Proliferative phenotype of pulmonary microvascular endothelial cells plays a critical role in the overexpression of CTGF in the bleomycin-injured rat.

The pathogenesis of idiopathic pulmonary fibrosis (IPF) is not very clear, with evidence for the involvement of both inflammation and aberrant vascular remodeling (associated with angiogenesis). Pulmonary microvascular endothelial cells (PMVECs), which play a major role in inflammation, secrete cytokines that promote the transformation and collagen synthesis of fibroblasts. Moreover, angiogenesis is characterized by PMVEC proliferation. The main aim of this study was to confirm the role of PMVECs in pulmonary fibrosis. Accordingly, we observed the functional changes in PMVECs in bleomycin (BLM)-treated rats (pulmonary fibrosis model) in vivo, and compared them with those of rats with pneumonia. The proliferation phenotype and intracellular ionized calcium concentration ([Ca(2+)]i) of PMVECs from BLM-treated rats were also investigated. The functioning of PMVECs was abnormal in BLM-injured rats, particularly with regard to their proliferation and secretion of connective tissue growth factor (CTGF). [Ca(2+)]i was increased in the proliferated PMVECs from BLM-treated rats. The findings suggest that dysfunction of PMVECs characterized by overexpression of CTGF is critical in rat pulmonary injury induced by BLM, and is probably related with the proliferative phenotype and [Ca(2+)]i overload. It can be concluded from the results that proliferation of PMVECs plays an important role in the pathogenesis of BLM-induced PF.

Pulmonary microvascular endothelial cells from bleomycin-induced rats promote the transformation and collagen synthesis of fibroblasts.

Accumulation and activation of myofibroblasts are the hallmark of progressive pulmonary fibrosis, and the resident fibroblasts are the major source of myofibroblasts. However, the key factors involved in the transformation of fibroblasts are unknown. Pulmonary microvascular endothelial cells (PMVECs), major effector cells against pathogenesis in early stages of the disease, can secrete cytokines to induce the differentiation of mesenchymal cells. We speculated that PMVECs could secrete pro-fibrotic cytokines and promote the transformation of fibroblasts into myofibroblasts. Accordingly, we established a co-culture system with PMVECs and fibroblasts to examine the specific transformation and collagen synthesis of the co-cultured fibroblasts by FACS and Western blot, prior to and after treatment with neutralizing antibodies against transforming growth factor-beta1 (TGF-ß1) and connective tissue growth factor (CTGF). We also analyzed expression of TGF-ß1 and CTGF in PMVECs. The synthesis and secretion of TGF-ß1 and CTGF protein were up-regulated in PMVECs isolated from bleomycin (BLM)-treated rats, most prominently at 7 days post-instillation. We showed that the PMVECs isolated from BLM-induced rats could induce the transformation of normal fibroblasts and their secretion of collagen I, which was inhibited by both neutralizing anti-TGF-ß1 and anti-CTGF antibodies. Therefore, up-regulation of TGF-ß1 and CTGF in PMVECs plays an important role in activation, transformation, and collagen synthesis of fibroblasts; in particular, these effects in PMVECs are likely to be the key factors for activation and stimulation of static fibroblasts in lung interstitium in early stages of pulmonary fibrosis disease.

Promoter cloning and characterization of the rabbit BK channel beta1 subunit gene.

The beta1 subunit of the voltage-dependent and Ca(2+)-activated large-conductance K(+) channel (BK) in mammalian smooth muscle cells (SMCs) plays an important role in regulating smooth muscle tone and is closely linked with a series of smooth muscle tone associated diseases. However, knowledge of the transcriptional regulation of the BK beta1 is still largely unclear. For the first time, we cloned and characterized the full-length genomic sequence of the rabbit BK beta1 containing a 5'-flanking region of 2021 bp. The full-reading frame of the BK beta1 spans ~7.7 kb and is organized into 4 exons and 3 introns. All of the exon/intron junction sequences contain the GT/AG consensus junction sequence. The transcription initiation site (+1G) is located at 447 bp upstream of the translation initiation codon. Bioinformatics analysis indicated that, without any canonical TATA-box, the 5'-flanking region possesses a high GC content and contains a number of putative transcription factor binding sites. 5'-deletion analysis demonstrated that the region of -93/+30 potentially functions as a core promoter region. A gel mobility shift assay and chromatin immunoprecipitation assay revealed that Sp1 specifically interacts with a putative Sp1-binding site (-91/-85) in vitro and in vivo. Mutation of this site significantly diminished the promoter activities. Over-expression of Sp1 in smooth muscle cells of rabbit sphincter of Oddi enhanced the promoter activities of the BK beta1 in a dose-dependent manner. Thus, we suggest that the Sp1-binding site (-91/-85) is essential to the basal transcription of the rabbit BK beta1. Our studies provide a basic knowledge of the transcription regulation of the rabbit BK beta1.

Sphincter of Oddi dysfunction in hypercholesterolemic rabbits.

BACKGROUND AND OBJECTIVES: The mechanisms that trigger gallbladder evacuation dysfunction, the key risk factor for gallstone formation, have not yet been fully elucidated. The sphincter of Oddi (SO) plays important roles in the regulation of gallbladder evacuation and maintenance of normal hydraulic pressure of the biliary tract. The aim of our study was to investigate the effects of hypercholesterolemia on the motility function of SO and the underlying mechanisms of SO dysfunction (SOD). METHODS: Forty New Zealand white rabbits were divided randomly into the control group fed with standard chow and the experimental (Ch) group fed with a high-cholesterol diet for 8 weeks. Changes in the maximal gallbladder emptying rate, gallbladder evacuation with cholecystokinin-octapeptide (CCK-8) stimulation and SO functions of both groups were measured in vivo; B ultrasound examination was used for dynamic observation of peristaltic movements in vivo; SO pressure was measured using manometry; morphological characteristics were observed by electronic microscope; laser scanning confocal fluorescence microscopy was used to identify changes in [Ca]i and Ca oscillation in primary SO smooth muscle cells (SMCs). RESULTS: Gallbladder cholestasis was observed during early stages of gallstone formation in Ch rabbits. CCK-8 could not improve the gallbladder cholestatic state in Ch group. Passive dilation of SO significantly improved the cholestatic state in Ch rabbits (P<0.05), although the maximal gallbladder emptying rate was still lower than that of the control group. Manometry data indicted a significant increase in the base pressure of the SO low-pressure ampulla segment and high-pressure segment (P<0.05) in Ch group. laser scanning confocal fluorescence microscopy assay data indicated that [Ca]i in SO cells of Ch group significantly increased and were in a state of overload (P<0.05); Ca oscillation signals in SO cells of Ch group were also abnormal. CONCLUSION: Hypercholesterolemia initially induced SOD, leading to increased gallbladder evacuation resistance and cholestasis. We suggested that [Ca]i overload and/or Ca oscillation abnormality potentially play important roles in the pathogenesis of SOD.

Molecular cloning, tissue distribution and bioinformatics analyses of the rabbit BK channel beta1 subunit gene.

Large-conductance, voltage-dependent and Ca(2+)-sensitive K(+) (BK) channels are composed of pore-forming alpha subunits and the modulatory beta subunits. In smooth muscle, the modulatory beta1 subunits are vital in rendering BK channels function as an important regulator of smooth muscle tone and excitability. In this study, we cloned and characterized the BK beta1 subunit gene from rabbits (New Zealand white) and observed its tissue distribution pattern. The full-length cDNA of the BK beta1 subunit, amplified by 5'-RACE and 3'-RACE, is 1,437 bp in nucleotide containing a 447 bp 5'-UTR, a 385 bp 3'-UTR and a 576 bp open reading frame (ORF) which encodes a peptide of 191 amino acids. Sequence analyses showed that the rabbit BK beta1 subunit cDNA is 90, 84 and 82% homologous with that of human, mouse and rat respectively. The similarity is 86, 83, and 83% at the deduced amino acids level with human, mouse and rat beta1 subunit gene, respectively. Northern blots indicated that the rabbit BK beta1 subunit gene is highly expressed in sphincter of Oddi (SO) and aortal smooth muscle tissues, whereas with relatively lower level of expression in heart and skeletal muscle tissues and with no expression found in tissues of liver, lung, kidney and brain. Bioinformatics analyses indicated that the encoded protein is a membrane protein with two transmembrane helical regions containing four functional domains, one possible PKA phosphorylation site (T14) at the N-terminal and two N-glycosylation sites (N80 and N142) at the extracellular loop. For the first time, we identified and characterized the full-length cDNA sequence of the rabbit BK channel beta1 subunit gene, which will set the basis for further investigation in the transcriptional regulation of this gene.

Down regulated expression of the beta1 subunit of the big-conductance Ca2+ sensitive K+ channel in sphincter of Oddi cells from rabbits fed with a high cholesterol diet.

Hypercholesterolemia, which is closely related to gallbladder bile stasis, can cause sphincter of Oddi dysfunction (SOD) by increasing the tension of sphincter of Oddi (SO). Intracellular calcium ion concentration ([Ca(2+)](i)) could influence the tension of SO. The beta1 subunit of the big-conductance Ca(2+) sensitive K(+) channel (BK(Ca)) can enhance the sensitivity of the BK(Ca) channel to [Ca(2+)](i). Absence and decline of the BKCa channel subunit beta1 could lead to many diseases. However, the relationship between hypercholesterolemia and the expression of beta1 subunit is not well understood. In this study, we successfully expressed and purified the rabbit BK(Ca) beta1 subunit protein and prepared its polyclonal antibody. The specificity of the prepared antibody was determined by Western blotting. A SOD rabbit model induced by a high cholesterol diet was established and the expression of the beta1 subunit of SO was determined by immunohistochemical staining and western blotting. Compared with the controls, our results demonstrated that hypercholesterolemia could decrease the expression of the beta1 subunit in the SO cells from rabbits. This indicates that lower expression of BKCa channel beta1 subunit might induce SOD.

[The influence on the BK channel beta1 subunit expression of SO caused by high cholesterol in rabbits].

AIM: To investigate the effects on protein expression of big-conductance Ca(2+)-sensitive K(+) channel (BKca) beta1 subunit caused by high cholesterol in Rabbit Oddi's sphincter (SO) cells. METHODS: The rat-anti-rabbit polyclonal antiserum against beta1 subunits of BKca channel of SO cell was prepared. And the protein expression of BKca channel beta1 subunit of SO tissue was detected by semi-quantitative immunohistochemical staining. RESULTS: The protein expression of BKca channels beta1 subunit of SO tissue in HC group was reduced, and there's statistically significant difference between the HC group and the control group. CONCLUSION: High cholesterol can reduce the protein expression of BK Channel's beta1 subunit in Rabbits' SO which suggests high cholesterol can affect the function of BKca channel.

[Changes of tight junction and Cx43 expression in microvessel endothelial cells of mouse lung induced by bleomycin].

OBJECTIVE: To investigate the changes of expression of connexin-43 (Cx43) and the tight junction of microvessel endothelial cells (EC) to approach the effects in bleomycin (BLM) induced pulmonary fibrosis (PF). METHODS: Forty healthy rats were equally and randomizedly divided into the control group and the experiment group. In both group, vWf in blood serum was measured with ELISA method on 3rd, 7th, 14th, 28th day after BLM treatment. Rats in each group were infused with lanthanum nitrate on 3rd, 7th, 14th, 28th day after BLM treatment. The lung samples were made and the tight junction and the distribution of the granules of lanthanum in the microvessel EC were observed with transmission electron microscopy in the control and experiment groups. The lung microvessel EC of the rats in each group were preserved by tissue culture methods at the same time, and the expression of Cx43 were observed by laser scanning confocal microscopy. RESULTS: The serum vWf in the peripheral blood of the experiment group was significantly higher than that of the control group, and was the highest on 3rd day after BLM treatment (P < 0.01). The blood vessel EC of the control group were intact. The basement membrane was uninterrupted. Granules of lanthanum nitrate did not penetrate the tight junction of EC. The width of junction gap in the experimental group was increased and the lanthanum granules of high density were found deposited in the linear form in the gap junction. Low expression of Cx43 was observed in experiment group. The expression rate of Cx43 was 25%, 38%, 45% and 71% respectively on 3rd, 7th, 14th, 28th day, significantly less than those in the control group (P < 0.05). CONCLUSION: It may be the important pathological basis for the BLM induced abnormality of the interstitial tissues in the lung that the tight junction of EC is continuously in the open state, which causes the effusions of inflammatory cells and the corresponding cytokine secretion, and thus initiates the overproliferation of fibroblasts.

[Effects of sense vascular endothelial growth factor cDNA transfection on mononuclear chemotaxis protein-1 expression in rat pulmonary microvessel endothelial cells].

OBJECTIVE: To study the transformation characteristics of tight junction of microvessel endothelial cells in bleomycin (BLM) induced pulmonary fibrosis (PF), and the effects of vascular endothelial growth factor (VEGF) on mononuclear chemotaxis protein-1 (MCP-1) mRNA expression in pulmonary microvessel endothelial cells (EC). METHODS: Forty healthy rats were equally divided into a control and an experimental group in random. In the experimental group, PF were induced by BLM application. In each group, 10 rats were instilled by lanthanum sal at 3, 7, 14 and 28 d after BLM application, and lung samples were made and observed by transmission electron microscopy. The pulmonary microvessel EC of the other 10 rats in each group were preserved by tissue culture methods at the same time, and the cells were transfected with sense VEGF cDNA, and then MCP-1 mRNA expression was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) technique. RESULTS: Blood vessel endothelial cells of the control group were intact. The basement membrane was shown as a continuous line. Granules of lanthanum sal did not cross the tight junction of endothelial cells. The width of the junction gap in rats of the experimental group treated at different times was increased and lanthanum particles of high density were seen in the gap junction, particularly on day 3, and were distributed in the area of subendothelium. The MCP-1 mRNA expression in VEGF transfected microvessel EC was significantly higher than that of the control group (1.21 +/- 0.22 vs 0.36 +/- 0.06, P < 0.05). CONCLUSION: In BLM induced PF, the opening of the tight junction of EC, and the high expression of MCP-1 induce inflammatory cell infiltration and cytokine over-secretion, which in turn enhances proliferation of fibroblasts, one of the important factors underlying lung fibrosis.

[Preparation of polyclonal antiserum against beta subunit of rabbit BK channel].

AIM: To prepare polyclonal antiserum against beta subunit of rabbit BK channel in mice. METHODS: Gene encoding the intracellular fragment of rabbit BK channel's beta subunit was amplified by RT-PCR. The GST-beta fusion protein was expressed in E. coli. The fusion protein from PAGE gel was used to immunize BALB/c mice and prepare polyclonal antiserum. The specificity of antiserum was identified by ELISA and Western blot. RESULTS: A unique band about 300 bp was amplified by RT-PCR and was verified to be BK channel beta subunit by DNA sequencing. The SDS-PAGE analysis showed that the M(r) of the fusion protein was about 37,000. The purity of GST-beta fusion protein was over 95%. The polyclonal antiserum against GST-beta fusion protein could recognize both GST-beta fusion protein and beta protein in rabbit tissues. The highest titer of the antiserum was about 1:128,000, as shown by Western blot and ELISA, respectively. CONCLUSION: The gene encoding the intracellular fragment of rabbit BK channel's beta subunit has been cloned. The polyclonal antiserum against beta subunit of rabbit BK channel with high titer and specificity has been prepared successfully.

[Relationship between bleomycin-induced pulmonary fibrosis and vascular endothelial cell injury].

OBJECTIVE: To explore the relationship between the injury of vascular endothelial cells and formation of lung fibrosis by bleomycin (BLM) in rats. METHODS: The rats of experimental groups were treated with bleomycin intratracheally to induce pulmonary fibrosis. The expression of vascular endothelial growth factor (VEGF) in pulmonary tissues were analyzed qualitatively and quantitatively by immunohistochemistry and image analysis system. RESULTS: (1) HISTOLOGY: Edema in rat alveoli and alveolar septum, inflammatory cells exudation, degeneration and necrosis of type I and type II alveolar epithelial cells (AETI and AETII), ruptured alveolar basement membrane, as well as swollen vascular endothelial cells and karyopyknosis were observed in 3 d and 7 d after treatment with BLM. AETII proliferation, with more fibroblasts in alveolar septum, and new capillary vessel formation in 7, 14 d, as well as thickened alveolar septum, damaged alveolar structure, and obvious pulmonary tissue fibrosis in 28 d after treatment with BLM were observed. (2) Immunohistochemistry: in normal control, VEGF expressed weakly in pulmonary tissue distributing mainly in AETII, bronchial epithelial cells, alveolar macrophages and leydig's cells. While in bleomycin treated groups, the expression of VEGF increased markedly. The expression in AETII, and pulmonary macrophage were significantly higher than that in control in 3 d to 28 d (P < 0.05, P < 0.01). The rat leydig's cells also had higher expression of VEGF in 7, 14, 28 d (P < 0.05, P < 0.01). CONCLUSION: The high expression of VEGF is related to vascular endothelial cells injury which may be one of important factors in the formation of bleomycin-induced pulmonary fibrosis.

Effects of cholesterol on proliferation and functional protein expression in rabbit bile duct fibroblasts.

AIM: To investigate the effect of cholesterol (Ch) on the growth and functional protein expression of rabbit bile duct fibroblasts. METHODS: The cultured bile duct fibroblasts were divided randomly into two groups: the control group and the experiment group (fibroblasts were incubated respectively with 0.6 g/L Ch for 12, 24, 36 and 48 h). The growth and DNA synthesis of bile duct fibroblasts were measured by the means of (3)H-TdR incorporation. The total protein content of fibroblast was measured by BSA protein assay reagent kit, then the expression of alpha-actin was analyzed semi-quantitatively by Western blot. RESULTS: After treatment with 0.6 g/L Ch for 12, 24, 36 and 48 h, the values of (3)H-TdR incorporation of bile duct fibroblasts were respectively 3.1+/-0.39, 3.8+/-0.37, 4.6+/-0.48 and 5.2+/-0.56 mBq/cell, and the values of the corresponding control groups were 3.0+/-0.33, 3.2+/-0.39, 3.7+/-0.49 and 4.3+/-0.43 mBq/cell. After comparing the values of experiment groups and their corresponding control groups, it was found that the (3)H-TdR incorporation of bile duct fibroblasts after treatment with 0.6 g/L Ch for 24, 36 and 48 h were significantly increased (P<0.05, P<0.01, P<0.01), while the (3)H-TdR incorporation of 12-h group was not different statistically from its control group. Ch had no obvious effect on the total protein content of fibroblasts. After incubated with 0.6 g/L Ch for 12, 24, 36 and 48 h, the total protein content of each experiment group was not altered markedly compared with its corresponding control group. The values of experiment groups were 0.246+/-0.051, 0.280+/-0.049, 0.263+/-0.044 and 0.275+/-0.056 ng/cell, and those of corresponding control groups were 0.253+/-0.048, 0.270+/-0.042, 0.258+/-0.050 and 0.270+/-0.045 ng/cell. Western blot analysis revealed that the alpha-actin expression in fibroblasts affected by Ch for 12 and 24 h was not markedly changed compared with their corresponding control groups (P>0.05), the values of total gray scale of 12- and 24-h groups were 1748+/-185 and 1756+/-173, respectively. But after stimulation with Ch for 36 h, the total gray scale of fibroblasts (1923+/-204) was significantly higher than that of control group (1734+/-197). When the time of Ch treatment was lengthened to 48 h, the alpha-actin expression was markedly elevated, the total gray scale was 2 189+/-231 (P<0.01 vs control group). CONCLUSION: Moderately concentrated Ch can promote the proliferation of bile duct fibroblasts at early stage. With the prolongation of Ch treatment, the alpha-actin expression of fibroblasts was also increased, but the hypertrophy of fibroblasts was not observed.

The study between the dynamics and the X-ray anatomy and regularizing effect of gallbladder on bile duct sphincter of the dog.

AIM: To study the relationship between the radiological anatomy and the dynamics on bile duct sphincter in bile draining and regularizing effect of gallbladder. METHODS: Sixteen healthy dogs weighing 18 kg to 25 kg were divided randomly into control group and experimental group (cholecystectomy group). Cineradiography, manometry with perfusion, to effect of endogenous cholecystokinin and change of ultrastructure were employed. RESULTS: According to finding of the choledochography and manometry, in control group the intraluminal basal pressure of cephalic cyclic smooth muscle of choledochal sphincter cCS was 9.0+/-2.0 mmHg and that of middle oblique smooth muscle of choledochal sphincter (mOS) was 16.8+/-0.5 mmHg, the intraluminal basal pressure of cCS segment was obviously lower than that of mOS (P<0.01) in the interval period of bile draining, but significative difference of intraluminal basal pressure of the mOS segment was not found between the interval period of bile draining (16.8+/-0.5 mmHg) and the bile flowing period (15.9+/-0.9 mmHg) (P>0.05). The motility of cCS was mainly characterized by rhythmically concentric contraction, just as motility of cCS bile juice was pumped into the mOS segment in control group. And motility of mOS segment showed mainly diastolic and systolic activity of autonomically longitudinal peristalsis. There was spasmodic state in cCS and mOS segment and reaction to endogenous cholecystokinin was debased after cholecystectomy. The change of ultrastructure of cCS portion showed mainly that the myofilaments of cell line in derangement and mitochondria is swelling. CONCLUSION: During fasting, the cCS portion has a function as similar cardiac "pump" and it is main primary power source in bile draining, and mOS segment serves mainly as secondary power in bile draining. The existence of the intact gallbladder is one of the important factors in guaranteeing the functional coordination between the cCS and mOS of bile duct sphincter. There is dysfunction in the cCS and mOS with cholecystectomy.

Effects of cholesterol on the phenotype of rabbit bile duct fibroblasts.

AIM: To investigate how cholesterol (Ch) can affect the phenotype of bile duct fibroblasts of New Zealand rabbits. METHODS: 16 rabbits were divided randomly into two groups: the control group and the experiment group. The rabbits in experiment group were fed with hypercholesterol diet for 8 weeks. Bile duct was dissociated from rabbits and prepared for transmission electron microscopy. The purified bile duct fibroblasts were cultured and divided randomly into three groups: control group, Ch middle concentration group (0.6 g/L), Ch high concentration group (1.2 g/L). After incubated for 72 h, the fibroblasts were made into specimens for transmission electron microscopy. The expression of alpha-actin in bile duct fibroblasts was measured by means of laser scanning confocal microscopy. RESULTS: With the transmission electron microscopy, the normal bile duct fibroblasts were shuttle-shaped, and there were abundant rough endoplasmic reticulums (RER), but few mitochondria or microfilaments in cytoplasm. This is the typical phenotype of fibroblasts. Bile duct fibroblasts of hypercholesterolemic rabbits were observed. by the transmission electron microscopy Rough endoplasmic reticulums were significantly reduced, with a lot of microfilament bundles or stress fibers appeared in cytoplasm, especially under plasma membrane. Dense bodies were scattered within these bundles. Macula densas and discontinuous sarcolemma were found under plasma membrane. It suggested that the bile duct fibroblasts of hypercholesterolemic rabbits presented the phenotype of smooth muscle cell. The cultured bile duct fibroblasts also had typical phenotype of fibroblasts. After stimulated by middle concentration cholesterol (0.6 g/L) for 72 h, there appeared lots of microfilaments in cytoplasm, but without dense body, macula densa and discontinuous sarcolemma. Observed with confocal microscopy, there were many regular bundles of microfilaments in fibroblasts treated with middle concentration ch (0.6 g/L) and the expression of alpha-actin was significantly increased. The average fluorescence value of middle concentration group was 1 628+/-189 (P<0.01 vs control group). Microfilaments and the expression of alpha-actin were greatly decreased in fibroblasts of high concentration group (1.2 g/L). The average fluorescence value of high concentration group was 1 427+/-153 (P<0.05 vs middle concentration group). There were a lower expression of alpha-actin and few microfilaments in bile duct fibroblasts of control group with an average fluorescence value of 1 224+/-138. CONCLUSION: Cholesterol can make bile duct fibroblasts have the phenotypic characteristics of smooth muscle cell both in vitro and in vivo and this effect is more significant in vivo. The effect is probably associated with some other factors besides cholesterol.

Effect of cholesterol liposomes on calcium mobilization in muscle cells from the rabbit sphincter of Oddi.

AIM: To analyze the influence of cholesterol liposome on the Ca2+ mobilization of cultured muscle cells in the rabbit sphincter of Oddi's. METHODS: New Zealand rabbit was sacrificed and the sphincter of Oddi (SO) segment was obtained aseptically. The SO segment was cut into pieces and cultured in DMEM solution. Then the smooth muscle cells were subcultured, and the 4th-7th passage cells were used for further investigation. The intracellular Ca2+ increase was measured under confocal microscope after the addition of 20 mmol x L(-1) KCl, 10(-7) mol x L(-1) acetylcholine and 10(-7) mol x L(-1) cholecystokinin, and different antagonists were added to analyze the Ca2+ mobilization pathway. After the cells were incubated with 1g x L(-1) cholesterol liposome (CL)(molar ratio was -2:1), the intracellular Ca2+ increase was measured again to determine the effect of CL on cellular Ca(2+) mobilization. RESULTS: The resting cellular calcium concentration of cultured SO cell was 108+/-21 nmol x L(-1).The intracellular Ca2+ increases induced by 20 mmol x L(-1) KCl, 10(-7) mol x L(-1) ACh and 10(-7) mol x L(-1) CCK were 183+/-56% 161+/-52% and 130+/-43%, respectively. When the extracellular Ca2+ was eliminated by 2 mmol x L(-1) EGTA and 5 micromol x L(-1) verapamil, the intracellular Ca2+ increases induced by KCl, ACh and CCK were 20+/-14%,82+/-21% and 104+/-23%, respectively. After the preincubation with heparin, the Ca2+ increases were 62+/-23% and 23+/-19% induced by ACh and CCK, as for preincubation with procaine they were 72+/-28% and 85+/-37% induced by ACh and CCK, respectively. Pretreatment with CL for 18 h, the resting cellular Ca2+ concentration elevated to 152+/-26 nmol x L(-1), however, the cellular Ca2+ increase percentages in response to these agonists were 67+/-32%,56+/-33% and 34+/-15%. CONCLUSION: KCl elicit the SO cellular Ca2+ increase depends on influx of extracellular Ca2+, ACh evoked the SO cellular Ca2+ increase is through the mobilization of intracellular Ca2+ pool and influx of extracellular Ca2+ as well, CCK excites the SO cells mainly through mobilization of intracellular IP3-sensitive Ca2+ store. After the incorporation with cholesterol liposome, KCl,ACh and CCK induced cellular Ca2+ increase percentages decreased.

Dynamic and ultrastructural study of sphincter of Oddi in early-stage cholelithiasis in rabbits with hypercholesterolemia.

AIM:To study the relationship between pre-formation of gallstone and the kinetics and ultra-structure of sphincter of Oddi.METHODS:Adult female rabbits were used and divided into 3 groups,and fed with either normal or high cholesterol diet for four or eight weeks.Each group contained eight rabbits. The manometry of sphincter of Oddi, biliary cineradiography, gallbladder volume measurement and ultrastructure observation under electron microscope were performed.RESULTS:In groups I and II,the basal pressure in low-pressure ampulla or high pressure zone of sphincter of Oddi was elevated,the amplitude of phasic contraction was decreased and the volume of gallbladder were increased, with a significant difference (P < 0.01) from those of control. Gallstones were found in group II rabbits (7/8). Under cineradiography, low-pressure ampulla showed a spasmodic status without apparent peristaltic contraction. Under electron microscope, inside the muscular cells of sphincter of Oddi, loosening of micro filament and swelling of plasmosomes which congregated at the top were observed. The amount showed no obvious change under nitric oxide synthase (NOS) stain.CONCLUSION:Twisting of the microfilament and disarrangement of kink macula densa inside the muscular cells suggested that the sphincter of Oddi was under spasmodic status. The impaired diastolic function caused and aggravated the stasis of cystic bile. The swelling plasmosome could be one of the important factors in elevating the tonic pressure of sphincter of Oddi.