Morphological changes in rat pancreatic slices associated with inhibition of enzyme secretion by high concentrations of secretagogues.

Stimulation of enzyme secretion in rat pancreatic slices by cholinergic agonists or by cholecystokinin-pancreozymin (CCK-PZ) and its peptide analogs showed a biphasic dose response curve. The optimal concentrations eliciting an efficient rate of enzyme secretion were 1 microM for carbamylcholine or acetylcholine, and 5 nM and 20 nM for CCK-PZ octapeptide and CCK-PZ, respectively. At higher concentrations of secretagogues, however, the rate of secretion progressively declined, and almost complete inhibition was achieved at 1 mM of carbamylcholine or acetylcholine and at 0.1 microM of CCK-PZ or its octapeptide analog. Atropine displaced the dose-response curve for carbamylcholine to the right so that in the presence of 7 microM atropine a concentration of 1 mM carbamylcholine now gave an optimal rate of enzyme secretion. The ionophore A-23187 which bypasses the receptor and elicits enzyme secretion did not relieve the inhibition caused by supraoptimal concentrations of secretagogues, indicating that the inhibition occurs at the cellular rather than at the receptor level. Secretin had no effect on the inhibition of enzyme secretion by a high concentration of carbamylcholine, indicating that the inhibition was not caused by lack of water and electrolyte secretion. The energy-producing metabolism was not affected since the ATP level in the pancreatic slices was the same in the presence of either inhibitory or optimal concentrations of secretagogues. The inhibition of enzyme secretion was reversible since restoration of efficient enzyme secretion occurred after removal of carbamylcholine (1 mM) by washing, followed by addition of an optimal concentration of CCK-PZ octapeptide. Morphological studies revealed that the presence of inhibitory concentrations of secretagogues caused severe distortion of the lumen structure: disruption of the filamentous system surrounding the lumen, disappearance of microvilli, and production of distended evaginations of the luminal membrane containing cellular material. These changes eventually caused a reduction in the size of the lumen which becomes plugged with secretory material. It is suggested that these changes in the microtubular microfilamentous system could account for the inhibition of enzyme secretion.

Modulation of collagen synthesis by a growth factor and by the extracellular matrix: comparison of cellular response to two different stimuli.

Cultured bovine corneal endothelial cells can be grown in three ways: on plastic, on plastic with fibroblast growth factor present in the media, and on their own preformed extracellular matrix. On plastic alone, cells grow in a disorderly fashion and secrete matrix on all cell surfaces. Cells grown on plastic with growth factor or on a matrix, at confluence, have matrix deposition only on the basal surface of the cells and an orderly contact-inhibited pattern of growth. This correlates with the polarity they demonstrate histologically. This cell-matrix pattern resembles the pattern observed in vivo. Both the soluble growth factor and the extracellular matrix are able to modulate the pattern of collagen synthesis and deposition by cells, but they do so in two entirely different ways. In cells grown on the extracellular matrix, total collagen synthesis is lower but more efficient. Collagen is deposited primarily into the cell layer even at the early sparse stage of culture. In cells grown on plastic with growth factor in the media, collagen is initially secreted into the media and does not become incorporated into the matrix. The deposition of collagen on the basal surface of cell occurs only late in the culture, and is achieved by increments in a stepwise manner. The in vivo-like pattern is not manifest until confluence has been reached. Thus, the extracellular matrix functions not only as a structural support, but is also instructional to the cells plated on it. In this case, the matrix regulates the level of collagen synthesis in the cells and modulates the pattern of collagen deposition. Soluble growth factors may act in part by enhancing a cell's ability to elaborate an appropriate matrix pattern necessary for the cell's own growth and accurate function.

Effect of cell density on thrombin binding to a specific site on bovine vascular endothelial cells.

We studied thrombin binding to proliferating and confluent endothelial cells derived from bovine vascular endothelium. [125]thrombin was incubated with nonconfluent or confluent endothelial cells and both the total amount bound and the amount linked in a 77,000-dalton thrombin-cell complex were determined. Approximately 230,000 molecules of thrombin bound per cell in nonconfluent cultures compared to 12,800 molecules per cell in confluent cultures. Approximately 67,7000 thrombin molecules were bound in an apparently covalent complex, Mr = 77,000, with each cell in sparse cultures, whereas only 4,600 thrombin molecules per cell were bound in this complex with confluent cultures. Similar studies with [125I]thrombin and endothelial cells derived from bovine cornea revealed no difference either in the total amount of thrombin bound or in the amount bound in the 77,000-dalton complex using sparse or confluent cultures. When confluent vascular endothelial cultures were wounded, additional cellular binding sites for the 77,000-dalton complex with thrombin appeared within 24 h. A 237% increase in the amount of thrombin bound to these sites was induced by a wound which resulted in a 20% decrease in cell number in the monolayer. There was no significant increase in thrombin binding to other cellular sites at 24 h. These experiments provide evidence that the first change in thrombin binding after injury is an increase in the cellular sites involved in the 77,000-dalton complex, and suggest that thrombin binding to endothelial cells may be important in the vascular response to injury.

New approaches in the measurement of coagulation.

In this session contributors present recent developments in laboratory tools for investigation of haemorrhagic disorders as well as their relative utility in clinical research. In an overview of B. Sørensen the present knowledge is summarized on the dynamic properties of whole blood fibrin formation as studied by changes in whole blood elasticity on a thrombelastometry system. Additionally, fibrin formation dynamics using simple APTT methods are presented. G. Castaman reviews the pathophysiology of von Willebrand's disease (VWD) and explains which tests are best used in diagnosis and subclassification of VWD accounting for recent developments. This presentation also describes the treatment technologies available today and their implications in clinical management of bleeding episodes in VWD. J. Lloyd addresses the assay discrepancy phenomenon that is found in some of our patients suffering from mild haemophilia A. Assay discrepancy most often means a much lower factor VIII:C value by a two-stage or chromogenic assay for factor VIII:C compared to the activity recorded by the one-stage clotting system for factor VIII:C. In rare cases, the opposite phenomenon exist. The presentation includes data from 16 Australian families with discrepant results. D. Varon reports on an assay for study of platelet function in whole blood under flow conditions. The equipment is described as a cone-and-plate(let) analyser in which the adhesion and aggregation of platelets onto a polystyrene surface is studied under arterial flow conditions. Basically, it is anticipated that proteins such as VWF and fibrinogen of flowing blood is attached to the polystyrene surface where they build up a thrombogenic surface. In the study of the author platelets were pre-activated with agonists and platelet deposits were determined after passage of whole blood for a pre-set time interval. Data presented suggest that the assay is sensitive to platelet numbers as well as qualitative changes in platelets themselves, and several examples of disorders characterized by enhanced as well as reduced platelet aggregating activities illustrates the sensitivity of the method.

Aspirin responsiveness in acute brain ischaemia: association with stroke severity and clinical outcome.

PURPOSE: Platelets play a critical role in the pathogenesis of acute brain ischaemia. We studied the association between the degree of inhibition of platelet function by aspirin (ASA) and the severity and outcome of acute brain ischaemia. METHODS: Platelet responsiveness to ASA was assessed in patients with acute brain ischaemia, treated with ASA since hospital admission. The degree of ASA responsiveness was assessed by optical aggregometry and categorized into patients with good response, partial response and complete unresponsiveness to ASA (good responders, partial responders and non-responders, respectively). An additional evaluation of responsiveness to ASA was performed by Impact-R (cone and platelet analyzer). Patients underwent serial clinical assessment during hospitalization, at discharge and during follow-up. RESULTS: Among 105 patients (mean age 63 +/- 12 years; 66% men), impaired ASA responsiveness at baseline as assessed by aggregometry was associated with increased stroke severity at baseline, unfavourable clinical course, and poor functional outcome during follow-up (p < 0.05 for all). Age-adjusted odds ratios in non-responders compared to good responders were 9.8 for severe stroke on admission (95% CI 2.8-34.9), 3.1 for lack of early clinical improvement (95% CI 1.1-8.8) and 8.6 for poor functional outcome during follow-up (95% CI 2.4-30.4). Less robust trends were observed with the Impact-R. CONCLUSIONS: Impaired responsiveness to ASA in acute brain ischaemia is common and is associated with worse neurological deficits at stroke onset, early clinical deterioration and poorer functional outcome. The clinical significance of these findings requires further evaluation in larger longitudinal studies.

Testing agonist-induced platelet aggregation by the Impact-R [Cone and plate(let) analyzer (CPA)].

The Impact-R [Cone and plate(let) analyzer (CPA)] is useful to assess platelet adhesion in different diseases and to monitor antiplatelet therapy. The purpose of the present study was to adapt this system to test agonist-induced platelet aggregation. Blood samples were tested by light transmission platelet aggregometry (LTA), Impact-R regular test and Impact-R agonist-response test. In the latter, samples were pre-incubated for 1 min with an agonist leading to platelet activation, micro-aggregates formation and reduced adhesion. Impact-R regular test of ten healthy volunteers demonstrated platelet adhesion (surface coverage, SC) of 11.2 +/- 2.6% while LTA induced by ADP, ristocetin, epinephrine, collagen and arachidonic acid (AA) yielded maximal aggregation (81% to 93%). In the Impact-R agonist-response test, SC was reduced to 2.2 +/- 1.0%, 1.2 +/- 0.9%, 2.3 +/- 1.0%, 2.2 +/- 0.8% and 2.4 +/- 0.4%, respectively. Prostaglandin E(1) treatment weakened SC reduction in response to ADP and epinephrine (SC of 8.8 +/- 1.8% and 9.5 +/- 2.0%, respectively). Inhibition of P2Y(12) receptor with 2MeSAMP resulted in a dose-dependent decrease in maximal aggregation in the ADP-induced test, which inversely correlated to SC in the Impact-R ADP-response test. The Impact-R agonist-response tests detected aggregation defects in patients with storage pool disease, severe von Willebrand disease and epinephrine response deficiency, and may be useful to assess the effect of different agonists on platelet aggregation.

[Influence of laser radiation of the whole blood in vitro on adhesion and aggregation of blood platelets].

The authors revealed dependence of reaction blood plates to photoeffect on the dose and rate of blood movement at laser radiation of donor blood in vitro. The red light decreases adhesion and aggregation of blood plates both at high and low rate of shift. Infrared laser radiation is effective only at high rate of shift leading to increase of adhesion and decrease of aggregation of blood plates. Blue laser is effective in small doses only and at low rate of sift it leads to decrease of adhesion and at high rate it provokes increase of adhesion. Blue laser do not have a significant influence on aggregation of blood plates. These results make possible to suppose ambiguity of biological response of venous and arterial blood to radiation.

Effect of basic fibroblast growth factor on the growth and differentiation of adult stromal bone marrow cells: enhanced development of mineralized bone-like tissue in culture.

Rat stromal bone marrow cells (SBMC) were shown to produce mineralized bone-like tissue in culture in the presence of dexamethasone, ascorbic acid, and beta-glycerophosphate. The addition of 3 ng/ml of basic fibroblast growth factor (bFGF) resulted in a significant increase in formation of mineralized tissue. The present study was aimed at assessing the effect of bFGF on the proliferation and differentiation of SBMC and on the sequential development of mineralized bone-like tissue in culture. Transmission electron microscopy of bFGF-treated cultures demonstrated the development of a multilayered structure resembling mineralized bone tissue consisting of cell layers embedded within a heavy extracellular matrix. The matrix was rich in bundles of collagen fibers associated with extensive mineral deposits consisting of hydroxyapatite as determined by infrared spectrophotometry. The addition of 3 ng/ml of bFGF resulted in significant enhancement of [3H]thymidine and [3H]proline incorporation and protein accumulation by 12-, 2.5-, and 2.5-fold, respectively. bFGF treatment increased cAMP responsiveness, alkaline phosphatase activity, osteocalcin level, 45Ca2+ deposition, and mineralized-like tissue formation and induced the earlier expression of these markers in the treated culture. A biphasic sequence of events was observed during the development of mineralized bone-like tissue in bFGF-treated and control cultures. The first phase is characterized by cell proliferation and matrix accumulation and is reflected by a progressive increase in [3H]thymidine and [3H]proline incorporation until day 11. The second phase, which follows, is characterized by a sharp decline in cell proliferation and matrix accumulation and a concomitant expression of osteoblast differentiation as reflected by the progressive increase in alkaline phosphatase activity, mineral deposition, and osteocalcin expression. Treatment of cultures with bFGF accentuated this biphasic sequence of events. These results indicate that bFGF has the capacity to stimulate both the growth and the biochemical functions of SBMC obtained from a young adult animal.

A collagenous cementum-derived attachment protein is a marker for progenitors of the mineralized tissue-forming cell lineage of the periodontal ligament.

The periodontal ligament (PDL) is a fibrous and cellular connective tissue that mediates tooth attachment to bone, and it comprises fibroblastic and mineralized tissue-forming (MTF) progenitors. The MTF progenitors are believed to give rise to the cementoblastic and osteoblastic lineages. Cementum attachment protein (CAP) is a collagenous cementum-derived protein which binds strongly to osteoblasts, moderately to PDL cells, and weakly to gingival fibroblasts. The aim of the present study was to determine the relationship between the capacity of PDL progenitors to bind CAP and their potential to express alkaline phosphatase (ALP) and form mineralized-like tissue in culture. Cloned human PDL progenitor populations obtained from nine human donors were assayed for their constitutive capacity to bind CAP and express ALP, and for the dexamethasone-induced potential to form mineralized-like tissue in culture in the presence of ascorbic acid and beta-glycerophosphate. Forty percent of the progenitor clones produced mineralized-like tissue. Two patterns of mineralization were observed: a spread and flat pattern similar to that produced by human bone cells in culture and a nodular ridge-like type resembling that formed by human cementoma-derived cells. A direct correlation was found between the percentage of ALP positive cells in each progenitor clone and the amount of mineralized-like tissue formed (r = 0.565). Similar correlations were found between the number of ALP positive cells and the binding capacity of each clone (r = 0.392) and between the CAP binding capacity and mineralized-like tissue formation (r = 0.584). Multiple regression analysis indicated that the constitutive capacity of a clone to bind CAP and express ALP is directly correlated to its dexamethasone-induced potential to form mineralized tissue (r = 0.675). These results indicate that CAP binding and ALP expression can serve as markers for the identification of MTF progenitors in the heterogeneous cultured population of the human periodontal ligament. These data show for the first time that binding capacity to extracellular components of mineralized tissues can be a marker for mineralized tissue-forming progenitors.

Anti-thrombin activity in cultured aortic and capillary endothelial cells: binding, internalization and degradation of thrombin.

Thrombin (Th) binds specifically to confluent cultures of adult bovine aortic (ABAE) and bovine brain capillary (BBC) endothelial cells. Saturation of 125I-Th binding is observed after 1 h exposure to the ligand and at an extracellular concentration of 0.5 and 1.0 microgram/ml for ABAE and BBC cells, respectively. Under optimal conditions both ABAE and BBC cultures bind about 2 to 5 ng/10(6) cells, which represents about 20% of Th binding to bovine corneal endothelial (BCE) cells. Under optimal conditions less than 30% of the total cell associated 125I-Th is internalized in ABAE and BBC cells, while in BCE cells the extent of internalization is more than 50%. The internalized 125I-Th is degraded both in ABAE and BBC cells as previously demonstrated in BCE cells. As analyzed by SDS-PAGE, 17%, 22% and 77% of the bound 125I-Th is in complex with anti-thrombins (anti-Ths) in BBC, ABAE and BCE cultures, respectively. ABAE cells possess 3 types of complexes, one which appears only on the cell surface with a molecular weight of 78 kDa, and two others which appear only in the conditioned medium (CM) with molecular weights of 84 and 85 kDa. BBC and BCE cells demonstrate only one type of complex with a molecular weight of 77 kDa which appears both on the cell surface and in the CM. The 125I-Th 77 kDa complex formed in the CM of BCE cells is recognized and bound by BBC cells and ABAE cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolism of cholesterol and phospholipids in cultured human vascular smooth muscle cells: differences between artery and vein-derived cells and the effect of oxygen partial pressure.

Human smooth muscle (SM) cells derived from vena saphena magna, aorta abdominalis and arteria mamaria were grown in culture under 40 or 145 mmHg oxygen partial pressure (pO2) and their lipid metabolism studied. Esterification of the cellular [3H]cholesterol was higher by 2.5-fold in artery derived than in vein-derived cells and was slightly higher in cultures exposed to 145 mmHg than to 40 mmHg pO2. Cholesterol efflux in the presence of high density lipoprotein (HDL) in the incubation medium was higher in artery-derived than vein-derived cells. Apolipoprotein (apo) AI also supported cholesterol efflux to a higher extent in artery than in vein-derived cells. Cholesterol efflux in the presence of apo AI was accompanied by a decrease of 50% in cellular [3H]cholesteryl ester in both cell types. SM cultures exposed to [3H]choline incorporated about 90% of the radioactivity to phosphatidylcholine (PC) and 10% to sphingomyelin (SPM). During 5 days exposure to [3H]choline, 10 to 15% and 20 to 30% of the newly synthesized PC and SPM, respectively, were released by vein-derived cells into the incubation medium. The relative amount of SPM of the total radioactive phospholipids released by vein-derived cultures was significantly higher in cultures growing under 40 mmHg than 145 mmHg pO2 reaching a value of up to 33% of the radioactive phospholipids in the incubation medium. HDL was shown to serve as an acceptor for phospholipids released by both vein and artery-derived SM cells, while free apo AI supported phospholipid efflux in artery but not in vein-derived SM cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Patterns of cellular peptide synthesis by cultured bovine granulosa cells.

The electrophoretic distribution of the polypeptides synthesized by bovine granulosa cell cultures after metabolic labeling with [35S]methionine has been analyzed by double gel electrophoresis. The fluctuations of 35 polypeptides have been followed as a function of the size of the follicles from which cultures originated, as a function of the cultures' proliferative stage (sparce, actively growing vs. confluent, resting cultures), and finally as a function of whether cells were exposed to either epidermal or fibroblast growth factor. When the patterns of protein synthesis in sparce vs. confluent granulosa cell cultures derived from small-sized follicles were compared, only a few differences were observed. In confluent cultures, 6 new peptides appeared, while 1 peptide present in sparce cultures disappeared. Cultures maintained in the presence of fibroblast or epidermal growth factor synthesized 20 new peptides upon reaching confluence. Among these were the 6 new peptides present in confluent but not in sparse granulosa cell cultures maintained in the absence of growth factors. The changes in protein synthesis observed in cultures grown in the presence of growth factors may reflect their direct effect on the cellular metabolism. A comparison between the protein distribution in cells derived from small- vs. large-sized follicles showed that fewer proteins were ultimately produced at confluence in cells derived from large-size follicles than in cells derived from small-sized follicles. This could be related to the process of cellular differentiation taking place within granulosa cells. The patterns of [35S]methionine-labeled proteins secreted by the granulosa cells into the incubation medium were analyzed and found to be similar regardless of the size of the follicle from which the culture originated. Little similarity between the proteins present in the follicular fluid and the pattern of the labeled proteins secreted into the incubation medium by cultured granulosa cells was observed. Only three proteins were identified which comigrate with proteins present in the follicular fluid. One of these was identified as fibronectin. This raises the possibility that the fibronectin present in the follicular fluid originated from granulosa cells and is not derived from plasma.

Factors controlling proliferation and progesterone production by bovine granulosa cells in serum-free medium.

Bovine granulosa cells seeded in the presence of serum on extracellular matrix-coated dishes proliferate actively when exposed to serum-free medium supplemented with insulin (2 microgram/ml), fibroblast growth factor (FGF, 100 ng/ml), and high density lipoprotein (HDL, 30 microgram protein/ml). The final density of the cultures is 80-120% that of cultures grown in the presence of medium supplemented with optimal concentration (10%) of calf serum. Insulin has the greatest effect on cell proliferation when added alone to serum-free medium, since it induced an increase in cell number that was 35-60% that observed with optimal serum concentration. Somatomedin C can replace insulin when added alone. FGF, epidermal growth factor, or HDL had no significant effect on cell proliferation by themselves. When these factors were added together with insulin, they acted synergistically in stimulating cell proliferation. When cultures were seeded in the total absence of serum, the addition of transferrin (10 microgram/ml) to serum-free medium was required in order for insulin and FGF to be mitogenic. Cultures maintained on extracellular matrix and exposed to serum-free medium alone have a lifespan in culture of 4 generations. Addition of insulin, FGF, and HDL increases the lifespan of the cultures to 12 generations. Bovine granulosa cells, which proliferate in a defined medium, respond to dibutyryl cAMP by releasing progesterone into the medium. Addition of FSH to the defined medium resulted in a 30% decrease in cell proliferation and in a 2.1-fold increase in the amount of progesterone released into the medium in response to dibutyryl cAMP. This release of progesterone reached a level similar to that observed with cultures grown in medium supplemented with optimal concentration of serum and exposed or not to FSH during their growth phase and at confluence. These results demonstrate that bovine granulosa cells can actively proliferate in a serum-free medium and maintain their differentiated function, as indicated by their ability to produce progesterone.

Role of lipoproteins and 3-hydroxy-3-methylglutaryl coenzyme A reductase in progesterone production by cultured bovine granulosa cells.

The relative contributions of lipoproteins and 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase to progesterone production by bovine granulosa cells exposed to plasma or liquor folliculi (LF) were studied. LF did not contain and very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), or low density lipoprotein (LDL). These lipoproteins were present in the plasma at concentrations of 92 micrograms protein/ml for VLDL and IDL together and 139 micrograms protein/ml for LDL. In contrast, high density lipoprotein (HDL) was present in LF at a concentration (763 micrograms protein/ml) that was 59% of that in plasma (1293 micrograms protein/ml). Bovine granulosa cells exposed to human plasma produce progesterone in response to dibutyryl cAmP. Sixty-three percent of the progesterone released by the cells was dependent on LDL but not HDL derived from human plasma. When cells were exposed to bovine plasma, 75% of the progesterone release was dependent on the presence of lipoproteins in the medium. Both LDL and HDL of bovine origin were able to support progesterone production, although LDL was effective at concentrations (on a molar basis) 20-fold lower than HDL. The LF was able to support progesterone production 45% as well as bovine plasma. The differences between the greater ability of the whole fractions and the lesser ability of their respective lipoprotein-deficient derivatives to support progesterone synthesis were 4-fold for bovine plasma, 2.7-fold for human plasma, and 1.7-fold for LF. The relative abilities of equivalent concentrations of LDL to restore the rate of progesterone synthesis seen in the lipoprotein-deficient fraction toward that seen in the whole fraction were greatest in the LF, intermediate in human plasma, and least in bovine plasma. These observations taken together suggest that the low level of support of progesterone synthesis that is offered by LF is due to its deficiency in LDL. HMG CoA reductase, the regulated and rate-limiting enzyme of cholesterol synthesis, was induced (2- to 3-fold) by dibutyryl cAMP and was suppressed by both human and bovine LDL and to a lesser extent by bovine HDL. Compactin, a competitive inhibitor of HMG CoA reductase, inhibited progesterone production relatively little when cells were exposed to complete plasma or LF. However, when cells were exposed to a lipoprotein-deficient bovine plasma or LF, compactin was very efficient in reducing (by 76%) progesterone release. Bovine granulosa cells exposed to plasma primarily use cholesterol derived from LDL in order to produce progesterone. Their ability to produce progesterone when exposed to LF was limited, and the cells were probably more dependent on de novo cholesterol synthesis than cells exposed to plasma.

Basic fibroblast growth factor enhances the capacity of bone marrow cells to form bone-like nodules in vitro.

The role of basic fibroblast growth factor (bFGF) in the proliferation and differentiation of rat bone marrow cells in culture was studied. bFGF stimulated [3H]thymidine incorporation into these cells by 4-fold at a concentration of 0.3 ng/ml and half-maximal effect was observed at a concentration of 15 pg/ml. In addition to its mitogenic effect, bFGF stimulated alkaline phosphatase activity by 3.6-fold. Continuous treatment with bFGF (for 21 days) resulted in a 6.3-fold increase in the culture dish surface area covered by bone-like mineralized tissue. Maximal bone-like tissue formation was observed in the presence of 3 ng/ml bFGF with half-maximal effect at a concentration of 0.3 ng/ml. These results indicate the possible role of bFGF in the proliferation of osteogenic rat bone marrow cells and their differentiation into cells of osteoblast-like phenotype.

Differential response of platelets to chemokines: RANTES non-competitively inhibits stimulatory effect of SDF-1 alpha.

BACKGROUND: Among the chemokines related to CXC and CC receptor groups and released from platelets, leukocytes and endothelial cells, SDF-1, TARC and MDC have been found to be platelet agonists. Platelets do not contain SDF-1 alpha. In contrast, RANTES is constitutively present in platelet alpha-granules and released upon platelet activation. OBJECTIVES: To study a possible role of RANTES as a modulator of SDF-1 alpha effect on platelets, in relation to CXCR4 and various CC receptors. METHODS: CXCR-4 (CXCL12) receptor expression and platelet activation were evaluated by flow cytometry, platelet deposition was studied by cone and plate(let) analyzer, and platelet aggregation by turbidometric aggregometry. RESULTS: Flow cytometry studies revealed similar expression of CXCR-4, the specific receptor of SDF-1 alpha on intact, inactivated, and activated platelets. Preincubation of platelets with RANTES affected neither CXCR-4 expression, nor SDF-1 alpha binding to the platelet membrane. In the presence of fibrinogen, SDF-1 alpha activated gel-filtered platelets. RANTES did not activate platelets, but substantially (by 70%) inhibited SDF-1 alpha-induced fibrinogen binding. Similarly, RANTES abrogated the promoting effect of SDF-1 alpha on whole blood platelet adhesion to endothelial cell monolayer under venous flow conditions. In platelet-rich plasma, RANTES moderately inhibited SDF-1 alpha-induced platelet aggregation, while it did not affect aggregation induced by thrombin-receptor activation peptide, adenosine diphosphate, or phorbol 12-myristate 13-acetate. A synergistic inhibitory effect of RANTES and prostaglandin E1 used at subthreshold concentrations, on SDF-1 alpha-induced aggregation and SDF-1 alpha-induced fibrinogen binding to platelets was observed, which may suggest involvement of RANTES in a cAMP-dependent signal transduction pathway. CONCLUSIONS: RANTES non-competitively inhibits activation of platelets by SDF-1 alpha, and thus may play a regulatory role in platelet response to inflammation.

Effect of maturation on the osteogenic response of cultured stromal bone marrow cells to basic fibroblast growth factor.

Formation of bone-like tissue in culture by stromal bone marrow cells (SBMC) derived from young growing rats is dependent on dexamethasone (Dex) (Cell Tissue Res 254:317; 1988) and is significantly enhanced by basic fibroblast growth factor (bFGF) (J Bone Miner Res 8:919; 1993). The aim of this study was to examine the effect of maturation on the osteogenic potential and the response to Dex and bFGF of SBMC by using cultures derived from young growing (6 weeks old) and adult (9 months old) rats. SBMC cultures were grown in the presence of Dex (10(-8) or 10(-7) mol/L) at both P(0) and P(1) and either in the presence or absence of bFGF. The effect of Dex and bFGF on mineralized bone-like tissue (MBT) formation was assessed at P(1). The highest levels of mineralized tissue formation in P(1) subcultures in the absence of bFGF were obtained when cultures derived from young rats (6 weeks old) were treated with Dex 10(-7) and 10(-8) mol/L at P(0) and P(1), respectively, and when cultures derived from adult rats were exposed to Dex 10(-8) mol/L both at P(0) and P(1). Under these optimal Dex concentrations, the amount of MBT formed by adult rat-derived cultures was 15-fold lower than that of young rat-derived ones. The addition of bFGF to P(0) cultures or to P(1) cultures grown under optimal Dex conditions enhanced MBT formation in P(1) cultures derived from both young and adult rats, but this effect was considerably more pronounced in the adult rat-derived cultures. The maximal levels of MBT formation were produced by cultures derived from adult rats treated with bFGF at both P(0) and P(1), whereas in cultures derived from young rats, the addition of bFGF at P(0) was not necessary for maximal MBT production. This stimulating effect of bFGF on MBT formation by adult rat-derived cultures was accompanied by a 2.2-, 1.8-, and 4.3-fold increase in proliferation, alkaline phosphatase activity, and Ca(2+) deposition rate, respectively. bFGF increased the level of glucocorticoid receptor by approximately 2. 3-fold in Dex-treated cultures derived from young animals. These results indicate that maturation is associated with a decrease in the proportion of osteoprogenitor cells in the stromal bone marrow and in their capacity to express the osteogenic phenotype. They further point to the significant role of bFGF in stimulating proliferation and osteogenic expression of stromal bone marrow osteoprogenitors derived from adult rats.

Basic fibroblast growth factor enhances the growth and expression of the osteogenic phenotype of dexamethasone-treated human bone marrow-derived bone-like cells in culture.

Basic fibroblast growth factor (bFGF) was shown to enhance rat stromal bone marrow cells in culture to produce mineralized bone-like tissue in response to dexamethasone (Dex) treatment (Pitaru et al., J Bone Miner Res 8:919; 1993). The purpose of this study was to explore the effect of bFGF on Dex-treated human stromal bone marrow cells (hSBMC) in culture. Human SBMC from 6 patients were cultured for 14 days (P0) and then subcultured and grown for 28 days in the presence of Dex (10(-8) mol/L). The effect of bFGF on cell proliferation at P0 and protein content, DNA content, alkaline phosphatase activity (ALP), osteocalcin secretion, and formation of mineralized bone-like tissue (MBT) at P1 was analyzed. bFGF treatment resulted in a 2.4-fold increase in cell number at P0 and a concentration-dependent increase in [3H]-thymidine incorporation at P1, reaching a maximum increase of 3.7-fold at a concentration of 0.3 ng/mL. Furthermore, bFGF significantly increased both DNA content (two- to threefold), protein content (five- to sixfold), and the amount of MBT (up to 20-fold) at P1 cultures. Morphological evaluation of the MBT at the electron microscope level revealed a mineralization process along collagen fibrils similar to the natural process. The osteogenic nature of the bFGF-treated cultures was further shown by their ALP activity, as well as osteocalcin secretion in response to 1,25-dihydroxyvitamin D3. In conclusion, bFGF demonstrated a stimulatory effect on the proliferation of Dex-treated hSBMC-derived osteoprogenitors while maintaining their capacity to fully differentiate and form bone-like tissue in culture.

Characterization of bicarbonate-dependent potassium uptake in cultured corneal endothelial cells.

Bovine corneal endothelial (BCE) cells in culture demonstrated 86Rb+ uptake which was mostly ouabain-sensitive with some (15 to 50%) ouabain-insensitive uptake that was dependent on the presence of bicarbonate in the incubation medium. Bovine smooth muscle (SM) cells demonstrated ouabain-sensitive 86Rb+ uptake but the ouabain-insensitive 86Rb+ uptake was not bicarbonate-dependent. Although omission of bicarbonate from the incubation buffer resulted in some reduction in the pH, this change was not responsible for the reduction in the ouabain-insensitive 86Rb+ uptake. Furthermore, the removal of bicarbonate decreased the 86Rb+ influx but not its efflux. This ouabain-insensitive and bicarbonate-dependent 86Rb+ influx in BCE cells proceeded at a linear rate for at least 60 min and increased as a function of bicarbonate concentration such that almost maximal uptake was observed at a concentration of about 10 to 15 mM. Saturation of the bicarbonate-dependent 86Rb+ pump in BCE cells occurred at a concentration of 2 mM Rb+ in the incubation buffer, similar to the previously observed value for the Na+, K+-ATPase. Competition experiments with both unlabeled Rb+ and K+ demonstrated that likewise in the Na+, K+-ATPase the 86Rb+ influx represented physiological influx of K+. Furthermore, the energy requirements of the bicarbonate-dependent 86Rb+ uptake were similar to those of the 86Rb+ uptake via the Na+, K+-ATPase. The results described in this work demonstrated a novel bicarbonate-dependent K+ pump in addition to the Na+, K+-ATPase pump.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of blood components in ocular silicone oil emulsification. Studies on an in vitro model.

PURPOSE: To develop an in vitro model for silicone oil emulsification and to explore the blood components involved in this process. METHODS: The capacity of various blood components to support silicone oil (1000 CS) emulsification was studied by applying 0.5 ml oil on top of 0.5 ml saline containing various blood components. Each tube was sonicated for 150 seconds and centrifuged at 5000 g for 20 minutes. Three phases were noted in the tube: At the top was clear silicone oil, in the middle was emulsified silicone oil, and at the bottom was aqueous solution. The tubes were photographed, and the percentage of the phase length containing emulsified silicone oil (middle) of the total length of the three phases was calculated from the projected image of each tube. RESULTS: Emulsified silicone oil in plasma or serum was initiated after 100 seconds of sonication and quickly reached maximum (approximately 80%) at 120 seconds. The size of these oil droplets prepared in vitro was 0.0467 +/- 0.028 mm, closely resembling that observed in oil samples removed from a patient's anterior chamber (0.038 +/- 0.018 mm). Under these conditions, silicone oil emulsified in the presence of whole blood cells occurred only at a concentration of 120 micrograms protein/ml; in the presence of red blood cell membranes, it occurred at a concentration of 60 micrograms protein/ml. Lipoprotein-deficient serum failed to support emulsification; however, samples of high-density lipoprotein and low-density lipoprotein supported this process. Purified high-density lipoprotein-apolipoproteins supported oil emulsification. The addition of phosphatidylcholine further enhanced this process, but phosphatidylcholine alone failed to support emulsification. CONCLUSIONS: A simple and fast in vitro model to study factors affecting silicone oil emulsification was developed. Using this model, red blood cell membranes, plasma lipoproteins, and purified HDL-apolipoproteins supported silicone oil emulsification. Lipids did not, but they had the capacity to enhance the apolipoprotein-supported emulsification.