Age-related alterations in the biochemical and functional properties of the bladder in type 2 diabetic GK rats

Previous studies have demonstrated that experimental type 1 diabetes induced by streptozotocin causes alterations in the biochemical and functional properties of several receptor systems in the rat bladder. However, the exact mechanism involved in the pathophysiology of voiding dysfunction in type 2 diabetic patients is unknown. Because the GK rat is a widely accepted genetically determined rodent model for human type 2 diabetes, we investigated diabetes-induced changes in the bladder smooth muscle of the GK rats at several time points. Male GK rats and age-matched Wistar rats, as controls, were maintained for 4, 8, 16, and 32 weeks. Contractile responses to KCl, carbachol, ATP, and electrical field stimulation (EFS) were measured by using the isolated muscle bath techniques. Acetylcholine (ACh) release induced by EFS from bladder muscle strips was measured by using high-performance liquid chromatography coupled with a microdialysis procedure. Maximum contractile responses to carbachol and ATP, the release of ACh, and tissue sorbitol levels were similar in bladders from GK and control rats until 8 weeks of age. At 16 weeks of age, however, the contractile responses to carbachol and ATP, and tissue sorbitol levels were increased, and the EFS-induced ACh release was decreased in GK rats compared with controls. Although the maximum contractile responses to EFS were unchanged until 16 weeks of age, they were decreased in 32-week-old GK rats, compared with controls. Our data indicate the presence of age-related alterations in the biochemical and functional properties of the bladder in type 2 diabetic GK rats.     

Inhibitory effects of botulinum toxin on pyloric and antral smooth muscle

Botulinum toxin injection into the pylorus is reported to improve gastric emptying in gastroparesis. Classically, botulinum toxin inhibits ACh release from cholinergic nerves in skeletal muscle. The aim of this study was to determine the effects of botulinum toxin on pyloric smooth muscle. Guinea pig pyloric muscle strips were studied in vitro. Botulinum toxin type A was added; electric field stimulation (EFS) was performed every 30 min for 6 h. ACh (100 microM)-induced contractile responses were determined before and after 6 h. Botulinum toxin caused a concentration-dependent decrease of pyloric contractions to EFS. At a low concentration (2 U/ml), botulinum toxin decreased pyloric contractions to EFS by 43 +/- 9% without affecting ACh-induced contractions. At higher concentrations (10 U/ml), botulinum toxin decreased pyloric contraction to EFS by 75 +/- 7% and decreased ACh-induced contraction by 79 +/- 9%. In conclusion, botulinum toxin inhibits pyloric smooth muscle contractility. At a low concentration, botulinum toxin decreases EFS-induced contractile responses without affecting ACh-induced contractions suggesting inhibition of ACh release from cholinergic nerves. At higher concentrations, botulinum toxin directly inhibits smooth muscle contractility as evidenced by the decreased contractile response to ACh.     

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.     

Coupled nitric oxide and autonomic receptor functional responses in the normal and inflamed urinary bladder of the rat

Both divisions of the autonomic nervous system are involved in regulation of urinary bladder function. Several substances, other than noradrenaline and acetylcholine, seem to play important roles in physiology and pathophysiology of lower urinary tract. In the current study, we aimed to examine if there exist interplays between nitric oxide (NO) and autonomic transmitters and if such interactions vary in different parts of the urinary bladder in healthy and cyclophosphamide (CYP)-induced cystitic rats; when administered to the animals (100 mg/kg; i.p.), the cytotoxic CYP metabolite acrolein induces bladder inflammation. In the current study a series of in vitro functional studies were performed on detrusor muscle strip preparations. Stimulation with electrical field stimulation (EFS), methacholine, adenosine 5′-triphosphate (ATP), and adrenaline evoked contractile responses in isolated bladder preparations that were significantly reduced in cyclophosphamide (CYP)-treated rats. While the nitric oxide synthase inhibitor N(omega)-nitro-L-arginine (L-NNA; 10(-4) M) did not affect contractile responses in normal, healthy strip preparations, it significantly increased the contractile responses to EFS, methacholine and adrenaline, but not to ATP, in the bladders from the CYP-treated rats. In the CYP-treated rats, the ATP-evoked relaxatory part of its dual response (an initial contraction followed by a relaxation) was 6-fold increased in comparison with that of normal preparations, whereas the isoprenaline relaxation was halved in the CYP-treated. While L-NNA (10(-4) M) had no effect on the isoprenaline-evoked relaxations, it reduced the ATP-evoked relaxations in strip preparations from the bladder body of CYP-treated rats. Stimulation of beta(2)- and beta(3)-adrenoceptors evoked relaxations and both responses were reduced in cystitis, the latter to a larger extent. In the trigone, the reduced ATP-evoked contractile response in the inflamed strips was increased by L-NNA, while L-NNA had no effect on the ATP-evoked relaxations, neither on the relaxations in healthy nor on the larger relaxations in the inflamed trigone. The study shows that both contractile and relaxatory functions are altered in the state of inflammation. The parasympathetic nerve-mediated contractions of the body of the bladder, evoked by the release of ATP and acetylcholine, were substantially reduced in cystitis. The relaxations to beta-adrenoceptor and purinoceptor stimulation were also reduced but only the ATP-evoked relaxation involved NO.     

Proinflammatory cytokines alter/reduce esophageal circular muscle contraction in experimental cat esophagitis

Cholinergic mechanisms are largely responsible for esophageal contraction in response to swallowing or to in vitro electrical field stimulation (EFS). After induction of experimental esophagitis by repeated acid perfusion, the responses to swallowing and to EFS were significantly reduced but contraction in response to ACh was not affected, suggesting that cholinergic mechanisms are damaged by acid perfusion but that myogenic mechanisms are not. Measurements of ACh release in response to EFS confirmed that release of ACh was reduced in esophagitis compared with normal controls. To examine factors contributing to this neuropathy, normal esophageal strips were incubated for 1-2 h with the proinflammatory cytokines IL-1beta (100 U/ml), IL-6 (1 ng/ml), or TNF-alpha (1 ng/ml). IL-1beta and IL-6 levels, measured by Western blot analysis, increased in esophagitis compared with normal circular muscle. IL-1beta and IL-6 reduced contraction in response to EFS (2-10 Hz, 0.2 ms) but did not affect ACh-induced contraction, suggesting that these cytokines inhibit ACh release without affecting myogenic contractile mechanisms. EFS-induced ACh release was significantly reduced in normal esophageal strips by incubation in IL-1beta or IL-6, suggesting that they may contribute to the contractility changes. TNF-alpha at 1 ng/ml, however, did not affect the response to ACh or to electrical stimulation but inhibited both at higher concentrations. TNF-alpha levels were low in normal muscle and did not increase with esophagitis. The data suggest that the proinflammatory cytokines IL-1beta and IL-6 contribute to reduced esophageal contraction by inhibiting release of ACh from myenteric neurons.     

Abnormal modulation of cholinergic neurotransmission by endomorphin 1 and endomorphin 2 in isolated bronchus of type 1 diabetic rats

To assess whether diabetes alters the regulatory effects of mu-opioid receptor (MOR) agonists on the cholinergic bronchoconstriction, we investigated the inhibitory effects of endomorphins (EMs) on the electrical field stimulation (EFS)-induced cholinergic bronchoconstriction in type 1 diabetic rats. At 4 weeks after the onset of diabetes, both the EFS- and exogenous acetylcholine (ACh)-induced bronchoconstriction in diabetes in vitro were greater than those in non-diabetes rats. Furthermore, endomorphin 1 (EM1) and endomorphin 2 (EM2) inhibited the response to EFS in diabetic rat isolated bronchus in a concentration- and frequency-dependent manner, which is in agreement with that in non-diabetes. However, the inhibitory effects of EMs on the EFS-induced bronchoconstriction in diabetes were significantly weaker than those in non-diabetes. Both EM1 and EM2 (1 microM) had no effect on the contractile response to exogenous ACh, indicating a prejunctional effect. Furthermore, the inhibitory effect on the EFS-induced bronchoconstriction was blocked by naloxone (10 microM). Eight weeks after the induction of diabetes, both the EFS- and exogenous ACh-induced bronchoconstrictions in diabetes were further enhanced compared to those in short-time (4 weeks) diabetic rats. Moreover, the inhibitory effects of EMs on the EFS-induced bronchoconstriction were further attenuated. These results suggest that dysfunction of presynaptic inhibitory modulation through opioid receptor by EMs may take place in the bronchus of diabetic rats.     

Effects of magnolol (5,5'-diallyl-2,2'-dihydroxybiphenyl) on diabetic nephropathy in type 2 diabetic Goto-Kakizaki rats

We investigated the effect of magnolol (5,5'-diallyl-2,2'-dihydroxybiphenyl), a marker compound isolated from the cortex of Magnolia officinalis, in non-obese type 2 diabetic Goto-Kakizaki (GK) rats. The rats were treated orally with magnolol (100 mg/kg body weight) once a day for 13 weeks. In magnolol-treated GK rats, fasting blood glucose and plasma insulin were significantly decreased, and the pancreatic islets also showed strong insulin antigen positivity. Urinary protein and creatinine clearance (Ccr) were significantly decreased. Pathological examination revealed the prevention of the glomeruli enlargement in magnolol-treated GK rats. The overproduction of renal sorbitol, advanced glycation endproducts (AGEs), type IV collagen, and TGF-beta1 mRNA were significantly reduced in magnolol-treated GK rats. Thus based on our findings, the use of magnolol could result in good blood glucose control and prevent or retard development of diabetic complications such as diabetic nephropathy.     

Smooth muscle and purinergic contraction of the human, rabbit, rat, and mouse testicular capsule

The smooth-muscle cells of the testicular capsule (tunica albuginea) of man, rat, and mouse were examined by electron microscopy. They were characteristically flattened, elongated, branching cells and diffusely incorporated into the collagenous matrix and did not form a compact muscle layer. Contractile and synthetic smooth-muscle cell phenotypes were identified. Nerve varicosities in close apposition to smooth muscle were seen in human tissue. Contractions induced by adenosine 5'-triphosphate (ATP), alpha, beta-methylene ATP, noradrenaline (NA), acetylcholine (ACh), and electrical field stimulation (EFS) of autonomic nerves were investigated. Nerve-mediated responses of the rabbit and human tunica albuginea were recorded. The EFS-induced human responses were completely abolished by prazosin. In the rabbit, EFS-induced contractile responses were reduced by pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid by 36% and by prazosin by 77%. Both antagonists together almost completely abolished all EFS-induced contractions. The human tunica albuginea was contracted by NA, ATP, and alpha, beta-methylene ATP, but not by ACh. The rabbit and rat tunica albuginea were contracted by NA, ATP, alpha, beta-methylene ATP, and ACh. The mouse tunica albuginea was contracted by ACh, ATP, and alpha, beta-methylene ATP, but relaxed to NA. Immunohistochemical studies showed that P2X1 (also known as P2RX1) and P2X2 (also known as P2RX2) receptors were expressed on the smooth muscle of the rodent testicular capsule, expression being less pronounced in man. The testicular capsule of the rat, mouse, rabbit, and man all contain contractile smooth muscle. ATP, released as a cotransmitter from sympathetic nerves, can stimulate the contraction of rabbit smooth muscle. Human, rat, and mouse testicular smooth muscle demonstrated purinergic responsiveness, probably mediated through the P2X1 and/or P2X2 receptors.     

Transcutaneous Electrical Nerve Stimulation (TENS) Improves the Diabetic Cytopathy (DCP) via Up-Regulation of CGRP and cAMP

The objective of this study was to investigate the effects and mechanism of Transcutaneous Electrical Nerve Stimulation (TENS) on the diabetic cytopathy (DCP) in the diabetic bladder. A total of 45 rats were randomly divided into diabetes mellitus (DM)/TENS group (n = 15), DM group (n = 15) and control group (n = 15). The rats in the DM/TENS and TENS groups were electronically stimulated (stimulating parameters: intensity-31 V, frequency-31 Hz, and duration of stimulation of 15 min) for three weeks. Bladder histology, urodynamics and contractile responses to field stimulation and carbachol were determined. The expression of calcitonin gene-related peptide (CGRP) was analyzed by RT-PCR and Western blotting. The results showed that contractile responses of the DM rats were ameliorated after 3 weeks of TENS. Furthermore, TENS significantly increased bladder wet weight, volume threshold for micturition and reduced PVR, V% and cAMP content of the bladder. The mRNA and protein levels of CGRP in dorsal root ganglion (DRG) in the DM/TENS group were higher than those in the DM group. TENS also significantly up-regulated the cAMP content in the bladder body and base compared with diabetic rats. We conclude that TENS can significantly improve the urine contractility and ameliorate the feeling of bladder fullness in DM rats possibly via up-regulation of cAMP and CGRP in DRG.     

Loss of caveolin-1 expression is associated with disruption of muscarinic cholinergic activities in the urinary bladder

Caveolin-1 (Cav1), a structural protein of caveolae, plays cell- and context-dependent roles in signal transduction pathway regulation. We have generated a knockout mouse homozygous for a null mutation of the Cav1 gene. Cav1 knockout mice exhibited impaired urinary bladder contractions in vivo during cystometry. Contractions of male bladder strips were evoked with electric and pharmacologic stimulation (5–40 Hz, 1–10 μM carbachol, 10 mM ,β-methylene ATP, 100 mM KCl). Acetylcholine (ACh) and norepinephrine (NE) release from bladder strips were measured with a radiochemical method by incubating the strips with ¹⁴C-choline and ³H-NE prior to electric stimulation, whereas ATP release was measured using the luciferin-luciferase assay with a luminometer. A 60–75% decline in contractility was observed when Cav1 knockout muscle strips were stimulated with electric current or carbachol, compared to wildtype muscle strips. No difference in contractility was noted when contractions were evoked either by the purinergic agonist ,β-methylene ATP, or by extracellular potassium. To investigate the relative contribution of non-cholinergic activity to bladder contractility, the amplitude of the electric stimulation-evoked contractions was compared in the presence of the muscarinic antagonist atropine (1 μM). While the non-muscarinic (purinergic) response was unaltered, muscarinic cholinergic response was principally disrupted in Cav1 knockout mice. The loss of Cav1 gene expression was also associated with a 70% reduction in ACh release. NE and ATP release was not altered. It is concluded that the loss of caveolin-1 is associated with disruption of M₃ muscarinic cholinergic activity in the bladder. Both pre-junctional (acetylcholine neurotransmitter release from neuromuscular junctions) and post-junctional (M₃ receptor-mediated signal transduction in bladder smooth muscles) mechanisms are disrupted, resulting in impaired bladder contraction.     

Potentiation of contraction of rabbit airway smooth muscle by some cyclooxygenase products

An alteration in smooth muscle sensitivity may be one of the mechanisms of the airway hyperresponsiveness observed in asthma. Indomethacin inhibits experimentally induced airway hyperresponsiveness. We thus examined the effects of the cyclooxygenase products PGD2, PGF2 alpha and a thromboxane A2 analogue U46619 on contractile responses of rabbit airway smooth muscle to histamine, carbachol and electrical field stimulation (EFS). PGD2 did not potentiate any contractile responses. When PGF2 alpha (1 microM) was administered 30 min before cumulative concentration-response curves to histamine and carbachol, no potentiation was observed. However, PGF2 alpha (1 microM) added immediately before EFS and bolus doses of histamine potentiated the contractile responses. U46619 increased the cumulative concentration-responses to both histamine and carbachol. The fact that we could alter smooth muscle sensitivity in vitro with PGF2 alpha and a thromboxane analogue suggests that these mediators may be involved in the airway hyperresponsiveness observed in asthma.     

Sodium selenate partially corrects impaired functional responses in detrusor muscle in streptozotocin-induced diabetic rats

Selenium selenate was administered to streptozotocin-induced diabetic rats to assess its effects on the detrusor muscle. Thirty-two rats were divided into four groups of eight subjects each. The study animals were made diabetic by means of a single intravenous injection of streptozotocin (STZ). The responsiveness of the detrusor was improved in the group injected with sodium selenate. Diabetes caused significant increases in carbachol and β,γ-MeATP-evoked contractions and significant decrease of contractions induced by electrical stimulation. Isoprenaline-induced relaxation of the detrusor muscle was diminished by diabetes, whereas ATP relaxation appeared to be increased. Although adenosine-induced relaxations in controls and in diabetic rats were accompanied by unchanged responses in normoxic conditions, a significant enhancement in the detrusor muscle was observed during hypoxia. This enhancement of adenosine responsiveness in hypoxic conditions is inhibited in diabetes. Treatment with sodium selenate prevented alterations of both carbachol-induced contractility and isoprenaline-evoked relaxation, whereas nerve-mediated contractions and purinergic responses were not improved in diabetic rats after treatment. Our data suggest that changes in cholinergic and adrenergic responses were the result of selenium deficiency in diabetic rats.     

Elevated expression and activity of protein-tyrosine phosphatase 1B in skeletal muscle of insulin-resistant type II diabetic Goto-Kakizaki rats

We investigated the cellular mechanism(s) of insulin resistance associated with non-insulin dependent diabetes mellitus (NIDDM) using skeletal muscles isolated from non-obese, insulin resistant type II diabetic Goto-Kakizaki (GK) rats, a well known genetic rat model for type II diabetic humans. Relative to non-diabetic control rats (WKY), insulin-stimulated insulin receptor (IR) autophosphorylation and insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation were significantly inhibited in GK skeletal muscles. This may be due to increased dephosphorylation by a protein tyrosine phosphatase (PTPase). Therefore, we measured skeletal muscle total PTPase and PTPase 1B activities in the skeletal muscles isolated from control rats (WKY) and diabetic Goto-Kakizaki (GK) rats. PTPase activity was measured using a synthetic phosphopeptide, TRDIY(P)ETDY(P)Y(P)RK, as the substrate. Basal PTPase activity was 2-fold higher (P < 0.001) in skeletal muscle of GK rats when compared to WKY. Insulin infusion inhibited skeletal muscle PTPase activity in both control (26.20% of basal, P < 0.001) and GK (25.35% of basal, P < 0.001) rats. However, PTPase activity in skeletal muscle of insulin-stimulated GK rats was 200% higher than hormone-treated WKY controls (P < 0.001). Immunoprecipitation of PTPase 1B from skeletal muscle lysates and analysis of the enzyme activity in immunoprecipitates indicated that both basal and insulin-stimulated PTPase 1B activities were significantly higher (twofold, P < 0.001) in skeletal muscle of diabetic GK rats when compared to WKY controls. The increase in PTPase 1B activity in diabetic GK rats was associated with an increased expression of the PTPase 1B protein. We concluded that insulin resistance of GK rats is accompanied atleast by an abnormal regulation of PTPase 1B. Elevated PTPase 1B activity through enhanced tyrosine dephosphorylation of the insulin receptor and its substrates, may lead to impaired glucose tolerance and insulin resistance in GK rats.     

Age-related changes in urethrovesical coordination in male rats: relationship with bladder instability?

The micturition profile in conscious animals and the urethrovesical coordination in anesthetized conditions were investigated in 6- and 24-mo-old male Sprague-Dawley rats. The in vitro pharmacological responses to KCl, electrical field stimulation (EFS), carbachol, phenylephrine, and isoprenaline were determined in the isolated bladder body, the bladder neck, and urethra. A morphometric and immunohistological study has been included. During conscious cystomanometry, 63% of the aging rats but only 25% of the adult rats showed spontaneous contractions during the bladder-filling phase. In conscious aging rats, basal pressure, threshold pressure, and micturition pressure were also significantly increased. In anesthetized aging rats, a decrease in resting urethral pressure at micturition threshold and the occurrence of a significant delay in urethral relaxation during micturition were associated with an increased residual volume. In all isolated tissues, contractile response to KCl was not modified with aging, whereas age-related decreases in maximal responses to carbachol in the bladder body and to phenylephrine and carbachol in the urethra were observed. In the bladder neck only, we found a significant decrease in the amplitude of neurogenic contractions associated with fibrosis but without decrease in nerve density. These experiments show significant modifications in the voiding pattern of aging rats associated with urethral dysfunction and with regionally specific pharmacological and structural changes of the urinary tract. We propose that aging in rats is characterized by an impairment of the urethrovesical coordination, leading to bladder dysfunctions similar to those induced by bladder outlet obstruction.     

Alterations in vascular endothelial function in the aorta and mesenteric artery in type II diabetic rats

We used the partial protection exerted by suitable dosages of nicotinamide against the beta-cytotoxic effect of streptozotocin (STZ) to create an experimental diabetic syndrome in adult rats that appears closer to type II diabetes mellitus than other available animal models. The dosage of 230 mg/kg of nicotinamide given intraperitoneally 15 min before STZ administration (65 mg/kg i.v.) yielded animals with hyperglycemia (187.8 +/- 17.8 vs. 103.8 +/- 2.8 mg/dL in controls; P < 0.001) and preservation of plasma insulin levels. This study assessed the relationship between endothelial dysfunction and agonist-induced contractile responses in such rats. In the thoracic aorta, the acetylcholine (ACh) induced relaxation was significantly reduced and the noradrenaline (NA) induced contractile response was significantly increased in diabetic rats compared with age-matched control rats. In the superior mesenteric artery, the ACh-induced relaxation was similar in magnitude between diabetic and age-matched control rats; however, the ACh-induced endothelium-derived hyperpolarizing factor (EDHF) type relaxation was significantly weaker in diabetic rats than in the controls. The phenylephrine (PE) induced contractile response was not different between the two groups. The plasma concentration of NOx (NO2- + NO3-) was significantly lower in diabetic rats than in control rats. We conclude that vasomotor activities in conduit arteries are impaired in this type II diabetes model.     

K+-induced alterations in airway muscle responsiveness to electrical field stimulation

We investigated possible pre- and postsynaptic effects of K+-induced depolarization on ferret tracheal smooth muscle (TSM) responsiveness to cholinergic stimulation. To assess electromechanical activity, cell membrane potential (Em) and tension (Tm) were simultaneously recorded in buffer containing 6, 12, 18, or 24 mM K+ before and after electrical field stimulation (EFS) or exogenous acetylcholine (ACh). In 6 mM K+, Em was -58.1 +/- 1.0 mV (mean +/- SE). In 12 mM K+, Em was depolarized to -52.3 +/- 0.9 mV, basal Tm did not change, and both excitatory junctional potentials and contractile responses to EFS at short stimulus duration were larger than in 6 mM K+. No such potentiation occurred at a higher K+, although resting Em and Tm increased progressively above 12 mM K+. The sensitivity of ferret TSM to exogenous ACh appeared unaffected by K+. To determine whether the hyperresponsiveness in 12 mM K+ was due, in part, to augmented ACh release from intramural airway nerves, experiments were done using TSM preparations incubated with [3H]choline to measure [3H]ACh release at rest and during EFS. Although resting [3H]ACh release increased progressively in higher K+, release evoked by EFS was maximal in 12 mM K+ and declined in higher concentrations. We conclude that small elevations in the extracellular K+ concentration augment responsiveness of the airways, by increasing the release of ACh both at rest and during EFS from intramural cholinergic nerve terminals. Larger increases in K+ appear to be inhibitory, possibly due to voltage-dependent effects that occur both pre- and postsynaptically.     

Preventive effects of cyclohexenonic long-chain fatty alcohol on diabetic cystopathy in the rat

In this study, we investigated the preventive effect of n-hexacosanol on diabetes-induced bladder dysfunction in the rat. Diabetes was induced in 8-week-old male Sprague-Dawley rats by administering an injection of streptozotocin (50 mg/kg, i.p.). The rats were randomly divided into 4 groups (age-matched control rats, diabetic rats without treatment with n-hexacosanol, and diabetic rats treated with n-hexacosanol (2 and 8 mg/kg, i.p. every day)) and maintained for 4 weeks. The serum glucose and serum insulin levels were determined, and the functions of bladder were estimated by voiding behavior, cystometric, and functional studies to carbachol and KCl. Furthermore, we examined possible diabetic induced histological changes in these rats. Treatment with n-hexacosanol did not alter diabetic status including body mass, bladder mass, and serum glucose and serum insulin levels, but significantly improved the maximum contraction pressure of the detrusor and residual urine volume in cystometric studies and Emax values to carbachol in functional studies in a dose-dependent manner. Diabetes induced bladder smooth muscle hypertrophy, which tended to be ameliorated by treatment with n-hexacosanol in a dose-dependent manner. Treatment with n-hexacosanol did not alter the diabetic status, but significantly improved diabetic cystopathy in a dose-dependent manner.     

Receptor activated bladder and spinal ATP release in neurally intact and chronic spinal cord injured rats

Neurally intact (NI) rats and chronic spinal cord injured (SCI) rats were studied to determine how activation of mechanosensory or cholinergic receptors in the bladder urothelium evokes ATP release from afferent terminals in the bladder as well as in the spinal cord. Spinal cord transection was performed at the T(9)-T(10) level 2-3 weeks prior to the experiment and a microdialysis fiber was inserted in the L(6)-S(1) lumbosacral spinal cord one day before the experiments. Mechanically evoked (i.e. 10 cm/W bladder pressure) ATP release into the bladder lumen was approximately 6.5-fold higher in SCI compared to NI rats (p<0.05). Intravesical carbachol (CCh) induced a significantly greater release of ATP in the bladder from SCI as compared to NI rats (3424.32+/-1255.57 pmol/ml versus 613.74+/-470.44 pmol/ml, respectively, p<0.05). However, ATP release in NI or SCI rats to intravesical CCh was not affected by the muscarinic antagonist atropine (Atr). Spinal release of ATP to bladder stimulation with 10 cm/W pressure was five-fold higher in SCI compared to NI rats (p<0.05). CCh also induced a significantly greater release of spinal ATP in SCI rats compared to controls (4.3+/-0.9 pmol versus 0.90+/-0.15 pmol, p<0.05). Surprisingly, the percent inhibitory effect of Atr on CCh-induced ATP release was less pronounced in SCI as compared to NI rats (49% versus 89%, respectively). SCI induces a dramatic increase in intravesical pressure and cholinergic receptor evoked bladder and spinal ATP release. Muscarinic receptors do not mediate intravesical CCh-induced ATP release into the bladder lumen in NI or SCI rats. In NI rats sensory muscarinic receptors are the predominant mechanism by which CCh induces ATP release from primary afferents within the lumbosacral spinal cord. Following SCI, however, nicotinic or purinergic receptor mechanisms become active, as evidenced by the fact that Atr was only partially effective in inhibiting CCh-induced spinal ATP release.     

Involvement of Rho/Rho-kinase signalling in the contractile activity and acetylcholine release in the mouse gastric fundus

In this study effects of Rho kinase inhibitors have been examined on the mouse gastric fundal smooth muscle reactivity and neurotransmitter (acetylcholine) release. Two Rho-kinase inhibitors, Y-27632 and fasudil (HA-1077), conspicuously suppressed the contractile responses to carbachol (CCh) and KCl as well as electrical field stimulation (EFS, 40 V, 0.5 ms, and 20 s). pEC(50) value for CCh and EC(50) value for KCl were 6.68+/-0.15 M and 10.4+/-2.8 mM, respectively. EFS induced reproducible contraction (38.3+/-4.75 mN/g tissue) which was almost abolished and potentiated in the presence of atropine (10(-6)M) and eserine (10(-6)M), respectively. The Rho-kinase inhibitors relaxed the fundic strips preconstricted by submaximal concentration of CCh or KCl in a concentration dependent manner. With CCh-elicited contraction, the pEC(50) values of Y-27632 and fasudil were 5.45+/-0.14 and 5.11+/-0.14 M, respectively (p>0.05). However, the pEC(50) values for Y-27632 and fasudil on KCl-induced tone were 6.09+/-0.1 and 5.35+/-0.06 M (p<0.001), respectively. Moreover, [3H]acetylcholine ([3H]ACh) release upon EFS from the gastric fundus was measured and it was found that Y-27632 (10(-4)M) significantly impaired the release. At 3 Hz the radioactivity ratio obtained after and before EFS (S(2)/S(1) ratio) was 0.88+/-0.03 in control but 0.63+/-0.08 in the presence of 10(-4)M Y-27632 (p<0.05). These results suggest that Rho kinase inhibitors can not only relax the gastric fundus but also modulate CCh, cholinergic nerve stimulation, and KCl-induced contraction. Furthermore, Rho/Rho kinase signalling may play a role in the neurotransmitter (ACh) release in the mouse gastric fundus.