The caring doctor is an oxymoron. General practice will develop best if "caring" is replaced by professionalism.
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Family Practice↗
Biomedical subjects
A D Short
Publications and source records attributed to A D Short.
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↗
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↗
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↗
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↗
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↗
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↗
Early management of myocardial infarction. The challenge for the GPs is to change.
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Aspirin↗
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↗
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↗
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↗
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↗
Function and organization of the inositol 1,4,5-trisphosphate-sensitive calcium pool.
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Animals↗
Persistent intracellular calcium pool depletion by thapsigargin and its influence on cell growth.
The intracellular Ca2+ pump inhibitor, thapsigargin, added to DDT1MF-2 smooth muscle cells in culture, irreversibly inhibited accumulation of Ca2+ within cells, permanently emptied the inositol 1,4,5-trisphosphate (InsP3)-sensitive Ca2+ pool, and simultaneously induced profound alteration of cell growth. After only a brief (30-min) treatment of cultured cells with 3 microM thapsigargin followed by extensive washing, the total releasable InsP3-sensitive Ca2+ pool remained entirely empty, even after 7 days of culture without thapsigargin. After thapsigargin treatment, cells retained viability, usual morphology, and normal mitochondrial function. Despite the otherwise normal appearance and function of thapsigargin-treated cells, cell division was completely blocked by thapsigargin. DNA synthesis was completely inhibited when thapsigargin was added immediately after passaging, but was suppressed only slowly (4-6 h) when added to rapidly synthesizing cells (24 h after passaging). Protein synthesis was reduced by approximately 70% in thapsigargin-treated cells. The sensitivity of thapsigargin-mediated inhibition of cell division, DNA synthesis, protein synthesis, and Ca(2+)-pumping activity were all similar with the EC50 values for thapsigargin in each case being close to 10 nM. Upon application to DDT1MF-2 cells, thapsigargin transiently increased resting cytosolic Ca2+ (0.15 microM) to a peak of 0.3 microM within 50 s; thereafter, free Ca2+ declined to 0.2 microM by 150 s and continued to slowly decline toward resting levels. Cells treated with thapsigargin for 1-72 h in culture displayed normal resting cytosolic Ca2+ levels. However, application of thapsigargin or epinephrine to such cells resulted in no change in the intracellular Ca2+, indicating that the internal Ca2+ pool remained completely empty. These results suggest that emptying of Ca2+ from intracellular thapsigargin-sensitive Ca(2+)-pumping pools induces profound alteration of cell proliferation.
Animals↗
Direct observations of synapses between GABA-immunoreactive boutons and identified spinocervical tract neurons in the cat's spinal cord.
Three spinocervical tract neurons in adult cats were physiologically characterized and intracellularly labelled with horseradish peroxidase. The neurons were reconstructed and examined with the light microscope and were prepared for postembedding immunochemical analysis by using an antiserum which specifically recognizes GABA in glutaraldehyde-fixed tissue. Semithin sections were tested and examined with the light microscope. Somata, proximal, and distal dendrites of all three cells were associated with numerous punctate GABA-immunoreactive structures. Immunoreactive perikarya of small neurons in the vicinity of spinocervical tract cells were also observed. Ultrastructural analysis, with the immunogold technique, revealed that somata and proximal dendrites of all three neurons received synaptic contacts (about 37% of total synapses) from GABA-immunoreactive boutons and that distal dendrites were also associated with substantial numbers of immunoreactive structures (about 27% of synapses). Immunoreactive boutons were small (about 1 micron in diameter), contained irregularly shaped agranular vesicles, and formed symmetrical synaptic junctions with identified neurons. An additional group of immunoreactive boutons was observed to be associated with one of the cells only; these contained many large dense-core vesicles in addition to small agranular vesicles. Boutons containing round agranular vesicles and flattened agranular vesicles were not observed to be immunoreactive. The evidence supports the idea that much of the postsynaptic inhibition observed in spinocervical tract neurons is mediated by GABA and that even the most distal dendrites of these neurons receive inhibitory inputs.
Animals↗
The effect of retinoic acid on parathyroid hormone- and parathyroid hormone-related peptide-induced intracellular calcium in a rat osteosarcoma cell line, UMR106.
We have examined the effects of parathyroid hormone (PTH) and PTH-related peptide (PTH-rP) on intracellular calcium (Ca2+i) in a rat osteogenic sarcoma cell line, UMR106. Synthetic bovine (b)PTH(1-34) caused a small inconsistent rise in Ca2+i in UMR106 cells, whilst cells pretreated with retinoic acid (RA, 1 mumol/l) for 18 h exhibited reproducible, significant and dose-dependent increases in Ca2+i levels in response to bPTH. The effect of RA on PTH-induced changes in Ca2+i were dependent upon both dose and time. Purified human (h)PTH-rP(1-34) increased Ca2+i in the absence of RA in the same cells. However, RA increased the magnitude of PTH-rP-stimulated changes in Ca2+i without affecting the concentration required for a maximal response. RA also prolonged the delay before the Ca2+i response was observed. Maximal responses to PTH-rP were greater in magnitude than those to PTH. These changes appeared not to be due to cyclic AMP (cAMP), since neither dibutyryl cAMP (1 mmol/l) nor forskolin (15 mumol/l) affected Ca2+i. PTH- and PTH-rP-mediated Ca2+i transients were not completely abolished by the absence of extracellular calcium, and both peptides increased basal levels of inositol trisphosphate. PTH and PTH-rP were subject to mutual desensitization, but were not desensitized by prostaglandin E2. PTH(7-34) antagonized PTH- but not PTH-rP-mediated Ca2+i transients. We conclude that there may be some important differences in the mechanism of action of PTH and PTH-rP.
Animals↗
Direct observations of synapses between GABA-immunoreactive boutons and muscle afferent terminals in lamina VI of the cat's spinal cord.
Single group Ia muscle afferent fibres in the lumbar spinal cord of the cat were impaled with microelectrodes and labelled with horseradish peroxidase. Two collateral axons were prepared for combined light and electron microscopy. Arbors selected from lamina VI were processed by the postembedding immunogold technique with antiserum which specifically recognizes GABA in glutaraldehyde-fixed tissue. Twelve Ia boutons were examined through series of thin sections with the electron microscope and all of them were associated with presynaptic axon terminals which were positively labelled for GABA. Some Ia boutons received synaptic contacts from several GABAergic terminals. The present study establishes that a GABA-like substance is present in axon terminals presynaptic to Ia afferent boutons in lamina VI of the spinal cord. This evidence provides a morphological basis for presynaptic inhibition of Ia afferent input into lamina VI.
Animals↗
Interleukin-1 stimulates diglyceride accumulation in the absence of protein kinase C activation.
Despite advances in the knowledge of the intracellular signalling in response to extracellular messengers, the mechanism of action of interleukin-1 (IL-1) has remained an enigma. In the present study, we have employed human dermal fibroblasts (Detroit 532 cells) to investigate IL-1 beta-induced changes in intracellular signals. Both recombinant human IL-1 beta and a native preparation purified from human placental tissue were employed. Cyclic AMP levels in cell monolayers were unaltered by IL-1 beta. Also, IL-1 beta did not influence significantly the levels of phosphatidylinositol, phosphatidylinositol 4-monophosphate, and phosphatidylinositol 4,5-bisphosphate in the membrane, nor the water-soluble inositol phosphates, inositol monophosphate, inositol bisphosphate and inositol trisphosphate, in cells prelabelled with myo-[3H]inositol. In addition, intracellular calcium as measured by Quin2 was unaffected by interleukin-1. However, in cells labelled with [3H]glycerol or [3H]arachidonic acid, IL-1 beta caused an immediate rise in diglyceride (DG) accumulation. As the effects of IL-1 beta have been reported to be mimicked by tumour-promoting phorbol esters, this rise in DG suggested the involvement of protein kinase C (PKC). However, repeated experiments failed to reveal any acute effect of IL-1 beta on the activity of this enzyme. Furthermore, IL-1 beta did not cause the translocation of PKC between the membrane and the cytosol as has been found in response to other extracellular signals. Rather, IL-1 beta appeared to increase the synthesis of PKC in both membrane and cytosol preparations, an effect which could be prevented by coincubation with cycloheximide. These findings suggest that the diglyceride formed in response to IL-1 beta does not activate protein kinase C.
Calcium↗