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A prompt gamma-ray neutron activation analysis (PGNAA) system was used to calibrate and validate a Monte Carlo model as a proof of principle for the quantification of chlorine in soil. First, the response of an n-type HPGe detector to point sources of 60Co and 152Eu was determined experimentally and used to calibrate an MCNP4a model of the detector. The refined MCNP4a detector model can predict the absolute peak detection efficiency within 12% in the energy range of 120–1400 keV. Second, a PGNAA system consisting of a light-water moderated 252Cf (1.06 μg) neutron source, and the shielded and collimated HPGe detector was used to collect prompt gamma-ray spectra from Savannah River Site (SRS) soil spiked with chlorine. The spectra were used to calculate the minimum detectable concentration (MDC) of chlorine and the prompt gamma-ray detection probability. Using the 252Cf based PGNAA system, the MDC for Cl in the SRS soil is 4400 μg/g for an 1800-second irradiation based on the analysis of the 6110 keV prompt gamma-ray. MCNP4a was used to predict the PGNAA detection probability, which was accomplished by modeling the neutron and gamma-ray transport components separately. In the energy range of 788 to 6110 keV, the MCNP4a predictions of the prompt gamma-ray detection probability were generally within 60% of the experimental value, thus validating the Monte Carlo model.

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.

Proinsulin conversion to insulin was studied using electrically permeabilized rat islets of Langerhans. Using high-performance liquid chromotography separation of radiolabelled islet proteins, we have demonstrated that conversion was dependent upon temperature, sensitive to pH and regulated by MgATP. Ammonium acetate was used to collapse the granular pH gradient, over a pH range of 3·5–7·4. Conversion was optimum at pH 4·5–5·5 and was reduced, but not abolished, at pH 7·4. Ca2+ (10 μm) and 4β-phorbol 12-myristate 13-acetate (500 nm), which are insulin secretagogues in permeabilized islets, caused no significant stimulation of conversion.

Research-based pain assessment and management innovations are not fully utilized in clinical nursing practice. Thus children continue to suffer despite strategies that could eliminate or significantly reduce their pain. An educational program was developed and implemented to integrate state-of-the-art pain assessment and management strategies into the clinical practice of pediatric nurses. This article reports on evaluation of the research utilization process during development and implementation of the program. The program included formal classes, development of instruments for pain assessment and documentation, ongoing consultation on pain management strategies, and designation of a unit-based staff nurse liaison. Findings illuminated the process through which nursing staff on one unit came to learn about new ideas, try those ideas in their clinical practice, re-invent certain strategies to better meet their needs, and, ultimately, to adopt innovations deemed helpful in their work.

Findings from a grounded theory research study provide insights into making health care practices more beneficial for the needs and concerns of women with chronic pain. This article focuses on how healthy women advantageously used natural/alternative health care and pain management practices. The "healthiest" women primarily used self-care with a wide, holistic range of natural/alternative modalities.

We have investigated the effects of isoflurane on insulin secretion in vitro from rat isolated islets of Langerhans and found a significant, dose-related and reversible inhibition of insulin secretion. Isoflurane 2% decreased insulin secretion stimulated by glucose 20 mmol litre-1 to basal, nonstimulated values. In other studies to identify the stage in the stimulus secretion pathway for insulin at which the anaesthetic may exert an inhibitory action, we have stimulated insulin release using glyceraldehyde and a phorbol ester. Insulin secretion induced by these secretagogues was also blocked by isoflurane. This suggests that the inhibitory effect of the anaesthetic agent may be at a site distal to stimulation of insulin secretion by glyceraldehyde and phorbol esters.

Possible interactions between polymerized (F-) actin and insulin-storage granules from rat islets of Langerhans were examined in vitro by comparing the sedimentation of the granules in the presence of various actin concentrations. Actin in the concentration range 0.1–0.5 mg/ml produced a retardation in granule-sedimentation rates consistent with binding of the granules to the actin filaments. The interaction was increased by addition of ATP (2mM), but was decreased by CaCl2 (0.1 mM). Binding of granules to actin was unaffected by cyclic AMP or by preincubation of the granules with phospholipase C. Specificity of the interaction was confirmed by the use of depolymerized (G-) actin and of myosin to provide a solution of comparable viscosity; neither of these caused any alteration of granule sedimentation. Possible implications of this interaction of insulin-storage granules with actin for the mechanism of insulin secretion are briefly discussed.

Monensin, a univalent ionophore, is a carboxylic acid produced by Streptomyces cinnamonensis. It will complex various alkali-metal ions, but most readily binds Na+. Because of interest in the possible role of Na+ in the regulation of insulin secretion, we examined its effects on several aspects of the metabolism of isolated rat islets of Langerhans. The ionophore inhibited glucose-stimulated insulin release in a concentration-dependent manner, completely inhibiting secretion evoked by 20 mM-glucose at concentrations as low as 0.1 microM in static incubations. In perifusion experiments, both phases of insulin release were equally affected. Monensin (0.1 microM) had no significant effect on glucose oxidation as measured by the generation of 14CO2 from [14C]glucose. Monensin increased the rate of 22Na+ efflux from preloaded islets and net 22Na+ uptake over 30 min, in the absence of changes in islet volume or extracellular space. The ionophore increased the Rb+/K+ permeability of islet cells, as shown by its inhibition of 86Rb+ retention and stimulation of 86Rb+ efflux. At 0.1 microM, monensin abolished glucose-stimulated 45Ca2+ uptake by islets during 5 min incubations, and stimulated 45Ca2+ efflux from preloaded islets perifused with Ca2+-free medium, even in the complete absence of extracellular Na+. Studies of the uptake of 14C-labelled 5,5-dimethyloxazolidine-2,4-dione showed that 0.1 microM-monensin increased net intracellular pH from 7.05 to 7.13. 7 Monensin has widespread, complex, effects on the secretory responses and ion handling by the B cells, which are difficult to interpret in terms solely of actions as a Na+ ionophore.

Infection of DBA2 mice with the M strain of EMC virus was used to study the effects of virusinduced diabetes on the A and B cells of the islets of Langerhans. A transient hypoglycaemia was seen in 48% of mice 2–3 days after infection and probably resulted from increased serum insulin concentrations together with inhibition of glucagon secretion at that time. Islets from hypoglycaemic mice showed no significant alterations from control level in basal or fluoride-stimulated adenylate cyclase activity. Overall, 70% of infected mice became hyperglycaemic with a maximum incidence 6 days after infection. Hyperglycaemia was accompanied by a dramatic reduction in the total pancreatic insulin content and in insulin secretory responses to glucose and theophylline, while A-cell structure and function appeared relatively unaffected in diabetic animals. Basal adenylate cyclase activity was increased in hyperglycaemic mice at 7 days after infection, while fluoride-stimulated adenylate cyclase activity was normal throughout the course of infection. Ultrastructural alterations were observed in a small proportion of B cells from two days after infection and included abnormalities of mitochondrial structure and increased electron opacity of the cytoplasm of affected cells, which subsequently led to complete necrosis. The results suggest that EMC virus specifically affects the B cells of the islets and that disturbances of A cell function may be secondary to B cell damage.

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 intra-cellular distribution of divalent cations may represent one important mechanism of action of cyclic AMP in regulating insulin secretion.

Dynorphin-[1-13], at concentrations of 5.8 × 10−12 to 5.8 × 10−9 M, stimulated insulin secretion from isolated islets of Langerhans of the rat, in medium containing 6 mM glucose. Higher concentrations of dynorphin had no significant effect on secretion. Dynorphin (5.8 × 10−9 M) was unable to initiate insulin release from islets in the presence of 2 mM glucose, or to increase insulin secretion further in the presence of 20 mM glucose or 6 and 12 mM glyceraldehyde. Dynor-phin-induced insulin secretion from islets was blocked by verapamil (5 μM) or by chlorpropamide (72 μM), but not by a mu opiate receptor antagonist, naloxone (0.11 μM), or by ICI 154129, a specific antagonist for the delta receptor (0.25 μM). Dynorphin had no effect on islet somatostatin secretion, under conditions in which insulin secretion was greatly stimulated. Glucose (20 mM) and glyceraldehyde (6 and 12 mM) significantly increased both insulin and somatostatin secretion. Dynorphin (5.8×10−9 M) increased 45Ca2+ uptake into islets, and also increased intracellular islet c-AMP levels. These changes persisted when higher concentrations of dynorphin were used. These results suggest that (1) dynorphin is a very potent stimulus for insulin secretion; (2) dynorphin does not affect somatostatin secretion in static incubations of islets, in the same way as does glucose and glyceraldehyde; (3) dynorphin's effects may involve increased calcium ion movement and can be blocked by verapamil; (4) dynorphin can also increase islet c-AMP, and could thereby modulate the responsiveness of other secretagogues; (5) the actions of dynorphin on insulin secretion are not mediated by delta or mu opiate receptors in islets.

A procedure is described for the preparation of ultrathin frozen sections of glutaraldehyde-fixed or unfixed islets of Langerhans by cryo-ultramicrotomy. Freezing of the tissue was accomplished by direct immersion of isolated islets in liquid nitrogen. Sectioning was performed at a specimen temperature of -80 °C and a knife temperature of -40 °C, the ribbon of sections being collected on a trough containing 60 % dimethyl sulphoxide. Staining was accomplished with 4 % silicotungstic acid and sections were protected from drying artifacts by rinsing with 0.5% polyethylene glycol. Even in tissue not subjected to prior glutaraldehyde fixation, most of the structural features of A and B cells were well preserved in frozen sections, which were obtained in a number and quality which should render them suitable for ultrastructural, cytochemical or radioautographic studies.

Studies of the role of the microtubule-microfilamentous system in insulin secretion have been widened by continuing experimentation and analysis to provide a comprehensive working hypothesis which embraces ideas of the way in which the polymerization of microtubules and microfilaments may be regulated and how these cytoskeletal components may act together to enhance the process of granule movement. It is also possible to speculate about, but not yet to demonstrate, the way in which the activities of this effector system could be regulated by calcium and by cyclic AMP, which are essentially involved in the regulation of rates of secretion.

Islets of Langerhans isolated from normal female rats have been used in studies of3H-progesterone uptake by intact islet cells. The intracellular sites which are involved in binding of the hormones have been determined by subcellular fractionation and autoradiography. Uptake of3H-progesterone into islets occurred in a temperature and concentration dependent manner. The uptake increased rapidly in the first 30 min, and could be partially displaced by addition of excess unlabelled progesterone.3H-progesterone uptake was lowered by incubation of the islets in the absence of Ca++, at 0° compared to 37° C, or to a much lesser extent when islet cycilic AMP levels were raised by addition of 3-isobutyl-l-methylxanthine. However, uptake was unaffected by prior treatment of the islets with neuraminidase orp-hydroxymercuribenzoate. Differential centrifugation of islets which had previously been incubated with3H-progesterone showed the highest specific activity of binding in the nuclei and debris fraction. Isolation of a purified nuclear fraction by sedimentation through sucrose solutions confirmed that binding was present in the nuclear component of this heterogeneous fraction, while autoradiographic studies suggested both nuclear and cytosolic localisation of3H-progesterone. In a separate series of experiments, evidence was obtained for the existence of saturable cytosolic binding of progesterone, and for movement of labelled hormone from the cytosol to the nuclear fraction. It is suggested that, in the islets of Langerhans as in other target tissues, the action of progesterone involves penetration into the cells, binding to a cytosolic receptor protein, and subsequent transfer to the nucleus. The nuclear events which lead to subsequent alteration of rates of insulin secretion remain to be determined.

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.

Islets of Langerhans, isolated from normal or 19-day pregnant rats, were cultured for 20 h at 37 degrees C in tissue culture medium 199. When islets were cultured in medium containing low glucose (5.5 mM), the higher adenylate cyclase activity and insulin secretory responses characteristic of islets from pregnant rats were maintained during the test period of 29 h. Islets from normal and pregnant rats were also cultured for 20 h in medium containing a very high glucose concentration (83.3 mM) in order to load the B cells with glycogen. It was found, after glycogen loading, that, while adenylate cyclase activity increased to a greater extent in islets from pregnant rats than controls, this activity was not increased in proportion to the striking changes in insulin release rate observed in pregnant rat islets. The results show that the difference in insulin secretory response between islets from normal and pregnant rats may be preserved when the islets are cultured for 20 h, and that these differences are enhanced for a variety of reasons after culture of islets in 83.3 mM glucose.

Interactions between actomyosin and insulin storage granules isolated from rat islets of Langerhans have been examined in a simple system in vitro, which allows comparison of the sedimentation of the granules in the presence of absence of actomyosin in various conditions. Actomyosin altered granule-sedimentation rates in a manner consistent with the binding of the granules of actomyosin filaments. This interaction was enhanced by addition of ATP (1.5 mM) but unaltered by addition of CaCl2, by calmodulin or by calmodulin in the presence of 10 microM-CaCl2. Addition of EGTA (0.1 mM), cyclic AMP (10 microM) of cytochalasin B (10 microgram/ml) were also without effects in these conditions. Pre-incubation of granules with phospholipase c did not affect granule-actomyosin interaction. Ultrastructural studies showed close contacts between the membranes of the granules and actomyosin filaments. The results indicate the possibility that actomyosin might provide the motile force for granule translocation during the insulin secretory process.

A partially purified secretory granule fraction, isolated from rat islets of Langerhans by differential centrifugation, was used for investigating the stability of the beta granules during incubation in various conditions. Effects of pH, temperature, and time were studied; the granules possessed optimal stability at 4° and pH 6.0, and could be solubilized at pH 4.0 or 8.5, or in the presence of sodium deoxycholate, but not by phospholipase c, ouabain, or alloxan. Incubation with glucose or some of its metabolites, or with tolbutamide, ATP, or cyclic 3',5'-AMP did not alter the stability of the beta granules Exogenous insulin-131I was not bound by the isolated granules under the conditions used; no specific insulin-degrading activity could be detected in subcellular fractions of the islets. These findings indicate that intracellular solubilization of the granules with subsequent diffusion of the insulin into the extracellular space is not a likely mode of insulin secretion in vivo, and suggest that a crystalline zinc-insulin complex may exist in the matrix of the beta granules.

The distribution of radioautographic grains over organelles within the beta cells of rat islets of Langerhans was investigated at various times after pulse labeling of the isolated islets with tritium-labeled amino acids. Ten minutes after the start of labeling most of the grains were situated over the endoplasmic reticulum and cytoplasm; by contrast, 60 min from the start of labeling the majority of the grains were associated with the beta granules. At 20, 30, and 45 minutes after pulse labeling the proportion of grains associated with the Golgi complex was increased two- to three-fold over the 10- or 60-minute values. The distribution of radioautographic grains over granules in the intact cells did not suggest that the electron-lucent type of secretory granules were precursors of the electron-opaque granules. Furthermore, studies of the pattern of grains over granules isolated by centrifugation 60 min after pulse labeling showed no preferential labeling of the electron-lucent type of granule. It is concluded that labeled amino acids are incorporated initially in the endoplasmic reticulum, and that the label subsequently appears in the beta granules. The Golgi complex participates either in the formation of the beta granule or in the translocation of the granule through the cytoplasm of the cell.

The intracellular processes involved in synthesis, transport and storage of newly synthesized proteins in the rat somatotroph, together with their time course and metabolic requirements, have been investigated in a quantitative electron-microscopic radioautography study of the tissue following pulse labelling with tritium-labelled amino acids and chase incubations in various conditions. Proteins are synthesized initially on the rough-surfaced elements of the endoplasmic reticulum and are transported within 10 min after their synthesis to transitional areas between the rough-surfaced endoplasmic reticulum and Golgi complex. Transfer to the Golgi lamellae is achieved, probably via transfer vesicles, within about 60 min after synthesis, while formation of mature storage granules occurs within 2 h following protein synthesis. Further experiments utilizing cycloheximide or ouabain during the chase incubations showed that the intracellular transport of newly synthesized protein and its time course are not significantly affected by inhibitors of protein synthesis, or by inhibition of sodium-potassium dependent ATPase by ouabain. Inhibitors of oxidative phosphorylation (250 µM 2,4-dinitrophenol) or of respiration (10 µM antimycin A) markedly reduced intracellular ATP levels and inhibited the intracellular transport processes. The requirement for ATP appeared to be operative at 2 stages: in the movement of transfer vesicles to the Golgi complex and in the formation of storage granules; possible roles of ATP in these processes are discussed.

A cytochemical method has been used to investigate the localization of adenyl cyclase activity in A and B cells of isolated rat islets of Langerhans. Adenosine triphosphate was initially utilized as substrate, the pyrophosphate liberated being precipitated by lead ions at its site of production. The specificity of the method was increased by the use of adenylyl-imidodiphosphate as an alternative substrate; this adenosine triphosphate analogue was not hydrolyzed by adenosine triphosphatase but provided an effective substrate for adenyl cyclase. Adenyl cyclase activity, which was found to retain its glucagon and fluoride sensitivity in glutaraldehyde-fixed tissue, was found exclusively and almost uniformly in the plasma membranes of A and B cells. Storage granule membrane, incorporated into the plasma membrane during secretion of the granule content by exocytosis, appeared to be devoid of adenyl cyclase activity.

1. A protein was demonstrated in mammalian islets of Langerhans that after purification appeared as a single component possessing both cyclic-AMP (adenosine 3′:5′-cyclic monophosphate)-binding and cyclic-AMP-dependent protein phosphokinase activities. 2. The protein had an intrinsic association constant for cyclic AMP of 1.15×10−8m, which was similar to the Km for cyclic AMP (1.11×10−8m) of the protein phosphokinase activity. 3. Incubation of the protein in the presence of cyclic AMP resulted in its dissociation into cyclic-AMP-independent protein phosphokinase (catalytic) and cyclic-AMP-binding (receptor) subunits, which could be separated on Sephadex G-200. 4. The cyclic-AMP-dependent protein phosphokinase was capable of phosphorylating a variety of proteins, the most readily phosphorylated being histone, casein and protein components of sub-cellular fractions prepared from islets of Langerhans. 5. The cyclic-AMP-dependent phosphorylation of histone had a Km for ATP of 1.1×10−5m. 6. The endogenous protein phosphokinase activity in rat islets incubated with agents that are known to alter the intracellular concentration of cyclic AMP was investigated. Theophylline and 3-isobutyl-1-methylxanthine, agents that raise cyclic AMP concentrations in islets, increased the activity of the protein phosphokinase, whereas adrenaline, which lowers islet cyclic AMP concentrations, decreased its activity. 7. It is suggested that cyclic AMP may exert its effects on insulin release by increasing the activity of a protein phosphokinase and may thereby promote the phosphorylation and activity of a rate-determining component of the secretory mechanism.

Partially purified storage granule fractions obtained from rat anterior pituitaries have been utilized to study some properties of the isolated growth hormone granules during incubation in vitro. The isolated granules showed optimal stability at room temperature in the pH range 5.5-6.5 and dissolved progressively with time at a rate which was unaffected by the presence of EDTA, EGTA, ouabain, magnesium ions, or by resuspension in hypotonic media. However, the granules were partially stabilized by the presence of calcium, tyramine or adenosine phosphates; ATP was most effective of the agents tested and induced almost complete stability. Agents known to stimulate the secretion of growth hormone from the intact cells, cyclic 3',5'-AMP, dibutyryl cyclic 3',5'-AMP, theophylline or high potassium concentrations, were each without significant effect on the isolated granules. The granule fraction was ineffective indegrading added 125I-growth hormone over a pH range of 4.5-8.5 and was unable specifically to bind exogenous 125I-growth hormone. Further experiments to elucidate a possible cytological role of nucleotides in stabilization of growth hormone during storage granule formation demonstrated that adenine nucleotides were not constituents of the isolated granules, nor were they secreted into the incubation medium concomitantly with growth hormone. Depletion of intracellular ATP levels has previously been shown to prevent the formation of growth-hormone storage granules, and it is suggested that one role of the nucleotide may be to facilitate the formation and stabilization of storage granules in the Golgi complex by interacting with the growth hormone to induce its precipitation.

1. The concentrations of cyclic AMP were compared in islets of Langerhans isolated from the pancreases of normal female and pregnant rats and were higher in islets in pregnancy. 2. There was also a significant increase in adenylate cyclase activity in homogenates of islets from pregnant rats compared with those from normal rats. 3. Increased cyclic AMP concentration in islets from pregnant rats was reflected in increased protein kinase activity. When the cyclic AMP-dependent protein kinase activity was increased by 3-isobutyl-1-methylxanthine this stimulated activity was significantly greater in pregnancy. 4. Insulin-secretion studies with islets from normal and pregnant rats showed that theophylline or 3-isobutyl-1-methylxanthine, which raise intracellular cyclic AMP concentrations, caused a significantly greater insulin secretion in pregnancy. 5. It was also found that in the presence of a glucose concentration too low to stimulate insulin secretion, the latter could be induced if the cyclic AMP concentrations were raised sufficiently with 3-isobutyl-1-methylxanthine. 6. It is suggested that the higher cyclic AMP concentrations observed in islets in pregnancy mediate the greater insulin-secretory capacity, as well as the greater sensitivity of these islets to low glucose concentrations.

A DNAase-inhibition assay was used to determine the proportions of globular (G-) and filamentous (F-) actin in islets of Langerhans after incubation in various conditions, or after subcellular fractionation. Stimulation of insulin secretion resulted in an ATP-dependent increase in the proportion of F-actin present; fractionation showed 80-90% of the actin to be present in the final supernatant.

It was possible to vary the replication rate of cells in the islets of Langerhans of adult rats. The rate of incorporation of [3H]thymidine into islet DNA was increased at 12 days of pregnancy to 2·3-fold and at 19 days of pregnancy to 1·3-fold that in control rats. Ovariectomy, which leads to lowered plasma levels of ovarian steroids, induced a significant and unexpected increase in the rate of thymidine incorporation into islets; treatment of ovariectomized rats with 2 μg oestradiol/rat per day for 3 days reversed this upward trend. When islets from normal rats were cultured with certain combinations of steroid hormones including progesterone and oestradiol or with insulin secretagogues, with the exception of glucose, a decreased rate of DNA synthesis was usually found compared with that in control rats. Since treatment with steroid hormones inhibited incorporation of [3H]thymidine into islets from ovariectomized rats and directly reduced incorporation into tissue-cultured islets from normal rats in vitro, it was concluded that increased levels of steroid hormones were not responsible for the higher rate of regeneration of islet cells in pregnant rats. However, a striking correlation between levels of blood glucose in vivo and DNA synthesis in islets in vitro has been observed.

Isolated rat islets of Langerhans which had been pretreated with 200 nM-phorbol 12-myristate 13-acetate (PMA) for 20-24 h, a treatment reported in other cell types to deplete cells of protein kinase C activity, were found not to contain detectable Ca2+/phospholipid-dependent protein kinase activity. These islets did not secrete insulin in response to a subsequent exposure to PMA (0.1 or 1 microM) during a 30 min incubation, although insulin secretion could be stimulated by 20 mM-glucose, a response which was enhanced by 20 microM-forskolin. PMA-pretreated islets that had been permeabilized by high-voltage discharge showed unimpaired secretory responses to an increase in Ca2+ concentration, cyclic AMP and forskolin. These results suggest that (i) pretreatment of islets with tumour-promoting phorbol esters may be a useful means of investigating the role of protein kinase C in stimulus-secretion coupling in the pancreatic beta-cell and (ii) protein kinase C may not play an essential role in glucose-induced insulin secretion.

Binding of filamentous actin to isolated insulin storage granules has been demonstrated in an in vitro system; this binding is enhanced by 2 mM ATP, but is diminished in the presence of 1 mM CaCl2. It is unaffected by glucose or by cyclic AMP. Estimation of the actin content of isolated rat islets by DNAase inhibition indicates that G-actin constitutes 1 to 2% of the protein content of the islets, that 50 to 70% of the actin content of the islets is present as the depolymerized globular from (G-actin), and that the proportion of F-actin found may be increased in islets incubated in the presence of 20mM glucose. Thus both the proportions of G- and F-actin, and the binding of granules to F-actin may be important in the regulation of rates of insulin secretion.

Pulse-chase experiments utilising(3H)leucine have been used to study the effects of colchicine and vinblastine on intracellular transport and secretion of newly synthesised growth hormone from rat anterior pituitary fragments. Growth hormone was isolated from medium and fragments by polyacrylamide gel electrophoresis. When colchicine or vinblastine, which disrupt microtubules, were added immediately after pulse labelling, inhibition of the subsequent secretion of newly synthesised growth hormone was detected throughout the succeeding 5 h. Similar inhibition was seen if the drugs were added after a 1 h delay. However, if colchicine or vinblastine were added only after a 2 h chase incubation, then no significant effect on subsequent release of labelled growth hormone was seen. The results suggest that these agents may inhibit the transport of newly formed growth hormone storage granules from the Golgi complex to the cytoplasmic pool. Microtubules do not appear to be involved in the mechanism of the final secretion of newly synthesised hormone by exocytosis.

Studies of the cellular mechanisms of insulin secretion from intact β-cells are hampered, to some extent, by the existence of an intact plasma membrane which limits the degree to which the intracellular environment can be experimentally manipulated. One means of circumventing this problem is to permeabilise the plasma membrane of the β-cells by a high voltage discharge technique.