Oscar A. Gende

Hydrogen peroxide activates calcium influx in human neutrophils

The correlation between an increased production of reactive oxygen species (ROS) and an enhanced calcium entry in primed neutrophils stimulated with fMLP suggests that endogenous ROS could serve as an agonist to reinforce calcium signaling by positive feedback. This work shows that exogenous H2O2 produced a rapid influx of Mn2+ and an increase of intracellular calcium. The H2O2 was insufficient to produce significant changes in the absence of extracellular calcium but addition of Ca2+ to H2O2-treated cells suspended in a free Ca2+/EGTA buffer resulted in a great increase in [Ca2+]i reflecting influx of Ca2+ across the cell membrane. The increase of intracellular calcium was inhibited by Ni2+, La3+, and hyperosmotic solutions of mannitol and other osmolytes. This raises the possibility that the secretion of H2O2 by activated neutrophils could act as an autocrine regulator of neutrophil function through the activation of calcium entry.

Increased sensitivity to nifedipine of smooth muscle from hypertensive rats.

Summary: The sensitivity of smooth muscle from aortas of spontaneously hypertensive rats (SHR), renal hypertensive rats: two kidney-one clip and one kidney-one clip (2K 1C, 1K 1C) and DOCA salt hypertensive rats to the relaxant effect of nifedipine (NIF) was studied. A parallel leftward shift of the concentration-relaxation curve was detected in the K-precontracted aortic smooth muscle from hypertensive rats. This increased sensitivity seems to be related to the degree of hypertension and independent of the experimental method used to produce the high blood pressure level. No change in sensitivity was detected either in SHR or in renal hypertensive rats when mitroglycerin was used as a vasodilator.

Identification of a sodium-bicarbonate symport in human platelets

Intracellular pH (pH(i)) was measured in human platelets using fluorescent probes. Basal pH(i) was higher in HC(O3-)- buffered solutions (7(7.33 +/- 0.01) than in nominally HCO3- free, Hepes buffered solutions (7.16 +/- 0.01, P < 0.05). Addition of EIPA caused to fall in Hepes, but did not inhibit the increase of pH(i) when platelets maintained in Hepes were transferred to a CO2/HCO3- buffer. After an intracellular acidosis induced by an NH4Cl prepulse, the initial velocity of recovery (d(pH)/dt(i), in pH units/min) was 3.32 +/- 0.69 in Hepes-buffered solution and 2.85 +/- 0.88 in HCO3- media. Taking into account the differences in buffer capacity, the efflux of acid equivalents after 1.2 min was twice as much in the presence of bicarbonate. The addition of 30 mumol/1 EIPA effectively blocked acid efflux (d(pH)/dt(i) = 0.08 +/ 0.04) in a nominally HCO3- free solution, whereas the recovery was reduced but not abolished (d(pH)/dt(i) = 0.37 +/- 0.10, P < 0.05) in the presence of bicarbonate. The stilbene derivative SITS further inhibited the EIPA-resistant pH(i) recovery. Removal of external Na+ inhibited the HCO(3-)-dependent recovery whereas depletion of internal Cl-, did not suppress it. Depolarization of the membrane had no effect on this recovery. The results suggest the contribution of an electroneutral Na+/HCO3- cotransport in the recovery of pH(i) following an acid load. Both the Na+/H+ antiport and the HCO(3-)-dependent mechanism contribute approx. 50% each to the total acid equivalent efflux during the recovery from a pH(i) 6.46 +/- 0.14 to the basal pH(i) in human platelets.

Chelerythrine Inhibits Na+-H+ Exchange in Platelets From Spontaneously Hypertensive Rats

Hypertension has been associated with increased activity of the Na(+)-H+ exchanger. To study the role played by protein kinase C in this process, we used chelerythrine, a potent and specific inhibitor of the kinase. After an acid load by ammonium chloride preincubation, platelets isolated from spontaneously hypertensive rats showed a faster and larger increase in intracellular pH than platelets from Wistar-Kyoto rats. The initial rate of intracellular pH recovery was 2.46 +/- 0.26 pH units per minute in spontaneously hypertensive rats and 1.74 +/- 0.19 in Wistar-Kyoto rats. For protein kinase C inhibition, platelets were incubated for 30 minutes with 10 mumol/L chelerythrine. This treatment induced a significant reduction in the recovery rate only in spontaneously hypertensive rat platelets, indicating that a pathway involving protein kinase C participates in the prestimulation of the exchanger in cells from this rat strain. Addition of chelerythrine reduced the baseline intracellular pH of platelets. No significant difference was found between the decrease of steady-state intracellular pH induced by chelerythrine in either rat strain. These findings indicate that this model of hypertension is characterized by increased Na(+)-H+ activity mediated by protein kinase C stimulation.

Hypertonic saline solutions attenuate agonist-induced changes of intracellular calcium

The proper fluid for resuscitation of hemorrhagic shock is still controversial. Hypertonic saline solutions would cause an impairment of platelet function, aggravating blood loss in case of uncontrolled hemorrhage. This work examines the in vitro effect of hypertonic NaCl solutions on the changes in [Ca2+]i induced by 100 μM ADP, 0.1 IU/ml thrombin or 0.5 μM ionomycin in human platelets. Furthermore, it was investigated if the addition of NaCl reduces the mobilization from intracellular stores or the calcium entry from extracellular media. In a solution containing 1 mM CaCl2, the increase of [Ca2+]i produced by thrombin was 93, 75 or 55% of the 300 mosM control when osmolarity of the solution was 350, 400, or 500 mosM, respectively. The calcium signal induced by ADP decreased more rapidly in hypertonic media than in isotonic solution. In a calcium-free solution, the mobilization of stored calcium produced by thrombin was reduced when osmolarity was increased from 300 mosM to 350, 400 or 500 mosM by 86, 75 or 45% of the control, respectively. The increase of [Ca2+]i produced by subsequent introduction of 1 mM extracellular calcium was also reduced. Similar effects were found when platelets were stimulated by ADP. Instead, the capacitative calcium entry induced by ionomycin was increased in 500 mosM media by a 138% of the isotonic control. The decrease in the Ca2+ signal produced by receptor agonists in hypertonic media may play a role in the reduction of platelet responses such as aggregation or shape change when hypertonic resuscitation fluids are utilized.

Characterization of the Na+/HCO3- Cotransport in Human Neutrophils

Background: Bicarbonate transport has crucial roles in regulating intracellular pH (pHi) in a variety of cells. The purpose of this study was to evaluate its participation in the regulation of pHi in resting and stimulated human neutrophils. Methods: Freshly isolated human neutrophils acidified by an ammonium prepulse were used in this study. Results: We demonstrated that resting neutrophils have a bicarbonate transport mechanism that prevents acidification when the Na+/H+ exchanger is blocked by EIPA. Neutrophils acidified by an ammonium prepulse showed an EIPA-resistant recovery of pHi that was inhibited by the blocker of the anionic transporters SITS or the Na+/HCO3- cotransporter (NBC) selective inhibitor S0859, and abolished when sodium was removed from the extracellular medium. In western blot and RT-PCR analysis the expression of NBCe2 but not NBCe1 or NBCn1 was detected in neutrophils Acidified neutrophils increased the EIPA-insensitive pHi recovery rate when its activity was stimulated with fMLF/ cytochalasin B. This increase in the removal of acid equivalents was insensitive to the blockade of the NADPH oxidase with DPI. Conclusion: It is concluded that neutrophils have an NBC that regulates basal pHi and is modulated by chemotactic agents.

Inhibition of cytochalasin-primed neutrophils by hyperosmolarity.

Experimental and clinical investigations using hyperosmotic solutions for resuscitation of hemorrhagic shock demonstrated modulation of the inflammatory response. Decreased postinjury hyperinflammation has been attributed to a reduction in neutrophil-mediated tissue damage. This study shows that cytoskeletal disruption with cytochalasinB did not reverse or prevent the inhibitory effect of an osmolarity increase on the neutrophil cytotoxic response to a formyl peptide. In cytochalasin-primed neutrophils, the hyperosmolarity-dependent inhibition promptly reversed after returning to iso-osmotic levels. Paradoxically, an increase in osmolarity after stimulation produced an increase in the release of reactive oxygen species to the extracellular medium. The inhibitory effect of hyperosmotic NaCl can be reproduced by solutions of similar osmolarity containing N-methyl glucamine or sucrose, but solutions containing mannitol allowed an almost complete response to N-formyl methionyl leucyl phenylalanine. The effects on the release of reactive oxygen species to the extracellular media found with the OxyBURST-bovine serum albumin assay correlated with the changes of the intracellular calcium signal, indicating that the inhibition by hyperosmolarity occurs near the receptor level.

Paradoxical effect of calcium on some cardiovascular effects of verapamil in the rat

The ability of calcium infusions to reverse some cardiovascular effects of verapamil was tested in anesthetized rats and on isolated rat atria. Intravenous infusions of 1.88 mg/kg verapamil increased the cycle length (CL), lengthened the PR interval (PRi), and decreased the mean arterial pressure (Pa). After these effects were stabilized, a series of infusions of calcium chloride (338 mumol/kg each) were performed. Calcium on the one hand promoted the return of Pa to its basal value; on the other hand, it failed to reverse the effect of verapamil on AV conduction and the first infusion of calcium produced an additional increase in CL of 55 +/- 16 ms. This paradoxical effect of calcium was prevented by previous infusion of atropine (0.2 mg/kg). On isolated atria, the increase of calcium concentration in the media ([Ca2+]0) from 1.0 to 6.0 mM increased the concentration frequency by approximately 40 beats/min, both in atropinized and in nonatropinized preparations. After verapamil, however, the same increase in [Ca2+]0 decreased atrial rate in 50 +/- 15 beats/min in nonatropinized atria. The results obtained indicate that extra calcium can overcome the hypotensive effect of verapamil, whereas it paradoxically increases its negative chronotropic effect and fails to reverse its effect on AV conduction. This paradoxical effect of calcium can be prevented, both in vivo and in vitro, by atropine blockade of muscarinic receptors.

Effect of changes in CO2 tension upon hydrogen ion activity of the skeletal muscle of the dog.

The effect of graded changes in PCo2 upon intracellular hydrogen ion activity [H+]i of skeletal muscle of nephrectomized dogs was studied. [H+]i was calculated by the distribution of 5, 5-Dimethyloxazolidine-2, 4-Dione (DMO).

Cl−/HCO3- exchange activity in fMLP-stimulated human neutrophils

It is well known that chemotactic agents active Na(+)/H(+) exchanger, increasing intracellular pH of neutrophils, but their effect on bicarbonate transporters have not been established yet. To study the effect of fMLP on the activity of Cl(-)/HCO(3)(-) exchange, the rate of pH recovery after acute Cl(-) readmission in cell subjected to an alkaline load by CO(2) washout in a Cl-free medium was measured. The activity of the exchanger was reduced to 72% of control when cells were pre-incubated for 5 min with 0.1 μM fMLP and reached 48% of control in steady state after acute exposure. After extracellular bicarbonate or TMA addition the rate recovery of intracellular pH was reduce at 72% and at 84%, respectively. The inhibitory effect on the intracellular pH recovery was not affected by blockers of Na(+)/H(+) exchange. We conclude from these studies that an increase of pH(i) produced for this chemotactic agent is facilitated by the simultaneous activation of Na(+)/H(+) exchange and inhibition of Cl(-)/HCO(3)(-) exchange in neutrophils.