Biomedical subjects

Donald T Ward

Publications and source records attributed to Donald T Ward.

13 recordsLinked to original sources

PubMed2006

Ca2+-sensing receptor induces Rho kinase-mediated actin stress fiber assembly and altered cell morphology, but not in response to aromatic amino acids.

The Ca2+-sensing receptor (CaR) is a pleiotropic, type III G protein-coupled receptor (GPCR) that associates functionally with the cytoskeletal protein filamin. To investigate the effect of CaR signaling on the cytoskeleton, human embryonic kidney (HEK)-293 cells stably transfected with CaR (CaR-HEK) were incubated with CaR agonists in serum-free medium for up to 3 h. Addition of the calcimimetic NPS R-467 or exposure to high extracellular Ca2+ or Mg2+ levels elicited actin stress fiber assembly and process retraction in otherwise stellate cells. These responses were ablated by cotreatment with the calcilytic NPS 89636 and were absent in vector-transfected HEK-293 cells. Cotreatment with the Rho kinase inhibitors Y-27632 and H1152 attenuated the CaR-induced morphological change but not intracellular Ca2+ (Ca2+(i)) mobilization or ERK activation, although transfection with a dominant-negative RhoA-binding protein also inhibited calcimimetic-induced actin stress fiber assembly. CaR effects on morphology were unaffected by inhibition of G(q/11) or G(i/o) signaling, epidermal growth factor receptor, or the metalloproteinases. In contrast, CaR-induced cytoskeletal changes were not induced by the aromatic amino acids, treatments that also failed to potentiate CaR-induced ERK activation despite inducing Ca2+(i) mobilization. Together, these data establish that CaR can elicit Rho-mediated changes in stress fiber assembly and cell morphology, which could contribute to the receptor's physiological actions. In addition, this study provides further evidence that aromatic amino acids elicit differential signaling from other CaR agonists.

Actins↗

PubMed2005

Evidence in favor of a calcium-sensing receptor in arterial endothelial cells: studies with calindol and Calhex 231.

Small increases in extracellular Ca2+ dilate isolated blood vessels. In the present study, the possibility that a vascular, extracellular Ca2+-sensing receptor (CaSR) could mediate these vasodilator actions was investigated. Novel ligands that interact with the CaSR were used in microelectrode recordings from rat isolated mesenteric and porcine coronary arteries. The major findings were that (1) raising extracellular Ca2+ or adding calindol, a CaSR agonist, produced concentration-dependent hyperpolarizations of vascular myocytes, actions attenuated by Calhex 231, a negative allosteric modulator of CaSR. (2) Calindol-induced hyperpolarizations were inhibited by the intermediate conductance, Ca2+-sensitive K+ (IKCa) channel inhibitors, TRAM-34, and TRAM-39. (3) The effects of calindol were not observed in the absence of endothelium. (4) CaSR mRNA and protein were present in rat mesenteric arteries and in porcine coronary artery endothelial cells. (5) CaSR and IKCa proteins were restricted to caveolin-poor membrane fractions. We conclude that activation of vascular endothelial CaSRs opens endothelial cell IKCa channels with subsequent myocyte hyperpolarization. The endothelial cell CaSR may have a physiological role in the control of arterial blood pressure.

Animals↗

PubMed2005

Aminoglycosides induce acute cell signaling and chronic cell death in renal cells that express the calcium-sensing receptor.

The aminoglycoside antibiotics (AGAs) are calcium-sensing receptor (CaR) agonists that are toxic to the renal proximal tubule. Proximal tubule-derived opossum kidney (OK) cells express CaR-like proteins and respond to AGAs with intracellular Ca2+ mobilization and extracellular regulated protein kinase (ERK) phosphorylation. To examine the possible cellular basis of AGA toxicity, acute and chronic responses to AGA treatment in OK cells and in CaR stably transfected HEK-293 cells (CaR-HEK) were studied. Changes in cell-fate signaling, proliferation, and cell death were detected by semiquantitative Western blotting, Hoechst staining, cell counting, and FACS analysis. Confocal microscopy was used to study the relative internalization of fluorophore-labeled gentamicin in CaR-transfected and -nontransfected cells. Here it is reported that the AGA neomycin and gentamicin elicit acute, phosphatidylinositol-3 kinase-dependent phosphorylation of Akt, glycogen synthase kinase 3beta, and p38 mitogen-activated protein kinase. After 24 h of gentamicin treatment, OK cell proliferation was observed, whereas after 4 d, the OK cells underwent cell death, an effect that was mimicked by the CaR agonists spermine and polyarginine. Furthermore, gentamicin elicited substantially more cell death in CaR-HEK cells than in nontransfected HEK-293 cells. The pan-specific caspase inhibitor Z-VAD significantly inhibited cell death in both OK and CaR-HEK cells. Finally, the intracellular uptake of Texas Red-labeled gentamicin was equivalent in both CaR-transfected and vector-transfected HEK-293 cells, suggesting that the CaR does not enhance drug uptake. Together, these observations demonstrate that the AGAs induce both acute and chronic cell fate changes in OK cells and CaR-HEK cells and that the proximal tubular CaR is likely to contribute to signaling underlying the renal toxicity of the AGAs.

Aminoglycosides↗

PubMed2004

Evidence for a functional calcium-sensing receptor that modulates myogenic tone in rat subcutaneous small arteries.

Myogenic tone of small arteries is dependent on the presence of extracellular calcium (Ca(o)(2+)), and, recently, a receptor that senses changes in Ca(2+), the calcium-sensing receptor (CaR), has been detected in vascular tissue. We investigated whether the CaR is involved in the regulation of myogenic tone in rat subcutaneous small arteries. Immunoblot analysis using a monoclonal antibody against the CaR demonstrated its presence in rat subcutaneous arteries. To determine whether the CaR was functionally active, segments of artery (< 250 microm internal diameter) mounted in a pressure myograph with an intraluminal pressure of 70 mmHg were studied after the development of myogenic tone. Increasing Ca(o)(2+) concentration ([Ca(2+)](o)) cumulatively from 0.5 to 10 mM induced an initial constriction (0.5-2 mM) followed by dilation (42 +/- 5% loss of tone). The dose-dependent dilation was mimicked by other known CaR agonists including magnesium (1-10 mM) and the aminoglycosides neomycin (0.003-10 mM) and kanamycin (0.003-3 mM). PKC activation with the phorbol ester phorbol-12,13-dibutyrate (20nM) inhibited the dilation induced by high [Ca(2+)](o) or neomycin, whereas inhibition of PKC with GF109203X (10 microM) increased the responses to Ca(o)(2+) or neomycin, consistent with the role of PKC as a negative regulator of the CaR. We conclude that rat subcutaneous arteries express a functionally active CaR that may be involved in the modulation of myogenic tone and hence the regulation of peripheral vascular resistance.

Aminoglycosides↗

PubMed2004

Differential expression of divalent metal transporter DMT1 (Slc11a2) in the spermatogenic epithelium of the developing and adult rat testis.

Iron is essential for male fertility, and disruptions in iron balance lead to impairment of testicular function. The divalent metal transporter DMT1 is a key modulator of transferrin- and non-transferrin-bound iron homeostasis. As a first step in determining the role of DMT1 in the testis, we have characterized the pattern of DMT1 expression in the developing and adult rat testis. Northern blot analysis and RT-PCR of testis polyadenylated RNA revealed the presence of iron-responsive element (IRE) and non-IRE transcripts. Semiquantitative immunoblotting of immature and adult rat testis uncovered the expression of two distinct DMT1 protein species. Immunohistochemistry showed that DMT1 was widespread throughout each seminiferous tubule and was expressed in the intracellular compartment. In the adult rat testis, DMT1 was immunolocalized to both the Sertoli and germ cells. In contrast to the immature testis, expression was dependent on the stage of the spermatogenic cycle. DMT1 was not detected on any plasma membranes in either the developing or the adult testis, suggesting that DMT1 is not primarily responsible for translocating iron across this epithelium. Our data suggest an important role for DMT1 in intracellular iron handling during spermatogenesis and imply that germ cells have a need for a precisely targeted and timed supply of iron. We suggest that DMT1 may, as it does in other tissues, play a role in transporting iron between intracellular compartments and thus may play an important role in male fertility.

Age Factors↗

PubMed2004

Physiological changes in extracellular calcium concentration directly control osteoblast function in the absence of calciotropic hormones.

We investigated the direct effects of changes in free ionized extracellular calcium concentrations ([Ca2+]o) on osteoblast function and the involvement of the calcium-sensing receptor (CaR) in mediating these responses. CaR mRNA and protein were detected in osteoblast models, freshly isolated fetal rat calvarial cells and murine clonal osteoblastic 2T3 cells, and in freshly frozen, undecalcified preparations of human mandible and rat femur. In fetal rat calvarial cells, elevating [Ca2+]o and treatment with gadolinium, a nonpermeant CaR agonist, resulted in phosphorylation of the extracellular signal-regulated kinases 1 and 2, Akt, and glycogensynthase kinase 3beta, consistent with signals of cell survival and proliferation. In agreement, cell number was increased under these conditions. Expression of the osteoblast differentiation markers core binding factor alpha1, osteocalcin, osteopontin, and collagen I mRNAs was increased by high [Ca2+]o, as was mineralized nodule formation. Alkaline phosphatase activity was maximal for [Ca2+]o between 1.2 and 1.8 mM. Inhibition of CaR by NPS 89636 blocked responses to the CaR agonists. In conclusion, we show that small deviations of [Ca2+]o from physiological values have a profound impact on bone cell fate, by means of the CaR and independently of systemic calciotropic peptides.

Animals↗

PubMed2004

Calcium receptor-mediated intracellular signalling.

As a G protein-coupled receptor (GPCR), the extracellular calcium-sensing receptor (CaR) responds to changes in extracellular free calcium concentration by inducing intracellular signalling. These CaR-induced signals then specifically modulate cellular functions such as parathyroid hormone secretion from the parathyroid glands and calcium reabsorption in the kidney and thus to understand how the CaR functions one must understand how it signals. CaR-induced signalling involves intracellular Ca2+ mobilisation/oscillations as well as the activation of various phospholipases and protein kinases and the suppression of cAMP formation. This review will detail the intracellular pathways by which the CaR is believed to elicit its physiological functions and summarises the evidence for cell- and agonist-specific differential signalling.

Animals↗

PubMed2003

Human calcium-sensing receptor can be suppressed by antisense sequences.

We have evaluated the ability of an antisense cDNA sequence, directed to the amino-terminus of the human calcium-sensing receptor (CaR), to reduce the expression and function of an EGFP-tagged CaR (CaR-EGFP) in HEK293 cells. Confocal microscopy and Western blot analysis showed a significant and selective reduction of the expression of CaR-EGFP by the antisense construct. Measurements of changes in intracellular calcium induced by CaR agonists showed that CaR-EGFP function was significantly reduced by the antisense sequence, as was agonist-evoked phosphorylation of extracellular signal-regulated protein kinases (ERK1,2). A sense construct directed to the same region of the receptor had no effect, confirming the specificity of the antisense construct. Our results indicate that a CaR antisense cDNA reduces both the expression and function of the receptor. In the absence of strong, specific pharmacological inhibitors of CaR, the antisense approach will be helpful to elucidate contributions of the CaR to cell physiology.

Blotting, Western↗

PubMed2003

Calpain-mediated AQP2 proteolysis in inner medullary collecting duct.

Vitamin D-elicited hypercalcemia/hypercalciuria is associated with polyuria in humans and in animal models. In rats, dihydrotachysterol (DHT) induces AQP2 water channel downregulation despite unaltered AQP2 mRNA expression and thus we investigated the mechanism of AQP2 degradation. Incubation of AQP2-containing inner medullary collecting duct (IMCD) endosomes with Ca(2+) or calpain elicited AQP2 proteolysis, an effect abolished by leupeptin. This endogenous, Ca(2+)-sensitive protease activity exhibited a different proteolytic digest pattern from trypsin, which also degraded AQP2 in vitro. IMCDs contain abundant micro-calpain protein and functional calpain proteolytic activity as demonstrated by immunohistochemistry, immunoblotting, and gel zymography. Furthermore, by small particle flow cytometry we demonstrated that micro-calpain colocalizes with apical IMCD endosomes. DHT does not appear to elicit general proteolysis, however, in addition to AQP2 degradation, DHT treatment also diminished micro-calpain and calpastatin expression although whether these changes contributed to the AQP2 instability remains unclear. Together, these data show for the first time that AQP2 is a substrate for calpain-mediated proteolysis and that furthermore, micro-calpain, like AQP2, is both highly expressed in renal inner medulla and localized to apical IMCD endosomes.

Animals↗

PubMed2002

Renal physiology of the extracellular calcium-sensing receptor.

The kidney is a fundamental component of the Ca2+ homeostatic system and its ability to sense extracellular Ca2+ ([Ca2+]o) levels in the urinary filtrate and the interstitial fluid is an important ionic feedback mechanism in achieving normocalcaemia. The protein responsible for the measurement of [Ca2+]o is the extracellular Ca2+-sensing receptor (CaR), which is expressed in multiple sites along the nephron. Its cellular localization and apparent function(s) appear to depend upon the region of the nephron in which it is expressed. The renal expression of CaR will be detailed in this review and its role in integrating [Ca2+]o with the calciotropic signals responsible for Ca2+ homeostasis will be discussed.

Absorption↗

PubMed2002

Aminoglycosides increase intracellular calcium levels and ERK activity in proximal tubular OK cells expressing the extracellular calcium-sensing receptor.

Aminoglycoside antibiotics (AGAs) are nephrotoxic, with most of the damage confined to the proximal tubule, but the mechanism for cellular toxicity is not clear. It has been previously shown that the extracellular-calcium sensing receptor (CaR) is expressed in intact rat proximal tubule and can be stimulated by the AGA neomycin. To investigate whether CaR could contribute to AGA-induced nephrotoxicity, the acute responses to various AGAs in the proximal tubule-derived opossum kidney (OK) cell line were examined. The presence in OK cells of CaR-related transcripts and protein was demonstrated by northern analyses, reverse transcriptase-PCR, immunocytochemistry, and immunoblotting. OK cells responded to elevated extracellular calcium (Ca(2+)(o)) and neomycin but also to gentamicin and tobramycin with an increase in cytosolic [Ca(2+)]. Ca(2+)(o), neomycin, and gentamicin also activated the extracellular signal-regulated kinases, ERK1 and ERK2. Neomycin-induced ERK activation was both dose- and time-dependent and was attenuated by inhibitors of phosphatidylinositol 3-kinase, phosphatidylinositol bisphosphate (PIP(2))-specific phospholipase C, and MEK1, but not of protein kinase C. Thus, proximal tubular OK cells express a CaR that mediates Ca(2+)(i) mobilization and PIP(2)-PLC-dependent ERK activation in response to AGAs and thus could play a role in AGA-induced nephrotoxicity.

Animals↗

PubMed2001

Functional, molecular, and biochemical characterization of streptozotocin-induced diabetes.

Altered divalent cation homeostasis with bone mineral loss, hypercalciuria, and hypomagnesemia have been associated consistently with human diabetes mellitus. This study investigated functional, molecular, and biochemical determinants that accompany this condition in chronically (2 wk) streptozotocin (STZ)-diabetic rats. Catheterized, conscious, diabetic rats on servo-controlled fluid replacement exhibited an increased GFR (+70%) and a substantially raised urinary calcium output (+568%) when compared with control rats. In addition, fractional calcium reabsorption was reduced, indicating that the hypercalciuria was not due solely to an osmotic effect but may involve an actual tubular defect. The expression of proteins involved in renal distal Ca2+ and water transport in STZ-diabetic rats were then studied by Western analysis and immunofluorescence microscopy to investigate the molecular basis of the hypercalciuria. Extracellular Ca2+-sensing receptor abundance was reduced to 52% of control in STZ-diabetes, whereas thiazide-sensitive NaCl cotransporter expression was increased by 192%. Subcutaneous insulin implant rectified both functional and molecular parameters. The levels of calbindin D(28k), plasma membrane Ca2+ ATPase, and aquaporin 1 in whole kidney and of aquaporin 2 in inner medulla were unchanged in diabetic and/or insulin replacement. Blood levels of 1,25(OH)(2)D(3) were reduced in diabetes as were levels of osteocalcin, a marker of bone formation. It is concluded that diabetic hypercalciuria in rats involves elevated GFR with raised urinary output, reduced Ca2+ reabsorption, and impaired bone deposition. Changes in Ca2+-sensing receptor and NaCl cotransporter protein expression could account for the altered divalent cation homeostasis seen during diabetes mellitus.

Animals↗

PubMedDate not supplied

Noradrenaline-induced paxillin phosphorylation, ERK activation and MEK-regulated contraction in intact rat mesenteric arteries.

In rat mesenteric arteries, noradrenaline (NA) induces a time-dependent increase in tyrosine phosphorylation of a number of proteins, one of which was identified as paxillin. NA-induced protein tyrosine phosphorylation was ablated by tyrosine kinase inhibition, virtually unaffected by protein kinase C (PKC) inhibition or PKC downregulation and was mimicked by KCl. NA also caused a time-dependent activation of the extracellular signal-regulated kinases (ERK)1 and ERK2. These responses were blocked by the ERK-activating kinase (MEK) inhibitor PD98059 and by tyrosine kinase inhibition but only modestly attenuated by PKC downregulation or inhibition. Pretreatment of cannulated mesenteric arteries (50 mm Hg internal pressure) with PD98059 significantly reduced the contractile responsiveness of the vessels to NA (1.56 +/- 0.14 microM, EC(50) control; 3.32 +/- 0.49 microM, EC(50) + PD98059, p < 0.01). Thus, NA induces time-dependent increases in protein-tyrosine phosphorylation and ERK activation in rat mesenteric arteries that could suggest a role for Ca(2+)-dependent non-receptor tyrosine kinases and ERKs in the response of small arteries to NA. In addition, the modulation of NA-induced mesenteric artery contraction by inhibition of the MEK/ERK pathway further implicates ERK in the regulation of, though perhaps not the mediation of NA-induced small artery contraction.

Animals↗