Fast exocytosis with few Ca(2+) channels in insulin-secreting mouse pancreatic B cells.

The association of L-type Ca(2+) channels to the secretory granules and its functional significance to secretion was investigated in mouse pancreatic B cells. Nonstationary fluctuation analysis showed that the B cell is equipped with <500 alpha1(C) L-type Ca(2+) channels, corresponding to a Ca(2+) channel density of 0.9 channels per microm(2). Analysis of the kinetics of exocytosis during voltage-clamp depolarizations revealed an early component that reached a peak rate of 1.1 pFs(-1) (approximately 650 granules/s) 25 ms after onset of the pulse and is completed within approximately 100 ms. This component represents a subset of approximately 60 granules situated in the immediate vicinity of the L-type Ca(2+) channels, corresponding to approximately 10% of the readily releasable pool of granules. Experiments involving photorelease of caged Ca(2+) revealed that the rate of exocytosis was half-maximal at a cytoplasmic Ca(2+) concentration of 17 microM, and concentrations >25 microM are required to attain the rate of exocytosis observed during voltage-clamp depolarizations. The rapid component of exocytosis was not affected by inclusion of millimolar concentrations of the Ca(2+) buffer EGTA but abolished by addition of exogenous L(C753-893), the 140 amino acids of the cytoplasmic loop connecting the 2(nd) and 3(rd) transmembrane region of the alpha1(C) L-type Ca(2+) channel, which has been proposed to tether the Ca(2+) channels to the secretory granules. In keeping with the idea that secretion is determined by Ca(2+) influx through individual Ca(2+) channels, exocytosis triggered by brief (15 ms) depolarizations was enhanced 2.5-fold by the Ca(2+) channel agonist BayK8644 and 3.5-fold by elevating extracellular Ca(2+) from 2.6 to 10 mM. Recordings of single Ca(2+) channel activity revealed that patches predominantly contained no channels or many active channels. We propose that several Ca(2+) channels associate with a single granule thus forming a functional unit. This arrangement is important in a cell with few Ca(2+) channels as it ensures maximum usage of the Ca(2+) entering the cell while minimizing the influence of stochastic variations of the Ca(2+) channel activity.

Body centre of mass movement in the lame horse.

The body centre of mass (BCM) is a key factor in the analysis of equine gait, as its position and movement determines the distribution and magnitude of loads on the limbs. Changes in the BCM movement are proposed to be important factors in the lameness management of horses. In this study, changes in the position and the 3-dimensional (3-D) movement of the BCM in horses with induced forelimb lameness were studied using a kinematic, segmental method. The kinematics of 30 markers representing 20 body segments were recorded in 12 horses while trotting (3.9 m/s) on a treadmill using a high speed video system. A transient lameness model, evoking pressure-induced pain on the hoof sole, was used to induce 2 degrees of forelimb lameness. Based on segmental inertial data from Buchner et al, (1997) 3-D segmental centres of mass as well as the BCM were calculated. The changes in BCM movement due to lameness and the influence of selected segments on the total centre of mass were analysed. During moderate forelimb lameness, vertical displacement of the BCM showed a 34% reduced amplitude during the stance phase of the lame limb and a 9% increased amplitude during the stance phase of the sound forelimb. In the sagittal direction, the BCM during midstance moved 9 mm backwards during the stance phase of the lame forelimb. Transversal movements showed a slight, but significant shift of the BCM to the side of the sound forelimb. Head/neck segment movement changed in a sagittal as well as in a vertical direction, but only a small amount, insufficient for considerable BCM position changes. The results show a similar pattern of BCM and trunk movement and only small adaptations of BCM position due to lameness. The influence of the sagittal position of the BCM has to be seen as a minor factor in lameness management compared to the dynamic influences of a changed vertical BCM movement and the moment caused by the typical head and neck movement.

Priming of insulin granules for exocytosis by granular Cl(-) uptake and acidification.

ATP-dependent priming of the secretory granules precedes Ca(2+)-regulated neuroendocrine secretion, but the exact nature of this reaction is not fully established in all secretory cell types. We have further investigated this reaction in the insulin-secreting pancreatic B-cell and demonstrate that granular acidification driven by a V-type H(+)-ATPase in the granular membrane is a decisive step in priming. This requires simultaneous Cl(-) uptake through granular ClC-3 Cl(-) channels. Accordingly, granule acidification and priming are inhibited by agents that prevent transgranular Cl(-) fluxes, such as 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and an antibody against the ClC-3 channels, but accelerated by increases in the intracellular ATP:ADP ratio or addition of hypoglycemic sulfonylureas. We suggest that this might represent an important mechanism for metabolic regulation of Ca(2+)-dependent exocytosis that is also likely to be operational in other secretory cell types.

Body centre of mass movement in the sound horse.

The body centre of mass (BCM) is a key factor in the analysis of equine locomotion, as its position and movement determines the distribution and magnitude of loads on the limbs. In this study, the three-dimensional (3D) movement of the BCM in walking and trotting horses was assessed using a kinematic, segmental method. Thirty markers representing 20 body segments were recorded in 12 sound horses while standing, walking and trotting on a treadmill using a high-speed video system. Based on segmental inertial data, 3D positions of the segmental centres of mass as well as the total BCM were calculated. The position within the trunk during square standing and the movements of the BCM were determined for the three planes. The position of the BCM in the standing horse is presented relative to external reference points. At the trot, vertical displacement amplitude of the BCM amounted to 53 (6) mm as mean (sd), which was 27% smaller than external trunk movement. Medio-lateral displacement amplitude of the BCM was 19 (4) mm, 34% less than trunk amplitude. Sagittal forward-backward oscillations of the BCM independent from general forward movement were 13 (3) mm, being 24% less than trunk movements. At the walk, vertical, medio-lateral and sagittal BCM movements were smaller than trunk movements by 43, 65 and 65% respectively. The results show reduced and efficient BCM movements compared to the trunk and form a basis for the assessment of various clinical conditions such as lameness, the influence of a rider and various dressage performances.

The dileucine motif within the tail of MPR46 is required for sorting of the receptor in endosomes.

The cytoplasmic tail of MPR46 carries a leucine-based motif that is required for the sorting of lysosomal enzymes by the receptor. In addition, it is one of three independent, but functionally redundant, internalization signals present in the cytoplasmic tail of MPR46. We have analyzed a mutant of MPR46, in which the dileucine pair was replaced by alanines (MPR46 LL/AA) with respect to its intracellular distribution and trafficking. Ultrastructural analysis of cells expressing the MPR46 LL/AA mutant revealed that the substitution of the dileucine pair causes a shift of the receptor distribution from the TGN, where it is packaged into AP1-containing vesicles, to vesicular structures distributed throughout the cytoplasm. The vesicles could be identified as early endosomes with internalized BSA-gold and rab5 as markers. By analyzing the receptor trafficking biochemically, we found that return of the LL/AA mutant receptor from the plasma membrane/endosome pool back to the TGN was impaired, while recycling from endosomes to the plasma membrane was enhanced. In conclusion, our data indicate that the dileucine motif in the MPR46 tail is required for a sorting event in endosomes.