Tetrahydrobiopterin (BH4), a cofactor for nNOS, restores gastric emptying and nNOS expression in female diabetic rats

Abstract

Gastroparesis is a debilitating disease predominantly affecting young women. Recently, dysregulation of neuronal nitric oxide synthase (nNOS) in myenteric plexus neurons has been implicated for delayed solid gastric emptying/gastroparesis in diabetic patients. In this study, we have explored the role of tetrahydrobiopterin (BH4), a major cofactor for nNOS activity and NO synthesis in diabetic gastroparesis. Diabetes was induced with single injection of streptozotocin (55 mg/kg body wt, ip) in female rats, with experiments performed on week 3 or 9 following induction, with or without 3-wk BH4 supplementation. Gastric pyloric BH4 levels were significantly decreased in diabetic female rats compared with control (18.6 +/- 1.45 vs. 31.0 +/- 2.31 pmol/mg protein). In vitro studies showed that 2,4-diamino-6-hydroxypyrimidine (DAHP), an inhibitor of BH4 synthesis, significantly decreased gastric NO release and nitrergic relaxation. Three-week dietary supplementation of BH4 either from day 1 or week 6 significantly attenuated diabetes-induced delayed gastric emptying for solids (3 wk: BH4, 67 +/- 6.7 vs. diabetic, 36.05 +/- 7.09; 9 wk: BH4, 57 +/- 8.45 vs. diabetic, 33 +/- 9.91) and diabetes-induced reduction in pyloric nNOS-alpha protein expression in female rats. Supplementation of BH4 significantly restored gastric nNOS-alpha dimerization in 9-wk-old diabetic female rats. In addition, BH4 treatment reversed (17.23 +/- 5.81 vs. 42.0 +/- 2.70 mmHg x s) the diabetes-induced changes in intragastric pressures (IGP) and gastric pyloric nitrergic relaxation (-0.62 +/- 0.01 vs. -0.22 +/- 0.07). BH4 deficiency plays a critical role in diabetes-induced alterations including delayed solid gastric emptying, increased IGP, reduced pyloric nitrergic relaxation, and nNOS-alpha expression in female rats. Supplementation of BH4 accelerates gastric emptying by restoring nitrergic system in diabetic female rats. Therefore, BH4 supplementation is a potential therapeutic option for female patients of diabetic gastroparesis.

Fig. 1.

Effect of diabetes on gastric pyloric tetrahydrobiopterin (BH4) content in female rats. A: changes in total biopterin and BH4 content in gastric pylorus from nondiabetic (ND) and diabetic (DM) female rats. B: ratio of total biopterin and BH4 in ND and DM female gastric pylorus. The values are means ± SE for 4–6 animals in each group. *P < 0.05 compared with ND group.

Fig. 2.

Effect of 2, 4-diamino-6-hydroxypyrimidine (DAHP), a selective inhibitor of GTP cyclohydrolase-1, the rate-limiting enzyme for de novo BH4 synthesis, on nitric oxide (NO) production (A) and nitrergic relaxation (B) in rat gastric pyloric neuromuscular tissue. A: gastric neuromuscular pyloric tissues were incubated in the presence or absence of 10 mmol DAHP either for 24 or 48 h. B: Nonadrenergic, noncholinergic (NANC) relaxation in response to transmural nerve stimulation was measured in control and DAHP (10 mM, 3 h preincubation)-treated circular neuromuscular pyloric strips (electric-field stimulation, 90 V, 2 Hz, 1-ms pulse for duration of 1 min). The values are means ± SE for 6–8 strips. *P < 0.05 compared with control group; AUC, area under the curve.

Fig. 3.

A: effect of BH4 on solid gastric emptying in ND and DM female rats. Groups (n = 4–6) of diabetic rats were given BH4 (BH4DM) tablets daily for 3 wk either from day 1 (5 mg/kg body wt per day) or 6 wk (20 mg/kg body wt per day) after diabetic induction with single injection of streptozotocin (STZ: 55 mg/kg body wt ip). The values are means ± SE for 6–8 animals. *P < 0.05 compared with ND group. #P < compared with DM group. B: effect of BH4 on intragastric pressure (IGP; mmHg × s) in DM female rats. IGPs were measured using ambulatory telemetric devices (see materials and methods and Ref. 14) in groups of freely moving ND and DM female rats. A group of DM female rats received BH4 tablets (BH4DM) for 3 wk after 6 wk of diabetic induction by single injection of STZ (55 mg/kg body wt ip). The values are means ± SE for 4–6 animals. *P < 0.05 compared with ND group. #P < 0.05 compared with DM group.

Fig. 4.

Effect of BH4 on nitrergic relaxation in diabetic rat gastric muscular tissues. A group of DM female rats received BH4 tablets (BH4DM) for 3 wk after 6 wk of diabetic induction by single injection of STZ (55 mg/kg body wt ip). The values are means ± SE for 4–6 animals. *P < 0.05 compared with ND group. #P < 0.05 compared with DM group.

Fig. 5.

Effect of BH4 on constitutive nitric oxide synthase (cNOS) activity in diabetic rat gastric tissues. A: cNOS activity in gastric fundus, antrum, and pyloric tissues in healthy (control) female rats (n = 4 in each group). B: cNOS activity in BH4-treated diabetic female rat gastric pyloric tissues. A group of DM female rats received BH4 tablets (BH4DM) for 3 wk after 6 wk of diabetic induction. The ND group received citrate buffer (vehicle). The values are means ± SE for 4–6 animals. *P < 0.05 compared with ND group. #P < 0.05 compared with DM group.

Fig. 6.

Effect of BH4 in the expression and dimerization of neuronal NO synthase (nNOS) in diabetic rat gastric tissues. A: representative immunoblot and densitometric analysis data for nNOS protein expression in ND and 9-wk DM female rat gastric pylorus. The values are means ± SE for 4 samples in each group. B: representative immunoblot and densitometric analysis data for nNOS-α protein expression in ND and DM female rat gastric pylorus. A group of DM female rats received BH4 tablets (BH4DM) for 3 wk after day 1 (5 mg/kg body wt per day) or 6 wk (20 mg/kg body wt per day) of diabetic induction by single injection of STZ (55 mg/kg body wt ip). The values are means ± SE for 4 samples in each group. *P < 0.05 compared with ND group. #P < compared with untreated (0) diabetic group. C: representative immunoblot and densitometric analysis data for nNOS-α protein expression in ND and 9-wk DM female rat gastric pylorus. D: representative immunoblot and densitometric analysis data for nNOS-α dimerization in ND and 9-wk DM female rat gastric pylorus. A group of DM female rats received BH4 tablets (BH4DM) for 3 wk after 6 wk (20 mg/kg body wt per day) of diabetic induction. The values are means ± SE for 3–4 samples in each group. *P < 0.05 compared with ND group. #P < 0.05 compared with untreated (0) diabetic group.