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Fibroblast growth factor 23 (FGF23) is secreted primarily by osteocytes and regulates phosphate and vitamin D metabolism. Elevated levels of FGF23 are clinically associated with endothelial dysfunction and arterial stiffness in chronic kidney disease (CKD) patients; however, the direct effects of FGF23 on endothelial function are unknown. We hypothesized that FGF23 directly impairs endothelial vasorelaxation by hindering nitric oxide (NO) bioavailability. We detected expression of all four subtypes of FGF receptors ( Fgfr1–4) in male mouse aortas. Exogenous FGF23 (90–9,000 pg/ml) did not induce contraction of aortic rings and did not relax rings precontracted with PGF2α. However, preincubation with FGF23 (9,000 pg/ml) caused a ∼36% inhibition of endothelium-dependent relaxation elicited by acetylcholine (ACh) in precontracted aortic rings, which was prevented by the FGFR antagonist PD166866 (50 nM). Furthermore, in FGF23-pretreated (9,000 pg/ml) aortic rings, we found reductions in NO levels. We also investigated an animal model of CKD ( Col4a3 −/− mice) that displays highly elevated serum FGF23 levels and found they had impaired endothelium-dependent vascular relaxation and reduced nitrate production compared with age-matched wild types. To elucidate a mechanism for the FGF23-induced impairment, we measured superoxide levels in endothelial cells and aortic rings and found that they were increased following FGF23 treatment. Crucially, treatment with the superoxide scavenger tiron reduced superoxide levels and also restored aortic relaxation to ACh. Therefore, our data suggest that FGF23 increases superoxide, inhibits NO bioavailability, and causes endothelial dysfunction in mouse aorta. Together, these data provide evidence that high levels of FGF23 contribute to cardiovascular dysfunction.

Autosomal recessive hypophosphatemic rickets (ARHR) is a heritable disorder characterized by hypophosphatemia, osteomalacia, and poor bone development. ARHR results from inactivating mutations in the DMP1 gene with the human phenotype being recapitulated in the Dmp1 null mouse model which displays elevated plasma fibroblast growth factor 23. While the bone phenotype has been well-characterized, it is not known what effects ARHR may also have on skeletal, cardiac, or vascular smooth muscle function, which is critical to understand in order to treat patients suffering from this condition. In this study, the extensor digitorum longus (EDL-fast-twitch muscle), soleus (SOL-slow-twitch muscle), heart, and aorta were removed from Dmp1 null mice and ex-vivo functional tests were simultaneously performed in collaboration by three different laboratories. Dmp1 null EDL and SOL muscles produced less force than wildtype muscles after normalization for physiological cross sectional area of the muscles. Both EDL and SOL muscles from Dmp1 null mice also produced less force after the addition of caffeine (which releases calcium from the sarcoplasmic reticulum) which may indicate problems in excitation contraction coupling in these mice. While the body weights of the Dmp1 null were smaller than wildtype, the heart weight to body weight ratio was higher. However, there were no differences in pathological hypertrophic gene expression compared to wildtype and maximal force of contraction was not different indicating that there may not be cardiac pathology under the tested conditions. We did observe a decrease in the rate of force development generated by cardiac muscle in the Dmp1 null which may be related to some of the deficits observed in skeletal muscle. There were no differences observed in aortic contractions induced by PGF2α or 5-HT or in endothelium-mediated acetylcholine-induced relaxations or endothelium-independent sodium nitroprusside-induced relaxations. In summary, these results indicate that there are deficiencies in both fast twitch and slow twitch muscle fiber type contractions in this model of ARHR, while there was less of a phenotype observed in cardiac muscle, and no differences observed in aortic function. These results may help explain skeletal muscle weakness reported by some patients with osteomalacia and need to be further investigated.

We have recently reported that a novel muscle-specific inositide phosphatase (MIP/MTMR14) plays a critical role in [Ca2+]i homeostasis through dephosphorylation of sn-1-stearoyl-2-arachidonoyl phosphatidylinositol (3,5) bisphosphate (PI(3,5)P2). Loss of function mutations in MIP have been identified in human centronuclear myopathy. We developed a MIP knockout (MIPKO) animal model and found that MIPKO mice were more susceptible to exercise-induced muscle damage, a trademark of muscle functional changes in older subjects. We used wild-type (Wt) mice and MIPKO mice to elucidate the roles of MIP in muscle function during aging. We found MIP mRNA expression, MIP protein levels, and MIP phosphatase activity significantly decreased in old Wt mice. The mature MIPKO mice displayed phenotypes that closely resembled those seen in old Wt mice: i) decreased walking speed, ii) decreased treadmill activity, iii) decreased contractile force, and iv) decreased power generation, classical features of sarcopenia in rodents and humans. Defective Ca2+ homeostasis is also present in mature MIPKO and old Wt mice, suggesting a putative role of MIP in the decline of muscle function during aging. Our studies offer a new avenue for the investigation of MIP roles in skeletal muscle function and as a potential therapeutic target to treat aging sarcopenia.

The neuroprotective effects of exogenous erythropoietin (EPO) in animals and humans after brain injury may be afforded, in part, by the influence of EPO on cerebral arteries. We tested (1) if EPO itself is vasoactive and (2) if EPO enhances endothelium-mediated dilations, specifically those mediated by endothelium-derived hyperpolarizing factor (EDHF). Immunoblotting and reverse transcriptase-polymerase chain reaction (RT-PCR) were used to detect EPO receptor. Rat middle cerebral arteries (MCAs) were isolated, pressurized, and perfused in vitro. EPO was directly applied to MCAs to test its vasoactivity. Endothelium-mediated dilations were elicited by UTP, whereas EDHF-mediated dilations were elicited by UTP after inhibition of endothelial nitric oxide synthase and cyclooxygenase. mRNA and protein for EPO receptor was found in rat MCA. Abluminal application of 0.001-10 U/mL EPO, which is selective for vascular smooth muscle, did not alter vessel diameter. In contrast, luminal application of EPO, which is selective for endothelium, resulted in concentration-dependent dilations of up to 39 +/- 16% at 10 U/mL (p = 0.0018), though responses were variable. A single dose of EPO (1,000 U/kg) administered to rats 24 h prior to examining vascular function potentiated dilations to UTP 2.6-fold (p0.0001). EDHF-mediated dilations were potentiated 2.1-fold following in vivo EPO treatment (p = 0.0034). This study demonstrates that EPO can directly dilate rat MCAs via the endothelium, though not all vessels are responsive. Additionally, pre-treatment with EPO for 24 h in vivo potentiates endothelium-mediated dilations, specifically those mediated by EDHF. Thus, enhanced endothelium-mediated dilations may partially underlie the neuroprotective effects of EPO after brain injury.

Objective— The goal of this study was to compare vascular function, superoxide levels, and MnSOD protein expression in young (4 to 7 months) and old (22 to 24 months) MnSOD +/+ and MnSOD-deficient (MnSOD +/− ) mice. Methods and Results— Relaxation of aorta in vitro to the endothelium-dependent dilator acetylcholine (ACh) was similar in young MnSOD +/+ (n=9) and young MnSOD +/− (n=6) mice. This response was impaired in old MnSOD +/+ (n=8) mice and old MnSOD +/− mice (n=14), with dysfunction being greater in old MnSOD-deficient mice (eg, 100 μmol/L ACh produced 77±3% [mean±SE], 77±3%, 70±4%, and 57±4% relaxation in young MnSOD +/+ , young MnSOD +/− , old MnSOD +/+ , and old MnSOD +/− mice, respectively). The endothelial dysfunction was similar in mice on both C57BL/6 and CD-1 genetic backgrounds. In contrast to ACh, responses to the endothelium-independent dilator sodium nitroprusside were enhanced in old MnSOD +/+ and MnSOD +/− mice compared with both groups of young mice ( P +/− mice compared with old MnSOD +/+ and young mice ( P Conclusions— These data provide the first direct evidence that MnSOD halpoinsufficiency results in increased vascular oxidative stress and endothelial dysfunction with aging.

Peroxisome proliferator activated receptor alpha (PPARα) is one of the PPAR isoforms belonging to the nuclear hormone receptor superfamily that regulates genes involved in lipid and lipoprotein metabolism. PPARαis present in the vascular wall and is thought to be involved in protection against vascular disease. To determine if PPARαcontributes to endothelial function, conduit and cerebral resistance arteries were studied inPparα−/−mice using isometric and isobaric tension myography, respectively. Aortic contractions to PGF2αand constriction of middle cerebral arteries to phenylephrine were not different between wild type (WT) andPparα−/−; however, relaxation/dilation to acetylcholine (ACh) was impaired. There was no difference in relaxation between WT andPparα−/−aorta to treatment with a nitric oxide (NO) surrogate indicating impairment in endothelial function. Endothelial NO levels as well as NO synthase expression were reduced inPparα−/−aortas, while superoxide levels were elevated. Two-week feeding with the reactive oxygen species (ROS) scavenger, tempol, normalized ROS levels and rescued the impaired endothelium-mediated relaxation inPparα−/−mice. These results suggest thatPparα−/−mice have impaired endothelial function caused by decreased NO bioavailability. Therefore, activation of PPARαreceptors may be a therapeutic target for maintaining endothelial function and protection against cardiovascular disease.

Carbon monoxide (CO) has been postulated to be a signaling molecule in many tissues, including the vasculature. We examined vasomotor responses of adult rat and mouse cerebral arteries to both exogenously applied and endogenously produced CO. The diameter of isolated, pressurized, and perfused rat middle cerebral arteries (MCAs) was not altered by authentic CO (10−6to 10−4M). Mouse MCAs, however, dilated by 21 ± 10% at 10−4M CO. Authentic nitric oxide (NO·, 10−10to 10−7M) dilated both rat and mouse MCAs. At 10−8M NO·, rat vessels dilated by 84 ± 4%, and at 10−7M NO·, mouse vessels dilated by 59 ± 9%. Stimulation of endogenous CO production through heme oxygenase (HO) with the heme precursor δ-aminolevulinic acid (10−10to 10−4M) did not dilate the MCAs of either species. The metalloporphyrin HO inhibitor chromium mesoporphyrin IX (CrMP) caused profound constriction of the rat MCA (44 ± 2% at 3 × 10−5M). Importantly, this constriction was unaltered by exogenous CO (10−4M) or CO plus 10−5M biliverdine (both HO products). In contrast, exogenous CO (10−4M) reversed CrMP-induced constriction in rat gracilis arterioles. Control mouse MCAs constricted by only 3 ± 1% in response to 10−5M CrMP. Magnesium protoporphyrin IX (10−5M), a weak HO inhibitor used to control for nonspecific effects of metalloporphyrins, also constricted the rat MCA to a similar extent as CrMP. We conclude that, at physiological concentrations, CO is not a dilator of adult rodent cerebral arteries and that metalloporphyrin HO inhibitors have nonspecific constrictor effects in rat cerebral arteries.

The cerebrovascular endothelium exerts a profound influence on cerebral vessels and cerebral blood flow. This review summarizes current knowledge of various dilator and constrictor mechanisms intrinsic to the cerebrovascular endothelium. The endothelium contributes to the resting tone of cerebral arteries and arterioles by tonically releasing nitric oxide (NO•). Dilations can occur by stimulated release of NO•, endothelium-derived hyperpolarization factor, or prostanoids. During pathological conditions, the dilator influence of the endothelium can turn to that of constriction by a variety of mechanisms, including decreased NO• bioavailability and release of endothelin-1. The endothelium may participate in neurovascular coupling by conducting local dilations to upstream arteries. Further study of the cerebrovascular endothelium is critical for understanding the pathogenesis of a number of pathological conditions, including stroke, traumatic brain injury, and subarachnoid hemorrhage.

MnSOD is the only mammalian isoform of SOD that is necessary for life. MnSOD−/−mice die soon after birth, and MnSOD+/−mice are more susceptible to oxidative stress than wild-type (WT) mice. In this study, we examined vasomotor function responses in aortas of MnSOD+/−mice under normal conditions and during oxidative stress. Under normal conditions, contractions to serotonin (5-HT) and prostaglandin F2α(PGF2α), relaxation to ACh, and superoxide levels were similar in aortas of WT and MnSOD+/−mice. The mitochondrial inhibitor antimycin A reduced contraction to PGF2αand impaired relaxation to ACh to a similar extent in aortas of WT and MnSOD+/−mice. The Cu/ZnSOD and extracellular SOD inhibitor diethyldithiocarbamate (DDC) paradoxically enhanced contraction to 5-HT and superoxide more in aortas of WT mice than in MnSOD+/−mice. DDC impaired relaxation to ACh and reduced total SOD activity similarly in aortas of both genotypes. Tiron, a scavenger of superoxide, normalized contraction to 5-HT, relaxation to ACh, and superoxide levels in DDC-treated aortas of WT and MnSOD+/−mice. Hypoxia, which reportedly increases superoxide, reduced contractions to 5-HT and PGF2αsimilarly in aortas of WT and MnSOD+/−mice. The vasomotor response to acute hypoxia was similar in both genotypes. In summary, under normal conditions and during acute oxidative stress, vasomotor function is similar in WT and MnSOD+/−mice. We speculate that decreased mitochondrial superoxide production may preserve nitric oxide bioavailability during oxidative stress.

Abstract —We sought to determine whether adenovirus-mediated gene transfer in vivo of calcitonin gene–related peptide (CGRP), a potent vasodilator, ameliorates cerebral vasoconstriction after experimental subarachnoid hemorrhage (SAH). Arterial blood was injected into the cisterna magna of rabbits to mimic SAH 5 days after injection of AdRSVCGRP (8×10 8 pfu), AdRSVβgal (control virus), or vehicle. After injection of AdRSVCGRP, there was a 400-fold increase in CGRP in cerebrospinal fluid. Contraction of the basilar artery to serotonin in vitro was greater in rabbits after SAH than after injection of artificial cerebrospinal fluid ( P P P P P 1 receptor antagonist. To determine whether gene transfer of CGRP after SAH may prevent cerebral vasoconstriction, we constructed a virus with a cytomegalovirus (CMV) promoter, which results in rapid expression of the transgene product. Treatment of rabbits with AdCMVCGRP after experimental SAH prevented constriction of the basilar artery 2 days after SAH. Thus, gene transfer of CGRP prevents cerebral vasoconstriction in vivo after experimental SAH.

The pulvinar nucleus is a major source of input to visual cortical areas, but many important facts are still unknown concerning the organization of pulvinocortical (PC) connections and their possible interactions with other connectional systems. In order to address some of these questions, we labeled PC connections by extracellular injections of biotinylated dextran amine into the lateral pulvinar of two monkeys, and analyzed 25 individual axons in several extrastriate areas by serial section reconstruction. This approach yielded four results: (1) in all extrastriate areas examined (V2, V3, V4, and middle temporal area [MT]/V5), PC axons consistently have 2-6 multiple, spatially distributed arbors; (2) in each area, there is a small number of larger caliber axons, possibly originating from a subpopulation of calbindin-positive giant projection neurons in the pulvinar; (3) as previously reported by others, most terminations in extrastriate areas are concentrated in layer 3, but they can occur in other layers (layers 4,5,6, and, occasionally, layer 1) as collaterals of a single axon; in addition, (4) the size of individual arbors and of the terminal field as a whole varies with cortical area. In areas V2 and V3, there is typically a single principal arbor (0.25-0.50 mm in diameter) and several smaller arbors. In area V4, the principal arbor is larger (2.0- to 2.5-mm-wide), but in area MT/V5, the arbors tend to be smaller (0.15 mm in diameter). Size differences might result from specializations of the target areas, or may be more related to the particular injection site and how this projects to individual cortical areas. Feedforward cortical axons, except in area V2, have multiple arbors, but these do not show any obvious size progression. Thus, in areas V2, V3, and especially V4, PC fields are larger than those of cortical axons, but in MT/V5 they are smaller. Terminal specializations of PC connections tend to be larger than those of corticocortical, but the projection foci are less dense. Further work is necessary to determine the differential interactions within and between systems, and how these might result in the complex patterns of suppression and enhancement, postulated as gating mechanisms in cortical attentional effects, or in different states of arousal.

Hyperthermia is an important approach for the treatment of several diseases. Hyperthermia is also thought to induce hypertrophy of skeletal muscles in vitro and in vivo, and has been used as a therapeutic tool for millennia. In the first part of our work, we revise several relevant patents related to the utilization of hyperthermia for the treatment and diagnostic of human diseases. In the second part, we present exciting new data on the effects of forced and natural overexpression of HSP72, using murine in vitro (muscle cells) and ex vivo (primary skeletal muscles) models. These studies help to demonstrate that hyperthermia effects are orchestrated by tight coupling between gene expression, protein function, and intracellular Ca(2+) signaling pathways with a key role for calcium-induced calcium release. We hope that the review of current patents along with previous unknown information on molecular signaling pathways that underlie the hypertrophy response to hyperthermia in skeletal muscles may trigger the curiosity of scientists worldwide to explore new inventions that fully utilize hyperthermia for the treatment of muscle diseases.

Store-operated Ca(2+) entry (SOCE) has recently been shown to be of physiological and pathological importance in the heart, particularly during cardiac hypertrophy. However, measuring changes in intracellular Ca(2+) during SOCE is very difficult to study in adult primary cardiomyocytes. As a result there is a need for a stable and reliable in vitro model of SOCE which can be used to test cardiac drugs and investigate the role of SOCE in cardiac pathology. HL-1 cells are the only immortal cardiomyocyte cell line available that continuously divides and spontaneously contracts while maintaining phenotypic characteristics of the adult cardiomyocyte. To date the role of SOCE has not yet been investigated in the HL-1 cardiac cell line. We report for the first time that these cells expressed stromal interaction molecule 1 (STIM1) and the Ca(2+) release-activated Ca(2+) (CRAC) channel Orai1, which are essential components of the SOCE machinery. In addition, SOCE was tightly coupled to sarcoplasmic reticulum (SR)-Ca(2+) release in HL-1 cells, and such response was not impaired in the presence of voltage dependent Ca(2+) channels (L-type and T-type channels) or reverse mode Na(+)/Ca(2+) exchanger (NCX) inhibitors. We were able to abolish the SOCE response with known SOCE inhibitors (BTP-2 and SKF-96365) and by targeted knockdown of Orai1 with RNAi. In addition, knockdown of Orai1 resulted in lower baseline Ca(2+) and an attenuated response to thapsigargin (TG) and caffeine, indicating that SOCE may play a role in Ca(2+) homeostasis during unstressed conditions in cardiomyocytes. Currently, there is little knowledge about SOCE in cardiomyocytes, and the present results suggest that HL-1 cells will be of great utility in investigating the role of SOCE in the heart.

Phosphoinositide (3,5)-bisphosphate [PI(3,5)P2] is a newly identified phosphoinositide that modulates intracellular Ca2+by activating ryanodine receptors (RyRs). Since the contractile state of arterial smooth muscle depends on the concentration of intracellular Ca2+, we hypothesized that by mobilizing sarcoplasmic reticulum (SR) Ca2+stores PI(3,5)P2would increase intracellular Ca2+in arterial smooth muscle cells and cause vasocontraction. Using immunohistochemistry, we found that PI(3,5)P2was present in the mouse aorta and that exogenously applied PI(3,5)P2readily entered aortic smooth muscle cells. In isolated aortic smooth muscle cells, exogenous PI(3,5)P2elevated intracellular Ca2+, and it also contracted aortic rings. Both the rise in intracellular Ca2+and the contraction caused by PI(3,5)P2were prevented by antagonizing RyRs, while the majority of the PI(3,5)P2response was intact after blockade of inositol (1,4,5)-trisphosphate receptors. Depletion of SR Ca2+stores with thapsigargin or caffeine and/or ryanodine blunted the Ca2+response and greatly attenuated the contraction elicited by PI(3,5)P2. The removal of extracellular Ca2+or addition of verapamil to inhibit voltage-dependent Ca2+channels reduced but did not eliminate the Ca2+or contractile responses to PI(3,5)P2. We also found that PI(3,5)P2depolarized aortic smooth muscle cells and that LaCl3inhibited those aspects of the PI(3,5)P2response attributable to extracellular Ca2+. Thus, full and sustained aortic contractions to PI(3,5)P2required the release of SR Ca2+, probably via the activation of RyR, and also extracellular Ca2+entry via voltage-dependent Ca2+channels.

Phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) is the most recently identified phosphoinositide, and its functions have yet to be fully elucidated. Recently, members of our muscle group have shown that PI(3,5)P2 plays an important role in skeletal muscle function by altering Ca(2+) homeostasis. Therefore, we hypothesized that PI(3,5)P2 may also modulate cardiac muscle contractility by altering intracellular Ca(2+) ([Ca(2+)](i)) in cardiac myocytes. We first confirmed that PI(3,5)P2 was present and increased by insulin treatment of cardiomyocytes via immunohistochemistry. To examine the acute effects of PI(3,5)P2 treatment, electrically paced left ventricular muscle strips were incubated with PI(3,5)P2. Treatment with PI(3,5)P2 increased the magnitude of isometric force, the rate of force development, and the area associated with the contractile waveforms. These enhanced contractile responses were also observed in MIP/Mtmr14(-/-) mouse hearts, which we found to have elevated levels of PI(3,5)P2. In cardiac myocytes loaded with fura-2, PI(3,5)P2 produced a robust elevation in [Ca(2+)](i). The PI(3,5)P2-induced elevation of [Ca(2+)](i) was not present in conditions free of extracellular Ca(2+) and was completely blocked by ryanodine. We investigated whether the phosphoinositide acted directly with the Ca(2+) release channels of the sarcoplasmic reticulum (ryanodine receptors; RyR2). PI(3,5)P2 increased [(3)H]ryanodine binding and increased the open probability (P(o)) of single RyR2 channels reconstituted in lipid bilayers. This strongly suggests that the phosphoinositide binds directly to the RyR2 channel. Thus, we provide inaugural evidence that PI(3,5)P2 is a powerful activator of sarcoplasmic reticulum Ca(2+) release and thereby modulates cardiac contractility.

Heat shock proteins (Hsp's) are molecular chaperones that are critical for the maintenance of cellular homeostasis and have been shown to protect cells and tissues from a large variety of damaging insults. Conflicting reports have been published on the effects of HS on whole animal models of hypertrophy. Some reports suggest that HS inhibits skeletal muscle growth, while others show an increase in the size and proliferative potential of intact skeletal muscles. However recent studies have suggested that heat shock can induce hypertrophy in cardiac muscle cell lines. The cellular response of skeletal muscle cells exposed to heat shock (HS) is to our knowledge an untested treatment method for studying muscle hypertrophy. Therefore, our main goal was to examine the hypertrophic reactions to a mild HS treatment (43°C for 20 min) in C2C12 muscle cells. C2C12's are widely considered to be very representative of skeletal muscle cells in vivo, and can therefore serve as a useful model of physiological hypertrophy. We found that our mild HS treatment induced significant hypertrophy (> 30% of cell area growth) of differentiated C2C12 myotubes, indicating that HS is an effective treatment for induction of hypertrophy in C2C12 muscle cells. We are now investigating the mechanisms underlying HS-induced hypertrophy in C2C12 muscle cells. We believe that these studies will be useful in establishing the foundation for a better and in-depth understanding of HS-induced stress in skeletal muscles. (Support:American Heart Association 0535355N, Missouri Life Sciences Research Board & NIH Opportunities Grant/Recovery Act (GO Grant) to MB.

Trichloroacetaldehyde monohydrate [chloral hydrate (CH)] is a sedative/hypnotic that increases cerebral blood flow (CBF), and its active metabolite 2,2,2-trichloroethanol (TCE) is an agonist for the nonclassical two-pore domain K+ (K2P) channels TREK-1 and TRAAK. We sought to determine whether TCE dilates cerebral arteries in vitro by activating nonclassical K+ channels. TCE dilated pressurized and perfused rat middle cerebral arteries (MCAs) in a manner consistent with activation of nonclassical K+ channels. Dilation to TCE was inhibited by elevated external K+ but not by an inhibitory cocktail (IC) of classical K+ channel blockers. Patch-clamp electrophysiology revealed that, in the presence of the IC, TCE increased whole-cell currents and hyperpolarized the membrane potential of isolated MCA smooth muscle cells. Heating increased TCE-sensitive currents, indicating that the activated channel was thermosensitive. Immunofluorescence in sections of the rat MCA demonstrated that, like TREK-1, TRAAK is expressed in the smooth muscle of cerebral arteries. Isoflurane did not, however, dilate the MCA, suggesting that TREK-1 was not functional. These data indicate that TCE activated a nonclassical K+ channel with the characteristics of TRAAK in rat MCA smooth-muscle cells. Stimulation of K+ channels such as TRAAK in cerebral arteries may therefore explain in part how CH/TCE increases CBF.

Little is known about the presence and function of two-pore domain K+ (K2P) channels in vascular smooth muscle cells (VSMCs). Five members of the K2P channel family are known to be directly activated by arachidonic acid (AA). The purpose of this study was to determine 1) whether AA-sensitive K2P channels are expressed in cerebral VSMCs and 2) whether AA dilates the rat middle cerebral artery (MCA) by increasing K+ currents in VSMCs via an atypical K+ channel. RT-PCR revealed message for the following AA-sensitive K2P channels in rat MCA: tandem of P domains in weak inward rectifier K+ (TWIK-2), TWIK-related K+ (TREK-1 and TREK-2), TWIK-related AA-stimulated K+ (TRAAK), and TWIK-related halothane-inhibited K+ (THIK-1) channels. However, in isolated VSMCs, only message for TWIK-2 was found. Western blotting showed that TWIK-2 is present in MCA, and immunohistochemistry further demonstrated its presence in VSMCs. AA (10–100 μM) dilated MCAs through an endothelium-independent mechanism. AA-induced dilation was not affected by inhibition of cyclooxygenase, epoxygenase, or lipoxygenase or inhibition of classical K+ channels with 10 mM TEA, 3 mM 4-aminopyridine, 10 μM glibenclamide, or 100 μM Ba2+. AA-induced dilations were blocked by 50 mM K+, indicating involvement of a K+ channel. AA (10 μM) increased whole cell K+ currents in dispersed cerebral VSMCs. AA-induced currents were not affected by inhibitors of the AA metabolic pathways or blockade of classical K+ channels. We conclude that AA dilates the rat MCA and increases K+ currents in VSMCs via an atypical K+ channel that is likely a member of the K2P channel family.

Background and Purpose— Superoxide may play an important role in cerebral vasospasm after subarachnoid hemorrhage (SAH). Our first goal was to determine the effect of gene transfer of extracellular superoxide dismutase (ECSOD) on vasospasm after experimental SAH. Our second goal was to determine whether tissue binding of ECSOD via the heparin-binding domain (HBD) is important for the effect of the enzyme. Thus, we examined effects of gene transfer of ECSOD with the HBD deleted (ECSODΔHBD). Methods— Adenovirus expressing human ECSOD (AdECSOD), ECSODΔHBD (AdECSODΔHBD), or no transgene (AdBglII) was injected into the cisterna magna of anesthetized rabbits 30 minutes after induction of experimental SAH. Cerebral angiography, an assay for ECSOD activity in cerebrospinal fluid (CSF), and Western blotting for human ECSOD in the basilar artery were performed. Results— Baseline diameter of the basilar artery averaged 0.77±0.01 mm (mean±SEM) and was similar in all treatment groups. Decreases in diameter of the basilar artery 2 days after SAH were smaller after AdECSOD (11±3%; n=10) than after AdBglII (25±4%; n=7; P Conclusions— Gene transfer of ECSOD reduces cerebral vasospasm after experimental SAH. Tissue binding of the enzyme is essential for cerebral vascular effects of ECSOD.

Adenovirus (Ad)-mediated gene transfer to blood vessels is relatively inefficient, probably because binding of adenovirus to the endothelium and adventitia seems to be limited. Association of calcium phosphate (CaPi) precipitates with adenovirus improves efficiency of gene transfer to some cells in culture and to mouse lung in vivo. In this study, we tested the hypothesis that CaPi is useful for adenovirus-mediated gene transfer to blood vessels. In fibroblast and endothelial cells in culture, Ad:CaPi coprecipitates greatly increased transgene expression. Ad:CaPi also enhanced transgene expression in both adventitia and endothelium of carotid arteries and aortae from rabbits studied ex vivo. After injection of Ad:CaPi into the cisterna magna of rabbits in vivo, the transgene product was markedly increased in leptomeninges of the ventral brain stem, including the adventitia of the basilar artery. We also examined mechanisms of enhanced gene transfer. Binding of adenovirus to fibroblast and endothelial cells in culture, and to the basilar artery in vivo, as determined using Southern blot analysis, was augmented by CaPi. Antibody to adenoviral fiber knob did not inhibit augmented transgene expression by Ad:CaPi. The finding suggests that improved adenoviral binding occurs primarily via a fiber-independent pathway. Thus, CaPi precipitates are useful for improvement of adenovirus-mediated gene transfer to blood vessels in vitro and in vivo.