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Originally published In Press as doi:10.1074/jbc.M401084200 on February 29, 2004

J. Biol. Chem., Vol. 279, Issue 20, 21461-21468, May 14, 2004
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Ca2+-sensing Transgenic Mice

POSTSYNAPTIC SIGNALING IN SMOOTH MUSCLE*

Guangju Ji{ddagger}, Morris E. Feldman{ddagger}, Ke-Yu Deng{ddagger}, Kai Su Greene{ddagger}, Jason Wilson{ddagger}, Jane C. Lee{ddagger}, Robyn C. Johnston{ddagger}, Mark Rishniw{ddagger}, Yvonne Tallini{ddagger}, Jin Zhang§, Winthrop G. Wier§, Mordecai P. Blaustein§, Hong-Bo Xin{ddagger}, Junichi Nakai¶, and Michael I. Kotlikoff{ddagger}||

From the {ddagger}Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853, the §Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201, and the Laboratory for Neuronal Circuit Dynamics, RIKEN Brain Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan

Received for publication, January 30, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Genetically encoded signaling proteins provide remarkable opportunities to design and target the expression of molecules that can be used to report critical cellular events in vivo, thereby markedly extending the scope and physiological relevance of studies of cell function. Here we report the development of a transgenic mouse expressing such a reporter and its use to examine postsynaptic signaling in smooth muscle. The circularly permutated, Ca2+-sensing molecule G-CaMP (Nakai, J., Ohkura, M., and Imoto, K. (2001) Nat. Biotechnol. 19, 137-141) was expressed in vascular and non-vascular smooth muscle and functioned as a lineage-specific intracellular Ca2+ reporter. Detrusor tissue from these mice was used to identify two separate types of postsynaptic Ca2+ signals, mediated by distinct neurotransmitters. Intrinsic nerve stimulation evoked rapid, whole-cell Ca2+ transients, or "Ca2+ flashes," and slowly propagating Ca2+ waves. We show that Ca2+ flashes occur through P2X receptor stimulation and ryanodine receptor-mediated Ca2+ release, whereas Ca2+ waves arise from muscarinic receptor stimulation and inositol trisphosphate-mediated Ca2+ release. The distinct ionotropic and metabotropic postsynaptic Ca2+ signals are related at the level of Ca2+ release. Importantly, individual myocytes are capable of both postsynaptic responses, and a transition between Ca2+-induced Ca2+ release and inositol trisphosphate waves occurs at higher synaptic inputs. Ca2+ signaling mice should provide significant advantages in the study of processive biological signaling.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Genetically encoded cell reporters of cell signaling hold great promise for the study of in vivo cell physiology. This field has advanced rapidly recently with the development of fluorescence resonance energy transfer-based probes such as cameleons (2), fluorescent proteins that can be modified following expression (3), and re-engineered fluorescent proteins (1, 4) (for review, see Ref. 5). The use of these tools as transgenes in mammals has been quite limited, however, because of difficulties associated with a limited signal/noise ratio of the probes and toxic consequences of cellular expression. Circularly permutated Ca2+-sensing molecules such as G-CaMP (1) and pericam (4) display fluorescence increases ~3-fold over the physiological range of [Ca2+]i and Ca2+ binding is minimal in cells at resting levels of [Ca2+]i, important experimental advantages for in vivo imaging. Here we report the production of transgenic mice expressing G-CaMP under control of the smooth muscle myosin heavy chain promoter and the use of these mice to resolve nerve-evoked, postsynaptic signaling in smooth muscle. Postsynaptic Ca2+ signaling shows a graded shift from ryanodine receptor to inositol trisphosphate receptor signaling; these postsynaptic signals have discreet thresholds and are mediated by different neurotransmitters. G-CaMP-signaling mice provide an effective tool to examine Ca2+ signaling in single lineages from complex tissues.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Generation of smGC Mice—pGEX-4T3-smooth muscle isoform of myosin heavy chain (smMHC)1 was created by subcloning the 16.1-kb smMHC promoter construct, including the first intron from pAUGSAHE-SM-MHCP (6) (SalI digestion) into pGEX-4T3 (SalI/XhoI). G-CaMP (GenBankTM accession number AH550759, previously termed GCaMP1.3) was first released from pN1-GCaMP1.3 (1) by AflII (blunt end) and BglII and subcloned into pBluescript digested with XbaI (blunt end) and BamHI to form pBS-GCaMP1. G-CaMP was released with SacII (blunt end) and SmaI from pBS-GCaMP1.3 and subcloned into pGEX-4T3-smMHC digested with SalI to create pGEX-smMHCGCaMP1. The transgene (17.6 kb) was released by NotI, purified using a QIAEX II kit, and injected into fertilized mouse eggs by standard methods. Animals were genotyped by PCR using primers specific for the circularly permutated eGFP, using an N-terminal probe (758-778: 5'-CTT CAG CTC GAT GCG GTT CAC) and a C-terminal probe (286-306: 5'-GAG CAA AGA CCC CAA CGA GAA G-3') to amplify a 493-bp fragment (Fig. 1A). Tail DNA was extracted by standard methods and 1 µl of the digest used as a template.



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  		FIG. 1.
smGC transgenic mice. A, diagram of transgene. The rat smooth muscle myosin heavy chain (smMHC) promoter construct consisted of ~4 kb of 5'-untranslated sequence (5' UT), the noncoding first exon (E1), and the roughly 12-kb first intron. The G-CaMP construct consisting of the myosin light chain kinase peptide (M13), circularly permutated eGFP (eGFPn and eGFPc, N and C-terminal fragments, respectively), and calmodulin (CaM), was placed directly downstream of a Kozak sequence (linkers are indicated by red bars). Arrows show the position of primers used for genotyping, which are specific for circularly permutated eGFP. Unique restriction sites are shown. B, genotyping. PCR of litter identifying the 423-bp G-CaMP fragment in one founder (smGC-2). The PCR fragment identifies a circularly permutated GFP sequence. C, transgene expression. Sections from smGC3 mice were processed with pre-immune (left) or anti-GFP (right) antisera, demonstrating robust expression of the transgene confined to smooth muscle tissues. Magnifications are urinary bladder x4, coronary artery x40, bronchus x40, and colon x20.

 
Immunostaining—Formalin-fixed, paraffin-embedded tissues were immunostained with rabbit polyclonal anti-GFP antibodies, 1:50 dilution (Chemicon International, Temecula, CA), and counterstained by standard methods (7). Briefly, tissues were deparaffinized and rehydrated, blocked with 0.5% H2O2 in methanol for 10 min at room temperature, incubated with Pronase for 20 min at room temperature, and blocked with normal goat serum (prediluted; Zymed Laboratories Inc.) and 5% bovine serum albumin. Tissues were then incubated with the primary antibody for 2 h at 37 °C and the secondary antibody (biotinylated goat anti-rabbit, Zymed Laboratories Inc., prediluted) for 15 min at room temperature, followed by incubation with Streptavidin peroxidase (Zymed Laboratories Inc., prediluted) and then aminoethyl carbazole substrate (Zymed Laboratories Inc. kit) for 5 min at room temperature. Sections were then washed and counterstained with hematoxylin before mounting with fluoromount.

Imaging—smGC mice were euthanized with CO2 and tissues rapidly removed and dissected on ice. Segments of the urinary bladder running along the axis from the neck to the fundus were cut to preserve nerve conduction pathways. The segments were placed with the serosal surface on the bottom of an optical recording chamber and a retaining clip containing a stimulating electrode and kevlar fibers placed on top to maintain physiological length of the tissue. The muscle segments were perfused with buffered extracellular solution consisting of (mM): 137 NaCl, 5.4 KCl, 1.8 CaCl2, 1.0 MgCl2, 10 glucose, 10 HEPES, pH 7.4). Stimulation pulses were generated by a Grass S48 stimulator controlled by computer-generated TTL pulses and confocal images obtained with a Bio-Rad Radiance 2000 confocal head attached to a Nikon TMIII inverted microscope. Confocal x-y images were obtained at 71-ms intervals and the stimulation signal encoded in the confocal image. Transmitted light images indicated contractions in individual myocytes, particularly during sustained or high frequency stimulations. The effect of individual cell movement on fluorescence in the isometric preparation was minimized by using low frequency stimulations and excluding from analysis myocytes that undergo prominent shortening. Data were analyzed and pseudolinescans generated by custom-written Matlab routines or by ImageJ. Pseudocolor images were scaled linearly, using the lowest and highest pixel values for the entire image series; pseudolinescans are presented as F/Fo by dividing all pixels by the mean value for that pixel before the event and then scaled between 0.5 and 3. Descriptive parameters for Ca2+ flashes and waves were obtained by least squares fit using custom-written routines in Matlab. An event was considered a Ca2+ wave if wave propagation could be distinguished in consecutive images. Single cells were dissociated as previously described (8). Mesenteric artery images were recorded at 2 frames/s using a Nipkow spinning disk confocal head. Images showing cross-sections of the arterial walls were captured through the center, parallel to the long axis, of an intact pressurized (70 mm Hg) mesenteric artery at 35 °C. The lung slice preparation was similar to that previously described (9). Briefly, the trachea was cannulated and the lungs filled with 2% agarose (type VII-A: low gelling temperature) before removal. Following cooling, single lobes were sectioned at 100 µm using a vibrated tissue sectioner (OTS-5000; Electron Microscopy Sciences). Confocal images were obtained using a Bio-Rad Radiance 2000 confocal scan head coupled to a Nikon inverted microscope. In both cases, fluorescence profiles were normalized by dividing the time-dependent fluorescence (F) by the average prestimulation fluorescence (Fo).

In Situ Calibration of G-CaMP—Individual muscle segments from smGC mice were incubated in 100 µg/ml saponin in physiological solution at room temperature for 1 h. Segments were mounted on the microscope and fluorescence measured at fixed excitation strength and photomultiplier gain during exposure to solutions in which EGTA and Ca2+ were added at mM concentrations to fix the [Ca2+]free, using the WINMX programs (10). Mean fluorescence was averaged for 3-5 myocytes within each image for each solution condition, these values were averaged, and the means of all experiments were fit by least squares to the Hill equation: ((F - Fo)/(Fmax - Fo) = 1/(1 + 10n(x-Kd)), where F, Fo, and Fmax are the observed, zero Ca2+, and saturating Ca2+ fluorescence, respectively, n the Hill coefficient, Kd the affinity constant, and x the pCa.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Production of Smooth Muscle G-CaMP Transgenic Mice—The Ca2+-sensitive, circularly permutated eGFP variant G-CaMP (1) was used to construct a transgene by insertion downstream of the transcriptionally active first intron of the smMHC promoter (6, 11) (Fig. 1A), which we have previously used to direct robust, smooth muscle-specific eGFP expression in mice (7). The G-CaMP Ca2+ reporter utilizes a circularly permutated eGFP fusion protein that obtains a photoactive conformation upon binding of the C-terminal Ca2+/calmodulin with the N-terminal M13 recognition peptide from myosin light chain kinase. Three founders were crossed with C57BL/6 mice and experiments conducted in two of these lines with the highest expression (smGC-2 and smGC-3, Fig. 1B). Hemizygous smGC mice produce litters of normal size and sex ratio and have expected longevity; histopathological analysis revealed no discernable phenotype associated with the expression of the Ca2+-sensing reporter. Transgene expression was examined by immunostaining with an anti-eGFP polyclonal antibody that recognizes the circularly permutated G-CaMP. As shown in Fig. 1C, the transgene is robustly expressed in vascular and nonvascular smooth muscle and expression is confined to tissues of smooth muscle lineage. Importantly, expression is observed in virtually all cells within a muscle tissue, an obvious advantage in the context of examining cell signaling.

Ca2+-sensing in Transgenic Mouse Cells—We examined the function of G-CaMP in a range of smooth muscle tissues. To confirm Ca2+ signaling under optimal optical conditions, G-CaMP was first examined in single myocytes dispersed from the urinary bladder of smGC mice. As shown in Fig. 2A, enzymatically dispersed myocytes display a low but detectable level of fluorescence at rest and respond to Ca2+-mobilizing stimuli (application of 100 µM methacholine) with a greater than 2-fold increase in fluorescence. The Ca2+ reporter is diffusely distributed in the cytosol with no evidence of organellar accumulation, in organelles, which is a common problem associated with membrane-permeant small molecule indicators, and the indicator reports typical Ca2+ oscillations and waves in myocytes despite the slower fluorescence kinetics than achieved, for example, with small organic molecules with low Kd, such as Fluo-4 (1, 12).



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  		FIG. 2.
Function of G-CaMP expressed in transgenic mice. A, Ca2+ fluorescence in single smooth muscle cells dissociated from an smGC transgenic mouse. Above, confocal fluorescent images from a series taken before and after application of 100 µM methacholine to a urinary bladder myocyte from an smGC-2 mouse. Middle and below, fluorescence profile and pseudolinescan from the same experiment. The profile represents the average value of the pixels, indicated by the red circle, and the pseudolinescan, the values of the pixels indicated by the dotted line, taken from 500 sequential images. B, calibration of G-CaMP in situ. Top, confocal images of saponin, permeabilized urinary bladder smooth muscle segments incubated in solutions clamped at 100 nM and 1 µM free Ca2+ as shown and transmitted light image of muscle. Bottom, mean data from three such experiments and the fit to a Hill equation. C, G-CaMP Ca2+ signaling in bronchial smooth muscle in the mouse airway. Top, confocal images from a lung slice before stimulation and at peak response to application of 50 µM methacholine. Bottom, normalized fluorescence from bronchial myocytes (red circle) shows marked oscillatory responses. D, G-CaMP Ca2+ signaling in arterial vascular smooth muscle during vasocontraction. Top, selected confocal images before and during application of 10 µM phenylephrine to an isolated, pressurized mesenteric artery. Bottom left, normalized arterial myocyte fluorescence from the entire image series. Bottom right, higher resolution confocal image of single layer of arterial myocytes during phenylephrine vasoconstriction. All calibration bars are 10 µm.

 
A significant advantage of genetically encoded reporters is the ability to monitor intracellular signaling events in defined cell lineages within complex multicellular tissues without disrupting cell-cell contacts or permeabilizing membranes, a frequent necessity for efficient loading of small molecule reporters. Because G-CaMP does not retain fluorescence emission at 37 °C (1), experiments were conducted at room temperature in ex vivo tissues. The Ca2+ dependence of G-CaMP fluorescence was calibrated in saponin-permeabilized urinary bladder muscle segments exposed to solutions with fixed free Ca2+ ranging from 50-1.8 mM and 1 µM Mg2+. As shown in Fig. 2B, in three such experiments the fluorescence of individual cells within muscle segments increased sharply over the physiological range of cellular free Ca2+. The fit of these data to the Hill equation yielded a Kd of 328 nM and a Hill coefficient of 2.9, which may be compared with the values of 235 nM and 3.3 for the recombinant protein measured in vitro (1). There is a well documented increase in the apparent Kd of Ca2+ indicators measured in the presence of cellular proteins compared with in vitro measurements in physiological salt solutions, probably due to substantial protein binding of the indicator molecules (13-15). Given the numerous cellular calmodulin-binding proteins and the effect of protein binding on the Ca2+ affinity of calmodulin (16), it is likely that interactions between G-CaMP and cellular proteins markedly influence the apparent affinity of the complex for Ca2+. The apparent Kd and Hill coefficient of G-CaMP in smGC mice indicate that ~93% of the total Ca2+-dependent fluorescence occurs over the range between 100 nM and 1 µM free [Ca2+]i. Thus, G-CaMP is a useful tool for the measurement of cytosolic Ca2+ signaling, although the high Hill coefficient relative to small molecule dual wavelength (17) or single wavelength (12) Ca2+ indicators indicates that G-CaMP will be fully bound (and fluorescence thus saturate) under conditions of maximal Ca2+ signaling.

The function of G-CaMP was verified in lung, arterial, and urinary bladder smooth muscle. As shown in Fig. 2, Ca2+ signaling was observed in bronchial smooth muscle during constriction of a small airway produced by application of 50 µM methacholine and in arterial myocytes during adrenergic vasoconstriction. Dynamic changes in [Ca2+]i in myocytes within these complex tissues could easily be resolved, and Ca2+ oscillations in individual myocytes were routinely observed. These measurements confirm the utility of smGC mice for the examination of dynamic Ca2+ signaling in a range of physiological preparations. Moreover, Ca2+ signaling can be measured in what is often a single layer of smooth muscle during physiological contractions without disturbing the essential integrity of the tissues.

Dual Modes of Postsynaptic Ca2+ Signaling in Urinary Bladder Smooth Muscle—Neuromuscular coupling in many smooth muscles, including the urinary bladder, is known to result from the co-release of acetylcholine and ATP (for review, see Ref. 18), although little is known about the nature of nerve-evoked Ca2+ signals or the precise role of specific neurotransmitters in mediating distinct postsynaptic Ca2+ events. We used smGC mice to examine postsynaptic neuromuscular signaling in the intact mouse detrusor (urinary bladder) by field-stimulating intrinsic nerves at a distance of several centimeters from imaged myocytes (Fig. 3A). All postsynaptic Ca2+ signaling was eliminated by application of 1 µM tetrodotoxin (data not shown), confirming that the responses consisted of conducted action potentials in intrinsic nerves. Low frequency (0.5-1.0 Hz) stimulation evoked random Ca2+ responses in individual myocytes (Fig. 3B) rather than synchronous responses in all cells, which likely reflects the low probability of neurotransmitter release at individual synapses (19). Postsynaptic Ca2+ responses resulted in 1.5-3-fold increases in fluorescence of the genetically encoded reporter in situ, similar to the responses observed in single cells (Fig. 2).



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  		FIG. 3.
Postsynaptic Ca2+ signaling in smGC mice. A, recording preparation used to identify nerve-evoked postsynaptic Ca2+ responses in smooth muscle. B, postsynaptic Ca2+ responses in detrusor muscle. Upper and lower figure sets are taken from the same series of images during which the tissue was stimulated at 1 Hz and confocal images obtained every 71 ms. Top, the x-y image pairs of each set are the images at the time of the stimulus (red box) and at the peak response. Middle, the average fluorescence profile (F/Fo) from the pixels identified by the white circle in the stimulation image. Dotted lines show position of images in full experiment linescan. Bottom, pseudolinescan from the pixels indicated by the white line in the stimulation image. The point of occurrence of the x-y images on the profile and linescan is indicated by the dotted lines. LSCM, laser scanning confocal microscope.

 
Interestingly, two distinct postsynaptic Ca2+ transients were observed. The most common events, which we term Ca2+ flashes, were transient increases in [Ca2+]i confined to individual myocytes, or occasionally 2-3 myocytes, characterized by a rapid, almost simultaneous rise in [Ca2+]i throughout the entire myocyte (Fig. 3B). This mode of Ca2+ signaling is quite similar to spontaneous events reported in arterial smooth muscle loaded with fluo-3 (20). Less frequently, a second pattern of Ca2+ firing was observed, consisting of slowly propagating, asynchronous Ca2+ waves. Asynchronous Ca2+ waves dominated the postsynaptic response at higher stimulation frequencies (4-10 Hz), suggesting different thresholds for the modes of Ca2+ signaling. Examples of these markedly different postsynaptic Ca2+ signals within the same tissue segment are shown in Fig. 4; pseudolinescans constructed from a line of pixels drawn along the length of myocytes demonstrate slow Ca2+ waves proceeding from one end of a cell to the other at a propagation rate of roughly 15 µm/s, whereas in Ca2+ flashes wave propagation was not detected in x-y image sets acquired at ~14 Hz.



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  		FIG. 4.
Dual modes of postsynaptic Ca2+ signaling in smGC mice. A, examples of a Ca2+ flashes (top) and asynchronous Ca2+ waves (bottom) in the same muscle segment, taken from a series of 300 images obtained every 71 ms. The image at which the stimulus was applied is shown at left and indicated as time 0, and the timing of subsequent issues is shown above each image. At right pseudolinescans constructed from pixels indicated by the dotted line in the first image demonstrate the rapid rise of Ca2+ along the length of the myocyte above and the propagation of the wave in the images shown below. B, fluorescence profile for the Ca2+ flash (red) and Ca2+ wave (gray) in panel A; mean value of pixels indicated by the red circles. Arrows above indicate the timing of the stimuli in each experiment. Note the rapid rise and exponential decay of the Ca2+ flash and the more complex decay kinetics of the Ca2+ wave.

 
Pharmacologic Dissection of Postsynaptic Ca2+ Responses—We reasoned that the two distinct modes of postsynaptic Ca2+ signaling likely reflect distinct neurotransmitter responses, which have been related to specific contractile responses in the detrusor (21). Previous reports in fluo-4-loaded smooth muscle tissues have identified nerve-evoked or spontaneous asynchronous, propagating Ca2+ waves (22-25), and purinergic, postsynaptic subcellular junctional Ca2+ transients (19, 24) in smooth muscle cells in situ. Asada et al. (20) have reported spontaneous Ca2+ flashes in arterial myocytes. Dual modes of nerve-evoked Ca2+ signaling in the same myocytes have not been reported in smooth muscle, however, and we therefore further investigated the nature of these Ca2+ signals. Ca2+ flashes occurred with a shorter lag time following stimulation, the kinetics of the rise and decay of the [Ca2+]i transients were markedly faster, and the peak [Ca2+]i obtained was significantly lower than observed in Ca2+ waves (Fig. 5A). Nerve-evoked postsynaptic Ca2+ flashes were resistant to atropine (1 µM; Fig. 5B) (although the frequency of events was decreased) and to xestospongin (a selective inhibitor of InsP3 receptors) (1 µM, not shown), and the atropine-resistant Ca2+ flashes were completely blocked by ryanodine (10 µM), indicating that they resulted from a rapid Ca2+ release from ryanodine receptors (Fig. 5, B and C). Thus Ca2+ flashes do not arise from the release of acetylcholine or the generation of InsP3. Conversely, Ca2+ waves were never observed in the presence of atropine (1 µM; Fig. 5C) and were virtually abolished following the application of xestospongin (not shown), indicating that cholinergic signaling through InsP3 Ca2+ release results in asynchronous Ca2+ waves, consistent with previous reports (22, 24) that these signals arise from cholinergic signaling. Conversely, the purinergic receptor antagonist suramin blocked all Ca2+ flashes, and all postsynaptic responses were also abolished by the combined action of atropine and the non-selective purinergic receptor antagonist suramin (data not shown). Selective desensitization of P2X receptors with {alpha},{beta} methyleneATP ({alpha},{beta} meATP) further demonstrated that Ca2+ flashes arise by stimulation of P2X receptors (see below). The rapid and synchronous nature of Ca2+ flashes suggested that they arise through P2X-mediated depolarization and Ca2+-induced Ca2+ release, rather than simply by Ca2+ influx through nonselective P2X channels. This hypothesis was confirmed in two ways. First, incubation with 10 µM ryanodine abolished virtually all Ca2+ flashes without decreasing the probability of Ca2+ waves (Fig. 5D), indicating that ryanodine receptor-mediated Ca2+-release results in Ca2+ flashes. Second, rapid inhibition of voltage-dependent Ca2+ channels with nitrendipine (10 µM) also almost completely eliminated Ca2+ flashes (Fig. 5D) without diminishing the probability of postsynaptic asynchronous Ca2+ waves.



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  		FIG. 5.
Mechanism of Ca2+ release in postsynaptic Ca2+ flashes and Ca2+ waves. A, distinct properties of Ca2+ flashes and Ca2+ waves. Mean amplitude (F/Fo) and kinetics (s) for Ca2+ flashes and waves and frequency of events before and after exposure to 1 µM atropine. B, Ca2+ flashes are not associated with cholinergic neurotransmission. Mean Ca2+ fluorescence within a single myocyte undergoing repeated nerve stimulation (arrow) demonstrates virtually identical postsynaptic Ca2+ flashes in the same cell before and after application of 1 µM atropine. Following perfusion with ryanodine, repeated stimulations no longer elicit the Ca2+ flash. C, Ca2+ flash and wave probability from experiments as in panel B. The probability of evoking Ca2+ flashes is not significantly affected by application of atropine but is almost abolished following exposure to ryanodine. Conversely, Ca2+ waves are not observed in the presence of atropine. Note the higher probability of Ca2+ flashes relative to Ca2+ waves in control stimulations. D, Ca2+ flashes occur through CICR. In the presence of 10 µM ryanodine (left) or 10 µM nitrendipine (right), the probability of observing postsynaptic Ca2+ flashes is markedly reduced, whereas an increase in the average number of evoked Ca2+ waves was observed (not significant). Experiment number indicated by parentheses. * and ** indicate significance at the 0.05 and 0.01 levels, respectively.

 
Relationship between Postsynaptic Ca2+ Responses—Several aspects of the myocyte Ca2+ signals were striking relative to the relationship between the two types of neurotransmitter responses. First, as stated, the probability of observing Ca2+ flashes was markedly higher than that of Ca2+ waves at low stimulation frequencies, which may result largely from prejunctional effects related to the stimulus-dependent release of ATP and acetylcholine (26) but may also relate to the intrinsic efficiency of the downstream coupling events. Second, we commonly observed Ca2+ flashes followed by a Ca2+ wave in the same cell (Fig. 6A), whereas Ca2+ waves were never followed by Ca2+ flashes during a single stimulation train, suggesting that Ca2+ release by P2X/CICR occurred at a lower threshold of neural input and that muscarinic/InsP3 release depleted Ca2+ stores sufficiently to render myocytes refractory to subsequent CICR. Third, low frequency stimulation commonly evoked repeated Ca2+ flashes, whereas higher frequency stimulation resulted in a wave in the same cell (Fig. 6B), further indicating a higher threshold for muscarinic postsynaptic Ca2+ responses.



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  		FIG. 6.
Relationship between modes of postsynaptic Ca2+ signaling. A, individual myocytes are capable of both postsynaptic responses. Left, initial fluorescence of myocytes with the responding cell outlined. Right, pseudocolor images (cropped and rotated for clarity) show an initial Ca2+ flash followed by a Ca2+ wave in the same myocyte. The image at which the stimulus was applied is shown at left and indicated as time 0. Below, the average fluorescence values from the pixels identified by the white circle in the stimulus image are plotted, and the pseudolinescan from the pixels indicated by the dotted line in the stimulus image is shown. The pseudolinescan indicates the lower amplitude, simultaneous rise in [Ca2+]i followed by an asynchronous Ca2+ wave that propagates parallel to the axis of the dotted line. Arrows indicate stimulation pulses. B, frequency dependence of postsynaptic signaling modes. Selected images at left show stimulus (red box) and subsequent Ca2+ flash during 1-Hz stimulation. Group at right from subsequent 5-Hz stimulation, showing conversion of response to a slow Ca2+ wave. Below, the average fluorescence from all the pixels within the white circle in the first image (red line and arrows indicate first scan at 1 Hz-stimulation, black line indicates second scan at 5-Hz stimulation; stimulations shown until postsynaptic response occurs). Note that the postsynaptic response in the same cell has shifted mode from a Ca2+ flash (red) to an asynchronous Ca2+ wave (black) and that the postsynaptic Ca2+ signal occurs earlier in the series at higher frequency stimulation. C, desensitization of P2X receptors converts postsynaptic responses to asynchronous Ca2+ waves. Selected images from tissue exposed to {alpha},{beta} meATP (50 µM) show the development of synchronous Ca2+ waves after stimulation (red box). Note that all responses in this experiment are Ca2+ waves. D, probability of Ca2+ flashes and Ca2+ waves in the presence of {alpha},{beta} meATP. ** indicates significance at the 0.01 level.

 
The role of P2X receptors in rapid Ca2+ flashes and the relationship between postsynaptic signaling modes in individual cells was established by using the selective P2X agonist {alpha},{beta} meATP, which rapidly desensitizes P2X1 receptors, highly expressed in urinary bladder smooth muscle (27, 28). In the presence of {alpha},{beta} meATP, Ca2+ flashes were almost completely abolished and all postsynaptic signaling shifted to prominent, asynchronous Ca2+ waves, the probability of which was markedly enhanced by P2X receptor desensitization (Fig. 6, C and D). The augmented probability of Ca2+ waves at low stimulation frequency under conditions of P2X1 desensitization (and the increase in the probability of Ca2+ waves in the presence of ryanodine or nitrendipine (Fig. 5D)) further suggest that the rapid release of Ca2+ by ionotropic receptor stimulation and CICR raises the threshold for the slower activating and more sustained Ca2+ release occurring through InsP3R gating. Thus, these data suggest a degree of postsynaptic signal integration within myocytes.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The combination of transgenic, molecular engineering, and imaging technologies provides remarkable opportunities to design and target the expression of molecules that can be used to report critical cellular events in vivo (29-32). smGC mice reported here provide a means to efficiently examine physiological, postsynaptic signaling in vascular and nonvascular smooth muscle, and additional lines of mice with lineage-restricted expression of G-CaMP are likely to provide an important experimental advantage for the study of multicellular physiological processes in mammals. Moreover, crosses of these and other signaling mice with gene-targeted mice will provide a powerful tool to evaluate mammalian gene function in vivo. G-CaMP is robustly expressed in smGC mice and can be used to examine vascular and nonvascular Ca2+ signaling in situ.

We have used G-CaMP transgenic mice to investigate postsynaptic Ca2+ responses in individual myocytes in intact muscle of the urinary bladder. Our results indicate that individual visceral smooth muscle cells respond to motor nerve depolarization by two distinct modes of postsynaptic Ca2+ responses: rapid Ca2+ flashes that occur through CICR mediated by ATP binding to P2X receptors and slower, InsP3-mediated Ca2+ waves mediated by acetylcholine-activating muscarinic receptors. These data are consistent with numerous previous studies identifying cholinergic and purinergic excitatory neurotransmission and a prominent P2X component in visceral smooth muscles (27, 28, 33-35), extending information about these processes to the postsynaptic Ca2+ responses within individual myocytes in intact tissues. Evoked Ca2+ responses arising from the separate actions of ATP and acetylcholine on ionotropic (P2X) and metabotropic (muscarinic) receptors are remarkably distinct at the level of intracellular Ca2+ release; rapid purinergic Ca2+ flashes result from CICR, mediated by P2X and channel gating and ryanodine receptor gating. Although the CICR process likely initiates with one or more localized Ca2+ sparks (36), the imaging bandwidth employed, as well as the poor quantum efficiency and slow transition kinetics of G-CaMP at low Ca2+ concentration (1), precluded identification of such events in these experiments. Measurements of Ca2+ signals in single myocytes correspond extremely well with a previous study of the neural basis of contractile responses in the urinary bladder in which atropine inhibited high frequency contractions in the rat and guinea pig detrusor but had little effect at low stimulation frequencies, whereas {alpha},{beta} meATP maximally inhibited low frequency stimulations (21). Thus, distinct modes of postsynaptic Ca2+ release in single myocytes strongly correlate with force production in the intact muscle.

Our findings also highlight the specific relationship between the different modes of postsynaptic Ca2+ signaling in smooth muscle. The rapid ionotropic response occurs with a distinctly lower stimulus threshold than metabotropic Ca2+ signaling, and the occurrence of Ca2+ release through an ionotropic response appears to increase the threshold for the metabotropic response, indicating an interdependence at the level of intracellular Ca2+ release. That is, by releasing Ca2+ from the sarcoplasmic reticulum, rapid Ca2+ flashes appear to decrease the probability of Ca2+ waves, thereby modulating the kinetics and intensity of muscle contraction. Our data do not exclude other interactions, however, such as the participation of ryanodine receptor Ca2+ release channels in the propagation of Ca2+ waves. Importantly, individual myocytes are capable of both postsynaptic responses, which are functionally linked at the level of Ca2+ release. These findings suggest a general mechanism by which a low frequency of firing of autonomic motor nerves results in a rapid mobilization of intracellular Ca2+ and graded, transient contractions, whereas increases in motor nerve depolarizations, through presynaptic mechanisms that result in augmented release of acetylcholine and shifts in the mode of postsynaptic signaling, result in slower Ca2+ waves and sustained contractions. This mechanism likely facilitates the rapid activation of detrusor muscle.

In summary, we report the production and use of transgenic mice for the investigation of Ca2+ signaling in living tissues. Advances in the quantum yield and stability of protein-based signaling molecules, as well as the generalization of this approach to the detection of other intracellular molecules, will likely markedly extend the scope and physiological relevance of studies of cell function.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants HL045239, D58795 [GenBank] , and DK065992 (to M. I. K.), HL45215 (to M. P. B.), and HL64708 (to W. G. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| To whom correspondence should be addressed: Dept. of Biomedical Sciences, College of Veterinary Medicine, Cornell University, T4018 VRT, Box 11, Ithaca, NY 14853-6401. Tel.: 607-253-3336; Fax: 607-253-4447; E-mail: mik7{at}cornell.edu.

1 The abbreviations used are: smMHC, smooth muscle isoform of myosin heavy chain; CICR, calcium-induced calcium release; GFP, green fluorescent protein; {alpha},{beta} meATP, {alpha},{beta} methyleneATP; InsP3, inositol trisphosphate. Back


   ACKNOWLEDGMENTS
 
We thank Dr. G. K. Owens for providing the smMHC promoter construct, Pat Fisher for help with tissue preparation and immunostaining, and Dr. Gwen Spizz for critically reading the manuscript.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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