| Gastroparesis (delayed gastric emptying) is frequent in diabetic patients. It is a well-recognized complication of longstanding diabetes. The symptom complex typically associated with gastroparesis, occur in 25%-55% of patients with long-standing type 1 or type 2 diabetes. Symptoms of diabetic gastropathy can range from mild dyspepsia to recurrent vomiting and abdominal pain and may progress to irreversible end-stage gastric failure known as gastroparesis. Gastroparesis seriously affects the quality of life. However, the pathophysiology of diabetic gastropathy and gastroparesis is still not delineated. It is generally considered that diabetic gastropathy and gastroparesis may be due to visceral autonomic neuropathy, remodeling of the interstitial cells network of Cajal, hyperglycemia and degeneration of smooth muscle. Several physiological studies have reported that dysfunction of the gastric smooth muscle in diabetes is associated not only with neural factors but also with intracellular signaling pathways.Since antrial natriuretic peptide (ANP) was isolated from atrium by de Bold et al in 1981, brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), dendroaspis (DNP), micrurus natriuretic peptide (MNP), and ventricular natriuretic peptide (VNP) were found in succession. Natriuretic peptides (NPs) are distributed in all over the body besides heart and exerted natriuretic-diuretic, vasorelaxation, and other functions designed to decrease blood pressure and to control electrolyte homeostasis and so on.Three types of single-transmembrane natriuretic peptid receptors (NPRs) for ANP, BNP and CNP have been identified, for example, NPR type A (NPR-A), type B (NPR-B) and type C (NPR-C). NPR-A and NPR-B have membrane-bound particulate guanylate cyclase (pGC) which catalyses the formation of cyclic guanosine monophosphate (cGMP) from guanosine triphosphate (GTP). NPR-A preferentially binds ANP and BNP, but has a low affinity for CNP, NPR-B has a much higher affinity for CNP than either ANP or BNP.NPs was also secreted from gastric mucosa. The previous research of our indicated that CNP relaxed the gastric circular and longitudinal smooth muscles in human, rat and guinea-pig stomach, and natriuretic peptide receptors (NPRs) were distributed in rat gastric smooth muscle layer. In smooth muscle, CNP activates its cognate NPR-B, which include intracellular particulate guanalyl cyclase (pGC) domain and catalyzes the synthesis of cyclic guanosine monophosphate (cGMP) within the cytosol. CNP relaxed the gastric smooth muscle by increasing Ik(Ca) which is activated by IICR through the cGMP pathway to hyperpolarize the membrane potential. CNP and NPR-B have been detected in the stomach. CNP mRNA expression was increased in kidney of streptozotocin (STZ)-induced diabetic r33ats and NPR-B expression was enhanced in vascular smooth muscle in diabetic mouse. However, it is not clear that the relationship between diabetic gastroparesis and natriuretic peptide signal pathway. So the present study was designed to investigate the effect of CNP-NPR-B-pGC-cGMP signal pathway involve in diabetic gastropathy or gastroparesis was investigated in STZ-induced rats by organ bath study, immunohistochemistry, radioimmunoassay and RT-PCR.The results were as follows:1. Rats were used to experiment at 4 weeks after injection STZ. At the time of the experiment all STZ-treated rats exhibited hyperglycemia, their blood glucose concentrations (478.0±27.9mg/dl) were significantly higher than those of the nondiabetic control rats (108.9±11.4mg/dl, n=8, P<0.001) and the body weights of the diabetic rats (209.7±8.0g) were significantly lower than those of the control rats (247.4±13.1g, n=8, P<0.05 ) and their urine volume of the diabetic rats (145.0±8.0ml/d) were significantly more than those of the nondiabetic control rats (15.2±1.2ml/d, n=8, P<0.001).2. The frequency of spontaneous contraction was significantly decreased in diabetic rats while the amplitude of spontaneous contraction was not significantly affected in diabetic rats. The frequency of spontaneous contraction was decreased from 3.10±0.14 cycle/min of control rat to 2.23±0.13cycle/min diabetic rat (n=8, P<0.01), however, the contractilities were 115.18±8.69 and 109.34±6.54 in normal and diabetic rats, respectively (n=8, P>0.05).3. The inhibitory effect of CNP on spontaneous contraction was revealed a dose-dependency, and the inhibitory percentages were 25.5±1.7%, 43.6±3.2%, 85.1±2.5% in diabetic rats and 20.5±1.5%, 31.1±1.7%, 58.9±3.7% in control group at the concentrations of 0.01μmol/L, 0.03μmol/L, and 0.1μmol/L, respectively (n=6, P<0.05). The frequency of spontaneous contraction was decreased by 26.95±2.82% and 53.33±2.03% by CNP (0.1μmol/L) in normal and diabetic rats, respectively (n=8, P<0.01), and the inhibition time was prolonged from 1.43±0.80min in control to 8.95±2.07min by CNP (0.1μmol/L ) in diabetic rats(n=8, P<0.01). It is indicated that gastric smooth muscle was more sensitive to CNP in diabetic rats than control group.4. cGMP production in gastric smooth muscle tissue, pretreated with 0.1μmol/L CNP, was examined. The cGMP production in gastric smooth muscle was significantly potentiated in diabetic rats, and the cGMP contents were 2.70±0.32 pmol/mg protein and 4.42±0.23 pmol/mg protein in control and diabetic groups, respectively (n=8, P < 0.05).5. The activity of particulate GC was measured by radioimmunoassay to determine whether the cGMP production induced by CNP via activation of particulate GC in gastric muscle tissue. The basal rate of cGMP production by pGC activation were 0.74±09pmol/mg protein/min and 0.89±13 pmol/mg protein/min in control and diabetic rats, respectively (n=6, P>0.05). By the addition of 0.1μmol/L CNP in the muscle tissue membrane, the rates of cGMP production were 3.22±32pmol/mg protein/min, 5.92±23pmol/mg protein/min in control and diabetic rats, respectively (n=6, P<0.01 vs control group, P<0.01 vs Vehicle).6. The NPR-B immunopositive brown granules were expressed in gastric antrial smooth muscle in normal and diabetic rats, however, the staining was more deep in diabetic rats. The staining indexes were increased from 17.63±1.49 of control to 30.67±1.59 in diabetic rats, and there was significant difference between normal and diabetic rats (n=9, P<0.01). In succession NPR-B gene expression was investigated in gastric smooth muscle by RT-PCR. The densities of expression of NPR-B mRNA were 0.13±0.03 and 0.34±0.10 in control and diabetic rats, respectively. The expression of NPR-B mRNA was more significantly increased in the diabetic rats than that in the control (n=3, P<0.01).7. the CNP immunopositive brown granules were mainly expressed in gastric muscle layers of normal and diabetic rats, and the staining indexes in gastric muscle were 41.26±0.71% and 41.71±0.21% in control and diabetic rats (n=9, P>0.05 ).The results suggest that: 1. After 4 weeks induced diabetes, the gastric smooth muscle motility begin to appear disorder, that mostly exhibit slow rhythm and turbulence, however, the amplitude of spontaneous contraction is not significantly affected in diabetic rats. 2. After 4 weeks induced diabetes, the sensitivity of the gastric smooth muscle to CNP-cGMP signal pathway is significantly potentiated, it is mediated by increase of NPR-B expression and pGC activity in diabetic rats. 3. After 4 weeks induced diabetes, the CNP content in gastric smooth muscle is not significantly changed in the diabetic rat. 4. CNP-NPR-B-pGC-cGMP signal pathway may be involved in the pathogenesis and development of diabetic gastropathy or gastroparesis.In summary, the CNP-cGMP signal pathway is up regulated in diabetic gastric smooth muscle and CNP-NPR-B-pGC-cGMP signal pathway may be involved in the process of diabetic gastropathy or gastroparesis. The results would theoretically be significant in pathogenesis of diabetic gastropathy or gastroparesis, and might experimentally be an important foundation for therapy and prevention of diabetic gastropathy or gastroparesis.
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