Weeks In Diabetic Rats Research Articles

Treatment with Cyclohexenonic Long-Chain Fatty Alcohol Reverses Diabetes-Induced Tracheal Dysfunction in the Rat

In this study, we tried to elucidate the effect of cyclohexenonic long-chain fatty alcohol (N-hexacosanol) on tracheal dysfunction in diabetic rats. Diabetes was induced in 8-week-old male Sprague-Dawley rats by administering an intraperitoneal injection of 50 mg/kg streptozotocin. Non-diabetic control rats received an injection of citrate-phosphate buffer alone. Four weeks after the induction of diabetes, rats were randomly divided into 5 groups: age-matched non-diabetic control rats (group A); 4-week diabetic rats without N-hexacosanol treatment (group B); diabetic rats treated with vehicle (group C), and diabetic rats treated with N-hexacosanol at a dose of 2 or 8 mg/kg i.p. every day for the following 4 weeks (group D and group E, respectively; n = 6–8 animals in each group). Serum glucose and insulin levels were determined, as were the contractile responses induced by carbachol and 100 mmol/l KCl. The participation of M₂ and M₃ receptors was investigated in the trachea by real-time polymerase chain reaction (PCR), hematoxylin and eosin (HE) and immunohistochemical staining. Hypertrophy of airway smooth muscle was observed in diabetic rats, and was ameliorated by treatment with N-hexacosanol. Treatment with either 2 or 8 mg/kg N-hexacosanol did not alter diabetic rat status, i.e., body weight, serum glucose or serum insulin levels, but it significantly reversed the decrease in tracheal wall thickness and diabetes-induced hypercontractility in the rat trachea. In the immunohistochemical studies, muscarinic M₂ and M₃ receptors were expressed in the airway smooth muscle, the elastic fibers, the fibroblast and the surface of epithelium, and these expressions were not altered by either induction of diabetes or N-hexacosanol treatment. The expression of M₃ muscarinic receptor mRNAs in the trachea tended to be increased by the induction of diabetes and normalized when treated with N-hexacosanol. Our data indicate that N-hexacosanol could reverse diabetes-induced hypercontractility in the rat trachea.

Enhancement of methoxamine‐induced contractile responses of rat ventricular muscle in streptozotocin‐induced diabetes is associated with α1A adrenoceptor upregulation

To clarify the time-related changes in cardiac function and the mechanism underlying the cardiac dysfunction present in diabetes mellitus, we studied mechanical responses induced by alpha(1)- and beta-adrenoceptors, the Ca(2+)-entry promoter Bay K 8644- and ryanodine (an agent known to inhibit Ca(2+) release from the sarcoplasmic reticulum) in papillary muscles from streptozotocin (STZ)-induced diabetic and age-matched control rats. Male Wistar rats received a single injection of STZ (60 mg kg(-1)) via the tail vein to induce diabetes. For the mechanical studies, papillary muscle preparations were suspended in an organ bath and isometric contractions were measured in 1-, 4-, and 10-week STZ-induced diabetic and age-matched control rats. In 1-week diabetic rats, the contractions induced by isoproterenol, methoxamine and Bay K 8644 were unchanged (vs. age-matched controls). In 4-week diabetic rats, (a) the isoproterenol- and Bay K 8644-induced contractions were impaired, (b) sensitivity to ryanodine was reduced, whereas (c) the methoxamine-induced contraction was unchanged. In 10-week diabetic rats, the isoproterenol- and Bay K 8644-induced contractile responses were impaired and the sensitivity to ryanodine was reduced, but in sharp contrast the methoxamine-induced contraction was enhanced. Both the mRNA level for the alpha(1A) adrenoceptor (but not the alpha(1B) or alpha(1D) mRNAs) and alpha(1A) adrenoceptor protein were increased in 10-week diabetic rats (vs. age-matched controls). These results suggest that impairments of beta-adrenergic and Ca(2+)-handling mechanisms occur early in the development of cardiomyopathy in STZ-induced diabetic rats, and that this is followed by augmentation of alpha(1A) adrenoceptor-mediated inotropy due to alpha(1A) adrenoceptor upregulation.

High glucose increases B1-kinin receptor expression and signaling in endothelial cells

The loss of endothelial function is the initiating factor in the development of diabetic vascular disease. Kinins control endothelial function by the activation of two receptors: the B2 which is constitutively expressed, and the B1 which is highly induced in pathological conditions. In the present study, we observed that the levels of B1-receptor mRNA and protein are induced in endothelial cells incubated in high glucose. An increase in B1-receptor was also observed in the endothelial layer of aortas, from 4-week diabetic rats. When cells were grown in high glucose, the B1 agonist des-Arg 9-BK increased nitrite levels, whereas in normal glucose nitrite levels were unchanged. Nitrite increase was blocked by L-NAME and 1400W indicating the participation of the inducible Nitric Oxide Synthase (iNOS). iNOS protein levels were also increased in high glucose. These results demonstrate the participation of the B1 receptor in the signaling pathways mediated by kinins in high glucose.

Arterial stiffening and cardiac hypertrophy in a new rat model of type 2 diabetes

We determined the effects of NIDDM on haemodynamic parameters describing arterial wall elasticity and cardiac hypertrophy in rats administered streptozotocin (STZ) and nicotinamide (NA), using the aortic impedance analysis. Male Wistar rats at 2 months were administered intraperitoneally 180 mg kg(-1) of NA, 30 min before an intravenous injection of 50 mg kg(-1) STZ, to induce type 2 diabetes. The STZ-NA rats were divided into two groups, 4 weeks and 8 weeks after induction of diabetes, and compared with untreated age-matched controls. Pulsatile aortic pressure and flow signals were measured by a high-fidelity pressure sensor and electromagnetic flow probe, respectively, and were then subjected to Fourier transformation for the analysis of aortic input impedance. In each diabetic group, the experimental syndrome was characterized by a moderate and stable hyperglycaemia and a relative deficiency of insulin secretion. However, the 8-week but not the 4-week STZ-NA diabetic rats showed a decrease in cardiac output in the absence of any significant changes in mean aortic pressure, having increased total peripheral resistance. The diabetic syndrome at 8 weeks also contributed to an increase in aortic characteristic impedance, from 1.49 +/- 0.33 (mean +/- SD) to 1.95 +/- 0.28 mmHg s mL(-1) (P < 0.05), suggesting a detriment to the aortic distensibility in NIDDM. Meanwhile, the STZ-NA diabetic animals after 8 weeks had an increased wave reflection factor (0.46 +/- 0.09 vs. 0.61 +/- 0.13, P < 0.05) and decreased wave transit time (25.8 +/- 3.8 vs. 20.6 +/- 2.8 ms, P < 0.05). Ratio of the left ventricular weight to body weight was also enhanced in the 8-week STZ-NA diabetic rats. The heavy intensity with early return of the pulse wave reflection may augment systolic load of the left ventricle coupled to the arterial system, leading to cardiac hypertrophy in the rats at 8 weeks after following STZ and NA administration.

Effects of a selective bradykinin B 1 receptor antagonist on increased plasma extravasation in streptozotocin-induced diabetic rats: Distinct vasculopathic profile of major key organs

Diffuse vasculopathy is a common feature of the morbidity and increased mortality associated with insulino-dependent type 1 diabetes. Increased vascular permeability leading to plasma extravasation occurs in surrounding tissues following endothelial dysfunction. Such micro- and macro-vascular complications develop over time and lead to oedema, hypertension, cardiomyopathy, renal failure (nephropathy) and other complications (neuropathy, retinopathy). In the present investigation, we studied the effect of a selective bradykinin B 1 receptor antagonist, R-954, on the enhanced vascular permeability in streptozotocin (STZ)-induced diabetic Wistar rats compared with age-matched controls. Plasma extravasation was determined using Evans blue dye in selected target tissues (left and right heart atria, ventricles, lung, abdominal and thoracic aortas, liver, spleen, renal cortex and medulla), at 1 and 4 weeks following STZ administration. The vascular permeability was significantly increased in the aortas, cortex, medulla, and spleen in 1-week STZ rats and remained elevated at 4 weeks of diabetes. Both atria showed an increased vascular permeability only after 4-week STZ-administration. R-954 (2 mg/kg, bolus, s.c.), given 2 h prior to Evans blue dye, to 1- and 4-week diabetic rats significantly inhibited (by 48–100%) plasma leakage in most tested tissues affected by diabetes with no effect in healthy rats. These results showed that the inducible bradykinin B 1 receptor subtype participates in the modulation of the vascular permeability in diabetic rats and suggest that selective bradykinin B 1 receptor antagonism could have a beneficial role in reducing diabetic vascular complications.

Role of protein kinase C on the alteration of retinal endothelin-1 in streptozotocin-induced diabetic rats

Activation of protein kinase C (PKC) has been implicated in the pathogenesis of diabetic retinopathy. The purpose of this study was to investigate the role of PKC on the alteration of retinal endothelin-1 in 2-week streptozotocin (STZ)-induced diabetic rats. The measurement of retinal PKC activities from membranous and cytosolic fractions was conducted by ELISA. Retinal tissues were analysed for the expression of endothelin-1 (ET-1), endothelin-3 (ET-3), endothelin-A (ET-A), and endothelin-B (ET-B) mRNA by means of semi-quantitative RT-PCR. Retinal vasculature isolated by trypsin digest technique was immunostained for ET-1. We followed the alteration of retinal ET-1 after intravitreal injection of a general PKC inhibitor, GF109203X, in 2-week diabetic rats. Retinal PKC specific activities were significantly increased by 37% ( P=0·027) in the membranous fraction in diabetic rats compared with normal rats, whereas PKC specific activities in the cytosolic fraction were unchanged. The retina from the diabetic rats showed increased ET-1 mRNA expression after 2 weeks, while no changes were found for ET-3, ET-A and ET-B. ET-1 immunoreactivity was also increased in the retinal vasculature of diabetic rats. Retinal ET-1 expression was decreased after intravitreal injection of GF109203X (10 −5, 10 −6, 10 −7 m) in a dose-dependent manner. The results from this study showed that the enhanced ET-1 expression associated with the activation of PKC has occurred in early diabetes, and PKC inhibitor could reverse the up regulation of ET-1.

β-Adrenergic activation reveals impaired cardiac calcium handling at early stage of diabetes

Cardiac function is known to be impaired in diabetes. Alterations in intracellular calcium handling have been suggested to play a pivotal role. This study aimed to test the hypothesis that β-adrenergic activation can reveal the functional derangements of intracellular calcium handling of the 4-week diabetic heart. Langendorff perfused hearts of 4-week streptozotocin-induced diabetic rats were subjected to the β-adrenoceptor agonist isoproterenol. Cyclic changes in [Ca 2+] i levels were measured throughout the cardiac cycle using Indo-1 fluorescent dye. Based on the computational analysis of the [Ca 2+] i transient the kinetic parameters of the sarcoplasmic reticulum Ca 2+-ATPase and the ryanodine receptor were determined by minimizing the squared error between the simulated and the experimentally obtained [Ca 2+] i transient. Under unchallenged conditions, hemodynamic parameters were comparable between control and diabetic hearts. Isoproterenol administration stimulated hemodynamic function to a greater extent in control than in diabetic hearts, which was exemplified by more pronounced increases in rate of pressure development and decline. Under unchallenged conditions, [Ca 2+] i amplitude and rate of rise and decline of [Ca 2+] i as measured throughout the cardiac cycle were comparable between diabetic and control hearts. Differences became apparent under β-adrenoceptor stimulation. Upon β-activation the rate-pressure product showed a blunted response, which was accompanied by a diminished rise in [Ca 2+] i amplitude in diabetic hearts. Computational analysis revealed a reduced function of the sarcoplasmic reticulum Ca 2+-ATPase and Ca 2+-release channel in response to β-adrenoceptor challenge. Alterations in Ca 2+ i handling may play a causative role in depressed hemodynamic performance of the challenged heart at an early stage of diabetes.

Biomechanical properties of the rat oesophagus in experimental type-1 diabetes.

The gastrointestinal tract remodels its morphology and circumferential stress-strain properties in diabetes mellitus. This study adds one more piece of mechanical knowledge, namely the oesophageal shear modulus and its dependence on the circumferential and longitudinal stresses and strains of oesophagus in diabetic rats and control rats. Diabetes was induced by a single intraperitoneal injection of streptozotocin (50 mg kg(-1) body weight). The diabetic rats lived up to 28 days after the induction of diabetes. The oesophagus was studied in vitro using a triaxial machine. Stepwise elongation and inflation plus continuous twist were carried out with measurement of the resultant forces. Circumferential and longitudinal stresses and strains were computed from steady-state values of longitudinal force, outer diameter and the applied pressure. The shear modulus was computed from the twist angle-torque relationship. The circumferential and longitudinal stress-strain relationships were exponential. The circumferential stiffness increased 1 week after induction of diabetes (P < 0.001). The longitudinal stiffness increased in the 4-week diabetic rats (P < 0.001). The shear modulus varied as function of longitudinal strain and pressure. The oesophagus became stiffer in shear 4 weeks after the induction of diabetes (P < 0.001).

Diabetes Mellitus Increases the Rate of Development of Decompensation in Rats With Outlet Obstruction

We investigated the effects of the combination of bladder outlet obstruction and diabetes mellitus on in vitro rat bladder body strip function. Longitudinal strips were removed from ventral and dorsal detrusor of age matched control, 2-week diabetic, 2-week obstructed and 2-week obstructed diabetic rats. Contractile responses to electrical field stimulation, carbachol, adenosine triphosphate, KCl and phenylephrine, and relaxations in response to norepinephrine and isoproterenol were measured. Bladders from diabetic, obstructed and obstructed diabetic rats were 1.6-fold, 2.6-fold and 3.6-fold heavier than those from controls. Responses of bladder strips from diabetics to all stimuli were similar to those of controls. Strips from obstructed rats were significantly less responsive to norepinephrine than those from controls or diabetics and strips from obstructed diabetics were significantly less responsive to norepinephrine and isoproterenol than those from all other groups. Strips from obstructed diabetics had significantly decreased responses to field stimulation, while responses to carbachol were decreased to a lesser extent. Responses of strips from obstructed rats to field stimulation were also decreased compared with controls but were significantly greater than those of the obstructed diabetic group. Responses to adenosine triphosphate, KCl and phenylephrine were similar in all groups. The combination of outlet obstruction and diabetes mellitus causes significant increases in bladder mass compared with either diabetes or obstruction alone. Bladder strips from obstructed diabetics show characteristics of denervation accompanied by alterations in beta-adrenergic function, suggesting that the coexistence of outlet obstruction and diabetes increases the rate of development of bladder decompensation.

Diabetes increases formation of advanced glycation end products on Sarco(endo)plasmic reticulum Ca2+-ATPase.

Prolongation of relaxation is a hallmark of diabetic cardiomyopathy. Most studies attribute this defect to decreases in sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2a) expression and SERCA2a-to-phospholamban (PLB) ratio. Since its turnover rate is slow, SERCA2a is susceptible to posttranslational modifications during diabetes. These modifications could in turn compromise conformational rearrangements needed to translocate calcium ions, also leading to a decrease in SERCA2a activity. In the present study one such modification was investigated, namely advanced glycation end products (AGEs). Hearts from 8-week streptozotocin-induced diabetic (8D) rats showed typical slowing in relaxation, confirming cardiomyopathy. Hearts from 8D animals also expressed lower levels of SERCA2a protein and higher levels of PLB. Analysis of matrix-assisted laser desorption/ionization time-of-flight mass data files from trypsin-digested SERCA2a revealed several cytosolic SERCA2a peptides from 8D modified by single noncrosslinking AGEs. Crosslinked AGEs were also found. Lysine residues within actuator and phosphorylation domains were cross-linked to arginine residues within the nucleotide binding domain via pentosidine AGEs. Two weeks of insulin-treatment initiated after 6 weeks of diabetes attenuated these changes. These data demonstrate for the first time that AGEs are formed on SERCA2a during diabetes, suggesting a novel mechanism by which cardiac relaxation can be slowed during diabetes.

Blood-retinal barrier breakdown induced by activation of protein kinase C via vascular endothelial growth factor in streptozotocin-induced diabetic rats

Purpose. To investigate (1) the mechanism of blood-retinal barrier breakdown induced by protein kinase C (PKC) activation (2) the relationship between PKC activation and vascular endothelial growth factor (VEGF) in 2-week streptozotocin-induced diabetes. Methods. Retinal PKC activities, retinal and vitreous concentration of VEGF protein were conducted by enzyme-linked immunoabsorbent assay (ELISA). Retinal VEGF mRNA expression was analyzed by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). Blood-retinal barrier breakdown was quantified using the Evans blue technique. Alteration of retinal VEGF and blood-retinal barrier were observed after intravitreal injection of PKC inhibitor, GF109203X, in 2-week diabetic rats. Results. Two weeks of diabetes in rats resulted in elevation of retinal PKC specific activities, retinal and vitreous VEGF, and retinal vascular permeability. Retinal VEGF and retinal vascular permeability were decreased after intravitreal injection of GF109203X (10 -5, 10 -6 mol/L) in a dose-dependent manner. Conclusions. The results from this study showed that enhanced expression of VEGF and PKC in early diabetes and the blood-retinal barrier breakdown of early diabetic retinopathy induced by PKC activation may be partly due to the upregulation of VEGF. PKC inhibitor could reverse the blood-retinal barrier breakdown.

Effect of Streptozotocin-Induced Diabetes on Rat Brain Mitochondria

This study evaluated several parameters related to mitochondrial function and oxidative stress in streptozotocin (STZ)-treated rats, a model of type 1 diabetes. For this purpose, the respiratory indexes (RCR and ADP/O ratio), mitochondrial transmembrane potential (ΔΨm), repolarization lag phase, repolarization level, mitochondrial enzymatic activities, ATP and malondialdehyde (MDA) levels, reduced glutathione (GSH), vitamin E and cardiolipin contents were determined in rat brain mitochondria isolated after 4 and 9 weeks after STZ treatment. Brain mitochondria isolated from citrate (vehicle)-treated Wistar rats were used as control. We observed that STZ-induced diabetes did not substantially affect brain mitochondrial function. Instead, 4-week diabetic rats presented higher mitochondrial respiratory chain enzymatic activities, especially succinate-cytochrome C reductase activity, compared to 4-week control rats. In 9-week diabetic rats, only a significant decrease in cardiolipin content was observed; however, a significant increase in mitochondrial GSH content occurred. All other parameters analysed remained statistically unchanged. From these results, we conclude that STZ-induced diabetes did not promote brain mitochondrial dysfunction, suggesting that oxidative stress associated with type 1 diabetes is not directly related to mitochondrial dysfunction, but probably is related to extramitochondrial factor(s).

Effect of Streptozotocin‐Induced Diabetes on Rat Brain Mitochondria

This study evaluated several parameters related to mitochondrial function and oxidative stress in streptozotocin (STZ)-treated rats, a model of type 1 diabetes. For this purpose, the respiratory indexes (RCR and ADP/O ratio), mitochondrial transmembrane potential (DeltaPsim), repolarization lag phase, repolarization level, mitochondrial enzymatic activities, ATP and malondialdehyde (MDA) levels, reduced glutathione (GSH), vitamin E and cardiolipin contents were determined in rat brain mitochondria isolated after 4 and 9 weeks after STZ treatment. Brain mitochondria isolated from citrate (vehicle)-treated Wistar rats were used as control. We observed that STZ-induced diabetes did not substantially affect brain mitochondrial function. Instead, 4-week diabetic rats presented higher mitochondrial respiratory chain enzymatic activities, especially succinate-cytochrome C reductase activity, compared to 4-week control rats. In 9-week diabetic rats, only a significant decrease in cardiolipin content was observed; however, a significant increase in mitochondrial GSH content occurred. All other parameters analysed remained statistically unchanged. From these results, we conclude that STZ-induced diabetes did not promote brain mitochondrial dysfunction, suggesting that oxidative stress associated with type 1 diabetes is not directly related to mitochondrial dysfunction, but probably is related to extramitochondrial factor(s).

Diabetes can alter the signal transduction pathways in the lens of rats.

Diabetes is known to affect cataract formation by means of osmotic stress induced by activated aldose reductase in the sorbitol pathway. In addition, alterations in the bioavailability of numerous extralenticular growth factors has been reported and shown to result in various consequences. We have found that the basic fibroblast growth factor (bFGF) accumulates in the vitreous humor of 3- and 8-week diabetic rats. Consequently, the associating signal transduction cascades were severely disrupted, including upregulated phosphorylation of extracellular signal-regulated kinase (ERK) and the common stress-associated mitogen-activated protein kinases p38 and SAPK/JNK. Conversely, under diabetic condition, we observed a dramatic inhibition of phosphatidylinositol-3 kinase activity in lenses obtained from the same animal. Rats treated with the aldose reductase inhibitor AL01576 for the duration of the diabetic condition showed that the diabetes-induced lenticular signaling alterations were normalized, comparable to controls. However, treatment of AL01576 in vitro was ineffective at normalizing the altered constituents in extracted diabetic vitreous after the onset of diabetes. The effect of AL01576 in the high galactose-induced cataract model in vitro was also examined. Administration of AL01576 to lens organ culture normalized the aberrant signaling effects and morphological characteristics associated with in vitro sugar cataract formation. In conclusion, our findings demonstrate diabetes-associated alterations in the lens signal transduction parameters and the effectiveness of AL01576 at normalizing such alterations. The causes for these alterations can be attributed to elevated vitreal bFGF in conjunction with osmotic stress and associated attenuation in redox status of the lens.

Role of Protein Kinase C on the Expression of Platelet-Derived Growth Factor and Endothelin-1 in the Retina of Diabetic Rats and Cultured Retinal Capillary Pericytes

Increased expression of endothelin-1 (ET-1) is associated with diabetic retinopathy and vasculopathy, although the molecular explanation has not been defined. The effects of high glucose and protein kinase C (PKC) activation on platelet-derived growth factor (PDGF)-BB and of ET-1 expression in the retina of streptozotocin (STZ)-induced diabetic rats and bovine retinal pericytes (BRPC) were examined. In 4-week diabetic rats, PDGF-B and prepro-ET-1 (ppET-1) mRNA levels increased significantly by 2.8- and 1.9-fold, respectively, as quantified by RT-PCR. Treatment with PKC-beta isoform-specific inhibitor (LY333531) or insulin normalized retinal ET-1 and PDGF-B expression. In BRPC, high glucose levels increased ppET-1 and PDGF-B mRNA expression by 1.7- and 1.9-fold, respectively. The addition of PDGF-BB but not PDGF-AA increased expression of ppET-1 and vascular endothelial growth factor mRNA by 1.6- and 2.1-fold, respectively, with both inhibited by AG1296, a selective PDGF receptor kinase inhibitor. A general PKC inhibitor, GF109203X, suppressed PDGF-BB's induction of ET-1 mRNA. Thus, increased ET-1 expression in diabetic retina could be due to increased expression of PDGF-BB, mediated via PDGF-beta receptors in part by PKC activation. The novel demonstration of elevated expression of PDGF-B and its induction by PKC activation identifies a potential new molecular step in the pathogenesis of diabetic retinopathy.

Chronic diabetes alters function and expression of ryanodine receptor calcium-release channels in rat hearts.

Alteration in cardiac function is one of the hallmarks of diabetes and in late stage is manifested as a decrease in contractility. While it is established that the release of calcium ions from internal sarcoplasmic reticulum via type 2 ryanodine receptor calcium-release channels (RyR2) is vital for efficient contraction, the relationship between diabetes-induced decrease in cardiac performance and alterations in expression and/or function of RyR2 is not well delineated. The present study was designed to address this question and to determine whether changes to RyR2 induced by chronic diabetes could be minimized with insulin-treatment. When paced at 3.3 Hz (200 beats per minute), hearts from 8-week streptozotocin-induced diabetic rats showed decreased responsiveness to isoproterenol stimulation; +dT/dt and -dT/dt were 56.5 +/- 11.4% and 42.1 +/- 12.1% that of control, respectively. Hearts from 8-week diabetic rats expressed 51.2% less RyR2 than controls, In addition, RyR2 from diabetic rats also showed decreased ability to bind the specific ligand [3H]ryanodine (22.4 +/- 1.8% less [3H]ryanodine per microg of RyR2 protein), suggesting dysfunction. Two-weeks of insulin treatment, initiated after 6 weeks of untreated diabetes was able to minimize loss in function and expression of RyR2. Taken collectively, these data suggest that the decrease in cardiac contractility induced by chronic diabetes results in part from decreases in expression and alteration in function of RyR2 and these changes could be attenuated with insulin treatment.

Effects of Ba-Wei-Die-Huang-Wan on the cholinergic function and protein expression of M 2 muscarinic receptor of the urinary bladder in diabetic rats

The study investigated the effect of a traditional Chinese herbal preparation ‘Ba-Wei-Die-Huang-Wan’ on the cholinergic function of the urinary bladder in diabetic rats, as well as its influence on the protein expression of muscarinic M 2 receptors. Wistar rats were divided into three groups: (1) control rats; (2) 2-week streptozotocin-induced diabetic rats; and (3) diabetic rats treated with Ba-Wei-Die-Huang-Wan 26 mg/kg thrice daily for 3 days. A dose-response study was performed for bladder muscle strip contractile response to a muscarinic agonist, arecaidine propargyl ester (APE). The amount of M 2 receptor protein in the bladder was measured by Western immunoblotting using monoclonal antibodies. The bladder contractile response to APE was significantly increased in the diabetic rats compared to the control. The M 2 receptor protein density was also significantly higher by 106% ( P<0.01, n=8) in diabetes. Ba-Wei-Die-Huang-Wan treatment significantly lowered the plasma glucose level of the diabetic rats. The increases in contractile response to APE as well as the M 2 receptor protein density were partially reversed by the treatment. In conclusion, there was an over-expression of M 2 receptor resulting in hyper-contractility in the bladder of diabetic rats. Ba-Wei-Die-Huang-Wan significantly alleviated the alterations.

Systolic elastance and resistance in the regulation of cardiac pumping function in early streptozotocin-diabetic rats.

We determined the roles of maximal systolic elastance (E(max)) and theoretical maximum flow ((max)) in the regulation of cardiac pumping function in early streptozotocin (STZ)-diabetic rats. Physically, E(max) can reflect the intrinsic contractility of the myocardium as an intact heart, and (max) has an inverse relation to the systolic resistance of the left ventricle. Rats given STZ 65 mg/kg i.v. (n = 17) were divided into two groups, 1 week and 4 weeks after induction of diabetes, and compared with untreated age-matched controls (n = 15). Left ventricular (LV) pressure and ascending aortic flow signals were recorded to calculate E(max) and (max), using the elastance-resistance model. After 1 or 4 weeks, STZ-diabetic animals show an increase in effective LV end-diastolic volume (V(eed)), no significant change in peak isovolumic pressure (P(iso)(max)), and a decline in effective arterial volume elastance (E(a)). The maximal systolic elastance E(max) is reduced from 751.5 +/- 23.1 mmHg/ml in controls to 514.1 +/- 22.4 mmHg/ml in 1- and 538.4 +/- 33.8 mmHg/ml in 4-week diabetic rats. Since E(max) equals P(iso)(max)/V(eed), an increase in V(eed) with unaltered P(iso)(max) may primarily act to diminish E(max) so that the intrinsic contractility of the diabetic heart is impaired. By contrast, STZ-diabetic rats have higher theoretical maximum flow (max) (40.9 +/- 2.8 ml/s in 1- and 44.5 +/- 3.8 ml/s in 4-week diabetic rats) than do controls (30.7 +/- 1.7 ml/s). There exists an inverse relation between (max) and E(a) when a linear regression of (max) on E(a) is performed over all animals studied (r = 0.65, p < 0.01). The enhanced (max) is indicative of the decline in systolic resistance of the diabetic rat heart. The opposing effects of enhanced (max) and reduced E(max) may negate each other, and then the cardiac pumping function of the early STZ-diabetic rat heart could be preserved before cardiac failure occurs.

Alteration of M3 Subtype Muscarinic Receptors in the Diabetic Rat Urinary Bladder

The M₃ receptor (M₃-mAChR) is the major muscarinic subtype in the animal bladder responsible for detrusor contraction. The alterations in its protein quantity and biosynthesis during diabetic cystopathy were investigated. 3-month-old male Wistar rats were divided into two groups: (1) 2-week diabetic rats and (2) normoglycemic control rats. Diabetes was induced by a single intravenous injection of 60 mg/kg streptozotocin. The amount of M₃ receptor protein in the rat bladder body tissue was measured by Western immunoblotting using monoclonal antibodies. For determination of M₃ muscarinic receptor mRNA in the bladder tissue, the method of Northern blotting was employed. The results of the Western immunoblotting showed that the amount of M₃-mAChR protein in the diabetic bladder was significantly increased by about 70.2 ± 8.5% when compared to the control bladder (p < 0.05, n = 8). Northern blotting demonstrated a 54.7 ± 6.0% increase of M₃-mAChR mRNA in the diabetic bladder (p < 0.05, n = 8). The findings of the present study demonstrated an upregulation of M₃-mAChR biosynthesis in the diabetic urinary bladder. This phenomenon offers an explanation of the increased contractility after muscarinic stimulation of the detrusor muscle of diabetic animals.

Effects of nerve growth factor on expression of GAP-43 in right atria after sympathectomy in diabetic rats.

The present study investigated the role of nerve growth factor (NGF) in the regeneration of noradrenergic nerves of right atria (following 6-hydroxydopamine; 6-OHDA, 100 mg/kg, i.p.) from non-diabetic and 8-week diabetic rats. In cryostat sections of the right atria, GAP-43 immunoreactivity was concentrated in nerve terminals, preterminal axons of the endocardium, epicardium and myocardium, as well as in nerve fibres innervating the blood vessels and ganglionic cells. In serial sections, all positive staining for GAP-43 showed immunoreactivity for the neuronal marker PGP-9.5. In untreated non-diabetic rats, the total GAP-43 immunoreactivity was reduced to 60% relative to pretreatment levels, at day 14 after 6-OHDA, as quantified by Western blotting. In diabetic rats, 6-OHDA treatment produced a marked increase in the levels of total GAP-43 at days 28 and 49. NGF treatment (1 mg/kg, s.c., 3 times/week, for 2 weeks) had no effect on the level of total GAP-43 in right atria from non-diabetic and diabetic rats before treatment with 6-OHDA. However, it normalized the reduced GAP-43 immunoreactivity observed in 6-OHDA-treated non-diabetic rats. Interestingly, NGF treatment alone produced an increase in GAP-43 phosphorylation relative to total GAP-43 in right atria from both non-diabetic (44%) and diabetic groups (42%). These findings suggest that nerve terminals of the right atria retain, in the mature adult, the capacity for structural and functional plasticity. The expression of GAP-43 in right atria of control and diabetic rats was differentially affected by 6-OHDA treatment. In injured noradrenergic neurones of the right atria, NGF modified the expression of GAP-43 only in non-diabetic rats and not in diabetic rats.