Blood Vessel Tone Research Articles

Thrombospondin-1 and CD47 regulate blood pressure and cardiac responses to vasoactive stress

Nitric oxide (NO) locally regulates vascular resistance and blood pressure by modulating blood vessel tone. Thrombospondin-1 signaling via its receptor CD47 locally limits the ability of NO to relax vascular smooth muscle cells and increase regional blood flow in ischemic tissues. To determine whether thrombospondin-1 plays a broader role in central cardiovascular physiology, we examined vasoactive stress responses in mice lacking thrombospondin-1 or CD47. Mice lacking thrombospondin-1 exhibit activity-associated increases in heart rate, central diastolic and mean arterial blood pressure and a constant decrease in pulse pressure. CD47-deficient mice have normal central pulse pressure but elevated resting peripheral blood pressure. Both null mice show exaggerated decreases in peripheral blood pressure and increased cardiac output and ejection fraction in response to NO. Autonomic blockade also induces exaggerated hypotensive responses in awake thrombospondin-1 null and CD47 null mice. Both null mice exhibit a greater hypotensive response to isoflurane, and autonomic blockage under isoflurane anesthesia leads to premature death of thrombospondin-1 null mice. Conversely, the hypertensive response to epinephrine is attenuated in thrombospondin-1 null mice. Thus, the matricellular protein thrombospondin-1 and its receptor CD47 serve as acute physiological regulators of blood pressure and exert a vasopressor activity to maintain global hemodynamics under stress.

Vascular smooth muscle dysfunction induced by monomethylarsonous acid (MMA III): A contributing factor to arsenic-associated cardiovascular diseases

While arsenic in drinking water is known to cause various cardiovascular diseases in human, exact mechanism still remains elusive. Recently, trivalent-methylated arsenicals, the metabolites of inorganic arsenic, were shown to have higher cytotoxic potential than inorganic arsenic. To study the role of these metabolites in arsenic-induced cardiovascular diseases, we investigated the effect of monomethylarsonous acid (MMA III), a major trivalent-methylated arsenical, on vasomotor tone of blood vessels. In isolated rat thoracic aorta and small mesenteric arteries, MMA III irreversibly suppressed normal vasoconstriction induced by three distinct agonists of phenylephrine (PE), serotonin and endothelin-1. Inhibition of vasoconstriction was retained in aortic rings without endothelium, suggesting that MMA III directly impaired the contractile function of vascular smooth muscle. The effect of MMA III was mediated by inhibition of PE-induced Ca 2+ increase as found in confocal microscopy and fluorimeter in-lined organ chamber technique. The attenuation of Ca 2+ increase was from concomitant inhibition of release from intracellular store and extracellular Ca 2+ influx via L-type Ca 2+ channel, which was blocked by MMA III as shown in voltage-clamp assay in Xenopus oocytes. MMA III did not affect downstream process of Ca 2+, as shown in permeabilized arterial strips. In in vivo rat model, MMA III attenuated PE-induced blood pressure increase indeed, supporting the clinical relevance of these in vitro findings. In conclusion, MMA III-induced smooth muscle dysfunction through disturbance of Ca 2+ regulation, which results in impaired vasoconstriction and aberrant blood pressure change. This study will provide a new insight into the role of trivalent-methylated arsenicals in arsenic-associated cardiovascular diseases.

Endothelium and nitric oxide

Studied nature of the "blood vessels relaxing factor" derived from endothelium that was identified as nitric oxide caused intensive scientific research on nitric oxide regarding some aspects of its impact on human physiological and pathological processes. The objective of this short review is to discuss widely used (in the clinical practice) direct and indirect donors of nitric oxide and/or other agents, increasing nitric oxide concentration in human body, and their beneficial role for the prevention of atherosclerosis. Under physiological conditions, endothelium regulates the tone of blood vessels, homeostasis of which is maintained by endothelium-generated vasoconstrictors and vasodilators. The most important vasodilator and the main substance produced by the endothelium is nitric oxide. The failure of synthesis and/or the lost of nitric oxide bioavailability is the major feature of endothelial dysfunction and key factor initiating progression of atherosclerosis. The endothelial dysfunction initiates the series of events, which stimulate and aggravate the course of atherosclerosis by increasing endothelial permeability, platelet aggregation, and leukocyte adhesion, and cytokine expression. Further, the review deals with the mechanisms of action of statins, angiotensin-converting enzyme inhibitors, L-arginine, direct nitric oxide donors (nitroglycerin, isosorbide mononitrate and isosorbide dinitrate), and indirect nitric oxide donors (phosphodiesterase-V inhibitors, K(ATP) openers).

Targeting by myosin phosphatase‐RhoA interacting protein mediates RhoA/ROCK regulation of myosin phosphatase

Vascular smooth muscle cell contractile state is the primary determinant of blood vessel tone. Vascular smooth muscle cell contractility is directly related to the phosphorylation of myosin light chains (MLCs), which in turn is tightly regulated by the opposing activities of myosin light chain kinase (MLCK) and myosin phosphatase. Myosin phosphatase is the principal enzyme that dephosphorylates MLCs leading to relaxation. Myosin phosphatase is regulated by both vasoconstrictors that inhibit its activity to cause MLC phosphorylation and contraction, and vasodilators that activate its activity to cause MLC dephosphorylation and relaxation. The RhoA/ROCK pathway is activated by vasoconstrictors to inhibit myosin phosphatase activity. The mechanism by which RhoA and ROCK are localized to and interact with myosin light chain phosphatase (MLCP) is not well understood. We recently found a new member of the myosin phosphatase complex, myosin phosphatase-rho interacting protein, that directly binds to both RhoA and the myosin-binding subunit of myosin phosphatase in vitro, and targets myosin phosphatase to the actinomyosin contractile filament in smooth muscle cells. Because myosin phosphatase-rho interacting protein binds both RhoA and MLCP, we investigated whether myosin phosphatase-rho interacting protein was required for RhoA/ROCK-mediated myosin phosphatase regulation. Myosin phosphatase-rho interacting protein silencing prevented LPA-mediated myosin-binding subunit phosphorylation, and inhibition of myosin phosphatase activity. Myosin phosphatase-rho interacting protein did not regulate the activation of RhoA or ROCK in vascular smooth muscle cells. Silencing of M-RIP lead to loss of stress fiber-associated RhoA, suggesting that myosin phosphatase-rho interacting protein is a scaffold linking RhoA to regulate myosin phosphatase at the stress fiber.

Kv1.5 potassium channel gene regulation by Sp1 transcription factor and oxidative stress

K(V)1.5, a voltage-gated potassium channel, has functional importance in regulating blood vessel tone and cardiac action potentials and is a target for numerous therapeutic drug development programs. Despite the importance of K(V)1.5, there is little knowledge of the mechanisms controlling expression of its underlying gene, Kcna5. We identified a 5' flanking region of the murine Kcna5 gene that drives expression of a luciferase reporter gene in primary smooth muscle cells and a smooth muscle cell line. The promoter contained CACCC nucleotide motifs, which we have shown to bind the Sp1 transcription factor in the aorta under physiological conditions in vivo. Inhibition of Sp1-Kcna5 promoter interactions using mithramycin A, a dominant-negative Sp1 mutant, or disruption of the CACCC boxes by mutagenesis inhibited promoter activity. Conversely, expression of exogenous Sp1 augmented promoter activity. Sp1 has known sensitivity to oxidative stress and, consistent with this property, Kcna5 promoter activity was suppressed by hydrogen peroxide-induced oxidative stress. Our results show that Kcna5 promoter activity in vascular smooth muscle is critically dependent on Sp1 regulation via CACCC box motifs and identify mechanisms that potentially influence the expression of K(V)1.5 channel expression in physiological or pathological conditions.

Reaction mechanisms of 15-hydroperoxyeicosatetraenoic acid catalyzed by human prostacyclin and thromboxane synthases

Prostacyclin synthase (PGIS) and thromboxane synthase (TXAS) are atypical cytochrome P450s. They do not require NADPH or dioxygen for isomerization of prostaglandin H 2 (PGH 2) to produce prostacyclin (PGI 2) and thromboxane A 2 (TXA 2). PGI 2 and TXA 2 have opposing actions on platelet aggregation and blood vessel tone. In this report, we use a lipid hydroperoxide, 15-hydroperoxyeicosatetraenoic acid (15-HPETE), to explore the active site characteristics of PGIS and TXAS. The two enzymes transformed 15-HPETE not only into 13-hydroxy-14,15-epoxy-5,8,11-eicosatrienoic acid (13-OH-14,15-EET), like many microsomal P450s, but also to 15-ketoeicosatetraenoic acid (15-KETE) and 15-hydroxyeicosatetraenoic acid (15-HETE). 13-OH-14,15-EET and 15-KETE result from homolytic cleavage of the O–O bond, whereas 15-HETE results from heterolytic cleavage, a common peroxidase pathway. About 80% of 15-HPETE was homolytically cleaved by PGIS and 60% was homolytically cleaved by TXAS. The V max of homolytic cleavage is 3.5-fold faster than heterolytic cleavage for PGIS-catalyzed reactions (1100 min −1 vs. 320 min −1) and 1.4-fold faster for TXAS (170 min −1 vs. 120 min −1). Similar K M values for homolytic and heterolytic cleavages were found for PGIS (∼60 μM 15-HPETE) and TXAS (∼80 μM 15-HPETE), making PGIS a more efficient catalyst for the 15-HPETE reaction.

Effect of nitric-oxide synthesis on tumour blood volume and vascular activity: a phase I study

Nitric oxide has been implicated in tumour angiogenesis and in the maintaining of vasodilator tone of tumour blood vessels. The tumour vascular effects of inhibition of nitric-oxide synthesis have not been investigated in patients with cancer. Seven women and 11 men (12 with non-small-cell lung cancer, five prostate cancer, and one cervical cancer) were recruited onto a phase I dose-escalation study and received a single dose of the nitric oxide synthase inhibitor, N-nitro-L-arginine (L-NNA). Dose escalation was done by a modified Fibonacci scale with three patients at each dose level, starting with 0.1 mg/kg. Changes in dynamic contrast-enhanced CT measures of tumour relative blood volume and transfer constant (K) were measured at 1 h and 24 h after L-NNA administration. In the 18 patients, toxic effects were self-limiting cardiovascular changes: three patients had Common Toxicity Criteria version 2.0 grade 1 hypertension; two had grade 1 sinus bradycardia; and one had grade 1 palpitation. L-NNA area under the curve (AUC) increased linearly with dose from 163 micromol min(-1) L(-1) at 0.1 mg/kg L-NNA to 2150 micromol min(-1) L(-1) at 0.9 mg/kg L-NNA. In eight patients that underwent dynamic CT scanning, tumour blood volume decreased 1 h after L-NNA treatment (mean 42.9% [range 12.0-62.1]; paired t test p=0.0070), which was sustained for up to 24 h (mean 33.9% [range 6.5-64.9]; p=0.035). This decrease in blood volume was associated with an increase in the number of non-perfused pixels from 7.3% (SD 5.5) at baseline to 25.1% (15.3; p=0.0089) at 1 h, and 18.2% (12.9; p=0.050) at 24 h. There was a significant correlation between L-NNA plasma AUC and the reduction in tumour blood volume at 24 h after L-NNA (r=0.83; p=0.010). We have shown in vivo in patients with cancer that nitric oxide has a role in maintaining tumour blood supply, and we provide early clinical evidence that inhibition of nitric-oxide synthesis has tumour antivascular activity.

A novel role for endothelial KATP channels in regulating coronary blood vessel tone

ATP-sensitive K+(KATP) channels couple cellular energy status to membrane excitability. Although it is established that KATP channels regulate coronary blood vessel tone, the specific role of endothelial KATP channels is unknown. We transgenically targeted endothelial KATP channels by expressing a dominant-negative Kir6.1 subunit under the control of the Tek promoter. Histologically, the coronary endothelium was preserved in transgenic (TG) mice. Ergonovine did not induce coronary vasospasm and the mean blood pressure was not detectably different in these mice. The nitric oxide pathway was intact since bradykinin decreased the coronary perfusion pressure (CPP). Levcromakalim also decreased the CPP, most likely by acting on smooth muscle KATP channels. In contrast, the CPP of isolated hearts was substantially elevated in TG mice. Pre-treatment of mice with bosentan [an endothelin-1 (ET-1) receptor antagonist] normalized the CPP, suggestive of an enhanced ET-1 release. Indeed, ET-1 levels (measured by RIA) were elevated in the coronary effluent of TG hearts. KATP channel blockers (e.g. tolbutamide) similarly elevated ET-1 release; both from isolated rat hearts and from cultured human coronary artery endothelial cells. In conclusion, our data suggest a novel role for endothelial KATP channels in regulating coronary vessel tone by controlling regulated exocytosis of the vasoconstrictor ET-1.

Sirolimus Causes Relaxation of Human Vascular Smooth Muscle: A Novel Action of Sirolimus Mediated via ATP-Sensitive Potassium Channels

Little is known about the vasomotor effects of sirolimus, and preliminary studies using animal models have provided conflicting results. The present study was designed to determine the effects of sirolimus on vasomotor tone in human blood vessels. Human radial artery segments were cut into rings, denuded of endothelium, and placed into organ chambers for isometric tension recording. Sirolimus (10(-10) to 10(-6) M) caused concentration-dependent relaxation of human arteries contracted with U46619 (9,11-dideoxy-11alpha,9alpha-epoxymethano-prostaglandin F(2alpha); 10(-8) M) [-log (M) EC(50) (pD(2)) = 7.28 +/- 0.1; E(max) = 57 +/- 6%] or phenylephrine (10(-6) M) (pD(2) = 7.16 +/- 0.4; E(max) = 45 +/- 9%). Sirolimus-induced relaxation was unaffected by treatment with indomethacin (10(-5) M) but was nearly abolished in tissues contracted by depolarization with elevated K(+) (60 mM). In U46619-contracted rings, the response to sirolimus was markedly inhibited in the presence of the specific ATP-sensitive potassium (K(ATP)) channel blocker, glyburide (10(-6) M), but was unaffected by treatment with blockers of large conductance, calcium-activated potassium channel (iberiotoxin, 10(-7) M), small conductance, calcium-activated potassium channel (apamin, 10(-6) M), or voltage-gated potassium channel (4-aminopyridine, 10(-3) M). The K(ATP) channel opener, aprikalim (10(-7) to 10(-5) M), caused concentration-dependent relaxations that were inhibited by glyburide (10(-6) M) and abolished in tissues contracted with elevated K(+) (60 mM), thus confirming that K(ATP) channel opening causes relaxation of these arteries. These data suggest that sirolimus, at concentrations attained in vivo, causes relaxation of human arteries, and this effect is mediated by opening of K(ATP) channels in vascular smooth muscle. Reduced vasomotor tone is a heretofore unrecognized action of sirolimus that could potentially contribute to its efficacy in drug-eluting stents.

ADMA Increases Arterial Stiffness and Decreases Cerebral Blood Flow in Humans

Preclinical studies have revealed that the endogenous nitric oxide synthase inhibitor, asymmetric dimethylarginine (ADMA), increases vascular tone in cerebral blood vessels. Marked elevations of ADMA blood levels were found in patients with diseases characterized by decreased cerebral perfusion, such as ischemic stroke. Arterial stiffness is an independent predictor of stroke and other adverse cardiovascular events. The aim of this study was to investigate the influence of a systemic subpressor dose of ADMA on arterial stiffness and cerebral perfusion in humans. Using a double-blind, vehicle-controlled study design, we allocated 20 healthy men in random order to infusion of either ADMA (0.10 mg ADMA/kg per min) or vehicle over a period of 40 minutes. Arterial stiffness was assessed noninvasively by pulse wave analysis. All volunteers underwent measurement of cerebral perfusion by dynamic contrast-enhanced perfusion magnetic resonance imaging of the brain. Infusion of ADMA significantly decreased total cerebral perfusion by 15.1+/-4.5% (P=0.007), whereas blood flow in the vehicle group increased by 7.7+/-2.8% (P=0.02). ADMA also increased arterial stiffness as assessed by measurement of the augmentation index (-12.6+/-1.9 to -9.6+/-1.5, P=0.007). Our results document for the first time that subpressor doses of ADMA increase vascular stiffness and decrease cerebral perfusion in healthy subjects. Thus, ADMA is an important endogenous modulator of cerebral vascular tone and may be involved in the pathogenesis of cerebrovascular disease.

Adventitia contribution in vascular tone: insights from adventitia‐derived cells in a tissue‐engineered human blood vessel

Whether the adventitia component of blood vessels directly participates in the regulation of vascular tone remains to be demonstrated. We have recently developed a human tissue-engineered blood vessel comprising the three tunicae of a native blood vessel using the self-assembly approach. To investigate the role of the adventitia in the modulation of vascular tone, this tissue-engineering method was used to produce three vascular constructs from cells explanted and proliferated from donor vessel tunicae 1) an adventitia + a media, or only 2) an adventitia, or 3) a media. The vasoconstriction responses of these 3 constructs to endothelin, the most potent vasopressor known up-to-date, as well as to nonselective and selective agonists and antagonists, were compared. The adventitia contracted to endothelin-1, -2, whereas the media and the media+adventitia contracted to all three endothelins. Endothelin-induced contraction of the adventitia was dependent on ET(A) receptors, whereas that of the media and the adventitia+media was ET(A) and ET(B) receptor-dependent. RT-PCR studies corroborated these results. SNP induced a dose-dependent relaxation of the three tissue constructs. We also demonstrated that the endothelin-converting enzyme, responsible for the formation of the active endothelin peptides, was present and functional in the adventitia. In conclusion, this is the first direct demonstration that the adventitia has the capacity to contract and relax in response to vasoactive factors. The present study suggests that the adventitia of a blood vessel could play a greater role than expected in the modulation of blood vessel tone.

Pressure Under Pressure

GENETICS Hypertension is an extremely common disorder that, left untreated, can lead to stroke, heart disease, and kidney failure. Individuals of African descent are at greater risk of developing high blood pressure than are those of European descent, and this may reflect adaptations to distinct environmental selection pressures experienced by ancestral populations. For example, ancient human populations living in hot humid climates where salt was scarce would likely have a physiology adapted to maximize salt retention (which would concomitantly increase blood vessel tone), but this selective pressure would be lost once populations moved to cooler regions. Young et al. present genetic data consistent with the hypothesis that differential susceptibility to hypertension among modern humans is due to climatic adaptation during the out-of-Africa expansion. Studying worldwide variation in five genes implicated in blood pressure regulation, they find that the prevalence of allelic variants that would increase heat adaptation (and hence hypertension susceptibility) is significantly greater in populations living at low latitudes or in hot wet climates than in those at high latitudes or cold dry climates. In addition, using data from an epidemiologic study of blood pressure in 52 different populations, they conclude that a major portion of the worldwide variation in blood pressure can be accounted for by latitude and a variant allele of GNB3, the beta-3 subunit of guanine nucleotide-binding protein. — PAK PLoS Genet. 10.1371/journal.pgen.0010082.eor (2005).

Part 10.6: Anaphylaxis

Anaphylaxis is a severe, systemic allergic reaction characterized by multisystem involvement, including the skin, airway, vascular system, and gastrointestinal tract. Severe cases may result in complete obstruction of the airway, cardiovascular collapse, and death. The term classic anaphylaxis refers to hypersensitivity reactions mediated by the subclass of antibodies immunoglobulins IgE and IgG. Prior sensitization to an allergen has occurred, producing antigen-specific immunoglobulins. Subsequent reexposure to the allergen provokes the anaphylactic reaction. Many anaphylactic reactions, however, occur without a documented prior exposure. Anaphylactoid or pseudoanaphylactic reactions display a similar clinical syndrome, but they are not immune-mediated. Treatment for the two conditions is similar. The inciting allergen binds to antigen-specific IgE that has accumulated on previously sensitized basophils and mast cells. These cells almost immediately release a series of mediators, including histamines, leukotrienes, prostaglandins, thromboxanes, and bradykinins. When released locally and systemically, these mediators cause increased mucous membrane secretions, increased capillary permeability and leak, and markedly reduced smooth muscle tone in blood vessels (vasodilation) and bronchioles. Any antigen capable of activating IgE can be a trigger for anaphylaxis. In terms of etiology, researchers generally list the following categories of causes: pharmacologic agents, latex, stinging insects, and foods. In up to 5% of cases the antigenic agent cannot be identified.

Reduced vascular reactivity after chronic nitroglycerine administration: EDHF mechanism is also downregulated

British Journal of Pharmacology (2005) 146, 479–480. doi:10.1038/sj.bjp.0706364

PARAMETERS OF BRACHIAL ARTERY REACTIVE HYPEREMIA TEST IN TYPE 2 DIABETIC PATIENTS

Purpose: Impaired endothelium-dependent vasodilatation is a diffuse disease process resulting in abnormal regulation of blood vessel tone and loss of several atheroprotective effects of the normal endothelium. Systemic vascular endothelial function can be evaluated through measuring brachial arterial vasodilatation (physiologic answer to reactive hyperemia after transient ischemia), using high-resolution ultrasonography. The aim of the present study was to investigate the parameters of endothelial function in diabetic patients.

The NH2 Terminus of RCK1 Domain Regulates Ca2+-dependent BKCa Channel Gating

Large conductance, voltage- and Ca2+-activated K+ (BKCa) channels regulate blood vessel tone, synaptic transmission, and hearing owing to dual activation by membrane depolarization and intracellular Ca2+. Similar to an archeon Ca2+-activated K+ channel, MthK, each of four α subunits of BKCa may contain two cytosolic RCK domains and eight of which may form a gating ring. The structure of the MthK channel suggests that the RCK domains reorient with one another upon Ca2+ binding to change the gating ring conformation and open the activation gate. Here we report that the conformational changes of the NH2 terminus of RCK1 (AC region) modulate BKCa gating. Such modulation depends on Ca2+ occupancy and activation states, but is not directly related to the Ca2+ binding sites. These results demonstrate that AC region is important in the allosteric coupling between Ca2+ binding and channel opening. Thus, the conformational changes of the AC region within each RCK domain is likely to be an important step in addition to the reorientation of RCK domains leading to the opening of the BKCa activation gate. Our observations are consistent with a mechanism for Ca2+-dependent activation of BKCa channels such that the AC region inhibits channel activation when the channel is at the closed state in the absence of Ca2+; Ca2+ binding and depolarization relieve this inhibition.

Functional role of TRPC proteins in native systems: implications from knockout and knock‐down studies

Available data on transient receptor potential channel (TRPC) protein functions indicate that these proteins represent essential constituents of agonist-activated and phospholipase C-dependent cation entry pathways in primary cells which contribute to the elevation of cytosolic Ca2+. In addition, a striking number of biological functions have already been assigned to the various TRPC proteins, including mechanosensing activity (TRPC1), chemotropic axon guidance (TRPC1 and TRPC3), pheromone sensing and the regulation of sexual and social behaviour (TRPC2), endothelial-dependent regulation of vascular tone, endothelial permeability and neurotransmitter release (TRPC4), axonal growth (TRPC5), modulation of smooth muscle tone in blood vessels and lung and regulation of podocyte structure and function in the kidney (TRPC6). The lack of compounds which specifically block or activate TRPC proteins impairs the analysis of TRPC function in primary cells. We therefore concentrate in this contribution on (i) studies of TRPC-deficient mouse lines, (ii) data obtained by gene-silencing approaches using antisense oligonucleotides or RNA interference, (iii) expression experiments employing dominant negative TRPC constructs, and (iv) recent data correlating mutations of TRPC genes associated with human disease.

The Three-Dimensional Structure of the cGMP-Dependent Protein Kinase I - α Leucine Zipper Domain and Its Interaction with the Myosin Binding Subunit.

Vascular smooth muscle contractile state is tightly coupled to myosin light chain phosphorylation. Myosin light chain phosphorylation is regulated by the counter regulatory activities of myosin light chain kinase and myosin phosphatase. Nitric oxide (NO) and nitrovasodilators induce vascular smooth muscle cell relaxation in part by cGMP-dependent protein kinase (cGK)-mediated activation of myosin phosphatase. This in turn functions to dephosphorylate myosin light chains. Mechanistically this involves a protein-protein interaction mediated by a leucine zipper (LZ) domain that is localized within the N-terminus of cGK1α and the C-terminus of myosin-binding subunit (MBS). To better understand the importance of this interaction in regulating smooth muscle vascular tone we have determined the three-dimensional structure of the LZ domain of cGK1α, (cGKIα1–59) using triple resonance, multidimensional nuclear magnetic resonance (NMR) spectroscopy. We have expressed the LZ domain (cGKIα1–59) in E. coli under conditions facilitating the uniform incorporation of 13C/15N and 2H isotopic labels and purified this expressed protein. Circular dichroism data supported that cGK1α is comprised of an α-helix, while our NMR data identified that this helix commences at residue Ala9 and extends through to residue Leu47 of the LZ domain. 15N backbone longitudinal and transverse relaxation rates are best fit to a rotational diffusion anisotropy model that is consistent with a coiled-coil dimmer structure, not a monomeric helix. This suggests that cGKIα1–59 is comprised of a coiled-coil conformation over this same region. A time course investigation carried out with the LZ domain and a homobifunctional amine reactive crosslinker [dithio-bis(succinimidyl propionate)] (DSP) supported the presence of a homodimer. Furthermore, 15N heteronuclear single quantum correlation (HSQC) chemical shift mapping data has been used to identify residues that are critical for interacting with the C-terminal region of the myosin binding subunit (MBS). These data have allowed us to identify those residues necessary for cGK1α dimerization, while simultaneously permitting a better understanding of the critical determinants of cGK1α and MBS that are involved in regulating the contractile state of smooth muscles within blood vessels. The spatial and temporal contractile state of these blood vessels is important in the physiologic control of blood vessel tone and plays a role in the pathogenesis of hypertension and atherosclerosis a leading cause of death in the US and worldwide.

Functional role of TRPC proteins in vivo: lessons from TRPC-deficient mouse models

In order to elucidate the functional role of TRPC genes, in vivo, the targeted inactivation of these genes in mice is an invaluable technique. In this review, we summarize the currently available results on the phenotype of TRPC-deficient mouse lines. The analysis of mice with targeted deletion in three TRPC genes demonstrates that these proteins represent essential constituents of agonist-activated and phospholipase C-dependent Ca 2+ entry channels in primary cells. Furthermore, from the deficits observed in these TRPC-deficient mouse lines a striking number of biological functions could already be ascribed to TRPC2, TRPC4, and TRPC6, not only on the cellular level but also for complex organ functions and integrative physiology. Accordingly, TRPC2 proteins are critically involved in pheromone sensing by neurones of the vomeronasal organ and, thereby, in the regulation of sexual and social behavior of mice, TRPC4 proteins are essential determinants of endothelial-dependent regulation of vascular tone, endothelial permeability, and neurotransmitter release from thalamic interneurones, and TRPC6 proteins are supposed to have a fundamental role in the regulation of smooth muscle tone in blood vessels and lung.

Non-invasive evaluation of endothelial function in hypertensive elderly patients

Impaired endothelium-dependent vasomotion is a diffuse disease process resulting in abnormal regulation of blood vessel tone and loss of several atheroprotective effects of the normal endothelium. The aim of the present study was to investigate the effects of aging and hypertension on endothelial function. Sixty-six geriatric subjects with ages over 60 (48 hypertensive and 18 healthy) and 40 middle-aged subjects (16 hypertensive and 24 healthy) were included in the study. Systemic vascular endothelial function was evaluated through measuring brachial arterial vasodilation, a physiologic answer to reactive hyperemia occured with increased blood flow in the vessel after transient ischemia (flow-mediated dilation, FMD%), and with carotid artery intima-media thickness (IMT) measurement, using high-resolution ultrasonography. Endothelial independent vasodilation was also measured after administration of sublingual isosorbide dinitrate (isosorbide dinitrate mediated dilation, IDNMD%). FMD% was significantly decreased in elderly and/or hypertensive (HT) patients (geriatric HT: 9.5 ± 4.7%, geriatric non-HT: 12.7 ± 5.5%, middle-aged HT: 12.9 ± 4.3% and middle-aged non-HT: 18.9 ± 8.1%) (geriatric HT versus geriatric non-HT ( P = 0.02), geriatric HT versus middle-aged HT ( P = 0.01), geriatric non-HT versus middle-aged non-HT ( P = 0.008)). Both FMD% and IDNMD% were inversely correlated with age, baseline vessel diameter and carotid artery intima-media thickness. FMD% was also inversely correlated with diastolic blood pressure. No correlation was found between FMD% and systolic blood pressure, serum cholesterol and triglyceride levels. Endothelium dependent (EDD) and independent dilatation of large arteries decreased with aging even in the healthy elderly, and FMD further declined in HT elderly patients, indicating that age and hypertension independently impair endothelial function. Positive correlations with age and hypertension, and significant inverse correlation with FMD, makes carotid artery IMT a possible indicator of endothelial function.