Biomedical subjects

R A Hodin

Publications and source records attributed to R A Hodin.

At least 19 recordsLinked to original sources

PubMed2001

p21 gene regulation during enterocyte differentiation.

BACKGROUND: Enterocyte differentiation is associated with a withdrawal from the cell cycle and the transcriptional activation of the cell cycle inhibitor, p21. We sought to define the molecular mechanisms involved in p21 gene activation in an in vitro system. METHODS: Transient transfections were performed in HT-29 cells with plasmids containing various 5' deletions of the p21 promoter upstream of the luciferase reporter -/+ the histone deacetylase 1 (HDAC1) expression plasmid. After 24 h, cells were treated -/+ 5 mM sodium butyrate (NaBu) or another histone hyperacetylating agent, trichostatin A (TSA, 0.3 microM) for 24 h. After protein extraction, luciferase activity was measured. Acid/urea/triton gel electrophoresis was performed to examine histone acetylation in cells. RESULTS: NaBu and TSA both caused histone H4 hyperacetylation. Both NaBu and TSA caused a marked increase in the transactivation of plasmids containing 291 bp of the p21 promoter upstream of the transcriptional start site, similar to that previously seen for a 2.4-kb construct. A decrease in reporter gene induction was seen between 173 and 153 bp. This was followed by a marked increase in promoter induction from 143 to 117 bp. Finally, only low basal activity was seen in the case of the 93-bp plasmid. HDAC1 blocked NaBu-mediated induction of all plasmids. CONCLUSIONS: p21 gene activation during HT-29 cell differentiation occurs via at least two regions of cis-acting elements: one located between -93 and -117 bp, and the other between -173 and -291 bp. Histone hyperacetylation likely plays a role in this activation.

Acetylation↗

PubMed2001

Laparoscopic splenectomy for massive splenomegaly using a Lahey bag.

BACKGROUND: Although the recent development of hand-assisted laparoscopic surgery (HALS) has made the laparoscopic retraction of large spleens feasible, the laparoscopic removal of massively enlarged spleens (>1,000 g) remains a significant problem because these spleens do not fit into endoscopic bags. Consequently, in order to remove massive spleens either a large abdominal incision or morcellation of the spleen outside of an endoscopy bag is required. METHODS: Two patients, with spleens weighing 2,510 g and 1,720 g, underwent laparoscopic splenectomy using a hand port to ensure safe retraction. The massive spleen was placed into a Lahey bag that was inserted into the abdomen through the hand port site. While in the Lahey bag, the spleen was removed piecemeal through the hand port site. RESULTS: Both operations were completed laparoscopically without complications. The patients were discharged on postoperative day 2 and experienced minimal morbidity. CONCLUSIONS: The Lahey bag facilitates laparoscopic splenectomy for massive splenomegaly as even the most massive spleens will fit into a Lahey bag. A massive spleen may be removed piecemeal from the Lahey bag through the small hand port incision without risking a large abdominal incision, splenosis, or the insertion of a morcellator.

Adult↗

PubMed2001

Transient vs. prolonged histone hyperacetylation: effects on colon cancer cell growth, differentiation, and apoptosis.

The role of histone hyperacetylation in regard to growth, differentiation, and apoptosis in colon cancer cells was assessed in an in vitro model system. HT-29 cells were grown in +/-10% fetal bovine serum with either 5 mM sodium butyrate or 0.3 microM trichostatin A [single dose (T) or 3 doses 8 h apart (TR)] for 24 h. Serum-starved HT-29 cells were further treated with epidermal growth factor or insulin-like growth factor I for an additional 24 h. Apoptosis was quantified with propidium iodide and characterized by electron microscopy. Northern blot analyses were performed with cDNA probes specific for intestinal alkaline phosphatase, Na-K-2Cl cotransporter, the cell cycle inhibitor p21, and the actin control. Flow cytometric analysis revealed a time-dependent growth suppression along with early induction of p21 mRNA in the butyrate, T, and TR groups. Histone hyperacetylation, assessed by acid-urea-triton gel electrophoresis, was transient in the T group but persisted for up to 24 h in the butyrate and TR groups. Induction of apoptosis, growth factor unresponsiveness, and differentiation occurred in the butyrate- and TR-treated cells but not those treated with a single dose of trichostatin A. Thus transient hyperacetylation of histones is sufficient to induce p21 expression and produce cellular growth arrest, but prolonged histone hyperacetylation is required for induction of the programs of differentiation, apoptosis, and growth factor unresponsiveness.

Acetylation↗

PubMed2001

American ginseng transcriptionally activates p21 mRNA in breast cancer cell lines.

American ginseng (AG) has been demonstrated to inhibit breast cancer cell growth in vitro. p21 protein, a universal cell cycle inhibitor, binds cyclin-CDK complexes, an important mechanism in cell cycle regulation. The purpose of this investigation was to determine if AG induces p21 gene expression in hormone sensitive (MCF-7) and insensitive (MDA-MB-231) breast cancer cell lines. Cells grown in steroid stripped medium (SSM) were treated with AG, 17-beta-estradiol (E2), genistein or cycloheximide (CHX). Northern blot analyses were performed using human p21Cip1 and 36B4 cDNA probes. Cell lines were transiently transfected with select mouse p21 CAT reporter constructs, including those lacking a p53 binding site. Cell cycle analyses was performed by FACScan. The results revealed that AG induced p21 mRNA expression in MCF-7 and MDA-MB-231 cells (p=0.0004; p < or =0.0001, respectively). Neither E2 nor genistein alter p21 mRNA expression. CHX, a protein synthesis inhibitor, did not block p21 mRNA expression induced by AG, indicating that p21 is induced as an immediate early gene. AG activated p21 reporter constructs in transfected cells, independent of p53 binding sites. The cell cycle proliferative phase was significantly decreased by AG and increased by E2 (p < or =0.0001). AG may inhibit breast cancer cell growth by transcriptional activation of the p21 gene, independent of p53.

Animals↗

PubMed2000

Short-stay laparoscopic adrenalectomy.

BACKGROUND: We performed a consecutive series of unilateral laparoscopic adrenalectomies (LA) with the expectation of short (less than 24 h) hospital stay. Results were compared with those from laparoscopic cholecystectomy (LC) and unilateral open adrenalectomy (OA). METHODS: A combination of chart review and patient questionnaires was used to compare LA (n = 19) to LC (n = 20) regarding length of stay (LOS), narcotic requirements, and time to full recovery. Chart reviews also were used to compare LA to OA (n = 48) regarding operating room time (OR time), LOS, and surgical morbidity. RESULTS: All of the LC patients as compared with 47% of the LA patients were discharged within 24 h. The reason for additional hospitalization in the LA group was pain control. After discharge, the narcotic requirement lasted 6.6 days in the LA group as compared with 3.4 days in the LC group (p < 0.01), but the times until full recovery were not significantly different (12.2 vs 11.3 days respectively). Operating room times did not differ significantly between the LA and OA groups (3.3 and 3.8 h, respectively), but there were fewer postoperative complications and much shorter LOS in the LA group (1.5 vs 6.3 days; p < 0.001), a difference that remained significant even when cases from the same time period were compared. CONCLUSIONS: Increased pain in LA as compared with LC patients may result in a slightly longer LOS and higher narcotic requirement during the early postoperative period, but time to full recovery between the two groups is the same. As compared with its open counterpart, LA offers a significant reduction in LOS and morbidity with no increase in OR time.

Adrenal Gland Diseases↗

PubMed2000

Laparoscopically assisted ileocolectomy for Crohn's disease through a pfannenstiel incision.

Recently, laparoscopically assisted bowel resections have been shown to be less morbid than the traditional laparotomy, especially for benign conditions such as Crohn's disease. While reports describing laparoscopically assisted bowel resections use a small midline or right transverse incision, we describe a novel laparoscopically assisted approach employing a Pfannenstiel incision for Crohn's patients. We attempted the Pfannenstiel incision since it is well known to be associated with less postoperative pain, decreased ileus and hospital stay, and low rates of wound infection and incisional hernia, compared with midline or right transverse incisions. Furthermore, we found that the Pfannenstiel incision offers additional advantages that may be uniquely suited for Crohn's patients. First, the cosmetic position of the incision is particularly attractive to the young population affected by Crohn's. Second, the Pfannenstiel incision preserves fresh tissue in the midline, right, and left lower quadrants in the event that reoperation or stoma placement are required in the future owing to recurrent disease. We describe our technique in 10 consecutive patients undergoing ileocolectomy for Crohn's disease. Our patients experienced minimal morbidity and were pleased with the cosmetic results of their incisions.

Adult↗

PubMed1999

Protein kinase C activation downregulates the expression and function of the basolateral Na+/K+/2Cl(-) cotransporter.

The basolateral Na+/K+/2Cl(-) cotransporter (NKCC1) has been shown to be an independent regulatory site for electrogenic Cl(-) secretion. The proinflammatory phorbol ester, phorbol 12-myristate 13-acetate (PMA), which activates protein kinase C (PKC), inhibits basal and cyclic adenosine monophosphate (cAMP)-stimulated NKCC1 activity in T84 intestinal epithelial cells and decreases the steady state levels of NKCC1 mRNA in a time- and dose-dependent manner. The levels of NKCC1 protein also fall in accordance with the NKCC1 mRNA transcript and these levels are unaffected by 4alpha-phorbol, which does not activate PKC. Inhibition of maximal (cAMP-stimulated) NKCC1 functional activity by PMA was first detected by 1 h, whereas decreases in the steady state levels of NKCC1 mRNA were not detectable until 4 h. NKCC1 mRNA expression recovers toward control levels with extended treatment of cells with PMA suggesting that the PMA effects on NKCC1 expression are mediated through activation of PKC. Although NKCC1 mRNA and protein levels return to control values after extended PMA exposure, NKCC1 functional activity does not recover. Immunofluorescence imaging suggest that the absence of functional recovery is due to failure of newly synthesized NKKC1 protein to reach the cell surface. We conclude that NKCC1 has the capacity to be regulated at the level of de novo expression by PKC, although decreased NKCC1 expression alone cannot account for either early or late loss of NKCC1 function.

Carrier Proteins↗

PubMed1999

Videoscopic harvest of the inferior epigastric artery.

The inferior epigastric artery has been found to be a useful conduit for performing arterial coronary revascularization. The present report describes a minimally invasive port access technique for harvesting the inferior epigastric artery.

Coronary Artery Bypass↗

PubMed1999

Histone acetylation and cancer.

In the past year, several papers have been published which implicate a link between alterations in chromatin structure and the development of cancer. Both histone hyperacetylation and hypoacetylation appear to be important in the neoplastic process, depending on the target gene involved. In the case of colon cancer, induction of the p21 gene by histone hyperacetylation may be a mechanism by which dietary fiber prevents carcinogenesis.

Acetylation↗

PubMed1999

Is sestamibi-guided parathyroidectomy really cost-effective?

BACKGROUND: Sestamibi-guided limited neck explorations are an alternative to the standard bilateral neck exploration for patients with primary hyperparathyroidism. A recently published meta-analysis by Denham and Norman (JACS vol.186, 1998) suggested that a sestamibi-directed approach offers a cost benefit because it decreases operative and recovery room times, hospital stay, and the number of frozen sections needed. METHODS: We reviewed 41 bilateral neck explorations for primary hyperparathyroidism and compared our results with those reported by the meta-analysis to determine whether a sestamibi-directed approach is cost effective. RESULTS: Operative and recovery room times averaged 60.3 +/- 19.3 and 45 minutes, respectively. Forty six percent of the patients were treated as outpatients, and 1.21 +/- 0.57 frozen sections were obtained per case. Our standard bilateral exploration cost 47% less than the bilateral approach and 17% less than the sestamibi-directed operation calculated in the meta-analysis. There were no cases of nerve injury or permanent hypocalcemia, 98% of patients were cured, and 61% of patients did not require narcotics postoperatively. CONCLUSIONS: Sestamibi-guided parathyroidectomy may not offer any advantage over the standard bilateral exploration. In our experience, a bilateral neck exploration can be performed on an outpatient basis and at low cost, with a high success rate and minimal morbidity. Most patients do not require narcotics, and the cosmetic results are excellent.

Ambulatory Surgical Procedures↗

PubMed1999

Acidic conditions exacerbate interferon-gamma-induced intestinal epithelial hyperpermeability: role of peroxynitrous acid.

OBJECTIVE: Nitric oxide (NO*) derived from exogenous donors has been shown to increase the permeability of cultured intestinal epithelial monolayers, an effect that is augmented by mildly acidic conditions. Because interferon-gamma (IFN-gamma) also increases intestinal epithelial permeability, at least partly by an NO*-dependent mechanism, we sought to determine whether IFN-gamma-induced hyperpermeability is increased under acidic conditions. METHODS: Human intestinal epithelial (Caco-2BBe) cells were grown as monolayers on permeable supports in bicameral chambers. Permeability was assessed by measuring transepithelial electrical resistance (TER) or the transepithelial passage of fluorescein disulfonic acid. Inducible nitric oxide synthase (iNOS) messenger RNA expression was determined by northern blot analysis. Concentrations of nitrite and nitrate (NO2-/NO3-), stable oxidation products of NO*, were determined using the Greiss reaction. Cellular adenosine triphosphate (ATP) levels were determined using the luciferin/luciferase method. MEASUREMENTS AND MAIN RESULTS: Incubation of Caco-2BBe monolayers with INF-gamma (1000 units/mL) at an extracellular pH (pHo) of 7.4 increased permeability to fluorescein disulfonic acid and decreased TER. However, incubation of monolayers with IFN-gamma under mildly acidic conditions (i.e., pHo 7.0-6.6) accelerated the decrease in TER and augmented the increase in permeability induced by the cytokine. IFN-gamma-induced iNOS messenger RNA expression and NO2-/NO3- accumulation in medium were unaffected by acidic conditions. At pHo 7.4, incubation of Caco-2BBe monolayers with IFN-gamma (1000 units/mL) for 72 hrs had no effect on intracellular ATP content compared with monolayers simultaneously incubated under the same conditions but in the absence of the cytokine. However, when the cells were incubated for 72 hrs with the same concentration of IFN-gamma under mildly acidic conditions (i.e., pHo 7.0 or 6.6), ATP levels were significantly decreased. At pHo 7.0, IFN-gamma-induced increases in permeability were ameliorated by addition of the following agents: 2-phenyl-4,4,5,5- tetramethylimidazoline-1-oxyl-3-oxide (a NO* scavenger), N(G)-monomethyl-L-arginine (a iNOS inhibitor), dimethyl sulfoxide (a hydroxyl radical scavenger), and ascorbate (a peroxynitrous acid scavenger). CONCLUSION: Mild acidosis augments IFN-gamma-induced intestinal epithelial hyperpermeability and ATP depletion, possibly by fostering the formation of peroxynitrous acid and/or hydroxyl radical.

Adenosine Triphosphate↗

PubMed1999

Cytokine-induced intestinal epithelial hyperpermeability: role of nitric oxide.

OBJECTIVE: Incubation of enterocytic monolayers with interferon (IFN)-gamma increases nitric oxide (NO) production and permeability, but NO synthesis inhibitors ameliorate the development of IFN-gamma-induced hyperpermeability. Induction of inducible nitric oxide synthase (iNOS), an isoform of the enzyme responsible for NO biosynthesis, is often enhanced by the synergistic effects of multiple cytokines. Moreover, many of the cytopathic effects of NO are mediated by peroxynitrite, which is produced by the reaction of NO with superoxide radical anion. In the present study, we sought to determine whether combinations of several proinflammatory cytokines, including IFN-gamma, interleukin-1beta, and tumor necrosis factor-alpha, have synergistic effects on the induction of iNOS expression and/or hyperpermeability to hydrophilic solutes in cultured enterocytic monolayers. We also assessed the effects of aminoguanidine (a relatively selective iNOS inhibitor), L-N(G)-monomethyl arginine (an isoform-nonselective NO synthase inhibitor), and Tiron (a superoxide radical anion scavenger) on the development of cytokine-induced hyperpermeability. DESIGN: Caco-2 monolayers were incubated under control conditions or with IFN-gamma, interleukin-1beta, or tumor necrosis factor-alpha alone, pairwise combinations of these cytokines, or all three cytokines together (cytomix; CM). iNOS messenger RNA (mRNA) expression was assessed using Northern blot analysis. The permeability of Caco-2 monolayers growing on permeable supports in bicameral chambers was assessed by measuring the apical-to-basolateral flux of fluorescein disulfonic acid. The concentration of nitrate plus nitrite in culture supernatants, an indirect measure of NO production, was determined using the Griess reaction. RESULTS: After 24 hrs of incubation, up-regulation of iNOS mRNA expression was evident only in cells exposed to IFN-gamma-containing formulations. Expression of iNOS mRNA was far greater in cells incubated with CM than in cells treated with IFN-gamma alone or either of the two-component IFN-gamma-containing cytokine combinations. Compared with IFN-gamma, CM resulted in a higher rate of NO production over 48 hrs of incubation. The permeability of Caco-2 monolayers increased by approximately six-fold and approximately 20-fold after incubation for 48 hrs with IFN-gamma alone and CM, respectively. The increase in permeability induced by incubation with CM was significantly ameliorated by the addition of aminoguanidine, L-N(G)-monomethyl arginine, or Tiron. CONCLUSIONS: IFN-gamma-containing combinations of cytokines are potent inducers of iNOS in cultured enterocytic monolayers. Peroxynitrite may be an important mediator of cytokine-induced gut epithelial hyperpermeability.

1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium ↗

PubMed1999

Acquired interferon gamma responsiveness during Caco-2 cell differentiation: effects on iNOS gene expression.

BACKGROUND: Impairment of intestinal barrier function occurs under a variety of inflammatory conditions and is mediated at least in part by interferon gamma (IFN-gamma) induced nitric oxide (NO) production. Previous in vivo studies have shown that systemic lipopolysaccharide treatment caused an induction of the rat inducible nitric oxide synthase (iNOS) mRNA primarily in villus cells, rather than in undifferentiated crypt cells. AIMS: To examine iNOS induction by IFN-gamma in vitro as a function of enterocyte differentiation. METHODS: Preconfluent and postconfluent Caco-2 cells were treated with IFN-gamma in the presence or absence of various inhibitors. Northern analyses were performed to assess the magnitude of iNOS mRNA induction. IFN-gamma receptor mRNA and protein levels were determined. RESULTS: iNOS mRNA induction by IFN-gamma occurred at two hours and was not blocked by cycloheximide, indicating that it is an immediate early response. iNOS induction and nitrite/nitrate increases were inhibited by dexamethasone and pyrrolidine dithiocarbamate, supporting an important role for the NF-kappaB transcription factor in this process. The stimulated iNOS induction was seen almost exclusively under conditions of cellular differentiation-that is, in postconfluent Caco-2 cells. This increased IFN-gamma responsiveness seen in postconfluent Caco-2 cells correlated with an increased expression of IFN-gamma receptor, whereas T84 and HT-29 cells did not show any significant alterations in either iNOS induction or IFN-gamma receptor levels as a function of postconfluent growth. CONCLUSIONS: With regard to iNOS mRNA induction, IFN-gamma responsiveness is acquired during Caco-2 cell differentiation, perhaps related to an increase in the numbers of IFN-gamma receptors.

Blotting, Northern↗

PubMed1999

A novel Sp1-related cis element involved in intestinal alkaline phosphatase gene transcription.

We have used sodium butyrate-treated HT-29 cells as an in vitro model system to study the molecular mechanisms underlying intestinal alkaline phosphatase (IAP) gene activation. Transient transfection assays using human IAP-CAT reporter genes along with DNase I footprinting were used to localize a critical cis element (IF-III) corresponding to the sequence 5'-GACTGGGCGGGGTCAAGATGGA-3'. Deletion of the IF-III element resulted in a dramatic reduction in reporter gene activity, and IF-III was shown to function in the context of a heterologous (SV40) promoter in a cell type-specific manner, further supporting its functional role in IAP transactivation. Electrophoretic mobility shift assays revealed that IF-III binds Sp1 and Sp3, but these factors comprise only a portion of the total nuclear binding and appear to mediate only a small portion of its transcriptional activity. IF-III does not correspond to any previously characterized regulatory region from other intestine-specific genes. We have thus identified a novel, Sp1-related cis-regulatory element in the human IAP gene that appears to play a role in its transcriptional activation during differentiation in vitro.

Alkaline Phosphatase↗

PubMed1999

Short-chain fatty acids and thyroid hormone interact in regulating enterocyte gene transcription.

BACKGROUND: Enterocyte differentiation is known to be regulated by a variety of extracellular compounds, among which are triiodothyronine (T3) and the short-chain fatty acids (SCFAs). Because several SCFAs are known to induce histone hyperacetylation, and T3 action has been recently linked to chromatin structure, we sought to investigate the interplay between SCFAs and T3 in regard to the enterocyte differentiation marker, intestinal alkaline phosphatase (IAP). METHODS: Caco-2 cells were transiently transfected with a reporter construct containing 2.4 kb of the human IAP gene 5' flanking region (IAP2.4CAT). Cotransfections were carried out with and without thyroid hormone receptor-1 (TR beta-1) or histone deacetylase-1 (HDAC-1) expression plasmids. Cells were treated with 5 mmol/L SCFAs (propionic, butyric, valeric, or caproic acids as propionate, butyrate, valerate, or caproate, respectively), with and without 10 nmol/L T3. Reporter gene activity was measured and the level of histone acetylation assessed by means of acid-urea-triton (AUT) gel assays. RESULTS: TR beta-1 cotransfection caused a marked decrease in IAP reporter gene activity, which is consistent with the well-known phenomenon of ligand independent repression (LIR), whereas T3 treatment reversed the LIR and caused further reporter gene activation. Treatment with SCFAs similarly resulted in a complete blockage of LIR, and, in fact, turned the TR beta-1 into a transcriptional activator, even in the absence of T3. Concomitant treatment with T3 and butyric acid produced an additive effect on IAP transactivation. In contrast, cotransfection with HDAC-1 attenuated the effects of SCFAs on IAP gene activation. AUT gel studies demonstrated histone hyperacetylation in response to SCFA treatment. CONCLUSION: One or more DNA cis-elements in the human IAP gene mediate ligand independent repression by the TR beta-1, an effect that can be entirely reversed by those SCFAs that induce histone hyperacetylation. In addition T3 and SCFAs can act in concert to induce IAP gene transcription, demonstrating an important link between triiodothyronine and histone hyperacetylation in regard to enterocyte-specific gene expression.

Alkaline Phosphatase↗

PubMed1998

p21(WAF1) is required for butyrate-mediated growth inhibition of human colon cancer cells.

A diet high in fiber is associated with a decreased incidence and growth of colon cancers. Butyrate, a four-carbon short-chain fatty acid product of fiber fermentation within the colon, appears to mediate these salutary effects. We sought to determine the molecular mechanism by which butyrate mediates growth inhibition of colonic cancer cells and thereby to elucidate the molecular link between a high-fiber diet and the arrest of colon carcinogenesis. We show that concomitant with growth arrest, butyrate induces p21 mRNA expression in an immediate-early fashion, through transactivation of a promoter cis-element(s) located within 1.4 kb of the transcriptional start site, independent of p53 binding. Studies using the specific histone hyperacetylating agent, trichostatin A, and histone deacetylase 1 indicate that growth arrest and p21 induction occur through a mechanism involving histone hyperacetylation. We show the critical importance of p21 in butyrate-mediated growth arrest by first confirming that stable overexpression of the p21 gene is able to cause growth arrest in the human colon carcinoma cell line, HT-29. Furthermore, using p21-deleted HCT116 human colon carcinoma cells, we provide convincing evidence that p21 is required for growth arrest to occur in response to histone hyperacetylation, but not for serum starvation nor postconfluent growth. Thus, p21 appears to be a critical effector of butyrate-induced growth arrest in colonic cancer cells, and may be an important molecular link between a high-fiber diet and the prevention of colon carcinogenesis.

Butyrates↗

PubMed1998

Na-K-2Cl cotransporter gene expression and function during enterocyte differentiation. Modulation of Cl- secretory capacity by butyrate.

The basolateral Na-K-2Cl cotransporter (NKCC1) is a key component of the intestinal crypt cell secretory apparatus. Its fate during the transition to absorptive enterocyte and the potential impact of its altered expression on secretory output have not been addressed. In this report, NKCC1 mRNA was found to be expressed in rat jejunal crypt but not villus cells. Butyrate treatment of intestinal epithelial HT29 cells induced a differentiation pattern that recapitulated the rat intestinal crypt-villus axis, with NKCC1 mRNA levels decreasing in a time- and dose-dependent fashion in parallel with upregulation of apical brush-border markers. Butyrate but not acetate or proprionate decreased basal and cAMP-stimulated bumetanide-sensitive K+ (86Rb) uptake in both HT29 cells and the Cl--secreting T84 line. Butyrate markedly decreased transepithelial Cl- secretion in confluent T84 monolayers without effect on cAMP-regulated apical Cl- efflux. We conclude that NKCC1 regulation during enterocyte differentiation occurs at the level of gene expression, and that selective downregulation of NKCC1 gene expression and function by butyrate leads to a profound decrease in transepithelial Cl- secretion. These data emphasize the importance of NKCC1 in determining epithelial secretory capacity and suggest the possibility of modulation of the enterocytic transport phenotype as therapy for diarrheal disorders.

Animals↗

PubMed1998

Differential expression of inducible nitric oxide synthase messenger RNA along the longitudinal and crypt-villus axes of the intestine in endotoxemic rats.

OBJECTIVES: To characterize the mechanisms leading to excessive production of nitric oxide within the gut as a consequence of endotoxemia. We sought to: a) determine the time course of inducible nitric oxide synthase (iNOS) messenger RNA (mRNA) expression in the intestine after challenging rats with lipopolysaccharide (LPS); and b) investigate whether there is differential expression of iNOS in enterocytes along the longitudinal or crypt-villus axes of the intestine in rats after LPS administration. DESIGN: Prospective, randomized, unblinded study. SETTING: Research laboratories at a large university-affiliated medical center. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: At T = 0 hr, rats were injected with O111:B4 Escherichia coli LPS (5 mg/kg) or a similar volume of the saline vehicle. At various time points thereafter, samples of duodenum, jejunum, ileum, colon, and liver were harvested for subsequent extraction of RNA. In some cases, populations of enterocytes enriched in either crypt or villus cells were harvested from the ileum. In some studies, rats were injected with cycloheximide (25 mg i.p.) 15 mins before being challenged with LPS or dexamethasone (2 mg i.p.) 30 mins before being injected with LPS. MEASUREMENTS AND MAIN RESULTS: iNOS mRNA was undetectable in ileal tissue from rats under basal conditions, but was evident by T = 1 hr and was maximal at T = 2 hrs after injection of LPS. Thereafter, ileal iNOS mRNA concentrations decreased and were undetectable again at T = 24 hrs. At T = 2 hrs after LPS injection, there was marked expression of iNOS mRNA in the ileum, whereas much lower concentrations of iNOS mRNA were detected in the jejunum and colon, and no iNOS mRNA was detected in the duodenum. At T = 3 hrs after LPS injection, expression of iNOS mRNA was up-regulated in both villus and crypt cells, although LPS-induced iNOS mRNA was more prominent in the former than the latter cell type. Pretreatment of rats with dexamethasone virtually abrogated the expression of iNOS mRNA in ileal samples obtained 3 hrs after the injection of LPS. Prior treatment of rats with the protein synthesis inhibitor, cycloheximide, also blunted LPS-induced iNOS mRNA expression. CONCLUSIONS: LPS-induced iNOS expression is differentially regulated along both the longitudinal and crypt villus axes of the intestinal mucosa, being most prominent in the villus cells of the ileum. LPS-induced iNOS expression is blunted by pretreating rats with dexamethasone or cycloheximide. The latter finding suggests that LPS-induced expression of iNOS mRNA in the gut requires new protein synthesis. Differential regulation of nitric oxide production along the longitudinal and crypt-villus axes of the gut may be a determinant of the pattern of sepsis-induced intestinal damage.

Animals↗