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. 2012 Oct 19;303(12):H1418–H1425. doi: 10.1152/ajpheart.00211.2012

Melatonin inhibits nitric oxide signaling by increasing PDE5 phosphorylation in coronary arteries

Praveen Shukla 1, Chengwen Sun 1, Stephen T O'Rourke 1,✉
PMCID: PMC3532536  PMID: 23086989

Abstract

Melatonin inhibits nitric oxide (NO)-induced relaxation of coronary arteries. We tested the hypothesis that melatonin increases the phosphorylation of phosphodiesterase 5 (PDE5), which increases the activity of the enzyme and thereby decreases intracellular cGMP accumulation in response to NO and inhibits NO-induced relaxation. Sodium nitroprusside (SNP) and 8-Br-cGMP caused concentration-dependent relaxation of isolated coronary arteries suspended in organ chambers for isometric tension recording. In the presence of melatonin, the concentration-response curve to SNP, but not 8-Br-cGMP, was shifted to the right. The effect of melatonin on SNP-induced relaxation was abolished in the presence of the PDE5 inhibitors zaprinast and sildenafil. Melatonin markedly inhibited the SNP-induced increase in intracellular cGMP in coronary arteries, an effect that was also abolished by zaprinast. Treatment of coronary arteries with melatonin caused a nearly fourfold increase in the phosphorylation of PDE5, which increased the catalytic activity of the enzyme and thereby increased the degradation of cGMP to inactive 5′-GMP. Melatonin-induced PDE5 phosphorylation was markedly attenuated in the presence of the PKG1 inhibitors DT-2 or Rp-8-Br-PET-cGMPS and in those arteries in which PKG1 expression was first downregulated by 24-h incubation with SNP before exposure to melatonin. The selective MT2 receptor antagonist 4-phenyl-2-propionamidotetralin completely blocked the stimulatory effect of melatonin on PDE5 phosphorylation as well as the inhibitory effect of melatonin on SNP-induced relaxation and intracellular cGMP. Thus, in coronary arteries, melatonin acts via MT2 receptors and PKG1 to increase PDE5 phosphorylation, resulting in decreased cGMP accumulation in response to NO and impaired NO-induced vasorelaxation.

Keywords: cGMP, melatonin MT2 receptors, protein kinase G, smooth muscle relaxation, phosphodiesterase 5

an increasing body of evidence supports a role for melatonin in the local regulation of vascular tone (33, 43); however, the mechanisms underlying the vasomotor effects of the hormone are not yet clear. Melatonin has both direct and indirect effects on the vasculature and may cause either vasoconstriction or vasodilation depending on the origin of the blood vessel under investigation. For example, melatonin directly contracts rat cerebral arteries (9, 25, 56), whereas in isolated caudal arteries of the rat, melatonin has no direct action but potentiates contractile responses induced by other vasoconstrictors (18, 24, 32, 35, 52, 55). Conversely, melatonin dilates the rat and rabbit aorta, iliac, renal, and basilar arteries (49, 57). In humans, melatonin increases blood flow in certain vascular beds (e.g., forearm) while decreasing flow in others (e.g., renal) (10). The vasomotor effects of melatonin are mediated primarily via the activation of two distinct receptor subtypes, termed MT1 and MT2 (16), which are present in the vasculature. Thus, the heterogeneity in responses to melatonin may be dependent, in part, on the relative distribution and function of specific melatonin receptor subtypes in individual blood vessels.

MT2 receptors are present in human coronary arteries, and, although their function is unknown, MT2 receptor expression is altered in patients with coronary artery disease (17). In porcine coronary arteries, melatonin impairs vascular smooth muscle relaxation in response to nitric oxide (NO) (53, 59). This inhibitory effect of melatonin on NO-induced vasorelaxation is mediated by activation of MT2 melatonerigic receptors, which are expressed in coronary vascular smooth muscle cells (53). Since it is widely held that NO-induced relaxation of vascular smooth muscle results from increased intracellular accumulation of cGMP, which, in turn, activates downstream signaling events that lead to relaxation (28, 29, 34), interference with the NO/cGMP signaling cascade may provide a link by which MT2 receptor activation is coupled to impaired NO-induced responses in coronary arteries.

Intracellular levels of cGMP are tightly regulated by the opposing activities of guanylyl cyclase and cGMP-specific phosphodiesterase (PDE) (22). There are 11 different families of PDE enzymes, with multiple isoforms within each family, including PDE5, which is the major cGMP-hydrolyzing enzyme expressed in most smooth muscle cells (3). PDE5 is highly specific for cGMP and plays a pivotal role in NO/cGMP signaling in vascular smooth muscle cells (3, 37, 39). A net decrease in intracellular cGMP levels may result from increased degradation by PDE (22); thus, increased PDE5 activity represents a potential mechanism by which melatonin could inhibit responses to NO. In vascular smooth muscles cells, PDE5 activity is primarily regulated by its phosphorylation status, whereby phosphorylation of PDE5 at Ser92 markedly enhances the catalytic activity of the enzyme (12, 37, 46). At present, the role of PDE5, if any, in the modulation of cGMP levels by melatonin in vascular smooth muscle is not known. Therefore, we tested the hypothesis that melatonin increases the phosphorylation of PDE5, which increases the activity of the enzyme and thereby inhibits intracellular cGMP accumulation in response to NO and inhibition of NO-induced relaxation.

MATERIALS AND METHODS

Materials

The following drugs were used: bradykinin, DT-2, melatonin, Rp-8-Br-PET-cGMPS, sildenafil, and sodium nitroprusside (SNP) (Sigma Chemical, St. Louis, MO); 4-phenyl-2-propionamidotetralin (4P-PDOT), 8-Br-cGMP, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), and zaprinast (Tocris, Ellisville, MO); and 9,11-dideoxy-11α,9α-epoxymethano-PGF2α (U-46619) (Cayman Chemical, Ann Arbor, MI). Drug solutions were prepared daily, kept on ice, and protected from light until used. All drugs were dissolved initially in double-distilled water (DDW) with the exception of melatonin and 4P-PDOT, which were dissolved in ethanol, and sildenafil and zaprinast, which were dissolved in DMSO, before further dilution in distilled water. Drugs were added to the organ chambers in volumes not greater than 0.2 ml. Drug concentrations are reported as final molar concentrations in the organ chamber. The composition of physiological salt solution (PSS) was as follows (in mM): 118.3 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25.0 NaHCO3, and 11.1 glucose.

Methods

Tissue preparation.

Fresh porcine hearts were obtained from a local abattoir and were immediately immersed in cold PSS. After transfer to the laboratory, the left anterior descending coronary artery was dissected free from the surrounding myocardium, cleaned of adherent fat and connective tissue, and cut into rings of 4–5 mm in length. Experiments were performed in rings denuded of the endothelium, which was removed by gently rubbing the intimal surface with a fine forceps, to eliminate any potential confounding effects of vasoactive mediators released from endothelial cells and since we have previously shown that the inhibitory effect of melatonin on NO-induced relaxation occurs at the level of the vascular smooth muscle (53, 59). Four to eight coronary arterial rings were prepared from each heart.

Organ chamber experiments.

Coronary arterial rings were suspended in water-jacketed organ chambers filled with 25 ml PSS, as previously described (59). The organ chamber solution was aerated with a mixture of 95% O2-5% CO2, and the temperature was maintained at 37°C throughout the experiment. Each ring was suspended by means of two fine stainless steel wire clips passed through the lumen; one clip was anchored inside the organ chamber, and the other was connected to a force transducer (model FT03, Grass Instrument, Quincy, MA). Isometric tension was measured and recorded on a Grass polygraph. Tissues were stretched progressively to the optimal point of their length-tension relationship, using KCl (20 mM) to generate a standard contractile response. After this procedure, preparations were equilibrated at their optimal length for at least 30 min before further exposure to any vasoactive substances. Removal of the endothelium was confirmed in each preparation by the absence of relaxation to the endothelium-dependent vasodilator bradykinin (10−7 M).

Relaxation of coronary arteries was studied in rings contracted with U-46619 (3 × 10−9 M), a thromboxane A2 mimetic. After the U-46619-induced contraction had reached a stable plateau, relaxation responses to increasing concentrations of SNP (10−9–10−5 M) or 8-Br-cGMP (10−9–10−4 M) were obtained in the presence or absence of melatonin (10−9–10−7 M), which was added to the organ chamber immediately before the addition of U-46619. In some experiments, preparations were incubated with either the PDE5 inhibitors zaprinast (10−5 M) or sildenafil (10−7 M) or the MT2 receptor antagonist 4P-PDOT (10−7 M) for 30 min before exposure to melatonin. These inhibitors remained in contact with the tissues throughout the remainder of the experiment.

cGMP estimation.

ELISA was used to measure cGMP levels in porcine coronary arteries. Coronary arterial rings were suspended in water-jacketed organ chambers filled with 25 ml PSS and allowed to equilibrate for at least 1 h at 37°C. After the equilibration period, coronary artery rings were treated with SNP (5 min, 10−5 M) in the absence or presence of melatonin (5 min, 10−7 M). In some experiments, rings were pretreated for 20 min with zaprinast (10−5 M) or 4P-PDOT (10−7 M) before being exposed to melatonin and SNP. After drug treatments, rings were frozen in liquid nitrogen and homogenized with an IKA Ultra, Turrax-T8 homogenizer (IKA Works, Wilmington, NC) at 4°C in 0.1 N hydrochloric acid. Tissue homogenates were centrifuged for 10 min at ≥600 g. cGMP and total protein content were determined in the supernatant as per the direct cGMP enzyme immunoassay kit (Assay Design, Ann Arbor, MI) and a Bio-Rad Dc protein assay kit (Bio-Rad Laboratories, Hercules, CA), respectively. cGMP levels are expressed as picomoles per microgram of protein.

PKG1 downregulation.

Coronary arterial rings were incubated for 24 h in serum-free DMEM (supplemented with 100 U/ml penicillin and 100 mg/ml streptomycin, 37 ± 0.5°C, 20% O2-5% CO2, pH 7.4) in the presence of solvent (DMSO or DDW) or SNP (10−5 M). In some experiments, tissues were first incubated with ODQ (10−5 M), an inhibitor of soluble guanylyl cyclase, before being exposed SNP (10−5 M). After drug treatments, rings were frozen in liquid nitrogen, and immunoblot analysis was performed using anti-PKG1 antibody as described below.

PDE5 and phospho-PDE5 protein expression.

After incubation with SNP (10−5 M) for 24 h, coronary arterial rings were repeatedly rinsed and suspended in water-jacketed organ chambers filled with 25 ml PSS and allowed to equilibrate for at least 1 h at 37°C. In control experiments, coronary arterial rings that had not been previously exposed to SNP (10−5 M) but were otherwise treated identically for the 24-h incubation period were also suspended in organ chambers for equilibration. After the equilibration period, coronary arterial rings were treated with melatonin (5 min, 10−7 M). In a separate set of experiments, coronary arterial rings were incubated for 30 min with two chemically unrelated, selective PKG inhibitors, DT2 (10−5 M) (13, 51) or Rp-8-Br-PET-cGMPS (3 × 10−5 M) (14, 45), before being exposed to melatonin (10−7 M).

After drug treatments, rings were frozen in liquid nitrogen and homogenized with an IKA Ultra, Turrax-T8 homogenizer (IKA Works) at 4°C in lysis buffer supplemented with a protease and phosphatase inhibitor cocktail (Santa Cruz Biotechnology, Santa Cruz, CA). Tissue homogenates were kept on ice for 10 min and afterward centrifuged for 10 min at 10,000 g. Supernatants were collected for protein determination and Western blot analysis. Protein concentrations were assayed using a Bio-Rad Dc Protein assay kit (Bio-Rad Laboratories). Aliquots of supernatants containing equal amounts of protein (80–200 μg) were separated on a 7% polyacrylamide gel by SDS-PAGE, and proteins were electrophoretically transferred onto a polyvinylidene difluoride membrane (Bio-Rad Laboratories). Blots were blocked with 5% nonfat dry milk in PBS (pH 7.4) and then incubated overnight at 4°C with a primary antibody specific for PKG1 (Santa Cruz Biotechnology) or PDE5 or phospho-PDE5 (FabGennix, Frisco, TX) using dilutions of 1:1,000, 1:1,000, or 1:200, respectively. Membranes were washed two times for 15 min using PBS-Tween 20 and incubated with a horseradish peroxidase-linked secondary antibody (Santa Cruz Biotechnology). To ensure equal loading, blots were analyzed for β-tubulin expression using an anti-β-tubulin antibody (Santa Cruz Biotechnology). Immunodetection was performed using an enhanced chemiluminescence light-detecting kit (Thermo Scientific, Rockford, IL). In preliminary control experiments, no immunoreactive bands were observed in samples treated with loading buffer alone or with specific blocking peptides for the primary antibodies (provided by the manufacturer). Images are representative of six different samples.

Data analysis.

Relaxation responses are expressed as percentages of the initial tension induced by U-46619. For each vasodilator, both the maximal percent relaxation and the concentration necessary to produce 50% of its own maximal response (EC50) were determined. EC50 values were converted to negative logarithms and expressed as −log molar EC50 (pD2). Results are expressed as means ± SE, and n refers to the number of animals from which blood vessels were taken. Immunoblots were analyzed to determine the density of the individual protein band and normalized with respect to the density of the corresponding PDE5 or β-tubulin protein band. Values were compared by Student's t-test for paired or unpaired observations or by ANOVA and a post hoc Bonferroni's multiple-comparison analysis to determine significance between groups, as appropriate. Values were considered to be significantly different when P < 0.05.

RESULTS

Organ Chamber Experiments

SNP (10−9–10−5 M), a NO donor (31), and 8-Br-cGMP (10−6–10−4 M), a stable, cell-permeable analog of cGMP (11, 21), each caused concentration-dependent relaxations in isolated porcine coronary artery rings contracted with the thromboxane A2 mimetic U-46619 (Fig. 1). In the presence of melatonin, the concentration-response curve to SNP (Fig. 1A), but not 8-Br-cGMP (Fig. 1B), was shifted to the right in a parallel manner. pD2 values for SNP in the absence and presence of melatonin (10−8 and 10−7 M) were 7.64 ± 0.1, 7.32 ± 0.1, and 6.64 ± 0.2, respectively (P < 0.05 vs. control), whereas melatonin (10−9 M) had no significant effect (pD2: 7.70 ± 0.1). SNP and 8-Br-cGMP each caused complete (i.e., 100%) relaxation in both untreated and melatonin-treated rings. Melatonin itself had no direct effect on resting tension or -46619-induced contraction, as previously reported (59).

Fig. 1.

Fig. 1.

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Log concentration-response curves for sodium nitroprusside (SNP; A) or 8-Br-cGMP (B) in producing relaxations of isolated porcine coronary arteries (without endothelium) in the absence and presence of melatonin (Mel). Data are expressed as percentages of the U-46619 (3 × 10−9 M)-induced increase in tension, which averaged 5.83 ± 0.6 g in control rings and did not differ significantly in rings incubated with Mel. Each point represents the mean ± SE; n = 9. *Statistically significant difference from control in the presence of Mel (P < 0.05).

Incubation of coronary arterial rings with the selective MT2 receptor antagonist 4P-PDOT (10−7 M) (5, 15) had no effect on the concentration-response curve to SNP but abolished the inhibitory effect of melatonin on SNP-induced relaxation (Fig. 2A). Moreover, in the presence of the PDE5 inhibitor zaprinast (10−5 M) (3, 37), melatonin had no effect on the SNP concentration-response curve (Fig. 2B). Similarly, in the presence of sildenafil (10−7 M) to inhibit PDE5 (2, 4), melatonin had no effect on the concentration-response curve to SNP [pD2: 7.80 ± 0.2 and 7.65 ± 0.2 for SNP in the absence and presence of melatonin (10−7 M), respectively, n = 6, P > 0.05]. The contractile response to U-46619 was unaffected by the presence of 4P-PDOT or the PDE5 inhibitors (Fig. 2).

Fig. 2.

Fig. 2.

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A: effect of Mel (10−7 M) on SNP-induced relaxation of isolated porcine coronary artery arteries (without endothelium) in the absence and presence of the selective MT2 receptor antagonist 4-phenyl-2-propionamidotetralin (4P-PDOT; 10−7 M). B: effect of Mel (10−7 M) on SNP-induced relaxation of isolated porcine coronary arteries in the presence of the phosphodiesterase 5 (PDE5) inhibitor zaprinast (Zap; 10−5 M). Data are expressed as percentages of the U-46619 (3 × 10−9 M)-induced increase in tension, which averaged 2.65 ± 0.3 g in control rings and did not differ significantly in rings incubated with either 4P-PDOT or Zap. Each point represents the mean ± SE; n = 6–8. *Statistically significant difference in the presence of Mel (P < 0.05).

cGMP Experiments

SNP produced a significant increase in intracellular cGMP levels in coronary arteries (Fig. 3). Melatonin had no effect on basal cGMP levels but markedly inhibited the SNP-induced increase in intracellular cGMP (Fig. 3). Similar to the results obtained in the vasorelaxation experiments described above, the effect of melatonin on SNP-induced increases in intracellular cGMP levels was abolished in the presence of either the PDE5 inhibitor zaprinast (10−5 M) or the MT2 receptor antagonist 4P-PDOT (10−7 M; Fig. 3).

Fig. 3.

Fig. 3.

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Effect of the PDE5 inhibitor Zap (10−5 M) and the selective MT2 receptor antagonist 4P-PDOT (10−7 M) on the SNP (10−5 M)-induced increase in cGMP levels in the presence and absence of Mel (10−7 M). cGMP levels are expressed as picomoles per microgram of protein; n = 6–7. *Statistically significant difference between groups (P < 0.05).

PDE Expression Experiments

The expression of PDE5 protein in porcine coronary arteries was determined by immunoblot analysis. A strong immunoreactive band at 95 kDa, corresponding to PDE5 (36, 42), was detected in immunoblots of coronary artery homogenates (Fig. 4A). Immunoblot analysis of coronary arteries exposed to melatonin (10−7 M) demonstrated a significant increase in PDE5 phosphorylation compared with untreated controls (Fig. 4, B and C). The MT2 receptor antagonist 4P-PDOT (10−7 M) had no effect on basal phospho-PDE5 levels but completely abolished the stimulatory effect of melatonin (10−7 M) on PDE5 phosphorylation (Fig. 4, B and C).

Fig. 4.

Fig. 4.

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A: PDE5 protein detected as an intense immunoreactive band at 95 kDa in porcine coronary artery homogenates from four different animals (lanes 1–4). B and C: effect of Mel on PDE5 phosphorylation in the presence and absence of the selective MT2 receptor antagonist 4P-PDOT (10−7 M, n = 6). C shows the immunodensity of phospho-PDE5 normalized to the corresponding PDE5 protein band immunodensity. *Statistically significant difference between groups (P < 0.05).

PKG1 Downregulation and Pharmacological Inhibition

Incubation of porcine coronary artery rings with SNP (10−5 M, 37°C in serum-free DMEM, 24 h) caused a significant decrease in PKG1 protein expression (Fig. 5). The effect of SNP on PKG1 expression was abolished by ODQ (10−5 M), a selective soluble guanylyl cyclase inhibitor (23). In control rings not subjected to PKG1 downregulation, melatonin (10−7 M) caused a significant increase in PDE5 phosphorylation (Fig. 6). The effect of melatonin on PDE5 phosphorylation was markedly attenuated in arteries that had been first treated with SNP (10−5 M, 24 h) to induce the downregulation of PKG1 (Fig. 6).

Fig. 5.

Fig. 5.

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A: effect of SNP on PKG1 protein expression in porcine coronary arteries in the presence and absence of a soluble guanylyl cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10−5 M, n = 5). Double-distilled water (DDW) and DMSO were the solvents for SNP and ODQ, respectively. B: immunodensity of PKG1 normalized to the corresponding β-tubulin protein band immunodensity. *Statistically significant difference between groups (P < 0.05).

Fig. 6.

Fig. 6.

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A: Mel-induced (10−7 M) PDE5 phosphorylation was significantly attenuated in porcine coronary arteries that had been first treated with SNP (10−5 M, 24 h) to induce the downregulation of PKG1 (n = 5). B: immunodensity of phospho-PDE5 normalized to the corresponding PDE5 protein band immunodensity. *Statistically significant difference between groups (P < 0.05).

In a separate set of experiments, the melatonin-induced increase in PDE5 phosphorylation was abolished in coronary arterial rings incubated with the PKG inhibitors DT-2 (10−5 M) or Rp-8-Br-PET-cGMPS (3 × 10−5 M) before exposure to melatonin (10−7 M; Fig. 7).

Fig. 7.

Fig. 7.

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A: Mel-induced (10−7 M) PDE5 phosphorylation was significantly attenuated in porcine coronary arteries that had been first treated with Rp-8-Br-PET-cGMPS (3 × 10−5 M, 30 min) or DT-2 (10−5 M, 30 min) to inhibit PKG1 activity in arterial rings (n = 4). B: immunodensity of phospho-PDE5 normalized to the corresponding PDE5 protein band immunodensity. *Statistically significant difference between groups (P < 0.05).

DISCUSSION

The major finding of the present study is that the cGMP-hydrolyzing enzyme PDE5 plays a key role in the inhibitory effect of melatonin on NO-induced relaxation of coronary arteries. This conclusion is supported by the observations that 1) melatonin stimulates the phosphorylation of PDE5 in coronary arteries, which markedly enhances the catalytic activity of the enzyme and thereby increases the degradation of intracellular cGMP (12, 37, 46); 2) the inhibitory effects of melatonin on NO-induced relaxation and increased intracellular cGMP accumulation are abolished by inhibition of PDE5; and 3) the effects of melatonin on PDE5 phosphorylation, cGMP accumulation, and vasorelaxation are all blocked by the selective MT2 receptor antagonist 4P-PDOT. The results further suggest that melatonin-induced phosphorylation of PDE5 may be mediated, in part, by the activation of PKG1 since incubation with selective PKG inhibitors or downregulation of the kinase significantly attenuated melatonin-induced PDE5 phosphorylation. These findings provide a novel mechanistic basis for the inhibitory action of melatonin on NO-induced vasorelaxation and provide new insights into potential mechanisms by which melatonin may regulate blood vessel diameter.

The NO/cGMP signaling pathway plays a central role in maintaining coronary vascular tone. In vascular smooth muscles cells, NO activates soluble guanylyl cyclase to stimulate the biosynthesis of cGMP, which, in turn, activates PKG (28, 29). Activated PKG phosphorylates numerous ion channels and pumps, many of which promote a reduction in cytosolic Ca2+ (8, 34) as well as membrane hyperpolarization and relaxation of arterial smooth muscle cells (30, 41, 58, 60). Impaired signaling via the NO/cGMP pathway ultimately results in inhibition of NO-induced vascular smooth muscle relaxation and is associated with several cardiovascular disorders (e.g., hypertension, vasospasm, etc.) (6, 54).

In the present study, melatonin impaired porcine coronary arterial relaxation evoked by the NO donor SNP but had no effect on the response to 8-Br-cGMP, a stable, cell-permeable analog of cGMP that increases the phosphorylation of PKG1 but is not a substrate for PDE5 (11, 21). The lack of effect of melatonin on 8-Br-cGMP-induced relaxation is consistent with the notion that melatonin increases the degradation of intracellular cGMP in response to NO, perhaps by increasing PDE5 catalytic activity. This hypothesis is further supported by the observations showing that the inhibitory effect of melatonin on SNP-induced relaxation was abolished in the presence of two different PDE5 inhibitors, sildenafil and zaprinast, and that zaprinast markedly attenuated the SNP-induced increase in intracellular cGMP levels. Since PDE5 is the major cGMP-hydrolyzing enzyme expressed in vascular smooth muscle cells (3), these data strongly suggest that the activation of PDE5 may account, at least in part, for the effect of melatonin on NO-induced relaxation, although we cannot completely exclude a role for other cGMP-hydrolyzing PDEs that may be present in vascular smooth muscle cells, e.g., PDE1.

In vascular smooth muscles cells, PDE5 activity is primarily regulated by its phosphorylation status (48, 50). Phosphorylation of PDE5 at Ser92 markedly enhances cGMP-binding affinity in the regulatory domain of PDE5 and increases the catalytic activity of the enzyme by severalfold, leading to a decrease in intracellular cGMP levels (26, 38, 47). Thus, measuring PDE5 phosphorylation is a useful surrogate marker for increased PDE5 activity in intact tissues (e.g., isolated arteries), albeit an indirect indicator. Since the inhibitory effects of melatonin on both SNP-induced relaxations and intracellular cGMP accumulation were abolished by inhibitors of PDE5, we hypothesized that an increase in PDE5 phosphorylation by melatonin may underlie the inhibitory effect on NO-induced responses. In support of this hypothesis, immunoblot analysis identified the expression of PDE5 protein in porcine coronary arteries, and exposure to melatonin indeed caused a significant increase in PDE5 phosphorylation. Moreover, PDE5 protein expression did not differ between melatonin-treated and untreated tissues, indicating that the increase in PDE5 activity is the result of a posttranslational increase in PDE5 protein phosphorylation (7). The fact that the stimulatory effect of melatonin on PDE5 phosphorylation was abolished in the presence of the MT2 receptor antagonist 4P-PDOT further supports the hypothesis that the impairment of NO-induced relaxation of porcine coronary arteries involves MT2 receptor-dependent stimulation of PDE5 phosphorylation and the ensuing decrease in intracellular cGMP accumulation in response to NO.

The exact signal transduction pathway by which MT2 receptors are coupled to PDE5 phosphorylation in coronary artery smooth muscle cells remains to be elucidated. MT2 receptors belong to the transducin family of G protein-coupled receptors; thus, one possibility is that the signal transduction pathway coupled to MT2 receptors may be similar to that in the retinal rhodopsin-transducin (Gtαβγ) system. Activation of the heterotrimeric G protein transducin (Gtαβγ) leads to its dissociation into an active GTP-bound α (GtαGTP)-subunit, which, in turn, enhances PDE activity and results in a decrease in intracellular cGMP levels (1, 40). Another possibility is that the activation of MT2 receptors by melatonin may result in the activation of an intermediate signaling molecule (e.g., kinase), which then phosphorylates and activates PDE5. In vascular smooth muscles cells, PKG is the primary enzyme that phosphorylates PDE5 (46). Indeed, PDE5 is such a selective substrate for PKG that it is considered one of the best markers for the activation of PKG (46). There are two families of PKG, i.e., PKG1 and PKG2, which are derived from separate genes, Prkg1 and Prkg2 (19). In blood vessels, PKG1 is the predominant form (19, 20, 27, 44) and is more commonly involved in NO/cGMP signaling (19). Hence, a pharmacological model of PKG1 downregulation was used to determine the role of PKG1 in the stimulatory effect of melatonin on PDE5 phosphorylation in porcine coronary arteries. Incubation of porcine coronary artery rings with SNP for 24 h caused a significant decrease in PKG1 protein expression, an effect that was abolished by the soluble guanylyl cyclase inhibitor ODQ, suggesting that elevated cGMP levels present during continuous exposure to NO suppress PKG1 expression. Notably, the melatonin-induced increase in PDE5 phosphorylation was nearly abolished in those arteries in which PKG1 expression was downregulated. When taken together with the observation that the melatonin-induced increase in PDE5 phosphorylation was also suppressed in the presence of two chemically distinct PKG inhibitors, the results support the hypothesis that PKG1 activation mediates the stimulatory effect of melatonin on PDE5 phosphorylation in porcine coronary arteries. Additional studies will be necessary to confirm this hypothesis and to clarify the molecular mechanisms involved.

In summary, the present study demonstrates that the activation of MT2 receptors increases PDE5 phosphorylation in coronary arteries, likely via the activation of PKG1, which results in the inhibition of NO-mediated increases in intracellular cGMP and smooth muscle relaxation. Although our understanding of the role of specific melatonin receptor subtypes in cardiovascular homeostasis and disease is still evolving, the present findings shed new light on the interactions between melatonin and NO signaling in the vasculature.

GRANTS

This work was supported in part by National Heart, Lung, and Blood Institute Grant HL-77204.

DISCLOSURES

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

AUTHOR CONTRIBUTIONS

Author contributions: P.S., C.S., and S.T.O. conception and design of research; P.S. performed experiments; P.S., C.S., and S.T.O. analyzed data; P.S., C.S., and S.T.O. interpreted results of experiments; P.S. prepared figures; P.S. and S.T.O. drafted manuscript; P.S. and S.T.O. edited and revised manuscript; P.S., C.S., and S.T.O. approved final version of manuscript.

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