Biomedical subjects

I S Ambudkar

Publications and source records attributed to I S Ambudkar.

At least 19 recordsLinked to original sources

PubMed2000

Expression of truncated transient receptor potential protein 1alpha (Trp1alpha ): evidence that the Trp1 C terminus modulates store-operated Ca2+ entry.

Transient receptor potential protein 1 (Trp1) has been proposed as a component of the store-operated Ca(2+) entry (SOCE) channel. However, the exact mechanism by which Trp1 is regulated by store depletion is not known. Here, we examined the role of the Trp1 C-terminal domain in SOCE by expressing hTrp1alpha lacking amino acids 664-793 (DeltaTrp1alpha) or full-length hTrp1alpha in the HSG (human submandibular gland) cell line. Both carbachol (CCh) and thapsigargin (Tg) activated sustained Ca(2+) influx in control (nontransfected), DeltaTrp1alpha-, and Trp1alpha-expressing cells. Sustained [Ca(2+)](i), following stimulation with either Tg or CCh in DeltaTrp1alpha-expressing cells, was about 1.5-2-fold higher than in Trp1alpha-expressing cells and 4-fold higher than in control cells. Importantly, (i) basal Ca(2+) influx and (ii) Tg- or CCh-stimulated internal Ca(2+) release were similar in all the cells. A similar increase in Tg-stimulated Ca(2+) influx was seen in cells expressing Delta2Trp1alpha, lacking the C-terminal domain amino acid 649-793, which includes the EWKFAR sequence. Further, both inositol 1,4,5-trisphosphate receptor-3 and caveolin-1 were immunoprecipitated with DeltaTrp1alpha and Trp1alpha. In aggregate, these data suggest that (i) the EWKFAR sequence does not contribute significantly to the Trp1-associated increase in SOCE, and (ii) the Trp1 C-terminal region, amino acids 664-793, is involved in the modulation of SOCE.

Calcium↗

PubMed2000

Assembly of Trp1 in a signaling complex associated with caveolin-scaffolding lipid raft domains.

Trp1 has been proposed as a component of the store-operated Ca(2+) entry (SOC) channel. However, neither the molecular mechanism of SOC nor the role of Trp in this process is yet understood. We have examined possible molecular interactions involved in the regulation of SOC and Trp1 and report here for the first time that Trp1 is assembled in signaling complex associated with caveolin-scaffolding lipid raft domains. Endogenous hTrp1 and caveolin-1 were present in low density fractions of Triton X-100-extracted human submandibular gland cell membranes. Depletion of plasma membrane cholesterol increased Triton X-100 solubility of Trp1 and inhibited carbachol-stimulated Ca(2+) signaling. Importantly, thapsigargin stimulated Ca(2+) influx, but not internal Ca(2+) release, and inositol 1,4,5-triphosphate (IP(3))-stimulated I(soc) were also attenuated. Furthermore, both anti-Trp1 and anti-caveolin-1 antibodies co-immunoprecipitated hTrp1, caveolin-1, Galpha(q/11), and IP(3) receptor-type 3 (IP(3)R3). These results demonstrate that caveolar microdomains provide a scaffold for (i) assembly of key Ca(2+) signaling proteins into a complex and (ii) coordination of the molecular interactions leading to the activation of SOC. Importantly, we have shown that Trp1 is also localized in this microdomain where it interacts with one or more components of this complex, including IP(3)R3. This finding is potentially important in elucidating the physiological function of Trp.

Calcium↗

PubMed2000

Trp1, a candidate protein for the store-operated Ca(2+) influx mechanism in salivary gland cells.

The trp gene family has been proposed to encode the store-operated Ca(2+) influx (SOC) channel(s). This study examines the role of Trp1 in the SOC mechanism of salivary gland cells. htrp1, htrp3, and Trp1 were detected in the human submandibular gland cell line (HSG). HSG cells stably transfected with htrp1alpha cDNA displayed (i) a higher level of Trp1, (ii) a 3-5-fold increase in SOC (thapsigargin-stimulated Ca(2+) influx), determined by [Ca(2+)](i) and Ca(2+)-activated K(+) channel current measurements, and (iii) similar basal Ca(2+) permeability, and inhibition of SOC by Gd(3+) but not by Zn(2+), as compared with control cells. Importantly, (i) transfection of HSG cells with antisense trp1alpha cDNA decreased endogenous Trp1 level and significantly attenuated SOC, and (ii) transfection of HSG cells with htrp3 cDNA did not increase SOC. These data demonstrate an association between Trp1 and SOC and strongly suggest that Trp1 is involved in this mechanism in HSG cells. Consistent with this suggestion, Trp1 was detected in the plasma membrane region, the proposed site of SOC, of acinar and ductal cells in intact rat submandibular glands. Based on these aggregate data, we propose Trp1 as a candidate protein for the SOC mechanism in salivary gland cells.

Animals↗

PubMed2000

Construction and function of a recombinant adenovirus encoding a human aquaporin 1-green fluorescent protein fusion product.

Transfer of the human aquaporin 1 (hAQP1) gene provides a novel way to potentially correct the severe salivary hypofunction associated with therapeutic radiation for head and neck cancer. To facilitate the study of individual cells transduced with this gene, we have designed a fusion product of the hAQP1 and jellyfish green fluorescent protein (GFP) cDNAs. An expression plasmid, pACCMVhAQP1GFP, and a recombinant adenovirus, AdhAQP1GFP, encoding this fusion product were constructed. Both the recombinant plasmid and virus directed the expression of the encoded, 55-kDa fusion protein (hAQP1GFP), which was detected in the plasma membranes of several epithelial cell lines (293, SMIE, and A5). hAQP1GFP was functionally active and facilitated fluid movement across a polarized salivary epithelial cell monolayer (approximately 5-fold noninfected controls) in response to an osmotic gradient. In response to a hypotonic challenge, individual epithelial cells expressing the fusion protein exhibited significantly more capacitance (used herein as an indicator of cell swelling) than control cells. Conversely, in response to a hypertonic challenge, individual infected cells shrunk more rapidly (approximately 2- to 3-fold) and to a greater extent than control cells. We conclude that AdhAQP1GFP is a useful experimental tool to identify and study individual cells expressing a water channel transgene.

Adenoviridae↗

PubMed2000

Regulation of calcium in salivary gland secretion.

Neurotransmitter-regulation of fluid secretion in the salivary glands is achieved by a coordinated sequence of intracellular signaling events, including the activation of membrane receptors, generation of the intracellular second messenger, inositol 1,4,5, trisphosphate, internal Ca2+ release, and Ca2+ influx. The resulting increase in cytosolic [Ca2+] ([Ca2+]i) regulates a number of ion transporters, e.g., Ca2+-activated K+ channel, Na+/K+/2Cl- co-transporter in the basolateral membrane, and the Ca2+-activated Cl- channel in the luminal membrane, which are intricately involved in fluid secretion. Thus, regulation of [Ca2+]i is central to the regulation of salivary acinar cell function and is achieved by the concerted activities of several ion channels and Ca2+-pumps localized in various cellular membranes. Ca2+ pumps, present in the endoplasmic reticulum and the plasma membrane, serve to remove Ca2+ from the cytosol. Ca2+ channels present in the endoplasmic reticulum and the plasma membrane facilitate rapid influx of Ca2+ into the cytosol from the internal Ca2+ stores and from the external medium, respectively. It is well-established that prolonged fluid secretion is regulated via a sustained elevation in [Ca2+]i that is primarily achieved by the influx of Ca2+ into the cell from the external medium. This Ca2+ influx occurs via a putative plasma-membrane-store-operated Ca2+ channel which has not yet been identified in any non-excitable cell type. Understanding the molecular nature of this Ca2+ influx mechanism is critical to our understanding of Ca2+ signaling in salivary gland cells. This review focuses on the various active and passive Ca2+ transport mechanisms in salivary gland cells--their localization, regulation, and role in neurotransmitter-regulation of fluid secretion. In addition to a historical perspective of Ca2+ signaling, recent findings and challenging problems facing this field are highlighted.

Animals↗

PubMed1999

ATP-dependent activation of K(Ca) and ROMK-type K(ATP) channels in human submandibular gland ductal cells.

[Ca(2+)](i) and membrane current were measured in human submandibular gland ductal (HSG) cells to determine the regulation of salivary cell function by ATP. 1-10 microM ATP activated internal Ca(2+) release, outward Ca(2+)-dependent K(+) channel (K(Ca)), and inward store-operated Ca(2+) current (I(SOC)). The subsequent addition of 100 microM ATP activated an inwardly rectifying K(+) current, without increasing [Ca(2+)](i). The K(+) current was also stimulated by ATP in cells treated with thapsigargin in a Ca(2+)-free medium and was blocked by glibenclamide and tolbutamide, but not by charybdotoxin. This suggests the involvement of a Ca(2+)-independent, sulfonylurea-sensitive K(+) channel (K(ATP)). UTP mimicked the low [ATP] effects, while benzoyl-ATP activated internal Ca(2+) release, a Ca(2+) influx pathway, and K(Ca). Thus, ATP acts via P(2U) (P2Y(2)) and P(2Z) (P2X(7)) receptors to increase [Ca(2+)](i) and activate K(Ca), but not K(ATP). Importantly, (i) ROMK1 and the cystic fibrosis transmembrane regulator protein (but not SUR1, SUR2A, or SUR2B) and (ii) cAMP-stimulated Cl(-) and K(+) currents were detected in HSG cells. These data demonstrate for the first time that a ROMK-type K(ATP) channel is present in salivary gland duct cells that is regulated by extracellular ATP and possibly by the cystic fibrosis transmembrane regulator. This reveals a potentially novel mechanism for K(+) secretion in these cells.

Adenosine Triphosphate↗

PubMed1999

Cloning of Trp1beta isoform from rat brain: immunodetection and localization of the endogenous Trp1 protein.

The Trp gene product has been proposed as a candidate protein for the store-operated Ca2+ channel, but the Trp protein(s) has not been identified in any nonexcitable cell. We report here the cloning of a rat brain Trp1beta cDNA and detection and immunolocalization of the endogenous and expressed Trp1 protein. A 400-bp product, with >95% homology to mouse Trp1, was amplified from rat submandibular gland RNA. Rat-specific primers were used for cloning of a full-length rat brain Trp1beta cDNA (rTrp1), encoding a protein of 759 amino acids. Northern blot analysis demonstrated the transcript in several rat and mouse tissues. The peptide (amino acids 523-536) was used to generate a polyclonal antiserum. The affinity-purified antibody 1) immunoprecipitated human Trp1 (hTrp1) from transfected HEK-293 cells, 2) reacted with a protein of approximately 92 kDa, but not with hTrp3, in membranes of hTrp3-expressing HEK-293 cells, and 3) reacted with proteins of 92 and 56 kDa in human and rat brain membranes. Confocal microscopy and cell fractionation demonstrated that endogenous and expressed hTrp1 and expressed hTrp3 proteins were localized in the plasma membrane of HEK-293 cells, consistent with their proposed role in Ca2+ influx. The data demonstrate for the first time the presence of Trp1 protein in a nonexcitable cell.

Amino Acid Sequence↗

PubMed1999

Radiation-induced progressive decrease in fluid secretion in rat submandibular glands is related to decreased acinar volume and not impaired calcium signaling.

The mechanism(s) of radiation-induced salivary gland dysfunction is poorly understood. In the present study, we have assessed the secretory function (muscarinic agonist-stimulated saliva flow, intracellular calcium mobilization, Na+/K+/2Cl- cotransport activity) in rat submandibular glands 12 months postirradiation (single dose, 10 Gy). The morphological status of glands from control and irradiated rats was also determined. Pilocarpine-stimulated salivary flow was decreased by 67% at 12 months (but not at 3 months) after irradiation. This was associated with a 47% decrease in the wet weight of the irradiated glands. Histological and morphometric analysis demonstrated that acinar cells were smaller and occupied relatively less volume and convoluted granular tubules were smaller but occupied the same relative volume, while intercalated and striated ducts maintained their size but occupied a greater relative volume in submandibular glands from irradiated compared to control animals. In addition, no inflammation or fibrosis was observed in the irradiated tissues. Carbachol- or thapsigargin-stimulated mobilization of Ca2+ was similar in dispersed submandibular gland cells from control and irradiated animals. Further, [Ca2+]i imaging of individual ducts and acini from control and irradiated groups showed, for the first time, that mobilization of Ca2+ in either cell type was not altered by the radiation treatment. The carbachol-stimulated, bumetanide-sensitive component of the Na+/K+/ 2Cl- cotransport activity was also similar in submandibular gland cells from control and irradiated animals. These data demonstrate that a single dose of gamma radiation induces a progressive loss of submandibular gland tissue and function. This loss of salivary flow is not due to chronic inflammation or fibrosis of the gland or an alteration in the neurotransmitter signaling mechanism in the acinar or ductal cells. The radiation-induced decrease in fluid secretion appears to be related to a change in either the water-handling capacity of the acini or the number of acinar cells in the gland.

Animals↗

PubMed1998

Ca2+-dependent inactivation of a store-operated Ca2+ current in human submandibular gland cells. Role of a staurosporine-sensitive protein kinase and the intracellular Ca2+ pump.

Stimulation of human submandibular gland cells with carbachol, inositol trisphosphate (IP3), thapsigargin, or tert-butylhydroxyquinone induced an inward current that was sensitive to external Ca2+ concentration ([Ca2+]e) and was also carried by external Na+ or Ba2+ (in a Ca2+-free medium) with amplitudes in the order Ca2+ > Ba2+ > Na+. All cation currents were blocked by La3+ and Gd3+ but not by Zn2+. The IP3-stimulated current with 10 microM 3-deoxy-3-fluoro-D-myo-inositol 1,4,5-triphosphate and 10 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid in the pipette solution, showed 50% inactivation in <5 min and >5 min with 10 and 1 mM [Ca2+]e, respectively. The Na+ current was not inactivated, whereas the Ba2+ current inactivated at a slower rate. The protein kinase inhibitor, staurosporine, delayed the inactivation and increased the amplitude of the current, whereas the protein Ser/Thr phosphatase inhibitor, calyculin A, reduced the current. Thapsigargin- and tert-butylhydroxyquinone-stimulated Ca2+ currents inactivated faster. Importantly, these agents accelerated the inactivation of the IP3-stimulated current. The data demonstrate that internal Ca2+ store depletion-activated Ca2+ current (ISOC) in this salivary cell line is regulated by a Ca2+-dependent feedback mechanism involving a staurosporine-sensitive protein kinase and the intracellular Ca2+ pump. We suggest that the Ca2+ pump modulates ISOC by regulating [Ca2+]i in the region of Ca2+ influx.

Calcium↗

PubMed1998

Characteristics of a low affinity passive Ca2+ influx component in rat parotid gland basolateral plasma membrane vesicles.

We have previously reported the presence of two Ca2+ influx components with relatively high (KCa = 152 +/- 79 microM) and low (KCa = 2.4 +/- 0.9 mM) affinities for Ca2+ in internal Ca2+ pool-depleted rat parotid acinar cells [Chauthaiwale et al. (1996) Pfluegers Arch. 432: 105-111]. We have also reported the presence of a high affinity Ca2+ influx component with KCa = 279 +/- 43 microM in rat parotid gland basolateral plasma membrane vesicles (BLMV). [Lockwich, Kim & Ambudkar (1994) J. Membrane Biol. 141:289-296]. The present studies show that a low affinity Ca2+ influx component is also present in BLMV with KCa = 2.3 +/- 0.41 mM (Vmax = 16.36 +/- 4.11 nmoles of Ca2+/mg protein/min). Our data demonstrate that this low affinity component is similar to the low affinity Ca2+ influx component that is activated by internal Ca2+ store depletion in dispersed parotid gland acini by the following criteria: (i) similar KCa for calcium flux, (ii) similar IC50 for inhibition by Ni2+ and Zn2+; (iii) increase in KCa at high external K+, (iv) similar effects of external pH. The high affinity Ca2+ influx in cells is different from the low affinity Ca2+ influx component cells in its sensitivity to pH, KCl, Zn2+ and Ni2+. The low and high affinity Ca2+ influx components in BLMV can also be distinguished from each other based on the effects of Zn2+, Ni2+, KCl, and dicyclohexylcarbodiimide. In aggregate, these data demonstrate the presence of a low affinity passive Ca2+ influx pathway in BLMV which displays characteristics similar to the low affinity Ca2+ influx component detected in parotid acinar cells following internal Ca2+ store depletion.

Animals↗

PubMed1998

Gamma-irradiation-induced cell cycle arrest and cell death in a human submandibular gland cell line: effect of E2F1 expression.

This study examined the effect of gamma-irradiation (5 and 10 Gy) on the human submandibular cell line (HSG). Radiation treatment (5 Gy and 10 Gy) induced a dose-dependent decrease in cell proliferation, with a G2/M arrest of the cell cycle, and an increase in cell death (cells with <2n DNA increased from 7% in control cells to 34% and 40% in 5 and 10 Gy irradiated cells, respectively). [Ca2+]i measurements demonstrated that the status of internal Ca2+ stores, and muscarinic receptor-mediated Ca2+ mobilization, in irradiated cells was comparable to that in non-irradiated cells. These data suggest that 1) irradiated HSG cells maintain normal physiology and 2) internal Ca2+ store depletion does not account for the decreased cell proliferation. To manipulate the radiation-induced cell cycle arrest, we examined the effect of the transcription factor E2F1, which has been shown to induce cell cycle progression in HSG cells (Lillibridge and O'Connell, 1997, J. Cell. Physiol., 1 72:343-350). The ability of irradiated HSG cells to express and appropriately route proteins was demonstrated by using adenovirus-mediated expression of beta-galactosidase, alpha1-antitrypsin, and aquaporin-1. Infection of HSG cells with an adenoviral vector encoding E2F1, either 12 h before or immediately following irradiation, but not post-irradiation, induced maintenance of cells in the S phase of the cell cycle, reduced the number of cells arrested at G2/M, and decreased the rate of appearance of cells with <2n DNA. While the mechanism of irradiation-induced cell death has not yet been confirmed, these data suggest that expression of the E2F1 gene product in HSG cells can be a useful strategy to manipulate cell cycle events and reduce the initial loss of cells due to radiation.

Adenoviridae↗

PubMed1998

Regulation of KCa current by store-operated Ca2+ influx depends on internal Ca2+ release in HSG cells.

This study examines the Ca2+ influx-dependent regulation of the Ca2+-activated K+ channel (KCa) in human submandibular gland (HSG) cells. Carbachol (CCh) induced sustained increases in the KCa current and cytosolic Ca2+ concentration ([Ca2+]i), which were prevented by loading cells with 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid (BAPTA). Removal of extracellular Ca2+ and addition of La3+ or Gd3+, but not Zn2+, inhibited the increases in KCa current and [Ca2+]i. Ca2+ influx during refill (i.e., addition of Ca2+ to cells treated with CCh and then atropine in Ca2+-free medium) failed to evoke increases in the KCa current but achieved internal Ca2+ store refill. When refill was prevented by thapsigargin, Ca2+ readdition induced rapid activation of KCa. These data provide further evidence that intracellular Ca2+ accumulation provides tight buffering of [Ca2+]i at the site of Ca2+ influx (H. Mogami, K. Nakano, A. V. Tepikin, and O. H. Petersen. Cell 88: 49-55, 1997). We suggest that the Ca2+ influx-dependent regulation of the sustained KCa current in CCh-stimulated HSG cells is mediated by the uptake of Ca2+ into the internal Ca2+ store and release via the inositol 1,4,5-trisphosphate-sensitive channel.

Atropine↗

PubMed1997

Role for protein kinase in Ca2+-dependent feedback modulation of divalent cation influx in internal-Ca2+-store-depleted rat parotid gland cells.

Divalent cation (Ca2+ and Mn2+) influx, stimulated by internal Ca2+ store depletion, into rat parotid acinar cells is inhibited by conditions which increase protein phosphorylation [T. Sakai and I.S. Ambudkar (1996) Am J Physiol 271:C284-C294]. The present study examines the involvement of this protein phosphorylation and Ca2+ in the store-dependent inactivation of divalent cation entry. Internal Ca2+ store depletion, achieved by incubation (30 min) of cells in nominally Ca2+-free medium containing either carbachol or thapsigargin, stimulated Ca2+, and Mn2+, influx into cells. In either case, inclusion of 1.5 mM Ca2+ for the last 5 min of incubation resulted in a decrease in Ca2+ (33-41%) and Mn2+ (50%) influx, which could not be accounted for by internal Ca2+ store refill. The inhibition was prevented when internal-store-depleted cells were treated (prior to incubation with Ca2+) with either staurosporine or K-252a, but not with H-7 or KN-93. Refilling of internal Ca2+ store(s) in carbachol-treated cells (incubation with Ca2++atropine) induced complete inhibition of divalent cation influx, which was not prevented by treatment with protein kinase inhibitors. These data suggest the staurosporine-sensitive (and K-252a-sensitive) protein phosphorylation is not involved in Ca2+-store-refilling-dependent inactivation of Ca2+ influx but mediates a Ca2+-dependent feedback modulation of divalent cation influx in rat parotid gland acinar cells.

Animals↗

PubMed1997

Interferon-gamma induces a decrease in the intracellular calcium pump in a human salivary gland cell line.

Interferon-gamma (IFN-gamma) +/- tumor necrosis factor-alpha (TNF-alpha) induces antiproliferation and intracellular Ca2+ store depletion in a human submandibular ductal cell line (HSG), which can be reversed on cytokine removal [A. J. Wu, G. C. Chen, B. J. Baum, and I. S. Ambudkar. Am. J. Physiol. 270 (Cell Physiol. 39): C514-C521, 1996]. Here we have examined a possible mechanism for the IFN-gamma-induced intracellular Ca2+ store depletion. There was a time-dependent decrease in thapsigargin-dependent internal Ca2+ release after exposure of the cells to the cytokines. The intracellular Ca2+ pump [sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)] protein in lysates and membranes of cells treated with IFN-gamma +/- TNF-alpha, but not with TNF-alpha alone, showed a similar time-dependent decrease (examined using a SERCA2 antibody). Removal of the cytokines, which resulted in recovery of cell growth and refill of internal Ca2+ stores, also increased the level of SERCA protein. The decrease in SERCA is not a result of decreased cell proliferation, since thapsigargin, 2,5-di-(t-butyl)-1,4-hydroquinone, or serum-free growth conditions induced antiproliferative effects on HSG cells without any corresponding decrease in SERCA. We suggest that the IFN-gamma-induced decrease in the level of SERCA accounts for the depleted state of internal Ca2+ stores in cytokine-treated HSG cells. These data suggest a novel mechanism for the inhibition of HSG cell growth by IFN-gamma.

Calcium↗

PubMed1996

Adenovirus-mediated expression of aquaporin-5 in epithelial cells.

A recombinant adenovirus coding for rat aquaporin-5 was constructed and plaque purified. The recombinant adenovirus (AdrAQP5) mediated the expression of aquaporin-5 in rat and human salivary cell lines and in dog kidney cells in vitro as demonstrated by Northern blot and Western blot analyses, and by confocal microscopy after immunofluorescent labeling. In kidney cells, expression of the transgene was optimal if cells were infected at their basolateral surface, a phenomenon associated with the distribution of integrin receptors on these cells. The expressed aquaporin-5 protein was functionally active because viral-mediated gene transfer resulted in a significant increase in the osmotically directed net fluid secretion rate across monolayers of kidney cells. AdrAQP5 should provide an efficient and useful means to impart facilitated water permeability to cells lacking such a pathway.

Adenoviridae↗

PubMed1996

Interferon-gamma induced cell death in a cultured human salivary gland cell line.

Increased levels of several cytokines, including interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha), have been demonstrated in the salivary gland microenvironment of patients with Sjögren's syndrome (SS). How these cytokines may be contributing to the pathogenesis of the disease is not well understood. This study examined the role of IFN-gamma +/- TNF-alpha on cellular death in a cultured human salivary gland cell line (HSG). Cells treated long-term with IFN-gamma +/- TNF-alpha demonstrate a profound antiproliferative effect with a decrease in cell number to below that initially plated. Treatment of HSG cells with TNF-alpha alone did not have any significant effects on growth but did increase the expression of the IFN-gamma receptor. Cells labelled with propidium iodide and anti-digoxigenin dUTP/dATP were examined by flow cytometry to determine the percentage of cells exhibiting low DNA content and DNA strand breaks. The percentage of cells exhibiting subdiploid DNA and DNA strand breaks increased with increased time of exposure to the cytokines. The maximum percentage of cells exhibiting DNA degradation at 12 days was 58% for cells treated with IFN-gamma + TNF-alpha, 31% for IFN-gamma treated cells, and < 5% for TNF-alpha-treated and untreated cells. The cells with subdiploid ( < 2n) DNA were subsequently demonstrated to represent two populations, both with evidence of increased DNA strand breaks but with differing light scatter characteristics. One population had features of cells undergoing necrosis, whereas the second population exhibited features of apoptosis. These findings were confirmed by transmission electron microscopy. Cells not exposed to cytokines did not exhibit significant evidence of either death process. We conclude that long-term exposure of a human salivary gland epithelial cell line to IFN-gamma +/- TNF-alpha leads to increased DNA degradation and subsequent cell death. This suggests a potential SS disease mechanism and implicates the role of the epithelial cell in this disease as an important area for future study.

Cell Count↗

PubMed1996

Presence of two Ca2+ influx components in internal Ca2+-pool-depleted rat parotid acinar cells.

The molecular mechanism(s) involved in mediating Ca2+ entry into rat parotid acinar and other non-excitable cells is not known. In this study we have examined the kinetics of Ca2+ entry in fura-2-loaded parotid acinar cells, which were treated with thapsigargin to deplete internal Ca2+ pools (Ca2+-pool-depleted cells). The rate of Ca2+ entry was determined by measuring the initial increase in free cytosolic [Ca2+] ([Ca2+]i) in Ca2+-pool-depleted, and control (untreated), cells upon addition of various [Ca2+] to the medium. In untreated cells, a low-affinity component was detected with KCa = 3. 4 +/- 0.7 mM (where KCa denotes affinity for Ca2+) and Vmax = 9.8 +/- 0.4 nM [Ca2+]i /s. In thapsigargin-treated cells, two Ca2+ influx components were detected with KCa values of 152 +/- 79 microM (Vmax = 5.1 +/- 1.9 nM [Ca2+]i/s) and 2.4 +/- 0.9 mM (Vmax = 37.6 +/- 13.6 nM [Ca2+]i/s), respectively. We have also examined the effect of Ca2+ and depolarization on these two putative Ca2+ influx components. When cells were treated with thapsigargin in a Ca2+-free medium, Ca2+ influx was higher than into cells treated in a Ca2+-containing medium and, while there was a 46% increase in the Vmax of the low-affinity component (no change in KCa), the high-affinity component was not clearly detected. In depolarized Ca2+-pool-depleted cells (with 50 mM KCl in the medium) the high-affinity component was considerably decreased while there was an apparent increase in the KCa of the low-affinity component, without any change in the Vmax. These results demonstrate that Ca2+ influx into parotid acinar cells (1) is increased (four- to five-fold) upon internal Ca2+ pool depletion, and (2) is mediated via at least two components, with low and high affinities for Ca2+.

Animals↗

PubMed1996

Interferon-gamma induces persistent depletion of internal Ca2+ stores in a human salivary gland cell line.

Interferon-gamma (IFN-gamma), in the presence of tumor necrosis factor-alpha (TNF-alpha), decreases proliferation of a human salivary gland ductal cell line, HSG (Wu, A., R. Kurrasch, J. Katz, P. Fox, B. Baum, and J. Atkinson. J. Cell. Physiol. 161:217-226, 1994). We examined the possible effects of these cytokines (1,000 U/ml IFN-gamma +/- 20 U/ml TNF-alpha for 7 days) on Ca2+ mobilization in HSG cells. In HSG cells, fetal bovine serum (10%) or carbachol (100 microM) stimulated rapid increases in cytosolic Ca2+ concentration ([Ca2+]i), apparently mobilized from different thapsigargin-sensitive intracellular Ca2+ stores. Serum induced a proliferative effect on HSG cells, which was suppressed (> 90%) by treatment with IFN-gamma +/- TNF-alpha, but not with TNF-alpha alone. Serum-, carbachol-, and thapsigargin-stimulated [Ca2+]i elevations were reduced by 90, 60, and > 65%, respectively, in cells treated with IFN-gamma +/- TNF-alpha and 30, 45, and 45%, respectively, in cells treated with TNF-alpha. Removal of the cytokines from the growth medium induced recovery of both cell proliferation and Ca2+ mobilization responses within 7 days. Treatment of HSG cells with thapsigargin (0.02-2 nM) induced a dose-dependent decrease in cell proliferation. Additionally, acute treatment (< 10 min) of cells with IFN-gamma did not affect [Ca2+]i or alter carbachol-, thapsigargin-, or serum-induced changes in [Ca2+]i. These data demonstrate that prolonged treatment of HSG cells with IFN-gamma +/- TNF-alpha leads to a persistent depletion of intracellular Ca2+ stores. We suggest that this may have a role in cell growth.

Blood Physiological Phenomena↗