Regulation of phosphofructokinase-1 on submandibular salivary glands of rats after isoproterenol administration

The purpose of this investigation was to study the effect of isoproterenol (IPR) treatment on the regulation of phosphofructokinase-1 of submandibular salivary glands of rats. The animals were divided into control and experimental groups. In the first set of experiments, the rats received 5 mg of IPR/kg b.w. and were sacrificed at 24 hours after 1, 2, 3 and 4 doses. The content of fructose-2,6-bisphosphate (Fru-2,6-P(2)) and the activity of 6-phosphofructo-2-kinase (PFK-2) (active and total) were determined. The Fru-2,6-P(2) content was found to be reduced and the activity of PFK-2 (active and total) showed differences from the control. The active/total ratio, was higher for the group of one dose sacrificed 12 hours after the agonist injection as compared to the control. In the other groups, there were reductions which varied from 25 to 33%. In the second set of the experiment, the animals were injected with 23.0 mg of IPR/kg b.w. and were sacrificed from 5 up to 720 minutes after the administration of the agonist. After the sacrifice, salivary gland samples were analyzed for Fru-2,6-P(2). Again, a reduction in the metabolite content was observed. Using beta and alpha receptor blockers, it was found that both inhibited only partially the effect of IPR. The purification of PFK-1 up to homogeneity, from submandibular glands of rats which received 5 mg of IPR/mg b.w. as well as from the control, was performed and the Km and state of phosphorylation were determined. Rats from the group sacrificed 12 hours after the injection of the agonist showed the lowest Km for Fru-6-P. Animals which received 3 doses of IPR showed the highest phosphate content/mol of enzyme. Experiments of dephosphorylation of the purified PFK-1 from this latter group revealed that the presence of the phosphate groups influence the kinetic properties of the enzyme.     

Enzymatic sulfation of mucus glycoprotein in rat submandibular salivary gland

1. The enzymic activity which catalyzes transfer of sulfate ester group from 3'-phosphoadenosine-5'-phosphosulfate to mucus glycoprotein was found associated with Golgi-rich membrane fraction of rat submandibular salivary gland. 2. Optimum enzyme activity was obtained with 0.5% Triton X-100, 4 mM MgCl2 and 25 mM NaF at a pH of 6.8 using desulfated submandibular salivary mucus glycoprotein. The apparent Km of the enzyme for mucus glycoprotein was 11.1 mg/ml. 3. Alkaline borohydride reductive cleavage of the synthesized 35S-labeled glycoprotein led to the liberation of the label into reduced oligosaccharides. A 75.4% of the label was found incorporated in four oligosaccharides. These were identified in order of abundance as sulfated penta-, tri-, hepta- and nonsaccharides. 4. Based on the results of chemical and enzymatic analyses of the intact and desulfated compounds the pentasaccharide was characterized as SO3H----GlcNAc beta----Gal beta----GlcNAc(NeuAc alpha----)GalNAc-ol and the trisaccharide as SO3H----GlcNAc beta----Gal beta----GalNAc-ol.     

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.     

Compensatory proliferative response of the rat submandibular salivary gland to unilateral extirpation

The present experiment was conducted in order to identify the progenitor compartment of the submandibular salivary gland (SSG) and to explore the proliferative activity of this gland in response to unilateral extirpation. Left submandibular and retrolingual glands were extirpated in 30 rats (B.W. 200 +/- 12 g). The rats were killed 0, 1, 2, 3, 5 and 7 days after surgery. Five intact rats served as controls. The animals were given intraperitoneal injections of 3HTdR (0.5 microCi/grB. W) 1 h before they were killed. The contralateral SSG's were subjected to routine histological procedures and embedded in glycol methacrylate. Selected sections (2 micron thickness) were processed for autoradiography. In each gland, labelled and unlabelled nuclei were counted in 50 random microscopic fields and sorted according to their parenchymal histomorphological features and "nuclear class" (number of nuclei/cross section/feature). In the control glands the total labelling index (LI) was 0.18%; during acute compensatory stimulation, however, the total LI reached a maximum of 0.86% on day 3 after surgery. suggesting that the SSG, which normally undergoes a slow turnover, is capable of elevated proliferation in response to a stimulus. In both normal and stimulated glands, the LI was higher in the intercalated ducts (1.1%-5.85%) than in the granular ducts (0.17%-0.93%) and acini (0.05%-0.36%). This consistency of LI ratio between the various histomorphological features in the normal and experimental glands indicates that the glandular progenitor compartment is located in the intercalated ducts, which supply cells to both the ductal system and acini.(ABSTRACT TRUNCATED AT 250 WORDS)     

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

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.     

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.     

Influence of dietary omega-3 fatty acids on transmembrane signalling in rat submandibular salivary gland

We have previously shown the incorporation of dietary omega-3 and omega-6 fatty acids from menhaden oil and corn oil, respectively, into membrane phospholipids of submandibular salivary gland (SMSG) of rat [Alam S. Q. and Alam B. S. (1988) Arch. Oral Biol. 33, 295-299]. We now demonstrate the influence of such incorporation on the regulation of G proteins and adenylate cyclase activity. Cholera toxin ribosylated three protein peptides (Mr 42,000, 44,000 and 46,000) to different extents in the two groups. We found 4.9- and 2.6-fold higher and 0.4-fold lower ribosylation of Mr 42,000, 44,000 and 46,000 peptides, respectively, in SMSG membranes of rats fed a diet containing 10% menhaden oil (group II) compared to those fed 10% corn oil (group I). Functional distinctions between different forms of these peptides are not known. Cholera toxin also exhibited radiolabelling of three peptides in the SMSG membranes from normal or fasting rats. In these membranes inhibitory G proteins were not detected by pertussis toxin dependent ADP ribosylation or by a low concentration of guanylyl 5-imidodiphosphate (10(-8) M), which selectively activates inhibitory G proteins which inhibit forskolin stimulated activity of adenylate cyclase. In group II membranes both basal and fluoride stimulated activities of adenylate cyclase were found to be significantly higher than the corresponding values in group I (P less than 0.02). In cholera toxin dependent ribosylated membranes of group I, basal and fluoride stimulated activities of adenylate cyclase were significantly higher than those obtained in the absence of cholera toxin (P less than 0.02). Surprisingly, corresponding values were found to be lower in ribosylated membranes of group II. This could be due either to conformational changes in heavily ribosylated G proteins, which alters coupling with the catalytic subunit of adenylate cyclase, or due to dissociation of excessive inhibitory beta gamma complex from alpha beta gamma complex upon the activation of G proteins.     

Inducing effects of chronic administration of isoproterenol on 1-acyl-sn-glycero-3-phosphate and 1-acyl-sn-glycero-3-phosphocholine acyltransferases in rat parotid salivary gland

1. In rat parotid gland, chronic administration of isoproterenol caused significant increase of linoleic acid and decrease of arachidonic acid at the sn-2 position of phosphatidylcholine. 2. The activities of 1-acyl-sn-glycero-3-phosphate and 1-acyl-sn-glycero-3-phosphocholine acyltransferases were increased 3-8-fold and 2-fold, respectively, in the parotid gland microsomes of isoproterenol-treated rat. 3. Furthermore, the specificity of these two enzymes for various acyl-CoAs was also changed by administration of isoproterenol. 4. The alteration of unsaturated fatty acid composition at the sn-2 position of phosphatidylcholine was at least in part due to the change of activity and substrate specificity of lysophospholipid acyltransferases.     

Potassium release from submandibular salivary gland in vitro

Rat submandibular gland slices, incubated in continuously-gassed Krebs-Ringer bicarbonate buffer, were shown to release K⁺ in response to α-adrenergic and muscarinic cholinergic stimulation. The system employed the specific α-, β-adrenergic and cholinergic receptor-blocking agents phentolamine, propranolol and atropine, respectively, in combination with the agonists l-epinephrine and carbamylcholine both of which required the presence of Ca²⁺ for their effect. The introduction of Ca²⁺ into the cell via the ionophore A23187, with all neurotransmitter receptors blocked, resulted in K⁺ release. Ouabain also allowed extensive K⁺ release which was in addition to, and hence independent of, that elicited by epinephrine and carbamylcholine. Ethacrynic acid, a potent inhibitor of salivary secretion in vivo, had no influence on K⁺ movement. K⁺ was released by both physalaemin and an eledoisin-related peptide independently of normal neurotransmitter receptors. The activity of the eledoisin-related peptide did not require the presence of extracellular Ca²⁺. The implication of cyclic GMP at some stage of K⁺ release was suggested by experiments with a phosphodiesterase inhibitor.The results support an hypothesis where the initial stimulus at either α-adrenergic or muscarinic cholinergic receptors causes an immediate permeability change such that Ca²⁺ enters the cells resulting in K⁺ release. The loss of K⁺ is quickly countered by the ouabain-sensitive ($\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}$) ATPase which would be activated by the lowered intracellular K⁺ levels.     

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.     

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.     

A method for the isolation of rat submandibular salivary gland polysomes on linear sucrose density gradients

A procedure for isolating rat submandibular salivary gland polysomes on sucrose density gradients has been described. Electron micrographs of the gland revealed the existence of a very well-developed system of membranebound ribosomes. These morphological findings dictated the use of vigorous homogenization of tissue, a detergent, and low g-forces during tissue fractionation procedures for maximal recovery of this cellular organelle. The methodology allowed for the isolation of polysomes susceptable to RNase degradation and able to incorporate labeled amino acids into protein in a cell-free system.