Purification, characterization, and localization of two ATP diphosphohydrolase isoforms in bovine heart.

Two ATP diphosphohydrolase (ATPDase) isoforms have been purified from the bovine heart ventricle. The purification procedure includes the following steps: differential centrifugation, sucrose cushion centrifugation, solubilization with Triton X-100, DEAE agarose ion exchange, and Affi-Gel blue-Sepharose and concanavalin A (con A)-Sepharose chromatographies. The purified enzyme has an optimum pH of catalysis of 7.5 and requires Ca2+ or Mg2+. The apparent Michaelis constant of the enzyme, with ADP as the substrate, is 29 microM, and the apparent maximal velocity is 1.6 mumol.min-1.mg protein-1. Substrate specificity, heat-inactivation curves, and copurification of adenosinetriphosphatase (ATPase) and adenosinediphosphatase (ADPase) activities confirmed the identity of the purified enzyme as an ATPDase. In addition, polyacrylamide gel electrophoresis, under nondenaturing conditions, showed identical migration patterns for the protein involved in ATPase and ADPase activities. Western blot analysis, with an antibody that specifically recognizes the NH2-terminal sequence of pig pancreas ATPDase and specifically reacts with bovine and human ATPDases, showed cross-reactivity with the purified ATPDase isoforms from the bovine heart. Immunocytochemical localization in the ventricle produced strong reactions with the plasma membrane of Purkinje fiber cells and the majority of myocardial cells. Immunoreactivity was variable, producing a mosaic-like aspect. As expected, smooth muscle cells and endothelial cells of coronary vessels were highly reactive. This ectoenzyme could play a protective role against the potentially deleterious effects of extracellular ATP. In tandem with 5'-nucleotidase, it produces adenosine, a powerful vasodilator, especially in hypoxic or ischemic conditions that favor the release of ATP.

Purification of the blood vessel ATP diphosphohydrolase, identification and localisation by immunological techniques.

ATP diphosphohydrolase (ATPDase) or apyrase (EC 3.6.1.5), an enzyme that hydrolyses the gamma and beta phosphate residues of triphospho- and diphosphonucleosides, has been purified from the bovine aorta media. A particulate fraction was isolated by differential, and sucrose cushion centrifugations, producing a 33-fold enrichment in ADPase activity. Solubilization of the enzyme from the particulate fraction with Triton X-100 caused a partial loss of activity. The solubilized enzyme was purified by DEAE-agarose, Affi-Gel blue and Concanavalin A column chromatographies yielding an additional 138-fold enrichment of the enzyme. The enzyme preparation was further purified by PAGE under non-denaturing conditions, followed by its detection on the gel. The active band was cut out and separated by SDS/PAGE. Overstaining with silver nitrate revealed a single band corresponding to a molecular mass of 78000. Presence of an ATP binding site on the latter protein was demonstrated by labelling with 5'-p-fluorosulfonylbenzoyladenosine (FSBA), an analogue of ATP, followed by its detection by a Western blot technique. Labelling specificity was demonstrated by competition experiments with Ca-ATP and Ca-ADP. An antiserum directed against the N-terminal sequence of the pig pancreas ATPDase (54 kDa) cross-reacted with the bovine aorta ATPDase at 78 kDa. Digestion of the ATPDase with N-glycosidase F caused a marked shift of the molecular mass, thereby showing multiple N-oligosaccharide chains. Immunohistochemical localisation confirmed the presence of ATPDase on both endothelial and smooth muscle cells.

Preservation of pointing accuracy toward moving targets after extensive visual cortical ablations in cats.

Impairments in reaching toward stationary and moving targets were studied in cats after restricted or extensive removal of visual cortical areas (areas 17, 18 and 19 and lateral suprasylvian visual areas). Regardless of the extent of the cortical lesion, cats were at first unable to localise and reach for a stationary target whereas they were soon able to detect and accurately point toward a mobile one. Moreover, the onset latency of such movements was dramatically increased. During post-operative re-training, the cats were unable to improve their accuracy scores when reaching towards stationary targets. In contrast, full compensation was observed for the accuracy of reaching movements directed toward moving targets. A partial recovery was observed for movement latency values that progressively decreased but left a permanent 30-40 ms impairment following extensive lesions. The role of extrageniculate messages and alternative routes involving other cortical areas in taking in charge the visuomotor activity is discussed.

Laterality in cats: paw preference and performance in a visuomotor activity.

Laterality in paw use was investigated over a period of 6 years in 44 domestic cats trained to perform a reaching movement toward a moving spot of light. Both paw preference and paw performance were recorded. At a 50 percent criterion, no significant paw preference was found at the level of the group. When a 90 percent criterion was considered, 23 subjects had a significant preference for one paw. Among these strongly lateralized animals, there were more left- (N = 17) than right-pawed (N = 6) cats. The analysis of visuo-motor performances included reaction time, movement time, and reaching accuracy. Lateralized cats had a faster reaction time than nonlateralized cats. The more-used paw had a shorter reaction time, a shorter movement time, and was also more accurate than the less-used paw. Thus, the findings demonstrate a functional advantage of being lateralized. Moreover, the results confirm the existence of an asymmetry of paw preference in cats and show a consistent relation between paw preference and performance.

Visuomotor relearning after brain damage crucially depends on the integrity of the ventrolateral thalamic nucleus.

The role of the ventrolateral thalamic nucleus (VL) has been analyzed in the phase of motor recovery that takes place when the execution of a previously learned movement has been perturbed by damage to another brain area. Cats were trained to perform a reaching movement toward a moving target-spot; they underwent bilateral brain lesions after performance had stabilized. A VL lesion induced a very transient increase of reaction time. The lesion of the main thalamic relay of the visual extrageniculate pathway (LP) severely disrupted accuracy and reaction time but was followed by full recovery. When both lesions were performed together, the deficits were similar to those induced by LP lesion alone, although they were more pronounced. However, the functional compensation was incomplete, and furthermore, the recovery of accuracy crucially depended on regular testing. These results support the involvement of VL in motor learning or relearning.

Long-term effects of dark rearing on a visually guided reaching movement in cats.

The existence of long-term effects of dark-rearing on visuo-motor coordination is still controversial. In this study 2 dark-reared (DR) cats were trained, after 5-6 years of recovery, to perform a reaching movement towards a stationary or a moving target. The accuracy, and the reaction and movement times were evaluated. The scores obtained by the DR cats were compared to those of normal subjects after a similar period of training. The results showed that while the accuracy of DR cats was not impaired, the performance of their reaching movements was slower than normal and its triggering was delayed. These data are discussed with regards to electrophysiological and behavioural data obtained on analogous DR cats after long-term recovery.

Visual 'cortical-recipient' and 'tectal-recipient' pontine zones play distinct roles in cat visuomotor performance.

In order to test the hypothesis that visual information reaching the cerebellum through the pontine nuclei is involved in the control of visually guided movements, the effects of bilateral kainic acid pontine lesions have been analysed in cats performing a reaching movement towards a spot of light that was either stationary or moving. In 4 cats, the lesion was restricted either to the ventromedian region (cortical-recipient zone) or to the dorsolateral nucleus (tectal-recipient zone) of the pons. A major and persistent impairment was seen when the cerebellum was deprived of the pontine information influenced by the colliculus. While cats displayed no impairment when reaching towards a stationary target, they exhibited a strong accuracy deficit associated with an increased reaction time when reaching towards a moving target. In contrast, lesioning the pontine zone influenced by the visual cortex induced a transient accuracy deficit with moving targets and a transient delay in movement onset whatever the mode of target presentation. These results emphasise the involvement of visual pontine regions in the guidance of movements; they also confirm previous results showing that tectal visual information plays a more important role than that originating in the visual cortex when movements are directed towards moving targets.

Motor deficit induced by red nucleus lesion: re-appraisal using kainic acid destructions.

The motor deficits induced by red nucleus lesions have been well documented but so far based upon approaches destroying both cells and fibres of passage. In the present study we used kaïnic lesions, which are known to spare, at least partially, the fibres of passage in order to re-investigate the motor deficit induced by rubral lesion. Five cats were fully trained to perform a forepaw pointing movement towards a moving spot of light. Four of them underwent a bilateral neurotoxic lesion of the red nucleus; a fifth one, was used as a control, with the lesion being done electrolytically. Kaïnic lesions induced strong dysmetria with a tendency to over-reaching, and a delayed movement onset; after postoperative training, the dysmetria only persisted when reaching towards targets moving at high speed. The electrolytic lesion led to a much stronger deficit with an additional lengthening of the execution phase duration. Moreover, although overall accuracy could recover, time taken for movement initiation and execution stayed permanently impaired. In the light of these results a distinction can be made between the red nucleus syndrome per se and the one due to damage to fibres of passage.

[Modification of the strategy of intentional movement after lesions of the substantia nigra in the cat]. / Modification de la stratégie d'un mouvement intentionnel après lésion de la substance noire chez le chat.

Bilateral lesions of the substantia nigra were carried out in cats previously overtrained at performing a visually guided forepaw movement towards a moving target. Both their reaction time (RT) and movement time (MT) were impaired postoperatively. On the other hand, their pointing precision was unimpaired after lesion and even improved relative to the preoperative level. This improved accuracy persisted even when the duration of the movement had returned to normal. It is suggested that lesioned animals develop and keep a new strategy using visual feed-back throughout the movement, while normal cats, even on such a complex pointing task, mainly use visual information to trigger their movement.

Brachium pontis lesions in cats partly reproduce the cerebellar dysfunction of voluntary reaching movements.

The pontocerebellar pathway in the brachium pontis (BP), is known to convey signals from various cortical and subcortical visual structures to the cerebellum. Recently, a cortico-pontocerebellar pathway involving the BP has been implicated in the control of visually guided movements, on the basis of anatomical and physiological data. To further test this hypothesis, using behavioural methods, we studied the effects of a bilateral interruption of these projections in the BP, on 5 cats fully trained to perform a forepaw movement towards a moving target-light. The postoperative deficit consisted of an impairment in precision, with a strong tendency to over-reach and and increase in reaction time, contrasting with an unimpaired movement time. Although there was some initial recovery, performance soon stabilized with a permanent impairment in accuracy and reaction time. These results are discussed in relation to the various sensory signals processed at the pontine level and forwarded to the cerebellum, and compared with the effects of motor dysfunction of cerebellar origin.

Glucose tolerance and erythrocyte insulin receptors in Friedreich's ataxia.

Detailed in vivo and in vitro studies of glucose and insulin metabolism in Friedreich's ataxia patients and unaffected family members have further defined the extent of the abnormalities in carbohydrate metabolism. The high incidence of glucose intolerance and a hyperinsulinemic response to a glucose challenge in a high percentage of Friedreich's ataxia patients has been confirmed. An increased incidence of glucose intolerance among heterozygotes is suggested, while the siblings show a more normal distribution of diabetes and a nearly normal insulin response to the glucose tolerance test. Human growth hormone patterns are normal for all groups. Preliminary studies of insulin binding to erythrocytes suggest a difference in the binding characteristics among diabetic Friedreich's ataxia patients, while the binding in the non-diabetic Friedreich's ataxia group is similar to that of non-diabetic controls. Results from a small group of non-diabetic siblings suggest a normal insulin binding, while a tendency toward increased binding at low insulin concentrations among diabetic family members is noted.