Arteriolar Vascular Smooth Muscle Cells Research Articles

Extracellular matrix-specific focal adhesions in vascular smooth muscle produce mechanically active adhesion sites

Integrin-mediated mechanotransduction in vascular smooth muscle cells (VSMCs) plays an important role in the physiological control of tissue blood flow and vascular resistance. To test whether force applied to specific extracellular matrix (ECM)-integrin interactions could induce myogenic-like mechanical activity at focal adhesion sites, we used atomic force microscopy (AFM) to apply controlled forces to specific ECM adhesion sites on arteriolar VSMCs. The tip of AFM probes were fused with a borosilicate bead (2 ~ 5 microm) coated with fibronectin (FN), collagen type I (CNI), laminin (LN), or vitronectin (VN). ECM-coated beads induced clustering of alpha(5)- and beta(3)-integrins and actin filaments at sites of bead-cell contact indicative of focal adhesion formation. Step increases of an upward (z-axis) pulling force (800 ~ 1,600 pN) applied to the bead-cell contact site for FN-specific focal adhesions induced a myogenic-like, force-generating response from the VSMC, resulting in a counteracting downward pull by the cell. This micromechanical event was blocked by cytochalasin D but was enhanced by jasplakinolide. Function-blocking antibodies to alpha(5)beta(1)- and alpha(v)beta(3)-integrins also blocked the micromechanical cell event in a concentration-dependent manner. Similar pulling experiments with CNI, VN, or LN failed to induce myogenic-like micromechanical events. Collectively, these results demonstrate that mechanical force applied to integrin-FN adhesion sites induces an actin-dependent, myogenic-like, micromechanical event. Focal adhesions formed by different ECM proteins exhibit different mechanical characteristics, and FN appears of particular relevance in its ability to strongly attach to VSMCs and to induce myogenic-like, force-generating reactions from sites of focal adhesion in response to externally applied forces.

Localization and function of connexin 45 in the renal cortical vasculature

Connexins (Cx) play an important role in cell to cell communication via intercellular gap junction channels or transiently open pores (hemichannels). We studied Cx45 in the mouse kidney using conditional Cx45‐deficient (Cx45fl/fl:Nestin‐Cre) mice expressing EGFP in place of Cx45 in cells that during development expressed the intermediate filament nestin. EGFP fluorescence was found in the afferent and efferent arteriolar vascular smooth muscle (VSMC) and renin producing JG cells and in the extra and intraglomerular mesangium indicating that Cx45 is normally expressed in the juxtaglomerular apparatus (JGA). Immunoblotting detected a 1.5‐fold increase in renal cortical renin content in Cx45−/− mice. Wild type and Cx45−/− VSMCs were cultured from dissected afferent arteriole explants. Cells were loaded with Fluo‐4/Fura red and ratiometric [Ca2+]i imaging was performed using fluorescence confocal microscopy. Mechanical stimulation of a single VSMC caused a 5‐fold increase in [Ca2+]i which propagated from cell to cell throughout the monolayer (12.6±1.1 μm/s) over distances >400 μm. In VSMCs from Cx45−/− mice Ca2+ wave propagation was significantly inhibited with velocity of 7±1 μm/s and peak [Ca2+]i =56.21±15 (n=6). Addition of either the purinergic receptor antagonist suramin or the Cx blocker 18‐alpha GA reduced Ca2+ wave propagations. Cx45 plays an important role in renal vascular control, renin release and JGA function.

Activation of Phospholipase Cγ1 Protects Renal Arteriolar VSMCs from H2O2-Induced Cell Death

Background: We evaluated the effect of hydrogen peroxide (H₂O₂) on viability of vascular smooth muscle cells (VSMCs) of renal resistance arterioles and determined whether responses are modulated by activation of PLCγ₁. Methods: Phospholipase C (PLC)-isozyme protein levels and activity were measured using Western blot analysis and enzymatic production of phosphoinositol 1,4,5-trisphosphate (IP₃), respectively. Stimulation of PLCγ₁ was assessed by immunoblots of tyrosine phosphorylation. Results: Cytotoxicity of H₂O₂ exposure was concentration-dependent (30% death with 250 µM; 87% with 500 µM at 8 h) and time-dependent (7% at 1 h; 30% at 8 h with 250 µM H₂O₂). Catalase abolished such relations. H₂O₂increased PLCγ₁ expression more than that of PLCδ₁ and almost doubled total PLC enzymatic activity between 2 and 8 h, changes prevented by catalase. The PLC inhibitor U73112 (3 µM) enhanced the cytotoxic concentration and time effects of H₂O₂. In acute studies, H₂O₂ rapidly caused tyrosine phosphorylation of PLCγ₁. Conclusion: H₂O₂ increased PLCγ₁ expression and almost doubled total PLC activity, changes abolished by catalase. We conclude that H₂O₂ is cytotoxic to cultured VSMCs of renal preglomerular arterioles, a process that is attenuated by compensatory increases in PLCγ₁ protein level, tyrosine phosphorylation of PLCγ₁ and PLC enzymatic activity to generate IP₃.

Phospholipase C Expression and Activity in Smooth Muscle Cells of Renal Arterioles and Aorta of Young, Spontaneously Hypertensive Rats During Culture

Phospholipase C (PLC)-beta(1) and -delta(1), but not -gamma(1), protein expressions in fresh renal arterioles and aorta are greater in 6-week-old, spontaneously hypertensive rats (SHRs) versus normotensive Wistar-Kyoto rats (WKYs). This PLC activity is also greater in both vessels of SHRs. In the present study, we tested whether cultured vascular smooth muscle cells (VSMCs) of preglomerular arterioles and aorta accurately reflect strain differences observed in fresh vessels, with VSMCs of SHRs predicted to have higher levels of PLC isozymes and enzyme activity. We assessed the stability of variables over passages 3 to 11. The VSMCs were isolated and cultured using standard techniques. The PLC-isozyme protein levels and catalytic activity were determined by Western blot analysis and inositol 1,4,5-trisphosphate (IP(3)) production, respectively. Immunoblots showed expression of PLC-gamma(1) and -delta(1), but not PLC-beta(1), in VSMCs from both vessels. Arteriolar VSMCs of SHRs had three-to-fivefold higher levels of PLC-gamma(1) and -delta(1) during passages 3 to 8. Enzymatic activity in these VSMCs was higher in SHRs versus WKYs, especially during passages 6 to 11. In contrast, cultured aortic VSMCs of SHRs had two-to-threefold lower densities of PLC-gamma(1) and -delta(1) protein. Compared with fresh resistance arterioles and aorta, cultured VSMCs exhibit changes in PLC-isozyme protein levels and enzyme activity that vary with passage. The differences between cultured VSMCs of SHRs and WKYs do not accurately reflect those in fresh resistance and conduit vessels, either qualitatively or quantitatively. The results of VSMC culture studies should be interpreted with caution and should ideally be compared with more physiologically relevant fresh preparations.

Angiotensin II, reactive oxygen species, and Ca2+signaling in afferent arterioles

In afferent arteriolar vascular smooth muscle cells, ANG II induces a rise in cytosolic Ca(2+) ([Ca(2+)](i)) via inositol trisphosphate receptor (IP(3)R) stimulation and by activation of the adenine diphosphate ribose (ADPR) cyclase to form cyclic ADPR, which sensitizes the ryanodine receptor (RyR) to Ca(2+). We hypothesize that ANG II stimulation of NAD(P)H oxidases leads to the formation of superoxide anion (O(2)-*, which, in turn, activates ADPR cyclase. Afferent arterioles were isolated from rat kidney with the magnetized microsphere and sieving technique and loaded with fura-2 to measure [Ca(2+)](i). ANG II rapidly increased [Ca(2+)](i) by 124 +/- 12 nM. In the presence of apocynin, a specific inhibitor of NAD(P)H oxidase assembly, the [Ca(2+)](i) response was reduced to 35 +/- 5 nM (P < 0.01). Tempol, a superoxide dismutase mimetic, did not alter the [Ca(2+)](i) response to ANG II at a concentration of 10(-4) M (99 +/- 12 nM), but 10(-3) M tempol reduced the response to 32 +/- 3 nM (P < 0.01). The addition of nicotinamide, an inhibitor of ADPR cyclase, to apocynin or tempol (10(-3) M) resulted in no further inhibition. Measurement of superoxide production with the fluorescent probe tempo 9-AC showed that ANG II caused an increase of 48 +/- 20 arbitrary units; apocynin or diphenyl iodonium (an inhibitor of flavoprotein oxidases) inhibited the response by 94%. Hydrogen peroxide (H(2)O(2)) was studied at physiological (10(-7) M) and higher concentrations. In the presence of H(2)O(2) (10(-7) M), neither baseline [Ca(2+)](i) nor the response to ANG II was altered (125 +/- 15 nM), whereas H(2)O(2) (10(-6) and 10(-5) M) inhibited the [Ca(2+)](i) response to ANG II by 35 and 46%, respectively. We conclude that ANG II rapidly activates NAD(P)H oxidases of afferent arterioles, leading to the formation of O(2)-*, which then stimulates ADPR cyclase to form cADPR. cADPR, by sensitizing the RyR to Ca(2+), augments the Ca(2+) response (calcium-induced calcium release) initiated by activation of the IP(3)R.

Romancing the macula densa at UAB

The Nephrology Research and Training Center, established in 1977 at the University of Alabama at Birmingham by Thomas E. Andreoli, served as a catalyst to stimulate multiple areas of investigations in renal physiology and nephrology. Individuals with backgrounds in biophysics, membrane transport, renal hemodynamics, structural biology, and nephrology interacted with each other, thus providing an exciting and collegial environment. The laboratory of renal hemodynamics focused on the control of renal blood flow, glomerular filtration rate in normal and hypertensive models, and on the important role of the macula densa in providing communication from the tubules to the vascular elements. Studies initially focused on the role of the macula densa feedback mechanism in mediating renal autoregulatory behavior. Subsequent experiments examined various aspects of the feedback system, including the identification and characterization of membrane transport events that sense changes in tubular fluid concentration and transfer information to intracellular signaling mechanisms. More recent investigations have focused on the capability of the macula densa cells to synthesize and release various vasoactive mediators that can influence vascular tone of the glomerular arterioles. In particular, the ability of the macula densa cells to secrete ATP has stimulated continued interest in the hypothesis that ATP may serve an important role in mediating signals to afferent arteriolar vascular smooth muscle cells.

Imaging remodeling of the actin cytoskeleton in vascular smooth muscle cells after mechanosensitive arteriolar constriction.

Experiments were performed to determine whether remodeling of the actin cytoskeleton contributes to arteriolar constriction. Mouse tail arterioles were mounted on cannulae in a myograph and superfused with buffer solution. The alpha1-adrenergic agonist phenylephrine (0.1-1 micromol/l) caused constriction that was unaffected by cytochalasin D (300 nmol/l) or latrunculin A (100 nmol/l), inhibitors of actin polymerization. In contrast, each compound abolished the mechanosensitive constriction (myogenic response) evoked by elevation in transmural pressure (PTM; 10-60 or 90 mmHg). Arterioles were fixed, permeabilized, and stained with Alexa-568 phalloidin and Alexa-488 DNAse I to visualize F-actin and G-actin, respectively, using a Zeiss 510 laser scanning microscope. Elevation in PTM, but not phenylephrine (1 micromol/l), significantly increased the intensity of F-actin and significantly decreased the intensity of G-actin staining in arteriolar vascular smooth muscle cells (VSMCs). The increase in F-actin staining caused by an elevation in PTM was inhibited by cytochalasin D. In VSMCs at 10 mmHg, prominent F-actin staining was restricted to the cell periphery, whereas after elevation in PTM, transcytoplasmic F-actin fibers were localized through the cell interior, running parallel to the long axis of the cells. Phenylephrine (1 micromol/l) did not alter the architecture of the actin cytoskeleton. In contrast to VSMCs, the actin cytoskeleton of endothelial or adventitial cells was not altered by an elevation in PTM. Therefore, the actin cytoskeleton of VSMCs undergoes dramatic alteration after elevation in PTM of arterioles and plays a selective and essential role in mechanosensitive myogenic constriction.

The future of pharmacologic stress: selective a 2a adenosine receptor agonists

Adenosine and dipyridamole, the currently available vasodilators for myocardial perfusion imaging, produce hyperemic coronary flow by stimulating A 2A adenosine receptors on arteriolar vascular smooth muscle cells. However, both vasodilators nonselectively activate A 1, A 2B, and A 3 adenosine receptors, which contributes to common undesirable effects. In the development of a novel pharmacologic stress agent, more selective agonism of the A 2A receptor subtype would be desirable. Currently, 2 selective A 2A adenosine receptor agonists are being evaluated in phase 3 studies as pharmacologic stress agents. The highly selective, potent, low-affinity A 2A adenosine agonist regadenoson (also known as CVT-3146) holds significant potential as a pharmacologic stress agent, based on available results from experimental and clinical trials. Regadenoson produces maximal hyperemia quickly and maintains it for an optimal duration that is practical for radionuclide myocardial perfusion imaging. Regadenoson's simple rapid bolus administration and short duration of hyperemic effect point to an advantage of enhanced control for the clinician.

CADASIL Notch3 mutant proteins localize to the cell surface and bind ligand.

Cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a vascular dementia arising from abnormal arteriolar vascular smooth muscle cells. CADASIL results from mutations in Notch3 that alter the number of cysteine residues in the extracellular epidermal growth factor-like repeats, important for ligand binding. It is not known whether CADASIL mutations lead to loss or gain of Notch3 receptor function. To examine the functional consequences of CADASIL mutations, we engineered 4 CADASIL-like mutations into rat Notch3 and have shown that the presence of an unpaired cysteine does not impair cell-surface expression or ligand binding.

Redox signaling of the arteriolar myogenic response.

Arteriolar vascular smooth muscle cells (VSMCs) are mechanosensitive, constricting to elevations in transmural pressure (P(TM)). The goal of the present study was to determine using mouse isolated tail arterioles and arteries whether oxidant signaling regulates this myogenic response. In response to P(TM) elevation, VSMCs of arterioles but not arteries generated constriction and increased reactive oxygen species (ROS) activity (using the H(2)O(2)-sensitive probe dichlorodihydrofluorescein). Arterioles had increased expression of NADPH oxidase components compared with arteries. Inhibition of NADPH oxidase, using mice with targeted impairment of enzyme components (p47(phox) or rac1) or diphenyleneiodonium, prevented the pressure-induced generation of ROS. When ROS activity was inhibited, either by inhibiting NADPH oxidase or with N-acetylcysteine, the myogenic constriction was abolished. The myogenic constriction was also inhibited by catalase, which inactivates H(2)O(2), but was unaffected by a cell-permeant mimic of superoxide dismutase (MnTMPyP). alpha(1)-Adrenergic constriction was not associated with altered ROS activity and was not affected by inhibition of NADPH oxidase or ROS. Exogenous H(2)O(2) constricted VSMCs of arterioles but not arteries. Thus, NADPH oxidase and ROS, in particular H(2)O(2), contribute to the myogenic response of arteriolar VSMCs.

Phosphodiesterases in the rat renal vasculature.

The objective this investigation was to determine the relative importance of type I, III, and IV phosphodiesterases in the regulation of cyclic adenosine monophosphate (cAMP) in the renal circulation. In the first experimental series, four groups of isolated rat kidneys perfused with Tyrode's solution were stimulated with isoproterenol (3 microM) and then treated with increasing concentrations (from the approximately IC50 to 30 times the approximately IC50 in threefold increments) of one of four phosphodiesterase inhibitors: group 1, 3-isobutyl-1-methylxanthine, a "broad-spectrum" phosphodiesterase inhibitor (10-300 microM); group 2, Ro 20-1724, a selective type IV phosphodiesterase inhibitor (3-100 microM); group 3, 8-methoxymethyl-3-isobutyl-1-methylxanthine, a selective type I phosphodiesterase inhibitor (3-100 microM); and group 4, milrinone, a selective type III phosphodiesterase inhibitor (0.3-10 microM). In the second experimental series, five groups of cultured preglomerular (interlobular and afferent arteriolar) vascular smooth-muscle cells were stimulated with isoproterenol (1 microM) and treated with vehicle or supramaximal concentrations (30 times IC50) of either 3-isobutyl-1-methylxanthine (300 microM), Ro 20-1724 (100 microM), 8-methoxymethyl-3-isobutyl-1-methylxanthine (100 microM), or milrinone (10 microM). In perfused kidneys and cultured pre-glomerular vascular smooth-muscle cells, 3-isobutyl-1-methylxanthine and Ro 20-1724 similarly increased renal cAMP release and total cellular (extracellular + intracellular) cAMP levels, respectively. In contrast, neither 8-methoxymethyl-3-isobutyl-1-methylxanthine nor milrinone affected renal cAMP release or total cellular cAMP levels. These data indicate that in the renal circulation, type IV phosphodiesterase is the predominant phosphodiesterase isozyme.

Angiotensin II-receptor stimulation of cytosolic calcium concentration in cultured renal resistance arterioles

This study provides an initial characterization of basic morphological properties of cultures of vascular smooth muscle cells (VSMC) from rat preglomerular resistance vessels and of the functional coupling of angiotensin II (ANG II) receptors to cytosolic free calcium concentration ([Ca2+]i (fura 2 fluorescence photometry). Renal VSMC were isolated from interlobular arteries and afferent arterioles (< 50 microns) using an iron oxide sieving method and compared with rat aortic VSMC cultured under similar conditions. Quiescent monolayers maintained uniform morphology and [Ca2+]i signaling profile between passages 3 and 10. Arteriolar and aortic VSMC were spindle shaped and expressed smooth muscle-specific alpha-actin and myosin heavy chains SM-1 and SM-2. ANG II caused a rapid increase in [Ca2+]i, followed by a sustained plateau phase at 50-60% of the peak value. The initial maximum [Ca2+]i responses were dose dependent and of similar magnitude in renal arteriolar and aortic VSMC. ANG II (10(-7) M) increased [Ca2+]i from 50 to 240 nM in arteriolar and from 57 to 201 nM in aortic VSMC (P < 0.001 for both). Inhibition of ANG II effects on [Ca2+]i revealed significant signaling through distinct AT-receptor subtypes (losartan and PD-123319 sensitive) in renal arteriolar VSMC. In contrast, only losartan was effective in aortic VSMC. The AT2-receptor ligand CGP-42112 had no effect in either vessel type. Our results demonstrate that cultured arteriolar VSMC have anatomical similarities to aortic VSMC and functional differences in AT-receptor signaling in response to ANG II. This novel preparation should provide a useful approach with which to investigate cellular mechanisms concerning receptor coupling to signaling pathways involved in vascular reactivity of arteriolar VSMC in the microcirculation in general and the kidney in particular.

Paracrine regulation of the renal microcirculation

There has been an explosive growth of interest in the multiple interacting paracrine systems that influence renal microvascular function. This review first discusses the membrane activation mechanisms for renal vascular control. Evidence is provided that there are differential activating mechanisms regulating pre- and postglomerular arteriolar vascular smooth muscle cells. The next section deals with the critical role of the endothelium in the control of renal vascular function and covers the recent findings related to the role of nitric oxide and other endothelial-derived factors. This section is followed by an analysis of the roles of vasoactive paracrine systems that have their origin from adjoining tubular structures. The interplay of signals between the epithelial cells and the vascular network to provide feedback regulation of renal hemodynamics is developed. Because of their well-recognized contributions to the regulation of renal microvascular function, three major paracrine systems are discussed in separate sections. Recent findings related to the role of intrarenally formed angiotensin II and the prominence of the AT1 receptors are described. The possible contribution of purinergic compounds is then discussed. Recognition of the emerging role of extracellular ATP operating via P2 receptors as well as the more recognized functions of the P1 receptors provides fertile ground for further studies. In the next section, the family of vasoactive arachidonic acid metabolites is described. Possibilities for a myriad of interacting functions operating both directly on vascular smooth muscle cells and indirectly via influences on endothelial and epithelial cells are discussed. Particular attention is given to the more recent developments related to hemodynamic actions of the cytochrome P-450 metabolites. The final section discusses unique mechanisms that may be responsible for differential regulation of medullary blood flow by locally formed paracrine agents. Several sections provide perspectives on the complex interactions among the multiple mechanisms responsible for paracrine regulation of the renal microcirculation. This plurality of regulatory interactions highlights the need for experimental strategies that include integrative approaches that allow manifestation of indirect as well as direct influences of these paracrine systems on renal microvascular function.