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P Whiting

Publications and source records attributed to P Whiting.

At least 55 records · Page 3Linked to original sources
PubMed1995

Isolation and characterisation of human proximal tubular cells derived from kidney cortical segments.

1. Human renal proximal tubular cells (HPTC) were isolated by collagenase digestion and purified following filtration and isopycnic Percoll density centrifugation. This method used cortical tissue obtained from surgical nephrectomies and was both rapid and simple, providing a preparation of cells with high viability (> 93 +/- 3%) and recovery (16 +/- 7 x 10(6) cells g-1 of cortical tissue). 2. Characterisation of the isolated cells showed that, in terms of morphology, enzyme profile, transport systems and hormonal responsiveness, they were > 95% proximal tubular. The transport systems obeyed Michaelis-Menten kinetics, with the kinetic parameters of the glucose transport system (Km = 2.5mM, Vmax = 7.7 nmol min-1 mg-1 protein) suggesting a higher proportion of PT cells originating from the S1-S2 segment of the nephron. Isolated HPTC also maintained levels of reduced glutathione (GSH) (11.9 +/- 3.2 nmol mg-1 protein) and exhibited cytochrome P450-dependent activity, levels of spectrally determined P450 being 0.22 +/- 0.07 nmol mg-1 protein. 3. These results demonstrate the isolation of a viable and functioning homogeneous preparation of HPTC from cortical tissue, with potential for use in short term pharmacological, physiological and toxicological studies.

Alanine Transaminase↗
PubMed1994

Comparison of the binding of nicotinic agonists to receptors from human and rat cerebral cortex and from chick brain (alpha 4 beta 2) transfected into mouse fibroblasts with ion channel activity.

(-)-Nicotine, cytisine and carbachol evoked 86Rb efflux from mouse fibroblasts stably transfected with alpha 4 beta 2 chick brain nicotinic subunits. This response to (-)-nicotine was inhibited by mecamylamine and dihydro-beta-erythroidine and was mirrored by a rise in intracellular Ca2+ measured by microspectrofluorimetry. Lobeline and isoarecolone methiodide evoked no significant 86Rb from cells and unlike the above agonists displayed significantly different IC50 values for the displacement of [3H]nicotine from mammalian (rat and human cerebral cortex) and transfected fibroblast membranes.

Animals↗
PubMed1994

Physostigmine and galanthamine: probes for a novel binding site on the alpha 4 beta 2 subtype of neuronal nicotinic acetylcholine receptors stably expressed in fibroblast cells.

In the present study, we demonstrated that the chicken alpha 4 beta 2 neuronal nicotinic receptor stably expressed in transfected mouse fibroblasts (M10 cells) can be activated via the acetylcholine-binding site or via a site that is distinct from that for acetylcholine and recognizes physostigmine and galanthamine as agonists. In outside-out patches excised from dexamethasone-induced M10 cells, (+)-anatoxin-a, physostigmine and galanthamine (each at 1 microM) activated single channels with conductances of 18 and 30 pS. Dihydro-beta-erythroidine (1-30 nM), but not the nicotinic receptor-specific monoclonal antibody FK1, reduced the frequency of channels activated by anatoxin (1 microM). On the other hand, the frequency of channel activity induced by physostigmine (1 microM) was unaffected by dihydro-beta-erythroidine and was markedly decreased by FK1. In uninduced M10 cells and in dexamethasone-treated untransfected fibroblasts, we observed that physostigmine, galanthamine and nicotinic agonists did not evoke whole-cell or single-channel currents. Also, neither [3H]L-nicotine nor FK1 was able to bind to uninduced M10 cells. In dexamethasone-induced M10 cells, the nicotinic agonists acetylcholine, anatoxin, 1,1-dimethyl-4-phenylpiperazinium, (-)-nicotine, and cytisine (each at 100 microM) activated whole-cell currents that showed a marked inward rectification and were sensitive to blockade by dihydro-beta-erythroidine (100 nM). However, neither galanthamine nor physostigmine could evoke whole-cell currents in cells that were responsive to nicotinic agonists. Other effects of physostigmine and galanthamine on the nicotinic receptor that outweight the agonist properties of these compounds could account for their inability to evoke whole-cell currents.

Animals↗
PubMed1993

Molecular determinants of general anesthetic action: role of GABAA receptor structure.

Using receptors expressed from mouse brain mRNA in Xenopus oocytes, we found that enhancement of type A gamma-aminobutyric acid (GABAA) receptor-gated Cl- channel response is a common action of structurally diverse anesthetics, suggesting that the GABAA receptor plays an important role in anesthesia. To determine if GABAA receptor subunit composition influences actions of anesthetics, we expressed subunit cRNAs in Xenopus oocytes and measured effects of enflurane on GABA-activated Cl- currents. Potentiation of GABA-activated currents by enflurane was dependent on the composition of GABAA receptor protein subunits; the order of sensitivity was alpha 1 beta 1 > alpha 1 beta 1 gamma 2S = alpha 1 beta 1 gamma 2L > total mRNA. The results suggest that anesthetics with simple structures may act on the GABAA receptor protein complex to modulate the Cl- channel activity and provide a molecular explanation for the synergistic clinical interactions between benzodiazepines and general anesthetics.

Anesthesia↗
PubMed1993

(+)-Anatoxin-a is a potent agonist at neuronal nicotinic acetylcholine receptors.

The effects of the nicotinic agonist (+)-anatoxin-a have been examined in four different preparations, representing at least two classes of neuronal nicotinic receptors. (+)-Anatoxin-a was most potent (EC50 = 48 nM) in stimulating 86Rb+ influx into M10 cells, which express the nicotinic receptor subtype comprising alpha 4 and beta 2 subunits. A presynaptic nicotinic receptor mediating acetylcholine release from hippocampal synaptosomes was similarly sensitive to (+)-anatoxin-a (EC50 = 140 nM). alpha-Bungarotoxin-sensitive neuronal nicotinic receptors, studied using patch-clamp recording techniques, required slightly higher concentrations of this alkaloid for activation: Nicotinic currents in hippocampal neurons were activated by (+)-anatoxin-a with an EC50 of 3.9 microM, whereas alpha 7 homooligomers reconstituted in Xenopus oocytes yielded an EC50 value of 0.58 microM for (+)-anatoxin-a. In these diverse preparations, (+)-anatoxin-a was between three and 50 times more potent than (-)-nicotine and approximately 20 times more potent than acetylcholine, making it the most efficacious nicotinic agonist thus far described.

Acetylcholine↗
PubMed1993

The influence of the gamma 2L subunit on the modulation of responses to GABAA receptor activation.

1. Whole-cell patch clamp recordings were made from L-cells transfected with 2 combinations of subunits of the GABAA receptor. Log concentration-response curves were constructed to gamma-aminobutyric acid (GABA) on alpha 1,beta 1,gamma 2L containing cells and compared to those from alpha 1,beta 1 containing cells. The effects of flunitrazepam, pentobarbitone and alphaxalone on the concentration-response relationships were also examined. 2. From the log concentration-response curves, GABA had a mean (+/- s.e. mean) pEC50 = 5.2 +/- 0.09 and slope factor = 1.7 +/- 0.02 on alpha 1,beta 1,gamma 2L cells which were significantly different from the values obtained from alpha 1,beta 1 cells where the pEC50 = 5.6 +/- 0.02 and the slope = 1.5 +/- 0.02. 3. Flunitrazepam produced a parallel leftward shift of GABA concentration-response curves on alpha 1,beta 1,gamma 2L cells. The EC50 for flunitrazepam = 6.3 +/- 2.7 nM. No increase in the maxima of the GABA concentration-response curves was found in the presence of flunitrazepam. Flunitrazepam did not potentiate responses from alpha 1,beta 1 cells. 4. The log concentration-response curves from both populations of cells were shifted to the left by equal amounts by pentobarbitone. A significant increase in the maximal response to GABA was also produced by pentobarbitone. This occurred at lower concentrations of pentobarbitone on alpha 1,beta 1 cells. 5. Alphaxalone produced leftward shifts of GABA log concentration-response curves of similar magnitudes in both populations of cells. Significant increases in the maxima were found at 100 nM in alpha 1, beta 1 cells but not up to 1 microM in alpha 1,beta 1,gamma 2L cells.6. These results provide further evidence of the modulatory role of the gamma 2L subunit of the GABAA receptor containing alpha 1 and beta 2 subunits. As well as influencing the apparent affinity of GABA and conferring benzodiazepine modulation, it also appeared to regulate the increase in maximal response produced in the presence of barbiturates and steroids. This latter effect may imply that barbiturates and steroids increase the channel open-state probability in the presence of GABA and that this effect is diminished by the presence of the gamma 2L subunit.

Anesthetics↗
PubMed1993

Pharmacology of GABA rho 1 and GABA alpha/beta receptors expressed in Xenopus oocytes and COS cells.

1. The rho 1 protein, which we previously cloned from retina, assembles as a homooligomer that transduces the binding of gamma-aminobutyric acid (GABA) into robust chloride currents. However, its insensitivity to bicuculline, pentobarbitone and benzodiazepines, all potent agents at typical GABAA receptors, suggested that it may react atypically to other GABA agonists and antagonists. 2. cDNAs for the rho 1 and the alpha 5 beta 1 receptors for GABA were expressed as homo- and heterooligomers, respectively, in Xenopus oocytes. The selectivities of the respective receptors for various agonists were investigated using concentration-response experiments in voltage clamped cells. 3. The most potent agonists at the rho 1 receptor were trans-4-aminocrotonic acid (TACA) > GABA > muscimol; at the alpha 5 beta 1 receptor the rank order was muscimol > GABA > 4,5,6,7-tetrahydroisoxazole[4,5-c]pyridine-3-ol (THIP). The most specific agonists were cis-(2-(aminomethyl)-cyclopropyl-carboxylic acid (CAMP) and THIP for the rho 1 and the alpha 5 beta 1 receptors, respectively. 4. Comparing GABA, TACA and cis-aminocrotonic acid (CACA) at rho 1 receptors expressed in COS cells gave results almost indistinguishable from those found at oocytes; the pharmacology of rho 1 seems independent of the expression system. 5. Agonists THIP, piperidine-4-sulphonic acid (P4S), and isoguvacine, whose C-C-C-N chains are constrained by rings into a folded conformation and were potent at the alpha 5 beta 1 receptor, were among the weakest at the rho 1 receptor. However CACA and CAMP, which align better with the extended than the folded conformation, were weakest at the alpha 5 beta 1 receptor but moderately potent at the pl receptor. These findings suggest that the rho l receptor recognizes agonists in the extended conformation, in contrast to GABAA receptors, which are believed to recognize agonists in the partially folded conformation.6. In contrast to the alpha 5 beta 1 receptor, gradations in maximum responses were apparent in the rho l receptor,suggesting various degrees of partial agonism. In particular, imidazole-4-acetic acid (I4AA), whose maximum response was only 3% of GABA's maximum, had an apparent Kd for activating the rho l receptor of 16 microM; but it had an apparent Kd for competitively blocking the receptor of 0.64 microM. This difference suggests that steric constraints in the activated (open channel) receptor are tighter than in the resting receptor.7. Hill coefficients approached 2 at the rho l receptor, but were closer to unity at the alpha 5 beta 1 receptor. Thus,the rho l receptor displayed higher cooperativity.8. Unlike typical GABAA receptors, the rho l receptor was insensitive to the competitive antagonists bicuculline, SR95531, securinine, and (+)-tubocurarine.

Animals↗
PubMed1993

Three subtypes of alpha-bungarotoxin-sensitive nicotinic acetylcholine receptors are expressed in chick retina.

A recent report described the isolation of cDNA clones encoding alpha 7 and alpha 8 subunits of alpha-bungarotoxin-sensitive nicotinic ACh receptors (alpha BgtAChRs) from chick brain and demonstrated that they were related to, but distinct from, the alpha subunits of nicotinic ACh receptors (nAChRs) from muscles and neurons. Monoclonal antibodies against the two alpha BgtAChR subunits were used to demonstrate that at least two subtypes are present in embryonic day 18 chicken brain. The predominant brain subtype contains alpha 7 subunits, while a minor subtype contains both alpha 7 and alpha 8 subunits. Both subtypes may also contain other subunits. Here we report the results of immune precipitation studies and immunohistochemical studies of alpha BgtAChRs in the chick retina. In addition to the two subtypes found in brain, a new alpha BgtAChR subtype that contains alpha 8 subunits, but not alpha 7 subunits, was identified and was found to be the major subtype in chick retina. This subtype has a lower affinity for alpha-bungarotoxin (alpha Bgt) than does the subtype containing only alpha 7 subunits. Small amounts of this alpha 8 subtype were also detected in brain by labeling with higher concentrations of 125I-alpha Bgt than had been used previously. The subtype containing only alpha 7 subunits comprised 14% of the alpha BgtAChRs in hatchling chick retina. The subtype containing alpha 8 subunits (but no alpha 7 subunits) accounted for 69%, and the alpha 7 alpha 8 subtype accounted for 17%. Amacrine, bipolar, and ganglion cells displayed alpha 8 subunit immunoreactivity, and a complex pattern of labeling was evident in both the inner and outer plexiform layers. In contrast, only amacrine and ganglion cells exhibited alpha 7 subunit immunoreactivity, and the pattern of alpha 7 subunit labeling in the inner plexiform layer differed from that of alpha 8 subunit labeling. These disparities suggest that the alpha BgtAChR subunits are differentially expressed by different populations of retinal neurons. In addition, the distribution of alpha BgtAChR subunit immunoreactivity was found to differ from that of alpha-Bgt-insensitive nAChR subunits.

Animals↗
PubMed1992

Human chromosomal localization of genes encoding the gamma 1 and gamma 2 subunits of the gamma-aminobutyric acid receptor indicates that members of this gene family are often clustered in the genome.

The gamma-aminobutyric acid (GABA) receptors are the major inhibitory neurotransmitter receptors in the brain and the site of action of a number of important pharmacological agents including barbiturates, benzodiazepines, and ethanol. The gamma 1 and gamma 2 subunits have been shown to be important in mediating responses to benzodiazepines, and a splicing variant of the gamma 2 subunit, gamma 2L, has been shown to be necessary for ethanol actions on the receptor, raising the possibility that the gamma 2 gene may be involved in human genetic predisposition to the development of alcoholism. We have assigned the human genes encoding the gamma 1 and gamma 2 subunits of the GABAA receptor to chromosomes 4 and 5, respectively, by PCR amplification of human-specific products from human-hamster somatic cell hybrid DNAs. Using panels of chromosome-specific natural deletion hybrids, we have further localized the gamma 1 gene (GABRG1) to 4p14-q21.1 and the gamma 2 gene (GABRG2) to 5q31.1-q33.2. These data indicate that the gamma 1 gene may be clustered together with the previously mapped alpha 2 and beta 1 genes on chromosome 4 and that the gamma 2 gene may be close to the previously localized alpha 1 gene on chromosome 5. To further examine the latter possibility the alpha 1 gene was mapped using the chromosome 5 deletion hybrids and shown to be within the same region as the gamma 2 gene, 5q31.1-q33.2. A PCR-based screening strategy was used to isolate a 450-kilobase human genomic yeast artificial chromosome clone containing both the alpha 1 and gamma 2 genes. Pulsed-field gel restriction mapping of the yeast artificial chromosome indicates that the two genes are within 200 kilobases of each other. The data presented here provide further evidence for the nonrandom organization of the human genome by demonstrating that members of the GABAA receptor gene family often occur in small gene clusters widely distributed in the genome.

Base Sequence↗
PubMed1992

cDNA cloning of human and rat brain myo-inositol monophosphatase. Expression and characterization of the human recombinant enzyme.

Inositol monophosphatase (EC 3.1.3.25) is a key enzyme in the phosphoinositide cell-signalling system. Its role is to provide inositol required for the resynthesis of phosphatidylinositol and polyphosphoinositides. It is the probable pharmacological target for lithium action in brain. Using probes derived from the bovine inositol monophosphatase cDNA we have isolated cDNA clones encoding the human and rat brain enzymes. The enzyme is highly conserved in all three species (79% identical). The coding region of the human cDNA was inserted into a bacterial expression vector. The expressed recombinant enzyme was purified and its biochemical properties examined. The human enzyme is very similar to the bovine enzyme.

Amino Acid Sequence↗
PubMed1992

Molecular cloning of a serotonin receptor from human brain (5HT1E): a fifth 5HT1-like subtype.

Degenerate primers, suitable for polymerase chain reaction studies and based on the conserved structure of G protein-coupled receptors, were used to isolate cDNA clones encoding putative G protein-coupled receptors from a human hippocampal cDNA library. One clone isolated by this approach (AC1) encoded a putative receptor with 39% amino acid sequence identity to the serotonin 5HT1A receptor and 47% identity to the 5HT1D receptor. When expressed transiently in the human embryonic kidney cell line 293, AC1 cDNA-encoded receptor displayed high affinity (Kd = 15 nM) and saturability for [3H]serotonin, suggesting that AC1 encodes a 5HT1-like receptor. However, 5-carboxamidotryptamine demonstrated low affinity (pKi = 5.15) compared with serotonin (pKi = 8.14), consistent with the observed binding of the putative 5HT1E receptor. The excellent correlation observed between the pharmacology of the expressed receptor encoded by AC1 and the human brain 5HT1E binding site confirms that AC1 encodes a 5HT1E receptor and establishes a fifth 5HT1-like receptor subtype.

Adenylyl Cyclases↗
PubMed1992

The pharmacology of recombinant GABAA receptors containing bovine alpha 1, beta 1, gamma 2L sub-units stably transfected into mouse fibroblast L-cells.

1. Responses to gamma-aminobutyric acid (GABA) were evoked in mouse fibroblast L-cells stably transfected with bovine, alpha 1, beta 1, gamma 2L sub-units of the GABAA receptor. Expression was stimulated via a steroid-inducible promoter system. 2. In near symmetrical intracellular and extracellular chloride concentrations, GABA evoked inward currents at negative holding potentials that reversed at +5 mV and displayed slight outward rectification. Concentration-response curves were fitted well by the logistic equation. GABA had a pEC50 = 5.1 +/- 0.1 and the curves had a slope of 1.9 +/- 0.1. 3. Responses to GABA were antagonized by bicuculline, picrotoxin and penicillin. The action of bicuculline was competitive (pA2 = 6.4) whilst the block by picrotoxin was uncompetitive and strongly agonist-dependent. 4. Benzodiazepine receptor agonists potentiated responses to 3 microM GABA. The rank order of potency was FG 8205 > flunitrazepam > zolpidem > C1218872. FG 8205 and C1218872 produced markedly lower maximal potentiations with efficacies 0.4 and 0.6 x that of flunitrazepam, respectively. The potencies of zolpidem and C1218872 observed are in agreement with the BZ1 type pharmacology of this sub-unit combination. The potentiation of GABA by flunitrazepam was antagonized by flumazenil with a Ki of 3.8 nM. 5. GABA responses were potentiated in the presence of pentobarbitone and alphaxalone. The response was also noticeably broadened by these compounds due to a decrease in the response decay rate. Concentrations of pentobarbitone of 100 microM and above evoked an inward current in the absence of GABA. Alphaxalone up to 10 microM did not evoke a direct response. 6. This expression system produced functional receptors that behaved in a fashion analogous to those found endogenously in other preparations. Thus, this system appears to provide a useful and versatile preparation for the analysis of sub-unit regulation of GABAA receptor pharmacology.

Animals↗
PubMed1992

The use of lithium clearance studies in the early detection of cyclosporin A (CsA) nephrotoxicity: a protocol of renal function assessment with CsA therapy.

Cyclosporin A nephrotoxicity was studied with the use of lithium and creatinine clearance tests in 18 patients with chronic intraocular inflammation (duration of treatment 3-48 months). In 11 patients lithium clearance and fractional excretion of lithium were significantly reduced (compared with pretreatment levels) within the first six months of treatment. There was no significant change in either serum creatinine or creatinine clearance within this period. In 14/18 patients there was a significant reduction in lithium clearance and fractional excretion of lithium during the treatment period. 7 patients whose therapy was stopped because of continuing nephrotoxicity despite dose reduction, demonstrated some reversibility of renal function on cessation of cyclosporin A. We propose a protocol for the assessment of renal function in these patients so that with dose modulation the changes in these parameters can be minimised, reducing the risk of renal impairment, whilst maximising immunosuppressive treatment.

Chronic Disease↗
PubMed1992

Activation of protein kinase C selectively inhibits the gamma-aminobutyric acidA receptor: role of desensitization.

The effects of protein kinase C (PKC) activators on gamma-aminobutyric acidA (GABAA) receptor function were studied by two-electrode voltage-clamp in Xenopus oocytes expressing brain mRNA or subunit cDNAs and in isolated mouse brain cerebellar membrane vesicles (microsacs), using 36Cl- uptake. Both oocytes and microsacs showed transient (desensitizing) and sustained (nondesensitizing) GABAA receptor responses. In oocytes expressing brain mRNA, the PKC activator phorbol myristoyl acetate (PMA), but not the inactive analog phorbol 12-monomyristate, inhibited both transient and sustained GABA-gated chloride currents. The inhibition by PMA was concentration dependent, with an EC50 of approximately 5 nM, and resulted in a decrease in the efficacy, but not the potency, of GABA. Additionally, PMA inhibited GABA-gated chloride currents in oocytes expressing alpha 1 beta 1 gamma 2L subunit cDNAs. The effect of PMA on recombinant receptors was significantly antagonized by PKC inhibitory peptide (PKCI). In the microsac preparation, the PKC activators (-)-7-octylindolactam V and PMA inhibited the sustained phase of 36Cl- flux without altering the transient phase. The action of PMA was blocked by kinase inhibitors and by depletion of Mg-ATP and was mimicked by protein phosphatase inhibitors. These results demonstrate that activation of PKC inhibits GABAA receptor function, and the results from the microsac experiments suggest that PKC-dependent phosphorylation preferentially inactivates a nondesensitized form or state of the receptor.

Animals↗
PubMed1991

Differential expression of GABAA receptor alpha-subunits in rat brain during development.

Unique cytoplasmic loop regions of the alpha 1, alpha 2, alpha 3, and alpha 5 subunits of the GABAA receptor have been expressed in E. coli and used to generate polyclonal antisera specific for these subunits. The antibodies identify proteins by SDS-polyacrylamide gel electrophoresis and western blotting of molecular size 51 kDa, 53 kDa, 59 kDa and 55 kDa, respectively, which show differential patterns of expression during development. Whereas the alpha 2 and alpha 3 subunits are present at early stages, the expression of alpha 1 and alpha 3 subunits is low at birth and increases with age. This differential expression could be correlated with previous studies examining the developmental expression of BZ1 and BZ2 benzodiazepine binding sites.

Animals↗