Survival of encapsulated human primary fibroblasts and erythropoietin expression under xenogeneic conditions.

Allogeneic cells are the most attractive source for cell transplantation, as the use of xenogeneic cells is hampered by safety concerns and the use of autologous cells involves practical difficulties. The immune rejection of allogeneic cells can be overcome by physical immunoprotection provided by polymer encapsulation. To study the variability of cell and donor sources, we compared different primary human cells as candidates for gene therapy-mediated delivery of human erythropoietin (hEpo). DARC-3.1 fibroblasts, MDX-01 fibroblasts, and ARPE-19 retinal pigment epithelial cells were encapsulated into polyethersulfone hollow fibers and implanted for 1 month in nude mice as well as in immunocompetent and FK506-immunosuppressed mice to test their in vivo resistance, with the assumption that xenogeneic conditions constitute a stringent model for human application. DARC-3.1 fibroblasts showed the best survival, prompting us to evaluate cell lineages from the same donor (DARC-3.2) or another donor (DARC-4.3 and DARC-4.4). With the exception of DARC-4.3, the remaining three lineages showed comparable survival in immunocompetent C3H and DBA/2J mice. DARC-3.1 fibroblasts were retrovirally engineered with hEpo cDNA, reaching a secretion level of 170 IU of hEpo per 10(6) cells per day. Encapsulated DARC-3.1-hEpo cells led to significantly increased hematocrits in the various hosts and under various transplantation conditions. The present study shows that encapsulated primary human DARC-3.1 fibroblasts are able to survive under xenogeneic conditions and, once engineered with hEpo cDNA, to increase the hematocrit of transplanted mice.

In vivo calcium deposition on polyvinyl alcohol matrix used in hollow fiber cell macroencapsulation devices.

The encapsulation of genetically modified cells represents a promising approach for the delivery of therapeutic proteins. The functionality of the device is dependent on the characteristics of the biomaterials, the procedures used in its confection and the adaptability of the encapsulated cells in the host. We report conditions leading to the development of calcifications on the polyvinyl alcohol (PVA) matrix introduced in hollow fiber devices for the encapsulation of primary human fibroblasts implanted in mice. The manufacturing procedures, batches of PVA matrix and cell lineages were assessed for their respective role in the development of the phenomenon. The results showed that the calcification is totally prevented by substituting phosphate-buffer saline with ultra-pure sterile water in the rinsing procedure of the matrix. Moreover, a positive correlation was found, when comparing two fibroblast cell lineages, between the level of lactate dehydrogenase (LDH) activity measured in the cells and the degree of calcium deposition. Higher LDH activity may decrease calcium depositions because it generates in the device a more acidic microenvironment inhibiting calcium precipitation. The present study defines optimized conditions for the encapsulation of primary human fibroblasts in order to avoid potentially detrimental calcifications and to allow long-term survival of encapsulated cells.

Glucose-6-phosphatase in the insulin secreting cell line INS-1.

The glucose-6-phosphatase system of the glucose sensitive insulin secreting rat insulinoma cells (INS-1) was investigated. INS-1 cells contain easily detectable levels of glucose-6-phosphatase enzyme protein (assessed by Western blotting) and have a very significant enzymatic activity. The features of the enzyme (Km and Vmax values, sensitivity to acidic pH, partial latency, and double immunoreactive band) are similar to those of the hepatic form. On the other hand, hardly detectable levels of glucose-6-phosphatase activity and protein were present in the parent glucose insensitive RINm5F cell line. The mRNA of the glucose-6-phosphate transporter was also more abundant in the INS-1 cells. The results support the view that the glucose-6-phosphatase system of the beta-cell is associated with the regulation of insulin secretion.

Human gingival crevicular fluid contains MRP8 (S100A8) and MRP14 (S100A9), two calcium-binding proteins of the S100 family.

Human gingival crevicular fluid contains unidentified proteins which might play a role as markers in periodontal diseases. Therefore, low-molecular-weight proteins found in human gingival crevicular fluid (GCF), but absent from serum, were identified in the present study by means of two-dimensional electrophoresis (2-D PAGE) analysis. GCF, serum, and whole saliva were collected from periodontitis and healthy subjects, as well as from edentulous and newborn subjects. Protein samples were separated by two-dimensional polyacrylamide gel electrophoresis, stained with silver, and compared with reference protein maps in the SWISS-2D PAGE database. In GCF and saliva from periodontitis patients and healthy subjects, four dominant low-molecular-mass (from 8 to 14 kDa) acidic spots were observed. They were not found in serum and were less visible in saliva from edentulous and newborn subjects. From N-terminal amino acid sequencing, the two 2-D protein spots of 8 kDa and isoelectric points between 6.5 and 7.0 were both identified as protein MRP8 (SI00A8), a member of the S100 family of calcium-binding proteins. Using peptide mass fingerprinting and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS), we identified the other two protein spots, with mass of 14 kDa and isoelectric points between 5.5 and 6.0, as protein MRP14 (S100A9), also belonging to the S100 family. The presence of MRP8 and MRP14 in GCF was confirmed by Western blot, with monoclonal antibodies. The two polypeptides, MRP8 and MRP14, identified in GCF represent the major difference between the 2-D PAGE patterns of serum and GCF, and we hypothesize that they may play an important role in the gingival sulcus and could represent possible markers for periodontal diseases.

Lentivirus-mediated Bcl-2 expression in betaTC-tet cells improves resistance to hypoxia and cytokine-induced apoptosis while preserving in vitro and in vivo control of insulin secretion.

betaTC-tet cells are conditionally immortalized pancreatic beta cells which can confer long-term correction of hyperglycemia when transplanted in syngeneic streptozocin diabetic mice. The use of these cells for control of type I diabetes in humans will require their encapsulation and transplantation in non-native sites where relative hypoxia and cytokines may threaten their survival. In this study we genetically engineered betaTC-tet cells with the anti-apoptotic gene Bcl-2 using new lentiviral vectors and showed that it protected this cell line against apoptosis induced by hypoxia, staurosporine and a mixture of cytokines (IL-1beta, IFN-gamma and TNF-alpha). We further demonstrated that Bcl-2 expression permitted growth at higher cell density and with shorter doubling time. Expression of Bcl-2, however, did not inter- fere either with the intrinsic mechanism of growth arrest present in the betaTC-tet cells or with their normal glucose dose-dependent insulin secretory activity. Furthermore, Bcl-2 expressing betaTC-tet cells retained their capacity to secrete insulin under mild hypoxia. Finally, transplantation of these cells under the kidney capsule of streptozocin diabetic C3H mice corrected hyperglycemia for several months. These results demonstrate that the murine betaTC-tet cell line can be genetically modified to improve its resistance against different stress-induced apoptosis while preserving its normal physiological function. These modified cells represent an improved source for cell transplantation therapy of type I diabetes.

Short-term insulin-induced glycogen formation in primary hepatocytes as a screening bioassay for insulin action.

We describe a novel bioassay to measure specific insulin-like activity in primary cultures of rat hepatocytes by determination of [3H]glycogen from d-[6-3H]glucose. The dose-response curve of insulin in this assay exhibited an EC50 of 0.42 (+/-0.04) nM, which is comparable to the dissociation constant of insulin from its receptor in hepatocytes. We used this assay to examine possible residual insulin-like activity of the four major fragments formed upon insulin degradation by insulin protease. Fragments A1-13B1-9, A1-14B1-9,and A14-21B14-30 showed no measurable activity. Although preparations of fragment A14-21B10-30 displayed dose-dependent agonist activity with an EC50 of 380 (+/-40) nM, we conclude that this was due to an insulin-like impurity since the chemically synthesized fragment showed no such activity. In summary, this bioassay demonstrates the action of insulin on glycogen formation in hepatocytes and provides a rapid and sensitive measurement of insulin-like activity which could facilitate screening studies.

Pancreatic beta-cell regeneration after 48-h glucose infusion in mildly diabetic rats is not correlated with functional improvement.

We investigated the effect of glucose infusion on beta-cell regeneration in rats made mildly diabetic by a single injection of low dosage (35 mg/kg) streptozotocin (STZ). Nondiabetic (ND) and STZ rats were submitted to a 48-h glucose infusion (hyperglycemia approximately 22 mmol/l in both groups: ND and STZ hyperglycemic-hyperinsulinemic [ND HG-HI and STZ HG-HI rats]). Before infusion, beta-cell mass was 65% lower in STZ rats than in ND rats (2.0 +/- 0.02 vs. 5.5 +/- 0.6 mg), 1.6-fold increased in ND HG-HI rats (8.7 +/- 1.7 mg), and 2.7-fold increased in STZ HG-HI rats (5.4 +/- 0.9 mg). In ND HG-HI rats, beta-cell enlargement was related to an increase in beta-cell responsiveness to nutrient secretagogues both in vivo and in vitro, whereas in STZ HG-HI rats, no significant improvement in insulin secretion could be noticed. To determine the respective role of hyperglycemia and hyperinsulinemia on beta-cell area changes, ND and STZ rats were submitted to a 48-h hyperinsulinemic-euglycemic clamp. No modification of beta-cell mass was detected in either group. In conclusion, 48-h superimposed hyperglycemia was enough to restore beta-cell mass previously reduced by STZ injection. This effect seemed to be due to hyperglycemia rather than hyperinsulinemia alone. The data stress the dissociation between beta-cell regeneration and improvement in islet function in diabetic rats. Our model seems suitable for studying factors that can improve the plasticity and function of the pancreas in NIDDM.

Histones and basic polypeptides activate Ca2+/cation influx in various cell types.

Histone H2A (1-10 microg/ml) added to Ehrlich ascite cell suspensions promoted: (i) Ca2+ influx, but no apparent intracellular Ca2+ mobilization; (ii) plasma-membrane depolarization and Na+ influx in Ca2+-free medium, which were recovered by Ca2+ readmission; (iii) influx of other cations such as Ba2+, Mn2+, choline+ and N-methyl-d-glucamine+, but not of propidium+, ethidium bromide and Trypan Blue. H2A-induced Ca2+ influx and cell depolarization were: (i) blocked by La3+ and Gd3+, but not by various inhibitors of receptor-activated Ca2+-influx pathways/channels; (ii) mimicked by various basic polypeptides, with Mr>4000; (iii) prevented or reversed by polyanions such as polyglutamate or heparin; (iv) present in other cell types, such as Jurkat, PC12 and Friend erythroleukaemia cells, but virtually absent from rat hepatocytes and thymocytes. We conclude that cationic proteins/polypeptides, by interacting in a cell-specific manner with the cell surface, can activate in those cells putative non-selective Ca2+ channels and membrane depolarization.

Glucose-induced insulin secretion in INS-1 cells depends on factors present in fetal calf serum and rat islet-conditioned medium.

To study the regulation of growth and differentiated function of insulin-secreting cells, the rat insulinoma cell line INS-1 was cultured in a defined serum-free medium containing prolactin, IGF-I, and triiodothyronine, which was originally reported to maintain insulin secretion of islet cells. Growth and viability, as well as cellular insulin content of INS-1 cells in the defined medium, were comparable to the control cells cultured in the complete medium containing 10% fetal calf serum. However, after a 3-day culture in this medium, insulin secretion in response to glucose, pyruvate, and leucine was markedly blunted compared with the control cells (-78, -68, and -56%, respectively), whereas the response to 30 mmol/l K+ was only slightly decreased. In these cells: 1) nutrient metabolism assessed by tetrazolium salt reduction was reduced in response to pyruvate and leucine, which are mainly metabolized in the mitochondria; 2) oxidation of both [3,4-(14)C]glucose and [1-(14)C]pyruvate was decreased (-22 and -32%, respectively); 3) glucose failed to depolarize the membrane potential, whereas tolbutamide was fully active; 4) video imaging analysis of cytosolic Ca2+ showed a decrease in the population of glucose-responsive cells, while the response to 30 mmol/l K+ was preserved; 5) serum replenishment for 3 days restored glucose-induced insulin secretion. Interestingly, conditioned serum-free medium from rat islets maintained the insulin secretory function of INS-1 cells, although glucagon, somatostatin, and some other factors failed to restore the function. In contrast, conditioned media from HepG2, PC12, and human umbilical vein endothelial cells did not substitute for serum. Thus, the impaired insulin secretion of the cells cultured in the defined medium is best explained by defective mitochondrial metabolism. Islet cells, but not INS-1 cells, produce factors required for normal signal generation by nutrient secretagogues.

Glucose-stimulated insulin secretion correlates with changes in mitochondrial and cytosolic Ca2+ in aequorin-expressing INS-1 cells.

Nutrient-stimulated insulin secretion is dependent upon the generation of metabolic coupling factors in the mitochondria of the pancreatic B cell. To investigate the role of Ca2+ in mitochondrial function, insulin secretion from INS-1 cells stably expressing the Ca2+-sensitive photoprotein aequorin in the appropriate compartments was correlated with changes in cytosolic calcium ([Ca2+]c) and mitochondrial calcium ([Ca2+]m). Glucose and KCl, which depolarize the cell membrane, as well as the Ca2+-mobilizing agonist, carbachol (CCh), cause substantial increases in [Ca2+]m which are associated with smaller rises in [Ca2+]c. The L-type Ca2+-channel blocker, SR7037, abolished the effects of glucose and KCl while attenuating the CCh response. Glucose-induced increases in [Ca2+]m, [Ca2+]c, and insulin secretion all demonstrate a pronounced initial peak followed by a sustained plateau. All three parameters are increased synergistically when glucose and CCh are combined. Finally, [Ca2+]m, [Ca2+]c, and insulin secretion also display desensitization phenomena following repeated additions of the three stimuli. The high sensitivity of [Ca2+]m to Ca2+ influx and the desensitization-resensitization effects can be explained by a model in which the mitochondria of INS-1 cells are strategically located to sense Ca2+ influx through plasma membrane Ca2+ channels. In conclusion, the correlation of [Ca2+]m and [Ca2+]c with insulin secretion may indicate a fundamental role for Ca2+ in the adaptation of oxidative metabolism to the generation of metabolic coupling factors and the energy requirements of exocytosis.

Functional N-methyl-D-aspartate receptors in O-2A glial precursor cells: a critical role in regulating polysialic acid-neural cell adhesion molecule expression and cell migration.

The capacity for long-distance migration of the oligodendrocyte precursor cell, oligodendrocyte-type 2 astrocyte (O-2A), is essential for myelin formation. To study the molecular mechanisms that control this process, we used an in vitro migration assay that uses neurohypophysial explants. We provide evidence that O-2A cells in these preparations express functional N-methyl-D-aspartate (NMDA) receptors, most likely as homomeric complexes of the NR1 subunit. We show that NMDA evokes an increase in cytosolic Ca2+ that can be blocked by the NMDA receptor antagonist AP-5 and by Mg2+. Blocking the activity of these receptors dramatically diminished O-2A cell migration from explants. We also show that NMDA receptor activity is necessary for the expression by O-2A cells of the highly sialylated polysialic acid-neural cell adhesion molecule (PSA-NCAM) that is required for their migration. Thus, glutamate or glutamate receptor ligands may regulate O-2A cell migration by modulating expression of PSA-NCAM. These studies demonstrate how interactions between ionotropic receptors, intracellular signaling, and cell adhesion molecule expression influence cell surface properties, which in turn are critical determinants of cell migration.

Oscillations of cytosolic free calcium in bombesin-stimulated HIT-T15 cells.

The mechanism underlying the generation of cytosolic free Ca2+ ([Ca2+]i) oscillations by bombesin, a receptor agonist activating phospholipase C, in insulin secreting HIT-T15 cells was investigated. At 25 microM, 61% of cells displayed [Ca2+]i oscillations with variable patterns. The bombesin-induced [Ca2+]i oscillations could last more than 1 h and glucose was required for maintaining these [Ca2+]i fluctuations. Bombesin-evoked [Ca2+]i oscillations were dependent on extracellular Ca2+ entry and were attenuated by membrane hyperpolarization or by L-type Ca2+ channel blockers. These [Ca2+]i oscillations were apparently not associated with fluctuations in plasma membrane Ca2+ permeability as monitored by the Mn2+ quenching technique. 2,5-di-(tert-butyl)-1,4-benzohydroquinone (tBuBHQ) and 4-chloro-m-cresol, which interfere with intracellular Ca2+ stores, respectively, by inhibiting Ca(2+)-ATPase of endoplasmic reticulum and by affecting Ca(2+)-induced Ca2+ release, disrupted bombesin-induced [Ca2+]i oscillations. 4-chloro-m-cresol raised [Ca2+]i by mobilizing an intracellular Ca2+ pool, an effect not altered by ryanodine. Caffeine exerted complex actions on [Ca2+]i. It raised [Ca2+]i by promoting Ca2+ entry while inhibiting bombesin-elicited [Ca2+]i oscillations. Our results suggest that in bombesin-elicited [Ca2+]i oscillations in HIT-T15 cells: (i) the oscillations originate primarily from intracellular Ca2+ stores; and (ii) the Ca2+ influx required for maintaining the oscillations is in part membrane potential-sensitive and not coordinated with [Ca2+]i oscillations. The interplay between intracellular Ca2+ stores and voltage-sensitive and voltage-insensitive extracellular Ca2+ entry determines the [Ca2+]i oscillations evoked by bombesin.

Postreceptor signalling of growth hormone and prolactin and their effects in the differentiated insulin-secreting cell line, INS-1.

Signal transduction of two mitogens for pancreatic beta-cells, GH and PRL, was investigated using the differentiated insulin-secreting cell line, INS-1. Addition of human GH (hGH) or ovine PRL in a serum-substitute medium increased growth, insulin content, and nutrient metabolism evaluated by tetrazolium salt reduction. hGH, bovine GH (bGH), and PRL also stimulated [3H]thymidine incorporation in a dose-dependent manner (1 pM - 1 nM). hGH induced cytosolic Ca2+ ([Ca2+]i) rises, which were transient, dependent on the presence of extracellular Ca2+, blocked by verapamil, calciseptine, and the hyperpolarizing agent diazoxide, suggesting that hGH stimulates Ca(2+)-influx through L-type Ca(2+)-channels. Similar effects on [Ca2+]i were observed with bGH or PRL. hGH caused membrane depolarization in a small proportion of the cells ( < 25%) as detected by cell-attached patch-clamp analysis. However, hGH failed to stimulate acute insulin secretion. hGH, bGH, and PRL promoted tyrosine phosphorylation of JAK2 tyrosine kinase. Verapamil inhibited neither [3H]thymidine incorporation nor JAK2 phosphorylation stimulated by hGH, whereas a tyrosine kinase inhibitor, lavendustin A, blocked the mitogenic effect. Involvement of cAMP is suggested because Rp-cyclic adenosine-3', 5'-monophosphorothioate, a competitive inhibitor of protein kinase A, abolished hGH-induced [Ca2+]i rises and DNA synthesis. cAMP appears to play a permissive role, although hGH failed to raise cellular cAMP levels. These results support the idea that activation of JAK2 is a major signaling event, whereas the [CA2+]i rise is not a prerequisite, for the mitogenic effects of GH and PRL in insulin-secreting cells.

Activity-dependent mobilization of the adhesion molecule polysialic NCAM to the cell surface of neurons and endocrine cells.

The alpha-2,8-linked sialic acid polymer (PSA) on the neural cell adhesion molecule (NCAM) is an important regulator of cell surface interactions. We have examined the translocation of PSA-NCAM to the surface of cultured cortical neurons and insulin secreting beta cells under different conditions of cell activity. Endoneuraminidase N, an enzyme that specifically cleaves PSA chains, was used to remove pre-existing PSA from the plasma membrane and the re-expression of the molecule was monitored by immunocytochemistry. Punctate PSA immunostaining was restored on the surface of 68% of neurons within 1 h. This recovery was almost completely prevented by tetrodotoxin, suggesting that spontaneous electrical activity is required. K+ depolarization (50 mM) allowed recovery of PSA surface staining in the presence of tetrodotoxin and this effect required the presence of extracellular Ca2+. Rapid redistribution of PSA-NCAM to the surface of beta cells was observed under conditions that stimulate insulin secretion. Ca2+ channel inhibition decreased both PSA-NCAM expression and insulin secretion to control, non-stimulated levels. Finally, subcellular fractionation of an insulin-secreting cell line showed that the secretory vesicle fraction is highly enriched in PSA-NCAM. These results suggest that PSA-NCAM can be translocated to the cell surface via regulated exocytosis. Taken together, our results provide unprecedented evidence linking cell activity and PSA-NCAM expression, and suggest a mechanism for rapid modulation of cell surface interactions.

Dynamic pacing of cell metabolism by intracellular Ca2+ transients.

During cell activation, Ca2+, by stimulating the NADH-producing mitochondrial dehydrogenases, triggers the generation of reducing equivalents whereby ATP production is sustained. In cell populations, [Ca2+] changes in the mitochondrial matrix were demonstrated to parallel rapidly those in the cytosol ([Ca2+]i). There is still no indication as to whether metabolic activation follows oscillatory patterns similar to those of [Ca2+]i. Therefore, changes in NAD(P)H were monitored in single pancreatic beta-cells, adrenal glomerulosa cells, and liver cells during oscillatory [Ca2+]i transients. Rapid NAD(P)H and [Ca2+]i oscillations with similar frequency and sensitive both to changes in glucose concentration and to extracellular Ca2+ removal were identified in a subpopulation of pancreatic beta-cells in primary culture. Furthermore, Ca(2+)-dependent oscillatory NAD(P)H formation could be evoked by the pulsatile application of depolarizing [K+], demonstrating the pacing effect of increased [Ca2+]i on beta-cell metabolism. In adrenal glomerulosa cells, angiotensin II, a physiological stimulator of aldosterone production, could be shown to elicit the oscillatory formation of mitochondrial NAD(P)H through frequency modulation of [Ca2+]i transients. In contrast to the two former endocrine cell types, in hepatocytes, [Arg8]vasopressin and epinephrine caused the amplitude modulation of NAD(P)H formation. Taken together, these results provide unprecedented evidence for a cell-specific pacing of metabolism by [Ca2+]i transients coordinated with cell activation and function.

In vivo phosphorylation of the Na,K-ATPase alpha subunit in sciatic nerves of control and diabetic rats: effects of protein kinase modulators.

The phosphorylation state of the Na,K-ATPase alpha subunit has been examined in 32P-labeled sciatic nerves of control and streptozotocin-treated diabetic rats. Intact nerves were challenged with protein kinase (PK) modulators and alpha-subunit 32P labeling was analyzed after immunoprecipitation. In control nerves, the PKC activator phorbol 12-myristate 13-acetate (PMA) had little effect on alpha-subunit 32P labeling. In contrast, staurosporine, a PKC inhibitor, and extracellular calcium omission decreased it. In Ca(2+)-free conditions, PMA restored the labeling to basal levels. The cAMP-raising agent forskolin reduced the 32P labeling of the alpha subunit. The results suggest that nerve Na,K-ATPase is tonically phosphorylated by PKC in a Ca(2+)-dependent manner and that PKA modulates the phosphorylation process. In nerves of diabetic rats, PMA increased 32P labeling of the alpha subunit. In contrast to staurosporine or extracellular calcium omission, the decreased state of phosphorylation seen with forskolin was no longer significant in diabetic nerves. No change in the level of alpha-subunit isoforms (alpha 1 or alpha 2) was detected by Western blot analysis in such nerves. In conclusion, the altered effect of PK activators on Na,K-ATPase phosphorylation state is consistent with the view that a defect in PKC activation exists in diabetic nerves.

Alpha, beta I, beta II, delta, and epsilon protein kinase C isoforms and compound activity in the sciatic nerve of normal and diabetic rats.

Defective protein kinase C (PKC) has been implicated in impaired Na+,K(+)-ATPase activity in the sciatic nerve of streptozotocin-induced diabetic rats. In the present study, alpha, beta I, beta II, gamma, delta, and epsilon isoform-specific antibodies were used in parallel to the measurement of compound PKC activity for the characterization of PKC distribution and isoform expression in sciatic nerves of normal and diabetic rats. To distinguish isoform expression between the axonal and glial compartments, PKC isoforms were evaluated in nerves subjected to Wallerian degeneration and in a pure primary Schwann cell culture. alpha, beta I, beta II, delta, and epsilon but no gamma isoforms were detected in sciatic nerve. Similar immunoreactivity was observed in degenerated nerves 3-4 days after transection except for diminished beta I and epsilon species; in Schwann cell cultures, only alpha, beta II, delta, and epsilon were detected. In normal nerves, two-thirds of PKC compound activity was found in the cytosol and 50% of total enzyme activity translocated to the Na+,K(+)-ATPase-enriched membrane fraction with phorbol myristate acetate. Similar redistribution patterns were observed for the immunoreactivity of all isoforms with the exception of delta, which did not translocate to the membrane with phorbol myristate acetate. No abnormality in compound PKC activity, in the immunoreactive intensity, or in the distribution of PKC isoforms could be detected in rat sciatic nerve after 6-12 weeks of diabetes. Thus, defective activation rather than decreased intrinsic PKC activity may occur in diabetic neuropathy.

Regulation of mitochondrial glycerol-phosphate dehydrogenase by Ca2+ within electropermeabilized insulin-secreting cells (INS-1).

(1) A new insulin-secreting cell line (INS-1; Asfari et al. (1992) Endocrinology 130, 167-178) has been used to study the regulation by Ca2+ of mitochondrial FAD-linked glycerol-phosphate dehydrogenase (FAD-GPDH) in situ. (2) Enzyme activity was examined on-line in electropermeabilized cells by a new, sensitive, assay. This involved the reduction of the artificial electron acceptor, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), monitored by the quenching of the fluorescence of rhodamine-18. Using this approach, similar total levels of FAD-GPDH activity (nmol/min per 10(6) cells) were measured in INS-1 cells (1.35 +/- 0.22) and isolated rat islet cells (1.63 +/- 0.02) (3) Ca2+ ions markedly activated the enzyme, lowering the apparent Km-value for added DL-glycerophosphate from 8.8 +/- 1.4 mM to 1.0 +/- 0.1 mM. Ca2+ had no effect on the apparent Vmax. The enzyme displayed cooperative kinetics with respect to DL-glycerophosphate (Hill coefficient of 2.0 +/- 0.2 and 1.6 +/- 0.2 in the absence and presence respectively of Ca2+). Half-maximal effects of Ca2+ were observed in the range 30-130 nM, depending on the concentration of glycerol phosphate. (4) Enzyme activity was weakly (30%) inhibited by diazoxide, but not by the diabetogenic drug, streptozotocin. (5) The data indicate that INS-1 cells represent an excellent model for studying the rôle of FAD-GPDH in the control of insulin secretion.

Video imaging of cytosolic Ca2+ in pancreatic beta-cells stimulated by glucose, carbachol, and ATP.

In order to define the differences in the distribution of cytosolic free Ca2+ ([Ca2+]i) in pancreatic beta-cells stimulated with the fuel secretagogue glucose or the Ca(2+)-mobilizing agents carbachol and ATP, we applied digital video imaging to beta-cells loaded with fura-2.83% of the cells responded to glucose with an increase in [Ca2+]i after a latency of 117 +/- 24 s (mean +/- S.E., 85 cells). Of these cells, 16% showed slow wave oscillations (frequency 0.35/min). In order to assess the relationship between membrane potential and the distribution of the [Ca2+]i rise, digital image analysis and perforated patch-clamp methods were applied simultaneously. The system used allowed sufficient temporal resolution to visualize a subplasmalemmal Ca2+ transient due to a single glucose-induced action potential. Glucose could also elicit a slow depolarization which did not cause Ca2+ influx until the appearance of the first of a train of action potentials. [Ca2+]i rose progressively during spike firing. Inhibition of Ca2+ influx by EGTA abolished the glucose-induced rise in [Ca2+]i. In contrast, the peak amplitude of the [Ca2+]i response to carbachol was not significantly different in normal or in Ca(2+)-deprived medium. Occasionally, the increase of the [Ca2+]i rise was polarized to one area of the cell different from the subplasmalemmal rise caused by glucose. The amplitude of the response and the number of responding cells were significantly increased when carbachol was applied after the addition of high glucose (11.2 mM). ATP also raised [Ca2+]i and promoted both Ca2+ mobilization and Ca2+ influx. The intracellular distribution of [Ca2+]i was homogeneous during the onset of the response. A polarity in the [Ca2+]i distribution could be detected either in the descending phase of the peak or in subsequent peaks during [Ca2+]i oscillations caused by ATP. In the absence of extracellular Ca2+, the sequential application of ATP and carbachol revealed that carbachol was still able to raise [Ca2+]i after exhaustion of the ATP response. This may be due to desensitization to the former agonist, since the response occurred in the same area of the cell. These results reveal subtle differences in [Ca2+]i distribution following membrane depolarization with glucose or the application of Ca(2+)-mobilizing agonists.

Inositol tetrakisphosphate isomers and elevation of cytosolic Ca2+ in vasopressin-stimulated insulin-secreting RINm5F cells.

Signal generation during the stimulation of insulin secretion by arginine vasopressin (AVP) was investigated in RINm5F cells. AVP (0.1 microM) caused a biphasic cytosolic Ca2+ ([Ca2+]i) rise, namely a rapid transient marked elevation after stimulation followed by a series of oscillations. In the absence of extracellular Ca2+, the sustained oscillations were abolished, while the initial [Ca2+]i transient was only partly decreased, indicating that the former are due to Ca2+ influx and the latter due mainly to mobilization from internal Ca2+ stores. AVP also evoked a transient depolarization of the average membrane potential. AVP-induced Ca2+ influx during the sustained phase, which was strictly dependent on receptor occupancy, was attenuated by membrane hyperpolarization with diazoxide. However, blockade of Ca2+ channels of the L- or T-type was ineffective. AVP stimulated production of diacylglycerol and inositol phosphates; for the latter both [3H] inositol labeling and mass determinations were performed. A transient increase in Ins(1,4,5)P3 was followed by a marked enhancement of Ins(1,3,4,5)P4 (8-fold) peaking at 15 s and gradually returning to basal values. Ins(1,3,4,6)P4 and Ins(3,4,5,6)P4 exhibited the most long-lasting augmentation (4- and 1.7-fold, respectively), and therefore correlated best with the period of sustained [Ca2+]i oscillations. InsP5 and InsP6 were not elevated. The effects of AVP, including the stimulation of insulin secretion from perifused cells, were obliterated by a V1 receptor antagonist. In conclusion, AVP induces protracted [Ca2+]i elevation in RINm5F cells which is associated with long-lasting increases in InsP4 isomers. The accumulation of InsP4 isomers reflects receptor occupancy and accelerated metabolism of the inositol phosphates. Activation of second messenger-operated Ca2+ channels is not necessarily implicated because of the attenuating effect of membrane hyperpolarization.