Rajender Nandigama

Feed Forward Cycle of Hypotonic Stress-induced ATP Release, Purinergic Receptor Activation, and Growth Stimulation of Prostate Cancer Cells

ATP is released in many cell types upon mechanical strain, the physiological function of extracellular ATP is largely unknown, however. Here we report that ATP released upon hypotonic stress stimulated prostate cancer cell proliferation, activated purinergic receptors, increased intracellular [Ca2+]i, and initiated downstream signaling cascades that involved MAPKs ERK1/2 and p38 as well as phosphatidylinositol 3-kinase (PI3K). MAPK activation, the calcium response as well as induction of cell proliferation upon hypotonic stress were inhibited by preincubation with the ATP scavenger apyrase, indicating that hypotonic stress-induced signaling pathways are elicited by released ATP. Hypotonic stress increased prostaglandin E2 (PGE2) synthesis. Consequently, ATP release was inhibited by antagonists of PI3K (LY294002 and wortmannin), phospholipase A2 (methyl arachidonyl fluorophosphonate (MAFP)), cyclooxygenase-2 (COX-2) (indomethacin, etodolac, NS398) and 5,8,11,14-eicosatetraynoic acid (ETYA), which are involved in arachidonic acid metabolism. Furthermore, ATP release was abolished in the presence of the adenylate cyclase (AC) inhibitor MDL-12,330A, indicating regulation of ATP-release by cAMP. The hypotonic stress-induced ATP release was significantly blunted when the ATP-mediated signal transduction cascade was inhibited on different levels, i.e. purinergic receptors were blocked by suramin and pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS), the Ca2+ response was inhibited upon chelation of intracellular Ca2+ by 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), and ERK1,2 as well as p38 were inhibited by UO126 and SB203580, respectively. In summary our data demonstrate that hypotonic stress initiates a feed forward cycle of ATP release and purinergic receptor signaling resulting in proliferation of prostate cancer cells.

Bladder outlet obstruction influences mRNA expression of cholinergic receptors on sensory neurons in mice.

AIMS In patients with bladder outlet obstruction (BOO), dysregulation of bladder afferent neurons seems to contribute to irritative symptoms. Cholinergic receptors, addressed by both neuronal and non-neuronal (urothelial) acetylcholine, can alter neuronal excitability. Thus we investigated the influence of BOO on the expression of muscarinic (mAChR) and nicotinic (nAChR) acetylcholine receptors in the lumbosacral dorsal root ganglia (DRG) of mice. MAIN METHODS BOO was induced in 13 C57/BL6 mice by partial suturing of the urethra. Eleven mice were sham-operated (loose/freely movable suture around the urethra), and eleven untreated mice served as controls. Cystometry was performed five weeks later in conscious mice. DRG at segmental levels L5-S2 were dissected and real-time quantitative PCR was performed. Expression of mAChR subtypes M1-M5 and nAChR subunits α2-7, α9-10, β2-4 was examined. KEY FINDINGS Expression of all mAChR subtypes and nAChR subunits α3-7, α10, β2-4 was detected. Expression of α2 and α9 was absent. Rank order of expression was M2>M4>M3>M5>M1, α3≥α6>α7>α4>α10>α5 and β2>β4>β3 in untreated animals. BOO mice presented distinct obstruction with development of residual urine. Sham mice showed only minimal BOO. Relative mRNA expression of nAChR subunits revealed significant reduction of α3, α5, α6, α10 and β4 in sham-operated vs. untreated mice. In BOO vs. sham-operated mice, reduction of nAChR subunits α10 (p=0.038) and α5 (p=0.053) was found. SIGNIFICANCE BOO has a considerable impact on nAChR, but not on mAChR mRNA expression in sensory neurons. We hypothesize that a reduction in mRNA expression of nAChR subunits represents a link to altered sensitivity under non-obstructive conditions.

Expression of nicotinic acetylcholine receptor subunit mRNA in mouse bladder afferent neurons

Nicotinic acetylcholine receptors (nAChR) influence bladder afferent activity and reflex sensitivity, and have been suggested as potential targets for treating detrusor overactivity. Mechanisms may include indirect effects, e.g. involving the urothelium, and direct action on nAChR expressed by afferent neurons. Here we determined the nAChR repertoire of bladder afferent neurons by retrograde neuronal tracing and laser-assisted microdissection/reverse transcriptase polymerase chain reaction (RT-PCR), and quantified retrogradely labelled nAChRα3-subunit-expressing neurons by immunohistochemistry in nAChR α3β4α5 cluster enhanced green fluorescent protein (eGFP) reporter mice. Bladder afferents distinctly expressed mRNAs encoding for nAChR-subunits α3, α6, α7, β2-4, and weakly α4. Based upon known combinatorial patterns of subunits, this predicts the expression of at least three basically different subunits of nAChR - α3(∗), α6(∗) and α7(∗) - and of additional combinations with β-subunits and α5. Bladder afferents were of all sizes, and their majority (69%; n=1367) were eGFP-nAChRα3 positive. Immunofluorescence revealed immunoreactivities to neurofilament 68 (NF68), transient receptor potential cation channel vanilloid 1 (TRPV1), substance P (SP) and calcitonin gene-related peptide (CGRP) in eGFP-nAChRα3-positive and -negative neurons. For each antigen, all possible combinations of colocalisation with eGFP-nAChRα3 were observed, with eGFP-nAChRα3-positive bladder neurons without additional immunoreactivity being most numerous, followed by triple-labelled neurons. In conclusion, more than one population of bladder afferent neurons expresses nAChR, indicating that peripheral nicotinic initiation and modulation of bladder reflexes might result, in addition to indirect effects, from the direct activation of sensory terminals. The expression of multiple nAChR subunits offers the potential of selectively addressing functional aspects and/or sensory neuron subpopulations.