Tumor microenvironment conditions alter Akt and Na+/H+ exchanger NHE1 expression in endothelial cells more than hypoxia alone: implications for endothelial cell function in cancer.

BACKGROUND: Chronic angiogenesis is a hallmark of most tumors and takes place in a hostile tumor microenvironment (TME) characterized by hypoxia, low nutrient and glucose levels, elevated lactate and low pH. Despite this, most studies addressing angiogenic signaling use hypoxia as a proxy for tumor conditions. Here, we compared the effects of hypoxia and TME conditions on regulation of the Na+/H+ exchanger NHE1, Ser/Thr kinases Akt1-3, and downstream effectors in endothelial cells. METHODS: Human umbilical vein endothelial cells (HUVEC) and Ea.hy926 endothelial cells were exposed to simulated TME (1% hypoxia, low serum, glucose, pH, high lactate) or 1% hypoxia for 24 or 48 h, with or without NHE1 inhibition or siRNA-mediated knockdown. mRNA and protein levels of NHE1, Akt1-3, and downstream effectors were assessed by qPCR and Western blotting, vascular endothelial growth factor (VEGF) release by ELISA, and motility by scratch assay. RESULTS: Within 24 h, HIF-1α level and VEGF mRNA level were increased robustly by TME and modestly by hypoxia alone. The NHE1 mRNA level was decreased by both hypoxia and TME, and NHE1 protein was reduced by TME in Ea.hy926 cells. Akt1-3 mRNA was detected in HUVEC and Ea.hy926 cells, Akt1 most abundantly. Akt1 protein expression was reduced by TME yet unaffected by hypoxia, while Akt phosphorylation was increased by TME. The Akt loss was partly reversed by MCF-7 human breast cancer cell conditioned medium, suggesting that in vivo, the cancer cell secretome may compensate for adverse effects of TME on endothelial cells. TME, yet not hypoxia, reduced p70S6 kinase activity and ribosomal protein S6 phosphorylation and increased eIF2α phosphorylation, consistent with inhibition of protein translation. Finally, TME reduced Retinoblastoma protein phosphorylation and induced poly-ADP-ribose polymerase (PARP) cleavage consistent with inhibition of proliferation and induction of apoptosis. NHE1 knockdown, mimicking the effect of TME on NHE1 expression, reduced Ea.hy926 migration. TME effects on HIF-1α, VEGF, Akt, translation, proliferation or apoptosis markers were unaffected by NHE1 knockdown/inhibition. CONCLUSIONS: NHE1 and Akt are downregulated by TME conditions, more potently than by hypoxia alone. This inhibits endothelial cell migration and growth in a manner likely modulated by the cancer cell secretome.

Treatment of knee osteoarthritis with pulsed electromagnetic fields: a randomized, double-blind, placebo-controlled study.

OBJECTIVE: The investigation aimed at determining the effectiveness of pulsed electromagnetic fields (PEMF) in the treatment of osteoarthritis (OA) of the knee by conducting a randomized, double-blind, placebo-controlled clinical trial. DESIGN: The trial consisted of 2h daily treatment 5 days per week for 6 weeks in 83 patients with knee OA. Patient evaluations were done at baseline and after 2 and 6 weeks of treatment. A follow-up evaluation was done 6 weeks after treatment. Activities of daily living (ADL), pain and stiffness were evaluated using the Western Ontario and McMaster Universities (WOMAC) questionnaire. RESULTS: Within group analysis revealed a significant improvement in ADL, stiffness and pain in the PEMF-treated group at all evaluations. In the control group there was no effect on ADL after 2 weeks and a weak significance was seen after 6 and 12 weeks. Significant effects were seen on pain at all evaluations and on stiffness after 6 and 12 weeks. Between group analysis did not reveal significant improvements over time. Analysis of ADL score for the PEMF-treated group revealed a significant correlation between less improvement and increasing age. Analysis of patients <65 years using between group analysis revealed a significant improvement for stiffness on treated knee after 2 weeks, but this effect was not observed for ADL and pain. CONCLUSIONS: Applying between group analysis we were unable to demonstrate a beneficial symptomatic effect of PEMF in the treatment of knee OA in all patients. However, in patients <65 years of age there is significant and beneficial effect of treatment related to stiffness.

Nitric oxide-induced signalling in rat lacrimal acinar cells.

The aim of the present study was to investigate the physiological role of nitric oxide (NO) in mediating secretory processes in rat lacrimal acinar cells. In addition, we wanted to determine whether the acinar cells possess endogenous nitric oxide synthase (NOS) activity by measuring NO production using the fluorescent NO indicator 4,5-diaminofluorescein (DAF-2). We initiated investigations by adding NO from an external source by means of the NO-donor, S-nitroso-N-acetyl-penicillamine (SNAP). Cellular concentrations of cyclic guanosine 5'-phosphate (cGMP) ([cGMP]) were measured by radioimmunoassay (RIA), and we found that SNAP induced a fast increase in the [cGMP], amounting to 350% of the [cGMP] in resting cells. Moreover, addition of SNAP and elevating [cGMP] in fura-2 loaded lacrimal acinar cells, resulted in a cGMP-dependent protein kinase-mediated release of Ca2+ from intracellular stores, leading to a rise in the intracellular free Ca2+ concentration ([Ca2+]i). The Mn2+ quenching studies revealed that the Ca2+ release was not accompanied by Ca2+ influx. Finally, we demonstrate that lacrimal acinar cells possess endogenous NOS activity, which is activated by beta-adrenergic stimulation and not by a rise in [Ca2+]i alone. We show that in rat lacrimal acinar cells, NO and cGMP induce Ca2+ release from intracellular stores via G kinase activation. However, the changes in [Ca2+]i are relatively small, suggesting that this pathway plays a modulatory role in Ca2+ signalling, thus not by itself causing fast transient increases in [Ca2+]i. In addition, we suggest that endogenously produced NO activated by beta-adrenergic receptor stimulation, plays an important role in signalling to the surrounding tissue.

Reduction in the rate of inositol 1,4,5-trisphosphate synthesis in rat parotid acini by lithium.

Stimulation of muscarinic cholinergic receptors on rat parotid acinar cells causes a rapid production of inositol phosphates, with the key metabolic event being the breakdown of phosphatidylinositol 4,5-bisphosphate into inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and diacylglycerol. Here a high-performance liquid chromatographic technique was used to measure the effects of intracellular lithium ions on the amount of various inositol phosphates produced. When acini were stimulated maximally with acetylcholine (ACh), the sum of all inositol phosphates produced followed a monoexponential function with a production rate constant for Ins(1,4,5)P3 of 0.07 +/- 0.01 solidus/sec. The presence of 23 mM LiCl intracellularly reduced the production rate constant of Ins(1,4,5)P3 induced by ACh to 0.03 +/- 0.01 solidus/sec, resulting in a decrease in the Ins(1,4,5)P3 production as well as in the magnitude of the rise in the intracellular free Ca2+ concentration. The lithium ion (Li+) did not affect the rate of conversion of Ins(1,4,5)P3 to either inositol 1,4-bisphosphate or inositol 1,3,4,5-tetrakisphosphate. The rate of the inositol phosphate production after the addition of the Ca2+ ionophore ionomycin was unaffected by intracellular Li+ (23 mM), which implies that the action of Li+ was at the muscarinic cholinergic receptor, on G-protein or on the interactions between G-proteins and phospholipase C. Thus, in the early events after receptor stimulation with ACh, Li+ causes a reduction in the concentration of the cellular messengers Ins(1,4,5)P3 and Ca2+.

Nitric oxide and cGMP activate Ca2+-release processes in rat parotid acinar cells.

We characterized the enzymic properties of ADP-ribosyl cyclase in rat parotid acinar cells by using a fluorescence technique. ADP-ribosyl cyclase is capable of synthesizing the Ca2+ -mobilizing nucleotide cADP-ribose (cADPR) from NAD(+) and has previously been shown to be regulated by cGMP via a cGMP-dependent protein kinase (G kinase). We therefore investigated whether NO/cGMP-activated pathways are present in rat parotid acinar cells and whether NO/cGMP signalling exerts control over cellular Ca2+ signalling processes. Our results showed that stimulation of acinar cells with adrenaline, isoproterenol, substance P and NO resulted in a rise in the [cGMP]. In addition, NO induced a release of Ca2+ from intracellular ryanodine-sensitive stores via a cGMP/G-kinase-mediated process. Thus our data reveal that a rise in [cGMP], caused by either neurotransmitter or NO activation, activates a G kinase, which in turn controls Ca2+ release from ryanodine-sensitive stores. Since parotid acinar cells possess ADP-ribosyl cyclase activity, we propose a model in which cADPR is the link between NO/cGMP signalling pathways and release of Ca2+ from ryanodine-sensitive stores.

Nitric oxide synthesis causes inositol phosphate production and Ca2+ release in rat parotid acinar cells.

The activity of nitric oxide synthase (NOS) in rat parotid acinar cells was measured using a newly synthesized fluorescent NO indicator DAF-2/DA. Our results show that NO production is most effectively stimulated by activation of the beta-adrenergic receptor, and to a minor extent by substance P (SP). NO activates the production of cGMP, an intracellular messenger that has been shown to release Ca2+ from ryanodine-sensitive intracellular stores. We found that cGMP is also able to release Ca2+ from ryanodine-insensitive intracellular stores. Our data show that a rise in the cGMP concentration induces inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] synthesis and Ca2+ release from intracellular stores.

Adrenoceptor-activated nitric oxide synthesis in salivary acinar cells.

We investigated the cellular regulation of nitric oxide synthase (NOS) activity in isolated acinar cells from rat parotid and human labial salivary glands, using the newly developed fluorescent nitric oxide (NO) indicator, DAF-2. We found that sympathetic stimulation with norepinephrine (NE) caused a strong increase in NO synthesis that was not seen after parasympathetic stimulation with acetylcholine. In rat parotid acinar cells, we furthermore investigated to which extent the NOS activity was dependent on the intracellular free Ca2+ concentration ([Ca2+]i) by simultaneously measuring NO synthesis and [Ca2+]i. It was found that a simple correlation between the rise in [Ca2+]i and the rate of NO production following NE stimulation does not exist, and studies in which [Ca2+]i was elevated by means of the Ca2+ ionophore, ionomycin, further established that even a very large rise in [Ca2+]i did not cause significant NO synthesis. We furthermore found that activating adrenoceptors with NE causes synthesis of cGMP by activating a guanylyl cyclase, and that an enhanced [cGMP] evoked by use of caged cGMP causes Ca2+ release from internal stores. Thus, upon sympathetic stimulation, salivary gland acini synthesize NO that, in addition to playing a role in controlling intracellular [Ca2+]i, also might play a role in retrograde signaling processes to the surrounding tissue.

Activation of P2z purinoceptors diminishes the muscarinic cholinergic-induced release of inositol 1,4,5-trisphosphate and stored calcium in rat parotid acini. ATP as a co-transmitter in the stimulus-secretion coupling.

The effect of extracellular ATP on the intracellular free Ca2+ concentration ([Ca2+]i) and inositol phosphate production following stimulation with the muscarinic cholinergic agonist acetylcholine (ACh) was investigated in isolated rat parotid acinar cells. Stimulation of rat parotid acinar cells with ATP4- results in a rise in [Ca2+]i that is due to influx of extracellular Ca2+ and mobilization of Ca2+ from intracellular stores. Stimulation with purinergic agonists revealed that both influx as well as Ca2+ release from intracellular stores was mediated through activation of P2z receptors. The Ca2+ mobilization from intracellular stores was due to production of Ins(1,4,5)P3 and was inhibited by U73122, an inhibitor of phospholipase C-coupled processes. Under Ca(2+)-free conditions ATP4- caused a dose-dependent inhibition (IC50 = 8 microM) of the ACh-evoked Ca2+ release. The inhibitory effect of ATP4- is due to activation of the P2z purinoceptors, which results in a strong reduction in the ACh-induced inositol phosphate production. Prestimulation with 100 microM ATP4- reduced the amount of Ins(1,4,5)P3 formed after maximal ACh stimulation by 91%. In conclusion, the inhibitory effect of ATP4- on the ACh-mediated response is due to interactions of the activated P2z receptor with the phospholipase C-coupled processes underlying the muscarinic cholinergic response.

Ca2+ signalling in exocrine acinar cells: the diffusional properties of cellular inositol 1,4,5-trisphosphate and its role in the release of Ca2+.

The correlation between acetylcholine induced changes in the intracellular free, Ca2+ concentration ([Ca2+]i), and the inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) content in isolated acini from the rat parotid and lacrimal glands was investigated. Applying digital image processing on Fura-2 loaded acini, we observed that Ca2+ increases either simultaneously throughout the acinar configurations or that occasionally, the rise near the lumen can precede the rise near the basal part by 50-100 ms. Measurements on cell suspensions revealed a correlation between changes in [Ca2+]i and changes in the cellular Ins(1,4,5)P3 content, and it is concluded that in the individual cells Ins(1,4,5)P3 is released to the cytosol within the first second after stimulation. Applying a diffusion coefficient for cytoplasmic Ins(1,4,5)P3 of 2.83 x 10(-6) cm2/s (Allbritton et al., 1992, Science, 258, 1812-1815), we have calculated the concentration profile for this messenger in a sphere with a radius of 10 microns where Ins(1,4,5)P3 is released in the center following a monoexponential function with a rate constant of 4 s-1. Assuming that Ins(1,4,5)P3 concentrations of 1 or 5% of the maximum value is able to release Ca2+, we calculated that Ca2+ waves can appear at a rate of 100 or 40 microns/s. The present data are consistent with Ins(1,4,5)P3 being a cellular messenger, that by diffusion, initiates the Ca2+ release from the cellular pools within the first fraction of a second.