Diabetes induces sex-dependent changes in neuronal nitric oxide synthase dimerization and function in the rat gastric antrum

Abstract

Diabetic gastroparesis is a disorder that predominantly affects women. However, the biological basis of this sex bias remains completely unknown. In this study we tested the hypothesis that a component of this effect may be mediated by the nitrergic inhibitory system of the enteric nervous system. Age-matched male and female Sprague-Dawley rats were studied 8 or 12 wk after streptozotocin (55 mg/kg body wt ip)-induced sustained hyperglycemia and compared with controls. Solid gastric emptying (GE) studies were performed in all the groups. Changes in gastric antrum neuronal nitric oxide synthase (nNOS) mRNA and protein levels were analyzed by real-time PCR and Western immunoblotting, respectively. nNOS dimerization studies were performed using low-temperature SDS-PAGE. In vitro nitrergic relaxation (area under curve/mg tissue wt) was studied after the application of electric field stimulation in an organ bath. Changes in intragastric pressure (mmHg.s) in freely moving rats in the presence or absence of N(G)-nitro-l-arginine methyl ester (nitric oxide synthase inhibitor) were examined by an ambulatory telemetric method. After diabetes induction, GE is delayed in both male and female rats. However, diabetic females exhibited significant delayed GE than in diabetic males. Compared with male controls, gastric nNOS expression and nitrergic relaxation were substantially elevated in healthy female control rats, accompanied by significantly reduced intragastric pressure. The active dimeric form and dimer-to-monomer ratio of nNOSalpha were also higher in healthy females compared with male rats (P < 0.05). Diabetic females, but not males, showed significant (P < 0.05) impairment in both gastric nNOSalpha dimerization and nitrergic relaxation, accompanied by an increase in intragastric pressure. Our data provide evidence that females may have a greater dependency on the nitrergic mechanisms in health. Furthermore, diabetes seems to affect the nitrergic system to a greater extent in females than in males. Together, these changes may account for the greater vulnerability of females to diabetic gastric dysfunction.

Fig. 1

Intragastric pressure (IGP) contractions were observed using implanted telemetric IGP devices. Contractions were defined as rapidly increasing pressure events, 5 mmHg or more in amplitude, with a duration of 5 s or more. These pressure curves were isolated and an integral from baseline was calculated. Ten such consecutive contractions were evaluated in this way to obtain a mean IGP for each animal for each time point. Means and SE of the IGP were then found for each group of animals at each time point.

Fig. 2

Sex-dependent changes in solid gastric emptying in healthy and diabetic rats. Diabetes was induced with a single injection of streptozotocin (STZ; 55 mg/kg body wt ip), and all studies were performed after 8 wk of diabetes induction. The control group received vehicle (citrate buffer). Fasted rats were exposed to normal rat chow ad libitum for a 3-h period and gastric emptying was monitored 4 h later. Data are means ± SE of 4–7 animals per group. *P < 0.05 (ANOVA).

Fig. 3

Effect of diabetes on gastric antrum nonadrenergic and noncholinergic (NANC) relaxation in response to transmural nerve stimulation (90 V, 2 Hz) in age-matched male and female rats. Gastric antrum muscle tissues obtained from 12-wk-old rats were preincubated with atropine, phentolamine, and propranolol (10 µM each) and active tone was induced initially with 100 µM 5-hydroxytryptamine. The nitric oxide (NO) dependence of the NANC relaxations in control and diabetic groups was confirmed by preincubation with the NO inhibitor nitro-l-arginine methyl ester (l-NAME; 100 µM). Each point represents means ± SE from 4–6 animals in each group. *Significant inhibition with l-NAME; #P < 0.05 for female control compared with male control; $P < 0.05 for female diabetic compared with female control.

Fig. 4

A: comparison of IGP (mmHg•s) for male control vs. female control animals. We noticed that the male animals tend to have more powerful contractions in general, but roughly the same number of contractions per unit time. In fact, the integral of this activity is significantly higher for the male controls compared with female controls. B: comparison of control vs. nitric oxide synthase inhibitor, l-NAME-treated (200 mg/kg body wt sc per rat for 4 days) female animals. Notice that the l-NAME-treated animals tend to have generally more powerful and more frequent contractions. In fact, the integral of this activity is significantly higher for the l-NAME-treated animals compared with female controls. C and D: effect of diabetes on IGP in age-matched male (C) and female (D) rats. IGP were measured using ambulatory telemetric devices (see MATERIALS AND METHODS) in the presence or absence of l-NAME (200 mg/kg body wt sc per rat for 4 days) administration by osmotic minipumps in conscious, unrestrained animals. Each point represents means ± SE from 3–4 animals in each group. *P < 0.05.

Fig. 5

Sex- and diabetes-related changes in the expression of neuronal nitric oxide synthase (nNOS) in the gastric distal antrum of male control, female control, male diabetic, and female diabetic rats. nNOS mRNA (A) and protein (B) levels were measured from gastric antrum by quantitative RT-PCR and Western blot analysis, respectively. Diabetes was induced with a single injection of STZ (55 mg/kg body wt ip) and all studies were performed after 12 wk of diabetes induction. The control group received vehicle (citrate buffer). A: nNOS and 18S mRNA expressions were measured by real-time RT-PCR. Relative abundance of nNOS mRNA normalized by 18S was calculated from threshold cycles (CT), i.e., 2^−ΔΔCT. B: representative immunoblot analysis of gastric distal antrum homogenates shows the expression of nNOS (155 kDa) and γ-tubulin (48 kDa) (top). Equal amounts of protein (40 µg) were loaded on each lane and detected with monoclonal antibody specific for the COOH-terminal domain of nNOS. Densitometric analysis followed by ratio of nNOS to γ-tubulin are calculated (bottom). Bars represent means ± SE (n = 3–4). Significant differences from control vs. diabetes are noted. *P < 0.05 for male diabetic compared with male control; #P < 0.05 for female control compared with male control.

Fig. 6

Sex- and diabetes-related changes in the expression of neuronal nitric oxide-α dimerization (nNOSα) in the gastric distal antrum of male control, female control, male diabetic, and female diabetic rats. Equal amounts of protein (40 µg) were loaded on each lane and detected with polyclonal antibody specific for the exon 2 encoded NH₂-terminal domain of nNOSα. Top: representative immunoblot of gastric distal antrum homogenates (n = 3–4) showing the ratio of nNOSα dimers (310 kDa) to monomers (155 kDa). Bottom: densitometric analysis followed by a ratio of nNOSα dimerization to γ-tubulin is calculated. Bars represent means ± SE. Significant differences from control vs. diabetes are noted. #P < 0.05 for female control compared with male control and $P < 0.05 for female diabetic compared with female control.

Fig. 7

Sex-dependent changes in solid gastric emptying (A), gastric antrum nitrergic relaxation (B) and gastric antrum nNOS dimer levels (C) in healthy and diabetic rats. Diabetes was induced with a single injection of STZ (55 mg/kg body wt ip). The control group received vehicle (citrate buffer). Diabetes reduced solid gastric emptying (A), nitrergic relaxation (B), and nNOSα protein dimer levels (C) in diabetic female gastric tissues compared with diabetic male rats. The changes in the above parameters were normalized with their respective control group. Data were presented as % change over control. *P < 0.05 (ANOVA).