Michael R. Knowles

Stable expression of human D3 dopamine receptors in GH4C1 pituitary cells

Human D3 dopamine receptor DNA was stably transfected into GH4C1 pituitary cells. Displacement of iodosulpiride binding in hD3 transfected cells (Kd = 0.3 nM, B max = 89 fmol/mg protein) by dopaminergic ligands was indistinguishable from that of hD3 receptors in CHO cells. Only two clonal cell lines exhibited weak GppNHp‐dependent shifts in [3H]N‐0437 binding, and these were used for functional assays. Neither arachidonic acid metabolism, cAMP levels, inositol phosphate turnover, intracellular calcium, or potassium currents were consistently affected by dopamine (1–10 μM). The paucity of responses indicates that human D3 receptors do not couple efficiently to these second messengers in GH4C1 cells.

Pharmacology of high‐threshold calcium currents in GH4C1 pituitary cells and their regulation by activation of human D2 and D4 dopamine receptors

1 The objective of this study was to characterize the pharmacology of calcium currents in GH4C1 pituitary cells and determine whether activation of heterologously expressed human dopamine receptors can regulate their function. Human D2(short), D3 and D4,2 receptor cDNAs were separately transfected into GH4C1 cells and whole cell calcium currents were recorded by use of nystatin‐perforated patch clamp techniques. 2 High‐threshold calcium currents were antagonized in a biphasic manner by the dihydropyridine, nisoldipine. The half‐maximally effective concentration for each site was 0.2 nm (pIC50 = 9.78 ± 0.21, n = 4) and 339 nm (pIC50 = 6.47 ± 0.12, n = 4). The component of current inhibited by 10 nm nisoldipine was also blocked by ω‐conotoxin GVIA (30 ± 9% at 30 nm, n = 6) or by ω‐agatoxin IVA (34 ± 7% at 100 nm, n = 4). 3 Activation of either D2 or D4 receptors by dopamine (10 μm) or quinpirole (0.1 to 10 μm) reduced the peak calcium current by ca. 20% in the majority of cells studied. No inhibition was observed in control or D3 transfected GH4C1 cell lines. 4 The mobilisation of intracellular calcium by thyrotropin releasing hormone in hD4‐GH4C1 cells was also studied using Fura‐2 AM microspectrofluorimetry. Thyrotropin releasing hormone caused a concentration‐dependent increase in calcium mobilisation with an EC50 of 7 nm. D4 receptor activation had no effect upon either basal or hormone‐induced [Ca2+]i transients. 5 These results demonstrate that GH4C1 pituitary cells have at least two types of dihydropyridine‐sensitive high‐threshold calcium currents and that like D2 receptors, human D4 receptors can also regulate calcium channel function.

Alterations of stress related proteins in genetically altered mice revealed by two-dimensional differential in-gel electrophoresis analysis

Transgenic, knockout and knockin mice are useful tools for linking specific genes with behaviour and other complex biological processes. However, complications arising due to compensatory changes, genetic background differences and other factors could lead to difficulty in interpreting the resulting changes in phenotype. We have used fluorescence two‐dimensional differential in‐gel electrophoresis in combination with matrix‐assisted laser desorption/ionization‐time of flight mass fingerprinting to investigate the possibility that distinct genetic alterations can lead to common protein expression changes in genetically modified mice. Brain proteomes were compared from two transgenic mouse strains (Tg2576 × TgPS1 and Tg2576), two knockout mouse strains (5‐HT7R –/– and GABAARα5 –/–) and one knockin mouse strain (GABAARα1‐H101R). Both of the transgenic models showed an isoform change in the heat shock 70 related protein, mortalin. The knockout and knockin models showed similar changes in mortalin expression along with an alteration of the anti‐oxidant protein 2. The observed proteomic alterations indicate that stress‐responsive protein pathways may be altered artefactually in all of the mouse models used in this study and highlights an area where caution is needed in interpreting proteomic changes in genetically modified mice.

Characterisation of a chimeric hD3/D2 dopamine receptor expressed in CHO cells

The D2 dopamine receptor is known to be functionally coupled when expressed in CHO cells, whereas the effector systems for the D3, dopamine receptor remain unclear. A chimeric, human D3/D2 receptor (hD3/D2) was constructed containing the third intracellular loop region of the D2 receptor. CHO cells stably expressing the D2, D3, or hD3/D2 receptors were created and the pharmacology of the receptors was examined. The chimeric hD3/D2 receptor retained D3‐like affinities for dopaminergic ligands. However, in contrast to the D2 receptor neither the D3 receptor nor the hD3/D2 receptor could functionally couple to the adenylate cyclase or arachidonic acid release mechanisms.

Mechanisms of action of the antidepressants fluoxetine and the substance P antagonist L-000760735 are associated with altered neurofilaments and synaptic remodeling

Antidepressants are widely prescribed in the treatment of depression, although the mechanism of how they exert their therapeutic effects is poorly understood. To shed further light on their mode of action, we have attempted to identify a common proteomic signature in guinea pig brains after chronic treatment with two different antidepressants. Both fluoxetine and the substance P receptor (NK(1)R) antagonist (SPA) L-000760735 altered cortical expression of multiple heat shock protein 60 forms along with neurofilaments and related proteins that are critical determinants of synaptic structure and function. Analysis of NK(1)R-/- mice showed similar alterations of neurofilaments confirming the specificity of the effects observed with chronic NK(1)R antagonist treatment. To determine if these changes were associated with structural modification of synapses, we carried out electron microscopic analysis of cerebral cortices from fluoxetine-treated guinea pigs. This showed an increase in the percentage of synapses with split postsynaptic densities (PSDs), a phenomenon that is characteristic of activity-dependent synaptic rearrangement. These findings suggest that cortical alterations of the neurofilament pathway and increased synaptic remodeling are associated with the mechanism of these two antidepressant drug treatments and may contribute to their psychotherapeutic actions.

Functional Coupling of Human D2, D3, and D4 Dopamine Receptors in HEK293 Cells

The D2 dopamine receptor is known to be functionally coupled to the inhibition of adenylate cyclase when expressed in a number of mammalian cell lines. However, functional coupling of the recently discovered D3 and D4 dopamine receptor subtypes has been more difficult to demonstrate. In this study, human D2, D3 and D4 receptors were stably expressed separately in human embryonic kidney cells (HEK 293). In these cells, activation of D2, D3 or D4 receptors resulted in the inhibition of forskolin-stimulated adenylate cyclase activity in a dose responsive manner. This activation was prevented by pre-incubation of the cells expressing these receptors with the dopaminergic antagonist haloperidol. Radioligand binding studies using [3H]spiperone confirmed that the atypical neuroleptic clozapine has higher affinity for the human D4 receptor than the D3 or D4 receptors, although only 6-fold higher than the D2 receptor in this study. In addition, ribonuclease protection studies demonstrated the presence of D4 dopamine receptor mRNA in human brain regions.

Depression of high‐threshold calcium currents by activation of human D2 (short) dopamine receptors expressed in differentiated NG108‐15 cells

1 This study examined the regulation of calcium currents in differentiated NG108‐15 cells that had been stably transfected with cDNA encoding the short isoform of the human D2 dopamine receptor. Whole cell calcium currents were recorded by nystatin‐perforated patch clamp recording. 2 Transient low‐threshold calcium currents elicited by depolarizations from − 100 mV to − 20 mV were reversibly depressed by NiCl2 (84 ± 8% at 30 μm; n = 3) and by ω‐agatoxin IVA (15 ± 5%; 100 nm, n = 7). These currents were unaffected by hD2 receptor activation. 3 High‐threshold calcium currents elicited by depolarizations from − 80 mV to 0 mV were partly blocked by ω‐conotoxin GVIA (67 ± 6% at 100 nm, n = 4) and by the subsequent addition of the dihydropyridine, nisoldipine (94 ± 3% at 1 μm). Consistent with the presence of at least two distinct types of high‐threshold calcium channels, nisoldipine alone (38 ± 15% at 1 μm, n = 6) did not preclude the inhibition caused by ω‐conotoxin GVIA (69 ± 13% at 100 nm, n = 4). The residual current was completely blocked by 100 μm CdCl2 (98.8 ± 0.4%, n = 7). 4 In hD2‐transfected cells, but not untransfected cells, high‐threshold currents were depressed by quinpirole (30 ± 4% at 100 nm; n = 15) with a pEC50 of 8.61 ± 0.22 (n = 5), as well as by (−)‐noradrenaline (28 ± 5% at 1 μm, n = 9). Responses to both agonists were selectively antagonized by S‐(−)sulpiride (100 nm) but not by the α‐adrenoceptor antagonist, phentolamine (10 μm). The depression caused by (−)‐noradrenaline was positively correlated with that of quinpirole for each cell (r2= 0.91, slope = 0.99). 5 hD2‐receptor‐mediated inhibition of high‐threshold calcium currents was abolished by pretreatment of cells with ω‐conotoxin GVIA (100 nm; n = 4). However, a component of the high‐threshold current was reversibly depressed by ω‐conotoxin GVIA (67% to 45% depression after 10 min wash). This current was also depressed by hD2 receptor activation (59 ± 9% depression in 100 nm quinpirole, n = 3), and was completely blocked by nisoldipine (95 ± 2% at 1 μm). 6 These data demonstrate that activation of hD2(short) dopamine receptors can regulate both ω‐conotoxin GVIA, and dihydropyridine‐sensitive high‐threshold calcium currents in neuroblastoma cells. Morever, the ability of human D2 dopamine receptors to regulate more than one type of calcium current supports the notion that these receptors have a diverse functional role in the central nervous system.

Proteomic analysis identifies alterations in cellular morphology and cell death pathways in mouse brain after chronic corticosterone treatment.

Some patients with Major Depression and other neurological afflictions display hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis. HPA hyperactivity may be due to impaired feedback inhibition and manifested as increased levels of circulating cortisol. Subcutaneous implants of corticosterone pellets were used to mimic this situation in mice to gain insight into any effects on brain function by comparative proteomic analysis using two-dimensional Differential In-Gel Electrophoresis. A total of 150 different protein spots were altered by corticosterone treatment in the hypothalamus, hippocampus and cerebral cortex. Of these, 117 spots were identified by matrix-assisted laser desorption/ionization-time of flight mass fingerprinting equating to 51 different proteins. Association of these corticosterone-modulated proteins with biological functions using the Ingenuity Pathways Analysis tool showed that cell morphology was significantly altered in the hippocampus and cerebral cortex, whereas the hypothalamus showed significant changes in cell death. Ingenuity Pathways Analysis of the canonical signaling pathways showed that glycolysis and gluconeogenesis were altered in the hypothalamus and the hippocampus and all three brain regions showed changes in phenylalanine, glutamate and nitrogen metabolism. Further elucidation of these pathways could lead to identification of biomarkers for the development of pharmacological therapies targeted at neuropsychiatric disorders.

The effects of substrates, products and other ligands on the susceptibility of inositol monophosphatase to proteolysis by endoprotease lys-C

Inositol monophosphatase is cleaved by endoprotease lys‐C at a single site (Lys36—Ser37). The rate of proteolysis is greatly reduced in the presence of substrate (D,L‐Ins(I)P) and Mg2+, and less so in the presence of Pi and Mg2+, consistent with protection of the susceptible bond in the E—P or E·Pi states of the enzyme. Potentiation by Li+ of the protection afforded by a substrate analogue, 1S‐phosphoryloxy‐2R,4S‐dihydroxycyclohexane, and Mg2+ supports the idea that Li+ binds to the E—P state.