Depletion of ryanodine-sensitive Ca2+ store activates Ca2+ entry in rat submandibular gland acinar cells

The existence of ryanodine-sensitive Ca2+ stores and their role in the Ca2+ entry mechanism were examined in the rat submandibular gland acinar cells, using the microfluorimetry of intracellular Ca2+ concentration ([Ca2+]i). In the presence of thapsigargin, a Ca(2+)-ATPase inhibitor of inositol (1, 4, 5) triphosphate (InsP3)-sensitive Ca2+ stores, caffeine caused an increase in [Ca2+]i, which was inhibited by treatment with ryanodine (a ligand to the Ca(2+)-induced Ca2+ release channels). In the cells treated with ryanodine, 1 mM Ca2+ addition to a Ca(2+)-free solution caused a marked increase in [Ca2+]i, which was eliminated by application of Ni2+ or SK & F 96365, suggesting a Ca2+ entry triggered by ryanodine. The maximal change in the net increase in [Ca2+]i caused by the ryanodine-coupled Ca2+ entry, was 104.0 +/- 16.0 nM, which intense was caused by 10 microM ryanodine. Emptying the InsP3-sensitive stores by treatment with thapsigargin also caused Ca2+ entry, which maximally changed [Ca2+]i by 349.6 +/- 15.1 nM. Ten mumol/liter ryanodine was confirmed to cause a release of 45Ca2+ from the parotidic microsomal fraction enriched in endopalsmic reticulum. We propose that ryanodine-sensitive Ca2+ stores are present in rat submandibular gland acinar cells. We further propose that release of Ca2+ from the ryanodine-sensitive stores, which means eventually depletion of the ryanodine-sensitive Ca2+ stores, can activate the Ca2+ entry. The ability for Ca2+ entry coupled with the ryanodine-sensitive Ca2+ stores seems to be about 30% of the ability for Ca2+ entry coupled with the thapsigargin-sensitive Ca2+ stores.     

Postnatal development of acinar cells in rat submandibular gland as revealed by electron microscopic staining for carbohydrates

The postnatal differentiation of acinar cells in rat submandibular gland was studied by staining with periodic acid-thiosemicarbazide-silver proteinate to identify carbohydrate-containing macromolecules in the electron microscope. This method revealed glycogen particles and internal substructure in the secretory granules of developing acinar cells. On the basis of morphologic and histochemical criteria three phases of acinar cell development were defined. In the pro-acinar phase, during the first week after birth, pro-acinar cells and terminal tubular cells were the main components of the terminal tubules in the rudimentary gland. The secretory granules of the pro-acinar cells contained speckled or rod-like substructures which stained intensively for carbohydrates and were digested by proteolytic enzymes. During the second to third week after birth, which is the immature-acinar-cell phase, thread-like substructures were seen in the secretory granules. These structures, which were not digested by proteolytic enzymes, disappeared gradually. The acinar cells of 4-week-old or older rats displayed no particular substructure in the secretion granules and represented the final, mature phase of development.     

Distribution and translocation of isoforms of protein kinase C in rat submandibular acinar cells

The distribution of six isoforms of protein kinase C (PKC) in seromucous acinar cells of rat submandibular gland was examined and their translocation from the cytosolic- to the membrane fraction after different stimuli investigated. Western blotting, immunostaining with isoform-specific antibodies and scanning densitometry showed that PKC-alpha and epsilon were distributed fairly evenly between the cytosol and membranes in resting cells, while isoforms- beta, delta and zeta were all predominantly localized (over 80%) in membranes. PKC-gamma was not detected. PKC-alpha was mobilized to the membrane fraction by the phorbol ester, TPA, but not by the phosphoinositide-coupled agonists carbachol, methoxamine and substance P (SP). PKC-epsilon was translocated by TPA and carbachol but not by SP or methoxamine. Biochemical assay of total PKC confirmed that cytosolic enzyme activity was significantly reduced by TPA and carbachol to 29% and 75% respectively of control levels. These results suggest that muscarinic regulation of the mucosecretory response in the rat submandibular gland may be mediated by the PKC-epsilon isoform.     

Expression and localization of human kallistatin in rat submandibular gland after intracapsular gene injection

Gene delivery into rat submandibular gland in vivo by direct intracapsular injection has been studied. After the administration of adenovirus constructs, Ad-RSV-LacZ and Ad-CMV-LacZ, beta-galactosidase expression was localized in the granular convoluted tubular and striated duct cells of rat submandibular gland by in situ enzyme histochemistry. Adenovirus-mediated delivery of the human kallistatin gene (Ad-RSV-HKBP) into rat submandibular gland results in the expression of human kallistatin in a time-dependent manner. The expression of immunoreactive kallistatin in submandibular gland was detected 1 day after the Ad-RSV-HKBP injection and it reached a plateau (1-2 ng/mg protein) 2 days after gene delivery. Higher levels of human kallistatin were found in the submandibular gland of 6-month-old rats than in one-month-old rats. After direct gene injection, human kallistatin was localized mainly in cells of the granular convoluted tubules and striated ducts of rat submandibular gland using a specific monoclonal antibody to human kallistatin. The results indicate that direct intracapsular gene delivery into the submandibular gland provides a simple and reliable method for introducing foreign genes into the gland. This method can be used for studying gene regulation in vivo and may have potential for gene therapy in oral diseases.     

Ultrastructural localization of P2X3 receptors in rat sensory neurons

We used isolated IgG antibodies selective for P2X3 receptors to study the ultrastructural distribution of these receptors in rat sensory neurons. In trigeminal ganglia, P2X3 receptor immunoreactivity occurred in small and large nerve cell bodies and their processes. Endoplasmic reticulum and Golgi apparatus were heavily stained; cytoplasmic matrix was faintly to moderately stained. In synaptic glomeruli in lamina II of cervical dorsal horn, P2X3 receptor-immunoreactive core terminals were postsynaptic to unlabelled vesicle-containing dendrites and axons. In the nucleus of the solitary tract, receptor-positive boutons synapsed on dendrites and cell bodies and had complex synaptic relationships with other axon terminals and vesiculated dendrites. These observations identify sites from which ATP could be released to influence sensory signalling within the central nervous system.     

Differentiating myoepithelial and acinar cells in rat neonatal parotid gland and histogenetic concepts for salivary gland tumors

Histogenetic concepts for salivary gland tumors are predicated on the presence of reserve or undifferentiated cells in normal glands, presumably the source for cell renewal and induction of tumors. Developing rat parotid gland, which remains fetal-like at birth, provides the opportunity to study differentiation and observe whether cytologically undifferentiated cells do or do not have functional indicators of specific differentiation pathways. Immunohistochemistry and immuno-electron microscopy, when applied to parotid gland at birth, at 12 days of age and in the adult gland, indicate that commitment to myoepithelial cell differentiation occurs prior to development of structural changes characteristic of these cells. Conversely, secretory granules are evident in differentiating acinar cells prior to synthesis of amylase. The results suggest that an appearance of undifferentiation does not confer reserve cell status either in the normal salivary gland or their tumors.     

Alterations in Ca2+ storage and mobilization in submandibular acinar cells of reserpine-treated rats

For cosmetic reasons, hand prostheses are provided with cosmetic gloves. Their pleasing appearance, however, is accompanied by poor mechanical behavior, resulting in a negative influence on prosthesis operation. Glove stiffness is high and nonlinear, and internal friction in the glove material causes energy dissipation (hysteresis). In this article, two methods for reducing hysteresis in cosmetic gloves are proposed, that may be applied independently or in combination. Glove modification. Altering the mechanical properties of the glove itself is the first method that is presented. It was found possible to reduce both stiffness and hysteresis about 50% by forming grooves into the inside of the glove. Together with the evaluation of this method, several properties of the cosmetic glove were determined. Motion optimization. Additionally, a second method for reducing hysteresis was developed. The amount of hysteresis is influenced by the way the glove is forced to deform. The prosthesis mechanism, determining this deformation, was designed for minimum hysteresis and maximum cosmesis. For the prosthesis-glove combination used in this study, thumb motion optimization reduced hysteresis by about 65%.     

Adrenergic receptors and secretory responses of the rat submandibular salivary gland after periodic incisor reduction

While loss-of-function mutations in Gsalpha are invariably associated with the short stature and brachydactyly of Albright hereditary osteodystrophy (AHO), the association with hormone resistance (to parathyroid hormone and thyrotropin) typical of pseudohypoparathyroidism type Ia (PHP-Ia) is much more variable. Observational studies and DNA polymorphism analysis suggest that maternal transmission of the Gsalpha mutation may be required for full expression of clinical hormone resistance. To test this hypothesis, we studied transmission of a frameshift mutation in Gsalpha through three generations of a pedigree affected by AHO and PHP-Ia. While all family members carrying this loss-of-function mutation in one Gsalpha allele had AHO, neither the presence of the mutation nor the degree of reduction of erythrocyte Gsalpha bioactivity allowed prediction of phenotype (AHO alone versus AHO and PHP-Ia). Paternal transmission of the mutation (from the patriarch of the first generation to three members of the second generation) was not associated with concurrent PHP-Ia, but maternal transmission (from two women in the second generation to four children in the third generation) was invariably associated with PHP-Ia. No expansion of an upstream short CCG nucleotide repeat region was detected, nor was there evidence of uniparental disomy by polymorphism analysis. This report, the first to document the effects across three generations of both paternal and maternal transmission of a specific Gsalpha mutation, strongly supports the hypothesis that a maternal factor determines full expression of Gsalpha dysfunction as PHP-Ia.     

Occurrence and nuclear localization of cAMP response element-binding protein in the post-natal development of the rat submandibular gland

Cyclic AMP response element-binding protein (CREB) is a 43-kDa polypeptide that binds a cAMP response element located at the 5' promoter region of cAMP regulatory genes. The spatial and temporal distribution of CREB in the postnatal development of the rat submandibular gland was investigated using immunohistochemistry with a specific antibody. At birth, cells of the terminal tubules and ducts in the submandibular gland showed a nuclear CREB immunoreactivity of moderate intensity. At 1-2 weeks after birth, an intense CREB immunoreactivity was localized primarily to acinar cells. When the beta-adrenergic agonist isoproterenol was administered to 2-week-old rats, a twofold transient increase in the number of immunoreactive acinar cells was induced. Beginning 3 weeks after birth, CREB immunoreactivity shifted from acini to the duct system and showed a clear localization in the cells of the intercalated ducts and distal portions of striated ducts, where the granular convoluted tubule develops after 4 weeks. Immunopositive materials were localized exclusively in the nuclei of both acinar and ductal immunoreactive cells. After the development of the granular convoluted tubules, CREB immunoreactivity was absent in the tubule cells and was gradually reduced in intensity over the entire gland. In order to examine a hypothesis that CREB is involved in the initial differentiation of the granular convoluted tubular cells, testosterone was administered to hypophysectomized adult rats. Whereas the tubular cells of hypophysectomized rats showed a complete regression, and no CREB immunoreactivity was found in any acinar or duct cells, administration of testosterone for a few days induced an intense CREB immunoreactivity in the nuclei of duct cells, followed by their differentiation into the granular convoluted tubular cells. These results suggested that CREB is involved not only in the growth and differentiation of acinar cells that are regulated by beta-adrenergic nerves but also in those of the duct system, and especially in the androgen-regulated differentiation of the granular convoluted tubular cells, during the post-natal development of the rat submandibular gland.     

Substructures of the acinar basement membrane of rat submandibular gland as shown by alcian blue staining and cryo-fixation followed by freeze-substitution

The distribution of surface-exposed antigenic glycolipids in seven recent clinical isolates of Mycobacterium tuberculosis was established. Thin-layer and liquid chromatographies revealed a uniformity in the glycolipid pattern. Chemical analysis of the individual glycolipids of a selected strain enabled the identification of glycolipids of serological interest in all the other clinical isolates. Phenolic glycolipid-Tb1 (PGL-Tb1) was lacking in all strains, but appreciable amounts of a partially deglycosylated version (PGL-Tb1D) were present in the seven isolates. Diacyltrehaloses (DATs) were detected in all strains, showing themselves to be major glycolipids. Lipooligosaccharides (LOS-II) were present in the seven strains studied though only in trace amounts. These results shed new light on the open debate on the distribution of these interesting glycolipids in typical clinical isolates of M. tuberculosis. In the search for a serological test for tuberculosis, and in accordance with our observations, we believe that PGL-Tb1 and LOS-II should not be the target molecules for serology and that it is worthwhile to continue investigating the value of DATs as antigens. We also believe that it would be of interest to undertake research to assess the usefulness of PGL-Tb1D as an antigen.     

Promiscuous coupling of receptors to Gq class alpha subunits and effector proteins in pancreatic and submandibular gland cells

Mice with deficiencies in one or more Gq class alpha subunit genes were used to examine the role of the alpha subunit in regulating Ca2+ signaling in pancreatic and submandibular gland cells. Western blot analysis showed that these cells express three of the four Gq class subunits, Galphaq, Galpha11, and Galpha14 but not Galpha15. Surprisingly, all parameters of Ca2+ signaling were identical in cells from wild type and four lines of mutant mice: 1) Galpha11-/-, 2) Galpha11-/-/Galpha14-/-, 3) Galpha14-/-/Galpha15-/-, and 4) Galphaq-/-/Galpha15-/-. These parameters included the Kapp for several Gq class coupled receptors, induction of [Ca2+]i oscillations by weak stimulation, and a biphasic [Ca2+]i response by strong stimulation. Furthermore, Ca2+ release from internal stores and Ca2+ entry were not affected in cells from any of the mutant mice. We conclude that Galphaq, Galpha11, and Galpha14 promiscuously couple several receptors (m3 muscarinic, bombesin, cholecystokinin, and alpha1 adrenergic) to effector proteins that activate both Ca2+ release from internal stores and Ca2+ entry.     

Endothelin receptors in rat pituitary gland

An algorithm is described for reducing ghost artifacts in echo planar imaging (EPI) using phase corrections derived from images reconstructed using only even or odd k-space lines. The N/2 ghost, that arises principally from time-reversal of alternate k-space lines, was significantly reduced by this algorithm without the need for a calibration scan. In images obtained in eight subjects undergoing EPI for auditory functional MRI (fMRI) experiments, N/2 ghost intensity was reduced from 10.3% +/- 2.1% (range: 7.9-14.1%) to 4.5% +/- 0.2% (range: 4.1-4.9%) of parent image intensity, corresponding to a percent reduction in ghost intensity of 54% +/- 9% (range: 43-65%), and the algorithm restored this intensity to the parent image. It provided a significant improvement in image appearance, and increased the correlation coefficients related to neural activation in functional MRI studies. The algorithm provided reduction of artifacts from all polynomial orders of spatial phase errors in both spatial directions. The algorithm did not eliminate N/2 ghost intensity contributed by field inhomogeneities, susceptibility, or chemical shift.     

P2X receptors in cochlear Deiters'' cells

The performance of different anion-exchange media have been compared for the isolation of plasmid DNA and genomic DNA from bacterial cells and human whole blood. Whatman DEAE-Magarose, based on an agarose bead containing a paramagnetic component, has been compared with prepacked gravity-flow columns containing a derivatised silica matrix. In each case the DNA isolation at various scales of operation was similar both in terms of yield and quality. The magnetic susceptibility of DEAE-Magarose is very high, facilitating the use of this separation technique for rapid flexible batch chromatographic processes, a limitation of the prepacked column techniques.     

Laminin-1 and laminin-2 G-domain synthetic peptides bind syndecan-1 and are involved in acinar formation of a human submandibular gland cell line

The culture of human submandibular gland (HSG) cells on laminin-1 induces acinar differentiation. We identified a site on laminin involved in acinar differentiation using synthetic peptides derived from the C-terminal G-domain of the laminin alpha1 and alpha2 chains. The alpha1 chain peptide AG73 (RKRLQVQLSIRT) decreases the size of acini formed on laminin-1. Cells cultured with either AG73 or the homologous alpha2 chain peptide MG73 (KNRLTIELEVRT) form structures that appear acinar-like, but the cell nuclei are not polarized to the basal surface and no lumen formation occurs, indicating that additional sites on laminin are required for complete differentiation. The G-domain of laminin-1 contains both integrin and heparin binding sites, and anti-beta1-integrin antibodies disrupt acinar formation. Cell adhesion to the peptides and to E3, an elastase digest fragment of laminin-1 containing AG73, is specific, since other laminin peptides or EDTA do not compete the binding. Heparin and heparan sulfate decrease cell adhesion to AG73 and MG73 but anti-beta1-integrin antibodies have no effect. Treating the cell surface with heparitinase inhibits adhesion to both AG73 and MG73. We isolated cell surface ligands using both peptide affinity chromatography and laminin-1 affinity chromatography. Treating the material bound to the affinity columns with heparitinase and chondroitinase enriches for a core protein identified as syndecan-1 by Western blot analysis, thus identifying a syndecan-1 binding site in the globular domain of laminin-1 and laminin-2. In summary, multiple interactions between laminin and HSG cells contribute to acinar differentiation, involving both beta1-integrins and syndecan-1.     

Fine structure of the acinar and duct cell components in the parotid and submandibular salivary glands of the rat: a TEM, SEM, and HRSEM study

We have developed a process for producing fine, very flexible microwires suitable for use as small signal leadwires or nerve electrodes. The process incorporates metallization of high-performance monofilament polymer fibers to yield electrically conductive fibers with greatly improved flexibility over solid metal wires of similar strength. The metallization layers are produced by serial vacuum deposition of a 0.3 micron thick coating of three metals, titanium-tungsten (Ti/W), gold (Au), and platinum (Pt), onto monofilament, poly-p-phenyl-terephthalate aramid fibers (Kevlar). The metallized fibers are then insulated with an approx. 1 micron thick layer of silicone elastomer. The result is a microlead with high electrical conductivity (linear resistance = 30 omega/cm), desirable interfacial properties, excellent mechanical stability and extremely high flexibility. These physical characteristics are appropriate for application as signal leadwires or recording/stimulating electrodes where small size and high flexibility are paramount. In this paper we report on the electrical and mechanical properties of these metallized fibers and demonstrate their use as intrafascicular electrodes for recording multi-unit neural activity in feline peripheral nerves.     

Characterization and localization of galanin receptors in human entorhinal cortex

The neuropeptide galanin (GAL) has a widespread distribution throughout the human cortex. The entorhinal cortex (ENT) plays a crucial role in the transfer of cortico-cortical information related to memory and displays severe degeneration in Alzheimer's disease (AD). However, very little is known about the pharmacology of the GAL receptor (GALR) in normal human ENT. Therefore, we pharmacologically visualized their distribution and characterized GALRs using in vitro receptor autoradiography and radioligand binding assays. Autoradiograms revealed intense GALR labeling, mainly in the substantia innominata, hypothalamus, the bed nucleus of the stria terminalis and within layers 2 and 4 of the ENT. Kinetic experiments showed that saturation of GALR sites by [125I]GAL (human) (hGAL) occurred within 2 h and that this binding readily reversed in the presence of a GTP analog, but not in the presence of excess unlabeled hGAL. Analysis of [125I]hGAL binding data from saturation experiments gave KD values of 98.6+/-21.6 pM, Bmax values of 52.9+/-32.4 fmol/mg protein and identified a high and low affinity state of the GALR. The presence of 5'-guanylylimidodiphosphate (GppNHp) or NaCl reduced the agonist labeling of hGALR in ENT membranes.