Donna Sellers

Diabetes Induced Changes in Rat Mesenchymal Stem Cells

Diabetes mellitus, the single most important cause of vascular disease in the industrialized world, is also associated with bone loss and impaired fracture healing. Mesenchymal stem cells (MSCs) have the potential to differentiate into osteoblasts, chondrocytes and adipocytes and other mesenchymal cells and play a central role in bone formation and repair. Because of this, we have investigated the possibility that diabetes has direct effects on MSCs in vivo and that this might represent a cellular basis for diabetes-induced osteoporosis. We isolated MSCs from rats with streptozotocin-induced diabetes and analysed them ex vivo for their ability to proliferate and differentiate in the fibroblastic colony-forming unit assay. Effects of diabetes on bone metabolism in vivo were determined by analysing tibiae from control and diabetic animals by quantitative computerized tomography. The total number of colonies and osteoblastic colonies staining positive for alkaline phosphatase were quantified and both colony size and number were found to be significantly reduced in diabetic rats. The changes appear to be mediated by the induction of apoptosis and senescence by advanced glycation end products (AGEs), together with an increase in the receptor for AGEs (RAGE). These changes were paralleled by extensive loss of trabecular bone in the tibiae of the diabetic animals. These data suggest that MSCs become exhausted during diabetes and lose their differentiation potential, leading to a net loss of trabecular bone. Therefore, direct effects on MSCs may be responsible for some of the orthopaedic effects associated with diabetes.

The role of the urothelium in mediating bladder responses to isoprenaline.

To investigate whether the responses of the pig bladder to isoprenaline (a nonselective β‐adrenoceptor agonist) are influenced by the presence of an intact urothelium and whether any influence might be attributed to the release of nitric oxide (NO), since stimulation of β‐adrenoceptors induces a direct relaxation of detrusor smooth muscle and β‐adrenoceptors are also present on the urothelium.

OnabotulinumtoxinA significantly attenuates bladder afferent nerve firing and inhibits ATP release from the urothelium

To investigate the direct effect of onabotulinumtoxinA (OnaBotA) on bladder afferent nerve activity and release of ATP and acetylcholine (ACh) from the urothelium.

The β3-adrenoceptor mediates the inhibitory effects of β-adrenoceptor agonists via the urothelium in pig bladder dome†‡

Relaxation of detrusor muscle via β‐adrenoceptors may contribute to urine storage during bladder filling. Thus there is increasing interest in β‐adrenoceptor agonists as a potential treatment for detrusor overactivity. The role of the urothelium in bladder responses to β‐adrenoceptor agonists is not yet clear, although we have shown that these agonists have a greater inhibitory effect on detrusor contraction when the urothelium is intact. The aim was to determine which β‐adrenoceptor subtype is involved in this effect.

The role of c-kit-positive interstitial cells in mediating phasic contractions of bladder strips from streptozotocin-induced diabetic rats

What’s known on the subject? and What does the study add?

(−)-(9S)-9-(3-Bromo-4-fluorophenyl)-2,3,5,6,7,9-hexahydrothieno[3,2-b]quinolin-8(4H)-one 1,1-Dioxide (A-278637): A Novel ATP-Sensitive Potassium Channel Opener Efficacious in Suppressing Urinary Bladder Contractions. I. In Vitro Characterization

Alterations in the myogenic activity of the bladder smooth muscle are thought to serve as a basis for the involuntary detrusor contractions associated with the overactive bladder. Activation of ATP-sensitive K+ (KATP) channels has been recognized as a potentially viable mechanism to modulate membrane excitability in bladder smooth muscle. In this study, we describe the preclinical pharmacology of (−)-(9S)-9-(3-bromo-4-fluorophenyl)-2,3,5,6,7,9-hexahydrothieno[3,2-b]quinolin-8(4H)-one 1,1-dioxide (A-278637), a novel 1,4-dihydropyridine KATPchannel opener (KCO) that demonstrates enhanced bladder selectivity for the suppression of unstable bladder contractions in vivo relative to other reference KCOs. A-278637 activated KATP channels in bladder smooth muscle cells in a glyburide (glibenclamide)-sensitive manner as assessed by fluorescence membrane potential assays using bis-(1,3-dibutylbarbituric acid)trimethine oxonol (EC50 = 102 nM) and by whole cell patch clamp. Spontaneous (myogenic) phasic activity of pig bladder strips was suppressed (IC50 = 23 nM) in a glyburide-sensitive manner by A-278637. A-278637 also inhibited carbachol- and electrical field-stimulated contractions of bladder strips, although the respective potencies were 8- and 13-fold lower compared with inhibition of spontaneous phasic activity. As shown in the accompanying article [Brune ME, Fey TA, Brioni JD, Sullivan JP, Williams M, Carroll WA, Coghlan MJ, and Gopalakrishnan M (2002)J Pharmacol Exp Ther 303:387–394], A-278637 suppressed myogenic contractions in vivo in a model of bladder instability with superior selectivity compared with other KCOs, WAY-133537 [(R)-4-[3,4-dioxo-2-(1,2,2-trimethyl-propylamino)cyclobut-1-enylamino]-3-ethyl-benzonitrile] and ZD6169 [(S)-N-(4-benzoylphenyl)3,3,3-trifluro-2hydroxy-2-methyl-priopionamide]. A-278637 did not interact with other ion channels, including L-type calcium channels or other neurotransmitter receptor systems. The pharmacological profile of A-278637 represents an attractive basis for further investigations of selective KATP channel openers for the treatment of overactive bladder via myogenic etiology.

Testosterone suppresses the expression of regulatory enzymes of fatty acid synthesis and protects against hepatic steatosis in cholesterol-fed androgen deficient mice

AIMS Non-alcoholic fatty liver disease and its precursor hepatic steatosis is common in obesity and type-2 diabetes and is associated with cardiovascular disease (CVD). Men with type-2 diabetes and/or CVD have a high prevalence of testosterone deficiency. Testosterone replacement improves key cardiovascular risk factors. The effects of testosterone on hepatic steatosis are not fully understood. MAIN METHODS Testicular feminised (Tfm) mice, which have a non-functional androgen receptor (AR) and very low serum testosterone levels, were used to investigate testosterone effects on high-cholesterol diet-induced hepatic steatosis. KEY FINDINGS Hepatic lipid deposition was increased in Tfm mice and orchidectomised wild-type littermates versus intact wild-type littermate controls with normal androgen physiology. Lipid deposition was reduced in Tfm mice receiving testosterone treatment compared to placebo. Oestrogen receptor blockade significantly, but only partially, reduced the beneficial effects of testosterone treatment on hepatic lipid accumulation. Expression of key regulatory enzymes of fatty acid synthesis, acetyl-CoA carboxylase alpha (ACACA) and fatty acid synthase (FASN) were elevated in placebo-treated Tfm mice versus placebo-treated littermates and Tfm mice receiving testosterone treatment. Tfm mice on normal diet had increased lipid accumulation compared to littermates but significantly less than cholesterol-fed Tfm mice and demonstrated increased gene expression of hormone sensitive lipase, stearyl-CoA desaturase-1 and peroxisome proliferator-activated receptor-gamma but FASN and ACACA were not altered. SIGNIFICANCE An action of testosterone on hepatic lipid deposition which is independent of the classic AR is implicated. Testosterone may act in part via an effect on the key regulatory lipogenic enzymes to protect against hepatic steatosis.

Spontaneous contractions of the pig urinary bladder : the effect of ATP-sensitive potassium channels and the role of the mucosa

To investigate the influence of the mucosa on the inhibitory effects of the ATP‐sensitive potassium channel (KATP channel) opener, cromakalim, on the spontaneous contractions of pig bladder strips from the bladder dome and trigone. Little is known about the influence of the mucosa on spontaneous contractions and whether the nature of these contractions differs between the bladder dome and trigone.

Muscarinic agonists and antagonists: Effects on the urinary bladder

Voiding of the bladder is the result of a parasympathetic muscarinic receptor activation of the detrusor smooth muscle. However, the maintenance of continence and a normal bladder micturition cycle involves a complex interaction of cholinergic, adrenergic, nitrergic and peptidergic systems that is currently little understood. The cholinergic component of bladder control involves two systems, acetylcholine (ACh) released from parasympathetic nerves and ACh from non-neuronal cells within the urothelium. The actions of ACh on the bladder depend on the presence of muscarinic receptors that are located on the detrusor smooth muscle, where they cause direct (M₃) and indirect (M₂) contraction; pre-junctional nerve terminals where they increase (M₁) or decrease (M₄) the release of ACh and noradrenaline (NA); sensory nerves where they influence afferent nerve activity; umbrella cells in the urothelium where they stimulate the release of ATP and NO; suburothelial interstitial cells with unknown function; and finally, other unidentified sites in the urothelium from where prostaglandins and inhibitory/relaxatory factors are released. Thus, the actions of muscarinic receptor agonists and antagonists on the bladder may be very complex even when considering only local muscarinic actions. Clinically, muscarinic antagonists remain the mainstay of treatment for the overactive bladder (OAB), while muscarinic agonists have been used to treat hypoactive bladder. The antagonists are effective in treating OAB, but their precise mechanisms and sites of action (detrusor, urothelium, and nerves) have yet to be established. Potentially more selective agents may be developed when the cholinergic systems within the bladder are more fully understood.

Potential therapeutic targets for treatment of the overactive bladder

Abstract Muscarinic receptor antagonists remain the main therapy for the treatment of the overactive bladder yet severe adverse effects make them unsuitable for a large number of patients. The development of new drugs with novel mechanisms of action for the treatment of this condition is therefore essential. This article considers some of the targets currently under investigation for the development of such compounds. β-adrenoceptor agonists and KATP channel openers inhibit detrusor muscle activity and remain targets for drug development. There is also evidence that α-adrenoceptor antagonists may be effective in the overactive bladder, but the mechanism involved in this action is unclear. Finally the role of tachykinins in regulating bladder function through both the sensory and the motor innervation make them a potential target for drug development, but as with the the others, a selective action on the bladder must remain the goal of drug development.