Spontaneous Transient Depolarizations Research Articles

Cardiac PDGFRα+ interstitial cells generate spontaneous inward currents that contribute to excitability in the heart.

The cell types and conductance that contribute to normal cardiac functions remain under investigation. We used mice that express an enhanced green fluorescent protein (eGFP)-histone 2B fusion protein driven off the cell-specific endogenous promoter for Pdgfra to investigate the distribution and functional role of PDGFRα+ cells in the heart. Cardiac PDGFRα+ cells were widely distributed within the endomysium of atria, ventricle, and sino-atrial node (SAN) tissues. PDGFRα+ cells formed a discrete network of cells, lying in close apposition to neighboring cardiac myocytes in mouse and Cynomolgus monkey (Macaca fascicularis) hearts. Expression of eGFP in nuclei allowed unequivocal identification of these cells following enzymatic dispersion of muscle tissues. FACS purification of PDGFRα+ cells from the SAN and analysis of gene transcripts by qPCR revealed that they were a distinct population of cells that expressed gap junction transcripts, Gja1 and Gjc1. Cardiac PDGFRα+ cells generated spontaneous transient inward currents (STICs) and spontaneous transient depolarizations (STDs) that reversed at 0 mV. Reversal potential was maintained when ECl = -40 mV. [Na+ ]o replacement and FTY720 abolished STICs, suggesting they were due to a non-selective cation conductance (NSCC) carried by TRPM7. PDGFRα+ cells also express β2 -adrenoceptor gene transcripts, Adrb2. Zinterol, a selective β2 -receptor agonist, increased the amplitude and frequency of STICs, suggesting these cells could contribute to adrenergic regulation of cardiac excitability. PDGFRα+ cells in cardiac muscles generate inward currents via an NSCC. STICs generated by these cells may contribute to the integrated membrane potentials of cardiac muscles, possibly affecting the frequency of pacemaker activity.

Ca2+ -activated Cl- channels (TMEM16A) underlie spontaneous electrical activity in isolated mouse corpus cavernosum smooth muscle cells.

Penile detumescence is maintained by tonic contraction of corpus cavernosum smooth muscle cells (CCSMC), but the underlying mechanisms have not been fully elucidated. The purpose of this study was to characterize the mechanisms underlying activation of TMEM16A Ca2+ -activated Cl- channels in freshly isolated murine CCSMC. Male C57BL/6 mice aged 10-18 weeks were euthanized via intraperitoneal injection of sodium pentobarbital (100 mg.kg-1 ). Whole-cell patch clamp, pharmacological, and immunocytochemical experiments were performed on isolated CCSM. Tension measurements were performed in whole tissue. TMEM16A expression in murine corpus cavernosum was confirmed using immunocytochemistry. Isolated CCSMC developed spontaneous transient inward currents (STICs) under voltage clamp and spontaneous transient depolarizations (STDs) in current clamp mode of the whole cell, perforated patch clamp technique. STICs reversed close to the predicted Cl- equilibrium potential and both STICs and STDs were blocked by the TMEM16A channel blockers, Ani9 and CaCC(inh)-A01. These events were also blocked by GSK7975A (ORAI inhibitor), cyclopiazonic acid (CPA, sarcoplasmic reticulum [SR] Ca2+- ATPase blocker), tetracaine (RyR blocker), and 2APB (IP3 R blocker), suggesting that they were dependent on Ca2+ release from intracellular Ca2+ stores. Nifedipine (L-type Ca2+ channel blocker) did not affect STICs, but reduced the duration of STDs. Phenylephrine induced transient depolarizations and transient inward currents which were blocked by Ani9. Similarly, phenylephrine induced phasic contractions of intact corpus cavernosum muscle strips and these events were also inhibited by Ani9. This study suggests that contraction of CCSM is regulated by activation of TMEM16A channels and therefore inhibition of these channels could lead to penile erection.

Pacemaker Mechanisms Driving Pyeloureteric Peristalsis: Modulatory Role of Interstitial Cells.

The peristaltic pressure waves in the renal pelvis that propel urine expressed by the kidney into the ureter towards the bladder have long been considered to be 'myogenic', being little affected by blockers of nerve conduction or autonomic neurotransmission, but sustained by the intrinsic release of prostaglandins and sensory neurotransmitters. In uni-papilla mammals, the funnel-shaped renal pelvis consists of a lumen-forming urothelium and a stromal layer enveloped by a plexus of 'typical' smooth muscle cells (TSMCs), in multi-papillae kidneys a number of minor and major calyces fuse into a large renal pelvis. Electron microscopic, electrophysiological and Ca2+ imaging studies have established that the pacemaker cells driving pyeloureteric peristalsis are likely to be morphologically distinct 'atypical' smooth muscle cells (ASMCs) that fire Ca2+ transients and spontaneous transient depolarizations (STDs) which trigger propagating nifedipine-sensitive action potentials and Ca2+ waves in the TSMC layer. In uni-calyceal kidneys, ASMCs predominately locate on the serosal surface of the proximal renal pelvis while in multi-papillae kidneys they locate within the sub-urothelial space. 'Fibroblast-like' interstitial cells (ICs) located in the sub-urothelial space or adventitia are a mixed population of cells, having regional and species-dependent expression of various Cl-, K+, Ca2+ and cationic channels. ICs display asynchronous Ca2+ transients that periodically synchronize into bursts that accelerate ASMC Ca2+ transient firing. This review presents current knowledge of the architecture of the proximal renal pelvis, the role Ca2+ plays in renal pelvis peristalsis and the mechanisms by which ICs may sustain/accelerate ASMC pacemaking.

Effects of new-generation TMEM16A inhibitors on calcium-activated chloride currents in rabbit urethral interstitial cells of Cajal.

Interstitial cells of Cajal (ICC) isolated from the rabbit urethra exhibit Ca2+-activated Cl- currents (I ClCa) that are important for the development of urethral tone. Here, we examined if TMEM16A (ANO1) contributed to this activity by examining the effect of "new-generation" TMEM16A inhibitors, CACCinh-A01 and T16Ainh-A01, on I ClCa recorded from freshly isolated rabbit urethral ICC (RUICC) and on contractions of intact strips of rabbit urethra smooth muscle. Real-time quantitative PCR experiments demonstrated that TMEM16A was highly expressed in rabbit urethra smooth muscle, in comparison to TMEM16B and TMEM16F. Single-cell RT-PCR experiments revealed that only TMEM16A was expressed in freshly isolated RUICC. Depolarization-evoked I ClCa in isolated RUICC, recorded using voltage clamp, were inhibited by CACCinh-A01 and T16Ainh-A01 with IC50 values of 1.2 and 3.4μM, respectively. Similarly, spontaneous transient inward currents (STICs) recorded from RUICC voltage clamped at -60mV and spontaneous transient depolarizations (STDs), recorded in current clamp, were also inhibited by CACCinh-A01 and T16Ainh-A01. In contrast, spontaneous Ca2+ waves in isolated RUICC were only partially reduced by CACCinh-A01 and T16Ainh-A01. Finally, neurogenic contractions of strips of rabbit urethra smooth muscle (RUSM), evoked by electric field stimulation (EFS), were also significantly reduced by CACCinh-A01 and T16Ainh-A01. These data are consistent with the idea that TMEM16A is involved with CACCs in RUICC and in contraction of rabbit urethral smooth muscle.

Role of prostatic interstitial cells in prostate motility.

The prostate is a gland whose secretions contribute to the seminal fluids ejaculated upon activation of autonomic sympathetic nerves. In elder males, the prostate undergoes an increase in stroma mass and myogenic tone, leading to benign prostatic hyperplasia that occludes the proximal urethra and the presentation of various lower urinary tract symptoms that decrease their quality of life. This review summarises the role of prostatic interstitial cells (PICs) in the generation of the spontaneous tone in the prostate. It presents current knowledge of the role of Ca2+ plays in PIC pacemaking, as well as the mechanisms by which this spontaneous activity triggers slow wave generation and stromal contraction. PICs display a small T-type Ca2+ current (ICaT) and a large L-type Ca2+ current (ICaL). In contrast to other interstitial cells in the urinary and gastrointestinal tracts, spontaneous Ca2+ signalling in PICs is uniquely dependent on Ca2+ influx through ICaL channels. A model of prostatic pacemaking is presented describing how ICaL can be triggered by an initial membrane depolarization evoked upon the selective opening of Ca2+-activated Cl– channels by Ca2+ flowing only through ICaT channels. The resulting current flow through ICaL results in release of Ca2+ from internal stores and the summation of Cl–-selective spontaneous transient depolarizations (STDs) to form pacemaker potentials that propagate passively into the prostatic stroma to evoke regenerative action potentials and excitation-contraction coupling.

Satellite microglia show spontaneous electrical activity that is uncorrelated with activity of the attached neuron.

Microglia are innate immune cells of the brain. We have studied a subpopulation of microglia, called satellite microglia. This cell type is defined by a close morphological soma-to-soma association with a neuron, indicative of a direct functional interaction. Indeed, ultrastructural analysis revealed closely attached plasma membranes of satellite microglia and neurons. However, we found no apparent morphological specializations of the contact, and biocytin injection into satellite microglia showed no dye-coupling with the apposed neurons or any other cell. Likewise, evoked local field potentials or action potentials and postsynaptic potentials of the associated neuron did not lead to any transmembrane currents or non-capacitive changes in the membrane potential of the satellite microglia in the cortex and hippocampus. Both satellite and non-satellite microglia, however, showed spontaneous transient membrane depolarizations that were not correlated with neuronal activity. These events could be divided into fast-rising and slow-rising depolarizations, which showed different characteristics in satellite and non-satellite microglia. Fast-rising and slow-rising potentials differed with regard to voltage dependence. The frequency of these events was not affected by the application of tetrodotoxin, but the fast-rising event frequency decreased after application of GABA. We conclude that microglia show spontaneous electrical activity that is uncorrelated with the activity of adjacent neurons.

Spatiotemporal dynamics of synaptic drive in urinary bladder syncytium: A computational investigation.

The urinary bladder wall is composed of the detrusor smooth muscle (DSM) in which adjacent cells are electrically coupled to form a three dimensional syncytium. The structural complexity of the tissue is further enhanced by a distributed innervation pattern. Experimental techniques employed in order to analyze detrusor excitability have been unable as yet to provide a satisfactory understanding of either the normal electrical functioning of the tissue, or of the changes that come about in pathological conditions. Our work aims at exploring the interplay between factors that determines the spread of junction potentials in the tissue which is critical for generation of action potential and subsequent contraction of the bladder wall. Results from our model suggest that in the detrusor syncytium, the mean interval between subsequent spontaneous neurotransmitter releases at a single varicosity is about 91.5 seconds such that in the normally functioning tissue, spontaneous transient depolarizations (STDs) occur with a mean interval of 2-7 seconds. Increase in neurotransmitter release frequency might result in higher excitability of the tissue, leading to bladder instability. Results also indicate that increase in intercellular coupling is another probable cause for such a pathophysiological scenario.

Intracellular Ca2+release from endoplasmic reticulum regulates slow wave currents and pacemaker activity of interstitial cells of Cajal

Interstitial cells of Cajal (ICC) provide pacemaker activity in gastrointestinal muscles that underlies segmental and peristaltic contractions. ICC generate electrical slow waves that are due to large-amplitude inward currents resulting from anoctamin 1 (ANO1) channels, which are Ca(2+)-activated Cl(-) channels. We investigated the hypothesis that the Ca(2+) responsible for the stochastic activation of ANO1 channels during spontaneous transient inward currents (STICs) and synchronized activation of ANO1 channels during slow wave currents comes from intracellular Ca(2+) stores. ICC, obtained from the small intestine of Kit(+/copGFP) mice, were studied under voltage and current clamp to determine the effects of blocking Ca(2+) uptake into stores and release of Ca(2+) via inositol 1,4,5-trisphosphate (IP3)-dependent and ryanodine-sensitive channels. Cyclocpiazonic acid, thapsigargin, 2-APB, and xestospongin C inhibited STICs and slow wave currents. Ryanodine and tetracaine also inhibited STICs and slow wave currents. Store-active compounds had no direct effects on ANO1 channels expressed in human embryonic kidney-293 cells. Under current clamp, store-active drugs caused significant depolarization of ICC and reduced spontaneous transient depolarizations (STDs). After block of ryanodine receptors with ryanodine and tetracaine, repolarization did not restore STDs. ANO1 expressed in ICC has limited access to cytoplasmic Ca(2+) concentration, suggesting that pacemaker activity depends on Ca(2+) dynamics in restricted microdomains. Our data from studies of isolated ICC differ somewhat from studies on intact muscles and suggest that release of Ca(2+) from both IP3 and ryanodine receptors is important in generating pacemaker activity in ICC.

ANO1‐ther brick in the wall – role of Ca2+‐activated Cl− channels of interstitial cells of Cajal in cholinergic motor control of gastrointestinal smooth muscle

ANO1‐ther brick in the wall – role of Ca²⁺‐activated Cl⁻ channels of interstitial cells of Cajal in cholinergic motor control of gastrointestinal smooth muscle

Muscarinic activation of Ca2+‐activated Cl− current in interstitial cells of Cajal

Interstitial cells of Cajal (ICC) provide pacemaker activity and functional bridges between enteric motor nerve terminals and gastrointestinal smooth muscle cells. The ionic conductance(s) in ICC that are activated by excitatory neural inputs are unknown. Transgenic mice (Kit(copGFP/+)) with constitutive expression of a bright green fluorescent protein were used to investigate cellular responses of ICC to cholinergic stimulation. ICC displayed spontaneous transient inward currents (STICs) under voltage clamp that corresponded to spontaneous transient depolarizations (STDs) under current clamp. STICs reversed at 0 mV when E(Cl) = 0 mV and at -40 mV when E(Cl) was -40 mV, suggesting the STICs were due to a chloride conductance. Carbachol (CCh, 100 nm and 1 μm) induced a sustained inward current (depolarization in current clamp) and increased the amplitude and frequency of STICs and STDs. CCh responses were blocked by atropine (10 μm) or 4-DAMP (100 nm), an M(3) receptor antagonist. STDs were blocked by niflumic acid and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (both 100 μm), and CCh had no effect in the presence of these drugs. The responses of intact circular muscles to CCh and stimulation of intrinsic excitatory nerves by electrical field stimulation (EFS) were also compared. CCh (1 μm) caused atropine-sensitive depolarization and increased the maximum depolarization of slow waves. Similar atropine-sensitive responses were elicited by stimulation of intrinsic excitatory neurons. Niflumic acid (100 μm) blocked responses to EFS but had minor effect on responses to exogenous CCh. These data suggest that different ionic conductances are responsible for electrical responses elicited by bath-applied CCh and cholinergic nerve stimulation.

The functional role of intramuscular interstitial cells of Cajal in the stomach

Intramuscular interstitial cells of Cajal (ICC-IM) are found within the smooth muscle layers of the stomach. ICC-IM are mainly spindle shaped cells with bipolar processes orientated along the long axis of surrounding smooth muscle cells. ICC-IM make close contacts with nerve varicosities and form gap junctions with neighbouring smooth muscle cells, indicating that ICC-IM mediate enteric motor neurotransmission. These morphological properties of ICC-IM are similar throughout the stomach. However, the electrical properties of these cells differ from region to region. In the fundus, ICC-IM generate spontaneous transient depolarizations (STDs), resulting in an ongoing discharge of unitary potentials in the smooth muscle cells. ICC-IM in the corpus generate slow waves and as they fire at the highest frequency they serve as the dominant pacemaker cells in the stomach. On the other hand, ICC-IM in the antrum generate the secondary component of slow waves triggered by the initial component that propagates passively from myenteric ICC (ICC-MY). Thus, the different electrical properties of ICC-IM play a critical role in creating the distinct functions of the proximal and distal regions of the stomach such that the fundus acts as a reservoir of food, the corpus as a dominant pacemaker region, while the antrum acts as a region for mixing and propulsion of food.

Functions of ICC‐like cells in the urinary tract and male genital organs

Interstitial cells of Cajal (ICC)-like cells (ICC-LCs) have been identified in many regions of the urinary tract and male genital organs by immunohistochemical studies and electron microscopy. ICC-LCs are characterized by their spontaneous electrical and Ca2+ signalling and the cellular mechanisms of their generation have been extensively investigated. Spontaneous activity in ICC-LCs rises from the release of internally stored Ca2+ and the opening of Ca2+-activated Cl− channels to generate spontaneous transient depolarizations (STDs) in a manner not fundamentally dependent on Ca2+ influx through L-type voltage-dependent Ca2+ channels. Since urogenital ICC-LCs have been identified by their immunoreactivity to Kit (CD117) antibodies, the often-used specific marker for ICC in the gastrointestinal tract, their functions have been thought likely to be similar. Thus ICC-LCs in the urogenital tract might be expected to act as either electrical pacemaker cells to drive the smooth muscle wall or as intermediaries in neuromuscular transmission. However, present knowledge of the functions of ICC-LCs suggests that their functions are not so predetermined, that their functions may be very region specific, particularly under pathological conditions. In this review, we summarize recent advances in our understanding of the location and function of ICC-LCs in various organs of the urogenital system. We also discuss several unsolved issues regarding the identification, properties and functions of ICC-LCs in various urogenital regions in health and disease.

Spontaneous electrical and Ca2+ signals in the mouse renal pelvis that drive pyeloureteric peristalsis

1. Peristalsis in the smooth muscle cell (SMC) wall of the pyeloureteric system is unique in physiology in that the primary pacemaker resides in a population of atypical SMCs situated near the border of the renal papilla. 2. Atypical SMCs display high-frequency Ca(2+) transients upon the spontaneous release of Ca(2+) from inositol 1,4,5-trisphosphate (IP(3))-dependent stores that trigger cation-selective spontaneous transient depolarizations (STDs). In the presence of nifedipine, these Ca(2+) transients and STDs seldom propagate > 100 mum. Synchronization of STDs in neighbouring atypical SMCs into an electrical signal that can trigger action potential discharge and contraction in the typical SMC layer involves a coupled oscillator mechanism dependent on Ca(2+) entry through L-type voltage-operated Ca(2+) channels. 3. A population of spindle- or stellate-shaped cells, immunopositive for the tyrosine receptor kinase kit, is sparsely distributed throughout the pyeloureteric system. In addition, Ca(2+) transients and action potentials of long duration occurring at low frequencies have been recorded in a population of fusiform cells, which we have termed interstitial cells of Cajal (ICC)-like cells. 4. The electrical and Ca(2+) signals in ICC-like cells are abolished upon blockade of Ca(2+) release from either IP(3)- or ryanodine-dependent Ca(2+) stores. However, the spontaneous Ca(2+) signals in atypical SMCs or ICC-like cells are little affected in W/W(-v) transgenic mice, which have extensive lesions of their intestinal ICC networks. 5. In summary, we have developed a model of pyeloureteric pacemaking in which atypical SMCs are indeed the primary pacemakers, but the function of ICC-like cells has yet to be determined.

Interstitial cells of Cajal generate spontaneous transient depolarizations in the rat gastric fundus

Intracellular recordings were made from isolated circular muscle bundles of rat gastric fundus. The majority of cells generated an ongoing discharge of electrical activity that were <or=10 mV in amplitude (unitary potentials). A second pattern of electrical activity was recorded in less than 1% of all impalements. This electrical activity was characterized by high frequency, large amplitude spontaneous transient depolarizations (STDs) with a maximum rate of rise (dV/dt(max)) of 0.5 V/s. Injection of the fluorescent dye propidium iodide into cells and double labeling with an antibody against the Kit receptor revealed that unitary potentials were recorded from circular smooth muscle cells (CSMC), whereas STDs were generated by intramuscular interstitial cells of Cajal (ICC-IM). Sustained injection periods (>15 min) resulted in the spread of dye between CSMC, between ICC-IM, and between CSMC and ICC-IM. Two types of STDs were observed, regularly occurring continuous STDs and irregular noisy bursting STDs. The amplitude of STDs varied between the two types of STDs. Single units summed to develop STDs with a maximum amplitude of 30 mV. Sodium nitroprusside (3 microM) induced membrane hyperpolarization and abolished unitary potentials generated by CSMC. In contrast, the amplitude of STDs generated by ICC-IM was increased with membrane hyperpolarization. Hyperpolarization induced by pinacidil (10 microM) also increased the amplitude of STDs and enhanced dV/dt(max). These observations indicate that STDs generated in ICC-IM spread passively to the adjacent CSMC to evoke the discharge of unitary potentials in the gastric fundus.

KCNQ Currents and Their Contribution to Resting Membrane Potential and the Excitability of Interstitial Cells of Cajal From the Guinea Pig Bladder

PurposeThe presence of novel KCNQ currents was investigated in guinea pig bladder interstitial cells of Cajal and their contribution to the maintenance of the resting membrane potential was assessed.Materials and MethodsEnzymatically dispersed interstitial cells of Cajal were patch clamped with K+ filled pipettes in voltage clamp and current clamp modes. Pharmacological modulators of KCNQ channels were tested on membrane currents and the resting membrane potential.ResultsCells were stepped from −60 to 40 mV to evoke voltage dependent currents using a modified K+ pipette solution containing ethylene glycol tetraacetic acid (5 mM) and adenosine triphosphate (3 mM) to eliminate large conductance Ca activated K channel and Kadenosine triphosphate currents. Application of the KCNQ blockers XE991, linopirdine (Tocris Bioscience, Ellisville, Missouri) and chromanol 293B (Sigma®) decreased the outward current in concentration dependent fashion. The current-voltage relationship of XE991 sensitive current revealed a voltage dependent, outwardly rectifying current that activated positive to −60 mV and showed little inactivation. The KCNQ openers flupirtine and meclofenamic acid (Sigma) increased outward currents across the voltage range. In current clamp mode XE991 or chromanol 293B decreased interstitial cell of Cajal resting membrane potential and elicited the firing of spontaneous transient depolarizations in otherwise quiescent cells. Flupirtine or meclofenamic acid hyperpolarized interstitial cells of Cajal and inhibited any spontaneous electrical activity.ConclusionsThis study provides electrophysiological evidence that bladder interstitial cells of Cajal have KCNQ currents with a role in the regulation of interstitial cell of Cajal resting membrane potential and excitability. These novel findings provide key information on the ion channels present in bladder interstitial cells of Cajal and they may indicate relevant targets for the development of new therapies for bladder instability.

Spontaneous Electrical Waveforms in Aging Guinea Pig Prostates

We characterized spontaneous electrical activity in the aging guinea pig prostate. Membrane potential recordings were made using conventional single microelectrode recording techniques. Three types of spontaneous waveforms were recorded, including spikes, slow waves and spontaneous transient depolarizations. Spikes were classified as hyperactive or active. Active cells showed a mean +/- SEM frequency of 5.06 +/- 0.63 minutes(-1), significantly different from that in hyperactive cells (362.05 +/- 151.82 minutes(-1), p <0.05). After hyperpolarization amplitude was also significantly different in the active and hyperactive groups (17.80 +/- 1.98 vs 9.96 +/- 1.05 mV). Spike activity was abolished by 1 microM nifedipine in 7 preparations (p <0.05). Slow wave activity occurred at a frequency of 5.2 +/- 0.5 minutes(-1). The spike component of slow wave activity was abolished by 1 microM nifedipine, although the depolarizing transient remained unaltered from control values (8.1 +/- 3.1 mV, paired Student's t test p >0.05). Spontaneous transient depolarizations were recorded in the presence of slow waves in 10 preparations and of spikes in 13, and in quiescent cells in 9. Spontaneous transient depolarization frequency was highest in otherwise quiescent cells (24.55 +/- 6.48 minutes(-1)) compared to that in the presence of slow waves or spikes. Adding 1 microM nifedipine in 5 preparations did not significantly affect any measured parameters (p >0.05). Pacemaker potentials were not recorded in the aging prostate. With increased age there is an increase in spike activity, which could conceivably explain the increased prostatic tone that accompanies aging. Spike activity and the spike component of the slow wave were abolished by nifedipine, suggesting a role for L-type channels. Finally, spontaneous transient depolarizations were unaffected by nifedipine, suggesting that mechanisms other than Ca(2+) entry via L-type channels are responsible for their generation and maintenance.

Spontaneous transient depolarizations in lymphatic vessels of the guinea pig mesentery: pharmacology and implication for spontaneous contractility

Guinea pig mesenteric lymphatic vessels exhibit rhythmic constrictions induced by action potential (AP)-like spikes and initiated by entrainment of spontaneous transient depolarizations (STDs). To characterize STDs and the signaling mechanisms responsible for their occurrence, we used intracellular microelectrodes, Ca2+ imaging, and pharmacological agents. In our investigation of the role of intracellular Ca2+ released from Ca2+ stores, we observed that intracellular Ca2+ transients accompanied some STDs, although there were many exceptions where Ca2+ transients occurred without accompanying STDs. STD frequency and amplitude were markedly affected by activators/inhibitors of inositol 1,4,5-trisphosphate receptors (IP3Rs) but not by treatments known to alter Ca2+ release via ryanodine receptors. A role for Ca2+-activated Cl(-) (Cl(Ca)) channels was indicated, as STDs were dependent on the Cl(-) but not Na+ concentration of the superfusing solution and were inhibited by the Cl(Ca) channel blockers niflumic acid (NFA), anthracene 9-carboxylic acid, and 5-nitro-2-(3-phenylpropylamino)benzoic acid but not by the volume-regulated Cl(-) blocker DIDS. Increases in STD frequency and amplitude induced by agonist stimulation were also inhibited by NFA. Nifedipine, the hyperpolarization-activated inward current blocker ZD-7288, and the nonselective cation/store-operated channel blockers SKF-96365, Gd3+, and Ni2+ had no or marginal effects on STD activity. However, nifedipine, 2-aminoethoxydiphenyl borate, NFA, SKF-96365, Gd3+, and Ni2+ altered the occurrence of spontaneous APs. Our findings support a role for Ca2+ release through IP3Rs and a resultant opening of Cl(Ca) channels in STD generation and confirm the importance of these events in the initiation of lymphatic spontaneous APs and subsequent contractions. The abolition of spontaneous APs by blockers of other excitatory ion channels suggests a contribution of these conductances to lymphatic pacemaking.

Spontaneous NA+ transients in individual mitochondria of intact astrocytes

Mitochondria in intact cells maintain low Na(+) levels despite the large electrochemical gradient favoring cation influx into the matrix. In addition, they display individual spontaneous transient depolarizations. The authors report here that individual mitochondria in living astrocytes exhibit spontaneous increases in their Na(+) concentration (Na(mit)(+) spiking), as measured using the mitochondrial probe CoroNa Red. In a field of view with approximately 30 astrocytes, up to 1,400 transients per minute were typically detected under resting conditions. Na(mit)(+) spiking was also observed in neurons, but was scarce in two nonneural cell types tested. Astrocytic Na(mit)(+) spikes averaged 12.2 +/- 0.8 s in duration and 35.5 +/- 3.2 mM in amplitude and coincided with brief mitochondrial depolarizations; they were impaired by mitochondrial depolarization and ruthenium red pointing to the involvement of a cation uniporter. Na(mit)(+) spiking activity was significantly inhibited by mitochondrial Na(+)/H(+) exchanger inhibition and sensitive to cellular pH and Na(+) concentration. Ca(2+) played a permissive role on Na(mit)(+) spiking activity. Finally, the authors present evidence suggesting that Na(mit)(+) spiking frequency was correlated with cellular ATP levels. This study shows that, under physiological conditions, individual mitochondria in living astrocytes exhibit fast Na(+) exchange across their inner membrane, which reveals a new form of highly dynamic and localized functional regulation.

Role of Ca2+ entry and Ca2+ stores in atypical smooth muscle cell autorhythmicity in the mouse renal pelvis

Electrically active atypical smooth muscle cells (ASMCs) within the renal pelvis have long been considered to act as pacemaker cells driving pelviureteric peristalsis. We have investigated the role of Ca2+ entry and uptake into and release from internal stores in the generation of Ca2+ transients and spontaneous transient depolarizations (STDs) in ASMCs. The electrical activity and separately visualized changes in intracellular Ca2+ concentration in typical smooth muscle cells (TSMCs), ASMCs and interstitial cells of Cajal-like cells (ICC-LCs) were recorded using intracellular microelectrodes and a fluorescent Ca2+ indicator, fluo-4. In 1 microM nifedipine, high frequency (10-30 min(-1)) Ca2+ transients and STDs were recorded in ASMCs, while ICC-LCs displayed low frequency (1-3 min(-1)) Ca2+ transients. All spontaneous electrical activity and Ca2+ transients were blocked upon removal of Ca2+ from the bathing solution, blockade of Ca2+ store uptake with cyclopiazonic acid (CPA) and with 2-aminoethoxy-diphenylborate (2-APB). STD amplitudes were reduced upon removal of the extracellular Na+ or blockade of IP3 dependent Ca2+ store release with neomycin or U73122. Blockade of ryanodine-sensitive Ca2+ release blocked ICC-LC Ca2+ transients but only reduced Ca2+ transient discharge in ASMCs. STDs in ASMCS were also little affected by DIDS, La3+, Gd3+ or by the replacement of extracellular Cl(-) with isethionate. ASMCs generated Ca2+ transients and cation-selective STDs via mechanisms involving Ca2+ release from IP3-dependent Ca2+ stores, STD stimulation of TSMCs was supported by Ca2+ entry through L type Ca2+ channels and Ca2+ release from ryanodine-sensitive stores.

Spontaneous electrical and Ca2+ signals in typical and atypical smooth muscle cells and interstitial cell of Cajal‐like cells of mouse renal pelvis

Electrical rhythmicity in the renal pelvis provides the fundamental drive for the peristaltic contractions that propel urine from the kidney to bladder for storage until micturition. Although atypical smooth muscles (ASMCs) within the most proximal regions of the renal pelvis have long been implicated as the pacemaker cells, the presence of a sparsely distributed population of rhythmically active Kit-positive interstitial cells of Cajal-like cells (ICC-LCs) have confounded our understanding of pelviureteric peristalsis. We have recorded the electrical activity and separately visualized changes in intracellular Ca(2+) concentration in typical smooth muscle cells (TSMCs), ASMCs and ICC-LCs using intracellular microelectrodes and a fluorescent Ca(2+) indicator, fluo-4. Nifedipine (1-10 microm)-sensitive driven action potentials and Ca(2+) waves (frequency 6-15 min(-1)) propagated through the TSMC layer at a velocity of 1-2 mm s(-1). High frequency (10-40 min(-1)) Ca(2+) transients and spontaneous transient depolarizations (STDs) were recorded in ASMCs in the absence or presence of 1 microm nifedipine. ICC-LCs displayed low frequency (1-3 min(-1)) Ca(2+) transients which we speculated arose from cells that displayed action potentials with long plateaus (2-5 s). Neither electrical activity propagated over distances > 50 microm. In 1 microm nifedipine, ASMCs or ICC-LCs separated by < 30 microm displayed some synchronicity in their Ca(2+) transient discharge suggesting that they may well be acting as 'point sources' of excitation to the TSMC layer. We speculate that ASMCs act as the primary pacemaker in the renal pelvis while ICC-LCs play a supportive role, but can take over pacemaking in the absence of the proximal pacemaker drive.