Expression of cyclin D1a and D1b as predictive factors for treatment response in colorectal cancer.

BACKGROUND: The aim of this study was to investigate the value of the cyclin D1 isoforms D1a and D1b as prognostic factors and their relevance as predictors of response to adjuvant chemotherapy with 5-fluorouracil and levamisole (5-FU/LEV) in colorectal cancer (CRC). METHODS: Protein expression of nuclear cyclin D1a and D1b was assessed by immunohistochemistry in 335 CRC patients treated with surgery alone or with adjuvant therapy using 5-FU/LEV. The prognostic and predictive value of these two molecular markers and clinicopathological factors were evaluated statistically in univariate and multivariate survival analyses. RESULTS: Neither cyclin D1a nor D1b showed any prognostic value in CRC or colon cancer patients. However, high cyclin D1a predicted benefit from adjuvant therapy measured in 5-year relapse-free survival (RFS) and CRC-specific survival (CSS) compared to surgery alone in colon cancer (P=0.012 and P=0.038, respectively) and especially in colon cancer stage III patients (P=0.005 and P=0.019, respectively) in univariate analyses. An interaction between treatment group and cyclin D1a could be shown for RFS (P=0.004) and CSS (P=0.025) in multivariate analysis. CONCLUSION: Our study identifies high cyclin D1a protein expression as a positive predictive factor for the benefit of adjuvant 5-FU/LEV treatment in colon cancer, particularly in stage III colon cancer.

Episiotomy characteristics and risks for obstetric anal sphincter injuries: a case-control study.

OBJECTIVES: To investigate the association between the geometrical properties of episiotomy and obstetric anal sphincter injuries (OASIS) because episiotomies angled at 40-60° are associated with fewer OASIS than episiotomies with more acute angles. DESIGN: Case-control study. SETTING: University Hospital of North Norway, Tromsø and Nordland Hospital, Bodø, Norway. SAMPLE: Seventy-four women who had one vaginal birth and episiotomy. Cases (n = 37) have sustained OASIS at birth, while controls (n = 37) had not. The groups were matched for instrumental delivery. METHODS: Two groups of women with history of only one vaginal birth were compared. Episiotomy scar was identified and photographed and relevant measures were taken. Data were analysed using conditional logistic analysis. MAIN OUTCOME MEASURES: Mean episiotomy angle, length, depth, incision point. RESULTS: The risk of sustaining OASIS decreased by 70% (odds ratio [OR] 0.30; 95% CI 0.14-0.66) for each 5.5-mm increase in episiotomy depth, decreased by 56% (OR 0.44; 95% CI 0.23-0.86) for each 4.5-mm increase in the distance from the midline to the incision point of the episiotomy, and decreased by 75% (OR 0.25; 95% CI 0.10-0.61) for each 5.5-mm increase in episiotomy length. Lastly, there was no difference in mean angle between groups but there was a "U-shaped" association between angle and OASIS (OR 2.09; 95% CI 1.02-4.28) with an increased risk (OR 9.00; 95% CI 1.1-71.0) of OASIS when the angle was either smaller than 15° or >60°. CONCLUSION: The present study showed that scarred episiotomies with depth > 16 mm, length > 17 mm, incision point > 9 mm lateral of midpoint and angle range 30-60° are significantly associated with less risk of OASIS. Shrinkage of tissue must be considered.

Incontinence after primary repair of obstetric anal sphincter tears is related to relative length of reconstructed external sphincter: a case-control study.

OBJECTIVE: To determine if anatomic primary repair with end-to-end reconstruction of the external anal sphincter (EAS) in its full length combined with separate repair of coexisting internal anal sphincter (IAS) tear, when present, results in less incontinence and better anal sphincter integrity compared with conventional primary end-to-end repair in which the IAS is not actively reconstructed. METHODS: Women who sustained third- or fourth-degree obstetric tears were included prospectively in the study following anatomic primary repair. Women treated with conventional primary repair prior to the study period comprised the control group. Three-dimensional endoanal ultrasonography (3D-EAUS) images were classified according to the EAUS defect score, and incontinence according to St Mark's score. RESULTS: Sixty-three women were included in the study group and 61 in the control group, with mean follow-up times of 11 and 21 months, respectively. Among women who had not delivered vaginally prior to the tear, St Mark's score ≥ 3 was reported by 9.6% (5/52) in the study group and 37.5% (15/40) in the control group at follow-up (P = 0.002). The corresponding numbers among women who had previously delivered vaginally were 36.4% (4/11) and 42.9% (9/21), respectively (non-significant). St Mark's score correlated with the EAUS defect score (P = 0.017). An EAS defect exceeding 50% of the sphincter length was significantly less common in the study group, and in a multivariable logistic regression model, mode of repair (anatomic vs conventional) was the only factor explaining the difference in EAS sphincter length between the two groups (P = 0.007). CONCLUSION: Improved continence status after anatomic primary repair was associated with a better longitudinal reconstruction of the EAS, while the integrity of the IAS did not differ between the groups. Women with a history of vaginal delivery prior to the sphincter tear had an inferior outcome regardless of mode of repair.

Spigelian-cryptorchidism syndrome: a case report and discussion of the basic elements in a possibly new congenital syndrome.

Pediatric cases of Spigelian hernias are rare. Only a few reports on this condition, in combination with ipsilateral cryptorchidism and testis in the hernia sac, have been published. We report on Spigelian hernia in a 3-week-old boy containing both the ipsilateral testis, without a gubernaculum and an incarcerated loop of the small intestine. It has been suggested that the combination of Spigelian hernia and ipsilateral cryptorchidism is part of a new syndrome. We discuss whether the lack of a gubernaculum and an inguinal canal reported in other similar cases may be additional elements of this new syndrome. We present a comprehensive overview of pediatric patients with Spigelian-cryptorchidism syndrome reported in the English language literature. In 75% of male infants with Spigelian hernia, there is an associated ipsilateral cryptorchidism, and in 87% of these patients, the testis is found inside the hernia sac. Thus, the surgeon dealing with a congenital Spigelian hernia should look for an undescended testis and be prepared to find it in the hernia sac.

Expression of EZH2 and Ki-67 in colorectal cancer and associations with treatment response and prognosis.

BACKGROUND: Enhancer of zeste homologue 2 (EZH2) is a member of the Polycomb group of genes that is involved in epigenetic silencing and cell cycle regulation. METHODS: We studied EZH2 expression in 409 patients with colorectal cancer stages II and III. The patients were included in a randomised study, and treated with surgery alone or surgery followed by adjuvant chemotherapy. RESULTS: EZH2 expression was significantly related to increased tumour cell proliferation, as assessed by Ki-67 expression. In colon cancer, strong EZH2 expression (P=0.041) and high proliferation (>or=40%; P=0.001) were both associated with better relapse-free survival (RFS). In contrast, no such associations were found among rectal cancers. High Ki-67 staining was associated with improved RFS in colon cancer patients who received adjuvant chemotherapy (P=0.001), but not among those who were treated by surgery alone (P=0.087). In colon cancers stage III, a significant association between RFS and randomisation group was found in patients with high proliferation (P=0.046), but not in patients with low proliferation (P=0.26). Multivariate analyses of colon cancers showed that stage III (hazard ratio (HR) 4.00) and high histological grade (HR 1.80) were independent predictors of reduced RFS, whereas high proliferation indicated improved RFS (HR 0.55). CONCLUSION: Strong EZH2 expression and high proliferation are associated features and both indicate improved RFS in colon cancer, but not so in rectal cancer.

Locally recurrent rectal cancer in Norway.

BACKGROUND: The purpose of this study was to describe management and outcome in patients with locally recurrent rectal cancer based on data from the Norwegian Colorectal Cancer Registry. METHODS: This was a prospective national cohort study of 577 patients with local recurrence after major resection (R0/R1) for rectal cancer between November 1993 and December 2001 (initial cohort of 4504 patients). RESULTS: Of the 577 patients, 185 (32.1 per cent) had curative resections (R0/R1), 203 (35.2 per cent) had palliative radiotherapy with or without palliative surgery and chemotherapy, and 189 (32.8 per cent) received no treatment at all or only palliative surgery or chemotherapy. The overall 5-year survival rate was 14.9 per cent. Ninety-seven patients had an R0 resection, and 88 had an R1 resection, with 5-year overall survival of 55 and 20 per cent respectively. This outcome reflected surgical treatment in 33 different hospitals. Some 274 patients (47.5 per cent) had metastases. The 5-year survival rate after R0 resection was 62 per cent in patients without metastases. CONCLUSION: Obtaining an R0 resection is the most important prognostic factor in treating recurrent rectal cancer.

Three-dimensional endoanal ultrasonography: intraobserver and interobserver agreement using scoring systems for classification of anal sphincter defects.

OBJECTIVES: To determine the degree of intraobserver and interobserver agreement for an experienced and an inexperienced sonologist using two scoring systems for ultrasonographic assessment of anal sphincter defects. METHODS: The study sample comprised the datasets of all women aged between 20 and 40 years who had attended our outpatient clinic and undergone a complete three-dimensional (3D) endoanal ultrasound (EAUS) examination in the period from January 2003 to December 2005. The EAUS datasets were assessed twice independently by two sonologists: one with experience of > 400 3D EAUS assessments and one inexperienced sonologist who had performed approximately 50 assessments before the study. Cases with intraobserver disagreement were resolved by a third (final) assessment. The final assessment from each observer was used to determine the degree of interobserver agreement. Sphincter defects were classified according to our EAUS defect score and the Starck score. RESULTS: EAUS datasets of 55 women were included. Based on first vs. second assessments, intraobserver agreement for the experienced sonologist was good for our EAUS defect score (weighted kappa, 0.75) and the Starck score (weighted kappa, 0.73). Intraobserver agreement for the inexperienced sonologist was moderate for our EAUS defect score (weighted kappa, 0.58) and good for the Starck score (weighted kappa, 0.62). Interobserver agreement was good for both our EAUS defect score (weighted kappa, 0.65) and the Starck score (weighted kappa, 0.74). CONCLUSIONS: Intraobserver and interobserver agreement was acceptable for both scoring systems. The experienced sonologist obtained a higher degree of intraobserver agreement than did the inexperienced sonologist.

Long-term results after anterior sphincteroplasty for anal incontinence.

OBJECTIVE: To assess the long term incontinence and quality of life (Qol) results after elective anterior sphincteroplasty for anal incontinence. MATERIALS AND METHODS: Short and long term follow-up included respectively 28 and 25 of the 29 patients who were operated between 1989 and 1998 in our institution. Qol was assessed with gastro intestinal quality of life index (GIQLI). Incontinence was graded according to Parks score supplied with St Mark's score at long term follow-up. RESULTS: 21 (73%) patients had a history of obstetric sphincter tears. Mean age at operation was 45 years (range 6-77). Median time from operation to short term follow-up was 26 months (mean 38 months, range 2-113) and 84 months (mean 105, range 74-185) to long term follow-up. At short term follow-up 19 of 28 patients (68%) were continent for stool compared with nine of 25 patients (36%) at long term follow-up. Nine of 17 (53%) who were continent for stool at short term follow-up remained continent for stool at long term follow-up. Patients with a history of obstetric sphincter tear had less severe incontinence at long term follow-up compared to women with other causes of incontinence (St. Mark's score 8 and 16 respectively, p = 0,015). Patients with no incontinence or gas incontinence only, had higher quality of life score at both follow-ups than those who where incontinent for stool (p = 0,007 and p= 0,014 respectively). CONCLUSION: More than half of the patients remained continent for stool at long term follow-up. Continence for stool was associated with high Qol score.

Quality of life in Dukes' B and C colorectal carcinoma.

Adjuvant chemotherapy (ACT) is standard therapy in Dukes' C colorectal carcinoma (CRC) in several countries. Several studies have documented a significant improvement in overall survival, but the clinical efficacy on quality of life (QoL) has been doubted. To clarify this item, we performed a study on QoL in 94 CRC patients diagnosed between 1993-96 and randomised to surgery with or without ACT. 82 'survivors' were mailed the EORTC QLQ C-30. Sixty-two patients (76%) responded. They reported a high level of functioning and a low frequency of symptoms. ACT did not improve global QoL, and tended to cause less pain (p=0.023) and more nausea/vomiting (p=0.065).

Adenomas with adenocarcinoma: a study evaluating the risk of residual cancer and lymph node metastasis.

BACKGROUND AND AIMS: The increasing number of cases with colorectal adenomas with adenocarcinoma necessitates renewed evaluation of classification systems and risk factors. The aim for this retrospective study was to evaluate the potential risk of residual cancer and lymph node metastasis in patients with colorectal adenomas with adenocarcinoma. MATERIAL AND METHODS: An investigation of adenomas with adenocarcinoma in 74 patients was performed on histological slides and compared with clinical characteristics. A total of 44 of the samples were from macroscopically and microscopically completely resected lesions, and cancer at extended surgery was compared with pathology reports, classifications, and histopathological features. RESULTS: In all, 26 cases of adenomas with adenocarcinoma in the rectum and rectosigmoid were among women and 11 in men while 22 men as opposed to 15 women had primary lesions in colon, giving a significant association between gender and localization (p = 0.01). For macroscopically and microscopically fully resected lesions, Haggitt classification or submucosal invasion did not correlate with cancer at extended surgery. The lack of information on resection margins in the primary pathology reports was found to correlate significantly with residual cancer at extended surgery (p < 0.001) with residual cancer in 3 out of the 10 cases with no information, 1 out of the 5 where the resection margins were uncertain, 1 out of the 4 where the resection margins were not free, and none of the 25 cases when the resection margins were reported as free. In colon, 1 case out of the 6 with extended surgery (16.7%) was diagnosed with residual cancer compared with 4 out of the 10 (40%) from rectum. CONCLUSIONS: Haggitt or submucosal classifications were not found to be predictors for residual cancer in the remaining bowel tissue or lymph node metastasis. The only significant factor indicating increased risk of residual cancer was the lack of information on resection margins in the pathology report. Surgeons should therefore be alert when adenomas with adenocarcinomas are not confirmed as microscopically free in the pathology report.

Translation and validation of the Norwegian version of the fecal incontinence quality-of-life scale.

BACKGROUND AND AIM: Fecal incontinence quality-of-life scale (FIQLS) is a condition-specific health-related quality-of-life questionnaire composed of four scales: lifestyle, coping/behaviour, depression/self-perception and embarrassment. It has been widely translated and used as an evaluation tool for patients with fecal incontinence. Our aim was to translate the FIQLS, and to test some of the psychometric properties of the Norwegian version of the questionnaire. MATERIAL AND METHODS: The FIQLS was translated to Norwegian, and administered to a sample of 76 patients (73 women) who completed the questionnaire at baseline and again after three weeks. In addition, the severity of incontinence was assessed by phone-interviews (St. Mark's score). RESULTS: Three of four domains had good internal consistency in terms of Cronbach's alpha (.83-.91), the fourth (embarrassment) somewhat lower (.64). Stability over time was acceptable for all domains with ICC ranging from .74 to .86. Correlation with severity of incontinence (St. Mark's score) was medium to large for all four domains (-.46 to -.63) supporting the construct validity of the Norwegian FIQLS. CONCLUSION: The Norwegian version of fecal incontinence quality-of-life scale has been successfully translated and tested.

Correlation between anal sphincter defects and anal incontinence following obstetric sphincter tears: assessment using scoring systems for sonographic classification of defects.

OBJECTIVE: To determine if there is a correlation between the sonographic extent of anal sphincter defects revealed by three-dimensional endoanal sonography (EAUS) and the degree of anal incontinence following primary repair of obstetric sphincter tears. METHODS: This was a follow-up study of women who had suffered anal sphincter tearing during vaginal delivery at Aalesund Hospital between January 2002 and July 2004. Incontinence was assessed by St Mark's score. The anal canal was assessed with three-dimensional endoanal sonography (EAUS). Sphincter defects were classified according to the Starck score and our new EAUS defect score. The EAUS images were interpreted by an observer blinded to other patient data. RESULTS: Sixty-one women were included in this study. Incontinence was reported by 32 (52%) women at a median of 21 (range, 9-35) months after delivery. Three-dimensional EAUS datasets were obtained in 55 women. There was a significant correlation between St Mark's score and our EAUS defect score (P = 0.034), and correlation approached but did not reach significance between St Mark's score and the Starck score (P = 0.053). There was a strong correlation between our EAUS defect score and the Starck score (P < 0.001). CONCLUSIONS: There is a positive correlation between the extent of sphincter defects and the degree of anal incontinence following primary repair of obstetric sphincter tears. Our findings highlight the importance of adequate reconstruction of the anal sphincters during primary repair.

Impact of radiotherapy on local recurrence of rectal cancer in Norway.

BACKGROUND: The purpose of this study was to analyse the impact of radiotherapy on local recurrence of rectal cancer in Norway after the national implementation of total mesorectal excision (TME). METHODS: This was a prospective national cohort study of 4113 patients undergoing major resection of rectal carcinoma between November 1993 and December 2001. RESULTS: The proportion of patients who had radiotherapy before or after operation increased from 4.6 per cent in 1994 to 23.0 per cent in 2001. The cumulative 5-year local recurrence rate decreased from 16.2 to 10.7 per cent. Multivariable analysis showed that preoperative radiotherapy significantly reduced local recurrence (hazard ratio 0.59 (95 per cent confidence interval 0.39 to 0.87)). The use of preoperative radiotherapy in patients from a local hospital offering radiotherapy was 50 per cent higher than that for patients from a hospital without such services (P = 0.003); cumulative 5-year local recurrence rates for these patients were 10.6 and 15.8 per cent respectively (P < 0.001). CONCLUSION: Following national implementation of TME for rectal cancer, increased use of preoperative radiotherapy appeared to reduce recurrence rates further.

Improvement of real-time polymerase chain reaction for quantifying TNF-alpha mRNA expression in inflamed colorectal mucosa: an approach to optimize procedures for clinical use.

OBJECTIVE: The precise measurement of local tumor necrosis factor alpha (TNF-alpha) expression in tissue is important in understanding the pathogenesis of inflammatory bowel diseases (IBD). Real-time polymerase chain reaction (PCR) is a sensitive, versatile method and is becoming a commonly used tool for the quantification of gene expression. The aim of this study was to optimize the laboratory procedure for biopsy sampling, storage and calibration of result for TNF-alpha mRNA quantification with real-time PCR of colorectal biopsies. MATERIAL AND METHODS: Endoscopic biopsies from the colorectum were obtained from 18 patients with ulcerative colitis (UC), 11 patients with Crohn's disease (CD) and 18 normal controls. Optimization of procedures for real-time PCR performance was carried out. RESULTS: The transport medium, RNAlater, exhibited a high preservation effect against RNA degradation even after 8 days of storage at room temperature; one biopsy from each patient was sufficient for RNA extraction, cDNA synthesis and TNF-mRNA quantification. An assay was established with a technical reproducible sensitivity of 100 copies/microL. The observed interassay variations were 7.4 % coefficient of variation (CV) and 7.2 % CV in low and high TNF-alpha mRNA expression biopsies, respectively. TNF-alpha mRNA levels in colorectal biopsies from patients with either CD or moderate to severe UC were markedly increased, and 8 approximately 9-fold higher than those in healthy controls. CONCLUSIONS: This optimization improves the clinical use of real-time PCR for quantification of TNF-alpha gene expression in colorectal biopsies and provides a sensitive reproducible assay.

[Use of personal computers for statistical analyses]. / Bruk av personlige datamaskiner til statistiske analyser.

We report the range in elapsed time for various statistical analyses and for the same logistic regression analysis in 23 different personal computers (PCs). Without a floating-point coprocessor, the elapsed time increased from ten to 113 minutes in the same PC. Elapsed time ranged from two to 45 minutes from 80486 to 8086 PCs with a floating-point coprocessor. The PC equipment we need strongly depends on the type and frequency of the statistical analyses we are to perform. If a floating-point coprocessor is installed, the 80286/80287 PC family may be fast enough for most users.

Penicillin toxicity in isolated rat hepatocytes revealed by decreased incorporation of valine into proteins.

Rat liver parenchymal cells were isolated from normal and barbiturate pretreated rats. Cells from untreated animals were exposed to penicillin over a concentration range from 0.14 mM to 14.0 mM (50-5000 micrograms/ml). An inhibition of the incorporation of 14C valine into stationary and medium proteins, ranging from 23% at 0.28 mM to 90% at 14.0 mM, was observed. The effect of a single dose penicillin (1.4 mM) on protein incorporation, enzyme leakage and viability was compared to the effect of paracetamol (6.6 mM) and tertiary butanol (10.9 mM). In these concentrations paracetamol and penicillin both inhibited the incorporation of valine into cell and medium protein in hepatocytes from untreated rats. Tertiary butanol showed no such effect. No drug affected the viability or the leakage of enzymes from the hepatocytes. In cells from barbiturate treated animals both paracetamol, penicillin and tertiary butanol had a significant inhibitory effect on the incorporation of radioactive labelled precursor into cell and medium proteins, but no effect on the leakage of enzymes or viability. The ratio between labelled medium and cell proteins was 31% lower in suspensions of control cells from barbiturate treated animals than in cells from untreated rats. It was concluded that penicillin may exert marked effects on protein metabolism in the frequently used isolated rat hepatocyte system, especially if the drug concentration well exceeds the usual cell culture concentration of 30-60 micrograms/ml.

Isolated rat hepatocytes in suspension: potential hepatotoxic effects of six different drugs.

Isolated rat hepatocytes in suspension were studied with regard to various measures of hepatic toxicity. We compared enzyme leakage (ASAT, ALAT, LDH), cell viability (trypan blue exclusion), intracellular ATP content, and incorporation of 14C-valine into stationary and export proteins while the cells were exposed to six different drugs at two different concentrations. The drugs were oxytetracycline, paracetamol, carbon tetrachloride, ethanol, methotrexate and fentanyl. The results were compared to known in vivo responses, in particular to see whether concentrations resulting in dose-related in vivo effects would similarly affect the functions tested in vitro. Leakage of enzymes exhibited a graded increase with a corresponding rise in the concentration of oxytetracycline and carbon tetrachloride. Reduction in incorporation of 14C-valine into cell and medium proteins showed a similar graded effect with rising concentrations of paracetamol, carbon tetrachloride, and ethanol. Intracellular levels of ATP gradually decreased with increasing concentrations of carbon tetrachloride and ethanol. An obvious reduction in viability was only registered with increasing concentrations of carbon tetrachloride, while paracetamol tended to give a similar response. We found no major discrepancies between already known in vivo effects and our in vitro results when testing paracetamol, carbon tetrachloride, ethanol, methotrexate, and fentanyl. We could not, however, demonstrate inhibition of protein synthesis by oxytetracycline at the concentrations tested. No single measurement was adequate for testing all drugs. The test of 14C-valine incorporation into hepatocyte export proteins plus LDH leakage seemed to constitute a useful combination in detecting drug toxicity in hepatocyte suspensions.

Effect of cholecystokinin on the accumulation of inositol phosphates in isolated pancreatic islets.

Sulfated cholecystokinin octapeptide (CCK-8S) potentiated glucose-induced secretion in isolated pancreatic islets with a maximal effect at 12 mM glucose, whereas no effect was observed at 3.3 and 25 mM glucose. This effect of CCK-8S was maximal at 10(-7) M. Anion-exchange fast-protein liquid chromatography analysis of [3H]inositol phosphates derived from islets prelabeled with myo-[3H]inositol showed that glucose induced accumulation of the 1,4,5-isomer of inositol trisphosphate and of inositol tetrakisphosphate. At 3.3 mM glucose, CCK-8S stimulated accumulation of inositol trisphosphate and inositol tetrakisphosphate to levels induced by 25 mM glucose alone. The net effect of CCK-8S on the accumulation of the inositol phosphates was maximal at 12 mM glucose and decreased at higher glucose concentrations. At 12 mM glucose the accumulation of inositol phosphates increased gradually up to 10(-7) M CCK-8S. This study indicates that CCK-8S potentiates glucose-induced insulin secretion through a mechanism involving the hydrolysis of polyphosphoinositides and the generation of inositol phosphates. However, activation of the inositol cycle per se did not seem to induce insulin secretion.

Sorbitol in isolation of rat pancreatic islets. Effects on islet yield, insulin secretion and accumulation of inositol phosphates.

Tissue aggregation and exocrine contamination are problems encountered in gradient separation of pancreatic islets. Here we report that sorbitol used as an osmotic component in Percoll gradients gives a low ionic strength gradient with improved purity of islet fraction, less islet aggregation and reduced time for final manual rinsing following separation in gradients with NaCl as osmotic component. Previous reports have indicated that long-term (weeks) exposure to high sorbitol concentrations leads to low intracellular levels of inositol phosphates and subsequent effects on the intracellular signal transduction in cells. In our model, short-term exposure to high sorbitol concentrations had no effect on the accumulation of the inositol phosphates or insulin secretion caused by glucose. On the other hand, sorbitol increased the basal insulin secretion three-fold, apparently via a non-stimulatory mechanism. Therefore, we conclude that sorbitol is preferable to NaCl as the osmotic component in Percoll gradient separation of rat pancreatic islets, although long-term exposure should be avoided due to potential toxic effects.

Cholecystokinin and somatostatin modulate the glucose-induced insulin secretion by different mechanisms in pancreatic islets. A study on phospholipase C activity and calcium requirement.

To study the interaction between the phospholipase C activation and the insulin secretion, isolated pancreatic islets were stimulated with glucose and the sulfated cholecystokinin octapeptide (CCK). To discriminate between intracellular mechanisms, experiments with agents inhibiting adenylyl cyclase and calcium-channels like somatostatin and verapamil, were performed. The phospholipase C activity, i.e. the accumulation of inositol phosphates, was increased by CCK (100 nmol l-1) at 3.3 mmol l-1 glucose. This effect of CCK did not require extracellular Ca2+, was not inhibited by somatostatin (100 nmol l-1), and no concomitant increase in the insulin secretion was observed. Both the phospholipase C activity and the insulin secretion increased in response to 12 mmol l-1 glucose. Somatostatin was able in some extent to inhibit these effects of glucose. At 12 mmol l-1 glucose, the phospholipase C activity and the insulin secretion were potentiated by CCK. CCK also counteracted the effect of somatostatin on the phospholipase C activity and the insulin secretion. Verapamil (2.5 umol l-1) more or less completely inhibited both the glucose-induced phospholipase C activity and the insulin secretion. Moreover, whereas the CCK-induced increase in the phospholipase C activity was unaffected, verapamil blocked the CCK-induced increase in the insulin secretion. We conclude that CCK directly activates phospholipase C, whereas glucose and somatostatin modulates phospholipase C via a Ca(2+)-dependent mechanism. CCK potentiates the insulin secretion by increased phospholipase C activity, but with a requirement of glucose at an apparent threshold level of Ca(2+)-influx.