Department of Physiology, University of South Alabama College of Medicine, Mobile, Alabama 36688
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ABSTRACT |
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We examined whether milrinone-mediated attenuation of small mesenteric artery vasoconstriction results predominantly from the activation of vascular smooth muscle K+ channels. Resistance arteries (~150 µm) were dissected from rat mesentery and were mounted on a wire myograph. Isometric force development in response to increasing concentrations of norepinephrine (NE) was monitored before and after treatment with the type 3 phosphodiesterase inhibitor milrinone. Milrinone significantly reduced NE-induced vasoconstriction, attenuating both NE sensitivity and maximal tension generation. Inhibition of ATP-sensitive K+ channels or voltage-gated K+ channels did not prevent the milrinone-induced attenuation of NE responses. Blockade of inwardly rectifying K+ channels or Ca2+-sensitive K+ channels prevented the milrinone-mediated reduction in NE sensitivity, but this effect was apparently due to direct enhancement of vasoconstrictor responsiveness rather than interference with the mechanism of milrinone action. In addition, milrinone elicited substantial relaxation in vessels preconstricted with 100 mM KCl. This effect was mimicked by the adenylyl cyclase activator forskolin and was reversed by the Rp diastereomer of cAMP, which is a cAMP-dependent protein kinase (PKA) inhibitor. Our results indicate that cAMP/PKA-mediated impairment of vasoconstriction may occur without the contribution of K+ channel regulation.
vascular smooth muscle; adenosine 3',5'-cyclic monophosphate; phosphodiesterase inhibitor; vasodilation; type 3 phosphodiesterase
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INTRODUCTION |
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CYCLIC NUCLEOTIDE-DEPENDENT signaling pathways are believed to impede vasoconstriction largely by reducing the intracellular Ca2+ concentration ([Ca2+]i) in vascular smooth muscle cells (VSMC; see Refs. 4, 10, 13). Although the mechanism of [Ca2+]i regulation has not been fully elucidated, it appears to derive at least in part from inhibition of Ca2+ influx through voltage-sensitive L-type Ca2+ channels on the VSMC plasmalemma. Some investigators have reported attenuation of L-type Ca2+ entry as a result of cAMP elevation or activation of cAMP-dependent protein kinase (PKA) in vascular smooth muscle (VSM; see Refs. 18 and 24). Xiong and Sperelakis (24) showed a reduction of L-type Ca2+ currents in VSMCs resulting from treatment with forskolin, cyclic nucleotide analogs, or the catalytic subunit of PKA. It remains to be seen whether this control of Ca2+ influx results directly from L-type Ca2+ channel inhibition or from activation of one or more membrane K+ channels. Opening of membrane K+ channels elicits cell hyperpolarization due to increased outward K+ current. This hyperpolarization leads to the closing of voltage-sensitive L-type Ca2+ channels, which reduces net Ca2+ influx and favors VSM relaxation.
Four K+ channels have been
identified in VSM (16). Voltage-gated
K+ channels
(KV) are stimulated by
depolarization of the plasma membrane, whereas
Ca2+-sensitive
K+ channels
(KCa) are opened by
intracellular Ca2+ and membrane
depolarization. These channels may contribute significantly to basal
vascular tone and may offer a mechanism for feedback regulation of
vasoconstriction. ATP-sensitive K+
channels (KATP) are activated
under conditions of low intracellular ATP, providing metabolic
regulation of vascular contractile state. Inwardly rectifying
K+ channels
(KIR) are activated by
hyperpolarization or extracellular K+, usually yielding an inward
K+ current when membrane potential
becomes excessively negative. Recent evidence from various patch-clamp
and isolated vessel studies has revealed potentially relevant cyclic
nucleotide-dependent regulation of VSM
K+ channels (2, 5, 9, 14, 16, 21,
22, 25). cAMP and PKA as well as
Gs activation have been linked
to enhancement of current through
KCa channels (14, 21, 22). Similar
regulation of KV (2) and
KATP (5, 9, 25) by vasodilator
agonists or PKA stimulation has been reported. At present, little is
known about the regulation of KIR
channel function, although it may also be a target of cyclic nucleotide
signaling. Despite recent reports, the overall role of
K+ channel regulation in cyclic
nucleotide-dependent mechanisms of vasodilation remains unclear.
In this study, a small-wire myograph was used to assess norepinephrine (NE)-induced isometric force generation in small mesenteric resistance arteries. Vasoconstrictor responses were reassessed after pretreatment with the type 3 phosphodiesterase (PDE3) inhibitor milrinone. The effects of milrinone on NE responses were also evaluated in the presence of various K+ channel blockers to determine the role of each K+ channel in the milrinone-induced inhibition of vasoconstriction. Finally, we tested the ability of milrinone and the adenylyl cyclase activator forskolin to relax vessels under conditions in which opening of K+ channels would have minimal influence on vascular tone. Our findings indicate that modulation of vasoconstrictor response by cAMP-elevating agents may occur without K+ channel regulation.
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METHODS |
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Male Sprague-Dawley rats (200-300 g) were anesthetized with
isoflurane and were killed by thoracotomy and removal of the heart. Small artery segments (150-200 µm) were dissected from the
mesentery and bathed in pH-adjusted (7.4) physiological saline solution (PSS) containing (in mM): 120 NaCl, 5 KCl, 2.5 CaCl2, 1.2 MgSO4, 25 NaHCO3, 1.2 NaH2PO4,
0.02 EDTA, and 5.5 glucose. The vessels were mounted on a small vessel
wire myograph for isometric force measurement (15). The normalization
procedure of Mulvany and Halpern (15) was employed to optimize vessel
internal circumference for tension development [set at 0.9 × internal circumference when internal pressure is 100 mmHg (0.9 × IC100)]. The
arteries were allowed to equilibrate for 60 min and were exposed to at
least two consecutive priming doses of NE
(104 M) to ensure that
maximal responses were reproducible. After thorough washing with normal
PSS, the vessels were subjected to one of the protocols described
below. NE, glibenclamide, 4-aminopyridine, iberiotoxin, apamin, and
forskolin were purchased from Research Biochemicals International
(Natick, MA). Milrinone, BaCl2,
and the Rp diastereomer of cAMP (Rp-cAMPS) were
purchased from Sigma (St. Louis, MO), and 8-(4-chlorophenylthio)-cGMP
Rp diastereomer (8-CPT-Rp-cGMPS) was purchased from
Calbiochem (San Diego, CA).
NE concentration-effect curves.
A cumulative concentration-effect curve to NE
(108 to
10
3.5 M) was generated for
each vessel segment. After thorough rinsing with PSS, vessels were
either exposed to 100 µM milrinone alone or to a combination of
milrinone and one or more K+
channel blocker(s) for 1 h before generating a second
concentration-effect curve to NE. This dose of milrinone was previously
found to maximally inhibit NE-induced peak tension generation. Drug
administrations used to achieve specific
K+ channel blockade were as
follows: 10 µM glibenclamide
(KATP inhibition), 3 mM
4-aminopyridine (KV inhibition),
100 µM BaCl2
(KIR inhibition), 0.1 µM
iberiotoxin (large-conductance KCa
inhibition), and 0.1 µM iberiotoxin plus 0.3 µM apamin (large- and
small-conductance KCa inhibition).
As a control for each of the aforementioned protocols, a second vessel
segment from each rat was simultaneously monitored. Each of these
vessels was exposed to vehicle (0.1% DMSO) or treated with the same
regimen of K+ channel inhibitors
listed above in the absence of milrinone before generating the second
NE concentration-effect curve.
Contraction and relaxation of arterial
segments. Vessels were contracted by replacing the
normal PSS in the myograph chamber (5 ml) with PSS containing 100 mM
KCl (i.e., same as PSS above except KCl was increased to 100 mM and
NaCl was reduced to 25 mM). After tension had stabilized, the vessels
were exposed to either 50 µM milrinone or 5 µM forskolin. Similar
milrinone or forskolin treatment of vessels contracted with
105 M NE was performed.
These concentrations of milrinone and forskolin were found to elicit
maximal relaxation of NE-induced tension in preliminary experiments.
Some milrinone-relaxed vessels were subsequently exposed to
Rp-cAMPS (400 µM) or 8-CPT-Rp-cGMPS (10 µM).
Data analysis. In total, 40 rats were
used for NE concentration-effect experiments consisting of milrinone
(n = 12), milrinone plus glibenclamide
(n = 6), milrinone plus
4-aminopyridine (n = 6), milrinone
plus BaCl2
(n = 6), milrinone plus iberiotoxin (n = 5), milrinone plus iberiotoxin
plus apamin (n = 5), and
appropriate vehicle controls. Ten rats were used for the
contraction/relaxation protocol. For concentration-effect curves,
isometric force (mN) was measured and normalized for vessel length and
basal tone, yielding the net change in tension per millimeter vessel
(mN/mm). Nonlinear regression curves were fit to individual sets of
concentration-effect data, from which
pD2 and maximal tension values
were calculated and expressed as means ± SE. Also, individual
shifts in pD2 value after
experimental perturbation were expressed as change from control
(pD2 after treatment
pD2 before treatment) for each curve. Similarly, changes in maximal tension generation were expressed relative to control responses (maximal tension generated after treatment
maximal tension generated before treatment) within individual vessels. Shifts in pD2
(
pD2) and maximal tension
(
max) were averaged for each experiment and were presented as means ± SE. It should be noted that a minus (
) sign denotes a
decrease in sensitivity or maximal tension, whereas a plus (+)
indicates an increase. Relaxation of prestimulated vessels
(n = 8) was expressed as percent
change from the total generated tension. Unless otherwise noted, for
comparisons of individual data sets, a value of
P < 0.05 was considered
statistically significant.
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RESULTS |
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Effects of milrinone on NE concentration-effect
curves. Milrinone (100 µM) had no effect on resting
vascular tension but reduced NE sensitivity and maximal tension
generation in rat small mesenteric arteries as indicated by a
rightward, downward shift in the NE concentration-effect curve (Fig.
1). The
pD2
(log[EC50]) value decreased significantly from 5.88 ± 0.09 before milrinone to 5.57 ± 0.06 (P < 0.05) after 60 min
of milrinone exposure (P < 0.05). NE-induced maximal tension was also significantly reduced from 8.13 ± 0.46 before to 5.39 ± 0.47 mN/mm after milrinone treatment (P < 0.05). No vehicle effects were
observed in control vessels (data not shown). It was also noted that
milrinone-treated vessels developed a persistent rhythmic oscillation
of tension after NE exposure.
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NE concentration-effect curves in the presence of milrinone and
K+ channel
inhibitors.
Before generating the second NE concentration-effect curve, vessels
were exposed to milrinone along with one or more
K+ channel blocker(s) (Fig.
2). For reference purposes, Table
1 summarizes the concentrations and
intended sites of action for the
K+ channel blockers employed. In
vessels pretreated with the combination of milrinone and glibenclamide,
pD2 shifted from a control value of 5.93 ± 0.09 to 5.59 ± 0.068 (P < 0.05), and maximal tension decreased from 7.88 ± 0.73 to 4.76 ± 0.39 mN/mm
(P < 0.05). Similarly, in vessels
exposed to the combination of milrinone and 4-aminopyridine, both
pD2 [5.75 ± 0.10 vs.
4.94 ± 0.06 (P < 0.05)]
and maximal tension [8.68 ± 0.79 vs. 6.58 ± 0.58 mN/mm
(P < 0.05)] were reduced relative to control. In the next group of vessels,
pD2 values were not different
before and after exposure to the combination of milrinone and
Ba2+ (5.90 ± 0.09 vs. 5.92 ± 0.05), whereas maximal tension fell from 7.98 ± 0.80 to 6.52 ± 0.70 mN/mm (P < 0.05). Thus blockade of Ba2+-sensitive
K+ channels restored normal NE
sensitivity in milrinone-treated arteries. Vessels treated with
milrinone and iberiotoxin showed a slight albeit significant reduction
in pD2 from 5.83 ± 0.05 to
5.71 ± 0.01 (P < 0.05), and
maximal tension in these vessels shifted from 10.10 ± 0.39 to 7.58 ± 0.34 mN/mm (P < 0.05).
Finally, pD2 was not different
before (6.21 ± 0.09) and after (6.25 ± 0.10) exposure to the
combination of milrinone, iberiotoxin, and apamin, although maximal
tension decreased from 6.61 ± 0.48 to 5.55 ± 0.45 mN/mm
(P < 0.05). Therefore, blockade of
iberiotoxin- and apamin-sensitive
K+ channels restored normal NE
sensitivity in milrinone-treated vessels. Table
2 summarizes the effects of milrinone on NE
concentration-effect curves in the absence or presence of various
K+ channel blockers. No
K+ channel blocker fully restored
maximal NE responsiveness in milrinone-treated vessels.
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NE concentration-effect curves in the presence of
K+ channel
inhibitors alone.
To perform the appropriate controls for the previous set of
experiments, vessels were exposed to one or more
K+ channel blocker(s) but not to
milrinone before the second NE concentration-effect curve was generated
(Fig. 3). Transient contraction was
observed with administration of 4-aminopyridine,
Ba2+, or the combination of
iberiotoxin and apamin, but no increase in tension was noted at the end
of the incubation period. Glibenclamide decreased
pD2 from 5.88 ± 0.13 to 5.72 ± 0.13 (P < 0.05) and reduced maximal tension from 8.33 ± 0.78 to 7.23 ± 0.67 mN/mm
(P < 0.05). Exposure to
4-aminopyridine was associated with decreased
pD2 from 5.90 ± 0.08 to
5.42 ± 0.10 (P < 0.05), although maximal tension generation was not different from
control (7.09 ± 0.50 vs. 6.83 ± 0.68 mN/mm).
Ba2+ elicited a left shift in the
concentration-effect curve with an increase in
pD2 from 5.78 ± 0.13 to 6.14 ± 0.14, but maximal tension was not significantly changed (8.20 ± 0.35 vs. 7.85 ± 0.59 mN/mm). Iberiotoxin alone increased
pD2 from 5.89 ± 0.06 to 6.21 ± 0.08 (P < 0.05) but had no
significant effect on maximal tension (6.86 ± 0.57 vs. 6.79 ± 0.64 mN/mm). Finally, the combination of iberiotoxin and apamin
resulted in a significant left shift in
pD2 from 5.97 ± 0.11 to 6.62 ± 0.23 (P < 0.05) without
significantly altering maximal tension (6.58 ± 1.13 vs. 6.76 ± 1.46 mN/mm). Table 3 summarizes the effects
of K+ channel blockers on NE
concentration-effect curves in the absence of milrinone. All of the
K+ channel blockers altered NE
sensitivity compared with paired control
pD2 values. Glibenclamide and
4-aminopyridine reduced NE sensitivity, whereas
Ba2+, iberiotoxin, and the
combination of iberiotoxin and apamin increased NE sensitivity. Only
glibenclamide significantly affected maximal tension generation,
reducing it compared with controls. These findings underscore the need
for proper control data when interpreting drug effects. In this case,
comparison of milrinone-treated K+
channel-blocked vessels with untreated
K+ channel-blocked vessels allows
for the K+ channel-dependent
effects of milrinone to be specifically addressed.
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Effects of cAMP elevation on
K+- or
NE-contracted vessels.
Milrinone relaxed vessels preconstricted with NE (56 ± 4%) and
induced rhythmic oscillations in tension. Milrinone caused a similar
relaxation (52 ± 6%) in vessels contracted with 100 mM KCl but
failed to induce oscillations of vascular tone (Fig. 4). Forskolin also relaxed both NE- and
K+-contracted vessels by 95 ± 2 and 91 ± 1%, respectively. As with milrinone,
forskolin evoked oscillations in vessels contracted with NE but not
those contracted with KCl. In KCl-contracted, milrinone-relaxed
vessels, maximal force was restored by the PKA inhibitor
Rp-cAMPS (400 µM) but not by the cGMP-dependent protein kinase (PKG) inhibitor 8-CPT-Rp-cGMPS (10 µM;
Fig. 5).
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DISCUSSION |
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VSMC K+ channels are known to play an important role in the regulation of vascular tone (1, 16) inasmuch as gating of K+ channels alters membrane potential, thereby modulating influx of Ca2+ through voltage-sensitive L-type Ca2+ channels. Under the influence of vasoconstrictor stimuli, the regulation of K+ current through these channels may be important in determining the magnitude and duration of contraction. Recently, cAMP elevation has been linked to the activation of membrane K+ channels (2, 5, 9, 14, 16, 21, 22, 25), perhaps resulting directly from PKA-dependent phosphorylation of the channels themselves. Although cAMP is associated with reduced [Ca2+]i and attenuation of vasoconstriction, the functional significance of K+ channel regulation under conditions of elevated cAMP has not been clearly defined. In this study, we examined the effects of the PDE3 inhibitor milrinone on NE-induced VSM contraction and explored the potential significance of K+ channels in the manifestation of these effects.
Inhibition of the cGMP-inhibitable cAMP phosphodiesterase (i.e., PDE3) is known to cause relaxation of VSM and attenuation of vasoconstriction (3, 7, 8, 12, 19, 20). This action is associated with dose-dependent increases in intracellular cAMP, although slight increases in cGMP have also been reported (11). Furthermore, in separate studies by Haynes et al. (3) and Komas et al. (8), impairment of vasoconstriction resulting from PDE3 inhibition was shown to be independent of the endothelium. In the present study, we show that pretreatment with the PDE3 inhibitor milrinone reduces both NE sensitivity and maximal tension generation in mesenteric resistance arteries. These actions are presumably related to the ability of milrinone to elevate cAMP in VSM. The inhibitory effects of milrinone on vasocontrictor effectiveness were evident during both the onset and sustained phases of the NE contraction (Fig. 1). Also, in most vessels (>90%) pretreated with milrinone, NE induced large (2-4 mN) rhythmic oscillations of isometric force. Preliminary studies in our laboratory have shown that oscillations in tension follow oscillations in [Ca2+]i (unpublished observation). There are several possible mechanisms whereby milrinone could cause oscillations in agonist-induced tension. One possibility is that depolarization and [Ca2+]i elevation are offset by the periodic opening of K+ channels. The intermittent hyperpolarization would rhythmically influence Ca2+ influx through L-type Ca2+ channels. Another possibility is that the dynamics of agonist-induced sarcoplasmic reticulum Ca2+ release and sequestration are altered by milrinone. Both of these interpretations are consistent with the observation that milrinone induced tension oscillations in vessels contracted with NE but not in vessels contracted with high KCl. Further investigation of these potential mechanisms is warranted.
After initial observations, we postulated that a major mechanism of milrinone action might be an increase in the outward VSMC K+ current and consequential cell hyperpolarization resulting from cAMP elevation and PKA- and/or PKG-dependent phosphorylation of certain membrane K+ channels. As mentioned earlier, cyclic nucleotide-dependent activation of some classes of K+ channels has been reported, but the physiological role of this regulation in the control of VSM contraction is not clear. Coadministration of various K+ channel inhibitors with milrinone in attempt to prevent its suppression of NE concentration-effect curves yielded a number of interesting results. Blockade of KATP or KV with glibenclamide or 4-aminopyridine, respectively, did not prevent the effects of milrinone on NE responses. In fact, both glibenclamide and 4-aminopyridine tended to augment the milrinone-induced right shift in the NE concentration-effect curve. The cause of this potentiation of NE desensitization is unknown but might result from the compensatory opening of other K+ channels after KV or KATP inhibition (i.e., opening of KCa channels as a result of membrane depolarization). Other possibilities include nonspecific effects of these blockers or direct effects of glibenclamide or 4-aminopyridine on the endothelium. Although blockade of large-conductance KCa with iberiotoxin did not counter the effects of milrinone, simultaneous blockade of both large- and small-conductance KCa with the combination of iberiotoxin and apamin did prevent the milrinone-induced right shift, restoring normal NE sensitivity. Ba2+ also restored NE sensitivity to control levels in milrinone-treated vessels, presumably through inhibition of VSM KIR. Together, these findings suggest that cAMP-mediated reduction of tension development may be partially mediated via opening of KCa or KIR. However, it was noted in these experiments that no K+ channel blockade achieved full restoration of both NE sensitivity and maximal tension development, arguing against the actions of milrinone being manifested entirely through the regulation of K+ channels.
After the experiments with milrinone in the presence of
K+ channel blockers, it was still
unclear whether the displacement of milrinone-treated NE
concentration-effect curves by
Ba2+ or iberiotoxin/apamin was due
to direct inhibition of milrinone action or simply due to the
introduction of a separate depolarizing influence. In other words, the
NE concentration-effect curve generated in the presence of milrinone
and a K+ channel blocker may
coincidentally represent the net effect of two unrelated but opposing
influences: reduced NE responsiveness due to milrinone and enhanced NE
responsiveness due to the K+
channel blocker. If the two were equal and opposite then it is conceivable that no change in response would be detected. To evaluate this possibility, we examined the effects of the
K+ channel blockers alone on NE
concentration-effect curves. Both Ba2+ and the combination of
iberiotoxin and apamin significantly enhanced NE sensitivity compared
with paired control concentration-effect curves (Table 3 and Fig. 3).
The net shift in pD2 from control (pD2) was 0.37 ± 0.13 in
the presence of Ba2+ compared with
0.07 ± 0.06 (P < 0.05) in
vessels treated with Ba2+ and
milrinone (P < 0.05, unpaired
t-test). The net shift in
pD2 from control was 0.63 ± 0.31 in the presence of iberiotoxin and apamin compared with
0.07 ± 0.07 (P < 0.05) in
vessels treated with iberiotoxin, apamin, and milrinone
(P < 0.05, unpaired
t-test). Because responses in the
presence of these K+ channel
blockers were depressed when milrinone was also present, we conclude
that the inhibitory effects of milrinone on NE sensitivity are
essentially independent of KIR or
KCa function.
It is interesting to note that increased NE sensitivity in the presence of iberiotoxin and apamin was observed in the present study. This may reflect an enhanced depolarization and the loss of negative feedback regulation of [Ca2+]i. NE-induced Ca2+ mobilization and cell depolarization would normally be expected to evoke the opening of membrane KCa channels. Under conditions of KCa channel blockade, this mechanism of feedback hyperpolarization is prevented. As a result, depolarization is sustained and [Ca2+]i is elevated, allowing lower concentrations of NE to produce increases in tension.
The potentiation of NE sensitivity resulting from Ba2+ treatment is difficult to interpret. NE-induced contraction is associated with membrane depolarization. Because KIR channels should theoretically be closed during depolarization, it is not clear how inhibition of these channels influences vascular tension. It has been reported that extracellular K+ concentration of ~5 mM (i.e., that of the PSS used in our experiments) increases the open probability of KIR channels, allowing for hyperpolarization and relaxation of VSM. In fact, these channels normally carry an outward current under resting conditions (16). Therefore, blocking these channels with Ba2+ may favor depolarization and perhaps enhance vasoconstrictor sensitivity.
To determine the overall dependence of milrinone-induced attenuation of vasoconstriction on changes in transmembrane K+ flux, we assessed the ability of milrinone to relax arteries that were preconstricted with 100 mM KCl. In addition to depolarizing and constricting VSM, 100 mM KCl abolishes the transmembrane K+ gradient. Under these conditions, opening of membrane K+ channels is ineffective at eliciting cell hyperpolarization and VSM relaxation. Milrinone caused substantial relaxation of tension induced by high K+, which was comparable to that observed in vessels preconstricted with NE. These experiments were repeated using the adenylyl cyclase activator forskolin, which also relaxed both K+- and NE-contracted vessels. Furthermore, addition of the PKA inhibitor Rp-cAMPS (400 µM) after relaxation by milrinone fully restored the contraction induced by high K+. Notably, the PKG inhibitor 8-CPT-Rp-cGMPS (10 µM) failed to restore tension in KCl-constricted, milrinone-relaxed vessels. Collectively, these results support the contention that cAMP can interfere with VSM contraction through a PKA-dependent mechanism that is essentially independent of K+ channel regulation.
A number of recent studies support the idea of cAMP/PKA-mediated
augmentation of K+ currents,
particularly through KCa,
KATP, and
KV. Unfortunately, very few
studies have assessed the functional role of
K+ channel regulation on vascular
contractile state and responsiveness to vasoconstrictor agonists. Our
data suggest that cAMP-dependent events can oppose VSM contraction
through a mechanism that is essentially independent of
K+ channel regulation. Jackson (5)
reported that the action of a number of vasodilators, including
-agonists, adenosine, and prostacyclin, involves the opening of VSM
KATP, although agents that are not
dependent on receptor occupation such as forskolin and dibutyryl-cAMP
manifest their dilatory effects without
KATP channel regulation. These
findings suggest that Gs
stimulation may directly elicit the opening of
KATP, whereas cAMP may cause vascular relaxation via a separate mechanism.
In our study, the attenuation of NE responsiveness by the cAMP-elevating agent milrinone could not be directly linked to the regulation of any of the four known types of K+ channels. In a study by Lindgren et al. (11), milrinone (up to 10 µM) was found to only moderately relax human mesenteric arteries (400-700 µm) constricted by 127 mM K+. These findings suggest that K+ channel regulation may play a role in milrinone-induced relaxation. In our experiments, 50 µM milrinone was an effective relaxing agent in arteries constricted with high K+ (100 or 120 mM), indicating that milrinone can relax depolarized vessels without the involvement of K+ channel activation. McDaniel et al. (13) found that swine carotid arteries constricted with 109 mM K+ could be relaxed by an ~10-fold elevation in cAMP concentration. Interestingly, this relaxation was not accompanied by a decrease in [Ca2+]i, suggesting that cAMP can elicit dilatory effects in VSM through a mechanism that does not necessarily require regulation of K+ channels or [Ca2+]i.
In summary, the present study reveals that the PDE3 inhibitor milrinone reduces NE-induced isometric force generation in small mesenteric arteries. Regulation of KATP or KV does not appear to be an important mechanism of milrinone-induced attenuation of the NE response. Although blockade of KCa or KIR opposes the effects of milrinone, neither channel appears to play an essential role in the milrinone-induced reduction in NE sensitivity. Furthermore, the cAMP-elevating agents milrinone and forskolin can evoke relaxation of vascular tension induced by high K+. Therefore, cAMP-mediated control of vascular contraction may ultimately depend on regulation of processes other than transmembrane K+ flux, including direct inhibition of Ca2+ mobilization or reduction in Ca2+ sensitivity.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-51430.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: J. N. Benoit, Dept. of Physiology, Univ. of South Alabama College of Medicine, MSB 3024, Mobile, AL 36688 (E-mail: jbenoit{at}jaguar1.usouthal.edu).
Received 6 April 1998; accepted in final form 3 March 1999.
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