Muscarinic stimulation increases basal Ca2+ and inhibits spontaneous Ca2+ transients in murine colonic myocytes

2001 ◽  
Vol 280 (3) ◽  
pp. C689-C700 ◽  
Author(s):  
Orline Bayguinov ◽  
Brian Hagen ◽  
Kenton M. Sanders
Keyword(s):  
Nonselective Cation Channel ◽  
Inhibitory Effects ◽  
Patch Clamp Recording ◽  
Fk 506 ◽  
Whole Cell Patch Clamp ◽  
Outward Currents ◽  
Receptor Interactions ◽  
Muscarinic Stimulation ◽  
Spontaneous Transient Outward Currents ◽  
Stimulation Of

Localized Ca2+ transients in isolated murine colonic myocytes depend on Ca2+ release from inositol 1,4,5-trisphosphate (IP3) receptors. Localized Ca2+ transients couple to spontaneous transient outward currents (STOCs) and mediate hyperpolarization responses in these cells. We used confocal microscopy and whole cell patch-clamp recording to investigate how muscarinic stimulation, which causes formation of IP3, can suppress Ca2+ transients and STOCs that might override the excitatory nature of cholinergic responses. ACh (10 μM) reduced localized Ca2+ transients and STOCs, and these effects were associated with a rise in basal cytosolic Ca2+. These effects of ACh were mimicked by generalized rises in basal Ca2+ caused by ionomycin (250–500 nM) or elevated external Ca2+ (6 mM). Atropine (10 μM) abolished the effects of ACh. Pretreatment of cells with nicardipine (1 μM), or Cd2+ (200 μM) had no effect on responses to ACh. An inhibitor of phospholipase C, U-73122, blocked Ca2+ transients and STOCs but did not affect the increase in basal Ca2+ after ACh stimulation. Xestospongin C (Xe-C; 5 μM), a membrane-permeable antagonist of IP3 receptors, blocked spontaneous Ca2+ transients but did not prevent the increase of basal Ca2+ in response to ACh. Gd3+(10 μM), a nonselective cation channel inhibitor, prevented the increase in basal Ca2+ after ACh and increased the frequency and amplitude of Ca2+ transients and waves. Another inhibitor of receptor-mediated Ca2+ influx channels, SKF-96365, also prevented the rise in basal Ca2+after ACh and increased Ca2+ transients and development of Ca2+ waves. FK-506, an inhibitor of FKBP12/IP3 receptor interactions, had no effect on the rise in basal Ca2+ but blocked the inhibitory effects of increased basal Ca2+ and ACh on Ca2+transients. These results suggest that the rise in basal Ca2+ that accompanies muscarinic stimulation of colonic muscles inhibits localized Ca2+ transients that could couple to activation of Ca2+-activated K+channels and reduce the excitatory effects of ACh.

Serotonergic modulation of neuronal activity in rat midbrain periaqueductal gray

2013 ◽  
Vol 109 (11) ◽  
pp. 2712-2719 ◽  
Author(s):  
Hyo-Jin Jeong ◽  
Karen Lam ◽  
Vanessa A. Mitchell ◽  
Christopher W. Vaughan
Keyword(s):  
Brain Slices ◽  
Periaqueductal Gray ◽  
Membrane Excitability ◽  
Inhibitory Effects ◽  
Whole Cell Patch Clamp ◽  
Outward Currents ◽  
Selective Ligands ◽  
Inward Currents ◽  
Subtype Selective ◽  
Serotonergic Modulation

Serotonin (5-HT) modulates pain and anxiety from within the midbrain periaqueductal gray (PAG). In the present study, the effects of 5-HT- and 5-HT1/2 subtype-selective ligands on rat PAG neurons were examined using whole cell patch-clamp recordings in brain slices. In voltage clamp, 5-HT produced outward and inward currents in distinct subpopulations of neurons that varied throughout different subregions of the PAG. The 5-HT1A agonist R(+)-8-OH-DPAT (1 μM) produced outward currents in subpopulations of PAG neurons. By contrast, sumatriptan (1 μM) and other 5-HT1B, -D, and -F subtype agonists had little or no postsynaptic activity. The 5-HT2A/C agonists DOI (3 μM) and TCB-2 (1 μM) produced inward currents in subpopulations of PAG neurons, and DOI enhanced evoked inhibitory postsynaptic currents via a presynaptic mechanism. In current clamp, both R(+)-8-OH-DPAT and sumatriptan produced an excitatory increase in evoked mixed postsynaptic potentials (PSPs). In addition, R(+)-8-OH-DPAT, but not sumatriptan, directly hyperpolarized PAG neurons. By contrast, the 5-HT2 agonist DOI depolarized subpopulations of neurons and produced an inhibitory decrease in evoked mixed PSPs. These findings indicate that 5-HT1A and 5-HT1B/D ligands have partly overlapping inhibitory effects on membrane excitability and synaptic transmission within the PAG, which are functionally opposed by 5-HT2A/C actions in specific PAG subregions.

scholarly journals Trigeminal Neuralgia TRPM8 Mutation

2021 ◽  
Vol 7 (1) ◽  
pp. e550
Author(s):  
Roberta Gualdani ◽  
Jun-Hui Yuan ◽  
Philip R. Effraim ◽  
Giulia Di Stefano ◽  
Andrea Truini ◽  
...  
Keyword(s):  
Trigeminal Neuralgia ◽  
Transient Receptor Potential ◽  
Receptor Potential ◽  
Nonselective Cation Channel ◽  
Patch Clamp Recording ◽  
Mutant Channel ◽  
Whole Cell Patch Clamp ◽  
Transient Receptor Potential Melastatin ◽  
Intracellular Ca ◽  
Transient Receptor

ObjectiveTo assess the functional effects of a variant, c.89 G > A (p.Arg30Gln), in the transient receptor potential melastatin 8 (TRPM8) cold-sensing, nonselective cation channel, which we have previously identified in a patient with familial trigeminal neuralgia.MethodsWe carried out Ca2+ imaging and whole-cell patch-clamp recording.ResultsThe TRPM8 mutation enhances channel activation, increases basal current amplitude and intracellular [Ca2+] in cells carrying the mutant channel, and enhances the response to menthol.ConclusionsWe propose that Arg30Gln confers gain-of-function attributes on TRPM8, which contribute to pathogenesis of trigeminal neuralgia in patients carrying this mutation.

Activation of subfornical organ neurons in rats through pre- and postsynaptic α-adrenoceptors

2006 ◽  
Vol 290 (6) ◽  
pp. R1646-R1653 ◽  
Author(s):  
Eiko Honda ◽  
Kentaro Ono ◽  
Shinji Kataoka ◽  
Kiyotoshi Inenaga
Keyword(s):  
Gaba Receptor ◽  
Membrane Conductance ◽  
Subfornical Organ ◽  
Patch Clamp Recording ◽  
Pcr Assay ◽  
Whole Cell Patch Clamp ◽  
Outward Currents ◽  
Current Clamp Mode ◽  
Inward Currents ◽  
Pulse Stimulation

The effects of noradrenaline (NA) and its analogs on subfornical organ (SFO) neurons in rat slice preparations were investigated by using whole cell patch-clamp recording. In the current-clamp mode, the application of NA at 10–100 μM produced membrane depolarization (63%, 17 responsive neurons/27 neurons tested) and hyperpolarization (22%, 6/27 neurons). In the voltage-clamp mode, NA application at 1–100 μM produced inward currents (69%, 42/61 neurons) and outward currents (23%, 14/61 neurons). These currents remained in the presence of TTX or both glutamate and GABA receptor antagonists. In most of the neurons (25/31 neurons) showing inward currents in the presence of NA, the membrane conductance was not changed by voltage ramps or hyperpolarizing pulse stimulation. Similar responses were obtained by the application of the α1-agonist phenylephrine. The phenylephrine-induced inward currents were inhibited by the α1-antagonist prazosin. The α2-agonist clonidine decreased the frequency of spontaneous GABAergic inhibitory postsynaptic currents (4/10 neurons). In addition, RT-PCR assay and immunohistochemical staining showed the existence of α1-adrenoceptors in the SFO. The results suggest that SFO neurons in rats are activated postsynaptically through α1-adrenoceptors and that the activation is enhanced by suppressing GABAergic inhibitory synaptic inputs through presynaptic α2-adrenoceptors.

scholarly journals Large-Conductance Calcium-Activated Potassium Channels and Voltage-Dependent Sodium Channels in Human Cementoblasts

2021 ◽  
Vol 12 ◽  
Author(s):  
Satomi Kamata ◽  
Maki Kimura ◽  
Sadao Ohyama ◽  
Shuichiro Yamashita ◽  
Yoshiyuki Shibukawa
Keyword(s):  
Alveolar Bone ◽  
Attachment Site ◽  
Ionic Currents ◽  
Ionic Channels ◽  
Patch Clamp Recording ◽  
Physiological Processes ◽  
Whole Cell Patch Clamp ◽  
Outward Currents ◽  
Voltage Dependent ◽  
Inward Currents

Cementum, which is excreted by cementoblasts, provides an attachment site for collagen fibers that connect to the alveolar bone and fix the teeth into the alveolar sockets. Transmembrane ionic signaling, associated with ionic transporters, regulate various physiological processes in a wide variety of cells. However, the properties of the signals generated by plasma membrane ionic channels in cementoblasts have not yet been described in detail. We investigated the biophysical and pharmacological properties of ion channels expressed in human cementoblast (HCEM) cell lines by measuring ionic currents using conventional whole-cell patch-clamp recording. The application of depolarizing voltage steps in 10 mV increments from a holding potential (Vh) of −70 mV evoked outwardly rectifying currents at positive potentials. When intracellular K+ was substituted with an equimolar concentration of Cs+, the outward currents almost disappeared. Using tail current analysis, the contributions of both K+ and background Na+ permeabilities were estimated for the outward currents. Extracellular application of tetraethylammonium chloride (TEA) and iberiotoxin (IbTX) reduced the densities of the outward currents significantly and reversibly, whereas apamin and TRAM-34 had no effect. When the Vh was changed to −100 mV, we observed voltage-dependent inward currents in 30% of the recorded cells. These results suggest that HCEM express TEA- and IbTX-sensitive large-conductance Ca2+-activated K+ channels and voltage-dependent Na+ channels.

scholarly journals Local circuit input to the medullary reticular formation from the rostral nucleus of the solitary tract

2008 ◽  
Vol 295 (5) ◽  
pp. R1391-R1408 ◽  
Author(s):  
J. Nasse ◽  
D. Terman ◽  
S. Venugopal ◽  
G. Hermann ◽  
R. Rogers ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Reticular Formation ◽  
Calcium Imaging ◽  
Neonatal Rat ◽  
Solitary Tract ◽  
Patch Clamp Recording ◽  
Whole Cell ◽  
Cell Patch Clamp ◽  
Whole Cell Patch Clamp ◽  
Stimulation Of

The intermediate reticular formation (IRt) subjacent to the rostral (gustatory) nucleus of the solitary tract (rNST) receives projections from the rNST and appears essential to the expression of taste-elicited ingestion and rejection responses. We used whole cell patch-clamp recording and calcium imaging to characterize responses from an identified population of prehypoglossal neurons in the IRt to electrical stimulation of the rNST in a neonatal rat pup slice preparation. The calcium imaging studies indicated that IRt neurons could be activated by rNST stimulation and that many neurons were under tonic inhibition. Whole cell patch-clamp recording revealed mono- and polysynaptic projections from the rNST to identified prehypoglossal neurons. The projection was primarily excitatory and glutamatergic; however, there were some inhibitory GABAergic projections, and many neurons received excitatory and inhibitory inputs. There was also evidence of disinhibition. Overall, bath application of GABAA antagonists increased the amplitude of excitatory currents, and, in several neurons, stimulation of the rNST systematically decreased inhibitory currents. We have hypothesized that the transition from licks to gapes by natural stimuli, such as quinine monohydrochloride, could occur via such disinhibition. We present an updated dynamic model that summarizes the complex synaptic interface between the rNST and the IRt and demonstrates how inhibition could contribute to the transition from ingestion to rejection.

Coupling strength between localized Ca2+transients and K+ channels is regulated by protein kinase C

2001 ◽  
Vol 281 (5) ◽  
pp. C1512-C1523 ◽  
Author(s):  
Orline Bayguinov ◽  
Brian Hagen ◽  
James L. Kenyon ◽  
Kenton M. Sanders
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Coupling Strength ◽  
Single Channel ◽  
Bk Channels ◽  
Kinase C ◽  
Whole Cell Patch Clamp ◽  
Outward Currents ◽  
Gf 109203X ◽  
Spontaneous Transient Outward Currents

Localized Ca2+ transients resulting from inositol trisphosphate (IP3)-dependent Ca2+ release couple to spontaneous transient outward currents (STOCs) in murine colonic myocytes. Confocal microscopy and whole cell patch-clamp techniques were used to investigate coupling between localized Ca2+ transients and STOCs. Colonic myocytes were loaded with fluo 3. Reduction in external Ca2+([Ca2+]o) reduced localized Ca2+transients but increased STOC amplitude and frequency. Simultaneous recordings of Ca2+ transients and STOCs showed increased coupling strength between Ca2+ transients and STOCs when [Ca2+]o was reduced. Gd3+ (10 μM) did not affect Ca2+ transients but increased STOC amplitude and frequency. Similarly, an inhibitor of Ca2+influx, 1–2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl)propoxy]ethyl-1H-imidazole (SKF-96365), increased STOC amplitude and frequency. A protein kinase C (PKC) inhibitor, GF-109203X, also increased the amplitude and frequency of STOCs but had no effect on Ca2+ transients. Phorbol 12-myristate 13-acetate (1 μM) reduced STOC amplitude and frequency but did not affect Ca2+ transients. 4α-Phorbol (1 μM) had no effect on STOCs or Ca2+ transients. Single channel studies indicated that large-conductance Ca2+-activated K+ (BK) channels were inhibited by a Ca2+-dependent PKC. In summary 1) Ca2+ release from IP3 receptor-operated stores activates Ca2+-activated K+ channels; 2) Ca2+ influx through nonselective cation channels facilitates activation of PKC; and 3) PKC reduces the Ca2+ sensitivity of BK channels, reducing the coupling strength between localized Ca2+ transients and BK channels.

Purinergic activation of spontaneous transient outward currents in guinea pig taenia colonic myocytes

2000 ◽  
Vol 278 (2) ◽  
pp. C352-C362 ◽  
Author(s):  
In Deok Kong ◽  
Sang Don Koh ◽  
Kenton M. Sanders
Keyword(s):  
Guinea Pig ◽  
Pyridoxal Phosphate ◽  
Receptor Blocker ◽  
Disulfonic Acid ◽  
Sk Channels ◽  
Patch Clamp Technique ◽  
Whole Cell Patch Clamp ◽  
Outward Currents ◽  
Relaxation Responses ◽  
Spontaneous Transient Outward Currents

Spontaneous transient outward currents (STOCs) were recorded from smooth muscle cells of the guinea pig taenia coli using the whole cell patch-clamp technique. STOCs were resolved at potentials positive to −50 mV. Treating cells with caffeine (1 mM) caused a burst of outward currents followed by inhibition of STOCs. Replacing extracellular Ca2+ with equimolar Mn2+ caused STOCs to “run down.” Iberiotoxin (200 nM) or charybdotoxin (ChTX; 200 nM) inhibited large-amplitude STOCs, but small-amplitude “mini-STOCs” remained in the presence of these drugs. Mini-STOCs were reduced by apamin (500 nM), an inhibitor of small-conductance Ca2+-activated K+ channels (SK channels). Application of ATP or 2-methylthioadenosine 5′-triphosphate (2-MeS-ATP) increased the frequency of STOCs. The effects of 2-MeS-ATP persisted in the presence of charybdotoxin but were blocked by combination of ChTX (200 nM) and apamin (500 nM). 2-MeS-ATP did not increase STOCs in the presence of pyridoxal phosphate 6-azophenyl-2′,4′-disulfonic acid, a P2 receptor blocker. Similarly, pretreatment of cells with U-73122 (1 μM), an inhibitor of phospholipase C (PLC), abolished the effects of 2-MeS-ATP. Xestospongin C, an inositol 1,4,5-trisphosphate (IP3) receptor blocker, attenuated STOCs, but these events were not affected by ryanodine. The data suggest that purinergic activation through P2Y receptors results in localized Ca2+ release via PLC- and IP3-dependent mechanisms. Release of Ca2+ is coupled to STOCs, which are composed of currents mediated by large-conductance Ca2+-activated K+ channels and SK channels. The latter are thought to mediate hyperpolarization and relaxation responses of gastrointestinal muscles to inhibitory purinergic stimulation.

Unitary excitatory synaptic currents in preganglionic neurons mediated by two distinct groups of interneurons in neonatal rat sacral parasympathetic nucleus

1996 ◽  
Vol 76 (1) ◽  
pp. 215-226 ◽  
Author(s):  
I. Araki ◽  
W. C. De Groat
Keyword(s):  
Nmda Receptor Antagonist ◽  
Neonatal Rat ◽  
Patch Clamp Recording ◽  
Lumbosacral Spinal Cord ◽  
Postsynaptic Currents ◽  
Preganglionic Neurons ◽  
Sacral Parasympathetic Nucleus ◽  
Whole Cell Patch Clamp ◽  
Two Populations ◽  
Stimulation Of

1. Excitatory postsynaptic currents (EPSCs) in parasympathetic preganglionic neurons (PGNs) were examined by the use of the whole cell patch-clamp recording technique in slice preparations of the neonatal rat lumbosacral spinal cord. Synaptic responses were evoked in PGNs by extracellular stimulation of a neighboring interneuron. 2. Stimulation of interneurons medial to the sacral parasympathetic nucleus (SPN) elicited EPSCs or inhibitory postsynaptic currents in 58 and 11%, respectively, of PGNs. Stimulation of interneurons dorsal to the SPN evoked EPSCs in 70% of PGNs. 3. EPSCs occurred at short latency (2.1 ms) and were usually elicited in an all-or-none manner, indicating that they were monosynaptic and mediated by a single interneuron (i.e., unitary). 4. EPSCs were mediated by both non-N-methyl-D-aspartate (non-NMDA) and NMDA receptors. 5. Unitary excitatory postsynaptic potentials evoked by single stimuli did not induce action potentials in PGNs, but repetitive stimulation (> 20 Hz) of the single interneurons could evoke firing of PGNs. 2-Amino-5-phosphonovalerate, an NMDA receptor antagonist, reduced the synaptic depolarization induced in PGNs by high-frequency interneuronal impulses. 6. EPSCs mediated by dorsal interneurons were smaller in amplitude (36.3 +/- 15.7 pA, mean +/- SD) than EPSCs mediated by medial interneurons (88.4 +/- 45.7 pA). 7. Paired-pulse facilitation of EPSCs was observed in PGNs (147.2 +/- 26.2%). The degree of facilitation was higher in dorsal (174.6 +/- 10.3%) than in medial interneuronal pathways (120.9 +/- 3.6%). Within each of interneuronal pathways the degree of facilitation was independent of the magnitude of the unitary EPSC. 8. The results show that PGNs receive monosynaptic glutamatergic excitatory inputs from two distinct populations of interneurons in the dorsal and medial regions of the SPN. These two populations of interneurons are likely to have different functions in the regulation of the preganglionic outflow to the pelvic organs.

Inhibitory effects of pirenzepine on muscarinic stimulation of rat pancreas

1983 ◽  
Vol 92 (3-4) ◽  
pp. 259-264 ◽  
Author(s):  
Jean-Paul Dehaye ◽  
Jacques Winand ◽  
Laurent Hermans ◽  
Piotr Poloczek ◽  
Jean Christophe
Keyword(s):  
Inhibitory Effects ◽  
Rat Pancreas ◽  
Muscarinic Stimulation ◽  
Stimulation Of

Plasma membrane IP3 receptors

2006 ◽  
Vol 34 (5) ◽  
pp. 910-912 ◽  
Author(s):  
C.W. Taylor ◽  
O. Dellis
Keyword(s):  
Plasma Membrane ◽  
Cell Receptor ◽  
Ip3 Receptors ◽  
Animal Cells ◽  
Patch Clamp Recording ◽  
Comparable Amount ◽  
Whole Cell Patch Clamp ◽  
Functional Consequences ◽  
Dt40 Cells ◽  
Stimulation Of

IP3Rs (inositol 1,4,5-trisphosphate receptors) are expressed in the membranes of non-mitochondrial organelles in most animal cells, but their presence and role within the plasma membrane are unclear. Whole-cell patch–clamp recording from DT40 cells expressing native or mutated IP3Rs has established that each cell expresses just two or three functional IP3Rs in its plasma membrane. Only approx. 50% of the Ca2+ entry evoked by stimulation of the B-cell receptor is mediated by store-operated Ca2+ entry, the remainder appears to be carried by the IP3Rs expressed in the plasma membrane. Ca2+ entering the cell via just two large-conductance IP3Rs is likely to have very different functional consequences from the comparable amount of Ca2+ that enters through the several thousand low-conductance store-operated channels.

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