Biomedical subjects

D L Gill

Publications and source records attributed to D L Gill.

At least 19 recordsLinked to original sources

PubMed2004

Sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) gene silencing and remodeling of the Ca2+ signaling mechanism in cardiac myocytes.

Transient elevations of cytosolic Ca2+ are a common mechanism of cellular signaling. In striated muscle, the sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) plays an important role in terminating Ca2+ transients by returning cytosolic Ca2+ to intracellular stores. Stored Ca2+ can then be released again for subsequent signaling. We down-regulated SERCA2 gene expression in cultured cardiac myocytes by means of endogenous transcription of small interfering RNA encoded by an exogenous cDNA template. The cDNA template was delivered by adenovirus vector. Reduction of SERCA expression in all myocytes in culture was documented by immunochemistry, real-time RT-PCR, and determination of ATP-dependent Ca2+ transport. The reduction of SERCA2 expression was associated with the up-regulation of transient receptor potential (TRP) channel proteins (TRPC4 and TRPC5) and Na+/Ca2+ exchanger, indicating that intracellular store deficiency was compensated for by Ca2+ fluxes through the plasma membrane. In fact, SERCA silencing was followed by increased transcription of Na+/Ca2+ exchanger, TRPC4, TRPC5, and related transcriptional factors, such as stimulating protein 1, myocyte enhancer factor 2, and nuclear factor of activated cells 4, through activation of calcineurin. This finding demonstrates that the observed compensation occurs through transcriptional crosstalk and the remodeling of Ca2+ signaling pathways. The wide significance of this regulatory mechanism is related to its general involvement in Ca2+ signaling dynamics and in cardiac development and hypertrophy.

Animals↗

PubMed2001

Regulation of melastatin, a TRP-related protein, through interaction with a cytoplasmic isoform.

The TRP (transient receptor potential) superfamily includes a group of subfamilies of channel-like proteins mediating a multitude of physiological signaling processes. The TRP-melastatin (TRPM) subfamily includes the putative tumor suppressor melastatin (MLSN) and is a poorly characterized group of TRP-related proteins. Here, we describe the identification and characterization of an additional TRPM protein TRPM4. We reveal that TRPM4 and MLSN each mediate Ca(2+) entry when expressed in HEK293 cells. Furthermore, we demonstrate that a short form of MLSN (MLSN-S) interacts directly with and suppresses the activity of full-length MLSN (MLSN-L). This suppression seems to result from the inhibition of translocation of MLSN-L to the plasma membrane. We propose that control of translocation through interaction between MLSN-S and MLSN-L represents a mode for regulating ion channel activity.

Amino Acid Sequence↗

PubMed2001

Expression of functional receptor-coupled TRPC3 channels in DT40 triple receptor InsP3 knockout cells.

The TRPC3 channel, an intensively studied member of the widely expressed transient receptor potential (TRP) family, is a Ca(2+)-conducting channel activated in response to phospholipase C-coupled receptors. Despite scrutiny, the receptor-induced mechanism to activate TRPC3 channels remains unclear. Evidence indicates TRPC3 channels interact directly with intracellular inositol 1,4,5-trisphosphate receptors (InsP(3)Rs) and that channel activation is mediated through coupling to InsP(3)Rs. TRPC3 channels were expressed in DT40 chicken B lymphocytes in which all three InsP(3)R genes were deleted (DT40InsP(3)R-k/o). Endogenous B-cell receptors (BCR) coupled through Syk kinase to phospholipase C-gamma (PLC-gamma) activated the expressed TRPC3 channels in both DT40w/t and DT40InsP(3)R-k/o cells. The diacylglycerol (DAG) analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) also activated TRPC3 channels independently of InsP(3)Rs. BCR-induced TRPC3 activation was blocked by the PLC enzymic inhibitor, U-73122, and also blocked by wortmannin-induced PLC substrate depletion. Neither U-73122 nor wortmannin modified either OAG-induced TRPC3 activation or store-operated channel activation in DT40 cells. Cotransfection of cells with both G protein-coupled M5 muscarinic receptors and TRPC3 channels resulted in successful M5 coupling to open TRPC3 channels mediated by PLC-beta. We conclude that TRPC3 channels are activated independently of InsP(3)Rs through DAG production resulting from receptor-mediated activation of either PLC-gamma or PLC-beta.

Androstadienes↗

PubMed2001

A common mechanism underlies vertebrate calcium signaling and Drosophila phototransduction.

Drosophila phototransduction is an important model system for studies of inositol lipid signaling. Light excitation in Drosophila photoreceptors depends on phospholipase C, because null mutants of this enzyme do not respond to light. Surprisingly, genetic elimination of the apparently single inositol trisphosphate receptor (InsP(3)R) of Drosophila has no effect on phototransduction. This led to the proposal that Drosophila photoreceptors do not use the InsP(3) branch of phospholipase C (PLC)-mediated signaling for phototransduction, unlike most other inositol lipid-signaling systems. To examine this hypothesis we applied the membrane-permeant InsP(3)R antagonist 2-aminoethoxydiphenyl borate (2-APB), which has proved to be an important probe for assessing InsP(3)R involvement in various signaling systems. We first examined the effects of 2-APB on Xenopus oocytes. We found that 2-APB is efficient at reversibly blocking the robust InsP(3)-mediated Ca(2+) release and store-operated Ca(2+) entry in Xenopus oocytes at a stage operating after production of InsP(3) but before the opening of the surface membrane Cl(-) channels by Ca(2+). We next demonstrated that 2-APB is effective at reversibly blocking the response to light of Drosophila photoreceptors in a light-dependent manner at a concentration range similar to that effective in Xenopus oocytes and other cells. We show furthermore that 2-APB does not directly block the light-sensitive channels, indicating that it operates upstream in the activation of these channels. The results indicate an important link in the coupling mechanism of vertebrate store-operated channels and Drosophila TRP channels, which involves the InsP(3) branch of the inositol lipid-signaling pathway.

Animals↗

PubMed2001

Assessment of the role of the inositol 1,4,5-trisphosphate receptor in the activation of transient receptor potential channels and store-operated Ca2+ entry channels.

The mechanism for coupling between Ca(2+) stores and store-operated channels (SOCs) is an important but unresolved question. Although SOCs have not been molecularly identified, transient receptor potential (TRP) channels share a number of operational parameters with SOCs. The question of whether activation of SOCs and TRP channels is mediated by the inositol 1,4,5-trisphosphate receptor (InsP(3)R) was examined using the permeant InsP(3)R antagonist, 2-aminoethoxydiphenyl borate (2-APB) in both mammalian and invertebrate systems. In HEK293 cells stably transfected with human TRPC3 channels, the actions of 2-APB to block carbachol-induced InsP(3)R-mediated store release and carbachol-induced Sr(2+) entry through TRPC3 channels were both reversed at high agonist levels, suggesting InsP(3)Rs mediate TRPC3 activation. However, electroretinogram recordings of the light-induced current in Drosophila revealed that the TRP channel-mediated responses in wild-type as well as trp and trpl mutant flies were all inhibited by 2-APB. This action of 2-APB is likely InsP(3)R-independent since InsP(3)Rs are dispensable for the light response. We used triple InsP(3)R knockout DT40 chicken B-cells to further assess the role of InsP(3)Rs in SOC activation. (45)Ca(2+) flux analysis revealed that although DT40 wild-type cells retained normal InsP(3)Rs mediating 2-APB-sensitive Ca(2+) release, the DT40InsP(3)R-k/o cells were devoid of functional InsP(3)Rs. Using intact cells, all parameters of Ca(2+) store function and SOC activation were identical in DT40wt and DT40InsP(3)R-k/o cells. Moreover, in both cell lines SOC activation was completely blocked by 2-APB, and the kinetics of action of 2-APB on SOCs (time dependence and IC(50)) were identical. The results indicate that (a) the action of 2-APB on Ca(2+) entry is not mediated by the InsP(3)R and (b) the effects of 2-APB provide evidence for an important similarity in the function of invertebrate TRP channels, mammalian TRP channels, and mammalian store-operated channels.

Adenosine↗

PubMed2000

Ca2+ entry mediated by store depletion, S-nitrosylation, and TRP3 channels. Comparison of coupling and function.

The mechanism for coupling between Ca(2+) stores and store-operated channels (SOCs) is an important but unresolved question. SOC-mediated Ca(2+) entry is complex and may reflect more than one type of channel and coupling mechanism. To assess such possible divergence the function and coupling of SOCs was compared with two other distinct yet related Ca(2+) entry mechanisms. SOC coupling in DDT(1)MF-2 smooth muscle cells was prevented by the permeant inositol 1,4,5-trisphosphate (InsP(3)) receptor blockers, 2-aminoethoxydiphenyl borate (2-APB) and xestospongin C. In contrast, Ca(2+) entry induced by S-nitrosylation and potentiated by store depletion (Ma, H-T., Favre, C. J., Patterson, R. L., Stone, M. R., and Gill, D. L. (1999) J. Biol. Chem. 274, 35318-35324) was unaffected by 2-APB, suggesting that this entry mechanism is independent of InsP(3) receptors. The cycloalkyl lactamimide, MDL-12, 330A (MDL), prevented SOC activation (IC(50) 10 micrometer) and similarly completely blocked S-nitrosylation-mediated Ca(2+) entry. Ca(2+) entry mediated by the TRP3 channel stably expressed in HEK293 cells was activated by phospholipase C-coupled receptors but independent of Ca(2+) store depletion (Ma, H.-T., Patterson, R. L., van Rossum, D. B., Birnbaumer, L., Mikoshiba, K., and Gill, D. L. (2000) Science 287, 1647-1651). Receptor-induced TRP3 activation was 2-APB-sensitive and fully blocked by MDL. Direct stimulation of TRP3 channels by the permeant diacylglycerol derivative, 1-oleoyl-2-acetyl-sn-glycerol, was not blocked by 2-APB, but was again prevented by MDL. The results indicate that although the activation and coupling processes for each of the three entry mechanisms are distinct, sensitivity to MDL is a feature shared by all three mechanisms, suggesting there may be a common structural feature in the channels themselves or an associated regulatory component.

Animals↗

PubMed2000

Requirement of the inositol trisphosphate receptor for activation of store-operated Ca2+ channels.

The coupling mechanism between endoplasmic reticulum (ER) calcium ion (Ca2+) stores and plasma membrane (PM) store-operated channels (SOCs) is crucial to Ca2+ signaling but has eluded detection. SOCs may be functionally related to the TRP family of receptor-operated channels. Direct comparison of endogenous SOCs with stably expressed TRP3 channels in human embryonic kidney (HEK293) cells revealed that TRP3 channels differ in being store independent. However, condensed cortical F-actin prevented activation of both SOC and TRP3 channels, which suggests that ER-PM interactions underlie coupling of both channels. A cell-permeant inhibitor of inositol trisphosphate receptor (InsP3R) function, 2-aminoethoxydiphenyl borate, prevented both receptor-induced TRP3 activation and store-induced SOC activation. It is concluded that InsP3Rs mediate both SOC and TRP channel opening and that the InsP3R is essential for maintaining coupling between store emptying and physiological activation of SOCs.

Actins↗

PubMed2000

Perceived stress and blood pressure in early adolescent children.

The objective of this investigation was to determine the individual contributions of perceived daily, major, and total stressors to blood pressure in early adolescent children. Toward this goal, cardiovascular risk factors were assessed in 74 6th-grade students. Height and body weight, measured in standard fashion, were used to calculate body mass index (BMI). Waist and hip circumferences and triceps and calf skinfolds were taken to determine the distribution and percentage of body fat, respectively. Seated resting blood pressure was obtained using a mercury sphygmomanometer. The dietary sodium-to-potassium ratio was calculated from a food intake questionnaire. Family history of hypertension was self-reported by participant's parents, and physical activity and perceived stress levels were determined by questionnaire. When added to the hierarchical regression models, the perceived stress variables did not significantly predict any additional variance in systolic or diastolic blood pressure in this early adolescent sample. Additionally, bivariate correlations between the stress variables and blood pressure were nonsignificant. The nonpsychological hypertension risk factors accounted for 25%-35% of the total variance in systolic and diastolic blood pressure. Further, regression analyses revealed that with the exception of BMI and the sodium-to-potassium ratio, no other risk factors were independent predictors of systolic or diastolic blood pressure. Further identification and understanding of environmental precursors of childhood hypertension is recommended.

Adolescent↗

PubMed1999

Ca(2+) entry activated by S-nitrosylation. Relationship to store-operated ca(2+) entry.

The coupling between Ca(2+) pools and store-operated Ca(2+) entry channels (SOCs) remains an unresolved question. Recently, we revealed that Ca(2+) entry could be activated in response to S-nitrosylation and that this process was stimulated by Ca(2+) pool emptying (Favre, C. J., Ufret-Vincenty, C. A., Stone, M. R., Ma, H-T. , and Gill, D. L. (1998) J. Biol. Chem. 273, 30855-30858). In DDT(1)MF-2 smooth muscle cells and DC-3F fibroblasts, Ca(2+) entry activated by the lipophilic NO donor, GEA3162 (5-amino-3-(3, 4-dichlorophenyl)1,2,3,4-oxatriazolium), or the alkylator, N-ethylmaleimide, was observed to be strongly activated by transient external Ca(2+) removal, closely resembling activation of SOC activity in the same cells. The nonadditivity of SOC and NO donor-activated Ca(2+) entry suggested a single entry mechanism. Calyculin A-induced reorganization of the actin cytoskeleton prevented SOC but had no effect on GEA3162-induced Ca(2+) entry. However, a single entry mechanism could account for both SOC and NO donor-activated entry if the latter reflected direct modification of the entry channel by S-nitrosylation, bypassing the normal coupling process between channels and pools. Small differences between SOC and GEA3162-activated Ba(2+) entry and sensitivity to blockade by La(3+) were observed, and in HEK293 cells SOC activity was observed without a response to thiol modification. It is concluded that in some cells, S-nitrosylation modifies an entry mechanism closely related to SOC and/or part of the regulatory machinery for SOC-mediated Ca(2+) entry.

Animals↗

PubMed1999

Store-operated Ca2+ entry: evidence for a secretion-like coupling model.

The elusive coupling between endoplasmic reticulum (ER) Ca2+ stores and plasma membrane (PM) "store-operated" Ca2+ entry channels was probed through a novel combination of cytoskeletal modifications. Whereas coupling was unaffected by disassembly of the actin cytoskeleton, in situ redistribution of F-actin into a tight cortical layer subjacent to the PM displaced cortical ER and prevented coupling between ER and PM Ca2+ entry channels, while not affecting inositol 1,4,5-trisphosphate-mediated store release. Importantly, disassembly of the induced cortical actin layer allowed ER to regain access to the PM and reestablish coupling of Ca2+ entry channels to Ca2+ store depletion. Coupling is concluded to be mediated by a physical "secretion-like" mechanism involving close but reversible interactions between the ER and the PM.

Actins↗

PubMed1998

Fatty acid-mediated calcium sequestration within intracellular calcium pools.

Intracellular Ca2+ pools play an essential role in generating Ca2+ signals. The heterogeneity of intracellular Ca2+ pools reflects the complex and dynamic character of the endoplasmic reticulum within which they reside. Translocation of Ca2+ between distinct subcompartments of the endoplasmic reticulum is mediated by a sensitive and specific GTP-activated process involving formation of reversible communicating junctions (Rys-Sikora, K. E., Ghosh, T. K., and Gill, D. L. (1994) J. Biol. Chem. 269, 31607-31613). In the presence of palmitate at 10 microM or above, this GTP-activated mechanism mediates substantial Ca2+ accumulation within a specific Ca2+-pumping pool. The fatty acid- and GTP-dependent accumulation of Ca2+ was highly chain length-specific; pentadecanoate (C15) and palmitate (C16) were equally effective, whereas fatty acids of shorter or longer chain length were either marginally effective or devoid of effect. Fatty acids with one or more unsaturated carbons were without effect, regardless of chain length. Palmitate-induced Ca2+ accumulation was immediately terminated with 2 microM palmitoyl-CoA, a blocker of the GTP-activated Ca2+-translocating mechanism. The anion transport inhibitor 4, 4'-diisothiocyanostilbene-2,2'-disulfonic acid completely prevented both palmitate- and oxalate-mediated GTP-dependent Ca2+ accumulation, with EC50 approximately 30 microM. Ca2+ sequestered in the presence of palmitate and GTP could be immediately and completely released by A23187, whereas the sequestered Ca2+ was remarkably resistant to release induced by inositol 1,4,5-trisphosphate (InsP3). In contrast, oxalate-sequestered Ca2+ within the same pool could be effectively released by either ionophore or InsP3. The results indicate that fatty acids are specifically transported into the lumen of a subset of Ca2+ pools, wherein they mediate substantial sequestration of Ca2+ in a distinct membrane-associated substate that is not readily releasable by opened InsP3-sensitive Ca2+ channels.

Animals↗

PubMed1998

Ca2+ pool emptying stimulates Ca2+ entry activated by S-nitrosylation.

The entry of Ca2+ following Ca2+ pool release is a major component of Ca2+ signals; yet despite intense study, how "store-operated" entry channels are activated is unresolved. Because S-nitrosylation has become recognized as an important regulatory modification of several key channel proteins, its role in Ca2+ entry was investigated. A novel class of lipophilic NO donors activated Ca2+ entry independent of the well defined NO target, guanylate cyclase. Strikingly similar entry of Ca2+ induced by cell permeant alkylators indicated that this Ca2+ entry process was activated through thiol modification. Significantly, Ca2+ entry activated by either NO donors or alkylators was highly stimulated by Ca2+ pool depletion, which increased both the rate of Ca2+ release and the sensitivity to thiol modifiers. The results indicate that S-nitrosylation underlies activation of an important store-operated Ca2+ entry mechanism.

Alkylating Agents↗

PubMed1998

Multidimensional correlates of falls in older women.

Older women who had fallen within the last year (n = 63) were compared with those who had not fallen (n = 67) on several psychological and motor measures. Both fallers and nonfallers demonstrated high levels of functioning. Discriminant analysis results indicated that a combination of variables, including physical activity and both psychological (general well-being, self-efficacy) and motor (functional reach, mobility) measures differentiated fallers and nonfallers. Results suggest that falling is a multidimensional phenomenon, that small declines on multiple factors may increase risk of falls, and that multifaceted interventions may help maintain high levels of functioning and prevent declines often associated with increased age.

Accidental Falls↗

PubMed1998

Gender and competitive motivation: from the recreation center to the Olympic arena.

Gender makes a difference; we do gender in sport. Gender is a pervasive social force in society, and the sport world reflects society's gender hierarchy in the extreme. Gender is so ingrained in our sport structure and practice that we cannot simply treat all athletes the same. Nor can we assume that male and female athletes are dichotomous opposites, and treat all males one way and all females another way. Biology is part of the mix, but biology is not destiny. Gender is a dynamic, social influence that varies with the individual, situation, and time.

Female↗

PubMed1997

Recovery of Ca2+ pools and growth in Ca2+ pool-depleted cells is mediated by specific epoxyeicosatrienoic acids derived from arachidonic acid.

Depletion of Ca2+ pools using the irreversible Ca2+ pump blocker, thapsigargin, induces DDT1MF-2 smooth muscle cells to enter a stable nonproliferative state. Reversal of this state can be mediated by high (20%) serum treatment, which induces new Ca2+ pump protein, return of Ca2+ pools, and reentry of cells into the cell cycle; the effect of serum can be mimicked by the essential fatty acids (EFA), arachidonic, linoleic, and alpha-linolenic acids (Graber, M.N., Alfonso, A., and Gill, D.L., (1996) J. Biol. Chem. 271, 883-888). The possible requirement for EFA metabolism in inducing recovery of Ca2+ pool-depleted growth-arrested cells was investigated. Neither cyclooxygenase or lipoxygenase inhibitors had any effect on arachidonic acid-induced growth recovery of thapsigargin-treated cells. In contrast, the cytochrome P-450 epoxygenase inhibitors, SKF525A and metyrapone, substantially reduced arachidonic acid-induced recovery of growth while having minimal effects on control cell growth. Both epoxygenase inhibitors completely prevented the arachidonic acid-induced recovery of bradykinin-releasable Ca2+-pumping pools, whereas cyclooxygenase and lipoxygenase inhibitors had no effect. The effectiveness of the four cytochrome P-450 metabolites of arachidonic acid on recovery of Ca2+ pools were compared; 8,9- and 11,12-epoxyeicosatrienoic acid (EET) at 1.5 microM were completely effective in recovering agonist-sensitive Ca2+ pools, whereas the 5,6- and 14,15-EETs were without effect. SKF525A did not block the action of 8,9- or 11, 12-EET indicating further P-450 metabolism was not required. Hydration of the active EET molecules prevented Ca2+ pool recovery since the dihydroxy-derivatives of both 8,9- and 11,12-EET were ineffective. The specificity of effectiveness among EET molecules for subsequent resumption of growth of thapsigargin-treated cells was the same as for Ca2+ pool recovery. Significantly, the P-450 inhibitors, SKF525A and metyrapone, both prevented the action of 20% serum in inducing recovery of thapsigargin-treated cells, whereas cyclooxygenase and lipoxygenase inhibitors were ineffective, indicating that EFAs are the active component within serum that is responsible for recovery of Ca2+ pool-depleted cells. The specific action of EETs in mediating recovery of Ca2+ pools and growth of thapsigargin-treated cells represents not only a novel action of epoxygenase products from EFAs, but also a potentially significant new signaling pathway that may effect translational control and regulate transition from a stationary to proliferative growth state.

8,11,14-Eicosatrienoic Acid↗

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↗

PubMed1996

Ca2+ pools and cell growth: arachidonic acid induces recovery of cells growth-arrested by Ca2+ pool depletion.

The intracellular Ca2+ pump blocker, thapsigargin, induces emptying of Ca2+ pools and entry of DDT1MF-2 smooth muscle cells into a quiescent G(0)-like growth state. Although thapsigargin blocks pumps essentially irreversibly, high serum (20%) induces appearance of new pump protein, return of functional pools, and reentry of cells into the cell cycle (Waldron, R. T., Short, A. D., Meadows, J.J., Ghosh, T. K., and Gill, D. L. (1994) J. Biol. Chem. 269, 11927-11933). Through analysis of the effects of defined serum components and growth supplements, we reveal here that the factors in serum responsible for inducing recovery of Ca2+ pools and growth in thapsigargin-arrested DDT1MF-2 cells are exactly mimicked by the three essential fatty acids, arachidonic, linoleic, and alpha-linolenic acids. The EC50 values for arachidonic and linoleic acids on growth induction of thapsigargin-arrested cells were the same, approximately 5 microM. Nonessential fatty acids, including myristic, palmitic, stearic, oleic, and arachidic acids, were without any effect. Although not proven to be the active component of serum, levels of arachidonic and linoleic acids in serum were sufficient to explain serum-induced growth recovery. Significantly, arachidonic or linoleic acids induced complete recovery of bradykinin-sensitive Ca2+ pools within 6 h of treatment of thapsigargin-arrested cells. Protein synthesis inhibitors (cycloheximide or puromycin) completely blocked the appearance of serum-induced or arachidonic acid-induced agonist-sensitive pools. The sensitivity and fatty acid specificity of Ca2+ pool recovery in thapsigargin-arrested cells were almost identical to that for growth recovery. No pool or growth recovery was observed with 5,8,11,14-eicosatetraynoic acid, the nonmetabolizable analogue of arachidonic acid, suggesting that conversion to eicosanoids underlies the pool and growth recovery induced by essential fatty acids. The results provide not only further information on the link between Ca2+ pools and cell growth but also evidence for a potentially important signaling pathway involved in inducing transition from a stationary to a proliferative growth state.

Animals↗

PubMed1996

Calcium pools, calcium entry, and cell growth.

The Ca2+ pump and Ca2+ release functions of intracellular Ca2+ pools have been well characterized. However, the nature and identity of Ca2+ pools as well as the physiological implications of Ca2+ levels within them, have remained elusive. Ca2+ pools appear to be contained within the endoplasmic reticulum (ER); however, ER is a heterogeneous and widely distributed organelle, with numerous other functions than Ca2+ regulation. Studies described here center on trying to determine more about subcellular distribution of Ca2+ pools, the levels of Ca2+ within Ca2+ pools, and how these intraluminal Ca2+ levels may be physiologically related to ER function. Experiments utilizing in situ high resolution subcellular morphological analysis of ER loaded with ratiometric fluorescent Ca2+ dyes, indicate a wide distribution of inositol 1,4,5-trisphosphate (InsP3)-sensitive Ca2+ pools within cells, and large changes in the levels of Ca2+ within pools following Insp3-mediated Ca2+ release. Such changes in Ca2+ may be of great significance to the translation, translocation, and folding of proteins in ER, in particular with respect to the function of the now numerously described luminal Ca(2+)-sensitive chaperonin proteins. Studies have also focussed on the physiological role of pool Ca2+ changes with respect to cell growth. Emptying of pools using Ca2+ pump blockers can result in cells entering a stable quiescent G(o)-like growth state. After treatment with the irreversible pump blocker, thapsigargin, cells remain in this state until they are stimulated with essential fatty acids whereupon new pump protein is synthesized, functional Ca2+ pools return, and cells re-enter the cell cycle. During the Ca2+ pool-depleted growth-arrested state, cells express a Ca2+ influx channel that is distinct from the store-operated Ca2+ influx channels activated after short-term depletion of Ca2+ pools. Overall, these studies indicate that significant changes in intraluminal ER Ca2+ do occur and that such changes appear linked to alteration of essential ER functions as well as to the cell cycle-state and the growth of cells.

Animals↗