Endothelial cell production of nitrogen oxides in response to interferon gamma in combination with tumor necrosis factor, interleukin-1, or endotoxin.

Clinical studies using biological response modifiers in cancer therapy have shown that the major dose-limiting toxic effects are hypotension and diffuse microvascular leakage. The cause and pathophysiology of this hypotension remains unknown. Previous experiments have demonstrated that a number of cell types, including endothelial cells, neutrophils, and macrophages, can secrete a potent hypotensive agent--endothelium-derived relaxing factor, which has recently been identified as nitric oxide. In this study, we tested interferon gamma, tumor necrosis factor, interleukin-1, interleukin-2, muramyl dipeptide, and endotoxin for their effects on production of nitrogen oxides by endothelial cells. Interferon gamma, in combination with tumor necrosis factor, interleukin-1 (IL-1), or endotoxin, induced murine brain endothelial cells to secrete nitrites (20-45 microM within 48 hr), which are breakdown products of nitric oxide. Nitrite production was blocked by incubation of endothelial cells in medium without L-arginine, a substrate for nitric-oxide synthase. Accumulation of nitrites was also inhibited by addition of NG-monomethyl-L-arginine (L-NMMA), which acts as a competitive inhibitor of this enzyme. The inhibitory effects of L-NMMA were reversed by addition of excess L-arginine. These results suggest (a) that endothelial cells produce nitric oxide in response to immunomodulators and (b) that endothelial cell-derived nitric oxide plays a role in the development of hypotension in patients treated with tumor necrosis factor or interleukins. Furthermore, administration of substrate analogues such as L-NMMA may favorably alter the toxicity associated with these immunomodulators and result in a higher maximum tolerated dose, with subsequent improvement in the antitumor activity.

Inhibition of interleukin-1-alpha-induced nitric oxide synthase in vascular smooth muscle and full reversal of interleukin-1-alpha-induced hypotension by N omega-amino-L-arginine.

BACKGROUND: Interleukin-1-alpha (IL-1) is a cytokine with potentially therapeutic immunoproliferative and tumoricidal activities. Preliminary clinical studies suggest that use of IL-1 may be restricted by dose-limiting hypotension. PURPOSE: The purpose of this study was to investigate the role of nitric oxide (NO.) as a possible mediator of this hypotension. METHODS: Cytokine-treated rat aortic smooth muscle cells were assayed for nitrite production, a stable breakdown product of nitric oxide. Nitric oxide synthase from smooth muscle cells was partially characterized in cytosol preparations using a novel Fe(2+)-myoglobin method to test for nitric oxide production. To determine the role of NO. on the immunorestorative and antineoplastic activity of IL-1, N omega-amino-L-arginine (NAA) or N omega-monomethyl-L-arginine (NMA), inhibitors of nitric oxide synthase, were added to either cultures of IL-1-dependent T cells or A375 melanoma cells exposed to IL-1. To investigate the effects of NAA in vivo, pentobarbital anesthetized dogs, which were made hypotensive by administration of IL-1, received a single intravenous bolus dose of NAA. The effects of NAA were then reversed by the administration of L-arginine. RESULTS: Our results show that cultured IL-1-activated rat aortic smooth muscle cells synthesize nitric oxide, a potent vasodilator. Induction of nitric oxide synthase is augmented by interferon-gamma and blocked by IL-1 receptor antagonist and by inhibitors of RNA or protein synthesis. Nitric oxide synthesis by IL-1-activated smooth muscle cells is inhibited by NAA, NMA, and N omega-nitro-L-arginine (NNA) with ED50 (i.e., effective dose for 50% inhibition) values of 20, 60, and 1000 microM, respectively; this rank order of inhibition is characteristic of an agonist-unregulated, inducible isoform of nitric oxide synthase. In smooth muscle cells, inhibition of NO. synthesis by NAA is reversed by excess L-arginine. Consistent with the induction of unregulated NO. synthesis in vascular smooth muscle in vivo, administration of IL-1 (50 micrograms/kg) to dogs caused a 33.5% decrease in systemic vascular resistance and a 28% decrease in blood pressure within 3 hours. Subsequent administration of NAA (20 mg/kg) rapidly and completely reversed the hypotension and increased systemic vascular resistance; these effects of NAA were reversed by L-arginine. Neither the immunoproliferative nor the tumoricidal activity of IL-1 was diminished by NAA. CONCLUSIONS: Our results indicate that (a) vascular smooth muscle is a likely source as well as a target of IL-1-induced NO. synthesis, causing vasodilatation and hypotension, (b) nitric oxide synthase inhibitors can fully reverse this hypotension, and (c) the therapeutically useful properties of IL-1 are not diminished by nitric oxide synthase inhibitors. IMPLICATIONS: Administration of inhibitors of nitric oxide synthase can reverse the pathological cardiovascular effects of IL-1 at concentrations that do not interfere with the potentially useful immunoproliferative or tumoricidal effects of this cytokine. In the context of the current clinical trials of IL-1, this finding would represent a very significant advantage.

Escalated therapy for refractory urothelial tumors: methotrexate-vinblastine-doxorubicin-cisplatin plus unglycosylated recombinant human granulocyte-macrophage colony-stimulating factor.

Thirty-two assessable patients with metastatic urothelial tumors refractory to standard chemotherapy with methotrexate, vinblastine, doxorubicin, and cisplatin (MVAC) were treated with escalated doses of MVAC plus unglycosylated recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF). Results of this phase I trial revealed that escalated MVAC (30 mg of methotrexate/m2, 4 mg of vinblastine/m2, 60 mg of doxorubicin/m2, and 100 mg of cisplatin/m2) can be tolerated by heavily pretreated patients. The side effects of rhGM-CSF are dose- and schedule-dependent. The maximum tolerated dose is 250 micrograms/m2 per day as a single dose administered subcutaneously (SC) for 10 consecutive days. This dose is well tolerated in outpatients, resulting in only modest fever and few side effects. The same dose delivered as a continuous infusion or a higher dose delivered either as a continuous infusion or SC caused significant side effects. For phase II trials, the starting dose of rhGM-CSF when combined with escalated MVAC is 120 micrograms/m2 per day SC for 10 consecutive days. forty percent of the treated patients responded, seven (23%) with complete remission and five (17%) with partial remission. This response rate is higher than anticipated from such a modest dosage escalation in chemotherapy-refractory patients.

Role of nitric oxide in lysis of tumor cells by cytokine-activated endothelial cells.

The purpose of these studies was to determine whether nitric oxide produced by cytokine-activated murine lung vascular endothelial cells plays a role in their lytic destruction of M-5076 reticulum cell sarcoma. Vascular endothelial cells harvested from perfused lungs of mice were adapted to grow in culture. Cloned lines ascertained to be of endothelial origin were incubated in vitro with interferon gamma and tumor necrosis factor. Lysis of radiolabeled tumor cells and accumulation of nitrite in the culture medium were determined at several time points. The concentration of nitrite in the culture medium directly correlated with endothelial cell-mediated tumor cell lysis. Endothelial cells cultured in L-arginine-free medium did not produce significant tumor cell lysis nor accumulation of nitrite in the medium. Both tumor cell lysis and nitrite accumulation were observed when the deficient medium was reconstituted with L-arginine, suggesting that endothelial cell-mediated tumor lysis was dependent on L-arginine, a precursor of nitric oxide. Moreover, specific inhibition of nitric oxide synthesis by NG-methyl-L-arginine resulted in complete inhibition of endothelial cell-mediated lysis of the M-5076 reticulum cell sarcoma. Similarly, treatment of cytokine-activated endothelial cells with dexamethasone inhibited both target cell lysis and production of nitrite. Collectively, these results suggest that nitric oxide plays a major role in the lysis of tumor cells mediated by cytokine-activated endothelial cells.

Escalated MVAC with or without recombinant human granulocyte-macrophage colony-stimulating factor for the initial treatment of advanced malignant urothelial tumors: results of a randomized trial.

PURPOSE: Hematopoietic growth factors have been shown to ameliorate the side effects of chemotherapy. Here we assess the ability of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) to increase the dose-intensity and reduce the side effects of escalated methotrexate, vinblastine, doxorubicin, and cisplatin (MVAC) chemotherapy. PATIENTS AND METHODS: A prospective randomized trial to compare escalated MVAC versus escalated MVAC with rhGM-CSF was conducted. All patients were treated at The University of Texas M.D. Anderson Cancer Center (UTMDACC) and had a metastatic or unresectable urothelial tumor. Forty-eight patients were randomized (25 to MVAC with rhGM-CSF and 23 to escalated MVAC alone). The clinical characteristics of the study populations were similar (ie, degree of tumor dissemination and performance status). RESULTS: The dose-intensity in the two arms of the study did not differ significantly. No difference in the frequency of bacteriologically documented infections occurred between the two study arms. CONCLUSION: The use of the hematopoietic growth factor rhGM-CSF did not result in an increased dose-intensity of escalated MVAC. The inability to increase the dose-intensity of MVAC further was a result of nonhematologic side effects of the chemotherapy. Escalation of treatment delivered at its median-tolerated dose is unlikely to result in additional therapeutic benefit for patients with common solid tumors. Future development of therapy may require the development of new agents or concepts, rather than modification of existing therapies.

A prospective randomized trial comparing MVAC and CISCA chemotherapy for patients with metastatic urothelial tumors.

To evaluate the relative efficacy of cisplatin, cyclophosphamide, and Adriamycin (doxorubicin; Adria Laboratories, Columbus, OH) (CISCA) versus methotrexate, vinblastine, Adriamycin, and cisplatin (MVAC), a prospective randomized trial was performed in patients with advanced metastatic urothelial tumors. Patients were stratified by histologic disease type and degree of tumor dissemination. Equal distribution of the clinical characteristics was achieved. One hundred ten patients with metastatic disease of the urinary tract (86 bladder, 16 renal pelvis, seven ureter, one prostatic urethra) met eligibility criteria and were enrolled on study. These represented 82% of the total patients seen during the study period in the Section of Genitourinary Oncology who met the eligibility criteria. The combined complete and partial response rate was significantly higher for patients treated with MVAC than for those treated with CISCA (65% v 46%; P less than .05). The survival duration of MVAC-treated patients was significantly longer than that of CISCA-treated patients (mean, 62.6 weeks; median, 48.3; range, 5.0+ to 162.3+ v mean, 40.4 weeks; median, 36.1; range, 7+ to 147.1+). We conclude that MVAC chemotherapy is superior to CISCA chemotherapy, achieving a higher response rate and a longer survival for equivalent patients with metastatic urothelial tumors.

Cationic liposome-mediated E1A gene transfer to human breast and ovarian cancer cells and its biologic effects: a phase I clinical trial.

PURPOSE: Preclinical studies have demonstrated that the adenovirus type 5 E1A gene is associated with antitumor activities by transcriptional repression of HER-2/neu and induction of apoptosis. Indeed, E1A gene therapy is known to induce regression of HER-2/neu-overexpressing breast and ovarian cancers in nude mice. Therefore, we evaluated the feasibility of intracavitary injection of E1A gene complexed with DC-Chol cationic liposome (DCC-E1A) in patients with both HER-2/neu-overexpressing and low HER-2/neu-expressing breast and ovarian cancers in a phase I clinical trial. PATIENTS AND METHODS: An E1A gene complexed with DCC-E1A cationic liposome was injected once a week into the thoracic or peritoneal cavity of 18 patients with advanced cancer of the breast (n = 6) or ovary (n = 12). RESULTS: E1A gene expression in tumor cells was detected by immunohistochemical staining and reverse transcriptase-polymerase chain reaction. This E1A gene expression was accompanied by HER-2/neu downregulation, increased apoptosis, and reduced proliferation. The most common treatment-related toxicities were fever, nausea, vomiting, and/or discomfort at the injection sites. CONCLUSION: These results argue for the feasibility of intracavitary DCC-E1A administration, provide a clear proof of preclinical concept, and warrant phase II trials to determine the antitumor activity of the E1A gene.

Potent inhibition of inducible nitric oxide synthase by geldanamycin, a tyrosine kinase inhibitor, in endothelial, smooth muscle cells, and in rat aorta.

We have examined whether specific protein tyrosine kinase (PTK) inhibitors (genistein, tyrphostin, or geldanamycin) prevent nitric oxide (NO.) production in rat smooth muscle cells (SMC), in murine brain endothelial cells (MBE), and in isolated rat aortas treated with endotoxin (LPS) and/or cytokines. Tyrphostin failed to inhibit either the release of nitrite in both endothelial and smooth muscle cells or vascular hyporeactivity in rat aorta, caused by immunostimulants. Genistein decreased nitrite production in MBE only at high concentration but had no effect on nitrite production in SMC and on the hypocontractility in aortic rings. In contrast, low concentrations of geldanamycin abolished the release of nitrite in MBE and in SMC treated with endotoxin and/or cytokines. Geldanamycin inhibited also the hypocontractility to phenylephrine in aortic rings treated with LPS or interleukin-1. This inhibitor failed to inhibit the release of nitrite and the vascular hyporeactivity once nitric oxide synthase (NOS) was induced by immunostimulants whereas methyl-L-arginine, an inhibitor of NOS, had significant effects. These data suggest that LPS- and cytokines-induced NO. production initiate a common signaling pathway involving a PTK that is inhibited by geldanamycin but not or slightly by tyrphostin or genistein at a point that precedes the induction of NOS.

Rapid, quantitative microassay for the monokine respiration inhibitory factor.

The murine monokine respiration inhibitory factor (RIF) induces lesions at Complex I and Complex II of the mitochondrial electron transport chain (ETC) of tumor cells; these lesions in the ETC appear closely linked with cytostasis of the targets. In this report we describe the use of the sensitive murine mammary adenocarcinoma line EMT-6 in a colorimetric microassay for the effects of RIF on the ETC and target replication. The participation of cytolytic molecules in this assay system was excluded because of the resistance of the target to their effects. The endpoint for the assay was the ETC-mediated reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to its colored formazan. The two major coupling sites of MTT in the ETC of EMT-6 cells are shown to be proximal to Coenzyme Q, detected either by malate oxidation through Complex I or succinate oxidation through Complex II. The assay was sensitive to both RIF-induced lesions at these dehydrogenases and to the cytostasis-linked reduction in target cell number. The assessment of ETC lesions by this microassay correlated directly with that determined by the less sensitive polarimetric assay based on oxygen consumption. We demonstrate the application of this microassay to parameters for the production of RIF by activated murine macrophages, and to initial molecular characterization of this mediator.

Characterization of the effects of two new arginine/citrulline analogues on constitutive and inducible nitric oxide synthases in rat aorta.

1. New potent inhibitors of nitric oxide synthase (NOS) may be useful in the treatment of septic shock, a disorder characterized by a vascular hyporeactivity to catecholamines caused by an overproduction of nitric oxide (NO-). We examined the effects of L-thiocitrulline (L-TC) and S-methyl-L-thiocitrulline (L-SMTC), novel NOS inhibitors, on the constitutive and inducible NOS in rat aorta and compared those effects with inhibition due to NG-methyl-L-arginine (L-NMA). 2. Phenylephrine evoked similar concentration-contraction curves in the control rings and in the rings treated with these different NOS inhibitors (10 microM), whereas 100 microM of L-NMA, L-TC or L-SMTC increased significantly, and to a similar extent, contractions evoked by phenylephrine in aortic rings with endothelium without significantly affecting the maximal responses. 3. Relaxations evoked by acetylcholine, adenosine triphosphate, or calcium ionophore were significantly inhibited in a dose-dependent manner by L-NMA, L-SMTC, or L-TC (10-100 microM). The potencies of these inhibitors in reducing the relaxations of these vasodilators were not significantly different. 4. In endotoxin-treated preparations with endothelium, the three L-arginine analogues (10 microM) significantly potentiated contractile responses to phenylephrine (pEC50: 6.73 +/- 0.12 and 7.3 +/- 0.12, 7.34 +/- 0.13, or 7.22 +/- 0.14; in the absence and the presence of L-NMA, L-TC, or L-SMTC respectively) and increased maximal contractions from 1.53 +/- 0.15 g to 1.95 +/- 0.13 g, 2.08 +/- 0.12 g, and 2.03 +/- 0.13 g with L-NMA, L-TC, and L-SMTC respectively. A higher concentration of these NOS inhibitors (100 microM)further increased contractions evoked by this alpha1-agonist without further enhancing the maximal contractions; however, contractions evoked by 10 nM phenylephrine were significantly greater in the presence of L-SMTC or L-TC than in the presence of L-NMA (100 microM) (L-NMA: 0.4 +/- 0.11 g; L-TC:0.78 +/- 0.14 g and L-SMTC: 0.82+/-0.17 g). The effects of these inhibitors on NO- synthesis induced by endotoxin were significantly reversed by addition of L-arginine (1 mM) but not by L-citrulline (1 mM). InLPS-treated rings with endothelium, all three NOS inhibitors (100 microM) shifted the concentration contraction curves evoked by phenylephrine significantly to the left (pEC5o: 7.19 +/- 0.03 and 7.79 +/- 0.08,8.01 +/- 0.07, or 8.02 +_ 0.07, in the absence and the presence of L-NMA, L-TC, or L-SMTC, respectively)and increased significantly maximal contractions from 2.05 +/- 0.05 g to 2.38 +/- 0.14 g, 2.5 +/- 0.12 g, and 2.4 +_ 0.21 g with L-NMA, L-TC, and L-SMTC, respectively. L-TC and L-SMTC were significantly more potent than L-NMA in potentiating contractions evoked by 10 nM and 30 nM phenylephrine.5. L-TC and L-SMTC produced dose-dependent increases in tone in LPS-treated aortic rings with and without endothelium. In LPS-treated rings with endothelium, L-NMA induced contractions but in preparations without endothelium low concentrations of L-NMA induced small contractions while high concentrations of this inhibitor evoked relaxations. In both preparations L-TC and L-SMTC were significantly more potent than L-NMA in increasing vascular tone.6. These results suggest that L-SMTC, L-TC and L-NMA were equipotent on basal and agonist stimulated NO- synthesis produced by the constitutive isoform of NOS, whereas the two new L-arginine analogues were more potent than L-NMA in inhibiting the production of NO- induced by endotoxin in rat aorta.

Beneficial versus detrimental effects of nitric oxide synthase inhibitors in circulatory shock: lessons learned from experimental and clinical studies.

Septic shock is a physiological derangement of the cardiovascular system characterized by pathological vasodilation. Recent studies have established a role for nitric oxide, previously known as endothelium-derived relaxing factor, in the vascular dysfunction of sepsis. This finding suggests that inhibition of nitric oxide synthase (NOS), the enzyme responsible for nitric oxide production, could be a target for therapeutic intervention. Animal studies have provided conflicting results, demonstrating both beneficial and detrimental effects. We provide here an overview of the preclinical studies of NOS inhibitors and an update of the clinical studies. The low toxicity and marked antihypotensive activity of NOS inhibitors in humans highlight some of the drawbacks of certain animal models and provide important insights into the experimental study of septic shock.

Cardiovascular effects of NG-methyl-L-arginine in chronically instrumented conscious dogs.

The cardiovascular effects of nitric oxide blockade were examined in five conscious chronically instrumented dogs. The hypothesis tested was that nitric oxide release plays a role in vascular tone and regional organ blood flow under physiological conditions. Aortic pressures; the first derivative of the left ventricular pressure; cardiac output (CO); heart rate; and carotid, coronary, renal, hepatic, and portal blood flows were recorded before and after bolus injection of 5, 10, and 20 mg/kg of NG-methyl-L-arginine (L-NMA). In response to L-NMA, mean arterial pressure increased by 7, 20, and 35%, respectively, in a dose-dependent manner, whereas CO decreased. CO reduction was sustained at the highest dose, whereas peripheral blood flows were not altered. These data suggest that blocking basal nitric oxide synthesis by administering L-NMA leads to a modest dose-dependent pressor response despite a marked and sustained reduction in CO recorded at the highest dose of L-NMA. Moreover, within our dose range, although the nitric oxide synthase inhibition provides a significant pressor response, it does not alter the resting carotid, coronary, renal, hepatic, and portal blood flows.

NG-methyl-L-arginine, an inhibitor of nitric oxide formation, reverses IL-2-mediated hypotension in dogs.

The effects of NG-methyl-L-arginine (L-NMA), an inhibitor of nitric oxide formation, were studied in dogs treated with interleukin-2 (IL-2). The administration of IL-2 to dogs resulted in hypotension within 3 days of treatment. The development of hypotension correlated with accumulation in the serum of nitrate, which is a stable breakdown product of nitric oxide. Administration of L-NMA decreased serum nitrate levels and increased the mean arterial pressure. The antihypotensive effect was dose dependent with a maximum effect observed at a dose of 20 mg/kg. Administration of a continuous infusion of L-NMA (5 mg.kg-1.h-1) maintained the mean arterial pressure for 48 h with concurrent administration of IL-2. Evaluation of IL-2-induced lymphokine-activated killer cell proliferation and tumoricidal activity toward a canine glioblastoma target cell line was unaffected by L-NMA. These studies imply that L-NMA may effectively ameliorate the dose-limiting hypotension associated with administration of IL-2 without adversely affecting the antitumor effects.

Design of nitric oxide synthase inhibitors and their use to reverse hypotension associated with cancer immunotherapy.

It is now just 10 years since it was first appreciated that NO is endogenously synthesized in mammals. In this period, two constitutive and one inducible isoform of NOS have been isolated, sequenced, and characterized with respect to their protein chemistry and catalytic mechanism. A wide variety of NOS inhibitors, most targeted to the arginine binding site in the oxygenase domain, have been synthesized and used to elucidate the physiological and pathophysiological roles of NO. It is now clear that NO is involved in signal transduction (e.g., in neurotransmission and blood pressure homeostasis), and that these roles are mediated by low concentrations of NO synthesized by nNOS or eNOS. The NO receptor is the heme cofactor of soluble isoform of guanylyl cyclase. Higher amounts of NO, typically but not always synthesized by iNOS, are often cytotoxic. At a minimum, high concentrations of NO derange the signal transduction pathways normally served by nNOS or eNOS. In addition, NO or its nitrosative products (RSNO, N2O3, or ONOO-) inhibit or damage cellular constituents, interfering with DNA synthesis, energy metabolism, and the structural integrity of the cell. Such cytotoxicity can be beneficial to the host if pathogens or tumor cells are destroyed, but is detrimental to the host if it results in inappropriate inflammation, hypotension, or immunosuppression. Therapeutic utility of NOS inhibitors has been demonstrated in sepsis and cytokine-induced hypotension; additional applications are being identified in a treatment of inflammatory and autoimmune disorders.

A phase I study of FAP (5-Fluorouracil, α-interferon and cisplatin) combined with methotrexate for the treatment of advanced urothelial malignancies.

Combination chemotherapy using 5-fluorouracil and α-interferon is active against advanced urothelial cancers. Its toxcity profile appears favorable such that addition of other active agents (i.e., cisplatin and methotrexate) may improve its therapeutic window. Our goal was to identify the starting dose of FAP (5-fluorouracil-α-interferon-cisplatin) that will permit delivery of methotrexate in patients with advanced chemotherapy-refractory urothelial cancers. This is a phase 1 study in which the dose level of FAP that permitted delivery of two doses of methotrexate in 28 days will be considered the maximum tolerated dose (MTD). This is based on experiences with MVAC chemotherapy in which only half of the patients received two doses of methotrexate in a 28 days cycle. The dose level identified as the MTD is as follows: 5-FU 500 mg/m(2) by intravenous continuous infusion daily for 5 days in weeks 1 and 4; α-interferon 5 MU/m(2) subcutaneously daily for 5 days simultaneously with 5-FU infusion in weeks 1 and 4 and thrice weekly during weeks 2 and 3; and cisplatin 25 mg/m(2) plus methotrexate 30 mg/m(2) intravenously once weekly for 4 weeks. The treatment will be repeated every 6 weeks. Most of the significant toxic effects, including mucositis, neutropenia, and thrombocytopenia, are encountered during weeks 3 and 4. This study showed that the MTD for FAP combined with methotrexate has been determined for phase II studies. Exploration of alterative regimen such as FAP in the treatment of advanced urothelial cancer is warranted. FAP has a unique mechanism of action and acceptable toxicity profile that may allow novel drug combinations and improved therapeutic efficacy.

Effects of N-methyl-L-arginine on cardiac and regional blood flow in a dog endotoxin shock model.

PURPOSE: Indirect evidence suggests a decrease in organ perfusion as a result of nitric oxide (NO) inhibition in endotoxic shock. Cardiac and regional hemodynamic responses to N-methyl-L-arginine (L-NMA), a nonspecific inhibitor of constitutive and inducible nitric oxide synthase (NOS), were assessed in nine conscious dogs subjected to endotoxin. MATERIALS AND METHODS: Lipopolysaccharide (LPS) was titrated to a maximum of 200 microg/kg, IV, over 45 minutes. L-NMA was given in a dose of 20 mg/kg, IV. Hemodynamic parameters were recorded for 6 hours following L-NMA administration. RESULTS: LPS induced significant decreases in mean arterial blood pressure (MAP), cardiac output (CO), first derivative of left ventricular pressure (dP/dt), coronary blood flow, carotid blood flow, mesenteric blood flow, renal blood flow, and a significant hepatic vasodilation. L-NMA fully reversed the effects of LPS on MAP, heart rate, dP/dt, coronary and carotid blood flow, and reversed mesenteric blood flow and hepatic blood flow at 1 and 3 hours, respectively. L-NMA partially overcame the LPS-induced decrease in renal blood flow at 30 minutes and 1 hour. Except for mesenteric and carotid circulation, L-NMA did not change regional vascular resistance. CONCLUSIONS: It is likely that constitutive NOS is implicated in immediate cardiac, carotid, mesenteric, and renal vascular changes, whereas inducible NOS accounted for delayed responses in hepatic and coronary circulation.

Nitric oxide and shock.

Shock can be defined as the failure of the circulatory system to provide necessary cellular nutrients, including oxygen, and to remove metabolic wastes. Although it is now recognized that more than 100 different forms of shock exist, this recognition is more a reflection of the widespread use of the term to describe a variety of disease states. For the purpose of this monograph, we concentrate on various forms of cardiovascular shock, in particular, shock that may be linked to inappropriate vasodilation from overproduction of the endogenous vasodilator, nitric oxide. Some forms of shock have been extensively studied, and convincing evidence exists for the role of nitric oxide. Other disease states have been less well characterized in terms of their association with excess nitric oxide production. Available evidence of a role for nitric oxide is discussed in the hope of stimulating the interest of investigators to explore these areas more thoroughly.

Drug-induced vasodilation in an in vitro and in vivo study: the effects of nicardipine, papaverine, and lidocaine on the rabbit carotid artery.

Extreme arterial vasoconstriction (vasospasm) is a common problem encountered in microvascular surgery. An ideal pharmacologic tool able to counteract ischemia during microsurgery should be easy to apply and exert its action both locally and distally in the microcirculation of the flap. We have compared in vitro and in vivo vascular properties of nicardipine, papaverine, and lidocaine in the rabbit carotid artery. In vitro, rings from the rabbit carotid artery (n = 7) were bathed in Krebs-Ringers solution and stretched progressively to an optimal tension of 3.7 to 4.2 g. The specimens were contracted with norepinephrine (1 microM), and a cumulative dose response curve was established. In vivo, microvascular anastomoses were performed bilaterally in the rabbit carotid artery in 35 animals using 9-0 nylon suture and standard microsurgical techniques. During and after the anastomoses, nicardipine (0.1, 0.01 mg topical, or 0.1 mg/hour IV), papaverine (30 mg/cc topical), and lidocaine (2% with and without epinephrine) were applied (blinded) at the anastomotic site in five rabbits each. Heparinized sodium chloride was used as topical irrigation for control and to clean the anastomosis. Blood flow changes were monitored continuously with the transonic Doppler for 30 minutes after the procedure. The systemic blood pressure was also monitored in a group of pilot experiments. A documented decrease in blood flow was noted in all animals after the microvascular anastomosis. Nicardipine and papaverine evoked a concentration-dependent relaxation to precontracted rings to norepinephrine. Nicardipine was greater than papaverine in inducing relaxation. Lidocaine demonstrated a biphasic response with low concentrations potentiating contraction. Systemic nicardipine and papaverine significantly increased the blood flow in the rabbit carotid artery. Topical application of nicardipine and lidocaine did not significantly alter the blood flow; however, the application of nicardipine demonstrates a trend toward increased flow. Lidocaine with epinephrine significantly decreased the blood flow. No drug was found to alter the blood pressure of the animals. Our results demonstrate that nicardipine and papaverine seem to be pharmacologic tools able to increase the blood flow in anastomotic arteries. In contrast, the use of 2% lidocaine as a spasmolytic agent should be re-evaluated, since this substance may act as a partial agonist.