Insulin binding monitored by fluorescence correlation spectroscopy.

AIM/HYPOTHESIS: The characteristics of insulin binding to its receptors have been extensively studied by the radioligand binding assay. We used fluorescence correlation spectroscopy to determine the distribution of diffusion times and further novel data on the kinetics of insulin's binding to its receptor. METHODS: Cultured human renal tubular cells (HRTC) were incubated with tetramethyl rhodamine labelled insulin (Rh-Ins) for 60 min. Fluorescence intensity fluctuations and autocorrelation functions for Rh-Ins, free in the incubation medium and bound to the cell membrane, were studied at single-molecule detection sensitivity in a 0.2 fL confocal volume. RESULTS: Measurements at the cell membrane revealed Rh-Ins binding with at least two diffusion components (diffusion times tauD1 = 0.8 ms, tauD2 = 20 ms) and corresponding weight fractions of y1 = 0.43 and y2 = 0.42. Specificity of the binding was shown by the dislocation of bound Rh-Ins when excess unlabelled insulin was added. Scatchard analysis showed a nonlinear plot, revealing two binding processes with different affinities (Kass approximately 2 x 10(10) M(-1) and approximately 1 x 10(9) M(-1), respectively). CONCLUSION/INTERPRETATION: The fluorescence correlation spectroscopy results show two classes of binding sites with different affinities for insulin, or interactions between receptor sites consistent with negative cooperativity. This conclusion is in agreement with studies of insulin binding using radioligand binding assays. Because of its high sensitivity (single molecule detection), FCS, provides additional data allowing a more precise evaluation of the kinetics of ligand-receptor interactions at low expression levels in living cells.

Bacillus Calmette-Guerin induces long-term local formation of nitric oxide in the bladder via the induction of nitric oxide synthase activity in urothelial cells.

PURPOSE: Bladder instillation of bacillus Calmette-Guerin (BCG) is effective therapy for recurrent superficial bladder cancer and carcinoma in situ. BCG induces nitric oxide synthase activity in the bladder. Nitric oxide is formed from L-arginine by nitric oxide synthase. We investigated nitric oxide formation and its localization in bladder cancer patients treated with intravesical BCG instillation. MATERIALS AND METHODS: The L-citrulline conversion assay was done to assess nitric oxide synthase activity in BCG treated T24 human bladder cancer cells and cultured normal human urothelial cells. Nitrite and nitrate in cell culture medium, urine and plasma were measured by capillary electrophoresis. Nitric oxide formation in the bladder was measured by chemiluminescence. RESULTS: A 24-hour treatment with BCG induced calcium independent nitric oxide synthase activity in T24 cells in a dose dependent manner. Nitrite and nitrate production by T24 cells also increased in a dose dependent manner after 24-hour BCG treatment. BCG treatment of cultured normal human urothelial cells resulted in the induction of calcium dependent and independent nitric oxide synthase activity. Nitrite in the urine of patients receiving BCG for the first time was increased 5-fold 24 hours after instillation. Furthermore, BCG increased luminal nitric oxide in the bladder. The increase was noted after a single treatment and sustained for 6 months. No changes in plasma nitrite or nitrate were observed after BCG treatment. CONCLUSIONS: BCG induces the local formation of nitric oxide in the bladder, whereas no evidence for systemic nitric oxide formation was noted. Increased nitric oxide production in the bladder is likely due to the induction of nitric oxide synthase activity in urothelial cells.

Specific binding of proinsulin C-peptide to human cell membranes.

Recent reports have demonstrated beneficial effects of proinsulin C-peptide in the diabetic state, including improvements of kidney and nerve function. To examine the background to these effects, C-peptide binding to cell membranes has been studied by using fluorescence correlation spectroscopy. Measurements of ligand-membrane interactions at single-molecule detection sensitivity in 0.2-fl confocal volume elements show specific binding of fluorescently labeled C-peptide to several human cell types. Full saturation of the C-peptide binding to the cell surface is obtained at low nanomolar concentrations. Scatchard analysis of binding to renal tubular cells indicates the existence of a high-affinity binding process with K(ass) > 3.3 x 10(9) M(-1). Addition of excess unlabeled C-peptide is accompanied by competitive displacement, yielding a dissociation rate constant of 4.5 x 10(-4) s(-1). The C-terminal pentapeptide also displaces C-peptide bound to cell membranes, indicating that the binding occurs at this segment of the ligand. Nonnative D-C-peptide and a randomly scrambled C-peptide do not compete for binding with the labeled C-peptide, nor were crossreactions observed with insulin, insulin-like growth factor (IGF)-I, IGF-II, or proinsulin. Pretreatment of cells with pertussis toxin, known to modify receptor-coupled G proteins, abolishes the binding. It is concluded that C-peptide binds to specific G protein-coupled receptors on human cell membranes, thus providing a molecular basis for its biological effects.

Endogenously formed nitric oxide modulates cell growth in bladder cancer cell lines.

OBJECTIVES: Nitric oxide (NO) is formed in many mammalian tissues, and a growing body of evidence suggests that NO is involved in cell growth and cell differentiation. Low concentrations of NO can stimulate cell growth; high concentrations result in cytostatic/cytotoxic effects. It has previously been shown that intravesical treatment with bacille Calmette-Guérin (BCG) for bladder cancer increases NO production in the human urinary bladder and that NO inhibits bladder cancer cell growth in vitro. In this study, we investigated nitric oxide synthase (NOS) activity in different bladder cancer cells and the role of the NO precursor L-arginine in cell proliferation. METHODS: NOS activity was assessed by citrulline assay in cultured normal human urothelial cells and bladder cancer cell lines T24 and MBT-2 before and after treatment with cytokines. We also measured cell growth at various L-arginine concentrations and after addition of the NOS inhibitor N(G)-nitro-L-arginine (L-NNA) in unstimulated and cytokine-stimulated cells. RESULTS: Normal urothelial cells, as well as T24 and MBT-2 cells, showed calcium-dependent NOS activity under basal conditions. The bladder cancer cell lines also showed calcium-independent NOS activity in contrast to the normal cells. After cytokine treatment, both the normal cells and the cancer cell lines showed a marked increase in calcium-independent NOS activity. There was a dose-dependent stimulation of cell growth in the cancer cell lines after L-arginine addition, and this effect could be antagonized by L-NNA. Cytokine treatment inhibited cell growth, and this inhibition was partly reversed by L-NNA. CONCLUSIONS: Normal urothelial cells and bladder cancer cell lines MBT-2 and T24 show NOS activity, and cytokine treatment induces calcium-independent NOS activity. Our results suggest that endogenous activity of the constitutively expressed form of NOS in unstimulated cells promotes cell proliferation, and NO production secondary to increased activity of the inducible form of NOS after cytokine treatment inhibits cell growth.

The role of nitric oxide in bacillus Calmette-Guérin mediated anti-tumour effects in human bladder cancer.

Bacillus Calmette-Guérin (BCG) has been used for many years to treat cancer of the urinary bladder. It constitutes effective intravesical therapy of carcinoma in situ and recurrent superficial bladder cancer. Although the mechanism of action is unknown, most evidence suggests an immune-mediated mechanism. BCG treatment is known to increase cytokine production in the urinary bladder. As cytokines may induce nitric oxide synthase (NOS) activity and as nitric oxide (NO) exerts cytotoxic effects on tumour cells, we investigated the role of NO in BCG-mediated anti-tumour activity. Here we demonstrate a marked induction of both calcium-dependent and calcium-independent NOS activity in the human urinary bladder after BCG treatment. The presence of NOS in the urothelial cells was also demonstrated by the use of immunohistochemistry. Furthermore, patients treated with BCG showed a 30 times higher production of gaseous NO as measured in the urinary bladder by chemiluminescence. Finally, NO donors exerted cytotoxic effects on bladder cancer cell lines. These findings suggest that NO synthesis may be an important mechanism in BCG-mediated anti-tumour therapy.

Nitric oxide synthase activity in human renal cell carcinoma.

PURPOSE: Nitric oxide (NO) is generated in mammalian tissue by the conversion of L-arginine to L-citrulline. The reaction is catalyzed by nitric oxide synthase (NOS). NO has been suggested to have a dual role in tumor biology with both antitumor and tumor promoter activity. Furthermore, it has been proposed that NO contributes to interleukin-2-induced antitumor activity. Since interleukin-2 is used in the treatment of renal cell carcinoma (RCC) it was of interest to study the NOS activity in the human kidney and in RCC and its correlation to tumor grade. Furthermore, the effect of cytokine treatment on NOS activity and the effect of NO donor application was studied in cultured cells. MATERIALS AND METHODS: The effect of cytokine treatment on NOS activity and the effect of NO donor application on cell proliferation was studied in cultured human proximal tubular cells and in RCC cell lines HN4 and HN51. NOS activity was measured by the L-arginine to L-citrulline conversion assay. RESULTS: Calcium-dependent NOS activity was found in all non-malignant kidney tissues (486+/-63 pmol. min(-1) g(-1) tissue). The activity was significantly lower in RCC (24+/-6 pmol. min(-1) g(-1) tissue) and correlated with tumor grade; thus high grade tumors showed lower activity than low grade tumors. Calcium-independent NOS activity was not detected in non-malignant kidney tissue or in RCC tissue. In cultured proximal tubular cells and RCC cell lines HN4 and HN51, cytokine treatment induced a marked increase in NOS activity and NO exerted cytostatic effects on these cell lines. CONCLUSIONS: The NOS activity was higher in non-malignant kidney tissue than in RCC tissue and was inversely correlated with tumor grade. Furthermore, cytokine treatment induced a marked increase in NOS activity and NO exerted cytostatic effects on cultured proximal tubular cells and RCC cell lines.