Mark Taylor - Academia.edu (original) (raw)

Papers by Mark Taylor

International Journal of Molecular Sciences, 2021

Uterine contractions prior to 37 weeks gestation can result in preterm labor with significant ris... more Uterine contractions prior to 37 weeks gestation can result in preterm labor with significant risk to the infant. Current tocolytic therapies aimed at suppressing premature uterine contractions are largely ineffective and cause serious side effects. Calcium (Ca2+) dependent contractions of uterine smooth muscle are physiologically limited by the opening of membrane potassium (K+) channels. Exploiting such inherent negative feedback mechanisms may offer new strategies to delay labor and reduce risk. Positive modulation of small conductance Ca2+-activated K+ (KCa2.3) channels with cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine (CyPPA), effectively decreases uterine contractions. This study investigates whether the receptor agonist oxytocin might solicit KCa2.3 channel feedback that facilitates CyPPA suppression of uterine contractions. Using isometric force myography, we found that spontaneous phasic contractions of myometrial tissue from nonpregnant mice wer...

International Journal of Molecular Sciences, 2020

Transient receptor potential vanilloid 4 channels (TRPV4) are pivotal regulators of vascular home... more Transient receptor potential vanilloid 4 channels (TRPV4) are pivotal regulators of vascular homeostasis. Altered TRPV4 signaling has recently been implicated in various cardiovascular diseases, including hypertension and atherosclerosis. These versatile nonselective cation channels increase endothelial Ca2+ influx in response to various stimuli including shear stress and G protein-coupled receptor (GPCR) activation. Recent findings suggest TRPV4 channels produce localized Ca2+ transients at the endothelial cell plasma membrane that may allow targeted effector recruitment and promote large-scale Ca2+ events via release from internal stores (endoplasmic reticulum). However, the specific impact of TRPV4 channels on Ca2+ signaling in the intact arterial intima remains unknown. In the current study, we employ an endothelium-specific TRPV4 knockout mouse model (ecTRPV4−/−) to identify and characterize TRPV4-dependent endothelial Ca2+ dynamics. We find that carotid arteries from both ecTR...

American journal of physiology. Regulatory, integrative and comparative physiology, Jan 22, 2017

Long-term hypoxia (LTH) has a profound effect on pulmonary arterial vasoconstriction in the fetus... more Long-term hypoxia (LTH) has a profound effect on pulmonary arterial vasoconstriction in the fetus and adult. Dysregulation in Ca2+ signaling is important during the development of LTH induced pulmonary hypertension. In the present study we tested the hypothesis that L-type Ca2+ channels, which are voltage-dependent and found in smooth, skeletal, and cardiac muscle, are important in the adaptation of pulmonary arterial contractions in post-natal maturation and in response to LTH. Pulmonary arteries were isolated from fetal or adult sheep maintained at low- or high-altitude (3,801 m) for >100 days. The effects of a L-type Ca2+ channel opener FPL 64176 (FPL) in the presence or absence of an inhibitor Nifedipine (NIF) on arterial contractions, intracellular Ca2+ oscillations, and ryanodine receptor-driven Ca2+ sparks were measured. FPL induced pulmonary arterial contractions in all groups that were sensitive to NIF. However, when compared to 125 mM K+, FPL contractions were greater i...

Microcirculation (New York, N.Y. : 1994), Apr 20, 2017

Disruption of blood flow promotes endothelial dysfunction and predisposes vessels to remodeling a... more Disruption of blood flow promotes endothelial dysfunction and predisposes vessels to remodeling and atherosclerosis. Recent findings suggest spatial and temporal tuning of local Ca(2+) signals along the endothelium is vital to vascular function. In the current study, we examined whether chronic flow disruption causes alteration of dynamic endothelial Ca(2+) signal patterning associated with changes in vascular structure and function. For these studies, we performed surgical partial-ligation (PL) of the left carotid arteries of mice to establish chronic low flow for 2 weeks; right carotid arteries remained open and served as controls (C). Histological sections showed substantial remodeling of PL compared to C arteries, including formation of neointima. Isometric force measurements revealed increased phenylephrine-induced contractions and decreased KCl-induced contractions in PL verses C arteries. Endothelium-dependent vasorelaxation in response to acetylcholine (ACh; 10(-8) - 10(-5) ...

American journal of physiology. Heart and circulatory physiology, May 4, 2016

Activation of vascular endothelial small- (KCa2.3, SK3) or intermediate- (KCa3.1, IK1) conductanc... more Activation of vascular endothelial small- (KCa2.3, SK3) or intermediate- (KCa3.1, IK1) conductance Ca(2+)-activated potassium channels induces vasorelaxation via an endothelium-derived hyperpolarization (EDH) pathway. Although the activation of SK3 and IK1 channels converges on EDH, their subcellular effects on signal transduction are different and not completely clear. In this study a novel endothelium-specific SK3 knockout (SK3(-/-)) mouse model was utilized to specifically examine the contribution of SK3 channels to mesenteric artery vasorelaxation, endothelial Ca(2+) dynamics, and blood pressure. The absence of SK3 expression was confirmed using real-time qPCR and western blot analysis. Functional studies showed impaired EDH-mediated vasorelaxation in SK3(-/-) small mesenteric arteries. Immunostaining results from SK3(-/-) vessels confirmed the absence of SK3 and further showed altered distribution of TRPV4 (transient receptor potential channels, type 4). Electrophysiological re...

Oxidative Medicine and Cellular Longevity, 2015

We hypothesized that transgenic mice overexpressing thep22phoxsubunit of the NADPH oxidase select... more We hypothesized that transgenic mice overexpressing thep22phoxsubunit of the NADPH oxidase selectively in smooth muscle (Tgp22smc) would exhibit an exacerbated response to transluminal carotid injury compared to wild-type mice. To examine the role of reactive oxygen species (ROS) as a mediator of vascular injury, the injury response was quantified by measuring wall thickness (WT) and cross-sectional wall area (CSWA) of the injured and noninjured arteries in bothTgp22smcand wild-type animals at days 3, 7, and 14 after injury. Akt, p38 MAPK, and Src activation were evaluated at the same time points using Western blotting. WT and CSWA following injury were significantly greater inTgp22smcmice at both 7 and 14 days after injury while noninjured contralateral carotids were similar between groups. Apocynin treatment attenuated the injury response in both groups and rendered the response similar betweenTgp22smcmice and wild-type mice. Following injury, carotid arteries fromTgp22smcmice dem...

Pulmonary Circulation, 2015

Our previous work has shown that the increased lung endothelial permeability response to 14,15-ep... more Our previous work has shown that the increased lung endothelial permeability response to 14,15-epoxyeicosatrienoic acid (14,15-EET) in rat lung requires Ca 2+ entry via vanilloid type-4 transient receptor potential (TRPV4) channels. Recent studies suggest that activation of TRPV4 channels in systemic vascular endothelium prolongs agonist-induced hyperpolarization and amplifies Ca 2+ entry by activating Ca 2+-activated K + (K Ca) channels, resulting in vessel relaxation. Activation of endothelial K Ca channels thus has potential to increase the electrochemical driving force for Ca 2+ influx via TRPV4 channels and to amplify permeability responses to TRPV4 activation in lung. To examine this hypothesis, we used Western blot analysis, electrophysiological recordings, and isolated-lung permeability measurements to document expression of TRPV4 and K Ca channels and the potential for functional coupling. The results show that rat pulmonary microvascular endothelial cells express TRPV4 and 3 K Ca channels of different conductances: large (BK), intermediate (IK), and small (SK3). However, TRPV4 channel activity modulates the IK and SK3, but not the BK, channel current density. Furthermore, the TRPV4-mediated permeability response to 14,15-EET in mouse lung is significantly attenuated by pharmacologic blockade of IK and SK3, but not BK, channels. Collectively, this functional coupling suggests that endothelial TRPV4 channels in rodent lung likely form signaling microdomains with IK and SK3 channels and that the integrated response dictates the extent of lung endothelial injury caused by 14,15-EET.

PloS one, 2014

Mesenteric artery endothelium expresses both small (SK3)- and intermediate (IK1)-conductance Ca(2... more Mesenteric artery endothelium expresses both small (SK3)- and intermediate (IK1)-conductance Ca(2+)-activated K(+) (KCa) channels whose activity modulates vascular tone via endothelium-dependent hyperpolarization (EDH). Two other major endothelium-dependent vasodilation pathways utilize nitric oxide (NO) and prostacyclin (PGI2). To examine how ovariectomy (ovx) affects the basal activity and acetylcholine (ACh)-induced activity of each of these three pathways to vasorelaxation, we used wire myograph and electrophysiological recordings. The results from functional studies using isolated murine mesenteric arteries show that ovx reduces ACh-induced endothelium-dependent vasodilation due to decreased EDH and NO contributions, although the contribution of PGI2 is upregulated. Both endothelial SK3 and IK1 channels are functionally coupled to TRPV4 (transient receptor potential, vanilloid type 4) channels: the activation of TRPV4 channels activates SK3 and IK1 channels, leading to EDH-medi...

Frontiers in physiology, 2014

Although acute and chronic vasoregulation is inherently driven by endothelial Ca(2+), control and... more Although acute and chronic vasoregulation is inherently driven by endothelial Ca(2+), control and targeting of Ca(2+)-dependent signals are poorly understood. Recent studies have revealed localized and dynamic endothelial Ca(2+) events comprising an intricate signaling network along the vascular intima. Discrete Ca(2+) transients emerging from both internal stores and plasmalemmal cation channels couple to specific membrane K(+) channels, promoting endothelial hyperpolarization and vasodilation. The spatiotemporal tuning of these signals, rather than global Ca(2+) elevation, appear to direct endothelial functions under physiologic conditions. In fact, altered patterns of dynamic Ca(2+) signaling may underlie essential endothelial dysfunction in a variety of cardiovascular diseases. Advances in imaging approaches and analyses in recent years have allowed for detailed detection, quantification, and evaluation of Ca(2+) dynamics in intact endothelium. Here, we discuss recent insights i...

Journal of Visualized Experiments, 2014

Intracellular Ca 2+ signals are commonly studied with fluorescent Ca 2+ indicator dyes and micros... more Intracellular Ca 2+ signals are commonly studied with fluorescent Ca 2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca 2+ imaging data is time consuming and subject to bias. Automated signal analysis algorithms based on region of interest (ROI) detection have been implemented for one-dimensional line scan measurements, but there is no current algorithm which integrates optimized identification and analysis of ROIs in two-dimensional image sequences. Here an algorithm for rapid acquisition and analysis of ROIs in image sequences is described. It utilizes ellipses fit to noise filtered signals in order to determine optimal ROI placement, and computes Ca 2+ signal parameters of amplitude, duration and spatial spread. This algorithm was implemented as a freely available plugin for ImageJ (NIH) software. Together with analysis scripts written for the open source statistical processing software R, this approach provides a high-capacity pipeline for performing quick statistical analysis of experimental output. The authors suggest that use of this analysis protocol will lead to a more complete and unbiased characterization of physiologic Ca 2+ signaling.

Proceedings of the National Academy of Sciences, 2000

Arrays of octameric peptide libraries on cellulose paper were screened by using 32 P-autophosphor... more Arrays of octameric peptide libraries on cellulose paper were screened by using 32 P-autophosphorylated cGMP-dependent protein kinase Iα (cGPK) to identify peptide sequences with high binding affinity for cGPK. Iterative deconvolution of every amino acid position in the peptides identified the sequence LRK 5 H (W45) as having the highest binding affinity. Binding of W45 to cGPK resulted in selective inhibition of the kinase with K i values of 0.8 μM and 560 μM for cGPK and cAMP-dependent protein kinase (cAPK), respectively. Fusion of W45 to membrane translocation signals from HIV-1 tat protein (YGRKKRRQRRRPP-LRK 5 H, DT-2) or Drosophila Antennapedia homeo-domain (RQIKIWFQNRRMKWKK-LRK 5 H, DT-3) proved to be an efficient method for intracellular delivery of these highly charged peptides. Rapid translocation of the peptides into intact cerebral arteries was demonstrated by using fluorescein-labeled DT-2 and DT-3. The inhibitory potency of the fusion peptides was even greater than that...

Proceedings of the National Academy of Sciences, 2008

Calcium (Ca 2+ ) release through inositol 1,4,5-trisphosphate receptors (IP 3 Rs) regulates the f... more Calcium (Ca 2+ ) release through inositol 1,4,5-trisphosphate receptors (IP 3 Rs) regulates the function of virtually every mammalian cell. Unlike ryanodine receptors, which generate local Ca 2+ events (“sparks”) that transmit signals to the juxtaposed cell membrane, a similar functional architecture has not been reported for IP 3 Rs. Here, we have identified spatially fixed, local Ca 2+ release events (“pulsars”) in vascular endothelial membrane domains that project through the internal elastic lamina to adjacent smooth muscle membranes. Ca 2+ pulsars are mediated by IP 3 Rs in the endothelial endoplasmic reticulum of these membrane projections. Elevation of IP 3 by the endothelium-dependent vasodilator, acetylcholine, increased the frequency of Ca 2+ pulsars, whereas blunting IP 3 production, blocking IP 3 Rs, or depleting endoplasmic reticulum Ca 2+ inhibited these events. The elementary properties of Ca 2+ pulsars were distinct from ryanodine-receptor-mediated Ca 2+ sparks in sm...

Circulation Research, 2003

The endothelium is a critical regulator of vascular tone, and dysfunction of the endothelium cont... more The endothelium is a critical regulator of vascular tone, and dysfunction of the endothelium contributes to numerous cardiovascular pathologies. Recent studies suggest that apamin-sensitive, small-conductance, Ca 2+ -activated K + channels may play an important role in active endothelium-dependent vasodilations, and expression of these channels may be altered in disease states characterized by vascular dysfunction. Here, we used a transgenic mouse (SK3 T/T ) in which SK3 expression levels can be manipulated with dietary doxycycline (DOX) to test the hypothesis that the level of expression of the SK subunit, SK3, in endothelial cells alters arterial function and blood pressure. SK3 protein was elevated in small mesenteric arteries from SK3 T/T mice compared with wild-type mice and was greatly suppressed by dietary DOX. SK3 was detected in the endothelium and not in the smooth muscle by immunohistochemistry. In whole-cell patch-clamp experiments, SK currents in endothelial cells from ...

Arteriosclerosis, Thrombosis, and Vascular Biology, 2013

Objective— Intermediate and small conductance K Ca channels IK1 (K Ca 3.1) and SK3 (K Ca 2.3) are... more Objective— Intermediate and small conductance K Ca channels IK1 (K Ca 3.1) and SK3 (K Ca 2.3) are primary targets of endothelial Ca 2+ signals in the arterial vasculature, and their ablation results in increased arterial tone and hypertension. Activation of IK1 channels by local Ca 2+ transients from internal stores or plasma membrane channels promotes arterial hyperpolarization and vasodilation. Here, we assess arteries from genetically altered IK1 knockout mice (IK1 −/− ) to determine whether IK1 channels exert a positive feedback influence on endothelial Ca 2+ dynamics. Approach and Results— Using confocal imaging and custom data analysis software, we found that although the occurrence of basal endothelial Ca 2+ dynamics was not different between IK1 −/− and wild-type mice ( P >0.05), the frequency of acetylcholine-stimulated (2 μmol/L) Ca 2+ dynamics was greatly decreased in IK1 −/− endothelium (515±153 versus 1860±319 events; P <0.01). In IK1 −/− /SK3 T/T mice, ancillary ...

American Journal of Physiology-Cell Physiology, 2012

Ca2+ signals are commonly measured using fluorescent Ca2+ indicators and microscopy techniques, b... more Ca2+ signals are commonly measured using fluorescent Ca2+ indicators and microscopy techniques, but manual analysis of Ca2+ measurements is time consuming and subject to bias. Automated region of interest (ROI) detection algorithms have been employed for identification of Ca2+ signals in one-dimensional line scan images, but currently there is no process to integrate acquisition and analysis of ROIs within two-dimensional time lapse image sequences. Therefore we devised a novel algorithm for rapid ROI identification and measurement based on the analysis of best-fit ellipses assigned to signals within noise-filtered image sequences. This algorithm was implemented as a plugin for ImageJ software (National Institutes of Health, Bethesda, MD). We evaluated the ability of our algorithm to detect synthetic Gaussian signal pulses embedded in background noise. The algorithm placed ROIs very near to the center of a range of signal pulses, resulting in mean signal amplitude measurements of 99...

American Journal of Physiology-Cell Physiology, 2012

When arteries constrict to agonists, the endothelium inversely responds, attenuating the initial ... more When arteries constrict to agonists, the endothelium inversely responds, attenuating the initial vasomotor response. The basis of this feedback mechanism remains uncertain, although past studies suggest a key role for myoendothelial communication in the signaling process. The present study examined whether second messenger flux through myoendothelial gap junctions initiates a negative-feedback response in hamster retractor muscle feed arteries. We specifically hypothesized that when agonists elicit depolarization and a rise in second messenger concentration, inositol trisphosphate (IP3) flux activates a discrete pool of IP3 receptors (IP3Rs), elicits localized endothelial Ca2+ transients, and activates downstream effectors to moderate constriction. With use of integrated experimental techniques, this study provided three sets of supporting observations. Beginning at the functional level, we showed that blocking intermediate-conductance Ca2+-activated K+ channels (IK) and Ca2+ mobili...

The American Journal of Pathology, 2013

Microcirculation, 2012

The endothelium is vital to normal vasoregulation. Although acute vasodilation associated with br... more The endothelium is vital to normal vasoregulation. Although acute vasodilation associated with broad endothelial Ca 2+ elevation is well-known, the control and targeting of Ca 2+ dependent signals in the endothelium is poorly understood. Recent studies have revealed localized IP 3motivated Ca 2+ events occurring basally along the intima that may provide the fundamental basis for various endothelial influences. Here, we provide an overview of dynamic endothelial Ca 2+ signals and discuss the potential role of these signals in constant endothelial control of arterial tone and the titration of functional responses in vivo. In particular, we focus on the functional architecture contributing to the properties and ultimate impact of these signals and explore new avenues in evaluating their prevalence and specific modalities in intact tissue. Finally, we discuss spatial and temporal effector recruitment through modification of these inherent signals. It is suggested that endothelial Ca 2+ signaling is a continuum in which the specific framework of storerelease components and cellular targets along the endothelium allows for differential modes of Ca 2+ signal expansion and distinctive profiles of effector recruitment. The precise composition and distribution of these inherent components may underlie dynamic endothelial control and specialized functions of different vascular beds.

American Journal of Physiology-Cell Physiology, 2007

Mechanisms regulating uterine contractility are poorly understood. We hypothesized that a specifi... more Mechanisms regulating uterine contractility are poorly understood. We hypothesized that a specific isoform of small conductance Ca2+-activated K+ (SK) channel, SK3, promotes feedback regulation of myometrial Ca2+ and hence relaxation of the uterus. To determine the specific functional impact of SK3 channels, we assessed isometric contractions of uterine strips from genetically altered mice (SK3T/T), in which SK3 is overexpressed and can be suppressed by oral administration of doxycycline (SK3T/T+Dox). We found SK3 protein in mouse myometrium, and this expression was substantially higher in SK3T/T mice and lower in SK3T/T+Dox mice compared with wild-type (WT) controls. Sustained contractions elicited by 60 mM KCl were not different among SK3T/T, SK3T/T+Dox, and WT mice. However, the rate of onset and magnitude of spontaneously occurring phasic contractions was muted significantly in isolated uterine strips from SK3T/T mice compared with those from WT mice. These spontaneous contracti...

AJP: Cell Physiology, 2011

Organized uterine contractions, including those necessary for parturition, are dependent on calci... more Organized uterine contractions, including those necessary for parturition, are dependent on calcium entry through voltage-gated calcium channels in myometrial smooth muscle cells. Recent evidence suggests that small-conductance Ca2+-activated potassium channels (KCa2), specifically isoforms KCa2.2 and 2.3, may control these contractions through negative feedback regulation of Ca2+ entry. We tested whether selective pharmacologic activation of KCa2.2/2.3 channels might depress uterine contractions, providing a new strategy for preterm labor intervention. Western blot analysis and immunofluorescence microscopy revealed expression of both KCa2.2 and KCa2.3 in the myometrium of nonpregnant (NP) and pregnant (gestation day 10 and 16; D10 and D16, respectively) mice. Spontaneous phasic contractions of isolated NP, D10, and D16 uterine strips were all suppressed by the KCa2.2/2.3-selective activator CyPPA in a concentration-dependent manner. This effect was antagonized by the selective KCa...

International Journal of Molecular Sciences, 2021

Uterine contractions prior to 37 weeks gestation can result in preterm labor with significant ris... more Uterine contractions prior to 37 weeks gestation can result in preterm labor with significant risk to the infant. Current tocolytic therapies aimed at suppressing premature uterine contractions are largely ineffective and cause serious side effects. Calcium (Ca2+) dependent contractions of uterine smooth muscle are physiologically limited by the opening of membrane potassium (K+) channels. Exploiting such inherent negative feedback mechanisms may offer new strategies to delay labor and reduce risk. Positive modulation of small conductance Ca2+-activated K+ (KCa2.3) channels with cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine (CyPPA), effectively decreases uterine contractions. This study investigates whether the receptor agonist oxytocin might solicit KCa2.3 channel feedback that facilitates CyPPA suppression of uterine contractions. Using isometric force myography, we found that spontaneous phasic contractions of myometrial tissue from nonpregnant mice wer...

International Journal of Molecular Sciences, 2020

Transient receptor potential vanilloid 4 channels (TRPV4) are pivotal regulators of vascular home... more Transient receptor potential vanilloid 4 channels (TRPV4) are pivotal regulators of vascular homeostasis. Altered TRPV4 signaling has recently been implicated in various cardiovascular diseases, including hypertension and atherosclerosis. These versatile nonselective cation channels increase endothelial Ca2+ influx in response to various stimuli including shear stress and G protein-coupled receptor (GPCR) activation. Recent findings suggest TRPV4 channels produce localized Ca2+ transients at the endothelial cell plasma membrane that may allow targeted effector recruitment and promote large-scale Ca2+ events via release from internal stores (endoplasmic reticulum). However, the specific impact of TRPV4 channels on Ca2+ signaling in the intact arterial intima remains unknown. In the current study, we employ an endothelium-specific TRPV4 knockout mouse model (ecTRPV4−/−) to identify and characterize TRPV4-dependent endothelial Ca2+ dynamics. We find that carotid arteries from both ecTR...

American journal of physiology. Regulatory, integrative and comparative physiology, Jan 22, 2017

Long-term hypoxia (LTH) has a profound effect on pulmonary arterial vasoconstriction in the fetus... more Long-term hypoxia (LTH) has a profound effect on pulmonary arterial vasoconstriction in the fetus and adult. Dysregulation in Ca2+ signaling is important during the development of LTH induced pulmonary hypertension. In the present study we tested the hypothesis that L-type Ca2+ channels, which are voltage-dependent and found in smooth, skeletal, and cardiac muscle, are important in the adaptation of pulmonary arterial contractions in post-natal maturation and in response to LTH. Pulmonary arteries were isolated from fetal or adult sheep maintained at low- or high-altitude (3,801 m) for >100 days. The effects of a L-type Ca2+ channel opener FPL 64176 (FPL) in the presence or absence of an inhibitor Nifedipine (NIF) on arterial contractions, intracellular Ca2+ oscillations, and ryanodine receptor-driven Ca2+ sparks were measured. FPL induced pulmonary arterial contractions in all groups that were sensitive to NIF. However, when compared to 125 mM K+, FPL contractions were greater i...

Microcirculation (New York, N.Y. : 1994), Apr 20, 2017

Disruption of blood flow promotes endothelial dysfunction and predisposes vessels to remodeling a... more Disruption of blood flow promotes endothelial dysfunction and predisposes vessels to remodeling and atherosclerosis. Recent findings suggest spatial and temporal tuning of local Ca(2+) signals along the endothelium is vital to vascular function. In the current study, we examined whether chronic flow disruption causes alteration of dynamic endothelial Ca(2+) signal patterning associated with changes in vascular structure and function. For these studies, we performed surgical partial-ligation (PL) of the left carotid arteries of mice to establish chronic low flow for 2 weeks; right carotid arteries remained open and served as controls (C). Histological sections showed substantial remodeling of PL compared to C arteries, including formation of neointima. Isometric force measurements revealed increased phenylephrine-induced contractions and decreased KCl-induced contractions in PL verses C arteries. Endothelium-dependent vasorelaxation in response to acetylcholine (ACh; 10(-8) - 10(-5) ...

American journal of physiology. Heart and circulatory physiology, May 4, 2016

Activation of vascular endothelial small- (KCa2.3, SK3) or intermediate- (KCa3.1, IK1) conductanc... more Activation of vascular endothelial small- (KCa2.3, SK3) or intermediate- (KCa3.1, IK1) conductance Ca(2+)-activated potassium channels induces vasorelaxation via an endothelium-derived hyperpolarization (EDH) pathway. Although the activation of SK3 and IK1 channels converges on EDH, their subcellular effects on signal transduction are different and not completely clear. In this study a novel endothelium-specific SK3 knockout (SK3(-/-)) mouse model was utilized to specifically examine the contribution of SK3 channels to mesenteric artery vasorelaxation, endothelial Ca(2+) dynamics, and blood pressure. The absence of SK3 expression was confirmed using real-time qPCR and western blot analysis. Functional studies showed impaired EDH-mediated vasorelaxation in SK3(-/-) small mesenteric arteries. Immunostaining results from SK3(-/-) vessels confirmed the absence of SK3 and further showed altered distribution of TRPV4 (transient receptor potential channels, type 4). Electrophysiological re...

Oxidative Medicine and Cellular Longevity, 2015

We hypothesized that transgenic mice overexpressing thep22phoxsubunit of the NADPH oxidase select... more We hypothesized that transgenic mice overexpressing thep22phoxsubunit of the NADPH oxidase selectively in smooth muscle (Tgp22smc) would exhibit an exacerbated response to transluminal carotid injury compared to wild-type mice. To examine the role of reactive oxygen species (ROS) as a mediator of vascular injury, the injury response was quantified by measuring wall thickness (WT) and cross-sectional wall area (CSWA) of the injured and noninjured arteries in bothTgp22smcand wild-type animals at days 3, 7, and 14 after injury. Akt, p38 MAPK, and Src activation were evaluated at the same time points using Western blotting. WT and CSWA following injury were significantly greater inTgp22smcmice at both 7 and 14 days after injury while noninjured contralateral carotids were similar between groups. Apocynin treatment attenuated the injury response in both groups and rendered the response similar betweenTgp22smcmice and wild-type mice. Following injury, carotid arteries fromTgp22smcmice dem...

Pulmonary Circulation, 2015

Our previous work has shown that the increased lung endothelial permeability response to 14,15-ep... more Our previous work has shown that the increased lung endothelial permeability response to 14,15-epoxyeicosatrienoic acid (14,15-EET) in rat lung requires Ca 2+ entry via vanilloid type-4 transient receptor potential (TRPV4) channels. Recent studies suggest that activation of TRPV4 channels in systemic vascular endothelium prolongs agonist-induced hyperpolarization and amplifies Ca 2+ entry by activating Ca 2+-activated K + (K Ca) channels, resulting in vessel relaxation. Activation of endothelial K Ca channels thus has potential to increase the electrochemical driving force for Ca 2+ influx via TRPV4 channels and to amplify permeability responses to TRPV4 activation in lung. To examine this hypothesis, we used Western blot analysis, electrophysiological recordings, and isolated-lung permeability measurements to document expression of TRPV4 and K Ca channels and the potential for functional coupling. The results show that rat pulmonary microvascular endothelial cells express TRPV4 and 3 K Ca channels of different conductances: large (BK), intermediate (IK), and small (SK3). However, TRPV4 channel activity modulates the IK and SK3, but not the BK, channel current density. Furthermore, the TRPV4-mediated permeability response to 14,15-EET in mouse lung is significantly attenuated by pharmacologic blockade of IK and SK3, but not BK, channels. Collectively, this functional coupling suggests that endothelial TRPV4 channels in rodent lung likely form signaling microdomains with IK and SK3 channels and that the integrated response dictates the extent of lung endothelial injury caused by 14,15-EET.

PloS one, 2014

Mesenteric artery endothelium expresses both small (SK3)- and intermediate (IK1)-conductance Ca(2... more Mesenteric artery endothelium expresses both small (SK3)- and intermediate (IK1)-conductance Ca(2+)-activated K(+) (KCa) channels whose activity modulates vascular tone via endothelium-dependent hyperpolarization (EDH). Two other major endothelium-dependent vasodilation pathways utilize nitric oxide (NO) and prostacyclin (PGI2). To examine how ovariectomy (ovx) affects the basal activity and acetylcholine (ACh)-induced activity of each of these three pathways to vasorelaxation, we used wire myograph and electrophysiological recordings. The results from functional studies using isolated murine mesenteric arteries show that ovx reduces ACh-induced endothelium-dependent vasodilation due to decreased EDH and NO contributions, although the contribution of PGI2 is upregulated. Both endothelial SK3 and IK1 channels are functionally coupled to TRPV4 (transient receptor potential, vanilloid type 4) channels: the activation of TRPV4 channels activates SK3 and IK1 channels, leading to EDH-medi...

Frontiers in physiology, 2014

Although acute and chronic vasoregulation is inherently driven by endothelial Ca(2+), control and... more Although acute and chronic vasoregulation is inherently driven by endothelial Ca(2+), control and targeting of Ca(2+)-dependent signals are poorly understood. Recent studies have revealed localized and dynamic endothelial Ca(2+) events comprising an intricate signaling network along the vascular intima. Discrete Ca(2+) transients emerging from both internal stores and plasmalemmal cation channels couple to specific membrane K(+) channels, promoting endothelial hyperpolarization and vasodilation. The spatiotemporal tuning of these signals, rather than global Ca(2+) elevation, appear to direct endothelial functions under physiologic conditions. In fact, altered patterns of dynamic Ca(2+) signaling may underlie essential endothelial dysfunction in a variety of cardiovascular diseases. Advances in imaging approaches and analyses in recent years have allowed for detailed detection, quantification, and evaluation of Ca(2+) dynamics in intact endothelium. Here, we discuss recent insights i...

Journal of Visualized Experiments, 2014

Intracellular Ca 2+ signals are commonly studied with fluorescent Ca 2+ indicator dyes and micros... more Intracellular Ca 2+ signals are commonly studied with fluorescent Ca 2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca 2+ imaging data is time consuming and subject to bias. Automated signal analysis algorithms based on region of interest (ROI) detection have been implemented for one-dimensional line scan measurements, but there is no current algorithm which integrates optimized identification and analysis of ROIs in two-dimensional image sequences. Here an algorithm for rapid acquisition and analysis of ROIs in image sequences is described. It utilizes ellipses fit to noise filtered signals in order to determine optimal ROI placement, and computes Ca 2+ signal parameters of amplitude, duration and spatial spread. This algorithm was implemented as a freely available plugin for ImageJ (NIH) software. Together with analysis scripts written for the open source statistical processing software R, this approach provides a high-capacity pipeline for performing quick statistical analysis of experimental output. The authors suggest that use of this analysis protocol will lead to a more complete and unbiased characterization of physiologic Ca 2+ signaling.

Proceedings of the National Academy of Sciences, 2000

Arrays of octameric peptide libraries on cellulose paper were screened by using 32 P-autophosphor... more Arrays of octameric peptide libraries on cellulose paper were screened by using 32 P-autophosphorylated cGMP-dependent protein kinase Iα (cGPK) to identify peptide sequences with high binding affinity for cGPK. Iterative deconvolution of every amino acid position in the peptides identified the sequence LRK 5 H (W45) as having the highest binding affinity. Binding of W45 to cGPK resulted in selective inhibition of the kinase with K i values of 0.8 μM and 560 μM for cGPK and cAMP-dependent protein kinase (cAPK), respectively. Fusion of W45 to membrane translocation signals from HIV-1 tat protein (YGRKKRRQRRRPP-LRK 5 H, DT-2) or Drosophila Antennapedia homeo-domain (RQIKIWFQNRRMKWKK-LRK 5 H, DT-3) proved to be an efficient method for intracellular delivery of these highly charged peptides. Rapid translocation of the peptides into intact cerebral arteries was demonstrated by using fluorescein-labeled DT-2 and DT-3. The inhibitory potency of the fusion peptides was even greater than that...

Proceedings of the National Academy of Sciences, 2008

Calcium (Ca 2+ ) release through inositol 1,4,5-trisphosphate receptors (IP 3 Rs) regulates the f... more Calcium (Ca 2+ ) release through inositol 1,4,5-trisphosphate receptors (IP 3 Rs) regulates the function of virtually every mammalian cell. Unlike ryanodine receptors, which generate local Ca 2+ events (“sparks”) that transmit signals to the juxtaposed cell membrane, a similar functional architecture has not been reported for IP 3 Rs. Here, we have identified spatially fixed, local Ca 2+ release events (“pulsars”) in vascular endothelial membrane domains that project through the internal elastic lamina to adjacent smooth muscle membranes. Ca 2+ pulsars are mediated by IP 3 Rs in the endothelial endoplasmic reticulum of these membrane projections. Elevation of IP 3 by the endothelium-dependent vasodilator, acetylcholine, increased the frequency of Ca 2+ pulsars, whereas blunting IP 3 production, blocking IP 3 Rs, or depleting endoplasmic reticulum Ca 2+ inhibited these events. The elementary properties of Ca 2+ pulsars were distinct from ryanodine-receptor-mediated Ca 2+ sparks in sm...

Circulation Research, 2003

The endothelium is a critical regulator of vascular tone, and dysfunction of the endothelium cont... more The endothelium is a critical regulator of vascular tone, and dysfunction of the endothelium contributes to numerous cardiovascular pathologies. Recent studies suggest that apamin-sensitive, small-conductance, Ca 2+ -activated K + channels may play an important role in active endothelium-dependent vasodilations, and expression of these channels may be altered in disease states characterized by vascular dysfunction. Here, we used a transgenic mouse (SK3 T/T ) in which SK3 expression levels can be manipulated with dietary doxycycline (DOX) to test the hypothesis that the level of expression of the SK subunit, SK3, in endothelial cells alters arterial function and blood pressure. SK3 protein was elevated in small mesenteric arteries from SK3 T/T mice compared with wild-type mice and was greatly suppressed by dietary DOX. SK3 was detected in the endothelium and not in the smooth muscle by immunohistochemistry. In whole-cell patch-clamp experiments, SK currents in endothelial cells from ...

Arteriosclerosis, Thrombosis, and Vascular Biology, 2013

Objective— Intermediate and small conductance K Ca channels IK1 (K Ca 3.1) and SK3 (K Ca 2.3) are... more Objective— Intermediate and small conductance K Ca channels IK1 (K Ca 3.1) and SK3 (K Ca 2.3) are primary targets of endothelial Ca 2+ signals in the arterial vasculature, and their ablation results in increased arterial tone and hypertension. Activation of IK1 channels by local Ca 2+ transients from internal stores or plasma membrane channels promotes arterial hyperpolarization and vasodilation. Here, we assess arteries from genetically altered IK1 knockout mice (IK1 −/− ) to determine whether IK1 channels exert a positive feedback influence on endothelial Ca 2+ dynamics. Approach and Results— Using confocal imaging and custom data analysis software, we found that although the occurrence of basal endothelial Ca 2+ dynamics was not different between IK1 −/− and wild-type mice ( P >0.05), the frequency of acetylcholine-stimulated (2 μmol/L) Ca 2+ dynamics was greatly decreased in IK1 −/− endothelium (515±153 versus 1860±319 events; P <0.01). In IK1 −/− /SK3 T/T mice, ancillary ...

American Journal of Physiology-Cell Physiology, 2012

Ca2+ signals are commonly measured using fluorescent Ca2+ indicators and microscopy techniques, b... more Ca2+ signals are commonly measured using fluorescent Ca2+ indicators and microscopy techniques, but manual analysis of Ca2+ measurements is time consuming and subject to bias. Automated region of interest (ROI) detection algorithms have been employed for identification of Ca2+ signals in one-dimensional line scan images, but currently there is no process to integrate acquisition and analysis of ROIs within two-dimensional time lapse image sequences. Therefore we devised a novel algorithm for rapid ROI identification and measurement based on the analysis of best-fit ellipses assigned to signals within noise-filtered image sequences. This algorithm was implemented as a plugin for ImageJ software (National Institutes of Health, Bethesda, MD). We evaluated the ability of our algorithm to detect synthetic Gaussian signal pulses embedded in background noise. The algorithm placed ROIs very near to the center of a range of signal pulses, resulting in mean signal amplitude measurements of 99...

American Journal of Physiology-Cell Physiology, 2012

When arteries constrict to agonists, the endothelium inversely responds, attenuating the initial ... more When arteries constrict to agonists, the endothelium inversely responds, attenuating the initial vasomotor response. The basis of this feedback mechanism remains uncertain, although past studies suggest a key role for myoendothelial communication in the signaling process. The present study examined whether second messenger flux through myoendothelial gap junctions initiates a negative-feedback response in hamster retractor muscle feed arteries. We specifically hypothesized that when agonists elicit depolarization and a rise in second messenger concentration, inositol trisphosphate (IP3) flux activates a discrete pool of IP3 receptors (IP3Rs), elicits localized endothelial Ca2+ transients, and activates downstream effectors to moderate constriction. With use of integrated experimental techniques, this study provided three sets of supporting observations. Beginning at the functional level, we showed that blocking intermediate-conductance Ca2+-activated K+ channels (IK) and Ca2+ mobili...

The American Journal of Pathology, 2013

Microcirculation, 2012

The endothelium is vital to normal vasoregulation. Although acute vasodilation associated with br... more The endothelium is vital to normal vasoregulation. Although acute vasodilation associated with broad endothelial Ca 2+ elevation is well-known, the control and targeting of Ca 2+ dependent signals in the endothelium is poorly understood. Recent studies have revealed localized IP 3motivated Ca 2+ events occurring basally along the intima that may provide the fundamental basis for various endothelial influences. Here, we provide an overview of dynamic endothelial Ca 2+ signals and discuss the potential role of these signals in constant endothelial control of arterial tone and the titration of functional responses in vivo. In particular, we focus on the functional architecture contributing to the properties and ultimate impact of these signals and explore new avenues in evaluating their prevalence and specific modalities in intact tissue. Finally, we discuss spatial and temporal effector recruitment through modification of these inherent signals. It is suggested that endothelial Ca 2+ signaling is a continuum in which the specific framework of storerelease components and cellular targets along the endothelium allows for differential modes of Ca 2+ signal expansion and distinctive profiles of effector recruitment. The precise composition and distribution of these inherent components may underlie dynamic endothelial control and specialized functions of different vascular beds.

American Journal of Physiology-Cell Physiology, 2007

Mechanisms regulating uterine contractility are poorly understood. We hypothesized that a specifi... more Mechanisms regulating uterine contractility are poorly understood. We hypothesized that a specific isoform of small conductance Ca2+-activated K+ (SK) channel, SK3, promotes feedback regulation of myometrial Ca2+ and hence relaxation of the uterus. To determine the specific functional impact of SK3 channels, we assessed isometric contractions of uterine strips from genetically altered mice (SK3T/T), in which SK3 is overexpressed and can be suppressed by oral administration of doxycycline (SK3T/T+Dox). We found SK3 protein in mouse myometrium, and this expression was substantially higher in SK3T/T mice and lower in SK3T/T+Dox mice compared with wild-type (WT) controls. Sustained contractions elicited by 60 mM KCl were not different among SK3T/T, SK3T/T+Dox, and WT mice. However, the rate of onset and magnitude of spontaneously occurring phasic contractions was muted significantly in isolated uterine strips from SK3T/T mice compared with those from WT mice. These spontaneous contracti...

AJP: Cell Physiology, 2011

Organized uterine contractions, including those necessary for parturition, are dependent on calci... more Organized uterine contractions, including those necessary for parturition, are dependent on calcium entry through voltage-gated calcium channels in myometrial smooth muscle cells. Recent evidence suggests that small-conductance Ca2+-activated potassium channels (KCa2), specifically isoforms KCa2.2 and 2.3, may control these contractions through negative feedback regulation of Ca2+ entry. We tested whether selective pharmacologic activation of KCa2.2/2.3 channels might depress uterine contractions, providing a new strategy for preterm labor intervention. Western blot analysis and immunofluorescence microscopy revealed expression of both KCa2.2 and KCa2.3 in the myometrium of nonpregnant (NP) and pregnant (gestation day 10 and 16; D10 and D16, respectively) mice. Spontaneous phasic contractions of isolated NP, D10, and D16 uterine strips were all suppressed by the KCa2.2/2.3-selective activator CyPPA in a concentration-dependent manner. This effect was antagonized by the selective KCa...