1. Extracellular polyamines regulate fluid secretion in rat colonic crypts via the extracellular calcium-sensing receptor 
Sam X. Cheng, John P. Geibel, Steven C. Hebert 
Gastroenterology 
Volume 126, Issue 1, Pages (January 2004)
DOI: /j.gastro

2. Figure 1
Time-dependent changes in IP3 accumulation in isolated rat colonic epithelial cells elicited by either additions of 1 mmol/L spermine alone, 2 mmol/L Ca2+o alone, or in combination. The incubations were performed at 37°C for indicated periods of time before measurements of cellular IP3 were made. Data shown are mean + SEM of 4–6 experiments. The 2 initial peak responses to Ca2+o and/or spermine were observed at 15 and 60 seconds, respectively. The 60-second IP3 peak (indicated by arrow) was used in subsequent experiments shown in Figures 2–4.
Gastroenterology  , DOI: ( /j.gastro )

3. Figure 2
Dose-response relationship for spermine-induced increases in IP3 accumulation in the absence and presence of different concentrations of Ca2+o. Cells were incubated with indicated drugs for 60 seconds at 37°C before changes in IP3 were measured. Data shown are representative of 3 separate experiments. Note that Ca2+o appears to be required for and sensitizes the action of spermine. With the presence of 2 mmol/L Ca2+o, addition of as little as 25 μmol/L spermine was able to elicit a rise in IP3.
Gastroenterology  , DOI: ( /j.gastro )

4. Figure 3
Effects of polyamines spermine, spermidine, and putrescine on IP3 accumulation. All experiments were performed in the presence of 0.5 mmol/L Ca2+o. Data are average of 3 or 4 separate experiments. For comparison, changes in IP3 were expressed as fractional IP3 accumulation by normalizing to control IP3 responses elicited by 0.5 mmol/L Ca2+o alone without polyamines. IP3 responses elicited by 0.5 mmol/L spermine were mimicked by spermidine and putrescine but at higher concentrations. ∗P < 0.05 vs. control (i.e., 0.5 mmol/L Ca2+o alone).
Gastroenterology  , DOI: ( /j.gastro )

5. Figure 4
Dose-response relationship for Ca2+o-elicited IP3 responses in the absence and presence of 100 μmol/L spermine. Cells were incubated with indicated agonists for 60 seconds at 37°C before changes in IP3 were measured. Data shown are mean + SEM of 4–7 separate experiments and plotted either in arithmetic (A) or semilog (B) scale. With the presence of spermine, cells show greater sensitivity to Ca2+o. The differences between the 2 curves were statistically significant (F tests: F = (A) and (B), P = and , respectively).
Gastroenterology  , DOI: ( /j.gastro )

6. Figure 5
Dependence of Ca2+o and Ca2+o-sensing mechanism for spermine-elicited Ca2+i responses. Epithelial cells (∼0.25–0.5 mg protein/mL) were freshly isolated from rat distal colon and loaded with Fluo-3, and changes in Ca2+i were then recorded by spectrofluorometry. The tracings shown are representative of 3 separate Ca2+i responses to additions (arrows) of Ca2+o alone (A) or spermine in the absence of Ca2+o (B) or presence of physiologic 2 mmol/L Ca2+o (C) or maximal doses of Ca2+o (D). In these experiments, the initial Ca2+o was 0 (B) or 0.1 mmol/L (A, C, D). Note that all the tracings shown in A–D were performed under same dye-loading conditions on cells from the same preparations (see Materials and Methods section). Ca2+i responses to spermine require presence of Ca2+o (B and C), and no additional spermine effect on Ca2+i was found when maximal Ca2+i response was achieved by stimulation with a cumulative dose of 5 mmol/L Ca2+o (D).
Gastroenterology  , DOI: ( /j.gastro )

7. Figure 6
Effects of spermine on basal netJv of isolated perfused colonic crypts. In these experiments, netJv was measured in the absence of exogenous influences on cell cyclic AMP. Crypts were perfused symmetrically (i.e., both apically and basolaterally) with Ringer’s solutions containing 1.0 mmol/L Ca2+o with the presence (A and C) or absence (B) of 50 μmol/L basolateral bumetanide and/or presence (B and C) or absence (A) of 5 mmol/L basolateral spermine. The first and second series of data in panel B showed Jv under the same experimental conditions before and after solution change. Note that spermine increased basal netJv and that the spermine effect was primarily achieved via modulation of a bumetanide-sensitive, secretory component of netJv. Unless otherwise indicated, the data shown are the distribution (open boxes and dashed lines) and average (solid boxes and solid lines) of 5 separate, paired experiments, each performed on 3 crypts of distal colons of 1 animal. ∗P < 0.05; NS, not significant; secJv, secretory flux of netJv; absJv, absorptive flux of netJv.
Gastroenterology  , DOI: ( /j.gastro )

8. Figure 7
Effects of apical spermine on secretory Jv in isolated perfused colonic crypts stimulated by forskolin (FSK), applied basolaterally. In these experiments, crypts were perfused symmetrically (i.e., both apically and basolaterally) with Ringer’s solutions containing either 1.0 mmol/L (A), 0.5 mmol/L (B) or 0.1 mmol/L (C) of Ca2+o. The first bars in each panel showed Jv under basal conditions. A positive value indicates net fluid absorption, and a negative value indicates secretion. Note that addition of spermine to the luminal perfusate dose dependently diminished the forskolin effect in stimulating net fluid secretion and that as little as 1 nanomolar spermine was effective in significantly reducing the secretory Jv at 0.5 or 1.0 mmol/L Ca2+o (P < 0.01). Data shown are the average of 5–45 separate experiments, each performed on 3 to 6 crypts of distal colons of 1 or 2 animals.
Gastroenterology  , DOI: ( /j.gastro )

9. Figure 8
Effects of basolateral spermine on secretory Jv in isolated perfused colonic crypts stimulated by forskolin (FSK), applied basolaterally. In these experiments, crypts were perfused symmetrically (i.e., both apically and basolaterally) with Ringer’s solutions containing either 1.0 mmol/L (A), 0.5 mmol/L (B), or 0.1 mmol/L (C) of Ca2+o. The first bars in each panel showed Jv under basal conditions. Similar to what was observed for apical spermine (see Figure 7), addition of spermine to the basolateral bath dose dependently diminished the forskolin effect in stimulating net fluid secretion. As little as 1 nanomolar spermine was effective in significantly reducing the secretory Jv at 0.5 or 1.0 mmol/L Ca2+o. Data shown are the average of 5–60 separate experiments, each performed on 3 to 6 crypts of distal colons of 1 or 2 animals.
Gastroenterology  , DOI: ( /j.gastro )

10. Figure 9
Dose-response relationship for spermine-elicited Jv responses in the presence of 0.1, 0.5, or 1.0 mmol/L Ca2+o. Spermine was applied apically (A) or basolaterally (B). Other experimental conditions are detailed in the legends to Figures 7 and 8. Individual Jv responses were normalized and plotted in semilog scale. The sensitivity to spermine is left shifted by increasing basolateral or apical Ca2+o from 0.1 mmol/L to 0.5 mmol/L (F tests [basolateral/apical]: F = 28.80/36.88; P = /0.0001) or from 0.1 mmol/L to 1.0 mmol/L (F = 6.348/78.43; P = 0.02/0.0001). The sensitivity to spermine is also slightly greater in the presence of 1.0 mmol/L Ca2+o than 0.5 mmol/L Ca2+o, even if this difference did not reach statistical significance (F = 3.27/3.24; P = 0.07/0.08). Also, cells show greater sensitivity to basolateral spermine than apical spermine in the presence of 0.1 mmol/L Ca2+o (F = 12.26, P = ), but this difference was not apparent when Ca2+o was at 0.5 mmol/L (F = 0.02, P = 0.99) or 1.0 mmol/L (F = 0.32, P = 0.86).
Gastroenterology  , DOI: ( /j.gastro )