Ghrelin-Related Peptides Exert Protective Effects in the Cerebral Circulation of Male Mice Through a Nonclassical Ghrelin Receptor(s)

Abstract

The ghrelin-related peptides, acylated ghrelin, des-acylated ghrelin, and obestatin, are novel gastrointestinal hormones. We firstly investigated whether the ghrelin gene, ghrelin O-acyltransferase, and the ghrelin receptor (GH secretagogue receptor 1a [GHSR1a]) are expressed in mouse cerebral arteries. Secondly, we assessed the cerebrovascular actions of ghrelin-related peptides by examining their effects on vasodilator nitric oxide (NO) and superoxide production. Using RT-PCR, we found the ghrelin gene and ghrelin O-acyltransferase to be expressed at negligible levels in cerebral arteries from male wild-type mice. mRNA expression of GHSR1a was also found to be low in cerebral arteries, and GHSR protein was undetectable in GHSR-enhanced green fluorescent protein mice. We next found that exogenous acylated ghrelin had no effect on the tone of perfused cerebral arteries or superoxide production. By contrast, exogenous des-acylated ghrelin or obestatin elicited powerful vasodilator responses (EC₅₀ < 10 pmol/L) that were abolished by the NO synthase inhibitor N^ω-nitro-L-arginine methyl ester. Furthermore, exogenous des-acylated ghrelin suppressed superoxide production in cerebral arteries. Consistent with our GHSR expression data, vasodilator effects of des-acylated ghrelin or obestatin were sustained in the presence of YIL-781 (GHSR1a antagonist) and in arteries from Ghsr-deficient mice. Using ghrelin-deficient (Ghrl^−/−) mice, we also found that endogenous production of ghrelin-related peptides regulates NO bioactivity and superoxide levels in the cerebral circulation. Specifically, we show that NO bioactivity was markedly reduced in Ghrl^−/− vs wild-type mice, and superoxide levels were elevated. These findings reveal protective actions of exogenous and endogenous ghrelin-related peptides in the cerebral circulation and show the existence of a novel ghrelin receptor(s) in the cerebral endothelium.

Ghrelin is a 28-amino acid peptide hormone produced primarily by the stomach and small intestine. In the plasma, ghrelin circulates in 2 distinct forms, acylated ghrelin and des-acylated ghrelin. The acylated form of ghrelin is generated through the addition of an octanoyl group to proghrelin by the enzyme ghrelin O-acyltransferase (GOAT) (1, 2). Acylated ghrelin is an orexigenic (“appetite stimulating”) hormone, and numerous studies have demonstrated its importance in stimulating GH release and food intake to regulate energy homeostasis and body weight (3). These effects are driven largely by the high expression of the only functional ghrelin receptor so far been characterized, GH secretagogue receptor 1a (GHSR1a), in the hypothalamus and pituitary gland (4, 5). Des-acylated ghrelin is the dominant form of ghrelin in the plasma but is devoid of GHSR1a binding and does not stimulate GH release (6). In fact, it has been proposed that des-acylated ghrelin might be a functional inhibitor of acylated ghrelin via a novel, yet to be identified ghrelin receptor(s) (7). More recently, the 23-amino acid peptide obestatin was identified as a product of the ghrelin gene, and like the 2 ghrelin forms, it is primarily produced by the stomach and exerts metabolic actions (8). Obestatin was originally identified as the endogenous ligand for the orphan receptor, G protein-coupled receptor 39 (8); however, this has since been disputed (9). Recent evidence suggests that obestatin may bind with the glucagon-like peptide 1 receptor (GLP-1R) to exert its metabolic actions. Indeed, competitive binding studies have shown that obestatin interacts with GLP-1R in pancreatic β-cells and adipocytes and may trigger survival and metabolic signaling pathways in these cell types through activation of GLP-1R (10, 11).

Ghrelin and GHSR1a are expressed in the cardiovascular system, including endothelial cells of systemic arteries (12, 13). Furthermore, exogenous ghrelin-related peptides exert a number of protective effects on endothelial and vascular smooth muscle (VSM) cells of systemic blood vessels, suggesting cardiovascular functions of the ghrelin system (14). For example, studies show that acylated ghrelin stimulates the production of vasoprotective nitric oxide (NO) by cultured endothelial cells and endothelial cells of intact systemic arteries (14–16) and inhibits apoptosis of cultured endothelial and VSM cells (17, 18). In the rat aorta, acylated ghrelin inhibits the activity of the prooxidant enzymes, the nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (19), which are expressed in both endothelial and VSM cells, and antagonises the vasoconstrictor effect of the renin-angiotensin system peptide, angiotension II, in cultured aortic VSM cells (20). Much less is known about the vascular effects of des-acylated ghrelin and obestatin. However, there is evidence that exogenous des-acylated ghrelin or obestatin also exert vasodilator effects on intact systemic arteries (21–23). Although the GHSR1a is expressed in numerous extrahypothalamic areas of the brain (24), it is unknown whether the “ghrelin system” (ie, ghrelin, GHSR, and GOAT) is expressed and therefore active in the cerebral vasculature. Furthermore, whether ghrelin-related peptides exert protective effects on cerebral arteries remains to be investigated. This is important to test given that recent evidence suggests that the production of these peptides may be altered in patients with stroke risk factors (eg, ageing, obesity, hypertension) and after stroke (25). Therefore, in the present study, we firstly examined whether ghrelin, GOAT, and GHSR are expressed in mouse cerebral arteries. Secondly, we investigated whether exogenous acylated ghrelin, des-acylated ghrelin, and obestatin exert protective effects on cerebral arteries by examining their effects on vasodilator NO bioactivity and superoxide production. Finally, using ghrelin-deficient mice, we evaluated the potential actions of endogenously produced ghrelin-related peptides in the cerebral circulation.

Materials and Methods

All experimental procedures were approved by the Monash University Animal Ethics Committee. Ghrelin-deficient (Ghrl^−/−) (5) and Ghsr-deficient mice (Ghsr^−/−) (26) were obtained from Regeneron Pharmaceuticals. Nox2-deficient mice (Nox2^−/−) mice (27) were originally generated in the laboratory of Professor Mary Dinauer (28). All mice were then rederived in our animal facility and then backcrossed onto C57Bl6/J mice for at least 10 generations. For studies using Ghrl^−/− and Ghsr^−/− mice, genetically related (ie, generated from same heterozygous breeding pairs) wild-type (WT) (C57Bl6/J) mice were used as controls. Genotypes were determined by PCR amplification of tail DNA. In all other experiments, WT mice (C57Bl6/J) were obtained from our own breeding colony. GHSR-enhanced green fluorescent protein (eGFP) reporter mice (26) were obtained from the Mouse Mutant Regional Resource Center at University of California at Davis. This mouse was generated by the GENSATproject at The Rockefeller University and contains a modified bacterial artificial chromosomes, in which a GFP reporter is inserted immediately upstream of the coding sequence for the Ghsr gene. Mice were identified by genotyping using forward 5′-GGACCTCCTCAGGGGACCAGAT-3′ and reverse 5′-GGTCGGGGTAGCGGCTGAA-3′ primers. All mice were male and 8–12 weeks old. In total, 105 WT (25.7 ± 0.3 g), 25 Ghrl^−/− (26.0 ± 1.8 g), 4 Ghsr^−/− (27.5 ± 2.5 g), 3 GHSR-eGFP (28 ± 2.1 g), and 3 Nox2^−/− (26 ± 2.2 g) mice were used. Mice were euthanized by inhalation of isofluorane followed by decapitation.

Real-time PCR

Purified RNA was extracted from cerebral arteries (pooled middle cerebral arteries [MCAs] and basilar arteries) of WT mice and from the stomach (positive control for ghrelin and GOAT) and pituitary gland (positive control for GHSR) of WT mice. cDNA was synthesized using the iScript cDNA Synthesis kit (number 170–8890; Bio-Rad Laboratories). Real-time quantitative PCR was then used to measure mRNA for ghrelin and GHSR1a using TaqMan Gene Expression Master Mix (Applied Biosystems) and TaqMan primers (ghrelin: forward 5′-ACCCAGAGGACAGAGGACAA-3′ and reverse 5′-TCGAAGGGAGCATTCAACCTG-3′, NM_021488.4; GHSR1a: forward 5′-GCTGCTCACCGTGATGGTAT-3′ and reverse 5′-ACCACAGCAAGCATCTTCACT-3′, NM_177330.4). To measure GOAT mRNA, we used Quantifast SYBR Green PCR Mastermix (QIAGEN) and a Quantifast SYBR Green primer (Mm_Mboat4_1_SG, catalog number QT00307069, NM_001126314). Data were normalized to ribosomal 18s and represented relative to expression levels in either the stomach (positive control for ghrelin and GOAT) or pituitary gland (GHSR1a) using the 2^−ΔΔCt method (29).

Double-label immunofluorescence

GHSR-eGFP reporter mice were deeply anesthetized with isoflurane and then perfused with 0.05 mol/L PBS, followed by 4% paraformaldehyde. Brains were removed and postfixed in 4% paraformaldehyde overnight at 4°C and placed in 30% sucrose solution. Brains were then sectioned (30 μm) using a cryostat, and sections were stored in cryoprotectant at −20°C until use. To determine whether GHSR is expressed in cerebral arteries and arterioles, double-labeled immunofluorescence was performed for eGFP and the endothelial cell marker, von Willebrand factor (vWF). Briefly, sections were washed with 0.1 mol/L PBS (3 × 10 min), followed by 1% hydrogen peroxide for 15 minutes to block endogenous peroxidase activity, and then washed with 0.1 mol/L PBS (3 × 10 min). Sections were subsequently blocked for 30 minutes with 10% goat serum and 4% horse serum and then incubated with chicken anti-eGFP (1:1000; Aves Laboratories) and rabbit anti-vWF (1:1000; Abcam) primary antibodies overnight at 4°C (Supplemental Table 1). The day after, tissues were washed with 0.1 mol/L PBS (3 × 10 min) and incubated with goat antichicken Alexa Fluor 488 (1:400; Invitrogen) and goat antirabbit Alexa Fluor 594 (1:1000; Zymed Laboratories) secondary antibodies for 1.5 hours at room temperature. Sections were then washed in 0.1 mol/L PBS (3 × 10 min), mounted, and coverslipped. Tissue-mounted slides were viewed and photographed at magnification of ×20 or ×10 with an Olympus fluorescence microscope.

Perfusion myography

Preparation of isolated cannulated MCA segments

MCAs were mounted between 2 microcannulae in a pressure myograph (Living Systems Instrumentation, Inc) as previously described (27). Arteries were constantly superfused with warm (37°C), carbogen-bubbled (95% O₂, 5% CO₂) Krebs-bicarbonate solution (118 mmol/L NaCl, 4.5 mmol/L KCl, 0.45 mmol/L MgSO₄, 1.03 mmol/L KH₂PO₄, 25 mmol/L NaHCO₃, 11.1 mmol/L glucose, and 2.5 mmol/L CaCl₂). The intraluminal pressure was gradually increased to 60 mm Hg and maintained at this level using a pressure servo unit without further intraluminal perfusion. MCAs were allowed to equilibrate for 10 minutes before baseline diameters were measured. Arteries were then exposed to a high potassium physiological saline solution (KPSS) containing 122.7 mmol/L KCl (equimolar replacement of NaCl with KCl) to induce maximal vascular contraction. MCAs were then washed with Krebs-bicarbonate solution and allowed to equilibrate for a further 10 minutes before experimental protocols were performed.

Experimental protocol

To assess the effect of exogenous ghrelin-related peptides on vasodilator NO bioactivity, MCAs from WT mice were preconstricted with the thromboxane A₂ mimetic U46619 (10 nmol/L to 1 μmol/L) to approximately 30%–40% of maximal response to KPSS. Once contractile responses were stable, cumulative doses of acylated ghrelin, des-acylated ghrelin, or obestatin (100 fmol/L to 10 nmol/L for all peptides) were applied extraluminally to MCA and responses measured after 15 minutes. In addition, “time control” experiments were performed, whereby MCAs were preconstricted with U46619, but no peptides were applied. Any changes in diameter were then recorded every 15 minutes for 1.5 hours. In some experiments, concentration-response curves to the peptides were repeated in the presence of the NO synthase (NOS) inhibitor, N^ω-nitro-L-arginine methyl ester (L-NAME) (100 μmol/L; 30-min preincubation), or the GHSR1a antagonist, YIL-781 (0.5 μmol/L; 15-min preincubation) (30). Finally, the effect of des-acylated ghrelin (100 fmol/L to 10 nmol/L) on cerebral artery tone was assessed in U46619-preconstricted MCA from Ghsr^−/− and genetically related WT mice. In all experiments, papaverine (100 μmol/L) was applied to MCA after completion of concentration-response curves to ensure maximal relaxation could be achieved. Only one concentration-response curve was performed on each MCA.

To assess the effect of endogenous ghrelin-related peptides on vasodilator NO bioactivity, we measured constrictor responses (measured from baseline diameter) to L-NAME (100 μmol/L) in MCA from Ghrl^−/− and genetically related WT mice. Inhibiting endothelial NOS (eNOS) activity with L-NAME unmasks the basal relaxant effects of NO generation in the artery wall (manifested as a contraction in response to L-NAME) and is a commonly used approach to assess NO bioactivity (31). Constrictor responses were recorded once they reached a steady level (∼45 min).

Quantification of superoxide production

All experiments were carried out using pooled MCA and basilar arteries. Superoxide production was measured using either L-012 (100 μmol/L)- or lucigenin (5 μmol/L)-enhanced chemiluminescence as previously described (32, 33). In all experiments, superoxide counts were measured for 30 minutes, background counts were then subtracted and normalized to dry tissue weight.

The effect of exogenous acylated ghrelin (10 nmol/L), des-acylated ghrelin (0.1–10 nmol/L), or obestatin (10 nmol/L) on superoxide production in cerebral arteries from WT mice was measured in response to either phorbol-12,13 dibutyrate (PDBu) (10 μmol/L) or angiotensin II (1 nmol/L). We have previously shown that both PDBu and angiotensin II increase cerebral artery superoxide production through activation of Nox2 oxidase (27, 31), which is a key enzymatic source of superoxide in cerebral arteries. Furthermore, we also examined the effect of des-acylated ghrelin (10 nmol/L) on NADPH (Nox2 oxidase substrate; 100 μmol/L)-stimulated superoxide production. Lastly, we measured the effect of des-acylated ghrelin on NADPH (100 μmol/L)-stimulated superoxide production in the presence of L-NAME (100 μmol/L; 30-min preincubation). In experiments using NADPH, we used an alternative chemiluminescent probe, lucigenin (5 μmol/L), to rule out any potential L-012 artifacts.

The effect of endogenous ghrelin-related peptides on superoxide production was assessed by measuring PDBu (10 μmol/L)-stimulated superoxide levels in cerebral arteries from Ghrl^−/− and WT mice.

Western blotting

Protein expression of eNOS and the Nox2 catalytic subunit was measured in cerebral arteries (pooled basilar and MCA) from Ghrl^−/− and genetically related WT mice using Western blotting. Spleen from WT and Nox2-deficient (Nox2^−/−) mice was used as a positive and negative control for Nox2, respectively. Anti-eNOS and anti-Nox2 mouse monoclonal antibodies were purchased from BD Biosciences (North Ryde) (Supplemental Table 1). Arteries and spleen were excised, snap frozen in liquid nitrogen, and homogenized in Laemmli buffer (25% glycerin, 12.5% β-mercaptoethanol, 7.5% sodium docedyl sulfate, 25% 1 mol/L Tris-HCl [pH 8.0], and 0.25 mg/mL bromophenol blue) over liquid nitrogen. Protein concentration was determined using the reducing agent and detergent compatible (RCDC) assay (Bio-Rad). Equal amounts of protein were loaded onto a 10% polyacrylamide gel and transferred to a nitrocellulose membrane. Membranes were blocked in 5% skim milk for 1 hour and then incubated overnight at 4°C with the appropriate primary antibody (1:1000 for eNOS and Nox2, 1:2000 for β-actin) in 5% skim milk. Membranes were then incubated with a horseradish peroxidase-conjugated antimouse IgG for 1 hour. Immunoreactive bands were detected by enhanced chemiluminescence, quantified using a ChemiDoc XRS molecular imager (Bio-Rad), and normalized to intensity of corresponding bands for β-actin.

Drugs

Human acylated ghrelin (SC1357), des-acylated ghrelin (SC1483), and obestatin (SC1509) were purchased from Polypeptide Laboratories and dissolved in saline. All subsequent dilutions were made in either Krebs-bicarbonate or Krebs-HEPES solution. U46619 (Sapphire Bioscience) was prepared at 10 mmol/L in 100% ethanol and then diluted in Krebs-bicarbonate solution. YIL-781 (Tocris Bioscience) was dissolved in 100% dimethyl sulfoxide as a 0.5 mmol/L stock solution and diluted in Krebs-bicarbonate solution such that the final concentration of dimethyl sulfoxide was less than or equal to 0.1%. All other drugs were purchased from Sigma and dissolved in either Krebs-bicarbonate or Krebs-HEPES solution.

Data analysis

All results are presented as mean ± SEM. Statistical comparisons were performed using one or two-way ANOVA with a Bonferroni multiple comparison post hoc test, or unpaired t test, as appropriate. P < .05 was considered statistically significant.

Results

Expression of ghrelin and GHSR1a in mouse cerebral arteries

Using RT-PCR, we found that ghrelin and GOAT were expressed at negligible levels in cerebral arteries from WT mice relative to levels in stomach (ghrelin: 1000-fold lower in arteries vs stomach, P < .05 [Figure 1A]; GOAT: 100-fold lower, P < .05 [Figure 1B]), whereas expression of GHSR1a was low relative to pituitary (4.5-fold lower, P < .05) (Figure 1C). Next, using GHSR-eGFP mice and double-label immunofluorescence, we identified GHSR protein in several brain areas, including the hypothalamus, hippocampus, amygdala, thalamic nuclei (Figure 1Di), and cortex (Figure 1Div). Furthermore, vWF was observed in endothelial cells of parenchymal (Figure 1Dii) and pial (Figure 1Dv) cerebral vessels. However, when these images were overlaid, the pattern of GHSR was not colocalized with vWF (Figure 1D, iii and vi), indicating the GHSR is not expressed at the protein level in cerebral vessels.

Figure 1. RT-PCR detection of ghrelin (A), GOAT (B), and GHSR1a (C) mRNA in cerebral arteries from WT mice relative to expression levels in stomach/pituitary.

Values are given as mean ± SEM (A, n = 3–5; B, n = 6–7; C, n = 5). *, P < .05 vs stomach or pituitary (unpaired t test). Also shown are representative photomicrographs (D, n = 3) showing the expression of GHSR (i, iv) or the endothelial cell marker, vWF (ii, v), in the thalamic nuclei (top panel) and cortex (bottom panel) of GHSR-eGFP mice. Merged images (iii, vi) revealed that GHSR is not colocalized with vWF. White arrows indicate pial (cortex) and parenchymal (thalamic nuclei) cerebral vessels. Magnification, ×20 (A–C) and ×10 (D–F). Scale bar, 100 μm.

Effect of exogenous ghrelin-related peptides on vasodilator NO bioactivity in mouse cerebral arteries

Baseline diameters of MCA and the level of U46619 preconstriction were similar between experimental groups (data not shown). Acylated ghrelin appeared to elicit modest constrictions of MCA from WT mice; however, responses were unaffected by the GHSR1a antagonist YIL-781 (0.5 μmol/L) (Figure 2A). Furthermore, comparable decreases in vessel diameters were observed in time control experiments (Figure 2A), indicating that acylated ghrelin does not modulate mouse cerebral artery tone. By contrast, des-acylated ghrelin (100 fmol/L to 10 nmol/L) (Figure 2B) and obestatin (100 fmol/L to 10 nmol/L) (Figure 2C) were both found to be highly potent vasodilator agents eliciting concentration-dependent dilatations, with EC₅₀ values of 1–10 pmol/L and maximal effects at approximately 1 nmol/L (R_max: des-acylated ghrelin, 70 ± 10%; obestatin, 88 ± 5%, n = 5–8). Vasodilator responses to des-acylated ghrelin were abolished in the presence of L-NAME (100 μmol/L; P < .05) (Figure 3B), and responses to obestatin were markedly attenuated (P < .05) (Figure 2C). Consistent with our expression data for GHSR1a, dilator responses to either des-acylated ghrelin or obestatin were sustained in the presence of YIL-781 (0.5 μmol/L; P > .05) (Figure 2, B and C). Furthermore, in MCA from Ghsr^−/− mice, the vasodilator responses to des-acylated ghrelin were similar to responses in arteries from genetically related WT mice (Figure 2D), indicating that the actions of des-acylated ghrelin were not mediated by the GHSR1a.

Figure 2. Cumulative concentration-response curves showing responses to acylated ghrelin (A), des-acylated ghrelin (B), or obestatin (C) in MCAs from WT mice in the absence and presence of either L-NAME (100 μmol/L) or YIL-781 (0.5 μmol/L).

Also shown are cumulative concentration-response curves to des-acylated ghrelin in MCA from Ghsr^−/− and genetically related WT mice (D). Responses are expressed as change in vessel diameter (% response to papaverine) and given as mean ± SEM (A, n = 4–6; B, n = 4–8; C, n = 4–5; D, n = 4). *, P < .05 vs control (two-way ANOVA with Bonferroni post hoc test).

Figure 3. The effect of acylated ghrelin (10 nmol/L) (A), des-acylated ghrelin (10 nmol/L) (B) and obestatin (10 nmol/L) (C) on PDBu (10 μmol/L; Nox2 activator)-stimulated superoxide production by cerebral arteries from WT mice.

Superoxide production was measured by L-012 (100 μmol/L)-enhanced chemiluminescence. All results are expressed as 10³ counts/s·mg of dry tissue weight and given as mean ± SEM (A and C, n = 5; B, n = 4). *, P < .05 vs control (paired t test).

Effect of exogenous ghrelin-related peptides on superoxide production in mouse cerebral arteries

Neither acylated ghrelin (10 nmol/L) nor obestatin (10 nmol/L) modulated superoxide production in cerebral arteries from WT mice generated in response to the Nox2 oxidase activator PDBu (10 μmol/L) (Figure 3, A and C). Des-acylated ghrelin (10 nmol/L), however, caused a concentration-dependent (0.1–10 nmol/L) attenuation of PDBu (10 μmol/L)-stimulated superoxide production (data shown for 10 nmol/L) (Figure 3B), such that at 0.1 nmol/L, 1 nmol/L, and 10 nmol/L, des-acylated ghrelin reduced superoxide production by 52%, 38%, and 35%, respectively (P < .05). We also found that des-acylated ghrelin (10 nmol/L) attenuated angiotensin II (1 nmol/L)- or NADPH (100 μmol/L)-stimulated superoxide production by 45% and 34%, respectively (P < .05) (Figure 4, A and B), and that the effect of des-acylated ghrelin on NADPH-stimulated superoxide production was sustained in the presence of L-NAME (P > .05) (Figure 4C).

Figure 4. The effect of des-acylated ghrelin (10 nmol/L) on angiotensin II (1 nmol/L, A)-stimulated superoxide production in cerebral arteries from WT mice.

Also shown is the effect of des-acylated ghrelin (10 nmol/L) on NADPH (100 μmol/L)-stimulated superoxide production in the absence (B) and presence of L-NAME (100 μmol/L) (C). All results are expressed as 10³ counts/s · mg of dry tissue weight and given as mean ± SEM (A, n = 7; B, n = 7; C, n = 8). *, P < .05 vs control (paired t test).

Vasodilator NO, superoxide production, and eNOS/Nox2 protein expression in ghrelin-deficient mice

Baseline diameters were similar between genotypes and experimental groups (WT, 94 ± 7 μm; Ghrl^−/−, 95 ± 2 μm). The magnitude of L-NAME-induced constrictions (measured from baseline) of MCA from Ghrl^−/− mice were approximately 37% lower than responses in genetically related WT mice (P < .05) (Figure 5A), whereas constrictor responses to KPSS were similar between genotypes (Figure 5A), indicative of reduced NO bioactivity and/or production. PDBu-stimulated superoxide production was approximately 62% greater in cerebral arteries from Ghrl^−/− than in WT mice (P < .05) (Figure 6A). Cerebral artery expression of eNOS and Nox2 was similar in Ghrl^−/− and WT mice (Figures 5B and 6B).

Figure 5. A, Constrictor responses to L-NAME (100 μmol/L) and KPSS (122.7 mmol/L K⁺) of isolated MCAs from Ghrl^−/− and genetically related WT mice. B, Representative Western blots showing protein expression of eNOS in cerebral artery (pooled basilar and middle cerebral) homogenates from Ghrl^−/− and WT mice, and summary of immunoreactive band intensity. Results are expressed as percent change from baseline diameter (A), or relative intensity normalized to β-actin intensity (B) and are given as mean ± SEM (A, n = 6; B, n = 3). P < .05 vs WT (unpaired t test).

Figure 6. A, PDBu (10 μmol/L; Nox2 activator) (A)-stimulated superoxide production in cerebral arteries from Ghrl^−/− and genetically related WT mice. B, Representative Western blots showing protein expression of Nox2 in cerebral artery (pooled basilar and middle cerebral) homogenates from Ghrl^−/− and WT mice, and summary of immunoreactive band intensity. Values are expressed as 10³ counts/s·mg of dry tissue weight (A), or relative intensity normalized to β-actin intensity (C), and are given as mean ± SEM (A and B, n = 6; B, n = 3). *, P < .05 vs WT; #, P < .05 vs control (one-way ANOVA with Bonferroni post hoc test).

Discussion

The major novel findings of this study are: 1) mouse cerebral arteries express negligible amounts of ghrelin and GOAT genes and low levels of the gene for GHSR1a but do not express GHSR at the protein level; 2) exogenous des-acylated ghrelin or obestatin are highly potent cerebral vasodilators, whereas acylated ghrelin has no effect; 3) the powerful vasodilator responses to des-acylated ghrelin or obestatin are dependent on the production of NO and occur independently of GHSR1a; 4) exogenous des-acylated ghrelin suppresses superoxide production, whereas neither acylated ghrelin nor obestatin has any effect; and 5) NO bioactivity is markedly reduced in cerebral arteries from ghrelin-deficient mice, and superoxide production is elevated. Collectively, these data provide the first demonstration that exogenous and endogenous ghrelin-related peptides exert protective effects on cerebral arteries and show the existence of a novel ghrelin receptor(s) in cerebral endothelial cells that are not recognized by acylated ghrelin.

GHSR1a is broadly expressed in the cardiovascular system of humans and rodents, including endothelial cells of systemic arteries (13). Moreover, ghrelin mRNA is expressed in cultured human endothelial cells (human umbilical vein endothelial cells), suggesting that ghrelin-related peptides might be synthesized by vascular cells (12). Indeed, small amounts of ghrelin have been detected in endothelial cells of human arteries (23). Taken together with their protective effects on systemic arteries, these findings suggest a role for ghrelin-related peptides as paracrine/autocrine mediators in the systemic vasculature. Conversely, very little is known about the functions of these peptides in the cerebral circulation. Thus, in our initial experiments, we investigated whether the ghrelin system is expressed in the mouse cerebral circulation. Using RT-PCR, we found that ghrelin and GOAT mRNA were expressed at negligible levels in mouse cerebral arteries relative to the stomach, indicating that ghrelin-related peptides are not produced locally. Two forms of GHSR have been identified thus far: a functional 7 transmembrane receptor, GHRS1a, and a truncated form, GHSR1b. GHSR1b was considered in the past to be functionally inactive. However, it is now believed to heterodimerize with GHSR1a and reduce its constitutive activity (34). Here, we found evidence that although GHSR1a mRNA was expressed at low levels in mouse cerebral arteries, neither GHSR form was expressed at the protein level. Indeed, using novel GHSR-eGFP reporter mice, we found abundant GHSR in several brain regions, whereas there was no evidence of protein for GHSR in either pial or parenchymal cerebral vessels. Thus, in contrast to systemic arteries but consistent with a recent study (35), these results indicate that GHSR is not expressed in cerebral arteries.

Endothelial-derived NO has a number of protective roles in the vasculature, including inhibiting vascular contraction. Recent studies show that acylated ghrelin stimulates NO production in human, bovine, and rat aortic endothelial cells using a signaling pathway involving eNOS and GHSR1a and elicits NO-dependent relaxation of intact systemic arteries in vitro (14). In addition, acylated ghrelin has been reported to elicit vasodilator effects via activation of calcium-activated potassium channels and independently of GHSR1a (22), inferring that blood vessels may express a novel, nonclassical ghrelin receptor(s) (22). Indeed, des-acylated ghrelin and obestatin also exert vasodilator effects on human and rodent systemic arteries (21–23). To our knowledge, the present study is the first to show that physiologically relevant concentrations of ghrelin-related peptides exert vasoactive effects on cerebral arteries. Specifically, we found that application of des-acylated ghrelin or obestatin to mouse MCAs in vitro caused concentration-dependent vasodilator responses, whereas acylated ghrelin had no effect on tone. Remarkably, maximum vasodilator responses to either peptide occurred at nanomolar concentrations, and EC₅₀ values were in the picomolar range. Well-characterized cerebral vasodilators, such as acetylcholine, substance P, and bradykinin, typically cause maximal dilator responses of mouse cerebral vessels in the micromolar range with nanomolar EC₅₀ values (36). Thus, des-acylated ghrelin and obestatin are highly potent cerebral vasodilators and perhaps the most potent ever described. In rat systemic arteries, the vasodilator actions of des-acylated ghrelin involve activation of endothelial calcium-activated potassium channels (22). In contrast, we found here that the NOS inhibitor L-NAME abolished responses to des-acylated ghrelin, indicating that cerebral vasodilatation is dependent on the activity of NOS and the subsequent production of NO from the endothelial lining. Thus, different signaling mechanisms contribute to the vasodilator effects of des-acylated ghrelin in cerebral vs systemic vascular beds. Obestatin has been described to cause NO-dependent relaxation of rat systemic arteries (21). Our finding that responses to obestatin were markedly attenuated by L-NAME indicates that NO also primarily mediates dilator responses in cerebral arteries. In accordance with the lack of GHSR in cerebral arteries, the vasodilator responses to both peptides were sustained in the presence of the selective GHSR1a antagonist YIL-8781. Similarly, dilator responses to des-acylated ghrelin were sustained in arteries from Ghsr-deficient mice. Thus, des-acylated ghrelin and obestatin, but not acylated ghrelin, acutely stimulate vasoprotective NO production in cerebral endothelial cells through a nonclassical ghrelin receptor(s).

There is ample evidence that low levels of reactive oxygen species (ROS) (eg, superoxide) are important signaling molecules in blood vessels (37). However, when ROS production is enhanced and/or their metabolism by antioxidants is impaired (ie, oxidative stress), ROS exert a number of damaging effects on blood vessels including inactivation of NO (38). Thus, the delicate balance between the production and scavenging of ROS determines the impact of ROS on vascular function. The NADPH oxidases are the only enzymes yet discovered with the primary function of producing superoxide (37). The Nox2-NADPH oxidase (or “Nox2 oxidase”) isoform is predominantly expressed in the endothelial cell layer of cerebral arteries and is a major source of superoxide under physiological conditions (27). Moreover, the activity of this isoform is elevated in cerebral vessels during disease and is a key contributor to the development of oxidative stress (38). In this study, we found novel evidence that exogenous des-acylated ghrelin modulates superoxide levels in cerebral vessels. Indeed, using the using the chemiluminescent probe, L-012, we demonstrate that des-acylated ghrelin markedly attenuated superoxide levels in response to 2 stimuli of Nox2 oxidase, ie, PDBu and angiotensin II (27, 31), whereas neither acylated ghrelin nor obestatin had any effect. Furthermore, using an alternative luminescent probe (lucigenin), we found similar effects of des-acylated ghrelin on NADPH-stimulated superoxide levels, thus ruling out potential L-012 artifacts. Thus, using 2 separate superoxide detection techniques, these findings collectively show that des-acylated ghrelin can suppress cerebral artery superoxide levels in response to 3 stimuli of Nox2 oxidase. Importantly, the ability of des-acylated ghrelin to suppress NADPH-stimulated superoxide levels was sustained in the presence of L-NAME, indicating that increased stoichiometric removal of superoxide by NO is unlikely to explain its effect on cerebral artery superoxide levels. Moreover, ghrelin does not modulate superoxide levels generated in a xanthine/xanthine oxidase cell-free assay (19), thus it is unlikely that des-acylated ghrelin is acting as an antioxidant in cerebral vessels. Instead, we propose that des-acylated ghrelin attenuates superoxide levels by suppressing the activity of Nox2 oxidase.

Numerous studies over the past decade reinforce the idea that GHSR1a is not the sole ghrelin receptor. Perhaps the most convincing evidence comes from studies showing similar functional responses to acylated and des-acylated ghrelin in cells/tissues (eg, pancreas, skeletal muscle, and liver) that do not express GHSR1a, or in Ghsr-deficient mice (39). Furthermore, there are examples of responses to des-acylated ghrelin that are not mirrored by acylated ghrelin (eg, endothelial progenitor cells, neurons, and pancreatic β-cells) (39). Thus, at least 2 families of receptors have been proposed: those specific for des-acylated ghrelin or acylated ghrelin (eg, GHSR1a) and receptors common to both forms (39). Our findings here add to this growing list of novel ghrelin receptors. Specifically, we show functional evidence for the existence of non-GHSR receptors in cerebral vessels that are not recognized by acylated ghrelin. Less is known about the biological actions of obestatin or whether it shares a receptor(s) with the ghrelin forms. However, our finding that des-acylated ghrelin and obestatin have similar effects on vascular tone but not on superoxide levels infers that, at least in cerebral arteries, these 2 peptides are unlikely to share a common receptor but instead bind to distinct receptors. Moreover, these findings suggest that although both receptors trigger downstream signaling mechanisms that lead to eNOS activation, the signaling pathways activated by the receptor for obestatin do not couple to Nox2 oxidase. None of the novel receptors accounting for the actions of ghrelin or obestatin have been identified. It is probable, however, that, like GHSR1a, at least some of these receptors are G protein coupled. Indeed, the actions of both ghrelin forms on the pancreas and the liver have been linked with the G protein, Gαs (40, 41). Similarly, obestatin has been reported to exert vasodilator effects on systemic blood vessels through activation of an adenylate cyclase-linked G-protein coupled receptor (21). Importantly, both eNOS and Nox2 oxidase activity are modulated by various G-protein coupled receptors.

Although studies, including the present one, have shown that exogenous ghrelin-related peptides exert protective effects on blood vessels, there is a lack of research describing the physiologic function(s) of endogenously produced ghrelin-related peptides in any vascular bed. Thus, using ghrelin-deficient mice, which lack all ghrelin-related peptides, we tested whether such endogenous peptides might regulate NO bioactivity and superoxide production in cerebral arteries. Consistent with our earlier findings with exogenous peptides, we found that constrictions to L-NAME but not KPSS (a control vasoconstrictor) were significantly smaller in MCAs from ghrelin-deficient mice than in genetically related WT mice, indicating a lower level of NO bioactivity. Furthermore, we found evidence that Nox2-derived superoxide production is elevated in cerebral vessels from ghrelin-deficient mice. Acylated ghrelin potently stimulates the GH/IGF-I axis, which in turn has favorable effects on NO bioactivity and superoxide production (42). However, we have previously reported that the GH/IGF-I axis is preserved in ghrelin-deficient mice (43). Thus, our findings likely relate to the lack of circulating ghrelin-related peptides rather than a perturbation of the GH/IGF-I axis. Moreover, given that expression levels of eNOS and Nox2 were similar between genotypes, we propose that ghrelin-related peptides presumably modulate NO bioactivity and superoxide production in vivo by modulating the catalytic activities of eNOS and Nox2, respectively. Consistent with this interpretation is our earlier findings that des-acylated ghrelin/obestatin have direct, albeit acute, actions on eNOS and Nox2 activity in cerebral vessels. Multiple signal transduction pathways converge to regulate eNOS activity in blood vessels such as the PI3K/Akt and MAPK pathways (44). Similarly, numerous factors regulate the activity of Nox2 oxidase, including its association with several regulatory subunits (eg, p47phox, p67phox, p22phox, and Rac) (37). Thus, ghrelin-related peptides might influence eNOS and Nox2 activity in vivo by modulating the expression and/or activity of 1 or more of these molecules. As discussed, when generated in excess, superoxide reacts avidly with NO. Thus, it is conceivable that this mechanism may also contribute to the lower NO bioactivity in ghrelin-deficient mice.

This study provides the first evidence of protective actions of exogenous and endogenous ghrelin-related peptides on the cerebral circulation, and it provides evidence of an important role for and the existence of novel ghrelin receptors in cerebral arteries. Furthermore, given that the peptides modulate NOS and Nox2, which are both primarily expressed in endothelial cells, it is highly likely that these receptors are expressed in the endothelium. The discovery of cerebral vascular responses to des-acylated ghrelin and obestatin gives new perspective to the relative physiological importance of these peptides in regulating cerebral vascular function and potentially brain perfusion, especially given that des-acylated ghrelin may be the dominant form of ghrelin in the blood (45). Furthermore, our findings may prove to be all the more crucial when we consider that the ghrelin system appears to be suppressed after stroke and in patients with stroke risk factors (eg, diabetes, obesity, hypertension, and ageing) (25). Thus, it is fascinating to contemplate whether a deficiency in ghrelin-related peptides is an underlying contributor to the impaired cerebral artery NO bioactivity and oxidative stress that often accompanies these conditions and whether these peptides could be exploited therapeutically.