Biomedical subjects
Publications and source records attributed to J R Garrett.
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Submandibular gland homogenates from age-matched male and female rats were focused on pH 4-6.5 isoelectric focusing gels. At least 14 bands (A to N) showing amidolytic activity against three different oligopeptide-7-amino-4-trifluoromethylcoumarin (AFC) derivatives, immobilized on cellulose diacetate overlay membranes, were observed under ultraviolet light on focused gels. All the proteinases were optimally eluted, from excised gel pieces, into 20 mM ammonium bicarbonate buffer, pH 9.8, containing 0.1% Triton X-100; the protein content of the band eluants was measured after selective precipitation of protein from interfering substances. Differences were observed in the substrate specificities of the proteinases such that enzymes in bands A and K showed increased reactivity to ZVKKR-AFC, and those in bands H and J to ZR-AFC and DVLR-AFC, respectively. While the ability of aprotinin to inhibit the enzymes showed an inverse relationship to their isoelectric points, soybean trypsin inhibitor was most potent against bands B to I and least effective against bands K to N. Extracts from the glands of female rats contained the same mixture of proteinases as their male counterparts but the concentrations of all the proteinases apart from band K were reduced by approximately 25-50%. Where suitable substrates are available these methods have general applications for the rapid identification and characterization of other enzymes fractionated on IEF gels.
Microliths in Araldite-embedded pieces of submandibular and sublingual glands of cat were stained in semithin sections by Methylene Blue and Azure II followed by Basic Fuchsin, and were examined in ultrathin sections by electron-microscopical X-ray microanalysis. Calcium and phosphorus were detected in substantial aggregates of crystals that were stained by Basic Fuchsin and appeared to be hydroxyapatite, but were not detected in granular material that was stained by Methylene Blue and Azure II and appeared to be organic. The polychromatic stain thus appears to be a useful indicator of calcified material. The majority of microliths in acini contained substantial aggregates of crystals, whereas the majority of those in ducts did not. This corresponds to the distribution of the glandular calcium, and suggests that microliths are variously enriched with calcium according to its local level.
This investigation concerns the natural history of microlith in the salivary glands of cat. Microliths were detected in more sublingual than submandibular glands and were almost absent in the parotid. They were found intraparenchymally, intraluminally and interstitially, and ultrastructurally in phagosomes of acinar, ductal and myoepithelial cells, intermixed with the cytoplasm of degenerate acinar cells, and in intraparenchymal macrophages and a multinuclear giant cell. They appear to form in healthy acinar cells during autophagocytosis, and possibly to be discharged luminally, laterally or basally, and to form in the debris of degenerate cells intraparenchymally and intraluminally. They appear to be removed by expulsion in the saliva, scavenging macrophages, and possible eventual degradation in the parenchymal phagosomes. The greater occurrence of microliths in the sublingual gland may relate to a low level of secretory activity, and the near absence of microliths in the parotid to a low level of calcium. The feline salivary glands were found to be an outstanding model for the investigation of microlithiasis.
Submandibular gland responses to sympathetic and parasympathetic nerve stimulation were studied in streptozotocin-diabetic rats. Morphologically, the acinar cells in control glands were relatively uniform in size and contained electron-lucent granules. The granular ducts were distinguished by the presence of electron-dense granules. With the exception of intracellular lipid droplets and the presence of a few autophagosomes in diabetic glands, no consistent differences in acinar cell structure were observed. In contrast, the diameter of the granular ducts and the granule content of their cells were less in diabetic glands. At 3 weeks sympathetic flow rate, salivary protein concentration, and total protein output were unaffected by diabetes. Sympathetic flow rate was greater at 3 months, and the concentration of protein in the saliva was lower. In 6-month diabetic rats flow rate remained increased, but protein concentration and total protein output were reduced. The decrease in salivary protein concentration at 3 and 6 months was accompanied by a reduction in secretory granule release from acinar and granular duct cells. No consistent differences in flow rate, protein concentration, protein output, or secretory granule release were observed following parasympathetic stimulation. We conclude that the effects of diabetes on nerve-stimulated flow rate and protein release depend on the duration of diabetes and the type of stimulation, and are independent of one another.
Although feline salivary glands have been used in investigations on secretion and microlithiasis and both processes involved calcium, nothing is known about its distribution in these glands. Therefore we have demonstrated the presence of calcium by a histochemical technique using glyoxal bis(2-hydroxyanil) and a biochemical technique using dry ashing. The histochemical technique stained serous acinar cells weakly and rarely found mucous acinar cells strongly in the parotid gland, mucous acinar cells moderately to strongly and serous acinar cells weakly in the sublingual gland, and central and demilunar acinar cells moderately to strongly in the submandibular gland. The biochemical technique revealed less calcium in the parotid than in the submandibular and sublingual glands. Both techniques revealed a decrease of calcium in submandibular and sublingual glands following parasympathetic stimulation. The histochemical distribution of calcium, which corresponds to that of acinar secretory glycoprotein, and the loss of calcium following parasympathetic stimulation, which causes release of secretory granules, indicate the presence of calcium in secretory granules. The concentration of calcium in the different types of acinar cell corresponds to the acidity of the secretory glycoprotein and suggests that calcium is present as a cationic shield to allow the condensation of polyionic glycoprotein in secretory granules.
Obstruction and increased secretory activity are considered to be important aetiological factors of salivary microlithiasis, which may itself be an aetiological factor of sialadenitis. However, there is a lack of substantial evidence for the importance of obstruction, and investigations on increased secretory activity used pathological doses of pharmacological agents. Therefore further investigation of these factors is essential. Feline parotid, submandibular and sublingual salivary glands, in which microliths occur normally as in man, were examined after ductal ligation to produce obstruction, electrical stimulation of the glandular nerves to produce increased secretory activity, or both. Microliths were detected in: 0 out of 38 untreated, 1 out of 55 ligated, 2 out of 17 stimulated and 2 out of 17 stimulated ligated submandibular glands; 6 out of 29 untreated, 7 out of 46 ligated, 3 out of 12 stimulated and 4 out of 14 stimulated ligated sublingual glands; and no parotid glands. The chi 2 test confirmed that the experimental procedures did not produce an increased occurrence of microliths. Microliths were detected in parenchymal cells, intercellularly in atrophic parenchyma, intraluminally, interstitially and in macrophages. The present investigation indicates that obstruction and increased secretory activity are not important aetiological factors of salivary microlithiasis.
Time scales for the reformation of the secretory granules in granular tubules and their constituent proteinases were assessed after inducing a massive degranulation by intraperitoneal injection of cyclocytidine in conscious animals. The minimum working dose of cyclocytidine to produce the maximum degranulation and depletion of proteinase activity, at 3 h after injection, was 75 mg/kg. Histologically, although most granular tubule cells then appeared to be extensively degranulated, isolated individual cells showing little or no degranulation always persisted. Acinar cells also showed some depletion of secretory material. At 15 h after injecting cyclocytidine the formation of new granules had begun in the granular tubule cells, but it was not extensive or uniform in adjacent cells; however, the acinar cells already appeared to be regranulated. The pattern of granule reformation in granular tubule cells progressed gradually, so that 7-10 days after cyclocytidine-induced degranulation the cells were mostly packed with granules and showed similar appearances to those of normal resting control glands. Individual proteinases in extracts of the glands were assayed specifically using fluorogenic oligopeptide amidase substrates, with and without appropriate inhibitors. This revealed a 95% reduction in total proteinase activity 3 h after cyclocytidine (75 mg/kg). In the same extracts, acinar peroxidase was reduced by 28%. Peroxidase levels recovered to control values within 15 h after cyclocytidine but recovery of proteinases progressed more gradually and did not occur uniformly for the different constituent proteinases. Tissue kallikrein (rK1) showed the most rapid recovery and had reached levels approaching normal within 3 days.(ABSTRACT TRUNCATED AT 250 WORDS)
Parasympathectomy is followed by a greatly increased occurrence of microliths in the feline submandibular gland, which appears to be because of secretory inactivity. The sympathetic nerves are also important in secretory processes, and so feline submandibular, sublingual and parotid glands subjected to postganglionic sympathectomy for periods from 1 day to 1 yr have now been investigated. Microliths were detected in two out of 28 sympathectomized submandibular glands and four out of 27 untreated glands, and in 10 out of 22 sympathectomized sublingual glands and seven out of 19 untreated glands. There were no significant differences between the occurrence of microliths in sympathectomized and untreated glands. Microliths were not detected in any of 29 sympathectomized and 30 untreated parotid glands. The appearance of the sympathectomized glands was similar to that of the untreated glands. The failure of sympathectomy to affect the occurrence of microliths or the appearance of the glands is possibly because of parasympathetic nerve impulses, which produce continuing secretory activity, and also the spontaneous secretion of the sublingual gland. The results support the concept that secretory inactivity is an aetiological factor of microlithiasis.
Although microliths occur in normal human salivary glands and may be an aetiological factor of sialadenitis, little is known of their natural history. In an attempt to remedy this, we investigated a large archival collection of normal and experimental feline parotid, submandibular and sublingual salivary glands. In submandibular and sublingual glands, microliths were detected ultrastructurally in: all types of acinar secretory cells; myoepithelial cells; ductal cells; lumina; intercellular spaces; basement membrane; stroma; macrophages; multinuclear giant cells; and neutrophils. Microliths were not detected ultrastructurally in parotid glands. Microliths appear to form in acinar cells during autophagy and in stagnant secretory material in lumina. Microliths appear to be removed by secretion in the saliva, discharge from cells laterally and basally, and engulfment by macrophages. There appears to be a turnover of microliths, which possibly is upset by secretory inactivity with a resulting accumulation that leads to localized obstruction and sialadenitis.
1. The extent to which parasympathetic vasodilator responses, in the submandibular gland of the cat, depend upon release of nitric oxide related (NO chi) or endothelium-derived relaxing factor (EDRF) within the gland has been investigated in anesthetized cats given N omega-nitro-L-arginine methyl ester (L-NAME) which specifically blocks the synthesis of EDRF from arginine. 2. Close intra-arterial infusions of L-NAME (> or = 100 mg kg-1) produced a steady and significant rise in mean aortic pressure together with a steady increase in basal submandibular vascular resistance over the next 20-30 min, which persisted thereafter. 3. In cats pretreated with propranolol, to block beta-adrenoceptor-mediated vasodilatation, salivation and vasodilatation in response to stimulation of the chorda-lingual nerve were reduced but not abolished by L-NAME (> or = 100 mg kg-1, I.A.). Subsequent administration of atropine (> or = 1 mg kg-1 I.V.) completely suppressed the secretory response and virtually eliminated the vascular response. 4. In cats pretreated with atropine (> or = 1.0 mg kg-1 I.V.) administration of L-NAME (> or = 100 mg kg-1 I.A.) effectively suppressed the vasodilator response to chorda-lingual stimulation at 2 Hz continuously, or at 20 Hz for 1 s at 10 s intervals. 5. Administration of L-NAME (> or = 100 mg kg-1 I.A.) effectively suppressed the submandibular vasodilator response to infusions of VIP (10 and 20 ng I.A.) and significantly reduced, but did not abolish that to acetylcholine (100 ng min-1 I.A.). 6. These results provide further support for the view that both acetylcholine and vasoactive intestinal polypeptide-like immunoreactivity (VIP) are released from the postganglionic parasympathetic nerve terminals and produce effects on the blood vessels in submandibular glands of the cat. They also provide evidence for a direct vascular action of acetylcholine, independent of NO chi, but VIP appears to act indirectly via NO chi formation.
The levels of tissue kallikrein, tonin, and other kallikrein-like proteinases were determined in extracts of rat submandibular glands 3 mo after the induction of diabetes with STZ (65 mg/kg i.v.). Total kallikrein-like proteinase activity was assayed catalytically with the fluorogenic substrate DVLR-AFC. Tissue kallikrein was assayed by using the same substrate in the presence of SBTI. Activity of other kallikrein-like proteinases was defined as the difference between the total kallikrein-like activity and that of tissue kallikrein. Tonin was assayed by using the substrate ZVKKR-AFC in the presence of aprotinin. Results were compared with age-matched controls and with diabetic rats that had received daily insulin injections for the last week of the test period. The results showed that all activities were significantly reduced in diabetic glands compared with controls. Insulin treatment restored concentrations of tissue kallikrein activity, whereas the activities of tonin and other kallikrein-like proteinases were unchanged. RIA supported these findings. The results indicate that in rat submandibular glands, insulin affects the synthesis of kallikrein-like proteinases in different ways and, allowing for the slowness of the processes involved, insulin may exert a direct influence on the regulation of tissue kallikrein synthesis but only have indirect effects on the synthesis of tonin and the closely related kallikrein-like proteinases.
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Salivary glands of cat were examined from 1 to 42 days following parasympathectomy and compared with contralateral normal control glands. Microliths were detected by light microscopy in none of 11 parotid, 31 out of 41 submandibular and four out of 22 sublingual glands following parasympathectomy, and one out of 19 parotid, five out of 28 submandibular and four out of 15 sublingual normal control glands. The greatly increased occurrence of microliths in the submandibular gland was statistically significant. Microliths in the parasympathectomized submandibular glands were detected by light microscopy most often in ductal lumina, followed by acinar lumina, ductal parenchyma, interstitial stroma, and acinar parenchyma. They were detected by electron microscopy also in the basement membrane overlying protruding processes of myoepithelial cells and in intraparenchymal macrophages. Intracellular microliths were in phagosomes. In the parasympathectomized submandibular glands, parenchymal atrophy was seen and particularly involved the striated ducts; secretory material and cellular debris were seen in lumina; and macrophages and neutrophils were more apparent than normally. The great increase of microlithiasis in the submandibular gland appears to be the result of secretory inactivity, and microliths appear to form in stagnant secretory material and cellular debris in lumina and in phagosomes of parenchymal cells.
At least fourteen separate bands of proteinase activity, labelled A-N, were identified by an enzyme overlay membrane technique, using oligopeptide-7-amino-4-trifluoromethylcoumarin (AFC) substrates in rat submandibular gland extracts fractionated on pH 4-6.5 isoelectric focusing gels. The proteinases were eluted into an ammonium bicarbonate buffer pH 9.8 containing 0.1% Triton X-100 and the relative contribution of each band to total activity evaluated using D-Val-Leu-Arg-AFC (DVLR-AFC) and Z-Val-Lys-Lys-Arg-AFC (ZVKKR-AFC) as substrates. Immunoblotting of band eluants run on sodium dodecyl sulphate gels with antibodies showed that band A was identical with tonin and bands K-N contained tissue kallikrein. Tonin was found to hydrolyse ZVKKR-AFC but not DVLR-AFC. Estimates of the Km values of tissue kallikrein for DVLR-AFC and tonin for ZVKKR-AFC were found to be similar (approx. 20 microM) yet the former enzyme hydrolysed its substrate five times faster. Tonin was inhibited by soybean trypsin inhibitor (SBTI) but not by aprotinin. Tissue kallikrein, on the other hand, was inhibited by aprotinin but was considerably more resistant to inhibition by SBTI. In tissue extracts 95% of the ZVKKR-AFC lytic activity in the presence of 1 microM aprotinin is due to tonin and a similar percentage of the DVLR-AFC hydrolysing activity in the presence of 10 microM SBTI is due to tissue kallikrein. These findings were used for the specific measurement of these two proteinases in submandibular gland extracts and in saliva without prior purification. Using these inhibitor based assays we revealed qualitative differences in the composition of proteinases secreted into saliva during parasympathetic versus sympathetic stimulation of the submandibular gland. The distribution of proteinases in sympathetic saliva is very similar to that found in submandibular extracts but on parasympathetic stimulation, although much less proteinase is released, the contributions of the more acidic isomers of tissue kallikrein are increased and that of tonin and other proteinases dramatically decreased. The data suggest that parasympathetic and sympathetic nerves induce proteinase secretion via different pathways.
Sections from normal resting submandibular glands were incubated in a p-nitrophenylphosphate substrate mixture to demonstrate Na,K-ATPase activity by light and electron microscopy. Dense deposits of reaction product occurred within the basal and lateral infoldings of striated duct cells. A similar, less heavy deposition occurred in demilune cells. Only scattered, infrequent pockets of staining occurred in association with the basal plasma membrane of central acinar cells, supporting the belief that these cells do not contribute greatly to the secretion of fluid. No reaction was detected on luminal membranes. Glands tested after sympathetic or parasympathetic nerve stimulation showed no obvious differences from resting glands. These results contrast with the previously reported paucity of immunostaining in resting demilunes with an antibody to Na,K-ATPase and the large increase in the immunostaining by these cells after parasympathetic stimulation. It is inferred that there must be an inaccessibility to immunobinding sites of Na,K-ATPase in resting glands, and that the increase in binding which occurs after parasympathetic stimulation represents a conformational change that did not affect its enzyme catalytic reaction with this substrate.
The lectin-horseradish peroxidase conjugate of GSA I-B4 (Griffonia simplicifolia isolectin) has some binding affinity with capillary walls in sections of the major salivary glands from normal rats. After inducing diabetes with streptozotocin a conspicuous increase in the staining intensity with GSA I-B4 was already evident in parotid capillaries at 3 weeks and this increase persisted at 3 and 6 months. In submandibular capillaries, however, an increased uptake of GSA I-B4 was evident only at 6 months after inducing diabetes. The reasons for these different time scales in the two glands are not known but the increased uptake of GSA I-B4, which is due to an increase in carbohydrate-containing sites with available terminal alpha-D-galactose, is considered to reflect a pathological change.
Two separate sets of experiments were performed on female Wistar rats made diabetic with streptozotocin: (1) a time-course study where groups of three animals were removed at weekly intervals, up to 4 weeks after induction of diabetes, with an age-matched group of control (normal) animals kept for 4 weeks; (2) six further animals were made diabetic and kept for 7 weeks; three of these were given insulin in the final week. At the required time the animals were anaesthetized and the salivary glands removed and preserved by fixation or freezing. The frozen tissues were later homogenized and the protein and lipid content analysed. Histologically, intracellular lipid droplets had accumulated in the majority of the diabetic salivary glands. In the time-course experiment, the visible amount of intracellular lipid reached a maximum after 2 weeks and then decreased, with a concomitant disappearance of interstitial lipid. The increased lipid content was not attributable to any one class. The fatty acid profiles of the glands showed an increase in the percentages of C18:0 (stearic acid) and C18:2w6 (linoleic acid) and a decrease in the percentages of C18:1w9 (oleic acid) and C20:4w6 (arachidonic acid). After 1 week of insulin treatment the lipid content and the fatty acid profiles returned to normal. Thus the effect of insulin on salivary gland lipid metabolism is rapid both in its occurrence and reversibility. The effects seen in the diabetic rats are considered to be due to a lack of insulin and not to the presence of streptozotocin.