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Am J Physiol Regul Integr Comp Physiol 277: R1453-R1461, 1999;
0363-6119/99 $5.00
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Vol. 277, Issue 5, R1453-R1461, November 1999

Tachyphylaxis to PACAP-27 after inhibition of NO synthesis: a loss of adenylate cyclase activation

Erin J. Whalen, Alan Kim Johnson, and Stephen J. Lewis

Departments of Pharmacology and Psychology and The Cardiovascular Center, The University of Iowa, Iowa City, Iowa 52242


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The vasodilator effects of pituitary adenylate cyclase activating polypeptide (PACAP-27) are subject to tachyphylaxis in rats treated with the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME). This study examined whether this tachyphylaxis is due to the loss of vasodilator potency of cAMP generated by activation of the Gs protein-coupled PACAP receptors. Five successive treatments with PACAP-27 (2 nmol/kg iv) produced pronounced vasodilator responses in saline-treated rats that were not subject to tachyphylaxis. The first injection of PACAP-27 (2 nmol/kg iv) in L-NAME (50 µmol/kg iv)-treated rats produced vasodilator responses of similar magnitude to those in saline-treated rats, whereas four subsequent injections produced progressively and markedly smaller responses. The hemodynamic effects of the membrane-permeable cAMP analog 8-(4-chlorophenylthiol)-cAMP (8-CPT-cAMP; 5-15 µmol/kg iv) were similar in L-NAME-treated rats and in L-NAME-treated rats that had received the five injections of PACAP-27. In addition, five injections of 8-CPT-cAMP (10 µmol/kg iv) produced pronounced vasodilator responses in saline- and L-NAME-treated rats that were not subject to the development of tachyphylaxis. These results suggest that a loss of biological potency of cAMP is not responsible for tachyphylaxis to PACAP-27 in L-NAME-treated rats. This tachyphylaxis may be due to the inability of the Gs protein-coupled PACAP receptor to activate adenylate cyclase.

Gs protein-coupled receptors; vasodilation; adenosine 3',5'-cyclic monophosphate; nitrosyl factors; nitric oxide


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE PITUITARY ADENYLATE cyclase activating polypeptides (PACAP-27 and -38; 1, 15, 16) exert their effects by activation of Gs protein-coupled PACAP receptors (2, 23). PACAP-27 and -38 relax isolated vessels in an endothelium-independent manner, and this relaxation is associated with increases in cAMP levels (30). In addition, the vasodilator effects of PACAP-27 in cats are not attenuated by the nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME; 12). These findings suggest that the vasodilation produced by systemically injected PACAP polypeptides involves the PACAP receptor-mediated activation of adenylate cyclase (AC) within vascular smooth muscle (VSM; 12, 13, 18) rather than the release of endothelium-derived NO or NO-containing factors (together referred to as nitrosyl factors; 10, 17, 25).

Five injections of PACAP-27 (2 nmol/kg iv) produced pronounced and equivalent vasodilator responses in saline-treated rats (28, 31). The first injection of this dose of PACAP-27 in L-NAME-treated rats produced vasodilator responses similar to those in saline-treated rats (28, 31). However, subsequent injections of PACAP-27 produced progressively and markedly smaller responses (28, 31). This demonstrates that the vasodilator effects of PACAP-27 are subject to rapid tachyphylaxis after inhibition of NO synthesis. Administration of the endothelium-derived S-nitrosothiol, L-S-nitrosocysteine (L-SNC; 10, 17) prevented tachyphylaxis to PACAP-27 in L-NAME-treated rats, whereas administration of the NO donor sodium nitroprusside or the membrane-permeable cGMP analog 8-(4-chlorophenylthiol)-cGMP (8-CPT-cGMP) did not (31). This suggests that L-SNC prevented tachyphylaxis to PACAP-27 by mechanisms other than its decomposition to NO and the generation of cGMP in VSM (10, 17). S-nitrosothiols exert their effects by nitrosation of amino acids, especially cysteine residues in G proteins (11, 14), enzymes, and receptor-operated ion channels (25). Accordingly, endothelium-derived S-nitrosothiols may prevent tachyphylaxis to PACAP-27 by nitrosating amino acids in PACAP receptors or other components of the signal transduction cascade.

The rapid desensitization of Gs protein-coupled beta -adrenoceptors is due to phosphorylation of these receptors by cAMP-dependent protein kinase (PKA) and G protein-coupled receptor kinases (GRKs; 9, 19, 22). To our knowledge, there is no evidence that PACAP receptors are phosphorylated by PKA or GRKs, although these receptors contain amino acids that would be subject to phosphorylation in other receptors (24). The ability of nitrosyl factors to nitrosate amino acids in PACAP receptors may prevent the phosphorylation of these receptors by PKA or GRKs. The nitrosation of amino acids would decrease in a time-dependent manner after inhibition of release of endothelium-derived nitrosyl factors (25). PACAP receptor activation would then allow PKA or GRKs to desensitize these receptors. Alternatively, tachyphylaxis to PACAP-27 may involve 1) enhanced degradation of cAMP generated by Gs protein-coupled receptors, 2) diminished capacity of cAMP to activate PKA, which is the main mechanism by which cAMP relaxes VSM (9, 19, 22), or 3) diminished ability of PKA to relax VSM.

The goal of this study was to determine whether tachyphylaxis to PACAP-27 in L-NAME-treated rats is due to a decrease in cAMP-mediated relaxation of VSM rather than to the inability of Gs protein-coupled receptors to activate AC. The specific aims were to determine 1) whether tachyphylaxis occurs to the hemodynamic effects of five injections of the membrane permeable cAMP analog 8-(4-chlorophenylthiol)-cAMP (8-CPT-cAMP; 10 µmol/kg iv) in L-NAME (50 µmol/kg iv)-treated rats, and 2) whether the dose-dependent effects of 8-CPT-cAMP (5-15 µmol/kg iv) are diminished in L-NAME-treated rats that received five injections of PACAP-27 (2 nmol/kg iv) compared with those treated with L-NAME only.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Rats. The protocols were approved by the University of Iowa Animal Care and Use Committee. Male Sprague-Dawley rats (250-300 g; n = 25) were used in these studies.

Surgical procedures. The rats were anesthetized with an injection of pentobarbital sodium (50 mg/kg ip). Polyethylene catheters were surgically 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. Supplemental doses of pentobarbital sodium (5 mg/kg iv) were given as needed during the surgical procedures and the experiments. A midline laparotomy was performed, and pulsed Doppler flow probes were placed around the superior mesenteric arteries and the lower abdominal aorta to measure blood flow velocities to determine mesenteric (MR) and hindquarter (HQR) vascular resistances, respectively (3-7). 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 (8). 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 95% O2-5% CO2 via a face mask.

Experimental protocols. In the following protocols, the hemodynamic responses in the hindquarter and mesenteric beds were examined to determine whether the mechanisms underlying tachyphylaxis to PACAP-27 were similar in these beds. The first group of rats (n = 5) received an injection of saline (0.9% NaCl wt/vol) and, after 15-20 min, the rats received five injections of PACAP-27 (2 nmol/kg iv) each given about 10 min apart to allow sufficient time for 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. The second group of rats (n = 5) received an injection of L-NAME (50 µmol/kg iv). After 15-20 min, at which time the hypertensive and vasoconstrictor effects of the NO synthesis inhibitor had reached their plateau levels (see RESULTS), the rats received five injections of PACAP-27 (2 nmol/kg iv) each given 10 min apart. These rats then received injections of 8-CPT-cAMP (5-15 µmol/kg iv). The third group of rats (n = 5) received an injection of L-NAME (50 µmol/kg iv), and, after 15-20 min, the rats received five injections of saline given at equivalent intervals as the injections of PACAP-27 in the previous group. These rats then received injections of 8-CPT-cAMP (5-15 µmol/kg iv). The fourth group of rats (n = 5) received an injection of saline, and, after 15-20 min, the rats received five consecutive injections of 8-CPT-cAMP (10 µmol/kg iv) given ~10 min apart to allow sufficient time for the hypotensive and vasodilator effects of each injection to return to preinjection levels or to new plateau levels before the next injection was given. The fifth group of rats (n = 5) received an injection of L-NAME (50 µmol/kg iv), and, after 15-20 min, the rats received five consecutive injections of 8-CPT-cAMP (10 µmol/kg iv) given ~10 min apart. The maximal falls in vascular resistances and the falls in MAP associated with these falls in vascular resistances were determined for each injection of PACAP-27 or 8-CPT-cAMP. The maximal PACAP-27-induced falls in MR occurred 10-20 s before the maximal falls in HQR, although the falls in MAP were similar at these times.

Drugs. L-NAME and 8-CPT-cAMP were from Sigma Chemical (St. Louis, MO). Pentobarbital sodium was from Abbott (Chicago, IL). PACAP-27 was from Bachem (Torrance, CA).

Statistical analyses. The data are presented as the mean ± SE. The data were analyzed by repeated measures analysis of variance (32) followed by Student's modified t-test with the Bonferroni correction for multiple comparisons between means (29).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Hemodynamic effects of five successive injections of PACAP-27 in saline- or L-NAME-treated rats. The maximal falls in HQR produced by five injections of PACAP-27 (2 nmol/kg iv) in saline- or L-NAME (50 µmol/kg iv)-treated rats are summarized in Fig. 1. The falls in MAP are also shown. The first injection of PACAP-27 (injection 1) produced pronounced vasodilator and hypotensive responses in saline-treated rats (P < 0.05 for both responses). Injections 2-5 of PACAP-27 produced similar responses (P > 0.05, for all comparisons to first injection response). Injection 1 of PACAP-27 produced pronounced falls in HQR and MAP in L-NAME-treated rats (P < 0.05 for both responses). These responses were similar to those in saline-treated rats (P > 0.05 for both comparisons). However, unlike the saline-treated rats, injections 2-4 of PACAP-27 produced progressively smaller vasodilator responses. The responses produced by injections 3-5 of PACAP-27 were smaller than those produced by injection 1 (P < 0.05 for all comparisons). The hypotensive response produced by injection 2 of PACAP-27 was markedly smaller than that produced by injection 1 (P < 0.05 for all responses). The hypotensive responses produced by injections 3-5 of PACAP-27 were similar to that of injection 2 (P > 0.05 for all comparisons).


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  		Fig. 1.   Summary of maximal falls in hindquarter vascular resistance (HQR) produced by 5 injections of pituitary adenylate cyclase activating polypeptide (PACAP-27; 2 nmol/kg iv) in saline-treated rats (A; n = 5) and NG-nitro-L-arginine methyl ester (B; L-NAME; 50 µmol/kg iv)-treated rats (n = 5). Falls in mean arterial blood pressure (MAP) at point of maximal fall in HQR are also shown. Data are expressed as means ± SE of percent changes from resting values. * P < 0.05, 2nd-5th injections vs. 1st injection of PACAP-27.

The maximal falls in MR produced by five injections of PACAP-27 (2 nmol/kg iv) in saline- or L-NAME (50 µmol/kg iv)-treated rats are summarized in Fig. 2. The falls in MAP at the time of maximal falls in MR are also shown. Injection 1 of PACAP-27 produced pronounced vasodilator and hypotensive responses in saline-treated rats (P < 0.05 for both responses). Each subsequent injection of PACAP-27 produced very similar responses (P > 0.05, for all comparisons to injection 1 response). Injection 1 of PACAP-27 produced pronounced falls in MR and MAP in L-NAME-treated rats (P < 0.05 for both responses). These responses were similar to those in saline-treated rats (P > 0.05 for both comparisons). However, unlike the saline-treated rats, subsequent injections of PACAP-27 produced progressively smaller vasodilator and hypotensive responses. The responses produced by injections 2-5 of PACAP-27 were smaller than those produced by injection 1 (P < 0.05 for all comparisons).


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  		Fig. 2.   Summary of maximal falls in mesenteric vascular resistance (MR) produced by 5 injections of PACAP-27 (2 nmol/kg iv) in saline-treated rats (A; n = 5) and L-NAME (B; 50 µmol/kg iv)-treated rats (n = 5). Falls in mean arterial blood pressure (MAP) at point of maximal fall in MR are also shown. Data are expressed as means ± SE of percent changes from resting values. * P < 0.05, 2nd-5th injections vs. 1st injection of PACAP-27. dagger  P < 0.05 1st injection of PACAP-27 in L-NAME-treated rats vs. 1st injection of PACAP-27 in saline-treated rats.

Hemodynamic effects of 8-CPT-cAMP in L-NAME-treated rats that received five successive doses of saline or PACAP-27. The maximal falls in HQR and MR produced by 8-CPT-cAMP (5-15 µg/kg iv) in L-NAME (50 µmol/kg iv)-treated rats that received five injections of saline (n = 5) or five injections of PACAP-27 (2 nmol/kg iv) are summarized in Fig. 3. The falls in MAP at the point of maximal hindquarter and mesenteric vasodilation are also shown. 8-CPT-cAMP produced dose-dependent falls in HQR and MR that were associated with dose-dependent falls in MAP in both groups of L-NAME-treated rats. These 8-CPT-cAMP-induced responses were similar in both groups (P > 0.05 for all comparisons).


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  		Fig. 3.   Summary of maximal falls in HQR (A) and MR (B) produced by 8-(4-chlorophenylthiol)-cAMP (8-CPT-cAMP; 5-15 µmol/kg iv) in L-NAME (50 µmol/kg iv)-treated rats (n = 5) that received 5 injections of saline (post-L-NAME + saline) and in L-NAME (50 µmol/kg iv)-treated rats (n = 5) that received five injections of PACAP-27 (2 nmol/kg iv; post-L-NAME + PACAP-27). Falls in MAP at point of maximal falls in HQR or MR are also shown. Data are expressed as means ± SE of percent changes from resting values. Note that each injection of 8-CPT-cAMP produced significant falls in MAP, HQR, and MR in both groups (P < 0.05 for all comparisons). Also note that there were no between group differences in effects of 8-CPT-cAMP at the P < 0.05 level.

Baseline hemodynamic values in saline-treated rats that received five successive injections of PACAP-27. The resting hemodynamic values of the saline-treated rats that received five injections of PACAP-27 (2 nmol/kg iv) are summarized in Table 1. Saline did not affect resting MAP (0 ± 1%, P > 0.05), HQR (-2 ± 2%, P > 0.05), or MR (2 ± 2%, P > 0.05). Resting MAP and MR remained constant over the time in which the injections of PACAP-27 were given. That is, MAP and MR returned to preinjection values after recovery from the hypotensive and vasodilator effects of each injection of PACAP-27 (P > 0.05 for all comparisons). Resting HQR returned to lower resting levels after recovery from injection 3 of PACAP-27 (preinjection 4 value, -24 ± 7% of postsaline values, P < 0.05) and remained at these levels after recovery from injection 4 (preinjection 5 value, -18 ± 6% of postsaline values, P < 0.05).

                              
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  		Table 1.   Resting hemodynamic parameters before each injection of PACAP-27

Baseline hemodynamic values in L-NAME-treated rats that received five successive injections of saline or PACAP-27 and then injections of 8-CPT-cAMP. The resting MAP, HQR, and MR values in the two groups of L-NAME (50 µmol/kg iv)-treated rats that received five injections of saline or PACAP-27 (2 nmol/kg iv) followed by injections of 8-CPT-cAMP (5-15 µmol/kg iv) are summarized in Table 2. The preinjection values were similar in the two groups of rats (P > 0.05 for all comparisons). L-NAME increased resting MAP in rats that received injections 1-5 of saline (24 ± 5%, P < 0.05) and in those that received injections 1-5 of PACAP-27 (21 ± 4%, P < 0.05). These increases were similar in both groups (P > 0.05). Resting MAP remained constant over the time injections 1-5 of saline or PACAP-27 were given and over the time the three injections of 8-CPT-cAMP were administered (P > 0.05 for all comparisons). More specifically, these values returned to preinjection values after recovery from the hypotensive effects of each injection of PACAP-27 or 8-CPT-cAMP.

                              
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  		Table 2.   Resting hemodynamic parameters before injections of saline or PACAP-27 followed by 8-CPT-cAMP in L-NAME-treated rats

The preinjection HQR values were similar in these two groups of rats (P > 0.05). L-NAME increased resting HQR in rats that received injections 1-5 of saline (124 ± 17%, P < 0.05) and in rats that received injections 1-5 of PACAP-27 (136 ± 21%, P < 0.05). These increases were similar in both groups (P > 0.05). Resting HQR remained constant over the time injections 1-5 of saline were administered (P > 0.05 for all comparisons). Baseline HQR returned to lower values after recovery from injection 2 of 8-CPT-cAMP (in Table 2, see rows designated third pre-8-CPT-cAMP injection value, P < 0.05). Resting HQR returned to lower values after recovery from the vasodilator effects of injections 3-5 of PACAP-27 (in Table 2, see rows designated pre-fourth and -fifth PACAP-27 injections and the row designated first pre-8-CPT-cAMP injection, P < 0.05 for all comparisons). The values before each injection of 8-CPT-cAMP were similar to one another (P > 0.05 for all comparisons). In addition, all pre-PACAP-27 and pre-8-CPT-cAMP injection values were higher than the pre-L-NAME-values (P < 0.05 for all comparisons).

The preinjection MR values were similar in these two groups of rats (P > 0.05). L-NAME increased resting MR in the rats that received injections 1-5 of saline (208 ± 34%, P < 0.05) and in rats that received injections 1-5 of PACAP-27 (192 ± 24%, P < 0.05). These L-NAME-induced responses were similar in both groups (P > 0.05). The resting MR remained constant over the time injections 1-5 of saline were given (P > 0.05 for all comparisons). In these rats, MR returned to a lower resting value after recovery from the vasodilator effects of the second (10 µg/kg) dose of 8-CPT-cAMP (P < 0.05, pre-third dose of 8-CPT-cAMP vs. pre-first dose of 8-CPT-cAMP) and remained at these levels before the injection of the third dose (15 µg/kg) of 8-CPT-cAMP. In contrast, resting MR returned to higher values after recovery from the vasodilator effects of injections 2-5 of PACAP-27 (in Table 2, see rows designated third-fifth pre-PACAP-27 injections and the row designated pre-first-8-CPT-cAMP injection, P < 0.05 for all comparisons to first pre-PACAP-27). In these rats, resting MR returned to lower values after recovery from the vasodilator effects of injections 1-2 of 8-CPT-cAMP (in Table 2, see rows designated second and third pre-8-CPT-cAMP injections, P < 0.05 for both comparisons to first pre-8-CPT-cAMP value). In addition, all pre-PACAP-27 and pre-8-CPT-cAMP injection values were higher than the pre-L-NAME-values (P > 0.05 for all comparisons).

Maximal hemodynamic effects produced by five consecutive injections of 8-CPT-cAMP in saline- and L-NAME-treated rats. A summary of the maximal falls in HQR produced by five successive injections of 8-CPT-cAMP (10 µmol/kg iv) in saline- or L-NAME-treated rats is shown in Fig. 4. The falls in MAP at the time of the maximal falls in HQR are also shown. Injection 1 of 8-CPT-cAMP in saline-treated rats produced a pronounced vasodilator response and a fall in MAP (P < 0.05 for both responses). Each subsequent injection of 8-CPT-cAMP produced similar hemodynamic responses (P > 0.05 for all between injection comparisons). Injection 1 of 8-CPT-cAMP in L-NAME-treated rats produced a pronounced vasodilator response and a fall in MAP (P < 0.05 for both responses). These responses were similar to those in saline-treated rats (P > 0.05 for both comparisons). The subsequent injections of 8-CPT-cAMP in L-NAME-treated rats produced similar vasodilator responses to those produced by the first injection (P < 0.05 for all comparisons). These responses were similar to those observed in the saline-treated rats (P < 0.05 for all comparisons). However, the falls in MAP associated with injections 3-5 of 8-CPT-cAMP in L-NAME-treated rats were larger than those produced by injection 1 (P < 0.05 for all comparisons). Nevertheless, each injection of 8-CPT-cAMP produced similar falls in MAP in comparison to saline-treated rats (P > 0.05 for all comparisons).


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  		Fig. 4.   Summary of maximal falls in HQR produced by 5 injections of 8-CPT-cAMP (10 µmol/kg iv) in saline-treated rats (n = 5) and L-NAME (50 µmol/kg iv)-treated rats (n = 5). Falls in MAP at point of maximal fall in HQR are also shown. Data are expressed as means ± SE of percent changes from resting values. * P < 0.05, 3rd-5th injections vs. 1st injection of 8-CPT-cAMP. Note that there were no differences in responses produced by 8-CPT-cAMP in saline-treated animals compared with L-NAME-treated rats at P < 0.05 level.

A summary of the maximal falls in MR produced by five successive doses of 8-CPT-cAMP (10 µmol/kg iv) in saline- or L-NAME-treated rats is shown in Fig. 5. The falls in MAP at the time of the maximal falls in MR are also shown. Injection 1 of 8-CPT-cAMP in saline-treated rats produced a pronounced vasodilator response and a fall in MAP (P < 0.05 for both responses). Each subsequent injection of 8-CPT-cAMP produced similar responses (P > 0.05 for all between injection comparisons). Injection 1 of 8-CPT-cAMP in L-NAME-treated rats produced a pronounced vasodilator response and a fall in MAP (P < 0.05 for both responses). The maximal falls in MR produced by each injection of 8-CPT-cAMP were greater than those in saline-treated rats (P > 0.05 for all comparisons). The falls in MAP produced by injections 1-4 of CPT-cAMP in L-NAME-treated rats were similar to those in saline-treated rats (P > 0.05 for all comparisons). However, the fall in MAP produced by injection 5 of 8-CPT-cAMP in L-NAME-treated rats was greater than that in saline-treated rats (P < 0.05).


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  		Fig. 5.   Summary of maximal falls in MR produced by 5 injections of 8-CPT-cAMP (10 µmol/kg iv) in saline-treated rats (n = 5) and L-NAME (50 µmol/kg iv)-treated rats (n = 5). Falls in MAP at point of maximal fall in MR are also shown. Data are expressed as means ± SE of percent changes from resting values. * P < 0.05, 5th injection vs. 1st injection of 8-CPT-cAMP. dagger  P < 0.05, responses in L-NAME-treated-rats vs. saline-treated rats.

Resting hemodynamic parameters over the course of administration of five consecutive injections of 8-CPT-cAMP in saline- and L-NAME-treated rats. Resting MAP, HQR, and MR values before the injection of saline or L-NAME (50 µmol/kg iv) and before each injection of the five injections of 8-CPT-cAMP (10 µmol/kg iv) are summarized in Table 3. Saline did not affect MAP (0 ± 2%, pre- vs. postsaline, P > 0.05). Resting MAP returned to preinjection levels after recovery from the hypotensive effects of injection 1 of 8-CPT-cAMP (pre-second injection of 8-CPT-cAMP, -3 ± 2% of pre-first injection/postsaline levels, P > 0.05). However, baseline MAP returned to lower resting values after recovery from the hypotensive effects of injection 2 of 8-CPT-cAMP (pre-third injection of 8-CPT-cAMP, -8 ± 2% of postsaline levels, P < 0.05). The resting MAP values remained at these lower values after recovery from injection 3 (pre-fourth injection of 8-CPT-cAMP, -8 ± 3% of postsaline levels, P < 0.05) and injection 4 (pre-fifth injection of 8-CPT-cAMP, -11 ± 3% of postsaline levels, P < 0.05) of 8-CPT-cAMP. L-NAME increased resting MAP (26 ± 6%, pre- vs. post-L-NAME, P < 0.05). Unlike the saline-treated rats, baseline MAP in the L-NAME-treated rats returned to original baseline levels after recovery from injections 1-4 of 8-CPT-cAMP (P > 0.05, for all comparisons to postsaline levels).

                              
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  		Table 3.   Resting hemodynamic parameters before each injection of 8-CPT-cAMP

Saline did not affect HQR (1 ± 2%, pre- vs. postsaline, P > 0.05). Resting HQR returned to preinjection levels after recovery from the vasodilator effects of injections 1-3 of 8-CPT-cAMP (P > 0.05, for all comparisons to postsaline levels). Resting HQR returned to a lower value than postsaline values after recovery from the vasodilator effects of injection 4 of 8-CPT-cAMP (pre-fifth injection of 8-CPT-cAMP, -12 ± 2% of first injection/postsaline values, P < 0.05). L-NAME increased resting HQR (140 ± 17%, pre- vs. post- L-NAME, P < 0.05). Resting HQR returned to preinjection levels after recovery from the vasodilator effects of injections 1-3 of 8-CPT-cAMP (P > 0.05, for all comparisons to postsaline levels). HQR returned to a lower value than the postsaline value after recovery from the vasodilator effects of injection 4 of 8-CPT-cAMP (pre-fifth injection of 8-CPT-cAMP, -11 ± 3% of first injection/postsaline values, P < 0.05). However, this value was still higher than pre-L-NAME values (84 ± 14%, P < 0.05).

Saline did not affect MR (14 ± 8%, pre- vs. postsaline, P > 0.05). Resting MR returned to the original values after recovery from the vasodilator effects of injections 1-4 of 8-CPT-cAMP (P > 0.05, for all comparisons). L-NAME increased resting MR (162 ± 23%, pre- vs. post-L-NAME, P < 0.05). Resting MR returned to preinjection levels after recovery from the vasodilator effects of injections 1-2 of 8-CPT-cAMP (P > 0.05, for both comparisons to postsaline levels). Resting MR in L-NAME-treated rats returned to lower baseline values after recovery from the vasodilator effects of injection 3 of 8-CPT-cAMP (pre-fourth injection of 8-CPT-cAMP, -17 ± 3% of post-L-NAME values, P < 0.05) and was still evident after recovery from injection 4 (pre-fifth injection of 8-CPT-cAMP, -25 ± 5% of post-L-NAME values, P < 0.05). However, these values were still higher than pre-L-NAME values (P < 0.05 for all comparisons).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Successive injections of PACAP-27 (2 nmol/kg iv) produced similar hypotensive and vasodilator responses in saline-treated pentobarbital sodium-anesthetized rats, whereas tachyphylaxis associated with the hemodynamic effects of PACAP-27 in pentobarbital sodium-anesthetized L-NAME-treated rats developed rapidly. The above findings are similar to those observed in urethan-anesthetized rats (28, 31). This suggests that the development of tachyphylaxis to PACAP-27 in L-NAME-treated rats is not due to the presence of a particular anesthetic. The tachyphylaxis to PACAP-27 in L-NAME-treated rats may be due to the desensitization of PACAP receptors. However, it is possible that the PACAP receptor is fully able to activate AC and it is the loss of potency of cAMP that underlies the diminished responses to PACAP-27 in L-NAME-treated rats. The loss of cAMP signaling may be due to 1) enhanced degradation of cAMP generated by activation of the Gs protein-coupled receptor, 2) diminished capacity of cAMP to activate PKA, or 3) diminished capacity of activated PKA to exert its relaxant effects in VSM. Falls in MAP are due to the sum of the falls in resistances in all vascular beds. The maximal PACAP-27-induced falls in MR occurred several seconds before the maximal falls in HQR. The falls in MAP associated with the maximal PACAP-27-induced falls in MR and presumably in other vascular beds declined in parallel with the falls in MR in L-NAME-treated rats. The falls in MAP associated with the maximal PACAP-27-induced falls in HQR in L-NAME-treated rats declined very rapidly, whereas the falls in HQR declined relatively slowly. The rapid loss of PACAP-27-induced vasodilation in the mesenteric and other peripheral circulations would contribute to the rapid loss of PACAP-27-mediated falls in MAP despite the gradual falls in HQR. At present, we have no data to explain why PACAP-27-mediated vasodilation declines more slowly than in other vascular beds. It is possible that the density of PACAP-27 receptors is higher in the hindquarter bed than in other vascular beds. This would require more exposure to PACAP-27 before enough PACAP receptors are desensitized such that a loss of response could be observed.

One principal finding of this study was that the membrane-permeable cAMP analog 8-CPT-cAMP (5-15 µmol/kg iv) produced dose-dependent hypotensive and vasodilator actions in L-NAME-treated rats and that these responses were similar in L-NAME-treated PACAP-27-tolerant rats. These findings suggest that tachyphylaxis to PACAP-27 was not due to the loss of potency of cAMP. Accordingly, the tachyphylaxis associated with the hemodynamic effects of PACAP-27 may involve the diminished capacity of Gs protein-coupled PACAP receptors to activate AC. The resting baseline HQR values returned to slightly lower values after recovery from the vasodilator effects of the later injections of PACAP-27 in the L-NAME-treated rats, whereas the resting MR values rose slightly after recovery from the vasodilator effects of PACAP-27 in these rats. Although these changes in resting baseline resistances may have contributed to the development of tachyphylaxis to PACAP-27, this tachyphylaxis occurred before any significant changes in resting vascular resistances were observed. The hemodynamic effects of the Gs protein-coupled beta -adrenoceptor agonist isoproterenol are markedly diminished in L-NAME-treated PACAP-27-tolerant rats (28). It is possible that the gradual increase in resting MR is due to a reduction in vasodilator influence of neurogenically derived PACAP-27 or catecholamines in this bed.

The first injection of 8-CPT-cAMP (10 µmol/kg iv) produced pronounced vasodilator responses in the mesenteric and hindquarter beds of saline-treated rats. The hindquarter vasodilation produced by the first injection of this dose of 8-CPT-cAMP in L-NAME-treated rats was similar to that in saline-treated rats. In contrast, the mesenteric vasodilation was somewhat greater than that in saline-treated rats. These results suggest that the vasodilator effects of 8-CPT-cAMP are mediated by direct actions in VSM rather than by the release of endothelium-derived relaxing factors. In addition, it appears that the vasorelaxant effects of 8-CPT-cAMP are exaggerated in the mesenteric bed after inhibition of NO synthesis. This would suggest that nitrosyl factors inhibit the mechanisms by which cAMP relaxes VSM in this bed.

Another important finding of this study was that the hemodynamic effects of five successive injections of 8-CPT-cAMP (10 µmol/kg iv) were not subject to tachyphylaxis in saline- or L-NAME-treated rats. This suggests that the vasorelaxant potency of cAMP in VSM does not change as a result of the absence of exposure to endothelium-derived nitrosyl factors. More specifically, it appears that the loss of these nitrosyl factors does not result in the progressive diminution in cAMP-mediated activation of PKA or PKA-mediated relaxation of VSM. These findings further support the possibility that tachyphylaxis to PACAP-27 in L-NAME-treated rats is due to the reduced PACAP receptor-mediated generation of cAMP rather than to the diminished vasodilator potency of the cyclic nucleotide. The resting baseline HQR and MR values returned to slightly lower values after recovery from the vasodilator effects of the later injections of 8-CPT-cAMP in the saline- and L-NAME-treated rats. It therefore appears that that our injection protocol did not allow sufficient time for the hemodynamic effects of the later injections of 8-CPT-cAMP to completely subside before the subsequent injections were given. This suggests that the vasodilator effects of the later injections of 8-CPT-cAMP were superimposed on the residual effects of the preceding injections. Nevertheless, the percentages of decrease in HQR and MR produced by each injection of 8-CPT-cAMP were similar to one another.

It is tempting to assume that tachyphylaxis to PACAP-27 in L-NAME-treated rats may be due to the desensitization of PACAP receptors. As mentioned, tachyphylaxis associated with the hemodynamic effects of PACAP-27 did not occur in saline-treated rats. However, these findings do not preclude the possibility that PACAP-27 desensitizes PACAP receptors or their signal transduction processes. The rapid desensitization of Gs protein-coupled beta -adrenoceptors in reconstituted membrane preparations is due to the phosphorylation of these receptors by PKA and GRKs (9, 19, 22). The phosphorylated receptor is unable to couple to Gs proteins and activate AC (9, 19, 22). The desensitized receptor is sequestered and then dephosphorylated by phosphatases before reincorporation into the membrane (9). At present, there is no evidence that PACAP receptors are phosphorylated by PKA or GRKs, although these receptors contain amino acids that may be subject to phosphorylation (24). The lack of tachyphylaxis to PACAP-27 in saline-treated rats may be because 1) PACAP-27 does not desensitize PACAP-sensitive 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) the receptors desensitized by PACAP-27 are rapidly dephosphorylated and reincorporated in the plasma membranes such that the next injection of PACAP-27 has a full complement of receptors to activate. The tachyphylaxis to PACAP-27 in L-NAME-treated rats is prevented by the administration of the S-nitrosothiol L-SNC before each injection of PACAP-27 (31). Although S-nitrosothiols increase cGMP levels in VSM (26), the systemic administration of the NO donor sodium nitroprusside or the membrane-permeable cGMP analog 8-CPT-cGMP did not prevent tachyphylaxis to PACAP-27 in L-NAME-treated rats (31). This suggests that L-SNC may prevent tachyphylaxis to PACAP-27 by cGMP-independent mechanisms (31). On the basis of existing knowledge (11, 14, 25), it is possible that L-SNC prevents tachyphylaxis to PACAP-27 by nitrosating elements in the PACAP receptor signal transduction cascade.

Perspectives

This study provides important evidence that tachyphylaxis to PACAP-27 in L-NAME-treated rats is not due to the loss of biological potency of cAMP in VSM. It is possible that tachyphylaxis to PACAP-27 in L-NAME-treated rats may be due to 1) the PKA- or GRK-mediated phosphorylation of PACAP receptors that prevents the receptor from coupling to Gs proteins, 2) the inability of Gs proteins to activate AC, or 3) diminished resensitization of the phosphorylated PACAP-27 receptors. Moreover, our results (28, 31) suggest that endothelium-derived (3) and neurogenically derived (4-6, 20) nitrosyl factors may prevent tachyphylaxis to PACAP-27 by cGMP-independent mechanisms. It is possible that the ability of S-nitrosothiols such as L-SNC to prevent tachyphylaxis to PACAP-27 may be due to their capacity to nitrosate amino acids or to activate stereoselective recognition sites in the vasculature (7, 26, 27). The present findings raise the possibility that other Gs protein-coupled receptors may rapidly desensitize after inhibition of NO synthesis. We have not examined whether PACAP-38 or vasoactive intestinal polypeptide, which exert their effects by activation of type I and type II PACAP receptors, respectively (1, 2), are subject to tachyphylaxis in L-NAME-treated rats. However, we have obtained preliminary evidence that the vasodilator actions of the Gs protein-coupled receptor agonist isoproterenol are subject to rapid development of tachyphylaxis after administration of L-NAME (unpublished observations). These findings suggest that endothelium-derived nitrosyl factors may regulate the function of a variety of Gs protein-coupled receptors. Disease states such as diabetes are associated with compromised endothelial cell function and a loss of Gs protein-coupled receptor-mediated vasodilation (21). Our findings raise the possibility that the loss of vasodilator potency of Gs protein-coupled receptor agonists in diabetes may involve the loss of PACAP receptor signal transduction due to the absence of endothelium-derived nitrosyl factors.


    ACKNOWLEDGEMENTS

We acknowledge the expert assistance of Mike Burcham in the preparation of the figures.


    FOOTNOTES

This work was supported in part by National Institutes of Health Grants HL-14388, HL-57472, and DK-54759; National Aeronautics and Space Administration NAG5-6171; and 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 15 March 1999; accepted in final form 1 July 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Am J Physiol Regul Integr Compar Physiol 277(5):R1453-R1461
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society


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