Phosphodiesterase 5 inhibitor suppresses prostate weight increase in type 2 diabetic rats.

AIMS: Hyperinsulinemia is an important causative factor of prostate enlargement in type 2 diabetes (T2D), however, clinically prostate weight increases during hypoinsulinemic condition. To investigate the pathogenesis of prostate enlargement and effects of phosphodiesterase 5 inhibitor (PDE5i), male Otsuka Long-Evans Tokushima Fatty (OLETF) and Long-Evans Tokushima Otsuka (LETO) rats were used as T2D and control, respectively. MATERIALS AND METHODS: OLETF and LETO rats were treated with oral tadalafil (100 µg/kg/day) or vehicle for 12 wks from at the age of 36 wks. KEY FINDINGS: Prostate weight of OLETF rats was significantly higher than that of LETO at 36 wks, and increased at 48 wks. In OLETF rats, prostate blood flow was significantly lower at 48 wks versus 36 wks. Twelve-week-tadalafil treatment increased prostate blood flow and suppressed prostate weight increase in both strains. This change was inversely correlated with changes in prostate expressions of hypoxia-inducible factor-1 alpha (HIF-1α) and 8-hydroxy-2'-deoxyguanosine (8-OHdG). Increases with age were observed in mRNA and/or protein levels of cytokines interleukin (IL)-6, IL-8, and tumor necrosis factor-alpha (TNF-α) and cell growth factors insulin-like growth factor-1 (IGF-1), basic fibroblast growth factor (bFGF), and transforming growth factor-beta (TGF-ß); especially IL-6, TNF-α, IGF-1, bFGF and TGF-ß increased with T2D. Tadalafil suppressed these cytokines and growth factors. SIGNIFICANCE: These data suggest chronic ischemia caused by T2D leads to oxidative stress, resulting in prostate enlargement through upregulation of several cytokines and growth factors. Treatment with PDE5i improves prostate ischemia and might prevent enlargement via suppression of cytokines and growth factors in T2D.

Urinary reabsorption in the rat kidney by anticholinergics.

Anticholinergics, therapeutic agents for overactive bladder, are clinically suggested to reduce urine output. We investigated whether this effect is due to bladder or kidney urine reabsorption. Various solutions were injected into the bladder of urethane-anesthetized SD rats. The absorption rate for 2 h was examined following the intravenous administration of the anticholinergics imidafenacin (IM), atropine (AT), and tolterodine (TO). The bilateral ureter was then canulated and saline was administered to obtain a diuretic state. Anticholinergics or 1-deamino-[8-D-arginine]-vasopressin (dDAVP) were intravenously administered. After the IM and dDAVP administrations, the rat kidneys were immunostained with AQP2 antibody, and intracellular cAMP was measured. The absorption rate was ~ 10% of the saline injected into the bladder and constant even when anticholinergics were administered. The renal urine among peaked 2 h after the saline administration. Each of the anticholinergics significantly suppressed the urine production in a dose-dependent manner, as did dDAVP. IM and dDAVP increased the intracellular cAMP levels and caused the AQP2 molecule to localize to the collecting duct cells' luminal side. The urinary reabsorption mechanism through the bladder epithelium was not activated by anticholinergic administration. Thus, anticholinergics suppress urine production via an increase in urine reabsorption in the kidneys' collecting duct cells via AQP2.

Correcting imbalance of sex hormones by a phosphodiesterase 5 inhibitor improves copulatory dysfunction in male rats with type 2 diabetes.

INTRODUCTION: Sexual dysfunction is a common complication in men with type 2 diabetes and is often refractory to treatment. This study investigated the long-term influence of the phosphodiesterase 5 inhibitor (PDE5I) tadalafil on the level of sex hormones and sexual function in male Otsuka Long-Evans Tokushima Fatty (OLETF) rats as an animal model of spontaneous type 2 diabetes. RESEARCH DESIGN AND METHODS: We treated 36-week-old male OLETF and non-diabetic Long-Evans Tokushima Otsuka (LETO) rats with oral tadalafil (100 µg/kg/day) for 12 weeks; sham groups received vehicle for 12 weeks. Before and after tadalafil treatment, serum levels of total and free testosterone, estradiol, luteinizing hormone (LH), follicle-stimulating hormone and proinflammatory cytokines were compared among four treatment groups. Copulatory function was examined by matching each rat to an estrous female. After completion of the experiment, total fat mass in the abdomen was measured. RESULTS: Testosterone levels were significantly lower in OLETF versus LETO rats at 36 weeks. After 12 weeks of tadalafil treatment, levels of testosterone were significantly increased both in OLETF-tadalafil and LETO-tadalafil groups versus vehicle groups. Tadalafil decreased estradiol levels both in OLETF and LETO rats. Furthermore, tadalafil increased serum LH levels with a reduction of proinflammatory cytokines. Total fat mass was significantly lower in the OLETF-tadalafil group versus the OLETF-vehicle group. A significant suppression of copulatory behavior, that is, elongation of intromission latency was found in OLETF rats. However, tadalafil treatment for 12 weeks shortened the intromission latency. CONCLUSION: Our results indicate that tadalafil treatment might improve copulatory disorder in the type 2 diabetic model via improvement of an imbalance in sex hormones and an increase in LH levels.

Role of corticotropin-releasing factor on bladder function in rats with psychological stress.

Stress-related peptide corticotropin-releasing factor (CRF) and CRF-related peptides are distributed in the peripheral viscera such as the bladder. We investigated the contribution of psychological stress (PS) and CRF on bladder function. Male rats received sham stress (SS) or PS using a communication box method for 120 min every day for 7 days. One group of rats received the intraperitoneal CRF-R1 antagonist antalarmin for 7 days during stress exposure. Mean voided volume per micturition was significantly lower in PS rats compared to SS rats, which was antagonized by antalarmin treatment. Increases in plasma and bladder CRF, and mRNA expressions of bladder CRF, CRF-R1, and M2/3 muscarinic receptors, were found in PS rats. CRF did not influence bladder contraction in itself; however, stress increased the response of muscarinic contraction of bladder strips. These changes were antagonized by antalarmin treatment. In conclusion, PS reinforces M3 receptor-mediated contractions via CRF-R1, resulting in bladder storage dysfunction.

Castration increases PGE2 release from the bladder epithelium in male rats.

AIMS: Androgen deprivation therapy has been widely used for the treatment of prostate cancer. While sexual side effects including decreased sexual desire and function are well studied, there are only limited reports about its influences on lower urinary tract symptoms. The aim of this study is to clarify the influences of castration in male rats. METHODS: Ten-week-old male rats were divided into treatment group (bilateral orchiectomy) and control group (sham surgery). Two-months after the surgery, adenosine triphosphate (ATP), prostaglandin E2 (PGE2), and nerve growth factor (NGF) released from stretched bladder epithelium were measured by luciferin-luciferase assay or ELISA. The mRNA levels of bladder cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) were determined by real-time PCR. The protein level of bladder COX-2 was analyzed by western blot analysis. Bio-Plex Pro cytokine assay was performed to quantify the level of proinflammatory cytokine interleukin (IL)-1ß in the bladder. RESULTS: The PGE2 release from stretched bladder epithelium was significantly increased after castration, which increased more than 50% compared with control. On the other hand, those of ATP and NGF were not different from those of the controls. Testosterone replacement restored the PGE2 increase. Castration significantly increased bladder IL-1ß protein level and COX-2 at both mRNA and protein levels, whereas caused no marked changes in the COX-1 mRNA level. CONCLUSIONS: These findings suggest that castration induces inflammation in the rat bladder, which causes elevated PGE2 release from bladder epithelium and may finally contribute to the disruption of bladder storage function.

Nerve growth factor release from the urothelium increases via activation of bladder C-fiber in rats with cerebral infarction.

AIMS: There are some reports that bladder C-fibers are partially involved in detrusor overactivity in patients with brain lesions. We investigated the contribution of bladder C-fiber to decreased bladder capacity in rats with cerebral infarction. METHODS: Cerebral infarction was induced under halothane anesthesia by left middle cerebral artery occlusion with 4-0 nylon thread in female Sprague-Dawley rats. Intramural amounts of ATP and prostaglandin E2 , in vivo and in vitro ATP, NGF, and prostaglandin E2 release from the distended bladder urothelium, and changes in mRNA expressions of sensor molecules and receptors were monitored 6 h after the occlusion. Cystometry was performed in rats with or without resiniferatoxin pretreatment. RESULTS: Overexpression of sensor molecule, transient receptor potential vanilloid-type channel 1, acid-sensing ion channel 2, purinergic receptors P2X3 , and M2 /M3 muscarinic receptors was found in the bladder. These changes were accompanied by increases in ATP and NGF release from the urothelium. In contrast, when bladder C-fibers were desensitized by resiniferatoxin, no increase in NGF release from the urothelium was found either in vivo or in vitro. There was no difference in the percentage decrease in bladder capacity between cerebral infarction rats pretreated with resiniferatoxin and cerebral infarction rats without pretreatment. CONCLUSIONS: Results indicate that expression of sensor molecules in the bladder is altered by distant infarction in the brain. ATP and NGF release from the urothelium also increased. NGF release was related to activation of bladder C-fibers. Bladder C-fibers might not contribute much to decreased bladder capacity caused by cerebral infarction.

Underlying mechanisms of urine storage dysfunction in rats with salt-loading hypertension.

AIMS: Spontaneous hypertensive rats provide a genetic model for exploring the pathogenesis of urine storage dysfunction related to hypertension (HT). In humans, however, HT develops by both genetic and environmental factors including lifestyle factors such as a high-calorie diet, excessive salt intake and stress. We investigated the influence of salt-loading on bladder function and the underlying mechanisms of storage dysfunction related to HT. MAIN METHODS: Six-week-old male Dahl salt-sensitive (DS) and Dahl salt-resistant (DR) rats were fed with a normal or high-salt diet for 12weeks. Micturition parameters were obtained from a metabolic cage. Whole bladders were excised from 18-week-old rats and distended in an organ bath. The releases of adenosine triphosphoric acid (ATP) and prostaglandin E2 (PGE2) from the distended bladder epithelia were measured. Changes in bladder blood flow (BBF) were determined with a laser-speckle-blood-flow imaging system. KEY FINDINGS: An increase in mean blood pressure (BP) was noted only in DS rats after salt-loading. During the inactive (sleeping) period, voided volume per micturition gradually increased in DR rats fed a normal or high-salt diet and normal-diet DS rats, while it did not change in the DS rats fed a high-salt diet. Bladder distension significantly increased ATP and PGE2 release from the urothelium in DS rats fed a high-salt diet. BBF was significantly decreased in high-salt-diet DS rats. SIGNIFICANCE: One mechanism behind the relationship between salt-sensitive HT and urine storage dysfunction may be an increase in ATP and PGE2 release from the urothelium via suppression of BBF.

Effects of the 5α-reductase inhibitor dutasteride on rat prostate α1A-adrenergic receptor and its mediated contractility.

OBJECTIVE: To clarify the possible interference of the 5α-reductase inhibitor dutasteride with α-adrenergic blockers, whose action is mainly mediated by α1A-adrenergic receptor. METHODS: Male rats were divided into dutasteride and vehicle-treated groups. The drug treatment group was treated with oral dutasteride 0.5 mg/kg/d, and the control group received vehicle only for 2 months. After the 2-month treatment, the rats' ventral prostate weight changes and the testosterone and dihydrotestosterone levels in the serum were measured. In vitro organ-bath studies, real-time polymerase chain reaction, and tissue-segment binding were performed to determine the expression of α1A-adrenergic receptors and its mediated contractility. RESULTS: Dutasteride treatment significantly decreased the rats' ventral prostate weight, increased their testosterone levels, and decreased the dihydrotestosterone levels in their serum. There were no marked changes in the α1A-adrenergic receptor messenger ribonucleic acid expression, relative phenylephrine-induced contractility, or nerve-mediated contractility between the groups. Dutasteride treatment caused no marked changes in the relative binding capacity of α1A-adrenergic receptor, whereas it greatly decreased the total protein expression of this subtype and its mediated maximal contraction in the whole ventral prostate. CONCLUSION: These results suggest that dutasteride does not interfere with α-adrenergic blockers but otherwise has beneficial effects on their actions. Therefore, the long-term administration of the combination of dutasteride with an α-adrenergic blocker might be a better choice for the treatment of lower urinary tract symptoms due to benign prostatic hyperplasia.

Synthesis of silk fibroin-insulin bioconjugates and their characterization and activities in vivo.

The regenerated liquid silk fibroin with an average molecular mass of about 60 kDa consists of 18 kinds of amino acids containing approximately 10% of polar amino acids with hydroxyl and amino groups such as serine and lysine. The liquid silk fibroin is coupled covalently with insulin molecules through these strongly polar side groups by using glutaraldehyde. The physicochemical properties of the silk fibroin-insulin (SF-Ins) bioconjugates were investigated by enzyme-linked immunosorbent assay for the quantitative measurement of insulin. The biological activities of the insulin bioconjugates were characterized in vitro and in vivo. The SF-Ins constructs obtained by 5 h of covalent crosslinking showed much higher recovery (about 70%) and in vitro stability in human serum than bovine serum albumin-insulin (BSA-Ins) derivatives. The results in human serum indicated that the half-life in vitro of the biosynthesized SF-Ins derivatives was 2.1 and 1.7 times more than that of BSA-Ins conjugates and native insulin, respectively. The immunogenicity of the regenerated silk fibroin and the antigenicity of silk fibroin-modified insulin were not observed in both rabbits and rats. The pharmacological activity of the SF-Ins bioconjugates in diabetic rats evidently lengthened and was about 3.5 times as long as that of the native insulin, nearly 21 h. The bioconjugation of insulin with the regenerated silk fibroin greatly improved its physicochemical and biological stability.

Synthesis, characterization and immunogenicity of silk fibroin-L-asparaginase bioconjugates.

L-asparaginase (ASNase) is one basic drug in the treatment of acute lymphoblastic leukemia (ALL). Because its half-life time is too short and it is easy to arouse allergic reaction, use in practical clinic is considerably limited. Silk fibroin (SF) with different molecular mass from 40 to 120 kDa is a natural biocompatible protein and could be used as a novel bioconjugate for enzyme modification to overcome its usual shortcomings mentioned above. When the enzyme was bioconjugated covalently with the water-soluble fibroin by glutaraldehyde, the enzyme kinetic properties and immune characteristics in vivo of the resulting silk fibroin-L-asparaginase (SF-ASNase) bioconjugates were investigated in detail. The results show that the modified ASNase was characterized by its higher residual activity (nearly 80%), increased heat and storage stability and resistance to trypsin digestion, and its longer half-life (63 h) than that of intact ASNase (33 h). The abilities of intact and modified ASNases to arouse allergic reaction are 2(4) and 2(1) antibody titers, respectively. Bioconjugation of silk fibroin significantly helps to reduce the immunogenicity and antigenicity of the enzyme. The apparent Michaelis constants of the modified ASNase (K(m(app))=0.844 x 10(-3)mol L(-1)) was approximately six times lower than that of enzyme alone, which suggests that the affinity of the enzyme to substrate l-asparagine elevated when bioconjugated covalently with silk fibroin. SF-ASNase bioconjugates could overcome the common shortcomings of the native form. Therefore, the modified ASNase coupled with silk fibroin has the potential values of being studied and developed as a new bioconjugate drug.

[Cloning and expression of Buthus martensii Karsch scorpion toxin gene (BmK IT3) in Escherichia coli].

According to the reported sequence of Buthus martensii Karsch scorpion toxin gene (BmK IT3), we synthesized two primers, which were complementary in a region. By the means of PCR, we got the gene. The gene was fused in expression vector pET-28a, which gave rise to a recombinant plasmid pET(IT3R). Then it was transformed into E. coli BL21 (DE3). With IPTG induction, the gene was efficiently expressed. And the fusion product was soluble.

[Package of Bombyx mori nuclear polyhedrosis virus with heterogoneous polyhedring protein].

Cloning the segment of polyhedrin gene which was separated from AcMNPV genome DNA by PCR method into transfer vector pBacPAK8, we got recombinant transfer vector pOAc, then contransfecting BmN cells with the vector and linear virus Bm-BacPAK6, harvested recombinant virus hp-BmNPV which can produce polyhedrin but no blue plaques. Analysing of recombinant virus from polyhedrin, recombinant viral DNA and polyhedra, we confirmed that polyhedring of AcMNPV can be not only high-level expressed in BmN cells, But can recognize recombinant BmNPV DNA and assemble them to polyhedra. We observed that the pattern of restriction enzyme changed due to DNA recombination, the size of recombinant viral polyhedron (1.2-2.9 microns) was obviously smaller than those of wild-type BmNPV by electron microscopy.