Biomedical subjects
Publications and source records attributed to G B Proctor.
Feeding hard chow to rats for 1 h caused an increase in parotid-type amylase in the serum. The increase was largely inhibited by parasympathetic or sympathetic denervation of the parotid glands and also by beta-adrenoceptor blockade. The results suggest that movement of amylase into the blood is influenced not only by the volume of saliva being formed but also by its amylase content.
The enzyme Na+, K(+)-ATPase was localized immunohistochemically in major salivary glands of the cat before and after autonomic nerve stimulation. Immunostaining was limited to basolateral plasma membranes. Cells lining striated and excretory ducts contained abundant Na+, K(+)-ATPase and showed no changes with neural stimulation. Serous-type cells in resting glands varied in reactivity, showing weak to moderate staining intensity in the parotid gland and more uniform staining of greater intensity in the sublingual gland. In contrast, demilune cells in the resting submandibular gland showed little if any staining. Mucous-type cells were negative in all glands. Parasympathetic stimulation promoted a gradual increase in immunostaining of submandibular demilune cells, which became marked with time. Sympathetic stimulation produced no detectable changes in Na+, K(+)-ATPase immunoreactivity in any site. These results support the concept that basolateral Na+, K(+)-ATPase is essential to the formation of a near-isotonic primary saliva by serous-type cells. The mechanism whereby parasympathetic stimulation evokes a marked flow of submandibular saliva remains unexplained, but has now been shown to involve a marked increase in the immunoreactivity of Na+, K(+)-ATPase at the base of the gland's demilune cells.
Homogenates of rat submandibular glands were electrophoresed on isoelectric focussing gels and bands of proteinase activity demonstrated by means of overlay membranes impregnated with fluorogenic oligopeptide substrates. Inhibition characteristics of individual proteinase bands were tested within the gels and after excision and subsequent elution from the gels. Some bands of activity were resistant to inhibition by pre-incubation of the gel with certain inhibitors, but were subsequently found to be inhibited when tested with the same substrate after elution. These inconsistencies were influenced by the use of different substrates. The results indicate that, though some useful screening information may be obtained by applying inhibitors to the gels, this system does not permit a reliable assessment of inhibition characteristics which require subsequent elution and assay.
Stimulation of the nerves to the rat parotid gland demonstrated that increases in serum amylase levels can originate exclusively from this gland. However, whilst parasympathetic nerve stimulation caused a significant movement of parotid amylase into the blood, sympathetic nerve stimulation did not, despite the higher concentration of amylase in the saliva evoked. Experiments in which conscious animals were fed for a short period showed that parotid amylase reaches the blood during normal activity. The mechanisms underlying the movement of parotid amylase into the circulation are still unknown but there appears to be some dependence on the volume of saliva being produced.
1. Parotid salivas were collected from rats following electrical stimulation of either the sympathetic or parasympathetic nervous supplies. 2. The protein compositions of these salivas were compared using high performance ion-exchange chromatography (FPLC), SDS electrophoresis and biochemical assay. 3. Chromatography and electrophoresis indicated differences between the protein compositions of sympathetic and parasympathetic salivas. 4. Protein secretion derived solely from the exocytosis of parotid acinar cells cannot account for these differences.
1. The output and composition of proteins in nerve stimulated saliva samples were compared. 2. Protein output upon parasympathetic stimulation was higher than following sympathetic stimulation and was accompanied by an obvious degranulation of acini with the former but not the latter. These events are the converse of those in the rat parotid gland. 3. Superimposition of sympathetic upon parasympathetic stimulation caused an augmented output of salivary protein. 4. Electrophoresis of salivas revealed differences between individual cats in protein composition but not between differently stimulated salivas.
Observations were made on amylase secretion from the rat parotid gland induced by parasympathetic nerve stimulation in vivo. In spite of a low salivary amylase concentration a large total amylase output, equivalent to 36% of initial gland content, was found on prolonged activation (5 Hz, 120 min). However, no decrease in gland amylase content was found, which indicates that the output of amylase was balanced by synthesis. We suggest that prolonged, parasympathetically induced amylase secretion is dependent on activation of amylase synthesis and that the secretion is mainly of non-granular origin. Parasympathetic nerve stimulation produced an even larger amylase output from sympathectomized glands. Even under these conditions gland amylase content was unchanged and no obvious degranulation was found when the glands were examined after stimulation. This is in marked contrast to the decrease in amylase content and to the degranulation which are found after sympathetic activation.
One week after surgical sympathectomy the amylase and protein concentrations of parotid glands were increased by 250 and 51% respectively. Whilst these concentration changes had partly returned to normal after 12 weeks, major differences still remained in the protein composition of parotid glands. Anion-exchange chromatography showed that proline-rich proteins were progressively reduced and that the relative proportions of other proteins changed variably. These results indicate that normal synthesis of proline-rich proteins requires on-going stimulation of the cells via the sympathetic nerve. In the absence of sympathetic nerve activity there was initially an increased synthesis of amylase which then returned towards normal.
Diabetes was induced with streptozocin in male Wistar rats. After 4-6 mo, parotid salivary flow was induced by stimulating bipolar electrodes (3-5 V for 2 ms) on either the auriculotemporal nerve (5 Hz) or the cervical sympathetic trunk (10 Hz). During parasympathetic stimulation, flow rates were 443 +/- 287 microliters/30 min in diabetic animals compared to 657 +/- 134 microliters/30 min in controls (P less than .01). Although parotid flow rates during parasympathetic stimulation were well maintained in controls, they decreased rapidly in most diabetic rats. Parotid secretory response to sympathetic stimulation was also reduced in diabetic compared with control animals (40 +/- 16 microliters/30 min and 82 +/- 50 microliters/30 min, respectively, P less than .01). In contrast to results obtained with direct neural stimulation, no differences in threshold dose or total saliva were observed when cholinergic (methacholine) and peptidergic (physalaemin) agonists were administered intravenously. Response threshold for an adrenergic agonist (epinephrine) was significantly increased in diabetes. Electron microscopy showed axoplasmic abnormalities in nonmyelinated axons in diabetic glands but not in control glands. Although most nerve profiles appeared normal, neuroaxonal abnormalities were found in every diabetic animal. The ultrastructural findings and the similar responses to methacholine and physalaemin suggest that autonomic neuropathy may be a factor in the abnormal response of the parotid gland to parasympathetic nerve stimulation.
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The protein constituents in parasympathetically evoked saliva from normal and short-term sympathectomized parotid glands were compared. There was a reduction in all proline-rich proteins (PRP) in the saliva following sympathectomy. The decrease was quantified for acidic PRP by high-performance ion-exchange chromatography, which showed an increase in the ratio of amylase to other proteins. These results suggest that sympathetic impulses influence the synthesis of PRP and amylase in opposite directions.
Electrical stimulation of the cervical sympathetic nerve trunk delivered at 50 Hz in bursts of 1 s every 10 s, evoked a more copious, uniform and reproducible flow of saliva than when delivered at 10 Hz continuously. This advantage of burst stimulation occurred with parotid secretion and was especially evident with secretion from submandibular glands, where the oedema, commonly seen after stimulating the sympathetic nerve continuously, was avoided. Therefore stimulation in bursts is recommended for obtaining sympathetic salivary responses in rats.
Chemical properties of Weigert's resorcin-fuchsin, orcinol-new fuchsin, Sheridan's crystal violet and their resorcinol-free analogues were investigated using reverse-phase and gel filtration chromatography, electrophoresis, and visible light spectroscopy. Their staining properties were also studied. It was concluded that 1) the staining components of Weigert's resorcin-fuchsin, orcinol-new fuchsin and their resorcinol-free analogues are all indamine oligomers, 2) resorcinol is required for the production of Sheridan's crystal violet, the staining components of which consist of crystal violet substituted by varying numbers of resorcinyl substituents, 3) the staining components of all preparations are cationic (i.e., basic) dyes, 4) iron is present in staining solutions as the tetrachloroferrate anion (FeCl4-) and not as Fe or as a dye-chelate, and 5) since even the smallest Weigert's resorcin-fuchsin, orcinol-new fuchsin or Sheridan's crystal violet component has a conjugated bond number of 32, the observed staining of elastic fibers is only as expected.
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A total of ninety acid, basic, and non-ionic dyes were screened for fluorescent staining of various Carnoy fixed rat tissues. It was found that the fluorescence/non-fluorescence of a dye could be predicted using a conjugated bond number (CBN) cut-off value. Thus 90% of dyes with CBNs of 29 or less were fluorescent; whilst 70% of dyes whose CBNs exceeded 30 were nonfluorescent. The cut-off value was not significantly influenced by the charge, or the hydrophobic-hydrophilic character of the dye; though fluorescence was greatly influenced by the mode of fixation. The CBN cut-off value proved surprisingly robust. Thus most fluorochromes found in the histochemical literature have small conjugated systems, with CBNs less than the cut-off value. This includes labels of immunoglobulins, vital stains of neurones, and fluorescent Schiff reagents. Conversely several dyes used to quench background autofluorescence have large conjugated systems, with CBNs substantially above the cut-off value.
The suitability of reverse-phase thin layer chromatography using a commercial adsorbant and aqueous methanol as an analytical tool for biological stains was investigated. The wide range of applicability of this technique is shown by the fact that of 120 dyes used as biological stains, 84 of diverse chemical character were successfully chromatographed by varying only the water content of the eluent. Unsuccessful chromatography was due either to immobility or streaking. Dyes exhibiting this behavior can be identified prior to chromatography by inspection of their structural formulae. Rf values were found to be significantly correlated with the calculated partition coefficients. This relationship provides information for the identification of dye components revealed by chromatography and a discussion of its use in the chemical characterization of various dyestuffs is presented.
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