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Biomedical subjects

J J Singer

Publications and source records attributed to J J Singer.

At least 37 records · Page 2Linked to original sources
PubMed1991

Direct regulation of ion channels by fatty acids.

A variety of fatty acids regulate the activity of specific ion channels by mechanisms not involving the enzymatic pathways that convert arachidonic acid to oxygenated metabolites. Furthermore, these actions of fatty acids occur in patches of membrane excised from the cell and are not mediated by cellular signal transduction pathways that require soluble factors such as nucleotides and calcium. Thus, fatty acids themselves appear to regulate the action of channels directly, much as they regulate the action of several purified enzymes, and might constitute a new class of first or second messengers acting on ion channels.

Animals↗
PubMed1990

Dual regulation of M current in gastric smooth muscle cells: beta-adrenergic-muscarinic antagonism.

The effects of the beta-adrenergic agent isoproterenol on membrane currents were studied in freshly dissociated gastric smooth muscle cells of Bufo marinus. Voltage-clamp experiments were carried out with patch pipettes in the tight-seal, whole-cell recording mode or with conventional microelectrodes. Isoproterenol induced a current identified as M current by the following criteria: the induced current is outward and carried by K+ ions, is suppressed by muscarine or acetylcholine, remains steadily activated, turns off with hyperpolarization, and exhibits slow relaxations in response to voltage jumps. In contrast to endogenous M current, isoproterenol-induced M current usually exhibited slower relaxations on hyperpolarizing voltage commands and displayed a steady-state conductance/voltage relationship that was shifted in the negative direction along the voltage axis. M current was also induced by either forskolin or phosphodiesterase-resistant cAMP analogs. In all cases, muscarinic agonists suppressed the M current, apparently by acting at a locus downstream from regulation of cAMP levels by adenylate cyclase and phosphodiesterase. beta-Adrenergic agents may act to increase the number of M channels available to be opened and also modify their kinetics.

Acetylcholine↗
PubMed1990

Membrane currents and cholinergic regulation of K+ current in esophageal smooth muscle cells.

The tight-seal whole cell recording technique with patch pipettes was used to study membrane currents of smooth muscle cells freshly dissociated from the esophagus of cats. Under voltage clamp with K+ in the pipette, depolarizing commands elicited an initial inward current followed by a transient outward current that peaked and then declined to reveal spontaneous outward currents (SOCs). SOCs were evident at -60 mV and more positive potentials. The reversal of SOCs at the K+ equilibrium potential and their suppression by tetraethylammonium chloride lead to the conclusion that they represent the activity of K+ channels. Acetylcholine (ACh) caused reversible contraction of these cells and had two successive effects on membrane currents, causing transient activation of K+ current followed by suppression of SOCs. Both of these effects were blocked by atropine. Consistent with these observations, in current clamp, ACh caused a transient hyperpolarization followed by depolarization. The inward current activated by depolarization was blocked by external Cd2+, consistent with the inward current being a voltage-activated calcium current. Two types of Ca2+ current could be distinguished on the basis of voltage-activation range, time course of inactivation and "run-down" during whole cell recording.

Acetylcholine↗
PubMed1989

Arachidonic acid and other fatty acids directly activate potassium channels in smooth muscle cells.

Arachidonic acid, as well as fatty acids that are not substrates for cyclooxygenase and lipoxygenase enzymes, activated a specific type of potassium channel in freshly dissociated smooth muscle cells. Activation occurred in excised membrane patches in the absence of calcium and all nucleotides. Therefore signal transduction pathways that require such soluble factors, including the NADPH-dependent cytochrome P450 pathway, do not mediate the response. Thus, fatty acids directly activate potassium channels and so may constitute a class of signal molecules that regulate ion channels.

Animals↗
PubMed1989

Regulation of calcium concentration in voltage-clamped smooth muscle cells.

The regulation of intracellular calcium concentration in single smooth muscle cells was investigated by simultaneously monitoring electrical events at the surface membrane and calcium concentration in the cytosol. Cytosolic calcium concentration rose rapidly during an action potential or during a voltage-clamp pulse that elicited calcium current; a train of voltage-clamp pulses caused further increases in the calcium concentration up to a limit of approximately 1 microM. The decline of the calcium concentration back to resting levels occurred at rates that varied with the calcium concentration in an apparently saturable manner. Moreover, the rate of decline at any given calcium concentration was enhanced after a higher, more prolonged increase of calcium. The process responsible for this enhancement persisted for many seconds after the calcium concentration returned to resting levels. Thus, the magnitude and duration of a calcium transient appear to regulate the subsequent calcium removal.

Action Potentials↗
PubMed1989

Substance P, like acetylcholine, augments one type of Ca2+ current in isolated smooth muscle cells.

Electrophysiological recordings from freshly-dissociated smooth muscle cells from toad stomach revealed that substance P enhances one of two types of Ca2+ currents. That is, substance P enhances the slowly inactivating, high-threshold current but not the fast inactivating, low-threshold current. Acetylcholine has the same effect, but the acetylcholine action is blocked by atropine whereas the substance P action is not, indicating that the two agents act at different receptor sites. Thus, substance P, like acetylcholine, has a dual excitatory action on the smooth muscle cells employed in these studies, enhancing a specific type of Ca2+ current, as demonstrated here, and suppressing a voltage-sensitive K+ conductance, as previously described [Sims, S.M., Walsh, J.V., Jr. & Singer, J.J. (1986) Am. J. Physiol. 251, C580-C587].

Acetylcholine↗
PubMed1988

Antagonistic adrenergic-muscarinic regulation of M current in smooth muscle cells.

The beta-adrenergic agonist isoproterenol and analogs of adenosine 3',5'-monophosphate (cAMP) induced a potassium current, M current, in freshly dissociated gastric smooth muscle cells. Muscarinic agonists suppress this current, apparently by acting at a locus downstream from regulation of cAMP levels by adenylate cyclase and phosphodiesterase. Thus, M current can be induced by an agent and regulated in antagonistic fashion by beta-adrenergic and muscarinic systems.

Acetylcholine↗
PubMed1988

Stretch-activated ion channels in smooth muscle: a mechanism for the initiation of stretch-induced contraction.

As in many smooth muscle tissue preparations, single smooth muscle cells freshly dissociated from the stomach of the toad Bufo marinus contract when stretched. Stretch-activated channels have been identified in these cells using patch-clamp techniques. In both cell-attached and excised inside-out patches, the probability of the channel being open (Po) increases when the membrane is stretched by applying negative pressure to the extracellular surface through the patch pipette. The increase in Po is mainly due to a decrease in closed time durations, but an increase in open time duration is also seen. The open-channel current-voltage relationship shows inward rectification and is not appreciably altered when K+ is substituted for Na+ as the charge-carrying cation in Ca2+-free (2 mM EGTA) pipette solutions bathing the extracellular surface of the patch. The inclusion of physiological concentrations of Ca2+ (1.8 mM) in pipette solutions (containing high concentrations of Na+ and low K+) significantly decreases the slope conductance as well as the unitary amplitude. The channel also conducts Ca2+, since inward currents were observed using pipette solutions in which Ca2+ ions were the only inorganic cations. When simulating normal physiological conditions, we find that substantial ionic current is conducted into the cell when the channel is open. These characteristics coupled with the high density of the stretch-activated channels point to a key role for them in the initiation of stretch-induced contraction.

Animals↗
PubMed1988

Regulation of one type of Ca2+ current in smooth muscle cells by diacylglycerol and acetylcholine.

Electrophysiological recordings from freshly dissociated smooth muscle cells from the stomach of the toad Bufo marinus revealed two types of Ca2+ currents. One has a low threshold of activation and inactivates rapidly; the other has a high threshold of activation and inactivates more slowly. Acetylcholine (ACh) increased the high-threshold current but not the low-threshold current. The synthetic diacylglycerol analog sn-1,2-dioctanoylglycerol, an activator of protein kinase C (PKC), mimicked these effects of ACh on Ca2+ currents. However, another diacylglycerol analog, 1,2-dioctanoyl-3-thioglycerol, which has a closely related structure but does not activate PKC, failed to increase the Ca2+ current. The same was true of 1,2-dioctanoyl-3-chloropropanediol, an analog that even at high concentrations only minimally activates PKC. These results suggest that diacylglycerol may be the second messenger mediating the effects of ACh on one type of voltage-activated Ca2+ channel, possibly by activating PKC.

Acetylcholine↗
PubMed1987

Identification and characterization of major ionic currents in isolated smooth muscle cells using the voltage-clamp technique.

Voltage-clamp experiments were carried out on freshly dissociated single vertebrate smooth muscle cells from the stomach muscularis of Bufo marinus. Conventional two-microelectrode methodology was used, thus avoiding rapid dialysis of the cytosol. Four major phases of current were identified upon voltage jumps from negative holding levels to more positive levels. The first phase of current was an initial, inward current. This current was blocked by external Mn2+ and was of the correct magnitude to account for the rising phase of the Ca2+-dependent, TTX-independent action potentials found in these cells. Following this initial, inward Ca2+ current, a large outward current was observed which reached its peak over a period of hundreds of milliseconds and then decayed over a period of seconds to a steady-state level. The peak outward current and the steady-state outward current constitute the second and third major currents. The peak outward current was the largest current observed, with a magnitude as large as tens of nanoamps whereas the inward current was at most about one nanoamp. The peak outward current was reduced more than tenfold in the presence of external TEA. It was also decreased or abolished when the preceding inward current was diminished or eliminated by using external Mn2+ or less negative holding potentials. In this way the peak outward current was identified as a Ca2+-activated K+ current whose slow decay was hypothesized to result from removal of internal Ca ions by cellular mechanisms following the initial rise in [Ca2+]i resulting from the inward current. A fourth major current was an early transient outward current observed most clearly upon voltage jumps to more positive potentials when the inward current was eliminated by using less negative holding potentials or external Mn2+. A classical steady-state inactivation relationship as a function of membrane potential was constructed for the inward current. A substantial portion of this inactivation curve lies at potentials negative to the apparent threshold for activation of inward current, suggesting a true voltage-dependent inactivation. Although additional Ca2+-dependent inactivation could not be ruled out, neither could evidence for it be found.

Action Potentials↗
PubMed1987

Characterization of calcium-activated potassium channels in single smooth muscle cells using the patch-clamp technique.

Single-channel currents were recorded with the patch-clamp technique from freshly dissociated vertebrate smooth muscle cells from the stomach of Bufo marinus. Of the variety of channels observed, one displayed a large linear conductance of 250 pS (in symmetric 130 mM KCl) which in excised patches was shown to be highly K+ selective. The probability of the channel being open (Po) increased when [Ca2+]i was elevated and/or when the membrane potential was made more positive. Thus, the features of this channel resemble the large-conductance Ca2+-activated K+ channel found in a wide variety of cell types. The voltage sensitivity of the channel was studied in detail. For patches containing a single large-conductance channel a plot of Po versus membrane potential followed the Boltzmann relationship. Increasing [Ca2+]i shifted this plot to the left along the voltage axis to more negative potentials. Both the mean closed time and mean open time varied with potential as a single exponential with almost all of the voltage sensitivity of Po residing in the mean closed time. These results were verified with a series of experiments carried out at low Po (less than 0.1) in patches containing multiple (N) large-conductance channels. Here the ln (NPo) was a linear function of potential with an inverse slope of 9 mV. Almost all of the potential sensitivity lay in the mean closed time the natural log of which was also a linear function of potential with an inverse slope 11 mV in magnitude. The characteristics of this channel as well as the appearance of several of them in almost every patch suggest that they underlie the large peak outward macroscopic current found with whole-cell voltage-clamp studies.

Animals↗
PubMed1987

Acetylcholine increases voltage-activated Ca2+ current in freshly dissociated smooth muscle cells.

The regulation of voltage-activated Ca2+ current by acetylcholine was studied in single freshly dissociated smooth muscle cells from the stomach of the toad Bufo marinus by using the tight-seal whole-cell recording technique. Ca2+ currents were elicited by positive-going command pulses from a holding level near -80 mV in the presence of internal Cs+ to block outward K+ currents. Ca2+ current was greatest in magnitude at command potentials near 10 mV. At such command potentials, acetylcholine increased the magnitude of the inward current and slowed its decay. The effects of acetylcholine were seen in the absence of external Na+ or with low Cl- (aspartate replacement) in the bathing solution and could be mimicked by muscarine. The peak of the current-voltage relationship for the Ca2+ current was not discernibly shifted along the voltage axis by acetylcholine. These results demonstrate that activation of muscarinic receptors not only suppresses a K+ current (M-current), as we have previously demonstrated [Sims, S. M., Singer, J. J. & Walsh, J. V., Jr. (1985) J. Physiol. (London) 367, 503-529], but also increases the magnitude and slows the decay of Ca2+ current.

Acetylcholine↗
PubMed1986

An automated technique for analysis of current transitions in multilevel single-channel recordings.

Detailed kinetic studies of ion channel gating are best carried out using the patch-clamp technique which permits the measurement of the ionic current through individual channels. Typical patch-clamp recordings show the current signal, in the form of a sequence of rectangular pulses (analogous to a random telegraph signal), riding on slow baseline drift, partially obscured by high-frequency noise and distorted by filtering. In order to analyze such recordings, we have developed a set of interactive Pascal programs based on a feature-detection algorithm capable of identifying current transitions in multiple-channel recordings in the presence of substantial levels of noise and drift. Software operation is largely automated but includes provisions for examination and correction of the output. The software was optimized and systematically evaluated using simulated data with variable amounts of noise and drift. Results indicate that satisfactory performance is obtained for signal-to-noise ratio as low as four even with uncommonly large baseline drift. Steady-state processing speeds varied from 1,000 to 4,000 samples per second depending on data complexity.

Automation↗
PubMed1986

Multiple neuropeptides exert a direct effect on the same isolated single smooth muscle cell.

The contractile effect of various neuropeptides was examined by pressure ejecting these agents from a pipette onto single smooth muscle cells freshly dissociated from the stomach of Bufo marinus. Substance P, cholecystokinin-octapeptide, and bombesin caused contraction, whereas vasoactive intestinal peptide, secretin, and dopamine inhibited acetylcholine-induced contractions. Acetylcholine and the three peptides which produced contraction were found in some instances to act on the same cell, suggesting that receptors for these agents exist on one and the same cell.

Acetylcholine↗
PubMed1986

Substance P and acetylcholine both suppress the same K+ current in dissociated smooth muscle cells.

The effect of substance P on freshly dissociated gastric smooth muscle cells was examined electrophysiologically. Substance P caused depolarization, associated with a membrane conductance decrease, which led to the generation of action potentials and contraction. When the membrane potential was held constant under voltage clamp, substance P induced a net inward current, also associated with a conductance decrease. The net inward current resulted from suppression of an outward K+ current, one which resembled the acetylcholine-sensitive M-current in these cells. When substance P maximally suppressed this outward K+ current, acetylcholine (ACh) had no additional effect. Conversely, when ACh fully suppressed the M-current, substance P was without additional effect. These results indicate that substance P suppresses the same outward K+ current affected by ACh. Suppression of M-current by substance P was observed in approximately half (44 of 85) of the cells studied in these experiments. In those cells that did not respond to substance P, ACh was nevertheless capable of suppressing the M-current. Thus both substance P and cholinergic agonists appear to exert their excitatory effects on smooth muscle cells by inhibiting a common K+ current.

Acetylcholine↗
PubMed1986

Large-conductance Ca2+-activated K+ channels in freshly dissociated smooth muscle cells.

Freshly dissociated cells from the stomach muscularis of the toad Bufo marinus have been employed to carry out a systematic set of electrophysiological studies on the membrane properties of smooth muscle. The existence of Ca2+-activated K+ channels became apparent during the first studies under current clamp. In subsequent studies under voltage clamp, a Ca2+-activated. TEA-sensitive outward current was evident, and it was more than an order of magnitude larger than any other current observed in the cells. The channel responsible, at least in part, for this large outward current has been identified on the basis of single-channel records, and some of its main characteristics have been studied. It is similar in many respects to the large-conductance, Ca2+-activated K+ channel seen in other preparations. This channel has now been found in a considerable diversity of smooth muscle types.

Animals↗