Kevin Monaghan

Pacemaking in interstitial cells of Cajal depends upon calcium handling by endoplasmic reticulum and mitochondria

1 Pacemaker cells, known as interstitial cells of Cajal (ICC), generate electrical rhythmicity in the gastrointestinal tract. Pacemaker currents in ICC result from the activation of a voltage‐independent, non‐selective cation conductance, but the timing mechanism responsible for periodic activation of the pacemaker current is unknown. 2 Previous studies suggest that pacemaking in ICC is dependent upon metabolic activity 1y1yand1 Ca2+ release from intracellular stores. We tested the hypothesis that mitochondrial Ca2+ handling may underlie the dependence of gastrointestinal pacemaking on oxidative metabolism. 3 Pacemaker currents occurred spontaneously in cultured ICC and were associated with mitochondrial Ca2+ transients. 4 Inhibition of the electrochemical gradient across the inner mitochondrial membrane blocked Ca2+ uptake and pacemaker currents in cultured ICC and blocked slow wave activity in intact gastrointestinal muscles from mouse, dog and guinea‐pig. 5 Pacemaker currents and rhythmic mitochondrial Ca2+ uptake in ICC were also blocked by inhibitors of IP3‐dependent release of Ca2+ from the endoplasmic reticulum and by inhibitors of endoplasmic reticulum Ca2+ reuptake. 6 Our data suggest that integrated Ca2+ handling by endoplasmic reticulum and mitochondria is a prerequisite of electrical pacemaking in the gastrointestinal tract.

Novel voltage-dependent non-selective cation conductance in murine colonic myocytes

Two components of voltage‐gated, inward currents were observed from murine colonic myocytes. One component had properties of L‐type Ca2+ currents and was inhibited by nicardipine (5 × 10−7m). A second component did not ‘run down’ during dialysis and was resistant to nicardipine (up to 10−6m). The nicardipine‐insensitive current was activated by small depolarizations above the holding potential and reversed near 0 mV. This low‐voltage‐activated current (ILVA) was resolved with step depolarizations positive to ‐60 mV, and the current rapidly inactivated upon sustained depolarization. The voltage of half‐inactivation was ‐65 mV. Inactivation and activation time constants at ‐45 mV were 86 and 15 ms, respectively. The half‐recovery time from inactivation was 98 ms at ‐45 mV. ILVA peaked at ‐40 mV and the current reversed at 0 mV. I lva was inhibited by Ni2+ (IC50= 1.4 × 10−5m), mibefradil (10−6 to 10−5m), and extracellular Ba2+. Replacement of extracellular Na+ with N‐methyl‐d‐glucamine inhibited ILVA and shifted the reversal potential to ‐7 mV. Increasing extracellular Ca2+ (5 × 10−3m) increased the amplitude of ILVA and shifted the reversal potential to +22 mV. ILVA was also blocked by extracellular Cs+ (10−4m) and Gd3+ (10−6m). Warming increased the rates of activation and deactivation without affecting the amplitude of the peak current. We conclude that the second component of voltage‐dependent inward current in murine colonic myocytes is not a ‘T‐type’ Ca2+ current but rather a novel, voltage‐gated non‐selective cation current. Activation of this current could be important in the recovery of membrane potential following inhibitory junction potentials in gastrointestinal smooth muscle or in mediating responses to agonists.

Contribution of Kv4 channels toward the A-type potassium current in murine colonic myocytes

A rapidly inactivating K+ current (A‐type current; IA) present in murine colonic myocytes is important in maintaining physiological patterns of slow wave electrical activity. The kinetic profile of colonic IA resembles that of Kv4‐derived currents. We examined the contribution of Kv4 α‐subunits to IA in the murine colon using pharmacological, molecular and immunohistochemical approaches. The divalent cation Cd2+ decreased peak IA and shifted the voltage dependence of activation and inactivation to more depolarized potentials. Similar results were observed with La3+. Colonic IA was sensitive to low micromolar concentrations of flecainide (IC50= 11 μM). Quantitative PCR indicated that in colonic and jejunal tissue, Kv4.3 transcripts demonstrate greater relative abundance than transcripts encoding Kv4.1 or Kv4.2. Antibodies revealed greater Kv4.3‐like immunoreactivity than Kv4.2‐like immunoreactivity in colonic myocytes. Kv4‐like immunoreactivity was less evident in jejunal myocytes. To address this finding, we examined the expression of K+ channel‐interacting proteins (KChIPs), which act as positive modulators of Kv4‐mediated currents. Qualitative PCR identified transcripts encoding the four known members of the KChIP family in isolated colonic and jejunal myocytes. However, the relative abundance of KChIP transcript was 2.6‐fold greater in colon tissue than in jejunum, as assessed by quantitative PCR, with KChIP1 showing predominance. This observation is in accordance with the amplitude of the A‐type current present in these two tissues, where colonic myocytes possess densities twice that of jejunal myocytes. From this we conclude that Kv4.3, in association with KChIP1, is the major molecular determinant of IA in murine colonic myocytes.

Identification of PDGFRα Positive Populations of Interstitial Cells in Human and Guinea Pig Bladders

PURPOSE The bladder wall comprises a complex array of cells, including urothelium, smooth muscle, nerves and interstitial cells. Interstitial cells have several subtypes based on site, morphology and differential expression of markers such as anti-vimentin and anti-KIT. We examined whether a subpopulation of interstitial cells immunopositive for PDGFRα exists in human and guinea pig bladders. MATERIALS AND METHODS Human and guinea pig bladder tissues were processed for immunohistochemistry and examined by bright field or confocal microscopy. Whole mount tissues and paraffin sections were labeled with antibodies to PDGFRα, vimentin, KIT and PGP9.5. Protein expression was assessed by Western blot. RESULTS PDGFRα(+) cells were present in human and guinea pig bladders. In the guinea pig PDGFRα(+) cells had a branched stellate morphology and formed networks in the lamina propria. In human and guinea pig detrusors PDGFRα(+) cells were elongated on the boundary of smooth muscle bundles or were seen as groups of stellate cells in the interbundle spaces. PDGFRα(+) cells were located close to nerves labeled by PGP9.5. Double labeling revealed that PDGFRα(+) cells were a subgroup of the vimentin(+) population. A significant proportion of PDGFRα(+) cells were also KIT(+). Bands corresponding to PDGFRα, KIT and vimentin proteins were detected on Western blot. CONCLUSIONS To our knowledge this study is the first to identify PDGFRα(+)/KIT(+) cells in the bladder lamina propria and detrusor layers. These cells are a subgroup of the vimentin(+) population, showing the complexity of bladder interstitial cells. PDGFRα(+) cells are apparently structurally associated with intramural nerves, indicating integration with bladder control mechanisms.

A pH-sensitive potassium conductance (TASK) and its function in the murine gastrointestinal tract

The excitability of smooth muscles is regulated, in part, by background K+ conductances that determine resting membrane potential. However, the K+ conductances so far described in gastrointestinal (GI) muscles are not sufficient to explain the negative resting potentials of these cells. Here we describe expression of two‐pore K+ channels of the TASK family in murine small and large intestinal muscles. TASK‐2, cloned from murine intestinal muscles, resulted in a pH‐sensitive, time‐dependent, non‐inactivating K+ conductance with slow activation kinetics. A similar conductance was found in native intestinal myocytes using whole‐cell patch‐clamp conditions. The pH‐sensitive current was blocked by local anaesthetics. Lidocaine, bupivacaine and acidic pH depolarized circular muscle cells in intact muscles and decreased amplitude and frequency of slow waves. The effects of lidocaine were not blocked by tetraethylammonium chloride, 4‐aminopyridine, glibenclamide, apamin or MK‐499. However, depolarization by acidic pH was abolished by pre‐treatment with lidocaine, suggesting that lidocaine‐sensitive K+ channels were responsible for pH‐sensitive changes in membrane potential. The kinetics of activation, sensitivity to pH, and pharmacology of the conductance in intestinal myocytes and the expression of TASK‐1 and TASK‐2 in these cells suggest that the pH‐sensitive background conductance is encoded by TASK genes. This conductance appears to contribute significantly to resting potential and may regulate excitability of GI muscles.

The stretch‐dependent potassium channel TREK‐1 and its function in murine myometrium

Non‐technical summary  During pregnancy the uterus must maintain a low contractile state to permit growth of the fetus and inhibt premature delivery. We show uterine smooth muscle cells express specific potassium channels (called stretch‐dependent potassium channels; TREK‐1). These channels are activated by stretch, stabilize resting membrane potentials of cells at negative potentials, and reduce excitability. During pregnancy the expression of TREK‐1 channels increases, and this may contribute to reduced excitability. Near the onset of labour, TREK‐1 expression declines, and this may promote the transition to a contractile state. Thus, our data suggest dynamic regulation of TREK‐1 channel expression in the uterus contributes to the maintenance of pregnancy.

Hyperglycemia and Diabetes Downregulate the Functional Expression of TRPV4 Channels in Retinal Microvascular Endothelium

Retinal endothelial cell dysfunction is believed to play a key role in the etiology and pathogenesis of diabetic retinopathy. Numerous studies have shown that TRPV4 channels are critically involved in maintaining normal endothelial cell function. In the current paper, we demonstrate that TRPV4 is functionally expressed in the endothelium of the retinal microcirculation and that both channel expression and activity is downregulated by hyperglycaemia. Quantitative PCR and immunostaining demonstrated molecular expression of TRPV4 in cultured bovine retinal microvascular endothelial cells (RMECs). Functional TRPV4 activity was assessed in cultured RMECs from endothelial Ca2+-responses recorded using fura-2 microfluorimetry and electrophysiological recordings of membrane currents. The TRPV4 agonist 4α-phorbol 12,13-didecanoate (4-αPDD) increased [Ca2+]i in RMECs and this response was largely abolished using siRNA targeted against TRPV4. These Ca2+-signals were completely inhibited by removal of extracellular Ca2+, confirming their dependence on influx of extracellular Ca2+. The 4-αPDD Ca2+-response recorded in the presence of cyclopiazonic acid (CPA), which depletes the intracellular stores preventing any signal amplification through store release, was used as a measure of Ca2+-influx across the cell membrane. This response was blocked by HC067047, a TRPV4 antagonist. Under voltage clamp conditions, the TRPV4 agonist GSK1016790A stimulated a membrane current, which was again inhibited by HC067047. Following incubation with 25mM D-glucose TRPV4 expression was reduced in comparison with RMECs cultured under control conditions, as were 4αPDD-induced Ca2+-responses in the presence of CPA and ion currents evoked by GSK1016790A. Molecular expression of TRPV4 in the retinal vascular endothelium of 3 months’ streptozotocin-induced diabetic rats was also reduced in comparison with that in age-matched controls. We conclude that hyperglycaemia and diabetes reduce the molecular and functional expression of TRPV4 channels in retinal microvascular endothelial cells. These changes may contribute to diabetes induced endothelial dysfunction and retinopathy.

GABAergic control of arteriolar diameter in the rat retina

PURPOSE To investigate the role of γ-aminobutryic acid (GABA) in the regulation of arteriolar diameter in the rat retina. METHODS The actions of GABA on arteriolar diameter were examined using ex vivo retinal whole-mount preparations and isolated vessel segments. In most experiments, arterioles were partially preconstricted with endothelin (Et)-1. The expression levels of GABAA and GABAB receptors on isolated rat retinal Müller cells were assessed by immunohistochemistry. RESULTS GABA (0.1-1 mM) evoked vasodilation or vasoconstriction of arterioles in whole-mount preparations. No such effects were observed with isolated vessel segments. In whole mount samples, the GABAA receptor agonist muscimol caused vasomotor responses in only a small proportion of vessels. In contrast, arteriolar responses to the GABAB receptor agonists baclofen and SKF97541 more closely resembled those observed with GABA. No responses were seen with the GABAC receptor agonist 5-methylimidazoleacetic acid. GABA-induced vasodilator responses were, for the most part, repeatable in the presence of the GABAA receptor antagonist bicuculline. These responses, however, were completely blocked in the presence of the GABAB receptor inhibitor 2-hydroxysaclofen. Strong immunolabeling for both GABAA and GABAB receptors was detected in isolated Müller cells. In the absence of Et-1-induced preconstriction, most vessels were unresponsive to bicuculline or 2-hydroxysaclofen. CONCLUSIONS GABA exerts complex effects on arteriolar diameter in the rat retina. These actions appear largely dependent upon the activation of GABAB receptors in the retinal neuropile, possibly those located on perivascular Müller cells. Despite these findings, endogenous GABA appears to contribute little to the regulation of basal arteriolar diameter in the rat retina.

Mucosal modulation of contractility in bladder strips from normal and overactive rat models and the effect of botulinum toxin A on overactive bladder strips

To investigate the local, regulatory role of the mucosa on bladder strip contractility from normal and overactive bladders and to examine the effect of botulinum toxin A (BoNT‐A).

TRPA1 activation in a human sensory neuronal model: Relevance to cough hypersensitivity?

The cough reflex becomes hyperresponsive in acute and chronic respiratory diseases, but understanding the underlying mechanism is hampered by difficulty accessing human tissue containing both nerve endings and neuronal cell bodies. We refined an adult stem cell sensory neuronal model to overcome the limited availability of human neurones and applied the model to study transient receptor potential ankyrin 1 (TRPA1) channel expression and activation. Human dental pulp stem cells (hDPSCs) were differentiated towards a neuronal phenotype, termed peripheral neuronal equivalents (PNEs). Using molecular and immunohistochemical techniques, together with Ca2+ microfluorimetry and whole cell patch clamping, we investigated roles for nerve growth factor (NGF) and the viral mimic poly I:C in TRPA1 activation. PNEs exhibited morphological, molecular and functional characteristics of sensory neurons and expressed functional TRPA1 channels. PNE treatment with NGF for 20 min generated significantly larger inward and outward currents compared to untreated PNEs in response to the TRPA1 agonist cinnamaldehyde (p<0.05). PNE treatment with poly I:C caused similar transient heightened responses to TRPA1 activation compared to untreated cells. Using the PNE neuronal model we observed both NGF and poly I:C mediated sensory neuronal hyperresponsiveness, representing potential neuro-inflammatory mechanisms associated with heightened nociceptive responses recognised in cough hypersensitivity syndrome. Development of a novel human adult stem cell neuronal model to investigate neural hyperresponsiveness in cough http://ow.ly/Ilvb30eokUC