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Biomedical subjects

J A Thompson

Publications and source records attributed to J A Thompson.

At least 109 records · Page 6Linked to original sources
PubMed1997

Immunolocalization of acidic fibroblast growth factor and receptors in the tubulointerstitial compartment of chronically rejected human renal allografts.

Tubular damage and loss associated with interstitial inflammation and fibrosis may be the most important determinants in chronic renal allograft rejection. To elucidate potential pathophysiologic mechanisms associated with tubulointerstitial lesions, we examined the expression of a fibrogenic cytokine, acidic fibroblast growth factor (FGF-1) and its high-affinity receptors, in both relevant renal transplant controls (n=5) and tissue from patients (n=19) who underwent nephrectomy after graft loss, secondary to chronic rejection. In situ hybridization and immunohistochemical analyses demonstrated minimal expression of FGF-1 mRNA and protein in the tubulointerstitial compartment of the normal human kidney. In contrast, tubulointerstitial lesions in kidney allografts experiencing chronic rejection demonstrated the exaggerated appearance of both FGF-1 protein and mRNA in resident inflammatory and tubular epithelial cells. Patterns of staining were consistent throughout tubular compartments and did not appear to be localized to any particular region. The tubulointerstitium in kidneys with findings of chronic rejection also exhibited increased immunodetection of proliferating cell nuclear antigen in the tubular epithelium, inflammatory cell infiltrate, and neovascular structures. The enhanced appearance of FGF-1 and readily detectable fibroblast growth factor receptors suggests that this polypeptide mitogen may serve as an important mediator of growth and repair responses, associated with development of angiogenesis and tubulointerstitial lesions during chronic rejection of human renal allografts.

Biomarkers↗
PubMed1997

Adenoviral vector infection of the human exocrine pancreas.

OBJECTIVE: To determine if a viable cadaveric pancreas might be used to study viral transfection efficacy in a manner precisely mimicking in vivo human studies. DESIGN: Ex vivo gene transfer to an intact human pancreatic duct. SETTING: Molecular biology laboratory and organ procurement center. INTERVENTION: The recombinant adenoviral vector that contains the Escherichia coli beta-galactosidase (LacZ) gene driven by the human cytomegalovirus promoter, ie, AdCMVLacZ, was used to transfect the epithelial cells of the pancreatic ductal system. A human pancreas (150 g wt/wt) procured for transplantation, but subsequently found unsuitable, was used for the study. The splenic, superior mesenteric arteries and portal vein were cannulated and perfused in a heat-controlled organ procurement perfusion system. A segment of vascularized, perfused distal pancreatic duct was isolated with a balloon occlusion catheter. The recombinant adenoviral vector AdCMVLacZ was introduced into the lumen of the distal segment of the pancreatic duct and incubated for 6 hours at 25 degrees C. The proximal segment of the pancreatic duct was not exposed to the vector and served as control tissue. Tissue was harvested and processed for evaluation of beta-galactosidase activity. RESULTS: Adenoviral vector-infected pancreatic ducts exhibited intense blue staining, indicative of reporter gene expression in the epithelial cells of the pancreatic duct. The phenotype of these cells was confirmed by immunohistochemical studies using anti-annexin III polyclonal antibody. Control tissue not exposed to the adenoviral vector was subjected to an identical analysis and did not reveal evidence of expression of the reporter gene. CONCLUSIONS: This study demonstrates the first successful transfection of epithelial cells of the pancreatic duct from normal human pancreas with a recombinant adenovirus. This system will provide not only information on the efficacy of transfection but also a novel gene therapeutic approach to target pancreatic ductal adenocarcinoma.

Adenoviruses, Human↗
PubMed1997

Predicting end-of-test semen quality in bulls prior to performance testing.

The first objective of this study was to determine if serum concentrations of specific hormones (testosterone, progesterone and androstenedione) in bulls at the start of performance testing could predict semen quality at the end-of-test when used in a multivariate model. The second objective was to evaluate other clinical measurements (breed, age, body weight, hip height and scrotal circumference) for predicting end-of-test semen quality. End-of-test semen quality was related to steroid concentrations and several pre-testing measurements, including age, body weight, hip height and scrotal circumference (SC). Combining the 3 steroid concentrations into a predictive test had a sensitivity of 0.6 and specificity of 0.5 at its most accurate point. The repeatability of the test result was extremely low (r(2) = 0.16; P < 0.05). In multivariate analyses, breed and start-of-test SC remained significant predictors of end-of-test semen quality (P < 0.05) while the other variables were nonsignificant (P > 0.1), suggesting that start-of-test SC was the most accurate predictor of end-of-test semen quality. Removing bulls at the start-of-test that had scrotal measurements of less than 20 cm, 24 cm, 28 cm or 32 cm resulted in sensitivities and specificities of 0.19, 0.94; 0.41, 0.81; 0.64, 0.56; and 0.94, 0.12, respectively. No cut-point had both adequate sensitivity and specificity. Because clinical tests were correlated, combining the tests to improve accuracy was not justified.

Journal Article↗
PubMed1997

Interleukin-2 after autologous stem cell transplantation for hematologic malignancy: a phase I/II study.

The success of autologous stem cell transplantation (ASCT) for hematologic malignancy is limited largely by a high relapse rate. It is postulated that IL-2 administered after ASCT may eliminate minimal residual disease and thereby reduce relapses. A phase I/II study was performed to identify a regimen of IL-2 (Chiron) that could be given early after ASCT in phase III trials. In the phase I study, beginning a median of 46 days after ASCT for hematologic malignancy, cohorts of three to four patients received escalating doses of 'induction' IL-2 of 9, 10, or 12 x 10(6) IU/m2/day for 4 or 5 days by continuous i.v. infusion (CIV), followed by a 4-day rest period, and then 1.6 x 10(6) IU/m2/day of maintenance IL-2 by CIV for 10 days. The maximum tolerated dose (MTD) of induction IL-2 was 9 x 10(6) IU/m2/day x 4. In the phase II study, 52 patients received the MTD. Eighty percent of patients completed induction IL-2. Most patients exhibited some degree of capillary leak. One patient died of CMV pneumonia and one died of ARDS. Maintenance IL-2 was well tolerated. In the phase I/II study, 16 of 31 patients with non-Hodgkin lymphoma (NHL), 3/8 with Hodgkin disease (HD), 4/17 with AML, and 4/5 with ALL remain in CR. Two of six multiple myeloma (MM) patients remain in PR. Although the regimen of IL-2 identified had significant side-effects in some patients, it was well tolerated in the majority of patients. Phase III prospectively randomized clinical trials are in progress to determine if this IL-2 regimen will decrease the relapse rate after ASCT for AML and NHL.

Adjuvants, Immunologic↗
PubMed1997

Interleukin-12 induced cytolytic activity in lymphocytes from recipients of autologous and allogeneic stem cell transplants.

Interleukin-12 (IL-12) has been reported to enhance the cytolytic activity of NK and activated T cells and to induce low levels of lymphokine-activated killer (LAK) activity in normal human lymphocytes. Therapy with IL-12 has induced tumor eradication in murine models. These observations suggest that IL-12 might have a role as treatment for minimal residual disease following transplantation. To determine whether PBL from recipients of autologous (autoSCT) and allogeneic (alloSCT) bone marrow or peripheral blood stem cell transplants respond to IL-12 with generation of LAK activity, PBL were incubated with IL-12 for 5 days, then tested in a 51Cr release assay for lysis of Daudi. PBL from 17 normal 'control' individuals were similarly tested and lysis was observed in only 3/17 (mean 16.9% of the three). By contrast, PBL from 10/12 patients obtained a median of 30 days after autoSCT, exhibited significant IL-12-induced LAK activity (mean lysis, 35.3%, P < 0.005 vs controls). PBL from 18 of 20 patients tested a median of 44 days after alloSCT also exhibited significant LAK activity (mean lysis, 30.0%, P < 0.005 vs controls). In autoSCT recipients, IL-12 and IL-2 at high concentrations (1000 U/ml each) were additive for induction of LAK activity, whereas low, suboptimal concentration of IL-12 (250 U/ml) and IL-2 (1 U/ml) were synergistic in 3/5 experiments. The percentage of PBL expressing IL-12 receptor beta 1 chain (IL-12r beta 1) was higher in stem cell recipients than in normal individuals, P < 0.05. Moreover, a higher percentage of IL-12r beta 1-positive PBL was associated with greater IL-12-induced LAK activity in transplant recipients. These studies demonstrate that PBL obtained early after stem cell transplantation have a higher percentage of cells expressing IL-12r beta 1 and respond to IL-12 with significantly greater LAK cytotoxicity than PBL from normal controls. These results suggest that IL-12 is a potentially attractive candidate for study as consolidative immunotherapy after stem cell transplantation.

Cells, Cultured↗
PubMed1997

Decreased zinc affinity of amyotrophic lateral sclerosis-associated superoxide dismutase mutants leads to enhanced catalysis of tyrosine nitration by peroxynitrite.

Mutations to Cu/Zn superoxide dismutase (SOD) linked to familial amyotrophic lateral sclerosis (ALS) enhance an unknown toxic reaction that leads to the selective degeneration of motor neurons. However, the question of how >50 different missense mutations produce a common toxic phenotype remains perplexing. We found that the zinc affinity of four ALS-associated SOD mutants was decreased up to 30-fold compared to wild-type SOD but that both mutants and wild-type SOD retained copper with similar affinity. Neurofilament-L (NF-L), one of the most abundant proteins in motor neurons, bound multiple zinc atoms with sufficient affinity to potentially remove zinc from both wild-type and mutant SOD while having a lower affinity for copper. The loss of zinc from wild-type SOD approximately doubled its efficiency for catalyzing peroxynitrite-mediated tyrosine nitration, suggesting that one gained function by SOD in ALS may be an indirect consequence of zinc loss. Nitration of protein-bound tyrosines is a permanent modification that can adversely affect protein function. Thus, the toxicity of ALS-associated SOD mutants may be related to enhanced catalysis of protein nitration subsequent to zinc loss. By acting as a high-capacity zinc sink, NF-L could foster the formation of zinc-deficient SOD within motor neurons.

Amyotrophic Lateral Sclerosis↗
PubMed1997

The relation of neuropsychological measures to levels of cognitive functioning in elderly individuals: a discriminant analysis approach.

This study addresses the question of which tests in a comprehensive neuropsychologicaI test battery best discriminate normal, mildly, and moderate to severely cognitiveIy impaired older participants. Sixty-six geriatric participants were administered a battery of neuropsychological tests as part of an outpatient geriatric clinic evaluation. A discriminant analysis procedure using a consensus rating of global cognitive impairment as the grouping variable was employed to analyze the data. From 15 neuropsychological test measures, five representing the domains of learning and memory, visuospatial and executive functioning were identified as the best predictors of level of cognitive impairment. Findings demonstrate the utility of discriminant function analysis (DFA) procedures for developing reduced-length cognitive batteries that accurately classify participants in terms of levels of cognitive impairment and identify mild dysfunction in participants at risk of cognitive impairment. Further cross-validation studies are needed to confirm the utility of these more circumscribed batteries.

Journal Article↗
PubMed1997

Outpatient chemoimmunotherapy for the treatment of metastatic melanoma.

The prognosis for most patients with metastatic melanoma is poor, as the median survival is only 7 months. Phase II trials of chemoimmunotherapy have, however, reported response rates of 40% to 70% and prolonged disease-free survival in small subgroups of patients. Most chemoimmunotherapy trials have used high doses of recombinant interleukin-2 (rIL-2) and recombinant interferon alpha (rIFN-alpha) that produce significant toxicity and require prolonged hospitalization to manage side effects. To develop a treatment regimen for metastatic melanoma that would minimize costly hospitalization, we initiated a phase II trial of outpatient chemoimmunotherapy. Patients were treated with monthly cycles of intravenous carmustine, dacarbazine, cisplatin, and tamoxifen plus self-administered subcutaneous rIL-2 and rIFN-alpha as outpatients. To date, 32 patients have received 94 cycles of therapy. The most common toxicity was nausea and vomiting. Hospitalization for the management of toxicity was required in only seven cycles (7%). Thirty patients have been assessed for clinical response. The overall response rate was 43% (13% complete and 30% partial response). This phase II trial has established a tolerable regimen of outpatient chemoimmunotherapy, which has shown significant antitumor activity.

Adult↗
PubMed1997

Heparin-dependent fibroblast growth factor activities: effects of defined heparin oligosaccharides.

Heparin and related molecules have been identified as important participants in fibroblast growth factor (FGF) signaling although the mechanisms of action remain unclear. We have used heparin oligosaccharides to examine steps in the signaling process which could be affected by the polysaccharide. Immobilized FGF-1 and FGF-2 bound all sizes of oligosaccharides tested, ranging from tetrasaccharide to decasaccharide, at physiological salt concentration. Each group of oligosaccharide was eluted from the FGF affinity columns in several peaks, and larger oligosaccharides showed higher apparent affinity for the immobilized growth factors compared to the shorter ones. Heparin hexasaccharides were the smallest fragments providing complete protection of FGF-1 and FGF-2 against trypsin digestion. Tetrasaccharides, however, were able to provide partial protection. The requirement of heparin for ligand-receptor interaction was evaluated in receptor binding assays using Sf9 insect cells engineered to overexpress different recombinant FGF receptor (FGFR) species including FGFR1 beta, FGFR1 alpha or FGFR4 at the cell surface. In these assays hexasaccharides were the smallest fragments capable of stimulating FGF-receptor interaction. Over the range of concentrations examined, neither hexasaccharides nor octasaccharides were able to stimulate receptor binding to the level attained by intact heparin. In fact, these oligosaccharides interfered with the ability of intact heparin in promoting FGF-receptor binding. The presence of both stimulatory and inhibitory activities in hexasaccharide and octasaccharide populations could be attributed to structural heterogeneity within the oligosaccharide preparations. However, similar observations were obtained with "highly-sulfated" structurally homogeneous preparations of hexasaccharide and octasaccharide, although these molecules generally had greater stimulatory and less inhibitory activity than their structurally heterogeneous counterparts. Hexasaccharides were found to be the smallest fragments able to potentiate the FGF-1-induced 3T3 cell proliferation while their effect on FGF-2 signaling was less clear. These observations suggest that heparin can modulate FGF-signaling at several stages with different end results.

3T3 Cells↗
PubMed1997

Updated analysis of an outpatient chemoimmunotherapy regimen for treating metastatic melanoma.

PURPOSE: Aggressive inpatient chemoimmunotherapy protocols for metastatic melanoma have yielded encouraging response rates but have required lengthy hospitalizations. To reduce or eliminate the need for hospitalization, we have developed an outpatient chemoimmunotherapy regimen and assessed its efficacy and toxicity in 53 patients treated at the University of Washington Medical Center. PATIENTS AND METHODS: Eligible patients with measurable metastatic melanoma received carmustine (150 mg/m2 every 6-8 weeks) and dacarbazine (660 mg/m2) and cisplatin (75 mg/m2) every 3 to 4 weeks in an infusion center plus tamoxifen (20 mg/day). Patients self-administered subcutaneous recombinant interleukin-2 (rIL-2) at 3 MIU/m2/day on days 3 to 9, and recombinant interferon alfa-2a (rIFN-alpha 2a) at 3 MIU on day 3 and at 5 MIU/m2/day on days 5, 7, and 9. Maintenance rIFN-alpha 2a was self-administered subcutaneously at 5 MIU/m2 tiw for 12 months after complete or stable partial response. Response and survival were assessed. RESULTS: Fifty-three patients (median age = 49 years) have received 181 cycles. To date, there have been 10 complete responses (19%) lasting 2 to 28+ months and 12 partial responses (23%) lasting 2 to 11 months, for an overall response rate of 42% (95% confidence interval, 28%-55%). The median overall survival was 12 months. Grade 3/4 vomiting occurred in 32% of cycles, but hospitalization for supplemental intravenous fluids was required in only 11% of cycles for a median of 3 days. Grade 4 thrombocytopenia and neutropenia occurred in 9% and 8% of cycles, respectively. Grade 3 renal dysfunction occurred in only one cycle and was reversible. CONCLUSION: A chemoimmunotherapy regimen for patients with metastatic melanoma has been defined that is well tolerated on an outpatient basis and is associated with a median survival comparable to that with aggressive inpatient chemoimmunotherapy regimens.

Adult↗
PubMed1997

Interleukin-2 therapy after bone marrow or stem cell transplantation for hematologic malignancies.

PURPOSE: Autologous or allogeneic bone marrow transplantation (BMT) or stem cell transplantation (SCT) for advanced hematologic malignancies is associated with a high relapse rate. It has been postulated that recombinant interleukin-2 (rIL-2) administered as consolidative immunotherapy early after BMT or SCT, at a time of minimal residual disease, might reduce the relapse rate. We review here preliminary results from a series of studies designed to investigate the safety, immunomodulatory effects, and clinical benefits of rIL-2 therapy following autologous and allogeneic BMT and SCT. PATIENTS AND METHODS: Patients with hematologic malignancies underwent autologous or allogeneic BMT or SCT and received rIL-2 by continuous intravenous infusion a median of 33 to 56 days later. In all trials, the rIL-2 regimen consisted of a moderate induction dose for 4 to 5 days in the hospital, 4 to 6 days of rest, and a low maintenance dose for 10 days in the outpatient setting. A phase I trial of Roche rIL-2 after autoBMT, a feasibility trial of autologous lymphokine-activated killer cells with rIL-2, and another phase I/II trial of Chiron rIL-2 after autoBMT were performed. A similar phase I trial of IL-2 after alloBMT was also performed in children with acute leukemia beyond first complete remission. RESULTS: An rIL-2 regimen has been identified that can be tolerated early after transplantation. Administration of this rIL-2 regimen induces marked increases in CD3+CD8+ T lymphocytes and CD3-CD56+ natural killer cells and enhances their antitumor cytolytic activity. Encouraging but somewhat inconsistent clinical outcomes were noted in phase I/II trials in patients with lymphoma and acute myeloid leukemia. CONCLUSIONS: The results of phase I/II trials are sufficiently encouraging to justify prospectively randomized phase III trials to determine whether rIL-2 after autologous SCT will reduce the rate of posttransplantation relapse and improve survival in patients with advanced hematologic malignancies.

Adolescent↗
PubMed1997

Metastatic renal cell carcinoma: long-term survival after therapy with high-dose continuous-infusion interleukin-2.

PURPOSE: This article undertakes to define the response rate, long-term survival, and toxicity in patients with metastatic renal cell carcinoma (MRCC) treated with high-dose continuous intravenous infusion (CIV) recombinant interleukin-2 (rIL-2) with or without lymphokine-activated killer (LAK) cells. PATIENTS AND METHODS: One hundred twenty-three consecutive patients received CIV rIL-2 (18-22 MIU/m2/day on days 1-5, and 6-8 MIU/m2/day on days 10-19) on one of five sequential protocols at the University of Washington between 1988 and 1995. The first 76 patients received LAK cells. The median age was 55 years (range, 32-76 years), and 71% had undergone prior nephrectomy. RESULTS: Nine patients achieved a complete response (7.3%) and 14 patients achieved a partial response (11.4%) for an overall response rate of 19% (95% confidence interval, 12%-26%). The median survival was 19 months, and the 5-year survival was 20%. Seven of nine complete responders (78%) remain in continuing complete response at 43+ to 109+ months. Intensive care unit and vasopressor support were required in 42% and 23% of patients, respectively, who received rIL-2 + LAK cells, and in 18% and 4% of those who received rIL-2 alone. There was one treatment-related death. CONCLUSION: We report the largest single-institution experience and the longest survival for patients with MRCC treated with CIV rIL-2. The administration of rIL-2 by CIV is associated with less frequent intensive care unit and vasopressor support than with high-dose intravenous bolus regimens, and hence may enhance the therapeutic index in patients with MRCC.

Adult↗
PubMed1996

Acidic fibroblast growth factor enhances peroxynitrite-induced apoptosis in primary murine fibroblasts.

Oxidative stress is considered a major mediator of apoptosis in several cellular systems. Peroxynitrite is a highly toxic oxidant formed by the reaction of nitric oxide with superoxide. Primary embryonic murine fibroblasts, exposed to 1 mM peroxynitrite, resulted in delayed cell death characterized by membrane blebbing, cytoplasmic shrinkage, nuclear condensation, and DNA fragmentation that were more characteristic of apoptosis than necrosis. In addition, both morphological alterations and DNA fragmentation were inhibited by the endonuclease inhibitor aurintricarboxylic acid. Pretreatment of fibroblasts with acidic fibroblast growth factor (FGF-1) markedly enhanced peroxynitrite-induced apoptosis, an observation restricted to immediate-early transcriptional and activated tyrosine phosphorylation processes. FGF-1 pretreatment had no modulatory effect on cell death elicited by other reactive oxygen species, suggesting that enhancement of apoptosis involves a unique relationship between peroxynitrite and the growth factor. Exposure of cells to peroxynitrite resulted in immediate tyrosine nitration of several polypeptides, including major targets with estimated molecular masses of 62, 68, and 77 kDa. Pretreatment with FGF-1 did not alter targets of peroxynitrite-mediated tyrosine nitration, but rather increased the total amount of this amino acid modification. Treatment with other reactive oxygen species failed to induce tyrosine nitration. Collectively, these efforts demonstrate that FGF-1 transiently renders primary fibroblasts more sensitive to peroxynitrite-induced apoptosis. In addition, results presented here predict a pivotal role for FGF-1 and peroxynitrite-induced cytotoxicity during the resolution of inflammation and repair processes in vivo.

Animals↗
PubMed1996

Isolation of tyrosinase-specific CD8+ and CD4+ T cell clones from the peripheral blood of melanoma patients following in vitro stimulation with recombinant vaccinia virus.

The identification of Ags expressed by tumor cells and recognized by autologous T cells has led to the prospect of treating cancer by adoptive transfer of tumor-reactive T cells selected for Ag specificity. Tyrosinase is an Ag expressed by normal melanocytes as well as melanoma cells for which responses by autologous T cells have been detected. To evaluate the frequency with which tyrosinase-specific T cells can be isolated from melanoma patients for potential use in therapy, a recombinant vaccinia virus expressing tyrosinase was constructed for infection of autologous APCs that could be used to stimulate T cells reactive with this protein. Eight patients were studied, with peripheral blood serving as the source of both responder T cells and autologous APCs. Tyrosinase-specific CD8+ CTL clones were isolated from five of the eight patients with melanoma. The tyrosinase-specific CTL generated in this manner recognized autologous tumor cells as well as targets expressing the recombinant virus vector. CTL clones from three of the individuals were restricted to HLA-A28, -B8, and -B60, which have not previously been identified as alleles that can present immunogenic tyrosinase peptides. Tyrosinase-specific CD4+ T cell clones were isolated from six of the eight patients by stimulation with autologous APCs infected with recombinant vaccinia virus, and all these CD4+ clones were capable of recognizing autologous tumor cells. These studies demonstrate a high prevalence of CD4+ and CD8+ tyrosinase-specific responses in peripheral blood and support the feasibility of using peripheral blood to generate T cells for tumor therapy without the requirement for isolating T cells that have infiltrated tumor sites.

Animals↗
PubMed1996

Nitration and inactivation of manganese superoxide dismutase in chronic rejection of human renal allografts.

Inflammatory processes in chronic rejection remain a serious clinical problem in organ transplantation. Activated cellular infiltrate produces high levels of both superoxide and nitric oxide. These reactive oxygen species interact to form peroxynitrite, a potent oxidant that can modify proteins to form 3-nitrotyrosine. We identified enhanced immunostaining for nitrotyrosine localized to tubular epithelium of chronically rejected human renal allografts. Western blot analysis of rejected tissue demonstrated that tyrosine nitration was restricted to a few specific polypeptides. Immunoprecipitation and amino acid sequencing techniques identified manganese superoxide dismutase, the major antioxidant enzyme in mitochondria, as one of the targets of tyrosine nitration. Total manganese superoxide dismutase protein was increased in rejected kidney, particularly in the tubular epithelium; however, enzymatic activity was significantly decreased. Exposure of recombinant human manganese superoxide dismutase to peroxynitrite resulted in a dose-dependent (IC50 = 10 microM) decrease in enzymatic activity and concomitant increase in tyrosine nitration. Collectively, these observations suggest a role for peroxynitrite during development and progression of chronic rejection in human renal allografts. In addition, inactivation of manganese superoxide dismutase by peroxynitrite may represent a general mechanism that progressively increases the production of peroxynitrite, leading to irreversible oxidative injury to mitochondria.

Amino Acid Sequence↗
PubMed1996

Hydralazine prevents nitroglycerin tolerance by inhibiting activation of a membrane-bound NADH oxidase. A new action for an old drug.

Hydralazine has been shown to reduce mortality in patients with congestive heart failure when given concomitantly with isosorbide dinitrate. Recently, we demonstrated that nitrate tolerance is in part due to enhanced vascular superoxide .O2- production. We sought to determine mechanisms whereby hydralazine may prevent tolerance. Rabbits either received no treatment, nitroglycerin patches (1.5 micrograms/kg/min x 3 d), hydralazine alone (10 mg/kg/d in drinking water), or hydralazine and nitroglycerin. Aortic segments were studied in organ chambers and relative rates of vascular .O2- production were determined using lucigenin-enhanced chemiluminescence. Nitroglycerin treatment markedly inhibited relaxations to nitroglycerin (maximum relaxations in untreated: 92 +/- 1 vs. 64 +/- 3% in nitroglycerin-treated patients and increased vascular .O2- production by over two-fold (P < 0.05). Treatment with hydralazine in rabbits not receiving nitroglycerin significantly decreased .O2- production in intact rabbit aorta and increased sensitivity to nitroglycerin. When given concomitantly with nitroglycerin, hydralazine completely prevented the development of nitrate tolerance and normalized endogenous rates of vascular .O2- production. Studies of vessel homogenates demonstrated that the major source of .O2- was an NADH-dependent membrane-associated oxidase displaying activities of 67 +/- 12 vs. 28 +/- 2 nmol .O2-.min-1.mg protein-1 in nitroglycerin-treated vs. untreated aortic homogenates. In additional studies, we found that acute addition of hydralazine (10 microM) to nitroglycerin-tolerant vessels immediately inhibited .O2- production and NADH oxidase activity in vascular homogenates. The chemiluminescence signal was inhibited by a recombinant heparin-binding superoxide dismutase (HBSOD) demonstrating the specificity of this assay for .O2-. These observations suggest that a specific membrane-associated oxidase is activated by chronic nitroglycerin treatment, and the activity of this oxidase is inhibited by hydralazine, providing a mechanism whereby hydralazine may prevent tolerance. The ability of hydralazine to inhibit vascular .O2- anion production represents a novel mechanism of action for this drug.

Animals↗