Glucosidase and mannosidase inhibitors mediate increased secretion of mutant alpha1 antitrypsin Z.

It is now well known that the addition and trimming of oligosaccharide side chains during post-translational modification play an important role in determining the fate of secretory, membrane, and lysosomal glycoproteins. Recent studies have suggested that trimming of oligosaccharide side chains also plays a role in the degradation of misfolded glycoproteins as a part of the quality control mechanism of the endoplasmic reticulum (ER). In this study, we examined the effect of several inhibitors of carbohydrate processing on the fate of the misfolded secretory protein alpha1 antitrypsin Z. Retention of this misfolded glycoprotein in the ER of liver cells in the classical form of alpha1 antitrypsin (alpha1-AT) deficiency is associated with severe liver injury and hepatocellular carcinoma and lack of its secretion is associated with destructive lung disease/emphysema. The results show marked alterations in the fate of alpha1 antitrypsin Z (alpha1-ATZ). Indeed, one glucosidase inhibitor, castanospermine (CST), and two mannosidase inhibitors, kifunensine (KIF) and deoxymannojirimycin (DMJ), mediate marked increases in secretion of alpha1-ATZ by distinct mechanisms. The effects of these inhibitors on secretion have interesting implications for our understanding of the quality control apparatus of the ER. These inhibitors may also constitute models for development of additional drugs for chemoprophylaxis of liver injury and emphysema in patients with alpha1-AT deficiency.

Transepithelial electrical responses to sodium and potassium of nonsensory region of gerbil utricle.

Sheets of utricular epithelium from gerbil were mounted in a micro-Ussing chamber. Step changes in [Na] and [K] on each side of the epithelium led to changes in transepithelial potential difference (Vt) and resistance (Rt) which were consistent with the presence of ionic conductances for these two ions. Responses of Vt to 20 mM Rb steps were similar to those to 20 mM K steps. Several common Na and K channel blockers were applied: No evidence for the presence of amiloride-sensitive Na channels was found. At 10(-6) M, amiloride caused no significant changes in Vt but at 10(-3) M it initially decreased or increased Vt when applied from the apical or the basolateral side, respectively. Ba and quinidine each reduced Vt, quinidine more strongly from the basolateral than from the apical side. Tetraethylammonium, another K channel blocker, had no significant effect from either side. These data suggest that in spite of the low value of Rt, the cellular pathway contributes significantly to the voltage response to cation concentration steps in this epithelium. The mode of action of 10(-3) M amiloride and the K channel blockers remains uncertain.

Transepithelial electrical responses to Cl- of nonsensory region of gerbil utricle.

Sheets of utricular epithelium from gerbil were mounted in a micro-Ussing chamber in order to identify and localize chloride conductances. The [Cl-] was rapidly reduced (substituted with isethionate) in the apical or basolateral perfusate and the transepithelial potential difference (Vt) and transepithelial resistance (Rt) were monitored continuously. In addition, agents known to inhibit anion transport in other epithelia were applied. The direction of all initial changes in Vt and Rt due to Cl- substitutions were consistent with the presence of ionic conductances for Cl- on both sides of the epithelium. The time-courses and magnitudes of the fall in Vt and increase in Rt during apical [Cl-] steps in the presence and absence of basolateral bumetanide were monophasic and identical in the two cases. The response of Vt to basolateral [Cl-] steps was biphasic and the initial response was greatly attenuated by bumetanide. These findings demonstrate that the largest conductance for Cl- is in the basolateral cell membrane, but that the paracellular and/or apical pathway also possess a finite Cl- conductance. All three agents tested, 3',5-dichlorodiphenylamine-2-carboxylic acid (DCDPC), 5-nitro-2(3-phenylpropylamino)benzoic acid (NPPB) and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), caused an increase in Vt. NPPB and DIDS were more effective from the apical side. DCDPC and DIDS administered from the apical side led to a decrease in Rt. These results suggest that these agents act in this tissue by enhancing a conductive pathway on the apical membrane rather than blocking the basolateral Cl- conductance.

Potassium secretion by nonsensory region of gerbil utricle in vitro.

The isolated nonsensory region of the gerbil utricle in vitro produced a lumen-positive transepithelial electrical potential difference (VT) of +5.7 mV and a luminal fluid containing 106 mM K when bathed in mammalian Ringer solution (5 mM K and 150 mM Na). The lumen of this region was perfused in vitro with K-free solution and the luminal [K], VT, and transepithelial resistance (RT) were measured before and following perfusion under control conditions and after addition of bumetanide (0.1 mM) or ouabain (1 mM) to the bath. The perfusate contained a reduced [Ca], since the average value of utricular endolymph in vivo (0.28 +/- 0.03 mM) measured with Ca-selective microelectrodes was 38% of that in perilymph. Under control conditions, the luminal [K] initially increased at a rate of 2.13 mumol X cm-2 X h-1 after perfusion; net secretion continued until the luminal [K] returned to its preperfusion level. This flux rate corresponds to 57 microA/cm2. The "equivalent short-circuit current" (Equiv. Isc; VT/RT) was found to average 61 microA/cm2. Both K secretion and VT were fully inhibited by bumetanide and by ouabain. Luminal application of Ba (5 mM) in K-free solution had no effect on the initial rate of K secretion, but did prevent full recovery of luminal [K] to the control level. These results are the first estimates of K secretion by the nonsensory cells of the utricle and are the first to directly demonstrate inhibition of K secretion in the inner ear by bumetanide and in the nonsensory tissue of the utricle by ouabain.

Sidedness of action of loop diuretics and ouabain on nonsensory cells of utricle: a micro-Ussing chamber for inner ear tissues.

It is known that nonsensory tissues of the utricle produce a lumen-positive transepithelial electrical potential difference (VT). This potential has been shown previously to be inhibited by ouabain and bumetanide applied to the bathing medium in vitro. In order to more fully characterize the origin of this potential we mounted the utricle as a flat sheet in a newly designed Ussing chamber and measured the VT and transepithelial resistance (RT) while perfusing the endolymphatic and perilymphatic surfaces independently with identical solutions. The aperture of the chamber was 1.5 X 10(-4) cm2. VT averaged 5.6 +/- 0.46 mV and RT was 24.0 +/- 2.43 omega X cm2 (n = 45). Ouabain and loop diuretics of the furosemide family were found to inhibit the VT only from the serosal side. The KI for ouabain was 7.63 X 10(-5) M. The loop diuretics tested inhibited the VT in the same order as in other tissues known to contain a Na/2 Cl/K cotransporter (KI: 2-benzylamino-4-cyclohexylsulfonyl-5-sulfamoylbenzolsulfonate++ + (BCSB), 1.72 X 10(-7) M; bumetanide, 1.10 X 10(-6) M; piretanide, 5.67 X 10(-6) M; furosemide, 4.14 X 10(-5) M). It is concluded that this tissue produces a lumen-positive VT (i) in the absence of a transepithelial chemical gradient; the generation of which is dependent upon the activity of (ii) Na,K-ATPase and (iii) a Na/2 Cl/K cotransporter; (iv) in the basolateral membranes of the nonsensory cells; (v) which is not depressed by luminal application of inhibitors of these transporters.

Transepithelial cation movements in gerbil utricles.

The inner ear epithelium secretes potassium (K+) and absorbs sodium (Na+). The authors' experiments utilized, for the first time, isolation of the nonsensory from the sensory regions of the utricle in the vestibular labyrinth of the gerbil by means of injecting insulating seals. It was found that the isolated nonsensory region accumulated rubidium (Rb+) (as a marker for K+) in the endolymph to a level of eight times (41 mmol/l) that in the incubation medium (5 mmol/l) over a 90-minute period. However, Na+ rose during this period, suggesting that cells excluded from this preparation (sensory regions) may be involved in Na+ reabsorption. Further observations of the time course of changes in the luminal concentrations of cations suggested that the endpoints observed at 90 minutes represented a new steady state in which both Na+ and K+ concentrations were higher and lower, respectively, than under control conditions (by about 40 mmol/l each), but they were still each displaced from electrochemical equilibrium. This implies that the processes of Na+ absorption and/or K+ secretion may be shared in some way between the two regions. This finding is unexpected, based on previous models of endolymph production, in which ionic regulation has been assigned to nonsensory regions alone.

Support of cochlear metabolic and ion transport processes solely by perilymphatic perfusion.

Morphologic considerations would seem to suggest that the cochlear duct could not be maintained in a fully functional state in the absence of a blood supply. We found, however, that perilymphatic perfusion could be used as a substitute for the normal vascular circulation. The criteria used to determine cochlear function included (1) normal endocochlear potential, (2) normal net secretory flux of rubidium (as a tracer for K), and (3) normal levels of ATP in both the organ of Corti and the stria vascularis. All criteria were satisfied by our perfusion regimen.

Minimal concentrations of metabolic substrates capable of supporting cochlear potentials.

The objective of this study was to determine the capability of glucose analogues, as well as lactate and pyruvate, to maintain the endolymphatic potential and the cochlear microphonics. In addition, the minimum concentration at which different substrates (including D-glucose) were able to sustain the potentials ("critical' concentration) was determined. Synthetic blood containing various substrates at different concentrations was perfused via the anterior inferior cerebellar artery. The critical concentration for D-glucose was found to be 15 mg% (0.83 mM). L-Glucose, galactose and fructose were not able to support the potentials at concentrations as high as 200 mg%. On the other hand, mannose was capable of supporting the potentials; however, the critical concentration (50 mg% or 2.8 mM) was substantially higher than that of D-glucose. Both lactate and pyruvate could support the potentials, but the critical concentrations (8.5 mM and 6.5 mM, respectively) were markedly higher than in the case of glucose, even when the difference of carbon equivalents was taken into consideration. The data are discussed in the context of the intermediary metabolism and possible carrier systems of the stria vascularis.

Effect of substrate-free vascular perfusion upon cochlear potentials and glycogen of the stria vascularis.

The effect of vascular perfusion of the anterior inferior cerebellar artery with synthetic blood containing no metabolic substrates upon the endolymphatic potential (EP) and the cochlear microphonics (CM) was determined in the guinea pig. In substrate-free perfusion the potentials were maintained for an average of 84 min. Subsequently, the EP declined at an average rate of 1.4 mV/min until a new steady-state level was temporarily established when the potential had dropped to about 30 mV. The decline of the CM appeared to be accounted for largely by the decline of the EP. During substrate-free perfusion prior to the onset of the decline of the potentials, the level of strial glycogen remained unchanged; glycogen decreased significantly only after the potentials had started to decline. When substrate-free vascular perfusion was accompanied by simultaneous substrate-free perilymphatic perfusion, the potentials started to decline immediately. On the basis of these data, we conclude that strial glycogen plays no role in the prolonged maintenance of the EP during substrate-free perfusion; rather, the potential seems to be maintained by entry of glucose (and presumably other substrates) from perilymph into the stria vascularis.

Adenine nucleotides of the organ of Corti under metabolic stress.

The purpose of the reported experiments was to measure the concentrations of adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) in the organ of Corti in order to arrive at estimates of three commonly used adenylate ratios. Under normal conditions the concentrations of ATP, ADP, and AMP amounted to 15.8, 3.9, and 0.53 mmoles/kg dry weight, respectively. Of the three substances, AMP is the most sensitive indicator of metabolic stress, since ischemia of 65 seconds leads to an increase of 155%. Under normal conditions the adenylate energy charge, the energy status, and the phosphorylation state amounted to 0.83, 4.1, and 2.5 gram wet weight/mumole, respectively. Within 10 minutes of ischemia the energy charge had declined by 26%, the energy status by 50%, and the phosphorylation state by 76%. The apparent equilibrium constant of adenylate kinase of the organ of Corti was found to be 0.55. The potential significance of these data and their relationship to the situation in the stria vascularis are discussed.

Changes in cation contents of stria vascularis with ouabain and potassium-free perfusion.

Perfusion of the perilymphatic space of guinea pig cochleae with K-free medium leads to a gradual decline of the endocochlear potential (EP) over 30-50 min to a negative value (mean: -12 mV). The input resistance of scala media does not decrease during this time. The ATP and K content of the stria vascularis are reduced by similar amounts (26 and 34%, respectively) during this period. Perfusion of 1 mM ouabain produces a different pattern of response: strial ATP remains normal while strial K content is strongly reduced (by 77%). Strial Na rises in a complementary way to the K loss. These results demonstrate that a reduction of the K concentration of the perilymph leads to an inhibition of the generator of the positive component of the EP rather than to a general increase of cochlear duct membrane conductance. In addition, they suggest, in concert with other considerations (such as the slower rate of decline of the EP during K-free vascular perfusion (Wada, J., Kambayashi, J., Marcus, D.C. and Thalmann, R (1979): Arch. Otorhinolaryngol. 225, 79-81)), that the mode of action may be different from that of ouabain. In spite of the lack of teleological support, we offer the hypothesis that the strial generator of the EP may primarily utilize K from perilymph and that vascular K may not have access to the generator.

Adenine nucleotides of the stria vascularis.

The levels of the adenine nucleotides ATP, ADP, and AMP in the stria vascularis were measured under normal conditions, and following various durations of ischemia. The concentrations of these compounds were used for the calculation of the adenylate energy charge, the energy status and the phosphorylation state of the stria. Following 10 min of ischemia the adenylate energy charge had decreased three fold, the energy status seven fold and the phosphorylation state 14 fold. To study the potential for recovery of strial function following various brief and prolonged ischemic intervals, a method for the perfusion of the ear via the anterior inferior cerebellar artery was developed. For various reasons it was found advantageous to use "artifical blood" as perfusate, relying upon fluorocarbons as oxygen carriers. The endolymphatic potential was used as electrical indicator of strial function. Recovery of the endolymphatic potential following brief periods of ischemia was paralleled by a corresponding increase of the ATP levels and a drastic decrease of the AMP levels of the stria vascularis. Preliminary results on the effects of substrate-free perfusion are presented.

Adenylate energy charge, energy status, and phosphorylation state of stria vascularis under metabolic stress.

The purpose of the reported experiments was to measure the strial concentrations of adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) in order to arrive at estimates of three commonly used adenylate ratios. Under normal conditions, the concentrations of ATP, ADP, and AMP were found to be 11.4, 3.7, and 0.6 mmoles/kg dry weight, respectively. Of the three substances, AMP is the most sensitive indicator of metabolic stress, since its concentration doubles within 6 sec. of ischemia and reaches a peak level of about 1500% of the control following 65 sec. of ischemia. Under normal conditions, the "adenylate energy charge," the "energy status," and the "phosphorylation state" amount to 0.84, 3.0, and 1.52 gram wet weight/mumole, respectively. In ischemia of 10 min. duration, the adenylate energy charge decreases 3 fold, the energy status 7 fold and the phosphorylation state 14 fold. The size of the adenylate pool shows a slight increase in the earliest stage of ischemia, but declines progressively thereafter. The apparent equilibrium constant of strial adenylate kinase was found to be 0.48. The advantages and limitations of the different adenylate ratios, as indicators of metabolic health and as regulatory parameters, are discussed.

Respiratory rate and ATP content of stria vascularis of guinea pig in vitro.

Stria vascularis from guinea pig cochleae was incubated in vitro to determine its metabolic response to variations in substrate and ion composition of the incubation medium. The respiratory rate at 37 degrees C in a medium containing glucose and pyruvate as substrate was 17.3 +/- 1.33 (SEM, n = 51) microliter O2/mg dry weight-hour. The stria could not maintain constant respiration by relying solely upon endogenous fuel stores. With substrate supplied, the ATP level could be maintained at about 73% of that existing in vivo. Glucose appears to be an adequate substrate for stria in vitro since glutamate, pyruvate, and fumarate did not increase the respiratory rate. Succinate increased respiration markedly but did not increase the ATP level. Ouabain (10(-4) M) caused a 48% decrease in the respiratory rate. Incubation in Na+-free and K"-free medium, each resulted in irreversible decrease of respiratory rate comparable to (or greater than) that caused by ouabain. These data are in accord with the high activity of Na+-K+-ATPase in the stria and the pronounced sensitivity of the endolymphatic potential to ouabain.

Respiratory quotient of stria vascularis of guinea pig in vitro.

The respiratory quotient of the stria vascularis was measured in vitro by means of Cartesian diver microgasometry. A value of about 1.2 was found when the incubation medium was phosphate-buffered serum substitute with glucose as the sole substrate. This value suggests that endogenous lipids and amino acids do not contribute significantly to strial respiratory metabolism and that carbohydrate is the primary fuel in vitro. A high activity of the hexose monophosphate pathway may be responsible for raising the respiroatyr quotient above unity.