General biological features of ETB(−/−)R
The ETB(−/−)R had a unique appearance, with their almost white coat (caused by a reduced number of pigment cells in the coat) and abdominal distension. The ETB(+/+)R had a black coat limited to the head and part of the body. The physique of the ETB(−/−)R was poor with severe emaciation and growth retardation. The ETB(−/−)R hair was lustreless and rough. The death rate of the ETB(−/−)R was significantly greater than that of the ETB(+/+)R. The shortest life span of the ETB(−/−)R in the present study was three days and the longest was 34 days (mean 18.1 days).
Figure 1A shows the abdominal dissection and digestive tract from the stomach to the anus. The ETB(−/−)R had intestinal dilation in the same location with abdominal distention. In the ETB(−/−)R, a constricted bowel region was observed throughout the colon, including the caecum and lower part of the ileum, followed by a dilated region with faecal stasis. Therefore, megaileum was usually observed. These pathological features resemble those of human long segment Hirschsprung's disease; they appeared to be more severe than those of general short segment Hirschsprung's disease in which the constricted portion of the bowel region is generally in the colon.
| Figure 1(A) Dissection of the entire gastrointestinal tract. The severe intestinal enlargement is shown in the homozygous endothelin ETB receptor null rat (ETB(−/−)R) compared with the homozygous wild type ETB(+/+)R. Dilation of the proximal ileum (more ...) |
Figure 1B shows the structure of the myenteric plexus in the colon and ileum of the ETB(+/+)R and ETB(−/−)R. Although there were some individual differences, the myenteric plexus in the colon and ileum of the ETB(+/+)R showed a similar shape and size of ganglia with a regular meshwork pattern (fig 1B ). In the dilated (proximal) portion of the ETB(−/−)R ileum however the myenteric plexus appeared to be decreased in density and stretched in the shape of meshwork compared with that in the ETB(+/+)R ileum (fig 1B ). In the constricted (distal) part of the ETB(−/−)R colon, the myenteric ganglia were completely absent, and only some extrinsic nerve fibres or bundles were observed (fig 1B ).
Contractions
Carbachol (0.001–100 μM), a muscarinic receptor agonist, produced concentration dependent contractions both in the constricted (distal) and dilated (proximal) portions of ileum from the ETB(+/+)R and ETB(−/−)R (fig 2
). The contractions generated by carbachol in the constricted (distal) part of the ETB(−/−)R ileum (100 μM carbachol: 5.88 (1.39) mN/mg ww; n=8) were significantly greater than those of the corresponding part of the ETB(+/+) ileum (1.99 (0.39) mN/mg ww; n = 8) (fig 2B
). However, the contractile responses induced by carbachol in the dilated (proximal) parts of the ETB(−/−)R ileum (2.60 (0.64) mN/mg ww; n=8) were almost the same as those of the corresponding part of the ETB(+/+)R (2.25 (0.19) mN/mg ww; n=8) (fig 2A
). The constricted (distal) ileum in the ETB(−/−)R showed greater contractility than the dilated (proximal) ileum.
| Figure 2Contractile effect of carbachol on the proximal (A) and distal (B) parts of the ileum in the homozygous endothelin ETB receptor null rat (ETB(−/−)R) compared with the homozygous wild type ETB(+/+)R. Contractile responses are expressed (more ...) |
Addition of KCl (5.4–72.7 mM) produced concentration dependent contractions in both the constricted (distal) and dilated (proximal) ileum from the ETB(+/+)R and ETB(−/−)R (fig 3 ). The amplitude of the contractions generated by KCl of constricted (distal) parts of the ETB(−/−)R ileum (72.7 mM KCl: 3.87 (0.99) mN/mg ww; n=8) was significantly greater than that of corresponding parts of the ETB(+/+)R ileum (1.75 (0.30) mN/mg ww; n=8) (fig 3B ). However, the contractile responses induced by KCl in the dilated (proximal) parts of the ileum in the ETB(−/−)R ileum (1.86 (0.41) mN/mg ww; n=8) were almost the same as those of corresponding parts of the ETB(+/+)R (1.56 (0.22) mN/mg ww; n=8) (fig 3A ). The contraction generated by KCl in the constricted (distal) part of the ETB(−/−)R ileum was greater than that of the dilated (proximal) part.
| Figure 3Contractile effects of KCl on the proximal (A) and distal (B) parts of the ileum in the homozygous endothelin ETB receptor null rat (ETB(−/−)R) compared with the homozygous wild type ETB(+/+)R. Contractile responses are expressed in mN (more ...) |
Addition of endothelin-1 (0.1–3×100 nM), a non-selective agonist of endothelin ETA and ETB receptors, produced concentration dependent contractions in both the constricted (distal) and dilated (proximal) ileum from the ETB(+/+)R and ETB(−/−)R (fig 4 ). The amplitude of contractions generated by endothelin-1 of the constricted (distal) parts of the ETB(−/−)R ileum (100 nM endothelin-1: 7.45 (1.76) mN/mg ww; n=9) was significantly higher than that of the corresponding parts of the ETB(+/+)R ileum (1.21 (0.31) mN/mg ww; n=10) (fig 4B ). In addition, the contractile responses induced by endothelin-1 at higher concentrations (10–100 nM) in the dilated (proximal) parts of the ileum in the ETB(−/−)R (100 nM endothelin-1: 2.84 (0.75) mN/mg ww; n=9) were also significantly higher than those of the corresponding parts of the ETB(+/+)R (0.67 (0.18) mN/mg ww; n=10) (fig 4A ). The constricted (distal) ileum of the ETB(−/−)R showed greater contractility than the dilated (proximal) ileum.
| Figure 4Contractile effects of endothelin-1 on the proximal (A) and distal (B) parts of the ileum in the homozygous endothelin ETB receptor null rat (ETB(−/−)R) compared with the homozygous wild type ETB(+/+)R. Contractile responses are expressed (more ...) |
Contractions induced by 10 μM carbachol in the muscle strips of the constricted regions (colon, caecum, and distal ileum) from the ETB(−/−)R were significantly greater than those of the corresponding parts of the ETB(+/+)R (8.04 (1.42) v 4.22 (0.56) mN/mg ww; 7.79 (1.30) v 4.84 (0.54) mN/mg ww, and 5.35 (1.36) v 1.46 (0.33) mN/mg ww, respectively; n=9–11) whereas contractions of muscle strips of the dilated ileum and jejunum from the ETB(−/−)R were the same as those of the corresponding parts of the ETB(+/+)R (2.08 (0.61) v 2.01 (0.13) mN/mg ww and 3.17 (0.48) v 3.44 (0.37) mN/mg ww, respectively; n=9–11) (fig 5 )
| Figure 5Comparison of the contractile effects of 10 μM carbachol on intestinal smooth muscle of the homozygous endothelin ETB receptor null rat (ETB(−/−)R) compared with the homozygous wild type ETB(+/+)R. Contractile responses are expressed (more ...) |
Possible involvement of inhibitory substances
The ETB(−/−)R has few nerves in the aganglionic segments but the wild type has normal enteric neurones present. Regarding the increased contractility in ETB(−/−)R, it is possible that release of transmitters such as nitric oxide and ATP could inhibit the contractility of the intestine in ETB(+/+)R more than ETB(−/−)R. Contraction induced by carbachol (0.1 μM) was augmented following addition of
l-NAME (100 μM) in the proximal and distal ileum of ETB(+/+) and ETB(−/−) rats. However, we observed no significant differences between responses in ETB(+/+) and ETB(−/−), which implies that nitric oxide may not be involved in the increased contractility in ETB(−/−) rats. In contrast, suramine (300 μM), a non-competitive agonist of P
2X and P
2Y receptors, was unable to modify the carbachol induced contractions in the ileal strips, indicating that ATP is not responsible for the increased contractility of the ETB(−/−)R intestine.
Changes in the ETA receptor
RT-PCR analysis of RNA extracted from the muscle tissue of ETB(+/+)R and ETB(−/−)R was carried out. As shown in fig 6
, expression of the RT-PCR product encoding the housekeeping gene GAPDH (308 bp) was identical in controls and ETB(−/−)R. ETB receptor (586 bp) mRNAs were strongly expressed in the normal ETB(+/+)R. It has been reported that a 301 bp region intervening between direct repeat sequences was deleted in the ETB(−/−)R, and that the deletion produces various transcripts due to aberrant splicing.
10 Consistent with this observation, we observed two bands in an area approximately 300 bp shorter than in normal rats. In contrast, ETA receptor (205 bp) mRNAs were expressed both in ETB(+/+)R and ETB(−/−)R, with no significant difference between the two. There was also no significant difference in ETA receptor mRNA between the constricted (distal) and dilated (proximal) ileum from the ETB(−/−)R (ETA mRNA/GAPDH mRNA: distal ileum in ETB(+/+)R 0.41 (0.09); distal ileum in ETB(−/−)R 0.63 (0.2); proximal ileum in ETB(−/−)R 0.61 (0.08); n=3 each).
| Figure 6Expression of ETA and ETB receptor mRNAs in the ileal smooth muscle of the homozygous endothelin ETB receptor null rat (ETB(−/−)R) compared with the homozygous wild type ETB(+/+)R. Typical results after 45 cycles of reverse transcription-polymerase (more ...) |
Histology
A histological profile of thickness of the smooth muscle layer can be obtained by determining the number of piling smooth muscle cells of each muscle layer of the ETB(+/+)R and ETB(−/−)R. In general, both the longitudinal and circular smooth muscle layers were thicker in the ETB(−/−)R than in the ETB(+/+)R although the diameter of each smooth muscle cell around the nuclear region was not different, as shown in fig 7A
.
| Figure 7Thickness of the muscle layer indicated by the number of piling cells in the muscle layers of the homozygous endothelin ETB receptor null rat (ETB(−/−)R) compared with the homozygous wild type ETB(+/+)R. (A) Cross section of the caecum (more ...) |
To obtain more numerical data on the thickness of the smooth muscle layer, the smooth muscle cells in the circular or longitudinal muscle layers were counted in the ETB(−/−)R and ETB(+/+)R on the longitudinal or circular section, respectively. As shown in fig 7B , the number of cells piled in the longitudinal smooth muscle layers of both the caecum and colon of the ETB(−/−)R was significantly greater than that in the corresponding muscle layers of the ETB(+/+)R. However, the number of cells in the circular smooth muscle in the proximal ileum (dilated segment) and that of longitudinal smooth muscle of the caecum (constricted segment) of the ETB(−/−)R was not statistically different from that of the ETB(+/+)R.