1. Volume 122, Issue 4, Pages 1088-1100 (April 2002)
The type II inositol 1,4,5-trisphosphate receptor can trigger Ca2+ waves in rat hepatocytes 
Keiji Hirata, Thomas Pusl, Allison F. O'Neill, Jonathan A. Dranoff, Michael H. Nathanson 
Gastroenterology 
Volume 122, Issue 4, Pages (April 2002)
DOI: /gast
Copyright © 2002 American Gastroenterological Association Terms and Conditions

2. Fig. 1
Hepatocytes express the type I and II, but not the type III, InsP3R. Western analysis using type I– and type II–specific InsP3R antibodies identifies single bands of the appropriate sizes in hepatocyte lysates (60 μg). In contrast, Western analysis using a monoclonal type III–specific InsP3R receptor antibody fails to identify a band in hepatocyte lysates, although type III InsP3R is identified in lysates from the positive control, RIN-5F cells (60 μg each).
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

3. Fig. 2
The type I InsP3R is uniformly distributed in hepatocytes. Confocal fluorescence image of a section of rat liver labeled with a polyclonal antibody directed against the type I InsP3 receptor, plus rhodamine phalloidin. (A) Staining for the type I InsP3R is diffusely distributed (bar = 20 μm). (B) Same section, labeled with rhodamine phalloidin. The staining is specific for filamentous actin, which outlines the plasma membrane in hepatocytes. The staining is most intense along the canalicular membrane, where actin is most concentrated. (C) Merged image of panels A and B, which reveals that the type I InsP3R is distributed throughout the cytosol of hepatocytes. (D) Negative control image labeled with secondary antibody alone plus rhodamine phalloidin.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

4. Fig. 3
The type II InsP3R is concentrated in the apical region of hepatocytes. Confocal fluorescence image of a section of rat liver labeled with antibody CT2 directed against the type II InsP3 receptor, plus rhodamine phalloidin. (A) Staining for the type II InsP3R is most intense along discrete portions of the plasma membrane (bar = 20 μm). (B) Same section, labeled with rhodamine phalloidin. (C) Merged image of panels A and B, which reveals that the type II InsP3R is most concentrated in the pericanalicular region (arrowheads). The negative control is shown in Figure 2D, because the same secondary antibody was used to stain InsP3R-1 and InsP3R-2. (D) Higher magnification image of a separate liver section labeled with CT2 (green), plus the nuclear stain propidium iodide (red). Note that InsP3R-2 labeling closely outlines the canaliculus (arrows), consistent with panel C (bar = 10 μm).
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

5. Fig. 4
Distribution of InsP3R isoforms in isolated rat hepatocyte multiplets. (A) Confocal immunofluorescence demonstrates that the type I InsP3R is diffusely distributed in isolated hepatocytes, with some scattered punctate staining. Cells labeled with secondary antibody alone revealed no such staining (not shown; bar = 10 μm). (B) In contrast, the type II InsP3R is concentrated in the pericanalicular region. Note that cells bordered by 2 hepatocytes have 2 such pericanalicular regions. Cells labeled with secondary antibody alone revealed no such staining (not shown). (C) Live hepatocytes labeled with mitotracker red show that mitochondria are distributed throughout the cytosol in these cells.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

6. Fig. 5
The ryanodine receptor is not expressed in rat liver. By use of RT-PCR, a single 530-bp product common to all known RyR isoforms was identified in brain (lane 1), but not in liver (lane 2) or in reverse transcriptase–free negative controls (lane 3).
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

7. Fig. 6
Cyclic ADPR does not increase Ca2+ in hepatocytes. NPE-cADPR was microinjected into the middle cell of isolated rat hepatocyte triplets, and then Cai2+ was monitored by confocal microscopy as the cADPR was uncaged. (A) Transmission image of an isolated rat hepatocyte triplet. (B) Confocal image of the same triplet, loaded with the fluorescent Ca2+ indicator fluo-4. The nucleus (n) and cytosol (c) of the middle cell are indicated. This and subsequent images of cells loaded with fluo-4 are pseudocolored according to the color scale shown. (C) Simultaneous confocal image of Texas red, which was co-injected with caged cADPR into the middle cell (bar = 10 μm). (D) Tracing of nuclear and cytosolic fluorescence at baseline, after flash photolysis of cADPR, and after stimulation with vasopressin (10 nmol/L). Photorelease of caged cADPR does not increase Ca2+ in either the cytosol or nucleus, but stimulation with vasopressin promptly increases Ca2+ in both compartments. To ensure maximal uncaging, cells were exposed to UV light for 100–200 ms. For comparison, increases in Cai2+ were observed after exposure to UV light for as briefly as 2 ms in cells injected with caged InsP3 (range of exposures, 2–100 ms in Figures 7 and 8). This tracing is representative of that observed in 5 separate hepatocytes.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

8. Fig. 7
InsP3-induced Ca2+ signals in hepatocytes. Microinjected NPE-InsP3 was injected into the middle cell of isolated rat hepatocyte triplets, then uncaged by UV flash photolysis as Cai2+ was monitored by fluo-4 fluorescence by using time-lapse confocal microscopy. Cells were exposed to UV light for 2–100 ms, although most were exposed for <10 ms. (A) Transmission image, followed by serial confocal images obtained before and 160, 640, and 1280 ms after a 5-ms UV flash. The apical (a) and basolateral (b) regions of the middle cell are indicated in the transmission image. The pseudocolor scale used to reflect increases in fluo-4 fluorescence is shown at the bottom (bar = 10 μm). (B) Corresponding tracing, which shows the apical and basolateral increase in fluo-4 fluorescence observed after InsP3 is uncaged. The tracing is scaled by its baseline value so that initial fluorescence is 100%, and arrowheads are inserted to indicate the time points shown in panel A. The peak increase in apical and basolateral fluorescence is attained at 1280 ms and is readily determined. However, note that other kinetic parameters (the time delay until Cai2+ begins to increase, time until Cai2+ reaches its peak, and the rate at which Cai2+ increases from 20% to 80% of its peak) are difficult to measure with this collection rate. (C) Cai2+ increases to the same extent in the apical and basolateral region. Hepatocytes were subjected to a range of flash durations to examine responses to a range of InsP3 concentrations. Each data point represents an individual uncaging experiment (n = 60). The solid line here and in subsequent figures is the linear regression curve for these data, and the dashed lines represent the 95% confidence intervals for the regression curve. The slope for this regression curve is 0.997, indicating that apical and basolateral Cai2+ increase to the same extent.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

9. Fig. 8
InsP3-induced Ca2+ signals examined by confocal line scanning microscopy. (A) Confocal fluorescence image of an isolated rat hepatocyte triplet loaded with fluo-4. The line scan was collected along the yellow line. The apical (a) and basolateral (b) portions of the middle hepatocyte that are along the scan line are indicated. (B) Corresponding line scan obtained along the long axis of the triplet after uncaging of InsP3. The horizontal gray bar indicates the UV flash (bars = 10 μm [horizontal] and 1 second [vertical]). This corresponds to a spatial resolution (along the horizontal axis) of 0.32 μm/pixel and a temporal resolution (along the vertical axis) of 6 ms/pixel (3.6 seconds from top to bottom). The apical (a) and basolateral (b) regions of the middle hepatocyte are indicated here as well. (C) Graphical representation of the increase in fluorescence detected in the apical and basal regions of the middle cell in a similar experiment. In contrast to the tracing in Figure 6B, the time delay until the onset of the Cai2+ signal, the time delay until Cai2+ reaches its peak, and the rate at which Cai2+ increases from 20% to 80% of its peak each are readily measured here. (D) The apical delay from uncaging of InsP3 to onset of Cai2+ signaling is shorter than the basolateral delay. Each data point represents an individual uncaging experiment (n = 32). The slope for this regression curve is 1.44, which shows that Cai2+ begins to increase in the apical region sooner than in the basolateral region over the entire range of increases in Cai2+ that are elicited. The unity line (slope = 1) is shown in gray in this and subsequent panels for comparison. (E) Cai2+ signals increase more quickly in the apical region than in the basolateral region after flash photolysis of InsP3. The slope for this regression curve is 0.435, indicating that the rise time is more brief for apical Cai2+ signals. The inset shows the initial portion of the curve in greater detail. (F) Apical Cai2+ peaks sooner than basolateral Cai2+ after flash photolysis of InsP3. The slope for this regression curve is 1.20, which shows that the time required for apical Cai2+ to reach its peak is less than that required for basolateral Cai2+ over the entire range of increases in Cai2+ that are elicited.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

10. Fig. 9
Vasopressin induces Ca2+ waves in isolated hepatocyte triplets. (A) Confocal fluorescence image of an isolated rat hepatocyte triplet loaded with fluo-4. (B) Corresponding line scan obtained along the long axis of the triplet during stimulation with vasopressin (10 nmol/L). (C) Apical increases in Cai2+ began sooner than basolateral increases in Cai2+ after stimulation with vasopressin. Tracing indicates the apical and basolateral increases in Cai2+ in the middle cell of the triplet shown in the previous panels. The increases begin at the arrows. Vasopressin was added ~20 seconds before the increases in Cai2+ shown here; this reflects the lag time that is known to occur between vasopressin stimulation and the onset of Cai2+ signaling in hepatocytes.22 This result is representative of that observed in 27 separate experiments. (D) Comparison of the rate of the apical and basolateral Cai2+ increase induced by vasopressin and InsP3. The regression line and the corresponding 95% confidence intervals are those calculated for InsP3 (Figure 8E). Note that most values obtained for vasopressin are within this 95% confidence region.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions