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S W Jones

Publications and source records attributed to S W Jones.

At least 55 records · Page 3Linked to original sources
PubMed1993

Intracellular ATP and GTP are both required to preserve modulation of N-type calcium channel current by norepinephrine.

Norepinephrine (NE) inhibits voltage-dependent calcium channels of sympathetic neurons. We investigated the role of intracellular nucleotides in this inhibition for clues to receptor-channel coupling mechanisms. Both ATP and GTP are required to preserve NE responsiveness during whole-cell dialysis. The response to NE was gradually lost in bullfrog sympathetic neurons dialyzed with GTP as the only nucleotide, ATP only, or no nucleotides. Replacing ATP with ATP[gamma-S] resulted in spontaneous modulation of calcium channel current, possibly because of production of GTP[gamma-S]. The nonhydrolyzable ATP analog p[NH]ppA could substitute for ATP to preserve NE responsiveness. The protein phosphatase inhibitors okadaic acid and calyculin-A did not affect NE inhibition of calcium channel current, or recovery from that inhibition. These results suggest protein phosphorylation is not involved in the inhibition of calcium channel current, but binding of ATP to some intracellular site is required for the coupling of adrenergic receptors to calcium channels.

Adenosine Triphosphate↗
PubMed1993

Effects of temperature on calcium current of bullfrog sympathetic neurons.

1. The temperature dependence of whole-cell calcium current was studied in bullfrog sympathetic neurons. 2. The factor by which the peak calcium current increased upon a 10 degrees C increase in temperature (Q10) was 1.6 +/- 0.1 (mean +/- S.E.M., n = 9), for a change from 20 to 30 degrees C. 3. Activation and deactivation were more rapid at a higher temperature. The Q10 was approximately 7 in the middle of the voltage range (near -10 mV), where the kinetics were slowest. Time constants were less temperature dependent at more positive or negative voltages (Q10 approximately 2 at -70 mV). 4. Near -10 mV, activation and deactivation were associated with a large enthalpy and a large positive entropy change. Deactivation at -70 mV reflected a smaller enthalpy change, and almost no change in entropy. 5. Activation is only slightly more temperature dependent than deactivation, when both are measured at the same voltage. 6. The peak current shifts slightly (approximately 5 mV) to more negative voltages upon a change from 20 to 30 degrees C. 7. Inactivation has a Q10 of approximately 2 at -10 mV. 8. Changes in the kinetics of activation or inactivation could be observed during recording at a constant temperature. These changes were generally small, especially for activation kinetics, and could be distinguished from temperature-dependent changes. 9. The changes in entropy and Q10 with voltage suggest that the rate-limiting steps for activation and deactivation are different at extreme voltages vs. the middle of the voltage range.

Animals↗
PubMed1992

Cadmium block of calcium current in frog sympathetic neurons.

Cd2+ blocks whole-cell calcium currents in frog sympathetic neurons by 50% at approximately 300 nM. Strong depolarizations rapidly reverse that blockade (tau = 1.3 ms at +120 mV). Reblocking follows bimolecular kinetics (rate = 1.2 x 10(8) M-1 s-1) at voltages where channels are mostly open (0 to +30 mV). The unblocking rate is approximately 50 s-1, so the dissociation constant calculated from the rate constants is approximately 400 nM. Steady-state block is strong at -80 mV, so closed channels can also be blocked. However, reblocking is extremely slow (tau = 1-2 s) at voltages where the channels are mostly closed. The rates for Cd2+ entry and exit are greater than 100-fold lower for closed channels than for open channels, and closed channels appear to be closed at both ends.

Animals↗
PubMed1992

Calcium currents in the A7r5 smooth muscle-derived cell line. An allosteric model for calcium channel activation and dihydropyridine agonist action.

We have investigated the gating kinetics of calcium channels in the A7r5 cell line at the level of single channels and whole cell currents, in the absence and presence of dihydropyridine (DHP) calcium channel agonists. Although latencies to first opening and macroscopic currents are strongly voltage dependent, analysis of amplitude histograms indicates that the primary open-closed transition is voltage independent. This suggests that the molecular mechanisms for voltage sensing and channel opening are distinct, but coupled. We propose a modified Monod-Wyman-Changeux (MWC) model for channel activation, where movement of a voltage sensor is analogous to ligand binding, and the closed and open channels correspond to inactive (T) and active (R) states. This model can account for the activation kinetics of the calcium channel, and is consistent with the existence of four homologous domains in the main subunit of the calcium channel protein. DHP agonists slow deactivation kinetics, shift the activation curve to more negative potentials with an increase in slope, induce intermingled fast and slow channel openings, and reduce the latency to first opening. These effects are predicted by the MWC model if we make the simple assumption that DHP agonists act as allosteric effectors to stabilize the open states of the channel.

Action Potentials↗
PubMed1992

Calcium current modulation in frog sympathetic neurones: L-current is relatively insensitive to neurotransmitters.

1. Neurotransmitters (noradrenaline, NA; chicken II luteinizing hormone-releasing hormone, LHRH) and activators of G proteins (GTP-gamma-S and AlF3) partially inhibit calcium current in bullfrog sympathetic neruones. Activation of the remaining current is slowed and shifted to more positive voltages. 2. The N-type calcium current appears to be the type modulated, since approximately 90% of peak current is blocked by omega-conotoxin (omega CgTx) and modulation is not affected by nisoldipine. 3. Calcium current at relatively negative voltages (-30 to -50 mV) is resistant to transmitter modulation. The current at such voltages is also resistant to omega CgTx, suggesting that it results from a different type of calcium channel. 4. The omega CgTx-resistant current includes dihydropyridine (DHP)-sensitive and DHP-resistant components. The omega CgTx- and DHP-resistant current is inhibited by transmitter agonist, but the DHP-sensitive (L-type) current is not. 5. In cells dialysed with a low concentration of calcium buffer (0.1 mM-BAPTA), transmitters still inhibit N-current incompletely. However, L-current was partially inhibited (approximately 10%) by LHRH, NA and the muscarinic agonist oxotremorine-M (OXO-M).

Aluminum↗
PubMed1992

Separation and modulation of calcium currents in bullfrog sympathetic neurons.

The calcium current of frog sympathetic neurons has relatively rapid activation kinetics (tau < 3 ms) in response to changes in voltage. Pharmacologically, the current is blocked approximately 90% of omega-conotoxin, but < 10% by dihydropyridine antagonists. This suggests that nearly all of the current is N type. However, inactivation is slow and incomplete even for depolarizations lasting > 1 s, consistent with recent evidence that N-type channels do not always inactivate rapidly. The calcium current is partially inhibited via receptors for acetylcholine, luteinizing hormone releasing hormone, substance P, ATP, and norepinephrine. These effects are mimicked by internal dialysis with GTP-gamma-S, suggesting involvement of a G protein. The transmitters affect the activation kinetics of the calcium current in a voltage-dependent manner, which can be modeled as a reversible shift of some channels to "reluctant" states in which strong depolarization is needed to produce channel opening. The effects of transmitters develop and recover with t1/2 approximately 1-2 s, so if a second messenger is involved in receptor-calcium channel coupling, it must act rapidly.

Animals↗
PubMed1991

Time course of receptor-channel coupling in frog sympathetic neurons.

The M-type potassium current and the N-type calcium current are inhibited by several different neurotransmitters in frog sympathetic neurons. These effects seem to be mediated via G proteins, but it is not clear whether diffusible second messengers are involved. Using a rapid (approximately 100 ms) flow tube perfusion system to apply agonists, the inhibition of calcium current develops and recovers rapidly but not instantaneously (t1/2 = 1-2 s). M-current inhibition is considerably slower, with t1/2 approximately 30 s for recovery from inhibition. At least for M-current inhibition, there appears to be sufficient time for involvement of an enzymatic cascade in receptor-channel coupling.

Animals↗
PubMed1991

Calcium currents in the A7r5 smooth muscle-derived cell line. Calcium-dependent and voltage-dependent inactivation.

Inactivation of a dihydropyridine-sensitive calcium current was studied in a cell line (A7r5) derived from smooth muscle of the rat thoracic aorta. Inactivation is faster with extracellular Ca2+ than with Ba2+. In Ba2+, inactivation increases monotonically with depolarization. In Ca2+, inactivation is related to the amount of inward current, so that little inactivation is seen in Ca2+ for brief depolarizations approaching the reversal potential. Longer depolarizations in Ca2+ reveal two components of inactivation, the slower component behaving like that observed in Ba2+. Furthermore, lowering extracellular Ca2+ slows inactivation. These results are consistent with the coexistence of two inactivation processes, a slow voltage-dependent inactivation, and a more rapid current-dependent inactivation which is observable only with Ca2+. Ca(2+)-dependent inactivation is decreased but not eliminated when intracellular Ca2+ is buffered by 10 mM BAPTA, suggesting that Ca2+ acts at a site on or near the channel. We also studied recovery from inactivation after either a short pulse (able to produce significant inactivation only in Ca2+) or a long pulse (giving similar inactivation with either cation). Surprisingly, recovery from Ca(2+)-dependent inactivation was voltage dependent. This suggests that the pathways for recovery from inactivation are similar regardless of how inactivation is generated. We propose a model where Ca(2+)- and voltage-dependent inactivation occur independently.

Animals↗
PubMed1991

Calcium currents in the A7r5 smooth muscle-derived cell line. Increase in current and selective removal of voltage-dependent inactivation by intracellular trypsin.

We studied the effects of trypsin on L-type calcium current in the A7r5 smooth muscle cell line. Intracellular dialysis with trypsin increased the whole-cell current up to fivefold. The effect was concentration dependent, and was prevented by soybean trypsin inhibitor. Ensemble analysis indicated an increase in the number of functional channels, and possibly a smaller increase in the open probability, with no change in the single channel current. The shape of the current-voltage curve was unaffected. Trypsin also nearly eliminated inactivation of currents carried by Ba2+, but had little or no effect on the rapid inactivation process in Ca2+, This indicates that trypsin removes voltage-dependent but not Ca(2+)-dependent inactivation, suggesting the existence of distinct protein domains for these two mechanisms of calcium channel inactivation.

Animals↗
PubMed1990

Calcium currents in the A7r5 smooth muscle-derived cell line.

We have studied voltage-dependent calcium channels in the A7r5 smooth muscle cell line by measuring the high-affinity binding of radiolabelled dihydropyridines (DHPs), whole-cell and single-channel currents in patch-clamped cells, as well as cytosolic calcium ([Ca2+]i) in fura-2-loaded cell suspensions and monolayers. Intact A7r5 cells express saturable, high-affinity, voltage-sensitive DHP binding sites with pharmacological properties characteristic of L-type calcium channels. When cells were voltage clamped in the whole-cell configuration with near normal intra- and extracellular solutions, a DHP-sensitive inward current resembling the L-type calcium current was dominant. With barium (10 mM) as the charge carrier, peak inward currents were typically recorded at test potentials between 0 and +20 mV. Currents were blocked by extracellular cadmium with a half-maximal inhibitory concentration of approximately 1 microM. Isoproterenol (1 microM) or forskolin (10 microM) increased currents in approximately half of the cells tested. Forskolin (10 microM) increased single-channel activity in five of eight cell-attached patches. After cells had been quiescent for several weeks, cell suspensions showed changes in resting [Ca2+]i in response to DHPs and increased potassium. Most confluent monolayers of cells showed spontaneous transient elevations in [Ca2+]i. Bath application of Bay K 8644 increased the frequency and magnitude of these [Ca2+]i transients, whereas nifedipine abolished the transients. These data suggest that the [Ca2+]i transients were due to synchronous action potentials in electrically coupled cell monolayers.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1990

LHRH and GTP-gamma-S modify calcium current activation in bullfrog sympathetic neurons.

LHRH (chicken II luteinizing hormone-releasing hormone) partially reduces calcium currents and slows the activation kinetics of part of the remaining current in frog sympathetic neurons. The effects of LHRH are mimicked by intracellular dialysis with GTP-gamma-S. A strong depolarization can temporarily reverse the effects of LHRH or GTP-gamma-S: activation kinetics return to normal, and the amplitude of the current is increased (facilitation). Facilitation develops rapidly (tau = 4-6 ms at greater than +30 mV) and decays more slowly (t 1/2 = 60 ms at -80 mV). Tail currents in LHRH are smaller and faster than in the control, and these effects are partially reversed by facilitation. These results can be explained by a model in which a fraction of the channels is shifted into a "reluctant" gating mode, where opening requires stronger depolarization. If this mechanism is at the root of presynaptic inhibition, our results predict that inhibition of transmitter release would be overcome during bursts of high frequency activity.

Animals↗
PubMed1990

Identification of mitogen-responsive ribosomal protein S6 kinase pp90rsk, a homolog of Xenopus S6 kinase II, in chicken embryo fibroblasts.

Antiserum raised against recombinant Xenopus ribosomal protein S6 kinase (rsk) was used to identify a 90,000-Mr ribosomal S6 kinase, pp90rsk, in chicken embryo fibroblasts. Adding serum to cells stimulated the phosphorylation of pp90rsk on serine and threonine residues and increased the activity of S6 kinase measured in immune complex assays. Xenopus S6 kinase II and chicken embryo fibroblast pp90rsk had nearly identical phosphopeptide maps.

Animals↗
PubMed1990

Dihydropyridine actions on calcium currents of frog sympathetic neurons.

Dihydropyridines (DHPs) generally have little effect on whole-cell calcium currents of neurons, even at concentrations far higher than those effective on muscle. Either neuronal calcium currents are much less sensitive to DHPs, or only a small proportion of the current is DHP-sensitive. We find that DHP agonists and antagonists act at low concentration on calcium currents in frog sympathetic neurons but that the effects are small even at optimal concentrations. The half-maximal dose (EC50) of the agonist Bay K 8644 is approximately 50 nM, and the effect of Bay K 8644 is blocked by 50% at approximately 300 nM nifedipine, from a holding potential of -80 mV. Nifedipine is more effective from a holding potential of -50 mV. These results suggest the presence of an L-type calcium current, with DHP sensitivity similar to L-currents in cardiac muscle. The predominant (greater than 90%) calcium current in frog sympathetic neurons is a DHP-resistant N-type current. However, high concentrations of DHPs (10 microM) partially block N-type calcium current, as well as voltage-dependent sodium and potassium currents.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1989

Phosphotyrosyl-protein phosphatases. I. Separation of multiple forms from bovine brain and purification of the major form to near homogeneity.

Seven Tyr-protein phosphatase activities were isolated from bovine brain using phosphotyrosyl-casein as a model substrate. The activities were resolved from the cytosolic fraction by a three-step procedure employing successive DEAE-cellulose, phosphocellulose, and gel permeation chromatography steps. The seven activities accounted for 70% of the Tyr-protein phosphatase activity in bovine brain extracts and were distinct from type 1 and type 2 Ser/Thr-protein phosphatases and from the major alkaline phosphatase activities. Apparent molecular weights of the activities by gel permeation chromatography were: phosphotyrosyl-protein phosphatase (PTP)-1A (Mr 86,000), PTP-1B (Mr 24,000), PTP-2 (Mr 88,000), PTP-3 (Mr 90,000), PTP-4 (Mr 80,000), PTP-5 (Mr 48,000), and PTP-6 (Mr 104,000). PTP-5 was the major activity accounting for 26% of total while the remaining activity was divided rather evenly among the other six activities. PTP-5 was further purified to near homogeneity by additional chromatographies on Affi-Gel Blue, heparin-agarose, and Mono S giving an overall purification of 50,000-fold and a yield of 5.8%. One of two major polypeptides (Mr 46,000) in the preparation was identified as PTP-5 since it alone expressed protein phosphatase activity when protein-staining bands were eluted from sodium dodecyl sulfate-polyacrylamide gels and renatured. PTP-5 had a neutral pH optimum, and using phosphotyrosyl-casein as substrate it had a Km of 130 nM and a Vmax of 10 mumol Pi released.min-1.mg protein-1. These kinetic parameters are well within the range of values obtained for other pure protein phosphatases. PTP-5 also dephosphorylated pp60v-src (autophosphorylated at Tyr-416) at 10% of the rate observed with phosphotyrosyl-casein. Additionally the ratio of phosphotyrosyl-casein/pp60v-src phosphatase activity was relatively constant throughout the PTP-5 purification procedure. These results indicate that PTP-5 is able to bind and efficiently dephosphorylate phosphotyrosyl-proteins and suggest that it is a physiologically relevant Tyr-protein phosphatase.

Alkaline Phosphatase↗