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R J Lang

Publications and source records attributed to R J Lang.

At least 19 recordsLinked to original sources
PubMed2004

Electrical and neurotransmitter activity of mature neurons derived from mouse embryonic stem cells by Sox-1 lineage selection and directed differentiation.

Sx1TV2/16C is a mouse embryonic stem (ES) cell line in which one copy of the Sox1 gene, an early neuroectodermal marker, has been targeted with a neomycin (G418) selection cassette. A combination of directed differentiation with retinoic acid and G418 selection results in an enriched neural stem cell population that can be further differentiated into neurons. After 6-7 days post-plating (D6-7PP) most neurons readily fired tetrodotoxin (TTX)-sensitive action potentials due to the expression of TTX-sensitive Na(+) and tetraethylammonium (TEA)-sensitive K(+) channels. Neurons reached their maximal cell capacitance after D6-7PP; however, ion channel expression continued until at least D21PP. The percentage of cells receiving spontaneous synaptic currents (s.s.c.) increased with days in culture until 100% of cells received a synaptic input by D20PP. Spontaneous synaptic currents were reduced in amplitude and frequency by TTX, or upon exposure to a Ca(2+)-free, 2.5 mm Mg(2+) saline. S.s.c. of rapid decay time constants were preferentially blocked by the nonNMDA glutamatergic receptor antagonists CNQX or NBQX. Ca(2+) levels within ES cell-derived neurons increased in response to glutamate receptor agonists l-glutamate, AMPA, N-methyl-d-aspartate (NMDA) and kainic acid and to acetylcholine, ATP and dopamine. ES cell-derived neurons also generated cationic and Cl(-)-selective currents in response to NMDA and glycine or GABA, respectively. It was concluded that ES-derived neurons fire action potentials, receive excitatory and inhibitory synaptic input and respond to various neurotransmitters in a manner akin to primary central neurons.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed2002

Modulators of internal Ca2+ stores and the spontaneous electrical and contractile activity of the guinea-pig renal pelvis.

1. The role of internal Ca(2+) stores in the generation of the rhythmic electrical and contractile activity in the guinea-pig proximal renal pelvis was examined using intracellular microelectrode and muscle tension recording techniques. 2. Ryanodine (30 microM) transiently increased contraction amplitude, while caffeine (0.5 - 3 mM) reduced contraction amplitude and frequency. Contractility was also reduced by 2-aminoethoxy-diphenylborate (2-APB 60 microM), xestospongin C (1 microM), U73122 (5 microM) and neomycin (4 mM), blockers of IP(3)-dependent release from Ca(2+) stores. 3. 60 mM K(+) saline-evoked contractions were reduced by caffeine (1 mM), U73122 (5 microM) and neomycin (4 mM), but little affected by ryanodine or 2-APB (60 microM). 4. Spontaneous action potentials consisting of an initial spike followed by a long plateau were recorded (frequency 8.6+/-1.0 min(-1)) in small urothelium-denuded strips of proximal renal pelvis. 5. Action potential discharge was blocked in 75 and 35% of cells by 2-APB (60 microM) and caffeine (1 mM), respectively. In the remaining cells, only a truncation of the plateau phase was observed. 6. Cyclopiazonic acid (CPA 10 microM for 10 - 180 min), blocker of CaATPase, transiently increased contraction frequency and amplitude. Action potential durations were increased 3.6 fold. Contraction amplitude and frequency slowly declined during a prolonged (>60 min) CPA exposure. 7. We conclude that the action potential in caffeine-sensitive cells and the shoulder component of caffeine-insensitive action potential arise from the entry of Ca(2+) through Ca(2+) channels. The inhibitory actions of modulators of internal Ca(2+) release were partially explained by a blockade of Ca(2+) entry.

Animals↗
PubMed2002

Thiol reagents and nitric oxide modulate the gating of BKCa channels from the guinea-pig taenia caeci.

1. The site of the direct modulation of the gating of BKCa channels by the nitric oxide donor s-nitroso-l-cysteine (NOCys) was examined in excised membrane patches of the guinea-pig taenia caeci by the use of various thiol (sulphydryl)-specific reagents, including N-ethylmaleimide (NEM) and three charged methanethiosulphonate (MTS) reagents, namely positively charged 2-aminoethyl MTS hydrobromide (MTSEA) and [2-(trimethylammonium)ethyl] MTS bromide (MTSET) and negatively charged sodium (2-sulphonatoethyl) MTS (MTSES), which all specifically convert sulphydryls to a disulphide. 2. At 10 micro mol/L, NOCys transiently increased the probability of opening (N.Po) of the BKCa channels (at 0 mV) after a delay of 1-2 min. 3. Disulphide-reducing agents, such as dithiothreitol (10 micro mol/L), increased N.Po in a manner that was reversed by the sulphide-oxidizing agent thimerosal (10 micro mol/L). Both positively charged MTSET (2.5 mmol/L) and negatively charged MTSES (2.5 mmol/L) rapidly increased N.Po. However, only the MTSES-evoked increase in N.Po remained after a prolonged washout period. 4. The specific alkylating agent of cysteine thiols NEM (1 mmol/L) and the positively charged, but membrane permeable, MTSEA (2.5 mmol/L) decreased N.Po (at 0 mV). 5. Pre-exposure of excised membrane patches to NEM or MTSES prevented the excitatory actions of NOCys (10 micro mol/L). 6. We conclude that MTSES and NOCys must modify thiols on cysteine residues within basic regions of the channel protein that would electrostatically exclude MTSEA and MTSET. A consensus sequence of various mammalian alpha-subunits of the BKCa channel reveals two pairs of cysteine residues surrounded by basic amino acids that could be the site of action for NOCys and MTSES.

Animals↗
PubMed2001

Characterization of the spontaneous electrical and contractile activity of smooth muscle cells in the rat upper urinary tract.

PURPOSE: We morphologically and electrophysiologically identified the cells that generate the electrical activity underlying the peristaltic contractions of the rat upper urinary tract. MATERIALS AND METHODS: Electron microscopy and tension recording techniques were used to characterize the smooth muscle cells underlying spontaneous contractions in the wall of the rat ureter, and proximal and distal renal pelvis. Intracellular microelectrodes, containing 4% neurobiotin were used to record data from the cells of the renal pelvis, which were later viewed on a confocal microscope. RESULTS: Spontaneous myogenic contractions (average 22.3 +/- 2.2 minutes(-1)) originated in the proximal renal pelvis and propagated into the distal renal pelvis and ureter in 6 preparations. Smooth muscle cells in the renal pelvis and ureter were typical in appearance with greater than 85% of their sectional area containing clumped contractile filaments. In contrast, contractile fibrils occupied only 65% of the sectional area of the smooth muscle cells within the most proximal region of the renal pelvis (pelvicaliceal junction). In strips of the renal pelvis spindle shaped cells 83 to 200 microm. long fired spontaneous action potentials (6 minutes(-1)) consisting of an initial spike, a quiescent plateau phase and abrupt hyperpolarization to a peak diastolic potential of -60 mV. Other spindle shaped cells 94 to 112 microm. long displayed small membrane transients (15 minutes(-1)) 9 to 19 mV. in amplitude, firing from a diastolic potential of -40 mV. CONCLUSIONS: It is likely that the spontaneous contractile activity of the rat upper urinary tract arises from the discharge of action potentials in typical smooth muscle cells of the proximal renal pelvis that are directly driven by the spontaneous membrane oscillations of atypical smooth muscle cells.

Action Potentials↗
PubMed2000

Nitric oxide and thiol reagent modulation of Ca2+-activated K+ (BKCa) channels in myocytes of the guinea-pig taenia caeci.

The modulation of large conductance Ca2+-activated K+ (BKCa) channels by the nitric oxide (NO) donors S-nitroso-L-cysteine (NOCys) and sodium nitroprusside (SNP) and agents which oxidize or reduce reactive thiol groups were compared in excised inside-out membrane patches of the guinea-pig taenia caeci. When the cytosolic side of excised patches was bathed in a physiological salt solution (PSS) containing 130 mM K+ and 15 nM Ca2+, few BKCa channel openings were recorded at potentials negative to 0 mV. However, the current amplitude and open probability (NPo) of these BKCa channels increased with patch depolarization. A plot of ln(NPo) against the membrane potential (V) fitted with a straight line revealed a voltage at half-maximal activation (V0.5) of 9.4 mV and a slope (K) indicating an e-fold increase in NPo with 12.9 mV depolarization. As the cytosolic Ca2+ was raised to 150 nM, V0.5 shifted 11.5 mV in the negative direction, with little change in K (13.1 mV). NOCys (10 microM) and SNP (100 microM) transiently increased NPo 16- and 3. 7-fold, respectively, after a delay of 2-5 min. This increase in NPo was associated with an increase in the number of BKCa channel openings evoked at positive potentials by ramped depolarizations (between -60 and +60 mV). Moreover, this NOCys-induced increase in NPo was still evident in the presence of 1H-[1,2,4]oxadiazolo[4, 3-a]quinoxalin-1-one (ODQ; 10 microM), the specific blocker of soluble guanylyl cyclase. The sulfhydryl reducing agents dithiothreitol (DTT; 10 and 100 microM) and reduced glutathione (GSH; 1 mM) also significantly increased NPo (at 0 mV) 7- to 9-fold, as well as increasing the number of BKCa channel openings evoked during ramped depolarizations. Sulfhydryl oxidizing agents thimerosal (10 microM) and 4,4'-dithiodipyridine (4,4DTDP; 10 microM) and the thiol-specific alkylating agent N-ethylmaleimide (NEM; 1 mM) significantly decreased NPo (at 0 mV) to 40-50% of control values after 5-10 min. Ramped depolarizations to +100 mV evoked relatively few BKCa channel openings. The effects of thimerosal on NPo were readily reversed by DTT, while the effects of NOCys were prevented by NEM. It was concluded that both redox modulation and nitrothiosylation of cysteine groups on the cytosolic surface of the alpha subunit of the BKCa channel protein can alter channel gating.

Animals↗
PubMed2000

Effects of selective inhibitors of cyclo-oxygenase-1 (COX-1) and cyclo-oxygenase-2 (COX-2) on the spontaneous myogenic contractions in the upper urinary tract of the guinea-pig and rat.

The role of cyclo-oxygenase-1 (COX-1) and cyclo-oxygenase-2 (COX-2) in the upper urinary tract of the guinea-pig and rat was examined using simultaneous tension recordings of the proximal and distal regions of the renal pelvis and the ureter. The guinea-pig upper urinary tract contracted at a frequency (7.52+/-0.3 min(-1) at 35 degrees C) significantly lower than the frequency in the proximal renal pelvis (21.6+/-1.3 min(-1)) and in the distal renal pelvis and ureter (20.2+/-1.4 min(-1)) of the rat (at 30 degrees C). Indomethacin (>/=1 microM for 60 min), decreased the motility index (amplitudexfrequency) (MI) in all three regions of the guinea-pig upper urinary tract, an effect which mainly arose from a decrease in the frequency of contractions. In the rat, indomethacin (1 - 30 microM for 60 min) significantly decreased the MI calculated in the proximal renal pelvis (>/=30 microM indomethacin), and in the distal renal pelvis (>/=10 microM indomethacin), arising from a significant decrease in the amplitude of contractions. The COX-1 inhibitor, valeryl salicylate (VSA) (5 - 100 microM for 60 min), had no effect on either the amplitude or frequency of contractions in the guinea-pig upper urinary tract. In contrast, VSA increased the force of contractions in the proximal and distal renal pelvis of the rat, whilst having little effect on the frequency of contractions. The COX-2 inhibitor, NS-398 (10 - 100 nM for 60 min) reduced the MI in the guinea-pig upper urinary tract in a concentration-dependent manner. The MIs calculated for the proximal renal pelvis, distal renal pelvis and ureter, were decreased by 72, 64 and 72% respectively, in 100 nM NS-398. NS-398 (10 - 100 nM) had no effect on any of the three parameters measured in either the proximal or distal renal pelvis of the rat. These data suggest that endogenously-released prostaglandins (PGs) maintain the myogenic contractility of the upper urinary tract in both the guinea-pig and rat. Moreover COX-2 is the primary enzyme involved in synthesizing PGs in the guinea-pig upper urinary tract, while COX-1 appears to be the predominantly active enzyme in the rat.

Animals↗
PubMed1999

Molecular cloning and characterization of the intermediate-conductance Ca(2+)-activated K(+) channel in vascular smooth muscle: relationship between K(Ca) channel diversity and smooth muscle cell function.

Recent evidence suggests that functional diversity of vascular smooth muscle is produced in part by a differential expression of ion channels. The aim of the present study was to examine the role of Ca(2+)-activated K(+) channels (K(Ca) channels) in the expression of smooth muscle cell functional phenotype. We found that smooth muscle cells exhibiting a contractile function express predominantly large-conductance ( approximately 200 pS) K(Ca) (BK) channels. In contrast, proliferative smooth muscle cells express predominantly K(Ca) channels exhibiting a much smaller conductance ( approximately 32 pS). These channels are blocked by low concentrations of charybdotoxin (10 nmol/L) but, unlike BK channels, are insensitive to iberiotoxin (100 nmol/L). To determine the molecular identity of this K(+) channel, we cloned a 1.9-kb cDNA from an immature-phenotype smooth muscle cell cDNA library. The cDNA contains an open reading frame for a 425 amino acid protein exhibiting sequence homology to other K(Ca) channels, in particular with mIK1 and hIK1. Expression in oocytes gives rise to a K(+)-selective channel exhibiting intermediate-conductance (37 pS at -60 mV) and potent activation by Ca(2+) (K(d) 120 nmol/L). Thus, we have cloned and characterized the vascular smooth muscle intermediate-conductance K(Ca) channel (SMIK), which is markedly upregulated in proliferating smooth muscle cells. The differential expression of these K(Ca) channels in functionally distinct smooth muscle cell types suggests that K(Ca) channels play a role in defining the physiological properties of vascular smooth muscle.

Amino Acid Sequence↗
PubMed1999

Identification of the cells underlying pacemaker activity in the guinea-pig upper urinary tract.

1. The varying profile of cell types along the muscle wall of the guinea-pig upper urinary tract was examined electrophysiologically, using intracellular microelectrodes, and morphologically, using both electron and confocal microscopy. 2. Simple 'pacemaker' oscillations (frequency of 8 min-1) of the membrane potential were recorded in both the pelvi-calyceal junction (83 % of cells) and the proximal renal pelvis (15 % of cells), but never in the distal renal pelvis or ureter. When filled with the cell marker, neurobiotin, 'pacemaker' cells were spindle shaped and approximately 160 microm in length. 3. In most cells of the ureter (100 %) and in both the proximal (75 %) and distal (89 %) renal pelvis, spontaneous action potentials (frequency of 3-5 min-1) consisted of an initial spike, followed by a number of potential oscillations superimposed on a plateau phase. When filled with neurobiotin, cells firing these 'driven' action potentials, were spindle shaped and > 250 microm in length. 4. Greater than 80 % of smooth muscle cells in the pelvi-calyceal junction were 'atypical', having < 40 % of their sectional areas occupied by loosely packed contractile filaments. Most of the smooth muscle cells in the ureter (99.7 %) and both the proximal (83 %) and distal (97.5 %) renal pelvis were of 'typical' appearance in that they contained cytoskeletal and contractile elements occupying > 60 % of cross-sectional area. 5. A third type of spontaneously discharging cell fired 'intermediate' action potentials (3-4 min-1), consisting of a single spike followed by a quiescent plateau and an abrupt repolarization. These cells were morphologically similar to interstitial cells of Cajal (ICC). However, these 'ICC-like' cells were not immuno-reactive for c-Kit, the proto-oncogene for tyrosine kinase. 6. In summary, 'atypical' smooth muscle cells were predominant in the pelvi-calyceal junction and fired 'pacemaker' potentials at a frequency significantly higher than 'driven' action potentials recorded in 'typical' smooth muscle cells throughout the renal pelvis and ureter. 'Intermediate' action potentials were recorded in 'ICC-like' cells in both the pelvi-calyceal junction and renal pelvis. We suggest that these 'ICC-like' cells act as a preferential pathway, conducting and amplifying pacemaker signals to initiate action potential discharge in the driven areas of the upper urinary tract.

Action Potentials↗
PubMed1999

K(+) channel blocker modulation of the refractory period in spontaneously active guinea-pig ureters.

The effects of various K(+) channel blockers on the spontaneous electrical activity of the smooth muscle cells of the ureter still attached to its primary pacemaker regions were investigated using standard intracellular microelectrode recording techniques. Spontaneous action potentials in the ureter were complex, consisting of an initial rapidly rising spike which was followed by a period of membrane oscillation, a quiescent plateau phase and terminated by an abrupt repolarisation and an after-hyperpolarisation with a peak "diastolic" potential of -66 mV. This after-hyperpolarization decayed slowly over 5-20 s until the underlying triggering potentials achieved threshold for another action potential discharge. Application of the Ca(2+)-entry blocker, nifedipine (1 mu;M), blocked action potential discharge within 2-5 min, after which the membrane settled at a potential of -55 mV. 4-Aminopyridine (4-AP)(1 mM for 2 min) and Ba(2+) (100 mu;M for 2 min) both depolarized significantly the diastolic potential. In 4-AP, this membrane depolarisation was associated with a decreased amplitude of the initial spike and an increase in the half-amplitude duration. In contrast, tetraethylammonium (TEA) (0.5 mM for 2 min) only increased the frequency and half-amplitude duration of these ureteric action potentials. Apamin (200 nM), Cs(+) (1 mM) and glibenclamide (1 microM) had no significant effects on any parameters of the ureteric action potential. It was concluded that the refractory period of the spontaneous action potentials in the whole-mount preparation of the upper urinary tract was determined by the opening of at least three K(+) channel populations: large conductance ('maxi K') Ca(2+)-activated K(+) channels; Ca(2+)-insensitive transiently opening K(+) (I(Kto)) channels and K(+)-selective inward rectifier channels.

Action Potentials↗
PubMed1999

Effects of nerve stimulation on spontaneously active preparations of the guinea pig ureter.

The effects of intrinsic nerve stimulation on the spontaneous electrical activity of the smooth muscle cells of the guinea pig ureter still attached to its renal pelvis were investigated using standard intracellular microelectrode techniques. Action potentials discharged spontaneously at a frequency of 3.3 +/- 0.2 min(-1) (n = 67) and consisted of an initial rapidly rising spike, followed by a variable period (0.2-5 s) of membrane potential oscillation and a quiescent plateau phase which was terminated by an abrupt repolarisation and after-hyperpolarisation to -66 mV. Transmural electrical stimulation (20-50 Hz for 2 s) transiently decreased the frequency of action potential discharge; the half-amplitude duration of the following action potentials, however, was transiently increased to 156 +/- 12% of control. Substance P (1 microM applied for 2 min) or neurokinin A (100 nM for 2 min) transiently increased the frequency of action potential discharge to 155 +/- 19% and 142 +/- 21%, respectively, of control. The excitatory actions of nerve stimulation or agonist application were reduced by the tachykinin antagonist, MEN 10,627 (1-3 microM), while the inhibitory actions of nerve stimulation were enhanced by MEN 10,627 (1 microM) or thiorphan (1 microM). Capsaicin (10 microM for 10-15 min) also evoked a transient increase in the frequency and half-amplitude duration of the ureteric action potentials, in a manner blocked by MEN 10,627 (3 microM), which was followed by a long period of membrane potential quiescence. Human calcitonin gene related peptide (hCGRP) (100 nM applied for 2-5 min) induced a time-dependent decrease in the frequency amplitude and duration of the spontaneous action potentials, in a manner blocked by glibenclamide (1 microM). It was concluded that the nerve-evoked excitatory and inhibitory changes in the parameters of the spontaneous ureteric action potentials arise from the release of the sensory neuropeptides, tachykinins and CGRP, respectively.

Action Potentials↗
PubMed1998

Inhibition of voltage-activated K+ currents in smooth muscle cells of the guinea pig proximal colon by noradrenergic agonists.

1. The effects of noradrenaline and isoprenaline on the Ca2+i-insensitive, voltage-activated K+ current in smooth muscle cells from the circular muscle layer of the guinea pig proximal colon were investigated by using standard whole-cell patch-clamp techniques at room temperature (22-24 degrees C). 2. The Ca2+-activated K+ current was eliminated by bathing cells in tetraethylammonium (TEA;2-5 mM) and a Ca2+-entry blocker (Cd2+, 0.1 mM) or nifedipine, 2-10 microM) and by internally perfusing cells with 3 mM EGTA. 3. Two Ca2+i-insensitive, voltage-activated K+ currents were recorded at potentials positive to -50 mV: (a) a transient K+ current (IKto) that was blocked by 4-aminopyridine (5 mM) and (b) a delayed rectifier-type K+ current (IKdel) that was blocked by TEA (>10 mM). 4. Both noradrenaline (10-50 microM) and isoprenaline (5-50 microM) reduced the amplitudes of IKto and IKdel irreversibly after a slow onset (2-5 min). This reduction was mimicked by forskolin (50-100 microM) and by 8 bromo-c-AMP (500 microM). 5. The voltage of half-maximal availability (V0.5) of IKto (-74.6+/-2.3 mV) was unaffected by isoprenaline (10 microM) (-76.7+/-3.6 mV, n=4), but the background "leak" current (Ileak) was increased from -48+/-9 to -70+/-20 pA. 6. Our data suggest that stimulation of beta-adrenoceptors in the circular muscle layer of the guinea pig proximal colon inhibits voltage-activated Ca2+i-insensitive K+ currents.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1998

Effects of nitric oxide donors, S-nitroso-L-cysteine and sodium nitroprusside, on the whole-cell and single channel currents in single myocytes of the guinea-pig proximal colon.

1. The nature of the membrane channels underlying the membrane conductance changes induced by the nitric oxide (NO) donors, S-nitroso-L-cysteine (NOCys) and sodium nitroprusside (SNP) were investigated in single myocytes isolated from the circular muscle layer of the guinea-pig proximal colon, by use of standard whole-cell and single channel recording techniques. 2. Under voltage clamp, depolarizing steps from -60 mV elicited a rapidly-developing, little-inactivating outward K+ current (IK) at potentials positive to -40 mV (at 20-25 degrees C). The steady-state level (ISS) of this K current increased in amplitude as the step potential was made to more positive potentials. If the depolarizing steps were made from a holding potential of -80 mV an additional rapidly activating and inactivating outward K+ current was also elicited, superimposed on IK. 3. At 20-25 degrees C, NOCys (2.5 microM), SNP (100 microM) and 8-bromo-cyclic GMP (500 microM) increased the amplitude of ISS of IK elicited from a holding potential of -60 mV. In contrast, NOCys (2-5 microM) had little effect on ISS at 35 degrees C. Higher concentrations (> or = 5 microM at 20-25 degrees C and > or = 10 microM at 35 degrees C) of NOCys decreased the peak amplitude (I[Peak]) and ISS of IK in a concentration-dependent manner. This blockade of IK with NOCys was always associated with an increase of the holding current (IHold), due to the activation of a membrane conductance with a reversal potential between 0 and + 30 mV and which was reduced approximately 50% upon the addition of Cd2+ (1 mM). 4. NOCys (2.5 to 10 microM) or SNP (100 microM) increased the activity of large conductance Ca2+-activated (BK) K' channels in both cell-attached and excised inside-out patches, bathed in either a symmetrical high K+ (130 mM) or an asymmetrically K+ (6 mMout: 130 mMin) physiological saline. Increases in BK channel activity in NOCys (10 microM) or SNP (100 microM) were associated with an increase in the probability of BK channel opening (N.Po), and with a negative shift of the plots of ln(N.Po) against the patch potential, with little change in the slopes of these plots. In cell-attached patches, the increase in N.Po with NOCys was often associated with a decrease in the BK single channel conductance. 5. In both cell-attached and excised patches, NOCys (2.5 to 10 microM) also activated an additional population of channels which allowed inward current flow at potentials positive to EK. In excised inside-out patches bathed in asymmetrical K+ physiological saline, these single channel currents were 2-3 pA in amplitude at -30 mV and reversed in direction near + 10 mV, even if the NaCl (126 mM) concentration in the pipette solution had been replaced with an equimolar concentration of Na gluconate. 6. Under current clamp, NOCys (2.5 microM) and SNP (100 microM) had variable effects on the membrane potential of colonic myocytes, inducing either a small membrane hyperpolarization of <5 mV, or a slowly-developing membrane depolarization of about 5 mV. In contrast, NOCys (5 microM) produced a transient membrane hyperpolarization which was followed by a large depolarization of the membrane potential to positive potentials. The electrotonic potentials elicited in response to an injection of constant hyperpolarizing current (10 pA for 400 ms) were little changed during the NOCys (5 PM)-induced membrane hyperpolarization, but significantly reduced (to 61% of control) during the periods of membrane depolarization. 7. It was concluded that NOCys and SNP, directly increased the number of active BK channels in the membrane of colonic myocytes which leads to a small rapidly oscillating membrane hyperpolarization. The following rebound depolarization in NOCys arises from both the direct opening of a population of cationic channels and the blockade of voltage- and Ca-activated K+ conductances. Finally, the apamin-sensitive K+channels underlying the initial transient hyperpolarization recorded in the intact proximal colon, in response to nerve-released or directly-applied NO, have yet to be identified at the single channel or whole-cell current level.

Animals↗
PubMed1998

Stretch-evoked inhibition of spontaneous migrating contractions in a whole mount preparation of the guinea-pig upper urinary tract.

1. The effects of circumferentially-applied stretch on the spontaneous contractility of a whole mount preparation of the guinea-pig upper urinary tract (UUT) (renal pelvis and ureter) were investigated by use of standard isometric tension recording techniques. 2. Simultaneous tension recordings of the proximal and distal portions of the renal pelvis (RP) and ureter revealed that spontaneous contractions, in 79% (n = 66) of preparations, originated in the proximal RP (at a frequency of 4.5 min(-1)) and propagated to the distal RP and ureter at a velocity of 1-3 cm s(-1). Pretreatment with tetrodotoxin (TTX) (3-10 microM) or N(G)-nitro-L-arginine (100 microM) had little effect on the spontaneous contractility of the UUT, motility indexes (MIs) (contraction amplitude x contraction frequency) calculated after 20 min exposure were little affected by TTX or N(G)-nitro-L-arginine (L-NOARG). Omega-conotoxin GVIA (100 nM) significantly reduced MI values in both the proximal RP and ureter. 3. Exposure of the spontaneously-active UUT to capsaicin (10 microM for 15 min) induced a transient increase in UUT contractility, followed by a prolonged negative inotropic effect. The MI values, calculated 60 min after the washout of capsaicin, for the proximal and distal RP and ureter were reduced to 56%, 53% (n = 18) and 61% (n = 16), respectively, of their control values. This capsaicin pretreatment blocked the positive inotropic effects of transmural electrical nerve stimulation on UUT contractility to reveal a small inhibitory effect which was readily blocked by tetrodotoxin (3 microM) (n = 3). The excitatory and inhibitory actions of nerve stimulation were both blocked by TTX (3 microM). 4. A second exposure to capsaicin (10 microM for 15 min), further reduced the MI values (calculated 60 min after washout) in the proximal and distal RP to 41% and 31%, respectively (n = 6; P<0.05), of the initial control values. 5. In 61% (n = 99) of preparations, the application of stretch to the proximal RP (0.5 to 2 mm) evoked a decrease in the amplitude of the contractions recorded in the distal RP, but not in the ureter. Stretch applied to the distal RP or ureter had no effect on the contractions recorded in the other regions of the UUT. 6. In 5 out of 6 preparations, a single application of capsaicin (10 microM for 15 min) had little effect on the change in contractile force of the distal RP evoked upon stretch of the proximal RP. 7. The inhibition of the distal RP upon stretch of the proximal RP was partially reduced (P<0.05) when the UUT was pretreated with the calcitonin gene-related peptide (CGRP) receptor antagonist, hCGRP (8-37) (1 microM). 8. The application of the CGRP receptor agonist, hCGRP (100 nM) inhibited contractility in the UUT in a region dependent manner. The MI of the proximal RP was decreased 32% after 6 min; while the MIs of the distal RP and ureter were reduced 83% and 63%, respectively, within 5 min of the application of hCGRP. 9. Glibenclamide (1 microM) had little effect on the spontaneous contractility of the UUT, but significantly reduced the inhibition of the distal RP evoked upon stretch (0.5 to 2 mm) of the proximal RP. TTX (3-10 microM), L-NOARG (100 microM) or omega-conotoxin GVIA (100 nM) had little effect on the stretch-evoked inhibition of the distal RP. 10. It was concluded that circumferential stretch of the proximal RP inhibits the contractility of the distal RP and that a component of this inhibition involves the activation of a glibenclamide-sensitive mechanism via the release of endogenous CGRP, possibly from the varicosities of intramural sensory nerves.

Animals↗
PubMed1998

Electrical basis of peristalsis in the mammalian upper urinary tract.

1. Peristalsis in the mammalian upper urinary tract (UUT) is mostly myogenic in origin, originating predominately in the proximal pelvicalyceal regions of the renal pelvis, an area that is enriched with specialized smooth muscle cells termed 'atypical' smooth muscle cells. Propagating peristaltic contractions are little affected by blockers of either autonomic nerve function or nerve impulse propagation; however, blockers of sensory nerve function or prostaglandin synthesis reduce both the frequency and the strength of the spontaneous contractions underlying peristalsis. 2. The electrical drive for these peristaltic contractions has long been considered to involve mechanisms analogous to the heart, such that 'atypical' smooth muscle cells generate spontaneous 'pacemaker' action potentials. These pacemaker potentials trigger the firing of action potentials and contraction in the muscular regions of the renal pelvis, which propagate distally to the ureter, propelling urine towards the bladder. 3. Recent intracellular microelectrode and single cell/channel patch-clamp studies have revealed that the ionic conductances underlying the action potentials recorded in the UUT are likely to involve the opening and slow closure of voltage-activated 'L-type' Ca2+ channels, offset by the time-dependent opening and closure of both voltage- and Ca(2+)-activated K+ channels. 4. In the present review we summarize the current knowledge of the ionic mechanisms underlying action potential discharge in the UUT, as well as present our view on how this electrical activity supports the initiation and conduction of UUT peristalsis.

Action Potentials↗
PubMed1996

Characterization of the membrane conductance changes underlying the apamin-resistant NANC inhibitory junction potential in the guinea-pig proximal and distal colon.

The nature of the electrically- or stretch-evoked nonadrenergic, noncholinergic (NANC) inhibitory junction potentials (IJPs) in circular smooth muscle cells of the guinea-pig proximal and distal colon were investigated using standard intracellular microelectrode recording techniques. We have confirmed that the NANC IJP, recorded in the presence of hyoscine (1 microM) and nifedipine (1 microM), can be divided into two components with apamin (250 nM), a blocker of the small conductance Ca2(+)-activated K+ channels. Both the apamin-sensitive and the apamin-resistant components of the IJP were blocked by tetrodotoxin (1.6 microM) or by lowering the external Ca2+ concentration (to 0.25 mM). The apamin-sensitive IJP was also blocked by omega-conotoxin GVIA (100 nM), a blocker of 'N-type' Ca2+ channels. The apamin-resistant IJP and rebound post-stimulus depolarization (PSD) were reduced upon exposure to either NG-L-arginine (NOLA), an inhibitor of nitric oxide synthase (NOS), or the nitric oxide (NO) scavenger, haemoglobin. The effects of NOLA were partially reversed in the presence of excess L-arginine, a substrate for NOS, suggesting that NO, or a related NO-donor compound, is likely to be the apamin-resistant inhibitory transmitter. Blockade of either the apamin-sensitive or apamin-resistant IJP was associated with membrane depolarization and a decrease in the membrane conductance in the absence of nerve stimulation. In the proximal colon, the apamin-resistant IJP and PSD could both be demonstrated to arise from an increase in the membrane conductance after subtraction of a non-linear background conductance. The hyperpolarization upon repetitive NANC nerve stimulation was mimicked by the NO donor, S-nitroso-L-cysteine (2.5-25 microM), which evoked a transient apamin-sensitive, but omega-conotoxin GVIA resistant, component followed by a slower apamin-resistant component. These results suggest that neurally-released NO has a number of actions in the guinea-pig colon, causing apamin-resistant hyperpolarization and depolarization, as well as directly opening apamin-sensitive K+ channels.

Animals↗
PubMed1996

Effects of nitric oxide (NO) and NO donors on the membrane conductance of circular smooth muscle cells of the guinea-pig proximal colon.

1. The membrane conductance changes underlying the membrane hyperpolarizations induced by nitric oxide (NO), S-nitroso-L-cysteine (NC) and sodium nitroprusside (SNP) were investigated in the circular smooth muscle cells of the guinea-pig proximal colon, by use of standard intracellular microelectrode recording techniques. 2. NO (1%), NC (2.5-25 microM) and SNP (1-1000 microM) induced membrane hyperpolarization in a concentration-dependent manner, the hyperpolarizations to NO and NC developing more rapidly than those to SNP. The slower-developing responses to SNP were mimicked by the membrane permeable analogue of guanosine 3':5' cyclic-monophosphate (cyclic GMP), 8-bromo-cyclic GMP (500 microM), and by isoprenaline (10 microM). 3. The hyperpolarizations to NC and SNP were reduced in a low Ca2+ (0.25 mM) saline and upon the addition of haemoglobin (20 microM), but were not effected by NG-nitro-L-arginine (L-NOARG) (100 microM) or omega-conotoxin GVIA (100 nM). the hyperpolarizations to SNP were also significantly reduced by methylene blue (50 microM). 4. Apamin (250 nM) depolarized the membrane potential approximately 10 mV and reduced the initial transient component of the hyperpolarization to NO (1%) and NC (25 microM), but had no effects on the hyperpolarizations to SNP and cyclic GMP. Tetraethylammonium (TEA) (5-15 mM), had little effect on the membrane responses to NO(1%), NC(2.5-25 microM), SNP(100(-1000) microM) or cyclic GMP(500 microM). However, TEA (5-15 mM) reduced the membrane hyperpolarizations to SNP (10 microM) and isoprenaline (10 microM) in a concentration-dependent manner. The hyperpolarization to isoprenaline (10 microM) remaining in the presence of 15 mM TEA was blocked by ouabain (10 microM). 5. The amplitude of electronic potentials (1 s duration) elicited during NO donor hyperpolarizations were little changed or only slightly reduced (5-25%). However, the amplitude of the electrotonic potentials elicited during maintained electrically-induced hyperpolarizations of similar amplitude were significantly increased (30-150%), suggesting that the non-linear membrane properties of the proximal colon partially mask an increase in membrane conductance elicited during the NO donor hyperpolarizations. 6. Membrane hyperpolarization in the presence of an NO donor, 8-bromo-cyclic GMP, isoprenaline, or upon application of a maintained hyperpolarizing electrical current, often evoked oscillations of the membrane potential. These oscillations were prevented by Cs+ (1 mM). 7. These results indicate that NO and NC hyperpolarize the circular muscle of the proximal colon by activating at least two TEA-resistant membrane K+ conductances, one of which is sensitive to apamin blockade. The K+ conductance increases activated by SNP or 8-bromo-cyclic GMP were little effected by apamin, perhaps suggesting a common mechanism. In contrast, the hyperpolarization to isoprenaline appears to involve the activation of TEA-sensitive Ca2(+)-activated K+ ('BK') channels, as well as a Na:K ATPase. Finally, the 'background' membrane conductance of the circular muscle cells of the proximal colon decreased upon membrane hyperpolarization to reveal oscillations of the membrane potential which may well represent 'pacemaker' or 'slow wave' activity.

Animals↗
PubMed1996

The effects of K+ channel blockers on the spontaneous electrical and contractile activity in the proximal renal pelvis of the guinea pig.

PURPOSE: The role of various K+ channel populations in determining the time course and amplitude of the spontaneous action potentials and contractions in the smooth muscle cells of the guinea pig renal pelvis were investigated by standard electrophysiological and tension recording techniques. MATERIALS AND METHODS: Electrical recordings in the proximal renal pelvis were used to demonstrate the presence of 3 cell populations based on the waveform of their Ca(2+)-dependent action potentials which we have termed "pacemaker," "intermediate" and "driven" action potentials. RESULTS: Blockade of large conductance (BK) or small conductance (SK) Ca(2+)-activated K+ channels with charybdotoxin (30 to 60 nM.) and tetraethylammonium (TEA) (0.5 to 2 mM.) or apamin (200 nM.) increased the duration of the action potentials recorded in "driven" cells. Tetraethylammonium (2 mM.) and 4-aminopyridine (2 mM.) increased the frequency of action potential discharge in both "driven" and "pacemaker" cells, as well as having a positive inotropic and chronotropic effect on the spontaneous contractions of the renal pelvis. In contrast, glibenclamide (0.1 to 1 microM.), Cs2+ (1 mM.) and Ba2+ (100 microM.) had little effect on either the contractile or electrical activity of the renal pelvis. CONCLUSION: It was concluded that the frequency of contraction in the proximal renal pelvis is determined by the frequency of action potential discharge in "pacemaker" cells. On the other hand, increases in the amplitude of pelvic contractions may well be correlated with membrane depolarization and/or increases in the duration of the action potentials recorded in "driven" cells.

4-Aminopyridine↗
PubMed1995

Effects of nerve stimulation on the spontaneous action potentials recorded in the proximal renal pelvis of the guinea-pig.

The effects of nerve stimulation on the electrical and mechanical activity of the smooth muscle of the proximal renal pelvis of the guinea-pig were investigated using standard tension and microelectrode recording techniques. Spontaneous action potentials were deemed to have been recorded from three cell types: (1) "pacemaker" cells (9 of > 120) had membrane potentials (MPs) of -42.1 +/- 2.9 mV and fired action potentials of a simple waveform; (2) "driven" cells (> 100) had more stable MPs of -56.1 +/- 1.2 mV (n = 36) and more complex "ureter-like" action potentials; (3) the remaining cells had MPs of -45.5 +/- 1.7 mV (n = 15) and action potentials with a waveform "intermediate" to groups (1) and (2). Nifedipine (0.1-1 microM) and Cd2+ (0.1-1 mM) blocked all spontaneous action potential discharge and depolarized the membrane to near -40 mV. Intramural nerve stimulation (10-50 Hz for 1-10 s) increased both the amplitude and frequency of the spontaneous contractile activity, this increase peaked in about 30 s and decayed slowly over several minutes. Nerve stimulation depolarized pacemaker and driven cells 9.1 +/- 3.5 (n = 3) and 1.6 +/- 0.7 (n = 6) mV, respectively; the frequency of their action potential discharge increased from 7.6 +/- 2.7 and 9.9 +/- 1.1/min to 17.3 +/- 0.5 and 11.1 +/- 1.4/min, respectively. The duration of the action potentials in driven cells also increased significantly for several minutes. All these effects were blocked by tetrodotoxin (TTX) (1.6 microM). It was concluded that the positive chronotropic and inotropic effects of nerve stimulation on renal pelvis contractility can be correlated with the changes in the frequency and duration of the action potentials recorded in driven cells.

Action Potentials↗