Electron Microscopic Histochemistry Research Articles

Biochemical characterization of mitochondria from adult worms and plerocercoid larvae of Spirometra mansoni shows mixed populations of anaerobic and aerobic mitochondria

The mitochondria of adult and plerocercoid Spirometra mansoni were characterized in isolated mitochondria and in situ by electron microscopic histochemistry with special attention to the respiratory chain. Although the specific activities of the constituent enzyme complexes of succinate oxidase are fairly similar in adult and plerocercoid mitochondria, those of succinate oxidase and NADH-FRD are approximately 4- and 25-fold higher in adult mitochondria than in plerocercoid mitochondria, respectively. Quinone analysis by high performance liquid chromatography and mass spectrometry showed that adult and plerocercoid mitochondria contained both rhodoquinone-10 and ubiquinone-10 at concentrations of 4.98 and 0.106 nmol mg−1 for adult, and 0.677 and 0.137 nmol mg−1 for plerocercoid, respectively. Inhibition studies on the succinate-oxidase system of adult mitochondria showed that they possessed both cyanide-sensitive and -insensitive succinate oxidases, the latter of which produces hydrogen peroxide. Adult mitochondria, when NADH was used as a substrate, were shown to produce hydrogen peroxide, and the production of hydrogen peroxide decreased to undetectable levels in the presence of fumarate. The specific activities of NADH-fumarate reductase and cytochrome c oxidase were significantly higher in mature proglottids than in immature and gravid proglottids. Isopycnic density-gradient centrifugation analyses and in situ electron microscopic histochemistry revealed that both adult and plerocercoid mitochondria were heterogeneous in terms of respiratory function and physicochemical properties. The physiological significance of adult and plerocercoid mitochondria is discussed in relation to the oxygen tension of their parasitic habitats.

Perineural capsaicin induces the uptake and transganglionic transport of choleratoxin b subunit by nociceptive c-fiber primary afferent neurons

The distribution of spinal primary afferent terminals labeled transganglionically with the choleratoxin B subunit (CTB) or its conjugates changes profoundly after perineural treatment with capsaicin. Injection of CTB conjugated with horseradish peroxidase (HRP) into an intact nerve labels somatotopically related areas in the ipsilateral dorsal horn with the exceptions of the marginal zone and the substantia gelatinosa, whereas injection of this tracer into a capsaicin-pretreated nerve also results in massive labeling of these most superficial layers of the dorsal horn. The present study was initiated to clarify the role of C-fiber primary afferent neurons in this phenomenon. In L5 dorsal root ganglia, analysis of the size frequency distribution of neurons labeled after injection of CTB-HRP into the ipsilateral sciatic nerve treated previously with capsaicin or resiniferatoxin revealed a significant increase in the proportion of small neurons. In the spinal dorsal horn, capsaicin or resiniferatoxin pretreatment resulted in intense CTB-HRP labeling of the marginal zone and the substantia gelatinosa. Electron microscopic histochemistry disclosed a dramatic, ∼10-fold increase in the proportion of CTB-HRP-labeled unmyelinated dorsal root axons following perineural capsaicin or resiniferatoxin. The present results indicate that CTB-HRP labeling of C-fiber dorsal root ganglion neurons and their central terminals after perineural treatment with vanilloid compounds may be explained by their phenotypic switch rather than a sprouting response of thick myelinated spinal afferents which, in an intact nerve, can be labeled selectively with CTB-HRP. The findings also suggest a role for GM1 ganglioside in the modulation of nociceptor function and pain.

Distribution of gastrin‐releasing peptide in the rat trigeminal and spinal somatosensory systems

Gastrin-releasing peptide (GRP) has recently been identified as an itch-specific neuropeptide in the spinal sensory system in mice, but there are no reports of the expression and distribution of GRP in the trigeminal sensory system in mammals. We characterized and compared GRP-immunoreactive (ir) neurons in the trigeminal ganglion (TG) with those in the rat spinal dorsal root ganglion (DRG). GRP immunoreactivity was expressed in 12% of TG and 6% of DRG neurons and was restricted to the small- and medium-sized type cells. In both the TG and DRG, many GRP-ir neurons also expressed substance P and calcitonin gene-related peptide, but not isolectin B4 . The different proportions of GRP and transient receptor potential vanilloid 1 double-positive neurons in the TG and DRG imply that itch sensations via the TG and DRG pathways are transmitted through distinct mechanisms. The distribution of the axon terminals of GRP-ir primary afferents and their synaptic connectivity with the rat trigeminal sensory nuclei and spinal dorsal horn were investigated by using light and electron microscopic histochemistry. Although GRP-ir fibers were rarely observed in the trigeminal sensory nucleus principalis, oralis, and interpolaris, they were predominant in the superficial layers of the trigeminal sensory nucleus caudalis (Vc), similar to the spinal dorsal horn. Ultrastructural analysis revealed that GRP-ir terminals contained clear microvesicles and large dense-cored vesicles, and formed asymmetric synaptic contacts with a few dendrites in the Vc and spinal dorsal horn. These results suggest that GRP-dependent orofacial and spinal pruriceptive inputs are processed mainly in the superficial laminae of the Vc and spinal dorsal horn.

Light and Electron Microscopic Histochemistry of the Peroxidase Activity in the Secretory Granules of the Developing Hamster Submandibular Gland

On the basis of light and electron microscopic observations of the post natal development of the hamster submandibular gland, granules in the acinar cells showed considerably variations in size and shape, as well as electron density of the peroxidase-positive reaction. The present study shows that secretory granules of the hamster submandibular gland undergo changes of area and of intensity for peroxidase activity 6 months after birth.

Changes of scleral sulfated proteoglycans in three cases of nanophthalmos

To examine the composition of scleral sulfated proteoglycans in three nanophthalmic eyes by electron microscopic histochemistry. Scleral tissues from three cases of nanophthalmos were collected from the Niigata University Hospital for transmission electron microscopic examination. Sulfated proteoglycans were studied using the cuprolinic blue (CB) dye binding method in conjunction with enzymatic digestion. Compared with the controls, the nanophthalmic sclerae were much thicker, and the collagen fibrils showed a less orderly arrangement. Twisting or fraying of collagen fibrils was noted in some areas, and fine fibrous materials surrounded the abnormal fibrils. CB-positive proteoglycan filaments were found around the collagen fibrils and in the fine fibrous materials in the nanophthalmic specimens. The number of proteoglycan filaments was smaller than that in the normal specimens. After enzymatic digestion, the CB-positive filaments were identified as dermatan/chondroitin sulfate proteoglycans. Although the number of dermatan sulfate filaments in nanophthalmos sclerae was similar to that in the controls, there were many fewer chondroitin sulfate filaments. A loss of chondroitin sulfate proteoglycan was observed in nanophthalmic sclerae. The alteration may be related to the abnormal organization of collagen fibrils and the onset of uveal effusion.

Cytochrome c oxidase activity in mitochondria of cardiomyocytes from isolated cardiac tissue incubated under long-term hypoxic conditions

Electron microscopic histochemistry based upon the oxidative polymerization of 3,3′-diaminobenzidine (DAB) was applied to identify cytochrome c oxidase activity. We found that the incubation of isolated small pieces of cardiac tissue over 72 h under hypoxic conditions caused changes in the mitochondrial ultrastructure and disorders in the functional activity of mitochondria, particularly in the IV complex of respirator chain. Small, electron-dense mitochondria appeared inside electron-light mitochondria (“mitochondria inside mitochondria”) stained positively for cytochrome c oxidase activity along the full length of crista. The results are discussed in connection with the concept of intracellular regeneration and mitochondria structural transformations during mitoptosis.

Cellular and subcellular localizations of nonheme ferric and ferrous iron in the rat brain: a light and electron microscopic study by the perfusion-Perls and -Turnbull methods

Iron in the brain is utilized for cellular respiration, neurotransmitter synthesis/degradation, and myelin formation. Iron, especially its ferrous form, also has the potential for catalyzing the Fenton reaction to generate highly cytotoxic hydroxyl radicals. The amount of iron in the brain must therefore be strictly controlled. In this study, we focused on the cellular and subcellular localizations of nonheme ferric (Fe(III)) and ferrous (Fe(II)) iron in the adult female rat brain using light and electron microscopic histochemistry. Although Fe(II) deposition was much less dominant than Fe(III), the brain contained iron in both forms. Among the cellular elements of the brain, oligodendrocytes were numerically the most prominent and heavily iron-storing cells. Pericapillary astrocytes and sporadic microglial cells also showed dense iron accumulation. Large neurons involved in the motor system were relatively strongly iron-positive. Subcellularly, Fe(III) and Fe(II) were mainly localized in lysosomes, and occasionally in the cytosol and mitochondria. Furthermore, capillary endothelial cells had Fe(III)-positive reactions in lysosomes and the cytosol, with Fe(II)-positive reactions on the luminal membrane. With advancing age, both Fe(III) and Fe(II) became more extensively distributed and accumulated more numerously in oligodendrocytes and astrocytes. These findings suggest that age-related increases in Fe(II) accumulation may raise the risk of tissue damage in the normal brain.

Time‐Specific Effects of Perinatal Glucocorticoid Treatment on Anterior Pituitary Morphology, Annexin 1 Expression and Adrenocorticotrophic Hormone Secretion in the Adult Female Rat

Perinatal glucocorticoid (GC) treatment is increasingly associated with long-term disturbances in hypothalamo-pituitary-adrenocortical function. In the male rat, such treatment induces profound molecular, morphological and functional changes in the anterior pituitary gland at adulthood. To determine whether these effects are sex-specific, we have examined the effects of perinatal dexamethasone treatment on the female pituitary gland, focusing on (i) the integrity of the annexin 1 (ANXA1) dependent regulatory effects of GCs on adrenocorticotrophic hormone (ACTH) release and (ii) corticotroph and folliculo-stellate (FS) cell morphology. Dexamethasone was given to pregnant (gestational days 16-19) or lactating (days 1-7 post partum) rats via the drinking water (1 microg/ml); controls received normal drinking water. Pituitary tissue from the female offspring was examined ex vivo at adulthood (60-90 days). Both treatment regimes reduced the intracellular and cell surface ANXA1 expression, as determined by western blot analysis and quantitative immunogold electron microscopic histochemistry. In addition, they compromised the ability of dexamethasone to suppress the evoked release of ACTH from the excised tissue in vitro, a process which requires the translocation of ANXA1 from the cytoplasm to the cell surface of FS cells. Although neither treatment regime affected the number of FS cells or corticotrophs, both altered the subcellular morphology of these cells. Thus, prenatal dexamethasone treatment increased while neonatal treatment decreased FS cell size and cytoplasmic area. By contrast, corticotroph size was unaffected by either treatment, as also was the size of the secretory granules. Corticotroph granule density and margination were, however, increased markedly by the prenatal treatment, while the neonatal treatment had no effect on granule density but decreased granule margination. Thus, perinatal dexamethasone treatment exerts long-term effects on the female pituitary gland, altering gene expression, cell morphology and the ANXA1-dependent GC regulation of ACTH secretion. The changes are similar but not identical to those reported in the male.

Axotomy induces contrasting changes in calcium and calcium-binding proteins in oculomotor and hypoglossal nuclei of Balb/c mice

Motor neurons with different susceptibility to degeneration have been identified in amyotrophic lateral sclerosis (ALS). Increase of intracellular calcium has been proposed as a mediator, amplifying the damage through a positive feedback of the known pathological processes. Accordingly, the potential of motor neurons to limit calcium increases during injury might be proportional to their viability. A basic mechanism of reducing calcium amplitudes depends on the calcium-buffering capacity, determined by the calcium-binding protein content. In this study, oculomotor and hypoglossal neurons, prototypes of resistant and vulnerable motor neurons in ALS were examined in axotomy experiments. Total calcium-, parvalbumin-, and calbindin-D28k levels of motor neurons of adult mice were characterized by electron microscopic histochemistry and light microscopic immunostaining. In hypoglossal neurons, compared with oculomotor neurons, larger and more enduring increases of calcium were detected. The perikarya of hypoglossal neurons remained immunonegative for both parvalbumin and calbindin-D28k. Qualitatively, no major cell loss was noted after axotomy, but a decreased neuronal marker staining at days 1-14 suggested a reversible injury of hypoglossal neurons. Oculomotor neurons were not stained for calbindin-D28k but stained for parvalbumin in control conditions, staining which increased at postoperative days 7-14 before returning to baseline. Neuronal marker staining did not change in these cells during the observed period. The higher level of parvalbumin in resistant motor neurons and their ability to up-regulate parvalbumin after injury, paralleled by a smaller increase of intracellular calcium suggest that parvalbumin may have a protective effect in these cells.

Sublethal dose of 4-hydroxynonenal reduces intracellular calcium in surviving motor neurons in vivo

4-Hydroxynonenal (4-HNE), a major lipid peroxidation product, induces oxidative stress, acts as an autonomous effector of cell death in motor neuron hybrid cell cultures, and is elevated in the cerebrospinal fluid (CSF) of patients with amyotrophic lateral sclerosis (ALS). Elevation of the total intracellular calcium has also been demonstrated in motor axon terminals of ALS patients as well as in spinal motor neurons of animal models of familial and sporadic ALS. Since the association of intracellular calcium and oxidative stress has been suggested in ALS, the in vivo effect of intrathecally administered 4-HNE on the motor neuronal calcium level was examined in the spinal cord of rats. After 12 days of treatment, total intracellular calcium was assayed by electron microscopic histochemistry using the oxalate-pyroantimonate method. Morphology of spinal motor neurons was characterized by light and electron microscopy. In rats, 4-HNE treatment induced a mild impairment of gait, elevation of 4-HNE in the CSF, loss of spinal motor neurons, and reduction of total calcium in the surviving, structurally intact motor neurons. 4-HNE could only cause a lesion if glutathione synthesis was concomitantly inhibited in the animals. The results suggest that upstream components of the oxidative injury in relation to lipid peroxidation are necessary to compromise the glutathione system in ALS, allowing an increase of 4-HNE in the CSF, which further aggravates the primary oxidative lesion. The reduced intracellular calcium in the surviving motor neurons with no morphological features of degeneration may reflect an impaired ionic homeostasis, which may indicate a residual damage of an incomplete degenerative process.

Atypical Composition and Ultrastructure of Proteoglycans in the Mouse Corneal Stroma

Recently, gene-targeted strains of mice with null mutations for specific proteoglycans (PGs) have been used for investigations of the functional role of these molecules. In the present study, the corneal stroma of the mouse was examined to provide some baseline PG morphologies in this species. Monoclonal antibodies to specific glycosaminoglycan (GAG) chain sulfation patterns were used to characterize PG composition in corneal extracts by SDS-PAGE and Western blot analysis and to identify their tissue distribution by immunofluorescence microscopy. PGs were also visualized by transmission electron microscopy after contrast enhancement with cationic dye fixation. Western blot analysis of pooled corneal extracts and immunofluorescence of tissue sections identified 4-sulfated, but not 6-sulfated, chondroitin sulfate/dermatan sulfate (CS/DS). Keratan sulfate (KS) was present only as a low-sulfated moiety. Electron microscopic histochemistry disclosed a complex array of corneal PGs present as (1) fine filaments radiating from collagen fibrils, and (2) elongate, straplike structures, running either along the fibril axis or weaving across the primary fibril orientation. These large structures were digested by chondroitinase ABC, but not by keratanase. KS in the mouse is predominantly undersulfated and generates an immunostaining pattern that differs from that observed in corneas of other mammalian species thus far investigated. The mouse cornea resembles other mammalian corneas in the presence of filamentous arrays of small, collagen-associated stromal PGs visualized by cationic dye staining. However, large dye-positive structures with a CS/DS component are also present and appear to be unique to the cornea of this species.

Expression, subcellular localization and phosphorylation status of annexins 1 and 5 in human pituitary adenomas and a growth hormone‐secreting carcinoma

Annexin 1 (ANXA1), a 37-kDa protein, plays an important role as a mediator of glucocorticoid action in the anterior pituitary gland and has been implicated in the processes of tumorigenesis in a number of other tissues. As a prelude to examining the potential role of ANXA1 in the pathophysiology of pituitary tumours, this study examined the expression, phosphorylation status and distribution of ANXA1 and the closely related protein, annexin 5 (ANXA5), in a series of pituitary adenomas and in two carcinomas. Forty-two human pituitary adenomas were examined. Parallel studies were performed on normal pituitary tissue, obtained postmortem, a GH-secreting carcinoma and a grade 4 astrocytoma. The tissue was processed for analysis of ANXA1 mRNA and protein expression by reverse transcriptase polymerase chain reaction (RT-PCR), Western blot analysis and immunogold electron-microscopic histochemistry. Parallel measures of ANXA5 mRNA and protein were also made. ANXA1 mRNA and protein were detected in all but three adenomas studied; the protein was localized mainly, but not exclusively, to nonendocrine cells. ANXA5 expression was more variable and was contained within both endocrine and nonendocrine cells of these tumours. In comparison with the adenomas, the GH-secreting carcinoma showed abundant expression of both ANXA1 and ANXA5, with intense ANXA1 staining in some but not all tumour/endocrine cells. A serine-phosphorylated species of ANXA1 was detected in all pituitary tumours studied; by contrast, tyrosine-phosphorylated ANXA1 was detected in only four adenomas and in the GH carcinoma. ANXA1 and ANXA5 were also expressed in abundance in the astrocytoma. The results demonstrate expression of both ANXA1 and ANXA5 in human pituitary tumours and thus raise the possibility that these proteins influence the growth and/or functional activity of the tumours.

Immunohistochemical characterization of a polyclonal antibody against Sphaerospora dicentrarchi (Myxosporea: Bivalvulida), a parasite from sea bass (Dicentrarchus labrax L.) (Teleostei: Serranidae).

Rabbit antibody was raised against Sphaerospora dicentrarchi, a histozoic parasite of sea bass (Dicentrarchus labrax L.). Light and electron immunohistological staining were used to characterize its specificity and possible reactivity toward other fish parasites. In light immunohistochemistry the polar capsules and valves of the S. dicentrarchi spores appeared strongly stained, whereas developmental stages were not. Electron microscopic histochemistry revealed intense labeling in valves and some developmental stages. Cross-reaction was observed with all the myxosporean parasites assayed, even with those belonging to other genera. Polar capsules of all the myxosporean species except Polysporoplasma sparis were the main structures stained by the polyclonal antibody. These observations could reveal the existence of conserved antigenic epitopes in polar capsules of different Myxosporea.

Uptake of neutrophils and neutrophilic components by macrophages in the inflamed peritoneal cavity of rainbow trout (Oncorhynchus mykiss)

The interaction between neutrophils and macrophages in peritoneal inflammatory exudates in rainbow trout injected intraperitoneally with several bacterial and non-bacterial phlogistic agents was studied by light and electronmicroscopic histochemistry. The transfer of neutrophilic components to macrophages was demonstrated by the presence in macrophages of myelo-peroxidase, an enzyme of the neutrophil not produced by rainbow trout monocytes and macrophages. Two mechanisms observed for the transfer were phagocytosis of intact neutrophils and phagocytosis of apoptotic bodies released from inflammatory neutrophils. Neutrophils with phagocytosed bacteria were also found to be ingested by macrophages, with the consequent transfer of neutrophilic components and bacteria to the macrophages. Two other mechanisms of transfer are considered. One involves the intake by the macrophage of neutrophilic granules or granular molecules released by lysing or degranulating neutrophils, respectively. The other may be through gap junctions which were observed between adhering inflammatory neutrophils and macrophages.

Post-fertilization changes in the zona pellucida glycoproteins of rat eggs.

The zona pellucida (ZP) is the extracellular coat surrounding the mammalian egg. Numerous evidence supports the role of ZP carbohydrate residues as the specific sperm receptors. In this study we used lectins to study different distribution patterns of carbohydrate residues in the rat ZP, and to follow changes at fertilization. ZP were collected from follicular, ovulated, and fertilized eggs, incubated with one of 11 different biotin-labeled lectins, followed by avidin-fluorescein isothiocyanate (FITC) complex, and visualized by epifluorescent microscopy. For electron microscope (EM) histochemistry, eggs were embedded in LR white and ultrathin sections were stained with the complex Ricinus communis lectin (RCA-1)-colloidal gold. Some lectins (RCA-I, Glycine max) bound to the entire ZP while others were restricted to the inner or outer zones [Griffonia simplicifolia, Concanovalia ensiformis, Triticum vulgaris (WGA), succinyl-WGA]. Other lectins (Lens culinaris, Ulex europhaeus) were totally excluded. The RCA-1 binding pattern changed following sperm penetration, from homogeneous in ZP of ovulated eggs (57%) to uneven in ZP of fertilized (71%) or activated (68%) eggs. Our results demonstrate an uneven distribution of different sugar residues in the rat ZP, and a post-fertilization change in the distribution of beta-galactose, which is specifically recognized by RCA-I, presumably correlated with other changes in the ZP that lead to the block to polyspermy.

Electron microscopic histochemistry for acetylcholinesterase in the dorsal cochlear nucleus of rat

Acetylcholinesterase (AChE), the enzyme which catalyzes hydrolysis of the neurotransmitter acetylcholine, is located in cholinergic neurons, cholinoceptive neurons, and extracellularly in the synaptic clefts of cholinergic synapses. In the rat cochlear nucleus, high activities of AChE have been found in locations rich in granule cells. In the rat dorsal cochlear nucleus (DCN), light microscopic histochemistry for AChE is characterized by stained dots and some patches, especially in its middle, fusiform soma layer, which contains a dense population of granule cells. The fine structure of AChE staining in the DCN has not previously been described. Electron microscopic histochemistry for AChE was performed on perfusion-fixed rat brains. Preliminary results indicate that AChE is distributed differentially across DCN layers. In the molecular layer, AChE staining was found in the extracellular space between nerve fibers and inside the cytoplasm of some dendrites. In deeper layers, extracellularly located AChE was less common, but staining was found inside some of the larger dendrites, some large neuron somata (probably fusiform cells), and some glomeruluslike structures. There appeared to be some labeled fibers coursing in the acoustic striae. [Supported by NIH Grant No. DC00172.]

Cholinoceptive neurons without acetylcholinesterase activity and enzyme-positive neurons without cholinergic synaptic innervation are present in the main olfactory bulb of adult rat.

Light and electron microscopic histochemistry revealed acetylcholinesterase-positive and acetylcholinesterase-negative neurons in the main olfactory bulb of adult rat. Their distribution patterns on various neuron types have been analysed in detail. (1) No acetylcholinesterase staining could be demonstrated in the granule cells which receive a large number of the cholinergic synapses. (2) In contrast, enzyme activity was present in the soma and dendrites in most of the non-cholinergic and non-cholinoceptive relay cells (mitral cells and tufted cells) and in a subset of short-axon interneurons, where cholinergic synapses could not be detected. (3) Within the neuropil of glomeruli, two compartments were present, one of which was free of acetylcholinesterase-positive structures, while many enzyme-positive neuronal elements were seen in the other. (4) Characteristically, cholinergic and non-cholinergic neuronal structures showed triadic arrangements. (5) The axonal release of acetylcholinesterase from cholinergic axons is probable. It is suggested that, in the olfactory bulb, acetylcholinesterase is release by cholinergic afferent axons, and it is the cholinergic synapses that determine which postsynaptic neurons are cholinoceptive rather than the intraneuronal presence of acetylcholinesterase. In the main olfactory bulb, the acetylcholinesterase present in the relay cells therefore appears to have functions other than the hydrolysis of acetylcholine.

Study of Histriculus cavicola Cyst Wall Using Different Lectins

Summary Histriculus cavicola resting cyst walls have been examined with a variety of lectins binding specifically to certain sugar residues. Light and electron microscopic lectin histochemistry shows that the endocyst is the cyst wall layer with a greater and more varied saccharide residue content since it binds to the majority of lectins tested. The mesocyst contains some saccharide residues although these have not been detected in previous cytochemistry studies. The ectocyst is not stained with any of the lectins tested. None of the cyst wall layers were stained with MAA and UEA-I lectins, however we cannot rule out the possibility of the presence of sialic acid residues and/or fucose linked oligosaccharides from other linkages. Lectin blottings of isolated cyst wall proteins show the presence of several glycoproteins with molecular weights of 210-200, 190, 110-105, 95-85, 76, 73-68, 55, 50, 45, 40-36 kDa. The 190 kDa glycoprotein is located in the endocyst since this is the only band stained with PNA and DBA, lectins that bind strongly and only to the endocyst layer. This glycoprotein is the only one in which we have detected complex N and 0 linked glycans. The other glycoproteins present N linked glycans, although some of these may have 0 linked glycans, so far not detected by us.

Horseradish Peroxidase Permeability across Rat Nasal Mucosa in Selective Stimulation of Substance P Innervation with Capsaicin

To investigate the physiological role of substance P innervation (SPI) in the nasal mucosa from inhaled irritant macromolecules, SPI in the rat nasal mucosa was selectively stimulated with capsaicin and the degree of horseradish peroxidase (HRP) permeability across the nasal epithelium was evaluated by enzyme immunoassay or by histochemical electron microscopy. The serum level of HRP was reduced significantly in capsaicin-administered rats compared to control rats along the time course with quantitative analysis of the enzyme immunoassay. Control rats showed heavy permeation of HRP across the epithelium, but capsaicin-administered rats showed weak permeation of HRP across the epithelium with histochemical electron microscopy. The epithelial lining and its tight junctions were left intact as judged by electron microscopy. In conclusion, selective stimulation of SPI of the rat nasal mucosa with capsaicin decreased the epithelial absorption permeability to HRP without compromising the epithelial integrity. These findings imply that the physiological role of capsaicin-sensitive SPI in the rat mucosa is to protect the airway from inhaled irritant macromolecules.

Peroxisomes and sterol carrier protein-2 in luteal cell steroidogenesis: a possible role in cholesterol transport from lipid droplets to mitochondria

In the present investigation, we have studied peroxisomes and sterol carrier protein-2 (SCP 2) in control and luteinizing hormone stimulated rat luteal cells. Superovulated immature rats in mid-luteal phase (8 days after ovulation) were divided into two groups (n=4/group) and treated with vehicle (0.2 ml saline), or luteinizing hormone (LH, 20 μg/rat). In this animal model, LH acutely stimulates steroidogenesis. Thirty minutes later, corpora lutea were fixed by whole body perfusion and processed for (1) electron microscopic immunocytochemistry to localize SCP 2 via the protein A gold immunolabeling technique, and for (2) electron microscopic histochemistry to stain peroxisomal catalase via the alkaline 3,3′-diaminobenzidine tetrahydrochloride method. In the steroidogenic, mid-phase luteal cells of vehicle treated rats (controls), SCP 2 was highly concentrated in peroxisomes and sparsely scattered on mitochondria, but no labeling was observed in lipid droplets. In the luteal cells of rats acutely stimulated with LH, peroxisomes immunolabeled for SCP 2 were observed within the luteal cell lipid droplets and mitochondria, and in union with lipid droplets and mitochondria. Moreover, in contrast to control luteal cells, significant immunolabeling for SCP 2 was detected within the lipid droplets and mitochondria in luteal cells of LH-treated rats. As SCP 2 binds cholesterol to 1:1 molar ratio and is known to be involved in the intracellular movement of cholesterol, these findings suggest that peroxisomes and SCP 2 may possibly be involved in delivering cholesterol from lipid droplets to the mitochondria when luteal cell steroidogenesis is acutely stimulated by LH.