Compartmentalized cAMP signalling regulates vasopressin-mediated water reabsorption by controlling aquaporin-2.

The cAMP/PKA (protein kinase A) signalling pathway is activated by a plethora of stimuli. To facilitate the specificity of a cellular response, signal transduction complexes are formed and segregated to discrete sites (compartmentalization). cAMP/PKA signalling compartments are maintained by AKAPs (A-kinase anchoring proteins) which bind PKA and other signalling proteins, and by PDEs (phosphodiesterases). The latter hydrolyse cAMP and thus limit its diffusion and terminate PKA activity. An example of a cAMP-dependent process requiring compartmentalization of cAMP/PKA signals is arginine-vasopressin-regulated water reabsorption in renal principal cells. A detailed understanding of the protein interactions within a signal transduction complex offers the possibility to design agents influencing PKA binding to a specific AKAP, the targeting of an AKAP or the interactions of AKAPs with other signalling molecules. The ability to specifically modulate selected branches of a signal transduction pathway would greatly advance basic research, and may lead to new drugs suitable for the treatment of diseases caused by dysregulation of anchored PKA signalling (e.g. renal and cardiovascular diseases).

Three-dimensional vestibular eye and head reflexes of the chameleon: characteristics of gain and phase and effects of eye position on orientation of ocular rotation axes during stimulation in yaw direction.

We investigated gaze-stabilizing reflexes in the chameleon using the three-dimensional search-coil technique. Animals were rotated sinusoidally around an earth-vertical axis under head-fixed and head-free conditions, in the dark and in the light. Gain, phase and the influence of eye position on vestibulo-ocular reflex rotation axes were studied. During head-restrained stimulation in the dark, vestibulo-ocular reflex gaze gains were low (0.1-0.3) and phase lead decreased with increasing frequencies (from 100 degrees at 0.04 Hz to < 30 degrees at 1 Hz). Gaze gains were larger during stimulation in the light (0.1-0.8) with a smaller phase lead (< 30 degrees) and were close to unity during the head-free conditions (around 0.6 in the dark, around 0.8 in the light) with small phase leads. These results confirm earlier findings that chameleons have a low vestibulo-ocular reflex gain during head-fixed conditions and stimulation in the dark and higher gains during head-free stimulation in the light. Vestibulo-ocular reflex eye rotation axes were roughly aligned with the head's rotation axis and did not systematically tilt when the animals were looking eccentrically, up- or downward (as predicted by Listing's Law). Therefore, vestibulo-ocular reflex responses in the chameleon follow a strategy, which optimally stabilizes the entire retinal images, a result previously found in non-human primates.

Ht31: the first protein kinase A anchoring protein to integrate protein kinase A and Rho signaling.

In an attempt to isolate protein kinase A anchoring proteins (AKAPs) involved in vasopressin-mediated water reabsorbtion, the complete sequence of the human AKAP Ht31 was determined and a partial cDNA of its rat orthologue (Rt31) was cloned. The Ht31 cDNA includes the estrogen receptor cofactor Brx and the RhoA GDP/GTP exchange factor proto-lymphoid blast crisis (Lbc) sequences. The Ht31 gene was assigned to chromosome 15 (region q24-q25). It encodes Ht31 and the smaller splice variants Brx and proto-Lbc. A protein of the predicted size of Ht31 (309 kDa) was detected in human mammary carcinoma and HeLa cells. Anti-Ht31/Rt31 antibodies immunoprecipitated RhoA from primary cultured rat renal inner medullary collecting duct cells, indicating an interaction between the AKAP and RhoA in vivo. These results suggest that Ht31/Rt31 represent a new type of AKAP, containing both an anchoring and a catalytic domain, which appears to be capable of modulating the activity of an interacting partner. Ht31/Rt31 have the potential to integrate Rho and protein kinase A signaling pathways, and thus, are prime candidates to regulate vasopressin-mediated water reabsorbtion.

Ocular counterroll modulates the preferred direction of saccade-related pontine burst neurons in the monkey.

Saccade-related burst neurons in the paramedian pontine reticular formation (PPRF) of the head-restrained monkey provide a phasic velocity signal to extraocular motoneurons for the generation of rapid eye movements. In the superior colliculus (SC), which directly projects to the PPRF, the motor command for conjugate saccades with the head restrained in a roll position is represented in a reference frame in between oculocentric and space-fixed coordinates with a clear bias toward gravity. Here we studied the preferred direction of premotor burst neurons in the PPRF during static head roll to characterize their frame of reference with respect to head and eye position. In 59 neurons (short-lead, burst-tonic, and long-lead burst neurons), we found that the preferred direction of eye displacement of these neurons changed, relative to head-fixed landmarks, in the horizontal-vertical plane during static head roll. For the short-lead burst neurons and the burst-tonic group, the change was about one-fourth of the amount of ocular counterroll (OCR) and significantly different from a head-centered representation. In the long-lead burst neurons, the rotation of the preferred direction showed a larger trend of about one-half of OCR. During microelectrical stimulation of the PPRF (9 sites in 2 monkeys), the elicited eye movements rotated with about one-half the amount of OCR. In a simple pulley model of the oculomotor plant, the noncraniocentric reference frame of the PPRF output neurons could be reproduced for recently measured pulley positions, if the pulleys were assumed to rotate as a function of OCR with a gain of 0.5. We conclude that the saccadic displacement signal is transformed from a representation in the SC with a clear bias to gravity to a representation in the PPRF that is closely craniocentric, but rotates with OCR, consistent with current concepts of the oculomotor plant.

The inflammatory action of CD40 ligand (CD154) expressed on activated human platelets is temporally limited by coexpressed CD40.

Recently, we have demonstrated that human platelets carry preformed CD40 ligand (CD154) molecules, which rapidly appear on the platelet surface following stimulation by thrombin. Once on the surface, platelet CD154 induces an inflammatory reaction of CD40-bearing endothelial cells. This study shows that strong platelet agonists other than thrombin also lead to the expression of CD154 on the platelet surface. At the same time, several lines of evidence are presented that together indicate that thrombotic events in the vasculature are generally accompanied by activation of the inflammatory potential of platelet CD154. This study also reports the constitutive expression of CD40, the receptor for CD154, on platelets. The binding of CD154 to coexpressed CD40 in the platelet aggregate leads within minutes to hours to the cleavage of membrane-bound surface CD154 and the release of an 18-kd soluble form of the molecule. Soluble CD154 (sCD154), in contrast to transmembrane CD154, can no longer induce an inflammatory reaction of endothelial cells. These findings indicate that the interaction of platelet CD154 with CD40 on neighboring cells is temporally limited to prevent an uncontrolled inflammation at the site of thrombus formation. Thus, similar to the very tight regulation of the CD154-CD40 interaction in the immune system, an effective mechanism controls the inflammatory potential of platelet CD154 in the vascular system. (Blood. 2001;98:1047-1054)

Chameleon eye position obeys Listing's law.

Listing's law (LL) states that 3D-eye positions lie in a plane, when they are described as single-axis rotations from the primary position. This implies that the degrees of freedom of eye movements are reduced from three to two. Various hypotheses exist, regarding the implementation of LL. These include facilitation of binocular vision, optimization of oculomotor control, and mechanical constraints in the orbit. We recorded the 3D-eye position during saccadic scanning in the chameleon, to investigate whether LL is valid in an animal with different anatomical and behavioral characteristics compared to primates. We show that in chameleons, the eye position obeys LL with a high precision. Since the anatomical arrangement of the orbit in chameleons is very different from that in primates, and binocular fused vision is virtually absent, we suggest that in the chameleon, LL mainly optimizes oculomotor control.

Effects of alertness on three-dimensional eye movements.

PURPOSE: To investigate effects of alertness on three-dimensional (3D) eye movements. METHODS: During drowsy and alert periods, 3D eye movements were recorded with dual search coils in three normal rhesus monkeys, and in two of these monkeys after placement of bilateral kainic acid lesions of the rostral interstitial nucleus of the medial longitudinal fasciculus (riMLF). RESULTS: When the three monkeys were in the normal state, the average plane thickness (standard deviation of all rotation vectors from the regressed plane) during drowsy periods (range, 1.2-1.9 degrees ) increased significantly compared with the plane thickness during alert periods (range, 0.7-0.9 degrees ) in the light (P <.05). The plane thickness increased significantly in the monkey with asymmetric bilateral riMLF-lesions (P <.05); however, the increase was not significant in the monkey with more symmetric bilateral riMLF-lesions. After the bilateral riMLF-lesions, the plane thickness also increased with drowsiness in both monkeys (P <.02), as it had in the normal state. CONCLUSION: The implementation of Listing's law is independent of the presence of vertical-torsional burst neurons in the riMLF. The increase in thickness of Listing's plane during drowsy periods is not due to an imprecise signal from saccadic burst neurons to the 3D velocity-to-position integrator. It is possible that this integrator itself controls Listing's law, depending on the state of alertness.

Effects of optokinetically induced rotatory self-motion on spatial perception and representation.

OBJECTIVE: Our aim was to investigate the influence of optokinetically induced rotatory self-motion sensation (circular vection [CV]) on asymmetries in real and representational space in normal subjects. BACKGROUND: Vestibular and optokinetic stimulation (particularly when accompanied by rightward CV) can reduce left-sided hemineglect in patients. METHOD: Twenty healthy right-handed men were administered a line bisection (LB) task and a stimulus-response compatibility task monitoring mental representation of space (the RULER task). The RULER task required speeded unimanual decisions ("smaller than 6?" vs. "larger than 6?") to foveally presented numbers between 1 and 11. Both tasks were performed in a baseline condition (no stimulation) and with full-field optokinetic stimulation to induce CV to either side. RESULTS: The bisection marks of both hands were shifted significantly to the left during leftward CV, introducing a pseudoneglect for the left and right hands. Rightward CV did not influence LB. In the RULER task, we found a stimulus-response compatibility, namely, faster right-hand responses to large numbers (i.e., 7-11) and faster left-hand responses to small numbers (i.e., 1-5). Although optokinetic stimulation did not significantly affect subjects' representation of space, the overall pattern of observed deviations was strikingly similar to that obtained in LB. CONCLUSIONS: Optokinetic stimulation affects healthy subjects' exploration and, to a lesser extent, their representation of space. In contrast to previous studies in neglect patients and healthy subjects, we found that leftward CV rather than rightward CV induced a leftward deviation of the subjective midpoint. This discrepancy is most likely a consequence of the exclusively peripheral visual field stimulation in our experiment. We suggest that the leftward deviation during rightward CV described in former studies may be due to the cuing effect of the leftward moving dots in the central visual field. In the absence of these central cues, the direction of CV seems to be the main determining factor for observed hemispatial effects.

[Morphology and phenomenology of death by kicking (II)]. / Zur Morphologie und Phänomenologie des Tottretens (II).

In the years from 1982 to 1995, 20,504 autopsy cases were examined at the Departments of Forensic Medicine in Hamburg and Greifswald. In 103 cases death was caused by kicking. In the Greifswald area, the occurrence of kicking as the cause of death could be demonstrated in 68 cases (30% of all homicides), which is in contrast to the observations in Hamburg, were kicking amounted to only 2.5% of the homicides. Most of the victims (average age 44 years) were under the influence of alcohol (mean blood alcohol concentration 1.75/1000), and most were on social support. The victims often showed other signs of blunt trauma such as blows with the fist and strangulation. In a few cases, cutting and stabbing wounds were also found. In the majority of cases death was due to severe haemorrhages and aspiration of blood in addition to the direct effects of the head injuries. The crimes were committed by one person in 46 cases, two in 20 and three in 4 cases. The perpetrators (average age 27.6 years) were always younger than the victims. Case reports show that fatal kicking was mostly triggered by relatively trivial arguments. None of the incidents were preplanned by the perpetrators. In contrast to an expected higher incidence of kicking deaths in the urban area of Hamburg, these crimes occurred ten times more frequently in the rural area of Greifswald.

Three-dimensional eye movement recording for clinical application.

Methods to measure eye rotations in 3D have developed to a stage where routine clinical application is realistic. Besides the equipment, it requires a basic understanding of 3-dimensional geometry for calibration and interpretation. Relevant parameters are orientation, displacement and thickness of Listing's plane for spontaneous or goal-directed eye movements, and counterrolling or nystagmus with a roll component for vestibular function. The method with the highest temporal and spatial resolution is the magnetic search coil technique. Video-based systems are still slow and cannot be used to characterize saccades. Often, the task of reconstructing the 3-dimensional eye position from a 2-dimensional image of the eye is underestimated. Search coil measurements have shown no firm correlation between the orientation of Listing's plane and "classical" landmarks like stereotaxic head position, emphasizing that Listing's plane is functionally, and not anatomically, determined.

Projections from the superior colliculus motor map to omnipause neurons in monkey.

Descending projections from the superior colliculus (SC) motor map to the saccadic omnipause neurons (OPNs) were examined in monkeys by using anterograde transport of tritiated leucine. The SC was divided into three zones: the rostral pole of the motor map, a small horizontal saccade zone in central SC, and a large horizontal saccade zone in caudal SC. Tracer injections into the intermediate layers of the three zones led to different patterns of silver grain deposits in and around nucleus raphe interpositus (RIP), which contains the OPNs: 1) From the rostral pole of the motor map, coarse axon branches of the crossed predorsal bundle spread medially into the RIP, branched, and terminated predominantly unilaterally over cells on the same side. 2) From the small horizontal saccade zone, the axon branches were of a finer caliber and terminated diffusely in the RIP, mainly on the same side. 3) From the large horizontal saccade zone, no terminal labeling was found within the RIP. 4) From the rostral pole of the motor map and small horizontal saccade zone, fiber branches from the ipsilateral descending pathway terminated diffusely over RIP. 5) In addition, terminal labeling in reticulospinal areas of the pons and medulla increased in parallel with the size of the saccade according to the SC motor map. The results suggest that there are multiple projections directly onto OPNs from the rostral SC but not from the caudal SC associated with large gaze shifts. The efferents from the rostral pole of the motor map may subserve the suppression of saccades during visual fixation, and those from the small horizontal saccade zone could inhibit anatagonist premotor circuits.

Vestibular stimulation affects dichotic lexical decision performance.

We report an experimental attempt to shift, by vestibular stimulation, healthy subjects' right ear advantage (REA) in a dichotic listening (DL) task with words and nonwords as stimuli. Forty right-handed men performed the task under two different conditions, once while sitting in a stationary turning chair (baseline) and once during sinusoidal rotation. In this latter condition, every other stimulation was received during maximal left-to-right (i.e., clockwise), every other during maximal right-to-left (i.e., counterclockwise) acceleration. There was a reliable REA for lexical decision accuracy in the baseline and right-to-left trials but not during left-to-right rotation. While right ear performance was unaffected by rotation, there were more correct lexical decisions to left ear targets exclusively during left-to-right turns (one-tailed P = 0.05). Since there were no parallel shifts in auditory thresholds under the different conditions, this effect is not due to any hypothetical auditory-vestibular interactions on a primary sensory level. The improvement in left ear DL performance, although small in our study, is comparable to the symptom-alleviating effect of caloric vestibular stimulation in patients with left-sided hemispatial neglect and interpreted as a consequence of a rotation-induced attentional shift towards the left hemispace.

Three-dimensional extraocular motoneuron innervation in the rhesus monkey. I: Muscle rotation axes and on-directions during fixation.

The rotation axis for each of the six extraocular muscles was determined in four eyes from three perfused rhesus monkeys. Measurements of the locations of muscle insertions and origins were made in the stereotaxic reference frame with the x-y plane horizontal and the x-z plane sagittal. The computed rotation axes of the horizontal recti were close to being in the x-z plane at an angle of about 15 degrees to the z axis. The rotation axes of the vertical recti and the obliques were close to being in the x-y plane at an angle of about 30 degrees to the y axis. In five alert rhesus monkeys, we simultaneously recorded extraocular motoneuron activity and eye position in three dimensions (3D). The activity of 51 motoneuron axons was obtained from the oculomotor (n=34), trochlear (n=11), and abducens nerve (n=6) during spontaneous eye movements. To extend the torsional range of eye position, the animals were also put in different static roll positions, which induced ocular counterroll without dynamic vestibular stimulation. Periods of 100 ms during fixation or slow eye movements (<10 degrees/s) were chosen for analysis. For each motoneuron, a multiple linear regression was performed between firing frequency and 3D eye position, expressed as a rotation vector, in both stereotaxic and Listing's reference frame. The direction with the highest correlation coefficient (average R=0.94+/-0.07 SD) was taken as the on-direction. Each unit's activity could be unequivocally attributed to one particular muscle. On-directions for each motoneuron were confined to a well-defined cone in 3D. Average on-directions of motoneurons differed significantly from the corresponding anatomically determined muscle rotation axes expressed in the stereotaxic reference frame (range of deviations: 11.9 degrees to 29.0 degrees). This difference was most pronounced for the vertical recti and oblique muscles. The muscle rotation axes of the vertical rectus pair and the oblique muscle pair form an angle of 58.3 degrees, whereas the corresponding angle for paired motoneuron on-directions was 105.6 degrees. On-directions of motoneurons were better aligned with the on-directions of semicircular canal afferents (range of deviation: 9.4-18.9 degrees) or with the anatomically determined sensitivity vectors of the semicircular canals (range of deviation: 3.9-15.9 degrees) than with the anatomically determined muscle rotation axes, but significant differences remain to be explained. The on-directions of motoneurons were arranged symmetrically to Listing's plane, in the sense that the torsional components for antagonistically paired muscles were almost equal, but of opposite sign. Thus, the torsional components of motoneuron on-directions cancel when eye movements are confined to Listing's plane. This arrangement simplifies the neuronal transformations for conjugate head-fixed voluntary eye movements, while the approximate alignment with the semicircular canal reference frame is optimal for generating compensatory eye movements.

Canal and otolith afferent activity underlying eye velocity responses to pitching while rotating.

Pitching the head while rotating (PWR) combines periodic activation of the semicircular canals and the otoliths to generate pitch and roll eye deviations and continuous horizontal nystagmus. Monkeys were tested after individual pairs of semicircular canals were plugged and single units were recorded in the vestibular nerve while the animals were sinusoidally pitched 20-40 deg about a spatial horizontal axis with 5- and 16-s periods and simultaneously rotated about a spatial vertical axis at 30-120 deg/s. As previously shown, the steady-state horizontal response disappeared after plugging the vertical semicircular canals, but was maintained when the lateral canals were plugged. When the left anterior and right posterior canal (LARP) pair was left intact, the steady-state response depended on the axis about which the pitching took place. When the axis was normal to the LARP plane, there was no steady-state response. When the pitching axis was perpendicular to the LARP normal, the response was maximal. Firing rates of otolith units were approximately in phase with pitch position, and the addition of rotation about a vertical axis did not change the response. Lateral canal units did not have a steady-state modulation during pitch or constant velocity rotation. During PWR, they oscillated at twice the pitch frequency. This corresponded to the frequency at which the canal was maximally activated as it aligned with the plane of rotation. The amplitude of modulation increased proportionally to rotational velocity, but the phase remained the same. These characteristics were unchanged during roll while rotating (RWR), which induces little continuous nystagmus. Anterior and posterior canal units were maximally excited near pitch-velocity maxima and minima, respectively, during pure pitching. During PWR, however, the phases of both components simultaneously shifted toward each other and toward being in phase with otolith units. The peak excitation tended toward a forward-pitch position when the rotation was to the ipsilateral side, and toward a backward pitch position when the rotation was to the contralateral side. With 120-deg/s rotation during a 16-s pitch period, the phase difference between anterior and posterior canal units was as small as 17 deg. These data support the postulate that the correlation between vertical canal and otolith units is the critical factor in generating continuous unidirectional horizontal nystagmus during PWR.

Activated platelets induce tissue factor expression on human umbilical vein endothelial cells by ligation of CD40.

CD40 is a type I member of the tumour necrosis factor (TNF) receptor superfamily of proteins, and is present on a wide variety of cells including vascular endothelial cells. Ligation of this receptor on endothelial cells is known to increase expression of inflammatory adhesion molecules. We have recently demonstrated that platelets express the ligand of CD40 (CD154) within seconds of exposure to agonist, and interact with endothelial cells to participate directly in the induction of an inflammatory response. Here we show that activated platelets induce tissue factor (TF) expression on endothelial cells in a CD40/CD154-dependent manner, and that the magnitude of this response can equal that induced by TNFe. Moreover, CD40 ligation on endothelial cells downregulates the expression of thrombomodulin. We also show that CD40-mediated TF expression is less sensitive to inhibition with the oxidative radical scavenger pyrrolidine dithiocarbamate than is that mediated by TNFalpha, indicating that CD40 has a distinct signalling pathway. Tissue factor is a cell membrane protein which functions as the main trigger of the extrinsic pathway of blood coagulation, and its expression on endothelial cells is implicated in wound healing and angiogenesis. Since platelets are among the first cells involved in haemostasis following tissue injury, our data showing that ligation of CD40 by CD154 induces a procoagulant phenotype on vascular endothelial cells suggests that platelets may play an important role in the induction of wound healing.

The collicular code of saccade direction depends on the roll orientation of the head relative to gravity.

This paper investigates the influence of static head tilt on the relation between activity in the motor layers of the superior colliculus (mSC) and saccadic oculomotor output. Based on single-unit recordings and electrical microstimulation in awake rhesus monkeys, we report that head roll changes the direction of the saccade vector generated by the mSC, with respect to a head-fixed coordinate system. Typically, the vector rotates in a direction that is opposite to the head roll direction. This rotation exceeds the amount of ocular counterroll that is observed as a result of difference in static head roll positions. We find that the mSC works in an oculocentric coordinate system that is biased in the direction of gravity. It is argued that the modification is a result of processes that occur downstream of or parallel to the mSC.

CD40 ligand on activated platelets triggers an inflammatory reaction of endothelial cells.

CD40 ligand (CD40L, CD154), a transmembrane protein structurally related to the cytokine TNF-alpha, was originally identified on stimulated CD4+ T cells, and later on stimulated mast cells and basophils. Interaction of CD40L on T cells with CD40 on B cells is of paramount importance for the development and function of the humoral immune system. CD40 is not only constitutively present on B cells, but it is also found on monocytes, macrophages and endothelial cells, suggesting that CD40L has a broader function in vivo. We now report that platelets express CD40L within seconds of activation in vitro and in the process of thrombus formation in vivo. Like TNF-alpha and interleukin-1, CD40L on platelets induces endothelial cells to secrete chemokines and to express adhesion molecules, thereby generating signals for the recruitment and extravasation of leukocytes at the site of injury. Our results indicate that platelets are not only involved in haemostasis but that they also directly initiate an inflammatory response of the vessel wall.

Induction, regulation, and function of soluble TRAP (CD40 ligand) during interaction of primary CD4+ CD45RA+ T cells with dendritic cells.

To assess the induction, regulation, and the relative roles of cell surface tumor necrosis factor-related activation protein (TRAP; CD40 ligand) and the soluble form of TRAP (sTRAP) in the initial phase of T cell activation, primary CD4+ CD45RA+ (naive) T cells were co-cultured with mature Langerhans' cells (mLC) in the presence of superantigen. In this cell system, TRAP was very efficiently induced in T cells at both the mRNA and protein levels. After appearing on the cell surface, TRAP was rapidly down-regulated by a mechanism triggered through interaction of TRAP with CD40 on mLC. Co-culture of T cells with mLC led to the release of sTRAP, an 18-kDa protein capable of binding to CD40. Experimental data strongly suggest that sTRAP is not released by proteolytic cleavage of TRAP on the cell surface, but is generated in an intracellular compartment. Release of sTRAP and induction of TRAP cell surface expression were found to be regulated independently. In terms of function, sTRAP cannot compete with cell surface TRAP for ligation of CD40 on mLC, indicating that sTRAP release is not a mechanism for termination of the TRAP/CD40 interaction. However, sTRAP on its own rapidly down-regulates CD40 expression on mLC and has long-lasting anti-apoptotic effects on dendritic cells. Thus, we infer from our results obtained in vitro that primary activation of CD4+ T cells by dendritic cells in the lymphoid tissues leads to release of sTRAP, which may act on CD40+ bystander cells in a cytokine-like fashion.