Daniel Giglio

Altered muscarinic receptor subtype expression and functional responses in cyclophosphamide induced cystitis in rats.

In the in vitro study, it was investigated whether the expression of muscarinic receptors and cholinergic responses were altered in the situation of experimental cystitis. Rats were treated with cyclophosphamide intraperitoneally and the bladders were excised 36-100 h later. Immunohistochemistry and immunoblotting showed all subtypes of the muscarinic receptor (M1-M5) to be present in the specimens from inflamed urinary bladders and controls. In the cyclophosphamide-treated rats, the expression of muscarinic M5 receptors was increased by more than 40 times (p<0.01; n=8) both in the smooth muscle and the urothelium. Both the maximal contractile response to carbachol and to a high potassium concentration was approximately halved in cyclophosphamide-treated tissues, whereas the reduction was substantially greater in response to low carbachol concentrations (<EC(50)). The administration of 4-DAMP inhibited the carbachol-induced contractile responses of inflamed strips less potently than of controls, whereas pirenzepine and methoctramine showed equipotency in the two groups. The nitric oxide synthase inhibitor l-NNA increased the contractile effect of carbachol in inflamed detrusor strips, while it had no effect in controls. Immunoblotting showed endothelial nitric oxide synthase (eNOS) to be up-regulated in cystitis, and immunohistochemistry revealed the change to occur in the urothelium and in the suburothelial layer. The alteration of cholinergic detrusor responses in cyclophosphamide-treated rats depends mainly on a general detriment of contractility but also on indirect effects possibly via nitric oxide synthesis. The most prominent histological alterations occurred in the urothelium in which muscarinic M5 receptors increased in particular. The study further underlines that the urothelium may play significant roles in the pathogenesis of urinary bladder disorders such as interstitial cystitis.

Muscarinic Receptor Subtypes in the Lower Urinary Tract

Acetylcholine acting on muscarinic M3 receptors on the detrusor muscle is the principal stimulus for inducing the contractile response for urinary bladder voiding. The urinary bladder expresses, however, all cloned muscarinic receptor subtypes (M1–M5). In terms of quantity, the M2 subtype dominates over the M3 subtype in the detrusor, and its role in contraction seems to be primarily indirect, by blocking stimuli from cAMP-coupled receptors that induce relaxation. The excitatory M1 and inhibitory M2 and/or M4 subtypes are also expressed prejunctionally. Muscarinic M1 and M2/M4 autoreceptors facilitate and inhibit, respectively, the release of acetylcholine. The urothelium had been considered to be a passive barrier; however, during the last decade, it has been shown that the urothelium is of importance for bladder function. In a state of bladder pathology, muscarinic receptor changes occur in the detrusor, prejunctionally, and in the urothelium, but the character of the change differs between disorders. The urothelium expresses all subtypes of muscarinic receptors, and upon stimulation it releases factors affecting bladder afferents and smooth muscle. During inflammation, the expression of muscarinic M5 receptors is increased, particularly in the urothelium, together with a cholinergic-induced production of nitric oxide in the mucosa. The present review describes signalling mechanisms, expression and functional effects of muscarinic receptors in the lower urinary tract. Their roles in physiological and pathophysiological conditions, as well as clinical implications of the occurrence of different muscarinic receptors, are discussed.

Studies of muscarinic receptor subtypes in salivary gland function in anaesthetized rats.

The in vivo study aimed to examine whether muscarinic receptor subtypes other than muscarinic M3 receptors exert exocrine functional roles in the rat salivary glands. The effects of pirenzepine, methoctramine and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) were examined on secretion from the major salivary glands evoked by acetylcholine (0.001-10 micromol kg(-1) i.v.) in pentobarbitone-anaesthetized rats. Observations were occasionally made on glandular blood flow. 4-DAMP (0.1-100 nmol kg(-1) i.v.) markedly and equipotently inhibited the acetylcholine-evoked fluid responses in all glands. Pirenzepine (0.1 micromol kg(-1) i.v.-10 mmol kg(-1) i.v.) showed significantly lower inhibitory potency than 4-DAMP, most conspicuously in the parotid, while methoctramine (0.1 micromol kg(-1) i.v.-10 mmol kg(-1) i.v.) exerted an even lesser inhibitory effect. Also against acetylcholine-evoked blood flow increases, 4-DAMP showed a conspicuous potency. At 1 and 10 micromol kg(-1) i.v. of pirenzepine, the antagonist reduced the protein concentration in the submandibular saliva, but not in the parotid saliva. While 4-DAMP (1 and 10 nmol kg(-1) i.v.) significantly inhibited acetylcholine-evoked protein secretory responses in the submandibular glands, methoctramine (below 10 micromol kg(-1) i.v.) affected the responses in neither gland. The reduction of the protein concentration in submandibular saliva caused by 4-DAMP and pirenzepine was inhibited by N(omega)-nitro-L-arginine methyl ester (L-NAME; 30 mg kg(-1) i.p.), while L-NAME had no or only minute effects on the parotid protein secretion. Thus, in addition to muscarinic M3 receptors, other muscarinic receptors contribute to in vivo functional responses in rat submandibular and sublingual glands. While these other receptors are muscarinic M1 receptors in the sublingual gland, they may be a different subtype, possibly muscarinic M5 receptors, in the submandibular gland. However, muscarinic M1 receptors may induce indirect effects via nitric oxide in the submandibular gland.

Cholinergic nitric oxide release from the urinary bladder mucosa in cyclophosphamide-induced cystitis of the anaesthetized rat

Background and purpose: Previous reports have suggested that nitric oxide (NO) may be released by cholinergic stimuli in the rat bladder in cyclophosphamide‐induced cystitis, affecting bladder function. In the current study, we evaluated the effects of cyclophosphamide‐induced cystitis on muscarinic whole bladder contractile responses in vivo, and further, if NO might be released from the mucosa by cholinergic stimuli.

Assessment and Characterization of Purinergic Contractions and Relaxations in the Rat Urinary Bladder

The aim of the present study was to assess the purinoceptor functional responses of the urinary bladder by using isolated rat urinary bladder strip preparations. ATP elicited a transient bladder contraction followed by a sustained relaxation and ADP, UDP and UTP generated predominantly potent relaxations (relaxatory potencies: ADP = ATP > UDP = UTP). The ATP contractions were desensitized with the P2X(1/3) purinoceptor agonist/desensitizer alpha,beta-meATP and reduced by the P2 purinoceptor antagonist PPADS but unaffected by the P2 purinoceptor antagonist suramin. Electrical field stimulation (1-60 Hz) evoked frequency-dependent bladder contractions that were decreased by incubation with alpha,beta-meATP but not further decreased by PPADS. Suramin antagonized relaxations generated by UDP but not those by ADP, ATP or UTP. PPADS antagonized and tended to antagonize UTP and UDP relaxations, respectively, but did neither affect ADP nor ATP relaxations. ADP relaxations were insensitive to the P2Y(1) purinoceptor antagonist MRS 2179 and the ATP-sensitive potassium channel antagonist glibenclamide. The ATP relaxations were inhibited by the P1 purinoceptor antagonist 8-p-sulfophenyltheophylline but unaffected by the A2A adenosine receptor antagonist 8-(3-chlorostyryl)caffeine and glibenclamide. Adenosine evoked relaxations that were antagonized by the A2B adenosine receptor antagonist PSB 1115. Thus, in the rat urinary bladder purinergic contractions are elicited predominantly by stimulation of the P2X(1) purinoceptors, while UDP/UTP-sensitive P2Y purinoceptor(s) and P1 purinoceptors of the A2B adenosine receptor subtype are involved in bladder relaxation.

Effects of amphetamine on salivary secretion

Amphetamine induces xerogenic effects, but its mechanism of action and xerogenic potency are unknown. In the current in vivo study on the rat parotid gland, the effects of amphetamine on reflex-evoked and acetylcholine-evoked salivation were examined in the absence and presence of adrenergic and dopaminergic antagonists. Under anaesthesia, amphetamine increased the secretion of salivary fluid and the amount of protein therein in response to acetylcholine. Phentolamine abolished the increase in salivary flow and had no effect on the salivary protein concentration, whereas propranolol only reduced the salivary protein concentration. Reflex activation of the secretion evoked a well-maintained level of secretion that was reduced by amphetamine [50% inhibitory dose (ID(50)) 1.9 +/- 0.1 mg kg(-1) intravenously); the salivary protein concentration was increased in the presence of amphetamine. Phentolamine and haloperidol reduced the amphetamine-inhibitory effect on the reflex-evoked fluid response, whereas propranolol had no effect on the fluid response. The xerogenic effect of amphetamine is mainly exerted by central mechanisms involving alpha-adrenoceptors, while, indirectly, amphetamine causes secretion of protein by inducing the release of noradrenaline from glandular nerve terminals.

Urokinase Plasminogen Activator Colocalizes with CD25+ Cells in Atherosclerotic Vessels

Proteolytic activity in vascular tissue is necessary for cellular migration, remodelling of extracellular matrix and the development of atherosclerotic lesions. Inflammatory cells, mainly macrophages, are numerous in atherosclerotic plaques and may synthesize and secrete proteolytic enzymes. The principal activator of plasminogen in tissues is urokinase plasminogen activator (u-PA). To determine if an activated phenotype of inflammatory cells colocalizes with local expression of u-PA in atherosclerotic vessels, vascular biopsies from 15 patients with peripheral atherosclerotic disease were analyzed by immunohistochemistry on consecutive sections. Anti-CD68 antibodies were used as markers for macrophages and were positive in 14/15 specimens. Anti-CD25 (interleukin-2 receptor-α) antibodies were used to identify inflammatory cells with an activated phenotype and were positive in 9/14 CD68+ specimens. The same 9 specimens were positive for u-PA. A positive reaction for u-PA was found only in specimens with CD25+ cells. Specimens with positive reactions for all three antibodies were further analyzed with computer-assisted image analysis. The colocalization with u-PA was higher for CD25 compared to CD68 in all specimens. Mean percentage of the u-PA-positive area in regions positive for cellular markers was 52% (SEM 6%) for CD25 and 19% (SEM 5%) for CD68 (p < 0.01). The results indicated that the activation of macrophages in atherosclerotic vessels may modulate local proteolysis and be of importance in plaque development and stability.

Changes in muscarinic receptors in the toad urothelial cell line TBM-54 following acrolein treatment.

1 In cyclophosphamide‐induced cystitis in the rat, cholinergic function of the bladder and muscarinic receptor expression are altered. In the present study, we investigated whether the toad urothelial cell line TBM‐54 expresses functional muscarinic receptors and whether changes in muscarinic receptors can be induced in vitro by treating cells with acrolein, a metabolite of cyclophosphamide causing cystitis. 2 The occurrence of muscarinic receptors on cells was assessed by microphysiometry, a method analysing receptor function by measuring changes in the extracellular acidity rate (ECAR) in response to receptor stimulation. 3 Challenging untreated cells with the muscarinic receptor agonist carbachol gave rise to a concentration‐dependent increase in changes in ECAR, with a maximal response at 1 mmol/L carbachol of 51 ± 6%. Pre‐incubating cells with different muscarinic receptor antagonists (i.e. pirenzepine (M1 receptor selective), methoctramine (M2/M4 receptor selective) and 4‐diphenylacetoxy‐N‐methylpiperidine methobromide (4‐DAMP; M3/M1/M5 receptor selective)), gave rise to a concentration‐dependent decrease in the effects of carbachol (0.5 mmol/L) on changes in ECAR. 4 Western blot analysis was used to determine the expression of all muscarinic receptor subtypes (M1–M5) by the cell line. Following acrolein treatment, cells were markedly less sensitive to carbachol and the expression of muscarinic M2 receptors was decreased, whereas the expression of muscarinic M3 receptors was increased. 5 In conclusion, the urothelial cell line TBM‐54 expresses functional muscarinic receptors and exposure to acrolein leads to a modulation in the expression of muscarinic receptors. Consequently, acrolein may have direct effects on muscarinic receptor function and expression that contribute to the pathogenesis of cyclophosphamide‐induced cystitis.

Downregulation of toll-like receptor 4 and IL-6 following irradiation of the rat urinary bladder.

The pathophysiology behind radiation cystitis is poorly understood. Here we investigated whether bladder irradiation affects the immune system of the rat urinary bladder. Female rats were sedated and exposed to one single radiation dose of 20 Gy or only sedated (controls) and killed 16 h to 14 days later. Rats were placed in a metabolic cage at 16 h, 3 days, 7 days and 14 days following bladder irradiation. The urinary bladders were harvested and analysed with qPCR, immunohistochemistry and/or Western blot for the expression of interferon (IFN)‐γ, interleukin (IL)‐1β, IL‐2, IL‐4, IL‐5, IL‐6, IL‐10, IL‐13, nitric oxide synthases (eNOS, iNOS and nNOS), tumour necrosis factor (TNF)‐α and toll‐like receptor 4 (TLR4). Urine was collected and analysed for IL‐6 and nitrite (reflecting nitric oxide activity) with ELISA and the Griess reaction, respectively. Irradiation increased bladder frequency and decreased voiding volumes 14 days following bladder irradiation. Bladder irradiation increased the expression of IL‐10 and collagen in the bladder, while TLR4 and IL‐6 expressions were decreased in the urothelium concomitantly with a decrease in mast cells in the submucosa and urine levels of IL‐6 and nitrite. The present findings show that bladder irradiation leads to urodynamic changes in the bladder and may suppress important immunoregulatory pathways in the urinary bladder.

Screening for human papillomavirus, cervical cytological abnormalities and associated risk factors in HIV-positive and HIV-negative women in Rwanda

Cervical cancer is the major cause of death from cancer in Africa. We wanted to assess the prevalence of human papillomavirus (HPV) infections and associated risk factors and to determine whether HPV testing could serve as a screening method for squamous intraepithelial lesions (SILs) in Rwanda. We also wanted to obtain a broader understanding of the underlying risk factors for the establishment of HPV infection in Rwanda.