Cytoskeleton-dependent activation of the inducible nitric oxide synthase in cultured aortic smooth muscle cells.

1. Vascular endothelial and smooth muscle cells generate nitric oxide (NO) via different nitric oxide synthase (NOS) isozymes. Activation of the endothelial constitutive NOS (ecNOS) contributes to the maintenance of cardiovascular homeostasis, whereas expression of the endotoxin- and cytokine-inducible pathway (iNOS) within the vascular smooth muscle is thought to be responsible for the cardiovascular collapse which occurs during septic shock and antitumour therapy with cytokines. Since the cytoskeleton is involved in the activation of certain genes and in some effects of endotoxin in macrophages, we investigated the role of microtubules and microfilaments in the activation of the NO pathway in cultured vascular cells. 2. Depolymerization of microtubules by either nocodazole or colchicine prevented lipopolysaccharide (LPS)- and interleukin-1 beta-induction of NO-dependent cyclic GMP accumulation. Steady state levels of iNOS mRNA, assessed by Northern blot and RT-PCR, and iNOS protein, assessed by Western blotting, were also decreased by either colchicine or nocodazole treatment. 3. Taxol enhanced microtubule polymerization alone, and prevented microtubule depolymerization elicited by nocodazole and colchicine. Associated with its effect on microtubule assembly, taxol prevented the inhibitory effects of nocodazole and colchicine on cyclic GMP accumulation and iNOS mRNA levels. 4. Disruption of microfilaments by cytochalasins had no inhibitory effect on the activation of the inducible NO pathway. 5. In contrast to cytokine-stimulated smooth muscle cells, modulation of either microtubule or microfilament assembly did not affect the constitutive NO pathway in endothelial cells, as endothelial cell- and NO-dependent cyclic GMP accumulation in endothelial-smooth muscle co-cultures remained unchanged. 6. Our findings demonstrate that microtubules play a prominent role in the activation of the inducible NO pathway in response to inflammatory mediators in smooth muscle cells but not of the constitutive synthesis of NO in endothelial cells.     

A comparison of the in vitro and in vivo activities of IgG and F(ab')2 fragments of a mixture of three monoclonal anti-Her-2 antibodies.

PURPOSE: We have demonstrated previously that a mixture of three anti-Her-2 monoclonal antibodies (MAbs) that bind to different epitopes on the extracellular domain of Her-2 expressed on a human breast cancer cell line has more potent antitumor activity than the individual MAbs both in vitro and in xenografted severe combined immunodeficient mice. Because the activity of Herceptin is Fc dependent, we determined whether this would also be the case when a mixture of these three anti-Her-2 MAbs was used. EXPERIMENTAL DESIGN: IgG and highly purified F(ab')(2) fragments of the anti-Her-2 MAbs and Herceptin were prepared and evaluated for their ability to induce cell death, inhibit vascular endothelial growth factor secretion, and mediate antibody-dependent cellular cytotoxicity and complement-mediated cytotoxicity in vitro. They were also compared for their abilities to induce regression of large BT474 tumors in severe combined immunodeficient mice. RESULTS: All of the F(ab')(2) fragments were >95% pure and, as expected, did not mediate antibody-dependent cellular cytotoxicity or complement-dependent cytotoxicity in vitro. The in vitro antiproliferative and proapoptotic effects of the IgGs and F(ab')(2) fragments were similar. In contrast, the IgGs had significant antitumor activity in vivo, whereas their F(ab')(2) fragments were only marginally effective even at 5-fold higher doses to offset their shorter half-lives. CONCLUSIONS: These results confirm the importance of the Fc portion of Herceptin for optimal in vivo activity and demonstrate that even a mixture of three anti-Her-2 MAbs that are highly effective at inducing cell death in vitro requires Fc-mediated effector function for optimal in vivo activity.     

Pharmacological tools for hydrogen sulphide research: a brief,introductory guide for beginners

The purpose of this brief review is to help researchers in their initial approach to the H₂S field and to provide answers for the most frequently posed questions by newcomers to the topic related to H₂S donors and inhibitors of H₂S synthesis, as well as methods to measure H₂S production. Here the reader will find a practical guide that provides fast and to the point information on how to (i) deliver H₂S to cells; (ii) modulate its endogenous production; and (iii) measure its levels in fluids, cells and tissues in order to gain an understanding of its role in health and disease.

Linked Articles

This article is part of a themed section on Pharmacology of the Gasotransmitters. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-6     

PDE5 inhibition against acute renal ischemia reperfusion injury in rats: does vardenafil offer protection?

Purpose

To evaluate the effect of vardenafil on renal function after renal ischemia–reperfusion (IR) injury (IRI) in a rat model.

Materials and methods

Seventy-one Wistar rats were divided into 7 groups including (1) a vehicle-treated group, (2) a vehicle pretreated-IR group, (3–6) vardenafil pretreated-IR groups in doses of 0.02, 0.2, 2 and 20 μg/kg, respectively, (7) a group of IR followed by treatment with 2 μg/kg of vardenafil. Vardenafil or vehicle solution was administered one hour before unilateral nephrectomy and the induction of 45 min of ischemia on the contralateral kidney by clamping of renal pedicle. Four hours of reperfusion were allowed after renal ischemia. Studied parameters were serum creatinine, fractional excretion of sodium (FENa), and histological evaluation of renal specimens. In addition, renal tissue cGMP levels, ERK1/2 phosphorylation as well as renal function by renal scintigraphy were also evaluated.

Results

Administration of vardenafil before the induction of ischemia resulted in a significant reduction in creatinine and FENa levels as well as in less histological lesions observed in treated kidneys in comparison with the vehicle-treated group. The underlying mechanism of cytoprotection was cGMP depended and involved the phosphorylation of ERK proteins. Renal scintigraphy confirmed that PDE5 inhibition attenuates renal IRI.

Conclusions

Vardenafil attenuates renal IRI. Based on similar results from relevant studies on other PDE-5 inhibitors in renal and cardiac IRI, it can be assumed that all PDE-5 inhibitors share a common mechanism of cytoprotection.     

Unexpected toxicity after low-dose docetaxel treatment of a cancer patient with clinically latent HCV-positive hepatic cirrhosis

We report a case with unexpected toxicity after low-dose docetaxel chemotherapy. The patient had a history of clinically latent HCV-positive hepatic cirrhosis when she presented with inoperable pulmonary adenocarcinoma. She was recruited in a protocol combining standard radiotherapy (RT) with docetaxel (30 mg/m2 week). On day 7, after the 1st docetaxel infusion, grade III neutropenia (980 neutrophils), grade II platelet toxicity (90,000/ml) and lymphopenia (486/ml) had developed. Chemotherapy and RT were interrupted and the neutrophil counts were partially restored (1400/ml), while the platelet counts were back to normal (140,000/ml) and the lymphocyte counts were further reduced (320/ml), on day 15. Bilateral leg oedema and hair loss appreared. On day 21, there was a full restoration of neutrophil counts (1890/ml), while there was persistent lymphocytopenia (300/ml). Alopecia grade III was now evident. Dysphagia grade II complicated with fungal oropharyngeal infection appeared on day 24 (24 Gy of RT). One more dose of docetaxel of 30 mg/m2 was given on day 36 Grade II neutropenia (1050/ml) and grade III platelet toxicity (48,000/ml) were observed 14 days after the second docetaxel dose, while dysphagia grade II appeared once again. After a one-week delay, RT was continued to a total dose of 54 Gy. Liver function tests remained unchanged throughout the treatment. Post-RT CT-scan of the chest and upper abdomen showed complete response of the lung lesion. We suggest that, when docetaxel is chosen to treat cancer patients with HCV-positive hepatic cirrhosis, a starting dose schedule reduced by at least 50% should be considered before escalating to the standard dose.     

Feasibility of azithromycin prophylaxis during a pertussis outbreak among healthcare workers in a university hospital in Paris.

The objective of the present study was to evaluate the feasibility of azithromycin prophylaxis with respect to tolerability and compliance during a pertussis outbreak among healthcare workers in a university hospital ward. Compliance with the prophylaxis regimen was 89%; compliance was 75% from intent-to-treat perspective. The rate of adverse events was 33%. Female sex was associated with reporting of adverse events. Nonstudents and healthcare workers who reported adverse events were less compliant with the prophylaxis regimen.