inhibition of nitric oxide synthesis. 'N.A. Herity, *J.D. Allen, **B. Silke & A.A.J. Adgey. Regional Medical Cardiology Centre, Royal Victoria Hospital,Belfast BT12 ...
Br. J. Pharmacol. (1994), 112, 423-428
kWMacmillan Press Ltd, 1994
Comparison of the ability of nicardipine, theophylline and zaprinast to restore cardiovascular haemodynamics following inhibition of nitric oxide synthesis 'N.A. Herity, *J.D. Allen, **B. Silke & A.A.J. Adgey Regional Medical Cardiology Centre, Royal Victoria Hospital, Belfast BT12 6BA and Departments of *Physiology and **Therapeutics and Clinical Pharmacology, Queen's University, Belfast BT9 7BL 1 The use of pharmacological inhibitors of nitric oxide (NO) synthesis to treat patients with septic shock is limited by the observation that they cause a fall in cardiac output in some subjects. The aim of this work was to investigate this fall and to test whether it was reversible by subsequent administration of nicardipine, theophylline or the cyclic GMP-selective phosphodiesterase inhibitor, zaprinast (M&B 22948). 2 In pentobarbitone-anaesthetized pigs, haemodynamic indices were measured before and after intravenous administration of NG-nitro-L-arginine methyl ester (L-NAME) in a dose-response protocol (0.2-20 mg kg-'; n = 6) and as a single bolus of 10 mg kg-' either alone or followed by increasing doses of nicardipine, theophylline or zaprinast (n = 8 in each group). 3 L-NAME caused a dose-dependent rise in systemic vascular resistance and mean systemic arterial pressure and a dose-dependent fall in cardiac output. A single bolus of L-NAME (10 mg kg-') produced these effects within 15 min. 4 Subsequent administration of nicardipine (0.05-0.2 mg kg-') caused complete reversal of systemic vasoconstriction and hypertension and in doing so completely restored cardiac output. Theophylline (7.5-1O mg kg-') partially reversed the rise in systemic vascular resistance and partially restored cardiac output but the effect was small compared to that of nicardipine. Zaprinast (1-5 mg kg-') had no significant effect on any of these variables. 5 These results suggest that reduced cardiac output following inhibition of NO synthesis is an effect of increased afterload on the heart and is reversible by nicardipine and to a lesser extent by theophylline. These findings may have potential value for those using NO synthase inhibitors to treat patients with septic shock. Keywords: Nitric oxide; nicardipine; theophylline; zaprinast; N0-nitro-L-arginine methyl ester; cardiovascular haemodynamics
Introduction Endothelium-derived relaxing factor (EDRF) is a physiological vasodilator thought to be the nitric oxide radical (NO; for review see Moncada et al., 1991) or a closely related molecule, perhaps the nitroxyl radical (Fukuto et al., 1992) or both. NO is synthesized from its precursor L-arginine in a reaction catalysed by NO synthase of either the constitutive or inducible form. It activates guanylate cyclase accelerating formation of guanosine 3':5'-cyclic monophosphate (cyclic GMP) in vascular smooth muscle cells thus causing relaxation and vasodilatation (Moncada et al., 1991). Although the details of this mechanism remain uncertain the evidence suggests that a cyclic GMP-dependent protein kinase activates a calcium transport protein causing a reduction in intracellular free calcium concentration and thus relaxation (Lincoln, 1989). NO synthesis is competitively inhibited by L-arginine analogues such as N0-monomethyl-L-arginine and N0-nitroL-arginine methyl ester (L-NMMA and L-NAME; Rees et al., 1990). Administration of such agents to experimental animals causes widespread vasoconstriction, increased peripheral vascular resistance and arterial hypertension (Moncada et al., 1991) and a fall in cardiac output in rats (Gardiner et al., 1990; Amrani et al., 1992), rabbits (Persson et al., 1990), dogs (Kilbourn et al., 1990) and sheep (Tresham et al., 1991). In the present study we have documented the effects of intravenous administration of L-NAME to pentobarbitoneanaesthetized pigs in a dose-response protocol (0.2-20mg kg-') and as a single large bolus (10mgkg-'). A fall in I
Author for correspondence.
cardiac output has also been observed in human subjects receiving NO synthase inhibitors (Petros et al., 1991) or the guanylate cyclase inhibitor, methylene blue (Schneider et al., 1992) as treatment for septic shock resulting in limitation of their use to treat this condition. The reason for the fall in cardiac output is unknown although increased afterload (Gardiner et al., 1990; Persson et al., 1990), reduced preload (Persson et al., 1990) direct myocardial action (Gardiner et al., 1990; Persson et al., 1990; Schulz et al., 1992), coronary vasoconstriction (Gardiner et al., 1990; Persson et al., 1990; Amrani et al., 1992; Schulz et al., 1992) and activation of baroreceptor reflexes (Kilbourn et al., 1990) have all been suggested. Inhibition of endocardial (Schulz et al., 1991) or myocardial (Schulz et al., 1992) NO synthesis are further potential mechanisms. We have investigated the observed reduction in cardiac output by testing the hypothesis that it could be restored by subsequent administration of vasodilator agents. The agents used, nicardipine (a highly vasoselective calcium channel blocker), theophylline (a non-selective phosphodiesterase inhibitor) and zaprinast (M&B 22948; a cyclic GMP-selective phosphodiesterase inhibitor) were chosen because increased cyclic GMP levels and a fall in intracellular free calcium concentration are central to NO-mediated vasodilatation and thus we felt that each had the potential to mimic restoration of NO to the vascular smooth muscle cell.
Methods Adult pigs of either sex (32-52 kg) were sedated with azaperone (Stresnil 160 mg, i.m.) and anaesthetized with pen-
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tobarbitone (Sagatal, May and Baker, 30 mgkg-' i.v. initially and supplemented as required) via an ear vein. They tvere intubated and ventilated with room air by a Palmer Ideal pump at a rate and tidal volume adjusted according to nd-tidal carbon dioxide concentration (ETCO2). A warming lanket maintained body temperature above 36.50C as measured by a rectal thermistor (Harvard). A cannula was inserted into a forelimb vein for drug administration.
Haemodynamic measurements The right common carotid artery was cannulated and connected via a transducer (Druck PDCR 75) to a 4-channel electronic recorder (Gould 2400S) to register arterial blood pressure. A continuous surface electrocardiogram (lead III) was recorded with electronic measurement of heart rate (Neurolog). A transcutaneous pulse oximeter (Ohmeda 4700 Oxicap) measured tissue oxygen saturation (SaO2) and
FTCO2.
For measurement of cardiac output, a triple lumen balloon-tipped thermodilution catheter (Edwards Laboratories or Baxter) of either 7.0 or 7.5 French gauge was passed
through the right external jugular vein to the main pulmonary artery under fluoroscopic control. The proximal lumen was used for delivery of the injectate and for measurement of right atrial pressure. One litre of injectate (5% dextrose in water cooled to 0-0.50C) was stored in a flask over ice. A thermistor probe inside the flask measured injectate temperature. A short length of plastic tubing connected the solution via a three way tap to the injectate syringe For each measurement 10 ml of injectate was drawn PBraun). into the syringe and infused by a gas-powered injector gun (USCI No. 372000) through the proximal port of the catheter. The resultant change in blood temperature was measured by a thermistor 4 cm from the catheter tip, the cardiac output was calculated by an on line cardiac output computer (American Edwards Laboratories 9520A) and the thermodilution curve recorded (American Edwards Laboratories 9812). This procedure was repeated eight times for each data point and the first two readings disregarded to correct for catheter warming between measurements. Cardiac output was calculated as the mean of the remaining six readings. Systemic vascular resistance (SVR) was calculated from the
formula: SVR =
(MAP-MRAP) x 8
kPalI-'s
~~~CO yWhere MAP represents mean arterial pressure, MRAP tepresents mean right atrial pressure, CO represents cardiac output, 8 (=60/7.5) converts mmHg 1' min to kPa Is.
Experimental protocols (a) Dose-response effects of L-NAME (n = 6) Following the initial preparation, two pretreatment sets of haemodynamic measurements were made 15 min apart to ensure stability. L-NAME was then administered intravenously initially as a bolus of 0.2 mg kg-'. The cumulative dose was increased every 20min to 0.5 mgkg-', 1 mgkg-', 2mg kg-', 5mg kg-', 10mgkg-1; 20mgkg-' in three of the six pigs. Haemodynamic measurements were recorded 15 min after each dose. The results obtained were compared with those in a control group of six pigs who followed the same protocol but received equal volumes of isotonic saline rather than L-NAME. (b) Effects of a single bolus of L-NAME alone (n = 8) Following the initial two pretreatment sets of measurements, LNAME (10 mg kg-', i.v.) was administered as a single bolus. Hiaemodynamic measurements were recorded 15, 30, 45 and 60 min later and the results compared with the second pretreatment value.
(c) Effects of a single bolus of L-NAME followed by nicardipine (n = 8) Following the initial two pretreatment sets of measurements, L-NAME (10 mg kg', i.v.) was administered as a single bolus. Haemodynamic measurements were recorded 15 min later. Nicardipine was then administered intravenously initially at a dose of 0.05 mg kg-'. The cumulative dose was increased at 15 min intervals to 0.1 mg kg' and then to 0.2 mg kg-'. Haemodynamic measurements were repeated 10 min after each dose. The results obtained were compared with those in the group of eight pigs who received only the bolus of L-NAME and no further drug (group (b) above).
(d) Effects of a single bolus of L-NAME followed by theophylline (n = 8) Following the initial two pretreatment sets of measurements, L-NAME (10 mg kg', i.v.) was administered as a single bolus. Haemodynamic measurements were recorded 15 min later. Theophylline was then administered intravenously initially at a dose of 7.5 mg kg'. The cumulative dose was increased after 15 min to 10mg kg-'. Haemodynamic measurements were repeated 10 min after each dose. The results obtained were compared with those in the group of eight pigs receiving only the bolus of L-NAME (group (b) above). (e) Effects of a single bolus of L-NAMEfollowed by zaprinast (n = 8) Following the initial two pretreatment sets of measurements L-NAME (10 mg kg', i.v.) was administered as a single bolus. Haemodynamic measurements were recorded 15 min later. Zaprinast was then administered intravenously initially at a dose of 1 mg kg'. The cumulative dose was increased at 15 min intervals to 3 mg kg-' and then to S mg kg'. Haemodynamic measurements were repeated 10 min after each dose. The results obtained were compared with those in the group of eight pigs receiving only the bolus of L-NAME (group (b) above).
Drugs used L-NAME (Sigma) was administered as a solution of 10 mg
ml-' dissolved in normal saline. Nicardipine (a gift from Syntex Research) was administered as the manufacturer's solution of 2.5 mg ml-'. Theophylline (Sigma) was administered as a solution of 2 mg ml-' in normal saline. Zaprinast (M&B 22948; a gift from Rhone-Poulenc Rorer) was administered as a solution of 10 mg ml' in 5% triethanolamine in normal saline.
Statistical analysis Results are expressed as mean ± standard error of the mean (s.e.mean). The Mann-Whitney U test (SPSS) analysed the change produced in a given haemodynamic variable caused by administration of each active drug by comparison with that produced by its corresponding control (viz: effects of L-NAME compared with those of normal saline; effects of L-NAME followed by nicardipine, theophylline and zaprinast compared with those of L-NAME followed by no further drug). The null hypothesis stated that there was no difference between drug groups and was rejected if P
