PubMed Health⌕ Search

Biomedical subjects

S M Sims

Publications and source records attributed to S M Sims.

At least 55 records · Page 3Linked to original sources
PubMed1994

Mammalian osteoclasts express a transient potassium channel with properties of Kv1.3.

Previous studies have revealed that expression of K+ channels in osteoclasts correlates with cell morphology and is influenced by interaction with the extracellular matrix. In this study, we investigated the electrophysiological properties of an outwardly rectifying K+ channel in rat and mouse osteoclasts using patch-clamp techniques. Cell-attached patch recordings revealed a channel of approximately 14 pS conductance that opened upon depolarization, and had a reversal potential close to that predicted for a K+ channel. Channel activity was transient; inactivation of ensemble currents, like that of whole-cell currents, occurred as a single exponential process. Both single-channel and macroscopic currents exhibited use-dependent inactivation in response to repetitive depolarizations. Two scorpion toxins, margatoxin and charybdotoxin, blocked this transient K+ channel, with half-maximal inhibition at 200 pM and 5 nM, respectively. In contrast, dendrotoxin (500 nM) had little effect. In summary, the outwardly rectifying K+ channel in osteoclasts resembles the Shaker-related K+ channel, Kv1.3. When membrane potential was recorded in whole-cell configuration, charybdotoxin (50 nM) caused a depolarization of 5 to 10 mV from resting levels of -50 mV or more positive; therefore this K+ channel contributes to the membrane potential of osteoclasts under some conditions. To investigate the molecular nature of osteoclast K+ channels, we performed RT-PCR on osteoclast RNA using primers for Kv1.3 and the inward rectifier, IRK1. mRNA encoded by Kv1.3 and IRK1 was detected and message identity confirmed by restriction enzyme digestion and sequence analysis. We conclude that osteoclasts exhibit, in addition to the previously described inward rectifier, an outwardly rectifying K+ conductance with properties of the Kv1.3. channel.

Animals↗
PubMed1994

Lamellipod extension and K+ current in osteoclasts are regulated by different types of G proteins.

Osteoclasts are the cells responsible for the resorption of bone and other mineralized tissues. GTP-binding proteins (G proteins) play important roles in regulating the activity of many cell types; however, there is limited knowledge of their functions in osteoclasts. We used the patch-clamp technique in the whole-cell configuration to introduce either hydrolysis-resistant guanosine triphosphate analogues or fluoroaluminate into single rat osteoclasts, and examined the effects of G protein activation on cell morphology and ionic conductances. Guanosine 5'-O-(3-thiotriphosphate) or 5'-guanylyl-imidodiphosphate, but not the control compounds adenosine 5'-O-(3-thiotriphosphate) or guanosine 5'-O-(2-thiodiphosphate), induced: (1) prompt spreading due to extension of lamellipodia; and (2) after a latency of several minutes, complete suppression of the inwardly rectifying K+ current. Pertussis toxin did not alter either spreading or suppression of K+ current induced by guanosine 5'-O-(3-thiotriphosphate). Cytochalasin D, but not colchicine, prevented guanosine 5'-O-(3-thiotriphosphate)-induced spreading, consistent with actin polymerization underlying lamellipod extension. Whole-cell capacitance did not change during guanosine 5'-O-(3-thiotriphosphate)-induced spreading, which is consistent with a lack of change in total plasma membrane area. Fluoroaluminate did not induce spreading, but it did suppress the K+ current. The differential effects of fluoroaluminate and guanosine 5'-O-(3-thiotriphosphate) suggest that lamellipod extension is regulated by a small molecular mass, monomeric G protein, whereas the inwardly rectifying K+ current is regulated by a large molecular mass, heterotrimeric G protein. Thus, osteoclast motility and ion transport are regulated by separate G protein-coupled pathways.

Actins↗
PubMed1993

Haemonchus contortus: ivermectin-induced paralysis of the pharynx.

How the avermectins cause the elimination of gastrointestinal nematodes from host animals has not yet been clearly identified. Using visual and radiometric parameters to measure oral ingestion in Haemonchus contortus, we showed that ivermectin (IVM) rapidly inhibited ingestion at concentrations > or = 10(-10) M. Motility, monitored quantitatively with an automated motility meter, was unaffected by IVM at concentrations < or = 10(-8) M, while ATP levels were unaffected at concentrations < or = 10(-6) M. Since motility and ATP levels, independent measures of short-term viability, are unaffected by concentrations of IVM that effectively block oral ingestion, the drug can be used as a chemical ligature. Although H. contortus was shown to be dependent upon an exogenous supply of glucose for survival in culture, IVM (10(-9) and 10(-7) M) altered neither the uptake of 3-O-[3H]methylglucose nor the metabolism of [13C]glucose by the parasite. These data suggest that H. contortus depends upon the transcuticular uptake of glucose in culture. If oral ingestion of other nutrients is essential for long-term survival in vivo, disruption of this process may represent the primary mechanism of IVM action.

3-O-Methylglucose↗
PubMed1993

Histamine activates Cl- and K+ currents in guinea-pig tracheal myocytes: convergence with muscarinic signalling pathway.

1. We investigated the effects of histamine on membrane currents and contractile state of isolated guinea-pig tracheal myocytes using perforated patch and whole-cell recording techniques. The effects of histamine were compared to those of acetylcholine (ACh) and caffeine. 2. During voltage clamp (Vhold = -60 mV), histamine elicited contraction and an inward current (Ihist) which was often followed by current oscillations. Ihist had a reversal potential (Vrev) of -9 +/- 3 mV. 3. Ihist was dependent on the Cl- gradient and was antagonized by the Cl- channel blocker niflumic acid. Vrev was more positive (+2 +/- 1 mV) when K(+)-selective currents were blocked by Cs+ and TEA. When all external Na+ was replaced with N-methyl-D-glucamine, there was a small reduction in the amplitude of Ihist. 4. The histamine-induced current was similar to that elicited by ACh and by caffeine with respect to time course, amplitude, and current-voltage relationship. Responses to histamine and to ACh were non-additive, consistent with a convergence of histaminergic and cholinergic signalling pathways. Ihist was antagonized by the H1 histaminergic receptor antagonist astemizole, but not by atropine. 5. When recorded using the perforated patch configuration, Ihist could be elicited repeatedly for more than 30 min. When cells were studied in the whole-cell configuration using a pipette solution containing 0.025 mM EGTA, the amplitude of Ihist was initially the same as that obtained using perforated patch but then decreased; the time required for the responses to decrease to 50% (t1/2) was 8.2 +/- 1.0 min. When 1 mM EGTA was included in the pipette solution (whole-cell configuration), the initial response to histamine was significantly decreased in size and t1/2 was reduced to 3.3 +/- 0.7 min. 6. The characteristics of the signalling pathway were examined in cells studied using the whole-cell configuration with 0.025 mM EGTA in the recording pipette. Heparin significantly reduced t1/2 to 4.3 +/- 0.8 min. GTP gamma S elicited inward current and oscillations; both effects were enhanced by histamine. GTP gamma S also reduced t1/2 to 1.4 +/- 0.1 min. Pertussis toxin did not alter the amplitude or time course of Ihist. 7. We conclude that in guinea-pig tracheal myocytes, binding of histamine to H1 receptors leads to release of Ca2+ from intracellular stores and subsequent activation of Cl- and K+ conductances as well as contraction. Furthermore, we demonstrate that ACh elicits similar physiological responses due to a convergence of the histaminergic and muscarinic signalling pathways.

Acetylcholine↗
PubMed1993

Muscarinic stimulation of tracheal smooth muscle cells activates large-conductance Ca(2+)-dependent K+ channel.

We investigated the regulation of the large-conductance Ca(2+)-dependent K+ (KCa) channel by acetylcholine (ACh) in freshly dissociated tracheal smooth muscle cells. Channels were recorded in the cell-attached patch configuration, and cells were stimulated with ACh, muscarine, or caffeine. We identified KCa channel activity based on 1) the voltage dependence of channel opening; 2) the large unitary conductance (242 +/- 5 pS with symmetrical 135 mM K+); 3) dependence of the reversal potential on the [K+] gradient, shifting 56 +/- 3 mV/10-fold change in extracellular [K+]; and 4) opening of channels after elevation of cytosolic free Ca2+ concentration ([Ca2+]i) using the Ca2+ ionophore A23187. When cells were bathed either in a physiological saline solution or a solution containing 135 mM K+ (to clamp cell membrane potential near 0 mV), ACh caused contraction of cells and activation of voltage-dependent channels. With 135 mM extracellular K+, the channels activated by ACh had a unitary conductance of 247 +/- 10 pS, and currents reversed near the K+ equilibrium potential (-1 +/- 1 mV). The effects of ACh were reversible, blocked by atropine, and mimicked by muscarine. From these characteristics we conclude that muscarinic stimulation of canine tracheal smooth muscle cells leads to activation of the large-conductance KCa channel. Because the KCa channels were isolated from ACh by the patch pipette, the increased channel activity was probably mediated by a cytosolic second messenger. ACh shifted the threshold for KCa channel opening to less positive membrane potentials, similar to that seen with elevation of [Ca2+]i.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗
PubMed1993

Emptying and refilling of Ca2+ store in tracheal myocytes as indicated by ACh-evoked currents and contraction.

Membrane currents and contractions evoked by acetylcholine (ACh) in freshly dissociated canine tracheal myocytes were investigated using the nystatin perforated-patch recording technique. In cells held at -60 mV in the presence of nifedipine, ACh evoked inward current (IACh) and contraction. Caffeine mimicked the effects of ACh. IACh and contractions could be evoked 3-4 min after removing external Ca2+ but were abolished by prolonged exposure to Ca(2+)-free media. Both responses were restored within minutes of reintroduction of Ca2+, even though the cells were held at -60 mV in the presence of nifedipine. IACh and ACh-evoked contractions were also reversibly abolished by continued exposure to caffeine. Cyclopiazonic acid (CPA), a blocker of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase, reduced IACh by > 95% within 15 min but had little or no effect on the contractile responses evoked by ACh. IACh was restored after washout of CPA even though cells were held at -60 mV. After depleting the Ca2+ store with the use of CPA, depolarization of the membrane to +10 mV immediately before application of ACh led to a partial restoration of IACh. This restorative effect of depolarization was potentiated by Bay K 8644 and antagonized by nifedipine. In conclusion, IACh and contractions in canine tracheal myocytes are mediated by Ca2+ released from an internal store that can be depleted by prolonged removal of extracellular Ca2+, prolonged exposure to caffeine, or by blockade of the SR Ca(2+)-ATPase. At least two Ca2+ influx pathways appear to contribute to refilling of the internal store: one pathway that is not activated by depolarization or ACh and a second involving dihydropyridine-sensitive voltage-activated Ca2+ channels that may be in direct contact with the SR (i.e., conduct extracellular Ca2+ directly into the SR, bypassing the cytosol).

Acetylcholine↗
PubMed1993

Platelet-activating factor stimulates resorption by rabbit osteoclasts in vitro.

We have shown previously that platelet-activating factor (PAF), a potent inflammatory mediator, acts directly on isolated rat osteoclasts to elevate cytosolic free Ca2+ concentration ([Ca2+]i). The purpose of this study was to examine the effects of PAF on osteoclast function. Osteoclasts were isolated from the long bones of neonatal rabbits and studied in three ways. [Ca2+]i of fura-2-loaded osteoclasts was monitored by microspectrofluorimetry. In 9 out of 16 cells tested, PAF (10-100 nM) caused elevation of [Ca2+]i that peaked then returned to baseline. In contrast, the biologically inactive precursor and metabolite of PAF, lyso-PAF, was without effect. Using time-lapse videomicroscopy, we found that PAF elicited retraction of peripheral pseudopods. Although calcitonin induced sustained retraction and immobility, the response to PAF was transient and, within 30 min, pseudopods reformed. To assess effects of PAF on resorptive activity, osteoclasts were cultured on dentin slices for 48 h in the presence of vehicle, PAF (200 nM), or calcitonin (100 ng/ml). PAF increased the area of individual resorption pits (from control values of 1,660 +/- 110 to 2,240 +/- 200 microns2, P < 0.05) and the total planar area resorbed per unit area of substrate (from 7.6 +/- 1.6 to 14.5 +/- 3.1 x 10(4) microns2/cm2, P < 0.025). As expected, calcitonin significantly decreased resorptive activity. These data indicate that PAF activates osteoclastic resorption. PAF may play a role in mediating the resorption of bone and mineralized cartilage in inflammatory diseases such as rheumatoid arthritis and periodontitis.

Animals↗
PubMed1992

Inwardly rectifying potassium current in rabbit osteoclasts: a whole-cell and single-channel study.

Ionic conductances of rabbit osteoclasts were investigated using both whole-cell and cell-attached configurations of the patch-clamp recording technique. The predominant conductance found in these cells was an inwardly rectifying K+ conductance. Whole-cell currents showed an N-shaped current-voltage (I-V) relation with inward current activated at potentials negative to EK. When external K+ was varied, I-V curves shifted 53 mV/10-fold change in [K+]out, as predicted for a K(+)-selective channel. Inward current was blocked by Ba2+ and showed a time-dependent decline at negative potentials, which was reduced in Na(+)-free external solution. Inward single-channel currents were recorded in the cell-attached configuration. Single-channel currents were identified as inward-rectifier K+ channels based on the following observations: (i) Unitary I-V relations rectified, with only inward current resolved. (ii) Unitary conductance (gamma) was 31 pS when recorded in the cell-attached configuration with 140 mM K+ in the pipette and was found to be dependent on [K+]. (iii) Addition of Ba2+ to the pipette solution abolished single-channel events. We conclude that rabbit osteoclasts possess inwardly rectifying K+ channels which give rise to the inward current recorded at negative potentials in the whole-cell configuration. This inwardly rectifying K+ current may be responsible for setting the resting membrane potential and for dissipating electrical potential differences which arise from electrogenic transport of protons across the osteoclast ruffled border.

Animals↗
PubMed1992

Mechanisms of microenvironmental pH regulation in the cuticle of Ascaris suum.

The excretion kinetics of various organic acids by Ascaris suum were quantified to determine if the excretion of these metabolic end-products could generate and maintain a microclimate pH within the aqueous compartment of the cuticle. Ligated and nonligated A. suum were incubated in media buffered with 0.25 or 2.5 mM Hepes (initial pH 7.5) or 0.5 or 5 mM glycine (initial pH 3.25). The concentration of organic acids and the pH of the media were followed for 24 h. Several volatile fatty acids, including acetic, 2-methylbutyric, 2-methylvaleric, n-valeric, and n-butyric, were excreted at relatively high rates. Propionic, n-caproic, 2-methylcaproic, tiglic acid, and the non-volatile organic acids, lactic and succinic, were excreted more slowly. The organic acids were excreted at a constant rate and in apparently fixed molar concentration ratios. The accumulation of organic acids was associated with changes in pH of the medium until a limiting constant pH, in the vicinity of the pKa of the volatile fatty acids, was reached. The rate of organic acid excretion was not affected by initial medium pH, buffer capacity, or parasite ligation. The rate of pH change induced by the excretion of organic acids was also insensitive to whether ligated or nonligated A. suum were used, but was dependent on the initial buffer capacity of the medium. These results suggest that A. suum excrete the end-products of carbohydrate metabolism across the cuticle. The presence of organic acids in the aqueous pores of the cuticle creates and maintains a microclimate pH of about 5.0 +/- 0.3. This pH will influence the transport properties of weak acids and bases and should be considered in the design of delivery systems for anthelmintics.

Acids↗
PubMed1992

Mechanistic studies in the transcuticular delivery of antiparasitic drugs. II: Ex vivo/in vitro correlation of solute transport by Ascaris suum.

Using live, intact Ascaris suum and a closed perfusion system, the absorption kinetics and tissue distribution of selected radiolabeled permeants were measured to determine the importance of the transcuticular pathway for drug absorption. The data support the conclusions established by previous in vitro transport studies which utilized excised cuticle-hypocuticle tissue preparations. The external surface of A. suum can be breached by drugs and the rate-determining barrier is the lipoidal hypocuticle tissue, provided the permeant is sufficiently small to traverse the aqueous-filled, negatively charged collagen matrix of the cuticle. The ex vivo permeability coefficients of the model permeants for the cuticle-hypocuticle barrier were in good quantitative agreement with the in vitro permeability coefficients. The lipophilic permeants hydrocortisone and p-nitrophenol were preferentially distributed in the gut tissue, whereas the hydrophilic permeant urea was distributed evenly throughout the organism and was extensively metabolized. Ligated and nonligated A. suum showed no significant differences in either uptake kinetics or tissue distribution of the permeants. This indicates that the transcuticular pathway is the major route of drug absorption as compared to oral ingestion.

Absorption↗
PubMed1992

Skin alteration and convective solvent flow effects during iontophoresis. II. Monovalent anion and cation transport across human skin.

Total flux enhancement of ions during iontophoresis is due primarily to the electrochemical potential gradient. However, secondary effects such as convective solvent flow and, in biological membranes, permeability increases as a result of applied field may also contribute to flux enhancement. The modified Nernst-Planck theory includes a solvent flow velocity term and predicts that the flux of uncharged molecules is enhanced or retarded depending on the polarity of the applied field. Polarity-dependent solvent flow velocity, as measured by the flux enhancement of mannitol, has been demonstrated in human epidermal membrane during iontophoresis. In the present study, the solvent flow velocity effects on the flux enhancement of a model cation (tetraethylammonium ion) and a model anion (salicylate ion) across human epidermal membrane were examined. The contribution of membrane alterations, due to the applied field, on overall ion flux was also considered. Solvent flow was found to have a small effect on the flux enhancement of both ions. However, membrane alterations were found to increase greatly the flux of the ionic species. Alterations in the epidermal membrane occurred at the highest voltage investigated (1000 mV) and appeared to reverse over time as indicated by the current and transport data.

Anions↗
PubMed1992

Cholinergic activation of a non-selective cation current in canine gastric smooth muscle is associated with contraction.

1. The effects of acetylcholine on membrane electrical properties of single smooth muscle cells dissociated from the circular layer of the canine gastric corpus were investigated using the nystatin perforated patch technique. Cells retained their ability to contract during recording, making it possible to correlate changes in membrane potential and membrane currents with contractions. 2. Acetylcholine caused depolarization from -59 +/- 8 mV to -18 +/- 7 mV (means +/- S.D., n = 12) with no generation of action potentials. The depolarization was associated with a membrane conductance increase, consistent with acetylcholine activating an inward current. In addition, acetylcholine caused contraction of cells to 58% of initial length. Both depolarization and contraction were reversible and were antagonized by atropine. 3. Under voltage clamp, acetylcholine activated inward current associated with increased current noise. The current-voltage relationship of the acetylcholine-induced current was studied at steady-state voltages and with voltage ramp commands. The inward current was largest between -40 and -20 mV and reversed direction to outward close to 0 mV (reversal potential, Erev = +3 +/- 9 mV). Reduction of external Na+ concentration to 21 mM shifted Erev to -42 +/- 5 mV, as predicted for a non-selective cation current. The conductance activated by acetylcholine (gACh) increased sigmoidally with depolarization, with about 2.5 nS activated at 0 mV. 4. Cells consistently contracted upon stimulation with acetylcholine, even when studied under voltage clamp at potentials as negative as -100 mV. This was consistent with muscarinic receptor activation causing release of Ca2+ from internal stores. When cells were bathed in Ca(2+)-free solutions, the first application of acetylcholine elicited normal inward current and contraction. Thereafter, both inward current and contractions were greatly diminished or absent, suggesting that the stores of Ca2+ had been depleted. 5. Caffeine caused reversible contraction and activation of inward current similar to that elicited by acetylcholine. 6. It is concluded that muscarinic stimulation of canine gastric smooth muscle cells involves activation of a non-selective cation conductance and is consistently accompanied by contraction. The release of Ca2+ from internal stores may be a common trigger for both events.

Acetylcholine↗
PubMed1992

Acetylcholine activates non-selective cation and chloride conductances in canine and guinea-pig tracheal myocytes.

1. Membrane currents activated by acetylcholine (ACh) were investigated in isolated canine and guinea-pig tracheal myocytes using the nystatin perforated patch configuration of whole-cell recording. ACh caused depolarization accompanied by a membrane conductance increase. 2. When cells were held under voltage clamp (holding potential, Vh = -60 mV), ACh elicited inward current (IACh) of up to 3900 pA, with a reversal potential (Erev) of approximately -20 mV. 3. Removal of extracellular Na+ (Na+o) reduced but did not eliminate IACh. IACh remaining in the absence of Na+ reversed direction close to the predicted equilibrium potential for Cl-. Erev shifted 32 +/- 4 mV per 10-fold change of [Cl-]i. Increasing external [K+] caused Erev to shift in the positive direction. These results suggest that ACh activated chloride and non-selective cation conductances. 4. In the absence of Na+o, the Cl- channel blockers SITS or niflumic acid reversibly antagonized IACh. 5. Caffeine and ryanodine elicited currents both in the presence and absence of Na+o; these currents had a reversal potential similar to that of IACh. Caffeine applied before ACh occluded the response to ACh. 6. We also observed two types of spontaneous membrane currents. Spontaneous transient outward currents (STOCs) may represent Ca(2+)-activated K+ currents. Spontaneous inward currents were also observed which were reduced in magnitude (but not eliminated) by removal of Na+o and reversed direction at approximately the Cl- equilibrium potential. The spontaneous inward currents and STOCs were coincident and were reversibly suppressed by ACh. 7. ACh elicited contractions of cells under voltage clamp at -60 mV, an effect also observed in the absence of extracellular Ca2+ or when IACh was reduced by omission of Na+o and exposure to Cl- channel blockers. The number of cells which did contract in response to ACh decreased, however, when the concentration of internal Cl- decreased. 8. All effects of ACh on contraction and membrane currents were antagonized by atropine. 9. We conclude that activation of muscarinic receptors in mammalian tracheal myocytes causes release of Ca2+ from intracellular stores and subsequent activation of Cl- and non-selective cation conductances. This is the first direct demonstration of these conductances in tracheal smooth muscle cells. Activation of these conductances does not appear to be required for contraction. However, regulation of cytosolic Cl- levels may be important for release and uptake of Ca2+ from internal stores.

Acetylcholine↗
PubMed1992

Substrate influences rat osteoclast morphology and expression of potassium conductances.

1. We studied the electrophysiological properties of freshly isolated rat osteoclasts using the whole-cell configuration of the patch-clamp technique. Membrane currents were recorded from cells plated on three substates: dentine, type I collagen and glass. 2. Based on their morphology, we defined two categories of osteoclasts. 'Rounded' osteoclasts were dome-shaped and lacked lamellipodia. 'Spread' osteoclasts were flattened and had lamellipodia. The proportion of 'rounded' osteoclasts was significantly greater when cells were plated on dentine or type I collagen than when cells were plated on glass. 3. 'Spread' osteoclasts expressed an inwardly rectifying K+ conductance regardless of the substrate on which they were plated. 4. 'Rounded' osteoclasts, on all substrates, expressed a transient, outwardly rectifying conductance that was selective for K+ based on: reversal of deactivation tail currents at -74 mV; a 60 mV shift in tail current reversal potential for 10-fold change in [K+]o; and blockade of outward current by extracellular 4-aminopyridine, charybdotoxin, and intracellular Cs+. The outward K+ current had an activation threshold of approximately -50 mV, with half-activation at -29 mV. The current also exhibited voltage-dependent inactivation, with half-inactivation at approximately -40 mV. 5. Outward K+ current in 'rounded' osteoclasts was reduced when extracellular Ca2+ was removed and upon addition of Ni2+, but was unaffected by Cd2+ or nifedipine. 6. 'Rounded' osteoclasts had large whole-cell capacitance for their apparent surface area. Capacitance was positively correlated with K+ conductance. The additional surface membrane we detected through capacitance measurements may be the 'ruffled border' of actively resorbing osteoclasts. 7. We conclude that substrate influences the expression of osteoclast phenotype, as defined by morphology and K+ conductances. 'Rounded' osteoclasts express an outwardly rectifying K+ conductance, with no apparent inwardly rectifying K+ conductance. In contrast, 'spread' osteoclasts exhibit an inwardly rectifying K+ conductance with no outwardly rectifying K+ conductance. The 'spread' phenotype may represent a motile phase, while the 'rounded' phenotype may represent a resorptive phase of osteoclastic activity.

Animals↗
PubMed1992

Role for diacylglycerol in mediating the actions of ACh on M-current in gastric smooth muscle cells.

The role of the second messenger diacylglycerol (DAG) in mediating muscarinic suppression of M-current, a type of a voltage-gated K+ current that is suppressed by acetylcholine (ACh), was examined in freshly isolated smooth muscle cells from toad stomach. Currents were recorded using a single electrode voltage clamp employing conventional microelectrodes. Extracellular application of 1,2-dioctanoyl-sn-glycerol (DiC8), a synthetic DAG that is a potent activator of protein kinase C (PKC), reversibly suppressed M-current. Current relaxations, representing the voltage-dependent closure of K+ channels underlying M-current, were also decreased by DiC8, although suppression was not always as complete as it was with ACh. In contrast, another DAG analogue, 1,2-dioctanoyl-3-thioglycerol, which has a structure closely related to DiC8 but does not activate PKC, failed to inhibit M-current. Furthermore, M-current induced by the beta-agonist isoproterenol, by a mechanism apparently mediated by adenosine 3',5'-cyclic monophosphate (S. M. Sims, L. H. Clapp, J. V. Walsh, Jr., and J. J. Singer. Pflugers Arch. 417: 291, 1990), was also suppressed by DiC8. Both ACh and DiC8 were found to suppress endogenous and isoproterenol-induced M-current without altering the time course of M-current deactivation, suggesting that these agents act by decreasing the number of channels available to be opened. These results provide evidence that muscarinic regulation of M-current is mediated by DAG.

Acetylcholine↗
PubMed1992

Calcium and potassium currents in canine gastric smooth muscle cells.

Membrane ionic currents were recorded in single smooth muscle cells dissociated from circular muscle of dog stomach (corpus region). When studied under voltage clamp with K+ in the patch electrode, depolarization to potentials more positive than -40 mV, from a holding potential of -70 or -80 mV, evoked transient inward current followed by outward current. Evidence that the outward current was due to K+ came from analysis of deactivation tail currents, which reversed direction close to the K+ equilibrium potential. In addition, the outward current was reduced by tetraethylammonium (TEA, 1-5 mM) applied to the external surface of cells. The Ca(2+)-channel blocker Cd2+ blocked the inward current and also reduced outward current, suggesting Ca(2+)-activated K+ current contributed to the outward current. The voltage-activated inward current was studied in isolation with Cs+ and TEA in the recording electrode to block K+ current. In standard bathing solution containing 2.5 mM Ca2+, the inward current activated between -50 and -40 mV, with peak inward current at +10 mV. The depolarization-activated inward current was blocked by nifedipine and enhanced by BAY K 8644, providing evidence that it was Ca2+ current. The Ca2+ current showed transient and sustained components, both of which showed similar voltage activation and inactivation ranges. The half-inactivation potential was approximately -37 mV. These results provide evidence that smooth muscle cells from the canine gastric corpus possess K+ and Ca2+ channels. Based on the voltage dependence of activation and inactivation and sensitivity to dihydropyridines, L-type Ca2+ channels predominate in canine gastric corpus smooth muscle.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗