INTRODUCTION

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THE VASCULAR ENDOTHELIUM takes part in several processes, including vasoconstriction and vasodilatation. A polypeptide with vasoconstrictor properties named endothelin, later called endothelin-1 (ET-1), was isolated from cultured porcine aortic endothelial cells, sequenced, and cloned in 1988 (29). ET-1 has been shown to be the most potent vasopressor peptide characterized (23), with long-lasting action in rats (29). We have previously shown that intravenous infusion of ET-1 in humans is followed by long-lasting vasoconstriction in the splanchnic and renal circulations (2, 27), as well as reduction in splanchnic glucose output (2, 3). Whereas plasma disappearance curves showed two half-lives of ET-1 of ~1.5 and 35 min, the duration of vasoconstriction was 1 h in the splanchnic and 3 h in the renal circulation (27), respectively. These long-lasting effects have been ascribed to the tight binding of ET-1 to its receptors (15). The difference in vascular responses to ET-1 may be explained by different populations of ET receptors.

Two types of ET receptors belonging to the G-protein-coupled superfamily have been cloned, the ET_A-type (5), which is expressed in vascular smooth muscle, and the ET_B-type, expressed in the endothelium (see Ref. 23). ET_A-receptor activation stimulates phospholipase C and formation of inositol trisphosphate, which, in turn, mobilizes intracellular calcium and diacylglycerol, resulting in phosphorylation of contractile proteins and smooth muscle contraction (18, 21). ET_A-receptor stimulation is also followed by influx of extracellular calcium (26). Activation of the endothelial ET_B receptor results in release of nitric oxide (NO) (12, 17) and prostaglandin I₂ (PGI₂) (12), which, by acting on smooth muscle, is followed by formation of cGMP, cAMP, reduced intracellular calcium, and vasodilatation. Furthermore, a smooth muscle ET receptor with ET_B-like characteristics has been described on human vascular smooth muscle, where it mediates vasoconstriction (10). Thus ET-1 can act via different receptors and different signal transduction pathways to bring about two diametrically opposed effects: vasoconstriction and vasodilatation.

Increased plasma levels of ET-1 have been described for virtually all pathophysiological conditions in the cardiovascular system, e.g., myocardial infarction (19), cardiogenic shock (8), essential hypertension (22), as well as pulmonary hypertension (24). Cyclooxygenase (COx) inhibition increases the pressor response to ET-1 in guinea pigs (12), indicating a moderating effect of prostaglandins on ET-1-induced vasoconstriction. The present study was undertaken to investigate the effect of COx inhibition per se on cardiac output (CO) and splanchnic and renal vasoactivity and to determine whether COx inhibition alters the response of these variables to a subsequent ET-1 infusion in healthy male volunteers.

	METHODS

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Subjects

Procedure

Teflon catheters were introduced in the femoral vein and advanced to the hepatic vein in all eight subjects and to a renal vein in four of these subjects, under fluoroscopic control. In the other four subjects, a balloon-tipped catheter was introduced into an antecubital vein and advanced to a branch of the pulmonary artery under fluoroscopic control. Pulmonary capillary wedge pressure was monitored by pressure recording and fluoroscopy. Diclofenac (Voltaren, Ciba-Geigy, Basel, Switzerland) dissolved in saline was administered intravenously over a 5-min period at a dose of 75 mg. Pulmonary oxygen uptake was determined and blood samples were drawn simultaneously from the brachial and pulmonary arteries and the hepatic vein in the basal state and at 10, 20, and 30 min after the diclofenac administration for determinations of dye concentrations (in the brachial artery and hepatic vein), oxygen content, and arterial ET-1 levels. Thirty minutes after administration of diclofenac, a 20-min ET-1 (Peninsula Laboratory, Belmont, CA) infusion was initiated. The infusion rate was 4 pmol · kg¹ · min¹. Pulmonary oxygen uptake was determined and blood samples were drawn from all sites at 20 min of ET-1 infusion.

Continuous recording of heart rate (HR) and mean arterial pressure (MAP) was performed in all subjects. Mean pulmonary arterial pressure (MPAP) and mean pulmonary capillary wedge pressure (PCWP) were registered, and CO was estimated according to the Fick principle on the basis of pulmonary oxygen uptake and systemic arterial-pulmonary arterial oxygen difference [(a-pa)_O2] in four subjects. Splanchnic and renal blood flows were determined in all subjects by constant infusions of cardiogreen and p-aminohippurate as previously described (1). For calculation of renal blood flow, the fractional extraction of p-aminohippurate (the arterial minus renal venous concentration divided by arterial concentration) was assumed to be 0.9 in those four subjects who were not fitted with a renal vein catheter. In all subjects, arterial insulin, glucagon, and glucose concentrations as well as hepatic vein glucose concentrations were determined before and after diclofenac administration, as well as during and after the ET-1 infusion.

Analyses

Calculations

Data are presented as means ± SE. Because of the experimental setup, the postdiclofenac data were treated separately. ANOVA, according to the repeated-measures design, was used for basal, 10-, 20-, and 30-min postdiclofenac data, followed by Fisher's protected least-significant difference test, i.e., the interpretation of a significant effect by computing Student's t-test between all means in the effect; "protected" indicates that the test is preceded by a significant F-test. When the variances differed too much, giving F-values not allowing for ANOVA, logarithms were used to achieve homogeneous variances (see Ref. 1). The ET-1 effects (at 20 min of ET-1 infusion) were compared with the 30-min postdiclofenac data by using Student's paired t-test (see Table 1). For comparisons with previous results with ET-1 infusion, Student's unpaired t-test was used (see Table 1 and Fig 1).

View this table: [in this window] [in a new window]   Table 1.   HR, pulmonary O2, (a-pa)O2, CO, BFSpl, RBF, 2:Spl, 2:renal, MAP, MPAP, PCWP, SVR, PVR, VRSpl, RVR, and arterial levels of ET-1, insulin, glucagon, and glucose as well as SGO before and up to 30 min after diclofenac and at 20 min of ET-1 infusion (50 min after diclofenac)

View larger version (40K): [in this window] [in a new window]   Fig. 1.   Comparison of effects of endothelin-1 (ET-1) infusion with and without (controls, open bars) prior blockade of cyclooxygenase by diclofenac (control values are taken from Ref. 2). Left: *** and * indicate significantly different from basal value ( P < 0.001 and P < 0.05, respectively) according to ANOVA, repeated measures for the time points before and up to 30 min after diclofenac followed by Fisher paired least significant difference test. Values at 20 min of ET-1 are compared with values 30 min postdiclofenac or basal value in the controls, according to Student's paired t-test. Right: values at 20 min of ET-1 infusion with and without (controls) prior blockade expressed in percentage of basal value. P value indicates difference between groups based on Student's unpaired t-test.

	RESULTS

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Effects of Diclofenac Administration

HR, pulmonary oxygen uptake, (a-pa)_O2, CO, and splanchnic and renal blood flow and oxygen uptakes (Table 1). HR did not show a consistent change after diclofenac administration. Similarly, pulmonary oxygen uptake remained unchanged. (a-pa)_O2 rose from 45 ± 2 to 49 ± 3 ml/l at 20 min after the administration of diclofenac (P < 0.01) and then fell. CO fell after diclofenac from 5.89 ± 0.32 to 5.45 ± 0.35 l/min at 20 min (P < 0.01), followed by an increase (P < 0.01) back to basal value 30 min after the administration. Splanchnic and renal blood flow had dropped by an average 14 and 12%, respectively, at 10 min after diclofenac (P < 0.001). Splanchnic blood flow increased (P < 0.05) toward basal level 30 min after diclofenac, whereas the suppression of renal blood flow was unchanged and 15% below basal value at that time (P < 0.001). The splanchnic and renal oxygen extractions changed in parallel with the blood flow changes, and consequently the splanchnic and renal oxygen uptakes remained unchanged after diclofenac.

Systemic and pulmonary blood pressure and vascular resistance (Table 1). MAP increased from the basal value of 92 ± 2 to 96 ± 3 mmHg at 10 min after diclofenac (P < 0.01), was still elevated at 20 min (P < 0.05), but did not differ from basal value 30 min after diclofenac. SVR changes did not, because of the few observations (4 subjects), attain statistical significance (repeated-measures ANOVA, P < 0.1). MPAP, PCWP, and PVR were unchanged after diclofenac. Splanchnic (VR_Spl) and renal (RVR) vascular resistance increased within 10 min after diclofenac (P < 0.001). Although RVR was unchanged at 30 min after diclofenac, VR_Spl had decreased (P < 0.01) and almost returned to basal value.

Arterial levels of plasma ET-1 and catecholamines. The arterial ET-1 level was 5.53 ± 0.36 pmol/l in the basal state and unchanged at 30 min after diclofenac (6.75 ± 0.77 pmol/l). Neither epinephrine nor norepinephrine had changed from basal values (0.13 ± 0.04 and 1.02 ± 0.07 nmol/l, respectively) at 30 min after diclofenac administration (0.13 ± 0.04 and 1.07 ± 0.12 nmol/l, respectively).

Glucose, insulin, and glucagon (Table 1). Basal insulin and glucagon concentrations were 7.33 ± 1.22 µU/ml and 67.3 ± 4.3 pg/ml, respectively, and did not change after diclofenac administration. Arterial glucose showed a transient small increase. Splanchnic glucose production fell from the basal value 0.85 ± 0.13 to 0.65 ± 0.07 mmol/min (P < 0.01) at 10 min, with no further change at 30 min after diclofenac, when the value was still below basal (P < 0.05).

Effects of ET-1 Infusion

Cardiovascular variables (Table 1). Twenty-minute administration of ET-1, starting 30 min after diclofenac, caused no significant change in HR or pulmonary oxygen uptake. MAP increased by 4%, and CO fell by 13% (P < 0.05). SVR increased (P < 0.05). MPAP fell during the ET-1 infusion (P < 0.05), whereas PCWP and PVR remained unchanged. Splanchnic blood flow fell by 38 ± 3.5%, and renal blood flow fell by 33 ± 3.2% during the ET-1 infusion (P < 0.001). VR_Spl and RVR increased during the ET-1 infusion (P < 0.001).

Arterial levels of plasma ET-1 and catecholamines. Arterial ET-1 levels rose to 78.3 ± 8.3 pmol/l at 20 min of infusion. Epinephrine and norepinephrine levels were unchanged at 20 min of ET-1 infusion (0.10 ± 0.01 and 0.97 ± 0.13 nmol/l, respectively).

Glucose, insulin, and glucagon (Table 1). Arterial levels of glucose, insulin, and glucagon fell during the ET-1 infusion compared with 30 min after diclofenac (P < 0.05-0.001). Splanchnic glucose production did not show a significant change.

	DISCUSSION

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Cardiovascular Effects

The vasoconstrictor response to COx inhibition after diclofenac was proportionally similar in the splanchnic and renal vascular beds but was transient in the splanchnic vascular bed, with a return to basal level within 30 min after diclofenac (Fig. 1). Concomitant with a rapid return of splanchnic blood flow, CO also increased from 20 to 30 min and MAP had returned to basal value at 30 min after diclofenac. The reversal of the effect of diclofenac on splanchnic blood flow is noteworthy, considering the long plasma half-life of diclofenac (1-2 h), but it could be due to more rapid elimination of diclofenac at the site of action. Another possibility is other local compensatory mechanisms in the splanchnic as opposed to the renal vascular bed.

PGI₂ synthesis has been suggested to exert negative feedback and suppress ET-1 release (11), suggesting that an additional possible cause of the reduction in blood flow after COx inhibition could be increased ET-1 release. The present responses to COx inhibition are not what would be expected of an ET-1-mediated response. Accordingly, our previous results with intravenous ET-1 administration (0.2-4 pmol · kg¹ · min¹) have shown that the vasoconstrictor response is approximately twofold higher in the splanchnic than the renal circulation (2, 4, 27). Furthermore, these suppressions of splanchnic as well as renal blood flow still persisted 60 min after the ET-1 infusions (4, 27), in accordance with the almost irreversible binding of ET-1 to its receptors (15).

Nor is it likely that decreased NO production could have been caused by COx inhibition because this would be expected to result in a decreased inhibition of preproendothelin-1 proteolysis and hence increased ET-1 formation (6, 30), followed by increased plasma levels of ET-1. In accordance with such a mechanism, our previous results have shown that administration of the endogenous NO synthase inhibitor N^G-monomethyl-L-arginine is followed by increased pulmonary ET-1 release, prolonged splanchnic and renal vasoconstriction, and significantly reduced response to exogenous ET-1 (1). Had the effect of N^G-monomethyl-L-arginine been mainly withdrawal of the inhibitory NO influence on smooth muscle contraction, then the ET-1 response would have been increased vasoconstriction.

The present renal vasoconstrictor response to diclofenac still persisted 30 min after diclofenac. That this effect was not ET-1 elicited is further supported by the subsequent ET-1 infusion results demonstrating an even more pronounced renal blood flow reduction (33 ± 3%) compared with those in our previous control subjects (23 ± 2%, P < 0.02) (4, 27) who received ET-1 in the same way (4 pmol · kg¹ · min¹ for 20 min) as in the present study (Fig. 1). The total decrease in renal blood flow with COx inhibition and ET-1 infusion in the present study was 43 ± 3% compared with the basal value and was thus twofold higher than that previously found with ET-1 alone (P < 0.01). Taken together with the lack of changes in plasma ET-1 levels after COx inhibition, and the same proportional increase in arterial ET-1 levels with ET-1 infusion (12-fold) as previously reported (2, 27, 28), the results indicate that basal PGI₂ synthesis, as opposed to NO (1), does not suppress ET-1 synthesis.

The subsequent ET-1 infusion caused reductions in splanchnic blood flow (38%), CO (13%), and increase in MAP (4%) of a magnitude similar to those previously reported for splanchnic blood flow (34-42%) (4, 27), CO (14%) (28), and MAP (6-7%) (27, 28) with only ET-1 infusion administered in the same way as in the present study.

Metabolic Effects

As to the mechanism for the reduced splanchnic glucose output, the levels of the glucoregulatory hormones insulin, glucagon, epinephrine, and norepinephrine were unchanged. Although the hormonal findings are in agreement with those reported by others in healthy subjects using indomethacin as a COx inhibitor (9), the others reported a simultaneous increase in splanchnic glucose output (9). Attempts to explain the difference in splanchnic glucose output can only be speculative. Interestingly, in vitro studies have shown that prostaglandins stimulate hepatic glycogenolysis (7), suggesting that our finding of reduced splanchnic glucose output after diclofenac might be due to withdrawal of prostaglandin stimulation of glycogenolysis. ET-1, on the other hand, has been shown to stimulate glycogenolysis in perfused rat livers or rat hepatocytes (13, 25), whereas our studies with ET-1 infusion in healthy subjects have demonstrated reduced splanchnic glucose output. The results illustrate the difficulties encountered when in vitro and in vivo data, as well as data from different species, are compared.

The ET-1 infusion after diclofenac administration did not cause a similar proportional decrease (50-55%) in splanchnic glucose output, as previously found with only ET-1 infused in the same way as in the present study (2, 3) or at a rate of 0.2 pmol · kg¹ · min¹ (42 ± 6%) (4). This could be because splanchnic glucose output was already markedly reduced (to 0.68 mmol/l) before the ET-1 infusion. The splanchnic glucose production at 20 min of ET-1 infusion was too low to maintain the arterial glucose level (Table 1), which fell. This was accompanied by a reduction in arterial insulin levels, as previously found with ET-1 infusion (2, 3). Similarly, arterial glucagon fell as previously reported (2, 3). Because these hormonal changes occurred to the same extent as previously reported, but with a much smaller effect on splanchnic glucose output, the results suggest that ET-1 has a direct suppressive effect on glucagon release.

In summary, COx inhibition causes increased MAP mainly due to reductions in renal and splanchnic blood flows, indicating a basal vasodilating effect mediated by PGI₂. The longer duration of renal blood flow suppression, demonstrating that the kidney is more susceptible to COx inhibition by diclofenac, suggests that the kidney is more dependent on PGI₂ synthesis for maintaining basal vascular tone. Compensatory mechanisms to counteract the effects of diclofenac seem to operate in the splanchnic but not the renal vascular bed, rendering the latter more sensitive to circulating ET-1.

The results imply that whenever COx inhibitors such as diclofenac are administered to patients with elevated ET-1 levels, the potentiated effect of these two on renal blood flow has to be taken into consideration. This is especially important in pathophysiological conditions (with reduced renal blood flow) as seen in renal insufficiency (16).