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Departments of Pharmacology and Psychology and Cardiovascular Center, University of Iowa, Iowa City, Iowa 52242
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The vasodilator effects of pituitary adenylate cyclase-activating polypeptide (PACAP)-27 are subject to tachyphylaxis in rats treated with the nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME). We examined whether this tachyphylaxis could be prevented by administration of the putative endothelium-derived nitrosyl factor S-nitroso-L-cysteine (L-SNC) and whether L-SNC may exert its effects via increases in cGMP levels in vascular smooth muscle. Five doses of PACAP-27 (2 nmol/kg iv) produced pronounced vasodilator responses in saline-treated rats. These responses were not subject to tachyphylaxis. The first injection of PACAP-27 (2 nmol/kg iv) in L-NAME-treated (50 µmol/kg iv) rats produced vasodilator responses similar to those in saline-treated rats, whereas subsequent injections produced progressively smaller responses. The injection of L-SNC (1,200 nmol/kg iv) before each injection of PACAP-27 prevented tachyphylaxis to the Gs protein-coupled receptor agonist in L-NAME-treated rats, whereas equihypotensive doses of the NO donor sodium nitroprusside (100 µg/kg iv) did not. The injection of the membrane-permeant cGMP analog 8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphate (8-CPT-cGMP; 30 µmol/kg iv) to L-NAME-treated rats restored resting hemodynamic values to pre-L-NAME levels but did not prevent the development of tachyphylaxis to PACAP-27. These results suggest that nitrosyl factors prevent the development of tachyphylaxis to the hemodynamic actions of PACAP-27. These nitrosyl factors may act independently of their ability to generate cGMP in vascular smooth muscle.
Gs protein-coupled receptors; vasodilation
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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THE PITUITARY adenylate cyclase-activating polypeptides (PACAP)-27 and -38 exert their effects by activation of G protein-coupled type I and type II PACAP-sensitive receptors (1, 2, 16, 17, 23, 24). The relaxant effects of PACAP-27 and -38 in isolated vessels are not affected by endothelium denudation, and these responses are associated with increases in cAMP levels (29). In addition, the vasodilator effects of PACAP-27 in cats are not diminished by the nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME; Ref. 13). Taken together, these findings suggest that PACAP exerts its effects by activation of Gs protein-coupled PACAP receptors on vascular smooth muscle (VSM) rather than by the release of endothelium-derived NO and NO-containing factors (together referred to as nitrosyl factors; Refs. 9, 18, 25).
The hypotensive and vasodilator effects of PACAP-27 (2 nmol/kg iv) are subject to tachyphylaxis in L-NAME-treated rats (28). This suggests that endothelium-derived nitrosyl factors may prevent the development of tachyphylaxis to PACAP-27. Endothelium-derived NO exerts its vasorelaxant effects primarily by the generation of cGMP in VSM (9). The putative endothelium-derived S-nitrosothiol S-nitroso-L-cysteine (L-SNC; Ref. 18) readily decomposes to NO in biological preparations (9, 18, 25). S-nitrosothiols also alter the activities of G proteins (10, 15), fusion proteins involved in vesicular exocytosis (12), and enzymes and receptor-operated ion channels (25) by the nitrosation of amino acids in these proteins. Therefore, endothelium-derived nitrosyl factors may prevent the development of tachyphylaxis to PACAP-27 by cGMP-dependent or cGMP-independent mechanisms. The aim of this study was to determine whether the development of tachyphylaxis to PACAP-27 in L-NAME-treated rats is prevented by the administration of L-SNC, the NO donor sodium nitroprusside (SNP), or the membrane-permeant cGMP analog 8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphate (8-CPT-cGMP).
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Rats. The protocols were approved by the University of Iowa Animal Care and Use Committee. Male Sprague-Dawley rats (Harlan, Indianapolis, IN; 250-300 g) were used in these studies.
Surgical procedures. Some rats were anesthetized with an initial injection of pentobarbital sodium (25 mg/kg ip) followed by an injection of urethan (500 mg/kg ip). These rats were supplemented with urethan (100 mg/kg iv) during the surgical procedures and the experiments as needed. Other rats were anesthetized with an injection of pentobarbital (50 mg/kg ip), and supplemental doses of pentobarbital (5 mg/kg iv) were given as needed. Polyethylene catheters were placed in the femoral vein for the administration of drugs and in the lower abdominal aorta via the femoral artery for the direct measurement of pulsatile and mean (MAP) arterial blood pressure. A midline laparotomy was performed, and pulsed Doppler flow probes were placed around the left renal artery, the superior mesenteric artery, and the lower abdominal aorta to measure blood flow velocities to determine renal (RR), mesenteric (MR), and hindquarter (HQR) vascular resistances, respectively (3-5). The hemodynamic responses in the hindquarter, renal, and mesenteric beds were examined to determine whether the mechanisms underlying tachyphylaxis to PACAP-27 were similar in these beds. Details of the Doppler technique, including construction of the probes, the reliability of the method for the estimation of blood flow velocity, and the quantitative determination of percent changes in resistance, have been described previously (7). During surgery and experimentation, animal body temperature was maintained at 37°C via a thermostat-controlled heating pad. The rats were allowed to breathe room air supplemented with carboxygen (95% O2-5% CO2) via a face mask.
Experimental protocol 1. Five injections of PACAP-27 (2 nmol/kg iv) were given to urethan-anesthetized rats before and 15-20 min after injection of saline (0.9% NaCl; n = 5) or L-NAME (25 µmol/kg iv; n = 7). The hemodynamic changes produced by L-NAME had reached their plateaus at this time. The injections of PACAP-27 were given ~10 min apart to allow the hypotensive and vasodilator effects of each injection of PACAP-27 to return to preinjection levels or to new plateau levels before the next injection was given.
Experimental protocol 2. One group of urethan-anesthetized rats (n = 5) received L-NAME (50 µmol/kg iv), and after 15-20 min (the time at which the hemodynamic values had reached their plateau values), a dose of L-SNC (1,200 nmol/kg iv) was given. Once the hemodynamic effects of L-SNC had subsided, the first dose of PACAP-27 (2 nmol/kg iv) was given. This procedure was repeated until five doses of L-SNC and PACAP-27 were given. The higher dose of L-NAME used in these studies was given to ensure adequate blockade of NO synthesis in these longer protocols.
Experimental protocol 3. One group of urethan-anesthetized rats (n = 5) received L-NAME (50 µmol/kg iv), and after 15-20 min a dose of SNP (100 µg/kg iv) was given. Once the hemodynamic effects of SNP had subsided, the first dose of PACAP-27 (2 nmol/kg iv) was given. This procedure was repeated until five doses of SNP and PACAP-27 were given. The higher dose of L-NAME used in these studies was given to ensure adequate blockade of NO synthesis in these longer protocols.
Experimental protocol 4. Two groups of pentobarbital-anesthetized rats received L-NAME (50 µmol/kg iv), and after 15-20 min one group (n = 5) received a bolus injection of saline and the other group (n = 5) received a bolus injection of 8-CPT-cGMP (30 µmol/kg iv). This dose of 8-CPT-cGMP caused the resting baseline parameters to return to pre-L-NAME levels (see RESULTS). After 15-20 min (at which time the hemodynamic effects of 8-CPT-cGMP had reached plateau values), these two groups received five injections of PACAP-27 (2 nmol/kg iv) given ~10 min apart. The rats that received 8-CPT-cGMP (30 µmol/kg iv) were supplemented with 8-CPT-cGMP (15 µmol/kg iv) halfway through the PACAP-27 injection protocol to keep baseline values at pre-L-NAME levels. The effects of 8-CPT-cGMP on the development of tachyphylaxis to PACAP-27 were also examined in urethan-anesthetized rats (n = 3). The protocol was identical to that described for pentobarbital-anesthetized rats.
Drugs. L-Cysteine, urethan, sodium nitrite, L-NAME, and 8-CPT-cGMP were obtained from Sigma Chemical (St. Louis, MO). Pentobarbital sodium was obtained from Abbott (Chicago, IL). PACAP-27 was obtained from Bachem (Torrance, CA). L-SNC was prepared by reacting 1 ml of 0.2 M sodium nitrite and 1 ml of 0.2 M L-cysteine (containing 100 µl of 1 N HCl), which resulted in a stable (pH ~ 3) 0.1 M stock solution (5). The stock solution was dissolved 1,000 times in saline to yield a weakly acidic injectate (pH 5-6).
Statistical analyses. The data are presented as means ± SE. The data were analyzed by repeated-measures analysis of variance followed by Student's modified t-test with the Bonferroni correction for multiple comparisons between means (3-5).
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Maximal hemodynamic effects produced by 5 consecutive injections of
PACAP-27 in urethan-anesthetized rats before and after administration
of L-NAME.
The hemodynamic effects produced by five injections of PACAP-27 (2 nmol/kg iv) in urethan-anesthetized rats before and after administration of L-NAME (25 µmol/kg iv) are summarized in Fig. 1.
Before administration of L-NAME,
injection 1 of PACAP-27 produced pronounced falls in MAP, HQR, and MR
but relatively minor falls in RR. Each subsequent injection of PACAP-27
produced similar responses (P > 0.05 for all comparisons). After administration of
L-NAME,
injection 1 of PACAP-27 produced
hypotensive and vasodilator responses that were similar to those before
administration of L-NAME
(P > 0.05 for all comparisons). The
vasodilator response produced by the first injection of PACAP-27 in the
renal bed was greater (P < 0.05)
after administration of L-NAME.
However, this renal vasodilation was similar
(P > 0.05) to the vasodilation produced by the fifth injection of PACAP-27 before administration of
L-NAME. Each subsequent
injection of PACAP-27 produced progressively and substantially smaller
responses after administration of
L-NAME. The hemodynamic
effects of five injections of PACAP-27 (2 nmol/kg iv) were similar
before and after administration of saline
(P > 0.05 for all comparisons, data
not shown).
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Time course of hemodynamic effects produced by 5 injections of
PACAP-27 before and after administration of
L-NAME.
The time course of the hemodynamic effects produced by the first,
second, and fifth injections of PACAP-27 (2 nmol/kg iv) before and
after administration of L-NAME
(25 µmol/kg iv) is summarized in Fig. 2.
Before administration of L-NAME,
injection
1 of PACAP-27 produced decreases in
MAP, HQR, and MR that lasted >4 min. Each subsequent injection of
PACAP-27 produced responses of similar duration
(P > 0.05 for all comparisons),
except for the renal vasodilation produced by
injection 5 of PACAP-27, which was of longer duration than that produced by
injection
1 (P < 0.05). After administration of
L-NAME, the duration of the
PACAP-27-induced responses progressively diminished with each injection
of PACAP-27.
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Resting hemodynamic parameters before each injection of PACAP-27
before and after administration of
L-NAME.
The resting hemodynamic parameters before each of the five injections
of PACAP-27 (2 nmol/kg iv) before and after administration of
L-NAME (50 µmol/kg iv) are
summarized in Table 1.
L-NAME increased MAP (+34 ± 6%, P < 0.05), HQR (+182 ± 17%, P < 0.05), RR (+87 ± 12%,
P < 0.05), and MR (+107 ± 14%,
P < 0.05). The pre-PACAP-27 injection values remained at similar levels after administration of
L-NAME
(P > 0.05 for all comparisons), with
some exceptions. The preinjection HQR values for
injection
5 of PACAP-27 were lower than those of
injection
1 (
24 ± 6%,
P < 0.05). The preinjection MR
values for injection
4 were higher than those of
injection 1 (+28 ± 7%,
P < 0.05) because the baseline MR
returned to higher values after recovery from the vasodilator effects
of injection 3. The preinjection MR values for the
fifth injection of PACAP-27 were higher than the preinjection values
for the fourth injection of PACAP-27 (+14 ± 3%,
P < 0.05).
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Effects of L-SNC on
PACAP-27-induced tachyphylaxis in
L-NAME-treated, urethan-anesthetized
rats.
Urethan-anesthetized rats received
L-NAME (50 µmol/kg iv) and
then five injections of PACAP-27 (2 nmol/kg iv), each preceded by an
injection of L-SNC (1,200 nmol/kg iv). The hemodynamic effects of
L-SNC or PACAP-27 were allowed
to return to preinjection levels or to a new resting plateau (see next
section) before the next injection was given. The maximal effects
produced by the five injections of
L-SNC are shown in the left
panels of Fig. 3, and the hemodynamic
effects produced by the five injections of PACAP-27 (2 nmol/kg iv) are
shown in the right panels. The first injection of
L-SNC produced pronounced falls
in MAP and vascular resistances (P < 0.05 for all comparisons). Each subsequent injection of
L-SNC produced similar responses
(P > 0.05 for all between-injection comparisons). The first injection of PACAP-27 produced pronounced falls
in MAP and vascular resistances (P < 0.05 for all comparisons) similar to those produced in
L-NAME-treated rats that did not receive L-SNC
(P > 0.05 for all comparisons). The
hemodynamic effects produced by the subsequent injections of PACAP-27
were not subject to tachyphylaxis in the
L-NAME-treated rats that
received L-SNC before each
injection of PACAP-27 (P > 0.05 for
all between-injection comparisons).
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Resting parameters in
L-NAME-treated rats that received
L-SNC before each of 5 injections of
PACAP-27.
The resting parameters in the
L-NAME-treated (50 µmol/kg iv)
rats that received L-SNC (1,200 nmol/kg iv) before each of the five injections of PACAP-27 (2 nmol/kg
iv) are summarized in Table 2.
L-NAME produced a substantial
increase in MAP (+44 ± 5% of 1st
pre-L-SNC injection value,
P < 0.05), HQR (+136 ± 18%,
P < 0.05), RR (+141 ± 15%,
P < 0.05) and MR (+236 ± 28%,
P < 0.05). The
pre-L-SNC and pre-PACAP-27
injection values remained at similar levels in these
L-NAME-treated rats
(P > 0.05 for all comparisons), with
one exception. The preinjection HQR values for the fifth injection of
L-SNC and PACAP-27 were lower
than the preinjection values for the first injection of
L-SNC (17 ± 5%,
P < 0.05) and PACAP-27 (16 ± 4%, P < 0.05). This is
because the resting HQR returned to a lower resting value after
recovery from the vasodilator effects of the fourth injection of
PACAP-27.
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Effects of SNP on PACAP-27-induced tachyphylaxis in
L-NAME-treated, urethan-anesthetized
rats.
A group of urethan-anesthetized rats received
L-NAME (50 µmol/kg iv) and
then five injections of PACAP-27 (2 nmol/kg iv), each preceded by an
injection of SNP (100 µg/kg iv). The maximal hemodynamic effects
produced by the five injections of SNP are shown in the left panels of
Fig. 4, and the effects of the five injections of PACAP-27 (2 nmol/kg iv) are shown in the right panels of
this figure. The first injection of SNP produced pronounced falls in
MAP and resistances in each bed
(P < 0.05 for all
comparisons). These responses were similar to or greater in magnitude
than those produced by L-SNC
(see Fig. 3). Each subsequent injection of SNP produced similar
responses (P > 0.05 for all
between-injection comparisons). The first injection of PACAP-27
produced pronounced falls in MAP and vascular resistances
(P < 0.05 for all comparisons) that
were similar to those in rats that received
L-NAME only
(P > 0.05 for all comparisons).
However, SNP did not prevent the development of tachyphylaxis to the
hemodynamic effects of subsequent doses of PACAP-27.
L-NAME produced increases in
resting MAP and vascular resistances similar to those described above
(P > 0.05 for all comparisons). The
pre-SNP and pre-PACAP-27 injection values remained constant in these
L-NAME-treated rats
(P < 0.05 for all comparisons). More
specifically, the resting hemodynamic values returned to preinjection
values after recovery from the hemodynamic effects of these compounds
(P > 0.05 for all comparisons).
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Maximal vasodilator responses produced by 5 injections of PACAP-27
in L-NAME-treated rats that received
saline or 8-CPT-cGMP before injection of PACAP-27.
The maximal changes in HQR produced by five consecutive injections of
PACAP-27 (2 nmol/kg iv) in two groups of pentobarbital-anesthetized rats before and after administration of
L-NAME (50 µmol/kg iv) are
summarized in Fig. 5. One group of
L-NAME-treated rats received a
bolus injection of saline before administration of the five injections
of PACAP-27. The other group of
L-NAME-treated rats (n = 5) received a bolus injection of
8-CPT-cGMP (30 µmol/kg iv) before administration of the five
injections of PACAP-27. This dose of 8-CPT-cGMP lowered MAP and HQR to
pre-L-NAME values (see next
section). The first injection of PACAP-27 produced pronounced falls in
HQR and MAP in the
L-NAME-treated rats that
received an injection of saline before the first injection of PACAP-27 (P < 0.05 for both responses). As
can be seen, tachyphylaxis readily occurred to the hemodynamic effects
of PACAP-27 on repeated administration of this peptide. The maximal
falls in HQR produced by the first injection of PACAP-27 in
L-NAME-treated rats that
received 8-CPT-cGMP were similar to those in rats that received saline
before the first injection of the peptide
(P > 0.05 for all comparisons). As
can be seen, 8-CPT-cGMP did not prevent the development of tachyphylaxis to the hindquarter vasodilator effects of PACAP-27 in
L-NAME-treated rats.
More specifically, the vasodilator responses produced by each injection
of PACAP-27 were similar in saline- and 8-CPT-cGMP-treated rats
(P > 0.05 for all comparisons).
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Resting hemodynamic values before each of 5 consecutive injections
of PACAP-27 in L-NAME-treated rats
that received bolus injection of saline or 8-CPT-cGMP before injection
of PACAP-27.
The resting hemodynamic parameters before each of five injections of
PACAP-27 (2 nmol/kg iv) in pentobarbital-anesthetized, L-NAME-treated (50 µmol/kg iv)
rats that received an injection of saline before PACAP-27 are
summarized in Table 3.
L-NAME increased MAP (+39 ± 6%, P < 0.05), HQR (+131 ± 23%, P < 0.05), and MR (+177 ± 19%, P < 0.05). The
pre-PACAP-27 MAP values were similar in these
L-NAME-treated rats
(P > 0.05 for all comparisons). More specifically, resting MAP returned to preinjection values after recovery from the hypotensive effects of each injection of PACAP-27. The preinjection HQR values for
injection
4 of PACAP-27 were lower than those of
injection
1 (16 ± 4%,
P < 0.05). This is because the
resting HQR returned to a lower resting value after recovery from the
vasodilator effects of injection
3 of PACAP-27. The preinjection HQR
values for injection
5 of PACAP-27 were lower than those of injection
1 (19 ± 4%,
P < 0.05). This is because the
resting HQR remained at lower resting values after recovery from the
vasodilator effects of injection
4. The preinjection MR values for
injection 4 of PACAP-27 were higher than the
preinjection values of injection 1 (+32 ± 8%,
P < 0.05). This is because the
resting MR returned to a higher resting value after recovery from the
vasodilator effects of injection
3. The preinjection MR values for
injection 5 of PACAP-27 were also higher than
the preinjection values of injection
1 (+45 ± 9%,
P < 0.05). This is
because the resting MR remained at higher resting values after recovery
from the vasodilator effects of
injection
4 of PACAP-27.
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35 ± 7%, P < 0.05), HQR (41 ± 6%,
P < 0.05), and MR (58 ± 9%, P < 0.05). Resting
MAP and resistance values after the injection of 8-CPT-cGMP were
similar to the
pre-L-NAME values
(P > 0.05 for all comparisons). The
MAP and HQR values did not change over the time in which the five
injections of PACAP-27 were given (P > 0.05 for all comparisons). However, the preinjection MR values for
injections
4 and
5 of PACAP-27 were higher than those
before injection
1 (+56 ± 18% and +98 ± 27%,
respectively; P < 0.05). This steady
increase in baseline MR occurred even though a 15 µmol/kg dose of
8-CPT-cGMP was given before the third injection of PACAP-27 was administered.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The hypotensive and vasodilator effects of PACAP-27 (2 nmol/kg iv) were not subject to tachyphylaxis in saline-treated, urethan-anesthetized rats. The hemodynamic effects of the first injection of PACAP-27 in L-NAME-treated rats were similar to those in saline-treated rats. This supports evidence that the vasodilator effects of PACAP-27 are mediated by Gs protein-coupled receptors rather than by endothelium-derived relaxing factors (13, 14, 19, 29). Tachyphylaxis rapidly developed to the hemodynamic effects of PACAP-27 in L-NAME-treated rats. In addition, the injection of L-SNC before each injection of PACAP-27 prevented tachyphylaxis to PACAP-27. This suggests that tachyphylaxis to PACAP-27 in L-NAME-treated rats was caused by the loss of exposure of VSM to endothelium-derived nitrosyl factors. A high dose of L-SNC was given to generate as much cGMP in VSM as possible. In addition, the pronounced hemodynamic effects of each injection of L-SNC were allowed to subside before the subsequent injection of PACAP-27 was given. This was done to determine whether the mechanisms by which L-SNC relaxes VSM (5, 9) could be separated from those that prevent tachyphylaxis to PACAP-27. The vasorelaxant effects of S-nitrosothiols in vitro involve their decomposition to NO, which relaxes VSM by the generation of cGMP (9, 26). In theory, nitrosyl factors may prevent tachyphylaxis to PACAP-27 by activation of a cGMP-dependent protein kinase (6). The finding that L-SNC prevented tachyphylaxis to PACAP-27 even though the hemodynamic effects of the S-nitrosothiol had subsided suggests that this process was cGMP independent. The changes in intracellular cGMP levels should parallel the L-SNC-mediated falls in vascular resistances. Accordingly, cGMP levels should peak at the time of maximal vasodilation and should return to preinjection values after recovery from the vasodilator effects of L-SNC.
The injection of a large dose of the NO donor SNP before each injection of PACAP-27 did not prevent tachyphylaxis to the Gs protein-coupled receptor agonist. Each dose of SNP produced pronounced hemodynamic responses (equal to or greater than those produced by L-SNC) that were presumably caused by the generation of cGMP in VSM (26). It should be noted that SNP is not simply an NO donor (26), and the results with this compound should be taken with some caution. However, it was also evident that the administration of the membrane-permeant cGMP analog 8-CPT-cGMP was also unable to prevent the development of tachyphylaxis to PACAP-27 in the L-NAME-treated rats. These results suggest that L-SNC prevented tachyphylaxis to PACAP-27 by cGMP-independent mechanisms. The 8-CPT-cGMP experiments also provided evidence that the L-NAME-induced increases in vascular resistances were not responsible for the development of tachyphylaxis to PACAP-27. More specifically, resting HQR remained at pre-L-NAME levels during the injections of PACAP-27 in these 8-CPT-cGMP-treated rats. Tachyphylaxis to the vasodilator effects of PACAP-27 occurred in these rats even though baseline HQR was at pre-L-NAME levels. The preinjection MR levels for the first three injections of PACAP-27 were similar to pre-L-NAME levels. This is important because near-maximal tachyphylaxis was obtained with the third injection of PACAP-27. However, the resting preinjection MR values for the fourth and fifth injections of PACAP-27 were higher than pre-L-NAME levels. This rise in baseline MR may not have been caused by the loss of potency of 8-CPT-cGMP, because another injection of 8-CPT-cGMP given about halfway through the PACAP-27 injection protocol did not prevent the increase in baseline MR. The increase in baseline MR may have been caused by some active vasoconstrictor process.
Although tachyphylaxis to PACAP-27 was not observed in saline-treated
rats, this does not preclude the possibility that PACAP receptors were
desensitized. The rapid desensitization of
Gs protein-coupled 
-adrenoceptors in membrane preparations is caused by the
phosphorylation of the receptors by cAMP-dependent protein kinase (PKA)
and by G protein-coupled receptor kinases (GRK; Refs. 8, 20, 22). The
phosphorylated receptor cannot couple to
Gs proteins (8, 20, 22). The
desensitized receptor is sequestered and dephosphorylated before
reincorporation into the plasma membrane (8). At present, there is no
evidence that PACAP receptors are phosphorylated by PKA or GRK,
although these receptors contain amino acids that are subject to
phosphorylation in other receptors (24). The lack of tachyphylaxis to
PACAP-27 in saline-treated rats may be because
1) PACAP-27 does not desensitize
PACAP receptors under normal conditions;
2) five injections of PACAP-27 (2 nmol/kg iv) do not desensitize enough receptors for a loss of response
to be evident; or 3) PACAP receptors
desensitized by each injection of PACAP-27 are rapidly dephosphorylated
and reincorporated into the plasma membranes so that the next injection
of PACAP-27 has a full complement of receptors to activate.
The processes responsible for tachyphylaxis to PACAP-27 in L-NAME-treated rats and the mechanisms by which nitrosyl factors prevent this tachyphylaxis remain to be determined. We have obtained preliminary evidence that tachyphylaxis to PACAP-27 is not caused by the loss of biological activity of cAMP generated within VSM (30). It is possible that tachyphylaxis to PACAP-27 is caused by the failure of the agonist-occupied receptor to generate cAMP. This may be caused by 1) PKA- or GRK-mediated phosphorylation of PACAP receptors that prevents the receptor from coupling to Gs proteins, 2) inability of Gs proteins to activate adenylate cyclase, or 3) inhibition of intracellular processes that dephosphorylate and reincorporate the receptor into the plasma membrane (8, 20, 22). It is possible that endothelium-derived and neurogenically derived nitrosyl factors (3, 4, 21) prevent tachyphylaxis to PACAP-27 by the activation of stereoselective recognition sites in the vasculature (5, 26, 27) or by nitrosation of amino acids in the receptor or other elements in the signal transduction cascade (10, 12, 15, 25). Although it is possible that NO synthase directly synthesizes S-nitrosothiols and that NO may be oxidized to a nitrosating agent (i.e., NO+; Refs. 9, 18, 25), there is no conclusive evidence that this occurs under physiological conditions. Moreover, there is no evidence that the PACAP receptor is nitrosated. However, this receptor does contain cysteine residues that, in theory, would be susceptible to nitrosation (24). The nitrosation of serine, threonine, and tyrosine residues in Gs protein-coupled receptors may prevent their phosphorylation. In addition, the nitrosation of cysteine residues may prevent PKA or GRK from coupling to the receptor and phosphorylating amino acids. The possible mechanisms by which S-nitrosothiols prevent tachyphylaxis to PACAP-27 include the following: 1) nitrosation of the PACAP receptor may prevent PKA or GRK from phosphorylating the receptor; 2) nitrosation of Gs proteins or adenylate cyclase may be important in the coupling of these proteins; and 3) nitrosation events regulate the phosphatase activity or the processes that reincorporate the receptor into plasma membranes.
In summary, this study provides evidence that tachyphylaxis to PACAP-27 in L-NAME-treated rats may be caused by the loss of influence of endothelium-derived nitrosyl factors on the processes that desensitize PACAP receptors. We have obtained preliminary evidence that the vasodilator actions of isoproterenol are subject to tachyphylaxis in L-NAME-treated rats and that L-SNC prevents this tachyphylaxis from occurring (31, 32). It therefore appears that nitrosyl factors may prevent the desensitization of a variety of Gs protein-coupled receptors in vivo. Disease states such as hypertension (11) are associated with compromised endothelial cell function and a loss of Gs protein-coupled receptor-mediated vasodilation. Our findings suggest that the loss of vasodilator potency of Gs protein-coupled receptor agonists in disease states may be caused by desensitization of these receptors as a consequence of reduced release of endothelium-derived nitrosyl factors.
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ACKNOWLEDGEMENTS |
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The authors acknowledge the expert assistance of Mike Burcham in the preparation of the figures.
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FOOTNOTES |
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This work was supported in part by grants from the National Institutes of Health (HL-14388, HL-57472, and DK-54759), from the National Aeronautics and Space Administration (NAG5-6171), and from the Office of Naval Research (N00014-97-1-0145).
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: A. K. Johnson, Dept. of Psychology, Univ. of Iowa, 11 Seashore Hall E., Iowa City, IA 52242-1407.
Received 18 May 1998; accepted in final form 16 February 1999.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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1.
Arimura, A.
Pituitary adenylate cyclase activating polypeptide (PACAP): discovery and current status of research.
Regul. Pept.
37:
287-303,
1992[Medline].
2.
Christophe, J.
Type I receptors for PACAP (a neuropeptide even more important than VIP?).
Biochim. Biophys. Acta
1154:
183-199,
1993[Medline].
3.
Davisson, R. L.,
J. N. Bates,
A. K. Johnson,
and
S. J. Lewis.
Use-dependent loss of acetylcholine- and bradykinin-mediated vasodilation after nitric oxide synthase inhibition: evidence for preformed stores of nitric oxide-containing factors in vascular endothelial cells.
Hypertension
28:
354-360,
1996
4.
Davisson, R. L.,
O. S. Possas,
S. P. Murphy,
and
S. J. Lewis.
Neurogenically derived nitrosyl factors mediate sympathetic vasodilation in the hindlimb vasculature of the rat.
Am. J. Physiol.
272 (Heart Circ. Physiol. 41):
H2369-H2376,
1997
5.
Davisson, R. L.,
M. D. Travis,
J. N. Bates,
and
S. J. Lewis.
Hemodynamic effects of L- and D-S-nitrosocysteine in the rat: stereoselective S-nitrosothiol recognition sites.
Circ. Res.
79:
256-262,
1996
6.
Girault, J.-A.
Protein phosphorylation and dephosphorylation in mammalian central nervous system.
Neurochem. Int.
23:
1-25,
1993[Medline].
7.
Haywood, J. R.,
R. A. Shaffer,
C. Fastenow,
G. D. Fink,
and
M. J. Brody.
Regional blood flow measurement with pulsed Doppler flowmeter in conscious rat.
Am. J. Physiol.
241 (Heart Circ. Physiol. 10):
H273-H278,
1981
8.
Hausdorff, W. P.,
M. G. Caron,
and
R. J. Lefkowitz.
Turning off the signal: desensitization of -adrenergic receptor function.
FASEB J.
4:
2881-2889,
1990[Abstract].
9.
Ignarro, L. J.
Nitric oxide: a novel signal transduction mechanism for transcellular communication.
Hypertension
16:
477-483,
1990
10.
Lander, H. M.,
P. K. Sehajpal,
and
A. Novogrodsky.
Nitric oxide signaling: a possible role for G-proteins.
J. Immunol.
151:
7182-7187,
1993[Abstract].
11.
Lüscher, T. F.,
and
P. M. Vanhoutte.
The Endothelium: Modulator of Cardiovascular Function. Boca Raton, FL: CRC, 1990, p. 111-164.
12.
Meffert, M. K.,
N. C. Calakos,
R. H. Scheller,
and
H. Schulman.
Nitric oxide modulates synaptic vesicle docking/fusion reactions.
Neuron
16:
1229-1236,
1996[Medline].
13.
Minkes, R. K.,
T. J. McMahon,
T. R. Higuera,
W. A. Murphy,
D. H. Coy,
and
P. J. Kadowitz.
Analysis of systemic and pulmonary vascular responses to PACAP and VIP: role of adrenal catecholamines.
Am. J. Physiol.
263 (Heart Circ. Physiol. 32):
H1659-H1669,
1992
14.
Minkes, R. K.,
T. J. McMahon,
J. S. Hood,
W. A. Murphy,
D. H. Coy,
D. B. McNamara,
and
P. J. Kadowitz.
Differential effects of PACAP and VIP on the pulmonary and hindquarters vascular beds of the cat.
J. Appl. Physiol.
72:
1212-1217,
1992
15.
Miyamoto, A.,
U. Laufs,
C. Pardo,
and
J. K. Liao.
Modulation of bradykinin receptor ligand binding affinity and its coupled G-proteins by nitric oxide.
J. Biol. Chem.
272:
19601-19608,
1997
16.
Miyata, A.,
A. Arimura,
R. R. Dahl,
N. Minamino,
A. Uehara,
L. Jiang,
M. D. Culler,
and
D. H. Coy.
Isolation of a novel 38 residue-hypothalamic polypeptide which stimulates adenylate cyclase in pituitary cells.
Biochem. Biophys. Res. Commun.
164:
567-574,
1989[Medline].
17.
Miyata, A.,
L. Jiang,
R. R. Dahl,
C. Kitada,
K. Kubo,
M. Fujino,
N. Minamino,
and
A. Arimura.
Isolation of a neuropeptide corresponding to the N-terminal 27 residues of the pituitary adenylate cyclase-activating polypeptide with 38 residues (PACAP-38).
Biochem. Biophys. Res. Commun.
170:
643-648,
1990[Medline].
18.
Myers, P. R.,
R. L. Minor, Jr.,
R. Guerra, Jr.,
J. N. Bates,
and
D. G. Harrison.
Vasorelaxant properties of the endothelium-derived relaxing factor more closely resemble S-nitrosocysteine than nitric oxide.
Nature
345:
161-163,
1990[Medline].
19.
Nandha, K. A.,
M. A. Benito-Orfila,
D. M. Smith,
M. A. Ghatei,
and
S. R. Bloom.
Action of pituitary adenylate cyclase-activating polypeptide and vasoactive intestinal polypeptide on the rat vascular system: effects on blood pressure and receptor binding.
J. Endocrinol.
129:
69-73,
1991
20.
Palczewski, K.,
and
J. L. Benovic.
G-protein-coupled receptor kinases.
Trends Biochem. Sci.
16:
387-391,
1991[Medline].
21.
Possas, O. S.,
and
S. J. Lewis.
NO-containing factors mediate hindlimb vasodilation produced by superior laryngeal nerve stimulation.
Am. J. Physiol.
273 (Heart Circ. Physiol. 42):
H234-H243,
1997
22.
Premont, R. T.,
J. Inglese,
and
R. J. Lefkowitz.
Protein kinases that phosphorylate activated G protein-coupled receptors.
FASEB J.
9:
175-182,
1995[Abstract].
23.
Shivers, B. D.,
T. J. Gorcs,
P. E. Gottschall,
and
A. Arimura.
Two high affinity binding sites for pituitary adenylate cyclase-activating polypeptide have different tissue distributions.
Endocrinology
128:
3055-3065,
1991[Abstract].
24.
Spengler, D.,
C. Waeber,
C. Pantaloni,
F. Holsboer,
J. Bockaert,
P. H. Seeburg,
and
L. Journot.
Differential signal transduction by five splice variants of the PACAP receptor.
Nature
365:
170-175,
1993[Medline].
25.
Stamler, J. S.,
D. J. Singel,
and
J. Loscalzo.
Biochemistry of nitric oxide and its redox-activated forms.
Science
258:
1898-1902,
1992
26.
Travis, M. D.,
L. L. Stoll,
J. N. Bates,
and
S. J. Lewis.
L- and D-S-nitroso-,-dimethylcysteine differentially increase cGMP in cultured vascular smooth muscle cells.
Eur. J. Pharmacol.
318:
47-53,
1996[Medline].
27.
Travis, M. D.,
R. L. Davisson,
J. N. Bates,
and
S. J. Lewis.
Hemodynamic effects of L- and D-S-nitroso-,-dimethylcysteine in rat.
Am. J. Physiol.
273 (Heart Circ. Physiol. 42):
H1493-H1501,
1997
28.
Travis, M. D.,
E. J. Whalen,
and
S. J. Lewis.
Heterologous desensitization of -adrenoceptor signal transduction in vivo.
Eur. J. Pharmacol.
328:
R1-R3,
1997[Medline].
29.
Warren, J. B.,
L. E. Donnelly,
S. Cullen,
B. E. Robertson,
M. A. Ghatei,
S. R. Bloom,
and
J. MacDermot.
Pituitary adenylate cyclase-activating polypeptide: a novel, long-lasting, endothelium-independent vasorelaxant.
Eur. J. Pharmacol.
197:
131-134,
1991[Medline].
30.
Whalen, E. J.,
A. K. Johnson,
and
S. J. Lewis.
Nitrosyl factors regulate Gs protein-coupled receptors (Abstract).
FASEB J.
12:
A697,
1998.
31.
Whalen, E. J.,
and
S. J. Lewis.
Tachyphylaxis of -adrenoceptor function occurs in vivo following inhibition of nitric oxide synthesis (Abstract).
Hypertension
28:
551,
1996.
32.
Whalen, E. J.,
and
S. J. Lewis.
Tachyphylaxis of -adrenoceptor-mediated vasodilation after nitric oxide synthase (NOS) inhibition (Abstract).
FASEB J.
11:
A10,
1997.
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) in mean arterial pressure (MAP) and hindquarter
(HQR), renal (RR), and mesenteric (MR) vascular resistances produced by
5 successive injections of pituitary adenylate cyclase-activating
polypeptide (PACAP)-27 (2 nmol/kg iv) in urethan-anesthetized rats
(n = 5) before
(A) and after
(B) injection of
NG-nitro-L-arginine methyl ester
(L-NAME; 25 µmol/kg iv). Data
are expressed as means ± SE of percent changes from preinjection
values. * P < 0.05, 2nd-5th
injections vs. 1st injection of PACAP-27. 
P < 0.05, 1st
injection post-L-NAME vs. 1st injection
pre-L-NAME.
