Michael Humphreys-Beher

Inhibition of isoproterenol-induced DNA and RNA synthesis in rat parotid and pancreas following removal of submandibular-sublingual glands

Summary[3H] thymidine incorporation into DNA of the parotid (PA) gland of adult and 20-day-old rats and into DNA of the pancreas (PANC) of 20-day-old rats was increased markedly following a 2-day regimen of isoproterenol (ISO) administration. However, when the submandibular-sublingual (SM-SL) glands had been removed just prior to initiation of the ISO injections, the [3H] thymidine incorporation into PA and PANC was inhibited, and cpm/mg protein of these organs was even lower than that of organs of untreated rats with SM-SL glands present. Removal of the PA glands just prior to initiation of the ISO regimen had no effect on the ISO-induced [3H] thymidine incorporation into DNA of PANC but partially inhibited that of the submandibular (SM) gland. It is suggested that the inhibitory effects on DNA and RNA synthesis that follow removal of SM-SL glands are attributable to the growth factors (epidermal growth factor and nerve growth factor) found in the rat SM gland. These factors appear to regulate normal DNA synthetic activity of exocrine glands as well as β1-adrenoceptor mediated DNA synthesis. Cellular hypertrophy induced by the ISO was less markedly affected by absence of the SM glands, but a partial inhibition of [3H] uridine incorporation into RNA of PA of adult rats also occurred when SM-SL glands were removed prior to initiation of the ISO-regimen.

β-Adrenergic receptors and cAMP levels of rat parotid and submandibular glands during chronic isoproterenol treatment

The number of cell surface beta-adrenergic receptors and the level of cyclic AMP of the parotid and the submandibular gland were examined in rats treated for up to 10 days with twice daily injections of the beta-adrenergic agonist, isoproterenol. Receptor densities of 125 +/- 8.7 fmol/mg membrane protein for the parotid and 60.1 +/- 5.6 fmol/mg for the submandibular glands were found with [3H]dihydroalprenolol (beta-adrenergic receptor antagonist) binding of glands from control rats. No change from levels of controls was found in the number of beta-receptors of the submandibular gland with chronic isoproterenol stimulation; the parotid glands, on the other hand, showed a 22% decrease in dihydroalprenolol binding from the 4th until the 8th day of treatment. By day 10 of isoproterenol treatment the parotid gland demonstrated a shift from a population consisting of primarily beta-adrenergic receptors to one consisting of equal numbers of beta 1- and beta 2-adrenoceptors. The basal level of cAMP present in cell lysates remained unchanged in the isoproterenol-treated submandibular gland while the parotid gland showed a 30-40% decrease. Control and isoproterenol-treated animals demonstrated the same time course of cAMP accumulation after a single challenge with isoproterenol.

Analysis of protein synthesis in rat salivary glands after chronic treatment with β-receptor agonists and phosphodiesterase inhibitors

Chronic administration of the beta-adrenergic receptor agonist isoproterenol (5 mg/200 g animal for 10 days) resulted in rat parotid and submandibular gland hypertrophy, and it induced synthesis of a series of proline-rich proteins (PRPs) and glycoproteins. Treated parotid glands additionally exhibit an increase in activity for the Golgi membrane enzyme UDP-galactose; N-acetylglucosamine 4 beta-galactosyltransferase. A series of beta-receptor agonists and phosphodiesterase inhibitors were examined for their abilities to influence salivary gland protein biosynthesis in a fashion similar to that observed with chronic isoproterenol treatment. beta 1/beta 2-Adrenergic-receptor agonists exhibited the greatest effects on parotid gland hypertrophy and PRP biosynthesis. These beta-agonists were also able to increase 4 beta-galactosyltransferase activity, but they did not promote the synthesis of a 220,000 dalton glycoprotein. Terbutaline (beta 2-receptor agonist) induced parotid gland hypertrophy but was only able to induce protein biosynthesis at higher drug concentrations. Finally, methoxyphenamine was unable to produce the observed changes in protein synthesis even at increased drug dosages. The phosphodiesterase inhibitors (theophylline and caffeine) were able to induce de novo PRP biosynthesis at drug doses of 20 mg/200 g animal. However, while causing mild gland hypertrophy, there was no observable change in 4 beta-galactosyltransferase activity with either phosphodiesterase inhibitor. This same regimen of beta-receptor agonists was unable to induce submandibular gland hypertrophy, PRP or glycoprotein biosynthesis in the same animals. This was also true for the two phosphodiesterase inhibitors. Co-injection of a beta 1 antagonist along with isoproterenol blocked the above protein changes in both the submandibular and parotid glands, suggesting that the stimulation of protein synthesis takes place by beta 1-type receptors on the gland cell surfaces.

β Adrenergic and Muscarinic Receptor Densities of Rat Submandibular Main Duct

Abstract [3H]DHA binding studies show that main duct of rat submandibular gland has both β1 and β2 adrenoceptors, with the percentages of each being 69 and 31%, respectively, whereas whole submandibular gland has 90% β1 and 10% β2 adrenoceptors. Muscarinic receptors of main duct are 25% less than that of whole submandibular gland.

Neural and dietary modulation of proline-rich protein and 4β-galactosyltransferase biosynthesis in rat parotid glands

Maintenance of rats on a bulk diet (50 per cent inert cellulose and 50 per cent laboratory chow) before or after the removal of the submandibular-sublingual glands resulted in hypertrophy of the parotid gland, and the induction of basic proline-rich proteins in that gland with apparent molecular weights similar to those found after chronic administration of isoproterenol. Surgical removal of either the sympathetic or parasympathetic nerve, or of both, impeded both gland hypertrophy and the gene expression for proline-rich protein in the denervated gland. Rats fed normal chow did not have induction of these proteins in the innervated control parotid nor in the denervated contralateral gland. The experimental regimen also showed in-vitro translation of mRNA with electrophoretic protein patterns similar to those with isoproterenol treatment. There was also increased synthesis of the enzyme 4 beta-galactosyltransferase (EC 2.4.1.38).

Effects of electrical stimulation of the sympathetic nerve on the levels of β-adrenergic and cholinergic muscarinic receptors and cyclic nucleotides in rat salivary glands

After 30 and 60 min of stimulation, there were decrease of 16 to 19 per cent in beta-adrenoceptors in rat submandibular and parotid glands; a 10-min stimulation caused no change. Pre-incubation of the reaction mixtures (stimulated glands) with atenolol, a beta 1-antagonist, prevented most dihydroalprenolol (DHP) binding, but with butoxamine, a beta 2-antagonist, DHP binding was nearly complete. Thus the beta-receptor was of the beta 1-subtype. Muscarinic receptors of parotid gland showed no change after 10 min stimulation; after 30 min there was an increase of 12 per cent, and after 60 min, of 28 per cent. With submandibular gland, there was also no change at 10 min but, at 30 min, there was a 25 per cent increase, and at 60 min, a decrease of 18 per cent. Cyclic-AMP levels of parotid gland were markedly elevated after 10 min of stimulation (9-fold increase above controls) but, at 30 and 60 min, there was only a 1.6-fold increase. In submandibular gland, cyclic-AMP increased 10-fold at 10 min; at 30 min it was 2.5 times control levels and at 60 min, 1.9 times. Cyclic-GMP levels of parotid gland increased 34-fold after 30 or 60 min of nerve stimulation, but only 1.6-fold at 10 min. With submandibular gland, there was a 22-fold increase at 10 min, but at 30 and 60 min this was 15- and 12-fold, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Beta-adrenergic and muscarinic receptors of parotid gland following maintenance of rats on liquid diet.

Parotid gland of adult rats maintained exclusively on liquid (milk) diet for 7 or 13 days showed a 25% reduction in number of †-adrenoceptors, and after 21 days, the reduction was 33%; with maintenance of rats on Metrecal for 7 days, the decrease was 24% for female rats and 22% for male rats. The decrease in number of muscarinic receptors after 7 or 13 days on milk was 32%, and 38% after 21 days; the decreases for rats on Metrecal for 7 days were 32% for females and 35% for males. In rats maintained on liquid (milk) diet for 7 days, and then denervated by unilateral removal of the parasympathetic and sympathetic innervations to parotid there were decreases of 39–42% in number of β-receptors and 50–52% in muscarinic receptors at 6 or 14 days after denervation (maintenance on liquid diet for 13 and 21 days, respectively) from those of innervated glands of chow-fed rats; denervated glands of rats on chow diet showed the same reduction. Thus, it was concluded that absence of neurally mediated glandular activity, imposed by either diet or surgical removal of the nerves, caused marked decreases in number of both β-adrenergic and muscarinic receptors, but that the presence of the nerves, even though inactive (liquid diet), provided a trophic influence that prevented the more marked decreases seen in the absence (surgical removal) of the nerves.

β-Adrenoceptors in the rat parotid gland enlarged by salivariectomy and bulk diet. Effects of denervation

The numbers of beta-adrenergic receptors and level of cyclic AMP (cAMP) of the parotid gland of adult female rats were determined 4 weeks after introduction of a regimen that induced a 2-fold increase in gland weight. This regimen consisted of ablation of the submandibular-sublingual glands and substitution of the normal chow diet with a bulk diet consisting of 50% inert cellulose and 50% ground solid chow. There was a 2.4-fold increase in number (density) of beta-adrenoceptors in the enlarged parotid gland when comparison was made with parotid glands of control rats. The beta-adrenoceptor present in the enlarged and normal glands was of the beta 1 subtype. Removal of either autonomic pathway at the time of partial salivariectomy and dietary substitution was followed by a small reduction in number of beta-adrenoceptors (4-9% with either denervation), but when both nerves were removed the reduction was 25%; in magnitude, these changes were generally similar to those observed with denervated parotid glands of chow-fed rats. The norepinephrine concentration of the enlarged gland was much less than that of normal glands (reduced 38%); sympathectomy of normal or enlarged parotid glands resulted in a marked lowering of norepinephrine concentrations (to 1-5% of control levels); parasympathectomy had no effect on norepinephrine concentration of enlarged parotid glands but caused a decrease in that of the parotid of normal size. Apparently, the number of beta-adrenoceptors depends on the degree of activity of both the parasympathetic and sympathetic nerves to the parotid.(ABSTRACT TRUNCATED AT 250 WORDS)

Alpha-lactalbumin acts as a bimodal regulator of rat parotid acinar cell growth.

Isoproterenol, a beta-adrenergic receptor agonist, causes hypertrophy and hyperplasia of the rat parotid gland. The stimulation of parotid acinar cells to a growth phase is accompanied by a cell surface localization of the enzyme 4 beta-galactosyltransferase. Alpha-lactalbumin, a specific modifier protein for 4 beta-galactosyltransferase, when given subsequent to the initiation of isoproterenol treatment and the commencement of parotid enlargement, resulted in a termination of gland hypertrophy and DNA synthesis. Gland size did not, however, return to control levels with the continued injection of isoproterenol and alpha- lactalbumin. In contrast, the injection of alpha-lactalbumin in neonatal rats (7-14 days post-partum) stimulated parotid gland hypertrophy and DNA synthesis. This treatment also lead to the precocious expression of the major parotid gland salivary enzyme, amylase.

Muscarinic receptors of rat parotid gland enlarged by gland ablation and bulk diet. Effects of denervation

The density of muscarinic binding sites was increased 10% in the rat parotid gland enlarged (2 times control) as a result of ablation of the submandibular-sublingual glands and maintenance of rats on bulk diet (50% inert cellulose plus 50% solid chow) for 4 weeks. When either the parasympathetic or sympathetic innervation to the gland was unilaterally removed at the time of submandibular-sublingual ablation and introduction of the bulk diet, the density of muscarinic receptors showed an even greater increase from levels of innervated glands of chow-fed controls (29%); with removal of both nerves, the increase was 39%. A 36% increase in cyclic guanosine monophosphate levels accompanied the increase in receptors of the enlarged gland, but when the parotid was denervated, there was no change in cyclic GMP. Absence of either or both nerves led to a maximal decrease of 24-29% in density of muscarinic receptors of parotid gland of chow-fed controls, but to no change in cyclic GMP levels. While autonomic influences mediate the changes in density of muscarinic receptors of parotid gland of chow-fed rats, some additional factor is apparently involved in their increase in the enlarged gland.