Better Understanding of Phosphoinositide 3-Kinase (PI3K) Pathways in Vasculature: Towards Precision Therapy Targeting Angiogenesis and Tumor Blood Supply.

The intracellular PI3K-AKT-mTOR pathway is involved in regulation of numerous important cell processes including cell growth, differentiation, and metabolism. The PI3Kα isoform has received particular attention as a novel molecular target in gene therapy, since this isoform plays critical roles in tumor progression and tumor blood flow and angiogenesis. However, the role of PI3Kα and other class I isoforms, i.e. PI3Kß, γ, δ, in the regulation of vascular tone and regional blood flow are largely unknown. We used novel isoform-specific PI3K inhibitors and mice deficient in both PI3Kγ and PI3Kδ (Pik3cg(-/-)/Pik3cd(-/-)) to define the putative contribution of PI3K isoform(s) to arterial vasoconstriction. Wire myography was used to measure isometric contractions of isolated murine mesenteric arterial rings. Phenylephrine-dependent contractions were inhibited by the pan PI3K inhibitors wortmannin (100 nM) and LY294002 (10 µM). These vasoconstrictions were also inhibited by the PI3Kα isoform inhibitors A66 (10 µM) and PI-103 (1 µM), but not by the PI3Kß isoform inhibitor TGX 221 (100 nM). Pik3cg(-/-)/Pik3cd(-/-)-arteries showed normal vasoconstriction. We conclude that PI3Kα is an important downstream element in vasoconstrictor GPCR signaling, which contributes to arterial vasocontraction via α1-adrenergic receptors. Our results highlight a regulatory role of PI3Kα in the cardiovascular system, which widens the spectrum of gene therapy approaches targeting PI3Kα in cancer cells and tumor angiogenesis and regional blood flow.

Elevated vascular level of ortho-tyrosine contributes to the impairment of insulin-induced arterial relaxation.

Previous studies have shown that in diabetes mellitus, insulin-induced relaxation of arteries is impaired and the level of ortho-tyrosine (o-Tyr), an oxidized amino acid is increased. Thus, we hypothesized that elevated vascular level of o-Tyr contributes to the impairment of insulin-induced vascular relaxation. Rats were fed with o-Tyr for 4 weeks. Insulin-induced vasomotor responses of isolated femoral artery were studied using wire myography. Vascular o-Tyr content was measured by HPLC, whereas immunoblot analyses were preformed to detect eNOS phosphorylation. Sustained oral supplementation of rats with o-Tyr increased the content of o-Tyr in the arterial wall and significantly reduced the relaxations to insulin. Sustained supplementation of cultured endothelial cells with o-Tyr increased the incorporation of o-Tyr and mitigated eNOS Ser (1 177) phosphorylation to insulin. Increasing arterial wall o-Tyr level attenuates insulin-induced relaxation - at least in part - by decreasing eNOS activation. Elevated level of o-Tyr could be an underlying mechanism for vasomotor dysfunction in diabetes mellitus.

Simple 2,4-diacylphloroglucinols as classic transient receptor potential-6 activators--identification of a novel pharmacophore.

The naturally occurring acylated phloroglucinol derivative hyperforin was recently identified as the first specific canonical transient receptor potential-6 (TRPC6) activator. Hyperforin is the major antidepressant component of St. John's wort, which mediates its antidepressant-like properties via TRPC6 channel activation. However, its pharmacophore moiety for activating TRPC6 channels is unknown. We hypothesized that the phloroglucinol moiety could be the essential pharmacophore of hyperforin and that its activity profile could be due to structural similarities with diacylglycerol (DAG), an endogenous nonselective activator of TRPC3, TRPC6, and TRPC7. Accordingly, a few 2-acyl and 2,4-diacylphloroglucinols were tested for their hyperforin-like activity profiles. We used a battery of experimental models to investigate all functional aspects of TRPC6 activation, including ion channel recordings, Ca(2+) imaging, neurite outgrowth, and inhibition of synaptosomal uptake. Phloroglucinol itself was inactive in all of our assays, which was also the case for 2-acylphloroglucinols. For TRPC6 activation, the presence of two symmetrically acyl-substitutions with appropriate alkyl chains in the phloroglucinol moiety seems to be an essential prerequisite. Potencies of these compounds in all assays were comparable with that of hyperforin for activating the TRPC6 channel. Finally, using structure-based modeling techniques, we suggest a binding mode for hyperforin to TRPC6. Based on this modeling approach, we propose that DAG is able to activate TRPC3, TRPC6, and TRPC7 because of higher flexibility within the chemical structure of DAG compared with the rather rigid structures of hyperforin and the 2,4-diacylphloroglucinol derivatives.

Raloxifene protects endothelial cell function against oxidative stress.

BACKGROUND AND PURPOSE: Maintaining a delicate balance between the generation of nitric oxide (NO) and removal of reactive oxygen species (ROS) within the vascular wall is crucial to the physiological regulation of vascular tone. Increased production of ROS reduces the effect and/or bioavailability of NO, leading to an impaired endothelial function. This study tested the hypothesis that raloxifene, a selective oestrogen receptor modulator, can prevent endothelial dysfunction under oxidative stress. EXPERIMENTAL APPROACH: Changes in isometric tension were measured in rat aortic rings. The content of cyclic GMP in aortic tissue was determined by radioimmunoassay. Phosphorylation of endothelial NOS (eNOS) and Akt was assayed by Western blot analysis. KEY RESULTS: In rings with endothelium, ACh-induced relaxations were attenuated by a ROS-generating reaction (hypoxanthine plus xanthine oxidase, HXXO). The impaired relaxations were ameliorated by acute treatment with raloxifene. HXXO suppressed the ACh-stimulated increase in cyclic GMP levels; this effect was antagonized by raloxifene. The improved endothelial function by raloxifene was abolished by ICI 182,780, and by wortmannin or LY294002. Raloxifene also protected endothelial cell function against H2O2. Raloxifene increased the phosphorylation of eNOS at Ser-1177 and Akt at Ser-473; this effect was blocked by ICI 182,780. Finally, raloxifene was not directly involved in scavenging ROS, and neither inhibited the activity of xanthine oxidase nor stimulated that of superoxide dismutase. CONCLUSION AND IMPLICATIONS: Raloxifene is effective against oxidative stress-induced endothelial dysfunction in vitro through an ICI 182,780-sensitive mechanism that involves the increased phosphorylation and activity of Akt and eNOS in rat aortae.

Renoprotection: focus on TRPV1, TRPV4, TRPC6 and TRPM2.

Members of the transient receptor potential (TRP) cation channel receptor family have unique sites of regulatory function in the kidney which enables them to promote regional vasodilatation and controlled Ca2+ influx into podocytes and tubular cells. Activated TRP vanilloid 1 receptor channels (TRPV1) have been found to elicit renoprotection in rodent models of acute kidney injury following ischaemia/reperfusion. Transient receptor potential cation channel, subfamily C, member 6 (TRPC6) in podocytes is involved in chronic proteinuric kidney disease, particularly in focal segmental glomerulosclerosis (FSGS). TRP vanilloid 4 receptor channels (TRPV4) are highly expressed in the kidney, where they induce Ca2+ influx into endothelial and tubular cells. TRP melastatin (TRPM2) non-selective cation channels are expressed in the cytoplasm and intracellular organelles, where their inhibition ameliorates ischaemic renal pathology. Although some of their basic properties have been recently identified, the renovascular role of TRPV1, TRPV4, TRPC6 and TRPM2 channels in disease states such as obesity, hypertension and diabetes is largely unknown. In this review, we discuss recent evidence for TRPV1, TRPV4, TRPC6 and TRPM2 serving as potential targets for acute and chronic renoprotection in chronic vascular and metabolic disease.

Ignition of calcium sparks in arterial and cardiac muscle through caveolae.

Ca(2+) sparks are localized intracellular Ca(2+) events released through ryanodine receptors (RyRs) that control excitation-contraction coupling in heart and smooth muscle. Ca(2+) spark triggering depends on precise delivery of Ca(2+) ions through dihydropyridine (DHP)-sensitive Ca(2+) channels to RyRs of the sarcoplasmic reticulum (SR), a process requiring a very precise alignment of surface and SR membranes containing Ca(2+) influx channels and RyRs. Because caveolae contain DHP-sensitive Ca(2+) channels and may colocalize with SR, we tested the hypothesis that caveolae are the structural element necessary for the generation of Ca(2+) sparks. Using methyl-ss-cyclodextrin (dextrin) to deplete caveolae, we found that dextrin dose-dependently decreased the frequency, amplitude, and spatial size of Ca(2+) sparks in arterial smooth muscle cells and neonatal cardiomyocytes. However, temporal characteristics of Ca(2+) sparks were not significantly affected. We ruled out the possibility that the decreases in Ca(2+) spark frequency and size are caused by changes in DHP-sensitive L-type channels, SR Ca(2+) load, or changes in membrane potential. Our results suggest a novel signaling model that explains the formation of Ca(2+) sparks in a caveolae microdomain. The transient elevation in [Ca(2+)](i) at the inner mouth of a single caveolemmal Ca(2+) channel induces simultaneous activation and thus opens several RyRs to generate a local Ca(2+) release event, a Ca(2+) spark. Alterations in the molecular assembly and ultrastructure of caveolae may lead to pathophysiological changes in Ca(2+) signaling. Thus, caveolae may be intimately involved in cardiovascular cell dysfunction and disease.

Regulation of calcium sparks and spontaneous transient outward currents by RyR3 in arterial vascular smooth muscle cells.

Intracellular Ca(2+) levels control both contraction and relaxation in vascular smooth muscle cells (VSMCs). Ca(2+)-dependent relaxation is mediated by discretely localized Ca(2+) release events through ryanodine receptor (RyR) channels in the sarcoplasmic reticulum (SR). These local increases in Ca(2+) concentration, termed sparks, stimulate nearby Ca(2+)-activated K(+) (BK) channels causing BK currents (spontaneous transient outward currents or STOCs). STOCs are hyperpolarizing currents that oppose vasoconstriction. Several RyR isoforms are coexpressed in VSMCs; however, their role in Ca(2+) spark generation is unknown. To provide molecular information on RyR cluster function and assembly, we examined Ca(2+) sparks and STOCs in RyR3-deficient freshly isolated myocytes of resistance-sized cerebral arteries from knockout mice and compared them to Ca(2+) sparks in cells from wild-type mice. We used RT-PCR to identify RyR1, RyR2, and RyR3 mRNA in cerebral arteries. Ca(2+) sparks in RyR3-deficient cells were similar in peak amplitude (measured as F/F(0)), width at half-maximal amplitude, and duration compared with wild-type cell Ca(2+) sparks. However, the frequency of STOCs (between -60 mV and -20 mV) was significantly higher in RyR3-deficient cells than in wild-type cells. Ca(2+) sparks and STOCs in both RyR3-deficient and wild-type cells were inhibited by ryanodine (10 micromol/L), external Ca(2+) removal, and depletion of SR Ca(2+) stores by caffeine (1 mmol/L). Isolated, pressurized cerebral arteries of RyR3-deficient mice developed reduced myogenic tone. Our results suggest that RyR3 is part of the SR Ca(2+) spark release unit and plays a specific molecular role in the regulation of STOCs frequency in mouse cerebral artery VSMCs after decreased arterial tone.

Mice with disrupted BK channel beta1 subunit gene feature abnormal Ca(2+) spark/STOC coupling and elevated blood pressure.

Large-conductance potassium (BK) channels in vascular smooth muscle cells (VSMCs) sense both changes in membrane potential and in intracellular Ca(2+) concentration. BK channels may serve as negative feedback regulators of vascular tone by linking membrane depolarization and local increases in intracellular Ca(2+) concentration (Ca(2+) sparks) to repolarizing spontaneous transient outward K(+) currents (STOCs). BK channels are composed of channel-forming BKalpha and auxiliary BKbeta1 subunits, which confer to BK channels an increased sensitivity for changes in membrane potential and Ca(2+). To assess the in vivo functions of this ss subunit, mice with a disrupted BKbeta1 gene were generated. Cerebral artery VSMCs from BKbeta1 -/- mice generated Ca(2+) sparks of normal amplitude and frequency, but STOC frequencies were largely reduced at physiological membrane potentials. Our results indicate that BKbeta1 -/- mice have an abnormal Ca(2+) spark/STOC coupling that is shifted to more depolarized potentials. Thoracic aortic rings from BKbeta1 -/- mice responded to agonist and elevated KCl with a increased contractility. BKbeta1 -/- mice had higher systemic blood pressure than BKbeta1 +/+ mice but responded normally to alpha(1)-adrenergic vasoconstriction and nitric oxide-mediated vasodilation. We propose that the elevated blood pressure in BKbeta1 -/- mice serves to normalize Ca(2+) spark/STOC coupling for regulating myogenic tone. The full text of this article is available at http://www.circresaha.org.

Inhibition of voltage-dependent Ca2+ currents and activation of pertussis toxin-sensitive G-proteins via muscarinic receptors in GH3 cells.

In the rat pituitary cell line GH3, carbachol inhibits PRL secretion in a pertussis toxin-sensitive manner. For elucidation of the underlying mechanisms, we studied the effect of carbachol on voltage-dependent Ca2+ currents. Under voltage-clamp conditions, carbachol inhibited whole-cell Ca2+ currents by about 25%. This inhibitory action of carbachol was not observed in cells treated with pertussis toxin, indicating the involvement of a pertussis toxin-sensitive G-protein. In membranes of GH3 cells, carbachol stimulated a pertussis toxin-sensitive high-affinity GTPase. In immunoblot experiments with peptide antisera, we identified two forms of the Gi alpha-subunit (41 and 40 kDa) and two forms of the Go alpha-subunit (40 and 39 kDa). The 40-kDa Gi alpha-subunit was recognized by an antibody specific for the Gi2 alpha-subunit, and the 39-kDa Go alpha-subunit was detected by an antibody specific for the Go2 alpha-subunit. Incubation of membranes with the photoreactive GTP analog [alpha-32P]GTP azidoanilide resulted in photo-labelling of 40- and 39-kDa pertussis toxin substrates comigrating with G-protein alpha-subunits of the corresponding molecular masses. Carbachol dose-dependently stimulated incorporation of the photoreactive GTP analog into the 39-kDa pertussis toxin substrate and, to a lesser extent, into 40-kDa pertussis toxin substrates. The data indicate that muscarinic receptors of GH3 cells couple preferentially to Go, which is likely to be involved in the inhibition of secretion, possibly by conferring an inhibitory effect to voltage-dependent Ca2+ channels.

Functional transient receptor potential vanilloid 1 and transient receptor potential vanilloid 4 channels along different segments of the renal vasculature.

AIM: Transient receptor potential vanilloid 1 (TRPV1) and vanilloid 4 (TRPV4) cation channels have been recently identified to promote endothelium-dependent relaxation of mouse mesenteric arteries. However, the role of TRPV1 and TRPV4 in the renal vasculature is largely unknown. We hypothesized that TRPV1/4 plays a role in endothelium-dependent vasodilation of renal blood vessels. METHODS: We studied the distribution of functional TRPV1/4 along different segments of the renal vasculature. Mesenteric arteries were studied as control vessels. RESULTS: The TRPV1 agonist capsaicin relaxed mouse mesenteric arteries with an EC50 of 25 nm, but large mouse renal arteries or rat descending vasa recta only at >100-fold higher concentrations. The vasodilatory effect of capsaicin in the low-nanomolar concentration range was endothelium-dependent and absent in vessels of Trpv1 -/- mice. The TRPV4 agonist GSK1016790A relaxed large conducting renal arteries, mesenteric arteries and vasa recta with EC50 of 18, 63 nm and ~10 nm respectively. These effects were endothelium-dependent and inhibited by a TRPV4 antagonist, AB159908 (10 µm). Capsaicin and GSK1016790A produced vascular dilation in isolated mouse perfused kidneys with EC50 of 23 and 3 nm respectively. The capsaicin effects were largely reduced in Trpv1 -/- kidneys, and the effects of GSK1016790A were inhibited in Trpv4 -/- kidneys. CONCLUSION: Our results demonstrate that two TRPV channels have unique sites of vasoregulatory function in the kidney with functional TRPV1 having a narrow, discrete distribution in the resistance vasculature and TRPV4 having more universal, widespread distribution along different vascular segments. We suggest that TRPV1/4 channels are potent therapeutic targets for site-specific vasodilation in the kidney.

Ca2+ channels, 'quantized' Ca2+ release, and differentiation of myocytes in the cardiovascular system.

The application of confocal microscopy to cardiac and skeletal muscle has resulted in the observation of transient, spatially localized elevations in [Ca2+]i, termed 'Ca2+ sparks'. Ca2+ sparks are thought to represent 'elementary' Ca2+ release events, which arise from one or more ryanodine receptor (RyR) channels in the sarcoplasmic reticulum. In cardiac muscle, Ca2+ sparks appear to be key elements of excitation-contraction coupling, in which the global [Ca2+]i transient is thought to involve the recruitment of Ca2+ sparks, each of which is controlled locally by single coassociated L-type Ca2+ channels. Recently, Ca2+ sparks have been detected in smooth muscle cells of arteries. In this review, we analyse the complex relationship of Ca2+ influx and Ca2+ release with local, subcellular Ca2+ microdomains in light of recent studies on Ca2+ sparks in cardiovascular cells. We performed a comparative analysis of 'elementary' Ca2+ release units in mouse, rat and human arterial smooth muscle cells, using measurements of Ca2+ sparks and plasmalemmal K(Ca) currents activated by Ca2+ sparks (STOCs). Furthermore, the appearance of Ca2+ sparks during ontogeny of arterial smooth muscle is explored. Using intact pressurized arteries, we have investigated whether RyRs causing Ca2+ sparks (but not smaller 'quantized' Ca2+ release events, e.g. hypothetical 'Ca2+ quarks') function as key signals that, through membrane potential and global cytoplasmic [Ca2+], oppose arterial myogenic tone and influence vasorelaxation. We believe that voltage-dependent Ca2+ channels and local RyR-related Ca2+ signals are important in differentiation, proliferation, and gene expression. Our findings suggest that 'elementary' Ca2+ release units may represent novel potent therapeutic targets for regulating function of intact arterial smooth muscle tissue.

Calcium sparks in human coronary artery smooth muscle cells resolved by confocal imaging.

OBJECTIVE: The observation of local 'elementary' Ca2+ release events (Ca2+ sparks) through ryanodine receptor (RyR) channels in the sarcoplasmic reticulum (SR) has changed our understanding of excitation-contraction (EC) coupling in cardiac and smooth muscle. In arterial smooth muscle, Ca2+ sparks have been suggested to oppose myogenic vasoconstriction and to influence vasorelaxation by activating co-localized Ca2+ activated K+ (K(Ca)) channels (STOCs). However, all prior studies on Ca2+ sparks have been performed in non-human tissues. METHODS: In order to understand the possible significance of Ca2+ sparks to human cardiovascular function, we used high spatial resolution confocal imaging to record Ca2+ sparks in freshly-isolated, individual myocytes of human coronary arteries loaded with the Ca2+ indicator fluo-3. RESULTS: Local SR Ca2+ release events recorded in human myocytes were similar to 'Ca2- sparks' recorded previously from non-human smooth muscle cells. In human myocytes, the peak [Ca2+]i amplitudes of Ca2+ sparks (measured as F/F0) and width at half-maximal amplitude were 2.3 and 2.27 microm, respectively. The duration of Ca2+ sparks was 62 ms. Ca2+ sparks were completely inhibited by ryanodine (10 micromol/l). Ryanodine-sensitive STOCs could be identified with typical properties of K(Ca) channels activated by Ca2+ sparks. CONCLUSION: Our data implies that modern concepts suggesting an essential role of Ca2+ spark generation in EC coupling recently derived from non-human muscle are applicable to human cardiovascular tissue. Although the basic properties of Ca2+ sparks are similar, our results demonstrate that Ca2+ sparks in coronary arteries in humans, have features distinct from non-arterial smooth muscle cells of other species.

beta(1)-Subunit of BK channels regulates arterial wall[Ca(2+)] and diameter in mouse cerebral arteries.

Mice with a disrupted beta(1) (BK beta(1))-subunit of the large-conductance Ca(2+)-activated K(+) (BK) channel gene develop systemic hypertension and cardiac hypertrophy, which is likely caused by uncoupling of Ca(2+) sparks to BK channels in arterial smooth muscle cells. However, little is known about the physiological levels of global intracellular Ca(2+) concentration ([Ca(2+)](i)) and its regulation by Ca(2+) sparks and BK channel subunits. We utilized a BK beta(1) knockout C57BL/6 mouse model and studied the effects of inhibitors of ryanodine receptor and BK channels on the global [Ca(2+)](i) and diameter of small cerebral arteries pressurized to 60 mmHg. Ryanodine (10 microM) or iberiotoxin (100 nM) increased [Ca(2+)](i) by approximately 75 nM and constricted +/+ BK beta(1) wild-type arteries (pressurized to 60 mmHg) with myogenic tone by approximately 10 microm. In contrast, ryanodine (10 microM) or iberiotoxin (100 nM) had no significant effect on [Ca(2+)](i) and diameter of -/- BK beta(1)-pressurized (60 mmHg) arteries. These results are consistent with the idea that Ca(2+) sparks in arterial smooth muscle cells limit myogenic tone through activation of BK channels. The activation of BK channels by Ca(2+) sparks reduces the voltage-dependent Ca(2+) influx and [Ca(2+)](i) through tonic hyperpolarization. Deletion of BK beta(1) disrupts this negative feedback mechanism, leading to increased arterial tone through an increase in global [Ca(2+)](i).

Dent's disease: identification of a novel mutation in the renal chloride channel CLCN5.

Dent's disease is an inherited tubulopathy caused by a mutation in the CLCN5 chloride channel gene. It is characterized by low-molecular weight proteinuria, hypercalciuria, nephrolithiasis or nephrocalcinosis, rickets and eventual-progressive renal failure. Onset of clinical symptoms show a great variability, making a diagnosis at an early stage of the disease often difficult. Given the variably clinical picture, genetic analysis can provide a reliable method to confirm the diagnosis. Here, we report on the case of a patient with progressive renal failure showing signs of a tubular lesion and symptoms of Dent's disease. Although this rare disease was suspected by means of the clinical features, it was genetic analysis that confirmed the diagnosis and revealed a novel mutation in the CLCN5 gene.

On the mechanisms of action of PAF on the myocardium.

The effects of PAF (1-2 X 10(-7) M) on intracellular slow Ca-mediated action potentials (Ca-AP) and isometric contractions of guinea-pig auricle muscles and ionic currents of frog atrial trabeculae were investigated. PAF decreased the amplitude, duration and upstroke velocity of the slow Ca-AP mediated via the L-type Ca channels in guinea-pig auricle muscles without changes in the resting potential. The PAF receptor antagonist U-66985 (1-5 X 10(-6) M), histamine (10(-4) M) and 4-aminopyridine (3 mM) inhibited the depressant effects of PAF on the slow AP and contractions. PAF reduced the peak of the slow inward Ca current (ICa) via the L-type Ca channels of frog atrial trabeculae. Simultaneously, the delayed outward K current (IK) was increased by PAF. Pretreatment with the PAF receptor antagonist BN 52021 (4 X 10(-6) M) protected the effects of PAF on ICa and IK. Our data demonstrate both by direct and indirect methods of ICa registration an inhibiting effect of PAF on the ICa via the L-type Ca channels and make probable a similarity in PAF signalling in cardiac cells of cold- and warm-blooded animals. The coupling of changes of the ICa with the augmentation of IK accentuate the important role of myocardial L-type Ca and K channels in mediating the negative inotropic action of PAF. The ability of U-66985 and BN 52021 to inhibit the electrophysiological effects of PAF hint at a membrane signalling coupled by specific binding sites for PAF in the myocardium.

Increase in insulin-induced relaxation of consecutive arterial segments toward the periphery: Role of vascular oxidative state.

RATIONALE: The oxidative state has been implicated in the signaling of various vasomotor functions, yet its role regarding the vasomotor action of insulin is less known. OBJECTIVE: To investigate the insulin-evoked relaxations of consecutive arterial segments of different oxidative state and the role of extracellular signal-regulated kinase (ERK) pathway. METHODS AND RESULTS: The oxidative state, as assessed by the level of ortho-tyrosine, was higher in the thoracic aorta of rats than in the abdominal aorta, and was the lowest in the femoral artery. The vasomotor function of vessels of same origin was studied using a small-vessel myograph. Insulin-induced relaxations increased toward the periphery (i.e., thoracic < abdominal < femoral). Aortic banding and hydrogen peroxide/aminotriazole increased the oxidative state of the thoracic aorta that was accompanied by ERK activation and decreased relaxation to insulin, and vice versa, acutely lowered oxidative state by superoxide dismutase/catalase improved relaxation. In contrast, insulin-induced relaxation of the femoral artery could be enhanced with a higher oxidative state, and reduced with a lower state. CONCLUSIONS: Oxidative state of vessels modulates the magnitude of vasomotor responses to insulin, which appears to be mediated via the ERK signaling pathway.

Transient receptor potential vanilloid 1 (TRPV1), TRPV4, and the kidney.

Recent preclinical data indicate that activators of transient receptor potential channels of the vanilloid receptor subtype 1 (TRPV1) may improve the outcome of ischaemic acute kidney injury (AKI). The underlying mechanisms are unclear, but may involve TRPV1 channels in dorsal root ganglion neurones that innervate the kidney. Recent data identified TRPV4, together with TRPV1, to serve as major calcium influx channels in endothelial cells. In these cells, gating of individual TRPV4 channels within a four-channel cluster provides elementary calcium influx (calcium sparklets) to open calcium-activated potassium channels and promote vasodilation. The TRPV receptors can also form heteromers that exhibit unique conductance and gating properties, further increasing their spatio-functional diversity. This review summarizes data on electrophysiological properties of TRPV1/4 and their modulation by endogenous channel agonists such as 20-HETE, phospholipase C and phosphatidylinositide 3-kinase (PI3 kinase). We review important roles of TRPV1 and TRPV4 in kidney physiology and renal ischaemia reperfusion injury; further studies are warranted to address renoprotective mechanism of vanilloid receptors in ischaemic AKI including the role of the capsaicin receptor TRPV1 in primary sensory nerves and/or endothelium. Particular attention should be paid to understand the kidneys' ability to respond to ischaemic stimuli after catheter-based renal denervation therapy in man, whereas the discovery of novel pharmacological TRPV modulators may be a successful strategy for better treatment of acute or chronic kidney failure.

Multiple pathways for ATP-induced intracellular calcium elevation in pheochromocytoma (PC12) cells.

Extracellular ATP excites neurons in both the peripheral and central nervous system. To elucidate the mechanisms involved, we used spectrofluorometric analysis to study the pathways by which extracellular ATP elevates the intracellular Ca2+ concentration ([Ca2+]i) in individual, fura-2-loaded, rat pheochromocytoma PC12 cells. ATP (> 1 microM) increased [Ca2+]i. The ATP effect on [Ca2+]i was completely abolished by a nominally Ca(2+)-free extracellular medium, which indicates that the ATP-induced increase in [Ca2+]i was due to an influx of extracellular Ca2+. We next applied specific blockers of voltage-dependent Ca2+ channels and used experimental protocols with depolarizing external K(+)-rich solutions. Our results show that ATP induces influx of extracellular Ca2+ through (a) dihydropyridine-sensitive (Ln-type) Ca2+ channels, (b) Ca(2+)-permeable, voltage-independent, Cd(2+)-insensitive cation channels, and (c) an as yet unidentified, voltage-dependent, Cd(2+)-sensitive Ca2+ influx system.

Voltage-dependent Ca2+ channels in arterial smooth muscle cells.

The past years have seen some significant advances in our understanding of the functional and molecular properties of voltage-dependent Ca2+ channels in arterial smooth muscle. Molecular cloning and expression studies together with experiments on native voltage-dependent Ca2+ channels revealed that these channels are built upon a molecular structure with properties appropriate to function as the main source for Ca2+ entry into arterial smooth muscle cells. This Ca2+ entry regulates intracellular free Ca2+, and thereby arterial tone. We summarize several avenues of recent research that should provide significant insights into the functioning of voltage-dependent Ca2+ channels under conditions that occur in arterial smooth muscle. These experiments have identified important features of voltage-dependent Ca2+ channels, including the steep steady-state voltage-dependence of the channel open probability at steady physiological membrane potentials between -60 and -30 mV, and a relatively high permeation rate at physiological Ca2+ concentrations, being about one million Ca2+ ions/s at -50 mV. This calcium permeation rate seems to be a feature of the pore-forming Ca2+ channel alpha1 subunit, since it was identical for native channels and the expressed alpha1 subunit alone. The channel activity is regulated by dihydropyridines, vasoactive hormones and intracellular signaling pathways. While the membrane potential of smooth muscle cells primarily regulates arterial muscle tone through alterations in Ca2+ influx through dihydropyridine-sensitive voltage-dependent ('L-type') Ca2+ channels, the role of these channels in the differentiation and proliferation of vascular smooth muscle cells is less clear. We discuss recent findings suggesting that other Ca2+ permeable ion channels might be important for the control of Ca2+ influx in dedifferentiated vascular smooth muscle cells.

Thyrotropin-releasing hormone induces opposite effects on Ca2+ channel currents in pituitary cells by two pathways.

Thyrotropin-releasing hormone (TRH) stimulates pituitary secretion by steps involving a cytosolic Ca2+ rise. We examined various pathways of Ca2+ elevation in pituitary GH3 cells. By using the patch clamp technique in the whole-cell configuration and Ba2+ as divalent charge carrier through Ca2+ channels, TRH (1 microM) reversibly reduced the current by about 55%. This hormonal effect was prevented by infusing guanine 5'-[beta-thio]diphosphate (GDP[beta S]) intracellularly but not by pretreating the cell with pertussis toxin (PT). Since PT-insensitive guanine nucleotide-binding regulatory (G) proteins are known to mediate a hormone-stimulated inositol trisphosphate-mediated Ca2+ release from intracellular stores, we assume that the inhibitory effect of TRH on Ba2+ currents through Ca2+ channels is caused by the increased intracellular Ca2+. To prevent a Ca(2+)-release-dependent inhibition of Ca2+ channels, we preincubated GH3 cells in a medium free of divalent charge carriers and measured the Na+ current through Ca2+ channels. When fura-2 was used as indicator for the cytosolic Ca2+, TRH induced a release from intracellular stores only once and had no effect on the intracellular Ca2+ concentration during further applications. In line with this observation, TRH initially reduced the Na+ current through Ca2+ channels but stimulated it during subsequent applications. The stimulation was sensitive to GDP[beta S] and was abolished by pretreatment with PT, suggesting that the stimulatory action of TRH is mediated by a G protein different from the one that functionally couples the receptor to phosphatidylinositol 4,5-bisphosphate hydrolysis. In conclusion, the present data suggest that TRH increases the intracellular Ca2+ concentration by two interacting pathways, that release from intracellular stores causes a secondary blockage of Ca2+ channels, and that, especially with empty intracellular Ca2+ stores, Ca2+ channels are stimulated by a PT-sensitive G protein.