886 prevented the endothelium-dependent contractions, while dazoxiben reduced them in rings from streptozotocin-treated rats, suggesting that thromboxane A2, activating TP- receptors, is involved. Twelve weeks after the injection of streptozotocin, the changes in endothelium-dependent relaxations and contractions to A23187 were even more noticeable. The protein expression of COX-1 was increased in femoral arteries of the diabetic rats. Valeryl salicylate and SC560 inhibited the contractions, suggesting that the EDCFs are produced by COX-1. At that time, a combination of S18886 with EP1-blockers was required to abolish the contractions, suggesting that the EDCFs involved act at both TP- and EP-receptors. Rings without endothelium from streptozotocin-treated rats exhibited a reduced maximal contraction to potassium chloride and U46619, combined with hyper-responsiveness to the latter, suggesting that more prolonged diabetes also alters the responsiveness of vascular smooth muscle. Conclusion and Implications: The production of EDCFs is progressively increased in the course of type I diabetes. Eventually, the disease also damages vascular smooth muscle. British Journal of Pharmacology (2007) 150, 624–632. doi:10.1038/sj.bjp.0706999

Endothelium-dependent relaxations are attributed to the release of various factors, such as nitric oxide, carbon monoxide, reactive oxygen species, adenosine, peptides and arachidonic acid metabolites derived from the cyclooxygenases, lipoxygenases, and cytochrome P450 monooxygenases pathways. The hyperpolarization of the smooth muscle cell can contribute to or be an integral part of the mechanisms underlying the relaxations elicited by virtually all these endothelial mediators. These endothelium-derived factors can activate different families of K(+) channels of the vascular smooth muscle. Other events associated with the hyperpolarization of both the endothelial and the vascular smooth muscle cells (endothelium-derived hyperpolarizing factor (EDHF)-mediated responses) contribute also to endothelium-dependent relaxations. These responses involve an increase in the intracellular Ca(2+) concentration of the endothelial cells followed by the opening of Ca(2+)-activated K(+) channels of small and intermediate conductance and the subsequent hyperpolarization of these cells. Then, the endothelium-dependent hyperpolarization of the underlying smooth muscle cells can be evoked by direct electrical coupling through myoendothelial junctions and/or the accumulation of K(+) ions in the intercellular space between the two cell types. These various mechanisms are not necessarily mutually exclusive and, depending on the vascular bed and the experimental conditions, can occur simultaneously or sequentially, or also may act synergistically.

In mature spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY), acetylcholine and the calcium ionophore A-23187 release endothelium-derived contracting factors (EDCFs), cyclooxygenase derivatives that activate thromboxane-endoperoxide (TP) receptors on vascular smooth muscle. The EDCFs released by acetylcholine are most likely prostacyclin and prostaglandin (PG)H2, whereas those released by A-23187 remain to be identified. Isometric tension and the release of PGs were measured in rings of isolated aortas of WKY and SHR. A-23187 evoked the endothelium-dependent release of prostacyclin, thromboxane A2, PGF2α, PGE2, and possibly PGH2 (PGI2 ≫ thromboxane A2 = PGF2α = PGE2). In SHR aortas, the release of prostacyclin and thromboxane A2 was significantly larger in response to A-23187 than to acetylcholine. In response to the calcium ionophore, the release of thromboxane A2 was significantly larger in aortas of SHR than in those of WKY. In both strains of rat, the inhibition of cyclooxygenase-1 prevented the release of PGs and the occurrence of endothelium-dependent contractions. Dazoxiben, the thromboxane synthase inhibitor, abolished the A-23187-dependent production of thromboxane A2 and inhibited by approximately one-half the endothelium-dependent contractions. U-51605, an inhibitor of PGI synthase, reduced the release of prostacyclin elicited by A-23187 but induced a parallel increase in the production of PGE2 and PGF2α, suggestive of a PGH2 spillover, which was associated with the enhancement of the endothelium-dependent contractions. These results indicate that in the aorta of SHR and WKY, the endothelium-dependent contractions elicited by A-23187 involve the release of thromboxane A2 and prostacyclin with a most likely concomitant contribution of PGH2.

Endothelial cells synthesize and release various factors that regulate angiogenesis, inflammatory responses, hemostasis, as well as vascular tone and permeability. Endothelial dysfunction has been associated with a number of pathophysiological processes. Oxidative stress appears to be a common denominator underlying endothelial dysfunction in cardiovascular diseases. However, depending on the pathology, the vascular bed studied, the stimulant, and additional factors such as age, sex, salt intake, cholesterolemia, glycemia, and hyperhomocysteinemia, the mechanisms underlying the endothelial dysfunction can be markedly different. A reduced bioavailability of nitric oxide (NO), an alteration in the production of prostanoids, including prostacyclin, thromboxane A2, and/or isoprostanes, an impairment of endothelium-dependent hyperpolarization, as well as an increased release of endothelin-1, can individually or in association contribute to endothelial dysfunction. Therapeutic interventions do not necessarily restore a proper endothelial function and, when they do, may improve only part of these variables.

The endothelium controls vascular tone not only by releasing nitric oxide (NO) and prostacyclin but also by other pathways causing hyperpolarization of the underlying smooth muscle cells. This characteristic was at the origin of the denomination endothelium-derived hyperpolarizing factor (EDHF). We know now that this acronym includes different mechanisms. In general, EDHF-mediated responses involve an increase in the intracellular calcium concentration, the opening of calcium-activated potassium channels of small and intermediate conductance and the hyperpolarization of the endothelial cells. This results in an endothelium-dependent hyperpolarization of the smooth muscle cells, which can be evoked by direct electrical coupling through myo-endothelial junctions and/or the accumulation of potassium ions in the intercellular space. Potassium ions hyperpolarize the smooth muscle cells by activating inward rectifying potassium channels and/or Na + /K + -ATPase. In some blood vessels, including large and small coronary arteries, the endothelium releases arachidonic acid metabolites derived from cytochrome P450 monooxygenases. The epoxyeicosatrienoic acids (EET) generated are not only intracellular messengers but also can diffuse and hyperpolarize the smooth muscle cells by activating large conductance calcium-activated potassium channels. Additionally, the endothelium can produce other factors such as lipoxygenases derivatives or hydrogen peroxide (H 2 O 2 ). These different mechanisms are not necessarily exclusive and can occur simultaneously.

Unlike sodium, potassium is vasoactive; for example, when infused into the arterial supply of a vascular bed, blood flow increases. The vasodilation results from hyperpolarization of the vascular smooth muscle cell subsequent to potassium stimulation by the ion of the electrogenic Na+-K+pump and/or activating the inwardly rectifying Kir channels. In the case of skeletal muscle and brain, the increased flow sustains the augmented metabolic needs of the tissues. Potassium ions are also released by the endothelial cells in response to neurohumoral mediators and physical forces (such as shear stress) and contribute to the endothelium-dependent relaxations, being a component of endothelium-derived hyperpolarization factor-mediated responses. Dietary supplementation of potassium can lower blood pressure in normal and some hypertensive patients. Again, in contrast to NaCl restriction, the response to potassium supplementation is slow to appear, taking ∼4 wk. Such supplementation reduces the need for antihypertensive medication. “Salt-sensitive” hypertension responds particularly well, perhaps, in part, because supplementation with potassium increases the urinary excretion of sodium chloride. Potassium supplementation may even reduce organ system complications (e.g., stroke).

The present experiments were designed to determine whether or not endothelium-dependent contractions can be evoked in the aorta of the mouse, and if so, whether or not deleting the COX1 gene affects the response. Sex differences in the response were also examined. Rings of murine aorta were suspended in a Halpern-Mulvany myograph for recording of isometric force. In the aorta of the male wild type C57BL/b6 mice (36-40 weeks old), both acetylcholine and the calcium ionophore caused endothelium-dependent increases in force in the presence of L-NAME, and these were inhibited by valeryl salicylate (a selective COX1 inhibitor) and S18886 (a selective antagonist of TP receptors). Such endothelium-dependent contraction was absent in the aorta of COX1 knockout mice and present in that of COX2 knockout mice. Similar results were obtained in aortas of female wild-type, COX2 and COX1 knockout mice. These experiments reveal the existence of EDCF-mediated contractions in arteries of the mouse. These contractions, as in the aorta of the spontaneously hypertensive rat, are caused by endogenous agonists(s) of TP receptors produced by cyclooxygenase 1, because they are observed in the aortas of COX2 knockout mice but not in aortas of COX1 knockout mice. The present study provides direct evidence that COX1 is indeed the isoform of cyclooxygenase responsible for the production of EDCF.

In the spontaneously hypertensive rat (SHR) and aging Wistar-Kyoto rats (WKY), acetylcholine releases an endothelium-derived contracting factor (EDCF) produced by endothelial cyclooxygenase-1, which stimulates thromboxane A2 receptors (TP receptors) on vascular smooth muscle. The purpose of the present study was to identify this EDCF by measuring changes in isometric tension and the release of various prostaglandins by acetylcholine. In isolated aortic rings of SHR, U 46619, prostaglandin (PG) H2, PGF2alpha, PGE2, PGD2, prostacyclin (PGI2) and 8-isoprostane, all activate TP receptors of the vascular smooth muscle to produce a contraction (U 46619>>8-isoprostane=PGF2alpha=PGH2>PGE2=PGD2>PGI2). The contractions produced by PGH2 and PGI2 were fast and transient, mimicking endothelium-dependent contractions. PGI2 did not relax isolated aortic rings of WKY and SHR. Acetylcholine evoked the endothelium-dependent release of thromboxane A2, PGF2alpha, PGE2, PGI2 and most likely PGH2 (PGI2>>PGF2alpha>or=PGE2>TXA2>8-isoprostane, PGD2). Dazoxiben abolished the production of thromboxane A2, but did not influence the endothelium-dependent contractions to acetylcholine. The release of PGI2 was significantly larger in the aorta of SHR than in WKY, and the former was more sensitive to the contractile effect of PGI2 than the latter. The inhibition of PGI-synthase was associated with an increase in PGH2 spillover and the enhancement of acetylcholine-induced endothelium-dependent contractions. Thus, in the aorta of SHR and aging WKY, the endothelium-dependent contractions elicited by acetylcholine most likely involve the release of PGI2 with a concomitant contribution of PGH2.

1 In coronary arteries, bradykinin opens endothelial intermediate- and small-conductance Ca2+-sensitive K+ channels (IKCa and SKCa) and, additionally, releases epoxyeicosatrienoic acids (EETs) from the endothelium. To clarify the involvement of these pathways in endothelium-dependent myocyte hyperpolarization, bradykinin-induced electrical changes in endothelial cells and myocytes of porcine coronary arteries (following nitric oxide (NO) synthase and cyclooxygenase inhibition) were measured using sharp microelectrodes. 2 Hyperpolarization of endothelial cells by bradykinin (27.0±0.9 mV, n=4) was partially inhibited (74%) by blockade of IKCa and SKCa channels using 10 μM TRAM-39 (2-(2-chlorophenyl)-2,2-diphenylacetonitrile) plus 100 nM apamin (leaving an iberiotoxin-sensitive component), whereas the response to substance P was abolished. 3 After gap junction blockade with HEPES, (N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulphonic acid)) hyperpolarization of the endothelium by 100 nM bradykinin was abolished by TRAM-39 plus apamin, whereas myocyte hyperpolarization still occurred (12.9±1.0 mV, n=4). The residual hyperpolarizations to 100 nM bradykinin were antagonized by the EET antagonist, 14,15-EEZE (14,15-epoxyeicosa-5(Z)-enoic acid) (10 μM), and abolished by iberiotoxin. Bradykinin-induced myocyte hyperpolarizations were also reduced by 14,15-EEZE-mSI (14,15-EEZE-methylsulfonylimide) (5,6- and 14,15-EET antagonist), whereas those to exogenous 11,12-EET were unaffected. 4 These data show that bradykinin-induced hyperpolarization of endothelial cells (due to the opening of IKCa and SKCa channels) is electrotonically transferred to the myocytes via gap junctions. Bradykinin (but not substance P) also hyperpolarizes myocytes by a mechanism (independent of endothelial cell hyperpolarization) which involves endothelial cell production of EETs (most likely 14,15- and/or 11,12-EET). These open endothelial IKCa and SKCa channels and also activate large-conductance calcium-sensitive K+ channels (BKCa) on the surrounding myocytes. British Journal of Pharmacology (2005) 145, 775–784. doi:10.1038/sj.bjp.0706256

1. Endothelial cells, under given circumstances, can initiate contraction (constriction) of the vascular smooth muscle cells that surround them. Such endothelium-dependent, acute increases in contractile tone can be due to the withdrawal of the production of nitric oxide, to the production of vasoconstrictor peptides (angiotensin II, endothelin-1), to the formation of oxygen-derived free radicals (superoxide anions) and/or the release of vasoconstrictor metabolites of arachidonic acid. The latter have been termed endothelium-derived contracting factor (EDCF) as they can contribute to moment-to-moment changes in contractile activity of the underlying vascular smooth muscle cells. 2. To judge from animal experiments, EDCF-mediated responses are exacerbated by aging, spontaneous hypertension and diabetes. 3. To judge from human studies, they contribute to the blunting of endothelium-dependent vasodilatations in aged subjects and essential hypertensive patients. 4. Since EDCF causes vasoconstriction by activation of the TP-receptors on the vascular smooth muscle cells, selective antagonists at these receptors prevent endothelium-dependent contractions, and curtail the endothelial dysfunction in hypertension and diabetes.

1 This study was designed to determine whether the endothelium-dependent hyperpolarizations evoked by acetylcholine in guinea-pig carotid artery involve a cytochrome P450 metabolite and whether they are linked to the activation of two distinct populations of endothelial KCa channels, SKCa and IKCa. 2 The membrane potential was recorded in the vascular smooth muscle cells of the guinea-pig isolated carotid artery. All the experiments were performed in the presence of Nω-L-nitro arginine (100 μM) and indomethacin (5 μM). 3 Under control conditions (Ca2+: 2.5 mM), acetylcholine (10 nM to 10 μM) induced a concentration- and endothelium-dependent hyperpolarization of the vascular smooth muscle cells. Two structurally different specific blockers of SKCa, apamin (0.5 μM) or UCL 1684 (10 μM), produced a partial but significant inhibition of the hyperpolarization evoked by acetylcholine whereas charybdotoxin (0.1 μM) and TRAM-34 (10 μM), a nonpeptidic and specific blocker of IKCa, were ineffective. In contrast, the combinations of apamin plus charybdotoxin, apamin plus TRAM-34 (10 μM) or UCL 1684 (10 μM) plus TRAM-34 (10 μM) virtually abolished the acetylcholine-induced hyperpolarization. 4 In the presence of a combination of apamin and a subeffective dose of TRAM-34 (5 μM), the residual hyperpolarization produced by acetylcholine was not inhibited further by the addition of either an epoxyeicosatrienoic acid antagonist, 14,15-EEZE (10 μM) or the specific blocker of BKCa, iberiotoxin (0.1 μM). 5 In presence of 0.5 mM Ca2+, the hyperpolarization in response to acetylcholine (1 μM) was significantly lower than in 2.5 mM Ca2+. The EDHF-mediated responses became predominantly sensitive to charybdotoxin or TRAM-34 but resistant to apamin. 6 This investigation shows that the production of a cytochrome P450 metabolite, and the subsequent activation of BKCa, is unlikely to contribute to the EDHF-mediated responses in the guinea-pig carotid artery. Furthermore, the EDHF-mediated response involves the activation of both endothelial IKCa and SKCa channels, the activation of either one being able to produce a true hyperpolarization. British Journal of Pharmacology (2005) 144, 477–485. doi:10.1038/sj.bjp.0706003

In the aorta of the spontaneously hypertensive rat (SHR), endothelium-dependent contractions are enhanced by inhibitors of NO synthase and scavengers of NO, but not by methylene blue, an inhibitor of guanylyl cyclase, suggesting that the endothelium-derived contracting factor (EDCF) interacts chemically with NO and is inactivated by the latter. However, in view of the relative lack of specificity of methylene blue this hypothesis was re-examined. Acetylcholine-induced endothelium-dependent contractions of isolated rings of SHR aorta were significantly and similarly potentiated by two NOS inhibitors, by two structurally different NO scavengers, by two inhibitors of guanylate cyclase ODQ and NS2028, but to a lesser extent by methylene blue. The contraction of the isolated rat trachea in response to methacholine and the contraction of the rat aorta in response to both 8-isoprostane and KCl were inhibited significantly by methylene blue. Methylene blue binds to the M3 muscarinic receptor subtype but not to the TP receptor. Therefore, methylene blue is an antagonist of the M3 muscarinic receptor subtype, involved in the release of EDCF, and a non-specific inhibitor of TP receptor-mediated contractions, the receptor involved in the action of EDCF. These inhibitory effects of methylene blue are likely to counteract the effect of the inhibition of soluble guanylate cyclase. These results rule out the hypothesis according to which NO would chemically inactivate EDCF.

AIM: These studies were performed to test the hypothesis that endogenous neuropeptide Y (NPY) acting on the NPY Y5 receptor subtype contributes to the control of food intake. The hypothesis was tested using S 25585—a newly synthesized NPY Y5 receptor antagonist. METHODS AND RESULTS: S 25585 was shown to be a high-affinity antagonist of the NPY Y5 receptor subtype (IC50 5 nM) with no significant affinity toward other NPY receptor subtypes and over 40 other receptors, channels or uptake systems. S 25585 (7.5 mg/kg, i.p.) did not induce a conditioned taste aversion, significantly alter need-induced sodium appetite or induce pica, suggesting that at this dose the compound did not induce illness or malaise. In satiated rats, S 25585 (5.0 and 7.5 mg/kg, i.p.) significantly decreased the overfeeding induced by i.c.v. injection of NPY (1 μg) and the highly selective NPY Y5 receptor agonist [hPP1−17, Ala31, Aib32]NPY (0.7 μg). In rats fasted for 4 h immediately before the dark phase, analysis of the microstructure of feeding behavior revealed that S 25585 significantly increased latency to eat and significantly decreased the duration and size of the meals without altering the meal number or eating rate. Analysis of the behavioral satiety sequence at this time revealed that the animals passed through the normal pattern of feeding, grooming and resting. Although S 25585 appeared to be influencing a physiological system controlling appetite, this does not involve the NPY Y5 receptor since the antagonist also markedly reduced food intake in the NPY Y5 knockout mouse. CONCLUSIONS: The results presented do not support a role for the NPY Y5 receptor in the control of food intake. The results further illustrate that it is imperative that the activity of any new NPY Y5 antagonist be assessed in the NPY Y5 knockout mouse before assuming that its effect on food intake is due to blockade of this receptor.

Endothelial dysfunction is now associated with countless cardiovascular and noncardiovascular pathologies, and has been referred to in the scientific literature more than 65,000 times since 1980 (PubMed search, December 2015). The term was coined in the mid-80s, following the major breakthrough by

Retinal pericytes are key cells involved in the regulation of retinal blood flow. The purpose of this work was to identify the K+ channel population expressed in cultured bovine retinal pericytes and to determine whether beta-adrenergic stimulation alters the activity of these channels. Isolated pericytes were obtained by homogenization and filtration of bovine retina and K+ channels were studied with the whole-cell configuration of the patch-clamp technique on 3-5 passaged pericytes. Pericytes expressed an inward current dependent on extracellular K+ concentration which was sensitive to micromolar concentrations of barium, a characteristic of an inward-rectifying K+ current. Furthermore, two voltage-dependent outward currents were also observed. Their activation and inactivation properties, as well as their respective sensitivity to 4-aminopyridine and iberiotoxin, were indicative of voltage-sensitive and large-conductance calcium-activated K+ channels (BKCa). Isoproterenol and dibutyryl cyclic adenosine monophosphate enhanced the activity of BKCa without affecting the other potassium currents. In conclusion, bovine retinal pericytes express mainly two outward potassium currents, KV and BKCa, as well as an inward rectifying K+ current, Kir. Physiologic stimuli such as an increase in extracellular potassium concentration or beta-adrenergic receptor stimulation enhance the activity of Kir and BKCa, respectively, suggesting a potential role for these channels in the control of retinal blood flow.

1. Endothelin-1 (ET-1) and tumor necrosis factor alpha (TNFalpha) by their action on adipocytes have been independently linked to the pathogenesis of insulino-resistance. In isolated adipocytes, TNFalpha induces the expression of the inducible nitric oxide synthase (iNOS). The purpose of the present work was, in the 3T3-F442A adipocyte cell line, to characterise TNFalpha-induced iNOS expression and to determine whether or not ET-1 could influence TNFalpha-induced iNOS expression and NO production. 2. In differentiated 3T3-F442A, treatment with TNFalpha (20 ng ml(-1)) induced the expression of a functional iNOS as demonstrated by nitrite assay, Western blot, reverse transcription-polymerase chain reaction and Northern blot analysis. TNFalpha-induced iNOS expression requires nuclear factor kappaB activation, but does not necessitate the activation of the PI-3 kinase/Akt and P38-MAP kinase pathways. 3. ET-1, but not ET-3, inhibited the TNFalpha-induced expression of iNOS protein and mRNA as well as nitrite production. The effects of ET-1 were blocked by a specific ETA (BQ123, pA(2) 7.4) but not by a specific ETB receptor antagonist (BQ788). 3T3-F442A adipocytes express the mRNAs for prepro-ET-1 and the ET-A receptor subtype, but not for the ET-B subtype. 4. The inhibitory effect of ET-1 was not affected by bisindolylmaleimide, SB 203580 or indomethacin, inhibitors of protein kinase C, p38-MAP kinase and cyclooxygenase, respectively, and was not associated with cAMP production. However, the effect of ET-1 was partially reversed by wortmannin, suggesting the involvement of PI3 kinase in the transduction signal of ET-1. 5. Differentiated 3T3-F442A adipocytes did not release ET-1 with or without exposure to TNFalpha, although the mRNA for preproET-1 was detected in both pre- and differentiated adipocytes. 6. Thus, these results confirm that adipocytes are a target for circulating ET-1 and demonstrate that the activation of the ETA receptor subtype can prevent TNFalpha-induced iNOS expression.

This study characterizes the K+ channel(s) underlying charybdotoxin-sensitive hyperpolarization of porcine coronary artery endothelium. Two forms of current-voltage (I/V) relationship were evident in whole-cell patch-clamp recordings of freshly-isolated endothelial cells. In both cell types, iberiotoxin (100 nM) inhibited a current active only at potentials over +50 mV. In the presence of iberiotoxin, charybdotoxin (100 nM) produced a large inhibition in 38% of cells and altered the form of the I/V relationship. In the remaining cells, charybdotoxin also inhibited a current but did not alter the form. Single-channel, outside-out patch recordings revealed a 17.1±0.4 pS conductance. Pipette solutions containing 100, 250 and 500 nM free Ca2+ demonstrated that the open probability was increased by Ca2+. This channel was blocked by charybdotoxin but not by iberiotoxin or apamin. Hyperpolarizations of intact endothelium elicited by substance P (100 nM; 26.1±0.7 mV) were reduced by apamin (100 nM; 17.0±1.8 mV) whereas those to 1-ethyl-2-benzimidazolinone (1-EBIO, 600 μM, 21.0±0.3 mV) were unaffected (21.7±0.8 mV). Substance P, bradykinin (100 nM) and 1-EBIO evoked charybdotoxin-sensitive, iberiotoxin-insensitive whole-cell perforated-patch currents. A porcine homologue of the intermediate-conductance Ca2+-activated K+ channel (IK1) was identified in endothelial cells. In conclusion, porcine coronary artery endothelial cells express an intermediate-conductance Ca2+-activated K+ channel and the IK1 gene product. This channel is opened by activation of the EDHF pathway and likely mediates the charybdotoxin-sensitive component of the EDHF response. Keywords: Endothelium, EDHF, hyperpolarization, calcium-activated potassium channels, charybdotoxin, iberiotoxin, apamin, IK1 gene product Introduction The vascular endothelium controls vessel tone by releasing nitric oxide (Furchgott & Zawadzki, 1980) and prostacyclin (Moncada & Vane, 1979) as well as by a third pathway which involves hyperpolarization of the vascular smooth muscle (reviewed by Busse et al., 2002). This ‘endothelium-dependent hyperpolarizing factor (EDHF)' pathway is inhibited by a combination of the toxins apamin and charybdotoxin, but not apamin and iberiotoxin (Corriu et al., 1996; Zygmunt & Hogestatt, 1996; Petersson et al., 1997; Chataigneau et al., 1998; Edwards et al., 1998; 2000). Given the specificities of these toxins (reviewed by Garcia et al., 1991; Castle, 1999), small- and intermediate-conductance Ca2+-activated K+ channels (SKCa and IKCa, respectively) but not large-conductance Ca2+-activated K+ channels (BKCa) are implicated in the EDHF pathway. In the vasculature, SKCa and IKCa are expressed in endothelial cells (Sakai, 1990; Marchenko & Sage, 1996; Kohler et al., 2000; Burnham et al., 2002) but not in smooth muscle cells with the contractile phenotype while BKCa are mainly expressed in myocytes (Zygmunt et al., 1997; Neylon et al., 1999; Quignard et al., 2000). Furthermore, the combination of apamin and charybdotoxin blocks EDHF-mediated vasodilatation if selectively applied to the endothelium and inhibits the hyperpolarization of the endothelial cells produced by acetylcholine or bradykinin (Edwards et al., 1998; 2000; Doughty et al., 1999; Ohashi et al., 1999). Finally, the increase in endothelial intracellular calcium concentration, provoked by the agonist, is not inhibited by the two toxins (Ghisdal & Morel, 2001). Altogether, these experimental results suggest that the hyperpolarization of the endothelial cells is the critical initiating step in the EDHF-mediated responses (Quignard et al., 2000; Edwards et al., 2000; Busse et al., 2002) and that apamin plus charybdotoxin exert their effects at this site (Edwards et al., 1998). In an earlier study, in porcine coronary artery endothelial cells, an apamin-sensitive K+ channel which is likely to be involved in the EDHF response was proposed to be an SKCa containing the SK3 subunit (Burnham et al., 2002). The purpose of the present study was, in the same cells, to characterize the K+ channels sensitive to charybdotoxin which are also involved in EDHF-mediated responses.

Summary: This study was designed to determine whether lipoxygenase-dependent metabolites of arachidonic acid are involved in the endothelium-dependent hyperpolarization of the guinea pig carotid artery. The membrane potential of vascular smooth muscle cells was measured with intracellular microelectrodes and potassium channels were studied on freshly isolated cells with the patch-clamp technique. Acetylcholine-induced hyperpolarizations were not affected by arachidonyl trifluoromethyl ketone (AACOCF3), quinacrine (phospholipase A2 inhibitors), or eicosatetraenoic acid (nonspecific inhibitor of lipoxygenase, cytochrome P450, and cyclooxygenase). In contrast, cinnamyl-3,4 dihydroxy-α-cyanocinnamate (CDC) and AA861 (lipoxygenase inhibitors) as well as 1-(6-(17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino) hexyl)-1H-pyrrole-2,5-dione (U-73122) (phospholipase C inhibitor) produced a significant inhibition of the hyperpolarization. An opener of intermediate conductance calcium-activated potassium channels, 1-ethyl-2-benzamidazolinone (1-EBIO), induced a hyperpolarization that was unaffected by AACOCF3, CDC, AA861, or U-73122 but was inhibited by charybdotoxin. (±)12-hydroxy-eicosatetraenoic acid (12-HETE) and 12(S)-hydroperoxy-eicosatetraenoic acid (12(S)-HpETE) did not induce any significant changes in membrane potential. CDC inhibited the voltage-gated potassium current and increased the large conductance calcium-activated potassium current whereas AA861 inhibited both potassium currents. These results confirm that, in the isolated carotid artery of the guinea pig, stimulation of endothelial muscarinic receptors involves phospholipase C activation and indicate that the activation of phospholipase A2 and the release of lipoxygenase metabolites is unlikely to explain endothelium-dependent hyperpolarization.

Mechanisms underlying K+-induced hyperpolarizations in the presence and absence of phenylephrine were investigated in endothelium-denuded rat mesenteric arteries (for all mean values, n=4). Myocyte resting membrane potential (m.p.) was −58.8±0.8 mV. Application of 5 mM KCl produced similar hyperpolarizations in the absence (17.6±0.7 mV) or presence (15.8±1.0 mV) of 500 nM ouabain. In the presence of ouabain +30 μM barium, hyperpolarization to 5 mM KCl was essentially abolished. In the presence of 10 μM phenylephrine (m.p. −33.7±3 mV), repolarization to 5 mM KCl did not occur in the presence or absence of 4-aminopyridine but was restored (−26.9±1.8 mV) on addition of iberiotoxin (100 nM). Under these conditions the K+-induced repolarization was insensitive to barium (30 μM) but abolished by 500 nM ouabain alone. In the presence of phenylephrine + iberiotoxin the hyperpolarization to 5 mM K+ was inhibited in the additional presence of 300 nM levcromakalim, an action which was reversed by 10 μM glibenclamide. RT–PCR, Western blotting and immunohistochemical techniques collectively showed the presence of α1-, α2- and α3-subunits of Na+/K+-ATPase in the myocytes. In K+-free solution, re-introduction of K+ (to 4.6 mM) hyperpolarized myocytes by 20.9±0.5 mV, an effect unchanged by 500 nM ouabain but abolished by 500 μM ouabain. We conclude that under basal conditions, Na+/K+-ATPases containing α2- and/or α3-subunits are partially responsible for the observed K+-induced effects. The opening of myocyte K+ channels (by levcromakalim or phenylephrine) creates a ‘K+ cloud’ around the cells which fully activates Na+/K+-ATPase and thereby abolishes further responses to [K+]o elevation. British Journal of Pharmacology (2002) 136, 918–926. doi:10.1038/sj.bjp.0704787

The apamin-sensitive small-conductance Ca2+-activated K+ channel (SKCa) was characterized in porcine coronary arteries. In intact arteries, 100 nM substance P and 600 μM 1-ethyl-2-benzimidazolinone (1-EBIO) produced endothelial cell hyperpolarizations (27.8±0.8 mV and 24.1±1.0 mV, respectively). Charybdotoxin (100 nM) abolished the 1-EBIO response but substance P continued to induce a hyperpolarization (25.8±0.3 mV). In freshly-isolated endothelial cells, outside-out patch recordings revealed a unitary K+ conductance of 6.8±0.04 pS. The open-probability was increased by Ca2+ and reduced by apamin (100 nM). Substance P activated an outward current under whole-cell perforated-patch conditions and a component of this current (38%) was inhibited by apamin. A second conductance of 2.7±0.03 pS inhibited by d-tubocurarine was observed infrequently. Messenger RNA encoding the SK2 and SK3, but not the SK1, subunits of SKCa was detected by RT – PCR in samples of endothelium. Western blotting indicated that SK3 protein was abundant in samples of endothelium compared to whole arteries. SK2 protein was present in whole artery nuclear fractions. Immunofluorescent labelling confirmed that SK3 was highly expressed at the plasmalemma of endothelial cells and was not expressed in smooth muscle. SK2 was restricted to the peri-nuclear regions of both endothelial and smooth muscle cells. In conclusion, the porcine coronary artery endothelium expresses an apamin-sensitive SKCa containing the SK3 subunit. These channels are likely to confer all or part of the apamin-sensitive component of the endothelium-derived hyperpolarizing factor (EDHF) response. British Journal of Pharmacology (2002) 135, 1133–1143; doi:10.1038/sj.bjp.0704551

AIM: The aim of this review is to critically assess available evidence that blockade of the actions of NPY at one of the five NPY receptor subtypes represents an attractive new drug discovery target for the development of an appetite suppressant drug. RESULTS: Blockade of the central actions of NPY using anti-NPY antibodies, antisense oligodeoxynucleotides against NPY and NPY receptor antagonists results in a decrease in food intake in energy-deprived animals. These results appear to show that endogenous NPY plays a role in the control of appetite. The fact that NPY receptors exist as at least five different subtypes raises the possibility that the actions of endogenous NPY on food intake can be adequately dissociated from other effects of the peptide. Current drug discovery has produced a number of highly selective NPY receptor antagonists which have been used to establish the NPY Y1 receptor subtype as the most critical in regulating short-term food intake. However, additional studies are now needed to more clearly define the relative contribution of NPY acting through the NPY Y2 and NPY Y5 receptors in the complex sequence of physiological and behavioral events that underlie the long-term control of appetite. CONCLUSIONS: Blockade of the NPY receptor may produce appetite-suppressing drugs. However, it is too early to state with certainty whether a single subtype selective drug used alone or a combination of NPY receptor selective antagonists used in combination will be necessary to adequately influence appetite regulation.

© Springer-Verlag Berlin Heidelberg 2014. Angiotensin-II and oxidative stress are involved in the genesis of aortic aneurysms, a phenomenon exacerbated by endothelial nitric oxide synthase (eNOS) deletion or uncoupling. The purpose of this work was to study the endothelial function in wild-type C57BL/6 (BL) and transgenic mice expressing the h-angiotensinogen and h-renin genes (AR) subjected to either a control, or a high-salt diet plus a treatment with a NO-synthase inhibitor, N-ω-nitro-L-argininemethyl- ester (L-NAME; BLSL and ARSL). BLSL showed a moderate increase in blood pressure, while ARSL became severely hypertensive. Seventy-five percent of ARSL developed aortic aneurysms, characterized by major histomorphological changes and associated with an increase in NADP(H) oxidase-2 (NOX2) expression. Contractile responses (KCl, norepinephrine, U-46619) were similar in the four groups of mice, and relaxations were not affected in BLSL and AR. However, in ARSL, endothelium-dependent relaxations (acetylcholine, UK-14304) were significantly reduced, and this dysfunction was similar in aortae without or with aneurysms. The endothelial impairment was unaffected by catalase, superoxide-dismutase mimetic, radical scavengers, cyclooxygenase inhibition, or TP-receptor blockade and could not be attributed to sGC oxidation. Thus, ARSL is a severe hypertension model developing aortic aneurysm. A vascular dysfunction, involving both endothelial (reduced role of NO) and smooth muscle cells, precedes aneurysms formation and, paradoxically, does not appear to involve oxidative stress.

Background Lipocalin‐2 is a proinflammatory adipokine upregulated in obese humans and animals. A pathogenic role of lipocalin‐2 in hypertension has been suggested. Mice lacking lipocalin‐2 are protected from dietary obesity‐induced cardiovascular dysfunctions. Administration of lipocalin‐2 causes abnormal vasodilator responses in mice on a high‐fat diet (HFD). Methods and Results Wild‐type and lipocalin‐2 knockout mice were fed with standard chow or HFD. Immunoassays were performed for evaluating the circulating and tissue contents of lipocalin‐2. The relaxation and contraction of arteries were studied using a wire myograph. Blood pressure was monitored with implantable radio telemetry. Dietary obesity promoted the accumulation of lipocalin‐2 protein in blood and arteries. Deficiency of this adipokine protected mice from dietary obesity‐induced elevation of blood pressure. Mass spectrometry analysis revealed that human and murine lipocalin‐2 were modified by polyamination. Polyaminated lipocalin‐2 was rapidly cleared from the circulation. Adipose tissue was a major site for lipocalin‐2 deamidation. The circulating levels and the arterial accumulation of deamidated lipocalin‐2 were significantly enhanced by treatment with linoleic acid (18:2n−6), which bound to lipocalin‐2 with high affinity and prevented its interactions with matrix metalloproteinase 9 ( MMP 9). Combined administration of linoleic acid with lipocalin‐2 caused vascular inflammation and endothelial dysfunction and raised the blood pressure of mice receiving standard chow. A human lipocalin‐2 mutant with cysteine 87 replaced by alanine (C87A) contained less polyamines and exhibited a reduced capacity to form heterodimeric complexes with MMP 9. After treatment, C87A remained in the circulation for a prolonged period of time and evoked endothelial dysfunction in the absence of linoleic acid. Conclusions Polyamination facilitates the clearance of lipocalin‐2, whereas the accumulation of deamidated lipocalin‐2 in arteries causes vascular inflammation, endothelial dysfunction, and hypertension.

In intact mesenteric arteries, increasing [K+]o by 5 mM hyperpolarized both endothelial and smooth muscle cells. Subsequent exposure to 10 μM phenylephrine depolarized both cell types which were then repolarized by a 5 mM increase in [K+]o. In endothelium-denuded vessels, increasing [K+]o by 5 mM hyperpolarized the smooth muscle but K+ had no effect after depolarization by 10 μM phenylephrine. On subsequent exposure to iberiotoxin plus 4-aminopyridine, the repolarizing action of 5 mM K+ was restored. In endothelium-intact vessels exposed to phenylephrine, pretreatment with a gap junction inhibitor (gap 27) reduced K+-mediated smooth muscle repolarization without affecting the endothelial cell response. It is concluded that phenylephrine-induced efflux of K+via smooth muscle K+ channels produces a local increase in [K+]o which impairs repolarization to added K+. Thus, studies involving vessels precontracted with agonists which increase [K+]o maximize the role of gap junctions and minimize any contribution to the EDHF pathway from endothelium-derived K+. British Journal of Pharmacology (2001) 134, 1–5; doi:10.1038/sj.bjp.0704256

In porcine coronary arteries, smooth muscle hyperpolarizations produced by the nitric oxide donor, NOR-1, and the prostacyclin analogue, iloprost, were compared with those induced by substance P and bradykinin and attributed to the endothelium-derived hyperpolarizing factor (EDHF). In the presence of 300 microM L-nitroarginine and 10 microM indomethacin, iloprost-induced hyperpolarizations were partially inhibited by 10 microM glibenclamide whereas those to NOR-1, substance P and bradykinin were unaffected. Hyperpolarizations produced by maximally-effective concentrations of NOR-1 and NS1619 were identical (to -65 mV). They were significantly less than those generated by either substance P or bradykinin (to approximately -80 mV) and were abolished by iberiotoxin 100 nM, a concentration which had essentially no effect on responses to substance P or bradykinin. Incubation of segments of intact arteries for 16 - 22 h in bicarbonate-buffered Krebs solution had little effect on EDHF responses to substance P or bradykinin. In contrast, after incubation for this period of time in HEPES-buffered Tyrode solution or Krebs containing 10 mM HEPES the EDHF response to substance P was abolished and that to bradykinin was markedly reduced. The residual bradykinin-induced hyperpolarization following incubation in Tyrode solution was inhibited by iberiotoxin and by 10 microM 17-octadecynoic acid. We conclude that substance P activates only the EDHF pathway in the presence of nitric oxide synthase and cyclo-oxygenase inhibitors. Incubation in HEPES-buffered Tyrode solution abolishes the EDHF responses to substance P and bradykinin to reveal an additional hyperpolarizing mechanism, associated with the opening of K(+) channels, activated only by bradykinin.

The purpose of this study in the hamster cheek pouch was to determine whether or not vascular endothelial growth factor (VEGF) induced changes in plasma extravasation and if so, the mechanism(s) involved. The cheek pouch microcirculatory bed of the anaesthetized hamster was directly observed under microscope and the number of vascular leakage sites, as shown by fluorescein isothiocyanate (FITC-dextran, 150 kD) extravasation, was counted. Drugs and VEGF were applied topically. VEGF from 0.05 to 0.5 μg ml−1 (1.2 to 12 nM) produced a dose-dependent increase in the number of microvascular leakage sites from virtually none in basal conditions to up to 250 in some pouches. The effects of VEGF (0.1 μg ml−1 or 2.4 nM) were blocked in a concentration-dependent manner by the non-specific heparin growth factor antagonist TBC-1635 (0.1, 1 and 3μM). The placenta growth factor (PlGF-1: 0.1 and 0.5 μg ml−1 or 3.4 and 17 nM) did not increase plasma extravasation, per se, but abolished the effects of VEGF (2.4 nM). The increases in microvascular leakage produced by VEGF (2.4 nM) were partially but significantly (P

Experiments were performed to identify the potassium channels involved in the acetylcholine-induced endothelium-dependent hyperpolarization of the guinea-pig internal carotid artery. Smooth muscle and endothelial cell membrane potentials were recorded in isolated arteries with intracellular microelectrodes. Potassium currents were recorded in freshly-dissociated smooth muscle cells using patch clamp techniques. In single myocytes, iberiotoxin (0.1 μM)-, charybdotoxin (0.1 μM)-, apamin (0.5 μM)- and 4-aminopyridine (5 mM)-sensitive potassium currents were identified indicating the presence of large- and small-conductance calcium-sensitive potassium channels (BKCa and SKCa) as well as voltage-dependent potassium channels (KV). Charybdotoxin and iberiotoxin inhibited the same population of BKCa but a conductance specifically sensitive to the combination of charybdotoxin plus apamin could not be detected. 4-aminopyridine (0.1–25 mM) induced a concentration-dependent inhibition of KV without affecting the iberiotoxin- or the apamin-sensitive currents. In isolated arteries, both the endothelium-dependent hyperpolarization of smooth muscle and the hyperpolarization of endothelial cells induced by acetylcholine or by substance P were inhibited by 5 mM 4-aminopyridine. These results indicate that in the vascular smooth muscle cells of the guinea-pig carotid artery, a conductance specifically sensitive to the combination of charybdotoxin plus apamin could not be detected, comforting the hypothesis that the combination of these two toxins should act on the endothelial cells. Furthermore, the inhibition by 4-aminopyridine of both smooth muscle and endothelial hyperpolarizations, suggests that in order to observe an endothelium-dependent hyperpolarization of the vascular smooth muscle cells, the activation of endothelial potassium channels is likely to be required. Keywords: 4-Aminopyridine, apamin, charybdotoxin, endothelium, EDHF, iberiotoxin, potassium channels, smooth muscle Introduction The vascular endothelium controls tone in the underlying smooth muscle cells by releasing various factors including nitric oxide (NO) (Furchgott & Zawadzki, 1980), prostacyclin (Moncada & Vane, 1979) and an unidentified endothelium-derived hyperpolarizing factor (EDHF) (Feletou & Vanhoutte, 1988; Taylor & Weston, 1988). Many observations combine to suggest that the EDHF-induced hyperpolarization of the vascular smooth muscle involves the opening of potassium channels. Thus, it is associated with a decrease in membrane resistance (Bolton et al., 1984; Chen & Suzuki 1989a,1989b), inversely related to the extracellular K+ concentration and cannot be observed at K+ concentrations higher than 25 mM (Chen & Suzuki, 1989a; Nagao & Vanhoutte, 1992; Corriu et al., 1996a). Furthermore, endothelium-dependent hyperpolarizations are associated with an increase in rubidium efflux (Taylor et al., 1988) and prevented by non-selective inhibitors of potassium channels such as tetraethylammonium and tetrabutylammonium (Chen et al., 1991; Nagao & Vanhoutte, 1992; Van de Voorde et al., 1992). In various tissues, apamin (a specific inhibitor of small conductance calcium-activated potassium channels) alone or in combination with charybdotoxin (a non-specific inhibitor of calcium-activated potassium channels), inhibits the responses attributed to EDHF (Murphy & Brayden, 1995; Garland & Plane, 1996; Corriu et al., 1996a; Zygmunt & Hoggestatt, 1996; Chataigneau et al., 1998; Yamanaka et al., 1998; Quignard et al., 1999a). These toxins seem selective as they inhibit EDHF-mediated responses without affecting relaxations or hyperpolarizations produced by endothelial nitric oxide or prostacyclin. Furthermore, the increase in endothelial intracellular calcium produced by acetylcholine was not affected by the two toxins (Yamanaka et al., 1998). Therefore, it has been assumed that the target(s) for the two toxins is on the vascular smooth muscle. However, calcium-activated potassium channels are also expressed in endothelial cells (Marchenko & Sage, 1996) and in the rat hepatic artery and the rabbit aortic valve, the combination of charybdotoxin plus apamin inhibits endothelial cell hyperpolarization produced by acetylcholine (Edwards et al., 1998; Ohashi et al., 1999). Furthermore, in rat mesenteric artery, charybdotoxin and apamin block EDHF responses if selectively applied to the endothelium (Doughty et al., 1999). Thus, the endothelial actions of the toxins might be responsible for the inhibition of EDHF. Finally, another potassium channel blocker, 4-aminopyridine, inhibits acetylcholine-induced endothelium-dependent hyperpolarization in the isolated coronary artery of the guinea-pig (Eckman et al., 1998). However, in the same blood vessel, 4-aminopyridine inhibits also acetylcholine-induced endothelial cell hyperpolarization (Chen & Cheung 1992a), suggesting again that the endothelial action of the potassium channel blocker could be responsible for the inhibition of EDHF. The present experiments were therefore designed to study the different types of potassium channels expressed in freshly isolated smooth muscle cells from the guinea-pig carotid artery and to determine the role of endothelial cells in the inhibitory effect of potassium channel blockers.

1. The effects of endothelium-derived hyperpolarizing factor (EDHF: elicited using substance P or bradykinin) were compared with those of 11,12-EET in pig coronary artery. Smooth muscle cells were usually impaled with microelectrodes through the adventitial surface. 2. Substance P (100 nM) and 11,12-EET (11,12-epoxyeicosatrienoic acid; 3 microM) hyperpolarized endothelial cells in intact arteries. These actions were unaffected by 100 nM iberiotoxin but were abolished by charybdotoxin plus apamin (each 100 nM). 3. Substance P (100 nM) and bradykinin (30 nM) hyperpolarized intact artery smooth muscle; Substance P had no effect after endothelium removal. 11,12-EET hyperpolarized de-endothelialized vessels by 12.6+/-0.3 mV, an effect abolished by 100 nM iberiotoxin. 4. 11,12-EET hyperpolarized intact arteries by 18.6+/-0.8 mV, an action reduced by iberiotoxin, which was ineffective against substance P. Hyperpolarizations to 11, 12-EET and substance P were partially inhibited by 100 nM charybdotoxin and abolished by further addition of 100 nM apamin. 5. 30 microM barium plus 500 nM ouabain depolarized intact artery smooth muscle but responses to substance P and bradykinin were unchanged. 500 microM gap 27 markedly reduced hyperpolarizations to substance P and bradykinin which were abolished in the additional presence of barium plus ouabain. 6. Substance P-induced hyperpolarizations of smooth muscle cells immediately below the internal elastic lamina were unaffected by gap 27, even in the presence of barium plus ouabain. 7. In pig coronary artery, 11,12-EET is not EDHF. Smooth muscle hyperpolarizations attributed to 'EDHF' are initiated by endothelial cell hyperpolarization involving charybdotoxin- (but not iberiotoxin) and apamin-sensitive K(+) channels. This may spread electrotonically via myoendothelial gap junctions but the involvement of an unknown endothelial factor cannot be excluded.

1. In guinea-pig internal carotid arteries with an intact endothelium, acetylcholine (10 microM) and levcromakalim (10 microM) each hyperpolarized the smooth muscle whereas a 5 mM elevation of extracellular K(+) was without effect. 2. Incubation of the carotid artery with the gap junction inhibitors carbenoxolone (100 microM) or gap 27 (500 microM) essentially abolished the hyperpolarization to acetylcholine but it was without effect on that to levcromakalim. Carbenoxolone had no effect on the acetylcholine-induced endothelial cell hyperpolarization but inhibited the smooth muscle hyperpolarization induced by the endothelial cell K(+) channel opener, 1-ethyl-2-benzimidazolinone (600 microM). 3. In rat hepatic and mesenteric arteries with endothelium, carbenoxolone (100 or 500 microM) depolarized the smooth muscle but did not modify hyperpolarizations induced by KCl or levcromakalim. In the mesenteric (but not the hepatic) artery, the acetylcholine-induced hyperpolarization was inhibited by carbenoxolone. 4. Phenylephrine (1 microM) depolarized the smooth muscle cells of intact hepatic and mesenteric arteries, an effect enhanced by carbenoxolone. Gap 27 did not have a depolarizing action. In the presence of phenylephrine, acetylcholine-induced hyperpolarization of both hepatic and mesenteric artery myocytes was partially inhibited by each of the gap junction inhibitors. 5. Collectively, the data suggest that gap junctions play some role in the EDHF (endothelium-derived hyperpolarizing factor) response in rat hepatic and mesenteric arteries. However, in the guinea-pig internal carotid artery, electrotonic propagation of endothelial cell hyperpolarizations via gap junctions may be the sole mechanism underlying the response previously attributed to EDHF.

The characteristics of endothelium-dependent hyperpolarization in rat hepatic artery have been further investigated in the presence of inhibitors of cyclo-oxygenase and nitric oxide synthase. Using sharp micro-electrodes, the smooth muscle hyperpolarization induced by acetylcholine, KCl or 1-ethyl-2-benzimidazolinone (1-EBIO) in intact hepatic arteries was abolished by 30 μM barium plus 500 nM ouabain. In vessels without endothelium, the smooth muscle hyperpolarization induced by KCl was not reduced by 30 μM barium alone. However, in the presence of barium the effects of KCl were partially inhibited by 100 nM ouabain and essentially abolished by 500 nM ouabain. Using sharp micro-electrodes, the hyperpolarization of both the smooth muscle and the endothelium induced by 1-EBIO or by acetylcholine was unaffected by 100 nM iberiotoxin. However, in the presence of 100 nM charybdotoxin, the effects of 1-EBIO were abolished whereas those of acetylcholine were only partially reduced. The hyperpolarization induced by levcromakalim was unaffected by either charybdotoxin or iberiotoxin. Under whole-cell patch-clamp recording conditions, 1-EBIO induced a voltage-insensitive, charybdotoxin-sensitive K+ current in cultured endothelial cells but was without effect on K+ currents in smooth muscle cells isolated from hepatic arteries. It is concluded that the endothelium-dependent hyperpolarization of smooth muscle induced by either acetylcholine or by 1-EBIO in rat hepatic artery is initially associated with the opening of endothelial calcium-sensitive K+-channels insensitive to iberiotoxin. The resulting accumulation of K+ in the myoendothelial space activates an isoform of Na+/K+-ATPase which is sensitive to low concentrations of ouabain. British Journal of Pharmacology (1999) 128, 1064–1070; doi:10.1038/sj.bjp.0702916

1. The purpose of this work was to verify the hypothesis that the rabbit ileum is a selective preparation for the NPY Y5 receptor by using new selective antagonists recently synthesized. Spontaneous contractions of the rabbit isolated ileum were recorded and binding experiments were performed in cells expressing the human NPY Y1, Y2, Y4 or Y5 receptor subtype. 2. NPY analogues produced a concentration-dependent transient inhibition of the spontaneous contractions of the rabbit ileum with the following order of potency hPP > rPP > PYY > or = [Leu31,-Pro34]-NPY > NPY >> NPY13-36. Pre-exposure to rPP, PYY, [Leu31,Pro34]-NPY or NPY (but not NPY13-36) inhibited the effect of subsequent administration of hPP suggesting cross-desensitization of the preparation. The apparent affinity of the various agonists studied was correlated to the affinity reported for the human Y4 receptor subtype (and to a lesser extent for the rat Y4 subtype) but not to the affinity for the Y5 receptor subtype. 3. BIBO 3304, a selective NPY Y1 receptor antagonist, and CGP 71683A, a selective NPY Y5 receptor antagonist, did not affect the response to hPP. JCF 109, another NPY Y5 receptor antagonist, produced an inhibition of the response to hPP but only at the highest dose tested (10 microM) which also, by itself, produced intrinsic inhibitory effects. 4. 1229U91, a non-selective ligand for Y1, Y2, Y4 and Y5 receptors with high affinity toward the Y1 and Y4 receptor subtypes, produced a concentration-dependent transient inhibition of the spontaneous contractions of the rabbit ileum and a dose-dependent inhibition of the response to hPP (apparent pKB: 7.2). 5. These results suggest that in the rabbit ileum, the NPY receptor involved in the inhibition of the spontaneous contractile activity is a NPY Y4 receptor subtype.

Experiments were designed to determine in two arteries (the guinea-pig carotid and the porcine coronary arteries) whether or not the endothelium-derived hyperpolarizing factor (EDHF) can be identified as potassium ions, and to determine whether or not the inwardly rectifying potassium current and the Na+/K+ pump are involved in the hyperpolarization mediated by EDHF. The membrane potential of vascular smooth muscle cells was recorded with intracellular microelectrodes in the presence of Nω-L-nitro-arginine (L-NA) and indomethacin. In vascular smooth muscle cells of guinea-pig carotid and porcine coronary arteries, acetylcholine and bradykinin induced endothelium-dependent hyperpolarizations (−18±1 mV, n=39 and −19±1 mV, n=7, respectively). The hyperpolarizations were not affected significantly by ouabain (1 μM), barium chloride (up to 100 μM) or the combination of ouabain plus barium. In both arteries, increasing extracellular potassium concentration by 5 or 10 mM induced either depolarization or in a very few cases small hyperpolarizations which never exceeded 2 mV. In isolated smooth muscle cells of the guinea-pig carotid artery, patch-clamp experiments shows that only 20% of the vascular smooth muscle cells expressed inwardly rectifying potassium channels. The current density recorded was low (0.5±0.1 pA pF−1, n=8). These results indicate that, in two different vascular preparations, barium sensitive-inwardly rectifying potassium conductance and the ouabain sensitive-Na+/K+ pump are not involved in the EDHF-mediated hyperpolarization. Furthermore, potassium did not mimic the effect of EDHF pointing out that potassium and EDHF are not the same entity in those arteries. British Journal of Pharmacology (1999) 127, 27–34; doi:10.1038/sj.bjp.0702493

1. Isometric tension was recorded in isolated rings of aorta, carotid, coronary and mesenteric arteries taken from endothelial nitric oxide synthase knockout mice (eNOS(-/-) mice) and the corresponding wild-type strain (eNOS(+/+) mice). The membrane potential of smooth muscle cells was measured in coronary arteries with intracellular microelectrodes. 2. In the isolated aorta, carotid and coronary arteries from the eNOS(+/+) mice, acetylcholine induced an endothelium-dependent relaxation which was inhibited by N(omega)-L-nitro-arginine. In contrast, in the mesenteric arteries, the inhibition of the cholinergic relaxation required the combination of N(omega)-L-nitro-arginine and indomethacin. 3. The isolated aorta, carotid and coronary arteries from the eNOS(-/-) mice did not relax in response to acetylcholine. However, acetylcholine produced an indomethacin-sensitive relaxation in the mesenteric artery from eNOS(-/-) mice. 4. The resting membrane potential of smooth muscle cells from isolated coronary arteries was significantly less negative in the eNOS(-/-) mice (-64.8 +/- 1.8 mV, n = 20 and -58.4 +/- 1.9 mV, n = 17, for eNOS(+/+) and eNOS(-/-) mice, respectively). In both strains, acetylcholine, bradykinin and substance P did not induce endothelium-dependent hyperpolarizations whereas cromakalim consistently produced hyperpolarizations (- 7.9 +/- 1.1 mV, n = 8 and -13.8 +/- 2.6 mV, n = 4, for eNOS(+/+) and eNOS(-/-) mice, respectively). 5. These findings demonstrate that in the blood vessels studied: (1) in the eNOS(+/+) mice, the endothelium-dependent relaxations to acetylcholine involve either NO or the combination of NO plus a product of cyclo-oxygenase but not EDHF; (2) in the eNOS(-/-) mice, NO-dependent responses and EDHF-like responses were not observed. In the mesenteric arteries acetylcholine releases a cyclo-oxygenase derivative.

1. The purpose of these experiments was to determine whether or not the endothelium-dependent hyperpolarizations of the vascular smooth muscle cells (observed in the presence of inhibitors of nitric oxide synthase and cyclo-oxygenase) can be attributed to the production of an endogenous cannabinoid. 2. Membrane potential was recorded in the guinea-pig carotid, rat mesenteric and porcine coronary arteries by intracellular microelectrodes. 3. In the rat mesenteric artery, the cannabinoid receptor antagonist, SR 141716 (1 microM), did not modify either the resting membrane potential of smooth muscle cells or the endothelium-dependent hyperpolarization induced by acetylcholine (1 microM) (17.3 +/- 1.8 mV, n = 4 and 17.8 +/- 2.6 mV, n = 4, in control and presence of SR 141716, respectively). Anandamide (30 microM) induced a hyperpolarization of the smooth muscle cells (12.6 +/- 1.4 mV, n = 13 and 2.0 +/- 3.0 mV, n = 6 in vessels with and without endothelium, respectively) which could not be repeated in the same tissue, whereas acetylcholine was still able to hyperpolarize the preparation. The hyperpolarization induced by anandamide was not significantly influenced by SR 141716 (1 microM). HU-210 (30 microM), a synthetic CB1 receptor agonist, and palmitoylethanolamide (30 microM), a CB2 receptor agonist, did not influence the membrane potential of the vascular smooth muscle cells. 4. In the rat mesenteric artery, the endothelium-dependent hyperpolarization induced by acetylcholine (1 microM) (19.0 +/- 1.7 mV, n = 6) was not altered by glibenclamide (1 microM; 17.7 +/- 2.3 mV, n = 3). However, the combination of charybdotoxin (0.1 microM) plus apamin (0.5 microM) abolished the acetylcholine-induced hyperpolarization and under these conditions, acetylcholine evoked a depolarization (7.7 +/- 2.7 mV, n = 3). The hyperpolarization induced by anandamide (30 microM) (12.6 +/- 1.4 mV, n = 13) was significantly inhibited by glibenclamide (4.0 +/- 0.4 mV, n = 4) but not significantly affected by the combination of charybdotoxin plus apamin (17.3 +/- 2.3 mV, n = 4). 5. In the guinea-pig carotid artery, acetylcholine (1 microM) evoked endothelium-dependent hyperpolarization (18.8 +/- 0.7 mV, n = 15). SR 141716 (10 nM to 10 microM), caused a direct, concentration-dependent hyperpolarization (up to 10 mV at 10 microM) and a significant inhibition of the acetylcholine-induced hyperpolarization. Anandamide (0.1 to 3 microM) did not influence the membrane potential. At a concentration of 30 microM, the cannabinoid agonist induced a non-reproducible hyperpolarization (5.6 +/- 1.3 mV, n = 10) with a slow onset. SR 141716 (1 microM) did not affect the hyperpolarization induced by 30 microM anandamide (5.3 +/- 1.5 mV, n = 3). 6. In the porcine coronary artery, anandamide up to 30 microM did not hyperpolarize or relax the smooth muscle cells. The endothelium-dependent hyperpolarization and relaxation induced by bradykinin were not influenced by SR 141716 (1 microM). 7. These results indicate that the endothelium-dependent hyperpolarizations, observed in the guinea-pig carotid, rat mesenteric and porcine coronary arteries, are not related to the activation of cannabinoid CB1 receptors.

Using intracellular microelectrodes, we investigated the effects of 17-octadecynoic acid (17-ODYA) on the endothelium-dependent hyperpolarization induced by acetylcholine in the guinea-pig isolated internal carotid artery with endothelium. In the presence of Nω-nitro-L-arginine (L-NOARG, 100 μM) and indomethacin (5 μM) to inhibit nitric oxide synthase and cyclo-oxygenase, acetylcholine (1 μM) evoked an endothelium-dependent hyperpolarization which averaged −16.4 mV starting from a resting membrane potential of −56.8 mV. There was a negative correlation between the amplitude of the hyperpolarization and the absolute values of the resting membrane potential. The acetylcholine-induced endothelium-dependent hyperpolarization was not altered by charybdotoxin (0.1 μM) or iberiotoxin (30 nM). It was partially but significantly reduced by apamin (0.5 μM) to −12.8±1.2 mV (n=10) or the combination of apamin plus iberiotoxin (−14.3±3.4 mV, n=4). However, the combination of charybdotoxin and apamin abolished the hyperpolarization and under these conditions, acetylcholine evoked a depolarization (+7.1±3.7 mV, n=8). 17-ODYA (10 μM) produced a significant hyperpolarization of the resting membrane potential which averaged −59.6 mV and a partial but significant inhibition of the acetylcholine-induced endothelium-dependent hyperpolarization (−10.9 mV). Apamin did not modify the effects of 17-ODYA but in the presence of charybdotoxin or iberiotoxin, 17-ODYA no longer influenced the resting membrane potential or the acetylcholine-induced hyperpolarization. When compared to solvent (ethanol, 1% v/v), epoxyeicosatrienoic acids (EpETrEs) (5,6-, 8,9-, 11,12- and 14,15-EpETrE, 3 μM) did not affect the cell membrane potential and did not relax the guinea-pig isolated internal carotid artery. These results indicate that, in the guinea-pig internal carotid artery, the involvement of metabolites of arachidonic acid through the cytochrome P450 pathway in endothelium-dependent hyperpolarization is unlikely. Furthermore, the hyperpolarization mediated by the endothelium-derived hyperpolarizing factor (EDHF) is probably not due to the opening of BKCa channels. British Journal of Pharmacology (1998) 123, 574–580; doi:10.1038/sj.bjp.0701629