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Department of Clinical Physiology, Huddinge University Hospital, S-141 86 Huddinge; and Department of Physiology and Division of Pharmacology, Karolinska Institute, S-171 77 Stockholm, Sweden
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
ACKNOWLEDGEMENTS
FOOTNOTES
REFERENCES
ABSTRACT 
Ahlborg, Gunvor, and Jan M. Lundberg. Nitric
oxide-endothelin-1 interaction in humans. J. Appl.
Physiol. 82(5): 1593-1600, 1997.
Healthy men
received NG-monomethyl-L-arginine
(L-NMMA) intravenously to study
cardiovascular and metabolic effects of nitric oxide synthase blockade
and whether this alters the response to endothelin-1 (ET-1) infusion.
Controls only received ET-1.
L-NMMA effects were that heart
rate (17%), cardiac output (17%), and splanchnic and renal blood flow
(both 33%) fell promptly (all P < 0.01). Mean arterial blood pressure (6%), and systemic (28%) and
pulmonary (40%) vascular resistances increased
(P < 0.05 to 0.001). Arterial ET-1
levels (21%) increased due to a pulmonary net ET-1 release
(P < 0.05 to 0.01). Splanchnic glucose output (SGO) fell (26%, P < 0.01). Arterial insulin and glucagon were unchanged. Subsequent ET-1
infusion caused no change in mean arterial pressure, heart rate, or
cardiac output, as found in the present controls, or in splanchnic and
renal blood flow or splanchnic glucose output as previously found with
ET-1 infusion (G. Ahlborg, E. Weitzberg, and J. M. Lundberg.
J. Appl. Physiol. 79: 141-145,
1995
[Medline]
). In conclusion, L-NMMA
like ET-1, induces prolonged cardiovascular effects and suppresses SGO.
L-NMMA causes pulmonary ET-1
release and blocks responses to ET-1 infusion. The results indicate
that nitric oxide inhibits ET-1 production and thereby interacts with
ET-1 regarding increase in vascular tone and reduction of SGO in
humans.
NG-monomethyl-L-arginine and
endothelin-1 infusion; cardiac output; splanchnic and renal blood flow; systemic and pulmonary vascular resistance; arterial levels and
pulmonary release of endothelin-1; insulin and splanchnic glucose
output
INTRODUCTION EVER SINCE the endothelium-dependent vasodilatory
effect of acetylcholine (14) was shown to be mediated by nitric oxide (NO) (23), there has been an increasing interest in the possible role
of NO in health and disease. The systemic hypotension seen during
endotoxic shock has been ascribed to an excess formation of NO (20). In
patients with acute respiratory distress syndrome, NO administration
causes reduction of the pulmonary hypertension (26). Other studies have
shown that hypoxic vasoconstriction in healthy subjects is reversed by
NO inhalation (12). However, there is no direct evidence that NO is
involved in the regulation of pulmonary vascular resistance in healthy
humans. NO is formed by two principally different types of cytosolic NO
synthases (NOS). One type, which is found in neurons and endothelial
cells, is constitutive, is Ca2+
calmodulin dependent, and releases picomoles of NO on receptor stimulation; the other type is induced, e.g., by immunological stimuli,
is Ca2+ independent, and releases
hundred- to thousand-fold higher amounts of NO. The substrate for NOS
is L-arginine (22). The
L-arginine analog
NG-monomethyl-L-arginine
(L-NMMA), which occurs naturally
in human blood (29), is a competitive inhibitor of NOS (24). Previous studies in healthy humans have shown that administration of
L-NMMA into the brachial artery
causes vasoconstriction in the forearm (28). Administration into a
systemic vein causes increased systemic arterial blood pressure and
reduces heart rate and cardiac index as estimated by noninvasive
methods (15). The influence of NO synthesis in different systemic
vascular beds in humans, with the exception of muscle, is incompletely
known.
Apart from NO, vascular endothelial cells have been shown to synthesize
a potent pressor substance called endothelin-1 (ET-1) (35). Previous
studies of the effects of ET-1 infusion in healthy humans have shown
that ET-1 causes vasoconstriction in the splanchnic and renal vascular
beds as well as reduction of cardiac output and splanchnic glucose
production (1-3, 32, 33). Several substances stimulate the
expression of preproendothelin mRNA (36). NO has been shown to suppress
ET-1 release (8, 27, 38) by inhibition of preproendothelin synthesis
(38). Furthermore, the ET-receptor antagonist bosentan has recently
been shown to attenuate the pressor effects of NOS inhibition in rats
(25). These observations suggest that NO might promote its vasodilatory effects by blocking the formation of ET-1. On the other hand, ET-1
stimulates NO formation via endothelial
ETB-receptor activation (11, 31).
If NO and ET-1 interact at the smooth muscle contraction level,
withdrawal of NO production would result in increased sensitivity to
exogenous ET-1. In this study we have, therefore, examined the effects
of intravenous administration of
L-NMMA on cardiac output and
vascular resistance in pulmonary, splanchnic, and renal vascular beds
in healthy human subjects. We also studied the effects of the NOS
blocker on ET-1 levels in plasma and splanchnic glucose output.
Moreover, we examined whether
L-NMMA changes the responses to
infused exogenous ET-1.
METHODS Subjects
Procedure
All subjects were studied in the supine position after an overnight fast. All catheters were inserted percutaneously. One thin Teflon catheter was introduced into an antecubital vein for infusion of dye indicators for determination of blood flow. Another was inserted into the brachial artery for blood sampling and blood pressure measurements. 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.In group 1, another catheter (Cournand
no. 7) was positioned in the hepatic vein, under fluoroscopic control.
L-NMMA (Calbiochem-Novabiochem, Läufelfinger, Switzerland) dissolved in saline was administered intravenously during 5 min at a dose of 3 mg/kg as previously described
(15). Pulmonary O2 uptake
(
O2) 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 L-NMMA infusion
for determinations of dye concentrations (in the brachial artery and
hepatic vein), O2 contents, and
plasma ET-1 levels. Thirty minutes after
L-NMMA, a 20-min ET-1 (Peninsula
Laboratory, Belmont, CA) infusion was initiated. In two subjects, the
infusion rate was 0.2 pmol · kg
1 · min1
for 20 min. In three subjects, the rate was 0.2 pmol · kg1 · min1
during the first 10 min and 1 pmol · kg1 · min1
for the last 10 min. Pulmonary
O2 was determined, and
blood samples were drawn from all sites at 20 min of ET-1 infusion.
In group 2, ET-1, 0.2 pmol · kg1 · min1,
was infused for 60 min. Pulmonary
O2 was determined, and
blood samples were drawn simultaneously from the catheters in the basal
state repeatedly during and up to 90 min after the infusion (see Table
2).
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In both groups, heart rate (HR), mean arterial pressure (MAP), mean
pulmonary artery pressure (MPAP) and mean pulmonary capillary wedge
pressure (PCWP) were recorded continuously during the studies. Cardiac
output (CO) was estimated according to the Fick principle on the basis
of pulmonary O2 and
systemic arterial-pulmonary arterial
O2 difference
[(a-pa)DO2].
In group 1, splanchnic and renal blood
flow were determined by constant infusions of cardiogreen and
p-aminohippurate (PAH) as previously
described (1). For calculation of renal blood flow, the fractional
extraction of PAH (the arterial minus renal venous concentration
divided by arterial concentration) was assumed to be 0.9. In
group 1, arterial insulin, glucagon
and glucose concentrations, as well as glucose concentration in the
hepatic vein were determined before and after L-NMMA administration as well as
during and after the ET-1 infusion.
Analyses
Plasma ET-1 concentrations were assessed by radioimmunoassay technique. Plasma aliquots (1 ml) were extracted with acid ethanol and dried under a nitrogen stream. For determination of ET-1, ET-1 antiserum (E1) and 125I-labeled ET-1 (Amersham) were used. The detection limit for the assay was 0.40 fmol/tube. Expressing the ET-1 value as 100%, the cross-reactivity of the used antiserum is 16% for ET-1 (1621), 27% for ET-2, 8% for ET-3, and 0.045% for
Big ET-1 (138). No cross-reactivity with Big ET-1 (2238) was
observed at concentrations up to 1 µM. The intra- and interassay
variations were 6 and 14%, respectively (16). For characterization of
the immunoreactivity in the extracted samples, extra samples were
subjected to reverse phase high-performance liquid chromatography.
O2 saturation and hemoglobin
concentration in blood were determined with an OSM 3 radiometer and
blood gases with an ABL 520 radiometer (Radiometer, Copenhagen,
Denmark). O2 content in expired
air was determined with a zirconium oxide cell (S-3A/I)
O2 analyzer (Ametek, Pittsburgh, PA) and CO2 content by the
infrared technique (Ametek CD-3A
CO2 analyzer). The
radioimmunological techniques used to analyze insulin and glucagon as
well as the glucose dehydrogenase method for glucose measurement have
been described previously (1).
Calculations
Systemic vascular resistance (SVR) was calculated as MAP/CO and pulmonary vascular resistance (PVR) as (MAP
PCWP)/CO.
Data are means ± SE. Analysis of variance (ANOVA), according to the repeated-measures design, was used followed by the Fisher protected least-significant difference test (PLSD), 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 homogenous variances (35).
RESULTS
Group 1
Effects of L-NMMA administration. HR, PULMONARYO2, PULMONARY AND
SPLANCHNIC ARTERIOVENOUS O2
DIFFERENCES, CO, AND SPLANCHNIC AND RENAL BLOOD FLOW (TABLE
1).
HR decreased from 58 ± 4 beats/min in the basal state to 48 ± 4 beats/min within 3 min after the start of the
L-NMMA administration (P < 0.001). From 10 to 20 min after
L-NMMA there was a small increase, but HR was still below basal values 30 min after
L-NMMA. Pulmonary
O2 remained unchanged after
L-NMMA administration (Table 1).
(a-pa)DO2
rose from 46 ± 2.0 to 56.0 ± 2.3 ml/l within 10 min after
L-NMMA
(P < 0.001), with no further change
at 30 min after the administration. CO fell from 5.57 ± 0.25 to
4.64 ± 0.23 l/min at 10 min after
L-NMMA
(P < 0.01) and was still below basal
value 30 min after the administration. Stroke volume (SV) did not
change significantly during the study. The systemic arterial-hepatic venous O2 difference had increased
from 45 ± 4 to 64 ± 5 ml/l at 10 min after the
L-NMMA administration
(P < 0.001) and was still elevated
20 min later (P < 0.05). Splanchnic
and renal blood flow had dropped by an average of 33% at 10 min after
L-NMMA
(P < 0.001) and, although the values
increased from 10 up to 30 min (P < 0.01) after L-NMMA, they were
still 16-23% below basal values (P < 0.01). Splanchnic
O2 was unchanged during the
study.
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1
in the basal state to 20.9 ± 1.1 mmHg · min · l1
(P < 0.001) at 10 min. PVR increased
after L-NMMA from 0.72 ± 0.09 to 1.01 ± 0.11 mmHg · min · l1
at 20 min after the L-NMMA
administration (P < 0.05). Neither SVR or PVR showed any further change. Splanchnic
(VRSpl) and renal (RVR) vascular
resistances increased within 10 min after
L-NMMA (P < 0.001).
VRSpl decreased from 10 to 20 min
(P < 0.01), and RVR showed a
progressive decrease from 10 to 30 min
(P < 0.05) after
L-NMMA. Both
VRSpl and RVR were above basal
levels 30 min after the infusion (range
P < 0.01-0.001).
VENTILATION.
There was no difference in respiratory rate or ventilation before (13.8 ± 1.8 breaths/min and 8.02 ± 0.59 l/min, respectively) compared
with after L-NMMA (14.2 ± 1.7 breaths/min and 7.86 ± 0.42 l/min, respectively),
as estimated from collection of expired air during mouth breathing with
a noseclip.
ARTERIAL LEVELS AND PULMONARY AND SPLANCHNIC EXCHANGES OF PLASMA
ET-1.
Arterial ET-1 levels rose slightly (ANOVA, repeated measures,
P < 0.05) after the
L-NMMA-administration from 7.57 ± 0.56 pmol/l in the basal state to maximally 9.56 ± 0.74 pmol/l at 20 min after the
L-NMMA administration
(P < 0.05). Ten minutes later,
arterial plasma ET-1 concentration had returned to basal value.
Pulmonary arterial plasma ET-1 concentration was 7.42 ± 0.93 pmol/l
in the basal state and did not show a significant change after
L-NMMA. There was no pulmonary
arterial-arterial plasma ET-1 difference except at 20 min
after the administration of
L-NMMA (2.00 ± 0.38 pmol/l, P < 0.01), corresponding to
a net ET-1 release of 5.30 ± 0.93 pmol/min
(P < 0.005) in the pulmonary
vascular bed. There was no arterial-hepatic venous ET-1 difference at
any time, indicating that splanchnic release did not differ from local
uptake of ET-1; i.e., there was no net production.
GLUCOSE, INSULIN, AND GLUCAGON (FIG.
1).
Basal arterial glucose and glucagon concentrations were 4.82 ± 0.16 mmol/l and 65 ± 6.6 pg/ml, respectively, and did not change during
the study (P < 0.57 and
P < 0.81, respectively; ANOVA, repeated measures). Neither did arterial insulin show a significant change (P < 0.11, ANOVA, repeated
measures). Splanchnic glucose production fell after
L-NMMA
(P < 0.01, ANOVA, repeated measures) from 0.85 ± 0.14 to 0.63 ± 0.09 mmol/min
(P < 0.01) at 10 min after
with no further change up to 30 min after
L-NMMA.
Effects of ET-1 infusion after L-NMMA administration. There was no difference in responses between those receiving the higher compared with those receiving the lower ET-1 dose. Therefore they were treated as one group. CARDIOVASCULAR VARIABLES (TABLE 1). The administration of ET-1, 30 min after L-NMMA, caused no further change in HR, pulmonary
O2, CO, SVR,
or PVR. In addition, there was no change in pulmonary arterial
pressures during the ET-1 infusion. Nor did splanchnic blood flow or
VRSpl change. The renal blood flow
was unchanged 10 min after the onset of the ET-1 infusion (1.191 ± 0.112 l/min) and then showed a small decrease, which at the end of
infusion corresponded to a further reduction of 0.111 ± 0.036 l/min
(P < 0.05).
ET-1 LEVELS.
Arterial ET-1 levels rose to 24.9 ± 6.7 pmol/l at 20 min of
infusion. At 20 min of ET-1 infusion, the concentration of ET-1 in the
pulmonary artery was 56.8 ± 18.2 pmol/l.
VENTILATION.
Neither respiratory rate nor ventilation changed during ET-1 infusion.
GLUCOSE, INSULIN, AND GLUCAGON (FIG. 1).
There was no change in arterial levels of glucose, insulin, or glucagon
during ET-1 infusion compared with 30 min after
L-NMMA (values at 20 min of ET-1
infusion corresponding to 4.18 ± 0.18 mmol/l, 7.5 ± 1.1 µU/ml, and 63 ± 5.9 pg/ml, respectively). Nor did splanchnic
glucose production show a significant change (the value corresponding
to 0.55 ± 0.07 mmol/min at 20 min of ET-1 infusion).
Group 2
Effects of ET-1 infusion. HR , PULMONARYO2,
PULMONARY
(A-PA)DO2,
AND CO (TABLE 2).
HR showed a transient decrease from 58 ± 3 beats/min in the basal
state to 55 ± 4 beats/min at 20 min of the ET-1 infusion (P < 0.05). Pulmonary
O2 did not change during the
ET-1 infusion. (a-pa)DO2
rose from 45 ± 2 to 49 ± 2 ml/l within 3 min of infusion (P < 0.001) and was still elevated
90 min after the infusion (P < 0.05). CO fell from 5.97 ± 0.18 to 5.28 ± 0.20 l/min at 10 min of the infusion (P < 0.001) and then
remained unchanged during and up to 30 min after the infusion. SV
decreased from 103 ± 5 to 95 ± 5 ml/min
(P < 0.05) at 10 min of the
infusion. CO and SV had returned to basal values 90 min after the
infusion.
SYSTEMIC AND PULMONARY BLOOD PRESSURE AND VASCULAR RESISTANCES
(TABLE 2).
MAP increased during ET-1 infusion from 93 ± 3 mmHg within 10 min
to 95 ± 4 mmHg (P < 0.05), with
a further increase up to 60 min of infusion
(P < 0.001). Ninety minutes after
the infusion, MAP was still significantly increased
(P < 0.01). SVR increased from 15.8 ± 0.7 mmHg · min · l1
in the basal state to 18.1 ± 0.8 mmHg · min · l1
at 10 min of infusion (P < 0.001).
Basal SVR was restored 90 min after infusion. MPAP did not change,
whereas mean PCWP fell during and was still decreased at 90 min after
the infusion (P < 0.01). PVR
increased from 0.86 ± 0.08 mmHg · min · l1
at 20 min to 1.10 ± 0.06 mmHg · min · l1
at 30 min of infusion (P < 0.05) and
was elevated up to 30 min postinfusion
(P < 0.01).
ARTERIAL LEVELS OF PLASMA ET-1.
Arterial ET-1 was 4.87 ± 0.55 pmol/l in the basal state and rose
during the ET-1 infusion (P < 0.001, ANOVA, repeated measures) to 5.72 ± 0.5 pmol/l at 2 min and 7.74 ± 0.88 pmol/l at 10 min (P < 0.05), with a further increase to 11.2 ± 2.13 pmol/l at 60 min of
infusion. The value was still elevated at 60 min (6.78 ± 1.01 pmol/l, P < 0.01) but not 90 min
after ET-1 infusion.
DISCUSSION
The present results demonstrate that intravenous administration of a NOS blocker, L-NMMA, to healthy humans induces marked cardiovascular effects as indicated by decreased HR, CO, and splanchnic and renal blood flow and splanchnic glucose output. In addition, MAP, SVR, and PVR increased. No effects were seen on pulmonary pressures. Furthermore, L-NMMA abolished the effects of subsequently infused ET-1 on these variables.
Cardiovascular Effects
The effects of L-NMMA on MAP agreed well with those reported in a previous study, in which a similar dose of L-NMMA was administered (15). These results indicate that NO synthesis is involved in the maintenance of basal vascular tone. The increase in MAP in this and the previous study (15) was associated with decreases in HR and CO. These were most likely baroreceptor reflex-mediated responses because neither SV nor left ventricular filling pressure, as represented by PCWP, changed in the present study. Thus it is unlikely that L-NMMA reduced the contractility of the heart. Furthermore, NO itself, if anything, reduces the contractility of cardiac myocytes in vitro (9).The administration of L-NMMA has been shown to cause constriction of the human forearm vasculature (28). The present results show that both splanchnic and renal blood flow also fall in response to L-NMMA. Thus it is clear that effects in these vascular beds also contribute to the increased SVR. In fact, at the presently administered dose of L-NMMA, the reduction in splanchnic and renal blood flow totaled 0.9 l/min. The corresponding decrease in CO was ~l l/min. Thus little of the reduction in CO could be due to flow reduction in other vascular beds such as muscle. In other words, the vasoconstriction in the splanchnic and renal vascular beds seems to be the most important determinant for the increased MAP after L-NMMA administration. To our knowledge, there are no previous reports, from in vivo human studies, that these vascular beds respond to NO or NO synthesis blockade, although indirect evidence from animal studies has suggested that this is the case (for a review, see Ref. 34). In addition, humans have been shown to synthesize more NO3 from intragastric vs. intravenous administration of arginine (10), suggesting that NO has a regulatory function in the splanchnic region.
The total mass of skeletal muscle is ~40-50% of body weight
(21). However, the basal blood flow in skeletal muscle is only ~20-50
ml · kg1 · min1
(5). The reduction in CO measured in the present study allows for a
blood flow reduction of ~0.3 l/min in the musculature. This corresponds to a reduction by ~30%, i.e., the same proportional reduction in skeletal muscle as in the splanchnic and renal tissues, indicating that all these vascular beds are equally dependent on NO
synthesis for maintenance of basal tone.
When L-NMMA was infused, we noticed a maximal increase in PVR (40%), which was of a magnitude similar to that for SVR (28%). We could not demonstrate any change in the pulmonary blood pressure. This could be due to the fact that basal pulmonary pressure is too low to allow a similar proportional increase in MPAP as that seen for MAP (5%) to be detected. Absence of a pulmonary pressure response after L-NMMA administration has also been noticed in isolated normoxic, but not hypoxic, rat lungs (6). Previous results also indicate that preconstriction of the pulmonary vessels is necessary to elicit NO release and pressure response (6). This could explain why inhalation of NO together with air has no effect on MPAP or CO in healthy humans (13) and suggests that endogenous NO has optimal vasodilatory effects under basal normoxic conditions.
Previous data indicate that NO inhibits synthesis of ET-1 (8, 27, 38) at the preproendothelin level in endothelial cells. Inhibition of NO synthesis would be expected to lead to increased ET-1 synthesis and possibly an overflow to the blood. This is consistent with our finding that the systemic arterial concentration of ET-1 was increased 20 min after L-NMMA infusion. A further support for this theory is the observation that ET-1 concentration was more greatly increased in systemic than in pulmonary arterial blood. Thus, although the para- and autocrine events elicited by ET-1 need not necessarily be accompanied by changes in circulating levels of ET-1, we noticed a net pulmonary release of ET-1 at 20 min after L-NMMA corresponding to 5.30 ± 0.93 pmol/min (P < 0.005).
In a previous study, we found that intravenous administration of ET-1 at doses that evoked a 37% elevation of systemic arterial ET-1 levels was accompanied by decreases in splanchnic and renal blood flow corresponding to 18 and 10%, respectively (3). In the present study, we observed only a 26% increase in systemic arterial ET-1 levels. However, because the present circulating ET-1 levels represent an "overflow" of ET-1 synthesized by the endothelium, the local concentration at the site of action can be assumed to be higher, especially because the main ET-1 release seems to occur abluminally (30). In experiments that used even higher ET-1 doses, leading to 10-fold increases in systemic arterial concentration, the reduction in splanchnic and renal blood flow was 34 and 26%, respectively (32). Consequently, it seems possible that the reductions in splanchnic and renal blood flow (~33%) noted in the present study (Table 1) on infusion of L-NMMA could, at least partly, be ET-1 mediated and that a significant part of the dilating effect of NO on the vasculature might be due to inhibition of ET-1 synthesis. This hypothesis is supported by the findings that NO stimulates cGMP formation (18) and that a cGMP-dependent mechanism or NO formation inhibits ET-1 formation in cultured endothelial cells (8, 27, 38).
To further test our hypothesis that NO exerts a physiological effect in vivo by interfering with ET-1 formation, the NOS blockade was followed by intravenous infusion of ET-1 30 min after L-NMMA. The rate of ET-1 infusion was the same (3) as has previously been shown to cause significant, prolonged (>1- to 3-h) reductions in splanchnic and renal blood flow (3, 32) and, in the present control group (group 2), to cause prolonged increase in MAP and decrease in CO. No further fall in splanchnic blood flow was seen on ET-1 infusion after L-NMMA in the present study. At 20 min of ET-1 infusion, the fall in renal blood flow in the present study corresponded to 0.11 ± 0.04 l/min, which is significantly smaller (P < 0.05) than our previously noted reduction of 0.21 ± 0.04 l/min (3) with the same ET-1 dose. In fact, the small drop in renal blood flow at 20 min of ET-1 infusion after L-NMMA in the present study is in agreement with our previous results in resting control subjects, who did not receive L-NMMA or ET-1 but showed a slow progressive reduction in renal blood flow with time (3).
Our previous data have shown that infusion of ET-1 at 4 pmol · kg1 · min1
causes reduction of CO (33). The present data show that infusion of
ET-1 alone at 0.2 pmol · kg1 · min1
(group 2) causes an 11% fall in CO within 10 min of infusion (P < 0.001), whereas
the same dose of ET-1 infused after
L-NMMA caused no further effects
on CO or blood pressure. The prompt CO response (within 3 min) in
group 2, which received only ET-1 infusion at a dose leading to an increase of arterial ET-1 (47%), comparable to the physiological increase previously seen during physical exercise (2) and the prolonged suppression of CO (Table 2),
emphasizes the potency of ET-1 and also the potential importance of
circulating ET-1. Both the observation that the vascular effect of ET-1
infusion was nearly abolished and the prolonged duration of blood flow
reductions in lung, splanchnic, and renal tissues after
L-NMMA infusion support
interaction of NO with ET-1 by suppressing ET-1 synthesis. Had NO
mainly exerted its vasodilatory effect by acting directly on smooth
muscle contraction, the withdrawal of NO production would be expected
to potentiate the vasoconstrictor response to ET-1 infusion. The
explanation underlying the surprising observation that the
vasoconstrictor effects of exogenous ET-1 were virtually abolished up
to 50 min after L-NMMA may be
related to long-term occupancy of
ETA and
ETB receptors by excess
endogenously released peptide (or internalization of ET receptors),
which prevents receptor access for the circulating exogenous ET
counterpart. The notion that part of the vasodilatory action of
nitrovasodilatators (27) or NO (8, 38) might be due to suppression of
ET-1 production has previously been suggested on the basis of studies in cultured human endothelial cells. Conversely, ET-1 has been shown to
release NO from mesenteric arteries (11, 31). ET-1 stimulates NO
formation (11, 31) presumably via
ETB-receptor stimulation and
intracellular Ca2+ release (36).
Support for this theory could be the unexplained increase in ET-1
levels found in patients with chronic heart failure, treated with the
ET-1-receptor antagonist bosentan (19). Blockade of the
ETB receptor would be expected to
offset the stimulating effect of ET-1 on NO synthesis and,
consequently, the inhibitory effect of NO on ET-1 synthesis
and/or release, resulting in increased ET-1 levels. On the
basis of these studies and the present data, we would like to suggest
that there is a feedback mechanism between ET-1 and NO synthesis that
acts reciprocally to regulate vascular tone in intact humans in vivo,
at least in the basal state.
Splanchnic Glucose Output
Synthesis of NO does not only take place in the vascular endothelium. Consequently, for example, not only endothelial but also neuronal NOS have been identified in the human kidney (4), indicating the potential importance of NO in different signalling events. Interestingly, NOS blockade in the present study also caused a prolonged (>60-min) decrease in splanchnic glucose output, a phenomenon we have previously seen during and after ET-1 infusion in humans (1-3). That splanchnic glucose output is not dependent on the blood flow is shown by findings during exercise when glucose output may increase fivefold while splanchnic blood flow falls by 50% (2). Also, the hepatic venous-arterial glucose concentration difference falls with a higher rate of ET-1 infusion at rest (1). Furthermore, ET-1 infusion before exercise causes suppressed splanchnic glucose output during exercise compared with control exercise that is not directly related to the reductions in splanchnic blood flow. Thus exercise immediately after ET-1 infusion results in further depression of splanchnic blood flow but an increasing splanchnic glucose output (2). L-NMMA did not lead to any consistent change in arterial insulin or glucagon levels but to a persistent suppression of splanchnic glucose output, with no further change during the subsequent ET-1 infusion (Fig. 1). Similar changes in splanchnic glucose output with no or a minor change in arterial glucose concentrations indicating reduced peripheral utilization were seen in our previous ET-1 infusion studies (1-3). Therefore, the present results concerning the effect of NOS blockade on splanchnic glucose output and arterial glucose concentrations are also consistent with an ET-1-mediated effect. Lower insulin and glucagon plasma levels have been found in vivo in dogs after NOS blockade (7). In addition, suppression of arterial insulin and glucagon levels was also seen in humans after infusion of ET-1 at rates higher than those used in the present study (1, 2). Our present results do not, however, allow an interpretation of the mechanism underlying the reduction in splanchnic glucose output. In freshly isolated islets, NO does not seem to mediate arginine- or glucose-stimulated insulin secretion because this occurs without changes in NO release or cGMP content and is not prevented by NOS blockade (18). In fact, our results show the same pattern of decreased splanchnic glucose output with NOS blockade as with previous ET-1 infusion without blockade of the NO synthesis (1-3). This observation favors a mediator other than NO, possibly ET-1, for the reduction in splanchnic glucose output.In summary, the results show that NOS blockade was followed by increased arterial ET-1 levels, pulmonary ET-1 release, MAP, and SVR and PVR but reduced HR, CO, splanchnic and renal blood flow, and splanchnic glucose production. The pattern was similar to that previously reported for splanchnic and renal blood flow and splanchnic glucose output (3) and in the present control group for HR, MAP, CO, and SVR and PVR with increases in ET-1 levels 80 (3) to 130% above basal value. Although L-NMMA caused only a 25% increase in ET-1 levels, the intra- and paracellular concentrations are probably higher. This and the fact that the subsequent ET-1 infusion in the L-NMMA-treated subjects did not cause any further cardiovascular change or reduction in splanchnic glucose output support that NO suppresses ET-1 production and, thereby, interacts with ET-1 regarding vascular tone and splanchnic glucose output in humans. The results raise the question of whether pathologically increased ET-1 levels, which have been described for many pathological conditions in the cardio-pulmonary-vascular system, might, at least in part, be due to defective NO synthesis, which would be expected to disturb the normal control of vascular tone and splanchnic glucose output.
ACKNOWLEDGEMENTS
This study was supported by Swedish Medical Research Council Grants 14X-10374 and 14X-6554, the King Gustav V and Queen Victoria Foundation, and the Clas Groschinsky Foundation.
FOOTNOTES
Address for reprint requests: G. Ahlborg, Dept. of Clinical Physiology, Huddinge Univ. Hospital, S-141 86 Huddinge, Sweden.
Received 10 April 1996; accepted in final form 24 January 1997.
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