# Presentation: 639208

## The Use of Fluorescent Resonance Energy Transfer to Visualize Nitric Oxide Based Signaling Events in Living Systems

- Department of Environmental and Occupational Health
- University of Pittsburgh Graduate School of Public Health

- Claudette M. St. Croix, PhD

**Notes:**
Work describes the use of a variety of multimodal optical imaging technologies to investigate an intracellular signaling pathway involving S-nitrosation of zinc-sulfur clusters in the metal-binding protein, metallothionein, and the resultant effects on zinc homeostasis in pulmonary endothelium. In the last part of the talk I will introduce our new data implicating a role for NO-induced zinc release in mediating pulmonary vasoregulation.

## S-Nitrosation and Cell Signaling

- Broillet Cell Mol Life Sci 55:1036, 1999

**Notes:**
NO is formed endogenously from L-arginine by the action of NO synthase. As we are all now aware, NO exerts a number of diverse biological activities Although multiple chemical mechanisms exist by which nitric oxide can exert its effects the most commonly recognized interaction involves the formation of a bond between the free radical NO and ferrous heme, as is best exemplified by the activation of sGC. A second well recognized, but even more biochemically complex pathway for NO-induced protein modification, is via the addition or transfer of nitrosonium (NO+). The most abundant targets for these nitrosation events are cysteine thiols. Proteins in which such functional modulation by NO has been characterized include the NMDA receptor coupled ion channel, the cardiac ryanodine receptor, hemoglobin, Ras and cJNK. Thus, S-nitrosation affects many intracellular signaling and metabolic pathways. The first series of experiments I will present today will describe our attempts to discriminate between S-nitrosation and free radical based events in living cell culture.

## Metallothionein

- Kägi & Vasak, University of Zürich

**Notes:**
Contains 61 aa residues, 20 of which are cysteines which can bind up to 7 Zn atoms. Molecule is composed of two clusters N-terminal (alpha) cluster contains 3 Zn and C-terminal (beta) cluster contains 4. Each metal is tetrahedrally coordinated to 4 thiolate bonds with some of the thiolate ligands sharing the Zn atom. The effects of NO on the purified protein have been long appreciated. A number of biochemical techniques including ESR (electron spin resonance), NMR, and circular dichroism have been used to show that NO can cause unfolding and release of metal from MT.  

## Cellular Function of Metallothionein

*Metal Ion Homeostasis*: sequestering and transferring Zn, Cu, Cd.
*Antioxidant*:
- Elevated oxidative stress
- Intercepts O2•-, HO•, •NO, ONOO-
- Genetic manipulation studies show that MT can protects cells and tissues against oxidative and nitrosative damage.

**Notes:**
There are four MT genes in mice , III and IV confined to CNS. I and II ubiquitous. Although precise physiological roles for members of this multigene family remain unknown, it has been suggested that major functions of MT-I and -II are to detoxify heavy metals, regulate zinc and copper homeostasis and limit oxidative damage 
Metallothionein is a redox-sensitive gene that is elevated during various types of oxidative stress and is highly upregulated by tissue injury, infection, inflammation and neoplastic disease.
 The in vitro data shows that MT can intercept hydroxyl radicals and superoxide anion (Fliss, 1992; Sato, 1993; Thomas, 1986; Thornally, 1985) and can react with NO by forming EPR-detectable iron-dinitrosyl-sulfur complex (Kennedy, 1993; Schwarz, 1995).
 Overexpression of MT protects cells and tissues against oxidative and nitrosative damage.  Alternatively, underexpression of MT via antisense or targeted gene disruption approaches enhances the sensitivity of cells and tissues to oxidative stress including pro-apoptotic stimuli.  Therefore, a compelling case is apparent that MT participates in cellular defense against partially reduced oxygen and nitrogen species. 

## Non-radiative transfer of excited state energy of the donor to a second light absorbing molecule (acceptor).
 Acceptor releases energy through its characteristic fluorescent emission. 

- Non-radiative transfer of excited state energy of the donor to a second light absorbing molecule (acceptor).
-  Acceptor releases energy through its characteristic fluorescent emission.

**Notes:**
We utilized fluorescence resonance energy transfer techniques  to monitor the interactions of NO with GFP-modified target proteins in live cells. FRET is a non-destructive, spectroscopic method that can monitor the proximity and relative angular orientation of fluorophores in living cells.
This cartoon illustrates the basic principles involved. The donor fluorophore (in this case cyan) is excited by light of a specific wavelength (450 nm). The excited fluorphore transfers its excited state energy to the acceptor (in this case yellow). Having been excited by the donor fluorophore, the acceptor will then emit light at its characteristic wavelength. This is monitored by examining the emission intensities of the donor (which is quenched or reduced) and acceptor which is increased when FRET efficiency is high.

## Fluorescence Resonance Energy Transfer

- Detection of NO using metallothionein and two GFP mutants: In the presence of metal, MT folds allowing efficient FRET between ECFP and EYFP fused at either end of MT.  In the presence of NO (or a chelating agent) the protein unfolds and FRET is reduced.

**F****luorescence ****R****esonance ****E****nergy ****T****ransfer**

## Wide-field FRET

- Widefield FRET using standard Cyan/Yellow fluorescent proteins  is difficult because of bleed-through from Cyan emission  into yellow FP (25%)
- While controls such as acceptor photobleaching are critical, methods such as spectral unmixing have proven invaluable.
- (3 confocal options and one wide field option currently)

## Spectral Confocal 

- Spectral Confocal
- and FRET
- This is the Zeiss META design, equivalent though different systems are available from Leica and Olympus

**Notes:**
The unique scanning module is the core of the LSM 510 META. It contains motorized collimators, scanning mirrors, individually adjustable and positionable pinholes, and highly sensitive detectors including the META detector. All these components are arranged to ensure optimum specimen illumination and efficient collection of reflected or emitted light. A highly efficient optical grating provides an innovative way of separating the fluorescence emissions in the META detector. The grating projects the entire fluorescence spectrum onto the 32 channels of the META detector. Thus, the spectral signature is acquired for each pixel of the scanned image and subsequently can be used for the digital separation into component dyes. 

## S-nitroso-L-cysteine modifies metallothionein

**Control****	****      L-SNCEE**

**Cyan**
**Emission**

**Yellow **
**Emission**

**Both**

- FRET-MT Reporter

- St Croix CM. *Free Radic Biol Med*. 37:785-92, 2004

## L-SNCEE increases labile Zn

- in pulmonary endothelial cells

**Notes:**
We utilized multiphoton laser scanning microscopy with the zinc-specific fluorophore, Zinquin, to show that the conformational changes in MT in response to L-SNCEE were also associated with increases in labile zinc. Zinquin-E is quinoline-based a non-fluorescent membrane-permeant fluorophore with a specific affinity for Zn. Under these conditions, we observed significant time-dependent increases in Zinquin fluorescence in response to 50 uM SNCEE.

## S-nitroso-L-cysteine-EE mediated changes in FRET-MT are reversible

- St Croix CM. *Free Radic Biol Med*. 37:785-92, 2004

**Notes:**
In separate experiments, the L-SNCEE induced changes in FRET were reversed by the reducing agent dithiothreitol (50 uM) suggesting nitrosothiol-mediated modification of cysteine residues in MT. 

## cGMP Reporter (cygnet-2)

- Honda et al. *PNAS* 98:2437, 2001

**Notes:**
The 2nd FRET based reporter was obatain from roger Tsien.  The reporter is based on a truncated form of Protein kinase I and was designed to visualized the cGMP related pathway in living cells.  The lower panel you see the domain structure of the reporter which consists of the cGMP binding and catalytic domains of PKG 1 alpha sandwiched between cyan and yellow fluorescent protein. 
This cartoon mimics the conformation of the protein in the inactive state. When we activate sGC with NO we will increase cGMP which will bind to the reporter which will change conformation – the cyan and yellow will change distance and or angular orientation and cause a change in FRET.

Direct binding of cGMP to the PKG domain of cyg2 induces conformational change  that can be recorded as decrease in FRET

sGC is a heterodimeric protein with both  and  subunits required for catalytic activity.
NO directly binds to the heme group forming a ferrous nitrosyl-heme complex. This causes a conformational change in the planar porphyrin ring which activates sGC to catalyze the hydrolysis of GTP to cGMP.

## S-nitroso-L-cysteine activates sGC 

**Cyan**
**Emission**

**Yellow **
**Emission**

**Both**

**Control**

**L-SNCEE**

- cGMP Reporter

- S-nitroso-L-cysteine activates sGC

**Notes:**
SPAEC were transfected with cDNA encoding a fluorescent cGMP indicator (cygnet-2).  cGMP induces conformational changes in PKG Iα, detectable as decreases in FRET between the cyan and the yellow protein with the cygnet-2 reporter [10]. 
Cyan and yellow emissions at baseline and after application of L-SNCEE in live endothelial cells that transfected with cygnet-2 reporter.  Spectral reports for individual endothelial cells expressing cygnet-2.  Both L-SNCEE (top) and D-SNCEE (bottom) (50 μM) induced conformational changes in the cygnet-2 (cGMP) reporter as shown by a decrease in energy transfer with an increase in the peak emission intensity of the donor (cyan, ~485 nm) and a decrease in that of the acceptor (yellow, ~525 nm) 

## Effects of SNCEE on FRET-MT are unaffected by HbO2 

- Effects of SNCEE on FRET-MT are unaffected by HbO2

- St Croix CM. *Free Radic Biol Med*. 37:785-92, 2004

**Notes:**
Oxyhemoglobin which would be expected to block any process involving ∙NO liberated by decomposition of nitrosothiol, via its potent scavenging ability, but would not necessarily affect transnitrosation. In contrast to the effects of L-SNCEE on FRET-MT, there was no significant change in FRET in response to equimolar concentrations of D-SNCEE  to the media, demonstrating that the effects were likely mediated by transnitrosation of MT by S-nitrosothiol. In contrast, as might be expected, HbO2 had profound effects on response of the cGMP reporter to L-SNCEE.   

## Imaging isolated, perfused mouse lung

**20X**

**Notes:**
Currently establishing an intact, organ fluorescent microscopy techniques to visual these NO mediated signaling pathways in situ in the isolated, perfused mouse lung in real time.
Cannula inserted in pulmonary artery through a puncture in the right ventricle and another in the left atrium.
Perfused with Krebs-Ringer bicarbonate buffer with 5% dextran, pH 7.4. Continuous measurements of airway pressure (maintained at 10 cmH2O) and perfusion pressure – estimates pulmonary artery pressure when flow is held constant. 
Maintained for about 3-4 hours.  

## Slide 16

## Expression of FRET-MT in fixed mouse pulmonary endothelium

- Pulmonary gene transfer via tail vein injection of DOTAP:cholesterol liposomes followed by adenovirus containing cDNA for FRET-MT.

**Notes:**
In collaboration with Song Li and Annette Wilson, we have been successful in directing an adenoviral vector encoding the FRET.MT reporter to pulmonary endothelium of mouse using preinjection of cationic liposomes.  

## Expression of FRET-MT in isolated perfused mouse lung

**FRET-MT**

**Cy5-Albumin**

**Overlay**

## Acceptor photo-bleaching confirms that 

- Acceptor photo-bleaching confirms that
- FRET-MT is functional in the perfused lung

- FRET-MT is regulated by NO in pulmonary endothelium of the intact, perfused mouse lung

**Notes:**
Carbachol – activates muscarinic receptors – increases intracellular Ca, activates eNOS and relaxes vasculature. PI 3-kinase/Akt-dependent eNOS activation plays a role in carbachol-induced NO generation in the pulmonary artery 

##  NO donors also increase labile zinc in the intact perfused mouse lung

- A: Zinquin, pretreatment, pseudocolored 2P section in *ex vivo *perfused murine lung.
- B: ZnCl2 (+ the zinc ionophore, pyrithione) to  perfusate
- C: attenuation by the zinc chelator, TPEN
- D: addition of NO donor PAPA nonoate was added to the perfusate (500 M)

**Pearce, LL *****Proc Natl Acad Sci***** 97: 477-482, 2000.**

**Notes:**
Pressurized arteriography and videomicroscopy revealed no difference between the diameter pressure curves of vessels under passive conditions. Furthermore, myogenic tone developed similarly in vessels from either types of mice after distention in the presence of L-NAME (Fig. 4A). In contrast, myogenic tone in MT-/-  vessels was significantly less than MT+/+ vessels when endogenous NO was generated (Fig. 4B), as confirmed by its partial reversibility with L-arginine (not shown). Myogenic reflexes were similar in wild-type mice whether they were backbred or directly crossed between OLA129 and C57BL6. Therefore, the interaction between NO and MT facilitates this important vascular reflex. This enhanced relaxing effect of NO during myogenic reflex in MT-/- vessels may be direct via increased availability of NO to affect presumptive targets such as soluble guanylate cyclase or calcium activated potassium channels (Pearce, 2000). Alternatively, use of the zinc-specific chelator, TPEN suggested that the enhanced relaxing effect of NO during myogenic reflex in MT/ vessels may be indirect due to the absence of releasable metals (i.e. Zn) from MT that would normally produce contraction. 
Myogenic tone refers to the ability of vascular smooth muscle to alter its state of contractility in response to changes of intraluminal pressure; the vessel constricts in opposition to an increase in intravascular pressure and dilates when the pressure decreases (Davis, 1999). Myogenic behavior has been observed in a variety of vascular beds, including veins and conduit arteries, but is especially prevalent in the resistance vasculature (Dessy, 2000) and is modified by the silmultaneous production of NO. 
Mesenteric resistance arteries [diameters of 200-250 microns at 60 mmHg (1 mmHg = 133 Pa)] were removed from wild-type and MT knockout mice. Vessels were mounted on glass cannulae in pairs (one wild type; one null mutant) in a dual chamber pressurized arteriograph. The arteriograph was placed on the stage of a compound microscope. The proximal cannula was joined in series with a pressure transducer connected to a servo-controlled peristaltic pump. This allowed a desired intraluminal pressure to be established, maintained, and recorded in the absence of flow. The vessels were maintained in Hepes-buffered salt solution and were equilibrated for 1 h at 60 mmHg. At 45 min into the equilibrium, a conditioning stretch was performed from 60 to 100 mmHg. A video camera on the microscope provided an image of the artery on a television monitor, and measurements of lumen diameter and wall thickness were continuously made by using a video dimension analyzer (17). Myogenic tone was assessed by decreasing the intraluminal pressure to 20 mmHg for 10 min, after which pressure was rapidly (2 s) increased by 20 mmHg at 4-min intervals up to 120 mmHg. L-nitro-arginine-methyl-ester (L-NAME) (0.25 mM) was added to the bath to inhibit nitric oxide synthase for 15 min before repeating the changing pressure protocol. To reverse inhibition of nitric oxide synthase, L-arginine was given at a concentration of 2.5 mM for 15 min, and the protocol was repeated. The smooth muscle was then inactivated in a solution of calcium-free Hepes saline solution with papaverine (0.1 mM) and ethylene-bis(oxyethylenenitrilo)tetraacetic acid (EGTA), and the protocol was repeated. Percent tone was calculated as [(Dr   Dpss)/Dr] × 100, where Dr is the passive (smooth muscle inactivated) diameter and Dpss is the internal diameter in buffered saline solution at each pressure. 

## Acute hypoxia and pulmonary vasoconstriction

- von Euler & Liliestrand, 1946

**Notes:**
HPV is unique to the pulmonary vascular bed...This shows the original pressure trace published in 1946 by von Euler. Data obtained in closed-chested, spontaneously breathing cats. This was the first observation of an acute increase in pulmonary arterial pressure in respondse to low PO2. HPV teleologically – automatically increases pulmonary vascular resistance in poorly aerated regions of the lung thereby redirecting pulmonary blood flow to regions richer in oxygen content. Despite intensive research since the original observations of von Euler, the mechanism responsible for HPV remains enigmatic. There is strong evidence that hypoxia causes inhibition of K channels in PASM with subsequent depolarization and Ca influx through voltage-activated Ca channels 
Cat anaethetized with chloralase open thorax, bottom trace is systemic blood pressure.  6 – is where they used 10.5% O2 in N2. 
HPV mediated appears to be uniform across the lung with a prominent role for small resistance vessels.

## Small vessels of the subpleural vasculature constrict in response to hypoxia 

- Small vessels of the subpleural vasculature constrict in response to hypoxia

**Notes:**
We used laser scanning confocal microscopy (Zeiss 510Meta) of the subpleural vasculature of a perfused lung isolated from a Tie2-GFP mouse (Jackson Laboratories). The The TIE2-GFP mouse expresses GFP under the direction of the endothelial-specific receptor tyrosine kinase (Tie2) promoter and can therefore be used to visualize pulmonary vessels. Images were collected using a 40X PlanNeoFluar (1.3NA) oil immersion optic at 512X512 pixels. We observed a 10-15% decrease in vessel diameter in small vessels (<60 mm) of the subpleural vasculature in response to hypoxia. These changes in diameter were reversed upon return to normoxia. Such reversibility suggest that changes in diameter are not caused by compression of the vessels due to edema formation. 
It has been long recognized that the constriction of small (>100 mm) muscular pulmonary arteries contributes to the increase in PVR in hypoxic regions of the lung whereas smaller arterioles thatwhich tend to lack a smooth muscle layer were largely assumed to be passive. Evidence obtained using X-ray angiograms (Al-Tinawa, 1994) combined with hemodynamic modeling of the lung vascular bed (Haworth, 1991), however, suggested that pulmonary capillaries contribute substantially to the total change in pulmonary vascular resistance during hypoxia. There is also direct evidence using videomicroscopy to measure vessel diameter demonstrating that hypoxia induces active constriction of 30-50 mm pulmonary microvessels. Active capillary constriction has also been reported in the pancreas rete capillaries and the spleen and indirect evidence using vasoactive chemicals to induce reorganization of the endothelial microfilament system suggests that capillary endothelial cells contribute to capillary constriction. In particular, the endothelial contractile apparatus was shown to be involved in reducing capillary cross-sectional area during ischemia-reperfusion in the coronary vasculature. Our preliminary data shows that small (> 50 mm) pulmonary vessels constrict in response to hypoxia(Figure 9) . As vessels of this size are generally not associated  with smooth muscle these data suggest that endothelial contraction contributes to HPV in the isolated perfused mouse lung. 

## MT -/- mice show no increase in labile zinc in response to hypoxia and have a blunted HPV 

- MT -/- mice show no increase in labile zinc in response to hypoxia and have a blunted HPV

**Notes:**
In contrast to the hypoxia-induced increases in labile zinc shown in MT wild-type mice, hypoxia has no effect on FluoZin-3 fluorescence in the IPL of MT -/- animals. Furthermore, the MT -/- mice showed a significantly blunted hypoxic pressor response (n=2 per group).
Also note data with zinc chelation

## Hypoxia modifies FRET.MT in cultured endothelial cells

**FRET.MT Reporter**

**Emission Wavelength (nm)**

**Emission Wavelength (nm)**

## Hypoxia regulates FRET.MT in the IPL of MT wild-type mice

**Normalized Emission Intensity**

## Hypoxia-induced  increases in zinc are dependent on NO  

- Hypoxia-induced  increases in zinc are dependent on NO

**Notes:**
While the increases in intracellular calcium in response to hypoxia are well documented, little is know regarding hypoxia and zinc flux.
Primary cultures of mouse lung endothelial cells were loaded with the zinc-sensitive fluorophore, FluoZin-3 (5 mM, Molecular Probes) and imaged in a closed perfusion chamber using epifluorescent microscopy. After collection of baseline images, cells were exposed to perfusate that had been bubbled with anoxic gas (95% N2, 5% CO2) that acutely reduces oxygen tension to approximately 15 mmHg in this system. Under these conditions, we observed a time dependent increase in FluoZin fluorescence as shown in figure 1. Furthermore the hypoxia-induced changes in fluorescence were reversed by the zinc chelator, TPEN. Furthermore, colleagues at the University of Pittsburgh have recently confirmed that FluoZin-3 is not sensitive to changes in intracellular calcium (I.J. Reynolds, personal communication). 

## Hpoxia increases labile zinc in the isolated perfused lung of MT wild-type mice 

- Hpoxia increases labile zinc in the isolated perfused lung of MT wild-type mice

**Notes:**
We used confocal microscopy to image FluoZin-3 fluorescence in the reconstructed vasculature of the isolated, perfused mouse lung. The preparation was perfused with a peristaltic pump (0.5-2 lm/min) with a modified Krebs Henseleit solution with 4% dextran. The lung was perfused with FluoZin-3 (5 mM) for 20 min prior to the start of the experiment. The lungs were held statically inflated during imaging. Images were collected using a PlanNeofluor 20X dry objective using sequential XYZ-sections (512X512 pixels, at Nyquist axial frequency(250nm)) the three-dimensional anatomy of the vasculature was  reconstructed.  Individual XYZ-stacks were collected at one minute intervals during each hypoxic exposure to build a temporal model of vessel size change. Figure 2 shows that baseline fluorescence was significantly increased during hypoxia. The largest increases in fluorescence appear to be within the endothelium of this 40 mm vessel. 

## Summary

- Hypoxia induces increases in labile zinc in the endothelium of small diameter vessels.
- Pharmacologic (e.g. zinc-specific chelator, TPEN) and genetic (targeted ablation of zinc regulatory protein, MT) inhibition of hypoxic mediated elevations in free Zn significantly blunt HPV.
- Hypoxia-induced increases in NO synthesis contribute to HPV via formation of S-nitrosothiol in the metal binding center of MT and resultant changes in zinc homeostasis.

## Acknowledgements

- _EOH_
- Bruce R. Pitt
- Karanee Leelavanichkul
- Zi-Lue Tang
- Karla J. Wasserloos
- Molly S. Stitt
- Xianghong Lui
- _Pharmacy_
- Song Li
- Annette Wilson

- _Center for Biological Imaging_
- Simon C. Watkins
- Glenn Papworth
- _Surgery_
- Detcho Stoyanovsky
- Roger Y. Tsien (UC-San Diego)