Tonin, an esteroprotease from rat submaxillary glands

Tonin is an enzyme found in the rat submaxillary glands which liberates angiotensin II from angiotensinogen, the Skeggs tetradecapeptide renin substrate, and angiotensin I. Tonin hydrolyzes benzoyl-arginine ethyl ester, benzoyl-arginine methyl ester, tosyl-arginine methyl ester, benzoyl-arginine p-nitroanilide and other small synthetic substrates at an optimum ph of 9.0. Tonin shows, however, a great specificity with respect to angiotensin I. Tonin is inhibited by diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride at high concentrations (greater than 10(-2) M) and by soybean trypsin inhibitor and aprotinin. Tonin is thus an esteroprotease of the class of the serine protease with trypsin- and chymotrypsin-like activity. Tonin belongs to the same family of enzyme as glandular kallikrein and the gamma subunit of the nerve growth factor.     

Long-term cold adaptation in the rat

1. To determine whether long-term cold exposure induces insulative adaptation in the rat, two groups of eight adult animals each were exposed to 4 and 25 degrees C, respectively, for 18 months. 2. At any ambient temperature between -5 and 30 degrees C, the cold adapted animals had a higher rate of oxygen uptake, and higher unfurred skin temperatures than the controls. 3. At ambient temperatures below thermoneutrality, whole body thermal resistance increased continuously in both groups of animals. 4. It is concluded that long-term exposure does not induce insulative adaptation, and that thermal resistance is not maximal at the lower critical temperature.     

Extreme water efficiency of Cape gannet Morus capensis chicks as an adaptation to water scarcity and heat stress in the breeding colony

Cape gannet Morus capensis chicks depend entirely on fish prey and metabolic water for water requirements during development. Water loss through evaporative cooling due to heat stress is substantial. We measured water flux and field metabolic rates (FMR) of Cape gannet chicks and adults to determine if gannets developed water saving strategies. The water economy index (WEI, g kJ^?1) decreased with chick age according to the model WEI = 0.676 – 0.272 × log₁₀(t), indicating that water efficiency increased with age. At fledging, the WEI of chicks was at the level expected of adult desert birds. Desert birds maintain a low WEI by also having a low FMR, whereas Cape gannet chicks have FMR comparable to other seabird species’ nestling requirements. We propose that maintaining low WEI is adaptive for Cape gannets because (1) chicks need to balance water loss through evaporative cooling, (2) fledglings need to overcome a period of up to a week when they cannot ingest any water and (3) adults spend extended periods in the breeding colony during which water can become a limiting factor. Understanding the physiological mechanism of maintaining low WEI will become increasingly important with future rising temperatures.     

Certain characteristic features of the lipolytic factor from rat submaxillary salivary glands

The aqueous extract of rat salivary submaxillary gland was found to contain three protein fractions activating the release of free fatty acids (FFA) and glycerol from rat epididymal adipose tissue in vitro. Physico-chemical investigations of these proteins demonstrated certain common features: all three fractions were albumins having a common isoelectric point, and their aqueous solutions absorbed light at the same wavelength. The use of lipolysis activators and inhibitors (theophylline, propranolol, insulin) for investigating their effects on FFA and glycerol release produced by these protein fractions explained the mechanism of the lipolytic action of the protein fractions from rat submaxillary glands.     

Tolerance to heat of rats treated by beta-blocker. Role of submaxillary salivary glands

Effect of beta-adrenoceptor blockade on the changes in rectal temperature (RT), heat production (HP) and noradrenaline (NA) concentration in the submaxillary salivary glands (SMSG) of rats exposed to heat (45 degrees C) was studied. Propranolol (P) (15 mg/kg i.p.) decreased the tolerance to heat. The survival time of propranolol-treated (PT) rats was 30 min shorter. The temperature curve of control rats exposed to heat can be divided into three phases: a rapid rise in RT; a plateau (TP) and a prelethal increase. In PT animals, under identical conditions, TP disappears and RT further rises accelerating death. In the initial phase of heat exposure, HP was markedly decreased to the same extent in both experimental groups; but after 20 min HP increases in PT rats. The content of NA from SMSG both in the initial phase and in the TP phase is modified in PT rats.     

beta-Adrenergic and muscarinic cholinergic receptors in rat submaxillary glands. Effects of thyroidectomy.

Administration of triiodothyronine to thyroidectomized rats increased the density of beta-adrenergic receptors in rat submaxillary gland without significantly changing the density of muscarinic cholinergic receptors. Thus, thyroid hormone appears to regulate beta-adrenergic sensitivity in the rat salivary gland.     

Influence of thyroid function upon "Substance P" induced secretion of saliva by submaxillary glands

The effects of changes in thyroid function on the action of "Substance P" upon the secretion of saliva by the submaxillary glands was studied in male Wistar rats, with parasympathetic decentralization on the left side. The dose-response curves to increasing doses of "Substance P" showed in hyperthyroid animals increased salivary secretion while in hypothyroid animals the dose-response curve to the drug was decreased. Every animal showed supersentivity to "Substance P" in the decentralized gland. The influence of changes in thyroid function in the denervated glands was the same as that in the unoperated side, increased salivary secretion in hyperthyroidism and decreased in hypothyroidism.     

Signaling pathways involved in pilocarpine-induced mucin secretion in rat submandibular glands

We have studied the signaling pathways involved in pilocarpine-induced mucin release in rat submandibular slices. Pilocarpine produced a significant increment of PGE2 levels and a positive (r=0.8870) and significant (p=0.0077) correlation between PGE2 production and mucin released was determined. The participation of PGE2 was confirmed by the use of indomethacin (indo) and of acetyl salicylic acid (ASA), cyclooxygenase inhibitors, which inhibited pilocarpine-induced mucin release. The muscarinic receptors involved in the regulation of mucin release were identified as M1 and M4 by the use of the selective acetylcholine receptors (mAChR) antagonists, pirenzepine, AF-DX 116, 4-DAMP and tropicamide. The secretory process was dependent on both, intracellular and extracellular calcium pools since it was inhibited by thapsigargin and verapamil. Cyclic AMP, nitric oxide synthase and PKC also participated in pilocarpine-induced mucin release. It is concluded that pilocarpine, by activation the M1 and M4 mAChR subtypes induces an increase of intracellular Ca2+ concentration ([Ca2+]I) and elevates cAMP levels, which in turn stimulates COX, PKC and NOS and promotes mucin exocytosis. PGE2 released induces cAMP accumulation which, together with PKC are involved in the PGE2 increased Ca2+/cAMP-regulated exocytosis. Thus, cAMP accumulation induced by cholinergic stimulation is, in part, the result of PGE2 production.     

Topology of glucosylceramide synthesis in Golgi membranes from porcine submaxillary glands

The topology of ceramide glucosyltransferase and de novo synthesized glucosylceramide was studied in sealed and 'right-side-out' vesicles of porcine submaxillary glands derived from Golgi apparatus. Pronase treatment which did not cause any breakdown of the luminal glycoprotein galactosyltransferase activity, inhibited the ceramide glucosyltransferase to more than 50% at a ratio proteinase to Golgi protein 1:100. Trypsin at the same concentration, while producing no inactivation of luminal galactosyltransferase, caused a complete loss of ceramide glucosyltransferase activity. The membrane-impermeable compound, DIDS, which did not cause any inhibition of the galactosyltransferase, inhibited the ceramide glucosyltransferase (70% reduction at 80 microM DIDS). Thus, the enzyme ceramide glucosyltransferase is accessible from the cytoplasmic side of the Golgi vesicles. The orientation of the newly synthesized glucosylceramide is studied by the ability of the enzyme glucosylceramidase to hydrolyse this compound both on intact and on disrupted vesicles. The same percentage (respectively, 36 and 30%) of hydrolysis was obtained during an incubation of 3 h, showing that glucosylceramide is not at all protected from external hydrolysis. Pronase-treated vesicles revealed an increase in glucosylceramidase hydrolysis (up to 45%), which indicates that glucosylceramide that glucosylceramide may be cryptic. All these results indicate that the ceramide glucosyltransferase, as well as related glucosylceramide, are cytoplasmically oriented in Golgi vesicles from porcine submaxillary glands.     

II. Ultrastructure of duct cell granules in mammalian submaxillary glands

Summary Discrete, PAS-positive granules of relatively uniform electron-density and size characterise the intercalated duct cells of mammalian submaxillary glands. Smaller, electron-dense organelles are seen in the cells at the junction of the intercalary-striated duct region in the guinea-pig. The large granules of variable electron-density which are observed in the proximal, modified intercalary cells in the rabbit closely resemble the granules in the acinar cells of the guinea-pig. Several populations of granules differing in size are found in the striated granular tubules of the rat and hamster; the organelles in the rat show two grades of electron-density whereas those in the hamster are uniformly dense. Numerous small granules with compactly arranged intragranular material occupy the apical part of the striated ducts of the cat, dog and rabbit. The chemical composition of each population of duct cell granules is unknown. The question whether granules containing kallikrein, trypsin-like enzymes and amylase are stored in the duct cells is discussed.We wish to thank The Wellcome Trust for a travel grant to attend the International Symposium on Vasopeptides (Florence, July 1971) where this work was briefly reported.