An evaluation of the effect of non-setting calcium hydroxide on human dentine: a pilot study.

AIM: To evaluate the effect of non-setting calcium hydroxide (NSCH) on the hardness and elastic modulus of dentine from extracted permanent premolar human teeth. METHODS: 30 freshly extracted single rooted human premolar teeth were decoronated and the roots then sectioned longitudinally into equal halves. In the experimental group a thin layer of NSCH was applied whilst the control group had no medicament. After 1, 3 and 6 months, nanoindentation was used to assess dentine hardness and the modulus of elasticity. Scanning Electron Microscopy (SEM) was used to visualize the depth of penetration of NSCH into the dentinal tubules. RESULTS: SEM images showed that there were no structural changes in the dentine slabs that had NSCH application after 1, 3 or even 6 months. However, penetration of NSCH into the dentine tubules was seen at both 3 and 6 months with a significant reduction in the hardness of dentine observed at 3 (p<0.02) and 6 months (p<0.01). The modulus of elasticity was significantly lower (p<0.01) at 6 months. CONCLUSION: It appears that there is a significant reduction in the hardness of dentine with increasing periods of calcium hydroxide application. Prolonged application of NSCH could have a detrimental effect on dentine, making the dentine more prone to fracture.

Molar-incisor-hypomineralisation: a literature review.

BACKGROUND: Molar-Incisor-Hypomineralisation (MIH) is a qualitative defect of 1-4 first permanent molars with or without the maxillary and mandibular permanent incisors. It seems to have been recognised first in the 1970s and prevalence varies between 2.8% and 25%, dependent upon the study. METHODS: The dental literature was searched using a number of key terms entered into MEDLINE, the reference list of each paper as located was examined for further papers that had been missed in the initial search. RESULTS: The review of the literature showed that teeth that are affected indicate a systemic cause at around the time of birth; investigators have put forward a number of possible causes; asthma, pneumonia, upper respiratory tract infections, otitis media, antibiotics, dioxins in mother's milk, tonsillitis and tonsillectomy and exanthamatous fevers of childhood. However, at the present time the aetiology remains unclear. Treatment of the affected permanent first molars can include restorations using adhesive intra-coronal restorations to extra-coronal restorations (e.g. preformed metal crowns). There is little evidence to support one option over another. In severe cases extraction at the optimum time may be the best option; allowing the permanent second molars to come forwards. There is little improvement in affected anterior teeth with microabrasion and direct or indirect composite resin restorations may be appropriate in some children. Ultrastructural and biochemical make up of MIH affected teeth seem to have been investigated less than other areas. CONCLUSION: It is important that children with MIH are diagnosed as early as possible and managed appropriately; this will involve multidisciplinary input.

UK National Clinical Guidelines in Paediatric Dentistry: stainless steel preformed crowns for primary molars.

This revised Clinical Guideline in Paediatric Dentistry replaces the previously published sixth guideline (Fayle SA. Int J Paediatr Dent 1999; 9: 311-314). The process of guideline production began in 1994, resulting in first publication in 1997. Each guideline has been circulated widely for consultation to all UK consultants in paediatric dentistry, council members of the British Society of Paediatric Dentistry (BSPD), and to people of related specialities recognized to have expertise in the subject. The final version of this guideline is produced from a combination of this input and thorough review of the published literature. The intention is to encourage improvement in clinical practice and to stimulate research and clinical audit in areas where scientific evidence is inadequate. Evidence underlying recommendations is scored according to the SIGN classification and guidelines should be read in this context. Further details regarding the process of paediatric dentistry guideline production in the UK is described in the Int J Paediatr Dent 1997; 7: 267-268.

Case report: scurvy in an epileptic child on a ketogenic diet with oral complications.

BACKGROUND: Epilepsy is a symptom of cerebral dysfunction, where there is a sudden and disorganised discharge of electrical activity from a group of neurones, producing symptoms that range from sensory absences to convulsive movements and unconsciousness. Fasting is recognised as reducing the frequency of epileptic seizures in difficult to control patients. The ketogenic diet is a high fat, low carbohydrate and adequate protein diet that mimics the biochemical effects of fasting. It is deficient in some essential elements that require supplementation. CASE REPORT: A 9-year old girl with learning difficulties, developmental delay and refractory epilepsy was placed on the ketogenic diet in 2003. Prior to starting the diet she had had as many as 12 tonic seizures/day, with prolonged periods of non-convulsive status epilepticus. Subsequent to being placed on the diet, the frequency of her seizures reduced markedly; there were long periods during which she had none. In late 2006, the patient inhaled a primary molar. This was retrieved by emergency bronchoscopy and at the same time the remaining primary teeth were extracted. Three weeks later she was admitted to hospital with low-grade fever, persistently bleeding sockets, oedema of her hands and feet, a petechial rash and bruising. A differential diagnosis included: liver disease, bleeding dyscrasia, oncological pathology or scurvy. The most striking finding amongst a number of investigations was a vitamin C level of 0.7 micromol/l (Deficiency: < 11 micromol/l). Accordingly a diagnosis of scurvy was made. TREATMENT: The patient was prescribed ascorbic acid 500 mg twice/day. Three weeks later the patient's vitamin C level was 141.5 micromol/l; the dose was therefore reduced to 250 mg once/day. FOLLOW-UP: At two-month review, the signs and symptoms of scurvy had resolved. CONCLUSION: Inhaling a tooth and scurvy are both rare occurrences. Paediatric dentists should be aware of the possible implications of a ketogenic diet.

An X-ray microtomography study on the mineral concentration of carious dentine removed during cavity preparation in deciduous molars.

Dentists use a number of criteria in order to assess when a cavity is caries free, amongst which hardness is probably the most widely used. However, the judgement is subjective. X-ray microtomography (XMT) is a non-destructive microscopic technique that allows in vitro specimens to be scanned, manipulated and then rescanned. In this study, a high-definition XMT scanner was used to determine the mineral distribution of carious dentine in 10 deciduous molars, and the extent of dentine removed by an experienced clinician was investigated. For each tooth, after an initial XMT scan, caries was removed using a steel bur in a slow hand-piece. The tooth was then repositioned and rescanned. Mineral concentrations were calculated from the linear attenuation coefficients assuming the mineral phase to be hydroxyapatite and the organic phase to be collagen. The volume of dentine tissues removed was calculated by subtracting data of the second scan from the first. The results showed that the mean modal mineral concentration for the 10 teeth was 1.42 g x cm(-3) for sound dentine. Because of uncertainty about collagen concentration in carious dentine, the mean modal mineral concentration for the carious dentine had a range of 0.37-0.5 g x cm(-3). It was found that the subjective criteria used by the operator could lead to inconsistency of cavity preparation. The cavities could be overprepared by 8.5-44.3% in volume. However, the overpreparation was not uniform throughout the cavity: residual demineralised dentine could still be detected in the postoperative scan in isolated regions.

Dentinal carious lesion in three dimensions.

AIM: The aim of this study was to show the morphology of the carious lesion in dentine in three dimensions (3D). DESIGN: A novel high-definition X-ray microtomography (XMT) scanner was used to scan 10 carious primary molars at a resolution of 15 x 15 x 15 microm3. A stack of approximately 640 XMT slices were recorded for each tooth. Using this data set and a volume rendering algorithm, each tooth was reconstructed in 3D. The VG Studio Max 1.0 visualization software package was used to make normal enamel and dentine transparent to show the carious lesions in 3D. A video film, comprised of the rendered images from 60 viewing angles rotating through 360 degrees , was produced to show the carious lesion and its relation to the pulp in a three-dimensional perspective (http://www.smd.qmul.ac.uk/dental/oralgrowdev/biophysics/xmt/images/carious.mpg). RESULTS: These images showed that carious lesions in dentine were bowl-shaped. The pulp adjacent to the carious lesion was also observed to mimic the base of the bowl-shaped lesion. CONCLUSIONS: It was concluded from the teeth studied that the shape at the base of the carious lesion in dentine is curved in 3D, rather than conical, as traditionally believed from two-dimensional image interpretation. Further 3D studies are needed to investigate whether the bowl-shaped carious lesions in dentine also apply to caries in other types of teeth.

Differential pH and capsaicin responses of Griffonia simplicifolia IB4 (IB4)-positive and IB4-negative small sensory neurons.

Protons play a key role in nociception caused by inflammation and ischaemia, but little is known about the relative sensitivities of different dorsal root ganglion (DRG) neurons. We have therefore examined the responses in vitro of rat DRG cells classified according to whether or not they bind Griffonia simplicifolia IB4 (IB4), a lectin which is widely used to distinguish between two major populations of small diameter neurons. Under voltage-clamp conditions, proton-activated inward currents were found in approximately 90% of small DRG neurons and showed one of three waveforms: transient, sustained or mixed. The majority of IB4-positive (IB4+) neurons (63%) gave rise to sustained inward currents that were sensitive to capsazepine. In contrast, the most prevalent waveform in small IB4-negative (IB4-) neurons (69%) was a mixed response containing transient and sustained components. The transient component was inhibited by amiloride whilst the sustained component showed a variable sensitivity to capsazepine. We also found that more IB4+ cells responded to capsaicin and, on average, gave rise to a larger magnitude of response than small IB4- neurons, consistent with their higher prevalence and greater amplitude of vanilloid receptor 1 (TRPV1)-like acid responses. The increase in intracellular Ca(2+) induced by capsaicin was also slightly greater in IB4+ neurons and in these cells its magnitude correlated with the level of TRPV1 immunoreactivity. Our data suggest that acid-sensing ion channels (ASICs) and TRPV1 are the major acid-sensitive receptors in small IB4- neurons, whilst TRPV1 is the predominant one in IB4+ neurons. Because ASIC-like responses were approximately 10-fold more sensitive to changes in H(+) than TRPV1-like responses, we speculate that small IB4- rather than IB4+ neurons play an essential role in sensing acid. Our results also highlight differences in capsaicin responses between IB4+ and IB4- small neurons and reveal the close link between capsaicin responses and levels of TRPV1 expression.

Regulation of nociceptive neurons by nerve growth factor and glial cell line derived neurotrophic factor.

Nociceptive dorsal root ganglion (DRG) cells can be divided into three main populations, namely (1) small diameter non-peptide-expressing cells, (2) small-diameter peptide-expressing (calcitonin gene related peptide (CGRP), substance P) cells, and (3) medium-diameter peptide-expressing (CGRP) cells. The properties of these cell populations will be reviewed, with a special emphasis on the expression of the vanilloid (capsaicin) receptor VR1 and its regulation by growth factors. Cells in populations 1 and 2 express VR1, a nonselective channel that transduces certain nociceptive stimuli and that is crucial to the functioning of polymodal nociceptors. Cells in population 1 can be regulated by glial cell line derived neurotrophic factor (GDNF) and those in populations 2 and 3 by nerve growth factor (NGF). In vivo, DRG cells express a range of levels of VR1 expression and VR1 is downregulated after axotomy. However, treatment with NGF or GDNF can prevent this downregulation. In vitro, DRG cells also show a range of VR1 expression levels that is NGF and (or) GDNF dependent. Functional studies indicate that freshly dissociated cells also show differences in sensitivity to capsaicin. The significance of this is not known but may indicate a difference in the physiological role of cells in populations 1 and 2.

A fundamental role for the nitric oxide-G-kinase signaling pathway in mediating intercellular Ca(2+) waves in glia.

In this study, we highlight a role for the nitric oxide-cGMP-dependent protein kinase (NO-G-kinase) signaling pathway in glial intercellular Ca(2+) wave initiation and propagation. Addition of the NO donor molsidomine (100-500 microM) or puffing aqueous NO onto primary glial cell cultures evoked an increase in [Ca(2+)](i) in individual cells and also local intercellular Ca(2+) waves, which persisted after removal of extracellular Ca(2+). High concentrations of ryanodine (100-200 microM) and antagonists of the NO-G-kinase signaling pathway essentially abrogated the NO-induced increase in [Ca(2+)](i), indicating that NO mobilizes Ca(2+) from a ryanodine receptor-linked store, via the NO-G-kinase signaling pathway. Addition of 10 microM nicardipine to cells resulted in a slowing of the molsidomine-induced rise in [Ca(2+)](i), and inhibition of Mn(2+) quench of cytosolic fura-2 fluorescence mediated by a bolus application of 2 microM aqueous NO to cells, indicating that NO also induces Ca(2+) influx in glia. Mechanical stress of individual glial cells resulted in an increase in intracellular NO in target and neighboring cells and intercellular Ca(2+) waves, which were NO, cGMP, and G-kinase dependent, because incubating cells with nitric oxide synthase, guanylate cyclase, and G-kinase inhibitors, or NO scavengers, reduced Delta[Ca(2+)](i) and the rate of Ca(2+) wave propagation in these cultures. Results from this study suggest that NO-G-kinase signaling is coupled to Ca(2+) mobilization and influx in glial cells and that this pathway plays a fundamental role in the generation and propagation of intercellular Ca(2+) waves in glia.

Intercellular Ca(2+) waves in rat hippocampal slice and dissociated glial-neuron cultures mediated by nitric oxide.

Nitric oxide (NO) may participate in cell-cell communication in the brain by generating intercellular Ca(2+) waves. In hippocampal organotypic and dissociated glial-neuron (>80% glia) cultures local applications of aqueous NO induced slowly propagating intercellular Ca(2+) waves. In glial cultures, Ca(2+) waves and Mn(2+) quench of cytosolic fura-2 fluorescence mediated by NO were inhibited by nicardipine, indicating that NO induces Ca(2+) influx in glia which is dihydropyridine-sensitive. As NO treatments also depolarised the plasma membrane potential of glia, the nicardipine-sensitive Ca(2+) influx might be due to the activation of dihydropyridine-sensitive L-type Ca(2+) channels. Both nicardipine-sensitive intercellular Ca(2+) waves and propagating cell depolarisation induced by mechanical stress of individual glia were inhibited by pretreating cultures with either an NO scavenger or N(G)-methyl-L-arginine. Results demonstrate that NO can induce Ca(2+) waves in hippocampal slice cultures, and that Ca(2+) influx coupled to NO-mediated membrane depolarisation might assist in fashioning their spatio-temporal dynamics.

Effects of dexamethasone and phorbol ester on P2 receptor-coupled Ca2+ signalling and lipocortin 1 presentation in U937 cells.

1. Cell surface bound lipocortin 1 (LC1) is a putative mediator of the antiproliferative and anti-inflammatory effects of glucocorticoids. This study assessed the hypothesis that the glucocorticoid, dexamethasone-phosphate (dex-p), might exert the above effects via an LC1-mediated downregulation of receptor-coupled Ca2+ signalling, using P2-receptor mediated intracellular Ca2+ accumulation in U937 cells as an appropriate model. 2. Addition of ATP (1-100 microM) to cells resulted in a transient increase in cytosolic Ca2+ ([Ca2+]i). Prior treatment of cells with dex-p (3-24 h) increased the magnitude of this Ca2+ transient at high, but not low concentrations of ATP, and increased thapsigargin (Tg)-induced Ca2+ influx, indicating that store-operated Ca2+ influx was potentiated in these cells. For cells treated with dex-p for 24 h, cell surface levels of LC1 were significantly reduced by 63%. 3. Differentiation of cells with 1 nM phorbol ester (PMA) for 24 h resulted in a 2.4 fold increase in the cell surface level of LC1 and inhibition of the ATP-induced Ca2+ response. However, the Tg-induced Ca2+ response was unaffected by long-term PMA treatment, and incubating cells with LC1 did not alter Tg-induced Ca2+ mobilization and influx, or the ATP-mediated Ca2+ response. 4. Data from this study suggest that: (1) dex-p does not inhibit P2-receptor-coupled Ca2+ signalling in this cell line, (2) the observed modulation of the ATP-induced increase in [Ca2+]i by dex-p and PMA, and store-operated Ca2+ influx by dex-p, is not linked to an increase in the cell surface level of LC1, and (3) differentiation of U937 cells with PMA downregulates the ATP-induced Ca2+ response, but does not affect the thapsigargin-sensitive Ca2+ pool or store-operated Ca2+ influx of these cells.

Conversion of highly malignant colon cancer from an aggressive to a controlled disease by oral administration of a metalloproteinase inhibitor.

In this study, we describe the activity of CT1746, an orally-active synthetic MMP inhibitor that has a greater specificity for gelatinase A, gelatinase B and stromelysin than for interstitial collagenase and matrilysin, in a nude mouse model that better mimics the clinical development of human colon cancer. The model is constructed by surgical orthotopic implantation (SOI) of histologically-intact tissue of the metastatic human colon tumor cell line Co-3. Animals were gavaged with CT1746 twice a day at 100 mg/kg for 5 days after the SOI of Co-3 for 43 days. In this model CT1746 significantly prolonged the median survival time of the tumor-bearing animals from 51 to 78 days. Significant efficacy of CT1746 was observed on primary tumor growth (32% reduction in mean tumor area at day 36), total spread and metastasis (6/20 treated animals had no detectable spread and metastasis at autopsy compared to 100% incidence of secondaries in control groups). Efficacy of CT1746 could also be seen on reducing tumor spread and metastasis to individual organ sites such as the abdominal wall, cecum and lymph nodes compared to vehicle and untreated controls. We conclude that chronic administration of a peptidomimetic MMP inhibitor via the oral route is feasible and results in inhibition of solid tumor growth, spread and metastasis with increase in survival in this model of human cancer, thus converting aggressive cancer to a more controlled indolent disease.

Non-retinal abnormalities associated with progressive retinal atrophy (PRA) in the miniature poodle.

Osmotic fragility was examined in red blood cells taken from miniature poodles with progressive retinal atrophy (PRA). In this study, the median osmotic fragility for erythrocytes (concentration of NaCl required for 50% haemolysis) was found to be significantly lower for clinically affected, compared to unaffected animals. These differences were maintained regardless of changes in erythrocyte incubation pH (7.0, 7.5). There was no difference in the slope parameter of erythrocyte osmotic fragility profiles for both sets of animals, indicating a similar variability of erythrocyte age. In groups of animals maintained with the same diet and fasted for 12 hr, and in groups of animals with no dietary constraints, total plasma cholesterol was significantly lower for affected, compared to unaffected animals. Red blood cell indices were also assessed; mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), red blood cell count (RBC) and blood haemoglobin concentration (Hb) were normal, while erythrocyte packed cell volume (PCV) was significantly reduced for affected, compared to unaffected. Data from this study suggest that the abnormal erythrocyte osmotic fragility of affected animals is unlikely to be a consequence of a higher erythrocyte surface area/volume ratio, higher plasma cholesterol concentration, or greater proportion of new to old erythrocytes, and that a general membrane defect might be associated with prcd in the miniature poodle.

Functional importance of the dihydropyridine-sensitive, yet voltage-insensitive store-operated Ca2+ influx of U937 cells.

The Ca2+ current activated by Ca2+ store depletion in non-excitable cells is classically regarded as being dihydropyridine-insensitive, suggesting that store-operated Ca2+ channels (SOCs) are dissimilar to voltage-gated Ca2+ channels (VGCs) of excitable-cells. Here, we demonstrate dihydropyridine-sensitivity for the store-operated Ca2+ influx induced by ATP and thapsigargin (Tg) in the non-excitable U937 cell-line. Ca2+ store depletion by prior treatment of cells with either Tg or ATP, stimulated a Ca2+ entry mechanism that was inhibited by nicardipine, nifedipine, and the specific L-type Ca2+ channel blocker, calciseptine. A functional requirement for this Ca2+ influx mechanism in agonist-induced mitogenesis seemed likely, since nicardipine, a particularly potent inhibitor of store-operated Ca2+ influx in these cells, suppressed ATP- and Tg-stimulated cell proliferation. Depolarisation of cells with KCl, or gramicidin failed to elicit an increase in cytosolic Ca2+, suggesting that while the store-operated Ca2+ influx channel of U937 cells shares pharmacologic properties with the L-type Ca2+ channel, it is voltage-insensitive and therefore may resemble an L-type Ca2+ channel lacking a voltage sensor.

Calcium store depletion potentiates a phosphodiesterase inhibitor- and dibutyryl cGMP-evoked calcium influx in rat pituitary GH3 cells.

A role for cGMP in the control of capacitative Ca2+ influx was identified in rat pituitary GH3 cells. Application of 50 microM - 1 mM of the non-specific phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), or the specific cGMP-phosphodiesterase inhibitor, zaprinast, induced a dose-dependent increase in the intracellular free Ca2+ concentration [Ca2+]i of the pituitary cell line, as assessed by video ratio imaging using fura-2. Response onset times were identical and response profiles were similar in all cells analysed. Application of 50 microM dibutyryl cGMP to GH3 cells resulted in heterogeneous Ca2+ responses, consisting of single or multiple transients with varying onset times. In all cases, increases in [Ca2+]i were predominantly due to Ca2+ influx, since no responses were detected in low Ca2+ medium, or following pre-incubation of cells with 1 microM verapamil, or nicardipine. Depleting intracellular Ca2+ stores by prior treatment of cells with 1 microM thapsigargin resulted in a dramatic potentiation in the Ca2+ influx mediated by both phosphodiesterase inhibitors and dibutyryl cGMP, suggesting that cGMP modulates a dihydropyridine-sensitive Ca2+ entry mechanism in GH3 cells which is possibly regulated by the state of filling of Ca2+ stores.

Effect of matrix metalloproteinase inhibitors on tumor growth and spontaneous metastasis.

Four potent, synthetic inhibitors of matrix metalloproteinases (MMPs) were assessed as inhibitors of tumor growth and spontaneous metastasis to the lung. Mat Ly Lu rat prostate tumor, LOX human melanoma and M27 murine Lewis lung tumor were implanted subcutaneously (s.c.) in mice and allowed to grow for 3-12 days. The lungs of the tumor-bearing mice were then removed and implanted s.c. into untreated mice, and the outgrowth of secondary tumors from the implanted lungs measured. The incidence and rate of outgrowth of secondary tumors increased with the length of primary tumor growth, validating these measurements as indices of spontaneous metastasis to the lung. Compounds were tested by s.c. implantation of minipumps which delivered compound throughout the period of primary tumor growth and spontaneous metastasis to the lung at steady-state drug concentrations orders of magnitude greater than the concentrations needed to either inhibit collagenase, gelatinase or stromelysin in vitro. Inhibitor treatment slowed the growth of primary s.c. Mat Ly Lu and LOX tumors by 40-60% but had no significant effect on the growth of primary M27 tumors. Surprisingly, inhibitor treatment had no significant effect on the ability of the lung to generate secondary tumors when reimplanted s.c. in untreated mice. Because of the possible importance of cathepsins B, H and L in tumor growth and metastasis, the irreversible inhibitor E-64 was also infused by s.c. minipump. E-64 had no effect on the growth or spontaneous metastasis of Mat Ly Lu or M27 tumors.

Anti-Ig-induced calcium influx in rat B lymphocytes mediated by cGMP through a dihydropyridine-sensitive channel.

In contrast to excitable tissues where calcium channels are well characterized, the nature of the B lymphocyte calcium channel is unresolved. Here, we demonstrate by single cell analysis of freshly isolated rat B cells that the anti-immunoglobulin (Ig)-induced calcium influx takes place through a channel which shares pharmacologic and serologic properties with the L-type calcium channel found in excitable tissues. It is sensitive to the dihydropyridines nicardipine and Bay K 8644, to calciseptine, and to an anti-peptide antibody raised against the alpha1 subunit of the L-type calcium channel, but is voltage-insensitive. Anti-alpha1 and anti-alpha2 antibodies stain B but not T lymphocytes. Application of a cGMP agonist, measurement of cGMP levels in anti-Ig-stimulated B cells, and examining the effect of a guanylyl cyclase inhibitor on the anti-Ig response show that cGMP mediates the influx. This possibly involves a cGMP-dependent protein kinase. The anti-Ig-induced response is not abolished by prior treatment of B cells with a high dose of thapsigargin. These findings undermine the widely held belief of a categorical divide between excitable and non-excitable tissue calcium channels, demonstrate the limitations of the capacitative calcium influx theory, and point to a distinction between the calcium response mechanisms utilized by B and T lymphocytes.

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.

A cADP-ribose antagonist does not inhibit secretagogue-, caffeine- and nitric oxide-induced Ca2+ responses in rat pancreatic beta-cells.

It is controversial whether the Ca2+ mobilizing agent, cADP-ribose (cADPR), is implicated in secretagogue-mediated intracellular Ca2+ responses of pancreatic beta-cells. In this study we utilised a potent antagonist of cADPR, 8-amino-cADPR, to determine whether cADPR is involved in glucose-, acetylcholine-, caffeine- and nitric oxide-induced intracellular Ca2+ responses of isolated rat beta-cells. The antagonist was found to be effective in the complete inhibition of cADPR-induced Ca2+ release from sea urchin egg microsome preparations, when used at equivalent concentrations to cADPR (between 0.1-10 microM) in the assay. Isolated beta-cells were co-loaded with up to 50 microM 8-amino-cADPR, and Fura-2 or Fluo-3, by the whole-cell patch technique. At this concentration, the antagonist failed to affect standard glucose- and acetylcholine-induced increases in the intracellular free Ca2+ ([Ca2+]i) of isolated rat pancreatic beta-cells, as assessed by video ratio imaging and single wavelength microfluorimetry. Applying the same methodology, the antagonist also failed to affect NO- and caffeine-induced intracellular Ca2+ responses of rat beta-cells. These results suggest that cADPR does not appear to play a fundamental role in beta-cell Ca2+ signalling. As a control, patch-loading with heparin (2 mg/ml) however, abolished the acetylcholine response but neither affected the NO- or caffeine-induced mobilization of intracellular Ca2+. These results support the involvement of the IP3-receptor in acetylcholine-induced mobilization of intracellular Ca2+, but not that invoked by caffeine.