The tactile motion aftereffect suggests an intensive code for speed in neurons sensitive to both speed and direction of motion.

Neurophysiological studies in primates have found that direction-sensitive neurons in the primary somatosensory cortex (SI) generally increase their response rate with increasing speed of object motion across the skin and show little evidence of speed tuning. We employed psychophysics to determine whether human perception of motion direction could be explained by features of such neurons and whether evidence can be found for a speed-tuned process. After adaptation to motion across the skin, a subsequently presented dynamic test stimulus yields an impression of motion in the opposite direction. We measured the strength of this tactile motion aftereffect (tMAE) induced with different combinations of adapting and test speeds. Distal-to-proximal or proximal-to-distal adapting motion was applied to participants' index fingers using a tactile array, after which participants reported the perceived direction of a bidirectional test stimulus. An intensive code for speed, like that observed in SI neurons, predicts greater adaptation (and a stronger tMAE) the faster the adapting speed, regardless of the test speed. In contrast, speed tuning of direction-sensitive neurons predicts the greatest tMAE when the adapting and test stimuli have matching speeds. We found that the strength of the tMAE increased monotonically with adapting speed, regardless of the test speed, showing no evidence of speed tuning. Our data are consistent with neurophysiological findings that suggest an intensive code for speed along the motion processing pathways comprising neurons sensitive both to speed and direction of motion.

An in vitro investigation of the cardiovascular effects of the 5-HT(4) receptor selective agonists, velusetrag and TD-8954.

The 5-HT(4) receptor agonists, and gastrointestinal (GI) prokinetic agents, cisapride and tegaserod, lack selectivity for the 5-HT(4) receptor. Cisapride is a potent human ether-à-go-go-related gene (hERG) potassium channel inhibitor while cisapride and tegaserod have significant affinity for 5-HT(1) and 5-HT(2) receptor subtypes. Marketing of both compounds was discontinued due to cardiovascular concerns (cardiac arrhythmias with cisapride and ischemic events with tegaserod). The reported association of tegaserod with ischemia has been postulated to involve coronary artery constriction or augmentation of platelet aggregation. This in vitro study investigated the effects of two of the new generation of highly selective 5-HT(4) receptor agonists, velusetrag and TD-8954, on canine, porcine and human coronary artery tone, human platelet aggregation and hERG potassium channel conductance. No significant off-target actions of velusetrag or TD-8954 were identified in these, and prior, studies. While cisapride inhibited potently the hERG channel currents, tegaserod failed to affect platelet aggregation, and had only a small contractile effect on the canine coronary artery at high concentrations. Tegaserod inhibited the 5-HT-induced contractile response in the porcine coronary artery. New generation 5-HT(4) receptor agonists hold promise for the treatment of patients suffering from GI motility disorders with a reduced cardiovascular risk.

Fra-1 controls motility of bladder cancer cells via transcriptional upregulation of the receptor tyrosine kinase AXL.

Fos-related antigen 1 (Fra-1) is a Fos family member overexpressed in several types of human cancers. Here, we report that Fra-1 is highly expressed in the muscle-invasive form of the carcinoma of the bladder (80%) and to a lesser extent in superficial bladder cancer (42%). We demonstrate that in this type of cancer Fra-1 is regulated via a C-terminal instability signal and C-terminal phosphorylation. We show that manipulation of Fra-1 expression levels in bladder cancer cell lines affects cell morphology, motility and proliferation. The gene coding for AXL tyrosine kinase is directly upregulated by Fra-1 in bladder cancer and in other cell lines. Importantly, our data demonstrate that AXL mediates the effect of Fra-1 on tumour cell motility but not on cell proliferation. We suggest that AXL may represent an attractive therapeutic target in cancers expressing high Fra-1 levels.

Multichannel surface recordings on the visual cortex: implications for a neuroprosthesis.

Using a multi-channel platinum surface electrode array, recordings from cat primary visual cortex were obtained in response to visual stimuli, and electrical stimuli delivered using the elements of the array itself. Neural responses to electrical stimuli were consistent, regardless of stimulus polarity or leading phase (biphasic), although thresholds were lower for monophasic than biphasic pulses. Both visual and electrical stimuli reliably evoked responses with characteristic components, which interacted with each other in a nonlinear summation showing first facilitation then suppression during the window of interaction. The chronaxie for eliciting threshold cortical responses was about 100 mus, and the charge density with a pulse width of 50-100 mus was around 55 muC cm(-2). These data form the basis of understanding the types of cortical responses to stimuli delivered by devices suitable for chronic implantation.

The in vitro pharmacological profile of TD-5108, a selective 5-HT(4) receptor agonist with high intrinsic activity.

The in vitro pharmacological profile of TD-5108, a novel, selective 5-HT(4) receptor agonist, was compared to that of clinically efficacious gastroprokinetic 5-HT(4) receptor agonists. TD-5108 produced an elevation of cyclic adenosine monophosphate in human embryonic kidney 293 cells expressing the human recombinant 5-HT(4(c)) (h5-HT(4(c))) receptor (pEC(50) = 8.3) and 5-HT(4) receptor-mediated relaxation of the rat esophagus (pEC(50) = 7.9) and contraction of the guinea pig colon (pEC(50) = 7.9). In all in vitro assays, TD-5108 was a high intrinsic activity agonist, unlike tegaserod, mosapride, and cisapride which, in the majority of test systems, had lower intrinsic activity. TD-5108 had high affinity (pK (i) = 7.7) and selectivity (> or =25-fold) for h5-HT(4(c)) receptors over other biogenic amine receptors. TD-5108 was >500-fold selective over other 5-HT receptors (including h5-HT(2B) and h5-HT(3A)) and, at 3 microM, had no effect on human ether-à-go-go-related gene K+ channels. In conclusion, TD-5108 is a selective 5-HT(4) receptor agonist in vitro. The high intrinsic activity and preferential binding of TD-5108 to 5-HT4 over other 5-HT receptors may result in an improved clinical profile for the treatment of gastrointestinal disorders of reduced motility.

Suppression of vibrotactile discrimination by transcranial magnetic stimulation of primary somatosensory cortex.

A number of human and animal studies have reported a differential representation of the frequency of vibrotactile stimuli in the somatosensory cortices: neurons in the primary somatosensory cortex (SI) are predominantly responsive to lower frequencies of tactile vibration, and those in the secondary somatosensory cortex (SII) are predominantly responsive to higher frequencies. We employed transcranial magnetic stimulation (TMS) over SI in human subjects to investigate the extent to which the inactivation of SI disrupted the discrimination of vibrotactile stimulation at frequencies that give rise to the tactile sensations of flutter (30 Hz) and vibration (200 Hz). Frequency discrimination around the 30-Hz standard following application of TMS to SI was reduced in seven of the eight subjects, and around the 200-Hz standard was reduced in all eight subjects. The average change in discrimination following TMS was about 20% for both low and high frequencies of vibrotactile stimulation. These data suggest that disruption of SI: (1) has a direct effect on the discrimination of both low and high frequencies of vibrotactile stimuli, consistent with a serial model of processing, or (2) has a direct effect on low-frequency vibrotactile stimuli and an indirect effect on the processing of high-frequency vibrotactile stimuli by SII via cortico-cortical connections between the two regions.

The in vitro pharmacology of the peripherally restricted opioid receptor antagonists, alvimopan, ADL 08-0011 and methylnaltrexone.

This study characterized the pharmacology of the peripherally restricted opioid receptor antagonists, alvimopan, its metabolite, ADL 08-0011, and methylnaltrexone. The activities of the compounds were investigated with respect to human or guinea pig opioid receptor binding and function in recombinant cell lines and mechanical responsiveness of the guinea pig ileum. Alvimopan and ADL 08-0011 had higher binding affinity than methylnaltrexone at human mu opioid receptors (pK (i) values of 9.6, 9.6, and 8.0, respectively). The compounds had different selectivities for the mu receptor over human delta and guinea pig kappa opioid receptors. ADL 08-0011 had the highest mu receptor selectivity. With respect to their mu opioid receptor functional activity ([(35)S]GTPgammaS incorporation), methylnaltrexone had a positive intrinsic activity, consistent with partial agonism, unlike alvimopan and ADL 08-0011, which had negative intrinsic activities. Alvimopan, ADL 08-0011, and methylnaltrexone antagonized inhibitory responses mediated by the mu opioid agonist, endomorphin-1 (pA (2) values of 9.6, 9.4, and 7.6, respectively) and by U69593, a kappa opioid agonist (pA (2) values of 8.4, 7.2, and 6.7, respectively). In morphine-naive guinea pig ileum, methylnaltrexone reduced, while alvimopan and ADL 08-0011 increased, the amplitude of electrically evoked contractions and spontaneous mechanical activity. In tissue from morphine-dependent animals, alvimopan and ADL 08-0011 increased spontaneous activity to a greater degree than methylnaltrexone. The data suggested that alvimopan-induced contractions resulted predominantly from an interaction with kappa opioid receptors. It is concluded that alvimopan, ADL 08-0011, and methylnaltrexone differ in their in vitro pharmacological properties, particularly with respect to opioid receptor subtype selectivity and intrinsic activity. The clinical significance of the data from this study remains to be determined.

A comparison of the pharmacological properties of guinea-pig and human recombinant 5-HT4 receptors.

BACKGROUND AND PURPOSE: 5-HT(4) receptor agonists are used therapeutically to treat disorders of reduced gastrointestinal motility. Since such compounds are evaluated in guinea-pigs, we cloned, expressed and pharmacologically characterized the guinea-pig 5-HT(4) and human 5-HT(4(b)) splice variant, which share 95% homology. The functional properties of guinea-pig 5-HT(4(b)) receptors were compared with native receptors in guinea-pig colon. EXPERIMENTAL APPROACH: Membrane radioligand binding and whole cell cAMP accumulation assays were used to determine the affinities, potencies and intrinsic activities (IA). Contraction of the guinea-pig distal colon longitudinal muscle myenteric plexus preparation (LMMP) was monitored to evaluate functional activity. KEY RESULTS: pK(i) values for guinea-pig and human recombinant receptors, and guinea-pig striatum 5-HT(4) receptors, were in agreement, as were the potency and IA values for guinea-pig and human 5-HT(4) receptors expressed at a similar density ( approximately 0.2 pmol mg(-1) protein). Tegaserod was a potent (pEC(50)=8.4 and 8.7, respectively), full agonist at both guinea-pig and human 5-HT(4) receptors. In contrast, in the LMMP preparation, tegaserod was a potent, partial agonist (pEC(50)=8.2; IA=66%). CONCLUSIONS AND IMPLICATIONS: Close agreement between the pharmacological properties of guinea-pig and human 5-HT(4) receptors support the use of guinea-pig model systems for the identification of 5-HT(4) receptor therapeutics. However, the mechanisms underlying the different agonist properties of tegaserod in recombinant and isolated tissue preparations, and the extent to which these impact the clinical efficacy of tegaserod as a prokinetic agent, remain to be determined.

Evidence for a multivalent interaction of symmetrical, N-linked, lidocaine dimers with voltage-gated Na+ channels.

The interaction of symmetrical lidocaine dimers with voltage-gated Na+ channels (VGSCs) was examined using a FLIPR membrane potential assay and voltage-clamp. The dimers, in which the tertiary amines of the lidocaine moieties are linked by an alkylene chain (two to six methylene units), inhibited VGSC activator-evoked depolarization of cells heterologously-expressing rat (r) Na(v)1.2a, human (h) Na(v)1.5, and rNa(v)1.8, with potencies 10- to 100-fold higher than lidocaine (compound 1). The rank order of potency (C4 (compound 4) > C3 (compound 3) > or = C2 (compound 2) = C5 (compound 5) = C6 (compound 6) >> compound 1) was similar at each VGSC. Compound 4 exhibited strong use-dependent inhibition of hNa(v)1.5 with pIC50 values < 4.5 and 6.0 for tonic and phasic block, respectively. Coincubation with local anesthetics but not tetrodotoxin attenuated compound 4-mediated inhibition of hNa(v)1.5. These data suggest that the compound 4 binding site(s) is identical, or allosterically coupled, to the local anesthetic receptor. The dissociation rate of the dimers from hNa(v)1.5 was dependent upon the linker length, with a rank order of compound 1 > compound 5 = compound 6 > compound 2 >> compound 3. The observation that both the potency and dissociation rate of the dimers was dependent upon linker length is consistent with a multivalent interaction at VGSCs. hNa(v)1.5 VGSCs did not recover from inhibition by compound 4. However, "chase" with free local anesthetic site inhibitors increased the rate of dissociation of compound 4. Together, these data support the hypothesis that compound 4 simultaneously occupies two binding sites on VGSCs, both of which can be bound by known local anesthetic site inhibitors.

The 5-HT4 receptor agonist, tegaserod, is a potent 5-HT2B receptor antagonist in vitro and in vivo.

1 Tegaserod (Zelnorm) is a potent 5-hydroxytryptamine4 (5-HT4) receptor agonist with clinical efficacy in disorders associated with reduced gastrointestinal motility and transit. The present study investigated the interaction of tegaserod with 5-HT2 receptors, and compared its potency in this respect to its 5-HT4 receptor agonist activity. 2 Tegaserod had significant binding affinity for human recombinant 5-HT2A, 5-HT2B and 5-HT2C receptors (pKi=7.5, 8.4 and 7.0, respectively). The 5-HT2B receptor-binding affinity of tegaserod was identical to that at human recombinant 5-HT4(c) receptors (mean pKi=8.4) in human embryonic kidney-293 (HEK-293) cells stably transfected with the human 5-HT4(c) receptor. 3 Tegaserod (0.1-3 microm) inhibited 5-HT-mediated contraction of the rat isolated stomach fundus potently (pA2=8.3), consistent with 5-HT(2B) receptor antagonist activity. Tegaserod produced, with similar potency, an elevation of adenosine 3',5' cyclic monophosphate in HEK-293 cells stably transfected with the human 5-HT4(c) receptor (mean pEC50=8.6), as well as 5-HT4) receptor-mediated relaxation of the rat isolated oesophagus (mean pEC50=8.2) and contraction of the guinea-pig isolated colon (mean pEC50=8.3). 4 Following subcutaneous administration, tegaserod (0.3 or 1 mg kg(-1)) inhibited contractions of the stomach fundus in anaesthetized rats in response to intravenous dosing of alpha-methyl 5-HT (0.03 mg kg(-1)) and BW 723C86 (0.3 mg kg(-1)), selective 5-HT2B receptor agonists. At similar doses, tegaserod (1 and 3 mg kg(-1) subcutaneously) evoked a 5-HT4 receptor-mediated increase in colonic transit in conscious guinea-pigs. 5 The data from this study indicate that tegaserod antagonizes 5-HT2B receptors at concentrations similar to those that activate 5-HT4 receptors. It remains to be determined whether this 5-HT2B receptor antagonist activity of tegaserod contributes to its clinical profile.

Comparison of the pharmacological properties of rat Na(V)1.8 with rat Na(V)1.2a and human Na(V)1.5 voltage-gated sodium channel subtypes using a membrane potential sensitive dye and FLIPR.

A novel, membrane potential sensitive dye and a fluorescence imaging plate reader (FLIPR) have been used to characterize the pharmacological properties of rat Na(v)1.8 voltage-gated sodium channels (VGSC) in parallel with rat Na(v)1.2a and human Na(v)1.5 VGSC subtypes, respectively. The sensitivity of recombinant Na(v)1.2a-CHO, Na(v)1.5-293-EBNA, and Na(v)1.8-F-11 cells to VGSC activators was subtype dependent. Veratridine evoked depolarization of Na(v)1.2a-CHO and Na(v)1.5-293-EBNA cells with pEC(50) values of 4.78 +/- 0.13 and 4.84 +/- 0.12, respectively (n = 3), but had negligible effect on Na(v)1.8-F-11 cells (pEC(50) < 4.5). Type I pyrethroids were without significant effect at all subtypes. In contrast, the type II pyrethroids deltamethrin and fenvalerate evoked direct depolarization of Na(v)1.8-F-11 and Na(v)1.5-293-EBNA cells. Deltamethrin potentiated the veratridine-evoked response in Na(v)1.8-F-11 cells by > or =20-fold, in contrast to a

Coding of disparity information in extrastriate cortex of the cat.

We have used information theory to analyse the responses of neurons in area 21a of the cat to disparity stimuli. Visual stimuli consisted of drifting sinusoidal gratings presented simultaneously to each eye. The relative spatial phase of the gratings varied between stimulus periods in a pseudo-random sequence of 45 degrees increments that covered the full 360 degrees. The mean information content of the responses of all neurons across all phases was 0.72 bits (+/-0.10, SE, n=29). The information conveyed by each neuron was well correlated with the extent to which the interocular phase difference modulated the response of the cell. However, information content was not simply related to firing rate, as there was usually significant information content in the neuronal responses to phase differences that elicited the minimum firing rate. In general, burst responses (impulse intervals <4 ms) did not convey more information than that conveyed by the total response. The contribution to the cumulative information of the response in successive 100-ms segments decreased over the course of the 1-s stimulus. The ratio of information transmitted at 200 ms to that transmitted over the full second had a median of 0.30 while the ratio of 500 ms to 1 s was 0.68.

New applications of electron diffraction in the pharmaceutical industry: polymorph determination by using a combination of electron diffraction and synchrotron X-ray powder diffraction techniques.

Electron diffraction has been recently used in the pharmaceutical industry to study the polymorphism in crystalline drug substances. While conventional X-ray diffraction patterns could not be used to determine the cell parameters of two forms of the microcrystalline GP IIb/IIIa receptor antagonist roxifiban, a combination of electron single-crystal and synchrotron powder diffraction techniques were able to clearly distinguish the two polymorphs. The unit-cell parameters of the two polymorphs were ultimately determined using new software routines designed to take advantage of each technique's unique capabilities. The combined use of transmission electron microscopy (TEM) and synchrotron patterns appears to be a good general approach for characterizing complex (low-symmetry, large-unit-cell, micron-sized) polymorphic pharmaceutical compounds.

SH3 ligands in the dopamine D3 receptor.

It has recently been observed that G protein-coupled receptors (GPCRs) can interact with SH3 domains through polyproline motifs. These interactions appear to be involved in receptor internalization and MAPK signalling. Here we report that the third cytoplasmic loop of the dopamine D3 receptor can interact in vitro with the adaptor protein Grb2. While the amino- and carboxy-terminal SH3 domains of Grb2 separately did not interact with the D3 receptor loop, the interaction is at least partially maintained with a Grb2 mutant for the amino-terminal SH3 domain, but disrupted for a Grb2 mutant with a nonfunctional carboxy-terminal SH3 domain. The data indicate the need of structural integrity of the entire Grb2 protein for the interaction and dominant role of the carboxy-terminal SH3 domain in the interaction. Disruption of the PXXP motifs in the D3 receptor did not affect the interaction with Grb2. These results indicate that GPCRs may contain SH3 ligands that do not contain the postulated minimal consensus sequence PXXP.

Crystalline versus amorphous content of lumaxistrade mark analog XP280 using X-ray and electron diffraction methods.

In the course of the development of Lumaxistrade mark (roxifiban), the physical state of XP280 (the besylate salt of the active metabolites of roxifiban) and SC887 (the mesylate salt of the free base of roxifiban) were characterized. Powder X-ray diffraction patterns of XP280 were ambiguous in that a high degree of background signal was present and potentially indicative of the existence of an amorphous phase. Herein the results of combined synchrotron X-ray diffraction and electron microscopy (diffraction and imaging) studies on XP280 and SC887 are reported. The combination of these two techniques allowed an unambiguous assessment of the crystallinity, as well as determination of four of the unit cell parameters of XP280 and complete determination of the unit cell parameters for SC887.

Binocular interactions in area 21a of the cat.

We investigated binocular suppression in area 21a cells of the anaesthetized cat using drifting sinusoidal gratings presented simultaneously to each eye. The grating presented to the dominant eye was always oriented optimally and the grating presented to the non-dominant eye was either at the same orientation, but at the least effective relative spatial phase, or orthogonal. The binocular response of approximately 80% of cells was less than the monocular dominant eye response when there was a mismatch in orientation or spatial location between stimuli. Response suppression in the two binocular stimulus conditions had a correlation coefficient (r) of 0.55. We propose a parsimonious model to account for the response facilitation and suppression by binocular stimulation of area 21a neurons.

Polymorph determination for the GP IIb/IIIa antagonist, roxifiban, using a combination of electron diffraction and synchrotron X-ray powder diffraction techniques.

Unit cell parameters of two polymorphs of roxifiban have been determined by a combination of transmission electron microscopy (TEM) single-crystal and synchrotron X-ray powder diffraction techniques. While it was difficult to differentiate the two forms by their standard X-ray diffraction patterns, the high-resolution synchrotron patterns clearly showed striking differences. Unit cells for the two forms required the use of cell parameters derived from TEM diffraction patterns. The two unit cells are, not surprisingly, very similar except for a doubling of one of the axes for form II. The combined use of TEM and synchrotron patterns appears to be a good general approach for characterizing complex (low-symmetry, large unit cell) polymorphs.

Distinct dynamin-dependent and -independent mechanisms target structurally homologous dopamine receptors to different endocytic membranes.

D1 and D2 dopamine receptors are structurally homologous G protein-coupled receptors that serve distinct physiological functions both in neurons and nonneural cell types. We have observed that these receptors are selectively endocytosed in HEK293 cells by distinct dynamin-dependent and -independent mechanisms. Although these endocytic mechanisms operate with similarly rapid kinetics, they differ in their regulation by agonist and deliver D1 and D2 receptors specifically to different primary endocytic vesicles. After this segregation into different endocytic membranes, both D1 and D2 receptors recycle to the plasma membrane. Similar results are observed in Neuro2A neuroblastoma cells coexpressing both receptors at high levels. These findings establish that "classical" dynamin-dependent and "alternative" dynamin-independent endocytic mechanisms differ in their physiological regulation, sort structurally homologous signaling receptors in the plasma membrane, and mediate distinct early endocytic pathways leading to recycling endosomes. Our results also refute the previous hypothesis that dynamin-independent endocytosis targets G protein-coupled receptors selectively to lysosomes, and they suggest a new role of endocytic sorting mechanisms in physically segregating structurally homologous signaling receptors at the cell surface.

Binocular phase interactions in area 21a of the cat.

1. Binocular interactions related to retinal disparity were investigated in single neurons in area 21a of extrastriate cortex in the anaesthetized cat using sinusoidal luminance gratings. 2. The responses of approximately two-thirds of neurons were profoundly modulated by a relative phase difference between identical drifting gratings presented to each eye. This modulation included both facilitatory and inhibitory interocular interactions. The selectivity for binocular disparity was about twice as sharp as the selectivity for monocular spatial position. 3. Significant phase modulation was retained in many neurons at interocular orientation differences exceeding 45 deg. The response suppression associated with stimulation at a phase shift 180 deg from the optimum was stronger than the response suppression to an interocular orientation difference of 90 deg. 4. The proportion of phase modulated neurons and the potency of modulation in area 21a neurons exceed that reported for phase-selective complex cells in area 17. Neurons in area 21a show sharp disparity tuning that is relatively insensitive to changes in orientation and monocular position, which suggests that this extrastriate region has a role in stereoscopic depth perception.

Utility of microcalorimetry in the characterization of the browning reaction.

An isothermal microcalorimeter was utilized to characterize a model solid-state interaction. The degradation of the HIV protease inhibitor, DMP 450, in a binary mixture with hydrous lactose was followed in the presence of 5% additional water. Heat produced in the microcalorimeter sample vessel from either chemical or physical change is channeled through extremely sensitive thermopile blankets and is measured as it flows into infinite heat sinks. Solid-state 1:1 mixtures of DMP 450 and hydrous lactose each with 5% water added were analyzed in the microcalorimeter at 50, 60 and 65 degrees C. The resulting heat flow profiles were consistent with an autocatalytic rate law. An activation energy of 26.12 kcal mol(-1) for the DMP 450:lactose mixture was determined from the slope of the Arrhenius plot of the microcalorimetry heat flow maximum value versus the reciprocal of the absolute temperature. The activation energy determined by the traditional method with HPLC analysis was found to be in excellent agreement with the microcalorimetry value at 26.38 kcal mol(-1).