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S W Jones

Publications and source records attributed to S W Jones.

At least 37 records · Page 2Linked to original sources
PubMed1997

Bradykinin antagonists in human systems: correlation between receptor binding, calcium signalling in isolated cells, and functional activity in isolated ileum.

The determination of the relationship between ligand affinity and bioactivity is important for the understanding of receptor function in biological systems and for drug development. Several physiological and pathophysiological functions of bradykinin (BK) are mediated via the B2 receptor. In this study, we have examined the relationship between B2 receptor (soluble and membrane-bound) binding of BK peptidic antagonists, inhibition of calcium signalling at a cellular level, and in vitro inhibition of ileum contraction. Only human systems were employed in the experiments. Good correlations between the studied activities of BK antagonists were observed for a variety of different peptidic structures. The correlation coefficients (r) were in the range of 0.905 to 0.955. In addition, we analyzed the effect of the C-terminal Arg9 removal from BK and its analogs on B2 receptor binding. The ratios of binding constants (Ki(+Arg)/Ki(-Arg)) for the Arg9 containing compounds and the corresponding des-Arg9 analogs varied from about 10 to 250,000. These ratios strongly depend on the chemical structures of the compounds. The highest ratios were observed for two natural agonist pairs, BK/des-Arg9-BK and Lys0-BK/des-Arg9-Lys0-BK.

Bradykinin↗
PubMed1997

Delayed rectifier current of bullfrog sympathetic neurons: ion-ion competition, asymmetrical block and effects of ions on gating.

1. The delayed rectifier (DR) K+ channel pore was probed using different permeant and blocking ions applied intra- and extracellularly. Currents were recorded from bullfrog sympathetic neurons using whole-cell patch-clamp techniques. 2. With intra- and extracellular Cs+ (0 K+), there were large, tetraethylammonium (TEA)-sensitive currents. Adding K+ back to the extracellular solution revealed that the current with Cs+i was K+ selective (permeability ratio PCs/PK = 0.17 +/- 0.02, n = 4) and showed a strong anomalous mole fraction effect. 3. There were also large non-inactivating currents with Na+i and Na+o (0 K+). The current with Na+i was K+ selective (Na+o vs. K+o: PNa/PK = 0.022 +/- 0.005, n = 5), and was TEA sensitive with K+o but not with Na+o. 4. Permeant ions affected gating kinetics. DR currents activated faster in K+ than in Cs+, and activated faster with increasing concentrations of either K+ or Cs+. Deactivation was slowed by increased K+ or Cs+ concentration, with no difference between K+ and Cs+. 5. The pore was also characterized using intracellular blocking ions. A wide variety of monovalent cations (TEA, N-methyl-D-glucamine, arginine, choline, CH3NH3+, Li+, Cs+ and Na+) blocked DR channels from the inside in a voltage-dependent manner: KD at 0 mV was 2.9 mM for TEA and 134-487 mM for the others, at apparent electrical distances (delta) of 0.33-0.79. There was no detectable block by 10 mM Mgi2+. Apart from TEA, the organic cations did not block from the outside. 6. The permeability to Na+ in the absence of K+, and the strong anomalous mole fraction effects observed for Cs+o + K+o mixtures, suggest that DR channels select for K+ using ion-ion competition. The block by large intracellular cations shows that the pore is asymmetrical. The loss of high affinity TEAo block with Na+i and Na+o, and the effects of permeant ions on gating, suggest that channel conformation may be affected by ions in the pore.

Animals↗
PubMed1997

High-voltage-activated calcium currents in neurons acutely isolated from the ventrobasal nucleus of the rat thalamus.

We studied the high-voltage-activated (HVA) calcium currents in cells isolated from the ventrobasal nucleus of the rat thalamus with the use of the whole cell patch-clamp technique. Low-voltage-activated current was inactivated by the use of long voltage steps or 100-ms prepulses to -20 mV. We used channel blocking agents to characterize the currents that make up the HVA current. The dihydropyridine (DHP) antagonist nimodipine (5 microM) reversibly blocked 33 +/- 1% (mean +/- SE), and omega-conotoxin GVIA (1 microM) irreversibly blocked 25 +/- 5%. The current resistant to DHPs and omega-conotoxin GVIA was inhibited almost completely by omega-conotoxin MVIIC (90 +/- 5% at 3-5 microM) and was partially inhibited by omega-agatoxin IVA (54 +/- 4% block at 1 microM). We conclude that there are at least four main HVA currents in thalamic neurons: N current, L current, and two omega-conotoxin MVIIC-sensitive currents that differ in their sensitivity to omega-agatoxin IVA. We also examined modulation of HVA currents by strong depolarization and by G protein activation. Long (approximately 1 s), strong depolarizations elicited large, slowly deactivating tail currents, which were sensitive to DHP antagonists. With guanosine 5'-0-(3-thiotriphosphate) (GTP-gamma-S) in the intracellular solution, brief (approximately 20 ms), strong depolarization produced a voltage-dependent facilitation of the current (44 +/- 5%), compared with cells with GTP (22 +/- 7%) or guanosine 5'-O-(2-thiodiphosphate) (7 +/- 4%). However, the HVA current was inhibited only weakly by 100 microM acetylcholine (8 +/- 4%). Effects of the gamma-aminobutyric acid-B agonist baclofen were variable (3-39% inhibition, n = 12, at 10-50 microM).

Animals↗
PubMed1997

Generation of multiple mRNA fingerprints using fluorescence-based differential display and an automated DNA sequencer.

Differential display is a method for the survey, analysis and comparison of gene expression in eukaryotic cells and tissues. Differential display involves isolation of high-quality nondegraded RNA, selective reverse transcription of polyadenylated mRNA using specific anchored oligopolydeoxythymidine [oligo(dT)] primers, and the subsequent PCR amplification of the cDNA with the same oligo(dT), an arbitrary upstream primer and radioisotopes for labeling the PCR products. The radioisotopically labeled products are then separated on a sequencing gel. In this report, we describe a rapid, specific, nonradioactive fluorescent differential display methodology in which fluorescently differentially labeled anchored oligo(dT) downstream primers are used in the reaction, with subsequent analysis of fluorescently labeled PCR products on an automated sequencer. Complete gene expression profiles, containing multiple mRNA fingerprints are possible by the simultaneous comparison of the multicolored banding patterns of the fluorescently differentially labeled products from several primer combinations. This modification of the differential display technique simplifies the assay and increases the throughput of high sample volumes required for comparative gene expression studies in various clinical applications.

Antibodies↗
PubMed1996

The effects of external pH on calcium channel currents in bullfrog sympathetic neurons.

We have investigated the effects of external pH (pHo) on whole-cell calcium channel currents in bullfrog sympathetic neurons. The peak inward current increased at alkaline pHo and decreased at acidic pHo. We used tail currents to distinguish effects of pHo on channel gating and permeation. There were large shifts in the voltage dependence of channel activation (approximately 40 mV between pHo and 9.0 and pHo 5.6), which could be explained by binding of H+ to surface charge according to Gouy-Chapman theory. To examine the effects of pHo on permeation, we measured tail currents at 0 mV, following steps to + 120 mV to maximally activate the channels. Unlike most previous studies, we found only a approximately 10% reduction in channel conductance from pHo 9.0 to pHo 6.4, despite a approximately 25 mV shift of channel activation. At lower pHo the channel conductance did decrease, which could be described by binding of H+ to a site with pKa = 5.1. In some cells, there was a separate slow decrease in conductance at low pHo, possibly because of changes in internal pH. These results suggest that changes in current at pHo > 6.4 result primarily from a shift in the voltage dependence of channel activation. A H(+)-binding site can explain a rapid decrease in channel conductance at lower pHo. The surface charge affecting gating has little effect on the local ion concentration near the pore, or on the channel conductance.

Animals↗
PubMed1996

Ion permeation and block of M-type and delayed rectifier potassium channels. Whole-cell recordings from bullfrog sympathetic neurons.

Ion permeation and conduction were studied using whole-cell recordings of the M-current (I(M)) and delayed rectifier (IDR), two K+ currents that differ greatly in kinetics and modulation. Currents were recorded from isolated bullfrog sympathetic neurons with 88 mM [K+]i and various external cations. Selectivity for extracellular monovalent cations was assessed from permeability ratios calculated from reversal potentials and from chord conductances for inward current. PRb/PK was near 1.0 for both channels, and GRb/GK was 0.87 +/- 0.01 for IDR but only 0.35 +/- 0.01 for I(M) (15 mM [Rb+]o or [K+]o). The permeability sequences were generally similar for I(M) and IDR: K+ approximately Rb+ > NH4+ > Cs+, with no measurable permeability to Li+ or CH3NH3+. However, Na+ carried detectable inward current for IDR but not I(M). Nao+ also blocked inward K+ current for IDR (but not IM), at an apparent electrical distance (delta) approximately 0.4, with extrapolated dissociation constant (KD) approximately 1 M at 0 mV. Much of the instantaneous rectification of IDR in physiologic ionic conditions resulted from block by Nao+. Extracellular Cs+ carried detectable inward current for both channel types, and blocked I(M) with higher affinity (KD = 97 mM at 0 mV for I(M), KD) approximately 0.2 M at 0 mV for IDR), with delta approximately 0.9 for both. IDR showed several characteristics reflecting a multi-ion pore, including a small anomalous mole fraction effect for PRb/PK, concentration-dependent GRb/GK, and concentration-dependent apparent KD's and delta's for block by Nao+ and Cso+. I(M) showed no clear evidence of multi-ion pore behavior. For I(M), a two-barrier one-site model could describe permeation of K+ and Rb+ and block by Cso+, whereas for IDR even a three-barrier, two-site model was not fully adequate.

Animals↗
PubMed1995

Isolation and characterization of a new basic antigen from short ragweed pollen (Ambrosia artemisiifolia).

A new basic antigen with a molecular weight of 36.5 kDa and a pI of 8.65 was purified from short ragweed pollen using ammonium sulfate precipitation, Q-sepharose chromatography and S-sepharose chromatography. The purified protein, herein referred to as Ambrosia artemisiifolia basic antigen (AaBA), migrated as a single band in SDS-polyacrylamide gel electrophoresis and isoelectric focusing, and as a single peak on reverse phase HPLC. AaBA reacted with sera from humans allergic to ragweed pollen including pooled NIH serum. AaBA and two major acidic allergens, Amb a I (antigen E) and Amb a II (antigen K), displayed immunological cross-reactivity inasmuch as they competed significantly with each other in a competition ELISA. This suggests that at least some major epitopes in all three antigens are similar; however, other possibilities like adjacent location of different epitopes cannot be ruled out at this time. Molecular weight, amino acid composition and sequence data suggest that AaBA is very similar to Amb a II whereas it is significantly different from other basic antigens purified from ragweed pollen.

Allergens↗
PubMed1995

Surface charge and calcium channel saturation in bullfrog sympathetic neurons.

Currents carried by Ba2+ through calcium channels were recorded in the whole-cell configuration in isolated frog sympathetic neurons. The effect of surface charge on the apparent saturation of the channel with Ba2+ was examined by varying [Ba2+]o and ionic strength. The current increased with [Ba2+]o, and the I-V relation and the activation curve shifted to more positive voltages. The shift of activation could be described by Gouy-Chapman theory, with a surface charge density of 1 e-/140 A2, calculated from the Grahame equation. Changes in ionic strength (replacing N-methyl-D-glucamine with sucrose) shifted the activation curve as expected for a surface charge density of 1 e-/85 A2, in reasonable agreement with the value from changing [Ba2+]o. The instantaneous I-V for fully activated channels also changed with ionic strength, which could be described either by a low surface charge density (less than 1 e-/1,500 A2), or by block by NMG with Kd approximately 300 mM (assuming no surface charge). We conclude that the channel permeation mechanism sees much less surface charge than the gating mechanism. The peak inward current saturated with an apparent Kd = 11.6 mM for Ba2+, while the instantaneous I-V saturated with an apparent Kd = 23.5 mM at 0 mV. This discrepancy can be explained by a lower surface charge near the pore, compared to the voltage sensor. After correction for a surface charge near the pore of 1 e-/1,500 A2, the instantaneous I-V saturated as a function of local [Ba2+]o, with Kd = 65 mM. These results suggest that the channel pore does bind Ba2+ in a saturable manner, but the current-[Ba2+]o relationship may be significantly affected by surface charge.

Animals↗
PubMed1994

Concentration dependence of neurotransmitter effects on calcium current kinetics in frog sympathetic neurones.

1. Noradrenaline (NA) slows the activation kinetics of N-type calcium channels, via G proteins. It has been suggested that the G proteins act by binding directly to the calcium channels. If the slow kinetics reflect binding and unbinding of G proteins, the rates should depend on the concentration of activated G protein. 2. We used different concentrations of NA, and increasing durations of intracellular dialysis with GTP-gamma-S, to vary the concentration of activated G protein. 3. At depolarized potentials (-20 or -10 mV), the slow activation kinetics showed no detectable concentration dependence. This analysis required correction for effects of inactivation on the measured time constants. 4. At -80 mV, reinhibition of calcium channel current was more rapid for larger responses. Thus, the effect appears to be concentration dependent at -80 mV, but not at more depolarized voltages. 5. This voltage dependence is actually expected from kinetic principles: the binding step is rate limiting when the position of equilibrium is toward the bound state (at -80 mV), but not when equilibrium favours unbinding (when the channel is open). 6. During inhibition, the channel appears to 'sense' directly the concentration of the modulator, possibly active G proteins.

Animals↗
PubMed1994

Reevaluation of Ca2+ channel types and their modulation in bullfrog sympathetic neurons.

With 90 mM Ba2+, the main Ca2+ current in frog sympathetic neurons peaks near +30 mV and is blocked by omega-conotoxin GVIA (omega-CgTx). It is modulated by norepinephrine (NE) in a voltage-dependent manner via a membrane-delimited mechanism. Surprisingly, a different current dominates at more negative voltages (-30 to +10 mV). That novel current is not sensitive to selective blockers of L- or N-type channels (respectively, dihydropyridines or omega-CgTx) and is inhibited weakly if at all by NE. It is selectively inactivated at -40 mV and is selectively blocked by Ni2+, whereas Cd2+ is slightly more potent against the main current. The novel current is associated with a 19 pS channel (0.6 pA at 0 mV). This channel may have been misidentified as the single-channel correlate of the whole-cell N-type Ca2+ current in some previous studies.

Animals↗
PubMed1993

Effects of dexamethasone on L-type calcium currents in the A7r5 smooth muscle-derived cell line.

Patch clamp experiments were used to characterize the effect of dexamethasone on calcium currents in A7r5 cells. Pretreatment for 48 h with 200 nM dexamethasone did not affect the single channel conductance, the voltage dependence of channel opening, or the voltage-dependent inactivation of L-type channels. However, dexamethasone caused an approximately 2-fold increase in the amplitude of L-type calcium currents in 5 out of 9 experiments, suggesting an increase in the number of active channels. The effect of dexamethasone appeared to be greatest on batches of cells with low control current density. The amplitude of T-type calcium current was not affected by dexamethasone.

Animals↗
PubMed1993

Purification and characterization of a novel zinc-proteinase from cultures of Aeromonas hydrophila.

While searching for an enzyme capable of breaking epsilon-(gamma-Glu)-Lys isopeptide bonds cross-linking protein chains, we purified a metallo-proteinase which mimics the action of an isopeptidase on the gamma-chain dimers of cross-linked fibrin. The enzyme is present in the growth medium of the bacterium Aeromonas hydrophila, isolated from the intestinal tract of the leech Hirudo medicinalis. It is a 19-kDa protein which specifically hydrolyzes the Gly-Ala peptide bond within the Gly-Gly-Ala sequence, located near the cross-link site in the gamma-chain dimer of fibrin. Substrate specificity studies with a number of synthetic peptides suggest that the enzyme prefers Gly-Gly or acetyl-Gly in the P2 and P1 positions, respectively (Schecter, I., and Berger, A. (1967) Biochem. Biophys. Res. Commun. 27, 157-162). Nonpolar amino acid residues seem to be favored in the P1' and P2' positions. The enzyme contains one atom of zinc and is inhibited by 1,10-phenanthroline, but not by EDTA. Iodoacetate, leupeptin, diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride, pepstatin, and alpha 2-macroglobulin have no effect on enzyme activity. Disulfide reducing reagents, such as dithiothreitol or 2-mercaptoethanol, inactivate the enzyme completely. The partial amino-terminal sequence shows 46% identity with a zinc metallo-proteinase from a strain of Lysobacter enzymogenes and 69% identity with the LasA protein from Pseudomonas aeruginosa.

Aeromonas hydrophila↗
PubMed1993

Phosphorylation enhances inactivation of N-type calcium channel current in bullfrog sympathetic neurons.

We have investigated the effects of phosphatase and protein kinase inhibitors on calcium channel currents of bullfrog sympathetic neurons using the whole cell configuration of the patch clamp technique. Intracellular dialysis with the phosphatase inhibitors okadaic acid and calyculin A markedly enhanced the decline of inward current during a depolarizing voltage step. Tail current analysis demonstrated that this was genuine inactivation of calcium channel current, not activation of an outward current. The rapidly inactivating current is N-type calcium current (blocked by omega-conotoxin and resistant to nifedipine). Staurosporine, a nonselective protein kinase inhibitor, prevented the action of okadaic acid, suggesting that protein phosphorylation is involved. Under control conditions, the time course of inactivation could be described by the sum of two exponentials (tau = 150 ms and 1200 ms), plus a constant (apparently noninactivating) component, during depolarizations lasting 2 s. Okadaic acid induced a rapid inactivation process (tau = 15 ms) that was absent or negligible under control conditions, without obvious effect on the two slower time constants. As in control cells, inactivation in okadaic-acid-treated cells was strongest near -20 mV, with less inactivation at more positive voltages. However, inactivation did not depend on calcium influx. Modulation of calcium channel activity by phosphorylation may underly the spontaneous shift between inactivating and noninactivating modes recently observed for N-type calcium channels. Differences in basal phosphorylation levels could also explain why N-type calcium channels, originally described as rapidly and completely inactivating, inactivate slowly and incompletely in many neurons.

Animals↗