Liver embolization with trisacryl gelatin microspheres (embosphere) in patients with neuroendocrine tumors.

PURPOSE: To report our experience of liver embolization with trisacryl gelatin microspheres (Embospheretrade mark) in patients with metastatic neuroendocrine tumors. MATERIAL AND METHODS: Fifteen patients underwent selective embolization of the right or left hepatic artery with Embosphere. One lobe was embolized in seven patients and both lobes, on separate occasions, in eight patients. Seven patients had midgut carcinoids, two had lung carcinoids, one suffered from a thymic carcinoid, and five had endocrine pancreatic tumors. Eight patients suffered from endocrine symptoms, seven of whom had carcinoid syndrome and one WDHA (watery diarrhea, hypokalemia, achlorhydria) syndrome. RESULTS: Partial radiological response was seen after eight embolizations (in six different patients), stable disease was observed after 13 embolizations (after three of these, necroses occurred), while radiological progression was noted after only two embolizations. Only two patients experienced a biochemical response. Clinical improvement of carcinoid syndrome was observed after five embolizations. There were no major complications. Fever >38 degrees C was seen after all but four embolizations, and urinary tract infections were diagnosed after eight embolizations. CONCLUSION: Selective hepatic artery embolization with Embosphere particles is a safe treatment for patients with metastatic neuroendocrine tumors and may lead to partial radiological response as well as symptomatic improvement of disabling endocrine symptoms.

Extracellular calcium increases free cytoplasmic calcium and DNA synthesis in human osteoblasts.

A high concentration of extracellular calcium (8 mM) induced an increase in free cytoplasmic calcium, a lower cyclic AMP level and increased DNA synthesis in primary cultures of human osteoblast-like cells. Inhibition of protein kinase C with bisindolylmaleimide I inhibited the stimulatory effect of extracellular calcium on DNA synthesis in human osteoblast-like cells, whereas inhibition of protein kinase A with Rp-cAMPs had no effect on DNA synthesis. This indicates that protein kinase C, possibly via increased free cytoplasmic calcium, mediates the effect of extracellular calcium on DNA synthesis in osteoblast-like cells rather than a relative decrease in cyclic AMP and protein kinase A activity. Furthermore, a low concentration (0.5 mM) of extracellular calcium decreased DNA synthesis. In conclusion, these data suggest that a high extracellular calcium level may be a coupling factor that recruits osteoblasts in the bone remodeling process, and that a low level of extracellular calcium may also regulate osteoblast function.

Weak evidence of thyrotropin receptors in primary cultures of human osteoblast-like cells.

Extra-thyroidal thyrotropin (TSH) receptors (TSHRs) have been demonstrated in several tissues and cells, including human and rat osteosarcoma cell lines. We have explored whether human TSHR (hTSHRs) also are present in primary cultures of human osteoblast-like (hOB) cells. [(125) I]TSH binding was limited in hOB cells, but somewhat higher in UMR 106-01 cells and considerably higher in hTSHR-transfected CHO cells. In hOB cells, the basal intracellular cAMP levels increased 282% after stimulation with 10 U/L TSH. In the hTSHR-transfected CHO cells, the cAMP increase was 3030% in response to 10 U/L TSH and 1240% after 1 U/L TSH. Free cytoplasmic calcium did not change in response to TSH in hOB cells. HTSHR mRNA was detected in hOB cells from 3/4 bone by reverse transcriptase polymerase chain reaction RT-PCR and nucleotide sequencing HTSHR mRNA, but could not be demonstrated with the RNase protection technique in hOB cells from 5 different donors. In conclusion, even after the use of several methods, we have found only weak evidence for expression and presence of functionally active hTSHR in hOB cells. Given the low level of expression, specific binding and cAMP signaling, we suggest that it is unlikely that circulating TSH plays a physiological role for bone metabolism mediated through osteoblasts.

Glucose-induced oscillations in cytoplasmic free Ca2+ concentration precede oscillations in mitochondrial membrane potential in the pancreatic beta-cell.

Using dual excitation and fixed emission fluorescence microscopy, we were able to measure changes in cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)) and mitochondrial membrane potential simultaneously in the pancreatic beta-cell. The beta-cells were exposed to a combination of the Ca(2+) indicator fura-2/AM and the indicator of mitochondrial membrane potential, rhodamine 123 (Rh123). Using simultaneous measurements of mitochondrial membrane potential and [Ca(2+)](i) during glucose stimulation, it was possible to measure the time lag between the onset of mitochondrial hyperpolarization and changes in [Ca(2+)](i). Glucose-induced oscillations in [Ca(2+)](i) were followed by transient depolarizations of mitochondrial membrane potential. These results are compatible with a model in which nadirs in [Ca(2+)](i) oscillations are generated by a transient, Ca(2+)-induced inhibition of mitochondrial metabolism resulting in a temporary fall in the cytoplasmic ATP/ADP ratio, opening of plasma membrane K(ATP) channels, repolarization of the plasma membrane, and thus transient closure of voltage-gated L-type Ca(2+) channels.

Glucose-dependent insulinotropic hormone potentiates the hypoglycemic effect of glibenclamide in healthy volunteers: evidence for an effect on insulin extraction.

Glucose-dependent insulinotropic hormone (GIP) is an intestinal hormone considered to be an important mediator of the incretin effect, i.e. the augmented insulin release observed in response to orally, compared with iv, administered glucose, despite isoglycemic glucose profiles. Stimulation of beta-cell secretion of insulin by GIP is seen both in vitro and in vivo at permissive extracellular glucose concentrations (> 6 mmol/L). It has also been claimed that part of the incretin effect is due to decreased insulin extraction. We now show that an infusion of GIP in healthy volunteers in whom blood glucose levels were maintained at 5 mmol/L, increased glibenclamide-stimulated levels of plasma insulin without significantly changing the C peptide profile. The increased plasma insulin levels necessitated extra glucose infusion to maintain euglycemia, demonstrating the biological significance of the elevated insulin levels. Infusion of GIP alone caused neither glucose changes nor elevation of C peptide or insulin levels. Hence, our results show that at a blood glucose concentration of 5 mmol/L, GIP augments the increase in plasma insulin levels stimulated by glibenclamide, possibly acting through a mechanism involving decreased insulin extraction in the liver or peripheral tissues, thus increasing insulin availability.

Spontaneous and stimulated transients in cytoplasmic free Ca(2+) in normal human osteoblast-like cells: aspects of their regulation.

We characterize two patterns of transients in cytoplasmic free calcium ([Ca2+]i) in normal human osteoblast-like cells (hOB cells). Firstly, spontaneous oscillations in [Ca2+]i were found to be common. The [Ca2+]i oscillations were completely inhibited by thapsigargin, indicating that Ca2+ fluxes between intracellular Ca2+ pools and the cytosol contributed to the generation of the [Ca2+]i oscillations. Removing extracellular Ca2+ either attenuated or completely inhibited spontaneous [Ca2+]i oscillations. Gadolinium, an inhibitor of stretch activated cation channels (SA-cat channels), reduced the frequency of [Ca2+]i oscillations. Hence, entry of calcium from the extracellular space, possibly through SA-cat channels also seemed to be of importance in the regulation of these [Ca2+]i oscillations. The role of the observed spontaneous [Ca2+]i oscillations in hOB cell function is not clear. Secondly, a decrease in pericellular osmolality, which causes the plasma membrane to stretch, transiently increased [Ca2+]i in hOB cells. This effect was also observed in a Ca2+ free extracellular environment, suggesting that osmotic stimuli release Ca2+ from intracellular pools. This finding indicates a possible signaling pathway by which mechanical strain can promote anabolic effects on the human skeleton.

Short- and long-term effects of beta-cellulin and transforming growth factor-alpha on beta-cell function in cultured fetal rat pancreatic islets.

The polypeptide beta-cellulin, identified in conditioned media from insulinoma cell cultures and produced by pancreatic islet cells, was recently identified as a possible autocrine growth factor for the pancreatic islet beta-cell. In this study, we investigated the short- and long-term actions of beta-cellulin, and the structurally related transforming growth factor-alpha (TGF-alpha), on beta-cell function in fetal rat pancreatic islets in vitro. We found that neither beta-cellulin nor TGF-alpha (10 nM each), in contrast to glucose (20 mM), acutely influenced beta-cell levels of cytosolic-free Ca2+. Additionally, whereas glucose markedly increased short-term (60-min) insulin release, neither beta-cellulin nor TGF-alpha (10 nM each) influenced the rate of hormone secretion at basal (3 mM) or stimulatory (20 mM) concentrations of glucose. Likewise, long-term (24-h) exposure of islets to a high glucose concentration significantly augmented the secretion of insulin. This effect was slightly potentiated by TGF-alpha (10 nM), but not beta-cellulin (10 nM), at high (but not low) glucose concentrations. Conversely, the islet insulin content was not significantly affected by beta-cellulin or TGF-alpha at any glucose concentration tested. We conclude that, although beta-cellulin is produced by islet cells, the peptide does not seem to be of importance for the regulation of insulin production by isolated pancreatic beta-cells.

[Mechanisms of action of peroral antidiabetics. Sulfonylurea preparations block the ATP-dependent potassium channels]. / Perorala antidiabetikas verkningsmekanism. Sulfonylureapreparat stänger ATP-reglerad kaliumkanal.

Although hypoglycaemic sulphonylureas have been used to treat non-insulin-dependent diabetes mellitus (NIDDM) for the past forty years, their mechanisms of action at the molecular level have only recently been elucidated. A combination of electrophysiological and molecular biological techniques showed the target of sulphonylureas to be a sulphonylurea receptor (SUR1) and potassium channel (Kir6.2) complex. Together, these two proteins form the ATP-dependent potassium (KATP) channel occurring in insulin-secreting cells. An increase in the blood glucose level triggers a chain of events in insulin-secreting cells and K(ATP) channel closure which is a prerequisite for insulin secretion. In NIDDM, however, an increase in blood glucose fails to close the K(ATP) channel satisfactorily, but this can be remedied by the administration of sulphonylureas.

Oscillations in KATP channel activity promote oscillations in cytoplasmic free Ca2+ concentration in the pancreatic beta cell.

Pancreatic beta cells exhibit oscillations in electrical activity, cytoplasmic free Ca2+ concentration ([Ca2+](i)), and insulin release upon glucose stimulation. The mechanism by which these oscillations are generated is not known. Here we demonstrate fluctuations in the activity of the ATP-dependent K+ channels (K(ATP) channels) in single beta cells subject to glucose stimulation or to stimulation with low concentrations of tolbutamide. During stimulation with glucose or low concentrations of tolbutamide, K(ATP) channel activity decreased and action potentials ensued. After 2-3 min, despite continuous stimulation, action potentials subsided and openings of K(ATP) channels could again be observed. Transient suppression of metabolism by azide in glucose-stimulated beta cells caused reversible termination of electrical activity, mimicking the spontaneous changes observed with continuous glucose stimulation. Thus, oscillations in K(ATP) channel activity during continuous glucose stimulation result in oscillations in electrical activity and [Ca2+](i).

Dissociation between exocytosis and Ca(2+)-channel activity in mouse pancreatic beta-cells stimulated with calmidazolium (compound R24571).

Calmidazolium, a calmodulin inhibitor, suppressed influx of Ca2+ through voltage-gated Ca2+ channels in mouse pancreatic beta-cells. Despite this fact, calmidazolium stimulated insulin release from beta-cells at basal glucose concentration. This effect was not mediated by protein kinase C (PKC), since it persisted in PKC-depleted cells. RpcAMPS significantly attenuated the calmidazolium-stimulated insulin secretion, indicating that calmidazolium acts, at least partly, through PKA. The compound also stimulated insulin secretion from electropermeabilized beta-cells, indicating effects on distal steps in the stimulus-secretion coupling. The use of calmidazolium offers possibilities to investigate the mechanisms activating exocytosis under conditions where the cytoplasmic-free Ca2+ concentration does not increase.

Measurements of cytoplasmic free Ca2+ concentration in human pancreatic islets and insulinoma cells.

In human pancreatic islets an increase in the glucose concentration from 3 to 20 mM raised the free cytoplasmic Ca2+ concentration [( Ca2+]i), an effect being reversible upon withdrawal of the sugar. Depolarization with a high concentration of K+ or the sulphonylurea tolbutamide also raised [Ca2+]i. Addition of extracellular ATP produced a transient rapid rise in [Ca2+]i. Oscillations in [Ca2+]i were observed in the presence of 10 mM glucose. Insulinoma cells responded to glucose and tolbutamide with increases in [Ca2+]i, whereas the sulphonamide diazoxide caused a decrease in [Ca2+]i. These findings confirm previous results obtained in rodent beta-cells.

Inositol trisphosphate-dependent periodic activation of a Ca(2+)-activated K+ conductance in glucose-stimulated pancreatic beta-cells.

Glucose-stimulated insulin secretion is associated with the appearance of electrical activity in the pancreatic beta-cell. At intermediate glucose concentrations, beta-cell electrical activity follows a characteristic pattern of slow oscillations in membrane potential on which bursts of action potentials are superimposed. The electrophysiological background of the bursting pattern remains unestablished. Activation of Ca(2+)-activated large-conductance K+ channels (KCa channel) has been implicated in this process but seems unlikely in view of recent evidence demonstrating that the beta-cell electrical activity is unaffected by the specific KCa channel blocker charybdotoxin. Another hypothesis postulates that the bursting arises as a consequence of two components of Ca(2+)-current inactivation. Here we show that activation of a novel Ca(2+)-dependent K+ current in glucose-stimulated beta-cells produces a transient membrane repolarization. This interrupts action potential firing so that action potentials appear in bursts. Spontaneous activity of this current was seen only rarely but could be induced by addition of compounds functionally related to hormones and neurotransmitters present in the intact pancreatic islet. K+ currents of the same type could be evoked by intracellular application of GTP, the effect of which was mediated by mobilization of Ca2+ from inositol 1,4,5-trisphosphate (InsP3)-sensitive intracellular Ca2+ stores. These observations suggest that oscillatory glucose-stimulated electrical activity, which is correlated with pulsatile release of insulin, results from the interaction between the beta-cell and intraislet hormones and neurotransmitters. Our data also provide evidence for a close interplay between ion channels in the plasma membrane and InsP3-induced mobilization of intracellular Ca2+ in an excitable cell.

Extracellular ATP increases cytoplasmic free Ca2+ concentration in clonal insulin-producing RINm5F cells. A mechanism involving direct interaction with both release and refilling of the inositol 1,4,5-trisphosphate-sensitive Ca2+ pool.

Effects of extracellularly applied ATP (added as disodium salt) on stimulus-secretion coupling were investigated in clonal insulin-producing RINm5F cells. Cytoplasmic free Ca2+ concentration [( Ca2+]i), electrical activity, membrane potential, formation of InsP3 and insulin release were measured. Addition of ATP in a Ca2(+)-containing medium promoted a rapid rise in [Ca2+]i, which was followed by a slow decline towards the basal level. In a Ca2(+)-free medium, the ATP-induced increase in [Ca2+]i was smaller, but still enough to elicit insulin secretion. Upon normalization of the extracellular Ca2+ concentration, the response to ATP recovered instantaneously. The presence of glucose in the incubation medium was a prerequisite to obtain a pronounced effect of ATP in the absence of extracellular Ca2+. However, glucose did not enhance the response to ATP in a Ca2(+)-containing medium. The effect of ATP was dose-dependent, with a clearly detectable increase in [Ca2+]i at 1 microM and a maximal response being obtained at 200 microM-ATP. The response to ATP was unaffected by activating adenylate cyclase by forskolin, but was abolished by 10 nM of the phorbol ester phorbol 12-myristate 13-acetate. The effects of ATP on [Ca2+]i could not be accounted for by a generalized increase in plasma-membrane permeability, as evident from the failure of the nucleotide to increase the fluorescence of the nuclear stain ethidium bromide. After stimulation with ATP there was an increase in membrane potential, in both the absence and the presence of extracellular Ca2+. Blockage of the voltage-activated Ca2+ channals with D-600, in a Ca2(+)-containing medium, decreased the effect of ATP on [Ca2+]i slightly. Patch-clamp measurements using the cell-attached patch configuration revealed that the RINm5F cells produce spontaneous action potentials, the frequency of which increased markedly on addition of ATP. Whole-cell recordings demonstrated that the increase in spike frequency was not associated with the development of an inward current, but was rather accountable for by a decrease in the activity of the ATP-regulated K+ channels. Addition of 200 microM-ATP stimulated phospholipase C activity, as evident from the formation of InsP3, both in the absence and in the presence of extracellular Ca2+. Thus in the absence of extracellular Ca2+ the stimulatory effect of ATP on insulin release can be explained by InsP3-induced mobilization of intracellularly bound Ca2+. Hence, in the RINm5F cells extracellular ATP acts in a manner similar to other Ca2(+)-mobilizing agents.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibition of inositol trisphosphate-stimulated calcium mobilization by calmodulin antagonists in rat liver epithelial cells.

Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), an intracellular second messenger produced from the hydrolysis of phosphatidylinositol 4,5-bisphosphate, interacts with cytoplasmic membrane structures to elicit the release of stored Ca2+. Ins(1,4,5)P3-induced Ca2+ mobilization is mediated through high affinity receptor binding sites; however, the biochemical mechanism coupling receptor occupation with Ca2+ channel opening has not been identified. In studies presented here, we examined the effects of naphthalenesulfonamide calmodulin antagonists, W7 and W13, and a new selective antagonist, CGS 9343B, on Ca2+ mobilization stimulated by Ins(1,4,5)P3 in neoplastic rat liver epithelial (261B) cells. Intact fura-2 loaded cells stimulated by thrombin, a physiological agent that causes phosphatidylinositol 4,5-bisphosphate hydrolysis and Ins (1,4,5)P3 release, responded with a rise in cytoplasmic free Ca2+ levels that was dose dependently inhibited by W7(Ki = 25 microM), W13 (Ki = 45 microM), and CGS 9343B (Ki = 110 microM). Intracellular Ca2+ release stimulated by the addition of Ins(1,4,5)P3 directly to electropermeabilized 261B cells was similarly inhibited by pretreatment with anti-calmodulin agents. W7 and CGS 9343B, which potently blocked Ca2+/calmodulin-dependent protein kinase, had no significant effect on protein kinase A or C in dose range required for complete inhibition of Ca2+ mobilization. Ca2+ release channels and Ca2+-ATPase pump activity were also unaffected by calmodulin antagonist treatment. These results indicate that calmodulin is tightly associated with the intracellular membrane mechanism coupling Ins(1,4,5)P3 receptors to Ca2+ release channels

Epidermal growth factor-stimulated DNA synthesis requires an influx of extracellular calcium.

The dependency of normal cell proliferation on adequate extracellular Ca2+ levels was further investigated by determining the role of Ca2+ influx in epidermal growth factor (EGF)-induced rat liver epithelial (T51B) cell DNA synthesis. Fura-2-loaded T51B cells responded with an increase in [Ca2+]i to EGF (5-50 ng/ml) that was blocked by low (25 microM) extracellular Ca2+ or by pretreatment with 50 microM La3+ to inhibit plasma membrane Ca2+ flux. Confluent T51B cells treated for 24 h with EGF (0.1-50 ng/ml) dose-dependently incorporated [3H]-thymidine into cell nuclei. Low extracellular Ca2+ or addition of La3+ prevented the EGF-stimulated rise in labeled nuclei, indicating that a movement of Ca2+ into the cell was required for DNA synthesis. This was supported by our findings that bradykinin, which induced a rise in [Ca2+]i by opening plasma membrane Ca2+ channels in T51B cells (but not A23187, thrombin or ATP, which raise [Ca2+]i primary through mobilization of intracellular Ca2+ stores), potentiated DNA synthesis stimulated by submaximal doses of EGF. Potentiation of the action of EGF by the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA), indicates that activation of protein kinase C and an influx of Ca2+ share a common mechanism for initiating DNA synthesis.