J.R. Martinez

Autonomic receptors in the developing submandibular glands of neonatal rats

The functional maturation of the gland during the neonatal period involves specific temporal sequences in the appearance of autonomic membrane receptors and associated changes in the secretory response to receptor stimulation. The density and affinity of cholinergic muscarinic and of α- and β-adrenergic receptors were determined in the glands of 1-, 7-, 14- and 21-day-old rats, using the radioligands [3H]-quinuclidinyl benzylate, [3H]-prazosin and [3H]-dihydroalprenolol for the measurement of muscarinic cholinergic, α1-adrenergic and β-adrenergic receptors, respectively. The density of binding sites followed similar developmental courses, whether expressed as pmolg tissue or pmolg protein. The densities (pmolg protein) of β-adrenergic and muscarinic receptors were low in 1-day-old animals, but increased rapidly; adult levels were reached by 2 and 3 weeks of age, respectively. α1-Adrenergic receptor binding was barely detectable at birth, increased slightly during the first week, dramatically by 14 days and approached adult levels by 21 days. The number of receptors per gland for these 3 autonomic receptor-binding sites increased 50 – 100-fold during the first 3 weeks of postnatal development. The affinities (KD) for each of the three ligands did not differ significantly with age. Surprisingly, the α2-adrenergic receptor density ([3H]-p-aminoclonidine binding) was high in 1-day-old animals and increased significantly during the first 2 weeks of life. The binding declined after 3 weeks and was nearly undetectable by 6 weeks. Frozen submandibular glands had markedly lower α2-adrenergic binding compared to fresh glands. These findings suggest that each of the autonomic receptors in rat submandibular glands follows its own specific developmental pattern, and that the appearance of individual receptor types may correlate with the ability of the developing gland to respond to specific stimulants. A K+-release response can be elicited by epinephrine at 2 weeks of age, when α1 receptor binding sites appear, while both the receptor binding sites and the response to muscarinic agonists are present at birth. As β-adrenergic receptor-binding sites may develop simultaneously with isoproterenol-stimulated adenylate-cyclase activity, it seems that receptor density is important in this response. The functional significance of the high density of α2-adrenergic receptors in the submandibular gland of neonatal animals is not clear.

Fluid and electrolyte secretion from the isolated, perfused submandibular and sublingual glands of the rat

Abstract Isolated preparations of the rat submandibular gland secreted when perfused at a rate of 3 ml/min with physiological salt solutions containing glucose and either acetylcholine (10 −9 to 10 −4 M) or pilocarpine (10 −7 to 10 −3 M). The maximum secretory response was 250 μl g −1 min −1 with acetylcholine (10 −6 M) and 130 μl g −1 min −1 with pilocarpine (10 −6 M). Higher agonist concentrations usually resulted in smaller secretory responses. The response to continued stimulation showed a slow decline, although it was more sustained than with a similar preparation of the rabbit submandibular gland. The excretion curves for Na, K, Ca, Cl and HCO 3 evoked by the two parasympatho-mimetic agonists were similar to those reported from in-vivo experiments. Ouabain (10 −3 M), the substitution of Na by Li or the omission of K in the perfusion solution markedly inhibited the secretory response from the isolated, perfused-gland preparation. Partial substitution of the perfusate Na with choline caused only partial inhibition of secretion, but decreased Na and increased Ca and K concentrations in saliva. It is concluded that the perfused-gland preparation functions normally with respect to the secretion of fluid and electrolytes, and offers a suitable vehicle for investigation of the salivary secretory process. The sublingual gland secreted in response to pilocarpine. The maximum secretory rate was 130 μl g −1 min −1 and the saliva was poor in Na and Cl and rich in K, HCO 3 and Ca.

Progressive secretory dysfunction in the rat submandibular gland after excretory duct ligation

Unilateral ligation of the main excretory duct of the rat submandibular gland caused a progressive deterioration of secretory function characterized by: (1) the secretion of progressively smaller volumes of saliva in response to a standard maximal intraperitoneal (i.p.) dose of pilocarpine. Saliva volume was reduced 69.4, 88.8 and 95.9 per cent, respectively, 1, 3 and 7 days after duct ligation. By 2 weeks, the contralateral, non-ligated gland had an enhanced response to pilocarpine and the ligated gland a 96.8 per cent reduction in the volume of saliva secreted; (2) a progressive reduction in the maximum flow rates attained upon stimulation with pilocarpine, which were 23.4, 10.1 and 5.1 per cent of those attained in the contralateral gland at, respectively, 1, 3 and 7 days after ligation; (3) a progressive increase in the sodium concentrations of saliva, which became plasma-like 2 weeks after ligation; (4) a significantly reduced secretory response to standard doses of isoproterenol, phenylephrine, methoxamine and substance P 2 weeks after ligation. In addition, both cholinergic and adrenergic receptors were significantly reduced in number 2 weeks after ligation. Administration of daily injections of pilocarpine (10 mg, i.p.) after duct ligation reduced the gland atrophy observed 3 days (by 17 per cent) and 1 week (by 9 per cent) later, but did not prevent the reduction in volume or flow rats observed after duct ligation alone. Thus ligation of the main excretory duct causes a progressive dysfunction in acinar and duct cells in the rat submandibular gland and alters their responsiveness to physiological regulators of secretion. The alterations in glandular secretory function and in the composition of saliva which occur in diseases causing partial or complete obstruction to the flow of saliva are likely to be similar to the changes described.

In-vitro release of K+ from the developing submandibular gland of early postnatal rats

The release studied by incubating tissue slices in an enriched, oxygenated medium in the presence and absence of secretagogues indicated that slices from 1-day-old rats released K+ in response to carbamylcholine, but not in response to epinephrine. The response to carbamylcholine was dose-related, with a 12.6 +/- 1.3 per cent release at a concentration of 2 X 10(-5) M. Epinephrine did not induce K+ release in concentrations from 2 X 10(-7) to 2 X 10(-4) M. Glands from 7- and 14-day-old rats released a similar amount as those of newborns in response to carbamylcholine and by 21 days of age the glands released 21.5 +/- 1.9 per cent of their K+ content upon exposure to 2 X 10(-5) M carbamylcholine. A response to epinephrine (2 X 10(-5) M) was first detected at 14 days of age, when the slices released 8.3 +/- 1.2 per cent of their K+. Slices of 21-day-old rats showed a 17.9 +/- 1.3 per cent K+ release in response to this dose. As in adult glands, the net amount of K+ released by the developing postnatal gland seems to be the result of two opposing mechanisms, a passive efflux and an active re-uptake which depends on the activation of an ouabain-sensitive Na+ -K+ -ATPase. The passive efflux component was similar in glands from different postnatal ages and was enhanced by secretagogues. The extent of active uptake, on the other hand, decreased with increasing postnatal age. As in the adult, the net release of K+ depended on the presence of Ca2+ in the incubation medium, but the sensitivity of the response to Ca2+ omission varied in accord with the age of the animals and with the stimulant used. It is speculated that proacinar and terminal tubular cells may be involved in K+ release in the early postnatal period.

Ca2+ mobilization and Cl efflux in submandibular salivary cells of adult and newborn rats.

These cells were isolated by enzymatic digestion from glands of 1-day-old and fully mature rats. The effects of exposure to acetylcholine on cytosolic Ca2+ and Cl efflux were studied by, respectively, spectrofluorimetry of fura-2 and the net efflux of the isotopic tracer 36Cl from preloaded cells. In both types of cells, when incubated in Ca2+-containing solutions, acetylcholine initially caused a rapid, significant increase in cytosolic Ca2+ (from approx. 90 to 480-570 nmol), followed by a slower decrease to a plateau value of 280-290 nmol. The initial peak persisted (315-339 nmol) in Ca2+-free solutions but the cytosolic Ca2+ concentrations decreased rapidly to levels below prestimulation values (30 nmol). 36Cl efflux in tracer preloaded cells incubated in Ca2+-containing medium in the presence of acetylcholine was 18% in cells of new born animals and 35% in adult cells. In Ca2+-free medium, mature cells showed a transient but significant (26%) efflux of 36Cl. Cells of 1-day-old rats did not show a net efflux of 36Cl under these conditions, but subsequent addition of Ca2+ caused a 15% reduction (i.e. efflux) in tracer content. The antagonist 3,4,5-trimethoxybenzoate-8(N,N-dithylamino)octyl ester (TMB 8), which blocks internal Ca2+ release, inhibited both the initial increase in cell Ca2+ in both types of cells and the transient efflux of 36Cl seen in mature cells when incubated in Ca2+-free solutions. At high concentrations (5 mM), LaCl3 inhibited efflux of 36Cl in mature cells but not in those of newborn rats. However, at lower concentrations (0.1 mM), which do not interfere with fluorescence spectra, LaCl3 did not inhibit the effect of acetylcholine on cell Ca2+. These results suggest that Cl efflux in adult submandibular cells is regulated by an increase in cell Ca2+ arising from release of internal Ca2+ and from influx of external Ca2+. Both of these responses are evident in cells of newborn animals but Cl efflux is either decreased or absent. This is likely to be associated with a deficiency in the Cl channels or in the coupling between Ca2+ and the channel substrate through regulatory molecules associated with phosphorylation of the channel protein.

Secretory responses of the rat submandibular and parotid glands to sequential stimulation with pilocarpine and isoproterenol

Abstract Administration of a maximal secretory intraperitoneal (i.p.) dose of pilocarpine (10mg/kg body wt) resulted in a four-fold increase in the volume of saliva secreted by the rat submandibular gland in response to a subsequent i.p. injection of isoproterenol (10 mg/rat), compared to the volume elicited by isoproterenol alone. The enhanced response was inhibited when atropine (5 mg/kg body wt, i.p.) was administered after the pilocarpine, but before the isoproterenol. In contrast, previous stimulation with isoproterenol significantly inhibited the response to subsequent pilocarpine and only 34 per cent of the volume of saliva elicited by pilocarpine alone was observed, an inhibition not modified by intervening injection of propranolol (5 mg/kg body wt). The mean protein concentration of submandibular saliva was similar (≈ 11 mg/ml) when the two secretagogues were administered sequentially in alternating order. Total protein output correlated with saliva volume under each condition. Thus, protein output during the response to isoproterenol administered after a previous pilocarpine injection was similar to that observed with isoproterenol alone (2453 and 2674 μg/30 min, respectively). Protein output was 1826μg/30 min when pilocarpine was administered following isoproterenol. Atropine between sequential pilocarpine and isoproterenol stimuli resulted in a protein output of 2033 μg/30 min; propranolol between successive isoproterenol and pilocarpine stimuli reduced protein output to 792 μg/30 min. Observations in respect of parotid gland were similar. Previous administration of either secretagogue did not modify the Na, K and Ca excretory patterns of submandibular saliva elicited by the second agent, compared with the second stimulant alone.

In vivo effects of prostaglandin E1 and lysine-bradykinin on rat salivary secretions elicited by parasympathomimetic stimulation

Intravenous injections of either prostaglandin E1 or lysine bradykinin (kallidin) modified the secretory response of the submandibular, parotid and sublingual glands of the rat to intravenous infusions of acetylcholine. The two substances caused reductions (of from 16 to 67 per cent) in salivary flow rates when administered in concentrations ranging from 10 to 50 micrograms/kg body weight. The effect lasted for 20-30 min, followed by a return to pre-injection levels and in general, depended on the concentration of the secretory stimulator and on the dose of test substance used. In the submandibular and sublingual gland, both substances generally caused a concomitant increase in the salivary Na+ concentrations. This effect depended on the concentrations of acetylcholine and of test substance and varied from 10 to 117 per cent. The effect was more marked in submandibular saliva. Absolute increases in salivary Na+ concentration were not observed in the parotid gland, but the reductions in salivary Na+ concentrations (from 2.5 to 31.7 per cent) were smaller and did not parallel the simultaneous reduction in flow rate, which was between 16.4 and 60.4 per cent. As both kinins and prostaglandins are present in the glands and may be activated as a result of secretory or metabolic activity, the results suggest that they act as modulators of the secretory response to cholinergic stimulation. The divergent effects on flow rate and on salivary Na+ concentration suggest that kinins and prostaglandins have specific and independent effects on acinar and duct cells.

An examination of functional linkage between K efflux and 36Cl efflux in rat submandibular salivary gland acini in vitro.

Suspensions of dispersed acini isolated by enzymatic (collagenase) digestion were used to investigate possible interactions between transmembrane K and Cl movements. The isotopic tracer 36Cl monitored uptake and efflux of Cl under conditions where K efflux was either stimulated or inhibited. Uptake (accumulation) of 36Cl in the absence of experimental manipulation was time-dependent, resulting in a steady-state isotope content of 8.9 +/- 0.2 nmol/mg protein after 3-5 min of incubation. This content was reduced 28 per cent by the K-ionophore, valinomycin (10 microM), which also caused a net efflux of 36Cl (28 per cent) from tracer-preloaded acini. Valinomycin also released 38 per cent of the cellular K content and caused efflux of 86Rb from acini preloaded with this tracer. The efflux of 36Cl induced by 1 microM acetylcholine (23 per cent) was blocked by the K-channel blocker, quinidine (1 mM), and incompletely by apamin (1 mM). Efflux of 36Cl was also blocked by the chloride-channel blocker, 3,5-dichlorophenyl-2-amine-carboxylic acid. Thus, induction of K release (efflux) in these acini is balanced by a parallel efflux of Cl, and blockade of K release inhibits acetylcholine-induced 36Cl efflux, which suggests a functional linkage between these two events. According to current opinion, these ion movements occur, respectively, in the basolateral (K) and apical (Cl) cell membranes, so any linkage implies that the apical Cl conductance can be regulated, at least in part, by changes in membrane potential which are secondary to secretagogue-induced changes in K conductance.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of acetylcholine and transport inhibitors on K content in dispersed submandibular salivary cells of newborn and adult rats

These isolated cells were used to measure uptake of the radioactive tracer 86Rb as an index of changes in K+ content. There was time-dependent tracer uptake in the absence of secretagogue in both one-day-old and adult cells. Kinetic analysis of this uptake revealed two components, a fast one during the first 5 min of incubation and a slower one between 5 and 30 min. The early (fast) component was slower in the newborn cells (0.084 nmol mg-1 min-1) than in adult cells (0.116 nmol mg-1 min-1). Values for the second component of uptake were, respectively, 0.015 and 0.009 nmol mg-1 min-1. Basal uptake of K+ was inhibited by both ouabain and furosemide (at 1 mM concentrations) but, after 5 min of incubation, the effect was more pronounced in adult cells. The K+ content (i.e. K+ uptake) was increased after exposure to 1 microM acetylcholine in both cell types, but the effect was larger in those from one-day-old rats after 5 min incubation (16 per cent increase, compared to 9 per cent increase in adult cells). The secretagogue-induced increase in K+ uptake or content was inhibited by ouabain (57 per cent in adult cells, 24 per cent in newborn cells), and by furosemide (25 and 37 per cent, respectively). These findings suggest that K+ uptake in submandibular cells occurs by both an ouabain-sensitive Na, K pump and a diuretic-sensitive transport system. Both appear to have lower resting activities in the cells of newborn rats but can be significantly stimulated cholinergically.(ABSTRACT TRUNCATED AT 250 WORDS)

36Cl fluxes in dispersed-cell aggregates of the submandibular salivary gland of early postnatal rats

Salivary cells were isolated from the gland of rats (1, 7, 14 and 21 days old) by enzymic digestion. They formed small aggregates with typical morphological characteristics and were functional units with characteristic 36Cl fluxes. The rates of isotopic uptake in the absence of secretagogue (basal uptake) were similar at all ages. Uptake was time-dependent, leading to a steady-state isotope content of 6.7-8.9 nmol/mg protein after 3-5 min of incubation. 36Cl accumulation (uptake) in the unstimulated condition was inhibited 33-39 per cent by exposure to 1 mM furosemide at all ages, but was reduced significantly less (50 per cent) by 1 microM acetylcholine in cells of 1-day-old animals than in older ones. In aggregates exposed to 36Cl for 12 min (preloaded with tracer), acetylcholine caused a net efflux of isotope which was significantly less in cells of 1-day-old rats (18 versus 35 per cent in cells of older animals). Thus it was shown (1) that functional cell aggregates can be obtained from early postnatal rats; (2) that when not stimulated, their time-dependent Cl accumulation is partially inhibited by loop diuretics, and so likely to involve a cation/anion cotransport system; and (3) that Cl efflux, which may occur by way of membrane channels, is significantly reduced early in the postnatal period. The putative Cl channels are therefore either not fully developed, or they do not become responsive to stimulation until later. This reduced Cl efflux could be related to differences in Ca2+ mobilization at early and late phases of postnatal glandular development as Cl efflux is a Ca2+-dependent phenomenon in salivary cells.