Effect of PSC 833, verapamil and amiodarone on adriamycin toxicity in cultured rat cardiomyocytes.

Primary cultures of heart myocytes from neonatal rats were used as an in vitro cardiac cell system to study the effects of the p-170kDa glycoprotein (Pgp) blockers PSC 833 [(3'-keto-Bmt1)-(Val2)-cyclosporine], verapamil and amiodarone on adriamycin cardiotoxicity. Immunostaining revealed the presence of Pgp in the cardiomyocytes. Adriamycin induced a concentration-dependent increase in creatine kinase (CK) leakage, a parameter indicating cell death. None of the Pgp blockers was toxic up to 10 microM, but amiodarone markedly increased CK leakage at 25 microM. 1 microM of the Pgp blockers did not increase adriamycin induced CK leakage, whereas 10 microM of the Pgp blockers significantly augmented adriamycin-induced CK leakage. In parallel, cytoplasmic vacuolization and plasma membrane disruptions were observed. Frequency of contraction of cardiomyocytes, as determined by digital image analysis, was concentration-dependently decreased by adriamycin. 1 microM PSC 833 had no additional effect on contractility, only 10 microM PSC 833 enhanced the impairment of contractility induced by adriamycin. Amiodarone and verapamil alone and in combination with adriamycin already at concentrations of 1 microM completely blocked contractility of cardiomyocytes. The results suggest that the increased toxicity of adriamycin in the presence of amiodarone, verapamil and PSC 833 is mediated by an effective blockage of the Pgp efflux pump. The results further indicate that the combination of adriamycin and PSC 833 might be better tolerated with regard to cardiac side-effects, than the combination of adriamycin and verapamil or amiodarone.

The antineoplastic drug vinorelbine activates non-immunological histamine release from rat mast cells.

OBJECTIVE AND DESIGN: We explore the mechanism of the antineoplastic drug vinorelbine activation in its rat mast cell exocytosis. MATERIALS: The study was carried out on mast cells obtained from Sprague-Dawley rats. TREATMENT: Vinorelbine (5-100 micrograms/mL), cholera toxin (200 ng/mL), pertussis toxin (100 ng/mL), benzalkonium chloride (10 micrograms/mL), compound 48/80 (1 microgram/mL), okadaic acid (1 microM), 12-tetradecanoate-acetate (50 ng/ml), perphenazine (1 microgram/ml), theophylline (10 mM), IBMX (1 mM), rolipram (15 microM). METHODS: Histamine release was measured fluorimetrically. RESULTS: The drugs that modify G-protein activity, cholera toxin, pertussis toxin or benzalkonium chloride, do not modify the response profile. The exocytosis elicited by compound 48/80 is decreased by Gs or Gi modulation, which suggests that G proteins are not involved in vinorelbine stimulated secretion. The phosphatase inhibitor okadaic acid shows no effect on vinorelbine-stimulated release, nor on the activation or inhibition of protein kinase C with phorbol 12-tetradecanoate-acetate or perphenazine. The unspecific phosphodiesterase inhibitors theophylline and IBMX inhibited histamine release, but not the phosphodiesterase IV inhibitor rolipram. CONCLUSIONS: The overall results show that vinorelbine activates histamine release through a rather selective mechanism that may be mediated by certain phosphodiesterase-dependent transduction pathways.

Mitoxantrone induces nonimmunological histamine release from rat mast cells.

The antineoplastic drug mitoxantrone (MTX) elicits a fast noncytotoxic and nonimmunological histamine release from peritoneal and pleural rat mast cells. The non specific phosphodiesterase inhibitor isobuthyl-methylxantine (1 mM) decreases the potency of MTX. Theophylline (10 mM) decreases both the potency and the efficacy of MTX-induced histamine secretion. The protein kinase C (PKC) activator, tetradecanoyl-phorbol-13-acetate (50 ng/mL), enhances the effect of MTX, whereas the non specific PKC inhibitor trifluoperazine (10 microM) exerts no effect. Histamine release was also unaffected by substances acting on G-proteins, namely pertussis toxin (200 ng/mL), cholera toxin (300 mg/mL) and benzalkonium chloride (10 micrograms/ mL). The inhibition of protein phosphatases 1 and 2A by okadaic acid (1 microM) does not modify the response. The results indicate that mitoxantrone elicits the exocytosis in mast cells by a mechanism similar to the parent compound adriamycine, but different to the polyamine compound 48/80.

Study of the activation mechanism of human GRF(1-29)NH2 on rat mast cell histamine release.

Human growth releasing factor (GRF) (1-29)NH2 releases histamine from pleural and peritoneal rat mast cells by a non cytotoxic and non immunological mechanism. Pretreatment of cells with pertussis toxin markedly inhibits the secretion, suggesting a possible function of a Gi-protein in the activation pathway. In order to determine the role of cAMP on GRF mediated secretion, mast cells were preincubated with isobutylmethylxanthine (IBMX) or cholera toxin, since both drugs greatly and enhance cAMP levels. IBMX inhibits mediator secretion while, in contrast, cholera toxin is ineffective to modify histamine release. The PKC activator TPA amplifies the response of mast cells to human GRF, shifting the dose-response curve to the left. The pretreatment of mast cells with the phosphatase inhibitor okadaic acid exerts no effect on the dose-response function curve to GRF. The response to human GRF does not depend on extracellular calcium, but there is a good correlation between the percent of histamine released and 45calcium uptake. The kinetic of calcium uptake is fast, maximum uptake being reached in 30 seconds.

Study of the activation mechanism of adriamycin on rat mast cells.

Adriamycin (ADR) induces nonimmunological and noncytotoxic histamine release from peritoneal and pleural rat mast cells. This secretion is unaffected by the pretreatment with pertussis toxin, cholera toxin and benzalkonium chloride. Histamine release induced by compound 48/80, was markedly inhibited by pertussis toxin, cholera toxin, benzalkonium chloride and neuraminidase. The ADR dose-response curve is significantly shifted to the right when cells were preincubated with the unspecific phosphodiesterase inhibitor IBMX. The activation of protein kinase C (PKC) with the phorbol esther TPA increases the response to ADR, while PKC inhibition with trifluoperazine decreases histamine release. The pretreatment of mast cells with okadaic acid did not modify the response to ADR. These results suggest that ADR elicits histamine release with a mechanism notably different from compound 48/80.

Effect of okadaic acid on immunologic and non-immunologic histamine release in rat mast cells.

We have studied the effect of the protein phosphatase (PP) inhibitor, okadaic acid (OKA) on histamine release elicited by immunologic and non-immunologic stimuli in peritoneal and pleural rat mast cells. When cells were stimulated with antigen (egg albumin), OKA strongly inhibited histamine release. This finding suggests that PP1 and PP2A substrates mediate immunoglobulin E-(IgE) dependent secretion in mast cells. In contrast, after non-immunologic activation of mast cells with different drugs, such as the neuropeptide growth hormone releasing factor (GRF) and the cytostatic agents Adriamycin, navelbine and mitoxantrone, there is no effect of OKA on histamine secretion. These results indicate that IgE-dependent secretion is mediated by substrates of PP1 and PP2A, whereas following non-immunological stimuli, they activate pathways that lead to protein phosphorylation; these proteins are not substrates of PP1 and PP2A.

Histamine release on rat pleural and peritoneal mast cells elicited by human GRF(1-29)NH2.

We have studied the release of histamine elicited by human GRF(1-29)NH2 on rat peritoneal and pleural mast cells. The secretion is temperature dependent, the release being optimal at 37 degrees C, and it can be inhibited with antimycin A. The activation of pleural and peritoneal mast cells is similar, but there is a slight inhibition for both populations after purification with Percoll.