Inhibition Of Na-H Exchanger Research Articles

Exacerbated brain edema in a rat streptozotocin model of hyperglycemic ischemic stroke: Evidence for involvement of blood–brain barrier Na–K–Cl cotransport and Na/H exchange

Cerebral edema is exacerbated in diabetic ischemic stroke through poorly understood mechanisms. We showed previously that blood–brain barrier (BBB) Na–K–Cl cotransport (NKCC) and Na/H exchange (NHE) are major contributors to edema formation in normoglycemic ischemic stroke. Here, we investigated whether hyperglycemia-exacerbated edema involves changes in BBB NKCC and NHE expression and/or activity and whether inhibition of NKCC or NHE effectively reduces edema and injury in a type I diabetic model of hyperglycemic stroke. Cerebral microvascular endothelial cell (CMEC) NKCC and NHE abundances and activities were determined by Western blot, radioisotopic flux and microspectrofluorometric methods. Cerebral edema and Na in rats subjected to middle cerebral artery occlusion (MCAO) were assessed by nuclear magnetic resonance methods. Hyperglycemia exposures of 1-7d significantly increased CMEC NKCC and NHE abundance and activity. Subsequent exposure to ischemic factors caused more robust increases in NKCC and NHE activities than in normoglycemic CMEC. MCAO-induced edema and brain Na uptake were greater in hyperglycemic rats. Intravenous bumetanide and HOE-642 significantly attenuated edema, brain Na uptake and ischemic injury. Our findings provide evidence that BBB NKCC and NHE contribute to increased edema in hyperglycemic stroke, suggesting that these Na transporters are promising therapeutic targets for reducing damage in diabetic stroke.

Hyperglycemia‐induced Elevation of NKCC1 Expression in Blood Brain Barrier Endothelial Cells: Greater Effect of Intermittent Versus Sustained Exposures

Diabetics with accompanied hyperglycemia (HG) suffer from exacerbated cerebral edema and worsened neurological outcomes following ischemic stroke. We have demonstrated previously that hypoxia and other factors present during cerebral ischemia stimulate cerebral microvascular endothelial cell (CMEC) Na‐K‐Cl (NKCC) cotransport and Na/H exchange (NHE) activities. We also found that inhibition of these blood‐brain barrier (BBB) Na transporters reduces brain Na uptake and edema in the rat middle cerebral artery occlusion (MCAO) model of ischemic stroke. In more recent studies focusing on diabetic stroke, we demonstrated that sustained 6‐hour to 7‐day HG exposures increase expression and activity of CMEC NKCC and NHE, resulting in more pronounced responses to ischemic factor stimulation. Further, we found that inhibition of BBB NKCC and NHE effectively reduces edema and infarct in hyperglycemic rats subjected to MCAO. Diabetics often experience postprandial excursions of blood glucose. Studies have provided evidence that while sustained HG can induce stress/injury responses in endothelial cells, intermittent HG can exaggerate those responses. The present study was conducted to investigate the effects of intermittent HG exposures on BBB Na transporter expression and activity. For this, cultured bovine CMEC were exposed daily over 2 to 5 days to HG medium (DMEM with 30 mM glucose, 320 mOsm) or to osmotic control medium (DMEM with 30 mM mannitol, 320 mOsm) for 6 hours, followed by exposure to normoglycemic medium (DMEM with 5 mM glucose, 295 mOsm) for 18 hours. Quantitative Western blot analysis was then used to assess NKCC1 protein abundance in CMEC lysates. We found that CMEC NKCC1 abundance increased in response to intermittent HG exposures in a graded manner with larger increases observed following greater number of successive exposures. No change in NKCC1 abundance occurred with repeated intermittent exposures to normoglycemic medium. In addition, our results show that 5 days of intermittent HG exposures caused a 124% increase in NKCC1 abundance compared to 40% and 65% increases following sustained 2‐day and 7‐day exposures, respectively. In the present study we also investigated the effects of varying HG levels on CMEC Na transporter abundance. Here, we exposed CMEC for 48 hours to HG media containing 10, 20 and 30 mM glucose, to osmotic control media (with mannitol) or to normoglycemic medium (with 5 mM glucose). We found that NKCC1 and NHE1 protein abundances exhibited a graded response to increasing HG levels. Our findings support the hypothesis that BBB endothelial NKCC1 and NHE1 are highly sensitive to increasing levels of HG and, in particular, that clinically relevant repeated intermittent exposures result in more pronounced BBB Na transporter responses than sustained exposures.Support or Funding InformationSupported by NIH, ADA and AHA

Diminished natriuretic response to dopamine in old rats is due to an impaired D1-like receptor-signaling pathway

Dopamine (DA) causes natriuresis and diuresis, which results from activation of D1-like receptor (D1R) located on proximal tubules. Earlier, we reported that DA failed to inhibit Na,K-ATPase in proximal tubules of old Fischer 344 rats. The present study was designed to investigate the functional consequence of this phenomenon. Measurements of the functional (natriuretic and diuretic) response to intravenously infused DA and SKF 38393 (D1R agonist) in adult (6 month) and old (24 month) Fischer 344 rats were taken. Biochemical measurements were carried out to determine the potential defects in D1R and its signaling pathway in proximal tubules of old rats. We found that intravenous infusion of DA and SKF 38393 caused natriuresis and diuresis in adult rats, but this response was blunted in old rats. In the isolated proximal tubules, DA and SKF 38393 inhibited Na,H-exchanger (NHE) in adult rats; however, this inhibition was attenuated in old rats. Radioligand binding revealed approximately 46% reduction in D1R binding sites in brush border membranes (BBMs) in old compared with adult rats. SKF 38393 stimulated [35S]GTPgammaS binding in BBM in adult rats, but not in old rats, suggesting an impaired D1R-G protein coupling. DA and SKF 38393 stimulated adenylyl cyclase (AC) activity in adult but not in the old rats. Forskolin and NaF stimulated AC activity in a comparable manner in adult and old rats, indicating no defect in AC and G proteins. DA and SKF 38393 failed to stimulate protein kinase A (PKA) activity in proximal tubules of old rats. Dibutyryl-cAMP-mediated PKA activation was also absent in old rats. A decrease in D1R binding sites, a coupling defect with G proteins, and a defect in PKA activation lead to diminished DA-mediated inhibition of NHE in old rats, which may contribute to the blunted natriuretic response to DA in these animals.

Regulation of Connexin 43 Gene Expression by Cyclical Mechanical Stretch in Neonatal Rat Cardiomyocytes

Myocardial cells respond to changes in the mechanical forces imposed on them with changes in myocardial tension in the short term and with structural remodeling in the long term. Since these responses involve intercellular communication, we have investigated regulation of the gap junction proteins, connexin 43 (Cx43), connexin 40 (Cx40) and connexin 37 (Cx37), by cyclical mechanical stretch. Results were compared with parallel experiments on c-fos and GAPDH. Twenty percent stretch of cultured rat cardiomyocytes caused a 3-fold increase in Cx43 mRNA levels by 2 h. c-fos mRNA levels increased after 30 min of stretch, whereas Cx40, Cx37, and GADPH mRNA did not change. Protein levels of Cx43 increased by 4 h and remained elevated for 16 h. New protein synthesis was not a requirement for the stretch-induced rise in Cx43 expression, since mRNA levels were unaffected by treatment with cycloheximide. In addition, mechanical stretch induced alkalization of cardiomyocytes that was antagonized by inhibiting Na-H exchanger (NHE). Gap junction potential (Gj) was concomitantly elevated. Chemical closure of Cx channels by insulin was followed by inhibition of NHE. In conclusion, cyclical mechanical stretch caused increased expression of the gap junction protein Cx43 in cardiomyocytes and also the Gj. The augmentation of Cx43 mRNA expression and its functional status were associated with activation of NHE.

Mechanisms of protection of the ischemic and reperfused myocardium by sodium-hydrogen exchange inhibition.

Sodium-hydrogen exchange (Na-H exchange) is a major regulator of intracellular pH and is one of the major mechanisms for restoring pH after ischemia-induced intracellular acidosis. However, activation of Na-H exchange during ischemia and reperfusion is also involved in paradoxical induction of cell injury. This likely reflects the fact that activation of the exchanger is closely coupled to sodium influx and, as a consequence, to elevation in intracellular calcium concentrations through sodium-calcium exchange. In addition to intracellular acidosis, other factors can also stimulate the exchanger, including various autocrine and paracrine factors, such as endothelin-1, angiotensin II, alpha(1)-adrenergic agonists, as well as toxic agents, such as hydrogen peroxide and lysophosphatidylcholine. Although at least six Na-H exchange isoforms have thus far been identified, it appears that the 1 subtype, termed NHE1, is the predominant isoform in the mammalian myocardium. Effective pharmacological inhibitors of Na-H exchange, including those that are NHE1 specific, have been extensively demonstrated to protect the ischemic and reperfused myocardium in terms of improved systolic and diastolic function, preservation of cellular ultrastructure, attenuation of the incidence of arrhythmias, and reduction of apoptosis. Moreover, the salutary effects of these agents have been demonstrated using a variety of experimental models as well as animal species, suggesting that the role of Na-H exchange in mediating injury is not species specific. Thus, Na-H exchange represents an important target for pharmacological intervention in attenuation of ischemia and reperfusion-induced cardiac injury. Coupled with the low potential for toxicity of the agents, Na-H exchange inhibition could emerge as an effective therapeutic strategy in cardiac disorders, particularly involving conditions associated with ischemia and reperfusion.

Na-H exchange inhibition with cariporide limits functional impairment caused by repetitive ischemia.

Intracellular calcium ([Ca]i) overload on reperfusion may be one of the mechanisms responsible for ischemia-induced regional myocardial dysfunction. Because inhibiting the Na-H exchanger (NHE) limits intracellular sodium ([Na]i) and subsequent [Ca]i accumulation, we hypothesized that NHE inhibition would attenuate regional dysfunction in response to 25 cycles of ischemia (I, 2-min) and reperfusion (R, 8-min) of the left circumflex coronary artery (LCx) in conscious swine. Six animals were instrumented to measure arterial pressure, regional myocardial blood flow (colored microspheres), systolic wall thickening (WTh) in the normally perfused (left anterior descending, LAD) and LCx regions (sonomicrometry), LCx blood flow velocity (Doppler), and to reversibly occlude the LCx (hydraulic occluder). Each animal completed three protocols separated by 7 days: ISC, 25 I/R cycles; CAR, 25 I/R cycles + NHE inhibition (cariporide); and VEH, vehicle administration for 4.2 h. Regional myocardial blood flow was measured during LCx occlusion in the first protocol and 10 min after I/R 25 in all protocols. Systemic hemodynamics were similar among and within each protocol. Blood flow measured during LCx occlusion confirmed that perfusion was reduced (p < 0.05) to this compared with the LAD region. During ISC, LCx WTh was reduced (p < 0.05) after five IR cycles, and a stable reduction (approximately 55% of baseline; p < 0.05) was present after 20 I/R cycles. During CAR, LCx systolic WTh was reduced (p < 0.05) only after 15 and 25 I/R cycles (approximately 80 and 72%, respectively). The decrease in LCx WTh was greater in ISC than in CAR (p < 0.05). LCx WTh was not altered during VEH, while LAD WTh was similar within and among all protocols. Regional blood flow measured after 25 I/R cycles was not different among protocols. Our results indicate that NHE inhibition delays the onset and limits the degree of regional dysfunction in response to repeated bouts of ischemia and reperfusion.

ETAReceptor Mediated Inhibition of Intracellular pH Regulation in Cultured Bovine Corneal Epithelial Cells

The contributions were determined in primary cultures of bovine corneal epithelial cells (BCEC) of Na:H exchange (NHE) and vacuolar H+-ATPase (i.e. V-type) activity to the regulation of intracellular pH (pHi). Furthermore, we characterized the effects on pHiregulation of exposure to 1 μmET-1 under control and acid loaded conditions. With the pH sensitive dye, 2′,7′ Bis (carboxyethyl)-5,6-carboxyfluorescein acetoxymethyl ester (BCECF-AM), the control pHiwas 7.1 in NaCl (nominally HCO3-free) Ringers. Inhibition of NHE with 100 μmdimethylamiloride (DMA) rapidly decreased pHiby 0.37 units. Similarly, selective inhibition of V-type H+-ATPase with 10 μmbafilomycin A1decreased pHiby 0.22 units. Following acid loading in NaCl Ringers with a 20 mmNH4Cl prepulse, pHirecovery was partially inhibited by exposure to either Na-free (NMGCl) Ringers, 100 μmDMA or 20 μmbafilomycin A1. Based on decreases in H+efflux resulting from selective inhibition of NHE and V-type H+pump activity, NHE activity accounts for 76% of the pHirecovery following acid loading. Under control conditions, ET-1 (1 μm) had no effect on pHiwhereas ET-1 completely suppressed pHirecovery following acid loading in NaCl or NMGCl Ringers. This inhibitory effect was largely due to stimulation of ETAbecause in the presence of BQ-123 (10 μm), a selective ETAreceptor antagonist, pHirecovery was completely restored. Suppression of pHirecovery also occurred following stimulation of protein kinase C (PKC) with 10−7mphorbol myristate (PMA) whereas 10−7m4 α phorbol 12,13 didecanoate (PDD) had no effect. ET-1 failed to suppress pHirecovery after inhibition of PKC with 0.5 μmcalphostin C suggesting that the inhibition of pHirecovery by ET-1 is a consequence of PKC stimulation. Similarly, inhibition of Ca2+-dependent calmodulin stimulated CaM II kinase with KN-62 (10 μm) reversed the suppression of pHirecovery by ET-1. Preinhibition of either protein phosphatase (PP), PP-1, PP-2A or PP-2B activity with 1 μmphenylarsine oxide, 10 nmokadaic acid, 10 μmcyclosporin A1or 20 μmBAPTA, also obviated the suppression of pHirecovery by ET-1. Therefore ETAreceptor mediated inhibition of pHiregulation following acid loading could be a consequence of either PKC or CaMII kinase stimulation. Each one of these kinases may in turn phosphorylate and thereby stimulate the activities of PP-1, PP-2A or PP-2B. An increase in the activity of any one of these protein phosphatases could lead to dephosphorylation of the NHE and V-type H+pump. This alteration may prevent them from becoming adequately stimulated to elicit pHirecovery in response to acid loading.

A Rapid Ischemia-induced Apoptosis in Isolated Rat Hearts and its Attenuation by the Sodium–Hydrogen Exchange Inhibitor HOE 642 (Cariporide)

Apoptosis is a potentially important myocardial response to pathology including ischemia and reperfusion. Na–H exchange (NHE) represents an important mechanism for mediating such injury. The present study was done to determine if NHE inhibition can affect early apoptosis in an acute model of ischemia and reperfusion. Isolated rat hearts were subjected to zero-flow ischemia for various durations with or without subsequent 30 min of reperfusion. Nick-end-labelling of biotin-dUTP (TUNEL staining), as well as DNA extraction followed by agarose gel electrophoresis, were used to semiquantify apoptotic cells and identify DNA laddering, respectively. Apoptosis first appeared after 10 min of ischemia and reached a maximum level after 30 min. The number of apoptotic cells after 30 min of ischemia was 31 ± 3 per 100 high power microscopic fields, whereas in reperfused hearts the number of cells was 34 ± 3. To determine the effect of NHE inhibition, hearts were pretreated 15 min prior to ischemia with HOE 642, a potent and specific inhibitor of the isoform (NHE-1) found in myocardium. HOE 642 significantly reduced the number of apoptotic cells in the ischemic and reperfused heart to 2 ± 1 and 6 ± 1, respectively (P<0.05 from untreated hearts). DNA laddering was not observed with electrophoretic DNA analysis, likely owing to the small number of apoptotic cells involved. Hearts recovered nearly 100% of function in both groups, although there was a significantly higher recovery after 1 and 2 min of reperfusion in those hearts treated with HOE 642. Our study shows that apoptosis, albeit very mild in nature, can be rapidly induced in isolated hearts by a relatively brief period of ischemia without reperfusion, which can be markedly attenuated by the NHE inhibitor HOE 642. The ability of HOE 642 to markedly attenuate apoptosis may be important in terms of understanding the drug's cardioprotective properties as well as the overall role of NHE in heart disease.

Chloride-dependent Na-H Exchange: A NOVEL MECHANISM OF SODIUM TRANSPORT IN COLONIC CRYPTS

The mechanism of sodium movement across apical membrane of colonic crypt cells of rat distal colon was examined in studies of both 22Na uptake by apical membrane vesicles (AMV) and the rate of intracellular pH (pHi) recovery from an acid load by the addition of lumen sodium. In the presence of chloride but not in its absence, 22Na uptake in crypt AMV was stimulated by an outward gradient of either [H+] or [Na+]. 22Na uptake stimulated by an outward [Na+] gradient was also observed in the presence of other halides in the order of chloride > bromide > fluoride > iodide. pHi recovery from an acid load was both lumen sodium- and chloride-dependent, and the rate of pHi recovery by lumen sodium in the presence of chloride was 65-fold greater than that in the absence of chloride (dpH/dt is 655.4 and 10.2 in the presence and absence of chloride, respectively). One mM amiloride inhibited both [H+] gradient-stimulated 22Na uptake in the presence of chloride in crypt AMV (80%) and lumen sodium- and chloride-dependent pHi recovery in crypt cells (96%). [H+] gradient stimulation of 22Na uptake by crypt AMV in the presence of chloride was less sensitive to amiloride than amiloride inhibition of Na-H exchange in colonic surface AMV. These studies provide compelling evidence that a chloride-dependent Na-H exchange that is relatively amiloride-resistant is present in the apical membrane of colonic crypt cells. As prior studies have not identified a chloride-dependent Na-H exchange, the molecular and functional basis of this novel transport process is not known.

Modulation of Na-H exchange activity by angiotensin II in opossum kidney cells.

Angiotensin II (ANG II) was shown to modulate transport in the renal proximal tubule through both inhibition of adenylate cyclase and protein kinase C (PKC) activation. We evaluated the effects of ANG II on adenosine 3',5'-cyclic monophosphate (cAMP) content and Na-H exchange activity (amiloride-sensitive Na influx) in two strains of opossum kidney (OK) cells originating from different sources, OK-VD and OK-RR cells. In OK-VD cells, ANG II inhibited basal and parathyroid hormone (PTH)-induced cAMP generation in a pertussis toxin-sensitive manner and reversed PTH inhibition of Na-H exchange. These effects of ANG II were prevented by PD 123319, a selective nonpeptide antagonist of AT2 receptors. In contrast, DuP 753, which antagonizes selectively AT1 receptors, had no effect. In OK-RR cells, ANG II had no effect on cAMP content and decreased Na-H exchange activity. The effect of ANG II persisted in the presence of PTH but was abolished by PKC downregulation and by DuP 753, but not by PD 123319. In conclusion, two types of ANG II receptors, coupled to distinct signaling pathways, were expressed independently in OK cells originating from two different sources and mediated opposite effects of ANG II on Na-H exchange activity. Those models provide a powerful tool for studying the intracellular steps involved in the tubular effects of ANG II and to evaluate the effect of pharmacological inhibitors of ANG II binding to its receptors.

Effect of partial Na pump and Na-H exchange inhibition on [Ca]i during acidosis in cardiac cells.

This study investigated the effects of partial Na pump inhibition and Na-H exchange inhibition on contractile function, intracellular pH (pHi), and intracellular Ca concentration ([Ca]i) during intracellular acidosis, using the fluorescent dyes 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein and fura-2 in isolated cardiac cells of adult rabbits. Intracellular acidosis with normal extracellular pH was induced by an NH4Cl (10 mM) prepulse technique. A nontoxic concentration (0.5 microM) of ouabain was used to inhibit the Na pump. 5-(N-ethyl-N-isopropyl)amiloride (EIPA) was used to inhibit Na-H exchange. In both the absence and presence of ouabain, pHi fell transiently and then recovered after removal of NH4Cl. Ouabain did not alter the pHi changes observed after removal of NH4Cl. Diastolic and systolic [Ca]i increased during acidosis after NH4Cl removal. In the presence of ouabain, the increase in [Ca]i during acidosis was greater than that in the absence of this drug. Ouabain enhanced the recovery of contractile function during acidosis. In both the absence and presence of ouabain, Na-H exchange inhibition by EIPA reduced the recovery of pHi and mechanical function and the increase in [Ca]i, which were normally observed after NH4Cl removal. These data suggest that in adult rabbit myocytes the Na pump inhibition enhances the increase in [Ca]i during acidosis, and the Na-H exchange inhibition reduces it. The [Ca]i increase during acidosis may be in part due to the altered Na-Ca exchange, which in turn results from the increased Na-H exchange.

β-Adrenergic and Somatostatin Receptors Regulate Na-H Exchange Independent of cAMP

Abstract Activation of beta-adrenergic and somatostatin receptors increases and attenuates, respectively, cAMP. We have determined, however, that in enteric endocrine cells beta-adrenergic and somatostatin receptors also regulate Na-H exchange activity, independent of their effects on cAMP. In cells loaded with a pH-sensitive dye, epinephrine, acting at a beta 2-adrenergic receptor induced an alkalinization while somatostatin caused an acidification of intracellular pH (pHi). These pHi changes were dependent on extracellular Na+ and inhibited by amiloride. Forskolin, dibutyryl-cAMP and 8-bromo-cAMP, however, had no effect on pHi. Cholera toxin, while decreasing the EC50 for epinephrine-stimulated increases in cAMP, had no effect on epinephrine-induced alkalinization, suggesting receptor coupling to Na-H exchange was not mediated by a cholera toxin-sensitive stimulatory GTP-binding protein (Gs). Additionally, epinephrine stimulated Na-H exchange in cyc- variants of S49 lymphoma cells, which lack a fundamental Gs. In the presence of pertussis toxin, somatostatin attenuation of cAMP was completely reversed; however, somatostatin inhibition of Na-H exchange was not affected. We suggest that beta-adrenergic and somatostatin receptors regulate Na-H exchange independent of changes in cAMP and possibly independent of GTP-binding proteins previously described as being coupled to these receptors.

Endothelin-induced contraction of human peripheral resistance vessels is partly dependent on stimulation of sodium-hydrogen exchange

The role of Na-H exchange in endothelin-induced contraction of human peripheral resistance vessels was investigated. Endothelin produced a dose-dependent contraction which was greatly attenuated in the presence of a low extracellular sodium concentration. Inhibition of Na-H exchange by the amiloride analogue 5-(N,N-hexamethylene) amiloride (5-NNHA) resulted in a greater than 65% relaxation of a maximal endothelin-induced contraction in the presence of normal extracellular sodium. However, in the presence of a low extracellular sodium concentration, inhibition of Na-H exchange only resulted in a 25% relaxation. These data suggest that endothelin-induced vasoconstriction of human peripheral resistance vessels is mediated in part by stimulation of Na-H exchange.

Calcium-mediated cyclic AMP inhibition of Na-H exchange in small intestine.

8-Bromo cyclic AMP (cAMP) (10(-4) M) inhibits Na absorption in isolated chicken enterocytes as has been reported previously. Direct measurements of intracellular pH (pHi) using 5,6-carboxyfluorescein diacetate showed that both 8-bromo cAMP and the diuretic amiloride (10(-3) M) stimulated a persistent decrease in pHi of approximately 0.1 pH units, effects that were Na dependent and were not additive when cells were stimulated with both agents. These results suggest inhibition of an amiloride-sensitive Na/H exchange by cAMP. Direct measurements of intracellular Ca [Ca]i were also made using quin 2. 8-Bromo cAMP (10(-4) M) stimulated an immediate and persistent (greater than 10 min) increase in [Ca]i of approximately 20 nM, an effect that was not dependent on extracellular Ca. Pretreatment of cells with the specific calmodulin inhibitor calmidazolium (10(-7) M) and the intracellular Ca-buffering agent MAPTAM blocked cAMP's effects on pH and Na uptake, but did not interfere with amiloride's effects. An increase in [Ca]i stimulated by the Ca ionophore A23187 (10(-6) M) was sufficient by itself to decrease pHi and inhibit amiloride-sensitive Na influx in isolated enterocytes. We conclude that cAMP stimulates the release of endogenous Ca in isolated enterocytes. This increase in [Ca]i appears to be essential for inhibition of amiloride-sensitive Na-H exchange by this cyclic nucleotide.

Phorbol ester inhibition of Na-H exchange in rabbit proximal colon.

In rabbit proximal colon, in vitro addition of phorbol 12,13-dibutyrate (PDB, 10(-7) M) to the serosal bathing medium inhibits mucosal (m)-to-serosal (s) unidirectional Na flux (JsmNa) without altering JsmNa or unidirectional Cl fluxes. Similar results were obtained when amiloride (2 X 10(-4) M) was added to the mucosal bathing medium. No additivity of effect was seen when tissues were exposed to both agents. Measurements with carboxyfluorescein reveal that the two agents cause equal decreases of intracellular pH (pHi), an effect that is dependent on the presence of extracellular Na (Na replacement also decreases pHi). No additivity of pHi effects is seen when both agents are added together. To determine the membrane site of this PDB-inhibitable Na-H exchange, Na influx across the luminal border of proximal colon was measured and was found to be inhibited equally by PDB and amiloride. We conclude that PDB, by activation of protein kinase C, inhibits electro-neutral amiloride-sensitive Na-H exchange in the luminal membrane of proximal colon.

Ion transport in rabbit ileal mucosa. I. Na and Cl fluxes and short-circuit current.

Ion transport in rabbit ileal mucosa. I. Na and Cl fluxes and short-circuit current.