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American Journal of Physiology - Heart and Circulatory Physiology logoLink to American Journal of Physiology - Heart and Circulatory Physiology
. 2010 Apr 9;298(6):H2093–H2101. doi: 10.1152/ajpheart.01045.2009

Sympathetic nerves and the endothelium influence the vasoconstrictor effect of low concentrations of ouabain in pressurized small arteries

Hema Raina 1, Qingli Zhang 2, Albert Y Rhee 1, Thomas L Pallone 2, W Gil Wier 1,✉
PMCID: PMC2886643  PMID: 20382851

Abstract

We hypothesized that in salt-dependent forms of hypertension, endogenous ouabain acts on arterial smooth muscle to cause enhanced vasoconstriction. Here, we tested for the involvement of the arterial endothelium and perivascular sympathetic nerve terminals in ouabain-induced vasoconstriction. Segments of rat mesenteric or renal interlobar arteries were pressurized to 70 mmHg at 37°C and exposed to ouabain (10−11–10−7 M). Removal of the endothelium enhanced ouabain-induced vasoconstriction by as much as twofold (at an ouabain concentration of 10−9 M). A component of the ouabain-induced vasoconstriction is due to the enhanced spontaneous release of norepinephrine (NE) from nerve terminals in the arterial wall. The α1-adrenoceptor blocker prazosin (10−6 M) decreased ouabain-induced vasoconstrictions by as much as 50%. However, neither the contraction induced by sympathetic nerve activity (SNA) nor the NE release evoked by SNA (measured directly by carbon fiber amperometry) was increased by ouabain (<10−7 M). Nevertheless, the converse case was true: after brief bursts of SNA, vasoconstrictor responses to ouabain were transiently increased (1.75-fold). This effect may be mediated by neuropeptide Y and Y1 receptors on smooth muscle. In arteries lacking the endothelium and exposed to prazosin, ouabain (10−11 M and greater) caused vasoconstriction, indicating a direct effect of very “low” concentrations of ouabain on arterial smooth muscle. In conclusion, in intact arteries, the endothelium opposes ouabain (10−11–10−7M)-induced vasoconstriction, which is caused by both enhanced spontaneous NE release and direct effects on smooth muscle. Ouabain (<10−7M) does not enhance SNA-mediated contractions, but SNA enhances ouabain-induced contractions. The effects of endogenous ouabain may be accentuated in forms of hypertension that involve sympathetic nerve hyperactivity and/or endothelial dysfunction.

Keywords: mesenteric, sympathetic nerve activity, neuropeptide Y, renal interlobar artery, neurotransmitters

human essential hypertension is a multifactorial disorder involving many of the homeostatic mechanisms that control mean arterial blood pressure (27). Despite its complexity, hypertension always involves some increase in vascular resistance. The mechanisms that increase vascular resistance likely include alterations of circulating and neurally released vasoconstrictors and/or vasodilators, altered endothelial function, and changes in the molecular machinery of smooth muscle cells (SMCs). A hypothesis receiving much current attention is that, in salt-dependent forms of hypertension, elevated levels of endogenous ouabain (EO) in the blood plasma act to enhance the myogenic tone of small arteries and raise vascular resistance (56). In this hypothesis, circulating EO inhibits the α2-isoform of Na+-K+ pumps of arterial smooth muscle to increase intracellular [Na+] and [Ca2+] (via Na+/Ca2+ exchange 1) and promote vasoconstriction (55, 56). Indeed, ouabain is an arterial vasoconstrictor (3, 49) and produces hypertension when administered chronically to rodents (35, 55). Plasma levels of EO are elevated in normal humans on a high-salt diet (34), in ∼50% of human essential hypertensives (42), and in several salt-dependent animal models of hypertension (44).

Despite the above evidence, the putative role and mechanisms of action of EO in salt-dependent hypertension remain controversial. For example, 1) other endogenous Na+ pump inhibitors, such as marinobufagenin, are also elevated under similar conditions (18, 22) and might account for the vasoconstrictor effect, instead of EO; 2) the site of action of EO may be the hypothalamus (30) or sympathetic nerve terminals (1, 2); 3) ouabain binding to Na+-K+-ATPase stimulates protein tyrosine kinases, such as Src (31); and 4) the levels of circulating EO are low (<5 × 10−9 M), raising the possibility that EO is not a significant arterial vasoconstrictor. Several other factors are also relevant. The effects of ouabain have not often been examined in small arteries that have myogenic tone (as a result of being pressurized during study). Myogenic tone is extremely important because cytoplasmic [Ca2+] is elevated, and the “Ca2+ sensitivity” of contraction is enhanced when myogenic tone has developed. Both factors tend to allow very small changes in cytoplasmic [Ca2+] to cause contraction. Furthermore, in hypertension induced by the administration of ouabain, sympathetic nerve activity (SNA) is significantly increased (1), implying that the rise in vascular resistance may be at least partially due to elevated SNA and/or enhanced sympathetic neuromuscular transmission rather than a direct action of ouabain on arterial smooth muscle per se. Finally, most forms of hypertension are characterized by endothelial dysfunction; if the endothelium is involved in ouabain-induced vasoconstriction, it may also be involved in the role of EO in hypertension.

In the present study, we investigated several factors that might participate as mechanisms by which EO increases vascular resistance. We examined the concentration dependence of ouabain in arterial vasoconstriction, establishing that ouabain does induce significant vasoconstriction at low concentrations. We measured the influence of ouabain on sympathetic neuromuscular transmission and neurogenic contraction of small arteries that have myogenic tone. Since arteries are exposed to tonic SNA in vivo, we examined the effects of SNA on the vasoconstrictor effects of ouabain. In particular, we sought to determine if SNA potentiates the effects of ouabain. This might be expected as a result of the sympathetic cotransmitter neuropeptide Y (NPY), which is known to potentiate the actions of certain other vasoconstrictors and neurotransmitters. To perform these experiments, we used pressurized (isobaric) mesenteric arteries or renal interlobar arteries (when specified), both of which are well innervated by sympathetic nerves (12). Mesenteric small arteries are widely regarded as representative “resistance” arteries and, thus, are highly appropriate for a study relevant to hypertension (10). Although renal interlobar arteries certainly do not play exactly the same role in total peripheral resistance that mesenteric arteries do, they also exhibit myogenic tone (25) and are highly innervated by sympathetic nerves. When appropriate, we removed the endothelium of these arteries to examine its influence on ouabain-induced vasoconstriction.

MATERIALS AND METHODS

Preparation of Arteries and Experimental Solutions

All experiments were approved by and carried out according to the guidelines of the International Animal Care and Use Committee of the University of Maryland School of Medicine. Animals were maintained on a 12:12-h light-dark schedule at 22–25°C and 45–65% humidity and fed ad libitum on a standard rodent diet and tap water.

The mesenteric arcade/kidney was dissected from the abdominal cavity of Sprague-Dawley rats (100–150 g, Harlan) that had been killed by inhalation of CO2. The tissue was rinsed free of blood and placed in a temperature-controlled dissection chamber (5°C) containing a solution of the following composition (in mmol/l): 3.0 MOPS, 145.0 NaCl, 5.0 KCl, 2.5 CaCl2, 1.0 MgSO4, 1.0 KH2PO4, 0.02 EDTA, 2.0 sodium pyruvate, and 5.0 glucose (pH 7.4). Segments of fourth-order mesenteric arteries were dissected free. Alternatively, kidneys were hemisected, and interlobar arteries (0.5–1 mm in length, without side branches) were dissected from the surrounding kidney tissue as previously described (26).

Arteries were transferred to a recording chamber, and the proximal end was cannulated with a tapered glass pipette, secured with a single strand of 10-0 Ethilon ophthalmic nylon suture (Ethicon, Somerville, NJ), and gently flushed to remove any blood from the lumen. The distal end of the vessel was then cannulated, and the artery was gently stretched to approximate its in situ length and pressurized to 70 mmHg. One pipette was attached to a servo-controlled pressure-regulating device (Living Systems, Burlington, VT), whereas the other was attached to a closed stopcock. Arterial segments were continuously superfused with gassed Krebs solution containing (in mmol/l) 112.0 NaCl, 25.7 NaHCO3, 4.9 KCl, 2.0 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 11.5 glucose, and 10.0 HEPES (pH 7.4, gas composition: 5% O2-5% CO2-90% N2) at 37°C and were studied in the absence of intraluminal flow. Arteries were viewed with a ×10 objective with bright-field illumination on a Nikon TMS microscope (Nikon, Melville, NY) equipped with a monochrome video charge-coupled device camera. Outside diameter was continuously monitored with an online edge detection system that uses a video frame grabber and custom LabView software (National Instruments, Austin, TX). Passive diameter was determined by incubating the arteries with Ca2+-free solution for 10 min at the end of each experiment. Arteries that developed significant leaks were discarded (41).

To remove endothelial cells, an air bubble was introduced into the lines by opening the distal stopcock and allowing air to enter. Once the air bubble reached the lumen of the artery, the distal stopcock was closed to stop the solution flow. The air bubble was pushed through the lumen by opening the distal stopcock, and an additional 15-min waiting period was allowed before measurements were taken. The adequacy of endothelial removal was judged by verifying a lack of response to ACh (10 μM). Passive diameters were recorded by superfusing the blood vessel with 0 mM Ca2+, Krebs bicarbonate buffer solution containing 2 mM EGTA for 20 min. Adequate removal of the endothelium was indicated by relaxation of not more than 10% of the passive diameter. Perfusion with the air bubble was repeated as necessary to achieve this.

Electrophysiology

Electrical field stimulation (EFS) was applied via two platinum wires placed in the bath parallel to the long axis of the artery and connected to a stimulator (model S48, Astro-Med). The frequency range of EFS was 0.3–30 Hz, the pulse duration was 0.15 ms, and the intensity was 30 V. Tetrodotoxin (TTX; 0.1 μM) was used to confirm that the responses produced were neurogenic and not produced by direct electrical stimulation of SMCs.

We used carbon fiber electrodes and constant voltage amperometry to measure norephinephrine (NE) release at the adventitial surface of rat mesenteric small arteries using the technique previously described by Dunn et al. (15). Perivascular sympathetic nerves (28) were stimulated with a suction electrode, into which one end of the artery was drawn. The carbon fiber electrodes were positioned against the surface of the artery at an angle of 30° to the horizontal plane with an ∼100-μm length being in contact with the surface of the artery. A potential difference of +0.3 V was applied and maintained between the recording electrode connected to an AMU130 Nano amperometer and an Ag-AgCl pellet placed in the recording chamber. The resulting NE oxidation currents were constantly monitored.

We used sharp microelectrodes to measure excitatory junction potentials (EJPs) as a measure of ATP release. EJP recordings were made from the surface of the arteries 1–3 mm distal to the mouth of the suction electrode. The connective tissue and fat cells were carefully removed to expose the nerves and SMCs near the surface of the artery. Intracellular recordings were made using the technique previously described by Brock et al. (5). Glass microelectrodes (71.5 ± 2.72 MΩ, n = 39) back filled with 3 M KCl were used to measure both the membrane potential and EJPs of SMCs. EJPs were elicited by brief trains of stimuli at low frequencies (duration: 0.15 ms, voltage: 20 V, 5 pulses at 1 or 2 Hz).

Reagents

Ouabain, ACh, phenylephrine (PE), NPY, NPY fragment 24–36, prazosin, and TTX were from Sigma (St. Louis, MO). BIBP3226 was either a gift of Boehringer Ingelheim Pharmaceuticals or purchased from American Peptide and Bachem Biosciences. Stock reagents were stored at −20°C. Reagents were thawed and diluted 1:1,000 on the day of the experiment.

Statistics

Data in the text and figures are reported as means ± SE. One-way ANOVA was used to establish if significant differences existed between groups (one-way ANOVA, Sidak-Holmes post hoc treatment). Intragroup and intergroup comparisons were evaluated with paired and unpaired t-tests as appropriate. P values of <0.05 were used to reject the null hypothesis. The significance of differences was evaluated with SigmaStat 3.11 (Systat Software, Point Richmond, CA) using parametric or nonparametric tests as appropriate for the data.

RESULTS

Ouabain Concentration Effect on Pressurized Arteries

Control (endothelium intact) arteries.

We examined the effects of ouabain over the range of concentrations (10−11–10−7 M) that is believed to span that of EO present normally in plasma and when EO is elevated in hypertension (20, 48) (Figs. 1, 2, and 3). In these experiments, artery external diameters were continuously monitored. The effects of ouabain on artery diameter were measured and analyzed in two ways: 1) as the absolute magnitude of the change in diameter (in μm; Figs. 1A, 2A, and 3A) and 2) as a fraction of the initial diameter just before the exposure to ouabain (Figs. 1B, 2B, and 3B). The second method has the effect of normalizing for the variations in the initial diameters of different arteries and also presents the ouabain-induced vasoconstriction as the percent change in a physiologically relevant diameter, viz. at 70 mmHg, the pressure at which these arteries have myogenic tone. Concentration-effect curves for ouabain on “control” mesenteric arteries are shown in Figs. 1, 2, and 3. Ouabain induced concentration-dependent vasoconstriction, and the highest concentration used was 10−7 M. The effect was not maximal at that concentration, but higher concentrations were not used, since these would not be relevant physiologically.

Fig. 1.

Fig. 1.

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The endothelium (Endo) influences the vasoconstrictor effect of ouabain in mesenteric arteries. Shown is a summary of the results of the concentration-effect curves for ouabain obtained from control mesenteric arteries and arteries from which the endothelium was removed. A and B: results are plotted as absolute magnitudes of the change in diameter (in μm; A) and changes in diameter as a fraction of the initial diameter just before the exposure to ouabain (in %; B). Removal of the endothelium caused a significant shift of both concentration-effect curves to the left. n = 4–8. *P < 0.001 vs. controls.

Fig. 2.

Fig. 2.

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Prazosin caused a significant attenuation of ouabain-induced vasoconstriction in mesenteric arteries. Isolated mesenteric arteries were acutely exposed to increasing concentrations of ouabain in the presence or absence of prazosin (an α1-adrenoceptor blocker, 10−6 M, n = 4–5) to obtain concentration-effect curves. A: absolute magnitude of the change in diameter (ΔD, in µm). B: ΔD as a fraction of the initial diameter (Di) just before exposure to ouabain. Prazosin caused a significant attenuation of ouabain-induced vasoconstriction at ouabain concentrations of 10−10 and 10−7 M. *P < 0.001 vs. controls. The endothelium was not removed in this set of experiments.

Fig. 3.

Fig. 3.

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Direct effects of ouabain on smooth muscle of pressurized mesenteric arteries. To measure the effects of ouabain directly on smooth muscle, we obtained ouabain concentration-effect curves in mesenteric arteries in which the endothelium had been removed and prazosin (10−6 M) was present. A: absolute magnitude of the change in diameter (ΔD, in µm). B: ΔD as a fraction of the initial diameter (Di) just before exposure to ouabain. Ouabain induced significant vasoconstriction over the range of concentrations examined. The vasoconstriction was not, however, significantly different than in control arteries (intact endothelium, no prazosin, n = 4–5). Thus, the effects of endothelial removal (which increases ouabain-induced vasoconstriction) and block of α1-adrenoceptors (which decreases ouabain-induced vasoconstriction) were almost perfectly offset. See text for details.

Influence of the endothelium.

Ouabain blunts PE-induced contraction of aortic rings through an endothelium-dependent mechanism (14). Similarly, low concentrations of ouabain (e.g., 10−10 M) increase cytosolic and stored [Ca2+] in endothelial cells of the descending vasa recta (38). This raises the possibility that the vasoconstrictor effects of ouabain on small arteries might be opposed or masked by Ca2+-dependent vasodilators released from the endothelium. Thus, we obtained ouabain concentration-effect curves in arteries in which the endothelium had been removed by the passage of air (see materials and methods). One-way ANOVA showed that the removal of endothelium caused a significant upward shift of the ouabain concentration-effect curve (n = 4–8, P < 0.001; Fig. 1, A and B). Removal of the endothelium also increased the vasoconstrictor effect of ouabain (10−7 M) in rat renal interlobar arteries (n = 7, P < 0.05 by the Mann-Whitney rank sum test; Fig. 4); data from mesenteric arteries are also shown again at an ouabain concentration of 10−7 M for comparison (n = 6, P < 0.01 by a paired t-test; Fig. 4).

Fig. 4.

Fig. 4.

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Role of the endothelium in ouabain-induced vasoconstriction of renal interlobar and mesenteric arteries. A: representative recording showing that 10−7 M ouabain-induced vasoconstriction was enhanced after endothelial denudation in interlobar arteries. B: summary of the results showing that 10−7 M ouabain-induced vasoconstriction was significantly increased after endothelial denudation in rat interlobar arteries (n = 7, *P < 0.05 vs. controls by the Mann-Whitney rank sum test) and mesenteric arteries (n = 6, *P < 0.05 vs. controls by a paired t-test).

Influence of spontaneous neurotransmitter release.

Low concentrations of ouabain increase the spontaneous release of the sympathetic neurotransmitter NE in the rat aorta (3, 17), causing vasoconstriction through neurally released NE rather than simply a direct effect on smooth muscle. Presumably, this occurs through an effect of ouabain to increase cytoplasmic [Ca2+] in the nerve terminals, increasing the spontaneous release of NE-containing synaptic vesicles. To determine whether such an effect might exist in small arteries, we obtained ouabain concentration-effect curves in the presence of the highly specific α1-adrenoceptor blocker prazosin (10−6M; Fig. 2, A and B).

One-way ANOVA showed that the presence of prazosin caused a significant attenuation of ouabain-induced vasoconstriction of the ouabain concentration-effect curve (n = 4–5, P < 0.001; Fig. 2, A and B) in endothelium-intact mesenteric arteries. Similarly, prazosin reduced the ouabain-induced vasoconstriction (percent change in diameter) of renal interlobar arteries from 3.14 ± 0.7% to 2.22 ± 0.7% (n = 5). Thus, it appears that a significant component of the ouabain-induced vasoconstriction of both rat mesenteric and renal interlobar arteries is due to the activation of α1-adrenoceptors on smooth muscle, presumably by neurally released NE.

Ouabain concentration effect on arterial smooth muscle.

The data described above indicate that any direct vasoconstrictor effects of ouabain on arterial smooth muscle cannot be observed in the presence of the endothelium and functional α1-adrenoreceptors in intact arteries. To measure the effects of ouabain directly on smooth muscle, we obtained ouabain concentration-effect curves in mesenteric arteries in which the endothelium had been removed and prazosin (10−6 M) was present (Fig. 3). These data revealed a relatively small vasoconstrictor effect of ouabain. Interestingly, the vasoconstriction in these arteries was not statistically different from that of arteries with the endothelium and with α1-adrenoceptors intact (n = 4–5; Fig. 3, A and B); thus, it appears that the opposing influences of the endothelium and α1-adrenoreceptor normally “cancel” each other nearly exactly, leaving a net effect (vasoconstriction) that is identical to that produced by ouabain acting on smooth muscle alone.

Effects of Ouabain on Evoked Sympathetic Teurotransmitter Release

As shown above, ouabain appears to increase the spontaneous release of NE in rat mesenteric and renal interlobar arteries, as judged by the effects of prazosin on ouabain-induced artery contractions. In vivo, both mesenteric and interlobar arteries are subject to tonic SNA. Thus, it was interesting to determine if ouabain also increased evoked neurotransmitter release, defined here as that caused by the depolarization of sympathetic axons and perivascular nerve terminals present in isolated arteries. It is known that higher concentrations of Na+-K+ pump inhibitors increase both spontaneous and evoked neurotransmitter release at many types of neuronal and neuromuscular junctions, probably by increasing nerve terminal [Ca2+] (47, 57) both at rest and during repetitive activity. Here, we first sought the possible effects of low concentrations of ouabain on the neural release of the major contractile activator NE (Fig. 5A). In rat mesenteric small arteries, ouabain increased the release of NE from sympathetic perivascular nerve terminals as evoked by trains of electrical stimuli, but only at supraphysiological concentrations of greater than ∼3 × 10−5 M (Fig. 5B). The effects of lower concentrations of ouabain were not statistically significant (at P > 0.05). Neurally released ATP also activates contraction in these arteries and may be the predominant sympathetic neurotransmitter in small arteries at physiological intraluminal pressures (43). Thus, we also observed the effects of ouabain on ATP release, as judged by the amplitude of EJPs during trains of stimuli (Fig. 6A) [EJPs result from inward Na+ and Ca2+ current through ATP-gated channels that contain P2X1 subunits (37)]. At two concentrations, 10−7 and 10−6 M, ouabain did not affect either the 1st or 10th EJP evoked by a train of electrical stimuli (Fig. 6B).

Fig. 5.

Fig. 5.

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At high but not at low concentrations, ouabain increased summated norepinephrine (NE) release from perivascular sympathetic nerve terminals in mesenteric arteries. NE release was measured using a carbon fiber electrode placed on the surface of the artery, and perivascular nerve terminals were stimulated by electrical field stimulation (EFS). A: summated NE oxidation currents (NEOCs) in response to stimulation at 10 Hz for 2.5 s. Stimulus artifacts on the carbon fiber electrode output are visible as the thick black trace during the period of stimulation. B: ouabain concentration-effect curve over the range of 10−7–3 × 10−4 M. Ouabain increased NE release only at concentrations of >3 × 10−5 M.

Fig. 6.

Fig. 6.

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Effect of ouabain on excitatory juctional potential (EJP) amplitudes in mesenteric arteries. A: EJPs were measured with sharp microelectrode impalements of smooth muscle cells in mesenteric arteries. Brief bursts of EFS at low frequencies (2.0 Hz) were given, resulting in a train of EJPs that summated and exhibited synaptic potentiation. Vm, membrane potential. B: effects of ouabain on ATP release, as assessed by the amplitudes of EJPs at the first, maximum, and summated EJP, as indicated in A. Neither 10−7 nor 10−4 M ouabain had a significant effect on the amplitudes of EJPs. The first EJP was unaffected, as was the final level of depolarization reached (the “summated amplitude”).

Sympathetically mediated contraction.

As described above, 10−7 M ouabain did not increase either evoked NE or ATP release, as judged by NE at the surface of the artery and by the amplitude of EJPs, respectively. Due to a requirement to eliminate motion in the experiments involving microelectrodes, arteries were pinned down in the recording chamber. Thus, any effects of ouabain on neurally evoked contraction could not be determined. Therefore, we tested, in separate experiments, whether ouabain can enhance sympathetically evoked vasoconstriction of pressurized renal interlobar arteries. Over the range of frequencies and train durations examined, 10−7 M ouabain did not affect neurogenic contractions, either of normal arteries or those subjected to endothelial removal (Fig. 7C).

Fig. 7.

Fig. 7.

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Effect of 10−7 M ouabain on sympathetically mediated (neurogenic) contraction in interlobar arteries. A and B: neurogenic contractions over the range of frequencies examined in the absence (A) and presence (B) of 10−7 M ouabain. C: summary of the results showing that at the range of frequencies and train durations examined, 10−7 M ouabain did not affect neurogenic contractions (n = 7).

Effects of Sympathetic Nerves on the Actions of Ouabain

The data above indicate that ouabain, at physiologically relevant concentrations, does not potentiate neurogenic contractions (i.e., those elicited by SNA) of intact small arteries. This is despite an apparent increase in spontaneous sympathetic neurotransmitter release. It has been known that SNA does potentiate contractions to certain vasoconstrictors, such as PE and angiotensin (36). As discussed below, it is thought that such effects are mediated by neurally released NPY (6, 11). Thus, we examined whether SNA (to which small arteries are tonically exposed in vivo) might similarly affect ouabain-induced vasoconstriction. EFS (30 V × 0.15 ms at a frequency of 30 Hz for 2.5 min) potentiated vasoconstriction caused by 10−7 M ouabain (n = 6; Fig. 8, A and B). In contrast, the repetitive application of 10−7 M ouabain without EFS failed to show such facilitation (data not shown). PE also failed to cause facilitation of ouabain-induced vasoconstriction (not shown).

Fig. 8.

Fig. 8.

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Bursts of sympathetic nerve activity (SNA) increased the vasoconstrictor actions of ouabain in interlobar arteries. A: ouabain (10−7 M) was given before and after a burst of SNA (EFS: 30 Hz, 2.5 min). Vasoconstriction in response to ouabain was significantly increased after the burst of SNA. B: summary of the results showing that SNA significantly potentiated the vasoconstrictor effect of 10−7 M ouabain (n = 6, *P < 0.05 vs. before EFS by a paired t-test).

NPY Potentiates Ouabain-Induced Vasoconstriction

NPY is a sympathetic neurotransmitter that regulates diverse functions of the central and peripheral nervous systems, cardiovascular system, and immune systems (58). In the vasculature, it has been proposed that NPY both facilitates smooth muscle reactivity (32) and inhibits prejunctional release of its cotransmitters (9, 33). Although NPY itself can cause vasoconstriction, it can exert facilitatory effects at concentrations that do not themselves cause contraction, implying a modulation of signal transduction pathways. NPY potentiated the vasoconstrictor effect of 10−7 M ouabain (n = 5, P < 0.01 by a paired t-test; Fig. 9, A and B) in rat mesenteric arteries. Similarly, in rat interlobar arteries, NPY potentiated the vasoconstrictor effect of 10−7 M ouabain (n = 4, P < 0.05 by a paired t-test; Fig. 9C) an effect that could be blocked by 3 × 10−7 M BIBP3226, a specific NPY Y1 receptor antagonist (n = 4, P < 0.05 by a paired t-test; Fig. 9C). We next tested whether Y2 receptors were also involved in mediating these effects. In contrast, 10−8 M NPY fragment 24–36, a specific NPY Y2 receptor agonist, failed to potentiate vasoconstriction by 10−7 M ouabain (n = 4, P > 0.05 by a paired t-test; Fig. 9D).

Fig. 9.

Fig. 9.

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Neuropeptide Y (NPY) potentiates the vasoconstrictor effect of 10−7 M ouabain in mesenteric and renal interlobar arteries. A: representative recording of the vasoconstrictor effect of ouabain measured in the absence and presence of 10−8 M NPY in mesenteric arteries. In the presence of NPY, ouabain-induced vasoconstriction was larger than in its absence. B: 10−8 M NPY significantly potentiated the vasoconstrictor effect of 10−7 M ouabain in mesenteric arteries (n = 5, *P < 0.01 vs. ouabain by a paired t-test). C: in rat interlobar arteries, NPY (3 × 10−8 M) significantly potentiated the vasoconstrictor effect of 10−7 M ouabain (n = 4, *P < 0.05 vs. ouabain by a paired t-test), which could be blocked by 3 × 10−7 M BIBP3226 (BIBP), a specific NPY Y1 receptor antagonist (n = 4, δP < 0.05 vs. NPY + ouabain by a paired t-test). D: graph showing that 10−8 M NPY fragment 24–36, a specific NPY Y2 receptor agonist, failed to potentiate vasoconstriction by 10−7 M ouabain in rat mesenteric arteries (n = 4, P > 0.05 vs. ouabain by a paired t-test).

DISCUSSION

EO, a steroid secreted by the adrenal gland (4, 23, 29), circulates in low concentrations (23, 34) and is believed to act by binding to the α-subunit of Na+-K+-ATPase, either inhibiting Na+ transport or activating signal transduction pathways (52). In the former mechanism, the inhibition of Na+ transport leads to the elevation of local concentrations of Na+ in SMCs and thus to the accumulation of intracellular Ca2+ via decreased efflux or increased influx of Ca2+ through the Na+/Ca2+ exchanger (2). The major new results of the present study are that 1) ouabain-induced vasoconstriction of rat mesenteric and renal interlobar arteries is increased by endothelial removal; 2) a component of ouabain-induced vasoconstriction is attributable to NE, since it is blocked by the specific α1-adrenoreceptor blocker prazosin; and 3) SNA increases ouabain-induced vasoconstriction, possibly via the sympathetic neurotransmitter NPY, acting through the Y1 receptor.

Influence of the Endothelium in Ouabain-Induced Vasoconstriction

Previous experimental studies have demonstrated that the endothelium may either increase (14) or decrease ouabain-induced vasoconstriction. For example, it has been suggested that the endothelium of Wistar Kyoto rats releases a factor in response to ouabain that decreases the smooth muscle vasoconstrictor response to ouabain (39, 40), whereas the endothelium of spontaneously hypertensive rats and old Wistar Kyoto rats releases a factor that increases the vasoconstrictor effect of ouabain. It was thus suggested (40) that the inhibitory effect of the endothelium on contractions induced by ouabain (in young normal animals) might be lost in aged and/or hypertensive animals, having been replaced by an enhancement of ouabain-induced vasoconstriction. This would be the result of the release of an endothelial vasoconstrictor in old/hypertensive animals instead of a vasodilator. Recently, it has been shown that very low concentrations of ouabain, as used here (10−10 M), elevate cytosolic [Ca2+] in endothelial cells of the renal vasa recta (38). However, whether this effect of ouabain also occurs in the vascular endothelium, and what its consequences might be, is not known at present. Our observations on young normal rats clearly indicate an influence of the endothelium, possibly mediated by a Ca2+-dependent vasodilator, that opposes the direct smooth muscle-mediated effects of ouabain.

Increased Spontaneous Release of NE Is a Component of Ouabain-Induced Vasoconstriction

Our data indicated that a component of ouabain-induced vasoconstriction requires α1-adrenoceptors, an effect most easily accounted for by the hypothesis that ouabain increased the release of NE from sympathetic nerve terminals in the artery wall. In human mesenteric arteries, very low concentrations of ouabain (10−9 M) are indeed thought to have such an effect (3). It has been suggested that ouabain inhibits the α3-subunit of Na+-K+ pumps in sympathetic nerve terminals, raising their cytoplasmic [Ca2+], to promote a rise in NE release via increased Ca2+-dependent exocytosis. However, because the neuronal NE transporter uses the electrochemical gradient of Na+, inhibition of nerve terminal Na+-K+ pumps may also increase NE efflux via reversal of the NE transporter, resulting in nonexocytotically released NE (45). In heart muscle, the effects of ouabain on in situ sympathetic nerve endings have been attributed (in equal portions) to both increased exocytototic and nonexocytotic mechanisms (53). Our data do not allow us to distinguish between these possibilities. Increased Ca2+-dependent vesicular exocytosis of NE would be expected to be evident as an increased frequency of spontaneous miniature NE oxidation currents, but spontaneous miniature oxidation currents were not routinely detected by our carbon fiber electrodes. Possibly consistent with a transporter-mediated increase in NE release is the fact that ouabain, in physiologically relevant concentrations (<10−7 M), did not enhance the evoked release of NE (i.e., during stimulated SNA). Consistent with this, it did not increase EJPs (a manifestation of neurally released ATP), nor did it increase neurally stimulated contractions (at concentrations of <10−7 M). High concentrations of ouabain did increase NE release evoked by SNA. A Ca2+-dependent exocytotic increase in NE release might still have occurred in theory, however, because there is some evidence that the Ca2+ sensitivity of spontaneous and evoked sympathetic neurotransmitter release is differentially controlled (46). The presumed increase in nerve terminal cytoplasmic [Ca2+] that is caused by ouabain inhibition of Na+-K+-ATPase might have different effects than the localized increase in [Ca2+] that is caused by the entry of Ca2+ through voltage-dependent Ca2+ channels during stimulated nerve terminal activity.

SNA Potentiates the Vasoconstrictor Effects of Ouabain

It has been previously shown that SNA potentiates the vasoconstrictor effect of NE (36). It is now known that this effect is mediated by the sympathetic neurotransmitter NPY, which is coreleased with NE and ATP and acts on smooth muscle Y1 receptors (54). Only the adrenergic component, not the purinergic component, of the arterial contraction is facilitated. In arteries, we reported that (exogenous) NPY increased the frequency of α1-adrenoreceptor agonist-induced Ca2+ waves (51), thus potentiating agonist-induced contraction. Here, we asked whether SNA might also potentiate the vasoconstrictor effects of ouabain. Such a phenomenon would be important, given that many arteries in vivo are subject to tonic SNA, and increased SNA occurs in hypertension. Indeed, SNA did potentiate the vasoconstrictor effect of ouabain, in a similar manner to the way that SNA potentiates the PE-induced contraction, and the mechanism involved the Y1 receptor, implicating NPY. In support of this, exogenously applied NPY also potentiated ouabain-induced vasoconstriction. The mechanism(s) of this effect is not known at present, but the action of NPY to potentiate the action of vasoconstrictors (such as NE) has been attributed to a reduction of cAMP, which is a potent vasorelaxant, or to the ability to elevate [Ca2+] directly. In our experiments, a reduction in cAMP seems unlikely, given that cAMP should not be elevated by the conditions of the experiment. Alternately, if NPY also increased [Ca2+] by a separate mechanism, this would tend to increase ouabain-induced vasoconstriction by several mechanisms: 1) it would allow an ouabain-induced increase in [Ca2+] to produce a larger increase in contractile force (due to the nonlinear relationship between [Ca2+] and force and a higher overall [Ca2+]) or 2) it might stimulate Ca2+-dependent PKC to inhibit myosin light chain phosphatase (i.e., an increase in “Ca2+ sensitivity”) (13). The present experiments do not distinguish between these possibilities. Finally, one might expect neurally released NE to exert a facilitatory effect on ouabain-induced vasoconstriction, since the activation of α1-adrenoreceptors does increase the Ca2+ sensitivity of contraction (by ultimately inhibiting myosin light chain phosphatase) (50). Such a facilitatory effect was not observed, however, in the rat mesenteric vascular bed (36). There, injections of NE neither affected the responses to SNA nor any subsequent responses to NE. We speculate that any facilitatory effect of NE due to Ca2+ sensitization might be evident during continuous SNA, when NE is continuously present to exert a Ca2+-sensitizing effect.

Possible Physiological Significance

The present results have implications for the role or importance of EO in normal and hypertensive organisms. Importantly, the effects we observed are all mediated by concentrations of ouabain (10−10–10−7 M; Figs. 1 and 2) that are similar to those found in plasma. This establishes that the influences of sympathetic nerve terminals and the endothelium on ouabain-induced vasoconstriction that we observed here may be relevant to the living animal. The effects of ouabain on mesenteric small arteries and renal interlobar arteries appeared similar, as far as could be investigated here. Nevertheless, these arteries serve quite different functions in the organism, and, therefore, the effects of ouabain on them, in hypertension, may be different.

With respect to SNA, many small arteries in living animals are subjected to repetitive bursts of SNA, and the resulting neurogenic vascular contraction contributes to total peripheral resistance. Indeed, it has been recently concluded that “a balance between cardiac output and sympathetically mediated vasoconstriction contribute importantly to normal regulation of arterial pressure in humans” (8). Thus, in vivo, the effects of EO on artery contraction may be even greater than those observed here for comparable concentrations of ouabain on isolated arteries where SNA is absent. Importantly, most forms of hypertension have been shown to involve sympathetic hyperactivity, as judged by muscle SNA and NE spillover (16, 21). Thus, we speculate that the vasoconstrictor effect of EO may be even greater in hypertension, when sympathetic nervous system hyperactivity is present.

We (56) have previously noted that the vasoconstrictor effect of ouabain, as observed in isolated arteries, is dependent on the presence of myogenic tone. Myogenic tone or myogenic responsiveness is jointly governed by cytoplasmic [Ca2+] and the “Ca2+ sensitivity of contraction.” The latter is probably regulated by PKC and Rho kinase-mediated inhibition of myosin light chain phosphatase. Thus, hypertensive arteries in situ may be more sensitive to ouabain than isolated arteries because, in hypertension, intraluminal pressure leads to increased Ca2+ sensitivity and increased background cytoplasmic [Ca2+] (which would increase the contractile activation resulting from further changes in [Ca2+]).

In contrast to SNA and myogenic tone, the presence of a normal endothelium in arteries in situ might be expected to reduce the vasoconstrictor effects of EO, according to our results. In hypertension, however, endothelial dysfunction is often present, with reduced production of vasodilators and/or enhanced production of vasoconstrictors (7). Thus, the vasoconstrictor effects of EO may be greater in hypertension, if endothelial dysfunction is present.

Conclusions

In summary, in both mesenteric small arteries and renal interlobar arteries, the vasoconstrictor effect of low concentrations of ouabain involves NE release from sympathetic nerves as well as a direct effect on vascular smooth muscle. A normal endothelium blunts, and bursts of SNA enhance, ouabain-induced arterial vasoconstriction. We suggest that the vasoconstrictor effect of EO may be potentiated in hypertension by the presence of sympathetic hyperactivity [which itself may be a result of ouabain acting centrally (19, 24)], endothelial dysfunction, and enhanced myogenic tone (itself a result of increased intravascular pressure).

GRANTS

This work was supported by National Heart, Lung, and Blood Institute Grants R01-HL-073094 and P01-HL-078870.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

ACKNOWLEDGMENTS

The authors thank Dr. M. P. Blaustein for critical discussions.

Present address of Q. Zhang: Active Diagnostics Incorporated, Davis, CA 95616.

Present address of A. Y. Rhee: Synchrogenix Information Strategies Incorporated, Wilmington, DE 19801.

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