Changes in glycoconjugates in rat submandibular gland after chronic treatment with reserpine and isoproterenol

Summary Chronic treatment of rats with reserpine, isoproterenol, or a combination of these two agents has been suggested as a means to produce an experimental animal model for the chronic exocrinopathy cystic fibrosis. The effect of these treatments on glycoconjugate distribution in rat submandibular gland acinar cells was investigated by quantitative lectin cytochemistry. Significant changes in wheat-germ agglutinin (WGA), soy bean agglutinin (SBA) and concanavalin A (Con A) binding sites in the mucus granules were observed, but peanut agglutinin (PNA) binding was not significantly affected. The quantitative changes in glycoconjugates in the acinar cells of the submandibular gland could be a possible explanation for the increased binding of calcium by the intracellular mucus noted in previous studies on these animal models.     

Differential effects of chronic reserpine exposure on Ca2+ sequestering mechanisms in rat submandibular gland vesicles

To determine the effects of exposure to reserpine on subcellular Ca2+ transporting systems, active Ca2+ uptake was measured with and without ruthenium red in submandibular gland vesicles obtained from rats after chronic treatment with reserpine. The properties of ruthenium red-sensitive Ca2+ uptake were similar to those measured in submandibular gland vesicles from untreated rats: it was abolished by the dye, was relatively low at 1 microM Ca2+ but increased markedly at millimolar Ca2+ levels and was positively and significantly correlated with the mitochondrial membrane marker, cytochrome-C oxidase activity, in membrane subfractions obtained by differential centrifugation (r = 0.67, p = 0.0005, n = 29). On the other hand, ruthenium red-insensitive Ca2+ uptake, though stimulated at submicromolar Ca2+ concentrations, was reduced by a mean of 54% compared to preparations from untreated animals and particulate RNA content was 18% of that found in control preparations. Moreover, the distributions of ruthenium red-insensitive Ca2+ uptake and particulate RNA (which are closely correlated in vesicles from untreated rats) were not significantly related when measured in vesicles of submandibular glands from reserpine treated rats. Other membrane markers and overall membrane protein content were not significantly altered after chronic reserpine exposure. We conclude that reserpine treatment has little effect on mitochondrial Ca2+ uptake capacity but abolishes or drastically reduces the high affinity Ca2+-sequestering activity which, in submandibular gland vesicles from untreated rats, is apparently associated with the endoplasmic reticulum.     

Effect of acute administration of isoproterenol on submandibular salivary gland of female rat

The functions of salivary glands are under the regulation of both sympathetic as well as parasympathetic nerve fibers. Further, it has also been demonstrated that chronic administration of a beta-adrenergic agonist isoproterenol (IPR) results in hypertrophy and hyperplasia of submandibular gland [Schneyer C A, Am J Physiol, 203 (1962) 232]. Specific purpose of the present attempt was to look for metabolic responses of submandibular gland of oestrous female rats at very short intervals after 10 min of administration of 5, 10 and 15 micrograms of IPR to females in oestrous condition; pharmacological action and clearance time being only 8 min. The results indicated significant reduction in case of enzymic activities of phosphorylase, total ATPase and Na(+)-K+ ATPase. Cyclic AMP-specific phosphodiesterase and succinate dehydrogenase activities were suppressed only with 5 micrograms dose, but with rising dose levels the effect was not so apparent. Protein content of the gland was reduced slightly by administration of IPR. Hence, it became clear that submandibular gland responds rapidly to IPR administration. Implications of these observations are discussed.     

Dose-dependent morphological changes of intercellular canaliculi during stimulation with carbachol and isoproterenol in the isolated rat submandibular gland

Intercellular canaliculi (IC) form a primary mixing reservoir for transcellularly and paracellularly secreted saliva whose composition depends on the degree of elevation of cytosolic Ca2+ and of cytosolic cyclic AMP concentrations caused by the secretagogues employed. In perfused rat submandibular gland (SMG), appearance of exocytosis on IC reflected the quantity of secreted mucin. Morphological observations were carried out by HR-SEM using a modified osmium maceration method on specimens treated with CCh and/or ISP. Mild secretory stimulation revealed that exocytosis did not occur simultaneously, even along the same intercellular canaliculus. Higher doses did not alter the spatial distribution of exocytosis along intercellular canaliculi but increased its temporal frequency, dose dependently. These findings lead us to conclude that, under low levels of secretory stimulation, exocytosis does not show a dose-dependent change, but that its spatial and temporal frequency changes in a dose-dependent manner.     

Quantitative immunocytochemistry of rat submandibular secretory proteins during chronic isoproterenol administration and recovery

We utilized quantitative electron microscopic immunogold labeling procedures to follow changes in the intragranular content of five secretory proteins of the rat submandibular gland (SMG) during and after chronic treatment with the beta-adrenergic agonist isoproterenol (IPR). Labeling intensities (gold particles/microns2) of acinar cell secretory granules for mucin and glutamine/glutamic acid-rich proteins, major secretory proteins of the normal SMG, showed opposite responses to IPR. Labeling intensities increased for mucin and decreased for glutamine/glutamic acid-rich proteins immediately after IPR injections began, then rapidly returned to control levels after cessation of IPR treatment. SMG Protein C immunoreactivity, found in both acinar and intercalated duct granules, was less affected by IPR. However, opposite changes in labeling intensity were observed between acinar and intercalated duct granules. Labeling intensities for proline-rich proteins, IPR-inducible secretory proteins, increased only after 10 days of stimulation and maintained a high level even after cessation of drug treatment. Type 2 cystatin, another IPR-inducible protein, increased gradually with chronic IPR treatment and decreased slowly during the recovery phase. These results suggest that chronic beta-adrenergic stimulation affects the expression of genes for several rat SMG secretory proteins in a different manner.     

Short-term inhibition of cellular autophagy by isoproterenol in the submandibular gland

The present study examines the short-term (i.e. 10 min after injection) influence of isoproterenol on cellular autophagy in the rat submandibular gland. The volume fraction of autophagic vacuoles was significantly reduced, suggesting that an anticatabolic reaction, namely inhibition of cellular autophagy, is an early and significant event in the growth of the submandibular gland which is known to occur with long-term isoproterenol treatment.     

Effects of carbamylcholine and isoproterenol on electrolyte fluxes in perfused main duct of submandibular gland of reserpinized rat

Administration of reserpine (RES) at a dosage of 0.5 mg/kg body wt, ip daily for 7 days was found to lower the dose of carbamylcholine and isoproterenol that alters sodium and potassium transport by cells of the main duct of rat submandibular gland. In the perfused main excretory duct of the submandibular gland of the RES rat, administration of carbamylcholine at a dosage of 1 microgram/kg body wt, inhibited net efflux of sodium (17%) and administration of isoproterenol at a dosage of 2 micrograms/kg body wt increased net efflux of sodium (20%); these drugs, at the same dosages, did not induce significant change in electrolyte flux of normal rat. At a dosage of 5 micrograms/kg body wt, carbamylcholine decreased net influx of potassium (15%) in the RES rat but was without effect on normal rat. Isoproterenol at the dosage of 5 micrograms/kg body wt significantly inhibited net influx of potassium in both the RES rat and normal rat. The data suggested that the duct cells developed supersensitivity to sympathomimetic and parasympathomimetic stimulation after chronic RES treatment.     

Sulpholipid formation in rat submandibular gland.

Comparisons of nuclease digestions of chromatin in nuclei from rye embryos and rat liver show that their nucleosomal DNA is similar, i.e. DNA subunits consist of 200 base-pair repeats with 140 base-pair cores of identical substructure. The identical nucleosome structure is present in nuclei from cells of rye embryos that have been non-viable (i.e. dead) for more than 7 years. These findings indicate a high degree of stability of the DNA-histone complex and are consistent with conservation of the nucleosomal structure of chromatin during evolution.     

Parasympathetic reflex vasodilatation in rat submandibular gland

The present study was designed to investigate 1) whether parasympathetic reflex vasodilatation occurs in the submandibular gland (SMG) in deeply urethan-anesthetized, cervically vagotomized, and sympathectomized rats when the central cut end of the lingual nerve (LN) is electrically stimulated and 2) to what extent the neural mechanisms underlying such responses are the same as those involved in the response to direct stimulation of the chorda-LN (CLN). Stimulation of each nerve separately elicited a marked blood flow increase in SMG. Section of the chorda tympani abolished the SMG blood flow response but had no effect on the lip blood flow increase evoked by LN stimulation. Section of the CLN abolished the SMG blood flow increases evoked by stimulation of either nerve. The SMG blood flow increases (regardless of whether they were evoked by LN or CLN stimulation) were markedly reduced by the autonomic cholinergic ganglion blocker hexamethonium. The present study demonstrates that a parasympathetic reflex vasodilator mechanism is present in the rat SMG and that it can express its effects under deep general anesthesia.     

Experimental calcification in rat submandibular gland.

The present study describes the phenomenon of calciphylaxis, rapid calcification due to treatment with sensitizer dihydrotachysterol (DHT) and challenging agent 5-hydroxytryptamine (5-HT) in the rat submandibular gland (SMG) in terms of light and electron microscopy, and histochemistry. For biophysical analysis of the calcified bodies, X-ray microanalysis (XMA) and X-ray powdered diffraction methods were used. The calcified lesions in the salivary glands were histologically divided into 3 types: type 1, calcification of basal membranes in duct-like structures; type 2, granular calcified materials with remarkable necrotic changes in cell, containing 3 kinds of small vesicular structures observed in electron microscopy; and type 3, von Kossa's positive structures containing needle-like crystalline and electron-dense amorphous materials. Con A and UEA-1 lectin staining reactions were strong in the type 1 and 2 lesions. These findings suggest that the calcification matrix may contain mannose, fucose and glucose. The X-ray microanalysis of calcified materials revealed the magnesium whitelockite pattern, the type 3 displayed high quantities of Ca, P, and Mg ions comparing with the type 1 and 2, and the X-ray diffraction showed the hydroxyapatite pattern. We suggest that the above changes may be categorized as dystrophic calcification due to necrotic alterations brought about by the hypercalcaemic condition.     

Alteration of tight junctional permeability in the rat parotid gland after isoproterenol stimulation

The permeability of junctional complexes to ultrastructural tracers of different molecular weight and the freeze-fracture appearance of junctional structure were investigated in the resting and stimulated rat parotid gland. Tracers were administered retrogradely via the main excretory duct, and allowed to flow by gravity (16 mmHg) into the gland for 15-60 min. Secretion was induced in some animals by intraperitoneal injection of isoproterenol. In resting glands, the tracers microperoxidase , cytochrome c, myoglobin, tyrosinase (subunits), and hemoglobin were restricted to the luminal space of the acini and ducts. In glands stimulated 1-4 h before tracer administration, reaction product for microperoxidase , cytochrome c, myoglobin, and tyrosinase was found in the intercellular and interstitial spaces, whereas hemoglobin was usually retained in the lumina. In contrast, horseradish peroxidase and lactoperoxidase appeared to penetrate the tight junctions and reaction product was localized in the extracellular spaces in both resting and stimulated glands. Diffuse cytoplasmic staining for horseradish peroxidase and lactoperoxidase was frequently observed in acinar and duct cells. The distribution of horseradish peroxidase was similar in both Sprague-Dawley and Wistar-Furth rats, and at concentrations of 0.1-10 mg/ml in the tracer solution. Freeze- fracture replicas of stimulated acinar cells revealed an increased irregularity of the tight junction meshwork, but no obvious gaps or discontinuities were observed. These findings indicate that (a) tight junctions in the resting rat parotid gland are impermeable to tracers of molecular weight greater than or equal to 1,900; (b) stimulation with isoproterenol results in a transient increase in junctional permeability allowing passage of tracers of molecular weight less than or equal to 34,500; (c) junctional permeability cannot be directly correlated with junctional structure; and (d) the behavior of horseradish peroxidase and lactoperoxidase in the rat parotid gland is inconsistent with their molecular weights. Cell membrane damage due to the enzymatic activity or binding of these two tracers may account for the observed distribution.     

Biochemical and physical analysis of subcellular membranes in rat parotid gland enlarged by chronic isoproterenol administration

1. Chronic administration of isoproterenol caused similar alterations of membrane lipid profile in at least two rat parotid subcellular fractions, secretory granular and microsomal. 2. Typical changes in phospholipid classes and fatty-acyl chain groups were an increase of phosphatidylcholine and a decrease of sphingomyelin, and an increase of octadecadienoyl chain and a decrease of eicosatetraenoyl chain, respectively. 3. Electron spin resonance study showed that the isoproterenol-treatment also affected a membrane physical property, which may be through these compositional changes in membrane constituents.     

Label-retaining cells in the rat submandibular gland.

To identify stem cells in salivary glands, label-retaining cells (LRCs) were established in rat submandibular glands. Developing and regenerating glands were labeled with bromodeoxyuridine (BrdU). To cause gland regeneration, the glands were injured by duct obstruction. BrdU LRCs were observed in all the parenchymal structures except for the acinus of the glands labeled during regeneration. Among these LRCs, a few, but not many, expressed neither keratin18 (K18; an acinar/duct cell marker) nor alpha-smooth muscle actin (alphaSMA; a myoepithelial cell marker), and thus were putative stem cells. These (K18 and alphaSMA)(neg) LRCs were invariably observed in the intercalated duct and the excretory duct. In the intercalated duct, they were at the proximal end bordering the acinus (the neck of the intercalated duct). Next, to test the above identification, gland extirpation experiments were performed. LRCs were established by labeling developing glands with iododeoxyuridine (IdU) in place of BrdU. Removal of one submandibular gland forced the IdU-LRCs in the remaining gland to divide. They were labeled with chlorodeoxyuridine (CldU). The (K18 and alphaSMA)(neg) LRCs in the neck of the intercalated duct and in the excretory duct did not change in number or in IdU label. The CldU label appeared in these cells and then disappeared. These results indicate that the (K18 and alphaSMA)(neg) LRCs have divided asymmetrically and are thus considered salivary gland stem cells.     

Microvascular transplantation of the rat submandibular gland

Xerostomia results from salivary gland irradiation during treatment of head and neck malignancies. In addition to having difficulty with speech and swallowing, these patients experience loss of taste, dental caries, and chronic fungal infections. The paired submandibular glands provide 70 percent of the normal salivary flow and are difficult to shield during radiation therapy. Another sicca condition, xerophthalmia, may result from facial nerve injury or other medical disorders and results in pain, corneal ulceration, and possible vision loss. Treatment options for xerostomia are limited, and management of xerophthalmia usually focuses on the eyelids, rather than the fundamental problem of inadequate secretory protection. In this study, a rat model for submandibular gland microvascular transplantation was developed to assess the feasibility of salivary tissue transfer. Sixteen rats underwent submandibular gland transplantation from the neck to the groin. Fourteen of these rats underwent microvascular anastomosis of the vascular pedicle. Ten glands were assessed for viability at 4 days after transplantation, and four glands were examined after 7, 10, 14, or 21 days. By gross and histologic examination, 93 percent of transplanted glands showed expected long-term viability after at least 4 postoperative days. Microvascular techniques were shown to be applicable to the transplantation of submandibular gland salivary tissue. This has not previously been shown in a rat model. It is possible that submandibular glands could be transplanted to the eye for treatment of xerophthalmia and out of the neck during irradiation of the head and neck, with subsequent replantation after treatment as a means of preventing permanent xerostomia.     

Cholecystokinin modulates isoproterenol induced changes in rat parotid gland

1. Parotid gland secretory function and activity of several enzymes involved in intracellular second messenger signalling were measured in rats receiving 0.5 ml i.p. injections of saline (control), isoproterenol, CCK or both drugs.2. Isoproterenol caused a 2.5-fold increase in parotid gland wet weight compared to control. Chronic administration of CCK alone has no effect on gland weight. A combination of CCK and isoproterenol did not alter the hypertrophy of the gland observed with isoproterenol alone.3. Isoproterenol administration caused a 74% decrease in parotid gland amylase enzyme activity. While CCK alone did not influence the enzyme activity, it depressed amylase mRNA steady state levels and had an additive effect on further decreasing mRNA levels when administered in combination with β-agonist.4. Phospholipase C registered an increase ranging from 22 to 38% in all experimental groups as compared to control.5. Parotid gland protein kinase C and PdtIns 3-kinase activity were not altered in response to CCK alone, but in combination with isoproterenol, appeared to moderate β-agonist signal transduction responses.     

Ribonucleic acid association with androgen receptor from rat submandibular gland

The transformed androgen receptor from rat submandibular gland converts to a faster sedimenting form (6-8S) on a glycerol gradient centrifugation after withdrawal of a transformation-inducing reagent (KCl or ATP). In this report, the association of cytosolic RNA with the transformed androgen receptor was investigated as a possible mechanism of molecular conversion of the androgen receptor. When the transformed and converted androgen receptors were treated with RNase A, these receptors sedimented at 4.5S in a low-salt glycerol gradient. Addition of RNA from rat submandibular gland to the RNase-Sepharose-treated transformed receptor caused a shift of receptor peak from 4.5S to 5.8S. RNA from rat submandibular gland, yeast RNA and E. coli rRNA inhibited DNA-cellulose binding of a RNase-treated transformed receptor in the absence of molybdate. These observations suggest that conversion from the transformed 4S androgen receptor to a 6-8S form resulted from the association of RNA(s) with the transformed receptor.     

Demonstration of reduced levels of zinc in rat brain after treatment with d-amphetamine, but not after treatment with reserpine

Histochemical and atomic absorption spectrophotometric methods were used to study the effects of reserpine and d-amphetamine on the neuronal trace metal distribution in various regions of the central nervous system (hippocampus, parietal cortex, cerebellum). Six hours after single d-amphetamine treatment (15 mg/kg i.p.), the neuronal zinc level was significantly decreased in the hippocampus and in the parietal cortex. The intensity of sulphide silver staining was similarly greatly decreased in all layers of the hippocampus and the parietal cortex. Such a change was not observed when d-amphetamine was administered in a lower dose (5 or 10 mg/kg i.p.). Twenty hours after single reserpine treatment (10 mg/kg i.p.), there were no changes in the tissue levels and distribution of zinc, copper, iron and manganese. In animals treated with reserpine on five consecutive days, in a dose of 10 mg/kg/day i.p., the trace metal distribution twenty hours following the final treatment was essentially the same as in the control. The results strongly suggest that zinc does not play a direct role in vivo in the storage and mobilization processes of the catecholamines. A high dose of d-amphetamine, however, has a non-specific, toxic effect that is not interrelated with the catecholaminergic neuronal function; this effect is manifested in a diminished intensity of sulphide silver staining and in a reduction of the tissue zinc level.