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Biomedical subjects

G S Oxford

Publications and source records attributed to G S Oxford.

At least 19 recordsLinked to original sources
PubMed2005

Genetic drift within a protected polymorphism: enigmatic variation in color-morph frequencies in the candy-stripe spider, Enoplognatha ovata.

The candy-stripe spider, Enoplognatha ovata, exhibits a striking color polymorphism comprising three morphs. A number of lines of evidence strongly suggest that this polymorphism is maintained by natural selection: its presence in a sister species, E. latimana; the physical nature of the variation; the virtual lack of monomorphic populations; the highly consistent rank-order of morphs within populations; and the presence of large-scale clines associated with climatic variables. However, the absence of selection is equally strongly suggested by very local surveys of morph frequencies over space and time, perturbation experiments, and a variance in morph frequency between populations that is virtually independent of spatial scale. In addition, local spatial patterns in one study site (Nidderdale, Yorkshire, England) have been explained in terms of intermittent drift over half a century ago, a hypothesis supported here by the distributions of four other genetic markers (two allozyme and two visible polymorphisms). A heuristic model is suggested that reconciles these apparently contradictory messages regarding the importance of drift and selection in this system. It is proposed that when allele frequencies of the color morph redimita lie between approximately 0.05 and 0.3, the deltaq on q plot is very shallow, so that within this region, where the majority of populations lie, selection is weak and drift is the major force determining local morph frequencies. However, outside this range of frequencies, powerful selection acts to protect the polymorphism. This model may apply to polymorphisms in other species and explain why evidence of selection in natural populations is often elusive.

Animals↗
PubMed2001

Functional analysis of capsaicin receptor (vanilloid receptor subtype 1) multimerization and agonist responsiveness using a dominant negative mutation.

The recently cloned vanilloid receptor subtype 1 (VR1) is a ligand-gated channel that is activated by capsaicin, protons, and heat. We have attempted to develop a dominant negative isoform by targeting several mutations of VR1 at highly conserved amino acids or at residues of potential functional importance and expressing the mutants in Chinese hamster ovary cells. Mutation of three highly conserved amino acid residues in the putative sixth transmembrane domain disrupts activation of the VR1 receptor by both capsaicin and resiniferatoxin. The vanilloid binding site in this mutant is intact, although the affinity for [(3)H]resiniferatoxin (RTX) is diminished by nearly 40-fold. Interestingly, this mutant retains a significant but diminished response to protons, supporting the existence of multiple gating mechanisms for different stimuli. The mutant appears to function by interfering with the gating induced by vanilloids rather than the expression level or permeability of the receptor. In addition, this mutant was found to function as a strong dominant negative mutation when coexpressed with wild-type VR1, providing functional evidence that the VR1 receptor forms a multimeric complex. Analysis of both current density and [(3)H]RTX affinity in cells cotransfected with different ratios of wild-type and mutant VR1 is consistent with tetrameric stoichiometry for the native capsaicin receptor.

Amino Acid Substitution↗
PubMed2001

Sucrase-isomaltase is an adenosine 3',5'-cyclic monophosphate-dependent epithelial chloride channel.

BACKGROUND & AIMS: We previously isolated a monoclonal antibody against a Necturus gallbladder epitope that blocks native adenosine 3',5'-cyclic monophosphate (cAMP)-dependent chloride channels in intestine, gallbladder, urinary bladder, and airway epithelia in various animals. METHODS: Using this antibody, we purified a 200-kilodalton protein that, when reconstituted in lipid bilayers, forms 9-pS chloride channels that are blocked by the antibody. RESULTS: Amino acid sequencing of the purified protein showed strong homology to rabbit sucrase-isomaltase, an abundant intestinal enzyme. Western blot analysis of the in vitro-translated sucrase-isomaltase was indistinguishable from that of the protein used in the lipid bilayer studies. Expression of this protein in Chinese hamster ovary cells and in Xenopus laevis oocytes yielded cAMP-dependent chloride currents that in the latter system were blocked by the antibody. CONCLUSIONS: Because the monoclonal antibody blocks cAMP-dependent currents in epithelia as well as those produced both by the reconstituted and by the heterologously expressed protein, sucrase-isomaltase is a cAMP-dependent epithelial chloride channel. Thus an enzyme that can also function as an ion channel has been described for the first time.

Amino Acid Sequence↗
PubMed2001

Critical issues in PhD training for biomedical scientists.

The rapidly changing world of modern biomedical research is raising important new issues for traditional PhD training programs and is creating concern among young PhD scientists about their futures. Specifically, the United States is producing substantially more biomedical PhDs than can be accommodated in professional positions that truly require the PhD as a credential. The "surplus" PhD population is being relegated to poorly paid, unstable, and increasingly unsatisfying jobs. In addition, many current graduate and postdoctoral training programs may not be adequately preparing young scientists for the more complex, more quantitative biological science of the future. Finally, many current graduate training programs are not attracting a sufficient portion of the most talented young people in the nation. To ameliorate these problems in the training and early career paths of basic biomedical scientists, the authors make specific recommendations, such as urging (1) that graduate trainees should be supported exclusively by competitive individual fellowships, training grants, or institutional funds and not by RO1s or similar research awards; (2) that graduate and postdoctoral stipends be increased so that they provide a reasonable living wage; and (3) that research-intensive academic institutions create a career path for biomedical PhDs other than that designed for the traditional tenure-track, grant-funded principal investigator and faculty member. They conclude that it is in the interest of faculty and institutions to make these and other drastic changes because the current system is both inherently unfair and self-destructive.

Biomedical Technology↗
PubMed2000

Dominant-negative mutants identify a role for GIRK channels in D3 dopamine receptor-mediated regulation of spontaneous secretory activity.

The human D3 dopamine receptor can activate G-protein-coupled inward rectifier potassium channels (GIRKs), inhibit P/Q-type calcium channels, and inhibit spontaneous secretory activity in AtT-20 neuroendocrine cells (Kuzhikandathil, E.V., W. Yu, and G.S. Oxford. 1998. Mol. Cell. Neurosci. 12:390-402; Kuzhikandathil, E.V., and G.S. Oxford. 1999. J. Neurosci. 19:1698-1707). In this study, we evaluate the role of GIRKs in the D3 receptor-mediated inhibition of secretory activity in AtT-20 cells. The absence of selective blockers for GIRKs has precluded a direct test of the hypothesis that they play an important role in inhibiting secretory activity. However, the tetrameric structure of these channels provides a means of disrupting endogenous GIRK function using a dominant negative approach. To develop a dominant-negative GIRK mutant, the K(+) selectivity amino acid sequence -GYG- in the putative pore domain of the human GIRK2 channels was mutated to -AAA-, -GLG-, or -GFG-. While the mutation of -GYG- to -GFG- did not affect channel function, both the -AAA- and -GLG- GIRK2 mutants were nonfunctional. This suggests that the aromatic ring of the tyrosine residue rather than its hydroxyl group is involved in maintaining the pore architecture of human GIRK2 channels. When expressed in AtT-20 cells, the nonfunctional AAA-GIRK2 and GLG-GIRK2 acted as effective dominant-negative mutants and significantly attenuated endogenous GIRK currents. Furthermore, these dominant-negative mutants interfered with the D3 receptor-mediated inhibition of secretion in AtT-20 cells, suggesting they are centrally involved in the signaling pathway of this secretory response. These results indicate that dominant-negative GIRK mutants are effective molecular tools to examine the role of GIRK channels in vivo.

Action Potentials↗
PubMed2000

Voltage-dependent ion channels in CAD cells: A catecholaminergic neuronal line that exhibits inducible differentiation.

Cell lines derived from tumors engineered in the CNS offer promise as models of specific neuronal cell types. CAD cells are an unusual subclone of a murine cell line derived from tyrosine hydroxylase (TH) driven tumorigenesis, which undergoes reversible morphological differentiation on serum deprivation. Using single-cell electrophysiology we have examined the properties of ion channels expressed in CAD cells. Despite relatively low resting potentials, CAD cells can be induced to fire robust action potentials when mildly artificially hyperpolarized. Correspondingly, voltage-dependent sodium and potassium currents were elicited under voltage clamp. Sodium currents are TTX sensitive and exhibit conventional activation and inactivation properties. The potassium currents reflected two pharmacologically distinguishable populations of delayed rectifier type channels while no transient A-type channels were observed. Using barium as a charge carrier, we observed an inactivating current that was completely blocked by nimodipine and thus associated with L-type calcium channels. On differentiation, three changes in functional channel expression occurred; a 4-fold decrease in sodium current density, a 1.5-fold increase in potassium current density, and the induction of a small noninactivating barium current component. The neuronal morphology, excitability properties, and changes in channel function with differentiation make CAD cells an attractive model for study of catecholaminergic neurons.

Action Potentials↗
PubMed1999

Association of the D2 dopamine receptor third cytoplasmic loop with spinophilin, a protein phosphatase-1-interacting protein.

Signaling through D2 class dopamine receptors is crucial to correct brain development and function, and dysfunction of this system is implicated in major neurological disorders such as Parkinson's disease and schizophrenia. To investigate potential novel mechanisms of D2 receptor regulation, the third cytoplasmic loop of the D2 dopamine receptor was used to screen a rat hippocampal yeast two-hybrid library. Spinophilin, a recently characterized F-actin and protein phosphatase-1-binding protein with a single PDZ domain was identified as a protein that specifically associates with this region of D2 receptors. A direct interaction between spinophilin and the D2 receptor was confirmed in vitro using recombinant fusion proteins. The portion of spinophilin responsible for interacting with the D2 third cytoplasmic loop was narrowed to a region that does not include the actin-binding domain, the PDZ domain, or the coiled-coil. This region is distinct from the site of interaction with protein phosphatase-1, and both D2 receptors and protein phosphatase-1 may bind spinophilin at the same time. The interaction is not mediated via the unique 29-amino acid insert in D2long; both D2long and D2short third cytoplasmic loops interact with spinophilin in vitro and in yeast two-hybrid assays. Expression of D2 receptors containing an extracellular hemagglutinin epitope in Madin-Darby canine kidney cells results in co-localization of receptor and endogenous spinophilin as determined by immunocytochemistry using antibodies directed against spinophilin and the HA tag. We hypothesize that spinophilin is important for establishing a signaling complex for dopaminergic neurotransmission through D2 receptors by linking receptors to downstream signaling molecules and the actin cytoskeleton.

Actins↗
PubMed1999

Activation of human D3 dopamine receptor inhibits P/Q-type calcium channels and secretory activity in AtT-20 cells.

The D3 dopamine receptor is postulated to play an important role in the regulation of neurotransmitter secretion at both pre- and postsynaptic terminals. However, this hypothesis and the underlying mechanisms remain untested because of the lack of D3-selective ligands, paucity of appropriate model secretory systems, and the weak and inconsistent coupling of D3 receptors to classical signal transduction pathways. The absence of ligands that selectively discriminate between D3 and D2 receptors in vivo precludes the study of D3 receptor function in the brain and necessitates the use of heterologous expression systems. In this report we demonstrate that activation of the human D3 dopamine receptor expressed in the AtT-20 neuroendocrine cell line causes robust inhibition of P/Q-type calcium channels via pertussis toxin-sensitive G-proteins. In addition, using the vesicle trafficking dye FM1-43, we show that D3 receptor activation significantly inhibits spontaneous secretory activity in these cells. Our results not only support the hypothesis that the D3 receptor can regulate secretory activity but also provide insight into the underlying signaling mechanisms. We propose a functional model in which the D3 receptor tightly regulates neurotransmitter release at a synapse by only allowing the propagation of spikes above a certain frequency or burst-duration threshold.

Animals↗
PubMed1998

Human dopamine D3 and D2L receptors couple to inward rectifier potassium channels in mammalian cell lines.

The molecular mechanisms coupling the D3 dopamine receptor to downstream effectors have neither been well defined nor well characterized. Here we examine the coupling of the human D3 receptor to G-protein coupled inward rectifier potassium channels (GIRKs) in mammalian cells. Human D3 receptors couple strongly to homomeric human GIRK2 channels coexpressed in Chinese hamster ovary (CHO) cells, with a coupling efficiency comparable to that of D2L receptors. The coupling between D3 receptors and native GIRK channels was examined in an AtT-20 mouse pituitary cell line stably expressing the human D3 receptor. AtT-20 cells endogenously express somatostatin and muscarinic receptors coupled to GIRK channels. RT-PCR and Western blot analyses revealed that AtT-20 cells natively express Kir3.1 and Kir3.2 channel isoforms, but not D2 or D3 dopamine receptors. In D3 receptor expressing AtT-20 cells, application of the D2/D3 receptor agonist, quinpirole, induces pertussis toxin-sensitive inward rectifying K+ currents that are blocked by barium. Activation of D3 receptors leads to both homologous desensitization of this receptor and an unusual unidirectional heterologous desensitization of somatostatin receptors. AtT-20 cells may be a good model to examine the functional role of D3 dopamine receptors in regulating neurotransmitter secretion.

Adenosine Triphosphate↗
PubMed1998

Evolution and ecology of spider coloration.

Genetic color variation provides a tangible link between the external phenotype of an organism and its underlying genetic determination and thus furnishes a tractable system with which to explore fundamental evolutionary phenomena. Here we examine the basis of color variation in spiders and its evolutionary and ecological implications. Reversible color changes, resulting from several mechanisms, are surprisingly widespread in the group and must be distinguished from true genetic variation for color to be used as an evolutionary tool. Genetic polymorphism occurs in a large number of families and is frequently sex limited: Sex linkage has not yet been demonstrated, nor have the forces promoting sex limitation been elucidated. It is argued that the production of color is metabolically costly and is principally maintained by the action of sight-hunting predators. Key avenues for future research are suggested.

Journal Article↗
PubMed1997

The role of calcium in the desensitization of capsaicin responses in rat dorsal root ganglion neurons.

Capsaicin (Cap) is a pungent extract of the Capsicum pepper family, which activates nociceptive primary sensory neurons. Inward current and membrane potential responses of cultured neonatal rat dorsal root ganglion neurons to capsaicin were examined using whole-cell and perforated patch recording methods. The responses exhibited strong desensitization operationally classified as acute (diminished response during constant Cap exposure) and tachyphylaxis (diminished response to successive applications of Cap). Both acute desensitization and tachyphylaxis were greatly diminished by reductions in external Ca2+ concentration. Furthermore, chelation of intracellular Ca2+ by addition of either EGTA or bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid to the patch pipette attenuated both forms of desensitization even in normal Ca2+. Release of intracellular Ca2+ by caffeine triggered acute desensitization in the absence of extracellular Ca2+, and barium was found to effectively substitute for calcium in supporting desensitization. Cap activated inward current at an ED50 of 728 nM, exhibiting cooperativity (Hill coefficient, 2.2); however, both forms of desensitization were only weakly dependent on [Cap], suggesting a dissociation between activation of Cap-sensitive channels and desensitization. Removal of ATP and GTP from the intracellular solutions resulted in nearly complete tachyphylaxis even with intracellular Ca2+ buffered to low levels, whereas changes in nucleotide levels did not significantly alter the acute form of desensitization. These data suggest a key role for intracellular Ca2+ in desensitization of Cap responses, perhaps through Ca2+-dependent dephosphorylation at a locus that normally sustains Cap responsiveness via ATP-dependent phosphorylation. It also seems that the signaling mechanisms underlying the two forms of desensitization are not identical in detail.

Adenosine Triphosphate↗
PubMed1994

Absence of coupling between D2 dopamine receptors and calcium channels in lactotrophs from cycling female rats.

Recent evidence suggests that an important mechanism underlying the inhibition of PRL secretion by dopamine in the anterior pituitary is a direct inhibition of current through voltage-gated calcium channels. An alternative mechanism involves the activation of G protein-coupled potassium channels by D2 receptor activation, subsequent hyperpolarization of the lactotroph membrane, and an indirect inhibition of calcium influx as spontaneous electrical activity is reduced. Using patch voltage clamp methods, we have reexamined the effect of D2 receptor activation on calcium currents (ICa) in pituitary cells from normal cycling female rats and in GH4Cl pituitary tumor cells expressing cloned D2 receptors. Furthermore, we have examined secretory responses using a single cell immunoblot method. Dopamine (0.1-10 microM) failed to significantly inhibit ICa in either GH4Cl cells or normal female lactotrophs. Similarly, the D2 agonist quinpirole (20-100 microM) did not reduce ICa in lactotrophs. No responses to D2 agonists were seen when barium was substituted for calcium or when experiments were performed using the nystatin-permeabilized patch technique to avoid loss of intracellular macromolecules. Quinpirole also failed to inhibit ICa in lactotrophs isolated from lactating female rats. We have thus far been unable to observe a significant inhibition of ICa by activation of D2 receptors. PRL secretion assessed by immunoblotting methods was dramatically inhibited by quinpirole at normal (5 mM) extracellular K+. However, in elevated (50 mM) K+ that depolarizes the cells and activates calcium channels, quinpirole produced only a very modest inhibition of secretion. We conclude that direct inhibition of ICa by D2 receptor activation is not a major mechanism underlying the dopaminergic inhibition of PRL, secretion in normal female lactotrophs.

Animals↗
PubMed1993

Amylin modulates beta-cell glucose sensing via effects on stimulus-secretion coupling.

The release of insulin from the pancreatic beta cell is dependent upon a complex interplay between stimulators and inhibitors. Recently, amylin, a peptide secreted by pancreatic beta cells, has been implicated in the development of type II (noninsulin dependent) diabetes through its modulation of the peripheral effects of insulin. However, the effect of amylin on insulin secretion from the beta cell has remained controversial. It is reported here that in single beta cells exhibiting normal glucose sensing, amylin causes membrane hyperpolarization, increases in net outward current, and reductions in insulin secretion. In contrast, in cells with abnormal glucose sensing (e.g., from db/db diabetic mice), amylin has no effect on electrical activity or secretion. Thus, amylin's effects on excitation-secretion coupling in the beta cell of the pancreas appear to be linked to the cell's capacity for normal glucose sensing.

Amyloid↗
PubMed1993

Modulation of ion channels underlying excitation-secretion coupling in identified lactotrophs and gonadotrophs.

The role of ion channel activity in the response of rat pituitary lactotrophs and gonadotrophs to dopamine (DA) and GnRH, respectively, was investigated. Single lactotrophs and gonadotrophs were unambiguously identified with the reverse hemolytic plaque assay, and recordings of membrane potential and current were obtained using whole-cell and single-channel patch-clamp techniques. In lactotrophs, DA inhibited spontaneous electrical activity by activating a K+ conductance that hyperpolarized the cells. A 50 pS K+ channel underlies this response and was activated following agonist binding to a D2 type receptor via a "direct" interaction with a pertussis toxin-sensitive G-protein. In gonadotrophs, GnRH triggers rhythmic hyperpolarizations due also to a K+ conductance increase. The K+ channel underlying the GnRH response is an apamin-sensitive, Ca(2+)-activated channel. Although both agonists produce hyperpolarizations in their respective target cells via K+ channel activation, differences in intracellular calcium response probably discriminate the stimulatory (GnRH) and inhibitory (DA) actions on hormone secretion. Each K+ channel type plays a different role in modulating the intracellular Ca2+ levels to yield these actions.

Action Potentials↗
PubMed1993

Guanine nucleotide binding proteins mediate D2 dopamine receptor activation of a potassium channel in rat lactotrophs.

1. The involvement of guanine nucleotide binding proteins in the coupling of D2 dopamine (DA) receptors to single potassium channels was examined in rat pituitary lactotrophs. 2. Lactotrophs were unambiguously identified by the reverse haemolytic plaque assay (RHPA) and membrane potentials, whole-cell and single channel currents recorded using patch electrode methods. 3. DA or the D2 selective agonist, quinpirole, induced the opening of single K+ channels in cell-attached patches underlying robust hyperpolarizations of membrane potential in single cells. 4. Both whole-cell and single channel responses were independent of Ca2+ or cAMP concentrations. 5. Pertussis toxin (PTX) pretreatment (50-250 ng/ml, 6-12 h) blocked the action of DA on lactotroph membrane potential and uncoupled D2 receptors from single K+ channels in cell-attached patches. 6. Internal dialysis with GDP beta S (guanosine 5'-O-(2-thiodiphosphate) greatly reduced whole-cell responses to DA in a dose-dependent manner. 7. Internal dialysis of lactotrophs with GTP gamma S (guanosine 5'-O-(3-thiotriphosphate) potentiated DA responses in a dose-dependent manner while rendering the responses irreversible at higher doses. 8. DA (100 nM) or quinpirole (10 microM) activated K+ channels in excised outside-out membrane patches that were identical to those identified in cell-attached patches in terms of conductance and gating kinetics. 9. It is proposed that D2 receptors are coupled to non-voltage-dependent K+ channels by G proteins of the Gi/Go class and that this coupling is via a direct, membrane delimited pathway.

Animals↗
PubMed1992

Involvement of the nitric oxide-cyclic GMP pathway in the desensitization of bradykinin responses of cultured rat sensory neurons.

Bradykinin (BK) excites a subset of dorsal root ganglion neurons by inducing an inward cation current (IBK) that strongly desensitizes and is accompanied by elevations in cGMP. We have examined the links between cGMP metabolism and IBK. The BK dose dependencies of IBK activation, desensitization, and cGMP production are comparable. Stimulation (with sodium nitroprusside [NP] or 8-bromo-cGMP [8Br-cGMP]) or inhibition (with methylene blue, hemoglobin, and nitric oxide synthase [NOS] inhibitors) of cGMP levels did not mimic or diminish IBK. However, desensitization was affected by the following agents: first, desensitization was enhanced by NP and reduced by NOS inhibitors. Second, the effects of NOS inhibitors could be overcome by 8Br-cGMP or L-arginine. Third, 8Br-cGMP modification of desensitization required receptor occupancy. We conclude that the NO-cGMP pathway affects a component of IBK desensitization at the receptor or G protein level.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1992

Differential responses of Ca-activated K channels to bradykinin in sensory neurons and F-11 cells.

The nonapeptide bradykinin (BK) excites a subset of dorsal root ganglion (DRG) neurons with putative nociceptive functions by stimulating an inward cation current. In addition, BK stimulates various intracellular signaling pathways including an elevation of intracellular Ca2+. In a DRG neuron x neuroblastoma hybrid cell (F-11), BK stimulates similar increases in intracellular [Ca2+] and inward current but also elicits a large transient outward current through Ca(2+)-activated K channels. We have investigated the mechanisms underlying differential expression of outward current responses in the two cell types at the single channel level. Although K(Ca) channel activity appears in inside-out patches from both cells exposed to Ca2+, BK applied to the extrapatch membrane of cell-attached patches activates K(Ca) channels in F-11 but not DRG neurons. Whereas single K(Ca) channels are quantitatively similar in terms of conductance, voltage-dependence, and sensitivity to tetraethylammonium, they differ in sensitivity to intracellular Ca2+. Channel activation in both cells requires at least four Ca2+ ions, but half-maximal activation occurs at slightly higher [Ca2+] for DRG neurons. The shift in the Ca2+ dose-response curve combined with the steep [Ca2+] dependence of channel open probability makes it less likely that a BK-induced rise in internal [Ca2+] induced will trigger a transient outward current and resultant hyperpolarization in a DRG neuron.

Animals↗