A portable shield for a neutron howitzer used for instructional and research purposes.

Neutron howitzers are routinely used in universities to activate samples for instructional laboratory experiments on radioactivity. They are also a convenient source of neutrons and gammas for research purposes, but they must be used with caution. This paper describes the modeling, design, construction, and testing of a portable, economical shield for a 1.0 Curie neutron howitzer. The Monte Carlo N Particle Transport Code (MCNP5) has been used to model the (239)PuBe source and the howitzer and to design the external neutron and gamma shield.

Physiology and pathophysiology of Na+/H+ exchange and Na+ -K+ -2Cl- cotransport in the heart, brain, and blood.

Maintenance of a stable cell volume and intracellular pH is critical for normal cell function. Arguably, two of the most important ion transporters involved in these processes are the Na+/H+ exchanger isoform 1 (NHE1) and Na+ -K+ -2Cl- cotransporter isoform 1 (NKCC1). Both NHE1 and NKCC1 are stimulated by cell shrinkage and by numerous other stimuli, including a wide range of hormones and growth factors, and for NHE1, intracellular acidification. Both transporters can be important regulators of cell volume, yet their activity also, directly or indirectly, affects the intracellular concentrations of Na+, Ca2+, Cl-, K+, and H+. Conversely, when either transporter responds to a stimulus other than cell shrinkage and when the driving force is directed to promote Na+ entry, one consequence may be cell swelling. Thus stimulation of NHE1 and/or NKCC1 by a deviation from homeostasis of a given parameter may regulate that parameter at the expense of compromising others, a coupling that may contribute to irreversible cell damage in a number of pathophysiological conditions. This review addresses the roles of NHE1 and NKCC1 in the cellular responses to physiological and pathophysiological stress. The aim is to provide a comprehensive overview of the mechanisms and consequences of stress-induced stimulation of these transporters with focus on the heart, brain, and blood. The physiological stressors reviewed are metabolic/exercise stress, osmotic stress, and mechanical stress, conditions in which NHE1 and NKCC1 play important physiological roles. With respect to pathophysiology, the focus is on ischemia and severe hypoxia where the roles of NHE1 and NKCC1 have been widely studied yet remain controversial and incompletely elucidated.

Age-related differences in Na+-dependent Ca2+ accumulation in rabbit hearts exposed to hypoxia and acidification.

In this study, we test the hypothesis that in newborn hearts (as in adults) hypoxia and acidification stimulate increased Na(+) uptake, in part via pH-regulatory Na(+)/H(+) exchange. Resulting increases in intracellular Na(+) (Na(i)) alter the force driving the Na(+)/Ca(2+) exchanger and lead to increased intracellular Ca(2+). NMR spectroscopy measured Na(i) and cytosolic Ca(2+) concentration ([Ca(2+)](i)) and pH (pH(i)) in isolated, Langendorff-perfused 4- to 7-day-old rabbit hearts. After Na(+)/K(+) ATPase inhibition, hypoxic hearts gained Na(+), whereas normoxic controls did not [19 +/- 3.4 to 139 +/- 14.6 vs. 22 +/- 1.9 to 22 +/- 2.5 (SE) meq/kg dry wt, respectively]. In normoxic hearts acidified using the NH(4)Cl prepulse, pH(i) fell rapidly and recovered, whereas Na(i) rose from 31 +/- 18.2 to 117.7 +/- 20.5 meq/kg dry wt. Both protocols caused increases in [Ca](i); however, [Ca](i) increased less in newborn hearts than in adults (P < 0.05). Increases in Na(i) and [Ca](i) were inhibited by the Na(+)/H(+) exchange inhibitor methylisobutylamiloride (MIA, 40 microM; P < 0.05), as well as by increasing perfusate osmolarity (+30 mosM) immediately before and during hypoxia (P < 0.05). The data support the hypothesis that in newborn hearts, like adults, increases in Na(i) and [Ca](i) during hypoxia and after normoxic acidification are in large part the result of increased uptake via Na(+)/H(+) and Na(+)/Ca(2+) exchange, respectively. However, for similar hypoxia and acidification protocols, this increase in [Ca](i) is less in newborn than adult hearts.

Hypertonic perfusion inhibits intracellular Na and Ca accumulation in hypoxic myocardium.

Much evidence supports the view that hypoxic/ischemic injury is largely due to increased intracellular Ca concentration ([Ca](i)) resulting from 1) decreased intracellular pH (pH(i)), 2) stimulated Na/H exchange that increases Na uptake and thus intracellular Na (Na(i)), and 3) decreased Na gradient that decreases or reverses net Ca transport via Na/Ca exchange. The Na/H exchanger (NHE) is also stimulated by hypertonic solutions; however, hypertonic media may inhibit NHE's response to changes in pH(i) (Cala PM and Maldonado HM. J Gen Physiol 103: 1035-1054, 1994). Thus we tested the hypothesis that hypertonic perfusion attenuates acid-induced increases in Na(i) in myocardium and, thereby, decreases Ca(i) accumulation during hypoxia. Rabbit hearts were Langendorff perfused with HEPES-buffered Krebs-Henseleit solution equilibrated with 100% O(2) or 100% N(2). Hypertonic perfusion began 5 min before hypoxia or normoxic acidification (NH(4)Cl washout). Na(i), [Ca](i), pH(i), and high-energy phosphates were measured by NMR. Control solutions were 295 mosM, and hypertonic solutions were adjusted to 305, 325, or 345 mosM by addition of NaCl or sucrose. During 60 min of hypoxia (295 mosM), Na(i) rose from 22+/-1 to 100+/-10 meq/kg dry wt while [Ca](i) rose from 347+/-11 to 1,306+/-89 nM. During hypertonic hypoxic perfusion (325 mosM), increases in Na(i) and [Ca](i) were reduced by 65 and 60%, respectively (P<0.05). Hypertonic perfusion also diminished Na uptake after normoxic acidification by 87% (P<0.05). The data are consistent with the hypothesis that mild hypertonic perfusion diminishes acid-induced Na accumulation and, thereby, decreases Na/Ca exchange-mediated Ca(i) accumulation during hypoxia.

Malignant gliomas display altered pH regulation by NHE1 compared with nontransformed astrocytes.

Malignant gliomas exhibit alkaline intracellular pH (pH(i)) and acidic extracellular pH (pH(e)) compared with nontransformed astrocytes, despite increased metabolic H(+) production. The acidic pH(e) limits the availability of HCO(-)(3), thereby reducing both passive and dynamic HCO(-)(3)-dependent buffering. This implies that gliomas are dependent upon dynamic HCO(-)(3)-independent H(+) buffering pathways such as the type 1 Na(+)/H(+) exchanger (NHE1). In this study, four rapidly proliferating gliomas exhibited significantly more alkaline steady-state pH(i) (pH(i) = 7.31-7.48) than normal astrocytes (pH(i) = 6.98), and increased rates of recovery from acidification, under nominally CO(2)/HCO(-)(3)-free conditions. Inhibition of NHE1 in the absence of CO(2)/HCO(-)(3) resulted in pronounced acidification of gliomas, whereas normal astrocytes were unaffected. When suspended in CO(2)/HCO(-)(3) medium astrocyte pH(i) increased, yet glioma pH(i) unexpectedly acidified, suggesting the presence of an HCO(-)(3)-dependent acid loading pathway. Nucleotide sequencing of NHE1 cDNA from the gliomas demonstrated that genetic alterations were not responsible for this altered NHE1 function. The data suggest that NHE1 activity is significantly elevated in gliomas and may provide a useful target for the development of tumor-selective therapies.

Na-dependent changes in intracellular Ca in spontaneously hypertensive rat hearts.

To determine whether Na/Ca exchange is altered in primary hypertension, Na-dependent changes in intracellular Ca, ([Ca]i), were measured in isolated perfused hearts from Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats. Intracellular Na, (Nai, mEq/kg dry wt), and [Ca]i were measured by NMR spectroscopy. Control [Ca]i was less in WKY than SHR (176 +/- 18 vs 253 +/- 21 nmol/l; mean +/- S.E., P < 0.05), whereas Nai was not significantly different. One explanation for this is that net Na/Ca exchange flux is decreased in SHR. If this hypothesis is correct, the rate of Ca uptake in SHR should be less than WKY when Na/Ca exchange is reversed by decreasing the transmembrane Na gradient. The Na gradient was reduced by decreasing extracellular Na, ([Na]o) and/or by increasing [Na]i. To increase [Na]i, Na uptake was stimulated by acidification while Na extrusion by Na/K ATPase was inhibited by K-free perfusion. Seventeen minutes after acidification, Nai had increased but was not significantly different in SHR and WKY (18.0 +/- 2.3 to 57.4 +/- 7.6 vs 20.3 +/- 0.6 to 66.5 +/- 4.8 mEq/kg dry wt, respectively). Yet [Ca]i was greater in WKY than SHR (1768 +/- 142 vs 1201 +/- 90 nmol/l; P < 0.05). [Ca]i was also measured after decreasing [Na]o from 141 to 30 mmol/l. Fifteen minutes after reducing [Na]o, [Ca]i was greater in WKY than SHR (833 +/- 119 vs 425 +/- 94 nmol/l; P < 0.05). Thus for both protocols, decreasing the transmembrane Na gradient led to increased [Ca]i in both SHR and WKY, but less increase in SHR. The results are consistent with the hypothesis that Na/Ca exchange activity is less in SHR than WKY myocardium.

Cloning and expression of the Na+/H+ exchanger from Amphiuma RBCs: resemblance to mammalian NHE1.

The cDNA encoding the Na+/H+ exchanger (NHE) from Amphiuma erythrocytes was cloned, sequenced, and found to be highly homologous to the human NHE1 isoform (hNHE1), with 79% identity and 89% similarity at the amino acid level. Sequence comparisons with other NHEs indicate that the Amphiuma tridactylum NHE isoform 1 (atNHE1) is likely to be a phylogenetic progenitor of mammalian NHE1. The atNHE1 protein, when stably transfected into the NHE-deficient AP-1 cell line (37), demonstrates robust Na+-dependent proton transport that is sensitive to amiloride but not to the potent NHE1 inhibitor HOE-694. Interestingly, chimeric NHE proteins constructed by exchanging the amino and carboxy termini between atNHE1 and hNHE1 exhibited drug sensitivities similar to atNHE1. Based on kinetic, sequence, and functional similarities between atNHE1 and mammalian NHE1, we propose that the Amphiuma exchanger should prove to be a valuable model for studying the control of pH and volume regulation of mammalian NHE1. However, low sensitivity of atNHE1 to the NHE inhibitor HOE-694 in both native Amphiuma red blood cells (RBCs) and in transfected mammalian cells distinguishes this transporter from its mammalian homologue.

Ischemic preconditioning: effects on pH, Na and Ca in newborn rabbit hearts during Ischemia/Reperfusion.

In adult hearts, ischemic preconditioning (PC) has been shown to decrease ischemia-induced changes in intracellular pH (pHi) and [Ca] ([Ca]i) and decrease associated injury. These results are consistent with the interpretation that PC decreases the stimulus for Na uptake via Na/H exchange, thereby decreasing intracellular Na (Nai) accumulation, and thus decreasing the change in force driving Na/Ca exchange, which otherwise contributes to ischemia-induced increases in [Ca]i. Given documented age-related differences in myocardial responses to ischemia, we tested the hypothesis that in newborn hearts, PC will diminish intracellular [H], Nai, and [Ca]i during ischemia/reperfusion. NMR was used to measure pHi, Nai, [Ca]i, ATP, and PCr in isolated newborn (4-7 days) rabbit hearts Langendorff-perfused with Krebs-Henseleit solution equilibrated with 95% O2/5% CO2 at 36+/-1 degrees C. Control hearts were perfused 30 min before initiating 40 min global ischemia followed by 40 min reperfusion. PC hearts were treated the same except four 5-min intervals of ischemia each followed by 10 min of perfusion which preceded global ischemia. At end ischemia, pHi was higher in PC than control hearts (6.31+/-0.03 v 5.83+/-0.05; P<0.05). Similarly, PC diminished Nai-accumulation during ischemia and reperfusion (P<0.05). Control Nai rose from 16.2+/-2.6 to 108.8+/-10.3 (mEq/kg dry weight) and recovered to 55.2+/-10.1 and the corresponding values for PC hearts were 25.6+/-6.2, 70.0+/-7.9 and 21.9+/-5.2. PC also improved [Ca]i recovery during reperfusion (P<0.05). Control [Ca]i rose from 418+/-43 to 1100+/-78 (nm/l) and recovered to 773+/-63, whereas in PC hearts the values were 382+/-40, 852+/-136 and 371+/-45, respectively. In addition, PC decreased coronary resistance during reperfusion (P<0.05) as reflected by lower perfusion pressures under constant flow conditions (65.9+/-1.5 v 56. 1+/-4.1 mmHg at end of reperfusion). Finally, PC improved recovery of left-ventricular developed pressure (LVDP-43.8+/-12.0 v 17.2+/-3. 0% of control; P<0.05) and diminished CK release (607+/-245 v 2432+/-639 IU/g dry weight; P<0.05) during reperfusion. The results are consistent with the hypothesis.

Ethylisopropylamiloride diminishes changes in intracellular Na, Ca and pH in ischemic newborn myocardium.

Numerous studies suggest that in adult hearts myocardial ischemic injury is in part the result of proton stimulation of Na/H exchange which increases intracellular Na (Nai) and thus leads to increases in intracellular Ca concentration ( [Ca]i) due to changes in Na/Ca exchange flux. Corollary to the hypothesis, inhibition of Na/H exchange diminishes Na and Ca accumulation and improves heart function after ischemia. To test this hypothesis and its corollary in newborn hearts, NMR spectroscopy was used to measure intracellular pH (pHi), Nai, [Ca]i, and high energy phosphates in isolated, 4-7-day-old rabbit hearts, Langendorff-perfused with Krebs-Henseleit solution at pH 7.4+/-0.5 equilibrated with 95% O2/5% CO2 at 36+/-1 degrees C. Control hearts were perfused for 30 min before initiating 40 min of global ischemia followed by 40 min of reperfusion. In a second group of hearts ethylisopropylamiloride (EIPA-10 microM) was added to the perfusate 20 min before global ischemia to inhibit Na/H exchange. After 15 min ischemia, pHi in EIPA-treated hearts (6.41+/-0.04) was higher than that of the control hearts (6.20+/-0.08; P<0.05). EIPA also limited the increase in Nai and [Ca]i during ischemia and improved Nai and [Ca]i recovery during reperfusion (P<0.05). Nai (mEq/kg dry weight) rose from 18. 1+/-3.2 to 110.6+/-14.0 and recovered to 53.3+/-12.3 in the control group. The corresponding Nai values for EIPA-treated hearts were 16. 2+/-2.4, 39.6+/-9.6 and 12.6+/-3.5, respectively. In control hearts [Ca]i (nM/l) rose from 332+/-42 to 1157+/-89 and recovered to 842+/-55, whereas in EIPA-treated hearts the values were 255+/-32, 616+/-69 and 298+/-34, respectively. EIPA also preserved cellular ATP during ischemia and reperfusion and diminished inorganic phosphate during reperfusion (P<0.05). Finally, EIPA treatment improved recovery of left ventricular developed pressure (68.2+/-8.9 v 16.2+/-3.6% of control) and limited myocardial injury as indicated by decreased total creatine kinase release during reperfusion (348+/-132 v 2432+/-639 IU/g dry weight). Thus, as in adults, the results from newborn hearts are consistent with the hypothesis.

Effects of Na-K-2Cl cotransport inhibition on myocardial Na and Ca during ischemia and reperfusion.

In the context of the "pump-leak" hypothesis (37), changes in myocardial intracellular Na (Nai) during ischemia and reperfusion have historically been interpreted to be the result of changes in Na efflux via the Na-K pump. We investigated the alternative hypothesis that changes in Nai during ischemia are the result of changes in the Na "leak" rather than changes in the pump. More specifically, we hypothesize that the increase in Nai during ischemia is in part the result of increased Na uptake mediated by Na/H exchange. Furthermore, we present data consistent with the interpretation that the Na-K-2Cl cotransporter is active (or, alternatively, displaced from equilibrium) during ischemia and may contribute an additional Na efflux pathway during reperfusion. Thus inhibition of Na efflux via Na-K-2Cl cotransport during ischemia and reperfusion could result in increased Nai and therefore decreased force driving Ca efflux via Na/Ca exchange and ultimately increased intracellular Ca concentration ([Ca]i). Nai (in meq/kg dry wt) and [Ca]i (in nM) were measured in isolated Langendorff-perfused rabbit hearts using nuclear magnetic resonance spectroscopy. Except, during the 65 min of ischemia, hearts were perfused with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-buffered Krebs-Henseleit solution equilibrated with 100% O2 at 23 degrees C and pH 7.4 +/- 0.05. During ischemia, Nai rose from 16.6 +/- 0.3 to 62.9 +/- 5.1 (delta Nai approximately 46) meq/kg dry wt and decreased during subsequent reperfusion (mean +/- SE, n = 3 hearts). To measure Na uptake ("leak") in the absence of efflux via the Na-K pump, in all of the protocols described below, the perfusate was nominally K-free solution containing 1 mM ouabain for 10 min before ischemia and during the 30-min reperfusion. After K-free perfusion, Nai rose from 20.2 +/- 0.5 to 79.1 +/- 5.3 (delta Nai approximately 59) meq/kg dry wt (n = 3) during ischemia and decreased during K-free reperfusion. When amiloride (1 mM) was added to the K-free perfusate to inhibit Na/H exchange, Nai rose from 16.3 +/- 0.9 to 44.7 +/- 5.1 (delta Nai approximately 28) meq/kg dry wt (n = 3) during ischemia; i.e., amiloride decreased Na uptake. When bumetanide (20 microM) was added to the nominally K-free perfusate to inhibit Na-K-2Cl contransport, Nai rose from 22.5 +/- 3.9 to 83.8 +/- 13.9 (delta Nai approximately 61 meq/kg dry wt (n = 3) during ischemia and did not decrease during reperfusion; i.e., bumetanide inhibited Na recovery during reperfusion (P < 0.05 compared with bumetanide free). For the same protocol, the presence of bumetanide resulted in increased [Ca]i during ischemia and reperfusion (P < 0.05); these increases in [Ca]i are interpreted to be the result of increased Nai. Thus the results are consistent with the hypotheses.

Mechanisms of regulatory volume decrease in nonpigmented human ciliary epithelial cells.

To study the net solute and water efflux pathways of the ciliary epithelium we employed a cultured human NPE cell line. Because of the possible relationship between transepithelial ion and water flux and cell volume regulation, the ion efflux pathways mediating regulatory volume decrease (RVD) were investigated. Osmotic swelling of NPE cells was followed by a volume recovery. Volume recovery was K+ dependent and inhibited by K+ channel blockers such as quinine (1 mM). After osmotic swelling, a Cl(-)-dependent membrane depolarization occurred that was inhibited by Cl- channel blockers such as 5-nitro-2-(3-phenylpropylamino)benzoic acid (100 microM) or Ca2+ chelators such as ethylene glycolbis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA, 2.0 mM). Cell swelling was also accompanied by an increase in intracellular Ca2+ concentration ([Ca2+]i) of approximately 200 nM. The swelling-induced rise in [Ca2+]i and RVD were diminished in the presence of 10 microM La3+, 50 nM 12-O-tetradecanoylphorbol 13-acetate, and nominally Ca(2+)-free medium. Near total blockage of RVD occurred after pretreatment of NPE cells with Ca(2+)-free EGTA-1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) acetoxymethyl ester-containing solutions. The inhibition of RVD by EGTA-BAPTA treatment was overcome by increasing K+ conductance with gramicidin. The above findings indicate that RVD in NPE cells is mediated by separate K+ and Cl- conductances (channels). These data also show that swelling-induced increases in [Ca2+]i help modulate net ion efflux during regulation.

Labeling of the Amphiuma erythrocyte K+/H+ exchanger with H2DIDS.

Subsequent to swelling, the Amphiuma red blood cells lose K+, Cl-, and water until normal cell volume is restored. Net solute loss is the result of K+/H+ and Cl-/HCO3- exchangers functionally coupled through changes in pH and therefore HCO3-. Whereas the Cl-/HCO3- exchanger is constitutively active, K+/H+ actively is induced by cell swelling. The constitutive Cl-/HCO3- exchanger is inhibited by low concentrations (< 1 microM) of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) or H2DIDS, yet the concentration of H2DIDS > 25 microM irreversibly modifies the K+/H+ exchanger in swollen cells. We exploited the volume-dependent irreversible low-affinity reaction between H2DIDS and the K+/H+ to identify the protein(s) associated with K+/H+ exchange activity. Labeling of the membrane proteins of intact cells with 3H2DIDS results in high-affinity labeling of a broad 100-kDa band, thought to be the anion exchanger. Additional swelling-dependent low-affinity labeling at 110 kDa suggests the possibility of a volume-induced population of anion exchangers. Finally, the correlation between volume-sensitive K+/H+ modification and low-affinity labeling suggests that transport activity is associated with a protein of approximately 85 kDa. Although a 55-kDa protein is also labeled, it is a less likely candidate, since label incorporation and transport modification are less well correlated than that of the 85- and 110-kDa proteins.

pH regulatory Na/H exchange by Amphiuma red blood cells.

In Amphiuma red blood cells, the Na/H exchanger has been shown to play a central role in the regulation of cell volume following cell shrinkage (Cala, P. M. 1980. Journal of General Physiology. 76:683-708.) The present study was designed to evaluate the existence of pH regulatory Na/H exchange in the Amphiuma red blood cell. The data illustrate that when the intracellular pHi was decreased below the normal value of 7.00, Na/H exchange was activated in proportion to the degree of acidification. Once activated, net Na/H exchange flux persisted until normal intracellular pH (6.9-7.0) was restored, with a half time of approximately 5 min. These observations established a pHi set point of 7.00 for the pH-activated Na/H exchange of Amphiuma red blood cell. This is in contrast to the behavior of osmotically shrunken Amphiuma red blood cells in which no pHi set point could be demonstrated. That is, when activated by cell shrinkage the Na/H exchange mediated net Na flux persisted until normal volume was restored regardless of pHi. In contrast, when activated by cell acidification, the Na/H exchanger functioned until pHi was restored to normal and cell volume appeared to have no effect on pH-activated Na/H exchange. Studies evaluating the kinetic and inferentially, the molecular equivalence of the volume and pHi-induced Amphiuma erythrocyte Na/H exchanger(s), indicated that the apparent Na affinity of the pH activated cells is four times greater than that of shrunken cells. The apparent Vmax is also higher (two times) in the pH activated cells, suggesting the involvement of two distinct populations of the transporter in pH and volume regulation. However, when analyzed in terms of a bisubstrate model, the same data are consistent with the conclusion that both pH and volume regulatory functions are mediated by the same transport protein. Taken together, these data support the conclusion that volume and pH are regulated by the same effector (Na/H exchanger) under the control of as yet unidentified, distinct and cross inhibitory volume and pH sensing mechanisms.

Cell volume and pH regulation by the Amphiuma red blood cell: a model for hypoxia-induced cell injury.

The Amphiuma red blood cell is one of the model systems employed early in the study of vertebrate cell volume regulation. Following both cell swelling and shrinkage the Amphiuma red blood cell demonstrates volume regulation to virtual completion in 90-120 min. When swollen the Amphiuma red blood cell loses K, Cl and osmotically obliged water, while following shrinkage volume regulation is the result of Na, Cl and therefore water uptake. The main contribution of the Amphiuma red cell as a model is that it was the first cell in which volume regulation was demonstrated to be electroneutral and more specifically that K/H and Na/H exchangers were responsible for regulation following cell swelling and shrinkage, respectively. Additionally, the Amphiuma red blood cell K/H and Na/H exchangers have been demonstrated to function in a pH regulatory capacity. The latter observation in turn led to the demonstration of the mutually exclusive and contradictory nature of volume and pH regulation predicted upon Na/H exchanger activity. These observations prompted our recent investigations of the Na/H exchanger as a contributor to hypoxia-induced cell damage, using the rabbit heart as a model. These studies illustrated that Na, and Ca imbalances characteristic of hypoxia-induced cell damage are ultimately referable to the Na/H exchanger's function in a pH regulatory capacity, which contributes fundamentally to cell volume and Ca derangement and ultimately cell injury.

Na-H exchange in myocardium: effects of hypoxia and acidification on Na and Ca.

Historically, increase in cell Na content during ischemic and hypoxic episodes were thought to result from impaired ATP production causing decreased Na(+)-K(+)-ATPase activity. Here we report the results of testing the alternate hypothesis that hypoxia-induced Na uptake is 1) the result of increased entry, as opposed to decreased extrusion 2) via Na-H exchange operating in a pH regulatory capacity and that cell Ca accumulation occurs via Na-Ca exchange secondary to collapse of the Na gradient. We used 23Na-, 19F-, and 31P-nuclear magnetic resonance (NMR) to measure intracellular Na content (Nai), Ca concentration [( Ca]i), pH (pHi), and high-energy phosphates in Langendorff-perfused rabbit hearts. When the Na(+)-K(+)-ATPase was inhibited by ouabain and/or K-free perfusion, hearts subjected to hypoxia gained Na at a rate greater than 10 times that of normoxic controls [during the first 12.5 min Nai increased from 7.9 +/- 5.8 to 34.9 +/- 11.0 (SD) meq/kg dry wt compared with 11.1 +/- 16.3 to 13.6 +/- 9.0 meq/kg dry wt, respectively]. When normoxic hearts were acidified using a 20 mM NH4Cl prepulse technique, pHi rapidly fell from 7.27 +/- 0.24 to 6.63 +/- 0.12 but returned to 7.07 +/- 0.10 within 20 min, while Na uptake was similar in rate and magnitude to that observed during hypoxia (24.5 +/- 13.4 to 132.1 +/- 17.7 meq/kg dry wt). During hypoxia and after NH4Cl washout, increases in [Ca]i were similar in time course to those observed for Na.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulatory volume decrease in alveolar macrophages: cation loss is not correlated with changes in membrane recycling.

Alveolar macrophages regain their normal volume after swelling in hypo-osmotic solutions. This process, termed regulatory volume decrease (RVD), is initiated 3-5 minutes after exposure of cells to hypo-osmotic solutions, and by 30 min, near-normal volumes are attained. Volume decrease does not occur at 0 degrees C or in solutions in which Na+ has been replaced by K+, or Cl- by the impermeant anion gluconate. These results, as well as direct measurement of intracellular cations, indicate that decreases in cell volume result primarily from the loss of K+ and Cl- and are similar to RVD in lymphocytes. Kinetic analysis of cation loss, both by directly measuring changes in intracellular cation content and by assaying rubidium efflux, showed that cation loss occurred immediately upon media dilution. The rate of cation loss fit first-order kinetics and preceded both the initiation of volume decrease and the maximum increase in surface receptor number. These results suggest that the cation transporters responsible for RVD are located at the cell surface and that regulation of activity is not dependent on alterations in membrane movement.

Dynamic NMR measurement of volume regulatory changes in Amphiuma RBC Na+ content.

23Na nuclear magnetic resonance (NMR) and conventional chemical methods were employed to measure Na+ fluxes in Amphiuma red blood cells (RBC) during volume regulation. Paramagnetic shift reagents [dysprosium triethylenetetraminehexaacetic acid (DyTTHA) and dysprosium tripolyphosphate (Dy(TPP)2)] were used to alter extracellular Na+ magnetic resonance. Data are presented describing 23Na resonance dependence on shift reagent, sodium and calcium concentration. We confirmed that the shift reagents neither enter the cells nor alter intracellular Na+, K+, and Cl-concentrations under control conditions when extracellular calcium was maintained greater than 0.5 mM. We also confirmed that the shift reagent complexes chelate calcium [Dy(TPP)2 much more so than DyTTHA] and that their toxic effects could be alleviated by adjusting calcium in the cell's suspension medium to control levels. In parallel experiments, where volume-activated Na+ fluxes ranged from 0.3 to 3 mmol Na+/kg dry cell solid (DCS) x minute in cells containing from 30 to 150 mmol Na+/kg DCS, changes in intracellular sodium measured by 32Na NMR were within 4% of those measured by conventional destructive methods. Finally, we present data that are consistent with the interpretation that 6 mmol Na+/kg DCS plus 16% of intracellular Na+ is NMR invisible.

Na/H exchange-dependent cell volume and pH regulation and disturbances.

1. The role of Na/H exchange in cell volume and pH regulation is discussed. In addition the roles of Cl/HCO3 exchange and system buffers are evaluated as they relate to Na/H exchange-dependent changes in cell salt and water content and intracellular pH. 2. Data obtained from studies of Amphiuma red blood cells showed that in addition to previously reported Na/H exchange dependent volume regulation the pathway is also involved in regulating cell pH. 3. These data showed that in contrast to volume activated Na/H exchange, when the pathway is pH activated it does not deactivate as a function of cell volume. 4. Given what appeared to be mutually exclusive volume and pH regulatory functions of the Na/H exchange, we hypothesized that the pathway might play a role in hypoxic cell swelling (cytotoxic edema). 5. In studies performed on perfused rabbit hearts employing 23Na NMR we were able to observe that relative to normoxic controls hypoxic hearts exhibited a five-fold increase in intracellular Na content when the Na-K pump was inhibited by ouabain and/or K-free perfusate. 6. These studies lead us to conclude that hypoxia-induced Na uptake is the result of an increased inward Na leak as opposed to decreased Na pumping. 7. Based upon studies with a variety of inhibitors of dissipative Na transport, we conclude that the increased inward Na leak in hypoxic hearts is via Na/H exchange.