Alvaro Munoz

ABCC8 and ABCC9: ABC transporters that regulate K+ channels

The sulfonylurea receptors (SURs) ABCC8/SUR1 and ABCC9/SUR2 are members of the C-branch of the transport adenosine triphosphatase superfamily. Unlike their brethren, the SURs have no identified transport function; instead, evolution has matched these molecules with K+ selective pores, either KIR6.1/KCNJ8 or KIR6.2/KCNJ11, to assemble adenosine triphosphate (ATP)-sensitive K+ channels found in endocrine cells, neurons, and both smooth and striated muscle. Adenine nucleotides, the major regulators of ATP-sensitive K+ (KATP) channel activity, exert a dual action. Nucleotide binding to the pore reduces the activity or channel open probability, whereas Mg-nucleotide binding and/or hydrolysis in the nucleotide-binding domains of SUR antagonize this inhibitory action to stimulate channel openings. Mutations in either subunit can alter this balance and, in the case of the SUR1/KIR6.2 channels found in neurons and insulin-secreting pancreatic β cells, are the cause of monogenic forms of hyperinsulinemic hypoglycemia and neonatal diabetes. Additionally, the subtle dysregulation of KATP channel activity by a KIR6.2 polymorphism has been suggested as a predisposing factor in type 2 diabetes mellitus. Studies on KATP channel null mice are clarifying the roles of these metabolically sensitive channels in a variety of tissues.

Botulinum toxin type A normalizes alterations in urothelial ATP and NO release induced by chronic spinal cord injury

The purpose of this paper was to simultaneously examine changes in urothelial ATP and NO release in normal and spinal cord injured animals as well as in spinal cord injured animals treated with botulinum toxin type A (BoNT-A). Furthermore we correlated changes in transmitter release with functional changes in bladder contraction frequency, and determined the effects of BoNT-A on bladder efferent nerve function. Normal and spinal cord injured rat bladders were injected on day 0 with either vehicle (saline containing bovine serum albumin) or BoNT-A. On day 2, in vitro neurotransmitter release and bladder strip contractility studies as well as in vivo cystometrographic studies were conducted. Resting ATP release was significantly enhanced following spinal cord injury (i.e. 57% increase, p<0.05) and was unaffected by BoNT-A treatment. SCI increased hypoosmotic evoked urothelial ATP release by 377% (p<0.05). BoNT-A treatment reduced evoked ATP release in SCI bladders by 83% (p<0.05). In contrast, hypoosmotic stimulation induced NO release was significantly inhibited following SCI (i.e. 50%, p<0.05) but recovered in SCI rats treated with BoNT-A (i.e. 195% increase in NO release in SCI-BTX-treated rats compared to SCI controls, p<0.01). Changes in urothelial transmitter release coincided with a significant decrease in non-voiding bladder contraction frequency (i.e. 71%, p<0.05) in SCI-BTX rats compared to SCI rats. While no difference was measured between neurally evoked contractile amplitude between SCI and SCI-BTX animals, atropine (1 microM) inhibited contractile amplitude to a greater extent (i.e. 76%, p<0.05) in the SCI-BTX group compared to the SCI group. We hypothesize that alterations in the ratio of excitatory (i.e. ATP) and inhibitory (i.e. NO) urothelial transmitters promote bladder hyperactivity in rat bladders following SCI that can be reversed, to a large extent, by treatment with BoNT-A.

Removal of urothelium affects bladder contractility and release of ATP but not release of NO in rat urinary bladder

BackgroundThe objective of our work was to investigate both the contractile function and the release of ATP and NO from strips of bladder tissue after removal of the urothelium.MethodsThe method of removal was a gentle swabbing motion rather than a sharp surgical cutting to separate the urothelium from the smooth muscle. The contractile response and ATP and NO release were measured in intact as well as on swabbed preparations. The removal of the urothelial layer was affirmed microscopically.ResultsAfter the swabbing, the smaller contractions were evoked by electrical as well as by chemical stimulation (50 μM carbachol or 50 μM α, β meATP). Electrical stimulation, carbachol and substance P (5 μM) evoked lower release of ATP in the swabbed strips than in intact strips. Although release of NO evoked by electrical stimulation or substance P was not changed, release of NO evoked by carbachol was significantly less in the swabbed preparations.ConclusionSince swabbing removes only the urothelium, the presence of the suburothelial layer may explain the difference between our findings and those of others who found an increase in contractility. Evoked release of ATP is reduced in swabbed strips, indicating that ATP derives solely from the urothelium. On the other hand, electrical stimulation and substance P evoke identical degrees of NO release in both intact and swabbed preparations, suggesting that NO can be released from the suburothelium. Conversely, carbachol-induced release of NO is lower in swabbed strips, implying that the cholinergic receptors (muscarinic or nicotinic) are located in the upper layer of the urothelium.

Modulation of bladder afferent signals in normal and spinal cord-injured rats by purinergic P2X3 and P2X2/3 receptors

Whats known on the subject? and What does the study add?

Central inhibitory effect of intravesically applied botulinum toxin A in chronic spinal cord injury

We evaluated a putative central inhibitory effect of intravesical botulinum toxin A (BoNT‐A) on the activity of lumbosacral spinal neurons in a chronic spinal cord injury (SCI) model of bladder overactivity.

Activation of cholinergic receptors blocks non-adrenergic non-cholinergic contractions in the rat urinary bladder

In the present study, the plasticity of the non-adrenergic non-cholinergic (NANC) response was investigated. Isolated rat bladder strips were electrically stimulated and the evoked contractions were isometrically recorded. The NANC part of the contractions were unmasked by applying 500 nM 4-DAMP, a potent muscarinic antagonist. Treatment of the bladder strips with 10 microM carbachol (a cholinergic agonist) increased the muscle tone but did not alter the neurally evoked contractions. However, carbachol decreased: (1) the NANC response from 74.6% to 33.3% of control and (2) the purinergic contractile response to alpha,beta-methylene ATP (alpha,beta-mATP) (10 microM) from 97.0% to 43.4% (p<0.05). Treatment with the cholinesterase inhibitor eserine (10 microM) also significantly decreased the NANC response to 21.1% (p<0.0001). The purinergic receptor antagonist suramin (100 microM) did not affect the neurally evoked contractions, however; subsequent addition of 4-DAMP decreased the contractions to 31%. Activation of the smooth muscle cholinergic receptors (with carbachol or eserine) and purinergic receptors (with alpha,beta-mATP) decreased the NANC contractions and the direct contractile response to alpha,beta-mATP. When the electrically evoked contractions were facilitated by the L-type Ca2+ channel activator, Bay-K 8644 the subsequent application of 4-DAMP did not unmask inhibited NANC contractions. We conclude that activation of muscarinic receptors by cholinergic agonist, carbachol or by endogenous acetylcholine (ACh) induce a cascade of events that leads to diminished purinergic response and consequently an inhibition of the bladder NANC response.

Lumbosacral sensory neuronal activity is enhanced by activation of urothelial purinergic receptors.

Urothelial purinergic receptors are important for the regulation of afferent sensory pathways in bladder pain and overactivity. Using in vivo electrophysiological recordings we evaluated the activity of spinal dorsal horn neurons in female rats at the L6/S1 level when urinary bladder pressure was abruptly increased. Intravesical infusion of ATP and systemic application of suramin allowed us to evaluate the contribution of urothelial purinergic receptors. Rats were anesthetized with isofluorane. Suprapubic, venous and tracheal catheters were implanted. Laminectomy was performed at the L6-S1 spinal levels. The cervical spinal cord was transected, and rats were mechanically pithed. Anesthesia was stopped, rats were ventilated, and a muscle relaxant was administered. The frequency of spinal neural activity was recorded via tungsten electrodes inserted into the dorsal horn at the L6-S1 level. The signal was amplified, filtered and recorded with a data acquisition system at 10 kHz sampling rate. Vital signs as well as bladder pressure were monitored in real time. We evaluated field potentials during intravesical pressure steps ranging from 0 to 60 cm H(2)O in (A) control (saline in the bladder), (B) after stimulation of urothelial purinergic receptors (1mM vesical ATP), and (C) after the intravenous application of the non-specific purinergic antagonist suramin (100mg/kg). Pressure steps were maintained for 1 min followed by 3 min for recovery. Only neurons that showed an increased activity during bladder distention were evaluated. Under control conditions, the generation of field potentials increased concomitantly with bladder pressure steps, showing an activity change threshold between 20 and 40 cm H(2)O. Intravesical application of 1mM ATP produced an increase in baseline activity, indicative of noxious stimulation, and spinal neuronal activity markedly increased above 40 cm H(2)O pressure. Systemic suramin prevented the increase in neural activity in response to pressure changes, even after intravesical ATP. These results suggest that urothelial purinergic receptors are important modulators of lumbosacral dorsal spinal neuronal activity. The inhibitory effects of suramin imply that enhanced lumbosacral neuronal signals result from activation of C-fibers during noxious bladder stimulation.

Plasticity of non-adrenergic non-cholinergic bladder contractions in rats after chronic spinal cord injury

The purpose of this study was to examine the pharmacologic plasticity of cholinergic, non-adrenergic non-cholinergic (NANC), and purinergic contractions in neurogenic bladder strips from spinal cord injured (SCI) rats. Bladder strips were harvested from female rats three to four weeks after T(9)-T(10) spinal cord transection. The strips were electrically stimulated using two experimental protocols to compare the contribution of muscarinic and NANC/purinergic contractions in the presence and the absence of carbachol or muscarine. The endpoints of the study were: (1) percent NANC contraction that was unmasked by the muscarinic antagonist 4-DAMP, and (2) P2X purinergic contraction that was evoked by α,β-methylene ATP. NANC contraction accounted for 78.5% of the neurally evoked contraction in SCI bladders. When SCI bladder strips were treated with carbachol (10 μM) prior to 4-DAMP (500 nM), the percent NANC contraction decreased dramatically to only 13.1% of the neurally evoked contraction (P=0.041). This was accompanied by a substantial decrease in α,β-methylene ATP evoked P2X contraction, and desensitization of purinergic receptors (the ratio of subsequent over initial P2X contraction decreased from 97.2% to 42.1%, P=0.0017). Sequential activation of the cholinergic receptors with carbachol (or with muscarine in neurally intact bladders) and unmasking of the NANC response with 4-DAMP switched the neurally evoked bladder contraction from predominantly NANC to predominantly cholinergic. We conclude that activation of muscarinic receptors (with carbachol or muscarine) blocks NANC and purinergic contractions in neurally intact or in SCI rat bladders. The carbachol-induced inhibition of the NANC contraction is expressed more in SCI bladders compared to neurally intact bladders. Along with receptor plasticity, this change in bladder function may involve P2X-independent mechanisms.

Diabetic plasticity of non-adrenergic non-cholinergic and P2X-mediated rat bladder contractions

We investigated the plasticity effects of diabetes mellitus and diuresis on the non-adrenergic non-cholinergic (NANC) and purinergic (P2X-type) contractile responses in longitudinal rat bladder strips. Female Sprague-Dawley rats received streptozotocin to induce diabetes, or sucrose in water to induce diuresis as a control condition for polyuria. Experiments were carried out at four weeks after treatments, using bladders from non-treated rats as control. Urinary bladder strips were electrically stimulated throughout the experiments to generate neurally evoked contractions (NEC). In all cases, P2X-mediated purinergic contractions were evaluated at the beginning and end of the stimulations with α,β-methylene-adenosine triphosphate (α,βMeATP). The NANC responses were assessed by using two independent protocols. First, cholinergic receptors were activated with carbachol (CCh), followed by inhibition of the muscarinic component with atropine. In the second protocol, the application order for CCh and atropine was reversed. The NANC response, unmasked with the application of atropine, and the P2X purinergic contractions were analyzed. NANC contractions in diabetic bladder strips are more resistant to the desensitizing effects caused by activation of cholinergic receptors. In early stages of experimental diabetes, NANC responses in diabetic strips are less sensitive to functional inhibition mediated by the cholinergic activation. However, P2X-mediated purinergic contractions are more sensitive to desensitization in diabetic or diuretic bladders. For instance preventing muscarinic receptor activation with atropine does not counteract the desensitization of purinergic contractions in either diabetic or diuretic strips. We suggest that diabetes may induce a plasticity of the NANC and P2X-mediated bladder contractile responses. The first one may be associated with diabetic neuropathic damage to bladder nerves, while impaired P2X purinergic contractions might be associated with detrusor hypertrophy observed in diabetic and diuretic strips.

RELEASE OF ATP AND NO FROM RAT PROSTATE: AN IN VITRO AND IN VIVO ASSESSMENT OF ADRENERGIC RECEPTOR STIMULATION

Hypothesis / aims of study Benign prostatic hyperplasia (BPH) and concomitant lower urinary tract symptoms (LUTS) affect millions of men worldwide. Recently, there has been an increasing effort to understand neurotransmitter mechanisms related to voiding dysfunction symptoms in BPH. Several studies have suggested changes in ATP and nitric oxide (NO) release in models of voiding dysfunction. The current literature suggests that ATP can often lead to overactivity in the genitourinary tract, and NO leads to relaxation. Our experiments aim to explore the relationship between ATP and NO release in prostate tissue when alpha-1 adrenergic receptors are activated with the agonist phenylephrine. We do this using both an in vitro organ bath system measuring stromal and capsular release, as well as an in vivo system which uses a microdialysis probe to isolate and sample the stromal contribution.