Fungicide prochloraz induces oxidative stress and DNA damage in vitro.

Prochloraz is widely used in horticulture and agriculture, e.g. as a post-harvest anti-mold treatment. Prochloraz is a known endocrine disruptor causing developmental toxicity with multiple mechanisms of action. However, data are scarce concerning other toxic effects. Since oxidative stress response, with formation of reactive oxygen species (ROS), is a common mechanism for different toxic endpoints, e.g. genotoxicity, carcinogenicity and teratogenicity, the aim of this study was to investigate if prochloraz can induce oxidative stress and/or DNA damage in human cells. A cell culture based in vitro model was used to study oxidative stress response by prochloraz, as measured by the activity of the nuclear factor erythroid 2-related factor 2 (Nrf2), a key molecule in oxidative defense mechanisms. It was observed that prochloraz induced oxidative stress in cultured human adrenocortical H295R and hepatoma HepG2 cells at non-toxic concentrations. Further, we used Comet assay to investigate the DNA damaging potential of prochloraz, and found that non-toxic concentrations of prochloraz induced DNA damage in HepG2 cells. These are novel findings, contradicting previous studies in the field of prochloraz and genotoxicity. This study reports a new mechanism by which prochloraz may exert toxicity. Our findings suggest that prochloraz might have genotoxic properties.

Frondoside A enhances the antiproliferative effects of gemcitabine in pancreatic cancer.

Pancreatic cancer has a very poor prognosis. While gemcitabine is the mainstay of therapy and improves quality of life, it has little impact on survival. More effective treatments are desperately needed for this disease. Frondoside A is a triterpenoid glycoside isolated from the Atlantic sea cucumber, Cucumaria frondosa. Frondoside A potently inhibits pancreatic cancer cell growth and induces apoptosis in vitro and in vivo. The aim of the present study was to investigate whether frondoside A could enhance the anti-cancer effects of gemcitabine. Effects of frondoside A and gemcitabine alone and in combination on proliferation were investigated in two human pancreatic cancer cell lines, AsPC-1 and S2013. To investigate possible synergistic effects, combinations of low concentrations of the two drugs were used for a 72 h treatment period in vitro. Growth inhibition was significantly greater with the drug combinations than their additive effects. Combinations of frondoside A and gemcitabine were tested in vivo using the athymic mouse model. Xenografts of AsPC-1 and S2013 cells were allowed to form tumours prior to treatment with the drugs alone or in combination for 30 days. Tumours grew rapidly in placebo-treated animals. Tumour growth was significantly reduced in all treatment groups. At the lowest dose tested, gemcitabine (4 mg/kg/dose), combined with frondoside A (100 µg/kg/day) was significantly more effective than with either drug alone. To conclude: The present data suggest that combinations of frondoside A and gemcitabine may provide clinical benefit for patients with pancreatic cancer.

Glucose-induced inhibition of insulin secretion.

Increase in glucose is known to elevate the concentration of cytoplasmic Ca(2+) ([Ca(2+) ]i ) in pancreatic ß-cells and stimulate insulin secretion. However, rise of glucose can also lower [Ca(2+) ]i and inhibit insulin release. In the present review, we examine the mechanisms for this inhibition and highlight its importance for the healthy ß-cell and the development of diabetes. It is possible to distinguish between 60 and 90 s of prompt inhibition and the late inhibition seen after the first-phase peak of insulin release. The introductory inhibition is characteristic of the healthy ß-cell and mediated by sequestration of [Ca(2+) ]i in the endoplasmic reticulum. This inhibition is easily seen in studies of isolated islets but too brief to be detected in a conventional intravenous glucose tolerance test. Coupled to simultaneous rise of glucagon, the introductory suppression of insulin release is the starting point for the antiphase relation between the subsequent insulin and glucagon pulses. Another effect of the initial suppression is to increase the pool of readily releasable granules responsible for the first-phase release of insulin. The presence of late inhibition of insulin release is an indicator of ß-cell dysfunction. Patients with type 2 diabetes often respond to intravenous bolus injection of glucose with 5-10 min of late suppression of circulating insulin.

Effect of ß-carotene on catechol-induced genotoxicity in vitro: evidence of both enhanced and reduced DNA damage.

Intake of antioxidants from the diet has been recognized to have beneficial health effects, but the potential benefit of taking antioxidants such as ß-carotene as supplements is controversial. The aim of the present study was to evaluate the potential protective effects of a physiologically relevant concentration (2 µM) of ß-carotene on the DNA damaging effects of catechol in mouse lymphoma L5178Y cells. Two different exposure protocols were used: simultaneous exposure to ß-carotene and catechol for 3 h; and exposure to catechol for 3 h after 18 h pre-treatment with the vitamin. DNA damage was evaluated using the comet assay (employing one procedure for general damage, and another procedure, which also included oxidative DNA damage). Independent of exposure protocol and procedure for comet assay, ß-carotene did not increase the basal level of DNA damage. However, at the highest concentration of catechol (1 mM), ß-carotene was found to clearly increase the level of catechol-induced DNA damage, especially in the pre-treated cells. Interestingly, an opposite effect was observed at lower concentrations of catechol, but the ß-carotene related reduction of catechol-induced genotoxicity was significant (P < 0.05) only for the procedure including oxidative damage induced by 0.5 mM catechol. Taken together our results indicate that ß- carotene can both reduce and enhance the DNA damaging effects of a genotoxic agent such as catechol. This indicates that it is the level of catechol-induced DNA damage that seems to determine whether ß-carotene should be regarded as a beneficial or detrimental agent when it comes to its use as a dietary supplement.

Potential genotoxicity of plant extracts used in Ethiopian traditional medicine.

AIM OF THE STUDY: Although traditional herbal medicines are widely used in Ethiopia, no information is available on their potential genotoxicity. In the present study, hydroalcoholic extracts of Glinus lotoides, Plumbago zeylanica, Rumex steudelii and Thymus schimperi were evaluated for their DNA damaging effects using the comet assay. MATERIAL AND METHODS: Mouse lymphoma L5178Y cells were exposed to different concentrations of the extracts for 3h with and without metabolic activation (S9-mix) using 4-nitroquinoline-N-oxide and benzo(a)pyrene as positive controls, and vehicles as negative controls. RESULTS: In the absence of S9, all extracts were found to induce significant DNA damage without affecting the cell viability. T. schimperi and R. steudelii were the most potent DNA-damaging extracts, and G. lotoides and P. zeylanica the least potent. The addition of S9 had different effects on the DNA damage induced by the extracts: it lowered the DNA damaging effect of P. zeylanica, did not affect the DNA damaging effect of T. schimperi, and increased the DNA damaging effects of R. steudelii and G. lotoides. CONCLUSION: The findings of the present study suggest that all extracts evaluated have a genotoxic potential in vitro which needs to be substantiated by further studies.

Interindividual differences in initial DNA repair capacity when evaluating H2O2-induced DNA damage in extended-term cultures of human lymphocytes using the comet assay.

It has been suggested that extended-term cultures of human lymphocytes could be used as a complement to cell lines based on transformed cells when testing the genotoxicity of chemicals. To investigate whether the pattern of induced DNA damage and its subsequent repair differs significantly between cultures based on different blood donors, hydrogen peroxide (H(2)O(2))-induced DNA damage was measured in cultures from four different subjects using the comet assay. The DNA damage was significantly increased in all cultures after 10 min exposure to 0.25 mmol/L H(2)O(2), and there was a significant decrease in the H(2)O(2)-induced DNA damage in all cultures after 30 min of DNA repair. The level of damage varied between the different donors, especially after the repair. Using PCR and DNA sequencing, exon 5 of the p53 gene was sequenced in the lymphocytes from the donors with the lowest and highest residual damage. No such mutation was found. Mouse lymphoma L5178Y cells carrying the p53 mutation in exon 5 were included as a reference. These cells were found to be less sensitive toward the H(2)O(2)-induced DNA damage, and they were also found to have a rather low DNA repair capacity. The demonstrated variation in H(2)O(2)-induced DNA damage and DNA repair capacity between the cultures from the different subjects may be important from a risk assessment perspective, but is obviously not of decisive importance when it comes to the development of a routine assay for genotoxicity.

External ATP triggers Ca2+ signals suited for synchronization of pancreatic beta-cells.

External ATP is supposed to trigger short-lived increases (transients) of cytoplasmic Ca2+ important for entraining insulin-secreting beta-cells into a common rhythm. To get insight into this process, rises of the cytoplasmic Ca2+ concentration ([Ca2+]i) induced by external ATP were compared with those obtained with acetylcholine, another neurotransmitter with stimulatory effects on the inositol trisphosphate (IP3) production. A ratiometric fura-2 technique was used for measuring [Ca2+]i in individual beta-cells and small aggregates isolated from ob/ob mouse islets and superfused with a medium containing methoxyverapamil. ATP and acetylcholine induced temporary rises of [Ca2+]I from a basal level manifested as solitary transients (<20 s) and bumps (> or =20 s) superimposed or not with transients. Addition of ATP (1-100 microM) usually triggered transients whereas acetylcholine induced bumps lacking superimposed transients. After the initial rise there was a steady-state elevation of [Ca2+]i in beta-cells exposed to acetylcholine but not to ATP. Similar differences were seen comparing the responses of rat beta-cells to 100 microM ATP and acetylcholine. Inhibition of the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) pump (with 50 microM cyclopiazonic acid) prevented both the ATP-induced rise of [Ca2+]i and the spontaneous firing of transients. Similar effects were seen after activation of protein kinase C (10 nM phorbol-12-myristate-13-acetate), whereas an inhibitor of this enzyme (2 microM bisindolylmaleimide) promoted the generation of transients. The results indicate that ATP fulfils the demands for a coordinator of the secretory activity of beta-cells by generating distinct [Ca2+]i transients without sustained elevation of basal [Ca2+]i.

Synchronization of pancreatic beta-cell rhythmicity after glucagon induction of Ca2+ transients.

Pancreatic beta-cells are biological oscillators requiring a coupling force for the synchronization of the cytoplasmic Ca(2+) oscillations responsible for pulsatile insulin release. Testing the idea that transients, superimposed on the oscillations, are important for this synchronization, the concentration of cytoplasmic Ca(2+) ([Ca(2+)](i)) was measured with ratiometric fura-2 technique in single beta-cells and small aggregates prepared from islets isolated from ob/ob-mice. Image analyses revealed asynchronous [Ca(2+)](i) oscillations in adjacent beta-cells lacking physical contact. The addition of glucagon stimulated the firing of [Ca(2+)](i) transients, which appeared in synchrony in adjacent beta-cells. Moreover, the presence of glucagon promoted synchronization of the [Ca(2+)](i) oscillations in beta-cells separated by a distance <100 microm but not in those >200 microm apart. The results support the proposal that the repolarizing effect of [Ca(2+)](i) transients provides a coupling force for co-ordinating the pulses of insulin release generated by pancreatic beta-cells.

Nitric oxide induces synchronous Ca2+ transients in pancreatic beta cells lacking contact.

AIMS: To evaluate the role of nitric oxide (NO) in the coordination of the Ca2+ signals generating pulsatile insulin release in pancreatic beta cells isolated from ob/ob mice. METHODOLOGY: Using ratiometric fura-2 technique for recording glucose-induced cytoplasmic Ca2+ transients, it was possible to demonstrate a synchronization of beta cells lacking contact. RESULTS: The frequency of the transients increased 10-fold in the presence of 20 n M glucagon. Additional increase in frequency with maintenance of synchronization was observed when the beta cells were exposed to 100 microM of the NO donors sodium nitroprusside and hydroxylamine. Bolus additions of 0.1-10 microM gaseous NO resulted in prompt appearance of cytoplasmic Ca2+ transients. An activator of soluble guanylate cyclase (mesoporphyrin) increased the frequency of the transients, and inhibition of this enzyme with 1H-(1,2,4) oxadiazolo [4,3-a] quinoxalin-1-one had the opposite effect. CONCLUSION: The results support the idea that nitrergic nerves generate beta-cell transients of Ca2+ synchronizing the activity of the numerous islets in the pancreas.

The endoplasmic reticulum is a glucose-modulated high-affinity sink for Ca2+ in mouse pancreatic beta-cells.

The regulation of organelle free Ca2+ was analysed in individual mouse pancreatic beta-cells loaded with the fluorescent low-affinity indicator furaptra. Removal of the cytoplasmic indicator by controlled digitonin permeabilization of the plasma membrane resulted in a sudden increase of the 340 nm/380 nm fluorescence excitation ratio followed by a gradual decay, reflecting the emptying of Ca2+ from organelle pools. Subsequent introduction of 3 mM ATP caused rapid refilling of a Ca2+ pool, which represented the endoplasmic reticulum (ER) in being mobilized with inositol 1,4,5-trisphosphate (IP3) and the sarco(endo)plasmic reticulum Ca2+-ATPase inhibitor thapsigargin. The concentration of Ca2+ in the ER observed immediately after permeabilization depended on the glucose concentration in a hyperbolic fashion with half-maximal filling at about 6 mM of the sugar. Glucose promotion of Ca2+ sequestration in the ER involved a high-affinity mechanism not requiring but accelerated by a rise of the cytoplasmic Ca2+ concentration. Glucose also exerted a long-term action on the ER storage of Ca2+, maintaining the set-point for its maximal concentration and preserving the response to IP3. The results indicate that the ER has an important role in the glucose-stimulated beta-cell by serving as a high-affinity sink for Ca2+, irrespective of the prevailing concentration of cytoplasmic Ca2+.

The role of growth hormone in adaptation to massive small intestinal resection in rats.

The residual small bowel undergoes profound adaptive alterations after surgical resection. GH is considered to have a role in regulation of these adaptive changes, but its precise role is unknown. We investigated the role of GH by studying the response to intestinal resection in rats with isolated GH deficiency. Spontaneous dwarf rats, a strain of rats with congenital isolated GH deficiency, underwent 60% resection of the small intestine and parameters of the response of the intestinal remnant were compared with age-matched GH-deficient rats undergoing transection, GH-normal rats undergoing 60% resection, and nonmanipulated GH-normal rats. Deficiency of GH did not inhibit hyperplasia of the mucosal mass of the intestinal remnant, indicating that GH is not required for regulation of this aspect of the adaptive response. However, GH deficiency resulted in lack of accumulation of mucosal protein, including lack of accumulation of digestive hydrolases. In addition, GH deficiency resulted in alterations in processing of digestive hydrolases of the distal intestine, indicating that GH may have region-specific effects on small intestinal function. We conclude that GH is required for the normal expression of specific components of the adaptive response to massive small intestinal resection, but not for all aspects. The aspects that require GH appear to involve protein synthesis and processing.

Evaluation of benzo(a)pyrene-induced DNA damage in human endothelial cells using alkaline single cell gel electrophoresis.

The alkaline version of the 'comet assay' was used to evaluate DNA damage in human umbilical vein endothelial cells (HUVEC) exposed to 0.1, 1.0, or 10 microM benzo(a)pyrene for 90min. The genotoxicity was monitored in HUVEC pretreated with the Ah-receptor agonist beta-naphthoflavone (BNF), previously shown to induce cytochrome P4501A1 (CYP1A1) activity in these cells, and in vehicle-treated HUVEC with only constitutive levels of this enzyme. Increased DNA damage was observed only in cells that had been exposed to 10 microM benzo(a)pyrene, cells exposed to BNF being subjected to the most extensive damage. The CYP1A/B-inhibitor alpha-naphthoflavone (ANF) reduced the benzo(a)pyrene-induced DNA-damage in the BNF-treated HUVEC to the same level as in the uninduced cells. The fact that benzo(a)pyrene induced DNA damage in vehicle-treated HUVEC suggests that there may be at least one alternative route of bioactivation for benzo(a)pyrene in these cells. Consequently, judging from the present results it seems as if tobacco-related polycyclic aromatic hydrocarbons (PAHs) may disrupt the function of the endothelial lining in blood vessels with low monooxygenase activity. It is proposed that exposure to Ah receptor agonists via, for example, tobacco smoke, may enhance the DNA-damaging effects of smoke-related genotoxic PAHs in human endothelial cells. The role of PAHs in endothelial dysfunction of tobacco smokers should therefore be further studied.

Measurement of DNA damage by the comet assay in rat embryos grown in media containing high concentrations of vitamin K(1).

It has been suggested that vitamin K(1) (phylloquinone) can cause genetic damage in rapidly dividing cells and that this should be considered in the risk/benefit analysis of the prophylactic use of vitamin K(1) in the newborn. Usual intramuscular administration of 1mg of vitamin K(1) to the newborn gives peak plasma levels of 1-2 microg/ml (approximately 2-4 microM). To investigate the possible harmful effects of high concentrations of vitamin K(1), rat embryos undergoing rapid cell division in the organogenic period were cultured for 46 hours in rat sera containing either 1, 10 or 100 microg of added vitamin K(1) per ml (2, 22 or 222 microM). At the end of the culture period the embryos were dissociated and the cells examined for evidence of DNA damage using the alkaline version of the comet assay. Control embryos were cultured in sera without added vitamin K(1) and positive controls were control embryos exposed to hydrogen peroxide at the end of the culture period. The results did not show any evidence of DNA damage in the vitamin K(1) exposed embryos. The positive controls showed a significant increase in tail length, moment and inertia. In conclusion, under the experimental conditions used, high concentrations of vitamin K(1) did not induce primary DNA damage in cells from rat embryos grown in vitro.

Mobilization of Ca2+ stores in individual pancreatic beta-cells permeabilized or not with digitonin or alpha-toxin.

The concentration of free Ca2+ in the cytoplasm and organelles of individual mouse pancreatic beta-cells was estimated with dual wavelength microfluorometry and the indicators Fura-2 and furaptra. Measuring the increase of cytoplasmic Ca2+ resulting from intracellular mobilization of the ion in ob/ob mouse beta-cells, most organelle calcium (92%) was found in acidic compartments released when combining the Ca2+ ionophore Br-A23187 with a protonophore. Only 3-4% of organelle calcium was recovered from a pool sensitive to the Ca(2+)-ATPase inhibitor thapsigargin. Organelle Ca2+ was also measured directly in furaptra-loaded beta-cells after controlled plasma membrane permeabilization. The permeabilizing agent alpha-toxin was superior to digitonin in preserving the integrity of intracellular membranes, but digitonin provided more reproducible access to intracellular sites. After permeabilization, the thapsigargin-sensitive fraction of Ca2+ detected by furaptra was as high as 90%, suggesting that the indicator essentially measures Ca2+ in endoplasmic reticulum (ER). Both alpha-toxin- and digitonin-permeabilized cells exhibited ATP-dependent uptake of Ca2+ into thapsigargin-sensitive stores with half-maximal and maximal filling at 6-11 microM and 1 mM ATP respectively. Most of the thapsigargin-sensitive Ca2+ was mobilized by inositol 1,4,5-trisphosphate (IP3), whereas caffeine, ryanodine, cyclic ADP ribose and nicotinic acid adenine dinucleotide phosphate lacked effects both in beta-cells from ob/ob mice and normal NMRI mice. Mobilization of organelle Ca2+ by 4-chloro-3-methylphenol was attributed to interference with the integrity of the ER rather than to activation of ryanodine receptors. The observations emphasize the importance of IP3 for Ca2+ mobilization in pancreatic beta-cells, but question a role for ryanodine receptor agonists.

Glucose regulation of free Ca(2+) in the endoplasmic reticulum of mouse pancreatic beta cells.

Free Ca(2+) was measured in organelles of individual mouse pancreatic beta cells loaded with the low affinity indicator furaptra. After removal of cytoplasmic indicator by controlled digitonin permeabilization the organelle Ca(2+) was located essentially in the endoplasmic reticulum (ER), >90% being sensitive to inhibition of sarco(endo)plasmic reticulum Ca(2+)-ATPases. The Ca(2+) accumulation in the ER of intact beta cells depended in a hyperbolic fashion on the glucose concentration with half-maximal and maximal filling at 5.5 and >20 mM, respectively. Also elevation of cytoplasmic Ca(2+) by K(+) depolarization significantly enhanced the Ca(2+) accumulation. In permeabilized beta cells 1-3 mM ATP caused rapid Ca(2+) filling of the ER reaching almost 500 microM. At 50 nM, Ca(2+) ER became half-maximally filled at 45 microM ATP, whereas only 3.5 microM ATP was required at 200 nM Ca(2+). Inositol 1,4,5-trisphosphate induced a rapid release of about 65% of the ER Ca(2+), and its precursor phosphatidylinositol 4,5-bisphosphate was found to slowly mobilize 75% by another mechanism. It is concluded that glucose is an efficient stimulator of Ca(2+) uptake in the ER of pancreatic beta cells both by increasing ATP and cytoplasmic Ca(2+). Because physiological concentrations of cytoplasmic ATP are in the mM range, Ca(2+) sequestration can be anticipated to be modulated by factors reducing its ATP sensitivity.

Ca2+ signaling in mouse pancreatic polypeptide cells.

Ca2+ signaling was studied in pancreatic polypeptide (PP)-secreting cells isolated from mouse islets of Langerhans. After measuring the cytoplasmic Ca2+ concentration ([Ca2+]i), the cells were identified by immunocytochemistry. Most PP-cells reacted to carbachol and epinephrine with prompt and reversible elevation of [Ca2+]i, often manifested as slow oscillations. The carbachol effect was muscarinic, because it was inhibited by atropine. Beta-adrenergic elevation of cAMP explains the epinephrine stimulation, which was mimicked by an activator of adenylate cyclase and blocked by an inhibitor of protein kinase A. The responses to carbachol and epinephrine apparently involve depolarization with opening of voltage-dependent Ca2+ channels, because the effects were prevented by the Ca2+ channel antagonist methoxyverapamil and by diazoxide, which activates ATP-dependent K+ (K(ATP)) channels. Being equipped with K(ATP) channels, the PP-cells often responded to tolbutamide or high concentrations of glucose with elevation of [Ca2+]i. Somatostatin reversed the [Ca2+]i elevation obtained by carbachol, epinephrine, tolbutamide, and glucose. These preliminary studies support the idea that glucose has a direct stimulatory effect on the PP-cells, which can be masked by locally released somatostatin. Expressing both K(ATP) channels and voltage-dependent Ca2+ channels, the PP-cells share fundamental regulatory mechanisms with other types of islet cells.

Alkaline single cell gel electrophoresis and human biomonitoring for genotoxicity: a study on subjects with residential exposure to radon.

Based on theoretical estimates and various correlation studies, it has been suggested that ingestion of radon in drinking water represents an increased risk for cancer. Such a risk has never been conclusively shown in epidemiological or experimental animal studies, however, and it has been questioned whether the radon level in the drinking water is of any significance in terms of overall radon exposure. Using primary DNA damage as a biological marker for an ongoing exposure to ionising radiation, the present study was undertaken to investigate whether people with different types of residential radon exposures differed with regard to their levels of DNA damage in circulating lymphocytes. DNA damage was measured in coded blood samples from 125 residents living in 45 households with different levels of radon-222 in the drinking water (10-2410 Bq/l) and indoor air (35-1025 Bq/m3) using alkaline single cell gel electrophoresis (the 'Comet' assay). Increased levels of radon in indoor air (>200 Bq/m3) were found to be associated with an increased level of DNA damage in peripheral lymphocytes (P

Voltage-dependent entry and generation of slow Ca2+ oscillations in glucose-stimulated pancreatic beta-cells.

The role of voltage-dependent Ca2+ entry for glucose generation of slow oscillations of the cytoplasmic Ca2+ concentration ([Ca2+]i) was evaluated in individual mouse pancreatic beta-cells. Like depolarization with K+, a rise of the glucose concentration resulted in an enhanced influx of Mn2+, which was inhibited by nifedipine. This antagonist of L-type Ca2+ channels also blocked the slow oscillations of [Ca2+]i induced by glucose. The slow oscillations occurred in synchrony with variations in Mn2+ influx and bursts of action currents, with the elevation of [Ca2+]i being proportional to the frequency of the action currents. A similar relationship was obtained when Ca2+ was replaced with Sr2+. Occasionally, the slow [Ca2+]i oscillations were superimposed with pronounced spikes temporarily arresting the action currents. It is concluded that the glucose-induced slow oscillations of [Ca2+]i are caused by periodic depolarization with Ca2+ influx through L-type channels. Ca2+ spiking, due to intracellular mobilization, may be important for chopping the slow oscillations of [Ca2+]i into shorter ones characterizing beta-cells situated in pancreatic islets.

Amino acid transformation of oscillatory Ca2+ signals in mouse pancreatic beta-cells.

Glucose-induced increase of cytoplasmic Ca2+ in pancreatic beta-cells is usually manifested as slow oscillations from the basal level. The significance of this rhythmicity for maintaining normal beta-cell function with periodic variations of circulating insulin made it of interest to investigate how the oscillatory Ca2+ signal was affected by various amino acids. Individual mouse beta-cells were very sensitive to alanine, glycine and arginine, sometimes responding with a transformation of the oscillations into sustained elevation of cytoplasmic Ca2+ at amino acid concentrations as low as 0.1 mM. Stimulation of the entry of Ca2+, obtained either by raising the extracellular concentration or by prolonging the open state of the voltage-dependent Ca2+ channels with BAY K 8644, resulted in reappearance of the rhythmic activity in the presence of the amino acids. Oscillatory Ca2+ signals in intact islets were more resistant to transformation by amino acids than those of individual beta-cells. It is therefore suggested that signals from the adjacent cells make it possible for beta-cells situated in islets to overcome a suppression of the oscillatory activity otherwise seen in the presence of alanine, glycine or arginine.