Barry Alexander

The Role of Nitric Oxide in Hepatic Metabolism

Nitric oxide (NO) may regulate hepatic metabolism directly by causing alterations in hepatocellular (hepatocyte and Kupffer cell) metabolism and function or indirectly as a result of its vasodilator properties. Its release from the endothelium can be elicited by numerous autacoids such as histamine, vasoactive intestinal peptide, adenosine, ATP, 5-HT, substance P, bradykinin, and calcitonin gene-related peptide. In addition, NO may be released from the hepatic vascular endothelium, platelets, nerve endings, mast cells, and Kupffer cells as a response to various stimuli such as endotoxemia, ischemia-reperfusion injury, and circulatory shock. It is synthesized by nitric oxide synthase (NOS), which has three distinguishable isoforms: NOS-1 (ncNOS), a constitutive isoform originally isolated from neuronal sources; NOS-2 (iNOS), an inducible isoform that may generate large quantities of NO and may be induced in a variety of cell types throughout the body by the action of inflammatory stimuli such as tumor necrosis factor and interleukin (IL)-1 and -6; and NOS-3 (ecNOS), a constitutive isoform originally located in endothelial cells. Another basis for differentiation between the constitutive and inducible enzymes is the requirement for calcium binding to calmodulin in the former. NO is vulnerable to a plethora of biologic reactions, the most important being those involving higher nitrogen oxides (NO2-), nitrosothiol, and nitrosyl iron-cysteine complexes, the products of which (for example, peroxynitrite), are believed to be highly cytotoxic. The ability of NO to react with iron complexes renders the cytochrome P450 series of microsomal enzymes natural targets for inhibition by NO. It is believed that this mechanism provides negative feedback control of NO synthesis. In addition, NO may regulate prostaglandin synthesis because the cyclooxygenases are other hem-containing enzymes. It may also be possible that NO-induced release of IL-1 inhibits cytochrome P450 production, which ultimately renders the liver less resistant to trauma. It is believed that Kupffer cells are the main source of NO during endotoxemic shock and that selective inhibition of this stimulation may have future beneficial therapeutic implications. NO release in small quantities may be beneficial because it has been shown to decrease tumor cell growth and levels of prostaglandin E2 and F2 alpha (proinflammatory products) and to increase protein synthesis and DNA-repair enzymes in isolated hepatocytes. NO may possess both cytoprotective and cytotoxic properties depending on the amount and the isoform of NOS by which it is produced. The mechanisms by which these properties are regulated are important in the maintenance of whole body homeostasis and remain to be elucidated.

Nitric oxide is the mediator of ATP‐induced dilatation of the rabbit hepatic arterial vascular bed

1 Livers of 10 New Zealand White rabbits were perfused in vitro with Krebs‐Bülbring buffer via the hepatic artery (HA) and portal vein (PV) at constant flows of 23 ± 1 and 77 + 1 ml min−1 100 g−1 respectively. The tone of the preparation was raised with noradrenaline (concentration: 10 μm). 2 Dose‐response curves for the vasodilatation produced by adenosine 5′‐triphosphate (ATP), acetylcholine (ACh), adenosine, and sodium nitroprusside (SNP) were obtained following injection into the HA supply. Injections were then repeated in the presence of the l‐arginine to nitric oxide pathway inhibitors N‐monomethyl‐l‐arginine (l‐NMMA, n = 6) and N‐nitro‐l‐arginine methyl ester (l‐NAME, n = 4) at concentrations of 30 μm and 100 μm for each inhibitor. 3 Both l‐NMMA and l‐NAME antagonized the responses to ATP and ACh; l‐NAME was 2–3 times more potent than l‐NMMA as an inhibitor of these endothelium‐dependent vasodilatations. Neither l‐NMMA nor l‐NAME attenuated responses of the endothelium‐independent vasodilators, adenosine and SNP. 4 These results indicate that nitric oxide is the mediator of ATP‐induced vasodilatation in the HA vascular bed of the rabbit and that the receptor responsible for the release of nitric oxide, the P2y‐purinoceptor, is located predominantly on the endothelium.

A simple and accurate mathematical method for calculation of the EC50

A simple, accurate, and speedy noncomputational technique for the calculation of the EC50 or any other concentration-related parameter of concentration-effect curves is presented. It avoids the necessity for graph construction or computational curve-fitting programs and allows accurate calculation of the EC50, where the value falls between two known concentrations The technique has been applied to a concentration-response curve constructed to hepatic arterial (HA) vasoconstrictor responses to HA injections of noradrenaline in an isolated dual-perfused rat liver preparation. EC50 values calculated by the new technique were compared to those calculated by conventional, established, noncomputational techniques. The new technique is faster, more accurate, and simpler to perform than other established noncomputational techniques used for the calculation of the EC50 and can be widely applied to many other pharmacological investigations.

Characterization of P2X‐ and P2Y‐purinoceptors in the rabbit hepatic arterial vasculature

1 Responses to adenosine 5′‐triphosphate (ATP) and its agonists were studied in the isolated liver of the rabbit dually perfused through the hepatic artery and the portal vein. 2 In the hepatic arterial vascular bed at basal tone, ATP and its agonists elicited vasoconstrictor responses with the rank order of potency α,β‐methylene ATP > 2‐methylthio ATP > ATP, consistent with their action at the P2X‐purinoceptor. 3 When tone was raised with noradrenaline (10−5 m), vasodilator responses were produced with ATP and 2‐methylthio ATP; α,β‐methylene ATP produced only further constriction. The rank order of vasodilator potency was 2‐methylthio ATP > ATP ≫ α,β‐methylene ATP, consistent with their action at the P2Y‐purinoceptor. 4 Methylene blue (10−5 m) antagonized vasodilator responses to acetylcholine and ATP, but not those to adenosine or sodium nitroprusside. Addition of 8‐phenyltheophylline (10−5 m) antagonized responses to adenosine but not those to sodium nitroprusside. Responses to ATP remaining after antagonism with methylene blue were not further antagonized by 8‐phenyltheophylline. 5 These results present evidence for discrete P2X‐ and P2Y‐purinoceptors in the rabbit hepatic arterial bed which mediate vasoconstrictor and vasodilator responses respectively. 6 Vasodilatation produced by ATP was entirely due to direct action at the P2Y‐purinoceptor, and not at a P1‐purinoceptor following breakdown to adenosine. The antagonism of these responses by methylene blue is consistent with the view that vasodilatation by ATP takes place largely via endothelial P2Y‐purinoceptors that lead to release of endothelium‐derived relaxing factor. However, we cannot exclude the possibility that P2Y‐purinoceptors located on the vascular smooth muscle play a contributory role in ATP‐induced vasodilatation.

NG-Nitro-l-arginine methyl ester attenuates vasodilator responses to acetylcholine but enhances those to sodium nitroprusside

Abstract— The effects of NG‐nitro‐l‐arginine methyl ester (l‐NAME), an inhibitor of the synthesis of the endothelium‐derived relaxing factor nitric oxide, were studied in two isolated perfused vascular beds: the rat mesenteric arterial bed and the hepatic arterial bed of the rabbit liver. The tone of both preparations was raised with noradrenaline (10 and 30 μm for rabbit and rat preparations, respectively). In both preparations, L‐NAME (30 μm) significantly attenuated vasodilator responses to the endothelium‐dependent vasodilator acetylcholine, but enhanced responses to sodium nitroprusside (a direct smooth muscle dilator). The evidence supports the view, previously established from work carried out in isolated vessels, that in addition to acting as an inhibitor of nitric oxide, l‐NAME enhances the responsiveness of smooth muscle to direct relaxation by nitrovasodilators.

Adenosine-induced dilatation of the rabbit hepatic arterial bed is mediated by A2-purinoceptors.

1 This study was carried out in order to identify the receptor responsible for adenosine‐induced dilatation of the hepatic arterial vascular bed. 2 Livers of 10 New Zealand White rabbits were perfused in vitro with Krebs‐Bülbring buffer via the hepatic artery and the portal vein at constant flows of 26 and 77 ml min−1 100 g−1 liver respectively. The tone of the preparation was raised by the presence of noradrenaline in the perfusate (concentration: 10−5 m). 3 Dose‐response curves for adenosine and its analogues 5′‐N‐ethyl‐carboxamido‐adenosine (NECA), the 2‐substituted NECA analogue CGS 21680C, and R‐ and S‐N6‐phenyl‐isopropyl‐adenosine (R‐ and S‐PIA) were obtained after their injection into the hepatic arterial supply. 4 The order of vasodilator potency of these agents was: NECA > CGS 21680C > adenosine > R‐PIA > S‐PIA. Their potency, expressed relative to that of adenosine, was in the approximate ratio 10:3:1:0.3:0.1, consistent with that resulting from activation of P1‐purinoceptors of the A2 sub‐type (which mediate vasodilatation due to adenosine). 5 The P1‐purinoceptor antagonist 8‐phenyltheophylline (10−5 m) caused significant attenuation of the vasodilatation to adenosine and analogues. 6 It is concluded that adenosine‐induced dilatation of the hepatic arterial vascular bed is mediated by P1‐purinoceptors of the A2 sub‐type.

An isolated dual-perfused rabbit liver preparation for the study of hepatic blood flow regulation

An original, isolated dual-perfused rabbit liver preparation was developed for investigations into mechanisms that control the hepatic vascular tone. The hepatic artery (HA) and portal vein (PV) were perfused at constant flows of 0.16 +/- 0.01 and 0.64 +/- 0.05 mL/g/min (n = 5), respectively. Responses of the hepatic arterial and portal venous vascular beds to noradrenaline (NA) were measured as changes in perfusion pressure. Noradrenaline injected directly into the hepatic artery and portal vein produced dose-dependent increases in pressure in the respective vascular beds, the maximum response in the hepatic arterial bed being two to three times greater than that in the portal venous bed. A restricted transmission of vasoconstrictor stimulus between the intrahepatic portal venous and hepatic arterial vasculature was demonstrated. The results demonstrate the suitability of the dual-perfused rabbit liver model for detailed studies of the control of hepatic vascular tone.

The transhepatic action of ATP on the hepatic arterial and portal venous vascular beds of the rabbit: the role of nitric oxide

1 The effect of bolus administration of adenosine 5′‐triphosphate (ATP) into the portal vein on hepatic arterial pressure (the transhepatic action of ATP) and portal venous pressure, and the contribution of nitric oxide towards these responses, was studied in the in vitro dual‐perfused rabbit liver. 2 At basal tone, hepatic arterial and portal venous vasoconstriction followed ATP injection, while at a tone raised with methoxamine (10−6‐10−5m) ATP caused hepatic arterial vasodilatation, and a phasic vasodilatation followed by vasoconstriction in the portal venous vascular bed. 3 To determine whether the transhepatic arterial dilatation was due to the diffusion of nitric oxide (NO) from the portal venous vasculature, NG‐nitro‐L‐arginine methyl ester (L‐NAME, 100 μm), an inhibitor of NO synthesis, was infused selectively into the portal vein. L‐NAME infusion potentiated portal venous vasoconstriction to ATP (‐log M ED50 5.32 ± 0.31 to 6.51 ± 0.43, P < 0.05, Students paired / test) indicating the possible inhibition of a NO‐mediated vasodilator component of the portal venous response to ATP. There was, however, no demonstrable difference in the transhepatic arterial vasodilatation induced by ATP during this infusion. 4 Simultaneous perfusion of both the hepatic arterial and portal venous inflows with L‐NAME (100 μm) resulted in a significant decrease in the amplitude of hepatic arterial responses to ATP demonstrating that these responses were ultimately mediated by an NO‐dependent mechanism. 5 This study has thus demonstrated a vasodilator component of the portal venous response to ATP that is NO‐mediated. It also provides evidence that it is not portally‐derived NO, but NO released from the hepatic arterial vascular bed, that accounts for the hepatic arterial vasodilatation to intra‐portal administration of ATP. This implies that ATP itself, and not a second messenger, diffuses from the portal venous to hepatic arterial vascular bed to elicit the hepatic arterial response.

Hepatic function during prolonged isolated rat liver perfusion using a new miniaturized perfusion circuit

Previous designs of isolated rat liver perfusion circuits used for toxicological investigations are often expensive, cumbersome, or traumatic in relation to hepatic biocompatibility following extended perfusion times. A new, miniaturized circuit that incorporates a novel design of organ bath, to maintain a buoyant preparation, and a high-efficiency miniaturized membrane tubing oxygenator is described. Livers from male Sprague-Dawley rats were perfused continuously for 6 hr in vitro using rat blood diluted to a perfusate hematocrit of 9.75 +/- 0.35% with Krebs-Henseleit buffer (KHB). Hepatic function was evaluated by measurement of perfusion pressure, flow rate, bile volume production, bile bilirubin content, hepatic oxygen uptake (HOU), bromosulphthalein (BSP) removal, hepatic enzyme activities, and electrolyte concentrations, and finally by histological examination. Perfusion pressure and flow rate remained stable at 8.7 +/- 1.7 mmHg and 1.92 +/- 0.06 mL min-1 g-1 liver, respectively. Bile volume production and HOU were maximal at 784 +/- 84 microL h-1 and 0.99 micromol/L min-1 g-1 respectively. Erythrocyte damage in the perfusate was evaluated by measurement of reduction in perfusate hematocrit from 9.75 +/- 0.35% to 9.27 +/- 0.24%, and increases in plasma free hemoglobin, which rose from 85.8 +/- 12.3 mg% to 650.1 +/- 53.3 mg% over the 6-hr perfusion period studied. Using bile volume production, hepatic oxygen uptake, and the liberation of plasma free hemoglobin as the most sensitive indices of adverse conditions, the new circuit was capable of supporting an isolated perfused rat liver for periods of up to 6 hr under close-to-physiological conditions.

Morphological changes during hepatocellular maturity in neonatal rats

Hepatocellular maturation is characterised by the progessive transition from an architecture in which hepatocyte plates are at least two cells thick to the familiar adult pattern in which liver cell plates are predominantly single‐cell in thickness. A similar process also has been noted during compensatory hyperplasia following damage, or destruction to the hepatic parenchyma. The pathological events that underlie these processes remain inadequately explained. A new morphological approach has been developed to study the maturity of rat neonatal livers in order to identify the factors which govern the structured morphogenesis of the liver in greater detail.