Glucose upregulates plasminogen activator inhibitor-1 gene expression in vascular smooth muscle cells

We investigated the effects of high concentrations of glucose on plasminogen activator inhibitor-1 (PAI-1) gene expression in cultured rat vascular smooth muscle cells (VSMC). In response to a high glucose concentration (27.5 mM), PAI-1 mRNA increased within 2 h, peaked at 4 h, remained elevated for another 4 h, then decreased to basal levels at 24 h. On the other hand, mannose at the same concentration (22.5 mM mannose plus 5.5 mM glucose) as an osmotic control had little effect on PAI-1 mRNA expression. The expression of PAI-1 mRNA that was also increased by H(2)O(2), angiotensin II, or phorbol myristate acetate, was reversed by the MAPK kinase (MEK) inhibitor PD98059 or the specific protein kinase C (PKC) inhibitor GF109203X. High glucose appeared to activate MAPK and PKC in VSMC judging from Elk-1 and AP-1 activation, respectively. PD98059 inhibited and GF109203X prevented subsequent PAI-1 induction by glucose. These results suggest that glucose at high concentrations induces PAI-1 gene expression in VSMC at least partially via MAPK and PKC activation. This direct effect of glucose might have important implications for the increased plasma concentrations of PAI-1 and possibly atherosclerosis that are associated with diabetes.     

Nonspecific inhibition of myogenic tone by PD98059, a MEK1 inhibitor, in rat middle cerebral arteries

Activation of MAP kinase kinase, also called ERK kinase (MEK), may lead to desinhibition of thin filament regulatory proteins and we therefore investigated the acute effects of the potent MEK inhibitor, PD98059 on the contractile properties of pressurized rat middle cerebral arteries. Cerebral arteries (diameter 100-150 microm) were mounted on a pressure myograph and PD98059 (10 microM, 40 microM) significantly inhibited (15% and 64%) myogenic tone (P < 0.001). At these concentrations, PD98059 also significantly reduced the vasopressin (0.1 microM)- and KCl (60 mM)-induced tone. Cumulative addition of exogenous Ca2+ (0.4-1.6 mM) increased myogenic tone to approximately 50% of constriction at 80 mmHg. This effect was inhibited by PD98059 (P < 0.001). These results demonstrate that pressure-induced myogenic tone is inhibited by PD98059 at the concentrations that have been reported to be selective for inhibition of MEK and the MAP kinase cascade. However, our results also demonstrate that PD98059 may have nonspecific effects on voltage-sensitive Ca2+ entry in vascular smooth muscle.     

ERK mediates anti-apoptotic effect through phosphorylation and cytoplasmic localization of p21Waf1/Cip1/Sdi in response to DNA damage in normal human embryonic fibroblast (HEF) cells

Since anti-apoptotic effect of ERK has not been elucidated clearly in DNA-damage-induced cell death, the role of ERK was examined in normal HEF cells treated with mild DNA damage using etoposide or camptothecin. ERK was activated by DNA damage in HEF cells. PD98059 increased apoptosis and reduced DNA-damage-induced p21Waf1/Cip1/Sdi level. Depletion of p21Waf1/Cip1/Sdi induced cell death and PD98059 induced additional cell death. DNA-damage-induced increase in cytoplasmic localization and phosphorylation of threonine residues of p21Waf1/Cip1/Sdi was reversed by PD98059. Thus, the results suggest that ERK pathway mediates anti-apoptotic effects through phosphorylation and cytoplasmic localization of p21Waf1/Cip1/Sdi in response to mild DNA damage.     

Taurine increases cell proliferation and generates an increase in [Mg2+]i accompanied by ERK 1/2 activation in human osteoblast cells

Taurine has been reported to influence bone metabolism, and its specific transport system, the taurine transporter, is expressed in osteoblasts. The mean [Mg2+]i was 0.51+/-0.01 mM in normal culture media. Taurine caused an increase in [Mg(2+)]i by 0.72+/-0.04 mM in human osteoblast (HOB) cells. This increment in [Mg2+]i was inhibited significantly by PD98059, nifedipine, lidocaine, and imipramine. Taurine was also shown to stimulate the activation of ERK 1/2. This taurine-stimulated ERK 1/2 activation was inhibited by PD98059. In the present study, taurine was shown to increase cell proliferation and generate an increase in [Mg2+]i accompanied by ERK 1/2 activation in HOB cells.     

Dual effect of glucose on cytoplasmic Ca2+ in single pancreatic beta-cells

The cytoplasmic Ca2+ concentration (Ca2+i) was measured in single pancreatic beta-cells from ob/ob-mice using the fluorescent indicator fura-2. Raising the glucose concentration from 3 to 20 mM resulted in 25% initial lowering of Ca2+i, followed by 250% rise above the basal level of 49 +/- 3 nM. Tolbutamide (100 microM) was as effective as glucose in increasing Ca2+i, although its action was more rapid and not preceded by any reduction. The results support the concept that stimulated removal of Ca2+ from the cytoplasm is an essential part of the physiological glucose effect on the pancreatic beta-cells.     

Hypotonic stress upregulates β- and γ-ENaC expression through suppression of ERK by inducing MKP-1

We investigated a physiological role for ERK, a member of the MAPK family, in the hypotonic stimulation of epithelial Na(+) channel (ENaC)-mediated Na(+) reabsorption in renal epithelial A6 cells. We show that hypotonic stress causes a major dephosphorylation of ERK following a rapid transient phosphorylation. PD98059 (a MEK inhibitor) increases dephosphorylated ERK and enhances the hypotonic-stress-stimulated Na(+) reabsorption. ERK dephosphorylation is mediated by MAPK phosphatase (MKP). Hypotonic stress activates p38, which in turn induces MKP-1 and to a lesser extent MKP-3 mRNA expression. Inhibition of p38 suppresses MKP-1 induction, preventing hypotonic stress from dephosphorylating ERK. Inhibition of MKP-1 and -3 by the inhibitor NSC95397 also suppresses the hypotonicity-induced dephosphorylation of ERK. NSC95397 reduces both β- and γ-ENaC mRNA expression and ENaC-mediated Na(+) reabsorption stimulated by hypotonic stress. In contrast, pretreatment with PD98059 significantly enhances mRNA and protein expression of β- and γ-ENaC even under isotonic conditions. However, PD98059 only stimulates Na(+) reabsorption in response to hypotonic stress, suggesting that ERK inactivation by itself (i.e., under isotonic conditions) is not sufficient to stimulate Na(+) reabsorption, even though ERK inactivation enhances β- and γ-ENaC expression. Based on these results, we conclude that hypotonic stress stimulates Na(+) reabsorption through at least two signaling pathways: 1) induction of MKP-1 that suppresses ERK activity and induces β- and γ-ENaC expression, and 2) promotion of translocation of the newly synthesized ENaC to the apical membrane.     

Glucose-induced oscillations of cytoplasmic Ca2+ in the pancreatic beta-cell

The cytoplasmic calcium concentration (Ca2+i) was measured in individual mouse pancreatic beta-cells loaded with fura-2 by recording the 340/380 nm fluorescence excitation ratio. An increase of the glucose concentration from 3 to 20 mM, caused initial lowering of Ca2+i followed by a rise with a peak preceding constant elevation at an intermediary level. However, at 11 mM glucose there were large Ca2+i oscillations with a frequency of 1 cycle per 2-6 min. The results indicate that both first and second phase secretion depend on elevated Ca2+i, and that many electrically coupled cells collectively determine the pace of rhythmic depolarization.     

Glucose-induced increase in cytoplasmic pH in pancreatic beta-cells is mediated by Na+/H+ exchange, an effect not dependent on protein kinase C.

Glucose-induced changes in cytoplasmic pH (pHi) were investigated using pancreatic beta-cells isolated from obese hyperglycemic mice. Glucose, at concentrations above 3-5 mM, depolarized the beta-cell and increased pHi, cytoplasmic free Ca2+ ([Ca2+]i), and insulin release. This increase in pHi was dependent on the presence of extracellular Na+ and was inhibited by 5-(N-ethyl-N-isopropyl) amiloride, a blocker of Na+/H+ exchange. Stimulation of protein kinase C with phorbol ester also induced an alkalinization. However, when protein kinase C activity was down-regulated, glucose stimulation still induced alkalinization. At 20 mM glucose, 10 mM NH4Cl induced a marked rise in pHi, paralleled by repolarization, inhibition of electrical activity, and decreases in both [Ca2+]i and insulin release. Reduction in [Ca2+]i was prevented by 200 microM tolbutamide, but not by 10 mM tetraethylammonium. At 4 mM glucose, NH4Cl induced a transient increase in insulin release, without changing [Ca2+]i. Exposure of beta-cells to 10 mM sodium acetate caused a persistent decrease in pHi, an effect paralleled by a small transient increase in [Ca2+]i. Acidification per se did not change the beta-cell sensitivity to glucose, not excluding that the activity of the ATP-regulated K+ channels may be modulated by changes in pHi.     

Nitric oxide-cyclic GMP system potentiates glucose-induced rise in cytosolic Ca2+ concentration in rat pancreatic beta-cells.

Involvement of nitric oxide (NO) in the regulation of insulin secretion from pancreatic beta-cells was investigated by measuring cytosolic Ca2+ concentration ([Ca2+]i) in isolated rat pancreatic beta-cells. At 7.0 mM glucose, L-arginine (0.1 mM) elevated [Ca2+]i in about 50% of the beta-cells examined. The response was partially inhibited by an NO synthase inhibitor, N(G)-monomethyl-L-arginine (L-NMA; 0.1 mM), suggesting that part of the response was mediated by the production of NO from L-arginine. D-Arginine at higher concentrations (3 or 10 mM) also increased [Ca2+]i at 7.0 mM glucose; however, the response was not affected by L-NMA (0.1 mM). Similar [Ca2+]i elevation was produced by NO (10 nM) and sodium nitroprusside (SNP; 10 microM) at 7.0 mM glucose. The SNP-induced increase in [Ca2+]i was abolished by nicardipine (1 microM), suggesting that the [Ca2+]i response is mediated by Ca2+ influx through L-type voltage-operated Ca2+ channels. In the presence of oxyhemoglobin (1 microM), the [Ca2+]i elevation induced by NO (10 nM) was abolished. Neither degradation products of NO, NO2- nor NO3-, caused any changes in [Ca2+]i. 8-Bromo-cyclic GMP (8-Br-cGMP; 3 mM) and atrial natriuretic peptide (0.1 microM) elevated [Ca2+]i at 7.0 mM glucose. We conclude that NO, which is produced from L-arginine in pancreatic islets, facilitates glucose-induced [Ca2+]i increase via the elevation of cGMP in rat pancreatic beta-cells. NO-cGMP system may physiologically regulate insulin secretion from pancreatic beta-cells.     

Glucose-mediated Ca(2+) signalling in single clonal insulin-secreting cells: evidence for a mixed model of cellular activation

Using clonal insulin-secreting BRIN-BD11 cells, we have assessed whether the graded response of the whole cell population to glucose can be accounted for by a dose-dependent recruitment of individual cells, an amplification of the response of the recruited cells or both. Cytosolic free Ca(2+) concentration ([Ca(2+)](i)) is an established index of beta-cell function. We used fura-2 microfluorescence techniques to assess the [Ca(2+)](i) responsiveness of single BRIN-BD11 cells to glucose and other secretagogues. Glucose (1-16.7 mM) evoked oscillatory [Ca(2+)](i) rises in these cells resembling those found in parental rat pancreatic beta-cells. The percentage of glucose-responsive cells was 11% at 1 mM and increased to 40-70% at 3-16.7 mM glucose, as assessed by a single-stimulation protocol. This profile was unrelated to possible differences in the cell cycle, as inferred from experiments where the cultured cells were synchronized by a double thymidine block protocol. Individual cells exhibited variable sensitivities to glucose (threshold range: 1-5 mM) and a variable dose-dependent amplification of the [Ca(2+)](i) responses (EC(50) range: 2-10 mM), as assessed by a multiple-stimulation protocol. Glyceraldehyde and alpha-ketoisocaproic acid had glucose-like effects on [Ca(2+)](i). The data support a mixed model for the activation of insulin-secreting cells. Specifically, the graded secretory response of the whole cell population is likely to reflect both a recruitment of individual cells with different sensitivities to glucose and a dose-dependent amplification of the response of the recruited cells.