Evidence for different functional properties of the neuronal calcium sensor proteins VILIP-1 and VILIP-3: from subcellular localization to cellular function.

The visinin-like-proteins VILIP-1 and -3 are EF-hand calcium-binding proteins and belong to the family of neuronal calcium sensor (NCS) proteins. Members of this family are involved in the calcium-dependent regulation of signal transduction cascades mainly in the nervous system. VILIP-1 and VILIP-3 are expressed in different populations of neuronal cells. To gain insights into the different functional characteristics of VILIP-1 and -3, we have compared the localization of the proteins in intact cells and the calcium-dependent membrane association in subcellular fractions. Furthermore, we have investigated the different functional properties of the two proteins in activating cGMP signal pathways and have defined different sets of protein interaction partners. Our data indicate that VILIP-3, which is mainly expressed in Purkinje cells, and VILIP-1, which is expressed in granule cells in the cerebellum, show a different calcium-dependent subcellular localization, may activate different cellular signaling pathways, and thus have signaling functions which seem to be cell-type specific.

Intracellular neuronal calcium sensor (NCS) protein VILIP-1 modulates cGMP signalling pathways in transfected neural cells and cerebellar granule neurones.

The family of intracellular neuronal calcium-sensors (NCS) belongs to the superfamily of EF-hand proteins. Family members have been shown by in vitro assays to regulate signal cascades in retinal photoreceptor cells. To study the functions of NCS proteins not expressed in photoreceptor cells we examined Visinin-like protein-1 (VILIP-1) effects on signalling pathways in living neural cells. Visinin-like protein-1 expression increased cGMP levels in transfected C6 and PC12 cells. Interestingly, in transfected PC12 cells stimulation was dependent on the subcellular localization of VILIP-1. In cells transfected with membrane-associated wild-type VILIP-1 particulate guanylyl cyclase (GC) was stimulated more strongly than soluble GC. In contrast, deletion of the N-terminal myristoylation site resulted in cytosolic localization of VILIP-1 and enhanced stimulation of soluble GC. To study the molecular mechanisms underlying GC stimulation VILIP-1 was examined to see if it can physically interact with GCs. A direct physical interaction of VILIP-1 with the recombinant catalytic domain of particulate GCs-A, B and with native GCs enriched from rat brain was observed in GST pull-down as well as in surface plasmon resonance interaction studies. Furthermore, following trituration of recombinant VILIP-1 protein into cerebellar granule cells the protein influenced only signalling by GC-B. Together with the observed colocalization of GC-B, but not GC-A, with VILIP-1 in cerebellar granule cells, these results suggest that VILIP-1 may be a physiological regulator of GC-B.

Abnormal localization of two neuronal calcium sensor proteins, visinin-like proteins (vilips)-1 and -3, in neocortical brain areas of Alzheimer disease patients.

The anatomical distribution of the neuronal calcium sensor proteins visinin-like protein-1 and -3 (VILIP-1 and -3) was investigated in various neocortical areas of Alzheimer's disease (AD) patients and controls. In AD and normal brains their cellular localization was confined to pyramidal and non-pyramidal neurons. In AD brains the intracellular immunostaining for VILIP-1 and to a lesser extent for VILIP-3 was found to be reduced in comparison to controls. Also, significantly less VILIP-1-immunoreactive neurons were found in the temporal cortex of AD patients as compared to normal brains. Accordingly, Western blot analysis revealed that immunoreactivity for VILIP-1 is less concentrated in tissue extracts of the temporal cortex of AD patients compared to controls. Extracellularly, VILIP-1 and VILIP-3 immunoreactive material was detected in close association with typical pathologic hallmarks of AD such as dystrophic nerve cell processes, amorphous and neuritic plaques, and extracellular tangles. In control brains an extraneuronal localization of VILIP-1 or VILIP-3 was never observed. Our morphological and neurochemical findings point to an involvement of these two neuronal calcium sensor proteins in pathology and possibly pathophysiology of changed calcium homeostasis in AD.

The neuronal EF-hand calcium-binding protein visinin-like protein-3 is expressed in cerebellar Purkinje cells and shows a calcium-dependent membrane association.

Visinin-like protein-3 is a member of the family of intracellular neuronal calcium sensors belonging to the superfamily of EF-hand proteins. Members of this family are involved in the calcium-dependent regulation of signal transduction cascades. To gain insights into the characteristics of visinin-like protein-3, we have generated specific antibodies against visinin-like protein-3 and determined the developmental and tissue distribution of the protein and its exact cellular and subcellular localization. Expression of visinin-like protein-3 protein appeared late in development mainly in the cerebellum. It is strongly expressed in cerebellar Purkinje cells. The protein expression results were further confirmed by in situ hybridization and compared with hippocalcin messenger RNA localization. Native cerebellar visinin-like protein-3 was shown to bind calcium and to associate in a calcium-dependent manner with membrane fractions during subcellular fractionation. Recombinant wild-type visinin-like protein-3 was shown to be N-terminally myristoylated in transfected cells. The membrane association was strongly reduced for the non-myristoylated mutant of visinin-like protein-3 in transfected cells. These results suggest that visinin-like protein-3, which is mainly expressed in Purkinje cells in vivo, shows a calcium-dependent association with cell membranes which is mediated by a calcium-myristoyl switch.

Modulation of polymyxin B effects on mammalian urinary bladder.

This report demonstrates that Ca2+, Mg2+, and protons alter the ability of polymyxin B (PX, a cationic antibiotic used clinically as a bactericidal agent) to increase the apical membrane conductance of the rabbit urinary bladder. Using electrophysiological methods, we determine that these alterations occur by two mechanisms. First, they blocked the PX-induced conductance in a rapid and reversible manner; second, they competed with PX for a membrane binding site. In addition, Ca2+ (but not Mg2+ or protons) altered the rate at which the induced conductance could be reversed. When solution pH was greater than 8.8, PX was not able to induce a conductance. This ability of high pH to inhibit the action of PX was due to a decrease in the number of positive charges on PX. Further studies demonstrated that for maximal activity, PX required its fatty acid tail. These data were used to develop a model describing the mechanism by which PX can induce a conductance in the apical membrane of the rabbit urinary bladder.

The neuronal calcium-sensor protein VILIP modulates cyclic AMP accumulation in stably transfected C6 glioma cells: amino-terminal myristoylation determines functional activity.

VILIP (visinin-like protein) is a member of the neuronal subfamily of EF-hand calcium sensor proteins. Members of this family are involved in the calcium-dependent regulation of the desensitization of signal cascades in retinal photoreceptors. To gain insight into the function of VILIP in cell signaling, we have transfected wild-type VILIP and mutant VILIP lacking the myristoylation consensus sequence into C6 glioma cells. Expression of wild-type VILIP did not significantly influence the desensitization of beta-adrenergic receptors, which are coupled to adenylyl cyclase in C6 cells. However, VILIP expression increased the beta-adrenergic receptor-stimulated cyclic AMP (cAMP) level in these cells severalfold. The stimulatory effect was also observed after direct stimulation of the adenylyl cyclase with forskolin, indicating that VILIP acts downstream of receptor and G protein in the beta-adrenergic signaling pathway in C6 cells. In contrast, the nonmyristoylated mutant of VILIP reduced cellular cAMP levels in C6 cells. Myristoylated wild-type VILIP was associated in a calcium-dependent manner with membrane fractions during subcellular fractionation, presumably owing to a calcium-myristoyl switch. In contrast, association of nonmyristoylated mutant VILIP with membranes was strongly reduced. Thus, myristoylation and most likely the calcium-dependent membrane association of VILIP are important prerequisites for the activating effect of wild-type VILIP on cAMP accumulation in C6 cells. These results suggest that VILIP acts as a calcium sensor molecule that modulates cell signaling cascades, possibly by direct or indirect regulation of adenylyl cyclase activity.

Calcium- and myristoyl-dependent subcellular localization of the neuronal calcium-binding protein VILIP in transfected PC12 cells.

Wild-type neuronal calcium-binding protein VILIP (visinin-like protein), and a myristoylation mutant of VILIP which lacks the consensus sequence for N-terminal myristoylation, have been stably transfected in PC12 cells. Immunocytochemical studies of VILIP-transfected PC12 cells have revealed the wild-type VILIP is strongly concentrated at the cell membrane, particularly at cell-cell contact sites, but is also distributed throughout the cytosol at moderate levels. In contrast, myristoylation-mutant VILIP shows a more even distribution, with significantly less association at cell-cell contact sites. Western blot analysis of subcellular fractions has shown that wild-type VILIP associates in a calcium-dependent manner with membrane fractions, whereas the myristoylation mutant only weakly associates with this fraction. Therefore, a calcium-myristoyl switch seems to be a major, but not sole determinant for the association of VILIP with membranes in living cells.

Effects of polymyxin B on mammalian urinary bladder.

Previous reports have demonstrated that large cationic polypeptides (of molecular mass 5,000 daltons or greater) cause an increase in the apical membrane conductance of the rabbit urinary bladder epithelium. This report investigates the effects of the small cationic molecule polymyxin B (PX: a 1,400 dalton antibiotic) on the permeability of the rabbit urinary bladder. The addition of micromolar concentrations of polymyxin B to the luminal solution of the rabbit urinary bladder resulted in an increase in the transepithelial conductance of the bladder. The magnitude of the increase in the conductance was dependent upon the concentration of PX, and the polarity and magnitude of the apical membrane potential. As the apical membrane potential was made more cell interior negative, the larger was the increase in the membrane conductance. This voltage-dependent increase in conductance was an exponential function of the applied voltage, with a negligible increase in conductance occurring when the membrane potential was cell interior positive. Upon changing the membrane voltage from cell interior positive to negative, there was a delay before there was a measurable change in the membrane conductance. The longer the apical membrane was exposed to PX, the more poorly reversible was its effect on the transepithelial conductance, suggesting a toxic effect of PX on this epithelium.

Intermittent enteral feeding in mechanically ventilated patients. The effect on gastric pH and gastric cultures.

OBJECTIVE: To evaluate the effect of intermittent (16 h/d) enteral feeding (IEF) on gastric pH and gastric microbial growth in mechanically ventilated patients. DESIGN: Prospective, case-controlled study. SETTING: Medical ICU and infectious disease research laboratory in a university hospital. PATIENT POPULATION: Thirteen mechanically ventilated patients receiving continuous enteral feeding (CEF). METHODS: Gastric pH and quantitative gastric cultures were obtained while patients received CEF. Each patient's feeding schedule was changed to IEF. Daily gastric pH and quantitative gastric cultures were obtained for 5 consecutive days. RESULTS: Gastric microbial growth was found in 85% (11/13) of patients receiving CEF. Implementation of IEF did not clear gastric microbial growth, as only one patient subsequently reverted to negative culture. Similar gastric microbial growth continued in 90% (10/11) of patients after institution of IEF. Gastric pH did not decrease with the administration of IEF (gastric pH with IEF, 3.8 +/- 0.6 vs 4.7 +/- 0.5 with CEF (not significant [NS]). The amount of microbial growth was also unchanged with IEF (total growth with IEF, 7.8 x 10(5) +/- 5.2 x 10(5) cfu/mL vs 8.7 x 10(5) +/- 4.6 x 10(5) cfu/mL with CEF) (NS). Thirty-eight percent (5/13) of patients developed new Gram-negative rod growth in gastric cultures while receiving IEF. Gram-negative rod isolates increased from 25% of total isolates (CEF) to 40% (IEF). CONCLUSION: Our preliminary data suggest gastric pH was not lowered and existing microbial growth was not cleared in ventilated patients receiving IEF after previously receiving CEF. Further controlled study in a larger group of patients is necessary to determine whether IEF is of benefit in decreasing gastric colonization and nosocomial pneumonia.

The ACR-NEMA Digital Imaging and communications Standard: a nontechnical description.

The purpose of this report is to present, in nontechnical terms, the purpose and history of the American College of Radiology and National Electrical Manufacturers Association (ACR-NEMA) Digital Imaging and Communications Standard (ACR-NEMA Standards Publication no. 300-1985), an explanation of what the standard is, the characteristics of the standard, possible applications of the standard, and ongoing standardization efforts. It is assumed that the reader has at least a basic understanding of the concepts and technologies described collectively as picture archiving and communication systems (PACS). For information on PACS, please refer to the NEMA PACS Primer and the list of suggested reading contained therein (for copies, write or call NEMA, 2101 L St, Suite 300, Washington, DC 20037, ATTN: Ms Cindy Fuscoe; telephone 202-457-1965).