In order to determine the effect of calcium mobilization on mitogen-activated protein (MAP) kinase activation, we have treated human foreskin fibroblasts (HSWP cells) and epidermal carcinoma (A431) cells with thapsigargin. Intracellular free was monitored by single cell image analysis using fura-2 correlated MAP stimulation as assessed immunoprecipitation, renaturation assays immunoblotting. Thapsigargin stimulated 44- 42-kDa isozymes in both types kinetics that were slightly delayed...

Pressor effects of the vasoconstrictor hormone arginine vasopressin (AVP), observed when systemic AVP concentrations are less than 100 pM, important for physiological maintenance blood pressure, and they also basis therapeutic use to restore pressure in hypotensive patients. However, mechanisms by which circulating induces arterial constriction unclear. We examined novel hypothesis that KCNQ potassium channels mediate actions AVP. Reverse transcriptase polymerase chain reaction revealed...

Abstract —Reperfusion of cardiac tissue after an ischemic episode is associated with metabolic and contractile dysfunction, including reduced tension development activation the Na + -H exchanger (NHE). Oxygen-derived free radicals are key mediators reperfusion abnormalities, although cellular mechanisms involved have not been fully defined. In present study, effects on mitogen-activated protein (MAP) kinase function were investigated using cultured neonatal rat ventricular myocytes. Acute...

Abstract —Angiotensin II (Ang II) induces vascular smooth muscle cell (VSMC) growth by activating G q -protein–coupled AT 1 receptors, which leads to elevation of cytosolic Ca 2+ ([Ca ] i ) and activation protein kinase C (PKC) mitogen-activated kinases. To assess the link between these Ang II–induced signaling events, we examined effect on proline-rich tyrosine (PYK2), previously found be activated a variety stimuli that increase [Ca or activate PKC. PYK2 distribution was demonstrated in...

The ATP-binding cassette family of transporter proteins, subfamily B (MDR/TAP), member 1 (ABCB1) (P-glycoprotein) is a key component the blood-brain barrier. Many antidepressants are subject to ABCB1 efflux. Functional polymorphisms may influence central nervous system bioavailability Single-nucleotide (SNPs) at rs1045642 (C3435T) have been associated with efflux pump efficiency. This explain part interindividual variation in antidepressant dose needed remit. Individuals (N=113) DSM-IV...

Endothelin-1 (ET) produces neonatal rat ventricular myocyte (NRVM) hypertrophy and activates focal adhesion kinase (FAK) in other cell types. In the present study, we examined whether ET activated FAK NRVM was necessary and/or sufficient for ET-induced hypertrophy. Chronic ET-1 stimulation (100 nM, 48 h) increased protein-to-DNA myosin heavy chain (MHC)-to-DNA ratios stimulated assembly of newly synthesized MHC into sarcomeres. also induced adhesions costameres, as evidenced by...

Capacitative Ca²⁺ entry (CCE) in vascular smooth muscle cells contributes to vasoconstrictor and mitogenic effects of vasoactive hormones. In A7r5 rat aortic cells, measurements cytosolic free concentration ([Ca²⁺]_i) have demonstrated that depletion intracellular stores activates CCE. However, there is disagreement published studies regarding the regulation this mechanism by hormone [Arg⁸]-vasopressin (AVP). We employed electrophysiological methods...

Celecoxib, rofecoxib, and diclofenac are clinically used cyclooxygenase-2 (COX-2) inhibitors, which have been under intense scrutiny because long-term rofecoxib (Vioxx; Merck, Whitehouse Station, NJ) treatment was found to increase the risk of adverse cardiovascular events. A differential profile for these drugs has emerged, but underlying mechanisms not fully elucidated. We investigated effects celecoxib, on ionic currents calcium signaling in vascular smooth muscle cells (VSMCs) using...

Current scientific literature generally attributes the vasoconstrictor effects of [Arg(8)]vasopressin (AVP) to activation phospholipase C (PLC) and consequent release Ca(2+) from sarcoplasmic reticulum. However, half-maximal PLC requires nanomolar concentrations AVP, whereas vasoconstriction occurs when circulating AVP are orders magnitude lower. Using cultured vascular smooth muscle cells, we previously identified a novel signaling pathway activated by 10-100 pM AVP. This is distinguished...

The Kv7 family (Kv7.1–7.5) of voltage-activated potassium channels contributes to the maintenance resting membrane potential in excitable cells. Previously, we provided pharmacological and electrophysiological evidence that Kv7.4 Kv7.5 form predominantly heteromeric activity is regulated by protein kinase C (PKC) response vasoconstrictors vascular smooth muscle Direct for Kv7.4/7.5 heteromer formation, however, lacking. Furthermore, it remains be determined whether both subunits are PKC....

Significance α 1 -Adrenergic receptors are important for the regulation of vascular function and targeted clinically blood pressure control. Here, we provide evidence that 1A/B -adrenergic (AR) form heteromeric complexes with chemokine (C-X-C motif) receptor 4 (CXCR4) on cell surface smooth muscle cells. We show disruption -AR:CXCR4 inhibits -AR–mediated functions in cells treatment CXCR4 agonists enhances potency -AR agonist phenylephrine to increase pressure. These findings extend current...

Kv7 (KCNQ) channels, formed as homo- or heterotetramers of Kv7.4 and Kv7.5 <i>α</i>-subunits, are important regulators vascular smooth muscle cell (VSMC) membrane voltage. Recent studies demonstrate that direct pharmacological modulation VSMC channel activity can influence blood vessel contractility diameter. However, the physiologic regulation is still poorly understood. Here, we study effect cAMP/protein kinase A (PKA) activation on whole K⁺ currents through endogenous channels...

Generation of reactive oxygen species (ROS) and intracellular Ca 2+ overload are key mechanisms involved in ischemia-reperfusion (I/R)-induced myocardial injury. The relationship between I/R injury has not been fully characterized. increase Na + /H exchanger (NHE-1) activity observed during is an attractive candidate to link increased ROS production with overload. We have shown that low doses H 2 O NHE-1 extracellular signal-regulated kinase (ERK)-dependent manner. In this study, we examined...

Lys-Bradykinin (BK), a mitogen for human foreskin fibroblasts (HSWP cells) (Owen, N. E., and Villereal, M. L. (1983) Cell 32, 979-985), elicits rapid, transient elevation of intracellular free Ca2+ concentration ([Ca2+]i) in these cells. We have used image analysis fura-2-loaded HSWP cells to examine the BK-induced [Ca2+]i changes individual BK-stimulated entry release stores can be distinguished by stimulating presence or absence extracellular Ca2+, inhibiting with 5 mM NiCl2. BK-sensitive...

Butyrate may have paradoxical effects on epithelial cells of similar origin. This study aimed to examine the hypothesis that one mechanism dictates a cell's response butyrate is its state activation. First, responses 24 h exposure (1–2 mM) normal and neoplastic human colonic activated by their isolation primary culture, colon cancer cell lines, LIM1215 Caco-2, were examined. In cultures cells, had no effect alkaline phosphatase activities but significantly suppressed urokinase receptor...

[Arg(8)]-vasopressin (AVP), at low concentrations (10-500 pM), stimulates oscillations in intracellular Ca(2+) concentration (Ca(2+) spikes) A7r5 rat aortic smooth muscle cells. Our previous studies provided biochemical evidence that protein kinase C (PKC) activation and phosphorylation of voltage-sensitive K(+) (K(v)) channels are crucial steps this process. In the present study, K(v) currents (I(Kv)) membrane potential were measured using patch clamp techniques. Treatment cells with 100 pM...

1. Arg8‐vasopressin (AVP)‐regulated Ca2+ transport were investigated in fura‐2‐loaded A7r5 cells using both single cell and population measurements. 2. AVP evokes an initial concentration‐dependent rise cytosolic free concentration ([Ca2+ ]i) to a peak which is independent of extracellular Ca2+, sustained signal that results from balance between stimulation entry efflux. 3. Depletion intracellular stores with thapsigargin, ionomycin, or prior treatment Ca2(+)‐free medium activates...

Abstract The mechanisms responsible for altered vascular smooth muscle cell (VSMC) function in hypertension remain unknown. In the spontaneously hypertensive rat (SHR) model of genetic hypertension, there are multiple abnormalities VSMC function, including increased growth, Na + -H exchange, and signal transduction by protein kinase C. family kinases termed mitogen-activated (MAP) has recently been shown to be essential mediators growth factor transduction. present study, alterations MAP...

Abstract Temporal changes in intracellular free Ca2+ concentration ([Ca2+]i) cultured human foreskin fibroblasts were investigated using image analysis techniques to simultaneously monitor Lys-bradykinin (BK)- or thrombin-induced elevations of [Ca2+]i each individual cell within a microscopic field. Responses BK are heterogeneous with respect the shapes time courses. Furthermore, onsets these responses follow variable lag period such that occur asynchronously. The asynchrony and...

KCNQ4 and KCNQ5 potassium channel subunits are expressed in vascular smooth muscle cells, although it remains uncertain how these assemble to form functional channels. Using patch-clamp techniques, we compared the electrophysiological characteristics effects of diclofenac, a known KCNQ activator, on human channels individually or together A7r5 rat aortic cells. The conductance curves overexpressed were fitted by single Boltzmann function each case (<i>V</i>_0.5 values: −31, −44,...

Expression and function of Kv7 (KCNQ) voltage-activated potassium channels in guinea pig human airway smooth muscle cells (ASMCs) were investigated by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), patch-clamp electrophysiology, precision-cut lung slices. qRT-PCR revealed expression multiple KCNQ genes both ASMCs. Currents with electrophysiological pharmacological characteristics currents measured freshly isolated In ASMCs, significantly suppressed application the...

Recent research suggests that smooth muscle cells express Kv7.4 and Kv7.5 voltage-activated potassium channels, which contribute to maintenance of their resting membrane voltage. New pharmacologic activators Kv7 ML213 (<i>N</i>-mesitybicyclo[2.2.1]heptane-2-carboxamide) ICA-069673 <i>N</i>-(6-chloropyridin-3-yl)-3,4-difluorobenzamide), have been reported discriminate among channels formed from different subtypes. We compared the effects on homomeric human Kv7.4, Kv7.5, heteromeric Kv7.4/7.5...

Abstract Monolayers of fura-2-loaded A7r5 cells, a cell line derived from rat embryonic aorta, generated spontaneous Ca2+ spikes that were synchronized within the population. These abolished by removal extracellular or addition nimodipine (50 nM), and their frequency was increased depolarization with high K+ treatment BAYK 8644 (1 microM), indicating entry through L-type channels is required for spiking. Several lines evidence indicate mobilization intracellular stores not necessary this 1)...