Parasympathetic denervation decreases muscarinic receptor binding in rat parotid.

After unilateral postganglionic parasympathetic denervation of rat parotid glands for 3, 6 or 16 days, the binding of [³H] quinuclidinylbenzilate, a potent and specific muscarinic antagonist, was decreased by 28–45%. The largest part of the decrease was already present at 3 days, and thus appeared to coincide with the loss of parasympathetic terminals (as shown by a 94% reduction in choline acetyltransferase activity). The decrease in binding was expressed as a reduced number of membrane sites, with no significant change in affinity, and could be accounted for only in part by a reduction in the size of denervated glands. The rapid loss of binding suggests the existence of muscarinic receptors on parasympathetic terminals. The clear absence of any receptor increases at longer periods of denervation, when parotid glands are reported to give supersensitive secretory responses, suggests that mechanisms other than receptor increases are important in denervation supersensitivity in exocrine glands.     

Effects of selective denervation and nerve growth factor on activity-mediated growth of rat parotid gland.

A dietary change from all liquid to solid food is followed by an average increase of 200% in [3H]thymidine uptake into the parotid gland of rat. However, removal of either the parasympathetic (Px) or the sympathetic (Sx) innervation to the parotid gland prior to the dietary change resulted in a partial inhibition of the increase; values for the parasympathectomized gland were 51% of those of the innervated gland, and values of the sympathectomized parotid gland were 42% of those of the innervated gland. Removal of both autonomic nerves caused a complete inhibition. Initiation of nerve growth factor (NGF) injection (1 microgram/kg body wt, two times daily for the 2 days of chow refeeding) at the time of chow refeeding had no effect on completely or partially denervated glands, and thymidine values for the denervated parotid gland of rats given NGF did not differ statistically from those of rats not given NGF. With parasympathectomy, sympathectomy, and complete denervation, weight of parotid gland was decreased from that of innervated glands, and administration of NGF had no effect on the denervation-induced decreases. The data show that both branches of the innervation to parotid gland must be intact to ensure a maximal increase in thymidine uptake with the dietary change from liquid to solid food. The level of the enzyme, beta 1-4 galactosyltransferase, involved in proliferation, also depended on the presence of intact nerves. Enzyme activity of innervated parotid gland showed an average increase of 200% with chow refeeding of rats previously on liquid diet, but with Px, the average increase was 51% of that of the innervated parotid, and with Sx, it was 41%. NGF administration did not cause any change in levels of this enzyme in any Px or PxSx parotid gland and only a small change in Sx parotid; it did increase levels of this enzyme in parotid of rats without submandibular-sublingual glands.     

Myenteric denervation differentially reduces enteroendocrine serotonin cell population in rats during postnatal development

Summary The enteric nervous and enteroendocrine systems regulate different processes in the small intestine. Ablation of myenteric plexus with benzalkonium chloride (BAC) stimulates epithelial cell proliferation, whereas endocrine serotonin cells may inhibit the process. To evaluate the connection between the systems and the influence of myenteric plexus on serotoninergic cells in rats during postnatal development, the ileal plexus was partially removed with BAC. Rats were treated at 13 or 21 days and sacrificed after 15 days. The cell bodies of myenteric neurons were stained by β NADH-diaphorase to detect the extension of denervation. The number of enteroendocrine cells in the ileum was estimated in crypts and villi in paraffin sections immunostained for serotonin. The number of neurons was reduced by 27.6 and 45% in rats treated on the 13th and 21st days, respectively. We tried to establish a correlation of denervation and the serotonin population according to the age of treatment. We observed a reduction of immunolabelled cells in the crypts of rats treated at 13 days, whereas this effect was seen in the villi of rats denervated at 21 days. These results suggest that the enteric nervous system might control the enteroendocrine cell population and this complex mechanism could be correlated to changes in cell proliferation.     

The importance of the prolonged postnatal period in determination of heart atrial muscarinic receptor and beta-adrenoceptor densities

Our previous work indicated that hyperstimulation of muscarinic receptors brought about profound changes not only in the density of the muscarinic receptors, but also of the beta-adrenoceptors both in vivo and in vitro. Now we try to identify the changes in receptor densities when the neuronal input from the autonomic nerves is disrupted, i.e. when neonatal rats were injected with 6-hydroxydopamine (6-OHDA; able to destroy sympathetic nerves). Although the interruption of neuronal input was successful as indicated by norepinephrine uptake, there was no change in receptor densities in the rat heart atria when 6-OHDA was applied in the first postnatal week only. When we repeated the 6-OHDA treatment consequently (on 14th, 21st and 28th postnatal day) there were clear increases of both receptor types (muscarinic receptors rise to 139% of control, beta-adrenoceptors to 134% of control). The atrial muscarinic receptor number was increased (to 146% of control) also when we combined the first week 6-OHDA application with another factor (when the pups abide with mother till the 56th day). The simple non-weaning of animals has no effect on the receptor densities. We can therefore conclude that the first week application of 6-OHDA was insufficient in changing the receptor number and that only repeated 6-OHDA administration was able to change them. Similarly, combination with other factor: non-weaning led to the muscarinic receptors increase. Our results can indicate the importance of the prolonged postnatal period for the heart muscarinic and beta-adrenergic receptor number determination.     

Phleichrome; A New Phytotoxic Compound Produced by Cladosporium phlei

Bottle choice tests using liquid diets were done with Sprague-Dawley (SD) rats. SD rats ingested more oil-and-sucrose-enriched milk (hi-fat) and less oil-enriched milk (hi-fat-no-carb) than sucrose-enriched (hi-carb) milk by two-bottle choice tests after they were habituated to liquid diets for 4 days. Chronic food restriction didn't increase hi-fat ingestion but hi-fat-no-carb. Rats ingested less without habituation, and overnight food deprivation increased intake. This increment was maintained after rats were free-fed. The difference in fat content of the maintenance diet had little effect on fat preference. These results showed SD rats prefer a sweet and fatty liquid diet than a sweet and lean liquid diet. Habituation and food restriction were more important than the composition of the maintenance diet to demonstrate a clear preference for the fatty liquid diet.     

Bottle-choice tests in Sprague-Dawley rats using liquid diets that differ in oil and sucrose contents

Bottle choice tests using liquid diets were done with Sprague-Dawley (SD) rats. SD rats ingested more oil-and-sucrose-enriched milk (hi-fat) and less oil-enriched milk (hi-fat-no-carb) than sucrose-enriched (hi-carb) milk by two-bottle choice tests after they were habituated to liquid diets for 4 days. Chronic food restriction didn't increase hi-fat ingestion but hi-fat-no-carb. Rats ingested less without habituation, and overnight food deprivation increased intake. This increment was maintained after rats were free-fed. The difference in fat content of the maintenance diet had little effect on fat preference. These results showed SD rats prefer a sweet and fatty liquid diet than a sweet and lean liquid diet. Habituation and food restriction were more important than the composition of the maintenance diet to demonstrate a clear preference for the fatty liquid diet.     

Modulation of pancreatic muscarinic receptors by weaning

We have investigated the effects of early and delayed weaning on the development of the rat pancreatic muscarinic receptors. Weaning after 12, 14 and 16 complete days resulted in significantly increased concentrations of muscarinic receptors. Milk feeding, as the exclusive source of food, from day 12 to 23, 25 or 28, was associated with slight non significant decreases in receptor concentration. In both instances, early or delayed weaning, the apparent KDs of (3H-QNB binding were not affected. It is suggested that early solid food intake can modulate rapidly the pancreatic muscarinic receptor population while exclusive milk feeding does not seem to influence the dynamic of the muscarinic receptor population.     

Influence of chronic ethanol consumption on the muscarinic cholinergic control of rat pancreatic acinar cells

There are a number of hypothetical explanations for the actions of ethanol on the exocrine pancreas; among them, the cholinergic hypothesis has received special attention. According to this hypothesis, chronic alcohol consumption induces alterations in the control of exocrine pancreatic function resulting in cholinergic hyperstimulation of pancreatic acinar cells and their muscarinic receptors. Our aim was to investigate the cholinergic control of pancreatic enzyme secretion and the number and affinity of muscarinic receptors in the pancreatic acinar cells of rats subjected to chronic ethanol ingestion. We also investigated whether a high-fibre diet modifies the actions of ethanol on these aspects of the exocrine pancreatic function. Four groups of rats received either a standard or a high fibre diet, and either water or 20% (v/v) ethanol. After 6 months of treatment, isolated pancreatic acini were used for the determination of carbachol-stimulated amylase secretion and for the analysis of muscarinic receptors, using 1-[N-methyl-3H]scopolamine as a radioligand. Neither chronic ethanol intake nor a high fibre diet caused any apparent alteration in pancreatic histology, neither did them modify plasmatic amylase levels. Chronic alcoholization resulted in a significant increase in the amylase released from pancreatic acini in response to carbachol stimulation, but it did not affect either the number or the affinity of pancreatic acinar muscarinic receptors. The actions of ethanol are not significantly modified by the simultaneous consumption of a high fibre diet.     

Alterations in Neurotransmitter Receptor Binding in Discrete Areas of the Copper-Deficient Rat Brain

Abstract: Neonatal copper deficiency produced alterations in central neurotransmitter receptors that were selective with respect both to brain region and to neurotransmitter receptor type. Both high- and low-affinity dopamine receptor densities in the corpus striatum were significantly lowered, 55% and 29%, respectively, when expressed on a wet weight basis. There was a significant decrease in the level of muscarinic receptors in the striatum whether expressed on the basis of wet weight (50%) or protein (27%). A smaller reduction in muscarinic receptor density was observed in the cortex, whereas there was no effect of copper deficiency in the cerebellum. The treatment did not change β-adrenergic receptor binding in either the cortex or cerebellum. The affinities of the receptors for the ligands was not affected by the low-copper diet. It was previously reported that copper deficiency produces regionally specific decreases in the concentrations of dopamine and norepinephrine. The greatest reduction occurred in the concentration of dopamine in the corpus striatum. The results from both studies suggest that copper deficiency in post-weanling rats may induce a selective morphological lesion.     

Absorption of 241Am in the gastrointestinal tract

In experiments with rats a study was made of a number of factors influencing the resorption of 241Am from the gastrointestinal tract (GIT). The resorption of 241Am from GIT was found to be 120-245 times more intensive in neonatal rats, during the first 21 days after birth (a milk diet), than in adult animals. A milk diet for adult rats produced a 5-fold increase in the resorption of 241Am from GIT. The additional administration of digestive enzymes, as a homogenate from pancreas and small intestine, produced a 7--9-fold increase in the rate of 241Am resorption from GIT.     

Adaptive changes in lipid composition of rat liver plasma membrane during postnatal development following maternal ethanol ingestion

The fatty acid composition of constituent phospholipids and the cholesterol content of rat liver plasma membranes were determined subsequent to maternal alcohol ingestion during pregnancy and lactation. The alcoholic group was given a liquid Metrecal diet containing 37% ethanol-derived calories. The control group was pair-fed an isocaloric sucrose/Metrecal diet. Litters were killed for lipid analyses at days 5, 15 and 25 after birth. These studies revealed that the total phospholipid phosphorus was similar and increased significantly with age in both groups. Cholesterol also increased significantly with age in both groups but was greater in the alcoholic pups, resulting in a higher cholesterol/phospholipid molar ratio. While the phosphatidylethanolamine (PE) content increased with age in both groups, that of sphingomyelin decreased. Phosphatidylserine + phosphatidylinositol (PS + PI) was significantly higher in the control group at all ages studied. A consistent increase of C22:6 in phosphatidylcholine (PC), sphingomyelin, PS + PI and in the total phospholipid fraction from alcoholic pups was observed. Although other fatty acid changes were found in PC, PS + PI and sphingomyelin, PE was not affected. These results suggest that specific adaptive changes were induced in the liver plasma membrane lipids of the progeny from alcoholic rats.     

Ultrastructural changes of rat parotid glands induced by a diet of liquid Metrecal

Summary The ultrastructure of parotid glands was studied in rats fed a diet of liquid Metrecal for two weeks and compared with that of parotid glands of control rats which received a diet of Purina lab chow. The liquid diet induced major alterations of acinar cells, but other parenchymal components were apparently unaffected.Most acinar cells of experimental rats were atrophic and some of these were undergoing necrosis. Lipid droplets and dense bodies (believed to be lysosomes) were numerous in atrophic cells. The Golgi apparatus, quantity of secretory granules, and intercellular canaliculi were smaller than in acinar cells of control rats.Such findings suggest that the secretory process was impaired and support the conclusion that parotid glands of rats maintained on a liquid diet are physiologically less active than those of chow fed rats. The decreased activity, as previously reported, may result from reduced masticatory activity.Supported by U.S.P.H.S. grant DE 02110.     

Immunohistochemical and ultrastructural investigation of acinar cells in submandibular and sublingual glands of rats fed a liquid diet

In atrophic parotid glands induced by liquid diet, acinar cell apoptosis is increased while proliferative activity is reduced. This study aimed to clarify how liquid diet affects submandibular and sublingual glands, including acinar cell apoptosis and proliferation. Seven-week-old male Wistar rats were fed either a liquid (experimental group) or pellet diet (control group) from 3 to 21 days, respectively. Submandibular and sublingual glands were weighed and examined histologically, ultrastructurally, and immunohistochemically using antibodies to cleaved caspase-3 (Casp-3) and 5-bromo-2′-deoxyuridine (BrdU). Weights of submandibular and sublingual gland from the experimental group were not significantly different from controls at any time point. Histological and ultrastructural characteristics of experimental acinar cells in both glands were normal. Acinar cells in control and experimental submandibular glands were positively stained with periodic acid Schiff (PAS) and weakly stained by alcian blue (AB). In control and experimental sublingual glands, mucous acinar cells were PAS-positive and strongly AB-positive. Although Casp-3- and BrdU-positive acinar cells were identified in both glands in the experimental group, their labeling indices were not significantly different from controls. In conclusion, liquid diet in rats does not induce atrophic alterations to acinar cells, including apoptosis and proliferative activity in submandibular and sublingual glands.     

Nerve growth factor preserves a critical motor period in rat striatum

We previously found the occurrence of a critical motor period during rat postnatal development where circling training starting the 7-day schedule at 30 days-but not before or after-induces a lifetime drop in the binding to cholinergic muscarinic receptors (mAChRs) in striatum. Here, we studied whether nerve growth factor (NGF) participates in this restricted period of muscarinic sensitivity. For this purpose, we administered mouse salival gland 2.5S NGF (1.4 or 0.4 microg/day, infused by means of ALZA minipumps) by intrastriatal unilateral route between days 25 and 39, and then trained rats starting at 40 days. Under these conditions, NGF induced a long-term reduction in the striatal [3H] quinuclidilbenzylate (QNB) binding sites despite the fact that motor training was carried out beyond the natural critical period. Thus, at day 70, measurement of specific QNB binding in infused striata of trained rats showed decreases of 42% (p < .0004) and 33% (p < .02) after administration of the higher and lower NGF doses, respectively, with respect to trained rats treated with cytochrome C, for control. Noncannulated striata of the NGF-treated rats also showed a decrease in QNB binding sites (44%; p < .0001) only at the higher infusion rate. This effect was not found in the respective control groups. Our observations show that NGF modulates the critical period in which activity-dependent mAChR setting takes place during rat striatal maturation.     

Bacteriological Studies of the Effects of Cow's Milk on Dental Plaque and Dental Caries in Rats

The effects of liquid milk and skim milk powder on the bacterial composition of rat dental plaque and dental caries were examined in two separate experiments. First, groups of rats fed a cariogenic diet plus one or other of three types of liquid milk had. after 28 d. similar plaque flora. Those rats receiving reconstituted spray-dried skim milk had a significantly higher incidence of dental caries than those receiving either pasteurized and homogenized milk or ultra-heat treated milk. The differences in caries scores may be related to differences in inorganic phosphate and calcium levels of the milks. Secondly, rats fed a diet containing 65% phosphate-free sucrose plus 32% spray-dried skim milk in powder form had a significantly lower dental caries score than did rats receiving a diet containing 65% sucrose plus 32% autoclaved roller-dried skim milk powder. At the end of this experiment, the plaque flora had changed significantly and the difference in caries incidence was related to the reduction in the percentage of potentially cariogenic bacteria.
These results indicate that the mechanism by which milk reduces dental caries incidence may be (a) by remineralizing incipient carious lesions or (b) by mediating changes in the bacterial composition of dental plaque.     

Cocaine Treatment Causes Early and Long-Lasting Changes in Muscarinic and Dopaminergic Receptors

1. The study of changes that persist after drug discontinuation could be fundamental to understand the mechanisms involved in craving and relapse. 2. In this work the changes occurring in muscarinic, D1- and D2-like receptors after 30 min (immediate), 1 day (early), 5 and 30 days (late) withdrawal periods were studied, in the striatum of rats treated once a day for 7 days with cocaine 20 and 30 mg/kg, i.p. 3. Binding assays were performed in 10% homogenates and ligands used were [3H]-N-methylscopolamine, [3H]-SCH 23390, and [3H]-spiroperidol for muscarinic (M1 + M2-like), D1-, and D2-like receptors, respectively. 4. Muscarinic receptors presented a downregulation at all doses and discontinuation times, while the dissociation constant (Kd) for this receptor decreased after 30 min, 5 and 30 days abstinence times. In relation to D1-like receptors we found an antagonistic effect with 100% increase in receptor number 30 min after the last cocaine injection, but after 1-day withdrawal a downregulation was observed with both doses that persisted up to 30 days, only with the higher dose. The dissociation constant value (Kd) for this receptor showed a decrease only with 5 and 30 days withdrawal. An increase occurred with D2-like receptors at all doses and withdrawal periods studied, while Kd increased in 30-min, 5, and 30 days withdrawal. 5. In this work we found that the subchronic cocaine treatment produces early and long-lasting modifications in cholinergic muscarinic as well in dopaminergic receptors that persist up to 30 days of cocaine withdrawal.     

Vitamin E and selenium regulate balance between beta-adrenergic and muscarinic responses in rat lungs

The effects of hydrogen peroxide on the beta-adrenergic and muscarinic responses of the rat trachea muscle were studied in vitro, after feeding rats, for 6 weeks, either a diet deficient in vitamin E and selenium or a control diet. In the control situation after incubation with 1 mM hydrogen peroxide for 30 min, a reduction of the maximal response to methacholine of 39% occurred whereas no pD2 shift could be demonstrated. Moreover, no response to isoprenaline after precontraction with 3 x 10(-7) M methacholine was left. In the deficient situation, we found a reduction to 64% of the response to methacholine after incubation with 1 mM hydrogen peroxide. Again isoprenaline became inactive, i.e. no relaxation with isoprenaline was observed after precontraction with 3 x 10(-7) M methacholine. We therefore conclude that vitamin E and selenium protect against oxidative stress in lung tissue and thus regulate the (patho-) physiological balance between adrenergic and muscarinic responses.     

Functional mediation of compensatory enlargement of the parotid gland

Summary The role of functional activity in mediating compensatory enlargement of the parotid gland after removal of the other major salivary glands was investigated. Increased levels of activity were achieved by feeding rats a bulk diet. Conversely, a liquid diet was used to reduce the functional demands on the parotid. It was found that the liquid diet completely prevented the compensatory response from occurring. Bulk diet, on the other hand, caused an even greater compensatory response than did the standard chow diet. Compensatory enlargement of the parotid, therefore, depends on its functional activity and not on other, e.g., humoral factors dissociated from function. The character of the cellular response in compensatory enlargement was also examined. The chow diet caused compensatory enlargement by an increase in cell size with little, if any, increase in cell number.Supported in part by the Veterans Administration and Grant DE 02110 of the U.S. Public Health Service     

Regulation of submaxillary gland muscarinic receptors during heat acclimation

Binding properties of submaxillary gland muscarinic receptors and agonist-induced saliva secretion were studied in rats subjected to heat acclimation. The maximal binding capacity for the muscarinic antagonist N-[3H]methyl-4-piperidyl benzilate was increased from control value of 0.21 to 0.40 pmol/mg protein within 1-2 days of heat acclimation. The increase in the number of muscarinic receptors per gland (100%) was by far higher than the increase in tissue weight (20%), indicating higher density of receptors in the acinar cells of the treated rats. High levels of receptors coincided with the appearance of high-affinity binding sites for muscarinic agonists (oxotremorine, pilocarpine and carbamylcholine), and with reduced tissue sensitivity to pilocarpine. After 4-8 weeks of heat acclimation, the number of receptors as well as tissue response to pilocarpine returned to control levels. These results suggest a functional correlation between the transient upregulation muscarinic receptors in the submaxillary gland and the physiological activity in salivary secretion, and indicate that the high-affinity muscarinic receptors may attenuate saliva secretion during the initial phase of heat acclimation.