Conversion of Ca2+ oscillation into propagative electrical signals by Ca2+-activated ion channels and connexin as a reconstituted Ca2+ clock model for the pacemaker activity.

Conversion of intracellular Ca2+ signals to electrical activity results in multiple and differing physiological impacts depending on cell types. In some organs such as gastrointestinal and urinary systems, spontaneous Ca2+ oscillation in pacermaker cells can function essentially as a Ca2+ clock mechanism, which has been originally found in pacemaking in sinoatrial node cell of the heart. The conversion of discrete Ca2+ clock events to spontaneous electrical activity is an essential step for the initiation and propagation of pacemaker activity through the multicellular organs resulting in synchronized physiological functions. Here, a model of intracellular signal transduction from a Ca2+ oscillation to initiation of electrical slow waves and their propagation were reconstituted in HEK293 cells. This was accomplished based on ryanodine receptor (RyR) type 3, Ca2+-activated ion channels, i.e. small conductance Ca2+-activated K+ channel (SK2) or Ca2+-activated Cl- channel (TMEM16A), and connexin43 being heterologously co-expressed. The propagation of electrical waves was abolished or substantially reduced by treatment with selective blockers of the expressed channels and 18ß-glycyrrhetinic acid, a gap junction inhibitor, respectively. Thus, we demonstrated that the conversion of Ca2+ oscillation to electrical signals with cell to cell propagation can be reconstituted as a model of Ca2+ clock pacemaker activity by combinational expression of critical elements in heterologous expression system.

Nitric oxide-induced oxidative stress impairs pacemaker function of murine interstitial cells of Cajal during inflammation.

The pacemaker function of interstitial cells of Cajal (ICC) is impaired during intestinal inflammation. The aim of this study is to clarify the pathophysiological mechanisms of ICC dysfunction during inflammatory condition by using intestinal cell clusters. Cell clusters were prepared from smooth muscle layer of murine jejunum and treated with interferon-gamma and lipopolysaccharide (IFN-γ+LPS) for 24h to induce inflammation. Pacemaker function of ICC was monitored by measuring cytosolic Ca(2+) oscillation in the presence of nifedipine. Treatment with IFN-γ+LPS impaired the pacemaker activity of ICC with increasing mRNA level of interleukin-1 beta, tumor necrosis factor-alpha and interleukin-6 in cell clusters; however, treatment with these cytokines individually had little effect on pacemaker activity of ICC. Treatment with IFN-γ+LPS also induced the expression of inducible nitric oxide synthase (iNOS) in smooth muscle cells and resident macrophages, but not in ICC. Pretreatment with NOS inhibitor, L-NAME or iNOS inhibitor, 1400W ameliorated IFN-γ+LPS-induced pacemaker dysfunction of ICC. Pretreatment with guanylate cyclase inhibitor, ODQ did not, but antioxidant, apocynin, to suppress NO-induced oxidative stress, significantly suppressed the impairment of ICC function induced by IFN-γ+LPS. Treatment with IFN-γ+LPS also decreased c-Kit-positive ICC, which was prevented by pretreatment with L-NAME. However, apoptotic ICC were not detected in IFN-γ+LPS-treated clusters, suggesting IFN-γ+LPS stimulation just changed the phenotype of ICC but not induced cell death. Moreover, ultrastructure of ICC was not disturbed by IFN-γ+LPS. In conclusion, ICC dysfunction during inflammation is induced by NO-induced oxidative stress rather than NO/cGMP signaling. NO-induced oxidative stress might be the main factor to induce phenotypic changes of ICC.

Assessment of gastric emptying and duodenal motility upon ingestion of a liquid meal using rapid magnetic resonance imaging.

Gastric emptying is achieved by co-operation between gastric and duodenal motor activity. Therefore, evaluation of gastric emptying and its associated mechanisms would benefit clinical therapy as well as medical research. Healthy volunteers underwent rapid magnetic resonance imaging (MRI) of the abdomen along the coronal plane after ingestion of a liquid meal. The gastric fundal and duodenal areas were quantified semi-automatically by self-developed segment software. The average gastric fundal area determined by the serosal end in 40 sequential images was reduced to ∼81% 30 min after and to ∼70% 60 min after ingestion of a liquid meal. The average duodenal area also decreased to ∼86% after 30 min and to 83% after 60 min. In contrast, changes in the centre of gravity increased to about fivefold after 30 min and to about threefold after 60 min. The mean velocity of the duodenal wall mimicked changes in the centre of gravity. The application of metoclopramide, a dopamine D(2) receptor antagonist, accelerated gastric emptying, presumably due to facilitated duodenal activity even immediately after liquid meal ingestion. The ingestion of water caused fast gastric emptying in 30 min, accompanied by high duodenal motility, but it ceased after 60 min, presumably reflecting complete gastric emptying. A rapid MRI scan visualized the association between gastric emptying and duodenal motility that could be modified by calories and dopaminergic neurotransmission. Changes in the centre of gravity and mean velocity of the duodenal wall appear to quantify the motility obtained from cine MRI accurately.

Dental pulp stem cells as a therapy for congenital entero-neuropathy.

Hirschsprung's disease is a congenital entero-neuropathy that causes chronic constipation and intestinal obstruction. New treatments for entero-neuropathy are needed because current surgical strategies have limitations5. Entero-neuropathy results from enteric nervous system dysfunction due to incomplete colonization of the distal intestine by neural crest-derived cells. Impaired cooperation between the enteric nervous system and intestinal pacemaker cells may also contribute to entero-neuropathy. Stem cell therapy to repair these multiple defects represents a novel treatment approach. Dental pulp stem cells derived from deciduous teeth (dDPSCs) are multipotent cranial neural crest-derived cells, but it remains unknown whether dDPSCs have potential as a new therapy for entero-neuropathy. Here we show that intravenous transplantation of dDPSCs into the Japanese Fancy-1 mouse, an established model of hypoganglionosis and entero-neuropathy, improves large intestinal structure and function and prolongs survival. Intravenously injected dDPSCs migrate to affected regions of the intestine through interactions between stromal cell-derived factor-1α and C-X-C chemokine receptor type-4. Transplanted dDPSCs differentiate into both pacemaker cells and enteric neurons in the proximal colon to improve electrical and peristaltic activity, in addition to their paracrine effects. Our findings indicate that transplanted dDPSCs can differentiate into different cell types to correct entero-neuropathy-associated defects.

Imipramine inhibition of TRPM-like plasmalemmal Mg2+ transport in vascular smooth muscle cells.

Depression is associated with vascular disease, such as myocardial infarction and stroke. Pharmacological treatments may contribute to this association. On the other hand, Mg(2+) deficiency is also known to be a risk factor for the same category of diseases. In the present study, we examined the effect of imipramine on Mg(2+) homeostasis in vascular smooth muscle, especially via melastatin-type transient receptor potential (TRPM)-like Mg(2+) -permeable channels. The intracellular free Mg(2+) concentration ([Mg(2+) ](i) ) was measured using (31) P-nuclear magnetic resonance (NMR) in porcine carotid arteries that express both TRPM6 and TRPM7, the latter being predominant. pH(i) and intracellular phosphorus compounds were simultaneously monitored. To rule out Na(+) -dependent Mg(2+) transport, and to facilitate the activity of Mg(2+) -permeable channels, experiments were carried out in the absence of Na(+) and Ca(2+) . Changing the extracellular Mg(2+) concentration to 0 and 6 mM significantly decreased and increased [Mg(2+) ](i) , respectively, in a time-dependent manner. Imipramine statistically significantly attenuated both of the bi-directional [Mg(2+) ](i) changes under the Na(+) - and Ca(2+) -free conditions. This inhibitory effect was comparable in influx, and much more potent in efflux to that of 2-aminoethoxydiphenyl borate, a well-known blocker of TRPM7, a channel that plays a major role in cellular Mg(2+) homeostasis. Neither [ATP](i) nor pH(i) correlated with changes in [Mg(2+) ](i) . The results indicate that imipramine suppresses Mg(2+) -permeable channels presumably through a direct effect on the channel domain. This inhibitory effect appears to contribute, at least partially, to the link between antidepressants and the risk of vascular diseases.

Diphosphate regulation of adenosine triphosphate sensitive potassium channel in human bladder smooth muscle cells.

PURPOSE: To clarify the properties of adenosine triphosphate sensitive K+ channel in human detrusor smooth muscle we examined the effect of the representative nicotinic acid derivatives ß-nicotinamide adenine dinucleotide, cyclic adenosine diphosphate ribose and nicotinic acid adenine dinucleotide phosphate (Sigma-Aldrich®) on human detrusor adenosine triphosphate sensitive K+ channels. MATERIALS AND METHODS: Patch clamp procedures were done in human detrusor cells. Reverse transcriptase and real-time polymerase chain reaction were performed to clarify the subunit components of adenosine triphosphate sensitive K+ channels. RESULTS: The K+ channel opener levcromakalim induced a long lasting outward current that was inhibited by glibenclamide (Sigma-Aldrich) under the whole cell configuration. The single channel study revealed that the unitary conductance of the adenosine triphosphate sensitive K+ channel in the human detrusor was 11 pS and nucleotide diphosphates increased its open probability. Applying ß-nicotinamide adenine dinucleotide also activated the adenosine triphosphate sensitive K+ channel but applying cyclic adenosine diphosphate ribose or nicotinic acid adenine dinucleotide phosphate had little effect on channel activation. Molecular studies indicated that Kir6.1 and SUR2B were the predominant components of the adenosine triphosphate sensitive K+ channel in the human detrusor. CONCLUSIONS: To our knowledge we report the first single channel study of the adenosine triphosphate sensitive K+ channel in the human detrusor. The properties of this channel, ie unitary conductance, adenosine triphosphate sensitivity and diphosphate activation, were consistent with those of other smooth muscle organs. ß-Nicotinamide adenine dinucleotide has the potency to activate adenosine triphosphate sensitive K+ channels in the human detrusor. This channel likely has some role during ischemic conditions as well as physiological muscle motion leading to the activation of cell metabolism.

Oscillatory membrane currents paradoxically induced via NO-activated pathways in detrusor cells.

Oscillatory inward membrane currents (I(oscil-in)) reflecting intracellular Ca(2+) ([Ca(2+)](i)) activity in detrusor cells, are thought to play an important role in producing tonic bladder contractions during micturition. The present patch clamp study revealed a new activation mechanism: sodium nitroprusside (SNP), a nitric oxide (NO) donor induced I(oscil-in) in a subpopulation of detrusor cells. The inhibitory effect of niflumic acid on SNP-induced I(oscil-in) suggests that Ca(2+)-activated Cl(-) channels are responsible for this current. In addition, SNP-induced I(oscil-in) required the cooperation of Ca(2+) influx through SK&F96365-sensitive channels and intracellular Ca(2+) release channels sensitive to ryanodine but insensitive to xestospongin C (XeC). This is also true for muscarinic agonist (carbachol: CCh)-induced I(oscil-in). However, 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a guanylyl cyclase inhibitor, suppressed SNP-induced I(oscil-in) but not CCh-induced I(oscil-in). The results suggest that a subpopulation of detrusor cells employ the NO/cGMP cascade to potentiate bladder contraction. Mechanisms underlying NO-induced I(oscil-in) are likely to contribute not only to the physiology but also to the pathophysiology of the lower urinary tract.

Levcromakalim and MgGDP activate small conductance ATP-sensitive K+ channels of K+ channel pore 6.1/sulfonylurea receptor 2A in pig detrusor smooth muscle cells: uncoupling of cAMP signal pathways.

Pharmacological studies have suggested the existence of ATP-sensitive K(+) (K(ATP)) channel as a therapeutic target in urinary bladders; however, electrical properties have not yet been shown. Patch-clamp techniques were applied to investigate the properties of K(ATP) channels in pig detrusor cells. In whole-cell configuration, levcromakalim, a K(ATP) channel opener, induced a long-lasting outward current in a concentration-dependent manner. The current-voltage curve of the levcromakalim-induced membrane current intersected at approximately -80 mV. This current was abolished by glibenclamide. Intracellular application of 0.1 mM GDP significantly enhanced the levcromakalim-induced membrane current, whereas cAMP did not. Furthermore, neurotransmitters related to cAMP signaling, such as calcitonin gene-related peptide, vasointestinal peptide, adenosine, and somatostatin, had little effect on the membrane current. In cell-attached configuration, levcromakalim activated K(+) channels with a unitary conductance of approximately 12 pS. When the patch configuration was changed to inside-out mode, the K(+) channel activity ran down. Subsequent application of 1 mM GDP reactivated the channels. The openings of the approximately 12 pS K(+) channels in the presence of 1 mM GDP was suppressed by ATP and glibenclamide. In reverse transcription-polymerase chain reaction, K(+) channel pore 6.1 and sulfonylurea receptor (SUR)2A were predominant in pig detrusor cells. The 12 pS K(+) channel activated by levcromakalim in pig detrusor smooth muscle cells is a K(ATP) channel. The predominant expression of SUR2A can account for the lack of effect of neurotransmitters related to cAMP.

Tyrosine kinase inhibitors and ATP modulate the conversion of smooth muscle L-type Ca2+ channels toward a second open state.

Properties of smooth and cardiac L-type Ca2+ channels differ prominently in several physiological aspects, including sympathetic modulation. To assess the possible underlying mechanisms, we applied the whole cell patch-clamp technique to guinea pig detrusor smooth muscle cells, in which only L-type Ca2+ channel currents are observed in practice. During depolarization to large positive potentials, the conformation of the majority of L-type Ca2+ channels is converted from the normal (O1) to a second open state (O2), which undergoes little inactivation during depolarization. Extracellular application of genistein, a known tyrosine kinase inhibitor, significantly attenuated the voltage-dependent conversion of Ca2+ channels to O2, accompanied by reduction of availability, whereas genistin, an inactive analog, had little effect. In the absence of ATP in the patch pipette, intracellular application of either genistein or tyrphostin-47 suppressed the conversion to O2. Computer calculation revealed that the acceleration of the O1 to an inactivated state qualitatively reconstructs the unique effects of PTK inhibitors antagonized by ATP. We concluded that under normal conditions smooth muscle L-type Ca2+ channels are already modulated by tyrosine-kinase and ATP-related mechanism(s) and thereby easily achieve the second conversion, which yields voltage-dependent modulation of L-type Ca2+ current analogous to that in cardiac myocytes during beta-adrenoceptor stimulation.

Dialysis membrane-enforced microelectrode array measurement of diverse gut electrical activity.

A variety of electrical activities occur depending on the functional state in each section of the gut, but the application of microelectrode array (MEA) is rather limited. We thus developed a dialysis membranes-enforced technique to investigate diverse and complex spatio-temporal electrical activity in the gut. Muscle sheets isolated from the gastrointestinal (GI) tract of mice along with a piece of dialysis membrane were woven over and under the strings to fix them to the anchor rig, and mounted on an 8×8 MEA (inter-electrode distance=150µm). Small molecules (molecular weight <12,000) were exchanged through the membrane, maintaining a physiological environment. Low impedance MEA was used to measure electrical signals in a wide frequency range. We demonstrated the following examples: 1) pacemaker activity-like potentials accompanied by bursting spike-like potentials in the ileum; 2) electrotonic potentials reflecting local neurotransmission in the ileum; 3) myoelectric complex-like potentials consisting of slow and rapid oscillations accompanied by spike potentials in the colon. Despite their limited spatial resolution, these recordings detected transient electric activities that optical probes followed with difficulty. In Addition, propagation of pacemaker-like potential was visualized in the stomach and ileum. These results indicate that the dialysis membrane-enforced technique largely extends the application of MEA, probably due to stabilisation of the access resistance between each sensing electrode and a reference electrode and improvement of electric separation between sensing electrodes. We anticipate that this technique will be utilized to characterise spatio-temporal electrical activities in the gut in health and disease.

Inherent pacemaker function of duodenal GIST.

Gastrointestinal stromal tumours (GIST) are thought to derive from interstitial cells of Cajal (ICCs), which are putative pacemaker cells for gut motility. Isolated cells were obtained by enzymatic treatment of human duodenum GIST tissue having a frequent gain-of-function gene mutation. After cell culturing, c-Kit immunoreactivity was preserved and the cells developed long processes. Whole cell patch clamp recordings revealed voltage-dependent outward currents, without transient inward currents. Intracellular Ca(2+) measurements showed oscillation-like spontaneous activity in some GIST cells. RT-PCR revealed expression of ion channels (Kv1.1, Kv1.6 and KCNH2; IP3R1, and IP3R2; TRPC1, 3, 6 and 7; Cx43), which have been suggested to play important roles in pacemaker activity. However, SCN5A, a TTX-resistant Na(+) channel known to be expressed in human ICCs, was below detectable levels. These data suggest that GIST cells appear to preserve some, but not all ionic mechanisms underlying pacemaker activity in ICC.

Co-contribution of IP3R and Ca2+ influx pathways to pacemaker Ca2+ activity in stomach ICC.

Intracellular Ca2+ oscillations in interstitial cells of Cajal (ICCs) are thought to be the primary pacemaker activity in the gut. In the present study, the authors prepared small tissues of 100-to 300-microm diameter (cell cluster preparation) from the stomach smooth muscle (including the myenteric plexus) of mice by enzymatic and mechanical treatments. After 2 to 4 days of culture, the intracellular Ca2+ concentration ([Ca2+]i) was measured. In the presence of nifedipine, a dihydropyridine Ca2+ channel antagonist, spontaneous [Ca2+]i oscillations were observed within limited regions showing positive c-Kitimmunoreactivity, a maker for ICCs. In the majority of cell cluster preparations with multiple regions of [Ca2+]i oscillations, [Ca2+]i oscillated synchronously in the same phase. A small number of cell clusters (8 of 53) showed multiple regions of [Ca2+]i oscillations synchronized but with a considerable phase shift. Neither tetrodotoxin (250 nM) nor atropine (10 microM) significantly affected [Ca2+]i oscillations in the presence of nifedipine. Low concentrations (40 microM) of Ni2+ had little effect on the spontaneous [Ca2+]i oscillation, but SK&F96365 (40 microM) and Cd2+ (120 microM) terminated it. Applications of either 2-aminoethoxydiphenyl borate (10 microM) or xestosponginC(10 microM) completely and rather rapidly (approximately 2 min) abolished the spontaneous [Ca2+]i oscillations. The results suggest that pacemaker [Ca2+]i oscillations in ICCs are produced by close interaction of intracellular Ca2+ release channels, especially inositol 1,4,5-trisphosphate receptor (InsP3R) and Ca2+ influx pathways, presumably corresponding to store-operated type channels. Reverse transcription polymerase chain reaction examinations revealed expression of TRPC2, 4, and 6, as well as InsP3R1 and 2 in ICCs.

Numerical Evaluation of Efficacy of Glutamate on Gastrointestinal Motility: Rapid MRI Study.

The umami taste amino acid, glutamate acts as a signaling molecule in multiple cellular systems in the body, including the brain and gastrointestinal tract. Therefore, glutamate may affect appetite by modulating gastrointestinal motility as well as through taste perception. In this study, we examined the effect of glutamate on gastric emptying and duodenal motility, by using rapid magnetic resonance imaging (MRI). Ten healthy male volunteers participated in the measurements. Abdominal coronal MR images were successively acquired after ingestion of a liquid meal with and without monosodium L-glutamate (MSG). Image analysis was performed with a homemade segment software, in which respiratory motions were cancelled automatically by minimizing an energy function, thereby allowing participants breathe freely during MRI measurements. In two out of 10 participants, gastric emptying slowed down, while in the remaining eight participants, gastric residual volume decreased to 84% without MSG, and to 73% with MSG after 60 min. The inclusion of MSG enhanced duodenal motility, judging from changes in, 1) the magnitude of the duodenal area, 2) the center of gravity, and 3) the mean velocity of the wall motions. The third parameter most significantly indicated the excitatory effect of MSG on duodenum motility (3-7 fold increase). In conclusion, the present observations of rapid MRI indicate that MSG accelerates gastric emptying by facilitating duodenal motility, at least in healthy subjects with positive responses to MSG. This suggests the possible use of MSG as a prokinetic nutrient for improving the quality of life in hospitalized patients after a clinical assessment.

Purinergic modulation of pacemaker Ca2+ activity in interstitial cells of Cajal.

Purinoceptors are widely distributed throughout the body, and are thought to have important contributions to numerous functions. In this study, we characterised the contribution of purinoceptors to the mechanisms underlying spontaneous rhythmicity of the gastro-intestinal tracts. Using cell cluster preparations (100-200 microm diameter) obtained from murine ileum, we measured spontaneous intracellular Ca2+([Ca2+]i) oscillations in the presence of nifedipine, as an index of pacemaker [Ca2+]i activity in interstitial cells of Cajal (ICCs, c-Kit-immunopositive cells), the pacemaker cells for gastrointestinal motility. This small preparation also contained smooth muscle and enteric neurones. Using various purinoceptor agonists and an antagonist, we characterised both TTX-sensitive and insensitive modulations of pacemaker [Ca2+]i activity in ICCs. Continuous application of either ATP, ATPgammaS, suramin or alpha,beta-methylene ATP (alpha,beta-meATP) suppressed pacemaker [Ca2+]i activity. The inhibitory effect of alpha,beta-meATP was completely abolished by a prior application of TTX. On the other hand, even in the presence of TTX, continuous application of 2-methylthio ATP (2-MeSATP) at concentrations greater than 30 microM caused a prompt rise followed by a slow decline of the baseline [Ca2+]i, and pacemaker [Ca2+]i oscillations were gradually suppressed during the decline. Neither UTP nor alpha,beta-meATP at high concentrations (30-100 microM) produced a similar [Ca2+]i response. These results suggest that the TTX-resistant, direct purinergic modulation of pacemaker [Ca2+]i activity in ICCs is mediated via P2X purinoceptors distinct from those involved in TTX-sensitive modulation. The slow decline may be attributed to desensitisation of these purinoceptors. The possible involvement of other purinoceptors is also discussed.

Ion transport regulated by protease-activated receptor 2 in human airway Calu-3 epithelia.

We examined the mechanisms underlying anion secretion mediated by protease-activated receptor 2 (PAR2) and its role in the regulation of ion transport, using polarized human airway Calu-3 cells. PAR2 stimulation by trypsin and a PAR2-activating peptide (PAR2AP), especially from the basolateral aspect, caused transient Cl(-) secretion due to cytosolic Ca(2+) mobilization. Antagonists of PI-PLC (U73122, ET-18-OCH(3)) and inositol 1,4,5-triphosphate (xestospongin C (Xest C)) were without effect on the PAR2AP-mediated Cl(-) secretion, whereas it was attenuated by D609 (a PC-PLC inhibitor) and phorbol 12-myristate 13 acetate (PMA, a PKC activator). Even 30 min after removal of PAR2AP after a 10-min-exposure, cells were still poorly responsive to PAR2 stimulation, but the reduced responsiveness was upregulated by a PKC inhibitor, GF109203X (GFX). Pretreatment with PAR2AP did not affect responses to anion secretagogues, such as isoproterenol, forskolin, thapsigargin, 1-ethyl-2-benzimdazolinone, and adenosine, but ATP-induced responses were significantly reduced. Nystatin permeabilization studies revealed that the presence of PAR2AP prevented ATP-induced increments in basolateral membrane K(+) conductance without affecting apical membrane Cl(-) conductance. ATP-elicited Ca(2+) mobilization, which was sensitive to D609 and PMA, was inhibited by the pretreatment with PAR2AP, and this inhibition was blunted by the presence of GFX. Collectively, stimulation of PAR2 generates a brief response of Cl(-) secretion through PC-PLC-mediated pathway, followed by not only auto-desensitization of PAR2 itself but also cross-desensitization of a PC-PLC-coupled purinoceptor. The two types of desensitization seem likely to have PKC-mediated downregulation of PC-PLC in common.

P2Y-mediated Ca2+ response is spatiotemporally graded and synchronized in sensory neurons: a two-photon photolysis study.

ATP is thought to be an initiator and modulator of noxious pain sensation. We employed two-photon photolysis to apply ATP locally and transiently, thus mimicking ATP release upon cell damage or exocytosis. Using this technique, an increase in intracellular Ca2+ concentration ([Ca2+]i) was induced via P2Y receptors in individual sensory neurons, or in a neurite region. The ATP-induced [Ca2+]i rise was attenuated by applications of either a phospholipase C inhibitor, or inhibitors for IP3 or ryanodine receptors. These results indicate that intracellular Ca2+ stores play a major role in contributing to the increase in [Ca2+]i. Spatiotemporal analysis revealed that local and transient applications of ATP increased [Ca2+]i by release from intracellular stores, but in a unique, graded, and synchronized manner. 1) As the duration of local ATP application was prolonged, the latency decreased and the magnitude of the [Ca2+]i rise increased; 2) The time course of the rising phase of the [Ca2+]i response to ATP was essentially the same over the cell body, once [Ca2+]i had started to rise. It is anticipated that sensory responses may be modulated variably, depending on the spatiotemporal characteristics of the ATP-related [Ca2+]i profile.

Real-time measurement of biomagnetic vector fields in functional syncytium using amorphous metal.

Magnetic field detection of biological electric activities would provide a non-invasive and aseptic estimate of the functional state of cellular organization, namely a syncytium constructed with cell-to-cell electric coupling. In this study, we investigated the properties of biomagnetic waves which occur spontaneously in gut musculature as a typical functional syncytium, by applying an amorphous metal-based gradio-magneto sensor operated at ambient temperature without a magnetic shield. The performance of differentiation was improved by using a single amorphous wire with a pair of transducer coils. Biomagnetic waves of up to several nT were recorded ~1 mm below the sample in a real-time manner. Tetraethyl ammonium (TEA) facilitated magnetic waves reflected electric activity in smooth muscle. The direction of magnetic waves altered depending on the relative angle of the muscle layer and magneto sensor, indicating the existence of propagating intercellular currents. The magnitude of magnetic waves rapidly decreased to ~30% by the initial and subsequent 1 mm separations between sample and sensor. The large distance effect was attributed to the feature of bioelectric circuits constructed by two reverse currents separated by a small distance. This study provides a method for detecting characteristic features of biomagnetic fields arising from a syncytial current.

Ca(2+) channel properties in smooth muscle cells of the urinary bladder from pig and human.

Ca(2+) channel properties of pig and human bladder smooth muscle were investigated utilizing standard whole-cell patch clamp techniques. Both the amplitude obtained and the current density of Ca(2+) channel current evoked by step depolarization were larger in human than in pig myocytes. The inward currents were sensitive to an L-type Ca(2+) channel antagonist, nifedipine, the effects of which were not significantly different between species. In both species, prior application of ATP (0.1 mM) had no effect on activation of this voltage-sensitive channel current, while a muscarinic receptor agonist, carbachol (0.1 mM), significantly attenuated the amplitude of this current. Furthermore, inclusion of GDP-beta-S or Heparin in the pipette abolished or had no effect on the suppression of Ca(2+) current by carbachol, respectively. These results forward the pig as a good model for the human in detrusor Ca(2+) channel properties, especially with regard to neural modulation, although voltage-sensitive Ca(2+) channels seem to make greater contribution in human bladder physiology.