New Drugs for the Na+/H+ Exchanger. Influence of Na+ Concentration and Determination of Inhibition Constants with a Microphysiometer

The NHE-1 isoform of the Na⁺/H⁺ exchanger is excessively activated in cardiac cells during ischemia. Hence NHE-1 specific inhibitors are being developed since they could be of beneficial influence under conditions of cardiac ischemia and reperfusion. In this study, the Cytosensor™ microphysiometer was used to measure the potency of four new drug molecules, i.e., EMD 84021, EMD 94309, EMD 96785 and HOE 642 which are inhibitors of the isoform 1 of the Na⁺/H⁺ exchanger. The experiments were performed with Chinese hamster ovary cells (CHO K1) which are enriched in the NHE-1 isoform of the Na⁺/H⁺ antiporter. The Na⁺/H⁺ exchanger was stimulated with NaCl and the rate of extracellular acidification was quantified with the Cytosensor. The proton exchange rate was measured as a function of the NaCl concentration in the range of 10–138 mm NaCl stimulation. The proton exchange rate followed Michaelis-Menten kinetics with a K _M = 30 ± 4 mm for Na⁺. Addition of either one of the four inhibitors decreased the acidification rate. The IC₅₀ values of the four compounds could be determined as 23 ± 7 nm for EMD 84021, 5 ± 1 nm for EMD 94309, 9 ± 2 nm for EMD 96785 and 8 ± 2 nm for HOE 642 at 138 mm NaCl, in good agreement with more elaborate biological assays. The IC₅₀ values increased with the NaCl concentration indicating competitive binding of the inhibitor. The microphysiometer approach is a fast and simple method to measure the activity of the Na⁺/H⁺ antiporter and allows a quantitative kinetic analysis of the proton excretion rate. Received: 3 September 1998/Revised: 20 November 1998     

Characteristics of acid extrusion from Chinese hamster ovary cells expressing different prostaglandin EP receptors

Acid extrusion responses to prostaglandin E2 were investigated in Chinese hamster ovary (CHO) cells heterologously expressing human EP1, EP2, and EP3I receptors (hEP1, hEP2 and hEP3I) by using a microphysiometer that detected small pH changes in the extracellular microenvironment. In the cells expressing hEP1, which is known to increase intracellular Ca2+, prostaglandin E2 (1 and 10 nM) slowly accelerated acid extrusion, but at higher concentrations an initial transient phase (approximately 5 times greater than the basal acidification) overlapped the slowly developing phase. In contrast, the cells expressing hEP2, which evokes cAMP production, showed dual responses to prostaglandin E2: an initial reduction followed by an acceleration of acid extrusion. In the cells expressing hEP3I, which is known to produce both a decrease in cAMP and a modest increase in intracellular Ca2+, acid extrusion was gradually accelerated by prostaglandin E2 and reached a plateau at around 2 min. Elimination of extracellular Ca2+ diminished the responses to prostaglandin E2 in hEP1 cells, but had little effect on the responses in hEP2 and hEP3I cells. Forskolin mimicked the dual effects of prostaglandin E2 observed in the hEP2 cells. Pretreatment with pertussis toxin inhibited the response to prostaglandin E2 in hEP3I cells, but the responses in hEP1 and hEP2 cells were not affected. Na+/H+ exchanger (NHE) inhibitors (EIPA and HOE642) suppressed all the responses induced by prostaglandin E2 in hEP1, hEP2, and hEP3I cells. These results suggest that EP receptor subtypes regulate acid extrusion mainly via NHE-1 through distinct signal transduction pathways in CHO cells.     

Receptor-independent effects of 2′(3′)-O-(4-benzoylbenzoyl)ATP triethylammonium salt on cytosolic pH

The effect of the relatively potent P2X7 receptor agonist 2′(3′)-O-(4-benzoylbenzoyl)adenosine 5′-triphosphate triethylammonium salt (BzATP-TEA) on cytosolic pH (pH_i) was studied using MC3T3-E1 osteoblast-like cells, which endogenously express P2X7 receptors. pH_i was measured fluorimetrically using the pH-sensitive dye 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. BzATP-TEA (0.3–1.5 mM) elicited fast-onset alkalinization responses. In contrast, adenosine 5′-triphosphate disodium salt (5 mM) failed to reproduce the BzATP-TEA-induced responses, indicating a P2 receptor-independent mechanism. We speculated that triethylamine, which is present in solutions of BzATP-TEA, permeates the plasma membrane, and is protonated intracellularly, leading to an increase in pH_i. Consistent with this hypothesis, triethylammonium (TEA) chloride mimicked the effects of BzATP-TEA on pH_i. Moreover, measurements using a Cytosensor microphysiometer revealed that TEA chloride transiently suppressed proton efflux from cells, whereas washout of TEA transiently enhanced proton efflux. BzATP-TEA also elicited a sustained increase in proton efflux that was blocked specifically by the P2X7 antagonist A-438079. Taken together, we conclude that BzATP-TEA-induced alkalinization is unrelated to P2X7 activation, but is due to the presence of TEA. This effect may confound assessment of the outcomes of P2X7 activation by BzATP-TEA in other systems. Thus, control experiments using TEA chloride are recommended to distinguish between receptor-mediated and nonspecific effects of this widely used agonist. We performed such a control and confirmed that BzATP-TEA, but not TEA chloride, caused the elevation of cytosolic free Ca²⁺ in MC3T3-E1 cells, ruling out the possibility that receptor-independent effects on pH_i underlie BzATP-TEA-induced Ca²⁺ signaling.     

Effects of norepinephrine or prostaglandin E2 on extracellular acidification rate of MCG 101, or K1735-M2 tumor cells

We studied by microphysiometry functional effects of two different signalling molecules in the murine tumor cell lines, MCG 101 and K1735-M2, namely norepinephrine (NE) and prostaglandin E2 (PGE2). This methodology implies estimation of intracellular metabolism by measurements of extracellular acidification rate (ECAR). MCG 101 (an undifferentiated, epithelial-like tumor), in contrast to K1735-M2 (a melanoma), has been found to produce great amounts of PGE2. Challenge of MCG 101 cells with PGE2 (0.284 and 2.84 microM for 9 min) elicited an increase in ECAR by about 10 and 41% above basal level, respectively. Pretreatment with indomethacin (0.5 microM) reduced the response to the two PGE2 concentrations by about 70 and 25%, respectively. In contrast, PGE2 caused virtually no response in K1735-M2 cells. Moreover, NE caused increases in ECAR in both cell types, possibly via beta3-adrenoceptors, as investigated pharmacologically in MCG 101, and by immunocytochemistry in both cell lines. The results obtained strongly suggest functional receptors for PGE2 in MCG 101, but not K1735-M2 tumor cells. Functional receptors for NE were demonstrated in both cell lines. There is possibly an autocrine loop in the MCG 101 cells, in which PGE2 activates cyclooxygenase.