Salah A. Baker

Electrical Slow Waves in the Mouse Oviduct Are Dependent upon a Calcium Activated Chloride Conductance Encoded by Tmem16a

ABSTRACT Myosalpinx contractions are critical for oocyte transport along the oviduct. A specialized population of pacemaker cells—oviduct interstitial cells of Cajal—generate slow waves, the electrical events underlying myosalpinx contractions. The ionic basis of oviduct pacemaker activity is unknown. We examined the role of a new class of Ca2+-activated Cl− channels (CaCCs)—anoctamin 1, encoded by Tmem16a—in oviduct slow wave generation. RT-PCR revealed the transcriptional expression of Tmem16a-encoded CaCCs in the myosalpinx. Intracellular microelectrode recordings were performed in the presence of two pharmacologically distinct Cl− channel antagonists, anthracene-9-carboxylic acid and niflumic acid. Both of these inhibitors caused membrane hyperpolarization, reduced the duration of slow waves, and ultimately inhibited pacemaker activity. Niflumic acid also inhibited propagating calcium waves within the myosalpinx. Slow waves were present at birth in wild-type and heterozygous oviducts but failed to develop by birth in mice homozygous for a null allele of Tmem16a (Tmem16atm1Bdh/tm1Bdh). These data suggest that Tmem16a-encoded CaCCs contribute to membrane potential and are responsible for the upstroke and plateau phases of oviduct slow waves.

Sulfur‐containing amino acids block stretch‐dependent K+ channels and nitrergic responses in the murine colon

1 Efforts to determine the role of stretch‐dependent K+ (SDK) channels in enteric inhibitory neural responses in gastrointestinal muscles are difficult due to a lack of blocking drugs for SDK channels. 2 SDK channels are blocked by sulfur‐containing amino acids. These compounds reduced the open probability of SDK channels in on and off‐cell patches of murine colonic myocytes. L‐Methionine was the most selective and had little or no effect on other known K+ conductances in colonic myocytes. 3 Application of L‐cysteine, L‐methionine or DL‐homocysteine depolarized intact muscles and enhanced spontaneous contractions. D‐Stereoisomers of these amino acids were less effective than L‐stereoisomers. 4 Pretreatment of muscles with tetrodotoxin, NW‐nitro‐L‐arginine or 1H‐[1,2,4] oxadiazolo [4,3‐a] quinoxalin‐1‐one reduced the depolarization responses to these compounds, suggesting that spontaneous neural activity and release of NO tonically activates SDK channels. 5 Nitrergic responses to nerve stimulation were reduced by sulfur‐containing amino acids. 6 These data suggest that nitrergic inhibitory junction potentials are mediated, in part, by activation of SDK channels in murine colonic muscles.

Distribution and Ca2+ signalling of fibroblast‐like (PDGFRα+) cells in the murine gastric fundus

•  A new class of interstitial cells, PDGFRα+ cells, is distributed densely in the proximal stomachs of mice. •  PDGFRα+ cells express the molecular apparatus necessary for transduction of inputs from enteric inhibitory motor neurons. •  PDGFRα+ cells generate spontaneous Ca2+ transients and display dynamic Ca2+ oscillations in response to purines. •  Purinergic responses are mediated by P2Y1 receptors and by Ca2+ release from intracellular Ca2+ stores. •  Ca2+ release in PDGFRα+ cells is the likely means by which purinergic neurotransmitters activate Ca2+‐activated K+ channels (SK) and hyperpolarization in GI muscles to elicit inhibitory motor responses. •  Spontaneous Ca2+ transients may be a means of regulating basal excitability of fundus muscles and release of purines from motor neurons may contribute to the control of pressure during filling in the proximal stomach.

Propagation of slow waves requires IP3 receptors and mitochondrial Ca2+ uptake in canine colonic muscles

In the gastrointestinal (GI) tract electrical slow waves yield oscillations in membrane potential that periodically increase the open probability of voltage‐dependent Ca2+ channels and facilitate phasic contractions. Slow waves are generated by the interstitial cells of Cajal (ICC), and these events actively propagate through ICC networks within the walls of GI organs. The mechanism that entrains spontaneously active pacemaker sites throughout ICC networks to produce regenerative propagation of slow waves is unresolved. Agents that block inositol 1,4,5‐trisphosphate (IP3) receptors and mitochondrial Ca2+ uptake were tested on the generation of slow waves in the canine colon. A partitioned chamber apparatus was used to test the effects of blocking slow‐wave generation on propagation. We found that active propagation occurred along strips of colonic muscle, but when the pacemaker mechanism was blocked in a portion of the tissue, slow waves decayed exponentially from the point where the pacemaker mechanism was inhibited. An IP3 receptor inhibitor, mitochondrial inhibitors, low external Ca2+, and divalent cations (Mn2+ and Ni2+) caused exponential decay of the slow waves in regions of muscle exposed to these agents. These data demonstrate that the mechanism that initiates slow waves is reactivated from cell‐to‐cell during the propagation of slow waves. Voltage‐dependent conductances present in smooth muscle cells are incapable of slow‐wave regeneration. The data predict that partial loss of or disruptions to ICC networks observed in human motility disorders could lead to incomplete penetration of slow waves through GI organs and, thus, to defects in myogenic regulation.

Characterization of the A-type potassium current in murine gastric antrum.

A‐type currents are rapidly inactivating potassium currents that operate at subthreshold potentials. A‐type currents have not been reported to occur in the phasic muscles of the stomach. We used conventional voltage‐clamp techniques to identify and characterize A‐type currents in myocytes isolated from the murine antrum. A‐type currents were robust in these cells, with peak current densities averaging 30 pA pF−1 at 0 mV. These currents underwent rapid inactivation with a time constant of 83 ms at 0 mV. Recovery from inactivation at −80 mV was rapid, with a time constant of 252 ms. The A‐type current was blocked by 4‐aminopyridine (4‐AP) and was inhibited by flecainide, with an IC50 of 35 μM. The voltage for half‐activation was −26 mV, while the voltage of half‐inactivation was −65 mV. There was significant activation and incomplete inactivation at potentials positive to −60 mV, which is suggestive of sustained current availability in this voltage range. Under current‐clamp conditions, exposure to 4‐AP or flecainide depolarized the membrane potential by 7‐10 mV. In intact antral tissue preparations, flecainide depolarized the membrane potential between slow waves by 5 mV; changes in slow waves were not evident. The effect of flecainide was not abolished by inhibiting enteric neurotransmission or by blocking delayed rectifier and ATP‐sensitive K+ currents. Transcripts encoding Kv4 channels were detected in isolated antral myocytes by RT‐PCR. Immunocytochemistry revealed intense Kv4.2‐ and Kv4.3‐like immunoreactivity in antral myocytes. These data suggest that the A‐type current in murine antral smooth muscle cells is likely to be due to Kv4 channels. This current contributes to the maintenance of negative resting membrane potentials.

Methionine and its derivatives increase bladder excitability by inhibiting stretch‐dependent K+ channels

During the bladder filling phase, the volume of the urinary bladder increases dramatically, with only minimal increases in intravesical pressure. To accomplish this, the smooth muscle of the bladder wall must remain relaxed during bladder filling. However, the mechanisms responsible for the stabilization of bladder excitability during stretch are unclear. We hypothesized that stretch‐dependent K+ (TREK) channels in bladder smooth muscle cells may inhibit contraction in response to stretch.

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.

Temporal sequence of activation of cells involved in purinergic neurotransmission in the colon

Platelet derived growth factor receptor α (PDGFRα+) cells in colonic muscles are innervated by enteric inhibitory motor neurons. PDGFRα+ cells generate Ca2+ transients in response to exogenous purines and these responses were blocked by MRS‐2500. Stimulation of enteric neurons, with cholinergic and nitrergic components blocked, evoked Ca2+ transients in PDGFRα+ and smooth muscle cells (SMCs). Responses to nerve stimulation were abolished by MRS‐2500 and not observed in muscles with genetic deactivation of P2Y1 receptors. Ca2+ transients evoked by nerve stimulation in PDGFRα+ cells showed the same temporal characteristics as electrophysiological responses. PDGFRα+ cells express gap junction genes, and drugs that inhibit gap junctions blocked neural responses in SMCs, but not in nerve processes or PDGFRα+ cells. PDGFRα+ cells are directly innervated by inhibitory motor neurons and purinergic responses are conducted to SMCs via gap junctions.

Role of TREK-1 potassium channel in bladder overactivity after partial bladder outlet obstruction in mouse.

PURPOSE Mouse models of partial bladder outlet obstruction cause bladder hypertrophy. Expression of a number of ion channels is altered in hypertrophic detrusor muscle, resulting in bladder dysfunction. We determined whether mechanosensitive TREK-1 channels are present in the murine bladder and whether their expression is altered in partial bladder outlet obstruction, resulting in abnormal filling responses. MATERIALS AND METHODS Partial bladder outlet obstruction was surgically induced in CD-1 mice and the mice recovered for 14 days. Cystometry was done to evaluate bladder pressure responses during filling at 25 microl per minute in partial bladder outlet obstruction mice and sham operated controls. TREK-1 channel expression was determined at the mRNA and protein levels by quantitative reverse transcriptase-polymerase chain reaction and Western blotting, respectively, and localized in the bladder wall using immunohistochemistry. RESULTS Obstructed bladders showed about a 2-fold increase in weight vs sham operated bladders. TREK-1 channel protein expression on Western blots from bladder smooth muscle strip homogenates was significantly decreased in obstructed mice. Immunohistochemistry revealed a significant decrease in TREK-1 channel immunoreactivity in detrusor smooth muscle in obstructed mice. On cystometry the TREK-1 channel blocker L-methioninol induced a significant increase in premature contractions during filling in sham operated mice. L-methioninol had no significant effect in obstructed mice, which showed an overactive detrusor phenotype. CONCLUSIONS TREK-1 channel down-regulation in detrusor myocytes is associated with bladder overactivity in a murine model of partial bladder outlet obstruction.