title: Effect of hyperosmolarity on agonist-induced increases of intracellular calcium in human platelets.
abstract: BACKGROUND
  Hypertonic solutions are useful for the management of hypovolemic shock but cause impairment in platelet function. This effect would reduce the ischemia/reperfusion damage caused by activated platelets, but it could be the cause of aggravating blood loss in case of uncontrolled hemorrhage. In this paper, it was studied if osmotic shrinkage of platelets affects the changes in intracellular calcium concentration ([Ca(2+)](i)) induced by thrombin or adenosine 5' diphosphate (ADP). Furthermore, it was investigated if hypertonic solutions change the mobilization from intracellular stores or the calcium entry. Previous reports from our laboratory described that the capacitative calcium entry is increased by alkalization and also that hyperosmolarity has an alkalinizing effect on human platelets so it was hypothesized that hyperosmolarity would be able to modify agonist-induced calcium entry.
  
  
  METHODS
  To study the response to agonists, platelets loaded with 2'7'-bis(carboxyethyl)-5(6)-carboxy-fluorescein (FURA 2) were incubated at 37 degrees C for 500 s in a N-2-hidroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered solution with 1 mM CaCl(2). The osmolarity of the solution was elevated by the addition of 200 mM mannitol or sucrose and after the stimulation with 0.1 IU/ml of thrombin or 100 microM ADP, [Ca(2+)](i) increases were compared. Platelets incubated in zero calcium/EGTA were stimulated with these agonists or with 0.1 microM thapsigargin to separately study the effect of hyperosmolarity on both calcium mobilization from intracellular stores and extracellular calcium entry.
  
  
  RESULTS
  It was found that hypertonic solutions decrease the [Ca(2+)](i) peak induced by the agonist: The increase of [Ca(2+)](i) in the presence of 200 mM mannitol produced by 100 microM ADP was 62+/-6% and response to 0.1 IU/ml thrombin was 74+/-7% of the increase in isotonic control solution. In the case of ADP, both mobilization and calcium entry were reduced to 66+/-3% and 65+/-6% of isotonic control, respectively. In the case of thrombin, only the mobilization showed a significant change (79+/-2% of parallel control). In platelets depleted by thapsigargin, the capacitative calcium entry was increased in hypertonic mannitol (174+/-25% of the isotonic control). Similar results were obtained with hypertonic sucrose solutions.
  
  
  CONCLUSIONS
  In spite of the stimulatory effect of hyperosmolarity on capacitative calcium entry observed in platelets in which the calcium stores were depleted with thapsigargin, the full response of intracellular calcium to the agonists tested (ADP and thrombin) was reduced by an increase in osmolarity. The decreased Ca(2+) mobilization observed may play a role in the reduction in hypertonic media of accompanying platelets responses such as aggregation or shape change.
authors.affiliation: Centro de Investigaciones Cardiovasculares, Facultad de Ciencias Médicas, Universidad Nacional de La Plata, 60 Y 120, 1900 La Plata, Argentina. ogende@atlas.med.unlp.edu.ar
authors.forename: Oscar A
authors.initials: OA
authors.lastname: Gende
mesh_terms: D000244:Adenosine Diphosphate / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects* / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects; D048429:Cell Size / Q000187:drug effects; D016257:Fura-2 / Q000378:metabolism; D006801:Humans; D006982:Hypertonic Solutions / Q000009:adverse effects / Q000494:pharmacology* / Q000627:therapeutic use; D009994:Osmolar Concentration; D015539:Platelet Activation / Q000187:drug effects*; D012769:Shock / Q000628:therapy; D019284:Thapsigargin / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology
chemical_list: D006982:Hypertonic Solutions; D000244:Adenosine Diphosphate; D019284:Thapsigargin; D013917:Thrombin; D002118:Calcium; D016257:Fura-2