FRET (Forster resonance energy transfer)-based biosensor molecules are powerful tools to reveal specific molecular interactions
in cells. Typically however, they are used in cultured cells that ...(inevitably) express different genes than their counterparts
in intact organisms. In such cells it may be impossible to administer physiological stimuli and measure physiological outputs.
Here, through the use of transgenic mice that express a FRET-based myosin light chain kinase (MLCK) biosensor molecule, we
report a technique for dynamically observing activation and regulation of MLCK within the smooth muscle cells of intact, functioning
small arteries, together with measurement of arterial force production and intracellular Ca 2+ .
Two-photon fluorescence microscopy and conscious, restrained optical biosensor mice were used to study smooth muscle Ca(2+) signaling in ear arterioles. Conscious mice were used in order to preserve ...normal mean arterial blood pressure (MAP) and sympathetic nerve activity (SNA). ExMLCK mice, which express a genetically-encoded smooth muscle-specific FRET-based Ca(2+) indicator, were equipped with blood pressure telemetry and immobilized for imaging. MAP was 101 ± 4 mmHg in conscious restrained mice, similar to the freely mobile state (107 ± 3 mmHg). Oscillatory vasomotion or irregular contractions were observed in most arterioles (71%), with the greatest oscillatory frequency observed at 0.25 s(-1). In a typical arteriole with an average diameter of ~35 μm, oscillatory vasomotion of a 5-6 μm magnitude was accompanied by nearly uniform Ca(2+) oscillations from ~0.1 to 0.5 μM, with maximum Ca(2+) occurring immediately before the rapid decrease in diameter. Very rapid, spatially uniform "Ca(2+) flashes" were also observed but not asynchronous propagating Ca(2+) waves. In contrast, vasomotion and dynamic Ca(2+) signals were rarely observed in ear arterioles of anesthetized exMLCK biosensor mice. Hexamethonium (30 μg/g BW, i.p.) caused a fall in MAP to 74 ± 4 mmHg, arteriolar vasodilation, and abolition of vasomotion and synchronous Ca(2+) transients.
MAP and heart rate (HR) were normal during high-resolution Ca(2+) imaging of conscious, restrained mice. SNA induced continuous vasomotion and irregular vasoconstrictions via spatially uniform Ca(2+) signaling within the arterial wall. FRET-based biosensor mice and two-photon imaging provided the first measurements of Ca(2+) in vascular smooth muscle cells in arterioles of conscious animals.
Departments of 1 Physiology and 2 Medicine, and the 3 Maryland Center for Heart, Hypertension and Kidney Disease, University of Maryland School of Medicine, Baltimore, Maryland
Submitted 11 May 2009
...; accepted in final form 13 July 2009
Prolonged ouabain administration to normal rats causes sustained blood pressure (BP) elevation. This ouabain-induced hypertension (OH) has been attributed, in part, to the narrowing of third-order resistance arteries ( 320 µm internal diameter) as a result of collagen deposition in the artery media (see Ref. 6). Here we describe the structural and functional properties of fourth-order mesenteric small arteries from control and OH rats, including the effect of low-dose ouabain on myogenic tone in these arteries. Systolic BP in OH rats was 138 ± 3 versus 124 ± 4 mmHg in controls ( P < 0.01). Pressurized (70 mmHg) control and OH arteries, with only a single layer of myocytes, both had 165-µm internal diameters and 20-µm wall thicknesses. Even after fixation, despite vasoconstriction, the diameters and wall thicknesses did not differ between control and OH fourth-order arteries, whereas in third-order arteries, both parameters were significantly smaller in OH than in controls. Myogenic reactivity was significantly augmented in OH fourth-order arteries. Nevertheless, phenylephrine- (1 µM) and high K + -induced vasoconstrictions and acetylcholine-induced vasodilation were comparable in control and OH arteries. Vasoconstrictions induced by 5 µM phenylephrine and by 10 mM caffeine in Ca 2+ -free media indicated that releasable sarcoplasmic reticulum Ca 2+ stores were normal in OH arteries. Importantly, 100 nM ouabain constricted both control and OH arteries by 26 µm, indicating that this response was not downregulated in OH rats. This maximal ouabain-induced constriction corresponds to a 90% increase in resistance to flow in these small arteries; thus ouabain at EC 50 of 0.66 nM should raise resistance by 35%. We conclude that dynamic constriction in response to circulating nanomolar ouabain in small arteries likely makes a major contribution to the increased vascular tone and BP in OH rats.
myogenic reactivity; resistance artery
Address for reprint requests and other correspondence: J. Zhang, Dept. of Physiology, Univ. of Maryland School of Medicine, 655 W. Baltimore St., Baltimore, MD 21201 (e-mail: jzhan002{at}umaryland.edu )
Store-operated Ca(2+) entry (SOCE) has recently been proposed to contribute to Ca(2+) influx in vascular smooth muscle cells (VSMCs). Adenosine is known for its protective role against hypoxia and ...ischemia by increasing nutrient and oxygen supply through vasodilation. This study was designed to examine the hypothesis that SOCE have a functional role in adenosine-induced vasodilation. Small mesenteric resistance arteries and mesenteric VSMCs were obtained from rats. Isometric tensions of isolated artery rings were measured by a sensitive myograph system. Laser-scanning confocal microscopy was used to determine the intracellular Ca(2+) concentration of fluo 3-loaded VSMCs. Adenosine (0.1-100 microM) relaxed artery rings that were precontracted by phenylephrine in a concentration-dependent manner. In cultured mesenteric VSMCs, passive store depletion by thapsigargin and active store depletion by phenylephrine both induced Ca(2+) influx due to SOCE. Adenosine inhibited SOCE-mediated increases in cytosolic Ca(2+) levels evoked by the emptying of the stores. In isolated artery rings, adenosine inhibited SOCE-induced contractions due to store depletion. A(2A) receptor antagonism with SCH-58261 and adenylate cyclase inhibition with SQ-22536 largely attenuated adenosine responses. The cAMP analog 8-bromo-cAMP mimicked the effects of adenosine on SOCE. Our results indicate a novel mechanism of vasodilatation by adenosine that involves regulation of SOCE through the cAMP signaling pathway due to activation of adenosine A(2A) receptors.
Confocal laser scanning microscopy and fluo 4 were used to visualize local and whole cell Ca(2+) transients within individual smooth muscle cells (SMC) of intact, pressurized rat mesenteric small ...arteries during activation of alpha1-adrenoceptors. A method was developed to record the Ca(2+) transients within individual SMC during the changes in arterial diameter. Three distinct types of "Ca(2+) signals" were influenced by adrenergic activation (agonist: phenylephrine). First, asynchronous Ca(2+) transients were elicited by low levels of adrenergic stimulation. These propagated from a point of origin and then filled the cell. Second, synchronous, spatially uniform Ca(2+) transients, not reported previously, occurred at higher levels of adrenergic stimulation and continued for long periods during oscillatory vasomotion. Finally, Ca(2+) sparks slowly decreased in frequency of occurrence during exposure to adrenergic agonists. Thus adrenergic activation causes a decrease in the frequency of Ca(2+) sparks and an increase in the frequency of asynchronous wavelike Ca(2+) transients, both of which should tend to decrease arterial diameter. Oscillatory vasomotion is associated with spatially uniform synchronous oscillations of cellular Ca(2+) and may have a different mechanism than the asynchronous, propagating Ca(2+) transients.
To investigate the cellular mechanisms for altered Ca homeostasis and contractility in cardiac hypertrophy, we measured whole-cell L-type Ca currents (ICa,L), whole-cell Ca transients (Cai), and Ca ...() sparks in ventricular cells from 6-month-old spontaneously hypertensive rats (SHRs) and from age- and sex-matched Wistar-Kyoto and Sprague-Dawley control rats. By echocardiography, SHR hearts had cardiac hypertrophy and enhanced contractility (increased fractional shortening) and no signs of heart failure. SHR cells had a voltage-dependent increase in peak Cai amplitude (at 0 mV, 1330 +/- 62 nmol/L SHRs versus 836 +/- 48 nmol/L controls, P<0.05) that was not associated with changes in ICa,L density or kinetics, resting Cai, or Ca () content of the sarcoplasmic reticulum (SR). SHR cells had increased time of relaxation. Ca sparks from SHR cells had larger average amplitudes (173 +/- 192 nmol/L SHRs versus 109 +/- 64 nmol/L control; P<0.05), which was due to redistribution of Ca sparks to a larger amplitude population. This change in Ca spark amplitude distribution was not associated with any change in the density of ryanodine receptors, calsequestrin, junctin, triadin 1, Ca, or phospholamban. Therefore, SHRs with cardiac hypertrophy have increased contractility, Ca (i) amplitude, time to relaxation, and average Ca spark amplitude ("big sparks"). Importantly, big sparks occurred without alteration in the trigger for SR Ca release (ICa,L), SR Ca content, or the expression of several SR Ca proteins. Thus, cardiac hypertrophy in SHRs is linked with an alteration in the coupling of Ca entry through L-type Ca channels and the release of Ca from the SR, leading to big sparks and enhanced contractility. Alterations in the microdomain between L-type Ca channels and SR Ca release channels may underlie the changes in Ca homeostasis observed in cardiac hypertrophy. Modulation of SR Ca release may provide a new therapeutic strategy for cardiac hypertrophy and for its progression to heart failure and sudden death. (Circ Res. 1999;84:424-434.)
Confocal microscopy of fluo-4 fluorescence in pressurized rat mesenteric small arteries subjected to low-frequency electrical field stimulation revealed Ca2+ transients in perivascular nerves and ...novel, spatially localized Ca2+ transients in adjacent smooth muscle cells. These muscle Ca2+ transients occur with a very brief latency to the stimulus pulse (most <3 ms). They are wider (approximately 5 micro m) and last longer (t(1/2), 145 ms) than Ca2+ sparks. They are abolished by the purinergic receptor (P2X) antagonist suramin, but they are totally unaffected by the alpha1 adrenoceptor antagonist prazosin or by capsaicin (which inhibits the function of perivascular sensory nerves). We conclude that these novel Ca2+ transients represent Ca2+ entering smooth muscle cells through P2X receptors activated by ATP released from sympathetic nerves, and we therefore call them "junctional Ca2+ transients" or jCaTs. As expected from spontaneous neurotransmitter release, jCaTs also occur spontaneously, with characteristics identical to evoked jCaTs. Visualization of sympathetic neurotransmission shows that purinergic components dominate at low frequencies of sympathetic nerve fiber activation.
Intercellular Ca2+ waves in rat heart muscle Lamont, Christine; Luther, Paul W.; Balke, C. William ...
The Journal of physiology,
11/1998, Letnik:
512, Številka:
3
Journal Article
Recenzirano
Odprti dostop
Confocal laser scanning microscopy was used to visualize intercellular transmission of Ca 2+ waves in intact rat ventricular trabeculae micro-injected with the calcium indicator fluo-3.
Ca 2+ waves ...usually failed to be transmitted from cell to cell. At identified individual end-to-end cell contacts, successful transmission
interspersed with failure, which sometimes occurred despite an apparent small spritz of Ca 2+ between cells. The probability of cell to cell transmission ( P tran ) was 0.13.
Ca 2+ waves arose preferentially near junctions of connected cells, where connexin-43 was found, but randomly in enzymatically
disconnected heart cells.
β-Adrenergic stimulation significantly increased P tran (to 0.22) and heptanol, an uncoupler of gap junction channels, significantly decreased it (to 0.045).
In regions of high Ca 2+ i due to damage, wave frequency decreased markedly with each cell-cell junction passed.
The Ca 2+ permeability of cardiac gap junctions may be regulated, and the low ability of cardiac gap junctions to transmit Ca 2+ may help control the spread of Ca 2+ from damaged regions.
Two-photon fluorescence microscopy and conscious, restrained optical biosensor mice were used to study smooth muscle Ca
2+
signaling in ear arterioles. Conscious mice were used in order to preserve ...normal mean arterial blood pressure (MAP) and sympathetic nerve activity (SNA). ExMLCK mice, which express a genetically-encoded smooth muscle-specific FRET-based Ca
2+
indicator, were equipped with blood pressure telemetry and immobilized for imaging. MAP was 101 ± 4 mmHg in conscious restrained mice, similar to the freely mobile state (107 ± 3 mmHg). Oscillatory vasomotion or irregular contractions were observed in most arterioles (71%), with the greatest oscillatory frequency observed at 0.25 s
−1
. In a typical arteriole with an average diameter of ~35 μm, oscillatory vasomotion of a 5–6 μm magnitude was accompanied by nearly uniform Ca
2+
oscillations from ~0.1 to 0.5 μM, with maximum Ca
2+
occurring immediately before the rapid decrease in diameter. Very rapid, spatially uniform “Ca
2+
flashes” were also observed but not asynchronous propagating Ca
2+
waves. In contrast, vasomotion and dynamic Ca
2+
signals were rarely observed in ear arterioles of anesthetized exMLCK biosensor mice. Hexamethonium (30 μg/g BW, i.p.) caused a fall in MAP to 74 ± 4 mmHg, arteriolar vasodilation, and abolition of vasomotion and synchronous Ca
2+
transients.
Summary
: MAP and heart rate (HR) were normal during high-resolution Ca
2+
imaging of conscious, restrained mice. SNA induced continuous vasomotion and irregular vasoconstrictions via spatially uniform Ca
2+
signaling within the arterial wall. FRET-based biosensor mice and two-photon imaging provided the first measurements of Ca
2+
in vascular smooth muscle cells in arterioles of conscious animals.
In most previous studies, ischaemia–reperfusion (I/R)‐induced vascular injury referred to injury in the tissue or blood vessel that was directly subjected to I/R. However, less attention has been ...focused on remote vascular injury that might be caused by cardiac I/R. In the present study, we aimed to assess whether cardiac I/R could affect vasoconstriction and vasodilatation in mesenteric arteries from Sprague–Dawley rats. Left anterior descending coronary arteries from adult male Sprague–Dawley rats were occluded (60 min) and then reperfused (120 min). Changes in haemodynamic parameters indicated that this procedure caused evident cardiac dysfunction. In mesenteric arteries isolated from the animals, cardiac I/R significantly increased the maximal contractions in response to KCl, 5‐hydroxytryptamine, phenylephrine and U46619 and decreased the maximal relaxation in response to acetylcholine, but not to sodium nitroprusside, compared with sham‐operated animals. The nitric oxide synthase inhibitor l‐NAME abolished differences of contractile responses to phenylephrine between sham‐operated and I/R rats. The antioxidant N‐acetyl‐l‐cysteine reversed the impairment of acetylcholine‐stimulated vasodilatation induced by regional cardiac I/R. However, l‐NAME caused a similar degree of inhibition of acetylcholine‐stimulated relaxation in mesenteric arteries from sham‐operated and I/R rats. Electron microscopy revealed that mesenteric arterial endothelial structure was degraded in the I/R group and that N‐acetyl‐l‐cysteine treatment prevented this structural damage. In conclusion, regional cardiac I/R caused by transient occlusion and reperfusion of the left anterior descending coronary artery results in peripheral vascular endothelial dysfunction.
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