Robert T. Mathie

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.

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.

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 blood flow measurement with inert gas clearance.

Inert gas clearance has been used for 20 years to measure hepatic blood flow. Injection of a saline solution of 85Kr or 133Xe is usually made via the PV, and the resulting hepatic clearance is monitored with a Geiger-Müller tube, scintillation crystal, or gamma camera. Complex slow components in 133Xe clearance curves, once believed to indicate a correspondingly complex hepatic microcirculation, are now considered to be caused by nonhepatic radioactivity. Normal liver is therefore believed to receive a homogeneous perfusion throughout the depth of tissue in any given region. HA blood and PV blood are normally completely mixed in the hepatic sinusoids. Macroscopic variations in tissue perfusion may exist in different lobes of the liver in both animals and man. The technique expresses flow in units of milliliters per minute per 100 g. Accurate and acceptably reproducible results have been obtained after PV injection of isotope; fast component analysis of 133Xe clearance is most appropriate, while beta detection of 85Kr yields a simple monoexponential curve. Normal hepatic blood flow in dogs and in man is 100-130 ml min-1 100 g-1. Employing sites of isotope administration other than the PV produces inaccurate results unless appropriate corrections are made. Accuracy of flow measurement is critically dependent on a knowledge of the partition coefficient of the gas used. Liver disease per se does not affect measurement accuracy, and many practical features make the technique an attractive tool for the measurement of hepatic hemodynamics in man. Nevertheless, it is essential that the investigator be aware of certain limitations of the method, and carefully apply current concepts of clearance curve analysis and interpretation, in order to derive maximum advantage.

The action of ATP on the hepatic arterial and portal venous vascular networks of the rabbit liver: the role of adenosine

ATP is released from blood vessels during periods of hypoxia and may be responsible for hepatic arterial vasodilatation during instances of reduced hepatic portal venous flow. The role of adenosine in ATP-induced vasodilator and vasoconstrictor responses of the hepatic arterial and portal venous vascular networks respectively was studied in the isolated dual-perfused rabbit liver in vitro to ascertain whether ATP could be catabolised to adenosine during transit through the hepatic parenchyma. Intra-arterial and intra-portal injections of ATP (-10 to -4 log mol/100 g liver) resulted in dose-dependent vasodilatation in the hepatic artery and vasoconstriction in the portal vein. Addition of 8-phenyltheophylline (10 microM), a non-selective P1-purinoceptor antagonist, to the hepatic arterial and portal venous perfusate significantly inhibited the hepatic arterial ED50 for responses to intra-arterial injected ATP from -8.70 +/- 0.22 to -7.63 +/- 0.28 log mol/100 g liver (P < 0.001); it also inhibited hepatic arterial responses to, mid-range, portal venous injections of ATP. The data suggest that the hepatic arterial vasodliatation to ATP is partly mediated via catabolism to adenosine and may be an important mechanism during periods of relative hepatic hypoxia associated with portal flow reduction.

Vasoconstrictor responsiveness of the rat mesenteric arterial bed in cirrhosis

1 The effects of cirrhosis on mesenteric vascular reactivity were assessed in constantly perfused mesenteric arterial beds isolated from cirrhotic rats (carbon tetrachloride with phenobarbitone, n=6), and from phenobarbitone‐treated and untreated age‐matched controls (n=4,5). 2 At a constant flow rate of 5 ml min−1 there was no difference in basal perfusion pressure between the groups. Electrical field stimulation (EFS; 4–32 Hz, 90V, 1 ms, 30 s) of perivascular nerves caused frequency‐dependent increases in perfusion pressure which were not different between the groups. Dose‐dependent vasoconstrictor responses to exogenous noradrenaline (NA), methoxamine (an α1‐adrenoceptor agonist), adenosine 5′‐triphosphate (ATP) and vasopressin were also similar between the groups. 3 The nitric oxide (NO) synthesis inhibitor NG‐nitro‐L‐arginine methyl ester (L‐NAME; 30 μm) augmented constrictor responses to NA, EFS, methoxamine and vasopressin in all groups, and as shown for EFS and NA, this was reversed by L‐arginine (300 μm). However, the maximum constrictor responses of cirrhotic preparations in the presence of L‐NAME were significantly lower than those of both groups of control animals at the highest frequency of EFS (32 Hz) and highest doses of NA (0.15 and 0.5 μmol) and, compared to phenobarbitone‐treated controls, methoxamine (5 μmol). Responses to ATP were significantly augmented by L‐NAME only in the cirrhotic group. 4 A step‐wise increase in perfusate flow to 10, 15 and 20 ml min−1 produced a broadly similar increase in perfusion pressure within each group. At increased flow rates, cirrhotic preparations were hyporesponsive to NA (15 nmol) compared to the phenobarbitone‐treated animals but not the untreated controls. Glibenclamide (5 μm) or L‐NAME (30 μm) had no significant effect on the relationship between flow and perfusion pressure or on responses to NA at the different flow rates. 5 We conclude that sympathetic neurotransmission is unchanged in cirrhosis. Endogenous NO is important in modulation of constriction in both normal and cirrhotic states. Changes in NO may occur in cirrhosis, although the role of this in hyporesponsiveness of cirrhotic preparations to NA at higher flow rates and to the greater potentiation of ATP‐mediated constriction in the presence of L‐NAME, together with the impact of factors such as changes in calcium and potassium channels, is not entirely clear.