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G B Proctor

Publications and source records attributed to G B Proctor.

At least 73 records · Page 4Linked to original sources
PubMed1993

Development and application of a method for the detection, elution, and characterization of rat submandibular proteinases separated on isoelectric focusing gels reveals male/female differences.

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.

Amino Acid Sequence↗
PubMed1993

In vivo secretory responses of submandibular glands in streptozotocin-diabetic rats to sympathetic and parasympathetic nerve stimulation.

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.

Animals↗
PubMed1993

Asynchronous reformation of individual kallikrein-related secretory proteinases in rat submandibular glands following degranulation by cyclocytidine.

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)

Ancitabine↗
PubMed1993

Protein synthesis in the major salivary glands of the rat and the effects of re-feeding and acute ethanol injection.

Rates of protein synthesis in the major salivary glands of the rat were accurately determined in vivo using the 'flooding dose' technique and the effects of reflex stimulation through feeding or acute ethanol treatment were assessed. Rats were divided into three groups. The control group was fasted overnight, the re-fed group was also fasted then re-fed the following morning and the third group received an intraperitoneal injection of ethanol after an overnight fast. In control rats the sublingual gland had a higher rate of protein synthesis than the other glands. After a feed the rate of protein synthesis in parotid glands was approximately double that of controls whilst that of the submandibular glands had increased by 24% and the sublingual glands remained unchanged. Acute ethanol injection led to a reduced rate of protein synthesis in all glands. The higher rate of protein synthesis in sublingual glands under fasting conditions may be related to the spontaneous secretion of fluid and protein from this gland, which would require a constant replenishment of secretory protein by synthesis. Protein synthesis in the parotid gland, like secretion, appears to be closely linked with the reflex stimulation caused by mastication. The smaller increase in rate of protein synthesis seen in the submandibular gland after a re-feed does not appear to have been recorded before. It could be mediated by the increased reflex stimuli caused by mastication or by some circulating factor. The mechanism by which ethanol reduces protein synthesis in all glands is uncertain.

Analysis of Variance↗
PubMed1993

Tissue kallikrein and tonin levels in submandibular glands of STZ-induced diabetic rats and the effects of insulin.

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.

Amino Acid Sequence↗
PubMed1992

New specific assays for tonin and tissue kallikrein activities in rat submandibular glands. Assays reveal differences in the effects of sympathetic and parasympathetic stimulation on proteinases in saliva.

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.

Amino Acid Sequence↗
PubMed1992

Na,K-ATPase in resting and stimulated submandibular salivary glands in cats, studied by means of ouabain-sensitive, K(+)-dependent p-nitrophenylphosphatase activity.

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.

4-Nitrophenylphosphatase↗
PubMed1992

Changes in the lectin-binding of capillaries in rat salivary glands after streptozotocin-induced diabetes.

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.

Animals↗
PubMed1992

Lipid analysis of the major salivary glands in streptozotocin-diabetic rats and the effects of insulin treatment.

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.

Animals↗
PubMed1992

Nerve-induced secretion of glycoconjugates from cat submandibular glands: a correlative study with lectin probes on tissues and saliva.

Horseradish peroxidase-conjugated lectins were used on tissue sections to localize the main secretory glycoproteins in cat submandibular glands and on Western blots to evaluate their movement into saliva with selective nerve stimulation. Central acinar cells bound lectins from Arachis hypogaea (PNA) specific for the terminal disaccharide Gal beta 1, 3GalNac, Griffonia simplifolia (GSA I-B4) specific for terminal alpha Gal, and Lotus tetragonolobus (LTA) specific for fucose. Lectins from Limax flavus (LFA) specific for sialic acid and Dolichos biflorus (DBA) specific for terminal alpha GalNac reacted preferentially with demilunar cells, whereas apical granules in striated ducts were recognized principally by LTA. Parasympathetic stimulation promoted the release of lectin-reactive glycoconjugates from both central and demilunar cells. In contrast, sympathetic stimulation caused almost complete release of LTA-reactive granules in striated ducts and only moderate secretion from demilunar cells. Lectin blots of stimulated saliva discriminated many of the constituent bands, providing information about their glycosylation. Several bands were common to both parasympathetic and sympathetic saliva, and many bands gave wider ranges of lectin binding than anticipated from the histochemistry. The major component in parasympathetic saliva was a glycoconjugate of less than 12 KD which reacted with every lectin tested. Lectin blots of sympathetic saliva showed a prominent diffuse LTA-reactive band around 33 KD, which was attributed to tissue kallikrein. The identity and cellular origin of most bands in stimulated submandibular saliva are still unclear but the technique shows considerable promise for improving the recognition and characterization of individual glycoconjugates.

Animals↗
PubMed1991

Secretory responses in granular ducts and acini of submandibular glands in vivo to parasympathetic or sympathetic nerve stimulation in rats.

The roles of sympathetic and parasympathetic nerves in the secretion of saliva from submandibular glands of rats have been tested by electrical stimulation of either nerve for 1 h unilaterally in separate animals. The flows of saliva thereby induced and their protein content were monitored. Structural changes in each gland were assessed by light- and electron microscopy and compared with the unstimulated contralateral control gland, and the extent of the changes was determined morphometrically. Sympathetic nerve stimulation induced a relatively low flow of saliva that was rich in protein and was accompanied by extensive degranulation from both acinar and granular duct cells. In contrast parasympathetic nerve stimulation induced a considerable flow of saliva that had a low protein content and no detectable degranulation occurred from the secretory cells. It is possible, therefore, that some protein in parasympathetic saliva may have arisen from a non-granular pathway.

Animals↗
PubMed1991

Proteinase activities in bovine atrium and the possible role of mast cell tryptase in the processing of atrial natriuretic factor (ANF).

1. Using low salt, Triton X-100 and high salt extracts of bovine atria, two main proteinases were identified by means of fluorogenic oligopeptide substrates. 2. An acidic proteinase, extracted in low salt and Triton X-100 was identified as cathepsin B, but it caused little hydrolysis of the Z-Gly-Pro-Arg- containing substrate that resembles the cleavage site for activation of pro-ANF. 3. An alkaline proteinase was extracted only with high salt and had characteristics of the serine proteinase tryptase. It cleaved Z-Gly-Pro-Arg- containing substrates more efficiently than others tested and was localized in and around mast cells histochemically. Previously, Imada et al., 1988 (J. biol. Chem. 263, 9515-9519) found an identical enzyme would cleave ANF from pro-ANF. 4. These results suggest therefore that mast cell tryptase may be involved in the activation of ANF from pro-ANF.

Amino Acid Sequence↗
PubMed1991

Serum amylase of non-parotid and non-pancreatic origin increases on feeding in rats and may originate from the liver.

1. In order to evaluate possible non-salivary contributions to the content of salivary-type amylase in the circulation, parotid glands--the only salivary source of amylase in rats--have been totally removed and the effects on serum amylase have been assessed, after fasting and at different times after feeding. 2. Despite the parotidectomy the resting level of salivary-type amylase remained the same and an increase was still found to occur on feeding. 3. Isoelectric focusing has identified additional isoforms of amylase in serum and liver distinct from those occurring in parotid saliva. 4. The liver therefore may be contributing to the fasting levels of serum amylase and to the increases that occur on feeding, in rats.

Amylases↗
PubMed1991

Parotid protein secretion from the rabbit during feeding.

Rabbits were trained to accept a standardized feeding regime. Under anaesthesia both parotid ducts were cannulated in a retrograde direction and saliva was subsequently collected in feeding sessions involving pellets and carrots before and after the administration of propranolol. Salivary total protein and amylase concentrations were assayed and protein analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Propranolol produced a reduction in protein secretion but not fluid secretion, indicating that protein secretion is partly under beta-adrenergic control. A transient increase in protein secretion was seen 8 h after the administration of propranolol and suggested the existence of different neural mechanisms involved in protein secretion compared to synthesis. Protein output (with high fluid secretion) during feeding on pellets was higher than on carrots (with high protein concentration) and suggested a significant role of the parasympathetic nervous system. The pattern of protein secretion as seen by SDS-PAGE was similar in individual rabbits and remained largely unchanged with the different foods and in the presence of propranolol.

Amylases↗
PubMed1990

Morphological effects of sympathetic nerve stimulation on rat parotid glands 3-4 weeks after the induction of streptozotocin diabetes.

Male Wistar rats were fasted overnight and anaesthetized 3-4 weeks after the induction of streptozotocin diabetes. The right parotid ducts were cannulated, and parotid salivary flow was induced by stimulating the sympathetic trunk in bursts (50 Hz, 1:10). Stimulated and unstimulated glands were weighed, fixed for morphometric analysis, and assayed for total protein and amylase. Gland weights did not differ between diabetic and control rats. Nevertheless, total protein (6.17 +/- 5.40 mg) and amylase (1.32 +/- 0.49 mg) output from the gland were reduced in diabetic animals compared with controls (13.73 +/- 2.81 and 3.41 +/- 0.51 mg, respectively). Morphometric analysis of unstimulated glands showed no differences in either acinar cell profile area, or in the number of secretory granule profiles/cell in unstimulated glands. Upon sympathetic stimulation, however, the depletion of granule profiles was significantly less in diabetic than in control rats. Finally, the mean diameter of secretory granules was significantly less in diabetic (0.89 +/- 0.05 microM) than in control (1.04 +/- 0.06 microM) glands.

Amylases↗
PubMed1990

Separation of the proteins of rat parotid saliva by hydrophobic-interaction chromatography.

Parasympathetically-evoked rat parotid salivary proteins were resolved into 11 peaks by hydrophobic-interaction chromatography. SDS-PAGE indicated that most peaks contained a single major component and that only one major protein was not chromatographed. The findings indicate that hydrophobic-interaction chromatography can be used in the quantification of changes in individual salivary proteins under different experimental conditions, and in the purification of individual salivary proteins.

Animals↗
PubMed1990

Effects of parasympathectomy on protein composition of sympathetically evoked parotid saliva in rats.

1. Male Wistar rats were given unilateral postganglionic parasympathectomies by sectioning the auriculo-temporal nerve. 2. Analyses of the protein compositions of sympathetically induced saliva from both glands 1 week later revealed changes in the proportions of different secretory proteins, in particular amylase and basic proline-rich proteins were decreased. 3. These results suggest that parasympathetic impulses are required for the normal synthesis of amylase by parotid parenchyma in rats. Basic proline-rich proteins, known to require a sympathetic drive for normal synthesis, appear to require a parasympathetic input as well.

Animals↗