Modulation of contractile function through NPY receptors during development of cardiomyocyte hypertrophy.

Severity of left ventricular hypertrophy (LVH) correlates with elevated plasma levels of neuropeptide Y (NPY) in hypertension. NPY elicits positive and negative contractile effects in cardiomyocytes through Y(1) and Y(2) receptors, respectively. This study tested the hypothesis that NPY receptor-mediated contraction is altered during progression of LVH. Ventricular cardiomyocytes were isolated from spontaneously hypertensive rats (SHRs) pre-LVH (12 weeks), during development (16 weeks), and at established LVH (20 weeks) and age-matched normotensive Wistar Kyoto (WKY) rats. Electrically stimulated (60 V, 0.5 Hz) cell shortening was measured using edge detection and receptor expression determined at mRNA and protein level. The NPY and Y(1) receptor-selective agonist, Leu(31)Pro(34)NPY, stimulated increases in contractile amplitude, which were abolished by the Y(1) receptor-selective antagonist, BIBP3226 [R-N(2)-(diphenyl-acetyl)-N-(4-hydroxyphenyl)methyl-argininamide)], confirming Y(1) receptor involvement. Potencies of both agonists were enhanced in SHR cardiomyocytes at 20 weeks (2300- and 380-fold versus controls). Maximal responses were not attenuated. BIBP3226 unmasked a negative contraction effect of NPY, elicited over the concentration range (10(-12) to 3 x 10(-9) M) in which NPY and PYY(3-36) attenuated the positive contraction effects of isoproterenol, the potencies of which were increased in cardiomyocytes from SHRs at 20 weeks (175- and 145-fold versus controls); maximal responses were not altered. Expression of NPY-Y(1) and NPY-Y(2) receptor mRNAs was decreased (55 and 69%) in left ventricular cardiomyocytes from 20-week-old SHRs versus age-matched WKY rats; parallel decreases (32 and 80%) were observed at protein level. Enhancement of NPY potency, producing (opposing) contractile effects on cardiomyocytes together with unchanged maximal response despite reduced receptor number, enables NPY to contribute to regulating cardiac performance during compensatory LVH.

Chemotherapy and TRAIL-mediated colon cancer cell death: the roles of p53, TRAIL receptors, and c-FLIP.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has recently attracted attention as a potential therapeutic agent in the treatment of cancer. We assessed the roles of p53, TRAIL receptors, and cellular Fas-associated death domain-like interleukin-1beta-converting enzyme inhibitory protein (c-FLIP) in regulating the cytotoxic effects of recombinant TRAIL (rTRAIL) alone and in combination with chemotherapy [5-fluorouracil (5-FU), oxaliplatin, and irinotecan] in a panel of colon cancer cell lines. Using clonogenic survival and flow cytometric analyses, we showed that chemotherapy sensitized p53 wild-type, mutant, and null cell lines to TRAIL-mediated apoptosis. Although chemotherapy treatment did not modulate mRNA or cell surface expression of the TRAIL receptors death receptor 4, death receptor 5, decoy receptor 1, or decoy receptor 2, it was found to down-regulate expression of the caspase-8 inhibitor, c-FLIP. Stable overexpression of the long c-FLIP splice form but not the short form was found to inhibit chemotherapy/rTRAIL-induced apoptosis. Furthermore, siRNA-mediated down-regulation of c-FLIP, particularly the long form, was found to sensitize colon cancer cells to rTRAIL-induced apoptosis. In addition, treatment of a 5-FU-resistant cell line with 5-FU down-regulated c-FLIP expression and sensitized the chemotherapy-resistant cell line to rTRAIL. We conclude that TRAIL-targeted therapies may be used to enhance conventional chemotherapy regimens in colon cancer regardless of tumor p53 status. Furthermore, inhibition of c-FLIP may be a vital accessory strategy for the optimal use of TRAIL-targeted therapies.

Differential Modulation of Transcriptional Activity of Estrogen Receptors by Direct Protein-Protein Interactions with the T Cell Factor Family of Transcription Factors

Two major signaling pathways, those triggered by estrogen (E(2)) and by the Wnt family, interact in the breast to cause growth and differentiation. The estrogen receptors ER(alpha) and ER(beta) are activated by binding E(2) and act as ligand-dependent transcription factors. The effector for the Wnt family is the Tcf family of transcription factors. Both sets of transcription factors recognize discrete but different nucleotide sequences in the promoters of their target genes. By using transient transfections of reporter constructs for the osteopontin and thymidine kinase promoters in rat mammary cells, we show that Tcf-4 antagonizes and Tcf-1 stimulates the effects of activated ER/E(2). For mutants of the former promoter, the stimulatory effects of ER(alpha)/E(2) can be made to be dependent on Tcf-1, and for the latter promoter the effects of the T cell factors (TCFs) are dependent on ER/E(2). Direct interaction between ERs and Tcfs either at the Tcf/ER(alpha)-binding site on the DNA or in the absence of DNA is established by gel retardation assays or by coimmunoprecipitation/biosensor methods, respectively. These results show that the two sets of transcription factors can interact directly, the interaction between ERs and Tcf-4 being antagonistic and that between ERs and Tcf-1 being synergistic on the activity of the promoters employed. Since Tcf-4 is the major Tcf family member in the breast, it is suggested that the antagonistic interaction is normally dominant in vivo in this tissue.

Limitations in using peptide drugs to characterize calcitonin gene-related peptide receptors

Calcitonin gene-related peptide (CGRP) is an endogenous vasodilator peptide that produces its effects by activation of CGRP(1) and CGRP(2) receptor subtypes, These receptor subtypes are characterized in functional studies using the agonist Cys(Acm)(2,7)-human-alpha-calcitonin gene-related peptide (Cys(ACM)(2,7)-h-alpha-CGRP), which activates CGRP(2) receptors, and the antagonist h-alpha CGRP(8-37) which has a high affinity for CGRP, receptors and a low affinity for CGRP(2) receptors. Our aim was to identify factors that may limit the use of these drugs to characterize CGRP receptor subtypes. We studied CGRP receptors using isolated ring segments of pig coronary and basilar arteries studied in vitro. The affinity of the antagonist h-alpha CGRP(8-37) for inhibiting h-alpha CGRP-induced relaxation of coronary arteries (log(10) of the antagonist equilibrium dissociation constant = -5.33) was determined from Schild plots that had steep slopes. Therefore, we used capsaicin to investigate the role of endogenous CGRP in confounding affinity measurements for h-alpha CGRP(8-37). After capsaicin treatment, the slopes of the Schild plots were not different from one, and a higher affinity of h-CGRP(8-37) in blocking relaxation was obtained (log(10) of the antagonist equilibrium dissociation constant = -6.01). We also investigated the agonist activity of the putative CGRP, receptor selective agonist Cys(Acm)(2,7)-h-alpha-CGRP. We found that maximal relaxation of coronary arteries caused by Cys(Acm)(2,7)-h-alpha CGRP was dependent upon the level of contractile tone induced by KCI. We also determined the K-A for Cys(Acm)(2,7)-h-alpha CGRP and found that the K-A (817 nM) was not significantly different from the EC50 (503 nM) for this drug in causing relaxation, indicating that Cys(Acm)(2,7)-h-alpha CGRP is a partial agonist. Because experimental conditions affect the actions of h-CGRP(8-37) and Cys(Acm)(2,7)-h-alpha CGRP, the conditions must be carefully controlled to reliably identify CGRP receptor subtypes.