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T F Walseth

Publications and source records attributed to T F Walseth.

At least 37 records · Page 2Linked to original sources
PubMed1996

Suppression of adenylate kinase catalyzed phosphotransfer precedes and is associated with glucose-induced insulin secretion in intact HIT-T15 cells.

Adenine nucleotide metabolism was characterized in intact insulin secreting HIT-T15 cells during the transition from non-stimulated (i. e. 0.2 mM glucose) to the glucose-stimulated secretory state. Metabolic dynamics were monitored by assessing rates of appearance of 18O-labeled phosphoryls of endogenous nucleotides in cells incubated in medium enriched in [18O]water. Most prominent of the metabolic alterations associated with stimulated insulin secretion was the suppression in the rate of adenylate kinase (AK)-catalyzed phosphorylation of AMP by ATP. This was manifest as a graded decrease of up to 50% in the rate of appearance of beta-18O-labeled species of ADP and ATP and corresponded to the magnitude of the secretory response elicited over a range of stimulatory glucose concentrations. The only nucleotide exhibiting a significant concentration change associated with suppression of AK activity was AMP, which decreased by about 50%, irrespective of the glucose concentration. Leucine-stimulated secretion also decreased the rate of AK-catalyzed phosphotransfer. This secretory stimulus-related suppression of AK-catalyzed phosphotransfer occurs within 45 s of glucose addition, precedes insulin secretion, depends on the internalization and metabolism of glucose, and is independent of membrane depolarization and the influx of extracellular calcium. The secretory stimulus-induced decrease in AK-catalyzed phosphotransfer, therefore occurs prior to or at the time of KATP+ channel closure but it is not associated with or a consequence of events occurring subsequent to KATP+ channel closure. These results indicate that AK-catalyzed phosphotransfer may be a determinant of ATP to ADP conversion rates in the KATP+ channel microenvironment; secretory stimuli-linked decreased rates of AK-catalyzed ADP generation from ATP (and AMP) would translate into an increased probability of ATP-liganded and, therefore, closed state of the channel.

Adenosine Diphosphate↗
PubMed1996

Activation and inactivation of Ca2+ release by NAADP+.

Nicotinic acid adenine dinucleotide phosphate (NAADP+) is a recently identified metabolite of NADP+ that is as potent as inositol trisphosphate (IP3) and cyclic ADP-ribose (cADPR) in mobilizing intracellular Ca2+ in sea urchin eggs and microsomes (Clapper, D. L., Walseth, T. F., Dargie, P. J., and Lee, H. C. (1987) J. Biol. Chem. 262, 9561-9568; Lee, H. C., and Aarhus, R. (1995) J. Biol. Chem. 270, 2152-2157). The mechanism of Ca2+ release activated by NAADP+ and the Ca2+ stores it acts on are different from those of IP3 and cADPR. In this study we show that photolyzing caged NAADP+ in intact sea urchin eggs elicits long term Ca2+ oscillations. On the other hand, uncaging threshold amounts of NAADP+ produces desensitization. In microsomes, this self-inactivation mechanism exhibits concentration and time dependence. Binding studies show that the NAADP+ receptor is distinct from that of cADPR, and at subthreshold concentrations, NAADP+ can fully inactivate subsequent binding to the receptor in a time-dependent manner. Thus, the NAADP+-sensitive Ca2+ release process has novel regulatory characteristics, which are distinguishable from Ca2+ release mediated by either IP3 or cADPR. This battery of release mechanisms may provide the necessary versatility for cells to respond to diverse signals that lead to Ca2+ mobilization.

Animals↗
PubMed1996

Nitric oxide-induced mobilization of intracellular calcium via the cyclic ADP-ribose signaling pathway.

Cyclic adenosine diphosphate ribose (cADPR) is a potent endogenous calcium-mobilizing agent synthesized from beta-NAD+ by ADP-ribosyl cyclases in sea urchin eggs and in several mammalian cells (Galione, A., and White, A. (1994) Trends Cell Biol. 4, 431 436). Pharmacological studies suggest that cADPR is an endogenous modulator of Ca2+-induced Ca2+ release mediated by ryanodine-sensitive Ca2+ release channels. An unresolved question is whether cADPR can act as a Ca2+-mobilizing intracellular messenger. We show that exogenous application of nitric oxide (NO) mobilizes Ca2+ from intracellular stores in intact sea urchin eggs and that it releases Ca2+ and elevates cADPR levels in egg homogenates. 8-Amino-cADPR, a selective competitive antagonist of cADPR-mediated Ca2+ release, and nicotinamide, an inhibitor of ADP-ribosyl cyclase, inhibit the Ca2+-mobilizing actions of NO, while, heparin, a competitive antagonist of the inositol 1,4,5-trisphosphate receptor, did not affect NO-induced Ca2+ release. Since the Ca2+-mobilizing effects of NO can be mimicked by cGMP, are inhibited by the cGMP-dependent-protein kinase inhibitor, Rp-8-pCPT-cGMPS, and in egg homogenates show a requirement for the guanylyl cyclase substrate, GTP, we suggest a novel action of NO in mobilizing intracellular calcium from microsomal stores via a signaling pathway involving cGMP and cADPR. These results suggest that cADPR has the capacity to act as a Ca2+-mobilizing intracellular messenger.

ADP-ribosyl Cyclase↗
PubMed1996

Fluorescent analogs of cyclic ADP-ribose: synthesis, spectral characterization, and use.

Cyclic ADP-ribose (cADPR) is a Ca(2+)-mobilizing cyclic nucleotide derived from NAD+. Accumulating evidence indicates that it is an endogenous modulator of the Ca(2+)-induced Ca2+ release mechanism in cells. In this study, we show that ADP-ribosyl cyclase catalyzes the cyclization of not only NAD+ but also several of its analogs with various purine bases (guanine, hypoxanthine, or xanthine) substituting for adenine. Unlike cADPR, the resulting cyclic products are fluorescent. Comparisons with various model compounds indicate that only 7-methyl substituted purine nucleosides and nucleotides are fluorescent, and the pH-dependence of their UV spectra is most similar to that of the fluorescent cADPR analogs, indicating that the site of cyclization of these analogs is at the N7-position of the purine ring. This finding is novel since the site of cyclization is at the N1-position for cADPR as determined by X-ray crystallography. That a single enzyme can cyclize a variety of substrates at two different sites has important implications mechanistically, and a model is proposed to account for these novel catalytic properties. Among the analogs synthesized, cyclic GDP-ribose is highly resistant to hydrolysis, while cyclic IDP-ribose can be readily hydrolyzed by CD38, a bifunctional enzyme involved in the metabolism of cADPR. These unique properties of the analogs can be used to develop fluorimetric assays for monitoring separately the cyclization and hydrolytic reactions catalyzed by the metabolic enzymes of cADPR. The convenience of the method in measuring kinetic parameters, pH-dependence, and modulator activity of the metabolic enzymes of cADPR is illustrated.

ADP-ribosyl Cyclase↗
PubMed1995

ADP-ribosyl cyclase and CD38 catalyze the synthesis of a calcium-mobilizing metabolite from NADP.

ADP-ribosyl cyclase catalyzes the cyclization of NAD+ to produce cyclic ADP-ribose (cADPR), which is emerging as an endogenous regulator of the Ca(2+)-induced Ca2+ release mechanism in cells. CD38 is a lymphocyte differentiation antigen which has recently been shown to be a bifunctional enzyme that can synthesize cADPR from NAD+ as well as hydrolyze cADPR to ADP-ribose. In this study, we show that both the cyclase and CD38 can also catalyze the exchange of the nicotinamide group of NADP+ with nicotine acid (NA). The product is nicotinic acid adenine dinucleotide phosphate (NAADP+), a metabolite we have previously shown to be potent in Ca2+ mobilization (Lee, H. C., and Aarhus, R. (1995) J. Biol. Chem. 270, 2152-2157). The switch of the catalysis to the exchange reaction requires acidic pH and NA. The half-maximal effective concentration of NA is about 5 mM for both the cyclase and CD38. In the absence of NA or at neutral pH, the cyclase converts NADP+ to another metabolite, which is identified as cyclic ADP-ribose 2'-phosphate. Under the same conditions, CD38 converts NADP+ to ADP-ribose 2'-phosphate instead, which is the hydrolysis product of cyclic ADP-ribose 2'-phosphate. That two different products of ADP-ribosyl cyclase and CD38, cADPR and NAADP+, are both involved in Ca2+ mobilization suggests a crucial role of these enzymes in Ca2+ signaling.

ADP-ribosyl Cyclase↗
PubMed1995

Cyclic ADP-ribose and its metabolic enzymes.

Cyclic ADP-ribose (cADPR) is a recently discovered cyclic nucleotide with Ca2+ signaling functions. There is a growing recognition that it is an endogenous modulator of the Ca(2+)-induced Ca2+ release mechanism in cells. The cyclic structure of cADPR has now been confirmed by x-ray crystallography. A series of analogs of cADPR has been synthesized, including antagonists and a novel analog, cyclic GDP-ribose. Considerable progress has been made in characterizing ADP-ribosyl cyclase, the synthetic enzyme, and cADPR hydrolase, the hydrolytic enzyme. A new class of bifunctional enzymes has been identified which catalyses both the synthesis and hydrolysis of cADPR. CD38, a lymphocyte differentiation antigen, is a member of this class. The understanding of the mechanisms of regulation of the metabolic enzymes and signaling by cADPR is likely to have important implications and several possibilities are discussed in this article.

ADP-ribosyl Cyclase↗
PubMed1995

Somatostatin selectively couples to G(o) alpha in HIT-T15 cells.

Previously, we have demonstrated that somatostatin mediates all of its inhibitory effects on glucose-induced insulin secretion from the HIT-T15 cell through pertussis toxin-sensitive G-proteins and that the membrane fraction of this clonal line of pancreatic beta-cells contains six such proteins: G(i) alpha 1, G(i) alpha 2, G(i) alpha 3, and three forms of G(o) alpha. To determine the specificity of somatostatin receptor-G-protein coupling in HIT-T15 cells, we examined the ability of antisera specific for the COOH-terminus of G alpha subtypes to inhibit somatostatin-induced augmentation of membrane GTPase activity. GTPase activity increased in membranes as a function of GTP. At all concentrations of GTP studied, 1 mumol/l somatostatin stimulated GTPase activity. Pertussis-toxin pretreatment prevented the effects of somatostatin. Antisera selective for G(o) alpha subtypes reduced the effects of somatostatin on GTPase activity (GTPase activity in absence of antisera, 125 +/- 3% of control; in the presence of antisera 976, 110 +/- 2% of control; n = 13, P < 0.001), whereas antisera directed against G(i) alpha 1, G(i) alpha 2, G(i) alpha 3, and Gs alpha were without effect. Somatostatin also significantly prevented cyclic AMP accumulation during perifusion with 11.1 mmol/l glucose through a pertussis toxin-sensitive mechanism. These data indicate that the somatostatin receptor couples to G(o) alpha in the HIT-T15 cell and suggest that G(o) alpha may link somatostatin to cyclic AMP metabolism in pancreatic beta-cells.

Animals↗
PubMed1995

Morphological and functional characterization of beta TC-6 cells--an insulin-secreting cell line derived from transgenic mice.

Morphological analysis of hormone content and functional assessment of hormone secretion were conducted in beta TC-6 cells, an insulin-secreting cell line derived from transgenic mice expressing the large T-antigen of simian virus 40 (SV40) in pancreatic beta-cells. We observed by immunohistochemistry and confocal microscopy that beta TC-6 cells contain abundant insulin and small amounts of glucagon and somatostatin (SRIF). Glucagon usually co-localized with insulin, whereas cells containing SRIF did not contain insulin or glucagon. Static incubation and perifusion experiments demonstrated that beta TC-6 cells at passage 30-45 secrete insulin in response to glucose. In static incubations, maximal stimulation was achieved for glucose concentrations > 2.8 mmol/l glucose, and the half-maximal effect was observed at 0.5 mmol/l. Maximal stimulation was four times greater than HIT-T15 cells at passage 72-81, although HIT cells had a greater response over their basal levels. The magnitude of the insulin response to glucose in perifusion was 1,734 +/- 384 pmol.l-1. min and was 4.6-fold greater in the presence of 3-isobutyl-1-methylxanthine. Low amounts of glucagon were released in response to amino acids. Epinephrine (EPI), and to a lesser extent SRIF, inhibited phasic glucose-induced insulin secretion. A major portion of these inhibitory effects was mediated by pertussis toxin-sensitive substrates. Immunoblots detected the presence of the G-proteins Gi alpha 2, Gi alpha 3, and Go alpha 2. These results indicate that beta TC-6 cells are a glucose-responsive cell line in which insulin exocytosis is physiologically regulated by EPI and SRIF through Gi/Go-mediated mechanisms.

1-Methyl-3-isobutylxanthine↗
PubMed1994

Enzymatic synthesis and characterizations of cyclic GDP-ribose. A procedure for distinguishing enzymes with ADP-ribosyl cyclase activity.

Cyclic nucleotides such as cAMP and cGMP are second messengers subserving various signaling pathways. Cyclic ADP-ribose (cADPR), a recently discovered member of the family, is derived from NAD+ and is a mediator of Ca2+ mobilization in various cellular systems. The synthesis and degradation of cADPR are, respectively, catalyzed by ADP-ribosyl cyclase and cADPR hydrolase. CD38, a differentiation antigen of B lymphocytes, has recently been shown to be a bifunctional enzyme catalyzing both the formation and hydrolysis of cADPR. The overall reaction catalyzed by CD38 is the formation of ADP-ribose and nicotinamide from NAD+, identical to that catalyzed by NADase. The difficulties in detecting the formation of cADPR have led to frequent identification of CD38 as a classical NADase. In this study, we show that both ADP-ribosyl cyclase and CD38, but not NADase, can cyclize nicotinamide guanine dinucleotide (NGD+) producing a new nucleotide. Analyses by high performance liquid chromatography and mass spectroscopy indicate the product is cyclic GDP-ribose (cGDPR) with a structure similar to cADPR except with guanine replacing adenine. Compared to cADPR, cGDPR is a more stable compound showing 2.8 times more resistance to heat-induced hydrolysis. These results are consistent with a catalytic scheme for CD38 where the cyclization of the substrate precedes the hydrolytic reaction. Spectroscopic analyses show that cGDPR is fluorescent and has an absorption spectrum different from both NGD+ and GDPR, providing a very convenient way for monitoring its enzymatic formation. The use of NGD+ as substrate for assaying the cyclization reaction was found to be applicable to pure enzymes as well as crude tissue extracts making it a useful diagnostic tool for distinguishing CD38-like enzymes from degradative NADases.

ADP-ribosyl Cyclase↗
PubMed1994

Cyclic ADP ribose activation of the ryanodine receptor is mediated by calmodulin.

Cyclic ADP-ribose (cADPR) is a newly identified nucleotide which can release calcium from a variety of cells, suggesting it is a messenger for mobilizing internal Ca2+ stores. Its cyclic structure has now been confirmed by X-ray crystallography. Available results are consistent with it being a modulator of Ca(2+)-induced Ca2+ release. Here we report that sea urchin egg microsomes purified by Percoll gradients lose sensitivity to cADPR, but the response can be restored by a soluble protein in the supernatant. Purification and characterization of the protein indicate that it is calmodulin. It appears to be sensitizing the Ca2+ release mechanism because caffeine and strontium, agonists of Ca(2+)-induced Ca2+ release, can also mimic calmodulin in conferring cADPR-sensitivity. Although evidence indicates that cADPR may be an activator of the ryanodine receptor, present results point to the importance of accessory proteins such as calmodulin in modulating its activity.

Adenosine Diphosphate Ribose↗
PubMed1993

Identification of cyclic ADP-ribose-binding proteins by photoaffinity labeling.

We have synthesized 8-azido-cyclic ADP-ribose (8N3-cADPR) and [32P]8-azido-cyclic ADP-ribose ([32P]8N3-cADPR) in order to characterize cyclic ADP-ribose-(cADPR) binding sites in sea urchin egg homogenates. 8N3-cADPR was an antagonist of cADPR since it did not induce Ca2+ release from egg microsomes but did inhibit the ability of cADPR to do so. The effect of 8N3-cADPR was reversible and could be overcome by high concentrations of cADPR, suggesting that both were acting on the same site. This was supported by the fact that 8N3-cADPR effectively competed for [32P]cADPR binding to microsomes. Reciprocally, binding of [32P]8N3-cADPR could also be selectively displaced by cADPR and 8N3-cADPR, but not by ADP-ribose. These results indicate that 8N3-cADPR binds specifically to the cADPR-binding sites and inhibits cADPR from releasing Ca2+. Photolysis of microsomes preincubated with [32P]8N3-cADPR resulted in specific labeling of proteins of 140 and 100 kDa, which could be prevented by 8N3-cADPR or nanomolar concentrations of cADPR, but not by micromolar concentrations of ADP-ribose, AMP, ADP, ATP, cyclic AMP or inositol 1,4,5-trisphosphate. Caffeine, an agonist of Ca(2+)-induced Ca2+ release, preferentially inhibited the labeling of the 100 kDa as compared to the 140-kDa protein. These results suggest that cADPR may not interact directly with the ryanodine receptor, but may instead, exert its effect through intermediate proteins.

Adenosine Diphosphate Ribose↗
PubMed1993

Formation and hydrolysis of cyclic ADP-ribose catalyzed by lymphocyte antigen CD38.

CD38 is a 42-kilodalton glycoprotein expressed extensively on B and T lymphocytes. CD38 exhibits a structural homology to Aplysia adenosine diphosphate (ADP)-ribosyl cyclase. This enzyme catalyzes the synthesis of cyclic ADP-ribose (cADPR), a metabolite of nicotinamide adenine dinucleotide (NAD+) with calcium-mobilizing activity. A complementary DNA encoding the extracellular domain of murine CD38 was constructed and expressed, and the resultant recombinant soluble CD38 was purified to homogeneity. Soluble CD38 catalyzed the formation and hydrolysis of cADPR when added to NAD+. Purified cADPR augmented the proliferative response of activated murine B cells, potentially implicating the enzymatic activity of CD38 in lymphocyte function.

ADP-ribosyl Cyclase↗
PubMed1993

Synthesis and characterization of antagonists of cyclic-ADP-ribose-induced Ca2+ release.

Cyclic ADP-ribose (cADPR) is a naturally-occurring metabolite of NAD+ that is as effective as inositol trisphosphate in mobilizing intracellular Ca2+. A series of analogs modified at the 8-position of the adenine group were synthesized for the investigation of the relationship between the structure of the metabolite and its Ca(2+)-mobilizing activity. Substitution with an amino group at the 8-position of the adenine ring produced an antagonist. The 1H-NMR spectrum of 8-amino-cADPR showed characteristics of that of cADPR and confirmed the replacement of the 8-proton. By itself, 8-amino-cADPR (150 nM) did not induce Ca2+ release from sea-urchin-egg homogenates but totally blocked cADPR (135 nM) from doing so. The effect was reversible, since high concentrations of cADPR could overcome the inhibition. Addition of 8-amino-cADPR to egg homogenates during the cADPR-induced Ca2+ release blocked the release immediately, demonstrating the effectiveness of the antagonist. Measurements of [32P]cADPR binding to its microsomal binding site showed that 8-amino-cADPR was as effective as cADPR itself in competing for the binding site. In addition to blocking cADPR from releasing Ca2+, 8-amino-cADPR also inhibited cADPR from potentiating Ca(2+)-release induced by either divalent cations or by caffeine. Two other 8-substituted analogs were also synthesized. Both 8-Br- and 8-azido-cADPR were also antagonists, although with less potency than 8-amino-cADPR. These results show that alterations at the 8-position of the adenine group do not inhibit cADPR from binding to its receptor but do eliminate the ability of the metabolite to activate the Ca(2+)-release mechanism.

Adenosine Diphosphate Ribose↗
PubMed1993

Calcium mobilization by dual receptors during fertilization of sea urchin eggs.

Fertilization is accompanied by a transient increase in the concentration of intracellular Ca2+, which serves as a signal for initiating development. Some of the Ca2+ appears to be released from intracellular stores by the binding of inositol trisphosphate (IP3) to its receptor. However, in sea urchin eggs, other mechanisms appear to participate. Cyclic adenosine diphosphate--ribose (cADPR), a naturally occurring metabolite of nicotinamide adenine dinucleotide, is as potent as IP3 in mobilizing Ca2+ in sea urchin eggs. Experiments with antagonists of the cADPR and IP3 receptors revealed that both Ca2+ mobilizing systems were activated during fertilization. Blockage of either of the systems alone was not sufficient to prevent the sperm-induced Ca2+ transient. This study provides direct evidence for a physiological role of cADPR in the Ca2+ signaling process.

Adenosine Diphosphate Ribose↗
PubMed1992

Preservation of insulin mRNA levels and insulin secretion in HIT cells by avoidance of chronic exposure to high glucose concentrations.

Glucose toxicity of the pancreatic beta cell is considered to play a secondary role in the pathogenesis of type II diabetes mellitus. To gain insights into possible mechanisms of action of glucose toxicity, we designed studies to assess whether the loss of insulin secretion associated with serial passages of HIT-T15 cells might be caused by chronic exposure to high glucose levels since these cells are routinely cultured in media containing supramaximal stimulatory concentrations of glucose. We found that late passages of HIT cells serially cultured in media containing 11.1 mM glucose lost insulin responsivity and had greatly diminished levels of insulin content and insulin mRNA. In marked contrast, late passages of HIT cells cultured serially in media containing 0.8 mM glucose retained insulin mRNA, insulin content, and insulin responsivity to glucose in static incubations and during perifusion with glucose. No insulin gene mutation or alteration of levels of GLUT-2 were found in late passages of HIT cells cultured with media containing 11.1 mM glucose. These data uniquely indicate that loss of beta cell function in HIT cells passed serially under high glucose conditions is caused by loss of insulin mRNA, insulin content, and insulin secretion and is preventable by culturing HIT cells under low glucose conditions. This strongly suggests potential genetic mechanisms of action for glucose toxicity of beta cells.

Animals↗