Gastrointestinal Physiology - Gianrico Farrugia

Figure 1.1.

Distribution of transient inward current recorded from human jejunal circular smooth muscle cells at a holding voltage of -100 mV. In 73 of 100 cells studied, two components of the inward current were identified (A1), a component with faster kinetics of activation and inactivation and a second component with slower kinetics of activation and inactivation carried by Ca2+ through L-type Ca2+ channels (8). The current-voltage relationship of the representative currents shown in A1 is shown in A2 with maximal peak inward current for the two components at -30 mV and -10mV. In 8 of 100 cells only the faster of the two components could be definitively made out. A representative example is shown in B1 with initial inward current seen at -60 mV and maximal peak inward current at -30 mV (B2). In 19 of 100 cells only the slower Ca2+ current could be definitively made out. A representative example is shown in C1 with initial inward current seen at -50 mV and maximal peak inward current at 0 mV (C2). The insets show current traces at -30 mV (solid line) and 0 mV (dashed line) from each of the three cells. The outward current seen at positive voltages (e.g. B1) is due to a non-selective cation current activated at voltages positive to 0 mV.

Figure 1.2.

Effect of removal of Na+ from the bath. Panel A shows representative current traces obtained using the pulse protocol shown in the inset. Both fast and slower components were present in this cell. Panel B shows the effect of substitution of Na+ with N-methyl-D-glucamine (NMDG) on inward currents. The current-voltage relationships are shown in panel C. The inset shows the mean maximal peak currents for both the fast and slow components in normal Ringer (black bars) and in NMDG (white bars). Removal of Na+ resulted in complete loss of the fast component (Na+) with no effect on the slow (Ca2+) current (inset, * P < 0.0001). Panel D shows the normalized mean current-voltage relationship obtained from human jejunal circular smooth muscle cells. Currents were recorded either from cells with no discernible Ca2+ current (n = 8) or in the presence of manganese replacing Ca2+ (n = 5) or in the presence of nifedipine (1 uM, n = 9). Inward Na+ current was first noted at about -60 mV with maximal peak inward current seen at -25 mV.

Figure 1.3.

Steady state activation and inactivation of the Na+ current obtained using the pulse protocols shown in the insets. Fits were obtained from the average V1/2 and slope (k) calculated for each experiment (n = 8 for the activation experiments, n = 7 for the inactivation experiments). V1/2 for activation was -47 mV and k 4.8. V1/2 for inactivation was -78 mV and k -3.2. A window current was present at voltages from -75 mV to -60 mV.

Figure 1.4.

Internal and External QX314 blocks the Na+ current. Panel A shows block of the Na+ current by internal QX314 (100 uM, circles, n=4). The data were fit with a single order exponential. In contrast there was no decrease in Na+ current in control records obtained in the absence of QX314 (triangles, n=3). Panel B shows the effect of external QX314 (500 uM, n=4). External QX314 also blocked the Na+ current.

Figure 1.5.

Slowing of inactivation by ATX II. Panel A shows a representative recording of Na+ current obtained from a sinlge smooth muscle using the pulse protocol shown in the inset. The cell was held at -100 mV. Panel B shows records from the same cell 10 min after addition of ATX II (10 uM). Inactivation was slowed by ATX II. Panel C shows the results of exponential fits to the inactivation curves (n=5). Control data were best fit with a single exponential with a tau of 4.8+/-0.4 ms at -35 mV (triangles). In contrast the data obtained in the presence of ATX II were best fit with a double exponential with taus of 3.9+/-0.8 ms and 50+/-19 ms at -35 mV (circles, squares).

Figure 1.6.

SCN5A is present in human jejunal smooth muscle cell libraries. Panel A shows cDNA bands obtained using PCR reactions with primers designed against portions of the published sequence of SCN5A. The bands were of the expected size for SCN5A and sequencing of the bands (two with overlapping sequence) revealed 99% homology with the published SCN5A sequence (panel B). Panel C shows cDNA bands obtained using RT-PCR from mRNA isolated from human jejunal circular smooth muscle cells collected by laser capture micro-dissection. Bands were the expected size for SCN5A and GADPH (control) and the products were confirmed by sequencing. No band was seen using c-kit primers.

Table 1:

Amino acid differences between HJSCN5A and the published sequence for human cardiac SCN5A. The human jejunal smooth muscle cell Na+ channel HJSCN5A has been deposited in the GenBank data libraries under the accession number AY038064.

Figure 2.1.

Expression of SCN5A in human jejunal smooth muscle. Gene-specific primers designed against Na+ channel alpha subunit SCN5A were used for RT-PCR amplification. The products were separated by 2 % agarose gel electrophoresis and visualized by ethidium bromide staining. RT-PCR showed that SCN5A was detected in human jejunal smooth muscle, freshly dissociated circular smooth muscle cells (about 100 cells) and human heart (positive control). There were no products in RT- heart sample with SCN5A primers.

Figure 2.2.

Expression of TTX-resistant sodium channels in human jejunal smooth muscle. cDNA libraries made from dissociated human jejunal circular smooth muscle cells were used to screen three known TTX-resistant Na+ channel alpha subunits (SCN5A, SCN10A and SCN11A). A product of the expected size was identified using oligonucleotide primers designed to amplify SCN5A. Products of the expected size were not amplified using primers designed against the sequences for SCN10A and SCN11A. No template with SCN5A primers provided a negative control.

Figure 2.3.

RT-PCR amplification of SCN5A from single human jejunal circular smooth muscle cells. Single smooth muscle cells were collected with patch pipettes under RNase-free conditions. Gene-specific primers for SCN5A were designed to span an intron to exclude contamination with genomic DNA. RT-PCR amplification showed that SCN5A was present in human jejunal circular smooth muscle cells. Automatic sequencing confirmed the identities of the purified cDNA products. Bath solution without aspiration of cells was used as a negative control. This figure is representative of results in a total of five independent experiments.

Figure 2.4.

SCN5A was amplified from circular smooth muscle cells collected by immuno-LCM. Approximately 1500 smooth muscle cells were collected from circular and longitudinal muscle layers of human jejunum and analyzed by RT-PCR. After reverse transcription, PCR was performed on multiple aliquots with gene specific primers. cKit was not detected in the collected samples indicating no contamination by ICC. An SCN5A cDNA band was detected in samples from circular muscle (Panel A) but not longitudinal muscle (Panel B).

Figure 2.5.

Immunolabeling of Na+ channel alpha subunit in human jejunum smooth muscle. Cryostat sections of human jejunum were immunolabeled with an anti-pan Na+channel antibody directed against sequences conserved in all known Na+ channel alpha subunits (panel A). Strong immunoreactivity was detected in circular smooth muscle layer (CM) and myenteric plexus (MP). Only background immunoreactivity was present in longitudinal muscle cells (LM) with punctate immunolabeling in the longitudinal muscle layer likely representing labeling of Na+ channels in nerve fibers. Panel B (control). Preabsorption of the primary antibody with antigen resulted in no immunoreactivity. Scale bar is 200 um.

Figure 2.6.

Full-length SCN5A mRNA was detected in human jejunal smooth muscle. 10 ug total RNA from human heart and 30 ug total RNA from human jejunal smooth muscle was used for Northern blot analysis. A cDNA probe, specific for SCN5A, hybridized to single target on both heart and jejunal smooth muscle RNA blots. The size of the mRNA was approximately 8.5 kb as reported in previous studies on SCN5A expression in human heart. The location of 28S on the same gel is indicated along the left side.

Figure 3.1.

Perfusion increases peak Na+ current. Panels A and B show representative Na+ current recordings obtained from a human jejunal circular smooth muscle cell at a holding voltage at -100 mV and using the pulse protocol in the inset. Recordings were taken in a still bath with Ringer?s solutions (A) and then during perfusion with the Ringer?s solution at 10 ml/min (B). Panel C shows the normalized current voltage relationships before and after perfusion and panel D the percent increase in maximal peak Na+ current evoked by perfusion (* p<0.01,n =41).

Figure 3.2.

Effect of perfusion on activation and inactivation. Panel A shows activation (time to peak) and panel B inactivation (single tau) for the Na+ current before and after perfusion. No changes in both activation and inactivation kinetics were seen.

Figure 3.3.

Cytochalasin D inhibits the perfusion-induced increase in peak Na+ current. Panels A and B show representative Na+ current recordings obtained using the pulse protocol in the inset. Panel A shows recordings were taken of a cell incubated in a solution of NaCl Ringer with 1 µM nifedipine and then the cell was perfused at 10ml/min with NaCl Ringer. Panel B shows recordings from the same cell after a 15 min incubation in NaCl Ringer solution containing 10 µM cytochalasin D and 1 µM nifedipine and then perfused with Ringer?s solution at 10ml/min. Panel C shows the normalized current voltage relationships and panel D displays the mean percent increase in maximal peak Na+ current evoked by perfusion (* p<0.01). Cytochalasin inhibited the perfusion-induced increase in Na+ current (n=6).

Figure 3.4.

Intracellular gelsolin inhibits perfusion-induced increase in Na+ current. Panels A and B show representative Na+ current recordings using the pulse protocol in the inset. Panel A shows currents obtained from a cell patch-clamped with 1 µM gelsolin in the intracellular solution and NaCl Ringer in the bath. Panel B shows a recording from the same cell perfused at 10ml/min with NaCl Ringer 15 min after break-in. Panel C shows the normalized current voltage relationships and panel D displays the mean percent increase in maximal peak Na+ current evoked by perfusion. Perfusion did not induce an increase in maximal peak Na+ currents in the presence of gelsolin (n=7).

Figure 3.5.

Phalloidin does not alter perfusion-induced increase in maximal peak Na+ current. Panels A and B show representative Na+ current recordings using the pulse protocol in the inset. Panel A shows control recordings of a cell in NaCl Ringer subsequently perfused with the same solution at 10ml/min. Panel B shows recordings of the same cell incubated for 15 min in Ringer?s solution with 25 µM phalloidin and then perfused at 10ml/min with NaCl Ringer alone. Panel C shows the normalized current voltage relationships and panel D displays the mean percent increase in maximal peak Na+ current evoked by perfusion (* p<0.01, n=7). Phalloidin incubation did not alter the perfusion-induced increase in Na+ current.

Figure 3.6.

Colchicine does not inhibit the perfusion-induced increase in Na+ current. Panels A and B show representative Na+ current recordings obtained using the pulse protocol in the inset. Panel A shows control recordings of a cell in NaCl Ringer which was then perfused with the same solution at 10ml/min. Panel B shows recordings of the same cell incubated for 15 min in NaCl Ringer with 10 µM colchicine and then perfused at 10ml/min. Panel C shows the normalized current voltage relationships and panel D displays the mean percent increase in maximal peak Na+ current evoked by perfusion (* p<0.05, n=10).

Figure 3.7.

Paclitaxel does not inhibit the perfusion-induced increase in Na+ current. Panels A and B show representative Na+ current recordings using the pulse protocol in the inset. Panel A shows control recordings from a cell in NaCl Ringer subsequently perfused with the same solution at 10ml/min. Panel B shows recordings of the same cell incubated for 15 min in NaCl Ringer with 25 µM paclitaxel and then perfused at 10ml/min with NaCl Ringer alone. Panel C shows the normalized current voltage relationships and panel D displays the mean percent increase in maximal peak Na+ current evoked by perfusion (* p<0.05, n=6).

Figure 3.8.

Acrylamide does not alter the perfusion-induced increase in maximal peak Na+ current. Panels A and B show representative Na+ current recordings obtained using the pulse protocol in the inset. Note the slower Ca2+ current present in these cells as nifedipine was not added to the bath. Panel A shows recordings from a cell incubated in NaCl Ringer with 5 mM acrylamide for 30 min. Panel B shows a recording from the same cell perfused at 10ml/min with NaCl Ringer. Panel C shows the normalized current voltage relationships and panel D displays the mean percent increase in maximal peak Na+ current evoked by perfusion (* p<0.01).

Figure 4.1.

Expression of Syntrophins in human intestinal smooth muscle. A, Gene specific primers designed against syntrophin a, B1,B2, y1, and y2 were used for PCR amplification from cDNA libraries. Single cDNA bands were obtained from syntrophin a, B1 and B2 primers but not y1. Three bands were observed on syntrophin y2 amplification. Sequence analysis showed that all three cDNA fragments were different transcripts of syntrophin y2. B, To determine anatomical localization of syntrophins in intestinal muscle layers approximately 1500 human jejunal smooth muscle cells from circular muscle and longitudinal muscle were collected by laser capture micro-dissection and syntrophin message amplified by RT-PCR on multiple aliquots with gene specific primers. Bands for syntrophin a, B2, and y2 were present in circular muscle and bands for syntrophin a, B1, and B2 in longitudinal muscle. C, Immunolabeling for syntrophin y2. Human jejunal sections were immunolabeled with an anti-syntrophin y2 antibody. Immunopositive smooth muscle cells were present in the circular but not longitudinal muscle layer. D, cellular localization of syntrophins. Primers designed to span introns to exclude genomic DNA contamination were used for 2-3 smooth muscle cell PCR amplification. Products of expected size for syntrophin a, B2, and y2 were 3 freshly dissociated human jejunal circular smooth muscle cells. Product identity was confirmed by band sequencing.

Figure 4.2.

Expression of splice variants of syntrophin y2 in human intestinal circular muscle. RT-PCR amplification showed at least 5 splice variants of syntrophin y2. Splice variant 1 was identical to the published sequence with 17 exons (Accession # NM_018968). Splice variant 2 had a 27 bp deletion in exon 9. Splice variant 3 had exons 3-6 deleted with a 222 bp insertion between exon 11 and 12. Splice variant 4 had a 256 bp insertion with a stop codon (|) between exon 9 and 10. Splice variant 5 had a 46 bp insertion with a stop codon (|) inserted between exon 14 and 15.

Figure 4.3.

Direct interaction between the PDZ domain of syntrophin y2 and the last 10 amino acids of SCN5A in vivo. A, Schematic diagram of baits and preys used in the yeast two hybrid system analysis (CT5A: last 100 AA of SCN5A, CT5A-10: CTS lacking the last 10 AA of SCN5A, Syn-y2-1: syntrophin-y2 splice variants 1 with an intact PDZ domain, Syn-y2-3: syntrophin-y2 splice variant 3 lacking a PDZ domain). B, Expression of the reporter gene HIS3. Each pair of constructs as indicated in A was co-transfected into the YRG-2 yeast strain. Yeast transformants were then selected on selective plates and tested for expression of reporter gene HIS3. Strong expression of HIS3 only occurred when the last 10AA of SCN5A and the PDZ domain of syntrophin y2 were both present. C, B-galactosidase activity. Colonies that grew on the selective plates were transferred onto the filter papers, and assayed for the B-galactosidase activity confirming that SCN5A and syntrophin y2 interact and that the interaction occurs through the C-terminus and the PDZ domain.

Figure 4.4.

Direct interaction between the PDZ domain of syntrophin y2 and the last 10 amino acids of C terminus of SCN5A in vitro. The full-length syntrophin y2 with a PDZ domain (Syn y2-1) and syntrophin y2 without a PDZ domain (Syn y2-3) were transfected into HEK 293 cells. The cell lysates were then incubated with GST, GST+CT5A (last 100 AA of the C-terminus of SCN5A, or GST+CT5A-10 (last 100 AA of the C-terminus except for the very last 10AA) beads. After washing, the proteins bound to the beads were resolved by 12% SDS-PAGE and identified by western blots using the anti-FLAG antibody as the probe. Specific binding was observed only between GST+CT5A and syntrophin y2-1.

Figure 4.5.

SCN5A C-terminus peptide and syntrophin y2 PDZ domain peptide block perfusion-induced increase in peak Na+ current. A. Control Na+ current obtained from a human jejunal circular smooth muscle cells using the pulse protocol in the inset 10 min after breaking in with 1 mM C-terminus peptide in the pipette solution and lack of activation of the Na+ current by perfusion. B. Control Na+ current obtained 10 min after breaking in with 1 mM C-terminus scrambled peptide in the pipette solution and activation of the Na+ current by perfusion. C and D. Mean current-voltage relationships for the effects of perfusion in the presence of the C-terminal peptide and the control scrambled peptide respectively. E. Mean peak inward Na+ currents. F. Coomassie Blue-stained recombinant purified GST and GST+PDZ shown on 15% SDS-PAGE. G. Control Na+ current 10 min after breaking in with 10 nM GST-PDZ peptide- in the pipette solution and lack of activation of the Na+ current by perfusion. H. Control Na+ current obtained 10 min after breaking in with just the GST peptide and activation of the Na+ current by perfusion. I and J. Mean current-voltage relationships for the effects of perfusion in the presence of GST-PDZ domain peptide and GST respectively. K. Mean peak inward Na+ currents.

Figure 4.6.

Effect of co-transfection of syntrophin y2 with SCN5A. A. Inward Na+ current at -40 mV for SCN5A alone and SCN5A+syntrophin y2. B. Steady state activation and inactivation curves for SCN5A alone and SCN5A+syntrophin y2 showing the right shift in activation and the smaller window current when syntrophin y2 was co-transfected. C. Time to peak (activation) was slower at all voltages tested when when syntrophin y2 was co-transfected with SCN5A. Fast inactivation was also slower at all voltages tested while slow inactivation was unchanged.

Figure 4.7.

Effect of truncation of SCN5A and of loss of the Syntrophin y2 PDZ domain on SCN5A kinetics. A, D. Whole cell current traces at -40 mV for SCN5A, SCN5A without the last 10 AA cotransfected with syntrophin y2 and SCN5A co-transfected with syntrophin y2 without the PDZ domain. B, E. Steady steady activation and inactivation curves. C, F. Activation and inactivation kinetics. Both truncation of SCN5A or absence of the PDZ domain of syntrophin y2 resulted in loss of the kinetic changes seen when the full length SCN5A was co-expressed with syntrophin y2.

Figure 5.1.

Single-cell RT-PCR amplifies ckit message from cells visually identified as interstitial cells of Cajal (ICC). A and B: examples of single cells displaying characteristic ICC morphology of 3 or more primary processes. C: single-cell RT-PCR was performed in 2 rounds by using nested primer pairs specific for c-kit mRNA. Amplification products are shown for 2 collected ICC (lanes 1 and 2), 1 smooth muscle cell (lane 3), and a human jejunal cDNA library (lane 4). A band of the correct size, confirmed by sequencing, was present in lanes 1, 2, and 4. No product for c-kit was present in lane 3, the smooth muscle cell. The 600-bp band is the result of genomic amplification.

Figure 5.2.

ICC express 2 transient inward currents. A: typical standard whole cell patch-clamp recording from a human small intestinal ICC held at -100 mV with Cs+ in the pipette to block K+ currents. Two transient components of inward current were evident, one with faster activation and inactivation kinetics that peaked at -30 mV and the other with slower activation and inactivation kinetics that peaked at 0 mV (A). The current-voltage relationships are shown in B. The pulse protocol is shown in the inset.

Figure 5.3.

Effect of nifedipine on transient inward current. A: representative current recordings from an ICC in NaCl Ringer solution and after nifedipine (1 uM) was added to the bath. The pulse protocol is shown in the inset. Nifedipine (1 uM) blocked the slow component that peaked at 0 mV but not the fast component that peaked at -30 mV. B: current-voltage relationships. Note the shift in membrane potential due to block of the Ca2+ current with a positive reversal potential, with a subsequent larger contribution to the reversal potential of the nonselective cation current present in these cells to the residual whole cell current (nonselective current reversing at +0mV and the Na+ current reversing at a positive voltage). C: mean maximal peak currents for Na+ (n = 8; P > 0.05) and Ca2+ (n = 6; P < 0.05) before and after nifedipine was added (*P < 0.05).

Figure 5.4.

Effect of N-methyl-D-glucamine (NMDG) on the fast component of the transient inward current. A: representative ICC current recordings using the pulse protocol in the inset with nifedipine (1 uM) in the bath to block L-type Ca2+ current. Replacement of bath solution Na+ with NMDG completely blocked the fast component of inward current. B: current-voltage relationships. C: mean maximal peak current of the fast component (n = 13; *P < 0.01).

Figure 5.5.

Effect of the selective Na+ channel blocker QX-314 on the fast inward component. A: typical Na+ current recordings from an ICC patched using the pulse protocol in the inset with nifedipine (1 uM) in the bath to block L-type Ca2+ current. Top records show currents recorded at t = 0 and t = 20 min from a control cell in NaCl Ringer bath. Bottom records show currents from an ICC 0 and 20 min after exposure to 500 uM QX-314. B: amplitude of normalized peak Na+ currents over time in the control (filled circles) and QX-314-exposed cell (open squares). C: mean normalized peak Na+ currents at 0 and 20 min for control (n = 3; P > 0.05) and QX-314-treated ICC (n = 4; P < 0.05).

Figure 5.6.

Reversal potential of instantaneous current-voltage relationships from the fast component of the inward current. Reversal potentials for the fast component of the inward current were recorded from ICC patched in the presence of 50mM intracellular Na+ and 150, 50, 5, and 1mM extracellular Na+, with NMDG substituting Na+. Nifedipine (1 uM) was present in the bath to block the L-type Ca2+ current. Reversal potentials (open circles) were plotted against the expected reversal potential of a perfectly selective Na+ channel current (solid line).

Figure 5.7.

Effect of perfusion on transient inward currents. Representative inward currents are displayed from an ICC before and during perfusion with NaCl Ringer solution. A: individual sweeps at -30 and 0 mV are shown to highlight the separate effects of perfusion on the Na+ and Ca2+ currents, respectively. B: current-voltage relationship for the peak inward currents. C: mean maximal peak current values for the Na+ (n = 7; *P < 0.05) and Ca2+ (n = 3; P > 0.05) currents before and during perfusion.

Figure 5.8.

SCN5A is expressed in single human intestinal ICC. Single ICC were collected from dissociated human jejunal circular smooth muscle strips. Single-cell RT-PCR was performed using primer pairs specific for SCN5A and designed to span an intron to detect genomic sequence. A band of the right product size was identified and sequenced (lane 2) to confirm that it was SCN5A. No band was seen from bath solution aspirated just above the cells (lane 1).

Figure 5.9.

Effect of lidocaine on smooth muscle strip electrical activity. Lidocaine (200 uM) was used as a Na+ channel blocker to determine the effect of Na+ channel block on the electrical slow wave (A, control before lidocaine; B, lidocaine; C, control after lidocaine). Lidocaine hyperpolarized the membrane potential by 10 mV, slowed the rate of rise of the slow wave, and decreased the slow wave frequency from 8/min to 7.5/min.

Figure 5.10.

Effect of Na+ removal on smooth muscle strip electrical activity. A human jejunal circular smooth muscle cell was impaled to record the electrical slow wave generated by ICC. Na+ was removed at the first * and replaced at the second *. Removal of Na+ resulted in an immediate hyperpolarization and a gradual disappearance of the electrical slow wave. The slow wave returned after Na+ was reintroduced in the bath.

Figure 5.11.

Effect of stretch on smooth muscle strip electrical activity. A human jejunal circular smooth muscle cell was impaled to record the electrical slow wave generated by ICC, and then 1.5 g of weight was added to stretch the muscle strip, which was fixed on one end. Stretch increased the slow wave frequency from 7/min to 7.3/min.