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Myocardial endotoxin tolerance may be induced in both males and females; however, it remains unknown whether there are mechanistic and threshold differences between the sexes. We hypothesized that endogenous estrogen mediates a higher threshold for endotoxin (ETX)-induced protection in females. Adult proestrus and ovariectomized (OVX) female rats were preconditioned (PC) with intraperitoneal injections of 125 (PC+125) or 500 (PC+500) μg/kg Salmonella typhimurium LPS (ETX) or normal saline (PC−). Twenty-four hours later, injury dose ETX (500 μg/kg) was injected. After 6 h, myocardial function was measured via Langendorff. p38 MAPK and JNK activation and TNF-α, IL-1, and IL-6 expression were evaluated. ETX injury significantly decreased left ventricular developed pressure in PC− groups vs. controls. PC+500 regimen protected against ETX injury, resulting in normal cardiac function. PC+125 regimen protected OVX but not proestrus females, which had diminished myocardial function. Activated JNK and TNF-α increased in PC− but were diminished in PC+500 animals. Importantly, activated JNK and TNF increased in PC+125 proestrus females, whereas PC+125 OVX females displayed decreases in these molecules. There were no differences in p38 MAPK activation or expression of IL-1 or IL-6. These results demonstrate that proestrus females require a higher stimulus (PC+500) to achieve myocardial protection against ETX injury. Removal of endogenous estrogen (OVX) lowered the preconditioning threshold (PC+125), resulting in protection after lesser injury. Additionally, myocardial JNK and TNF expression was decreased in OVX PC+125 females, which correlated with myocardial function differences. Therefore, we conclude that endogenous estrogen mediates a higher threshold for ETX tolerance in female myocardium.

Hypoxic pulmonary vasoconstriction is a challenging clinical problem with limited therapeutic options. Milrinone, a phosphodiesterase (PDE)-3 inhibitor, is frequently used to treat perioperative pulmonary hypertension. However, recent evidence suggests that the PDE-5 isoform may be more specific for lung tissue. We hypothesized that the PDE-5 inhibitor zaprinast has greater efficacy for pulmonary vasorelaxation, attenuation of hypoxic pulmonary vasoconstriction, and inhibition of hypoxia-induced pulmonary artery cytokine expression when compared with milrinone. To study this, isolated rat pulmonary artery and thoracic aorta rings suspended in physiologic organ baths for measurement of isometric force transduction were treated with vehicle (dimethyl sulfoxide), milrinone, or zaprinast to assess pulmonary artery relaxation, thoracic aorta relaxation, inhibition of hypoxic (pO2 = 30-35 mmHg) pulmonary vasoconstriction, and hypoxia-induced pulmonary artery TNF-alpha and IL-1beta expression (reverse transcriptase-PCR). Milrinone and zaprinast resulted in dose-dependent pulmonary artery and aortic relaxation, but zaprinast caused significantly less aortic relaxation compared with milrinone (50.12% +/- 3.36% versus 91.03% +/- 2.97%, P < 0.001). Zaprinast, but not milrinone, significantly inhibited hypoxic pulmonary vasoconstriction (zaprinast, 58.42% +/- 5.37%; milrinone, 77.65% +/- 4.42% versus vehicle: 74.42% +/- 7.54%). Hypoxia-induced upregulation of TNF-alpha and IL-1beta mRNA in pulmonary artery was decreased by zaprinast, but not milrinone, pretreatment. These results suggest that zaprinast, but not milrinone, preferentially vasodilates pulmonary artery over aorta, attenuates hypoxic pulmonary vasoconstriction, and inhibits hypoxia-induced pulmonary artery TNF-alpha and IL-1beta expression. Therefore, PDE-5 inhibition may be advantageous in the treatment of pulmonary hypertension.

Background Preconditioning is injury induced protection against subsequent insult. Studies have shown that both males and females may be preconditioned. Females appear to have an innate cardioprotection, therefore, we hypothesized that the preconditioning threshold may differ between males and females. Materials and methods Male and female rats were divided into five groups ( n = 4–9). Animals were given an intraperitoneal (i.p.) injection of 125, 250, or 500 micrograms/kilogram Salmonella typhimurium lipopolysaccharide (ETX) or 0.4-ml normal saline (NS). After 24-h incubation another i.p. injection of either 500 micrograms/kilogram ETX (injury dose) or NS was given and the animals incubated an additional 6 h. Shams received two injections of NS. Non-preconditioned rats (PC-) received NS followed by ETX. Preconditioned rats had i.p. injections of 125 μg/kg ETX (PC +125), 250 μg/kg ETX (PC +250), or 500 μg/kg ETX (PC +500) followed by the injury dose ETX. The rats were then anesthetized and myocardial function evaluated with the Langendorff perfusion model. Results PC +500 females were preconditioned and were able to maintain cardiac function similar to shams. Conversely, PC +125 females were not preconditioned with this stimulus and had a significant decrease in cardiac function similar to PC- rats. Male PC +500 and PC +125 rats, however, retained cardiac function comparable to shams, while PC- males showed a significant decrement. Conclusions Males and females may each be preconditioned by endotoxin; however, the preconditioning threshold is higher in females than males.

Pulmonary arteries exhibit a marked vasoconstriction when exposed to hypoxic conditions. Although this may be an adaptive response to match lung ventilation with perfusion, the potential consequences of sustained pulmonary vasoconstriction include pulmonary hypertension and right heart failure. Concomitant production of proinflammatory mediators during hypoxia may exacerbate acute increases in pulmonary vascular resistance. We hypothesized that acute hypoxia causes pulmonary arterial contraction and increases the pulmonary artery tissue expression of proinflammatory cytokines via a protein kinase C (PKC)-mediated mechanism. To study this, isometric force displacement was measured in isolated rat pulmonary artery rings during hypoxia in the presence and absence of the PKC inhibitors calphostin C or chelerythrine. In separate experiments, pulmonary artery rings were treated with the PKC activator thymeleatoxin for 60 min. After hypoxia, with or without PKC inhibition, or PKC activation alone, pulmonary artery rings were subjected to mRNA analysis for TNF-alpha and IL-1beta via RT-PCR. Our results showed that, in isolated pulmonary arteries, hypoxia caused a biphasic contraction and increased expression of TNF-alpha and IL-1beta mRNA. Both effects were inhibited by PKC inhibition. PKC activation resulted in pulmonary artery contraction and increased the pulmonary artery expression of TNF-alpha and IL-1beta mRNA. These findings suggest that hypoxia induces the expression of inflammatory cytokines and causes vasoconstriction via a PKC-dependent mechanism. We conclude that PKC may have a central role in modulating hypoxic pulmonary vasoconstriction, and further elucidation of its involvement may lead to therapeutic application.

Endotoxemia causes paradoxical effects on the systemic and pulmonary vasculature, resulting in systemic hypotension and increased pulmonary artery pressure. The local production of inflammatory mediators may have important effects on vascular tissue function. The purpose of this study was to delineate differences in function and the expression of tissue cytokine genes in the aorta and pulmonary artery after endotoxemia.Thoracic aorta and pulmonary artery branches were isolated from adult Sprague- Dawley rats (n = 4-6/group) 6 h after intraperitoneal injection of lipopolysaccharide (Salmonella typhimurium, 20 mg/kg) or vehicle (1.0 mL of saline). Arteries were suspended in perfused organ baths for measurement of isometric force transduction, and dose-response curves to phenylephrine (0.01-10 micromol/L), acetylcholine (0.01-10 micromol/L), and sodium nitroprusside (0.001-10 micromol/L) were generated. The vascular segments were also assessed for expression of tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta, IL-6, and inducible nitric oxide synthase (iNOS) messenger RNA (mRNA) by semiquantitative reverse transcriptase- polymerase chain reaction.Endotoxemia resulted in decreased contractility of the aorta (508.63 +/- 81.89 mg vs. 2544.16 +/- 142.05 mg in the vehicle group) and pulmonary artery (352.50 +/- 38.11 mg vs. 535.83 +/- 45.51 mg in the vehicle group) and decreased endothelium-dependent pulmonary artery relaxation (52.86 +/- 5.63% vs. 80.58 +/- 6.39% in the vehicle group). Expression of IL-1beta and iNOS mRNA by the pulmonary artery, but not the aorta, increased significantly in the endotoxintreated animals. Interleukin-6 was increased in both the pulmonary artery and the aorta during endotoxemia, whereas TNF concentrations were unchanged.Endotoxemia may cause aortic hypocontractility and impaired endothelium-dependent pulmonary artery vasorelaxation. Expression of inflammatory genes in vascular tissue may be site-specific and may contribute to the functional derangements associated with sepsis.

Background Mortality after acute respiratory distress syndrome is higher in males than in females. Gender differences in pulmonary vascular reactivity and local inflammatory response may explain this disparity. We hypothesized that endothelium-dependent pulmonary vasorelaxation is impaired in males and that this effect is related to differences in local inflammatory cytokine expression from the pulmonary vasculature. Methods Pulmonary artery (PA) rings (n = 12-16 per group) were isolated from adult male and female Sprague-Dawley rats treated with endotoxin (Salmonella typhimurium lipopolysaccharide, 20 mg/kg IP) or vehicle (0.9% normal saline), and connected to force transducers for measurement of isometric force displacement. Dose-response curves (0.01-10 μmol/L) to the endothelium-dependent vasodilator acetylcholine and the endothelium-independent vasodilator sodium nitroprusside were generated. PA rings were also evaluated for inducible nitric oxide synthase, tumor necrosis factor-alpha, and interleukin 1β messenger RNA (mRNA) by reverse transcriptase-polymerase chain reaction. Results Endotoxin had no effect on the maximum PA contraction in males (564.4 ± 37.37 mg vs 633.3 ± 54.67 mg vehicle) or females (446.3 ± 20.00 mg vs 444.2 ± 33.02 mg vehicle), but endothelium-dependent vasodilation was significantly decreased in males (47.49 ± 5.63% vs 77.61 ± 9.41% vehicle). Endothelium-independent vasodilation remained intact during endotoxemia. Endotoxin increased the PA expression of inducible nitric oxide synthase mRNA, but there was no gender difference. There was no change in expression of PA tumor necrosis factor, whereas endotoxemic males, but not females, had increased interleukin 1β mRNA, compared with vehicle. Conclusions These results suggest that sepsis-induced vascular dysfunction differs between males and females, and, therefore, treatment of acute lung injury may require gender-specific therapies.

Background Preconditioning is injury-induced protection from subsequent insult. Recent data indicates that males have lower preconditioning thresholds compared to females. Therefore, we hypothesized that testosterone may mediate the lower preconditioning threshold observed in males. Materials and methods Adult normal and castrated male Sprague-Dawley rats (n = 4–5) were given intraperitoneal (i.p.) injections of 125 or 500 μg/kg Salmonella typhimurium lipopolysaccharide (ETX) or 0.4 ml normal saline (NS). Another i.p. injection of 500 μg/kg ETX (injury dose) was given 24 h later. After 6 h, myocardial function was evaluated via the Langendorff perfusion model. Shams received only NS, while non-preconditioned rats (PC-) received NS followed by the 500 μg/kg ETX injury dose. Preconditioned rats received injections of 125 μg/kg ETX (PC +125) or 500 μg/kg ETX (PC +500), followed by the 500 μg/kg ETX injury dose. Results Normal PC +125 and PC +500 males were preconditioned and maintained cardiac function similar to shams (P > 0.05). Castrated PC +125 and PC +500 males were also preconditioned and maintained cardiac function similar to castrated shams (P > 0.05). Conversely, both normal and castrated PC-males showed significantly decreased cardiac function compared to shams (P Conclusions Endogenous testosterone does not mediate the lower preconditioning threshold in males.

Background Preconditioning is injury-induced protection against subsequent injury and may be induced by a variety of stimuli. Both males and females may be preconditioned; however, if females are relatively protected against the initial insult, is their preconditioning threshold higher? We hypothesized that preconditioning injury threshold differences may exist between genders, which may be associated with differences in myocardial inflammatory monokine production. Methods Male and female Sprague-Dawley rats (n = 3-5/group) were given intraperitoneal injections of 125 or 500 μg/kg Salmonella typhimurium lipopolysaccharide (ETX) or 0.4 mL normal saline (NS; 154 mmol/L NaCl). After 24 hours, another injection of 500 μg/kg ETX (injury dose) or NS was given, and the animals were incubated an additional 1 or 6 hours. The rats were anesthetized and myocardial function evaluated via the Langendorff perfusion model. Tumor necrosis factor-α (TNF-α), interleukin-(IL)-1β, and IL-6 were measured in 1-hour animals via an enzyme-linked immunosorbent assay. Nonpreconditioned rats (PC-) received NS followed by ETX. Preconditioned rats received either 125 μg/kg ETX (PC+125) or 500 μg/kg ETX (PC+500) followed by injury dose ETX. Results PC+125 and PC+500 males, as well as PC+500 females, were preconditioned and retained cardiac function similar to shams. PC+125 females were not preconditioned with this stimulus and had a decrease in cardiac function similar to PC- rats. Furthermore, PC+125 and PC+500 males, and PC+500 females had decreased release of TNF-α after preconditioning, while PC- animals and PC+125 females did not. Conclusions Males and females can be preconditioned by endotoxin; however the preconditioning threshold is higher in females than males.

Background. Pulmonary arteries exhibit a marked vasoconstriction when exposed to hypoxic conditions. Although this may be an adaptive response to match lung ventilation with perfusion, the potential consequences of sustained pulmonary vasoconstriction include pulmonary hypertension and right heart failure. The concomitant production of proinflammatory mediators by the pulmonary artery itself may exacerbate acute increases in pulmonary vascular resistance. We hypothesized that acute hypoxia causes pulmonary arterial contraction and increases the pulmonary artery tissue expression of proinflammatory cytokines via a protein kinase C (PKC) mediated mechanism. Methods. Isometric force displacement was measured in isolated rat pulmonary artery rings during hypoxia (95% N2/5% CO2, pO2 = 30–35 mmHg) in the presence and absence of the protein kinase C inhibitor chelerythrine (1 μmol/L). Following 60 min of hypoxia, pulmonary artery rings were subjected to mRNA analysis for TNF-μ, IL-1β, and iNOS via RT-PCR. Data were analyzed using two-way analysis of variance (ANOVA) with post-hoc Bonferonni test or unpaired t tests with alpha level less than 0.05 considered statistically significant. Results. Hypoxia caused a biphasic contraction: an early and delayed contraction which occurred 1–3 and 15–20 min, respectively, after the onset of hypoxia. Hypoxic pulmonary artery tissue had increased expression of TNF-μ, IL-1β, and iNOS mRNA compared to normoxic controls. PKC inhibition significantly (P