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Biomedical subjects

G B Proctor

Publications and source records attributed to G B Proctor.

At least 55 records · Page 3Linked to original sources
PubMed1996

Sequential secretion of rat submandibular kallikrein and peroxidase during intermittent sympathetic stimulation.

Secretion of peroxidase from acinar cells, true tissue kallikrein (rK1) from granular tubules and total protein have been assessed in sequential samples of rat submandibular saliva formed during intermittent periods of sympathetic stimulation in bursts of 50 Hz 1 s every 10 s at 5 V, for 1 or 2 min each with 2 min intervals between periods. This protocol was repeated twice after 1 h rest pauses. The salivary flow remained greater throughout than when using ongoing burst stimulation. Protein secretion was considerable in the first 2 samples (1 min followed by 2 min) reaching the remarkable concentration of 285 +/- 14.4 mg/ml, then it gradually decreased with little recovery after 1 h pauses. rK1 outputs followed a similar pattern. Peroxidase, however, showed no greater output in initial samples and continued steadily in similar amounts throughout. When intermittent stimulations were used for 1 sequence only (total stimulation time = 9 min) the glands showed 84 +/- 2.6% depletion of rK1 compared to the control glands. A correspondingly large depletion of granules occurred from the granular tubules on the stimulated side, which was greater than with ongoing burst stimulation for 1 h (Garrett et al., 1991). Thus, secretion of rK1 from the granules in granular tubules occurs most efficiently with short sharp bursts of high frequency sympathetic stimulation but soon diminishes. In contrast, peroxidase secretion from acinar cells continues steadily and more modestly for long periods of time undiminished.

Animals↗
PubMed1996

Glycoproteins in human parotid saliva assessed by lectin probes after resolution by sodium dodecyl sulphate-polyacrylamide gel electrophoresis.

Human parotid salivary glycoproteins separated by gradient sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto nitrocellulose have been investigated using a battery of biotinylated lectin probes of characterized sugar specificity. Lectin binding, detected on blots using avidin-biotin complex (ABC) and a chemiluminescence generating substrate, was recorded on photographic film and compared with the original fluorescein isothiocyanate (FITC) stained blots or with Coomassie Brilliant Blue R-250-stained gels run in parallel. A number of glycoprotein bands which were undetected by protein stains or the periodic acid Schiff reaction were revealed by lectins. Binding by lectins from Concanavalia ensiformis, Lens culinaris, Limax flavus, Phaseolus vulgaris, Ricinus communis, Triticum vulgaris, Lotus tetragonobulus and Ulex europaeus indicated that sialylated and fucosylated triantennary and bisected, N-linked complex sugar chains were present on many glycoproteins in addition to the major glycosylated proline-rich glycoprotein (GI). Binding with lectins from Arachis hypogaea and Dolichos biflorus indicated that the O-linked sugar chains were confined to the alpha-heavy chain of Ig A. Comparison of lectin binding in samples from five healthy individuals revealed differences in a number of glycoproteins in addition to the previously characterized G1 and CON 1/CON 2 polymorphisms and demonstrated that the H blood group antigen was expressed mainly on G1 in parotid saliva. This study will be used as a basis upon which to study salivary glycoproteins in diseases affecting parotid glands.

Adult↗
PubMed1996

Differences in the glycosylation of rat submandibular kallikreins.

The glycosylations of five different rat submandibular kallikreins, rK1, rK2, rK7, rK9 and rK10, vacuum-blotted onto nitrocellulose membranes, have been studied by means of labelled lectins using enhanced chemiluminescence detection. The results demonstrated the individual submandibular kallikreins are not heavily glycosylated in rats, but consistently show different patterns of glycosylation. Following digestion of slot-blotted enzymes with peptide-N-glycosidase F (PNGase): binding by lectin from Lens culinaris (alpha Man-directed) was abolished, whilst that of lectin from Maclura pomifera (Gal beta 1,3GalNAc-directed) persisted (but could be abolished by periodate oxidation and endo-alpha-N-acetylgalactosaminidase digestion), revealing that there are O- as well as N-linked sugar chains on the kallikreins; a novel observation for this family of enzymes. The presence of GalNAc in addition to GlcNAc, Fuc, Gal, and Man, in sugar chains of rK1 was confirmed by high pH anion exchange chromatography following acid hydrolysis. Different intensities of binding by lectin from Limax flavus (NeuNAc-directed) suggest that sialylation of individual kallikreins differs, whilst sialidase and PNGase digestions suggest that sialic acid is the terminal residue of some N-linked but not O-linked structures.

Amino Acid Sequence↗
PubMed1996

Apical secretion of rat submandibular tissue kallikrein continues in the absence of external stimulation: evidence for a constitutive secretory pathway.

Sequential samples of saliva evoked by low frequency parasympathetic nerve stimulation were collected from cannulated submandibular ducts of anaesthetized rats following periods of varying length without stimulation. After such rest periods the first samples of saliva were found to contain much higher levels of tissue kallikrein (rK1) activity and protein than the ensuing samples from the same stimulation period and the latter contained levels similar to those found previously in parasympathetically-evoked saliva. rK1 activity in first samples increased with the length of the preceding interval, indicating that a continuous secretion of rK1 occurs from ductal cells into glandular lumina and accumulates there in the absence of stimulation or fluid secretion. The protein secretory pattern following rest pauses was unaffected by alpha-adrenergic receptor blockade and a high percentage of the proteinase activity was resistant to soya bean trypsin inhibitor, showing that the secretion was not caused by exocytosis of storage granules from ductal cells and therefore was likely to be a result of a constitutive secretion of newly synthesized enzyme. The pattern of continuous secretion into lumina detected for total protein in the absence of stimulation, suggests that other secretory proteins may also be secreted similarly but at different rates.

Animals↗
PubMed1996

Glycosylation of secretory proteins in salivary glands and saliva studied by lectin-probes.

Lectin-probes were used histochemically to study glycosylation patterns in prepackaged secretory material in cat and rat submandibular glands and to assess the secretory changes induced by nerve stimulations. The same probes were also used for correlative biochemical assessments of the constituents in glandular extracts and in saliva from the same experiments, after electrophoretic separations and immobilization on nitrocellulose membranes. These studies provided a broader understanding than the use of either procedure alone. Lectin binding patterns in saliva were more complex than anticipated from the histochemistry and this probably relates to the presence of different constituents in secretory granules and a greater availability of binding sites biochemically. In cats, parasympathetic stimulation caused depletion of lectin staining in acini and to some extent in demilunes, whereas sympathetic stimulation caused depletion of lectin binding in striated ducts and reduction in demilunar size. Biochemically after SDS-PAGE of saliva similar secretory effects were observed but each nerve also evoked some secretion from the cells not showing histochemical change. In rats, sympathetic stimulation caused depletion of lectin positive granules from both acini and granular tubules. After SDS-PAGE of the saliva one zone of lectin binding 94-150 kD (Mr) was identified as acinar mucin. Granular tubule proteins consisting mainly of kallikreins occurred in a zone 25-35 kD (Mr) and lectin bindings suggested that different kallikreins may be differently glycosylated. This unexpected possibility was confirmed on slot-blot preparations of separated kallikreins, which also revealed the novel finding that some kallikreins are O- as well as N-glycosylated.

Animals↗
PubMed1996

Apical secretion of rat submandibular tissue kallikrein continues in the absence of external stimulation: evidence for a constitutive secretory pathway.

Sequential samples of saliva evoked by low frequency parasympathetic nerve stimulation were collected from cannulated submandibular ducts of anaesthetized rats following periods of varying length without stimulation. After such rest periods the first samples of saliva were found to contain much higher levels of tissue kallikrein (rK1) activity and protein than the ensuing samples from the same stimulation period and the latter contained levels similar to those found previously in parasympathetically evoked saliva rK1 activity in first samples increased with the length of the preceding interval, indicating that a continuous secretion of rK1 occurs from ductal cells into glandular lumina and accumulates there in the absence of stimulation or fluid secretion. The protein secretory pattern following rest pauses was unaffected by alpha-adrenergic receptor blockade and a high percentage of the proteinase activity was resistant to soya bean trypsin inhibitor, showing that the secretion was not caused by exocytosis of storage granules from ductal cells and therefore was likely to be a result of a constitutive secretion of newly synthesized enzyme. The pattern of continuous secretion into lumina detected for total protein in the absence of stimulation, suggests that other secretory proteins may also be secreted similarly but at different rates.

Animals↗
PubMed1996

Peroxidase and kallikrein in atropine-resistant secretion of submandibular saliva on parasympathetic nerve stimulation in anaesthetized rats.

Flow of submandibular saliva and the constituents secreted during chorda-lingual nerve stimulation at 40 Hz (3 x 10 min) were studied after alpha- and beta-adrenergic blockade, and in the absence (control) or presence of cholinergic blockade in anaesthetized rats. Peroxidase and true tissue kallikrein (rK1) were assessed to gain insight into the effects of the non-adrenergic, non-cholinergic (NANC) transmitters on protein secretion by acini and granular tubules, respectively. From control glands there was an overall mean flow of 181 +/- 15 microliters g-1 min-1, with no significant differences between the three periods. Secretion from atropinized glands was approximately 20% of that from control glands in the first 10 min, decreasing progressively to approximately 6% in the final period. Protein outputs from control glands showed no significant differences for the three periods (0.23 +/- 0.05 mg g-1 min-1). Protein outputs from atropinized glands were similar to controls in the first 10 min and then decreased significantly. Peroxidase output from control glands increased progressively (from 31 +/- 7 to 243 +/- 68 pmol dichlorofluorescein (DCF) g-1 min-1) but in saliva from atropinized glands the overall mean output was only 4.5 +/- 0.8 pmol DCF min-1 ml-1, with a progressive decrease between samples. Outputs of rK1 from control glands were similar for all samples (20.6 +/- 4.0 nmol AFC g-1 min-1), but there was a significantly smaller and decreasing output of rK1 from atropinized glands. In conclusion, NANC transmitters released from parasympathetic nerves during stimulation at high frequency appear to have little influence on the secretion of protein from rat submandibular acini and granular tubule cells when acting in isolation. This contrasts with their effects on amylase secretion from rat parotid glands under similar circumstances.

Adrenergic alpha-Antagonists↗
PubMed1996

Use of lectin-probes for correlative histochemical and biochemical assessments of the glycosylation patterns of secretory proteins, including kallikreins, in salivary glands and saliva.

Labelled lectins were used as probes to study the glycosylation and secretion of submandibular glycoproteins not only in sections of fixed glands but also in glandular extracts and in nerve-induced saliva, after electrophoretic separations and immobilization in nitrocellulose membranes. In cats the glycoproteins in sympathetic saliva differed considerably from those in parasympathetic saliva. In sympathetic saliva they were found to originate mainly from striated ducts, to some extent from demilunar cells and to a small extent from acinar cells, whereas in parasympathetic saliva they arose mainly from acinar cells and demilunes and only to a small extent from striated ducts. In rat submandibular glands sympathetic stimulation caused extensive depletion of lectin stainable granules from granular tubules. Corresponding strong binding occurred with the same lectins to constituents in saliva that ran between 25 and 35 kD on SDS gel electrophoresis and were shown to contain tissue kallikreins. Their binding patterns suggested that individual kallikreins from the same gland may be glycosylated in different ways. This possibility was tested on five different kallikreins after separation from submandibular extracts by isoelectric focussing. Lectin bindings on slot blot preparations of these kallikreins were tested before and after N-glycosidase F, sialidase or endo-alpha-N-acetylgalactosaminidase digestions. Results showed that, despite their close genetic and structural similarities, the kallikreins are in fact differently sialylated and fucosylated and the novel finding that some contain O-glycosidically linked side chains as well as the anticipated N-glycosidically linked side chains was revealed. Thus, correlative histochemical and biochemical assessments of bindings with lectin probes has provided important new information about differences in the glycosylation patterns of individual glycoproteins stored within the same secretory granules.

Animals↗
PubMed1995

Differential secretion of proteins by rat submandibular acini and granular ducts on graded autonomic nerve stimulations.

1. The influence of graded parasympathetic and sympathetic nerve stimulations on the secretion of protein from rat submandibular gland was studied. Peroxidase was used as a marker for the acini and rat tissue kallikrein (official nomenclature rK1) as the marker for granular ducts. Tonin (rK2) was also measured, and the ratio of rK2:rK1 was calculated as an indication of the cellular route of secretion. 2. Continuous parasympathetic nerve stimulation caused a copious flow of saliva that had a low protein content. The secretion of peroxidase (acini) showed a gradual moderate increase as the frequency increased. However, the concentrations of rK1 and rK2 (granular ducts) showed little change throughout, and the ratio of rK2:rK1 remained relatively constant. 3. Graded sympathetic stimulation was applied against a background of parasympathetic stimulation. Secretion of peroxidase was increased by the addition of 0.1 Hz continuous sympathetic stimulation. The amount increased thereafter up to 2 Hz, but showed no further increase if the stimulation was applied as bursts of 10 or 20 Hz. In comparison, the secretion of proteinase activity showed little change with superimposed continuous sympathetic stimulation, and the rK2:rK1 ratio was similar to that in saliva produced by parasympathetic stimulation alone. Sympathetic stimulation applied in bursts, however, caused a large increase in the secretion of proteinase activity, and with 20 Hz burst stimulation the rK2:rK1 ratio was indistinguishable from that of sympathetic saliva per se. There was an augmented secretion of both peroxidase and kallikrein when 20 Hz burst stimulation was combined with parasympathetic stimulation. The effects of sympathetic stimulation were abolished by alpha- and beta-adrenoceptor blockade.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Influences of secretory activities in rat submandibular glands on tissue kallikrein circulating in the blood.

Changes in serum levels of rat tissue kallikrein (rK1) in venous blood were measured, using a newly developed radioimmunoassay, before and after autonomic nerve stimulations of submandibular salivary secretion. rK1 secreted into saliva under these conditions was measured by radioimmunoassay and by enzymic activity assay, using the fluorogenic peptide substrate D-Val-Leu-Arg-7-amino-4-trifluoromethylcoumarin (AFC). Following an overnight fast, serum rK1 concentration was 30-40 ng ml-1. Unilateral electrical stimulation of the submandibular sympathetic nerve supply (at 50 Hz in bursts of 1 s every 10 s for 60 min) evoked a small flow of saliva with a very high rK1 concentration, resulting in a large output of rK1 of 2104.4 +/- 603.5 micrograms (n = 6). Such stimulation caused a large degranulation of granular duct cells and a corresponding reduction in glandular rK1 content. Unilateral electrical stimulation of the parasympathetic nerve supply (at 5 Hz continuously for 60 min) evoked a copious flow of saliva with a very low rK1 concentration, resulting in a low output of rK1 (18.1 +/- 4.9 micrograms; n = 6). Despite these large differences in salivary outputs of rK1, serum concentrations of rK1 were increased similarly following either sympathetic or parasympathetic stimulation by 48 and 46%, respectively. If the submandibular duct was briefly obstructed during sympathetic stimulation, inducing leakage and glandular oedema, then serum rK1 increased greatly (40-fold); a similar increase to that seen by others in previous studies without deliberate obstruction. Four days after bilateral submandibular-sublingual sialadenectomy serum rK1 concentration was reduced by approximately 50%. The results indicate that submandibular glands normally contribute to circulating levels of rK1 in rats, but this contribution is independent of the amounts of rK1 secreted into saliva by sympathetically induced exocytosis, and is likely to arise from basal vesicular transport. However, if glandular leakage occurs during sympathetic stimulation of submandibular secretion this then causes increases in the circulating levels of rK1 that correlate with the large amounts being secreted into saliva.

Animals↗
PubMed1994

A fluorometric assay of peroxidase activity utilizing 2',7'-dichlorofluorescein with thiocyanate: application to the study of salivary secretion.

A sensitive assay for secretory peroxidase activity has been developed utilizing the fluorogenic substrate 2',7'-dichlorofluorescin in the presence of thiocyanate. The assay has been characterized using bovine lactoperoxidase and used to determine the peroxidase activities of salivas and extracts obtained from rat submandibular glands. Comparison of the 2',7'-dichlorofluorescin-thiocyanate assay and the commonly used 2,2'-azinobis(3-ethylbenzthiazoline-6-sulphonic acid) colorimetric assay indicates that the new assay is approx. 50-fold more sensitive. This has enabled measurement of peroxidase activities present in parasympathetic saliva samples which were beyond the detection limit of the colorimetric assay. Despite great differences in the peroxidase activities and protein concentrations of parasympathetic and sympathetic salivas and tissue extracts, the activities per unit protein were very similar. Unlike most other published methods, prior dialysis of samples to remove interference by endogenous thiocyanate is not required. The assay is therefore convenient and will be particularly useful for applications in which sample volume or peroxidase activity is low.

Animals↗
PubMed1994

A fluorometric assay of peroxidase activity utilizing 2',7'-dichlorofluorescein with thiocyanate: application to the study of salivary secretion.

A sensitive assay for secretory peroxidase activity has been developed utilizing the fluorogenic substrate 2',7'-dichlorofluorescein in the presence of thiocyanate. The assay has been characterized using bovine lactoperoxidase and used to determine the peroxidase activities of salivas and extracts obtained from rat submandibular glands. Comparison of the 2',7'-dichlorofluorescein-thiocyanate assay and the commonly used 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) colorimetric assay indicates that the new assay is approx. 50-fold more sensitive. This has enabled measurement of peroxidase activities present in parasympathetic saliva samples which were beyond the detection limit of the colorimetric assay. Despite great differences in the peroxidase activities and protein concentrations of parasympathetic and sympathetic salivas and tissue extracts, the activities per unit protein were very similar. Unlike most other published methods, prior dialysis of samples to remove interference by endogenous thiocyanate is not required. The assay is therefore convenient and will be particularly useful for applications in which sample volume or peroxidase activity is low.

Animals↗
PubMed1994

Use of lectin probes on tissues and sympathetic saliva to study the glycoproteins secreted by rat submandibular glands.

We used a panel of nine lectins to detect the glycosylation patterns of rat submandibular glycoproteins. Binding of lectins was assessed on tissue sections and on Western blots of electrophoretically separated glycoproteins from glandular extracts or sympathetic saliva. Histochemical staining of tissue sections showed that two lectins (DBA and SBA) with affinity for terminal GalNAc residues were localized specifically to acinar cells. In contrast, LTA and UEA-I (alpha Fuc-directed) and LFA (NeuAc-directed) bound exclusively to granular tubule cells. PNA and MPA (beta Gal-directed), LCA (alpha Man- and alpha Glc-directed), WGA (beta GlcNAc- and NeuAc-directed), and sWGA (beta GlcNAc-directed) bound to both acinar and granular tubule cells. On electroblot preparations, LFA, PNA, WGA, DBA, and SBA reacted with high molecular weight acinar mucin components both in glandular extracts and in saliva. LTA, UEA-I, LFA, PNA, MPA, LCA, WGA, sWGA, and DBA bound to lower molecular weight bands on blots known to contain granular tubular proteinases. Lectin binding to acini and granular tubules was reduced in sections and in most bands from glandular extracts after sympathetic nerve stimulation. Our results show that (a) secretory glycoproteins from rat submandibular acinar cells are non-fucosylated and contain abundant alpha GalNAc and NeuAc and a small proportion of beta Gal in their oligosaccharide side chains, and (b) alpha Fuc, NeuAc, beta Gal, and alpha GalNAc form the major carbohydrate moieties of the secretory glycoproteins from granular tubules. The results confirm the considerable potential of lectin probes for studying glycoproteins in secretions and in their cells of origin.

Animals↗