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C M Yang

Publications and source records attributed to C M Yang.

At least 55 records · Page 3Linked to original sources
PubMed1998

Dissociation of intracellular Ca2+ release and Ca2+ entry response to 5-hydroxytryptamine in cultured canine tracheal smooth muscle cells.

The relationship between the agonist-sensitive Ca2+ pool and those discharged by the Ca2+ -ATPase inhibitor thapsigargin (TG) were investigated in canine tracheal smooth muscle cells (TSMCs). In fura-2-loaded TSMCs, 5-hydroxytryptamine (5-HT) stimulated a rapid increase in intracellular Ca2+ ([Ca2+]i), followed by a sustained plateau phase that was dependent on extracellular Ca2+. In such cells, TG produced a concentration-dependent increase in [Ca2+]i, which remained elevated over basal level for several minutes and was substantially attenuated in the absence of extracellular Ca2+. Application of 5-HT after TG demonstrated that the TG-sensitive compartment partly overlapped the 5-HT-sensitive stores. Pre-treatment of TSMCs with TG significantly inhibited the increase in [Ca2+]i induced by 5-HT in a time-dependent manner. Similar results were obtained with two other Ca2+ -ATPase inhibitors, cyclopiazonic acid and 2,5-di-t-butylhydroquinone. Although these inhibitors had no effect on phosphoinositide hydrolysis, Ca2+ -influx was stimulated by these agents. These results suggest that depletion of the agonist-sensitive Ca2+ stores is sufficient for activation of Ca2+ influx. Some characteristics of the Ca2+ -influx activated by depletion of internal Ca2+ stores were compared with those of the agonist-activated pathway. 5-HT-stimulated Ca2+ influx was inhibited by La3+, membrane depolarisation, and the novel Ca2+ -influx blocker 1-¿beta-[3-(4-methoxyphenyl) propoxy]-4-methoxyphenethyl¿-1H-imidazole hydrochloride (SKF96365). Likewise, activation of Ca2+ influx by TG also was blocked by La3+, membrane depolarisation, and SKF96365. These results suggest that (1) in the absence of PI hydrolysis, depletion of the agonist-sensitive internal Ca2+ stores in TSMCs is sufficient for activation of Ca2+ influx, and (2) the agonist-activated Ca2+ influx pathway and the influx pathway activated by depletion of the inositol 1,4,5-trisphosphate-sensitive Ca2+ pool are indistinguishable.

Animals↗
PubMed1998

Uncoupling of bradykinin-induced phosphoinositide hydrolysis and Ca2+ mobilization by phorbol ester in canine cultured tracheal epithelial cells.

1. Regulation of the increase in inositol phosphates (IPs) production and intracellular Ca2+ concentration ([Ca2+]i by protein kinase C (PKC) was investigated in canine cultured tracheal epithelial cells (TECs). Stimulation of TECs by bradykinin (BK) led to IPs formation and caused an initial transient [Ca2+]i peak in a concentration-dependent manner. 2. Pretreatment of TECs with phorbol 12-myristate 13-acetate (PMA, 1 microM) for 30 min attenuated the BK-induced IPs formation and Ca2+ mobilization. The maximal inhibition occurred after incubating the cells with PMA for 2 h. 3. The concentrations of PMA that gave half-maximal (pEC50) inhibition of BK-induced IPs accumulation and an increase in [Ca2+]i were 7.07 M and 7.11 M, respectively. Inactive phorbol ester, 4alpha-phorbol 12,13-didecanoate at 1 microM, did not inhibit these responses. Prior treatment of TECs with staurosporine (1 microM), a PKC inhibitor, inhibited the ability of PMA to attenuate BK-induced responses, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. 4. In parallel with the effect of PMA on the BK-induced IPs formation and Ca2+ mobilization, the translocation and down-regulation of PKC isozymes were determined. Analysis of cell extracts by Western blotting with antibodies against different PKC isozymes revealed that TECs expressed PKC-alpha, betaI, betaII, gamma, delta, epsilon, theta and zeta. With PMA treatment of the cells for various times, translocation of PKC-alpha, betaI, betaII, gamma, delta, epsilon and theta from cytosol to the membrane was seen after 5 min, 30 min, 2 h, and 4 h treatment. However, 6 h treatment caused a partial down-regulation of these PKC isozymes. PKC-zeta was not significantly translocated and down-regulated at any of the times tested. 5. Treatment of TECs with 1 microM PMA for either 30 min or 6 h did not significantly change the KD, and Bmax receptor for BK binding (control: KD=1.7+/-0.3 nM; Bmax=50.5+/-4.9 fmol/mg protein), indicating that BK receptors are not a site for the inhibitory effect of PMA on BK-induced responses. 6. In conclusion, these results suggest that activation of PKC may inhibit the phosphoinositide hydrolysis and consequently attenuate the [Ca2+]i increase or inhibit independently both responses to BK. The translocation of pKC-alpha, betaI, betaII, delta, epsilon, gamma, and theta induced by PMA caused an attenuation of BK-induced IPs accumulation and Ca2+ mobilization in TECs.

Animals↗
PubMed1998

Effect of forskolin on endothelin-induced phosphoinositide hydrolysis and calcium mobilization in cultured canine tracheal smooth muscle cells.

1. The effects of increase in intracellular adenosine 3':5'-cyclic monophosphate (cAMP) on endothelin-1 (ET-1)-induced generation of inositol phosphates (IPs) and increase in intracellular Ca2+ ([Ca2+]i) were investigated in canine cultured tracheal smooth muscle cells (TSMCs). 2. Pretreatment of TSMCs with either cholera toxin (CTX; 10 microg ml(-1), 4 h), forskolin (10 microM, 30 min), or dibutyryl cAMP (1 mM, 30 min) inhibited ET-1-stimulated Ca2+ mobilization (by 23 +/- 5%, n = 8) and IPs accumulation (by 32 +/- 6%, n = 4). While after treatment with forskolin for 24 h, the cells retained the ability to respond to ET-1-induced Ca2+ mobilization to the same extent as the control group. 3. Forskolin (1-100 microM) inhibited the ET-1-induced increase in [Ca2+]i, but the lower concentrations had little effect on this response. The inhibitory effects of these agents produced both depression of the maximal response and a shift to the right of the concentration-response curve of ET-1 without changing the -logEC50 values. 4. The water-soluble forskolin analogue L-858051, 7-deacetyl-7beta-(gamma-N-methylpiperazino)-butyryl forskolin, significantly inhibited ET-1-stimulated IPs accumulation. In contrast, the addition of 1,9-dideoxy forskolin, an inactive analogue of forskolin, had little effect on stimulated responses. Moreover, SQ-22536, 9-(tetrahydro-2-furanyl)-9H-purin-6-amine, an inhibitor of adenylate cyclase, and both H-89, N-(2-aminoethyl)-5-isoquinolinesulfonamide, and HA-1004, N-(2-guanidinoethyl)-5-isoquinolinesulfonamide, inhibitors of cAMP-dependent protein kinase (PKA), attenuated the ability of forskolin to inhibit ET-1-induced IPs accumulation. These results suggest that activation of cAMP/PKA was involved in these inhibitory effects of forskolin. 5. The locus of this inhibition of forskolin treatment on AlF4(-)-stimulated IPs accumulation was investigated in canine TSMCs. The AlF4(-)-induced IPs accumulation was inhibited by forskolin, supporting that G protein(s) are directly activated by AlF4- and uncoupled to phospholipase C by forskolin treatment. 6. We conclude that cAMP elevating agents inhibit ET-1-stimulated generation of IPs and Ca2+ mobilization in canine cultured TSMCs. Since generation of IPs and increases in [Ca2+]i are very early events in the activation of ET-1 receptors, attenuation of these events by cAMP elevating agents might well contribute to the inhibitory effect of cAMP on tracheal smooth muscle function.

Animals↗
PubMed1998

Rapid quantitation of thermal oxidation products in fats and oils by 1H-NMR spectroscopy.

This work describes the application of high-resolution proton nuclear magnetic resonance (1H-NMR) spectroscopy to the study of the thermal peroxidation of beef tallow and corn oil under standardized conditions. The approach provides a rapid, quantitative method for determining the degree of oxidation of unsaturated fatty acids in animal and vegetable fats and oils by quantitating the decreasing intensities of 1H-NMR peaks for allylic and olefinic protons in unsaturated fatty acid chains of triglycerides and the increasing peak intensities of hydroperoxide and saturated and alpha, beta-unsaturated aldehydic protons in relation to the less labile protons in the triglyceride molecule. Two-dimensional correlation spectroscopy analysis of highly oxidized beef tallow (180 degrees C for 24 h) suggested that the unsaturated aldehydes that persisted were apparently associated with carboxy groups.

Animals↗
PubMed1998

Thermally oxidized dietary fat and colon carcinogenesis in rodents.

Thermally oxidized animal fat (beef tallow) was assessed for colon cancer-promoting and -initiating activity in F-344 rats and CF-1 mice with the use of the aberrant crypt focus (ACF) assay. In two promotion studies, extensively oxidized beef tallow (110 degrees C for 144-168 h, peroxide value approx 200 meq/kg, with > 80% loss of allylic and olefinic protons) had relatively little effect on the growth of ACF in F-344 rats. The multiplication constant for treatment/control of ACF size in aberrant crypts per ACF at 100 days was 1.07 (95% confidence interval = 1.01-1.14) and 0.98 (95% confidence interval = 0.91-1.06). ACF size was not affected by less extensively oxidized beef tallow or by a 10-fold reduction of dietary alpha-tocopherol during the growth of the ACF. In initiation studies, extensively oxidized beef tallow administered by gavage increased the number of animals with ACF and the number of ACF per colon (11 of 23 and 5 of 29 animals with ACF; 1.09 +/- 0.29 and 0.21 +/- 0.09 ACF/colon, respectively). Less severely oxidized beef tallow was without effect. Further studies with CF-1 mice confirmed that extensively oxidized beef tallow increased numbers of animals with ACF and average ACF per colon. The unsaturated aldehyde acrolein was without effect in the ACF assay. These data suggest that highly thermolyzed beef tallow contains an uncharacterized initiator or leads to conditions in which spontaneously initiated ACF are increased.

Acrolein↗
PubMed1998

Effects of lid suturing and trans-scleral cryotherapy on ocular growth in a piglet model.

PURPOSE: To study whether lid suturing can induce axial myopia and explore whether trans-scleral cryotherapy can affect ocular growth in piglets or not. METHODS: A total of fourteen 2- to 3-week-old piglets were studied. Three groups were included: lid suture, cryotherapy, and both lid suture/cryotherapy groups. The lid suturing group (N = 6) was given lid suturing to produce visual deprivation. The cryotherapy group (N = 4) received trans-scleral cryotherapy 360 degrees to encircle anterior to the equator. Both treatment groups (N = 4) received both cryotherapy and lid suturing. The cycloplegic refraction, corneal power, biometric axial length, and intraocular pressure (IOP) were measured before the experiments and 4 months later. RESULTS: Mild axial myopia was induced in five lid-sutured eyes of the lid suturing group. The ocular refraction and eye size of cryotherapy eyes were not different from the control eyes in the cryotherapy group. More myopia was found in all four piglets that received both cryotherapy and lid suturing. CONCLUSIONS: Lid suturing can induce axial myopia; however, cryotherapy did not affect normal eye growth and did not prevent the development of lid suturing myopia in piglets.

Animals↗
PubMed1998

[Regulatory role of 5-hydroxytryptamine and motilin in migrating motor complex of small bowel in dogs].

Migrating motor complex (MMC) was recorded by chronically implanted strain gauge transducers on the serosa of small bowel in conscious dogs. Two silastic catheters for injecting drugs and taking blood samples were put respectively in a vena jugularis externa and in a jejunal artery which perfused a 10-15 cm intestinal segment. The present study was undertaken to observe the effect of drugs administered intravenously and intraarterially on MMC and alterations of plasma 5-hydroxytryptamine (5-HT) and motilin in different phases of MMC. The results showed: (1) Levels of plasma 5-HT and motilin showed cyclical fluctuations in different phases of MMC, and 5-HT reached peak before peak of motilin. Plasma 5-HT level reached a peak [(41.2 +/- 1.3) ng/ml] in the later period of phase II, which increased significantly as compared with phase I [(22.4 +/- 1.1) ng/ml] (P < 0.01), but plasma motilin level had a peak [(580.0 +/- 30.2) pg/ml] in phase III of MMC, which was higher than that in phase I [(140.0 +/- 21.0) pg/ml] (P < 0.01). (2) Intravenous motilin and intraarterial 5-HT and motilin could induce phase III contraction in the canine proximal small bowel and in the perfused segment respectively. Amplitude of contractions caused by motilin was higher than that by 5-HT in the perfused segment. (3) 5-HT receptor antagonist, motilin antiserum, 5,6-dihydroxytryptamine and tetrodotoxin perfused intra-arterially inhibited expression of MMC in the perfused segment and its caudal propagation. We conclude that plasma 5-HT and motilin may be dominant factors which initiate MMC of canine small bowel, and 5-HT peak appears before peak of motilin. 5-HT neurons and motilin neurons in enteric nervous system may play an important role in controlling MMC of canine small bowel.

Animals↗
PubMed1998

Automatic change detection: does the auditory system use representations of individual stimulus features or gestalts?

The effects of global and feature-specific probabilities of auditory stimuli were manipulated to determine their effects on the mismatch negativity (MMN) of the human event-related potential. The question of interest was whether the automatic comparison of stimuli indexed by the MMN was performed on representations of individual stimulus features or on gestalt representations of their combined attributes. The design of the study was such that both feature and gestalt representations could have been available to the comparator mechanism generating the MMN. The data were consistent with the interpretation that the MMN was generated following an analysis of stimulus features.

Adult↗
PubMed1998

Surgical treatment for diabetic retinopathy: 5-year experience.

To evaluate the surgical indications and results among patients undergoing pars plana vitrectomy for complications of diabetic retinopathy, the clinical records of all such patients treated by a single surgeon from January 1992 through August 1996 were reviewed. Pre- and postoperative visual acuity, indications for surgery, surgical methods, and anatomic outcomes were recorded and analyzed. A total of 106 eyes (92 patients) were included. Surgical indications included nonclearing vitreous hemorrhage (21 eyes), active fibrovascular proliferation (28 eyes), traction retinal detachment (42 eyes), premacular hemorrhages (3 eyes), combined traction and rhegmatogenous retinal detachment (7 eyes), severe macular edema (1 eye), and progressive macular or disc traction (4 eyes). Surgical techniques ranged from tissue segmentation to combined delamination and segmentation. Anatomical success was obtained in 90 eyes (84.9%). Visual acuity improved in 69 eyes (68.0%), stabilized in eight eyes (7.5%), and decreased in 29 eyes (28%). All 21 eyes with vitreous hemorrhage had anatomical success, while 10 of 42 (24%) eyes with traction retinal detachment eventually had retinal detachment. Fourteen eyes had postoperative persistent or recurrent vitreous hemorrhage requiring air-fluid exchange or vitreous lavage. Silicone oil was used in eight eyes. Surgical failure was mainly caused by iatrogenic breaks with persistent traction, or severe anterior or posterior reproliferation. These findings indicate that reasonably successful anatomical outcomes can be achieved with modern surgical methods. However, improvements in visual acuity may be less satisfactory. Eyes with widespread, thickened fibrovascular complexes with active neovascularization, and extensive traction retinal detachment have a poor prognosis.

Adult↗
PubMed1998

Optimal timing of retina examinations for premature infants.

Developments in modern neonatal intensive care have resulted in increased survival of very premature infants. Along with this increase in survival, there has been a concomitant increase in the frequency of retinopathy of prematurity (ROP). We investigated the relationship between the severity and the time course of ROP as well as the optimal time for retinal examinations of premature infants of various birth weights and gestational ages. A total of 80 premature infants were enrolled for analysis. The mean postnatal age of infants at the time of diagnosis was 7.3 +/- 3.4 weeks for stage 1 retinopathy, 9.2 +/- 5.8 for stage 2 retinopathy, 9.5 +/- 3.8 for stage 3 retinopathy, 10.7 +/- 3.7 for threshold disease, and 11.7 +/- 3.2 for stage 4 retinopathy. The mean postconceptional age of infants at the time of diagnosis was 35 +/- 3 for stage 1 retinopathy, 36.4 +/- 3 for stage 2 retinopathy, 37.6 +/- 3.5 for stage 3 retinopathy, 38.4 +/- 3.5 for threshold disease, and 40 +/- 3.7 for stage 4 retinopathy. The age at the time of the initial detection of ROP was from the ninth to 10th week among infants weighing less than 1,000 g at birth and from the fifth to seventh week among those weighing 1,000 g or more at birth. However, the postconceptional age at the time of initial detection of ROP for the whole group was 36 weeks and was not influenced by birth weight or gestational age. Therefore, we suggest that postconceptional age, rather than postnatal age, should be used to decide the timing of retinal examinations for premature infants.

Female↗
PubMed1998

Bullous retinal detachment in a patient with central serous chorioretinopathy.

Bullous retinal detachment, a severe and atypical presentation of central serous chorioretinopathy (CSCR), may develop in association with the use of systemic steroids. A 42-year-old man presented with a 2-month history of poor visual acuity in his right eye. A tentative diagnosis of Harada's syndrome was made and the patient was treated with large doses of systemic corticosteroids for 1 month, without improvement. Fundus examination showed bullous detachment with shifting subretinal fluid in both eyes. Fluorescein angiography demonstrated multiple leaking points with pigment epithelium detachment in both eyes. A diagnosis of bilateral severe CSCR was made and systemic steroid therapy was withdrawn. Several sessions of argon laser photocoagulation to the areas of fluorescein leakage in both eyes and transscleral drainage of subretinal fluid in the right eye were performed. Two months later, the retinae became attached in both eyes. Visual acuity in the right eye was limited by submacular fibrosis, while the visual acuity of the left eye was not affected. In summary, this case should alert physicians to suspect CSCR in patients with exudative retinal detachment, as the use of steroids is usually not only unnecessary but can also be harmful.

Adult↗
PubMed1997

Purinoceptor-stimulated phosphoinositide hydrolysis in Madin-Darby canine kidney (MDCK) cells.

Extracellular nucleotides, acting through P2-purinoceptors, have been implicated in the regulation of ion transport in epithelia, including Madin-Darby canine kidney (MDCK) cells. In this study, experiments were conducted to characterize the P2-purinoceptor subtype on MDCK cells responsible for stimulating inositol phosphate (IP) accumulation using a range of nucleotide analogues. In Ca2+- and Mg2+-free Krebs-Henseleit solution (KHS), ATP, UTP, and ATPgammaS caused an increase in IP accumulation as a function of concentration with comparable kinetics. The order of potency for the nucleotide analogues was UTP = ATPgammaS > ATP = 2-chloro ATP (Cl-ATP) >> alpha,beta-methylene ATP (alpha,beta-MeATP) = 2-methylthio ATP (2MeSATP). Selective agonists for P1-, P2X- and P2Y-purinoceptors, such as N6-cyclopentyl adenosine, AMP, alpha,beta-MeATP, and 2MeSATP, had little effect. Stimulation of MDCK cells with maximally effective concentrations of ATP and UTP showed no additive effect and furthermore, ATP, UTP, and ATPgammaS induced cross-desensitization of the IP response, suggesting that ATP and UTP act upon a common nucleotide receptor, i.e. a P2U-purinoceptor. In Ca2+- and Mg2+-containing KHS, the concentration-response curves of ATP, UTP, and ATPgammaS were shifted to the right of those obtained in Ca2+- and Mg2+-free buffer, and asymptotic maxima were not reached, indicating that ATP4- and not MgATP2- or CaATP2- was the active agonist. Pretreatment of MDCK cells with pertussis toxin (PTX) inhibited ATP- and UTP-induced IP accumulation in a concentration-dependent fashion but did not completely abolish the IP accumulation, indicating that a PTX-sensitive G protein was partially involved in the IP response. In conclusion, ATP- and UTP-stimulated IP accumulation in MDCK cells appears to be mediated through the activation of P2U-purinoceptors coupled to a G protein that is partially sensitive to PTX. A form of nucleotide uncomplexed with divalent ions such as ATP4- seems to be the preferential agonist form for the purinoceptors on MDCK cells.

Adenosine Triphosphate↗
PubMed1997

Changes in collagen phenotypes during progression and regression of cardiac hypertrophy.

OBJECTIVE: Excessive deposition of collagen has been implied to be responsible for abnormal stiffness and altered cardiac function during hypertrophy and heart failure. In the present paper we studied the changes in collagen and their phenotypes during development of cardiac hypertrophy in spontaneously hypertensive rats (SHR) compared to age- and sex-matched Wistar Kyoto (WKY). We also studied the changes in collagen after regression of hypertrophy, with antihypertensive therapy with ACE inhibitors, captopril (C) and lisinopril (L). METHOD: Collagen was extracted from the heart tissue by cyanogen bromide (CNBr) digestion. Collagen phenotypes were separated and quantified by SDS-polyacrylamide gel electrophoresis. The transcript levels(mRNA) of collagen phenotypes were determined by Northern analysis. RESULTS: Our studies showed that the ventricular collagen and their phenotypes did not alter in SHR during the first 6 months of progression of hypertrophy when compared to WKY. After 40 weeks, however, in SHR there was an unexpected rise in collagen content and the distribution of collagen phenotype differs compared to WKY, especially during the chronic phase of hypertrophy (65 weeks of age). In WKY during the aging process there was a gradual increase in type III collagen, whereas in SHR it plateaus after 40 weeks of age. Treatment with antihypertensive drugs captopril and lisinopril showed a similar degree of reduction in blood pressure (P < 0.001), regressed hypertrophy (P < 0.001), and reduced collagen, whereas decrease in type I to III ratio was found with captopril only, but not with lisinopril. This decrease in type I to III ratio due to captopril treatment is primarily due to an increase in type III collagen (both protein and transcript level) in SHR. CONCLUSION: Our data showed, for the first time, that during the chronic phase of hypertrophy in SHR there is a gradual reduction in type I to III ratio, primarily due to a lack of increase in type III collagen during chronic phase of hypertrophy. This suggests that quality of collagen is an important factor in determining the degree of cardiac stiffness. Our data also showed that not all ACE inhibitors have similar actions on collagen phenotype production. This suggests that perhaps the mechanism of action of ACE inhibitors on collagen are independent of its effect on angiotensin II formation.

Aging↗
PubMed1997

Effect of forskolin on bradykinin-induced calcium mobilization in cultured canine tracheal smooth muscle cells.

The effects of increases in intracellular adenosine 3':5'-cyclic monophosphate (cyclic AMP) on bradykinin (BK)-induced generation of inositol phosphates (IPs) and Ca2+ mobilization were investigated in canine cultured tracheal smooth muscle cells (TSMCs). Pretreatment of TSMCs with either forskolin or dibutyryl cyclic AMP attenuated BK-stimulated responses. The inhibitory effects of these agents produced both a depression of the maximal response and a shift to the right of the concentration-response curves of BK. The water-soluble forskolin analogue L-858051, 7-deacetyl-7 beta-(r-N-methylpiperazino)-butyryl forskolin, significantly attenuated BK-stimulated IPs accumulation, while 1,9-dideoxy forskolin, an inactive forskolin, had little effect on IPs response. Moreover, SQ-22536, 9-(tetrahydro-2-furanyl)-9-H-purin-6-amine, an inhibitor of adenylate cyclase, and both H-89, N-(2-aminoethyl)-5-isoquinolinesulfonamide, and HA-1004, N-(2-guanidinoethyl)-5-isoquinolinesulfonamide, inhibitors of cyclic AMP-dependent protein kinase (PKA), reversed the ability of forskolin to attenuate BK-stimulated IPs accumulation. The KD and Bmax, values of the BK receptor for [3H]BK binding were not significantly changed by forskolin treatment for 30 min and 4 h. The AlF4(-)-induced IPs accumulation was attenuated by forskolin, indicating that G protein(s) are directly activated by AlF4- and uncoupled to phospholipase C by forskolin treatment. These results suggest that activation of cyclic AMP/PKA might inhibit the BK-stimulated PI breakdown and consequently reduce the [Ca2+]i increases or inhibit independently both responses, which is distal to the BK receptor in canine cultured TSMCs.

Adenine↗
PubMed1997

Inhibition of 5-hydroxytryptamine-induced phosphoinositide hydrolysis and Ca2+ mobilization in canine cultured tracheal smooth muscle cells by phorbol ester.

1. Regulation of the increase in inositol-1,4,5-trisphosphate (IP3) production and intracellular Ca2+ concentration ([Ca2+]i by protein kinase C (PKC) was investigated in canine cultured tracheal smooth muscle cells (TSMCs). Stimulation of TSMCs by 5-hydroxytryptamine (5-HT) caused an initial transient [Ca2+]i peak followed by a sustained elevation of [Ca2+]i in a concentration-dependent manner. 2. Pretreatment of TSMCs with phorbol 12-myristate 13-acetate (PMA, 1 microM) for 30 min blocked the 5-HT-induced IP3 formation and Ca2+ mobilization. This inhibition was reduced after the cells had been incubated with PMA for 8 h, and within 48 h the 5-HT-induced Ca2+ mobilization reached the same extent as control cells. 3. The concentration of PMA that gave half-maximal inhibition of 5-HT-induced increase in [Ca2+]i was 4 nM. Pretreatment of TSMCs with staurosporine (1 microM) of GF109203X (0.1 microM), PKC inhibitors, inhibited the ability of PMA to attenuate 5-HT-induced responses, suggesting that the inhibitory effect of PMA was mediated through the activation of PKC. 4. In parallel with the effect of PMA on 5-HT-induced IP3 formation and Ca2+ mobilization, the translocation and down-regulation of PKC isozymes were determined by Western blot analysis in TSMCs. Analysis of cell extracts by Western blotting with antibodies against different PKC isozymes revealed that TSMCs expressed PKC-alpha, beta I, beta II, delta, epsilon, theta and zeta. With PMA treatment of the cells for various times, translocation of PKC-alpha, beta I, beta II, delta, epsilon, and theta from the cytosol to the membrane was seen after 5 min, 30 min, 2 h, and 4 h treatment. However, 24 h treatment caused a partial down-regulation of these PKC isozymes PKC-zeta was not significantly translocated and down-regulated at any of the times tested. 5. In conclusion, these results suggest that activation of PKC may inhibit the receptor-mediated phosphoinositide hydrolysis and consequently attenuate the [Ca2+]i increase or inhibit both responses independently. The translocation of PKC-alpha, beta I, beta II, delta, epsilon, and theta induced by PMA caused an attenuation of 5-HT-stimulated IP3 accumulation and Ca2+ mobilization in TSMCs.

Animals↗
PubMed1997

Inhibition of RPE cell-mediated matrix adhesion and collagen gel contraction by crovidisin, a collagen-binding snake venom protein.

PURPOSE: Cell-mediated collagen gel contraction plays an important role in the pathogenesis of proliferative vitreoretinopathy (PVR). Anti-adhesion therapy has been suggested as a promising strategy in the treatment of PVR. Crovidisin, a snake venom protein isolated from Crotalus viridis, has been shown to bind selectively to collagen and to inhibit collagen-induced platelet aggregation. In the present study, the effectiveness of crovidisin in inhibiting the attachment of retinal pigment epithelial (RPE) cells to collagen, and RPE cell-mediated collagen gel contraction, was evaluated. METHODS: Fluorescein isothiocyanate (FITC)-conjugated crovidisin was prepared and used to evaluate its binding affinity for collagen type I, fibronectin, vitronectin, and laminin. The inhibitory effect of crovidisin on RPE cell-mediated extracellular matrix attachment and collagen gel contraction was evaluated by cell adhesion and type I collagen gel contraction assays. The cytotoxic effect of crovidisin was examined with a cell proliferation assay, using the Alamar blue method. Flavoridin, an Arg-Gly-Asp-containing peptide from viper venom, was used for comparison. RESULTS: FITC-conjugated crovidisin bound selectively to collagen type I with high affinity. It did not bind to other matrix proteins, including fibronectin, vitronectin and laminin, nor to RPE cells. Crovidisin inhibited RPE cell attachment to type I collagen in a dose-dependent manner. This inhibitory effect was enhanced by the presence of flavoridin. Crovidisin also dose-dependently inhibited RPE cell-mediated type I collagen gel contraction. Crovidisin was non-toxic to RPE cells. CONCLUSIONS: Crovidisin, a snake venom-derived collagen-binding protein, possessing an inhibitory activity on RPE cell-collagen interaction and RPE cell-mediated collagen gel contraction, may be a useful tool for studying cell-collagen interaction, and a potential anti-adhesion therapeutic agent for ocular disorders in which cell-collagen interaction in involved, such as PVR.

Animals↗
PubMed1997

Pars plana vitrectomy in the treatment of combined rhegmatogenous retinal detachment and choroidal detachment in aphakic or pseudophakic patients.

BACKGROUND AND OBJECTIVE: The presence of extensive choroidal detachment (CD) in eyes with rhegmatogenous retinal detachment (RRD) is a major preoperative complication. Conventional treatment consists of 7 days to a few weeks of systemic and topical steroids followed by scleral buckling and drainage of suprachoroidal fluid. The author studied the usefulness of pars plana vitrectomy in the management of RRD combined with CD. PATIENTS AND METHODS: Ten consecutive patients with RRD complicated by CD were treated with pars plana vitrectomy. Nine had had previous cataract extraction, and one patient had pars plana lensectomy at the time of the surgery. The severity of the CD was evaluated with preoperative ophthalmologic echography. Sclerotomy sites for infusion cannulas were chosen in the area with less CD. Six-millimeter infusion cannulas were used. Pars plana vitrectomy, membrane peeling, and internal fluid-gas exchange with encircling scleral buckle placement were performed in a standard fashion. One patient had silicone oil injected. RESULTS: In each case, suprachoroidal fluid drained spontaneously through the sclerotomy sites around the vitrectomy instruments during the initial course of the operation without extra sclerotomy. Immediate intraoperative retinal reattachment was achieved in 10 cases. The retina remained attached in 9 cases during at least 6 months of follow-up without additional surgery. CONCLUSION: These cases demonstrate the usefulness of pars plana vitrectomy in treating RRD with CD in aphakic or pseudophakic eyes. This method may be an favorable alternative to conventional treatment.

Adult↗