P2X receptors in the rat duodenal villus.

Immunohistochemical techniques were performed on freshly frozen sections of the duodenum of the rat using specific polyclonal antibodies to unique peptide sequences of P2X1-7 receptors. Of the antibodies to the seven known P2X receptor subtypes that mediate extracellular signalling by nucleotides, three reacted with discrete structures in the duodenal villus of the rat. Anti-P2X1 reacted with the capillary plexus in the intestinal villus, which did not extend to the crypt region, suggesting that nucleotides may be involved in the uptake and transport of metabolites. Anti-P2X5 immunostained the membranes of the narrow "stem" of villus goblet cells, where the nucleus and cell organelles reside, possibly influencing synthesis and release of mucins. P2X7 receptor immunoreactivity was only seen in the membranes of enterocytes and goblet cells at the tip of the villus, where cells are exfoliated into the lumen, consistent with earlier findings that P2X7 is involved in apoptotic events. Thus, in complex structures such as the intestinal villus, purinoceptors appear to participate in several and diverse signalling functions.

Localisation of P2X5 and P2X7 receptors by immunohistochemistry in rat stratified squamous epithelia.

In order to investigate whether purinoceptors are involved in the physiological renewal and regeneration of epithelia, we used immunohistochemical techniques on fresh frozen sections of various stratified squamous epithelial tissues (cornea, tongue, soft palate, oesophagus, vagina and footpad) of the rat and specific polyclonal antibodies to unique peptide sequences of P2X1-7 receptors. Only two of the antibodies, anti-P2X5 and anti-P2X7, reacted with epithelial structures. P2X5 immunoreactivity was mainly associated with the membranes of the proliferating and differentiating cell layers (spinous and granular layer) in both keratinised and non-keratinised epithelia and growing hair follicles. In contrast, P2X7 immunoreactivity was clearly associated with the keratinisation process, the staining being most intense in the upper keratinised and the exfoliated layers. These findings suggest, for the first time, that P2X5 and P2X7 receptors play an important role in the physiological turnover of continuously regenerating cells, and further, raise the possibility that they represent novel targets for the development of pharmacological tools of potential benefit for diseases of epithelial dysfunction.

Expression of two ATP-gated ion channels, P2X5 and P2X6, in developing chick skeletal muscle.

Physiological and pharmacological studies have shown that ATP has potent effects on developing chick skeletal muscle. These effects have previously been shown to be developmentally regulated, and the responses were characteristic of activation of the P2X ligand-gated ion-channel family of ATP receptors. Here, using immunohistochemistry, we describe the expression patterns of two members of the P2X receptor family, P2X5 and P2X6, during development of skeletal muscle in the chick embryo. These receptors were first expressed at early stages of skeletal muscle development, and expression disappeared immediately before the stage at which fusion of myoblasts to form myotubes occurs. P2X5 was also demonstrated in nerves supplying developing skeletal muscle, in some dorsal root ganglion cells, and in dorsal and ventral spinal cord. No expression of the other five members of the P2X family were demonstrated in developing skeletal muscle.

Heat-treated myosin does not bind ATPase--inhibiting antibodies.

Polyclonal antibodies to native chicken pectoral fast-twitch myosin are directed to all subfragments of the molecule (S1, S2 and LMM), as seen in the ELISA and Western blotting techniques. The antibodies inhibit the Ca(2+)-activated myosin ATPase. Absorption of the antibodies with native myosin abolishes these reactions. Heat treatment of myosin for 2h at 40 degrees C will inactivate myosin ATPase and alter its antibody binding pattern: the binding of antibodies to the rod fractions is reduced, that to the globular head (S1) completely abolished. Thus, these antibodies are useful as sensitive probes for the structural integrity of the myosin head.

Immunochemical probing of the functional role of the 238-246 and 567-574 sequences of myosin heavy chain.

Polyclonal site-directed peptide antibodies were raised against the 567-574 and 238-246 sequences of the rabbit skeletal muscle myosin heavy chain. These sequences, which are located in the subfragment 1 (S1) segment of myosin, have been implicated by former studies in actin and nucleotide binding of the molecule and in the communication between the two binding sites. The antibodies obtained from rabbit sera were found to be conformation-sensitive since they specifically reacted with S1 in solid-phase binding assay but not in Western blot. The binding of both antibodies to S1 was strongly inhibited by actin. The antibody against the 567-574 sequence, Ab567-574, moderately decreased the binding of S1 to actin filaments in rigor but not in the weakly-attached state, while Ab238-246 did not influence the binding of S1 to actin under either conditions. Both antibodies inhibited the actin activation of the MgATPase of S1 but did not affect MgATPase without actin or the Ca- and K(EDTA)-activated ATPase activities of S1. The sliding velocity of actin filaments in the in vitro motility assays were also reduced in the presence of the antibodies. Ab567-574 had especially strong inhibitory effect on the movement of actin filaments. The results indicate that the binding of antibodies may induce conformational changes, which propagate in the S1 structure, perturb the coupling between the binding sites and impair the motor function of myosin.

Embryonic chicken gizzard: expression of the smooth muscle regulatory proteins caldesmon and myosin light chain kinase.

The patterns of expression of the smooth muscle regulatory proteins caldesmon and myosin light chain kinase were investigated in the developing chicken gizzard. Immunofluorescent studies revealed that both proteins were expressed as early as E5 throughout the mesodermal gizzard anlage, together with actin, alpha-actin-in and a small amount of nonmuscle myosin. These proteins appear to form the scaffold for smooth muscle development, defined by the onset of smooth muscle myosin expression. During E6, a period of extensive cell division, smooth muscle myosin begins to appear in the musculi laterales close to the serosal border and, later, also in the musculi intermedii. Until about E10, myosin reactivity expands into the pre-existing thin filament scaffold. Later in development, the contractile and regulatory proteins co-localize and show a regular uniform staining pattern comparable to that seen in adult tissue. By using immunoblotting techniques, the low-molecular mass form of caldesmon and myosin light chain kinase were detected as early as E5. During further development, the expression of caldesmon switched from the low-molecular mass to the high-molecular mass form; in neonatal and adult tissue, high-molecular mass caldesmon was the only isoform expressed. The level of expression of myosin light chain kinase increased continously during embryonic development, but no embryo-specific isoform with a different molecular mass was detected.

Fatty acid binding of myoglobin depends on its oxygenation.

In the present work we show with different binding assays chicken gizzard myoglobin is able to bind fatty acids and bromosulphophthalein (BSP) in vitro. The fatty acid binding depends on the oxygenation of the myoglobin. Freshly prepared chicken gizzard, chicken or bovine heart myoglobin have a high fatty acid binding capability. However, when oxy-myoglobin in converted to met-myoglobin by dialysis against acidic buffer at high ionic strength (or when commercially available myoglobins are used) a 60-70% lower fatty acid binding capacity is found. Like bovine serum albumin (BSA), gizzard myoglobin has the highest affinity for unsaturated fatty acids and a lower affinity for saturated fatty acids or dyes. Chicken gizzard smooth muscle myoglobin may function as an additional fatty acid binding protein in vivo.

Embryonic chicken gizzard: smooth muscle and non-muscle myosin isoforms.

Antibodies to smooth muscle and non-muscle myosin allow the development of smooth muscle and its capillary system in the embryonic chicken gizzard to be followed by immunofluorescent techniques. Although smooth muscle development proceeds in a serosal to luminal direction, angiogenetic cell clusters develop independently at the luminal side close to the epithelial layer, and the presumptive capillaries invade the developing muscle in a luminal to serosal direction. The smooth muscle and non-muscle myosin heavy chains in this avian system cannot be separated by SDS polyacrylamide gel electrophoresis and do not show isoform specificity in immunoblotting, unlike the system found in mammals. Only two myosin heavy chains with M(r) of 200 and 196 kDa were separable and considerable immunological cross-reactivity was found between the denatured myosin isoform heavy chains.

Embryonic chicken gizzard: immunolocalization of collagen and smooth muscle myosin.

Antibodies to chicken gizzard myosin and to chicken skin collagen type I allow the myofibrillar and connective tissue development in the embryonic chicken gizzard to be followed. Fibroblasts are assumed to synthesize collagen prior to the onset of smooth muscle cell development in the muscle primordium (day 5); they are presumably also responsible for collagen synthesis close to the presumptive lamina propria and in the developing tubular glands (day 14 to 17). From day 6 to 8, myosin and collagen are colocalized intracellularly, and from day 9 onward collagen fibers start to appear extracellularly, eventually forming the trellis-like connective tissue septa that give the rhomboid profile found in the adult muscle. The close association of collagen and myosin in early development suggests that the muscle cells themselves produce and export collagen.

Microcompartmentation of glycolytic enzymes in cultured cells.

The microcompartmentation of aldolase and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) was investigated in four different cell types (3T3 cells, SV 40 transformed 3T3 cells, mouse fibroblasts, chick embryo cardiomyocytes) combining cell permeabilization and indirect immunofluorescence technique. Permeabilization of the cells prior to fixation released the soluble fractions, whilst the total amount of enzymes was preserved in nonpermeabilized cells. Both enzymes exist in a soluble as well as in a structure-bound form. The soluble fraction of aldolase and GAPDH is distributed homogeneously throughout the cytoplasm, excluding the nucleus and vesicles. The permeabilization-resistant form is associated with the actin cytoskeleton. A considerable amount of both enzymes is located in the perinuclear region and cannot be attributed to a definite structure. Comparing the staining patterns of aldolase and GAPDH in four different cell types we found that the distribution of the enzymes corresponds with diverse forms of actin cytoskeletal organization of these cells. The codistribution is maintained in cells treated with cytochalasin D.

Basal cells of H-Dunning tumor are myoepithelial cells. A comparative immunohistochemical and ultrastructural study with male accessory sex glands and mammary gland.

Recent immunohistochemical studies have shown that basal cells in human prostatic epithelium are not myoepithelial cells. Since in the literature the Dunning tumor, originally described as a rat prostate carcinoma derived from the dorsolateral prostate of a Copenhagen rat, was reported to have myoepithelial cells, a comparative immunohistochemical and ultrastructural study was performed in the H-, HIF- and AT3-lines of the Dunning tumor, the male accessory sex glands (ventral, dorsal, lateral prostate, coagulating gland, bulbourethral gland) and the mammary gland of both Copenhagen and Wistar rats. Mono- and polyclonal antibodies directed against intermediate filament proteins (cytokeratin, desmin, vimentin) and the contractile proteins (alpha-actin, muscle type specific myosin, tropomyosin) were used along with phalloidin decoration of F-actin. As in the human prostate, none of the rat prostate lobes in either strains did contain basal cells expressing cytokeratin along with alpha-actin, myosin and tropomyosin Cells representing fully differentiated myoepithelial cells, however, were present as anticipated in the mammary gland, the bulbourethral gland and the H-tumor line of the Dunning tumor. This finding is difficult to reconcile with the contention of a prostatic origin of the H-Dunning tumor. Further studies are required to classify the epithelial parental tissue in order to define the true origin of the H-Dunning tumor and the tumor lines derived thereof.

Immunocytochemical demonstration of contractile cells in the human ovarian follicle.

Actin- and myosin-like immunoreactivity is found in cells located in the theca externa of the follicle wall of the human ovary, and corresponding to previously observed myoid cells. The immunocytochemical observation provides direct structural evidence that non-vascular contractile cells are also present in the follicle wall in humans. As expected, perifollicular blood vessels showed a positive immunoreaction for actin and myosin in their smooth muscle walls.

Immune response of rabbits to native G-actins.

Actins are highly conserved proteins and are therefore claimed to be not very immunogenic without prior denaturation or chemical modification. We have obtained in rabbits high-titered antibodies to "native" G-actins from chicken and man, and assayed their cross-reaction using an enzyme immunoassay, Western blotting and immunohistochemistry. The antigens differ in their ability to induce antibody formation (chicken gizzard actin [(beta), gamma] greater than chicken skeletal actin [alpha] = human platelet actin [beta, (gamma)]). Antibodies to skeletal actin [alpha] are muscle-specific and mainly directed against the homologous region comprising the N-terminus (residues 1-226). Antibodies to gizzard actin [(beta), gamma] cross-react, to a lesser extent, with the alpha and beta, (gamma) isoforms. They show no regional specificity within the homologous antigen. Antibodies to the tryptic core fragment (residues 69-374) of skeletal actin react with fragments comprising the C-terminal part of muscular actins. Antibodies to platelet actin [beta, (gamma)] cross-react with muscular actins, recognizing not the native, but slightly degraded molecules. Platelet actin induces the formation of high-titered albumin antibodies for hitherto unknown reasons.

Neuronal markers in the rodent pineal gland--an immunohistochemical investigation.

Although some embryological and morphological features speak in favour of a neuronal character of rodent pinealocytes, histochemistry and ultrastructure let this issue appear controversial. Using antibodies to different neurofilaments, the neural adhesion molecule L1, synaptophysin and tubulin as neuronal markers, the pineal glands of rat and guinea-pig were studied by means of immunofluorescence. Neurofilament-immunoreactivity was present in some rat pineal nerve fibers and in the majority of guinea-pig pinealocytes, L1 decorated rat intrapineal nerve fibers, synaptophysin was almost ubiquitously distributed in the pineal of both species, while tubulin-immunofluorescence was seen in nerve fibers of rat and guinea-pig pineal and in some pinealocytes of the latter. These findings speak in favour of the neuronal character of guinea-pig pinealocytes. The lack of neurofilament- and tubulin-immunoreactivity in rat pinealocytes might be attributable to very low concentrations of these proteins or species differences as to their expression. Further studies including in situ-hybridisation of relevant mRNAs will be necessary to answer these questions definitely.

Structural and immunological properties of myosin from human platelet external and internal membranes.

Our previous studies indicate that platelets contain two myosin isoforms, one of them localized in the membrane while the other in the cytoplasmic compartment. Structural and functional differences of these myosins have been characterized. In this study two platelet membrane subfractions, the external and the internal membranes, were isolated simultaneously from a crude membrane fraction and their purity was characterized using specific marker enzymes. Myosin was shown to be present in both membrane fractions and its structural and immunological properties were investigated. The electrophoretic mobilities of myosin in both membrane preparations were identical to the mobility of its cytoplasmic counterpart. Two-dimensional peptide mapping of the iodinated tryptic peptides of the myosin heavy chains indicated that at least one peptide is missing in the maps of the myosins from the external and internal membranes as compared to their soluble counterpart. Our data suggest that myosin is located in three distinct platelet compartments: cytosol, external and internal membranes. The same myosin isoform is located in the two membrane compartments, while the isoform found in the cytosol is different. The observed variations in the structure of the two isoforms may reflect differences in their respective physiological functions.

Pig brain homogenates contain smooth muscle myosin and cytoplasmic myosin isoforms.

Three myosin isoforms, two of smooth muscle and one of cytoplasmic origin, were found in porcine brain by Western blotting analysis with antibodies specific for smooth and cytoplasmic myosins. The smooth muscle isoforms comprise at least 30% of the total myosin present. Brain tissue is therefore not a suitable source for the isolation of pure cytoplasmic contractile proteins.

Histones bind to contractile proteins and inhibit actomyosin ATPase.

Nuclear histones bind to and precipitate the major contractile proteins, actin and myosin. The binding of histone to actin seems to reach saturation at 2:1 ratio, the interaction may serve some regulatory function(s) in intranuclear events. The binding of histone to myosin is not saturable, and, although it inhibits the actin-activated Mg2+-dependent myosin ATPase activity, does therefore not seem of physiological importance.

Isolation of two isoforms of a 21,000-dalton Ca2+-binding protein of bovine brain.

Using Ca2+-dependent hydrophobic interaction chromatography we have identified a novel bovine brain Ca2+-binding protein (CaBP) composed of 21 kDa and 23 kDa polypeptides. This calciprotein was further purified by heat-treatment in the presence of Ca2+ and ion-exchange chromatography. The isolated protein exhibits a number of properties in common with proteins belonging to the calmodulin family of CaBPs, including a Ca2+-dependent electrophoretic mobility shift on SDS-polyacrylamide gel electrophoresis, retention of the ability to bind 45Ca2+ after electrophoresis and Western blotting, and a high content of acidic amino acids. We have recently isolated and characterized a 21 kDa CaBP from bovine brain and conclude that the 21 kDa and 21/23 kDa CaBPs are isoforms since they have very similar U.V. absorption spectra and amino acid compositions, and polyclonal antibodies raised in rabbits against the 21 kDa CaBP cross-react to an identical degree with the 21/23 kDa CaBP as determined by the competitive enzyme-linked immunosorbent assay (ELISA). Both proteins contain carbohydrate, but they differ in the degree of glycosylation. Tissue distribution studies indicate the presence of both 21 kDa and 23 kDa Ca2+-binding polypeptides in bovine trachea, aorta, kidney, skeletal muscle and cardiac muscle, and chicken gizzard smooth muscle.

Myosin content and vasoconstrictive ability of the proximal and distal (renin-positive) segments of the preglomerular arteriole.

The PAP-technique and antibodies to myosin were used to demonstrate the prerequisites for vasoconstriction in the juxtaglomerular part of the preglomerular arteriole as compared with its proximal segment in rats and mice. In contrast with the myosin-positive/renin-negative proximal part of the afferent arteriole no myosin-like activity could be demonstrated in its distal, renin-positive part. In accordance, no thick myofilaments were found in fully differentiated juxtaglomerular epithelioid cells replete with mature secretory granules. Stimulation of the renin-angiotensin system was followed by an increase of the renin-positive/myosin-negative portions of the preglomerular arteriole. Marked interspecies and internephron variations in the length of this vessel segment under control and stimulated conditions were observed. The juxtaglomerular part of the preglomerular arteriole close to the macula densa seems therefore to have only limited capabilities for vasoconstriction. This finding may be of importance regarding the tubulo-glomerular feedback, a mechanism allegedly triggered by the so-called 'macula densa-signal'. It is suggested that this non-contractile segment of the afferent arteriole may represent the renal vascular receptor responsible for the increase of renin secretion during pressure reduction. Unlike the afferent arterioles, most of the efferent arterioles showed the highest level of their weak but distinct myosin-like immunoreactivity in the juxtaglomerular region, indicating some efferent juxtaglomerular vasoconstrictive ability.