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The influence of nitric oxide and cholecystokinin on tissue homeostasis in exocrine pancreas: an experimental study in rats
Linköping University, Department of Biomedicine and Surgery, Surgery. Linköping University, Faculty of Health Sciences.
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Growth of the pancreas is stimulated by cholecystokinin (CCK) in rats. Nitric oxide (NO), which is synthesized from the amino acid L-arginine by NO-synthases (NOS), interferes with CCK in modulating the pancreatic secretion. Both pro- and anti-apoptotic influences of NO have been observed in other tissues, but its importance for pancreatic tissue homeostasis has not been studied.

Cell proliferation and death rates were studied in the rat pancreas during different experimental conditions. CCK-octapeptide (CCK-8) was given in different doses either intermittently or continuously for three days and the pancreatic growth response was studied. Exogenous CCK-8 caused dose-dependent pancreatic atrophy when given intermittently and hyperplasia when given continuously.

The influence of NO on cell death and proliferation was studied during: 1) basal conditions, 2) CCK-8 induced hyperplasia, and 3) CCK-8 induced atrophy. The NO level was manipulated either by NOS inhibition (L-NNA) or by exogenous NO supply (SNAP). NO-metabolism was assessed in the basal situation by analysis of nitrite/nitrate excretion in urine (which was decreased by L-NNA and increased by SNAP), and L-arginine in serum (which increased by L-NNA) and L-citrulline in serum.

1) During basal conditions NOS inhibition (NO↓) increased apoptosis and decreased cell proliferation.

2) During CCK-8 induced hyperplasia NOS inhibition (NO↓) increased both apoptosis and cell proliferation. The apoptosis dominated as indicated by decreased DNA content. SNAP administration (NO↑) did neither influence apoptosis nor cell proliferation.

3) During CCK-8 induced atrophy (DNA↓, apoptosis↑, cell proliferation↑, and cytoplasmic vacuolization) NOS inhibition further increased apoptosis, reduced cell proliferation and abolished vacuole formation. SNAP administration (NO↑) decreased the DNA content, and increased both apoptosis and cell proliferation. The vacuole formation was still present. Hence, NO influences both the basal and the disturbed homeostasis in hyperplastic and atrophic rat pancreatic tissue.

Early events of a load of L-arginine, known to induce pancreatitis within 48 hrs, was studied at 8, 16 and 24 hrs by analysis of serum L-arginine and L-citrulline, pancreatic tissue ATP, apoptosis and cell proliferation. The initially increased serum L-arginine and L-citrulline decreased to levels below control at 24 hrs. Administration of L-arginine was correlated to a biphasic ATP production and formation of small vacuoles (mitochondrial swelling) in the acinar cells, most prominent at 8 hrs and followed by a gradually increased apoptosis rate. The cell proliferation decreased. At 24 hrs there was pronounced cell degeneration, but no evident necrosis. Another 20 amino acids in serum were also analysed at 24 hrs. Twelve amino acids (including the 'glutamate family') were significantly reduced. After an L-arginine load the augmented A TP production correlates to the initiation of pancreatic cell death. The disturbed amino acid metabolism seems to be of importance for development of experimental pancreatitis.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet , 2004. , 67 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 846
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-22407Local ID: 1619ISBN: 91-7373-820-4 (print)OAI: oai:DiVA.org:liu-22407DiVA: diva2:242720
Public defence
2004-05-06, Elsa Brändströmsalen, Hälsouniversitetet, Linköping, 13:00 (Swedish)
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2012-11-01Bibliographically approved
List of papers
1. Cholecystokinin octapeptide induces both proliferation and apoptosis in the rat pancreas
Open this publication in new window or tab >>Cholecystokinin octapeptide induces both proliferation and apoptosis in the rat pancreas
2001 (English)In: Regulatory Peptides, ISSN 0167-0115, E-ISSN 1873-1686, Vol. 98, no 1-2, 41-48 p.Article in journal (Refereed) Published
Abstract [en]

Cholecystokinin-8 (CCK-8) causes exocrine pancreatic hypertrophy and hyperplasia. High doses of the CCK analogue cerulein causes necrosis and an inflammatory response in the pancreas. We have studied the pancreatic growth response in rats after administration of CCK-8 for 3 days, given either intermittently (20–80 μg/kg) twice a day, or continuously (2.4–48 μg/kg per 24 h). Plasma CCK-8 levels, pancreatic wet weight, water, protein and DNA contents and the pancreatic caspase-3 activity were measured. Cell proliferation was visualized by [3H]thymidine incorporation and apoptosis by TUNEL reaction. Continuous administration of CCK-8 dose-dependently increased the plasma CCK levels, the pancreatic wet weight, protein and DNA contents as well as thymidine labeling index, apoptotic index and caspase-3 activity. Intermittent injections of CCK-8 caused transient raises in plasma CCK, increased apoptotic index and caspase-3 activity, a dose-dependent increase in thymidine labeling but caused a dose-dependent reduction of pancreatic wet weight, protein, and DNA contents. It is concluded that CCK-8 causes both increased proliferation and apoptosis in the pancreas. In case of continuous administration of CCK-8, the proliferation outweighs the apoptosis causing hyperplasia but in the case of intermittent administration the opposite effect is seen.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-25028 (URN)10.1016/S0167-0115(00)00223-8 (DOI)9451 (Local ID)9451 (Archive number)9451 (OAI)
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2017-12-13
2. The influence of nitric oxide on basal and cholecystokinin-8-induced proliferation and apoptosis in the rat pancreas
Open this publication in new window or tab >>The influence of nitric oxide on basal and cholecystokinin-8-induced proliferation and apoptosis in the rat pancreas
2002 (English)In: Regulatory Peptides, ISSN 0167-0115, E-ISSN 1873-1686, Vol. 106, no 1-3, 97-104 p.Article in journal (Refereed) Published
Abstract [en]

Nitric oxide (NO) is formed by different cell types in the pancreas. In this study, inhibition of endogenous nitric oxide by Nω-nitro-l-arginine (l-NNA) reduced the urinary excretion of NO2/NO3 and raised serum l-arginine and the NO donator S-nitroso-N-acetylpenicillamine (SNAP) increased the urinary excretion of NO2/NO3. The peptide cholecystokinin-8 (CCK-8) has a strong influence on exocrine pancreatic proliferation. Rat pancreas was excised and studied with regard to tissue weight, protein and DNA contents after 3 days of treatment with saline, l-NNA or SNAP given separately or combined with CCK-8. Further, proliferation of different pancreatic cells was studied with [3H]-thymidine incorporation and apoptotic activity was studied by analysing caspase-3 activity and histone-associated DNA fragments. The effects of l-NNA indicate that endogenous nitric oxide formation has a tonic inhibition on apoptosis in the pancreas during both basal condition and growth stimulation by CCK-8. In CCK-induced hyperplasia, NO inhibits the proliferation of acinar cells but stimulates ductal cells. Endogenous NO may regulate the balance between proliferation and apoptosis and in a situation of growth stimulation by CCK-8, it has a tonic inhibition on both mitogenesis and apoptosis thus slowing down the acinar cell turnover in the pancreas.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-25029 (URN)10.1016/S0167-0115(02)00056-3 (DOI)9452 (Local ID)9452 (Archive number)9452 (OAI)
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2017-12-13
3. Cholecystokinin-8-induced atrophy in the rat pancreas: influence of nitric oxide on proliferation, programmed cell death and NF-κB activation
Open this publication in new window or tab >>Cholecystokinin-8-induced atrophy in the rat pancreas: influence of nitric oxide on proliferation, programmed cell death and NF-κB activation
(English)Manuscript (preprint) (Other academic)
Abstract [en]

The mechanisms involved in cholecystokinin-8-induced atrophy in the pancreas are not known and in this study the roles of nitric oxide (NO) and NF-κB were studied in rats. CCK-8 was injected for 4 days, in a mode known to cause atrophy, and the NO formation was either decreased by Nω-nitro-L-arginine (L-NNA) or increased by S-nitroso-N-acetylpencillamine (SNAP). Activation of NF-κB was quantified by ELISA detection, apoptosis with caspase-3 and histone associated DNA-fragmentation and mitotic activity in the acinar, centroacinar and ductal cells was visualized by incorporation of [3H]-thymidine. Pancreatic histology, weight, protein- and DNA contents were studied.

Intermittent CCK injections reduced pancreatic weight, protein and DNA contents and increased apoptosis, acinar cell proliferation and NF-κB activation. It also caused vacuolisation of the acinar cells. Inhibition of endogenous NO formation by L-NNA further increased apoptosis and NF-κB activation but blocked the increased proliferation and vacuolisation of acinar cells. The DNA content was not further reduced. SNAP given together with CCK-8 increased both apoptosis and proliferation of acinar cells and strongly reduced the DNA content in the pancreas. Further, it caused vacuolisation in the acinar cells and these findings together indicate cell death by other pathways besides apoptosis. Histological examination showed no inflammation in any group.

During CCK-8 induced pancreatic atrophy endogenous NO suppresses apoptosis and stimulates regeneration of acinar cells but increases cell death by non-apoptotic pathways. Exogenous NO enhances the acinar cell turnover by increases of both proliferation and apoptotic and non-apoptotic cell deaths. NF-κB activation, in this situation, seems not to inhibit apoptosis or promote cell proliferation.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-85071 (URN)
Available from: 2012-11-01 Created: 2012-11-01 Last updated: 2012-11-01
4. The influence of a load of L-arginine on serum amino acids and pancreatic apoptosis/proliferation and ATP levels in the rat
Open this publication in new window or tab >>The influence of a load of L-arginine on serum amino acids and pancreatic apoptosis/proliferation and ATP levels in the rat
Show others...
2004 (English)In: Pancreas, ISSN 0885-3177, E-ISSN 1536-4828, Vol. 29, no 4, 113-120 p.Article in journal (Refereed) Published
Abstract [en]

OBJECTIVES:

Administration of high doses of amino acids like ethionine, methionine, and arginine causes pancreatic tissue damage. The initial mechanism behind these effects is not known. The aim of this study was to show the early effects of a load of L-arginine on programed cell death/proliferation and ATP levels in the pancreas.

METHODS:

We analyzed in rats the effects of intraperitoneal administration of L-arginine on serum amino acids, pancreatic cell apoptosis/proliferation, and ATP levels at 8, 16, and 24 hours. Serum amino acid concentrations were measured with HPLC, tissue ATP was measured fluorometrically, apoptosis was studied with caspase-3 activity and histone-associated DNA-fragments, and proliferation was studied with thymidine autoradiography.

RESULTS:

After a load of l-arginine, there were initially increased serum levels of L-arginine and L-citrulline, but these fell below control levels after 24 hours as well as amino acids in the glutamate family (ornithine, proline, histidine, and glutamine). Initially, increased ATP levels in the pancreatic tissue returned to control levels at 24 hours. The acinar cells proliferation was suppressed and the apoptosis rate strongly increased at 16 and 24 hours. Pancreatic histology showed vacuole formation in the acinar cells at 8 hours. At 16 hours, there was less vacuolization, but apoptotic bodies were seen, and at 24 hours there was cell degeneration but no necrosis.

CONCLUSIONS:

After a load of l-arginine, amino acid metabolism causes a high ATP production in the pancreatic tissue that may cause mitochondrial initiation of cell death.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-23748 (URN)15502637 (PubMedID)3258 (Local ID)3258 (Archive number)3258 (OAI)
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2017-12-13Bibliographically approved

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