Eric Seidlitz

Carbon monoxide formation in the ductus arteriosus in the lamb: implications for the regulation of muscle tone.

1 We have previously shown that carbon monoxide (CO) potently relaxes the lamb ductus arteriosus and have ascribed this response to inhibition of a cytochrome P450‐based mono‐oxygenase reaction controlling the formation of endothelin‐1 (ET‐1). In the present study, we have examined whether CO is formed naturally in the vessel. 2 The CO‐forming enzyme, haem oxygenase (HO), was identified in ductal tissue in its constitutive (HO‐2) and inducible (HO‐1) isoforms by Western immunoblotting and immunological staining procedures (both light and electron microscopy). HO‐1 was localized to endothelial and muscle cells, while HO‐2 was found only in muscle cells. Inside the muscle cells, HO‐1 and HO‐2 immunoreactivity was limited to the perinuclear region, and the Golgi apparatus in particular. However, upon exposure to endotoxin, HO‐1 became more abundant, and both HO isoforms migrated towards the outer region of the cytoplasm close to the sarcolemma. 3 CO was formed enzymatically from added substrate (hemin, 50 μm) in the 10,000 g supernatant of the ductus and its formation was inhibited by zinc protoporphyrin IX (ZnPP, 200 μM). 4 ZnPP (10 μM) had no effect on the tone of the ductus under normal conditions (2.5 to 95% O2), but it contracted the endotoxin‐treated ductus (at 2.5% O2). At the same concentration, ZnPP also tended to contract the hypoxic vessel (zero O2). 5 ZnPP (10 μm) curtailed the relaxant response of the oxygen (30%)/indomethacin (2.8 μM)‐contracted ductus to bradykinin (35 nM), while it left the sodium nitroprusside (35 nM) relaxation unchanged. 6 We conclude that CO is formed in the ductus and may exert a relaxing influence when its synthesis is upregulated by an appropriate stimulus.

Carbon monoxide-induced relaxation of the ductus arteriosus in the lamb: evidence against the prime role of guanylyl cyclase.

1 . We have previously found that carbon monoxide (CO) potently relaxes the lamb ductus arteriosus and have ascribed this response to inhibition of a cytochrome P450‐based mono‐oxygenase reaction which sustains contractile tone. Our proposal, however, has been questioned on the evidence of findings in other blood vessels implicating the guanylyl cyclase‐based relaxing mechanism as the target for CO. To investigate this issue further, we have carried out experiments in the isolated ductus from near‐term foetal lambs and have examined the effect of CO concomitantly on muscle tone and cyclic GMP content, both in the absence and presence of guanylyl cyclase inhibitors, or during exposure to monochromatic light at 450 nm. 2 . CO (65 μm) reversed completely, or nearly completely, the tone developed by the vessel in the presence of oxygen (30%) and indomethacin (2.8 μm). Cyclic GMP content tended to increase with the relaxation, but the change did not reach significance. Sodium nitroprusside (SNP), a NO donor, mimicked CO in relaxing the ductus. Contrary to CO, however, SNP caused a marked accumulation of cyclic GMP with levels being positively correlated with the relaxation. 3 . Methylene blue (10 μm) reduced marginally the CO relaxation, whilst LY‐83583 (10 μm) had an obvious, albeit variable, inhibitory effect. Basal cyclic GMP content was lower in tissues treated with either compound and rose upon exposure to CO. However, the levels attained were still within the range of values for tissues prior to any treatment. Furthermore, the elevation in cyclic GMP was not related to the magnitude of the CO relaxation. 4 . Illumination of the ductus with monochromatic light at 450 nm reversed the CO relaxation and any concomitant increase in cyclic GMP. In the absence of CO, light by itself had no effect. 5 . Ductal preparations with only muscle behaved as the intact preparations in reacting to CO, both in the absence and presence of guanylyl cyclase inhibitors, or during illumination. 6 . We conclude that the primary action of CO in the ductus arteriosus is not exerted on the guanylyl cyclase heme and that cyclic GMP may only have an accessory role in the relaxation to this agent. This finding reasserts the importance of a cytochrom P450‐based mono‐oxygenase reaction for generation of tone and as a target for CO in the ductus.

Re-expression of TSLC1 in a non-small-cell lung cancer cell line induces apoptosis and inhibits tumor growth

The TSLC1 tumor-suppressor gene is silenced in a number of human cancer tissues and cell lines, including lung, prostate, liver, stomach, pancreatic, and breast cancers. Expression of TSLC1 in a non-small-cell lung cancer (NSCLC) cell line A549 suppresses tumorigenicity in nude mice. However, the molecular mechanism of TSLC1 action is not yet elucidated. In the present study, we show that the expression of TSLC1 from a recombinant adenovirus vector (Ad-TSLC1) inhibited cell proliferation and induced apoptosis in the NSCLC cell line A549. We also demonstrated that subcutaneous tumor growth in nude mice induced by A549 cells was suppressed to the extent of 70–80% by intratumoral injection of Ad-TSLC1. Re-expression of TSLC1 also resulted in activation of the apoptotic protease caspase-3, accompanied by the cleavage of its substrate poly (ADP-ribose) polymerase (PARP). The antiproliferative and pro-apoptotic activity of TSLC1 required the presence of the FERM-binding and PDZ-interacting motifs located in the cytoplasmic domain. Our results demonstrate the pro-apoptotic and oncosuppressive activity of TSLC1 protein, and suggest the potential of TSLC1 for gene therapy.

Cancer cells release glutamate via the cystine/glutamate antiporter

Although the amino acid glutamate is used as an intercellular signaling molecule for normal bone homeostasis, little is known regarding its possible role in the metabolic disruption characteristic of bone metastasis. We have previously shown in vitro that cancer cell lines relevant to bone metastasis release glutamate into the extracellular environment. This study demonstrates the expression of multiple glutamate transporters in cancer cell lines of non-central nervous system origin. Furthermore, we identify the molecular mechanism responsible for glutamate export and show that this system can be inhibited pharmacologically. By highlighting that glutamate secretion is a common biological feature of cancer cells, this study suggests that tumor-derived glutamate could interfere with glutamate-dependent intercellular signaling in normal bone. Pharmacological interference with cancer cell glutamate release may be a viable option for limiting host bone response to invading tumor cells in bone metastasis.

Cancer cell lines release glutamate into the extracellular environment

Bone is one of the most frequent sites for metastasis of breast and prostate cancers. Bone metastases are associated with pathologic changes in bone turnover and severe pain. The mechanisms that trigger these effects are not well understood, but it is postulated that tumour cells release factors which interfere with signalling processes critical to bone homeostasis. We have identified that several cancer cell lines known to cause bone disruption in animal models of bone metastasis appear to secrete glutamate into their extracellular environment in vitro. Although these cells also express specific glutamate receptors, the implications of this potentially disruptive chemical signal are discussed in relation to normal glutamate-dependent communication processes in bone and a possible mechanistic connection is made between tumour cell glutamate release and the development of pathological changes in bone turnover.

Effect of zoledronic acid on the doxycycline-induced decrease in tumour burden in a bone metastasis model of human breast cancer

Bone is one of the most frequent sites for metastasis in breast cancer patients often resulting in significant clinical morbidity and mortality. Bisphosphonates are currently the standard of care for breast cancer patients with bone metastasis. We have shown previously that doxycycline, a member of the tetracycline family of antibiotics, reduces total tumour burden in an experimental bone metastasis mouse model of human breast cancer. In this study, we combined doxycycline treatment together with zoledronic acid, the most potent bisphosphonate. Drug administration started 3 days before the injection of the MDA-MB-231 cells. When mice were administered zoledronic acid alone, the total tumour burden decreased by 43% compared to placebo treatment. Administration of a combination of zoledronic acid and doxycycline resulted in a 74% decrease in total tumour burden compared to untreated mice. In doxycycline- and zoledronate-treated mice bone formation was significantly enhanced as determined by increased numbers of osteoblasts, osteoid surface and volume, whereas a decrease in bone resorption was also observed. Doxycycline greatly reduced tumour burden and could also compensate for the increased bone resorption. The addition of zoledronate to the regimen further decreased tumour burden, caused an extensive decrease in bone-associated soft tissue tumour burden (93%), and sustained the bone volume, which could result in a smaller fracture risk. Treatment with zoledronic acid in combination with doxycycline may be very beneficial for breast cancer patients at risk for osteolytic bone metastasis.

Function of cyclo‐oxygenase‐1 and cyclo‐oxygenase‐2 in the ductus arteriosus from foetal lamb: differential development and change by oxygen and endotoxin

Prenatal patency of the ductus arteriosus is maintained mainly by prostaglandin(PG) E2. Here we have examined the relative importance of cyclo‐oxygenase‐1 (COX1) and cyclo‐oxygenase‐2 (COX2) for PGE2 formation in the foetal lamb ductus (0.65 gestation onwards). Using fluorescence microscopy and immunogold staining, COX1 appeared more abundant than COX2 in endothelial and smooth muscle cells, and this difference was greater before‐term. Inside muscle cells, COX1 and COX2 immunoreactivity was located primarily in the perinuclear region. Endotoxin, given to the lamb in utero (∼0.1 μg kg−1), caused COX2 upregulation, while an opposite effect with disappearance of the enzyme followed endotoxin treatment in vitro (100 ng ml−1). COX1 immunoreactivity remained virtually unchanged with either treatment; however, this isoform as well as any induced COX2 migrated towards the outer cytoplasm. The COX2 inhibitor L‐745,337 (1 – 10 μM) contracted the isolated ductus at term, the response being almost as high as that to indomethacin (dual COX1/COX2 inhibitor) over the same dose‐range. Conversely, L‐745,337 was relatively less effective in the premature. Pretreatment of the premature in vivo with endotoxin enhanced the contraction of the ductus to L‐745,337, while in vitro endotoxin had a variable effect. The premature ductus exhibited a stronger contraction to L‐745,337 following exposure to oxygen. On the other hand, the oxygen contraction, which is modest before‐term, was enhanced by L‐745,337. We conclude that COX1 and COX2 develop unevenly in the ductus. While both enzymes contribute to PGE2 formation at term, COX1 is the major isoform in the premature. COX2, however, may acquire greater importance before‐term following physiological and pathophysiological stimuli.

Inhibition of the contraction of the ductus arteriosus to oxygen by 1-aminobenzotriazole, a mechanism-based inactivator of cytochrome P450.

1 We have proposed that contractile tension of the ductus arteriosus is sustained by a cytochrome P450‐linked mechanism acting as a limiting step in the synthesis of endothelin‐1 (ET‐1). In the present study, we have used the isolated ductus from near‐term foetal lambs and guinea‐pigs to investigate the effect on both muscle tone and ET‐1 formation of 1‐aminobenzotriazole (ABT), a suicide substrate for mono‐oxygenase reactions. 2 ABT relaxed the lamb ductus at rest (2.5% O2) and during the oxygen contraction (15 to 95% O2). The effect was seen at 40 μ, and at 0.8 mM active tone was almost completely abolished. ABT (1 mM) also reversed the oxygen contraction in the guinea‐pig ductus. 3 In the lamb ductus, the ABT response was not affected by removal of the endothelium or by treatment with 2.8 μ indomethacin (at 2.5% O2) and the ensuing contraction. 4 At both low and high concentration, ABT relaxed marginally, or not at all, the potassium‐contracted (55 mM) ductus from either species. 5 ET‐1 release from either the intact or endothelium‐denuded lamb ductus tended to decrease in the presence of ABT (1 mM), whilst during the same treatment cyclic GMP content of the tissue remained unchanged. 6 We conclude that ABT relaxation is due to suppression of a contractile mechanism and not to activation of prostaglandin‐ and NO‐mediated relaxing mechanisms. This contractile mechanism has a cytochrome P450‐based mono‐oxygenase reaction as a key component.

Post-activation potentiation and depression in the neocortex of the rat: II. Chronic preparations

Although long-term potentiation (LTP) has been demonstrated in a number of subcortical sites in chronic preparations, there have been no demonstrations of LTP in the neocortex of chronic preparations. Even neocortical slice and acute preparations often require a drug-induced suppression of inhibition before LTP effects can be reliably induced. We have attempted to induce LTP in neocortical sites in 7 different experiments using chronically prepared adult rats. We were unable to obtain any evidence, even a trend, for the induction of LTP. The following manipulations were tested: (1) standard stimulation train parameters that have been shown to be highly effective in subcortical and hippocampal sites; (2) a 10-fold increase in the intra-train pulse durations; (3) variations in train pulse frequency (1 Hz to 300 Hz) and train duration (100 ms to 15 min); (4) co-activation of multiple inputs by stimulation of combinations of cortical sites or cortical and thalamic sites; (5) reduction of inhibition by administration of picrotoxin; 5) Housing of animals in an enriched environment; (6) utilization of the neocortical stimulation trains as a cue in a learning task; (7) application of pilocarpine to co-activate cholinergic systems. Although none of these manipulations produced LTP, the application of pilocarpine did facilitate the induction of a long-lasting depression effect. These findings contrast with the results obtained from anesthetized rats and from studies using brain slices, where LTP can be reliably induced. These results are discussed in light of other recent findings with respect to LTP and LTD effects.

Inhibition of breast cancer-cell glutamate release with sulfasalazine limits cancer-induced bone pain.

Summary Inhibition of glutamate release via the system xC− cystine/glutamate antiporter with sulfasalazine reduces metastatic breast cancer‐induced bone pain in a novel animal behavioural model. ABSTRACT Cancer in bone is frequently a result of metastases from distant sites, particularly from the breast, lung, and prostate. Pain is a common and often severe pathological feature of cancers in bone, and is a significant impediment to the maintenance of quality of life of patients living with bone metastases. Cancer cell lines have been demonstrated to release significant amounts of the neurotransmitter and cell‐signalling molecule l‐glutamate via the system xC− cystine/glutamate antiporter. We have developed a novel mouse model of breast cancer bone metastases to investigate the impact of inhibiting cancer cell glutamate transporters on nociceptive behaviour. Immunodeficient mice were inoculated intrafemorally with the human breast adenocarcinoma cell line MDA‐MB‐231, then treated 14 days later via mini‐osmotic pumps inserted intraperitoneally with sulfasalazine, (S)‐4‐carboxyphenylglycine, or vehicle. Both sulfasalazine and (S)‐4‐carboxyphenylglycine attenuated in vitro cancer cell glutamate release in a dose‐dependent manner via the system xC− transporter. Animals treated with sulfasalazine displayed reduced nociceptive behaviours and an extended time until the onset of behavioural evidence of pain. Animals treated with a lower dose of (S)‐4‐carboxyphenylglycine did not display this reduction in nociceptive behaviour. These results suggest that a reduction in glutamate secretion from cancers in bone with the system xC− inhibitor sulfasalazine may provide some benefit for treating the often severe and intractable pain associated with bone metastases.