This review is focused on purinergic neurotransmission, i.e., ATP released from nerves as a transmitter or cotransmitter to act as an extracellular signaling molecule on both pre- and postjunctional membranes at neuroeffector junctions and synapses, as well as acting as a trophic factor during development and regeneration. Emphasis is placed on the physiology and pathophysiology of ATP, but extracellular roles of its breakdown product, adenosine, are also considered because of their intimate interactions. The early history of the involvement of ATP in autonomic and skeletal neuromuscular transmission and in activities in the central nervous system and ganglia is reviewed. Brief background information is given about the identification of receptor subtypes for purines and pyrimidines and about ATP storage, release, and ectoenzymatic breakdown. Evidence that ATP is a cotransmitter in most, if not all, peripheral and central neurons is presented, as well as full accounts of neurotransmission and neuromodulation in autonomic and sensory ganglia and in the brain and spinal cord. There is coverage of neuron-glia interactions and of purinergic neuroeffector transmission to nonmuscular cells. To establish the primitive and widespread nature of purinergic neurotransmission, both the ontogeny and phylogeny of purinergic signaling are considered. Finally, the pathophysiology of purinergic neurotransmission in both peripheral and central nervous systems is reviewed, and speculations are made about future developments.

/pdf/physiology-and-pathophysiology-of-purinergic-1z94g2hz0f.pdf

Physiology and Pathophysiology of Purinergic Neurotransmission

Micturition, or urination, occurs involuntarily in infants and young children until the age of 3 to 5 years, after which it is regulated voluntarily. The neural circuitry that controls this process is complex and highly distributed: it involves pathways at many levels of the brain, the spinal cord and the peripheral nervous system and is mediated by multiple neurotransmitters. Diseases or injuries of the nervous system in adults can cause the re-emergence of involuntary or reflex micturition, leading to urinary incontinence. This is a major health problem, especially in those with neurological impairment. Here we review the neural control of micturition and how disruption of this control leads to abnormal storage and release of urine.

/pdf/the-neural-control-of-micturition-1ofs2ivm8i.pdf

The neural control of micturition

The detrusor smooth muscle is the main muscle component of the urinary bladder wall. Its ability to contract over a large length interval and to relax determines the bladder function during filling and micturition. These processes are regulated by several external nervous and hormonal control systems, and the detrusor contains multiple receptors and signaling pathways. Functional changes of the detrusor can be found in several clinically important conditions, e.g., lower urinary tract symptoms (LUTS) and bladder outlet obstruction. The aim of this review is to summarize and synthesize basic information and recent advances in the understanding of the properties of the detrusor smooth muscle, its contractile system, cellular signaling, membrane properties, and cellular receptors. Alterations in these systems in pathological conditions of the bladder wall are described, and some areas for future research are suggested.

https://www.physiology.org/doi/pdf/10.1152/physrev.00038.2003

Urinary bladder contraction and relaxation: Physiology and pathophysiology

Evaluation of the human genome suggests that all members of the connexin family of gap-junction proteins have now been successfully identified. This large and diverse family of proteins facilitates a number of vital cellular functions coupled with their roles, which range from the intercellular propagation of electrical signals to the selective intercellular passage of small regulatory molecules. Importantly, the extent of gap-junctional intercellular communication is under the direct control of regulatory events associated with channel assembly and turnover, as the vast majority of connexins have remarkably short half-lives of only a few hours. Since most cell types express multiple members of the connexin family, compensatory mechanisms exist to salvage tissue function in cases when one connexin is mutated or lost. However, numerous studies of the last decade have revealed that mutations in connexin genes can also lead to severe and debilitating diseases. In many cases, single point mutations lead to dramatic effects on connexin trafficking, assembly and channel function. This review will assess the current understanding of wild-type and selected disease-linked mutant connexin transport through the secretory pathway, gap-junction assembly at the cell surface, internalization and degradation.

Life cycle of connexins in health and disease

The concept of a purinergic signaling system, using purine nucleotides and nucleosides as extracellular messengers, was first proposed over 30 years ago. After a brief introduction and update of purinoceptor subtypes, this article focuses on the diverse pathophysiological roles of purines and pyrimidines as signaling molecules. These molecules mediate short-term (acute) signaling functions in neurotransmission, mechanosensory transduction, secretion and vasodilatation, and long-term (chronic) signaling functions in cell proliferation, differentiation, and death involved in development and regeneration. Plasticity of purinoceptor expression in pathological conditions is frequently observed, including an increase in the purinergic component of autonomic cotransmission. Recent advances in therapies using purinergic-related drugs in a wide range of pathological conditions will be addressed with speculation on future developments in the field.

/pdf/pathophysiology-and-therapeutic-potential-of-purinergic-5eb03t8gkw.pdf

Pathophysiology and Therapeutic Potential of Purinergic Signaling

Objective  To determine whether suburothelial interstitial cells of the human bladder express gap junctions, and if so, to establish their extent and composition, using immunocytochemistry, confocal microscopy and electron microscopy.



Materials and methods  Bladder tissue was obtained at cystectomy; the tissue was: (i) frozen for cryosectioning and Northern blot analysis; (ii) fixed and embedded for standard thin-section electron microscopy; and (iii) processed using low-denaturation conditions in Lowicryl for immunogold-label electron microscopy. Cryosections were immunofluorescently labelled using antibodies against connexins 43, 40 and 45, vimentin, desmin and c-Kit ligand, and examined by confocal microscopy. Double labelling was used to determine the spatial relationship of labelling for connexin43 with that of vimentin and desmin. Thin-section electron microscopy was used to investigate interstitial cell ultrastructure and permit unequivocal identification of gap junctions, and immunogold labelling of Lowicryl sections was applied to localize connexin43.



Results  Immunoconfocal microscopy showed prominent labelling for the gap junction protein, connexin43, in a suburothelial band of cells that was also strongly positive for vimentin. The connexin43/vimentin-positive cells showed only weak labelling for desmin and c-Kit ligand, and were immunonegative for connexins 40 and 45. Northern blotting showed a corresponding abundance of connexin43 transcript in the mucosal layer but not the detrusor layer of the bladder wall. Electron microscopy revealed abundant gap junctions, recognized by their pentalaminar structure, between the cell processes of interstitial cells in the suburothelial zone. That these interstitial cell gap junctions were the source of the connexin43 immunolabelling observed by immunoconfocal microscopy was confirmed by immunogold labelling in sections of Lowicryl-embedded tissue examined by electron microscopy.



Conclusion  A network of interstitial cells, extensively linked by connexin43-containing gap junctions, is located beneath the urothelium in human bladder. As gap junctions provide pathways for direct cell-to-cell communication, the interstitial cellular network may operate as a functional syncytium, integrating signals and responses in the bladder wall.

Gap junctions and connexin expression in human suburothelial interstitial cells.

The Ca(2+)-regulating and electrophysiological properties of guinea-pig suburothelial myofibroblasts have been measured in order to investigate their potential role in the sensation of bladder fullness, due to their strategic position between the urothelium and afferent fibres. Previous work has shown that stretch of the bladder wall releases ATP. Cells that stain positively for vimentin were isolated. About 45% of cells (median membrane capacitance 13.3 pF) exhibited spontaneous depolarizations to about -25 mV with a physiological Cl(-) gradient (frequency 2.6 +/- 1.5 min(-1), duration 14.5 +/- 2.2 s, n= 15). Under voltage-clamp spontaneous inward currents (frequency 1.5 +/- 0.2 min(-1), duration 14.5 +/- 7.0 s, n= 18) were recorded, with a similar reversal potential. The spontaneous currents were preceded by intracellular Ca(2+) transients with a magnitude that was independent of membrane potential. All cells tested responded to ATP by generating an intracellular Ca(2+) transient, followed by inward currents; the currents had a similar reversal potential and slope conductance to their spontaneous counterparts. ATP-generated transients were mimicked by UTP and ADP but not by alpha,beta-methylene-ATP (1-10 microm) or CTP (30 microm), indicating that ATP acts via a P2Y receptor. Transients were partially attenuated by 1 mm suramin but PPADS (80 microm) had no effect. These data indicate that ATP acts via a P2Y receptor, but responses were resistant to the P2Y(1) antagonist MRS2179. ATP-generated transients were abolished by intracellular perfusion with heparin and TMB-8 indicating that IP(3) was the intracellular second messenger. The reversal potentials of the spontaneous and ATP-generated currents were shifted by about +45 mV by a 12-fold reduction of the extracellular [Cl(-)] and the currents were greatly attenuated by 1 mm DIDS. No transients were generated on exposure to the muscarinic agonist carbachol. We propose that these cells may play a regulatory step in the sensation of bladder fullness by responding to ATP. The precise mechanism whereby they couple urothelial ATP release to afferent excitation is the next step to be elucidated.

https://physoc.onlinelibrary.wiley.com/doi/pdf/10.1113/jphysiol.2004.067934

Purinergic regulation of guinea pig suburothelial myofibroblasts.

The properties of suburothelial myofibroblasts are described, and their possible role in shaping sensory responses from the bladder wall are discussed. Suburothelial myofibroblasts consist of long spindle-shaped cells that form a distinctive layer below the urothelium and are connected to each other through connexin 43 gap junctions. Isolated cells from guinea pig or human bladders display spontaneous fluctuations of membrane potential and intracellular [Ca2+], and respond in a similar way to exogenous application of adenosine triphosphate (ATP) or lowering of extracellular pH. ATP generates an intracellular Ca2+ transient via activation of a P2Y receptor, which in turn initiates a Ca2+-sensitive Cl− current inward at the normal membrane potential of −50 to −60 mV. Of the P2Y receptor subtypes identified by immunolabeling, the most prominent was the P2Y6 receptor. Cell pairs, without the formation of gap junctions, elicit augmented responses to exogenous agonists. Mechanical stimulation of the suburothelial layer in intact cross-sections of the bladder elicited Ca2+ waves that propagated across the suburothelial layer before invading the detrusor layer. This indicates that the suburothelial layer forms a discrete functional layer of cells capable of propagating signals over many cell lengths. A function for suburothelial myofibroblasts is proposed whereby they act as an amplification stage in the sensory response to bladder-wall stretch, as occurs during bladder filling. Neurourol. Urodynam. 26:914–919, 2007. © 2007 Wiley-Liss, Inc.

The function of suburothelial myofibroblasts in the bladder.

Electrical Characteristics of Suburothelial Cells Isolated From the Human Bladder

Bladder suburothelial myofibroblasts may modulate both sensory responses from the bladder wall and spontaneous activity. This study aimed to characterize further these cells in their response to exogenous agents implicated in mediating the above activity. Detrusor strips, with or without mucosa, and isolated suburothelial myofibroblasts were prepared from guinea pig bladders. Isometric tension, intracellular Ca2+, and membrane current were recorded. Cell pairs were formed by pushing two cells together. Tension, intracellular Ca2+, and membrane potential were also recorded from bladder sheets using normal or spinal cord-transected (SCT) rats. Spontaneous contractions were greater in detrusor strips with an intact mucosa and were augmented by 10 μM UTP. ATP, UTP, or reduced extracellular pH elicited Ca2+ transients and inward currents (Erev −30 mV) in isolated cells. Capsaicin (5–30 μM) reduced membrane current (37 ± 12% of control) with minor effects on Ca2+ transients: sodium nitroprusside reduced membrane currents (40 ± 21% of control). Cell pair formation, without an increase in cell capacitance, augmented ATP and pH responses (180 ± 58% of control) and reduced the threshold to ATP and acidosis. Glivec (20–50 μM) reversibly blocked the augmentation and also reduced spontaneous activity in bladder sheets from SCT, but not normal, rats. Glivec also disrupted the spread of Ca2+ waves in SCT sheets, generating patterns similar to normal bladders. Suburothelial myofibroblasts respond to exogenous agents implicated in modulating bladder sensory responses; responses augmented by physical intercellular contact. The action of glivec and its selective suppression of spontaneous activity in SCT rats identifies a possible pathway to attenuate bladder overactivity.

Guiping Sui

Papers

Gap junctions and connexin expression in human suburothelial interstitial cells.

Purinergic regulation of guinea pig suburothelial myofibroblasts.

The function of suburothelial myofibroblasts in the bladder.

Electrical Characteristics of Suburothelial Cells Isolated From the Human Bladder

Modulation of bladder myofibroblast activity: implications for bladder function