In vitro smooth muscle contractility before and after relief of experimental obstruction in the rat: application to the surgical management of ileal dilatation.

PURPOSE: Bowel dilatation occurs proximal to an obstruction and predisposes to intestinal dysmotility. The present study sought to determine whether or not changes in smooth muscle contractility and the thickness of the proximal, dilated bowel wall can be reversed following relief of the obstruction. MATERIALS AND METHODS: Three groups of seven male Wistar rats were studied. In 8-week-old animals in a control group and a sham-operated group, a small segment of bowel (designated as R1 for controls and R2 for shams) was resected 5.0 cm from the cecum. In the third (operated) group, a narrow, isoperistaltic intestinal loop was created proximal to an end-to-end anastomosis of the ileum in 4-week-old animals. When these animals were 6 weeks old, the loop was re-anastomosed to the distal small bowel (after resection of the loop's distal portion, referred to as R3). Two weeks later, a small segment of bowel was resected proximal to the anastomosis (R4). We evaluated the thickness of the smooth muscle layers and the in vitro contractile responses of circular smooth muscle ileal strips (R1-R4) to electrical stimulation and pharmacological stimulation (with KCl, acetylcholine (ACh), substance P, N(G)-nitro-l-arginine methyl ester (L-NAME) and histamine). RESULTS: The amplitudes of contraction in response to electrical and Ach-mediated stimulation were higher for R3 than for R4 (P<0.001), R1 and R2 (both P<0.05). Compared with R1 and R2, the smooth muscle layer was three times as thick in R3 (P<0.001) and 2.5 times as thick in R4 (P<0.01). CONCLUSION: Our study provides evidence of the possible recovery of intestinal motility (in response to neurotransmitters involved in gut function) after the relief of an obstruction. If ileal motility can conceivably return to normal values, conservative surgical procedures in pediatric patients should be preferred (in order to leave a sufficient length of bowel and avoid short bowel syndrome).

Intermediate conductance Ca2+ activated K+ channels are expressed and functional in breast adenocarcinomas: correlation with tumour grade and metastasis status.

K+ channels are key molecules in the progression of several cancer types and are considered to be potential targets for cancer therapy. In this study, we investigated the intermediate- conductance Ca2+-activated K+ channels (hKCa3.1) expression in both breast carcinoma (BC) specimens and human breast cancer epithelial primary cell cultures (hBCE) using immuno-histochemistry (60 samples), quantitative Real-Time RT-PCR (30 samples) and Western blot assay (30 samples). We also looked at whether or not the expression of these channels is correlated with breast carcinomas grade tumours and metastasis status. Furthermore, we characterized the hKCa3.1 channel activity in hBCE cells by using the Whole Cell Patch Clamp Technique. We found that hKCa3.1 transcripts and proteins were expressed in both BC samples and hBCE cells. Clinicopathologic evaluation indicated a significant correlation between hKCa3.1-expression and tumour grade. hKCa3.1 mRNA and protein were more highly expressed in grade III tumours than in both grades I and II. However, the hKCa3.1 expression-increase according to grade was only observed in tumours with negative metastasis status. Moreover, the hKCa3.1 channels expressed in hBCE cells are functional. This was attested by patch-clamp recordings showing typical hKCa3.1-mediated currents in these cells. In conclusion, these data suggest that hKCa3.1 might contribute to breast tumour-progression and can serve as a useful prognostic marker for breast cancer.

Expression of TRPC6 channels in human epithelial breast cancer cells.

BACKGROUND: TRP channels have been shown to be involved in tumour generation and malignant growth. However, the expression of these channels in breast cancer remains unclear. Here we studied the expression and function of endogenous TRPC6 channels in a breast cancer cell line (MCF-7), a human breast cancer epithelial primary culture (hBCE) and in normal and tumour breast tissues. METHODS: Molecular (Western blot and RT-PCR), and immunohistochemical techniques were used to investigate TRPC6 expression. To investigate the channel activity in both MCF-7 cells and hBCE we used electrophysiological technique (whole cell patch clamp configuration). RESULTS: A non selective cationic current was activated by the oleoyl-2-acetyl-sn-glycerol (OAG) in both hBCE and MCF-7 cells. OAG-inward current was inhibited by 2-APB, SK&F 96365 and La3+. TRPC6, but not TRPM7, was expressed both in hBCE and in MCF-7 cells. TRPC3 was only expressed in hBCE. Clinically, TRPC6 mRNA and protein were elevated in breast carcinoma specimens in comparison to normal breast tissue. Furthermore, we found that the overexpression of TRPC6 protein levels were not correlated with tumour grades, estrogen receptor expression or lymph node positive tumours. CONCLUSION: Our results indicate that TRPC6 channels are strongly expressed and functional in breast cancer epithelial cells. Moreover, the overexpression of these channels appears without any correlation with tumour grade, ER expression and lymph node metastasis. Our findings support the idea that TRPC6 may have a role in breast carcinogenesis.

Variability of the nonstructural 5A protein of hepatitis C virus type 3a isolates and relation to interferon sensitivity.

The nonstructural 5A (NS5A) protein of hepatitis C virus (HCV) genotype 1 is thought to interact with several cellular proteins, including the double-stranded RNA-dependent protein kinase (PKR) induced by interferon (IFN). The PKR-binding domain (PKR-BD; aa 2209-2274), including the IFN sensitivity-determining region (aa 2209-2248) and other regions, could be linked to IFN resistance. Thus, the entire NS5A sequence of 27 isolates of HCV genotype 3a was investigated in relation to the clinical response to IFN. The NS5A 3a protein presented a low variability with some specific variable regions. Differential analysis between IFN-resistant and -sensitive isolates identified 5 regions in NS5A, 2 of them inside the PKR-BD and another around the variable 3 region. However, using the yeast growth suppression assay, no interaction was found between 5 resistant NS5A 3a proteins and PKR. Some amino acid changes of the NS5A protein of genotype 3a seemed to relate to IFN resistance independently of the PKR pathway.