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Nitric oxide is believed to participate in nonspecific cellular immunity. Gram negative bacterial endotoxins increase the production of reactive nitrogen intermediates (RNI) in phagocytic cells by inducing the enzyme nitric oxide synthase II (NOS II). Anti-inflammatory glucocorticoids attenuate endotoxin-induced increases in RNI. This study evaluated the effect of in vivo administration of prednisolone on Escherichia coli lipopolysaccharide endotoxin (LPS)-induced increases in plasma RNI and neutrophil mRNA for NOS II and production of RNI in the rat. We show that LPS rapidly induces mRNA for NOS II and production of RNI (NO2- and NO3- anion) in rat neutrophils within 2 hr after in vivo administration of a sublethal dose of 0.5 mg/kg, i.v. A pharmacologic dose of prednisolone (50 micrograms/kg, im) given 15 min before LPS-attenuated production of NO2- and NO3- by neutrophils and suppressed LPS-stimulated mRNA for NOS II. 3-Amino, 1,2,4-triazine inhibited NO2- and NO3- production without affecting gene expression for NOS II. These data demonstrate that LPS rapidly induces functional gene expression for NOS II and prednisolone prevents induction of NOS II activity by inhibiting transcription of its mRNA.

Tumor necrosis factor-alpha (TNF-alpha) stimulates nitric oxide (NO) in vascular endothelium by induction of the enzyme NO synthase II (NOS II). We examined the effects of TNF-alpha on 1) endothelium-dependent (EDR) and endothelium-independent (EIR) relaxation and 2) contraction of bovine intralobar pulmonary arteries (BPA) and veins (BPV) in vitro. Acetylcholine (ACh), bradykinin (BK), histamine, and A23187 produced EDR of BPA contracted with a 50% effective concentration of U-46619 (15 nM), because relaxation was abolished by endothelium-rubbing and attenuated by L-NG-mono-methylarginine (L-NMMA; 300 microM). TNF-alpha (0.00417, 0.0417, 0.417, and 1.25 micrograms/ml) incubated with BPA for 60 min inhibited EDR of the BPA to ACh, BK, and histamine. The effects of TNF required 30 min for onset. Recovery of EDR occurred 3-4 h after washout of TNF-alpha. Pentoxifylline (1 microM) did not affect ACh-induced EDR but selectively reversed TNF-alpha-mediated inhibition of ACh-induced EDR. TNF-alpha-mediated inhibition of EDR was not reversible by L-NMMA, an inhibitor of NOS I and NOS II, the cyclooxygenase inhibitor ibuprofen, or CV-3908 (1 microM), a platelet-activating factor antagonist. The inhibitory effect of TNF-alpha on EDR was not mediated by nonspecific sensitization of the endothelium to human protein because recombinant human granulocyte colony-stimulating factor (10, 50, and 500 x 10(3) U/ml) did not affect EDR of BPA. The effect of TNF-alpha was specific for release of NO from the endothelium of BPA because TNF-alpha did not affect 1) EDR of BPV to ACh, BK, or ATP; 2) EIR of BPA or BPV to nitroprusside; and 3) contraction of either BPA or BPV to KCl, U-46619, histamine, norepinephrine, or serotonin. Thus TNF-alpha appears to selectively inhibit receptor-mediated EDR and NO release in BPA. TNF-alpha-mediated inhibition of EDR differs from that of L-arginine-based inhibitors and may represent an endogenous physiological mechanism of regulation of NO in the endothelium.

In isolated bath studies smooth muscle from the rat portal vein was evaluated for its reactivity and contractility, and the whole vessel wall was evaluated for its extensibility. Smooth muscle from the spontaneously hypertensive rat (SHR) had the following characteristics when compared with that from normotensive controls: (1) Spontaneous phasic contractions were more frequent and developed more tension; (2) threshold concentrations for responses to prostaglandins A-2 and B-2 were lower, but those for responses to epinephrine, norepinephrine, KCl, BaCl-2, and SrCl-2 were similar; (3) high concentrations of calcium had a less depressant action on the responses to the prostaglandins but not on the responses to the other agonists; (4) maximal contractile tensions to all agonists were greater; and (5) passive extensibility was less. These differences, because they are in the venous system, cannot be secondary to the increase in wall stress of arterial hypertension. The decreased passive extensibility in this vein in SHR creates a stiffer framework on which the active contractile process is able to develop greater tension. If this increase in active tension is generalized to all veins, it could be responsible for a decrease in venous capacity which increases the rate of venous return and, hence, increases cardiac output.

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Recent studies using chemiluminescence and spectrophotometry have shown that cultured and native endothelial cells release nitric oxide (NO). Pharmacological and biochemical evidence argue for and against the proposal that endothelium-derived relaxing factor (EDRF) is identical with free NO. In an attempt to identify EDRF as free NO, a bioassay technique was combined with an NO trap (hemoglobin bound to agarose; Ag-Hb), and electron paramagnetic resonance (EPR) spectroscopy was used to detect the resultant nitrosylhemoglobin (NO-Hb). Canine femoral arteries with or without endothelium were perfused with physiological saline solution containing superoxide dismutase and ibuprofen and were stimulated with acetylcholine. The relaxing activity of the effluent was monitored in canine coronary artery rings without endothelium (bioassay tissue) half-maximally contracted with U46619. Acetylcholine stimulated the release of EDRF from intact femoral arteries (but not from segments without endothelium), which relaxed the bioassay tissue by 63+±5%. NO (-1 and -10 nM) infused directly over the bioassay tissue produced 34±8% and 96±3% relaxation, respectively (ED%, --2 nM). Effluents were collected under vacuum in the absence of oxygen through a column containing Ag-Hb, and the samples were assayed for NO-Hb by EPR. Samples containing NO produced the triplet EPR signal characteristic of NO-Hb, but the effluent containing EDRF did not. Infusion of NO through the donor tissue in the presence of acetylcholine gave an EPR signal similar to that observed when NO had no contact with the tissue. Nitrite anion (up to 2.7X 102 M) produced no detectable NO-Hb in analogous experiments. Thus, EDRF released from the native endothelium of canine femoral artery cannot be identified as free NO. The present findings support a concept that EDRF may be a labile precursor of NO. (Circulation Research 1990;67:1446-1452 E ndothelium-derived relaxing factor (EDRF) is a potent vasodilator substance released by the endothelium.1-3 Recently, it has been proposed that EDRF may be identical with nitric oxide (NO).4-6 Both pharmacological and biochemical evidence support this proposal.4-11 NO and EDRF both are inactivated by superoxide anion and hemoglobin and are protected by superoxide dismutase.4-9 The biological half-life of EDRF and NO is similar.4-9 Finally, the release of EDRF by bradykiFrom the

Hyperglycemia can upregulate protein kinase C (PKC), which may be an important mediator of the progression from normal heart and muscle function to diabetic myopathy in the myocardium and skeletal muscle in type 1 insulin-dependent diabetes mellitus (IDM). We evaluated this possibility during the early stage of IDM in BB/Wor diabetic (D) rats and age-matched BB/Wor diabetes-resistant (DR) rats. Interventricular septal thickness, E wave peak velocity of tricuspid inflow (both minimum and maximum), and left ventricular (LV) weight index were increased, and the rate of change in LV pressure (LV dP/d t) decreased in D rats subjected to M-mode and two-dimensional echocardiography and hemodynamic recording of heart rate, LV pressure (LVP), +LV dP/d t, −LV dP/d t, and LV end-diastolic pressure (LVEDP) in vivo and in vitro 41 days after the onset of hyperglycemia. Whole ventricle basal PKC activity was increased by 44.4 and 18.4% in the particulate and soluble fractions, respectively, from D rats compared with that from DR rats using r-32P phosphorylation of appropriate peptide substrates. When measured by Western blot gel densitometry, particulate PKC-α and PKC-δ content increased by 89 and 24%, respectively, but soluble PKC-β and soluble and particulate PKC-ε were unchanged compared with that of DR rats. Similarly, gracilis muscle PKC activity and PKC-α and PKC-δ were elevated in the gracilis muscle, whereas that of the circulating neutrophil did not differ between the D and DR rats. Thus, in vivo, the early diabetic cardiomyopathy of the D rat is characterized by a restrictive LV with increased septal thickness and is associated with elevated PKC activity and increased amounts of myocardial particulate PKC-α and PKC-δ, which are also seen in the skeletal muscle. We conclude that increased PKC isozymes may play a pivotal role during IDM in the development of diabetic cardiomyopathy and skeletal muscle myopathy.