Studies on the role of angiotensin in experimental renovascular hypertension: an immunologic approach.

The role of angiotensin in three forms of experimental hypertension was assessed in rats. First, the acute blood pressure response to injected angiotensin amide and angiotensin acid was determined. Rats made hypertensive with deoxycorticosterone and saline showed exaggerated responses; rats made hypertensive by clipping one renal artery showed depressed responses; and rats made hypertensive by clipping one renal artery and contralateral nephrectomy showed normal responsivity to angiotensin amide but depressed responsivity to angiotensin acid. These findings suggested that different mechanisms may be involved in the three types of hypertension studied. To assess the role of angiotensin in these hypertensive rats the blood pressure response, the presence of antibodies determined by radioimmune techniques, and the degree of refractoriness to injected angiotensin after immunization with angiotensin were studied. None of six rats made hypertensive by deoxycorticosterone and saline, and none of five mock immunized rats with renal hypertension of both types had a fall in blood pressure. By contrast, of the 20 rats with both types of renal hypertension in which antibody determinations were made, 11 had developed a significant antibody titer, of which seven showed a significant reduction in blood pressure at the time of antibody determination, and three of the remaining four had a significant blood pressure reduction earlier in their course. None of the nine renal hypertensive rats without demonstrable antibodies had a reduced blood pressure at the time of antibody determination, and only one had an earlier reduction in blood pressure. The renal hypertensive rats were all refractory to injected angiotensin after immunization. These results suggest a primary role for angiotensin in both forms of renal hypertension.

pH modulates cAMP-induced increase in Na+ transport across frog skin epithelium.

Apical membrane potential (Va), fractional apical membrane resistance (FRa), and/or intracellular pH (pHi) were measured in principal cells of isolated frog (Rana pipiens) skin with microelectrodes under short-circuit conditions. Apical exposure to 0.33 mM 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (cAMP) depolarized Va, decreased FRa and increased short-circuit current (Isc). cAMP-induced 50% larger effects on Va and Isc at external pH (pHo) of 8.0 than at pHo 6.4. Increasing pHo from 6.4 to 8.0 in presence of cAMP further depolarized Va and increased Isc. cAMP-induced effects on Va and Isc were observed in the absence of Cl- and HCO3- and in the presence of 1 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) or 10 microM 5-(N-ethyl-N-isopropyl)amiloride (EIPA) or 1 microM 5-(N-methyl-N-isobutyl)amiloride (MIA). These data indicate that Na(+)-H+ exchange, Cl(-)-HCO3- exchange, and electrogenic Na(+)-(HCO3-)n cotransport are not involved in cAMP-induced increase in Isc. Apical exposure to 2 mM Cd2+ or Zn2+ depolarized Va, decreased FRa, increased Isc and increased pHi. In HCO(3-)-free solutions containing DIDS, unilateral replacement of apical Cl- by NO3- induced a fast transient depolarization of Va and an increase in Isc. These data suggest that potential-dependent changes in pHi are involved in increases in Isc. However, when changes in Va were minimized by pretreating the basolateral membrane with 25 or 75 mM K+, the cAMP-induced increase in Isc was not blocked. These data indicate that changes in pHi do not play a strict regulatory role but are only permissive in cAMP-induced effects on Isc.

Regulation of apical Na+ conductive transport in epithelia by pH.

Alterations in extracellular (pHo) and/or intracellular pH (pHi) have significant effects on the apical Na+ conductive transport in tight epithelia. They influence apical membrane Na+ conductance via a direct effect on amiloride-sensitive apical Na+ channel activity and indirectly through effects on the basolateral Na+/K(+)-ATPase. Changes in pH also modulate the hormonal regulation of apical Na+ conductive transport. The pH sensitive steps in hormone action include: (i) hormone-receptor binding, (ii) increase in intracellular cyclic 3',5'-adenosine monophosphate (cAMP), (iii) mobilization of intracellular free Ca2+ ([Ca2+]i), and (iv) incorporation of new channels into the apical membrane or recruitment of existing channels. Alternately, changes in pH induce secondary effects via alterations in [Ca2+]i. A reciprocal relationship between pHi and [Ca2+]i has been demonstrated in renal epithelial cells. Natriferic hormones induce a significant increase in pHi. There is a strong temporal relation between hormone-induced increase in pHi and overall increase in transepithelial Na+ transport. This suggests that changes in pHi act as an intermediate in the second messenger cascade initiated by the hormones. Several natriferic hormones activate Na(+)-H+ exchanger, H(+)-ATPase, H+/K(+)-ATPase, H+ conductive pathways in cell membranes or potential-induced changes in pHi. However, changes in pHi do not seem to be essential for the hormone effect on Na+ conductive transport. It is suggested that the role of pHi changes during hormone action is permissive rather than strictly obligatory.

Potential dependence of unidirectional chloride fluxes across isolated frog skin.

Isolated frog skins were voltage clamped at transepithelial potentials (Vt) ranging from -60 mV to 60 mV to measure transepithelial 36Cl- fluxes from the apical to the basolateral bathing solution (J13) and in the opposite direction (J31). The potential dependence of fluxes obtained in Na+-free choline Ringer's indicates the presence of conductive and nonconductive components that probably correspond to fluxes through paracellular and cellular pathways, respectively. Rectification of fluxes with reversal of the potential reflects a structural asymmetry, presumably in surface charge density. The data are consistent with a charge density of one negative charge per 280 A2 on the apical side. A new model for passive Cl- transport was developed that includes surface charge asymmetry and specifically accounts for the observed variation of conductance with potential. In normal frog Ringer's, J13 was larger than J31 at zero potential (active Cl- transport), J13 rose exponentially with increasing positive potential to reach a maximum at 40 mV (approximately open-circuit), and the predicted partial Cl- conductance exceeded the measured conductance leading to the conclusion that when J13 is largely driven by Na+ transport, much of the coupling occurs via nonconductive pathways. Theophylline stimulates Cl- transport that also occurs via nonconductive pathways as Vt becomes more positive.

The identity of the current carriers in canine lingual epithelium in vitro.

Ion transport across the lingual epithelium has been implicated as an early event in gustatory transduction. The fluxes of isotopically labelled Na+ and Cl- were measured across isolated canine dorsal lingual epithelium under short-circuit conditions. The epithelium actively absorbs Na+ and to a lesser extent actively secretes Cl-. Under symmetrical conditions with Krebs-Henseleit buffer on both sides, (1) Na+ absorption accounts for 46% of the short-circuit current (Isc); (2) there are two transcellular Na+ pathways, one amiloride-sensitive and one amiloride-insensitive; (3) ouabain, added to the serosal solution, inhibits both Isc and active Na+ absorption. When hyperosmotic (0.25 M) NaCl is placed in the mucosal bath, both Isc and Na+ absorption increase; net Na+ absorption is at least as much as Isc. Ion substitution studies indicate that the tissue may transport a variety of larger ions, though not as effectively as Na+ and Cl-. Thus we have shown that the lingual epithelium, like other epithelia of the gastrointestinal tract, actively transports ions. However, it is unusual both in its response to hyperosmotic solutions and in the variety of ions that support a transepithelial short-circuit current. Since sodium ion transport under hyperosmotic conditions has been shown to correlate well with the gustatory neural response, the variety of ions transported may likewise indicate a wider role for transport in taste transduction.

Effect of changes in transepithelial transport on the uptake of sodium across the outer surface of the frog skin.

The unidirectional sodium, uptake at the outer surface of the frog skin was measured by the method described by Biber and Curran (8). With bathing solutions containing 6 mM NaCl there is a good correlation between sodium uptake and short-circuit current (SCC) measured simultaneously except that the average uptake is about 40% higher than the average SCC. The discrepancy between uptake and SCC increases approximately in proportion to an increase in sodium concentration of the bathing solutions. Amiloride inhibits the unidirectional sodium uptake by 21 and 69% at a sodium concentration of 115 and 6 mM, respectively. This indicates that amiloride acts on the entry step of sodium but additional effects cannot be excluded. The sodium, uptake is not affected by 10(-4)M ouabain at a sodium concentration of 115 mM but is inhibited by 40% at a sodium concentration of 6 mM. Replacement of air by nitrogen leads to a 40% decrease of sodium uptake at a sodium concentration of 6 mM. The results support the view proposed previously (8) that the sodium uptake is made up of two components, a linear component which is, essentially, not involved in transepithelial movement of sodium and a saturating component which reflects changes in transepithelial transport. Amiloride, seems largely to affect the saturating component.

Coupled solute fluxes in toad skin.

Net inward flux of mannitol across toad skin induced by making the outside solution hypertonic with urea has been investigated. No significant relation between net mannitol flux and net Na flux could be detected when both fluxes were measured simultaneously. In addition, the net mannitol flux caused by hypertonic solution was not altered by inhibition of Na transport with ouabain or by replacement of all Na in the bathing solutions by choline. The rate of net mannitol flux was dependent on the magnitude of the urea concentration difference across the skin and the direction of net flux could be reversed by reversing the direction of the urea concentration difference. These observations suggest that the mannitol transfer is the result of a coupling between the flows of urea and mannitol.

Influence of transepithelial potential difference on the sodium uptake at the outer surface of the isolated frog skin.

The unidirectional uptake of sodium across the outer surface of the isolated frog skin (J(12) (Na)) was measured in the presence of transepithelial potential difference (Delta(psi)) ranging from +100 to -100 mV. With a sodium concentration of 115 mM in the bathing solutions J(12) (Na) increases significantly when the spontaneous Delta(psi) is reduced to zero by short-circuiting the skin. With an Na concentration of 6 mM a progressive increase J(12) (Na) can be observed when Delta(psi) is decreased in several steps from +100 to -100 mV (serosal side positive and negative, respectively). The observed change J(12) (Na) amounts to a fraction only of that predicted from the shift in Delta(psi). The results suggest that under open circuit conditions the potential step across the outside surface is at most one half of Delta(psi) and that the resistance across the outside and inside barrier of the skin is ohmic. This is in agreement with measurements of intracellular potentials in the frog skin and with resistance measurements carried out in the toad skin. The data strongly support the view that the saturating component of J(psi) proceeds via a charged carrier system. Exposure to negative values of Delta(psi) of 50 mV or more for times of 24 min or more result in a marked reduction of J(12) (Na) which shows only partial or no reversibility.

Direct measurement of uptake of sodium at the outer surface of the frog skin.

A combination of the methods described by Schultz et al. (6) and by Ussing and Zerahn (9) was used to measure directly the unidirectional uptake of sodium from the outside solution into the frog skin, under short-circuit conditions. The sodium uptake was determined at six sodium concentrations ranging from 3.4 to 114 mM. NaCl was replaced by choline chloride in the solutions bathing both sides of the skin. Sodium uptake is not a linear function of sodium concentration but appears to be composed of two components, a saturating one and one that varies linearly with concentration. The sodium uptake is inhibited by the addition of lithium to the outside solution. The effect appears to be primarily on the saturating component and has the characteristics of competitive inhibition. In addition, lithium uptake by the skin is inhibited by sodium. The effects of lithium cannot be ascribed to changes in electrical potential difference. Measurements with microelectrodes indicate that under short-circuit condition there is no change in the intracellular potential when lithium chloride is added to the outside solution.

A modified technique for the measurement of sulfhydryl groups oxidized by reactive oxygen intermediates.

This paper suggests a simple modification of the Ellman procedure when used to measure accurate changes in sulfhydryl (-SH) content induced by reactive oxygen intermediates (ROI). This modification became necessary when we found that the standard technique did not produce time invariant results in the presence of ROI-generating systems. Cysteine (cys; 20-100 microM) in 20 mM imidazole buffer (pH 7.0) containing 1.0 mM EDTA was reacted with excess (0.2 mM) 5,5'-dithiobis(2-nitrobenzoic acid), DTNB. The absorbance of the product (p-nitrothiophenol anion) was recorded at 412 nm (A412). This A412 was stable for 60 min and gave a linear relationship with cys concentrations used. ROI were generated either by 0.01 U xanthine oxidase (XO) + 0.01-1.0 mM hypoxanthine (HX), 0.01-1.0 mM H2O2, or H2O2 + 100 microM FeSO4. In the presence of ROI, A412 decreased with time and its rate of decrease was dependent upon the concentration of components of the ROI-generating system. This time-dependent decrease in A412 was prevented completely by the addition of 100 U of catalase (CAT). Therefore, we modified the DTNB method as follows: -SH groups were reacted with ROI for 30 min; this was followed by the addition of 100 U of CAT to scavenge the excess unreacted ROI before the addition of DTNB to generate the product. Using this modification the ROI-induced decrease in A412 was stable with time and was linearly related to the cys concentration. We further tested the modified procedure using metallothionein (MT) as a substrate for the ROI-induced changes in -SH content. MT, at concentrations of 2.5, 5.0, and 7.5 microM, was treated with XO + 100 microM HX.(ABSTRACT TRUNCATED AT 250 WORDS)

Sodium transport and distribution of electrolytes in frog skin.

The objective of this study on frog skin was to examine correlations between transepidermal active Na-transport and intracellular [Na]c, [K]c, [Cl]c homeostasis. Isolated, whole skins, and "split skins" were used in measurements of short-circuit current (SCC) and open skin potential (PD). Water and ion contents were estimated on split skins. Absolute [Na]c and [K]c varied over the range of 18 to 46, and 113 to 80 mM, respectively (Figure 7), but a complementary relationship existed between Na and K, such that [Na]c + [K]c remained approximately equal to 129 mM. Average values for [Na]c and [K]c were approximately equal to 31 and approximately equal to 96 mM, respectively. [Cl]c remained constant at approximately equal to 38 mM. This complementary relationship does not seem to be an artifact, caused by collagenase, used in the preparation of split skins. Whole skins and split skins in Ringer's solution, when treated with fluoroacetate (FAc), ouabain (Ou), or vanadate (Va) over wide ranges of concentrations, showed that FAc greatly depressed the SCC and the PD, without changing [Na]c, [K]c, [Cl]c. FAc acted only from the corium side of the skin. The decreasing SCC remained a Na-current, as in control skins. By comparison, such a separation of cellular functions could not be established with Ou, or Va. These inhibitors either affected SCC, PD, and cellular ion concentration, or they had no effect on any of these parameters. The complementary relationship between [Na]c and [K]c, with [Cl]c remaining again at approximately equal to 38 mM, was also found in tissues exposed to inhibitors. These results indicate that transcellular active Na transport and electrolyte homeostasis are not always rigidly coupled, suggesting that these processes may not be uniformly distributed within the epithelial cells, or among the interconnected cell layers of the frog skin epidermis.

Effect of external cation and anion substitutions on sodium transport in isolated frog skin.

Effects of changes in external ionic strength, external cation and/or anion substitution on transepithelial influx and efflux of sodium, short-circuit current and on transepithelial potential difference and resistance were studied in isolated frog skin. Active transport of Na was found to be highly dependent on both anionic and cationic composition of external medium. Relative abilities of external monovalent cations to inhibit active Na transport were H greater than Li greater than K greater than Rb greater than Cs greater than choline. Relative abilities of external monovalent anions to stimulate active Na transport were I greater than Br greater than Cl. Sequences of anion interaction and of resistance changes suggest that anionic stimulation of Na transport is not due to electrical coupling across outer cell membrane. The ability of different anions and cations to alter Na transport suggests that externally located charged groups act as important barriers or filters to ion movement. In addition, the experiments suggest that an increase in ionic strength of external medium has an effect on active transport of Na, a finding that indicates interference of surface charges with Na entry. Directional changes in efflux of Na due to changes in ionic composition of external medium usually paralleled changes in active Na transport. It is possible that the observed relationship between influx and efflux of Na is the result of common pathways and of interaction of the active transport system with Na efflux.

Active transport and exchange diffusion of Cl across the isolated skin of Rana pipiens.

Transepithelial Cl influx and efflux were measured in pairs of frog skin (Rana pipiens) matched according to short-circuit current, tissue conductance, and transepithelial potential (TEP). The skins were bathed symmetrically in NaCl Ringer and voltage clamped at TEP values ranging from -60 to +60 mV. At 0 TEP, Cl influx and net inward Cl movement (in neq X h-1 X cm-2) were, respectively, 961 +/- 116 and 463 +/- 68 in NaCl Ringer, 509 +/- 52 and 202 +/- 53 in amiloride-treated skins, 4,168 +/- 777 and 1,444 +/- 447 in theophylline-treated skins, and 587 +/- 38 and 97 +/- 44 in Na-free Ringer. A correlation was discovered between short-circuit current and Cl fluxes corresponding to a 2:6:1 relationship between changes in active Na transport and active Cl transport. Deviations from the predicted Cl flux ratio indicate the presence of exchange diffusion in the range of spontaneously occurring TEPs, in contrast to observations on R. temporaria and R. esculenta. The experiments indicate that a substantial portion of transepithelial Cl movement proceeds transcellularly 1) via active Cl transport that is Na dependent, amiloride sensitive, stimulated by theophylline, and apparently correlated with active Na transport, and 2) by means of exchange diffusion that not only occurs under short-circuit conditions but also at positive TEPs. It is possible to explain both the exchange diffusion and the properties of active Cl transport by a Cl-HCO3 exchange system at the apical side of the transporting cell that interacts with a Na-H exchange mechanism, a notion consistent with the recent observation of an amiloride-induced decrease in intracellular pH.

Potential-induced changes in intracellular pH.

In a variety of cell types and tissues there is a strong dependence of intracellular pH (pHi) on membrane potential (Vm). Since cell Vm values can be altered by hormones, ion concentrations, and changes in membrane conductances, the potential-dependent changes in pHi may serve as an important mechanism by which cells can alter their pHi to an environmental stimulus. The H+ flux across the cell membranes is thought to take place via putative H+ channels that are blocked by low concentrations of divalent metal ions. However, in Na(+)-transporting epithelia, a major part of the H+ flux seems to be via the amiloride-sensitive apical Na+ channels, which are not sensitive to divalent metal ions. The H+ flux via the Na+ channels can be modulated by natriferic hormones and intracellular second messengers. The H(+)-conductive pathways may play an important role in signal transduction in some cells.

Effect of inhibitors on transepithelial efflux of Na and nonelectrolytes in frog skin.

The transepithelial efflux of Na and several nonelectrolytes (mannitol, sucrose, and polyethylene glycol 900) were measured in the isolated frog skin under short-circuited conditions in chambers that had been specially designed to avoid edge damage. Ouabain (10(-3) and 10(-4) M) caused a dramatic increase in the efflux of Na, whereas the efflux of nonelectrolytes showed only a slight alteration. The efflux of Na increased after the application of dinitrophenol (10(-4) M), whereas the efflux of nonelectrolytes remained constant. Amiloride (10(-3) M) caused large variations in the efflux of Na, whereas the efflux of nonelectrolytes remained unchanged. The results provide evidence that these inhibitors do not alter the permeability of the paracellular pathway and that the total transepithelial efflux of Na or at least a very large portion of it, and possibly also the transepithelial efflux of Cl, proceeds via a transcellular pathway and not a paracellular pathway as has been widely accepted. The data further suggest that the Na proceeding via this pathway interacts directly or indirectly with the active transport step.

pH in principal cells of frog skin (Rana pipiens): effects of amiloride and potential.

Intracellular pH (pHi) and apical cell membrane potential (Va) were determined in principal cells of frog skin (Rana pipiens) with double-barrel micro-electrodes. In the Northern and Southern varieties, respectively, pHi is 0.38 and 0.26 pH units below bath pH. Amiloride, applied apically, causes reversible intracellular acidification at concentrations of 10(-5) M or higher. Voltage clamp-induced hyperpolarization and depolarization of Va result in intracellular acidification and alkalinization, respectively. This response of pHi is inhibited or abolished when the apical side is treated with 10(-3) M 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). Amiloride-induced intracellular acidification is not exclusively due to the hyperpolarization of Va that accompanies amiloride treatment since 1) amiloride causes greater acidification than equivalent voltage clamp-induced hyperpolarization of Va, 2) amiloride-induced acidification persists in DIDS-treated tissues, and 3) there is no correlation between hyperpolarization of Va and intracellular acidification occurring after amiloride. We conclude that pHi is below the extracellular pH. Amiloride causes intracellular acidification that may be in part connected with hyperpolarization of Va. However, a major component of amiloride-induced acidification is due to other factors, possibly inhibition of apical Na+-H+ exchange. The inhibitory effect of apically applied DIDS suggests that the voltage dependent changes in pHi are related to movement of HCO3 (or OH) ions across the apical cell membrane.

Effect of changes in extracellular Cl on intracellular Cl activity in frog skin.

Intracellular Cl activity was measured in isolated frog skin (Rana pipiens) with double-barrel microelectrodes. The initial rate of Cl uptake was measured in Cl-depleted cells on reexposure to Cl on apical or basolateral side. In skins with high and low conductance, cell CL activity increased 1.33 and 0.14 mM/s with apical reexposure and 5.03 and 0.30 mM/s with basolateral reexposure, respectively. The initial Cl uptake was reduced on the apical side by 93% with 10(-3) M DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid) and on the basolateral side by 99% with 10(-3) M SITS (4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid) plus 10(-5) M bumetanide. The initial rate of Cl loss was measured when Cl was removed from the bath: addition of HCO3 to Cl- and HCO3-free solution caused an acceleration of Cl loss in absence but not in presence of DIDS on apical side. In contrast, Cl loss across the basolateral side was not enhanced by HCO3. In conclusion, Na-transporting cells have a substantial Cl permeability on both sides. HCO3-stimulated Cl loss provides evidence for Cl-HCO3 exchange and permits localization of this process in apical cell membranes of granular cells.