BACKGROUND: A wide variety of neutrophil (PMN) functions are regulated by cytosolic calcium concentration. Calcium channel blockade might therefore decrease postshock inflammation but could also limit important cardiovascular compensations. PMN Ca2+ entry occurs, however, through store-operated calcium entry (SOCE) channels rather than the voltage operated (L-type) channels that regulate cardiovascular tone. We hypothesized that SOCE inhibition might suppress postshock PMN activation, lessening lung injury without compromising cardiovascular performance. METHODS: Human PMNs were treated in vitro with N-propargyl-nitrendipine (MRS1845 [MRS]) a dihydropyridine Ca2+ channel blocker with relative specificity for SOCE channels. Calcium flux was measured by fura fluorescence. Chemotaxis was studied in modified Boyden chambers. Respiratory burst was studied by dihydrorhodamine fluorescence. Exploratory studies were then performed where rats were subjected to trauma and hemorrhagic shock (T/HS) (laparotomy, then hemorrhage to a mean arterial pressure of 30-40 mm Hg for 90 minutes) after pretreatment with MRS or vehicle given intraperitoneally at laparotomy. In vivo PMN CD11b expression was then assayed by flow cytometry and lung injury was assessed as percentage Evans blue dye leak 3 hours after resuscitation. The shed blood volume required to achieve standardized hypotension was measured. RESULTS: In vitro, MRS suppressed human PMN SOCE without affecting calcium store release; it suppressed chemotaxis (60 +/- 6 vs. 150 +/- 15 x 10(3) PMNs/well, p = 0.002) and suppressed respiratory burst (62 +/- 11% vs. 100%, p < 0.05) at IC50 concentrations similar to those needed to suppress SOCE. In subsequent in vivo rat studies, MRS decreased postshock PMN CD11b expression from 397 +/- 93 to 268 +/- 39 MFU mean flourescent units (p < 0.05) and decreased lung Evans blue dye permeability from 8.1 +/- 1.9% to 3.4 +/- 0.1% (p < 0.05). MRS had no noticeable effect on the relationship between blood pressure and blood loss, with shed blood volume remaining almost identical (26 +/- 2 mL/kg vs. 27 +/- 3 mL/kg, p = not significant). CONCLUSION: Modulation of PMN Ca2+ entry by means of selective SOCE channel inhibition attenuates PMN inflammatory responses in vitro. In vivo, SOCE channel blockade attenuates trauma and hemorrhagic shock-induced PMN priming and lung injury without gross evidence of hemodynamic side effects. The relative specificity of SOCE channel blockade for "nonexcitable" cells such as PMNs may make it a valuable form of chemoprophylaxis for the inflammatory consequences of hemorrhagic shock in trauma patients.
BACKGROUND: A wide variety of neutrophil (PMN) functions are regulated by cytosolic calcium concentration. Calcium channel blockade might therefore decrease postshock inflammation but could also limit important cardiovascular compensations. PMN Ca2+ entry occurs, however, through store-operated calcium entry (SOCE) channels rather than the voltage operated (L-type) channels that regulate cardiovascular tone. We hypothesized that SOCE inhibition might suppress postshock PMN activation, lessening lung injury without compromising cardiovascular performance. METHODS:Human PMNs were treated in vitro with N-propargyl-nitrendipine (MRS1845 [MRS]) a dihydropyridine Ca2+ channel blocker with relative specificity for SOCE channels. Calcium flux was measured by fura fluorescence. Chemotaxis was studied in modified Boyden chambers. Respiratory burst was studied by dihydrorhodamine fluorescence. Exploratory studies were then performed where rats were subjected to trauma and hemorrhagic shock (T/HS) (laparotomy, then hemorrhage to a mean arterial pressure of 30-40 mm Hg for 90 minutes) after pretreatment with MRS or vehicle given intraperitoneally at laparotomy. In vivo PMN CD11b expression was then assayed by flow cytometry and lung injury was assessed as percentage Evans blue dye leak 3 hours after resuscitation. The shed blood volume required to achieve standardized hypotension was measured. RESULTS: In vitro, MRS suppressed human PMN SOCE without affecting calcium store release; it suppressed chemotaxis (60 +/- 6 vs. 150 +/- 15 x 10(3) PMNs/well, p = 0.002) and suppressed respiratory burst (62 +/- 11% vs. 100%, p < 0.05) at IC50 concentrations similar to those needed to suppress SOCE. In subsequent in vivo rat studies, MRS decreased postshock PMN CD11b expression from 397 +/- 93 to 268 +/- 39 MFU mean flourescent units (p < 0.05) and decreased lung Evans blue dye permeability from 8.1 +/- 1.9% to 3.4 +/- 0.1% (p < 0.05). MRS had no noticeable effect on the relationship between blood pressure and blood loss, with shed blood volume remaining almost identical (26 +/- 2 mL/kg vs. 27 +/- 3 mL/kg, p = not significant). CONCLUSION: Modulation of PMN Ca2+ entry by means of selective SOCE channel inhibition attenuates PMN inflammatory responses in vitro. In vivo, SOCE channel blockade attenuates trauma and hemorrhagic shock-induced PMN priming and lung injury without gross evidence of hemodynamic side effects. The relative specificity of SOCE channel blockade for "nonexcitable" cells such as PMNs may make it a valuable form of chemoprophylaxis for the inflammatory consequences of hemorrhagic shock in traumapatients.
Authors: Derayvia Grimes; Ryan Johnson; Madeline Pashos; Celeste Cummings; Chen Kang; Georgia R Sampedro; Eric Tycksen; Helen J McBride; Rajan Sah; Clifford A Lowell; Regina A Clemens Journal: Proc Natl Acad Sci U S A Date: 2020-09-14 Impact factor: 11.205