Eric G. Spokas

Role of endogenous nitric oxide in TNF-α and IL-1β generation in hepatic ischemia-reperfusion

In the present study, we examined the role of nitric oxide (NO) in early-response cytokine production by using a rat model of hepatic ischemia-reperfusion (HI/R). The left and median lobes of the liver were subjected to 30 min of ischemia, followed by 4 h of reperfusion. Group I and II rats were sham-operated controls that received saline (vehicle) or N(W)-nitro-L-arginine methylester (L-NAME) (10 mg/kg, iv); group III and IV rats were subjected to HI/R and received vehicle or L-NAME (10 mg/kg, iv, 10 min before reperfusion), respectively. Administration of L-NAME to rats subjected to I/R resulted in a fourfold decrease in plasma NO levels, accompanied by a marked increase of plasma alanine aminotransferase (ALT) activity relative to group III. These changes in group IV were associated with elevation of superoxide generation in ischemic liver lobes by 2.1-fold and circulating leukocyte number by 1.42-fold, compared with group III. Normalized for expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) messenger ribonucleic acid (mRNA), expression of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) mRNA in ischemic liver of group IV was augmented by 207% and 175% compared with Group III. The expression of (iNOS) mRNA was also increased (223%) relative to group III. Moreover, in group IV, plasma TNF-alpha levels at 4 h of reperfusion and IL-1beta levels at 90 min and 4 h of reperfusion were significantly increased compared with group III. No statistically significant changes were observed between groups I and II in plasma ALT activity, plasma NO levels, circulating leukocyte counts, superoxide generation in the ischemic lobes of liver, and plasma TNF-a and IL-1beta concentrations. The observed enhancement of I/R injury by L-NAME is consistent with the hypothesis that endogenous NO down-regulates TNF-alpha and IL1beta generation, thereby decreasing HI/R injury.

Presence of cytochrome P-450-dependent monooxygenase in intimal cells of the hog aorta.

Cytochrome P-450-dependent mixed function oxidase activity is present in vascular tissue; however, as far as we could determine, the distribution of monooxygenase activity across the blood vessel wall has not previously been assessed. The aryl-hydrocarbon hydroxylase activity was examined by metabolism of benzo[a]pyrene in microsomes prepared from intimal and smooth muscle cell scrapings of the hog thoracic aorta. Microsomes of intimal cells comprising 95% endothelial cells showed an approximately 2.5-fold increase in aryl-hydrocarbon hydroxylase activity compared with that in microsomes prepared from medial smooth muscle cells. Michaelis-Mentin kinetics for the intimal enzyme yielded an apparent Km value of 11.11 microM and an apparent Vmax of 3-OH benzo[a]pyrene of 40 pmol/mg protein/10 min. Aryl-hydrocarbon hydroxylase activity was dependent on nicotinamide adenine dinucleotide phosphate and was inhibited by 7,8 benzoflavone, SKF 525A, and carbon monoxide. The localization of cytochrome P-450-dependent mixed function oxidase primarily to the intimal surface of the aorta may indicate a role for this enzyme system in vasoregulation and the pathogenesis of atherosclerosis.

Intima-related vasodilatation of the perfused rat caudal artery

We examined the endothelial dependence of responses to ACh and some vasodilator drugs by using the central tail artery of the rat perfused with Krebs buffer. Perfusion with ACh (100 nM-100 microM) produced dose-dependent vasodilatation of arteries preconstricted with norepinephrine and antagonized pressor responses to periarterial electrical stimulation. Endothelium was removed by introducing a fine catheter through the lumen or a stream of gas (O2 95%-CO2 5%) intraluminally. Both procedures prevented the vasodilator effect of ACh. Gassing also abolished the vasodilatation in response to hydralazine 334 nM but not to equidilator amounts of papaverine 13 microM, or nitroglycerin 50 nM. These results indicating endothelial dependence of hydralazine and ACh responses are in accord with our previous studies on vascular rings.

STIMULATION OF RENIN RELEASE BY 6‐OXO‐PROSTAGLANDIN E1 AND PROSTACYCLIN

1 Renin release induced by 6‐oxo‐prostaglandin E1 (6‐oxo‐PGE1) was compared to release in response to prostacyclin (PGI2) and 6‐oxo‐PGF1α in slices of rabbit renal cortex. 2 Krebs‐Ringer medium bathing slices of renal cortex was collected for renin assay after four successive 20 min intervals (periods I‐IV). Renin release did not increase during periods I to IV in untreated slices. Agonists were added, only once, at the beginning of period III. Between periods III and IV, the incubation solution was aspirated and replaced with fresh medium. 3 PGI2 increased renin release during period III while 6‐oxo‐PGE1 stimulated release during periods III and IV. 6‐oxo‐PGE1 stimulated renin release (24%–74%) in concentrations ranging from 1–33 μm while PGI2 stimulated release at 10 μm (60%) but not at 5 μm. 6‐oxo‐PGF1α, 10 μm, did not release renin during period III (period III, 9%), but caused a small rise in period IV (29%). 4 6‐oxo‐PGE1, unlike PGI2, was stable under the incubation conditions (pH 7.4, 37°C) as indicated by recovery of undiminished platelet anti‐aggregatory material after 20 min. 5 In the rabbit kidney, activity of 9‐hydroxyprostaglandin dehydrogenase was greatest in the cortex and negligible in the papilla, corresponding to the zonal distribution of renin. 6 The prominent and sustained in vitro renin releasing effect of 6‐oxo‐PGE1, as well as the cortical localization of enzyme activity capable of generating this stable prostacyclin metabolite, suggest that formation of 6‐oxo‐PGE1 may contribute to PGI2‐induced renin release and may explain the delayed stimulation caused by 6‐oxo‐PGF1α.

Metabolism of prostaglandin E2 in the isolated perfused kidney of the rabbit

In the Krebs-perfused rabbit isolated kidney, [3H]PGE2 (5 microCi, 165 Ci/mmole) was infused intra-artially for 5 min; venous and urinary effluents were collected every 2 min for 20 min. Efflux of radioactive material peaked at 8 min and declined thereafter. The kidney retained 35% of the infused 3H. Samples were extracted for acidic lipids; PGE2, PGF2 alpha and metabolites were separated by TLC and quantified by a radiometric method. Efflux of [3H]PGF2 alpha into urinary and venous outflows increased progressively over the first 12 min and then plateaued for the remaining 4 min. By 12 min, conversion of [3H]PGE2 to [3H]PGF2 alpha was 70 and 80% as determined by radiolabeled products recovered in the urinary and venous effluents respectively. Estimates of total conversion of [3H]PGE2 to [3H]PGE2 alpha were 62 and 52% of the radiolabeled material exiting in the urinary and venous effluents respectively. The 15-keto and 13,14-dihydro-15-keto metabolites of [3H]PGF2 alpha appeared in the urine but were not found in the venous outflow. We conclude that PGE-9-ketoreductase (PGE-9KRD) activity is high in the rabbit isolated perfused kidney. Further, the extent of conversion of PGE2 to PGF2 alpha and metabolism of newly formed PGF2 alpha may differ within the vascular and tubular compartments of the kidney. PGE-9KRD activity may be important in the regulation of renal vascular tone, compliance of veins, and salt and water balance.

Position Paper: Regulation of Blood Pressure by Prostaglandin-Kinin Interactions

Functional coupling of the kallikrein-kinin system with prostaglandins amplifies the vasodilator and diuretic actions of kinins and may be essential to some of the effects of these peptide hormones on blood vessels and renal function (1). Together, prostaglandins and kinins constitute a major blood pressure-regulating system which opposes the effects of circulating hormones, such as angiotensins, ADH, epinephrine, and mineralocorticoids, and of excitation of the adrenergic nervous system (2). Important interactions of prostaglandins and kinins that can lower blood pressure occur within the kidney and blood vessels where they contribute to the regulation of extracellular fluid volume and vascular reactivity. A principal objective of this position paper is to examine the interrelationships of vasoactive polypeptides and arachidonic acid metabolites in terms of the regulation of blood pressure. The central nervous system has been neglected out of ignorance; some of the most important vasodepressor mechanisms involving kinins and prostaglandins presumably occur within the brain, an area of great potential for future studies.

Prostaglandin Mechanisms in Blood Pressure Regulation and Hypertension

In 1970, three studies were published which provided the basis for the proposal that prostaglandins are modulators of pressor hormones and adrenergic nervous activity (1,2,3). In the first two studies, infusion of either angiotensin II or norepinephrine was shown to cause release of PGE-like material into renal venous blood; the latter was associated with attenuation of the vasoconstrictor and anti-diuretic actions of these pressor hormones (1,2). In the third study, it was demonstrated that within minutes after induction of unilateral renal ischemia, prostaglandins were released into the venous blood of both kidneys. Release from the contralateral kidney was mediated by angiotensin II, which, presumably, also contributed to the release from the ischemic kidney (3). This study offered an explanation for the antihypertensive function of the contralateral kidney in terms of a possible prostaglandin mechanism. Thus, activation of the reninangiotensin system by renal ischemia can effect enhanced prostaglandin production in the ischemic and contralateral kidneys. A related study on the effects of norepinephrine infusion and renal nerve stimulation on renal function (4) strongly supported the proposal that renal prostaglandins act as a component of an intrarenal negative feedback control system which moderates anti-diuretic and vasoconstrictor systems.

6-Keto-Prostaglandin E1: Biosynthesis and Circulatory Effects

The changes in circulatory function induced by 6-keto-prostaglandin E1 (6-keto-PGE1) bear a close resemblance to the changes caused by prostacyclin (PGI2). Like PGI2, 6-keto-PGE1 is potent in stimulating renin secretion [l–3], inhibiting platelet aggregation [4], and reducing blood pressure and vascular resistance in diverse regional circulations [5, 6]. In this chaper, we present evidence that 6-keto-PGE1 may arise during the course of metabolic transformation of PGI2 through the activity of 9-hydroxyprostaglandin dehydrogenase (9-OH PGDH) identified in various tissues, including liver, blood platelets, and renal cortex. As PGI2 is unstable, having a half-life of approximately 3 min in aqueous solution [7], the identification of an active and stable metabolite of prostacyclin has important implications for understanding the time-dependency of prostacyclin-induced physiologic responses. In contrast to PGI2, when 6-keto-PGE1 is incubated in aqueous solution at pH 7.4, 37°C its platelet antiaggregatory activity remains undiminished for at least 20 min [3].