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Biomedical subjects

S L Howell

Publications and source records attributed to S L Howell.

At least 109 records · Page 6Linked to original sources
PubMed1977

Secretory capacity and ultrastructure of rat pancreatic islets after preservation of pancreas in different conditions.

Experiments were performed with rat pancreas to investigate optimum conditions for obtaining and preserving the pancreas for subsequent isolation of islets that were viable by both functional and morphological criteria. After only 30 min of warm ischaemia, the yield and viability of islets that could be isolated were poor. However, if the pancreas was removed and placed immediately in a cold bicarbonate-buffered medium, which was supplemented with HEPES to maintain the pH and Trasylol to inhibit proteolytic activity, then viable islets could be isolated consistently after over 8 hr of storage. These results demonstrate that even short periods of warm ischaemia will render the pancreas unsuitable for islet isolation. Once the pancreas is placed in a suitable cold medium, however, considerable delay may be permissible without adversely affecting the viability of the islets which can be obtained. The same conditions might prove to be applicable when human pancreas is obtained for the purpose of transplantation into diabetic subjects.

Animals↗
PubMed1977

Distribution of anionic sites on surface of B cell granule and plasma membranes: a study using cationic ferritin.

The distribution of anionic sites on the membranes of rat pancreatic B cells and of their storage granules has been studied by the use of a visual probe of cationic ferritin. Membranes of isolated storage granules possessed a net negative charge which was apparently evenly distributed; the number of anionic sites was not markedly altered by prior incubation of the granules with neuraminidase or with 10(-5) to 2 X 10(-3) M calcium chloride. Distribution of charges along B cell plasma membranes was less uniform but was similarly unaffected by alterations of calcium concentration, or by neuraminidase treatment. However, during the fusion of plasma membrane and granule membrane which occurs in exocytosis, the emerging granule membrane was found to be devoid of anionic sites. The implications of these findings for the regulation of insulin secretion by exocytosis are discussed.

Animals↗
PubMed1977

Regulation of insulin and glucagon secretion from a human islet cell adenoma.

The regulation of insulin biosynthesis, and insulin and glucagon secretion have been investigated in a human islet cell adenoma, by incubation of tumour fragments. Both biosynthesis and secretion of insulin were strongly stimulated by incubation of islet tumour cells in the presence of increasing glucose concentrations in the range 2-8 mmol/1. However, 20 mM-glucose or 20 mM-glucose plus isobutyl methylxanthine (IBMX), both of which provide potent secretagogues for normal B cells, failed to stimulate proinsulin biosynthesis and secretion from the tumour cells. Overall rates of secretion, expressed as a proportion of total insulin content, were up to 20-fold higher than those expected for normal pancreatic tissue. Glucagon secretion from the tumour was stimulated by low glucose concentrations; normal A cells also respond in this way under these conditions. However, no stimulation of glucagon secretion occurred in the presence of IBMX. There was therefore a major alteration in the regulation both of insulin and glucagon secretion, in that release of neither hormone was stimulated by cyclic AMP. Ultrastructural examination showed the tumour to be rather heterogeneous. A and B cells with normal storage granule content and structure were seen, as well as a rather larger number of B cells containing some granules of atypical appearance. The insulin content of the tumour (13 i.u./g wet wt) was consistent with 6-8% of the tumour cells being B cells.

Adenoma, Islet Cell↗
PubMed1976

The mode of action of adenosine 3':5'-cyclic phosphate in the regulation of insulin secretion.

Changes in the pancreatic beta-cell concentrations of adenosine 3':5'-cyclic phosphate (cyclic AMP) may lead to changes in rates of insulin release, although little is known of the exact mechanism by which this nucleotide may influence the secretory process. Previous studies indicated that in the beta-cell, as in other mammalian cell types, the effects of cyclic AMP may be exerted by the activation of a cyclic AMP-dependent protein kinase, and we have attempted to identify possible substrates for this enzyme in beta-cells. Cyclic AMP stimulated the phosphorylation of specific non-nuclear protein substrates; this effect was observed both in intact cells preincubated with sodium [32P]phosphate to label intracellular ATP and in broken cell preparations incubated with [gamma-32P]ATP. The substrates for protein kinase in islets are unknown but as in other tissues might include microtubular protein and specific proteins of the granule and plasma membranes. In separate experiments cyclic AMP stimulated the efflux of calcium from an organelle-bound (probably mitochondrial) pool, and this may result in rapid changes of intracellular calcium distribution in the beta-cell; these might in turn play an important role in the regulation of secretion. These results suggest that cyclic AMP may directly affect cytosolic calcium concentrations in the beta-cell, as well as promoting the phosphorylation and activity of other components which may be necessary for the maintenance of adequate secretory responses.

Animals↗
PubMed1976

Insulin release and the microtubular system of the islets of Langerhans: effects of insulin secretagogues on microtubule subunit pool size.

An assay system was devised to estimate the pool size of microtubule subunits in islet cells, and to study the importance of the equilibrium between polymerised microtubules and their subunits in the regulation of insulin release. The assay was based on the observation that colchicine binds specifically and quantitatively to microtubule protein subunits, but not to intact microtubules. Vinblastine and cold treatment, which have been shown to cause disaggregation of microtubules into subunits and to inhibit insulin release from islets, increased the number of colchicine binding subunits. D2O, which promotoes stability of microtubules and increases their number in islet cells, caused a decrease in subunit concentration. These results suggest that changes in the equilibrium between polymerised microtubules and their subunits could be studied by measuring the size of the subunit pool. When insulin release was stimulated, by incubating islets in high glucose concentrations or by increasing the intracellular concentration of cyclic AMP there was a reduction in the content of subunit protein. Conversely, when insulin release was inhibited by removal of calcium from the incubation medium there was a shift in the microtubule subunit equilibrium to give an increase in the number of subunits assayed. These results indicate that changes in the equilibrium between subunits and microtubules may play an important role in regulating rates of insulin secretion.

Animals↗
PubMed1976

45Calcium localization in islets of Langerhans, a study by electron-microscopic autoradiography.

Attempts were made to localize the sites of uptake of 45calcium in B cells of islets of Langerhans by electron-microscopic autoradiography. Despite the potential sources of error inherent in the partial loss of radioactivity during fixation, and in the relatively high energy of emission of this isotope, distribution of silver grains differed signficantly from random in all experiments. Grains were concentrated over mitochondria and to a lesser extent over storage granules. Incubation of islets in the presence of 10 mM glucose and isobutylmethyl-xanthine before fixation and autoradiography resulted in a small but not statistically significant reduction in silver grains associated with the mitochondria. These results further indicate a dominant role of mitochondria in the regulation of cytosolic calcium concentrations in pancreatic B cells.

Animals↗
PubMed1976

Barium accumulation in rat pancreatic B cells.

Barium has been used as an electron-opaque substitute for calcium in a study of the distribution of divalent cations between organelles in homogenates or intact rat islets of Langerhans. These were incubated in the presence of barium acetate. Accumulation of electron-opaque deposits was stimulated during incubation of islets in the presence of high glucose concentrations and was diminished in conditions in which intracellular cyclic AMP levels were raised. Mitochondria were found to be the principal sites of accumulation of electron-opaque deposits. Addition of dinitrophenol to homogenates or intact islets abolished mitochondrial barium accumulation. X-ray microanalysis of the deposits in frozen sections showed them to consist predominantly of barium and phosphate. These experiments serve to emphasize further the critical role of mitochondria in the regulation of divalent cation accumulation in B cells, and to confirm that a direct effect on intracellular distribution of divalent cations may represent one important mechanism of action of cyclic AMP in regulating insulin secretion.

Adenosine Triphosphate↗
PubMed1975

Insulin release and the microtubular system of the islets of Langerhans. Identification and characterization of tubulin-like protein.

1. Incubation of islets of Langerhans in vitro in the presence of colchicine produced a progressive inhibition of the insulin-secretory response to glucose, which was dependent on the time of incubation. 2. The uptake of [3-H]colchicine by islet cells was a rapid process, equilibrium being reached in less than 30 min. Part of the colchicine taken up was bound to protein material, which was recovered largely in a post-microsomal supernatant fraction prepared from the islets. In contrast with this rapid uptake, the binding of colchicine by islet-cell proteins in intact islets or in islet homogenates was a slow process, and equilibrium was not reached for 60-90 min. After an initial 30 min delay, the time-course of the binding of [3-H]colchicine to islet-cell proteins paralleled that for the inhibitory effect of colchicine on insulin release. 3. Some purification of the colchicine-binding material present in islet homogenates could be achieved by precipitation of the protein with 2mM-CaCl2 (2.8-fold). However, ion-exchange chromatography on DEAE-Sephadex produced a further 27-fold purification on elution with 0.6M-NaCl. 4. Colchicine-binding protein prepared from islets by ion-exchange chromatography showed an intrinsic association constant for colchicine of 1.4muM and an apparent molecular weight on gel filtration of 110000. 5. These results suggest that colchicine-binding protein in islet cells closely resembles tubulin extracted from the other tissues. The delayed effectiveness of colchicine in inhibiting insulin secretion is not due to poor penetration of colchicine into the cells but rather to slow binding of the alkaloid to islet-cell tubulin. It seems likely that, as in other tissues, this binding prevents polymerization of the tubulin into microtubules, and thus interferes with the release process.

Animals↗
PubMed1975

Calcium distribution in islets of Langerhans: a study of calcium concentrations and of calcium accumulation in B cell organelles.

Calcium concentrations of various pancreatic B cell organelles have been determined by X-ray microanalysis of areas of frozen sections of unfixed rat islets of Langerhans. Highest concentrations were detected in storage granules and in mitochondria, although calcium was also present in nuclei, in areas of endoplasmic reticulum and of cytoplasm. Accumulation of 45Ca by isolated organelles has been studied in homogenates and isolated subcellular fractions of rat islets of Langerhans. In the presence of a permeant anion (oxalate or phosphate), accumulation of 45Ca into mitochondria and microsomes was strongly stimulated by ATP. This net uptake was diminished during incubation of homogenates or of a mitochondria plus storage granule-rich fraction in the presence of cyclic AMP, dibutyryl cyclic GMP; 2:4-dinitrophenol or of ruthenium red. Investigations of the characteristics of 45Ca accumulation by homogenates prepared from storage granule-depleted islets showed no differences from those of normal islets, suggesting that the granules do not represent an important labile pool of calcium. With the exception of cyclic AMP and cyclic GMP none of the insulin secretagogues tested (glucose, leucine, arginine, adrenalin, noradrenalin, theophylline, glibenclamide) altered calcium accumulation by islet homogenates. On the basis of absolute calcium levels and of 45Ca uptake studies it is concluded that islet B cells contain a readily exchangeable mitochondrial calcium pool, and an endoplasmic reticulum pool containing a lower concentration of calcium which is also readily exchangeable. The storage granules, despite their high calcium content, do not appear to constitute a labile pool. It seems likely that the labile mitochondria and endoplasmic reticulum pools play a predominant role in the regulation of cytoplasmic free calcium levels, which may in turn be important in the regulation of rates of insulin secretion.

Adenosine Monophosphate↗
PubMed1974

Biosynthesis of glucagon in isolated pancreatic islets of guinea pigs.

1. The biosynthesis of glucagon in guinea-pig A(2) cells was investigated by incubation of isolated islets of Langerhans in the presence of [(3)H]tryptophan for periods of up to 14 days. Proteins were extracted from islets and incubation media and analysed by gel filtration. 2. In addition to very-high-molecular-weight (100000) proteins, the principal tryptophan-containing biosynthetic product after incubation for up to 17h was a protein of minimum mol.wt. 9000, which co-eluted on gel filtration with a peak of glucagon-like immunoreactivity, but was apparently devoid of biological activity in a fat-cell assay. A discrete peak of labelled glucagon was only recovered after incubation for at least 6 days. Losses of glucagon during the extraction and rapid secretion of newly synthesized glucagon into incubation media were excluded as reasons for the lack of recovery of labelled hormone from islets after shorter incubations. 3. The 9000-mol.wt. protein was localized to A(2) cells in experiments using B-cell-depleted islets, and to A(2)-cell granules by subcellular fractionation and electron-microscopic radioautography. Only glucagon was secreted into the incubation medium. 4. Possible relationships between the 9000-mol.wt. protein and glucagon are discussed in the light of postulated mechanisms of glucagon biosynthesis.

Adipose Tissue↗
PubMed1974

Regulation of guanylate cyclase in guinea-pig islets of Langerhans.

1. Guanylate cyclase activity was determined in homogenates of guinea-pig islets of Langerhans by measurement of the conversion of [alpha-(32)P]GTP into cyclic [(32)P]GMP, the reaction products being separated on columns of neutral alumina. 2. The pH optimum of the enzyme was 7.3; it showed a requirement for bivalent cations, the effectiveness of the cations tested being Mn(2+)>>Ca(2+)>Mg(2+). 3. About 70% of enzyme activity was sedimented by centrifugation at 105000g for 60min; activity was increased 2.3-fold by treatment of homogenates with 0.1% Triton X-100. 4. Guanylate cyclase activity of homogenates was increased by acetylcholine, secretin or pancreozymin, but was inhibited by adrenaline, noradrenaline or ATP. Insulin, glucagon, prostaglandins E(1) or E(2), glucose, F(-), diazoxide or glibenclamide were ineffective. 5. Determination of cyclic GMP amounts in islets by radioimmunoassay showed a basal concentration of 2.0pmol/mg of protein, which was increased by incubation of the islets in the presence of acetylcholine or the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, but was unaffected by glucose. 6. Dibutyryl cyclic GMP had significant stimulatory effects on rates of insulin biosynthesis in isolated rat islets of Langerhans. 7. These results suggest a possible role for cyclic GMP in the regulation of insulin biosynthesis and secretion.

Acetylcholine↗