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A D Short

Publications and source records attributed to A D Short.

At least 19 recordsLinked to original sources
PubMed2006

Identification of susceptibility and protective major histocompatibility complex haplotypes in canine diabetes mellitus.

Diabetes mellitus occurs spontaneously in dogs, which is believed to have an autoimmune component and to be a model of human latent autoimmune diabetes of adults (LADA). Some dog breeds (e.g. Samoyed) are particularly predisposed, whereas others (e.g. Boxer) are highly resistant. With the completion of the Dog Genome Assembly, comparative genomic studies of complex diseases in dogs, including diabetes, could provide an important investigative approach into such disorders. Type 1 diabetes in humans is strongly associated with major histocompatibility complex (MHC) class II polymorphisms. We have investigated whether canine dog leucocyte antigen (DLA) class II haplotypes are associated with diabetes. DNA from 460 cases and 1047 controls were genotyped for DLA-DRB1, DLA-DQA1 and DLA-DQB1 using sequence-based typing. Three DLA haplotypes, DRB1*009/DQA1*001/DQB1*008, DRB1*015/DQA1*0061/DQB1*023 and DRB1*002/DQA1*009/DQB1*001, were found at significantly increased frequency in cases with diabetes compared with controls. One DLA-DQ haplotype, DQA1*004/DQB1*013, was significantly reduced in cases with diabetes. Further analysis showed that DQA1 alleles carrying arginine at codon 55 of DQA1 were increased in dogs with diabetes. To our knowledge, this is the first report of a comparative study of MHC and diabetes in a non-rodent species. Since no laboratory model of LADA exists and dogs and humans share similar environments, further research into canine diabetes is warranted.

Alleles↗
PubMed2005

Canine DNA subjected to whole genome amplification is suitable for a wide range of molecular applications.

Molecular and genetic studies of canine disease phenotypes can be limited by the amount of DNA available for analysis. New methods have been developed to amplify the genomic DNA of a species producing large quantities of DNA from small starting amounts. Whole genome amplification (WGA) of DNA is now being used in human studies, although this technique has not been applied extensively in veterinary research. We evaluated WGA of canine DNA for suitability in a range of molecular tests. DNA from 93 canine blood extracted and 18 buccal swab samples was subjected to WGA using the GenomiPhi kit (Amersham). Genomic DNA was compared with WGA product using a range of techniques, including reference strand-mediated conformation analysis, denaturing high-performance liquid chromatography analysis, microsatellite genotyping, direct DNA sequencing, and single nucleotide polymorphism allelic discrimination. All samples amplified well, giving an average yield of 3 mug of DNA from 2.5 ng of starting material. Extremely high levels of experimental reproducibility and concordance were observed between source and WGA DNA samples for all analyses used: greater than 95% for blood extracted DNA and greater than 80% for buccal swab DNA. These studies clearly demonstrate the usefulness of WGA of canine DNA as a means of increasing DNA quantities for canine studies. This technique will have major implications for future veterinary research.

Animals↗
PubMed2000

Different receptors use inositol trisphosphate to mobilize Ca(2+) from different intracellular pools.

In cells expressing different receptors linked to Ins(1,4,5)P(3) formation, maximal stimulation of any one of them often releases all the Ins(1,4,5)P(3)-sensitive Ca(2+) stores, suggesting that Ins(1,4, 5)P(3) is used similarly by many receptors. In single HEK-293 cells, ATP and carbamylcholine (CCh) stimulated Ca(2+) release from intracellular stores via a pathway that was entirely dependent on Ins(1,4,5)P(3). After stimulation with maximal concentrations of ATP or CCh in Ca(2+)-free medium, there was no response to a second stimulation with the same agonist, indicating that each agonist had emptied the Ins(1,4,5)P(3)-sensitive stores to which it had access. However, the Ca(2+) release evoked by the second agonist was unaffected by prior stimulation with the first. We conclude that Ins(1,4,5)P(3) mediates the effects of both receptors, but Ins(1,4, 5)P(3) is more versatile than hitherto supposed, because the spatial organization of the signalling pathways apparently allows Ins(1,4, 5)P(3) made in response to each agonist to interact with different Ins(1,4,5)P(3) receptors.

Adenosine Triphosphate↗
PubMed2000

Parathyroid hormone controls the size of the intracellular Ca(2+) stores available to receptors linked to inositol trisphosphate formation.

In HEK 293 cells stably expressing type 1 parathyroid (PTH) receptors, PTH stimulated release of intracellular Ca(2+) stores in only 27% of cells, whereas 96% of cells responded to carbachol. However, in almost all cells PTH potentiated the response to carbachol by about 3-fold. Responses to carbachol did not desensitize, but only the first challenge in Ca(2+)-free medium caused an increase in [Ca(2+)](i), indicating that the carbachol-sensitive Ca(2+) stores had been emptied. Subsequent addition of PTH also failed to increase [Ca(2+)](i), but when it was followed by carbachol there was a substantial increase in [Ca(2+)](i). A similar potentiation was observed between ATP and PTH but not between carbachol and ATP. Intracellular heparin inhibited responses to carbachol and PTH, and pretreatment with ATP and carbachol abolished responses to PTH, suggesting that the effects of PTH involve inositol trisphosphate (IP(3)) receptors. PTH neither stimulated detectable IP(3) formation nor affected the amount formed in response to ATP or carbachol. PTH stimulated cyclic AMP formation, but this was not the means whereby PTH potentiated Ca(2+) signals. We suggest that PTH may regulate Ca(2+) mobilization by facilitating translocation of Ca(2+) between discrete intracellular stores and that it thereby regulates the size of the Ca(2+) pool available to receptors linked to IP(3) formation.

Adenosine Triphosphate↗
PubMed1999

Receptors linked to polyphosphoinositide hydrolysis stimulate Ca2+ extrusion by a phospholipase C-independent mechanism.

In A7r5 cells with empty intracellular Ca(2+) stores in which the cytosolic free Ca(2+) concentration ([Ca(2+)](i)) had been increased by capacitative Ca(2+) entry, stimulation of receptors linked to phospholipase C (PLC), including those for Arg(8)-vasopressin (AVP) and platelet-derived growth factor (PDGF), caused a decrease in [Ca(2+)](i.) This effect was further examined in a stable variant of the A7r5 cell line in which the usual ability of hormones to stimulate non-capacitative Ca(2+) entry is not expresssed. In thapsigargin-treated cells, neither AVP nor PDGF affected capacitative Mn(2+) or Ba(2+) entry, but both stimulated the rate of Ca(2+) extrusion, and their abilities to decrease [Ca(2+)](i) were only partially inhibited by removal of extracellular Na(+). These results suggest that receptors linked to PLC also stimulate plasma membrane Ca(2+) pumps. Activation of protein kinase C by phorbol 12, 13-dibutyrate (PDBu, 1 microM) also caused a decrease in [Ca(2+)](i) by accelerating Ca(2+) removal from the cytosol; the effect was again only partially inhibited by removal of extracellular Na(+). An inhibitor of PKC, Ro31-8220 (10 microM), abolished the ability of PDBu to decrease [Ca(2+)](i), without affecting the response to maximal or submaximal concentrations of AVP. Similar experiments with PDGF were impracticable because Ro31-8220, presumably by inhibiting the tyrosine kinase activity of the PDGF receptor, abolished all responses to PDGF. U73122 (10 microM), an inhibitor of PLC, completely inhibited PDGF- or AVP-evoked Ca(2+) mobilization, without preventing either stimulus from causing a decrease in [Ca(2+)](i). We conclude that receptors coupled to PLC, whether via G-proteins or protein tyrosine kinase activity, also share an ability to stimulate the plasma membrane Ca(2+) pump via a mechanism that does not require PLC activity.

Animals↗
PubMed1999

Psychosocial adjustment of children with chronic illness: an evaluation of three models.

This study was designed to assess social, emotional, and behavioral functioning of children with chronic illness and to evaluate three models addressing the impact of chronic illness on psychosocial functioning: discrete disease, noncategorical, and mixed. Families of children with cancer, sickle cell disease, hemophilia, and juvenile rheumatoid arthritis participated, along with families of classroom comparison peers without a chronic illness who had the closest date of birth and were of the same race and gender (COMPs). Mothers, fathers, and children provided information regarding current functioning of the child with chronic illness or the COMP child. Child Behavior Checklist and Children's Depression Inventory scores were examined. Results provided support for the noncategorical model. Thus, the mixed model evaluated in this study requires modifications before its effectiveness as a classification system can be demonstrated.

Adaptation, Psychological↗
PubMed1997

Store-operated Ca2+ entry and coupling to Ca2+ pool depletion in thapsigargin-resistant cells.

The release of Ca2+ from intracellular Ca2+ pumping pools and the entry of extracellular Ca2+ are tightly coupled events. The potent and specific intracellular Ca2+ pump inhibitor, thapsigargin, blocks Ca2+ accumulation and allows Ca2+ release from pools within mammalian cells, inducing major changes in endoplasmic reticulum function and cell growth. Recent studies characterized the pools of Ca2+ within permeabilized DC-3F/TG2 cells (a thapsigargin-resistant variant form of the DC-3F Chinese hamster lung fibroblast line, able to grow in 2 microM thapsigargin), revealing highly thapsigargin-resistant intracellular Ca2+ pumping activity capable of accumulating Ca2+ within an inositol 1,4,5-trisphosphate-releasable Ca2+ pool (Waldron, R. T., Short, A. D., and Gill, D. L. (1995) J. Biol. Chem. 270, 11955-11961). Using intact fura-2-loaded thapsigargin-resistant DC-3F/TG2 cells, the present study investigated the role of this unusual Ca2+ pumping activity in maintaining cytosolic Ca2+, generating Ca2+ signals, and mediating Ca2+ entry. The thapsigargin-resistant Ca2+ pumping pool was capable of generating rapid cytosolic Ca2+ signals in response to the phospholipase C-coupled agonist, oleoyl lysophosphatidic acid. The resting level of cytosolic Ca2+ in DC-3F/TG2 cells was 2-fold elevated compared with control cells (the parent DC-3F line), and transient extracellular Ca2+ removal induced a large "overshoot" in cytosolic Ca2+. The overshoot response was blocked by the Ca2+ influx inhibitor, SKF96365, and was kinetically identical to that induced in parent DC-3F cells after thapsigargin-induced Ca2+ pool emptying, indicating that the thapsigargin-resistant DC-3F/TG2 cells had "constitutively" opened Ca2+ entry channels coupled to an emptied or partially emptied thapsigargin-sensitive Ca2+ pumping pool. Even though oleoyl lysophosphatidic acid-mediated Ca2+ release induced little Ca2+ entry, complete ionomycin-activated emptying of the thapsigargin-resistant Ca2+ pool in DC-3F/TG2 cells induced a large, sustained entry of Ca2+ that was also completely blocked by SKF96365. The results revealed that the thapsigargin-resistant Ca2+ pump does maintain physiological Ca2+ levels, is able to fill an agonist-responsive Ca2+ pool in DC-3F/TG2 cells, and is likely responsible for the ability of these cells to function and grow in the presence of thapsigargin. In addition, Ca2+ influx in the resistant DC-3F/TG2 cells reflects emptying of pools that accumulate Ca2+ by both thapsigargin-sensitive and -resistant Ca2+ pumps; since these pumps accumulate Ca2+ in distinct pools in parent DC-3F cells, it is possible that more than one pool is coupled to Ca2+ influx in the resistant DC-3F/TG2 cells.

Animals↗
PubMed1997

Electrophysiological evidence that spinomesencephalic neurons in the cat may be excited via spinocervical tract collaterals.

Extracellular microelectrode recordings were made from spinomesencephalic tract (SMT) neurons in the lumbosacral spinal cord of cats anaesthetized with chloralose and paralysed with gallamine triethiodide. The SMT cells were antidromically fired from the posterolateral parts of the superior colliculus and the intercollicular region, were located in laminae IV to VIII, and had response properties and axonal conduction velocities similar to those described previously. The effects of stimulating the dorsolateral funiculus of the cervical cord at C3 and rostral C1, below and above the termination of spinocervical tract (SCT) axons in the lateral cervical nucleus, were examined on 33 SMT cells. The strength of stimulation was adjusted so that at C3 it was above threshold for antidromic activation of SCT cells and at C1 was below threshold for activation of the same cells. Seven (21%) SMT neurons were excited from C3 but not from C1. The remaining 26 (79%) were excited from both C3 and rostral C1 and 23 (70% of these) were excited significantly more from C3. That is, 91% of the total sample were either excited only from C3 or more strongly from C3 than from rostral C1. We discuss the possible neuronal systems involved and conclude that the greater excitatory effects from C3 are most likely due to antidromic activation of the SCT. The shortest latency effects from C3 indicate a monosynaptic linkage between SCT cells with the fastest axons and the SMT. The longer latency actions may be due to monosynaptic connexions from SCT cells with slower conducting axons, to di- or polysynaptic actions from SCT cells with fast axons, or a combination of both. SMT cells are another population of spinal neurons, in addition to postsynaptic dorsal column, spinothalamic and dorsal horn spinocerebellar neurons, which receive excitation via SCT collaterals.

Animals↗
PubMed1995

A novel Ca2+ entry mechanism is turned on during growth arrest induced by Ca2+ pool depletion.

Ca2+ pool depletion with Ca2+ pump blockers induces growth arrest of rapidly dividing DDT1MF-2 smooth muscle cells and causes cells to enter a stable, quiescent G0-like growth state (Short, A.D., Bian, J., Ghosh, T.K., Waldron, R.T., Rybak, S.L., and Gill, D.L. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 4986-4990). Here we reveal that induction of this quiescent growth state with the Ca2+ pump blocker, thapsigargin, is correlated with the appearance of a novel caffeine-activated Ca2+ influx mechanism. Ca2+ influx through this mechanism is clearly distinct from and additive with Ca2+ entry through store-operated channels (SOCs). Whereas SOC-mediated entry is activated seconds after Ca2+ pool release, caffeine-sensitive influx requires at least 30 min of pool emptying. Although activated in the 1-10 mM caffeine range, this mechanism has clearly distinct methylxanthine specificity from ryanodine receptors and is not modified by ryanodine. It is also unaffected by the Ca2+ channel blockers SKF96365 or verapamil and is independent of modifiers of cyclic nucleotide levels. Growth arrest by thapsigargin-induced Ca2+ pool depletion can be reversed by treatment with 20% serum (Waldron, R.T., Short, A.D., Meadows, J.J., Ghosh, T.K., and Gill, D.L. (1994) J. Biol. Chem. 269, 11927-11933). The serum-induced return of functional Ca2+ pools and reentry of cells into the cell cycle correlates exactly with the disappearance of the caffeine-sensitive Ca2+ influx mechanism. Therefore, appearance and function of this novel Ca2+ entry mechanism are closely tied to Ca2+ pool function and cell growth state and may provide an important means for modifying exit from or entry into the cell cycle.

Animals↗
PubMed1995

Thapsigargin-resistant intracellular calcium pumps. Role in calcium pool function and growth of thapsigargin-resistant cells.

Exposure of cells to the intracellular Ca2+ pump blocker, thapsigargin (TG), results in emptying of Ca2+ pools and termination of cell proliferation (Short, A. D., Bian, J., Ghosh, T. K., Waldron, R. T., Rybak, S. L., and Gill, D. L. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 4986-4990). DC-3F Chinese hamster lung cells were made resistant to TG by long-term stepwise exposure to increasing TG concentrations in culture (Gutheil, J. C., Hart, S. R., Belani, C. P., Melera, P. W., and Hussain, A. (1994) J. Biol. Chem. 269, 7976-7981). Since these cells (DC-3F/TG2) grow in the presence of TG, it was important to ascertain what Ca2+ pool function they retain. TG-resistant DC-3F/TG2 cells cultured with 2 microM TG had a doubling time (24 h) not significantly different from the parent DC-3F cells without TG. Analysis of TG-induced inhibition of 45Ca2+ uptake into permeabilized parent DC-3F cells revealed two distinct Ca2+ pump activities with 20,000-fold different sensitivities to TG; the IC50 values for TG were 200 pM and 4 microM, representing 80% and 20% of total pumping activity, respectively. Total pump activity in parent DC-3F and resistant DC-3F/TG2 cells was similar (0.23 +/- 0.10 and 0.18 +/- 0.08 nmol of Ca2+/10(6) cells, respectively). In DC-3F/TG2 cells, up to 100 nM TG had no effect on Ca2+ pumping; however, almost all pumping was blocked at higher TG concentrations with an IC50 of 5 microM. In both cell types, each Ca2+ pump activity (regardless of TG sensitivity) had high Ca2+ affinity (Km values congruent to 0.1 microM) and similar ATP dependence and vanadate sensitivity. In DC-3F cells, the TG-sensitive Ca2+ pool was releasable with inositol 1,4,5-trisphosphate (InsP3) or GTP and was oxalate-permeable; the TG-insensitive pool in these cells was not InsP3-releasable. GTP-induced Ca2+ uptake in the presence of oxalate indicated Ca2+ transfer between distinct pools in the DC-3F cells. In resistant DC-3F/TG2 cells, almost 50% of total TG-insensitive Ca2+ accumulation was releasable with InsP3; unlike the parent cells, this pool was not oxalate-permeable, and GTP induced no Ca2+ transfer between pools in the presence of oxalate. Thus, whereas InsP3 releases Ca2+ only from the high TG sensitivity Ca2+ pumping pool in parent DC-3F cells, in resistant DC-3F/TG2 cells the TG-resistant Ca2+ pumping pool now contains functional InsP3 receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1995

Effects of upper cervical spinal cord stimulation on neurons in the lumbosacral enlargement of the cat: spinothalamic tract neurons.

Extracellular microelectrode recordings were made from deep spinothalamic tract neurons in the lumbosacral spinal cord of cats anaesthetized with chloralose and paralyzed with gallamine triethiodide. The effects of upper cervical spinal cord stimulation were tested on 43 spinothalamic tract neurons, by stimulation of the ipsilateral dorsolateral funiculus at C3 and rostral C1 using five or six shocks at 333 Hz. The strength of cervical stimulation was adjusted so that the C3 shock was above threshold for antidromic activation of spinocervical tract neurons but the same strength of shock applied at C1 was below threshold for the same neurons. Four of the 43 spinothalamic cells (9%) were not influenced by upper cervical stimulation. The remaining 39 spinothalamic tract cells (91%) were all excited from the upper cervical cord. Twenty-seven of these (63%) were excited more strongly from C3 than from C1, 4 (9%) were excited more strongly from C1 than from C3, and the remaining eight cells (19%) showed no significant differences between their responses to stimulation at C1 and C3. There were no obvious differences between those spinothalamic tract neurons showing differential effects from C1 and C3 and those showing no such effects. The neuronal systems possibly responsible for the differential effects from C3 and C1 on spinothalamic tract neurons are discussed. We conclude that the most likely candidate system for the greater excitation from C3 compared with C1 is the subset of spinocervical tract neurons with axon collaterals in the lumbosacral enlargement and that the spinothalamic tract is a further ascending path, in addition to the postsynaptic dorsal column path, that receives excitatory input from spinocervical axon collaterals. The greater excitation from C1 compared with C3 is interpreted as due to excitation from C1 and a mixture of excitation and inhibition from C3. The responsible neuronal systems seem likely to be either the spinocervical neurons with axon collaterals operating on the spinothalamic tract via inhibitory interneurons, or cells in the lateral cervical nucleus with axons descending to the lumbosacral cord.

Animals↗
PubMed1994

The role of calcium, pH, and cell proliferation in the programmed (apoptotic) death of androgen-independent prostatic cancer cells induced by thapsigargin.

Calcium (Ca2+) accumulates within the endoplasmic reticulum of cells through function of the sarcoplasmic reticulum and endoplasmic reticulum Ca(2+)-dependent ATPase family of intracellular Ca(2+)-pumping ATPases. The resulting pools have important signaling functions. Thapsigargin (TG) is a sesquiterpene gamma-lactone which selectively inhibits the sarcoplasmic reticulum and endoplasmic reticulum Ca(2+)-dependent ATPase pumps with a 50% inhibitory concentration of approximately 30 nM. Treatment of androgen-independent prostate cancer cells of both rat and human origin with TG inhibits their endoplasmic reticulum Ca(2+)-dependent ATPase activity, resulting in a 3-4-fold elevation in the level of intracellular free Ca2+ (Cai) within minutes of exposure. Due to a secondary influx of extracellular Ca2+, this increase in Cai is sustained, resulting in morphological (cell rounding) and biochemical changes within 6-12 h (enhanced calmodulin, glucose regulated protein, and tissue transglutaminase expression, and decreased expression of the G1 cyclins). Within 24 h of exposure, androgen-independent prostatic cancer cells stop progression through the cell cycle, arrest out of cycle in G0, and irreversibly lose their ability to proliferate with a median effective concentration value of 31 nM TG. During the next 24-48 h, the genomic DNA of the G0-arrested cells undergoes double-strand fragmentation. This is followed by the loss of plasma membrane integrity and fragmentation of the cell into apoptotic bodies. During this process, there is no acidification in the intracellular pH. Using cells transfected with the avian M(r) 28,000 calbindin D Ca(2+)-buffering protein, it was demonstrated that the programmed death initiated by TG is critically dependent upon an adequate (i.e., 3-4-fold) sustained (> 1 h) elevation in Cai and not depletion of the endoplasmic reticulum pools of Ca2+. These results demonstrate that TG induces programmed cell death in androgen-independent prostatic cancer cells in a dose-dependent manner and that this death does not require proliferation or intracellular acidification but is critically dependent upon an adequate, sustained (i.e., > 1 h) elevation in Cai.

Androgens↗
PubMed1994

Endoplasmic reticulum calcium pump expression and control of cell growth.

Intracellular Ca2+ pump expression and Ca2+ pool function are shown to be closely associated with growth and proliferation of DDT1MF-2 hamster smooth muscle cells. The Ca2+ pump blocker thapsigargin induces sustained Ca2+ pool emptying and entry of cells into a quiescent G0-like state (Short, A. D., Bian, J., Ghosh, T. K., Waldron, R. T., Rybak, S. L., and Gill, D. L. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 4986-4990). Using DDT1MF-2 cells growth-arrested by exposure to 3 microM thapsigargin for 24 h, treatment with 20% serum for 6 h without thapsigargin induced expression of functional Ca2+ pump protein detected as a 110-kDa thapsigargin-sensitive phosphorylated intermediate; 2.5% serum treatment resulted in no functional pump expression. Western analysis revealed only a slight serum-induced increase in total Ca2+ pump protein. New functional Ca2+ pump protein could be detected within 1 h of high serum treatment of thapsigargin-arrested cells, increasing over a 6-h period and correlating with the appearance of new Ca2+ pools. Induction of Ca2+ pools required serum at 10% or higher; no pools appeared with 5% serum or less. Significantly, high serum was required for only a brief but precise period of time. Exposure of thapsigargin-arrested cells to a 45-min pulse of 20% serum followed by continued culture in 2.5% serum was sufficient for full induction of new functional Ca2+ pump protein and Ca2+ pools; in contrast, no pumps or pools were detected after a 30-min serum pulse. A 40-min high serum pulse resulted in arrested cells reentering the cell cycle, synthesizing DNA, and resuming normal proliferation; in contrast, 35 min of serum treatment resulted in cells remaining totally quiescent. The results provide important evidence for the necessity of functional endoplasmic reticulum Ca2+ pumps in serum-induced cell growth and reflect a remarkably precise signaling period during which quiescent cells become committed to a progression of events including Ca2+ pump expression, Ca2+ pool function, reentry into the cell cycle, and cell division.

Animals↗
PubMed1994

Differential ascending projections from neurons in the cat's lateral cervical nucleus.

Extracellular microelectrode recordings were made from single cells of the lateral cervical nucleus (LCN) in cats anaesthetized with chloralose and paralysed with gallamine triethiodide. The cells were tested for antidromic activation from the contralateral medial lemniscus and the contralateral tectum. Seventy-two LCN units were recorded which projected to one or both targets. Sixty (83%) projected through the medial lemniscus, and of these 36 (50% of the total) also projected to the tectum, whereas 24 (33%) projected through the medial lemniscus only; 12 (17%) projected only to the tectum. Twenty-nine units (40%) were excited by moving hairs of the coat but not by pinch of the skin, and 9 (31%) of these projected to the tectum, 11 (38%) through the medial lemniscus and 9 (31%) to both targets. Forty units (56%) were excited by hair movement and noxious pinch, and 3 (7%) of these projected to the tectum, 10 (25%) through the medial lemniscus and 27 (68%) to both targets. Three units (4%) had no discernible receptive fields and they all projected through the medial lemniscus, but not to the tectum. Of the 12 units projecting only to the tectum, 11 had receptive fields completely or partially on the trunk. Units projecting either through the medial lemniscus only, or through the medial lemniscus and also into the tectum, had receptive fields more widely distributed: these included small fields on the fore- and hind feet, on the limbs and also, a minority, on the trunk. Units with glove- or stocking-like receptive fields projected through the medial lemniscus. The results show that while most LCN cells project through the medial lemniscus, those excited by hair movement alone preferentially project either to the tectum or through the medial lemniscus, but not by both routes. The differences in receptive field properties of the differently projecting units are discussed in terms of the possible functions of the spinocervical system.

Afferent Pathways↗
PubMed1993

Inositol 1,4,5-trisphosphate-mediated quantal Ca2+ release measured by high resolution imaging of Ca2+ within organelles.

The distribution and operation of Ca2+ pools within cells has been directly studied in situ by monitoring the Ca2+ inside Ca2+ dye-loaded organelles using high resolution imaging procedures. Using DDT1MF-2 smooth muscle cells, loaded with fura-2 under conditions favoring dye entry into organelles and subjected to carefully controlled permeabilization still attached to coverslips, the Ca2+ within organelles was analyzed by high resolution, z axis-controlled imaging, and deblurring methods. Saturation analysis of entrapped fura-2 indicated that the dye reported Ca2+ identically to fura-2 in solution. Areas containing high Ca(2+)-sequestering organelles (> 5 microM free Ca2+) were observed to predominate around the nucleus and close to the periphery of the cell. Analysis of the actions of inositol 1,4,5-trisphosphate (InsP3) within small (3 microns 2) selected intracellular areas, revealed a "quantal" release phenomenon, with rapid attainment of limited stable release at submaximal InsP3 levels. The apparent EC50 for InsP3 was approximately 3 microns, higher than within suspensions of permeabilized cells. The action of InsP3 was competitively blocked by 10 micrograms/ml of the InsP3 antagonist, heparin. Applied after maximal InsP3-mediated Ca2+ release, heparin reversed InsP3-induced Ca2+ release resulting in reuptake of Ca2+ into Ca(2+)-pumping organelles with identical spatial distribution as before Ca2+ release. InsP3 released Ca2+ from all areas of high Ca(2+)-pumping organelles; extensive areas of high fura-2-loading, but low intraorganelle Ca2+, were unchanged by InsP3. GTP induced no alteration in Ca2+ release (in contrast to suspensions of permeabilized cells), suggesting that the InsP3-sensitive Ca2+ pool was functioning as a single homogeneous pool. Opening of InsP3-sensitive channels was also monitored by assessing InsP3-activated channel-mediated Mn2+ quenching of organelle-loaded fura-2; the results revealed a similar pattern of quantal release, with slightly increased apparent InsP3 sensitivity. The results provide the first high resolution in situ localization of Ca2+ signaling organelles and demonstrate the quantal operation of InsP3-sensitive Ca2+ pools within highly discrete subcellular loci.

Animals↗
PubMed1993

Intracellular Ca2+ pool content is linked to control of cell growth.

A close correlation was observed between intracellular Ca2+ pool depletion and refilling and the onset of DNA synthesis and proliferation of DDT1MF-2 smooth muscle cells. The intracellular Ca2+ pump inhibitors 2,5-di-tert-butyl-hydroquinone (DBHQ) and thapsigargin (TG) specifically emptied identical inositol 1,4,5-trisphosphate (InsP3)-sensitive Ca2+ pools and both arrested cell growth at concentrations corresponding to Ca2+ pump blockade. However, an important distinction was observed between the two inhibitors with respect to their reversibility of action. Upon removal of DBHQ from DBHQ-arrested cells, Ca2+ pools immediately refilled, and 14 hr later cells entered S phase followed by normal cell proliferation; the time for entry into S phase was identical to that for cells released from confluence arrest. Although TG irreversibly blocked Ca2+ pumping and emptied Ca2+ pools, high serum treatment of TG-arrested cells induced recovery of functional Ca2+ pools in 6 hr (via probable synthesis of new pump); thereafter cells proceeded to S phase and normal cell proliferation within the same time period (14 hr) as that following release of DBHQ-arrested cells. The precise relationship between Ca2+ pump blockade and growth arrest indicates that Ca2+ pool emptying maintains cells in a G0-like quiescent state; upon refilling of pools, normal progression into the cell cycle is resumed. It is possible that a specific cell cycle event necessary for G0 to G1 transition depends upon signals generated from the InsP3-sensitive Ca2+ pool.

Animals↗