Cristina Goso

Cardioprotective effects of zofenopril, a new angiotensin-converting enzyme inhibitor, on doxorubicin-induced cardiotoxicity in the rat.

We have studied the effect of zofenopril, a new angiotensin-converting enzyme inhibitor in preventing cardiac injury induced by chronic doxorubicin treatment in rats. Cardiac function was assessed by measuring changes in electrocardiogram (ECG) tracings, haemodynamics and cardiac responses in vivo to isoprenaline, 4 weeks after suspension of doxorubicin treatment, in vehicle-treated rats and in animals receiving zofenopril (15 mg/kg/os/day) alone, doxorubicin (1.5 mg/kg i.v. once a week for 5 weeks) or zofenopril+doxorubicin treatment. Doxorubicin induced a significant lengthening of the QalphaT interval, which was completely prevented by zofenopril treatment. The cardiac positive inotropic effect induced by i.v. isoprenaline was selectively depressed by doxorubicin (no changes in chronotropic responses) and this adverse effect of doxorubicin was also prevented in zofenopril+doxorubicin pretreated rats. Doxorubicin induced a significant increase in relative heart weight, which was likewise prevented in zofenopril+doxorubicin treated rats. In separate experiments, zofenopril did not interfere with the antitumor activity of doxorubicin (inhibition of tumor growth in nude mice xenografted with A2780 human tumor line). In conclusion, the oral administration of zofenopril is able to significantly ameliorate, up to 4 weeks after the end of doxorubicin administration, doxorubicin-induced cardiotoxicity without affecting the antitumor activity of this anthracycline.

Topical capsaicin administration protects against trinitrobenzene sulfonic acid-induced colitis in the rat

We used the [3H]resiniferatoxin binding assay to demonstrate for the first time the existence of vanilloid receptors in the rat colon and to explore their expression during trinitrobenzene sulfonic acid-induced colitis. Membranes obtained from control colon bound [3H]resiniferatoxin with an affinity of 3 nM; the receptor density was 450 fmol/mg protein or 9 fmol/mg wet weight. Capsaicin and capsazepine, a competitive antagonist of capsaicin, inhibited specific resiniferatoxin binding with Ki values of 3 microM and 0.1 microM, respectively. Trinitrobenzene sulfonic acid induced a very rapid ulceration in the colon: 1 h after treatment 90% of the colon showed ulcerative damage. Coadministration of 640 microM capsaicin diminished the ulcerative effect of trinitrobenzene sulfonic acid to 64% when examined 1 h after trinitrobenzene sulfonic acid challenge; however, this protective action was lost 23 h later. Colon samples obtained 4 h, 24 h, and 1 week after trinitrobenzene sulfonic acid challenge bound resiniferatoxin, capsaicin, and capsazepine with affinities similar to those of control samples. The receptor density remained at an essentially constant level when expressed in fmol/mg protein but, in keeping with the increased wet weights, showed a reduction when expressed in fmol/mg wet weight. We conclude that acute capsaicin administration protects against the ulcerative action of trinitrobenzene sulfonic acid, most likely via the release of protective neuropeptides from capsaicin-sensitive nerve endings. The loss of this protective action is presumably due to a depletion of the protective neuropeptides rather than to a loss of vanilloid (capsaicin) receptors.

Characterization of a peripheral vanilloid (capsaicin) receptor in the urinary bladder of the rat

Specific binding of [3H]resiniferatoxin (RTX) is thought to represent the vanilloid (capsaicin) receptor. In the present study, we have used this binding assay to identify for the first time a vanilloid receptor in the periphery and to compare it to central vanilloid receptors present in dorsal root ganglia (DRG) as well as in spinal cord of the rat. Rat urinary bladder membranes bound [3H]RTX with a Kd of 30 +/- 4 pM and a Bmax of 65 +/- 14 fmol/mg protein; the corresponding values were 19 +/- 3 pM and 104 +/- 14 fmol/mg protein in DRG, and 16 +/- 3 pM and 50 +/- 9 fmol/mg protein in spinal cord. Capsaicin inhibited [3H]RTX binding to membranes from urinary bladder, spinal cord, and DRG with similar potency (Ki values were 0.5 +/- 0.1 microM, 0.3 +/- 0.1, and 0.6 +/- 0.1 microM, respectively). Interestingly, [3H]RTX bound to urinary bladder in a non-cooperative fashion in contrast with the apparent positive cooperativity of [3H]RTX binding in both DRG and spinal cord (cooperativity index = 1.8 and 1.7, respectively). This finding suggests heterogeneity in the properties of the vanilloid receptors in the rat.

Chronic cardiotoxicity of anticancer anthracyclines in the rat: role of secondary metabolites and reduced toxicity by a novel anthracycline with impaired metabolite formation and reactivity

The anticancer anthracycline doxorubicin (DOX) causes cardiomyopathy upon chronic administration. There is controversy about whether DOX acts directly or after conversion to its secondary alcohol metabolite DOXol. Here, the role of secondary alcohol metabolites was evaluated by treating rats with cumulative doses of DOX or analogues – like epirubicin (EPI) and the novel disaccharide anthracycline MEN 10755 – which were previously shown to form less alcohol metabolites than DOX when assessed in vitro. DOX induced electrocardiographic and haemodynamic alterations, like elongation of QαT or SαT intervals and suppression of isoprenaline‐induced dP/dt increases, which developed in a time‐dependent manner and were accompanied by cardiomegaly, histologic lesions and mortality. EPI caused less progressive or severe effects, whereas MEN 10755 caused essentially no effect. DOX and EPI exhibited comparable levels of cardiac uptake, but EPI formed ∼60% lower amounts of its alcohol metabolite EPIol at 4 and 13 weeks after treatment suspension (P<0.001 vs DOX). MEN 10755 exhibited the lowest levels of cardiac uptake; hence, it converted to its alcohol metabolite MEN 10755ol ∼40% less efficiently than did EPI to EPIol at either 4 or 13 weeks. Cardiotoxicity did not correlate with myocardial levels of DOX or EPI or MEN 10755, but correlated with those of DOXol or EPIol or MEN 10755ol (P=0.008, 0.029 and 0.017, respectively). DOX and EPI inactivated cytoplasmic aconitase, an enzyme containing an Fe–S cluster liable to disassembly induced by anthracycline secondary alcohol metabolites. DOX caused greater inactivation of aconitase than EPI, a finding consistent with the higher formation of DOXol vs EPIol. MEN 10755 did not inactivate aconitase, which was because of both reduced formation and impaired reactivity of MEN 10755ol toward the Fe–S cluster. Aconitase inactivation correlated (P<0.01) with the different levels of cardiotoxicity induced by DOX or EPI or MEN 10755. These results show that (i) secondary alcohol metabolites are important determinants of anthracycline‐induced cardiotoxicity, and (ii) MEN 10755 is less cardiotoxic than DOX or EPI, a behaviour attributable to impaired formation and reactivity of its alcohol metabolite.

Role of NK-1 and NK-2 tachykinin receptor antagonism on the growth of human breast carcinoma cell line MDA-MB-231.

We demonstrate that neurokinin A (NKA) and substance P (SP) play a role in the proliferation of the estrogen receptor-negative (ER–) cell line MDA-MB-231, a human breast carcinoma expressing both NK-1 and NK-2 receptors. In vitro experiments showed that the specific receptor antagonists MEN 11467 (NK-1) and nepadutant (MEN 11420; NK-2) inhibited tumor cell proliferation, and blocked the stimulatory effect of SP and NKA. Anti-tumoral activity of NK-1 and NK-2 receptor antagonists was demonstrated in nude mice, measuring growth inhibition of MDA-MB-231 tumor cells xenografted s.c. and by using the hollow-fiber assay. In both systems a significant inhibition was found when compounds were administered at 5 mg/kg i.v. every day for 2 weeks. Results obtained from both these models suggest that the in vivo activity of NK-1 and NK-2 antagonists may be a result of a cytostatic effect rather than a cytotoxic effect. Our results suggest that the control of breast carcinoma (ER–) growth by tachykinin receptor antagonists may become a new form of targeted therapy for these human tumors.

Vanilloid receptors in the urinary bladder: regional distribution, localization on sensory nerves, and species-related differences

SummaryUsing selective surgical ablations we have investigated the localization of vanilloid receptors (specific [3H]resiniferatoxin binding sites) on terminals of the pelvic, hypogastric, and pudendal nerves in the rat urinary bladder. Pelvic and hypogastric nerve resections resulted in 90%6 and 25% loss of specific [3H]resiniferatoxin (RTX) binding sites, respectively, whilst pudendic nerve resection had no measurable effect on the binding. In control animals, the density of vanilloid receptors was 1.7-fold higher in the neck than in the dome of the urinary bladder; the Bmax values were 57±8 and 34±7 fmol/mg protein, respectively. The binding characteristics of the vanilloid receptor were similar in the urinary bladder of the rat and mouse: Kd values were 87±15 and 61±11 pM, Bmax values were 37±2 and 60±10 fmol/mg protein, respectively. In contrast to the findings for the rat and mouse, in the urinary bladder of the guinea pig and the hamster the low level of specific [3H]RTX binding prevented the detailed characterization of vanilloid receptors. Nonetheless, at a fixed (60 pM) concentration of [3H]RTX, specific binding both in the guinea pig and hamster urinary bladder was approximately 20% of that in the rat urinary bladder. In the urinary bladder of newborn rats, as in adults, a single class of specific [3H]RTX binding sites was found which bound RTX with an affinity of 110±20 pM and with a maximal binding capacity of 30±5 fmol/mg protein. We conclude that, in accord with the physiological findings, the majority of vanilloid receptors are located on terminals of the pelvic nerve in the rat urinary bladder with higher receptor density in the bladder neck as compared to the bladder dome. Whereas the comparably high density of vanilloid receptors in the rat and mouse urinary bladder and the low receptor density in the hamster are mirrored by the in vivo vanilloid-sensitivity of these species, the low level of vanilloid receptors in the urinary bladder of the guinea pig contrasts to the marked sensitivity of this species to capsaicin. We conclude that the level of vanilloid receptors is an important but not exclusive determinant of vanilloid-sensitivity.

Capsaicin-induced relaxation in the rat isolated external urethral sphincter: characterization of the vanilloid receptor and mediation by CGRP

1 The potential role of capsaicin‐sensitive nerves in the relaxation of the rat external urethral sphincter (REUS) was evaluated by demonstrating the existence of specific vanilloid (capsaicin) receptors and by investigating the sensory neurotransmitter(s) putatively involved in this relaxation. 2 Capsaicin (1 μm) relaxed REUS strips precontracted with noradrenaline (NA) (0.1 mm). This effect underwent desensitization and it was absent in preparations taken from adult capsaicin‐pretreated rats. 3 Capsaicin‐induced relaxation of NA‐precontracted REUS was mimicked by calcitonin gene‐related peptide (CGRP, 0.3–10 μm), but not by substance P (1 μm), vasoactive intestinal polypeptide (VIP, 1 μm), α‐β methylene ATP (10 μm), γ‐aminobutyric acid (GABA, 3 mm) or galanin (1 μm). A cross‐tachyphylaxis between capsaicin (1 μm) and CGRP (1 μm) was observed. Both capsaicin and CGRP‐induced relaxation were partially antagonized by the proposed CGRP antagonist, CGRP (8–37) (10 μm). 4 Electrical field stimulation (EFS, 2.5 Hz, 60 V, 1 ms, trains of 5 s every 5 min) of REUS evoked a contraction characterized by a largely adrenergic slowly developing tonic contraction with superimposed fast twitches due to the striated component of the strips. Both capsaicin (1 μm) and CGRP (0.01–1 μm) produced an almost complete inhibition of EFS‐induced tonic contraction. A cross‐tachyphylaxis between capsaicin and CGRP was observed. Furthermore, these inhibitory actions were unaffected by CGRP (8–37) (10 μm). 5 [3H]‐resiniferatoxin displayed specific, saturable binding to rat urethral membranes. Data were consistent with a single site with a Kd of 105 pm and a Bmax of 40 fmol mg−1 protein. This binding was inhibited by capsaicin with a Ki of 0.6 μm and it was reduced by approximately 80% in preparations taken from rats that had undergone surgical ablation of the major pelvic ganglion 4 days earlier. 6 In conclusion we have demonstrated the existence of vanilloid receptors on capsaicin‐sensitive nerves innervating the rat urethra mainly through the major pelvic ganglion. The activation of this set of nerves could lead to a local release of CGRP that in turn elicits a remarkable urethral relaxation. Such a mechanism could be of relevance in physiological conditions to facilitate urine expulsion during micturition and in pathological conditions to help removal of noxious stimuli following mechanical/chemical irritation of the lower urinary tract.

CD137 and CD137 ligand constitutively coexpressed on human T and B leukemia cells signal proliferation and survival.

CD137, a member of the tumor necrosis factor receptor family, provides expansion and survival signal to T cells. Its ligand, CD137L, in addition to its ability to costimulate T cells, signals back into antigen presenting cells promoting their activation and differentiation. Recently, CD137 has been proposed as a therapeutic target to improve and sustain anticancer immune response. Several activated T leukemia and B lymphoma cell lines expressed CD137 or CD137L, respectively, and soluble CD137L has been found in sera of leukemia patients. However, the functionality and role of these costimulatory molecules in hematologic malignancies are until now unknown. Interestingly, we observed constitutive CD137 and CD137L coexpression on both human T and B leukemia cell lines. The constitutive CD137 expression on unstimulated T or B leukemia cells presents some differences compared to CD137 expressed on PMA/ionomycin‐activated T leukemia cells. Surprisingly, in spite of the low expression level, both tumor CD137 and CD137L molecules signaled in T and B leukemia cells inducing proliferation and prolonging survival. In addition, CD137/CD137L system ligation opposed the anticancer drug cytotoxic effects, reducing the apoptotic DNA fragmentation and stimulating proliferation of doxorubicin‐escaped leukemia cells. Although the role of leukemia CD137/CD137L system in vivo is unknown, these data suggest that these costimulatory molecules might confer an advantage to hematologic tumors promoting survival, sustaining cellular growth and contributing to drug resistance.

Different susceptibility to neurokinin 1 receptor antagonists of substance P and septide-induced interleukin-6 release from U373 MG human astrocytoma cell line.

In a human astrocytoma cell line U373 MG, the activation of the neurokinin 1 (NK1) receptor by substance P (SP) increase, in a concentration-related manner (1 nM to 10 microM), the basal release of interleukin-6 (IL-6) as assayed by an ELISA method, in cell supernatants after 18 h of incubation. Septide, a selective NK1 receptor agonist, is equipotent to SP in inducing the IL-6 release showing similar Emax (2644 +/- 285 and 2830 +/- 271 pg/ml) and EC50 (15.6 +/- 3.6 and 13.8 +/- 3.2 nM). However, in binding assays on intact cells, septide was an about 50-fold weaker displacer of the binding of [3H][Sar9,Met(O2)11]SP than SP (Kis were 0.28 +/- 0.1 nM and 14.2 +/- 5.0 nM for SP and septide, respectively). NK2- and NK3-selective agonists (up to 1 microM) had no binding or functional effect. Highly selective non-peptide (CP96,345) or peptide (GR82,334) NK1 receptor antagonists were more effective in antagonizing septide-(IC50s 0.2 +/- 0.06 nM and 70 +/- 18 nM) than SP-(IC50s 6.7 +/- 1.3 nM and 1.95 +/- 0.4 microM) induced IL-6 secretion. These data support the existence, also in human U373 MG cells, of a septide-sensitive NK1 receptor subtype(s) and/or epitope(s) blocked with high affinity by NK1 antagonist.

Comparison of tachykinin NK1 receptors in human IM9 and U373 MG cells, using antagonist (FK888, (±)-CP-96,345, and RP 67580) binding

We have used one peptide (FK888) and two non-peptide ((+/-)-CP-96,345 and RP 67580) antagonists, along with the preferred endogenous agonist, substance P, to compare the pharmacological (binding) profile of NK1 receptors expressed by human B lymphoblastoma (IM9) and astrocytoma (U373 MG) cells. Of the ligands tested, substance P was the most potent in both cell lines: binding affinities were 0.1 nM for IM9 cells, and 0.3 nM for U373 MG cells, respectively. The high-affinity dipeptide antagonist, FK888, bound to NK1 receptors in both cell lines with similar potencies: Ki values were 1.2 nM and 3.6 nM for IM9 cells and U373 MG cells, respectively. Of the non-peptide antagonists, as expected, (+/-)-CP-96,345 displayed higher affinity (0.4 nM in IM9 cells, and 1.2 nM in U373 MG cells) than did RP 67580 (33 nM and 223 nM in IM9 cells and U373 MG cells, respectively) in both cell lines. We conclude that the pharmacological profile of NK1 receptors is similar in the human lymphoblastoma and astrocytoma cells, i.e. if NK1 receptor subtypes exist in humans, these cell lines are likely to express a similar subtype. Because IM9 cells grow faster and are easier to maintain, this cell line may be preferable to the astrocytoma cells as a primary screen to identify NK1 receptor antagonists.