Nitric oxide and nitric oxide synthase isoforms in the normal, hypertrophic, and failing heart

Increased oxidative stress is one of the basic contributors to the development of the cardiovascular complications in diabetes. Both endothelial and vascular smooth muscle cell dysfunctions are the main sign involved in the pathogenesis of diabetic cardiovascular dysfunction. Matrix metalloproteinases (MMPs) are expressed in the vasculature, and participate in tissue remodeling under pathological conditions such as increased oxidative stress, whereas little is known about effect of hyperglycemia on regulation of MMPs in vascular system. Therefore, we aimed to evaluate the effect of an antioxidant, sodium selenate treatment (0.3 mg/kg for 4 weeks) on function of streptozotocin-diabetic rat aorta. Sodium selenate treatment improved significantly impaired isoproterenol-induced relaxation responses and contraction responses of the aortic strips, and exhibited marked protection against diabetes-induced degenerative changes in the smooth muscle cell morphology. Biochemical data showed that sodium selenate treatment induced a significant regulation of MMP-2 activity and protein loss as well as normalization of increased levels of tissue nitrite and protein thiol oxidation. In addition, this treatment restored diabetes-induced increased levels of endothelin-1, PKC, and cAMP production in the aortic tissue. Taken together, our data demonstrate that these beneficial effects of sodium selenate treatment in diabetics are related to be not only inhibition of increased oxidative stress but also prevention of both receptor- and smooth muscle-mediated dysfunction of vasculature, in part, via regulation of MMP-2. Such an observation provides evidence for potential therapeutic usage of selenium compounds for the amelioration of vascular disorders in diabetes.     

Downregulation of Apoptosis and Modulation of TGF-β1 by Sodium Selenate Prevents Streptozotocin-Induced Diabetic Rat Renal Impairment

To investigate whether sodium selenate treatment would impact on the onset of diabetic nephropathy, we examined blood glucose, serum biochemical components, and interrelationship between oxidative stress, TGF-β1, and apoptosis in streptozotocin (STZ) induced diabetic rats. Sixty male Wistar rats were divided into six groups. Group I (n = 10), normal control; Group II (n = 10), diabetic control; Group III (n = 10), sodium selenate (16 μmoles/kg) + diabetic; Group IV (n = 10), sodium selenate (32 μmoles/kg) + diabetic; Group V (n = 10), sodium selenate (16 μmoles/kg) control; and Group VI (n = 10), sodium selenate (32 μmoles/kg) control. Sodium selenate was administered via orogastric route for 10 weeks. In the diabetic group, diabetes was induced by single intraperitoneal injection of STZ (50 mg/kg). The levels of blood glucose were estimated and total cholesterol, high-density lipoprotein (HDL) cholesterol, triglycerides, creatinine, urea, and albumin were detected in serum. Antioxidant status was examined by measuring the superoxide dismutase (SOD), catalase, glutathione, and lipid peroxidation in kidney tissues. Histopathological studies were performed in the kidney tissue sections. The expression of TGF-β1 was estimated by the immunohistochemical analysis in kidneys. Apoptotic study in kidney was performed using the TdT-mediated dUTP nick end labeling technique. It was observed that blood glucose, serum, total cholesterol, HDL cholesterol, triglycerides, creatinine, urea, and albumin were significantly higher in diabetic control groups. Diabetic + sodium selenate (16 and 32 μmoles/kg) significantly reduced blood glucose, serum, total cholesterol, HDL cholesterol, triglycerides, creatinine, urea, and albumin levels. Selenium-treated groups significantly increased antioxidant enzyme activities (SOD, catalase, and glutathione) in kidneys of diabetic rats. All enzyme activities of selenium control groups did not differ compared with the normal control. Sodium selenate reduces significantly lipid peroxidation in diabetic rats. Cellular architecture of the diabetic rats was altered whereas sodium selenate administration rectifies the degenerative changes of the kidney. Profound immunopositivity of TGF-β1 was observed in the glomerular and tubulointerstitial cells of diabetic rat kidney. Immunopositivity of TGF-β1 was significantly reduced in both low and high dose of sodium-selenate-treated rats (P < 0.05, P < 0.01). High numbers of apoptotic cells were observed in diabetic rats whereas sodium selenate in both doses significantly reduces the incidence of apoptosis (P < 0.05, P < 0.01). We conclude herein that sodium selenate has the potential to play a significant role in limiting the renal impairment by altering the apoptosis and TGF-β1 in experimental diabetic rats.     

Treatments with sodium selenate or doxycycline offset diabetes-induced perturbations of thioredoxin-1 levels and antioxidant capacity

Diabetes is associated with increased oxidative stress and impaired antioxidant defenses. Thioredoxin-1 (TRX-1) is a cytosolic thiol antioxidant and redox-active protein which plays a vital role in the maintenance of reduced intracellular redox state. In this study, the authors examined whether 4-week treatments with sodium selenate and doxycycline--a metalloproteinase-2 inhibitor which also has antioxidant-like effects--offset perturbations in oxidative stress and antioxidant protection in rat liver and skeletal muscle in streptozotocin-induced diabetes (SID) model. Experimental diabetes decreased TRX-1 levels in skeletal muscle and liver. On the other hand, SID increased oxidative stress marker protein carbonyl levels and decreased oxygen radical absorbance capacity (ORAC), an indicator of antioxidant capacity, in liver. A 4-week treatment of sodium selenate to diabetic rats decreased blood glucose levels moderately, while doxycycline treatment caused a reduction in weight loss of diabetic rats. Both doxycycline and sodium selenate prevented diabetes-induced decrease of TRX-1 levels in skeletal muscle, whereas only doxyxycline was effectively preventing diabetes-induced decrease of TRX-1 in liver. Furthermore, both treatments prevented diabetes-induced altered levels of protein carbonyls and ORAC in liver, and restored free and total protein thiol levels in both skeletal muscle and liver. In conclusion, the data of this study provides further evidence that sodium selenate and doxycycline treatments may control oxidative stress and improve antioxidant defense in diabetes.     

The effect of systemic leptin administration on aorta smooth muscle responses in diabetic rats

Leptin produces effects in central nervous system and peripheral tissues via its specific receptors. Leptin also stimulates nitric oxide release in a concentration-dependent manner. In this study, our aim was to test the hypothesis that whether leptin has a modulatory role on endothelium or smooth muscle function in streptozotocin (STZ)-induced diabetic rats. Wistar-Albino rats were divided into four groups: 1 – Control, 2 – Diabetic, 3 – Control + leptin and 4 – Diabetic + leptin. Experimental diabetes was produced by intraperitoneal injection of a single dose of STZ (55 mg/kg). Diabetes was determined by increased fasting blood glucose level on the 7th day of the experiment. Leptin (0.1 mg/kg/day) was administered intraperitoneally for 5 days. At the end of the 5th day, thoracic aortas were isolated and phenylephrine (Phe)-induced contractions and acetylcholine (ACh)-induced relaxations of each group were estimated. In diabetic rats, Phe-induced contractility was increased (p < 0.05). Leptin pre-treatment increased the Phe-induced contractility significantly in aortic rings obtained from diabetic rats (p < 0.05). In normal rats, leptin administration produced only a slight and non-significant increase in Phe-induced contractions. Although the relaxant responses were decreased in diabetic rats, leptin administration enhanced the ACh-induced relaxation in both normal and diabetic animals significantly. As a conclusion; chronic leptin pre-treatment caused a significant increase both in Phe-induced contractions and ACh-induced Endothelial-Derived Relaxing Factor (EDRF)/Nitric oxide-mediated relaxations in the aortic rings isolated from streptozotocin-induced diabetic rats. This peptide hormone caused a significant increase in the relaxations obtained by ACh while not inducing a significant alteration in the contractile effect of Phe in control rats.     

Effects of taurine on the reactivity of aortas from diabetic rats

The effects of the semi-essential amino acid-like nutrient, taurine, on alterations in the reactivities of aortas from male rats with chronic streptozotocin-induced diabetes were examined under in vitro conditions. In the absence of taurine, the contractile responsiveness of endothelium-denuded aortic rings from diabetic rats to norepinephrine, but not KCl, was enhanced compared to controls. This effect of norepinephrine on the diabetic rat aorta appeared to be associated with increased release of intracellular calcium, influx of extracellular calcium and protein kinase C-mediated responses. Incubation of endothelium-denuded aortic rings with 10 mM, but not 5 mM, taurine for 2 h reduced the augmented contractile responses of the tissues from diabetic rats to norepinephrine close to control levels, and this was associated with inhibition of responses linked to the release and influx of calcium, and protein kinase C activation. Endothelium-dependent relaxation of aortas from diabetic rats to acetylcholine was depressed relative to controls. This effect of diabetes was ameliorated close to control levels by incubating the tissues with 10 mM, but not 5 mM, taurine for 2 h. Incubation of nondiabetic rat aortic rings with 45 mM glucose for 3 h caused enhancement of contraction of the vascular smooth muscle to phenylephrine and impairment of endothelium-mediated vasorelaxation to acetylcholine, as compared to control responses. Co-incubation of the tissues with 5-10 mM taurine concentration-dependently reduced the alterations in both contractile and relaxant responses caused by high glucose. Overall, the data suggest that taurine ameliorates or prevents vascular reactivity alterations in diabetes. Such an observation provides preliminary evidence for taurine's potential as a therapeutic agent for the prevention or amelioration of vascular disorders in diabetes.     

Rapid pacing-induced preconditioning is recaptured by farnesol treatment in hearts of cholesterol-fed rats: Role of polyprenyl derivatives and nitric oxide

Sodium selenate, administered intraperitoneally (i.p.), resulted in an improvement in glucose tolerance in treated diabetic rats. Fed rat plasma glucose levels were reduced by selenate treatment in streptozotocin diabetic rats. The lowest values of blood glucose were reached within 3 weeks of beginning the treatment. Food and fluid consumption was reduced in treated compared to untreated diabetic rats. Diabetic treated rats did not release insulin in response to a glucose challenge and insulin release in response to a challenge was markedly reduced in control treated rats. Assessment of heart function using a working heart apparatus showed that treated diabetic rats with improved blood glucose levels had normal heart function at 8 weeks of diabetes in contrast to hearts from non-treated diabetics. This study extends previous observations on the in vivo insulin-like effects of sodium selenate.     

Time-dependent alteration in cromakalim-induced relaxation of corpus cavernosum from streptozocin-induced diabetic rats

The purpose of the present study was to investigate the relaxant responses to the ATP-sensitive potassium (K(ATP)) channel opener cromakalim in corpus cavernosum strips from 1-, 2-, 4-, 6-, and 8-week streptozocin-induced diabetic rats. Cromakalim (1 nM-0.1 mM) produced concentration-dependent relaxation in phenylephrine (7.5 microM)-precontracted isolated rat corporal strips. Compared with age-matched control animals, a significant enhancement in cromakalim-induced relaxation of corpus cavernosum was observed in 2-week diabetic animals, whereas the relaxant responses to cromakalim were decreased in 6-and 8-week diabetic animals. However, the cromakalim-induced relaxation was not altered in either 1-week or 4-week rat corporal strips in comparison with corresponding age-matched non-diabetic groups. Preincubation with the K(ATP) channel blocker glibenclamide (10 microM) significantly inhibited the cromakalim-induced relaxation in both non-diabetic and diabetic rat corpus cavernosum, but neither the voltage-dependent K(+) channel (K(V)) antagonist 4-aminopyridine (1 mM) nor the calcium-activated K(+) channel (K(Ca)) antagonist charybdotoxin (0.1 microM) had significant effect on cromakalim-induced relaxation in both control and diabetic rat corporal strips. Relaxation responses to the nitric oxide donor sodium nitroprusside (1 nM-0.1 mM) in diabetic rat corpus cavernosum were similar to that of age-matched controls. These data demonstrated that the relaxant responses to cromakalim were altered in diabetic cavernosal strips in a time dependent manner, suggesting that the period of diabetes mellitus may play a key role in the K(ATP) channels function in rat corpus cavernosum.     

Radiotelemetric monitoring of blood pressure and mesenteric arterial bed responsiveness in rats with streptozotocin-induced diabetes

We investigated the changes in arterial blood pressure (BP) and of mesenteric arterial bed (MAB) responsiveness that accompany streptozotocin (STZ)-induced diabetes. BP was recorded by radiotelemetry in conscious animals before and during a 4-week period following induction of the diabetic state with STZ. At the end of this period, the MAB was isolated and perfused under constant flow conditions: perfusion pressure (PP, mmHg) was taken as an index of arteriolar tone. BP was lower (P < 0.05) in STZ-treated diabetic rats (82.9+/-5.0 mmHg) than in vehicle-treated rats (108.9+/-6.3 mmHg). Basal perfusion pressure of the MAB was lower in STZ-treated rats than in control rats and inhibition of nitric oxide (NO) synthesis with N(G)-nitro-L-arginine-methyl-ester and N(G)-nitro-L-arginine (100 microM each) failed to change this relationship. Increases in PP of MAB to phenylephrine (Phe), norepinephrine (NE), and potassium chloride (KCl) were reduced in STZ-treated rats compared with control rats. Inhibition of NO synthesis reduced responses to Phe, NE, and KCL in both STZ and control rats. The reduced responsiveness of STZ rats to Phe, NE, and KCl persisted after inhibition of NO synthesis. Acetylcholine (ACh) evoked relaxation of the MAB in a dose-dependent fashion. Maximal responses to ACh, but not sodium nitroprusside, were lower in STZ rats than in vehicle treated rats. Inhibition of NO synthesis reduced responses to ACh in both STZ and control rats. The reduced responsiveness of STZ rats to ACh persisted after inhibition of NO synthesis. The data demonstrate that STZ-induced diabetes is associated with a fall in blood pressure when pressure is recorded with radiotelemetry. The fall in blood pressure may be related to a non-specific decrease in responsiveness to vasoconstrictor stimuli mediated at least in part by NO-independent mechanisms. A decrease in responsiveness to endothelial dependent vasodilator mechanisms appeared insufficient to restore responsiveness to vasoconstrictor stimuli.     

Decreased NO signaling leads to enhanced vasoconstrictor responsiveness in skeletal muscle arterioles of the ZDF rat prior to overt diabetes and hypertension

Approximately 40% of patients with type 2 diabetes present with concurrent hypertension at the time of diabetes diagnosis. Increases in peripheral vascular resistance and correspondingly enhanced vasoconstrictor capacity could have profound implications for the development of hypertension and the progression of insulin resistance to overt diabetes. The purpose of this study was to determine whether skeletal muscle arteriolar vasoconstrictor dysfunction precedes or occurs concurrently with the onset of diabetes and hypertension. Male Zucker diabetic fatty (ZDF) rats were studied at 7, 13, and 20 wk of age to represent prediabetic and short-term and long-term diabetic states, respectively. Conscious mean arterial pressure (MAP), fasted plasma insulin and glucose, vasoconstrictor responses, and passive mechanical properties of isolated skeletal muscle arterioles were measured in prediabetic, diabetic, and age-matched control rats. Elevated MAP was manifest in short-term diabetes (control 117 +/- 1, diabetic 135 +/- 3 mmHg) and persisted with long-term diabetes (control 113 +/- 2, diabetic 135 +/- 3 mmHg). This higher MAP was preceded by augmented arteriolar vasoconstrictor responses to norepinephrine and endothelin-1 and followed by diminished beta-adrenergic vasodilation and enhanced myogenic constriction in long-term diabetes. Furthermore, we demonstrate that diminished nitric oxide (NO) signaling underlies the increases in vasoconstrictor responsiveness in arterioles from prediabetic and diabetic rats. Arteriolar stiffness was not different between control and prediabetic or diabetic rats at any time point studied. Collectively, these results indicate that increases in vasoconstrictor responsiveness resulting from diminished NO signaling in skeletal muscle arterioles precede the development of diabetes and hypertension in ZDF rats.     

Exercise training ameliorates the impairment of endothelial and nitrergic corpus cavernosum responses in diabetic rats

AimsThe effect of exercise training (ET) on vascular responsiveness in diabetes mellitus has been largely well studied. However, limited studies have investigated the effects of ET on functional responses of the corpus cavernosum (CC) in diabetic animals. Therefore, the aim of this study was to investigate whether prior ET prevents the impairment of erectile function in streptozotocin-induced diabetic rats.Main methodsRats were exercised for four weeks prior to the induction of diabetes, and then again for another 4 weeks thereafter. Concentration–response curves to acetylcholine, sodium nitroprusside, Y-27632, BAY 412272 and phenylephrine (PE) were obtained in CC. The excitatory and inhibitory effects of electrical-field stimulation were also evaluated.Key findingsPlasma SOD levels were markedly decreased in the sedentary diabetic group (D-SD) as compared to control sedentary animals (C-SD), approximately 53% (P < 0.05) and this reduction was restored in trained diabetic animals. Physical training restored the impairment of endothelium-dependent and -independent relaxation responses seen in the D-SD group. The potency values for Y-27632 in the CC were significantly reduced in the D-SD group, which was reversed by physical training. The impairment of electrical-field stimulation (EFS)-induced relaxation seen in the D-SD group was restored by physical training. On the other hand, both EFS-induced contractions and concentration–response curves to PE in cavernosal strips were not modified by either diabetes or physical training.SignificancePractice of regular physical exercise may be an important approach in preventing erectile dysfunction associated with diabetes mellitus by re-establishment of the balance between NO production and its inactivation.     

Diabetes abolishes the vascular protective effects of estrogen in female rats

Estrogen is known to exert a protective effect against cardiovascular disease. However, women with diabetes have three times the risk as compared with age-matched non-diabetic women. Our previous study on aortic rings of ovariectomized (OVX) female rats treated with 17-beta-estradiol (E2) demonstrated that the beneficial effect of estrogen is related to the basal release of NO from endothelial cells. In the present study, in order to understand why estrogen protection is abolished in diabetes, we tested vascular responses in OVX, streptozotocin-diabetic female rats and their non-diabetic controls receiving or not E2 replacement. Concentration-response curves to norepinephrine (NE) showed attenuation of the contractile response in E2-treated diabetic, with respect to non-diabetic preparations. This response was further impaired in diabetic, E2-deprived rats. The basal release of NO, as evaluated by concentration-related responses to N(G)-methyl-L-arginine acetate in NE-precontracted aortic rings, was found to be impaired in E2-treated diabetic rats, no further effect being induced by E2 deprivation. The endothelium-dependent relaxation produced by carbachol did not change between groups, whereas the relaxation produced by histamine was enhanced by both diabetes and E2 deprivation. However, E2 treatment counteracted the response to histamine only in preparations from non-diabetic animals. Finally, the relaxation induced by sodium nitroprusside, an endothelium-independent relaxant agent, was comparable between groups. These findings suggest that the lack of protective effects of estrogen in diabetes may be mainly ascribed to the failure of estrogen to reverse the impaired basal release of NO and the abnormal relaxation to histamine, which are observed in the aorta of diabetic rats.     

The effect of ovariectomy on the contractile response of the rat isolated detrusor muscle and urethra

Contractile responses induced by carbachol on the detrusor muscle and by noradrenaline on the isolated urethra were compared between ovariectomized rats pretreated with estradiol (50 μg/animal s.c. twice daily for five days), untreated ovariectomized rats and intact animals. In the detrusor muscle, contractions induced by 30μM carbachol, when normalized with respect to KCl 100 mM-induced contraction, were similar for the three groups. Furthermore, contractions induced by 100 μM noradrenaline in the isolated urethra were not significatively different between groups. However, the pD₂ value for noradrenaline was greater in urethral tissue from ovariectomized rats compared with ovariectomized -estrogen treated and control rats. A similar result was found for pD₂ values for carbachol-induced contractions on the detrusor muscle. These results suggest that ovariectomy increases the sensitivity of the urinary bladder and urethra to the contractile effects of carbachol and noradrenaline, respectively and that this effect is reversed by in vivo estrogen pretreatment.     

Significant Improvement of Insulin Resistance of GK Rats by Treatment with Sodium Selenate

We studied the effect of sodium selenate on insulin resistance of Goto-Kakizaki (GK) rats. Rats were kept on standard laboratory chow with and without i.p. injections of sodium selenate (0.173 mg/kg body weight) for 14 days, and then subjected to the glucose clamp. The glucose clamp studies confirmed an improvement in insulin-stimulated glucose disposal in GK rats treated with sodium selenate, with respect to both insulin sensitivity and responsiveness. This amelioration of insulin resistance may be partly due to a direct effect of the sodium selenate on peripheral tissues. 2-Deoxyglucose uptake in sodium selenate-treated adipocytes was increased and the insulin findings suggest that sodium selenate increases not only insulin sensitivity but also insulin responsiveness. Sodium selenate also accelerated glucose incorporation into adipocytes of rats, suggesting that the action of sodium selenate is peripheral. Interestingly, sodium selenate at a high concentration (about 1 mmol/L) was more effective than insulin in enhancing glucose uptake. The present study suggested that sodium selenate treatment led to substantial improvement in peripheral insulin resistance.     

Influence of diabetes on norepinephrine-induced inositol 1,4,5-trisphosphate levels in rat aorta

The effects of norepinephrine on total tissue levels of inositol 1,4,5-trisphosphate were measured by protein binding assay in aortas from rats with chronic streptozotocin-induced diabetes and from age-matched control rats. In both control and diabetic aortas, norepinephrine induced a rapid, transient and concentration-dependent elevation of inositol 1,4,5-trisphosphate content during contraction. Maximum production of inositol 1,4,5-trisphosphate in response to norepinephrine was greater in diabetic than in control aortas. However, the sensitivities of control and diabetic aortas to norepinephrine for inositol 1,4,5-trisphosphate production were not significantly different. Enhanced norepinephrine-induced production of inositol 1,4,5-trisphosphate in diabetic aortas may contribute to the increased maximum contractile responsiveness of these arteries to the agonist. However, since enhanced contractile responses of diabetic aortas to norepinephrine were also detected at times when inositol 1,4,5-trisphosphate levels were not significantly increased, other factors also appear to be involved in mediating enhanced contractions of diabetic arteries to norepinephrine.     

Short-term insulin treatment and aortic expressions of IGF-1 receptor and VEGF mRNA in diabetic rats

We investigated the relationship between the changes in vascular responsiveness and growth factor mRNA expressions induced by 1-wk treatment with high-dose insulin in control and established streptozotocin (STZ)-induced diabetes. Aortas from diabetic rats, but not those from insulin-treated diabetic rats, showed impaired endothelium-dependent relaxation in response to ACh (vs. untreated controls). The ACh-induced nitrite plus nitrate (NOx) level showed no significant difference between controls and diabetics. Insulin treatment increased NOx only in diabetics. In diabetics, insulin treatment significantly increased the aortic expressions of endothelial nitric oxide synthase (eNOS) mRNA and VEGF mRNA. The expression of IGF-1 mRNA was unaffected by diabetes or by insulin treatment. In contrast, the mRNA for the aortic IGF-1 receptor was increased in diabetics and further increased in insulin-treated diabetics. In aortic strips from age-matched control rats, IGF-1 caused a concentration-dependent relaxation. This relaxation was significantly stronger in strips from STZ-induced diabetic rats. These results suggest that in STZ-diabetic rats, short-term insulin treatment can ameliorate endothelial dysfunction by inducing overexpression of eNOS and/or VEGF mRNAs possibly via IGF-1 receptors. These receptors were increased in diabetes, perhaps as result of insulin deficiency.     

Erigeron breviscapus prevents defective endothelium-dependent relaxation in diabetic rat aorta

We examined the endothelium-dependent relaxation response to acetylcholine (Ach) in streptozotocin-induced diabetic rat aorta at the stages of 2- and 6-wks' duration in vitro, and compared with another two groups which were treated with dietary supplement of 0.1% Aminoquanidine (AG) and 0.5% Erigeron breviscapus (EB) from 1-week of diabetes induction. At the stage of 2-wks' duration of diabetes, relaxation responses to lower concentrations of Ach in 0.3 uM phenylepherine-precontracted aortas were diminished significantly (P<0.05) compared with age-matched control, but the maximal relaxation of Ach remained unchanged. At the stage of 6-wks' duration, diabetes caused an approximately 60% (P<0.001) deficit in maximum relaxation, and this was significantly (P<0.001) prevented in AG and EB treated groups. There was an approximately 40% enhancement in the maximum contractile response to phenylepherine with diabetes (P<0.05), which was unaffected significantly by AG and EB treatments. The data suggest that the defective endothelium-dependent relaxation in diabetic rat aorta occurred as early as 2-wks' duration of diabetes, and the treatments of AG and EB could protect vascular endothelium although the deficits in vascular smooth muscle contractile responses were not protected.     

Diabetes-induced changes in responsiveness of rat bladder and vas deferens to peptides in vitro: susceptibility to reversal by insulin

Changes in responsiveness of the vas deferens and urinary bladder to bradykinin (BK) receptor agonists (Tyr8-BK and des-Arg9-BK), substance P (SP), and endothelin-1 (ET-1) were assessed 8 weeks after streptozotocin (STZ)-induced diabetes. Preparations from control or STZ-treated (60 mg/kg i.p.) male rats were tested for contractile and neurogenic twitch potentiating (TP, in VD only) effects of all four agonists (1 nM to 0.3 or 3 microM). In diabetic VD, contractile effects of Tyr8-BK, des-Arg9-BK, and SP were enhanced, but ET-1 effects were unchanged. In contrast, TP by des-Arg9-BK was unaffected, that by Tyr8-BK was decreased, and those by SP and ET-1 were increased. In diabetic UB, only contractions to des-Arg9-BK and SP were enhanced. Following insulin replacement (human, 1-3 U/day s.c.), starting 1 week after STZ, TP induced by Tyr8-BK and des-Arg9-BK in VD were further inhibited, but all other changes in both preparations were reversed at least partially. Insulin treatment of nondiabetic rats, however, also affected VD (but not UB) responsiveness, such that contractions to Tyr8-BK and TP by ET-1 were increased, but TP by Tyr8-BK was decreased. Thus, STZ-induced type I diabetes causes important alterations in responsiveness of non-vascular smooth muscle tissues of the rat to BK, SP, and ET-1. Long term insulin replacement, at doses normalising glycaemia, effectively reversed most changes in VD or UB responsiveness, but it is unclear if this is truly due to blocking of STZ-induced changes, since the treatment also affected responsiveness of nondiabetic tissues.     

Diabetes influences the effect of 17beta-estradiol on mechanical responses of rat urethra and detrusor strips

Estrogen deficiency is one of the factors involved in the stress incontinence in postmenopausal women, and estrogens have been used clinically in the treatment of urinary disorders during menopause. Sex hormones seem to be also involved in the diabetic changes of urinary bladder and urethra, because ovariectomy causes an increase in the micturition of streptozotocin-diabetic rats. In the present study diabetic and healthy female rats were used to investigate the effect of 17beta-estradiol on mechanical contractions to norepinephrine and to KCI and relaxations to ATP on isolated proximal urethral preparations as well as on contractions to ACh, ATP and KCl on detrusor smooth muscle strips. The data were compared with those obtained in OVX animals, with or without estradiol replacement. The present study showed that ovariectomy decreased the responses to ATP, NE and KCl in urethral preparations, and responses to ATP, ACh and KCl in bladder strips from both healthy and diabetic rats. Diabetes appeared to potentiate the effect of ovariectomy in both tissues. Estrogen replacement was able to recover functional responses in urethras of healthy rats. In diabetic rats, this treatment partially restored ATP-induced responses in both tissues, almost completely restored those to NE in urethra and those to ACh in bladder. This study clearly indicated that abnormalities of urethra and bladder function caused by ovariectomy can be restored by estrogen treatment also in diabetic animals, at least at an early stage of disease.     

The Dipeptidyl Peptidase-4 Inhibitor Linagliptin Preserves Endothelial Function in Mesenteric Arteries from Type 1 Diabetic Rats without Decreasing Plasma Glucose

The aim of the study was to investigate the effect of the DPP-4 inhibitor linagliptin on the mechanism(s) of endothelium-dependent relaxation in mesenteric arteries from STZ-induced diabetic rats. Both normal and diabetic animals received linagliptin (2 mg/kg) daily by oral gavage for a period of 4 weeks. To measure superoxide generation in mesenteric arteries, lucigenin-enhanced chemiluminescence was used. ACh-induced relaxation of mesenteric arteries was assessed using organ bath techniques and Western blotting was used to investigate protein expression. Pharmacological tools (1μM TRAM-34, 1μM apamin, 100 nM Ibtx, 100 μM L-NNA, 10 μM ODQ) were used to distinguish between NO and EDH-mediated relaxation. Linagliptin did not affect plasma glucose, but did decrease vascular superoxide levels. Diabetes reduced responses to ACh but did not affect endothelium-independent responses to SNP. Linagliptin improved endothelial function indicated by a significant increase in responses to ACh. Diabetes impaired the contribution of both nitric oxide (NO) and endothelium-dependent hyperpolarization (EDH) to endothelium-dependent relaxation and linagliptin treatment significantly enhanced the contribution of both relaxing factors. Western blotting demonstrated that diabetes also increased expression of Nox2 and decreased expression and dimerization of endothelial NO synthase, effects that were reversed by linagliptin. These findings demonstrate treatment of type 1 diabetic rats with linagliptin significantly reduced vascular superoxide levels and preserved both NO and EDH-mediated relaxation indicating that linagliptin can improve endothelial function in diabetes independently of any glucose lowering activity.     

Hyperglycemia does not increase basal hypothalamo-pituitary-adrenal activity in diabetes but it does impair the HPA response to insulin-induced hypoglycemia

Recently, we established that hypothalamo-pituitary-adrenal (HPA) and counterregulatory responses to insulin-induced hypoglycemia were impaired in uncontrolled streptozotocin (STZ)-diabetic (65 mg/kg) rats and insulin treatment restored most of these responses. In the current study, we used phloridzin to determine whether the restoration of blood glucose alone was sufficient to normalize HPA function in diabetes. Normal, diabetic, insulin-treated, and phloridzin-treated diabetic rats were either killed after 8 days or subjected to a hypoglycemic (40 mg/dl) glucose clamp. Basal: Elevated basal ACTH and corticosterone in STZ rats were normalized with insulin but not phloridzin. Increases in hypothalamic corticotrophin-releasing hormone (CRH) and inhibitory hippocampal mineralocorticoid receptor (MR) mRNA with STZ diabetes were not restored with either insulin or phloridzin treatments. Hypoglycemia: In response to hypoglycemia, rises in plasma ACTH and corticosterone were significantly lower in diabetic rats compared with controls. Insulin and phloridzin restored both ACTH and corticosterone responses in diabetic animals. Hypothalamic CRH mRNA and pituitary pro-opiomelanocortin mRNA expression increased following 2 h of hypoglycemia in normal, insulin-treated, and phloridzin-treated diabetic rats but not in untreated diabetic rats. Arginine vasopressin mRNA was unaltered by hypoglycemia in all groups. Interestingly, hypoglycemia decreased hippocampal MR mRNA in control, insulin-, and phloridzin-treated diabetic rats but not uncontrolled diabetic rats, whereas glucocorticoid receptor mRNA was not altered by hypoglycemia. In conclusion, despite elevated basal HPA activity, HPA responses to hypoglycemia were markedly reduced in uncontrolled diabetes. We speculate that defects in the CRH response may be related to a defective MR response. It is intriguing that phloridzin did not restore basal HPA activity but it restored the HPA response to hypoglycemia, suggesting that defects in basal HPA function in diabetes are due to insulin deficiency, but impaired responsiveness to hypoglycemia appears to stem from chronic hyperglycemia.