Effects of potassium and anion channel blockers on the cellular response of human osteoarthritic chondrocytes

The aim of this article is to determine to what extent the proliferation, CD44 expression, and apoptosis behavior of cells can be influenced by the modulation of ion channel activity on the cell membrane of human osteoarthritic chondrocytes. The potassium channel blocker 4-aminopyridine (4-AP) and the chloride and anion channel blocker 4-acetamido-4-isothiocyano-2,2-disulfonic acid stilbene (SITS) were used as ion channel modulators. Assessment of the proliferation was done by incorporation of ³H-thymidine. The detection of apoptotic cells and expression of the hyaluronic acid binding CD44-receptor were determined by flow cytometry. The results showed that 4-AP and SITS lead to a temporary increase in ³H-thymidine incorporation, followed by a suppression of proliferation after a 12-day incubation. 4-AP causes considerable cytotoxic effects. SITS leads to necrotic cell damage. CD44 expression is increased up to 43% after incubation with 4-AP for 24 or 48h, whereas prolonged incubation under SITS influence leads to a clear inhibition of CD44 expression. In conclusion, proliferation, CD44 expression, and apoptosis behavior of human chondrocytes can be influenced by modulation of ion channel activity. These results serve as a basis for further investigations to extend the therapeutic possibilities in the treatment of arthritis.     

Low-Flow Venovenous CO2 Removal in Association With Lung Protective Ventilation Strategy in Patients Who Develop Severe Progressive Respiratory Acidosis After Lung Transplantation

Background

Primary graft dysfunction (PGD) might occur after lung transplantation. In some severe cases, conventional therapies like ventilatory support, administration of inhaled nitric oxide (iNO), and intravenous prostacyclins are not sufficient to provide an adequate gas exchange. The aim of our study was to evaluate the use of a lung protective ventilation strategy associated with a low-flow venovenous CO₂ removal treatment to reduce ventilator-associated injury in patients that develop severe PGD after lung transplantation.

Methods

From January 2009 to January 2011, 3 patients developed PGD within 24 hours after lung transplantation. In addition to conventional medical treatment, including hemodynamic support, iNO and prostaglandin E1 (PGE1), we initiated a ventilatory protective strategy associated with low-flow venovenous CO₂ removal treatment (LFVVECCO2R). Hemodynamic and respiratory parameters were assessed at baseline as well as after 3, 12, 24, and 48 hours.

Results

No adverse events were registered. Despite decreased baseline elevated pulmonary positive pressures, application of a protective ventilation strategy with LFVVECCO2R reduced PaCO₂ and pulmonary infiltrates as well as increased pH values and PaO₂/FiO₂ ratios. Every patient showed simultaneous improvement of clinical and hemodynamic conditions. They were weaned from mechanical ventilation and extubated after 24 hours after the use of the low-flow venovenous CO₂ removal device.

Conclusion

The use of LFVVECCO2R together with a protective lung ventilation strategy during the perioperative period of lung transplantation may be a valid clinical strategy for patients with PGD and severe respiratory acidosis occured despite adequate mechanical ventilation.     

Potential of O6-methylguanine or O6-benzylguanine in the enhancement of chloroethylnitrosourea cytotoxicity on brain tumours

Summary The purine analogues O⁶-methylguanine and O⁶-benzylguanine are well-known as a chemical modulator of the DNA repair enzyme O⁶-methylguanine-DNA methyltransferase. Inactivation of the enzyme by O⁶-methylguanine or O⁶-benzylguanine is expected to enhance sensitivity of tumours to chloroethylnitrosoureas.We studied the effect of O⁶-methylguanine or O⁶-benzylguanine pretreatment on cytotoxity of 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) in brain tumour cells and transplanted brain tumours. Two-hour exposure of O⁶-methylguanine at higher concentrations (500 M, 1,000 M) increased ACNU cytotoxicity by only 2 times in ACNU-resistant C6-1 brain tumour cells. O⁶-Benzylguanine at concentrations between 10 and 100 M markedly enhanced the cytotoxic effecct. The ACNU sensitivity of the tumour cels pretreated with O⁶-benzylguanine was 5–40 times that of the cells without O⁶-benzylguanine. Neither O⁶-methylguanine nor O⁶-benzylguamne appreciably enhanced ACNU cytotoxicity of 9 L cells, which were origininally sensitive to ACNU. Intracarotid ACNU with O⁶-methylguanine or O⁶-benzylguanine decreased proliferating activity of transplanted C6-1 brain tumours significantly during 48 hours. O⁶-Benzylguanine pretreatment resulted in a greater degree of suppression for a long time. The C6-1 tumours treated only with intracarotid ACNU showed a transient inhibition and a rapid regrowth during 24 hours after the treatment.These results indicate that O⁶-methylguanine or O⁶-benzylguanine increases ACNU cytotoxicity and may be feasible for effective combination therapy with chloroethylnitrosourea in the chemotherapy of malignant brain tumours.     

Cyclosporin A inhibits prostaglandin E2 formation by rat mesangial cells in culture

A reversible reduction in glomerular filtration rate (GFR) is a frequent side effect in patients treated with the immunosuppressant cyclosporin A (CsA). The pathophysiology of acute CsA nephrotoxicity, however, is unclear. Since eicosanoids are local mediators of glomerular hemodynamics, they might be involved in CsA induced changes in GFR. We therefore studied the effect of CsA on prostaglandin E2 (PGE2) production by rat mesangial cells in culture. PGE2 production by mesangial cells following stimulation with angiotensin II (AII) (10(-6) M) or the Ca2+-ionophore A23187 (1 microgram/ml) was significantly inhibited when cells were grown for 24 hours in media which contained CsA (800 to 3200 ng/ml). CsA did not affect viability of mesangial cells as determined by 51Cr release or by cell proliferation measured by 3H-thymidine incorporation. CsA (3200 ng/ml) did not inhibit PGE2 formation by rat MC microsomes incubated with arachidonic acid. However, CsA, in a dose dependent manner, inhibited A23187 and angiotensin II induced release of 3H-labelled arachidonic acid from rat mesangial cells. These data demonstrate that CsA reduces PGE2 formation by rat mesangial cells in culture, probably by inhibiting the release of substrate arachidonic acid from cell membranes rather than by inhibition of cyclooxygenase. This effect may contribute to the reduction in GFR which accompanies CsA therapy.     

Photodynamic therapy of bladder cancer —uptake and phototoxicity of photosan in vitro

Summary The uptake of photosan and the intracellular sites of photoradiation-induced damage were investigated in vitro in bladder carcinoma cells and in normal bladder cells. Cells were examined by phase contrast, fluorescence and electron microscopy. The concentration of photosan, measured in g/10⁶ cells, showed a good correlation to the incubation time. At all incubation times, control cells showed a lower uptake when compared with tumor cells. Following photodynamic therapy (PDT), phase-contrast microscopy revealed marked changes in tumor cells, whereas only minor effects could be detected at the cell membrane of the control cells. Following PDT, most of the investigated cells showed onanges of the mitochondria and cytoplasma. These changes consisted of dissolution of the cristae, predominantly in the central part of the mitochondria. Twenty-four hours after PDT the shape of the mitochondria had changed markedly and the cristae were found to be completely destroyed. Moreover, the cystoplasma showed numerous vacuoles, and the number of mitochondria was decreased compared to non-treated cells.Supported by Deutsche Forschungsgemeinschaft Ha 131/1-2     

Decreased sup 3 H-Thymidine Incorporation By Human Bladder Epithelial Cells Following Exposure To Urine From Interstitial Cystitis Patients

Purpose

Interstitial cystitis (IC) is a chronic bladder disease of unknown etiology. We sought to determine whether a cytotoxin is present in the urine of IC patients that could cause the epithelial damage seen in this disease.

Materials and Methods

Evidence for a cytotoxin was sought using both a neutral red uptake viability assay in T24 bladder epithelial cells, and a³ H-thymidine incorporation assay in primary normal adult bladder epithelial cells and FHS 738 Bl human fetal bladder cells.

Results

The neutral red assay in T24 cells indicated the presence of a cytotoxin in 2 of 9 IC patient urine specimens. However, the more sensitive assay of cell proliferation (sup 3 H-thymidine incorporation) in normal adult human bladder epithelial cells revealed antiproliferative activity in urine from 10 of 13 (77 percent) IC patients vs. 3 of 19 (16 percent) controls (two-way analysis of variance, p = .019). The antiproliferative activity of IC urine specimens was confirmed using FHS 738 Bl human fetal bladder cells. The antiproliferative urinary substance(s) appeared to be a low molecular weight (less than 10,000 Da), heat stable, trypsin-sensitive factor(s).

Conclusions

Because a denuded or damaged bladder epithelium is a central finding in IC, it is possible that the antiproliferative protein(s) contributes to the pathogenesis of this disease.     

Effect of sex steroids on bone collagen synthesis in vitro

Summary Although sex steroids are known to affect skeletal metabolism, their effects on bone collagen synthesis have not been studied. We have examined the direct effects of progesterone, 17 estradiol, testosterone, 5 dihydrotestosterone and dehydroepiandrosterone on bone collagen and noncollagen protein synthesis in cultures of calvaria obtained from 21-day fetal rats. Bones were incubated for 24 to 168 h and³H-proline was added for the last 2 h of culture. Incorporation of the label into collagenase-digestible protein (CDP)¹ and noncollagen protein (NCP) was determined using repurified bacterial collagenase.Progesterone caused a dose dependent inhibition of the labeling of CDP at concentrations of 3×10^–7 M to 10^–5 M after 96 h of culture. A smaller inhibitory effect was observed on NCP. The inhibitory effect was slow in onset and persisted when bones were incubated for 48 h with progesterone and then transferred to control medium for 48 h. Progesterone also inhibited the incorporation of³H-thymidine and³H-uridine into fetal rat calvaria.After 24 h of culture, 17 estradiol and testosterone had no effects on the labeling of CDP and NCP. After 96 h, 17 estradiol had a small and inconsistent stimulatory effect on the labeling of CDP but testosterone had no effect. Neither hormone altered the inhibitory effects of parathyroid hormone, cortisol and progesterone. Dihydrotestosterone and dehydroepiandrosterone had no effects after 24 h and 96 h of culture. 17 estradiol, testosterone and dihydrotestosterone did not affect the incorporation of³H-uridine or³H-thymidine into fetal rat calvaria.Our studies indicate that progesterone is an inhibitor of bone collagen synthesis and estrogens and androgens are not major regulators of bone formation in vitro.     

Aromatase activity in human osteoblast-like osteosarcoma cell

Summary Aromatase activity was studied in cultured human osteosarcoma cell (HOS). HOS was incubated from 12 to 72 hours with 10^-10 M-10^-5 M dexamethasone. Aromatase activity was determined by measuring [³H]H₂O released upon the conversion of [1-³H]androstenedione to estrone. HOS showed aromatase activity, and apparent km for [1-³H]androstenedione was 4.46±0.98 nm (mean±SD). The aromatase activity was significantly increased by 10^-9 M-10^-5 M dexamethasone in a dose-dependent manner. Dexamethasone increased V_max of aromatase activity but did not change its km value. These results suggest that osteoblastic cells have aromatase activity which is regulated by glucocorticoid, and directly convert androgen to estrogen in itself.     

Thiamine (Vitamin B1) Protects against Glucose- and Insulin-Mediated Proliferation of Human Infragenicular Arterial Smooth Muscle Cells

2 . Cells were identified as ASMC by immunohistochemical analysis. Cells from passages 3-5 were exposed to glucose concentrations of 0.1 and 0.2% with and without insulin concentrations of 100 ng/mL and 1000 ng/mL, in the presence or absence of 200 μM of thiamine. Standard hemocytometry and ³H-thymidine incorporation quantified cell proliferation after incubation for 6 days and 24 hr, respectively. The data suggest that thiamine inhibits human infragenicular ASMC proliferation induced by high glucose and insulin. Vitamin B1 intake may prove important in delaying the atherosclerotic complications of diabetes.     

Adenovirus-mediated wild-typep53 tumor suppressor gene therapy induces apoptosis and suppresses growth of human pancreatic cancer

Background: Thep53 tumor suppressor gene is mutated in up to 70% of pancreatic adenocarcinomas. We determined the effect of reintroduction of the wild-typep53 gene on proliferation and apoptosis in human pancreatic cancer cells using an adenoviral vector containing the wild-typep53 tumor suppressor gene. Methods: Transduction efficiencies of six p53-mutant pancreatic cancer cell lines (AsPC-1, BxPC-3, Capan-1, CFPAC-1, MIA PaCa-2, and PANC-1) were determined using the reporter gene construct Ad5/CMV/-gal. Cell proliferation was monitored using a³H-thymidine incorporation assay, Western blot analysis forp53 expression was performed, and DNA laddering and fluorescence-activated cell sorter analysis were used to assess apoptosis.p53 gene therapy was tested in vivo in a subcutaneous tumor model. Results: The cell lines varied in transduction efficiency. The MIA PaCa-2 cells had the highest transduction efficiency, with 65% of pancreatic tumor cells staining positive for beta-galactosidase (-gal) at a multiplicity of infection (MOI) of 50. At the same MOI, only 15% of the CFPAC-1 cells expressed the -gal gene. Adenovirus-mediatedp53 gene transfer suppressed growth of all human pancreatic cancer cell lines in a dose-dependent manner. Western blot analysis confirmed the presence of the p53 protein product at 48 hours after infection. DNA ladders demonstrated increased chromatin degradation, and fluorescence-activated cell sorter analysis demonstrated a four-fold increase in apoptotic cells at 48 and 72 hours following infection with Ad5/CMV/p53 in the MIA PaCa-2 and PANC-1 cells. Suppression of tumor growth mediated by induction of apoptosis was observed in vivo in an established nude mouse subcutaneous tumor model following intratumoral injections of Ad5/CMV/p53. Conclusions: Introduction of the wild-typep53 gene using an adenoviral vector in pancreatic cancer withp53 mutations induces apoptosis and inhibits cell growth. These data provide preliminary support for adenoviral mediatedp53 tumor suppressor gene therapy of human pancreatic cancer.Presented at the 51st Annual Cancer Symposium of The Society of Surgical Oncology, San Diego, California, March 26–29, 1998.     

Calcitriol effect on natural killer cells from hemodialyzed and normal subjects

Patients with chronic renal failure have a decreased secretion of calcitriol (CTR). They also show an impaired cellular immune response including a defective natural killer (NK) cell-mediated activity. The aim of this study was to analyze, in vivo and in vitro, the effect of CTR on NK cell cytotoxicity in healthy control subjects and in hemodialyzed (HD) patients. Our results show that HD patients had baseline-depressed NK cell activity when compared with controls (P<0.001), which increased significantly after 1 month of oral CTR treatment (0.5 g/day) (P<0.001). Calcitriol treatment also induced a significant increase in CTR serum levels (P<0.001) and a significant decrease (P<0.001) in total parathyroid hormone (PTH). In vitro CTR treatment (10^-7 M) of peripheral blood mononuclear cells (PBMC) increased NK cell-mediated cytotoxicity after 24 hours of incubation with a maximum at 48 hours (P<0.001). In vitro CTR treatment at doses of 10^-11 and 10^-9 M did not significantly increase NK cytotoxic activity. The enhanced NK activity after CTR treatment was not the consequence of increased numbers of CD56 positive cells, nor to lymphocyte activation, as tested by the expression of the interleukin 2 receptor p55 chain (CD25) on their surface. In vitro treatment of PBMC from HD patients with CTR (10^-7 M, during 48 hours) also induced a strong increase in NK cell cytotoxicity (P<0.001). These results demonstrate a positive role of CTR in the stimulation of NK cell activity and support the hypothesis of a direct steroid-mediated action of CTR on NK cells, although an indirect effect mediated by the CTR-induced PTH decrease in vivo cannot be excluded. Our data also raise the possibility for potential therapeutic uses of this hormone in immunomodulation of patients with depressed NK cell activity.     

Growth-inhibitory effect of prostaglandin D2 on mouse glioma cells

The effects of prostaglandin D2 (PGD2) on the growth of mouse malignant glioma cells were studied in vitro and in vivo. The in vitro studies consisted of various concentrations of prostaglandins (PG's) being added to cultures of mouse glioma cells. At concentrations above 2.5 micrograms/ml, PGD2 strongly inhibited the proliferation of glioma cells, whereas PGE2 had no effect at the same value. Exposure to 5.0 micrograms/ml PGD2 for more than 2 hours resulted in inhibition of glioma cell proliferation. This growth-inhibitory effect of PGD2 was related to the inhibition of DNA synthesis of the cells. The in vivo studies were performed with a subcutaneously transplanted mouse glioma model. Injection of 0.5 mg/kg PGD2 into the tumor was more effective than the same concentration given by intraperitoneal injection. In mice with intracranially transplanted glioma, daily intraperitoneal injection of 0.5 mg/kg PGD2 had no significant effect on survival time.     

Effects of Inhaled Nitric Oxide on Primary Graft Dysfunction in Lung Transplantation

Introduction and Objectives

Inhaled nitric oxide (iNO) is a gaseous drug with known properties of specific pulmonary vasodilation and improved oxygenation. In some clinical trials on lung transplantation (LT) in animals, it has been demonstrated to reduce primary graft dysfunction (PGD) by limiting neutrophil adhesion and the inflammatory cascade. Our objective was to assess whether iNO showed this immunomodulatory effect by determining interleukin (IL)-6, -8, and -10 levels in blood and bronchoalveolar lavage (BAL) in LT patients, and its relationship with PGD incidence.

Materials and Methods

Forty-nine LT patients were recruited and included in the iNO or in the control group. Patients in the first group were given iNO (10 ppm) from the start of LT to 48 hours afterward. BAL and blood samples were taken preimplantation and at 12, 24, and 48 hours after graft reperfusion.

Results

The iNO group displayed a significantly lower incidence (P < .035) of PGD (17.2%) than the control group (45%). Significant differences (P < .05) were also observed in the iNO group with lower levels of IL-6 (in blood at 12 hours), IL-8 (in blood and BAL at 12 and 24 hours), and IL-10 (in blood at 12 and 24 hours and BAL at 24 hours).

Conclusions

PGD is associated with the development of an inflammatory process that is reduced by giving iNO to lung recipients. In our series, the iNO group displayed significantly lower content of IL-6, IL-8, and IL-10 in the majority of samples at 12 and 24 hours compared with the control group.     

Parathyroid hormone stimulates proliferation of chondroprogenitor cellsIn vitro

Summary Mandibular condylar explants of newborn ICR mice were maintained as serum-free organ culture systems and were used to study the effects of 0.1–10.0 U/ml parathyroid hormone (PTH) on the morphology of the organ and the ultrastructure of the chondroprogenitor cells. Parameters of proliferation such as³H-thymidine autoradiography and incorporation into the explants were also studied. The chondroprogenitoric zone gradually increased with increasing dosages of the hormone up to a maximum of 5-fold of the control with 5.0 U/ml PTH. Autoradiographic studies showed a 3-fold increase in the number of³H-thymidine-labeled cells in the chondroprogenitoric zone of PTH-treated explants. This was matched by a dose-dependent stimulation of³H-thymidine incorporation, reaching maximal values at 5.0 U/ml PTH. At this concentration, the stimulated incorporation of³H-thymidine was found to be dependent on the Ca²⁺ concentration of the medium. Chondroprogenitor cells located adjacent to the chondroblastic zone tended to pile up and aggregate in “syncytium”-like clusters, establishing intercellular gap junctions. All PTH-treated chondroprogenitor cells demonstrated large deposits of glycogen and highly elaborated stacks of their Golgi systems; the latter were associated with large numbers of vesicular elements. On the other hand, the chondroblastic zone was significantly reduced in size. Hence, it seems that PTH possesses a rather intense mitogenic effect upon chondroprogenitor cells and might possibly interfere with their normal pattern of differentiation into mature cartilage cells.     

Time-dependent effects of parathyroid hormone and prostaglandin E2 on DNA synthesis by periosteal cells from embryonic chick calvaria

Both PGE₂ and PTH (1-34) caused a time- and concentration-dependent stimulation of proliferation by embryonic chick periosteal cells. Cells were exposed to the agents for different periods of time, the medium was replaced with fresh medium, and ³H-TdR incorporation was measured after 16 hours. Challenge with 10^-6 M prostaglandin E₂ (PGE₂) or 10^-7 M parathyroid hormone (1-34) (PTH) for 5 minutes produced 4- and 5.5-fold increases in ³H-TdR incorporation, respectively. Longer exposures, however, produced diminishing responses and after 45 minutes, only minimal effects or slight inhibitions were seen. These timedependent effects were also seen with forskolin and dibutyryl-cAMP; TPA on the other hand stimulated DNA synthesis after both short- and long-term exposure. Both PGE₂ and PTH (1-34) stimulated cAMP synthesis in periosteal cells but neither could be shown to stimulate protein kinase-C (PKC) at concentrations required for stimulation of proliferation, and dibutyryl-cyclic AMP (cAMP) effectively inhibited endogenous PKC activity. It is possible that the stimulation of proliferation by short-term exposure to PGE₂ and PTH (1-34) is mediated by cAMP and that the time dependency possibly stems from the inhibition of endogenous PKC activity by increased intracellular cAMP levels.     

Proliferative responses to estradiol,IL-1α and TGFβ by cells expressing alkaline phosphatase in human osteoblast-like cell cultures

Summary The use of primary (nontransformed) bone cell cultures is hampered by their cellular heterogeneity. Primary cultures of osteoblast-like cells have been shown to proliferate in response to several osteotropic agents, but because mixed cell populations are present it is uncertain whether a true osteoblastic response was observed. By combining (1) localization of [³H]-thymidine incorporation into the nuclei of actively dividing cells by autoradiography with (2) subsequent induction of osteoblast differentiation by 1,25(OH)₂D₃ to optimize the number of cells expressing high alkaline phosphatase activity and (3) its localization by histochemical staining, it is possible to measure the proliferation of cells that are capable of expressing a more mature osteoblastic phenotype in heterogeneous human trabecular bone cell cultures.Over a 72-hour incubation period, rhIL-1 (0.2–2 ng/ml) exerted a dose-dependent stimulation of proliferation of cells expressing alkaline phosphatase. Purified human TGF₁ produced a biphasic increase in the proliferation of these cells (0.01–1 ng/ml) but 17 and 17-estradiol (10^-12–10^-8 M) failed to consistently regulate cell growth. Furthermore, 17-estradiol did not reproducibly modulate proliferation induced by IL-1 or TGF when added together in cultures. This procedure represents a more accurate method for the assessment of osteoblast proliferation in primary bone cell cultures and demonstrates that estrogen is not mitogenic for human osteoblasts and does not potentiate the actions of putative local stimulators of osteoblast replication.     

Fluoride increases net45Ca uptake by SaOS-2 cells: The effect is phosphate dependent

Summary Previousin vitro studies have shown that the effect of fluoride to increase avian osteoblast-like cell proliferation was dependent on the phosphate concentration.In vitro studies have further revealed that fluoride could also have direct effects on osteoblast-like cells to increase phosphate uptake and transiently increase cytosolic calcium. The current studies were intended to determine whether fluoride could increase net⁴⁵Ca uptake by human osteosarcoma (SaOS-2) cells and, if so, whether those effects would also be phosphate dependent. The results of these studies indicate that fluoride increased net⁴⁵Ca uptake by SaOS-2 cells, with biphasic dose and time dependencies. After 30 minutes of exposure, net⁴⁵Ca uptake was increased to a greater extent by 50 M fluoride (217 ± 16% of control,P < 0.001) than by 200 M fluoride; and the stimulatory effect of 100 M fluoride on net⁴⁵Ca uptake was greater after 20 minutes (187 ±22% of control,P < 0.001) than after 60 minutes (122 ± 7% of control,P < 0.05). These effects of fluoride to increase net⁴⁵Ca uptake were dependent on the phosphate concentration in the medium. Fluoride had no effect on net⁴⁵Ca uptake in medium containing 0.4 mM phosphate, but increased net⁴⁵Ca uptake in medium containing 1.2 or 2.0 mM phosphate (P < 0.005). As the phosphate concentration was increased, the biphasic fluoride dose-response curve was shifted to a lower range of fluoride concentrations. These effects of fluoride were not unique to SaOS-2 cells with very high steady-state levels of skeletal alkaline phosphatase; similar effects were seen in a subpopulation of SaOS-2 cells with much lower alkaline phosphatase levels. Further studies indicated that the effects of fluoride to increase SaOS-2 cell proliferation and skeletal alkaline phosphatase activity showed a similar pattern of phosphate dependency. As the fluoride-dependent increases in³[H]-thymidine incorporation and net⁴⁵Ca uptake were blocked by verapamil, these data are consistent with the general hypothesis that the osteogenic effects of fluoride are associated with acute effects to increase net Ca uptake.     

Analysis of Interleukin-6 and Interleukin-8 in Lung Transplantation: Correlation With Nitric Oxide Administration

Introduction and Objectives

Primary graft dysfunction (PGD) following lung transplantation (LT) is associated with an activation of the inflammatory cascade and release of cytokines. Inhaled nitric oxide (iNO) provides specific pulmonary vasodilatation and improves oxygenation. Our objective was to verify whether administering iNO to LT patients modified the blood and bronchoalveolar lavage (BAL) interleukin (IL)-6 and -8 levels in the event of PGD.

Materials and Methods

Thirty-two LT patients were randomized to the iNO treatment or the control group. Patients in the first group were given 10 ppm of iNO from the start of LT until 48 hours afterward. BAL and peripheral arterial blood samples were taken preimplantation as well as 12, 24, as and 48 hours postreperfusion.

Results

The iNO treatment group showed a lower incidence of PGD (29%) in comparison with the control group (40%). Significant differences (P < .05) were observed in the iNO group, with lower IL-6 levels at 12 hours in blood and BAL. A lower percentage of IL-8 was also detected in the iNO group at 24 hours in BAL and at 12 hours in blood and BAL.

Conclusions

Lung transplant recipients develop an inflammatory response following implantation with systemic elevation of IL-6 and significant local elevation of IL-8 within the first few hours, especially in the event of PGD. In our series, iNO appeared to modulate the inflammatory response by reducing IL concentrations found immediately after reimplantation, and this reduction was related to a lower incidence of PGD.     

Ca2+ uptake by endoplasmic reticulum of renal cortex. II. Effects of uninephrectomy and parathyroidectomy

Summary Calcium uptake by the endoplasmic reticulum (ER) is important for cellular calcium homeostasis, yet its regulation in nonmuscle cells is poorly understood. We reported that Ca²⁺ uptake by a light fraction of canine renal cortical ER (LER) is stimulated by protein kinase C in vitro. Here we describe conditions in vivo that stimulated renal cortical LER Ca²⁺ uptake. Thirty minutes after contralateral nephrectomy in the dog, ⁴⁵Ca²⁺ uptake into renal cortical LER was increased 42% above control LER. There was no difference in LER Ca²⁺ uptake 24 hours after uninephrectomy. Acute denervation did not reproduce the increase in LER ⁴⁵Ca²⁺ uptake seen at 30 minutes after uninephrectomy, nor did prior thyroparathyroidectomy abolish it. Forty-eight hours after thyroparathyroidectomy, ⁴⁵Ca²⁺ uptake activity into renal cortical LER was decreased sevenfold. In a proximal tubular cell line (LLC-PK₁), 30-minute incubation with 12-O-tetradecanoylphorbol-13-acetate doubled ⁴⁵Ca²⁺ uptake into a nonmitochondrial pool. Pretreatment with epidermal growth factor halved ER Ca²⁺ uptake, whereas insulin-like growth factor and growth hormone, alone or in combination, had no effect. Our data suggest that Ca²⁺ uptake into renal cortical ER is stimulated acutely during compensatory renal growth, perhaps through protein kinase C, and is stimulated chronically by parathyroid hormone. Offprint request to}: D. Moskowits, Nephrology Division 111B-JC Montreal Canada     

Nitric Oxide Mediates the Effect of Fluvastatin on Intercellular Adhesion Molecule-1 and Platelet EndothelialCell Adhesion Molecule-1 Expressionon Human Endothelial cells

Leukocyte and platelet adhesion to endothelial cells, an early step in the pathogenesis of atherosclerosis, is mediated through adhesion molecules. It has been shown that statins decrease adhesion molecule expression. We examined the hypothesis that fluvastatin decreased intercellular adhesion molecule-1 (ICAM-1) and platelet endothelial cell adhesion molecule-1 (PECAM-1) expression through a nitric oxide-mediated pathway. Human iliac artery endothelial cells were exposed to fluvastatin in the presence or absence of 2 mM N-monomethyl-L-arginine (L-NMMA). Flow cytometry analysis was used to measure ICAM-1 and PECAM-1 expression. In a separate experiment, confluent cell cultures were exposed in a serum-free medium to fluvastatin 20 M, and the supernatant was collected for nitrate/nitrite determination after 6 and 48 hr of incubation. Protein was isolated and processed for immunoblotting with monoclonal antibodies specific for endothelial nitric oxide synthase (eNOS), Ser¹¹⁷⁷–phosphorylated eNOS, and AMP kinase. Relative band intensity was assessed with densitometry. Results are presented as the mean ± standard deviation (SD), and p < 0.05 was considered significant. ICAM-1 and PECAM-1 were expressed constitutively. Human iliac artery endothelial cells (HIAECS) treated with 5 M fluvastatin did not exhibit reduced expression of PECAM-1 or ICAM-1. Incubation with 10 M fluvastatin reduced basal expression of both ICAM-1 and PECAM-1. Fluorescence intensity (FI) for these substance was as follows: 3638 ± 1671, p = 0.01 and PECAM-1 vs. control FI 276 ± 52 vs. 522 ± 78, p = 0.02. In the presence of 2 mM L-NMMA, fluvastatin failed to decrease the expression of ICAM-1 (fluvastatin 10 M + L-NMMA: FI was 3042 ± 1378 vs. 3638 ± 1671 for the control p = 0.01) or PECAM-1 (fluvastatin 10 M + L-NMMA: FI was 415 ± 188 vs. 522 ± 78 for the control, p = 0.1). Incubation with 20 M fluvastatin similarly reduced ICAM-1 expression (FI was 2014 ± 1595 vs. 3638 ± 1671 for the control, p = 0.02) and PECAM-1 expression (FI was 196 ± 109 vs. 522 ± 78 for the control, p = 0.02). This reduction was prevented in the presence of 2 mM L-NMMA. L-NMMA in a concentration of 2 mM had no significant effect on adhesion molecule expression (p > 0.05 for all comparisons of the control FI versus 2 mM L-NMMA mean FI). After a 48 hr incubation with 20 M fluvastatin there was a 219 ± 35% increase in the cell eNOS protein content (p = 0.01) and a 170 ± 26% increase in the cell AMPK protein content (p = 0.02). Ser¹¹⁷⁷-phosphorylated eNOS protein levels were increased by 41 ± 8% (p = 0.03). The nitric oxide concentration in the medium of the HIAEC treated with 20 M fluvastatin for 48 hr was significantly higher than that in the control (p = 0.0004), pointing to increased production during the incubation period. Fluvastatin thus decreases basal expression of ICAM-1 and PECAM-1. Competitive inhibition of eNOS with L-NMMA abolishes the effect of fluvastatin on ICAM-1 and PECAM-1 expression. The statin up-regulates eNOS and AMP kinase, one of the enzymes that activates eNOS via phosphorylation at Ser¹¹⁷⁷. We have shown that after a 48-hr exposure to fluvastatin there is an increased amount of the phosphorylated enzyme in the endothelial cells.