A7r5 Rat Aortic Smooth Muscle Research Articles

Molecular Composition of Functional KCNQ Channels in Vascular Smooth Muscle Cells Based on Effects of Diclofenac

KCNQ4 and KCNQ5 potassium channel subunits are expressed in vascular smooth muscle cells, though it remains uncertain how these subunits assemble to form functional channels. Using patch-clamp techniques, we compared the electrophysiological characteristics and effects of diclofenac, a known KCNQ channel activator, on human KCNQ4 and KCNQ5 channels expressed individually or together in A7r5 rat aortic smooth muscle cells. The conductance curves of the overexpressed channels were fit by a single Boltzmann function in each case (V0.5 values: −31 mV, −44 mV, and −38 mV for KCNQ4, KCNQ5, and KCNQ4/5, respectively). Diclofenac (100 μM) increased maximum conductance of KCNQ4 channels by 38%, but inhibited KCNQ5 and KCNQ4/5 channels, reducing maximum conductance by 53% and by 32% respectively. Differences in deactivation rates and distinct voltage-dependent effects of diclofenac on channel activation and deactivation observed with each of the subunit combinations (KCNQ4, KCNQ5 and KCNQ4/5) were used as diagnostic tools to evaluate native KCNQ currents in vascular smooth muscle cells. A7r5 cells express only KCNQ5 channels endogenously and their responses to diclofenac closely resembled those of the overexpressed KCNQ5 currents. In contrast, mesenteric artery myocytes, which express both KCNQ4 and KCNQ5 channels, displayed whole-cell KCNQ currents with properties and diclofenac responses characteristic of overexpressed heteromeric KCNQ4/5 channels.

Glucocorticoids Differentially Regulate the Expression of CRFR1 and CRFR2α in MIN6 Insulinoma Cells and Rodent Islets

Urocortin 3 (Ucn 3), member of the corticotropin-releasing factor (CRF) family of peptide hormones, is released from β-cells to potentiate insulin secretion. Ucn 3 activates the CRF type-2 receptor (CRFR2) but does not activate the type-1 receptor (CRFR1), which was recently demonstrated on β-cells. While the direct actions of Ucn 3 on insulin secretion suggest the presence of cognate receptors within the islet microenvironment, this has not been established. Here we demonstrate that CRFR2α is expressed by MIN6 insulinoma cells and by primary mouse and human islets, with no detectable expression of CRFR2β. Furthermore, stimulation of MIN6 cells or primary mouse islets in vitro or in vivo with glucocorticoids (GCs) robustly and dose-dependently increases the expression of CRFR2α, while simultaneously inhibiting the expression of CRFR1 and incretin receptors. Luciferase reporters driven by the mouse CRFR1 or CRFR2α promoter in MIN6 cells confirm these differential effects of GCs. In contrast, GCs inhibit CRFR2α promoter activity in HEK293 cells and inhibit the expression of CRFR2β in A7r5 rat aortic smooth muscle cells and differentiated C2C12 myotubes. These findings suggest that the GC-mediated increase of CRFR2α depends on the cellular context of the islet and deviates from the GC-mediated suppression of CRFR1 and incretin receptors. Furthermore, GC-induced increases in CRFR2α expression coincide with increased Ucn 3-dependent activation of cAMP and MAPK pathways. We postulate that differential effect of GCs on the expression of CRFR1 and CRFR2α in the endocrine pancreas represent a mechanism to shift sensitivity from CRFR1 to CRFR2 ligands.

Diclofenac Distinguishes among Homomeric and Heteromeric Potassium Channels Composed of KCNQ4 and KCNQ5 Subunits

KCNQ4 and KCNQ5 potassium channel subunits are expressed in vascular smooth muscle cells, although it remains uncertain how these subunits assemble to form functional channels. Using patch-clamp techniques, we compared the electrophysiological characteristics and effects of diclofenac, a known KCNQ channel activator, on human KCNQ4 and KCNQ5 channels expressed individually or together in A7r5 rat aortic smooth muscle cells. The conductance curves of the overexpressed channels were fitted by a single Boltzmann function in each case (V(0.5) values: -31, -44, and -38 mV for KCNQ4, KCNQ5, and KCNQ4/5, respectively). Diclofenac (100 μM) inhibited KCNQ5 channels, reducing maximum conductance by 53%, but increased maximum conductance of KCNQ4 channels by 38%. The opposite effects of diclofenac on KCNQ4 and KCNQ5 could not be attributed to the presence of a basic residue (lysine) in the voltage-sensing domain of KCNQ5, because mutation of this residue to neutral glycine (the residue present in KCNQ4) resulted in a more effective block of the channel. Differences in deactivation rates and distinct voltage-dependent effects of diclofenac on channel activation and deactivation observed with each of the subunit combinations (KCNQ4, KCNQ5, and KCNQ4/5) were used as diagnostic tools to evaluate native KCNQ currents in vascular smooth muscle cells. A7r5 cells express only KCNQ5 channels endogenously, and their responses to diclofenac closely resembled those of the overexpressed KCNQ5 currents. In contrast, mesenteric artery myocytes, which express both KCNQ4 and KCNQ5 channels, displayed whole-cell KCNQ currents with properties and diclofenac responses characteristic of overexpressed heteromeric KCNQ4/5 channels.

Diclofenac Activates Kv7.4 and Inhibits Kv7.5 Potassium Channels Heterologously Expressed in A7r5 Vascular Smooth Muscle Cells

Members of the KCNQ (Kv7) voltage-gated potassium channel gene family are differentially expressed through the body. Five Kv7 subtypes play major roles in regulation of membrane potential and cell excitability within different tissues. Well known functions of Kv7.1 in cardiac action potential duration, Kv7.2/7.3 in neuronal excitability, Kv7.4 in hearing and an emerging role of Kv7.5 in vascular tone, increases the demand for channel modulating drugs that exhibit selectivity among Kv7 subtypes. Diclofenac, an anti-inflammatory drug, was found to be a novel Kv7.2/7.3 channel opener and was used as a template to synthesize new activators and inhibitors of neuronal KCNQ channels (Kv7.2/7.3 and Kv7.4). We compare affects of diclofenac on human Kv7.4 and Kv7.5 currents using A7r5 rat aortic smooth muscle cells as an expression system and perforated patch-clamp techniques. Diclofenac, at 100∈1/4M, increased maximal conductance of Kv7.4 channels by 1.5-fold and induced a negative shift in the activation curve (by 9mV). Surprisingly, the same concentration of diclofenac (100∈1/4M) reduced maximal conductance of Kv7.5 channels by 2-fold, but also induced a robust negative shift in the activation curve (by 30mV). For the both Kv7.4 and Kv7.5, application of diclofenac (100∈1/4M) reduced the deactivation rate of the current. For Kv7.4, the deactivation rate was 1.7-fold slower in the presence of diclofenac, independent of the voltage in the range from −120mV to −90mV. In contrast, for Kv7.5 the reduced deactivation rate in the presence of diclofenac was voltage-dependent, changing linearly from 2-fold at −120mV to 4-fold at −90mV. These differences in diclofenac action on two members of the Kv7 channel family may reflect structural differences between Kv7.4 and Kv7.5 and make diclofenac a useful tool to distinguish between Kv7.4 and Kv7.5 currents in native tissues.

Inhibitory Effect on Thrombin-induced Myosin Light Chain Phosphorylation by Extract of a New Green Tea Cultivar in A7r5 Rat Aortic Smooth Muscle Cells

We show that an extract of a new green tea cultivar Cha Chuukanbohon Nou6 (Nou6), which rich in anthocyanin, has an inhibitory effect on myosin regulatory light chain (MRLC) phosphorylation in rat aortic smooth muscle cell line A7r5. The inhibitory effect of Nou6 extract was abolished when the extract was treated with polyvinylpyrrolidone which traps polyphenols.However, none of the major tea constituents tested had an inhibitory effect.In addition, we found that Nou6 extract had a strong effect on reducing the phosphorylation of myosin phosphatase targeting subunit 1 at Thr696. These results suggest that Nou6 extract inhibits MRLC phosphorylation induced by thrombin through activating myosin light chain phosphatase, and polyphenols in the tea extract may contribute to this effect.

Differential Effects of Selective Cyclooxygenase-2 Inhibitors on Vascular Smooth Muscle Ion Channels May Account for Differences in Cardiovascular Risk Profiles

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 risk profile for these drugs has emerged, but the underlying mechanisms have not been fully elucidated. We investigated the effects of celecoxib, rofecoxib, and diclofenac on ionic currents and calcium signaling in vascular smooth muscle cells (VSMCs) using patch-clamp techniques and fura-2 fluorescence and on arterial constriction using pressure myography. Celecoxib, but not rofecoxib or diclofenac, dramatically enhanced KCNQ (K(v)7) potassium currents and suppressed L-type voltage-sensitive calcium currents in A7r5 rat aortic smooth muscle cells (native KCNQ currents or overexpressed human KCNQ5 currents) and freshly isolated rat mesenteric artery myocytes. The effects of celecoxib were concentration-dependent within the therapeutic concentration range, and were reversed on washout. Celecoxib, but not rofecoxib, also inhibited calcium responses to vasopressin in A7r5 cells and dilated intact or endothelium-denuded rat mesenteric arteries. A celecoxib analog, 2,5-dimethyl-celecoxib, which does not inhibit COX-2, mimicked celecoxib in its enhancement of vascular KCNQ5 currents, suppression of L-type calcium currents, and vasodilation. We conclude that celecoxib inhibits calcium responses in VSMCs by enhancing KCNQ5 currents and suppressing L-type calcium currents, which ultimately reduces vascular tone. These effects are independent of its COX-2 inhibitory actions and may explain the differential risk of cardiovascular events in patients taking different drugs of this class.

FRET analysis of actin–myosin interaction in contracting rat aortic smooth muscle

We examined the interaction of smooth muscle myosin with alpha-actin and beta-actin isoforms during the contraction of A7r5 smooth muscle cells and rat aortic smooth muscle. The techniques of confocal microscopy and fluorescence resonance energy transfer (FRET) analysis were utilized in examining A7r5 cells and rat aortic rings contracted with phorbol 12,13-dibutyrate. Visual evaluation of confocal images of A7r5 smooth muscle cells contracted by phorbol 12,13-dibutyrate indicated significant disassociation of myosin from alpha-actin but not beta-actin. Whole-cell FRET analysis confirmed these observations (alpha-actin-myosin -67%, beta-actin-myosin -2%). Time course studies further showed that alpha-actin-myosin complex increased significantly (40%) within 1.5 min after the addition of phorbol 12,13-dibutyrate and then declined as contraction progressed. FRET analysis of rat aortic rings at different intervals of contraction indicated significant increases in alpha-actin-myosin at the initiation (79%) and plateau (67%) in force development, but not during the intermediate period of slowly developing tension (-4%). By comparison, beta-actin-myosin complex was unchanged except during slow force development, in which the association was significantly decreased (-30%). Similar to that of alpha-actin-myosin, Alexa 488 - phalloidin staining fluorescence indicated increased tissue F-actin content at the initiation (21%) and plateau (62%) in force. FRET images indicated the development of thickened cables and patches of alpha-actin-myosin in tissue throughout the interval of contraction. The results provide direct evidence of dynamic remodeling of the contractile protein during vascular smooth muscle contraction and suggest that FRET analysis may be a powerful tool for assessment of tissue protein-protein associations.

Opposite regulation of KCNQ5 and TRPC6 channels contributes to vasopressin-stimulated calcium spiking responses in A7r5 vascular smooth muscle cells

Physiologically relevant concentrations of [Arg 8]-vasopressin (AVP) induce repetitive action potential firing and Ca 2+ spiking responses in the A7r5 rat aortic smooth muscle cell line. These responses may be triggered by suppression of KCNQ potassium currents and/or activation of non-selective cation currents. Here we examine the relative contributions of KCNQ5 channels and TRPC6 non-selective cation channels to AVP-stimulated Ca 2+ spiking using patch clamp electrophysiology and fura-2 fluorescence measurements in A7r5 cells. KCNQ5 or TRPC6 channel expression levels were suppressed by short hairpin RNA constructs. KCNQ5 knockdown resulted in more positive resting membrane potentials and induced spontaneous action potential firing and Ca 2+ spiking. However physiological concentrations of AVP induced additional depolarization and increased Ca 2+ spike frequency in KCNQ5 knockdown cells. AVP activated a non-selective cation current that was reduced by TRPC shRNA treatment or removal of external Na +. Neither resting membrane potential nor the AVP-induced depolarization was altered by knockdown of TRPC6 channel expression. However, both TRPC6 shRNA and removal of external Na + delayed the onset of Ca 2+ spiking induced by 25 pM AVP. These results suggest that suppression of KCNQ5 currents alone is sufficient to excite A7r5 cells, but AVP-induced activation of TRPC6 contributes to the stimulation of Ca 2+ spiking.

Cycloxygenase-2 Inhibitor Celecoxib Is A Potent Activator Of Vascular KCNQ K+ Channels And An Inhibitor Of L-type Ca2+ Channels

Cyclooxygenase-2 (COX-2) inhibitors are important members of the family of non-steroidal anti-inflammatory drugs (NSAIDs). Celebrex® (celecoxib) and Vioxx® (rofecoxib) were introduced in 1999 and rapidly became the most frequently prescribed new drugs in the United States. Vioxx® was voluntarily withdrawn from the market because of a reported increased risk of myocardial infarction and stroke in patients taking the drug for prolonged periods of time. Celecoxib has been reported to inhibit several classes of ion channels, but its effects on vascular smooth muscle ion channels have not been described. Using whole-cell perforated patch clamp techniques we examined effects of celecoxib on K+ and Ca2+ currents in A7r5 rat aortic smooth muscle cells. Application of 10μM celecoxib enhanced K+ current by 2-3 fold and substantially inhibited Ca2+ currents with an apparent positive shift in the voltage-dependence of activation. Both effects were reversible on washout. Nether rofecoxib (10μM), another selective COX-2 inhibitor, nor diclofenac (10μM), a nonselective COX inhibitor, affected Ca2+ or K+ currents in A7r5 cells. We previously reported that KCNQ5 channels are the predominant K+ channels determining outward potassium current at negative membrane potentials in A7r5 cells. We estimated cumulative dose-response curve of celecoxib on isolated KCNQ5 currents. Celecoxib enhances KCNQ5 current in 3.5 fold with an EC50 of 6.9 ± 1.5 μM, without shifting the activation curve. Celecoxib (10μM) was unable to restore KCNQ current inhibited by 100 pM vasopressin (AVP, vasoconstrictor hormone) or 1nM PMA (PKC activator) but inhibition of L-type Ca2+ currents (with a positive shift of activation) persisted. The effects of celecoxib, but not rofecoxib, on vascular Ca2+ and K+ channels may explain the differential risks of cardiovascular diseases in patients treated with Celebrex® or Vioxx®.

Vasopressin stimulates action potential firing by protein kinase C-dependent inhibition of KCNQ5 in A7r5 rat aortic smooth muscle cells

[Arg(8)]-vasopressin (AVP), at low concentrations (10-500 pM), stimulates oscillations in intracellular Ca(2+) concentration (Ca(2+) spikes) in 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 in this process. In the present study, K(v) currents (I(Kv)) and membrane potential were measured using patch clamp techniques. Treatment of A7r5 cells with 100 pM AVP resulted in significant inhibition of I(Kv). This effect was associated with gradual membrane depolarization, increased membrane resistance, and action potential (AP) generation in the same cells. The AVP-sensitive I(Kv) was resistant to 4-aminopyridine, iberiotoxin, and glibenclamide but was fully inhibited by the selective KCNQ channel blockers linopirdine (10 microM) and XE-991 (10 microM) and enhanced by the KCNQ channel activator flupirtine (10 microM). BaCl(2) (100 microM) or linopirdine (5 microM) mimicked the effects of AVP on K(+) currents, AP generation, and Ca(2+) spiking. Expression of KCNQ5 was detected by RT-PCR in A7r5 cells and freshly isolated rat aortic smooth muscle. RNA interference directed toward KCNQ5 reduced KCNQ5 protein expression and resulted in a significant decrease in I(Kv) in A7r5 cells. I(Kv) was also inhibited in response to the PKC activator 4beta-phorbol 12-myristate 13-acetate (10 nM), and the inhibition of I(Kv) by AVP was prevented by the PKC inhibitor calphostin C (250 nM). These results suggest that the stimulation of Ca(2+) spiking by physiological concentrations of AVP involves PKC-dependent inhibition of KCNQ5 channels and increased AP firing in A7r5 cells.

Pharmacological and Electrophysiological Characterization of Store-Operated Currents and Capacitative Ca2+Entry in Vascular Smooth Muscle Cells

Capacitative Ca(2+) entry (CCE) in vascular smooth muscle cells contributes to vasoconstrictor and mitogenic effects of vasoactive hormones. In A7r5 rat aortic smooth muscle cells, measurements of cytosolic free Ca(2+) concentration ([Ca(2+)](i)) have demonstrated that depletion of intracellular Ca(2+) stores activates CCE. However, there is disagreement in published studies regarding the regulation of this mechanism by the vasoconstrictor hormone [Arg(8)]-vasopressin (AVP). We have employed electrophysiological methods to characterize the membrane currents activated by store depletion [store-operated current (I(SOC))]. Because of different recording conditions, it has not been previously determined whether I(SOC) corresponds to CCE measured using fura-2; nor has the channel protein responsible for CCE been identified. In the present study, the pharmacological characteristics of I(SOC), including its sensitivity to blockade by 2-aminoethoxydiphenylborane, diethylstilbestrol, or micromolar Gd(3+), were found to parallel the effects of these drugs on thapsigargin- or AVP-activated CCE measured under identical external ionic conditions using fura-2. Thapsigargin-stimulated I(SOC) was also measured in freshly isolated rat mesenteric artery smooth muscle cells (MASMC). Members of the transient receptor potential (TRP) family of nonselective cation channels, TRPC1, TRPC4, and TRPC6, were detected by reverse transcription-polymerase chain reaction and Western blot in both A7r5 cells and MASMC. TRPC1 expression was reduced in a stable A7r5 cell line expressing a small interfering RNA (siRNA) or by infection of A7r5 cells with an adenovirus expressing a TRPC1 antisense nucleotide sequence. Thapsigargin-stimulated I(SOC) was reduced in both the TRPC1 siRNA- and TRPC1 antisense-expressing cells, suggesting that the TRPC1 channel contributes to the I(SOC)/CCE pathway.

Evidence against reciprocal regulation of Ca2+ entry by vasopressin in A7r5 rat aortic smooth-muscle cells

Recent studies by Moneer and Taylor [(2002) Biochem. J. 362, 13-21] have proposed a reciprocal regulation of two Ca2+-entry pathways by AVP ([Arg8]-vasopressin) in A7r5 vascular smooth-amuscle cells. Their model proposes that AVP inhibits CCE (capacitative Ca2+ entry) and predicts a rebound of CCE after the removal of AVP. In the present study, we used whole-cell perforated patch-clamp techniques to measure ISOC (store-operated current) corresponding to CCE in A7r5 cells. When 100 nM AVP is present, it activates ISOC with no apparent rebound on removal of AVP. ISOC activated by thapsigargin or cyclopiazonic acid was not inhibited by 100 nM AVP. We also used fura 2 fluorescence techniques to re-examine the model of Moneer and Taylor, specifically focusing on the proposed inhibition of CCE by AVP. We find that 100 nM AVP activates capacitative Mn2+ entry and does not inhibit thapsigargin- or cyclopiazonic acid-activated Mn2+ entry. Moreover, Ca2+ entry after depletion of intracellular Ca2+ stores is enhanced by AVP and we detect no rebound of Ca2+ or Mn2+ entry after AVP removal. On the basis of these findings, we conclude that AVP does not inhibit CCE in A7r5 cells.

Cytotoxic actions of palytoxin on aortic smooth muscle cells in culture

Palytoxin (PTX), isolated from a zoanthid of the genus Palythoa, is the most potent marine toxin known. Intoxication by PTX leads to vasoconstriction, hemorrhage, ataxia, muscle weakness, ventricular fibrillation, pulmonary hypertension, ischemia and death. In this study, clonal A7r5 rat aortic smooth muscle cells were used to study the mechanism of PTX-mediated cytotoxicity. A7r5 cells exposed to PTX for > or = 15 min exhibited surface granularities, vacuoles and rounding. These alterations culminated in a loss of viability as indicated by marked increases in the release of lactate dehydrogenase. Electrophysiological recording from A7r5 cells disclosed a profound membrane depolarization and an increase in conductance to Na+ and K+. PTX-mediated cytotoxicity could not be reversed by washout or by the addition of 10 microM verapamil but was antagonized by 100 microM ouabain or by removal of extracellular Na+ or Ca2+. In light of the involvement of vascular smooth muscle in PTX poisoning, A7r5 cells could serve as a useful model to test specific drugs for treatment of PTX intoxication.

Multiple Promoters Direct Expression of Three AKAP12 Isoforms with Distinct Subcellular and Tissue Distribution Profiles

A Kinase Anchoring Protein 12 (AKAP12; also known as src-suppressed C kinase substrate (SSeCKS) and Gravin) is a multivalent anchoring protein with tumor suppressor activity. Although expression of AKAP12 has been examined in a number of contexts, its expression control remains to be elucidated. Herein, we characterize the genomic organization of the AKAP12 locus, its regulatory regions, and the spatial distribution of the proteins encoded by the AKAP12 gene. Using comparative genomics and various wet-lab assays, we show that the AKAP12 locus is organized as three separate transcription units that are governed by non-redundant promoters coordinating distinct tissue expression profiles. The proteins encoded by the three AKAP12 isoforms (designated alpha, beta, and gamma) share >95% amino acid sequence identity but differ at their N termini. Analysis of the targeting of each isoform reveals distinct spatial distribution profiles. An N-terminal myristoylation motif present in AKAP12alpha is shown to be necessary and sufficient for targeted expression of this AKAP12 isoform to the endoplasmic reticulum, a novel subcellular compartment for AKAP12. Our results demonstrate heretofore unrecognized complexity within the AKAP12 locus and suggest a mechanism for genetic control of signaling specificity through distinct regulation of alternately targeted anchoring protein isoforms.

Vascular Smooth Muscle Cells on Polyelectrolyte Multilayers: Hydrophobicity-Directed Adhesion and Growth

Polyelectrolyte multilayer films were employed to support attachment of cultured rat aortic smooth muscle A7r5 cells. Like smooth muscle cells in vivo, cultured A7r5 cells are capable of converting between a nonmotile "contractile" phenotype and a motile "synthetic" phenotype. Polyelectrolyte films were designed to examine the effect of surface charge and hydrophobicity on cell adhesion, morphology, and motility. The hydrophobic nature and surface charge of different polyelectrolyte films significantly affected A7r5 cell attachment and spreading. In general, hydrophobic polyelectrolyte film surfaces, regardless of formal charge, were found to be more cytophilic than hydrophilic surfaces. On the most hydrophobic surfaces, the A7r5 cells adhered, spread, and exhibited little indication of motility, whereas on the most hydrophilic surfaces, the cells adhered poorly if at all and when present on the surface displayed characteristics of being highly motile. The two surfaces that minimized cell adhesion consisted of two varieties of a diblock copolymer containing hydrophilic poly(ethylene oxide) and a copolymer bearing a zwitterionic group AEDAPS, (3-[2-(acrylamido)-ethyldimethyl ammonio] propane sulfonate). Increasing the proportion of AEDAPS in the copolymer decreased the adhesion of cells to the surface. Cells presented with micropatterns of cytophilic and cytophobic surfaces generated by polymer-on-polymer stamping displayed a surface-dependent cytoskeletal organization and a dramatic preference for adhesion to, and spreading on, the cytophilic surface, demonstrating the utility of polyelectrolyte films in manipulating smooth muscle cell adhesion and behavior.

Protein Kinase Cδ Induces Src Kinase Activity via Activation of the Protein Tyrosine Phosphatase PTPα

Previously we have shown that protein kinase C (PKC)-mediated reorganization of the actin cytoskeleton in smooth muscle cells is transmitted by the non-receptor tyrosine kinase, Src. Several authors have described how 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulation of cells results in an increase of Src activity, but the mechanism of the PKC-mediated Src activation is unknown. Using PKC isozymes purified from Spodoptera frugiperda insect cells, we show here that PKC is not able to activate Src directly. Our data reveal that the PKC-dependent Src activation occurs via the activation of the protein tyrosine phosphatase (PTP) PTP alpha. PTP alpha becomes activated in vivo after TPA stimulation. Further, we show that PKC delta phosphorylates and activates only PTP alpha in vitro but not any other of the TPA-responsive PKC isozymes that are expressed in A7r5 rat aortic smooth muscle cells. To further substantiate our data, we show that cells lacking PKC delta have a markedly reduced PTP alpha and Src activity after 12-O-tetradecanoylphorbol-13-acetate stimulation. These data support a model in which the main mechanism of 12-O-tetradecanoylphorbol-13-acetate-induced Src activation is the direct phosphorylation and activation of PTP alpha by PKC delta, which in turn dephosphorylates and activates Src.

Purification of phenylethanoids from Brandisia hancei and the antiproliferative effects on aortic smooth muscle.

The present study describes the isolation and purification of acteoside, 2'-acetylacteoside, poliumoside and brandioside, four phenylethanoid glycosides from Brandisia hancei. We examined their effects on the proliferation of cultured A7r5 rat aortic smooth muscle cells. The proliferative response was measured from the [(3)H]-thymidine incorporation into DNA. All four glycosides suppressed the proliferative response in the presence of 2 % or 5 % fetal bovine serum in a concentration-dependent manner. The rank order of effectiveness for inhibition of cell proliferation was: brandioside > or = poliumoside > 2'-acetylacteoside > or = acteoside. The acetyl group at position 2' of glucose does not seem necessary for the anti-proliferative effects of acteoside and 2'-acetylacteoside, while the hydroxy groups of the aromatic rings appear to play a role. Inhibition of smooth muscle cell proliferation by phenylethanoids indicates that these compounds may have preventative effects on arteriosclerosis.

Dual effects of endothelin-1 on extracellular acidification by A7r5 smooth muscle cells.

The effect of endothelin-1 on metabolic activity of A7r5 rat aortic smooth muscle cells was studied. Endothelin-1 (pEC(50) 7.5) elicited an increase in the rate of extracellular medium acidification of the A7r5 cells. The ETA receptor antagonist BQ-123 blocked the endothelin-1 effect completely (pA(2) 7.6). Ca2+ channel blockers affected the endothelin-1 induced response in different ways: diltiazem and nifedipine partially blocked the endothelin-1 induced response, whereas verapamil did not influence this endothelin-1 induced effect. However, upon removal of verapamil an endothelin-1 dependent rise in extracellular acidification occurred, apparently reflecting the lifting of the verapamil blockade of an endothelin-1 induced process. Thus, this study shows that the complex integrated cellular responses upon ET-1 receptor activation are reflected in metabolic activity.

Vasopressin Stimulates Ca 2+ Spiking Activity in A7r5 Vascular Smooth Muscle Cells via Activation of Phospholipase A 2

[Arg8]-vasopressin (AVP) is both a potent vasoconstrictor and a mitogen for vascular smooth muscle cells. AVP binds to a single class of receptors (V1a) in the A7r5 rat aortic smooth muscle cell line (Kd approximately 2 nmol/L). Stimulation of these cells with AVP results in an increase in cytoplasmic free Ca2+ concentration ([Ca2+]i) by releasing intracellular Ca2+ stores and increasing Ca2+ influx; the EC50 for these effects is approximately 5 nmol/L. AVP has recently been reported to stimulate arachidonic acid release in primary cultures of rat aortic smooth muscle over a much lower concentration range (EC50 approximately 0.05 nmol/L). The present study examined the effects of varying concentrations of AVP on spontaneous Ca2+ spiking activity in fura 2-loaded A7r5 cells. Frequency of CA2+ spiking increased with increasing [AVP] in the range of 10 to 500 pmol/L. Higher concentrations of AVP inhibited spiking but elicited the characteristic [Ca2+]i changes ascribed to the release of Ca2+ stores and increased Ca2+ entry. The effects of both low and high concentrations of AVP were inhibited by [1-(beta-mercapto-beta,beta,-pentamethylenepropionic acid),2-0-methyltyrosine]arginine vasopressin, a selective V1a vasopressin antagonist. Nimodipine (50 nmol/L), a blocker of L-type voltage-sensitive Ca2+ channels, abolished the Ca(2+)-spiking activity without inhibiting a maximal [Ca2+]i response to AVP (1 mumol/L). AVP-stimulated Ca2+ spiking, but not release of intracellular Ca2+ stores, was also abolished by ONO-RS-082 (1 mumol/L), an inhibitor of phospholipase A2. These results suggest that occupation of a small fraction of V1a vasopressin receptors by AVP results in stimulation of phospholipase A2 and leads to increased Ca(2+)-spiking activity. This effect may be important for fine tuning of vascular tone, whereas maximal stimulation by AVP (full receptor occupancy) may be required for more vigorous or sustained vasoconstriction or mitogenesis.

Structure-activity relationships of an endothelin ET A receptor antagonist, 50–235, and its derivatives

27- O-Caffeoyl myricerone (50–235) is a nonpeptide endothelin receptor antagonist which is highly selective for the endothelin ET A receptor subtype. In order to determine which functional groups in 50–235 are essential for its activity, we examined the potencies of 50–235 and its derivatives to inhibit [ 125I]endothelin-1 binding and endothelin-1-induced increase in the cytosolic Ca 2+ concentration in rat aortic smooth muscle A7r5 cells. The results suggest that the 3-keto, 17-carboxyl and 27-caffeoyl groups in 50–235 are important for ET A receptor blocking activity. Modifications of the catechol ring of the 27-caffeoyl group influenced the affinity and the functional antagonist activity, but the effects were not parallel.