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C J Lingle

Publications and source records attributed to C J Lingle.

At least 37 records · Page 2Linked to original sources
PubMed1996

[Ca2+]i elevations detected by BK channels during Ca2+ influx and muscarine-mediated release of Ca2+ from intracellular stores in rat chromaffin cells.

Submembrane [Ca2+]i changes were examined in rat chromaffin cells by monitoring the activity of an endogenous Ca(2+)-dependent protein: the large conductance Ca(2+)-and voltage-activated K+ channel (also known as the BK channel). The Ca2+ and voltage dependence of BK current inactivation and conductance were calibrated first by using defined [Ca2+]i salines. This information was used to examine submembrane [Ca2+]i elevations arising out of Ca2+ influx and muscarine-mediated release of Ca2+ from intracellular stores. During Ca2+ influx, some BK channels are exposed to [Ca2+]i of at least 60 microM. However, the distribution of this [Ca2+]i elevation is highly nonuniform so that the average [Ca2+]i detected when all BK channels are activated is only approximately 10 microM. Intracellular dialysis with 1 mM or higher EGTA spares only the BK channels activated by the highest [Ca2+]i during influx, whereas dialysis with 1 mM or higher BAPTA blocks activation of all BK channels. Submembrane [Ca2+]i elevations fall rapidly after termination of short (5 msec) Ca2+ influx steps but persist above 1 microM for several hundred milliseconds after termination of long (200 msec) influx steps. In contrast to influx, the submembrane [Ca2+]i elevations produced by release of intracellular Ca2+ by muscarinic actetylcholine receptor (mAChR) activation are much more uniform and reach peak levels of 3-5 microM. Our results suggest that during normal action potential activity only 10-20% of BK channels in each chromaffin cell see sufficient [Ca2+]i to be activated.

Animals↗
PubMed1996

Calcium-activated potassium channels in adrenal chromaffin cells.

Rat chromaffin cells express an interesting diversity of Ca(2+)-dependent K+ channels, including a voltage-independent, small-conductance, apamin-sensitive SK channel and two variants of voltage-dependent, large-conductance BK channels. The two BK channel variants are differentially segregated among chromaffin cells, such that BK current is completely inactivating in about 75-80% of rat chromaffin cells, while the remainder express a mix of inactivating and non-inactivating current or mostly non-inactivating BKs current. The single-channel conductance of BKi channels is identical to that of BKs channels. Although rates of current activation are similar in the two variants, the deactivation kinetics of the two channels also differ. Furthermore, BKi channels are somewhat less sensitive to scorpion toxins than BKs channels. The slow component of BKi channel deactivation may be an important determinant of the functional role of these channels. During blockade of SK current, cells with BKi current fire tonically during sustained depolarizing current injection, whereas cells with BKs current tend to fire only a few action potentials before becoming quiescent. The ability to repetitively fire requires functional BKi channels, since partial blockade of BKi channels by CTX makes a BKi cell behave much like a BKs cell. In contrast, the physiological significance of BKi inactivation may arise from the ability of secretagogue-induced [Ca2+]i elevations to regulate the availability of BKi channels during subsequent action potentials (Herrington et al., 1995). By reducing the number of BK channels available for repolarization, the time course of action potentials may be prolonged. This possibility remains to be tested directly. These results raise a number of interesting questions pertinent to the control of secretion in rat adrenal chromaffin cells. An interesting hypothesis is that cells with a particular kind of BK current may reflect particular subpopulations of chromaffin cells. These subpopulations might differ either in the nature of the material secreted from the cell (e.g., Douglass and Poisner, 1965) or in the responsiveness to particular secretagogues. The differences in electrical behavior between cells with BKi and BKs current suggest that the pattern of secretion that might be elicited by a single type of stimulus could differ. For BKi cells, secretion may occur in a tonic fashion during sustained depolarization, while secretion from cells with BKs current may be more phasic. In the absence of specific structural information about the domains responsible for inactivation of BKi channels, our understanding of the mechanism of inactivation remains indirect. BKi inactivation shares many features with N-terminal inactivation of voltage-dependent K+ channels. However, there are provocative differences between the two types of inactivation which require us to propose that the native inactivation domain of BKi channels may occlude access of permeant ions to the BK channel permeation pathway in a position at some distance from the actual mouth of the channel. Further understanding of the structural and mechanistic basis of inactivation of BKi channels promises to provide new insights into both the cytoplasmic topology of BK channels and the Ca(2+)- and voltage-dependent steps involved in channel activation.

Animals↗
PubMed1995

The suppression of Ca(2+)- and voltage-dependent outward K+ current during mAChR activation in rat adrenal chromaffin cells.

1. The mechanism by which muscarine, ionomycin or caffeine results in suppression of Ca(2+)- and voltage-dependent outward current in rat adrenal chromaffin cells was evaluated using both whole-cell voltage clamp and single channel recording. 2. The whole-cell current activated following the elevation of the cytosolic calcium concentration ([Ca2+]i) by muscarine inactivates with a time course comparable to that of single Ca(2+)- and voltage-dependent potassium (BK) channels. 3. The whole-cell inactivating current is pharmacologically similar to BK current. 4. The voltage dependence of inactivation and rate of recovery from inactivation are qualitatively similar for both whole-cell current and ensemble averages of single BK channels. Furthermore, changes in the rate of whole-cell current inactivation track expected changes in submembrane [Ca2+]. 5. The suppression of outward current can be accounted for solely by inactivation of BK channels and does not depend on the means by which [Ca2+]i is elevated. 6. Muscarinic acetylcholine receptor (mAChR) activation, changes in holding potential (-50 to -20 mV), and step depolarizations of different amplitude and duration were tested for their ability to elevate [Ca2+]i and thereby regulate the availability of BK current for activation. 7. Following muscarine-induced elevation of [Ca2+]i at holding potentials positive to -40 mV, the availability of BK current for activation was typically reduced by more than 50%. 8. Holding potentials in the range of -50 to -20 mV produced only slight alterations in the availability of BK current for activation. 9. Step depolarizations that cause maximal rates of Ca2+ influx (0 to +10 mV) must exceed 200 ms to reduce the availability of BK current by approximately 50%. 10. The results show that the muscarine-induced elevation of [Ca2+]i produces a profound reduction in the availability of BK channels for activation at membrane potentials likely to be physiologically meaningful. Although depolarization- induced Ca2+ influx can inactivate BK current, we propose that short duration depolarizations that occur during normal electrical activity will not significantly alter BK channel availability.

Action Potentials↗
PubMed1995

A human calcium-activated potassium channel gene expressed in vascular smooth muscle.

Large-conductance Ca(2+)-activated K+ (BK) channels are widespread and functionally heterogeneous. In other classes of K+ channels, functional heterogeneity derives from large gene families, alternative splicing, heterologous subunit composition, and functional modulation. The molecular basis of mammalian BK channel heterogeneity is unknown, since only a single gene (mSlo) has been identified. BK channels in native vascular smooth muscle have an apparent Ca2+ sensitivity approximately 10-fold greater than native brain or skeletal muscle channels, or cloned mSlo channels. Using mSlo as a low-stringency probe, we screened human arterial smooth muscle and genomic libraries extensively in search of genes or splice variants with novel properties. We isolated the human homologue of mSlo, including two novel splice variant forms, but found no other related genes. Electrophysiological characterization of the hSlo clones in Xenopus oocytes and Chinese hamster ovary cells gave BK currents that were not measurably different from mSlo currents. However, coexpression of hSlo with a recently cloned beta-subunit derived from smooth muscle dramatically increased apparent Ca2+ sensitivity. Thus alpha-subunits alone may not determine Ca2+ sensitivity of vascular smooth muscle BK channels. hSlo was mapped to human chromosome 10q23.1, and the genomic structure was analyzed. Immediately after the amino terminal, two unusual regions of trinucleotide repeating sequences are present. The first of these regions encodes polyglycine, and the second encodes polyserine. Both regions of repeated sequence are conserved between the mouse and human genome.

Amino Acid Sequence↗
PubMed1995

Inactivating and noninactivating Ca(2+)- and voltage-dependent K+ current in rat adrenal chromaffin cells.

The properties of Ca(2+)- and voltage-dependent K+ currents and their role in defining membrane potential were studied in cultured rat chromaffin cells. Two variants of large-conductance, Ca2+ and voltage-dependent BK channels, one noninactivating and one inactivating, were largely segregated among patches. Whole-cell noninactivating and inactivating currents resulting from each of these channels were segregated among different chromaffin cells. Cell-to-cell variation in the rate and extent of whole-cell current decay was not explained by differences in cytosolic [Ca2+] regulation among cells; rather, variation was due to differences in the intrinsic properties of the underlying BK channels. About 75% of rat chromaffin cells and patches express inactivating BK current (termed BKi) while the remainder express noninactivating BK current (termed BKs). The activation time course of both currents is similar, as is the dependence of activation on [Ca2+] and membrane potential. However, deactivation of BKi channels is slower than that of BKs channels. The functional role of these BK channel variants was studied in current-clamp recordings. Although both BKi and BKs currents contribute to action potential repolarization, cells expressing BKi current are better able to fire repetitively in response to constant current injection. Blockade of BKi current by charybdotoxin abolishes this behavior, showing that afterhyperpolarizations mediated by BKi current are permissive for repetitive firing. Thus, important properties of chromaffin cell membrane excitability are determined by the type of BK current expressed.

Adrenal Glands↗
PubMed1994

Multiple components of voltage-dependent potassium current in normal rat anterior pituitary cells.

1. Voltage-dependent K+ currents were studied in normal rat anterior pituitary cells using the patch-clamp technique. To obtain cultures enriched for lactotrophs, density gradient centrifugation was performed on pituitary cells isolated from lactating rats. 2. Depolarizations to about -30 mV from a holding potential of -80 mV activate a rapidly inactivating [time constant (tau) approximately 15-20 ms at -20 mV]K+ current. This transient current activated at low voltages (termed IA) is abolished by 5 mM external 4-aminopyridine (4-AP) but is largely resistant to external tetraethylammonium (TEA) (< or = 30 mM). 3. Recovery from inactivation of IA is fast, with a tau of 100-200 ms at -80 mV. Deactivation is also fast (tau approximately 2.2 ms at -50 mV). The voltage of half-activation of IA is approximately -20 mV. The current is completely inactivated at a holding potential of -40 mV. 4. Voltage-dependent K+ current activated by depolarizations from a holding potential of -40 mV was first detectable at about -20 mV (high voltage-activated) and had a time course that varied among cells. 5. Deactivation of high voltage-activated K+ current was best described by the sum of two exponentials, with tau of about 3.7 and 30 ms at -50 mV. Both components reversed close to the equilibrium potential for K+. 6. The amplitudes of the two tail currents were independent of each other when variable-duration commands were used to activate current. The amplitude of the fast component was largest with 10- to 20-ms commands to +40 mV and was reduced (< or = 50%) with 136-ms commands. The slow component amplitude reached a peak by 40 ms and remained constant for commands < or = 136 ms at +40 mV. 7. The contribution of each component to the total high voltage-activated tail current was variable among cells, with the amount of fast component correlating with the amount of inactivation produced by commands to +40 mV. 8. The two components of tail current activated by depolarizations from the -40 mV holding potential were abolished by external TEA (10 mM). 4-AP (5 mM externally) selectively abolished the fast component of high voltage-activated tail current while only partially reducing the slow component. 9. These results suggest that normal rat anterior pituitary cells possess at least three distinct types of voltage-dependent K+ current: a low voltage-activated, transient current (IA) and two high voltage-activated currents.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

Activation of nicotinic acetylcholine receptors on cultured Drosophila and other insect neurones.

1. Using whole-cell and single channel recordings, we have examined the properties of acetylcholine (ACh)-activated currents in neurones from larval and pupal Drosophila melanogaster (fruit fly), larval and embryonic Musca domestica (house fly), and nymphal Schistocerca gregaria (locust). 2. In all preparations, single channel recordings revealed two major classes of ACh-activated channels, with average conductances of approximately 32 and 59 pS. 3. At ACh concentrations from 1 to 10 microM, channel activity in Drosophila larval neurones occurs in bursts with an average of 1-2 openings. Open times and burst durations are described by one or two exponentials. Burst durations for the 32 pS channel (approximately 3 ms, slow component) were longer than those for the 59 pS channel (approximately 1.0 ms). The mean open interval duration for the 32 pS channel (slow component) was also longer than that of the 59 pS channel. 4. At high ACh (20-200 microM) concentrations, bursts of the smaller conductance channel occur in clusters separated by long-lived periods without channel activity. Considerable kinetic heterogeneity was observed among clusters. 5. The whole-cell dose-response curve suggests that activation of current by ACh increases up to at least 100 microM and that multiple ligand binding steps are involved. 6. Drosophila and Musca larval neuronal ACh-activated channels show some unique features in their cholinergic pharmacological properties: (a) they are only weakly activated by the potent neuromuscular nicotinic agonist suberyldicholine, (b) hexamethonium and decamethonium are weak, but approximately equi-effective blockers, and (c) alpha- and kappa-bungarotoxin (BTX) both blocked reversibly, though alpha-BTX appears to be the more potent inhibitor.

Acetylcholine↗
PubMed1992

Trypsin-sensitive, rapid inactivation of a calcium-activated potassium channel.

Most calcium-activated potassium channels couple changes in intracellular calcium to membrane excitability by conducting a current with a probability that depends directly on submembrane calcium concentration. In rat adrenal chromaffin cells, however, a large conductance, voltage- and calcium-activated potassium channel (BK) undergoes rapid inactivation, suggesting that this channel has a physiological role different than that of other BK channels. The inactivation of the BK channel, like that of the voltage-gated Shaker B potassium channel, is removed by trypsin digestion and channels are blocked by the Shaker B amino-terminal inactivating domain. Thus, this BK channel shares functional and possibly structural homologies with other inactivating voltage-gated potassium channels.

Adrenal Glands↗
PubMed1992

Activation of skeletal muscle nicotinic acetylcholine receptors.

Work over the past ten years has greatly increased our understanding of both the structure and function of the muscle nicotinic acetylcholine receptor. There is a strongly supported general picture of how the receptor functions: agonist binds rapidly to sites of low affinity and channel opening occurs at a rate comparable to the agonist dissociation rate. Channel closing is slow, so the channel has a high probability of being open if both agonist-binding sites are occupied by ACh. Results of expression studies have shown that each subunit can influence AChR activation and have given a structural basis for the major physiological change known for muscle AChR, the developmental change in AChR activation. These general statements notwithstanding, there are still major areas of uncertainty which limit our understanding. We have emphasized these areas of uncertainty in this review, to indicate what needs to be done. First, the quantitative estimates of rate constants are not as strongly supported as they should be. The major reasons are twofold--uncertainties about the interpretation of components in the kinetic data and difficulties of resolving brief events. As a result, any inferences about the functional consequences of structural alterations must remain tenuous. Second, the functional behavior of individual AChRs is not as well understood as it should be. The kinetic behavior of an individual receptor clearly can be complex (section II). In addition, there is evidence that superimposed on this complexity there may be stable and kinetically distinguishable populations of receptors (section III). Until the basis for the kinetically defined populations is clarified, kinetic parameters for receptors of defined structure cannot be unambiguously obtained. Finally, it is not surprising that the studies of AChR of altered structure have not given definitive results. Two reasons should be apparent from the preceding points: there is not a fully supported approach for kinetic analysis, and the "normal" population may not be clearly defined. An additional complication is also emerging, in that the available data support the idea that specific residues distributed over all subunits may influence AChR activation. This possibility renders the task of analysis that much more difficult. The muscle nicotinic AChR has served as a prototype for the family of transmitter-gated membrane channels, which includes the muscle and neuronal nicotinic receptors, the GABAA, the glycine and possibly the non-NMDA excitatory amino acid receptor (Stroud et al., 1990). It is interesting to note that the functional properties of the GABAA receptor, probably the best-studied of the other members of the family are rather similar.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1992

Two components of calcium-activated potassium current in rat adrenal chromaffin cells.

1. The activation of calcium (Ca2+)-dependent potassium (K+) currents in dissociated rat adrenal chromaffin cells was investigated using the dialysed cell recording technique. 2. Ca(2+)-dependent K+ current was the major component of outward current at command potentials from -30 mV to about +50 mV. 3. Two components of Ca(2+)-dependent outward current could be distinguished based on the voltage dependence of activation, the properties of tail currents following repolarization, and pharmacological properties. 4. One Ca(2+)-dependent current was similar to an after-hyperpolarization current (often termed IAHP) observed in other cell types. This current was largely blocked by 200 nM-apamin or 200 microM-curare, was associated with slow Ca(2+)-dependent tail current, and exhibited little dependence on voltage. In cells with cytosolic Ca2+ buffered to 500 nM-1 microM, curare-sensitive current accounted for most of the membrane current at potentials negative to about -40 mV. 5. A second component of Ca(2+)-activated K+ current exhibited voltage-dependent activation, was completely blocked by 1 mM-TEA, and turned off rapidly following repolarization. An unusual aspect of the TEA-sensitive currents was that they appeared to inactivate under conditions of constant cytosolic Ca2+. 6. A novel observation during these experiments was a slow hump of outward current which appears to result from a non-monotonic elevation in cytosolic Ca2+ during prolonged voltage jumps.

Adrenal Glands↗
PubMed1992

Effects of muscarine on single rat adrenal chromaffin cells.

1. The action of muscarine on membrane currents and cytosolic calcium (Ca2+) of dissociated rat adrenal chromaffin cells was investigated using standard whole-cell voltage-clamp techniques and microfluorimetry of unclamped single cells. 2. In cells held at a constant holding potential negative to -40 mV, brief (5-10 s) applications of muscarine produced a transient activation of outward current. The activation of this current by muscarine also occurs in the presence of 5 mM-Co2+. 3. The outward current activated by muscarine at holding potentials negative to about -40 mV is blocked over 90% by either 200 microM-curare or 200 nM-apamin. One millimolar TEA produces variable blocking effects at such potentials. 4. The outward current activated by muscarine is transient even in the continuing presence of muscarine. Complete recovery between pairs of muscarine applications occurs over a 1-2 min period. If sufficient time was allowed for recovery between muscarine applications, the muscarine-activated outward current could be reliably elicited in dialysed cells for periods of 20-30 min. 5. Voltage ramps were used to examine effects of muscarine on currents over a range of membrane potentials. Over all potentials, muscarine activates a relatively voltage-independent component which is blocked almost completely by 200 nM-apamin and by 200 microM-curare. At potentials negative to about -40 mV, the apamin- and curare-sensitive current accounts for virtually all muscarine-activated current. This current appears to correspond to a Ca(2+)-activated, voltage-independent current found in these cells. Effects of muscarine on currents activated at potentials positive to 0 mV are complex. At potentials above 0 mV, muscarine can produce either an activation or an inhibition of outward current. The outward current activated at positive potentials was primarily voltage dependent and blocked by 1 mM-TEA. However, in some cells activation of voltage-dependent current was not observed and, in such cases, muscarine produced an inactivation of the voltage-dependent component of current. The inactivation of outward current could also be observed in the presence of 5 mM-Co2+ indicating that the inactivation does not occur secondarily to an effect of muscarine on Ca2+ current. The possibility is discussed that the inactivation of outward current occurs as a result of intrinsic inactivation properties of the voltage-dependent Ca(2+)-dependent K+ current. According to this hypothesis, the extent to which inactivation of voltage-dependent outward current is observed depends on the magnitude of the muscarine-induced cytosolic Ca2+ elevation and the level of depolarization of the cell.(ABSTRACT TRUNCATED AT 400 WORDS)

Adrenal Glands↗
PubMed1992

Kinetic and pharmacological properties of low voltage-activated Ca2+ current in rat clonal (GH3) pituitary cells.

1. Low voltage-activated (LVA) Ca2+ current in clonal (GH3) pituitary cells was studied with the use of the whole-cell recording technique. The use of internal fluoride to facilitate the rundown of high voltage-activated (HVA) Ca2+ current allowed the study of LVA current in virtual isolation. 2. In 10 mM [Ca2+]o, detectable LVA current begins to appear at about -50 mV, with half-maximal activation occurring at -33 mV. The time course of activation was best described by a Hodgkin-Huxley expression with n = 3, suggesting that at least three closed states must be traversed before channel opening. 3. Deactivation was found to vary exponentially with membrane potential between -60 and -160 mV, indicating that channel closing is rate-limited by a single, voltage-dependent transition. 4. Onset and removal of inactivation between -40 and -130 mV were best described by the sum of two exponentials. Between -80 and -130 mV, both components of removal of inactivation showed little voltage dependence, with time constants of approximately 200-300 ms and 1-2 s. At membrane potentials above -40 mV, a single component of inactivation onset was detected. This component was voltage independent between -20 and +20 mV (tau = 22 ms). Thus inactivation of LVA current is best described by multiple, voltage-in-dependent processes. 5. Significant inactivation of LVA current occurred at -65 mV without detectable macroscopic current. This suggests that inactivation is not strictly coupled to channel opening. 6. Peak LVA current increased with increasing [Ca2+]o, with saturation approximately 50 mM. The Ca(2+)-dependence of peak LVA current was reasonably well described by a single-site binding isotherm with half-maximal LVA current at approximately 7 mM. 7. LVA current in GH3 cells was largely resistant to blockade by Ni2+. The relative potency of inorganic cations in blocking GH3 LVA current was (concentrations which produced 50% block): La3+ (2.4 microM) greater than Cd2+ (188 microM) greater than Ni2+ (777 microM). 8. Several organic agents, including putative LVA blockers, HVA current blockers and various anesthetic agents, were tested for their ability to block LVA current. The concentrations that produced 50% block are as follows: nifedipine (approximately 50 microM), D600 (51 microM), diltiazem (131 microM), octanol (244 microM), pentobarbital (985 microM), methoxyflurane (1.41 mM), and amiloride (1.55 mM). Phenytoin and ethosuximide produced 36 and 10% block at 100 microM and 2.5 mM, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Anesthetics↗
PubMed1991

Halothane inhibits two components of calcium current in clonal (GH3) pituitary cells.

The effect of halothane on isolated calcium (Ca2+) current of clonal (GH3) pituitary cells was investigated using standard whole-cell clamp techniques at room temperature. Halothane (0.1-5.0 mM) reversibly reduced both the low-threshold, transient [low-voltage-activated (LVA)] component and the high-threshold [high-voltage-activated (HVA)] component of Ca2+ current. Halothane had little effect on the voltage dependence of activation or inactivation of either component of Ca2+ current. Inhibition of the peak high-threshold Ca2+ current was half-maximal at about 0.8 mM halothane, with maximal inhibition (100%) occurring with 5 mM halothane. When measured at the end of a 190-msec command step, half-maximal reduction of high-threshold current occurred at less than 0.5 mM halothane. The low-threshold transient current was less sensitive to halothane, with half-maximal inhibition of peak transient current activated at -30 mV occurring at approximately 1.3 mM. The effect of halothane on the HVA current was apparently not mediated by changes in intracellular Ca2+ concentration. The ability of halothane to inhibit Ca2+ current was unaffected by either the inclusion of the rapid Ca2+ buffer 1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid (BAPTA) in the recording pipette or exposure of the cell to 10 mM caffeine. To assess the selectivity of the effect of halothane, the actions of halothane on two components of voltage-activated potassium (K+) current observed in the absence of extracellular Ca2+ and on voltage-dependent sodium (Na+) current were also examined. Halothane had no effect on the voltage-dependent, inactivating K+ current of GH3 cells at concentrations up to 1.2 mM. In contrast, the non-inactivating K+ current, though less sensitive to halothane than either Ca2+ current, was reduced by about 40% by 1.2 mM halothane at +20 mV. Peak Na+ current was also blocked by halothane, but 50% block required around 2.6 mM halothane with little effect at 1.6 mM. Reduction of Na+ current was associated with a substantial negative shift in the steady-state inactivation curve. Although the results indicate that a number of voltage-dependent ionic currents are sensitive to halothane, both components of Ca2+ current exhibit a greater sensitivity to halothane than any of three other voltage-dependent currents in GH3 cells. These results show that GH3 cell Ca2+ currents are selectively inhibited by clinically appropriate concentrations of halothane and that the reduction of Ca2+ current can account for the inhibition by halothane of TRH- or KCl-induced prolactin secretion in GH3 cells.

Calcium Channels↗
PubMed1991

The action of halothane on stimulus-secretion coupling in clonal (GH3) pituitary cells.

The effect of halothane on the physiological response to excitatory stimuli was assessed in clonal (GH3) pituitary cells. Halothane, at concentrations used to produce general anesthesia in animals (0.25-0.76 mM), inhibited thyrotropin-releasing hormone (TRH)-induced prolactin (PRL) secretion. The sustained (extracellular calcium-dependent) phase of PRL secretion was 70 +/- 7% inhibited by the highest concentration of halothane tested (0.76 mM); 50% inhibition was produced by approximately 0.4 mM halothane. The early (largely inositol trisphosphate-mediated) phase of secretion was less sensitive to halothane; 0.76 mM halothane produced 18 +/- 2% inhibition of the early phase of secretion. Consistent with these observations, halothane inhibited (IC50 approximately 0.45 mM) the sustained phase of the TRH-induced rise in intracellular calcium ([Ca2+]i) to a greater extent than the initial [Ca2+]i peak. The sustained phase of the [Ca2+]i elevation was inhibited by 75 +/- 7% at the highest concentration of halothane tested (0.76 mM), whereas the peak [Ca2+]i was only inhibited by 14 +/- 5%, consistent with the observation that halothane did not inhibit TRH-stimulated inositide hydrolysis in these cells. Halothane (0.5 mM) did not inhibit phorbol ester- or ionomycin-induced PRL secretion, indicating that halothane has inconsequential effects on the secretory apparatus. Halothane (0.5 mM) also inhibited KCl-induced PRL secretion by 50-80% and the corresponding KCl-induced rise in [Ca2+]i by 68 +/- 6%. These data indicate that halothane inhibits secretagogue-stimulated PRL secretion by reducing the elevation of [Ca2+]i produced by calcium (Ca2+) influx.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium↗
PubMed1989

Anomalous voltage dependence of channel blockade at a crustacean glutamate-mediated synapse.

1. The voltage dependence and concentration dependence of blockade of glutamate-activated currents by the diquaternary amine, chlorisondamine, were examined in a marine crustacean muscle. 2. Chlorisondamine results in the splitting of focally recorded synaptic current decays into two exponential components. The fast component becomes faster with increases in drug concentration and with hyperpolarization. The slow decay rate is unchanged or faster with hyperpolarization and the relative amplitude of the slow component is increased with hyperpolarization. 3. The alteration of synaptic current decay rates by chlorisondamine over the range of 5 to 100 microM and -80 to -140 mV is quantitatively consistent with a simple channel blockade model with a zero-voltage blocking rate of 6 x 10(5) M-1 s-1 at 12 degrees C with a voltage dependence of about 40 mV per e-fold change. The unblocking rate is about 5 s-1 at 0 mV and increases with hyperpolarization with a voltage dependence of about 30 mV per e-fold change. 4. The dose dependence and voltage dependence of blockade of ionophoretically activated glutamate currents by chlorisondamine are qualitatively consistent with the kinetic estimates. 5. The anomalous voltage dependence of the unblocking process is considered in terms of the possibility that the relief from blockade by chlorisondamine occurs by transit of chlorisondamine through the ion channel opened by glutamate.

Action Potentials↗
PubMed1988

Neuromuscular blocking agents.

Clearly, many aspects of the action of neuromuscular blockers remain poorly understood at the molecular level. In the case of competitive blockers, blockade of EPPs by competitive binding to the ACh receptor site accounts for the most clinically important aspect of blockade. Although train-of-four fade produced by curare and some other competitive agents probably represents a presynaptic action, the molecular mechanisms underlying this effect have not been addressed. Depolarizing blockade is inherently more complicated than competitive blockade. Simple depolarization and inactivation of the mechanism for generation of the action potential probably account for the major clinical effect seen in phase I block. Furthermore, the relative balance between activation of channels and desensitization may also provide a qualitative explanation for phase II block and tachyphylaxis. However, effects that are more likely to be explained by presynaptic actions of depolarizing blockers have also been described, and it is not yet possible to assess quantitatively whether the rates of onset of the different postsynaptic actions can account for most aspects of depolarizing block. This discussion has raised several issues which need to be addressed in future studies. 1. What are the presynaptic effects of cholinergic drugs? Do these drugs act through a specific receptor or, on other ion channels in the terminal membrane, or do they operate by mechanisms distinct from effects on membrane conductance? Can any of the observations be explained by indirect effects mediated through postsynaptic ACh receptors, e.g., K+ release? 2. What are the factors that influence variability in sensitivity to neuromuscular blockers among species, muscles within species, and during development? Many of the potential factors, e.g., differences in safety factor, resting conductances, ACh receptor type, and differences in the presence and absence of presynaptic receptors, have been outlined, but definitive tests of the contribution of any particular mechanisms are lacking. 3. Does modulation of desensitization play a role in any components of neuromuscular blockade? 4. Can trapping of blocking agents in ion channels in some cases account for slowly reversible components of blockade? 5. Can closed-channel block provide an effective mechanism of neuromuscular block?

Animals↗
PubMed1987

Activation of the primary kinetic modes of large- and small-conductance cholinergic ion channels in Xenopus myocytes.

1. The kinetic properties of single acetylcholine (ACh)-activated ion channels in tissue-cultured Xenopus myocytes have been examined in cell-attached patches. The rates of agonist binding and channel gating were inferred from the durations of open and closed intervals from channels exposed to 40 nM-200 microM-ACh. The predominant kinetic forms of large- (gamma 60) and small-conductance (gamma 40) cholinergic channels were compared. 2. At high [ACh], bursts were defined so that they primarily reflect sojourns in activatable states. The probability that a channel is open within a burst (Po) increases between 2 and 200 microM-ACh. Po is half-maximal at approximately 5 microM for gamma 40 channels and at approximately 25 microM for gamma 60 channels. 3. Open interval durations for gamma 40 channels are distributed as the sum of two exponentials, with the slow component (tau approximately 2.8 ms) accounting for greater than 80% of the total. Open interval durations for gamma 60 channels are often distributed as a single exponential with an apparent time constant of approximately 0.8 ms. For both conductance forms of channel, open interval durations show no significant dependence on [ACh] in the range 0.04-10 microM, but decrease at higher [ACh] in a manner consistent with channel block by agonist molecules. 4. Closed interval durations within bursts (2-100 microM-ACh) for gamma 60 or gamma 40 channels are described by the sum of two or three exponentials. For both conductance forms of channel the apparent time constant of the fastest component is approximately 40 microseconds and does not change significantly with [ACh], and the time constant of the predominant, slowest component (tau slow) decreases with increasing [ACh]. 5. For gamma 40 channels, at high [ACh] tau slow saturates at approximately 0.17 ms, while no saturation is apparent for gamma 60 channel tau slow values up to 200 microM-ACh. Below 50 microM-ACh, gamma 40 tau slow values are approximately 1.5 times shorter than gamma 60 values. 6. Estimates of rate constants for agonist binding and channel gating were obtained by fitting closed interval durations in the range 2-100 microM-ACh. Gamma 60 channels have a greater than 5-fold faster opening rate, approximately 10-fold faster closing rate, and approximately 3-fold lower affinity than do gamma 40 channels. There is some indication of positive co-operativity of ACh binding to gamma 40 channels.

Acetylcholine↗