Impaired functional activity of alveolar macrophages from GM-CSF-deficient mice.

We hypothesized that pulmonary granulocyte-macrophage colony-stimulating factor (GM-CSF) is critically involved in determining the functional capabilities of alveolar macrophages (AM) for host defense. To test this hypothesis, cells were collected by lung lavage from GM-CSF mutant mice [GM(-/-)] and C57BL/6 wild-type mice. GM(-/-) mice yielded almost 4-fold more AM than wild-type mice. The percentage of cells positive for the beta(2)-integrins CD11a and CD11c was reduced significantly in GM(-/-) AM compared with wild-type cells, whereas expression of CD11b was similar in the two groups. The phagocytic activity of GM(-/-) AM for FITC-labeled microspheres was impaired significantly compared with that of wild-type AM both in vitro and in vivo (after intratracheal inoculation with FITC-labeled beads). Stimulated secretion of tumor necrosis factor-alpha (TNF-alpha) and leukotrienes by AM from the GM(-/-) mice was greatly reduced compared with wild-type AM, whereas secretion of monocyte chemoattractant protein-1 was increased. Transgenic expression of GM-CSF exclusively in the lungs of GM(-/-) mice resulted in AM with normal or supranormal expression of CD11a and CD11c, phagocytic activity, and TNF-alpha secretion. Thus, in the absence of GM-CSF, AM functional capabilities for host defense were significantly impaired but were restored by lung-specific expression of GM-CSF.

Peroxynitrite-induced nitrotyrosination of proteins is blocked by direct 5-lipoxygenase inhibitor zileuton.

We have previously shown that the ability of overnight pretreatment with lipopolysaccharide (LPS) to suppress alveolar macrophage (AM) leukotrienes (LT) synthesis is explained by induction of nitric oxide (NO), and reactive oxygen intermediates (ROI). More recently we have demonstrated that the generation of peroxynitrite (ONOO-) from the combination of NO and ROI directly nitrotyrosinates the 5-lipoxygenase (5-LO) enzyme and reduces cell-free and intact AM 5-LO metabolism. This effect of ONOO- was associated with nitrotyrosination of the 5-LO enzyme in intact cells and after treatment of recombinant enzyme. We postulated that LPS treatment of cells resulted in activation of 5-LO with the generation of ROI, which in turn led to autoinactivation of the enzyme. In an effort to suppress ROI generated from activation of 5-LO we examined the effect of a direct 5-LO inhibitor on LPS-induced suppression of LT synthesis. Coincubation with the reversible 5-LO inhibitor zileuton during the LPS pretreatment of intact cells dose dependently blocked the inhibition of 5-LO metabolism by LPS. The effect of zileuton on LPS-induced suppression of LT synthesis was similar to that of N-monomethyl-L-arginine. Zileuton had no effect on inducible nitric-oxide synthase induction. Interestingly, zileuton blocked ONOO--induced nitrotyrosination of recombinant 5-LO in a cell-free system as well as of native enzyme in intact cells. Moreover, zileuton blocked the nitrotyrosination of other proteins. We conclude that the suppression of 5-LO activity occurring with LPS treatment can be blocked by zileuton. The mechanism by which zileuton is effective is in part explained by blocking nitrotyrosination of 5-LO.

Selective inhibition of COX-2 improves early survival in murine endotoxemia but not in bacterial peritonitis.

Prostaglandins of the E series are believed to act as important mediators of several pathophysiological events that occur in sepsis. Studies were performed to evaluate the effect of cyclooxygenase (COX)-2-specific inhibition on the outcome in murine endotoxemia and cecal ligation and puncture (CLP). We observed a significant time-dependent upregulation of PGE(2) production in both blood and lung homogenates of mice administered lipopolysaccharide intraperitoneally, which was nearly completely suppressed by the administration of the COX-2 inhibitor NS-398. Treatment with NS-398 significantly improved early but not late survival in lipopolysaccharide-challenged mice. On the contrary, elevated PGE(2) levels were found in bronchoalveolar lavage fluid but not in plasma of mice subjected to CLP (21 gauge). Pretreatment with NS-398 failed to significantly improve survival in CLP mice. No significant differences were noted in plasma or lung homogenate proinflammatory cytokine levels or lung neutrophil sequestration between the NS-398-treated and control groups. These results demonstrate that selective COX-2 inhibition confers early but not long-term benefits without affecting the expression of proinflammatory cytokines or the development of lung inflammation.

Pathogenic yeasts Cryptococcus neoformans and Candida albicans produce immunomodulatory prostaglandins.

Enhanced prostaglandin production during fungal infection could be an important factor in promoting fungal colonization and chronic infection. Host cells are one source of prostaglandins; however, another potential source of prostaglandins is the fungal pathogen itself. Our objective was to determine if the pathogenic yeasts Cryptococcus neoformans and Candida albicans produce prostaglandins and, if so, to begin to define the role of these bioactive lipids in yeast biology and disease pathogenesis. C. neoformans and C. albicans both secreted prostaglandins de novo or via conversion of exogenous arachidonic acid. Treatment with cyclooxygenase inhibitors dramatically reduced the viability of the yeast and the production of prostaglandins, suggesting that an essential cyclooxygenase like enzyme may be responsible for fungal prostaglandin production. A PGE series lipid was purified from both C. albicans and C. neoformans and was biologically active on both fungal and mammalian cells. Fungal PGE(x) and synthetic PGE(2) enhanced the yeast-to-hypha transition in C. albicans. Furthermore, in mammalian cells, fungal PGE(x) down-modulated chemokine production, tumor necrosis factor alpha production, and splenocyte proliferation while up-regulating interleukin 10 production. These are all activities previously documented for mammalian PGE(2). Thus, eicosanoids are produced by pathogenic fungi, are critical for growth of the fungi, and can modulate host immune functions. The discovery that pathogenic fungi produce and respond to immunomodulatory eicosanoids reveals a virulence mechanism that has potentially great implications for understanding the mechanisms of chronic fungal infection, immune deviation, and fungi as disease cofactors.

Prolonged exposure to lipopolysaccharide inhibits macrophage 5-lipoxygenase metabolism via induction of nitric oxide synthesis.

LPS from bacteria can result in the development of sepsis syndrome and acute lung injury. Although acute exposure to endotoxin primes leukocytes for enhanced synthesis of leukotrienes (LT), little is known about the effect of chronic exposure. Therefore, we determined the effect of prolonged LPS treatment on 5-lipoxygenase (5-LO) metabolism of arachidonic acid in alveolar macrophages (AM) and in peripheral blood monocytes. Pretreatment of AM with LPS caused time- and dose-dependent suppression of LT synthetic capacity. LPS pretreatment failed to inhibit arachidonic acid (AA) release. The fact that LPS inhibited LT synthesis from endogenous AA more than from exogenous AA suggested an effect on 5-LO-activating protein (FLAP). In addition, an inhibitory effect of LPS treatment on AM 5-LO activity was suggested by cell-free 5-LO enzyme assay. No effect on the expression of either 5-LO or FLAP proteins was observed. New protein synthesis was necessary for LPS-induced reduction of 5-LO metabolism in AM, and immunoblotting demonstrated marked induction of NO synthase (NOS). Inhibition by LPS was reproduced by an NO donor and was abrogated by inhibitors of constitutive and inducible NOS. Compared with AM, peripheral blood monocytes exhibited no suppression by LPS of 5-LO metabolism and no induction of inducible NOS. We conclude that prolonged exposure to LPS impairs AM 5-LO metabolism by NO-mediated suppression of both 5-LO and FLAP function. Because LT contribute to antimicrobial defense, this down-regulation of 5-LO metabolism may contribute to the increased susceptibility to pneumonia in patients following sepsis.

GM-CSF regulates bleomycin-induced pulmonary fibrosis via a prostaglandin-dependent mechanism.

To characterize the role of GM-CSF in pulmonary fibrosis, we have studied bleomycin-induced fibrosis in wild-type mice vs mice with a targeted deletion of the GM-CSF gene (GM-CSF-/- mice). Without GM-CSF, pulmonary fibrosis was worse both histologically and quantitatively. These changes were not related to enhanced recruitment of inflammatory cells because wild-type and GM-CSF-/- mice recruited equivalent numbers of cells to the lung following bleomycin. Interestingly, recruitment of eosinophils was absent in GM-CSF-/- mice. We investigated whether the enhanced fibrotic response in GM-CSF-/- animals was due to a deficiency in an endogenous down-regulator of fibrogenesis. Analysis of whole lung homogenates from saline- or bleomycin-treated mice revealed that GM-CSF-/- animals had reduced levels of PGE2. Additionally, alveolar macrophages were harvested from wild-type and GM-CSF-/- mice that had been exposed to bleomycin. Although bleomycin treatment impaired the ability of alveolar macrophages from wild-type mice to synthesize PGE2, alveolar macrophages from GM-CSF-/- mice exhibited a significantly greater defect in PGE2 synthesis than did wild-type cells. Exogenous addition of GM-CSF to alveolar macrophages reversed the PGE2 synthesis defect in vitro. Administration of the PG synthesis inhibitor, indomethacin, to wild-type mice during the fibrogenic phase postbleomycin worsened the severity of fibrosis, implying a causal role for PGE2 deficiency in the evolution of the fibrotic lesion. These data demonstrate that GM-CSF deficiency results in enhanced fibrogenesis in bleomycin-induced pulmonary fibrosis and indicate that one mechanism for this effect is impaired production of the potent antifibrotic eicosanoid, PGE2.

Regulation of 5-lipoxygenase metabolism in mononuclear phagocytes by CD4 T lymphocytes.

Alveolar macrophages (AM) are the primary resident effector cells in the alveolus. Leukotrienes (LT) are secreted by AM in their role as defender of the lung. 5-Lipoxygenase (5-LO) catalyzes the synthesis of LT in association with 5-LO-activating protein, termed "FLAP." AM demonstrate increased 5-LO metabolism compared to peripheral blood monocytes (PBM). Activated lymphocytes release mediators which upregulate 5-LO metabolism in PBM. The lymphocyte population of the lung consists predominantly of CD4+ helper constitutively "activated" T cells. We hypothesized that mediators released by pulmonary CD4+ T cells may upregulate and maintain of 5-LO metabolism in PBM as they enter the alveolar space and differentiate into AM. 5-LO metabolism in AM from CD4-depleted mice demonstrated reduced LT synthesis (LTC4: 66.9 +/- 8%; LTB4 61.4 +/- 6.2% control). The decrease in 5-LO metabolism was associated with reduced FLAP (30.1 +/- 14.5% of control) and 5-LO expression (49 +/- 13.7% of control). This defect in AM 5-LO metabolism in CD4-depleted mice was further associated with reduced LTC4 levels in bronchoalveolar lavage (BAL) fluid. In summary, factors secreted constitutively by lung lymphocytes, in particular CD4 cells, contribute to the increased 5-LO metabolism in AM.

Granulocyte-macrophage colony-stimulating factor upregulates reduced 5-lipoxygenase metabolism in peripheral blood monocytes and neutrophils in acquired immunodeficiency syndrome.

Leukotrienes (LT) are mediators derived from the 5-lipoxygenase (5-LO) pathway, which play a role in host defense, and are synthesized by both monocytes (peripheral blood monocyte [PBM]) and neutrophils (PMN). Because 5-LO metabolism is reduced in alveolar macrophages and PMN from acquired immunodeficiency syndrome (AIDS) subjects, we investigated the synthesis of LT by PBM and PMN from these subjects. There was a reduction (74.2% +/- 8.8% of control) in LT synthesis in PBM from human immunodeficiency virus (HIV)-infected compared with normal subjects. Expression of 5-LO (51.2% +/- 8.8% of control), and 5-LO activating protein (FLAP) (48.5% +/- 8.0% of control) was reduced in parallel. We hypothesized that this reduction in LT synthetic capacity in PBM and PMN was due to reduced cytokine production by CD4 T cells, such as granulocyte-macrophage colony-stimulating factor (GM-CSF). We treated 10 AIDS subjects with GM-CSF for 5 days. PBM 5-LO metabolism ex vivo was selectively increased after GM-CSF therapy and was associated with increased 5-LO and FLAP expression. PMN leukotriene B(4) (LTB(4)) synthesis was also augmented and associated with increased 5-LO, FLAP, and cytosolic phospholipase A(2) expression. In conclusion, as previously demonstrated for PMN, PBM from AIDS subjects also demonstrate reduced 5-LO metabolism. GM-CSF therapy reversed this defect in both PBM and PMN. In view of the role of LT in antimicrobial function, cytokine administration in AIDS may play a role as adjunct therapy for infections.

Alveolar lining fluid regulates mononuclear phagocyte 5-lipoxygenase metabolism.

The enzyme 5-lipoxygenase (5-LO) catalyses the synthesis of leukotrienes (LT), which are important in phagocytosis and killing of microorganisms. The alveolar macrophage (AM), the primary resident defender of the alveolar space, has a greater capacity for LT synthesis than its precursor, the peripheral blood monocyte (PBM). This study investigated whether the alveolar lining fluid (ALF) upregulates LT synthetic capacity in mononuclear phagocytes. Rat AM, peritoneal macrophages (PM) and ALF were obtained by lavage from pathogen-free animals. Human PBM were isolated from normal subjects. 5-LO metabolism and expression were measured with and without ALF. Rat ALF increased 5-LO metabolism (136.4+/-15.1% of control) in cultured PBM. This was associated with increased 5-LO activating protein (FLAP) (357+/-29.5 %), and 5-LO expression (188+/-31.3%). Culture of AM for 3 days resulted in a greater decrement in LTB4 synthesis (LTB4 15.4+/-6.9% of day 1) than in PM (54.7+/-8.3% of day 1), suggesting a greater dependence of AM 5-LO metabolism on ALF. 5-LO and FLAP expression decreased to a greater degree in AM than PM in culture. Furthermore, AM cultured with ALF maintained their LT synthetic capacity, FLAP and 5-LO expression compared with control cells cultured in medium alone. In conclusion, alveolar lining fluid increased 5-lipoxygenase metabolism in peripheral blood monocytes and maintained it in cultured alveolar macrophages, by a mechanism of increased 5-lipoxygenase and 5-lipoxygenase activating protein expression. This may boost host defence capabilities.

Granulocyte colony-stimulating factor administration to HIV-infected subjects augments reduced leukotriene synthesis and anticryptococcal activity in neutrophils.

Neutrophil (PMN) dysfunction occurs in HIV infection. Leukotrienes (LT) are mediators derived from the 5-lipoxygenase (5-LO) pathway that play a role in host defense and are synthesized by PMN. We investigated the synthesis of LT by PMN from HIV-infected subjects. There was a reduction (4.0+/-1.3% of control) in LT synthesis in PMN from HIV-infected compared with normal subjects. This was associated with reduced expression of 5-LO-activating protein (31.2+/-9.6% of normal), but not of 5-LO itself. Since HIV does not directly infect PMN, we considered that these effects were due to reduced release of cytokines, such as granulocyte colony-stimulating factor (G-CSF). We examined the effect of G-CSF treatment (300 microgram daily for 5 d) on eight HIV-infected subjects. PMN were studied in vitro before therapy (day 1) and on days 4 and 7. LTB4 synthesis was increased on day 4 of G-CSF treatment, and returned toward day 1 levels on day 7. 5-LO and 5-LO-activating protein expression were increased in parallel. As a functional correlate to this increase in PMN LT synthesis by G-CSF, we examined the effects on killing of Cryptococcus neoformans. Anticryptococcal activity of PMN from HIV-infected subjects was less than that of PMN from normal subjects. G-CSF treatment improved fungistatic activity of PMN. This increase in antifungal activity was attenuated by in vitro treatment with the LT synthesis inhibitor, MK-886. In conclusion, PMN from HIV-infected subjects demonstrate reduced 5-LO metabolism and antifungal activity in vitro, which was reversed by in vivo G-CSF therapy.

RANTES inhibits HIV-1 replication in human peripheral blood monocytes and alveolar macrophages.

Infection with human immunodeficiency virus (HIV)-1 most often leads to the development of acquired immune deficiency syndrome, which may manifest with opportunistic infections, many of which occur in the lung. Mononuclear phagocytes infected by HIV-1, being relatively resistant to its cytopathic effects, potentially act as a reservoir for the virus. The alveolar macrophage (AM), a differentiated lung tissue macrophage, is readily infected by HIV-1, after which the virus becomes relatively dormant. C-C chemokines, secreted by CD8 T lymphocytes and other cells, are known to suppress HIV replication in lymphocytes. In view of this observation, and the relative increase in CD8+ T lymphocytes during HIV-1 disease, particularly in the lung, we hypothesized that C-C chemokines might play a key role in suppressing HIV-1 replication in AM. We examined the effect of the C-C chemokines macrophage inflammatory protein (MIP)-1 alpha, MIP-1 beta, and regulated on activation normal T cell expressed and secreted (RANTES) singly and in combination on HIV-1 replication in peripheral blood monocytes (PBM) and AM infected in vitro. Our findings indicate that RANTES suppresses HIV-1 replication, as measured by reverse transcriptase activity, in PBM (41.3 +/- 15.2% of control, n = 3, P < 0.05) and AM (30.3 +/- 7.8% of control, n = 3, P < 0.05) in a dose-dependent manner. The other C-C chemokines had no significant effect singly (MIP-1 alpha PBM: 64.8 +/- 21.9%; AM: 115.0 +/- 2.4% of control; MIP-1 beta PBM: 68 +/- 19.6; AM: 63.3 +/- 26.2% of control) but modestly decreased HIV replication when incubated in addition to RANTES (24.5 +/- 6.5% of control). These observations suggest that RANTES plays a key role in modulating HIV-1 replication in mononuclear phagocytes in the blood and lung, and this may have therapeutic implications for prevention and/or treatment of HIV disease.

5-Lipoxygenase metabolism in alveolar macrophages from subjects infected with the human immunodeficiency virus.

Pulmonary infection represents a major source of morbidity and mortality in AIDS. One important component of pulmonary host defense is the elaboration by resident alveolar macrophages (AM) of proinflammatory leukotrienes (LT) and other 5-lipoxygenase (5-LO) metabolites of arachidonic acid (AA). In this study, we compared the 5-LO metabolic capacity of AM isolated from normal controls with two groups of HIV-infected subjects: (1) patients with low CD4 counts undergoing diagnostic evaluation for pulmonary indications, and (2) volunteers without pulmonary complaints stratified into normal (> 500) and low (< 200) CD4 count groups. Compared with AM from control subjects, AM from HIV-infected subjects with normal and low CD4 counts demonstrated a marked reduction in LT synthesis. This reduced metabolic capacity could not be attributed to in vivo activation because there was no increase in lavage fluid LTB4 levels. However, there was a reduction (approximately twofold) in 5-LO protein expression in both the normal and the low CD4 subsets. 5-LO-activating protein (FLAP) expression was unchanged in cells from the normal CD4 HIV group, but was decreased threefold in the two groups with low CD4 counts. These observations indicate that there is a graded defect in the 5-LO metabolic capacity of AM from HIV-infected subjects, with decreased expression of only 5-LO in the normal CD4 group, and decreased expression of both 5-LO and FLAP in the low CD4 group. This defect would be expected to compound the immunosuppression seen in these subjects.

Effects of granulocyte-macrophage colony-stimulating factor on eicosanoid production by mononuclear phagocytes.

Granulocyte-macrophage CSF (GM-CSF) primes granulocytes for leukotriene (LT) synthesis. Here, we examined the effects of GM-CSF on arachidonic acid (AA) metabolism in rat alveolar macrophages (AM), peritoneal macrophages, and human peripheral blood monocytes. Pretreatment of AMs with GM-CSF for 24 h significantly increased the synthesis of immunoreactive LTB4 upon subsequent stimulation with calcium ionophore. Enhanced LT synthesis required a minimum of 6 h of GM-CSF pretreatment, suggesting that protein synthesis was required for enhanced LT production; indeed, cycloheximide completely abolished the GM-CSF effect on LT synthesis. HPLC analysis confirmed that GM-CSF primed AMs for enhanced generation of LTB4, as well as the 5-lipoxygenase products LTC, and 5-HETE. Moreover, parallel increases in other AA metabolites and free AA were observed following GM-CSF pretreatment. The enhanced production of all AA metabolites indicated that GM-CSF up-regulated AA release. Consistent with this, whole cell lysates from GM-CSF-treated AMs demonstrated increased phospholipase A2 (PLA2) activity. The increased activity was resistant to DTT, indicating the involvement of a PLA2 other than the 14-kDa PLA2s. By immunoblot analysis, GM-CSF treatment caused an increase in the 85-kDa PLA2 protein comparable to the observed increase in PLA2 activity. Unlike AMs, neither peritoneal macrophages nor peripheral blood monocytes showed increased eicosanoid generation or increased expression of the 85-kDa PLA2 protein following GM-CSF pretreatment. These results indicate that GM-CSF increases the capacity of AMs, but not peritoneal macrophages or peripheral blood monocytes, to generate eicosanoids. This effect results from increased PLA2 activity, due at least in part to increased levels of the 85-kDa PLA2 protein.

Differential inhibitory effects of antidiabetic drugs on arterial smooth muscle cell proliferation.

We compared three drugs representing different classes of antidiabetic pharmacology (glyburide, a sulfonylurea; pioglitazone, a thiazolidinedione; and metformin, a biguanide) in terms of their direct effects on proliferation of cultured arterial smooth muscle cells (SMC). Rat aortic SMC were seeded at 4 x 10(4)/35 mm well. After 24 h, they were treated every 2 to 4 days for 2 weeks with 5% fetal bovine serum (FBS) in normal culture medium containing either drug vehicles or a low and a high but nontoxic level of glyburide (0.5 and 2.5 mumol/L), pioglitazone (1 and 5 mumol/L), and metformin (20 and 100 mumol/L). Vehicle-treated cells increased from 2 +/- 0 to 6 +/- 1 to 42 +/- 3 to 210 +/- 14 (cells per well x 10(4); 5 wells each) from day zero to 4 to 9 to 14. From day 9 to 14 these cell numbers were decreased an average of 20% by the 2.5 mumol/L glyburide (P < .05) and 43% by the 5 mumol/L pioglitazone (P < .05). The low levels of glyburide and pioglitazone and both the low and high levels of metformin failed to influence cell numbers. In a second experiment, even half the abovementioned high level of pioglitazone (2.5 mumol/L) still exerted a markedly greater antiproliferative effect on aortic SMC than a high level of 2.0 mumol/L glyburide (P < .05). In addition, neither drug's antiproliferative effect was influenced by a high level of insulin added to the medium (10 mU/mL). Similarly, a small but significant stimulatory effect of this high insulin on cell proliferation (P < .05) was not significantly affected by these two drugs (although pioglitazone tended to inhibit it). These results suggest that thiazolidinediones may be more useful antidiabetic agents than sulfonylureas and biguanides in inhibiting abnormal arterial SMC proliferation associated with atherosclerosis and postangioplastic restenosis which are common in diabetic patients.

Reduced 5-lipoxygenase metabolism of arachidonic acid in macrophages rrom 1,25-dihydroxyvitamin D3-deficient rats.

The peripheral blood monocyte (PBM) migrates into tissues and differentiates into mature tissue macrophages. Previous investigations from our laboratory have demonstrated that PBM have reduced 5-lipoxygenase (5-LO) metabolism of arachidonic acid (AA) and 5-LO activating protein (FLAP) expression as compared to differentiated alveolar macrophages (AM). Moreover, PBM differentiated with 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) displayed increased leukotriene synthesis and a parallel increase in FLAP expression. In the present study, we sought to examine the physiological role of 1,25-(OH)2D3 in the regulation of eicosanoid metabolism in terminally differentiated alveolar and peritoneal macrophages (PM), utilizing a well characterized rat model of vitamin D3-deficiency. AM from vitamin D3-deficient rats demonstrated reduced 5-LO metabolism of AA and a parallel reduction in FLAP expression compared to control rats. Similarly, PM from vitamin D3-deficient rats demonstrated reduced 5-LO metabolism of AA. The effect of vitamin D3 was specific for the 5-LO pathway, not affecting total release of AA or its metabolism via 12-lipoxygenase or cyclooxoygenase (COX) pathways in macrophages. Furthermore, it did not affect COX protein expression in macrophages or type II alveolar epithelial cells. In control animals, 1,25-(OH)2D3 concentrations were greater in bronchoalveolar lavage fluid (2.6-fold) and peritoneal lavage fluid (1.6-fold) than in serum, which may account for the greater FLAP expression in AM and PM than in PBM. These observations suggest that 1,25-(OH)2D3 plays a physiological role in upregulating the 5-LO pathway in tissue macrophages in vivo.

Sex-specific effects of an insulin secretagogue in stroke-prone hypertensive rats.

Glyburide, an insulin secretagogue and an insulin-sensitizing agent, lowers blood pressure in normal male and female dogs when administered acutely. Because insulin resistance may contribute to spontaneous hypertension in rats, we sought to determine if long-term administration of glyburide (5 mg/kg per day by diet, age 5 weeks to 5 months) would lower blood pressure in male and female stroke-prone spontaneously hypertensive rats. Arterial (aortic) rings from these rats were incubated with insulin in vitro (100 mU/mL) 1 hour before and during phenylephrine-induced contraction to determine if long-term glyburide administration improves vascular sensitivity to the intrinsic vasodilator action of insulin. Glyburide, however, significantly increased blood pressures and ratios of heart weight to body weight in 5-month-old female rats (+20 mm Hg diastolic, P < .05), with no significant change noted in male rats (+4 mm Hg diastolic). Glyburide increased plasma insulin levels (twofold, P < .04) in female but not in male rats. Glyburide did not affect plasma glucose or catecholamine levels. After incubation with insulin, aortic to rings from glyburide-treated female rats demonstrated more than 40% greater contractile responsiveness the phenylephrine compared with aortic rings from control female rats (P < .04). This insulin-dependent increase in phenylephrine-induced contraction consisted of a reversal in the in vitro action of insulin, from attenuation to accentuation of such contraction (P < .05). This change was not seen in male rats. Neither gender, glyburide, nor insulin influenced acetylcholine-induced relaxation of phenylephrine-induced contraction.(ABSTRACT TRUNCATED AT 250 WORDS)