Cygnets'. Intracellular Guanosine 3',5'- Cyclic Monophosphate Sensing in Primary Cells Using Fluorescence Energy Transfer
Guanosine 3',5'-cyclic monophosphate (cGMP) is a key player in the regulation of various physiological processes, including smooth muscle tone, neuronal excitability, epithelial electrolyte transport, phototransduction in the retina, and cell adhesion (Eigenthaler et al., 1999; Kaupp and Seifert, 2002; Lincoln et al., 2001; Schlossmann et al., 2003). Although the cGMP second messenger pathway has been steadily gaining recognition for its role in intracellular signaling, cGMP remains the least well understood member of the cyclic nucleotide family due to the following peculiarities of the cGMP signal transduction system. (1) The control of cGMP levels is complex, with formation of cGMP occurring through two different forms of guanylyl cyclases (Russwurm and Koesling, 2002; Wedel and Garbers, 2001) and degradation by a number of cGMP-specific phosphodiesterases (PDEs) (Rybalkin et al., 2003). (2) The intracellular actions of cGMP are mediated by cGMP-dependent protein kinases (PKGs) (Hofmann et al., 2000) and by several types of cyclic nucleotide-activated ion channels (Kaupp and Seifert, 2002). (3) The enzymes modulating cGMP levels are expressed differentially throughout mammalian tissues. Furthermore, a tightly controlled equilibrium of synthesis and breakdown generates highly flexible intracellular cGMP transients, contributing to the experimental and conceptual obstacles posed by the multiplicity in mechanisms of cGMP signaling and the difficulty of studying cGMP in broken cell preparations.
Genetically encoded indicators based on the insertion of conformationally sensitive domains between two mutants of green fluorescent protein (GFP) that participate in fluorescence resonance energy transfer (FRET) have proved to be powerful tools for observing the dynamics of intracellular signaling molecules noninvasively. Examples of such indicators include those that detect the second messengers Ca2+ (Miyawaki et al., 1997) and cAMP (Zaccolo and Pozzan, 2002). For the construction of our cGMP indicators, which we have named cygnets (cyclic GMP indicators using energy transfer), we chose PKG as the central cGMP sensor because it binds cGMP with high affinity, undergoes a conformational change in response to cGMP, and is not restricted to membranes (Pfeifer et al., 1999; Ruth et al., 1991; Zhao et al., 1997). We have demonstrated the validity of this intramolecular FRET approach and could show that cygnets (i) are exclusively selective for cGMP, (ii) allow detection of intracellular cGMP in single living cells, (iii) are fully reversible to monitor fast spatial and temporal cGMP changes, and (iv) are minimally invasive when analyzing intracellular cGMP signaling events (Honda et al., 2001; Sawyer et al., 2003). This article provides a detailed methodology for the use of our latest cGMP indicator version, Cygnet-2.1, in primary cell culture.
II. MATERIALS AND INSTRUMENTATION
The RFL-6 (CCL-192) established cell line is from American Type Culture Collection. The FuGENE 6 transfection reagent is from Roche (1814443). Cygnet- 2.1 DNA (pcDNA3.1(-)-Cygnet-2.1) is constructed as described previously (Honda et al., 2001).
Dulbecco's modification of Eagle's medium (DMEM, with 4.5g/liter glucose and L-glutamine, with sodium pyruvate; MT 10-013-CM), Ham's F-12 medium (MT 10-080-CM), fetal bovine serum (FBS, MT 35-010-CV), trypsin EDTA (MT 25-052-CI), amphotericin B (MT 30-003-CI), and gentamycin sulfate (30-005-CR) are from cellgro by Mediatech, Inc. Bovine serum albumin (BSA, A7638) and potassium phosphate [dibasic (K2HPO4, P-3786) and monobasic (KH2PO4, P-0662)] are from Sigma-Aldrich. Elastase (100617) is from ICN Biomedicals. Collagenase (199152) is from Worthington Biochemical Corporation. Penicillin / streptomycin / neomycin (15640-055) is from Gibco-BRL/Invitrogen. Collagen (354231), BD Falcon polystyrene 35 × 10-mm (353001) and 60 × 15- mm (353004) cell culture dishes are from BD Biosciences. Benzamidine hydrochloride (105240250) and EDTA (118430010) are from Fisher Scientific. The extracellular solution used for microscopic imaging consists of Hank's balanced salt solution (HBSS; cellgro by Mediatech, MT 21-020-CV) with 20mM HEPES (cellgro by Mediatech, MT 25-060-CI) and 2g/liter glucose (Sigma-Aldrich, G-7021).
cGMP (40732-48-7; reconstituted in distilled H2O) and fluorescence grade 8-(4-chlorophenylthio)guanosine- 3', 5'-cyclic monophosphate [8-pCPT-cGMP; 51239-26-0; reconstituted in dimethyl sulfoxide (DMSO)] are from BIOLOG Life Science Institute.
The following chemicals have been used to modulate intracellular cGMP levels: Nitric oxide donors S-nitrosoglutathione (GSNO; Calbiochem 487920, protect from light and reconstitute in cold distilled H2O free of divalent cations other than Ca2+; stock solution is stable for 2h at 4°C), N-(2-aminoethyl)-N-(2- hydroxy-2-nitrosohydrazino)-l,2-ethylenediamine [NOC-22, spermine NONOate; Calbiochem 567703; reconstitute in 0.1 N NaOH, ≥pH 10; t½ of NO release = 230min in phosphate-buffered saline (PBS), pH 7.4, 22°C] diethylamine NONOate (DEA NONOate; Calbiochem 292500, reconstitute in distilled H2O, t½ of NO release - 16 min in PBS, pH 7.4, 22°C), and (±)- S-nitroso-N-acetylpenicillamine (SNAP; Calbiochem 487910; protect from light and reconstitute in DMSO; tl/2 of NO release = 10 h) are reconstituted to 100× the final concentration of 100µM prior to use and stored on ice until needed.
Atrial [ANP (3-28), rat; 14-5-44A] and brain [BNP (1- 32), Rat; 14-5-11A] natriuretic peptides are from American Peptide Company. The C-type natriuretic peptide [CNP (6-22), human and porcine; 05-23-0310] is from Calbiochem. Peptides are reconstituted in distilled H2O to 100 µM stock solutions and stored at -20°C for several months until their use at a final concentration of 0.1-1 µM.
The phosphodiesterase inhibitors 3-isobutyl-1- methylxanthine (IBMX; 410957; nonspecific inhibitor of cAMP and cGMP phosphodiesterases, final concentration 0.6 mM), vinpocetine (677500; PDE I inhibitor; final concentration 0.2 mM), 8-methoxymethyl-3- isobutyl-l-methylxanthine (MM-IBMX; 454202; PDE I inhibitor; final concentration 40µM), erythro-9- (2-hydroxy-3-nonyl)adenine, HCl (EHNA; 324630; PDE II inhibitor; final concentration 8 µM; reconstitute in distilled H2O), 1,6-dihydro-2-methyl-6-oxo-(3,4'- bipyridine)-5-carbonitrile (Milrinone; 475840; PDE III inhibitor; final concentration 3 µM), 4-[3-(cyclopentyloxy)-4-methoxyphenyl]-2-pyrrolidinone (Rolipram; 557330; PDE IV inhibitor; final concentration 8 µM), and 1,4-dihydro-5-(2-propoxyphenyl)- 7H-1,2,3-triazolo[4,5-d]pyrimidine-7-one (Zaprinast; 684500; PDE V inhibitor; final concentration 4.5µM) are from Calbiochem. After reconstitution in DMSO (unless otherwise noted), stock solutions are stored at -20°C and used within 2 months. Sildenafil is a generous gift from Pfizer.
The fluorescence spectrometer F-4500 is from Hitachi. Quartz fluorescence cells (14-385-918A) for spectrophotometers and Dithiothreitol (DTT, 16568- 0050) are from Fisher Scientific. Dithiothreitol (DTT, 16568-0050),
A light-duty portable punch size XX (130010001) outfitted with a 0.5-in. round dye (Type O) is from Roper Whitney of Rockford, Inc. A Sylgard 184 silicone elastomer kit is from Dow Corning Corporation. Coverslips (22 × 22 mm, 1 thickness; 12-544-10) are acquired from Fisher Scientific.
An inverted Nikon Diaphot 200 microscope equipped with a Nikon Fluor 40/1.30 oil Ph4DL objective (Part 140010) is outfitted with an ORCA ER cooled charge-coupled device camera (Hamamatsu). Three filter wheels, one each for excitation, emission, and neutral density filters, and a shutter at the excitation filter wheel are controlled by Lambda 10-2 optical filter changers from Sutter Instruments. A lambda LS xenon arc lamp and power supply are also obtained from Sutter Instruments. The Cameleons 2 filter set (71007a) purchased from Chroma Technologies for dual emission consists of a D440/20x excitation filter, a 455DCLP dichroic, and two emission filters (D485/40m and D535/30m). Neutral density filters (0.1, 0.3, 0.5, 1, 2, 3) are also obtained from Chroma. Image acquisition is controlled by a computer loaded with Metamorph and Metafluor 4.64 software from Universal Imaging (Media, PA). A stage adaptor to hold 35-mm imaging dishes was constructed at the Instrumentation and Model Facility at the University of Vermont.
A. Cygnet Expression and Purification
Express recombinant Cygnet-2.1 protein in Spodoptera frugiperda (Sf9) cells using the Bac-to-Bac baculovirus system (GIBCO/BRL) and purify using cAMP-agarose as described earlier (Honda et al., 2001).
B. In Vitro cGMP Titration
Perform cGMP titrations of Cygnet-2.1 by adding 50nM of protein to a quartz fluorescence cell with buffer (50 mM KPO4, pH 6.8, 10 mM DTT, 10 mM benzamidine, and 5mM EDTA) for a final volume of 500 µl. Excite samples in a fluorescence spectrometer at 432nm and monitor emission intensities from 450 to 550nm. Plat the ratios of the 475 to 525 emission intensities against the concentration of cGMP added to the sample to generate a titration curve.
C. Cell Culture
The following cell types have been used successfully to express cygnets and monitor intracellular cGMP levels. Details on their preparation and handling are as follow.
1. Rat Fetal Lung Fibroblast Cells (RFL-6)
Cells should be cultured according to the supplier. Briefly, grow RFL-6 cells in Ham's F12 medium supplemented with 20% fetal bovine serum at 37°C, 5% CO2. Subculture every 5-6 days with 0.25% trypsin at a ratio of 1:4 and plate on glass-bottom dishes for imaging. RFL-6 cells should not used beyond passage number eight. Routinely verify cGMP responses with cygnet-transfected RFL-6 cells, an established cell line known to respond with high cGMP levels upon stimulation (Ishii et al., 1991).
2. Rat Aortic Smooth Muscle Cells (RASMC)
Preparation and Culture of RASMC
Alternatively, RASMC can be dissociated from the aorta using an isolation protocol adapted from Cornwell and Lincoln (1989) and Smith and Brock (1983).
D. Preparation of Imaging Dishes
E. Transfection of Cygnet-2.1
F. Data Acquisition and Analysis
Dual-Emission Imaging Protocol Using Metafluor
Making a Movie of a Pseudocolored Cell
G. Example of cGMP Sensing in Primary Rat Aortic Smooth Muscle Cells
At approximately 50-60% confluency, passage one cells were transfected with FuGENE 6 using a 3:1 ratio of FuGENE reagent to pcDNA3.1-Cygnet-2.1 DNA. Cells were imaged 24h posttransfection at 25°C using Hank's balanced salt solution with 20 mM HEPES (pH 7.35) and glucose (2g/liter) as the extracellular solution and 500-ms exposures at 10-s intervals.
Cygnet-2.1 expressed in both rat aortic smooth muscle cells (Figs. 1A and 1B) and RFL-6 cells (Honda et al., 2001) demonstrated cytosolic localization and nuclear exclusion when ECFP and citrine emissions were viewed individually. Pseudocoloring of the cell (Figs. 2C and 2D) correlates the ratio of the ECFP/citrine emissions to a color scale, with lower and higher ratios and cGMP levels represented by blue and red, respectively. A saturating dose of the cell membrane-permeable cGMP analog 8-pCPT-cGMP changed the pseudocoloring from blue to red (Fig. 1D) and correlates to a 30% increase in FRET ratio (Fig. 2A). However, in vitro results with purified Cygnet-2.1 showed that cGMP consistently causes a 40-50% ratio change. To analyze this apparent discrepancy, Cygnet- 2.1 purified from Sf9 cells was titrated with cGMP and 8-CPT-cGMP (Fig. 2B). The purified indicator's maximum ratio change attained with cGMP was approximately 45%, while the analog still only elicited a maximal ratio change of 30%. In fact, in cells permeabilized with 50µM digitonin in the presence of saturating concentrations of cGMP, cygnets demonstrated a 47% FRET ratio change as expected from the in vitro results (Fig. 2C).
Activating the particulate guanylate cyclase with 1 µM brain natriuretic peptide produced a FRET ratio change of 28% (Fig. 3A). Atrial natriuretic peptide produced a similar response, and both ANP- and BNPinduced cGMP accumulation could be washed out to near baseline (data not shown). Analysis of the individual emission intensities of ECFP and citrine demonstrates the mechanism of our cGMP indicator: cGMP generated in response to the activation of guanylate cyclase binds to the indicator and triggers a conformational change in the receptor. Subsequent alterations in the relative spatial arrangement of the two fluorophores result in a loss of energy transfer from ECFP to citrine and an increase in ECFP emission and a decrease in that of citrine (Fig. 3B).
Experiments acquiring images at 10-s intervals can run for greater than 30min. We have accumulated as many as 200 exposures before applying a stimulus and have observed no significant loss in indicator sensitivity. It should be noted that cells have the tendency to react quite differently to the same stimulus.
This work was supported by NSF Grant MCB-9983097 (WRGD) and the Totman Medical Research Trust.
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